Hot Spot-Based design and synthesis of small molecule inhibitors for β-Catenin/T-cell factor 4 protein-protein interaction and hemiporphyrin-like N-ACYL 2-aminoimidazoles: chemistry, biology and zinc binding properties

The β-Catenin/T-cell factor protein complex is a key component in the Wnt signaling pathway and is found to be dysregulated in cancer. This complex has been the target of many high throughput screening (HTS) campaigns. Although HTS leads have been identified, the lack of knowledge about binding modes of the complex prohibited structural optimizations. The Ji lab has analyzed its crystal structure and identified several key interactions. Using bioisosteric replacement, fragments matching the binding criteria found in the protein structure were designed to mimic the endogenous Tcf. Chapter 1 details the design, synthesis and biology of N-acyl sulfonamide bioisosteres. These molecules mimic 2 carboxylic acid side chains of Tcf with tetrazole and N-acyl sulfonamide moieties. Chapter 2 describes the structural modifications of the previous inhibitors, which allowed installation of other functionalities to engage additional binding elements. Furthermore, biochemical and celluar assays were performed to evaluate their efficacy. Naamidine A is a 2-aminoimidazole alkaloid from the marine sponge Leucetta chagosensis. It displays promising antitumor activities in vitro and in vivo. The Looper lab has reported a modular synthesis towards naamidine A, which allowed access to a range of N2-acyl-2-aminoimidazoles. One of them, termed ZNA, emerged as a hit during a screening effort. ZNA killed chemo-resistant cancer cells while leaving healthy cells unaffected. In collaboration with the Welm lab, we found that ZNA was an ionophore and shuttled Zn2+ iv ions across the cell membrane resulting in zinc dyshomeostasis and apoptosis. However, a preformed dimeric Zn-ZNA complex was biologically inactive, which could have been caused by the insolubility of the complex. In Chapter 3, we synthesized ZNA analogs with solubilizing side chains and evaluated these compounds in different cell lines. Chapter 4 details the biological importance of Zn/ZNA monomeric complexes. We generated several analogs with pendant C4-pyridine side chains that are capable of invoking a tridentate monomeric zinc complex. The preparation of these molecules revealed a mechanistically distinct and stereodefined amino-silylation reaction that has not been previously observed, offering access to new substitution patterns of the cyclic ene-guanidine scaffold. Chapter 5 details the reaction optimization and substrate scope of this transformation.

HOT SPOT-BASED DESIGN AND SYNTHESIS OF SMALL MOLECULE INHIBITORS FOR β-CATENIN/T-CELL FACTOR 4 PROTEINPROTEIN INTERACTION AND HEMIPORPHYRIN-LIKE N2-ACYL 2-AMINOIMIDAZOLES: CHEMISTRY, BIOLOGY AND ZINC BINDING PROPERTIES by Wenxing Guo A dissertation submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Chemistry The University of Utah May 2019 Copyright © Wenxing Guo 2019 All Rights Reserved The University of Utah Graduate School STATEMENT OF DISSERTATION APPROVAL The dissertation of Wenxing Guo has been approved by the following supervisory committee members: Ryan E. Looper , Chair 11/26/2018 Date Approved Matthew S. Sigman , Member Date Approved Janis Louie , Member 11/26/2018 Date Approved Andrew G. Roberts , Member 11/26/2018 Date Approved Carol S. Lim , Member Date Approved and by Cynthia J. Burrows the Department/College/School of and by David B. Kieda, Dean of The Graduate School. , Chair/Dean of Chemistry ABSTRACT The β-Catenin/T-cell factor protein complex is a key component in the Wnt signaling pathway and is found to be dysregulated in cancer. This complex has been the target of many high throughput screening (HTS) campaigns. Although HTS leads have been identified, the lack of knowledge about binding modes of the complex prohibited structural optimizations. The Ji lab has analyzed its crystal structure and identified several key interactions. Using bioisosteric replacement, fragments matching the binding criteria found in the protein structure were designed to mimic the endogenous Tcf. Chapter 1 details the design, synthesis and biology of N-acyl sulfonamide bioisosteres. These molecules mimic 2 carboxylic acid side chains of Tcf with tetrazole and N-acyl sulfonamide moieties. Chapter 2 describes the structural modifications of the previous inhibitors, which allowed installation of other functionalities to engage additional binding elements. Furthermore, biochemical and celluar assays were performed to evaluate their efficacy. Naamidine A is a 2-aminoimidazole alkaloid from the marine sponge Leucetta chagosensis. It displays promising antitumor activities in vitro and in vivo. The Looper lab has reported a modular synthesis towards naamidine A, which allowed access to a range of N2-acyl-2-aminoimidazoles. One of them, termed ZNA, emerged as a hit during a screening effort. ZNA killed chemo-resistant cancer cells while leaving healthy cells unaffected. In collaboration with the Welm lab, we found that ZNA was an ionophore and shuttled Zn2+ ions across the cell membrane resulting in zinc dyshomeostasis and apoptosis. However, a preformed dimeric Zn-ZNA complex was biologically inactive, which could have been caused by the insolubility of the complex. In Chapter 3, we synthesized ZNA analogs with solubilizing side chains and evaluated these compounds in different cell lines. Chapter 4 details the biological importance of Zn/ZNA monomeric complexes. We generated several analogs with pendant C4-pyridine side chains that are capable of invoking a tridentate monomeric zinc complex. The preparation of these molecules revealed a mechanistically distinct and stereodefined amino-silylation reaction that has not been previously observed, offering access to new substitution patterns of the cyclic ene-guanidine scaffold. Chapter 5 details the reaction optimization and substrate scope of this transformation. iv TABLE OF CONTENTS ABSTRACT .................................................................................................................. iii LIST OF ABBREVIATIONS....................................................................................... viii ACKNOWLEDGMENTS .............................................................................................. xi Chapters 1. COMPUTER-GUIDED DESIGN AND SYNTHESIS OF SMALL-MOLECULE INHIBITORS FOR CATENIN/TCF PROTEIN-PROTEIN INTERACTIONS USING BIOISOSTERE REPLACEMENT .................................................................................. 1 1.1 Introduction ...................................................................................................... 1 1.1.1 Brief History of Wnt Signaling Pathway ............................................... 1 1.1.2 Mechanism of Wnt Secretion and Signaling .......................................... 2 1.1.3 Wnt and Cancer..................................................................................... 4 1.1.4 Drugging Wnt Pathway and its Components ......................................... 5 1.1.5 β-Catenin/Tcf4 Complex as Therapeutic Target .................................... 7 1.1.6 Design and Syntheses of Inhibitors by Bioisostere Replacement............ 8 1.2 Results and Discussion ................................................................................... 12 1.2.1 Design, Synthesis and Biological Evaluation of N-acyl Sulfonamide Bioisosteres ................................................................................................. 12 1.2.2 Biological Evaluation and Discussion.................................................. 13 1.3 Conclusion ..................................................................................................... 16 1.4 References ...................................................................................................... 24 1.5 Supporting Information................................................................................... 32 1.5.1 Protein Structure for Computer Modeling............................................ 32 1.5.2 AutoDock4 Study................................................................................ 33 1.5.3 Fluorescence Polarization Assays ........................................................ 33 1.5.4 General Experimental Conditions (Chemistry) .................................... 35 1.5.5 Procedures and Characterizations ........................................................ 36 2. STRUCTURE-BASED MODIFICATION AND OPTIMIZATION OF 2,3 DISUBSTITUTED INDOLES AS POTENT INHIBITORS OF β-CATENIN/TCF PROTEIN-PROTEIN INTERACTIONS ....................................................................... 69 2.1 Introduction .................................................................................................... 69 2.1.1 Characteristics of Protein-Protein Interactions ..................................... 69 2.1.2 Design of 3-Substituted Indole N-acyl-sulfonamides and Related Compounds.................................................................................................. 71 2.2 Results and Discussion ................................................................................... 72 2.2.1 Design and Syntheses of 3-Substituted Indole N-acyl-sulfonamides .... 72 2.2.2 Biological Evaluations of 3-Substituted Indole N-acyl-sulfonamides ... 72 2.2.3 Design and Syntheses of 2,3-Disubstituted Indole-5-Carboxylic Acids 75 2.2.4 Biological Evaluation of 2,3-Disubstituted Indole-5-Carboxylic Acids 77 2.3 Conclusion ..................................................................................................... 79 2.4 References ...................................................................................................... 90 2.5 Supporting Information................................................................................... 94 2.5.1 Protein Structure for Computer Modeling............................................ 94 2.5.2 AutoDock4 Study................................................................................ 94 2.5.3 Fluorescence Polarization Assays ........................................................ 95 2.5.4 Site-Directed Mutagenesis Studies ...................................................... 97 2.5.5 MTS Cell Viability Assay ................................................................... 97 2.5.6 Cell Transfection and Luciferase Reporter Assay ................................ 98 2.5.7 General Experimental Conditions (Chemistry) .................................... 98 2.5.8 Procedures and Characterizations ........................................................ 99 3. SYNTHESIS AND BIOLOGICAL PROPERTIES OF ZNA-DERIVATIVES WITH WATER-SOLUBLE SIDE CHAINS .......................................................................... 251 3.1 Introduction .................................................................................................. 251 3.1.1 Naamidine and Naamine Natural Products from Leucetta Chagosensis ............................................................................................... 251 3.1.2 Biology of Naamidine Alkaloids ....................................................... 252 3.1.3 Syntheses and Structural Modifications of Naamidines ..................... 254 3.1.4 Biological Evaluations of Zinaamidole .............................................. 256 3.1.5 Zinc Ionophores in Medicinal Chemistry ........................................... 259 3.1.6 Mechanism of Action of ZNA ........................................................... 260 3.1.7 Biological Evaluation of ZNA Derivatives ........................................ 261 3.2 Results and Discussion ................................................................................. 262 3.2.1 ZNA-Derivatives with Water-Solubilizing Groups ............................ 262 3.2.2 Syntheses of ZNA with Solubilizing Groups ..................................... 263 3.2.3 Biological Evaluations and Discussions............................................. 264 3.3 Conclusion and Future Directions ................................................................. 266 3.4 References .................................................................................................... 277 3.5 Supporting Information................................................................................. 281 3.5.1 Experimental (Biology) ..................................................................... 281 3.5.2 General Experimental Conditions (Chemistry) .................................. 281 3.5.3 Procedures and Characterizations ...................................................... 283 4. SYNTHESES, BIOLOGICAL PROPERTIES AND ZINC BINDING MODE OF HEMIPORPHYRIN-LIKE ZNAS ............................................................................... 364 4.1 Introduction .................................................................................................. 364 4.2 Results and Discussion ................................................................................. 365 4.2.1 Syntheses of Hemiporphyrin-like ZNA Analogs................................ 365 4.2.2 Biological Evaluation and Discussion................................................ 367 vi 4.2.3 Zinc Binding Modes of ZNA 131, ZNA 148 and ZNA 194............... 369 4.3 Conclusion and Future Directions ................................................................. 372 4.4 References .................................................................................................... 380 4.5 Supporting Information………...……………………………………………..381 4.5.1 Experimental (Biology) ..................................................................... 381 4.5.2 General Experimental Conditions (Chemistry) .................................. 381 4.5.3 Procedures and Characterizations ...................................................... 382 4.5.4 Crystal Structure Report for C1 ......................................................... 515 4.5.5 Crystal Structure Report for C2 ......................................................... 536 4.5.6 Crystal Structure Report for C3 ......................................................... 564 4.5.7 Crystal Structure Report for C4 ......................................................... 581 5. STEREOSELECTIVE SYNTHESIS OF CYCLIC ENE-GUANIDINES USING A GUANYLATION/AMINOSILYLATION CASCADE REACTION ........................... 600 5.1 Introduction .................................................................................................. 600 5.2 Results and Discussion ................................................................................. 602 5.2.1 A Stereodefined Aminosilylation Reaction ........................................ 602 5.2.2 Reaction Optimization....................................................................... 603 5.2.3 Substrate Scope ................................................................................. 604 5.3 Putative Mechanism for the Guanylation/Aminosilylation Reaction .............. 606 5.4 Summary ...................................................................................................... 607 5.5 References .................................................................................................... 614 5.6 Supporting Information................................................................................. 616 5.6.1 General Experimental Conditions (Chemistry) .................................. 616 5.6.2 Procedures and Characterizations ...................................................... 617 5.6.3 Crystal Structure Report for 4.6.2 ...................................................... 715 5.6.4 Crystal Structure Report for 5.2.9 ...................................................... 735 vii LIST OF ABBREVIATIONS AcCl acetyl chloride AcOH acetic acid AgOAc silver(I) acetate AgOTf silver(I) triflate Bn benzyl Boc tert-butoxycarbonyl BSA N,O-(bistrimethylsilyl)acetamide CDCl3 deuterated chloroform CDI 1,1-carbodiimdazole CsF cesium(I) fluoride CH2Cl2 dichloromethane (COCl)2 oxalyl chloride CuBr copper(I) bromide CuI copper(I) iodide DBU 1,8-Diazabicyclo[5.4.0]undec-7-ene DIPEA N,N-diisopropylethylamine DMBA N,N-dimethylbarbituric acid DMF dimethylformamide DTAT Di-tert-butyl azodicarboxylate EDCI ethyl-1-dimethylaminopropylcarbodiimide hydrochloride Et2O diethylether Et3N triethylamine EtOAc EtOAc EtOH ethanol HCl hydrogen chloride HCOOH formic acid HgCl2 mercury(II) chloride HOAt 1-hydroxy-7-azabenzotriazole i-Pr isopropyl i-PrOH isopropanol K2CO3 potassium carbonate KF potassium fluoride Me methyl MeCN acetonitrile MeI iodomethane MeOH methanol MgSO4 magnesium sulfate Na2SO4 sodium sulfate NaHCO3 sodium bicarbonate NaBH4 sodium borohydride NaH sodium hydride NaI sodium iodide ix NaOH sodium hydroxide NH4OAc ammonium acetate NH2OH . HCl hydroxylamine hydrochloride n-BuLi n-butyllithium NH4Cl ammonium chloride Pd/C palladium on activated carbon Pd(OAc)2 palladium(II) acetate Pd(PPh3)4 tetrakis(triphenylphosphine)palladium(0) Ph phenyl PhMe toluene PPh3 triphenylphosphine n-Bu3SnN3 n-tributyl tin azide TBAF tetrabutyl ammonium fluoride TBDMSCl tert-butyldimethylsilyl chloride TMSCN trimethylsilyl cyanide THF tetrahydrofuran TFA trifluoroacetic acid TMS trimethylsilyl Zn(BF4)2 zinc(II) tetrafluoroborate ZnCl2 zinc chloride ZnSO4 zinc sulfate x ACKNOWLEDGMENTS First and foremost, I would like to express my deepest gratitude to my research advisor Dr. Ryan E. Looper for allowing me to be part of this lab. Looking back at the last six years, my PhD path has not been always very easy, but he has always been very supportive and provided guidance and advice along the way. I am very grateful to work with such an amazing crew of young and talented scientists in the Looper lab, Matthew Nelli, Chelsea Harmon, Chintelle James, Samuel Broadbent, Kendall Heitmeier, Kason Glover and all the former members. I also would like to give a special thanks to my bench mate Justin M. Salvant. He is a brilliant and skilled organic chemist and aspiring music producer, who helped me a lot on our ZNA project in the last two years. My gratitude also goes to Srinavas Reddy Paladugu who is an extremely knowledgeable chemist and helped me understand complex (and easy) organic mechanisms. I am grateful to have worked with all the members of the Curza team, Travis, Chad, Michael, Seth, Hari and especially Dr. Paul Sebahar, for being available any time I had questions regarding organic or medicinal chemistry, and for providing an “entertaining” research environment. I would like to thank Dr. Katrin Guillen from the Welm lab for performing all the biochemical and cellular assay and a special thanks to Dr. Ryan Vanderlinden for solving so many crystal structures. Let’s not forget my former lab mates from the Ji unit, Dr. Huang Zheng, Dr. Leon Catrow, Dr. Yongqiang Zhang, Dr. Binxun, Jack Wisniewski, Kevin Teuscher, Cory Jensen, Vanja Panic, and others. I was fortunate to work with all these talented and motivated chemists and biologists for four years. I would also like to thank my former advisor Dr. Haitao (Mark) Ji and his wife Dr. Min Zhang who have taught me the true definition of hard work. My gratitude also goes to all the staff members of the chemistry department who helped me along the way in the last six years: Jo Vallejo, Dr. Jim Mueller, Dr. Chen, Dr. Atta Arif, Dr. Peter Flynn and Dennis Edwards. I would also like to thank members of my committee, Dr. Janis Louie, Dr. Matthew Sigman, Dr. Andrew Roberts and Dr. Carol Lim for guidance, support and insightful discussions. Finally, I am very grateful for my girlfriend Yuqing Qiu for being by my side throughout grad school. xii CHAPTER 1 COMPUTER-GUIDED DESIGN AND SYNTHESIS OF SMALL-MOLECULE INHIBITORS FOR CATENIN/TCF PROTEIN-PROTEIN INTERACTIONS USING BIOISOSTERE REPLACEMENT 1.1 Introduction Signaling pathways are crucial for cellular function. These pathways control a variety of biological processes in the cell, including proliferation, apoptosis, and metabolism. Within a complex cellular network, a myriad of proteins are the workhorse of these pathways. Triggered by an external stimulus, the signal is propagated downstream by the interaction of specific proteins. On a molecular level, these binding events give rise to a succession of protein modifications, e.g., phosphorylations or acetylations. These modifications are interpreted as signals that elicit specific cellular responses, generally culminating in the activation or deactivation of gene expression. 1.1.1 Brief History of Wnt Signaling Pathway One such pathway is the Wnt signaling pathway, which is pivotal for cellular development and has been studied extensively.1-4 Wnt is divided into canonical (β-catenin dependent) and noncanonical (β-catenin independent) pathways. The canonical pathway is the focus of this dissertation. 2 The word Wnt is a portmanteau composed of two parts, W for wingless and nt for integration site. The latter part originated from the pioneering work of Nusse and Vamus.5 The studies focused on Mouse Mammary Tumour Virus (MMTV), a retrovirus known to induce breast cancer in certain laboratory murine strains.6 Although several strains of oncogenic retroviruses were known at the time, their cancer inducing mechanism of action remained elusive. However, they demonstrated that the MMTV provirus shared a common integration site within the mouse genome amongst different tumors. Moreover, upon insertion, a proto-oncogene was transcriptionally activated and dubbed as int1. In addition, it was found that Wingless, a mutant gene causing the loss of wing tissue in Drosophila melanogaster flies, is the homologue of int1.7 With regard to these discoveries, the two gene names were merged into Wnt1.8 Soon after, additional genes from the Wnt family were identified across the animal kingdom.9 The human and mouse genome contains 19 Wnt-related genes, whereas other species, such as Hydra vulgaris, only have 13. In contrast, single cell organisms, as well as plants, do not carry Wnt genes.10 1.1.2 Mechanism of Wnt Secretion and Signaling The Wnt gene encodes Wnt protein, a 40 kDa, evolutionarily conserved, cysteine rich growth factor. During post-translational modification, Wnt will be attached to a palmitoleic acid chain by porcupine, a palmitoyl transferase enzyme, harbored in the endoplasmic reticulum (ER)-membrane (Figure. 1.1).11-12 This process provides Wnt the necessary structural motif to execute the following events throughout its transport pathway.13 First, the transmembrane protein Wntless/Evi (Wls) residing in close proximity to porcupine in the ER will bind to the fatty acid chain of Wnt. Subsequently, with the aid 3 of secretory vesicles, the Wls/Wnt complex will be shuttled through the Golgi apparatus to the cell surface, where Wnt is released in a pH-dependent manner and secreted.14 Following this, it will be ferried by extracellular vesicles15-16 to reach the transmembrane receptor proteins, Lipoprotein related protein 5/6 (LRP 5/6), and Frizzled (Fzd) of a neighboring cell. The latter protein possesses a hydrophobic pocket,17 which interacts with the fatty acid chain of Wnt and thus significantly contributes to the overall binding affinity between ligand and receptor. Modified Wnts are hydrophobic in order to prevent long-range intercellular migrations. Therefore, the current hypothesis posits that the pathway operates in a paracrine and/or autocrine manner.4 β-Catenin is a 90 kDa multifunctional protein, which regulates cell-cell adhesion with E-cadherin.18 More importantly, it is a key component in the canonical Wnt pathway. The location and actions of β-catenin in the cell dictate the transcriptional output, in other words, whether Wnt signaling is on or off (Figure 1.2, adapted from reference 4). In the off-state (Wnt unbound), a multiprotein “destruction complex” (DC)19 targets excess cytosolic β-catenin for degradation (Figure. 1.2 left). This protein assembly contains tumor suppressor protein Axin, Adenomatous Polyposis Coli (APC), Ser/Thr kinases Glycogen Synthase Kinase 3 (GSK-3), Casein Kinase 1 (CK1) and E3-ubiquitin ligase F-box/WD repeat containing protein (β-TrCP). APC functions as a scaffold,20-21 using different domains to interact with Axin, β-catenin, GSK3, and CK1. The latter two kinases phosphorylate APC in order to enhance their target protein affinities.22-23 Simultaneously, a series of serine phosphorylations take place on β-catenin24-25 to create a binding motif for β-TrCP ubiquitination. Finally, the ubiquitinated β-catenin is degraded by proteasomes. Meanwhile, in the cell nucleus, the DNA-bound T-cell transcription factor (Tcf) in 4 complex with Groucho acts as a Wnt target gene repressor, thus preventing transcriptional activation.26 Upon receptor engagement by Wnt protein (Wnt-on, Figure 1.2 right),27 the destruction complex translocates upstream to bind to the phosphorylated tail of LRP. Consequently, GSK-3 is inhibited, dismantling DC from its task to down-regulate β-catenin levels in the cell. Therefore, β-catenin proteins accumulate in the cytoplasm and translocate into the cell nucleus. There, they replace Groucho and bind to Tcf (or Tcf4 in humans) to initiate gene transcription. On a cellular level, Wnt-mediated gene transcription is responsible for a broad spectrum of biological phenotypes such as cell proliferation, differentiation, polarity or apoptosis.28 These transcriptions can take place during embryogenesis, but are also implicated in adult tissue homeostasis. For instance, the Wnt signaling is engaged in almost all phases of the cell cycle.29 It invokes the expression of cyclin proteins, which serve as check point modulators (G1) and help organize and segregate chromosomes (mitosis). Moreover, the Wnt signal functions as an internal “compass” by establishing a vectorial factor during cell division and cell migration. This process establishes a certain degree of cellular asymmetry throughout morphogenesis, giving tissues the requisite shape and pattern to function properly.30 1.1.3 Wnt and Cancer Unsurprisingly, with Wnt signaling playing a fundamental role in all stages of cellular development, it is implicated in a panoply of diseases. These diseases are mostly caused by mutations of components within the pathway, leading to aberrant overactivation 5 of Wnt signaling. Neurological diseases,31 fibrosis32 and most importantly cancers have been studied in connection with Wnt. Table 1.133 summarizes affected components and their contribution towards tumorigenesis. A well-known example is the loss-of-function mutation of APC and its close relationship with colon cancer. It was the first mutant gene discovered in aberrantly overactivated Wnt pathway34 and is present in ~80% of sporadic colorectal tumors.40 This striking number, in addition to the fact that 93% of all types of colon cancers are caused by mutation of Wnt components,41 highlights the urgent need for the development of safe and effective therapeutics. 1.1.4 Drugging Wnt Pathway and its Components To date, no selective small-molecules Wnt pathway antagonists have been approved by the FDA. Development of drugs that target signaling pathways remains a significant challenge, largely due to risk of unintended interference with basic developmental processes, thus becoming a teratogenic toxin. Hence, it is critical to choose the right target in order to avoid creating a drug with antithetical qualities.42 In the modern world of drug discovery, fast-paced biochemical or virtual screening processes can be conducted to narrow down potential drug candidates from a library of chemical compounds. For instance, TOP-Flash transcriptional reporter assay (also termed luciferase reporter assay)43 has been frequently applied as a cell-based method to identify Wnt modulators.44-47 Some of them (Figure 1.3 left) are nontoxic polyphenol natural products isolated from plants. Despite exhibiting biological activity, no studies were conducted to elucidate mechanisms of action or molecular targets. Given the structural features, it was believed that they might function as ubiquitous redox active molecules48 6 and no following optimizations were pursued. Several active compounds targeting specific components of the Wnt pathway have been studied and developed. They can be divided into the following three classes: Fzd and porcupine inhibitors (upstream effector, Figure 1.3 right), Axin stabilizer (DS agonist, Figure 1.3 right) and β-catenin/Tcf antagonist (downstream effector, Figure 1.4). The Fzd/porcupine inhibitors have been found to suppress the initiation of signaling cascade by either blocking the receptor protein or disabling Wnt palmitoylation. Chen et al.49 have demonstrated that niclosamide, an anthelmintic used to treat tapeworm, promotes Fzd internalization, which leads to decreased Wnt-mediated gene expression. Furthermore, it exihibits antiproliferative effects in a Wnt-activated cancer cell line. Another report by Lum and co-workers50 described two groups of compounds, IWP and IWR, to be highly potent Wnt signaling antagonists. The former prevents Wnt production by binding the enzyme porcupine, whereas the latter prevents Wnt mediated response by inducing Axin expression. The authors suggested that higher Axin concentration could compensate for the loss of function in APC and down-regulation of β-catenin. However, targeting molecular components upstream of the signaling pathway might not be a safe strategy. For instance, niclosamide and IWP may shut down Wntmediated gene expression, but these drugs do not differentiate between canonical or noncanonical pathways. With the human genome encoding 10 Fzd and 19 Wnt protein isoforms,3 the number of potential combinations is numerous leading to a wide umbrella of downstream effects. For instance, noncanonical planar cell polarity (PCP) pathway governs the orientation of epithelial structures,51 whereas Wnt/Ca2+ pathway can act as a natural antagonist of β-catenin/Tcf4 signaling.52 Therefore, this approach contains 7 potential therapeutic risks and can lead to undesired side effects. The other option to stabilize Axin appears to be more advantageous, since the DS is not critical to the noncanonical pathways. But it will not be therapeutically effective if mutations occur further downstream in β-catenin for instance. Instead, targeting β-catenin/Tcf4 proteinprotein-interaction (PPI) could represent a viable strategy for the following reasons. First and foremost, overactivation of Wnt results in a gain-of-function for β-catenin. Consequently, developing inhibitors to disrupt interactions between β-catenin and Tcf4 would produce the desired therapeutic outcome. Second, almost all mutations within the pathway ultimately lead to increased formation of β-catenin/Tcf4 complex. Thus, counteracting this event could also block any dysregulation upstream from the nucleus. Lastly, it has been shown that deletion of the β-catenin gene reduces the growth of cancer cells.53 1.1.5 β-Catenin/Tcf4 Complex as Therapeutic Target Potential inhibitors from natural product library and NCI database have been tested for their Wnt specificity using a luciferase reporter assay. Those with low micromolar IC50 values were further validated in protein specific assays such as fluorescence polarizationassay (FP) or AlphaScreen. Figure 1.4 summarizes recently discovered inhibitors and their corresponding IC50 values.54 CGP049090 and PKF115-584,55 both high molecular weight compounds, showed high affinity towards β-catenin and disrupted its interaction with Tcf. However, these compounds interfered with APC/β-catenin PPI and DNA/Tcf binding. Due to their polycyclic aromatic systems, CGP049090 and PKF115-584 were likely acting as DNA 8 intercalators. Some other screening hits including quinones and toxoflavin (PKF118-310) were redox active and possibly interfered with many biochemical assays.56 Hence the results should be taken with skepticism due to lack of method to eliminate nonspecific binding effects (false positives). Li and co-workers57 showed that the natural product henryrin induced apoptosis specifically in Wnt dependent cancer cell lines such as SW480, whereas Wnt independent cancer cell line A549 and normal human cell lines were not affected. Immunoprecipitation experiments revealed its molecular target to be βcatenin/Tcf complex. But no biochemical assays were performed to determine specific binding modes. BC2158 was discovered from in silico screening targeting β-catenin/Tcf4 PPI. In biochemical assays, the compound inhibited the binding of β-catenin and Tcf4 proteins and suppressed β-catenin/Tcf4 driven gene transcription. However, copper ions are known to be cytotoxic and redox active. The application of copper complexes in cancer treatment is, unlike platinum-based drugs, still in its infancy.59 1.1.6 Design and Syntheses of Inhibitors by Bioisostere Replacement To date, there have been no strategies on structure-based design and synthesis of small molecules to disrupt β-catenin/Tcf4 PPI. Although compounds shown in Figure 1.4 are potential candidates and frequently found in review articles,54, 60 their structures have never been optimized due to lack of knowledge about binding modes. Additionally, specificity and undesired reactivity were the major flaws in high throughput screening (HTS) hits. In view of these shortfalls, the Ji lab approached the challenge by designing inhibitors from the bottom up. Our initial step was to identify and understand crucial amino acid residues responsible for the formation of β-catenin/Tcf complex. Subsequently those 9 residues were incorporated into the structure of a designed inhibitor, which in an ideal situation should disrupt the PPI. Autodock461 program was used to guide the drug design. Lastly, further structural optimization would improve potency, selectivity and druglikeness. β-Catenin possesses 781 amino acid (AA) residues grouped in three distinct sections. The N-terminal section contains about 130 AA residues, providing crucial phosphorylations sites for GSK3.62 The central section is grouped in 12 sequence repeats (armadillo repeats) each containing 42 residues. It interacts with E-cadherin,63 APC,64 and Tcf4, among others. Lastly, the C-terminal section with 100 AAs is responsible for the recruitment of transcriptional co-activators.65 The crystal structure of the armadillo repeat region in complex with Tcf4 was solved by Lepourcelet and co-workers (Figure 1.5 left).66 Mutational analysis revealed that not all AA residues were critical to forming the protein complex. Three specific regions (hot regions) contribute to the majority of the binding energy. Hot region 1 is located in a positively charged groove (repeat 4-9), where residues 13-25 of Tcf4 establish contact. In particular, D16/E17 of Tcf4 form two salt bridges with K435/K508 of β-catenin. The second hot region is comprised of residues 40-50 of Tcf4 and repeats 3-5 of β-catenin, with E24 forming another salt bridge with K312 respectively. Finally, the third hot region involves hydrophobic interactions between V44/L48 (Tcf4) and F253, I256, F293, A295, I296 (β-catenin). Within these hot regions, K435 and K508 were characterized as crucial “hot spots”.67-69 The Ji lab has applied SPR (surface plasmon resonance) to quantify the relative contribution of each hot spot.70 Consistent with earlier reports,71-72 β-catenin displayed high binding affinities towards wild-type Tcf (Table 1.2), but failed to bind to Tcf with 10 D16A/E17A double mutants. In contrast, little increase in Kd was observed when Tcf residues from hot regions 2 and 3 were mutated. This study highlighted the importance of D16/E17 which would serve as a template73 for inhibitor design. There are challenges associated with this approach. β-Catenin/Tcf PPI represents a tightly bound complex. The dissociation constant (Kd) ranges between 5 – 10 nM. The buried surface area between the two proteins is more than 2800 Å2.70 Designed inhibitors that only mimic D16/E17 will not outcompete Tcf-binding. Nevertheless, the crystal structure revealed multiple hydrophobic pockets surrounding K435/K508 unoccupied by Tcf4 (Figure 1.5 right). Thus, extending the side chain of an inhibitor into these pockets might exploit more binding elements and increase affinity toward β-catenin. In addition, the selectivity of an inhibitor towards hot region 1 also needs to be accounted for. Besides Tcf protein, E-cadherin and APC interact with β-catenin at the same binding site. K435 of β-catenin forms a salt bridge with D1486 (APC) and D830 (E-cadherin). Disrupting these interactions might lead to undesired blockage of cell adhesion and destruction complex in healthy cells. Yet binding modes adjacent to K435 differ significantly. While E17 of Tcf forms a salt bridge with K508 of β-catenin, the corresponding residues S831 (E-cadherin) and T1487 (APC) are not directly engaged in any bonding with β-catenin. In addition, quadruple mutations of P828YDS831 and D1484ADT1487 into alanine residues did not notably decrease their binding affinity towards β-catenin,67, 74 indicating that both APC and Ecadherin are not dependent on K435 for binding. Based on these conclusions, we proposed that mimicking D16/E17 would be a good starting point to generate selective inhibitors targeting β-catenin/Tcf4 PPI. Four well-defined pockets can be found surrounding K435/K508. They will be 11 termed pockets A, B, C, and D (Figure 1.6a). Pocket A contains the crucial binding element K435, but also C429, N430, H470 and S473. Pocket B is a hydrophobic pocket with V511, C573, L539, I569 and I507. Pocket C is a surface hydrophobic pocket with L519 and I579. Pocket D, a second surface pocket, is defined by C466, E462, L506 and A509. Pockets D and B are connected by K508 and R469. Pockets A, B, and C are connected through R474 and R515, which form an arginine channel between these pockets. Human Tcf4 segment G13ANDE17 binds to this site. The Ji lab has used a bioisostere replacement technique to mimic the carboxylic acid side chain of D16 and E17 with tetrazole and indazole-1-ol moieties, respectively (Figure 1.6c). Both functional groups have pKa values similar to carboxyl groups75 and are more hydrophobic, an attribute important for cell permeability. The tetrazole derivative also displays higher metabolic stability than its carboxylic acid counterpart. According to AutoDock4, the designed molecule, termed UU-T01, would form charge-charge interactions with K435, but additional hydrogen bonds (H-bonds) with N430 and H470 were predicted due to the presence of multiple nitrogen atoms. The D16 bioisostere was linked to indazole-1-ol, which engages in charge-charge interactions with K508. The electron rich cyclic core also enables a cation-π interaction with R469 (Figure 1.6b). Using this strategy, small molecule inhibitors (≤ 300 Da) like UU-T01 were synthesized. UUT01 inhibited β-catenin/Tcf PPI with Ki = 3.14 µM in FP-assays.70 Subsequent site-directed mutagenesis studies confirmed that K508, K435 and R469 were the critical residues interacting with UU-T01. 12 1.2 Results and Discussion 1.2.1 Design, Synthesis and Biological Evaluation of N-acyl Sulfonamide Bioisosteres Encouraged by the results of UU-T01, we envisioned extending this bioisostere replacement method in two directions (Figure 1.7a). First, the indazole-1-ol functionality was replaced by the 3-hydroxyisoxazole functionality. The former has rarely found application in medicinal chemistry. Only one previous report76 of its synthesis has been described. Contrarily, 3-hydroxyisoxazole has been extensively used as a carboxylic acid bioisostere (pKa = 4 - 5) in the development of GABA, NMDA, AMPA agonists.77 The second direction was to structurally simplify bicyclic heterocycles like UUT01 due to limited synthetic accessibility and diversification. We converted the scaffold into benzoic acid derivatives, which upon simple peptide coupling would give a variety of N-acyl-sulfonamides (Figure 1.7a). This carboxylic acid bioisostere (pKa » 4.0)78 provides a handle to extend aromatic hydrophobic groups R1 into pocket D. The goal was to establish a structure-activity relationship (SAR) for this pocket and simultaneously mimic D16/E17. AutoDock461 was employed to predict potential binding modes of the new scaffold. Molecules with aromatic R1 moieties were docked into hot region 1. Interestingly, such molecules display similar docking conformations compared to UU-T01. As shown in Figure 1.7b, the N-acyl-sulfonamide bearing a thiophene moiety is aligned towards K508 and occupies the hydrophobic pocket D. The benzene ring points towards R469, potentially engaging in a cation-π interaction, while tetrazole interacts with K435 via a H-bond. Synthesis (Figure 1.8a) of the 3-hydroxyisoxazole 1.19 commenced with a Heckcoupling between the aryl iodide 1.11 and acrylonitrile 1.12. The resulting E/Z isomers 13 1.13 were then reduced to 1.14 via hydrogenation. Subsequently, the methyl ester was converted into hydroxamic acid 1.15 under basic condition. Initial attempts to cyclize 1.14 with CDI in refluxing THF failed to produce the desired product.79 Adding Et3N or changing the solvent to PhMe was not successful. However, pretreatment of 1.14 with NaHCO3 in anhydrous DMF with 4Å molecular sieves, followed by addition of a solution of CDI in DMF, furnishes the heterocycle in 44% yield. Finally, the nitrile group was converted to the carboxylic acid 1.18 and its bioisostere 1.19. N-acyl-sulfonamides were prepared in 5 steps (Figure 1.8b), starting with a Heckcoupling between the aryl iodide 1.21 and acrylonitrile 1.12. Subsequent reduction of the cis/trans alkene afforded 1.22 in good yield. 1.23 was obtained by saponification. Next, the nitrile group underwent [2+3] cycloaddition with an azide to generate tetrazole 1.24 which was diversified into various aromatic sulfonamides 1.26 – 1.37. 1.2.2 Biological Evaluation and Discussion The compounds were evaluated by Dr. Min Zhang (Ji Lab) using a fluorescencepolarizaation (FP) assay.80 This assay (Figure 1.9) uses C-terminal fluorescein-labeled human Tcf4 (residues 7–51) as the fluorescence tracer and N-terminal His6 -tagged human β-catenin (residues 138–686) as the target protein. When the modified peptide in aqueous solution is excited by polarized light, the emitted light will be largely depolarized due to Brownian molecular motion. The motion will be significantly reduced when Tcf4 is bound to β-catenin, thus leading to a lower degree of polarization. This property can be used as a measure to quantify the effectiveness of small molecules to disrupt a PPI. In total 14 compounds, along with UU-T01, were tested. Throughout all in vitro evaluations, the Ki- 14 value of UU-T01 remained constant and was therefore used for comparison. The proof of concept studies associated with this molecule will be part of the following discussion. The results (Table 1.3) show a significant decrease in activity compared to UUT01. Interestingly, the 3-hydroxyisoxazole 1.19 experienced an almost 50-fold drop in activity although its structure differs only by one atom compared to 1.38. In addition, Nacyl-sulfonamides were more active when the R1 group was an unsubstituted or chlorinated thiophene. However, no promising results were observed for N-acyl-sulfonamides bearing benzene rings. These findings prompted us to reevaluate the approach from multiple perspectives, and we proposed two plausible theories. The first theory concerned the structure of the inhibitors. The decrease in potency of 1.19 relative to UU-T01 seemed unexpected since there was little structural change. However, Ji et al. previously described benzotriazole-1-ol 1.38 to be less potent than UUT01. The fused ring system of 1.38 has one more nitrogen atom, which might decrease the overall electron density of the molecule, leading to weaker cation-π interactions with R469. This structural feature can also be found in 3-hydroxyisoxazole 1.19, which possesses an electron withdrawing oxygen instead. As for the N-acyl-sulfonamides, we reasoned the likewise acyl group weakened the cation-π interactions between the benzene ring and R469. Our second theory concerned the binding of an inhibitor in hot region 1. The previous explanations pointed out flaws in structural designs with the presumption that Nacyl-sulfonamides and 3-hydroxyisoxazole did interact, although to a lesser extent, with K508/K435. This premise may be mistaken when considering the size of the target protein β-catenin. The armadillo repeat region contains over 500 AA residues and interacts with 15 Tcf4 on a surface of 2800 Å2. Thus, it raises the fundamental question of whether the low molecular weight compounds 1.26 – 1.37 were binding to K508/K435. Some biological activities observed in the in vitro assays could be caused by nonspecific interactions. Unlike UU-T01, no site-directed mutagenesis studies were performed for the N-acylsulfonamides. However, the ambiguous SAR (Table 1.3) may be an indication that these small molecules are not specific hot region 1 binders. If so, it challenges the initial approach to use AutoDock4 as a tool to guide structure-based drug design. At this point, a discussion about the drawbacks of using docking software is warranted. The main purpose of computational docking is to predict favorable conformations of a ligand in the receptor binding side. Docking is a routinely used strategy for traditional targets such as kinases, proteases and receptor proteins.81-84 In contrast, protein-protein interfaces are structurally more complex, and do not always offer welldefined and deep cavities for small molecule binding.85-86 The conformations are transient, meaning they differ depending on whether it is in an unbound state or bound to other protein/small molecules. Size, solvent exposure and flexibility are also important variables. Conventional docking programs are not optimized to model ligand interactions at proteinprotein interface.87-88 Autodock4 for example contains several specific features, which might not be well suited to model β-catenin-ligand interactions. 1) Autodock4 builds a confined search space, termed docking box,89 in which potential ligand poses are explored. It encompasses critical hot spots and some surrounding AA residues. The purpose is to shorten the processing time as well as decrease the number of irrelevant binding poses. However, problems may occur if the box is too restrictive to account for all possible structural conformations. In case of β-catenin, the box coordinates 16 were set arbitrarily. This could have led to misguided docking prediction. 2) β-Catenin was treated as a rigid macromolecule. This may be misleading and unrealistic considering the known dynamic behavior.90-91 Although Autodock4 enables side chains to undergo conformational changes, this feature was never applied to the docking studies of N-acyl-sulfonamides. 3) The program allows ligands to have torsional degree of freedom, while keeping bond angles and lengths constant.90 This feature enables rapid transformations of coordinates during docking, but may cause problems if a significant degree of distortion is required upon binding to the protein. 1.3 Conclusion β-Catenin/Tcf4 PPI plays a major role in the aberrantly activated Wnt signaling pathway. This PPI represents an attractive target for small molecule therapeutics. The Ji lab identified two critical hot spot residues responsible for the formation of the PPI and used a computer modeling-based approach to access novel PPI bioisosteres. In a previous study, UU-T01 emerged, serendipitously, to be a potent inhibitor of β-catenin/Tcf4 PPI. However, 3-hydroxyisoxazole and N-acyl-sulfonamides were not able to show similar or better biological activities in FP assays. It was later realized that a traditional docking program alone, optimized for enzyme modeling, was not able to reliably assist throughout the drug design process. This is largely attributed to the size, dynamic behavior, and complex surface morphology of a PPI interface and thus requires more sophisticated computational approaches to make dependable predictions. In silico screening is a viable strategy92 if physical properties of the PPI interface are accounted. Consensus docking93 17 and ensemble docking94 are common approximation methods which employ either multiple software or protein crystal structures. DARC (docking approach using ray-casting)95 and EleKit96 represent more customized methods which take shallow binding surfaces and flexibility into account. The design of potent inhibitors for PPI is certainly a challenging task. But a profound understanding of the protein combined with novel design strategies may lead to a drug molecule capable of disrupting PPIs. 18 Cell membrane Endoplasmic reticulum Cell membrane Golgi apparatus recycle pH 7.2 Wnt with palmitoleic acid chain 6.2 Porcupine 5.5 Frizzled Wls vesicle LRP Figure 1.1: Wnt protein migration through the cell and its secretion. Wnt ON Wnt OFF Wnt βcat Dvl Axin P βcat CK1 GSK3 βTrCP P P APC Ecad Ecad Cytosol P P Dvl Axin P βcat CK1 GSK3 P P APC βcat βcat βTrCP βcat βcat Nucleus Groucho Groucho βcat Tcf4 Tcf4 Wnt target gene Wnt target gene P Frizzled LRP Ubiquitin Phosphorylation Figure 1.2: Regulation of cytosolic β-catenin when the Wnt pathway is off (left). Increase of β-catenin concentration when Wnt is on (right). 19 HO OH OH Cl OH N H OH O OH OH O HO O O OH O O OH O EGCG Cl Niclosamide (Fzd inhibitor) OH HO Quercetin NO2 O HO O OH N S HO S N H O S HN H O O HO OH OMe OMe Curcumin IWR (Axin stabilizer) IWP (Porcupine inhibitor) Resveratrol Figure 1.3: Nonspecific Wnt inhibitors (left). Wnt inhibitors targeting upstream components of the pathway (right). OH O OH O OMe Me OMe OH Me Me O O MeO MeO Me OMe OH O N Me Me N N N O PKF118-310 (HTS) 0.8 µM O N N H S O O O Cl N H O BC-23 (virtual screening) 1.7 µM OAc H O O OH O PKF115-584 (HTS) 3.2 µM CGP049090 (HTS) 8.7 µM O OH O O OH OMe NH N OH HO O N N OH H Me Me OH O OH Henryin (bioassay screening) n.d O N Cu Cl Cl O Cu N BC21 (virtual screening) 5.0 µM Figure 1.4: PPI inhibitors discovered via biochemical assays. 20 A B Figure 1.5: Crystal structure of β-catenin/Tcf4. A) β-catenin/Tcf4 PPI with 3 hot regions of interactions. B) G13ANDE17 segement binding to β-catenin hot region 1. A B Figure 1.6: Hot region 1 of β-catenin/Tcf4 PPI. A) G13ANDE17 segement binding to β-catenin hot region 1. Predicted docking mode of UU-T01 (b). 21 A N N OH B O O O S N R 1 H OH N O Pocket D K508 + HN N N N UU-T01 Ki = 3.14 µM HN N N N HN N N N K435 N-acylsulfonamide 3-Hydroxyisoxazole A Figure 1.7: Development D16/E17 mimics. A) 3-hydroxyisoxazole and N-acyl sulfonamides. B) Docking mode of an N-acyl sulfonamides. A O O OMe I Pd(OAc)2, Et3N + CN DMF, 80 oC, 16 h NC 76% 1.12 OH 1.11 O NC OH OH N iPrOH, reflux, 12 h 68% O NC PhMe, reflux, 2 d 56% H N N N N O Pd(OAc)2, DIPEA DMF, 80 oC, 16 h; 1.12 N 1.19 O OMe 1.21 OH 1M NaOH THF, rt, 4 h quant. Pd/C, EtOH, rt, 1 h 62% over 2 steps I CN 1.23 O O O S N R 1 H CN 1.22 O OH O 1.17 + PhMe, reflux 2 d, 53% H 2N S 1.18 O O + O OH nBu3SnN3 1.17 O N HO 1.16 OMe dioxane, rt, 2 h 85% 1.14 3M NaOH 1.15 B NaOH NH2OH . HCl OH OH CDI, NaHCO3, 4Å MS DMF, 50 oC, 5 h 44% OMe MeOH, rt, 8 h NC 80% OH 1.13 O N H OH H2, Pd/C OMe O CDI, DBU R1 THF, reflux-rt 24 h, 55-99% HN N N N HN N N N 1.24 1.26 - 1.37 1.25 Cl R1 S S 1.26 Cl 1.27 S 1.28 Cl S 1.29 Cl NO2 F 1.33 1.34 1.31 1.30 F Me 1.32 Me Me 1.35 CF3 1.36 Cl 1.37 Figure 1.8: Synthesis of D16/E17 mimics. A) Synthetic route of 3-hydroxyisoxazoles. B) Synthetic route of N-acyl-sulfonamides. 22 Polarized light excitation FITC-Tcf4 𝛃-catenin High polarization Polarized light emission Small-molecule inhibitors Polarized light excitation FITC-Tcf4 𝛃-catenin Low polarization Polarized light emission Figure 1.9: Schematic illustration of the FP-assay (adopted from ref. 80). Table 1.1: Mutations of Wnt components and tumerogenesis. Mutated protein APC Axin GSK3 Tcf4 β-catenin LRP5 Cancer type FAP/sporadic colorectal34 heptatocellular/colorectal35 leukemia36 colorectal37 heptatocellular/medulloblastoma38 breast/parathyroid39 23 Table 1.2: SPR studies of β-catenin with wild-type and mutant Tcf.70 Peptides Tcf residues (7-51) Tcf residues (7-51, D16A, E17A) Tcf residues (7-51, E24A, E29A) Tcf residues (7-51, V44A, L48A) Kd (nM) 4.7 >100,000 28 89 Table 1.3: FP-Assay results for the synthesized molecules. Compound Ki ± SD (µM) Compound 1.19 141.50 ± 6.25 1.31 1.24 991.11 ± 36.45 1.32 1.26 87.34 ± 2.87 1.33 1.27 26.70 ± 2.25 1.34 1.28 77.79 ± 2.95 1.35 1.29 777.45 ± 15.74 1.36 1.30 > 2000 1.37 Ki ± SD (µM) > 2000 132.50 ± 4.89 888.30 ± 19.48 289.60 ± 10.51 235.85 ± 8.63 105.28 ± 5.45 192.80 ± 2.98 24 1.4 References 1. Logan, C. Y.; Nusse, R., The Wnt Signaling Pathway in Development and Disease. Annu. Rev. Cell Dev. Biol., 2004, 20, 781-810. 2. 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PLoS One, 2013, 8, e75762. 32 1.5 Supporting Information 1.5.1 Protein Structure for Computer Modeling The crystallographic coordinates for human β-catenin (PDB id, 2GL7, 2.60 Å resolution, Rcryst = 0.223) were obtained from the Research Collaboratory for Structural Bioinformatics (RCSB) protein database. All computational work was performed on Linux Red Hat 6.2 Workstations. The preparation of the crystal structure and molecular modeling were achieved with the commercially available Accelrys Discovery Studio 3.0 (http://accelrys.com/), Schrodinger (http://www.schrodinger.com/) and SYBYL X 2.0 (http://www.tripos.com) software packages. The missing side chains of β-catenin were added in SYBYL X2.0. The protonation states of the residues were set to pH 7.0 when adding the hydrogens. The AMBER 7 force field 99 within SYBYL X2.0 was used to optimize the orientation of hydrogen atoms and the missing side chains of the protein and of structural waters. After the protein structure was optimized, chains B (Tcf4), C (BCL9), D (the second monomer of β-catenin), E (the second monomer of Tcf4), F (the second monomer of BCL9), and solvent molecules were removed, leaving only one monomer of β-catenin for further calculation. The residues in the Tcf4 G13ANDE17 binding site of βcatenin include G422, S425-N426, T428-N430, K435, E462-P463, I465-C466, R469H470, S473-R474, Q482, L506-A509, V511-G512, R515-N516, L519, L536, L539, R565, E568-I569, E571-C573, G575-A576, H578-I579, R582, N609, R612-V613, E620, and Y654. 33 1.5.2 AutoDock4 Study AutoDock 4.2 was employed to perform the docking calculations. Only the polar hydrogen atoms remained on the protein structure, and Kollman united atom charges were assigned. The 3D structures of the ligands were built and partial atomic charges were also calculated using the Gasteiger–Marsili method. The rotatable bonds in the ligands were defined using AutoTors, which also unites the nonpolar hydrogens and partial atomic charges to the bonded carbon atoms. The grid maps were calculated using AutoGrid. The dimension of the grid box was 39 x 28.5 x 21.5 Å, and the grid spacing was set to 0.375 Å. Docking was performed using the Lamarckian genetic algorithm (LGA), and the pseudoSolis and Wets method were applied for the local search. Each docking experiment was performed 100 times, yielding 100 docked conformations. Parameters for the docking experiments were: initial population size of 150, a maximum of 1.5 x 106 energy evaluations; a maximum of 27,000 generations; a maximum of 1 top individual will automatically survive; random starting position and conformation. Other settings were the standard default parameters. All of the ligands followed the same docking protocol. The results of the docking experiments were evaluated by the auxiliary clustering analysis and/or by a visual inspection to match the proposed docking mode. 1.5.3 Fluorescence Polarization Assays Experiments were performed by Dr. Min Zhang (Ji lab) in 96-well Microfluor 2 black plates (Waltham, MA), and the samples were read with a Synergy 2 plate reader (Biotek, Winooski, VT). The polarization was measured at room temperature with an excitation wavelength at 485 nm and an emission wavelength at 535 nm. The FP saturation 34 experiments were performed in an assay buffer of 137 mM of NaCl, 2.7 mM of KCl, 10 mM of Na2HPO4, 2 mM of KH2PO4, 100 µg/mL of bovine gamma globulin, and 0.01% Triton-X 100. The final reaction volume was 100 µL. In the FP competitive inhibition assays, 10 nM of β-catenin (residues 142-686) was incubated with C-terminally fluorescein-labeled human Tcf4 (residues 7-51) for 30 min at 4 °C, and then different concentrations of the tested peptides or compounds in the assay buffer were added to make a final volume of 100 µL. Each assay plate was covered black and gently mixed on an orbital shaker for 3 h to reach equilibrium before polarization values were read. The background of the tested peptides or inhibitors was corrected by subtracting the raw intensity values of the sample background well (all components except probe) from the raw intensity values of the corresponding test wells (all components). The IC50 values were determined by nonlinear least-square analysis using GraphPad Prism 5.0. The Ki values were derived from the IC50 values by the reported method. Experiments were performed in triplicate and carried out in the presence of 1% DMSO. To evaluate the effects of the orders of adding proteins and compounds in the FP competitive inhibition assays, 10 nM of βcatenin (residues 142-686) was also incubate with different concentrations of the compounds in the assay buffer for 30 min at 4 °C, and then with C-terminally fluoresceinlabeled human Tcf4 (residues 7-51) in the assay buffer were added to make a final volume of 100 µL. Each assay plate was covered black and gently mixed on an orbital shaker for 3 h to reach equilibrium before polarization values were read. The background of the tested peptides or inhibitors was corrected by subtracting the raw intensity values of the sample background well (all components except probe) from the raw intensity values of the corresponding test wells (all components). The IC50 values were determined by nonlinear 35 least-square analysis using GraphPad Prism 5.0. The Ki values were derived from the IC50 values by the reported method. 1.5.4 General Experimental Conditions (Chemistry) All experiments were conducted under anhydrous conditions in an atmosphere of argon, using flame-dried apparatus and employing standard techniques in handling airsensitive materials. Dichloromethane (CH2Cl2), acetonitrile (CH3CN), tetrahydrofuran (THF), dimethylformamide (DMF) were degassed with nitrogen and passed through JC Meyer solvent systems. All reagents were used as received. Aqueous solutions of sodium bicarbonate, sodium chloride (brine), and ammonium chloride were saturated. Analytical thin layer chromatography was visualized by ultraviolet light. Flash chromatography was performed on SilicaFlash@ F60 silica gel (230 – 400 mesh). 1H NMR spectra were recorded using a Varian Unity Inova 500 (500 MHz) or a Varian Unity Inova 300 (300 MHz). 13 C NMR spectra were recorded using a Varian Unity Inova 500 (125 MHz) or Varian Unity Inova 300 (75 MHz). The 1H and 13 C NMR spectra are referenced to the residual solvent signals (7.26 ppm for 1H and 77.0 ppm for 13C in CDCl3; 2.05 ppm for 1H and 29.8 ppm for 13C in acetone-d6; 2.50 ppm for 1H and 39.5 ppm for 13C in DMSO-d6). Low (LRMS) and high (HRMS) mass spectra were determined on a Micromass Quattro II (ESI/APCI-TOF) at the University of Utah Mass Spectrometry Facility. 36 1.5.5 Procedures and Characterizations O OMe NC OH Methyl 4-(2-cyanovinyl)-2-hydroxybenzoate (1.13). Methyl 2-hydroxy-4iodobenzoate (100 mg, 0.36 mmol) and Pd(OAc)2 (8 mg, 0.036 mmol) were placed in a 20 mL oven-dried scintillation vial. The vial was evacuated and flushed three times with argon. Then acrylonitrile (0.12 mL, 1.79 mmol), Et3N (0.15 ml, 1.08 mmol) and 2 mL anhydrous DMF were added via syringe. The vial was sealed and heated at 80 oC for 16 h. Upon completion, the reaction was quenched with H2O and extracted two times with Et2O. After the removal of the organic solvent, the crude product was purified via flash column chromatography (5:1 hexanes/EtOAc) to obtainct a yellowish solid (56 mg, 76%). Rf = 0.6 (4:1 hexanes/EtOAc). The product was used for the alkene reduction without further characterization. O OMe NC OH Methyl 4-(2-cyanoethyl)-2-hydroxybenzoate (1.14). In a 100 mL round bottom flask, 1.13 (1.1 g, 5.41 mmol) was dissolved in 50 mL MeOH and 0.1 g of Pd/C (10 wt%) was added. The flask was evacuated and flushed three times with hydrogen gas. The reaction mixture was stirred at room temperature. After 8 h, the starting material was consumed and the reaction mixture was filtered through celite. The organic solvent of the filtrate was subsequently removed under reduced pressure and the crude product loaded on the column (6:1 hexanes/EtOAc). A yellow semisolid was isolated (884 mg, 80%). Rf = 0.5 (4:1 hexanes/EtOAc). 1H NMR (300 MHz, CDCl3): δ 10.76 (s, 1H), 7.77 (d, J = 8.1 Hz, 1H), 6.81 (s, 1H), 6.76 (dd, J = 1.5, 8.1 Hz, 1H), 3.92 (s, 3H), 2.92 (t, J = 7.5 Hz, 2H), 37 2.62 (t, J = 7.5 Hz, 2H). 13C NMR (75 MHz, CDCl3): δ 170.5, 162.0, 146.5, 130.7, 119.5, 118.9, 117.3, 111.6, 52.6, 31.7, 18.8. O NC N H OH OH 4-(2-Cyanoethyl)-N,2-dihydroxybenzamide (1.15). In a 100 mL round bottom flask, 1.14 (0.45 g, 2.19 mmol) and hydroxylamine hydrochloride (3.8 g, 54.8 mmol) were suspended in 10 mL dioxane and 36.5 mL of 3M aq. NaOH were added dropwise. The reaction was completed within 2 h, acidified to pH < 4 and extracted three times with EtOAc. Upon removal of the organic solvent, a pink solid (384 mg, 85%) was isolated and of suitable purity to be used for the next reaction without further purification. Rf = 0.15 (1:1 hexanes/EtOAc). 1H NMR (500 MHz, DMSO-d6): δ 12.32 (brs, 1H), 11.43 (brs, 1H), 9.31 (brs, 1H), 7.63 (d, J = 8.0 Hz, 1H), 6.84 (s, 1H), 6.79 (d, J = 8.0 Hz, 1H), 2.90 – 2.74 (m, 4H). 13 C NMR (125 MHz, DMSO-d6): δ 166.8, 160.1, 145.2, 127.4, 120.5, 119.4, 117.5, 112.7, 30.7, 18.0. OH N NC O 3-(3-Hydroxybenzo[d]isoxazol-6-yl)propanenitrile (1.16). Compound 1.15 (200 mg, 0.97 mmol), CDI (188.7 mg, 1.16 mmol), NaHCO3 (97.8 mg, 1.16 mmol) were placed in a 25 mL oven-dried round bottom flask with 4Å molecular sieves. The flask was flushed with argon and charged with 10 mL anhydrous DMF. The reaction was heated to 50 oC for 5 h. After the starting material was consumed as determined by TLC, the mixture was filtered through celite to remove the molecular sieves. The filtrate was cooled in an icewater bath and 1M HCl was added dropwise to adjust the pH to < 4. The color of the solution turned from red to yellow. Subsequently 50 mL EtOAc was added and the organic 38 layer was washed three times with brine. After the organic solvent was remove under reduced pressure, the crude product was purified by column chromatography (20:1 CH2Cl2/MeOH) to obtain a white solid (81 mg, 44%). Rf = 0.3 (15:1 CH2Cl2/MeOH). 1H NMR (300 MHz, CD3OD): δ 7.66 (d, J = 8.1 Hz, 1H), 7.38 (s, 1H), 7.25 (d, J = 8.1 Hz, 1H), 3.08 (t, J = 7.2 Hz, 2H), 2.81 (t, J = 7.2 Hz, 2H). 13C NMR (75 MHz, CD3OD): δ 164.2, 148.4, 142.9, 125.9, 123.9, 121.5, 119.2, 113.6, 109.7, 31.3, 18.3. OH N HO O O 3-(3-Hydroxybenzo[d]isoxazol-6-yl)propanoic acid (1.18). Compound 1.16 (73 mg, 0.38 mmol) was dissolved in 5 mL iPrOH and 5 mL of 3M aq. NaOH solution was added. The mixture was refluxed for 12 h. Upon consumption of the starting material, the reaction was cooled to room temperature and the organic solvent was removed under reduced pressure. The remaining aqueous layer was acidified with 1M HCl to pH < 4 until the desired product appeared as a white precipitate. The precipitate was filtered and washed with water and hexanes. The product 1.18 was isolated as a white solid (54 mg, 68%) and deemed to be of suitable purity for biochemical assays. Rf = 0.3 (10:1 CH2Cl2/MeOH). 1H NMR (500 MHz, DMSO-d6): δ 12.18 (brs, 2H), 7.62 (d, J = 8.0 Hz, 1H), 7.39 (s, 1H), 7.19 (d, J = 8.0 Hz, 1H), 2.96 (t, J = 7.5 Hz, 2H), 2.60 (t, J = 7.5 Hz, 2H). 13C NMR (125 MHz, DMSO-d6): δ 173.5, 165.1, 163.5, 144.5, 123.9, 120.9, 112.6, 109.3, 35.0, 30.5. 39 General procedure A1 for the synthesis of tetrazoles NC R nBu3SnN3, PhMe 110 oC H N N N N R In a 75 mL pressure flask, 1.16 mmol of alkyl nitrile was suspended in 25 mL anhydrous toluene, and nBu3SnN3 (1.54 g, 4.63 mmol) was added. The flask was sealed and heated to 110 oC. After 48 h, the reaction was cooled down to room temperature and 0.1 mL of AcOH was added. After stirring for 2 additional hours, a white precipitate formed and was filtered, washed with hexanes to obtain the tetrazole. OH N N HN O N N 6-(2-(2H-tetrazol-5-yl)ethyl)benzo[d]isoxazol-3-ol (1.19). The product 1.19 was isolated as a white solid (150 mg, 56%) and deemed to be of suitable purity for biochemical assays. Rf = 0.3 (10:1 CH2Cl2/MeOH). 1H NMR (500 MHz, DMSO-d6): δ 7.63 (d, J = 8.0 Hz, 1H), 7.40 (s, 1H), 7.16 (d, J = 8.5 Hz, 1H), 3.25 (t, J = 7.2 Hz, 2H), 3.18 (t, J = 7.5 Hz, 2H). 13C NMR (125 MHz, DMSO-d6): δ 165.3, 163.5, 155.4, 143.5, 123.9, 121.2, 113.1, 109.5, 32.9, 24.5. O OMe CN Methyl 3-(2-cyanoethyl)benzoate (1.22). To a solution of aryl iodide 1.21 (2.0 g, 7.63 mmol) in DMF, Pd(OAc)2 (170.62 mg, 0.76 mmol) was added at room temperature. The reaction mixture was flushed with argon and evacuated under vacuum three times. DIPEA (3.98 mL, 22.89 mmol) and acrylonitrile (2.5 mL, 38.15 mmol) were added while the reaction was heated to 80 ˚C. The reaction was cooled to room temperature after 16 h. 40 The mixture was filtered through celite and extracted two times with Et2O. The organic layer was dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The alkene isomers were isolated as yellow oil (1.2 g) and used for the next step without further purification. A solution of the alkene (1.2 g, 6.48 mmol) was dissolved in EtOH (1:1) in a round bottom flask and Pd/C (10 wt%, 100 mg) was added at room temperature. After evacuating and flushing the flask with hydrogen gas three times, the mixture was stirred under hydrogen atmosphere for 1 h. Upon completion, the reaction mixture was filtered through celite. The solvent of the filtrate was removed under reduced pressure to yield 1.22 as yellow viscous oil (0.9 g, 62% over two steps). Rf = 0.3 (4:1 hexanes/EtOAc). The product 1.22 was used for the ester hydrolysis without further purifications. 1H NMR (CDCl3, 500 MHz): δ 7.99 – 7.83 (m, 1H), 7.44 – 7.31 (m, 2H), 3.87 (s, 1H), 2.96 (t, J = 7.2 Hz, 2H), 2.62 (t, J = 7.2 Hz, 2H). 13C NMR (CDCl3, 75 MHz): δ 167.0, 138.6, 133.2, 130.9, 129.6, 129,2, 128.7, 119.1, 52.5, 31.5, 19.4. LRMS (ESI) [M + H]+ m/z = 190.1. O OH CN 3-(2-Cyanoethyl)benzoic acid (1.23). A solution of 1.22 (0.38 g, 2.0 mmol) in 10 mL THF was treated with 10 mL of 3M NaOH. The mixture was stirred at room temperature for 4 h. Then, cold 2M HCl was added dropwise until pH < 4. The resulting mixture was extracted with EtOAc, and the organic layer was washed with water and brine. After removing the solvent, a white solid (0.35 g, 99%) was obtained and used without further purification. Rf = 0.2 (1:1 hexanes/EtOAc). 1H NMR (CD3OD, 500 MHz): δ 7.96 (s, 1H), 7.92 (dd, J = 7.5, 1.0 Hz, 1H), 7.54 (d, J = 7.5 Hz, 1H), 7.44 (t, J = 7.5 Hz, 1H), 3.00 (t, J = 7.5 Hz, 2H), 2.77 (t, J = 7.5 Hz, 2H). 13C NMR (CDCl3, 125 MHz): δ 168.2, 41 139.1, 132.8, 130.9, 129.4, 128.5, 120.0, 30.7, 18.1. O OH HN N N N 3-(2-(2H-tetrazol-5-yl)ethyl)benzoic acid (1.24). Compound 1.24 was prepared from 1.23 (1.20 g, 6.85 mmol) following general procedure A1. The product was isolated as a white solid after filtration (0.79 g, 53%). Rf = 0.2 (20:1 CH2Cl2/MeOH). 1H NMR (DMSO-d6, 300 MHz): δ 7.75 – 7.72 (m, 2H), 7.43 (d, J = 6.9 Hz, 1H), 7.36 (t, J = 6.9 Hz, 1H), 3.23 (t, J = 7.8 Hz, 2H), 3.08 (t, J = 7.2 Hz, 2H). 13C NMR (DMSO-d6, 75 MHz): δ 168.0, 155.9, 141.1, 133.6, 131.6, 129.8, 129.3, 128.0, 33.1, 25.1. LRMS (ESI) [M+H]+ m/z = 219.1. General procedure A2 for the synthesis of N-acyl-sulfonamides O O O OH + HN N N N O R S O NH2 N H CDI, DBU S O R THF, reflux - r.t. HN N N N Carbonyldiimidazole (76 mg, 0.47 mmol) was dissolved in anhydrous THF and a solution of 1.24 (100 mg, 0.46 mmol) in anhydrous THF was added dropwise. The reaction was refluxed for 1 h and allowed to cool to room temperature. Aryl sulfonamide 1.25 (0.49 mmol) was added in one portion, followed by DBU (0.15 mL, 1.0 mmol). The reaction mixture was stirred for 36 h at room temperature. Upon completion, the solvent was evaporated, and the reaction was quenched by the dropwise addition of ice-cold 1M HCl. The aqueous layer was extracted two times with EtOAc, and the organic layer was dried 42 over anhydrous MgSO4. After removing the solvent, N-acyl sulfonamides were purified via flash column chromatography (75:24:1 hexanes/EtOAc/AcOH). O O O S N H S HN N N N 3-(2-(2H-tetrazol-5-yl)-ethyl)-N-(thiophen-2-ylsulfonyl)-benzamide (1.26). Compound 1.26 was prepared with 1.24 (100 mg, 0.46 mmol) following general procedure A2. The crude product was purified via flash column chromatography (75:24:1 hexanes/EtOAc/AcOH) to obtain a white solid (112 mg, 67%). Rf = 0.25 (75:24:1 hexanes/EtOAc/AcOH). 1H NMR (CD3OD, 300 MHz): δ 7.91 – 7.86 (m, 2H), 7.70 (s, 1H), 7.67 – 7.64 (m, 1H), 7.39 – 7.36 (m, 2H), 7.18 – 7.15 (m, 1H), 3.26 (t, J = 8.4 Hz, 2H), 3.16 (t, J = 8.7 Hz, 2H). 13C NMR (CD3OD, 75 MHz): δ 166.5, 155.8, 140.7, 140.0, 134.7, 134.0, 133.3, 132.6, 128.9, 128.1, 127.1, 126.4, 32.9, 24.6. LRMS (ESI) [M+H]+ m/z = 364.0. O O O S N H S Cl HN N N N 3-(2-(2H-tetrazol-5-yl)-ethyl)-N-((5-chlorothiopheyl)sulfonyl)-benzamide (1.27). Compound 1.27 was prepared with 1.24 (100 mg, 0.46 mmol) following general procedure A2. The crude product was purified via flash column chromatography (75:24:1 hexanes/EtOAc/AcOH) to obtain a white solid (100 mg, 55%). Rf = 0.2 (75:24:1 hexanes/EtOAc/AcOH). 1H NMR (CD3OD, 500 MHz): δ 7.75 (s, 1H), 7.69 (d, J = 4.0 Hz, 2H), 7.38 – 7.37 (m, 2H), 7.09 (d, J = 4.0 Hz, 1H), 3.27 (t, J = 7.5 Hz, 2H), 3.15 (t, J = 7.0 43 Hz, 2H). 13C NMR (CD3OD, 125 MHz): δ 169.3, 140.6, 139.0, 137.9, 133.6, 133.1, 128.8, 128.2, 126.8, 126.5, 33.0, 24.6. LRMS (ESI) [M+H]+ m/z = 398.0. O O O S N H S Cl Cl HN N N N 3-(2-(1H-tetrazol-5-yl)-ethyl)-N-((4,5-dichlorothiophen-2-yl)sulfonyl)benzamide (1.28). Compound 1.28 was prepared with 1.24 (100 mg, 0.46 mmol) following general procedure A2. The crude product was purified via flash column chromatography (75:24:1 hexanes/EtOAc/AcOH) to obtain a white solid (113 mg, 57%). Rf = 0.2 (75:24:1 hexanes/EtOAc/AcOH). 1H NMR (CD3OD, 300 MHz): δ 7.87 (s, 1H), 7.83 – 7.81 (m, 1H), 7.53 (s, 1H), 7.24 – 7.22 (m, 2H), 3.24 (t, J = 7.2 Hz, 2H), 3.09 (t, J = 7.8 Hz, 2H). 13C NMR (DMSO-d6, 75 MHz): δ 166.9, 141.2, 138.0, 134.1, 133.1, 132.3, 129.4, 129.3, 127.3, 124.3, 33.0, 25.0. HRMS (ESI) m/z calc. for C14H11Cl2N5O3S2Na [M+Na+]+ 453.9573, found 453.9583. O O O S N H S Me HN N N N 3-(2-(1H-tetrazol-5-yl)-ethyl)-N-((5-methylthiophen-2-yl)sulfonyl)-benzamide (1.29). Compound 1.29 was prepared with 1.24 (100 mg, 0.46 mmol) following general procedure A2. The crude product was purified via flash column chromatography (75:24:1 hexanes/EtOAc/AcOH) to obtain a white solid (107 mg, 62%). Rf = 0.4 (10:1 CH2Cl2/MeOH). 1H NMR (DMSO-d6, 500 MHz): δ 7.78 (s, 1H), 7.72 (t, J = 4.5 Hz, 2H), 7.25 (d, J = 3.5 Hz, 1H), 7.20 (d, J = 4.5 Hz, 2H), 6.64 (d, J = 3.0 Hz, 1H), 3.11 (t, J = 8.0 44 Hz, 2H), 3.00 (t, J = 8.0 Hz, 2H), 2.38 (s, 3H). 13C NMR (DMSO-d6, 125 MHz): δ 170.6, 157.0, 142.9, 140.2, 139.9, 130.6, 129.5, 129.0, 128.2, 127.0, 126.9, 124.9, 34.0, 26.0, 15.6. O O O S N H HN N N N 3-(2-(1H-tetrazol-5-yl)-ethyl)-N-(phenylsulfonyl)benzamide (1.30). Compound 1.30 was prepared with 1.24 (100 mg, 0.46 mmol) following general procedure A2. The crude product was purified via flash column chromatography (75:24:1 hexanes/EtOAc/AcOH) to obtain a white solid (98 mg, 60%). Rf = 0.5 (75:24:1 hexanes/EtOAc/AcOH). 1H NMR (CD3OD, 300 MHz): δ 8.09 (d, J = 7.2 Hz, 1H), 7.85 (s, 2H), 7.72 – 7.56 (m, 3H), 7.44 – 7.33 (m, 3H), 3.27 (t, J = 6.6 Hz, 2H), 3.15 (t, J = 6.9 Hz, 2H). 13 C NMR (CD3OD, 125 MHz): δ 169.3, 166.4, 140.7, 139.8, 133.6, 133.2, 132.6, 128.9, 128.8, 128.1, 128.1, 126.3, 32.9, 24.5. O O O S N H Me HN N N N 3-(2-(1H-tetrazol-5-yl)-ethyl)-N-(m-tolylsulfonyl)benzamide (1.31). Compound 1.31 was prepared with 1.24 (100 mg, 0.46 mmol) following general procedure A2. The crude product was purified via flash column chromatography (75:24:1 hexanes/EtOAc/AcOH) to obtain a white solid (114 mg, 67%). Rf = 0.5 (75:24:1 hexanes/EtOAc/AcOH). 1H NMR (CD3OD, 500 MHz): δ 7.90, (s, 1H), 7.86 (d, J = 7.5 Hz, 1H), 7.67, (s, 1H), 7.63 (d, J = 7.0 Hz, 1H), 7.50 (d, J = 7.5 Hz, 1H), 7.46 (t, J = 7.5 Hz, 1H), 7.39 (d, J = 7.5 Hz, 1H), 7.36 (t, J = 7.5 Hz, 1H) 3.26 (t, J = 7.5 Hz, 2H), 3.14 (t, J = 45 8.0 Hz, 2H), 2.43 (s, 3H). 13 C NMR (CD3OD, 125 MHz): δ 166.4, 155.8, 140.7, 139.6, 139.3, 134.3, 133.2, 132.5, 128.9, 128.7, 128.4, 128.1, 126.3, 125.3, 32.9, 24.54, 20.1. HRMS (ESI) m/z calc. for C17H17N5O3SN [M - H]- 370.0979, found 370.0980. O O O S N H Me HN N N N 3-(2-(1H-tetrazol-5-yl)ethyl)-N-tosylbenzamide (1.32). Compound 1.32 was prepared with 1.24 (100 mg, 0.46 mmol) following general procedure A2. The crude product was purified via flash column chromatography (75:24:1 hexanes/EtOAc/AcOH) to obtain a white solid (122 mg, 72%). Rf = 0.5 (75:24:1 hexanes/EtOAc/AcOH). 1H NMR (CD3OD, 300 MHz): δ 7.95 (d, J = 8.1 Hz, 2H), 7.67 (s, 1H), 7.63 (d, J = 8.7 Hz, 1H), 7.41 – 7.35 (m, 4H), 3.25 (t, J = 6.6 Hz, 2H), 3.14 (t, J = 6.9 Hz, 2H), 2.43 (s, 3H). 13C NMR (CD3OD, 75MHz): δ 166.4, 155.8, 145.0, 140.6, 136.8, 133.2, 132.6, 129.3, 128.9, 128.6, 128.3, 128.0, 126.3, 32.9, 24.6, 20.4. HRMS (ESI) m/z calc. for C17H17N5O3SN [M - H]370.0979, found 370.0994. O O O S N H F HN N N N 3-(2-(1H-tetrazol-5-yl)-ethyl)-N-((3-fluorophenyl)-sulfonyl)-benzamide (1.33). Compound 1.33 was prepared with 1.24 (100 mg, 0.46 mmol) following general procedure A2. The crude product was purified via flash column chromatography (75:24:1 hexanes/EtOAc/AcOH) to obtain a white solid (112 mg, 65%). Rf = 0.3 (75:24:1 hexanes/EtOAc/AcOH). 1H NMR (CD3OD, 500 MHz): δ 7.90 (d, J = 8.5 Hz, 1H), 7.82 (d, 46 J = 8.5 Hz, 1H), 7.81 (s, 1H), 7.66 – 7.61 (m, 2H), 7.46 (t, J = 8.5, 1H), 7.41-7.36 (m, 2H), 3.27 (t, J = 8.0 Hz, 2H), 3.15 (t, J = 7.5 Hz, 2H). 13C NMR (CD3OD, 125 MHz): δ 166.5, 163.3, 161.3, 141.9, 140.7, 133.3, 132.5, 131.0, 130.9, 128.9, 128.14, 126.4, 124.1, 120.7, 115.5, 32.9, 24.5. HRMS (ESI) m/z calc. for C16H14FN5O3S [M - H]- 374.0729, found 374.0727. O O O S N H F HN N N N 3-(2-(1H-tetrazol-5-yl)-ethyl)-N-((4-fluorophenyl)-sulfonyl)-benzamide (1.34). Compound 1.34 was prepared with 1.24 (100 mg, 0.46 mmol) following general procedure A2. The crude product was purified via flash column chromatography (75:24:1 hexanes/EtOAc/AcOH) to obtain a white solid (107 mg, 62%). Rf = 0.3 (75:24:1 hexanes/EtOAc/AcOH). 1H NMR (CD3OD, 500 MHz): δ 8.17 – 8.12 (dd, J = 8.5, 5.0 Hz, 2H), 7.68 (s, 1H), 7.64 (d, J = 7.0 Hz, 1H), 7.40 (d, J = 7.0 Hz, 1H), 7.37 (d, J = 7.0 Hz, 1H), 7.33 (t, J = 9.0 Hz, 2H), 3.26 (t, J = 7.5 Hz, 2H), 3.15 (t, J = 7.5 Hz, 2H). 13C NMR (CD3OD, 125 MHz): δ 167.0, 166.4, 164.9, 140.7, 135.9, 133.3, 132.5, 131.4, 131.3, 128.9, 128.1, 126.4, 116.0, 115.8. 32.9, 24.5. HRMS (ESI) m/z calc. for C16H14FN5O3S [M - H]374.0729, found 374.0727. O O O S N H F 3C HN N N N 3-(2-(1H-tetrazol-5-yl)ethyl)-N-((3-(trifluoromethyl)phenyl)sulfonyl)benzamide (1.35). Compound 1.35 was prepared with 1.24 (100 mg, 0.46 mmol) following 47 general procedure A2. The crude product was purified via flash column chromatography (75:24:1 hexanes/EtOAc/AcOH) to obtain a white solid (174 mg, 89%). Rf = 0.2 (75:24:1 hexanes/EtOAc/AcOH). 1H NMR (CD3OD, 300 MHz): δ 8.37 (s, 1H), 8.33 (d, J = 8.1 Hz, 1H), 7.96 (d, J = 7.8 Hz, 1H), 7.79 (t, J = 7.8 Hz, 1H), 7.68 (s, 1H), 7.63 (d, J = 7.5 Hz, 1H), 7.39 – 7.32 (m, 2H). 13 C NMR (CD3OD, 75 MHz): δ 166.5, 155.8, 140.9, 140.7, 133.5, 132.1, 131.9, 131.3, 130.8, 130.3, 130.3, 130.2, 129.0, 128.2, 126.5, 125.3, 125.3, 121.8, 32.9, 24.5. HRMS (ESI) m/z calc. for C17H14F3N5O3S [M - H]- 424.0697, found 424.0702. O O O S N H NO 2 HN N N N 3-(2-(1H-tetrazol-5-yl)ethyl)-N-((4-nitrophenyl)sulfonyl)benzamide (1.36). Compound 1.36 was prepared with 1.24 (100 mg, 0.46 mmol) following general procedure A2. The crude product was purified via flash column chromatography (75:24:1 hexanes/EtOAc/AcOH) to obtain a white solid (102 mg, 55%). Rf = 0.1 (10:1 CH2Cl2/MeOH). 1H NMR (CD3OD, 300 MHz): δ 8.34 (d, J = 9.0 Hz, 2H), 8.22 (d, J = 9.0 Hz, 2H), 7.80 (s, 1H), 7.77 – 7.72 (m, 1H), 7.29 – 7.22 (m, 1H), 3.25 (t, J = 6.9 Hz, 2H), 3.11 (t, J = 6.9 Hz, 2H). O O O S N H Cl Cl HN N N N 3-(2-(1H-tetrazol-5-yl)-ethyl)-N-((3,5-dichlorophenyl)sulfonyl)-benzamide (1.37). Compound 1.37 was prepared with 1.24 (100 mg, 0.46 mmol) following general 48 procedure A2. The crude product was purified via flash column chromatography (75:24:1 hexanes/EtOAc/AcOH) to obtain a white solid (121 mg, 62%). Rf = 0.4 (75:24:1 hexanes/EtOAc/AcOH). 1H NMR (CD3OD, 500 MHz): δ 8.01 (s, 2H), 7.80 (s, 1H), 7.72 (s, 1H), 7.67 (d, J = 7.5 Hz, 1H), 7.41 (d, J = 9.0 Hz, 1H), 7.37 (t, J = 9.0 Hz, 1H), 3.27 (t, J = 7.5 Hz, 2H), 3.15 (t, J = 7.0 Hz, 2H). 13C NMR (CD3OD, 125 MHz): δ 166.5, 155.8, 142.8, 140.8, 135.6, 133.5, 133.4, 132.2, 128.9, 128.2, 126.8, 126.5, 32.9, 24.5. HRMS (ESI) m/z calc. for C16H13Cl2N5O3S [M - H]- 424.0043, found 424.0048. 49 O OMe NC OH 12.0 11.5 11.0 10.5 10.0 9.5 180 170 160 150 9.0 140 8.5 130 8.0 120 7.5 110 7.0 6.5 6.0 5.5 f1 (ppm) 100 90 80 f1 (ppm) 5.0 70 4.5 60 4.0 3.5 50 3.0 40 1.99 2.00 3.04 0.87 1.00 0.90 0.73 1.14 2.5 30 2.0 20 1.5 1.0 10 0.5 0 0.0 -10 50 O N H OH NC OH 14.0 170 13.0 160 12.0 150 11.0 140 10.0 130 120 9.0 110 8.0 100 3.99 0.84 0.97 1.23 1.10 1.06 1.00 1.15 7.0 f1 (ppm) 6.0 5.0 90 f1 (ppm) 80 70 4.0 60 3.0 50 2.0 40 1.0 30 0.0 20 51 OH N O NC 8.0 180 7.5 170 7.0 160 150 6.5 140 6.0 130 5.5 120 5.0 4.5 4.0 f1 (ppm) 3.5 110 100 90 80 f1 (ppm) 2. 13 2. 06 1. 00 0. 98 1. 00 1.16 3.0 70 2.5 60 2.0 50 1.5 40 1.0 30 20 0.5 10 0.0 0 52 OH N HO O 2.14 2.13 1.00 0.84 1.03 1.18 1.92 O 13.0 12.5 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 f1 (ppm) 180 170 160 150 140 130 120 110 100 90 f1 (ppm) 80 70 60 50 40 30 20 53 OH H N N O 9.0 180 170 8.5 8.0 160 7.5 150 2.13 2.11 1.19 1.00 0.94 1.05 N N N 7.0 140 6.5 6.0 130 120 5.5 110 5.0 100 4.5 4.0 90 80 f1 (ppm) 3.5 3.0 70 2.5 60 2.0 1.5 1.0 50 40 30 0.5 20 0.0 10 -0.5 0 54 55 56 O OH HN N N N 9.0 8.5 180 8.0 170 7.5 160 1.96 2.23 2.00 1.11 1.22 1.24 7.0 150 6.5 140 6.0 130 120 5.5 110 5.0 100 4.5 4.0 f1 (ppm) 90 80 f1 (ppm) 3.5 70 3.0 60 2.5 50 2.0 40 1.5 30 20 1.0 10 0.5 0.0 0 -10 57 O O O S N H S HN N N N 1.26 58 O O O S N H S Cl 9.0 190 8.5 180 8.0 170 1.78 2.21 1.27 1.00 2.01 2.12 0.94 HN N N N 7.5 7.0 6.5 160 150 140 6.0 130 5.5 120 5.0 110 4.5 4.0 f1 (ppm) 100 90 f1 (ppm) 3.5 80 3.0 70 60 2.5 50 2.0 40 1.5 1.0 0.5 0.0 30 20 10 0 59 O O O S N H S Cl Cl 2.07 2.00 1.28 1.00 1.02 0.94 1.94 HN N N N 9.0 8.5 8.0 7.5 7.0 6.5 180 170 160 150 140 130 6.0 120 5.5 110 5.0 100 4.5 4.0 f1 (ppm) 90 80 f1 (ppm) 3.5 70 3.0 60 2.5 50 2.0 40 1.5 30 1.0 20 0.5 10 0.0 0 -10 60 O O O S N H S Me 8.0 180 7.5 170 7.0 160 6.5 150 6.0 140 5.5 130 5.0 120 110 4.5 100 3.24 2.13 2.23 1.04 1.05 2.18 1.29 1.00 1.07 HN N N N 4.0 3.5 f1 (ppm) 3.0 2.5 2.0 90 80 f1 (ppm) 70 60 50 1.5 40 1.0 30 0.5 20 0.0 10 -0.5 0 61 O O O S N H 9.0 8.5 170 160 8.0 150 7.5 140 3.67 3.17 1.30 1.00 1.92 2.78 3.10 HN N N N 7.0 130 6.5 120 6.0 5.5 110 5.0 100 4.5 4.0 f1 (ppm) 90 80 f1 (ppm) 3.5 70 3.0 60 2.5 2.0 1.5 50 40 30 1.0 20 0.5 10 0.0 0 62 O O O S N H Me 9.0 180 8.5 170 8.0 160 7.5 150 7.0 140 6.5 130 6.0 120 5.5 110 5.0 100 4.5 4.0 f1 (ppm) 90 f1 (ppm) 80 3.38 2.32 2.37 1.31 1.00 1.04 1.10 1.11 2.25 2.31 HN N N N 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 70 60 50 40 30 20 10 0 63 O O O S N H Me 9.0 180 8.5 170 8.0 160 7.5 150 7.0 140 6.5 130 6.0 120 5.5 110 5.0 100 4.5 4.0 f1 (ppm) 90 f1 (ppm) 3.5 80 3.06 2.14 2.15 1.32 2.00 0.99 1.00 4.19 HN N N N 3.0 70 2.5 60 2.0 50 1.5 40 1.0 30 0.5 20 0.0 10 -0.5 0 64 O O O S N H F 9.0 170 8.5 160 8.0 150 7.5 140 1.86 2.30 1.33 1.00 0.99 1.08 2.23 1.25 2.09 HN N N N 7.0 130 6.5 6.0 120 5.5 110 5.0 100 4.5 4.0 f1 (ppm) 90 80 f1 (ppm) 3.5 70 3.0 2.5 2.0 60 50 40 1.5 30 1.0 20 0.5 10 0.0 0 65 O O O S N H F HN N N N 8.5 8.0 190 180 7.5 2.32 2.25 1.00 1.01 1.05 0.95 2.15 1.88 1.34 7.0 6.5 6.0 5.5 5.0 4.5 4.0 f1 (ppm) 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 10 0 f1 (ppm) 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 66 O O O S N H F 3C 9.0 180 8.5 170 8.0 160 7.5 150 2.36 2.20 1.00 1.11 1.01 1.04 2.22 1.35 0.73 1.00 HN N N N 7.0 140 6.5 130 6.0 5.5 5.0 120 110 100 4.5 4.0 3.5 90 f1 (ppm) 80 70 f1 (ppm) 3.0 60 2.5 50 2.0 40 1.5 30 1.0 20 0.5 0.0 10 0 67 O O O S N H NO 2 9.0 8.5 8.0 7.5 1.85 2.09 2.01 1.00 0.99 1.36 1.94 1.95 HN N N N 7.0 6.5 6.0 5.5 5.0 4.5 4.0 f1 (ppm) 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 68 O O O S N H Cl Cl 9.0 190 8.5 180 2.68 2.69 1.37 2.03 1.00 1.21 1.22 HN N N N 8.0 7.5 7.0 6.5 170 160 150 140 6.0 130 5.5 120 5.0 110 4.5 4.0 f1 (ppm) 100 90 f1 (ppm) 3.5 80 3.0 70 2.5 60 2.0 50 40 1.5 30 1.0 20 0.5 10 0.0 0 CHAPTER 2 STRUCTURE-BASED MODIFICATION AND OPTIMIZATION OF 2,3 DISUBSTITUTED INDOLES AS POTENT INHIBITORS OF β-CATENIN/TCF PROTEIN-PROTEIN INTERACTIONS 2.1 Introduction Protein-protein interfaces represent an attractive target for medicinal chemists. These interactions are key components in signaling pathways and are dysregulated in many diseases. These types of macromolecular interactions are unrivaled in structural complexity and diversity.1-2 Hence, they were deemed “intractable” and “undruggable” for therapeutic interventions. 2.1.1 Characteristics of Protein-Protein Interactions The contacting surface between two proteins is large (1500 Å2 – 3000 Å2), flat and displays dynamic behavior.3 As a result, the design of potent and selective PPI modulators is a challenging task. In contrast, classic drug targets such as enzymes, interact on a relatively smaller area (600 Å2 – 1000 Å2) with deep, hydrophobic cavities suitable for small molecules binding.4 Nonetheless, mutational analyses revealed that not all residues of a PPI complex are critical for binding. Only certain amino acid residues contribute disproportionately to the overall binding energy. These residues are termed “hot spots”. A 70 hot spot is defined as a residue which, when substituted with an alanine, leads to a significant loss of binding energy (∆∆Gbinding > 1.5 kcal/mol).5 Hot spots are usually clustered in a hot region, the median PPI interface. Gestwicki and co-workers6 have characterized PPI types defined as four categories based on the buried surface area (BSA) and binding affinity (Figure 2.1): loose and narrow, loose and wide, tight and narrow, tight and wide. PPIs with loose binding affinities represent challenging targets due to weak or transient interactions and shallow surface topologies. Though few inhibitors were discovered through conventional high-throughput-screening7-8 or fragment-based screening,9-10 their binding affinities do not exceed the Kd-values of the PPI itself. In contrast, “tight and narrow” PPIs have become the most amenable drug targets. For instance, the interface of the Bcl2/BH3 protein complex resembles traditional enzymes with deeper binding pockets surrounded by a relatively small BSA.6 Using hot spot-based design strategy, numerous Bcl2/BH3 inhibitors were generated and show promising inhibitory potency in low nanomolar11 to picomolar range.12 More recently, a structural analogue of ABT-737 (Figure 2.1), Venetoclax, was FDA-approved for the treatment of chronic lymphocytic leukemia.13 It represents the first approved anticancer drug rationally designed to modulate PPI. β-Catenin/Tc4 PPI represents a tight and wide complex (Kd = 5 – 10 nM, BSA > 2800 Å2).14-16 These features pose a challenge for drug design because a potential inhibitor must outcompete Tcf4 with a slow dissociation rate and simultaneously overcome the large binding surface. The Ji lab has used two approaches, bioisostere replacement16 and peptidomimetic,17 to generate small molecules (UU-T01 and compound 19, Figure 2.1), 71 which showed low micromolar inhibition. However, given the strong affinity between βcatenin and Tcf4 proteins, there is still a significant disparity between inhibitory potency Ki and native dissociation constant Kd. Compared to compound 19, the small molecule UUT01 is only one-third its molecular weight but displays similar inhibitory potency. It also has higher ligand efficiency. Therefore, we believed that low molecular weight fragments such as UU-T01 would generally offer a good starting point for structural elaboration to increase binding affinity. 2.1.2 Design of 3-Substituted Indole N-acyl-sulfonamides and Related Compounds In earlier studies, the Ji lab identified D16/K435 and E17/K508 as critical interactions for the β-catenin/Tc4 complex. Subsequently, we designed and synthesized a series of N-acyl-sulfonamide bioisosteres to mimic D16 and E17 amino acid residues. This scaffold preserved the critical binding elements while allowing for structural expansion. Initial FP-assay results indicated poor binding affinity towards the β-catenin protein (Chapter 1), which we attributed to decreased cation-π interactions with R469. To address this shortcoming, the benzene ring was replaced with a more electron rich indole ring (Figure 2.2), capable of undergoing stronger cation-π interactions.18 Initial docking studies (Figure 2.3) with indole N-acyl-sulfonamides predicted this interaction with R469, while the sulfonamide and tetrazole moieties retained charge-charge interactions with K508 and K435. Interestingly, the indole ring was positioned adjacent to hydrophobic pocket B (Figure 2.3a). Functionalizing the indole C3-position would extend hydrophobic residues into pocket B and gain additional binding affinity. Autodock studies 72 revealed that a phenethyl chain had the right length and size to fit into pocket B while other critical interactions were maintained (Figure 2.3b). Therefore, we set to explore the druggability of pocket B using substituted phenethyl chains. 2.2 Results and Discussion 2.2.1 Syntheses of 3-Substituted Indole N-acyl-sulfonamides Syntheses of indole N-acyl-sulfonamides (Figure 2.4) commenced with the Nalkylation of methyl indole-5-carboxylate 2.11 to yield 2.13. Subsequent formylation under Vilsmeier-Haack condition formed aldehyde 2.14. Wittg olefination reaction of 2.14 with various aryl-triphenylphosphonium bromides resulted in a mixture of cis/trans alkenes, which upon reduction afforded 3-phenethyl indoles 2.15 – 2.19. Due to the electron-rich C5-position, standard basic hydrolysis conditions did not yield the desired product. Elevated temperature or prolonged reaction time with excess base led to nitrile reduction as well as N-dealkylation. However, a TMSCl mediated demethylation method19 formed the desired carboxylic acids 2.20 – 2.24. Coupling with different sulfonamides, followed by [2+3] cycloadditions, furnished 3-substituted indole N-acyl-sulfonamides 2.34 – 2.42. 2.2.2 Biological Evaluations of 3-Substituted Indole N-acyl-sulfonamides The Ki-values of the compounds were determined by using fluorescence polarization (FP) assay.20 To further evaluate the binding modes, the same assay was employed with mutant instead of wild-type β-catenin proteins. Selected molecules were also subjected to two cancer cell lines, SW480 (Wnt-active) and A549 (Wnt-inactive) to further assess their in vivo efficacy. Finally, we have also established a TOPFlash luciferase 73 assay with HEK293 or SW480 cell lines. This assay can be used to quantify intracellular Wnt signaling by transducing a luciferase-containing plasmid, which contains several Tcf binding sites. In the event of Wnt activation, β-catenins translocate into cell nucleus, bind to Tcf and induces luciferase production. Thus, a small molecule Wnt antagonist is expected to reduce the luciferase activity. Compounds 2.34 – 2.42 (Table 2.1) exhibited better Ki-values than N-acylsulfonamides 1.26 – 1.37 described in Chapter 1. No distinct SARs were observed for pocket B, but substrates with p-chloro substituted aryl chain were in general more potent. Compounds with mono- or di-chlorinated thiophenes were also more active than those with meta-fluoro benzene residues. For instance, compounds 2.35 and 2.36 were both almost 10 times more active than 2.38 and 2.39, respectively. Two inhibitors, 2.34 and 2.41, were evaluated by MTS assay and TOPFlash luciferase assay (Table 2.2). Both compounds inhibited the growth of SW480, an aberrantly Wnt activated colon cancer cell line, with moderate IC50 values. Unfortunately, luciferase activity in pcDNA3.1 – β-catenin transfected human embryonic kidney cell line (HEK293) could not be suppressed even at concentrations above 100 µM. The cell-based results were contradictory. Compounds 2.34 and 2.41 inhibited cancer cell proliferation, yet the luciferase assay suggested that they operated in a Wntpathway independent manner. This raised the question whether substrates of this scaffold were truly acting as modulators of the β-catenin/Tcf4 PPI. Next, we examined the following two aspects of the FP-assay in detail, which suggested that the 3-substituted indole N-acyl-sulfonamides might exhibit as promiscuous binders. We screened first nitrile-containing precursors, 2.25 and 2.27 – 2.33. The rationale was to compare activities 74 of those with the tetrazole containing compounds. The negative charge of a tetrazole should hypothetically engage in charge-charge interactions with K435, which is significantly stronger than a hydrogen bond formed by a nitrile group.21 Therefore, final compounds 2.34 – 2.43 with tetrazole moieties were anticipated to be more active than their nitrile precursors, if bound to K435. Against expectations, FP-assay revealed that nitrile precursors had at least similar, and in some cases better, activities than their tetrazole counterparts (Table 2.3). Next, we closely inspected the dose-response curves from the FP-assay. Most of the tested inhibitors 2.34 – 2.43 showed steep dose-response curves, whereas UU-T01 inhibited in a sigmoidal manner (Figure 2.5). This steepness is defined as Hill slope22 and it is quantified through the tangent slope of a sigmoidal curve at 50% inhibition. One challenge in drug discovery is the steep Hill slope exhibited by “hit molecules”, which inhibit the biological target at a faster pace than one would expect. For instance, UU-T01 increased inhibition from 10% to 90% over a 71-fold concentration range. However, 2.34 accomplished the same in a 20-fold concentration range (Figure 2.5). On a molecular level this phenomenon can be explained in two ways. First, if the tested macromolecular target had a simple kinetic binding mechanism, with presumably only one binding site and no allosteric site, then the Hill slope should be approximately equal to one. An increase of the slope is therefore generally associated with a higher number of protein-binding sites, meaning that the target, in our case β-catenin, interacted with multiple molecules of 2.34 in an unspecific fashion. Second, organic molecules with high sp2-carbon content are likely to undergo phase transition by forming colloidal aggregation. The surfaces of these aggregate species are 75 thought to adsorb proteins and thereby inhibit their activities nonspecifically.23-24 Shoichet and co-workers reported that aggregates disintegrated and lost potency in the presence of detergents.25 Especially assay conditions with 0.1% Triton X-100 as an additive have shown to effectively expose promiscuous aggregators but at the same time retain protein activity. We subsequently performed FP-assays with compound 2.34 using different concentrations of Triton X-100 and CHAPS. The results (Table 2.4) showed that the inhibitory ability of 2.34 was strongly attenuated with the addition of 0.1% Triton X-100 or 0.4% CHAPS. It corroborated the suspicion that 2.34 and likely other inhibitors from the same class act as aggregation inhibitors. Control studies confirm that different levels of detergents do not affect native β-catenin/Tcf4 binding (Table 2.4). Although this outcome confirmed our hypothesis about aggregate-driven inhibition, more biophysical analyses, such as dynamic light scattering or transmission electron microscopy,26 were required to further characterize aggregate species. Finally, the binding modes of 2.34 and 2.40 – 2.42 were evaluated by site-directed mutagenesis (Table 2.5). Comparison of the FP-assay results between the wild-type and mutant β-catenin proteins suggested that none of our designed molecules interacted with R469 or pocket B. Therefore, we reasoned that the inhibitory activities must stem from nonspecific interactions. 2.2.3 Design and Syntheses of 2,3-Disubstituted Indole-5-Carboxylic Acids A review27 by Huggins and co-workers states that PPI inhibitors inherently tend to be larger and more hydrophobic than drug molecules targeting traditional enzymes. Complex protein surface morphology and tight protein binding are reasons why drug 76 development in this area is difficult. Statistically, the average affinity contribution per nonhydrogen atom (ligand efficiency or LE) for PPI inhibitors is around 0.23 kcal/mol, whereas traditional drug leads have an LE of 0.32 kcal/mol. In terms of binding constants, if a PPI inhibitor with 30 nonhydrogen atoms binds to its target with 1 µM affinity, then a traditional lead can do the same with 90 nM affinity. Therefore, we envisioned further expanding the indole scaffold by incorporating additional hot spots in pocket C region. A more potent inhibitor would serve two purposes. First, aggregate-based inhibition would be less likely to occur at lower concentrations. Second, ligands with increased molecular weight would be prone to outcompete Tcf4 binding and achieve selectivity. We chose fused heterocycles such as benzothiazole or benzimidazole with a twocarbon linker to explore pocket C. These moieties were predicted via Autodock to interact with the arginine channel, composed of R515 and R474 and pocket C via cation-π and hydrophobic interactions. Furthermore, the N-acyl-sulfonamide moiety was removed due to its presumed hydrophobicity, which was suspected to contribute to the formation of potential aggregates. Instead, a carboxyl group served as a Tcf E17 replacement. For charge-charge interactions with K435, both tetrazole and carboxyl groups were incorporated into the scaffold (Figure 2.6). Syntheses of 2,3-disubstituted indole 5-carboxylic acids are depicted in Figure 2.7. Synthetic efforts were focused on functionalizing the 2-position of indole and installation of the heterocycles. Formylation of 2.17 under acid conditions resulted in recovery of the starting material, while basic conditions led to N-dealkylation. However, the left-hand fragments 2.43 – 2.46 could be accessed via AgOTf mediated iodination28 of 2.15 – 2.18. Attempts to couple 2-iodo indoles 2.45 with 2-vinyl benzimidazole SF1 were unsuccessful. 77 Given the nucleophilicity29 and electron-rich nature, coordination between the benzimidazole nitrogen and palladium could have led to stable and unreactive complexes.30-31 Consequently, most reactions resulted in the recovery of starting material as well as isolation of a N-dealkylated side product. Next, we screened several conditions (Table 2.6, Entry 1 – 9) for the Sonogashira reaction with 2-iodo indole 2.43 and alkyne S4.1. It was found that employing Pd(PPh3)4 and CuI as catalysts and Et3N as base/solvent at 60 oC gave the best yields (Table 2.6, Entry 9). Subsequent hydrogenation reactions afforded alkanes 2.56 – 2.64 (Figure 2.7). Azide-mediated cycloaddition, followed by ester hydrolysis, provided tetrazole-containing final compounds 2.73 – 2.80. Concurrently, several intermediates treated with aqueous NaOH in refluxing iPrOH yielded dicarboxylic acids 2.81 – 2.86 as another set of final compounds. 2.2.4 Biological Evaluation of 2,3-Disubstituted Indole-5-Carboxylic Acids We selected 2.73 and 2.85 and performed site-directed mutagenesis studies to validate the binding mode. Ki-values of 2.73 and 2.85 derived from FP-assay with native β-catenin/Tcf4 protein complex were compared with values from mutant β-catenin/Tcf4 complex (Table 2.7). Both compounds exhibited inhibitory activities in the lowmicromolar range, with Ki = 0.93 ± 0.51 µM and Ki = 1.98 ± 1.24 µM, respectively. In contrast, binding towards mutant proteins significantly dropped. For R469A, 2.73 has experienced a more than 40-fold and 2.85 a 19-fold decrease in activity. It demonstrated that R469 is an important hot spot and, presumably, responsible for an important cation-π interaction with the indole. Hydrophobic amino acid residues V511 and I569 from pocket B were mutated into hydrophilic serine residues. This change greatly affected the inhibition 78 of 2.73 (Ki = 48.75 ± 10.12 µM) and 2.85 (Ki = 65.70 ± 8.85 µM). Lastly, we examined whether benzothiazole and benzimidazole side chains were critical residues interacting with the arginine channel. Ki-values determined from FP-assay with the double-mutant protein R474A/R515A showed a significant decrease in binding affinity, with Ki = 25.52 ± 6.23 µM (2.73) and Ki = 56.75 ± 12.71 µM (2.85). Our results indicated that 2,3disubstituted indole-5-carboxylic acids inhibited β-catenin/Tcf4 PPI in a specific manner, namely by interacting with R469, pocket B and arginine channel in hot region 1. These results prompted us to further investigate the inhibitory activities of the final compounds 2.73 – 2.86. Ki-values derived from FP assays are shown in Table 2.8. Molecules with a tetrazole moiety were 2–4-fold more potent than their dicarboxylic acid counterparts. Due to the presence of several nitrogen atoms, tetrazoles were more likely to form additional hydrogen bonds with N430 and H470. Molecules with benzothiazole moiety were roughly 3–4-fold more potent than those with benzimidazole, suggesting stronger cation-π interactions. Further variations with electron-donating and withdrawing groups (2.74 vs. 2.76) did not result in notable changes of potency. We quantified the selectivity of 2.73 between β-catenin/Tcf4, β-catenin/APC, βcatenin/Ecadherin PPIs and compared the results with those from other known inhibitors (Table 2.9).16,32 Compound 2.73 exhibited good selectivity for β-catenin/Tcf4 over Ecadherin and APC. In contrast, both PKF compounds disrupted β-catenin/Ecadherin PPI. Compound UU-T01 showed similar selectivity compared to 2.73. MTS cell viability assays (Table 2.10) with SW480 (Wnt-active) and A549 (Wnt inactive) were performed to evaluate in vivo efficacy of 2.74M and 2.77M, the methyl ester derivatives of 2.74 and 2.77. The former inhibited cancer cell proliferation in the low 79 micromolar range with IC50 = 3.55 ± 1.54 µM for SW480 and IC50 = 12.12 ± 4.11 µM for A549. It displayed 4-fold selectivity for the Wnt-active over the Wnt inactive cell line. However, 2.77M showed significantly lower potency and selectivity, preventing cell growth in high micromolar range. A similar result for 2.74M was obtained in TOPFlash luciferase assay (Table 2.9). This compound suppressed Wnt-mediated gene transcription with IC50 = 4.77 ± 0.89 µM. Two known inhibitors, compound 1917 (Figure 2.1) and quercetin, a known Wnt modulator,33 were tested under the same conditions but were up to 48-fold less efficacious. Finally, we functionalized several 5-indole carboxylic acids into amide and ester derivatives 2.87 – 2.91 in order to probe pocket D. Preliminary results suggested that these compounds inhibited β-catenin/Tcf4 PPI with submicromolar affinities and had promising cell-based results (Table 2.11). In collaboration with the Welm lab from Huntsman Cancer Institute, 2.87, 2.89 and 2.90 were tested in a 3D branching assay34 using mammary epithelial cells (MEC) from female FVB mice. The organoid-based 3D MEC-assay was aimed to better replicate a cellular environment35 and evaluate phenotypical responses of normal Wnt-inactive cells towards our substrates. However, the tested compounds caused primarily cytotoxic phenotypes (Figure 2.9), which were attributed to the high hydrophobic nature (cLogP 8.6 – 9.5). 2.3 Conclusion β-Catenin/Tcf4 PPI is a downstream effector of the Wnt-signaling pathway and involved in a variety of diseases. In order to generate novel therapeutic PPI inhibitors, we used a hot spot-based design strategy previously validated with the development of UU- 80 T01. Coupled with extensive docking studies, we generated three series of inhibitors, Nacyl-sulfonamides (Chapter 1), 3-substituted indole N-acyl-sulfonamides, and 2,3disubstituted indole-5-carboxylic acids, which were biologically validated. Unfortunately, the first two series were found to be inactive. Only the highly functionalized indoles were determined to bind to the desired hot region and showed promising biological activities ex vivo. Our initial idea was to generate small molecule fragments (1st series) possessing the same binding epitope as UU-T01 and then gradually evolve the scaffold into more potent substrates (2nd and 3rd series) by capturing additional pharmacophores in the hot region 1. Yet this strategy has not entirely met with success as several factors of PPI drug design were not taken into consideration. First, we used a fragment-evolution strategy to increase potency/selectivity,36 without validating whether the fragment to begin with was proven biochemically, and not predicted by software, to bind to the active site. Second, drug discovery targeting PPI cannot solely rely on conventional docking software, which are not tailored for such complex macromolecular interactions. In a seminal review paper,37 Jim Wells once stated, “One should NOT presume that the most suitable binding site for small molecules is found in static protein X-ray structures.” Remarkably, for several frequently explored PPIs, conformational changes or cavity openings around the active site were observed when a small molecule was bound, but not in apo state or bound to another protein.38-39 Third, physio-chemical properties such as cLogP-values must be accounted for in the drug design in order to prevent aggreagation-based inhibition. Even though molecules from the third series have been biochemically validated 81 through site-directed mutagenesis to bind to the desired active site, questions still remain unanswered. How can the hydrophobicity be decreased? Does the potency still uphold when detergent is added? And most importantly, how to structurally modify an inhibitor which can effectively outcompete a tight-binding protein complex? Pharmaceutical industries have always relied on fragment-based screenings40 or high throughput screenings41 to discover new inhibitors of PPI. Therefore, it remains to be seen what contribution can be made in drug discovery using the bottom-up approach. 82 Loose and Narrow Loose and Wide N PDB: 1BKD RAS/SOS BSA = 3600 Å2 Kd = 3.6 µM NH2 N N NH2 N N PDB: 1F47 ZIPA/FTSZ BSA = 1196 Å2 Kd = 21.6 µM H N Me Pyrimidine-based inhibitor Ref. 7 Ki = 12 µM N N Me Compound 3 Ref. 8 Kd = 83.1 µM O DCAI Ref. 9 Kd = 1.5 mM H N O N N H N H BZIM Ref. 9 Kd = 1.1 mM N N N N N H Cl N H N Compound 2 Ref. 8 Kd = 73.9 µM N N 200 nM N H Me O Cl N H Me Me NH2 Compound 11 Ref. 10 Kd = 240 µM Tight and Wide Tight and Narrow PDB: 2XA0 Bcl-2/BAX-BH3 BSA = < 2500 Å2 Kd = 0.2 µM O O O S N H O NO2 PDB: 2GL7 N N N N NH Compound 33f Ref. 11 Ki = 2.4 nM Me N Me S 𝛃-catenin/Tcf4 OH BSA = > 2800 Å2 Kd = 5 -10 nM UU-T01 Ref. 16 Ki = 3.14 µM Compound 19 Ref. 17 Ki = 1.36 µM Cl O O O S N H Affinity of PPI Cl HN N N N NO2 O OH N N Buried Surface Area (BSA) Cl O ABT-737 Ref. 12 Ki = < 1 nM MeO NH S Me N Me O N H O O H N O N H N H OH 2500 Å2 Figure 2.1: Categorization of PPIs with one crystal structure and corresponding inhibitors in each category (adapted from Gestwiki et al., Expert Rev. Mol. Med., 2012, 14, e16). 83 R2 O O O O S N R 1 H OH O H N O previous work H N N H O R1 N HN N N N OH E17 O O O S N H this work HN N N N Indole N-acyl-sulfonamides N-acyl-sulfonamides Ki = 26.70 - 2000 µM D16 Figure 2.2: Scaffold development of N-acyl-sulfonamides to enhance the electron density. B A Pocket D Pocket B Pocket D Pocket B K508 K508 R469 R469 K435 K435 Figure 2.3: Autodock predicted binding modes for designed inhibitors A) Docking of an indole version of 1.28. B) Docking of 3-substituted indole N-acyl-sulfonamide 2.34. O MeO N H 6h, 72% 2.13 TMSCl, NaI MeO N 2.15 -2.19 R2 HO MeCN, reflux, 12 h 55-72% N CN S Cl Cl S Cl F 2.34 2.35 2.14 N CN n-Bu3SnN3, PhMe reflux, 2.5 - 3 d 25-85% O O O O O S N R1 H R2 N CN 2.25 - 2.33 HN 2.34 - 2.42 N N N Cl Cl S 2) H2, Pd/C THF/MeOH, rt, 3 h 42-83% R2 Cl Cl 1) Wittig salt, n-BuLi THF, -40 oC - rt, 3 h N CN O O O S CDI, DBU, 1.25 N R 1 H THF, reflux - rt, 2 d 37-82% H MeO NH4OAc, THF, rt-reflux overnight, 96% CN 2.20 - 2.24 Cl R1 N O R2 O O (COCl)2, DMF, DCM MeO 2.12 2.11 O CN + Br O NaH, DMF 0 cC - rt F F Cl F F O O S Cl Cl S Me Cl Cl S O O R2 2.36 2.37 2.38 2.39 2.40 2.41 2.42 Figure 2.4: Syntheses of 3-substituted indole N-acyl-sulfonamides 2.34 – 2.42. 84 100 UU-T01 Percent Inhibition (%) Percent inhibition (%) 100 80 90% inhibition 60 10% inhibition 40 20 0 60 0 1 2 3 10% inhibition 40 20 0 -1 2.34 80 90% inhibition 4 -1 0 1 2 3 Inhibitor log µM inhibitor µM Figure 2.5: Dose response curves of UU-T01 and 2.34. R2 O O O O S N R 1 H OH O H N O N H H N O D16 O R1 OH E17 O O O S N H HN N N N N-acyl-sulfonamides Ki = 26.70 - 2000 µM R2 HO N N N X HN N N N 3-Substituted indole N-acyl-sulfonamides Ki = 1.17 - 20.88 µM HO O HN N N N X = S or NH 2,3-Disubstituted indole-5-carboxylic acid Figure 2.6: Scaffold development towards 2,3-disubstituted indole 5-carboxylic acid. 85 O R2 O AgOTf, I2, CH2Cl2 MeO I + N N X = S or N O MeO N O X N HN aq. NaOH, i-PrOH reflux, 16 h, 43% - quant. R3 N N N N HN 2.65 - 2.72 N N N X = S or N 2.73 - 2.80 R2 HO X N reflux, 6 h 29 - 75% over 2 steps X = S or N R3 O rt, 48 h 47% - quant. R2 HO 3M NaOH, i-PrOH N X = S or N CN 2.56 - 2.64 R3 X = S or N 2.47 - 2.55 reflux, 48 h N N N R2 nBu3SnN3 PhMe X H2, Pd/C EtOH/THF X S4.1 - S4.5 MeO R2 R2 MeO CN CN 2.43 - 2.46 CN 2.15 - 2.18 O Pd(PPh3)4, CuI Et3N, DMF o R3 60 C, 24 h 57 - 90% X rt, 30 min 57 - 87% N O R2 MeO X N N R3 X = S or N 2.81 - 2.86 HO O dicarboxylic acid final compounds tetrazole-bearing final compounds R2 Cl Cl Cl Cl Cl R3 H X S S S 2.73 2.74 2.75 F Me H H H H R3 S N S S S X 2.76 2.77 2.78 OMe O(CH2)2OMe Cl 2.79 Cl Cl R2 H H 2.81 Me Me H H H S 2.86 N S S N 2.82 2.83 2.84 2.85 S 2.80 F H Figure 2.7: General synthesis of 2,3-disubstituted indole-5-carboxylic acids. R4 R2 X O N HO S N HN N N N R3 R2 R3 R4 X Compound F H F N 2.87 F H Cl N 2.88 Cl O(CH2)2OMe Cl N 2.89 Cl OMe Cl N 2.90 Cl OMe Cl O 2.91 R2 R4 O CDI, DBU THF, reflux - rt 2 d, 25 - 36% N X S N HN N N N R3 Figure 2.8: Amide or ester formation to functionalize C5-carboxyl group. R3 86 Figure 2.9: Results from 3D MEC assay after 144h. Compounds 2.87 (TG-5-23) solely displayed cytotoxic phenotype in organoids. 2.89 (TG-5-38) and 2.90 (TG-5-37) showed cytotoxicity in at least 50% of the organoids. Table 2.1: Ki for 3-substituted indoles determined by FP-assay. Compound 2.34 2.35 2.36 2.37 2.38 2.39 2.40 2.41 2.42 Ki ± SD (µM) 1.17 ± 0.60 2.00 ± 0.27 2.48 ± 0.25 6.60 ± 0.94 19.22 ± 1.52 20.88 ± 1.23 2.61 ± 0.51 2.38 ± 0.26 2.61 ± 0.21 Table 2.2: MTS cell proliferation and TOPFlash assays for 2.34 and 2.41. Compound 2.34 2.41 Cell proliferation (SW480) IC50 ± SD (µM) 33.43 ± 4.59 50.19 ±7.79 TOPFlash luciferase (HEK293) IC50 ± SD (µM) > 100 > 100 87 Table 2.3: Ki-values of some indole N-sulfonamides and their nitrile precursors. Final compound 2.34 2.36 2.37 2.38 2.42 Ki ± SD (µM) 1.17 ± 0.60 2.48 ± 0.25 6.60 ± 0.94 19.22 ± 1.52 2.61 ± 0.21 Nitrile precursor 2.25 2.27 2.28 2.29 2.33 Ki ± SD (µM) 0.57 ± 0.09 1.43 ± 0.60 5.05 ± 1.03 15.99 ± 1.32 6.60 ± 0.74 Table 2.4: Effect of Triton X-100 and CHAPS on binding affinity. Triton X-100 β-catenin/Tcf4 v/v% (. (Kd ± SD in nM) 0.0001 6.61 ± 1.64 0.01 3.09 ± 0.53 0.1 4.09 ± 0.56 2.34 (Ki ± SD in µM) 1.07 ± 0.80 0.63 ± 0.39 46.24 ± 4.15 v/v% 0.1 0.2 0.4 CHAPS β-catenin/Tcf4 (Kd ± SD in nM) 9.42 ± 1.79 7.87 ± 1.50 7.48 ± 1.43 2.34 (Ki ± SD in µM) 1.13 ± 0.38 1.42 ± 0.87 36.80 ± 3.99 Table 2.5: Mutation studies to evaluate binding mode of 2.34 and 2.40 – 2.42. β-catenin wild-type R469A V511S V511S/I569S 2.34 1.10 ± 0.71 1.77 ± 0.84 2.57 ± 0.88 1.20 ± 0.59 Ki ± SD (µM) 2.40 2.41 2.28 ± 0.69 2.38 ± 0.26 3.86 ± 0.97 3.51 ± 0.35 7.06 ± 1.19 2.38 ± 0.38 3.42 ± 1.19 3.40 ± 0.38 2.42 1.83 ± 0.24 4.28 ± 0.71 6.01 ± 1.30 5.23 ± 1.18 88 Table 2.6: Reaction optimization for the Sonogashira reaction. Cl Cl O MeO I N 2.43 Entry 1 2 3 4 5 6 7 8 9 CN + N Pd-catalyst, CuI Et3N, solvent S temperature, 18 h yield O N CN S4.1 Catalyst PdCl2(PPh3)2 PdCl2(PPh3)2 PdCl2(PPh3)2 Pd(OAc)2 Pd(P(tBu)3)3 Pd(PPh3)4 PdCl2(PPh3)2 Pd(PPh3)4 Pd(PPh3)4 N MeO Solvent DMF Et3N:THF 1:3 THF Et3N Et3N THF DMF DMF Et3N Temperature r.t. r.t. r.t. r.t. r.t. r.t. 60 oC 60 oC 60 oC S 2.47 Yield trace 13% 27% 16% trace 35% 52% 67% 85% Table 2.7: Mutation studies to evaluate binding mode of 2.73 and 2.85. β-catenin wild-type R469A V511S/I569S R474A/R515A 2.73 Mutant/WT Ki ± SD (µM) 0.93 ± 0.51 N.A. 39.34 ± 10.29 42 fold 48.75 ± 10.12 52 fold 25.52 ± 6.23 27 fold 2.85 Mutant/WT Ki ± SD (µM) 1.98 ± 1.24 N.A. 37.42 ± 8.38 19 fold 65.70 ± 8.856 33 fold 56.75 ± 12.71 28 fold Table 2.8: Ki-values for 2,3-disubstituted indoles-5-carboxylic acids. Final compound 2.73 2.74 2.75 2.76 2.77 2.78 2.79 Ki ± SD (µM) 2.78 ± 0.83 2.76 ± 0.58 5.76 ± 1.58 1.72 ± 0.79 8.28 ± 1.75 3.33 ± 0.59 1.57 ± 0.43 Final compound 2.80 2.81 2.82 2.83 2.84 2.85 2.86 Ki ± SD (µM) 7.63 ± 2.41 4.69 ± 0.97 23.31 ±5.88 19.02 ± 2.77 5.83 ± 1.31 15.47 ± 2.28 27.72 ± 6.84 89 Table 2.9: FP-assay to determine selectivity of inhibitors for β-catenin/Tcf4 PPI. Compound PKF115-584 PKF118-310 UU-T01 2.73 β-Cat./Tcf4 18 ± 2 5.8 ± 0.2 3.1 ± 0.5 0.93 ± 0.50 Ki ± SD (µM) β-Cat./Ecad. β-Cat./APC 13 ± 1 54 ± 1 13 ± 1 170 ± 10 100 ± 10 180 ± 10 25.52 ± 7.97 74.02 ± 11.46 Selectivity Tcf4/Ecad. Tcf4/APC 0.7 fold 3.0 fold 2.2 fold 29.3 fold 32.3 fold 58.1 fold 27 fold 79 fold Table 2.10: MTS assay and TOPFlash luciferase gene reporter assay. Compound comp. 19 quercetin 2.74M 2.77M Proliferation SW480 IC50 ± SD (µM) 152.6 ± 3.7 60.09 ± 4.75 3.55 ± 1.54 69.30 ± 10.74 Proliferation A549 IC50 ± SD (µM) N. D. N. D. 12.12 ± 4.11 120.5 ± 43.97 TOPFlash SW480 IC50 ± SD (µM) 231.9 ± 3.2 66.74 ± 3.64 4.77 ± 0.89 N. D. Table 2.11: MTS assay and TOPFlash luciferase gene reporter assay. Compound 2.87 2.89 2.91 Proliferation SW480 IC50 ± SD (µM) 12.45 ± 1.28 9.26 ± 4.91 164.10 ± 17.36 Proliferation A549 IC50 ± SD (µM) 41.55 ± 5.78 33.64 ± 10.64 311.50 ± 26.89 TOPFlash SW480 IC50 ± SD (µM) 16.23 ± 3.60 N. D. N. D. 90 2.4 References 1. Arkin, M. R.; Wells, J. A., Small-Molecule Inhibitors of Protein–Protein Interactions: Progressing towards the Dream. Nat. Rev. Drug Discov., 2004, 3, 301-317. 2. Arkin, Michelle R.; Tang, Y.; Wells, James A., Small-Molecule Inhibitors of Protein-Protein Interactions: Progressing toward the Reality. Chemistry & Biology, 2014, 21, 1102-1114. 3. Wells, J. A.; McClendon, C. L., Reaching for High-Hanging Fruit in Drug Discovery at Protein–Protein Interfaces. Nature, 2007, 450, 1001-1009. 4. Fuller, J. C.; Burgoyne, N. J.; Jackson, R. 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The preparation of the crystal structure and molecular modeling were achieved with the commercially available Accelrys Discovery Studio 3.0 (http://accelrys.com/), Schrodinger (http://www.schrodinger.com/) and SYBYL X 2.0 (http://www.tripos.com) software packages. The missing side chains of β-catenin were added in SYBYL X2.0. The protonation states of the residues were set to pH 7.0 when adding the hydrogens. The AMBER 7 force field 99 within SYBYL X2.0 was used to optimize the orientation of hydrogen atoms and the missing side chains of the protein and of structural waters. After the protein structure was optimized. Chains B (Tcf4), C (BCL9), D (the second monomer of β-catenin), E (the second monomer of Tcf4), F (the second monomer of BCL9), and solvent molecules were removed, leaving only one monomer of β-catenin for further calculation. The residues in the Tcf4 G13ANDE17 binding site of β-catenin include G422, S425-N426, T428-N430, K435, E462-P463, I465-C466, R469-H470, S473-R474, Q482, L506-A509, V511-G512, R515-N516, L519, L536, L539, R565, E568-I569, E571-C573, G575-A576, H578-I579, R582, N609, R612-V613, E620, and Y654. 2.5.2 AutoDock4 Study AutoDock 4.2 was employed to perform the docking calculations. Only the polar hydrogen atoms remained for the protein structure, and Kollman united atom charges were 95 assigned. The 3D structures of the ligands were built and partial atomic charges were also calculated using the Gasteiger–Marsili method. The rotatable bonds in the ligands were defined using AutoTors, which also unites the nonpolar hydrogens and partial atomic charges to the bonded carbon atoms. The grid maps were calculated using AutoGrid. The dimension of the grid box was 39 x 28.5 x 21.5 Å, and the grid spacing was set to 0.375 Å. Docking was performed using the Lamarckian genetic algorithm (LGA), and the pseudoSolis and Wets method were applied for the local search. Each docking experiment was performed 100 times, yielding 100 docked conformations. Parameters for the docking experiments were: initial population size of 150, a maximum of 1.5 x 106 energy evaluations; a maximum of 27,000 generations; a maximum of 1 top individual will automatically survive; random starting position and conformation. Other settings were the standard default parameters. All of the ligands followed the same docking protocol. The results of the docking experiments were evaluated by the auxiliary clustering analysis and/or by a visual inspection to match the proposed docking mode. 2.5.3 Fluorescence Polarization Assays All biological assays were performed by Dr. Min Zhang (Ji Lab). For FP-assay, experiments were performed in 96-well Microfluor 2 black plates (Waltham, MA), and the samples were read with a Synergy 2 plate reader (Biotek, Winooski, VT). The polarization was measured at room temperature with an excitation wavelength at 485 nm and an emission wavelength at 535 nm. The FP saturation experiments were performed in an assay buffer of 137 mM of NaCl, 2.7 mM of KCl, 10 mM of Na2HPO4, 2 mM of KH2PO4, 100 µg/mL of bovine gamma globulin, and 0.01% Triton-X 100. The final reaction volume was 96 100 µL. In the FP competitive inhibition assays, 10 nM of β-catenin (residues 142-686) was incubated with C-terminally fluorescein-labeled human Tcf4 (residues 7-51) for 30 min at 4 °C, and then different concentrations of the tested peptides or compounds in the assay buffer were added to make a final volume of 100 µL. Each assay plate was covered black and gently mixed on an orbital shaker for 3 h to reach equilibrium before polarization values were read. The background of the tested peptides or inhibitors was corrected by subtracting the raw intensity values of the sample background well from the raw intensity values of the corresponding test wells (all components). The IC50 values were determined by nonlinear least-square analysis using GraphPad Prism 5.0. The Ki values were derived from the IC50 values by the reported method. Experiments were performed in triplicate and carried out in the presence of 1% DMSO. To evaluate the effects of the orders of adding proteins and compounds in the FP competitive inhibition assays, 10 nM of β-catenin (residues 142-686) was also incubated with different concentrations of the compounds in the assay buffer for 30 min at 4 °C, and then with C-terminally fluorescein-labeled human Tcf4 (residues 7-51) in the assay buffer were added to make a final volume of 100 µL. Each assay plate was covered black and mixed on an orbital shaker for 3 h to reach equilibrium before polarization values were read. The background of the tested inhibitors was corrected by subtracting the raw intensity values of the sample background well (all components except probe) from the raw intensity values of the corresponding test wells (all components). The IC50 values were determined by nonlinear least-square analysis using GraphPad Prism 5.0. The Ki values were derived from the IC50 values by the reported method. 97 2.5.4 Site-Directed Mutagenesis Studies β-Catenin mutants V511S, V511S/I569S, R469A and R474A/R515A were generated using the overlapping PCR technique. The template for the mutagenesis reactions was the wild-type full-length β-catenin in pET-28a. KOD hot start DNA polymerase (Novagen) was used through all the experiments. Mutants were confirmed by direct sequencing (Core facility, University of Utah). The templates used for β-catenin double mutation were V511S for V511S/I569S and R474A for R474A/R515A, respectively. The double mutants were again confirmed by direct sequencing. Following the confirmation of the sequence, β-catenin mutants were cloned into a pET-28 vector and transformed into E. coli BL21 DE3. 2.5.5 MTS Cell Viability Assay Colorectal cancer cell lines, SW480 and A549 were seeded in 96 well plates at 4 Å~ 103 cells/well, maintained overnight at 37 °C, and incubated in the presence of inhibitor at various concentrations. Cell viability was monitored after 72 h using a freshly prepared mixture of one part phenazine methosulfate (PMS, Sigma) solution (0.92 mg/mL) and 19 parts 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium (MTs, Promega) solution (2mg/mL). Cells were incubated in 10 μL of this solution at 37 °C for 3 h and A490 was measured. The effect of each compound is expressed as the concentration required to reduce A490 by 50% (IC50) relative to vehicle-treated cells. Experiments were performed in triplicate. 98 2.5.6 Cell Transfection and Luciferase Reporter Assay Cell transfection and luciferase assay. FuGENE6 (Promega) 96 well plate format was used for the transfection of HEK293 and SW480 cells according to the manufacturer’s instructions. HEK293 cells were co-transfected with 45 ng of TOPFlash or FOPFlash reporter gene, 135 ng pcDNA3.1–β-catenin and 20 ng pCMV-RL normalization reporter gene. SW480 cells were co-transfected with 60 ng of TOPFlash or FOPFlash reporter gene and 40 ng pCMV-RL normalization reporter. Cells were cultured in DMEM and 10% fetal bovine serum (FBS) at 37 °C for 24 h, and the different concentrations of inhibitors or DMSO were then added. After 24 h, the luciferase reporter activity was measured using the Dual-Glo system (Promega). Normalized luciferase activity in response to treatment with small-molecule compounds was compared with that obtained from cells treated with DMSO. Experiments were performed in triplicate. 2.5.7 General Experimental Conditions (Chemistry) All experiments were conducted under anhydrous conditions in an atmosphere of argon, using flame-dried apparatus and employing standard techniques in handling airsensitive materials. Dichloromethane (CH2Cl2), acetonitrile (CH3CN), tetrahydrofuran (THF), dimethylformamide (DMF) were degassed with nitrogen and passed through JC Meyer solvent systems. All reagents were used as received. Aqueous solutions of sodium bicarbonate, sodium chloride (brine), and ammonium chloride were saturated. Analytical thin layer chromatography was visualized by ultraviolet light. Flash chromatography was performed on SilicaFlash@ F60 silica gel (230–400 mesh). 1H NMR spectra were recorded using a Varian Unity Inova 500 (500 MHz) or a Varian Unity Inova 300 (300 MHz). 13C 99 NMR spectra were recorded using a Varian Unity Inova 500 (125 MHz) or Varian Unity Inova 300 (75 MHz). The 1H and 13C NMR spectra are referenced to the residual solvent signals (7.26 ppm for 1H and 77.0 ppm for 13C in CDCl3; 2.05 ppm for 1H and 29.8 ppm for 13C in acetone-d6; 2.50 ppm for 1H and 39.5 ppm for 13C in DMSO-d6). Low (MS) and high (HRMS) mass spectra were determined on a Micromass Quattro II (ESI/APCI-TOF) at the University of Utah Mass Spectrometry Facility. 2.5.8 Procedures and Characterizations O MeO N CN Methyl 1-(2-cyanoethyl)-1H-indole-5-carboxylate (2.13). In a 50 mL oven-dried round bottom flask, methyl 1H-indole-5-carboxylate (0.3 g, 1.71 mmol) was dissolved in 20 mL anhydrous DMF and cooled down to 0 ˚C. NaH (0.10 g, 2.57 mmol) was added portionwise. The resulting heterogeneous mixture was stirred at 0 ˚C for 45 min. 3Bromopropanenitrile (0.21 mL, 2.57 mmol) was then added dropwise via syringe and the reaction mixture was allowed to warm up to room temperature. After 6 h, the reaction was quenched with 1M HCl, extracted two times with EtOAc and concentrated under reduced pressure. The crude product was purified via flash column chromatography (3:1 hexanes/EtOAc, followed by 2:1 hexanes/EtOAc) to obtain a yellow solid (0.28 g, 72%). Rf = 0.3 (2:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 8.39 (s, 1H), 7.93 (dd, J = 1.5, 8.7 Hz, 1H), 7.30 (d, J = 8.7 Hz, 1H), 7.20 (d, J = 3.3 Hz, 1H), 6.63 (d, J = 3.3 Hz, 1H), 4.42 (t, J = 6.6 Hz, 2H), 3.92 (s, 3H), 2.80 (t, J = 6.6 Hz, 2H). 13C NMR (CDCl3, 75 MHz): δ 168.1, 138.1, 129.1, 128.7, 124.5, 123.8, 122.5, 117.3, 108.6, 104.6, 52.2, 42.4, 100 19.4. LRMS (ESI) m/z = 229.2 [M+H]+. O O H MeO N CN Methyl 1-(2-cyanoethyl)-3-formyl-1H-indole-5-carboxylate (2.14). To a solution of oxalyl chloride (0.10 mL, 1.19 mmol) in 5 mL anhydrous CH2Cl2 at 0 ˚C was added dropwise 0.09 mL anhydrous DMF in 5 mL CH2Cl2. The heterogeneous mixture was stirred in an ice-water bath for 45 min. Compound 2.13 (0.26 g, 1.14 mmol) was added slowly while allowing the reaction mixture to warm up to room temperature. The reaction was completed after 6 h and the solvent was removed. The crude product was treated with a 20% aq. NH4OAc solution (10 mL) and THF (15 mL). The solution was refluxed for 30 min. Upon cooling down, the product was extracted two times with EtOAc and concentrated under reduced pressure. A yellow solid was isolated (0.28 g, 86%) and used without further purification. Rf = 0.2 (1:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 10.05 (s, 1H), 9.01 (s, 1H), 8.07 (dd, J = 1.5, 8.7 Hz, 1H), 7.89 (s, 1H), 7.38 (d, J = 8.7 Hz, 1H), 4.55 (t, J = 6.6 Hz, 2H), 3.95 (s, 3H), 2.95 (t, J = 6.6 Hz, 2H). 13C NMR (CDCl3, 75 MHz): δ 184.7, 167.5, 139.0, 126.3, 125.8, 125.3, 125.3, 120.1, 116.6, 109.3, 98.8, 52.4, 43.2, 19.4. LRMS (ESI) m/z = 257.2 [M+H]+. 101 General procedure B1 for the synthesis of 3-substituted indoles 2.15 – 2.19 via Wittig reaction and subsequent hydrogenation R O O MeO N H R O + R -40 oC - r.t. PPh3Br CN nBuLi, THF O Pd/C, H2 MeO N CN THF/ MeOH, r.t. MeO N CN A 50 mL oven-dried round bottom flask was charged with 1.18 mmol of the 4substituted benzyltriphenylphosphonium bromide and 10 mL anhydrous THF. The suspension was cooled down to –40 ˚C. Then 2.5M n-BuLi solution in hexanes (0.47 mL, 1.18 mmol) was added to the heterogeneous mixture via syringe. The resulting orange solution was stirred at –40 ˚C for 30 min, allowed to warm up to room temperature and stirred for another 30 min. 2.14 (250 mg, 0.98 mmol) was dissolved in 10 mL anhydrous THF and added via addition funnel into the stirring mixture. The resulting mixture was stirred for 3 h, quenched with sat. aq. NH4Cl solution, extracted two times with EtOAc and concentrated under reduced pressure. The crude product was purified via flash column chromatography to afford the E,Z-isomers as a pale yellow semisolid. In a 50 mL round bottom flask, the isolated alkenes were dissolved in THF (10 mL) and MeOH (10 mL). Then 10 wt. % Pd/C (25 mg) was added. After evacuating and flushing the flask with hydrogen gas three times, the reaction mixture was stirred under H2 atmosphere for 3 h. Upon completion, Pd/C was removed via filtration. The filtrate was concentrated under reduced pressure and the crude product was purified via column chromatography to afford 3-substituted indoles. 102 Cl O MeO N CN Methyl-3-(4-chlorophenethyl)-1-(2-cyanoethyl)-1H-indole-5-carboxylate (2.15). Compound 2.15 was prepared with 2.14 (250 mg, 0.98 mmol) following general procedure B1. The crude alkane product was purified via flash column chromatography (2:1 hexanes/EtOAc) to obtain a white solid (251 mg, 69% over two steps). Rf = 0.4 (2:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 8.31 (s, 1H), 7.95 (dd, J = 1.3, 8.4 Hz, 1H), 7.27 (d, J = 9.0 Hz, 1H), 7.25 (d, J = 8.4 Hz, 2H), 7.09 (d, J = 8.4 Hz, 2H), 6.87 (s, 1H), 4.37 (t, J = 6.6 Hz, 2H), 3.94 (s, 3H), 3.09 – 3.05 (m, 2H), 2.99 – 2.94 (m, 2H), 2.77 (t, J = 6.6 Hz, 2H). 13C NMR (CDCl3, 75 MHz): δ 168.2, 140.4, 138.5, 131.9, 130.1, 128.7, 128.6, 128.6, 128.2, 126.2, 123.9, 122.6, 121.9, 117.7, 117.3, 108.6, 52.2, 42.2, 35.9, 27.0, 19.4. LRMS (ESI) m/z = 367.2 [M+H]+. Me O MeO N CN Methyl 1-(2-cyanoethyl)-3-(4-methylphenethyl)-1H-indole-5-carboxylate (2.16). Compound 2.16 was prepared with 2.14 (2.50 g, 9.76 mmol) following general procedure B1. The crude alkane product was purified via flash column chromatography (3:1 hexanes/EtOAc) to obtain a white solid (2.49 g, 73% over two steps). Rf = 0.5 (2:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 8.35 (d, J = 1.2 Hz, 1H), 7.95 (dd, J = 103 1.6, 8.4 Hz, 1H), 7.27 (d, J = 8.7 Hz, 1H), 7.09 (s, 4H), 6.89 (s, 1H), 4.38 (t, J = 6.7 Hz, 2H), 3.94 (s, 3H), 3.10 – 3.01 (m, 2H), 3.00 – 2.92 (m, 2H), 2.77 (t, J = 6.7 Hz, 2H), 2.32 (s, 3H). 13C NMR (CDCl3, 75 MHz): δ 168.3, 139.0, 138.5, 135.7, 129.3, 128.6, 128.3, 126.1, 123.9, 123.9, 122.7, 121.9, 118.4, 117.4, 108.6, 52.2, 42.2, 36.1, 27.2, 21.3, 19.4. LRMS (ESI) m/z = 347.3 [M+H]+. F O MeO N CN Methyl 1-(2-cyanoethyl)-3-(4-fluorophenethyl)-1H-indole-5-carboxylate (2.17). Compound 2.17 was prepared with 2.14 (2.00 g, 7.81 mmol) following general procedure B1. The crude alkane product was purified via flash column chromatography (3:1 hexanes/EtOAc) to obtain a white solid (1.17 g, 43% over two steps). Rf = 0.5 (2:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 8.31 (s, 1H), 7.94 (d, J = 8.4 Hz, 1H), 7.27 (d, J = 8.7 Hz, 1H), 7.17 – 7.05 (m, 2H), 6.95 (t, J = 8.7 Hz, 2H), 6.87 (s, 1H), 4.35 (t, J = 6.3 Hz, 2H), 3.93 (s, 3H), 3.10 – 2.89 (m, 4H), 2.76 (t, J = 6.6 Hz, 2H). 13C NMR (CDCl3, 75 MHz): δ 168.2, 163.2, 160.0, 138.5, 137.7, 137.6, 130.2, 130.0, 128.2, 126.2, 123.9, 122.6, 121.9, 117.9, 117.4, 115.4, 115.1, 108.6, 52.2, 42.2, 35.8, 27.2, 19.4. LRMS (ESI) m/z = 351.2 [M+H]+. 104 O MeO N CN Methyl 1-(2-cyanoethyl)-3-phenethyl-1H-indole-5-carboxylate (2.18). Compound 2.18 was prepared with 2.14 (300 mg, 1.17 mmol) following general procedure B1. The crude alkane product was purified via flash column chromatography (3:1 hexanes/EtOAc) to obtain a white solid (274 mg, 71% over two steps). Rf = 0.5 (3:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 8.37 (s, 1H), 7.97 (dd, J = 1.5, 8.4 Hz, 1H), 7.34 – 7.26 (m, 3H), 7.24 – 7.16 (m, 3H), 6.89 (s, 1H), 4.39 (t, J = 6.9 Hz, 2H), 3.96 (s, 3H), 3.13 – 3.07 (m, 2H), 3.04 – 2.98 (m, 2H), 2.78 (t, J = 6.9 Hz, 2H). 13 C NMR (CDCl3, 75 MHz): δ 168.2, 142.0, 138.5, 128.8, 128.7, 128.6, 128.3, 126.2, 126.1, 123.9, 122.7, 121.9, 118.3, 117.3, 108.5, 52.2, 42.2, 36.6, 22.1, 19.4. O O O MeO N CN Methyl 3-(2-(benzo[d][1,3]dioxol-5-yl)ethyl)-1-(2-cyanoethyl)-1H-indole-5- carboxylate (2.19). Compound 2.19 was prepared with 2.14 (250 mg, 0.97 mmol) following general procedure B1. The crude alkane product was purified via flash column chromatography (2:1 hexanes/EtOAc) to obtain a white solid (220 mg, 62% over two steps). Rf = 0.4 (2:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 8.31 (s, 1H), 7.94 (d, J = 8.4 Hz, 1H), 7.27 (d, J = 8.4 Hz, 1H), 6.89 (s, 1H), 6.62 (dd, J = 1.2, 8.1 Hz, 1H), 6.67 (s, 1H), 6.63 (d, J = 7.8 Hz, 1H), 5.91 (s, 2H), 4.38 (t, J = 6.9 Hz, 2H), 3.94 (s, 3H), 3.03 105 (t, J = 7.2 Hz, 2H), 2.91 (t, J = 8.4 Hz, 2H), 2.77 (t, J = 6.6 Hz, 2H). 13C NMR (CDCl3, 75 MHz): δ 168.2, 147.7, 146.0, 138.4, 135.9, 128.3, 126.1, 123.9, 122.7, 121.9, 118.1, 117.3, 109.2, 108.5, 108.3, 101.0, 52.2, 42.2, 36.3, 27.4, 19.4. LRMS (ESI) m/z = 377.2 [M+H]+. General procedure B2 for the TMSCl-mediated demethylation R R O O TMSCl, NaI MeO HO MeCN, reflux N N CN CN A 50-mL oven-dried round bottom flask under nitrogen gas was charged with 2.34 mmol of the indole-5-methyl ester and 15 mL of anhydrous MeCN. To the stirring solution was added NaI (2.10 g, 14.06 mmol), followed by TMSCl (1.79 mL, 14.06 mmol). The reaction was refluxed for 36 h. Upon completion, the reaction was allowed to cool down and quenched with 10 mL water. The crude product was extracted with CH2Cl2, concentrated under reduced pressure and purified via flash column chromatography to obtain the indole-5-carboxylic acid. Cl O HO N CN 3-(4-Chlorophenethyl)-1-(2-cyanoethyl)-1H-indole-5-carboxylic acid (2.20). Compound 2.20 was prepared with 2.15 (860 mg, 2.34 mmol) following general procedure B2. The crude product was purified via flash column chromatography (100:1 CH2Cl2/MeOH) to obtain a white solid (0.5 g, 60%). Rf = 0.5 (40:1 CH2Cl2/MeOH). 1H 106 NMR (CD3OD, 300 MHz): δ 8.26 (s, 1H), 7.87 (d, J = 7.5 Hz, 1H), 7.48 (d, J = 8.4 Hz, 1H), 7.21 (d, J = 8.4 Hz, 2H), 7.13 (d, J = 8.4 Hz, 2H), 7.09 (s, 1H), 4.46 (t, J = 6.6 Hz, 2H), 3.06 – 2.99 (m, 4H), 2.92 (t, J = 6.6 Hz, 2H). 13C NMR (DMSO-d6, 75 MHz): δ 168.9, 141.4, 138.9, 131.1, 130.9, 128.78, 127.8, 123.2, 122.1, 122.0, 119.5, 116.4, 110.4, 41.8, 35.7, 26.9, 19.2. LRMS (ESI) m/z = 353.3 [M+H]+. Me O HO N CN 1-(2-Cyanoethyl)-3-(4-methylphenethyl)-1H-indole-5-carboxylic acid (2.21). Compound 2.21 was prepared with 2.16 (800 mg, 2.31 mmol) following general procedure B2. The crude product was purified via flash column chromatography (100:1 CH2Cl2/MeOH) to obtain a white solid (538 mg, 70%). Rf = 0.1 (100:1 CH2Cl2/MeOH). 1 H NMR (CD3OD, 300 MHz): δ 8.25 (s, 1H), 7.87 (d, J = 8.7 Hz, 1H), 7.46 (d, J = 8.7 Hz, 1H), 7.07 (s, 1H), 7.04 (s, 4H), 4.44 (t, J = 6.6 Hz, 2H), 3.02 – 2.87 (m, 6H), 2.26 (s, 3H). 13 C NMR (CD3OD, 75 MHz): δ 170.2, 139.0, 135.1, 128.7, 128.2, 128.1, 126.7, 123.2, 122.0, 121.4, 117.9, 117.3, 108.8, 41.6, 36.2, 27.1, 19.9, 18.4. LRMS (ESI) m/z = 355.2 [M+H]+. 107 F O HO N CN 1-(2-Cyanoethyl)-3-(4-fluorophenethyl)-1H-indole-5-carboxylic acid (2.22). Compound 2.22 was prepared with 2.17 (630 mg, 1.79 mmol) following general procedure B2. The crude product was purified via flash column chromatography (100:1 CH2Cl2/MeOH) to obtain a white solid (430 mg, 72%). Rf = 0.3 (40:1 CH2Cl2/MeOH). 1H NMR (acetone-d6, 300 MHz): δ 8.34 (s, 1H), 7.89 (dd, J = 1.5, 8.7 Hz, 1H), 7.60 (d, J = 8.4 Hz, 1H), 7.31 – 7.27 (m, 3H), 7.02 (t, J = 8.7 Hz, 2H), 4.60 (t, J = 6.6 Hz, 2H), 3.10 – 3.02 (m, 6H). 13C NMR (CD3OD, 75 MHz): δ 163.2, 160.0, 139.0, 138.0, 137.9, 130.0, 129.9, 128.0, 126.8, 123.2, 122.0, 117.9, 116.9, 114.7, 114.5, 108.8, 41.6, 35.7, 27.0, 18.4. LRMS (ESI) m/z = 337.2 [M+H]+. O O O HO N CN 3-(2-(Benzo[d][1,3]dioxol-5-yl)ethyl)-1-(2-cyanoethyl)-1H-indole-5-carboxylic acid (2.23). Compound 2.23 was prepared with 2.19 (100 mg, 0.27 mmol) following general procedure B2. The crude product was purified via flash column chromatography (100:1 CH2Cl2/MeOH) to obtain a white solid (52.8 mg, 55%). Rf = 0.2 (100:1 CH2Cl2/MeOH). 1H NMR (CD3OD, 300 MHz): δ 8.21 (s, 1H), 7.86 (d, J = 8.4 Hz, 1H), 7.46 (d, J = 8.4 Hz, 1H), 7.10 (s, 1H), 6.65 (d, J = 8.1 Hz, 2H), 6.69 (dd, J = 1.5, 8.1 Hz, 108 1H), 5.86 (s, 2H), 4.46 (t, J = 6.6 Hz, 2H), 3.02 (t, J = 6.6 Hz, 2H), 2.94 – 2.89 (m, 4H). 13 C NMR (CD3OD, 75 MHz): δ 169.0, 147.8, 146.0, 138.9, 136.3, 127.9, 127.7, 123.2, 122.0, 121.8, 119.5, 116.7, 110.3, 109.5, 108.6, 101.2, 41.8, 36.3, 27.4, 19.2. LRMS (ESI) m/z = 363.3 [M+H]+. General procedure B3 for the synthesis of N-acyl-sulfonamides R R O O HO + N CN O R S O NH2 CDI, DBU THF, reflux - r.t. R S O O N H N CN Carbonyldiimidazole (82.7 mg, 0.51 mmol) was dissolved in anhydrous THF and 0.43 mmol of the indol-5-carboxylic acid was added. The resulting mixture was refluxed for 1 h and allowed to cool to room temperature. Then 0.46 mmol of the aryl sulfonamiide 1.25 was added in one portion, followed by DBU (0.08 mL, 0.55 mmol). The reaction mixture was stirred for 1.5 d at room temperature and quenched by the dropwise addition of 20 mL ice-cold 1M HCl. The aqueous layer was extracted two times with 15 mL EtOAc, and the organic layer was dried over anhydrous MgSO4. After removing the solvent, the crude product was purified via flash column chromatography to obtain the N-acyl sulfonbamide. 109 Cl O Cl S S O O N H N Cl CN 3-(4-Chlorophenethyl)-1-(2-cyanoethyl)-N-((4,5-dichlorothiophen-2-yl)sulfonyl)-1H-indole-5-carboxamide (2.25). Compound 2.25 was prepared with 2.20 (150 mg, 0.43 mmol) following general procedure B3. The crude product was purified via flash column chromatography (30:1 CH2Cl2/MeOH) to obtain a white solid (163.8 mg, 68%). Rf = 0.2 (30:1 CH2Cl2/MeOH). 1H NMR (DMSO-d6, 300 MHz): δ 8.30 (s, 1H), 7.90 (s, 1H), 7.72 (d, J = 8.7 Hz, 1H), 7.63 (d, J = 9.0 Hz, 1H), 7.31 – 7.25 (m, 5H), 4.46 (t, J = 6.3 Hz, 2H), 3.05 – 2.94 (m, 6H). 13C NMR (DMSO-d6, 75 MHz): δ 167.2, 141.4, 139.2, 133.1, 131.1, 131.0, 128.8, 128.3, 127.6, 124.2, 122.5, 122.2, 121.7, 119.4, 116.9, 110.8, 41.8, 35.6, 27.0, 19.3. HRMS (ESI) m/z calcd. for C24H18Cl3N3O3S2Na [M + Na]+ 587.9747, found 587.9767. F O Cl S S O O N H N Cl CN 1-(2-Cyanoethyl)-N-((4,5-dichlorothiophen-2-yl)sulfonyl)-3-(4-fluorophenethyl)-1H-indole-5-carboxamide (2.26). Compound 2.26 was prepared with 2.22 (220 mg, 0.65 mmol) following general procedure B3. The crude product was purified via flash column chromatography (4:3:0.05 hexanes/THF/AcOH) to obtain a white solid (171 mg, 47%). Rf = 0.2 (4:3:0.05 hexanes/THF/AcOH). 1H NMR (DMSO-d6, 500 MHz): δ 8.28 (s, 110 1H), 7.88 (s, 1H), 7.71 (d, J = 1.5 Hz, 1H), 7.62 (d, J = 8.5 Hz, 1H), 7.29 – 7.25 (m, 3H), 7.06 (t, J = 5.4 Hz, 2H), 4.45 (t, J = 6.0 Hz, 2H), 2.99 – 2.96 (m, 6H). 13C NMR (DMSOd6, 300 MHz): δ 147.8, 145.9, 138.8, 136.3, 127.6, 121.9, 121.8, 119.4, 116.9, 116.9, 109.5, 108.7, 101.2, 41.8, 36.2, 27.5, 19.2. HRMS (ESI) m/z calcd. for C24H18Cl2FN3O3S2Na [M + Na]+ 572.0043, found 572.0064. O O O Cl S S O O N H N Cl CN 3-(2-(Benzo[d][1,3]dioxol-5-yl)ethyl)-1-(2-cyanoethyl)-N-((4,5-dichlorothiophen-2-yl)sulfonyl)-1H-indole-5-carboxamide (2.27). Compound 2.27 was prepared with 2.23 (250 mg, 0.72 mmol) following general procedure B3. The crude product was purified via flash column chromatography (4:3:0.05 hexanes/THF/AcOH) to obtain a white solid (338 mg, 82%). Rf = 0.25 (4:3:0.05 hexanes/THF/AcOH). 1H NMR (acetone-d6, 300 MHz): δ 8.37 (s, 1H), 7.83 – 7.80 (m, 2H), 7.65 (d, J = 8.7 Hz 1H), 7.31 (s, 1H), 6.78 (s, 1H), 6.74 – 6.67 (m, 2H), 5.93 (s, 2H), 4.61 (t, J = 6.6 Hz, 2H), 3.06 – 3.02 (m, 4H). 2.95 – 2.90 (m, 2H), 2.83 – 2.79 (m, 2H). 13C NMR (DMSO-d6, 75 MHz): δ 167.2, 147.8, 146.0, 139.2, 136.3, 133.1, 127.7, 124.3, 122.5, 121.9, 121.8, 119.4, 117.2, 110.79, 109.6, 108.7, 101.3, 41.9, 36.2, 27.5, 19.3. HRMS (ESI) m/z calcd. for C25H19Cl2N3O5S2Na [M + Na]+ 598.0035, found 598.0053. 111 Cl O S O O N H N F CN 3-(4-Chlorophenethyl)-1-(2-cyanoethyl)-N-((3-fluorophenyl)sulfonyl)-1Hindole-5-carboxamide (2.28). Compound 2.28 was prepared with 2.20 (220 mg, 0.62 mmol) following general procedure B3. The crude product was purified via flash column chromatography (4:3:0.05 hexanes/THF/AcOH) to obtain a white solid (164 mg, 52%). Rf = 0.25 (4:3:0.05 hexanes/THF/AcOH). 1H NMR (DMSO-d6, 500 MHz): δ 8.30 (s, 1H), 7.85 (d, J = 8.0 Hz, 1H), 7.78 (d, J = 8.0 Hz, 1H), 7.74 – 7.27 (m, 4H), 7.31 – 7.25 (m, 5H), 4.44 (t, J = 6.0 Hz, 2H), 3.00 – 2.95 (m, 6H). 13 C NMR (DMSO-d6, 125 MHz): δ 166.7, 163.0, 161.1, 142.5, 142.5, 141.4, 139.1, 132.3, 132.2, 131.1, 131.0, 129.1, 128.9, 128.8, 128.2, 127.6, 124.5, 124.5, 122.4, 122.4, 121.5, 121.4, 119.4, 116.8, 115.5, 115.3, 110.7, 41.8, 35.5, 26.9, 19.3. HRMS (ESI) m/z calcd. for C26H21ClFN3O3SNa [M + Na]+ 532.0868, found 532.0678. F O S O O N H N F CN 1-(2-Cyanoethyl)-3-(4-fluorophenethyl)-N-((3-fluorophenyl)sulfonyl)-1Hindole-5-carboxamide (2.29). Compound 2.29 was prepared with 2.22 (343 mg, 1.02 mmol) following general procedure B3. The crude product was purified via flash column chromatography (4:3:0.05 hexanes/THF/AcOH) to obtain a white solid (263 mg, 51%). Rf 112 = 0.2 (6:4:0.05 hexanes/THF/AcOH). 1H NMR (DMSO-d6, 500 MHz): δ 8.29 (s, 1H), 7.85 (d, J = 7.5 Hz, 1H), 7.78 (d, J = 7.5 Hz, 1H), 7.71 – 7.58 (m, 4H), 7.30 – 7.25 (m, 3H), 7.06 (t, J = 8.5 Hz, 2H), 4.44 (t, J = 6.0 Hz, 2H), 3.00 – 2.94 (m, 6H). 13C NMR (DMSOd6, 125 MHz): δ 168.5, 166.7, 163.0, 162.3, 161.1, 160.4, 142.6, 142.5, 139.1, 138.6, 138.5, 132.3, 132.2, 130.8, 130.8, 128.1, 127.6, 124.5, 124.5, 122.4, 122.4, 121.5, 121.3, 119.4, 117.0, 115.6, 115.5, 115.4, 115.3, 110.7, 41.8, 35.4, 27.2, 19.3. HRMS (ESI) m/z calcd. for C26H21F2N3O3SNa [M + Na]+ 516.1164, found 516.1183. O O O S O O N H N F CN 3-(2-(Benzo[d][1,3]dioxol-5-yl)-ethyl)-1-(2-cyanoethyl)-N-((3-fluoro-phenyl)sulfonyl)-1H-indole-5-carboxamide (2.30). Compound 2.30 was prepared with 2.23 (200 mg, 0.63 mmol) following general procedure B3. The crude product was purified via flash column chromatography (4:3:0.05 hexanes/THF/AcOH) to obtain a white solid (121 mg, 37%). Rf = 0.3 (4:3:0.1 hexanes/THF/AcOH). 1H NMR (DMSO-d6, 300 MHz): δ 8.27 (s, 1H), 7.84 (d, J = 7.5 Hz, 1H), 7.77 (d, J = 8.1 Hz, 1H), 7.73 – 7.55 (m, 4H), 7.31 (s, 1H), 6.88 (s, 1H), 6.78 (d, J = 7.8 Hz, 1H), 6.69 (d, J = 7.8 Hz, 1H), 5.94 (s, 2H), 4.44 (t, J = 6.3 Hz, 2H), 3.00 – 2.88 (m, 6H). 13C NMR (DMSO-d6, 125 MHz): δ 168.5, 163.0, 161.1, 147.8, 146.0, 139.1, 136.3, 132.2, 128.0, 127.7, 124.5, 122.4, 121.8, 121.5, 119.4, 117.1, 115.5, 115.3, 110.6, 109.5, 108.7, 101.2, 41.8, 36.2, 27.5, 19.2. HRMS (ESI) m/z calcd. for C27H22FN3O5SNa [M + Na]+ 542.1156, found 542.1158. 113 Cl O S S O O N H N Cl CN 3-(4-Chlorophenethyl)-N-((5-chlorothiophen-2-yl)-sulfonyl)-1-(2-cyanoethyl)-1H-indole-5-carboxamide (2.31). Compound 2.31 was prepared with 2.20 (200 mg, 0.57 mmol) following general procedure B3. The crude product was purified via flash column chromatography (4:3:0.05 hexanes/THF/AcOH) to obtain a white solid (197 mg, 58%). Rf = 0.2 (4:3:0.1 hexanes/THF/AcOH). 1H NMR (DMSO-d6, 300 MHz): δ 8.30 (s, 1H), 7.74 – 7.70 (m, 1H), 7.67 (s, 1H), 7.63 (d, J = 8.7 Hz, 1H), 7.31 – 7.24 (m, 6H), 4.45 (t, J = 6.0 Hz, 2H), 3.01 – 2.97 (m, 4H). 13C NMR (DMSO-d6, 125 MHz): δ 168.5, 141.4, 139.1, 131.1, 131.0, 128.8, 128.2, 127.6, 122.5, 121.6, 119.8, 119.4, 116.8, 110.74, 41.8, 35.6, 27.0, 19.3. HRMS (ESI) m/z calcd. for C24H19Cl2N3O3S2Na [M + Na]+ 554.0137, found 554.0160. Me O Cl S S O O N H N Cl CN 1-(2-Cyanoethyl)-N-((4,5-dichlorothiophen-2-yl)-sulfonyl)-3-(4-methyl-phenethyl)-1H-indole-5-carboxamide (2.32). Compound 2.32 was prepared with 2.21 (220 mg, 0.66 mmol) following general procedure B3. The crude product was purified via flash column chromatography (4:3:0.05 hexanes/THF/AcOH) to obtain a white solid (152 mg, 42%). Rf = 0.4 (4:3:0.05 hexanes/THF/AcOH). 1H NMR (acetone-d6, 300 MHz): δ 8.39 (s, 114 1H), 7.88 (dd, J = 1.5, 8.7 Hz, 1H), 7.71 (s, 1H), 7.59 (d, J = 8.7 Hz, 1H), 7.27 (s, 1H), 7.14 (d, J = 8.1 Hz, 2H), 7.08 (d, J = 8.4 Hz, 2H), 4.59 (t, J = 6.6 Hz, 2H), 3.05 –2.96 (m, 6H), 2.27 (s, 3H). 13C NMR (DMSO-d6, 125 MHz): δ 168.5, 139.4, 138.9, 135.3, 129.5, 128.9, 127.7, 127.7, 127.6, 123.7, 122.7, 121.3, 119.4, 117.0, 110.3, 95.0, 41.8, 36.1, 27.3, 21.3, 19.2. HRMS (ESI) m/z calcd. for C25H21Cl2N3O3S2Na [M + Na]+ 568.0294, found 568.0306. O O O S S O O N H N Cl CN 3-(2-(benzo[d][1,3]dioxol-5-yl)-ethyl)-N-((5-chlorothiophen-2-yl)sulfonyl)-1(2-cyanoethyl)-1H-indole-5-carboxamide (2.33). Compound 2.33 was prepared with 2.23 (200 mg, 0.51 mmol) following general procedure B3. The crude product was purified via flash column chromatography (4:3:0.05 hexanes/THF/AcOH) to obtain a white solid (141 mg, 50%). Rf = 0.2 (4:3:0.05 hexanes/THF/AcOH). 1H NMR (DMSO-d6, 300 MHz): δ 8.28 (s, 1H), 7.74 (d, J = 5.1 Hz, 1H), 7.64 (d, J = 9.0 Hz, 1H), 7.63 (d, J = 9.0 Hz, 1H), 7.32 (s, 1H), 7.28 (d, J = 3.9 Hz, 1H), 6.88 (s, 1H), 6.78 (d, J = 7.8 Hz, 1H), 6.68 (d, J = 8.1 Hz, 1H), 5.94 (s, 2H), 4.46 (t, J = 6.3 Hz, 2H), 3.01 – 2.87 (m, 6H). 13C NMR (DMSOd6, 75 MHz): δ 166.9, 147.8, 146.0, 139.2, 136.3, 134.6, 128.3, 127.7, 122.4, 121.9, 119.4, 117.2, 110.8, 109.6, 108.7, 101.3, 98.8, 41.8, 36.2, 27.5, 19.3. 115 General procedure B4 for the synthesis of tetrazoles R R O R S O O O N H R nBu3SnN3, PhMe N S O O N H N 110 oC CN HN N N N A 75 mL pressure flask was charged with a stir bar, 0.20 mmol of the alkyl nitrile, 20 mL anhydrous toluene and nBu3SnN3 (202 mg, 0.61 mmol). The flask was evacuated and flushed with nitrogen gas three time, sealed and refluxed for 48 h. Upon completion as indicated by TLC, the reaction was cooled down to room temperature and 0.1 mL of AcOH was added. After stirring for 2 additional hours, a white precipitate appeared. It was filtered, washed with hexanes and Et2O to isolate the desired tetrazole. Cl O Cl S S O O N H N Cl HN N N N 1-(2-(1H-Tetrazol-5-yl)ethyl)-3-(4-chlorophenethyl)-N-((4,5-dichlorothiophen-2-yl)-sulfonyl)-1H-indole-5-carboxamide (2.34). Compound 2.34 was prepared with 2.25 (115 mg, 0.20 mmol) following general procedure B4. The desired product was isolated as a white solid (76.5 mg, 62%). Rf = 0.1 (6:4:0.1 hexanes/EtOAc/AcOH). 1H NMR (DMSO-d6, 500 MHz): δ 8.25 (s, 1H), 7.91 (s, 1H), 7.66 (d, J = 9.0 Hz, 1H), 7.48 (d, J = 9.0 Hz, 1H), 7.29 (d, J = 8.5 Hz, 2H), 7.22 (d, J = 8.5 Hz, 2H), 7.20 (s, 1H), 4.56 (t, J = 7.0 Hz, 2H), 3.34 (t, J = 7.0 Hz, 2H), 2.94 – 2.92 (m, 4H). 13C NMR (DMSO-d6, 125 116 MHz): δ 168.5, 167.3, 141.4, 139.0, 138.3, 133.0, 132.0, 131.1, 130.9, 128.8, 128.3, 127.5, 124.2, 122.4, 122.1, 121.6, 116.6, 110.4, 44.0, 35.6, 26.9, 25.1. HRMS (ESI) m/z calcd. for C24H19Cl3N6O3S2 [M + H]+ 609.0098, found 608.9924. F O Cl S S O O N H N Cl HN N N N 1-(2-(1H-Tetrazol-5-yl)ethyl)-N-((4,5-dichlorothiophen-2-yl)sulfonyl)-3-(4fluoro-phenethyl)-1H-indole-5-carboxamide (2.35). Compound 2.35 was prepared with 2.26 (171 mg, 0.31 mmol) following general procedure B4. The desired product was isolated as a white solid (75mg, 41%). Rf = 0.1 (6:4:0.1 hexanes/EtOAc/AcOH). 1H NMR (DMSO-d6, 500 MHz): δ 8.08 (s, 1H), 7.78 (s, 1H), 7.67 (dd, J = 1.5, 8.5 Hz, 1H), 7.38 (d, J = 9.0 Hz, 1H), 7.12 (dd, J = 5.5, 8.5 Hz, 2H), 6.93 – 6.90 (m, 3H), 4.60 (t, J = 6.5 Hz, 2H), 3.40 (t, J = 6.5 Hz, 2H), 3.00 (t, J = 7.0 Hz, 2H), 2.92 (t, J = 8.0 Hz, 2H). 13C NMR (DMSO-d6, 125 MHz): δ 167.2, 162.3, 160.4, 139.1, 138.5, 133.0, 130.8, 130.7, 128.2, 127.6, 124.2, 122.4, 121.7, 116.7, 115.6, 115.4, 110.4, 44.0, 35., 27.2, 25.1. HRMS (ESI) m/z calcd. for C24H19Cl2FN6O3S2 [M - H]- 591.0248, found 591.0251. 117 O O O Cl S S O O N H N Cl HN N N N 1-(2-(1H-Tetrazol-5-yl)ethyl)-3-(2-(benzo[d][1,3]dioxol-5-yl)ethyl)-N-((4,5-dichloro-thiophen-2-yl)sulfonyl)-1H-indole-5-carboxamide (2.36). Compound 2.36 was prepared with 2.27 (200 mg, 0.34 mmol) following general procedure B4. The desired product was isolated as a white solid (157 mg, 73%). Rf = 0.1 (6:4:0.1 hexanes/EtOAc/AcOH). 1H NMR (DMSO-d6, 500 MHz): δ 8.25 (s, 1H), 7.91 (s, 1H), 7.91 (s, 1H), 7.66 (d, J = 8.5 Hz, 1H), 7.47 (d, J = 9.0 Hz, 1H), 7.21 (s, 1H), 6.85 (s, 1H), 6.77 (d, J = 8.0 Hz, 1H), 6.65 (d, J = 8.0 Hz, 1H), 5.95 (s, 2H), 4.56 (t, J = 6.5 Hz, 2H), 3.34 (t, J = 7.0 Hz, 2H), 2.91 (t, J = 7.0 Hz, 2H), 2.83 (t, J = 8.5 Hz, 2H). 13C NMR (DMSO-d6, 125 MHz): δ 168.5, 167.3, 147.8, 145.9, 139.0, 138.2, 133.0, 132.1, 128.2, 127.6, 124.2, 122.4, 122.0, 121.8, 121.7, 116.9, 110.3, 109.5, 108.6, 101.2. HRMS (ESI) m/z calcd. for C25H20Cl2N6O5S2 [M - H]- 617.0241, found 617.0234. Cl O S O O N H N F HN N N N 1-(2-(1H-Tetrazol-5-yl)-ethyl)-3-(4-chlorophenethyl)-N-((3-fluorophenyl)sulfonyl)-1H-indole-5-carboxamide (2.37). Compound 2.37 was prepared with 2.28 (200 mg, 0.39 mmol) following general procedure B4. The desired product was isolated as a 118 white solid (184 mg, 85%). Rf = 0.1 (6:4:0.1 hexanes/EtOAc/AcOH). 1H NMR (DMSOd6, 500 MHz): δ 8.25 (s, H), 7.84 (d, J = 7.5 Hz, H), 7.77 (d, J = 8.5 Hz, H), 7.70 – 7.69 (m, H), 7.66-7.58 (m, H), 7.45 (d, J = 8.5 Hz, H), 7.25 – 7.22 (m, H), 7.18 (s, H), 7.06 (t, J = Hz, H), 4.55 (t, J = 7.0 Hz, 2H), 3.33 (t, J = 6.5 Hz, 2H), 2.93 – 2.92 (m, 4H). 13C NMR (DMSO-d6, 125 MHz): δ 168.5, 166.7, 163.0, 161.1, 142.6, 142.5, 141.4, 139.0, 132.3, 132.2, 131.1, 130.9, 129.0, 128.9, 128.8, 128.2, 127.5, 124.5, 124.5, 122.4, 122.2, 121.5, 121.3, 116.5, 115.5, 115.3, 110.3, 44.0, 35.6, 26.9, 25.1. HRMS (ESI) m/z calcd. for C26H22ClFN6O3S [M - H]- 551.1074, found 551.1077. F O S O O N H N F HN N N N 1-(2-(1H-Tetrazol-5-yl)-ethyl)-3-(4-fluorophenethyl)-N-((3-fluorophenyl)sulfonyl)-1H-indole-5-carboxamide (2.38). Compound 2.38 was prepared with 2.29 (200 mg, 0.40 mmol) following general procedure B4. The desired product was isolated as a white solid (167 mg, 77%). Rf = 0.1 (6:4:0.1 hexanes/EtOAc/AcOH). 1H NMR (acetoned6, 500 MHz): δ 8.32 (s, 1H), 7.95 (d, J = 7.0 Hz, 1H), 7.86 (d, J = 9.0 Hz, 1H), 7.74 –7.68 (m, 2H), 7.53 – 7.48 (m, 2H), 7.23 (dd, J = 5.5, 9.0 Hz, 2H), 7.11 (s, 1H), 7.01 (t, J = 9.0 Hz, 2H), 4.69 (t, J = 7.0 Hz, 2H), 3.50 (t, J = 7.0 Hz, 2H), 3.02 – 2.93 (m, 4H). 13C NMR (DMSO-d6, 125 MHz): δ 166.7, 163.0, 162.3, 161.1, 160.4, 139.0, 138.6, 138.5, 132.3, 132.2, 130.8, 130.7, 128.2, 127.6, 124.5, 122.3, 121.5, 116.7, 115.6, 115.5, 115.4, 115.3, 110.3, 44.0, 35.5, 27.2, 25.1. HRMS (ESI) m/z calcd. for C26H22F2N6O3S [M - H]535.1369, found 535.1371. 119 O O O S O O N H N F HN N N N 1-(2-(1H-Tetrazol-5-yl)-ethyl)-3-(2-(benzo[d][1,3]dioxol-5-yl)ethyl)-N-((3fluoro-phenyl)-sulfonyl)-1H-indole-5-carboxamide (2.39). Compound 2.39 was prepared with 2.30 (105 mg, 0.20 mmol) following general procedure B4. The desired product was isolated as a white solid (28.4 mg, 25%). Rf = 0.1 (6:4:0.1 hexanes/EtOAc/AcOH). 1H NMR (DMSO-d6, 500 MHz): δ 8.23 (s, 1H), 7.83 (d, J = 7.0 Hz, 1H), 7.76 (d, J = 8.0 Hz, 1H), 7.68 (dd, J = 4.8, 8.4 Hz, 1H), 7.62 – 7.55 (m, 2H), 7.44 (d, J = 9.0 Hz, 1H), 7.19 (s, 1H), 6.85 (s, 1H), 6.77 (d, J = 7.5 Hz, 1H), 6.65 (d, J = 8.0 Hz, 1H), 5.93 (s, 2H), 4.55 (t, J = 6.0 Hz, 2H), 3.33 (s, 2H), 2.91 (t, J = 4.8 Hz, 2H), 2.82 (t, J = 4.8 Hz, 2H). 13C NMR (DMSO-d6, 125 MHz): δ 167.0, 163.0, 161.0, 147.8, 145.9, 138.9, 136.4, 132.2, 132.1, 128.0, 127.6, 124.4, 124.5, 122.4, 121.8, 121.5, 121.3, 121.1, 116.8, 115.5, 115.3, 110.2, 109.5, 108.6, 101.2, 44.0, 36.2, 27.4, 25.1. HRMS (ESI) m/z calcd. for C27H23FN6O5S [M - H]- 561.1362, found 561.1364. Cl O S S O O N H N Cl HN N N N 1-(2-(1H-tetrazol-5-yl)ethyl)-3-(4-chlorophenethyl)-N-((5-chlorothiophen-2yl)sulfonyl)-1H-indole-5-carboxamide (2.40). Compound 2.40 was prepared with 2.31 120 (140 mg, 0.26 mmol) following general procedure B4. The desired product was isolated as a pale-brown solid (134 mg, 90%). Rf = 0.1 (6:4:0.1 hexanes/EtOAc/AcOH). 1H NMR (DMSO-d6, 500 MHz): δ 8.24 (s, 1H), 7.69 (s, 1H), 7.65 (d, J = 9.0 Hz, 1H), 7.48 (d, J = 8.5 Hz, 1H), 7.29 (d, J = 8.5 Hz, 2H), 7.25 – 7.33 (m, 3H), 7.19 (s, 1H). 4.57 (t, J = 6.5 Hz, 2H), 3.34 (t, J = 6.5 Hz, 2H), 2.93 – 2.90 (m, 4H). 13 C NMR (DMSO-d6, 125 MHz): δ 168.5, 167.4, 141.4, 138.9, 134.0, 131.1, 130.9, 129.0, 128.9, 128.8, 128.1, 128.1, 127.5, 122.4, 121.4, 119.9, 116.5, 110.3, 44.0, 35.6, 26.9, 25.1. HRMS (ESI) m/z calcd. for C24H20Cl2N6O3S2Na [M – 2H+ + Na]+ 595.0162, found 597.0163. Me O Cl S S O O N H N Cl HN N N N 1-(2-(1H-tetrazol-5-yl)ethyl)-N-((4,5-dichlorothiophen-2-yl)-sulfonyl)-3-(4methyl phenethyl)-1H-indole-5-carboxamide (2.41). Compound 2.41 was prepared with 2.32 (140 mg, 0.26 mmol) following general procedure B4. The desired product was isolated as a white solid (110 mg, 73%). Rf = 0.1 (6:4:0.1 hexanes/EtOAc/AcOH). 1H NMR (DMSO-d6, 500 MHz): δ 8.24 (s, 1H), 7.89 (s, 1H), 7.67 (d, J = 9.0 Hz, 1H), 7.47 (d, J = 8.5 Hz, 1H), 7.20 (s, 1H), 7.10 (d, J = 8.0 Hz, 2H), 7.05 (d, J = 8.0 Hz, 2H), 4.56 (t, J = 7.0 Hz, 2H), 3.34 (t, J = 7.0 Hz, 2H), 2.92 – 2.85 (m, 4H), 2.23 (s, 3H). 13C NMR (DMSO-d6, 125 MHz): δ 168.5, 167.4, 139.3, 139.0, 138.5, 135.3, 132.8, 131.9, 129.5, 128.9, 128.1, 127.6, 124.1, 122.4, 122.3, 121.6, 117.0, 110.3, 44.0, 36.1, 27.3, 25.1, 21.3. HRMS (ESI) m/z calcd. for C25H22Cl2N6O3S2 [M - H]- 587.0499, found 587.0497. 121 O O O S S O O N H N Cl HN N N N 1-(2-(1H-tetrazol-5-yl)-ethyl)-3-(2-(benzo[d][1,3]-dioxol-5-yl)-ethyl)-N-((5chloro-thiophen-2-yl)-sulfonyl)-1H-indole-5-carboxamide (2.42). Compound 2.42 was prepared with 2.33 (140 mg, 0.26 mmol) following general procedure B4. The desired product was isolated as a brown solid (80 mg, 60%). Rf = 0.1 (6:4:0.1 hexanes/EtOAc/AcOH). 1H NMR (DMSO-d6, 500 MHz): δ 8.32 (s, 1H), 7.78 – 7.75 (m, 2H), 7.50 (d, J = 9.0 Hz, 1H), 7.18 (d, J = 4.5 Hz, 1H), 7.13 (s, 1H), 6.76 (s, 1H), 6.72 (d, J = 8.0 Hz, 1H), 6.66 (d, J = 7.5 Hz, 1H), 5.93 (s, 2H), 4.70 (t, J = 7.0 Hz, 2H), 3.51 (t, J = 6.5 Hz, 2H), 2.98 (t, J = 7.0 Hz, 2H), 2.87 (t, J = 7.0 Hz, 2H). 13C NMR (DMSO-d6, 125 MHz): δ 168.5, 167.0, 147.8, 145.9, 139.1, 139.0, 137.2, 136.4, 134.5, 128.2, 128.1, 127.6, 122.3, 122.1, 121.8, 121.6, 116.9, 110.3, 109.5, 108.6, 101.2, 44.0, 36.2, 27.4, 25.1. HRMS (ESI) m/z calcd. for C25H21ClN6O5S2 [M – 2H+ + Na]+ 605.0450, found 597.0443. General procedure B5 for the synthesis of 2-iodo indoles R R O O AgOTf, I2 MeO N CN THF, r.t. MeO I N CN A 100 mL round bottom flask was charged with 2.48 mmol of the 3-substituted indole, AgOTf (766.5 mg, 2.98 mmol) and 10 mL THF. A 5 mL solution of I2 (631 mg, 122 2.48 mmol) in THF was added dropwise via addition funnel. Then another portion of AgOTf (63.9 mg, 0.24 mmol) was added to the stirring mixture. Upon consumption of the starting material, the reaction was quenched with 10% aq. Na2S2O3 solution. The crude product was extracted two times with EtOAc, concentrated under reduced pressure and purified via column chromatography to obtain the 2-iodo indole. Cl O MeO I N CN Methyl 3-(4-chlorophenethyl)-1-(2-cyanoethyl)-2-iodo-1H-indole-5- carboxylate (2.43). Compound 2.43 was prepared with 2.15 (0.91 g, 2.48 mmol) following general procedure B5. The crude product was purified via flash column chromatography (3:1 hexanes/THF, followed by 2:1 hexanes/THF) to obtain a white solid (1.07 g, 57%). Rf = 0.3 (3:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 8.19 (s, 1H), 7.91 (dd, J = 1.5, 9.0 Hz, 1H), 7.36 (d, J = 8.5 Hz, 1H), 7.21 (d, J = 8.0 Hz, 2H), 7.06 (d, J = 8.0 Hz, 2H), 4.51 (t, J = 7.2 Hz, 2H), 3.94 (s, 3H), 3.02 (t, J = 7.5 Hz, 2H), 2.88 (t, J = 7.5 Hz, 2H), 2.75 (t, J = 7.0 Hz, 2H). 13 C NMR (CDCl3, 125 MHz): δ 167.6, 140.0, 139.6, 131.9, 130.1, 128.4, 127.7, 124.0, 123.5, 122.4, 121.3, 116.5, 109.0, 52.1, 42.9, 35.6, 29.6, 18.6. 123 Me O MeO I N CN Methyl 1-(2-cyanoethyl)-2-iodo-3-(4-methylphenethyl)-1H-indole-5- carboxylate (2.44). Compound 2.44 was prepared with 2.16 (150 mg g, 0.43 mmol) following general procedure B5. The crude product was purified via flash column chromatography (5:1 hexanes/EtOAc) to obtain a white solid (118 mg g, 57%). Rf = 0.3 (5:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 8.25 (s, 1H), 7.91 (d, J = 8.7 Hz, 1H), 7.36 (d, J = 8.7 Hz, 1H), 7.09 (s, 4H), 4.50 (t, J = 7.2 Hz, 2H), 3.95 (s, 3H), 3.03 (t, J = 7.2 Hz, 2H), 2.86 (t, J = 7.2 Hz, 2H), 2.76 (t, J = 7.2 Hz, 2H), 2.33 (s, 3H). 13C NMR (CDCl3, 75 MHz): δ 167.8, 140.2, 138.4, 135.8, 129.3, 128.7, 128.0, 124.3, 124.1, 122.4, 121.6, 116.8, 109.1, 86.9, 52.3, 43.1, 36.1, 30.0, 21.3, 18.8. LRMS (ESI) m/z = 495.2 [M + Na]+. F O MeO I N CN Methyl 1-(2-cyanoethyl)-3-(4-fluorophenethyl)-2-iodo-1H-indole-5- carboxylate (2.45). Compound 2.45 was prepared with 2.17 (150 mg g, 0.43 mmol) following general procedure B5. The crude product was purified via flash column chromatography (3:1 hexanes/EtOAc, followed by 2:1 hexanes/EtOAc to obtain a white solid (151 mg, 74%). Rf = 0.2 (2:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 8.19 124 (d, J = 0.9 Hz, 1H), 7.91 (dd, J = 1.5, 8.7 Hz, 1H), 7.36 (d, J = 8.7 Hz, 1H), 7.12 – 7.03 (m, 2H), 6.93 (t, J = 8.7 Hz, 2H), 4.49 (t, J = 6.9 Hz, 2H), 3.95 (s, 3H), 3.02 (t, J = 7.6 Hz, 2H), 2.88 (t, J = 7.5 Hz, 2H), 2.75 (t, J = 7.2 Hz, 2H). 13C NMR (CDCl3, 75 MHz): δ 167.8, 140.2, 137.0, 137.0, 130.4, 130.3, 127.9, 124.2, 123.8, 122.5, 121.5, 116.8, 115.4, 115.1, 109.2, 87.2, 52.3, 43.0, 35.7, 30.0, 18.8. O MeO I N CN Methyl 1-(2-cyanoethyl)-2-iodo-3-phenethyl-1H-indole-5-carboxylate (2.46). Compound 2.46 was prepared with 2.18 (275 mg g, 0.83 mmol) following general procedure B5. The crude product was purified via flash column chromatography (7:1 hexanes/EtOAc, followed by 6:1 and 5:1 hexanes/EtOAc) to obtain a white solid (262 mg, 70%). Rf = 0.25 (4:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 8.26 (s, 1H), 7.91 (d, J = 8.7 Hz, 1H), 7.36 (d, J = 8.7 Hz, 1H), 7.30 – 7.14 (m, 5H), 4.46 (t, J = 7.2 Hz, 2H), 3.95 (s, 3H), 3.09 – 2.98 (m, 2H), 2.95 – 2.85 (m, 2H), 2.72 (t, J = 7.2 Hz, 2H). 13C NMR (CDCl3, 75 MHz): δ 167.8, 141.5, 140.2, 128.9, 128.6, 127.9, 126.4, 124.1, 124.0, 122.4, 121.5, 117.0, 109.3, 87.2, 52.3, 43.0, 36.5, 29.9, 18.8. 125 General procedure B6 for the synthesis of 2-iodo-benzothiazoles from 2amino-benzothiazoles N R S NH2 p-TsOH, NaNO2, KI MeCN, 0 oC - r.t. N R I S A 100 mL round bottom flask was charged with 0.83 mmol of a 2-aminobenzothiazole, p-toluenesulfonic acid monohydrate (473.6 mg, 2.49 mmo) and 10 mL anhydrous MeCN. The suspension was cooled down to 0 oC. Then NaNO2 (114.8 mg, 1.66 mmol) and KI (358.56 mg, 2.16 mmol) were dissolved in 20 mL H2O and added to the mixture via addition funnel. The reaction was allowed to warm up to room temperature overnight. After 16 h, the reaction was quenched with 10% aq. Na2S2O3 solution, extracted two times with EtOAc and concentrated under reduced pressure. The crude product was purified via flash column chromatography to obtain the 2-iodo benzothizole. MeO N I S 2-Iodo-5-methoxybenzo[d]thiazole (S2.2). Compound S2.2 was prepared with 5methoxy-2-amino-benzothiazole (150 mg, 0.83 mmol) following general procedure B6. The crude product was purified via flash column chromatography (6:1 hexanes/EtOAc) to obtain a yellow solid (121.4 mg, 50%). Rf = 0.7 (5:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 7.66 (d, J = 9.0 Hz, 1H), 7.49 (d, J = 2.4 Hz, 1H), 7.01 (dd, J = 2.4, 8.7 Hz, 1H), 3.85 (s, 3H). 13C NMR (CDCl3, 75 MHz): δ 159.2, 155.5, 131.2, 120.8, 116.0, 106.4, 105.2, 55.9. Cl N I S 5-Chloro-2-iodobenzo[d]thiazole (S2.5). Compound S2.5 was prepared with 5chloro-2-amino-benzothiazole (150 mg, 0.81 mmol) following general procedure B6. The 126 crude product was purified via flash column chromatography (8:1 hexanes/EtOAc) to obtain a yellow solid (191.4 mg, 80%). Rf = 0.8 (2:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 8.01 (d, J = 1.8 Hz, 1H), 7.75 (d, J = 8.7 Hz, 1H), 7.38 (dd, J = 2.1 Hz, 8.4 Hz, 1H). 13C NMR (CDCl3, 75 MHz): δ 155.1, 137.7, 132.8, 126.4, 122.6, 121.3, 107.7. HO N I S 2-Iodobenzo[d]thiazol-5-ol (S2.3). Compound S2.2 (2.45 g, 8.41 mmol) was placed in a 100 mL oven-dried round bottom flask. The flask was evacuated and flushed three times with argon. Subsequently 30 mL anhydrous DCE was added via syringe. To this stirring solution was added portionwise AlCl3 (5.61 g, 42.08 mmol). The mixture was heated to 50 oC for 24 h. Upon completion, the reaction was cooled down to room temperature and the solids were removed via filtration. The filtrate was treated with sat. aq. NaHCO3-solution, extracted two times with EtOAc and concentrated under reduced pressure. The crude product was purified via flash column chromatography (5:1 hexanes/EtOAc, followed by 4:1 and 3:1 hexanes/EtOAc) to afford a yellow solid (1.53 g, 65%). Rf = 0.4 (3:1 hexanes/EtOAc). 1H NMR (CD3OD, 300 MHz): δ 7.74 – 7.67 (m, 1H), 7.35 – 7.29 (m, 1H), 6.98 – 6.91 (m, 1H). 13C NMR (CD3OD, 75 MHz): δ 157.1, 155.4, 129.6, 120.9, 115.7, 108.3, 106.7. MeO O N I S 2-Iodo-5-(2-methoxyethoxy)benzo[d]thiazole (S2.4). In a 25 mL round bottom flask, compound S2.3 (75 mg, 0.27 mmol) was dissolved in 5 mL anhydrous DMF. Cs2CO3 (263.9 mg, 0.81 mmol) and 1-bromo-2-methoxy-ethane (0.038 mL, 0.40 mmol) were added. The mixture was heated to 80 oC for 6 h and cooled to room temperature upon 127 completion. It was quenched with brine, extracted two times with EtOAc and concentrated under reduced pressure. The crude product was purified via flash column chromatography (3:1 hexanes/EtOAc) to isolate a white solid (49.7 mg, 55%). Rf = 0.4 (3:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 7.67 (d, J = 9.0 Hz, 1H), 7.50 (d, J = 2.4 Hz, 1H), 7.12 – 7.03 (m, 1H), 4.16 (t, J = 4.8 Hz, 2H), 3.77 (t, J = 4.5 Hz, 2H), 3.45 (s, 3H). 13C NMR (CDCl3, 75 MHz): δ 158.3, 155.4, 131.5, 120.8, 116.5, 106.4, 106.0, 71.1, 67.9, 59.5. General procedure B7 for Sonogashira-coupling reaction with 2-halo benzothiazoles and 2-bromo-benzimidazole N R I + S Me Si iPr Me Pd(PPh3)4, CuI Et3N:DMF (1:1) 50 oC, 16 h R N S Me Si iPr Me A 20 mL oven-dried scintillation vial was charged with 0.23 mmol of the 2halogenated heterocycle, Pd(PPh3)4 (26.98 mg, 0.023 mmol) and CuI (6.6 mg, 0.035 mmol). The vial was evacuated and flushed three times with argon. Then ethynyltriisopropylsilane (0.15 mL, 0.70 mmol), anhydrous Et3N (1 mL), DMF (1 mL) were added via syringe. The vial was sealed and the reaction mixture was stirred at 50 oC for 16 h. Upon completion, the reaction was quenched with brine, extracted two times with Et2O and concentrated under reduced pressure. The crude product was purified via flash column chromatography to obtain the TIPS-alkyne. N S Me Si iPr Me 2-((Triisopropylsilyl)ethynyl)benzo[d]thiazole (S3.1). Compound S3.1 was prepared with 2-bromo benzothiazole (50 mg, 0.23 mmol) following general procedure B7. 128 The crude product was purified via flash column chromatography (40:1 hexanes/EtOAc) to obtain a colorless liquid (64.6 mg, 87%). Rf = 0.7 (10:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 8.05 (d, J = 8.4 Hz, 1H), 7.84 (d, J = 8.4 Hz, 1H), 7.54 – 7.47 (m, 1H), 7.46 – 7.40 (m, 1H), 1.16 (s, 18H). 13C NMR (CDCl3, 75 MHz): δ 153.0, 148.7, 135.5, 126.9, 126.5, 124.0, 121.5, 101.0, 99.0, 18.8, 11.4. MeO Me Si iPr Me N S 5-Methoxy-2-((triisopropylsilyl)ethynyl)benzo[d]thiazole (S3.2). Compound S3.2 was prepared with 5-methoxy-2-iodo benzothiazole (60 mg, 0.21 mmol) following general procedure B7. The crude product was purified via flash column chromatography (20:1 hexanes/EtOAc) to obtain a colorless liquid (92 mg, 65%). Rf = 0.7 (5:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 7.65 (d, J = 8.7 Hz, 1H), 7.51 (d, J = 2.4 Hz, 1H), 7.07 (dd, J = 2.4, 8.7 Hz, 1H), 3.81 (s, 3H), 1.15 (s, 18H). 13C NMR (CDCl3, 75 MHz): δ 159.6, 154.3, 149.6, 127.4, 121.6, 117.1, 105.8, 100.6, 99.2, 55.8, 18.8, 11.4. MeO O N S Me Si iPr Me 5-(2-Methoxyethoxy)-2-((triisopropylsilyl)ethynyl)benzo[d]thiazole (S3.3). Compound S3.3 was prepared with S2.4 (49 mg, 0.14 mmol) following general procedure B7. The crude product was purified via flash column chromatography (7:1 hexanes/EtOAc) to obtain a reddish liquid (43 mg, 76%). Rf = 0.7 (7:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 7.66 (d, J = 9.0 Hz, 1H), 7.53 (d, J = 2.4 Hz, 1H), 7.13 (dd, J = 2.4, 9.0 Hz, 1H), 4.18 (t, J = 4.6 Hz, 2H), 3.78 (t, J = 4.6 Hz, 2H), 3.45 (s, 3H), 1.15 (s, 18H). 13C NMR (CDCl3, 75 MHz): δ 158.8, 154.1, 149.7, 127.5, 121.7, 117.7, 106.4, 101.0, 99.0, 71.1, 67.9, 59.5, 18.8, 11.4. 129 Cl Me Si iPr Me N S 5-Chloro-2-((triisopropylsilyl)ethynyl)benzo[d]thiazole (S3.4). Compound S3.4 was prepared with S2.5 (80 mg, 0.27 mmol) following general procedure B7. The crude product was purified via flash column chromatography (30:1 hexanes/EtOAc) to obtain a colorless liquid (92.1 mg, 97%). Rf = 0.7 (30:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 8.02 (d, J = 2.1 Hz, 1H), 7.73 (d, J = 8.4 Hz, 1H), 7.40 (dd, J = 2.1, 8.7 Hz, 1H), 1.16 (s, 18H). 13 C NMR (CDCl3, 75 MHz): δ 153.8, 150.4, 133.7, 133.1, 127.0, 123.6, 122.2, 102.2, 98.6. 18.8, 11.4. N N H Me Si iPr Me 2-((Triisopropylsilyl)ethynyl)-1H-benzo[d]imidazole (S3.5). Compound S3.5 was prepared with 2-bromo benzoimidazole (100 mg, 0.51 mmol) following general procedure B7. The crude product was purified via flash column chromatography (8:1 hexanes/EtOAc) to obtain a white solid (133 mg, 88%). Rf = 0.4 (7:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 11.23 (brs, 1H), 7.67 (dd, J = 3.0, 6.0 Hz, 2H), 7.28 (dd, J = 3.0, 6.0 Hz, 2H), 1.01 (s, 18H). 13C NMR (CDCl3, 75 MHz): δ 138.1, 135.4, 123.7, 115.7, 115.6, 96.6, 96.6, 18.6, 11.3. General procedure B8 for TIPS-deprotection using TBAF N R X Me 1M TBAF, THF Si iPr - 40 oC Me N R X X = S, NH A 25 mL round bottom flask was charged with 0.20 mmol of a TIPS-alkyne and 5 mL THF. The stirring solution was cooled down to –40 oC. Then 1M TBAF solution (0.41 mL, 0.41 mmol) was added dropwise. The reaction was allowed to warm up and completed 130 within 1 h. It was quenched with brine, extracted two times with EtOAc and concentrated under reduced pressure. The crude product was purified via column chromatography to afford the terminal alknyne. N S 2-Ethynylbenzo[d]thiazole (S4.1). Compound S4.1 was prepared with S3.1 (64.6 mg, 0.20 mmol) following general procedure B8. The crude product was purified via flash column chromatography (20:1 hexanes/EtOAc) to obtain a brown semisolid (32 mg, 99%). Rf = 0.4 (15:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 8.06 (d, J = 7.5 Hz, 1H), 7.84 (d, J = 8.1 Hz, 1H), 7.55 – 7.41 (m, 2H), 3.60 (s, 1H). 13C NMR (CDCl3, 75 MHz): δ 152.6, 147.7, 135.2, 127.0, 126.8, 123.9, 121.5, 84.7, 76.9, 18.0, 12.6. MeO N S 2-Ethynyl-5-methoxybenzo[d]thiazole (S4.2). Compound S4.2 was prepared with S3.2 (92 mg, 0.26 mmol) following general procedure B8. The crude product was purified via flash column chromatography (10:1 hexanes/EtOAc) to obtain a white solid (25 mg, 50%). Rf = 0.2 (20:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 7.70 (d, J = 8.7 Hz, 1H), 7.52 (d, J = 2.4 Hz, 1H), 7.11 (d, J = 2.4, 9.0 Hz, 1H), 3.88 (s, 3H), 3.57 (s, 1H). 13C NMR (CDCl3, 75 MHz): δ 159.7, 154.2, 148.6, 127.3, 121.7, 117.6, 105.9, 83.9, 77.1, 55.8. MeO O N S 2-Ethynyl-5-(2-methoxyethoxy)benzo[d]thiazole (S4.3). Compound S4.3 was prepared with S3.3 (1.35 g, 3.47 mmol) following general procedure B8. The crude product was purified via flash column chromatography (3:1 hexanes/EtOAc, followed by 2:1 hexanes/EtOAc) to obtain a brown solid (0.75 g, 93%). Rf = 0.2 (6:1 hexanes/EtOAc). 1H 131 NMR (CDCl3, 300 MHz): δ 7.65 (d, J = 9.0 Hz, 1H), 7.48 (d, J = 1.5 Hz, 1H), 7.12 (dd, J = 1.8, 8.7 Hz, 1H), 4.15 (t, J = 4.5 Hz, 2H), 3.75 (t, J = 4.5 Hz, 2H), 3.58 (s, 1H), 3.42 (s, 3H). 13C NMR (CDCl3, 75 MHz): δ 158.8, 154.1, 148.6, 127.5, 121.8, 118.0, 106.6, 84.1, 77.0, 71.0, 67.9, 59.4. Cl N S 5-Chloro-2-ethynylbenzo[d]thiazole (S4.4). Compound S4.4 was prepared with S3.4 (475 mg, 1.35 mmol) following general procedure B8. The crude product was purified via flash column chromatography (20:1 hexanes/EtOAc, followed by 15:1 hexanes/EtOAc) to obtain a brown solid (229 mg, 87%). Rf = 0.2 (20:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 8.00 (d, J = 1.5 Hz, 1H), 7.72 (d, J = 8.7 Hz, 1H), 7.39 (dd, J = 2.1, 8.7 Hz, 1H), 3.63 (s, 1H). 13C NMR (CDCl3, 75 MHz): δ 153.6, 149.4, 133.6, 133.2, 127.4, 123.7, 122.3, 85.0, 76.6. N N H 2-Ethynyl-1H-benzo[d]imidazole (S4.5). Compound S4.5 was prepared with S3.5 (1.26 g, 4.22 mmol) following general procedure B8. The crude product was purified via flash column chromatography 1:1 hexanes/EtOAc) to obtain a brown solid (594 mg, 99%). Rf = 0.5 (1:2 hexanes/EtOAc). 1H NMR (DMSO-d6, 300 MHz): δ 13.16 (brs, 1H), 7.54 (s, 2H), 7.31 – 7.18 (m, 2H), 4.65 (s, 1H). 13C NMR (DMSO-d6, 75 MHz): δ 134.7, 124.0, 123.8, 123.4, 83.9, 75.8. 132 General procedure B9 for Sonogashira coupling reaction R R O O MeO I N + N Pd(PPh3)4, CuI R Et3N, 60 X N MeO oC N CN X R X = S, NH CN A 20 mL oven-dried scintillation vial was charged with 0.20 mmol of a 2-iodo indole, 0.40 mmol of a terminal alkyne, Pd(PPh3)4 (23.1 mg, 0.02 mmol) and CuI (5.71 mg, 0.03 mmol). The vial was evacuated and purged three times with argon. Then 3 mL Et3N were added to the vial via syringe. The vial was sealed and heated at 60 oC. After 24 h the 2-iodo indole was consumed. The reaction was cooled down and the solvent was removed. The crude product was dissolved in CH2Cl2 and loaded on the column. Using CH2Cl2 as an eluent, colored impurities were removed. Then the solvent system was switched to hexanes/EtOAc to obtain the internal alkyne. Cl O N MeO N S CN Methyl 2-(benzo[d]thiazol-2-ylethynyl)-3-(4-chlorophenethyl)-1-(2- cyanoethyl)-1H-indole-5-carboxylate (2.47). Compound 2.47 was prepared with 2.43 (100 mg, 0.20 mmol) and S4.1 (64.4 mg, 0.40 mmol) following general procedure B9. The crude product was purified via flash column chromatography (CH2Cl2, then 3:1 hexanes/EtOAc, followed by 2:1 hexanes/EtOAc) to obtain a dark yellow solid (90.4 mg, 85%). Rf = 0.2 (2:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 8.29 (s, 1H), 8.12 (d, J = 7.8 Hz, 1H), 8.06 (dd, J = 1.5, 8.7 Hz, 1H), 7.92 (dd, J = 1.5, 7.8 Hz 1H), 7.62 – 7.47 133 (m, 2H), 7.38 (d, J = 8.7 Hz, 1H), 7.27 – 7.17 (m, 3H), 7.09 (d, J = 8.4 Hz, 2H), 4.61 (t, J = 6.9 Hz, 2H), 3.97 (s, 3H), 3.27 (t, J = 7.5 Hz, 2H), 3.04 (t, J = 7.5 Hz, 2H), 2.89 (t, J = 6.9 Hz, 2H). 13C NMR (CDCl3, 75 MHz): δ 167.6, 153.3, 147.4, 139.8, 139.4, 135.7, 132.2, 130.3, 128.9, 128.7, 127.3, 127.3, 127.2, 126.9, 126.7, 126.4, 123.3, 123.4, 121.7, 118.7, 116.9, 109.3, 92.7, 85.4. 52.4, 40.6, 36.2, 27.6, 19.1. Cl O N MeO N OMe S CN Methyl 3-(4-chlorophenethyl)-1-(2-cyanoethyl)-2-((5-methoxybenzo- [d]thiazol-2-yl)ethynyl)-1H-indole-5-carboxylate (2.48). Compound 2.48 was prepared with 2.43 (43.6 mg, 0.09 mmol) and S4.2 (25.1 mg, 0.13 mmol) following general procedure B9. The crude product was purified via flash column chromatography (CH2Cl2, then 2:1 hexanes/EtOAc, followed by 1:1 hexanes/EtOAc) to obtain a yellow solid (60.2 mg, 99%). Rf = 0.5 (1:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 8.27 (s, 1H), 8.04 (d, J = 8.4 Hz, 1H), 7.76 (d, J = 9.0 Hz, 1H), 7.56 (s, 1H), 7.37 (d, J = 8.4 Hz, 1H), 7.23 – 7.02 (m, 5H), 4.59 (t, J = 6.6 Hz, 2H), 3.96 (s, 3H), 3.91 (s, 3H), 3.25 (t, J = 7.6 Hz, 2H), 3.02 (t, J = 7.2 Hz, 2H), 2.88 (t, J = 6.0 Hz, 2H). 13C NMR (CDCl3, 75 MHz): δ 167.7, 159.9, 154.7, 148.3, 139.8, 139.3, 132.2, 130.3, 127.0, 126.7, 126.4, 123.4, 123.3, 123.2, 121.8, 118.8, 117.6, 117.0, 109.3, 105.7, 92.8, 85.3, 55.9, 52.4, 40.6, 36.2, 27.6, 19.1. 134 Cl O O N MeO N OMe S CN Methyl 3-(4-chlorophenethyl)-1-(2-cyanoethyl)-2-((5-(2-methoxyethoxy)- benzo-[d]thiazol-2-yl)ethynyl)-1H-indole-5-carboxylate (2.49). Compound 2.49 was prepared with 2.43 (80 mg, 0.16 mmol) and S4.3 (75.5 mg, 0.32 mmol) following general procedure B9. The crude product was purified via flash column chromatography (CH2Cl2, then 2:1 hexanes/EtOAc, followed by 1:1 hexanes/EtOAc and 1:2 hexanes/EtOAc) to obtain a yellow solid (63.8 mg, 66%). Rf = 0.2 (1:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 8.26 (s, 1H), 8.03 (dd, J = 1.2, 8.7 Hz, 1H), 7.73 (d, J = 9.0 Hz, 1H), 7.56 (s, 1H), 7.35 (d, J = 8.7 Hz, 1H), 7.23 – 7.14 (m, 3H), 7.06 (d, J = 8.1 Hz, 2H), 4.57 (t, J = 6.6 Hz, 2H), 4.21 (t, J = 4.5 Hz, 2H), 3.95 (s, 3H), 3.81 (t, J = 4.5 Hz, 2H), 3.47 (s, 3H), 3.24 (t, J = 7.3 Hz, 2H), 3.01 (t, J = 7.3 Hz, 2H), 2.86 (t, J = 6.6 Hz, 2H). 13C NMR (CDCl3, 75 MHz): δ 167.6, 159.0, 154.6, 148.3, 138.8, 139.3, 132.2, 130.3, 128.6, 127.0, 126.7, 126.4, 123.2, 123.2, 121.8, 118.75, 118.1, 116.9, 109.3, 106.5, 92.8, 85.3, 71.1, 68.0, 59.5, 52.3, 40.6, 36.2, 27.6, 19.1. Cl O N MeO N Cl S CN Methyl 2-((5-chlorobenzo[d]thiazol-2-yl)ethynyl)-3-(4-chlorophenethyl)-1-(2cyanoethyl)-1H-indole-5-carboxylate (2.50). Compound 2.50 was prepared with 2.43 (80 135 mg, 0.16 mmol) and S4.4 (62.7 mg, 0.32 mmol) following general procedure B9. The crude product was purified via flash column chromatography (CH2Cl2, then 3:1 hexanes/EtOAc, followed by 2:1 hexanes/EtOAc) to obtain a brown solid (63.1 mg, 72%). Rf = 0.1 (3:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 8.27 (s, 1H), 8.11 – 8.01 (m, 2H), 7.81 (d, J = 8.7 Hz, 1H), 7.45 (dd, J = 1.5, 8.7 Hz, 1H), 7.36 (d, J = 8.7 Hz, 1H), 7.17 (d, J = 8.1 Hz, 2H), 7.06 (d, J = 8.4 Hz, 2H), 4.58 (t, J = 6.6 Hz, 2H), 3.95 (s, 3H), 3.25 (t, J = 7.3 Hz, 2H), 3.02 (t, J = 7.3 Hz, 2H), 2.87 (t, J = 6.6 Hz, 2H). 13C NMR (CDCl3, 75 MHz): δ 167.6, 154.1, 149.1, 139.7, 139.4, 133.9, 133.5, 133.5, 132.2, 130.3, 128.7, 127.6, 127.3, 126.7, 123.7, 122.3, 118.5, 109.4, 92.4, 52.3, 40.7, 36.2, 27.6, 19.1. Cl O N MeO N N H CN Methyl 2-((1H-benzo[d]imidazol-2-yl)ethynyl)-3-(4-chlorophenethyl)-1-(2- cyano-ethyl)-1H-indole-5-carboxylate (2.51). Compound 2.51 was prepared with 2.43 (750 mg, 1.49 mmol) and S4.5 (422.5 mg, 2.98 mmol) following general procedure B9. The crude product was purified via flash column chromatography (CH2Cl2, then 1:1 hexanes/EtOAc, followed by 1:2 hexanes/EtOAc) to obtain a bright yellow solid, which was recrystallized from EtOH (562.2 mg, 73%). Rf = 0.2 (1:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 11.08 (brs, 1H), 8.19 (s, 1H), 8.01 (d, J = 9.0 Hz, 1H), 7.83 (brs, 1H), 7.50 (brs, 1H), 7.40 – 7.31 (m, 2H), 7.21 (d, J = 9.0 Hz, 1H), 7.19 (d, J = 8.4 Hz, 2H), 6.93 (d, J = 8.4 Hz, 2H), 4.35 (t, J = 6.0 Hz, 2H), 3.96 (s, 3H), 3.09 (t, J = 7.2 Hz, 2H), 2.89 (t, J = 7.2 Hz, 2H), 2.70 (t, J = 6.0 Hz, 2H). 13 C NMR (CDCl3, 75 MHz): δ 167.2, 139.9, 136 139.0, 134.6, 131.9, 130.3, 128.5, 128.8, 126.7, 126.2, 126.2, 125.1, 123.7, 123.2, 123.1, 119.1, 117.7, 109.0, 98.8, 89.7, 82.1, 52.4, 40.3, 36.1, 27.3, 19.1. F O N MeO N S CN Methyl 2-(benzo[d]thiazol-2-ylethynyl)-1-(2-cyanoethyl)-3-(4-fluorophen- ethyl)-1H-indole-5-carboxylate (2.52). Compound 2.52 was prepared with 2.45 (1.2 g, 2.52 mmol) and S4.1 (802 mg, 5.04 mmol) following general procedure B9. The crude product was purified via flash column chromatography (CH2Cl2, then 3:1 hexanes/EtOAc, followed by 2:1 and 1:1 hexanes/EtOAc) to obtain a yellow solid (992.5 mg, 77%). Rf = 0.2 (3:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 8.29 (s, 1H), 8.12 (d, J = 7.8 Hz, 1H), 8.06 (d, J = 8.4 Hz, 1H), 7.92 (d, J = 7.5 Hz, 1H), 7.63 – 7.44 (m, 2H), 7.37 (d, J = 8.4 Hz, 1H), 7.16 – 7.05 (m, 2H), 7.01 – 6.87 (m, 2H), 4.60 (t, J = 5.7 Hz, 2H), 3.96 (s, 3H), 3.26 (t, J = 6.6 Hz, 2H), 3.04 (t, J = 6.6 Hz, 2H), 2.89 (t, J = 6.0 Hz, 2H). 13C NMR (CDCl3, 75 MHz): δ 167.7, 153.3, 147.4, 139.4, 137.0, 135.7, 130.4, 130.3, 127.4, 126.9, 126.7, 126.4, 124.1, 123.3, 123.2, 121.7, 118.7, 117.0, 115.5, 115.2, 109.3, 92.6, 85.5, 52.3, 40.6, 36.1, 27.8, 19.1. 137 Me O N MeO N S CN Methyl 2-(benzo[d]thiazol-2-ylethynyl)-1-(2-cyanoethyl)-3-(4-methylphen- ethyl)-1H-indole-5-carboxylate (2.53). Compound 2.53 was prepared with 2.44 (600 mg, 1.26 mmol) and S4.1 (404 mg, 2.53 mmol) following general procedure B9. The crude product was purified via flash column chromatography (CH2Cl2, then 3:1 hexanes/EtOAc, followed by 1:1 hexanes/EtOAc) to obtain a brown solid (571.5 mg, 90%). Rf = 0.5 (1:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 8.34 (d, J = 1.5 Hz, 1H), 8.15 – 8.09 (m, 1H), 8.05 (dd, J = 1.8, 8.7 Hz, 1H), 7.95 – 7.89 (m, 1H), 7.60 – 7.46 (m, 2H), 7.38 (d, J = 9.0 Hz, 1H), 7.12 – 7.04 (m, 4H), 4.60 (t, J = 6.9 Hz, 2H), 3.96 (s, 3H), 3.26 (t, J = 7.2 Hz, 2H), 3.02 (t, J = 7.6 Hz, 2H), 2.88 (t, J = 6.9 Hz, 2H), 2.27 (s, 3H). 13C NMR (CDCl3, 75 MHz): δ 167.7, 153.3, 147.6, 139.4, 138.3, 135.8, 129.3, 128.7, 127.9, 127.3, 126.8, 126.4, 124.0, 123.4, 123.1, 121.6, 118.6, 117.0, 109.3, 92.7, 85.7, 52.3, 40.6, 36.5, 27.9, 21.3, 19.1. Me O N MeO N N H CN Methyl 2-((1H-benzo[d]imidazol-2-yl)ethynyl)-1-(2-cyanoethyl)-3-(4- methylphen-ethyl)-1H-indole-5-carboxylate (2.54). Compound 2.54 was prepared with 2.44 (100 mg, 0.21 mmol) and S4.5 (60.2 mg, 0.42 mmol) following general procedure B9. 138 The crude product was purified via flash column chromatography (80:1:0.2 CH2Cl2/MeOH/AcOH) to obtain a yellow solid (39.1 mg, 38%). Rf = 0.2 (1:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 10.07 (brs, 1H), 8.18 (s, 1H), 7.97 (d, J = 8.7 Hz, 1H), 7.61 – 7.60 (m, 2H), 7.27 – 7.24 (m, 2H), 7.17 (d, J = 8.7 Hz, 1H), 6.98 – 6.95 (m, 4H), 4.30 (t, J = 6.0 Hz, 2H), 3.95 (s, 3H), 3.04 (t, J = 7.2 Hz, 2H), 2.89 (t, J = 7.8 Hz, 2H), 2.66 (t, J = 6.0 Hz, 2H), 2.22 (s, 3H). 13C NMR (CDCl3, 75 MHz): δ 167.8, 139.0, 138.5, 135.7, 134.8, 132.3, 132.2, 129.1, 128.8, 128.7, 126.9, 126.7, 126.0, 124.6, 123.9, 118.8, 117.6, 109.0, 89.7, 82.3, 52.3, 40.6, 36.3, 27.4, 21.2, 19.0. O N MeO N S CN Methyl 2-(benzo[d]thiazol-2-ylethynyl)-1-(2-cyanoethyl)-3-phenethyl-1H- indole-5-carboxylate (2.55). Compound 2.55 was prepared with 2.46 (250 mg, 0.55 mmol) and S4.1 (175 mg, 1.1 mmol) following general procedure B9. The crude product was purified via flash column chromatography (CH2Cl2, then 2:1 hexanes/EtOAc, followed by 1:1 hexanes/EtOAc) to obtain a dark yellow solid (186.5 mg, 69%). Rf = 0.2 (3:2 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 8.35 (s, 1H), 8.11 (dd, J = 0.9, 8.1 Hz, 1H), 8.05 (dd, J = 1.5, 8.7 Hz, 1H), 7.91 (dd, J = 0.9 Hz, 8.1 Hz, 1H), 7.60 – 7.53 (m, 1H), 7.53 – 7.46 (m, 1H), 7.38 (d, J = 8.7 Hz, 1H), 7.28 – 7.21 (m, 2H), 7.20 – 7.13 (m, 3H), 4.60 (t, J = 6.9 Hz, 2H), 3.96 (s, 3H), 3.29 (t, J = 7.8 Hz, 2H), 3.06 (t, J = 7.8 Hz, 2H), 2.87 (t, J = 6.9 Hz, 2H), 13C NMR (CDCl3, 75 MHz): δ 167.7, 153.3, 147.5, 141.3, 139.4, 135.7, 128.9, 128.6, 127.7, 127.2, 126.8, 126.8, 126.4, 126.4, 124.0, 123.4, 123.2, 121.6, 118.6, 117.0, 109.3, 92.6, 85.6, 52.3, 40.6, 36.9, 27.7, 19.1. 139 General procedure B10 for the reduction of alkynes to alkanes R R O O N MeO N R X H2, Pd/C MeO N THF/EtOH (1:1) r.t. N S CN R X = S, NH CN In a 50 mL round bottom flask, 0.24 mmol of an internal alkyne was dissolved in THF (8 mL) and EtOH (8 mL). Then 10 wt. % Pd/C (12 mg) was added. After evacuating and flushing the flask with H2 gas three times, the reaction mixture was stirred under hydrogen atmosphere for 48 h. After the starting material was consumed, Pd/C was removed via filtration. The organic solvent was removed under reduced pressure and the crude product purified via flash column chromatography to obtain the alkane. Cl O MeO N N S CN Methyl 2-(2-(benzo[d]thiazol-2-yl)ethyl)-3-(4-chlorophenethyl)-1-(2- cyanoethyl)-1H-indole-5-carboxylate (2.56). Compound 2.56 was prepared with 2.47 (127 mg, 0.24 mmol) following general procedure B10. The crude product was purified via flash column chromatography (4:1 hexanes/EtOAc, followed by 3:1 and 2:1 hexanes/EtOAc) to obtain a yellow solid (65 mg, 50%). Rf = 0.5 (1:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 8.28 (s, 1H), 8.00 (d, J = 8.1 Hz, 1H), 7.95 (d, J = 8.7 Hz, 1H), 7.84 (d, J = 8.1 Hz, 1H), 7.49 (t, J = 7.6 Hz, 1H), 7.38 (t, J = 7.6 Hz, 1H), 7.26 – 7.16 (m, 3H), 6.99 (d, J = 8.1 Hz, 2H), 4.42 (t, J = 6.9 Hz, 2H), 3.96 (s, 3H), 3.20 (t, J = 7.6 Hz, 2H), 3.09 – 2.98 (m 4H), 2.96 – 2.89 (m, 2H), 2.72 (t, J = 6.9 Hz, 2H). 13C NMR (CDCl3, 140 75 MHz): δ 169.2, 168.2, 153.3, 141.98, 140.4, 138.4, 136.1, 135.3, 132.1, 130.3, 128.7, 128.1, 125.4, 123.7, 122.9, 122.3, 122.0, 121.9, 117.0, 114.4, 108.7, 52.2, 39.3, 36.3, 34.4, 27.0, 18.7. Cl O MeO N OMe N S CN Methyl 3-(4-chlorophenethyl)-1-(2-cyanoethyl)-2-(2-(5-methoxybenzo[d]- thiazol-2-yl)ethyl)-1H-indole-5-carboxylate (2.57). Compound 2.57 was prepared with 2.48 (50 mg, 0.09 mmol) following general procedure B10. The crude product was purified via flash column chromatography (1:1 hexanes/EtOAc) to obtain a yellow solid (52 mg, 99%). Rf = 0.2 (2:1 hexanes/EtOAc). 1H NMR (300 MHz, CDCl3): δ 8.26 (s, 1H), 7.93 (d, J = 8.7 Hz, 1H), 7.66 (d, J = 8.7 Hz, 1H), 7.45 (s, 1H), 7.28 – 7.14 (m, 3H), 7.06 – 6.93 (m, 3H), 4.41 (t, J = 6.9 Hz, 2H), 3.94 (s, 3H), 3.88 (s, 3H), 3.21 – 3.12 (m, 2H), 3.07 – 2.97 (m, 4H), 2.92 – 2.85 (m, 2H), 2,71 (t, J = 6.9 Hz, 2H). 13C NMR (75 MHz, CDCl3): δ 170.4, 168.2, 159.3, 154.5, 140.4, 138.4, 136.0, 132.1, 130.2, 128.1, 128.1, 126.9, 123.7, 122.3, 122.1, 122.0, 117.0, 115.5, 114.4, 108.6, 105.4, 55.8, 52.2, 39.3, 36.3, 34.4, 26.9, 23.9, 18.7. 141 Cl O MeO O N OMe N S CN Methyl 3-(4-chlorophenethyl)-1-(2-cyanoethyl)-2-(2-(5-(2-methoxy- ethoxy)benzo-[d]thiazol-2-yl)ethyl)-1H-indole-5-carboxylate (2.58). Compound 2.58 was prepared with 2.49 (152 mg, 0.25 mmol) following general procedure B10. The crude product was purified via flash column chromatography (1:1 hexanes/EtOAc, followed by 1:2 and 1:3 hexanes/EtOAc) to obtain a yellow solid (130 mg, 85%). Rf = 0.3 (1:1 hexanes/EtOAc). 1H NMR (300 MHz, CDCl3): δ 8.24 (s, 1H), 7.92 (d, J = 8.4 Hz, 1H), 7.64 (d, J = 9.0 Hz, 1H), 7.47 (s, 1H), 7.23 – 7.13 (m, 3H), 7.05 (d, J = 8.4 Hz, 1H), 6.97 (d, J = 8.1 Hz, 2H), 4.39 (t, J = 6.4 Hz, 2H), 4.17 (t, J = 3.9 Hz, 2H), 3.93 (s, 3H), 3.79 (t, J = 4.0 Hz, 2H), 3.46 (s, 3H), 3.16 (t, J = 4.2 Hz, 2H), 3.05 – 2.93 (m, 4H), 2.93 – 2.84 (m, 2H), 2.69 (t, J = 6.6 Hz, 2H). 13C NMR (75 MHz, CDCl3): δ 170.5, 168.2, 158.4, 154.5, 140.4, 138.4, 136.0, 132.0, 130.3, 130.2, 128.9, 128.7, 128.0, 127.2, 123.6, 122.3, 122.2, 122.1, 121.9, 117.1, 116.0, 114.4, 108.7, 106.2, 71.2, 67.9, 59.5, 52.2, 39.3, 36.3, 34.4, 26.9, 23.9, 18.7. Cl O MeO N Cl N S CN Methyl 2-(2-(5-chlorobenzo[d]thiazol-2-yl)ethyl)-3-(4-chlorophenethyl)-1-(2cyanoethyl)-1H-indole-5-carboxylate (2.59). Compound 2.59 was prepared with 2.50 (63 142 mg, 0.12 mmol) following general procedure B10. The crude product was purified via flash column chromatography (2:1 hexanes/EtOAc, followed by 1:1 hexanes/EtOAc) to obtain a yellow solid (53 mg, 83%). Rf = 0.4 (2:1 hexanes/EtOAc). 1H NMR (300 MHz, CDCl3): δ 8.26 (s, 1H), 8.01 – 7.90 (m, 2H), 7.72 (d, J = 8.4 Hz, 1H), 7.34 (dd, J = 1.8, 8.4 Hz, 1H), 7.23 (s, 1H), 7.19 (d, J = 8.1 Hz, 2H), 6.96 (d, J = 8.1 Hz, 2H), 4.41 (t, J = 6.6 Hz, 2H), 3.95 (s, 3H), 3.17 (t, J = 4.6 Hz, 2H), 3.04 – 2.95 (m, 4H), 2.95 – 2.87 (m, 2H), 2.72 (t, J = 6.9 Hz, 2H). 13 C NMR (75 MHz, CDCl3): δ 171.2, 168.1, 154.1, 140.3, 138.4, 135.8, 133.5, 132.5, 132.1, 130.2, 128.7, 125.9, 123.7, 122.8, 122.5, 122.4, 122.0, 116.9, 114.4, 108.6, 52.2, 39.3, 36.2, 34.4, 26.9, 23.7, 18.7. Cl O MeO N N N H CN Methyl 2-(2-(1H-benzo[d]imidazol-2-yl)ethyl)-3-(4-chlorophenethyl)-1-(2- cyano-ethyl)-1H-indole-5-carboxylate (2.60). Compound 2.60 was prepared with 2.51 (142.6 mg, 0.28 mmol) following general procedure B10. The crude product was purified via flash column chromatography (2:1 hexanes/acetone, followed by 1:1 hexanes/acetone) to obtain a white solid (101.9 mg, 71%). Rf = 0.2 (1:1 hexanes/EtOAc). 1H NMR (acetoned6, 300 MHz): δ 11.27 (brs, 1H), 8.17 (s, 1H), 7.87 – 7.78 (m, 1H), 7.60 (dd, J = 1.5, 8.7 Hz, 1H), 7.42 (brs, 2H), 7.27 – 7.20 (m, 2H), 7.20 – 7.11 (m, 4H), 4.71 (t, J = 6.7 Hz, 2H), 3.87 (s, 3H), 3.44 – 3.36 (m, 2H), 3.20 – 3.09 (m, 2H), 3.08 – 2.98 (m, 4H), 2.90 – 2.83 (m, 2H). 13C NMR (CDCl3, 75 MHz): δ 168.4, 153.1, 142.2, 140.5, 138.4, 136.4, 132.1, 131.8, 130.3, 129.1, 128.9, 128.6, 128.1, 126.3, 123.5, 122.9, 122.0, 117.4, 114.9, 114.3, 143 114.1, 108.7, 52.2, 38.9, 36.7, 29.8, 26.9, 23.1, 18.5. F O MeO N N S CN Methyl 2-(2-(benzo[d]thiazol-2-yl)ethyl)-1-(2-cyanoethyl)-3-(4-fluorophen- ethyl)-1H-indole-5-carboxylate (2.61). Compound 2.61 was prepared with 2.52 (0.99 g, 1.95 mmol) following general procedure B10. The crude product was purified via flash column chromatography (5:1 hexanes/EtOAc, followed by 4:1 and 3:1 hexanes/EtOAc) to obtain a yellow solid (0.83 g, 84%). Rf = 0.2 (2:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 8.28 (s, 1H), 7.99 (d, J = 8.4 Hz, 1H), 7.95 (d, J = 8.7 Hz, 1H), 7.82 (d, J = 7.8 Hz, 1H), 7.48 (t, J = 7.2 Hz, 1H), 7.37 (t, J = 7.5 Hz, 1H), 7.25 (d, J = 8.7, Hz, 1H), 7.05 – 6.98 (m, 2H), 6.97 – 6.87 (m, 2H), 4.41 (t, J = 6.7 Hz, 2H), 3.95 (s, 3H), 3.18 (t, J = 7.3 Hz, 2H), 3.08 – 3.00 (m, 4H), 2.96 – 2.90 (m, 2H), 2.71 (t, J = 6.7 Hz, 2H). 13 C NMR (CDCl3, 75 MHz): δ 169.2, 168.2, 160.0, 153.3, 138.4, 137.6, 136.1, 135.3, 130.3, 130.2, 128.1, 126.5, 125.4, 123.6, 122.9, 122.3, 122.0, 122.9, 117.9, 115.5, 115.2, 114.5, 108.7, 52.2, 39.3, 36.2, 34.4, 27.2, 23.9, 18.7. Me O MeO N N S CN Methyl 2-(2-(benzo[d]thiazol-2-yl)ethyl)-1-(2-cyanoethyl)-3-(4-methyl- phenethyl)-1H-indole-5-carboxylate (2.62). Compound 2.62 was prepared with 2.53 144 (0.57 g, 1.13 mmol) following general procedure B10. The crude product was purified via flash column chromatography (3:1 hexanes/EtOAc, followed by 2:1 hexanes/EtOAc) to obtain a yellow solid (0.46 g, 80%). Rf = 0.15 (3:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 8.37 (d, J = 0.9 Hz, 1H), 8.01 (d, J = 8.1 Hz, 1H), 7.96 (dd, J = 1.5, 8.4 Hz, 1H), 7.83 (d, J = 7.5 Hz, 1H), 7.53 – 7.44 (m, 1H), 7.43 – 7.34 (m, 1H), 7.25 (d, J = 8.7 Hz, 1H), 7.07 (d, J = 7.8 Hz, 2H), 6.97 (d, J = 8.1 Hz, 2H), 4.41 (t, J = 6.9 Hz, 2H), 3.97 (s, 3H), 3.19 (t, J = 7.6 Hz, 2H), 3.10 – 3.00 (m, 2H), 2.97 – 2.87 (m, 4H), 2.70 (t, J = 6.9 Hz, 2H), 2.31 (s, 3H). 13C NMR (CDCl3, 75 MHz): δ 169.5, 168.3, 153.3, 138.9, 138.5, 136.1, 135.8, 135.4, 129.4, 128.8, 128.2, 126.4, 125.3, 123.6, 122.9, 122.2, 122.1, 121.9, 117.15 114.8, 108.6, 52.2, 39.3, 36.6, 34.3, 27.3, 23.9, 21.3, 18.7. Me O MeO N N N H CN Methyl 2-(2-(1H-benzo[d]imidazol-2-yl)ethyl)-1-(2-cyanoethyl)-3-(4- methylphen-ethyl)-1H-indole-5-carboxylate (2.63). Compound 2.63 was prepared with 2.54 (61.6 mg, 0.13 mmol) following general procedure B10. The crude product was purified via flash column chromatography (80:1 CH2Cl2/MeOH) to obtain a white solid (34.6 mg, 56%). Rf = 0.1 (80:1 CH2Cl2/MeOH). 1H NMR (CDCl3, 300 MHz): δ 8.29 (s, 1H), 7.87 (d, J = 8.4, 1H), 7.48 (s, 2H), 7.22 – 7.18 (m, 2H), 7.09 (d, J = 8.7 Hz, 1H), 7.01 (d, J = 7.8 Hz, 2H), 6.91 (d, J = 7.8 Hz, 2H), 4.08 (t, J = 6.3 Hz, 2H), 3.93 (s, 3H), 3.13 (t, J = 7.2 Hz, 2H), 2.92 – 2.80 (m, 6H), 2.47 (t, J = 6.6 Hz, 2H), 2.25 (s, 3H). δ 13C NMR (CDCl3, 75 MHz): δ 168.4, 153.2, 139.1, 138.4, 136.4, 135.8, 129.2, 129.0, 128.1, 123.5, 145 122.8, 122.0, 117.3, 114.4, 108.7, 52.2, 38.8, 36.2, 30.0, 27.0, 23.2, 21.2, 18.5. MS (ESI) m/z = 491.4 [M+H]+. O MeO N N S CN Methyl 2-(2-(benzo[d]thiazol-2-yl)ethyl)-1-(2-cyanoethyl)-3-phenethyl-1H- indole-5-carboxylate (2.64). Compound 2.64 was prepared with 2.55 (186.5 mg, 0.38 mmol) following general procedure B10. The crude product was purified via flash column chromatography (5:1 hexanes/EtOAc, followed by 4:1 and 3:1 hexanes/EtOAc) to obtain a yellow solid (88.1 mg, 47%). Rf = 0.4 (3:2 hexanes/EtOAc). 1H NMR (300 MHz, CDCl3): δ 8.35 (s, 1H), 8.00 (d, J = 8.1 Hz, 1H), 7.95 (d, J = 8.4 Hz, 1H), 7.83 (d, J = 8.1 Hz, 1H), 7.48 (t, J = 7.6 Hz, 1H), 7.38 (t, J = 7.5 Hz, 1H), 7.29 – 7.16 (m, 4H), 7.07 (d, J = 7.2 Hz, 2H), 4.41 (t, J = 7.0 Hz, 2H), 3.96 (s, 3H), 3.14 (t, J = 7.8 Hz, 2H), 3.06 (t, J = 6.8 Hz, 2H), 3.02 – 2.89 (m, 4H), 2.70 (t, J = 6.9 Hz, 2H). 13C NMR (75 MHz, CDCl3): δ 169.4, 168.2, 153.3, 142.0, 138.4, 136.0, 135.3, 128.9, 128.7, 128.2, 126.4, 126.3, 125.3, 123.6, 122.9, 122.2, 122.0, 121.8, 117.0, 114.7, 108.6, 52.2, 39.3, 37.0, 34.4, 27.1, 23.9, 18.7. Cl O MeO N N S HN Methyl N N N 1-(2-(1H-tetrazol-5-yl)ethyl)-2-(2-(benzo[d]thiazol-2-yl)ethyl)-3-(4- chloro-phenethyl)-1H-indole-5-carboxylate (2.65). Compound 2.65 was prepared with 146 2.56 following general procedure B4. The product was used for the next step without further characterization. Cl O MeO N OMe N S HN N N N Methyl 1-(2-(1H-tetrazol-5-yl)ethyl)-3-(4-chlorophenethyl)-2-(2-(5-methoxybenzo[d] thiazol-2-yl)ethyl)-1H-indole-5-carboxylate (2.66). Compound 2.66 was prepared with 2.57 (210 mg, 0.37 mmol) following general procedure B4 to obtain the product as a yellow solid (149 mg, 66%). Rf = 0.1 (1:1 hexanes/EtOAc). 1H NMR (500 MHz, DMSO-d6): δ 8.04 (d, J = 1.5 Hz, 1H), 7.88 (d, J = 9.0 Hz, 1H), 7.71 (dd, J = 1.5, 9.0 Hz, 1H), 7.50 – 7.45 (m, 2H), 7.25 (d, J = 8.5 Hz, 2H), 7.07 (d, J = 8.0 Hz, 2H), 7.03 (dd, J = 2.0, 9.0 Hz, 1H), 4.60 (t, J = 7.0 Hz, 2H), 3.84 (s, 3H), 3.82 (s, 3H), 3.30 (t, J = 7.5 Hz, 2H), 3.14 (s, 4H), 2.91 (t, J = 7.5 Hz, 2H), 2.73 (t, J = 7.5 Hz, 2H). 13C NMR (125 MHz, DMSO-d6): δ 171.4, 167.6, 158.9, 154.5, 141.0, 138.6, 137.3, 131.0, 130.7, 128.5, 127.5, 126.9, 122.8, 122.4, 121.0, 120.9, 114.9, 113.1, 109.9, 105.7, 55.9, 52.1, 41.5, 36.2, 34.0, 26.4, 24.5, 23.5. 147 Cl O MeO O N OMe N S HN Methyl N N N 1-(2-(1H-tetrazol-5-yl)ethyl)-3-(4-chlorophenethyl)-2-(2-(5-(2- methoxy-ethoxy)benzo[d]thiazol-2-yl)ethyl)-1H-indole-5-carboxylate (2.67). Compound 2.67 was prepared with 2.58 following general procedure B4. The product was used for the next step without further characterization. Cl O MeO N Cl N S HN Methyl N N N 1-(2-(1H-tetrazol-5-yl)ethyl)-2-(2-(5-chlorobenzo[d]thiazol-2-yl)- ethyl)-3-(4-chlorophenethyl)-1H-indole-5-carboxylate (2.68). Compound 2.68 was prepared with 2.59 following general procedure B4. The product was used for the next step without further characterization. 148 Cl O MeO N N N H HN Methyl N N N 2-(2-(1H-benzo[d]imidazol-2-yl)ethyl)-1-(2-(1H-tetrazol-5-yl)-ethyl)- 3-(4-chlorophenethyl)-1H-indole-5-carboxylate (2.69). Compound 2.69 was prepared with 2.60 (184 mg, 0.36 mmol) following general procedure B4 to obtain the product as a yellow solid (150 mg, 75%). Rf = 0.4 (10:1 CH2Cl2/MeOH). 1H NMR (500 MHz, CD3OD): δ 8.15 (s, 1H), 7.80 (d, J = 9.5 Hz, 1H), 7.60 – 7.52 (m, 2H), 7.37 (t, J = 8.5 Hz, 1H), 7.32 – 7.29 (m, 2H), 7.13 (t, J = 8.0 Hz, 2H), 6.96 (d, J = 7.0 Hz, 1H), 6.92 (d, J = 8.5 Hz, 1H), 4.55 (t, J = 7.5 Hz, 2H), 3.89 (s, 3H), 3.12 – 2.94 (m, 4H), 2.87 – 2.69 (m, 4H). F O MeO N N S HN Methyl N N N 1-(2-(1H-tetrazol-5-yl)ethyl)-2-(2-(benzo[d]thiazol-2-yl)ethyl)-3-(4- fluoro-phenethyl)-1H-indole-5-carboxylate (2.70). Compound 2.70 was prepared with 2.61 following general procedure B4. The product was used for the next step without further characterization. 149 Me O MeO N N S HN Methyl N N N 1-(2-(1H-tetrazol-5-yl)ethyl)-2-(2-(benzo[d]thiazol-2-yl)ethyl)-3-(4- methyl-phenethyl)-1H-indole-5-carboxylate (2.71). Compound 2.71 was prepared with 2.62 following general procedure B4. The product was used for the next step without further characterization. O MeO N N S HN N N N Methyl 1-(2-(1H-tetrazol-5-yl)ethyl)-2-(2-(benzo[d]thiazol-2-yl)ethyl)-3-phenethyl-1H-indole-5-carboxylate (2.72). Compound 2.72 was prepared with 2.64 following general procedure B4. The product was used for the next step without further characterization. 150 General procedure B11 for ester hydrolysis R R O O MeO NaOH, iPrOH N N S HN R HO N reflux N S N N N HN R X = S, NH N N N A 50 mL round bottom flask was charged with 0.18 mmol of the methyl ester (crude product), iPrOH (10 mL) and 3M aq. NaOH (2 mL). The mixture was refluxed for 12 h. The reaction was then cooled down and the organic solvent was removed. To the remaining water layer was added dropwise 2M HCl until pH < 4. A precipitate formed was filtered, washed with water and cold Et2O to isolate the carboxylic acid. Cl O HO N N S HN N N N 1-(2-(1H-tetrazol-5-yl)-ethyl)-2-(2-(benzo[d]thiazol-2-yl)-ethyl)-3-(4chlorophen-ethyl)-1H-indole-5-carboxylic acid (2.73). Compound 2.73 was prepared with 2.65 (105 mg crude product) following general procedure B11 to obtain the product as a white solid (82 mg, 49% over two steps). Rf = 0.4 (15:1 CH2Cl2/MeOH). 1H NMR (500 MHz, DMSO-d6): δ 12.45 (brs, 1H), 8.11 (s, 1H), 8.04 (dd, J = 1.5, 8.0 Hz, 1H), 7.94 (dd, J = 1.5, 8.0 Hz, 1H), 7.71 (dd, J = 1.5, 8.5 Hz, 1H), 7.51 – 7.43 (m, 2H), 7.40 (td, J = 2.0, 8.0 Hz, 1H), 7.27 (dd, J = 3.0, 8.0 Hz, 2H), 7.09 (dd, J = 3.0, 8.0 Hz, 2H), 4.60 (t, J = 6.0 Hz, 2H), 3.15 (s, 4H), 2.90 (t, J = 6.5 Hz, 2H), 2.74 (t, J = 6.5 Hz, 2H). 13C NMR (125 151 MHz, DMSO-d6): δ 170.5, 169.0, 153.4, 141.3, 138.7, 137.2, 135.5, 131.2, 130.9, 129.0, 128.9, 128.8, 127.6, 126.8, 125.6, 123.0, 122.9, 122.2, 113.2, 109.9, 41.7, 36.4, 34.2, 26.8, 24.8, 23.7. Cl O HO N OMe N S HN N N N 1-(2-(1H-tetrazol-5-yl)-ethyl)-3-(4-chlorophenethyl)-2-(2-(5-methoxy-benzo[d]-thiazol-2-yl)ethyl)-1H-indole-5-carboxylic acid (2.74). Compound 2.74 was prepared with 2.66 (148 mg crude product) following general procedure B11 to obtain the product as a white solid (132 mg, 60% over two steps). Rf = 0.6 (6:1 CH2Cl2/MeOH). 1H NMR (500 MHz, DMSO-d6): δ 12.46 (brs, 2H), 8.11 (s, 1H), 7.89 (d, J = 8.5 Hz, 1H), 7.71 (d, J = 8.5 Hz, 1H), 7.49 – 7.44 (m, 2H), 7.26 (d, J = 8.5 Hz, 2H), 7.09 (d, J = 8.5 Hz, 2H), 7.03 (dd, J = 2.5, 8.5 Hz, 1H), 4.60 (t, J = 7.5 Hz, 2H), 3.82 (s, 3H), 3.16 – 3.10 (m, 4H), 2.90 (t, J = 7.5 Hz, 2H), 2.73 (t, J = 7.5 Hz, 2H). 13C NMR (125 MHz, DMSO-d6): δ 171.4, 168.7, 158.9, 154.5, 141.1, 138.5, 137.0, 130.9, 130.7, 128.5, 127.4, 126.9, 122.8, 122.7, 122.0, 121.1, 114.9, 113.0, 110.0, 109.7, 105.7, 55.9, 41.5, 36.2, 34.0, 26.5, 23.5. 152 Cl O HO O N OMe N S HN N N N 1-(2-(1H-tetrazol-5-yl)ethyl)-3-(4-chlorophenethyl)-2-(2-(5-(2-methoxyethoxy)-benzo[d]thiazol-2-yl)ethyl)-1H-indole-5-carboxylic acid (2.75). Compound 2.75 was prepared with 2.67 (150 mg crude product) following general procedure B11 to obtain the product as a yellow solid (110 mg, 60% over two steps). Rf = 0.5 (6:1 CH2Cl2/MeOH). 1H NMR (500 MHz, DMSO-d6): δ 8.11 (s, 1H), 7.88 (d, J = 8.5 Hz, 1H), 7.71 (dd, J = 1.0, 8.5 Hz, 1H), 7.48 (d, J = 2.0 Hz, 1H), 7.45 (d, J = 9.0 Hz, 1H), 7.26 (d, J = 8.0 Hz, 2H), 7.09 (d, J = 8.0 Hz, 2H), 7.04 (dd, J = 2.5, 9.0 Hz, 1H), 4.60 (t, J = 7.5 Hz, 2H), 4.16 (t, J = 4.5 Hz, 2H), 3.69 (t, J = 4.5 Hz, 2H), 3.32 (s, 3H), 3.30 (t, J = 7.5 Hz, 2H), 3.14 (s, 4H), 2.90 (t, J = 7.5 Hz, 2H), 2.73 (t, J = 7.5 Hz, 2H). 13C NMR (125 MHz, DMSOd6): δ 171.5, 168.7, 158.1, 154.4, 141.0, 138.5, 137.0, 130.9, 130.6, 128.5, 127.4, 127.0, 122.8, 122.7, 121.9, 121.1, 115.3, 113.0, 110.0, 109.7, 106.4, 70.8, 67.8, 58.6, 41.5, 36.1, 34.0, 26.4, 24.4, 23.5. Cl O HO N Cl N S HN N N N 1-(2-(1H-tetrazol-5-yl)ethyl)-2-(2-(5-chlorobenzo[d]thiazol-2-yl)ethyl)-3-(4chloro-phenethyl)-1H-indole-5-carboxylic acid (2.76). Compound 2.76 was prepared 153 with 2.68 (93 mg crude product) following general procedure B11 to obtain the product as a yellow solid (85 mg, 93% over two steps). Rf = 0.5 (6:1 CH2Cl2/MeOH). 1H NMR (500 MHz, DMSO-d6): δ 8.11 (d, J = 1.5 Hz, 1H), 8.07 (d, J = 8.5 Hz, 1H), 8.01 (d, J = 2.0 Hz, 1H), 7.71 (dd, J = 1.5, 8.5 Hz, 1H), 7.48 – 7.42 (m, 2H), 7.26 (d, J = 8.5 Hz, 2H), 7.08 (d, J = 8.5 Hz, 2H), 4.57 (t, J = 7.5 Hz, 2H), 3.25 (t, J = 7.5 Hz, 2H), 3.20 – 3.12 (m, 4H), 2.89 (t, J = 7.5 Hz, 2H), 2.73 (t, J = 7.5 Hz, 2H). 13C NMR (125 MHz, DMSO-d6): δ 173.1, 168.7, 154.9, 154.0, 141.0, 138.5, 136.8, 134.0, 131.3, 130.9, 130.6, 128.5, 127.4, 125.5, 124.0, 122.7, 122.2, 121.9, 121.1, 122.9, 109.7, 41.9, 36.2, 34.0, 26.4, 25.0, 23.4. Cl O HO N N N H HN N N N 2-(2-(1H-benzo[d]imidazol-2-yl)ethyl)-1-(2-(1H-tetrazol-5-yl)ethyl)-3-(4chloro-phenethyl)-1H-indole-5-carboxylic acid (2.77). Compound 2.77 was prepared with 2.69 (70 mg crude product) following general procedure B11 to obtain the product as a yellow solid (27 mg, 39% over two steps). Rf = 0.5 (6:1 CH2Cl2/MeOH). 1H NMR (500 MHz, DMSO-d6): δ 12.50, (brs, 1H), 8.11 (s, 1H), 7.78 (dd, J = 3.0, 5.5 Hz, 2H), 7.71 (t, J = 7.2 Hz, 1H), 7.53 – 7.46 (m, 3H), 7.26 (d, J = 8.5 Hz, 1H), 7.23 – 7.12 (m, 4H), 4.72 (t, J = 7.0 Hz, 2H), 3.39 – 3.26 (m, 4H), 3.22 (t, J = 7.5 Hz, 2H), 2.96 – 2.88 (m, 2H), 2.78 – 2.70 (m, 2H). 13C NMR (125 MHz, DMSO-d6): δ 168.7, 153.3, 142.0, 141.0, 138.7, 138.6, 135.7, 131.8, 130.8, 128.9, 128.5, 128.4, 128.4, 127.3, 127.3, 126.3, 125.7, 125.7, 122.9, 122.0, 121.2, 114.3, 113.7, 109.9, 41.4, 37.1, 27.6, 26.3, 24.6, 21.9. 154 F O HO N N S HN N N N 1-(2-(1H-tetrazol-5-yl)-ethyl)-2-(2-(benzo[d]thiazol-2-yl)-ethyl)-3-(4fluorophen-ethyl)-1H-indole-5-carboxylic acid (2.78). Compound 2.78 was prepared with 2.70 (146 mg crude product) following general procedure B11 to obtain the product as a yellow solid (132 mg, 72% over two steps). Rf = 0.5 (10:1 CH2Cl2/MeOH). 1H NMR (500 MHz, DMSO-d6): δ 12.46 (brs, 1H), 8.10 (s, 1H), 8.04 (d, J = 8.0 Hz, 1H), 7.94 (d, J = 8.5 Hz, 1H), 7.70 (d, J = 8.5 Hz, 1H), 7.51 – 7.45 (m, 2H), 7.39 (t, J = 8.5 Hz, 1H), 7.11 – 7.06 (m, 2H), 7.05 – 6.99 (m, 2H), 4.61 (t, J = 7.5 Hz, 2H), 3.30 (t, J = 7.5 Hz, 2H), 3.15 (s, 4H), 2.90 (t, J = 7.5 Hz, 2H), 2.74 (t, J = 7.5 Hz, 2H). 13C NMR (125 MHz, DMSO-d6): δ 170.3, 168.7, 162.1, 160.1, 153.1, 138.5, 138.2, 137.0, 135.2, 130.6, 130.5, 127.4, 126.5, 125.4, 122.7, 122.5, 122.0, 121.1, 115.3, 115.2, 113.1, 109.7, 41.5, 36.1, 34.0, 26.7, 24.5, 23.5. Me O HO N N S HN N N N 1-(2-(1H-tetrazol-5-yl)ethyl)-2-(2-(benzo[d]thiazol-2-yl)ethyl)-3-(4methylphen-ethyl)-1H-indole-5-carboxylic acid (2.79). Compound 2.79 was prepared with 2.71 (85 mg crude product) following general procedure B11 to obtain the product as 155 a yellow solid (69 mg, 75% over two steps). Rf = 0.5 (10:1 CH2Cl2/MeOH). 1H NMR (500 MHz, DMSO-d6): δ 12.50 (brs, 2H), 8.14 (d, J = 1.5 Hz, 1H), 8.04 (d, J = 8.0 Hz, 1H), 7.94 (d, J = 8.0 Hz, 1H), 7.72 (dd, J = 1.0, 8.5 Hz, 1H), 7.45 (q, J = 8.0 Hz, 2H), 7.39 (t, J = 8.0 Hz, 1H), 7.02 (d, J = 8.0 Hz, 2H), 6.96 (d, J = 8.0 Hz, 2H), 4.60 (t, J = 7.5 Hz, 2H), 3.30 (t, J = 7.5 Hz, 2H), 3.19 – 3.12 (m, 2H), 3.12 – 3.05 (m, 2H), 2.90 (t, J = 7.5 Hz, 2H), 2.72 (t, J = 7.5 Hz, 2H), 2.23 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ 169.8, 168.3, 152.7, 138.5, 138.0, 136.4, 134.7, 134.7, 128.8, 128.2, 128.2, 127.0, 126.0, 124.9, 122.2, 122.1, 122.0, 121.5, 120.6, 112.8, 109.2, 41.0, 36.1, 33.5, 26.5, 24.1, 23.0, 20.6. O HO N N S HN N N N 1-(2-(1H-tetrazol-5-yl)ethyl)-2-(2-(benzo[d]thiazol-2-yl)ethyl)-3-phen-ethyl1H-indole-5-carboxylic acid (2.80). Compound 2.80 was prepared with 2.72 (60 mg crude product) following general procedure B11 to obtain the product as a yellow solid (49 mg, 59% over two steps). Rf = 0.5 (10:1 CH2Cl2/MeOH). 1H NMR (500 MHz, DMSO-d6): δ 8.16 (s, 1H), 8.04 (d, J = 8.5 Hz, 1H), 7.94 (d, J = 7.5 Hz, 1H), 7.72 (d, J = 9.0 Hz, 1H), 7.51 – 7.44 (m, 2H), 7.40 (t, J = 8.0 Hz, 1H), 7.22 (t, J = 7.5 Hz, 2H), 7.15 (t, J = 7.5 Hz, 1H), 7.08 (d, J = 7.0 Hz, 2H), 4.60 (t, J = 7.5 Hz, 2H), 3.12 (s, 4H), 2.92 (t, J = 7.5 Hz, 2H), 2.76 (t, J = 7.5 Hz, 2H). 13 C NMR (125 MHz, DMSO-d6): δ 170.3, 168.7, 153.1, 142.1, 138.5, 136.9, 135.2, 128.8, 128.7, 127.5, 126.5, 126.3, 125.4, 122.7, 122.6, 122.5, 121.0, 113.3, 110.0, 109.7, 41.4, 37.0, 34.0 26.8, 24.3, 23.5. 156 Cl O HO N N S O HO 2-(2-(Benzo[d]thiazol-2-yl)ethyl)-1-(2-carboxyethyl)-3-(4-chlorophen-ethyl)1H-indole-5-carboxylic acid (2.81). Compound 2.81 was prepared with 2.57 (311 mg, 0.59 mmol) following general procedure B11 to obtain the product as a white solid (305 mg, 99%). Rf = 0.1 (1:1 hexanes/EtOAc). 1H NMR (500 MHz, DMSO-d6): δ 12.43 (brs, 2H), 8.12 (s, 1H), 8.03 (d, J = 8.0 Hz, 1H), 7.95 (d, J = 8.5 Hz, 1H), 7.72 (d, J = 8.5 Hz, 1H), 7.53 – 7.46 (m, 2H), 7.40 (t, J = 7.5 Hz, 1H), 7.26 (dd, J = 2.0, 8.5 Hz, 2H), 7.10 (dd, J = 2.5, 8.5 Hz, 2H), 4.42 (t, J = 7.0 Hz, 2H), 3.23 (t, J = 7.5 Hz, 2H), 3.17 – 3.12 (m, 2H), 2.91 (t, J = 7.5 Hz, 2H), 2.75 (t, J = 7.5 Hz, 2H), 2.66 (t, J = 7.5 Hz, 2H). 13C NMR (125 MHz, DMSO-d6): δ 172.6, 170.3, 168.8, 153.2, 141.1, 138.5, 137.1, 135.2, 130.9, 130.7, 128.5, 127.4, 126.5, 125.4, 122.7, 122.6, 122.5, 121.8, 121.0, 112.7, 109.9, 36.2, 34.8, 34.0, 26.5, 23.6. Cl O HO N N N H HO O 2-(2-(1H-benzo[d]imidazol-2-yl)ethyl)-1-(2-carboxyethyl)-3-(4-chlorophenethyl)-1H-indole-5-carboxylic acid (2.82). Compound 2.82 was prepared with 2.60 (150 mg, 0.29 mmol) following general procedure B11 to obtain the product as a white solid (150 mg, 99%). Rf = 0.1 (3:2 hexanes/acetone). 1H NMR (500 MHz, DMSO-d6): δ 157 12.45 (brs, 1H), 8.12 (s, 1H), 7.79 (dd, J = 3.0, 6.0 Hz, 2H), 7.74 (ddd, J = 1.5, 4.0, 8.5 Hz, 1H), 7.54 – 7.50 (m, 3H), 7.29 – 7.09 (m, 5H), 4.48 (t, J = 7.5 Hz, 2H), 3.21 (t, J = 7.5 Hz, 2H), 2.94 – 2.88 (m, 2H), 2.77 – 2.70 (m, 2H), 2.68 (t, J = 7.5 Hz, 2H). 13C NMR (125 MHz, DMSO-d6): δ 172.6, 168.7, 153.3, 142.0, 141.0, 138.6, 138.6, 135.8, 131.5, 130.8, 128.8, 128.5, 128.4, 127.3, 126.3, 125.8, 122.8, 121.9, 121.1, 114.2, 113.5, 113.2, 110.1, 37.1, 36.4, 34.9, 27.4, 26.3, 21.8. F O HO N N S HO O 2-(2-(Benzo[d]thiazol-2-yl)ethyl)-1-(2-carboxyethyl)-3-(4-fluorophen-ethyl)1H-indole-5-carboxylic acid (2.83). Compound 2.83 was prepared with 2.61 (120 mg, 0.23 mmol) following general procedure B11 to obtain the product as a white solid (109 mg, 90%). Rf = 0.1 (1:1 hexanes/EtOAc). 1H NMR (500 MHz, DMSO-d6): δ 12.49 (brs, 2H), 8.11 (s, 1H), 8.04 (d, J = 8.0 Hz, 1H), 7.95 (d, J = 7.5 Hz, 1H), 7.72 (d, J = 8.5 Hz, 1H), 7.52 – 7.47 (m, 2H), 7.40 (t, J = 7.5 Hz, 1H), 7.13 – 7.07 (m, 2H), 7.02 (t, J = 8.5 Hz, 2H), 4.41 (t, J = 7.0 Hz, 2H), 3.25 – 3.19 (m, 2H), 3.18 – 3.13 (m, 2H), 2.91 (t, J = 7.5 Hz, 2H), 2.75 (d, J = 7.5 Hz, 2H), 2.66 (d, J = 7.5 Hz, 2H). 13C NMR (125 MHz, DMSO-d6): δ 172.3, 170.0, 168.4, 152.7, 138.1, 137.8, 136.7, 134.8, 130.2, 130.1, 127.0, 126.1, 125.0, 122.3, 122.2, 121.4, 120.6, 115.0, 114.8, 112.4, 109.5, 35.7, 34.4, 33.6, 26.4, 23.2. 158 Me O HO N N S O HO 2-(2-(Benzo[d]thiazol-2-yl)ethyl)-1-(2-carboxyethyl)-3-(4-methylphen-ethyl)1H-indole-5-carboxylic acid (2.84). Compound 2.84 was prepared with 2.62 (120 mg, 0.23 mmol) following general procedure B11 to obtain the product as a white solid (95 mg, 78%). Rf = 0.5 (15:1 CH2Cl2/MeOH). 1H NMR (500 MHz, DMSO-d6): δ 8.14 (s, 1H), 8.04 (d, J = 8.0 Hz, 1H), 7.95 (d, J = 8.0 Hz, 1H), 7.72 (dd, J = 1.5, 8.5 Hz, 1H), 7.52 – 7.45 (m, 2H), 7.42 – 7.37 (m, 1H), 7.02 (d, J = 8.0 Hz, 2H), 6.96 (d, J = 7.5 Hz, 2H), 4.40 (t, J = 7.0 Hz, 2H), 3.23 (t, J = 7.5 Hz, 2H), 3.09 (t, J = 7.5 Hz, 2H), 2.89 (t, J = 7.5 Hz, 2H), 2.72 (t, J = 7.5 Hz, 2H), 2.63 (t, J = 7.5 Hz, 2H), 2.23 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ 172.4, 170.0, 168.4, 152.7, 138.6, 138.0, 136.6, 134.8, 134.7, 128.8, 128.8, 128.2, 126.9, 126.1, 124.92, 122.2, 122.18, 122.16, 121.4, 120.5, 112.5, 109.4, 36.2, 34.7, 33.6, 26.6, 23.2, 23.2, 20.7. Me O HO N N N H HO O 2-(2-(1H-benzo[d]imidazol-2-yl)ethyl)-1-(2-carboxyethyl)-3-(4-methyl-phenethyl)-1H-indole-5-carboxylic acid (2.85). Compound 2.85 was prepared with 2.63 (60 mg, 0.12 mmol) following general procedure B11 to obtain the product as a white solid (29 mg, 43%). Rf = 0.2 (10:1 CH2Cl2/MeOH). 1H NMR (500 MHz, DMSO-d6): δ 15.04 (brs, 159 1H), 12.48 (brs, 1H), 8.11 (s, 1H), 7.80 (dd, J = 3.0, 6.0 Hz, 2H), 7.74 (dd, J = 1.5, 8.5 Hz, 1H), 7.55 – 7.50 (m, 3H), 7.05 (d, J = 8.0 Hz, 2H), 7.01 (d, J = 7.5 Hz, 2H), 4.47 (t, J = 7.5 Hz, 2H), 3.19 (t, J = 7.5 Hz, 2H), 2.89 (t, J = 7.5 Hz, 2H), 2.72 – 2.63 (m, 4H), 2.20 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ 172.2, 169.1, 168.2, 152.9, 138.5, 138.1, 135.3, 134.7, 131.00, 130.99, 128.7, 128.6, 128.2, 126.9, 125.4, 121.4, 120.7, 113.8, 113.1, 36.3, 34.4, 27.0, 26.0, 23.3, 21.3, 20.6. O HO N N S HO O 2-(2-(Benzo[d]thiazol-2-yl)ethyl)-1-(2-carboxyethyl)-3-phenethyl-1H-indole-5 carboxylic acid (2.86). Compound 2.86 was prepared with 2.64 (90 mg, 0.18 mmol) following general procedure B11 to obtain the product as a white solid (125 mg, 99%). Rf = 0.2 (40:1 CH2Cl2/MeOH). 1H NMR (500 MHz, DMSO-d6): δ 8.12 (s, 1H), 7.99 (d, J = 8.0 Hz, 1H), 7.91 (d, J = 8.5 Hz, 1H), 7.68 (d, J = 8.0 Hz, 1H), 7.44 (d, J = 7.5 Hz, 1H), 7.41 (d, J = 9.0 Hz, 1H), 7.35 (t, J = 8.0 Hz, 1H), 7.18 (t, J = 7.5 Hz, 2H), 7.11 (t, J = 7.0 Hz, 1H), 7.04 (d, J = 7.5 Hz, 2H), 4.33 (t, J = 7.0 Hz, 2H), 3.17 (t, J = 7.5 Hz, 2H), 3.08 (t, J = 7.5 Hz, 2H), 2.87 (t, J = 7.5 Hz, 2H), 2.71 (t, J = 7.5 Hz, 2H). 13C NMR (125 MHz, DMSO-d6): δ 170.4, 153.2, 142.2, 138.4, 136.9, 135.3, 128.7, 128.6, 127.3, 126.5, 126.2, 125.3, 122.7, 122.6, 122.6, 122.5, 120.8, 112.7, 109.7, 37.1, 36.4, 34.1, 26.9, 23.8. 160 General procedure B12 for the functionalization of indole-5-carboxylic acid R R O O HO N N S HN X XH CDI, DBU, THF R+ reflux - r.t. R N N R S N N N HN N N N R X = O, NH A 50 mL oven-dried round bottom flask was charged with 0.28 mmol of an indole5-carboxylic acid, CDI (136.7 mg, 0.85 mmol) and 10 mL THF. The reaction mixture was refluxed until the carboxylic acid was consumed as indicated by TLC. The reaction mixture was cooled down to room temperature and 0.99 mmol of benzyl amine or benzyl alcohol was added via syringe, followed by DBU (0.99 mL, 0.99 mmol). The reaction mixture was stirred for 24 h and quenched with 15 mL of sat. aq. NH4Cl solution. The crude product was extracted twice with 10 mL CH2Cl2. The organic solvent was concentrated under reduced pressure and the crude product was purified via flash column chromatography to obtain the desired product. F O N H N N F S HN N N N 1-(2-(1H-tetrazol-5-yl)ethyl)-2-(2-(benzo[d]thiazol-2-yl)ethyl)-N-(4-fluorobenzyl) -3-(4-fluorophenethyl)-1H-indole-5-carboxamide (2.87). Compound 2.87 was prepared with 2.78 (153 mg, 0.28 mmol) following general procedure B12. The crude product was purified via flash column chromatography (30:1 CH2Cl2/MeOH, followed by 161 20:1 CH2Cl2/MeOH) to obtain the product as a white solid (50 mg, 27%). Rf = 0.3 (20:1 CH2Cl2/MeOH). 1H NMR (500 MHz, acetone-d6): δ 8.23 (s, 2H), 7.96 (d, J = 7.5 Hz, 1H), 7.94 (d, J = 8.0 Hz, 1H), 7.78 (dd, J = 1.5, 8.5 Hz, 1H), 7.51 – 7.36 (m, 5H), 7.14 – 7.09 (m, 2H), 7.06 (d, J = 8.5 Hz, 1H), 6.97 (t, J = 9.0 Hz, 2H), 4.74 (t, J = 7.0 Hz, 2H), 4.61 (s, 2H), 3.48 (t, J = 7.5 Hz, 2H), 3.22 (s, 4H), 2.97 (t, J = 7.5 Hz, 2H), 2.84 (t, J = 7.5 Hz, 2H). 13 C NMR (125 MHz, acetone-d6): δ 169.8, 167.5, 162.7, 162.2, 160.8, 160.3, 153.9, 153.3, 138.1, 138.1, 137.8, 136.3, 135.3, 130.2, 130.1, 129.5, 129.4, 127.6, 126.0, 124.9, 122.4, 121.7, 120.8, 118.1, 114.9, 114.8, 114.7, 114.6, 113.0, 109.0, 42.4, 41.3, 35.8, 33.8, 26.6, 24.3, 23.3. F O N H N N Cl S HN N N N 1-(2-(1H-tetrazol-5-yl)ethyl)-2-(2-(benzo[d]thiazol-2-yl)ethyl)-N-(4-chlorobenzyl)-3-(4-fluorophenethyl)-1H-indole-5-carboxamide (2.88). Compound 2.88 was prepared with 2.78 (66 mg, 0.12 mmol) following general procedure B12. The crude product was purified via flash column chromatography (30:1 CH2Cl2/MeOH, followed by 20:1 CH2Cl2/MeOH) to obtain the product as a white solid (20 mg, 25%). Rf = 0.5 (20:1 CH2Cl2/MeOH). 1H NMR (500 MHz, CD3CN): δ 7.94 (d, J = 1.0 Hz, 1H), 7.93 – 7.88 (m, 2H), 7.59 (dd, J = 1.5, 8.5 Hz, 1H), 7.55 (t, J = 6.0 Hz, 1H), 7.47 (ddd, J = 1.0, 7.0, 8.5 Hz, 1H), 7.40 – 7.31 (m, 4H), 7.28 (d, J = 8.5 Hz, 1H), 7.05 – 7.01 (m, 2H), 6.92 (t, J = 9.0 Hz, 2H), 4.58 – 4.50 (m, 4H), 3.33 (t, J = 7.0 Hz, 2H), 3.17 – 3.11 (m, 2H), 3.10 – 3.04 (m, 2H), 2.85 (t, J = 7.5 Hz, 2H), 2.73 (t, J = 7.5 Hz, 2H). 13C NMR (125 MHz, CD3CN): δ 162 170.2, 168.0, 162.1, 160.2, 153.8, 153.1, 138.9, 138.1, 138.1, 137.7, 136.4, 135.2, 132.0, 130.2, 130.1, 129.1, 128.3, 127.4, 126.1, 125.5, 125.0, 122.2, 121.9, 120.6, 117.9, 114.8, 114.6, 113.2, 109.1, 42.4, 41.3, 35.7, 33.7, 26.4, 24.2, 23.3. Cl O N H N N Cl O OMe S HN N N N 1-(2-(1H-tetrazol-5-yl)ethyl)-N-(4-chlorobenzyl)-3-(4-chlorophenethyl)-2-(2(5-(2-methoxyethoxy)benzo[d]thiazol-2-yl)-ethyl)-1H-indole-5-carboxamide (2.89). Compound 2.89 was prepared with 2.75 (63 mg, 0.10 mmol) following general procedure B12. The crude product was purified via flash column chromatography (20:1 CH2Cl2/MeOH, followed by 15:1 CH2Cl2/MeOH) to obtain the product as a white solid (22 mg, 30%). Rf = 0.4 (15:1 CH2Cl2/MeOH). 1H NMR (500 MHz, CD3CN): δ 7.93 (s, 1H), 7.73 (d, J = 8.5 Hz, 1H), 7.59 (dd, J = 1.5, 9.0 Hz, 1H), 7.54 (t, J = 6.9 Hz, 1H), 7.41 (d, J = 2.5 Hz, 1H), 7.38 – 7.30 (m, 4H), 7.28 (d, J = 8.5 Hz, 1H), 7.18 (d, J = 8.5 Hz, 2H), 7.03 – 6.97 (m, 2H), 4.56 – 4.50 (m, 4H), 4.16 – 4.11 (m, 2H), 3.73 – 3.68 (m, 2H), 3.36 (s, 3H), 3.32 (t, J = 7.5 Hz, 2H), 3.13 – 3.04 (m, 4H), 2.84 (t, J = 7.5 Hz, 2H), 2.71 (t, J = 7.5 Hz, 2H). 13 C NMR (125 MHz, CD3CN): δ 171.4, 168.0, 158.2, 154.4, 153.8, 141.0, 138.9, 137.7, 136.4, 132.0, 131.0, 130.2, 129.1, 128.3, 128.2, 128.1, 127.4, 127.0, 125.5, 122.2, 120.6, 117.9, 115.0, 113.1, 109.1, 105.92, 70.7, 67.7, 58.1, 42.4, 41.3, 35.8, 33.7, 26.1, 24.2, 23.4. 163 Cl O N H N OMe N Cl S HN N N N 1-(2-(1H-tetrazol-5-yl)ethyl)-N-(4-chlorobenzyl)-3-(4-chlorophenethyl)-2-(2(5-methoxybenzo[d]thiazol-2-yl)ethyl)-1H-indole-5-carboxamide (2.90). Compound 2.90 was prepared with 2.74 (90 mg, 0.15 mmol) following general procedure B12. The crude product was purified via flash column chromatography (20:1 CH2Cl2/MeOH) to obtain the product as a white solid (37 mg, 36%). Rf = 0.3 (20:1 CH2Cl2/MeOH). 1H NMR (500 MHz, acetone-d6): δ 8.31 (t, J = 6.0 Hz, 1H), 8.22 (d, J = 1.0 Hz, 1H), 7.82 – 7.74 (m, 2H), 7.47 – 7.42 (m, 2H), 7.41 (d, J = 8.5 Hz, 2H), 7.32 (d, J = 8.5 Hz, 2H), 7.22 (d, J = 8.5 Hz, 2H), 7.08 (d, J = 8.5 Hz, 2H), 7.01 (dd, J = 2.5, 8.5 Hz, 1H), 4.72 (t, J = 7.0 Hz, 2H), 4.61 (d, J = 5.5 Hz, 2H), 3.86 (s, 3H), 3.47 (t, J = 7.5 Hz, 2H), 3.24 – 3.20 (m, 2H), 3.18 – 3.15 (m, 2H), 2.95 (t, J = 7.5 Hz, 2H), 2.80 (t, J = 7.5 Hz, 2H). 13C NMR (125 MHz, DMSO-d6): δ 171.4, 167.6, 158.9, 154.5, 141.2, 139.7, 137.7, 136.6, 131.6, 130.9, 130.7, 129.5, 128.5, 127.3, 126.9, 125.6, 122.8, 121.1, 118.4, 114.9, 112,7, 109.5, 105.7, 55.9, 42.5, 41.6, 36.0, 34.1, 26.5, 24.7, 23.6. 164 Cl O O N OMe N Cl S HN N N N 4-Chlorobenzyl-1-(2-(1H-tetrazol-5-yl)ethyl)-3-(4-chlorophenethyl)-2-(2-(5methoxybenzo[d]thiazol-2-yl)ethyl)-1H-indole-5-carboxylate (2.91). Compound 2.91 was prepared with 2.74 (80 mg, 0.14 mmol) following general procedure B12. The crude product was purified via flash column chromatography (30:1 CH2Cl2/MeOH, followed by 15:1 CH2Cl2/MeOH) to obtain the product as a white solid (39 mg, 40%). Rf = 0.6 (10:1 CH2Cl2/MeOH). 1H NMR (500 MHz, DMSO-d6) δ 8.09 (s, 1H), 7.89 (d, J = 8.5 Hz, 1H), 7.73 (d, J = 8.5 Hz, 1H), 7.54 – 7.44 (m, 6H), 7.23 (d, J = 8.0 Hz, 2H), 7.06 (d, J = 8.5 Hz, 2H), 7.04 – 7.00 (m, 1H), 5.35 (s, 2H), 4.59 (t, J = 7.0 Hz, 2H), 3.82 (s, 3H), 3.15 (s, 4H), 2.90 (t, J = 7.5 Hz, 2H), 2.72 (t, J = 7.5 Hz, 2H). 13C NMR (125 MHz, acetone-d6) δ 170.8, 166.6, 159.1, 154.7, 140.9, 138.8, 136.9, 136.1, 133.2, 131.1, 130.2, 129.7, 128.5, 128.1, 127.6, 126.9, 122.4, 121.9, 121.1, 120.9, 114.7, 113.3, 109.2, 105.2, 64.9, 55.0, 41.5, 36.1, 33.8, 26.3, 24.5, 23.4. 165 O MeO N 2.13 9.0 190 8.5 180 8.0 170 7.5 160 7.0 150 6.5 140 6.0 130 5.5 120 5.0 110 4.5 4.0 f1 (ppm) 100 90 f1 (ppm) 2.00 2.89 2.00 0.97 1.13 0.99 0.96 0.87 CN 3.5 80 3.0 70 2.5 60 2.0 50 1.5 40 1.0 30 0.5 20 10 0.0 -0.5 0 166 O O H MeO N 10.5 10.0 9.5 9.0 200 180 170 190 8.5 160 8.0 150 7.5 140 7.0 130 6.5 120 6.0 5.5 110 5.0 4.5 f1 (ppm) 100 90 f1 (ppm) 4.0 80 2.33 3.01 2.05 1.03 0.98 0.95 0.81 CN 0.84 2.14 3.5 70 3.0 60 2.5 2.0 1.5 1.0 0.5 0.0 50 40 30 20 10 0 -0.5 -10 167 Cl O MeO N 2.15 9.0 180 8.5 170 8.0 160 7.5 150 7.0 140 6.5 130 6.0 120 2.31 2.21 2.18 3.01 2.18 1.26 1.99 1.90 1.03 1.01 1.00 CN 5.5 5.0 4.5 4.0 f1 (ppm) 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 110 100 90 80 f1 (ppm) 70 60 50 40 30 20 10 0 168 Me O MeO N 2.16 9.0 260 8.5 240 8.0 220 7.5 200 7.0 180 160 6.0 5.5 140 5.0 120 4.5 100 f1 (ppm) 4.0 80 3.5 3.0 60 2.97 2.06 1.99 1.95 f1 (ppm) 6.5 2.98 2.00 1.25 3.95 0.98 0.99 0.93 CN 2.5 40 2.0 20 1.5 0 1.0 -20 0.5 0.0 -40 -60 169 F O MeO N 2.17 9.0 190 8.5 180 8.0 170 7.5 160 150 7.0 140 6.5 130 6.0 120 5.5 110 5.0 4.5 4.0 f1 (ppm) 100 90 f1 (ppm) 80 2.01 4.19 2.96 2.00 1.05 1.97 1.93 0.98 0.93 0.86 CN 3.5 3.0 2.5 2.0 70 60 50 40 1.5 30 1.0 20 0.5 10 0.0 0 -10 170 O MeO N 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 f1 (ppm) 180 170 160 150 140 130 120 110 100 90 f1 (ppm) 80 3.5 70 2.08 4.16 2.98 2.00 3.06 3.04 0.97 0.99 CN 0.94 2.18 3.0 60 2.5 50 2.0 40 1.5 30 1.0 20 0.5 10 0.0 0 171 O O O MeO N 2.19 9.0 8.5 8.0 7.5 7.0 6.5 6.0 180 170 160 150 140 130 120 5.5 110 5.0 100 4.5 4.0 f1 (ppm) 90 80 f1 (ppm) 2.05 2.10 2.12 3.06 2.00 1.98 0.96 0.97 0.93 1.00 1.15 0.96 0.92 CN 3.5 70 3.0 60 2.5 50 2.0 40 1.5 30 1.0 20 0.5 10 0.0 0 172 Cl O HO N 2.20 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.30 2.13 2.00 0.99 1.81 1.83 1.01 0.94 0.86 CN 4.5 4.0 f1 (ppm) 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 1 90 180 170 160 150 140 130 120 110 100 90 f1 (ppm) 80 70 60 50 40 30 20 10 0 -0.5 173 Me O HO N 170 8.0 160 2.93 6.14 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 f1 (ppm) 3.5 3.0 2.5 2.0 1.5 1.0 140 130 120 110 100 90 80 f1 (ppm) 70 60 50 40 30 20 150 2.00 1.03 3.82 180 8.5 1.01 9.0 0.96 CN 0.95 2.21 0.5 10 0.0 0 174 F O HO N 2.22 9.0 170 8.5 160 8.0 150 7.5 140 7.0 130 6.5 120 6.0 110 5.5 100 5.0 90 4.5 4.0 f1 (ppm) 80 70 f1 (ppm) 6.23 2.00 0.97 1.01 2.77 1.91 0.83 CN 3.5 60 3.0 50 2.5 40 2.0 30 1.5 20 1.0 10 0.5 0 0.0 -10 175 O O O HO N 9.5 170 9.0 8.5 160 8.0 150 7.5 140 7.0 6.5 130 120 6.0 110 5.5 100 5.0 4.5 4.0 f1 (ppm) 90 80 f1 (ppm) 2.21 4.21 2.00 2.12 1.89 1.02 1.02 1.04 1.04 CN 1.05 2.23 3.5 70 3.0 60 2.5 2.0 50 40 1.5 30 1.0 20 0.5 0.0 10 0 176 Cl O Cl S S O O N H N Cl 6.16 2.00 4.92 CN 0.57 1.01 1.01 1.03 2.25 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 f1 (ppm) 4.0 3.5 3.0 2.5 170 160 150 140 130 120 110 100 90 80 f1 (ppm) 70 60 50 40 2.0 30 1.5 20 1.0 10 0.5 0 0.0 -10 177 F O Cl S S O O N H N Cl 8.5 180 8.0 170 7.5 160 7.0 150 6.5 140 6.0 130 5.5 120 5.0 110 100 4.5 4.0 f1 (ppm) 90 80 f1 (ppm) 6.54 2.00 1.92 2.93 CN 0.61 0.96 0.96 0.90 2.26 3.5 70 3.0 60 2.5 50 2.0 40 1.5 30 20 1.0 10 0.5 0 0.0 -10 178 O O O Cl S O O S N H N Cl 2.27 10.0 180 9.5 170 9.0 160 8.5 150 8.0 140 7.5 7.0 130 120 6.5 110 6.0 100 5.5 5.0 f1 (ppm) 90 80 f1 (ppm) 4.62 2.40 2.36 2.38 2.19 1.01 2.30 2.00 1.13 1.08 1.10 CN 4.5 4.0 70 3.5 60 3.0 50 2.5 40 2.0 1.5 1.0 0.5 30 20 10 0 0.0 -10 179 Cl O S O O N H N F 2.28 9.0 170 8.5 160 8.0 7.5 7.0 6.5 150 140 130 120 6.0 110 5.5 100 5.0 4.5 f1 (ppm) 90 80 f1 (ppm) 6.26 2.00 5.01 0.90 0.94 4.05 0.97 CN 4.0 3.5 70 3.0 60 2.5 50 2.0 40 1.5 30 1.0 20 0.5 10 0.0 0 180 F O S O O N H N F 180 9.5 170 9.0 160 8.5 150 8.0 140 7.5 7.0 130 6.5 120 6.0 110 5.5 100 5.0 4.5 f1 (ppm) 90 80 f1 (ppm) 6.21 2.00 0.94 10.0 0.91 0.92 4.05 2.98 1.93 CN 2.29 4.0 3.5 70 3.0 60 2.5 50 2.0 1.5 40 30 1.0 20 0.5 10 0.0 0 181 O O O S O O N H N F 10.0 180 9.5 170 9.0 160 8.5 150 8.0 7.5 140 130 7.0 120 6.5 110 6.0 100 5.5 5.0 f1 (ppm) 90 f1 (ppm) 4.5 80 6.15 2.00 2.06 0.96 1.00 0.95 0.91 1.05 4.11 0.95 CN 0.93 2.30 4.0 3.5 70 60 3.0 50 2.5 40 2.0 30 1.5 1.0 20 0.5 10 0.0 0 182 Cl O S S O O N H N Cl 8.5 190 8.0 180 170 7.5 160 7.0 150 6.5 140 6.0 130 5.5 120 4.06 2.00 CN 5.97 1.27 1.18 0.99 0.99 2.31 5.0 f1 (ppm) 4.5 4.0 3.5 110 100 90 f1 (ppm) 80 3.0 70 2.5 60 2.0 50 1.5 40 1.0 30 0.5 20 0.0 10 0 183 Me O Cl S S O O N H N Cl 9.0 170 8.5 160 8.0 150 7.5 140 7.0 6.5 130 6.0 120 5.5 110 5.0 4.5 4.0 f1 (ppm) 100 90 f1 (ppm) 3.5 80 3.0 70 3.07 6.36 2.00 CN 1.01 0.79 1.02 0.96 2.18 1.99 0.91 2.32 2.5 60 2.0 1.5 50 40 1.0 30 0.5 0.0 20 -0.5 10 184 O O O S S O O N H N Cl 2.33 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 f1 (ppm) 6.48 2.00 2.11 1.01 1.02 1.06 1.03 2.07 0.99 1.02 0.99 CN 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0 180 170 160 150 140 130 120 110 100 90 80 f1 (ppm) 70 60 50 40 30 20 10 0 -1 185 Cl O Cl S S O O N H N Cl 10.0 180 170 9.5 9.0 160 8.5 150 8.0 140 7.5 130 7.0 120 6.5 6.0 110 5.5 100 5.0 4.5 f1 (ppm) 90 80 f1 (ppm) 4.0 70 3.5 4.35 2.67 0.90 0.71 0.93 0.96 1.94 1.77 1.03 N N N 2.00 HN 2.34 3.0 60 2.5 50 2.0 1.5 40 30 1.0 20 0.5 10 0.0 -0.5 0 186 F O Cl S S O O N H N Cl 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 2.80 2.31 2.98 N N N 2.54 HN 0.88 0.76 1.00 0.98 2.19 3.27 2.35 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 80 70 60 50 40 30 20 10 0 f1 (ppm) f1 (ppm) 180 170 160 150 140 130 120 110 100 90 187 O O O Cl S S O O N H N Cl 9.5 180 9.0 170 8.5 8.0 7.5 7.0 6.5 160 150 140 130 120 6.0 110 5.5 100 5.0 4.5 f1 (ppm) 90 80 f1 (ppm) 4.0 3.5 70 2.09 2.14 2.11 2.04 2.06 0.91 0.75 0.97 0.97 0.98 N N N 0.97 1.00 1.00 HN 2.36 3.0 60 2.5 50 2.0 40 1.5 30 1.0 20 0.5 10 0.0 0 188 Cl O S O O N H N F 10.0 180 9.5 170 9.0 160 8.5 150 8.0 140 7.5 130 7.0 6.5 120 6.0 110 5.5 100 5.0 4.5 f1 (ppm) 90 80 f1 (ppm) 4.0 3.5 70 4.21 2.29 0.91 0.93 0.93 1.04 1.95 0.98 2.04 1.00 1.96 N N N 1.97 HN 2.37 3.0 60 2.5 50 2.0 40 1.5 30 1.0 0.5 20 0.0 10 -0.5 0 189 F O S O O N H N F 170 160 9.0 150 8.5 140 8.0 130 7.5 120 7.0 6.5 110 6.0 100 5.5 5.0 4.5 f1 (ppm) 90 80 f1 (ppm) 4.0 70 4.37 9.5 2.07 10.0 2.07 0.92 N N N 0.90 0.89 2.00 1.95 2.01 0.96 1.91 HN 2.38 3.5 3.0 60 50 2.5 2.0 40 1.5 30 1.0 20 0.5 10 0.0 0 190 O O O S O O N H N F 10.0 190 9.5 180 9.0 170 8.5 8.0 7.5 160 150 140 7.0 130 6.5 120 6.0 5.5 110 5.0 4.5 f1 (ppm) 4.0 3.5 100 90 f1 (ppm) 80 70 2.07 2.07 4.82 2.00 N N N 0.87 0.93 0.95 1.04 2.07 1.05 1.00 0.92 1.13 1.03 HN 2.21 2.39 3.0 60 2.5 50 2.0 1.5 40 1.0 30 0.5 20 0.0 10 -0.5 0 191 Cl O S S O O N H N Cl 10.0 9.5 9.0 180 170 160 8.5 150 8.0 140 7.5 130 7.0 6.5 120 6.0 110 5.5 100 5.0 f1 (ppm) 90 f1 (ppm) 4.5 80 4.0 70 3.5 60 4.23 3.35 2.00 N N N 0.92 HN 0.93 1.03 1.00 1.95 3.04 0.88 2.40 3.0 2.5 50 2.0 40 1.5 30 1.0 20 0.5 0.0 10 0 192 Me O Cl S S O O N H N Cl 9.5 180 9.0 170 8.5 8.0 160 7.5 150 7.0 140 6.5 130 6.0 5.5 5.0 4.5 f1 (ppm) 120 110 100 90 f1 (ppm) 4.0 80 3.5 70 3.0 60 2.97 4.26 2.12 2.06 N N N 0.96 HN 0.75 0.99 0.99 1.00 2.08 2.13 2.41 2.5 2.0 50 1.5 40 1.0 30 0.5 20 0.0 10 -0.5 0 193 O O O S O O S N H N Cl 9.0 180 8.5 170 8.0 160 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 f1 (ppm) 150 140 130 120 110 100 90 80 f1 (ppm) 3.5 70 1.98 1.92 2.18 2.04 2.14 0.91 N N N 0.91 0.97 1.00 0.90 0.97 0.96 1.09 1.03 HN 2.42 3.0 60 2.5 50 2.0 40 1.5 30 1.0 0.5 20 0.0 10 -0.5 0 194 Cl O MeO I N 2.43 9.0 180 8.5 170 8.0 160 7.5 150 7.0 140 2.18 2.20 2.10 2.96 2.08 1.07 4.09 2.17 1.03 0.92 CN 6.5 6.0 5.5 5.0 4.5 4.0 f1 (ppm) 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 130 120 110 100 90 80 f1 (ppm) 70 60 50 40 30 20 10 0 195 Me O MeO I N 2.44 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 f1 (ppm) 3.5 3.0 3.01 2.18 2.20 2.37 3.04 2.11 3.92 1.12 1.04 0.94 CN 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 196 F O MeO I N 9.5 9.0 190 180 8.5 170 8.0 160 7.5 150 7.0 140 6.5 130 120 6.0 110 5.5 100 2.05 2.07 2.04 2.90 2.00 1.01 2.01 1.99 0.97 CN 0.95 2.45 5.0 4.5 f1 (ppm) 4.0 3.5 90 80 f1 (ppm) 70 60 3.0 50 2.5 40 2.0 30 1.5 20 1.0 10 0.5 0 -10 0.0 -20 197 O MeO I N 2.46 9.5 200 9.0 190 8.5 180 8.0 170 7.5 160 7.0 6.5 6.0 5.5 5.0 150 140 130 120 110 4.5 4.0 f1 (ppm) 100 90 f1 (ppm) 2.00 2.02 2.00 2.74 1.94 6.17 0.94 0.90 CN 3.5 80 3.0 70 2.5 60 2.0 50 1.5 40 30 1.0 20 0.5 10 0.0 -0.5 0 -10 198 MeO N S2.2 S 9.0 200 8.5 190 8.0 180 7.5 170 160 3.00 0.95 0.97 0.97 I 7.0 150 6.5 140 6.0 130 5.5 120 5.0 110 4.5 4.0 f1 (ppm) 3.5 3.0 2.5 2.0 100 90 f1 (ppm) 80 70 60 50 1.5 40 1.0 30 0.5 20 0.0 10 -0.5 0 199 HO N S2.3 S 1.00 0.90 1.01 I 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 f1 (ppm) 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 180 170 160 150 140 130 120 110 100 90 80 f1 (ppm) 70 60 50 40 30 20 10 0 -10 200 O N I S2.4 9.0 180 8.5 170 8.0 160 7.5 150 7.0 140 6.5 130 6.0 120 5.5 110 5.0 100 4.5 4.0 f1 (ppm) 90 80 f1 (ppm) 2.96 2.05 2.06 1.00 0.94 S 0.99 MeO 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 70 60 50 40 30 20 10 0 201 Cl N S2.5 S 10.0 160 9.5 9.0 150 8.5 140 8.0 130 1.00 1.00 0.90 I 7.5 120 7.0 6.5 110 6.0 100 5.5 90 5.0 4.5 f1 (ppm) 4.0 80 f1 (ppm) 70 3.5 60 3.0 2.5 50 2.0 40 1.5 30 1.0 20 0.5 0.0 10 -0.5 0 202 Me Si iPr Me N S 160 8.5 8.0 7.5 150 140 19.15 2.06 9.0 0.98 0.96 S3.1 7.0 130 6.5 6.0 120 110 5.5 100 5.0 90 4.5 4.0 f1 (ppm) 80 f1 (ppm) 3.5 70 3.0 60 2.5 50 2.0 1.5 40 30 1.0 20 0.5 10 0.0 -0.5 0 203 9.0 160 8.5 8.0 150 7.5 140 2.98 1.03 0.98 S S3.2 19.04 Me Si iPr Me N 0.94 MeO 7.0 6.5 130 120 6.0 110 5.5 100 5.0 4.5 90 4.0 3.5 f1 (ppm) 3.0 80 70 f1 (ppm) 60 2.5 50 2.0 1.5 40 1.0 30 0.5 20 0.0 -0.5 10 0 -1.0 -10 204 O MeO Me Si iPr Me N S 9.0 180 8.5 170 8.0 160 7.5 150 7.0 140 6.5 130 6.0 120 5.5 110 5.0 100 4.5 4.0 f1 (ppm) 90 80 f1 (ppm) 18.38 2.76 1.93 1.93 0.93 1.00 0.98 S3.3 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 70 60 50 40 30 20 10 0 -0.5 -10 205 Cl Me Si iPr Me N S 1.00 1.15 1.24 19.51 S3.4 9.0 8.5 8.0 7.5 180 170 160 150 7.0 140 6.5 130 6.0 120 5.5 110 5.0 100 4.5 4.0 3.5 f1 (ppm) 90 80 f1 (ppm) 3.0 70 2.5 60 2.0 50 1.5 40 1.0 30 0.5 20 0.0 10 -0.5 -1.0 0 -10 206 Me Si iPr Me N 9.0 190 8.5 180 170 8.0 160 2.26 19.23 S3.5 2.00 N H 7.5 150 7.0 140 6.5 130 6.0 120 5.5 110 5.0 100 4.5 4.0 f1 (ppm) 3.5 3.0 2.5 2.0 90 80 f1 (ppm) 70 60 50 40 1.5 30 1.0 20 0.5 10 0.0 0 -0.5 -10 207 N S 10.0 180 9.5 9.0 8.5 170 160 150 8.0 140 0.82 0.87 0.88 2.00 S4.1 7.5 130 7.0 6.5 120 6.0 110 5.5 100 5.0 4.5 f1 (ppm) 4.0 90 80 f1 (ppm) 70 3.5 60 3.0 50 2.5 40 2.0 1.5 30 1.0 20 0.5 10 0.0 -0.5 0 -10 208 MeO N 9.0 180 8.5 170 8.0 160 7.5 150 7.0 140 6.5 6.0 5.5 5.0 4.5 4.0 f1 (ppm) 130 120 110 100 90 80 f1 (ppm) 0.91 3.27 1.02 1.00 1.05 S S4.2 3.5 70 3.0 60 2.5 50 2.0 40 1.5 1.0 30 0.5 20 0.0 10 -0.5 0 209 O MeO N S 9.0 210 200 8.5 190 8.0 180 7.5 7.0 170 160 6.5 150 6.0 140 5.5 130 5.0 120 2.14 0.87 2.95 2.04 1.01 1.02 0.97 S4.3 4.5 4.0 f1 (ppm) 110 100 90 f1 (ppm) 3.5 3.0 80 70 2.5 60 2.0 50 1.5 40 1.0 30 20 0.5 10 0.0 0 210 Cl N 13.5 190 180 12.5 170 11.5 160 10.5 150 140 9.5 130 8.5 120 7.5 110 0.77 1.84 2.00 0.71 S S4.4 6.5 f1 (ppm) 100 90 f1 (ppm) 5.5 80 4.5 70 60 3.5 50 2.5 40 1.5 30 20 0.5 10 -0.5 0 211 N 13.5 180 12.5 170 11.5 160 150 10.5 140 9.5 130 8.5 120 110 7.5 100 0.77 1.84 2.00 0.71 N H S4.5 6.5 f1 (ppm) 90 80 f1 (ppm) 5.5 70 4.5 60 3.5 50 2.5 40 30 1.5 20 0.5 10 -0.5 0 212 Cl O 2.47 10.0 190 180 9.5 170 9.0 160 8.5 150 8.0 140 7.5 7.0 130 6.5 120 6.0 110 5.5 100 5.0 4.5 f1 (ppm) 90 80 f1 (ppm) 4.0 70 2.00 2.05 2.01 CN 2.85 S 0.80 0.99 1.03 0.94 2.21 1.22 3.07 1.74 N 1.92 N MeO 3.5 60 3.0 50 2.5 40 2.0 30 1.5 1.0 0.5 0.0 20 10 0 -10 213 Cl O 2.48 10.5 10.0 9.5 9.0 190 170 160 180 8.5 150 8.0 140 7.5 130 7.0 120 6.5 6.0 110 5.5 100 5.0 4.5 f1 (ppm) 90 80 f1 (ppm) 4.0 70 2.04 2.07 1.71 CN 6.08 S 0.89 1.02 1.04 0.97 1.08 3.33 2.06 N 2.06 OMe N MeO 3.5 3.0 2.5 2.0 1.5 60 50 40 30 20 1.0 10 0.5 0 0.0 -10 -0.5 214 Cl O O N MeO N OMe S 0.89 1.03 1.05 1.11 1.15 3.23 1.95 10.0 180 9.5 9.0 170 160 8.5 150 8.0 140 7.5 130 7.0 1.97 1.96 2.94 2.12 3.05 2.37 1.99 2.11 2.49 CN 6.5 120 6.0 110 5.5 100 5.0 4.5 f1 (ppm) 4.0 90 80 f1 (ppm) 70 3.5 60 3.0 50 2.5 2.0 40 1.5 30 1.0 20 0.5 10 0.0 0 215 Cl O Cl N MeO N S 10.0 180 9.5 9.0 8.5 170 160 150 8.0 140 7.5 130 7.0 120 6.5 110 6.0 5.5 100 5.0 4.5 f1 (ppm) 90 80 f1 (ppm) 4.0 70 2.02 2.11 2.02 2.91 1.98 2.50 1.00 2.01 1.00 1.04 1.12 2.11 1.94 CN 3.5 3.0 2.5 60 50 40 2.0 30 1.5 20 1.0 10 0.5 0.0 0 -0.5 -10 216 Cl O N MeO N 2.21 2.19 2.01 2.85 1.94 0.83 1.00 0.97 0.97 1.98 1.25 2.08 1.73 2.51 0.83 CN N H 12.5 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 f1 (ppm) 190 180 170 160 150 140 130 120 110 100 90 f1 (ppm) 80 70 60 50 40 30 20 10 0 217 F O N MeO N 10.5 10.0 180 170 9.5 160 9.0 150 8.5 140 8.0 7.5 130 7.0 120 6.5 110 6.0 100 5.5 5.0 f1 (ppm) 4.5 90 80 f1 (ppm) 70 4.0 60 2.28 2.01 2.23 2.96 2.24 2.21 1.19 1.89 2.18 2.52 0.98 1.05 1.06 1.11 CN S 3.5 50 3.0 2.5 40 2.0 30 1.5 20 1.0 10 0.5 0.0 0 -10 218 Me O N MeO N S 10.0 180 9.5 170 9.0 8.5 160 150 8.0 140 7.5 130 7.0 6.5 120 6.0 110 5.5 100 5.0 4.5 f1 (ppm) 90 80 f1 (ppm) 4.0 70 3.5 60 3.0 2.91 2.01 2.03 2.03 2.90 1.98 2.09 1.06 4.05 2.53 0.97 2.00 0.98 CN 2.5 50 2.0 40 1.5 30 1.0 0.5 0.0 20 10 0 -0.5 -10 219 Me O N MeO N N H 11.0 10.5 10.0 9.5 180 170 160 9.0 150 8.5 140 8.0 130 7.5 120 7.0 110 6.5 6.0 100 5.5 5.0 f1 (ppm) 90 80 f1 (ppm) 4.5 70 4.0 3.5 60 3.0 50 2.5 40 2.87 1.99 2.02 1.97 2.86 1.89 0.95 0.98 2.03 2.26 1.02 3.82 2.54 1.46 CN 2.0 30 1.5 20 1.0 0.5 10 0.0 0 220 O N MeO S N 180 9.5 170 9.0 160 8.5 8.0 150 7.5 140 7.0 130 6.5 6.0 5.5 5.0 4.5 f1 (ppm) 120 110 100 90 80 f1 (ppm) 4.0 70 1.96 1.97 1.98 2.79 1.89 0.93 1.98 0.98 10.0 2.32 1.28 5.67 2.55 CN 3.5 3.0 60 2.5 50 2.0 40 1.5 30 1.0 0.5 0.0 20 10 0 -0.5 -10 221 Cl O MeO N N S CN 9.0 200 190 8.5 180 8.0 170 7.5 160 7.0 6.5 6.0 5.5 5.0 150 140 130 120 110 4.5 4.0 f1 (ppm) 100 90 f1 (ppm) 2.07 3.87 2.92 2.21 3.00 2.04 0.88 1.07 1.04 1.00 1.11 1.08 3.72 1.87 2.56 3.5 80 3.0 70 2.5 60 2.0 50 1.5 40 1.0 30 0.5 20 0.0 10 -0.5 0 -10 222 Cl O MeO OMe N N S CN 10.0 180 9.5 170 9.0 160 8.5 150 8.0 140 7.5 130 7.0 120 6.5 6.0 110 5.5 100 5.0 4.5 f1 (ppm) 90 80 f1 (ppm) 4.0 70 2.11 6.20 1.97 2.88 2.89 1.87 0.84 0.94 0.93 0.92 3.33 2.93 2.57 3.5 60 3.0 50 2.5 2.0 1.5 1.0 0.5 40 30 20 10 0 0.0 -10 223 Cl O MeO N O N OMe S CN 9.5 9.0 180 170 8.5 160 8.0 150 7.5 140 1.97 1.94 2.88 1.94 2.92 2.17 4.00 2.10 1.99 0.85 0.98 0.99 0.98 3.33 1.35 1.89 2.58 7.0 130 6.5 120 6.0 110 5.5 100 5.0 4.5 f1 (ppm) 90 80 f1 (ppm) 4.0 3.5 70 3.0 60 2.5 50 2.0 40 1.5 30 1.0 0.5 0.0 20 10 0 224 Cl O MeO Cl N N S CN 10.0 9.5 180 170 9.0 160 8.5 150 8.0 140 7.5 130 7.0 6.5 120 6.0 110 5.5 100 2.00 6.35 2.14 2.98 2.14 1.05 0.67 2.01 1.83 0.89 1.90 0.88 2.59 5.0 4.5 f1 (ppm) 4.0 3.5 90 80 f1 (ppm) 70 60 3.0 50 2.5 40 2.0 30 1.5 20 1.0 0.5 10 0.0 0 -10 225 Cl O MeO N N N H 12.0 11.5 11.0 10.5 10.0 9.5 180 170 160 150 140 9.0 8.5 130 8.0 120 7.5 110 7.0 6.5 100 6.0 5.5 f1 (ppm) 90 80 f1 (ppm) 5.0 70 4.5 4.0 60 2.34 1.96 4.31 3.64 2.93 2.00 1.00 1.00 1.31 1.72 2.09 4.34 0.77 CN 2.60 3.5 50 3.0 40 2.5 30 2.0 1.5 20 1.0 10 0.5 0 0.0 -0.5 -10 226 F O MeO N N S CN 9.0 210 200 8.5 8.0 190 180 7.5 170 7.0 160 6.5 150 140 6.0 130 5.5 5.0 120 110 2.08 4.17 1.84 1.91 2.88 1.90 0.93 1.97 1.02 0.98 1.06 1.19 2.03 2.09 2.61 4.5 4.0 f1 (ppm) 100 90 f1 (ppm) 3.5 80 3.0 70 60 2.5 50 2.0 1.5 40 30 1.0 20 0.5 10 0.0 0 -10 227 Me O MeO N N S 9.5 180 9.0 170 8.5 160 8.0 150 7.5 140 7.0 6.5 130 6.0 120 5.5 110 5.0 100 2.95 2.04 6.21 1.99 2.91 1.95 2.00 0.95 1.02 1.04 1.09 2.02 2.02 0.96 CN 2.62 4.5 4.0 f1 (ppm) 3.5 3.0 2.5 90 80 f1 (ppm) 70 60 50 2.0 40 1.5 30 1.0 20 0.5 0.0 10 -0.5 0 -10 228 Me O N MeO N H N 1.94 2.89 5.84 1.91 2.04 2.94 1.39 2.08 1.02 1.96 2.01 0.95 0.92 CN 2.63 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 f1 (ppm) 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 190 180 170 160 150 140 130 120 110 100 90 80 f1 (ppm) 70 60 50 40 30 20 10 0 -10 229 O MeO N N S CN 8.5 8.0 7.5 7.0 6.5 6.0 180 170 160 150 140 130 120 5.5 110 5.0 100 4.5 4.0 f1 (ppm) 90 80 f1 (ppm) 1.98 2.05 4.11 2.02 9.0 2.88 1.03 1.04 4.27 1.99 2.00 1.94 0.94 0.95 2.64 3.5 70 3.0 60 2.5 50 2.0 40 1.5 30 1.0 20 0.5 10 0.0 0 -10 230 Cl O MeO OMe N N S HN N N N 9.5 180 9.0 170 8.5 8.0 160 7.5 150 7.0 6.5 140 130 6.0 120 5.5 110 5.0 4.5 4.0 f1 (ppm) 100 90 f1 (ppm) 3.09 4.10 2.49 2.38 2.70 3.10 2.17 0.97 1.00 1.09 2.00 1.87 2.00 1.07 2.66 3.5 80 3.0 70 2.5 60 2.0 1.5 50 1.0 40 0.5 30 0.0 20 -0.5 231 Cl O MeO N N N H 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 f1 (ppm) 4.0 3.5 3.0 4.39 4.27 2.91 2.02 0.74 1.02 2.04 1.12 2.03 2.01 0.70 1.18 0.77 N HN N N 2.69 2.5 2.0 1.5 1.0 0.5 0.0 232 Cl O HO N N S HN N N N 13.5 180 12.5 170 160 11.5 150 10.5 140 9.5 130 8.5 120 110 7.5 100 6.5 f1 (ppm) 90 80 f1 (ppm) 5.5 3.94 2.20 2.21 2.21 0.87 0.92 0.96 0.99 2.18 1.07 1.76 2.15 0.75 2.73 4.5 70 60 3.5 50 2.5 40 1.5 30 0.5 20 10 -0.5 0 233 Cl O HO OMe N N S 13.5 200 190 12.5 180 11.5 170 10.5 160 150 9.5 140 8.5 130 7.5 120 6.5 f1 (ppm) 110 100 f1 (ppm) 5.5 90 4.5 80 3.96 2.19 2.08 2.99 2.00 0.96 1.01 1.04 1.94 2.20 2.04 1.08 N N N 2.74 1.11 HN 3.5 70 2.5 60 50 1.5 40 0.5 30 -0.5 20 234 Cl O HO N O N OMe S HN N N N 10.0 9.5 180 170 9.0 8.5 160 8.0 150 7.5 140 7.0 130 6.5 6.0 120 5.5 110 5.0 4.5 f1 (ppm) 100 90 f1 (ppm) 4.0 3.5 80 3.43 2.37 2.17 1.81 1.75 1.83 0.98 1.23 1.16 2.03 2.02 1.53 0.95 2.75 3.0 70 2.5 60 2.0 1.5 50 1.0 40 0.5 30 0.0 20 -0.5 235 Cl O HO N Cl N S HN N N N 9.0 8.5 180 170 8.0 160 7.5 150 7.0 140 6.5 6.0 130 5.5 120 5.0 110 4.5 4.0 f1 (ppm) 100 90 f1 (ppm) 2.88 4.33 2.20 2.09 2.04 0.83 0.91 0.74 0.93 1.81 1.73 1.82 2.76 3.5 80 3.0 70 2.5 2.0 60 1.5 50 1.0 40 0.5 0.0 30 20 -0.5 236 Cl O HO N N N H HN N N N 14.0 180 13.0 170 12.0 160 150 11.0 140 10.0 130 9.0 120 8.0 110 7.0 f1 (ppm) 100 90 f1 (ppm) 6.0 5.0 80 70 2.35 2.17 2.00 0.73 1.93 1.17 2.93 5.62 1.71 2.77 4.0 60 3.0 50 2.0 40 1.0 30 0.0 20 237 F O HO N N S HN N N N 14 13 12 180 170 160 11 150 10 140 9 8 130 7 120 6 110 5 f1 (ppm) 2.08 4.09 2.05 2.04 1.95 0.97 0.94 0.95 0.96 1.96 1.04 2.01 2.05 0.69 2.78 4 100 90 f1 (ppm) 3 2 1 80 70 60 0 50 -1 -2 40 -3 30 -4 20 238 Me O HO N N S HN N N N 2.12 2.26 2.28 2.05 2.05 3.08 1.99 0.93 0.93 0.95 0.94 2.01 1.05 1.94 1.96 1.12 2.79 14 13 12 11 10 9 8 7 6 5 f1 (ppm) 4 3 2 1 0 -1 -2 -3 -4 180 170 160 150 140 130 120 110 100 90 f1 (ppm) 80 70 60 50 40 30 20 10 0 239 O HO N N S HN N 13.5 180 170 12.5 160 11.5 150 10.5 140 9.5 130 8.5 120 7.5 110 6.5 f1 (ppm) 100 90 f1 (ppm) 5.5 80 4.5 70 4.22 2.20 2.08 1.99 0.95 0.92 0.92 0.98 2.03 1.09 2.13 1.22 1.88 1.12 N N 2.80 3.5 60 2.5 50 1.5 40 0.5 30 -0.5 20 240 Cl O HO N N S HO O 13.5 180 12.5 170 11.5 160 150 10.5 140 9.5 130 8.5 120 7.5 6.5 f1 (ppm) 110 100 90 f1 (ppm) 5.5 4.5 80 70 2.14 2.15 2.15 2.13 2.05 2.13 0.88 0.80 0.83 0.92 1.97 0.93 1.91 1.95 1.70 2.81 3.5 60 2.5 50 1.5 40 0.5 30 -0.5 20 241 Cl O HO N N N H O HO 13.5 180 12.5 170 11.5 160 150 10.5 140 9.5 130 8.5 120 7.5 110 6.5 f1 (ppm) 100 90 f1 (ppm) 5.5 4.5 80 70 2.27 2.20 2.17 2.00 0.70 1.84 0.95 2.87 4.70 2.33 2.82 3.5 60 2.5 50 1.5 40 0.5 30 -0.5 20 242 F O HO N N S HO O 13.5 180 170 12.5 160 11.5 150 10.5 140 9.5 130 120 8.5 110 7.5 100 6.5 f1 (ppm) 90 80 f1 (ppm) 5.5 4.5 70 60 1.94 2.12 2.05 2.04 2.28 1.97 0.95 0.89 0.89 0.95 1.97 1.01 2.05 1.99 2.14 2.83 3.5 50 2.5 40 1.5 30 0.5 20 10 -0.5 0 243 Me O HO N N S HO O 9.5 9.0 8.5 180 170 160 8.0 150 7.5 140 7.0 130 6.5 120 6.0 5.5 110 5.0 4.5 f1 (ppm) 100 4.0 90 80 f1 (ppm) 3.5 70 3.01 2.89 2.27 2.18 2.02 2.23 2.07 2.06 1.86 0.94 0.79 0.82 0.90 1.84 1.00 2.84 3.0 2.5 2.0 60 50 40 1.5 30 1.0 20 0.5 10 0.0 0 244 Me O HO N N N H 16 15 180 170 14 160 13 150 12 140 11 130 10 120 110 9 8 f1 (ppm) 7 6 100 90 f1 (ppm) 80 70 5 60 2.74 2.15 4.08 2.98 2.04 0.96 1.92 1.01 2.99 4.12 1.21 1.01 HO 2.85 O 4 50 3 40 2 30 20 1 0 10 0 245 O HO N N S O HO 10.0 180 9.5 170 9.0 160 8.5 150 8.0 140 7.5 7.0 130 6.5 120 6.0 110 5.5 5.0 4.5 100 90 f1 (ppm) 2.17 2.26 2.09 2.00 2.44 0.99 0.83 0.86 1.00 1.81 0.93 1.92 1.00 1.80 2.86 4.0 80 3.5 70 3.0 2.5 60 2.0 50 1.5 40 1.0 30 0.5 0.0 20 246 F O N H N N F S N HN N N 10.0 180 9.5 9.0 170 8.5 160 8.0 150 7.5 140 7.0 6.5 130 6.0 120 5.5 5.0 4.5 f1 (ppm) 110 100 90 f1 (ppm) 2.10 3.94 2.21 2.21 2.00 2.05 1.69 0.65 0.92 0.99 4.62 2.24 1.33 1.75 2.87 4.0 3.5 80 3.0 70 2.5 60 2.0 50 1.5 1.0 40 0.5 30 0.0 20 -0.5 247 F O N H N N Cl S HN N N N 9.5 180 9.0 170 8.5 8.0 160 7.5 150 7.0 140 6.5 130 6.0 120 5.5 5.0 110 4.5 4.0 f1 (ppm) 100 90 f1 (ppm) 2.66 2.67 2.45 2.51 2.47 4.02 1.05 2.00 1.22 1.25 1.15 5.10 1.23 2.10 2.30 2.88 3.5 80 3.0 2.5 70 2.0 60 1.5 50 1.0 40 0.5 30 0.0 -0.5 20 248 Cl O N H O N N Cl OMe S HN N N N 9.0 180 8.5 170 8.0 160 7.5 150 7.0 6.5 140 6.0 130 5.5 120 5.0 4.5 4.0 f1 (ppm) 110 100 90 f1 (ppm) 2.73 2.18 4.47 3.38 2.99 2.08 2.08 3.94 1.05 1.06 1.22 1.20 1.10 3.73 1.10 1.98 2.36 2.89 3.5 80 3.0 2.5 70 2.0 60 1.5 50 1.0 0.5 40 30 0.0 20 -0.5 249 Cl O N H OMe N N Cl S HN N N N 9.5 9.0 170 8.5 160 8.0 150 7.5 140 7.0 6.5 130 6.0 120 5.5 110 5.0 4.5 4.0 f1 (ppm) 100 90 f1 (ppm) 1.91 4.34 1.94 2.00 3.38 1.93 1.95 1.84 3.88 2.04 2.14 1.95 1.20 0.82 0.89 2.90 3.5 80 3.0 70 2.5 60 2.0 50 1.5 40 1.0 0.5 30 0.0 20 250 Cl O O OMe N N Cl S 9.0 170 8.5 160 8.0 150 7.5 7.0 140 6.5 130 6.0 120 5.5 110 5.0 4.5 4.0 f1 (ppm) 100 90 f1 (ppm) 4.00 2.17 2.12 3.13 2.01 2.05 0.89 0.89 1.00 5.75 1.92 1.99 1.06 2.91 N HN N N 3.5 80 3.0 70 2.5 2.0 60 1.5 50 1.0 40 0.5 30 0.0 -0.5 20 CHAPTER 3 SYNTHESIS AND BIOLOGICAL PROPERTIES OF ZNA-DERIVATIVES WITH WATER-SOLUBLE SIDE CHAINS 3.1 Introduction Marine sponges contain a wide range of structurally diverse and nitrogen-rich natural products displaying intriguing biological profiles. Among these sponges, the Leucetta genus has provided numerous examples of alkaloid compounds containing a 2aminoimidazole core.1 This core is considered a “privileged structure” due to its guanidinemimicking ability and pKa value (pKa ~ 7 – 9). Therefore, 2-aminoimidazole alkaloids have often served as valuable leads in drug discovery processes. 3.1.1 Naamidine and Naamine Natural Products from Leucetta Chagosensis In 1987, Carmely and Kashman reported the isolation and structural elucidation of four alkaloid natural products from the yellow marine sponge Leucetta chagosensis in Na’ama Bay, Egypt.2 In homage to the location of the sponge, they were termed naamidine A, naamine A, isonaamidine A and isonaamine A (Figure 3.1A). The natural products were constructed around the 2-aminoimidazole core. Naamine A and isonaamine A featured two benzylic substitutions at N1/C4 or C4/C5 positions. Naamidine A and isonaamidine A were further decorated with dehydrohydantoin moieties at the N2 position. Over the course of 252 next two decades, numerous other 2-aminoimidazol alkaloids were isolated from the Leucetta sponges, several of which were obtained as zinc complexes (Figure 3.1C).3-6 Pietra and co-workers have reported the isolation of naamidine A and G dimers in complex with Zn2+ as well as the A and G hybrid dimer complexes,7 in which two anionic naamidines acted as bidentate ligands to chelate to the metal center in a tetrahedral fashion. Lin and co-workers have described chagosendines, copper complexes of naamidine alkaloids isolated from Leucetta chagosensis.8 Despite plentiful work in isolating and characterizing these metal complexes, their exact biological roles are not well understood. Marine environment contains only traces of elements such as zinc, cooper or iron (below 10-6 M).9 Therefore these sponges might utilize organic compounds as siderophores to acquire a sufficient amount of metals for enzymatic activity or chemical defense mechanisms. 3.1.2 Biology of Naamidine Alkaloids 2-Aminoimidazoles (Figure 3.1) from the Leucetta sponges display a wide array of biological properties. For instance, isonaamidine E was found to inhibit the growth of HepG2, HM02 and Huh7 cancer cell lines at GI50-values ranging between 1.3 – 7.0 µg/mL.4 (2E,9E)-pyronaamidine 9-(N-methylimine) were mildly cytotoxicic towards A-549 (lung), MCF-7 (breast), and HT-29 (colon) cancer cell lines with GI50 values between 3 – 6 µg/mL. Pyronaamdine exhibited antimicrobial activities against Bacillus subtilis and Candida albicans at 100 µg/disk with 10 mm and 7 mm inhibition zones, respectively.10 Lovely and co-workers evaluated the anticancer activities of naamidine G and H in an MTT growth assay.11 Both compounds were cytotoxic against MCF-7 cancer cell line, with a value of 253 IC50 = 5.08 ± 0.05 and 29.9 ± 0.3 µM, respectively. The most prominent member in the naamidine family by far is naamidine A. Although the original isolation paper did not detail any biological properties, Ireland et al. later described its ability to antagonize epidermal growth factor (EGF) receptor signaling pathway, which is involved in the development of a number of human cancers.12 An in vitro EGF mitogenic assay with isonaamidine B, C and naamidine A revealed that only the latter showed selective inhibition of EGF-mediated over insulin receptor-mediated DNA synthesis (Table 3.1). Furthermore, athymic nude mice harboring squamous cell carcinoma (A431) with overexpressed EGF receptors were treated with naamidine A. After 7 days, it inhibited 85% tumor growth at a dose of 25 mg/kg. Doubling the dose to 50 mg/kg obtained near-complete inhibition of tumor growth. However, it also resulted in fatality of 33% of the tested mice (Table 3.2). The antitumor properties of naamidine A were later attributed to its ability to induce A431 cells arrest in G1-phase at low micromolar concentrations.13 More precisely, it changed the phosphorylation state of ERK1/2 enzymes and simultaneously potentiated their activities in a dose-dependent manner, leading to the hypothesis that a prolonged ERK signal was responsible for the observed cell cycle arrest.14 In a subsequent publication, Ireland also demonstrated that naamidine A was not only responsible for promoting cell cycle arrest, but also induced apoptosis by disrupting mitochondrial membrane potential and activating proapoptotic caspases 3, 8 and 9.15 Based on these results, there are potentially two independent mechanisms of action by which naamidine A exerts its antitumor, activity. 254 3.1.3 Syntheses and Structural Modifications of Naamidines Ireland’s study of naamidine A has received considerable attention from the synthetic and medicinal chemistry community. Several research groups have successfully accomplished total syntheses of this natural product and structurally related molecules (Figure 3.2). Most of the synthetic efforts have been focused on constructing the highly substituted heterocyclic ring structure as exemplified in the approaches made by Ohta16 and Lovely (Figure 3.2A/B).17 They identified functionalized imidazole rings 1 and 5 as useful synthons to install both benzylic groups at C5 and C4 positions via metalation, followed by quenching with the appropriate benzaldehyde. Introduction of an azide moiety and subsequent reduction yielded the 2-aminoimidazole core. Finally, the dehydrohydantoin functionality was installed via a TMS-activated N-methyl parabanic acid to obtain naamidine A or G. Watson’s synthetic strategy18 (Figure 3.2C) employed the Boc-protected tyrosine derivative 9, which was transformed into a Weinreb amide. Treatment with an aryl Grignard reagent accessed the a-amino ketone. This crucial intermediate was further condensed with cyanamide to form the 2-aminoimidazole core. Addition of TMS-activated N-methyl parabanic acid furnished the natural product naamidine G. The above-described methods either required multiple steps to introduce benzyl and amine functionalities or unstable a-amino ketones as precursors. In contrast, Looper and co-workers have developed an elegant hydroamination strategy in the total syntheses of naamine A and naamidine A (Figure 3.3A).19 A CuBr-catalyzed A3-coupling reaction accessed the allyl propargyl amine 14. Upon deprotection, guanylation20 of the resulting secondary amine 15 with Cbz-cyanamide potassium salt provided the N-acyl propargyl 255 guanidine 16. Treatment with AgNO3 led to the formation of N3-protected ene-guanidine 17 via regioselective 5-exo-dig cyclization. Subsequent global deprotection of Cbz- and Bn-groups yielded naamine A, which upon N2-functionalization delivered naamidine A. Concomitantly, the N3-protected ene-guanidine was also used to generate a library of N2acyl 2-aminoimidazoles via a 2-step acylation/Cbz-deprotection sequence (Figure 3.3B). More recently, the Looper group has developed a more concise and robust synthetic method, which mitigated the necessity of Cbz-deprotection (Figure 3.3C).21 Instead of Cbzcyanamide potassium salt, simple aryl cyanamides were utilized in the guanylation reaction. Subsequent treatment of N-acyl propargyl guanidine with NaH led to the desired N2-acyl 2-aminoimidazole via regioselective 5-exo-dig cyclization/isomerization. Looper’s approach accessed naamidine A on gram-scale and facilitated further biological testing for this natural product and its analogues. Despite naamidine A’s ability to inhibit EGF-mediated cell proliferation, it was never significantly selective when tested against MCF-7 and MCF-10A cell lines (EC50 = 5.9 and 8.1 µM, respectively). In addition, previous efforts to improve the potency of naamidine A have been unsuccessful.22-23 For instance, Watson and co-workers modified N2-, C4-, and C5-moieties as well as the heterocyclic core. In an EGF mitogenic assay none of the analogs exhibited improved potency. The authors noted that deletion of the dehydrohydantoin unit led to a decrease in antimitotic activity in almost all synthesized analogs. Based on these observations we thought to decouple the two distinct phenotypic activities of naamidine A (Figure 3.3D), namely kinase modulation and caspase activation via structural modification. It was hypothesized that the dehydrohydantoin unit acted as a two-point kinase binder, similar to 2-aminopyridines, and served also as a promiscuous binder, contributing to some off-target 256 effects. At the same time, we also recognize the ability of naamidine A to act as metal chelator, thus perturbing intracellular ion homeostasis and causing caspase-mediated cell death. To this end, we hoped to replace the dehydrohydantoin unit with a bioisoteric amide (Figure 3.3D). This scaffold change was aimed to eliminate the promiscuity of dehydrohydantoin while preserving the metal binding ability. 3.1.4 Biological Evaluations of Zinaamidole Researchers in Bryan Welm’s laboratory from Huntsman Cancer Institute performed a screen aiming to identify small molecules selectively targeting chemoresistant cancer cells over normal cells. They chose to measure the differential potencies of small molecules towards nontransformed mammary epithelial cell line (hTERT-HMEC) and patient-derived triple negative (ER-, PR, and HER2-) metastatic breast cancer cells (PE1007070).24 The tumor cells were collected from the pleural effusion of a 61-year-old female who relapsed after multiple rounds of chemotherapy (gemcitabine, carboplatin, doxorubicin, taxol and capecitabine). Several research groups from the Chemistry Department at the University of Utah (Looper, Sigman, Rainier and Burrows) contributed 560 compounds to the screening. Each cell type was treated with compounds at 20 µM in duplicate. After 4 days, the cellular viability was measured with an ATP-based luciferase assay. In order to further determine the selectivity for each compound, the percent viability of PE1007070 cells was subtracted from the viability of hTERT-HMEC cells. In total 15 compounds (3%) were found to have a high degree of selectivity for killing breast cancer cells and identified as “hits”. One N2-acyl 2-aminoimidazole analog, later dubbed 257 zinaamidole or ZNA (Figure 3.4), was among these 15 hits. To our surprise naamidine A did not exhibit any selectivity. In collaboration with Welm’s laboratory we further evaluated ZNA in a dosedependent manner using PE1005339, MCF-7 (ER+, PR-), T47D (ER+, PR+), MDA-MB231 (ER-, PR-, HER-) and MCF-10A (untransformed) cell lines. The results (Figure 3.5 a) demonstrated that ZNA was able to maintain potency against different cancer cell lines while leaving normal cells unaffected. On a molecular level, transcriptome analyses in MCF-7 cells identified the upregulation of metallothionine genes MT1F, MT1X, and MT2A and zinc transport protein genes SLC30A1 and SLC30A2 when treated with 30 µM ZNA for 3 h (Figure 3.5 b). In contrast, no changes were observed in MCF-10As. These metallothionines and transport proteins are known to be involved in maintaining intracellular zinc concentration by chelation (MTs), regulating influx and efflux across cell membranes (SLC30A1)25 or sequestering zinc within lysosomal compartments (SLC30A2).26 The resulting upregulation suggested that cancer cells were responding to a Zn2+ dyshomeostasis caused by the ZNA. FluoZin-3 staining experiment with MCF-7 and MCF-10A revealed that only the former experienced an increased fluorescence when treated with ZNA (Figure 3.6 left). This effect was significantly amplified up to 170-fold when 30 µM of ZnSO4 was added to the culture. In addition, we monitored the cellular zinc accumulation in MCF-7 and MCF-10A in a time dependent manner (Figure 3.6 right). After 48 h a 13-fold increase of intracellular zinc was detected for MCF-7 cell line, whereas the untransformed cells remained largely unaffected. The biological data undoubtedly pointed towards a correlation between upregulation of metal-trafficking proteins, ZNA-mediated zinc dyshomeostasis, and cell 258 death. We investigated potential mechanisms of action, which could lead to the observed cellular phenotypes. Naamidine A, which inspired the synthesis of ZNA and its derivatives, has been previously shown to induce apoptosis via caspase 3, 8 and 9 activation.15 However upon treatment with ZNA/ZnSO4 and staurosporine (STS), a known caspase activator,27 none of the tested cancerous cell lines displayed any change in caspase activity except STS (Figure 3.7). Next, we questioned whether cell death was directly linked to intracellular accumulation of zinc. Using fluorescence microscopy, we observed a significant amount of exogenous zinc in the lysosomes of MCF-7 accompanied with high cathepsin activity (Figure 3.8 left). However, the same effect was not seen in the untransformed MCF-10A when treated with ZNA/ZnSO4. These data suggested that ZNA acted as an ionophore and was causing lysosomal membrane permeabilization by flooding cancer cells with a superstoichiometric amount of zinc. The consequent leakage of lysosomal content into the cytosol led to cathepsin-mediated lysosomal cell death.28 The aforementioned results also showcased that normal cells have different ways to maintain zinc homeostasis, such as high expression of zinc export proteins or other endogenous mechanisms. Malignant breast tissues are known to contain high concentration of zinc (up to 72% more than in normal tissues).29 This is because cancer cells possess increased proliferation and metabolism rates in comparison to normal cells and are in constant demand of nutrients among which are zinc. A number of studies have reported aberrant gene expressions of zinc transport proteins in breast cancer cells. For instance, T47D cells were found to overexpress SLC30A2 and form intracellular zinc pools, whereas ZIP10 (SLC39A10) was linked with invasive behavior of breast cancer cells.30 In addition, ZIP7 (SLC39A7) has contributed to the tamoxifen-resistance properties in MCF-7 cells.31 We 259 believe these differences between malignant and healthy cells provide a therapeutic window to exploit novel cancer-selective ionophore drugs such as ZNA. 3.1.5 Zinc Ionophores in Medicinal Chemistry Ionophores are a subset of metal-binding drug molecules that repeatedly deliver and release metal ions into the cell.32 In contrast to chelators, which sequester metals, they increase the intracellular concentration of a metal, leading to metal ion dyshomeostasis and subsequently cell death. In addition, the biological effect of a compound can be further amplified when exogenous metal is added. This concept could be a useful approach in the context of cancer treatment. Zinc is an essential metal for cellular functions and is involved in many biological processes such as signaling pathways,33 gene transcription34 and protein-proteininteractions.35 The dyshomeostasis of zinc has been implicated with apoptotic cell death.36 To date, only a few compounds are known to be zinc ionophores (Figure 3.9): chloroquine,37 pyrrolidine dithiocarbamate,38 zincophorin39 and most notably clioquinol (CQ).40 It is currently in clinical trial for the treatment of Alzheimer’s disease and has been shown to exert anticancer properties by inducing a massive zinc influx into the cell. For instance, Lind and co-worker demonstrated that treatment of prostate cancer cell line DU145 with 10 µM CQ and 50 µM ZnCl2 enhanced cytotoxicity and intracellular zinc concentration. Subsequent lysosomal membrane disruption insulted in caspase-mediated apoptosis. Additionally, structural modifications to alter cLogP and pKa of the phenol had also an effect on the potency of CQ.40-41 When ZNA was tested alongside naamidine A and CQ on MCF-7 and MCF-10A 260 cell lines, only the former compound was selective and potent in favor of the cancer cells. CQ in combination with ZnSO4 was also cytotoxic towards healthy cells. On the contrary, the potency naamidine A diminished when co-treated with zinc, suggesting it acted as a chelator rather than an ionophore. 3.1.6 Mechanism of Action of ZNA ZNA selectively inhibited proliferation of MCF-7 cell line in a dose-dependent manner (Figure 3.10A). The activity was potentiated upon addition of exogenous ZnSO4. Interestingly, ZNA reached maximal inhibitory concentration at roughly 20 µM, matching the concentration of added ZnSO4. However, at higher concentrations an increase in cellular viability was observed. This phenomenon was later dubbed “rescue effect” and attributed to the formation of Zn(ZNA)2 dimer complex (Figure 3.10B) outside the cell. The complex itself, obtained as a precipitate by stirring a methanolic solution of ZNA with ZnSO4, was also tested and found to be biologically inactive. Given the preliminary biological data and observations, we hypothesized that ZNA entered the cell as a zinc monomer or free ligand (Figure 3.10C). In cytoplasm, N3-H became deprotonated due to the mild alkaline environment (pH = 7.2) and formed higher order zinc complexes. When the resulting complex entered the lysosome, the more acidic environment (pH = 4.5 – 5.0) likely led to protonation and release of a zinc ion. The free ligand could then be recycled to repeat another zinc uptake cycle. 261 3.1.7 Biological Evaluation of ZNA Derivatives We attribute the mode of action of ZNA to the N2-acyl 2-aminoimidazole functionality. Unlike naamidine A, ZNA does not chelate but weakly binds to zinc. Its ionophoric properties arise from a delicate interplay of acidity, binding affinity, and hydrophobicity, which governs potency and selectivity. This effect is amplified in a synergistic manner when exogenous zinc is added. We questioned whether structural modifications could further help us to better understand the properties of this molecule. Using chemistry developed in the Looper lab,21 a series of ZNA analogs with modifications at N1-, N2-, C4- and C5-positions were synthesized. (Figure 3.11). In collaboration with Dr. Katrin Guillen of the Welm laboratory, the potency and selectivity of those compounds were evaluated against MCF-7 and MCF-10A cell lines using an ATPlite assay. The potencies with and without exogenous zinc were expressed as EC50 values. In addition, we also examined the capability of each analog to induce zinc uptake at 25 µM in the absence and presence of ZnSO4 co-treatment (Table 3.3) using FluoZin-3 staining experiment. ZNA 4, 8 and 62 were modified at the R2 position, which in turn could have decreased the acidity of N3-H moiety and led to a stronger zinc binding. Co-treatment with ZnSO4 did not significantly potentiate zinc uptake. Instead a cellular rescue effect or increase in proliferation was observed implying these analogs possessed weak chelation properties. Therefore, electron poor moieties such as o-fluoro-aryl could be essential for retaining balanced zinc affinity. ZNA 60, bearing a larger R1 moiety, was relatively more potent than ZNA 62. However, this effect diminished when ZnSO4 was added as cotreatment. ZNA 3 and ZNA 28 both displayed greater ionophoric properties than ZNA, 262 suggesting that minor modifications at R4 group could generate more promising analogs. Although the healthy cells experienced a slightly higher zinc uptake, it did not influence the proliferation, underlining the fact that these types of cells were more capable of dealing with high zinc influx. Interestingly, ZNA 69 in combination with zinc co-treatment was capable of inducing high metal uptake. We believe the phenomenon is in some way associated with the chelating ability of the aliphatic amine. ZNA 47 decorated with aliphatic groups at C4-position have not shown to be biologically active. ZNA 49 contains an enzymatically cleavable prodrug motif at the N3-nitrogen and demonstrated similar activity compared to ZNA. This could indicate that ZNA in part targeted intracellular zinc. Despite some encouraging data collected from the new analogs, only a limited SAR was established. We questioned whether more profound structural changes could provide a better insight into the biology activity of ZNA. 3.2 Results and Discussion 3.2.1 ZNA-Derivatives with Water-Solubilizing Groups Previously we have described a “rescue effect” in the MCF-7 dose-response assay. Although this phenomenon illustrated the synergy between zinc ion and ZNA analogs, it might be deleterious to the overall potency due to potential precipitate or aggregate of the Zn(ZNA)2 dimer. In our in vivo mouse model, ZNA had to be administered as an aqueous suspension. The treatment led to an overall increase in survival advantage, but a high daily dosing of 100 mg/kg, IP, had to be maintained. To overcome the solubility issue, we proposed to introduce solubilizing moieties such as amino acid and ethylene glycol side chains through the modification of the C5-aryl 263 group. Our main goal was to identify a more polar analog of ZNA with similar potency and selectivity. Both moieties have been previously used in drug optimization processes. Sessler and co-workers have modified the zinc ionophore 1-hydroxypyridine-2-thione (ZnHPT) by adding mono-, di- and tri-(ethyleneglycol)-methyl ether.42 One of the water-soluble derivatives PCI-5002 has shown to inhibit A549 and PC3 cancer cell growth in xenograft models (Figure 3.12A). Researchers have also investigated the properties of amino acid (AA) conjugated drug molecules.43 Chemically, these compounds can be easily accessed via late-stage derivatization of the parent drug using standard peptide coupling reactions. They have shown to dramatically increase the water solubility and bioavailability. Cancer cells such as MCF-7 are well-known to overexpress various amino acid transporters, e.g., ASCT2 or LAT1.44 Taking advantage of these influx transporters, Shim and co-workers have developed a valine conjugated lapatinib analog (Figure 3.12A).45 This compound has shown enhanced potency towards several breast and lung cancer cells lines while leaving normal cells unaffected. Additional glutamine uptake experiments demonstrated that Vallapatinib entered the cancer cell via amino acid transporters. We questioned whether the same strategies could be applied to our lead compound. 3.2.2 Syntheses of ZNA with Solubilizing Groups ZNA analogs carrying natural and unnatural amino acid (AA) side chains were synthesized via standard peptide coupling conditions using ZNA 69, previously prepared by graduate student Justin Salvant. Subsequent Boc-group deprotection in HCl/MeOH yielded the desired ZNA analogs as water soluble HCl salts (Figure 3.13A). 264 Additional AA derivatives, ZNA 89, 90 and 91, were derived from the aniline analog 3.24, which was synthesized alongside ZNA 109 and 119 using procedures established in the Looper lab (Figure 3.13B and C).19 Finally, Mitsunobu reactions with ZNA 18 and primary alcohols allowed the transformation to ZNA analogs bearing different types of ethylene glycol side chains (Figure 3.13D). 3.2.3 Biological Evaluations and Discussions For the biological evaluations of ZNA analogs, two types of cellular assays were used to assess their in vitro efficacy and ionophoric properties (performed by Dr. K. Guillen from Welm Lab). First an ATPlite Luminescence Assay was employed with MCF-7 and MCF-10A cell lines in the presence and absence of additional 20 µM ZnSO4. This assay measures the concentration of ATP, a marker for cell viability. When the cells undergo apoptosis or necrosis, the amounts of ATP decline. The concentration of ATP can be determined by the reaction of ATP, luciferase and D-luciferin, resulting in the production of light. Second, FluoZin-3 staining experiment was used to quantifiy intracellular zinc ions uptaken by the tested compound. In particular, FluoZin-3 is a highly zinc-specific fluorescent dye which is applied to the cells upon drug treatment. Without ZnSO4 co-treatment, the biological results (Table 3.4) indicated that most of the amino acid (AA) analogs were less potent in comparison to ZNA. Most of the analogs were only slightly selective in favor of MCF-7 cell line. ZNA 76 and ZNA 94 were equipotent for both cell lines. Zinc uptake was notably reduced for all compounds. We suspect some AA moieties might be capable of metal sequestration, acting as zinc 265 chelators. It is known that zinc ion is essential to cellular functions and coordinates to numerous proteins via histidine, glutamate and aspartate binding motifs.46 Hong and coworkers found that vancomycin, for instance, induced zinc starvation in Staphylococcus aureus.47-48 Additional studies revealed that the N-methyl leucine moiety of vancomycin was chelating to zinc and formed Zn(Vanco)2 dimer complexes. However, addition of 25 µM exogenous zinc salt led to an increase in potency, suggesting that the new analogs, although not selective, were zinc synergistic. For ZNA 74, 75, 89, 90, and 94 high zinc uptake was observed, which to our surprise did not lead to an increase in cell death. We believe their hydrophobicity enabled zinc-assisted cell penetrance, but did not release it in the lysosome due to strong chelation. It is also worth noting that no “rescue effect” was observed in any of the dose response curves. When we subjected ZnSO4 to a solution of ZNA 74 in methanol, no precipitation was observed. Analysis of the crude reaction by 1H NMR in CD3OD showed a complex mixture of compounds, indicating that that the pendant AA side chain interfered with binding events between zinc ion and N2-acyl 2-aminoimidazole functionality. We observed the same results for ZNA 109 and 119. They were zinc synergistic and demonstrated exceptional zinc uptake capabilities in MCF-10A cell lines. ZNA analogs containing glycol ether side chains were slightly selective toward the transformed cell line but failed to retain selectively when additional zinc was added. For ZNA 117 and 118, MCF-7 cellular viabilities were restored in combination with the co-treatment. 266 3.3 Conclusion and Future Directions The Looper group has identified ZNA as an ionophore, which selectively induces zinc dyshomeostasis in MCF-7 breast cancer cells leading to lysosomal cell death. Despite promising biological activities and in vivo results, the ability of ZNA to form inactive and non-water-soluble zinc dimers has proven to be deleterious for potency. In Chapter 3, we addressed this problem by attaching polar functionalities to ZNA. Although our new compounds were more hydrophilic, many of them failed to show the desired potency, selectivity and zinc affinity in comparison to the original ZNA lead. The unique ability of ZNA to weakly bind to zinc and release it in the lysosome stemmed from a delicate interplay between acidity, zinc affinity and hydrophobicity. Minor structural changes are certainly tolerated and improved the potency, as exemplified in ZNA 3 and ZNA 28. Any major modifications involving the addition of nucleophilic heteroatoms could perturb binding equilibrium. Interestingly, some of the compounds induced high zinc uptake upon co-treatment with ZnSO4. This aspect can be further investigated. Here we need to answer several questions. First, at which concentration of exogenous zinc would we detect a notable elevation in zinc uptake? Second, are ZNA amino acid derivatives cell permeable given their hydrophilicity? We could address this issue by measuring the drug concentration in cell lysates. And finally, the fact that high zinc content did not lead to cell death could point towards the metal being sequestered inside the cell. Therefore, fluorescent microscopy studies could reveal a more diffuse distribution of zinc inside the cell. Taken together, these experiments will help us better understand whether amino acid derivatives operate with the same MOA as ZNA. 267 A HO HO N1 C5 C4 N Me C2 N NH N N3 N MeO HO N2 N O N O Me N NH2 N Me HO MeO N NH N N HO N Me isonaamidine A HO O naamidine A naamine A B R3 R2 R2 isonaamine A R1 R3 N MeO O Me OH MeO HO NH N NH N MeO N MeN MeO (2E,9E)-pyronaamidine 9-(N-methylimine) C N N N N N O O N N Me MeO O MeO O OMe Me Zn N O MeO Me N N O O N Me naamidine D Me OMe NH N naamidine C O Me NH N Me N Me MeO Me N O N HO O N R1 = H, R2 = OH, R3 = OMe, R4 = H; naamidine B R1 = OH, R2 = OMe, R3 = OMe, R4 = OH; naamidine E R1 = H, R2 = H, R3 = OMe, R4 = H; naamidine G R1 = H, R2 = OMe, R3 = OH, R4 = OMe; naamidine H R1 = H, R2 = OH, R3 = OMe, R4 = OMe; pyronaamidine R1 = OMe, R2 = H, R3 = OH; isonaamidine B R1 = OMe, R2 = H, R3 = OMe; isonaamidine C R1 = OMe, R2 = OMe, R3 = OMe; isonaamidine E MeO NH N O N Me O Me N NH N R1 N R4 N NH2 O N N N N N Me OMe O Cu N N O Zn-naamidine A dimer Me OH N O MeO Me N N N N Me MeO OMe O chagosendine C Figure 3.1: Natural products isolated from Leucetta Chargosensis (A). 2-Aminoimidazole alkaloids with different substitution pattern isolated from Leucetta sponges (B). Naamidine A dimer in complex with zinc and chagosendines C (C). 268 A Ohta’s approach toward naamidine A, 13 steps, 1.6% overall HO N NH N N O naamidine A N Me HO N Me N OMOM 2 3 SPh 1 Lovely’s approach towards naamidine G, 8 steps, 41% overall MeO N reduction, then hydantoin installation Me N MeO N Me NH N N MeO O N O Me 8 OH C5-formylation, aldehyde trapping N MeO I C N Me OMe OMe metalation& aldehyde trapping N HO N Me 7 C2azidation N3 OMe naamidine G I N I N Me 5 6 Watson’s approach towards naamidine G, 6 steps, 35% overall HO N N hydantoin installation Me HO N Me NH N N MeO metalation, then aldehyde trapping SPh N Me OMOM B N C5-bromination, metalation, aldehyde trapping SPh reduction& C2-functionalization N3 OTBS 4 N TBSO N Me Me OH MeO N MeO O N MeO reduction, then hydantoin installation Me O naamidine G NH2 Boc-deprotec., then cyanamide condensation NMeBoc BnO O O N OMe Me OMe 12 O Grignard reaction BnO N(OCH3)CH3 NMeBoc 11 O Weinreb amide formation OH NHBoc BnO 10 9 Figure 3.2: Synthetic approaches and key steps toward naamidine A and G. A) Total synthesis by Ohta, B) by Lovely and C) Watson. 269 A BnO BnO 13 N H 3.12 CuBr CHO Me BnO N MeO 14 BnO N N AgNO3 NH2 ene-guanidine 17 HO N MeOH, quant N O O Me + NH2 TMS HO PhMe N Me N N 110 oC 82% O N Cbz O naamidine A C N Cl N Cbz R1 = PMP or Ph R2 = Ar 18 MeO O Me NH F R1 R1 Et3N, CH2Cl2 NH Me 10% Pd/C H2 (60 psi) N O N H R2 MeO Me Me N O N2-acyl R2 2-aminoimidazole MeO N K+ N TMSCl, MeCN rt N MeOH 19 N O N MeO O R2 N N naamine A N Me N H MeO Me NH MeO 16 Pd(OH)2/C H2 (1 atm) Me N Cbz CH2Cl2 90% O BnO MeO R1 15 Me N TMSCl, MeCN quant. B Me BnO CN N- K+ N H CH2Cl2, r.t, 69% 3.13 O BnO Me MeCN 80 oC 65% MeO MeO Pd(PPh3)4 2-thiosalicylic acid N NaH Me N THF, rt NH2 N H O Me N F O 20 F 21 D N2-acyl 2-aminoimidazole MeO HO N Me N N H N MeO structural modification O N O N Me promiscous kinase binder N H Bioisostere replacement - hydantoin moiety removed - Zn-binding motif retained Me N R3 O Zn-binding motif N2-acyl 2-aminoimidazole Figure 3.3: Looper’s synthesic approach towards 2-aminoimidazoles. A) Total synthesis of naamine A and naamidine A. B) Synthesis of N2-acyl 2-aminoimidazoles from eneguanidines. C) NaH-mediated hydroamination using N2-acyl propargyl guanidine. D) Structural modification of naamidine A to retain Zn-binding motif and removal of dehydrohydantoin promiscuity. 270 MeO N N H Me N F O ZNA Figure 3.4: Screening effort identified ZNA (Hit 8) as a selective anti-breast-cancer reagent. a b Figure 3.5: Dose-response curves for breast cancer and normal cells treated with ZNA (a). Transcriptome analyses identified upregulation of metallothionines and zinc transport proteins in MCF-7 cell line while leaving MCF-10A unaffected (b). Figure 3.6: FluoZin-3 experiment revealed 170-fold increased zinc uptake in MCF-7 cells when ZnSO4 was added (left). After 48 h, MCF-7 cells showed a 13-fold increase in zinc uptake (right). 271 Figure 3.7: Studies of caspase activity with ZNA, ZnSO4 and STS as positive control. MCF-10A MCF-7 Figure 3.8: Fluorescence microscopy using FluoZin-3 (green) LysoTracker (red) and Hoechst (blue) identified zinc build-up in the lysosomes of MCF-7 cells. 272 A Me Et N HN S Et B Cl SH N I N OH N Cl pyrrolidine dithiocarbamate chloroquine Me OH OH OH O HO H Me O clioquinol (CQ) OH Me H Me Me Me Me Me Me zincophorin Figure 3.9: Biology of zinc ionophores. A) Structure of molecules with ionophoric properties. B) Cell viability after 48 h of treatment. ZNA A 0.5 -1 0 1 2 MeO 1.5 1.0 0.0 B MCF-10A rescue effect Fractional ATP content 1:1 1.5 Fractional ATP content ZNA + 20 µM ZnSO4 MCF-7 Me F N N H 0.5 0.0 3 N 1.0 O -1 0 log (µM) 1 2 3 log (µM) ZnSO4 MeOH, rt C + Zn2+ - H+ ZNA-Zn ZNA membrane penetrant + ZNA - H+ Zn(ZNA)2 - insoluble in aq. media - not membrane permeable MeO Me N F N N + ZNA ZNA Zn2+ lysosome ZNA-Zn ZNA O Zn(ZNA)2 cathepsin release and lysosomal cell death O N N F Zn2+ Zn N Me OMe Zn(ZNA)2 biologically inactive Figure 3.10: Properties of ZNA. (A) Dose-response curve in MCF-7. B) Reaction of ZNA with ZnSO4 led to the formation of Zn(ZNA)2 complex. C) Predicted MOA of ZNA. 273 MeO MeO N analog synthesis Me N R3 F N N H R4 ZNA 49 R1 N N N H O O Me ZNA# R1 4 Me 8 Me R2 O F N N O R2 Me O R4 R3 MeO Cl MeO OMe MeO Me 62 Br MeO Me 60 Br Cl Me 3 F Me 28 F O Me 69 NH2 F MeO Me 47 HO F Figure 3.11: Selected examples of ZNA analogs. A S N+ R O O R N+ Zn R=H S R = CH2(OCH2CH2)2OCH3 sufficiently soluble for in vivo studies 1-hydroxypyridine-2-thione N O Me N S O O HN Val-lapatinib analog: F N O Me B O NH2 insoluble in aq. media and poorly bioavailable Cl - easily derivatized in 2 steps - more potent than lapatinib - inhibited proliferation of MCF-7, MDA-MB-231 and A549 cell lines - entered the cancer cell via amino acid transporters Me MeO SG X N C5 Me O N C5 N N H X = O or NH SG = solubilizing groups C5-aryl group modification F Me N N H F O ZNA Figure 3.12: Enhancing the solubility of ionophores. A) Addition of solubilizing groups to the parent drug molecule. B) Modification of C5-aryl group to increase aqueous solubilty. 274 A O O H 2N N Me HN AA N DCM, 0 oC - rt, 16 h, 38 - 99% F N N H O HN AA Boc Boc-amino acid EDC . HCl, HOBt, Et3N O B 3.9 O O O O 4 H 2N 2 HCl NH2 H 2N HCl NH2 NH2 HCl O ZNA 76 ZNA 77 Me O ZNA 78 O O Me HCl NH2 ZNA 94 H 2N HCl HCl ZNA 95 O H S O O 3.10 O tBu 3.11 O NHR N 3.12 H DMBA Pd(PPh3)4 CuBr, MeCN 80 oC, 24 h 41 - 70% O Me N DCM, rt, 16 h 48 - 75% Me N H 3.13 3.14 - 3.16 H 2N N N H NaH, THF F N TMSCl, DIPEA DCM, rt, 3 h 75 - 84% H 2N Me N F N rt, 3 h 38 - 70% Me N Me N N H F tBu S O O ZNA 119 O 3.23 3.24 O AA NH Me N 2) HCl/MeOH 56 - 99% F N N H Me O Me O ZNA 109 1) Boc-amino acid HATU, DIPEA DMF, rt - 50 oC 24 h, 33 - 38% F N N rt, 6 h 99% O O H 2N TFA/DCM Me N H 3.20 - 3.22 Me 3.17 - 3.19 RHN RHN O NH ZNA 96 RHN Me C O NHR R: Me F N N H O Me ZNA 74 Me 3.1 - 3.8 Me HCl NH2 ZNA 75 HCl NH2 N rt, 6 h 55% - quant. F N N H ZNA 69 amino acids O Me HCl/MeOH Me Me N N H O F O O Me HCl NH2 ZNA 89 Me 4 O H 2N HCl NH2 2 HCl NH2 ZNA 91 ZNA 90 O 3.24 D HO EG: EG O N Me prim. alcohol DTAT, PPh3 N THF, 0 oC - rt 24 h N H O ZNA 18 F N HO Me HO N N H O ZNA 92 OH ZNA 117 MeO ZNA 118 ZNA 93 F OMe HO OMe MeO ZNA 116 Figure 3.13: Syntheses of hydrophilic ZNA derivatives. A) ZNAs with solubilizing side chains. B) Preparation of ZNA 109, 119 and aniline 3.24. C) Peptide coupling of 3.16 to access ZNA 89 – 91. D) Linking different ethylene glycol side chains to ZNA 18. 275 Table 3.1: Inhibition of isolated natural products on EGF and insulin mediated mitogenesis. IC50 (µM) compound naamidine A isonaamidine B isonaamidine C EGF 11.3 22.7 36.9 insulin 242 9.8 6.7 selectivity ratio 21 0.43 0.18 Table 3.2: In vivo evaluation of naamidine A against athymic nude mice harboring A431. dose (mg/kg) 50 25 12.5 3.13 % inhibition of tumor growth (number of deaths) 96.4 (2) 87.4 (1) 52 (0) 35 (0) Table 3.3: Biological evaluation of ZNA analogs. ZNA# EC50 in µM MCF-7 EC50 in µM with 20 µM Zn ZNA 4 8 62 60 3 28 69 47 49 21.0 35.4 34.8 20.1 21.4 13.4 25.4 32.8 N.A. 21.6 11.9 N.A. 75.6 N.A. N.A. 2.8 4.4 13.1 N.A. 12.4 MCF-10A EC50 EC50 in µM with in µM 20 µM Zn N.A. N.A. N.A. N.A. N.A. 26.9 N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. 12.4 N.A. N.A. MCF-7 Zn-uptake -Zn/+Zn MCF-10A Zn-uptake -Zn/+Zn 1.250/1.513 0.869/0.966 1.341/1.308 0.956/1.059 1.202/0.926 1.450/2.494 1.058/3.742 0.758/2.692 0.388/0.513 0.322/1.037 1.034/0.996 0.630/0.713 0.745/0.778 0.744/0638 0.824/0.649 1.016/1.658 0.718/2.606 0.738/5.610 0.587/0.666 0.605/1.276 276 Table 3.4: Biological results for ZNA derivatives with solubilizing groups. ZNA# EC50 in µM MCF-7 EC50 in µM with 20 µM Zn ZNA 74 75 76 77 78 94 95 96 89 90 91 109 119 92 93 116 117 118 21.0 N.A. 27.7 24.8 N.A. 59.2 24.3 50.6 23.1 36.7 38.5 27.9 33.7 32.9 45.0 44.9 N.A. 26.5 28.3 11.9 15.9 11.1 14.8 89.2 N.A. 9.1 27.0 5.8 11.2 12.8 29.8 9.3 10.7 14.2 14.9 16.7 78.7 52.2 MCF-10A EC50 EC50 in µM with in µM 20 µM Zn N.A. N.A. 93.6 28.4 N.A. N.A. 27.3 N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. 22.3 11.9 22.4 N.A. N.A. 8.8 27.5 22.3 11.9 22.0 24.3 12.7 13.7 23.0 22.3 24.4 N.A. N.A. MCF-7 Zn-uptake -Zn/+Zn MCF-10A Zn-uptake -Zn/+Zn 1.250/1.513 1.034/0.996 0.787/2.788 0.582/2.951 1.658/3.545 0.973/6.716 0.900/1.056 0.596/0.622 0.913/1.439 0.612/0.658 0.879/1.200 0.646/0.789 0.684/4.235 0.660/7.620 0.560/0.892 0.545/0.670 0.629/2.702 0.493/2.871 1.074/4.927 1.110/9.312 0.758/9.848 0.682/11.071 0.232/0.607 0.503/0.617 1.206/4.927 0.912/26.125 0.880/4.206 0.898/17.599 0.188/6.337 0.374/7.272 0.336/0.926 0.508/1.016 0.767/2.439 0.761/8.242 0.937/0.976 1.027/1.085 0.988/0.930 0.890/0.906 277 3.4 References 1. Sullivan, J. D.; Giles, R. L.; Looper, R. E., 2-Aminoimidazoles from Leucetta Sponges: Synthesis and Biology of an Important Pharmacophore. Curr. Bioact. Compd., 2009, 5, 39-78. 2. Carmely, S.; Kashman, Y., Naamines and Naamidines, Novel Imidazole Alkaloids from the Calcareous Sponge Leucetta Chagosensis. Tetrahedron Lett., 1987, 28, 30033006. 3. Carmely, S.; Ilanb, M.; Kashmana, Y., 2-Amino Imidazole Alkaloids from the Marine Sponge Leucetta Chagosensis. 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R.; Hong, H.-J., Zn(II) Mediates Vancomycin Polymerization and Potentiates its Antibiotic Activity Against Resistant Bacteria. Sci. Rep., 2017, 7, 4893. 281 3.5 Supporting Information 3.5.1 Experimental (Biology) All experiments were performed by Dr. Katrin P. Guillen (Welm Lab) at the Huntsman Cancer Institute. Dose response assays: For 5-day dose response assays, cells were seeded in 96-well plates in 100 µL standard culture media at a confluency to achieve 90% confluency at the completion of the 120-h assay. Following an overnight incubation, the media was replaced with 2% fetal bovine serum (FBS), small molecule- or vehicle control-containing media. The media was aspirated and replaced with fresh treatment media every 48 h during the experiment. Following the completion of the 120-h assay, cell viability was measured using an ATPlite assay (PerkinElmer, Waltham, MA, USA) following the manufacturer’s protocol. Measurement of intracellular Zn2+ by FluoZin-3 staining: At experimental endpoints, the low serum (2% FBS) drug-containing media was removed from cell culture plates and replaced with room temperature Hank’s Balanced Salt Solution (HBSS, Thermo Fisher Scientific) containing 2.5 µM FluoZin-3. The cells were incubated with the indicator for 30 min at room temperature in the dark. After staining, the cells were trypsinized, resuspended in ice-cold HBSS containing 2% FBS. Relative mean fluorescence was measured by flow cytometry (FACScan, BD Biosciences, San Jose, CA, USA). 3.5.2 General Experimental Conditions (Chemistry) All reactions requiring anhydrous conditions were performed under a positive pressure of nitrogen using flame-dried glassware. Commercially available reagents were 282 used as received or purified according to Purification of Laboratory Chemicals. Dimethylformamide (DMF), tetrahydrofuran (THF), acetonitrile (MeCN), and dichloromethane (CH2Cl2) were degassed with nitrogen and passed through a solvent purification system (Innovative Technologies Pure Solv). Methanol was distilled from magnesium turnings immediately prior to use. DIPEA was distilled from CaH2 immediately prior to use. Reactions were monitored to completion by TLC and visualized by a dual short/long wave UV lamp and stained with an aqueous solution of potassium permanganate. Flash chromatography was performed on silica gel SiliaFlash P60 (40-63 µm) from Silicycle. 1 H NMR spectra were recorded at 500 MHz as indicated. The chemical shifts (δ) of proton resonances are reported relative to the deuterated solvent peak: 7.26 for CDCl3 and 4.87 for H2O in CD3OD, using the following format: chemical shift [multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, app = apparent), coupling constant(s) (J in Hz), integral]. 13 C NMR spectra were recorded at 75 or 125 MHz. The chemical shifts of carbon resonances are reported relative to the deuterated solvent peak: 77.3 (center line) for CDCl3 and 49.0 (center line) for CD3OD. Mass spectra were obtained by ESI/APCI for LRMS or ESI/APCITOF for HRMS. 283 3.5.3 Procedures and Characterizations General procedure A1 for the peptide coupling with ZNA 69 O O H 2N Me N + N N H Boc-AA-OH F HN AA Boc EDC . HCl, HOAt N Et3N, CH2Cl2, r.t. Me N N H F O O A 25-mL oven-dried round bottom flask was equipped with a magnetic stirring bar and charged with 0.27 mmol of Boc-AA-OH and 5 mL CH2Cl2. The solution was cooled down to 0 oC in an ice-water bath. EDC . HCl (103.5 mg, 0.54 mmol) and HOBt (38.9 mg, 0.29 mmol) were added sequentially and the reaction mixture was stirred at the same temperature for 30 min. Then the primary amine ZNA 69 (80 mg, 0.18 mmol) was added in one portion followed by Et3N (0.1 mL, 0.72 mmol). The resulting mixture was allowed to warm up to room temperature and was stirred for 16 h. Upon consumption of the amine, the reaction was quenched with sat. NH4Cl and extracted 2x with CH2Cl2. After evaporation of the organic solvent, the crude product was purified via column chromatography) to yield the amide. Me BocHN O N H O Me N N H N O F Tert-butyl (S,E)-(1-((2-(4-(5-benzyl-2-((2-fluorobenzoyl)imino)-3-methyl-2,3di-hydro-1H-imidazol-4-yl)phenoxy)ethyl)amino)-1-oxopropan-2-yl)carbamate (3.1). Compound 3.1 was prepared with ZNA 69 (80 mg, 0.18 mmol) and Boc-L-alanine (51.1 mg, 0.27 mmol) following general procedure A1. The crude product was purified via flash 284 column chromatography (1:1 hexanes/EtOAc, followed by 1:2 hexanes/EtOAc) to yield a white solid (97.3 mg, 87%). Rf = 0.7 (15:1 CH2Cl2/MeOH). 1H NMR (CDCl3, 500 MHz): δ 8.02 (td, J = 7.5, 1.5 Hz, 1H), 7.36 – 7.32 (m, 1H), 7.25 – 7.22 (m, 4H), 7.16 (t, J = 7.5 Hz, 1H), 7.14 – 7.10 (m, 3H), 7.05 (dd, 11.0, 8.5 Hz, 1H), 6.95 (d, J = 8.5 Hz, 2H), 5.24 (brs, 1H), 4.19 (brs, 1H), 4.03 (t, J = 6.6 Hz, 2H), 3.80 (s, 2H), 3.64 (t, J = 6.6 Hz, 2H), 3.40 (s, 3H), 1.37 (s, 9H), 1.32 (d, J = 7.0 Hz, 3H). 13C NMR (CDCl3, 125 MHz): δ 173.2, 171.2, 161.4 (d, JCF = 253.7 Hz), 159.1, 155.5, 148.0, 138.0, 131.9 (d, JCF = 9.2 Hz), 131.7 (d, JCF = 2.5 Hz), 131.6, 128.8, 128.2, 126.7, 126.1 (d, JCF = 8.7 Hz), 124.9, 123.6 (d, JCF = 3.7 Hz), 120.4, 116.4 (d, JCF = 22.5 Hz), 115.0, 80.0, 66.7, 50.1, 38.8, 30.9, 30.2, 28.3, 18.4, 15.2. IR (thin film): 2983, 2945, 2876, 1668, 1566, 1509, 1494, 1453, 1364, 1286, 1247, 1173, 1094, 1030, 834, 758, 696 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C34H39FN5O5, 616.2935; found, 616.2932. Me Me BocHN O N H O Me N N N H O F Tert-butyl (S,E)-(1-((2-(4-(5-benzyl-2-((2-fluorobenzoyl)imino)-3-methyl-2,3di-hydro-1H-imidazol-4-yl)-phenoxy)ethyl)amino)-3-methyl-1-oxobutan-2-yl) carbamate (3.2). Compound 3.2 was prepared with ZNA 69 (50 mg, 0.11 mmol) and BocL-valine (36.5 mg, 0.17 mmol) following general procedure A1. The crude product was purified via flash column chromatography (1:2 hexanes/EtOAc) to yield a white solid (58.4 mg, 81%). Rf = 0.45 (10:1 CH2Cl2/MeOH). 1H NMR (CDCl3, 500 MHz): δ 8.06 (td, J = 8.0, 2.0 Hz, 1H), 7.39 – 7.35 (m, 1H), 7.30 – 7.26 (m, 4H), 7.20 (t, J = 8.0 Hz, 1H), 7.16 – 7.13 (m, 3H), 7.07 (dd, 11.0, 8.5 Hz, 1H), 6.98 (d, J = 9.0 Hz, 2H), 6.46 (brs, 1H), 5.07 (d, 285 J = 6.5 Hz, 1H), 4.07 (t, J = 5.5 Hz, 2H), 3,91 (t, J = 7.5 Hz, 1H), 3.81 (s, 2H), 3.75 – 3.64 (m, 2H), 3.43 (s, 3H), 2.19 – 2.13 (m, 1H), 1.40 (s, 9H), 0.95 (d, J = 6.5 Hz, 3H), 0.91 (d, J = 7.0 Hz, 3H). 13C NMR (CDCl3, 125 MHz): δ 171.9, 1601.5 (d, JCF = 232.5 Hz), 159.0, 155.9, 137.7, 131.9, 131.9 (d, JCF = 2.5 Hz), 131.7, 128.9, 128.2, 126.9, 123.6 (d, JCF = 3.7 Hz), 120.4, 116.5 (d, JCF = 23.7 Hz), 115.0, 80.0, 66.7, 60.1, 53.8, 38.8, 30.9, 30.1, 29.3, 19.3, 17.8. IR (thin film): 2968, 2934, 1692, 1655, 1562, 1547, 1363, 1238, 1218, 1175, 1071, 1015, 758, 738 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C36H43FN5O5, 644.3248; found, 644.3242. BocHN O N H O Me N N H N O F Tert-butyl (E)-(2-((2-(4-(5-benzyl-2-((2-fluorobenzoyl)imino)-3-methyl-2,3-dihydro-1H-imidazol-4-yl)phenoxy)ethyl)amino)-2-oxoethyl)carbamate (3.3). Compound 3.3 was prepared with ZNA 69 (80 mg, 0.18 mmol) and Boc-L-gylcine (47.3 mg, 0.27 mmol) following general procedure A1. The crude product was purified via flash column chromatography (1:5 hexanes/EtOAc, followed by 1:20 hexanes/EtOAc) to yield a white solid (93.7 mg, 86%). Rf = 0.2 (1:10 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 8.05 (t, J = 8.0 Hz, 1H), 7.34 (q, J = 7.0 Hz, 1H), 7.26 – 7.22 (m, 4H), 7.17 (t, J = 7.5 Hz, 1H), 7.14 – 7.10 (m, 3H), 7.04 (dd, J = 11.0, 8.5 Hz, 1H), 6.95 – 6.91 (m, 3H), 5.41 (brs, 1H), 4.03 (t, J = 6.6 Hz, 2H), 3.79 (s, 2H), 3.77 (brs, 2H), 3.64 (q, J = 6.6 Hz, 2H), 3.40 (s, 3H), 1.39 (s, 9H). 13 C NMR (CDCl3, 125 MHz): δ 169.8, 161.4 (d, JCF = 253.7 Hz), 159.0, 156.1, 137.8, 131.9 (d, JCF = 7.5 Hz), 131.7, 128.9, 128.2, 126.9, 123.7 (d, JCF = 2.5 Hz), 120.4, 116.5 (d, JCF = 22.5 Hz), 115.0, 80.3, 66.6, 44.4, 38.8, 30.9, 30.2, 28.3. 286 IR (thin film): 3061, 2957, 2930, 2871, 1733, 1716, 1699, 1674, 1653, 1646, 1606, 1575, 1512, 1456, 1363, 1288, 1248, 1227, 1179, 756, 696, 688 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C33H37FN5O5, 602.2770; found, 602.2764. H 2N BocHN O O N H O Me N N H N O F Tert-butyl methyl (S,E)-(4-amino-1-((2-(4-(5-benzyl-2-((2-fluorobenzoyl)imino)-3- -2,3-dihydro-1H-imidazol-4-yl)phenoxy)ethyl)amino)-1,4-dioxobutan-2-yl)- carbamate (3.4). Compound 3.4 was prepared with ZNA 69 (80 mg, 0.18 mmol) and BocL-asparagine (62.7 mg, 0.27 mmol) following general procedure A1. The crude product was purified via flash column chromatography (1:20 hexanes/EtOAc, followed by 1:30 hexanes/EtOAc) to yield a white solid (62.0 mg, 52%). Rf = 0.55 (10:1 CH2Cl2/MeOH). 1H NMR (CDCl3, 500 MHz): δ 8.04 (td, J = 7.5, 1.5 Hz, 1H), 7.40 – 7.38 (m, 1H), 7.29 – 7.23 (m, 4H), 7.18 (t, J = 7.5 Hz, 1H), 7.15 – 7.11 (m, 3H), 7.06 (dd, J = 10.5, 8.0 Hz, 1H), 6.96 (d, J = 8.5 Hz, 2H), 6.17 – 6.13 (m, 2H), 5.61 (brs, 1H), 4.45 (brs, 1H), 4.04 (t, J = 6.0 Hz, 2H), 3.80 (s, 2H), 3.64 (q, J = 5.5 Hz, 2H), 3.41 (s, 3H), 2.90 (d, J = 13.5 Hz, 1H), 2.53 (dd, J = 15.5, 6.0 Hz, 1H), 1.40 (s, 9H). 13 C NMR (CDCl3, 125 MHz): δ 173.4, 171.6, 163.9, 161.5 (d, JCF = 250.0 Hz), 160.4, 159.1, 155.8, 137.8, 131.9 (d, JCF = 7.5 Hz), 131.7, 128.8, 128.2, 126.8, 123.7 (d, JCF = 3.7 Hz), 120.4, 116.5 (d, JCF = 22.5 Hz), 115.1, 80.4, 66.6, 51.2, 38.9, 36.8, 30.9, 30.2, 28.3. IR (thin film): 2994, 2885, 1680, 1668, 1564, 1494, 1365, 1287, 1246, 1174, 1050, 835, 759, 696 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C35H40FN6O6, 659.2993; found, 659.2996. 287 O BocHN Boc NH N H O Me N N H N O F Di-tert-butyl (6-((2-(4-(5-benzyl-2-((2-fluorobenzoyl)imino)-3-methyl-2,3- dihydro-1H-imidazol-4-yl)phenoxy)ethyl)amino)-6-oxohexanes-1,5-diyl)-(S,E)dicarbamate (3.5). Compound 3.5 was prepared with ZNA 69 (110 mg, 0.25 mmol) and Boc-Lys-(Boc)-OH (128.6 mg, 0.37 mmol) following general procedure A1. The crude product was purified via flash column chromatography (1:1 hexanes/EtOAc, followed by 1:3 hexanes/EtOAc) to yield a white solid (184.8 mg, 96%). Rf = 0.4 (1:3 hexanes/EtOAc). 1 H NMR (CDCl3, 500 MHz): δ 8.04 (td, J = 7.5, 1.5 Hz, 1H), 7.37 – 7.33 (m, 1H), 7.28 – 7.24 (m, 4H), 7.18 (t, J = 7.5 Hz, 1H), 7.15 – 7.12 (m, 2H), 7.06 (dd, J = 11.0 Hz, 8.0 Hz, 1H), 6.97 (d, J = 8.5 Hz, 2H), 6.93 (t, J = 6.0 Hz, 1H), 5.34 (brs, 1H), 4.70 (brs, 1H), 4.12 – 4.08 (m, 1H), 4.05 (t, J = 5.5 Hz, 2H), 3.81 (s, 2H), 3.70 – 3.58 (m, 2H), 3.46 (q, J = 7.0 Hz, 1H), 3.42 (s, 3H), 3.10 – 2.98 (m, 2H), 1.84 (m, 1H), 1.68 (m, 1H), 1.41 (s, 9H), 1.38 (s, 9H), 1.29 – 1.20 (m, 2H), 1.19 (t, J = 7.0 Hz, 2H). 13 C NMR (CDCl3, 125 MHz): δ 172.5, 161.4 (d, JCF = 253.7 Hz), 159.1, 156.2, 155.8, 148.2, 137.9, 131.9 (d, JCF = 7.5 Hz), 131.6 (d, JCF = 1.8 Hz), 131.6, 128.8, 128.1, 126.7, 124.9, 123.6 (d, JCF = 3.6 Hz), 120.3, 116.5 (d, JCF = 23.2 Hz), 115.0, 79.9, 79.0, 66.7, 54.4, 39.8, 38.8, 32.0, 30.1, 29.6, 29.3, 28.4, 28.3, 22.6. IR (thin film): 2956, 2931, 2872, 1740, 1654, 1618, 1608, 1513, 1500, 1365, 1164, 667, 611 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C42H54FN6O7, 773.4038; found, 773.4037. 288 Me O Me BocHN N H O Me N N N H O F Tert-butyl (R,E)-(1-((2-(4-(5-benzyl-2-((2-fluorobenzoyl)imino)-3-methyl-2,3di-hydro-1H-imidazol-4-yl)phenoxy)ethyl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (3.6). Compound 3.6 was prepared with ZNA 69 (100 mg, 0.22 mmol) and Boc-D-valine (73.0 mg, 0.36 mmol) following general procedure A1. The crude product was purified via flash column chromatography (1:1 hexanes/EtOAc, followed by 1:2 Hexanes/EtOAc) to yield a white solid (136.8 mg, 95%). Rf = 0.5 (10:1 CH2Cl2/MeOH). 1 H NMR (CDCl3, 500 MHz): δ 8.04 (t, J = 7.5 Hz, 1H), 7.34 (q, J = 6.0 Hz, 1H), 7.26 – 7.22 (m, 4H), 7.17 (t, J = 7.5 Hz, 1H), 7.12 (t, J = 7.5 Hz, 3H), 7.05 (dd, J = 11.0, 9.0 Hz, 1H), 6.95 (d, J = 8.5 Hz, 2H), 6.76 (brs, 1H), 5.20 (d, J = 6.0 Hz, 1H), 4.03 (t, J = 5.5, 2H), 3.99 – 3.93 (m, 1H), 3.80 (s, 2H), 3.72 – 3.67 (m, 1H), 3.64 – 3.57 (m, 1H), 3.41 (s, 3H), 2.14 – 2.02 (m, 1H), 1.38 (s, 9H), 0.94 – 0.83 (m, 6H). 13C NMR (CDCl3, 125 MHz): δ 172.0, 161.4 (d, JCF = 252.5), 159.1, 155.9, 148.3, 137.9, 131.9 (d, JCF = 9.2 Hz), 131.6, 128.8, 128.2, 126.8, 126.3, 124.8, 123.6 (d, JCF = 3.7 Hz), 120.4, 116.5 (d, JCF = 23.1 Hz), 115.0, 79.8, 66.6, 60.0, 38.7, 30.9, 30.1, 28.3, 19.2, 17.8. IR (thin film): 2992, 2968, 2873, 1731, 1721, 1659, 1566, 1551, 1365, 1287, 1173, 1019, 760, 687 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C36H43FN5O5, 644.3248; found, 644.3256. 289 O BocHN N H O Me N N H N O F Tert-butyl (E)-(3-((2-(4-(5-benzyl-2-((2-fluorobenzoyl)imino)-3-methyl-2,3-di hydro-1H-imidazol-4-yl)phenoxy)ethyl)amino)-3-oxopropyl)carbamate (3.7). Compound 3.7 was prepared with ZNA 69 (100 mg, 0.22 mmol) and Boc-β-Ala-OH (63.6 mg, 0.37 mmol) following general procedure A1. The crude product was purified via flash column chromatography (1:3 hexanes/EtOAc, followed by 1:4 hexanes/EtOAc) to yield a white solid (139.2 mg, 99%). Rf = 0.15 (1:3 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 7.92 (t, J = 7.5 Hz, 1H), 7.24 – 7.20 (m, 1H), 7.14 – 7.09 (m, 4H), 7.06 (t, J = 7.5 Hz, 1H), 7.00 (t, J = 8.5 Hz, 3H), 6.93 (dd, J = 10.5, 9.0 Hz, 1H), 6.84 (d, J = 8.5 Hz, 2H), 6.51 (brs, 1H), 3.91 (t, J = 5.0 Hz, 2H), 3.68 (s, 2H), 3.51 (q, J = 5.0 Hz, 2H), 3.29 (s, 3H), 3.23 (q, J = 6.0 Hz, 2H), 2.26 (s, 2H), 1.26 (s, 9H). 13C NMR (CDCl3, 125 MHz): δ 171.8, 161.4 (d, JCF = 252.5 Hz), 159.1, 156.1, 148.1, 137.8, 131.9 (d, JCF = 7.6 Hz), 131.6, 131.6, 128.8, 128.1, 126.8, 124.9, 123.6 (d, JCF = 3.5 Hz), 120.3, 116.5 (d, JCF = 23.2 Hz), 115.0, 79.2, 66.7, 38.7, 36.7, 36.2, 30.9, 30.1, 28.3. IR (thin film): 2969, 2926, 1737, 1725, 1528, 1512, 1365, 1228, 1216, 1093, 799, 668 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C34H39FN5O5, 616.2935, found, 616.2929. 290 O N H N Boc O Me N N H N O F Tert-butyl (S,E)-2-((2-(4-(5-benzyl-2-((2-fluorobenzoyl)imino)-3-methyl-2,3- di-hydro-1H-imidazol-4-yl)phenoxy)ethyl)carbamoyl)azetidine-1-carboxylate (3.8). Compound 3.8 was prepared with ZNA 69 (100 mg, 0.22 mmol) and 1-Boc-L-azetidine2-carboxylic acid (67.6 mg, 0.37 mmol) following general procedure A1. The crude product was purified via flash column chromatography (1:20 hexanes/EtOAc) to yield a white solid (127.5 mg, 92%). Rf = 0.12 (1:3 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 8.04 (t, J = 7.5 Hz, 1H), 7.35 – 7.32 (m, 1H), 7.26 – 7.23 (m, 4H), 7.18 (t, J = 7.0 Hz, 1H), 7.13 – 7.10 (m, 3H), 7.05 (dd, J = 11.0, 8.5 Hz, 1H), 6.98 (d, J = 9.0 Hz, 2H), 4.63 (t, J = 6.5 Hz, 1H), 4.08 (t, J = 5.0 Hz, 2H), 3.88 – 3.86 (m, 1H), 3.82 – 3.39 (m, 5H), 3.40 (s, 3H), 2.40 (brs, 2H), 1.39 (s, 9H). 13C NMR (CDCl3, 125 MHz): δ 171.9, 171.6, 161.5 (d, JCF = 252.5 Hz), 159.1, 148.4, 137.8, 131.4 (d, JCF = 9.7 Hz), 131.7, 131.7, 131.6, 128.8, 128.2, 126.0, 126.3, 124.8, 123.6 (d, JCF = 3.5 Hz), 120.3, 116.5 (d, JCF = 23.2 Hz), 115.0, 80.9, 66.8, 62.2, 47.1, 38.6, 30.9, 30.1, 28.2, 19.7. IR (thin film): 2969, 2934, 1566, 1551, 1535, 1365, 1227, 1216, 1141, 896, 761 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C35H39FN5O5, 628.2935; found, 628.2939. 291 General procedure A2 for Boc-deprotection O O HN AA Boc N HCl/MeOH r.t. Me N F N N H HN AA Me N N H O F O To a vial with 2 mL MeOH was added 0.4 mL acetyl chloride dropwise. After 10 min, the resulting 3M methanolic hydrogen chloride solution was added to a 5 mL round bottom flask containing 0.16 mmol of the Boc-protected amino acid. After stirring the reaction mixture at room temperature for 6 h, the organic solvent was removed and the resulting crude product was partitioned between water and EtOAc. The aqueous layer was washed twice with EtOAc, frozen and concentrated under reduced pressure by lyophilization to yield the amino acid as a HCl salt. O Me H 2N N H . HCl O Me N N N H O F (S,E)-N-(5-(4-(2-(2-aminopropanamido)ethoxy)phenyl)-4-benzyl-1-methyl1,3-di-hydro-2H-imidazol-2-ylidene)-2-fluorobenzamide . HCl (ZNA 74). ZNA 74 was prepared with 3.1 (97.3 mg, 0.16 mmol) following general procedure A2 to yield the product as a white solid (78.1 mg, 89%). 1H NMR (CD3OD, 500 MHz): δ 7.94 (t, J = 7.0 Hz, 1H), 7.74 (q, J = 6.5 Hz, 1H), 7.47 – 7.41 (m, 3H), 7.38 (dd, J = 10.5, 8.5 Hz, 1H), 7.33 (t, J = 7.5 Hz, 2H), 7.26 (t, J = 7.5 Hz, 1H), 7.20 – 7.17 (m, 4H), 4.21 (t, J = 5.5 Hz, 2H), 4.05 (s, 2H), 4.02 (q, J = 7.0 Hz, 1H), 3.77 – 3.66 (m, 2H), 3.62 (s, 3H), 1.55 (d, J = 7.0 Hz, 3H). 13C NMR (CDCl3, 125 MHz): δ 176.1, 169.9, 166.3, 163.8, 160.3, 160.3 (d, 292 JCF = 251.25 Hz), 137.0, 136.5, 134.8 (d, JCF = 8.5 Hz), 132.0, 130.7 (d, JCF = 1.5 Hz), 129.2, 128.48, 127.8, 126.6, 126.3, 124.7 (d, JCF = 3.5 Hz), 120.6, 117.5, 116.2 (d, JCF = 22.1 Hz), 115.1, 66.2, 48.9, 38.8, 31.3, 29.3, 16.1. IR (thin film): 2930, 1674, 1558, 1512, 1455, 1287, 1249, 1177, 755, 721, 696, 667 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C29H31FN5O3, 516.2411; found, 516.2414. Me O Me H 2N N H . HCl O Me N N N H O F (S,E)-N-(5-(4-(2-(2-amino-3-methylbutanamido)ethoxy)phenyl)-4-benzyl-1methyl-1,3-dihydro-2H-imidazol-2-ylidene)-2-fluorobenzamide . HCl (ZNA 75). ZNA 75 was prepared with 3.2 (55.4 mg, 0.09 mmol) following general procedure A2 to yield the product as a white solid (50.5 mg, 99%). 1H NMR (CD3OD, 500 MHz): δ 7.89 (td, J = 7.5, 1.5 Hz, 1H), 7.72 – 7.67 (m, 1H), 7.42 – 7.37 (m, 3H), 7.34 (dd, J = 11.0, 9.0 Hz, 1H), 7.28 (t, J = 7.5 Hz, 2H), 7.21 (t, J = 7.5 Hz, 1H), 7.14 (s, 1H), 7.12 (d, J = 8.0 Hz, 2H), 4.22 – 4.13 (m, 2H), 4.01 (s, 2H), 3.82 – 3.77 (m, 1H), 3.67 (d, J = 6.0 Hz, 1H), 3.62 – 3.66 (m, 1H), 3.57 (s, 3H), 2.17 (sextet, J = 7.0 Hz, 1H), 1.04 (d, J = 2.5 Hz, 3H), 1.03 (d, J = 2.5 Hz, 3H). 13 C NMR (CDCl3, 125 MHz): δ 168.4, 163.7, 160.3 (d, JCF = 251.25 Hz), 160.3, 159.3, 137.0, 136.4, 134.8 (d, JCF = 8.5 Hz), 132.1, 130.8, 129.2, 128.5, 127.9, 126.7, 126.3, 124.7 (d, JCF = 3.1 Hz), 120.6 (d, JCF = 12.8 Hz), 117.4, 116.2 (d, JCF = 22.0 Hz), 115.1, 66.2, 58.4, 38.7, 31.6, 30.1, 29.4, 17.4, 16.8. IR (thin film): 3061, 2957, 2930, 2871, 1734, 1716, 1699, 1652, 1600, 1575, 1569, 1512, 1472, 1456, 1250, 757 cm-1. HRMS (ESITOF) [M + H]+ m/z: calcd for C31H35FN5O3, 544.2724; found, 544.2730. 293 O H 2N . HCl N H O Me N N H N O F (E)-N-(5-(4-(2-(2-aminoacetamido)ethoxy)phenyl)-4-benzyl-1-methyl-1,3. dihydro-2H-imidazol-2-ylidene)-2-fluorobenzamide HCl (ZNA 76). ZNA 76 was prepared with 3.3 (93.7 mg, 0.15 mmol) following general procedure A2 to yield the product as a yellow solid (71.1 mg, 85%). 1H NMR (CD3OD, 500 MHz): δ 7.89 (td, J = 8.0, 1.5 Hz, 1H), 7.73 – 7.66 (m, 1H), 7.41 – 7.36 (m, 3H), 7.33 (dd, J = 11.0, 8.5 Hz, 1H), 7.28 (t, J = 7.5 Hz, 2H), 7.21 (t, J = 7.5 Hz, 1H), 7.17 – 7.11 (m, 4H), 4.16 (t, J = 5.5 Hz, 2H), 4.00 (s, 2H), 3.71 (s, 2H), 3.68 (t, J = 5.5 Hz, 2H), 3,57 (s, 3H). 13C NMR (CDCl3, 125 MHz): δ 166.1, 163.7, 160.3, 160.3 (d, JCF = 250.0 Hz), 137.0, 136.4, 134.8 (d, JCF = 8.7 Hz), 132.0, 130.7 (d, JCF = 1.5 Hz), 129.2, 128.5, 127.8, 126.7, 126.3, 124.7 (d, JCF = 3.6 Hz), 120.6, 117.4, 116.2 (d, JCF = 22.1 Hz), 115.1, 66.3, 40.2, 38.7, 31.4, 29.3. IR (thin film): 3049, 2957, 2930, 2870, 1734, 1716, 1699, 1635, 1600, 1569, 1550, 1512, 1456, 1404, 1249, 1226 cm-1. HRMS (ESI-TOF) [M + MeOH + H]+ m/z: calcd for C28H29FN5O3, 556.2336; found, 556.2347. H 2N O N OH N H 2 . HCl O Me N N H N O F (S,E)-2-amino-N1-(2-(4-(5-benzyl-2-((2-fluorobenzoyl)imino)-3-methyl-2,3-dihydro-1H-imidazol-4-yl)phenoxy)ethyl)succinamide . HCl (ZNA 77). ZNA 77 was prepared with 3.4 (62.0 mg, 0.09 mmol) following general procedure A2 to yield the 294 product as a yellow solid (45.0 mg, 80%). 1H NMR (CD3OD, 500 MHz): δ 7.89 (t, J = 7.5 Hz, 1H), 7.73 – 7.66 (m, 1H), 7.39 (d, J = 8.0 Hz, 2H), 7.37 (s, 1H), 7.33 (dd, J = 11.0, 8.5 Hz, 1H), 7.28 (t, J = 7.5 Hz, 2H), 7.22 (t, J = 7.5 Hz, 1H), 7.17 – 7.08 (m, 4H), 4.33 – 4.19 (m, 2H), 4.16 (t, J = 5.0 Hz, 1H), 4.10 (s, 2H), 3.72 – 3.64 (m, 2H), 3.57 (s, 3H), 2.95 – 2.84 (m, 1H), 2.82 – 2.77 (m, 1H). 13C NMR (CDCl3, 125 MHz): δ 172.9, 169.9, 168.3, 167.8, 163.7, 160.3, 160.3 (d, JCF = 250.0 Hz), 159.9, 137.1, 136.4, 134.8 (d, JCF = 9.0 Hz), 132.0, 130.7, 129.2 (d, JCF = 8.5 Hz), 128.5, 127.8, 126.7, 126.3, 124.7 (d, JCF = 3.3 Hz), 120.6, 120.5, 117.6, 117.4, 116.2 (d, JCF = 22.1 Hz), 115.2, 115.1, 66.2, 51.1, 38.1, 34.9, 30.1, 31.4, 29.4. IR (thin film): 2929, 1716, 1683, 1652, 1599, 1550, 1540, 1512, 1496, 1456, 1362, 1291, 1250, 1180, 756 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C30H32FN6O4, 559.2469; found, 559.2460. O H 2N . 2 HCl H 2N N H O Me N N H N O F (S,E)-N-(4-benzyl-5-(4-(2-(2,6-diaminohexanamido)ethoxy)phenyl)-1-methyl1,3-dihydro-2H-imidazol-2-ylidene)-2-fluorobenzamide . 2 HCl (ZNA 78). ZNA 78 was prepared with 3.5 (184.8 mg, 0.24 mmol) following general procedure A2 to yield the product as a yellow solid (130.3 mg, 84%). 1H NMR (CD3OD, 500 MHz): δ 7.90 (td, J = 8.0, 1.5 Hz, 1H), 7.72 – 7.67 (m, 1H), 7.43 – 7.38 (m, 3H), 7.34 (dd, J = 11.0, 8.5 Hz, 1H), 7.29 (t, J = 7.5 Hz, 2H), 7.22 (t, J = 7.5 Hz, 1H), 7.17 – 7.12 (m, 4H), 4.19 (t, J = 5.0 Hz, 2H), 4.01 (s, 2H), 3.95 (t, J = 6.5 Hz, 1H), 3.69 (t, J = 5.0 Hz, 2H), 3.58 (s, 3H), 2.93 (t, J = 7.5 Hz, 2H), 1.98 (tt, J = 23.5, 7.5 Hz, 2H), 1.73 (quintet, J = 7.5 Hz, 2H), 1.57 – 1.49 (m, 2H). 13C NMR (CDCl3, 125 MHz): δ 168.9, 163.1, 160.3 (d, JCF = 251.2 Hz), 160.3, 295 137.0, 136.5, 134.8 (d, JCF = 8.7 Hz), 132.0, 130.7, 129.2, 128.5, 127.8, 126.7, 126.8, 124.7 (d, JCF = 3.5 Hz), 120.6 (d, JCF = 12.8 Hz), 117.4, 116.2 (d, JCF = 22.2 Hz), 66.2, 52.8, 38.9, 38.7, 31.4, 30.7, 29.3, 26.64 21.6. IR (thin film): 2956, 2930, 2871, 1771, 1733, 1716, 1683, 1646, 1600, 1575, 1540, 1512, 1472, 1456, 1436, 1250, 1228, 1180 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C32H38FN6O3, 573.2989; found, 573.2991. Me O Me H 2N N H O . HCl Me N N N H O F (R,E)-N-(5-(4-(2-(2-amino-3-methylbutanamido)ethoxy)phenyl)-4-benzyl-1methyl-1,3-dihydro-2H-imidazol-2-ylidene)-2-fluorobenzamide . HCl (ZNA 94). ZNA 94 was prepared with 3.6 (136.8 mg, 0.21 mmol) following general procedure A2 to yield the product as a yellow solid (114.0 mg, 93%). 1H NMR (CD3OD, 500 MHz): δ 7.90 (t, J = 7.0 Hz, 1H), 7.69 (dd, J = 13.0, 7.0 Hz, 1H), 7.43 – 7.37 (m, 3H), 7.33 (dd, J = 11.0, 8.5 Hz, 1H), 7.28 (t, J = 7.0 Hz, 2H), 7.21 (t, J = 7.0 Hz, 1H), 7.16 – 7.10 (m, 4H), 4.18 (s, 2H), 4.01 (s, 2H), 3.84 – 3.76 (m, 1H), 3.70 (d, J = 5.5 Hz, 1H), 3.63 – 3.53 (m, 4H), 2.18 (sextet, J = 6.5 Hz, 1H), 1.05 (d, J = 1.0 Hz, 3H), 1.03 (d, J = 1.0 Hz, 3H). 13 C NMR (CDCl3, 125 MHz): δ 168.4, 163.8, 160.3 (d, JCF = 251.2 Hz), 160.2, 137.1, 136.6, 134.8 (d, JCF = 9.0 Hz), 132.0, 130.7 (d, JCF = 1.5 Hz), 129.1, 128.5, 127.8, 126.6, 126.3, 124.7 (d, JCF = 3.5 Hz), 120.7, 120.6, 117.5, 116.2 (d, JCF = 22.1 Hz), 115.0, 66.2, 58.4, 38.7, 31.3, 30.1, 29.4, 17.4, 16.8. IR (thin film): 2930, 1749, 1699, 1652, 1635, 1608, 1550, 1540, 1512, 1456, 1288, 1250, 1178, 667 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C31H35FN5O3, 544.2724; found, 544.2730. 296 O H 2N O N H Me N . HCl N N H O F (E)-N-(5-(4-(2-(3-aminopropanamido)ethoxy)phenyl)-4-benzyl-1-methyl-1,3di-hydro-2H-imidazol-2-ylidene)-2-fluorobenzamide . HCl (ZNA 95). ZNA 95 was prepared with 3.7 (127.5 mg, 0.21 mmol) following general procedure A2 to yield the product as a yellow solid (76.8 mg, 67%). 1H NMR (CD3OD, 500 MHz): δ 7.90 (t, J = 7.5 Hz, 1H), 7.69 (dd, J = 13.0, 7.5 Hz, 1H), 7.44 – 7.37 (m, 3H), 7.34 (dd, J = 10.5, 8.5 Hz, 1H), 7.28 (t, J = 7.5 Hz, 2H), 7.21 (t, J = 7.5 Hz, 1H), 7.18 – 7.11 (m, 4H), 4.14 (t, J = 5.0 Hz, 2H), 4.01 (s, 3H), 3.63 (t, J = 5.0 Hz, 2H), 3.58 (s, 3H), 3.20 (t, J = 5.5 Hz, 2H), 2.66 (t, J = 6.0 Hz, 2H). 13C NMR (CDCl3, 125 MHz): δ 171.1, 163.7, 161.4, 160.3 (d, JCF = 251.2 Hz), 151.2, 137.0, 136.4, 134.8 (d, JCF = 34.0 Hz), 132.0, 130.8 (d, JCF = 5.0 Hz), 129.2, 128.5, 127.9, 126.7, 126.2, 124.7 (d, JCF = 3.5 Hz), 120.6 (d, JCF = 13.0 Hz), 117.3, 116.2 (d, JCF = 22.2 Hz), 115.1, 66.4, 38.5, 35.8, 31.5, 31.4, 29.3. IR (thin film): 3060, 2957, 2930, 2870, 1734, 1716, 1683, 1662, 1600, 1558, 1512, 1288, 1248, 1226, 1179, 754, 722, 668 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C29H31FN5O3, 516.2411; found, 516.2414. O N H . HCl N H O Me N N H N O F (S,E)-N-(2-(4-(5-benzyl-2-((2-fluorobenzoyl)imino)-3-methyl-2,3-dihydro-1Himida-zol-4-yl)phenoxy)ethyl)azetidine-2-carboxamide . HCl (ZNA 96). ZNA 96 was 297 prepared with 3.8 (139.2 mg, 0.22 mmol) following general procedure A2 to yield the product as a yellow solid (68.5 mg, 55%). 1H NMR (CD3OD, 500 MHz): δ 7.90 (t, J = 7.0 Hz, 1H), 7.69 (dd, J = 13.0, 7.0 Hz, 1H), 7.43 – 7.36 (m, 3H), 7.34 (dd, J = 10.5, 8.5 Hz, 1H), 7.28 (t, J = 7.5 Hz, 2H), 7.21 (t, J = 7.5 Hz, 1H), 7.17 – 7.12 (m, 4H), 5.05 (t, J = 8.5 Hz, 1H), 4.18 (t, J = 5.0 Hz, 2H), 4.12 (t, J = 9.0 Hz, 1H), 4.01 (s, 2H), 3.95 (dd, J = 15.5, 10.0 Hz, 1H), 3.70 (t, J = 5.0 Hz, 2H), 3.58 (s, J = 3H), 2.87 – 2.80 (m, 1H), 2.55 – 2.48 (m, 1H). 13C NMR (CDCl3, 125 MHz): δ 167.8, 163.9, 160.3 (d, JCF = 251.2 Hz), 160.3, 137.2, 136.6, 134.8 (d, JCF = 8.8 Hz), 132.0, 130.8 (d, JCF = 1.25 Hz), 129.1, 128.5, 127.9, 126.6, 126.3, 124.7 (d, JCF = 3.5 Hz), 120.7 (d, JCF = 12.8 Hz), 117.6, 116.2 (d, JCF = 22.1 Hz), 115.1, 66.3, 58.6, 43.7, 38.9, 31.4, 29.4, 23.5. IR (thin film): 3061, 2956, 2930, 2870, 1734, 1716, 1699, 1646, 1635, 1600, 1575, 1512, 1456, 1249, 1226, 756, 733 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C30H31FN5O3, 528.2411; found, 528.2418. General procedure A3 for the A3-coupling reaction NHR RHN H O N H Me CuBr, MeCN 80 oC N Me To a 25 mL pressure flask equipped with a stir bar were added 0.16 mmol of the aldehyde, N-allylmethylamine 3.12 (0.07 mL, 0.73 mmol), phenylacetylene 3.13 (0.09 mL, 0.85 mmol), CuBr (2.3 mg, 0.016 mmol), acetonitrile (5 mL), and 10 mg of oven-dried 4 Å molecular sieves. The flask was heated at 80 °C for 24 h and then allowed to cool to room temperature. The mixture was filtered through Celite and rinsed with EtOAc (20 mL). The organic layer was washed with sat. aq. NaHCO3 (15 mL), brine (15 mL) and dried 298 over Na2SO4. After filtration, the organic layer was concentrated under reduced pressure and purified via flash column chromatography to obtain the desired propargyl amine. H N Me O Me N N-(4-(1-(allyl(methyl)amino)-3-phenylprop-2-yn-1-yl)phenyl)-acetamide (3.14). Compound 3.14 was prepared with N-(4-formylphenyl)-acetamide 3.9 (100.0 mg, 0.16 mmol) following general procedure A3. The crude product was purified via flash column chromatography (1:1 hexanes/EtOAc) to yield a yellow semisolid (28.8 mg, 56%). Rf = 0.3 (1:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 8.51 (s, 1H), 7.59 – 7.56 (m, 4H), 7.54 – 7.52 (m, 2H), 7.36 – 7.30 (m, 3H), 5.91 (ddt, J = 16.7, 10.0, 7.0 Hz, 1H), 5.29 (d, J = 9.9 Hz, 1H), 5.17 (d, J = 6.0 Hz, 1H), 4.91 (s, 1H), 3.20 – 3.13 (m, 2H), 2.22 (s, 3H), 2.15 (s. 3H). 13 C NMR (CDCl3, 125 MHz): δ 169.2, 137.6, 136.4, 134.7, 131.84, 128.9, 128.4, 128.2, 123.2, 119.9, 117.7, 88.4, 84.9, 59.4, 57.8, 37.8, 24.4. IR (thin film): 3004, 2968, 2933, 1365, 1228, 1216, 1205, 896, 800, 706 cm-1. HRMS (ESI-TOF) [M + Na]+ m/z: calcd for C21H22N2ONa, 341.1630; found, 341.1628. Me O S H N O Me N N-(4-(1-(allyl-(methyl)amino)-3-phenylprop-2-yn-1-yl)phenyl)-methanesulfonamide (3.15). Compound 3.15 was prepared with N-(4-formylphenyl)methanesulfonamide 3.10 (840.0 mg, 4.21 mmol) following general procedure A3. The 299 crude product was purified via flash column chromatography (2:1 hexanes/EtOAc) to a yield yellow solid (635.0 mg, 41%). Rf = 0.5 (1:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 7.63 (d, J = 8.5, 2H), 7.53 – 7.51 (m, 2H), 7.34 (t, J = 3.5 Hz, 3H), 7.22 (d, J = 8.5, 2H), 6.81 (brs, 1H), 5.99 (ddt, J = 16.7, 10.0, 7.0 Hz, 1H), 5.28 (dd, J = 17.0, 1.0 Hz, 1H), 5.17 (d, J = 10.0 Hz, 1H), 4.94 (s, 1H), 3.17 (d, J = 7.0, 2H), 3.01 (s, 3H). 13C NMR (CDCl3, 125 MHz): δ 136.3, 135.99, 135.97, 131.8, 129.7, 128.3, 128.3, 123.0, 120.5, 117.8, 88.6, 84.4, 59.1, 57.8, 39.3, 37.7. IR (thin film): 3262, 3022, 2969, 1737, 1509, 1489, 1413, 1326, 1287, 1217, 1151, 1017, 967, 917, 756 cm-1. HRMS (ESI-TOF) [M + Na]+ m/z: calcd for C20H22N2O2SNa, 377.1300; found, 377.1306. BocHN Tert-butyl Me N (4-(1-(allyl(methyl)amino)-3-phenylprop-2-yn-1-yl)phenyl)- carbamate (3.16). Compound 3.16 was prepared with tert-butyl-(4-formylphenyl-) carbamate 3.11 (100.0 mg, 0.45 mmol) following general procedure A3. The crude product was purified via flash column chromatography (2:1 hexanes/EtOAc) to yield a yellow semisolid (119.4 mg, 70%). Rf = 0.5 (4:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 7.55 – 7.53 (m, 2H), 7.52 – 7.50 (m, 2H), 7.36 – 7.32 (m, 5H), 6.59 (s, 1H), 5.94 – 5.85 (ddt, J = 16.7, 10.0, 7.0 Hz, 1H), 5.26 (dd, J = 17.5, 1.5 Hz, 1H), 5.15 (d, J = 10.5 Hz, 1H), 4.91 (s, 1H), 3.18 – 3.11 (m, 2H), 2.21 (s, 3H), 1.52 (s, 9H). 13C NMR (CDCl3, 125 MHz): δ 153.0, 144.5, 138.0, 136.4, 132.0, 131.5, 129.3, 128.5, 128.4, 123.5, 118.1, 88.5, 85.3, 59.6, 57.9, 38.0, 28.6, 28.5. IR (thin film): 2977, 2893, 1691, 1592, 1528, 1412, 1316, 1261, 1156, 1053, 799, 667, 611 cm-1. HRMS (ESI-TOF) [M + Na]+ m/z: calcd for 300 C24H28N2O2Na, 399.2048; found 399.2055. General procedure A4 for the deallylation reaction NHR NHR DMBA Pd(PPh3)4 N Me CH2Cl2, r.t. N H Me To a 25-mL oven-dried round bottom flask equipped with a stir bar was added 0.09 mmol of the propargylamine, DMBA (28.4 mg, 0.18 mmol), Pd(PPh3)4 (5.26 mg, 0.0045 mmol) and 10 mL anhydrous CH2Cl2. The reaction mixture was allowed to stir at room temperature under N2 for 16 h. The reaction mixture was concentrated and redissolved in EtOAc (20 mL). The organic layer was washed with sat. aq. NaHCO3 (15 mL), brine (15 mL) and dried over Na2SO4. After filtration, the organic layer was concentrated under reduced pressure and purified via flash column chromatography. H N Me O Me NH N-(4-(1-(methylamino)-3-phenylprop-2-yn-1-yl)phenyl)acetamide (3.17). Compound 3.17 was prepared with 3.14 (29.1 mg, 0.09 mmol) following general procedure A4. The crude product was purified via flash column chromatography (1:1 hexanes/acetone, followed by 1:2 hexanes/acetone) to yield a white solid (19.1 mg, 75%). Rf = 0.5 (1:1 hexanes/acetone). 1H NMR (CDCl3, 300 MHz): δ 8.28 (s, 1H), 7.51 – 7.44 (m, 6H), 7.29 – 7.28 (m, 3H), 4.69 (s, 1H), 2.51 (s, 3H), 2.11 (s, 3H), 1.99 (brs, 1H). 13C NMR (CDCl3, 125 MHz): δ 169.0, 137.8, 135.6, 131.7, 128.3, 128.2, 128.2, 122.9, 120.1, 301 88.7, 85.8, 55.7, 33.6, 24.4. Me O S H N O Me NH N-(4-(1-(methylamino)-3-phenylprop-2-yn-1-yl)phenyl)-methanesulfonamide (3.18). Compound 3.18 was prepared with 3.15 (635.0 mg, 1.72 mmol) following general procedure A4. The crude product was purified via flash column chromatography (30:1 CH2Cl2/MeOH, followed by 15:1 CH2Cl2/MeOH) to yield a white solid (340.0 mg, 60%). Rf = 0.1 (1:1 Hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 7.55 (d, J = 8.0, 2H), 7.46 (t, J = 3.5, 2H), 7.32 – 7.31 (m, 3H), 7.20 (d, J = 8.0 Hz, 2H), 4.72 (s, 1H), 2.99 (s, 3H), 2.55 (s, 3H). 13 C NMR (CDCl3, 125 MHz): δ 137.3, 136.3, 131.7, 129.0, 128.3, 122.8, 120.8, 88.4, 86.0, 55.6, 39.4, 33.7. IR (thin film): 3311, 2969, 2933, 1486, 1339, 1321, 1287, 1225, 1146, 1080, 967, 912, 853, 750, 688 cm-1. HRMS (ESI-TOF) [M + Na]+ m/z: calcd for C17H18N2O2SNa, 337.0987; found, 337.0995. BocHN Tert-butyl Me NH (4-(1-(methylamino)-3-phenylprop-2-yn-1-yl)phenyl)carbamate (3.19). Compound 3.19 was prepared with 3.16 (119.0 mg, 0.31 mmol) following general procedure A4. The crude product was purified via flash column chromatography (2:1 hexanes/acetone) to yield a yellow solid (72.5 mg, 48%). Rf = 0.15 (1:1 hexanes/EtOAc). 1 H NMR (CDCl3, 300 MHz): δ 7.49 – 7.45 (m, 4H), 7.37 – 7.29 (m, 5H), 6.73 (brs, 1H), 4.70 (s, 1H), 2.53 (s, 3H), 1.51 (s, 9H). 13C NMR (CDCl3, 75 MHz): δ 153.0, 138.2, 134.9, 302 131.9, 128.5, 128.5, 128.4, 123.4, 118.8, 89.2, 85.8, 80.7, 55.9, 33.8, 28.6. General procedure A5 for the synthesis of N-acyl propargyl guanidines NHR RHN O N N H F N H Me N TMSCl, DIPEA CH2Cl2, r.t. H 2N Me N F O A 25-mL oven-dried round bottom flask was charged with N-cyano-2fluorobenzamide (42.3 mg, 0.26 mmol) and 5 mL anhydrous CH2Cl2. Then DIPEA (0.09 mL, 0.53 mmol) and TMSCl (0.034 mL, 0.27 mmol) were added via syringe. The reaction mixture was stirred for 20 min and 0.21 mmol of the amine dissolved in 5 mL CH2Cl2, was added to the stirring mixture. After 3 h, the reaction was quenched with sat. aq. NaHCO3 solution and extracted two times with 10 mL CH2Cl2. After removal of the organic solvent, the crude product was purified via column chromatography to yield the N-propargyl guanidine. H N Me O Me N N NH 2 O F (E)-N-(((1-(4-acetamidophenyl)-3-phenylprop-2-yn-1-yl)(methyl)amino)(amino)-methylene)-2-fluorobenzamide (3.20). Compound 3.20 was prepared with 3.17 (72.5 mg, 0.21 mmol) following general procedure A5. The crude product was purified via flash column chromatography (1:1 hexanes/EtOAc) to yield a yellow semisolid (81.4 mg, 75%). Rf = 0.25 (1:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 8.40 (s, 1H), 8.01 303 (td, J = 7.5, 1.5 Hz, 1H), 7.58 – 7.45 (m, 6H), 7.36 – 7.29 (m, 4H), 7.10 (t, J = 7.5 Hz, 1H), 7.02 (dd, J = 10.5, 8.5 Hz, 1H), 2.82 (s, 3H), 2.12 (s, 3H). 13C NMR (CDCl3, 125 MHz): δ 175.4, 175.4, 169.1, 161.6 (d, JCF = 255.0 Hz), 160.5, 138.2, 132.7, 132.0 (d, JCF = 8.7 Hz), 131.8, 131.7 (d, J = 1.5 Hz), 128.7, 128.4, 128.0, 127.6 (d, JCF = 9.0 Hz), 123.5 (d, JCF = 3.7 Hz), 122.3, 120.2, 116.6 (d, JCF = 23.1 Hz), 87.0, 84.8, 50.8, 31.6, 24.4. IR (thin film): 2968, 2930, 1511, 1365, 1216, 1205, 799, 761, 668 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C26H24FN4O2, 443.1883; found, 443.1878. Me O S H N O Me N N NH 2 O F (E)-N-(amino(methyl(1-(4-(methylsulfonamido)phenyl)-3-phenylprop-2-yn-1yl)-amino)methylene)-2-fluorobenzamide (3.21). Compound 3.21 was prepared with 3.18 (100.0 mg, 0.30 mmol) following general procedure A5. The crude product was purified via flash column chromatography (1:2 hexanes/EtOAc) to yield a white solid (121.8 mg, 84%). Rf = 0.25 (1:2 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 8.01 (t, J = 7.5 Hz, 1H), 7.65 (brs, 1H), 7.58 (d, J = 8.5 Hz, 2H), 7.53 – 7.47 (m, 2H), 7.36 – 7.31 (m, 4H), 7.26 (d, J = 8.5 Hz, 2H), 7.12 (t, J = 7.5 Hz, 1H), 7.04 (dd, J = 11.0, 8.5 Hz, 1H), 3.00 (s, 3H), 2.86 (s, 3H). 13C NMR (CDCl3, 125 MHz): δ 175.3, 175.3, 161.6 (d, JCF = 253.7 Hz), 160.4, 137.0, 134.1, 132.1 (d, JCF = 8.8 Hz), 131.9, 131.8 (d, JCF = 1.2 Hz), 128.9, 128.8, 128.5, 127.5 (d, JCF = 9.0 Hz), 123.3 (d, JCF = 3.7 Hz), 122.2, 120.7, 116.6 (d, JCF = 23.0 Hz), 87.2, 84.5, 65.9, 50.7, 39.4, 29.3. IR (thin film): 2969, 2925, 1587, 1555, 1530, 1425, 1355, 1324, 1216, 11148, 1056, 968, 896, 754, 689 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C25H24FN4O3S, 479.1553; found 479.1557. 304 BocHN Me N N NH 2 O Tert-butyl F (E)-(4-(1-(2-(2-fluorobenzoyl)-1-methylguanidino)-3-phenylprop- 2-yn-1-yl)phenyl)carbamate (3.22). Compound 3.22 was prepared with 3.19 (72.5 mg, 0.21 mmol) following general procedure A5. The crude product was purified via flash column chromatography (1:1 hexanes/EtOAc) to yield a yellow semisolid (81.4 mg, 75%). Rf = 0.2 (1:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 8.04 (td, J = 7.5, 1.5 Hz, 1H), 7.63 (brs, 1H), 7.53 (d, J = 8.5 Hz, 2H), 7.52 – 7.48 (m, 2H), 7.37 (d, J = 8.5 Hz, 2H), 7.35 – 7.31 (m, 3H), 7.12 (t, J = 7.5 Hz, 1H), 7.05 (dd, J = 11.0, 8.0 Hz, 1H), 6.69 (s, 1H), 2.84 (s, 3H), 1.50 (s, 9H). 13C NMR (CDCl3, 125 MHz): δ 175.6, 170.4, 162.0 (d, JCF = 255.0 Hz), 160.7, 153.0, 138.6, 132.2, 132.1, 131.9, 128.9, 128.6, 128.4, 127.9 (d, JCF = 8.7 Hz), 123.6 (d, JCF = 3.7 Hz), 122.6, 118.9, 116.8 (d, JCF = 23.1 Hz), 87.1, 85.2, 80.9, 51.0, 29.5, 28.5. IR (thin film): 2977, 2892, 1704, 1592, 1529, 1471, 1316, 1239, 1158, 1053, 1017, 897, 759 cm-1. HRMS (ESI-TOF) [M + Na]+ m/z: calcd for C29H29FN4O3Na, 523.2121; found, 523.2125. 305 General procedure A6 for the NaH-mediated cyclization of N-acyl propargyl guanidines RHN RHN NaH, THF N H 2N Me r.t. N F N Me N N H F O O In a 25 mL round-bottom flask containing a magnetic stir bar were added 0.66 mmol of the N-acyl propargyl guanidine and 10 mL THF under N2. To the solution was added NaH (32.0 mg, 0.80 mmol) at room temperature. The reaction mixture was stirred for 60 min, after which the solvent was removed under reduced pressure, and the crude product was redissolved in EtOAc (25 mL). The organic layer was washed with sat. aq. NH4Cl (10 mL) and brine (10 mL), dried over Na2SO4, filtered, and concentrated. The resulting crude product was purified via flash column chromatography to yield the desired N2-acyl 2-aminoimidazole. H N Me O Me N N H N O F (E)-N-(5-(4-acetamidophenyl)-4-benzyl-1-methyl-1,3-dihydro-2H-imidazol-2ylidene)-2-fluorobenzamide (ZNA 109). ZNA 109 was prepared with 3.20 (295.2 mg, 0.66 mmol) following general procedure A6. The crude product was purified via flash column chromatography (3:2 hexanes/acetone, followed by 1:1 hexanes/acetone) to yield a white solid (113.6 mg, 38%). Rf = 0.35 (1:1 hexanes/acetone). 1H NMR (CDCl3, 500 MHz): δ 8.08 (t, J = 7.5 Hz, 1H), 7.68 – 7.61 (m, 2H), 7.47 (t, J = 7.5 Hz, 1H), 7.41 – 7.38 306 (m, 1H), 7.36 – 7.28 (m, 5H), 7.22 (t, J = 7.5 Hz, 1H), 7.20 – 7.12 (m, 3H), 7.10 (dd, J = 11.0, 9.0 Hz, 1H), 3.84 (s, 2H), 3.47 (s, 3H), 2.23 (s, 3H). IR (thin film): 2956, 2930, 1675, 1606, 1514, 1463, 1319, 1257, 1179, 842, 756 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C26H24FN4O2, 443.1883; found, 443.1879. Me S H N O O Me N N H N O F (E)-N-(4-benzyl-1-methyl-5-(4-(methylsulfonamido)phenyl)-1,3-dihydro-2Himidazol-2-ylidene)-2-fluorobenzamide (ZNA 119). ZNA 119 was prepared with 3.21 (121.8 mg, 0.25 mmol) and 2.5 equiv. of NaH at 50 oC. The crude product was purified via flash column chromatography (40:1 CH2Cl2/MeOH) to yield a white solid (85.7 mg, 70%). Rf = 0.3 (30:1 CH2Cl2/MeOH). 1H NMR (CDCl3, 500 MHz): δ 12.12 (brs, 1H), 8.09 (t, J = 7.5 Hz, 1H), 7.41 (brs, 1H), 7.36 – 7.28 (m, 6H), 7.23 – 7.09 (m, 5H), 3.86 (s, 2H), 3.48 (s, 3H), 3.08 (s, 3H). IR (thin film): 2956, 2929, 2870, 1733, 17.16, 1699, 1652, 1608, 1559, 1539, 1514, 1525, 1475, 1366, 1332, 1301, 1225, 1141, 775, 760, 737 cm-1. HRMS (ESITOF) [M + H]+ m/z: calcd for C25H24FN4O3S, 479.1553; found, 479.1554. BocHN Me N N N H O F Tert-butyl (E)-(4-(5-benzyl-2-((2-fluorobenzoyl)imino)-3-methyl-2,3-dihydro1H-imidazol-4-yl)phenyl)carbamate (3.23). Copound 3.23 was prepared with 3.22 (81.4 mg, 0.16 mmol) following general procedure A6. The crude product was purified via flash column chromatography (2:1 hexanes/acetone) to yield a white solid (47.4 mg, 58%). Rf = 307 0.4 (1:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 8.07 (td, J = 8.0, 2.0 Hz, 1H), 7.49 (d, J = 8.5 Hz, 2H), 7.36 (q, J = 7.5 Hz, 1H), 7.30 – 7.24 (m, 4H), 7.20 (t, J = 7.5 Hz, 1H), 7.17 – 7.11 (m, 3H), 7.08 (dd, J = 11.5, 8.0 Hz, 1H), 6.80 (brs, 1H), 3.91 (s, 2H), 3.43 (s, 3H), 1.52 (s, 9H). 13 C NMR (CDCl3, 125 MHz): δ 161.5 (d, JCF = 253.7 Hz), 152.6, 139.3, 137.7, 131.9 (d, JCF = 8.7 Hz), 131.7 (d, JCF = 1.8 Hz), 131.0, 128.9, 128.2, 126.8, 123.6 (d, JCF = 3.3 Hz), 122.1, 118.7, 116.5 (d, JCF = 23.2 Hz), 81.0, 31.0, 30.2, 28.3. IR (thin film): 2956, 2928, 1723, 1703, 1568, 1525, 1482, 1366, 1315, 1237, 1159, 1056, 840, 758 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C29H30FN4O3, 501.2302; found, 501.2303. H 2N Me N N N H O F (E)-N-(5-(4-aminophenyl)-4-benzyl-1-methyl-1,3-dihydro-2H-imidazol-2ylidene) -2-fluorobenzamide (3.24). A 25-mL round bottom flask was charged with the Boc-protected aniline 3.23 (47.4 mg, 0.09 mmol) and 3 mL CH2Cl2. To the stirring mixture are room temperature was added 3 mL TFA. The reaction was completed after 2 h and quenched with aq. NaHCO3. The crude was extracted two times with CH2Cl2. After removing the organic solvent, the crude product was loaded on the column (30:1 CH2Cl2/MeOH) to yield a pale yellow solid (37.2 mg, 99%). Rf = 0.3 (1:1 hexanes/EtOAc). 1 H NMR (CDCl3, 500 MHz): δ 8.05 (t, J = 7.0 Hz, 1H), 7.39 (q, J = 6.0, 1H), 7.27 (t, J = 7.5 Hz, 2H), 7.20 (t, J = 7.0 Hz, 1H), 7.17 – 7.12 (m, 3H), 7.11 – 7.05 (m, 3H), 6.73 (d, J = 8.0 Hz, 2H), 3.81 (s, 2H), 3.43 (s, 3H). 13C NMR (CDCl3, 125 MHz): 172.1, 161.5 (d, JCF = 252.5 Hz), 148.9, 147.4, 137.9, 131.7, 131.7 (d, JCF = 1.8 Hz), 131.4, 128.9, 128.2, 308 126.8, 125.3, 123.6 (d, JCF = 3.6 Hz), 116.8, 116.5 (d, JCF = 23.1 Hz), 115.0, 30.8, 30.0. IR (thin film): 2969, 2945, 1737, 1612, 1563, 1518, 1451, 1355, 1287, 1215, 1178, 902, 830, 791, 756, 694, 667 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C24H22FN4O, 401.1778; found, 401.1783. General procedure A7 for the peptide coupling of 3.24 H 2N AA NH N Me N N H + Boc-AA-OH F HATU, DIPEA N DMF, r.t. - 50 oC Me N N H O F O 3.24 A 25-mL oven-dried round bottom flask was charged with Boc-AA-OH (97.5 mg, 0.45 mmol), 10 mL DMF, HATU (332.3 mg, 0.87 mmol) and DIPEA (0.17 mL, 0.99 mmol). The resulting reaction mixture was stirred for 30 min. Then 3.24 (100 mg, 0.25 mmol) was added in one portion. The reaction was heated to 50 oC for 24 h, quenched with 20 mL H2O and washed two times with 15 mL CH2Cl2. After removing the organic solvent, the crude product was purified via flash column chromatography to yield the amide. Me BocHN Me H N O Me N N N H O F Tert-butyl (S,E)-(1-((4-(5-benzyl-2-((2-fluorobenzoyl)-imino)-3-methyl-2,3-dihydro-1H-imidazol-4-yl)phenyl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (3.25). Compound 3.25 was prepared with 3.24 (100 mg, 0.25 mmol) and Boc-L-valine (97.5 mg, 0.45 mmol) following general procedure A7. The crude product was purified via flash 309 column chromatography (3:1 hexanes/acetone, followed by 2:1 hexanes/acetone) to yield a white solid (50.3 mg, 33%). Rf = 0.2 (3:1 hexanes/acetone). 1H NMR (CDCl3, 500 MHz): δ 8.91 (brs, 1H), 8.03 (td, J = 8.0, 1.5 Hz, 1H), 7.62 (d, J = 8.5 Hz, 2H), 7.38 – 7.33 (m, 1H), 7.26 – 7.19 (m, 4H), 7.19 – 7.09 (m, 4H), 7.06 (dd, J = 11.0, 8.5 Hz, 1H), 5.30 (s, 1H), 4.10 (s, 1H), 3.79 (s, 2H), 3.40 (s, 3H), 2.25 – 2.17 (m, 1H), 1.44 (s, 9H), 1.03 (d, J = 6.5 Hz, 3H), 1.00 (d, J = 7.0 Hz, 3H). IR (thin film): 2964, 2957, 2929, 2875, 1971, 846, 799, 772, 705 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C34H39FN5O4, 600.2986; found, 600.2986. Me BocHN O H N Me N N N H O F Tert-butyl (S,E)-(1-((4-(5-benzyl-2-((2-fluorobenzoyl)imino)-3-methyl-2,3-dihydro-1H-imidazol-4-yl)phenyl)amino)-1-oxopropan-2-yl)carbamate (3.26). Compound 3.26 was prepared with 3.24 (100 mg, 0.25 mmol) and Boc-L-alanine (84.9 mg, 0.45 mmol) following general procedure A7. The crude product was purified via flash column chromatography (50:1 CH2Cl2/MeOH) to yield a white solid (49.0 mg, 34%). Rf = 0.2 (40:1 CH2Cl2/MeOH). 1H NMR (CDCl3, 500 MHz): δ 12.07 (brs, 1H), 8.83 (brs, 1H), 8.05 (td, J = 7.5, 1.5 Hz, 1H), 7.64 (d, J = 8.5 Hz, 2H), 7.37 (q, J = 6.5 Hz, 1H), 7.33 – 7.24 (m, 4H), 7.20 (t, J = 7.5 Hz, 1H), 7.18 – 7.12 (m, 2H), 7.08 (dd, J = 11.0, 8.5 Hz, 1H), 5.04 (d, J = 7.0 Hz, 1H), 4.34 (s, 1H), 3.82 (s, 2H), 3.44 (s, 3H), 1.51 – 1.43 (m, 12H). IR (thin film): 2956, 2931, 2874, 1709, 1687, 1566, 1544, 1528, 1501, 1166, 826, 766, 668 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C32H35FN5O4, 572.2673; found, 572.2673. 310 BocHN H N BocHN Me N O N N H O F Di-tert-butyl (6-((4-(5-benzyl-2-((2-fluorobenzoyl)imino)-3-methyl-2,3- dihydro-1H-imidazol-4-yl)phenyl)amino)-6-oxohexanes-1,5-diyl)-(S,E)-dicarbamate (3.27). Compound 3.27 was prepared with 3.24 (100 mg, 0.25 mmol) and Boc-Lys-(Boc)OH (155.5 mg, 0.45 mmol) following general procedure A7. The crude product was purified via flash column chromatography (60:1 CH2Cl2/MeOH) to yield a white solid (70.0 mg, 38%). Rf = 0.2 (40:1 CH2Cl2/MeOH). 1H NMR (CDCl3, 500 MHz): δ 12.68 (brs, 1H), 8.70 (brs, 1H), 8.06 (td, J = 8.0, 1.5 Hz, 1H), 7.67 (d, J = 8.5 Hz, 2H), 7.39 – 7.35 (m, 1H), 7.34 – 7.26 (m, 4H), 7.20 (t, J = 7.5 Hz, 1H), 7.17 – 7.13 (m, 3H), 7.08 (dd, J = 11.0, 8.5 Hz, 1H), 5.24 (brs, 1H), 4.64 (brs, 1H), 4.20 (brs, 1H), 3.82 (s, 2H), 3.44 (s, 3H), 3.17 – 3.11 (m, 2H), 2.00 – 1.96 (m, 1H), 1.78 – 1.53 (m, 7H), 1.46 (s, 9H), 1.44 (s, 9H). Me H 2N . HCl Me H N O Me N N N H O F (S,E)-N-(5-(4-(2-amino-3-methylbutanamido)phenyl)-4-benzyl-1-methyl-1,3di-hydro-2H-imidazol-2-ylidene)-2-fluorobenzamide . HCl (ZNA 89). ZNA 89 was prepared with 3.25 (50.0 mg, 0.08 mmol) following general procedure A2 to yield the product as a yellow solid (25.3 mg, 56%). 1H NMR (CD3OD, 500 MHz): δ 7.90 (td, J = 7.5, 1.5 Hz, 1H), 7.86 (d, J = 8.5 Hz, 2H), 7.74 – 7.67 (m, 1H), 7.46 (d, J = 8.5 Hz, 2H), 7.39 (t, J = 7.5 Hz, 1H), 7.34 (dd, J = 11.0, 8.5 Hz 1H), 7.28 (t, J = 7.5 Hz, 2H), 7.21 (t, J 311 = 7.5 Hz, 1H), 7.15 (d, J = 7.0 Hz 2H), 4.04 (s, 2H), 3.91 (d, J = 6.0 Hz, 1H), 3.60 (s, 3H), 2.33 (sextet, J = 6.5 Hz, 1H), 1.14 (d, J = 7.0 Hz, 3H), 1.11 (d, J = 7.0 Hz, 3H). 13C NMR (CDCl3, 125 MHz): δ 167.0, 163.8, 160.3 (d, JCF = 250.0 Hz), 139.6, 137.1, 136.8, 134.8 (d, JCF = 9.0 Hz), 131.3, 130.7 (d, JCF = 1.3 Hz), 128.7, 128.5, 127.8, 126.7, 126.6, 124.7 (d, JCF = 3.5 Hz), 121.2, 120.7 (d, JCF = 13.0 Hz), 120.1, 116.2 (d, JCF = 22.0 Hz), 59.0, 31.4, 30.4, 29.4, 17.6, 16.4. IR (thin film): 3061, 2957, 2930, 2871, 1734, 1716, 1683, 1646, 1575, 1540, 1512, 1489, 1472, 1404, 1383, 1289, 1250, 754, 668 cm-1. HRMS (ESITOF) [M + H]+ m/z: calcd for C29H31FN5O2, 500.2462; found, 500.2470. Me H 2N . HCl O H N Me N N N H O F (S,E)-N-(5-(4-(2-aminopropanamido)phenyl)-4-benzyl-1-methyl-1,3-dihydro2H-imidazol-2-ylidene)-2-fluorobenzamide . HCl (ZNA 90). ZNA 90 was prepared with 3.26 (49.0 mg, 0.08 mmol) following general procedure A2 to yield the product as a yellow solid (40.6 mg, 94%). 1H NMR (CD3OD, 500 MHz): δ 7.90 (t, J = 7.0 Hz, 1H), 7.84 (d, J = 9.0 Hz, 2H), 7.69 (dd, J = 12.5, 7.0 Hz, 1H), 7.45 (d, J = 8.0 Hz, 2H), 7.39 (t, J = 8.0 Hz, 1H), 7.34 (dd, J = 11.0, 8.5 Hz, 1H), 7.28 (t, J = 7.5 Hz, 2H), 7.21 (t, J = 7.5 Hz, 1H), 7.15 (d, J = 7.5 Hz, 2H), 4.15 (q, J = 7.0 Hz, 1H), 4.03 (s, 2H), 3.60 (s, 3H), 1.63 (d, J = 7.0 Hz, 3H). 13C NMR (CDCl3, 125 MHz): δ 168.2, 163.7, 160.3 (d, JCF = 251.2 Hz), 139.9, 136.9, 136.7, 134.8 (d, JCF = 8.7 Hz), 131.2, 130.7 (d, JCF = 1.5 Hz), 128.8, 128.5, 127.8, 126.7, 126.6, 124.7 (d, JCF = 3.5 Hz), 120.9, 120.6 (d, JCF = 12.6 Hz), 120.1, 116.2 (d, JCF = 22.1 Hz), 49.6, 31.4, 29.3, 16.2. IR (thin film): 2929, 1699, 1625, 1600, 1558, 1539, 1513, 1499, 1456, 1288, 1252, 754, 697 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C27H27FN5O2, 312 472.2149; found, 472.2155. H 2N . 2 HCl H N H 2N O Me N N N H O F (S,E)-N-(4-benzyl-5-(4-(2,6-diaminohexanamido)phenyl)-1-methyl-1,3dihydro-2H-imidazol-2-ylidene)-2-fluorobenzamide . 2 HCl (ZNA 91). ZNA 91 was prepared with 3.27 (70.0 mg, 0.09 mmol) following general procedure A2 to yield the product as a yellow solid (57.1.1 mg, 99%). 1H NMR (D2O, 500 MHz): δ 7.70 (t, J = 7.0 Hz, 1H), 7.59 – 7.50 (m, 3H), 7.31 (d, J = 8.5 Hz, 2H), 7.24 (t, J = 7.5 Hz, 1H), 7.22 – 7.13 (m, 3H), 7.12 (t, J = 7.5 Hz, 1H), 7.01 (d, J = 7.0 Hz, 2H), 4.08 (t, J = 6.5 Hz, 1H), 3.88 (s, 2H), 3.41 (s, 3H), 2.88 (t, J = 7.5 Hz, 2H), 1.93 (q, J = 7.0 Hz, 2H), 1.62 (q, J = 7.5 Hz, 2H), 1.41 (q, J = 7.5 Hz, 2H). 13C NMR (CDCl3, 125 MHz): δ 167.4, 163.7, 160.3 (d, JCF = 250.0 Hz), 139.8, 137.0, 136.7, 134.8 (d, JCF = 8.6 Hz), 131.3, 130.7 (d, JCF = 1.25 Hz), 128.8, 128.5, 127.8, 126.7, 126.5, 124.7 (d, JCF = 3.3 Hz), 121.1, 120.6 (d, JCF = 13.1 Hz), 120.2, 116.4 (d, JCF = 22.1 Hz), 53.5, 38.9, 31.5, 30.8, 29.3, 26.7, 21.7. IR (thin film): 2956, 2930, 1699, 1608, 1513, 1289, 1252, 1180, 667 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C30H34FN6O2, 529.2727; found, 529.2732. 313 General procedure A8 for the Mitsunobu reaction HO ethylene glycol derived side chains (EG): EG O N prim. alcohol DTAT, PPh3 Me N N H TBSO F N THF, 0 oC - r.t. Me N N H O Me O F 3.28 Me O TBSO ZNA 18 ZNA 117 MeO ZNA 118 3.29 OMe O OMe MeO 3.30 A 50-mL oven-dried round bottom flask was charged with the PPh3 (66.7 mg, 0.26 mmol), DTAT (60.4 mg, 0.26 mmol) and 10 mL anhydrous THF. The solution was stirred at 0 oC. Then ZNA 18 (100 mg, 0.25 mmol) and 0.26 mmol of a primary alcohol dissolved in 5 mL THF were added via syringe. The reaction mixture was allowed to warm up to room temperature and stirred for 72 h. It was quenched with sat. aqueuos NaHCO3-solution and extracted two times with EtOAc. After removing the organic solvent, the crude product was purified via flash column chromatography. TBSO O Me N N H N O F (E)-N-(4-benzyl-5-(4-(2-((tert-butyldimethylsilyl)oxy)ethoxy)phenyl)-1methyl-1,3-dihydro-2H-imidazol-2-ylidene)-2-fluorobenzamide (3.28). Compound 3.28 was prepared with ZNA 18 (100 mg, 0.25 mmol) and 2-((tert-butyldimethylsilyl)oxy) ethanol (46.3 mg, 0.26 mmol) following general procedure A8. The crude product was purified via flash column chromatography (2:1 hexanes/EtOAc) to yield a colorless semisolid (125.4 mg, 90%). Rf = 0.6 (2:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 11.98 (brs, 1H), 8.07 (t, J = 7.5 Hz, 1H), 7.36 (q, J = 6.5 Hz, 1H), 7.31 – 7.24 (m, 4H), 7.20 (t, J = 7.5 Hz, 1H), 7.17 – 7.12 (m, 3H), 7.08 (dd, J = 11.0, 8.5 Hz, 1H), 7.01 (d, J = 8.5 314 Hz, 2H), 4.09 (t, J = 5.0 Hz, 2H), 4.00 (t, J = 5.0 Hz, 2H), 3.82 (s, 2H), 3.43 (s, 3H), 0.92 (s, 9H), 0.11 (s, 6H). 13 C NMR (CDCl3, 125 MHz): δ 171.9, 161.5 (d, JCF = 252.5 Hz), 159.3, 148.8, 137.7, 131.8 (d, JCF = 8.5 Hz), 131.7 (d, JCF = 1.7 Hz), 131.6, 128.9, 128.2, 126.9, 124.8, 123.6, 119.8, 116.5 (d, JCF = 23.1 Hz), 115.1, 69.4, 62.0, 31.6, 30.9, 30.1, 25.9, -5.71. IR (thin film): 2994, 2944, 1737, 1640, 1528, 1365, 1216, 1131, 1016, 829, 803, 667 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C32H39FN3O3Si, 560.2745; found, 560.2747. O O Me O Me Me N N H N O F (E)-N-(4-benzyl-5-(4-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)phenyl)-1methyl-1,3-dihydro-2H-imidazol-2-ylidene)-2-fluorobenzamide (3.29). Compound 3.29 was prepared with ZNA 18 (100 mg, 0.25 mmol) and (2,2-dimethyl-1,3-dioxolan-4yl)methanol (0.1 mL, 0.75 mmol) following general procedure A8. The crude product was purified via flash column chromatography (2:1 hexanes/acetone) to yield a white solid (74.5 mg, 58%). Rf = 0.4 (40:1 CH2Cl2/MeOH). 1H NMR (CDCl3, 500 MHz): δ 8.07 (td, J = 7.5, 2.0 Hz, 1H), 7.04 – 7.34 (m, 1H), 7.30 – 7.24 (m, 4H), 7.21 (t, J = 7.5 Hz, 1H), 7.18 – 7.12 (m, 3H), 7.08 (dd, J = 11.5, 8.5 Hz, 1H), 7.02 (d, J = 8.5 Hz, 2H), 4.51 (quintet, J = 6.0 Hz, 1H), 4.19 (dd, J = 8.5, 6.5 Hz, 1H), 4.01 (dd, J = 9.5, 5.5 Hz, 1H), 4.00 (dd, J = 9.5, 6.0 Hz, 1H), 3.93 (dd, J = 9.0, 6.0 Hz, 1H), 3.82 (s, 2H), 3,43 (s, 3H), 1.47 (s, 3H), 1.41 (s, 3H). 13C NMR (CDCl3, 125 MHz): δ 171.9, 161.5 (d, JCF = 252.5 Hz), 159.2, 137.7, 131.7 (d, JCF = 2.0 Hz), 131.6, 129.5, 128.9, 128.2, 126.8, 124.7, 123.6 (d, JCF = 3.5 Hz), 120.4, 116.5 (d, JCF = 23.3 Hz), 116.1, 115.1, 109.9, 73.9, 68.9, 66.8, 33.0, 30.1, 26.8, 25.3. 315 IR (thin film): 2986, 2932, 1735, 1699, 1478, 1365, 1242, 1147, 1049, 897, 764, 643 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C30H31FN3O4, 516.2299; found, 516.2302. TBSO OMe O Me N N H N O F (E)-N-(4-benzyl-5-(4-(3-((tert-butyldimethylsilyl)oxy)-2-methoxypropoxy)phenyl)-1-methyl-1,3-dihydro-2H-imidazol-2-ylidene)-2-fluorobenzamide (3.30). Compound 3.30 was prepared with ZNA 18 (150 mg, 0.37 mmol) and 3-((tertbutyldimethylsilyl)oxy)-2-methoxypropan-1-ol (86.4 mg, 0.39 mmol) following general procedure A8. The crude product was purified via flash column chromatography (3:2 hexanes/EtOAc) to yield a colorless semisolid (122.2 mg, 50%). Rf = 0.4 (40:1 CH2Cl2/MeOH). 1H NMR (CDCl3, 500 MHz): δ 11.94 (brs, 1H), 8.05 (t, J = 7.0 Hz, 1H), 7.34 (q, J = 6.5 Hz, 1H), 7.29 – 7.21 (m, 4H), 7.18 (t, J = 7.0 Hz, 1H), 7.16 – 7.09 (m, 3H), 7.07 (d, J = 11.0 Hz, 1H), 7.02 (d, J = 8.5 Hz, 2H), 3.75 – 3.68 (m, 2H), 3.66 – 3.60 (m, 2H), 3.51 (s, 3H), 3.43 (s, 2H), 3.42 (s, 3H), 3.30 (quintet, J = 5.0 Hz, 1H), 0.88 (s, 9H), 0.58 (s, 6H). 13C NMR (CDCl3, 125 MHz): δ 171.6, 161.5 (d, JCF = 252.5 Hz), 159.5, 148.6, 137.8, 131.8 (d, JCF = 9.0 Hz), 131.7 (d, JCF = 1.8 Hz), 131.6, 128.2, 128.2, 126.8, 124.9, 123.6 (d, JCF = 3.6 Hz), 120.0, 116.5 (d, JCF = 23.7 Hz), 115.1, 80.1, 67.6, 61.8, 57.9, 30.9, 30.1, 28.1, 25.8. IR (thin film): 2969, 1737, 1727, 1365, 1228, 1216, 1091, 898, 799, 668 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C34H43FN3O4Si, 604.3007; found, 604.3006. 316 MeO O Me N N H N O F (E)-N-(4-benzyl-5-(4-(2-methoxyethoxy)phenyl)-1-methyl-1,3-dihydro-2Himi-dazol-2-ylidene)-2-fluorobenzamide (ZNA 118). ZNA 118 was prepared with ZNA 18 (150 mg, 0.37 mmol) and 2-methoxyethan-1-ol (46.3 mg, 0.39 mmol) following general procedure A8. The crude product was purified via flash column chromatography (3:1 hexanes/acetone) to yield a white solid (29.8 mg, 36%). Rf = 0.4 (40:1 CH2Cl2/MeOH). 1H NMR (CDCl3, 500 MHz): δ 12.01 (brs, 1H), 8.07 (t, J = 7.5 Hz, 1H), 7.36 (q, J = 6.0 Hz, 1H), 7.31 – 7.23 (m, 4H), 7.20 (t, J = 7.0 Hz, 1H), 7.17 – 7.11 (m, 3H), 7.07 (dd, J = 11.0, 9.0 Hz, 1H), 7.03 (d, J = 8.5 Hz, 2H), 4.16 (t, J = 4.5 Hz, 2H), 3.81 (s, 2H), 3.77 (t, J = 4.5 Hz, 2H), 3.46 (s, 3H), 3.43 (s, 3H). 13C NMR (CDCl3, 125 MHz): δ 171.9, 161.5 (d, JCF = 252.5 Hz), 159.4, 148.9, 131.8 (d, JCF = 8.5 Hz), 131.7 (d, JCF = 1.7 Hz), 131.6, 128.9, 128.2, 126.9, 124.7, 123.6 (d, JCF = 3.5 Hz), 120.1, 116.5 (d, JCF = 23.0 Hz), 115.1, 70.9, 67.4, 59.3, 30.9, 30.1. IR (thin film): 2929, 1570, 1551, 1539, 1513, 1471, 1450, 1347, 1250, 1171, 1068, 861, 838, 764, 756, 709, 694 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C27H27FN3O3, 460.2053; found, 460.2052. OMe O MeO Me N N H N O F (E)-N-(4-benzyl-5-(4-(2,3-dimethoxypropoxy)phenyl)-1-methyl-1,3-dihydro2H-imidazol-2-ylidene)-2-fluorobenzamide (ZNA 117). ZNA 117 was prepared with 317 ZNA 18 (150 mg, 0.37 mmol) and 2,3-dimethoxypropan-1-ol (47.1 mg, 0.39 mmol) following general procedure A8. The crude product was purified via flash column chromatography (5:1 hexanes/acetone, followed by 4:1 hexanes/acetone) to yield a white solid (86.0 mg, 45%). Rf = 0.4 (40:1 CH2Cl2/MeOH). 1H NMR (CDCl3, 500 MHz): δ 12.07 (brs, 1H), 8.06 (td, J = 7.5, 1.5 Hz, 1H), 7.34 (q, J = 6.0 Hz, 1H), 7.29 – 7.23 (m, 4H), 7.19 (t, J = 7.5 Hz, 1H), 7,16 – 7.10 (m, 3H), 7.06 (dd, J = 11.0, 8.5 Hz, 1H), 7.02 (d, J = 8.5 Hz, 2H), 4.14 (dd, J = 9.5, 4.5 Hz, 1H), 4.08 (dd, J = 10.0, 5.5 Hz, 1H), 3.80 (s, 2H), 3.73 (quintet, J = 5.0 Hz, 1H), 3.59 (ddd, J = 19.5, 10.0, 5.0 Hz, 2H), 3.52 (s, 3H), 3.42 (s, 3H), 3.39 (s, 3H). 13C NMR (CDCl3, 125 MHz): δ 171.9, 161.5 (d, JCF = 252.5 Hz), 159.4, 148.8, 137.7, 131.8 (d, JCF = 8.5 Hz), 131.7 (d, JCF = 1.75 Hz), 131.6, 128.9, 128.2, 126.9, 124.7, 123.6 (d, JCF = 3.5 Hz), 120.1, 116.5 (d, JCF = 23.2 Hz), 115.1, 78.5, 71.6, 67.6, 59.4, 58.2, 30.9, 30.1. IR (thin film): 2950, 2930, 1693, 1559, 1512, 1456, 1361, 1287, 1249, 1178, 841, 758, 730 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C29H31FN3O4, 504.2299; found, 504.2308. General procedure A9 for the TBS-deprotection TBSO n O HO Me N n = 1 or 2 O Me N 1M TBAF N N H n N THF, 0 oC - r.t. O N H F n = 1 or 2 O F A 25-mL round bottom flask was charged with 0.13 mmol of a TBS-protected alcohol and 10 mL THF. The solution was cooled down to 0 oC and 1M TBAF in THF (0.24 mL, 0.24 mmol) was added dropwise via syringe. The reaction mixture was stirred for 2 h, quenched with sat. aq. NH4Cl-solution and extracted two times with 15 mL EtOAc. 318 After removing the organic solvent, the crude product was purified via flash column chromatography to yield the ethylene glycol-derived ZNA derivative. HO O Me N N H N O F (E)-N-(4-benzyl-5-(4-(2-hydroxyethoxy)phenyl)-1-methyl-1,3-dihydro-2Himi-dazol-2-ylidene)-2-fluorobenzamide (ZNA 92). ZNA 92 was prepared with 3.28 (74.1 mg, 0.13 mmol) following general procedure A9. The crude product was purified via flash column chromatography (1:1 hexanes/acetone) to yield a white solid (54.5 mg, 92%). Rf = 0.35 (1:1 hexanes/acetone). 1H NMR (CDCl3, 500 MHz): δ 11.97 (brs, 1H), 8.06 (td, J = 8.0, 2.0 Hz, 1H), 7.39 – 7.33 (m, 1H), 7.30 – 7.24 (m, 4H), 7.20 (t, J = 7.5 Hz, 1H), 7.17 – 7.12 (m, 3H), 7.07 (dd, J = 11.5, 8.5 Hz, 1H), 7.00 (d, J = 9.0 Hz, 2H), 4.09 (t, J = 4.5 Hz, 2H), 3.95 (t, J = 4.5 Hz, 2H), 3.81 (s, 2H), 3.41 (s, 3H). 13C NMR (CDCl3, 125 MHz): δ 171.5, 161.5 (d, JCF = 250.0 Hz), 159.3, 148.7, 137.8, 131.9 (d, JCF = 7.7 Hz), 131.7 (d, JCF = 1.8 Hz), 131.7, 128.9, 128.2, 126.9, 124.8, 123.7 (d, JCF = 3.5 Hz), 120.2, 116.5 (d, JCF = 23.2 Hz), 115.0, 69.4, 61.4, 31.0, 30.2. IR (thin film): 3394, 2932, 1557, 1542, 1514, 1477, 1361, 1247, 1213, 1187, 1157, 1044, 919, 836, 759, 736, 705, 690, 675 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C26H25FN3O3, 446.1880; found, 446.1888. 319 HO OMe O Me N N H N O F (E)-N-(4-benzyl-5-(4-(3-hydroxy-2-methoxypropoxy)phenyl)-1-methyl-1,3-dihydro-2H-imidazol-2-ylidene)-2-fluorobenzamide (ZNA 116). ZNA 116 was prepared with 3.30 (112.2 mg, 0.18 mmol) following general procedure A9. The crude product was purified via flash column chromatography (3:2 hexanes/acetone) to yield a white solid (57.8 mg, 64%). Rf = 0.3 (3:2 hexanes/acetone). 1H NMR (CDCl3, 500 MHz): δ 8.05 (td, J = 7.5, 1.5 Hz, 1H), 7.37 – 7.33 (m, 1H), 7.30 – 7.23 (m, 4H), 7.19 (t, J = 7.5 Hz, 1H), 7.16 – 7.10 (m, 3H), 7.06 (dd, J = 11.0, 8.5 Hz, 1H), 7.00 (d, J = 9.0 Hz, 2H), 4.09 (ddd, J = 13.5, 10.0, 5.0 Hz2H), 3.84 – 3.79 (m, 3H), 3.72 (dd, J = 11.5, 5.0 Hz, 1H), 3.66 (q, J = 5.0 Hz, 1H), 3.51 (s, 3H), 3.42 (s, 3H). 13 C NMR (CDCl3, 125 MHz): δ 161.5 (d, JCF = 250.0 Hz), 159.2, 148.4, 137.8, 131.9 (d, JCF = 8.8 Hz), 131.7 (d, JCF = 2.0 Hz), 131.6, 128.8, 128.2, 126.8, 124.8, 123.6 (d, JCF = 3.7 Hz), 120.2, 116.5 (d, JCF = 23.1 Hz), 115.0, 79.7, 67.2, 61.6, 58.2, 31.0, 30.2. IR (thin film): 2927, 1683, 1565, 1508, 1361, 1244, 1073, 834, 756 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C28H29FN3O4, 490.2142; found, 490.2144. HO OH O Me N N H N O F (E)-N-(4-benzyl-5-(4-(2,3-dihydroxypropoxy)phenyl)-1-methyl-1,3-dihydro2H-imidazol-2-ylidene)-2-fluorobenzamide (ZNA 93). ZNA 93 was prepared with 3.29 320 (74.5 mg, 0.14 mmol) following general procedure A2. The crude product was purified via flash column chromatography (3:1 hexanes/acetone) to yield a white solid (37.1 mg, 54%). Rf = 0.2 (3:1 hexanes/acetone). 1H NMR (CDCl3, 500 MHz): δ 8.05 (td, J = 8.0, 1.5 Hz, 1H), 7.40 – 7.34 (m, 1H), 7.30 – 7.23 (m, 4H), 7.20 (t, J = 7.5 Hz, 1H), 7.17 – 7.11 (m, 3H), 7.08 (dd, J = 11.0, 8.0 Hz, 1H), 6.99 (d, J = 8.5 Hz, 2H), 4.12 – 4.07 (m, 1H), 4.07 – 4.03 (m, 2H), 3.83 – 3.78 (m, 3H), 3.72 (dd, J = 11.0, 5.0 Hz, 1H), 3.63 (s, 1H), 3.42 (s, 3H). 13 C NMR (CDCl3, 125 MHz): δ 161.45 (d, JCF = 252.2 Hz), 159.1, 148.8, 137.7, 132.0, 131.6, 131.6, 128.9, 128.7, 128.2, 128.0, 126.8, 126.7, 124.9, 123.7, 120.4, 116.5 (d, JCF = 23.3 Hz), 115.0, 114.9, 70.2, 69.2, 63.5, 30.9, 30.2. IR (thin film): 3585, 3565, 2934, 2929, 2870, 1761, 1699, 1652, 1646, 1635, 1599, 1550, 1540, 1507, 1496, 1472, 1456, 1290, 1249 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C27H27FN3O4, 476.1986; found, 476.1986. 180 9.5 9.0 170 8.5 160 150 140 7.0 6.5 130 6.0 120 9.65 3.48 7.5 0.91 2.00 1.94 1.94 2.77 8.0 0.82 0.90 3.78 1.04 2.74 1.09 2.73 10.0 0.89 321 5.5 5.0 4.5 f1 (ppm) 110 100 f1 (ppm) 90 4.0 3.5 80 3.0 70 2.5 60 2.0 50 1.5 1.0 40 0.5 30 0.0 20 -0.5 190 9.0 180 8.5 170 6.0 5.5 5.0 150 140 130 120 110 100 160 0.92 2.00 1.06 1.99 2.23 4.5 f1 (ppm) 4.0 90 80 f1 (ppm) 3.5 3.0 70 60 2.5 50 2.0 40 1.5 30 3.12 3.44 6.5 9.62 7.0 1.14 7.5 2.78 8.0 0.89 0.98 4.03 0.89 2.80 0.97 1.96 9.5 0.93 322 1.0 20 0.5 10 0.0 0 -10 180 9.5 9.0 8.5 170 160 150 140 130 7.0 120 6.5 110 6.0 5.5 100 5.0 4.5 f1 (ppm) 90 80 f1 (ppm) 4.0 70 9.61 7.5 1.92 2.06 1.72 2.10 2.77 8.0 0.77 1.00 3.77 0.95 2.74 1.02 2.93 10.0 0.88 323 3.5 3.0 60 50 2.5 40 2.0 30 1.5 20 1.0 0.5 10 0.0 0 -0.5 -10 180 8.5 170 160 150 7.0 6.5 140 6.0 130 5.5 120 5.0 110 4.5 4.0 f1 (ppm) 100 90 f1 (ppm) 3.5 80 3.0 9.36 1.13 1.17 2.00 1.93 2.12 2.78 1.17 7.5 1.09 8.0 2.09 1.16 4.51 1.09 2.91 1.13 1.98 9.0 1.00 324 2.5 70 2.0 60 1.5 50 1.0 40 0.5 30 0.0 20 -0.5 9.0 180 8.5 170 8.0 160 7.5 7.0 150 140 6.5 130 6.0 120 5.5 5.0 110 4.5 4.0 f1 (ppm) 100 90 f1 (ppm) 3.5 80 1.22 1.31 8.73 9.15 2.06 2.12 2.05 0.93 2.10 1.87 2.27 1.29 2.57 1.22 1.04 1.03 3.72 1.01 2.63 0.99 1.82 0.99 1.00 325 3.0 70 2.5 2.0 60 1.5 50 1.0 40 0.5 30 0.0 20 -0.5 9.5 180 9.0 8.5 170 8.0 160 7.5 150 7.0 140 6.5 130 6.0 5.5 120 5.0 110 4.5 4.0 f1 (ppm) 100 90 f1 (ppm) 3.5 80 3.0 70 2.5 2.0 60 1.5 50 6.19 8.94 1.34 1.84 1.01 2.04 1.12 1.23 2.82 0.91 1.09 3.71 1.09 2.71 1.07 1.92 0.97 1.00 326 1.0 40 0.5 30 0.0 20 -0.5 9.0 180 8.5 170 8.0 160 7.5 150 7.0 140 6.5 130 6.0 5.5 120 5.0 110 4.5 4.0 f1 (ppm) 100 90 f1 (ppm) 3.5 80 3.0 70 2.5 9.09 2.01 1.92 1.89 1.87 2.87 2.08 0.78 1.07 3.72 1.04 2.74 1.15 1.86 0.92 0.98 327 2.0 60 1.5 50 1.0 40 0.5 30 0.0 20 -0.5 170 160 150 6.5 140 6.0 130 5.5 120 5.0 110 4.5 4.0 f1 (ppm) 100 90 f1 (ppm) 3.5 80 3.0 2.5 70 8.93 180 7.0 1.96 7.5 2.79 8.0 1.92 1.24 5.11 1.16 3.84 1.05 2.75 1.19 1.92 8.5 1.02 0.99 328 2.0 60 1.5 50 1.0 40 0.5 30 0.0 20 329 330 9.0 180 8.5 170 8.0 160 7.5 150 2.00 1.90 1.82 1.91 2.89 1.11 1.11 2.90 1.16 2.07 1.21 3.95 0.26 331 7.0 6.5 140 6.0 130 5.5 120 5.0 110 4.5 4.0 f1 (ppm) 3.5 3.0 100 90 f1 (ppm) 80 70 2.5 60 2.0 50 1.5 40 1.0 0.5 30 0.0 20 9.0 8.5 180 170 8.0 160 7.5 150 7.0 140 6.5 6.0 130 5.5 120 5.0 110 4.5 f1 (ppm) 4.0 100 90 f1 (ppm) 1.10 0.85 2.18 2.82 1.78 1.34 1.90 1.00 1.13 1.80 0.86 1.11 1.94 1.07 3.78 332 3.5 3.0 2.5 80 70 60 2.0 50 1.5 40 1.0 30 0.5 0.0 20 9.0 180 8.5 170 8.0 160 7.5 7.0 150 140 6.5 130 6.0 120 5.5 110 5.0 4.5 f1 (ppm) 4.0 100 90 f1 (ppm) 3.5 80 2.31 2.12 2.09 2.11 2.07 1.92 1.18 2.17 3.02 1.11 1.11 3.01 1.24 2.13 1.15 4.00 333 3.0 2.5 2.0 70 60 50 1.5 40 1.0 30 0.5 0.0 20 9.0 170 8.5 160 8.0 150 7.5 7.0 140 6.5 130 6.0 120 5.5 110 5.0 4.5 4.0 f1 (ppm) 100 90 f1 (ppm) 3.5 80 3.0 70 2.5 2.0 60 3.01 3.03 1.21 2.13 1.98 1.24 1.13 4.15 1.12 1.09 3.00 1.19 2.20 1.27 4.09 334 1.5 50 1.0 40 0.5 30 0.0 20 -0.5 9.5 9.0 170 8.5 160 8.0 150 7.5 140 7.0 6.5 130 6.0 120 5.5 110 5.0 4.5 f1 (ppm) 100 90 f1 (ppm) 4.0 3.5 80 2.13 2.32 2.18 3.31 2.10 2.11 1.13 1.15 3.00 1.36 2.19 1.35 4.06 335 3.0 70 2.5 60 2.0 50 1.5 1.0 40 0.5 30 0.0 20 -0.5 9.0 180 8.5 170 8.0 160 7.5 150 7.0 140 6.5 130 6.0 120 5.5 110 5.0 4.5 4.0 f1 (ppm) 3.5 3.0 100 90 80 f1 (ppm) 70 60 1.22 1.24 2.04 1.25 2.02 1.22 2.04 3.10 1.18 1.09 1.15 3.11 1.32 2.20 1.24 4.00 336 2.5 50 2.0 40 1.5 30 1.0 20 0.5 10 0.0 0 9.5 9.0 180 8.5 170 8.0 7.5 160 150 7.0 140 6.5 130 6.0 5.5 120 5.0 110 4.5 4.0 f1 (ppm) 3.5 100 90 f1 (ppm) 80 3.28 3.20 2.36 1.16 1.15 1.07 1.17 4.27 1.95 3.01 1.02 337 3.0 70 2.5 60 2.0 1.5 50 1.0 40 0.5 30 0.0 20 -0.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 f1 (ppm) 3.5 3.0 2.98 2.05 3.14 1.11 1.11 0.98 1.10 1.18 1.96 1.86 2.91 2.00 6.810 338 2.5 2.0 1.5 1.0 0.5 0.0 165 160 155 150 145 140 135 130 125 120 115 110 105 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 f1 (ppm) 9.5 160 9.0 8.5 150 8.0 140 7.5 130 7.0 120 6.5 110 6.0 5.5 100 5.0 90 4.5 f1 (ppm) 4.0 80 f1 (ppm) 70 3.5 60 3.0 50 2.5 9.95 3.13 2.06 1.08 1.07 1.02 1.00 2.15 1.85 4.76 339 2.0 40 1.5 30 1.0 20 0.5 10 0.0 0 180 170 8.0 160 7.5 150 0.94 7.0 140 6.5 6.0 130 5.5 120 5.0 110 4.5 4.0 f1 (ppm) 3.5 100 90 f1 (ppm) 80 3.0 70 3.08 1.10 8.5 3.02 9.0 6.22 2.78 1.00 340 2.5 2.0 60 1.5 50 1.0 40 0.5 30 0.0 20 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 f1 (ppm) 160 155 150 145 140 135 130 125 120 115 110 105 100 95 3.5 90 85 80 f1 (ppm) 75 3.10 8.5 3.10 9.0 1.03 2.04 1.93 2.76 2.05 341 3.0 2.5 70 65 60 2.0 55 1.5 50 45 1.0 40 0.5 35 30 0.0 25 -0.5 20 15 8.0 190 7.5 180 7.0 170 6.5 160 6.0 150 140 5.5 130 5.0 120 4.5 110 4.0 3.5 f1 (ppm) 100 90 f1 (ppm) 3.0 80 70 9.77 3.00 0.95 0.95 4.16 5.00 342 2.5 2.0 60 50 1.5 40 1.0 30 0.5 20 10 0.0 0 180 8.5 170 7.5 160 150 7.0 140 6.5 130 6.0 120 5.5 5.0 110 4.5 f1 (ppm) 4.0 100 90 f1 (ppm) 3.5 80 2.83 8.0 2.93 5.91 3.68 1.00 1.02 9.0 1.00 0.89 343 3.0 2.5 70 60 2.0 50 1.5 40 1.0 30 0.5 0.0 20 2.99 2.95 1.00 0.98 2.07 2.00 3.80 2.11 1.05 1.12 344 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 180 170 160 150 140 130 120 110 100 4.5 f1 (ppm) 4.0 90 80 f1 (ppm) 3.5 70 3.0 60 2.5 50 2.0 40 1.5 30 1.0 20 0.5 10 0.0 0 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 180 170 160 150 140 130 120 110 100 4.5 f1 (ppm) 4.0 90 80 f1 (ppm) 3.5 70 3.0 60 9.44 3.00 0.90 1.81 2.06 2.33 3.32 1.07 0.99 0.96 1.00 345 2.5 50 2.0 40 1.5 30 1.0 20 0.5 10 0.0 0 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 f1 (ppm) 3.5 3.07 2.88 2.01 2.33 0.87 1.22 4.91 1.28 2.91 1.04 1.00 346 3.0 2.5 2.0 1.5 1.0 0.5 0.0 2.01 2.80 2.90 0.98 5.80 5.33 1.00 0.90 347 13.5 13.0 12.5 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 f1 (ppm) 9.5 9.0 170 8.5 160 8.0 150 7.5 140 7.0 6.5 130 6.0 120 5.5 110 5.0 4.5 4.0 f1 (ppm) 100 90 f1 (ppm) 3.5 80 9.15 2.95 2.02 1.92 1.02 3.93 0.98 2.78 1.02 0.82 1.00 348 3.0 70 2.5 60 2.0 50 1.5 1.0 40 0.5 30 0.0 20 -0.5 9.0 170 8.5 160 8.0 150 7.5 140 7.0 130 6.5 120 6.0 110 5.5 100 5.0 4.5 4.0 f1 (ppm) 90 80 f1 (ppm) 2.92 2.00 1.00 2.10 1.00 2.97 2.98 1.99 1.00 349 3.5 70 3.0 60 2.5 50 2.0 40 1.5 30 1.0 20 0.5 10 0.0 0 8.5 8.0 7.0 6.5 6.0 5.5 5.0 4.5 4.0 f1 (ppm) 3.5 1.00 3.00 1.93 1.10 1.00 2.12 1.07 4.16 4.22 1.17 7.5 3.0 2.5 2.0 2.95 3.25 9.0 9.60 9.5 1.00 0.89 350 1.5 1.0 0.5 0.0 -0.5 11.84 2.71 1.90 0.83 0.78 0.80 1.88 1.00 3.88 1.04 2.25 0.76 0.90 0.98 351 13.0 12.5 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 f1 (ppm) 13.0 12.5 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 f1 (ppm) 5.5 5.0 4.5 4.0 3.5 1.17 6.82 8.89 8.38 1.99 2.98 2.12 1.02 1.05 1.01 0.99 2.00 1.12 4.18 0.99 2.59 0.93 1.16 1.01 352 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 9.5 170 9.0 160 8.5 8.0 150 7.5 140 7.0 130 6.5 6.0 5.5 120 110 100 5.0 4.5 f1 (ppm) 90 80 f1 (ppm) 4.0 3.5 70 2.95 2.88 1.11 2.10 1.03 3.23 1.25 1.72 0.97 2.07 1.16 1.14 1.99 1.07 2.00 353 3.0 60 2.5 50 2.0 40 1.5 1.0 30 20 0.5 10 0.0 0 9.0 170 8.5 160 8.0 7.5 150 7.0 140 6.5 130 6.0 120 5.5 110 5.0 4.5 4.0 f1 (ppm) 100 90 f1 (ppm) 3.5 80 2.92 2.91 1.12 1.99 1.14 2.00 1.13 2.01 1.17 1.11 2.19 1.06 1.97 354 3.0 70 2.5 60 2.0 50 1.5 40 1.0 30 0.5 0.0 20 9.0 8.5 8.0 180 170 160 7.5 150 7.0 140 6.5 130 6.0 5.5 120 5.0 110 4.5 4.0 f1 (ppm) 100 90 f1 (ppm) 3.5 80 2.10 2.29 2.13 2.19 2.90 1.07 1.98 1.14 2.94 1.88 1.16 3.05 1.00 2.00 355 3.0 2.5 70 60 2.0 50 1.5 40 1.0 30 0.5 0.0 20 13.0 12.5 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 f1 (ppm) 170 160 150 140 130 120 110 100 90 80 f1 (ppm) 70 60 50 40 30 20 10 5.64 9.38 2.00 1.98 2.06 3.10 1.09 1.09 4.13 1.20 3.11 1.12 1.96 1.08 356 0.5 0.0 -0.5 0 -10 9.0 180 8.5 170 8.0 160 7.5 150 7.0 140 6.5 130 6.0 120 5.5 110 5.0 100 4.5 4.0 f1 (ppm) 90 80 f1 (ppm) 3.09 2.88 3.07 0.98 1.07 0.96 1.03 1.95 0.99 1.15 4.00 1.24 2.97 1.16 1.91 0.98 357 3.5 70 3.0 60 2.5 2.0 50 1.5 40 1.0 30 0.5 20 0.0 10 -0.5 0 9.37 14.73 2.00 2.30 2.75 2.11 2.65 0.98 0.73 1.00 1.07 3.72 1.02 2.69 0.86 2.03 1.34 358 12.5 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 -1.0 f1 (ppm) 170 160 150 140 130 120 110 100 90 f1 (ppm) 80 70 60 50 40 30 20 12.5 12.0 11.5 11.0 10.5 10.0 9.5 180 170 160 150 140 9.0 130 8.5 120 8.0 110 7.5 7.0 100 6.5 6.0 f1 (ppm) 90 80 f1 (ppm) 2.02 1.97 2.05 2.78 2.84 1.01 3.82 0.92 2.81 1.03 1.89 1.00 1.33 359 5.5 70 5.0 4.5 60 4.0 50 3.5 40 3.0 2.5 30 2.0 20 1.5 10 1.0 0.5 0 0.0 -10 12.5 12.0 11.5 11.0 10.5 10.0 9.5 170 160 150 140 130 9.0 120 8.5 8.0 110 7.5 100 7.0 90 1.08 1.06 1.97 1.11 2.21 2.74 2.88 2.80 1.05 3.80 0.98 2.84 1.06 1.90 1.00 1.10 360 6.5 6.0 5.5 f1 (ppm) 80 f1 (ppm) 70 5.0 4.5 60 4.0 50 3.5 40 3.0 2.5 30 2.0 20 1.5 1.0 10 0.5 0 0.0 -10 2.10 2.10 2.08 3.11 1.00 1.03 3.94 0.96 2.92 1.06 2.04 1.43 361 13.0 12.5 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 f1 (ppm) 180 170 160 150 140 130 120 110 100 90 80 f1 (ppm) 70 60 50 40 30 20 10 0 -10 8.5 170 8.0 160 7.5 150 7.0 2.01 3.01 1.05 1.04 2.81 2.80 1.03 3.80 0.94 2.78 1.01 1.96 1.00 362 6.5 140 6.0 130 5.5 120 5.0 110 4.5 4.0 f1 (ppm) 100 90 f1 (ppm) 3.5 80 3.0 70 2.5 2.0 60 1.5 50 1.0 40 0.5 30 0.0 20 9.5 9.0 170 8.5 160 8.0 150 7.5 140 7.0 1.03 1.87 2.88 1.09 0.93 2.87 1.03 3.99 1.02 2.79 1.09 2.05 1.00 363 6.5 130 6.0 120 5.5 110 5.0 4.5 4.0 f1 (ppm) 100 90 f1 (ppm) 3.5 80 3.0 70 2.5 60 2.0 1.5 50 1.0 40 0.5 30 0.0 20 -0.5 CHAPTER 4 SYNTHESES, BIOLOGICAL PROPERTIES AND ZINC BINDING MODE OF HEMIPORPHYRIN-LIKE ZNA DERIVATIVES 4.1 Introduction A university-wide screening effort identified zinaamidole A (ZNA) as a new modulator of zinc ion homeostasis resulting in cancer cell death while leaving healthy cells unaffected. In particular, ZNA induced lysosomal membrane permeabilization in MCF-7 cells by delivering a super stoichiometric amount of zinc into the lysosome. The ionophoric properties stemmed from a balanced interplay between the N2-acyl 2-aminoimidazole functionality and zinc binding. The addition of exogenous zinc amplified the potency of ZNA. Maximum potency was achieved upon reaching equimolar concentration with the added ZnSO4 at roughly 20 µM (Figure 4.1A). Once this threshold was crossed, the formation of water insoluble zinc-ZNA dimers triggered a cellular rescue effect. In Chapter 3, this issue was approached by synthesizing and evaluating ZNA analogs with water-solubilizing functional groups such as amino acids or ethylene glycols. Although these analogs were more water-soluble than ZNA, the structural modifications were detrimental for ligand-zinc interactions, as characterized by the loss of potency and/or selectivity. In this work, we proposed to generate novel ZNA analogs with C4-pyridine units. 365 The addition of an sp2-hybridized nitrogen atom could lead to the formation of a hemiporphyrin-like tridentate ZNA-zinc monomer with better water solubility and biological activity. Chapter 4 details the synthesis, biology, and binding modes of those ZNA analogs. 4.2 Results and Discussion 4.2.1 Syntheses of Hemiporphyrin-like ZNA Analogs The Looper lab developed a 4-step synthesis to prepare N2-acyl 2-aminoimidazoles (discussed in Chapter 3).1 However, employing the same strategy to access pyridinecontaining analogs was not successful (Figure 4.2A). In our hands, procedures to prepare 4.1.0 were inadequate, resulting in poor yields. We decided not to pursue this chemistry. The low turnover was potentially due to coordination of the pyridine nitrogen to copper or palladium hindering the reactivity of the catalyst. This setback prompted us to revisit the synthesis of propargyl amine 4.1.0 and its derivatives. We thought to utilize a-amidoaryl sulfone 4.2 as our precursor (Figure 4.2B). Compound 4.2 has been shown to react with a variety of stabilized carbanions via a formal nucleophilic addition.2-3 First reported by Strating and Engberts,4 4.2 can be easily prepared on multigram scale using benzaldehyde 4.1.1, sodium benzenesulfinate 4.1.2, and tertbutyl carbamate 4.1.3 in the presence of formic acid. Deprotonation of the suitable terminal alkynes S1.2 – S7.2 with nBuLi at –78 oC and subsequent addition of 4.2 delivered N-Boc propargyl amines 4.3.1 – 4.3.6 in excellent yields. However, initial attempts to methylate 4.3.1 under basic conditions resulted in the formation of oxazole 4.3A (Figure 4.3A). We reasoned that the rapid cycloisomerization5-6 was promoted in the presence of the acidic 366 C5-proton and pyridine unit. To test this hypothesis, a small set of propargyl amine analogs (4.3B, 4.3C and 4.3D) lacking acidic C5-proton and/or the pyridine unit were generated. It was found that these compounds did not react to give the cyclized product upon treatment with NaH (Figure 4.3B). The undesired cycloisomerization could be circumvented by the addition of excess of MeI (Figure 4.4). Subsequent exposure to NaH gave the desired N-methyl propargyl amines 4.4.1 – 4.4.6. Boc-deprotection using 2M HCl in Et2O accessed the secondary amines 4.5.1 – 4.5.6. Next, we envisioned to guanylate the resultant secondary amines but to our surprise, guanylation7 of 4.51, 4.52 and 4.54 – 4.56 using cyanamides S8 – S10 yielded cyclic TMS-ene-guanidines 4.6.1 – 4.6.7 as a single cis-stereoisomer with moderate to good yields (Figure 4.4). However, when we subjected the secondary amine 4.5.3 to the same condition, the uncyclized N-acyl propargyl guanidine 4.6.8 was obtained as the major product. (Note: A more detailed discussion about this aminosilylation reaction, its conditions, substrate scope and limitations is in Chapter 5). TMS-deprotection was carried out using K2CO3/MeOH to generate the desired hemiporphyrin-like ZNA analogs (Figure 4.4). Concomitantly, 4.6.8 was converted into N2-acyl 2-aminoimdazole using NaH. During our synthetic efforts, we realized that N-methylation of substrates carrying pyridines with electron withdrawing groups sustained significantly lower yields due to competitive cycloisomerizations. For instance, 4.4.6 was isolated in only 23% yield along with 35% of the cyclized product 4.3E (Figure 4.5A). To overcome this, we first introduced the TMS-protected alkyne S11 to obtain 4.3.7, followed by N-methylation and subsequent TMS-deprotection which allowed for delayed installation of the substituted pyridines (S12 367 – S16) via Sonogashira coupling conditions (Figure 4.5B). This alteration in synthetic sequence avoided the undesired cycloisomerization products and enhanced the efficiency of the syntheses of the pyridine-containing propargyl amines 4.4.10 – 4.4.14. Following Boc-deprotection of these substrates, guanylation of the secondary amines 4.5.7 – 4.5.11 led to the formation of cyclic TMS-ene-guanidines 4.6.9 – 4.6.13. Finally, TMSdeprotection was carried out using methanolic K2CO3 to obtain the desired hemiporphyrinlike ZNA analogs (Figure 4.5B). 4.2.2 Biological Evaluation and Discussion The ZNA analogs prepared in this study (Figure 4.6) were evaluated by Dr. Katrin Guillen (Welm Lab) using ATPLite assays and MCF-7 and MCF-10A cell lines. Most of the tested analogs possessed EC50 values in the micromolar range, similar to or better than ZNA (Table 4.1). Unfortunately, the majority of the compounds were also cytotoxic toward untransformed MCF-10A cells, albeit at a higher concentration. The antiproliferative activity was greatly potentiated in both cell lines when exogenous zinc was added, demonstrating their ionophoric properties. ZNA 131 for instance was twice as active as ZNA. But in the presence of 20 µM Zn2+, the potency difference was elevated to more than 6-fold. Addition of a methoxy group to the C2-position of the pyridine unit slightly diminished its activity. Other regioisomers such as ZNA 176 and ZNA 177 were more potent or equipotent, respectively. A 12-point dose-response curve (Figure 4.7) of ZNA 176 illustrated the strong amplification of potency when the cells were co-treated with 20 µM ZnSO4. This effect persisted for drug concentrations between 1.5 µM – 60 µM. Only at higher dosing a cellular rescue effect was 368 only observed, indicating the formation of higher order zinc complexes. We also found that ZNA 176 could completely inhibit cell proliferation whereas ZNA in conjunction with ZnSO4 was not able to reach below 30% cell viability. 5-Methoxypyridine analogs 188, 186 and 187 displayed moderate activities in MCF-7 cell line but experienced more than 10-fold potentiation with zinc co-treatment. In addition, ZNA 186 also demonstrated 10fold selectivity for MCF-7 cell line. Expanding upon these results, we altered the nucleophilicity of the pyridine nitrogen by employing chlorine and methyl ester substitutions. Although no significant changes in activity were observed, it is worth mentioning that ZNA 175 showed almost no increase in potency with 20 µM ZnSO4. This could be attributed to the electronic property or the steric bulk of the ester group. A similar trend was observed for ZNA 197 and 198, suggesting that bulky substituents adjacent to the sp2-hybridized pyridine nitrogen preclude zinc ion binding. Another interesting comparison can be drawn between ZNA 131 and its elongated version ZNA 194. The latter molecule was approximately 3.5-fold less potent than 131 in MCF-7 cell line, suggesting that the eight-membered zinc metallocycle could be unfavorable. Our preliminary biological data highlighted the importance of a pendant C4pyridine unit and its role in leading to a significant increase in potency. This was corroborated by the negative outcome of control compounds ZNA 197 and ZNA 198. We believe that the heterocycle was involved in interactions with zinc and has potentially invoked an alternative binding motif from that observed in Zn(ZNA)2 dimer complex (Figure 4.1). 369 4.2.3 Zinc Binding Modes of ZNA 131, ZNA 148 and ZNA 194 In the original isolation report by Pietra and co-workers,8 they described the synthesis of zinc-naamidine G dimer complex by treating a chloroform solution of the natural product with 0.1M ZnSO4 in methanol. The desired compound was obtained in almost quantitative yield after stirring the mixture for 24 h at room temperature. Applying the same procedure to ZNA resulted in the formation of Zn(ZNA)2 dimer complex (Figure 4.8). The X-ray crystal structure showed two ZNA ligands binding to zinc via anionic N3nitrogen atoms, while the acyl group oxygens simultaneously coordinated to the metal center. The promising biological data encouraged us to explore potential binding motifs between hemiporphyrin-like ZNA analogs and zinc. We used Pietra’s reported procedure to treat ZNA 148 with ZnSO4 in methanol and obtained a white solid. However, we did not obtain the proposed tridentate zinc complex (Figure 4.1B). Instead, X-ray crystallography of this solid revealed a bidentate 2:2 ZNA-zinc complex (Figure 4.9). In crystal structure C1, the pendant C4-pyridine residue was engaged in zinc coordination. The complexation event also invoked an unusual endocyclic N3-imino tautomer, in which the N3-nitrogen atom was coordinated to the metal center. We determined the bond lengths of C2-N2 to be at 1.385 Å, whereas C2-N3 bond was only 1.326 Å. In comparison, ZNA-zinc complex was always characterized as an exocyclic N2-imino tautomer. Conversely, C1 showed no coordination between the N2-acyl groups and the metal, which led to the (misguided) assumption that the sterically bulky sulfates were alternating the binding motif. We theorized that switching to a smaller counter anion in our zinc source, such as ZnCl2, the classic N2-acyl coordinated tridentate binding motif could 370 be restored. Reaction of ZNA 148 and ZnCl2 yielded the complex C2 as a white powdery solid. Upon crystallization, the X-ray structure of C2 (Figure 4.10) showed a 1:1 ZNA-zinc bidentate complex. The ligand ZNA 148 occurred as a neutral species with two nitrogen atoms coordinating to the metal. Again, the N2-acyl group was not engaged in any binding events. This observation led us to correct our previous assumption that the sulfates were responsible for the altered binding exhibited by ZNA 148. Rather, the environment created by ZNA 148 ligand leads to zinc preferentially adopting tetrahedral coordination geometry. It is impossible for the ligand to twist either pyridine or the N2-acyl group (Figure 4.10, red) in such a way that the molecule can meet the requirement of a tridentate binding complex. Based on these observations, we aimed to achieve a tridentate complex by extending the C4-pyridine moiety by two methylene units, thus inducing more flexibility into the molecule. ZNA 194 formed with ZnCl2 complex C3, which was characterized via X-ray crystallography. Similar to C1 and C2, C3 retained the same binding mode. The pyridine and the N3-imino nitrogen atoms were coordinated to the metal center forming an eight-membered macrocyclic ring structure, whereas the acyl group was pointed away from the zinc metal (Figure 4.11). The reoccurring binding motifs observed in C2 and C3 can be explained both by the geometric nature of zinc cation as well as the nucleophilic nature of the chloride anion. Chloride clearly outcompetes the acyl group for metal binding. However, by exchanging zinc salt for one with noncoordinating counter-anions such as Zn(BF4)2, resulted in an octahedral zinc-ZNA dimer complex C4 (Figure 4.12) in which the N2-acyl group was 371 coordinated to the metal center. This study showcased some unexpected binding motifs of hemiporphyrin-like ZNA analogs with different zinc salts. Although the interesting motif variations contributed to our understanding of ZNA-zinc interactions, they do not necessarily reflect binding events taking place in the cellular environment. In order to mimic binding behaviors between ligand and metal, we performed Job-plot analyses. 9 This UV-Vis-based analytical method has been widely used to gain quantitative insights into stoichiometries underlying associations of two molecules in equilibrium. The measurements for compound ZNA 148 in aqueous in buffer solution were very challenging due to its poor solubility. However, when DMSO was used instead, a plot was obtained (Figure 4.13A) in which the inflection point occurred at X = 0.55, suggesting a 1:1 zinc:ligand ratio. In addition, measurements performed in EtOH/H2O showed similar results (X = 0.57), albeit with lower R2-values (Figure 4.13B). Taken together, the obtained data strongly suggested that ZNA 148 and zinc ion preferentially existed as monomeric complexes in nonaqueous solutions. This being said, in aqueous buffer solutions ZNA 148 and its analogs could still have interactions with other anionic species or water molecules. Besides, Zn2+ itself is known to coordinate to water,10 yet does not have a preference for the number of ligands surrounding the metal. It is also important to note that cysteine and histidine are known to coordinate with metals inside the cell and could also impact the in vivo ZNA-zinc binding interactions.11-12 372 4.3 Conclusion and Future Directions ZNA represents a novel class of zinc ionophore with therapeutic relevance for the treatment of breast cancer. The Looper group has previously elucidated its mechanism of action and identified the structural component responsible for zinc interactions. However, at high drug concentration, ZNA’s potency against cancer cells diminished due to the formation of inactive zinc dimers. This issue was addressed in Chapter 4 by generating hemiporphyrin-like compounds, which could invoke a different zinc binding motif. We developed a modular synthesis toward these new zinc-binding reagents by taking advantage of a-amidoaryl sulfone 4.2 as a valuable synthon and quickly generated a small library of ZNA analogs with different substitution patterns. Studies of binding modes between these new molecules and different zinc salts also revealed a new N,N-bidentate binding motif. The majority of the tested compounds have shown promising potency and zinc synergy in the MCF-7 cell line, yet they lacked selectivity. However, we believe additional analog syntheses could provide a more comprehensive understanding of the underlying mechanisms of hemiporphyrin-like ZNAs. For instance, further structural modifications could be made to coalesce the properties of ZNA 176 (most potent) and ZNA 187 (most selective) and provide a more holistically optimized drug lead. With regard to the diminished selectivity between MCF-7 and MCF-10A cell lines, we suspect that the hemiporphyrin-like ZNAs acted as very potent ionophores, delivering super stoichiometric amounts of zinc into the lysosomes. Although healthy cells possessed endogenous mechanisms to manage high zinc influx, a threshold could have been reached which overcame this protection. Here, we can use zinc uptake and fluorescence microscopy experiments to quantify and localize intramolecular zinc ions. 373 A Fractional ATP content 1.5 1:1 ZNA/Zn 1.0 0.5 0.0 ZNA ZNA + 20 µM ZnSO4 rescue effect -1 0 1 2 3 log (µM) B MeO Me N MeO N 2 Me N H N + ZnSO4 Zn O - H2SO4 F N F N O N N O N Me F MeO OMe MeO N N + ZnCl2 Me - HCl F N N H N Me F N N N Zn O O Cl Figure 4.1: ZNA and its pyridyl derivative. A) Dose-response curve in MCF-7 cell line. B) Water-insoluble ZNA-zinc complex and proposed structure of hemiporphyrin-like ZNA in complex with zinc (B). OMe A MeO O H + Me MeO N N S1.2 4.13 O MeO 4.1.1 S + O S OO S2.2 H N nBuLi, THF N R1 N MeO -78 3h 87 - 99% N MeO OMe S3.2 N R1 4.3.1 - 4.3.6 N OtBu O oC, S1.2 - S6.2 N OMe S1.2 O hemiporphyrin-like ZNAs OtBu 4.2 N F MeO HCOOH 4.1.2 N 4.1.0 H N MeOH, H2O rt, 48 h, 52% ONa N N H N MeO BocNH2 H N Me sluggish reaction O B N MeCN, 80 oC 3.12 4.1.1 Me CuBr N H Cl S4.2 S5.2 S6.2 Figure 4.2: Synthetic efforts towards hemiporphyrin-like ZNAs. A) A3-coupling with a pyridine-containing substrate. B) Syntheses of N-Boc 2-pyridine propargyl amine 4.3.1 – 4.3.6. 374 A MeO MeO H H N OtBu N NaH, MeI DMF O MeO Me OtBu O 0 oC, 5 min 86% N OtBu + O N N N 4.3A H: acidic C5-proton desired product was not formed 4.3.1 B cyclized product 4.3A was isolated No cyclization observed MeO H N Me Me H N OtBu H N OtBu O O N 4.3B N 4.3C OtBu O 4.3D Figure 4.3: Synthetic efforts towards hemiporphyrin-like ZNAs. A) Cycloisomerization with 4.3.1 when treated with NaH. B) Compounds without acidic C5-protons and/or pyridine units (4.3B, 4.3C and 4.3D) did not cyclize. MeO MeO H N OtBu O Me N NaH 10 equiv. MeI H N 2M HCl Et2O O DMF, 0 oC 2 h, yield CH2Cl2, rt, 3 h 31 - 84% R1 4.5.1: R1 = H, 92% 4.5.2: R1 = 2-OMe, 84% 4.5.3: R1 = 3-OMe, 68% N TMS N R2 K2CO3, MeOH N rt, 1 h O N R1 4.6.1 - 4.6.7 MeO S8: R2 = 2-F S9: R2 = 4-F S10: R2 = 4-Cl MeO N H Me N Me N N H R2 O MeO F NaH, THF rt, 2 h N N N 4.6.8 OMe 4.5.4: R1 = 4-OMe, 44% 4.5.5: R1 = 5-OMe, 64% 4.5.6: R1 = 2-Cl, 43% N NH2 O CN R2 N R1 4.4.1: R1 = H, 73% 4.4.4: R1 = 4-OMe, 67% 4.4.2: R1 = 2-OMe, 77% 4.4.5: R1 = 5-OMe, 67% 1 4.4.3: R = 3-OMe, 87% 4.4.6: R1 = 2-Cl, 23% Me N N H rt, 6 h yield MeO TMSCl, DIPEA O Me + N R1 N R1 4.3.1 - 4.3.6 MeO OtBu N H Me N F O MeO Figure 4.4: Syntheses of hemiporphyrin-like ZNA analogs. 375 MeO A MeO H N OtBu O MeO Me N NaH 10 equiv. MeI OtBu N + O DMF, 0 oC, 2 h OtBu Me N O N 23% Cl 4.3.6 N 35% Cl 4.4.6 4.3E B MeO OtBu H N nBuLi + O S OO TMS oC, -78 3h 4.3.7 4.2 Br Me N OtBu + S12: R1 = 2-Me S15: 2-OiPr S13: R1 = 2-iPr S16: 2-COOMe S14: R1 = 2-tBu 4.4.9 N H F Me N CN OtBu DCM, rt, 3 h 40 - 69% F N R1 N TMS N H 4.6.9 - 4.6.13 O TMS H N 2M HCl Et2O N 4.5.7 - 4.5.11 R1 MeO K2CO3, MeOH rt, 1 h 83 - 99% Me rt, 6 h 43 - 92% N R1 Me N TMSCl DIPEA rt, 1 h, 93% MeO O 4.4.10 - 4.4.14 K2CO3 MeOH OtBu O 4.4.7 MeO O S8 THF, 0 2 h, 75% THF, rt, 1 h 67 - 93% R1 O + PdCl2(PPh3)2 CuI, Et3N N oC TMS MeO Me N NaH, MeI OtBu O 96% S11 MeO MeO MeO H N Me N N R2 N H O N R1 Hemiporphyrin-like ZNA derivatives Figure 4.5: Synthetic efforts towards hemiporphyrin-like ZNAs. A) N-methylation of 4.3.6 with 2-chloro pyridine moiety. B) Alternative synthesis of ZNA analogs. 376 MeO Me N R2 N H N R1 O Hemiporphyrin-like ZNA ZNA# R1 R2 ZNA# F R1 R2 F 131 172 N N Cl F F 148 N 175 N OMe OMe O F 177 N F 3 N 194 OMe F F 176 N 195 N MeO Me F F 188 MeO N 196 N Me Me F 186 MeO F N 197 N tBu F 187 MeO Cl 198 N Figure 4.6: ZNA analogs with C4-pyridine substitutions. N Me O Me 377 MCF-10A MeO MCF-7 Me N N N H ZNA O F ZNA ZNA + 20 µM ZnSO4 MeO Me N N N H ZNA 176 O N F MeO Figure 4.7: Dose-response curves of ZNA and ZNA 176 in MCF-7 and MCF-10A cell lines. Me N MeO MeO N 2 ZnSO4 MeOH Me N rt, 24 h N N H F N O F Zn N N O F ZNA O N Me OMe Zn(ZNA)2 Figure 4.8: ZNA in complex with ZnSO4. OMe MeO N N Me ZnSO4 MeOH N rt, 16 h 61% N H F O OMe ZNA 148 MeO F O Me N N MeO N O N O S Me NH O Zn HN O O O N Zn O S O F O N OMe C1 steric repulsion ? Figure 4.9: ZNA 148 in complex with ZnSO4. 378 MeO MeO Me N ZnCl2 MeOH O N N N H Me N rt, 16 h 91% O N H N F Zn Cl Cl OMe F N OMe ZNA 148 C2 Figure 4.10: Reaction of ZNA 148 with ZnCl2 yielded complex C2. OMe HN N Me O N N OMe ZnCl2 MeOH rt, 16 h 60% N Me O HN Zn Cl F Cl N N F ZNA 194 C3 Figure 4.11: Reaction of ZNA 194 with ZnCl2 produced the bidentate zinc complex C3. H N Me N N N N 2 BF4- F MeO Me O N H F Zn(BF4)2 MeOH rt, 16 h 41% MeO N O N Zn N N O N H ZNA 131 F OMe N Me C4 Figure 4.12: Reaction of ZNA 131 with Zn(BF4)2 led to the octahedral zinc complex C4. B 0.3 0.25 0.2 0.15 0.1 0.05 0 0 0.2 0.4 0.6 0.8 Ratio of Zn/ZNA 148 in DMSO 1 Corrected absorbance at 284 nm A Corrected absorbance at 284 nm 379 0.4 0.3 0.2 0.1 0 0 0.2 0.4 0.6 0.8 1 Ratio of Zn/ZNA 148 in EtOH/H2O (1:2) Figure 4.13: Job plot analyses of ZNA 148 and ZnSO4 in A) DMSO with intercept at x = 0.55 and in B) EtOH/H2O (1:2) with x = 0.57. Table 4.1: EC50 values of ZNA analogs. ZNA# EC50 (µM) ZNA 131 148 177 176 188 186 187 172 175 194 195 196 197 198 21.0 10.7 23.0 12.2 9.8 44.7 22.7 28.7 27.1 12.7 38.4 33.9 12.9 N.A. 26.2 MCF-7 EC50 (µM) with 20 µM Zn 11.9 1.8 4.2 1.9 1.5 4.7 2.1 2.6 5.7 7.6 6.1 7.5 4.1 15.0 32.9 MCF-10A EC50 (µM) with EC50 in µM 20 µM Zn N.A. N.A. 23.7 2.0 N.A. 5.9 18.7 1.9 15.4 1.5 N.A. 8.5 54.9 3.0 40.2 24.1 N.A. 7.9 N.A. 10.5 N.A. 6.2 93.5 7.6 36.3 4.8 N.A. N.A. 74.2 N.A. 380 4.4 References 1. Salvant, J. M.; Edwards, A. V.; Kurek, D. Z.; Looper, R. E., Regioselective BaseMediated Cyclizations of Mono-N-acylpropargylguanidines. J. Org. Chem., 2017, 82, 6958-6967. 2. Morton, J.; Rahim, A.; Walker, E. R. H., A New General Method of α-AminoAlkylation. Tetrahedron Lett., 1982, 23, 4123-4126. 3. Mecozzi, T.; Petrini, M., Synthesis of Allylic and Propargylic Primary Amines by Reaction of Organometallic Reagents with α-Amidoalkyl Sulfones. J. Org. Chem., 1999, 64, 8970-8972. 4. Olijnsma, T.; Engberts, J.; Strating, J., The Mannich Condensation of Sulfinic Acid with Aldehydes and Carboxamides, Sulfonamides, or Lactams. Part IV. Recl. Trav. Chim. Pays-Bas, 1967, 86, 463-473. 5. Merkul, E.; Müller, T. J. J., A New Consecutive Three-Component Oxazole Synthesis by an Amidation–Coupling–Cycloisomerization (ACCI) Sequence. ChemComm., 2006, 4817-4819. 6. Görgen née Boersch, C.; Lutsenko, K.; Merkul, E.; Frank, W.; Müller, T. J. J., Catalytic One-Pot Synthesis of 4-(Hetero)aryl Substituted 5-(2-Oxoethyl) Oxazol-2(3H)ones by Coupling–Isomerization–Elimination (CIE) Sequence. Org. Chem. Front., 2016, 3, 887-896. 7. Looper, R. E.; Haussener, T. J.; Mack, J. B. C., Chlorotrimethylsilane Activation of Acylcyanamides for the Synthesis of Mono-N-acylguanidines. J. Org. Chem., 2011, 76, 6967-6971. 8. Ines, M.; Graziano, G.; Cécile, D.; Francesco, P., Novel Naamidine‐Type Alkaloids and Mixed‐Ligand Zinc(II) Complexes from a Calcareous Sponge, Leucetta sp., of the Coral Sea. Helv. Chim. Acta, 1995, 78, 1178-1184. 9. Olson, E. J.; Bühlmann, P., Getting More out of a Job Plot: Determination of Reactant to Product Stoichiometry in Cases of Displacement Reactions and n:n Complex Formation. J. Org. Chem., 2011, 76, 8406-8412. 10. Dudev, T.; Lim, C., Tetrahedral vs Octahedral Zinc Complexes with Ligands of Biological Interest: A DFT/CDM Study. J. Am. Chem. Soc., 2000, 122, 11146-11153. 11. Zoroddu, M. A.; Medici, S.; Peana, M.; Anedda, R., NMR Studies of Zinc Binding in a Multi-Histidinic Peptide Fragment. Dalton Trans., 2010, 39, 1282-1294. 12. Laitaoja, M.; Valjakka, J.; Jänis, J., Zinc Coordination Spheres in Protein Structures. Inorg. Chem., 2013, 52, 10983-10991. 381 4.5 Supporting Information 4.5.1 Experimental (Biology) All assay experiments were performed by Dr. Katrin P. Guillen (Bryan E. Welm Lab) at the Huntsman Cancer Institute. Dose response assays: For 5-day dose response assays, cells were seeded in 96-well plates in 100 µL standard culture media at a confluency to achieve 90% confluency at the completion of the 120-h assay. Following an overnight incubation, the media was replaced with 2% fetal bovine serum (FBS), small molecule- or vehicle control-containing media. The media was aspirated and replaced with fresh treatment media every 48 h during the experiment. Following the completion of the 120-h assay, cell viability was measured using an ATPlite assay (PerkinElmer, Waltham, MA, USA) following the manufacturer’s protocol. 4.5.2 General Experimental Conditions (Chemistry) All reactions requiring anhydrous conditions were performed under a positive pressure of nitrogen using flame-dried glassware. Commercially available reagents were used as received or purified according to Purification of Laboratory Chemicals. Dimethylformamide (DMF), tetrahydrofuran (THF), acetonitrile (MeCN), and dichloromethane (CH2Cl2) were degassed with nitrogen and passed through a solvent purification system (Innovative Technologies Pure Solv). Methanol was distilled from magnesium turnings prior to use. DIPEA was distilled from CaH2 immediately prior to use. Reactions were monitored by TLC and visualized by a dual short/long wave UV lamp and stained with an aqueous solution of potassium permanganate. Flash chromatography was performed on silica gel SiliaFlash P60 (40-63 µm) from Silicycle. 382 1 H NMR spectra were recorded at 500 MHz as indicated. The chemical shifts (δ) of proton resonances are reported relative to the deuterated solvent peak: 7.26 for CDCl3 and 4.87 for H2O in CD3OD, using the following format: chemical shift [multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, app = apparent), coupling constant(s) (J in Hz), integral]. 13C NMR spectra were recorded at 125 MHz. The chemical shifts of carbon resonances are reported relative to the deuterated solvent peak: 77.3 (center line) for CDCl3 and 49.0 (center line) for CD3OD. Mass spectra were obtained by ESI/APCI for LRMS or ESI/APCITOF for HRMS. 4.5.3 Procedures and Characterizations General procedure B1 for the Sonogashira coupling reactions of 2-bromo pyridines and alkynes R N Br + TMS PdCl2(PPh3)2 CuI, Et3N R THF, r.t. N TMS An oven-dried scintillation vial was charged with PdCl2(PPh3)2 (30 mg, 0.04 mmol), CuI (8 mg, 0.04 mmol) and 2 mL anhydrous THF. Then 0.83 mmol of a substituted 2-bromo-pyridine, TMS-acetylene (0.17 mL, 1.24 mmol) and Et3N (0.35 mL, 2.49 mmol) were added via syringe. The reaction mixture was stirred at room temperature for 1 h. Upon consumption of the pyridine, the solvent was removed. The crude product was purified via flash column chromatography to yield the TMS-alkyne. N OMe TMS 2-Methoxy-6-((trimethylsilyl)ethynyl)pyridine (S2.1). Compound S2.1 was prepared with 2-bromo-6-methoxypyridine (0.1 mL, 0.83 mmol) following general 383 procedure B1. The crude product was purified via flash column chromatography (30:1 hexanes/EtOAc) to yield a yellow oil (165 mg, 97%). Rf = 0.65 (20:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 7.46 (dd, J = 8.4, 7.8 Hz, 1H), 7.04 (dd, J = 7.2, 0.9 Hz, 1H), 6.66 (dd, J = 8.4, 0.6 Hz, 1H), 3.92 (s, 3H), 0.24 (s, 9H). 13C NMR (75 MHz, CDCl3): δ 164.0, 140.1, 138.6, 121.3, 111.6, 104.2, 94.1, 53.8, 0.0. IR (thin film): 2953, 2900, 1586, 1567, 1461, 1425, 1405, 1315, 1220, 1031, 982, 953, 843, 802 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C11H16NOSi, 206.1001; found, 206.1003. OMe N TMS 5-Methoxy-2-((trimethylsilyl)ethynyl)pyridine (S3.1). Compound S3.1 was prepared with 2-bromo-5-methoxypyridine (0.1 mL, 0.81 mmol) following general procedure B1. The crude product was purified via flash column chromatography (5:1 hexanes/EtOAc) to yield a brown oil (168 mg, 99%). Rf = 0.4 (4:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 8.16 (d, J = 2.7 Hz, 1H), 7.32 (d, J = 8.7 Hz, 1H), 7.03 (dd, J = 8.7, 3.0 Hz, 1H), 3.77 (s, 3H), 0.18 (s, 9H). 13C NMR (75 MHz, CDCl3): δ 155.3, 138.2, 135.3, 128.0, 120.2, 103.8, 93.0, 55.8, 0.0. IR (thin film): 2957, 1584, 1556, 1469, 1437, 1267, 1250, 1028, 867, 840, 761 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C11H16NOSi, 206.1001; found, 206.1013. OMe N TMS 4-Methoxy-2-((trimethylsilyl)ethynyl)pyridine (S4.1). Compound S4.1 was prepared with 2-bromo-4-methoxypyridine (0.1 mL, 0.83 mmol) following general procedure B1. The crude product was purified via flash column chromatography (5:1 384 hexanes/EtOAc) to yield a yellow oil (148.5 mg, 89%). Rf = 0.2 (7:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 8.27 (d, J = 5.7 Hz, 1H), 6.90 (d, J = 2.7 Hz, 1H), 6.67 (dd, J = 5.7, 2.7 Hz, 1H), 3.75 (s, 3H), 0.19 (s, 9H). 13C NMR (75 MHz, CDCl3): δ 165.7, 151.9, 144.4, 113.2, 110.6, 103.9, 94.4, 55.4, 0.1. IR (thin film): 2961, 1586, 1560, 1465, 1308, 1250, 1192, 1158, 1036, 942, 844, 668 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C11H16NOSi, 206.1001; found, 206.1005. MeO N TMS 3-Methoxy-2-((trimethylsilyl)ethynyl)pyridine (S5.1). Compound S5.1 was prepared with 2-bromo-3-methoxypyridine (3.0 g, 15.95 mmol) following general procedure B1. The crude product was purified via flash column chromatography (5:1 hexanes/EtOAc) to yield an orange oil (2.98 g, 91%). Rf = 0.3 (4:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 8.08 (dd, J = 4.5, 1.5 Hz, 1H), 7.16 – 7.08 (m, 2H), 3.80 (s, 3H), 0.20 (s, 9H). 13C NMR (75 MHz, CDCl3): δ 157.3, 141.8, 133.15 124.1, 117.9, 100.4, 99.7, 55.9, 0.0. IR (thin film): 2959, 1574, 1460, 1426, 1280, 1247, 1121, 870, 840, 760 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C10H16NOSi, 206.1001; found, 206.1011. N Cl TMS 2-Chloro-6-((trimethylsilyl)ethynyl)pyridine (S6.1). Compound S6.1 was prepared with 2-bromo-6-chloropyridine (0.1 g, 0.52 mmol) following general procedure B1. The crude product was purified via flash column chromatography (40:1 hexanes/EtOAc) to yield a white solid (101.6 mg, 93%). Rf = 0.5 (20:1 hexanes/EtOAc). 1 H NMR (CDCl3, 300 MHz): δ 7.59 (t, J = 7.8 Hz, 1H), 7.36 (dd, J = 7.8, 0.9 Hz, 1H), 7.26 (dd, J = 7.8, 0.9 Hz, 1H), 0.25 (s, 9H). 13C NMR (75 MHz, CDCl3): δ 151.4, 143.3, 138.7, 385 126.1, 124.2, 102.4, 96.9, 0.2. IR (thin film): 2958, 1558, 1429, 1249, 1139, 887, 844 cm1 . HRMS (ESI-TOF) [M + H]+ m/z: calcd for C10H13ClNSi, 210.0506; found, 210.0520. N TMS 2-(4-(Trimethylsilyl)-but-3-yn-1-yl)-pyridine (S7.1). A 25-mL oven-dried round bottom flask with a magnetic stirring bar was charged with 2-methylpyridine (0.21 mL, 2.14 mmol) and 5 mL of anhydrous THF. The solution was cooled down to - 78 oC and 2.5M n-BuLi solution in hexanes (0.86 mL, 2.14 mmol) was added via syringe dropwise. The resulting mixture was stirred at the same temperature for 30 min. Then 3-bromo-1(trimethylsilyl)-1-propyne (0.42 mL, 2.56 mol) was added slowly to the mixture. After 2 h, the reaction was quenched with sat. aq. NH4Cl solution. The crude product was extracted twice with EtOAc. Upon removal of the organic solvent, the crude product was loaded on the column (4:1 hexanes/EtOAc) and isolated as a colorless liquid (0.41 g, 95%). Rf = 0.4 (4:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 8.38 (d, J = 4.8 Hz, 1H), 7.44 (t, J = 7.8 Hz, 1H), 7.05 (d, J = 7.8 Hz, 1H), 6.96 – 6.94 (m, 1H), 2.84 (t, J = 7.5 Hz, 2H), 2.51 (t, J = 7.5 Hz, 2H), 0.01 (s, 9H). 13C NMR (75 MHz, CDCl3): δ 160.4, 149.4, 136.2, 123.3, 121.5, 106.5, 85.4, 37.3, 20.2, 0.2. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C12H18NSi, 204.1209; found, 204.1216. General procedure B2 for the TMS deprotection using K2CO3 K2CO3 R N MeOH, r.t. R N TMS A 50-mL round bottom flask was charged with 5.84 mmol of the TMS-alkyne and 20 mL MeOH. To the stirring mixture was added K2CO3 (0.85 g, 6.13 mmol) in one 386 portion. The reaction mixture was stirred at room temperature for 1 h, quenched with 50 mL sat. aq. NH4Cl solution and extracted with 50 mL EtOAc. After removing the organic solvent, the crude product was purified via flash column chromatography to yield the substituted 2-ethynyl pyridine. N OMe 2-Ethynyl-6-methoxypyridine (S2.2). Compound S2.2 was prepared with S2.1 (1.2 g, 5.84 mmol) following general procedure B2. The crude product was purified via flash column chromatography (50:1 hexanes/EtOAc, followed by 40:1 and 30:1 hexanes/EtOAc) to yield a yellow oil (0.77 g, 99%). Rf = 0.4 (30:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 7.46 (t, J = 7.8 Hz, 1H), 7.03 (d, J = 7.2 Hz, 1H), 6.67 (d, J = 8.4 Hz, 1H), 3.90 (s, 3H), 311 (s, 1H). 13C NMR (75 MHz, CDCl3): δ 164.0, 139.3, 138.7, 121.1, 112.1, 83.1, 76.7, 53.7. IR (thin film): 3284, 2950, 1598, 1569, 1463, 1424, 1312, 1257, 1212, 1028, 982, 801, 667 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C8H8NO, 134.0606; found, 134.0609. OMe N 2-Ethynyl-5-methoxypyridine (S3.2). Compound S3.2 was prepared with S3.1 (168 mg, 0.82 mmol) following general procedure B2. The crude product was purified via flash column chromatography (3:1 hexanes/EtOAc) to yield a white solid (100.6 mg, 92%). Rf = 0.25 (4:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 8.16 (d, J = 3.0 Hz, 1H), 7.32 (d, J = 8.7 Hz, 1H), 7.03 (dd, J = 8.7, 3.0 Hz, 1H), 3.76 (s, 3H), 3.00 (s, 1H). 13C NMR (75 MHz, CDCl3): δ 155.6, 138.4, 134.4, 128.2, 120.2, 82.8, 75.9, 55.8. IR (thin film): 387 3251, 1584, 1563, 1472, 1445, 1399, 1272, 1194, 1027, 907, 841 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C8H8NO, 134.0606; found, 134.0605. OMe N 2-Ethynyl-4-methoxypyridine (S4.2). Compound S4.2 was prepared with S4.1 (148.5 mg, 0.72 mmol) following general procedure B2. The crude product was purified via flash column chromatography (1:1 hexanes/EtOAc) to yield a reddish oil (75.3 mg, 78%). Rf = 0.1 (4:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 8.28 (d, J = 5.7 Hz, 1H), 6.91 (d, J = 2.7 Hz, 1H), 6.70 (dd, J = 5.7, 2.7 Hz, 1H), 3.75 (s, 3H), 3.06 (s. 1H). 13C NMR (75 MHz, CDCl3): δ 166.8, 151.1, 143.6, 113.7, 110.2, 82.9, 76.9, 55.5. IR (thin film): 3283, 1583, 1560, 1467, 1304, 1032, 921, 823 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C8H8NO, 134.0606; found, 134.0605. MeO N 2-Ethynyl-3-methoxypyridine (S5.2). Compound S5.2 was prepared with S5.1 (2.98 g, 14.50 mmol) following general procedure B2. The crude product was purified via flash column chromatography (1:1 hexanes/EtOAc) to yield a yellow oil (1.7 g, 88%). Rf = 0.2 (2:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 8.11 (dd, J = 4.2, 1.5 Hz, 1H), 7.23 – 7.13 (m, 2H), 3.84 (s, 3H), 3.37 (s, 1H). 13C NMR (75 MHz, CDCl3): δ 157.6, 142.0, 132.4, 124.5, 118.0, 81.7, 79.8, 56.0. IR (thin film): 3271, 2942, 2836, 1575, 1462, 1439, 1279, 1239, 1121, 1021, 796, 760, 668, 581 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C8H8NO, 134.0606; found, 134.0604. 388 N Cl 2-Chloro-6-ethynylpyridine (S6.2). Compound S6.2 was prepared with S6.1 (101.6 mg, 0.48 mmol) following general procedure B2. The crude product was purified via flash column chromatography (30:1 hexanes/EtOAc) to yield a white solid (66.0 mg, 99%). Rf = 0.3 (30:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 7.62 (t, J = 7.8 Hz, 1H), 7.39 (dd, J = 7.8, 0.9 Hz, 1H), 7.30 (dd, J = 8.1, 0.9 Hz, 1H), 3.19 (s, 1H). 13C NMR (75 MHz, CDCl3): δ 151.6, 142.5, 139.0, 126.2, 124.7, 81.7, 78.8. IR (thin film): 3273, 1570, 1557, 1429, 1142, 987, 855, 799, 676 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C7H5ClN, 138.0111; found, 138.0112. N 2-(But-3-yn-1-yl)pyridine (S7.2). Compound S7.2 was prepared with S7.1 (2.75 g, 13.52 mmol) following general procedure B2. The crude product was purified via flash column chromatography (4:1 hexanes/EtOAc) to yield a yellow oil (1.36 g, 77%). Rf = 0.3 (4:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 8.47 (d, J = 4.0 Hz, 1H), 7.53 (t, J = 7.5 Hz 1H), 7.13 (d, J = 7.5 H, 1H), 7.05 (t, J = 6.0 Hz, 1H), 2.94 (t, J = 7.5 Hz, 2H), 2.58 (t, J = 7.5 Hz, 2H), 1.89 (s, 1H). 13C NMR (75 MHz, CDCl3): δ 159.9, 149.5, 136.5, 123.2, 121.7, 83.8, 69.1, 37.1, 18.7. IR (thin film): 3297, 2917, 1589, 1569, 1475, 1434, 1243, 1146, 1049, 993, 749, 626 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C9H10N, 132.0813; found, 132.0814. 389 General procedure B3 for the preparation of N-Boc-propargyl amines MeO MeO NHBoc NHBoc O S O + nBuLi, THF N -78 oC R N R A 25-mL oven-dried round bottom flask with a magnetic stirring bar was charged with 0.79 mmol of a 2-ethynylpyridine derivative and 5 mL anhydrous THF. The stirring solution was cooled down to -78 oC and a 2.5M n-BuLi-solution in hexanes (0.3 mL, 0.74 mmol) was added via syringe. The resulting mixture was stirred at the same temperature for 30 min. Then a 5 mL THF solution of the amino sulfone 4.2 (100 mg, 0.26 mmol) was added to the mixture. The reaction was stirred for 3 h at -78 oC and then quenched with 15 mL sat. aq. NH4Cl solution. The crude product was extracted twice with 10 mL EtOAc. The organic layer was concentrated and the crude product was purified via flash column chromatography the Boc-protected propargyl amine. MeO NHBoc N Tert-butyl (1-(4-methoxyphenyl)-3-(pyridin-2-yl)prop-2-yn-1-yl)carbamate (4.3.1). Compound 4.3.1 was prepared with 4.2 (100 mg, 0.26 mmol) and 2-ethynyl pyridine S1.2 (0.08 mL, 0.79 mmol) following general procedure B3. The crude product was purified via flash column chromatography (3:1 hexanes/EtOAc, followed by 1:1 hexanes/EtOAc) to yield a white solid (78.3 mg, 87%). Rf = 0.5 (1:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 8.54 (d, J = 4.5 Hz, 1H), 7.60 (t, J = 7.8 Hz, 1H), 7.76 (d, J = 7.8 Hz, 2H), 7.40 (d, J = 8.1 Hz, 1H), 7.19 (t, J = 6.0 Hz, 1H), 7.86 (d, J = 8.7 Hz, 2H), 390 5.84 (d, J = 7.2 Hz, 1H), 5.23 (d, J = 6.0 Hz, 1H), 3.76 (s, 3H), 1.43 (s, 9H). 13C NMR (CDCl3, 75 MHz): δ 159.6, 154.9, 150.2, 143.1, 136.4, 131.4, 128.4, 127.4, 123.2, 114.3, 88.0, 84.1, 80.3, 55.5, 46.4, 28.6. IR (thin film): 2976, 2931, 1700, 1510, 1463, 1247, 1166, 1034, 779 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C20H23N2O3, 339.1709; found, 339.1711. MeO NHBoc N OMe Tert-butyl (1-(4-methoxyphenyl)-3-(6-methoxypyridin-2-yl)prop-2-yn-1-yl)- carbamate (4.3.2). Compound 4.3.2 was prepared with 4.2 (100 mg, 0.26 mmol) and 2ethynyl-6-methoxypyridine S2.2 (105.8 mL, 0.79 mmol) following general procedure B3. The crude product was purified via flash column chromatography (5:1 hexanes/EtOAc) to yield a white solid (92.8 mg, 95%). Rf = 0.4 (4:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 7.54 – 7.47 (m, 3H), 7.05 (d, J = 7.2 Hz, 1H), 6.90 (d, J = 8.7 Hz, 2H), 6.71 (d, J = 8.7 Hz, 1H), 5.86 (brs, 1H), 5.15 (brs, 1H), 3.94 (s, 3H), 3.81 (s, 3H), 1.47 (s, 9H). 13C NMR (CDCl3, 75 MHz): δ 164.0, 159.6, 154.9, 140.0, 138.6, 131.6, 128.4, 121.0, 114.2, 111.5, 87.3, 84.3, 80.4, 55.5, 53.8, 46.4, 28.6. IR (thin film): 2977, 1699, 1569, 1507, 1462, 1429, 1243, 1162, 1017, 801, 668 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C21H25N2O4, 369.1814; found, 369.1818. 391 MeO NHBoc N OMe Tert-butyl (1-(4-methoxyphenyl)-3-(5-methoxypyridin-2-yl)prop-2-yn-1-yl)- carbamate (4.3.3). Compound 4.3.3 was prepared with 4.2 (1.0 g, 2.65 mmol) and 2ethynyl-5-methoxypyridine S3.2 (1.1 g, 7.95 mmol) following general procedure B3. The crude product was purified via flash column chromatography (4:1 hexanes/EtOAc, followed by 3:1 and 2:1 hexanes/EtOAc) to yield a white solid (957.3 mg, 98%). Rf = 0.2 (3:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 8.22 (d, J = 3.0 Hz, 1H), 7.46 (d, J = 8.4 Hz, 2H), 7.35 (d, J = 8.7 Hz, 1H), 7.09 (dd, J = 8.4, 3.0 Hz, 1H), 6.85 (d, J = 8.4 Hz, 2H), 5.82 (brs, 1H), 5.28 (brs, 1H), 3.81 (s, 3H), 3.75 (s, 3H), 1.43 (s, 9H). 13C NMR (75 MHz, CDCl3): δ 159.6, 155.4, 138.3, 135.1, 131.7, 128.4, 128.0, 120.4, 114.2, 86.5, 83.9, 80.3, 55.9, 55.5, 46.4, 28.6. IR (thin film): 2969, 1611, 1586, 1565, 1509, 1485, 1475, 1440, 1365, 1267, 1242, 1162, 1030, 832, 762 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C21H25N2O4, 369.1814; found, 369.1814. MeO NHBoc N MeO Tert-butyl (1-(4-methoxyphenyl)-3-(4-methoxypyridin-2-yl)prop-2-yn-1-yl)- carbamate (4.3.4). Compound 4.3.4 was prepared with 4.2 (1.0 g, 2.65 mmol) and 2ethynyl-4-methoxypyridine S4.2 (1.1 g, 7.95 mmol) following general procedure B3. The crude product was purified via flash column chromatography (2:1 hexanes/EtOAc, 392 followed by 1:1 hexanes/EtOAc) to yield a yellow semisolid (858.5 mg, 88%). Rf = 0.1 (2:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 8.26 (d, J = 5.7 Hz, 1H), 7.40 (d, J = 8.1 Hz, 2H), 6.86 (d, J = 2.4 Hz, 1H), 6.78 (d, J = 8.1 Hz, 2H), 6.66 (dd, J = 5.7, 2.7 Hz, 1H), 5.79 (d, J = 8.4 Hz, 1H), 5.51 (d, J = 8.7 Hz, 1H), 3.71 (s, 3H), 3.66 (s, 3H), 1.37 (s, 9H). 13C NMR (75 MHz, CDCl3): δ 165.7, 159.5, 155.0, 151.2, 144.2, 131.4, 128.4, 114.2, 113.3, 110.0, 87.7, 84.0, 80.2, 55.4, 55.4, 46.3, 28.5. IR (thin film): 2969, 1708, 1610, 1585, 1560, 1508, 1468, 1365, 1304, 1240, 1161, 1036, 1019, 869, 830 cm-1. HRMS (ESITOF) [M + H]+ m/z: calcd for C21H25N2O4, 369.1814; found, 369.1814. MeO NHBoc MeO Tert-butyl N (1-(4-methoxyphenyl)-3-(3-methoxypyridin-2-yl)prop-2-yn-1-yl)- carbamate (4.3.5). Compound 4.3.5 was prepared with 4.2 (1.0 g, 2.65 mmol) and 2ethynyl-3-methoxypyridine S5.2 (882.1 mg, 6.62 mmol) following general procedure B3. The crude product was purified via flash column chromatography (3:2 hexanes/EtOAc) to yield a white semisolid (1.01 g, 99%). Rf = 0.25 (1:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 8.00 (s, 1H), 7.40 (d, J = 8.1 Hz, 2H), 7.02 (s, 2H), 6.73 (d, J = 8.7 Hz, 2H), 5.82 (d, J = 9.0 Hz, 1H), 5.51 (d, J = 8.7 Hz, 1H), 3.68 (s, 3H), 3.61 (s, 3H), 1.32 (s, 9H). 13 C NMR (75 MHz, CDCl3): δ 159.4, 157.2, 155.0, 141.6, 132.9, 131.6, 128.5, 124.1, 117.9, 114.0, 92.5, 80.9, 80.0, 55.8, 55.4, 46.4, 28.5. IR (thin film): 2972, 1679, 1511, 1464, 1276, 1240, 1162, 1121, 1012, 868, 838, 790, 584 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C21H25N2O4, 369.1814; found, 369.1823. 393 MeO NHBoc N Cl Tert-butyl (3-(6-chloropyridin-2-yl)-1-(4-methoxyphenyl)prop-2-yn-1-yl)- carbamate (4.3.6). Compound 4.3.6 was prepared with 4.2 (0.15 g, 0.39 mmol) and 2ethynyl-6-chloropyridine S6.2 (165.1 mg, 1.20 mmol) following general procedure B3. The crude product was purified via flash column chromatography (5:1 hexanes/EtOAc, followed by 4:1 hexanes/EtOAc) to yield a white solid (144.3 mg, 99%). Rf = 0.3 (4:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 7.55 (t, J = 7.8 Hz, 1H), 7,41 (d, J = 8.7 Hz, 2H), 7.29 (dd, J = 7.5, 0.9 Hz, 1H), 7.21 (dd, J = 8.1, 0.9 Hz, 1H), 6.83 (d, J = 8.7 Hz, 2H), 5.81 (d, J = 8.4 Hz, 1H), 5.26 (d, J = 8.4 Hz, 1H), 3.74 (s, 3H), 1.41 (s, 9H). 13C NMR (CDCl3, 75 MHz): δ 159.7, 154.9, 151.4, 143.3, 139.0, 130.9, 128.4, 126.0, 124.2, 114.3, 89.6, 82.8, 80.5, 55.5, 46.4, 28.6. IR (thin film): 2969, 1699, 1571, 1550, 1507, 1434, 1246, 1160, 1021, 797, 668 cm-1. HRMS (ESI-TOF) [M + Na]+ m/z: calcd for C20H21ClN2O3Na, 395.1138; found, 395.1134. MeO NHBoc TMS Tert-butyl (1-(4-methoxyphenyl)-3-(trimethylsilyl)prop-2-yn-1-yl)carbamate (4.3.7). Compound 4.3.7 was prepared with 4.2 (0.2 g, 0.53 mmol) and ethynyltrimethylsilane S11 (0.22 mL, 1.59 mmol) following general procedure B3. The crude product was purified via flash column chromatography (8:1 hexanes/EtOAc) to yield a white solid (169.2 mg, 96%). Rf = 0.5 (8:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): 394 δ 7.40 (d, J = 7.8 Hz, 2H), 6.85 (dd, J = 8.7, 1.5 Hz, 2H), 5.62 (s, 1H), 5.21 (s, 1H), 3.76 (s, 3H), 1.44 (s, 9H), 0.19 (s, 9H). 13 C NMR (75 MHz, CDCl3): δ 159.5, 155.0, 131.8, 128.4, 114.1, 104.3, 89.3, 80.2, 55.4, 46.6, 28.6, 0.1. IR (thin film): 3331, 2963, 1699, 1510, 1366, 1304, 1248, 1169, 1043, 843, 760 cm-1. HRMS (ESI-TOF) [M + Na]+ m/z: calcd for C18H27NO3SiNa, 356.1658; found, 356.1656. MeO NHBoc N Tert-butyl (1-(4-methoxyphenyl)-5-(pyridin-2-yl)pent-2-yn-1-yl)carbamate (4.3.8). Compound 4.3.8 was prepared with 4.2 (0.20 g, 0.53 mmol) and 2-(but-3-yn-1yl)pyridine S7.2 (0.16 mL, 1.22 mmol) following general procedure B3. The crude product was purified via flash column chromatography (3:2 hexanes/EtOAc) to yield a colorless oil (189.6 mg, 97%). Rf = 0.3 (1:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 8.48 (d, J = 4.2 Hz, 1H), 7.53 (td, J = 7.8, 1.8 Hz, 1H), 7.27 (d, J = 8.4 Hz, 2H), 7.14 – 7.06 (m, 2H), 6.76 (d, J = 8.7 Hz, 2H), 5.50 (s, 1H), 5.24 (s, 1H), 3.73 (s, 3H), 2.95 (t, J = 7.2 Hz, 2H), 2.64 (t, J = 7.2 Hz, 2H), 1.40 (s, 9H). 13C NMR (CDCl3, 75 MHz): δ 160.2, 159.3, 155.1, 149.5, 136.5, 132.4, 128.3, 123.4, 121.7, 113.9, 84.3, 80.0, 79.7, 55.5, 46.0, 37.3, 28.6, 19.2. IR (thin film): 3328, 2975, 2931, 1701, 1610, 1591, 1509, 1365, 1245, 1168, 1034, 883, 752, 668 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C22H27N2O3, 367.2022; found, 367.2019. 395 General procedure B4 for the methylation of N-Boc-propargyl amines MeO MeO NHBoc Me N MeI, NaH Boc DMF, 0 oC N R N R To an oven-dried 25-mL round-bottom flask was added 0.24 mmol of the Bocprotected propargyl amine and 5 mL anhydrous DMF. The stirring solution was cooled down to 0 oC and iodomethane (0.15 mL, 2.40 mmol) was added via syringe, followed by NaH (60wt% dispersed in mineral oil, 11.52 mg, 0.29 mmol). The reaction mixture was stirred at the same temperature for 2 h and quenched with sat. aq. NH4Cl solution. The crude product was extracted two times with 10 mL Et2O. The organic layer was concentrated and purified via flash column chromatography to yield the N-methyl propargyl amine. MeO Me N Boc N Tert-butyl (1-(4-methoxyphenyl)-3-(pyridin-2-yl)prop-2-yn-1-yl)(methyl)- carbamate (4.4.1). Compound 4.4.1 was prepared with 4.3.1 (82 mg, 0.24 mmol) following general procedure B4. The crude product was purified via flash column chromatography (3:1 hexanes/EtOAc) to yield a yellow oil (61.7 mg, 73%). Rf = 0.2 (3:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 8.58 (t, J = 2.1 Hz, 1H), 7.64 (t, J = 7.8 Hz, 1H), 7,45 (t, J = 7.2 Hz, 3H), 7.23 (t, J = 6.3 Hz, 1H), 6.87 (d, J = 8.7 Hz, 2H), 6.51 (d, 1H, rotamers), 3.78 (s, 3H), 2.74 (s, 3H), 1.50 (s, 9H). 13C NMR (75 MHz, CDCl3): δ 159.6, 150.3, 143.2, 136.4, 129.3, 128.8, 127.6, 123.3, 114.1, 86.1, 85.8, 80.5, 55.5, 50.8, 396 29.6, 28.7. IR (thin film): 2958, 1716, 1699, 1652, 1558, 1540, 1507, 1456, 668 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C21H25N2O3, 353.1865; found, 353.1860. MeO Me N Boc N OMe Tert-butyl (1-(4-methoxyphenyl)-3-(6-methoxypyridin-2-yl)prop-2-yn-1-yl)- (methyl)carbamate (4.4.2). Compound 4.4.2 was prepared with 4.3.2 (187.0 mg, 0.51 mmol) following general procedure B4. The crude product was purified via flash column chromatography (4:1 hexanes/EtOAc) to yield a yellow oil (149.0 mg, 77%). Rf = 0.5 (4:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 7.53 – 7.41 (m, 3H), 7.08 (d, J = 7.2 Hz, 1H), 6.89 (d, J = 8.7 Hz, 2H), 6.69 (d, J = 8.4 Hz, 1H), 6.51 (d, 1H, rotamers), 3.93 (s, 3H), 3.78 (s, 3H), 2.76 (s, 3H), 1.51 (s, 9H). 13C NMR (75 MHz, CDCl3): δ 164.1, 159.6, 140.0, 138.7, 129.6, 128.8, 121.0, 114.1, 111.5, 86.1, 85.4, 80.6, 55.5, 53.8, 50.8, 29.6, 28.7. IR (thin film): 2975, 1683, 1584, 1569, 1462, 1428, 1381, 1303, 1247, 1141, 1031, 877, 802, 765 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C22H27N2O4, 383.1971; found, 383.1972. MeO Me N Boc N OMe Tert-butyl (1-(4-methoxyphenyl)-3-(5-methoxypyridin-2-yl)prop-2-yn-1-yl)- (methyl)carbamate (4.4.3). Compound 4.4.3 was prepared with 4.3.3 (0.94 g, 2.55 mmol) following general procedure B4. The crude product was purified via flash column 397 chromatography (5:1 hexanes/EtOAc, followed by 4:1 and 3:1 hexanes/EtOAc) to yield a yellow oil (0.85 g, 87%). Rf = 0.3 (2:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 8.27 (d, J = 2.7 Hz, 1H), 7.52 – 7.40 (m, 3H), 7.14 (dd, J = 8.7, 3.0 Hz, 1H), 6,88 (d, J = 8.7 Hz, 2H), 6.50 (d, 1H, rotamers), 3.85 (s, 3H), 3.78 (s, 3H), 2.74 (s, 3H), 1.50 (s, 9H). 13 C NMR (75 MHz, CDCl3): δ 159.5, 155.4, 138.3, 135.2, 129.6, 128.9, 128.1, 120.5, 114.0, 85.6, 84.5, 80.4, 55.9, 55.5, 50.8, 29.6, 28.7. IR (thin film): 2972, 2838, 1683, 1585, 1509, 1473, 1438, 1382, 1365, 1302, 1267, 1246, 1172, 1140, 1031, 879, 850, 766, 590 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C22H27N2O4, 383.1971; found, 383.1976. MeO Me N Boc N MeO Tert-butyl (1-(4-methoxyphenyl)-3-(4-methoxypyridin-2-yl)prop-2-yn-1-yl)- (methyl)carbamate (4.4.4). Compound 4.4.4 was prepared with 4.3.4 (0.86 g, 2.33 mmol) following general procedure B4. The crude product was purified via flash column chromatography (3:1 hexanes/EtOAc, followed by 2:1 and 1:1 hexanes/EtOAc) to yield a yellow oil (0.66 g, 67%). Rf = 0.3 (1:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 8.33 (d, J = 3.6 Hz, 1H), 7.40 (s, 2H), 6.95 (s, 1H), 6.82 (d, J = 5.4 Hz, 2H), 6.72 (dd, J = 3.6 Hz, 1H), 6.46 (d, 1H, rotamers), 3.77 (s, 3H), 3.72 (s, 3H), 2.69 (s, 3H), 1.45 (s, 9H). 13 C NMR (75 MHz, CDCl3): δ 165.6, 159.3, 151.1, 144.0, 129.0, 128.6, 113.8, 113.3, 109.7, 85.4, 80.4, 80.2, 55.2, 55.2, 50.4, 29.2, 28.4. IR (thin film): 2973, 1686, 1587, 1510, 1465, 1383, 1305, 1249, 1174, 1143, 1036, 849, 815 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C22H27N2O4, 383.1971; found, 383.1967. 398 MeO Me N Boc MeO Tert-butyl N (1-(4-methoxyphenyl)-3-(3-methoxypyridin-2-yl)prop-2-yn-1-yl)- (methyl)carbamate (4.4.5). Compound 4.4.5 was prepared with 4.3.5 (1.0 g, 2.71 mmol) following general procedure B4. The crude product was purified via flash column chromatography (3:1 hexanes/EtOAc, followed by 2:1 and 3:2 hexanes/EtOAc) to yield a yellow oil (0.85 g, 82%). Rf = 0.3 (1:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 7.98 (s, 1H), 7.36 (d, J = 7.5 Hz, 2H), 7.02 (s, 2H), 6.72 (d, J = 8.4 Hz, 2H), 6.42 (d, 1H, rotamers), 3.69 (s, 3H), 3.59 (s, 3H), 2.63 (s, 3H), 1.34 (s, 9H). 13C NMR (75 MHz, CDCl3): δ 159.4, 157.4, 141.6, 132.9, 129.4, 129.8, 124.5, 124.1, 117.8, 113.9, 82.7, 81.8, 80.2, 55.8, 55.3, 50.8, 29.4, 28.5. IR (thin film): 2970, 1684, 1576, 1429, 1386, 1306, 1249, 1144, 1033, 760, 668 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C22H27N2O4, 383.1971; found, 383.1969. MeO Me N Boc N Cl Tert-butyl (3-(6-chloropyridin-2-yl)-1-(4-methoxyphenyl)prop-2-yn-1-yl)- (methyl)carbamate (4.4.6). Compound 4.4.6 was prepared with 4.3.6 (643.5 mg, 1.72 mmol) following general procedure B4. The crude product was purified via flash column chromatography (3:1 hexanes/Et2O, followed by 5:1 hexanes/EtOAc) to yield a yellow oil (153.6 mg, 23%). Rf = 0.5 (4:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 7.59 (t, J = 7.8 Hz, 1H), 7.45 – 7.33 (m, 3H), 7.25 (d, J = 8.1 Hz, 1H), 6.85 (d, J = 8.7 Hz, 2H), 6.51 399 (d, 1H, rotamers), 3.75 (s, 3H), 2.71 (s, 3H), 1.47 (s, 9H). 13C NMR (75 MHz, CDCl3): δ 159.6, 151.5, 143.1, 139.0, 128.8, 128.7, 126.2, 124.3, 114.1, 87.7, 84.6, 80.7, 50.8, 29.7, 28.6. IR (thin film): 2974, 2932, 1686, 1609, 1572, 1510, 1433, 1382, 1305, 1249, 1173, 1139, 1034, 882, 797 cm-1. HRMS (ESI-TOF) [M + Na]+ m/z: calcd for C21H23N2O4Na, 409.1295; found, 409.1295. MeO Me N Boc TMS Tert-butyl (1-(4-methoxyphenyl)-3-(trimethylsilyl)prop-2-yn-1-yl)(methyl)- carbamate (4.4.7). Compound 4.4.7 was prepared with 4.3.7 (165.7 mg, 0.49 mmol) following general procedure B4. The crude product was purified via flash column chromatography (3:1 hexanes/Et2O, followed by 5:1 hexanes/EtOAc) to yield a colorless oil (129.5 mg, 75%). Rf = 0.5 (8:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 7.37 (d, J = 8.4 Hz, 2H), 6.87 (d, J = 8.7 Hz, 2H), 6.24 (brs, 1H), 3.77 (s, 3H), 2.66 (s, 3H), 1.50 (s, 9H), 0.22 (s, 9H). 13 C NMR (75 MHz, CDCl3): δ 159.2, 155.6, 129.4, 128.5, 113.7, 102.0, 91.1, 80.0, 55.2, 50.7, 28.4, 0.1. IR (thin film): 2969, 1687, 1611, 1508, 1456, 1382, 1303, 1246, 1172, 1142, 1036, 842, 760 cm-1. HRMS (ESI-TOF) [M + Na]+ m/z: calcd for C19H29NO3SiNa, 370.1814; found, 370.1809. MeO Me N Boc N Tert-butyl (1-(4-methoxyphenyl)-5-(pyridin-2-yl)pent-2-yn-1-yl)-(methyl)- carbamate (4.4.8). Compound 4.4.8 was prepared with 4.3.8 (188.3 mg, 0.51 mmol) 400 following general procedure B4. The crude product was purified via flash column chromatography (3:2 hexanes/EtOAc) to yield a colorless oil (131.9 mg, 68%). Rf = 0.4 (1:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 8.56 (d, J = 3.9 Hz, 1H), 7.60 (td, J = 7.8, 1.8 Hz, 1H), 7.24 – 7.18 (m, 3H), 7.14 (dd, J = 7.5, 4.5 Hz, 1H), 6.81 (d, J = 8.4 Hz, 2H), 6.16 (d, 1H, rotamers), 3.79 (s, 3H), 3.05 (t, J = 7.2 Hz, 2H), 2.76 (t, J = 7.2 Hz, 2H), 2.53 (s. 3H), 1.48 (s, 9H). 13C NMR (75 MHz, CDCl3): δ 160.2, 159.2, 149.6, 136.5, 130.3, 128.7, 123.4, 121.7, 113.8, 85.8, 80.1, 55.4, 50.3, 37.4, 29.2, 28.7, 19.1. IR (thin film): 2975, 1683, 1589, 1508, 1435, 1385, 1365, 1304, 1245, 1173, 1127, 1032, 880, 850, 762 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C23H29N2O3, 381.2178; found, 381.2170. MeO Me N Boc Tert-butyl (1-(4-methoxyphenyl)prop-2-yn-1-yl)(methyl)carbamate (4.4.9). Compound 4.4.9 was prepared with 4.4.7 (1.20 g, 3.45 mmol) following general procedure B2. The crude product was purified via flash column chromatography (10:1 hexanes/EtOAc) to yield a pale yellow oil (0.89 g, 93%). Rf = 0.5 (10:1 hexanes/EtOAc). 1 H NMR (CDCl3, 300 MHz): δ 7.39 (d, J = 8.4 Hz, 2H), 6.87 (d, J = 8.4 Hz, 2H), 6.23 (d, 1H, rotamers), 3.78 (s, 3H), 2.67 (s, 3H), 2.54 (s, 1H), 1.50 (s, 9H). 13C NMR (75 MHz, CDCl3): δ 159.3, 154.8, 129.0, 128.5, 126.4, 113.8, 80.2, 74.3, 55.2, 49.9, 29.0, 28.4. IR (thin film): 3289, 2976, 1683, 1611, 1509, 1437, 1382, 1304, 1246, 1141, 1033, 879, 850, 766 cm-1. HRMS (ESI-TOF) [M + Na]+ m/z: calcd for C16H21NO3Na, 298.1419; found, 298.1425. 401 General procedure for the synthesis of Boc-protected propargyl amines via Sonogashira reaction MeO MeO Me N Boc PdCl2(PPh3)2 CuI, Et3N + R N Br Me N Boc THF, r.t. N R The preparation of Boc-protected propargyl amines followed general procedure B1. The reaction mixture was stirred at room temperature for 1 h, concentrated under reduced pressure and the crude product was purified via flash column chromatography. MeO Me N Boc N Me Tert-butyl (1-(4-methoxyphenyl)-3-(6-methylpyridin-2-yl)prop-2-yn-1-yl)- (methyl)carbamate (4.4.10). Compound 4.4.10 was prepared with 4.4.9 (0.30 g, 1.09 mmol) and 2-bromo-6-methylpyridine (0.14 mL, 1.20 mmol) following general procedure B1. The crude product was purified via flash column chromatography (7:2 hexanes/EtOAc) to yield a yellow oil (308.8 mg, 77%). Rf = 0.3 (3:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 7.52 (t, J = 7.5 Hz, 1H), 7.43 (d, J = 7.5 Hz, 2H), 7.29 (d, J = 7.5 Hz, 1H), 7.09 (d, J = 7.8 Hz, 1H), 6.86 (d, J = 8.7 Hz, 2H), 6.51 (d, 1H, rotamers), 3.77 (s, 3H), 2.73 (s, 3H), 2.53 (s, 3H), 1.48 (s, 9H). 13 C NMR (75 MHz, CDCl3): δ 159.5, 159.2, 136.6, 129.4, 129.0, 128.9, 128.7, 124.8, 123.1, 114.0, 85.9, 80.7, 55.5, 50.8, 31.1, 28.7, 24.8. IR (thin film): 2973, 2930, 1687, 1583, 1510, 1452, 1383, 1306, 1249, 1174, 1144, 1057, 850, 793 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C22H27N2O3, 367.2022; found, 367.2017. 402 MeO Me N Boc N Me Me Tert-butyl (3-(6-isopropylpyridin-2-yl)-1-(4-methoxyphenyl)prop-2-yn-1-yl)(methyl)carbamate (4.4.11). Compound 4.4.11 was prepared with 4.4.9 (0.30 g, 1.09 mmol) and 2-bromo-6-isopropylpyridine (0.18 mL, 1.20 mmol) following general procedure B1. The crude product was purified via flash column chromatography (7:2 hexanes/Et2O) to yield a yellow oil (358.8 mg, 83%). Rf = 0.5 (3:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 7.57 (t, J = 7.8 Hz, 1H), 7.44 (d, J = 7.8 Hz, 2H), 7.30 (d, J = 7.5 Hz, 1H), 7.13 (d, J = 7.8 Hz, 1H), 6.67 (d, J = 8.7 Hz, 2H), 6.51 (d, 1H, rotamers), 3.78 (s, 3H), 3.07 (septet, J = 6.9 Hz, 1H), 2.75 (s, 3H), 1.49 (s 9H), 1.28 (d, J = 6.9 Hz, 6H). 13 C NMR (75 MHz, CDCl3): δ 168.3, 159.5, 136.7, 129.6, 128.9, 128.8, 128.7, 125.3, 120.1, 114.0, 86.4, 80.6, 55.5, 50.9, 36.8, 31.1, 28.8, 22.8. IR (thin film): 2964, 2930, 1685, 1609, 1582, 1509, 1442, 1381, 1304, 1247, 1173, 1141, 1035, 880, 810 cm-1. HRMS (ESITOF) [M + H]+ m/z: calcd for C24H31N2O3, 395.2335; found, 395.2320. MeO Me N Boc N tBu Tert-butyl (3-(6-(tert-butyl)pyridin-2-yl)-1-(4-methoxyphenyl)prop-2-yn-1- yl)-(methyl)carbamate (4.4.12). Compound 4.4.12 was prepared with 4.4.9 (128.6 mg, 0.47 mmol) and 2-bromo-6-tert-butylpyridine (0.11 g, 0.51 mmol) following general procedure B1. The crude product was purified via flash column chromatography (7:2 403 hexanes/Et2O) to yield a yellow oil (0.13 g, 68%). Rf = 0.4 (10:1 hexanes/Et2O). 1H NMR (CDCl3, 300 MHz): δ 7.56 (t, J = 7.8 Hz, 1H), 7.47 (d, J = 7.8 Hz, 2H), 7.30 (d, J = 7.8 Hz, 2H), 6.90 (d, J = 8.7 Hz, 2H), 6.52 (d, 1H, rotamers), 3.80 (s, 3H), 2.78 (s, 3H), 1.52 (s, 9H), 1.36 (s, 9H). 13C NMR (75 MHz, CDCl3): δ 170.0, 159.5, 136.4, 129.7, 129.0, 128.8, 128.7, 124.8, 118.9, 114.0, 84.9, 80.5, 55.5, 52.0, 37.8, 30.4, 29.6, 28.7. IR (thin film): 2963, 1689, 1567, 1510, 1444, 1384, 1366, 1305, 1249, 1173, 1144, 1036, 812, 668 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C25H33N2O3, 409.2491; found, 409.2489. MeO Me N Boc Me N O Tert-butyl Me (3-(6-isopropoxypyridin-2-yl)-1-(4-methoxyphenyl)prop-2-yn-1- yl)-(methyl)carbamate (4.4.13). Compound 4.4.13 was prepared with 4.4.9 (0.30 g, 1.09 mmol) and 2-bromo-6-isopropoxypyridine (0.19 mL, 1.20 mmol) following general procedure B1. The crude product was purified via flash column chromatography (10:1 hexanes/Et2O, followed by 8:1 and 6:1 hexanes/Et2O) to yield a yellow oil (299.8 mg, 67%). Rf = 0.6 (4:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 7.52 – 7.39 (m, 3H), 7.04 (d, J = 7.2 Hz, 1H), 6.88 (d, J = 8.7 Hz, 2H), 6.63 (d, J = 8.1 Hz, 1H), 5.35 (septet, J = 6.3 Hz, 1H), 3.78 (s, 3H), 2.75 (s, 3H), 1.50 (s, 9H), 1.32 (d, J = 6.3 Hz, 6H). 13C NMR (75 MHz, CDCl3): δ 163.4, 159.5, 140.0 138.7, 129.6, 128.8, 120.6, 114.0, 112.1, 86.3, 85.0, 80.5, 68.3, 55.5, 50.8, 29.5, 28.7, 22.2. IR (thin film): 2975, 2932, 1686, 1609, 1566, 1439, 1381, 1303, 1247, 1140, 1033, 959, 559 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C24H31N2O4, 411.2284; found, 411.2290. 404 MeO Me N Boc N OMe O Methyl 6-(3-((tert-butoxycarbonyl)(methyl)amino)-3-(4-methoxyphenyl)prop1-yn-1-yl)picolinate (4.4.14). Compound 4.4.14 was prepared with 4.4.9 (0.1 g, 0.36 mmol) and methyl 6-bromopicolinate (78.4 mg, 0.36 mmol) following general procedure B1. The crude product was purified via flash column chromatography (40:1 hexanes/EtOAc) to yield a yellow oil (60.0 mg, 40%). Rf = 0.5 (20:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 8.08 (d, J = 7.8 Hz, 1H), 7.81 (t, J = 7.8 Hz, 1H), 7.66 (d, J = 7.8 Hz, 1H), 7.43 (d, J = 8.1 Hz, 2H), 6.88 (d, J = 8.1 Hz, 2H), 6.53 (d, 1H, rotamers), 4.00 (s, 3H), 3.80 (s, 3H), 2.75 (s, 3H), 1.50 (s, 9H). 13C NMR (75 MHz, CDCl3): δ 170.8, 165.0, 159.3, 155.4, 148.2, 143.0, 137.3, 130.5, 128.7, 128.5, 124.6, 113.8, 87.3, 84.8, 80.3, 55.1, 52.9, 50.5, 29.3, 28.3. HRMS (ESI-TOF) [M + Na]+ m/z: calcd for C23H26N2O5Na, 433.1739; found, 433.1738. General procedure B5 for Boc-deprotections MeO MeO Me N Boc 2M HCl in Et2O NHMe r.t. N R N R To 1.49 mmol of an N-Boc propargyl amine in a 50 mL round bottom flask with magnetic stir bar was added 10 mL of 2M HCl in Et2O via syringe. The resulting mixture was stirred at room temperature for 6 h and quenched with 30 mL of sat. aq. NaHCO3 solution. The crude product was extracted two times with 30 mL CH2Cl2. The organic layer 405 was concentrated and purified via flash column chromatography to yield the N-methyl amine. MeO NHMe N 1-(4-Methoxyphenyl)-N-methyl-3-(pyridin-2-yl)prop-2-yn-1-amine (4.5.1). Compound 4.5.1 was prepared with 4.4.1 (525.8 mg, 1.49 mmol) following general procedure B5. The crude product was purified via flash column chromatography (4:3 hexanes/acetone, followed by 3:2 and 1:1 hexanes/acetone) to yield a brown oil (350 mg, 92%). Rf = 0.1 (30:1 CH2Cl2/MeOH). 1H NMR (CDCl3, 300 MHz): δ 8.57 (d, J = 4.5 Hz, 1H), 7.62 (t, J = 7.8 Hz, 1H), 7.49 (d, J = 8.4 Hz, 2H), 7.43 (d, J = 7.8 Hz, 1H), 7.20 (dd, J = 6.9, 5.4 Hz, 1H), 6.88 (d, J = 8.4 Hz, 2H), 4.72 (s, 1H), 3.78 (s, 3H), 2.54 (s, 3H), 1.61 (s, 1H). 13 C NMR (75 MHz, CDCl3): δ 159.5, 150.2, 143.5, 136.3, 132.0, 129.0, 127.4, 123.0, 114.1, 89.7, 85.2, 55.7, 55.5, 34.0. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C16H17N2O, 253.1341; found, 253.1351. MeO NHMe N OMe 1-(4-Methoxyphenyl)-3-(6-methoxypyridin-2-yl)-N-methylprop-2-yn-1-amine (4.5.2). Compound 4.5.2 was prepared with 4.4.2 (149.0 mg, 0.39 mmol) following general procedure B5. The crude product was purified via flash column chromatography (5:2 hexanes/acetone) to yield a brown oil (77.5 mg, 70%). Rf = 0.3 (2:1 hexanes/acetone). 1H NMR (CDCl3, 300 MHz): δ 7.53 – 7.43 (m, 3H), 7.05 (dd, J = 7.2, 0.9 Hz, 1H), 6.88 (d, J 406 = 8.7 Hz, 2H), 6.67 (dd, J = 8.4, 0.9 Hz, 1H), 4.71 (s, 1H), 3.92 (s, 3H), 3.78 (s, 3H), 2.53 (s, 3H), 1.72 (s, 1H). 13 C NMR (75 MHz, CDCl3): δ 164.0, 159.5, 140.4, 138.7, 132.1, 129.1, 120.9, 114.1, 111.2, 88.9, 85.4, 55.8, 55.5, 53.8, 34.0. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C17H19N2O2, 283.1368; found 283.1447. MeO NHMe N OMe 1-(4-Methoxyphenyl)-3-(5-methoxypyridin-2-yl)-N-methylprop-2-yn-1-amine (4.5.3). Compound 4.5.3 was prepared with 4.4.3 (0.85 g, 2.22 mmol) following general procedure B5. The crude product was purified via flash column chromatography (1:1 hexanes/EtOAc, followed by 1:1 and 1:2 hexanes/acetone) to yield a red oil (0.43 g, 68%). Rf = 0.3 (20:1 CH2Cl2/MeOH). 1H NMR (CDCl3, 300 MHz): δ 8.17 (d, J = 3.0 Hz, 1H), 7.42 (d, J = 8.1 Hz, 2H), 7.30 (dt, J = 8.7, 0.9 Hz, 1H), 7.01 (dd, J = 8.7, 3.0 Hz, 1H), 6.79 (d, J = 8.1 Hz, 2H), 4.63 (s, 1H), 3.72 (s, 3H), 3.68 (s, 3H), 2.45 (s, 3H), 1.74 (brs, 1H). 13 C NMR (75 MHz, CDCl3): δ 159.4, 155.2, 138.2, 135.5, 132.1, 129.0, 127.9, 120.4, 114.0, 88.0, 84.9, 55.8, 55.7, 55.4, 33.9. MeO NHMe N MeO 1-(4-Methoxyphenyl)-3-(4-methoxypyridin-2-yl)-N-methylprop-2-yn-1-amine (4.5.4). Compound 4.5.4 was prepared with 4.4.4 (0.66 g, 1.72 mmol) following general procedure B5. The crude product was purified via flash column chromatography (1:1 407 hexanes/acetone, followed by 30:1 CH2Cl2/MeOH) to yield a dark yellow oil (0.22 g, 44%). Rf = 0.3 (20:1 CH2Cl2/MeOH). 1H NMR (CDCl3, 300 MHz): δ 8.27 (d, J = 5.7 Hz, 1H), 7.41 (d, J = 8.7 Hz, 2H), 6.89 (d, J = 2.4 Hz, 1H), 6.79 (d, J = 8.4 Hz, 2H), 6.65 (dd, J = 5.7, 2.4 Hz, 1H), 4.63 (s, 1H), 3.70 (s, 3H), 3.68 (s, 3H), 2.45 (s, 3H), 1.80 (brs, 1H). 13C NMR (75 MHz, CDCl3): δ 165.8, 159.4, 151.2, 144.6, 131.9, 128.9, 114.0, 113.2, 109.73, 89.2, 85.2, 55.6, 55.4, 33.9. MeO NHMe MeO N 1-(4-Methoxyphenyl)-3-(3-methoxypyridin-2-yl)-N-methylprop-2-yn-1-amine (4.5.5). Compound 4.5.5 was prepared with 4.4.5 (0.85 g, 2.22 mmol) following general procedure B5. The crude product was purified via flash column chromatography (50:1 CH2Cl2/MeOH, followed by 40:1 and 25:1 CH2Cl2/MeOH) to yield a dark yellow oil (402.3 mg, 64%). Rf = 0.15 (30:1 CH2Cl2/MeOH). 1H NMR (CDCl3, 300 MHz): δ 8.11 (s, 1H), 7.51 (d, J = 8.4 Hz, 2H), 7.16 – 7.07 (m, 2H), 6.82 (d, J = 8.4 Hz, 2H), 4.75 (s, 1H), 3.81 (s, 3H), 3.72 (s, 3H), 2.51 (s, 3H), 2.26 (brs, 1H). 13C NMR (75 MHz, CDCl3): δ 159.3, 157.2, 141.7, 133.5, 131.9, 129.2, 123.8, 117.8, 113.9, 94.1, 82.0, 55.9, 55.8, 55.5, 33.8. MeO NHMe N Cl 3-(6-Chloropyridin-2-yl)-1-(4-methoxyphenyl)-N-methylprop-2-yn-1-amine (4.5.6). Compound 4.5.6 was prepared with 4.4.6 (0.39 g, 1.02 mmol) following general procedure B5. The crude product was purified via flash column chromatography (1:1 408 hexanes/EtOAc, followed by 1:2 hexanes/EtOAc) to yield a dark yellow oil (0.12 g, 43%). Rf = 0.1 (1:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 7.54 (t, J = 7.5 Hz, 1H), 7.43 (d, J = 8.5 Hz, 2H), 7.32 (d, J = 7.5 Hz, 1H), 7.21 (d, J = 8.0 Hz, 1H), 6.85 (d, J = 8.5 Hz, 2H), 4.67 (s, 1H), 3.75 (s, 3H), 2.49 (s, 3H), 1.56 (s, 1H). 13C NMR (125 MHz, CDCl3): δ 159.3, 151.1, 143.2, 138.7, 131.4, 128.7, 125.7, 123.7, 113.9, 91.1, 83.8, 55.5, 55.3, 33.8. MeO NHMe N Me 1-(4-Methoxyphenyl)-N-methyl-3-(6-methylpyridin-2-yl)prop-2-yn-1-amine (4.5.7). Compound 4.5.7 was prepared with 4.4.10 (0.31 g, 0.84 mmol) following general procedure B5. The crude product was purified via flash column chromatography (2:1 hexanes/acetone, followed by 1:1 hexanes/acetone and 30:1 CH2Cl2/MeOH) to yield a dark yellow oil (0.16 g, 71%). Rf = 0.2 (1:1 hexanes/acetone). 1H NMR (CDCl3, 300 MHz): δ 7.47 – 7.39 (m, 3H), 7.21 (d, J = 7.5 Hz, 1H), 7.00 (d, J = 7.8 Hz, 1H), 6.83 (d, J = 8.7 Hz, 2H), 4.66 (s, 1H), 3.71 (s, 3H), 2.47 (s, 6H), 1.59 (brs, 1H). 13C NMR (75 MHz, CDCl3): δ 159.4, 159.0, 142.7, 136.5, 132.0, 129.0, 124.6, 122.8, 114.0, 89.3, 85.3, 55.7, 55.4, 34.1, 24.8. MeO NHMe N Me Me 3-(6-Isopropylpyridin-2-yl)-1-(4-methoxyphenyl)-N-methylprop-2-yn-1amine (4.5.8). Compound 4.5.8 was prepared with 4.4.11 (0.36 g, 0.90 mmol) following 409 general procedure B5. The crude product was purified via flash column chromatography (3:1 hexanes/acetone) to yield an orange oil (164.3 mg, 62%). Rf = 0.4 (30:1 CH2Cl2/MeOH). 1H NMR (CDCl3, 300 MHz): δ 7.49 – 7.37 (m, 3H), 7.20 (d, J = 7.5 Hz, 1H), 7.01 (d, J = 7.8 Hz, 1H), 6.81 (d, J = 8.7 Hz, 2H), 4.65 (s,1H), 3.68 (s, 3H), 2.99 (septet, J = 6.9 Hz, 1H), 2.45 (s, 3H), 1.61 (brs, 1H), 1.20 (d, J = 6.9 Hz, 6H). 13C NMR (75 MHz, CDCl3): δ 168.0, 159.4, 142.5, 136.7, 132.1, 129.0, 125.0, 119.7, 114.0, 89.0, 85.6, 55.8, 55.4, 36.7, 34.0, 22.8. MeO NHMe N tBu 3-(6-(Tert-butyl)pyridin-2-yl)-1-(4-methoxyphenyl)-N-methylprop-2-yn-1amine (4.5.9). Compound 4.5.9 was prepared with 4.4.12 (0.13 g, 0.32 mmol) following general procedure B5. The crude product was purified via flash column chromatography (6:1 hexanes/EtOAc, followed by 3:1, 1:1 hexanes/EtOAc and 2:1 hexanes/acetone) to yield a colorless oil (37.3 mg, 38%). Rf = 0.5 (2:1 hexanes/acetone). 1H NMR (CDCl3, 300 MHz): δ 7.58 – 7.49 (m, 3H), 7.26 (d, J = 7.8 Hz, 1H), 6.90 (d, J = 8.7 Hz, 2H), 4.73 (s, 1H), 3.80 (s, 3H), 2.54 (s, 3H), 1.58 (brs, 1H), 1.35 (s, 9H). 13C NMR (75 MHz, CDCl3): δ 169.9, 159.4, 142.3, 136.3, 132.2, 129.1, 124.7, 118.6, 114.0, 88.4, 86.0, 55.8, 55.5, 37.8, 34.0, 30.4. 410 MeO NHMe Me N Me O 3-(6-Isopropoxypyridin-2-yl)-1-(4-methoxyphenyl)-N-methylprop-2-yn-1amine (4.5.10). Compound 4.5.10 was prepared with 4.4.13 (0.30 g, 0.73 mmol) following general procedure B5. The crude product was purified via flash column chromatography (4:1 hexanes/acetone) to yield a yellow oil (0.11 g, 48%). Rf = 0.5 (1:1 hexanes/acetone). 1 H NMR (CDCl3, 300 MHz): δ 7.53 – 7.44 (m, 3H), 7.01 (d, J = 7.8 Hz, 1H), 6.89 (d, J = 8.7 Hz, 2H), 6.61 (d, J = 8.4 Hz, 1H), 5.35 (septet, J = 6.3 Hz, 1H), 4.71 (s, 1H), 3.78 (s, 3H), 2.53 (s, 3H), 1.73 (brs, 1H), 1.31 (d, J = 6.3 Hz, 6H). 13C NMR (75 MHz, CDCl3): δ 163.3, 159.4, 140.4, 138.6, 132.1, 129.1, 120.4, 114.0, 111.7, 88.4, 85.6, 68.2, 55.7, 55.5, 34.0, 22.2. HRMS (ESI-TOF) [M - H]+ m/z: calcd for C19H21N2O2, 309.1681; found 309.1603. MeO NHMe N OMe O Methyl 6-(3-(4-methoxyphenyl)-3-(methylamino)prop-1-yn-1-yl)picolinate (4.5.11). Compound 4.5.11 was prepared with 4.4.14 (0.6 g, 1.46 mmol) following general procedure B5. The crude product was purified via flash column chromatography (4:1 hexanes/acetone) to yield a yellow oil (46.4 mg, 10%). Rf = 0.2 (40:1 CH2Cl2/MeOH). 1H NMR (CDCl3, 300 MHz): δ 8.04 (d, J = 7.8 Hz, 1H), 7.78 (t, J = 7.8 Hz, 1H), 7.61 (d, J = 7.8 Hz, 1H), 7.47 (d, J = 8.7 Hz, 2H), 6.88 (d, J = 8.7 Hz, 2H), 4.71 (s, 1H), 3.98 (s, 3H), 411 3.78 (s, 3H), 2.53 (s, 3H), 1.71 (brs, 1H). 13 C NMR (75 MHz, CDCl3): δ 165.5, 159.5, 148.5, 143.7, 137.4, 131.7, 130.7, 129.0, 124.3, 114.1, 91.1, 84.5, 55.8, 55.5, 53.3, 34.1. MeO NHMe N 1-(4-Methoxyphenyl)-N-methyl-5-(pyridin-2-yl)pent-2-yn-1-amine (4.5.12). Compound 4.5.12 was prepared with 4.4.8 (0.13 g, 0.34 mmol) following general procedure B5. The crude product was purified via flash column chromatography (40:1 CH2Cl2/MeOH, followed by 20:1 and 10:1 CH2Cl2/MeOH) to yield a dark yellow oil (24.9 mg, 26%). Rf = 0.2 (10:1 CH2Cl2/MeOH). 1H NMR (CDCl3, 300 MHz): δ 8.53 (d, J = 4.2 Hz, 1H), 7.57 (td, J = 7.5, 1.8 Hz, 1H), 7.34 (d, J = 8.7 Hz, 2H), 7.19 (d, J = 7.8 Hz, 1H), 7.12 (dd, J = 7.2, 5.1 Hz, 1H), 6.83 (d, J = 8.7 Hz, 2H), 4.47 (s, 1H), 3.77 (s, 3H), 3.47 (brs, 1H), 3.01 (t, J = 7.5 Hz, 2H), 2.72 (d, J = 7.5 Hz, 2H), 2.35 (s, 3H). 13C NMR (75 MHz, CDCl3): δ 160.2, 159.5, 149.6, 136.5, 131.6, 129.2, 123.4, 121.7, 113.9, 86.0, 79.6, 55.5, 55.1, 37.5, 33.0, 19.2. 412 General procedure B6 for the preparation of TMS-ene-guanidine or N-acyl propargyl guanidine MeO MeO O NHMe + N H R CN Me N TMSCl, DIPEA CH2Cl2, r.t. N R N N R TMS N H O R A 50-mL oven-dried round bottom flask was charged with 1.73 mmol of a benzcyanamide and 10 mL anhydrous CH2Cl2 at room temperature. Then DIPEA (0.62 mL, 3.62 mmol) was added via syringe, followed by TMSCl (0.23 mL, 1.81 mmol). The reaction was stirred for 20 min and 0.21 mmol of a secondary propargyl amine, dissolved in 5 mL CH2Cl2, was added via syringe. The reaction was stirred for 3 h, quenched with sat. NaHCO3-solution and extracted two times with CH2Cl2. After removal of the organic solvent, the crude product was purified via column chromatography to obtain the TMSene-guanidine. MeO Me N N TMS N H N O F 2-Fluoro-N-((2E,4E)-5-(4-methoxyphenyl)-1-methyl-4-(phenyl(trimethylsilyl)methylene)imidazolidin-2-ylidene)benzamide (4.6.1). Compound 4.6.1 was prepared with amine 4.5.1 (0.18 g, 0.71 mmol) and N-cyano-2-fluorobenzamide S8 (279.7 mg, 1.73 mmol) following general procedure B6. The crude product was purified via flash column chromatography (3:2 hexanes/EtOAc, followed by 1:1 hexanes/EtOAc) to yield a white foam (0.22 g, 63%). Rf = 0.25 (1:2 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 10.97 (s, 1H), 8.42 (d, J = 5.0 Hz, 1H), 8.09 (td, J = 7.5, 2.0 Hz, 1H), 7.39 – 7.33 (m, 1H), 413 7.24 – 7.18 (m, 1H), 7.12 (t, J = 7.5 Hz, 1H), 7.06 (dd, J = 11.0, 8.5 Hz, 1H), 6.91 (t, J = 6.0 Hz, 1H), 6.62 (d, J = 8.5 Hz, 2H), 6.56 (d, J = 8.5 Hz, 2H), 6.39 (d, J = 7.5 Hz, 1H), 5.28 (s, 1H), 3.69 (s, 3H), 2.74 (s, 3H), 0.21 (s, 9H). 13C NMR (125 MHz, CDCl3): δ 176.0, 161.7 (d, JCF = 255.2 Hz), 159.9, 159.3, 159.2, 149.2, 148.3, 135.5, 132.3 (d, JCF = 8.8 Hz), 132.1, 128.7, 128.6, 126.7 (d, JCF = 8.8 Hz), 124.4, 123.4 (d, JCF = 3.1 Hz), 120.5, 116.6 (d, JCF = 22.7 Hz), 114.5, 113.7, 113.3, 64.8, 55.1, 28.3, 0.7. MeO Me N MeO N TMS N H N O F 2-Fluoro-N-((S,2E,4Z)-5-(4-methoxyphenyl)-4-((6-methoxypyridin-2-yl)(trimethylsilyl)methylene)-1-methylimidazolidin-2-ylidene)benzamide (4.6.2). Compound 4.6.2 was prepared with amine 4.5.2 (50.0 mg, 0.18 mmol) and N-cyano-2fluorobenzamide S8 (34.8 mg, 0.21 mmol) following general procedure B6. The crude product was purified via flash column chromatography (2:1 CH2Cl2/hexanes) to yield a white foam (52.1 mg, 57%). Rf = 0.3 (2:1 CH2Cl2/hexanes). 1H NMR (CDCl3, 500 MHz): δ 10.91 (s, 1H), 8.06 (td, J = 7.5, 2.0 Hz, 1H), 7.34 – 7.30 (m, 1H), 7.15 (t, J = 7.5 Hz, 1H), 7.09 (t, J = 7.5 Hz, 1H), 7.02 (dd, J = 10.5, 9.0 Hz, 1H), 6.63 (d, J = 8.5 Hz, 2H), 6.56 (d, J = 8.5 Hz, 2H), 6.32 (d, J = 8.0 Hz, 1H), 6.15 (d, J = 7.5 Hz, 1H), 5.23 (s, 1H), 3.67 (s, 3H), 3.65 (s, 3H), 2.72 (s, 3H), 0.19 (s, 9H). 13C NMR (125 MHz, CDCl3): δ 176.0, 170.9, 163.3, 161.7 (d, JCF = 255.1 Hz), 159.3, 159.2, 157.4, 147.2, 138.1, 132.3 (d, JCF = 8.7 Hz), 132.1, 132.0, 128.9, 126.9 (d, JCF = 9.0 Hz), 123.4 (d, JCF = 3.7 Hz), 116.6, 116.5, 116.5, 113.8, 113.7, 107.0, 65.3, 55.4, 53.3, 28.6, -0.3. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C28H32FN4O3Si, 519.2149; found 519.2228. 414 MeO Me N N MeO TMS N N H O F 2-Fluoro-N-((S,2E,4Z)-5-(4-methoxyphenyl)-4-((4-methoxypyridin-2-yl)(trimethylsilyl)methylene)-1-methylimidazolidin-2-ylidene)benzamide (4.6.3). Compound 4.6.3 was prepared with amine 4.5.4 (0.21 g, 0.74 mmol) and N-cyano-2fluorobenzamide S8 (292.1 mg, 1.78 mmol) following general procedure B6. The crude product was purified via flash column chromatography (2:1 CH2Cl2/hexanes) to yield a white foam (119.2 mg, 31%). Rf = 0.5 (20:1 CH2Cl2/MeOH). 1H NMR (CDCl3, 500 MHz): δ 10.93, 8.22 (d, J = 6.0 Hz, 1H), 8.08 (td, J = 8.0, 2.0 Hz, 1H), 7.38 – 7.32 (m, 1H), 7.11 (t, J = 7.5 Hz, 1H), 7.04 (dd, J = 11.0, 8.0 Hz, 1H), 6.65 (d, J = 8.5, 2H), 6.59 (d, J = 9.0 Hz, 2H), 6.47 (dd, J = 6.0, 2.0 Hz, 1H), 5.77 (d, J = 2.5 Hz, 1H), 5.21 (s, 1H), 3.69 (s, 3H), 3.50 (s, 3H), 2.73 (s, 3H), 0.19 (s, 9H). 13C NMR (125 MHz, CDCl3): δ 176.1, 165.4, 161.8 (d, JCF = 255.1 Hz), 161.3, 159.4, 159.3, 150.1, 148.1, 132.4 (d, JCF = 8.7 Hz), 132.1, 132.1, 128.9, 128.8, 126.7 (d, JCF = 9.0 Hz), 123.4 (d, JCF = 3.7 Hz), 116.6 (d, JCF = 22.7 Hz), 113.7, 113.4, 109.4, 107.9, 64.8, 55.2, 54.6, 28.4, -0.7. MeO N OMe TMS Me N N H N O F 2-Fluoro-N-((S,2E,4Z)-5-(4-methoxyphenyl)-4-((3-methoxypyridin-2-yl)(trimethylsilyl)methylene)-1-methylimidazolidin-2-ylidene)benzamide (4.6.4). Compound 4.6.4 was prepared with amine 4.5.5 (0.20 g, 0.71 mmol) and N-cyano-2fluorobenzamide S8 (0.28 g, 1.70 mmol) following general procedure B6. The crude product was purified via flash column chromatography (1:1 hexanes/EtOAc, followed by 415 1:1 hexanes/acetone) to yield a white solid (178.9 mg, 48%). Rf = 0.5 (20:1 CH2Cl2/MeOH). 1H NMR (CDCl3, 500 MHz): δ 10.96 (s, 1H), 8.09 (t, J = 7.5 Hz, 1H), 8.04 (d, J = 4.5 Hz, 1H), 7.40 – 7.33 (m, 1H), 7.12 (t, J = 7.5 Hz, 1H), 7.06 (dd, J = 10.5, 9.0 Hz, 1H), 6.91 (dd, J = 8.0, 5.0 Hz, 1H), 6.66 – 6.60 (m, 3H), 6.54 (d, J = 8.0 Hz, 2H), 5.24 (s, 1H), 3.69 (s, 3H), 3.33 (s, 3H), 2.74 (s, 3H), -0.17 (s, 9H). 13C NMR (125 MHz, CDCl3): δ 176.0, 161.8 (d, d, JCF = 255.0 Hz), 159.4, 159.3, 153.2, 150.8, 147.4, 140.6, 132.2 (d, JCF = 9.0 Hz), 132.1, 128.9, 128.5, 126.9 (d, JCF = 9.0 Hz), 123.4 (d, JCF = 3.7 Hz), 121.8, 116.6 (d, JCF = 23.0 Hz), 116.0, 113.3, 111.0, 64.9, 55.2, 54.2, 28.4, -1.2. MeO N OMe TMS Me N N H N F O 4-Fluoro-N-((S,2E,4Z)-5-(4-methoxyphenyl)-4-((3-methoxypyridin-2-yl)(trimethylsilyl)methylene)-1-methylimidazolidin-2-ylidene)benzamide (4.6.5). Compound 4.6.5 was prepared with amine 4.5.5 (0.10 g, 0.35 mmol) and N-cyano-4fluorobenzamide S9 (0.14 g, 0.85 mmol) following general procedure B6. The crude product was purified via flash column chromatography (1:1 hexanes/EtOAc) to yield a white solid (61.7 mg, 33%). Rf = 0.2 (1:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 11.07 (s, 1H), 8.33 – 8.28 (m, 2H), 8.06 (d, J = 4.5 Hz, 1H), 7.08 – 7.02 (m, 2H), 6.92 (dd, J = 8.5, 5.0 Hz, 1H), 6.67 – 6.61 (m, 3H), 6.54 (d, J = 8.5 Hz, 2H), 5.25 (s, 1H), 3.71 (s, 3H), 3.34 (s, 3H), 2.77 (s, 3H), 0.18 (s, 9H). 13C NMR (125 MHz, CDCl3): δ 176.6, 165.0 (d, JCF = 249.5 Hz), 159.5, 159.3, 153.2, 150.9, 147.5, 140.5, 134.2 (d, JCF = 2.8 Hz), 131.7 (d, JCF = 8.8 Hz), 128.9, 128.6, 121.8, 115.9, 114.6 (d, JCF = 21.3 Hz), 113.3, 110.6, 64.8, 55.2, 54.1, 29.4, -1.2. 416 MeO Me N N OMe TMS N H N Cl O 4-Chloro-N-((S,2E,4Z)-5-(4-methoxyphenyl)-4-((3-methoxypyridin-2-yl)(trimethyllsilyl)methylene)-1-methylimidazolidin-2-ylidene)benzamide (4.6.6). Compound 4.6.6 was prepared with amine 4.5.5 (0.10 g, 0.35 mmol) and N-cyano-4chlorobenzamide S10 (0.15 g, 0.85 mmol) following general procedure B6. The crude product was purified via flash column chromatography (1:1 hexanes/EtOAc) to yield a white solid (72.8 mg, 38%). Rf = 0.3 (1:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 11.05 (s, 1H), 8.22 (dd, J = 8.5, 2.5 Hz, 1H), 8.05 (d, J = 5.0 Hz, 1H), 7.34 (dd, J = 8.5, 2.0 Hz, 1H), 6.94 – 6.89 (m, 1H), 6.65 – 6.6 m, 3H), 6.53 (dd, J = 9.0, 2.0 Hz, 2H), 5.25 (s, 1H), 3.69 (s, 3H), 3.33 (s, 3H), 2.76 (s, 3H), 0.17 (s, 9H). 13C NMR (125 MHz, CDCl3): δ 176.6, 159.6, 159.3, 153.2, 150.8, 147.4, 140.5, 137.5, 136.5, 130.8, 128.9, 128.5, 128.0, 121.8, 116.0, 113.3, 110.8, 64.9, 55.2, 54.2, 28.4, -1.2. MeO Me N Cl N TMS N H N O F N-((S,2E,4Z)-4-((6-chloropyridin-2-yl)(trimethylsilyl)methylene)-5-(4methoxyphenyl)-1-methylimidazolidin-2-ylidene)-2-fluorobenzamide (4.6.7). Compound 4.6.7 was prepared with amine 4.5.6 (0.12 g, 0.43 mmol) and N-cyano-2fluorobenzamide S8 (0.17 g, 1.03 mmol) following general procedure B6. The crude product was purified via flash column chromatography (4:1 hexanes/EtOAc) to yield a yellow liquid (90.2 mg, 40%). Rf = 0.8 (1:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 11.01, 8.09 (t, J = 7.5 Hz, 1H), 7.38 – 7.34 (m, 1H), 7.16 (t, J = 7.5 Hz, 1H), 7.12 (t, J = 417 7.5 Hz, 1H), 7.05 (t, J = 9.5 Hz, 1H), 6.92 (d, J = 8.0 Hz, 1H), 6.72 (d, J = 8.0 Hz, 2H), 6.59 (d, J = 8.0 Hz, 2H), 6.31 (d, J = 7.5 Hz, 1H), 5.42 (s, 1H), 3.69 (s, 3H), 2.75 (s, 3H), 0.22 (s, 9H). 13C NMR (125 MHz, CDCl3): δ 176.1, 161.8 (d, JCF = 255.3 Hz), 160.9, 159.4, 159.0, 150.4, 149.6, 138.1, 132.5 (d, JCF = 8.8 Hz), 132.2 (d, JCF = 1.2 Hz), 129.0, 128.4, 126.6 (d, JCF = 8.7 Hz), 123.4 (d, JCF = 3.7 Hz), 122.8, 120.6, 116.6 (d, JCF = 22.8 Hz), 113.8, 111.5, 65.0, 55.2, 28.4, -0.6. MeO Me N N NH2 O F N OMe (E)-N-(amino((1-(4-methoxyphenyl)-3-(5-methoxypyridin-2-yl)prop-2-yn-1yl)-(methyl)amino)methylene)-2-fluorobenzamide (4.6.8). Compound 4.6.8 was prepared with amine 4.5.3 (0.42 g, 1.50 mmol) and N-cyano-2-fluorobenzamide S8 (592.5 mg, 3.61 mmol) following general procedure B6. The crude product was purified via flash column chromatography (1:1 hexanes/EtOAc, followed by 1:1 hexanes/acetone) to yield a yellow foam (287.0 mg, 43%). Rf = 0.2 (1:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 8.18 (d, J = 3.0 Hz, 1H), 7.98 (td, J = 7.5, 2.0 Hz, 1H), 7.67 (brs, 1H), 7.49 (d, J = 8.5 Hz, 2H), 7.36 (d, J = 8.5 Hz, 1H), 7.31 – 7.22 (m, 1H), 7.08 – 7.04 (m, 2H), 6.98 (dd, J = 11.0, 8.5 Hz, 1H), 6.81 (d, 8.5 Hz, 2H), 3.74 (s, 3H), 3.68 (s, 3H), 2.83 (s. 3H). 13C NMR (125 MHz, CDCl3): δ 175.0, 161.6 (d, JCF = 254.1 Hz), 160.5, 159.4, 155.4, 138.1, 134.4, 131.8 (d, JCF = 8.8 Hz), 131.8, 131.8, 128.7, 128.2, 127.7 (d, JCF = 8.8 Hz), 123.4 (d, JCF = 3.7 Hz), 120.2, 116.5 (d, JCF = 23.2 Hz), 113.9, 85.6, 84.1, 55.6, 55.2, 29.2. 418 MeO Me N N Me TMS N H N O F 2-Fluoro-N-((S,2E,4Z)-5-(4-methoxyphenyl)-1-methyl-4-((6-methylpyridin-2yl)(trimethylsilyl)methylene)imidazolidin-2-ylidene)benzamide (4.6.9). Compound 4.6.9 was prepared with amine 4.5.7 (0.16 g, 0.60 mmol) and N-cyano-2-fluorobenzamide S8 (236.6 mg, 1.44 mmol) following general procedure B6. The crude product was purified via flash column chromatography (3:1 hexanes/EtOAc) to yield a yellow semisolid (0.18 g, 60%). Rf = 0.4 (1:1 hexanes/EtOAc). Compound 4.6.9 was used for the synthesis of ZNA 195 without further characterization. MeO Me N Me N Me TMS N H N O F 2-Fluoro-N-((S,2E,4Z)-4-((6-isopropylpyridin-2-yl)(trimethylsilyl)methylene)5-(4-methoxyphenyl)-1-methylimidazolidin-2-ylidene)benzamide (4.6.10). Compound 4.6.10 was prepared with amine 4.5.8 (0.16 g, 0.56 mmol) and N-cyano-2-fluorobenzamide S8 (0.22 g, 1.34 mmol) following general procedure B6. The crude product was purified via flash column chromatography (7:2 hexanes/EtOAc) to yield a yellow semisolid (121.4 mg, 41%). Rf = 0.3 (3:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 10.93 (s, 1H), 8.09 (td, J = 7.5, 2.0 Hz, 1H), 7.39 – 7.34 (m, 1H), 7.17 – 7.12 (m, 2H), 7.06 (dd, J = 10.5, 8.5 Hz, 1H), 6.78 (d, J = 7.5 Hz, 1H), 6.63 (d, J = 9.0 Hz, 2H), 6.57 (d, J = 9.0 Hz, 2H), 6.25 (d, J = 8.0 Hz, 1H), 5.25 (s, 1H), 3.70 (s, 3H), 2.97 (septet, J = 7.0 Hz, 1H), 2.75 (s, 3H), 1.24 (d, J = 7.0 Hz, 6H), 0.21 (s, 9H). 13C NMR (125 MHz, CDCl3): δ 176.0, 166.9, 161.8 (d, JCF = 255.2 Hz), 159.3, 159.3, 158.9, 147.5, 135.8, 132.2 (d, JCF = 8.8 Hz), 132.1, 419 131.9, 128.9, 128.7, 126.9 (d, JCF = 9.1 Hz), 123.4 (d, JCF = 3.7 Hz), 121.4, 116.7, 116.5, 114.5 (d, JCF = 8.1 Hz), 113.7, 64.9, 55.3, 36.4, 28.3, 22.9, 22.7, -0.6. MeO Me N N tBu TMS N H N O F N-((S,2E,4Z)-4-((6-(tert-butyl)pyridin-2-yl)(trimethylsilyl)methylene)-5-(4methoxy-phenyl)-1-methylimidazolidin-2-ylidene)-2-fluorobenzamide (4.6.11). Compound 4.6.11 was prepared with amine 4.5.9 (37.0 mg, 0.09 mmol) and N-cyano-2fluorobenzamide S8 (36.1 mg, 0.22 mmol) following general procedure B6. The crude product was purified via flash column chromatography (3:1 hexanes/EtOAc) to yield a white solid (38.2 mg, 78%). Rf = 0.5 (3:1 heanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 10.91 (s, 1H), 8.08 (td, J = 7.5, 2.0 Hz, 1H), 7.40 – 7.36 (m, 1H), 7.20 (t, J = 7.5 Hz, 1H), 7.14 (t, J = 7.5 Hz, 1H), 7.08 (dd, J = 11.0, 8.5 Hz, 1H), 6.92 (d, J = 8.0 Hz, 1H), 6.64 (d, J = 8.5 Hz, 2H), 6.57 (d, J = 8.5 Hz, 2H), 6.36 (d, J = 7.5 Hz, 1H), 5.25 (s, 1H), 3.71 (s, 3H), 2.76 (s, 3H), 1.25 (s, 9H), 0.21 (s, 9H). MeO Me N O Me N Me TMS N H N O F 2-Fluoro-N-((S,2E,4Z)-4-((6-isopropoxypyridin-2-yl)(trimethylsilyl)methylene)-5-(4-methoxyphenyl)-1-methylimidazolidin-2-ylidene)benzamide (4.6.12). Compound 4.6.12 was prepared with amine 4.5.10 (0.11 g, 0.35 mmol) and Ncyano-2-fluorobenzamide S8 (0.14 mg, 0.85 mmol) following general procedure B6. The crude product was purified via flash column chromatography (5:1, followed by 4:1 hexanes/EtOAc) to yield a white solid (0.13 g, 67%). Rf = 0.4 (3:1 hexanes/EtOAc). 1H 420 NMR (CDCl3, 500 MHz): δ 10.90 (s, 1H), 8.08 (td, J = 7.5, 2.0 Hz, 1H), 7.40 – 7.35 (m, 1H), 7.19 (dd, J = 8.5, 7.5, Hz, 1H), 7.14 (td, J = 7.5, 1.0 Hz, 1H), 7.07 (ddd, J = 11.0, 8.0, 1.0 Hz, 1H), 6.67 (d, J = 8.5 Hz, 2H), 6.61 (d, J = 8.5 Hz, 2H), 6.30 (d, J = 8.5 Hz, 1H), 6.18 (d, J = 7.5 Hz, 1H), 5.24 (s, 1H), 4.98 (septet, J = 6.0 Hz, 1H), 3.71 (s, 3H), 2.77 (s, 3H), 1.28 (d, J = 6.0 Hz, 3H), 1.65 (d, J = 6.0 Hz, 3H), 0.21 (s, 9H). 13C NMR (125 MHz, CDCl3): δ 176.1, 162.8, 161.7 (d, JCF = 255.0 Hz), 159.4, 159.3, 157.5, 146.9, 138.1, 132.3 (d, JCF = 8.8 Hz), 132.0, 129.0, 128.7, 127.0 (d, JCF = 9.3 Hz), 123.4 (d, JCF = 3.7 Hz), 116.6 (d, JCF = 22.8 Hz), 116.0, 114.3, 113.7, 107.6, 67.3, 65.0, 55.1, 28.4, 22.3, 21.8, -0.6. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C30H36FN4O3Si, 547.2462; found, 547.2541. MeO Me N MeO N O Methyl TMS N H N O F 6-((Z)-((S,E)-2-((2-fluorobenzoyl)imino)-5-(4-methoxyphenyl)-1- methyl-imidazolidin-4-ylidene)(trimethylsilyl)methyl)picolinate (4.6.13). Compound 4.6.13 was prepared with amine 4.5.11 (0.15 g, 0.49 mmol) and N-cyano-2fluorobenzamide S8 (194.2 mg, 1.18 mmol) following general procedure B6. The crude product was purified via flash column chromatography (4:1 hexanes/EtOAc) to yield a yellow liquid (0.21 g, 78%). Rf = 0.7 (1:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 11.01 (s, 1H), 8.09 (td, J = 7.5, 2.0 Hz, 1H), 7.72 (d, J = 8.0 Hz, 1H), 7.39 – 7.34 (m, 1H), 7.31 (t, J = 7.5 Hz, 1H), 7.12 (t, J = 7.5 Hz, 1H), 7.06 (dd, J = 11.0, 8.0 Hz, 1H), 6.63 (d, J = 8.5 Hz, 2H), 6.54 (d, J = 8,5 Hz, 2H), 6.52 (d, J = 8.0 Hz, 1H), 5.44 (s, 1H), 3.99 (s, 3H), 3.68 (s, 3H), 2.74 (s, 3H), 0.23 (s, 9H). 13C NMR (125 MHz, CDCl3): δ 176.10, 166.06, 161.82 (d, JCF = 255.2 Hz), 160.6, 159.4, 159.1, 149.7, 147.6, 136.1, 132.4 (d, JCF = 8.8 Hz), 132.2, 132.2, 131.9, 128.8, 128.7, 127.8, 126.7 (d, JCF = 8.7 Hz), 123.4 (d, JCF 421 = 3.8 Hz), 121.9, 65.0, 55.2, 52.8, 28.3, -0.5. MeO Me N N NH2 O F N (E)-N-(amino((1-(4-methoxyphenyl)-5-(pyridin-2-yl)pent-2-yn-1-yl)(methyl)amino)methylene)-2-fluorobenzamide (4.6.14). Compound 4.6.14 was prepared with amine 4.5.12 (61.0 mg, 0.22 mmol) and N-cyano-2-fluorobenzamide S8 (85.3 mg, 0.52 mmol) following general procedure B6. The crude product was purified via flash column chromatography (2:1 hexanes/acetone) to yield a white solid (83.4 mg, 86%). Rf = 0.4 (1:1 hexanes/acetone). 1H NMR (CDCl3, 500 MHz): δ 8.55 (d, J = 5.0 Hz, 1H), 8.01 (t, J = 7.5Hz, 1H), 7.58 (t, J = 7.5 Hz, 1H), 7.33 (d, J = 8.0 Hz, 1H), 7.20 (d, J = 7.5 Hz, 1H), 7.17 – 7.09 (m, 2H), 7.05 (dd, J = 11.0, 8.5 Hz, 1H), 6.81 (d, J = 8.5 Hz, 2H), 3.78 (s, 3H), 3.05 (t, J = 7.0 Hz, 2H), 2.78 (t, J = 7.0 Hz, 2H), 2.65 (s, 3H). 13C NMR (125 MHz, CDCl3): δ 175.3, 161.7 (d, JCF = 254.6 Hz), 160.4, 159.9, 159.3, 149.4, 136.3, 131.9, 131.8, 129.5, 128.7, 127.6 (d, JCF = 8.7 Hz), 123.3 (d, JCF = 3.7 Hz), 123.2, 121.5, 86.4, 55.3, 50.2, 37.1, 29.3, 18.9. 422 General procedure for the preparation N2-acyl 2-aminoimidazole (ZNA) MeO MeO Me N K2CO3 N TMS N H N N H MeOH, r.t. N R Me N R O R N R O The preparation of hemiporphyrin-like N2-acyl 2-aminoimidazoles (ZNAs) followed general procedure B2 using 1.05 equiv. of K2CO3. The reaction mixture was stirred at room temperature for 1 h, quenched with sat. aq. NH4Cl-solution and extracted with CH2Cl2. After removing the organic solvent, the crude product was purified via flash column chromatography to yield the final compounds for biological evaluations. MeO Me N N H N N O F (E)-2-Fluoro-N-(5-(4-methoxyphenyl)-1-methyl-4-(pyridin-2-ylmethyl)-1,3dihydro-2H-imidazol-2-ylidene)benzamide (ZNA 131). ZNA 131 was prepared with 4.6.1 (0.21 g, 0.43 mmol) following general procedure B2. The crude product was purified via flash column chromatography (2:1 hexanes/acetone) to yield a yellow solid (0.14 g, 77%). Rf = 0.1 (2:1 hexanes/acetone). 1H NMR (CD3CN, 500 MHz): δ 12.26 (brs, 1H), 8.55 (d, J = 4.0 Hz, 1H), 7.98 (t, J = 7.0 Hz, 1H), 7.70 (td, J = 7.5, 1.5 Hz, 1H), 7.50 – 7.44 (m, 1H), 7.42 (d, J = 8.5 Hz, 2H), 7.23 (q, J = 7.0 Hz, 3H), 7.15 (dd, J = 11.0, 8.5 Hz, 1H), 7.07 (d, J = 8.5 Hz, 2H), 3.99 (s, 2H), 3.86 (s, 3H), 3.38 (s, 3H). 13 C NMR (125 MHz, CDCl3): δ 171.69, 161.41 (d, JCF = 252.3 Hz), 160.1, 157.7, 149.8, 148.5, 136.8, 131.8, 423 131.7, 131.7 (d, JCF = 1.8 Hz), 131.6, 126.4, 125.1, 123.6 (d, JCF = 3.5 Hz), 122.6, 121.9, 119.9, 116.4 (d, JCF = 23.2 Hz), 114.4, 55.3, 33.5, 30.2. IR (thin film): 2969, 1565, 1510, 1469, 1435, 1335, 1290, 1247, 1217, 1032, 835, 757 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C24H22FN4O2, 417.1727; found, 417.1729. MeO Me N N H N N O F OMe (E)-2-Fluoro-N-(5-(4-methoxyphenyl)-4-((6-methoxypyridin-2-yl)methyl)-1methyl-1,3-dihydro-2H-imidazol-2-ylidene)benzamide (ZNA 148). ZNA 148 was prepared with 4.6.2 (50.0 mg, 0.18 mmol) following general procedure B2. The crude product was purified via flash column chromatography (3:1 hexanes/EtOAc, followed by 2:1 hexanes/EtOAc) to yield a yellow solid (44.4 mg, 99%). Rf = 0.3 (1:1 hexanes/EtOAc). 1 H NMR (CD3CN, 500 MHz): δ 12.15 (brs, 1H), 7.97 (td, J = 8.0 Hz, 1H), 7.60 (dd, J = 8.5, 7.5 Hz, 1H), 7.47 – 7.42 (m, 3H), 7.22 (td, J = 7.5, 0.5 Hz, 1H), 7.14 (dd, J = 11.0, 8.5 Hz, 1H), 7.09 (d, J = 8.5 Hz, 2H), 4.01 (s, 3H), 3.88 (s, 2H), 3.87 (s, 3H), 3.38 (s, 3H). 13C NMR (125 MHz, CDCl3): δ 172.4, 164.1, 161.4 (d, JCF = 252.3 Hz), 160.2, 154.9, 149.1, 139.2, 131.6, 131.5 (d, JCF = 2.2 Hz), 131.4 (d, JCF = 8,6 Hz), 127.3, 124.2, 123.5 (d, JCF = 3.7 Hz), 119.8, 116.4 (d, JCF = 23.0 Hz), 114.8, 114.4, 109.3, 55.4, 53.8, 32.8, 30.0. IR (thin film): 2960, 1565, 1466, 1355, 1293, 1249, 1225, 1029, 835, 759 cm-1. HRMS (ESITOF) [M + H]+ m/z: calcd for C25H24FN4O3, 447.1832; found, 447.1835. 424 MeO Me N N N H O N F Cl (E)-N-(4-((6-chloropyridin-2-yl)methyl)-5-(4-methoxyphenyl)-1-methyl-1,3dihydro-2H-imidazol-2-ylidene)-2-fluorobenzamide (ZNA 172). ZNA 172 was prepared with 4.6.7 (90.2 mg, 0.17 mmol) following general procedure B2. The crude product was purified via flash column chromatography (3:1 hexanes/EtOAc, followed by 2:1 and 1:1 hexanes/EtOAc) to yield a yellow solid (44.8 mg, 58%). Rf = 0.25 (2:1 Et2O/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 12.11 (brs, 1H), 8.06 (t, J = 8.0 Hz, 1H), 7.52 (td, J = 7.5, 2.5 Hz, 1H), 7.41 – 7.36 (m, 1H), 7.31 (d, J = 8.5 Hz, 2H), 7.18 – 7.13 (m, 2H), 7.10 – 7.04 (m, 2H), 6.98 (d, J = 8.5 Hz, 2H), 3.95 (s, 2H), 3.84 (s, 3H), 3.42 (s, 3H). 13 C NMR (125 MHz, CDCl3): δ 161.3 (d, JCF = 251.8 Hz), 160.2, 159.2, 151.1, 139.3, 132.2 (d, JCF = 9.0 Hz), 131.7 (d, JCF = 1.8 Hz), 131.6, 126.5, 125.2, 123.8 (d, JCF = 3.6 Hz), 122.3, 121.0, 120.0, 116.4 (d, JCF = 23.5 Hz), 114.4, 55.3, 33.7, 30.4. IR (thin film): 1683, 1652, 1558, 1436, 1246, 1033, 834, 756, 682 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C24H21ClFN4O2, 451.1337; found, 451.1346. MeO Me N N H N N O OMe F O Methyl (E)-6-((2-((2-fluorobenzoyl)imino)-5-(4-methoxyphenyl)-1-methyl-2,3dihydro-1H-imidazol-4-yl)methyl)picolinate (ZNA 175). ZNA 175 was prepared with 4.6.13 (82.3 mg, 0.15 mmol) following general procedure B2. The crude product was purified via flash column chromatography (1:1 hexanes/EtOAc, followed by 1:2 425 hexanes/EtOAc and 40:1 CH2Cl2/MeOH) to yield a yellow solid (39.5 mg, 55%). Rf = 0.2 (1:1 hexanes/acetone). 1H NMR (CDCl3, 500 MHz): δ 8.03 (td, J = 8.0 Hz, 1H), 7.94 (d, J = 8.0 Hz, 1H), 7.70 (t, J = 8.0 Hz, 1H), 7.39 – 7.34 (m, 1H), 7.33 (d, J = 8.5 Hz, 2H), 7.29 (d, J = 7.5 Hz, 1H), 7.14 (t, J = 7.5 Hz, 1H), 7.06 (dd, J = 11.5, 8.0 Hz, 1H), 6.96 (d, J = 9.0 Hz, 2H), 4.08 (s, 2H), 3.98 (s, 3H), 3.82 (s, 3H), 3.41 (s, 3H). 13C NMR (125 MHz, CDCl3): δ 165.7, 161.3 (d, JCF = 251.8 Hz), 160.1, 158.7, 147.7, 137.8, 132.2 (d, JCF = 8.3 Hz), 131.7, 131.7, 131.7, 126.4, 125.9, 123.8 (d, JCF = 3.6 Hz), 123.2, 119.9, 116.4 (d, JCF = 23.5 Hz), 114.4, 55.3, 52.8, 34.0, 30.5. IR (thin film): 2950, 1712, 1685, 1561, 1353, 1286, 1246, 1175, 1032, 834, 753 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C26H24FN4O4, 475.1782; found, 475.1782. MeO Me N N H N N O F MeO (E)-2-Fluoro-N-(5-(4-methoxyphenyl)-4-((4-methoxypyridin-2-yl)methyl)-1methyl-1,3-dihydro-2H-imidazol-2-ylidene)benzamide (ZNA-176). ZNA 176 was prepared with 4.6.3 (116.2 mg, 0.22 mmol) following general procedure B2. The crude product was purified via flash column chromatography (1:2 hexanes/EtOAc, followed by 1:3 hexanes/EtOAc, then 30:1, 20:1 and 15:1 CH2Cl2/MeOH) to yield a white solid (67.0 mg, 68%). Rf = 0.1 (30:1 CH2Cl2/MeOH). 1H NMR (CDCl3, 500 MHz): δ 8.37 (d, J = 6.0 Hz, 1H), 8.30 (td, J = 7.5, 2.0 Hz, 1H), 7.36 – 7.28 (m, 3H), 7.12 (t, J = 7.5 Hz, 1H), 7.05 (dd, J = 11.5, 8.5 Hz, 1H), 6.98 (d, J = 8.5 Hz, 2H), 3.90 (s, 2H), 3.82 (s, 3H), 3.76 (s, 3H), 3.41 (s, 3H). 13C NMR (125 MHz, CDCl3): δ 171.5, 166.4, 161.4 (d, JCF = 252.2 Hz), 160.1, 159.2, 151.0, 148.2, 131.8, 131.7, 131.7 (d, JCF = 2.0 Hz), 131.6, 126.4, 125.1, 123.6 (d, 426 JCF = 3.3 Hz), 119.9, 116.4 (d, JCF = 23.2 Hz), 114.4, 109.0, 107.8, 55.3, 55.1, 33.5, 30.2. IR (thin film): 1683, 1593, 1564, 1510, 1481, 1355, 1289, 1247, 1176, 1033, 835, 759 cm1 . HRMS (ESI-TOF) [M + H]+ m/z: calcd for C25H24FN4O3, 447.1832; found, 447.1837. MeO Me N N H MeO N N F O (E)-4-Fluoro-N-(5-(4-methoxyphenyl)-4-((3-methoxypyridin-2-yl)methyl)-1methyl-1,3-dihydro-2H-imidazol-2-ylidene)benzamide (ZNA 186). ZNA 186 was prepared with 4.6.5 (61.7 mg, 0.12 mmol) following general procedure B2. The crude product was purified via flash column chromatography (2:1 Et2O/EtOAc) to yield a white solid (47.7 mg, 89%). Rf = 0.25 (2:1 Et2O/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 12.21 (brs, 1H), 8.27 (dd, J = 9.0, 6.0 Hz, 2H), 8.16 (dd, J = 4.5, 2.0 Hz, 1H), 7.40 (d, J = 8.5 Hz, 2H), 7.20 – 7.13 (m, 2H), 7.07 – 6.99 (m, 4H), 4.01 (s, 2H), 3.97 (s, 3H), 3.87 (s, 3H), 3.45 (s, 3H). 13C NMR (125 MHz, CDCl3): δ 173.6, 164.4 (d, JCF = 247.7 Hz), 160.0, 153.4, 150.3, 147.1, 141.1, 135.2 (d, JCF = 2.7 Hz), 131.8, 130.9 (d, JCF = 8.8 Hz), 123.9, 122.9, 119.9, 118.7, 117.3, 114.4 (d, JCF = 21.3 Hz), 114.3, 55.5, 55.3, 29.8, 28.2. IR (thin film): 2069, 1604, 1567, 1461, 1431, 1349, 1274, 1216, 1032, 1014, 854, 776 cm-1. HRMS (ESITOF) [M + H]+ m/z: calcd for C25H24FN4O3, 447.1832; found, 447.1837. 427 MeO Me N N N H MeO Cl O N (E)-4-Chloro-N-(5-(4-methoxyphenyl)-4-((3-methoxypyridin-2-yl)methyl)-1methyl-1,3-dihydro-2H-imidazol-2-ylidene)benzamide (ZNA 187). ZNA 187 was prepared with 4.6.6 (72.8 mg, 0.14 mmol) following general procedure B2. The crude product was purified via flash column chromatography (2:1 Et2O/EtOAc) to yield a white solid (28.6 mg, 45%). Rf = 0.25 (2:1 Et2O/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 12.39 (brs, 1H), 8.21 (d, J = 8.5 Hz, 2H), 8.17 (dd, J = 4.5, 1.5 Hz, 1H), 7.41 (d, J = 9.0 Hz, 2H), 7.34 (d, J = 8.5 Hz, 2H), 7.20 – 7.14 (m, 2H), 7.03 (d, J = 8.5 Hz, 2H), 4.01 (s, 2H), 3.98 (s, 3H), 3.88 (s, 3H), 3.45 (s, 3H). 13C NMR (125 MHz, CDCl3): δ 173.5, 160.7, 153.4, 150.2, 147.1, 141.1, 137.6, 136.3, 131.8, 130.2, 127.8, 123.9, 122.9, 119.8, 118.8, 117.3, 114.3, 55.5, 55.3, 29.8, 28.2. IR (thin film): 2932, 1635, 1567, 1456, 1349, 1290, 1248, 1177, 1013, 835, 770 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C25H24ClN4O3, 463.1537; found, 463.1538. MeO Me N N H MeO N N O F (E)-2-Fluoro-N-(5-(4-methoxyphenyl)-4-((3-methoxypyridin-2-yl)methyl)-1methyl-1,3-dihydro-2H-imidazol-2-ylidene)benzamide (ZNA 188). ZNA 188 was prepared with 4.6.4 (178.9 mg, 0.34 mmol) following general procedure B2. The crude product was purified via flash column chromatography (20:1 EtOAc/MeOH) to yield a white solid (75.3 mg, 49%). Rf = 0.15 (30:1 CH2Cl2/MeOH). 1H NMR (CDCl3, 500 MHz): 428 δ 12.21 (brs, 1H), 8.16, (q, 7.0 Hz, 1H), 8.00 (td, J = 8.0, 2.0 Hz, 1H), 7.40 (d, J = 8.5 Hz, 1H), 7.35 – 7.29 (m, 1H), 7.19 – 7.14 (m, 2H), 7.12 (t, J = 7.5 Hz, 1H), 7.08 – 7.00 (m, 3H), 4.01 (s, 2H), 3.97 (s, 3H), 3.86 (s, 3H), 3.42 (s, 3H). 13C NMR (125 MHz, CDCl3): δ 172.8, 161.3 (d, JCF = 251.2 Hz), 160.0, 153.4, 149.6, 147.2, 141.1, 131.8, 131.5 (d, JCF = 2.2 Hz), 131.1 (d, JCF = 8.6 Hz), 127.9, 124.1, 123.4 (d, JCF = 3.6 Hz), 122.9, 119.8, 117.3, 116.4 (d, JCF = 23.0 Hz), 114.3, 55.5, 55.3, 29.9, 28.4. IR (thin film): 1551, 1509, 1463, 1357, 1291, 1173, 1032, 907, 830, 756 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C25H24FN4O3, 447.1832; found, 447.1840. MeO Me N N H N N O F Me (E)-2-Fluoro-N-(5-(4-methoxyphenyl)-1-methyl-4-((6-methylpyridin-2yl)methyl)-1,3-dihydro-2H-imidazol-2-ylidene)benzamide (ZNA 195). ZNA 195 was prepared with 4.6.9 (0.18 g, 0.36 mmol) following general procedure B2. The crude product was purified via flash column chromatography (1:1 hexanes/EtOAc, followed by 40:1 CH2Cl2/MeOH) to yield a white solid (0.15 g, 99%). Rf = 0.15 (40:1 CH2Cl2/MeOH). 1 H NMR (CDCl3, 500 MHz): δ 12.26 (brs, 1H), 8.04 (t, J = 8.0 Hz, 1H), 7.42 (t, J = 7.5 Hz, 1H), 7.33 – 7.25 (m, 3H), 7.09 (t, J = 7.5 Hz, 1H), 7.02 (dd, J = 11.5, 8.5 Hz, 1H), 6.99 – 6.93 (m, 3H), 6.86 (d, J = 7.5 Hz, 1H), 3.89 (s, 2H), 3.80 (s, 3H), 3.39 (s, 3H), 2.54 (s, 3H). 13C NMR (125 MHz, CDCl3): δ 171.9, 161.4 (d, JCF = 252.5 Hz), 160.1, 158.6, 156.7, 148.8, 137.0, 131.6, 131.5 (d, JCF = 8.6 Hz), 127.0, 127.0, 124.6, 123.5 (d, JCF = 3.6 Hz), 121.3, 120.5, 119.9, 119.2, 116.4 (d, JCF = 23.1 Hz), 114.4, 55.3, 33.2, 30.0, 24.5. IR (thin film): 1624, 1564, 1555, 1468, 1442, 1382, 1361, 1342, 1288, 1216, 1109, 1017, 901, 845, 429 760 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C25H23FN4O2, 431.1883; found, 431.1879. MeO Me N N N H O N Me F Me (E)-2-Fluoro-N-(4-((6-isopropylpyridin-2-yl)methyl)-5-(4-methoxyphenyl)-1methyl-1,3-dihydro-2H-imidazol-2-ylidene)benzamide (ZNA 196). ZNA 196 was prepared with 4.6.10 (121.0 mg, 0.23 mmol) following general procedure B2. The crude product was purified via flash column chromatography (1:1 hexanes/EtOAc, followed by 40:1 CH2Cl2/MeOH) to yield a yellow solid (108.8 mg, 99%). Rf = 0.2 (40:1 CH2Cl2/MeOH). 1H NMR (CDCl3, 500 MHz): δ 12.16 (brs, 1H), 8.04 (td, J = 7.5, 1.0 Hz, 1H), 7.49 (t, J = 7.5 Hz, 1H), 7.36 (d, J = 8.5 Hz, 2H), 7.34 – 7.30 (m, 1H), 7.12 (t, J = 7.5 Hz, 1H), 7.05 (dd, J = 11.0, 9.0 Hz, 1H), 7.02 – 6.96 (m, 3H), 6.91 (d, J = 7.5 Hz, 1H), 3.92 (s, 2H), 3.84 (s, 3H), 3.43 (s, 3H), 3.07 (septet, J = 7.0 Hz, 1H), 1.31 (d, J = 7.0 Hz, 6H). 13C NMR (125 MHz, CDCl3): δ 172.1, 167.6, 161.4 (d, JCF = 252.6 Hz), 160.1, 159.5, 149.1, 137.1, 131.7, 131.6 (d, JCF = 2.1 Hz), 131.4 (d, JCF = 8.7 Hz), 127.2, 124.5, 123.5 (d, JCF = 3.7 Hz), 119.9, 119.5, 118.6, 116.4 (d, JCF = 23.2 Hz), 114.4, 55.3, 36.3, 33.4, 30.0, 22.6. IR (thin film): 2962, 1558, 1546, 1514, 1456, 1359, 1291, 1242, 1212, 837, 791, 760 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C27H28FN4O2, 459.2196; found, 459.2194. 430 MeO Me N N H N N O F tBu (E)-N-(4-((6-(tert-butyl)pyridin-2-yl)methyl)-5-(4-methoxyphenyl)-1-methyl1,3-dihydro-2H-imidazol-2-ylidene)-2-fluorobenzamide (ZNA 197). ZNA 197 was prepared with 4.6.11 (12.2 mg, 0.02 mmol) following general procedure B2. The crude product was purified via flash column chromatography (3:1 hexanes/EtOAc) to yield a yellow solid (10.5 mg, 99%). Rf = 0.4 (2:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 12.07 (brs, 1H), 8.04 (t, J = 8.0 Hz, 1H), 7.51 (t, J = 7.5 Hz, 1H), 7.41 (d, J = 7.5 Hz, 2H), 7.36 – 7.32 (m, 1H), 7.18 (d, J = 8.0 Hz, 1H), 7.14 (t, J = 7.5 Hz, 1H), 7.07 (dd, J = 10.5, 8.0 Hz, 1H), 7.01 (d, J = 7.5 Hz, 2H), 6.93 (d, J = 7.5 Hz, 1H), 3.92 (s, 2H), 3.87 (s, 3H), 3.45 (s, 3H), 1.37 (s, 9H). 13C NMR (125 MHz, CDCl3): δ 169.6, 161.4 (d, JCF = 252.5 Hz), 160.1, 156.0, 149.4, 136.9, 131.7, 131.6 (d, JCF = 2.0 Hz), 131.4 (d, JCF = 8.6 Hz), 127.5, 124.4, 123.5 (d, JCF = 3.7 Hz), 120.0, 119.2, 117.2, 116.4 (d, JCF = 23.2 Hz), 114.3, 55.3, 37.5, 33.5, 30.2, 30.0. IR (thin film): 2960, 1699, 1683, 1568, 1558, 1540, 1361, 1248, 836, 758, 668 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C28H30FN4O2, 473.2353; found, 473.2354. MeO Me N N N H O N Me O F Me (E)-2-Fluoro-N-(4-((6-isopropoxypyridin-2-yl)methyl)-5-(4-methoxyphenyl)1-methyl-1,3-dihydro-2H-imidazol-2-ylidene)benzamide (ZNA 198). ZNA 198 was prepared with 4.6.12 (0.13 g, 0.24 mmol) following general procedure B2. The crude 431 product was purified via flash column chromatography (2:1 hexanes/EtOAc) to yield a yellow solid (0.11 g, 98%). Rf = 0.4 (1:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 12.22 (brs, 1H), 8.06 (t, J = 8.0 Hz, 1H), 7.44 (t, J = 7.5 Hz, 1H), 7.38 – 7.31 (m, 3H), 7.13 (t, J = 7.5 Hz, 1H), 7.06 (t, J = 9.5 Hz, 1H), 6.99 (d, J = 7.0 Hz, 2H), 6.64 (d, J = 7.0 Hz, 1H), 6.52 (d, J = 8.0 Hz, 1H), 5.42 (septet, J = 5.0 Hz, 1H), 3.85 (s, 3H), 3.81 (s, 2H), 3.44 (s, 3H), 1.39 (d, J = 5.0 Hz, 6H). 13C NMR (125 MHz, CDCl3): δ 172.2, 163.4, 161.5 (d, JCF = 252.8 Hz), 160.1, 154.9, 149.3, 139.2, 131.6, 131.6, 131.5 (d, JCF = 8.6 Hz), 127.0, 124.3, 123.5 (d, JCF = 3.6 Hz), 119.5, 116.5 (d, JCF = 23.2 Hz), 114.4, 114.4, 109.8, 68.1, 55.3, 33.0, 30.0, 22.1. IR (thin film): 2974, 1630, 1557, 1509, 1449, 1333, 1288, 1247, 1109, 1033, 840, 759, 659 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C27H28FN4O3, 475.2145; found, 475.2144. General procedure B7 for the NaH-mediated cyclization of N-acyl propargyl guanidine MeO MeO N R H 2N NaH, THF Me N O N r.t. F R Me N N H F O In a 50 mL round-bottom flask containing a magnetic stir bar was added 0.64 mmol of the N-acyl propargyl guanidine and 10 mL THF under N2. To the solution was added NaH (31.0 mg, 0.77 mmol) at room temperature. The reaction mixture was stirred for 60 min, after which the solvent was removed under reduced pressure, and the crude product was redissolved in EtOAc (25 mL). The organic layer was washed with sat. aq. NH4Cl (10 432 mL) and brine (10 mL), dried over Na2SO4, filtered, and concentrated. The resulting crude product was purified via flash column chromatography to yield the desired N2-acyl 2aminoimidazole. MeO Me N N H N N O F OMe (E)-2-Fluoro-N-(5-(4-methoxyphenyl)-4-((5-methoxypyridin-2-yl)methyl)-1methyl-1,3-dihydro-2H-imidazol-2-ylidene)benzamide (ZNA 177). ZNA 177 was prepared with 4.6.8 (287.0 mg, 0.64 mmol) following general procedure B7. The crude product was purified via flash column chromatography (1:2 hexanes/EtOAc, followed by 1:3 hexanes/EtOAc, then 30:1 and 20:1 CH2Cl2/MeOH) to yield a white solid (205.6 mg, 71%). Rf = 0.2 (30:1 CH2Cl2/MeOH). 1H NMR (CDCl3, 500 MHz): δ 8.28 (d, J = 3.0 Hz, 1H), 8.04 (td, J = 7.5, 2.0 Hz, 1H), 7.36 – 7.28 (m, 3H), 7.12 (td, J = 7.5, 1.0 Hz, 1H), 7.10 – 7.01 (m, 3H), 6.98 (d, J = 8.5 Hz, 2H), 3.89 (s, 2H), 3.82 (s, 3H), 3.79 (s, 3H), 3.41 (s, 3H). 13C NMR (125 MHz, CDCl3): δ 171.7, 161.4 (d, JCF = 252.5 Hz), 160.7, 154.4, 149.5, 148.5, 137.1, 131.65 (d, JCF = 2.3 Hz), 131.64, 131.6, 126.6, 124.7, 123.5 (d, JCF = 3.5 Hz), 122.8, 121.5, 121.0, 119.8, 116.4 (d, JCF = 23.2 Hz), 114.8, 55.6, 55.3, 32.3, 30.1. IR (thin film): 2934, 1652, 1568, 1558, 1540, 1508, 1362, 1031, 835, 759, 667 cm-1. HRMS (ESITOF) [M + H]+ m/z: calcd for C25H24FN4O3, 447.1832; found, 447.1830. 433 MeO Me N N H N O F N (E)-2-Fluoro-N-(5-(4-methoxyphenyl)-1-methyl-4-(3-(pyridin-2-yl)propyl)1,3-dihydro-2H-imidazol-2-ylidene)benzamide (ZNA 194). ZNA 194 was prepared with 4.6.14 (83.0 mg, 0.19 mmol) following general procedure B7. The crude product was purified via flash column chromatography (80:1 CH2Cl2/MeOH, followed by 60:1, 50:1 and 40:1 CH2Cl2/MeOH) to yield a white solid (58.9 mg, 71%). Rf = 0.1 (30:1 CH2Cl2/MeOH). 1H NMR (CDCl3, 500 MHz): δ 8.53 (d, J = 4.5 Hz, 1H), 8.08 (td, J = 7.5, 1.5 Hz, 1H), 7.54 (td, J = 7.5, 1.5 Hz, 1H), 7.39 – 7.31 (m, 1H), 7.20 (d, J = 8.5 Hz, 2H), 7.15 (t, J = 7.5 Hz, 1H), 7.10 – 7.05 (m, 3H), 6.96 (d, J = 8.5 Hz, 2H), 3.85 (s, 3H), 3.41 (s, 3H), 2.78 (t, J = 7.5 Hz, 2H), 2.53 (t, J = 7.5 Hz, 2H), 2.01 (q, J = 7.5 Hz, 2H). 13 C NMR (125 MHz, CDCl3): δ 172.3, 161.5 (d, JCF = 252.6 Hz), 160.9, 160.0, 149.2, 136.4, 131.7 (d, JCF = 2.1 Hz), 131.6, 131.5, 127.0, 123.8, 123.5 (d, JCF = 3.7 Hz), 122.8, 121.2, 120.0, 116.5 (d, JCF = 23.1 Hz), 114.3, 55.4, 37.1, 29.9, 29.2, 23.7. IR (thin film): 2969, 2943, 1711, 1564, 1511, 1470, 1435, 1355, 1289, 1246, 1218, 1175, 1022, 886, 832, 755 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C26H26FN4O2, 445.2040; found, 445.2039. 434 General procedure B8 for synthesis of Zn-complexes MeO Me N N N H N R + Zn(II)-salt MeOH r.t. ZNA-Zinc complex R O A 20-mL scintillation vial with a magnetic stir bar was charged with 2 mL MeOH and 0.09 mmol of a hemiporphyrin-like ZNA. Then ZnSO4 . 7H2O (77.3 mg, 0.27 mmol) was added in one portion and a white precipitate appeared over a course of 16 h. It was collected via filtration, washed with MeOH and dried under reduced pressure to obtain the ZNA-zinc complex. F O Me N MeO MeO O N NH O S O Zn O N N Zn O O N S HN Me O O N OMe O OMe F ZNA 148 bound to ZnSO4 (C1). C1 was prepared with ZNA 148 (40 mg, 0.09 mmol) and ZnSO4 . 7H2O (77.3 mg, 0.27 mmol) following general procedure B8. The desired zinc complex was obtained as a white solid (58.1 mg, 61%). 1H NMR (CD3OD, 500 MHz): δ 7.76 (brs, 4H), 7.60 – 7.55 (m, 2H), 7.30 (d, J = 8.5 Hz, 4H), 7.26 (t, J = 7.5 Hz, 2H), 7.19 (dd, J = 11.0, 8.5 Hz, 2H), 7.10 (d, J = 8.5 Hz, 4H), 6.90 (d, J = 6.5 Hz, 2H), 6.82 (brs, 2H), 4.04 (s, 4H), 3.87 (s, 6H), 3.72 (brs, 6H), 3.46 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ 166.1, 163.3, 161.1 (d, JCF = 248.7 Hz), 159.8, 156.2, 149.7, 139.0, 132.2, 131.9, 131.6, 127.7, 127.1, 124.1, 121.1, 116.9 (d, J = 22.2 Hz), 115.2, 114.7, 108.4, 55.7, 53.1, 34.9, 30.7. IR (thin film): 2934, 1704, 1608, 1575, 1565, 1473, 1428, 1305, 1208, 1118, 1061, 1005, 799, 759, 656 cm-1. 435 MeO F N Me O N NH N Zn Cl OMe Cl ZNA 148 bound to ZnCl2 (C2). C2 was prepared with ZNA 148 (56.2 mg, 0.12 mmol) and ZnCl2 (54.4 mg, 0.36 mmol) following general procedure B8. The desired zinc complex was obtained as a white solid (64.1 mg, 92%). 1H NMR (CDCl3, 500 MHz): δ 10.17 (d, J = 5.5 Hz, 1H), 8.05 (t, J = 7.5 Hz, 1H), 7.87 (t, J = 7.5 Hz, 1H), 7.56 (dd, J = 13.0, 7.0 Hz, 1H), 7.29 (t, J = 7.5 Hz, 1H), 7.22 – 7.18 (m, 3H), 7.05 (d, J = 9.0 Hz, 2H), 6.95 (d, J = 7.5 Hz, 1H), 6.83 (d, J = 8.5 Hz, 1H), 4.03 (s, 3H), 3.98 (s, 2H), 3.88 (s, 3H), 3.39 (s, 3H). 13C NMR (125 MHz, CDCl3): δ 164.6, 164.8, 160.9 (d, JCF = 253.0 Hz), 160.5, 155.6, 143.9, 140.3, 134.7 (d, JCF = 9.1 Hz), 131.6 (d, JCF = 1,12 Hz), 131.5, 129.2, 128.5, 124.7 (d, JCF = 3.5 Hz), 120.2 (d, JCF = 11.5 Hz), 119.3, 117.2, 116.7 (d, JCF = 23.0 Hz), 114.7, 105.2, 55.6, 55.4, 33.1, 31.8. IR (thin film): 3239, 1703, 1575, 1564, 1457, 1436, 1302, 1253, 116, 1080, 910, 774, 756 cm-1. HRMS (ESI-TOF) [M - Cl]+ m/z: calcd for C25H23N4O3FClZn, 545.0734; found, 545.0745. MeO Me N N NH O Zn Cl Cl N F ZNA 194 bound to ZnCl2 (C3). C3 was prepared with ZNA 194 (55.2 mg, 0.12 mmol) and ZnCl2 (19.0 mg, 0.14 mmol) following general procedure B8. The desired zinc complex was obtained as a white solid (43.6 mg, 60%). 1H NMR (CDCl3, 500 MHz): δ 9.62 (d, J = 7.5 Hz, 1H), 9.10 (dd, J = 6.0, 1.5 Hz, 1H), 8.21 (t, J = 7.5 Hz, 1H), 7.94 (td, 436 J = 8.0, 1.5 Hz, 1H), 7.59 – 7.52 (m, 1H), 7.48 – 7.42 (m, 2H), 7.32 (t, J = 7.5 Hz, 1H), 7.24 – 7.21 (m, 3H), 7.01 (d, J = 8.5 Hz, 2H), 3.86 (s, 3H), 3.45 (s, 3H), 3.30 (t, J = 6.0 Hz, 2H), 2.24 (t, J = 6.0 Hz, 2H), 1.95 – 1.90 (m, 2H). 13C NMR (125 MHz, CDCl3): δ 168.5, 163.4, 161.9, 160.9 (d, JCF = 251.6 Hz), 160.3, 149.3, 140.9, 139.1, 134.5 (d, JCF = 9.0 Hz), 133.2, 131.8 (d, JCF = 1.3 Hz), 131.6, 129.5, 125.8, 124.8 (d, JCF = 3.5 Hz), 122.9, 120.1 (d, JCF = 11.8 Hz), 119.8, 116.5 (d, JCF = 23.1 Hz), 114.5, 55.4, 34.4, 32.5, 32.0, 21.4. IR (thin film): 3296, 2953, 1689, 1610, 1530, 1507, 1460, 1290, 1241, 1173, 1018, 888, 781, 759, 634 cm-1. HRMS (ESI-TOF) [M - Cl]+ m/z: calcd for C26H25N4O2FClZn, 543.0942; found, 543.0950. 2 BF4- MeO Me N N F NH O N Zn N O HN F N N Me OMe ZNA 131-Zinc dimer (C4). C3 was prepared with ZNA 131 (34.9 mg, 0.08 mmol) and Zn(BF4)2 (32.7 mg, 0.09 mmol) following general procedure B8. The desired zinc complex was obtained as a white solid (78.3 mg, 87%). 1H NMR (CD3CN, 500 MHz): δ 9.92 (s, 2H), 8.47 (dd, J = 5.5, 1.0 Hz, 2H), 7.97 (td, J = 7.5, 2.0 Hz, 2H), 7.66 (td, J = 7.5, 1.5 Hz, 2H), 7.64 – 7.60 (m, 2H), 7.48 (d, J = 8.0 Hz, 2H), 7.47 – 7.41 (m, 6H), 7.26 – 7.17 (m, 8H), 4.04 (d, 2H), 3.92 (s, 6H), 3.85 (d, 2H), 3.63 (s, 6H). 13 C NMR (125 MHz, CD3CN): δ 164.6, 160.7, 160.2 (d, JCF = 253.0 Hz), 159.2, 157.4, 149.3, 141.2, 136.0, 132.0, 131.3, 127.9, 127.4, 125.6, 125.5, 123.8, 118.6, 116.5 (d, JCF = 23.0 Hz), 114.6, 55.3, 31.6, 31.1. IR (thin film): 3336, 2839, 1670, 1653, 1577, 1535, 1468, 1299, 1253, 437 1179, 1060, 1014, 848, 786, 684 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C48H43B2N8O4F10Zn, 1071.2725; found, 1071.2928. 8.5 8.0 180 7.5 170 7.0 160 150 6.5 140 6.0 5.5 130 120 5.0 110 4.5 100 4.0 f1 (ppm) 90 80 f1 (ppm) 9.10 3.22 1.00 0.96 0.99 438 3.5 3.0 70 60 2.5 50 2.0 40 1.5 30 1.0 20 0.5 10 0.0 0 -0.5 -10 9.93 3.34 1.15 1.28 1.00 439 9.0 8.5 8.0 7.5 7.0 6.5 170 160 150 140 130 120 6.0 110 5.5 100 5.0 90 4.5 4.0 f1 (ppm) 80 f1 (ppm) 70 3.5 60 3.0 50 2.5 40 2.0 30 1.5 20 1.0 10 0.5 0 0.0 -10 -0.5 9.5 180 9.0 170 8.5 8.0 160 7.5 150 7.0 140 9.46 3.03 1.00 0.96 0.98 440 6.5 130 6.0 120 5.5 110 5.0 100 4.5 4.0 f1 (ppm) 3.5 3.0 90 80 f1 (ppm) 70 60 2.5 50 2.0 40 1.5 30 1.0 20 0.5 0.0 10 -0.5 0 -10 9.5 9.0 160 8.5 150 8.0 140 7.5 130 7.0 6.5 120 6.0 110 5.5 100 5.0 4.5 4.0 f1 (ppm) 90 80 f1 (ppm) 70 9.60 3.00 2.05 0.95 441 3.5 3.0 60 2.5 50 2.0 40 1.5 1.0 30 20 0.5 10 0.0 -0.5 0 1.00 0.97 1.04 9.40 442 9.0 160 8.5 8.0 7.5 150 140 130 7.0 120 6.5 110 6.0 100 5.5 5.0 90 4.5 4.0 f1 (ppm) 80 70 f1 (ppm) 3.5 60 3.0 2.5 2.0 50 40 30 1.5 20 1.0 10 0.5 0.0 0 -0.5 -10 9.0 170 8.5 160 8.0 150 6.5 130 120 140 6.0 110 5.5 100 5.0 90 4.5 4.0 f1 (ppm) 80 f1 (ppm) 3.5 70 3.0 60 9.20 7.0 1.93 7.5 2.01 1.16 0.84 9.5 1.00 0.91 443 2.5 50 2.0 40 1.5 30 1.0 20 0.5 0.0 10 0 -0.5 -10 8.5 180 8.0 170 7.5 160 7.0 150 0.88 3.11 0.99 0.98 1.05 444 6.5 6.0 5.5 140 130 120 5.0 110 4.5 100 4.0 f1 (ppm) 90 f1 (ppm) 3.5 80 3.0 70 2.5 60 2.0 50 1.5 40 1.0 30 20 0.5 10 0.0 0 10.5 180 10.0 9.5 170 9.0 160 8.5 150 8.0 140 7.5 130 0.98 3.27 1.11 1.10 0.96 445 7.0 120 6.5 6.0 110 5.5 100 5.0 4.5 f1 (ppm) 90 80 f1 (ppm) 4.0 70 3.5 3.0 60 2.5 50 2.0 40 1.5 30 1.0 20 0.5 0.0 10 -0.5 0 0.95 3.18 1.11 1.06 1.00 446 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 180 170 160 150 140 130 120 110 100 4.5 f1 (ppm) 4.0 90 80 f1 (ppm) 3.5 70 3.0 60 2.5 50 2.0 40 1.5 30 1.0 20 0.5 10 0.0 0 -10 9.0 8.5 160 8.0 7.5 150 140 0.86 3.17 1.98 0.93 447 7.0 130 6.5 120 6.0 110 5.5 5.0 100 4.5 4.0 f1 (ppm) 90 80 f1 (ppm) 3.5 70 3.0 60 2.5 2.0 50 40 1.5 30 1.0 20 0.5 0.0 10 0 8.5 8.0 7.5 0.91 1.04 0.99 1.06 448 7.0 6.5 6.0 5.5 5.0 4.5 4.0 f1 (ppm) 165 160 155 150 145 140 135 130 125 120 115 110 105 100 95 90 f1 (ppm) 3.5 85 3.0 80 75 2.5 70 65 2.0 60 1.5 55 50 1.0 45 0.5 40 35 0.0 30 25 -0.5 20 449 N 9.5 170 9.0 160 8.5 8.0 150 7.5 140 7.0 130 6.5 6.0 120 110 5.5 100 5.0 4.5 f1 (ppm) 90 80 f1 (ppm) 4.0 3.5 70 3.0 60 0.94 2.16 2.17 1.06 1.04 1.03 1.00 S7.2 2.5 2.0 50 40 1.5 30 1.0 0.5 20 0.0 10 -0.5 0 9.5 180 9.0 170 8.5 160 8.0 150 7.5 7.0 140 6.5 130 6.0 120 5.5 110 5.0 100 9.11 3.05 0.91 0.87 2.04 0.93 1.06 2.01 1.12 0.99 450 4.5 f1 (ppm) 4.0 3.5 3.0 2.5 90 f1 (ppm) 80 70 60 50 2.0 40 1.5 30 1.0 20 0.5 0.0 10 0 180 9.0 170 8.5 160 8.0 150 140 6.5 130 6.0 120 5.5 110 5.0 100 9.84 3.21 3.26 7.0 0.82 7.5 0.83 1.06 2.12 1.00 9.5 3.28 451 4.5 4.0 f1 (ppm) 3.5 90 80 f1 (ppm) 70 3.0 60 2.5 50 2.0 1.5 40 1.0 30 0.5 20 0.0 10 -0.5 0 180 8.5 170 8.0 160 7.5 7.0 150 140 6.5 130 6.0 120 5.5 110 9.43 3.22 3.43 0.71 1.90 1.08 1.04 2.00 0.87 9.0 0.88 452 5.0 100 4.5 4.0 f1 (ppm) 90 f1 (ppm) 3.5 80 3.0 70 2.5 60 2.0 50 1.5 1.0 40 30 0.5 20 0.0 10 -0.5 0 9.0 170 8.5 8.0 160 150 7.5 140 7.0 130 6.5 6.0 120 5.5 110 5.0 100 4.5 4.0 f1 (ppm) 90 80 f1 (ppm) 8.70 3.11 3.00 1.00 0.82 1.09 2.08 1.07 1.04 2.02 453 3.5 70 3.0 60 2.5 50 2.0 1.5 40 1.0 30 0.5 20 0.0 10 -0.5 0 9.20 3.21 3.16 0.97 0.74 2.01 1.98 1.84 0.95 454 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 f1 (ppm) 3.5 180 170 160 150 140 130 120 110 100 90 80 f1 (ppm) 70 \ 3.0 60 2.5 50 2.0 40 1.5 30 1.0 20 0.5 10 0.0 0 -0.5 -10 9.72 3.27 0.89 0.92 1.84 1.14 2.15 1.08 1.00 455 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 f1 (ppm) 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 180 170 160 150 140 130 120 110 100 90 80 f1 (ppm) 70 60 50 40 30 20 10 0 -10 9.0 190 180 8.5 170 8.0 7.5 7.0 160 150 140 6.5 130 6.0 120 5.5 110 5.0 100 4.5 f1 (ppm) 90 80 f1 (ppm) 4.0 3.5 70 60 3.0 50 2.5 40 9.75 8.95 3.03 0.86 0.88 2.11 2.00 456 2.0 1.5 1.0 0.5 30 20 10 0 0.0 -10 -20 9.0 180 8.5 170 8.0 160 7.5 150 7.0 140 6.5 130 6.0 120 5.5 110 5.0 100 8.84 2.03 2.06 2.92 0.89 0.86 2.00 2.05 1.93 1.13 0.85 457 4.5 4.0 f1 (ppm) 3.5 3.0 2.5 90 80 f1 (ppm) 70 60 50 2.0 40 1.5 30 1.0 20 0.5 10 0.0 0 10.0 180 9.5 170 9.0 8.5 160 150 8.0 140 7.5 130 7.0 6.5 120 6.0 110 5.5 100 5.0 f1 (ppm) 4.5 4.0 90 f1 (ppm) 80 70 3.5 60 3.0 50 9.58 3.09 3.27 0.98 2.03 0.91 1.00 2.92 1.03 458 2.5 2.0 40 1.5 30 1.0 20 0.5 10 0.0 0 180 8.5 170 8.0 160 150 140 6.5 130 6.0 120 5.5 110 9.38 7.0 3.01 7.5 3.18 2.99 1.07 2.11 1.00 1.03 9.0 3.34 459 5.0 4.5 f1 (ppm) 4.0 3.5 3.0 100 90 f1 (ppm) 80 70 60 2.5 50 2.0 40 1.5 30 1.0 20 0.5 10 0.0 0 9.0 170 8.5 160 8.0 150 7.5 140 7.0 130 6.5 6.0 120 110 5.5 100 5.0 90 4.5 4.0 f1 (ppm) 80 f1 (ppm) 3.5 70 3.0 60 9.31 2.97 2.85 2.88 1.02 2.00 0.98 0.82 3.05 460 2.5 50 2.0 40 1.5 30 1.0 0.5 20 10 0.0 0 -0.5 9.0 180 8.5 170 8.0 160 7.5 150 7.0 140 6.5 130 9.39 3.19 3.22 1.19 0.91 2.00 0.99 2.04 0.95 461 6.0 120 5.5 5.0 4.5 4.0 f1 (ppm) 3.5 110 100 90 80 f1 (ppm) 70 3.0 60 2.5 50 2.0 40 1.5 30 1.0 20 0.5 0.0 10 -0.5 0 9.5 180 9.0 170 8.5 160 8.0 7.5 150 7.0 140 6.5 130 6.0 120 5.5 110 5.0 100 9.08 3.03 3.14 2.90 0.98 2.00 2.10 2.00 0.93 462 4.5 f1 (ppm) 4.0 3.5 3.0 2.5 90 f1 (ppm) 80 70 60 50 2.0 40 1.5 30 1.0 20 0.5 10 0.0 0 9.0 180 8.5 170 8.0 160 7.5 150 7.0 6.5 6.0 5.5 5.0 4.5 4.0 f1 (ppm) 140 130 120 110 100 90 f1 (ppm) 80 9.29 3.27 3.25 1.05 2.00 0.89 1.05 463 3.5 70 3.0 60 2.5 50 2.0 40 1.5 1.0 30 0.5 20 0.0 10 0 9.5 170 9.0 8.5 160 8.0 150 7.5 140 7.0 130 6.5 120 6.0 110 5.5 100 5.0 90 4.5 4.0 f1 (ppm) 80 f1 (ppm) 3.5 70 3.0 60 9.02 9.12 2.97 3.02 1.08 2.08 2.00 464 2.5 50 2.0 1.5 40 30 1.0 20 0.5 10 0.0 -0.5 0 -10 9.0 170 8.5 8.0 160 7.5 150 7.0 140 6.5 130 6.0 120 5.5 110 5.0 100 4.5 4.0 f1 (ppm) 90 80 f1 (ppm) 3.5 70 3.0 60 2.5 50 9.31 3.07 2.20 2.10 2.97 1.22 2.00 2.92 1.20 0.81 1.00 465 2.0 40 1.5 30 1.0 20 0.5 10 0.0 0 9.0 8.5 170 160 8.0 150 7.5 140 7.0 130 6.5 6.0 120 5.5 110 5.0 100 4.5 4.0 f1 (ppm) 90 80 f1 (ppm) 3.5 3.0 70 60 9.06 2.85 0.84 3.04 0.86 2.00 1.90 466 2.5 50 2.0 40 1.5 1.0 30 0.5 20 0.0 10 -0.5 0 9.0 8.5 170 160 8.0 150 7.5 140 7.0 130 6.5 120 6.0 5.5 110 5.0 100 4.5 f1 (ppm) 90 80 f1 (ppm) 4.0 3.5 70 3.0 60 2.5 50 8.91 2.73 2.95 3.02 0.98 1.25 1.92 1.01 1.00 2.06 467 2.0 1.5 1.0 40 30 20 0.5 10 0.0 0 9.0 180 8.5 170 8.0 160 7.5 150 7.0 140 6.5 130 6.0 120 5.5 5.0 110 4.5 4.0 f1 (ppm) 100 90 f1 (ppm) 3.5 80 3.0 70 2.5 60 2.0 50 1.5 40 5.73 9.53 2.90 1.01 2.96 1.06 1.97 1.11 1.94 0.98 0.90 468 1.0 30 0.5 20 0.0 10 -0.5 0 9.0 180 8.5 170 8.77 8.91 3.01 3.17 2.00 1.24 2.17 1.99 469 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 f1 (ppm) 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 160 150 140 130 120 110 100 90 f1 (ppm) 80 70 60 50 40 30 20 10 0 9.5 9.0 180 8.5 170 8.0 160 7.5 150 7.0 140 6.5 130 6.0 120 5.5 110 5.0 4.5 f1 (ppm) 100 90 f1 (ppm) 4.0 80 3.5 70 3.0 60 2.5 50 2.0 40 6.12 9.41 2.95 2.93 0.96 1.00 2.01 1.14 3.03 470 1.5 30 1.0 20 0.5 10 0.0 0 9.0 180 8.5 170 8.0 160 7.5 7.0 6.5 6.0 5.5 5.0 150 140 130 120 110 100 4.5 4.0 f1 (ppm) 90 80 f1 (ppm) 9.09 2.98 3.14 3.03 1.00 2.13 1.00 1.06 1.09 2.14 471 3.5 70 3.0 60 2.5 50 2.0 40 1.5 30 1.0 20 0.5 10 0.0 0 -10 10.5 180 10.0 170 9.5 9.0 160 8.5 150 8.0 140 7.5 7.0 130 120 6.5 110 6.0 100 5.5 5.0 f1 (ppm) 90 80 f1 (ppm) 4.5 4.0 70 3.5 60 3.0 2.5 50 40 1.31 3.29 3.33 1.05 2.13 1.00 1.11 2.05 1.04 1.02 472 2.0 30 1.5 20 1.0 0.5 10 0.0 0 180 8.5 170 8.0 160 150 140 6.5 130 6.0 120 5.5 110 5.0 4.5 4.0 f1 (ppm) 3.5 100 90 80 f1 (ppm) 70 3.0 60 1.09 2.94 7.0 2.91 2.88 7.5 0.93 0.86 2.00 0.89 9.0 3.01 473 2.5 50 2.0 40 1.5 1.0 30 0.5 20 0.0 10 -0.5 0 10.0 180 9.5 170 9.0 8.5 160 8.0 150 7.5 140 7.0 130 6.5 120 6.0 110 5.5 100 5.0 4.5 f1 (ppm) 4.0 90 80 f1 (ppm) 3.5 70 3.0 2.5 60 50 0.97 2.89 2.95 2.90 0.95 0.92 2.00 0.98 1.02 2.00 474 2.0 40 1.5 30 1.0 0.5 20 0.0 10 -0.5 0 9.5 180 9.0 170 8.5 160 8.0 150 7.5 140 7.0 130 6.5 120 6.0 5.5 110 5.0 4.5 f1 (ppm) 100 90 f1 (ppm) 4.0 80 3.5 70 3.0 60 2.5 50 1.11 2.82 3.07 2.81 1.00 0.99 1.99 1.00 2.00 0.94 475 2.0 40 1.5 30 1.0 20 0.5 0.0 10 0 9.0 170 8.5 160 8.0 150 7.5 140 7.0 130 6.5 6.0 120 5.5 110 5.0 100 4.5 4.0 f1 (ppm) 90 80 f1 (ppm) 3.5 3.0 2.5 70 60 50 1.12 3.16 3.12 3.13 0.96 2.00 2.00 1.99 0.89 476 2.0 40 1.5 30 1.0 20 0.5 0.0 10 -0.5 0 8.0 7.5 7.0 6.5 180 170 160 150 6.0 140 5.5 130 5.0 120 4.5 110 4.0 3.5 f1 (ppm) 100 90 f1 (ppm) 3.0 80 1.15 3.10 3.18 1.09 2.00 1.04 2.01 0.92 0.88 477 2.5 70 2.0 60 1.5 1.0 50 0.5 40 0.0 30 -0.5 20 9.5 9.0 8.5 8.0 7.5 180 170 160 150 140 7.0 130 6.5 120 6.0 110 5.5 100 5.0 4.5 f1 (ppm) 90 f1 (ppm) 80 4.0 70 3.5 60 3.0 50 1.04 5.95 2.98 0.87 3.01 1.00 0.95 1.94 478 2.5 2.0 40 1.5 30 1.0 20 0.5 10 0.0 0 5.96 1.03 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 f1 (ppm) 4.0 3.5 3.0 2.5 2.0 170 160 150 140 130 120 110 100 90 f1 (ppm) 80 70 60 50 40 3.17 3.14 180 1.07 9.0 0.83 2.92 1.00 0.99 1.96 479 1.5 30 1.0 20 0.5 10 0.0 0 9.0 180 8.5 170 8.0 160 7.5 7.0 6.5 6.0 150 140 130 120 5.5 110 5.0 100 4.5 f1 (ppm) 4.0 90 80 f1 (ppm) 3.5 70 3.0 60 2.5 50 2.0 40 9.02 1.05 3.11 2.98 0.83 1.89 2.00 3.06 480 1.5 30 1.0 20 0.5 10 0.0 0 190 180 8.5 170 8.0 160 150 140 6.5 130 6.0 120 5.5 110 5.0 4.5 f1 (ppm) 100 90 f1 (ppm) 4.0 80 3.5 3.0 70 2.5 60 6.15 1.15 2.99 3.11 0.97 7.0 1.11 7.5 1.00 1.13 2.11 9.0 3.14 481 2.0 50 1.5 40 1.0 30 0.5 20 0.0 10 0 9.0 180 8.5 170 9.09 2.98 3.14 3.03 1.00 2.13 1.00 1.06 1.09 2.14 482 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 f1 (ppm) 3.5 160 150 140 130 120 110 100 90 80 f1 (ppm) 70 3.0 60 2.5 50 2.0 40 1.5 30 1.0 20 0.5 10 0.0 0 -10 170 8.5 160 8.0 7.5 150 7.0 140 6.5 130 6.0 120 5.5 110 5.0 3.5 3.0 100 90 f1 (ppm) 80 70 60 3.11 2.92 4.0 1.94 4.5 f1 (ppm) 0.92 2.05 180 9.0 1.23 9.5 2.04 0.88 1.00 2.02 0.90 0.87 483 2.5 50 2.0 40 1.5 1.0 30 0.5 20 0.0 10 0 12.0 180 11.5 11.0 170 10.5 10.0 160 150 9.5 9.0 140 8.5 130 8.0 120 7.5 110 7.0 6.5 100 6.0 5.5 f1 (ppm) 90 80 f1 (ppm) 5.0 4.5 70 4.0 60 3.5 50 3.0 8.41 2.96 3.00 0.97 0.96 1.20 1.12 1.10 1.04 1.98 2.01 1.06 1.08 1.05 0.98 484 2.5 40 2.0 30 1.5 20 1.0 0.5 10 0.0 0 -0.5 12.0 190 11.5 180 11.0 10.5 10.0 170 160 9.5 150 9.0 140 8.5 130 8.0 120 7.5 7.0 110 6.5 100 6.0 5.5 f1 (ppm) 90 80 f1 (ppm) 5.0 70 4.5 4.0 60 3.5 50 9.07 3.01 3.39 2.84 1.01 2.06 2.06 1.00 0.98 0.97 1.02 0.98 0.98 1.00 0.96 485 3.0 2.5 40 30 2.0 20 1.5 1.0 10 0.5 0 0.0 -10 -0.5 -20 11.5 180 11.0 10.5 170 10.0 160 9.5 150 9.0 140 8.5 130 8.0 7.5 120 7.0 110 6.5 6.0 5.5 5.0 f1 (ppm) 100 90 80 f1 (ppm) 4.5 70 4.0 60 3.5 50 3.0 40 8.26 2.95 2.99 2.95 0.90 0.94 2.00 2.11 1.00 1.06 1.10 0.97 1.09 0.96 1.24 486 2.5 2.0 30 1.5 20 1.0 10 0.5 0 0.0 -0.5 -10 12.0 11.5 11.0 10.5 10.0 9.5 180 170 160 150 9.0 140 8.5 130 8.0 7.5 120 7.0 110 6.5 6.0 100 5.5 5.0 f1 (ppm) 90 80 f1 (ppm) 4.5 4.0 70 3.5 60 3.0 8.77 2.87 3.02 3.04 1.06 1.00 0.99 1.02 1.07 3.00 1.95 1.09 0.97 1.04 487 2.5 50 2.0 40 1.5 30 1.0 0.5 20 0.0 -0.5 -1.0 -1.5 10 0 -10 11.5 11.0 180 10.5 170 10.0 160 9.5 150 9.0 8.5 140 8.0 130 7.5 120 7.0 110 6.5 6.0 100 5.5 5.0 f1 (ppm) 90 80 f1 (ppm) 4.5 70 4.0 3.5 60 8.64 2.81 2.92 3.02 1.02 2.16 1.16 2.95 2.00 0.99 1.15 2.02 488 3.0 50 2.5 40 2.0 30 1.5 1.0 20 0.5 10 0.0 0 -0.5 -10 11.5 11.0 180 10.5 170 10.0 160 9.5 150 9.0 140 8.5 8.0 130 7.5 120 7.0 110 6.5 100 6.0 5.5 5.0 f1 (ppm) 90 80 f1 (ppm) 4.5 70 4.0 60 3.5 50 8.97 3.01 2.89 3.00 1.01 1.09 3.03 2.03 2.02 0.91 2.06 1.04 489 3.0 2.5 40 2.0 30 1.5 20 1.0 10 0.5 0.0 0 -0.5 -10 12.0 11.5 11.0 10.5 10.0 9.5 180 170 160 150 9.0 140 8.5 130 8.0 120 7.5 7.0 110 6.5 100 6.0 5.5 5.0 f1 (ppm) 90 80 f1 (ppm) 4.5 70 4.0 3.5 60 8.58 2.84 2.83 1.05 1.00 1.05 0.99 1.01 0.97 1.88 1.82 0.98 1.00 0.95 490 3.0 50 2.5 40 2.0 1.5 30 1.0 20 0.5 0.0 -0.5 -1.0 10 0 -10 9.0 190 8.5 180 8.0 7.5 170 160 7.0 150 6.5 140 6.0 130 5.5 120 5.0 110 4.5 f1 (ppm) 100 90 f1 (ppm) 4.0 80 2.93 3.08 3.01 0.91 1.97 1.03 1.21 2.01 1.06 2.00 1.07 0.92 491 3.5 3.0 70 60 2.5 50 2.0 40 1.5 30 1.0 20 0.5 10 0.0 0 11.0 180 10.5 170 10.0 9.5 160 9.0 150 8.5 140 8.0 7.5 130 120 7.0 110 6.5 100 6.0 5.5 5.0 f1 (ppm) 4.5 4.0 90 80 f1 (ppm) 70 60 3.5 50 3.0 40 8.32 6.22 2.81 1.11 2.92 0.99 1.06 1.18 1.89 1.08 1.00 2.00 2.06 1.08 0.91 492 2.5 2.0 30 1.5 20 1.0 10 0.5 0 0.0 -10 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 f1 (ppm) 4.5 4.0 3.5 3.0 2.5 2.0 1.5 8.33 10.88 2.92 3.05 1.16 1.13 1.21 1.22 1.15 0.99 2.02 2.00 1.04 1.04 0.97 493 1.0 0.5 0.0 -0.5 -1.0 11.0 180 10.5 170 10.0 160 9.5 9.0 150 8.5 140 8.0 130 7.5 120 7.0 110 6.5 6.0 100 5.5 5.0 f1 (ppm) 4.5 90 80 f1 (ppm) 70 4.0 60 3.5 50 3.0 8.56 2.87 3.02 2.80 2.93 1.00 0.92 1.95 1.98 0.94 1.00 0.90 0.99 0.97 0.96 1.04 0.98 494 2.5 40 2.0 30 1.5 1.0 20 10 0.5 0 0.0 -10 12.0 11.5 180 11.0 10.5 10.0 170 160 150 9.5 140 9.0 8.5 130 8.0 120 7.5 110 7.0 6.5 100 6.0 5.5 f1 (ppm) 90 80 f1 (ppm) 5.0 4.5 70 4.0 60 3.5 50 3.0 40 8.78 2.93 3.14 3.00 1.08 1.21 1.25 1.23 1.22 2.02 1.83 1.04 0.98 1.08 1.00 495 2.5 2.0 30 1.5 20 1.0 10 0.5 0.0 0 -0.5 -10 180 7.5 170 160 150 7.0 140 6.5 130 6.0 120 5.5 110 5.0 100 4.5 f1 (ppm) 90 f1 (ppm) 4.0 80 3.5 70 2.01 2.95 8.0 2.08 1.03 3.09 1.07 1.99 1.00 1.85 8.5 3.00 1.04 9.0 0.95 496 3.0 60 2.5 50 2.0 40 1.5 30 1.0 20 0.5 10 0.0 0 13.0 12.5 12.0 11.5 11.0 10.5 10.0 9.5 180 170 160 150 140 130 9.0 8.5 120 8.0 110 7.5 100 7.0 6.5 6.0 f1 (ppm) 90 80 f1 (ppm) 5.5 5.0 70 4.5 60 4.0 50 3.00 1.97 2.98 1.00 1.00 1.01 1.97 2.89 1.08 2.05 1.22 0.86 497 3.5 3.0 40 2.5 30 2.0 1.5 20 1.0 10 0.5 0 0.0 -0.5 -10 13.0 12.5 12.0 11.5 11.0 10.5 10.0 180 170 160 150 140 9.5 9.0 130 8.5 120 8.0 7.5 110 7.0 6.5 6.0 f1 (ppm) 100 90 f1 (ppm) 5.5 5.0 80 4.5 70 4.0 3.06 2.84 2.08 3.14 1.08 2.90 1.05 1.03 2.06 0.99 0.94 1.12 1.00 498 3.5 60 3.0 2.5 50 2.0 40 1.5 1.0 30 0.5 0.0 20 -0.5 13.0 12.5 12.0 11.5 11.0 10.5 10.0 9.5 170 160 150 140 130 9.0 120 8.5 8.0 110 7.5 100 7.0 6.5 6.0 f1 (ppm) 90 80 f1 (ppm) 5.5 5.0 70 4.5 4.0 60 3.16 2.16 3.15 0.92 1.02 2.05 1.92 2.20 2.11 1.00 0.99 499 3.5 50 3.0 2.5 40 2.0 30 1.5 1.0 20 0.5 10 0.0 -0.5 0 180 8.5 170 7.5 160 150 7.0 140 2.80 8.0 1.92 2.75 2.91 0.85 1.95 0.97 1.08 1.06 1.92 9.0 1.00 0.92 0.99 500 6.5 130 6.0 120 5.5 110 5.0 100 4.5 f1 (ppm) 4.0 90 80 f1 (ppm) 3.5 70 3.0 60 2.5 2.0 1.5 1.0 0.5 0.0 50 40 30 20 10 0 9.0 180 8.5 170 8.0 160 7.5 150 7.0 140 6.5 130 6.0 120 5.5 110 5.0 100 2.86 1.93 2.93 2.93 1.05 1.01 3.07 1.12 1.17 2.02 1.07 1.02 501 4.5 f1 (ppm) 4.0 3.5 3.0 2.5 2.0 90 f1 (ppm) 80 70 60 50 40 1.5 30 1.0 20 0.5 10 0.0 0 9.0 170 8.5 160 8.0 150 7.5 7.0 140 6.5 130 6.0 120 5.5 110 5.0 100 4.5 f1 (ppm) 4.0 90 80 f1 (ppm) 2.89 1.95 2.90 2.91 3.04 1.02 3.08 1.94 1.05 0.99 502 3.5 70 3.0 60 2.5 50 2.0 40 1.5 1.0 0.5 0.0 30 20 10 0 180 170 160 150 140 130 120 8.5 8.0 110 7.5 100 7.0 6.5 f1 (ppm) 90 f1 (ppm) 6.0 80 5.5 5.0 70 4.5 60 3.00 9.0 2.11 2.99 3.04 13.5 13.0 12.5 12.0 11.5 11.0 10.5 10.0 9.5 2.06 2.14 4.04 1.08 1.90 0.87 503 4.0 3.5 50 3.0 40 2.5 30 2.0 1.5 20 1.0 10 0.5 0.0 0 180 170 160 150 140 9.5 130 9.0 8.5 120 8.0 110 7.5 100 7.0 6.5 6.0 f1 (ppm) 90 80 f1 (ppm) 5.5 70 5.0 4.5 60 3.03 11.0 10.5 10.0 2.15 3.11 3.00 13.0 12.5 12.0 11.5 2.10 2.02 2.09 2.10 1.39 2.00 0.96 504 4.0 3.5 50 40 3.0 2.5 30 2.0 20 1.5 1.0 10 0.5 0 0.0 12.5 12.0 180 11.5 170 11.0 160 10.5 10.0 150 9.5 140 9.0 130 8.5 8.0 120 7.5 110 7.0 100 6.5 6.0 f1 (ppm) 5.5 90 f1 (ppm) 80 5.0 70 4.5 4.0 60 2.88 2.05 2.94 2.99 2.00 1.04 1.89 1.09 3.00 1.08 0.86 1.00 505 3.5 50 3.0 40 2.5 2.0 30 1.5 20 1.0 0.5 10 0.0 0 180 170 8.0 160 150 7.0 140 6.5 130 6.0 120 5.5 110 5.0 100 4.5 f1 (ppm) 90 f1 (ppm) 4.0 80 3.5 70 2.10 2.99 3.05 1.01 1.13 1.99 1.03 2.91 2.01 7.5 3.0 60 2.11 8.5 2.04 9.0 1.00 0.92 506 2.5 2.0 50 40 1.5 30 1.0 20 0.5 10 0.0 0 13.0 12.5 12.0 11.5 11.0 10.5 10.0 170 160 150 140 9.5 130 9.0 120 8.5 8.0 110 7.5 100 7.0 6.5 6.0 f1 (ppm) 90 80 f1 (ppm) 5.5 5.0 70 4.5 60 4.0 3.5 50 2.91 3.06 2.08 3.09 1.04 2.98 1.15 1.12 2.76 1.04 1.00 1.00 507 3.0 40 2.5 2.0 30 1.5 20 1.0 0.5 10 0.0 0 13.0 12.5 12.0 11.5 11.0 10.5 10.0 9.5 180 170 160 150 140 130 9.0 8.5 120 8.0 110 7.5 100 7.0 6.5 6.0 f1 (ppm) 90 f1 (ppm) 80 5.5 5.0 70 4.5 60 4.0 3.5 50 5.98 1.12 2.97 2.05 2.94 1.01 2.09 1.02 1.10 0.99 2.99 0.97 1.00 0.92 508 3.0 40 2.5 2.0 30 1.5 20 1.0 0.5 10 0.0 0 13.0 12.5 12.0 11.5 11.0 10.5 10.0 170 160 150 140 9.5 130 9.0 120 8.5 8.0 110 7.5 100 7.0 6.5 6.0 f1 (ppm) 90 80 f1 (ppm) 5.5 5.0 70 4.5 60 4.0 8.63 2.62 1.92 2.87 1.24 1.00 1.06 1.92 1.17 0.99 1.10 1.21 1.87 0.95 509 3.5 50 3.0 40 2.5 2.0 30 1.5 20 1.0 0.5 10 0.0 0 170 160 150 140 130 9.0 120 8.5 110 100 7.0 6.5 6.0 f1 (ppm) 90 80 f1 (ppm) 5.5 70 5.0 4.5 60 4.0 50 3.5 5.92 3.04 7.5 2.99 2.08 8.0 0.97 1.12 2.93 1.11 1.08 1.89 0.94 0.88 13.0 12.5 12.0 11.5 11.0 10.5 10.0 9.5 1.00 1.01 510 3.0 40 2.5 30 2.0 1.5 20 1.0 10 0.5 0.0 0 10.0 180 9.5 9.0 160 8.5 8.0 140 7.5 7.0 120 2.02 3.10 2.97 3.02 2.00 1.09 1.89 1.12 1.09 2.00 1.08 0.98 511 6.5 6.0 100 5.5 5.0 f1 (ppm) 80 4.5 4.0 3.5 60 3.0 2.5 40 2.0 1.5 20 1.0 0.5 0.0 ppm 11.0 180 10.5 170 10.0 160 9.5 150 9.0 140 8.5 8.0 130 7.5 120 7.0 110 6.5 100 6.0 5.5 f1 (ppm) 90 f1 (ppm) 5.0 80 4.5 70 4.0 60 2.91 1.00 1.06 1.10 1.08 2.82 1.92 0.97 0.97 1.03 11.5 3.01 2.02 2.95 512 3.5 3.0 50 2.5 40 2.0 30 1.5 20 1.0 0.5 10 0.0 0 10.0 180 9.5 170 9.0 160 8.5 150 8.0 7.5 140 7.0 130 6.5 120 6.0 110 5.5 100 5.0 f1 (ppm) 4.5 4.0 90 f1 (ppm) 80 70 3.5 60 3.0 50 2.5 40 2.00 1.96 2.89 2.02 2.89 0.99 1.94 1.03 2.89 1.98 1.00 0.94 0.86 0.98 513 2.0 1.5 30 1.0 20 0.5 10 0.0 0 10.0 9.5 9.0 8.0 2.18 5.94 2.23 6.05 8.5 2.16 1.96 1.97 5.81 7.94 2.11 10.5 2.00 2.04 514 7.5 7.0 6.5 6.0 5.5 5.0 f1 (ppm) 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 515 4.5.4 Crystal Structure Report for C1 A clear colorless rhombic column-like specimen of C54H62F2N8O18S2Zn2, approximate dimensions 0.500 mm x 0.500 mm x 0.500 mm, was used for the X-ray crystallographic analysis. The X-ray intensity data were measured (λ = 0.71073 Å). A total of 2342 frames were collected. The total exposure time was 4.50 h. The frames were integrated with the Bruker SAINT software package using a narrow-frame algorithm. The integration of the data using a triclinic unit cell yielded a total of 73645 reflections to a maximum θ angle of 36.32° (0.60 Å resolution), of which 14172 were independent (average redundancy 5.197, completeness = 99.9%, Rint = 3.20%, Rsig = 2.53%) and 12027 (84.86%) were greater than 2σ(F2). The final cell constants of a = 8.9861(2) Å, b = 10.9663(3) Å, c = 15.6118(4) Å, α = 79.5730(10)°, β = 77.3840(10)°, γ = 80.7630(10)°, volume = 1464.74(6) Å3, are based upon the refinement of the XYZ-centroids of 9565 516 reflections above 20 σ(I) with 4.929° < 2θ < 74.15°. Data were corrected for absorption effects using the Multi-Scan method (SADABS). The ratio of minimum to maximum apparent transmission was 0.868. The calculated minimum and maximum transmission coefficients (based on crystal size) are 0.6410 and 0.6410. The structure was solved and refined using the Bruker SHELXTL Software Package, using the space group P -1, with Z = 1 for the formula unit, C54H62F2N8O18S2Zn2. The final anisotropic full-matrix least-squares refinement on F2 with 398 variables converged at R1 = 2.82%, for the observed data and wR2 = 7.75% for all data. The goodness-of-fit was 1.020. The largest peak in the final difference electron density synthesis was 0.668 e-/Å3 and the largest hole was -0.489 e-/Å3 with an RMS deviation of 0.066 e-/Å3. On the basis of the final model, the calculated density was 1.524 g/cm3 and F(000), 696 e-. Sample and crystal data for 170711REL. Identification code 170711REL Chemical formula C54H62F2N8O18S2Zn2 Formula weight 1343.97 g/mol Temperature 140(2) K Wavelength 0.71073 Å Crystal size 0.500 x 0.500 x 0.500 mm Crystal habit clear colourless rhombic column 517 Crystal system triclinic Space group P -1 Unit cell dimensions a = 8.9861(2) Å α = 79.5730(10)° b = 10.9663(3) Å β = 77.3840(10)° c = 15.6118(4) Å γ = 80.7630(10)° Volume 1464.74(6) Å3 Z 1 Density (calculated) 1.524 g/cm3 Absorption coefficient 0.975 mm-1 F(000) 696 Data collection and structure refinement for 170711REL. Theta range for data 2.16 to 36.32° collection Index ranges -14<=h<=14, -18<=k<=18, -26<=l<=26 Reflections collected 73645 Independent 14172 [R(int) = 0.0320] reflections Coverage of 99.9% independent reflections 518 Absorption correction Multi-Scan Max. and min. 0.6410 and 0.6410 transmission Structure solution direct methods technique Structure solution SHELXT 2014/5 (Sheldrick, 2014) program Refinement method Full-matrix least-squares on F2 Refinement program SHELXL-2017/1 (Sheldrick, 2017) Function minimized Σ w(Fo2 - Fc2)2 Data / restraints / 14172 / 1 / 398 parameters Goodness-of-fit on F2 1.020 Δ/σmax 0.001 12027 data; R1 = 0.0282, wR2 = I>2σ(I) 0.0725 Final R indices R1 = 0.0387, wR2 = all data 0.0775 w=1/[σ2(Fo2)+(0.0399P)2+0.4340P] Weighting scheme where P=(Fo2+2Fc2)/3 Largest diff. peak and 0.668 and -0.489 eÅ-3 519 hole R.M.S. deviation from 0.066 eÅ-3 mean Atomic coordinates and equivalent isotropic atomic displacement parameters (Å2) for 170711REL. U(eq) is defined as one third of the trace of the orthogonalized Uij tensor. x/a y/b z/c U(eq) Zn1 0.49686(2) 0.37422(2) 0.40468(2) 0.01155(3) S1 0.68684(2) 0.57392(2) 0.40706(2) 0.01185(4) F1 0.54414(10) 0.86577(8) 0.01805(6) 0.58788(8) 0.06442(5) 0.03793(19) O1 0.57950(10) 0.02623(17) O2 0.76452(8) 0.24613(7) 0.08396(5) 0.01891(13) O3 0.73373(9) 0.16548(7) 0.43406(6) 0.02193(14) O4 0.67969(8) 0.43817(7) 0.01977(13) 0.40878(5) 520 O5 0.55813(9) 0.64652(7) 0.37012(5) 0.01880(12) O6 0.83624(9) 0.60250(8) 0.35698(5) 0.02197(14) O7 0.67492(9) 0.59526(8) 0.49953(5) 0.02163(14) O8 0.01945(12) 0.75896(10) 0.38454(8) 0.96758(11) 0.27500(8) 0.0386(2) O9 0.11702(13) 0.0429(2) N1 0.49235(9) 0.60754(7) 0.21047(5) 0.01411(12) N2 0.29278(9) 0.50535(7) 0.18137(5) 0.01353(12) N3 0.39439(8) 0.42457(7) 0.29875(5) 0.01220(11) N4 0.50614(9) 0.18926(7) 0.39666(5) 0.01406(12) C1 0.67656(11) 0.73950(9) 0.12257(6) 0.24027(9) 0.36203(6) 0.01665(15) C00S 0.24913(11) 0.01634(15) 521 C2 0.65444(12) 0.85104(10) 0.06679(7) 0.94842(10) 0.05988(8) 0.93296(11) 0.11109(8) 0.82177(12) 0.16683(8) 0.72514(11) 0.17253(7) 0.63759(9) 0.12774(6) 0.51413(8) 0.22911(5) 0.59356(10) 0.10553(7) 0.40149(8) 0.22219(5) 0.36141(9) 0.18758(6) 0.33233(11) 0.10135(6) 0.02116(17) C3 0.73827(14) 0.0270(2) C4 0.85013(14) 0.0275(2) C5 0.87628(14) 0.0280(2) C6 0.79003(13) 0.02356(19) C7 0.58123(11) 0.01666(15) C8 0.39772(10) 0.01227(13) C9 0.25224(13) 0.02112(17) C10 0.21888(10) 0.01310(13) C11 0.09797(10) 0.01368(13) C12 0.12664(11) 0.01923(17) 522 C13 0.01317(11) 0.29422(11) 0.06907(6) 0.28461(9) 0.12230(6) 0.31349(10) 0.20797(6) 0.35192(10) 0.24003(6) 0.24326(13) 0.13324(8) 0.35351(8) 0.29486(5) 0.14803(9) 0.37175(6) 0.02611(10) 0.35705(8) 0.94602(10) 0.36689(9) 0.98713(10) 0.39226(8) 0.11067(9) 0.40731(6) 0.01998(17) C14 0.86790(10) 0.01468(14) C15 0.83748(10) 0.01822(16) C16 0.95281(10) 0.01688(15) C17 0.61179(12) 0.0277(2) C18 0.28212(10) 0.01276(13) C19 0.39028(11) 0.01612(15) C20 0.40175(15) 0.0255(2) C21 0.53505(17) 0.0316(2) C22 0.65396(15) 0.0269(2) C23 0.63309(11) 0.01745(15) 523 C24 0.87715(13) 0.09366(12) 0.44607(10) 0.0309(2) C25 0.1341(2) 0.66671(19) 0.41604(15) 0.0524(4) C26 0.2207(2) 0.9516(2) 0.19542(12) 0.0589(6) Bond lengths (Å) for 170711REL. Zn1-O4 1.9046(7) Zn1-O7 1.9334(7) Zn1-N3 2.0147(7) Zn1-N4 2.0419(7) S1-O6 1.4491(7) S1-O5 1.4607(8) S1-O7 1.4823(7) S1-O4 1.4954(7) F1-C2 1.3495(12) O1-C7 1.2161(11) O2-C14 1.3636(10) O2-C17 1.4214(13) O3-C23 1.3356(12) O3-C24 1.4292(14) O8-C25 1.426(2) O8-H8 0.84 O9-C26 1.401(2) O9-H9 0.84 N1-C7 1.3764(11) N1-C8 1.3860(11) N1-H1 0.951(8) N2-C10 1.3927(11) N2-C9 1.4598(12) N3-C8 1.3253(11) N3-C18 1.3875(10) N4-C23 1.3369(13) N4-C19 1.3532(11) N2-C8 1.3477(11) 524 C1-C2 1.3806(14) C1-C6 1.3871(14) C1-C7 1.4946(12) C00S-C18 1.4969(12) C00S-C19 1.5072(14) C00S-H00A 0.99 C00S-H00B 0.99 C3-C4 1.3876(17) C3-H3 0.95 C4-C5 1.3830(18) C4-H4 0.95 C5-C6 1.3896(15) C5-H5 0.95 C6-H6 0.95 C9-H9A 0.98 C9-H9B 0.98 C9-H9C 0.98 C10-C18 1.3625(12) C10-C11 1.4702(11) C11-C16 1.3889(12) C11-C12 1.3993(12) C12-C13 1.3805(12) C12-H12 0.95 C13-C14 1.3944(13) C13-H13 0.95 C14-C15 1.3891(12) C15-C16 1.3948(12) C15-H15 0.95 C16-H16 0.95 C17-H17A 0.98 C17-H17B 0.98 C17-H17C 0.98 C19-C20 1.3811(14) C20-C21 1.3869(19) C20-H20 0.95 C21-C22 1.3806(18) C21-H21 0.95 C22-C23 1.3929(14) C22-H22 0.95 C24-H24A 0.98 C24-H24B 0.98 C24-H24C 0.98 C25-H25A 0.98 C25-H25B 0.98 C25-H25C 0.98 C2-C3 1.3786(14) 525 C26-H26A 0.98 C26-H26C 0.98 C26-H26B 0.98 Bond angles (°) for 170711REL. O4-Zn1-O7 115.45(3) O4-Zn1-N3 121.40(3) O7-Zn1-N3 101.06(3) O4-Zn1-N4 119.28(3) O7-Zn1-N4 104.30(3) N3-Zn1-N4 91.30(3) O6-S1-O5 113.85(5) O6-S1-O7 108.00(4) O5-S1-O7 110.18(4) O6-S1-O4 107.92(5) O5-S1-O4 108.63(4) O7-S1-O4 108.09(5) C14-O2-C17 117.45(7) C23-O3-C24 118.00(9) S1-O4-Zn1 123.03(4) S1-O7-Zn1 133.37(5) C25-O8-H8 109.5 109.5 C7-N1-C8 122.37(7) C7-N1-H1 120.7(9) C8-N1-H1 114.9(9) C8-N2-C10 107.45(7) C8-N2-C9 127.05(8) C10-N2-C9 125.29(7) C8-N3-C18 106.12(7) C8-N3-Zn1 137.72(6) C18-N3-Zn1 116.08(6) C23-N4-C19 119.43(8) C23-N4-Zn1 120.12(6) C19-N4-Zn1 120.11(6) C2-C1-C6 118.02(9) C2-C1-C7 C6-C1-C7 121.52(9) C18-C00S-C19 113.21(7) C18-C00S-H00A 108.9 108.9 C26-O9-H9 120.46(9) C19-C00S-H00A 526 C18-C00S-H00B 108.9 C19-C00S-H00B 108.9 H00A-C00S-H00B 107.8 F1-C2-C3 118.74(10) F1-C2-C1 118.36(9) C3-C2-C1 122.89(10) C2-C3-C4 118.32(11) C2-C3-H3 120.8 C4-C3-H3 120.8 C5-C4-C3 120.17(10) C5-C4-H4 119.9 C3-C4-H4 119.9 C4-C5-C6 120.31(10) C4-C5-H5 119.8 C6-C5-H5 119.8 C1-C6-C5 120.29(10) C1-C6-H6 119.9 C5-C6-H6 119.9 O1-C7-N1 123.11(8) O1-C7-C1 123.27(8) N1-C7-C1 113.59(7) N3-C8-N2 111.14(7) N3-C8-N1 124.10(7) N2-C8-N1 124.71(8) N2-C9-H9A 109.5 N2-C9-H9B 109.5 H9A-C9-H9B 109.5 N2-C9-H9C 109.5 H9A-C9-H9C 109.5 H9B-C9-H9C 109.5 C18-C10-N2 105.82(7) C18-C10-C11 131.32(8) N2-C10-C11 122.86(8) C16-C11-C12 118.76(8) C16-C11-C10 120.47(7) C12-C11-C10 120.77(8) C13-C12-C11 120.70(8) C13-C12-H12 119.6 C11-C12-H12 119.6 C12-C13-C14 120.14(8) C12-C13-H13 119.9 C14-C13-H13 119.9 O2-C14-C15 124.80(8) O2-C14-C13 115.35(7) C15-C14-C13 119.85(8) C14-C15-C16 119.62(8) 527 C14-C15-H15 120.2 C16-C15-H15 120.2 C11-C16-C15 120.92(8) C11-C16-H16 119.5 C15-C16-H16 119.5 O2-C17-H17A109.5 O2-C17-H17B 109.5 H17A-C17-H17B 109.5 O2-C17-H17C 109.5 H17A-C17-H17C 109.5 H17B-C17-H17C 109.5 C10-C18-N3 109.46(7) C10-C18-C00S 129.17(7) N3-C18-C00S 121.33(7) N4-C19-C20 120.86(10) N4-C19-C00S 117.09(8) C20-C19-C00S 122.05(9) C19-C20-C21 119.17(10) C19-C20-H20 120.4 C22-C21-C20 120.43(10) C22-C21-H21 119.8 C20-C21-H21 119.8 C21-C22-C23 117.14(11) C21-C22-H22 121.4 C23-C22-H22 121.4 O3-C23-N4 111.62(8) O3-C23-C22 125.43(10) N4-C23-C22 122.94(9) O3-C24-H24A109.5 O3-C24-H24B 109.5 H24A-C24-H24B 109.5 O3-C24-H24C 109.5 H24A-C24-H24C 109.5 H24B-C24-H24C 109.5 O8-C25-H25A109.5 O8-C25-H25B 109.5 H25A-C25-H25B 109.5 O8-C25-H25C 109.5 H25A-C25-H25C 109.5 H25B-C25-H25C 109.5 O9-C26-H26A109.5 O9-C26-H26B 109.5 H26A-C26-H26B 109.5 O9-C26-H26C 109.5 H26A-C26-H26C 109.5 C21-C20-H20 120.4 528 H26B-C26-H26C 109.5 Torsion angles (°) for 170711REL. O6-S1-O4-Zn1 -144.35(5) O5-S1-O4-Zn1 -20.47(7) O7-S1-O4-Zn1 99.07(6) O6-S1-O7-Zn1 158.98(7) O5-S1-O7-Zn1 34.06(9) O4-S1-O7-Zn1 -84.50(8) C6-C1-C2-F1 -179.92(10) C6-C1-C2-C3 -1.01(16) C7-C1-C2-C3 179.52(10) F1-C2-C3-C4 179.21(11) C1-C2-C3-C4 0.30(18) C2-C3-C4-C5 0.47(19) C3-C4-C5-C6 -0.50(19) C2-C1-C6-C5 0.95(16) C7-C1-C6-C5 -179.58(10) C4-C5-C6-C1 -0.22(18) C8-N1-C7-O1 2.62(16) C8-N1-C7-C1 -179.01(8) C2-C1-C7-O1 60.74(15) C6-C1-C7-O1 -118.71(12) C6-C1-C7-N1 62.92(13) C18-N3-C8-N2 0.70(10) Zn1-N3-C8-N2 -175.69(7) C18-N3-C8-N1 178.04(8) Zn1-N3-C8-N1 1.65(14) -1.21(10) C9-N2-C8-N3 173.66(9) C10-N2-C8-N1 C9-N2-C8-N1 -3.66(14) C7-N1-C8-N3 131.14(10) C7-N1-C8-N2 -51.88(13) C8-N2-C10-C18 1.21(10) C9-N2-C10-C18 -173.78(9) C8-N2-C10-C11 -179.05(8) C9-N2-C10-C11 5.96(14) 57.64(14) C7-C1-C2-F1 0.61(15) C2-C1-C7-N1 -117.63(10) C10-N2-C8-N3 C18-C10-C11-C16 -178.54(8) 529 N2-C10-C11-C16 -122.03(10) C18-C10-C11-C12 - 122.47(11) N2-C10-C11-C12 57.86(13) C16-C11-C12-C13 -0.37(16) C10-C11-C12-C13 179.73(10) C11-C12-C13-C14 0.19(17) C17-O2-C14-C15 4.32(15) -175.70(10) C12-C13-C14-O2 -179.98(10) C17-O2-C14-C13 C12-C13-C14-C15 0.00(16) O2-C14-C15-C16 179.98(10) C13-C14-C15-C16 0.00(16) C12-C11-C16-C15 0.37(15) -179.73(9) C14-C15-C16-C11 -0.19(16) N2-C10-C18-N3 -0.81(10) C11-C10-C18-N3 179.48(9) N2-C10-C18-C00S -178.55(9) C11-C10-C18-C00S 1.74(16) 0.10(10) Zn1-N3-C18-C10 177.40(6) C8-N3-C18-C00S Zn1-N3-C18-C00S -4.66(11) C19-C00S-C18-C10 124.35(10) C19-C00S-C18-N3 -53.15(11) C23-N4-C19-C20 -0.14(14) Zn1-N4-C19-C20 173.22(8) C23-N4-C19-C00S 179.16(8) Zn1-N4-C19-C00S -7.48(11) C18-C00S-C19-N4 59.43(10) C18-C00S-C19-C20 -121.28(10) C10-C11-C16-C15 C8-N3-C18-C10 178.05(8) N4-C19-C20-C21 - 0.92(16) C00S-C19-C20-C21 179.82(10) C19-C20-C21-C22 0.89(19) C20-C21-C22-C23 0.14(19) C24-O3-C23-N4 -178.41(10) C24-O3-C23-C22 1.91(16) C19-N4-C23-O3 -178.43(8) Zn1-N4-C23-O3 8.20(11) C19-N4-C23-C22 1.26(15) 530 Zn1-N4-C23-C22 -172.10(8) C21-C22-C23-O3 C21-C22-C23-N4 -1.25(17) 178.40(11) Anisotropic atomic displacement parameters (Å2) for 170711REL. The anisotropic atomic displacement factor exponent takes the form: -2π2[ h2 a*2 U11 + ... + 2 h k a* b* U12 ] U11 U22 U33 U23 U13 U12 Zn1 0.01326(4) 0.01171(4) 0.00281(3) -0.00374(3) S1 0.01278(8) 0.01316(8) 0.00347(6) -0.00165(6) F1 0.0415(4) 0.0264(4) 0.0542(5) 0.0026(3) -0.0336(4) -0.0052(3) O1 0.0308(4) 0.0339(4) 0.0174(3) - 0.0105(3) 0.0032(3) -0.0173(3) O2 0.0147(3) 0.0267(3) 0.0191(3) - 0.0083(3) -0.0049(2) -0.0059(2) O3 0.0174(3) 0.0160(3) 0.0338(4) - 0.0031(3) -0.0106(3) 0.0008(2) O4 0.0151(3) 0.0126(3) 0.0340(4) - 0.0059(3) -0.0075(3) -0.0021(2) 0.01110(4) - -0.00263(3) 0.01083(8) - -0.00429(6) 531 O5 0.0216(3) 0.0157(3) 0.0209(3) - 0.0036(2) -0.0096(2) 0.0008(2) O6 0.0183(3) 0.0315(4) 0.0171(3) - 0.0036(3) 0.0017(2) -0.0133(3) O7 0.0180(3) 0.0362(4) 0.0131(3) - 0.0109(3) -0.0024(2) -0.0034(3) O8 0.0321(5) 0.0306(5) 0.0600(7) - 0.0042(4) -0.0232(5) -0.0080(4) O9 0.0389(5) 0.0433(6) 0.0474(6) - 0.0108(5) -0.0021(5) -0.0119(5) N1 0.0169(3) 0.0148(3) 0.0118(3) - 0.0015(2) -0.0025(2) -0.0066(2) N2 0.0143(3) 0.0150(3) 0.0122(3) - 0.0005(2) -0.0048(2) -0.0034(2) N3 0.0131(3) 0.0139(3) 0.0107(3) - 0.0014(2) -0.0033(2) -0.0042(2) N4 0.0173(3) 0.0114(3) 0.0143(3) - 0.0014(2) -0.0042(2) -0.0034(2) C1 0.0180(4) 0.0186(4) 0.0143(3) - 0.0018(3) -0.0017(3) -0.0080(3) C00S 0.0169(4) 0.0192(4) 0.0143(3) 0.0014(3) -0.0046(3) -0.0086(3) 532 C2 0.0215(4) 0.0184(4) 0.0258(4) - 0.0023(3) -0.0090(3) -0.0043(3) C3 0.0311(5) 0.0165(4) 0.0350(6) - 0.0007(4) -0.0090(4) -0.0076(4) C4 0.0275(5) 0.0243(5) 0.0343(5) - 0.0065(4) -0.0050(4) -0.0130(4) C5 0.0278(5) 0.0362(6) 0.0256(5) - 0.0024(4) -0.0101(4) -0.0161(5) C6 0.0266(5) 0.0292(5) 0.0174(4) 0.0030(3) -0.0078(3) -0.0139(4) C7 0.0180(4) 0.0184(4) 0.0143(3) - 0.0025(3) -0.0015(3) -0.0069(3) C8 0.0134(3) 0.0131(3) 0.0111(3) - 0.0022(2) -0.0027(2) -0.0034(3) C9 0.0250(4) 0.0198(4) 0.0199(4) 0.0045(3) -0.0120(3) -0.0047(3) C10 0.0119(3) 0.0163(3) 0.0120(3) - 0.0021(3) -0.0028(2) -0.0038(3) C11 0.0120(3) 0.0178(4) 0.0125(3) - 0.0032(3) -0.0034(2) -0.0032(3) C12 0.0141(3) 0.0324(5) 0.0128(3) - 0.0066(3) -0.0004(3) -0.0074(3) 533 C13 0.0162(4) 0.0334(5) 0.0130(3) - 0.0082(3) -0.0017(3) -0.0072(3) C14 0.0132(3) 0.0179(4) 0.0149(3) - 0.0039(3) -0.0047(3) -0.0032(3) C15 0.0125(3) 0.0284(5) 0.0157(3) - 0.0077(3) -0.0007(3) -0.0062(3) C16 0.0134(3) 0.0255(4) 0.0136(3) - 0.0076(3) -0.0016(3) -0.0041(3) C17 0.0147(4) 0.0404(6) 0.0336(5) - 0.0192(5) -0.0030(4) -0.0071(4) C18 0.0125(3) 0.0154(3) 0.0113(3) - 0.0016(3) -0.0026(2) -0.0048(3) C19 0.0219(4) 0.0141(3) 0.0139(3) 0.0002(3) -0.0051(3) -0.0073(3) C20 0.0377(6) 0.0161(4) 0.0274(5) - 0.0027(3) -0.0118(4) -0.0108(4) C21 0.0469(7) 0.0124(4) 0.0390(6) - 0.0070(4) -0.0132(5) -0.0041(4) C22 0.0334(6) 0.0123(4) 0.0348(5) - 0.0048(4) -0.0089(4) 0.0024(4) C23 0.0195(4) 0.0124(3) 0.0201(4) - 0.0017(3) -0.0043(3) -0.0012(3) 534 C24 0.0165(4) 0.0264(5) 0.0021(5) -0.0091(4) 0.0028(4) C25 0.0368(8) 0.0554(10) 0.0248(9) -0.0300(8) 0.0126(7) C26 0.0528(10) 0.0952(16) 0.0187(9) -0.0037(7) -0.0373(11) 0.0479(7) - 0.0735(12) - 0.0392(8) - Hydrogen atomic coordinates and isotropic atomic displacement parameters (Å2) for 170711REL. x/a y/b z/c U(eq) H8 -0.0508 0.7238 0.3755 0.058 H9 0.0954 0.8975 0.3026 0.064 H1 0.5131(16) 0.6333(14) H00A 0.1733 0.1982 0.3445 0.02 H00B 0.2023 0.2670 0.4203 0.02 H3 0.7199 1.0242 0.0210 0.032 H4 0.9089 0.9989 0.1078 0.033 H5 0.9536 0.8114 0.2014 0.034 H6 0.8089 0.6489 0.2108 0.028 H9A 0.3096 0.5647 0.0503 0.032 H9B 0.2782 0.6761 0.1081 0.032 H9C 0.1417 0.5991 0.1073 0.032 0.2612(7) 0.017 535 H12 0.2252 0.3389 0.0646 0.023 H13 0.0341 0.2745 0.0105 0.024 H15 -0.2613 0.3071 0.2445 0.022 H16 -0.0682 0.3719 0.2986 0.02 H17A -0.4296 0.3261 0.1494 0.042 H17B -0.3879 0.1818 0.1873 0.042 H17C -0.4524 0.2199 0.0970 0.042 H20 0.3194 -0.0025 0.3404 0.031 H21 0.5446 -0.1377 0.3561 0.038 H22 0.7461 -0.0665 0.3992 0.032 H24A 0.9342 0.0703 0.3891 0.046 H24B 0.8581 0.0180 0.4889 0.046 H24C 0.9374 0.1435 0.4684 0.046 H25A 0.1605 0.6008 0.3784 0.079 H25B 0.0945 0.6304 0.4774 0.079 H25C 0.2260 0.7055 0.4140 0.079 H26A 0.1705 0.9183 0.1564 0.088 H26B 0.3105 0.8930 0.2082 0.088 H26C 0.2537 1.0324 0.1660 0.088 536 4.5.5 Crystal Structure Report for C2 A translucent colorless Plate-like specimen of C25H23Cl2FN4O3Zn, approximate dimensions 0.100 mm x 0.250 mm x 0.500 mm, was used for the X-ray crystallographic analysis. The X-ray intensity data were measured (λ = 0.71073 Å). A total of 3676 frames were collected. The total exposure time was 5.11 h. The frames were integrated with the Bruker SAINT software package using a narrow-frame algorithm. The integration of the data using a triclinic unit cell yielded a total of 49898 reflections to a maximum θ angle of 27.10° (0.78 Å resolution), of which 10993 were independent (average redundancy 4.539, completeness = 99.7%, Rint = 4.83%, Rsig = 4.00%) and 537 8641 (78.60%) were greater than 2σ(F2). The final cell constants of a = 10.3998(5) Å, b = 14.3653(8) Å, c = 18.4744(9) Å, α = 68.023(2)°, β = 86.901(3)°, γ = 77.273(3)°, volume = 2495.3(2) Å3, are based upon the refinement of the XYZ-centroids of 9983 reflections above 20 σ(I) with 4.607° < 2θ < 56.11°. Data were corrected for absorption effects using the Multi-Scan method (SADABS). The ratio of minimum to maximum apparent transmission was 0.834. The calculated minimum and maximum transmission coefficients (based on crystal size) are 0.5760 and 0.8860. The structure was solved and refined using the Bruker SHELXTL Software Package, using the space group P -1, with Z = 4 for the formula unit, C25H23Cl2FN4O3Zn. The final anisotropic full-matrix least-squares refinement on F2 with 672 variables converged at R1 = 4.79%, for the observed data and wR2 = 13.77% for all data. The goodness-of-fit was 1.098. The largest peak in the final difference electron density synthesis was 1.348 e-/Å3 and the largest hole was -0.872 e-/Å3 with an RMS deviation of 0.105 e-/Å3. On the basis of the final model, the calculated density was 1.551 g/cm3 and F(000), 1192 e-. Sample and crystal data for 170720REL. Identification code 170720REL Chemical formula C25H23Cl2FN4O3Zn Formula weight 582.74 g/mol Temperature 150(2) K 538 Wavelength 0.71073 Å Crystal size 0.100 x 0.250 x 0.500 mm Crystal habit translucent colourless Plate Crystal system triclinic Space group P -1 Unit cell dimensions a = 10.3998(5) Å α = 68.023(2)° b = 14.3653(8) Å β = 86.901(3)° c = 18.4744(9) Å γ = 77.273(3)° Volume 2495.3(2) Å3 Z 4 Density (calculated) 1.551 g/cm3 Absorption coefficient 1.242 mm-1 F(000) 1192 Data collection and structure refinement for 170720REL. Theta range for data 2.01 to 27.10° collection Index ranges -13<=h<=13, -18<=k<=18, -23<=l<=23 Reflections collected 49898 Independent reflections 10993 [R(int) = 0.0483] 539 Coverage of 99.7% independent reflections Absorption correction Multi-Scan Max. and min. 0.8860 and 0.5760 transmission Structure solution direct methods technique Structure solution SHELXT 2014/5 (Sheldrick, 2014) program Refinement method Full-matrix least-squares on F2 Refinement program SHELXL-2017/1 (Sheldrick, 2017) Function minimized Σ w(Fo2 - Fc2)2 Data / restraints / 10993 / 609 / 672 parameters Goodness-of-fit on F2 1.098 Δ/σmax 0.001 8641 data; R1 = 0.0479, wR2 = I>2σ(I) 0.1252 Final R indices R1 = 0.0675, wR2 = all data 0.1377 Weighting scheme w=1/[σ2(Fo2)+(0.0779P)2+1.7098P] 540 where P=(Fo2+2Fc2)/3 Largest diff. peak and 1.348 and -0.872 eÅ-3 hole R.M.S. deviation from 0.105 eÅ-3 mean Atomic coordinates and equivalent isotropic atomic displacement parameters (Å2) for 170720REL. U(eq) is defined as one third of the trace of the orthogonalized Uij tensor. x/a y/b z/c U(eq) Zn01 0.76520(4) 0.74753(3) 0.07568(2) 0.02040(10) Zn02 0.23605(4) 0.83882(3) 0.67823(2) 0.02036(10) Cl1 0.88563(8) 0.85741(6) 0.00287(5) 0.02831(18) Cl2 0.79161(9) 0.70361(6) 0.20472(5) 0.02821(18) Cl3 0.09328(9) 0.78148(7) 0.62986(5) 0.03112(19) Cl4 0.40565(9) 0.71828(7) 0.74828(5) 0.0320(2) F1 0.91750(19) 0.47353(17) 0.33304(11) 0.0330(5) O1 0.0986(2) 0.38266(18) 0.16051(13) 0.0256(5) O2 0.6499(3) 0.3113(2) 0.81128(15) 0.0359(6) O3 0.5194(2) 0.88415(19) 0.09373(14) 0.0287(5) O4 0.3239(2) 0.9731(2) 0.36786(13) 0.0298(5) O5 0.6286(2) 0.41284(19) 0.51659(14) 0.0292(5) 541 O6 0.9990(2) 0.88110(18) 0.76435(14) 0.0260(5) N1 0.9178(3) 0.4915(2) 0.17966(15) 0.0218(5) N2 0.8344(3) 0.44624(19) 0.08039(15) 0.0192(5) N3 0.8002(3) 0.6103(2) 0.06265(15) 0.0200(5) N4 0.5806(3) 0.7933(2) 0.01855(15) 0.0206(5) N5 0.2010(3) 0.9519(2) 0.47627(15) 0.0222(6) N6 0.3044(3) 0.0867(2) 0.48086(15) 0.0209(5) N7 0.2970(3) 0.9535(2) 0.58909(15) 0.0206(5) N8 0.1842(3) 0.93822(19) 0.73764(15) 0.0189(5) C1 0.1111(3) 0.4345(2) 0.26701(17) 0.0194(6) C2 0.0503(3) 0.4574(3) 0.32876(19) 0.0240(7) C3 0.1199(4) 0.4627(3) 0.3877(2) 0.0317(8) C4 0.2558(4) 0.4472(3) 0.3847(2) 0.0311(8) C5 0.3200(3) 0.4242(3) 0.32399(19) 0.0268(7) C6 0.2480(3) 0.4172(2) 0.26653(18) 0.0225(6) C7 0.0436(3) 0.4315(2) 0.19864(18) 0.0208(6) C8 0.8543(3) 0.5152(2) 0.10881(18) 0.0195(6) C9 0.8653(3) 0.3350(2) 0.12100(19) 0.0251(7) C10 0.7623(3) 0.5015(2) 0.00963(17) 0.0191(6) C11 0.7296(3) 0.4519(2) 0.95858(17) 0.0191(6) C12 0.8247(3) 0.3777(2) 0.94171(19) 0.0227(6) C13 0.7951(4) 0.3333(3) 0.8927(2) 0.0279(7) C14 0.6692(3) 0.3610(3) 0.85890(19) 0.0252(7) 542 C15 0.5734(3) 0.4344(3) 0.87444(18) 0.0251(7) C16 0.6046(3) 0.4785(2) 0.92475(18) 0.0216(6) C17 0.5262(4) 0.3444(3) 0.7703(2) 0.0410(10) C18 0.7417(3) 0.6014(2) 0.00090(17) 0.0193(6) C19 0.6751(3) 0.6973(2) 0.93588(18) 0.0221(6) C20 0.5585(3) 0.7597(2) 0.96168(17) 0.0200(6) C21 0.4347(3) 0.7837(2) 0.9275(2) 0.0262(7) C22 0.3322(3) 0.8441(3) 0.9514(2) 0.0290(7) C23 0.3538(3) 0.8816(3) 0.0072(2) 0.0267(7) C24 0.4811(3) 0.8537(2) 0.03945(19) 0.0233(6) C25 0.4192(4) 0.9418(3) 0.1265(2) 0.0359(9) C26 0.1614(3) 0.8729(2) 0.38699(18) 0.0222(6) C27 0.0375(3) 0.8585(3) 0.4128(2) 0.0291(7) F2A 0.9702(3) 0.9109(2) 0.45218(18) 0.0359(9) C28 0.9745(4) 0.7950(3) 0.3943(2) 0.0373(9) C29 0.0362(4) 0.7438(3) 0.3480(2) 0.0413(9) C30 0.1590(4) 0.7565(3) 0.3203(2) 0.0379(9) C31 0.2197(3) 0.8210(3) 0.3390(2) 0.0302(8) F2B 0.3229(8) 0.8427(6) 0.3019(4) 0.037(2) C32 0.2374(3) 0.9371(2) 0.40777(18) 0.0221(6) C33 0.2697(3) 0.9955(2) 0.51331(18) 0.0208(6) C34 0.2684(3) 0.1621(3) 0.40162(18) 0.0264(7) C35 0.3579(3) 0.1049(2) 0.54102(18) 0.0198(6) 543 C36 0.4174(3) 0.1926(2) 0.52889(18) 0.0210(6) C37 0.5058(3) 0.2208(2) 0.46926(18) 0.0204(6) C38 0.5770(3) 0.2946(2) 0.46239(18) 0.0221(6) C39 0.5593(3) 0.3431(2) 0.51555(19) 0.0225(6) C40 0.4659(3) 0.3202(3) 0.57280(18) 0.0239(7) C41 0.3964(3) 0.2457(3) 0.57982(18) 0.0231(6) C42 0.7324(4) 0.4292(3) 0.4627(2) 0.0344(8) C43 0.3511(3) 0.0215(2) 0.60748(18) 0.0202(6) C44 0.3869(3) 0.9965(2) 0.69112(18) 0.0217(6) C45 0.2650(3) 0.0002(2) 0.73924(17) 0.0198(6) C46 0.2343(3) 0.0633(2) 0.78067(18) 0.0235(7) C47 0.1182(3) 0.0653(2) 0.82092(19) 0.0256(7) C48 0.0335(3) 0.0050(2) 0.81782(18) 0.0240(7) C49 0.0703(3) 0.9433(2) 0.77482(17) 0.0202(6) C50 0.8694(3) 0.8832(3) 0.7946(2) 0.0278(7) Bond lengths (Å) for 170720REL. Zn01-N3 2.026(3) Zn01-N4 2.092(3) Zn01-Cl1 2.2292(9) Zn01-Cl2 2.2416(9) Zn02-N7 2.036(3) Zn02-N8 2.075(3) Zn02-Cl3 2.2169(9) Zn02-Cl4 2.2379(9) F1-C2 1.352(4) O1-C7 1.210(4) 544 O2-C14 1.369(4) O2-C17 1.422(5) O3-C24 1.343(4) O3-C25 1.437(4) O4-C32 1.212(4) O5-C39 1.363(4) O5-C42 1.426(4) O6-C49 1.350(4) O6-C50 1.429(4) N1-C7 1.381(4) N1-C8 1.388(4) N1-H1N 0.984(10) N2-C8 1.339(4) N2-C10 1.403(4) N2-C9 1.457(4) N3-C8 1.323(4) N3-C18 1.381(4) N4-C24 1.335(4) N4-C20 1.358(4) N5-C32 1.379(4) N5-C33 1.394(4) N5-H5N 0.983(10) N6-C33 1.343(4) N6-C35 1.401(4) N6-C34 1.466(4) N7-C33 1.319(4) N7-C43 1.377(4) N8-C49 1.339(4) N8-C45 1.360(4) C1-C2 1.386(4) C1-C6 1.391(5) C1-C7 1.496(4) C2-C3 1.374(5) C3-C4 1.383(5) C3-H3 0.95 C4-C5 1.383(5) C4-H4 0.95 C5-C6 1.377(5) C5-H5 0.95 C6-H6 0.95 C9-H9A 0.98 C9-H9B 0.98 C9-H9C 0.98 C10-C18 1.352(4) C10-C11 1.469(4) C11-C16 1.384(4) 545 C11-C12 1.401(4) C12-C13 C12-H12 0.95 C13-C14 1.391(5) C13-H13 0.95 C14-C15 1.385(5) C15-C16 1.392(5) C16-H16 0.95 C17-H17A 0.98 C17-H17B 0.98 C17-H17C 0.98 C18-C19 1.500(4) C19-H19A 0.99 C20-C21 1.381(4) C21-H21 0.95 C22-C23 1.377(5) C22-H22 0.95 C23-C24 1.393(5) C23-H23 0.95 C25-H25A 0.98 C25-H25B 0.98 C25-H25C 0.98 C26-C27 1.382(5) C26-C31 1.395(5) C26-C32 1.498(5) C27-F2A 1.307(4) C27-C28 1.377(5) C27-H27 0.9502(10) C28-C29 1.375(6) C28-H28 0.95 C29-C30 1.375(6) C29-H29 0.95 C30-C31 1.378(5) C30-H30 0.95 C31-F2B 1.275(8) C31-H31 0.9501(11) C34-H34A 0.98 C34-H34B 0.98 C34-H34C 0.98 C35-C43 1.369(4) C35-C36 1.462(4) C15-H15 C19-C20 C19-H19B 1.368(5) 0.95 1.510(4) 0.99 C21-C22 C36-C37 1.383(5) 1.394(4) 546 C36-C41 1.398(5) C37-C38 C37-H37 0.95 C38-C39 1.385(5) C38-H38 0.95 C39-C40 1.393(5) C40-C41 1.383(5) C41-H41 0.95 C42-H42A 0.98 C42-H42B 0.98 C42-H42C 0.98 C43-C44 1.498(4) C44-H44A 0.99 C45-C46 1.369(4) C46-H46 0.95 C47-C48 1.382(5) C47-H47 0.95 C48-C49 1.382(4) C48-H48 0.95 C50-H50A 0.98 C50-H50B 0.98 C50-H50C 0.98 C40-H40 C44-C45 C44-H44B 1.387(4) 0.95 1.512(4) 0.99 C46-C47 1.383(5) Bond angles (°) for 170720REL. N3-Zn01-N4 91.78(10) N3-Zn01-Cl1 115.45(8) N4-Zn01-Cl1 106.43(8) N3-Zn01-Cl2 103.48(8) N4-Zn01-Cl2 123.37(8) Cl1-Zn01-Cl2 114.47(3) N7-Zn02-N8 88.78(10) N7-Zn02-Cl3 108.36(8) N8-Zn02-Cl3 124.49(8) N7-Zn02-Cl4 112.13(8) N8-Zn02-Cl4 104.91(7) Cl3-Zn02-Cl4 115.19(4) C14-O2-C17 117.0(3) C24-O3-C25 117.6(3) 547 C39-O5-C42 116.3(3) C49-O6-C50 118.7(3) C7-N1-C8 122.5(3) C7-N1-H1N C8-N1-H1N 116.(2) C8-N2-C10 C8-N2-C9 125.9(3) C10-N2-C9 C8-N3-C18 105.7(3) C8-N3-Zn01 133.4(2) C18-N3-Zn01 120.0(2) C24-N4-C20 118.9(3) C24-N4-Zn01 120.2(2) C20-N4-Zn01 120.9(2) C32-N5-C33 124.7(3) C32-N5-H5N 118.(2) 116.(2) 107.0(2) 126.6(3) C33-N5-H5N 111.(2) C33-N6-C35 107.0(3) C33-N6-C34 126.2(3) C35-N6-C34 125.7(3) C33-N7-C43 106.5(3) C33-N7-Zn02 133.3(2) C43-N7-Zn02 118.0(2) C49-N8-C45 118.5(3) C49-N8-Zn02 120.8(2) C45-N8-Zn02 120.7(2) C2-C1-C6 116.9(3) C2-C1-C7 126.4(3) C6-C1-C7 116.6(3) F1-C2-C3 117.7(3) F1-C2-C1 119.7(3) C3-C2-C1 122.6(3) C2-C3-C4 119.0(3) C2-C3-H3 120.5 C4-C3-H3 120.5 C5-C4-C3 120.0(3) C5-C4-H4 120.0 C3-C4-H4 120.0 C6-C5-C4 119.7(3) C4-C5-H5 120.1 C5-C6-C1 121.7(3) C5-C6-H6 119.2 C1-C6-H6 119.2 O1-C7-N1 122.3(3) C6-C5-H5 O1-C7-C1 120.1 121.7(3) 548 N1-C7-C1 115.9(3) N3-C8-N2 111.7(3) N3-C8-N1 123.2(3) N2-C8-N1 125.1(3) N2-C9-H9A 109.5 N2-C9-H9B 109.5 H9A-C9-H9B 109.5 N2-C9-H9C 109.5 H9A-C9-H9C 109.5 H9B-C9-H9C 109.5 C18-C10-N2 105.5(3) C18-C10-C11 131.7(3) N2-C10-C11 122.7(3) C16-C11-C12 118.2(3) C16-C11-C10 120.9(3) C12-C11-C10 120.8(3) C13-C12-C11 120.9(3) C13-C12-H12 119.6 C11-C12-H12 119.6 C12-C13-C14 120.3(3) C12-C13-H13 119.8 C14-C13-H13 119.8 O2-C14-C15 124.5(3) O2-C14-C13 115.5(3) C15-C14-C13 120.0(3) C14-C15-C16 119.1(3) C14-C15-H15 120.4 C16-C15-H15 120.4 C11-C16-C15 121.5(3) C11-C16-H16 119.3 C15-C16-H16 119.3 O2-C17-H17A109.5 O2-C17-H17B 109.5 H17A-C17-H17B 109.5 O2-C17-H17C 109.5 H17A-C17-H17C 109.5 H17B-C17-H17C 109.5 C10-C18-N3 110.1(3) C10-C18-C19 130.8(3) N3-C18-C19 118.9(3) C18-C19-C20 113.4(3) C18-C19-H19A 108.9 C20-C19-H19A 108.9 C18-C19-H19B 108.9 C20-C19-H19B 108.9 H19A-C19-H19B 107.7 549 N4-C20-C21 121.2(3) N4-C20-C19 117.5(3) C21-C20-C19 121.2(3) C20-C21-C22 118.9(3) C20-C21-H21 120.5 C22-C21-H21 120.5 C23-C22-C21 120.6(3) C23-C22-H22 119.7 C21-C22-H22 119.7 C22-C23-C24 117.3(3) C22-C23-H23 121.4 C24-C23-H23 121.4 N4-C24-O3 112.0(3) O3-C24-C23 125.0(3) N4-C24-C23 123.0(3) O3-C25-H25A109.5 O3-C25-H25B 109.5 H25A-C25-H25B 109.5 O3-C25-H25C 109.5 H25A-C25-H25C 109.5 H25B-C25-H25C 109.5 C27-C26-C31 116.3(3) C27-C26-C32 125.3(3) C31-C26-C32 118.4(3) F2A-C27-C28 116.5(3) F2A-C27-C26 120.7(3) C28-C27-C26 122.7(3) C28-C27-H27 118.8(8) C26-C27-H27 118.5(8) C29-C28-C27 119.3(4) C29-C28-H28 120.4 C27-C28-H28 120.4 C28-C29-C30 120.1(4) C28-C29-H29 120.0 C30-C29-H29 120.0 C29-C30-C31 119.7(4) C29-C30-H30 120.2 C31-C30-H30 120.2 F2B-C31-C30 116.8(5) F2B-C31-C26 120.6(5) C30-C31-C26 121.9(3) C30-C31-H31 119.4(9) C26-C31-H31 118.2(9) O4-C32-N5 O4-C32-C26 122.8(3) N5-C32-C26 114.5(3) 122.7(3) 550 N7-C33-N6 111.6(3) N7-C33-N5 121.7(3) N6-C33-N5 126.4(3) N6-C34-H34A109.5 N6-C34-H34B 109.5 H34A-C34-H34B 109.5 N6-C34-H34C 109.5 H34A-C34-H34C 109.5 H34B-C34-H34C 109.5 C43-C35-N6 105.6(3) C43-C35-C36 130.1(3) N6-C35-C36 124.1(3) C37-C36-C41 117.7(3) C37-C36-C35 120.5(3) C41-C36-C35 121.6(3) C38-C37-C36 122.0(3) C38-C37-H37 119.0 C36-C37-H37 119.0 C39-C38-C37 119.5(3) C39-C38-H38 120.3 C37-C38-H38 120.3 O5-C39-C38 124.4(3) O5-C39-C40 116.3(3) C38-C39-C40 119.3(3) C41-C40-C39 120.8(3) C41-C40-H40 119.6 C39-C40-H40 119.6 C40-C41-C36 120.6(3) C40-C41-H41 119.7 C36-C41-H41 119.7 O5-C42-H42A109.5 O5-C42-H42B 109.5 H42A-C42-H42B 109.5 O5-C42-H42C 109.5 H42A-C42-H42C 109.5 H42B-C42-H42C 109.5 C35-C43-N7 109.3(3) C35-C43-C44 131.9(3) N7-C43-C44 118.8(3) C43-C44-C45 110.9(3) C43-C44-H44A 109.4 C45-C44-H44A 109.4 C43-C44-H44B 109.4 C45-C44-H44B 109.4 H44A-C44-H44B 108.0 N8-C45-C46 121.2(3) 551 N8-C45-C44 115.8(3) C46-C45-C44 122.9(3) C45-C46-C47 119.7(3) C45-C46-H46 120.2 C47-C46-H46 120.2 C48-C47-C46 119.7(3) C48-C47-H47 120.2 C46-C47-H47 120.2 C49-C48-C47 117.8(3) C49-C48-H48 121.1 C47-C48-H48 121.1 N8-C49-O6 N8-C49-C48 123.1(3) 111.2(3) O6-C49-C48 125.8(3) O6-C50-H50A109.5 O6-C50-H50B 109.5 H50A-C50-H50B 109.5 O6-C50-H50C 109.5 H50A-C50-H50C 109.5 H50B-C50-H50C 109.5 Torsion angles (°) for 170720REL. C6-C1-C2-F1 -179.1(3) C7-C1-C2-F1 4.2(5) C6-C1-C2-C3 -0.4(5) C7-C1-C2-C3 -177.2(3) F1-C2-C3-C4 -179.6(3) C1-C2-C3-C4 1.7(5) C2-C3-C4-C5 -1.6(6) C3-C4-C5-C6 0.3(5) C4-C5-C6-C1 1.1(5) C2-C1-C6-C5 -1.1(5) C7-C1-C6-C5 176.0(3) C8-N1-C7-O1 -11.8(5) C8-N1-C7-C1 165.2(3) C2-C1-C7-O1 -157.4(3) C6-C1-C7-O1 25.8(4) C2-C1-C7-N1 25.6(5) C6-C1-C7-N1 -151.2(3) Zn01-N3-C8-N2 C18-N3-C8-N2 168.4(2) 0.2(4) C18-N3-C8-N1 -176.9(3) 552 Zn01-N3-C8-N1 -8.6(5) C10-N2-C8-N3 C9-N2-C8-N3 -171.5(3) C10-N2-C8-N1 0.5(4) 177.5(3) C9-N2-C8-N1 5.5(5) C7-N1-C8-N3 -127.9(3) C7-N1-C8-N2 55.5(5) C8-N2-C10-C18 -1.0(3) C9-N2-C10-C18 171.0(3) C8-N2-C10-C11 175.7(3) C9-N2-C10-C11 -12.4(5) C18-C10-C11-C16 -48.2(5) N2-C10-C11-C16 136.1(3) C18-C10-C11-C12 130.6(4) N2-C10-C11-C12 -45.1(4) C16-C11-C12-C13 0.4(5) C10-C11-C12-C13 -178.4(3) C11-C12-C13-C14 -0.2(5) C17-O2-C14-C15 5.6(5) C17-O2-C14-C13 C12-C13-C14-O2 -179.5(3) C12-C13-C14-C15 0.4(5) O2-C14-C15-C16 179.2(3) C13-C14-C15-C16 -0.8(5) C12-C11-C16-C15 -0.8(5) C10-C11-C16-C15 178.1(3) C14-C15-C16-C11 1.0(5) N2-C10-C18-N3 1.1(3) C11-C10-C18-N3 -175.1(3) C11-C10-C18-C19 1.4(6) C8-N3-C18-C10 Zn01-N3-C18-C10 -171.0(2) Zn01-N3-C18-C19 12.0(4) C10-C18-C19-C20 N3-C18-C19-C20 -63.2(4) C24-N4-C20-C21 -2.5(4) Zn01-N4-C20-C21 175.4(2) C24-N4-C20-C19 176.2(3) Zn01-N4-C20-C19 -6.0(4) C18-C19-C20-N4 C18-C19-C20-C21 -122.8(3) N4-C20-C21-C22 0.8(5) C19-C20-C21-C22 -177.8(3) C20-C21-C22-C23 1.3(5) -174.4(3) N2-C10-C18-C19 177.6(3) -0.8(4) C8-N3-C18-C19 -177.8(3) 120.6(4) 58.5(4) 553 C21-C22-C23-C24 -1.7(5) C20-N4-C24-O3 -177.3(3) Zn01-N4-C24-O3 4.9(4) C20-N4-C24-C23 2.0(5) Zn01-N4-C24-C23 -175.9(2) C25-O3-C24-C23 6.3(5) C22-C23-C24-N4 C22-C23-C24-O3 179.3(3) C32-C26-C27-F2A 7.5(5) C31-C26-C27-C28 C32-C26-C27-C28 -177.3(3) C26-C27-C28-C29 -0.3(6) C27-C28-C29-C30 -0.3(6) C28-C29-C30-C31 -0.2(6) C29-C30-C31-F2B -169.0(5) C29-C30-C31-C26 1.4(6) C27-C26-C31-F2B 168.1(5) C32-C26-C31-F2B -13.1(6) C27-C26-C31-C30 -2.0(5) C32-C26-C31-C30 176.8(3) C33-N5-C32-O4 -9.8(5) C33-N5-C32-C26 170.0(3) C27-C26-C32-O4 -157.1(3) C31-C26-C32-O4 24.2(5) C27-C26-C32-N5 C31-C26-C32-N5 -155.6(3) C43-N7-C33-N6 0.7(4) Zn02-N7-C33-N6 163.0(2) C43-N7-C33-N5 -173.3(3) Zn02-N7-C33-N5 -11.1(5) C35-N6-C33-N7 -0.3(4) C34-N6-C33-N7 -168.7(3) C35-N6-C33-N5 173.4(3) C34-N6-C33-N5 4.9(5) C32-N5-C33-N7 -133.3(3) C32-N5-C33-N6 53.7(5) C33-N6-C35-C43 -0.3(3) C34-N6-C35-C43 168.2(3) C33-N6-C35-C36 174.9(3) C34-N6-C35-C36 -16.5(5) C43-C35-C36-C37 128.5(4) N6-C35-C36-C37 -45.5(4) C43-C35-C36-C41 -46.4(5) C25-O3-C24-N4 -174.5(3) 0.1(5) C31-C26-C27-F2A -173.8(3) 1.4(5) F2A-C27-C28-C29 175.1(4) 23.0(4) 554 N6-C35-C36-C41 139.7(3) C41-C36-C37-C38 3.6(5) C35-C36-C37-C38 -171.4(3) C36-C37-C38-C39 -0.7(5) C42-O5-C39-C38 -5.2(5) C42-O5-C39-C40 C37-C38-C39-O5 176.6(3) C37-C38-C39-C40 -3.0(5) O5-C39-C40-C41 -175.9(3) C38-C39-C40-C41 3.7(5) C39-C40-C41-C36 -0.8(5) C37-C36-C41-C40 C35-C36-C41-C40 172.1(3) N6-C35-C43-N7 0.7(3) C36-C35-C43-N7 -174.1(3) N6-C35-C43-C44 -177.2(3) C36-C35-C43-C44 8.0(6) C33-N7-C43-C35 Zn02-N7-C43-C35 -166.3(2) Zn02-N7-C43-C44 11.9(4) C35-C43-C44-C45 N7-C43-C44-C45 -67.6(4) C49-N8-C45-C46 2.9(4) Zn02-N8-C45-C46 -177.5(2) C49-N8-C45-C44 -175.5(3) Zn02-N8-C45-C44 4.0(3) C43-C44-C45-N8 C43-C44-C45-C46 -121.9(3) N8-C45-C46-C47 -0.9(5) C44-C45-C46-C47 177.5(3) C45-C46-C47-C48 -1.1(5) C46-C47-C48-C49 0.9(5) C45-N8-C49-O6 176.9(2) Zn02-N8-C49-O6 -2.7(3) C45-N8-C49-C48 -3.1(4) Zn02-N8-C49-C48 177.3(2) C50-O6-C49-C48 5.1(4) C47-C48-C49-N8 C47-C48-C49-O6 -178.8(3) 174.4(3) -2.8(5) -0.9(4) C33-N7-C43-C44 177.3(3) 110.2(4) 56.5(3) C50-O6-C49-N8 1.2(5) -174.9(3) 555 Anisotropic atomic displacement parameters (Å2) for 170720REL. The anisotropic atomic displacement factor exponent takes the form: -2π2[ h2 a*2 U11 + ... + 2 h k a* b* U12 ] U11 U22 U33 Zn01 0.01867(19) U23 U13 U12 0.01969(19) 0.02434(19) -0.00954(14) -0.00023(14) - 0.01982(19) 0.02071(18) -0.00738(14) 0.00384(14) - 0.0296(4) 0.0285(4) -0.0093(3) 0.0040(3) - 0.0246(4) 0.0255(4) -0.0116(3) -0.0015(3) - 0.0371(5) 0.0336(4) -0.0168(4) 0.0058(3) - 0.0306(4) 0.0286(4) -0.0091(3) -0.0005(3) 0.0489(13) 0.0290(10) -0.0155(9) 0.0050(8) - 0.0322(13) 0.0282(12) -0.0166(10) 0.0016(9) - 0.0402(15) 0.0335(14) -0.0229(12) -0.0015(12) - 0.00454(14) Zn02 0.0203(2) 0.00484(14) Cl1 0.0281(4) 0.0121(3) Cl2 0.0361(5) 0.0048(3) Cl3 0.0300(4) 0.0166(4) Cl4 0.0297(5) 0.0050(3) F1 0.0189(10) 0.0019(9) O1 0.0199(12) 0.0032(10) O2 0.0441(16) 0.0126(12) 556 O3 0.0218(12) 0.0322(13) 0.0369(13) -0.0217(11) -0.0009(10) 0.0439(15) 0.0236(12) -0.0130(10) 0.0057(9) - 0.0334(13) 0.0376(13) -0.0171(11) 0.0050(10) - 0.0273(12) 0.0319(12) -0.0143(10) 0.0101(10) - 0.0239(14) 0.0232(13) -0.0118(11) -0.0015(10) - 0.0174(12) 0.0206(12) -0.0065(10) 0.0019(10) - 0.0224(13) 0.0229(13) -0.0114(10) 0.0004(10) - 0.0193(13) 0.0231(13) -0.0069(10) -0.0006(10) - 0.0215(13) 0.0205(13) -0.0080(10) 0.0036(11) - 0.0233(13) 0.0197(12) -0.0061(10) 0.0013(10) - 0.0224(13) 0.0198(12) -0.0075(10) 0.0031(10) - 0.0009(10) O4 0.0243(13) 0.0126(11) O5 0.0237(13) 0.0140(10) O6 0.0229(12) 0.0098(9) N1 0.0198(14) 0.0018(11) N2 0.0196(13) 0.0050(10) N3 0.0171(13) 0.0036(10) N4 0.0184(13) 0.0037(10) N5 0.0265(15) 0.0096(11) N6 0.0189(13) 0.0063(10) N7 0.0195(13) 0.0058(10) 557 N8 0.0165(13) 0.0174(12) 0.0212(12) -0.0056(10) 0.0023(10) - 0.0172(14) 0.0184(14) -0.0036(11) 0.0020(11) - 0.0260(16) 0.0234(15) -0.0074(13) 0.0021(12) - 0.038(2) 0.0235(16) -0.0128(15) -0.0003(14) - 0.0328(19) 0.0297(17) -0.0143(14) -0.0097(14) - 0.0281(17) 0.0276(16) -0.0054(13) -0.0017(13) - 0.0215(15) 0.0206(15) -0.0040(12) 0.0018(12) - 0.0182(14) 0.0225(15) -0.0053(12) 0.0006(12) - 0.0200(14) 0.0215(14) -0.0083(11) 0.0024(11) - 0.0165(15) 0.0272(16) -0.0054(12) -0.0016(14) - 0.0217(14) 0.0201(14) -0.0081(11) 0.0035(11) - 0.0033(10) C1 0.0208(15) 0.0060(12) C2 0.0201(16) 0.0034(13) C3 0.0312(19) 0.0005(15) C4 0.0317(19) 0.0017(15) C5 0.0210(17) 0.0060(13) C6 0.0217(16) 0.0041(12) C7 0.0202(16) 0.0054(12) C8 0.0172(15) 0.0041(11) C9 0.0300(18) 0.0062(13) C10 0.0167(15) 0.0064(12) 558 C11 0.0207(15) 0.0177(14) 0.0196(14) -0.0067(11) 0.0035(11) - 0.0226(15) 0.0254(16) -0.0095(12) 0.0010(12) - 0.0233(16) 0.0350(18) -0.0137(14) 0.0052(14) - 0.0273(16) 0.0222(15) -0.0103(13) 0.0033(13) - 0.0318(17) 0.0217(15) -0.0084(13) 0.0029(12) - 0.0231(15) 0.0221(15) -0.0065(12) 0.0036(12) - 0.050(2) 0.033(2) -0.0177(18) -0.0065(17) - 0.0227(15) 0.0201(14) -0.0096(12) 0.0035(11) - 0.0203(15) 0.0208(15) -0.0071(12) 0.0007(12) - 0.0143(14) 0.0206(14) -0.0029(11) -0.0008(12) - 0.0217(16) 0.0277(17) -0.0068(13) -0.0073(13) - 0.0067(12) C12 0.0205(16) 0.0044(12) C13 0.0271(18) 0.0047(13) C14 0.0298(18) 0.0125(14) C15 0.0227(17) 0.0109(13) C16 0.0187(15) 0.0067(12) C17 0.050(2) 0.024(2) C18 0.0166(15) 0.0048(12) C19 0.0235(16) 0.0026(12) C20 0.0223(16) 0.0043(11) C21 0.0267(17) 0.0027(13) 559 C22 0.0207(17) 0.0272(17) 0.0337(18) -0.0055(14) -0.0062(14) - 0.0250(16) 0.0295(17) -0.0060(13) -0.0001(13) 0.0198(15) 0.0253(16) -0.0072(12) 0.0020(12) 0.040(2) 0.043(2) -0.0266(17) 0.0007(16) 0.0230(15) 0.0201(15) -0.0063(12) -0.0047(12) 0.0290(17) 0.0354(18) -0.0144(14) -0.0004(14) 0.052(2) 0.0476(19) -0.0333(15) 0.0087(12) - 0.035(2) 0.055(2) -0.0160(17) -0.0049(16) - 0.036(2) 0.049(2) -0.0186(18) -0.0167(18) - 0.037(2) 0.036(2) -0.0219(16) -0.0062(17) - 0.0349(19) 0.0263(17) -0.0146(14) 0.0004(14) - 0.0032(13) C23 0.0196(16) 0.0000(13) C24 0.0229(16) - 0.0034(12) C25 0.030(2) 0.0028(16) C26 0.0203(15) 0.0001(12) C27 0.0224(17) 0.0003(13) F2A 0.0193(15) 0.0051(13) C28 0.0230(19) 0.0067(15) C29 0.042(2) 0.0047(17) C30 0.046(2) 0.0032(17) C31 0.0304(19) 0.0034(15) 560 C32 0.0173(15) 0.0261(16) 0.0199(14) -0.0077(12) -0.0007(12) - 0.0229(15) 0.0211(14) -0.0082(12) 0.0036(11) - 0.0275(17) 0.0226(16) -0.0058(13) -0.0017(13) - 0.0245(15) 0.0211(14) -0.0096(12) 0.0027(11) - 0.0226(15) 0.0221(15) -0.0059(12) -0.0003(12) - 0.0227(15) 0.0215(15) -0.0083(12) 0.0027(12) - 0.0214(15) 0.0244(15) -0.0065(12) 0.0039(12) - 0.0223(15) 0.0260(16) -0.0072(12) -0.0011(12) - 0.0295(17) 0.0223(15) -0.0135(13) 0.0016(12) - 0.0283(16) 0.0220(15) -0.0088(13) 0.0023(12) - 0.039(2) 0.046(2) -0.0191(17) 0.0084(15) - 0.0002(12) C33 0.0175(15) 0.0035(12) C34 0.0267(18) 0.0068(14) C35 0.0146(15) 0.0042(12) C36 0.0162(15) 0.0037(12) C37 0.0165(15) 0.0035(12) C38 0.0179(16) 0.0034(12) C39 0.0172(15) 0.0028(12) C40 0.0234(17) 0.0061(13) C41 0.0188(16) 0.0059(13) C42 0.0249(19) 0.0155(15) 561 C43 0.0150(15) 0.0231(15) 0.0225(14) -0.0091(12) 0.0024(11) - 0.0242(16) 0.0237(15) -0.0082(12) 0.0017(12) - 0.0209(15) 0.0167(14) -0.0051(11) -0.0019(11) - 0.0219(15) 0.0219(15) -0.0079(12) 0.0004(12) - 0.0229(16) 0.0253(16) -0.0119(13) 0.0026(13) - 0.0223(15) 0.0220(15) -0.0079(12) 0.0049(13) - 0.0178(14) 0.0193(14) -0.0047(11) 0.0010(11) - 0.0336(18) 0.0252(16) -0.0090(14) 0.0066(13) - 0.0033(12) C44 0.0177(15) 0.0070(12) C45 0.0196(15) 0.0025(12) C46 0.0270(17) 0.0066(13) C47 0.0305(18) 0.0049(13) C48 0.0257(17) 0.0026(13) C49 0.0205(15) 0.0023(12) C50 0.0243(17) 0.0106(14) Hydrogen atomic coordinates and isotropic atomic displacement parameters (Å2) for 170720REL. x/a y/b z/c U(eq) H1N 0.890(4) 0.542(2) 0.2048(19) 0.026 H5N 0.153(3) -0.096(2) 0.5141(16) 0.027 562 H3 1.0753 0.4768 0.4300 0.038 H4 1.3051 0.4524 0.4243 0.037 H5 1.4134 0.4133 0.3219 0.032 H6 1.2930 0.4001 0.2255 0.027 H9A 0.7956 0.3060 0.1095 0.038 H9B 0.8715 0.3183 0.1774 0.038 H9C 0.9497 0.3058 0.1034 0.038 H12 0.9109 0.3580 -0.0354 0.027 H13 0.8608 0.2833 -0.1183 0.033 H15 0.4875 0.4543 -0.1489 0.03 H16 0.5387 0.5281 -0.0639 0.026 H17A 0.5149 0.4168 -0.2644 0.062 H17B 0.4551 0.3367 -0.1923 0.062 H17C 0.5234 0.3025 -0.2608 0.062 H19A 0.6450 0.6780 -0.1055 0.026 H19B 0.7402 0.7405 -0.0871 0.026 H21 0.4201 0.7590 -0.1117 0.031 H22 0.2461 0.8600 -0.0709 0.035 H23 0.2848 0.9247 0.0231 0.032 H25A 0.3749 1.0050 0.0848 0.054 H25B 0.4598 0.9593 0.1650 0.054 H25C 0.3546 0.9003 0.1520 0.054 H27 -0.005(2) -0.108(3) 0.446(2) 0.043 563 H28 -0.1106 -0.2132 0.4134 0.045 H29 -0.0062 -0.3005 0.3351 0.05 H30 0.2019 -0.2790 H31 0.3081(19) 0.2884 0.046 -0.176(3) 0.324(3) H34A 0.3410 0.1541 0.3668 0.04 H34B 0.2513 0.2317 0.4023 0.04 H34C 0.1889 0.1510 0.3828 0.04 H37 0.5176 0.1885 0.4322 0.025 H38 0.6373 0.3118 0.4215 0.027 H40 0.4497 0.3562 0.6074 0.029 H41 0.3338 0.2304 0.6196 0.028 H42A 0.6958 0.4553 0.4093 0.052 H42B 0.7964 0.3642 0.4727 0.052 H42C 0.7764 0.4795 0.4690 0.052 H44A 0.4365 0.0464 0.6942 0.026 H44B 0.4447 -0.0730 0.7126 0.026 H46 0.2923 0.1054 0.7818 0.028 H47 0.0969 0.1080 0.8505 0.031 H48 -0.0473 0.0060 0.8444 0.029 H50A -0.1867 -0.0485 0.7684 0.042 H50B -0.1259 -0.1321 0.8509 0.042 H50C -0.1680 -0.1685 0.7854 0.042 0.036 564 4.5.6 Crystal Structure Report for C3 565 A clear colorless irregular piece from a rhombic crystal-like specimen of C26H25Cl2FN4O2Zn, approximate dimensions 0.300 mm x 0.400 mm x 0.400 mm, was used for the X-ray crystallographic analysis. The X-ray intensity data were measured. A total of 9567 frames were collected. The total exposure time was 20.76 h. The frames were integrated with the Bruker SAINT software package using a narrow-frame algorithm. The integration of the data using a monoclinic unit cell yielded a total of 196373 reflections to a maximum θ angle of 38.70° (0.57 Å resolution), of which 14693 were independent (average redundancy 13.365, completeness = 99.9%, Rint = 2.66%, Rsig = 1.15%) and 13519 (92.01%) were greater than 2σ(F2). The final cell constants of a = 7.7150(5) Å, b = 12.1937(8) Å, c = 27.3760(19) Å, β = 90.212(3)°, volume = 2575.4(3) Å3, are based upon the refinement of the XYZ-centroids of 9424 reflections above 20 σ(I) with 5.480° < 2θ < 77.17°. Data were corrected for absorption effects using the Multi-Scan method (SADABS). The ratio of minimum to maximum apparent transmission was 0.906. The structure was solved and refined using the Bruker SHELXTL Software Package, using the space group P 1 21/n 1, with Z = 4 for the formula unit, C26H25Cl2FN4O2Zn. The final anisotropic full-matrix least-squares refinement on F2 with 330 variables converged at R1 = 2.40%, for the observed data and wR2 = 6.30% for all data. The goodness-of-fit was 1.102. The largest peak in the final difference electron density synthesis was 0.637 e-/Å3 and the largest hole was -0.394 e-/Å3 with an RMS deviation of 0.061 e-/Å3. On the basis of the final model, the calculated density was 1.498 g/cm3 and F(000), 1192 e-. 566 Sample and crystal data for 20171031RL. Identification code 20171031RL Chemical formula C26H25Cl2FN4O2Zn Formula weight 580.79 g/mol Temperature 103(2) K Wavelength 0.71073 Å Crystal size 0.300 x 0.400 x 0.400 mm clear colourless irregular piece from a Crystal habit rhombic crystal Crystal system monoclinic Space group P 1 21/n 1 Unit cell a = 7.7150(5) Å α = 90° b = 12.1937(8) Å β = 90.212(3)° c = 27.3760(19) Å γ = 90° dimensions Volume 2575.4(3) Å3 Z 4 Density (calculated) 1.498 g/cm3 567 Absorption 1.200 mm-1 coefficient F(000) 1192 Data collection and structure refinement for 20171031RL. Theta range for data 2.74 to 38.70° collection Index ranges -13<=h<=13, -21<=k<=21, -48<=l<=48 Reflections collected 196373 Independent 14693 [R(int) = 0.0266] reflections Coverage of 99.9% independent reflections Absorption correction Multi-Scan Structure solution direct methods technique Structure solution SHELXT 2014/5 (Sheldrick, 2014) program Refinement method Full-matrix least-squares on F2 Refinement program SHELXL-2016/6 (Sheldrick, 2016) 568 Σ w(Fo2 - Fc2)2 Function minimized Data / restraints / 14693 / 0 / 330 parameters Goodness-of-fit on F2 1.102 Δ/σmax 0.003 13519 data; R1 = 0.0240, wR2 = I>2σ(I) 0.0617 Final R indices R1 = 0.0273, wR2 = all data 0.0630 w=1/[σ2(Fo2)+(0.0274P)2+0.7558P] Weighting scheme where P=(Fo2+2Fc2)/3 Largest diff. peak and 0.637 and -0.394 eÅ-3 hole R.M.S. deviation from 0.061 eÅ-3 mean Atomic coordinates and equivalent isotropic atomic displacement parameters (Å2) for 20171031RL. U(eq) is defined as one third of the trace of the orthogonalized Uij tensor. x/a y/b z/c Zn1 0.70037(2) U(eq) 0.20768(2) 0.68268(2) 0.01084(2) 569 Cl1 0.73278(3) 0.07633(2) 0.62695(2) 0.01957(3) Cl2 0.42352(2) 0.22646(2) 0.70853(2) 0.01621(3) F1 0.15627(7) 0.33546(6) 0.59563(2) 0.03115(13) O2 0.61763(8) 0.33687(6) 0.52609(2) 0.02219(11) O3 0.27456(8) 0.88837(5) 0.59107(2) 0.02253(11) N1 0.75287(7) 0.35697(4) 0.65509(2) 0.01144(8) N2 0.71771(7) 0.49713(5) 0.60523(2) 0.01248(8) N4 0.87032(7) 0.17241(4) 0.73850(2) 0.01187(8) N5 0.50525(7) 0.35149(5) 0.60188(2) 0.01251(8) C1 0.65565(8) 0.39983(5) 0.62000(2) 0.01158(9) C2 0.86611(8) 0.51827(5) 0.63269(2) 0.01178(9) C3 0.97557(8) 0.61500(5) 0.62396(2) 0.01189(9) C4 0.07501(9) 0.62190(6) 0.58139(2) 0.01593(10) C5 0.17504(10) 0.71347(6) 0.57202(3) 0.01722(11) C6 0.17532(9) 0.80133(6) 0.60467(3) 0.01463(10) C7 0.25722(12) 0.98794(7) 0.61824(3) 0.02466(15) C8 0.08129(9) 0.79480(6) 0.64776(3) 0.01597(10) C9 0.98300(9) 0.70108(5) 0.65715(2) 0.01455(10) C10 0.88519(8) 0.43135(5) 0.66374(2) 0.01156(9) C11 0.02648(8) 0.41111(6) 0.69984(3) 0.01453(10) C12 0.97655(10) 0.43339(6) 0.75301(3) 0.01687(11) C13 0.83816(9) 0.35583(5) 0.77378(2) 0.01415(10) C14 0.90344(8) 0.24033(5) 0.77628(2) 0.01184(9) 570 C15 0.93930(9) 0.07078(5) 0.73896(3) 0.01478(10) C16 0.04001(9) 0.03197(6) 0.77701(3) 0.01752(11) C17 0.07235(9) 0.10098(6) 0.81634(3) 0.01762(11) C18 0.00361(9) 0.20586(6) 0.81582(3) 0.01539(10) C19 0.64284(10) 0.56817(6) 0.56793(3) 0.01824(12) C20 0.49991(8) 0.31607(6) 0.55425(2) 0.01325(9) C21 0.34468(9) 0.25286(6) 0.53852(2) 0.01371(10) C22 0.18035(9) 0.26465(7) 0.55829(3) 0.01784(11) C23 0.03674(11) 0.20903(7) 0.54080(4) 0.02431(15) C24 0.05977(12) 0.13702(7) 0.50221(4) 0.02672(17) C25 0.22194(14) 0.12138(7) 0.48180(3) 0.02723(16) C26 0.36336(11) 0.17942(7) 0.49961(3) 0.02073(13) Bond lengths (Å) for 20171031RL. Zn1-N1 2.0127(6) Zn1-N4 2.0552(6) Zn1-Cl1 2.2267(2) Zn1-Cl2 2.2642(2) F1-C22 1.3516(10) O2-C20 1.2200(8) O3-C6 1.3613(8) O3-C7 1.4304(11) N1-C1 1.3242(8) N1-C10 N2-C1 1.3423(8) N2-C2 1.3914(8) 1.3852(8) N2-C19 1.4567(9) N4-C15 1.3486(8) N4-C14 1.3488(8) N5-C20 1.3742(8) 571 N5-C1 1.3913(8) C2-C10 N5-H5N 1.3664(9) 0.832(12) C2-C3 1.4707(9) C3-C9 1.3892(9) C3-C4 1.4002(9) C4-C5 1.3819(10) C4-H7 0.95 C5-C6 1.3953(10) C5-H1 0.95 C6-C8 1.3895(10) C7-H3 0.98 C7-H2 0.98 C7-H4 0.98 C8-C9 1.3959(9) C8-H6 0.95 C9-H5 0.95 C10-C11 1.4892(9) C11-C12 1.5313(10) C11-H17 C11-H16 0.99 C12-C13 1.5370(10) C12-H8 0.99 C12-H9 0.99 C13-C14 1.4972(9) C13-H15 0.99 C15-C16 1.3807(10) C15-H14 0.95 C16-C17 1.3884(11) C16-H13 0.95 C17-C18 1.3845(10) C17-H11 0.95 C18-H12 0.95 C19-H18 0.98 C19-H20 0.98 C19-H19 0.98 C20-C21 1.4865(9) C21-C22 1.3878(10) C21-C26 1.3994(10) C22-C23 1.3830(11) C23-C24 1.3856(14) C23-H21 0.95 C24-C25 1.3853(15) C24-H22 0.95 C13-H10 C14-C18 0.99 0.99 1.3926(9) 572 C25-C26 1.3875(12) C26-H24 0.95 C25-H23 0.95 Bond angles (°) for 20171031RL. N1-Zn1-N4 109.85(2) N1-Zn1-Cl1 111.726(18) N4-Zn1-Cl1 106.615(17) N1-Zn1-Cl2 102.543(17) N4-Zn1-Cl2 112.879(17) Cl1-Zn1-Cl2 113.289(8) C6-O3-C7 117.76(6) C1-N1-C10 C1-N1-Zn1 121.00(4) C10-N1-Zn1 132.63(4) C1-N2-C2 107.14(5) C1-N2-C19 C2-N2-C19 126.29(6) C15-N4-C14 118.91(6) C15-N4-Zn1 116.74(4) C14-N4-Zn1 124.07(4) C20-N5-C1 119.57(5) C20-N5-H5N 120.7(8) C1-N5-H5N 115.5(8) N1-C1-N2 111.45(5) N1-C1-N5 124.12(6) N2-C1-N5 124.40(6) C10-C2-N2 106.20(5) C10-C2-C3 131.50(6) N2-C2-C3 122.18(5) C9-C3-C4 118.52(6) C9-C3-C2 121.47(6) C4-C3-C2 120.00(6) C5-C4-C3 120.74(6) C5-C4-H7 119.6 C3-C4-H7 119.6 C4-C5-C6 120.07(6) C4-C5-H1 120.0 C6-C5-H1 120.0 O3-C6-C8 124.95(7) O3-C6-C5 106.30(5) 126.57(6) 115.03(6) 573 C8-C6-C5 120.02(6) O3-C7-H3 O3-C7-H2 109.5 H3-C7-H2 109.5 O3-C7-H4 109.5 H3-C7-H4 109.5 H2-C7-H4 109.5 C6-C8-C9 119.27(6) C6-C8-H6 120.4 C9-C8-H6 120.4 C3-C9-C8 121.28(6) C8-C9-H5 119.4 C2-C10-N1 C3-C9-H5 109.5 119.4 108.90(5) C2-C10-C11 128.20(6) N1-C10-C11 122.83(5) C10-C11-C12 114.54(6) C10-C11-H17 108.6 C12-C11-H17 108.6 C10-C11-H16 108.6 C12-C11-H16 108.6 H17-C11-H16 107.6 C11-C12-C13 114.84(5) C11-C12-H8 108.6 C13-C12-H8 108.6 C11-C12-H9 108.6 C13-C12-H9 108.6 H8-C12-H9 C14-C13-C12 111.21(6) 107.5 C14-C13-H10 109.4 C12-C13-H10 109.4 C14-C13-H15 109.4 C12-C13-H15 109.4 H10-C13-H15 108.0 N4-C14-C18 120.91(6) N4-C14-C13 118.68(5) C18-C14-C13 120.34(6) N4-C15-C16 122.89(6) N4-C15-H14 118.6 C16-C15-H14 118.6 C15-C16-C17 118.44(6) C15-C16-H13 120.8 C17-C16-H13 120.8 C18-C17-C16 118.96(6) C18-C17-H11 120.5 C16-C17-H11 120.5 574 C17-C18-C14 119.89(6) C17-C18-H12 120.1 C14-C18-H12 120.1 N2-C19-H18 109.5 N2-C19-H20 109.5 H18-C19-H20 109.5 N2-C19-H19 109.5 H18-C19-H19 109.5 H20-C19-H19 109.5 O2-C20-N5 120.96(6) O2-C20-C21 121.71(6) N5-C20-C21 117.32(6) C22-C21-C26 117.38(7) C22-C21-C20 124.72(6) C26-C21-C20 117.84(6) F1-C22-C23 117.58(7) F1-C22-C21 119.30(6) C23-C22-C21 123.10(7) C22-C23-C24 118.02(8) C22-C23-H21 121.0 C24-C23-H21 121.0 C25-C24-C23 120.88(7) C25-C24-H22 119.6 C23-C24-H22 119.6 C24-C25-C26 119.88(8) C24-C25-H23 120.1 C26-C25-H23 120.1 C25-C26-C21 120.72(8) C25-C26-H24 119.6 C21-C26-H24 119.6 Torsion angles (°) for 20171031RL. C10-N1-C1-N2 0.37(7) Zn1-N1-C1-N2 C10-N1-C1-N5 -177.76(6) Zn1-N1-C1-N5 C2-N2-C1-N1 0.25(7) C19-N2-C1-N1 C2-N2-C1-N5 178.37(6) C20-N5-C1-N1 4.93(9) -179.01(6) C19-N2-C1-N5 -114.85(7) -176.94(4) -0.89(11) C20-N5-C1-N2 67.26(9) 575 C1-N2-C2-C10 -0.77(7) C1-N2-C2-C3 175.73(6) C19-N2-C2-C10 C19-N2-C2-C3 178.49(6) -5.01(10) C10-C2-C3-C9 -74.49(10) N2-C2-C3-C9 110.01(8) C10-C2-C3-C4 105.66(9) N2-C2-C3-C4 -69.84(9) C9-C3-C4-C5 -1.56(11) C2-C3-C4-C5 178.30(7) C3-C4-C5-C6 -1.20(11) C7-O3-C6-C8 -11.90(11) C7-O3-C6-C5 169.32(7) C4-C5-C6-O3 -178.19(7) C4-C5-C6-C8 2.97(11) O3-C6-C8-C9 179.36(7) C5-C6-C8-C9 -1.92(11) C4-C3-C9-C8 2.62(10) C2-C3-C9-C8 -177.24(7) C6-C8-C9-C3 -0.89(11) N2-C2-C10-N1 1.00(7) C3-C2-C10-N1 -175.04(6) N2-C2-C10-C11 177.76(6) C3-C2-C10-C11 1.72(12) C1-N1-C10-C2 -0.86(7) Zn1-N1-C10-C2 176.01(5) C1-N1-C10-C11 -177.83(6) Zn1-N1-C10-C11 -0.96(10) C2-C10-C11-C12 103.77(8) N1-C10-C11-C12 -79.88(8) C15-N4-C14-C18 -1.54(9) Zn1-N4-C14-C18 172.07(5) C15-N4-C14-C13 175.29(6) Zn1-N4-C14-C13 -11.10(8) C14-N4-C15-C16 1.36(10) Zn1-N4-C15-C16 -172.72(6) N4-C15-C16-C17 -0.39(11) C15-C16-C17-C18 -0.38(11) C16-C17-C18-C14 0.19(11) N4-C14-C18-C17 0.79(10) C13-C14-C18-C17 -175.99(6) C1-N5-C20-O2 -8.02(10) C1-N5-C20-C21 172.64(6) O2-C20-C21-C22 -150.52(8) C20-C21-C22-C23 176.08(7) F1-C22-C23-C24 179.40(8) 576 C21-C22-C23-C24 1.05(13) C22-C23-C24-C25 -0.03(13) C23-C24-C25-C26 -0.89(14) C24-C25-C26-C21 0.83(13) C22-C21-C26-C25 0.13(12) C20-C21-C26-C25 -177.25(7) Anisotropic atomic displacement parameters (Å2) for 20171031RL. The anisotropic atomic displacement factor exponent takes the form: -2π2[ h2 a*2 U11 + ... + 2 h k a* b* U12 ] U11 U22 U33 Zn1 0.01232(3) U23 U13 U12 0.00874(3) 0.01145(3) 0.00061(2) -0.00176(2) - 0.01573(7) 0.01677(7) -0.00543(5) 0.00048(6) - 0.01598(6) 0.01946(7) 0.00362(5) 0.00116(5) - 0.0454(4) 0.0316(3) -0.0187(3) 0.00328(19) 0.0299(3) 0.0165(2) -0.0058(2) 0.00702(18) - 0.0181(2) 0.0258(3) 0.0040(2) 0.0041(2) - 0.01054(19) 0.01229(19) 0.00149(15) -0.00210(15) - 0.00118(2) Cl1 0.02620(8) 0.00240(6) Cl2 0.01320(6) 0.00009(5) F1 0.0165(2) 0.0003(2) O2 0.0201(2) 0.0084(2) O3 0.0236(2) 0.0101(2) N1 0.01149(18) 0.00160(15) 577 N2 0.0140(2) 0.0116(2) 0.01185(19) 0.00289(16) -0.00188(16) - 0.00957(19) 0.0138(2) 0.00022(15) -0.00167(16) 0.0157(2) 0.00928(18) 0.00024(16) -0.00092(15) - 0.0118(2) 0.0106(2) 0.00124(17) -0.00128(17) - 0.0109(2) 0.0122(2) 0.00171(17) -0.00055(17) - 0.0109(2) 0.0117(2) 0.00160(17) 0.00108(17) 0.0149(2) 0.0130(2) -0.00071(19) 0.0040(2) - 0.0180(3) 0.0143(2) 0.0016(2) 0.0053(2) - 0.0138(2) 0.0161(2) 0.00346(19) 0.00063(19) - 0.0154(3) 0.0294(4) 0.0046(3) -0.0049(3) - 0.0130(2) 0.0171(3) -0.0015(2) 0.0031(2) - 0.00168(16) N4 0.01224(19) 0.00017(15) N5 0.01253(19) 0.00346(16) C1 0.0124(2) 0.00170(17) C2 0.0122(2) 0.00179(17) C3 0.0131(2) - 0.00179(17) C4 0.0200(3) 0.0029(2) C5 0.0194(3) 0.0037(2) C6 0.0139(2) 0.00338(19) C7 0.0291(4) 0.0080(3) C8 0.0179(3) 0.0037(2) 578 C9 0.0160(2) 0.0134(2) 0.0143(2) -0.00081(19) 0.00420(19) - 0.0105(2) 0.0131(2) 0.00159(17) -0.00116(17) - 0.0141(2) 0.0172(3) 0.00332(19) -0.00384(19) - 0.0124(2) 0.0163(3) -0.00019(19) -0.0066(2) 0.0115(2) 0.0136(2) -0.00120(18) -0.00220(19) 0.0115(2) 0.0122(2) 0.00053(17) -0.00107(17) - 0.0101(2) 0.0197(3) 0.00006(19) -0.0028(2) 0.0130(2) 0.0245(3) 0.0033(2) -0.0040(2) 0.0189(3) 0.0193(3) 0.0045(2) -0.0041(2) 0.0175(3) 0.0140(2) 0.0010(2) -0.00323(19) 0.0169(3) 0.0167(3) 0.0069(2) -0.0048(2) 0.00265(19) C10 0.0111(2) 0.00162(16) C11 0.0122(2) 0.00265(18) C12 0.0219(3) - 0.0037(2) C13 0.0173(2) 0.00152(19) C14 0.0118(2) 0.00015(17) C15 0.0145(2) 0.00078(18) C16 0.0150(2) 0.00239(19) C17 0.0146(2) 0.0025(2) C18 0.0146(2) 0.0006(2) C19 0.0211(3) 0.0008(2) - 579 C20 0.0140(2) 0.0146(2) 0.0112(2) -0.00107(18) 0.00008(18) - 0.0144(2) 0.0112(2) 0.00003(18) -0.00140(18) - 0.0218(3) 0.0174(3) -0.0010(2) -0.0023(2) - 0.0270(4) 0.0292(4) 0.0039(3) -0.0065(3) - 0.0202(3) 0.0302(4) 0.0040(3) -0.0142(3) - 0.0202(3) 0.0234(3) -0.0051(3) -0.0078(3) - 0.0186(3) 0.0162(3) -0.0048(2) 0.0000(2) - 0.00144(18) C21 0.0155(2) 0.00263(19) C22 0.0144(2) 0.0019(2) C23 0.0167(3) 0.0060(3) C24 0.0296(4) 0.0102(3) C25 0.0380(4) 0.0087(3) C26 0.0274(3) 0.0046(3) Hydrogen atomic coordinates and isotropic atomic displacement parameters (Å2) for 20171031RL. x/a y/b z/c U(eq) H5N 0.4399(15) H7 1.0737 0.5630 0.5587 0.019 H1 1.2437 0.7166 0.5433 0.021 H3 1.1353 1.0105 0.6185 0.037 0.3243(10) 0.6227(4) 0.015 580 H2 1.3274 1.0454 0.6030 0.037 H4 1.2971 0.9761 0.6519 0.037 H6 1.0839 0.8535 0.6706 0.019 H5 0.9200 0.6961 0.6868 0.017 H17 1.1267 0.4580 0.6914 0.017 H16 1.0641 0.3338 0.6970 0.017 H8 0.9334 0.5096 0.7555 0.02 H9 1.0821 0.4278 0.7735 0.02 H10 0.8053 0.3806 0.8069 0.017 H15 0.7334 0.3586 0.7528 0.017 H14 0.9176 0.0238 0.7119 0.018 H13 1.0861 -0.0403 H11 1.1406 0.0766 0.8432 0.021 H12 1.0248 0.2542 0.8424 0.018 H18 0.6924 0.5498 0.5361 0.027 H20 0.6688 0.6449 0.5758 0.027 H19 0.5170 0.5576 0.5669 0.027 H21 -0.0744 0.2199 0.5548 0.029 H22 -0.0369 0.0979 0.4896 0.032 H23 0.2363 0.0710 0.4557 0.033 H24 0.4741 0.1692 0.4852 0.025 0.7763 0.021 581 4.5.7 Crystal Structure Report for C4 582 A clear colorless square bar-like specimen of C26H25BF5N4O2Zn0.5, approximate dimensions 0.100 mm x 0.100 mm x 0.400 mm, was used for the X-ray crystallographic analysis. The X-ray intensity data were measured. A total of 752 frames were collected. The total exposure time was 2.09 h. The frames were integrated with the Bruker SAINT software package using a narrow-frame algorithm. The integration of the data using a tetragonal unit cell yielded a total of 51342 reflections to a maximum θ angle of 33.14° (0.65 Å resolution), of which 8960 were independent (average redundancy 5.730, completeness = 100.0%, Rint = 5.89%, Rsig = 6.05%) and 6273 (70.01%) were greater than 2σ(F2). The final cell constants of a = 21.0647(16) Å, b = 21.0647(16) Å, c = 21.1775(16) Å, volume = 9396.9(16) Å3, are based upon the refinement of the XYZcentroids of 9328 reflections above 20 σ(I) with 4.719° < 2θ < 62.41°. Data were corrected for absorption effects using the Multi-Scan method (SADABS). The ratio of minimum to maximum apparent transmission was 0.923. The calculated minimum and maximum transmission coefficients (based on crystal size) are 0.7900 and 0.9410. The structure was solved and refined using the Bruker SHELXTL Software Package, using the space group I 41/a, with Z = 16 for the formula unit, C26H25BF5N4O2Zn0.5. The final anisotropic full-matrix least-squares refinement on F2 with 395 variables converged at R1 = 4.47%, for the observed data and wR2 = 12.07% for all data. The goodness-of-fit was 1.031. The largest peak in the final difference electron density synthesis was 0.902 e-/Å3 and the largest hole was -0.744 e-/Å3 with an RMS deviation of 0.069 e-/Å3. On the basis of the final model, the calculated density was 1.515 g/cm3 and F(000), 4384 e-. 583 Sample and crystal data for 20171204RL. Identification code 20171204RL Chemical formula C26H25BF5N4O2Zn0.5 Formula weight 564.00 g/mol Temperature 103(2) K Wavelength 0.71073 Å Crystal size 0.100 x 0.100 x 0.400 mm Crystal habit clear colourless square bar Crystal system tetragonal Space group I 41/a Unit cell dimensions a = 21.0647(16) Å α = 90° b = 21.0647(16) Å β = 90° c = 21.1775(16) Å γ = 90° Volume 9396.9(16) Å3 Z 16 Density (calculated) 1.515 g/cm3 Absorption coefficient 0.617 mm-1 F(000) 4384 584 Data collection and structure refinement for 20171204RL. Theta range for data 2.36 to 33.14° collection Index ranges -31<=h<=32, -32<=k<=27, -32<=l<=32 Reflections collected 51342 Independent reflections 8960 [R(int) = 0.0589] Coverage of 100.0% independent reflections Absorption correction Multi-Scan Max. and min. 0.9410 and 0.7900 transmission Structure solution direct methods technique Structure solution SHELXT 2014/5 (Sheldrick, 2014) program Refinement method Full-matrix least-squares on F2 Refinement program SHELXL-2016/6 (Sheldrick, 2016) Function minimized Σ w(Fo2 - Fc2)2 Data / restraints / 8960 / 55 / 395 parameters 585 Goodness-of-fit on F2 1.031 Δ/σmax 0.001 6273 data; R1 = 0.0447, wR2 = I>2σ(I) 0.1144 Final R indices R1 = 0.0716, wR2 = all data 0.1207 w=1/[σ2(Fo2)+(0.0567P)2+2.9158P] Weighting scheme where P=(Fo2+2Fc2)/3 Largest diff. peak and 0.902 and -0.744 eÅ-3 hole R.M.S. deviation from 0.069 eÅ-3 mean Atomic coordinates and equivalent isotropic atomic displacement parameters (Å2) for 20171204RL. U(eq) is defined as one third of the trace of the orthogonalized Uij tensor. x/a y/b z/c U(eq) Zn1 0.5 0.75 0.28821(2) 0.01268(7) F1 0.54462(6) 0.93795(7) 0.49160(6) 0.0448(4) O1 0.47182(6) 0.81035(6) 0.36924(5) 0.0191(2) O2 0.87495(7) 0.92712(7) 0.11360(7) 0.0334(3) 586 N1 0.55124(6) 0.68268(7) 0.22941(6) 0.0143(3) N2 0.58020(6) 0.80130(7) 0.29662(6) 0.0141(3) N3 0.56725(7) 0.85223(7) 0.39791(6) 0.0172(3) N4 0.65118(6) 0.87301(7) 0.32386(6) 0.0149(3) C1 0.53943(8) 0.62051(8) 0.23683(8) 0.0174(3) C2 0.57779(9) 0.57342(8) 0.21228(8) 0.0193(3) C3 0.63103(8) 0.59125(8) 0.17830(8) 0.0193(3) C4 0.64407(8) 0.65522(8) 0.17047(8) 0.0173(3) C5 0.60309(8) 0.70017(8) 0.19663(7) 0.0137(3) C6 0.61366(8) 0.77046(8) 0.18799(7) 0.0150(3) C7 0.62413(7) 0.80612(7) 0.24843(7) 0.0128(3) C8 0.66904(8) 0.85031(8) 0.26412(7) 0.0144(3) C9 0.72401(8) 0.87296(8) 0.22786(7) 0.0149(3) C10 0.76397(8) 0.82890(8) 0.19824(8) 0.0171(3) C11 0.81371(8) 0.84819(9) 0.16045(8) 0.0210(3) C12 0.82486(8) 0.91272(9) 0.15184(8) 0.0217(4) C13 0.78644(9) 0.95729(9) 0.18108(8) 0.0215(4) C14 0.73592(8) 0.93731(8) 0.21876(8) 0.0181(3) C15 0.88743(14) 0.99169(13) 0.10270(15) 0.0553(8) C16 0.68210(9) 0.92290(9) 0.36051(8) 0.0224(4) C17 0.59816(7) 0.84163(8) 0.34053(7) 0.0140(3) C18 0.50543(8) 0.83620(8) 0.40882(7) 0.0158(3) C19 0.47906(8) 0.84950(8) 0.47270(7) 0.0179(3) 587 C20 0.49774(9) 0.89865(9) 0.51164(9) 0.0245(4) C21 0.46905(10) 0.91110(11) 0.56912(9) 0.0328(5) C22 0.42118(11) 0.87177(12) 0.58897(9) 0.0380(5) C23 0.40094(12) 0.82185(12) 0.55143(10) 0.0396(6) C24 0.42923(10) 0.81159(10) 0.49328(9) 0.0297(4) B1A 0.7161(3) 0.8137(3) 0.4901(3) 0.0211(15) F2A 0.7285(4) 0.7793(4) 0.5437(4) 0.063(3) F3A 0.7728(2) 0.8427(4) 0.4746(3) 0.0507(16) F4A 0.6963(2) 0.77825(19) 0.44024(18) 0.0538(11) F5A 0.6686(3) 0.8582(3) 0.5028(2) 0.0481(19) B1B 0.7205(6) 0.8228(6) 0.5010(6) 0.029(5) F2B 0.7298(7) 0.7749(5) 0.5455(7) 0.043(4) F3B 0.7721(6) 0.8350(7) 0.4632(5) 0.048(3) F4B 0.6748(7) 0.7994(8) 0.4638(8) 0.126(9) F5B 0.7035(7) 0.8801(3) 0.5269(6) 0.074(5) B1C 0.7126(12) 0.8197(11) 0.4921(11) 0.06(4) F2C 0.6971(13) 0.7597(11) 0.5126(13) 0.085(9) F3C 0.7616(13) 0.8421(14) 0.5283(15) 0.113(12) F4C 0.7323(11) 0.8076(10) 0.4329(9) 0.055(6) F5C 0.6651(10) 0.8646(10) 0.4953(12) 0.008(4) 588 Bond lengths (Å) for 20171204RL. Zn1-N2 2.0133(13) Zn1-N2 2.0134(13) Zn1-N1 2.1740(14) Zn1-N1 2.1741(14) Zn1-O1 2.2165(12) Zn1-O1 2.2165(12) F1-C20 1.357(2) O1-C18 1.2250(19) O2-C12 1.364(2) O2-C15 1.404(3) N1-C1 1.342(2) N1-C5 1.346(2) N2-C17 1.315(2) N2-C7 1.3813(19) N3-C18 1.365(2) N3-C17 N3-H3N 0.88 1.397(2) N4-C17 1.345(2) N4-C8 1.404(2) N4-C16 1.460(2) C1-C2 1.381(2) C1-H1 0.95 C2-C3 1.384(3) C2-H2 0.95 C3-C4 1.385(2) C3-H3 0.95 C4-C5 1.396(2) C4-H4 0.95 C5-C6 1.508(2) C6-C7 1.501(2) C6-H6A C6-H6B 0.99 C7-C8 1.368(2) 0.99 C8-C9 1.469(2) C9-C14 1.392(2) C9-C10 1.401(2) C10-C11 1.380(2) C10-H10 0.95 C11-C12 1.392(3) C11-H11 0.95 C12-C13 1.386(3) C13-C14 1.395(2) 589 C13-H13 0.95 C14-H14 0.95 C15-H15A 0.98 C15-H15B 0.98 C15-H15C 0.98 C16-H16A 0.98 C16-H16B 0.98 C16-H16C 0.98 C18-C19 1.489(2) C19-C20 1.381(2) C19-C24 1.389(3) C20-C21 1.384(3) C21-C22 1.371(3) C21-H21 0.95 C22-C23 1.385(3) C22-H22 0.95 C23-C24 1.385(3) C23-H23 0.95 C24-H24 0.95 B1A-F2A 1.372(7) B1A-F3A 1.381(6) B1A-F5A 1.397(6) B1B-F4B 1.339(12) B1B-F5B 1.373(12) B1B-F3B 1.375(12) B1B-F2B 1.392(12) B1C-F4C 1.345(17) B1C-F3C 1.370(17) B1C-F2C 1.376(17) B1C-F5C 1.377(16) B1A-F4A 1.359(8) Bond angles (°) for 20171204RL. N2-Zn1-N2 169.85(7) N2-Zn1-N1 96.73(5) N2-Zn1-N1 89.10(5) N2-Zn1-N1 89.10(5) N2-Zn1-N1 96.73(5) N1-Zn1-N1 110.11(7) N2-Zn1-O1 90.84(5) N2-Zn1-O1 81.28(5) 590 N1-Zn1-O1 161.89(5) N1-Zn1-O1 86.35(5) N2-Zn1-O1 81.28(5) N2-Zn1-O1 90.84(5) N1-Zn1-O1 86.35(5) N1-Zn1-O1 161.90(5) O1-Zn1-O1 78.55(7) C18-O1-Zn1 129.05(11) C12-O2-C15 117.24(18) C1-N1-C5 118.55(14) C1-N1-Zn1 118.51(11) C5-N1-Zn1 121.32(11) C17-N2-C7 106.39(13) C17-N2-Zn1 130.59(11) C7-N2-Zn1 122.43(10) C18-N3-C17 123.55(14) C18-N3-H3N 118.2 C17-N3-H3N 118.2 C17-N4-C8 106.97(13) C17-N4-C16 125.78(14) C8-N4-C16 127.21(13) N1-C1-C2 N1-C1-H1 118.4 C2-C1-H1 C1-C2-C3 118.34(16) C3-C2-H2 120.8 C2-C3-C4 119.14(16) C2-C3-H3 120.4 C4-C3-H3 120.4 C3-C4-C5 119.32(16) C5-C4-H4 120.3 N1-C5-C4 N1-C5-C6 116.81(14) C4-C5-C6 121.76(14) C7-C6-C5 114.18(13) C7-C6-H6A 108.7 C5-C6-H6A 108.7 C7-C6-H6B C5-C6-H6B 108.7 H6A-C6-H6B 107.6 C8-C7-N2 109.50(13) C8-C7-C6 130.50(14) N2-C7-C6 119.67(14) C7-C8-N4 105.38(13) 123.25(16) 118.4 C1-C2-H2 C3-C4-H4 120.8 120.3 121.40(15) 108.7 591 C7-C8-C9 129.72(14) N4-C8-C9 124.88(14) C14-C9-C10 118.34(15) C14-C9-C8 122.04(15) C10-C9-C8 C11-C10-C9 121.40(16) 119.51(15) C11-C10-H10 119.3 C9-C10-H10 119.3 C10-C11-C12 119.46(17) C10-C11-H11 120.3 C12-C11-H11 120.3 O2-C12-C13 124.51(17) O2-C12-C11 115.18(17) C13-C12-C11 120.31(16) C12-C13-C14 119.80(16) C12-C13-H13 120.1 C14-C13-H13 120.1 C9-C14-C13 120.69(17) C9-C14-H14 119.7 C13-C14-H14 119.7 O2-C15-H15A109.5 O2-C15-H15B 109.5 H15A-C15-H15B 109.5 O2-C15-H15C 109.5 H15A-C15-H15C 109.5 H15B-C15-H15C 109.5 N4-C16-H16A109.5 N4-C16-H16B 109.5 H16A-C16-H16B 109.5 N4-C16-H16C 109.5 H16A-C16-H16C 109.5 H16B-C16-H16C 109.5 N2-C17-N4 111.76(14) N2-C17-N3 125.75(14) N4-C17-N3 122.49(14) O1-C18-N3 123.05(15) O1-C18-C19 119.33(15) N3-C18-C19 117.59(14) C20-C19-C24 117.32(16) C20-C19-C18 125.27(15) C24-C19-C18 117.31(16) F1-C20-C19 118.55(16) F1-C20-C21 118.51(17) C19-C20-C21 122.88(18) C22-C21-C20 118.45(19) C22-C21-H21 120.8 592 C20-C21-H21 120.8 C21-C22-C23 120.59(19) C21-C22-H22 119.7 C23-C22-H22 119.7 C24-C23-C22 119.7(2) C24-C23-H23 120.1 C22-C23-H23 120.1 C23-C24-C19 120.96(19) C23-C24-H24 119.5 C19-C24-H24 119.5 F4A-B1A-F2A 114.2(6) F4A-B1A-F3A 108.9(5) F2A-B1A-F3A 105.4(6) F4A-B1A-F5A 107.4(5) F2A-B1A-F5A 109.4(5) F3A-B1A-F5A 111.7(6) F4B-B1B-F5B 111.8(12) F4B-B1B-F3B 107.0(11) F5B-B1B-F3B 105.9(10) F4B-B1B-F2B 103.6(11) F5B-B1B-F2B 113.8(10) F3B-B1B-F2B 114.7(13) F4C-B1C-F3C 111.(2) F4C-B1C-F2C 101.2(18) F3C-B1C-F2C 109.(2) F4C-B1C-F5C 114.(2) F3C-B1C-F5C 106.4(19) F2C-B1C-F5C 116.(2) Torsion angles (°) for 20171204RL. C5-N1-C1-C2 -0.2(2) Zn1-N1-C1-C2 -165.96(13) N1-C1-C2-C3 0.0(3) C1-C2-C3-C4 0.3(2) C2-C3-C4-C5 -0.4(2) C1-N1-C5-C4 0.2(2) Zn1-N1-C5-C4 165.50(11) C1-N1-C5-C6 178.46(14) Zn1-N1-C5-C6 -16.23(18) C3-C4-C5-N1 0.1(2) C3-C4-C5-C6 -178.07(14) N1-C5-C6-C7 63.10(19) 593 C4-C5-C6-C7 -118.64(16) C17-N2-C7-C8 -0.06(18) Zn1-N2-C7-C8 171.97(11) C17-N2-C7-C6 Zn1-N2-C7-C6 -2.1(2) C5-C6-C7-C8 133.14(18) C5-C6-C7-N2 -54.2(2) N2-C7-C8-N4 -0.25(18) C6-C7-C8-N4 172.97(16) N2-C7-C8-C9 -178.41(16) C6-C7-C8-C9 -5.2(3) C17-N4-C8-C7 -174.12(14) 0.46(18) C16-N4-C8-C7 -177.27(16) C17-N4-C8-C9 178.74(15) C16-N4-C8-C9 1.0(3) C7-C8-C9-C14 N4-C8-C9-C14 -48.4(2) C7-C8-C9-C10 -46.8(2) N4-C8-C9-C10 135.41(17) C14-C9-C10-C11 -0.5(2) C8-C9-C10-C11 175.82(15) C9-C10-C11-C12 0.5(3) C15-O2-C12-C13 -1.0(3) C15-O2-C12-C11 C10-C11-C12-O2 -179.85(15) C10-C11-C12-C13 0.1(3) O2-C12-C13-C14 179.29(16) C11-C12-C13-C14 -0.7(3) C10-C9-C14-C13 0.0(2) C8-C9-C14-C13 -176.30(15) C12-C13-C14-C9 0.6(3) C7-N2-C17-N4 0.37(19) Zn1-N2-C17-N4 -170.77(11) Zn1-N2-C17-N3 9.0(3) C8-N4-C17-N2 C16-N4-C17-N2 177.24(16) C16-N4-C17-N3 -2.5(3) C18-N3-C17-N2 C18-N3-C17-N4 160.19(16) Zn1-O1-C18-N3 25.3(2) Zn1-O1-C18-C19 -152.87(12) C17-N3-C18-O1 0.9(3) C17-N3-C18-C19 179.10(15) O1-C18-C19-C20 -151.20(19) 129.46(19) 179.0(2) C7-N2-C17-N3 -179.88(16) -0.53(19) C8-N4-C17-N3 179.71(15) -19.5(3) 594 N3-C18-C19-C20 30.5(3) O1-C18-C19-C24 25.0(3) N3-C18-C19-C24 -153.34(18) C18-C19-C20-F1 -1.3(3) C24-C19-C20-C21 C18-C19-C20-C21 176.12(19) C19-C20-C21-C22 1.9(3) C20-C21-C22-C23 -1.8(4) C21-C22-C23-C24 -0.1(4) C22-C23-C24-C19 2.0(4) C20-C19-C24-C23 -1.9(3) C18-C19-C24-C23 -178.3(2) C24-C19-C20-F1 -177.45(19) -0.1(3) F1-C20-C21-C22 179.3(2) Anisotropic atomic displacement parameters (Å2) for 20171204RL. The anisotropic atomic displacement factor exponent takes the form: -2π2[ h2 a*2 U11 + ... + 2 h k a* b* U12 ] U11 U22 U33 U23 U13 U12 Zn1 0.01188(13) 0.01552(14) 0.01065(11) 0 F1 0.0419(8) 0.0492(8) 0.0434(7) -0.0300(6) 0.0193(6) - 0.0262(6) 0.0141(5) -0.0055(5) 0.0016(4) - 0.0370(8) 0.0358(8) 0.0085(6) 0.0138(6) - 0.0166(6) 0.0113(6) -0.0002(5) -0.0003(5) - 0 -0.00332(10) 0.0283(6) O1 0.0170(6) 0.0050(5) O2 0.0275(7) 0.0071(6) N1 0.0151(6) 0.0014(5) 595 N2 0.0139(6) 0.0177(7) 0.0107(6) 0.0005(5) 0.0009(5) - 0.0235(7) 0.0111(6) -0.0050(5) 0.0007(5) - 0.0174(7) 0.0125(6) -0.0013(5) -0.0007(5) - 0.0174(8) 0.0142(7) 0.0001(6) -0.0004(6) - 0.0172(8) 0.0161(7) -0.0016(6) -0.0048(6) 0.0230(9) 0.0154(7) -0.0054(6) -0.0051(6) 0.0249(9) 0.0135(7) -0.0035(6) -0.0007(6) - 0.0185(8) 0.0078(6) -0.0008(5) -0.0028(5) - 0.0187(8) 0.0097(6) 0.0007(6) 0.0004(6) - 0.0156(7) 0.0101(6) 0.0016(5) 0.0002(5) - 0.0170(7) 0.0113(6) 0.0020(6) -0.0007(5) - 0.0034(5) N3 0.0170(7) 0.0067(5) N4 0.0147(6) 0.0060(5) C1 0.0205(8) 0.0016(6) C2 0.0245(9) 0.0014(6) C3 0.0194(8) 0.0052(7) C4 0.0136(7) 0.0004(6) C5 0.0147(7) 0.0011(6) C6 0.0165(7) 0.0032(6) C7 0.0128(7) 0.0010(6) C8 0.0149(7) 0.0018(6) 596 C9 0.0129(7) 0.0190(8) 0.0129(7) 0.0041(6) -0.0020(6) - 0.0195(8) 0.0156(7) 0.0033(6) -0.0009(6) - 0.0280(9) 0.0186(8) 0.0020(7) 0.0013(6) 0.0318(10) 0.0173(8) 0.0057(7) 0.0006(6) - 0.0202(8) 0.0233(8) 0.0071(7) -0.0035(7) - 0.0204(8) 0.0182(7) 0.0028(6) -0.0016(6) - 0.0423(14) 0.0691(18) 0.0126(13) 0.0304(14) - 0.0259(9) 0.0187(8) -0.0047(7) -0.0008(7) - 0.0159(7) 0.0123(7) 0.0007(6) 0.0000(5) - 0.0165(7) 0.0134(7) -0.0003(6) 0.0014(6) - 0.0224(8) 0.0125(7) -0.0014(6) 0.0026(6) - 0.0035(6) C10 0.0163(8) 0.0021(6) C11 0.0164(8) 0.0009(7) C12 0.0159(8) 0.0053(7) C13 0.0209(9) 0.0066(7) C14 0.0158(8) 0.0023(6) C15 0.0543(16) 0.0163(12) C16 0.0227(9) 0.0126(7) C17 0.0138(7) 0.0032(6) C18 0.0174(8) 0.0030(6) C19 0.0189(8) 0.0029(6) 597 C20 0.0230(9) 0.0294(10) 0.0211(8) -0.0068(7) 0.0025(7) - 0.0441(13) 0.0213(9) -0.0152(9) 0.0024(8) - 0.0554(15) 0.0192(9) -0.0076(9) 0.0108(9) - 0.0460(14) 0.0279(10) -0.0055(10) 0.0205(10) - 0.0297(10) 0.0251(9) -0.0070(8) 0.0118(8) - 0.026(2) 0.016(2) 0.0046(19) 0.0032(16) 0.088(5) 0.034(4) 0.033(3) 0.023(3) 0.0343(19) 0.089(4) 0.013(2) -0.009(2) 0.0509(18) 0.0536(17) -0.0240(16) -0.0203(14) 0.061(3) 0.0251(19) 0.0055(16) 0.0056(15) 0.029(4) 0.031(7) 0.018(4) 0.010(5) 0.0083(7) C21 0.0330(11) 0.0051(9) C22 0.0395(12) 0.0057(11) C23 0.0450(13) 0.0169(11) C24 0.0342(11) 0.0120(8) B1A 0.022(2) 0.0011(17) F2A 0.066(5) 0.043(3) F3A 0.029(2) - 0.0181(16) F4A 0.057(2) 0.0048(16) F5A 0.059(3) 0.028(2) F2B 0.070(9) 0.007(4) - 598 F3B 0.062(7) 0.053(7) 0.028(3) 0.012(3) 0.036(4) 0.117(11) 0.166(14) 0.106(11) -0.104(10) 0.046(4) 0.078(7) 0.013(4) 0.060(7) 0.006(4) F4B 0.094(9) - 0.093(9) F5B 0.098(8) 0.015(4) Hydrogen atomic coordinates and isotropic atomic displacement parameters (Å2) for 20171204RL. x/a y/b z/c U(eq) H3N 0.5888 0.8702 0.4287 0.021 H1 0.5029 0.6083 0.2601 0.021 H2 0.5679 0.5299 0.2185 0.023 H3 0.6583 0.5600 0.1606 0.023 H4 0.6805 0.6684 0.1475 0.021 H6A 0.6510 0.7768 0.1603 0.018 H6B 0.5763 0.7887 0.1661 0.018 H10 0.7567 0.7848 0.2043 0.021 H11 0.8401 0.8177 0.1405 0.025 H13 0.7945 1.0013 0.1755 0.026 H14 0.7094 0.9679 0.2384 0.022 H15A 0.9238 0.9958 0.0741 0.083 599 H15B 0.8972 1.0127 0.1428 0.083 H15C 0.8501 1.0117 0.0836 0.083 H16A 0.6783 0.9133 0.4056 0.034 H16B 0.6617 0.9637 0.3516 0.034 H16C 0.7271 0.9252 0.3489 0.034 H21 0.4822 0.9461 0.5942 0.039 H22 0.4017 0.8788 0.6288 0.046 H23 0.3678 0.7948 0.5655 0.048 H24 0.4143 0.7781 0.4671 0.036 CHAPTER 5 STEREOSELECTIVE SYNTHESIS OF CYCLIC ENE-GUANIDINES USING A GUANYLATION/AMINOSILYLATION CASCADE REACTION 5.1 Introduction A mild and stereoselective guanylation/aminosilylation method for the synthesis of TMS-ene-guanidines is described herein. Guanylation of secondary propargyl amines bearing electron deficient alkynes with silyl carbodiimides led to the formation of N-acyl propargyl guanidines, which spontaneously underwent 5-exo-dig cyclization to yield silylated ene-guanidines. This stereodefined transformation provided the opposite alkene isomer of previously reported Lewis acid catalyzed hydroaminations, offering access to new substitution patterns of cyclic guanidine scaffolds. Cyclic guanidines represent an important class of nitrogen-containing organic compounds,1 which display a broad range of biological activities.2 Their abilities to establish strong charge-charge interactions, hydrogen bonds and cation-π stackings have spurred research efforts in the areas of medicinal chemistry,3 molecular recognition4 and organocatalysis.5 Traditional synthetic methods to prepare guanidines involves the activation of thiourea or S-methyl isothiourea using highly toxic mercury-(II) chloride (Figure 5.1A).6 An alternative approach involves the formation of the C-N bond using a preinstalled guanidine core and a corresponding carbon electrophile. This approach has 601 been used in the total syntheses of bioactive natural products such as cylindrospermopsin7 and batzelladine D (Figure 5.1B).8 In recent years, the preparation of such heterocycles from propargylic compounds using transition metal-mediated intramolecular hydroamination reactions has found wide applications in the synthetic community (Figure 5.1C).9 This process proceeds via the addition of an N-H or O-H bond across the π-system of alkynes, allowing not only versatile preparations of cyclic guanidines,10-11 but also oxazolidinones,12 oxazolidinthiones and imidazolidinthiones (Figure 5.1C).13 Looper and co-workers have developed an efficient hydroamination method to access 5- or 6-membered cyclic ene-guanidines using di-Boc protected propargyl guanidines (Figure 5.1C). Depending on the choice of the metal catalyst, the 5-exo-dig product (AgOAc) or the 6-endo-dig product (Rh2(oct)4) was obtained with high regioselectivity (>20:1 for either regioisomers). This method was later applied in the total synthesis of marine natural products naamine A, naamidine A14 and saxitoxin.15 Simultaneously, the Van der Eycken lab described the preparation of similar eneguanidines from secondary propargylamines via a one-pot guanylation/cyclization reaction (Figure 5.1C).10 Despite high yields and short reaction times, the usage of an excessive amount of heavy metals poses contaminatiom issues and could lead to altered biological activities.16 Later, the same research group reported a metal-free iodocyclization method to synthesize Boc-protected iodo-ene-guanidine, allowing subsequent introduction of additional functionalities (Figure 5.1C). 602 5.2 Results and Discussion 5.2.1 A Stereodefined Aminosilylation Reaction The Looper lab has a long-standing interest in alkaloids, natural products derived from marine sponges.17 In particular, the selective cytotoxicity profile of naamidine A18 has prompted the development of a concise and modular total synthesis of this natural product14 which also allows access to a library of diverse N2-acyl 2-aminoimidazoles. Initial screening has identified one such compound, zinaamidole A or ZNA, as a screening hit that showed antiproliferative activity against chemo-resistant primary plural effusion cells from breast cancer patients, while displaying minimal toxicity against untransformed breast cells. Further biological evaluation revealed that ZNA functioned as an ionophore, shuttling Zn2+ ions across the cell membrane which led to zinc dyshomeostasis and, ultimately, cellular apoptosis. In pursuance of ZNA analogs with enhanced ionophoric properties, we set out to prepare a library of hemiporphyrin-like compounds carrying pendant C4-pyridine side chains. During our synthetic efforts, we discovered the formation of an unusual cyclic TMS-ene-guanidine 4.6.2 upon treatment of the pyridine-2-propargyl amine 4.5.2 with Ncyanobenzamide S8 in the presence of silylating reagent TMSCl (Figure 5.2A). Presumably, the TMS-propargyl guanidine formed in situ underwent spontaneous aminosilylation in a 5-exo-dig fashion to give 4.6.2. The structure of this compound was unambiguously confirmed through X-ray crystallography (Figure 5.3B). Contrary to previously reported Lewis acid-mediated hydroamination reactions (Figure 5.1C), this transformation gave exclusively the opposite alkene isomer, in which the heteroaryl group R3 is positioned trans to the N3-nitrogen. To the best of our knowledge, there is no literature 603 precedent showing the preparation of these types of Z-alkenes in a stereodefined manner. Moreover, only a few reports have shown the addition of N-Si σ-bonds into C-C triple bonds (Figure 5.3). For example, Gotor and co-workers described the insertion of simple silyl imines into activated acetylenes to access azadienes.19 Kunai and co-workers reported the insertion of highly reactive benzyne intermediates into the N-Si σ-bond of aminosilanes, resulting in the formation of 2-silylanilines.21 To this end, we envisioned utilizing our newly discovered aminosilylation reaction to access a variety of cyclic TMSene-guanidines. 5.2.2 Reaction Optimization Optimization of the aminosilylation reaction began by evaluating silylation reagents TMSCl, TBDMSCl and N,O-(bistrimethylsilyl)acetamide (BSA)21 (Table 5.1). It was found that TMSCl and BSA were equally effective and provided the cyclized TMSene-guanidine 4.6.2 in under 3 h at room temperature (Table 5.1, entry 1 and 3). Contrarily, using TBDMSCl resulted only in the formation of N-acyl propargyl guanidine 5.2.3 in 88% yield (Table 5.1, entry 2). We suspected that the silyl group could not maintain bonded to the guanidine nitrogen due to steric hindrance, thus preventing cyclization. Next, 5.2.3 was used to investigate the effect of the base in the reaction (Table 5.2). In the absence of an external base, TMSCl failed to promote cyclization after 16 h (Table 5.2, entry 1); however, addition of the DIPEA restored the reactivity of TMSCl, resulting in moderate yields of the desired product 4.6.2 (Table 5.2, entry 2). In contrast, BSA induced the cyclization without any addition of exogenous base. Presumably, the transfer of the silyl group from BSA to N-cyano-benzamide S8 led to the formation of TMS-amidate,21 which functioned 604 as a base. 5.2.3 Substrate Scope Having identified two optimal conditions, we set out to explore the scope of this transformation employing a small library of 2-pyridine propargyl amines (prepared in Chapter 4). When TMSCl was first used as a silylating reagent, most of the substrates underwent guanylation/aminosilylation reactions in under 3 h at room temperature, resulting in the isolation of a single alkene isomer (Figure 5.4). Ene-guanidine 4.5.1 bearing an unsubstituted pyridine was isolated in good yield. Addition of an electron-donating methoxy group decreased the overall yields. Bulky substitutents adjacent to the pyridine nitrogen did not influence the efficiency of the reaction as shown in 4.6.11 and 4.6.12. However, unconjugated alkynes do not undergo aminosilylation under the given condition; instead, the N-acyl propargylguanidine 4.6.14 was obtained in high yield. Reaction of pyridine-3-propargyl amine yielded both TMS-ene-guanidine 5.2.1a and propargyl guanidine 5.2.1b in a 1:2 ratio, whereas pyridine-4-propargyl amine led exclusively to the ene-guanidine 5.2.2. Next, we used BSA reagent to probe several 2-pyridine propargyl amines and 2aryl propargyl amines bearing electron deficient groups (Figure 5.5). The latter class of compounds was assembled using chemistry described by Looper and van der Eycken.10, 22 Although all propargyl amines used in this study underwent initial guanylation reaction, it was in our interest to probe whether the resultant alkynes were susceptible to subsequent aminosilylation. We were pleased to find that our reaction is amenable for heteroaryl- and electron-deficient aryl propargyl amines. Introduction of a sterically hindered isopropoxy 605 group adjacent to the pyridine nitrogen afforded 4.6.12 in moderate yield. Better yield was obtained with ene-guanidine 5.2.4 and 5.2.5. Various aryl propargyl amines carrying electron withdrawing groups were also easily functionalized, providing the requisite eneguanidines 5.2.6-5.2.9 in 58%-80% yield. Notably, extended reaction time was required to obtain the para-chloro substrate 5.2.10, whereas the para-fluoro substituted derivative 5.2.11 was only isolated in trace amount. In contrast, meta-fluoro substituted ene-guanidine 5.2.12 was obtained in moderate yield in under 3 h. Another interesting outcome was observed when comparing ene-guanidine 5.2.14 and its regioisomer 5.2.15. The former substrate was isolated in 74% yield after 3 h, while the formation of 3n proceeded much slower, with only 12% isolated yield after 3 h and 65% after 24 h. These experimental data suggest that the R2-substituents are critical for rendering the electrophilicity of the alkynes and triggering the requisite aminosilylation to occur. In particular, the results related with the positional isomers (5.2.11, 5.2.12, 5.2.14 and 5.2.15) give us reasons to believe there is an adequate window for correlation between Hammett values (σ) and the yield of the reaction. To demonstrate the utility of our methodology, the resultant N2-acyl-eneguanidines were used for subsequent synthetic manipulations. Treatment of 4.6.2 with K2CO3/MeOH at ambient temperature resulted in a facile desilylation, accompanied by isomerization to afford the N2-acyl 2-aminoimidazole ZNA 148 (Figure 5.6a). However, when 5.2.4 was subjected to the same basic condition, a single Z-alkene isomer 5.2.20 was obtained of which the structure was confirmed by X-ray crystallography (Figure 5.6b). This outcome is interesting because vinyl anions are usually considered stereoretentive.23 However, Houk24 and Russel25 have shown in computational and experimental work that 606 these species could be stabilized by an adjacent electron-deficient group such as pyridine, thus leading to a lower barrier of stereoinversion. Bromination of 5.2.4 (Figure 5.6c) in the presence of Br2 and AgOAc provided bromo-ene-guanidine 5.2.21 as an E-alkene in 80% yield, which was unambiguously confirmed via NOE experiments. In contrast, using iodine as a halide source led to the formation of two alkene isomers 5.2.22 and 5.2.23 in a 1.7:1 ratio. Presumably, the reaction proceeded via a halogenated guanidium cation intermediacy of which its configuration is highly dependent on the steric effects between the pyridyl/iodide and the adjacent cyclobutyl moiety (Scheme 3d). Treating 5.2.4 with methanolic HCl at 50 oC resulted in the formation of the bicyclic guanidine 5.2.24 via spirocyclobutane ring expansion, which was previously reported in our laboratory (Scheme 3e).26 These types of fused 5-5 heterocycles represent a versatile synthon in the preparation of natural products such as nagelamide J. 5.3 Putative Mechanism for the Guanylation/Aminosilylation Reaction On the basis of the obtained results, a plausible mechanism (Figure 5.7) for the guanylation/aminosilylation sequence is proposed. Upon addition of a silylating reagent (BSA or TMSCl) to the N-cyanobenzamide S8 under basic conditions, the reactive N-silyl carbodiimide species M1 was formed in situ and became trapped by the secondary propargyl amine M2, resulting in TMS-propargyl guanidine M3. Thereafter, cyclization and TMS exchange led to the formation of the desired TMS-ene-guanidine. 607 5.4 Summary In conclusion, we have discovered a stereodefined hydroamination reaction for the preparation of N2-acyl-ene-guanidines. The presence of electron-deficient aryl- or heteroaryl groups in propargyl amines have uncovered previously unknown reactivities and allowed the facile preparation of guanidines bearing an exocyclic silylated Z-alkene. This unique reaction provides the opposite alkene stereoisomer of a Lewis-acid catalyzed hydroamination. Furthermore, we have also demonstrated the utility of these substrates in subsequent structural modifications, offering swift access to novel substitution patterns in cyclic guanidine scaffolds. 608 A Traditional approach S Boc N H N H R1 R1R2NH Boc Boc HgCl2, Et3N DMF Di-Boc thiourea B R2 N N H N Boc Di-Boc guanidine Synthesis of cyclic guanidine natural products OMe nC9H19 MeO H 2N O + NH OH O O Me N NH . HOAc H cyclic guanidine 22 O N OMe N N CbzN NHCBz OMe acyclic guanidine C Batzelladine D Me acyclic guanidine Br nC9H19 N CbzHN(H2C)4O O(CH2)4NHCBz NH . HCl H HO H H H H N NH N NH OMe Cylindrospermopsin N OMe cyclic guanidine Previous approaches in the preparation of 5-membered heterocycles from propargylic substrates Schmalz and co-workers, 2006 O N H O Bäckvall and co-workers, 2014 O Ph Au+ Ph N-propargyl carbamate O X N Ph R1 N H Y R2 Ph Y = O, X = O Oxazolidinone X R3 Pd2+ R1 Y Ph Y = O, X = S Oxazolinthione N R3 R2 Y = N, X = S Imidazolidinthione Ph Oxazolidinone Looper and co-workers, 2011 NBoc R1 N NHBoc AgOAc AcOH CH2Cl2, rt Rh2(oct)4 CH2Cl2, rt R2 R3 R1 N NBoc R1 N NHBoc SMe Boc R2 R3 N-propargyl amine or R1 N NBoc R2 R3 6-endo-dig product Boc-protected ene-guanidines van der Eycken and co-workers, 2010 NH NBoc R3 5-exo-dig product R3 Di-Boc protected propargyl guanidine R1 N Boc R2 R2 M NBoc Boc R2 N N H AgNO3 (1.4 equiv.) Et3N, MeCN, rt R3 R1 N van der Eycken and co-workers, 2016 R1 NBoc N Boc Boc-protected ene-guanidine 1) N,N’-Di Boc thiourea NH R2 R3 N-propargyl amine 2) I2, Et3N toluene, 16 h, rt R2 I R3 R1 N R3 = aryl or alkyl NBoc N Boc Boc-protected iodo-ene-guanidine Figure 5.1: Synthetic approaches towards guanidines. A) Traditional synthesis of guanidines. B) Total synthesis of guanidine containing natural products. C) Synthesis of 5-membered heterocycles using hydroamination strategy. 609 O A Me MeO N N H NH R3 4.5.2 MeO F TMSCl, DIPEA CH2Cl2, rt 3 h, 55% Me N N F 5-exo-dig cyclization H N MeO Me N O TMS R3 TMS R3 TMS-propargyl guanidine N H F N O 4.6.2 B 4.6.2 Figure 5.2: Unexpected formation of a TMS-ene guanidine. A) Guanylation of propargyl amine 4.5.2 yielded ene-guanidine 4.6.2. B) Crystal structure of 4.6.2. Gotor: R N R TMS H + N MeO2C CO2Me MeO2C CO2Me silyl imine Kunai R H TMS TMS + R2N SiMe2Ph KF N CsF MeO2C H H CO2Me azadiene SiMe2Ph NR2 OTf benzyne 2-silylaniline Figure 5.3: Previously reported aminosilylation reactions. 610 Me MeO O NH R1 MeO 2.5 equiv. DIPEA N H + CH2Cl2, rt, 3 h R2 TMS F F TMS N H N MeO O TMS 4.5.1 (84%) Me N OMe TMS N H N H Cl tBu MeO F Me N N 4.6.14 (86%) TMS N H N TMS N H Me N F Me 5.2.1a (30%) F N Me O Me N O N TMS N H O 4.6.12 (67%) MeO N NH2 O F O O MeO N N N H 4.6.4 (48%) 4.6.11 (69%) MeO F N OMe TMS N O Me N N O Me N 4.6.6 (38%) N NH2 O TMS N F 4.6.5 (33%) MeO F N Me N N O MeO Me N MeO N OMe TMS Product B 4.6.3 (31%) MeO Me N N H MeO O 4.6.2 (55%) MeO N N H R2 = o-F S8 R2 = p-F S9 R2 = p-Cl S10 R2 R1 R2 N N N NH2 O MeO Me N N N O Product A MeO Me N N H Me N + N R1 S8 - S10 MeO MeO Me N N 1.25 equiv. TMSCl F Me N N F N TMS N H O N 5.2.1b (64%) 5.2.2 (73%) Figure 5.4: Substrate scope for the guanylation/aminosilylation sequence using TMSCl. 611 O R3 R1 N H R2 N H CN Me N R1 1.25 equiv. BSA R2 N H 2.5 equiv. DIPEA TMS CH2Cl2, rt, 3 h N O R3 MeO MeO Me N N MeO Me TMS F Me N N H Me N O Me O 4.6.2 (57%) N TMS Me N F N N H MeO Me N TMS Me N NC F O TMS Cl TMS F N H N H O O TMS TMS N H N H F O EtO O O TMS Me N TMS Me TMS N H N H N H F N O Me N O EtO O TMS TMS N H TMS N H 5.2.18 (89%) O O N Cl O 5.2.16 (75%) Me N F N Me N O Me N N H 5.2.13 (74%) O OMe O TMS F N N H 5.2.9 (70%) EtO N NC N 5.2.17 (70%) F O 5.2.15 (65% after 24 h) Me N EtO N H 5.2.12 (53%) N 5.2.14 (74%) O N Me N F N Me N EtO Me N F 5.2.8 (58%) 5.2.11 (trace after 24 h) O O MeO TMS 5.2.10 (26% after 24 h) N H 5.2.5 (83%) F Me N EtO N Me N F N TMS F N MeO O F MeO Me N O 5.2.4 (91%) 5.2.7 (80%) 5.2.6 (58%) MeO N N H Me N F3C MeO N N H TMS 4.6.12 (72%) MeO F3C N MeO O F Me N F3C TMS N H N O 5.2.19 (57%) Figure 5.5: Substrate scope for the guanylation/aminosilylation sequence using BSA. 612 a) desilylation/isomerization MeO MeO Me N Me N N MeO TMS K2CO3, MeOH N N H rt, 1 h, 99% O N O F 4.2.6 N N H F OMe ZNA 148 Me N b) desilylation N N H K2CO3, MeOH rt, 1 h, 99% O N F OMe 5.2.20 X-ray structure of 5.2.20 Me N Me N c) bromination N MeO TMS N H N Br2, AgOAc O CH2Cl2, rt, 80% F 5.2.4 Br I2, AgOAc I N H CH2Cl2, rt N + I Me N e) ring expansion N TMS N H F X = Br, I N R= O N OMe F N HCl, MeOH O 50 oC, 16 h 45% F O 5.2.23 (30%) Me N N H N N H N MeO OMe MeO Me N N O F X TMS R F via guanidinium cation F OMe 5.2.21 (single E-alkene isomer) 5.2.22 (51%) steric hindrance Me N N R N TMS O H X O N Me N d) iodination N N H OMe Me N N O MeO 5.2.4 N N H F 5.2.24 Figure 5.6: Transformations of the resultant ene-guanidines. O O N N H + TMSCl N - HB F S8 R + B- C N TMS N H N O TMS F OMe M3 TMS-propargyl guanidine M3 + R N OMe secondary propargyl amine M2 5-exo-dig cyclization and TMS transfer concerted N Me F N-silyl carbodiimide M1 Me N HN R Me N F N MeO N TMS N H O TMS-ene guanidine Figure 5.7: Putative reaction pathways of ene-guanidine synthesis 613 Table 5.1: Initial reaction optimizations. Me O N MeO N H OMe + 4.5.2 1 2 3 Me N Silylating reagent N DIPEA CH2Cl2, rt, 3 h F Entry MeO MeO NH F N MeO N TMS N H Silylating reagent (1.25 equiv.) TMSCl TBDMSCl BSA N NH2 O + O F N 4.6.2 S8 Me N 5.2.3 OMe Product Yield (%) 4.6.2 5.2.3 4.6.2 55 88 57 Table 5.2: Effect of the base on the aminosilylation reaction. MeO Me N F MeO N NH2 O CH2Cl2, rt, time, yield N OMe Entry 1 2 3 4 Me N Conditions 5.2.3 Silylating reagent TMSCl (2 equiv.) TMSCl (1.25 equiv.) BSA (2 equiv.) BSA (1.25 equiv.) F N MeO N TMS N H O 4.6.2 Base none DIPEA (2.5 equiv.) none DIPEA (2.5 equiv.) Time (h) 16 16 3 3 Yield (%) 0 66 76 80 614 5.5 References 1. Coles, M. P., Bicyclic-Guanidines, -Guanidinates and -Guanidinium Salts: Wide Ranging Applications from a Simple Family of Molecules. ChemComm, 2009, 3659-3676. 2. Berlinck, R. G. S.; Romminger, S., The Chemistry and Biology of Guanidine Natural Products. Nat. Prod. Rep., 2016, 33, 456-490. 3. Greenhill, J. V.; Lue, P. Prog. Med. Chem., 1993, 30, 203-326. 4. Schug, K. A.; Lindner, W., Noncovalent Binding between Guanidinium and Anionic Groups: Focus on Biological- and Synthetic-Based Arginine/Guanidinium Interactions with Phosph[on]ate and Sulf[on]ate Residues. Chem. Rev., 2005, 105, 67-114. 5. Selig, P., Guanidine Organocatalysis. Synthesis, 2013, 45, 703-718. 6. Coppola, G. M.; Hardtmann, G. E.; Pfister, O. R., Chemistry of 2H-3,1Benzoxazine-2,4-(1H)-Dione (Isatoic Anhydride). Reactions with Thiopseudoureas and Carbanions. J. Org. Chem., 1976, 41, 825-831. 7. Xie, C.; Runnegar, M. T. C.; Snider, B. B., Total Synthesis of (±)Cylindrospermopsin. J. Am. Chem. Soc., 2000, 122, 5017-5024. 8. Cohen, F.; Overman, L. E.; Ly Sakata, S. K., Asymmetric Total Synthesis of Batzelladine D. Org. Lett., 1999, 1, 2169-2172. 9. Müller, T. E.; Hultzsch, K. C.; Yus, M.; Foubelo, F.; Tada, M., Hydroamination: Direct Addition of Amines to Alkenes and Alkynes. Chem. Rev., 2008, 108, 3795-3892. 10. Ermolat'ev, D. S.; Bariwal, J. B.; Steenackers, H. P.; De Keersmaecker, S. C.; Van der Eycken, E. V., Concise and Diversity-Oriented Route toward Polysubstituted 2Aminoimidazole Alkaloids and their Analogues. Angew. Chem. Int. Ed., 2010, 49, 94659468. 11. Gainer, M. J.; Bennett, N. R.; Takahashi, Y.; Looper, R.E., Regioselective Rhodium(II)-Catalyzed Hydroaminations of Propargylguanidines. Angew. Chem. Int. Ed., 2011, 50, 684-687. 12. Ritter, S.; Horino, Y.; Lex, J.; Schmalz, H.-G., Gold-Catalyzed Cyclization of OPropargyl Carbamates under Mild -Conditions: A Convenient Access to 4-Alkylidene-2Oxazolidinones. Synlett, 2006, 19, 3309-3313. 13. Alamsetti, S. K.; Persson, A. K. Å.; Bäckvall, J.-E., Palladium-Catalyzed Intramolecular Hydroamination of Propargylic Carbamates and Carbamothioates. Org. Lett., 2014, 16, 1434-1437. 615 14. Gibbons, J. B.; Salvant, J. M.; Vaden, R. M.; Kwon, K.-H.; Welm, B. E.; Looper, R. E., Synthesis of Naamidine A and Selective Access to N2-Acyl-2-Aminoimidazole Analogues. J. Org. Chem., 2015, 80, 10076-10085. 15. Bhonde, V. R.; Looper, R. E., A Stereocontrolled Synthesis of (+)-Saxitoxin. J. Am. Chem. Soc., 2011, 133, 20172-20174. 16. Fedoseev, P.; Sharma, N.; Khunt, R.; Ermolat'ev, D. S.; Van der Eycken, E. V., Iodine-Mediated Regioselective Guanylation-Amination of Propargylamines towards the Synthesis of Diversely Substituted 2-Aminoimidazoles. RSC Adv., 2016, 6, 75202-75206. 17. Sullivan, J. D.; Giles, R. L.; Looper, R. E., 2-Aminoimidazoles from Leucetta Sponges: Synthesis and Biology of an Important Pharmacophore. Curr. Bioact. Compd., 2009, 5, 39-78. 18. Copp, B. R.; Fairchild, C. R.; Cornell, L.; Casazza, A. M.; Robinson, S.; Ireland, C. M., Naamidine A Is an Antagonist of the Epidermal Growth Factor Receptor and an In Vivo Active Antitumor Agent. J. Med. Chem., 1998, 41, 3909-3911. 19. Barluenga, J.; Tomás, M.; Ballesteros, A.; Gotor, V., An Easy Synthesis of Electron-Withdrawing Substituted 2-Aza-1,3-Dienes and Their 1,4-Cycloaddition with Enamines. J. Chem. Soc., Chem. Commun., 1987, 1195-1196. 20. Yoshida, H.; Minabe, T.; Ohshita, J.; Kunai, A., Aminosilylation of Arynes with Aminosilanes: Synthesis of 2-Silylaniline Derivatives. ChemComm, 2005, 3454-3456. 21. Klebe, J. F.; Finkbeiner, H.; White, D. M., Silylations with Bis(trimethylsilyl)acetamide, a Highly Reactive Silyl Donor. J. Am. Chem. Soc., 1966, 88, 3390-3395. 22. Salvant, J. M.; Edwards, A. V.; Kurek, D. Z.; Looper, R. E., Regioselective BaseMediated Cyclizations of Mono-N-acylpropargylguanidines. J. Org. Chem., 2017, 82, 6958-6967. 23. Jenkins, P. R.; Symons, M. C. R.; Booth, S. E.; Swain, C. J., Why Is Vinyl Anion Configurationally Stable but a Vinyl Radical Configurationally Unstable? Tetrahedron Lett., 1992, 33, 3543-3546. 24. Caramella, P.; Houk, K. N., The Influence of Electron-Withdrawing Substituents on the Geometries and Barriers to Inversion of Vinyl Anions. Tetrahedron Lett., 1981, 22, 819-822. 25. Fawcett, J.; House, S.; Jenkins, P. R.; Lawrence, N. J.; Russell, D. R., Stereoselective Reactions of Lithio-Vinylsulf Oxides with Aldehydes. J. Chem. Soc. Perkin Ttrans. 1, 1993, 67-73. 26. Synthesis of C4-Aryl-N2-Acylaminoimidazoles. Justin M. Salvant, Emily K. Kirkeby, Wenxing Guo, Ryan E. Looper, Manuscript in preparation. 616 5.6 Supporting Information 5.6.1 General Experimental Conditions (Chemistry) All reactions requiring anhydrous conditions were performed under a positive pressure of nitrogen using flame-dried glassware. Commercially available reagents were used as received or purified according to Purification of Laboratory Chemicals. Dimethylformamide (DMF), tetrahydrofuran (THF), acetonitrile (MeCN), and dichloromethane (CH2Cl2) were degassed with nitrogen and passed through a solvent purification system (Innovative Technologies Pure Solv). Methanol was distilled from molecular sieves immediately prior to use. DIPEA was distilled from CaH2 immediately prior to use. Reactions were monitored to completion by TLC and visualized by a dual short/long wave UV lamp and stained with an aqueous solution of potassium permanganate. Flash chromatography was performed on silica gel SiliaFlash P60 (40-63 µm) from Silicycle. 1 H NMR spectra were recorded at 500 MHz as indicated. The chemical shifts (δ) of proton resonances are reported relative to the deuterated solvent peak: 7.26 for CDCl3 and 4.87 for H2O in CD3OD, using the following format: chemical shift [multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, app = apparent), coupling constant(s) (J in Hz), integral]. 13C NMR spectra were recorded at 125 MHz. The chemical shifts of carbon resonances are reported relative to the deuterated solvent peak: 77.3 (center line) for CDCl3 and 49.0 (center line) for CD3OD. Mass spectra were obtained by ESI/APCI for LRMS or ESI/APCITOF for HRMS. 617 5.6.2 Procedures and Characterizations General procedure C1 for the preparation of N-Boc-propargyl amines MeO MeO NHBoc O S O NHBoc N + MeO nBuLi or NHBoc or THF, -78 oC N N N A 100-mL oven-dried round bottom flask with a magnetic stirring bar was charged with 9.50 mmol of 3- or 4-ethynylpyridine and 30 mL of anhydrous THF. The stirring solution was cooled down to -78 oC and a 2.5M n-BuLi-solution in hexanes (3.50 mL, 8.70 mmol) was added via syringe. The resulting mixture was stirred at the same temperature for 30 min. Then the amino sulfone S4.2 (1.43 g, 3.8 mmol) in 25 mL THF was added to the mixture via syringe. The reaction mixture was stirred at -78 oC for 3 h and quenched with 100 mL of sat. aq. NH4Cl solution. The product was extracted twice with 100 mL EtOAc. The organic layer was concentrated and purified via flash column chromatography. MeO NHBoc N Tert-butyl (1-(4-methoxyphenyl)-3-(pyridin-3-yl)prop-2-yn-1-yl)carbamate (S17). Prepared with and S4.2 (1.43 g, 3.8 mmol) and 3-ethynylpyridine (0.98 mL, 9.50 mmol) following general procedure C1. The crude product was purified via flash column chromatography (7:2 hexanes/EtOAc, followed by 2:1 hexanes/EtOAc) to yield a white solid (1.176 g, 91%). Rf = 0.1 (4:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 8.63 (d, J = 2.0 Hz, 1H), 8.46 (d, J = 6.0 Hz, 1H), 7.67 (d, J = 8.0 Hz, 1H), 7.43 (d, J = 8.0 Hz, 2H), 7.17 (dd, J = 8.0, 5.0 Hz, 1H), 6.85 (d, J = 8.5 Hz, 2H), 5.82 (s, 1H), 5.76 (s, 1H), 618 3.75 (s, 3H), 1.43 (s, 9H). 13 C NMR (125 MHz, CDCl3): δ 159.4, 154.9, 152.3, 148.6, 138.6, 131.2, 128.1, 122.9, 119.9, 114.0, 91.6, 81.0, 80.1, 55.3, 46.2, 28.38. IR (thin film): 3202, 2969, 2929, 1704, 1540, 1511, 1366, 1304, 1253, 1170, 1024, 838, 735, 699 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C20H23N2O3, 339.1709; found, 339.1717. MeO NHBoc N Tert-butyl (1-(4-methoxyphenyl)-3-(pyridin-4-yl)prop-2-yn-1-yl)carbamate (S18). Prepared with and S4.2 (1.0 g, 2.65 mmol) and 4-ethynylpyridine (0.76 g, 7.42 mmol) following general procedure C1. The crude product was purified via flash column chromatography (3:1 hexanes/EtOAc, followed by 2:1 hexanes/EtOAc) to yield a white solid (0.78 g, 86%). Rf = 0.2 (1:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 8.55 (d, J = 5.5 Hz, 2H), 7.44 (d, J = 8.5 Hz, 2H), 7.30 (d, J = 5.5 Hz, 2H), 6.90 (d, J = 8.5 Hz, 2H), 5.83 (brs, 1H), 5.16 (brs, 1H), 3.80 (s, 3H), 1.46 (s, 9H). 13C NMR (125 MHz, CDCl3): δ 159.5, 154.7, 149.7, 130.9, 130.8, 128.2, 125.7, 114.1, 92.8, 81.9, 80.3, 55.3, 46.3, 28.3. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C20H23N2O3, 339.1630; found, 339.1709. General procedure C2 for the methylation of N-Boc-propargyl amines MeO MeO NHBoc MeI, NaH DMF, 0 Me N Boc oC To a solution of 3.47 mmol N-Boc propargyl amine in 15 mL anhydrous DMF at 0 o C was added iodomethane (2.2 mL, 34.7 mmol), followed by NaH (60wt% dispersed in mineral oil, 152.70 mg, 3.82 mmol). The reaction mixture was stirred at the same 619 temperature for 2 h and quenched with 50 mL of sat. aq. NH4Cl solution. The product was extracted two times with 60 mL Et2O. The organic layer was concentrated and purified via flash column chromatography. MeO Me N Boc N Tert-butyl (1-(4-methoxyphenyl)-3-(pyridin-3-yl)prop-2-yn-1-yl)(methyl)- carbamate (S19). Prepared with and S17 (1.17 g, 3.47 mmol) following general procedure C2. The crude product was purified via flash column chromatography (3:1 hexanes/EtOAc, followed by 2:1 hexanes/EtOAc) to yield a yellow oil (0.72 g, 59%). Rf = 0.25 (2:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 8.71 (s, 1H), 8.51 (d, J = 3.0 Hz, 1H), 7.74 (d, J = 7.5 Hz, 1H), 7.40 (d, J = 8.0 Hz, 2H), 7.23 (dd, J = 7.5, 5.0 Hz, 1H), 6.87 (d, J = 8.5 Hz, 2H), 6.59 (brs, 1H), 3.77 (s, 3H), 2.71 (s, 3H), 1.49 (s, 9H). 13C NMR (125 MHz, CDCl3): δ 159.4, 152.4, 148.8, 138.6, 129.1, 128.5, 123.0, 119.8, 113.9, 89.4, 82.8, 80.3, 55.2, 50.6, 29.3, 28.4. IR (thin film): 2931, 2836, 1684, 1585, 1509, 1474, 1364, 1304, 1246, 1172, 1142, 1024, 895, 759, 703 cm-1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C21H25N2O3, 353.1865; found, 353.1863. MeO Me N Boc N Tert-butyl (1-(4-methoxyphenyl)-3-(pyridin-4-yl)prop-2-yn-1-yl)(methyl)- carbamate (S20). Prepared with and S18 (0.76 g, 2.25 mmol) following general procedure C2. The crude product was purified via flash column chromatography (5:1 hexanes/EtOAc, 620 followed by 4:1 – 2:1 hexanes/EtOAc) to yield a yellow solid (0.24 g, 30%). Rf = 0.35 (1:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 8.55 (s, 2H), 7.38 (d, J = 5.5 Hz, 2H), 7.32 (s, 2H), 6.87 (d, J = 6.0 Hz, 2H), 6.59 (brs, 1H), 6.36 (brs, 1H), 3.76 (s, 3H), 2.71 (s, 3H), 1.49 (s, 9H). 13C NMR (125 MHz, CDCl3): δ 159.4, 149.8, 130.8, 128.8, 128.5, 125.8, 113.9, 90.8, 83.6, 80.4, 55.2, 50.6, 29.3, 28.4. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C21H25N2O3, 353.1787; found, 353.1865. General procedure C3 for the Boc-deprotections MeO MeO Me N Boc 2M HCl in Et2O NHMe r.t. To 2.04 mmol of an N-Boc propargyl amine in a 50 mL round bottom flask was added at 12 mL of 2M HCl in Et2O. The resulting mixture was stirred at room temperature for 6 h and quenched with 30 mL of sat. aq. NaHCO3 solution. The crude product was extracted two times with 30 mL CH2Cl2. The organic layer was concentrated and purified via flash column chromatography to yield the N-methyl amine. MeO NHMe N 1-(4-Methoxyphenyl)-N-methyl-3-(pyridin-3-yl)prop-2-yn-1-amine (5.1.1). Prepared with and S19 (718.6 mg, 2.04 mmol) following general procedure C3. The crude product was purified via flash column chromatography (2:1 hexanes/acetone) to yield a dark brown oil (434.6 mg, 84%). Rf = 0.3 (1:1 hexanes/acetone). 1H NMR (CDCl3, 500 MHz): δ 8.60 (s, 1H), 8.38 (s, 1H), 7.60 (d, J = 7.0 Hz, 1H), 7.37 (d, J = 8.0 Hz, 2H), 7.08 621 (dd, J = 7.5, 4.5 Hz, 1H), 6.79 (d, J = 8.5 Hz, 2H), 4.59 (s, 1H), 3.65 (s, 3H), 2.41 (s, 3H), 1.55 (s, 1H). 13 C NMR (125 MHz, CDCl3): δ 159.2, 152.3, 148.4, 138.4, 131.9, 128.6, 122.9, 120.2, 113.8, 93.0, 82.0, 55.6, 55.1, 33.7. MeO NHMe N 1-(4-Methoxyphenyl)-N-methyl-3-(pyridin-4-yl)prop-2-yn-1-amine (5.1.2). Prepared with and S20 (242.4 mg, 0.69 mmol) following general procedure C3. The crude product was purified via flash column chromatography (1:1 hexanes/acetone) to yield a yellow oil (132.2 mg, 76%). Rf = 0.30 (1:1 hexanes/EtOAc). It was used in the subesequent reaction without further purifications. General procedure C4 for the A3-coupling reactions OMe MeO H CuBr, MeCN O N H 80 oC N Me R Me R To a 25 mL pressure flask equipped with a stir bar were added 6.17 mmol of a 4substituted phenylacetylene, 4-methoxybenzaldehyde (0.5 mL, 4.11 mmol), Nallylmethylamine (0.94 mL, 5.76 mmol), CuBr (117.60 mg, 0.82 mmol), acetonitrile (10 mL), and 10 mg of oven-dried 4 Å molecular sieves. The flask was sealed and heated at 80 °C for 24 h and then allowed to cool to room temperature. The mixture was filtered through Celite and rinsed with EtOAc (20 mL). The organic layer was washed with sat. aq. NaHCO3 (15 mL), brine (15 mL) and dried over Na2SO4. After filtration, the organic layer was 622 concentrated under reduced pressure and purified via flash column chromatography to obtain the desired propargyl amine. MeO Me N CF3 N-(1-(4-methoxyphenyl)-3-(4-(trifluoromethyl)phenyl)prop-2-yn-1-yl)-Nmethyl-prop-2-en-1-amine (S21). Prepared with 1-ethynyl-4-(trifluoromethyl)benzene (0.58 mL, 6.17 mmol) following general procedure C4. The crude product was purified via flash column chromatography (15:1 hexanes/EtOAc, followed by 10:1 hexanes/EtOAc) to yield a yellow oil (1.09 g, 73%). Rf = 0.4 (8:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 7.68 – 7.59 (m, 4H), 7.57 – 7.53 (m, 2H), 6.98 – 6.90 (m, 2H), 6.00 – 5.89 (m, 1H), 5.31 (d, J = 16.5 Hz, 1H), 5.20 (d, J = 9.0 Hz, 1H), 4.96 (s, 1H), 3.82 (s, 3H), 3.26 – 3.12 (m, 2H), 2.25 (s, 3H). 13 C NMR (125 MHz, CDCl3): δ 159.2, 136.1, 132.1, 130.0, 129.9 (q, JCF = 32.3 Hz), 129.5, 128.6, 127.1, 125.4 (q, JCF = 3.8 Hz), 125.2 (q, JCF = 3.6 Hz), 123.0 (q, JCF = 257.0 Hz), 117.6, 113.6, 88.2, 86.8, 59.2, 57.7, 55.2. HRMS (ESITOF) [M + H]+ m/z: calcd for C21H21F3NO, 360.1497; found, 360.1575. MeO Me N Cl N-(3-(4-chlorophenyl)-1-(4-methoxyphenyl)prop-2-yn-1-yl)-N-methylprop-2en-1-amine (S22). Prepared with 1-chloro-4-ethynylbenzene (1.57 g, 11.52 mmol) following general procedure C4. The crude product was purified via flash column 623 chromatography (10:1 hexanes/EtOAc) to yield an orange oil (1.96 g, 73%). Rf = 0.4 (10:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 7.55 (dd, J = 8.7, 1.5 Hz, 2H), 7.45 (d, J = 8.4 Hz, 2H), 7.32 (d, J = 8.4 Hz, 2H), 6.92 (dd, J = 8.7, 1.2 Hz, 2H), 6.01 – 5.85 (m, 1H), 5.30 (d, J = 17.1 Hz, 1H), 5.19 (d, J = 10.2 Hz, 1H), 4.93 (s, 1H), 3.81 (s, 3H), 3.18 – 3.15 (m, 2H), 2.23 (s, 3H). 13C NMR (75 MHz, CDCl3): δ 159.3, 136.4, 134.3, 133.3, 130.9, 129.7, 128.9, 122.0, 117.9, 113.8, 87.3, 86.7, 59.4, 58.0, 55.5, 38.0. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C20H21ClNO, 326.1233; found, 326.1312. MeO Me N CN 4-(3-(Allyl(methyl)amino)-3-(4-methoxyphenyl)prop-1-yn-1-yl)benzonitrile (S23). Prepared with 4-ethynylbenzonitrile (0.99 g, 5.62 mmol) following general procedure C4. The crude product was purified via flash column chromatography (15:1 hexanes/EtOAc, followed by 10:1 hexanes/EtOAc) to yield a yellow oil (0.70 g, 40%). Rf = 0.2 (8:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 7.65 – 7.56 (m, 4H), 7.49 (d, J = 8.7 Hz, 2H), 6.90 (d, J = 8.7 Hz, 2H), 5.97 – 5.82 (m, 1H), 5.27 (d, J = 17.1 Hz, 1H), 5.17 (d, J = 10.2 Hz, 1H), 4.93 (s, 1H), 3.81 (s, 3H), 3.22 – 3.07 (m, 2H), 2.21 (s, 3H). 13C NMR (75 MHz, CDCl3): δ 159.4, 136.1, 132.6, 132.3, 130.4, 129.7, 128.3, 118.8, 118.1, 113.8, 111.7, 90.7, 86.8, 59.4, 58.0, 55.5, 38.0. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C21H21N2O, 317.1576; found, 317.1656. 624 MeO Me N F F N-(3-(3,5-difluorophenyl)-1-(4-methoxyphenyl)prop-2-yn-1-yl)-Nmethylprop-2-en-1-amine (S24). Prepared with 1-ethynyl-3,5-difluorobenzene (0.86 mL, 7.24 mmol) following general procedure C4. The crude product was purified via flash column chromatography (10:1 hexanes/EtOAc) to yield a yellow oil (1.22 g, 72%). Rf = 0.4 (8:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 1H NMR (CDCl3, 500 MHz): δ 7.51 (d, J = 8.5 Hz, 2H), 7.05 (dd, J = 7.5, 2.5 Hz, 2H), 6.91 (d, J = 8.5 Hz, 2H), 6.81 (tt, J = 9.0, 2.0 Hz, 1H), 5.96 – 5.87 (m, 1H), 5.29 (d, J = 18.0 Hz, 1H), 5.18 (d, J = 10.5 Hz, 1H), 4.92 (s, 1H), 3.83 (s, 3H), 3.18 (dd, J = 11.5, 5.5 Hz, 1H), 3.12 (dd, J = 13.5, 7.5 Hz, 1H), 2.22 (s, 3H). 13C NMR (125 MHz, CDCl3): 163.6 (d, JCF = 13.4 Hz), 161.7 (d, JCF = 13.4 Hz), 159.1, 136.0, 130.3, 129.4, 125.8 (t, JCF = 11.6 Hz), 117.8, 114.8 (d, JCF = 6.4 Hz), 113.6, 104.3 (t, JCF = 25.1 Hz), 87.8, 86.0 (t, JCF = 3.7 Hz), 59.1, 57.7, 55.3, 37.7. MeO Me N O OEt Ethyl 4-(3-(allyl(methyl)amino)-3-(4-methoxyphenyl)prop-1-yn-1-yl)benzoate (S25). Prepared from 4-methoxybenzaldehyde (0.45 mL, 3.75 mmol), ethyl 4ethynylbenzoate (0.85 g, 4.88 mmol), N-allylmethylamine (0.53 mL, 5.63 mmol) and CuBr (108 mg, 0.75 mmol) following general procedure C4. The crude product was purified via flash column chromatography (10:1 hexanes/EtOAc) to yield an orange oil 625 (0.98 g, 72%). Rf = 0.4 (6:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 8.02 (d, J = 8.0 Hz, 2H), 7.57 (d, J = 8.0 Hz, 2H), 7.53 (d, J = 8.5 Hz, 2H), 6.90 (d, J = 8.5 Hz, 2H), 6.96 – 6.87 (m, 1H), 5.90 (ddt, J = 16.5, 11.0, 6.5 Hz, 1H), 5.28 (d, J = 18.5 Hz, 1H), 5.17 (d, J = 10.0 Hz, 1H), 4.94 (s, 1H), 4.39 (q, J = 7.0 Hz, 2H), 3.81 (s, 3H), 3.23 – 3.13 (m, 2H), 2.23 (s, 3H), 1.40 (t, J = 7.5 Hz, 3H) ppm. 13C NMR (125 MHz, CDCl3): δ 166.0, 159.1, 136.1, 131.7, 130.6, 130.0, 129.8, 129.5, 129.4, 127.8, 122.6, 117.6, 113.6, 88.7, 87.5, 61.1, 59.3, 57.7, 55.2, 37.7, 14.3 ppm. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C23H26NO3, 364.1913; found, 364.1911. MeO Me N F N-(3-(4-Fluorophenyl)-1-(4-methoxyphenyl)prop-2-yn-1-yl)-N-methylprop-2en-1-amine (S26). Prepared from 4-methoxybenzaldehyde (0.72 mL, 5.94 mmol), 1fluoro-4-ethynylbenzene (1 g, 8.32 mmol), N-allylmethylamine (0.85 mL, 8.91 mmol) and CuBr (0.17 g, 1.64 mmol) following general procedure C4. The crude product was purified via flash column chromatography (10:1 hexanes/EtOAc) to yield an orange oil (1.24 g, 67%). Rf = 0.4 (15:1 hexanes/EtOAc, followed by 10:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 7.57 (d, J = 8.5 Hz, 2H), 7.52 (dd, J = 7.5, 5.5 Hz, 2H), 7.05 (t, J = 8.5 Hz, 2H), 6.93 (d, J = 8.5 Hz, 2H), 5.99 – 5.90 (m, 1H), 5.31 (d, J = 17.5 Hz, 1H), 5.19 (d, J = 10.0 Hz, 1H), 4.94 (s, 1H), 3.82 (s, 3H), 3.25 – 3.15 (m, 2H), 2.23 (s, 3H) ppm. 13C NMR (125 MHz, CDCl3): δ 162.4 (d, JCF = 248.4 Hz), 159.1, 136.3, 133.6 (d, JCF = 8.7 Hz), 130.9, 129.5, 119.4 (d, JCF = 3.7 Hz), 117.5, 115.5 (d, JCF = 21.2 Hz), 113.5, 87.0, 85.1, 59.3, 57.7, 55.2, 37.7 ppm. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C20H21FNO, 626 310.1607; found, 310.1608. MeO Me N F N-(3-(3-Fluorophenyl)-1-(4-methoxyphenyl)prop-2-yn-1-yl)-N-methylprop-2en-1-amine (S27). Prepared from 4-methoxybenzaldehyde (0.72 mL, 5.94 mmol), 1fluoro-4-ethynylbenzene (1 g, 8.32 mmol), N-allylmethylamine (0.85 mL, 8.91 mmol) and CuBr (0.17 g, 1.64 mmol) following general procedure C4. The crude product was purified via flash column chromatography (20:1 hexanes/EtOAc) to yield a yellow oil (1.23 g, 67%). Rf = 0.4 (8:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 7.54 (d, J = 8.5 Hz, 2H), 7.34 – 7.29 (m, 2H), 7.23 (d, J = 9.0 Hz, 1H), 7.08 – 7.04 (m, 1H), 6.93 (d, J = 8.5 Hz, 2H), 5.92 (ddt, J = 17.0, 11.0, 6.5 Hz, 1H), 5.29 (d, J = 17.0 Hz, 1H), 5.19 (d, J = 10.0 Hz, 1H), 4.93 (s, 1H), 3.83 (s, 3H), 3.22 – 3.13 (m, 2H), 2.23 (s, 3H) ppm. 13C NMR (125 MHz, CDCl3): δ 162.4 (d, JCF = 245.0 Hz), 159.1, 136.1, 130.6, 129.9 (d, JCF = 8.7 Hz), 129.5, 127.7 (d, JCF = 2.5 Hz), 125.0 (d, JCF = 10.0 Hz), 118.6 (d, JCF = 22.5 Hz), 117.7, 115.5 (d, JCF = 21.2 Hz), 113.5, 86.9, 86.4, 59.1, 57.7. 55.3, 37.7 ppm. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C20H21NOF, 310.1607; found, 310.1611. Me N O Ethyl OEt 4-(3-(allyl(methyl)amino)-4-phenylbut-1-yn-1-yl)benzoate (S28). Prepared from 2-phenylacetaldehyde (0.42 mL, 3.62 mmol), ethyl 4-ethynylbenzoate (0.82 g, 4.71 mmol), N-allylmethylamine (0.51 mL, 5.43 mmol) and CuBr (104 mg, 0.72 mmol) 627 following general procedure C4. The crude product was purified via flash column chromatography (20:1 hexanes/EtOAc, followed by 10:1 hexanes/EtOAc) to yield a yellow oil (0.56 g, 44%). Rf = 0.3 (8:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 7.96 (d, J = 8.5 Hz, 2H), 7.42 (d, J = 8.5 Hz, 2H), 7.35 – 7.28 (m, 4H), 7.26 – 7.21 (m, 1H), 5.85 (ddt, J = 16.5, 10.5, 6.5 Hz, 1H), 5.23 (dd, J = 17.0, 1.5 Hz, 1H), 5.15 (dd, J = 10.0, 1.5 Hz, 1H), 4.37 (q, J = 7.0 Hz, 2H), 3.90 (dd, J = 9.0, 5.5 Hz, 1H), 3.24 (dd, J = 13.5, 6.0 Hz, 1H), 3.12 (dd, J = 13.5, 7.0 Hz, 1H), 3.04 (dd, J = 13.0, 6.0 Hz, 1H), 2.98 (dd, J = 13.0, 9.5 Hz, 1H), 2.38 (s, 3H), 1.39 (t, J = 7.5 Hz, 3H) ppm. 13C NMR (125 MHz, CDCl3): δ 166.1, 138.5, 135.8, 131.5, 129.6, 129.40, 129.36, 128.2, 127.9, 126.5, 117.8, 90.0, 86.3, 61.1, 58.3, 58.2, 40.2, 37.8 ppm. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C23H26NO2, 348.1964; found, 348.1965. Me N O OEt Ethyl 3-(3-(allyl(methyl)amino)-4-phenylbut-1-yn-1-yl)benzoate (S29). Prepared from 2-phenylacetaldehyde (0.72 mL, 6.14 mmol), ethyl 3-ethynylbenzoate (1.39 g, 7.98 mmol), N-allylmethylamine (0.87 mL, 9.21 mmol) and CuBr (176 mg, 1.23 mmol) following general procedure C4. The crude product was purified via flash column chromatography (20:1 hexanes/EtOAc, followed by 15:1 hexanes/EtOAc) to yield a yellow oil (1.41 g, 63%). Rf = 0.1 (8:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 8.09 (s, 1H), 7.99 (d, J = 8.0 Hz, 1H), 7.57 (d, J = 7.5 Hz, 1H), 7.39 (t, J = 7.5 Hz, 1H), 7.36 – 7.32 (m, 4H), 7.30 – 7.25 (m, 1H), 5.92 – 5.83 (m, 1H), 5.27 (d, J = 17.0 Hz, 1H), 5.19 (d, J = 10.5 Hz, 1H), 4.41 (q, J = 7.0 Hz, 2H), 3.92 (dd, J = 9.0, 6.0 Hz, 1H), 3.28 (dd, J = 13.5, 628 5.5 Hz, 1H), 3.15 (dd, J = 13.5, 7.5 Hz, 1H), 3.09 – 2.99 (m, 2H), 2.41 (s, 3H), 1.44 (t, J = 7.0 Hz, 3H) ppm. 13C NMR (125 MHz, CDCl3): δ 166.0, 138.6, 135.9, 135.8, 132.7, 130.7, 129.5, 128.9, 128.34, 128.26, 126.5, 123.7, 117.8, 87.7, 85.9, 61.2, 58.4, 58.1, 40.3, 37.8, 14.4 ppm. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C23H26NO2, 348.1964; found, 348.1970. Me N Me Me CN 4-(3-(Allyl(methyl)amino)-5-methylhex-1-yn-1-yl)benzonitrile (S30). Prepared from 3-methylbutanal (1 mL, 9.11 mmol), 4-ethynylbenzonitrile (1.62 g, 12.8 mmol), Nallylmethylamine (1.3 mL, 13.67 mmol) and CuBr (261 mg, 1.82 mmol) following general procedure C4. The crude product was purified via flash column chromatography (10:1 hexanes/EtOAc) to yield a yellow oil (2.28 g, 94%). Rf = 0.3 (8:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 7.57 (d, J = 8.5 Hz, 2H), 7.48 (d, J = 8.5 Hz, 2H), 5.84 (ddt, J = 17.0, 11.0, 6.5 Hz, 1H), 5.22 (dd, J = 17.0, 1.0 Hz, 1H), 5.14 (d, J = 10.0 Hz, 1H), 3.73 (t, J = 7.5 Hz, 1H), 3.17 (dd, J = 13.5, 5.5 Hz, 1H), 3.03 (dd, J = 13.5, 7.5 Hz, 1H), 2.26 (s, 3H), 1.85 (septet, J = 6.5 Hz, 1H), 1.59 (m, 2H), 0.94 (t, J = 7.5 Hz, 6H) ppm. 13C NMR (125 MHz, CDCl3): δ 136.0, 132.2, 131.9, 128.4, 118.5, 117.5, 111.2, 92.8, 84.4, 58.3, 54.1, 42.4, 37.5, 25.1, 22.6, 22.3 ppm. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C18H23N2, 267.1861; found, 267.1864. 629 Me N CF3 N-Methyl-N-(3-(4-(trifluoromethyl)phenyl)prop-2-yn-1-yl)prop-2-en-1-amine (S31). Prepared from formaldehyde, 1-ethynyl-4-trifluoromethylbenzene, and Nallylmethylamine following general procedure C4.11 General procedure C5 for the deallylation reactions NHR NHR DMBA Pd(PPh3)4 N Me CH2Cl2, r.t. N H Me To a 100-mL oven-dried round bottom flask equipped with a stir bar was added 3.03 mmol of the N-allyl propargylamine, 1,3-dimethylbarbituric acid (0.95 g, 6.06 mmol), Pd(PPh3)4 (175.20 mg, 0.15 mmol) and 25 mL anhydrous CH2Cl2. The reaction mixture was allowed to stir at room temperature under N2 overnight. The reaction mixture was concentrated and redissolved in EtOAc (100 mL). The organic layer was washed with saturated NaHCO3 (100 mL), brine (100 mL) and dried over Na2SO4. After filtration, the organic layer was concentrated and purified via flash column chromatography to obtain the secondary propargyl amine. 630 MeO Me NH CF3 1-(4-Methoxyphenyl)-N-methyl-3-(4-(trifluoromethyl)phenyl)prop-2-yn-1amine (5.1.3). Prepared with S21 (1.09 g, 3.03 mmol) following general procedure C5. The crude product was purified via flash column chromatography (1:1 hexanes/EtOAc) to yield a yellow oil (566.1 mg, 58%). Rf = 0.1 (1:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 7.56 (s, 4H), 7.49 (d, J = 8.5 Hz, 2H), 6.92 (d, J = 8.5 Hz, 2H), 4.71 (s, 1H), 3.80 (s, 3H), 2.54 (s, 3H), 1.57 (brs, 1H). 13C NMR (125 MHz, CDCl3): δ 159.3, 132.0, 131.9, 129.8 (q, JCF = 32.5 Hz), 128.7, 127.0 (q, JCF = 1.5 Hz), 125.0 (q, JCF = 3.8 Hz), 122.9 (q, JCF = 270.8 Hz), 113.9, 92.0, 84.1, 55.7, 55.2, 33.7. HRMS (ESI-TOF) [M - H]+ m/z: calcd for C18H15F3NO, 318.1184; found, 318.1106. MeO Me NH Cl 3-(4-Chlorophenyl)-1-(4-methoxyphenyl)-N-methylprop-2-yn-1-amine (5.1.4). Prepared with S22 (1.96 g, 6.01 mmol) following general procedure C5. The crude product was purified via flash column chromatography (1:1 hexanes/EtOAc) to yield a yellow oil (1.24 g, 72%). Rf = 0.15 (1:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 7.46 (d, J = 7.2 Hz, 2H), 7.35 (d, J = 8.1 Hz, 2H), 7.23 (d, J = 7.5 Hz, 2H), 6.88 (d, J = 6.6 Hz, 2H), 4.65 (s, 1H), 3.74 (s, 3H), 2.49 (s, 3H), 1.52 (brs, 1H). 13C NMR (75 MHz, CDCl3): δ 159.5, 134.3, 133.2, 132.4, 129.0, 128.9, 121.9, 114.1, 90.7, 84.5, 55.9, 55.4, 34.0. HRMS (ESI- 631 TOF) [M - H]+ m/z: calcd for C17H15ClNO, 284.0920; found, 284.0842. MeO Me NH CN 4-(3-(4-Methoxyphenyl)-3-(methylamino)prop-1-yn-1-yl)benzonitrile (5.1.5). Prepared with S23 (695.3 mg, 2.19 mmol) following general procedure C5. The crude product was purified via flash column chromatography (1:1 hexanes/EtOAc) to yield a yellow oil (374.4 mg, 62%). Rf = 0.1 (1:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 7.59 (d, J = 8.4 Hz, 2H), 7.52 (d, J = 7.8 Hz, 2H), 7.45 (d, J = 8.7 Hz, 2H), 6.90 (d, J = 8.7 Hz, 2H), 4.70 (s, 1H), 2.87 (s, 3H), 2.52 (s, 3H), 1.49 (brs, 1H). 13C NMR (75 MHz, CDCl3): δ 159.6, 132.5, 132.4, 132.3, 132.2, 131.9, 128.9, 128.8, 128.7, 128.3, 118.7, 114.2, 111.7, 94.4, 84.1, 56.0, 55.6, 34.1. HRMS (ESI-TOF) [M - H]+ m/z: calcd for C18H15N2O, 275.1263; found, 275.1184. MeO Me NH F F 3-(3,5-Difluorophenyl)-1-(4-methoxyphenyl)-N-methylprop-2-yn-1-amine (5.1.6). Prepared with S24 (1.21 g, 3.66 mmol) following general procedure C5. The crude product was purified via flash column chromatography (1:1 hexanes/EtOAc) to yield a yellow oil (0.66 g, 55%). Rf = 0.2 (1:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 7.46 (d, J = 8.5 Hz, 2H), 7.00 – 6.96 (m, 2H), 6.92 (d, J = 8.5 Hz, 2H), 6.78 (tt, J = 9.0, 2.5 Hz, 1H), 4.69 (s, 1H), 3.82 (s, 3H), 2.53 (s, 3H), 1.51 (brs, 1H). 13 C NMR (125 MHz, 632 CDCl3): δ 162.7 (d, JCF = 13.2 Hz), 162.6 (d, JCF = 13.2 Hz), 159.3, 131.8, 128.7, 125.8 (t, JCF = 11.7 Hz), 114.68 (d, JCF = 26.5 Hz), 114.68 (d, JCF = 13.6 Hz), 113.9, 104.3 (t, JCF = 25.1 Hz), 91.6, 83.3, 55.6, 55.3, 33.8. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C17H16F2NO, 288.1122; found, 288.1200. MeO Me NH O Ethyl OEt 4-(3-(4-methoxyphenyl)-3-(methylamino)prop-1-yn-1-yl)benzoate (5.1.7). Prepared with S25 (0.93 g, 2.56 mmol) following general procedure C5. The crude product was purified via flash column chromatography (2:1 hexanes/EtOAc) to yield a yellow oil (0.78 g, 94%). Rf = 0.2 (1:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 87.00(d, J = 8.5 Hz, 2H), 7.52 (d, J = 8.5 Hz, 2H), 7.49 (d, J = 8.5 Hz, 2H), 6.91 (d, J = 8.5 Hz, 2H), 4.71 (s, 1H), 4.37 (q, J = 7.0 Hz, 2H), 3.81 (s, 3H), 2.55 (s, 3H), 1.63 (brs, 1H), 1.39 (t, J = 7.0 Hz, 3H) ppm. 13C NMR (125 MHz, CDCl3): δ 166.1, 159.3, 132.0, 131.6, 130.0, 129.8, 129.5, 129.4, 128.7, 127.8, 114.5, 113.9, 92.4, 84.8, 61.1, 55.7, 55.3, 33.8, 14.3 ppm. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C20H22NO3, 324.1600; found, 324.1609. 633 MeO Me NH F 3-(4-Fluorophenyl)-1-(4-methoxyphenyl)-N-methylprop-2-yn-1-amine (5.1.8). Prepared with S26 (1.24 g, 4.00 mmol) following general procedure C5. The crude product was purified via flash column chromatography (1:1 hexanes/EtOAc) to yield a yellow oil (0.86 g, 80%). Rf = 0.15 (1:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 7.49 (d, J = 8.5 Hz, 2H), 7.45 (dd, J = 8.5, 5.5 Hz, 2H), 7.01 (t, J = 8.5 Hz, 2H), 6.92 (d, J = 9.0 Hz, 2H), 4.69 (s, 1H), 3.81 (s, 3H), 2.54 (s, 3H), 1.64 (brs, 1H) ppm. 13 C NMR (75 MHz, CDCl3): δ 162.39 (d, JCF = 247.5 Hz), 159.2, 133.5 (d, JCF = 30.0 Hz), 132.3, 128.7, 119.2 (d, JCF = 3.7 Hz), 115.6 (d, JCF = 13.6 Hz), 113.9, 87.0, 84.3, 55.6, 55.3, 33.7 ppm. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C17H17NOF, 270.1294; found, 270.1294. MeO Me NH F 3-(3-Fluorophenyl)-1-(4-methoxyphenyl)-N-methylprop-2-yn-1-amine (5.1.9). Prepared with S27 (1.23 g, 3.98 mmol) following general procedure C5. The crude product was purified via flash column chromatography (3:1 hexanes/EtOAc, followed by 2:1 hexanes/EtOAc) to yield a dark yellow oil (0.43 g, 40%). Rf = 0.35 (1:1 hexanes/EtOAc). 1 H NMR (CDCl3, 500 MHz): δ 7.49 (d, J = 8.5 Hz, 2H), 7.29 – 7.23 (m, 2H), 7.17 (d, J = 9.5 Hz, 1H), 7.05 – 7.00 (m, 1H), 6.92 (d, J = 8.5 Hz, 2H), 4.70 (s, 1H), 3.82 (s, 3H), 2.55 (s, 3H), 1.66 (brs, 1H) ppm. 13C NMR (125 MHz, CDCl3): δ 162.3 (d, JCF = 245.0 Hz), 159.2, 132.1, 129.8 (d, JCF = 8.7 Hz), 128.7, 127,6 (d, JCF = 2.5 Hz), 118.5 (d, JCF = 22.5 634 Hz), 115.5 (d, JCF = 20.0 Hz), 113.9, 90.3, 84.3, 55.6, 55.3, 33.8 ppm. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C17H17NOF, 270.1294; found, 270.1299. Me NH O OEt Ethyl 4-(3-(methylamino)-4-phenylbut-1-yn-1-yl)benzoate (5.1.10). Prepared with S28 (0.55 g, 1.58 mmol) following general procedure C5. The crude product was purified via flash column chromatography (2:3 hexanes/EtOAc) to yield an orange oil (0.40 g, 82%). Rf = 0.1 (1:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 7.97 (d, J = 8.5 Hz, 2H), 7.43 (d, J = 8.5 Hz, 2H), 7.36 – 7.30 (m, 4H), 7.28 – 7.24 (m, 1H), 4.37 (q, J = 7.0 Hz, 2H), 3.80 (t, J = 6.5 Hz, 1H), 3.09 – 3-00 (m, 2H), 2.55 (s, 3H), 1.39 (t, J = 7.0 Hz, 3H), 1.34 (brs, 1H) ppm. 13C NMR (125 MHz, CDCl3): δ 166.1, 137.5, 131.5, 129.7, 129.4, 128.3, 127.9, 126.8, 93.2, 84.2, 61.1, 53.7, 41.9, 34.2, 14.3 ppm. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C20H22NO2, 308.1651; found, 308.1653. Me NH O OEt Ethyl 3-(3-(methylamino)-4-phenylbut-1-yn-1-yl)benzoate (5.1.11). Prepared with S29 (1.41 g, 3.85 mmol) following general procedure C5. The crude product was purified via flash column chromatography (3:2 hexanes/EtOAc) to yield an orange oil (0.91 g, 77%). Rf = 0.1 (1:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 8.06 (s, 1H), 7.96 (dd, J = 8.0, 1.5 Hz, 1H), 7.54 (dd, J = 8.0, 1.5 Hz, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.34 – 635 7.32 (m, 4H), 7.28 – 7.24 (m, 1H), 4.37 (q, J = 7.0 Hz, 2H), 3.78 (t, J = 7.0 Hz, 1H), 3.08 – 3.01 (m, 2H), 2.56 (s, 3H), 1.04 (t, J = 7.0 Hz, 3H) ppm. 13C NMR (125 MHz, CDCl3): 166.2, 137.8, 135.8, 132.9, 130.9, 129.9, 129.2, 128.6, 128.5, 127.0, 123.9, 91.3, 84.1, 61.4, 51.9, 42.2, 34.4, 14.5 ppm. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C20H22NO2, 308.1651; found, 308.1656. Me NH Me Me CN 4-(5-Methyl-3-(methylamino)hex-1-yn-1-yl)benzonitrile (5.1.12). Prepared with S30 (2.28 g, 8.56 mmol) following general procedure C5. The crude product was purified via flash column chromatography (2:3 hexanes/EtOAc) to yield a red oil (1.74 g, 90%). Rf = 0.1 (1:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 7.57 (d, J = 8.0 Hz, 2H), 7.48 (d, J = 8.0 Hz, 2H), 3.56 (t, J = 7.5 Hz, 1H), 2.54 (s, 3H), 1.90 (septet, J = 7.0 Hz, 1H), 1.63 – 1.50 (m, 3H), 1.06 (brs, 1H), 0.95 (t, J = 7.0 Hz, 6H) ppm. 13C NMR (125 MHz, CDCl3): δ 132.2, 131.9, 128.4, 118.5, 111.2, 95.9, 82.4, 50.9, 44.8, 34.0, 25.3, 23.0, 22.1 ppm. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C15H19N2, 227.1548; found, 227.1549. Me NH CF3 N-Methyl-3-(4-(trifluoromethyl)phenyl)prop-2-yn-1-amine (5.1.13). Prepared with S31 following general procedure C5.11 636 HO Me N Boc Tert-butyl (1-(hydroxymethyl)cyclobutyl)(methyl)carbamate (S32). A 25 mL round bottom flask was charged with 1-((tert-butoxycarbonyl)(methyl)- amino)cyclobutane-1-carboxylic acid (0.15 g, 0.65 mmol) and 5 mL anhydrous THF. To the stirring solution at room temperature was added 2M BH3 . DMS in THF (0.49 mL, 0.98 mmol). After 6 h, the reaction was quenched with sat. aq. NH4Cl solution and the crude product was extracted twice with 15 mL EtOAc. The organic layer was concentrated and purified via flash column chromatography (2:1 hexanes/EtOAc) to yield a colorless liquid (0.12 g, 87%). Rf = 0.3 (1:1 hexanes/EtOAc). 1H NMR (CD3OD, 500 MHz): δ 3.76 (s, 2H), 2.78 (s, 3H), 2.28 – 2.18 (m, 2H), 2.14 – 2.05 (m, 2H), 1.80 – 1.64 (m, 2H), 1.45 (s, 9H) ppm. 13C NMR (125 MHz, CD3OD): δ 155.6, 79.5, 65.0, 61.5, 30.9, 29.9, 29.0, 27.4, 13.1. HRMS (ESI-TOF) [M + Na]+ m/z: calcd for C11H21NO3Na, 238.1419; found, 238.1424. O Me N Boc Tert-butyl (1-formylcyclobutyl)(methyl)carbamate (S33). S32 (190 mg, 0.88 mmol) was dissolved in 5 mL CH2Cl2 and cooled down to 0 oC. Then Dess–Martin periodinane (748.60 mg, 1.76 mmol) was added in one portion. The reaction mixture was stirred for 2 h, quenched with sat. aq. NaHCO3 solution and extracted twice with CH2Cl2. After removing the organic solvent, the crude product was loaded on the column (3:1 hexanes/acetone). The aldehyde S33 was isolated as a colorless oil (136.20 mg, 72%). Rf = 0.7 (1:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 9.58 (s, 1H), 2.79 (s, 3H), 2.42 – 2.21 (m, 4H), 1.95 – 1.72 (m, 2H), 1.34 (s, 9H). 13C NMR (75 MHz, CDCl3): δ 199.8, 155.5, 87.0, 80.7, 76.9, 31.1, 29.4, 28.4, 13.8. HRMS (ESI-TOF) [M + Na]+ m/z: calcd for 637 C11H19NO3Na, 236.1365; found, 236.1263. Me N Boc Tert-butyl (1-ethynylcyclobutyl)(methyl)carbamate (S34). To a stirring solution the aldehyde S33 (136 mg, 0.64 mmol) in anhydrous MeOH at 0 oC was added dimethyl (diazomethyl)phosphonate (0.23 mL, 0.95 mmol). The reaction was allowed to warm up over a period of 16 h. Then it was quenched with sat. aq. NaHCO3 solution and extracted twice with CH2Cl2. The organic layer was concentrated and purified via flash column chromatography (6:1 hexanes/EtOAc) to yield a colorless liquid (91.70 mg, 68%). Rf = 0.65 (3:1 hexanes/EtOAc). 1H NMR (CDCl3, 300 MHz): δ 2.72 (s, 1H), 2.64 (s, 3H), 2.33 – 2.19 (m, 6H), 1.37 (s, 9H). 13C NMR (75 MHz, CDCl3): δ 155.8, 86.5, 80.1, 69.0, 54.9, 36.3, 28.6, 14.8. HRMS (ESI-TOF) [M + Na]+ m/z: calcd for C12H19NO2Na, 232.1416; found, 232.1313. Me N Boc N OMe Tert-butyl (1-((6-methoxypyridin-2-yl)ethynyl)cyclobutyl)(methyl)carbamate (S35). An oven-dried vial was charged with Pd(PPh3)4 (50 mg, 0.04 mmol), CuI (12.4 mg, 0.06 mmol) and 2 mL anhydrous THF. Then S34 (0.09 g, 0.43 mmol), 2-bromo-6methoxypyridine (0.05 mL, 0.43 mmol) and Et3N (0.16 mL, 1.30 mmol) were added via syringe. The reaction mixture was stirred at room temperature for 1 h. Upon completion, the solvent was removed. The crude product was loaded on the column (10:1 hexanes/EtOAc) and isolated as a yellow oil (102.1 mg, 74%). Rf = 0.55 (6:1 638 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 7.44 (td, J = 8.0, 2.0 Hz, 1H), 6.96 (dd, J = 7.5, 2.0 Hz, 1H), 6.63 (dd, J = 8.0, 1.5 Hz, 1H), 2.77 (s, 3H), 2.50 – 2.33 (m, 4H), 2.14 – 2.00 (m, 1H), 1.78 – 1.65 (m, 1H), 1.46 (s, 9H). 13C NMR (125 MHz, CDCl3): δ 163.7, 155.7, 140.4, 138.2, 120.6, 110.5, 91.3, 80.7, 79.9, 55.3, 53.4, 36.2, 30.1, 28.4, 14.73. HRMS (ESI-TOF) [M + Na]+ m/z: calcd for C18H24N2O3Na, 339.1787; found, 339.1685. Me N Boc CF3 Tert-butyl methyl(1-((4-(trifluoromethyl)phenyl)ethynyl)cyclobutyl)- carbamate (S36). Prepared from the Sonogashira coupling reaction between S34 (136 mg, 0.65 mmol) and 1-iodo-4-(trifluoromethyl)benzene (185 mg, 0.68 mmol). The crude product was purified via flash column chromatography (10:1 hexanes/EtOAc) to yield a colorless oil (155 mg, 68%). Rf = 0.5 (8:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 7.51 (dd, J = 21.0, 7.5 Hz, 4H), 2.78 (s, 3H), 2.50 – 2.37 (m, 4H), 2.12 – 2.01 (m, 1H), 1.83 – 1.72 (m, 1H), 1.47 (s, 9H) ppm. 13C NMR (125 MHz, CDCl3): δ 155.7, 131.9, 129.6 (q, JCF = 32.5 Hz), 127.2, 125.1 (q, JCF = 3.7 Hz), 124.4 (q, JCF = 270.0 Hz), 94.7, 80.0, 55.4, 36.3, 30.0, 28.4, 14.8 ppm. HRMS (ESI-TOF) [M + Na]+ m/z: calcd for C19H22NO2F3Na, 376.1500; found, 376.1510. 639 Me NH N OMe 1-((6-Methoxypyridin-2-yl)ethynyl)-N-methylcyclobutan-1-amine (5.1.14). Prepared with S35 (0.10 g, 0.32 mmol) following general procedure C3. The crude product was purified via flash column chromatography (1:1 hexanes/EtOAc, followed by 2:1 hexanes/acetone) to yield a yellow oil (58.2 mg, 84%). Rf = 0.3 (2:1 hexanes/acetone). 1H NMR (CDCl3, 500 MHz): δ 7.37 (t, J = 7.5 Hz, 1H), 6.91 (d, J = 7.5 Hz, 1H), 6.55 (d, J = 7.5 Hz, 1H), 3.82 (s, 3H), 2.35 – 2.30 (m, 5H), 2.06 – 1.98 (m, 2H), 1.94 – 1.84 (m, 2H), 1.58 (brs, 1H). 13 C NMR (125 MHz, CDCl3): δ 163.6, 140.4, 138.3, 120.5, 110.4, 92.7, 82.8, 54.8, 53.4, 34.5, 30.4, 15.1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C13H17N2O, 217.1263; found, 217.1341. Me NH CF3 N-Methyl-1-((4-(trifluoromethyl)phenyl)ethynyl)cyclobutan-1-amine (5.1.15). Prepared from the Boc-deprotection of S36 (155 mg, 0.44 mmol) following general procedure B. The crude product was purified via flash column chromatography (1:1 hexanes/EtOAc) to yield a yellow oil (102 mg, 92%). Rf = 0.1 (1:1 hexanes/EtAcO). 1H NMR (CDCl3, 500 MHz): δ 7.53 (dd, J = 17.0, 8.5 Hz, 4H), 2.45 (s, 3H), 2.44 – 2.35 (m, 2H), 2.17 – 2.09 (m, 2H), 2.07 – 1.96 (m, 2H), 1.72 (brs, 1H) ppm. 13C NMR (125 MHz, CDCl3): δ 131.8, 129.5 (q, JCF = 32.5 Hz), 127.3, 125.1 (q, JCF = 3.7 Hz), 123.9 (q, JCF = 270.0 Hz), 95.9, 81.9, 55.1, 34.9, 30.4, 15.1 ppm. HRMS (ESI-TOF) [M + H]+ m/z: calcd 640 for C14H15NF3, 254.1157; found, 254.1164. General procedure C6 for the guanylation/amino-silylation reaction using silyl chloride as an activation reagent O NHMe + R1 R2 R3 N H CN TMSCl or TBDMSCl DIPEA CH2Cl2, r.t. R1 Me N R1 R2 TMS N H N N O or R3 R2 Me N H 2N O R3 A 50-mL oven-dried round bottom flask was charged with N-cyano-2fluorobenzamide (338.8 mg, 2.06 mmol) and 15 mL anhydrous CH2Cl2. Then DIPEA (0.75 mL, 4.30 mmol) was added via syringe, followed by TMSCl (0.27 mL, 2.15 mmol). The reaction mixture was stirred for 20 min and 1.72 mmol of a secondary propargyl amine, dissolved in 5 mL CH2Cl2, was added via syringe. The reaction mixture was stirred for another 3 h, quenched with sat. aq. NaHCO3-solution and extracted two times with CH2Cl2. After removal of the organic solvent, the crude product was purified via column chromatography to obtain the TMS-ene-guanidine or N-acyl propargyl guanidine. MeO Me N N TMS N H N O F 2-Fluoro-N-((2E,4Z)-5-(4-methoxyphenyl)-1-methyl-4-(pyridin-3-yl(trimethylsilyl)methylene)imidazolidin-2-ylidene)benzamide (5.2.1a). Prepared with 5.1.1 (434.6 mg, 1.72 mmol) following general procedure C6. The crude product was purified via flash column chromatography (1:2 hexanes/EtOAc, followed by 1:3 hexanes/EtOAc) to yield a white foam (247.3 mg, 30%). Rf = 0.3 (1:2 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 10.96 (s, 1H), 8.26 (d, J = 3.5 Hz, 1H), 8.05 (td, J = 7.5, 1.0 641 Hz, 1H), 7.89 (brs, 1H), 7.38 – 7.32 (m, 1H), 7.11 (t, J = 7.5 Hz, 1H), 7.04 (dd, J = 10.5, 8.5 Hz, 1H), 6.94 – 6.87 (m, 1H), 6.73 (brs, 1H), 6.64 – 6.57 (m, 4H), 4.88 (s, 1H), 3.70 (s, 3H), 2.71 (s, 3H), 0.15 (s, 9H). 13C NMR (125 MHz, CDCl3): δ 176.2, 161.7 (d, JCF = 255.1 Hz), 159.6, 159.2, 149.3, 147.4, 146.7, 136.7, 136.5, 132.4 (d, JCF = 8.7 Hz), 132.0 (d, JCF = 1.3 Hz), 128.7, 128.7, 126.7 (d, JCF = 9.0 Hz), 123.4 (d, JCF = 3.8 Hz), 122.9, 116.6 (d, JCF = 22.8 Hz), 114.0, 110.3, 65.3, 55.2, 28.3, -0.9. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C27H30FN4O2Si, 489.2044; found, 489.2122. MeO Me N N NH2 O F N (E)-N-(amino((1-(4-methoxyphenyl)-3-(pyridin-3-yl)prop-2-yn-1-yl)(methyl)amino)methylene)-2-fluorobenzamide (5.2.1b). Prepared with 5.1.1 (434.6 mg, 1.72 mmol) following general procedure C6. The crude product was purified via flash column chromatography (1:2 hexanes/EtOAc, followed by 1:3 hexanes/EtOAc) to yield a white solid (416.5 mg, 64%). Rf = 0.2 (1:2 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 8.72 (d, J = 1.0 Hz, 1H), 8.53 (dd, J = 4.5, 1.5 Hz, 1H), 8.02 (td, J = 7.5, 2.0 Hz, 1H), 7.77 (dt, J = 8.0, 1.5 Hz, 1H), 7.69 (brs, 1H), 7.51 (d, J = 8.5 Hz, 2H), 7.37 – 7.31 m, 1H), 7.25 (dd, J = 8.0, 5.0 Hz, 1H), 7.11 (t, J = 8.0 Hz, 1H), 7.04 (dd, J = 11.0, 8.0 Hz, 1H), 6.89 (d, J = 8.5 Hz, 2H), 3.78 (s, 3H), 2.85 (s, 3H). 13C NMR (125 MHz, CDCl3): δ 175.3, 161.7 (d, JCF = 254.3 Hz), 160.5, 159.6, 152.4, 148.9, 138.8, 132.0 (d, JCF = 8.7 Hz), 131.8, 128.7, 128.7, 127.6 (d, JCF = 8.7 Hz), 123.4 (d, JCF = 3.7 Hz), 123.1, 119.7, 116.6 (d, JCF = 23.2 Hz), 114.1, 88.9, 83.2, 55.3, 50.5, 29.1. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C24H22FN4O2, 417.1727; found, 417.1731. 642 MeO Me N N TMS N N H O F 2-Fluoro-N-((2E,4Z)-5-(4-methoxyphenyl)-1-methyl-4-(pyridin-4-yl(trimethylsilyl)methylene)imidazolidin-2-ylidene)benzamide (5.2.2). Prepared with 5.1.2 (132.1 mg, 0.52 mmol) following general procedure C6. The crude product was purified via flash column chromatography (2:1 hexanes/acetone) to yield a yellow foam (185.8 mg, 73%). Rf = 0.25 (2:1 hexanes/acetone). 1H NMR (CDCl3, 500 MHz): δ 10.98 (s, 1H), 8.27 (d, J = 3.5 Hz, 2H), 8.09 (td, J = 7.5, 1.5 Hz, 1H), 7.45 – 7.38 (m, 1H), 7.16 (t, J = 7.5 Hz, 1H), 7.10 (dd, J = 11.0, 8.5 Hz, 1H), 6.71 – 6.62 (m, 4H), 6.49 (brs, 2H), 4.92 (s, 1H), 3.76 (s, 3H), 2.76 (s, 3H), 0.20 (s, 9H). 13C NMR (125 MHz, CDCl3): δ 176.3, 161.7 (d, JCF = 255.1 Hz), 159.7, 159.2, 150.7, 150.1, 149.6, 149.4, 146.1, 132.5 (d, JCF = 8.7 Hz), 132.0 (d, JCF = 1.5 Hz), 130.2, 128.9, 128.6, 126.7 (d, JCF = 9.1 Hz), 124.2, 123.5 (d, JCF = 3.7 Hz), 123.4, 116.7 (d, JCF = 22.7 Hz), 114.6, 114.1, 112.1, 65.1, 55.3, 28.3, 0.9. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C27H30FN4O2Si, 489.2044; found, 489.2122. MeO Me N N NH2 O F N OMe (E)-N-(amino((1-(4-methoxyphenyl)-3-(6-methoxypyridin-2-yl)prop-2-yn-1yl)-(methyl)amino)methylene)-2-fluorobenzamide (5.2.3). Prepared with 4.5.2 (0.10 g, 0.35 mmol) following general procedure C6. The crude product was purified via flash column chromatography (3:2 hexanes/EtOAc, followed by 1:1 hexanes/EtOAc) to yield a 643 yellow foam (0.14 g, 88%). Rf = 0.3 (1:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 8.06 (t, J = 7.5 Hz, 1H), 7.70 (brs, 1H), 7.57 (d, J = 8.5 Hz, 2H), 7.54 (t, J = 7.5 Hz, 1H), 7.40 – 7.33 (m, 1H), 7.17 – 7.06 (m, 3H), 6.91 (d, J = 8.5 Hz, 2H), 6.74 (d, J = 8.5 Hz, 1H), 3.96 (s, 3H), 3.81 (s, 3H), 2.90 (s, 3H). 13C NMR (125 MHz, CDCl3): δ 175.3, 163.9, 161.8 (d, JCF = 254.5 Hz), 160.4, 159.5, 139.5, 138.5, 131.9 (d, JCF = 8.7 Hz), 131.9 (d, JCF = 1.6 Hz), 128.9, 128.8, 127.5 (d, JCF = 8,8 Hz), 123.4 (d, JCF = 3.8 Hz), 120.9, 116.6 (d, JCF = 23.1 Hz), 114.0, 111.5, 86.2, 84.5, 55.3, 53.7, 50.5, 29.2. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C25H24FN4O3, 447.1754; found, 447.1832. General procedure C7 for the guanylation/amino-silylation reaction using BSA as an activation reagent O R1 R2 N H Me CN N H BSA, DIPEA CH2Cl2, rt, 3 h R1 R3 R2 TMS Me N N H N O R3 In a 20-mL oven-dried scintillation vial was added N-cyano-2-fluorobenzamide (30.8 mg, 0.19 mmol), 3 mL anhydrous CH2Cl2, DIPEA (0.07 mL, 0.39 mmol) and BSA (0.05 mL, 0.20 mmol). The reaction mixture was stirred at room temperature for 30 min and 0.16 mmol of the propargyl amine, dissolved in 1 mL CH2Cl2, was added to the mixture via syringe. After 3 h, it was quenched with 15 mL sat. aq. NaHCO3 solution and extracted with 15 mL CH2Cl2. The organic layer was concentrated and purified via flash column chromatography. 644 Me N MeO N TMS N H N O F 2-Fluoro-N-((6E,8Z)-8-((6-methoxypyridin-2-yl)(trimethylsilyl)methylene)-5methyl-5,7-diazaspiro[3.4]octan-6-ylidene)benzamide (5.2.4). Prepared with propargyl amine 5.1.14 (50.0 mg, 0.23 mmol) following general procedure C7. The crude product was purified via flash column chromatography (4:1 hexanes/EtOAc c) to yield a white foam (95.3 mg, 91%). Rf = 0.45 (1:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 10.72 (s, 1H), 8.03 (t, J = 7.5 Hz, 1H), 7.52 (t, J = 7.5 Hz, 1H), 7.41 – 7.33 (m, 1H), 7.13 (t, J = 7.5 Hz, 1H), 7.06 (t, J = 9.0 Hz, 1H), 6.78 (d, J = 7.5 Hz, 1H), 6.59 (d, J = 8.5 Hz, 1H), 3.90 (s, 3H), 3.17 (s, 3H), 2.66 (brs, 2H), 2.46 – 2.40 (m, 2H), 1.58 – 1.53 (m, 1H), 0.67 – 0.64 (m, 1H), 0.21 (s, 9H). 13C NMR (125 MHz, CDCl3): δ 175.9, 163.1, 161.5 (d, JCF = 254.3 Hz), 157.5, 156.7, 148.8, 137.9, 132.0 (d, JCF = 8.8 Hz), 131.9 (d, JCF = 1.8 Hz), 127.2 (d, JCF = 9.3 Hz), 123.4 (d, JCF = 3.5 Hz), 118.4, 116.5 (d, JCF = 23.0 Hz), 114.3, 107.3, 66.5, 53.3, 32.1, 25.4, 12.2, -0.8. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C24H30FN4O2Si, 453.2044; found, 453.2122. Me N F3C TMS N H N O F 2-Fluoro-N-((6E,8Z)-5-methyl-8-((4-(trifluoromethyl)phenyl)(trimethylsilyl)methylene)-5,7-diazaspiro[3.4]octan-6-ylidene)benzamide (5.2.5). Prepared with propargyl amine 5.1.14 (94 mg, 0.37 mmol) and N-cyano-2-fluorobenzamide (73.1 mg, 0.44 mmol) following general procedure C7. The crude product was purified via flash column chromatography (6:1 hexanes/EtOAc) to yield a white solid (151 mg, 83%). Rf = 0.3 (6:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 10.80 (s, 1H), 8.04 (td, J = 7.5, 645 1.5 Hz, 1H), 7.60 (d, J = 8.0 Hz, 2H), 7.43 – 7.37 (m, 1H), 7.32 (d, J = 8.0 Hz, 2H), 7.16 (t, J = 7.5 Hz, 1H), 7.09 (dd, J = 10.5, 8.5 Hz, 1H), 3.17 (s, 3H), 2.52 – 2.45 (m, 2H), 2.43 – 2.36 (m, 2H), 1.52 – 1.43 (m, 1H), 0.45 – 0.34 (m, 1H), 0.20 (s, 9H) ppm. 13C NMR (125 MHz, CDCl3): δ 176.1, 161.5 (d, JCF = 253.7 Hz), 157.4, 148.5, 143.4 (d, JCF = 1.2 Hz), 132.2 (d, JCF = 8.7 Hz), 131.9 (d, JCF = 2.5 Hz), 131.0, 128.2, 127.0 (d, JCF = 10.0 Hz), 124.5 (q, JCF = 3.7 Hz), 123.2 (q, JCF = 270.0 Hz), 116.6 (d, JCF = 22.5 Hz), 113.3, 66.2, 32.4, 25.4, 11.7, -0.8 ppm. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C25H28N3OF4Si, 490.1938; found, 490.1939. MeO Me N F3C TMS N H N O F 2-Fluoro-N-((2E,4Z)-5-(4-methoxyphenyl)-1-methyl-4-((4-(trifluoromethyl)phenyl)-(trimethylsilyl)methylene)imidazolidin-2-ylidene)benzamide (5.2.6). Prepared with propargyl amine 5.1.3 (50 mg, 0.16 mmol) and N-cyano-2-fluorobenzamide (30.8 mg, 0.19 mmol) following general procedure C7. The crude product was purified via flash column chromatography (4:1 hexanes/EtOAc) to obtain a white foam (50.2 mg, 58%). Rf = 0.55 (4:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 10.95 (s, 1H), 8.09 (t, J = 7.5 Hz, 1H), 7.41 – 7.37 (m, 1H), 7.29 (brs, 2H), 7.15 (t, J = 7.5 Hz, 1H), 7.08 (dd, J = 10.5, 8.5 Hz, 1H), 6.69 – 6.48 (m, 6H), 4.88 (s, 1H), 3.74 (s, 3H), 2.74 (s, 3H), 0.19 (s, 9H) ppm. 13C NMR (125 MHz, CDCl3): δ 176.2, 161.7 (d, JCF = 255.1 Hz), 159.6, 159.3, 146.4, 145.0, 132.4 (d, JCF = 8.8 Hz), 132.0 (d, JCF = 1.5 Hz), 129.2, 128.7, 127.8, 127.5, 126.9, 126.8, 124.9 (q, JCF = 3.7 Hz), 123.5 (d, JCF = 3.8 Hz), 123.2 (q, JCF = 270.1 Hz), 116.6 (d, JCF = 22.7 Hz), 113.9, 113.4, 65.2, 55.2, 28.3, -0.9 ppm. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C29H30F4N3O2Si, 556.1965; found, 556.2043. 646 MeO Me N NC TMS N N H O F N-((2E,4Z)-4-((4-cyanophenyl)(trimethylsilyl)methylene)-5-(4-methoxyphenyl)-1-methylimidazolidin-2-ylidene)-2-fluorobenzamide (5.2.7). Prepared with 5.1.5 (50.0 mg, 0.18 mmol) following general procedure C7. The crude product was purified via flash column chromatography (4:1 hexanes/EtOAc c) to yield a white foam (74.5 mg, 80%). Rf = 0.8 (1:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 10.98 (s, 1H), 8.08 (t, J = 7.5 Hz, 1H), 7.45 – 7.31 (m, 3H), 7.15 (t, J = 7.0 Hz, 1H), 7.09 (t, J = 9.5 Hz, 1H), 6.73 – 6.57 (m, 6H), 4.87 (s, 1H), 3.76 (s, 3H), 2.75 (s, 3H), 0.18 (s, 9H). 13 C NMR (125 MHz, CDCl3): δ 176.2, 161.8 (d, JCF = 255.5 Hz), 159.8, 159.2, 146.6, 146.5, 132.4 (d, JCF = 8.8 Hz), 132.0 (d, JCF = 1.6 Hz), 131.8, 129.7, 128.8, 128.6, 126.8 (d, JCF = 9.2 Hz), 123.4 (d, JCF = 4.0 Hz), 119.0, 116.7 (d, JCF = 23.2 Hz), 114.0, 113.2, 109.1, 65.2, 55.3, 28.3, -0.9. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C29H30FN4O2Si, 513.2044; found, 513.2122. MeO Me N O EtO TMS N H N O F Ethyl 4-((Z)-((E)-2-((2-fluorobenzoyl)imino)-5-(4-methoxyphenyl)-1-methylimida-zolidin-4-ylidene)(trimethylsilyl)methyl)benzoate (5.2.8). Prepared with propargyl amine 5.1.7 (50 mg, 0.15 mmol) and N-cyano-2-fluorobenzamide (30.3 mg, 0.18 mmol) following general procedure C7. The crude product was purified via flash column chromatography (4:1 hexanes/EtOAc) to yield a white foam (50.1 mg, 58%). Rf = 0.4 (4:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 10.94 (s, 1H), 8.10 (t, J = 7.5 Hz, 1H), 647 7.76 (brs, 2H), 7.44 – 7.38 (m, 1H), 7.16 (t, J = 7.5 Hz, 1H), 7.10 (dd, J = 11.0, 8.5 Hz, 1H), 6.62 (s, 6H), 4.93 (s, 1H), 4.37 (q, J = 7.0 Hz, 1H), 3.76 (s, 3H), 2.77 (s, 3H), 1.41 (t, J = 7.0 Hz, 3H), 0.19 (s, 9H) ppm. 13C NMR (125 MHz, CDCl3): δ 176.2, 166.7, 161.7 (d, JCF = 257.5 Hz), 159.6, 159.4, 146.2, 145.9, 132.4 (d, JCF = 8.7 Hz), 132.0 (d, JCF = 2.5 Hz), 129.4, 128.9, 128.8, 127.5, 126.8 (d, JCF = 10.0 Hz), 123.4 (d, JCF = 3.7 Hz), 116.6 (d, JCF = 23.7 Hz), 114.1, 113.9, 65.1, 60.8, 55.3, 28.4, 14.4, 0.8 ppm. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C31H35N3O4FSi, 560.2381; found, 560.2391. MeO Me N F F TMS N H N O F N-((2E,4Z)-4-((3,5-difluorophenyl)(trimethylsilyl)methylene)-5-(4-methoxyphenyl)-1-methylimidazolidin-2-ylidene)-2-fluorobenzamide (5.2.9). Prepared with 5.1.6 (50.0 mg, 0.17 mmol) following general procedure C7. The crude product was purified via flash column chromatography (4:1 hexanes/EtOAc c) to yield a white foam (63.8 mg, 70%). Rf = 0.8 (1:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 10.93 (s, 1H), 8.08 (td, J = 7.5, 1.5 Hz, 1H), 7.42 – 7.37 (m, 1H), 7.15 (t, J = 7.5 Hz, 1H), 7.09 (dd, J = 10.0, 8.5 Hz, 1H), 6.70 (s, 4H), 6.51 (t, J = 9.0 Hz, 1H), 6.05 (brs, 2H), 4.94 (s, 1H), 3.77 (s, 3H), 2.78 (s, 3H), 0.19 (s, 9H). 13C NMR (125 MHz, CDCl3): δ 176.2, 162.9 (d, JCF = 247.5 Hz), 162.8 (d, JCF = 247.6 Hz), 161.7 (d, JCF = 255.2 Hz), 159.9, 159.3, 146.6, 144.3 (t, JCF = 9.5 Hz), 132.4 (d, JCF = 8.7 Hz), 132.0 (d, JCF = 1.6 Hz), 128.7, 126.8 (d, JCF = 9.1 Hz), 123.4 (d, JCF = 3.7 Hz), 116.6 (d, JCF = 22.8 Hz), 114.1, 112.7 (t, JCF = 1.7 Hz), 111.6, 100.9 (t, JCF = 25.3 Hz), 65.1, 55.3, 28.3, -0.9. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C28H29F3N3O2Si, 524.1903; found, 524.1981. 648 MeO Me N Cl TMS N H N O F N-((2E,4Z)-4-((4-chlorophenyl)(trimethylsilyl)methylene)-5-(4-methoxyphenyl)-1-methylimidazolidin-2-ylidene)-2-fluorobenzamide (5.2.10). Prepared with 5.1.4 (50.0 mg, 0.17 mmol) following general procedure C7. After 3 h, another 2.5 equiv. of BSA were added and the reaction was allowed to stir for another 3 h. After work-up, the crude product was purified via flash column chromatography (4:1 hexanes/EtOAc c) to yield a white foam (37.7 mg, 41%). Rf = 0.8 (1:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 10.91 (s, 1H), 8.08 (td, J = 7.5, 1.0 Hz, 1H), 7.42 – 7.35 (m, 1H), 7.14 (t, J = 7.5 Hz, 1H), 7.08 (dd, J = 10.5, 8.5 Hz, 1H), 7.02 (brs, 2H), 6.67 – 6.63 (m, 4H), 6.46 (brs, 2H), 4.89 (s, 1H), 3.77 (s, 3H), 2,75 (s, 3H), 0.17 (s, 9H). 13C NMR (125 MHz, CDCl3): δ 176.1, 161.7 (d, JCF = 255.1 Hz), 159.6, 159.4, 146.3, 139.2, 132.3 (d, JCF = 8.8 Hz), 132.0 (d, JCF = 1.5 Hz), 131.3, 130.2, 129.1, 128.8, 128.2, 126.0 (d, JCF = 9.1 Hz), 123.4 (d, JCF = 3.8 Hz), 116.6 (d, JCF = 22.8 Hz), 113.9, 113.4, 65.2, 55.3, 28.3, -0.9. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C28H30ClFN3O2Si, 522.1702; found, 522.1780. MeO Me N F TMS N H N O F 2-Fluoro-N-((2E,4Z)-4-((3-fluorophenyl)(trimethylsilyl)methylene)-5-(4methoxy-phenyl)-1-methylimidazolidin-2-ylidene)benzamide (5.2.12). Prepared with propargyl amine 5.1.9 (50 mg, 0.18 mmol) and N-cyano-2-fluorobenzamide (36.4 mg, 0.22 mmol) following general procedure C7. The crude product was purified via flash column chromatography (5:1 hexanes/EtOAc) to yield a white foam (50.1mg, 55%). Rf = 0.4 (4:1 649 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 10.92 (s, 1H), 8.10 (td, J = 7.5, 1.5 Hz, 1H), 7.44 – 7.38 (m, 1H), 7.17 (t, J = 7.5 Hz, 1H), 7.10 (dd, J = 11.0, 8.5 Hz, 1H), 7.04 (brs, 1H), 6.77 (td, J = 8.5, 2.0 Hz, 1H), 6.69 – 6.62 (m, 4H), 6.32 (brs, 2H), 4.95 (s, 1H), 3.77 (s, 3H), 2.78 (s, 3H), 0.19 (s, 9H) ppm. 13C NMR (125 MHz, CDCl3): δ 176.2, 162.7 (d, JCF = 243.7 Hz), 161.7 (d, JCF = 255.0 Hz), 159.6, 159.4, 146.2, 142.9 (d, JCF = 7.5 Hz), 132.3 (d, JCF = 8.7 Hz), 132.0 (d, JCF = 1.2 Hz), 129.5 (d, JCF = 7.5 Hz), 129.0, 128.7, 126.9 (d, JCF = 8.7 Hz), 124.6, 123.5 (d, JCF = 3.7 Hz), 116.6 (d, JCF = 22.5 Hz), 115.7, 113.9, 113.6 d, JCF = 1.2 Hz), 112.3 (d, JCF = 20.0 Hz), 65.1, 55.3, 28.4, -0.8 ppm. HRMS (ESITOF) [M + H]+ m/z: calcd for C28H30N3O2F2Si, 506.2075; found, 506.2088. Me N O EtO TMS N H N O F Ethyl 4-((Z)-((E)-5-benzyl-2-((2-fluorobenzoyl)imino)-1-methylimidazolidin4-ylidene)(trimethylsilyl)methyl)benzoate (5.2.13). Prepared with propargyl amine 5.1.10 (50 mg, 0.16 mmoc7l) and N-cyano-2-fluorobenzamide (32.0 mg, 0.19 mmol) following general procedure C7. The crude product was purified via flash column chromatography (3:1 hexanes/EtOAc) to yield a white foam (64.1 mg, 74%). Rf = 0.4 (3:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 10.60 (s, 1H), 8.09 (d, J = 8.5 Hz, 2H), 8.01 (td, J = 7.5, 1.5 Hz, 1H), 7.41 – 7.36 (m, 1H), 7.26 (d, J = 8.0 Hz, 2H), 7.23 – 7.18 (m, 3H), 7.14 (t, J = 7.5 Hz, 1H), 7.07 (dd, J = 10.5, 8.5 Hz, 1H), 6.95 (d, J = 7.5 Hz, 2H), 4.59 (dd, J = 5.5, 3.0 Hz, 1H), 4.41 (q, J = 7.0 Hz, 2H), 2.92 (s, 3H), 2.79 (dd, J = 14.0, 3.5 Hz, 1H), 2.43 (dd, J = 14.0, 5.5 Hz, 1H), 1.43 (t, J = 7.0 Hz, 3H), 0.21 (s, 9H) ppm. 13C NMR (125 MHz, CDCl3): δ 175.9, 166.5, 161.6 (d, JCF = 255.0 Hz), 159.7, 146.6, 145.1, 650 135.4, 132.3 (d, JCF = 8.7 Hz), 132.0, 130.2, 129.1, 129.0, 128.4, 128.3, 127.1, 126.9 (d, JCF = 10.0 Hz), 123.4 (d, JCF = 3.7 Hz), 116.6 (d, JCF = 23.7 Hz), 113.0, 62.2, 61.0, 36.5, 29.9, 14.4, -0.7 ppm. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C31H35N3O3FSi, 544.2432; found, 544.2432. Me N O EtO TMS Ethyl N H N O 4-((Z)-((E)-2-(benzoylimino)-5-benzyl-1-methylimidazolidin-4- ylidene)(tri-methylsilyl)methyl)benzoate (5.2.14). Prepared with propargyl amine 5.1.10 (50 mg, 0.16 mmol) and N-cyano-benzamide (28.5 mg, 0.19 mmol) following general procedure C7. The crude product was purified via flash column chromatography (5:1 hexanes/EtOAc) to yield a white foam (63.8 mg, 74%). Rf = 0.5 (4:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 10.75 (s, 1H), 8.23 (d, J = 7.0 Hz, 2H), 8.08 (d, J = 8.5 Hz, 2H), 7.45 (tt, J = 7.0, 1.5 Hz, 1H), 7.38 (t, J = 7.5 Hz, 2H), 7.25 (d, J = 8.0 Hz, 2H), 7.22 – 7.15 (m, 3H), 6.94 (d, J = 8.0 Hz, 2H), 4.58 (dd, J = 5.5, 3.5 Hz, 1H), 4.40 (q, J = 7.0 Hz, 2H), 2.94 (s, 3H), 2.79 (dd, J = 14.5, 3.5 Hz, 1H), 2.43 (dd, J = 14.5, 5.5 Hz, 1H), 1.42 (t, J = 7.0 Hz, 3H), 0.21 (s, 9H) ppm. 13C NMR (125 MHz, CDCl3): δ 177.5, 166.4, 159.9, 146.7, 145.4, 137.9, 135.6, 131.3, 130.2, 129.3, 129.0, 128.4, 128.3, 127.8, 127.1, 112.4, 62.2, 61.0, 36.6, 29.8, 14.3, -0.7 ppm. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C31H36N3O3Si, 526.2526; found, 526.2532. 651 Me N O OEt TMS N H N O Ethyl 3-((Z)-((E)-2-(benzoylimino)-5-benzyl-1-methylimidazolidin-4-ylidene)(trimethylsilyl)methyl)benzoate (5.2.15). Prepared with propargyl amine 5.1.10 (50 mg, 0.16 mmol) and N-cyano-benzamide (28.5 mg, 0.19 mmol) following general procedure C7. The reaction was stirred for 16 h. The crude product was purified via flash column chromatography (6:1 hexanes/EtOAc) to yield a white foam (55.5 mg, 65%). Rf = 0.3 (6:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 10.75 (s, 1H), 8.24 (d, J = 7.5 Hz, 2H), 7.97 (d, J = 7.5 Hz, 1H), 7.87 (s, 1H), 7.52 – 7.44 (m, 2H), 7.43 – 7.36 (m, 3H), 7.24 – 7.16 (m, 3H), 6.94 (d, J = 6.5 Hz, 2H), 4.58 (dd, J = 5.5, 3.0 Hz, 1H), 4.42 (q, J = 7.0 Hz, 2H), 2.95 (s, 3H), 2.80 (dd, J = 14.5, 3.0 Hz, 1H), 2.43 (dd, J = 14.5, 6.0 Hz, 1H), 1.44 (t, J = 7.0 Hz, 3H), 0.22 (s, 9H) ppm. 13C NMR (125 MHz, CDCl3): δ 177.5, 166.6, 159.9, 145.6, 141.6, 137.9, 135.7, 133.7, 131.4, 131.2, 129.9, 129.4, 129.1, 128.9, 128.4, 127.8, 127.3, 127.0, 112.2, 62.2, 61.2, 36.7, 29.9, 14.4, -0.7 ppm. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C31H36N3O3Si, 526.2526; found, 526.2526. Me N O EtO TMS N H N Cl O Ethyl 4-((Z)-((E)-5-benzyl-2-((4-chlorobenzoyl)imino)-1-methylimidazolidin4-ylidene)(trimethylsilyl)methyl)benzoate (5.2.16). Prepared with propargyl amine 5.1.10 (50 mg, 0.16 mmol) and 4-chloro-N-cyanobenzamide (35.2 mg, 0.19 mmol) following general procedure C7. The crude product was purified via flash column 652 chromatography (5:1 hexanes/EtOAc) to yield a white foam (68.1 mg, 75%). Rf = 0.6 (3:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 10.69 (s, 1H), 8.18 (d, J = 8.5 Hz, 2H), 8.09 (d, J = 8.5 Hz, 2H), 7.36 (d, J = 8.5 Hz, 2H), 7.26 (d, J = 8.5 Hz, 2H), 7.24 – 7.17 (m, 3H), 6.95 (d, J = 8.0 Hz, 2H), 4.59 (dd, J = 5.5, 3.5 Hz, 1H), 4.41 (q, J = 7.0 Hz, 2H), 2.94 (s, 3H), 2.80 (dd, J = 14.5, 8.0 Hz, 1H), 2.44 (dd, J = 14.5, 6.0 Hz, 1H), 1.43 (t, J = 7.0 Hz, 3H), 0.22 (s, 9H) ppm. 13C NMR (125 MHz, CDCl3): δ 176.4, 166.5, 159.9, 146.6, 145.1, 137.6, 136.3, 135.5, 130.8, 130.2, 129.0, 128.9, 128.4, 128.3, 128.0, 127.1, 112.7, 62.2, 61.0, 36.5, 29.9, 14.4, -0.7 ppm. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C31H35N3O3SiCl, 560.2136; found, 560.2148. Me N O EtO TMS Ethyl N H N OMe O 4-((Z)-((E)-5-benzyl-2-((4-methoxybenzoyl)imino)-1-methyl- imidazolidin-4-ylidene)(trimethylsilyl)methyl)benzoate (5.2.17). Prepared with propargyl amine 5.1.10 (50 mg, 0.16 mmol) and 4-methoxy-N-cyanobenzamide (35.3 mg, 0.19 mmol) following general procedure C7. The crude product was purified via flash column chromatography (3:1 hexanes/EtOAc) to yield a white foam (62.3 mg, 70%). Rf = 0.4 (3:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 10.75 (s, 1H), 8.20 (d, J = 8.5 Hz, 2H), 8.07 (d, J = 8.5 Hz, 2H), 7.24 (d, J = 8.5 Hz, 2H), 7.21 – 7.14 (m, 3H), 6.93 (d, J = 6.5 Hz, 2H), 6.88 (d, J = 8.5 Hz, 2H), 4.56 (brs, 1H), 4.40 (q, J = 7.0 Hz, 2H), 3.84 (s, 3H), 2.92 (s, 3H), 2.79 (d, J = 14.0 Hz, 1H), 2.42 (dd, J = 14.5, 6.0 Hz, 1H), 1.42 (t, J = 7.0 Hz, 3H), 0.21 (s, 9H) ppm. 13C NMR (125 MHz, CDCl3): δ 177.0, 166.5, 162.4, 159.6, 146.8, 145.5, 135.7, 131.3, 130.6, 130.2, 129.0, 128.4, 128.2, 127.0, 113.0, 112.0, 62.1, 60.9, 55.3, 36.7, 29.8, 14.3, -0.7 ppm. HRMS (ESI-TOF) [M + H]+ m/z: calcd for 653 C32H38N3O4Si, 556.2632; found, 556.2637. Me Me Me N NC TMS N H N O N-((2E,4Z)-4-((4-Cyanophenyl)(trimethylsilyl)methylene)-5-isobutyl-1methylimidazolidin-2-ylidene)benzamide (5.2.18). Prepared with propargyl amine 5.1.12 (50 mg, 0.19 mmol) and N-cyano-benzamide (32.9 mg, 0.23 mmol) following general procedure C7. The crude product was purified via flash column chromatography (6:1 hexanes/EtOAc) to yield a white solid (86.3 mg, 89%). Rf = 0.5 (4:1 hexanes/EtOAc). 1 H NMR (CDCl3, 500 MHz): δ 11.02 (s, 1H), 8.28 (d, J = 7.5 Hz, 2H), 7.62 (d, J = 7.5 Hz, 2H), 7.47 (t, J = 7.5 Hz, 1H), 7.40 (t, J = 7.5 Hz, 2H), 7.16 (d, J = 8.5 Hz, 2H), 4.32 (t, J = 4.0 Hz, 1H), 3.05 (s, 3H), 1.64 – 1.56 (m, 1H), 1.31 (ddd, J = 15.0, 8.0, 3.5 Hz, 1H), 1.05 (dt, J = 15.0, 5.0 Hz, 1H), 0.78 (d, J = 6.5 Hz, 3H), 0.65 (d, J = 6.5 Hz, 3H), 0.23 (s, 9H) ppm. 13 C NMR (125 MHz, CDCl3): δ 177.7, 159.7, 147.2, 146.2, 137.6, 132.5, 131.5, 129.8, 129.4, 127.9, 118.9, 111.2, 109.8, 60.4, 38.4, 29.1, 24.2, 23.8, 22.4, -0.7 ppm. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C26H33N4OSi, 445.2424; found, 445.2431. Me N F3C TMS N H N O N-((2E,4Z)-1-methyl-4-((4-(trifluoromethyl)-phenyl)-(trimethylsilyl)methylene)-imidazolidin-2-ylidene)benzamide (5.2.19). Prepared with propargyl amine 5.1.13 (50 mg, 0.23 mmol) and N-cyano-benzamide (41.1 mg, 0.28 mmol) following general procedure C7. The crude product was purified via flash column chromatography (5:1 hexanes/EtOAc) to yield a white solid (57.7 mg, 57%). Rf = 0.5 (4:1 hexanes/EtOAc). 1 H NMR (CDCl3, 500 MHz): δ 10.98 (s, 1H), 8.29 (d, J = 7.0 Hz, 2H), 7.61 (d, J = 8.0 Hz, 654 2H), 7.49 (t, J = 7.5 Hz, 1H), 7.42 (t, J = 7.5 Hz, 2H), 7.13 (d, J = 7.5 Hz, 2H), 3.87 (s, 2H), 3.05 (s, 3H), 0.24 (s, 9H) ppm. 13C NMR (125 MHz, CDCl3): δ 177.5, 160.7, 146.1, 142.0, 137.8, 131.5, 129.4, 128.8, 128.2 (d, JCF = 32.56 Hz), 127.9, 125.9 (q, JCF = 3.7 Hz), 124.3 (q, JCF = 270.0 Hz), 111.3, 51.5, 30.6, -0.7 ppm. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C22H25F3N3OSi, 432.1719; found, 432.1714. Me N F N H N H N O OMe 2-Fluoro-N-((6E,8Z)-8-((6-methoxypyridin-2-yl)methylene)-5-methyl-5,7diazaspiro-[3.4]octan-6-ylidene)benzamide (5.2.20). To a solution of 5.2.4 (50 mg, 0.11 mmol) in 5 mL MeOH was added K2CO3 (16 mg, 0.12 mmol). The reaction mixture was stirred at room temperature for 1 h, quenched with sat. aq. NH4Cl solution and extracted twice with 10 mL CH2Cl2. After removing the organic solvent, the crude product was purified via flash column chromatography (3:1 hexanes/EtOAc) to obtain the Z-alkene as a white foam (44.4 mg, 99%). Rf = 0.5 (1:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 11.98, 8.05 (td, J = 7.5, 1.5 Hz, 1H), 7.50 (t, J = 7.5 Hz, 1H), 7.41 – 7.36 (m, 1H), 7.16 (t, J = 7.5 Hz, 1H), 7.08 (dd, J = 11.0, 8.5 Hz, 1H), 6.72 (d, J = 7.0 Hz, 1H), 6.53 (d, J = 8.5 Hz, 1H), 5.88 (s, 1H), 4.28 (s, 3H), 3.27 (s, 3H), 2.85 – 2.79 (m, 2H), 2.43 – 2.37 (m, 2H), 2.10 – 2.02 (m, 2H) ppm. 13C NMR (125 MHz, CDCl3): δ 174.7, 164.1, 161.6 (d, JCF = 254.5 Hz), 157.1, 154.0, 148.6, 138.6, 132.1 (d, JCF = 8.6 Hz), 131.9 (d, JCF = 1.7 Hz), 127.2 (d, JCF = 9.6 Hz), 123.4 (d, JCF = 3.7 Hz), 116.6 (d, JCF = 22.7 Hz), 115.2, 107.7, 97.1, 65.4, 54.8, 33.8, 26.0, 13.8 ppm. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C21H22FN4O2, 381.1649; found, 381.1727. 655 Me N Br N N H O N F OMe N-((6E,8E)-8-(bromo(6-methoxypyridin-2-yl)methylene)-5-methyl-5,7diazaspiro-[3.4]octan-6-ylidene)-2-fluorobenzamide (5.2.21). To a solution of 5.2.4 (30 mg, 0.07 mmol) in 3 mL anhydrous CH2Cl2 was added AgOAc (13.2 mg, 0.08 mmol) and 1M Br2 in CH2Cl2 (0.08 mL, 0.08 mmol). The reaction mixture was stirred at room temperature for 3 h, quenched with sat. aq. NaHCO3 solution and extracted twice with 10 mL CH2Cl2. After removing the organic solvent, the crude product was purified via flash column chromatography (5:1 hexanes/Et2O) to obtain a white solid (25.8 mg, 80%). 1H NMR (CDCl3, 500 MHz): δ 12.18 (s, 1H), 8.04 (td, J = 7.5, 2.0 Hz, 1H), 7.61 (t, J = 8.0 Hz, 1H), 7.44 (d, J = 8.0 Hz, 1H), 7.40 – 7.35 (m, 1H), 7.14 (td, J = 7.5, 1.0 Hz, 1H), 7.06 (ddd, J = 11.0, 8.5, 1.0 Hz, 1H), 6.62 (dd, J = 8.5, 1.0 Hz, 1H), 4.15 (s, 3H), 3.51 – 3.41 (m, 2H), 3.34 (s, 3H), 2.61 – 2.51 (m, 2H), 2.33 – 2.23 (m, 1H), 2.19 – 2.08 (m, 1H) ppm. 13 C NMR (125 MHz, CDCl3): δ 174.4, 163.1, 161.7 (d, JCF = 253.7 Hz), 155.6, 153.5, 143.0, 139.1, 132.1 (d, JCF = 8.7 Hz), 131.9 (d, JCF = 1.2 Hz), 126.9 (d, JCF = 8.7 Hz), 123.4 (d, JCF = 3.7 Hz), 116.6 (d, JCF = 22.5 Hz), 116.0, 109.5, 95.4, 68.2, 54.6, 29.2, 26.0, 13.3 ppm. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C21H21N4O2FBr, 459.0832; found, 459.0842. Me N I N H N N O F OMe 2-Fluoro-N-((6E,8E)-8-(iodo(6-methoxypyridin-2-yl)methylene)-5-methyl5,7-diaza-spiro[3.4]octan-6-ylidene)benzamide (5.2.22). To a solution of 5.2.4 (60 mg, 656 0.13 mmol) in 5 mL anhydrous CH2Cl2 was added AgOAc (26.5 mg, 0.16 mmol) and I2 (40.3 mg, 0.16 mmol) in one portion. The reaction mixture was stirred at room temperature for 3 h. Upon consumption of the starting material as indicated by TLC, the reaction was quenched with 10 mL 10% Na2S2O3 solution and extracted with 15 mL CH2Cl2. After removing the organic solvent, the crude product was purified via flash column chromatography (8:1 hexanes/EtOAc, followed by 6:1 hexanes/EtOAc) to obtain the Ealkene as a yellow solid (34.2 mg, 51%). Rf = 0.45 (4:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 12.18 (s, 1H), 8.05 (t, J = 7.5 Hz, 1H), 7.61 (t, J = 8.0 Hz, 1H), 7.44 (d, J = 7.5 Hz, 1H), 7.41 – 7.36 (m, 1H), 7.15 (t, J = 7.5 Hz, 1H), 7.08 (dd, J = 11.0, 9.0 Hz, 1H), 6.59 (d, J = 8.0 Hz, 1H), 4.12 (s, 3H), 3.58 – 3.47 (m, 2H), 3.37 (s, 3H), 2.58 – 2.48 (m, 2H), 2.36 – 2.25 (m, 1H), 2.22 – 2.11 (m, 1H) ppm. 13C NMR (125 MHz, CDCl3): δ 175.8, 163.0, 161.9 (d, JCF = 254.7 Hz), 156.9, 155.5, 146.8, 138.9, 132.5 (d, JCF = 8.8 Hz), 132.2 (d, JCF = 1.7 Hz), 126.5 (d, JCF = 9.3 Hz), 123.4 (d, JCF = 3.7 Hz), 119.0, 116.7 (d, JCF = 22.8 Hz), 110.3, 68.4, 65.6, 53.7, 32.0, 26.0, 12.5 ppm. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C21H21FIN4O2, 507.0615; found, 507.0693. Me N MeO N I N H N O F 2-Fluoro-N-((6E,8Z)-8-(iodo(6-methoxypyridin-2-yl)methylene)-5-methyl-5,7diaza-spiro[3.4]octan-6-ylidene)benzamide (5.2.23). Obtained as a white solid (20.1 mg, 30%) from the iodination of 5.2.4. Rf = 0.3 (4:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 10.74 (s, 1H), 8.13 (td, J = 7.5, 1.0 Hz, 1H), 7.62 (t, J = 7.5 Hz, 1H), 7.45 – 7.39 (m, 1H), 7.17 (t, J = 7.5 Hz, 2H), 7.10 (dd, J = 11.0. 8.5 Hz, 1H), 6.70 (d, J = 8.0 Hz, 1H), 3.95 (s, 3H), 3.20 (s, 3H), 2.76 – 2.69 (m, 2H), 2.55 – 2.47 (m, 2H), 1.70 – 1.63 (m, 1H), 657 0.90 – 0.82 (m, 1H) ppm. 13C NMR (125 MHz, CDCl3): δ 175.7, 163.0, 161.9 (d, JCF = 255.0 Hz), 156.9, 155.4, 146.8, 138.9, 132.5 (d, JCF = 10.0 Hz), 132.2 (d, JCF = 1.2 Hz), 126.5 (d, JCF = 7.5 Hz), 123.5 (d, JCF = 3.7 Hz), 119.1, 116.7 (d, JCF = 22.5 Hz), 110.4, 68.4, 65.6, 53.7, 32.0, 26.0, 12.5 ppm. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C21H21N4O2FI, 507.0693; found, 507.0703. OMe Me N N MeO N N H O F (E)-2-fluoro-N-(6a-methoxy-3a-((6-methoxypyridin-2-yl)methyl)-1-methylhexahydrocyclopenta[d]imidazol-2(1H)-ylidene)benzamide (5.2.24). To 5.2.4 (45 mg, 0.10 mmol) in a 10 mL flask with magnetic stir bar was added 3 mL of a 2M HCl solution in methanol, previously prepared from 1.43 mL acetyl chloride and 10 mL anhydrous MeOH. The resulting solution was heated under rigorous stirring for 16 h at 50 oC. Upon consumption of the starting material as indicated by TLC, the reaction was allowed to cool to room temperature. The solvent was removed under reduced pressure and 5 mL sat. aq. NaHCO3 solution was added. The crude product was extracted two times with 10 mL CH2Cl2. After removal of the organic solvent, the crude product was purified via flash column chromatography (4:1 hexanes/EtOAc) to obtain a white foam (18.4 mg, 45%). Rf = 0.4 (3:1 hexanes/EtOAc). 1H NMR (CDCl3, 500 MHz): δ 9.83 (s, 1H), 8.06 (t, J = 7.5 Hz, 1H), 7.51 (t, J = 7.5 Hz, 1H), 7.40 – 7.34 (m, 1H), 7.15 (t, J = 7.5 Hz, 1H), 7.07 (dd, J = 10.5, 8.5 Hz, 1H), 6.75 (d, J = 7.5 Hz, 1H), 6.65 (d, J = 8.0 Hz, 1H), 4.15 (s, 3H), 3.39 (s, 3H), 3.26 (d, J = 14.5 Hz, 1H), 3.05 – 2.99 (m, 4H), 2.23 (dd, J = 13.5, 5.5 Hz, 1H), 1.90 (td, J = 13.0, 6.0 Hz, 1H), 1.85 – 1.79 (m, 1H), 1.77 – 1.72 (m, 1H), 1.68 – 1.61 (m, 1H), 1.53 – 1.45 (m, 1H) ppm. 13C NMR (125 MHz, CDCl3): δ 164.0, 161.6 (d, JCF = 255.0 658 Hz), 160.5, 155.1, 138.9, 131.9, 131.8, 123.4 (d, JCF = 3.7 Hz), 116.7, 116.5, 109.3, 102.5, 68.9, 54.2, 52.2, 41.9, 40.9, 34.8, 29.7, 26.2, 21.4 ppm. HRMS (ESI-TOF) [M + H]+ m/z: calcd for C22H26N4O3F, 413.1989; found, 413.1989. 659 MeO NHBoc 160 9.0 8.5 150 8.0 140 7.5 130 7.0 120 6.5 6.0 110 5.5 100 5.0 90 4.5 f1 (ppm) 80 f1 (ppm) 4.0 70 9.12 3.07 1.04 1.21 2.13 1.01 0.96 9.5 1.10 2.06 1.12 N S17 3.5 3.0 60 2.5 50 2.0 40 1.5 30 1.0 20 0.5 0.0 10 0 660 MeO NHBoc S18 180 8.5 170 8.0 160 7.5 150 7.0 140 6.5 130 6.0 120 5.5 110 9.33 3.12 0.98 2.00 1.96 1.90 1.89 9.0 0.90 N 5.0 100 4.5 f1 (ppm) 4.0 90 80 f1 (ppm) 3.5 70 3.0 2.5 2.0 1.5 1.0 0.5 0.0 60 50 40 30 20 10 0 661 MeO Me N Boc 9.0 170 160 8.0 150 7.5 7.0 140 6.5 130 6.0 120 5.5 110 5.0 100 4.5 4.0 f1 (ppm) 90 80 f1 (ppm) 3.5 70 9.85 3.07 3.30 0.97 2.22 1.25 1.14 8.5 2.10 N 1.07 1.00 S19 3.0 60 2.5 50 2.0 40 1.5 30 1.0 20 0.5 10 0.0 0 662 MeO 9.0 170 8.0 7.5 160 150 140 7.0 130 6.5 120 6.0 110 5.5 100 5.0 4.5 f1 (ppm) 90 80 f1 (ppm) 4.0 3.5 70 3.0 60 9.32 3.28 3.01 0.72 0.97 1.89 1.95 8.5 1.81 N 2.00 S20 Me N Boc 2.5 50 2.0 40 1.5 30 1.0 20 0.5 0.0 10 0 663 MeO NHMe 9.0 8.5 8.0 170 160 150 7.5 140 7.0 130 6.5 120 6.0 110 5.5 5.0 100 4.5 f1 (ppm) 90 80 f1 (ppm) 4.0 3.5 70 3.0 60 2.5 50 1.28 2.93 3.38 1.04 1.98 1.02 1.99 0.91 0.86 0.99 N 5.1.1 2.0 40 1.5 1.0 0.5 0.0 30 20 10 0 664 MeO Me N 8.5 160 8.0 150 7.5 7.0 140 6.5 130 6.0 120 5.5 110 5.0 100 4.5 90 3.11 2.09 2.97 1.13 1.17 1.02 1.92 1.08 CF3 3.84 1.82 S21 4.0 f1 (ppm) 3.5 3.0 80 f1 (ppm) 70 60 2.5 50 2.0 40 1.5 30 1.0 0.5 20 0.0 10 0 665 MeO Me N S22 9.0 180 8.5 170 8.0 160 7.5 150 7.0 140 6.5 130 6.0 120 5.5 110 3.03 1.95 3.16 1.06 1.04 1.01 1.01 2.00 2.10 2.05 2.09 Cl 5.0 4.5 4.0 f1 (ppm) 3.5 3.0 2.5 2.0 100 90 f1 (ppm) 70 60 50 40 80 1.5 30 1.0 20 0.5 10 0.0 0 666 MeO Me N S23 9.0 170 8.5 160 8.0 150 7.5 140 7.0 130 6.5 120 6.0 110 5.5 5.0 4.5 f1 (ppm) 100 90 80 f1 (ppm) 4.0 70 3.5 60 3.15 1.98 3.01 1.32 1.04 0.96 1.12 2.00 4.06 1.95 CN 3.0 2.5 50 2.0 40 1.5 30 1.0 20 0.5 10 0.0 0 667 668 669 670 671 672 673 674 MeO Me NH 9.0 170 8.5 160 8.0 150 7.5 140 7.0 130 6.5 120 6.0 110 5.5 100 5.0 4.5 f1 (ppm) 90 80 f1 (ppm) 4.0 3.5 70 3.0 60 1.11 3.15 3.09 1.03 2.00 CF3 3.93 1.94 5.1.3 2.5 50 2.0 40 1.5 30 1.0 20 0.5 10 0.0 0 675 MeO Me NH 5.1.4 8.0 170 7.5 160 7.0 150 6.5 140 130 6.0 120 5.5 110 5.0 100 4.5 4.0 f1 (ppm) 90 80 f1 (ppm) 0.93 2.96 3.08 0.94 2.00 2.06 2.09 1.97 Cl 3.5 3.0 2.5 2.0 70 60 50 40 1.5 30 1.0 20 0.5 10 0.0 0 676 MeO Me NH 5.1.5 8.0 180 170 7.5 160 7.0 6.5 150 140 6.0 130 5.5 120 5.0 110 4.5 100 4.0 f1 (ppm) 90 80 f1 (ppm) 3.5 3.0 70 60 2.5 50 1.00 2.97 2.87 0.89 1.80 2.00 2.30 2.37 CN 2.0 40 1.5 30 1.0 20 0.5 10 0.0 0 677 MeO 8.0 170 7.5 160 7.0 150 140 6.0 130 5.5 120 5.0 110 100 4.5 90 4.0 f1 (ppm) 80 f1 (ppm) 3.18 3.21 1.04 6.5 0.93 F 1.79 1.95 0.89 F 2.00 5.1.6 Me NH 3.5 70 3.0 2.5 2.0 60 50 40 1.5 30 1.0 20 0.5 10 0.0 0 678 679 680 681 682 683 684 685 686 687 688 689 690 691 MeO Me N N O 11.0 180 10.5 170 10.0 160 9.5 9.0 150 8.5 140 8.0 130 7.5 120 7.0 110 6.5 6.0 100 2.94 1.04 11.5 5.5 5.0 f1 (ppm) 4.5 90 80 f1 (ppm) 70 4.0 60 9.08 F 1.00 1.09 1.05 5.2.1a 3.16 N H 1.10 TMS 1.07 1.13 1.19 1.28 1.16 4.14 N 3.5 50 3.0 2.5 40 2.0 30 1.5 20 1.0 10 0.5 0 0.0 -0.5 -10 692 MeO Me N N NH2 O F N 190 180 170 7.5 160 7.0 150 6.5 140 130 6.0 120 5.5 110 5.0 100 2.89 8.0 2.93 8.5 0.94 0.81 1.97 0.90 0.98 1.00 0.98 1.95 0.98 9.0 0.86 0.93 5.2.1b 4.5 f1 (ppm) 4.0 3.5 3.0 2.5 2.0 90 f1 (ppm) 80 70 60 50 40 1.5 30 1.0 20 0.5 10 0.0 0 693 MeO Me N N O 180 11.5 11.0 170 10.5 160 10.0 150 9.5 9.0 140 8.5 130 8.0 120 7.5 110 7.0 6.5 100 6.0 5.5 f1 (ppm) 90 80 f1 (ppm) 5.0 4.5 70 4.0 60 8.66 2.99 1.02 1.03 12.0 3.04 F 5.2.2 4.04 1.88 N H 1.00 1.06 1.15 TMS 1.97 1.03 N 3.5 50 3.0 2.5 40 2.0 30 1.5 20 1.0 10 0.5 0.0 0 694 MeO Me N N NH2 O F N OMe 9.0 180 8.5 170 8.0 160 7.5 150 7.0 140 3.02 3.10 3.22 3.11 2.07 0.90 1.02 1.95 1.11 1.17 1.00 5.2.3 6.5 130 6.0 120 5.5 110 5.0 100 4.5 4.0 f1 (ppm) 90 f1 (ppm) 80 3.5 70 3.0 60 2.5 50 2.0 40 1.5 30 1.0 20 0.5 10 0.0 0 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 5.6.3 Crystal Structure Report for Complex 4.6.2 An irregular prism-like specimen of C28H31FN4O3Si, approximate dimensions 0.200 mm x 0.700 mm x 1.000 mm, was used for the X-ray crystallographic analysis. The X-ray intensity data were measured. A total of 999 frames were collected. The total exposure time was 5.99 h. The frames were integrated with the Bruker SAINT software package using a narrow-frame algorithm. The integration of the data using a monoclinic unit cell yielded a total of 79356 reflections to a maximum θ angle of 35.63° (0.61 Å resolution), of which 12411 were independent (average redundancy 6.394, completeness = 100.0%, Rint = 3.04%, Rsig = 2.06%) and 10050 (80.98%) were greater than 2σ(F2). The final cell constants of a = 12.3958(15) Å, b = 12.0236(14) Å, c = 18.997(2) Å, β = 108.177(2)°, volume = 2690.1(5) Å3, are based upon the refinement of the XYZ-centroids of 9951 reflections above 20 σ(I) with 4.513° < 2θ < 72.62°. Data were corrected for absorption effects using the Multi-Scan method (SADABS). The ratio of minimum to 716 maximum apparent transmission was 0.900. The calculated minimum and maximum transmission coefficients (based on crystal size) are 0.8800 and 0.9740. The structure was solved and refined using the Bruker SHELXTL Software Package, using the space group P 1 21/n 1, with Z = 4 for the formula unit, C28H31FN4O3Si. The final anisotropic full-matrix least-squares refinement on F2 with 389 variables converged at R1 = 4.20%, for the observed data and wR2 = 12.06% for all data. The goodness-of-fit was 1.062. The largest peak in the final difference electron density synthesis was 0.565 e-/Å3 and the largest hole was -0.340 e-/Å3 with an RMS deviation of 0.058 e-/Å3. On the basis of the final model, the calculated density was 1.281 g/cm3 and F(000), 1096 e-. Sample and crystal data for 20180518RL. Identification code 20180518RL Chemical formula C28H31FN4O3Si Formula weight 518.66 g/mol Temperature 103(2) K Wavelength 0.71073 Å Crystal size 0.200 x 0.700 x 1.000 mm Crystal system monoclinic Space group P 1 21/n 1 Unit cell dimensions a = 12.3958(15) Å α = 90° 717 b = 12.0236(14) Å β = 108.177(2)° c = 18.997(2) Å γ = 90° Volume 2690.1(5) Å3 Z 4 Density (calculated) 1.281 g/cm3 Absorption coefficient 0.131 mm-1 F(000) 1096 Data collection and structure refinement for 20180518RL. Theta range for data 2.43 to 35.63° collection Index ranges -20<=h<=18, -19<=k<=19, -31<=l<=31 Reflections collected 79356 Independent 12411 [R(int) = 0.0304] reflections Coverage of independent 100.0% reflections Absorption correction Multi-Scan 718 Max. and min. 0.9740 and 0.8800 transmission Structure solution direct methods technique Structure solution SHELXT 2014/5 (Sheldrick, 2014) program Refinement method Full-matrix least-squares on F2 Refinement program SHELXL-2016/6 (Sheldrick, 2016) Function minimized Σ w(Fo2 - Fc2)2 Data / restraints / 12411 / 74 / 389 parameters Goodness-of-fit on F2 1.062 Δ/σmax 0.001 10050 data; R1 = 0.0420, wR2 = I>2σ(I) 0.1081 Final R indices R1 = 0.0569, wR2 = all data 0.1206 w=1/[σ2(Fo2)+(0.0559P)2+0.9323P] Weighting scheme where P=(Fo2+2Fc2)/3 Largest diff. peak and 0.565 and -0.340 eÅ-3 hole 719 R.M.S. deviation from 0.058 eÅ-3 mean Atomic coordinates and equivalent isotropic atomic displacement parameters (Å2) for 20180518RL. U(eq) is defined as one third of the trace of the orthogonalized Uij tensor. x/a y/b z/c U(eq) Si1 0.38635(2) 0.67225(2) 0.33068(2) 0.01849(6) O1 0.65047(7) 0.00416(6) 0.50152(4) 0.02311(13) O2 0.03705(6) 0.88074(6) 0.49686(4) 0.02088(12) O3 0.52079(6) 0.33379(5) 0.25287(4) 0.02186(13) N1 0.68218(6) 0.30706(5) 0.35493(4) 0.01431(11) N2 0.58168(6) 0.47714(5) 0.36249(4) 0.01370(11) N3 0.58207(6) 0.83038(5) 0.45796(4) 0.01437(11) N4 0.74205(6) 0.43835(6) 0.44935(4) 0.01536(12) C1 0.59225(7) 0.56337(6) 0.41380(4) 0.01282(12) C2 0.66827(7) 0.40256(6) 0.38570(4) 0.01284(12) C3 0.70661(7) 0.54449(6) 0.47313(4) 0.01355(12) C4 0.60320(7) 0.27761(6) 0.29015(4) 0.01397(12) C5 0.62065(7) 0.16329(6) 0.26402(4) 0.01436(12) C6 0.52870(9) 0.10533(8) 0.21746(5) 0.02332(17) F1B 0.43273(12) 0.14577(13) 0.19052(9) 0.0307(4) 720 C7 0.53885(12) 0.99788(9) 0.19369(7) 0.0343(2) C8 0.64329(13) 0.94601(8) 0.21572(7) 0.0343(3) C9 0.73702(10) 0.00142(8) 0.26050(7) 0.0295(2) C10 0.72514(8) 0.10857(7) 0.28436(6) 0.02133(16) F1A 0.81885(16) 0.15580(12) 0.31881(8) 0.0236(3) C11 0.84693(8) 0.38300(8) 0.48936(5) 0.02217(16) C12 0.79157(7) 0.63709(6) 0.47777(4) 0.01312(12) C13 0.81593(8) 0.67237(7) 0.41467(4) 0.01780(14) C14 0.89550(8) 0.75566(7) 0.41823(5) 0.01782(14) C15 0.95285(7) 0.80374(6) 0.48647(5) 0.01542(13) C16 0.92726(7) 0.77088(7) 0.54990(5) 0.01783(14) C17 0.84696(7) 0.68839(7) 0.54514(4) 0.01563(13) C18 0.06048(9) 0.92033(8) 0.43217(6) 0.02531(18) C19 0.51876(7) 0.64695(6) 0.41046(4) 0.01388(12) C20 0.55086(7) 0.72763(6) 0.47323(4) 0.01378(12) C21 0.54926(8) 0.69727(7) 0.54344(5) 0.01855(14) C22 0.58434(9) 0.77449(8) 0.60103(5) 0.02179(16) C23 0.61905(8) 0.87835(8) 0.58681(5) 0.02122(16) C24 0.61567(7) 0.90155(7) 0.51364(4) 0.01643(13) C25 0.66112(10) 0.02460(8) 0.42953(6) 0.02637(19) C26 0.4295(4) 0.6976(9) 0.2439(3) 0.0451(15) C27 0.2999(9) 0.5399(7) 0.3088(6) 0.0258(12) C28 0.3036(6) 0.7852(6) 0.3486(5) 0.0440(16) 721 C26B 0.4225(4) 0.7487(8) 0.2578(3) 0.0530(14) C27B 0.3093(9) 0.5418(7) 0.2969(5) 0.0330(15) C28B 0.2950(4) 0.7649(5) 0.3703(3) 0.0342(9) Bond lengths (Å) for 20180518RL. Si1-C28 1.798(6) Si1-C26B 1.829(4) Si1-C27B 1.845(7) Si1-C19 1.8793(8) Si1-C27 1.891(7) Si1-C28B 1.903(4) Si1-C26 1.909(4) O1-C24 1.3503(11) O1-C25 1.4355(12) O2-C15 1.3627(10) O2-C18 1.4296(12) O3-C4 1.2450(10) N1-C2 1.3240(10) N1-C4 1.3589(10) N2-C2 1.3620(10) N2-C1 1.4009(10) N2-H2 0.88 N3-C24 1.3228(10) N3-C20 1.3528(10) N4-C2 1.3393(10) N4-C11 1.4471(11) N4-C3 1.4662(10) C1-C19 1.3445(11) C1-C3 1.5280(11) C3-C12 1.5161(11) C3-H3 1.0 C4-C5 1.4999(11) C5-C10 C5-C6 1.3921(12) 1.3955(12) C6-F1B C6-C7 1.3872(14) C6-H1A C7-C8 1.379(2) C7-H7 0.95 0.95(2) 1.2384(18) 722 C8-C9 1.3786(19) C8-H8 0.95 C9-C10 1.3888(13) C9-H9 0.95 C10-F1A 1.275(2) C10-H1B C11-H11A 0.98 C11-H11B 0.98 C11-H11C 0.98 C12-C13 1.3907(11) C12-C17 1.3934(11) C13-H13 0.95 C14-C15 1.3946(11) C14-H14 0.95 C15-C16 1.3954(12) C16-C17 1.3881(12) C17-H17 0.95 C18-H18A 0.98 C18-H18B 0.98 C18-H18C 0.98 C19-C20 1.4917(11) C20-C21 1.3889(11) C21-C22 1.3968(13) C21-H21 0.95 C22-C23 1.3748(13) C22-H22 0.95 C23-C24 1.4054(12) C23-H23 0.95 C25-H25A 0.98 C25-H25B 0.98 C25-H25C 0.98 C26-H26A 0.98 C26-H26B 0.98 C26-H26C 0.98 C27-H27A 0.98 C27-H27B 0.98 C27-H27C 0.98 C28-H28A 0.98 C28-H28B 0.98 C28-H28C 0.98 C26B-H26D 0.98 C26B-H26E 0.98 C26B-H26F C27B-H27D 0.98 0.98 C13-C14 C16-H16 0.955(18) 1.3924(12) 0.95 723 C27B-H27E 0.98 C27B-H27F 0.98 C28B-H28D 0.98 C28B-H28E 0.98 C28B-H28F 0.98 Bond angles (°) for 20180518RL. C26B-Si1-C27B 112.7(3) C28-Si1-C19 112.1(2) C26B-Si1-C19 109.45(15) C27B-Si1-C19 C28-Si1-C27 111.1(4) 111.7(4) C19-Si1-C27 109.5(4) C26B-Si1-C28B 108.8(2) C27B-Si1-C28B C19-Si1-C28B 104.68(18) C28-Si1-C26 111.9(2) C19-Si1-C26 108.20(14) C27-Si1-C26 103.7(4) C24-O1-C25 116.62(7) C15-O2-C18 116.97(7) C2-N1-C4 118.00(7) C2-N2-C1 C2-N2-H2 124.2 C1-N2-H2 111.64(6) 124.2 C24-N3-C20 117.33(7) C2-N4-C11 125.10(7) C2-N4-C3 111.84(6) C11-N4-C3 123.02(7) C19-C1-N2 127.26(7) C19-C1-C3 127.22(7) N2-C1-C3 105.49(6) N1-C2-N4 122.07(7) N1-C2-N2 128.76(7) N4-C2-N2 109.15(7) N4-C3-C12 112.81(6) N4-C3-C1 101.55(6) C12-C3-C1 113.67(6) N4-C3-H3 109.5 C12-C3-H3 109.5 C1-C3-H3 109.5 109.2(3) 724 O3-C4-N1 127.25(7) O3-C4-C5 119.10(7) N1-C4-C5 113.64(7) C6-C5-C10 116.43(8) C6-C5-C4 119.67(8) C10-C5-C4 123.89(8) F1B-C6-C7 113.83(11) F1B-C6-C5 123.82(10) C7-C6-C5 122.25(10) C7-C6-H1A 111.(8) C5-C6-H1A 126.(8) C8-C7-C6 119.61(11) C8-C7-H7 120.2 C6-C7-H7 120.2 C9-C8-C7 119.96(9) C7-C8-H8 120.0 C8-C9-C10 119.66(10) C8-C9-H9 120.2 C10-C9-H9 120.2 C9-C8-H8 120.0 F1A-C10-C9 114.09(10) F1A-C10-C5 123.40(10) C9-C10-C5 C9-C10-H1B 120.(3) 122.05(10) C5-C10-H1B 117.(3) N4-C11-H11A109.5 N4-C11-H11B 109.5 H11A-C11-H11B 109.5 N4-C11-H11C 109.5 H11A-C11-H11C 109.5 H11B-C11-H11C 109.5 C13-C12-C17 118.65(7) C13-C12-C3 120.50(7) C17-C12-C3 120.85(7) C12-C13-C14 121.23(7) C12-C13-H13 119.4 C14-C13-H13 119.4 C13-C14-C15 119.43(8) C13-C14-H14 120.3 C15-C14-H14 120.3 O2-C15-C14 124.16(8) O2-C15-C16 115.94(7) C14-C15-C16 119.87(8) C17-C16-C15 119.85(7) C17-C16-H16 120.1 C15-C16-H16 120.1 725 C16-C17-C12 120.93(7) C16-C17-H17 119.5 C12-C17-H17 119.5 O2-C18-H18A109.5 O2-C18-H18B 109.5 H18A-C18-H18B 109.5 O2-C18-H18C 109.5 H18A-C18-H18C 109.5 H18B-C18-H18C C1-C19-Si1 109.5 C1-C19-C20 116.14(7) 124.58(6) C20-C19-Si1 119.17(6) N3-C20-C21 122.45(7) N3-C20-C19 116.44(7) C21-C20-C19 121.10(7) C20-C21-C22 118.90(8) C20-C21-H21 120.5 C22-C21-H21 120.5 C23-C22-C21 119.33(8) C23-C22-H22 120.3 C21-C22-H22 120.3 C22-C23-C24 117.39(8) C22-C23-H23 121.3 C24-C23-H23 121.3 N3-C24-O1 119.50(7) O1-C24-C23 115.94(7) N3-C24-C23 124.56(8) O1-C25-H25A109.5 O1-C25-H25B 109.5 H25A-C25-H25B 109.5 O1-C25-H25C 109.5 H25A-C25-H25C 109.5 H25B-C25-H25C 109.5 Si1-C26-H26A 109.5 Si1-C26-H26B 109.5 H26A-C26-H26B 109.5 Si1-C26-H26C 109.5 H26A-C26-H26C 109.5 H26B-C26-H26C 109.5 Si1-C27-H27A 109.5 Si1-C27-H27B 109.5 H27A-C27-H27B 109.5 Si1-C27-H27C 109.5 H27A-C27-H27C 109.5 H27B-C27-H27C 109.5 Si1-C28-H28A 109.5 726 Si1-C28-H28B 109.5 H28A-C28-H28B 109.5 Si1-C28-H28C 109.5 H28A-C28-H28C 109.5 H28B-C28-H28C 109.5 Si1-C26B-H26D 109.5 Si1-C26B-H26E 109.5 H26D-C26B-H26E 109.5 Si1-C26B-H26F 109.5 H26D-C26B-H26F 109.5 H26E-C26B-H26F 109.5 Si1-C27B-H27D 109.5 Si1-C27B-H27E 109.5 H27D-C27B-H27E 109.5 Si1-C27B-H27F 109.5 H27D-C27B-H27F 109.5 H27E-C27B-H27F 109.5 Si1-C28B-H28D 109.5 Si1-C28B-H28E 109.5 H28D-C28B-H28E 109.5 Si1-C28B-H28F 109.5 H28D-C28B-H28F 109.5 H28E-C28B-H28F 109.5 Torsion angles (°) for 20180518RL. C2-N2-C1-C19 -175.94(8) C4-N1-C2-N4 -179.77(7) C2-N2-C1-C3 5.75(8) C4-N1-C2-N2 2.23(12) C11-N4-C2-N1 3.96(13) C3-N4-C2-N1 -178.42(7) C11-N4-C2-N2 -177.69(8) C3-N4-C2-N2 -0.07(9) C1-N2-C2-N1 174.45(8) C2-N4-C3-C12 C1-N2-C2-N4 -3.76(9) -118.67(7) C11-N4-C3-C12 C2-N4-C3-C1 3.37(8) C11-N4-C3-C1 C19-C1-C3-N4 176.42(8) 59.01(10) -178.95(8) N2-C1-C3-N4 -5.27(7) 727 C19-C1-C3-C12 -62.13(10) N2-C1-C3-C12 C2-N1-C4-O3 4.73(13) C2-N1-C4-C5 -174.28(7) O3-C4-C5-C6 -23.82(12) N1-C4-C5-C6 155.28(8) 116.17(7) O3-C4-C5-C10 157.09(8) N1-C4-C5-C10 -23.81(11) C10-C5-C6-F1B -174.64(12) C4-C5-C6-F1B 6.20(16) C10-C5-C6-C7 1.53(14) C4-C5-C6-C7 -177.62(9) F1B-C6-C7-C8 175.74(13) C5-C6-C7-C8 -0.79(17) C6-C7-C8-C9 -0.67(17) C7-C8-C9-C10 1.28(16) C8-C9-C10-F1A -173.00(11) C8-C9-C10-C5 -0.48(15) C6-C5-C10-F1A 170.92(11) C4-C5-C10-F1A -9.97(15) C6-C5-C10-C9 -0.90(13) C4-C5-C10-C9 178.22(8) N4-C3-C12-C13 62.78(10) C1-C3-C12-C13 -52.16(10) N4-C3-C12-C17 -117.09(8) C1-C3-C12-C17 127.97(8) C17-C12-C13-C14 1.25(13) C3-C12-C13-C14 -178.62(8) C12-C13-C14-C15 0.76(13) C18-O2-C15-C14 5.31(12) C18-O2-C15-C16 -176.60(8) C13-C14-C15-O2 175.80(8) C13-C14-C15-C16 -2.23(13) O2-C15-C16-C17 -176.50(8) C14-C15-C16-C17 1.69(12) C15-C16-C17-C12 0.36(13) C13-C12-C17-C16 -1.81(12) C3-C12-C17-C16 178.06(7) N2-C1-C19-C20 179.62(7) C3-C1-C19-C20 -2.44(11) N2-C1-C19-Si1 -4.30(12) C3-C1-C19-Si1 173.65(6) C28-Si1-C19-C1 175.7(4) C26B-Si1-C19-C1 -82.6(4) C27B-Si1-C19-C1 43.0(3) C27-Si1-C19-C1 51.9(4) 728 C28B-Si1-C19-C1 161.0(2) C26-Si1-C19-C1 -60.5(4) C28-Si1-C19-C20 -8.4(4) C26B-Si1-C19-C20 C27B-Si1-C19-C20 -141.1(3) C27-Si1-C19-C20 -132.1(3) C28B-Si1-C19-C20 -23.0(2) C26-Si1-C19-C20 115.5(3) C24-N3-C20-C21 2.39(12) C24-N3-C20-C19 -177.07(7) C1-C19-C20-N3 108.99(8) Si1-C19-C20-N3 -67.31(9) C1-C19-C20-C21 -70.48(10) Si1-C19-C20-C21 113.22(8) N3-C20-C21-C22 -1.93(13) C19-C20-C21-C22 177.51(8) C20-C21-C22-C23 0.23(14) C21-C22-C23-C24 0.85(14) C20-N3-C24-O1 178.28(8) C20-N3-C24-C23 -1.23(13) C25-O1-C24-N3 -7.12(12) C25-O1-C24-C23 172.43(9) C22-C23-C24-N3 -0.37(14) C22-C23-C24-O1 -179.89(9) 93.4(4) Anisotropic atomic displacement parameters (Å2) for 20180518RL. The anisotropic atomic displacement factor exponent takes the form: -2π2[ h2 a*2 U11 + ... + 2 h k a* b* U12 ] U11 U22 U33 Si1 0.01697(11) U23 U13 U12 0.01763(11) 0.01939(11) 0.00466(8) 0.00352(8) 0.0149(3) 0.0201(3) -0.0023(2) 0.0073(3) 0.00164(8) O1 0.0337(4) 0.0057(2) - 729 O2 0.0198(3) 0.0178(3) 0.0235(3) -0.0015(2) 0.0047(2) - 0.0166(3) 0.0177(3) -0.0018(2) -0.0002(2) 0.0102(2) 0.0149(3) -0.0010(2) 0.0051(2) 0.0111(2) 0.0133(3) -0.0012(2) 0.0031(2) 0.0115(2) 0.0146(3) -0.0007(2) 0.0052(2) 0.0113(2) 0.0154(3) -0.0013(2) 0.0015(2) 0.0111(3) 0.0129(3) -0.0004(2) 0.0050(2) 0.0103(3) 0.0132(3) 0.0007(2) 0.0049(2) 0.0119(3) 0.0129(3) -0.0011(2) 0.0044(2) 0.0115(3) 0.0135(3) -0.0002(2) 0.0057(2) - 0.0119(3) 0.0145(3) -0.0010(2) 0.0078(3) - 0.0050(2) O3 0.0264(3) 0.0062(2) N1 0.0178(3) 0.0005(2) N2 0.0158(3) 0.0014(2) N3 0.0173(3) 0.0003(2) N4 0.0170(3) 0.0025(2) C1 0.0149(3) - 0.0003(2) C2 0.0153(3) 0.0002(2) C3 0.0158(3) 0.0008(2) C4 0.0175(3) 0.0001(2) C5 0.0185(3) 0.0003(2) 730 C6 0.0274(4) 0.0181(4) 0.0229(4) -0.0041(3) 0.0055(3) - 0.0276(7) 0.0448(9) -0.0119(6) 0.0055(5) - 0.0195(4) 0.0307(5) -0.0101(4) 0.0118(5) - 0.0139(4) 0.0363(5) -0.0053(4) 0.0311(6) - 0.0172(4) 0.0410(6) 0.0043(4) 0.0278(5) 0.0155(3) 0.0291(4) 0.0024(3) 0.0134(3) 0.0220(5) 0.0302(6) -0.0101(4) 0.0018(4) 0.0175(3) 0.0224(4) -0.0009(3) -0.0015(3) 0.0119(3) 0.0121(3) -0.0018(2) 0.0031(2) 0.0175(3) 0.0131(3) -0.0037(2) 0.0060(3) - 0.0167(3) 0.0162(3) -0.0029(3) 0.0074(3) - 0.0060(3) F1B 0.0167(6) 0.0012(5) C7 0.0518(7) 0.0120(4) C8 0.0635(8) 0.0002(4) C9 0.0407(6) 0.0091(4) C10 0.0231(4) 0.0029(3) F1A 0.0151(6) 0.0006(4) C11 0.0209(4) 0.0065(3) C12 0.0146(3) 0.0007(2) C13 0.0231(4) 0.0046(3) C14 0.0217(4) 0.0033(3) 731 C15 0.0148(3) 0.0121(3) 0.0183(3) -0.0018(2) 0.0036(3) 0.0171(3) 0.0143(3) -0.0031(3) 0.0015(3) 0.0155(3) 0.0122(3) -0.0016(2) 0.0031(2) 0.0227(4) 0.0307(5) -0.0010(3) 0.0118(4) 0.0115(3) 0.0150(3) -0.0004(2) 0.0059(2) 0.0124(3) 0.0148(3) -0.0001(2) 0.0067(2) 0.0169(3) 0.0182(3) 0.0008(3) 0.0124(3) 0.0229(4) 0.0161(3) 0.0002(3) 0.0120(3) 0.0204(4) 0.0154(3) -0.0037(3) 0.0076(3) 0.0136(3) 0.0159(3) -0.0013(2) 0.0051(3) 0.0194(4) 0.0222(4) 0.0026(3) 0.0092(4) 0.0009(2) C16 0.0195(3) - 0.0020(3) C17 0.0180(3) 0.0002(2) C18 0.0247(4) - 0.0062(3) C19 0.0159(3) 0.0000(2) C20 0.0155(3) 0.0009(2) C21 0.0244(4) - 0.0003(3) C22 0.0298(4) 0.0016(3) C23 0.0284(4) 0.0000(3) C24 0.0196(3) 0.0002(3) C25 0.0374(5) 0.0068(3) - 732 C26 0.0336(15) 0.073(4) 0.0270(16) 0.026(2) 0.0075(12) - 0.0175(16) 0.027(3) -0.0003(15) -0.0057(17) - 0.0263(19) 0.056(4) -0.014(2) -0.013(2) 0.076(4) 0.0327(17) 0.030(2) 0.0083(13) 0.030(2) 0.029(3) -0.0124(19) -0.0045(15) 0.0302(18) 0.043(2) -0.0137(13) 0.0005(13) 0.001(2) C27 0.0233(18) 0.0030(14) C28 0.031(2) 0.0148(15) C26B 0.0476(17) - 0.006(2) C27B 0.030(2) 0.0054(14) C28B 0.0225(10) 0.0088(11) Hydrogen atomic coordinates and isotropic atomic displacement parameters (Å2) for 20180518RL. x/a y/b z/c U(eq) H2 0.5260 0.4718 0.3205 0.016 H3 0.6947 0.5348 0.5225 0.016 H1A 0.454(4) H7 0.4742 -0.0398 0.1624 0.041 H8 0.6506 -0.1279 0.2001 0.041 H9 0.8094 -0.0335 0.2750 0.035 0.133(10) 0.196(3) 0.31(8) 733 H1B 0.790(2) 0.150(3) H11A 0.8469 0.3075 0.4699 0.033 H11B 0.8536 0.3792 0.5421 0.033 H11C 0.9113 0.4247 0.4832 0.033 H13 0.7776 0.6390 0.3683 0.021 H14 0.9106 0.7795 0.3746 0.021 H16 0.9647 0.8049 0.5962 0.021 H17 0.8295 0.6666 0.5884 0.019 H18A 1.0831 0.8578 0.4068 0.038 H18B 1.1221 0.9750 0.4466 0.038 H18C 0.9922 0.9553 0.3988 0.038 H21 0.5247 0.6252 0.5521 0.022 H22 0.5842 0.7554 0.6495 0.026 H23 0.6443 0.9323 0.6249 0.025 H25A 0.5869 1.0156 0.3918 0.04 H25B 0.7151 0.9716 0.4201 0.04 H25C 0.6886 1.1006 0.4275 0.04 H26A 0.4765 0.6356 0.2370 0.068 H26B 0.4729 0.7670 0.2496 0.068 H26C 0.3613 0.7032 0.2007 0.068 H27A 0.2327 0.5521 0.2657 0.039 H27B 0.2760 0.5188 0.3515 0.039 H27C 0.3461 0.4803 0.2979 0.039 0.3118(13) 0.020(15) 734 H28A 0.2847 0.7700 0.3941 0.066 H28B 0.2336 0.7930 0.3070 0.066 H28C 0.3476 0.8543 0.3547 0.066 H26D 0.4739 0.7037 0.2394 0.079 H26E 0.4599 0.8187 0.2780 0.079 H26F 0.3531 0.7645 0.2171 0.079 H27D 0.3144 0.4927 0.3390 0.05 H27E 0.3432 0.5048 0.2629 0.05 H27F 0.2294 0.5585 0.2708 0.05 H28D 0.3368 0.8331 0.3902 0.051 H28E 0.2763 0.7253 0.4101 0.051 H28F 0.2248 0.7841 0.3311 0.051 735 5.6.4 Crystal Structure Report for Complex 5.2.9 A clear colorless rectangular-like specimen of C21H21FN4O2, approximate dimensions 0.070 mm x 0.280 mm x 0.390 mm, was used for the X-ray crystallographic analysis. The X-ray intensity data were measured (λ = 0.71073 Å). A total of 726 frames were collected. The total exposure time was 8.07 h. The frames were integrated with the Bruker SAINT software package using a narrow-frame algorithm. The integration of the data using a triclinic unit cell yielded a total of 29796 reflections to a maximum θ angle of 29.54° (0.72 Å resolution), of which 5071 were independent (average redundancy 5.876, completeness = 99.9%, Rint = 3.25%, Rsig = 2.45%) and 4099 (80.83%) were greater than 2σ(F2). The final cell constants of a = 9.1800(13) Å, b = 10.0015(13) Å, c = 10.8632(15) 736 Å, α = 94.257(3)°, β = 109.040(3)°, γ = 102.930(3)°, volume = 907.1(2) Å3, are based upon the refinement of the XYZ-centroids of 9923 reflections above 20 σ(I) with 4.868° < 2θ < 68.27°. Data were corrected for absorption effects using the Multi-Scan method (SADABS). The ratio of minimum to maximum apparent transmission was 0.935. The calculated minimum and maximum transmission coefficients (based on crystal size) are 0.9620 and 0.9930. The structure was solved and refined using the Bruker SHELXTL Software Package, using the space group P -1, with Z = 2 for the formula unit, C21H21FN4O2. The final anisotropic full-matrix least-squares refinement on F2 with 255 variables converged at R1 = 4.17%, for the observed data and wR2 = 11.12% for all data. The goodness-of-fit was 1.031. The largest peak in the final difference electron density synthesis was 0.409 e/Å3 and the largest hole was -0.279 e-/Å3 with an RMS deviation of 0.051 e-/Å3. On the basis of the final model, the calculated density was 1.393 g/cm3 and F(000), 400 e-. Sample and crystal data for 20180718RL. Identification code 20180718RL Chemical formula C21H21FN4O2 Formula weight 380.42 g/mol Temperature 103(2) K Wavelength 0.71073 Å Crystal size 0.070 x 0.280 x 0.390 mm 737 Crystal habit clear colourless rectangular Crystal system triclinic Space group P -1 Unit cell dimensions a = 9.1800(13) Å α = 94.257(3)° b = 10.0015(13) Å β = 109.040(3)° c = 10.8632(15) Å γ = 102.930(3)° Volume 907.1(2) Å3 Z 2 Density (calculated) 1.393 g/cm3 Absorption coefficient 0.099 mm-1 F(000) 400 Data collection and structure refinement for 20180718RL. Theta range for data 2.55 to 29.54° collection Index ranges -12<=h<=12, -13<=k<=13, -15<=l<=15 Reflections collected 29796 Independent 5071 [R(int) = 0.0325] reflections 738 Coverage of 99.9% independent reflections Absorption correction Multi-Scan Max. and min. 0.9930 and 0.9620 transmission Structure solution direct methods technique Structure solution SHELXT 2014/5 (Sheldrick, 2014) program Refinement method Full-matrix least-squares on F2 Refinement program SHELXL-2017/1 (Sheldrick, 2017) Function minimized Σ w(Fo2 - Fc2)2 Data / restraints / 5071 / 0 / 255 parameters Goodness-of-fit on F2 1.031 4099 data; R1 = 0.0417, wR2 = I>2σ(I) 0.1024 Final R indices R1 = 0.0569, wR2 = all data 0.1112 w=1/[σ2(Fo2)+(0.0519P)2+0.4461P] Weighting scheme where P=(Fo2+2Fc2)/3 739 Largest diff. peak and 0.409 and -0.279 eÅ-3 hole R.M.S. deviation from 0.051 eÅ-3 mean Atomic coordinates and equivalent isotropic atomic displacement parameters (Å2) for 20180718RL. U(eq) is defined as one third of the trace of the orthogonalized Uij tensor. x/a y/b z/c U(eq) F1 0.80408(10) 0.94566(8) 0.46517(8) 0.02299(18) O1 0.57820(11) 0.69313(9) 0.39505(9) 0.0217(2) O2 0.18511(12) 0.66551(10) 0.02575(10) 0.0232(2) N1 0.70870(13) 0.53487(10) 0.49903(10) 0.0153(2) N2 0.51756(12) 0.42832(10) 0.28055(10) 0.0134(2) N3 0.66681(13) 0.30818(11) 0.40050(10) 0.0170(2) N4 0.29178(12) 0.47604(10) 0.04626(10) 0.0149(2) C1 0.79212(14) 0.77112(12) 0.60108(12) 0.0131(2) C2 0.84658(15) 0.90851(12) 0.58680(12) 0.0149(2) C3 0.94851(15) 0.01028(12) 0.69135(13) 0.0172(2) C4 0.99713(15) 0.97557(13) 0.81671(13) 0.0175(2) C5 0.94622(15) 0.83937(13) 0.83537(12) 0.0164(2) C6 0.84576(14) 0.73842(12) 0.72816(12) 0.0142(2) 740 C7 0.67970(14) 0.66217(12) 0.48617(12) 0.0143(2) C8 0.63200(14) 0.43192(12) 0.39898(12) 0.0136(2) C9 0.7754(2) 0.27144(15) 0.51373(14) 0.0320(4) C10 0.57457(14) 0.21309(12) 0.27760(11) 0.0127(2) C11 0.66496(15) 0.13378(12) 0.21231(13) 0.0159(2) C12 0.53308(16) 0.99790(13) 0.19057(13) 0.0190(3) C13 0.46889(15) 0.07001(12) 0.28482(13) 0.0163(2) C14 0.48661(14) 0.30591(12) 0.19592(12) 0.0126(2) C15 0.40005(14) 0.27568(12) 0.06645(12) 0.0141(2) C16 0.31323(14) 0.36178(12) 0.98540(12) 0.0138(2) C17 0.25398(14) 0.32530(13) 0.84848(12) 0.0164(2) C18 0.17055(15) 0.40790(14) 0.77201(13) 0.0184(2) C19 0.14693(15) 0.52300(14) 0.83187(13) 0.0191(3) C20 0.21077(14) 0.55152(13) 0.96989(13) 0.0168(2) C21 0.23671(17) 0.69064(14) 0.16655(14) 0.0225(3) Bond lengths (Å) for 20180718RL. F1-C2 1.3556(14) O2-C20 O1-C7 1.2315(15) 1.3544(15) O2-C21 N1-C8 1.3219(15) N1-C7 1.3658(15) N2-C8 1.3618(16) N2-C14 N2-H2 0.88 N3-C8 1.3455(15) 1.4297(17) 1.3898(14) 741 N3-C9 1.4397(17) N4-C20 N3-C10 1.3240(16) 1.4551(15) N4-C16 C1-C2 1.3918(17) C1-C6 1.3966(17) C1-C7 1.5050(16) C2-C3 1.3821(17) C3-C4 1.3846(18) C3-H3 0.95 C4-C5 1.3903(18) C4-H4 0.95 C5-C6 1.3908(17) C5-H5 0.95 1.3620(15) C6-H6 0.95 C9-H9A 0.98 C9-H9B 0.98 C10-C14 1.5137(16) C10-C11 1.5543(16) C10-C13 1.5621(17) C11-C12 1.5486(17) C11-H11A 0.99 C12-C13 1.5459(17) C12-H12B 0.99 C13-H13A 0.99 C13-H13B 0.99 C14-C15 1.3421(17) C15-C16 1.4540(16) C15-H15 0.95 C16-C17 1.3943(17) C17-C18 1.3890(18) C17-H17 0.95 C18-C19 1.3739(19) C18-H18 0.95 C19-C20 1.4004(18) C19-H19 0.95 C21-H21A 0.98 C21-H21B 0.98 C21-H21C 0.98 C9-H9C C11-H11B 0.98 0.99 C12-H12A 0.99 742 Bond angles (°) for 20180718RL. C20-O2-C21 116.98(10) C8-N1-C7 118.69(11) C8-N2-C14 111.10(10) C8-N2-H2 124.4 C14-N2-H2 124.4 C8-N3-C9 C8-N3-C10 112.21(10) 123.74(11) C9-N3-C10 123.95(10) C20-N4-C16 117.21(11) C2-C1-C6 116.82(11) C2-C1-C7 122.14(11) C6-C1-C7 121.04(11) F1-C2-C3 117.33(11) F1-C2-C1 119.66(10) C3-C2-C1 122.96(11) C2-C3-C4 118.98(11) C2-C3-H3 120.5 C4-C3-H3 C3-C4-C5 119.95(11) C5-C4-H4 120.0 C4-C5-C6 119.92(12) C4-C5-H5 120.0 C6-C5-H5 120.0 C5-C6-C1 121.34(11) C1-C6-H6 119.3 O1-C7-N1 O1-C7-C1 120.48(11) N1-C7-C1 111.64(10) N1-C8-N3 121.47(11) N1-C8-N2 130.04(11) N3-C8-N2 108.43(10) N3-C9-H9A 109.5 N3-C9-H9B 109.5 H9A-C9-H9B 109.5 N3-C9-H9C 109.5 H9A-C9-H9C 109.5 120.5 C3-C4-H4 C5-C6-H6 120.0 119.3 127.88(11) H9B-C9-H9C 109.5 N3-C10-C14 101.05(9) N3-C10-C11 118.00(10) C14-C10-C11 116.30(10) 743 N3-C10-C13 117.88(10) C14-C10-C13 115.87(10) C11-C10-C13 88.78(9) C12-C11-C10 89.44(9) C12-C11-H11A 113.7 C10-C11-H11A 113.7 C12-C11-H11B 113.7 C10-C11-H11B 113.7 H11A-C11-H11B 111.0 C13-C12-C11 89.58(9) C13-C12-H12A 113.7 C11-C12-H12A 113.7 C13-C12-H12B 113.7 C11-C12-H12B 113.7 H12A-C12-H12B 111.0 C12-C13-C10 89.26(9) C12-C13-H13A 113.8 C10-C13-H13A 113.8 C12-C13-H13B 113.8 C10-C13-H13B 113.8 H13A-C13-H13B 111.0 C15-C14-N2 127.48(11) C15-C14-C10 126.06(10) N2-C14-C10 106.44(10) C14-C15-C16 127.45(11) C14-C15-H15 116.3 C16-C15-H15 116.3 N4-C16-C17 121.93(11) N4-C16-C15 118.62(11) C17-C16-C15 119.45(11) C18-C17-C16 118.99(12) C18-C17-H17 120.5 C16-C17-H17 120.5 C19-C18-C17 119.82(12) C19-C18-H18 120.1 C17-C18-H18 120.1 C18-C19-C20 117.19(12) C18-C19-H19 121.4 C20-C19-H19 121.4 N4-C20-O2 N4-C20-C19 124.87(12) 119.45(11) O2-C20-C19 115.68(11) O2-C21-H21A109.5 O2-C21-H21B 109.5 H21A-C21-H21B 109.5 O2-C21-H21C 109.5 744 H21A-C21-H21C 109.5 H21B-C21-H21C 109.5 Torsion angles (°) for 20180718RL. C6-C1-C2-F1 177.32(11) C7-C1-C2-F1 -3.18(17) C6-C1-C2-C3 -0.18(18) C7-C1-C2-C3 179.32(11) F1-C2-C3-C4 -178.71(11) C1-C2-C3-C4 -1.16(19) C2-C3-C4-C5 1.43(18) C3-C4-C5-C6 -0.39(18) C4-C5-C6-C1 -1.00(18) C2-C1-C6-C5 1.26(17) C7-C1-C6-C5 -178.24(11) C8-N1-C7-O1 7.0(2) C8-N1-C7-C1 -172.37(10) C2-C1-C7-O1 -33.22(17) C6-C1-C7-O1 146.25(12) C2-C1-C7-N1 146.18(12) C6-C1-C7-N1 -34.35(15) C7-N1-C8-N3 173.32(11) C7-N1-C8-N2 -3.3(2) C9-N3-C8-N1 7.0(2) C10-N3-C8-N1 -176.69(11) C9-N3-C8-N2 -175.73(13) C10-N3-C8-N2 0.63(14) C14-N2-C8-N1 170.72(12) C14-N2-C8-N3 -6.29(14) C8-N3-C10-C14 4.57(13) C9-N3-C10-C14 -179.09(13) C8-N3-C10-C11 132.53(11) C9-N3-C10-C11 -51.12(18) C8-N3-C10-C13 -122.75(12) C9-N3-C10-C13 53.60(17) N3-C10-C11-C12 134.09(11) C14-C10-C11-C12 -105.59(11) C13-C10-C11-C12 12.86(9) C10-C11-C12-C13 -12.99(9) C11-C12-C13-C10 12.93(9) N3-C10-C13-C12 -134.21(11) C14-C10-C13-C12 105.96(11) 745 C11-C10-C13-C12 -12.88(9) C8-N2-C14-C15 -169.19(12) C8-N2-C14-C10 9.07(13) N3-C10-C14-C15 170.37(12) C11-C10-C14-C15 41.31(16) C13-C10-C14-C15 -61.01(16) N3-C10-C14-N2 -7.93(12) C11-C10-C14-N2 -136.98(10) C13-C10-C14-N2 120.70(10) N2-C14-C15-C16 -2.6(2) C10-C14-C15-C16 179.47(11) C20-N4-C16-C17 0.46(17) C20-N4-C16-C15 -179.29(11) C14-C15-C16-N4 -11.93(19) C14-C15-C16-C17 168.31(12) N4-C16-C17-C18 -0.05(18) C15-C16-C17-C18 179.70(11) C16-C17-C18-C19 -0.38(18) C17-C18-C19-C20 0.39(18) C16-N4-C20-O2 179.46(11) C16-N4-C20-C19 -0.46(18) C21-O2-C20-N4 -5.17(17) C21-O2-C20-C19 174.76(11) C18-C19-C20-N4 0.04(19) C18-C19-C20-O2 -179.88(11) Anisotropic atomic displacement parameters (Å2) for 20180718RL. The anisotropic atomic displacement factor exponent takes the form: -2π2[ h2 a*2 U11 + ... + 2 h k a* b* U12 ] U11 U22 U33 F1 0.0318(4) U23 U13 U12 0.0171(4) 0.0164(4) 0.0056(3) 0.0051(3) 0.0149(4) 0.0204(5) -0.0014(3) -0.0012(4) 0.0035(3) O1 0.0233(5) 0.0073(4) 746 O2 0.0304(5) 0.0197(5) 0.0216(5) 0.0050(4) 0.0060(4) 0.0109(5) 0.0138(5) -0.0003(4) 0.0041(4) 0.0102(4) 0.0143(5) -0.0004(4) 0.0039(4) 0.0117(5) 0.0128(5) -0.0013(4) 0.0006(4) 0.0126(5) 0.0167(5) 0.0026(4) 0.0047(4) 0.0110(5) 0.0143(5) -0.0005(4) 0.0056(4) 0.0143(5) 0.0133(5) 0.0025(4) 0.0053(4) 0.0109(5) 0.0217(6) -0.0001(4) 0.0060(5) 0.0160(6) 0.0179(6) -0.0041(4) 0.0033(5) 0.0190(6) 0.0136(6) 0.0009(4) 0.0042(4) 0.0123(5) 0.0167(6) 0.0021(4) 0.0064(4) 0.0149(4) N1 0.0197(5) 0.0043(4) N2 0.0149(5) 0.0046(4) N3 0.0232(5) 0.0073(4) N4 0.0146(5) 0.0036(4) C1 0.0144(5) 0.0044(4) C2 0.0175(5) 0.0052(4) C3 0.0171(5) 0.0023(4) C4 0.0154(5) 0.0041(4) C5 0.0169(5) 0.0072(4) C6 0.0152(5) 0.0053(4) 747 C7 0.0160(5) 0.0121(5) 0.0141(5) -0.0003(4) 0.0055(4) 0.0122(5) 0.0134(5) 0.0018(4) 0.0057(4) 0.0200(7) 0.0179(7) -0.0032(5) -0.0081(6) 0.0103(5) 0.0109(5) -0.0003(4) 0.0031(4) 0.0146(5) 0.0178(6) 0.0006(4) 0.0068(5) 0.0114(5) 0.0220(6) -0.0016(5) 0.0094(5) 0.0108(5) 0.0188(6) 0.0022(4) 0.0090(5) 0.0096(5) 0.0157(6) 0.0010(4) 0.0066(4) 0.0114(5) 0.0144(6) 0.0000(4) 0.0048(4) 0.0127(5) 0.0151(6) 0.0016(4) 0.0041(4) 0.0166(6) 0.0151(6) 0.0005(4) 0.0042(4) 0.0029(4) C8 0.0161(5) 0.0047(4) C9 0.0461(9) 0.0163(6) C10 0.0158(5) 0.0042(4) C11 0.0169(5) 0.0069(4) C12 0.0241(6) 0.0055(5) C13 0.0209(6) 0.0044(4) C14 0.0135(5) 0.0029(4) C15 0.0162(5) 0.0045(4) C16 0.0119(5) 0.0012(4) C17 0.0152(5) 0.0019(4) 748 C18 0.0148(5) 0.0223(6) 0.0152(6) 0.0044(5) 0.0037(5) 0.0205(6) 0.0199(6) 0.0090(5) 0.0041(5) 0.0145(5) 0.0207(6) 0.0036(5) 0.0060(5) 0.0181(6) 0.0231(7) 0.0015(5) 0.0079(5) 0.0012(5) C19 0.0164(6) 0.0053(5) C20 0.0152(5) 0.0038(4) C21 0.0273(7) 0.0100(5) Hydrogen atomic coordinates and isotropic atomic displacement parameters (Å2) for 20180718RL. x/a y/b z/c U(eq) H2 0.4695 0.4948 0.2603 0.016 H3 0.9847 1.1026 0.6774 0.021 H4 1.0652 1.0448 0.8899 0.021 H5 0.9800 0.8153 0.9212 0.02 H6 0.8130 0.6453 0.7416 0.017 H9A 0.7156 0.1993 0.5483 0.048 H9B 0.8300 0.3537 0.5820 0.048 H9C 0.8545 0.2364 0.4880 0.048 H11A 0.6717 0.1648 0.1294 0.019 H11B 0.7713 0.1318 0.2731 0.019 749 H12A 0.4568 -0.0296 0.0986 0.023 H12B 0.5749 -0.0808 0.2232 0.023 H13A 0.4990 0.0433 0.3739 0.02 H13B 0.3527 0.0613 0.2475 0.02 H15 0.3949 0.1880 0.0227 0.017 H17 0.2704 0.2452 -0.1919 0.02 H18 0.1299 0.3849 -0.3214 0.022 H19 0.0897 0.5808 -0.2182 0.023 H21A 0.2036 0.7707 0.1948 0.034 H21B 0.3533 0.7100 0.2038 0.034 H21C 0.1886 0.6085 0.1976 0.034