Part 1: Design and Synthesis of BRDT Selective Inhibitors As Male

Part 1: Design and Synthesis of BRDT Selective Inhibitors as Male Contraceptive Agents Part 2: Focused Library Synthesis for TGR5 (Takeda G Protein-Coupled Receptor 5) Antagonist

A Dissertation SUBMITTED TO THE FACULTY OF UNIVERSITY OF MINNESOTA BY Jiewei Jiang

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

Dr. Gunda I. Georg

September 2020

Acknowledgements I would like to express my sincere gratitude to my advisor Dr. Gunda Georg for her mentorship and guidance over the six years. Without her continuous support, I could not have accomplished this journey. Her dedication and enthusiasm to science will always be inspiring me in my future career. My appreciation also goes to Dr. Elizabeth Ambrose, Dr. Carrie Haskell-Luevano, and Dr. William Pomerantz for serving as my committee members. Their insightful suggestions as well as helpful critiques made me become a better scientific researcher. I am deeply grateful to people who made my work herein possible. Thank you to Dr. Timothy Ward and Dr. Peiliang Zhao for their contribution in the synthesis; Dr. Jon Hawkinson, Jonathan Solberg, Dr. Carolyn Paulson, Xianghong Guan for their expertise with fluorescence polarization assay; Dr. Jun Qi and Nana K. Offei-Addo for their expertise with AlphaScreen assay; Dr. Ernst Schönbrunn and Dr. Alice Chan for elegant co-crystal structures; Kristen John for her efforts and discussions about the TGR5 screening. I would like to thank all the Georg group and ITDD members for not only showing me how to do science but also making my everyday life colorful. Thank you to Dr. Sudhakar Jakkaraj and Dr. Narsihmulu Cheryala for generously sharing not only their lab supplies but also knowledge in chemistry; Dr. Sara Coulup, Dr. Erick Carlson, and Brain Gabet for the joyful conversations. A special thank you extends to Dr. Leigh Allen for her great efforts in reviewing, polishing, and finalizing my writings. I firmly appreciate her constructive suggestions and encouragements. I am indebted to Caitlin Boley for her prompt helps since my day one in the university. Last but not the least, I want to thank my parents for their unconditional love and support. No matter how far away we are geographically, my heart is always with you.

i Abstract Unintended pregnancies can have significant adverse socioeconomic effects and also health risks for women. One approach to reducing unintended pregnancies is the use of effective contraceptive methods. While women have multiple reversible contraceptive options available, there is an unmet need for safe and reversible male contraceptive methods. Two pharmacological approaches to male contraception have been pursued: disrupting the hormonal milieu (hormonal) or targeting key cellular components in sperm maturation and function (non-hormonal). Chapter 1 provides a brief overview of the current state of these approaches. Because of adverse side effects resulting from hormonal disruption, we aim to develop safe novel non-hormonal male contraceptive agents. To this end, we have targeted an epigenetic reader protein called the testis-specific bromodomain (BRDT), which plays an essential role in spermatogenesis. Chapter 2 describes the initial validation of a tricyclic dihydropyridine hit from a virtual screening campaign as a bromodomain inhibitor. Based on evidence from co-crystal structures and sequence alignments, we hypothesized that engaging the unique Arg54 in the first bromodomain of BRDT (BRDT-1), would achieve BRDT-1 selectivity over other bromodomain isoforms. Guided by this hypothesis, we explored three different structural modifications of the dihydropyridine scaffold: conversion its lactone functionality to a lactam, lactone ring-opening, and conformational restriction of the molecule by macrocyclization. Cellularly active analogs with a greater than 10-fold increase in affinity relative to the original hit compound were obtained. However, the desired BRDT-1 selectivity was not achieved for any of the three subsets. In addition, novel mechanisms of action for targeting BRDT were pursued. We converted potent analogs to proteolysis-targeted chimeras (PROTACs) for selective protein degradation and synthesized bivalent molecules for simultaneous occupancies of two bromodomains. Future work will involve binding assessments of the resultant compounds. Chapter 3 focuses on an inherited genetic disorder, polycystic liver disease (PLD), which currently lacks effective drug therapeutics to halt disease progression. The G protein-coupled receptor TGR5 was identified as strongly associated with PLD, indicating that a potent TGR5 antagonist could be a potential treatment for PLD patients. To develop ii a TGR5 antagonist, we hypothesized that known TGR5 agonists could be converted to antagonists via systematic structural modifications. After selecting the nicotinamide core as our starting point, we used combinatorial chemistry to generate a focused library with more than 100 analogs, which were screened for agonist and antagonist activity. However, the screening results revealed that this library yielded only TGR5 agonists rather than antagonists. Nevertheless, the results provide novel structure-activity relationship insight for TGR5 agonists based on the nicotinamide core. This experiment highlights the need to obtain additional information including the crystal structure of TGR5 and co-crystal structures for future TGR5 antagonist discovery efforts.

iii Table of Contents

List of Figures ...... ix List of Schemes ...... xii List of Tables ...... xiii List of Compounds ...... xiv List of Abbreviations...... xxiii

Chapter 1 Recent Progress in Male Contraception ...... 1

1.1. Social and Scientific Significance of Male Contraceptive Research ...... 1

1.2. Hormonal Male Contraception ...... 3

1.3. Non-hormonal Male Contraception ...... 7 1.3.1. Retinoic Acid Receptor α ...... 8 1.3.2. Cation Channels of Sperm ...... 8 1.3.3. Epididymal Protease Inhibitor ...... 9

1.4. Summary and Outlook...... 10

Chapter 2 Design and Synthesis of BRDT Selective Inhibitors as Male Contraceptive Agents...... 12

2.1. Review of Bromodomain and Extra Terminal Domain (BET) Proteins ...... 12 2.1.1. Epigenetics and Bromodomains ...... 12 2.1.2. The BET Family ...... 13 2.1.3. BRDT in Spermatogenesis ...... 16

2.2. Dihydropyridine Scaffold Identification ...... 18 2.2.1. Hit Identification ...... 18 2.2.2. Preliminary Structure-Activity Relationship (SAR) Exploration ...... 19 2.2.3. Binding Mode ...... 20 2.2.4. Design Rationale ...... 21

iv 2.3. Hit-to-Lead Optimization: Lactam Analogs ...... 23 2.3.1. Docking Prediction ...... 23 2.3.2. Chemistry ...... 24 2.3.3. Linker Optimization ...... 27 2.3.4. Substitution Effect ...... 29 2.3.5. Carboxylate Introduction ...... 30 2.3.6. Discussion ...... 32

2.4. Hit-to-Lead Optimization: Ring-Open Analogs ...... 33 2.4.1. Chemistry ...... 33 2.4.2. Ester Scaffold Exploration ...... 34 2.4.3. Amide Scaffold Exploration ...... 35 2.4.4. Co-crystal Structures ...... 36 2.4.5. Discussion ...... 37

2.5. Hit-to-Lead Optimization: Macrocyclic Analogs ...... 38 2.5.1. Chemistry ...... 39 2.5.2. SAR Discussion ...... 42 2.5.3. Co-crystal Structures ...... 44 2.5.4. Discussion ...... 45

2.6. Hit-to-Lead Optimization: PROTACs ...... 46 2.6.1. Brief Introduction of PROTACs ...... 46 2.6.2. BET PROTACs ...... 47 2.6.3. Design Rationale ...... 49 2.6.4. Chemistry ...... 49 2.6.5. Binding and Degradation Evaluation ...... 51 2.6.6. Discussion ...... 52

2.7. Hit-to-Lead Optimization: Bivalent Molecules ...... 53 2.7.1. Bivalent Strategy Introduction ...... 53 2.7.2. Design Rationale ...... 54 2.7.3. Chemistry ...... 55 v 2.7.4. Discussion ...... 56

2.8. Summary and Future Directions ...... 56

Chapter 3 Focused Library Synthesis for TGR5 (Takeda G Protein-Coupled Receptor 5) Antagonists ...... 58

3.1. PLD Introduction ...... 58 3.1.1. Genetic Mechanism ...... 58 3.1.2. Cholangiocyte Abnormalities ...... 59 3.1.3. Preclinical and Clinical Studies of Potential PLD Therapies ...... 60 3.1.4. Current Therapy ...... 61

3.2. TGR5 Introduction ...... 62 3.2.1. TGR5 Biological Functions ...... 63 3.2.2. Reported Agonists ...... 64 3.2.3. The Role of TGR5 in PLD ...... 65

3.3. Focused Library Syntheses and Biological Evaluation for TGR5 Antagonists 67 3.3.1. Design Rationale ...... 67 3.3.2. Scaffold Selection ...... 68 3.3.3. Library Generation ...... 69 3.3.4. Assay Development and Data Analysis ...... 71

3.4. Discussion ...... 78

Chapter 4 Experimental Data and Procedures ...... 80

4.1. Small Molecule Development-1: Lactams ...... 80 4.1.1. General Procedure for the Synthesis of Lactam Analogs ...... 84 4.1.2. General Procedure for the Synthesis of Lactam Analogs with Carboxylate Groups 98

4.2. Small Molecule Development-2: Ring Open Esters/Amides ...... 101 4.2.1. General Procedure for the Synthesis of Ring Open Ester Analogs ...... 101 4.2.2. General Procedure for the Synthesis of Ring Open Amide Analogs ...... 106 vi 4.3. Small Molecule Development-3: Macrocyclic Analogs ...... 111 4.3.1. General Procedure A ...... 111 4.3.2. General Procedure B ...... 114 4.3.3. General Procedure C ...... 117 4.3.4. General Procedure D ...... 121 4.3.5. General Procedure E ...... 125 4.3.6. General Procedure F ...... 126 4.3.7. General Procedure G ...... 128 4.3.8. General Procedure H ...... 132 4.3.9. General Procedure I ...... 137

4.4. Bifunctional Molecules: PROTACs ...... 140 4.4.1. General Procedure J ...... 143 4.4.2. General Procedure K ...... 144 4.4.3. General Procedure L ...... 146 4.4.4. General Procedure M ...... 147 4.4.5. General Procedure N ...... 149 4.4.6. General Procedure O (Click Reaction) ...... 151

4.5. Bifunctional Molecules: Bivalent Analog ...... 153

4.6. Analog Affinity Determinations ...... 156 4.6.1. AlphaScreen ...... 156 4.6.2. FP Assay ...... 156

4.7. Focused Library Syntheses ...... 157 4.7.1. General Procedure for the Synthesis of the Acid Intermediates ...... 157 4.7.2. General Procedure for the Amide Coupling Reaction ...... 165

4.8. TGR5 cAMP Assay ...... 221 4.8.1. Buffer Preparation ...... 221 4.8.2. Cell Plate Preparation ...... 221 4.8.3. Agonism Assay ...... 221 4.8.4. Antagonism Assay ...... 222 vii 4.8.5. Data Analysis ...... 222

References ...... 224

viii List of Figures

Figure 1.1. The lifespan of sperm...... 3 Figure 1.2. The design rationale of hormonal male contraceptives...... 4 Figure 1.3. Chemical structures of testosterone and androgen derivatives for hormonal contraceptive studies...... 5 Figure 1.4. Combination regimens and corresponding delivery modes for hormonal male contraception...... 6 Figure 1.5. Representative molecules from non-hormonal male contraceptive projects and corresponding targets...... 7 Figure 1.6. Retinoic acid receptor signaling cascade...... 8 Figure 1.7. Localization and function of CatSper...... 9 Figure 2.1. The role of bromodomains in chromatin remodeling...... 12 Figure 2.2. Structural information for BET proteins...... 14 Figure 2.3. Reported BET inhibitors and their corresponding categories...... 15 Figure 2.4. The essential roles of BRDT in spermatogenesis...... 16 Figure 2.5. Identification of the dihydropyridine scaffold...... 19 Figure 2.6. General synthetic route of the dihydropyridine scaffold and its SAR results from preliminary modifications...... 20 Figure 2.7. Binding pose of hit compound 2.1 with BRD4-1 (PDB ID: 5KDH)...... 21 Figure 2.8. Design rationale of the arginine hypothesis...... 22 Figure 2.9. Proposed modification parameters of the lactam analogs...... 23 Figure 2.10. Binding site analysis of BRDT-1...... 24 Figure 2.11. Binding and cellular profile of lactam analogs...... 30 Figure 2.12. Binding conformation of lactam analog 2.5j (yellow) with BRD4-1...... 32 Figure 2.13. Binding analysis of ring-open scaffold...... 36 Figure 2.14. Dihedral angle analysis of the ester analogs...... 37 Figure 2.15. Design rationale of macrocyclization on the benzyl ester analog 2.10e...... 38 Figure 2.16. The predicted binding conformation of 2.16d (purple) using the BRD4- 1/2.10e (yellow) complex as the docking template...... 39 ix Figure 2.17. SAR summary of macrocyclic analogs...... 43 Figure 2.18. Binding pose analyses of macrocyclic and ring-open analogs...... 44 Figure 2.19. Mechanism of action for PROTAC-mediated protein degradation...... 46 Figure 2.20. Representative BET PROTAC molecules derived from (+)-JQ1 and two different E3 ligase recruiting units...... 47 Figure 2.21. Example of BET degrader with superior antiproliferative activity...... 48 Figure 2.22. Example of BET degrader with isoform specificity...... 48 Figure 2.23. The phenethyl lactam 2.5c/BRD4-1 complex reveals that both the tolyl and phenethyl moieties are solvent accessible (dotted red circles)...... 49 Figure 2.24. Reported bivalent inhibitors...... 53 Figure 2.25. Superimposition of NCB6 (PDB ID: 5AD3) and 2.1 (PDB ID: 5KDH). ... 55 Figure 3.1. Genetic mutations responsible for ADPKD and ADPLD...... 59 Figure 3.2. Cellular alterations and molecular mechanisms involved in hepatic cystogenesis...... 60 Figure 3.3. Structure of octreotide and chemical entities used in preclinical/clinical studies for PLD...... 61 Figure 3.4. Therapeutic algorithm for PLD based on different therapeutic goals and clinical symptoms including cyst number and distribution as well as disease progression...... 62 Figure 3.5. Endogenous bile acids and their reported TGR5 agonism potency.170 ...... 63 Figure 3.6. TGR5 tissue distribution and corresponding biological outcomes upon its activation...... 64 Figure 3.7. Reported small molecule scaffolds and their TGR5 agonist potency values.187, 188...... 65 Figure 3.8. Opposite signaling outcome of TGR5 agonist in ciliary (left) and non-ciliary (right) cells...... 66 Figure 3.9. Reported TGR5 antagonist SBI-115 returned increased cAMP levels back to normal...... 67 Figure 3.10. Ten general modifications and corresponding examples of interconverting between GPCR agonist and antagonist...... 68

x Figure 3.11. Nicotinamide analogs with different GPCR functional activity...... 69 Figure 3.12. Phenol building blocks and their corresponding reasons for selection...... 69 Figure 3.13. Aniline library and their reason for selection...... 70 Figure 3.14. Proposed workflow for TGR5 antagonist identification...... 72 Figure 3.15. The SAR results that corroborate the published ones.183 ...... 73 Figure 3.16. Contradictory SAR conclusions regarding the 4-position of the aryl ether between our and reported study...... 74 Figure 3.17. New SAR results that expand upon previous structure-activity relationships...... 75 Figure 3.18. A-C: Histograms of the antagonism screening...... 77

xi List of Schemes

Scheme 2.1. Initial Synthetic Route for the Lactam Analog 2.5 ...... 25 Scheme 2.2. Proposed Mechanism of Dihydropyridine Formation ...... 26 Scheme 2.3. Optimized Route for the Synthesis of Lactam Analog 2.5 ...... 27 Scheme 2.4. General Synthetic Routes of Ring-open Esters 2.10 and Amides 2.11 ...... 33 Scheme 2.5. General Synthetic Route of Lactone Analogs 2.16a-i ...... 40 Scheme 2.6. General Synthetic Route of Lactone Analogs 2.20a-d ...... 41 Scheme 2.7. Synthetic Route of Lactam Analog 2.26 ...... 42 Scheme 2.8. Synthetic Routes for the Lactam Intermediates 2.31a, 2.31b, and 2.32 ...... 50 Scheme 2.9. Synthesis of PROTACs 2.36a-c ...... 51 Scheme 2.10. Synthetic Route of Bivalent Molecule 2.41 ...... 56 Scheme 3.1. Reported Synthetic Route and Its Optimization ...... 71

xii List of Tables

Table 2.1. Structure and Inhibitory Profile of the Primary Round of Lactam Modifications ...... 27 Table 2.2. Structure and Inhibitory Profile of the Second Round of Lactam Modifications ...... 29 Table 2.3. Structure and Inhibitory Profile of the Third Round of Lactam Modifications ...... 31 Table 2.4. Structure and Inhibitory Profile of the Ester Analogs ...... 34 Table 2.5. Structure and Inhibitory Profile of the Amide Analogs ...... 35 Table 2.6. Structure and Binding Profile of the WPF and ZA-derived PROTACs ...... 52

xiii List of Compounds

6-Amino-1-ethylpyrimidine-2,4(1H,3H)-dione (2.2)...... 80 Ethyl 4-Chloro-2-(4-methylbenzylidene)-3-oxobutanoate (2.3)...... 81 Ethyl 4-(Benzyloxy)-3-oxobutanoate (2.6)...... 81 Ethyl 4-(Benzyloxy)-2-(4-methylbenzylidene)-3-oxobutanoate (2.7)...... 82 Ethyl 7-((Benzyloxy)methyl)-1-ethyl-2,4-dioxo-5-(p-tolyl)-1,2,3,4,5,8- hexahydropyrido[2,3-d]pyrimidine-6-carboxylate (2.8)...... 82 Ethyl 1-Ethyl-7-(hydroxymethyl)-2,4-dioxo-5-(p-tolyl)-1,2,3,4,5,8-hexahydropyrido[2,3- d]pyrimidine-6-carboxylate (2.9)...... 83 Ethyl 7-(Chloromethyl)-1-ethyl-2,4-dioxo-5-(p-tolyl)-1,2,3,4,5,8-hexahydropyrido[2,3- d]pyrimidine-6-carboxylate (2.4)...... 84 7-Allyl-1-ethyl-5-(p-tolyl)-5,7,8,9-tetrahydro-1H-pyrrolo[3',4':5,6]pyrido[2,3- d]pyrimidine-2,4,6(3H)-trione (2.5a)...... 85 7-Benzyl-1-ethyl-5-(p-tolyl)-5,7,8,9-tetrahydro-1H-pyrrolo[3',4':5,6]pyrido[2,3- d]pyrimidine-2,4,6(3H)-trione (2.5b)...... 85 1-Ethyl-7-phenethyl-5-(p-tolyl)-5,7,8,9-tetrahydro-1H-pyrrolo[3',4':5,6]pyrido[2,3- d]pyrimidine-2,4,6(3H)-trione (2.5c)...... 86 1-Ethyl-7-(3-phenylpropyl)-5-(p-tolyl)-5,7,8,9-tetrahydro-1H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidine-2,4,6(3H)-trione (2.5d)...... 87 1-Ethyl-7-(2-phenoxyethyl)-5-(p-tolyl)-5,7,8,9-tetrahydro-1H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidine-2,4,6(3H)-trione (2.5e)...... 87 1-Ethyl-7-(1-methylpiperidin-4-yl)-5-(p-tolyl)-5,7,8,9-tetrahydro-1H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidine-2,4,6(3H)-trione (2.5f)...... 88 7-(1-Benzylpiperidin-4-yl)-1-ethyl-5-(p-tolyl)-5,7,8,9-tetrahydro-1H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidine-2,4,6(3H)-trione (2.5g)...... 88 7-(1-Benzoylpiperidin-4-yl)-1-ethyl-5-(p-tolyl)-5,7,8,9-tetrahydro-1H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidine-2,4,6(3H)-trione (2.5h)...... 89 7-((1-Benzylpiperidin-4-yl)methyl)-1-ethyl-5-(p-tolyl)-5,7,8,9-tetrahydro-1H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidine-2,4,6(3H)-trione (2.5i)...... 89 xiv 1-Ethyl-7-((1-(4-nitrobenzyl)piperidin-4-yl)methyl)-5-(p-tolyl)-5,7,8,9-tetrahydro-1H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidine-2,4,6(3H)-trione (2.5j)...... 90 1-Ethyl-7-(4-methylphenethyl)-5-(p-tolyl)-5,7,8,9-tetrahydro-1H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidine-2,4,6(3H)-trione (2.5k)...... 90 7-(4-Chlorophenethyl)-1-ethyl-5-(p-tolyl)-5,7,8,9-tetrahydro-1H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidine-2,4,6(3H)-trione (2.5l)...... 91 1-Ethyl-7-(4-hydroxyphenethyl)-5-(p-tolyl)-5,7,8,9-tetrahydro-1H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidine-2,4,6(3H)-trione (2.5m).113 ...... 91 1-Ethyl-7-(4-methoxyphenethyl)-5-(p-tolyl)-5,7,8,9-tetrahydro-1H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidine-2,4,6(3H)-trione (2.5n).113 ...... 92 1-Ethyl-7-(3-methoxyphenethyl)-5-(p-tolyl)-5,7,8,9-tetrahydro-1H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidine-2,4,6(3H)-trione (2.5o)...... 93 1-Ethyl-7-(2-methoxyphenethyl)-5-(p-tolyl)-5,7,8,9-tetrahydro-1H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidine-2,4,6(3H)-trione (2.5p)...... 93 7-(3,4-Dimethoxyphenethyl)-1-ethyl-5-(p-tolyl)-5,7,8,9-tetrahydro-1H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidine-2,4,6(3H)-trione (2.5q)...... 94 7-(2,3-Dimethoxyphenethyl)-1-ethyl-5-(p-tolyl)-5,7,8,9-tetrahydro-1H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidine-2,4,6(3H)-trione (2.5r)...... 94 7-(3,4-Dichlorophenethyl)-1-ethyl-5-(p-tolyl)-5,7,8,9-tetrahydro-1H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidine-2,4,6(3H)-trione (2.5s)...... 95 Ethyl 2-(4-(2-(1Ethyl-2,4,6-trioxo-5-(p-tolyl)-1,2,3,4,5,6,8,9-octahydro-7H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidin-7-yl)ethyl)phenoxy)acetate (2.5t ethyl ester). . 96 Methyl 4-((4-((1-Ethyl-2,4,6-trioxo-5-(p-tolyl)-1,2,3,4,5,6,8,9-octahydro-7H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidin-7-yl)methyl)piperidin-1-yl)methyl)benzoate (2.5u methyl ester)...... 96 Methyl 4-(2-(1-Ethyl-2,4,6-trioxo-5-(p-tolyl)-1,2,3,4,5,6,8,9-octahydro-7H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidin-7-yl)ethyl)benzoate (2.5v methyl ester)...... 97 Methyl 4-((1-Ethyl-2,4,6-trioxo-5-(p-tolyl)-1,2,3,4,5,6,8,9-octahydro-7H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidin-7-yl)methyl)benzoate (2.5w methyl ester)...... 97 Methyl 3-((1-Ethyl-2,4,6-trioxo-5-(p-tolyl)-1,2,3,4,5,6,8,9-octahydro-7H-

xv pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidin-7-yl)methyl)benzoate (2.5x methyl ester)...... 98 2-(4-(2-(1-Ethyl-2,4,6-trioxo-5-(p-tolyl)-1,2,3,4,5,6,8,9-octahydro-7H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidin-7-yl)ethyl)phenoxy)acetic Acid (2.5t)...... 99 4-((4-((1-Ethyl-2,4,6-trioxo-5-(p-tolyl)-1,2,3,4,5,6,8,9-octahydro-7H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidin-7-yl)methyl)piperidin-1-ium-1- yl)methyl)benzoate (2.5u)...... 99 4-(2-(1-Ethyl-2,4,6-trioxo-5-(p-tolyl)-1,2,3,4,5,6,8,9-octahydro-7H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidin-7-yl)ethyl)benzoic Acid (2.5v)...... 100 4-((1-Ethyl-2,4,6-trioxo-5-(p-tolyl)-1,2,3,4,5,6,8,9-octahydro-7H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidin-7-yl)methyl)benzoic Acid (2.5w)...... 100 3-((1-Ethyl-2,4,6-trioxo-5-(p-tolyl)-1,2,3,4,5,6,8,9-octahydro-7H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidin-7-yl)methyl)benzoic Acid (2.5x)...... 101 Methyl 1-Ethyl-7-methyl-2,4-dioxo-5-(p-tolyl)-1,2,3,4,5,8-hexahydropyrido[2,3- d]pyrimidine-6-carboxylate (2.10a)...... 102 Propyl 1-Ethyl-7-methyl-2,4-dioxo-5-p-tolyl-1,2,3,4,5,8-hexahydropyrido[2,3- d]pyrimidine-6-carboxylate (2.10b)...... 102 Allyl 1-Ethyl-7-methyl-2,4-dioxo-5-(p-tolyl)-1,2,3,4,5,8-hexahydropyrido[2,3- d]pyrimidine-6-carboxylate (2.10c)...... 103 tert-Butyl 1-Ethyl-7-methyl-2,4-dioxo-5-p-tolyl-1,2,3,4,5,8-hexahydropyrido[2,3- d]pyrimidine-6-carboxylate (2.10d)...... 103 Benzyl 1-Ethyl-7-methyl-2,4-dioxo-5-(p-tolyl)-1,2,3,4,5,8-hexahydropyrido[2,3- d]pyrimidine-6-carboxylate (2.10e)...... 104 4-Chlorobenzyl 1-Ethyl-7-methyl-2,4-dioxo-5-p-tolyl-1,2,3,4,5,8-hexahydropyrido[2,3- d]pyrimidine-6-carboxylate (2.10f)...... 104 4-Methoxybenzyl 1-Ethyl-7-methyl-2,4-dioxo-5-p-tolyl-1,2,3,4,5,8-hexahydropyrido[2,3- d]pyrimidine-6-carboxylate (2.10g)...... 105 3,4-Dimethoxybenzyl 1-Ethyl-7-methyl-2,4-dioxo-5-p-tolyl-1,2,3,4,5,8- hexahydropyrido[2,3-d]pyrimidine-6-carboxylate (2.10h)...... 105 4-Methoxyphenethyl 1-Ethyl-7-methyl-2,4-dioxo-5-p-tolyl-1,2,3,4,5,8- hexahydropyrido[2,3-d]pyrimidine-6-carboxylate (2.10i) ...... 106

xvi 1-Ethyl-7-methyl-2,4-dioxo-N-propyl-5-(p-tolyl)-1,2,3,4,5,8-hexahydropyrido[2,3- d]pyrimidine-6-carboxamide (2.11a)...... 107 N-Allyl-1-ethyl-7-methyl-2,4-dioxo-5-p-tolyl-1,2,3,4,5,8-hexahydropyrido[2,3- d]pyrimidine-6-carboxamide (2.11b)...... 108 N-Benzyl-1-ethyl-7-methyl-2,4-dioxo-5-p-tolyl-1,2,3,4,5,8-hexahydropyrido[2,3- d]pyrimidine-6-carboxamide (2.11c)...... 108 1-Ethyl-7-methyl-2,4-dioxo-N'-phenyl-5-p-tolyl-1,2,3,4,5,8-hexahydropyrido[2,3- d]pyrimidine-6-carbohydrazide (2.11d)...... 109 1-Ethyl-7-methyl-2,4-dioxo-N-phenethyl-5-p-tolyl-1,2,3,4,5,8-hexahydropyrido[2,3- d]pyrimidine -6-carboxamide (2.11e)...... 109 1-Ethyl-N-(4-methoxyphenethyl)-7-methyl-2,4-dioxo-5-p-tolyl-1,2,3,4,5,8- hexahydropyrido[2,3-d]pyrimidine-6-carboxamide (2.11f)...... 110 1-Ethyl-7-methyl-2,4-dioxo-N-(3-phenylpropyl)-5-p-tolyl-1,2,3,4,5,8- hexahydropyrido[2,3-d]pyrimidine-6-carboxamide (2.11g)...... 110 2-(2-Bromoethoxy)benzaldehyde (2.12a)...... 111 2-(3-Bromopropoxy)benzaldehyde (2.12b)...... 111 2-(4-Bromobutoxy)benzaldehyde (2.12c)...... 112 2-(2-Bromoethoxy)-4-methylbenzaldehyde (2.12d)...... 112 2-(3-Bromopropoxy)-4-methylbenzaldehyde (2.12e)...... 112 3-(2-Bromoethoxy)benzaldehyde (2.12f)...... 113 3-(2-Bromoethoxy)-4-methylbenzaldehyde (2.12g)...... 113 3-(3-Bromopropoxy)-4-methylbenzaldehyde (2.12h)...... 113 2-(2-(2-(Hydroxymethyl)phenoxy)ethoxy)benzaldehyde (2.13a)...... 114 2-(3-(2-(Hydroxymethyl)phenoxy)propoxy)benzaldehyde (2.13b)...... 114 2-(4-(2-(Hydroxymethyl)phenoxy)butoxy)benzaldehyde (2.13c)...... 115 2-(2-(2-(Hydroxymethyl)phenoxy)ethoxy)-4-methylbenzaldehyde (2.13d)...... 115 2-(3-(2-(Hydroxymethyl)phenoxy)propoxy)-4-methylbenzaldehyde (2.13e)...... 115 3-(2-(2-(Hydroxymethyl)phenoxy)ethoxy)benzaldehyde (2.13f)...... 116 3-(2-(2-(Hydroxymethyl)phenoxy)ethoxy)-4-methylbenzaldehyde (2.13g)...... 116 3-(3-(2-(Hydroxymethyl)phenoxy)propoxy)-4-methylbenzaldehyde (2.13h)...... 116

xvii 2-(3-(3-(Hydroxymethyl)phenoxy)propoxy)-4-methylbenzaldehyde (2.13i)...... 117 2-(2-(2-Formylphenoxy)ethoxy)benzyl 3-Oxobutanoate (2.14a)...... 118 2-(3-(2-Formylphenoxy)propoxy)benzyl 3-Oxobutanoate (2.14b)...... 118 2-(4-(2-Formylphenoxy)butoxy)benzyl 3-Oxobutanoate (2.14c)...... 118 2-(2-(2-Formyl-5-methylphenoxy)ethoxy)benzyl 3-Oxobutanoate (2.14d)...... 119 2-(3-(2-Formyl-5-methylphenoxy)propoxy)benzyl 3-Oxobutanoate (2.14e)...... 119 2-(2-(3-Formylphenoxy)ethoxy)benzyl 3-Oxobutanoate (2.14f)...... 119 2-(2-(5-Formyl-2-methylphenoxy)ethoxy)benzyl 3-Oxobutanoate (2.14g)...... 120 2-(3-(5-Formyl-2-methylphenoxy)propoxy)benzyl 3-Oxobutanoate (2.14h)...... 120 3-(3-(2-Formyl-5-methylphenoxy)propoxy)benzyl 3-Oxobutanoate (2.14i)...... 120 (Z)-16-Acetyl-6,7-dihydro-13H,15H-dibenzo[e,l][1,4,8]trioxacyclotridecin-15-one (2.15a)...... 121 (Z)-17-Acetyl-7,8-dihydro-6H,14H,16H-dibenzo[c,j][1,5,9]trioxacyclotetradecin-16-one (2.15b)...... 122 (Z)-18-Acetyl-6,7,8,9-tetrahydro-15H,17H-dibenzo[c,k][1,5,10]trioxacyclopentadecin- 17-one (2.15c)...... 122 (Z)-16-Acetyl-3-methyl-6,7-dihydro-13H,15H-dibenzo[e,l][1,4,8]trioxacyclotridecin-15- one (2.15d)...... 122 (Z)-17-Acetyl-3-methyl-7,8-dihydro-6H,14H,16H- dibenzo[c,j][1,5,9]trioxacyclotetradecin-16-one (2.15e)...... 123 (Z)-10-Acetyl-2,5,8-trioxa-1(1,3),6(1,2)-dibenzenacycloundecaphan-10-en-9-one (2.15f)...... 123 (Z)-10-Acetyl-16-methyl-2,5,8-trioxa-1(1,3),6(1,2)-dibenzenacycloundecaphan-10-en-9- one (2.15g)...... 124 (Z)-11-Acetyl-16-methyl-2,6,9-trioxa-1(1,3),7(1,2)-dibenzenacyclododecaphan-11-en-10- one (2.15h)...... 124 (Z)-11-Acetyl-15-methyl-2,6,9-trioxa-1(1,2),7(1,3)-dibenzenacyclododecaphan-11-en-10- one (2.15i)...... 124 2-(3-Hydroxypropoxy)benzaldehyde (2.17a)...... 125 2-(4-Hydroxybutoxy)benzaldehyde (2.17b)...... 125

xviii 2-(3-Hydroxypropoxy)-4-methylbenzaldehyde (2.17c)...... 126 2-(4-Hydroxybutoxy)-4-methylbenzaldehyde (2.17d)...... 126 3-(2-Formylphenoxy)propyl 3-oxobutanoate (2.18a)...... 127 4-(2-Formylphenoxy)butyl 3-oxobutanoate (2.18b)...... 127 3-(2-Formyl-5-methylphenoxy)propyl 3-oxobutanoate (2.18c)...... 127 4-(2-Formyl-5-methylphenoxy)butyl 3-oxobutanoate (2.18d)...... 128 (Z)-7-Acetyl-3,4-dihydro-2H,6H-benzo[f][1,5]dioxecin-6-one (2.19a)...... 129 (Z)-8-Acetyl-2,3,4,5-tetrahydro-7H-benzo[g][1,6]dioxacycloundecin-7-one (2.19b). .. 129 (Z)-7-Acetyl-11-methyl-3,4-dihydro-2H,6H-benzo[f][1,5]dioxecin-6-one (2.19c)...... 129 (Z)-8-Acetyl-12-methyl-2,3,4,5-tetrahydro-7H-benzo[g][1,6]dioxacycloundecin-7-one (2.19d)...... 130 2-(3-(2-Formylphenoxy)propoxy)benzonitrile (2.21)...... 130 (2-(3-(2-(Aminomethyl)phenoxy)propoxy)phenyl)methanol (2.22)...... 130 N-(2-(3-(2-(Hydroxymethyl)phenoxy)propoxy)benzyl)-3-oxobutanamide (2.23)...... 131 N-(2-(3-(2-Formylphenoxy)propoxy)benzyl)-3-oxobutanamide (2.24)...... 131 (E)-17-Acetyl-7,8,14,15-tetrahydro-6H,16H- dibenzo[f,m][1,5]dioxa[9]azacyclotetradecin-16-one (2.25)...... 132 3-Ethyl-1-methyl-3,6b,12,13-tetrahydro-4H,19H- dibenzo[5',6':12',13'][1,4,8]trioxacyclotridecino[11',10':4,5]pyrido[2,3-d]pyrimidine- 4,6,21(2H,5H)-trione (2.16a)...... 133 3-Ethyl-1-methyl-3,6b,13,14-tetrahydro-4H,12H,20H- dibenzo[6',7':13',14'][1,5,9]trioxacyclotetradecino[12',11':4,5]pyrido[2,3-d]pyrimidine- 4,6,22(2H,5H)-trione (2.16b)...... 133 3-Ethyl-1-methyl-3,6b,12,13,14,15-hexahydro-4H,21H- dibenzo[3',4':11',12'][1,5,10]trioxacyclopentadecino[13',14':4,5]pyrido[2,3-d]pyrimidine- 4,6,23(2H,5H)-trione (2.16c)...... 134 3-Ethyl-1,9-dimethyl-3,6b,12,13-tetrahydro-4H,19H- dibenzo[5',6':12',13'][1,4,8]trioxacyclotridecino[11',10':4,5]pyrido[2,3-d]pyrimidine- 4,6,21(2H,5H)-trione (2.16d)...... 134 3-Ethyl-1,9-dimethyl-3,6b,13,14-tetrahydro-4H,12H,20H-

xix dibenzo[6',7':13',14'][1,5,9]trioxacyclotetradecino[12',11':4,5]pyrido[2,3-d]pyrimidine- 4,6,22(2H,5H)-trione (2.16e)...... 135 21-Ethyl-27-methyl-21,22,23,24,25,28-hexahydro-4,7,10-trioxa-2(5,6)-pyrido[2,3- d]pyrimidina-1(1,3),6(1,2)-dibenzenacyclodecaphane-22,24,3-trione (2.16f)...... 135 21-Ethyl-14,27-dimethyl-21,22,23,24,25,28-hexahydro-4,7,10-trioxa-2(5,6)-pyrido[2,3- d]pyrimidina-1(1,3),6(1,2)-dibenzenacyclodecaphane-22,24,3-trione (2.16g)...... 136 21-Ethyl-14,27-dimethyl-21,22,23,24,25,28-hexahydro-4,7,11-trioxa-2(5,6)-pyrido[2,3- d]pyrimidina-1(1,3),6(1,2)-dibenzenacycloundecaphane-22,24,3-trione (2.16h)...... 136 21-Ethyl-14,27-dimethyl-21,22,23,24,25,28-hexahydro-4,7,11-trioxa-2(5,6)-pyrido[2,3- d]pyrimidina-1(1,2),6(1,3)-dibenzenacycloundecaphane-22,24,3-trione (2.16i)...... 137 4-Ethyl-6-methyl-5,10,11,16b-tetrahydro-1H,9H- benzo[9',10'][1,5]dioxecino[8',7':4,5]pyrido[2,3-d]pyrimidine-1,3,7(2H,4H)-trione (2.20a)...... 138 4-Ethyl-6-methyl-5,9,10,11,12,17b-hexahydro-1H- benzo[10',11'][1,6]dioxacycloundecino[9',8':4,5]pyrido[2,3-d]pyrimidine-1,3,7(2H,4H)- trione (2.20b)...... 138 4-Ethyl-6,14-dimethyl-5,10,11,16b-tetrahydro-1H,9H- benzo[9',10'][1,5]dioxecino[8',7':4,5]pyrido[2,3-d]pyrimidine-1,3,7(2H,4H)-trione (2.20c)...... 139 4-Ethyl-6,15-dimethyl-5,9,10,11,12,17b-hexahydro-1H- benzo[10',11'][1,6]dioxacycloundecino[9',8':4,5]pyrido[2,3-d]pyrimidine-1,3,7(2H,4H)- trione (2.20d)...... 139 3-Ethyl-1-methyl-3,6b,13,14,20,21-hexahydro-4H,12H- dibenzo[f,m]pyrimido[5',4':5,6]pyrido[3,4-k][1,5]dioxa[9]azacyclotetradecine- 4,6,22(2H,5H)-trione (2.26)...... 140 2-(2,6-Dioxopiperidin-3-yl)-4-hydroxyisoindoline-1,3-dione (2.33)...... 140 2-(2,6-Dioxopiperidin-3-yl)-4-((7-hydroxyheptyl)oxy)isoindoline-1,3-dione (2.34). ... 141 7-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)heptyl Methanesulfonate (2.35A)...... 141 4-((7-Azidoheptyl)oxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (2.35)...... 142

xx 2-Chloro-N-(prop-2-yn-1-yl)acetamide...... 142 2-(3-Formylphenoxy)-N-(prop-2-yn-1-yl)acetamide (2.27a)...... 143 2-(2-Formylphenoxy)-N-(prop-2-yn-1-yl)acetamide (2.27b)...... 144 Ethyl 7-((Benzyloxy)methyl)-1-ethyl-2,4-dioxo-5-(3-(2-oxo-2-(prop-2-yn-1- ylamino)ethoxy)phenyl)-1,2,3,4,5,8-hexahydropyrido[2,3-d]pyrimidine-6-carboxylate (2.28a)...... 145 Ethyl 7-((Benzyloxy)methyl)-1-ethyl-2,4-dioxo-5-(2-(2-oxo-2-(prop-2-yn-1- ylamino)ethoxy)phenyl)-1,2,3,4,5,8-hexahydropyrido[2,3-d]pyrimidine-6-carboxylate (2.28b)...... 145 Ethyl 1-Ethyl-7-(hydroxymethyl)-2,4-dioxo-5-(3-(2-oxo-2-(prop-2-yn-1- ylamino)ethoxy)phenyl)-1,2,3,4,5,8-hexahydropyrido[2,3-d]pyrimidine-6-carboxylate (2.29a)...... 146 Ethyl 1-Ethyl-7-(hydroxymethyl)-2,4-dioxo-5-(2-(2-oxo-2-(prop-2-yn-1- ylamino)ethoxy)phenyl)-1,2,3,4,5,8-hexahydropyrido[2,3-d]pyrimidine-6-carboxylate (2.29b)...... 147 Ethyl 7-(Chloromethyl)-1-ethyl-2,4-dioxo-5-(3-(2-oxo-2-(prop-2-yn-1- ylamino)ethoxy)phenyl)-1,2,3,4,5,8-hexahydropyrido[2,3-d]pyrimidine-6-carboxylate (2.30a)...... 148 Ethyl 7-(Chloromethyl)-1-ethyl-2,4-dioxo-5-(2-(2-oxo-2-(prop-2-yn-1- ylamino)ethoxy)phenyl)-1,2,3,4,5,8-hexahydropyrido[2,3-d]pyrimidine-6-carboxylate (2.30b)...... 148 2-(3-(1-Ethyl-7-(4-hydroxyphenethyl)-2,4,6-trioxo-2,3,4,5,6,7,8,9-octahydro-1H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidin-5-yl)phenoxy)-N-(prop-2-yn-1-yl)acetamide (2.31a)...... 149 2-(2-(1-Ethyl-7-(4-hydroxyphenethyl)-2,4,6-trioxo-2,3,4,5,6,7,8,9-octahydro-1H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidin-5-yl)phenoxy)-N-(prop-2-yn-1-yl)acetamide (2.31b)...... 150 1-Ethyl-7-(4-(prop-2-yn-1-yloxy)phenethyl)-5-(p-tolyl)-5,7,8,9-tetrahydro-1H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidine-2,4,6(3H)-trione (2.32)...... 150 N-((1-(7-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)heptyl)-1H-1,2,3-

xxi triazol-4-yl)methyl)-2-(3-(1-ethyl-7-(4-hydroxyphenethyl)-2,4,6-trioxo-2,3,4,5,6,7,8,9- octahydro-1H-pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidin-5-yl)phenoxy)acetamide (2.36a)...... 151 N-((1-(7-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)heptyl)-1H-1,2,3- triazol-4-yl)methyl)-2-(2-(1-ethyl-7-(4-hydroxyphenethyl)-2,4,6-trioxo-2,3,4,5,6,7,8,9- octahydro-1H-pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidin-5-yl)phenoxy)acetamide (2.36b) ...... 152 7-(4-((1-(7-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)heptyl)-1H-1,2,3- triazol-4-yl)methoxy)phenethyl)-1-ethyl-5-(p-tolyl)-5,7,8,9-tetrahydro-1H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidine-2,4,6(3H)-trione (2.36c)...... 153 6-Chloro-3-methoxy- [1,2,4]triazolo[4,3-b]pyridazine (2.37)...... 153 1-(3-Methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-ol (2.38)...... 154 2-(4-(1-Ethyl-2,4,6-trioxo-1,2,3,4,5,6,8,9-octahydrofuro[3',4':5,6]pyrido[2,3-d]pyrimidin- 5-yl)phenyl)acetic acid (2.40)...... 154 1-(3-Methoxy-[1,2,4]triazolo[4,3-a]pyridin-6-yl)piperidin-4-yl 2-(4-(1-Ethyl-2,4,6- trioxo-1,2,3,4,5,6,8,9-octahydrofuro[3',4':5,6]pyrido[2,3-d]pyrimidin-5-yl)phenyl)acetate (2.41)...... 155 Ethyl 2-Chloronicotinate...... 157 Desired Nicotinic Acid Intermediates ...... 158 Desired Nicotinamide Analogs ...... 166

xxii List of Abbreviations

11β-MNTDC 11β-Methyl-nortestosterone dodecyl carbonate

ADPLD Autosomal dominant polycystic liver disease

ADPKD Autosomal dominant polycystic kidney disease

ADMET Absorption, distribution, metabolism, excretion, and toxicity ARPKD Autosomal recessive polycystic kidney disease

ATRA All-trans-retinoic acid

BET Bromodomain and extra-terminal protein

BRDT Bromodomain testis-specific protein

CA Cholic acid

CatSper Cationic channel of sperm

CDCA Chenodeoxycholic acid

CDK2 Cyclin-dependent kinase 2

DCA Deoxycholic acid

DCM Dichloromethane

DIPEA N,N-Diisopropylethylamine

DMAU Dimethandrolone undecanoate

DMF Dimethylformamide

DSF Differential scanning fluorimetry

EC50 Half maximal effective concentration

EDCI N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride

xxiii EGF Epidermal growth factor

EGFR Epidermal growth factor receptor

EPPIN Epididymal protease inhibitor

FDA Food and Drug Administration

FP Fluorescence polarization

FSH Follicle-stimulating hormone

PrOF Protein-observed 19F NMR

GLP-1 Glucagon-like peptide-1

GnRH Gonadotropin-releasing hormone

GPCR G protein-coupled receptor

HPT Hypothalamic-pituitary-testicular

HATU 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5- b]pyridinium 3-oxid hexafluorophosphate HOBt Hydroxybenzotriazole

IC50 50% Inhibitory concentration

IGF1 Insulin-like growth factor 1

IUD Intrauterine device

Ki Inhibitory constant

LAH Lithium aluminum hydride

LCA Lithocholic acid

LH Luteinizing hormone

MEK MAPK/ERK kinase

MENT Methyl-19-nortestosterone

xxiv mp Melting point

MPA Medroxyprogesterone acetate

NICHD Eunice Kennedy Shiver National Institute of Child Health and Human Development NETE Norethisterone enanthate

NGO Non-governmental organization

NMR Nuclear magnetic resonance

PLD Polycystic liver diseases

PKD Polycystic kidney disease

PK/ PD Pharmacokinetics/pharmacodynamics

Pol II RNA polymerase II

PPI Protein-protein interaction ppm parts per million

PRORACs Proteolysis-targeted chimeras

PSA Prostate-specific antigen pTEFb Positive transcription elongation factor b qNMR Quantitative nuclear magnetic resonance

RARα Retinoic acid receptor α

RXR Retinoid-X receptor

SAR Structure-activity relationship

SAXS Small-angle X-ray scattering

SEMG1 Semenogelin 1

SNAr Nucleophilic aromatic substitution

xxv TCLA Taurine-conjugated lithocholic acid

TEA Triethylamine

THF Tetrahydrofuran

TLC Thin layer chromatography

TMD 2,2,6-Trimethyl-4H-1,3-dioxin-4-one

TR-FRET Time-resolved fluorescence energy transfer

TRPV4 Transient receptor potential cation channel subfamily V member 4 TSSK Testis-specific serine/threonine kinase

UPLC Ultra-performance liquid chromatography

VEGF Vascular endothelial growth factor

VEGFR Vascular endothelial growth factor receptor

WHO World Health Organization

xxvi Chapter 1 Recent Progress in Male Contraception 1.1. Social and Scientific Significance of Male Contraceptive Research Despite a slight decrease since 2008, nearly 45% of pregnancies in the United States are still unintended.1 These pregnancies can have significant societal impacts. First, unintended pregnancies can result in substantial costs for both maternity and infant care that are borne by federal and state governments. A study quantifying the cost of publicly- funded births in 2006 estimated that 64% of 1.6 million births from unintended pregnancies were funded by public insurance programs. Among the 2.0 million publicly funded births, 51% account for unintended pregnancies, occupying half of the total annual public expenditures on birth of $21.8 billion US dollars.2 Secondly, unintended pregnancies can contribute to profound sociological issues. For example, an unintended pregnancy can escalate the risk of union dissolution.3 In addition, two longitudinal studies concluded that unintended pregnancy was strongly associated with poorer maternal mental health in both their early (30s) and later (50s) life.4, 5 As suggested by the aforementioned studies, reducing the frequency of unintended pregnancies could have many beneficial societal outcomes. One way to reduce unintended pregnancies is the use of effective contraceptive methods. According to data spanning 2008 to 2011, it was the increased frequency of use and introduction of highly effective long- acting types of female contraceptives that ameliorated the rate of unintended pregnancy.1 The most common female contraceptives are pills, intrauterine device (IUDs), patches, and vaginal rings, all with greater than 90% success rates. However, these highly effective modalities are not suitable for every woman due to compliance issues or health conditions.6-8 For instance, female hormonal contraceptive pills may result in adverse effects including but not limited to depression, nausea, weight gain and cardiovascular problems.6 Unfortunately, contraceptive methods for males are relatively limited and unsatisfactory; the reversible options, condoms and withdrawal, have 13 and 20% failure rates,9 respectively, while vasectomy is considered permanent. Therefore, developing novel, safe, and reversible male contraceptives will not only help women who are unable to safely or acceptably take female contraceptives to achieve birth control but also address

1 the unmet need for men to pursue contraception. One question remains in the minds of many: will they take it? In fact, a survey in 2002 indicated that about 50% of interviewed men (1500 in total) in the US showed a willingness to use a reversible male contraceptive if available.10 Knowing that there is interest in this new family planning paradigm, it’s up to the researchers to discover potential drug-like compounds to control male fertility. In addition to its social significance, the development of male contraceptives offers an opportunity to showcase how academic labs/research institutes are at the leading edge of drug discovery and development. Male contraception programs have been virtually abandoned by the pharmaceutical industry since the 1990s. Under these circumstances, it is multidisciplinary collaborations among universities/research institutes that are conducting the preclinical studies through the pipeline with financial support from NICHD, WHO, and other NGOs.11-13 Furthermore, the unique strengths and features of academia that distinguish it from industry could be advantageous for male contraceptive discovery. As reviewed by Johnston and Goldberg, academic labs typically have a deeper understanding of the targeted biological process and a relatively flexible timeline for their milestones.14 What is also unique to academia is the ability to rekindle projects that were discontinued by one funding source to seek alternative financial support.14 Despite the paucity of funding available for this field, these strengths enable many academic male contraceptive programs to keep moving forward. To achieve male contraception, there are two general paradigms: targeted and non- targeted therapies. Targeted methods endeavor to manipulate an endogenous process that is critical for either sperm maturation or fertilization (Figure 1.1, orange and green flags). In these scenarios, cellular components (e.g., enzymes, ion channels, and transporters), which have been comprehensively validated for necessity, specificity, and druggability, are used for small molecule or antibody development.15, 16 Based on whether the hormonal milieu is disrupted, the small molecules can be further classified into the hormonal and non-hormonal categories.17, 18 In contrast, non-targeted methods do not rely on a specific endogenous process or modulator. Instead, they barricade the sperm from fertilizing the egg via a chemical or physical approach (Figure 1.1, red flag).19, 20 For instance, Amphora is a combination of citric acid, L-lactic acid, and potassium bitartrate that deactivates

2 sperm.20 The targeted male contraceptive paradigm has garnered a recent surge of interest and media attention as well as the formation of a nonprofit dedicated to advocacy and promotion of non-hormonal method development.21, 22 Therefore, the rest of this chapter will focus on current progress achieved with the hormonal contraceptives as well as the burgeoning non-hormonal contraceptive field.

Figure 1.1. The lifespan of sperm. Targeted male contraceptive therapy could take place during spermatogenesis via affecting key modulators (orange flags). After sperm maturation, there are also cellular components for targeted intervention (green flag) to immobilize the sperm. Non-targeted methods deactivate the spermatozoa via chemical or physical means (red flag).

1.2. Hormonal Male Contraception Given the great success of female hormonal contraceptives, researchers have sought the possibility of a “male pill” since the early 1970s.23, 24 It is the hypothalamic-pituitary- testicular (HPT) axis that gives rise to any potential hormonal male contraceptives.25 As depicted in Figure 1.2A, gonadotropin-releasing hormone (GnRH) from the hypothalamus stimulates the pituitary gland to release follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which further activate the Sertoli and Leydig cells in the testes, respectively, leading to testosterone secretion and sperm maturation. Importantly, a high testicular testosterone concentration (822 ± 136 nmol/L) is essential for spermatogenesis. As shown in Figure 1.2B, there are generally four stages in spermatogenesis, the first of which is the differentiation of the spermatogonia in mitosis. Then, FSH signals for the differentiated spermatogonia to undergo meiosis I to produce a spermatocyte (stage 2). Meiosis II reduces the diploid primary spermatocyte into a haploid secondary spermatocyte. Subsequently, testosterone (Figure 1.2B, purple rectangle) is required for the differentiation of secondary spermatocytes into round spermatids (stage 3). Then, spermatid elongation occurs in the process known as spermiogenesis, and finally mature spermatozoa (stage 4) develop. Meanwhile, a much lower serum testosterone level (22.8 ± 2 nmol/L) is responsible for libido, muscle mass maintenance, and negative feedback to the HPT axis (Figure 1.2A, red 3 arrows).26 Exogenous androgens can exploit this feedback loop to suppress testicular testosterone production (Figure 1.2A, orange dotted box), eventually resulting in reversible infertility.26 Simultaneously, exogenous androgens are still able to bind to peripheral androgen receptors, keeping functions other than spermatogenesis intact.

Figure 1.2. The design rationale of hormonal male contraceptives. A. Hypothalamic-pituitary-testicular (HPT) axis suppression loop. GnRH: gonadotropin-releasing hormone; FSH: follicle-stimulating hormone, LH: luteinizing hormone, T: testosterone. The mechanism of action of hormonal contraceptives is highlighted by the orange dotted box. B. Four differentiation stages of spermatogenesis. FSH regulates meiosis from differentiated spermatogonia to primary spermatocytes. Testosterone (T) is required to transition from secondary spermatocytes to spermatids (purple rectangle). 2n and n indicate the chromosome numbers inside the cells.

Given its necessity in spermatogenesis and role in negative feedback along the HPT axis, testosterone had been envisioned to be a potential contraceptive agent. Because of patient compliance and ease of administration, orally dosed testosterone was first evaluated. However, it was quickly apparent that testosterone had poor oral bioavailability (promptly

4 degraded by liver) and off-target effects (i.e., acne, high-density lipoprotein cholesterol suppression and lean body mass increase).27-29 To pursue an orally bioavailable compound and avoid the off-target effects of testosterone, follow-up investigations explored related androgen derivatives with better bioavailability as well as minimal side effects. 7α-Methyl-19-nortestosterone (MENT), dimethandrolone undecanoate (DMAU), and 11β-methyl-nortestosterone dodecyl carbonate (11β-MNTDC) are three next-generation representative androgens (Figure 1.3). They are resistant to the 5α-reductase that generates the metabolites responsible for adverse effects,30 thus displaying clean azoospermia profiles in animal models.31-33 MENT was formulated as an implant for better bioavailability and demonstrated contraceptive efficacy in human volunteers.34 However, inconsistent drug release was noticed during later evaluation,35 suggesting continuous improvement of the implant device is still needed. Meanwhile, DMAU and 11β-MNTDC, formulated as capsules, have recently completed early phase I clinical trials, both displaying effective serum testosterone suppression devoid of severe adverse effects.36, 37 Further studies will be towards long-term contraceptive efficacy and safety of these two drug candidates.

O O OH OH O O O H H H H H H H H H H H H H O O O H H O

Testosterone MENT DMAU 11β-MNTDC Figure 1.3. Chemical structures of testosterone and androgen derivatives for hormonal contraceptive studies.

In addition to implants and oral administration, transdermal formulation was also investigated, because of the lipophobicity of the androgen derivatives and the good patient compliance afforded by a gel formulation. Testosterone gel can be absorbed into the stratum corneum to form a reservoir of active component that acts as a rate-controlling membrane.38 The combination of testosterone gel and medroxyprogesterone acetate (MPA) pill, an oral progestin that augments the feedback inhibition of FHS and LH (Figure 1.4),39 resulted in sperm suppression (≤ 1 million/mL) with excellent reproducibility (27 out of 29 men).40 Another regimen under development is the daily combination of testosterone gel and Nestorone gel, a potent progestin with no androgenic, estrogenic, or glucocorticoid 5 activity,41 which led to azoospermia in 89% of men without severe adverse effects.42 In a phase II multisite international trial, norethisterone enanthate (NETE, Figure 1.4) and testosterone undecanoate were co-administered intramuscularly to assess efficacy and safety. Although the study was discontinued early due to the high incidence of mood disorders, injection site pain, and increased libido, the combination treatment did demonstrate adequate contraceptive effect (1.57% pregnancy) and reversibility (94.8% recovery).43

O O O O O O O H O HH H H H H HH H H O O O MPA (pill) Nestorone (gel) NETE (injection) + + + Testosterone (gel) Testosterone (gel) Testosterone undecanoate (injection) Figure 1.4. Combination regimens and corresponding delivery modes for hormonal male contraception.

Despite this progress, a male hormonal contraceptive is nowhere near the market yet. There are still several hurdles to overcome, the first of which are the side effects. Mood and libido changes were immediately noticed from preclinical and clinical evaluations of testosterone, which were presumably derived from its androgenic effects.44 Concerns regarding the long-term impact on metabolic changes, including serum cholesterol, blood pressure, and lean body mass, remain for hormonal therapy. Since male contraception is a novel therapy, it needs to be determined what side effects are tolerable or not for both regulatory agencies and potential users. Moreover, because of the sperm generation cascade, which takes approximately 72 days, contraceptives that disrupt these processes require at least 6 to 8 weeks of administration to be effective. Previous studies have indicated that the median time of effective sperm suppression was between 75 and 170 days.45 Such a long onset lag could be a potential impediment for hormonal treatments. Couples would have to rely on alternative contraceptive methods before the hormonal drug would be effective. In addition, the contraceptive effect of hormonal regimens showed racial variations. According to two WHO efficacy studies, Asian males were more susceptible to the hormonal regimen (89-100% azoospermia), compared with non-Asian

6 ones (45-70%).46, 47 These racial differences could also stymie further development of male hormonal contraceptives.

1.3. Non-hormonal Male Contraception In contrast to hormonal therapies that are disturbing the hormonal milieu, non- hormonal male contraceptives pursue reversible infertility via targeting key proteins in spermatogenesis or normal sperm functions. Consequently, the non-hormonal treatments inherently avoid hormone-related side effects. Therefore, any non-hormonal modality represents another opportunity to control fertility. If categorizing by mechanism of action, there are two classes of non-hormonal targets (Figure 1.5): (1) spermatogenesis-related proteins, such as bromodomain testis-specific protein (BRDT),48 cyclin-dependent kinase 2 (CDK2),49 testis-specific serine/threonine kinases (TSSKs),50 and retinoic acid receptor α (RARα);51 and (2) sperm motility/capacitation-orientated proteins, including Na, K-ATPase,12 cationic channels of sperm (CatSper),52 and the epididymal protease inhibitor (EPPIN).53 Below, three recent advancements in the non-hormonal contraceptive field are discussed.

Figure 1.5. Representative molecules from non-hormonal male contraceptive projects and corresponding targets. 7 1.3.1. Retinoic Acid Receptor α Vitamin A and its physiologically active metabolite all-trans-retinoic acid (ATRA) have long been known crucial for fertility.54, 55 ATRA serves as the endogenous ligand of the nuclear complex of retinoic acid receptors (RARs) and the retinoid-X receptors (RXRs). Further study unveiled that it was RARα activation that played an essential role in sperm maturation (Figure 1.6),56, 57 and RARα knock-out studies in mice yielded a sterile but healthy phenotype.51 Moreover, pan-RAR antagonist BMS-189453 (Figure 1.5) led to reversible infertility in a murine model, showing a failure of spermatid alignment and sperm release.58 Extensive SAR exploration around BMS-189453 was conducted to improve RARα selectivity and physicochemical properties.59 At present, several potent and selective inhibitors with reasonable PK/PD profiles have been synthesized and evaluated in mice, which are promising leads for future non-hormonal contraceptives.

Figure 1.6. Retinoic acid receptor signaling cascade. All-trans-retinoic acid (ATRA) binds to retinoic acid receptor (RAR)/RAR-retinoid X receptor (RXR) heterodimer complex bound to the RA response element (RARE). The RARE sits next to the target gene, resulting in activation of transcription for genes including Stra8, which is necessary for progression through meiosis.60

1.3.2. Cation Channels of Sperm As mentioned earlier, ion channels responsible for sperm motility/capacitation have been proposed as male contraceptive targets. The cation channels of sperm (CatSper) are pH-dependent and low voltage-dependent calcium channels specifically expressed on the sperm flagellum.52 Upon activation by the sex hormone progesterone, CatSper induces 8 calcium influx that eventually leads to hyperactivated motility (Figure 1.7),61 which is essential for sperm to penetrate the zona pellucida to form a zygote.62 Mice with the CatSper gene knocked out were completely infertile without other obvious defects.63 Despite several compounds (i.e., mibefradil64, Figure 1.5) being identified as CatSper blockers, the lack of specificity and potential toxicity hampered their further development.65 From an in-house high throughput screening campaign, the Georg group identified 7 structurally diverse novel hits as CatSper inhibitors.66 Iterative structural optimization yielded two lead compounds with submicromolar inhibitory potency.67 Future work will focus on selectivity assessment and potency improvement before moving to animal studies.

Figure 1.7. Localization and function of CatSper. Upon chemical activation by progesterone, CatSper in the principal piece of sperm induces calcium ion influx, which causes hyperactivated motility of the sperm.

1.3.3. Epididymal Protease Inhibitor Another validated contraceptive target is epididymal protease inhibitor (EPPIN). It is a testis-specific surface protein vital for sperm motility.53 Inside seminal vesicles, EPPIN exists in complex with semenogelin 1 (SEMG1), thus suppressing sperm motility.68 Once SEMG1 is cleaved from EPPIN by activated prostate-specific antigen (PSA) in the ejaculate,69 the spermatozoa gain progressive motility to swim in a viscous environment.66, 70 Small molecule EP055 (Figure 1.5) was developed to target EPPIN, which caused significant reduction of sperm motility (over 70% inhibition at 30 and 78 h after 75-80

9 mg/kg administration) in a macaque model.71 The molecule-induced loss of motility fully recovered 18 days postinfusion, indicating the reversibility of EP055. No further studies about EP055 have been disclosed, but it will be interesting to see how it behaves in long- term efficacy and safety investigations.

1.4. Summary and Outlook Great progress has been achieved in the targeted male contraceptive field as exemplified by the success of the projects highlighted. Several hormonal contraceptive candidates have entered clinical trials, which will pave the way for novel fertility control methods currently in late-stage evaluation as well as establish the regulatory framework for clinical trials and FDA approval. Although non-hormonal development remains in the early phase, various proposed targets have completed proof-of-concept studies and identified chemical entities for further development. The aforementioned programs reflect differing avenues to achieving male contraception, and it remains debatable which paradigm would be optimal: causing azoospermia (i.e., hormonal therapy) or inhibiting mature sperm (i.e., CatSper). While azoospermia is the most reliable and simple to measure in terms of clinical fertility assessment, these methods are accompanied by potential side effects as well as long on/off durations. In contrast, by selectively targeting the mature sperm, the time to establish and reverse infertility would be short and have limited peripheral impacts. One average (3.7 mL72) ejaculation contains a considerable number of sperm (around 15 to 200 million sperm per mL), so disrupting sperm motility could be an insurmountable goal. However, based on WHO criteria, the motility parameters for the 50th centile of men whose partner had a time-to-pregnancy of 12 months or less were 61 and 55% (total and progressive motility, respectively), whereas the 5th centile numbers that are considered the lower limit of fertility were 40 and 32%, respectively.72 Sperm motility does not need to be abolished but rather impeded, and progressive motility is most closely correlated with pregnancy rates.73, 74 While changing sperm morphology takes weeks, interfering with motility may be a sweet spot for interfering in sperm-oocyte fusion.

10 Like most medicinal chemistry-based programs, the quest for male contraceptive programs is high-risk, high-investment, and long-term. Unfortunately, limited financial support and pharmaceutical industry programmatic dormancy impede development and need to be properly addressed. With this in mind, the aforementioned non-targeted contraceptive programs are intriguing not only for their relatively high cost-effectiveness but also their emerging products could attract venture capital to fuel the pipeline and gain sustainable public awareness of male contraception.14 While the joke in the field has been that we’ve been 10 years away for the past 30 years, with the advancement of several products into later-stage clinical trials, the first novel male contraceptive product in the US is estimated to be approved by 2030.

11 Chapter 2 Design and Synthesis of BRDT Selective Inhibitors as Male Contraceptive Agents 2.1. Review of Bromodomain and Extra Terminal Domain (BET) Proteins 2.1.1. Epigenetics and Bromodomains ε-N-Lysine acetylation of histone tails in nucleosomes is considered an important 75 post-translational modification. The acetyl moiety on the lysine residue (KAc) neutralizes the positive charge and thus reduces its affinity for negatively charged DNA, resulting in a less closely packed DNA/histone complex that is ready for transcription (Figure 2.1A).76 For DNA to undergo gene transcription, it is the bromodomain-containing proteins that specifically recognize these KAc modifications and recruit the transcriptional machinery (Figure 2.1B).77 There are 46 proteins with 61 identified bromodomain(s), which are further classified into 8 subfamilies.78 Recent studies have shown that these bromodomains are involved in both endogenous and disease-related epigenetic processes.79-81 Consequently, modulation of bromodomain proteins has been proposed for various therapeutic areas, including cancer, cardiovascular disease, and male contraception.

Figure 2.1. The role of bromodomains in chromatin remodeling. A. Acetylation of lysines on the histone tails gives rise to a relatively loose, transcriptionally active DNA/histone complex. The bromodomain 12 proteins (reader) specifically recognize these epigenetic modifications. The acetylation level is regulated by histone acetyltransferases (writer) and deacetylases (eraser). B. After binding to acetylated lysines, the bromodomain protein (BRD) recruits positive transcription elongation factor b (pTEFb) and other components, which activate RNA polymerase II (Pol II) via phosphorylation to initiate gene transcription.

2.1.2. The BET Family Among the 8 bromodomain subfamilies, the bromodomain and extra-terminal (BET) family has attracted the most attention and has been extensively investigated. The BET family comprises four isoforms, BRD2, BRD3, and BRD4, and BRDT.78 The first three isoforms are universally detected in tissues, whereas BRDT is testis-restricted. Structurally, the BET proteins have two bromodomains (BD1 and BD2) in tandem (Figure

2.2A) for the recognition of KAc moieties with different affinities, which are mainly dictated by the locations as well as acetylation levels of lysine residues.78 There is an extra- terminal (ET) domain near the C-terminus, which is another region that interacts with chromatin and transcription proteins.82, 83 As shown in Figure 2.2B, each single bromodomain consists of four alpha helices (A, Z, B, and C) that are connected via two flexible loop regions (ZA and BC loops). Surrounded by two loops is a cavity in the middle of four helical bundles, inside of which there are five conserved water molecules stabilizing the protein folding via a hydrogen bond network.78 Notably, due to high sequence similarity (Figure 2.2C), the eight bromodomains in the BET subfamily share almost identical binding sites, which poses a challenge for domain/isoform specificity.

13 Figure 2.2. Structural information for BET proteins. A. Schematic figure of full-length BET proteins. BD1 and BD2 stand for the two bromodomains in tandem. ET represents the extra-terminal domain. The arrows above and below the schematic figure indicate the starting and ending sequence numbers of the bromodomains, respectively. B. The surface view of BRD4-1 (PDB ID: 3MXF). Four helices and two loops are listed with black text. Conserved water molecules are rendered in red spheres. C. Sequence identity between BET bromodomains.

Functionally, BET proteins preferentially bind to the di-acetylated lysine motifs on the histone tails.84 After recognition, BET proteins facilitate chromatin opening, recruit transcription factors, and activate paused RNA Pol II complexes to start transcriptional elongation (Figure 2.1B).85-87 In particular, BRD4, arguably the most comprehensively investigated BET protein, plays pivotal roles in regulating transcription of oncogenes, pro- inflammatory cytokines, and HIV gene expression.88-90 Consequently, BET inhibitors hold promise for anticancer, anti-inflammatory, and antiviral therapeutics.91-93

14 To achieve BET inhibition, researchers have developed multiple approaches. The first and most common method is competitive antagonism, wherein, for example, an acetylated lysine mimic of the inhibitor (Figure 2.3, highlighted in red) forms a conserved hydrogen bond with Asn140 (BRD4-1 numbering) and a hydrophobic moiety of the inhibitor (Figure 2.3, highlighted in blue) occupies the WPF shelf (Trp81, Pro82, and Phe83).94-96 Inspired by the fact that full-length BETs have two bromodomains in tandem, a bivalent strategy (exemplified by MT1) was proposed to occupy both KAc recognition sites simultaneously.97, 98 Another approach is to hijack the proteasome system and selectively degrade the BET proteins, which is accomplished via integrating a small molecule inhibitor with an E3 ligase recruiting motif (ARV-825).99, 100 Because of their excellent potencies, numerous BET inhibitors have entered the clinical evaluation stage.101 However, the observed toxicity from pan-BET inhibition has become a major obstacle for further development.102 Therefore, the next-generation BET inhibitors should possess better selectivity profiles for finer tuning of downstream transcription. Marked progress in domain-specific BET inhibitors has been accomplished via exploiting subtle structure differences between BD1 and BD2.103-106 Nonetheless, BET isoform selectivity remains challenging due to the structural conservation in the binding sites.

Monovalent inhibitors Bivalent inhibitors PROTACs O Cl O N N N N N N N H NH S N H N N N N O O N S N O S O N N O 7 N O O NH NH O O N N N O O N S O 3 N N Cl O Cl Cl (+)-JQ1 I-BET151 MT1 ARV-825

Domain Selective: BD-1 Domain Selective: BD-2

O O H HN O N NH O OH N HN HN N N O N O O F O N N NH O OH HN F O N N O O F O N H N N OH

GSK778 Compound V RVX-208 ABBV-744 GSK046

Figure 2.3. Reported BET inhibitors and their corresponding categories. The acetylated lysine mimic is highlighted in red, while the moiety that occupies the WPF shelf is marked in blue.

15 2.1.3. BRDT in Spermatogenesis From a contraceptive standpoint, we are particularly interested in BRDT, the testis- specific BET isoform. Its role in spermatogenesis was first investigated through targeted mutagenesis in mice. First BRDT bromodomain (BRDT-1) knock-out mice are viable, but produce fewer and morphologically abnormal spermatids.107 A systematic study of germ cell differentiation underscored BRDT’s indispensable role in sperm maturation.108, 109 mRNA accumulation of BRDT was first detectable at the second meiotic stage (Figure 2.4A) and the BRDT protein facilitated chromosome compaction as well as testis-specific gene expression in post-meiotic cells.

Figure 2.4 The essential roles of BRDT in spermatogenesis. A. BRDT expression is first detected at the spermatocyte stage. 2n and n indicate the chromosome numbers inside the cells. B. The comparison between wild-type and BRDT-1 knockout spermatids. The defects occur in the chromocenter and the domain localizations. C. Morphological comparison between the sperm generated by normal and BRDT-1 knockout mice. The abnormalities, indicated by red arrows, are observed in the head (H), mid-piece (M), or tail (T) parts of the mature spermatozoa.

16 When BRDT-1 was knocked out, several defects were observed, the first of which arose in the spermatid stage. Instead of a compact structure, there were multiple heterochromatic foci in the chromocenter (Figure 2.4B). The localization preference of BRDT around the single heterochromatin diminished as well;110 instead, BRDT was ubiquitously distributed inside the cells. During spermatid elongation, loss of the singular intact chromocenter resulted in the mislocalization of other domains. For instance, the testis-specific histone variant H1FNT was no longer restricted to the postacrosomal region, and the small heterochromatin foci disappeared from the nuclear periphery where H1FNT was not present (Figure 2.4B).107, 111 These ectopic domains were hypothesized to cause abnormal sperm morphology, including aberrant head and mid-piece as well as shortened tail (Figure 2.4C). In addition, the absence of BRDT resulted in global dysregulation of over 3,000 gene transcripts, where one-third of the genes were elevated and two-thirds were suppressed.108 This transcriptional defect also contributed to the abnormalities in spermiogenesis. The necessity of BRDT in human fertility was later confirmed via analyzing gene/protein expression from azoospermic patients’ sperm extractions.112 Furthermore, treatment with pan-BET inhibitor (+)-JQ1 resulted in a sterile phenotype with defective spermatids in a murine model.48 This small-molecule induced sterility was reversible after treatment was discontinued. Notably, the hormone levels and mating behavior remained intact throughout the study. Although testis weight decreased due to the lack of sperm, such weight loss was fully recovered to normal after 4 months of administration discontinuation.48 Taken together, the results above provided evidence that a selective and potent BRDT inhibitor could be a promising non-hormonal male contraceptive.

17 2.2. Dihydropyridine Scaffold Identification Adapted from Ayoub, A.; Hawk, M. L.; Herzig, R. J.; Wisniewski, A. J.; Gee, C. Zhu, J.- Y.; Berndt, N.; Scott, T. G.; Qi, J.; Jun, Q.; Bradner, J. E.; Ward, T. R.; Schönbrunn, E.; Georg, G. I.; Pomerantz, W. C. K. BET Bromodomain Inhibitors with One-step Synthesis Discovered from a Virtual Screen. J. Med. Chem. 2017, 60, 4805-4817.

BRDT inhibitors for male contraception, while effective, remain in the early stages of drug development. Most of the reported chemical scaffolds possess similar affinity towards all four BET isoforms, which is likely to cause unwanted toxicity if applied as male contraceptives. Therefore, the most urgent issue in BRDT inhibitor design is to achieve target selectivity over the structurally similar isoforms. The rest of this chapter pertains to attempts to introduce BRDT specificity.

2.2.1. Hit Identification In an effort to develop novel BRDT-selective compounds, we started from a virtual high throughput screen of a customized ZINC library (Figure 2.5A). From 6 million compounds, we picked compounds with the top 0.4% of docking scores and subjected them to secondary docking and computational filters, which eventually lowered the hit number to 200. Out of 200 compounds, 22 were selected based on commercial availability, structural novelty, and drug-like properties. To validate binding, we used a fluorescence polarization (FP) assay against both BRDT-1 and BRD4-1. A total of 9 compounds demonstrated unambiguous binding to both BET isoforms, among which dihydropyridine compound 2.1 was the most potent (BRDT-1: Ki = 690 nM, BRD4-1: Ki = 370 nM). To further confirm this result, we used differential scanning fluorimetry (DSF) and AlphaScreen experiments, both of which corroborated the FP data. Moreover, protein- observed 19F (PrOF) NMR was performed to generate evidence of direct binding. Accordingly, we confirmed dihydropyridine compound 2.1 as the hit compound (Figure 2.5B).113 However, the dihydropyridine scaffold was devoid of cellular activity or BRDT selectivity.

18 Figure 2.5. Identification of the dihydropyridine scaffold. A. Workflow of hit identification. B. Binding characterization of the hit compound in different assays.

2.2.2. Preliminary Structure-Activity Relationship (SAR) Exploration With the hit compound in hand, we conducted SAR studies, seeking potency and selectivity improvement. The synthesis was rapidly accomplished via a Hantzsch reaction from commercially available starting materials. Although none of the synthesized analogs showed any improvement over 2.1, we gained insight into the SAR patterns (Figure 2.6): (1) An ethyl group was optimal on the N-1 position (indicated as R1), as analogs bearing methyl, propyl, and cyclopropyl groups all experienced potency loss. (2) A free hydrogen on the N-3 position (indicated as R2) was essential for binding, since the corresponding methylation entirely eradicated activity. (3) A methyl motif was preferred on the para site of the aryl ring (indicated as R3). Chlorine and a trifluoromethyl groups at that position resulted in slightly decreased binding. (4) Oxidation of the dihydropyridine ring was deleterious for BET potency. (5) The lactone analog displayed the highest potency, compared to ketone (X = CH2) or N-Me lactam analogs.

19 R3 Methyl was optimal R3 CF3 and Cl were tolerated O R3 O R2 HOAc o N X 110 C O O O + + 2 Lactone was O R 2 H2N N O N more favored R X N Methylation was OH than N-Me lactam R1 CHO X not tolerated N N O or ketone H N N O R1 H R1 1 Oxidation R = Me, Et, CH2CF3, cyclopropyl, pyopyl abolished Moiety larger than ethyl 2 3 R = H, Me; R = H, Me, OMe, Cl, CF3; X = O, NMe, CH2 activity caused affinity decrease

Figure 2.6. General synthetic route of the dihydropyridine scaffold and its SAR results from preliminary modifications.

2.2.3. Binding Mode To elucidate the SAR patterns, our collaborator co-crystalized the lactone compound 2.1 with BRD4-1. Although the lactone was racemic, it was the S-stereoisomer that was observed in the complex. As shown in Figure 2.7A, only in the S configuration, is the tolyl group able to form a hydrophobic interaction with the WPF shelf. The NH group of the dihydropyridine core is the hydrogen bond donor and interacts with a conserved water molecule in the ZA channel (Figure 2.7B). If that position was oxidized, such an interaction would be abolished. Moreover, oxidation will convert the 4’ carbon from sp3 to sp2, which diminishes the contact between the tolyl group and the WPF shelf. The crystal structure revealed that the uracil motif formed the conserved hydrogen bond with Asn140, explaining why N-3 methylation abolished this key recognition element. The N-1 ethyl group occupied the hydrophobic area inside the binding pocket. Due to the importance of the network of conserved water molecules, larger substituents (propyl or cyclopropyl) were likely to have steric clash (Figure 2.7C).

20 Figure 2.7. Binding pose of hit compound 2.1 with BRD4-1 (PDB ID: 5KDH). A. The surface view of the binding site. Key residues are rendered in purple lines with black text. The hydrogen bond interactions are highlighted in yellow dotted lines. B. 2D binding diagram of 2.1. Hydrogen bonds are rendered in dotted lines, and water molecules are red dots. C. The cross-sectional view of the binding site. The water molecules are rendered in red dots, and the ethyl group is shown as spheres with appropriate Van der Waals radii.

2.2.4. Design Rationale To investigate the binding mode with BRDT-1, we docked 2.1 into the reported BRDT-1 crystal structure (Figure 2.8B). Considering the aforementioned sequence similarity, we envisioned a conformation very similar to the BRD4-1 complex (Figure 2.8A). Sequence alignment of the first bromodomains in the BET subfamily revealed that the main differences around the binding site lie in the ZA channel; BRDT-1 has a positively charged arginine (Arg54), whereas the other three have a neutral glutamine (Figure 2.8C). Arginine and negatively charged moieties such as carboxylates form an ionic interaction with covalent-like bond strength.114, 115 Meanwhile, electron-rich ring systems are prone to cation-pi interactions with the positive charge of arginine.116, 117 Therefore, we proposed the Arginine Hypothesis, where by engaging the arginine that flanks the ZA channel, we might be able to achieve BRDT-1 selectivity over other isoforms.118, 119 Herein, we propose either converting the lactone to a lactam or synthesizing ring-open ester/amide analogs, which would introduce an additional side chain that could occupy the ZA channel and engage with Arg54. Moreover, two aforementioned bifunctional approaches, targeted

21 protein degradation and the bivalent strategy, were also adopted to achieve BRDT-1 selectivity.

Figure 2.8. Design rationale of the arginine hypothesis. A&B. The co-crystal complex with BRD4-1 (A; PDB ID: 5KDH) and the predicted conformation in BRDT-1 (B; docking template: 4FLP). Key residues are rendered in purple lines with black text, and the unique arginine in BRDT-1 and corresponding glutamine in BRD4-1 are listed in blue text. C. Sequence alignment of BRDT-1 and BRD4-1. The conserved residues are highlighted in pink and similar residues are in blue. The target arginine in BRDT-1 and corresponding glutamine in BRD4-1 are indicated with red arrow. D. The proposed approaches for achieving BRDT-1 selectivity.

22 2.3. Hit-to-Lead Optimization: Lactam Analogs As planned, our first attempt at BRDT-1 selectivity was to convert the lactone to a lactam. For lactam side chain exploration, we pursued two directions as depicted in Figure 2.9, optimizing the linker region that determines the side chain orientation, and investigating the effects of groups attached to the linker that tunes interactions with the residues in the ZA channel. Moreover, to extensively investigate the arginine hypothesis, we also sought to introduce a carboxylic acid motif that could form an ionic interaction with Arg54.

Side chain optimization: 1. Substitution effect 2. Carboxylate introduction

Interaction with Arg54 O O R NH Linker N Linker optimization: N N O H 1. Length 2. Linear and cyclic

Side chain orientation

Figure 2.9. Proposed modification parameters of the lactam analogs.

2.3.1. Docking Prediction By visually inspecting the ZA channel, we noticed that this region had a funnel- shape (Figure 2.10A). To engage the target arginine, the lactam side chain needs to pass a narrow hydrophobic groove and extend into the wide solvent exposed area. Therefore, we selected the benzyl motif as the starting point for docking prediction. Glide (Maestro, Schrödinger) was able to flank the ZA channel with the benzyl moiety as well as anchor the dihydropyridine core in the binding site (Figure 2.10B). Moreover, the docking score of the benzyl lactam analog was higher than the lactone precursor due to the additional hydrophobic contacts between the benzyl group and surrounding residues in the ZA channel. However, no interaction with the Arg54 was observed in the prediction. Since the docking program treated the protein as a static structure, we envisioned that it was still possible for the small molecule to engage the target arginine residue.

23 Figure 2.10. Binding site analysis of BRDT-1. A. Front view of ZA channel. The beige trapezoid represents the channel shape, and the target Arg54 is highlighted in blue text. B. Predicted binding conformation of the benzyl lactam analog (yellow) in BRDT-1. The conserved residues are rendered in cyan lines, and the unique Arg54 in the ZA channel is listed with blue text.

2.3.2. Chemistry The initial method to prepare the desired lactam analogs is illustrated in Scheme 2.1. Ethyl urea and ethyl cyanoacetoacetate were first converted to 6-amino-1- ethylpyrimidine-2,4(1H,3H)-dione (2.2), employing sodium tert-butoxide as the base. Knoevenagel condensation between ethyl chloroacetoacetate and p-tolylaldehyde generated the other key intermediate 2.3 as a mixture of E/Z isomers. The mixture of 2.2 and 2.3 in methanol in the presence of magnesium sulfate generated bicyclic intermediate 2.4 via the Hantzsch dihydropyridine synthesis. A microwave-assisted reaction of intermediate 2.4 with primary amines generated targeted lactam analogs 2.5.

24 Scheme 2.1. Initial Synthetic Route for the Lactam Analog 2.5

O O O a NH + N N NH2 O H H2N N O

c O O d O O 2.2 NH NH O R N CHO O O Cl N N O N N O O O b O Cl H H Cl + O 2.4 2.5

2.3 Reagents and conditions: a) sodium tert-butoxide (2 equiv), ethanol, reflux, 91%; b) piperidine (0.1 equiv), ethanol, 25 C, 57%; c) magnesium sulfate, methanol, 45 C, 14%; d) primary amine (1.2 equiv), ethanol, microwave to 120 C.

It was envisioned that the low yield of the dihydropyridine synthesis step would make it challenging to efficiently prepare analogs. Therefore, we sought to first optimize the synthesis to improve the overall yield. The first yield-limiting factor was that uracil 2.2 had poor solubility in methanol. Although increasing temperature could improve its solubility, chloromethyl product 2.4 was not thermostable; it would undergo cyclization to form lactone 2.1. By analyzing the reaction mechanism, we speculated that low yield also arose from a side reaction of the dehydration step. Due to the electron-withdrawing nature of the chlorine, the olefin could be formed outside the dihydropyridine ring, generating regioisomer 2.4’ with similar polarity to that of target molecule 2.4 (Scheme 2.2).

25 Scheme 2.2. Proposed Mechanism of Dihydropyridine Formation

Taking these results into consideration, we designed an alternative route depicted in Scheme 2.3, in which the chlorine was introduced in the penultimate step of the reaction sequence so that the undesired dehydration is no longer possible. The reaction between ethyl 4-chloro-3-oxobutanoate and benzyl alcohol furnished benzyl ether 2.6, which was subjected to a Knoevenagel condensation to form intermediate 2.7. Next, a mixture of intermediate 2.7 and N-ethyluracil 2.2 in acetic acid was heated to reflux, providing Hantzsch product 2.8 in 42% yield. The higher yield in this case was a result of increased solubility of uracil 2.2 at higher temperature. A boron tribromide solution was used to cleave the ether bond and generate hydroxyl intermediate 2.9, which was subsequently converted into key chloromethyl intermediate 2.4 using sulfuryl chloride and imidazole. Despite an increase in the number of chemical steps, the optimized route simplified product purification and increased the overall yield.

26 Scheme 2.3. Optimized Route for the Synthesis of Lactam Analog 2.5

O O O O HO OBn O a O b O c O O + NH O O O Cl OBn N N O BnO H 2.6 2.7 2.8

d O O e O O f O O NH NH NH O O R N N N O N N O N N O HO H Cl H H

2.9 2.4 2.5

Reagents and conditions: a) sodium hydride (2.2 equiv), ethanol, 0 C to 25 C, 96%; b) piperidine (0.1 equiv), ethanol, 25 C, 56%; c) uracil 2.2 (1.0 equiv), acetic acid, reflux, 42%; d) 1 M boron tribromide solution in THF (2.2 equiv), dichloromethane, -78 C, 58%; e) sulfuric acid (1.2 equiv), imidazole (2.2 equiv), DMF, 0 C to 25 C, 56%; f) primary amine (1.2 equiv), ethanol, microwave to 120 C, 10-45%.

2.3.3. Linker Optimization We initially prepared ten lactam analogs with linear or cyclic linkers of varying lengths and tested them for inhibition of BRDT-1 and BRD4-1 using the AlphaScreen assay to characterize their selectivity profiles (Table 2.1).

Table 2.1. Structure and Inhibitory Profile of the Primary Round of Lactam Modifications

Compound IC50 (μM) R1 Linker Number BRDT-1 BRD4-1

2.5a -CH2- 2.2 0.63

2.5b -CH2- 0.55 0.24

2.5c -C2H4- 1.0 0.53

27 2.5d -C3H6- 5.2 0.87

2.5e -OC2H4- 0.94 0.28

2.5f Me 1.1 0.32

2.5g 1.1 0.34

2.5h 0.78 0.32

2.5i 1.0 0.30

2.5j O2N 0.64 0.17

2.1 - - - 5.4 4.1

(+)-JQ1* - - - 0.16 0.050

*(+)-JQ1 was used as the positive control. All compounds were tested once in duplicate.

Analog 2.5a, carrying an N-allyl group was found less active than the corresponding N-benzyl and phenethyl analogs 2.5b and 2.5c, respectively. The affinity increase of 2.5b, compared with lactone 2.1, corroborated previous docking predictions that the benzyl moiety was advantageous. Comparison between 2.5b and 2.5d underscored that a longer, linear, more flexible linker reduced potency, presumably as a result of an entropic penalty. Notably, the introduction of an oxygen atom into the linker (2.5e) rescued potency to some degree. Although a piperidine linker had little effect on inhibitory activity (compounds 2.5f-i), we believed that a piperidine moiety might improve solubility of the analogs. Nitro analog 2.5i showed the highest affinity against both BRD4-1 and BRDT-1. However, all modifications failed to provide any appreciable isoform selectivity.

28 2.3.4. Substitution Effect We then investigated the effect of substitution on the aryl ring using 2.5c as the template, expecting that perhaps an electron-rich aromatic ring could strengthen the potential cation-pi interaction with the unique Arg54 in BRDT-1. The results (Table 2.2), however, showed that collectively, aromatic substitution did not influence inhibitory potency significantly. Compounds with electron-donating groups (methyl and methoxy) were slightly more potent than those with electron-withdrawing groups (chlorine). Methoxy scanning on the aryl ring unveiled that 4-methoxy analog 2.5n produced the best results, followed by 2-methoxy (2.5p) and 3-methoxy (2.5o) substitution. Disubstitution (2.5q and 2.5r) failed to show any improvement of potency, and dichloro analog 2.5s showed the greatest loss of potency. In summary, none of the aryl modifications had an impact on BRDT-1 selectivity. On the contrary, all analogs showed higher affinity for BRD4-1 than BRDT-1, which was observed in other reported pan-BET inhibitors.94, 120

Table 2.2. Structure and Inhibitory Profile of the Second Round of Lactam Modifications

Compound IC50 (μM) R1 Number BRDT-1 BRD4-1

2.5c H 1.0 0.53

2.5k 4-Me 1.8 0.50

2.5l 4-Cl 4.1 1.1

2.5m 4-OH 0.76 0.39

2.5n 4-OMe 1.0 0.48

2.5o 3-OMe 2.9 0.48

2.5p 2-OMe 1.5 0.53

2.5q 3,4-diOMe 1.6 0.46

2.5r 2,3-diOMe 5.4 4.1

29 2.5s 3,4-diCl 12 3.3

(+)-JQ1* - - 0.16 0.050

*(+)-JQ1 was used as the positive control. All compounds were tested once in duplicate.

Analog 2.5c was co-crystalized with BRD4-1 (unpublished data from Schönbrunn group, Moffitt Cancer Center). As designed, the phenethyl side chain occupied the ZA channel and formed hydrophobic interactions with surrounding residues (Figure 2.11A). Nonetheless, low electron density for the phenethyl moiety was observed in the complex, indicating that the lactam side chain was rather flexible. Moreover, the potent lactam analog, 4-hydroxyl 2.5m, showed cellular activity, down-regulating cMyc expression, for the first time with a compound bearing the dihydropyridine scaffold (Figure 2.11B).

Figure 2.11. Binding and cellular profile of lactam analogs. A. Co-crystal structure of 2.5c (yellow) with BRD4-1. The phenethyl side chain was observed in the ZA channel. The key residues are shown in sticks. B. Antiproliferative activity and western blot analysis of 2.5c and 2.5m against multiple myeloma cell line MM.1S.

2.3.5. Carboxylate Introduction The lactam analogs in the primary optimization campaign were designed to explore the linker region, reach into the ZA channel, and interact with Arg54 via a cation-pi interaction. To further examine the Arginine Hypothesis, we sought to introduce a

30 carboxylate moiety to form an ionic interaction with the target Arg54. Analogs exhibiting sub-micromolar potency (Table 2.1) were selected as templates.

Table 2.3. Structure and Inhibitory Profile of the Third Round of Lactam Modifications

Compound IC50 (μM) R1 Number BRDT-1 BRD4-1

2.5t 2.2 0.89

O NH 2.5u 0.75 0.34

O O 2.5v NH 1.1 0.34 R1 N N N O H 2.5w 0.66 0.30

2.5x 0.50 0.29

(+)-JQ1* - - 0.16 0.050

*(+)-JQ1 was used as the positive control. All compounds were tested once in duplicate.

As shown in Table 2.3, we designed and prepared five carboxylic acid analogs (2.5t-x) with varied linker length of 6-12 atoms from the lactam nitrogen. A comparison of these analogs with corresponding ones devoid of carboxylic acids revealed that the carboxylate had little effect on potency and selectivity. We concluded that the orientation of the lactam side chain must not be optimal to engage Arg54, which was consistent with the crystallographic data. What was observed in the 2.5j/BRD4-1 complex (unpublished

31 data from Schönbrunn group, Moffitt Cancer Center) was that the lactam side chain was quite flexible and seemed to prefer a conformation that interacted with Trp81 (Figure 2.12). Ionic interaction with arginine requires a head-to-head orientation, which would necessitate conformational changes of the protein to allow interaction with the arginine residue. This movement could be challenging especially in the solvent exposed environment of Arg54.

Figure 2.12. Binding conformation of lactam analog 2.5j (yellow) with BRD4-1. In this co-crystal complex, the lactam side chain is in such an orientation that it forms a pi-pi interaction with Trp81, instead of extending into the ZA channel.

2.3.6. Discussion In this study, we conducted an exploration of the dihydropyridine lactam side chain to investigate its impact on BRDT-1 potency as well as selectivity. Based on the data, we concluded that the modifications of the lactam side chain did improve binding affinity resulting from hydrophobic interaction with the ZA channel. However, our attempts to engage the unique Arg54 of BRDT-1 achieved little success. Based on the crystallographic data, we speculated that the suboptimal orientation of the lactam side chain prevents interaction with the unique arginine residue. Future efforts should focus on introduction of rigidity to the side chain, directing the functional groups to the target arginine. 32 2.4. Hit-to-Lead Optimization: Ring-Open Analogs Besides lactamization, another way to introduce a side chain to the dihydropyridine ring is to open the lactone ring. The ring-open analogs discussed herein include both esters and amides. To explore the SAR patterns, we adopted the same modification strategy in the lactam series, which includes investigation into the linker region as well as the substitution effects of the ester/amide side chain. Additionally, we included the favored moieties in the lactam subset to generate head-to-head comparison between the lactam and ring-open subset.

2.4.1. Chemistry As depicted in Scheme 2.4, the target ester analogs 2.10a-i were rapidly generated via a multi-component reaction containing p-tolyl aldehyde, uracil 2.2, and corresponding substituted 3-oxobutanoate. Whereas, the amide analogs were synthesized in two steps. The methyl ester analog 2.10a was first hydrolyzed to generate the free acid intermediate. The subsequent coupling reactions with various amines yielded the target amide compounds 2.11a-g.

Scheme 2.4. General Synthetic Routes of Ring-open Esters 2.10 and Amides 2.11

Ring-open esters O O O R NH a O O + + R H2N N O O NH O CHO N N O H 2.2 2.10a-i Ring-open amides

O O O O O O b c R O NH HO NH N NH H N N O N N O N N O H H H

2.10a 2.11a-g

Reagents and conditions: a) AcOH, reflux, 27-46%; b) NaOH (4.0 equiv), 25 C; c) RNH2 (1.0 equiv), EDCI (1.0 equiv), HOBt (1.0 equiv), DIPEA (1.0 equiv), DCM, 25 C, 32-66% (over 2 steps). 33 2.4.2. Ester Scaffold Exploration In the ester subset, we first prepared nine analogs with various side chains (Table 2.4). As exemplified by analogs 2.10a-d, the linear aliphatic side chain was tolerated, whereas the sterically demanding t-butyl moiety caused a slight decrease in BRDT-1 affinity, which may be a result of the narrow groove in the ZA channel (Figure 2.10A). The introduction of a benzyl group (2.10e) gave rise to almost a five-fold increase in affinity, compared with hit 2.1, presumably due to hydrophobic contact with residues in the channel region. We then looked into the effect of substitution on the benzyl ring. As shown in Table 2.4, either electron-withdrawing (2.10f) or electron-donating (2.10g) groups on the aryl ring were not advantageous for potency. Moreover, di-substitution (2.10h) had minimal impact on BET binding. Further extension of the chain, albeit tolerated, failed to improve the binding affinity as seen by the comparison between 2.10g and 2.10i. Taken together, these data indicate that the ester side chain had marginal effect on isoform selectivity. Furthermore, the ester subset was less potent than the lactam one (by one order of magnitude). Table 2.4. Structure and Inhibitory Profile of the Ester Analogs

IC50 (μM) Compound R BRDT-1 BRD4-1

2.10a Me 5.9 5.4 2.10b 7.9 5.5

2.10c 4.7 3.1

2.10d 10 5.5

2.10e 0.79 0.97

Cl 2.10f 14 9.4

O 2.10g 15 11

34 O 2.10h O 7.0 4.5

2.10i 11 9.1

2.1 5.4 4.1 (+)-JQ1* 0.16 0.050 *(+)-JQ1 was used as the positive control. All compounds were tested once in duplicate.

2.4.3. Amide Scaffold Exploration In the amide subset, we observed greater affinity loss than with the ester subset, as shown in Table 2.5. The flexible and hydrophobic n-propyl moiety (2.11a) diminished binding. However, the introduction of an allyl group (2.11b) rescued this affinity loss. In contrast, the previously favored benzyl moiety (2.11c) or its surrogate phenylhydrazine moiety (2.11d) did not significantly improve potency. Likewise, an electron-donating group (2.11f), albeit tolerated, had minimal impact on binding. Linker extension (2.11c, 2.11e, and 2.11g) did not have significant effect on either affinity or selectivity.

Table 2.5. Structure and Inhibitory Profile of the Amide Analogs

IC50 (μM) Compound R BRDT-1 BRD4-1

2.11a 318 112

2.11b 11 18

2.11c 34 53

2.11d 49 49 HN

2.11e 51 50

2.11f 44 36

2.11g 51 33

35 2.1 - - 5.4 4.1 (+)-JQ1* - - 0.16 0.050 *(+)-JQ1 was used as the positive control. All compounds were tested once in duplicate.

2.4.4. Co-crystal Structures To rationalize the SAR patterns, analogs 2.10c and 2.10e were co-crystalized with BRD4-1 (unpublished data from Schönbrunn group, Moffitt Cancer Center). As shown in Figure 2.13, instead of occupying the ZA channel as initially designed, both the allyl and benzyl moieties were located next to the ZA loop, where hydrophobic interactions were formed between the allyl/benzyl ester side chain and two loop region leucine residues (Leu92 and Leu94) that had not been predicted computationally. Because of their position in the binding site, substitutions on the para position of benzyl ester ring are unlikely to form additional contacts with surrounding residues. In fact, the decreased affinity observed for 2.10f and 2.10g might arise from solvation penalty of para substitutions. Moreover, a longer flexible linker is likely to weaken hydrophobic interactions with the ZA loop. Because of the conformation, any interactions formed between the target arginine and the ring-open analogs are unlikely, explaining the lack of BRDT selectivity.

Figure 2.13. Binding analysis of ring-open scaffold. A. Binding pose of allyl analog 2.10c with BRD4-1. B. Binding pose of benzyl analog 2.10e with BRD4-1. The conserved binding residues are rendered in blue lines. The three conserved leucine residues on the ZA loop next to the benzyl group (purple) are listed with pink text.

36 The co-crystal complex also shed light on the potency loss seen with the amide analogs. Despite different metabolic stability, ester and amide bonds are usually considered interchangeable. However, in this case amide analogs suffered more affinity loss than ester ones. In the co-crystal structures, the dihedral angles (θ) of the allyl and benzyl esters (2.10c and 2.10e) were measured at 8.2 and 26.7 degrees, respectively (Figure 2.14 A&B). This dihedral angle might influence the hydrophobic interaction between the ester side chain and the ZA loop. Given that 2.10e was more potent than 2.10c, we speculated that a nonplanar conformation of the ester side chain (θ greater than 0 degrees) was preferred for binding. Meanwhile, the secondary amide bond is prone to a planar conformation (θ = 0 degrees for cis configuration), due to the delocalization of lone pair of electrons on the nitrogen. Furthermore, the comparison between 2.10c and 2.11b and 2.10e and 2.11c unveiled that differences in the dihedral angle were correlated with differences in the binding affinity. Therefore, we concluded that it was the planar configuration of the amide bond that led to a loss of affinity.

Figure 2.14. Dihedral angle analysis of the ester analogs. A&B. Measured dihedral angles of 2.10c (A) and 2.10e (B) by PyMOL are indicated by red double-headed arrows.

2.4.5. Discussion In this section, we exploited the ring-open strategy to improve BRDT-1 potency as well as selectivity. Among all synthesized analogs, benzyl ester analog 2.10e showed the highest potency, showing 0.97 and 0.79 µM IC50 values for BRD4-1 and BRDT-1, respectively. However, no BRDT selectivity was observed in this subset. The 37 crystallographic data revealed an unexpected binding conformation of the ring-open series, which rationalized both the SAR patterns and the lack of specificity. This optimization campaign also underscored the importance of conformational rigidity, as breaking the lactone ring into the ester/amide resulted in affinity loss. One can envision that follow-up modifications should append an additional side chain to the dihydropyridine core, so that the analogs will interact with both the ZA loop and ZA channel.

2.5. Hit-to-Lead Optimization: Macrocyclic Analogs The co-crystal structure of ring-open ester 2.10e also unveiled that its two aryl rings were spatially close (Figure 2.15A, 4.1 to 4.8 Å based on PyMOL measurement). Hence, we leveraged a macrocyclization approach to conformationally pre-organize the binding pose. Indeed, rational cyclization in drug discovery could increase binding affinity via decreasing conformational flexibility or improving membrane permeability and PK/PD profiles. For instance, a macrocyclic pyridone BET inhibitor was reported to have over a 30-fold affinity increase than its acyclic precursor, due to prioritized binding conformation (Figure 2.15B).121 Therefore, we sought to further optimize the ester subset through the macrocyclization method.

Figure 2.15. Design rationale of macrocyclization on the benzyl ester analog 2.10e. A. The measured distances between the two aryl rings are highlighted in cyan dotted lines. B. Reported macrocyclic BET inhibitor and its precursor.

Docking predictions of macrocyclic analogs generated binding poses similar to the ring-open precursor. For instance, designed analog 2.16d was predicted to have a hydrogen

38 bond with a conserved asparagine and a hydrophobic interaction with the WPF shelf (Figure 2.16). Nonetheless, the lactone aryl ring did not fully mimic the conformation of the ester side chain. We envisioned that such conformational differences might be due to the suboptimal ring size or the connection site on the aryl rings. Hence, we proposed the analogs below to investigate these two key parameters.

Figure 2.16. The predicted binding conformation of 2.16d (purple) using the BRD4-1/2.10e (yellow) complex as the docking template. There was slight deviation between the benzyl groups in ester and lactone scaffolds.

2.5.1. Chemistry Scheme 2.5 depicts the general synthetic route of macrocyclic analogs 2.16a-i. Hydroxybenzaldehydes (A ring precursor) and hydroxybenzyl alcohols (B ring precursor) were connected by a hydrocarbon linker with different lengths on the phenolic site (2.12a- i). Acylations of the primary hydroxyl group with 2,2,6-trimethyl-4H-1,3-dioxin-4-one (TMD) under microwave conditions provided beta keto ester intermediates (2.14a-i). Intramolecular Knoevenagel condensations constructed key macrocycles (2.15a-j) and the subsequent Hantzsch dihydropyridine synthesis gave rise to desired analogs 2.16a-j.

39 Scheme 2.5. General Synthetic Route of Lactone Analogs 2.16a-i

O R2 2 2 R2 R O R 3 1 3 1 R R R3 R1 R3 R1 a R R HO c O b A A O O Br R4 O 4 O 4 R4 O n R R OH n B n B

2.12a-h 2.13a-i 2.14a-i 2.14a R1 = CHO, R2 = H, R3 = H, R4 = H, n = 1, ortho R 2.14b R1 = CHO, R2 = H, R3 = H, R4 = H, n = 2, ortho 2.14c R1 = CHO, R2 = H, R3 = H, R4 = H, n = 3, ortho A 2.14d R1 = CHO, R2 = H, R3 = H, R4 = Me, n = 1, ortho O 2.15a R = H, n = 1 1 2 3 4 O 2.15b R = H, n = 2 2.14e R = CHO, R = H, R = H, R = Me, n = 2, ortho n 1 2 3 4 2.15c R = H, n = 3 2.14f R = H, R = H, R = H, R = CHO, n = 1, ortho B O 2.14g R1 = Me, R2 = H, R3 = H, R4 = CHO, n = 1, ortho O 2.15d R = Me, n = 1 2.14h R1 = Me, R2 = H, R3 = H, R4 = CHO, n = 2, ortho O 2.15e R = Me, n = 2 2.14i R1 = CHO, R2 = H, R3 = H, R4 = Me, n = 2, meta R O O n A d e O 2.16a-i B 2.15f R = H, n = 1 Structures shown O 2.15g R = Me, n = 1 in Figure 2.17 2.15h R = Me, n = 2 O

O O A B O 2.15i O

Reagents and conditions: a) dibromo alkane (2.0 equiv), K2CO3 (1.5 equiv), DMF, 75 C, 16-74%; b) hydroxybenzaldehyde (1.1 equiv), K2CO3 (1.5 equiv), DMF, 75 C, 46-99%; c) TMD (2.0 equiv), NaOAc (0.5 equiv), toluene, microwave to 120 C, 22-57%; d) piperidine (0.2 equiv), AcOH (0.1 equiv), ethanol, 75 C, 14-57%; e) uracil 2.2 (1.0 equiv), AcOH, reflux, 16-37%.

The investigation of linker length was achieved using 1,2-dibromoethane, 1,3- dibomopropane, and 1,4-dibromobutane. Replacement of 2-hydroxybenzaldehyde with 3- hydroxybenzaldehyde represented the study of substitution preference on the A ring. Meanwhile, switching from 2-hydroxybenzyl alcohol to 3-hydroxylbenzyl alcohol examined the effect of the B ring. Additionally, the para-methyl on the A ring was installed by using 2-hydroxy-4-methylbenzaldehyde as the starting material.

Analogs bearing only the A ring were designed to explore ring size and necessity of the B ring. As shown in Scheme 2.6, hydroxybenzaldehydes were alkylated with 3- Bromopropan-1-ol or 4-bromobutan-1-ol at the phenolic site (2.17a-d). The terminal alcohol then reacted with TMD to form beta-keto esters 2.18a-d. Ring closure and 40 dihydropyridine formation were accomplished through the same methods described above, which eventually yielded four analogs 2.20a-d for further comparison.

Scheme 2.6. General Synthetic Route of Lactone Analogs 2.20a-d

R R R R A O O a b c O A n OH O OH O O n n O CHO CHO CHO O O 2.17a-d 2.18a-d 2.19a-d R

A 2.20a n = 1, R = H d n O O 2.20b n = 2, R = H 2.20c n = 1, R = Me O NH O 2.20d n = 2, R = Me N N O H

Reagents and conditions: a) 3-Bromopropan-1-ol or 4-bromobutan-1-ol (1.0 equiv), K2CO3 (1.5 equiv), DMF, 75 C, 12-71%; b) TMD (2.0 equiv), NaOAc (0.5 equiv), toluene, microwave to 120 C, 21-66%; c) piperidine (0.2 equiv), AcOH (0.1 equiv), ethanol, 75 C, 16-28%; d) uracil 2.2 (1.0 equiv), AcOH, reflux, 8-30%.

In addition to the lactones, lactam analog 2.26 was designed and synthesized (Scheme 2.7). 2-Hydroxybenzaldehyde (A ring precursor) and 2-hydroxybenzonitrile (B ring precursor) were connected at the phenolic sites via 1,3-dibromopropane. Both the nitrile and the aldehyde groups were reduced by lithium aluminum hydride and the beta- keto amide 2.23 was formed selectively with TMD with the hydroxyl group intact. The benzaldehyde moiety was then re-established under mild oxidation conditions to form intermediate 2.24. Intramolecular Knoevenagel condensation yielded cyclic intermediate 2.25, which was converted into lactam analog 2.26 via Hantzsch synthesis.

41 Scheme 2.7. Synthetic Route of Lactam Analog 2.26

A B A B a A b c O O O Br O O O O OH O HN CHO CHO CN OH H2N O 2.12b 2.21 2.22 2.23

O O A B A O O O O O N d e H B f N NH O B H O H O O HN A N N O O O H

2.24 2.25 2.26

Reagents and conditions: a) 2-Hydroxybenzonitrile (1.2 equiv), K2CO3 (1.5 equiv), DMF, 75 C, 55%; b) LAH solution (2M in THF) (10 equiv), THF, 0 to 50 C, 98%; c) TMD (1.0 equiv), toluene, microwave to

180 C, 42%; s) MnO2 (10 equiv), DCM, 25 C, 72%; e) Piperidine (0.2 equiv), AcOH (0.1 equiv), ethanol, 75 C, 28%; f) uracil 2.2 (1.0 equiv), AcOH, reflux, 18%.

2.5.2. SAR Discussion The affinity and selectivity of the cyclic series were characterized by an in-house FP assay.122 As shown in Figure 2.17A, ring expansion via increasing the linker length had no significant effect on affinity, although 2.16c with the longest linker seemed to show a decrease in binding. Regardless of the ring size, 2.16a-c showed slight BRD4-1 preference over BRDT-1. Ring expansion via migrating of the linker from the ortho to the meta position on the A ring provided slightly better affinities for both constructs as exemplified by comparing 2.16b and 2.16f (Figure 2.17B). We speculated that these trends might have resulted from rigidity, as migration on the ring is likely to introduce less flexibility than linker extension. Meanwhile, there was no significant difference between attaching the linker on the ortho or meta position on either the A or B ring, given that 2.16h and 2.16i demonstrated similar affinities (Figure 2.17C).

42 Figure 2.17. SAR summary of macrocyclic analogs

Furthermore, the data for three analogs, 2.16d, 2.16e, and 2.16g, emphasized the benefit of para-methyl substitution on the A ring for binding. Especially BRDT binding increased (Figure 2.17D). This improved affinity might be derived from a better fit in the WPF shelf. Nonetheless, the migration on the A ring was not compatible with the para- methyl, given there was not significant activity increase observed. The analogs with only the A ring, 2.20a and 2.20b, displayed weaker binding with BRD4-1 than 2.16a and 2.16b, which emphasized the importance of the B ring (Figure 2.17E). The methyl effect was also observed in analogs devoid of the B ring. However, the improvement for 2.20c was not as strong as for 2.20d, despite the two molecules only having one carbon difference in the linker region. Additionally, lactam analog 2.26 failed to show superior affinity or selectivity compared with the corresponding lactone 2.16b (Figure 2.17F). Collectively, this series yielded 2.16e as the most potent analog, equipotent to acyclic precursor 2.10e (BRDT-1: 1.5 µM and BRD4-1: 0.23 µM).

43 2.5.3. Co-crystal Structures Our collaborators were able to co-crystalize 2.16b and 2.16d with BRD4-1 (unpublished data from Schönbrunn group, Moffitt Cancer Center). Despite different linker lengths, the two molecules adopted nearly identical conformations in the pocket (Figure 2.18A). This evidence illustrates that ring expansion via tuning linker length had limited impact on affinity, presumably due to similar poses of the rest of the molecule in the active site. Superimposition of 2.16d and 2.10e supported our initial design idea that a macrocyclic compound could mimic the conformation of the acyclic precursor (Figure 2.18B). However, no significant improvement in affinity was achieved. One explanation is that, compared with the ring-open analog, the preferred conformational pre-organization of the macrocyclic analog failed to compensate for the solvation penalty derived from the hydrocarbon linker exposed in the solvent accessible area. Another cause might be steric clash. As illustrated in Figure 2.17B, macrocyclic analog 2.16b (orange) perturbed the ZA loop (green ribbon) and caused a flip of Asn93 (N93 to N93’) compared to ester 2.10e (yellow) (salmon ribbon), which might negatively affect binding.

Figure 2.18. Binding pose analyses of macrocyclic and ring-open analogs. A. Superimposition of co- crystal complexes of BRD4-1 with 2.16b (cyan) and 2.16d (orange) reveals that despite spacer length, the two molecules have almost identical binding conformations. B. Overlay of 2.10e (yellow) and 2.16d (orange) in the active site of BRD4-1 shows that the aryl ring of the macrocyclic compound is situated closer to the ZA loop than the ester one, which leads to the perturbation of the loop region and flip of Asn93 (N93 to N93’).

Despite the loop perturbation, 2.16b and 2.16d displayed unexpected stabilization effects on BRDT-2 in the DSF assay. For instance, 2.16b induced a positive thermal shift 44 of 13.4 °C in BRDT-2, whereas the ∆Tm for BRD4-1 and BRDT-1 was 5.1 and 2.8 °C, respectively. Recent studies showed that inhibition of BD1 in BET proteins had profound impact on the global gene expression, whereas BD2 inhibition only affected stimulus- induced gene expression.106 These different outcomes might be a result of the 78 aforementioned KAc preferences of BD1 and BD2. Moreover, BD2 inhibitors were proposed to counter immunoinflammatory damages and have better specificity than pan inhibitors.105 Therefore, we will validate these results with the AlphaScreen and try to determine molecular determinants for the BD2 preference.

2.5.4. Discussion Inspired by the co-crystal structure, we constructed a macrocycle of the ring-open scaffold to pre-organize its binding conformation. We embarked on a systematic scan of both the ring size and the connection method, which yielded a macrocyclic analog 2.16e equipotent to its acyclic precursor. Co-crystallography greatly aided SAR pattern interpretation. Although most of the macrocyclic analogs failed to show BRDT-1 selectivity, analog 2.16b demonstrated unexpected BRDT-2 preference over other constructs. Further optimization is ongoing via introduction of solubilizing groups to the aryl ring as well as additional substituents into the ZA channel. Meanwhile, we will work on the validation and rationalization of BRDT-2 specificity.

45 2.6. Hit-to-Lead Optimization: PROTACs 2.6.1. Brief Introduction of PROTACs As mentioned earlier, targeted protein degradation, one of the bifunctional strategies, is an approach we exploited for BRDT-1 selectivity. Since the first description of heterobifunctional ligands that were able to induce protein degradation in 2001,99 recent years have witnessed a surge in exploration of targeted protein degradation as a novel therapeutic modality.123, 124 Such a strategy takes advantage of the endogenous protein homeostasis machinery. Proteins about to be degraded are labelled with chains of ubiquitin by the E3 ligases. The ubiquitin chains are specifically recognized by the proteasome, where labelled proteins are unfolded and digested.125 Well-designed small molecules, also termed proteolysis-targeted chimeras (PROTACs), can hijack the proteasome system to clear proteins of interest. A typical PROTAC degrader consists of a motif to bind the target protein, a linker, and an E3 ligase recruiter. The small molecule inhibitor and recruiter are able to recognize the protein of interest and the E3 ligase, respectively. After recognition, the PROTAC induces complex formation between the target protein and the ligase, which gives rise to ubiquitination and subsequent degradation by the proteasome (Figure 2.19).

Figure 2.19. Mechanism of action for PROTAC-mediated protein degradation. Step 1. PROTAC recognizes both the protein of interest and the E3 ubiquitin ligase; Step 2. Upon protein complex formation, the target protein is ubiquitylated; Step 3. Labelled protein is specifically degraded by proteasome and the PROTAC molecule is released. A PROTAC molecule consists of three components: a small molecule that binds the protein of interest (blue square), a E3 ligase recruiter (orange triangle), and a linker (black line) that integrates them together. 46 2.6.2. BET PROTACs Reported pan-BET inhibitors have also been re-purposed into PROTACs, obtaining excellent degradation potencies as well as superior antiproliferative activity.126, 127 Three aspects must be considered for PROTAC derivatization, where the first is to select the attachment site. An optimal site for linker connection is adjacent to a solvent accessible region. Crystallography is usually exploited to help identify these sites. The synthetic feasibility/simplicity of the proposed site requires considerations as well. The second factor is linker optimization, which plays an essential role in complex formation. Because of the suboptimal prediction of protein complex docking, linker optimization is usually conducted via screening parameters including lengths, types (hydrocarbon and PEG), and flexibility/rigidity. The third factor is the E3 ligase recruiting unit, two of which are cereblon- or VHL-recruiting ligands (Figure 2.20, highlighted in red and blue, respectively). These two types of ligands, albeit both effective in recruiting the ligases, might affect the cooperativity of the protein complex and the physicochemical properties of the macromolecule.

OH O O N N O O N N N NH S O N N N N O N H S O H 3 O O N HN NH N O O O 3 Cl S Cl N ARV-825 MZ1 Figure 2.20. Representative BET PROTAC molecules derived from (+)-JQ1 and two different E3 ligase recruiting units. Motifs in red and blue are capable of recruiting cereblon and VHL E3 ligases, respectively.

According to preclinical studies, BET PROTACs possessed hundreds of times higher antiproliferative potency than the corresponding BET inhibitors. For instance, ARV-771 was 10- to 500-fold more potent in apoptosis than its BET precursors (+)-JQ1 or OXT015 (Figure 2.21).128, 129 This boost in potency is a consequence of the unique mechanism of action. Instead of solely blocking the acetylated lysine pocket while leaving

47 other domains functional, BET degraders are able to wipe out the entire protein, thus completely diminishing BET-mediated transcription. Notably, OXT015 only achieved modest clinical activity in patients with advanced cancer, suggesting that occupancy-driven BET inhibition may be not sufficient for eradicating cancer cells.130 As such, the degradation-driven BET modulators (i.e., ARV-771) could be extremely attractive for anticancer therapy.131

OH N O N N N O N N S N O O N N N N N S S H H O N HN O N O N NH O O

S OH Cl Cl Cl N ARV-771 (+)-JQ1 OXT015 Figure 2.21. Example of BET degrader with superior antiproliferative activity.

Isoform selectivity enhancement is another potential feature of BET degraders. As mentioned earlier, BET isoform specificity remains challenging for small molecule inhibitors due to their conserved binding sites. Nonetheless, several BRD4-specific degraders derived from pan-BET inhibitors have been disclosed in the literature (exemplified by AT1, Figure 2.22).100, 132, 133 The unexpected selectivity was rationalized by the favored protein-protein interaction between the specific BET protein and the E3 ligase according to the crystallographic data.133 Nonetheless, it remains challenging to predict the selectivity profile of certain PROTAC molecules due to limited computational capacity.

N S N O N N HN S

N S NH O O O N HN

Cl OH AT1 Figure 2.22. Example of BET degrader with isoform specificity. AT1 induced selective BRD4 degradation,

48 while leaving BRD2 and BRD3 intact, in HeLa cells after 24 h (Figure 4 e-g in the original study133).

2.6.3. Design Rationale Interested in the reported superior potency and isoform selectivity of BET degraders, we endeavored to derivatize the dihydropyridine lactam into PROTACs. Starting from the phenethyl lactam analog, we identified that the tolyl group in the WPF shelf and the phenethyl side chain in the ZA channel were solvent accessible (Figure 2.23). From a chemistry perspective, the linker attachment on these two sites could be achieved via using modified benzaldehyde or phenethylamine as the starting materials. Because of their location, the tolyl group and lactam side chain modified analogs will be later referred to as WPF- and ZA-derived PROTACs, respectively.

Figure 2.23. The phenethyl lactam 2.5c/BRD4-1 complex reveals that both the tolyl and phenethyl moieties are solvent accessible (dotted red circles).

2.6.4. Chemistry Inspired by the reported study,134 we designed the PROTAC synthesis by preparing two fragments: the lactam intermediate with a terminal alkyne and a cereblon recruiting motif with an azide. These two parts could then be combined into the desired PROTACs via a final-stage click reaction. As depicted in Scheme 2.8, the synthesis of the lactam precursors for the WPF-derived PROTACs started with the synthesis of the modified benzaldehyde with a terminal alkyne attached to either the ortho or meta site (2.27a-b). The Hantzsch reaction constructed the dihydropyridine ring, followed by removal of the

49 benzyl ether and conversion of the hydroxyl group to chlorides 2.30a-b. Microwave- assisted reactions with tyramine yielded desired precursors 2.31a and 2.31b. For the synthesis of ZA-PROTAC intermediate 2.32, however, we directly coupled chloromethyl analog 1.5 with the alkyne-attached tyramine under the same microwave conditions as for analogs 2.31a and 2.31b.

Scheme 2.8. Synthetic Routes for the Lactam Intermediates 2.31a, 2.31b, and 2.32

WPF-derived PROTAC precursor

O HN O O O Cl OH a O HN b O c N + O NH H O BnO CHO CHO N N O H 2.27a-b 2.28a-b

O HN O HN O HN O O O O e O O O d O O HO O NH O NH NH N HO Cl N N O N N O N N O H H H 2.29a-b 2.30a-b 2.31a-b ZA-derived PROTAC precursor

O O O e O NH NH O N N N O O N N O Cl H H

2.4 2.32

Reagents and conditions: a) K2CO3 (1.1 equiv), NaI (0.1 equiv), acetonitrile, reflux, 56-63%; b) uracil 2.2

(1.0 equiv), 2.6 (1.0 equiv), AcOH, reflux, 16-29%; c) 1 M BBr3 in THF (2.5 equiv), DCM, -78 C, 49-56%; d) SO2Cl2 (2.0 equiv), imidazole (2.6 equiv), DMF, 0 C to 25 C, 22-76%; e) primary amine (1.1 equiv), ethanol, microwave to 120 C, 21-49%.

The cereblon recruiting motif was synthesized following a reported procedure (Scheme 2.9).127 Condensation between 3-hydroxyphthalic anhydride and 3- aminoperidine-2,6-dione hydrochloride yielded 4-hydroxyl thalidomide 2.33, which was alkylated with 7-bromo-1-heptanol at the phenolic site to provide intermediate 2.34. The

50 terminal hydroxyl group was then converted to azide 2.35. Cu(I)-catalyzed azide-alkyne cycloaddition between the azide and alkyne successfully yielded the target PROTAC analogs 2.36a-c.

Scheme 2.9. Synthesis of PROTACs 2.36a-c

Reagents and conditions: a) TEA (1.1 equiv), toluene, reflux, 47%; b) 7-bromo-1-heptanol (1.2 equiv), KI

(0.1 equiv), NaHCO3 (2.0 equiv), DMF, 60 C, 60%; c) (1) MsCl (2.0 equiv), TEA (3.0 equiv) DCM, (2)

NaN3 (7.0 equiv), DMF, 45 C, 31% over 2 steps; d) 2.31a, 2.31b, and 2.32 (1.0 equiv), CuSO4•H2O (0.1 equiv), sodium ascorbate (0.2 equiv), DMF/H2O, 19-33%.

2.6.5. Binding and Degradation Evaluation Assessment of the three PROTACs included evaluation of both binding affinity and degradation efficacy. As shown in Table 2.6, two WPF-derived analogs PROTACs, 2.36a and 2.36b, maintained BRD4-1 potency, while displaying slightly decreased potency towards BRDT-1. Whereas ZA-derived PROTAC 2.36c showed reduced affinity against both BRD4 and BRDT. This result might suggest that the WPF shelf region has a larger tolerance for structural modification than the ZA channel. We then used human multiple myeloma cells (MM.1S) with high BRD4 expression to determine the degradation efficacy. Unfortunately, none of the synthesized PROTACs were found to induce BRD4-specific degradation. Since the binding assay showed that three derivatives were able to bind BRD4 with reasonable affinity, we speculated that the lack of degradation efficacy could be due to poor ternary complex formation. Consequently, our next step was to further explore

51 different linker length and connection methods so that the molecules could more easily induce protein/ligase complex formation. Table 2.6. Structure and Binding Profile of the WPF and ZA-derived PROTACs

2.6.6. Discussion In an effort to explore BRDT selectivity, we exploited a targeted protein degradation approach and synthesized three dihydropyridine PROTACs. Binding characterization revealed that the three PROTACs had acceptable BRD4 affinities but lower affinity for BRDT. Unfortunately, the three analogs also failed to show any degradation efficacy in the cellular assay. We rationalized that the lack of cellular activity could be a result of poor membrane penetration or linker interference in the BET protein/E3 ligase complex formation. To further investigate the PROTAC-mediated BRDT specificity, our next step is to explore linker length as well as connection methods (PEG or amide) for more potent and selective BET degraders.

52 2.7. Hit-to-Lead Optimization: Bivalent Molecules 2.7.1. Bivalent Strategy Introduction Structurally, BET family members have two bromodomains in tandem (referred to as BRD2-, BRD3-, BRD4-, and BRDT-1 and BRD2-, BRD3-, BRD4- and BRDT-2) for acetylated lysine recognition. Although most of the reported BET inhibitors showed similar affinity to these 8 bromodomains, the binding events to both pockets in BET proteins are independent from each other. To pursue simultaneous occupancy of the tandem bromodomains, two groups reported a bivalent strategy at almost the same time in 2016. Waring and colleagues utilized the tool compound (+)-JQ1 and dimerized it by a PEG linker at the solvent exposed site (MT1, Figure 2.24A). Meanwhile, Tanaka and coworkers started from two acetylated lysine mimics (3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6- amines) and connected them via a 4-phenylpiperdine linker (NCB6, Figure 2.24A). Crystallographic data demonstrated that both molecules were able to dimerize two bromodomains and induce extensive intermolecular protein-protein interactions (PPIs), as shown in Figure 2.24B. Furthermore, small-angle X-ray scattering (SAXS) studies indicated that NCB6 was able to co-occupy both binding sites of tandem BRD4.

Figure 2.24. Reported bivalent inhibitors. A. The chemical structures of MT1 and NCB6, where the binding motifs of the two molecules are highlighted by green and brown ovals, respectively. B. The dimerized complex of two BRD4-1 monomers (green and cyan ribbons) induced by NCB6 (yellow) (PDB ID: 5AD3).

53 Inducing extensive intermolecular PPIs by bivalent molecules results in superior in vitro potency compared to the monovalent precursors. For instance, MT1 was 400-fold more active than (+)-JQ1 in a cellular assay (IC50s of MT1 and (+)-JQ1 in MV-4-11 cells were 0.170 and 72.2 nM, respectively) and showed promising antitumor efficacy in the xenograft model.98 Moreover, the bivalent inhibitors demonstrated appealing selectivity profiles. NCB6 displayed a tandem bromodomain (pKd: 11 for BRD4-1,2) preference over 97 either single construct (pKd: 8.1 and 7.3 for BRD4-1 and BRD4-2). Therefore, in addition to the arginine engagement and PROTAC strategies, we endeavored to functionalize the dihydropyridine scaffold into bivalent molecules for both potency and selectivity improvements.

2.7.2. Design Rationale Because the dihydropyridine scaffold was racemic, we envisioned that the self- dimerization strategy would result in multiple isomers, which could complicate interpretation of binding data. In contrast, the NCB6-type bivalent strategy uses an achiral acetylated lysine mimic. Additionally, triazolopyridazine addition will not dramatically increase the molecular weight, which might be advantageous for cellular penetration. As shown in Figure 2.25, superimposition of the co-crystal complexes of NCB6 and 2.1 with BRD4-1 revealed that both the 4-phenylpiperidine of NCB6 and the tolyl group of 2.1 occupied the WPF shelf, although their orientations differed slightly. Thus, we proposed a hybridization strategy, where one triazolopyridazine of NCB6 would be attached to the dihydropyridine at the tolyl site to generate the bivalent compound.

54 Figure 2.25. Superimposition of NCB6 (PDB ID: 5AD3) and 2.1 (PDB ID: 5KDH). Chain B of 5AD3 was overlaid with 5KDH. The three key residues of the WPF shelf are rendered in cyan lines. The two important aryl rings of NCB6 (green) and 2.1 (yellow) in the shelf region are indicated by blue and red arrows, respectively, as well as blue and red shading in the accompanying chemical structures.

2.7.3. Chemistry From the previous SAR results, methyl ester intermediate 2.39 showed moderate binding affinity (500 nM Ki for BRD4-1) and its phenylacetic methyl ester group, after hydrolysis, could be suitable for triazolopyridazine connection. Hence, we designed a convergent route as depicted in Scheme 2.10. Based on the reported procedures,97 triazolopyridazine 2.37 was generated from 6-chloro-6-hydrazinylpyridazine and tetramethoxymethane. A subsequent SNAr reaction with 4-hydoxylpyridine yielded the desired fragment of NCB6. Meanwhile, the dihydropyridine piece with phenylacetic acid 2.40 was generated via the Hantzsch reaction and ester hydrolysis in tandem. Intermediates 2.38 and 2.40 were integrated via a HATU-mediated coupling reaction to form 2.41. The characterization of both the fragments and the bivalent molecules is ongoing.

55 Scheme 2.10. Synthetic Route of Bivalent Molecule 2.41

N N O N N N Cl N N O O O O b N N a N NH + N N N N 2 O O N N H Cl N OH 2.37 O 2.38 e O O O O HO O O O O c O O d O O NH O NH NH N N O O O H CHO N N O N N O H H 2.41 2.39 2.40 Reagents and conditions: a) TEA (1.6 equiv), DME, 90 C, 85%; b) 4-hydroxypiperidine (3.0 equiv), DIPEA (2.0 equiv), ethanol, 55 C, 47%; c) Tetronic acid (1.0 equiv), 2.2 (1.0 equiv), AcOH, reflux; d) LiOH (7.0 equiv), THF/H2O, 52% for 2 steps; e) HATU (1.2 equiv), DIPEA (2.0 equiv), DMF, 13%.

2.7.4. Discussion In this section, we applied the bivalent approach to the dihydropyridine scaffold, seeking to introduce BRDT selectivity. At present, we have established the chemistry for the first NCB6-like bivalent molecule, and its binding characterization is still underway. Our future work will also include the synthesis and evaluation of MT1-like analogs.

2.8. Summary and Future Directions In conclusion, we identified tricyclic dihydropyridine scaffold 2.1 as a pan-BET inhibitor from a virtual high throughput screen. Based on preliminary SAR and its binding mode, we designed two sets of analogs, the lactams and the ring-open esters/amides. These modifications sought to explore the ZA channel to achieve isoform selectivity via interacting with the unique Arg54 in BRDT-1. The two series yielded 4-hydroxylphenethyl lactam 2.5m and benzyl ester 2.10e as the most potent analogs with about ten-fold improvement in potency and promising cellular activity compared with initial hit 2.1. However, none of the analogs displayed the desired BRDT-1 selectivity, presumably

56 because of suboptimal orientation of the analogs to interact with Arg54. One can envision that follow-up optimization will focus on the lactam side chain for better directing the functional groups to the target arginine of BRDT-1. Among the ring-open esters, an unexpected conformation was observed in the co- crystal structure, where the benzyl ester side chain was located outside the ZA channel. This discovery inspired the use of a macrocyclization approach for conformational pre- organization. Unfortunately, most macrocyclic analogs showed minimal improvement in either BRDT-1 affinity or selectivity. Nevertheless, macrocyclic analog 2.16b displayed unexpected BRDT-2 preference, which is worth further validation as well as optimization. Novel mechanisms of action for selective bromodomain inhibition were pursued as well. Targeted protein degradation has been recognized as a new avenue of selective BET inhibition. Therefore, potent lactam analog 2.5m was derivatized and three PROTACs were prepared in order to find the optimal connection site and linker length. Despite their reasonable affinity in the binding assay, all PROTACs failed to induce protein degradation in a cellular assay. We will endeavor to optimize the linker region for better induction of the protein/ligase complex formation. Meanwhile, a bivalent strategy was adopted for its potential to achieve outstanding potency as well as isoform selectivity. Proof-of-concept dihydropyridine molecule 2.41 was designed and synthesized following the reported method. Future work will include binding assessment as well as structural optimization of 2.41.

57 Chapter 3 Focused Library Synthesis for TGR5 (Takeda G Protein-Coupled Receptor 5) Antagonists 3.1. PLD Introduction Polycystic liver disease (PLD) is an inherited dysfunction of the biliary epithelium caused by genetic defects of proteins associated with intracellular organelles, mainly the primary cilium and endoplasmic reticulum. It is characterized by bile duct dilation and cyst development. The formation of multiple cysts on the liver alone is defined as autosomal dominant polycystic liver disease (ADPLD). This disease is often accompanied by autosomal dominant polycystic kidney disease (ADPKD) or autosomal recessive polycystic kidney disease (ARPKD). The occurrence of ADPKD is around 1:500 to 1:1,000 of the population,135 whereas the “isolated” ADPLD has a far lower morbidity (1:100,000).136

3.1.1. Genetic Mechanism The etiology of ADPLD remains elusive and most (~70%) of the related genetic alterations are still unresolved. According to several literature reports, mutations in three genes, PRKCSH,137 SEC63,138 and LRP5,139 have been identified as leading causes for ADPLD, with the highest frequency of mutation (~15%) in PRKCSH (Figure 3.1). PRKCSH and SEC63 encode proteins expressed in the endoplasmic reticulum for processing glycoproteins.137, 138 LRP5 is a transmembrane protein that acts as a co-receptor with Frizzled proteins to transduce Wnt signaling.139, 140 For ADPKD, the two leading causes are PKD1 and PKD2 mutations.141, 142 These genes encode mechanoreceptor polycystin-1 and nonselective calcium channel polycystin-2, respectively, which are coupled in the ciliary membrane for calcium uptake.143, 144 Besides intracellular signaling dysregulation, the aforementioned genetic alterations can lead to multiple pathophysiological defects, including alterations in ductal plate remodeling and primary cilium malformation. Together, these abnormalities contribute to hepatic cytogenesis.

58 Figure 3.1. Genetic mutations responsible for ADPKD and ADPLD. The width of each color along the x- axis represents the percentage of patients with a specific mutation. GUR stands for genetically unresolved. This figure was adapted from reference145.

3.1.2. Cholangiocyte Abnormalities Hepatic cystogenesis is thought to arise from a hyperproliferative phenotype of cholangiocytes, which are the epithelial cells that form the bile ducts. They play an essential role in the regulation of both bile flow and composition. To detect changes in bile flow, healthy cholangiocytes develop antenna-like single primary cilia that extend from the apical membrane into the bile duct lumen.146, 147 However, on cystic cholangiocytes, this sensory organelle appears shortened or entirely absent, due to aberrant expression of PLD- related proteins (Figure 3.2A).148, 149 The shortened cilia also deactivate the TRPV4 calcium channels, which function as osmosensors on these cilia.150 Despite its overexpression, TRPV4 fails to maintain the intracellular calcium concentration in cystic cholangiocytes (Figure 3.2C).151 Other key factors responsible for hyperproliferation are growth factors and hormones present in the fluid and secreted by cystic cholangiocytes. As illustrated in Figure 3.2D, vascular endothelial growth factor (VEGF) and its corresponding receptor VEGFR were upregulated in cholangiocytes isolated from ADPKD patients.152 Insulin-like growth factor 1 (IGF-1) was also reported to amplify the proliferative signal in cystic cholangiocytes (Figure 3.2E).153 Moreover, epidermal growth factor (EGF) induced a pronounced hyperproliferation in the cholangiocytes isolated from PCK rats.154

59 Figure 3.2. Cellular alterations and molecular mechanisms involved in hepatic cystogenesis. A. Due to genetic mutations in certain genes, the primary cilium undergoes morphological alteration, appearing shortened or entirely absent. B. Aberrant expression of polycystin-1 and 2 lead to high cAMP and low calcium concentration, which ultimately yields a hyperproliferative phenotype. C. The shorted cilium abolishes osmosensor TRPV4, which can’t be activated by anisosmotic conditions to intake extracellular calcium and suppress cAMP production. D. VEGF in the liver cyst fluids activates the MEK/ERK1/2 pathway and promotes proliferation. E. IGF-1 contributes to hyperproliferation via the PI3K/AKT/mTOR pathway. This figure was adapted from reference155.

3.1.3. Preclinical and Clinical Studies of Potential PLD Therapies Based on mechanistic studies, current therapeutic development for PLD is focused on antiproliferative strategies (Figure 3.3). Octreotide, a synthetic Somatostatin analog that lowers excess growth hormone secretion, was reported to reduce liver weight and cyst volume by decreasing cAMP levels in PCK rats.156 GSK1016790A, a TRPV4 activator, inhibited hyperproliferation of PCK rat cholangiocytes by escalating intracellular calcium levels. Unfortunately, it failed to show any potency in vivo.151 Semaxanib, a potent VEGFR inhibitor, blunted hepatic cystogenesis by blocking the VEGF cascade in an animal model.157 The mTOR inhibitor Rapamycin attenuated the proliferation stimulated by IGF-1 in Pkd2cKO mice but had minimal effects on cystogenesis in PCK rats.158, 159 Nonetheless, the EGFR inhibitor EKI-785 failed to show any protective effect on hepatic cystogenesis.160 At present, Somatostatin and its derivatives (Octreotide, Lanreotide,

60 and Pasireotide) have entered clinical trials for systematic evaluation against PLD.127, 161- 163

O Cl H N O N O O H2N S O H S N O Cl H N HO OH H OH N OH O OH H H HO S OH O N S H H GSK1016790A HO N OH N TRPV4 O O N N OH HO H O H N N O HO O O H H OH HN Br N H O H N HO HN O N N O Rapamycin NH2 H N mTOR Octreotide Semaxanib EKI-785 Hormone mimic VEGFR EGFR Figure 3.3. Structure of octreotide and chemical entities used in preclinical/clinical studies for PLD. Their corresponding mechanisms/targets are listed below the structures.

3.1.4. Current Therapy The primary symptom of PLD is the increased liver volume caused by multiple cysts scattered on the surface.164 Although not lethal, enlarged liver size (up to 10 times the normal size) might compress the abdominal and thoracic tissues, causing epigastric pain, abdominal distension, nausea, and vomiting. Other complications include infection, hemorrhage, and cyst rupture.136 The therapeutic regimen for PLD is predominantly based on the current disease progression as well as the patient-determined therapeutic goals (Figure 3.4). For asymptomatic circumstances, intermittent surveillance is suggested in PLD patients. Somatostatin, a natural growth hormone-inhibiting peptide, has been prescribed to slow cyst growth. Because of the high cost of Somatostatin ($7,000-11,000 per month), surgical intervention is another common option. PLD patients with one dominant cyst are eligible for aspiration sclerotherapy. For multiple cysts that are accessible on the anterior segment of the liver, fenestration is often highly recommended. If either aspiration or fenestration are not practical, hepatic resection is the leading option. For the most severe disease state, liver transplantation is the only curative method.145

61 Figure 3.4. Therapeutic algorithm for PLD based on different therapeutic goals and clinical symptoms including cyst number and distribution as well as disease progression. This figure was adapted from reference145.

3.2. TGR5 Introduction Given the high expense of Somatostatin and variable success with fenestration/resection because of cyst regrowth,165 there is an unmet need for potent and affordable PLD pharmaceuticals. Recent studies have suggested that TGR5 is an emerging target of interest for PLD treatment.166 TGR5 is a G protein-coupled receptor initially identified by Takeda in 2003.167 It is classified into the rhodopsin-like subfamily (Class A) and has relatively low sequence identity with known co-crystal structures of other Class A 168, 169 GPCRs (i.e. 20% sequence identify with adenosine A2A receptor). The endogenous ligands of TGR5 are various bile acids (Figure 3.5), including taurine-conjugated lithocholic acid (TCLA), lithocholic acid (LCA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA), and cholic acid (CA), among which TCLA has the highest potency.

62 Figure 3.5. Endogenous bile acids and their reported TGR5 agonism potency.170

3.2.1. TGR5 Biological Functions Although its mRNA is ubiquitously detected, TGR5 is highly expressed in the gall bladder, liver, brown adipose tissue, muscle, intestine, and kidney.171, 172 Upon ligand activation, TGR5 downstream effects are tissue dependent (Figure 3.6). For instance, TGR5 agonists increase energy expenditure in the muscle and brown adipose tissue.171 TGR5 suppresses the inflammation induced by lipopolysaccharide inside macrophages by inhibiting nuclear translocation of NFB and reducing the release of pro-inflammatory factors.173, 174 TGR5 stimulation in enteroendocrine cells releases glucagon-like peptide (GLP-1), which improves insulin secretion and sensitivity.175, 176 Because of these key players, an intense interest in developing TGR5 modulators for various therapeutic strategies has steadily increased in recent years. In particular, TGR5 agonists have been developed for treating obesity, atherosclerosis, and type 2 diabetes.173, 176

63 Figure 3.6. TGR5 tissue distribution and corresponding biological outcomes upon its activation. This figure was adapted from reference177.

3.2.2. Reported Agonists According to their structural features, the reported TGR5 agonists can be classified into two subsets: steroidal and non-steroidal ligands. The steroidal subset consists of endogenous bile acids and their derivatives. Semisynthetic bile acid libraries give rise to a plethora of potent TGR5 agonists with reasonable ADMET properties.176, 178-180 Meanwhile, non-steroidal ligands were initially discovered from compound library screenings. As shown in Figure 3.7, this subclass contains a wide range of structurally diverse scaffolds, including imidazole,181 quinoline,182 nicotinamide,183 and pyrazole.184 Because of the structural novelty and their higher potency compared to bile acid analogs, the non-steroidal ligands have garnered significant attention in the field. However, due to the lack of co- crystal complexes, it remains elusive whether the potency difference is derived from disparate binding modes between the two subsets. Thus far, limited data has been obtained from clinical trials with TGR5 agonist SB-756050 (Figure 3.7) for the treatment of type 2 diabetes mellitus.185, 186

64 Figure 3.7. Reported small molecule scaffolds and their TGR5 agonist potency values.187, 188

3.2.3. The Role of TGR5 in PLD In addition to the aforementioned clinical trials in type 2 diabetes, TGR5 has been proposed as a promising target for PLD. In the liver, TGR5 is detected in the cholangiocytes, Kupffer cells, and sinusoidal endothelial cells.189 Healthy cholangiocytes have TGR5 localized to various compartments, with its most abundant expression on the apical, ciliary, and nuclear membranes. Because of its physiological environment, TGR5 on the cilia is constantly exposed to bile acid agonists. As shown in Figure 3.8 (left, ciliary pathway), TGR5 activation by the bile acid agonist in the ciliary cholangiocyte decreased

166, cAMP production via the Gi protein, which resulted in genetic quiescence of the cells. 190 In the PLD disease state, mutation-induced cilia malformation or complete absence abolish the ciliary specificity of TGR5.148, 149 This alteration in distribution has further significant impact on the TGR5 signaling cascade. Inside the non-ciliary cholangiocytes, copy numbers of the TGR5 transcript were elevated over 20-fold and TGR5 protein 65 expression was increased by about 45%. In addition, there was simultaneous Gs protein increase (~67%) relative to ciliary cholangiocytes.166 This co-upregulation of TGR5 and

Gs indicated a different signaling process. As depicted in Figure 3.8 (right, non-ciliary pathway), instead of coupling with the Gi protein, TGR5 relied on the Gs protein to transmit the signal, increasing the cAMP production in the non-ciliary cells.166 This proposed pathway agreed with experimental observations. TGR5 agonists led to cAMP increases in cholangiocytes isolated from both PKC rats (~40%) and ADPKD mice (~45%), compared with wild type.166 The escalated cAMP level eventually resulted in the hyperproliferation phenotype and hepatic cystogenesis.191

Figure 3.8. Opposite signaling outcome of TGR5 agonist in ciliary (left) and non-ciliary (right) cells. This difference likely arises from the different G proteins, which dictate downstream cAMP production.

66 TGR5 as a potential PLD drug target was further validated by knock-out studies and small-molecule treatment. TGR5 shRNA lowered the cAMP level significantly in the PCK and ADPKD cholangiocytes. Furthermore, TGR5 shRNA transfection returned the agonist-induced cAMP increase and hyperproliferation to normal.166 Moreover, the TGR5- /-; Pkhd1del2/del2 double mutation successfully rescued the polycystic phenotype of Pkhd1del2/del2 mice, while leaving the animal healthy and fertile.166 The first reported TGR5 antagonist, SBI-115, was reported to ameliorate hepatic cystogenesis via decreasing cellular cAMP (Figure 3.9).166 Taken together, the available data indicate that TGR5 antagonists could be a safe and effective intervention for PLD.

Figure 3.9. Reported TGR5 antagonist SBI-115 returned increased cAMP levels back to normal.

3.3. Focused Library Syntheses and Biological Evaluation for TGR5 Antagonists 3.3.1. Design Rationale To evaluate their potential for PLD, we began by developing novel TGR5 antagonists. When we first initiated this project, no TGR5 antagonists had been disclosed and the lack of crystallographic data for TGR5 made it challenging to conduct virtual screening campaigns. Furthermore, high throughput screening is expensive and time- consuming for this preliminary study. Nonetheless, we noticed that in the literature there were published examples of small changes to scaffolds leading to major changes in functional activity at GPCRs. Dosa and Ambrose analyzed the reported cases and classified them into ten categories (Figure 3.10).192 This strategy was very compelling to us, and it was employed to frame our thinking about the plethora of known TGR5 agonists.

67 Figure 3.10. Ten general modifications and corresponding examples of interconverting between GPCR agonist and antagonist. The structural changes are highlighted in red.

3.3.2. Scaffold Selection According to the literature, there were more than 10 different scaffolds reported as TGR5 agonists. Initial scaffold selection is critical for success, so we devised two main criteria to guide selection of this starting point. We considered both structural and synthetic simplicity as the first rule to enable rapid exploration of chemical modifications. Secondly, we prioritized scaffolds with comprehensive SAR information so that we could both avoid redundant changes and use the documented data to validate our in-house assay. Following these rules, we eventually picked the nicotinamide scaffold for further modification. The SAR campaign around the nicotinamide core focused on modifying the phenolic and aniline moieties (Figure 3.11). More importantly, there seemed to be a trend that through modifying either the phenol or amide part, the functional activity started to invert among the reported analogs.183

68 R1 R2

R2 Cl Full agonist Full agonist N O EC50 10 nM N EC50 10 nM O Cl N O Cl N O Partial agonist 1 Partial agonist R Cl EC50 53 nM N EC50 67 nM CF3 F

Figure 3.11. Nicotinamide analogs with different GPCR functional activity.

3.3.3. Library Generation For nicotinamide library generation we first selected 3-phenyl, 4-chloro, and 2- chloro-5-flurophenols, because the corresponding analogs had been reported to show loss of activity and efficacy.183 Following the empirical rules of interconversion between GPCR agonism and antagonism,192 2-phenyl, 4-phenyl, and p-benzoyl phenols were included to add an aromatic group. Phenols with acetyl/propionyl groups were considered in order to add a hydrogen bond donor. To modify the substitutions on the phenol ring, we included eight phenols with functional groups that were not covered in the original publication183 (Figure 3.12).

Figure 3.12. Phenol building blocks and their corresponding reasons for selection.

As shown in Figure 3.13, the aniline library included reported moieties and those designed to accomplish the desired mode of action. N,2-Dimethylaniline, 2-methylaniline, 69 and 6-methyl-1,2,3,4-tetrahydroquinoline were reported to decrease potency in the initial study.183 Even though N-ethylaniline was previously advantageous for affinity, we included it and N-methylaniline to investigate changes in the aniline nitrogen substitution pattern. Moreover, 2-fluoro-N-methylaniline were selected to introduce a structural change near the amide bond.

NH NH Reported modifications Change in Aniline Nitrogen Substitution NH NH NH2 NH

NH F Change in Aromatic Substitution

Figure 3.13. Aniline library and their reason for selection.

After building block selection, we sought to elaborate a focused library in a rapid manner. The reported route included a microwave-assisted Ullmann-type coupling between 2-chloronicotinic acid with phenols, followed by amide coupling with the amine source. To accelerate the process and to allow a combinatorial approach, the Ullmann-type coupling was replaced with a base-catalyzed SNAr reaction. According to the new route (Scheme 3.1) 2-chloronicotinic acid was converted to the corresponding ethyl ester intermediate on a 10-gram scale, which was aliquoted into multiple fractions for the substitution reactions with various phenols. The sequent ester hydrolyses were conducted in a 24-well block to yield the corresponding acids, which were again split into aliquots for the final amide coupling with their respective amine building blocks. Notably, despite additional protection/deprotection steps, two functionalization steps were conducted in a high-throughput manner, which gave rise to a focused library with more than 130 analogs.

70 Scheme 3.1. Reported Synthetic Route and Its Optimization

Reagents and conditions: a) [Cu(CH3CN4)]PF6 (0.2 equiv), Cs2CO3 (2.5 equiv), toluene, microwave to 140 °C; b) substituted amine (1.2 equiv), HATU (1.3 equiv), DIPEA (2.0 equiv), DMF; c) conc. H2SO4 (0.1 equiv), ethanol, reflux, 85%; d) substituted phenol (1.1 equiv), K2CO3 (2.0 equiv), DMF, 90 C; e) NaOH (2.0 equiv),

H2O/1,4-dioxane, 21-60% in two steps; f) substituted amine (1.1 equiv), HATU (1.2 equiv), DIPEA (2.0 equiv), DMF, 10-99%.

3.3.4. Assay Development and Data Analysis To determine cAMP production inside the cell, we used the LANCE Ultra cAMP kit, which is a homogeneous time-resolved fluorescence resonance energy transfer (TR- FRET) assay. As depicted in Figure 3.14A, it is based on the competition between the cellular cAMP stimulated by TGR5 activation in CHO cells expressing recombinant human TGR5 (DiscoverX) and the europium-labeled cAMP tracer. This TR-FRET assay was embedded in our workflow at two stages, the first of which was to evaluate the agonist activity of the synthesized analogs (Figure 3.14B, step 1). Then the analogs unable to induce cAMP production were identified and subjected to another TR-FRET assay in which the TGR5 cells were incubated with these inactive analogs and an EC80 concentration of the known TGR5 agonist GPBAR-A (Figure 3.14B, step 2). Antagonist activity is indicated by a reduction of the GPBAR-A cAMP production. After antagonist hit identification, full dose-response curves were generated, followed by iterative structural modifications, seeking more potent TGR5 antagonists (Figure 3.14B, steps 3 and 4).

71 Figure 3.14. Proposed workflow for TGR5 antagonist identification. A. Mechanism of action of the LANCE Ultra cAMP assay. Europium (Eu) chelate-labeled cAMP tracer is recognized by the cAMP-specific monoclonal antibodies labeled with the Ulight™ dye. After the excitation of Eu, the emitted energy is transferred by fluorescence resonance energy transfer (FRET) to the Ulight™ molecule, which in turn emits light at 665 nm. The more cAMP produced by TGR5 activation in CHO cells expressing recombinant human TGR5, the higher competition with the Eu-labeled cAMP tracer, thus the lower light signal will be observed. B. Two-stage cell-based functional assay for identifying TGR5 antagonists.

Our data first validated some of the published SAR information, as shown in Figure 3.15. According to the literature, 3-phenyl and 4-chloro phenols were reported as not to be advantageous for binding. Our results not only confirmed this conclusion but also extrapolated it to all other synthesized amides. Secondly, sterically demanding substitution on the meta position of the phenoxy group was not favored. Our exploration showed that the 3-acetyl phenols turned out to be a less potent set of analogs. Moreover, it was reported that secondary amides resulted in loss of binding, presumably due to the planar configuration of secondary amide bonds not being preferred.183 We also observed that the 2-methylaniline-derived analogs underwent significant potency loss even when paired with favored phenol moieties, including 3-trifluoromethyl and 3-cyano phenol.

72 A. 3-Phenyl and 4-chloro phenols were detrimental

R2 R2 R2

O O Reported N O N O N inactive N N

F Ph N NH N Cl N

Tested inactive

B. Sterically demanding substitution C. Secondary amide resulted in activity loss was not favored

1 R1 R

Reported Cl Reported N inactive NH OEt inactive O O Cl

N O N O Tested Tested R1 R1 inactive inactive O CN CF3

Figure 3.15. The SAR results that corroborate the published ones.183

While some data corroborated the literature, we also noticed some discrepancies between our results and the published findings (Figure 3.16).183 According to the reported modeling, the putative binding site of TGR5 was able to tolerate para substitutions on the phenoxy moiety.183 The established activity data also suggested that polar side chains improved binding potency as well as aqueous solubility.183 However, what we concluded was that 4-acetyl, propionyl, phenyl, and benzoyl groups all eradicated TGR5 affinity. One explanation for this contradiction was that despite their similar size, the pocket was more accommodating to hydrophilic moieties, compared with hydrophobic ones. We observed another inconsistency regarding the analog with the 2-chloro-5-fluoro substitution on the phenoxy group, which was identified as completely inactive in the original study.183 Our SAR investigation, however, indicated that this modification was not detrimental to binding.

73 Figure 3.16. Contradictory SAR conclusions regarding the 4-position of the aryl ether between our and reported study. This difference might be reconciled by the hydrophilicity/hydrophobicity of the introduced moieties.

Our study expanded the reported scope of the substitution patterns around the nicotinamide scaffold. The ortho position of the phenoxy group did not tolerate bulky substitutions, as exemplified by the 2-phenyl subset that showed significantly lower potency even when paired with favored amino moieties (Figure 3.17A). This modification campaign also revealed that the cyano group was a surrogate of the 3-chloro group on the phenoxy ring (Figure 3.17B), presumably due to the similar electron-withdrawing and hydrophobic properties. 2,5-Dichloro substitution was reported to yield optimal binding, and replacement of the 2-chloro group with fluorine, methyl, or trifluoromethyl led to decreased potency.183 We found that 2,5-dichloro phenol could be replaced by 2-chloro-5- trifluoromethyl phenol, given that in the 2-chloro-5-trifluoromethyl subset all analogs showed full agonist potency, including the unfavored amides (Figure 3.17C). The 1,2,3,4- tetrahydroquinoline and 3-chloro-4-fluorophenol were reported to yield potent TGR5 agonists.183 As shown in Figure 3.17D, the combinations between favored 1,2,3,4- tetrahydroquinoline/N-ethylaniline and 3-methyl-4-fluoro or 3-trifluoromethyl-4-fluoro phenols resulted in analogs with reduced potency. Thus, we concluded that 3-chloro was not replaceable with either methyl or trifluoromethyl in this case. Although the 4-

74 chlorophenol analog was inactive, we noticed that the introduction of the preferred 2,5- dichloro substitution could rescue the agonist potency (Figure 3.17E). Combination of the 2-chloro and 3-trifluoromethyl moieties on the phenoxy group did not result in any synergistic effects (Figure 3.17F). Additionally, 2-fluoro-N-methylaniline-derived amides led to activity loss (Figure 3.17G), despite N-methylaniline being well-accepted in the original study.183

A. Phenyl at the ortho position was not tolerated B. Cl and CN were interchangeable at the para site

R2 R2 R1 O N N O N N N O Ph N O Cl CN Favored amino moieties R1

C. 2-Cl-5-CF3 phenol was preferred D. 3-Me-4-F and 3-CF3-4-F displayed reduced potencies R2

R2 R2 O R2

O N O

N O N NH N N Cl R1 F CF Unfavored amino moieties 1 Favored amino moieties 3 R = Me or CF3

E. 2,4,5-Trichlorophenol rescued the binding F. The combination of 3-Cl and 3-CF3 was not advantageous

2 R1 R2 R O N Cl N O O N N N Cl Cl N O Cl Cl Favored amino moieties R1 inactive active CF3

G. 2-F-N-methylaniline caused potency loss

F R1

O CN CF3 N O Favored phenolic R1 moieties

Figure 3.17. New SAR results that expand upon previous structure-activity relationships.

75 Most of synthesized analogs were characterized as potent or moderate TGR5 agonists. A total of 27 analogs showed low to no cAMP production. Therefore, we subjected the analogs to a sequential assay for antagonism evaluation, in which cells were co-incubated with the analog and known TGR5 agonist GPBAR-A (35 nM). Then the generated cAMP was measured via the LANCE Ultra cAMP kit. A potent TGR5 antagonist was expected to compete with GPBAR-A at the binding site, which would decrease cAMP production, compared to GPBAR-A alone. Meanwhile, a non-binder would have no competition with GPBAR-A, thus no signal change would be detected relative to GPBAR- A alone. According to the results obtained (Figure 3.18), most of the analogs failed to decrease the cAMP level at both concentrations tested. The only analog that appeared to antagonize the reference agonist GPBAR-A was analog 12 (10 µM), although at the higher concentration (100 µM) it increased cAMP production, indicating that it was likely a weak agonist. Additionally, cAMP increased slightly in some cases. Therefore, we concluded that the 27 analogs either did not bind to TGR5 or were weak agonists.

76 Figure 3.18. Histograms of the antagonism screening (A-C). Emax and EC80 stand for the cAMP levels induced by known TGR5 agonist GPBAR-A at 3 µM and 35 nM, respectively. Signal above Emax is considered out of the linear range of the assay. Blank is the DMSO only condition. All the analogs were tested once in duplicate at 10 and 100 µM. A potential antagonist is expected to lower cAMP below the red dashed line due to competition with GPBAR-A (35 nM). 77 3.4. Discussion To aid interpretation of the assay results, we sought to construct TGR5 homology models. However, the models generated failed to establish strong correlations between the docking scores and assay data, most likely due to the lack of crystal structures with high sequence identity to TGR5. Further optimization of the computational model via molecular dynamics did not yield satisfactory results either. Therefore, we discontinued these modeling studies after the first round of screening. In summary, starting from a known TGR5 agonist scaffold, a focused library was designed and synthesized with various minor modifications. More than 100 analogs were generated in a combinatorial fashion with the goal of converting the agonist to an antagonist. A two-phase functional assay was then developed to identify potential TGR5 antagonists. Most of the analogs were either potent or moderate agonists. Analogs filtered through the agonism assay did not show any inhibitory activity in the antagonism assay, indicating that the lack of agonism potency was a result of the compounds not binding to TGR5. From these results we obtained insights about the nicotinamide core as a TGR5 agonist, including both unfavored structural modifications and novel beneficial surrogates of reported substitutions. Moreover, we gained a deeper understanding of the interconversion of GPCR agonists to antagonists. To increase success of this experimental strategy, additional information is paramount, including crystallographic data of the protein structure as well as ligand binding modes. This investigation, however, did not indicate that it was impossible to develop TGR5 antagonists. After the conclusion of our study, Masyuk and coworkers disclosed m- tolyl 5-chloro-2-(ethylsulfonyl)pyrimidine-4-carboxylate (SBI-115, Fig. 3.9) as the first TGR5 antagonist. This compound was identified by high throughput screening of a 50K compound library in TGR5-expressing CHO-K1 cells. According to their studies, this antagonist reduced cyst growth (~30%) in cystic cholangiocytes via decreasing cAMP.166 Additionally, the combination of SBI-115 and Pasireotide, a Somatostatin analog, led to ~50% decrease in cyst growth. Neither binding characterization nor structural optimization of SBI-115 were pursued in this study. Considering its small size, SBI-115 can be used as 78 the starting point for structure-activity relationship explorations, seeking potent TGR5 antagonists.

79 Chapter 4 Experimental Data and Procedures General: All chemicals and solvents were purchased from commercial suppliers and directly used without further purification unless otherwise specified. Reactions performed under microwave irradiation utilized the Biotage Initiator. Reactions were monitored by

TLC on 0.2 mm silica gel plates (Merck Kieselgel GF254) and visualized under UV light (254 nm). Preparatory-scale flash column chromatography was conducted using medium- pressure liquid chromatography (MPLC) on a CombiFlash Companion (Teledyne ISCO, Inc.) with pre-pack silica columns (20–40 microns) and UV detection at 254 nm. 1H and 13C NMR spectra were performed on a Brucker Avance 400 MHz spectrometer. Chemical shifts were reported in parts per million (ppm) and coupling constants (J) were expressed in Hz. Splitting patterns were designed as s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; bs, broad singlet. Melting points were measured with a Kofler bench and are uncorrected. Purities of tested compounds were determined either by UPLC or qNMR, using DMSO2 as the internal standard and the qNMR protocol published in Journal of Medicinal Chemistry.193, 194

4.1. Small Molecule Development-1: Lactams 6-Amino-1-ethylpyrimidine-2,4(1H,3H)-dione (2.2).

H2N N O

Based on a reported procedure,195 ethyl 2-cyanoacetate (12.8 g, 113.5 mmol) and 1- ethylurea (10.0 g, 113.5 mmol) were dissolved in anhydrous ethanol (250 mL) in a 500 mL round-bottomed flask. Sodium tert-butoxide (21.8 g, 227.0 mmol) was then added and the mixture was heated to reflux for 24 h. After cooling to rt, the solvent was evaporated under reduced pressure, and the residue was dissolved in water (100 mL). The pH of the solution was adjusted to 1–2 with a 1M HCl solution. The precipitated solid was collected by filtration, washed with water, and dried under vacuum to afford the title compound as a 1 brown solid (16.0 g, 91%). H NMR (400 MHz, DMSO-d6) 10.28 (s, 1H), 6.78 (s, 2H), 4.52 (d, J = 2.1 Hz, 1H), 3.78 (q, J = 7.0 Hz, 2H), 1.08 (t, J = 7.0 Hz, 3H).

80 Ethyl 4-Chloro-2-(4-methylbenzylidene)-3-oxobutanoate (2.3).

O O Cl O

Ethyl 4-chloro-3-oxobutanoate (1.5 g, 9.1 mmol) and p-tolualdehyde (1.0 g, 8.7 mmol) were dissolved in ethanol (100 mL). Piperidine (74 mg, 0.87 mmol), and acetic acid (52 mg, 0.87 mmol) were added and the reaction was stirred at rt for 24 h. The volume of the solvent was reduced under reduced pressure, and the residue, dissolved in a small amount of solvent, was poured into water, and extracted with ethyl acetate. The organic phase was washed with brine, dried over anhydrous MgSO4, and concentrated under reduced pressure. The residue was purified by flash column chromatography (40 gram RediSep Gold silica gel column, 0–5% ethyl acetate in hexanes) to yield a yellow viscous oil as E/Z isomeric mixture (1.3 g, 57%). δ 7.81–7.35 (m, 1H), 7.28–7.25 (m, 2H), 7.19 (d, J = 8.2 Hz, 2H), 4.34–4.27 (m, 4H), 2.37 (d, J = 5.0 Hz, 3H), 1.35–1.23 (m, 3H).

Ethyl 4-(Benzyloxy)-3-oxobutanoate (2.6).

O OBn To a suspension of NaH (60%, 6.5 g, 162.8 mmol) in THF (100 mL) at 0 °C was added benzyl alcohol (8.4 g, 77.7 mmol) dropwise. The mixture was stirred at rt for an additional 2 h. To this mixture was added ethyl 4-chloroacetoacetate (12.2 g, 74.0 mmol) dropwise over 30 min. The resulting dark orange mixture was stirred at rt overnight before, cooled to 5 °C, acidified to pH 4 using a 1M HCl solution and extracted with ethyl acetate. The organic phase was washed with brine, dried over anhydrous MgSO4, and concentrated under reduced pressure. The residue was then purified by flash column chromatography (40 gram RediSep Gold silica gel column, 0–10% ethyl acetate in hexanes) to give the title 1 compound as light-yellow viscous oil (12.7 g, 73%). H NMR (400 MHz, CDCl3) δ 7.40– 7.28 (m, 5H), 4.59 (s, 2H), 4.18 (q, J = 7.1 Hz, 2H), 4.14 (s, 2H), 3.54 (s, 2H), 1.25 (t, J = 7.1 Hz, 3H).

81 Ethyl 4-(Benzyloxy)-2-(4-methylbenzylidene)-3-oxobutanoate (2.7).

O O OBn O

Ethyl 4-(benzyloxy)-3-oxobutanoate (12.7 g, 53.9 mmol) and p-tolualdehyde (6.5 g, 53.9 mmol) were dissolved in ethanol (100 mL). Piperidine (0.46 g, 5.4 mmol), and acetic acid (0.32 g, 5.39 mmol) were added and the reaction was stirred at rt for 24 h. The volume of the solvent was reduced under reduced pressure, and the residue, dissolved in a small amount of solvent, was poured into water, and extracted with ethyl acetate. The organic phase was washed with brine, dried over anhydrous MgSO4, and concentrated under reduced pressure. The residue was purified by flash column chromatography (40 gram RediSep Gold silica gel column, 0–10% ethyl acetate in hexanes) to yield a yellow viscous 1 oil as E/Z isomer mixture (8.1 g, 56%). H NMR (400 MHz, CDCl3) δ 7.73 (d, J = 21.1 Hz, 1H), 7.36 (d, J = 6.3 Hz, 2H), 7.31–7.27 (m, 5H),7.17 (t, J = 7.9 Hz, 2H), 4.60 (d, J = 5.5 Hz, 2H), 4.43–4.27 (m, 1H), 4.45–4.14 (m, 1H), 4.24–4.23 (m, 2H), 2.37 (d, J = 4.0 Hz, 3H), 1.28 (td, J = 7.2, 1.2 Hz, 3H).

Ethyl 7-((Benzyloxy)methyl)-1-ethyl-2,4-dioxo-5-(p-tolyl)-1,2,3,4,5,8- hexahydropyrido[2,3-d]pyrimidine-6-carboxylate (2.8).

O O NH O N N O BnO H Intermediates 2.2 (5.8 g, 37.5 mmol) and 2.7 (12.7 g, 37.5 mmol) were added into a 250 mL round-bottomed flask equipped with a reflux condenser. After the addition of acetic acid (100 mL), the mixture was heated to reflux overnight, during which the reaction turned into a clear light brown solution. After cooling to rt, most of the acetic acid was removed under reduced pressure. A saturated NaHCO3 solution (150 mL) was charged into the flask portionwise and the mixture was allowed to stir for an additional 0.5 h to neutralize the residual acid. A yellow solid precipitated from the aqueous solution and was collected by filtration. The solid was washed with hexanes and water twice. After drying under vacuum, 82 the crude the title compound (12.7 g, 71%) was used for the next step without further 1 purification. H NMR (400 MHz, DMSO-d6) δ 10.98 (s, 1H), 8.46 (s, 1H), 7.34 (dd, J = 22.3, 4.4 Hz, 5H), 7.08 (d, J = 8.0 Hz, 2H), 7.01 (d, J = 8.0 Hz, 2H), 4.97 (d, J = 13.9 Hz, 1H), 4.88 (s, 1H), 4.62–4.56 (m, 3H), 4.02 (qd, J = 7.2, 3.4 Hz, 3H), 3.90 (dt, J = 14.9, 7.0 Hz, 1H), 2.20 (s, 3H), 1.12 (t, J = 7.1 Hz, 3H), 1.08 (t, J = 7.1 Hz, 3H). 13C NMR (100

MHz, DMSO-d6) δ 165.8, 161.3, 149.8, 143.8, 143.7, 143.1, 137.7, 135.3, 128.6, 128.2, 127.69, 127.65, 127.1, 105.3, 89.7, 71.9, 65.5, 59.8, 40.1, 35.9, 20.5, 14.0, 13.2.

Ethyl 1-Ethyl-7-(hydroxymethyl)-2,4-dioxo-5-(p-tolyl)-1,2,3,4,5,8- hexahydropyrido[2,3-d]pyrimidine-6-carboxylate (2.9).

O O NH O N N O HO H Intermediate 2.8 (2.8 g, 6.0 mmol) was dissolved in dry DCM (50 mL) in a two-necked round-bottomed flask (250 mL) equipped with a nitrogen balloon and a septum. The resulting yellow solution was cooled to –78 °C before the addition of BBr3 (1 M solution, 18 mL, 18 mmol) dropwise. The solution was stirred for an additional 0.5 h after the removal of the dry ice bath. After the total consumption of the starting material monitored by TLC, the reaction was quenched by the addition of methanol and a saturated NaHCO3 solution. The mixture was extracted with ethyl acetate and the organic phase was washed with brine, dried over anhydrous MgSO4, and concentrate under reduced pressure. The residue was then purified by flash column chromatography (40 gram RediSep Gold silica gel column, DCM + 5.0% methanol) to yield the title compound as a white solid (1.5 g, 1 64%). H NMR (400 MHz, DMSO-d6) δ 10.99 (s, 1H), 8.36 (s, 1H), 7.08 (d, J = 7.9 Hz, 2H), 7.01 (d, J = 7.9 Hz, 2H), 6.01–5.95 (m, 1H), 4.86 (s, 1H), 4.79 (dd, J = 16.3, 4.6 Hz, 1H), 4.55 (dd, J = 16.3, 4.9 Hz, 1H), 4.07–4.01 (m, 3H), 3.90 (dq, J = 14.4, 7.0 Hz, 1H), 13 2.20 (s, 3H), 1.16 (dt, J = 16.0, 7.0 Hz, 6H). C NMR (100 MHz, DMSO-d6) δ 165.8, 161.3, 149.8, 147.2, 143.6, 143.4, 135.2, 128.5, 127.1, 101.9, 89.2, 59.5, 58.3, 54.9, 35.9, 20.5, 14.1, 13.2.

83 Ethyl 7-(Chloromethyl)-1-ethyl-2,4-dioxo-5-(p-tolyl)-1,2,3,4,5,8- hexahydropyrido[2,3-d]pyrimidine-6-carboxylate (2.4).

O O NH O N N O H Cl

A solution of SO2Cl2 (0.62 mL, 7.7 mmol) in DMF (5 mL) was added dropwise over 5 min to a stirred, ice-cooled solution of intermediate 2.9 (1.5 g, 3.8 mmol) and imidazole (0.68 g, 10 mmol) in DMF (20 mL). After the removal of the ice bath, the mixture was stirred at rt for 0.5 h, diluted with ethyl acetate, washed with water, and dried over anhydrous MgSO4. The solvent was removed under reduced pressure and the residue was purified by flash column chromatography (40 gram RediSep Gold silica gel column, DCM + 2.0% methanol) to yield the title compound as a light-yellow solid (0.87 g, 56%); mp 266–267 °C dec; 96% 1 purity determined by UPLC. H NMR (400 MHz, DMSO-d6) δ 11.03 (s, 1H), 8.99 (s, 1H), 7.07 (d, J = 8.0 Hz, 2H), 7.02 (d, J = 8.0 Hz, 2H), 5.17 (d, J = 11.0 Hz, 1H), 4.88 (s, 1H), 4.72 (d, J = 11.0 Hz, 1H), 4.14–4.04 (m, 3H), 3.96–3.86 (m, 1H), 2.20 (s, 3H), 1.16 (q, J = 13 7.0 Hz, 6H). C NMR (100 MHz, DMSO-d6) δ 165.2, 161.3, 149.8, 144.0, 143.2, 142.7, 135.5, 128.7, 127.1, 107.1, 89.8, 60.2, 48.6, 36.4, 35.7, 20.6, 13.9, 13.5. HRMS: calcd for + C20H22ClN3NaO4 [M + Na] , 426.1191; found 426.1199.

4.1.1. General Procedure for the Synthesis of Lactam Analogs

O O RNH2 O O NH NH O R N N N O N N O H H Cl The chloromethyl intermediate 2.4 (1 equiv) and the corresponding primary amine (1.1 equiv) were charged into a dry microwave tube (2 mL). After the addition of ethanol (1 mL), the tube was flushed with nitrogen gas and capped. The reaction was heated to 120 °C for 30 sec in the microwave reactor, which gave rise to a dark orange solution. The mixture was subsequently loaded into a cartridge and purified by flash column chromatography (4

84 gram RediSep Gold silica gel column, DCM + 10% methanol) to yield the desired compound.

7-Allyl-1-ethyl-5-(p-tolyl)-5,7,8,9-tetrahydro-1H-pyrrolo[3',4':5,6]pyrido[2,3- d]pyrimidine-2,4,6(3H)-trione (2.5a).

O O NH N N N O H

The title compound was synthesized via the general procedure for the synthesis of lactam analogs using chloromethyl intermediate 2.4 (50 mg, 0.12 mmol) and allylamine (7.8 mg, 0.14 mmol), which yielded a yellow solid (14 mg, 31%); mp 246–247 °C dec; 93% purity 1 determined by UPLC. H NMR (400 MHz, DMSO-d6) δ 10.39 (s, 1H), 8.89 (s, 1H), 7.15 (d, J = 7.9 Hz, 2H), 7.00 (d, J = 7.8 Hz, 2H), 5.78 (ddd, J = 15.3, 9.9, 4.8 Hz, 1H), 5.28 (d, J = 16.6 Hz, 1H), 5.09 (d, J = 16.6 Hz, 1H), 5.04 (d, J = 19.1 Hz, 1H), 4.93 (d, J = 19.1 Hz, 1H), 4.89 (s, 1H), 4.01 (q, J = 6.8 Hz, 2H), 3.86 (s, 2H), 2.20 (s, 3H), 1.09 (t, J = 6.8 Hz, 13 3H). C NMR (100 MHz, DMSO-d6) δ 175.4, 172.8, 162.0, 155.3, 150.9, 143.7, 134.8, 133.6, 128.3, 127.1, 115.9, 93.4, 90.0, 75.1, 43.3, 35.7, 32.3, 20.6, 14.0. HRMS: calcd for + C21H23N4O3 [M + H] , 379.1765; found 379.1771.

7-Benzyl-1-ethyl-5-(p-tolyl)-5,7,8,9-tetrahydro-1H-pyrrolo[3',4':5,6]pyrido[2,3- d]pyrimidine-2,4,6(3H)-trione (2.5b).

O O NH N N N O H

The title compound was synthesized via the general procedure for the synthesis of lactam analogs using chloromethyl intermediate 2.4 (50 mg, 0.12 mmol) and benzylamine (15 mg, 0.14 mmol), which yielded a yellow solid (18 mg, 35%); mp 223–224 °C dec; 97% purity 1 determined by UPLC. H NMR (400 MHz, DMSO-d6) δ 10.40 (s, 1H), 9.18 (s, 1H), 7.26 (d, J = 6.8 Hz, 3H), 7.17 (d, J = 7.9 Hz, 2H), 7.08 (d, J = 6.1 Hz, 2H), 7.02 (d, J = 7.9 Hz, 85 2H), 5.28 (d, J = 16.6 Hz, 1H), 5.12 (d, J = 16.6 Hz, 1H), 4.91 (s, 1H), 4.45 (d, J = 5.7 Hz, 2H), 4.02 (q, J = 6.8, 2H), 2.23 (s, 3H), 1.09 (t, J = 6.8 Hz, 3H). 13C NMR (100 MHz,

DMSO-d6) δ 175.6, 172.8, 162.1, 155.3, 150.9, 143.7, 137.4, 134.9, 128.4, 128.4, 127.3,

127.2, 126.8, 93.5, 90.0, 75.2, 44.5, 35.7, 32.4, 20.6, 14.0. HRMS: calcd for C25H25N4O3 [M + H]+, 429.1921; found 429.1929.

1-Ethyl-7-phenethyl-5-(p-tolyl)-5,7,8,9-tetrahydro-1H-pyrrolo[3',4':5,6]pyrido[2,3- d]pyrimidine-2,4,6(3H)-trione (2.5c).

O O NH N N N O H

The title compound was synthesized via the general procedure for the synthesis of lactam analogs using chloromethyl intermediate 2.4 (50 mg, 0.12 mmol) and phenethylamine (17 mg, 0.14 mmol), which yielded a yellow solid (14 mg, 25%); mp 252–253 °C dec; 93% 1 purity determined by UPLC. H NMR (400 MHz, DMSO-d6) δ 10.37 (s, 1H), 8.76 (s, 1H), 7.17 (s, 3H), 7.12 (d, J = 7.8 Hz, 2H), 7.01 (d, J = 7.8 Hz, 4H), 5.13 (d, J = 16.5 Hz, 1H), 5.00 (d, J = 16.5 Hz, 1H), 4.85 (s, 1H), 4.00 (dd, J = 14.6, 7.5 Hz, 2H), 3.46 (dd, J = 12.2, 6.1 Hz, 2H), 2.77–2.68 (m, 2H), 2.23 (s, 3H), 1.08 (t, J = 6.8 Hz, 3H). 13C NMR (100 MHz,

DMSO-d6) δ 175.1, 172.8, 162.1, 155.4, 150.9, 143.7, 138.3, 134.8, 128.7, 128.4, 128.2, 127.1, 126.2, 93.4, 89.9, 75.0, 43.0, 35.7, 35.2, 32.3, 20.6, 14.0. HRMS: calcd for + C26H27N4O3 [M + H] , 443.2078; found 443.2068.

86 1-Ethyl-7-(3-phenylpropyl)-5-(p-tolyl)-5,7,8,9-tetrahydro-1H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidine-2,4,6(3H)-trione (2.5d).

O O NH N N N O H

The title compound was synthesized via the general procedure for the synthesis of lactam analogs using chloromethyl intermediate 2.4 (50 mg, 0.12 mmol) and 3- phenylpropylamine (19 mg, 0.14 mmol), which yielded a yellow solid (15 mg, 28%); mp 1 244–245 °C dec; 98% purity determined by qNMR. H NMR (400 MHz, DMSO-d6) δ 10.38 (s, 1H), 8.68 (t, J = 6.0 Hz, 1H), 7.25 (t, J = 7.4 Hz, 2H), 7.17 (d, J = 7.5 Hz, 3H), 7.06 (d, J = 7.5 Hz, 2H), 7.01 (d, J = 7.8 Hz, 2H), 5.26 (d, J = 16.4 Hz, 1H), 5.09 (d, J = 16.5 Hz, 1H), 4.90 (s, 1H), 4.02 (q, J = 6.9 Hz, 2H), 3.23 (dd, J = 8.0, 4.7 Hz, 2H), 2.40 (t, J = 7.8 Hz, 2H), 2.19 (s, 3H), 1.73 (q, J = 7.4 Hz, 2H), 1.09 (t, J = 6.9 Hz, 3H). 13C NMR

(100 MHz, DMSO-d6) δ 175.1, 172.8, 162.1, 155.4, 150.9, 143.7, 141.0, 134.8, 128.3, 128.23, 128.17, 127.1, 125.8, 93.5, 89.8, 75.1, 40.8, 35.7, 32.4, 31.8, 30.6, 20.6, 14.0.

1-Ethyl-7-(2-phenoxyethyl)-5-(p-tolyl)-5,7,8,9-tetrahydro-1H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidine-2,4,6(3H)-trione (2.5e).

O O NH N O N N O H

The title compound was synthesized via the general procedure for the synthesis of lactam analogs using chloromethyl intermediate 2.4 (50 mg, 0.12 mmol) and 2- phenoxyethylamine (19 mg, 0.14 mmol), which yielded a yellow solid (15 mg, 27%); mp 1 248–249 °C dec; 90% purity determined by qNMR. H NMR (400 MHz, DMSO-d6) δ 10.40 (s, 1H), 8.91 (d, J = 6.2 Hz, 1H), 7.27 (dd, J = 8.5, 7.2 Hz, 2H), 7.13 (d, J = 7.7 Hz, 2H), 6.94 (d, J = 7.3 Hz, 3H), 6.86 (d, J = 8.1 Hz, 2H), 5.29 (d, J = 16.5 Hz, 1H), 5.10 (d, J = 16.5 Hz, 1H), 4.92 (s, 1H), 4.09–3.96 (m, 4H), 3.70–3.53 (m, 2H), 2.17 (s, 3H), 1.09 87 13 (t, J = 6.9 Hz, 3H). C NMR (100 MHz, DMSO-d6) δ 175.5, 173.1, 162.1, 158.0, 155.3, 150.9, 143.6, 134.7, 129.4, 128.4, 127.0, 120.8, 114.5, 93.4, 89.9, 75.1, 65.7, 41.1, 35.7, 32.1, 20.5, 14.0.

1-Ethyl-7-(1-methylpiperidin-4-yl)-5-(p-tolyl)-5,7,8,9-tetrahydro-1H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidine-2,4,6(3H)-trione (2.5f).

O O NH N N N N O H

The title compound was synthesized via the general procedure for the synthesis of lactam analogs using chloromethyl intermediate 2.4 (50 mg, 0.12 mmol) and 4-amino-1- methylpiperidine (16 mg, 0.14 mmol), which yielded a yellow solid (5.2 mg, 10%); mp 1 248–249 °C dec; 89% purity determined by qNMR. H NMR (400 MHz, DMSO-d6) δ 10.42 (s, 1H), 8.77 (s, 1H), 7.16 (d, J = 7.7 Hz, 2H), 7.01 (d, J = 7.8 Hz, 2H), 5.27 (d, J = 16.6 Hz, 1H), 5.09 (d, J = 16.5 Hz, 1H), 4.99 (s, 1H), 4.02 (q, J = 7.3 Hz, 2H), 3.55 (d, J = 33.2 Hz, 1H), 2.94–2.71 (m, 2H), 2.27 (s, 3H), 2.21 (s, 3H), 1.93–1.78 (m, 1H), 1.66 (d, J = 29.1 Hz, 3H), 1.34–1.19 (m, 2H), 1.10 (t, J = 6.9 Hz, 3H).

7-(1-Benzylpiperidin-4-yl)-1-ethyl-5-(p-tolyl)-5,7,8,9-tetrahydro-1H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidine-2,4,6(3H)-trione (2.5g).

O O NH N N N N O H

The title compound was synthesized via the general procedure for the synthesis of lactam analogs using chloromethyl intermediate 2.4 (50 mg, 0.12 mmol) and 4-amino-1- benzylpiperidine (27 mg, 0.14 mmol), which yielded a yellow solid (6.8 mg, 11%); mp 1 231–232 °C dec; 93% purity determined by qNMR. H NMR (400 MHz, DMSO-d6) δ 10.41 (s, 1H), 8.60 (s, 1H), 7.35–7.30 (m, 2H), 7.30–7.22 (m, 3H), 7.15 (d, J = 8.1 Hz, 2H), 7.01 (d, J = 7.7 Hz, 2H), 5.26 (d, J = 16.5 Hz, 1H), 5.08 (d, J = 16.4 Hz, 1H), 4.99 (s, 1H), 88 4.02 (q, J = 7.7, 7.0 Hz, 2H), 3.55 (s, 1H), 3.43 (s, 2H), 2.84–2.59 (m, 2H), 2.21 (s, 3H), 2.09–1.89 (m, 2H), 1.80 (s, 1H), 1.69–1.41 (m, 3H), 1.10 (t, J = 6.9 Hz, 3H).

7-(1-Benzoylpiperidin-4-yl)-1-ethyl-5-(p-tolyl)-5,7,8,9-tetrahydro-1H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidine-2,4,6(3H)-trione (2.5h).

O O NH N N O N N O H

The title compound was synthesized via the general procedure for the synthesis of lactam analogs using chloromethyl intermediate 2.4 (50 mg, 0.12 mmol) and (4-aminopiperidin- 1-yl)(phenyl)methanone (29 mg, 0.14 mmol), which yielded a yellow solid (20 mg, 32%); 1 mp 226–227 °C dec; 95% purity determined by qNMR. H NMR (400 MHz, DMSO-d6) δ 10.39 (s, 1H), 8.64 (s, 1H), 7.47–7.41 (m, 3H), 7.31 (dd, J = 6.5, 2.9 Hz, 2H), 7.14 (d, J = 7.8 Hz, 2H), 6.98 (d, J = 7.8 Hz, 2H), 5.26 (d, J = 16.5 Hz, 1H), 5.10 (d, J = 16.5 Hz, 1H), 4.89 (s, 1H), 4.39 (s, 1H), 4.06–3.94 (m, 2H), 3.47 (s, 1H), 3.15 (dq, J = 24.5, 6.9 Hz, 1H), 3.04–2.56 (m, 2H), 2.15 (s, 3H), 1.67 (s, 1H), 1.58–1.25 (m, 1H), 1.09 (t, J = 6.9 Hz, 3H), 1.01–0.93 (m, 2H).

7-((1-Benzylpiperidin-4-yl)methyl)-1-ethyl-5-(p-tolyl)-5,7,8,9-tetrahydro-1H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidine-2,4,6(3H)-trione (2.5i).

N O O NH N N N O H

The title compound was synthesized via the general procedure for the synthesis of lactam analogs using chloromethyl intermediate (50 mg, 0.12 mmol) and (1-benzylpiperidin-4- yl)methanamine (29 mg, 0.14 mmol), which gave rise to a yellow solid (13 mg, 20%); mp 1 216–217 °C dec; 89% purity determined by qNMR. H NMR (400 MHz, DMSO-d6) δ 10.39 (s, 1H), 8.63 (s, 1H), 7.36–7.27 (m, 2H), 7.29–7.23 (m, 3H), 7.14 (d, J = 8.1 Hz, 2H), 6.99 (d, J = 7.8 Hz, 2H), 5.26 (d, J = 16.4 Hz, 1H), 5.10 (d, J = 16.5 Hz, 1H), 4.90 (s, 1H), 89 4.07–3.96 (m, 2H), 3.40 (s, 2H), 3.11 (s, 2H), 2.71 (d, J = 10.9 Hz, 2H), 2.19 (s, 3H), 1.79 (t, J = 11.3 Hz, 2H), 1.37 (s, 3H), 1.16–0.97 (m, 5H).

1-Ethyl-7-((1-(4-nitrobenzyl)piperidin-4-yl)methyl)-5-(p-tolyl)-5,7,8,9-tetrahydro- 1H-pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidine-2,4,6(3H)-trione (2.5j).

O2N N O O NH N N N O H

The title compound was synthesized via the general procedure for the synthesis of lactam analogs using chloromethyl intermediate 2.4 (50 mg, 0.12 mmol) and (1-(4- nitrobenzyl)piperidin-4-yl)methanamine (35 mg, 0.14 mmol), which yielded a yellow solid (7.5 mg, 11%); mp 167–168 °C dec; 95% purity determined by qNMR. 1H NMR (400

MHz, DMSO-d6) δ 10.38 (s, 1H), 8.63 (t, J = 6.2 Hz, 1H), 8.18 (d, J = 8.4 Hz, 2H), 7.55 (d, J = 8.4 Hz, 2H), 7.13 (d, J = 7.9 Hz, 2H), 6.98 (d, J = 7.8 Hz, 2H), 5.25 (d, J = 16.5 Hz, 1H), 5.09 (d, J = 16.5 Hz, 1H), 4.89 (s, 1H), 4.01 (q, J = 6.9 Hz, 2H), 3.54 (s, 2H), 3.11 (q, J = 6.9 Hz, 2H), 2.68 (s, 2H), 2.17 (s, 3H), 1.85 (t, J = 11.2 Hz, 2H), 1.45–1.33 (m, 3H), 1.14–0.99 (m, 5H).

1-Ethyl-7-(4-methylphenethyl)-5-(p-tolyl)-5,7,8,9-tetrahydro-1H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidine-2,4,6(3H)-trione (2.5k).

O O NH N N N O H

The title compound was synthesized via the general procedure for the synthesis of lactam analogs using chloromethyl intermediate 2.4 (50 mg, 0.12 mmol) and 2-(p- tolyl)ethylamine (19 mg, 0.14 mmol), which yielded a yellow solid (17 mg, 31%); mp 189– 1 190 °C dec; 99% purity determined by qNMR. H NMR (400 MHz, DMSO-d6) δ 10.37 (s, 1H), 8.74 (t, J = 5.6 Hz, 1H), 7.12 (d, J = 7.8 Hz, 2H), 7.03–6.96 (m, 4H), 6.90 (d, J = 7.7

90 Hz, 2H), 5.17 (d, J = 16.5 Hz, 1H), 5.01 (d, J = 16.5 Hz, 1H), 4.86 (s, 1H), 4.01 (q, J = 6.9 Hz, 2H), 3.41–3.36 (m, 2H), 2.75–2.61 (m, 2H), 2.23 (s, 6H), 1.09 (t, J = 6.9 Hz, 3H).

7-(4-Chlorophenethyl)-1-ethyl-5-(p-tolyl)-5,7,8,9-tetrahydro-1H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidine-2,4,6(3H)-trione (2.5l).

O O NH N Cl N N O H

The title compound was synthesized via the general procedure for the synthesis of the lactam analogs using chloromethyl intermediate 2.4 (50 mg, 0.12 mmol) and 2-(4- chlorophenyl)ethylamine (22 mg, 0.14 mmol), which yielded a yellow solid (8.6 mg, 15%); 1 mp 159–161 °C dec; 98% purity determined by qNMR. H NMR (400 MHz, DMSO-d6) δ 10.38 (s, 1H), 8.70 (s, 1H), 7.20 (d, J = 8.0 Hz, 2H), 7.10 (d, J = 7.8 Hz, 2H), 7.04–6.98 (m, 4H), 5.16 (d, J = 16.5 Hz, 1H), 5.01 (d, J = 16.4 Hz, 1H), 4.84 (s, 1H), 4.00 (q, J = 6.9 Hz, 2H), 3.54–3.40 (m, 2H), 2.81–2.65 (m, 2H), 2.24 (s, 3H), 1.08 (t, J = 6.9 Hz, 3H). 13C

NMR (100 MHz, DMSO-d6) δ 175.1, 172.8, 162.0, 155.3, 150.9, 143.6, 137.3, 134.8, 130.8, 130.6, 128.3, 128.1, 127.1, 93.4, 89.9, 75.1, 42.7, 35.7, 34.2, 32.3, 20.6, 14.0.

1-Ethyl-7-(4-hydroxyphenethyl)-5-(p-tolyl)-5,7,8,9-tetrahydro-1H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidine-2,4,6(3H)-trione (2.5m).113

O O NH N HO N N O H

The title compound was synthesized via the general procedure for the synthesis of lactam analogs using chloromethyl intermediate 2.4 (50 mg, 0.12 mmol) and tyramine (19 mg, 0.14 mmol), which yielded a yellow solid (12 mg, 22%); mp 261–262 °C dec; 92% purity 1 determined by UPLC. H NMR (400 MHz, DMSO-d6) δ 10.37 (s, 1H), 9.16 (s, 1H), 8.73 (s, 1H), 7.12 (d, J = 7.9 Hz, 2H), 7.01 (d, J = 7.9 Hz, 2H), 6.79 (d, J = 8.2 Hz, 2H), 6.57 (d, J = 8.2 Hz, 2H), 5.15 (d, J = 16.4 Hz, 1H), 5.01 (d, J = 16.4 Hz, 1H), 4.86 (s, 1H), 4.00 91 (q, J = 6.8 Hz, 2H), 3.41–3.33 (m, 2H), 2.62–2.49 (m, 2H), 2.23 (s, 3H), 1.08 (t, J = 6.8 13 Hz, 3H). C NMR (100 MHz, DMSO-d6) δ 175.0, 172.8, 162.0, 155.7, 155.4, 150.9, 143.7, 134.8, 129.6, 128.3, 127.1, 115.0, 93.4, 89.8, 75.0, 43.4, 38.9, 35.6, 34.4, 32.3, 20.6, 14.0. + HRMS: calcd for C26H27N4O4 [M + H] , 459.2027; found 459.2038.

1-Ethyl-7-(4-methoxyphenethyl)-5-(p-tolyl)-5,7,8,9-tetrahydro-1H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidine-2,4,6(3H)-trione (2.5n).113

O O NH N O N N O H

The title compound was synthesized via the general procedure for the synthesis of lactam analogs using chloromethyl intermediate 2.4 (50 mg, 0.12 mmol) and 2-(4- methoxyphenyl)ethylamine (21 mg, 0.14 mmol), which yielded a yellow solid (12 mg, 22%); mp 222–223 °C dec; 96% purity determined by UPLC. 1H NMR (400 MHz, DMSO- d6) δ 10.37 (s, 1H), 8.73 (s, 1H), 7.12 (d, J = 7.9 Hz, 2H), 7.02 (d, J = 7.9 Hz, 2H), 6.92 (d, J = 8.4 Hz, 2H), 6.73 (d, J = 8.4 Hz, 2H), 5.17 (d, J = 16.5 Hz, 1H), 5.05–4.98 (d, J = 16.5 Hz, 1H), 4.85 (s, 1H), 4.00 (q, J = 6.7 Hz, 2H), 3.70 (s, 3H), 3.41 (dd, J = 13.6, 6.8 Hz, 2H), 2.70–2.62 (m, 2H), 2.23 (s, 3H), 1.08 (t, J = 6.7 Hz, 3H). 13C NMR (100 MHz, DMSO- d6) δ 175.0, 172.8, 162.0, 157.7, 155.4, 150.9, 143.7, 134.8, 130.2, 129.7, 128.4, 127.1, 113.6, 93.4, 89.8, 75.1, 54.9, 43.2, 35.7, 34.2, 32.3, 20.6, 14.0. HRMS: calcd for + C27H29N4O4 [M + H] , 473.2183; found 473.2180.

92 1-Ethyl-7-(3-methoxyphenethyl)-5-(p-tolyl)-5,7,8,9-tetrahydro-1H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidine-2,4,6(3H)-trione (2.5o).

O O O NH N N N O H

The title compound was synthesized via the general procedure for the synthesis of lactam analogs using chloromethyl intermediate 2.4 (50 mg, 0.12 mmol) and 2-(3- methoxyphenyl)ethylamine (21 mg, 0.14 mmol), which yielded a yellow solid (15 mg, 26%); mp 214–215 °C dec; 92% purity determined by qNMR. 1H NMR (400 MHz,

DMSO-d6) δ 10.38 (s, 1H), 8.77 (t, J = 5.8 Hz, 1H), 7.11 (d, J = 7.6 Hz, 3H), 6.99 (d, J = 7.8 Hz, 2H), 6.75 (dd, J = 11.3, 3.2 Hz, 2H), 6.58 (d, J = 7.5 Hz, 1H), 5.19 (d, J = 16.5 Hz, 1H), 5.01 (d, J = 16.4 Hz, 1H), 4.87 (s, 1H), 4.01 (q, J = 6.9 Hz, 2H), 3.69 (s, 3H), 3.52– 3.38 (m, 2H), 2.73 (t, J = 7.3 Hz, 2H), 2.21 (s, 3H), 1.08 (t, J = 6.9 Hz, 3H). 13C NMR (100

MHz, DMSO-d6) δ 175.1, 172.8, 162.0, 159.2, 155.4, 150.9, 143.6, 139.8, 134.8, 129.3, 128.4, 127.0, 120.9, 114.3, 111.7, 93.4, 89.8, 75.1, 54.8, 42.9, 35.7, 35.1, 32.2, 20.6, 14.0.

1-Ethyl-7-(2-methoxyphenethyl)-5-(p-tolyl)-5,7,8,9-tetrahydro-1H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidine-2,4,6(3H)-trione (2.5p).

O O O NH N N N O H The title compound was synthesized via the general procedure for the synthesis of lactam analogs using chloromethyl intermediate 2.4 (50 mg, 0.12 mmol) and 2-(2- methoxyphenyl)ethylamine (21 mg, 0.14 mmol), which yielded a yellow solid (20 mg, 36%); mp 254–255 °C dec; 93% purity determined by qNMR. 1H NMR (400 MHz,

DMSO-d6) δ 10.37 (s, 1H), 8.77 (t, J = 6.0 Hz, 1H), 7.18 (t, J = 7.9 Hz, 1H), 7.12 (d, J = 7.8 Hz, 2H), 7.00 (d, J = 7.8 Hz, 2H), 6.92 (d, J = 8.2 Hz, 1H), 6.85 (d, J = 7.3 Hz, 1H), 6.74 (t, J = 7.4 Hz, 1H), 5.14 (d, J = 16.5 Hz, 1H), 4.98 (d, J = 16.5 Hz, 1H), 4.84 (s, 1H), 4.00 (q, J = 6.9 Hz, 2H), 3.74 (s, 3H), 3.41 (q, J = 6.7 Hz, 2H), 2.79–2.66 (m, 2H), 2.22 (s, 93 13 3H), 1.08 (t, J = 6.9 Hz, 3H). C NMR (100 MHz, DMSO-d6) δ 175.0, 172.9, 162.0, 157.1, 155.4, 150.9, 143.7, 134.8, 130.2, 128.3, 127.8, 127.1, 125.9, 120.2, 110.5, 93.4, 89.8, 75.0, 55.2, 41.4, 35.6, 32.3, 30.2, 20.6, 14.0.

7-(3,4-Dimethoxyphenethyl)-1-ethyl-5-(p-tolyl)-5,7,8,9-tetrahydro-1H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidine-2,4,6(3H)-trione (2.5q).

O O NH N O N N O H O The title compound was synthesized via the general procedure for the synthesis of lactam analogs using chloromethyl intermediate 2.4 (50 mg, 0.12 mmol) and 3,4- dimethoxyphenethylamine (25 mg, 0.14 mmol), which yielded a yellow solid (10 mg, 16%); 1 mp 161–162 °C dec; 96% purity determined by qNMR. H NMR (400 MHz, DMSO-d6) δ 10.38 (s, 1H), 8.74 (t, J = 5.8 Hz, 1H), 7.11 (d, J = 7.8 Hz, 2H), 6.99 (d, J = 7.8 Hz, 2H), 6.77–6.70 (m, 2H), 6.53 (d, J = 8.1 Hz, 1H), 5.22 (d, J = 16.5 Hz, 1H), 5.02 (d, J = 16.5 Hz, 1H), 4.88 (s, 1H), 4.01 (q, J = 6.7 Hz, 2H), 3.69 (d, J = 8.6 Hz, 6H), 3.44 (q, J = 6.7 Hz, 2H), 2.69 (t, J = 7.1 Hz, 2H), 2.21 (s, 3H), 1.09 (t, J = 6.9 Hz, 3H). 13C NMR (100

MHz, DMSO-d6) δ 175.1, 172.8, 162.0, 155.4, 150.9, 148.5, 147.3, 143.7, 134.8, 130.7, 128.3, 127.0, 120.6, 112.5, 111.7, 93.4, 89.8, 75.1, 55.4, 55.3, 43.1, 35.6, 34.6, 32.2, 20.6, 14.0.

7-(2,3-Dimethoxyphenethyl)-1-ethyl-5-(p-tolyl)-5,7,8,9-tetrahydro-1H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidine-2,4,6(3H)-trione (2.5r).

O O O O NH N N N O H

The title compound was synthesized via the general procedure for the synthesis of lactam analogs using chloromethyl intermediate 2.4 (50 mg, 0.12 mmol) and 2,3- dimethoxyphenethylamine (25 mg, 0.14 mmol), which yielded a yellow solid (11 mg, 18%); 94 1 mp 255–256 °C dec; 97% purity determined by qNMR. H NMR (400 MHz, DMSO-d6) δ 10.37 (s, 1H), 8.79 (s, 1H), 7.11 (d, J = 7.8 Hz, 2H), 7.00 (d, J = 7.8 Hz, 2H), 6.87 (q, J = 8.4 Hz, 2H), 6.50 (d, J = 7.0 Hz, 1H), 5.18 (d, J = 16.5 Hz, 1H), 5.02 (d, J = 16.4 Hz, 1H), 4.84 (s, 1H), 4.01 (q, J = 6.9 Hz, 2H), 3.77 (s, 3H), 3.67 (s, 3H), 3.46–3.36 (m, 2H), 2.79– 13 2.64 (m, 2H), 2.22 (s, 3H), 1.09 (t, J = 6.9 Hz, 3H). C NMR (100 MHz, DMSO-d6) δ 175.1, 172.8, 162.0, 152.3, 151.0, 150.9, 146.7, 143.6, 134.8, 131.4, 128.3, 127.1, 123.7, 122.0, 111.4, 89.8, 75.1, 60.1, 55.5, 54.9, 42.10, 35.6, 32.3, 29.9, 20.6, 14.0.

7-(3,4-Dichlorophenethyl)-1-ethyl-5-(p-tolyl)-5,7,8,9-tetrahydro-1H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidine-2,4,6(3H)-trione (2.5s).

O O NH N Cl N N O H Cl The title compound was synthesized via the general procedure for the synthesis of lactam analogs using chloromethyl intermediate 2.4 (50 mg, 0.12 mmol) and 3,4- dichlorophenethylamine (27 mg, 0.14 mmol), which yielded a yellow solid (7.9 mg, 13%); 1 mp 189–190 °C dec; 97% purity determined by qNMR. H NMR (400 MHz, DMSO-d6) δ 10.39 (s, 1H), 8.68 (d, J = 6.7 Hz, 1H), 7.44 (s, 1H), 7.37 (d, J = 8.2 Hz, 1H), 7.08 (d, J = 7.8 Hz, 2H), 6.97 (dd, J = 11.6, 4.8 Hz, 3H), 5.19 (d, J = 16.5 Hz, 1H), 5.02 (d, J = 16.5 Hz, 1H), 4.85 (s, 1H), 4.00 (q, J = 6.9 Hz, 2H), 3.50 (h, J = 7.0 Hz, 2H), 2.76 (p, J = 7.1, 13 6.6 Hz, 2H), 2.22 (s, 3H), 1.08 (t, J = 6.9 Hz, 3H). C NMR (100 MHz, DMSO-d6) δ 175.2, 172.8, 162.0, 155.3, 150.9, 143.5, 139.6, 134.8, 130.8, 130.7, 130.2, 129.2, 128.9, 128.3, 127.0, 93.3, 89.8, 75.1, 42.3, 35.7, 33.8, 32.2, 20.6, 14.0.

95 Ethyl 2-(4-(2-(1Ethyl-2,4,6-trioxo-5-(p-tolyl)-1,2,3,4,5,6,8,9-octahydro-7H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidin-7-yl)ethyl)phenoxy)acetate (2.5t ethyl ester).

O O O NH N O O N N O H

The title compound was synthesized via the general procedure for the synthesis of the lactam analogs using chloromethyl intermediate 2.4 (50 mg, 0.12 mmol) and ethyl 2-(4-(2- aminoethyl)phenoxy)acetate (31 mg, 0.14 mmol), which yielded a yellow solid (25 mg, 1 37%). H NMR (400 MHz, DMSO-d6) δ 10.38 (s, 1H), 8.75 (s, 1H), 7.14 (d, J = 7.8 Hz, 2H), 7.04 (d, J = 7.8 Hz, 2H), 6.90 (d, J = 8.1 Hz, 2H), 6.72 (d, J = 8.2 Hz, 2H), 5.12 (d, J = 16.5 Hz, 1H), 5.00 (d, J = 16.5 Hz, 1H), 4.86 (s, 1H), 4.73 (s, 2H), 4.17 (q, J = 7.1 Hz, 2H), 4.02 (q, J = 7.1 Hz, 2H), 3.43 (d, J = 8.0 Hz, 2H), 2.74–2.63 (m, 2H), 2.25 (s, 3H), 1.22 (t, J = 7.1 Hz, 3H), 1.10 (t, J = 6.9 Hz, 3H).

Methyl 4-((4-((1-Ethyl-2,4,6-trioxo-5-(p-tolyl)-1,2,3,4,5,6,8,9-octahydro-7H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidin-7-yl)methyl)piperidin-1-yl)methyl)benzoate (2.5u methyl ester).

O N O O NH N N N O H

The title compound was synthesized via the general procedure for the synthesis of the lactam analogs using chloromethyl intermediate 2.4 (100 mg, 0.24 mmol) and methyl 4- ((4-(aminomethyl)piperidin-1-yl)methyl)benzoate (73 mg, 0.28 mmol), which yielded a 1 yellow solid (57 mg, 41%). H NMR (400 MHz, DMSO-d6) δ 10.39 (s, 1H), 8.64 (s, 1H), 7.92 (d, J = 7.9 Hz, 2H), 7.43 (d, J = 8.0 Hz, 2H), 7.14 (d, J = 7.7 Hz, 2H), 6.99 (d, J = 7.7 Hz, 2H), 5.24 (s, 1H), 5.10 (s, 1H), 4.90 (s, 1H), 4.02 (d, J = 7.7 Hz, 2H), 3.85 (s, 3H), 3.49 (s, 2H), 3.11 (s, 2H), 2.72 (s, 2H), 2.18 (s, 3H), 1.83 (t, J = 11.5 Hz, 2H), 1.39 (s, 3H), 1.10 (t, J = 6.9 Hz, 5H).

96 Methyl 4-(2-(1-Ethyl-2,4,6-trioxo-5-(p-tolyl)-1,2,3,4,5,6,8,9-octahydro-7H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidin-7-yl)ethyl)benzoate (2.5v methyl ester).

O O NH O N N N O H O The title compound was synthesized via the general procedure for the synthesis of the lactam analogs using chloromethyl intermediate 2.4 (50 mg, 0.12 mmol) and methyl 4-(2- aminoethyl)benzoate (25 mg, 0.14 mmol), which yielded a yellow solid (42 mg, 69%). 1H

NMR (400 MHz, DMSO-d6) δ 10.39 (s, 1H), 8.74 (s, 1H), 7.76 (d, J = 7.9 Hz, 2H), 7.12 (dd, J = 11.0, 7.9 Hz, 4H), 7.01 (d, J = 7.8 Hz, 2H), 5.17 (d, J = 16.5 Hz, 1H), 5.02 (d, J = 16.5 Hz, 1H), 4.85 (s, 1H), 4.01 (q, J = 6.8 Hz, 2H), 3.85 (s, 3H), 3.51 (dp, J = 27.8, 6.8 Hz, 2H), 2.82 (dq, J = 13.7, 7.3, 6.7 Hz, 2H), 2.26 (s, 3H), 1.10 (t, J = 6.9 Hz, 3H).

Methyl 4-((1-Ethyl-2,4,6-trioxo-5-(p-tolyl)-1,2,3,4,5,6,8,9-octahydro-7H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidin-7-yl)methyl)benzoate (2.5w methyl ester).

O O

O O NH N N N O H

The title compound was synthesized via the general procedure for the synthesis of the lactam analogs using chloromethyl intermediate 2.4 (100 mg, 0.24 mmol) and methyl 4- (aminomethyl)benzoate (46 mg, 0.28 mmol), which yielded a yellow solid (41 mg, 42%). 1 1 H NMR (400 MHz, DMSO-d6) H NMR (400 MHz, DMSO-d6) δ 10.44 (s, 1H), 9.21 (s, 1H), 7.87 (d, J = 8.0 Hz, 2H), 7.20 (t, J = 8.0 Hz, 4H), 7.05 (d, J = 7.7 Hz, 2H), 5.29 (d, J = 16.6 Hz, 1H), 5.13 (d, J = 16.6 Hz, 1H), 4.93 (s, 1H), 4.55 (d, J = 5.9 Hz, 2H), 4.03 (q, J = 6.8 Hz, 2H), 3.85 (s, 3H), 2.25 (s, 3H), 1.11 (t, J = 6.9 Hz, 3H).

97 Methyl 3-((1-Ethyl-2,4,6-trioxo-5-(p-tolyl)-1,2,3,4,5,6,8,9-octahydro-7H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidin-7-yl)methyl)benzoate (2.5x methyl ester).

O O O O NH N N N O H

The title compound was synthesized via the general procedure for the synthesis of the lactam analogs using chloromethyl intermediate 2.4 (100 mg, 0.24 mmol) and methyl 3- (aminomethyl)benzoate (46 mg, 0.28 mmol), which yielded a yellow solid (44 mg, 45%). 1 H NMR (400 MHz, DMSO-d6) δ 10.44 (s, 1H), 9.25 (s, 1H), 7.86 (d, J = 8.1 Hz, 2H), 7.45 (t, J = 7.7 Hz, 1H), 7.37 (d, J = 7.7 Hz, 1H), 7.17 (d, J = 7.7 Hz, 2H), 7.02 (d, J = 7.6 Hz, 2H), 5.30 (d, J = 16.6 Hz, 1H), 5.12 (d, J = 16.7 Hz, 1H), 4.93 (s, 1H), 4.54 (s, 2H), 4.03 (d, J = 7.3 Hz, 2H), 3.87 (s, 3H), 2.23 (s, 3H), 1.10 (t, J = 6.9 Hz, 3H).

4.1.2. General Procedure for the Synthesis of Lactam Analogs with Carboxylate Groups

The methyl or ethyl ester precursor (1 equiv) was dissolved in THF/H2O (1:1). After the addition of lithium hydroxide monohydrate (7 equiv), the mixture was stirred at rt overnight. Upon the consumption of the starting material monitored by TLC, the mixture was extracted with ethyl acetate and the aqueous layer was kept and acidified with a 1M HCl solution. The precipitated solid was collected via filtration and dried under high vacuum to yield the target carboxylic acid molecule.

98 2-(4-(2-(1-Ethyl-2,4,6-trioxo-5-(p-tolyl)-1,2,3,4,5,6,8,9-octahydro-7H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidin-7-yl)ethyl)phenoxy)acetic Acid (2.5t).

O O HO NH N O O N N O H

The title compound was synthesized via the general procedure for the synthesis of lactam analogs with carboxylate groups using ethyl 2-(4-(2-(1-ethyl-2,4,6-trioxo-5-(p-tolyl)- 1,2,3,4,5,6,8,9-octahydro-7H-pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidin-7- yl)ethyl)phenoxy)acetate (25 mg, 0.044 mmol), which yielded a yellow solid (10 mg, 43%); 1 mp 281–282 °C dec, 98% purity determined by qNMR. H NMR (400 MHz, DMSO-d6) δ 13.02 (s, 1H), 10.38 (s, 1H), 8.77 (t, J = 5.7 Hz, 1H), 7.14 (d, J = 7.8 Hz, 2H), 7.04 (d, J = 7.8 Hz, 2H), 6.90 (d, J = 8.1 Hz, 2H), 6.71 (d, J = 8.1 Hz, 2H), 5.17 (d, J = 16.5 Hz, 1H), 5.02 (d, J = 16.5 Hz, 1H), 4.87 (s, 1H), 4.60 (s, 2H), 4.02 (q, J = 7.0, 6.6 Hz, 2H), 3.50– 3.33 (m, 2H), 2.65 (dd, J = 13.1, 6.5 Hz, 2H), 2.25 (s, 3H), 1.10 (t, J = 6.9 Hz, 3H).

4-((4-((1-Ethyl-2,4,6-trioxo-5-(p-tolyl)-1,2,3,4,5,6,8,9-octahydro-7H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidin-7-yl)methyl)piperidin-1-ium-1- yl)methyl)benzoate (2.5u).

O NH O O NH N N N O H

The title compound was synthesized via the general procedure for the synthesis of lactam analogs with carboxylate groups using methyl 4-((4-((1-ethyl-2,4,6-trioxo-5-(p-tolyl)- 1,2,3,4,5,6,8,9-octahydro-7H-pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidin-7- yl)methyl)piperidin-1-yl)methyl)benzoate (57 mg, 0.098 mmol), which yielded a yellow solid (25 mg, 45%); mp 305–306 °C dec. 90% purity determined by UPLC.1H NMR (400

MHz, DMSO-d6) δ 13.21 (bs, 1H), 10.39 (s, 1H), 8.69 (s, 1H), 7.87 (d, J = 7.8 Hz, 2H), 7.33 (d, J = 7.8 Hz, 2H), 7.15 (d, J = 7.7 Hz, 2H), 6.99 (d, J = 7.7 Hz, 2H), 5.25 (d, J = 16.5 Hz, 1H), 5.10 (d, J = 16.4 Hz, 1H), 4.91 (s, 1H), 4.02 (q, J = 6.9 Hz, 2H), 3.46 (s, 2H), 99 3.12 (m, 2H), 2.71 (d, J = 11.3 Hz, 2H), 2.19 (s, 3H), 1.81 (t, J = 11.3 Hz, 2H), 1.45–1.30 (m, 3H), 1.10 (m, 5H).

4-(2-(1-Ethyl-2,4,6-trioxo-5-(p-tolyl)-1,2,3,4,5,6,8,9-octahydro-7H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidin-7-yl)ethyl)benzoic Acid (2.5v).

O O NH HO N N N O H O The title compound was synthesized via the general procedure for the synthesis of lactam analogs with carboxylate groups using methyl 4-(2-(1-ethyl-2,4,6-trioxo-5-(p-tolyl)- 1,2,3,4,5,6,8,9-octahydro-7H-pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidin-7- yl)ethyl)benzoate (42 mg, 0.084 mmol), which gave rise to a yellow solid (12 mg, 29%); 1 mp 316–317 °C dec; 98% purity determined by qNMR. H NMR (400 MHz, DMSO-d6) δ 12.83 (s, 1H), 10.39 (s, 1H), 8.75 (d, J = 5.9 Hz, 1H), 7.75 (d, J = 7.8 Hz, 2H), 7.11 (d, J = 7.8 Hz, 4H), 7.01 (d, J = 7.8 Hz, 2H), 5.17 (d, J = 16.5 Hz, 1H), 5.02 (d, J = 16.5 Hz, 1H), 4.85 (s, 1H), 4.01 (q, J = 6.9 Hz, 2H), 3.53 (ddq, J = 34.1, 19.6, 6.5 Hz, 2H), 2.82 (hept, J = 7.1 Hz, 2H), 2.25 (s, 3H), 1.09 (t, J = 6.9 Hz, 3H).

4-((1-Ethyl-2,4,6-trioxo-5-(p-tolyl)-1,2,3,4,5,6,8,9-octahydro-7H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidin-7-yl)methyl)benzoic Acid (2.5w).

O HO

O O NH N N N O H

100 12.92 (s, 1H), 10.44 (s, 1H), 9.21 (s, 1H), 7.84 (d, J = 8.0 Hz, 2H), 7.18 (dd, J = 7.9, 5.4 Hz, 4H), 7.04 (d, J = 7.7 Hz, 2H), 5.29 (d, J = 16.7 Hz, 1H), 5.13 (d, J = 16.6 Hz, 1H), 4.93 (s, 1H), 4.58–4.48 (m, 2H), 4.03 (d, J = 7.2 Hz, 2H), 2.25 (s, 3H), 1.10 (t, J = 6.9 Hz, 3H).

3-((1-Ethyl-2,4,6-trioxo-5-(p-tolyl)-1,2,3,4,5,6,8,9-octahydro-7H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidin-7-yl)methyl)benzoic Acid (2.5x).

O O O HO NH N N N O H

The title compound was synthesized via the general procedure for the synthesis of lactam analogs with carboxylate groups using methyl 4-((1-ethyl-2,4,6-trioxo-5-(p-tolyl)- 1,2,3,4,5,6,8,9-octahydro-7H-pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidin-7- yl)methyl)benzoate (44 mg, 0.090 mmol), which yielded a yellow solid (7.7 mg, 18%); mp 1 269–270 °C dec, 98% purity determined by qNMR. H NMR (400 MHz, DMSO-d6) δ 13.02 (s, 1H), 10.43 (s, 1H), 9.24 (s, 1H), 7.84 (d, J = 6.8 Hz, 2H), 7.42 (t, J = 7.8 Hz, 1H), 7.33 (d, J = 7.8 Hz, 1H), 7.17 (d, J = 7.7 Hz, 2H), 7.02 (d, J = 7.6 Hz, 2H), 5.30 (d, J = 16.6 Hz, 1H), 5.12 (d, J = 16.6 Hz, 1H), 4.93 (s, 1H), 4.54 (d, J = 5.7 Hz, 2H), 4.03 (q, J = 6.8 Hz, 2H), 2.23 (s, 3H), 1.10 (t, J = 6.9 Hz, 3H).

4.2. Small Molecule Development-2: Ring Open Esters/Amides 4.2.1. General Procedure for the Synthesis of Ring Open Ester Analogs

O O O R NH AcOH O O + + R H2N N O 110 ˚C O NH O CHO N N O H

Appropriately substituted 3-oxobutanoate (1 equiv), p-tolualdehyde (1 equiv), and intermediate 2.2 (1 equiv) were added to a dry microwave vial (5 mL). After adding AcOH (1 mL), the vial was flushed with nitrogen gas for 1 min and sealed. The mixture was heated at 110 °C for 6 h, which generated a clear yellow solution. Upon the completion of the

101 reaction, monitored by TLC, AcOH was evaporated under nitrogen flow and the resulting solid was purified by flash column chromatography (40 gram RediSep Gold silica gel column, DCM + 5% methanol) to give the targeted ring open ester analogs.

Methyl 1-Ethyl-7-methyl-2,4-dioxo-5-(p-tolyl)-1,2,3,4,5,8-hexahydropyrido[2,3- d]pyrimidine-6-carboxylate (2.10a).

O O

O NH

N N O H The title compound was synthesized via the general procedure for the synthesis of the ring open ester analogs using p-tolualdehyde (30 mg, 0.25 mmol), intermediate 2.2 (38 mg, 0.25 mmol), and methyl acetoacetate (29 mg, 0.25 mmol), which gave rise to a white solid (28 mg, 32%); mp 288–289 °C dec; with a purity 96% determined by qNMR. 1H NMR (400

MHz, DMSO-d6) δ 10.94 (s, 1H), 8.60 (s, 1H), 7.05 (d, J = 8.0 Hz, 2H), 7.00 (d, J = 8.0 Hz, 2H), 4.84 (s, 1H), 4.07–3.91 (m, 2H), 3.55 (s, 3H), 2.41 (s, 3H), 2.20 (s, 3H), 1.11 (t, 13 J = 8.0 Hz, 3H). C NMR (100 MHz, DMSO-d6) δ 166.8, 161.4, 149.9, 145.4, 143.9, 143.6, 135.1, 128.6, 126.9, 104.0, 90.1, 50.9, 36.2, 35.4, 20.5, 18.1, 13.7.

Propyl 1-Ethyl-7-methyl-2,4-dioxo-5-p-tolyl-1,2,3,4,5,8-hexahydropyrido[2,3- d]pyrimidine-6-carboxylate (2.10b).

O O

O NH

N N O H

The title compound was synthesized via the general procedure for the synthesis of the ring open ester analogs using p-tolualdehyde (30 mg, 0.25 mmol), intermediate 2.2 (38 mg, 0.25 mmol), and propyl acetoacetate (35 mg, 0.25 mmol), which yielded a white solid (52 mg, 55%); mp 178–180 °C dec; with a purity 96% determined by qNMR. 1H NMR (400 MHz,

DMSO-d6) δ 10.95 (s, 1H), 8.58 (s, 1H), 7.07–6.99 (m, 4H), 4.84 (s, 1H), 4.05–3.94 (m, 2H), 3.91 (t, J = 6.0 Hz, 2H), 2.42 (s, 3H), 2.20 (s, 3H), 1.55–1.50 (m, 2H), 1.12 (t, J = 8.0 102 13 Hz, 3H), 0.80 (t, J = 8.0 Hz, 3H). C NMR (100 MHz, DMSO-d6) δ 166.4, 161.4, 149.8, 145.2, 143.8, 143.7, 135.0, 128.4, 127.0, 104.2, 90.1, 64.9, 36.2, 35.6, 21.5, 20.5, 18.1, 13.7, 10.4.

Allyl 1-Ethyl-7-methyl-2,4-dioxo-5-(p-tolyl)-1,2,3,4,5,8-hexahydropyrido[2,3- d]pyrimidine-6-carboxylate (2.10c).

O O

O NH

N N O H The title compound was synthesized via the general procedure for the synthesis of the ring open ester analogs using p-tolualdehyde (30 mg, 0.25 mmol), intermediate 2.2 (38 mg, 0.25 mmol), and allyl acetoacetate (36 mg, 0.25 mmol), which yielded a white solid (26 mg, 27%); mp 226–227 °C dec; with a purity 92% determined by qNMR.1H NMR (400 MHz,

DMSO-d6) δ 10.97 (s, 1H), 8.64 (s, 1H), 7.06 (t, J = 4.0 Hz, 2H), 7.00 (d, J = 8.0 Hz, 2H), 5.90–5.83 (m, 1H), 5.19–5.12 (m, 2H), 4.87 (s, 1H), 4.50 (t, J = 4.0 Hz, 2H), 4.05–3.95 (m, 13 2H), 2.43 (s, 3H), 2.20 (s, 3H), 1.11 (t, J = 8.0 Hz, 3H). C NMR (100 MHz, DMSO-d6) δ 165.9, 161.4, 149.8, 145.8, 143.8, 143.6, 135.1, 132.9, 128.6, 126.9, 117.2, 103.9, 90.1, 63.9, 36.2, 35.4, 20.5, 18.2, 13.7. tert-Butyl 1-Ethyl-7-methyl-2,4-dioxo-5-p-tolyl-1,2,3,4,5,8-hexahydropyrido[2,3- d]pyrimidine-6-carboxylate (2.10d).

O O

O NH

N N O H

MHz, DMSO-d6) δ 10.90 (s, 1H), 8.46 (s, 1H), 7.08–7.00 (m, 4H), 4.77 (s, 1H), 4.08–3.93 103 (m, 2H), 2.37 (s, 3H), 2.21 (s, 3H), 1.33 (s, 9H), 1.12 (t, J = 6.0 Hz, 3H). 13C NMR (100

MHz, DMSO-d6) δ 165.8, 161.4, 149.9, 143.8, 143.8, 134.9, 128.4, 127.1, 105.9, 89.9, 79.2, 40.1, 36.2, 36.1, 27.8, 20.6, 18.1, 13.6.

Benzyl 1-Ethyl-7-methyl-2,4-dioxo-5-(p-tolyl)-1,2,3,4,5,8-hexahydropyrido[2,3- d]pyrimidine-6-carboxylate (2.10e).

O O

O NH

MHz, DMSO-d6) δ 10.95 (s, 1H), 8.64 (s, 1H), 7.31–7.27 (m, 3H), 7.18 (t, J = 4.0 Hz, 2H), 7.04 (d, J = 8.0 Hz, 2H), 6.98 (d, J = 8.0 Hz, 2H), 5.05 (d, J = 4.0 Hz, 2H), 4.87 (s, 1H), 4.08–3.91 (m, 2H), 2.43 (s, 3H), 2.20 (s, 3H), 1.12 (t, J = 6.0 Hz, 3H). 13C NMR (100 MHz,

DMSO-d6) δ 166.1, 161.4, 149.8, 145.9, 143.8, 143.6, 136.4, 135.1, 128.5, 128.2, 127.7, 127.6, 127.1, 103.9, 90.1, 65.0, 36.2, 35.6, 20.6, 18.2, 13.7.

4-Chlorobenzyl 1-Ethyl-7-methyl-2,4-dioxo-5-p-tolyl-1,2,3,4,5,8- hexahydropyrido[2,3-d]pyrimidine-6-carboxylate (2.10f).

O O

O NH

Cl N N O H The title compound was synthesized via the general procedure for the synthesis of ring open ester analogs using p-tolualdehyde (30 mg, 0.25 mmol), intermediate 2.2 (38 mg, 0.25 mmol), and 4-chlorobenzyl 3-oxobutanoate (57 mg, 0.25 mmol), which yielded a white solid (38 mg, 33%); mp 275–276 °C dec; with a purity 98% determined by qNMR. 1H

NMR (400 MHz, DMSO-d6) δ 10.96 (s, 1H), 8.64 (s, 1H), 7.37–7.34 (m, 2H), 7.20 (d, J =

104 8.0 Hz, 2H), 7.05–6.98 (m, 4H), 5.09–4.99 (m, 2H), 4.86 (s, 1H), 4.06–3.96 (m, 2H), 2.44 13 (s, 3H), 2.22 (s, 3H), 1.12 (t, J = 8.0 Hz, 3H). C NMR (100 MHz, DMSO-d6) δ 166.0, 161.3, 149.8, 146.1, 143.7, 143.6, 135.5, 135.1, 132.3, 129.6, 128.5, 128.2, 127.1, 103.7, 90.2, 64.2, 36.2, 35.6, 20.5, 18.2, 13.6.

4-Methoxybenzyl 1-Ethyl-7-methyl-2,4-dioxo-5-p-tolyl-1,2,3,4,5,8- hexahydropyrido[2,3-d]pyrimidine-6-carboxylate (2.10g).

O O

O NH

O N N O H The title compound was synthesized via the general procedure for the synthesis of ring open ester analogs using p-tolualdehyde (30 mg, 0.25 mmol), intermediate 2.2 (38 mg, 0.25 mmol), and 4-methoxybenzyl 3-oxobutanoate (56 mg, 0.25 mmol), which yielded a white solid (45 mg, 39%); mp 241–242 °C dec; with a purity 94% determined by qNMR. 1H

NMR (400 MHz, DMSO-d6) δ 10.94 (s, 1H), 8.61 (s, 1H), 7.18 (t, J = 4.0 Hz, 2H), 7.03– 6.97 (m, 4H), 6.89 (t, J = 4.0 Hz, 2H), 4.97 (s, 2H), 4.83 (s, 1H), 4.04–3.95 (m, 2H), 3.75 (s, 3H), 2.42 (s, 3H), 2.21 (s, 3H), 1.12 (t, J = 8.0 Hz, 3H). 13C NMR (100 MHz, DMSO- d6) δ 166.2, 161.3, 158.9, 149.9, 145.5, 143.9, 143.6, 135.0, 129.7, 128.5, 128.2, 127.1, 113.6, 104.1, 90.0, 64.9, 55.0, 36.2, 35.5, 20.5, 18.1, 13.6.

3,4-Dimethoxybenzyl 1-Ethyl-7-methyl-2,4-dioxo-5-p-tolyl-1,2,3,4,5,8- hexahydropyrido[2,3-d]pyrimidine-6-carboxylate (2.10h).

O O O O NH

O N N O H

The title compound was synthesized via the general procedure for the synthesis of ring open ester analogs using p-tolualdehyde (30 mg, 0.25 mmol), intermediate 2.2 (38 mg, 0.25 mmol), and 3,4-dimethoxybenzyl 3-oxobutanoate (63 mg, 0.25 mmol), which yielded a white solid (50 mg, 41%); mp 219–220 °C dec; with a purity 97% determined by qNMR. 105 1 H NMR (400 MHz, DMSO-d6) δ 10.95 (s, 1H), 8.62 (s, 1H), 7.03–6.96 (m, 4H), 6.90 (d, J = 8.0 Hz, 1H), 6.83 (d, J = 12.0 Hz, 2H), 4.97 (d, J = 4.0 Hz, 2H), 4.86 (s, 1H), 4.06– 3.95 (m, 2H), 3.74 (s, 3H), 3.69 (s, 3H), 2.43 (s, 3H), 2.21 (s, 3H), 1.12 (t, J = 6.0 Hz, 3H). 13 C NMR (100 MHz, DMSO-d6) δ 166.2, 161.4, 149.8, 148.5, 145.7, 143.9, 143.6, 135.0, 128.6, 128.5, 127.0, 120.6, 111.8, 111.4, 104.0, 90.1, 65.2, 55.4, 55.3, 36.2, 35.5, 21.0, 20.5, 18.1, 13.6.

4-Methoxyphenethyl 1-Ethyl-7-methyl-2,4-dioxo-5-p-tolyl-1,2,3,4,5,8- hexahydropyrido[2,3-d]pyrimidine-6-carboxylate (2.10i)

O O O

O NH

N N O H The title compound was synthesized via the general procedure for the synthesis of ring open ester analogs using p-tolualdehyde (30 mg, 0.25 mmol), intermediate 2.2 (38 mg, 0.25 mmol), and 4-methoxyphenethyl 3-oxobutanoate (59 mg, 0.25 mmol), which yielded a white solid (61 mg, 51%); mp 140–142 °C dec; with a purity 99% determined by qNMR. 1 H NMR (400 MHz, DMSO-d6) δ 10.94 (s, 1H), 8.57 (s, 1H), 7.09–6.98 (m, 6H), 6.82 (t, J = 6.0 Hz, 2H), 4.84 (s, 1H), 4.16–4.12 (m, 2H), 4.07–3.93 (m, 2H), 3.96 (s, 3H), 2.80– 2.76 (m, 2H), 2.36 (s, 3H), 2.21 (s, 3H), 1.11 (t, J = 6.0 Hz, 3H). 13C NMR (100 MHz,

DMSO-d6) δ 166.4, 161.4, 157.7, 149.9, 145.4, 143.9, 143.6, 135.0, 129.9, 129.7, 128.5, 127.0, 126.9, 113.6, 104.2, 90.2, 64.4, 36.2, 35.4, 33.4, 20.5, 18.1, 13.6.

4.2.2. General Procedure for the Synthesis of Ring Open Amide Analogs

O O NaOH O O RNH2 O O R O NH HO NH N NH EDCI, HOBt, H N N O N N O DIPEA N N O H H H

The amide analogs were derived from methyl ester analog 2.10a. NaOH (4 equiv) was added to the aqueous solution of 2.10a. After stirring for 18 h at rt, the resulting solution was acidified with AcOH and extracted with DCM three times. The combined organic 106 phases were washed with brine, dried over anhydrous MgSO4 and concentrated under vacuo to yield the crude acid intermediate, which was sequentially charged with DMF (10 mL) and cooled to 0 °C. DIPEA (1 equiv), EDCI (1 equiv), HOBt (1 equiv), and the amine (1 equiv) were added successively and stirred at rt for 3 h. Upon completion of the reaction, monitored by TLC, the mixture was poured into water and extracted with diethyl ether. The separated organic layer was washed with water and brine, dried over anhydrous

MgSO4 and concentrated to obtain the crude solid, which was purified by flash column chromatography (40 gram RediSep Gold silica gel column) eluting with a mixture of methanol and DCM to give the target dihydropyridines.

1-Ethyl-7-methyl-2,4-dioxo-N-propyl-5-(p-tolyl)-1,2,3,4,5,8-hexahydropyrido[2,3- d]pyrimidine-6-carboxamide (2.11a).

O O

N NH H N N O H The title compound was synthesized via the general procedure for the synthesis of ring open amide analogs using methyl ester analog 2.10a (88 mg, 0.25 mmol), which yielded a white solid (20 mg, 21%); mp 121–122 °C dec; with a purity 96% determined by qNMR. 1 H NMR (400 MHz, DMSO-d6) δ 10.78 (s, 1H), 8.11 (s, 1H), 7.65 (t, J = 6.0 Hz, 1H), 7.05–6.99 (m, 4H), 4.78 (s, 1H), 4.02–3.93 (m, 2H), 2.97 (t, J = 12.0 Hz, 2H), 2.21 (s, 3H), 2.09 (s, 3H), 1.37–1.31 (m, 2H), 1.12 (t, J = 8.0 Hz, 3H), 0.74 (t, J = 8.0 Hz, 3H). 13C NMR

(100 MHz, DMSO-d6) δ 167.6, 161.4, 149.9, 144.9, 143.0, 134.8, 132.9, 128.4, 127.1, 111.3, 88.0, 40.3, 37.6, 36.0, 22.2, 20.5, 17.2, 13.6, 11.3.

107 N-Allyl-1-ethyl-7-methyl-2,4-dioxo-5-p-tolyl-1,2,3,4,5,8-hexahydropyrido[2,3- d]pyrimidine-6-carboxamide (2.11b).

O O

N NH H N N O H

The title compound was synthesized via the general procedure for the synthesis of ring open amide analogs using methyl ester analog 2.10a (88 mg, 0.25 mmol), which yielded a white solid (30 mg, 32%); mp 115–116 °C dec; with a purity 97% determined by qNMR. 1 H NMR (400 MHz, DMSO-d6) δ 10.76 (s, 1H), 8.16 (s, 1H), 7.82 (t, J = 6.0 Hz, 1H), 7.05 (d, J = 8.0 Hz, 2H), 6.99 (d, J = 8.0 Hz, 2H), 5.74–5.64 (m, 1H), 4.95–4.86 (m, 2H), 4.79 (s, 1H), 4.04–3.89 (m, 2H), 3.70–3.57 (m, 2H), 2.20 (s, 3H), 2.10 (s, 3H), 1.11 (t, J = 8.0 13 Hz, 3H). C NMR (100 MHz, DMSO-d6) δ 167.5, 161.4, 145.0, 144.8, 143.0, 135.4, 134.9, 133.6, 128.5, 127.2, 114.7, 110.9, 88.1, 41.0, 37.6, 36.0, 20.6, 17.2, 13.6.

N-Benzyl-1-ethyl-7-methyl-2,4-dioxo-5-p-tolyl-1,2,3,4,5,8-hexahydropyrido[2,3- d]pyrimidine-6-carboxamide (2.11c).

O O

N NH H N N O H

The title compound was synthesized via the general procedure for the synthesis of ring open amide analogs using methyl ester analog 2.10a (88 mg, 0.25 mmol), which yielded a white solid (40 mg, 37%); mp 239–240 °C dec; with a purity 90% determined by qNMR. 1 H NMR (400 MHz, DMSO-d6) δ 10.79 (s, 1H), 8.21 (t, J = 6.0 Hz, 1H), 8.13 (s, 1H), 7.21–7.17 (m, 3H), 7.07–6.99 (m, 6H), 4.84 (s, 1H), 4.24 (t, J = 6.0 Hz, 2H), 4.02–3.95 (m, 13 2H), 2.24 (s, 3H), 2.12 (s, 3H), 1.13 (t, J = 6.0 Hz, 3H). C NMR (100 MHz, DMSO-d6) δ 167.7, 161.4, 150.0, 144.8, 143.0, 139.6, 134.9, 133.6, 128.4, 127.9, 127.4, 126.9, 126.4, 110.9, 88.1, 42.0, 37.7, 36.0, 20.6, 17.3, 13.6.

108 1-Ethyl-7-methyl-2,4-dioxo-N'-phenyl-5-p-tolyl-1,2,3,4,5,8-hexahydropyrido[2,3- d]pyrimidine-6-carbohydrazide (2.11d).

H O O N N NH H N N O H The title compound was synthesized via the general procedure for the synthesis of ring open amide analogs using methyl ester analog 2.10a (88 mg, 0.25 mmol), which yieled a white solid (17 mg, 16%), mp 164–165 °C dec, with a purity 94% determined by qNMR. 1 H NMR (400 MHz, DMSO-d6) δ 10.80 (s, 1H), 9.49 (d, J = 4.0 Hz, 1H), 8.19 (s, 1H), 7.61 (d, J = 4.0 Hz, 1H), 7.13 (d, J = 8.0 Hz, 2H), 7.06 (d, J = 8.0 Hz, 2H), 6.99–6.95 (m, 2H), 6.62 (t, J = 8.0 Hz, 1H), 6.36 (d, J = 8.0 Hz, 2H), 4.83 (s, 1H), 4.05–3.95 (m, 2H), 13 2.26 (s, 3H), 2.17 (s, 3H), 1.14 (t, J = 6.0 Hz, 3H). C NMR (100 MHz, DMSO-d6) δ 167.9, 161.3, 149.9, 149.3, 144.6, 142.9, 135.0, 133.8, 128.4, 128.3, 127.7, 118.1, 112.1, 109.4, 88.2, 37.9, 36.1, 20.6, 17.4, 13.6.

1-Ethyl-7-methyl-2,4-dioxo-N-phenethyl-5-p-tolyl-1,2,3,4,5,8-hexahydropyrido[2,3- d]pyrimidine -6-carboxamide (2.11e).

O O

N NH H N N O H

The title compound was synthesized via the general procedure for the synthesis of ring open amide analogs using methyl ester analog 2.10a (88 mg, 0.25 mmol), which yielded a white solid (62 mg, 56%); mp 194–195 °C dec; with a purity 97% determined by qNMR. 1 H NMR (400 MHz, DMSO-d6) δ 10.78 (s, 1H), 8.12 (s, 1H), 7.71 (t, J = 6.0 Hz, 1H), 7.25–7.15 (m, 3H), 7.11–6.98 (m, 6H), 4.76 (s, 1H), 4.02–3.91 (m, 2H), 3.25 (t, J = 8.0 Hz, 2H), 2.67–2.62 (m, 2H), 2.21 (s, 3H), 2.03 (s, 3H), 1.12 (t, J = 6.0 Hz, 3H). 13C NMR (100

MHz, DMSO-d6) δ 167.7, 161.4, 150.0, 144.8, 142.9, 139.4, 134.8, 133.6, 128.5, 128.4, 128.2, 127.1, 125.9, 110.9, 88.1, 40.2, 37.4, 36.0, 34.9, 20.6, 17.1, 13.6.

109 1-Ethyl-N-(4-methoxyphenethyl)-7-methyl-2,4-dioxo-5-p-tolyl-1,2,3,4,5,8- hexahydropyrido[2,3-d]pyrimidine-6-carboxamide (2.11f).

O O O

N NH H N N O H

The title compound was synthesized via the general procedure for the synthesis of ring open amide analogs using methyl ester analog 2.10a (88 mg, 0.25 mmol), which yielded a white solid (78 mg, 66%); mp 152–153 °C dec; with a purity 95% determined by qNMR. 1 H NMR (400 MHz, DMSO-d6) δ 10.81 (s, 1H), 8.13 (s, 1H), 7.71 (t, J = 6.0 Hz, 1H), 7.04–6.99 (m, 6H), 6.81–6.79 (m, 2H), 4.77 (s, 1H), 4.02–3.94 (m, 2H), 3.71 (s, 3H), 3.21 (t, J = 4.0 Hz, 2H), 2.61–2.56 (m, 2H), 2.22 (s, 3H), 2.05 (s, 3H), 1.12 (t, J = 6.0 Hz, 3H). 13 C NMR (100 MHz, DMSO-d6) δ 167.6, 161.3, 157.4, 150.0, 144.8, 143.0, 134.8, 133.6, 131.3, 129.4, 128.4, 127.1, 113.5, 110.9, 88.1, 54.9, 40.4, 37.4, 36.0, 34.0, 20.6, 17.2, 13.6.

1-Ethyl-7-methyl-2,4-dioxo-N-(3-phenylpropyl)-5-p-tolyl-1,2,3,4,5,8- hexahydropyrido[2,3-d]pyrimidine-6-carboxamide (2.11g).

O O

N NH H N N O H The title compound was synthesized via the general procedure for the synthesis of amide analogs using methyl ester analog 2.10a (88 mg, 0.25 mmol), which yielded a light-yellow solid (32 mg, 28%); mp 113–114 °C dec; with a purity 98% determined by qNMR. 1H

NMR (400 MHz, DMSO-d6) δ 10.79 (s, 1H), 8.12 (s, 1H), 7.71 (t, J = 4.0 Hz, 1H), 7.25 (t, J = 8.0 Hz, 2H), 7.17–7.05 (m, 5H), 6.99 (d, J = 8.0 Hz, 2H), 4.82 (s, 1H), 4.04–3.94 (m, 2H), 3.08–3.00 (m, 2H), 2.43–2.39 (m, 2H), 2.19 (s, 3H), 2.11 (s, 3H), 1.61 (t, J = 8.0 Hz, 13 2H), 1.13 (t, J = 6.0 Hz, 3H). C NMR (100 MHz, DMSO-d6) δ 167.6, 161.4, 149.8, 150.0, 144.9, 143.0, 141.8, 134.8, 133.0, 128.4, 128.1, 127.2, 125.6, 111.2, 88.0, 40.1, 37.7, 36.0, 32.5, 30.9, 20.5, 17.2, 13.6.

110 4.3. Small Molecule Development-3: Macrocyclic Analogs 4.3.1. General Procedure A

R R

K2CO3 + Br Br OH O Br n DMF, 70 oC n CHO CHO Based on a reported method,1 a solution of the hydroxybenzaldehyde (1 equiv) in DMF (5 mL) was added dropwise to a stirred suspension of 1,2-dibromoethane, 1,3- dibromopropane, or 1,4-dibromopropane (2 equiv) and K2CO3 (1.5 equiv) in DMF (20 mL). The reaction mixture was stirred at rt for 2 h, and an additional 2 h at 70 °C. Upon total consumption of the aldehyde, monitored by TLC, the mixture was poured into water and extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous MgSO4, and concentrated under reduced pressure. The residue was purified by flash column chromatography (40 gram RediSep Gold silica gel column, 0–15% ethyl acetate in hexanes) to give the title compound as a colorless oil.

2-(2-Bromoethoxy)benzaldehyde (2.12a).

Br O CHO The title compound was synthesized via general procedure A using 2- hydroxybenzaldehyde (1.0 g, 8.2 mmol) and 1,2-dibromoethane (3.1 g, 16.4 mmol), which 1 yielded a colorless oil (0.30 g, 16%). H NMR (400 MHz, CDCl3) δ 10.58 (s, 1H), 7.89 (dd, J = 7.7, 1.7 Hz, 1H), 7.58 (ddd, J = 8.8, 7.3, 1.6 Hz, 1H), 7.11 (t, J = 7.5 Hz, 1H), 6.99 (d, J = 8.4 Hz, 1H), 4.45 (t, J = 6.0 Hz, 2H), 3.74 (t, J = 6.1 Hz, 2H).

2-(3-Bromopropoxy)benzaldehyde (2.12b).

O Br CHO The title compound was synthesized via general procedure A using 2- hydroxybenzaldehyde (0.50 g, 4.1 mmol) and 1,3-dibromopropane (1.7 g, 8.2 mmol), 1 which yielded a colorless oil (0.73 g, 74%). H NMR (400 MHz, CDCl3) δ 10.54 (s, 1H),

111 7.86 (dq, J = 7.2, 3.4, 2.6 Hz, 1H), 7.62–7.54 (m, 1H), 7.04 (h, J = 8.4, 7.9 Hz, 2H), 4.27 (dd, J = 6.9, 5.1 Hz, 2H), 3.66 (td, J = 6.3, 1.8 Hz, 2H), 2.48–2.37 (m, 2H).

2-(4-Bromobutoxy)benzaldehyde (2.12c).

Br O CHO The title compound was synthesized via general procedure A using 2- hydroxybenzaldehyde (0.50 g, 4.1 mmol) and 1,4-dibromopropane (1.8 g, 8.2 mmol), 1 which yielded a colorless oil (0.24 g, 23%). H NMR (400 MHz, CDCl3) δ 10.53 (s, 1H), 7.87 (dd, J = 7.6, 1.8 Hz, 1H), 7.57 (td, J = 7.9, 7.1, 1.8 Hz, 1H), 7.06 (t, J = 7.5 Hz, 1H), 7.00 (d, J = 8.4 Hz, 1H), 4.16 (t, J = 5.8 Hz, 2H), 3.57–3.49 (m, 2H), 2.19–2.01 (m, 4H).

2-(2-Bromoethoxy)-4-methylbenzaldehyde (2.12d).

Br O CHO The title compound was synthesized via general procedure A using 2-hydroxy-4- methylbenzaldehyde (0.75 g, 5.5 mmol) and 1,2-dibromoethane (2.1 g, 11.0 mmol), which 1 yielded a colorless oil (0.40 g, 30%). H NMR (400 MHz, CDCl3) δ 10.49 (s, 1H), 7.78 (d, J = 7.8 Hz, 1H), 6.91 (d, J = 7.9 Hz, 1H), 6.78 (s, 1H), 4.43 (t, J = 6.1 Hz, 2H), 3.73 (t, J = 6.1 Hz, 2H), 2.43 (s, 3H).

2-(3-Bromopropoxy)-4-methylbenzaldehyde (2.12e).

O Br CHO The title compound was synthesized via general procedure A using 2-hydroxy- 4methylbenzaldehyde (0.75 g, 5.5 mmol) and 1,3-dibromopropane (2.2 g, 11.0 mmol), 1 which yielded a colorless oil (0.26 g, 18%). H NMR (400 MHz, CDCl3) δ 10.41 (s, 1H), 7.73 (dd, J = 7.9, 1.2 Hz, 1H), 6.86 (d, J = 7.9 Hz, 1H), 6.81 (s, 1H), 4.23 (td, J = 5.8, 1.2 Hz, 2H), 3.63 (td, J = 6.3, 1.2 Hz, 2H), 2.46–2.34 (m, 5H).

112 3-(2-Bromoethoxy)benzaldehyde (2.12f).

O Br

CHO The title compound was synthesized via general procedure A using 3- hydroxybenzaldehyde (0.75 g, 6.14 mmol) and 1,2-dibromoethane (2.31 g, 12.3 mmol), 1 which yielded a colorless oil (0.27 g, 19%). H NMR (400 MHz, CDCl3) δ 10.01 (d, J = 1.1 Hz, 1H) 7.55–7.47 (m, 2H), 7.45–7.40 (m, 1H), 7.24 (ddd, J = 7.6, 2.7, 1.5 Hz, 1H), 4.39 (td, J = 6.2, 1.1 Hz, 2H), 3.69 (td, J = 6.2, 1.1 Hz, 2H).

3-(2-Bromoethoxy)-4-methylbenzaldehyde (2.12g).

O Br

CHO The title compound was synthesized via general procedure A using 3-hydroxy- 4methylbenzaldehyde (0.50 g, 3.7 mmol) and 1,2-dibromoethane (1.4 g, 7.3 mmol), which 1 yielded a colorless oil (72 mg, 8%). H NMR (400 MHz, CDCl3) δ 9.95 (s, 1H), 7.43 (dq, J = 7.7, 1.5 Hz, 1H), 7.35 (dd, J = 7.8, 2.9 Hz, 1H), 7.33 (t, J = 1.7 Hz, 1H), 4.40 (td, J = 6.0, 3.1 Hz, 2H), 3.73 (td, J = 6.0, 3.2 Hz, 2H), 2.37 (d, J = 3.0 Hz, 3H).

3-(3-Bromopropoxy)-4-methylbenzaldehyde (2.12h).

Br CHO The title compound was synthesized via general procedure A using 3-hydroxy-4- methylbenzaldehyde (0.75 g, 5.5 mmol) and 1,3-dibromopropane (2.2 g, 11.0 mmol), 1 which yielded a colorless oil (0.74 g, 52%). H NMR (400 MHz, CDCl3) δ 9.93 (s, 1H), 7.41–7.29 (m, 3H), 4.19 (t, J = 5.8 Hz, 2H), 3.63 (t, J = 6.4 Hz, 2H), 2.38 (p, J = 6.2 Hz, 2H), 2.30 (s, 3H).

113 4.3.2. General Procedure B

R R OH OH K2CO3 Br O O + o O n DMF, 70 C n CHO OH CHO Bromoalkyl benzaldehyde (1 equiv) and 2-(hydroxymethyl) phenol (1 equiv) were dissolved in DMF (20 mL). K2CO3 (1.3 equiv) was then added into the reaction mixture, which was stirred at 70 °C overnight. Upon total consumption of the aldehyde monitored by TLC, the mixture was poured into water and extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous MgSO4, and concentrated under reduced pressure. The residue was purified by flash column chromatography (24 gram RediSep Gold silica gel column, 0–75% ethyl acetate in hexanes) to give a white solid.

2-(2-(2-(Hydroxymethyl)phenoxy)ethoxy)benzaldehyde (2.13a).

O O CHO The title compound was synthesized via general procedure B using 2.12a (0.30 g, 1.3 mmol) and 2-(hydroxymethyl) phenol (0.16 g, 1.3 mmol), which yielded a white solid (0.24 g, 1 67%). H NMR (400 MHz, CDCl3) δ 10.48 (s, 1H), 7.88 (dd, J = 7.7, 1.7 Hz, 1H), 7.60 (td, J = 7.9, 7.3, 1.7 Hz, 1H), 7.37–7.31 (m, 2H), 7.14–7.06 (m, 2H), 7.03 (t, J = 7.4 Hz, 1H), 6.97 (d, J = 8.1 Hz, 1H), 4.70 (d, J = 6.4 Hz, 2H), 4.56–4.47 (m, 4H), 2.31 (t, J = 6.5 Hz, 1H).

2-(3-(2-(Hydroxymethyl)phenoxy)propoxy)benzaldehyde (2.13b).

O O CHO HO The title compound was synthesized via general procedure B using 2.12b (0.79 g, 3.2 mmol) and 2-(hydroxymethyl) phenol (0.40, 3.2 mmol), which yielded a white solid (0.43 g, 46%). 1 H NMR (400 MHz, CDCl3) δ 10.52 (s, 1H), 7.86 (dd, J = 7.7, 1.8 Hz, 1H), 7.57 (ddd, J = 8.8, 7.3, 1.8 Hz, 1H), 7.35–7.28 (m, 2H), 7.09–7.02 (m, 2H), 6.99 (t, J = 7.4 Hz, 1H), 6.94

114 (d, J = 8.1 Hz, 1H), 4.72 (d, J = 6.1 Hz, 2H), 4.33 (t, J = 6.0 Hz, 2H), 4.29 (t, J = 6.1 Hz, 2H), 2.41 (p, J = 6.0 Hz, 2H), 2.22 (t, J = 6.4 Hz, 1H).

2-(4-(2-(Hydroxymethyl)phenoxy)butoxy)benzaldehyde (2.13c).

O O CHO The title compound was synthesized via general procedure B using 2.12c (0.24 g, 0.93 mmol) and 2-(hydroxymethyl) phenol (0.12 g, 0.93 mmol), which yielded a white solid 1 (0.29 g, 99%). H NMR (400 MHz, CDCl3) δ 10.53 (s, 1H), 7.86 (dd, J = 7.6, 1.7 Hz, 1H), 7.57 (td, J = 7.9, 7.2, 1.7 Hz, 1H), 7.34–7.27 (m, 2H), 7.06 (t, J = 7.6 Hz, 1H), 6.99 (dd, J = 17.4, 8.2 Hz, 2H), 6.91 (d, J = 8.2 Hz, 1H), 4.72 (d, J = 6.4 Hz, 2H), 4.24–4.18 (m, 2H), 4.14 (q, J = 7.4, 6.5 Hz, 2H), 2.23 (t, J = 6.4 Hz, 1H), 2.10 (p, J = 2.8 Hz, 4H).

2-(2-(2-(Hydroxymethyl)phenoxy)ethoxy)-4-methylbenzaldehyde (2.13d).

O O CHO The title compound was synthesized via general procedure B using 2.12d (0.40 g, 1.7 mmol) and 2-(hydroxymethyl) phenol (0.20 g, 1.7 mmol), which yielded a white solid (0.33 g, 1 69%). H NMR (400 MHz, CDCl3) δ 10.40 (s, 1H), 7.77 (d, J = 7.9 Hz, 1H), 7.37–7.29 (m, 2H), 7.02 (t, J = 7.5 Hz, 1H), 6.97 (d, J = 8.1 Hz, 1H), 6.91 (d, J = 7.9 Hz, 1H), 6.87 (s, 1H), 4.69 (d, J = 5.8 Hz, 2H), 4.49 (s, 4H), 2.44 (s, 3H), 2.33 (t, J = 6.5 Hz, 1H).

2-(3-(2-(Hydroxymethyl)phenoxy)propoxy)-4-methylbenzaldehyde (2.13e).

O O CHO HO The title compound was synthesized via general procedure B using 2.12e (0.930 g, 3.6 mmol) and 2-(hydroxymethyl) phenol (0.44 g, 3.6 mmol), which yielded a white solid (0.74 1 g, 69%). H NMR (400 MHz, CDCl3) δ 10.42 (s, 1H), 7.72 (d, J = 7.8 Hz, 1H), 7.32–7.27

115 (m, 2H), 6.96 (td, J = 7.5, 1.0 Hz, 1H), 6.91 (dd, J = 8.2, 1.0 Hz, 1H), 6.86–6.82 (m, 1H), 6.80 (s, 1H), 4.70 (d, J = 6.4 Hz, 2H), 4.27 (dt, J = 11.1, 6.0 Hz, 4H), 2.43–2.32 (m, 5H), 2.13 (t, J = 6.5 Hz, 1H).

3-(2-(2-(Hydroxymethyl)phenoxy)ethoxy)benzaldehyde (2.13f).

O O

HO CHO The title compound was synthesized via general procedure B using 2.12f (0.27 g, 1.2 mmol) and 2-(hydroxymethyl) phenol (0.15 g, 1.2 mmol), which yielded a white solid (0.16 g, 1 50%). H NMR (400 MHz, CDCl3) δ 10.01 (s, 1H), 7.55–7.49 (m, 2H), 7.49–7.46 (m, 1H), 7.32 (t, J = 7.5 Hz, 2H), 7.26 (ddd, J = 7.6, 2.8, 1.6 Hz, 1H), 7.01 (t, J = 7.5 Hz, 1H), 6.97 (d, J = 8.2 Hz, 1H), 4.70 (d, J = 6.6 Hz, 2H), 4.46 (s, 4H), 2.75 (t, J = 6.7 Hz, 1H).

3-(2-(2-(Hydroxymethyl)phenoxy)ethoxy)-4-methylbenzaldehyde (2.13g).

O O

HO CHO The title compound was synthesized via general procedure B using 2.12g (140 mg, 0.61 mmol) and 2-(hydroxymethyl) phenol (0.078 g, 0.61 mmol), which yielded a white solid 1 (60 mg, 35%). H NMR (400 MHz, CDCl3) δ 9.96 (s, 1H), 7.44–7.40 (m, 2H), 7.36–7.29 (m, 3H), 7.04–6.96 (m, 2H), 4.70 (s, 2H), 4.48 (s, 4H), 2.32 (s, 3H), 2.24 (d, J = 5.2 Hz, 1H).

3-(3-(2-(Hydroxymethyl)phenoxy)propoxy)-4-methylbenzaldehyde (2.13h).

O OH

O CHO The title compound was synthesized via general procedure B using 2.12h (0.74 g, 2.9 mmol) and 2-(hydroxymethyl) phenol (0.37 g, 2.9 mmol), which yielded a white solid (0.66 g, 1 76%). H NMR (400 MHz, CDCl3) δ 9.91 (s, 1H), 7.36 (d, J = 7.7 Hz, 2H), 7.30 (dq, J = 6.7, 2.0 Hz, 2H), 7.27 (s, 1H), 6.95 (td, J = 7.5, 1.0 Hz, 1H), 6.91 (d, J = 8.2 Hz, 1H), 4.70 116 (d, J = 6.2 Hz, 2H), 4.26 (td, J = 6.1, 3.7 Hz, 4H), 2.36 (p, J = 6.1 Hz, 2H), 2.29 (s, 3H), 2.04 (s, 1H).

2-(3-(3-(Hydroxymethyl)phenoxy)propoxy)-4-methylbenzaldehyde (2.13i).

O O CHO The title compound was synthesized via general procedure B using 2.12i (0.74 g, 2.9 mmol) and 3-(hydroxymethyl) phenol (0.37 g, 2.9 mmol), which yielded a white solid (0.74 g, 1 86%). H NMR (400 MHz, CDCl3) δ 10.41 (s, 1H), 7.72 (d, J = 7.8 Hz, 1H), 7.30–7.23 (m, 2H), 6.96–6.92 (m, 2H), 6.86–6.79 (m, 3H), 4.67 (d, J = 5.9 Hz, 2H), 4.28 (t, J = 6.1 Hz, 2H), 4.20 (t, J = 5.9 Hz, 2H), 2.38 (s, 3H), 2.33 (p, J = 6.0 Hz, 2H).

4.3.3. General Procedure C

O R R OH O O NaOAc O O O O O + O n toluene n CHO O MW to 130 oC CHO A mixture of substrate (1 equiv), 2,2,6-trimethyl-4H-1,3-dioxin-4-one (TMD) (1.3 equiv), and anhydrous NaOAc (1 equiv) were charged into a microwave tube (5 mL). After the addition of toluene (4 mL), the tube was sealed and heated to 130 °C for 20 min. After cooling to rt, the mixture was poured into water and extracted with ethyl acetate. The organic phase was washed with brine, dried over anhydrous MgSO4, and concentrated under reduced pressure. The residue was purified by flash column chromatography (24 gram RediSep Gold silica gel column, 0–50% ethyl acetate in hexanes) to yield a colorless oil.

117 2-(2-(2-Formylphenoxy)ethoxy)benzyl 3-Oxobutanoate (2.14a).

O O

O O CHO The title compound was synthesized via general procedure C using 2.13a (0.24 g, 0.88 mmol) and TMD (0.16 g, 1.2 mmol), which yielded a colorless oil (0.16 g, 50%). 1H NMR

(400 MHz, CDCl3) δ 10.47 (s, 1H), 7.87 (dd, J = 7.6, 1.8 Hz, 1H), 7.60 (td, J = 7.9, 7.2, 1.7 Hz, 1H), 7.37 (t, J = 8.7 Hz, 2H), 7.10 (t, J = 7.9 Hz, 2H), 7.03 (t, J = 7.5 Hz, 1H), 6.98 (d, J = 8.1 Hz, 1H), 5.24 (s, 2H), 4.51 (dd, J = 5.7, 3.9 Hz, 2H), 4.46 (dd, J = 6.1, 3.5 Hz, 2H), 3.47 (s, 2H), 2.24 (s, 3H).

2-(3-(2-Formylphenoxy)propoxy)benzyl 3-Oxobutanoate (2.14b).

O O CHO O O O The title compound was synthesized via general procedure C using 2.13b (0.42 g, 1.5 mmol) and TMD (0.27 g, 1.9 mmol), which yielded a colorless oil (0.12 g, 22%). 1H NMR (400

MHz, CDCl3) δ 10.52 (s, 1H), 7.85 (dd, J = 7.7, 1.8 Hz, 1H), 7.6–17.54 (m, 1H), 7.34 (t, J = 7.5 Hz, 2H), 7.06 (dd, J = 10.4, 8.1 Hz, 2H), 7.01–6.92 (m, 2H), 5.26 (s, 2H), 4.34 (t, J = 5.9 Hz, 2H), 4.25 (t, J = 5.9 Hz, 2H), 3.47 (s, 2H), 2.39 (p, J = 5.9 Hz, 2H), 2.25 (s, 3H).

2-(4-(2-Formylphenoxy)butoxy)benzyl 3-Oxobutanoate (2.14c).

O O

O O CHO The title compound was synthesized via general procedure C using 2.13c (0.29 g, 0.97 mmol) and TMD (0.18 g, 1.3 mmol), which yielded a colorless oil (0.18 g, 47%). 1H NMR

(400 MHz, CDCl3) δ 10.53 (s, 1H), 7.86 (dd, J = 7.6, 1.8 Hz, 1H), 7.57 (td, J = 8.0, 7.5, 1.8 Hz, 1H), 7.34 (td, J = 9.4, 8.3, 4.6 Hz, 2H), 7.08–7.02 (m, 2H), 6.98 (t, J = 7.5 Hz, 1H),

118 6.91 (d, J = 8.3 Hz, 1H), 5.27 (s, 2H), 4.20 (q, J = 6.8, 6.1 Hz, 2H), 4.11 (t, J = 5.6 Hz, 2H), 3.51 (s, 2H), 2.27 (s, 3H), 2.09–2.02 (m, 4H).

2-(2-(2-Formyl-5-methylphenoxy)ethoxy)benzyl 3-Oxobutanoate (2.14d).

O O

O O CHO The title compound was synthesized via general procedure C using 2.13d (0.33 g, 1.1 mmol) and TMD (0.21 g, 1.5 mmol), which yielded a colorless oil (0.19 g, 45%). 1H NMR (400

MHz, CDCl3) δ 10.39 (s, 1H), 7.76 (d, J = 7.7 Hz, 1H), 7.37 (t, J = 8.1 Hz, 2H), 7.03 (t, J = 7.5 Hz, 1H), 6.98 (d, J = 8.1 Hz, 1H), 6.90 (d, J = 8.3 Hz, 2H), 5.24 (s, 2H), 4.53– 4.43 (m, 4H), 3.47 (s, 2H), 2.44 (s, 3H), 2.24 (s, 3H).

2-(3-(2-Formyl-5-methylphenoxy)propoxy)benzyl 3-Oxobutanoate (2.14e).

O O CHO O O O The title compound was synthesized via general procedure C using 2.13e (0.74 g, 2.5 mmol) and TMD (0.46 g, 3.3 mmol), which yielded a colorless oil (0.57 g, 60%). 1H NMR (400

MHz, CDCl3) δ 10.42 (s, 1H), 7.71 (d, J = 8.1 Hz, 1H), 7.31 (t, J = 7.9 Hz, 2H), 7.00–6.88 (m, 2H), 6.83 (d, J = 7.4 Hz, 2H), 5.24 (s, 2H), 4.29 (t, J = 5.9 Hz, 2H), 4.22 (t, J = 5.9 Hz, 2H), 3.45 (s, 2H), 2.41–2.31 (m, 5H), 2.22 (s, 3H).

2-(2-(3-Formylphenoxy)ethoxy)benzyl 3-Oxobutanoate (2.14f).

O O

O CHO O O The title compound was synthesized via general procedure C using 2.13f (0.16 g, 0.59 mmol) and TMD (0.11 g, 0.76 mmol), which yielded a colorless oil (0.12 g, 57%). 1H NMR

(400 MHz, CDCl3) δ 10.02 (s, 1H), 7.51 (d, J = 7.3 Hz, 2H), 7.48 (d, J = 2.6 Hz, 1H), 7.36

119 (t, J = 7.9 Hz, 2H), 7.28–7.24 (m, 1H), 7.02 (t, J = 7.5 Hz, 1H), 6.98 (d, J = 8.1 Hz, 1H), 5.26 (s, 2H), 4.42 (q, J = 3.9, 3.2 Hz, 4H), 3.48 (s, 2H), 2.25 (s, 3H).

2-(2-(5-Formyl-2-methylphenoxy)ethoxy)benzyl 3-Oxobutanoate (2.14g).

O O

O CHO O O The title compound was synthesized via general procedure C using 2.13g (0.30 g, 1.3 mmol) and TMD (0.24 g, 1.7 mmol), which yielded a colorless oil (0.24 g, 67%). 1H NMR (400

MHz, CDCl3) δ 9.96 (s, 1H), 7.42 (d, J = 6.1 Hz, 2H), 7.39–7.32 (m, 3H), 7.05–6.98 (m, 2H), 5.26 (s, 2H), 4.44 (s, 4H), 3.47 (s, 2H), 2.31 (s, 3H), 2.24 (s, 3H).

2-(3-(5-Formyl-2-methylphenoxy)propoxy)benzyl 3-Oxobutanoate (2.14h).

O O

O O CHO The title compound was synthesized via general procedure C using 2.13h (0.66 g, 2.2 mmol) and TMD (0.41 g, 2.9 mmol), which yielded a colorless oil (35 mg, 41%). 1H NMR (400

MHz, CDCl3) δ 9.92 (s, 1H), 7.40–7.27 (m, 5H), 7.00–6.89 (m, 2H), 5.24 (s, 2H), 4.24 (dt, J = 11.9, 6.0 Hz, 4H), 3.44 (d, J = 3.1 Hz, 2H), 2.40–2.31 (m, 2H), 2.29 (d, J = 1.9 Hz, 3H), 2.22 (s, 3H).

3-(3-(2-Formyl-5-methylphenoxy)propoxy)benzyl 3-Oxobutanoate (2.14i).

O O

O O CHO The title compound was synthesized via general procedure C using 2.13i (0.73 g, 2.4 mmol) and TMD (0.45 g, 3.2 mmol), which yielded a colorless oil (0.18 g, 20%). 1H NMR (400

MHz, CDCl3) δ 10.43 (s, 1H), 7.72 (d, J = 7.9 Hz, 1H), 7.27 (s, 1H), 6.98–6.78 (m, 5H),

120 5.14 (s, 2H), 4.28 (t, J = 6.1 Hz, 2H), 4.20 (t, J = 5.9 Hz, 2H), 3.51 (s, 2H), 2.39 (s, 3H), 2.33 (p, J = 6.1 Hz, 2H), 2.25 (s, 3H).

4.3.4. General Procedure D

R O R O O O Piperidine n O O O O n O CHO EtOH O To an ethanol solution (30 mL) of the substrate (1 equiv), piperidine (0.1 equiv), and AcOH (0.1 equiv) were charged and the yellow solution was stirred at rt for 24 h. Upon total consumption of the starting material monitored by TLC, the mixture was poured into water and extracted with ethyl acetate. The organic phase was washed with brine, dried over anhydrous MgSO4, and concentrated under reduced pressure. The residue was purified by flash column chromatography (4 gram RediSep Gold silica gel column, 0–25% ethyl acetate in hexanes) on silica gel to yield a colorless wax.

(Z)-16-Acetyl-6,7-dihydro-13H,15H-dibenzo[e,l][1,4,8]trioxacyclotridecin-15-one (2.15a).

O O

O O O The title compound was synthesized via general procedure D using 2.14a (0.16 g, 0.45 1 mmol), which yielded a colorless wax (71 mg, 47%). H NMR (400 MHz, CDCl3) δ 7.39 (s, 1H), 7.32–7.24 (m, 3H), 7.24–7.20 (m, 1H), 6.99–6.91 (m, 3H), 6.85 (d, J = 8.4 Hz, 1H), 5.27 (s, 2H), 4.22 (q, J = 5.4, 4.9 Hz, 4H), 2.30 (s, 3H).

121 (Z)-17-Acetyl-7,8-dihydro-6H,14H,16H-dibenzo[c,j][1,5,9]trioxacyclotetradecin-16- one (2.15b).

O O

O O O The title compound was synthesized via general procedure D using 2.14b (0.12 g, 0.32 1 mmol), which yielded a colorless wax (16 mg, 14%). H NMR (400 MHz, CDCl3) δ 7.59 (s, 1H), 7.37 (dt, J = 15.4, 9.1 Hz, 3H), 7.28–7.25 (m, 1H),7.09 (t, J = 7.0 Hz, 2H), 6.99 (t, J = 7.9 Hz, 2H), 5.17 (s, 2H), 4.40 (t, J = 5.5 Hz, 2H), 4.15 (t, J = 5.5 Hz, 2H), 2.40 (s, 3H), 2.18 (p, J = 5.5 Hz, 2H).

(Z)-18-Acetyl-6,7,8,9-tetrahydro-15H,17H- dibenzo[c,k][1,5,10]trioxacyclopentadecin-17-one (2.15c).

O O O O O

The title compound was synthesized via general procedure D using 2.14c (0.17 g, 0.46 1 mmol), which yielded a colorless wax (77 mg, 46%). H NMR (400 MHz, CDCl3) δ 8.23 (s, 1H), 7.40–7.32 (m, 4H), 7.07 (d, J = 8.2 Hz, 1H), 6.99 (q, J = 7.9, 7.5 Hz, 3H), 5.32 (s, 2H), 4.23 (t, J = 7.1 Hz, 2H), 4.08 (t, J = 5.6 Hz, 2H), 2.39 (s, 3H), 2.22 (p, J = 6.8, 6.3 Hz, 2H), 1.96 (h, J = 6.4, 5.9 Hz, 2H).

(Z)-16-Acetyl-3-methyl-6,7-dihydro-13H,15H-dibenzo[e,l][1,4,8]trioxacyclotridecin- 15-one (2.15d).

O O

O O O The title compound was synthesized via general procedure D using 2.14d (0.19 g, 0.51 1 mmol), which yielded a colorless wax (101 mg, 57%). H NMR (400 MHz, CDCl3) δ 7.48

122 (s, 1H), 7.42–7.33 (m, 2H), 7.20 (d, J = 7.7 Hz, 1H), 7.07–6.98 (m, 2H), 6.85 (d, J = 7.7 Hz, 1H), 6.76 (s, 1H), 5.36 (s, 2H), 4.34–4.27 (m, 4H), 2.43–2.36 (m, 6H).

(Z)-17-Acetyl-3-methyl-7,8-dihydro-6H,14H,16H- dibenzo[c,j][1,5,9]trioxacyclotetradecin-16-one (2.15e).

O O

O O O The title compound was synthesized via general procedure D using 2.14e (0.57 g, 1.5 1 mmol), which yielded a colorless wax (20 mg, 4%). H NMR (400 MHz, CDCl3) δ 7.52 (s, 1H), 7.35 (d, J = 7.8 Hz, 2H), 7.12 (d, J = 8.0 Hz, 1H), 7.07 (t, J = 7.2 Hz, 2H), 6.77 (d, J = 6.7 Hz, 2H), 5.16 (s, 2H), 4.38 (t, J = 5.5 Hz, 2H), 4.12 (t, J = 5.3 Hz, 2H), 2.36 (d, J = 3.7 Hz, 6H), 2.14 (p, J = 5.6 Hz, 2H).

(Z)-10-Acetyl-2,5,8-trioxa-1(1,3),6(1,2)-dibenzenacycloundecaphan-10-en-9-one (2.15f).

O O

O O The title compound was synthesized via general procedure D using 2.14f (0.12 g, 0.34 1 mmol), which yielded a colorless wax (22 mg, 20%). H NMR (400 MHz, CDCl3) δ 7.75 (s, 1H), 7.45 (s, 1H), 7.37 (ddd, J = 13.8, 7.4, 1.6 Hz, 2H), 7.31 (d, J = 8.0 Hz, 1H), 7.08– 6.96 (m, 3H), 6.93 (d, J = 8.2 Hz, 1H), 5.47 (s, 2H), 4.64–4.58 (m, 2H), 4.41–4.34 (m, 2H), 2.46 (s, 3H).

123 (Z)-10-Acetyl-16-methyl-2,5,8-trioxa-1(1,3),6(1,2)-dibenzenacycloundecaphan-10- en-9-one (2.15g).

O O

O O The title compound was synthesized via general procedure D using 2.14g (0.22 g, 0.59 1 mmol), which yielded a colorless wax (50 mg, 24%). H NMR (400 MHz, CDCl3) δ 7.71 (s, 1H), 7.43–7.33 (m, 3H), 7.17 (d, J = 7.6 Hz, 1H), 7.03–6.91 (m, 3H), 5.48 (s, 2H), 4.70– 4.60 (m, 2H), 4.44–4.32 (m, 2H), 2.44 (s, 3H), 2.28 (s, 3H).

(Z)-11-Acetyl-16-methyl-2,6,9-trioxa-1(1,3),7(1,2)-dibenzenacyclododecaphan-11-en- 10-one (2.15h).

The title compound was synthesized via general procedure D using 2.14h (0.35 g, 0.90 1 mmol), which yielded a colorless wax (93 mg, 28%). H NMR (400 MHz, CDCl3) δ 7.49 (s, 1H), 7.43–7.34 (m, 2H), 7.31 (d, J = 1.6 Hz, 1H), 7.18 (d, J = 7.6 Hz, 1H), 6.99 (t, J = 7.4 Hz, 1H), 6.96–6.90 (m, 2H), 5.39 (d, J = 4.7 Hz, 2H), 4.53 (t, J = 7.2 Hz, 2H), 4.19– 4.11 (m, 2H), 2.43 (s, 3H), 2.25 (d, J = 10.4 Hz, 5H).

(Z)-11-Acetyl-15-methyl-2,6,9-trioxa-1(1,2),7(1,3)-dibenzenacyclododecaphan-11-en- 10-one (2.15i).

The title compound was synthesized via general procedure D using 2.14i (0.18 g, 0.47 1 mmol), which yielded a colorless wax (23 mg, 13%). H NMR (400 MHz, CDCl3) δ 8.75 (s, 1H), 7.47 (s, 1H), 7.39 (d, J = 7.9 Hz, 1H), 7.29 (d, J = 15.5 Hz, 1H), 6.91–6.77 (m, 124 3H), 6.71 (s, 1H), 5.38 (s, 2H), 4.56–4.47 (m, 2H), 4.11–4.06 (m, 2H), 2.50 (s, 3H), 2.40 (s, 3H), 2.34–2.29 (m, 2H).

4.3.5. General Procedure E

R R

K2CO3 + Br OH o OH n DMF, 70 C O OH n CHO CHO

Hydroxybenzaldehyde (1 equiv) and K2CO3 (1.5 equiv) were added into DMF (10 mL) and stirred at rt for 0.5 h. 3-Bromopropan-1-ol or 4-bromobutan-1-ol (1 equiv) in DMF was added dropwise into the mixture and stirred at 70 °C overnight. Upon total consumption of the starting material, monitored by TLC, the mixture was poured into water and extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous MgSO4, and concentrated under reduced pressure. The residue was purified by column chromatography (24 gram RediSep Gold silica gel column, 0–50% ethyl acetate in hexanes) to give a colorless oil.

2-(3-Hydroxypropoxy)benzaldehyde (2.17a).

O OH CHO The title compound was synthesized via general procedure E using 2- hydroxybenzaldehyde (0.50 g, 3.6 mmol) and 3-bromopropan-1-ol (0.50 g, 3.6 mmol), 1 which yielded a colorless oil (0.27 g, 42%). H NMR (400 MHz, CDCl3) δ 10.41 (s, 1H), 7.83 (dd, J = 7.7, 1.7 Hz, 1H), 7.57 (td, J = 7.9, 7.2, 1.8 Hz, 1H), 7.12–7.01 (m, 2H), 4.27 (t, J = 5.9 Hz, 2H), 3.93 (q, J = 5.6 Hz, 2H), 2.24–2.17 (m, 1H), 2.14 (q, J = 5.8 Hz, 2H).

2-(4-Hydroxybutoxy)benzaldehyde (2.17b).

OH O CHO The title compound was synthesized via general procedure E using 2- hydroxybenzaldehyde (0.75 g, 6.1 mmol) and 4-bromobutan-1-ol (0.93 g, 6.1 mmol),

125 1 which yielded a colorless oil (0.15 g, 12%). H NMR (400 MHz, CDCl3) δ 10.51 (s, 1H), 7.85 (dd, J = 7.7, 1.7 Hz, 1H), 7.56 (ddd, J = 8.8, 7.3, 1.7 Hz, 1H), 7.08–6.99 (m, 2H), 4.16 (t, J = 6.2 Hz, 2H), 3.78 (q, J = 6.0 Hz, 2H), 2.04–1.96 (m, 2H), 1.87–1.77 (m, 2H), 1.51 (t, J = 5.3 Hz, 1H).

2-(3-Hydroxypropoxy)-4-methylbenzaldehyde (2.17c).

O OH CHO The title compound was synthesized via general procedure E using 2-hydroxy-4- methylbenzaldehyde (0.75 g, 5.5 mmol) and 3-bromopropan-1-ol (0.76 g, 5.5 mmol), 1 which yielded a colorless oil (0.76 g, 71%). H NMR (400 MHz, CDCl3) δ 10.30 (s, 1H), 7.70 (d, J = 7.8 Hz, 1H), 6.88 (d, J = 7.9 Hz, 1H), 6.83 (s, 1H), 4.25 (t, J = 5.9 Hz, 2H), 3.92 (q, J = 5.2 Hz, 2H), 2.42 (s, 3H), 2.14 (p, J = 5.8 Hz, 2H), 1.68 (d, J = 4.3 Hz, 1H).

2-(4-Hydroxybutoxy)-4-methylbenzaldehyde (2.17d).

OH O CHO The title compound was synthesized via general procedure E using 2-hydroxy-4- methylbenzaldehyde (0.75 g, 5.5 mmol) and 4-bromobutan-1-ol (0.84 g, 5.5 mmol), which 1 yielded a colorless oil (0.14 g, 12%). H NMR (400 MHz, CDCl3) δ 10.43 (s, 1H), 7.74 (d, J = 7.8 Hz, 1H), 6.86 (d, J = 7.9 Hz, 1H), 6.80 (s, 1H), 4.14 (t, J = 6.2 Hz, 2H), 3.78 (q, J = 5.9 Hz, 2H), 2.42 (s, 3H), 2.04–1.94 (m, 2H), 1.87–1.77 (m, 2H), 1.54 (t, J = 5.3 Hz, 1H).

4.3.6. General Procedure F

R R O O O + O NaOAc O OH n toluene O O O o n CHO MW to 130 C CHO A mixture of substrate (1 equiv), 2,2,6-trimethyl-4H-1,3-dioxin-4-one (TMD) (1.3 equiv), and anhydrous NaOAc (1 equiv) were charged into a microwave tube (5 mL). After adding

126 toluene (4 mL), the tube was sealed and heated to 130 °C for 20 min. After cooling to rt, the mixture was poured into water and extracted with ethyl acetate. The organic phase was washed with brine, dried over anhydrous MgSO4, and concentrated under reduced pressure. The residue was purified by flash column chromatography (4 gram RediSep Gold silica gel column, 0–50% ethyl acetate in hexanes) yield a colorless oil.

3-(2-Formylphenoxy)propyl 3-Oxobutanoate (2.18a).

O O

O O CHO The title compound was synthesized via general procedure F using 2.17a (0.12 g, 0.67 mmol) and TMD (0.12 g, 0.87 mmol), which yielded a colorless oil (52 mg, 30%). 1H

NMR (400 MHz, CDCl3) δ 10.52 (s, 1H), 7.86 (dd, J = 7.7, 1.9 Hz, 1H), 7.57 (ddd, J = 8.8, 7.4, 1.9 Hz, 1H), 7.07 (t, J = 7.5 Hz, 1H), 7.02 (d, J = 8.4 Hz, 1H), 4.41 (t, J = 6.2 Hz, 2H), 4.21 (t, J = 6.1 Hz, 2H), 3.50 (s, 2H), 2.29 (s, 3H), 2.25 (t, J = 6.1 Hz, 2H). 4-(2-Formylphenoxy)butyl 3-Oxobutanoate (2.18b).

O O CHO O O The title compound was synthesized via general procedure F using 2.17b (0.15 g, 0.75 mmol) and TMD (0.14 g, 0.98 mmol), which yielded a colorless oil (120 mg, 57%). 1H

NMR (400 MHz, CDCl3) δ 10.53 (s, 1H), 7.86 (dd, J = 7.7, 1.7 Hz, 1H), 7.57 (td, J = 8.2, 7.8, 1.6 Hz, 1H), 7.05 (t, J = 7.5 Hz, 1H), 7.00 (d, J = 8.5 Hz, 1H), 4.27 (t, J = 6.1 Hz, 2H), 4.15 (t, J = 5.8 Hz, 2H), 3.49 (s, 2H), 2.30 (s, 3H), 2.04–1.87 (m, 4H).

3-(2-Formyl-5-methylphenoxy)propyl 3-Oxobutanoate (2.18c).

O O

O O CHO The title compound was synthesized via general procedure F using 2.17c (0.76 g, 3.9 mmol) and 2,2,6-trimethyl-4H-1,3-dioxin-4-one (0.71 g, 5.0 mmol), which yielded a colorless oil 1 (232 mg, 21%). H NMR (400 MHz, CDCl3) δ 10.44 (s, 1H), 7.76 (d, J = 8.0 Hz, 1H), 6.87

127 (d, J = 7.9 Hz, 1H), 6.81 (s, 1H), 4.41 (t, J = 6.1 Hz, 2H), 4.19 (t, J = 6.1 Hz, 2H), 3.51 (s, 2H), 2.43 (s, 3H), 2.29 (s, 3H), 2.24 (q, J = 6.1 Hz, 2H).

4-(2-Formyl-5-methylphenoxy)butyl 3-Oxobutanoate (2.18d).

O O CHO O O The title compound was synthesized via general procedure F using 2.17d (0.14 g, 0.65 mmol) and 2,2,6-trimethyl-4H-1,3-dioxin-4-one (0.12 g, 0.85 mmol), which yielded a 1 colorless oil (125 mg, 66%). H NMR (400 MHz, CDCl3) δ 10.44 (s, 1H), 7.75 (d, J = 7.9 Hz, 1H), 6.85 (d, J = 7.9 Hz, 1H), 6.79 (s, 1H), 5.32 (d, J = 1.1 Hz, 2H), 4.26 (t, J = 6.1 Hz, 2H), 3.49 (s, 2H), 2.42 (s, 3H), 2.29 (s, 3H), 1.94–1.89 (m, 4H).

4.3.7. General Procedure G

R R

O O Piperidine O n O O n EtOH O CHO O O To an ethanol solution (30 mL) of the substrate (1 equiv) piperidine (0.1 equiv), and AcOH (0.1 equiv) were added and the yellow solution was stirred at rt for 24 h. Upon total consumption of the starting material, monitored by TLC, the mixture was poured into water and extracted with ethyl acetate. The organic phase was washed with brine, dried over anhydrous MgSO4, and concentrated under reduced pressure. The residue was purified by flash column chromatography (4 gram RediSep Gold silica gel column, 0–25% ethyl acetate in hexanes) to yield a white solid.

128 (Z)-7-Acetyl-3,4-dihydro-2H,6H-benzo[f][1,5]dioxecin-6-one (2.19a).

O O O The title compound was synthesized via general procedure G using 2.18a (0.40 g, 1.5 1 mmol), which yielded a white solid (64 mg, 16%). H NMR (400 MHz, CDCl3) δ 7.51 (s, 1H), 7.42–7.32 (m, 2H), 7.05 (t, J = 7.5 Hz, 1H), 6.95 (d, J = 8.3 Hz, 1H), 4.73 (s, 2H), 4.40 (t, J = 5.0 Hz, 2H), 2.46 (d, J = 1.1 Hz, 3H), 2.14 (s, 2H).

(Z)-8-Acetyl-2,3,4,5-tetrahydro-7H-benzo[g][1,6]dioxacycloundecin-7-one (2.19b).

O O

O O The title compound was synthesized via general procedure G using 2.18b (0.12 g, 0.43 1 mmol), which yielded a white solid (30 mg, 27%). H NMR (400 MHz, CDCl3) δ 7.47 (s, 1H), 7.39 (dt, J = 7.8, 3.8 Hz, 2H), 7.01 (t, J = 7.4 Hz, 1H), 6.90 (d, J = 8.7 Hz, 1H), 4.39 (t, J = 5.7 Hz, 2H), 4.05 (t, J = 5.1 Hz, 2H), 2.45 (s, 3H), 2.16 (p, J = 5.5 Hz, 2H), 2.06 (d, J = 6.8 Hz, 2H).

(Z)-7-Acetyl-11-methyl-3,4-dihydro-2H,6H-benzo[f][1,5]dioxecin-6-one (2.19c).

O O O The title compound was synthesized via general procedure G using 2.18c (0.23 g, 0.84 1 mmol), which yielded a white solid (61 mg, 28%). H NMR (400 MHz, CDCl3) δ 7.49 (s, 1H), 7.23 (d, J = 7.8 Hz, 1H), 6.86 (d, J = 7.8 Hz, 1H), 6.76 (s, 1H), 4.72 (s, 2H), 4.38 (t, J = 5.0 Hz, 2H), 2.45 (s, 3H), 2.38 (s, 3H), 2.13 (s, 2H).

129 (Z)-8-Acetyl-12-methyl-2,3,4,5-tetrahydro-7H-benzo[g][1,6]dioxacycloundecin-7-one (2.19d).

O O

O O The title compound was synthesized via general procedure G using 2.18d (0.13 g, 0.43 1 mmol), which yielded a white solid (30 mg, 26%). H NMR (400 MHz, CDCl3) δ 7.35 (s, 1H), 7.17 (d, J = 7.7 Hz, 1H), 6.72 (d, J = 7.7 Hz, 1H), 6.61 (s, 1H), 4.29 (t, J = 5.7 Hz, 2H), 3.94 (t, J = 5.1 Hz, 2H), 2.34 (s, 3H), 2.29 (s, 3H), 2.05 (p, J = 5.3 Hz, 2H), 1.96 (dd, J = 10.7, 4.4 Hz, 2H).

2-(3-(2-Formylphenoxy)propoxy)benzonitrile (2.21).

K2CO3 + O Br HO DMF, 70 oC O O CHO CN CHO CN The title compound was synthesized via general procedure B using 2.12b (1.2 g, 5.0 mmol) and 2-hydroxybenzonitrile (0.60 g, 5.0 mmol), which yielded a white solid (0.92 1 g, 65%). H NMR (400 MHz, CDCl3) δ 10.50 (s, 1H), 7.84 (dd, J = 7.7, 1.7 Hz, 1H), 7.63–7.52 (m, 3H), 7.12–6.99 (m, 4H), 4.39 (t, J = 5.9 Hz, 2H), 4.33 (t, J = 5.8 Hz, 2H), 2.44 (p, J = 5.9 Hz, 2H).

(2-(3-(2-(Aminomethyl)phenoxy)propoxy)phenyl)methanol (2.22).

LAH O O O O THF CHO CN o 0 to 50 C OH NH2 To a flame-dried two-neck round bottom flask (100 mL) connected to a nitrogen balloon, lithium aluminum hydride solution (16 mL, 2M in THF) was charged and cooled to 0 C. The system was purged with nitrogen gas three times. Intermediate 2.21 (0.92 g, 3.3 mmol) solution in anhydrous THF (20 mL) was added dropwise over 20 min. The mixture was heated to 50 C for 12 h. Then the cloudy solution was cooled to 0 C before the addition of water and 3M NaOH solution dropwise. After filtration, the filter cake was washed with 130 methanol (10 mL) three times and the combined organic phase was dried over anhydrous

MgSO4, which under reduced pressure yielded the crude product as white solid (920 mg, 98%) without further purification.

N-(2-(3-(2-(Hydroxymethyl)phenoxy)propoxy)benzyl)-3-oxobutanamide (2.23).

O toluene MW to 180 oC O O + O O O O O

OH NH2 O OH N H Compound 2.22 (0.92 g, 3.1 mmol) and 2,2,6-trimethyl-4H-1,3-dioxin-4-one (0.45 g, 3.1 mmol) were charged into a 5 mL microwave tube. After the addition of toluene (4 mL), the mixture was heated up to 180 C for 10 min. The dark brown solution was poured into water, extracted with ethyl acetate, and dried over anhydrous MgSO4. The target compound was purified via flash column chromatography (24 gram RediSep Gold silica gel column, 0–50% ethyl acetate in hexanes) as a light yellow oil (500 mg, 42%).1H

NMR (400 MHz, CDCl3) δ 7.48 (s, 1H), 7.34–7.25 (m, 4H), 7.01–6.90 (m, 4H), 4.71 (s, 2H), 4.47 (d, J = 5.8 Hz, 2H), 4.28 (dt, J = 11.9, 6.0 Hz, 4H), 3.33 (s, 2H), 2.41 (p, J = 6.0 Hz, 2H), 2.21 (s, 3H), 1.87 (d, J = 6.5 Hz,1H).

N-(2-(3-(2-Formylphenoxy)propoxy)benzyl)-3-oxobutanamide (2.24).

MnO2 O O O O O O O O DCM OH N H O N H H Intermediate 2.23 (0.50 g, 1.4 mmol) was dissolved in DCM followed by the addition of

MnO2 (1.2 g, 13.5 mmol). The mixture was stirred at rt for 12 h. After the filtration through Celite, the organic phase was purified via the flash column chromatography (24 gram RediSep Gold silica gel column, 0–50% ethyl acetate in hexanes), which yielded the title 1 compound as a light-yellow oil (372 mg, 72%). H NMR (400 MHz, CD2Cl2) δ 10.53 (d, J = 0.7 Hz, 1H), 7.82 (dd, J = 7.7, 1.8 Hz, 1H), 7.60 (ddd, J = 8.8, 7.3, 1.9 Hz, 1H), 7.38– 7.25 (m, 3H), 7.13 (d, J = 8.5 Hz, 1H), 7.07 (t, J = 7.6 Hz, 1H), 6.99–6.93 (m, 2H), 4.47 (d, J = 5.9 Hz, 2H), 4.39 (t, J = 6.1 Hz, 2H), 4.29 (t, J = 6.0 Hz, 2H), 3.38 (s, 2H), 2.45 (p, J = 6.0 Hz, 2H), 2.22 (s, 3H). 131 (E)-17-Acetyl-7,8,14,15-tetrahydro-6H,16H- dibenzo[f,m][1,5]dioxa[9]azacyclotetradecin-16-one (2.25).

O O

Piperidine N O O O O H EtOH O H O N H O The title compound was synthesized via general procedure G using 2.24 (0.37 g, 0.97 1 mmol), which yielded a yellow oil (100 mg, 28%). H NMR (400 MHz, CD2Cl2) δ 7.88 (s, 1H), 7.56 (s, 1H), 7.38–7.27 (m, 2H), 7.22 (dd, J = 7.6, 1.8 Hz, 1H), 7.12 (dt, J = 7.4, 1.4 Hz, 1H), 7.07–6.99 (m, 2H), 6.97–6.91 (m, 2H), 4.43 (d, J = 5.1 Hz, 2H), 4.30–4.23 (m, 4H), 2.45–2.35 (m, 5H).

4.3.8. General Procedure H

R R

O O O O O O NH AcOH n n + O NH O H2N N O 110 ˚C O O N N O O H

The macrocyclic intermediate (1 equiv) and 2.2 (1 equiv) were added into a 20 mL scintillation vial followed by the addition of AcOH (5 mL). The mixture was heated to reflux overnight, during which the reaction turned into a clear light brown solution. After cooling to rt, the solution was poured into a saturated NaHCO3 solution (50 mL) portionwise, stirred for 0.5 h to neutralize the acid, and extracted with ethyl acetate. The organic phase was washed with brine, dried over anhydrous MgSO4, and concentrated under reduced pressure. The residue was purified by flash column chromatography (4 gram RediSep Gold silica gel column, DCM + 2.5% methanol) to yield a white solid.

132 3-Ethyl-1-methyl-3,6b,12,13-tetrahydro-4H,19H- dibenzo[5',6':12',13'][1,4,8]trioxacyclotridecino[11',10':4,5]pyrido[2,3-d]pyrimidine- 4,6,21(2H,5H)-trione (2.16a).

O O O

O NH O N N O H

The title compound was synthesized via general procedure H using 2.15a (71 mg, 0.21 mmol), which yielded a white solid (24 mg, 31%); mp 311–312 °C dec, with a purity 98% 1 determined by qNMR. H NMR (400 MHz, DMSO-d6) δ 10.81 (s, 1H), 8.52 (s, 1H), 7.44– 7.33 (m, 2H), 7.10 (dd, J = 13.1, 7.7 Hz, 2H), 7.05–7.00 (m, 1H), 6.95 (t, J = 7.4 Hz, 1H), 6.87 (d, J = 8.2 Hz, 1H), 6.80 (t, J = 7.4 Hz, 1H), 5.34 (s, 1H), 5.15 (d, J = 11.1 Hz, 1H), 4.84 (d, J = 11.1 Hz, 1H), 4.61–4.52 (m, 1H), 4.34 (dd, J = 9.0, 3.6 Hz, 1H), 4.28–4.18 (m, 2H), 4.13–3.95 (m, 2H), 2.22 (s, 3H), 1.15 (t, J = 6.9 Hz, 3H).

3-Ethyl-1-methyl-3,6b,13,14-tetrahydro-4H,12H,20H- dibenzo[6',7':13',14'][1,5,9]trioxacyclotetradecino[12',11':4,5]pyrido[2,3- d]pyrimidine-4,6,22(2H,5H)-trione (2.16b).

O O O

O NH O N N O H

The title compound was synthesized via general procedure H using 2.15b (16 mg, 0.045 mmol), which yielded a white solid (7.4 mg, 33%); mp 285–286 °C dec, with a purity 99% 1 determined by qNMR. H NMR (400 MHz, DMSO-d6) δ 10.79 (s, 1H), 8.51 (s, 1H), 7.36 (d, J = 7.6 Hz, 2H), 7.24 (d, J = 8.1 Hz, 1H), 7.09 (t, J = 7.8 Hz, 1H), 6.98 (q, J = 9.3, 8.3 Hz, 3H), 6.79 (t, J = 7.4 Hz, 1H), 5.22 (s, 1H), 5.04 (d, J = 10.7 Hz, 1H), 4.92 (d, J = 10.9 Hz, 1H), 4.38 (dq, J = 8.8, 4.1 Hz, 2H), 4.27–4.15 (m, 2H), 4.09–3.94 (m, 2H), 2.32 (s, 3H), 2.22–2.11 (m, 2H), 1.12 (t, J = 6.9 Hz, 3H).

133 3-Ethyl-1-methyl-3,6b,12,13,14,15-hexahydro-4H,21H- dibenzo[3',4':11',12'][1,5,10]trioxacyclopentadecino[13',14':4,5]pyrido[2,3- d]pyrimidine-4,6,23(2H,5H)-trione (2.16c).

O O O O NH O N N O H

The title compound was synthesized via general procedure H using 2.15c (77 mg, 0.21 mmol), which yielded a white solid (17 mg, 16%); mp 228–229 °C dec, with a purity 95% 1 determined by qNMR. H NMR (400 MHz, DMSO-d6) δ 10.85 (s, 1H), 8.54 (s, 1H), 7.35 (dt, J = 7.6, 3.7 Hz, 2H), 7.12–7.00 (m, 2H), 6.98–6.88 (m, 3H), 6.79 (t, J = 7.4 Hz, 1H), 5.33 (s, 1H), 5.16 (d, J = 11.1 Hz, 1H), 4.94 (d, J = 11.1 Hz, 1H), 4.28 (dq, J = 14.3, 6.5, 5.9 Hz, 2H), 4.22–3.91 (m, 4H), 2.18 (s, 4H), 2.06–1.97 (m, 1H), 1.93–1.86 (m, 1H), 1.72– 1.69 (m, 1H), 1.14 (t, J = 6.9 Hz, 3H).

3-Ethyl-1,9-dimethyl-3,6b,12,13-tetrahydro-4H,19H- dibenzo[5',6':12',13'][1,4,8]trioxacyclotridecino[11',10':4,5]pyrido[2,3-d]pyrimidine- 4,6,21(2H,5H)-trione (2.16d).

O O O

O NH O N N O H

The title compound was synthesized via general procedure H using 2.15d (101 mg, 0.29 mmol), which yielded a white solid (52 mg, 37%); mp 232–233 °C dec, with a purity 99% 1 determined by qNMR. H NMR (400 MHz, DMSO-d6) δ 10.80 (s, 1H), 8.49 (s, 1H), 7.39 (t, J = 7.9 Hz, 1H), 7.35 (d, J = 7.4 Hz, 1H), 7.11 (d, J = 8.1 Hz, 1H), 6.95 (t, J = 7.4 Hz, 1H), 6.89 (d, J = 7.7 Hz, 1H), 6.69 (s, 1H), 6.61 (d, J = 7.8 Hz, 1H), 5.28 (s, 1H), 5.13 (d, J = 11.1 Hz, 1H), 4.84 (d, J = 11.0 Hz, 1H), 4.61–4.51 (m, 1H), 4.37–4.29 (m, 1H), 4.22 (t, J = 7.4 Hz, 2H), 4.12–3.95 (m, 2H), 2.22 (d, J = 3.1 Hz, 6H), 1.14 (t, J = 6.9 Hz, 3H). 13 C NMR (100 MHz, DMSO-d6) δ 167.5, 160.9, 158.1, 153.6, 150.0, 145.0, 140.1, 136.8,

134 132.7, 131.6, 130.5, 128.3, 124.6, 121.2, 120.5, 113.4, 111.6, 107.2, 90.1, 67.0, 65.3, 62.5, 36.1, 28.2, 20.9, 17.3, 13.6.

3-Ethyl-1,9-dimethyl-3,6b,13,14-tetrahydro-4H,12H,20H- dibenzo[6',7':13',14'][1,5,9]trioxacyclotetradecino[12',11':4,5]pyrido[2,3- d]pyrimidine-4,6,22(2H,5H)-trione (2.16e).

O O O

O NH O N N O H

The title compound was synthesized via general procedure H using 2.15e (20 mg, 0.055 mmol), which yielded a white solid (7 mg, 26%); mp 244–245 °C dec, with a purity 99% 1 determined by qNMR. H NMR (400 MHz, DMSO-d6) δ 10.77 (s, 1H), 8.48 (s, 1H), 7.35 (t, J = 8.2 Hz, 2H), 7.23 (d, J = 8.0 Hz, 1H), 6.99 (t, J = 7.4 Hz, 1H), 6.83 (d, J = 7.7 Hz, 1H), 6.78 (s, 1H), 6.60 (d, J = 7.8 Hz, 1H), 5.16 (s, 1H), 5.04 (d, J = 10.7 Hz, 1H), 4.91 (d, J = 10.7 Hz, 1H), 4.36 (dt, J = 8.1, 3.5 Hz, 2H), 4.25–4.13 (m, 2H), 4.07–3.95 (m, 2H), 2.31 (s, 3H), 2.25–2.16 (m, 5H), 1.11 (t, J = 6.9 Hz, 3H).

21-Ethyl-27-methyl-21,22,23,24,25,28-hexahydro-4,7,10-trioxa-2(5,6)-pyrido[2,3- d]pyrimidina-1(1,3),6(1,2)-dibenzenacyclodecaphane-22,24,3-trione (2.16f).

O O

O NH

N N O H

The title compound was synthesized via general procedure H using 2.15f (22 mg, 0.065 mmol), which yielded a white solid (9.9 mg, 31%); mp 297–298 °C dec, with a purity 98% 1 determined by qNMR. H NMR (400 MHz, DMSO-d6) δ 10.99 (s, 1H), 8.68 (s, 1H), 7.44 (d, J = 7.3 Hz, 1H), 7.38 (t, J = 7.8 Hz, 1H), 7.24 (s, 1H), 7.07 (t, J = 8.8 Hz, 2H), 6.96 (t, J = 7.4 Hz, 1H), 6.67 (d, J = 8.1 Hz, 1H), 6.56 (d, J = 7.7 Hz, 1H), 5.17 (d, J = 11.1 Hz, 1H), 4.86 (d, J = 11.1 Hz, 1H), 4.74 (s, 1H), 4.65 (dd, J = 14.4, 7.0 Hz, 1H), 4.54 (dd, J =

135 12.6, 4.4 Hz, 1H), 4.38 (dd, J = 12.2, 6.9 Hz, 2H), 4.15–3.92 (m, 2H), 2.35 (s, 3H), 1.16 (t, J = 7.0 Hz, 3H).

21-Ethyl-14,27-dimethyl-21,22,23,24,25,28-hexahydro-4,7,10-trioxa-2(5,6)-pyrido[2,3- d]pyrimidina-1(1,3),6(1,2)-dibenzenacyclodecaphane-22,24,3-trione (2.16g).

O O

O NH

N N O H

The title compound was synthesized via general procedure H using 2.15g (50 mg, 0.14 mmol), which yielded a white solid (12 mg, 17%); mp 311–312 °C dec, with a purity 92% 1 determined by qNMR. H NMR (400 MHz, DMSO-d6) δ 10.97 (s, 1H), 8.64 (s, 1H), 7.43 (dd, J = 7.4, 1.8 Hz, 1H), 7.38 (td, J = 7.9, 1.8 Hz, 1H), 7.17 (d, J = 1.7 Hz, 1H), 7.08 (d, J = 8.3 Hz, 1H), 7.02–6.89 (m, 2H), 6.46 (dd, J = 7.6, 1.6 Hz, 1H), 5.15 (d, J = 11.1 Hz, 1H), 4.85 (d, J = 11.0 Hz, 1H), 4.67 (d, J = 23.1 Hz, 2H), 4.56 (dd, J = 12.5, 4.7 Hz, 1H), 4.37 (ddd, J = 20.0, 13.3, 5.7 Hz, 2H), 4.06 (dh, J = 28.4, 7.0 Hz, 2H), 2.34 (s, 3H), 2.09 (s, 3H), 1.15 (t, J = 6.9 Hz, 3H).

21-Ethyl-14,27-dimethyl-21,22,23,24,25,28-hexahydro-4,7,11-trioxa-2(5,6)-pyrido[2,3- d]pyrimidina-1(1,3),6(1,2)-dibenzenacycloundecaphane-22,24,3-trione (2.16h).

O O

O NH

N N O H

The title compound was synthesized via general procedure H using 2.15h (93 mg, 0.25 mmol), which yielded a white solid (27 mg, 21%); mp 213–214 °C dec, with a purity 90% 1 determined by qNMR. H NMR (400 MHz, DMSO-d6) δ 11.01 (s, 1H), 8.63 (s, 1H), 7.45– 7.39 (m, 2H), 7.17 (d, J = 8.2 Hz, 1H), 7.09 (d, J = 1.7 Hz, 1H), 7.03–6.92 (m, 2H), 6.39 (dd, J = 7.7, 1.6 Hz, 1H), 5.22 (d, J = 11.0 Hz, 1H), 4.97 (d, J = 11.0 Hz, 1H), 4.80 (s, 1H),

136 4.41–4.21 (m, 3H), 4.17–3.92 (m, 3H), 2.29 (s, 3H), 2.20–2.15 (m, 2H), 2.09 (s, 3H), 1.14 (t, J = 6.9 Hz, 3H).

21-Ethyl-14,27-dimethyl-21,22,23,24,25,28-hexahydro-4,7,11-trioxa-2(5,6)-pyrido[2,3- d]pyrimidina-1(1,2),6(1,3)-dibenzenacycloundecaphane-22,24,3-trione (2.16i).

The title compound was synthesized via general procedure H using 2.15i (23 mg, 0.063 mmol), which yielded a white solid (8 mg, 25%); mp 247–248 °C dec, with a purity of 95% 1 determined by qNMR. H NMR (400 MHz, DMSO-d6) δ 10.83 (s, 1H), 8.65 (s, 1H), 7.94– 7.78 (m, 1H), 7.19 (t, J = 7.8 Hz, 1H), 7.02 (d, J = 7.9 Hz, 1H), 6.82–6.76 (m, 2H), 6.74 (d, J = 1.6 Hz, 1H), 6.66 (dd, J = 8.0, 1.5 Hz, 1H), 5.65 (s, 1H), 5.09 (d, J = 14.4 Hz, 1H), 4.85 (d, J = 14.3 Hz, 1H), 4.78 (ddd, J = 12.3, 8.9, 6.2 Hz, 1H), 4.34 (ddd, J = 11.6, 7.7, 3.3 Hz, 1H), 4.11–3.98 (m, 4H), 2.51 (s, 3H), 2.18 (s, 5H), 1.12 (t, J = 6.9 Hz, 3H).

4.3.9. General Procedure I

R R

O NH AcOH O O n + n O H2N N O 110 ˚C O NH O O O N N O H

The macrocyclic intermediate (1 equiv) and 2.2 (1 equiv) were added into a 20 mL vial followed by the addition of AcOH (5 mL). The mixture was heated to reflux overnight, during which the reaction turned into a clear light brown solution. After cooling to rt, the solution was poured into a saturated NaHCO3 solution (50 mL) portionwise, stirred for 0.5 h to neutralize the acid, and extracted with ethyl acetate. The organic phase was washed with brine, dried over anhydrous MgSO4, and concentrated under reduced pressure. The residue was purified by flash column chromatography (4 gram RediSep Gold silica gel column, DCM + 2.5% methanol) to yield a white solid.

137 4-Ethyl-6-methyl-5,10,11,16b-tetrahydro-1H,9H- benzo[9',10'][1,5]dioxecino[8',7':4,5]pyrido[2,3-d]pyrimidine-1,3,7(2H,4H)-trione (2.20a).

O O

O NH O N N O H

The title compound was synthesized via general procedure I using 2.19a (64 mg, 0.26 mmol), which yielded a white solid (7 mg, 8%); mp 313–314 °C dec, with a purity 99% 1 determined by qNMR. H NMR (400 MHz, DMSO-d6) δ 10.90 (s, 1H), 8.41 (s, 1H), 7.10 (t, J = 7.6 Hz, 1H), 7.00–6.92 (m, 2H), 6.87 (t, J = 7.4 Hz, 1H), 5.40 (s, 1H), 4.72 (t, J = 9.4 Hz, 1H), 4.55–4.45 (m, 1H), 4.19–4.11 (m, 1H), 4.04 (m, 3H), 2.11 (s, 3H), 1.93 (d, J = 12.0 Hz, 1H), 1.81–1.71 (m, 1H), 1.18 (t, J = 7.0, 3H).

4-Ethyl-6-methyl-5,9,10,11,12,17b-hexahydro-1H- benzo[10',11'][1,6]dioxacycloundecino[9',8':4,5]pyrido[2,3-d]pyrimidine- 1,3,7(2H,4H)-trione (2.20b).

O O O NH O O N N H The title compound was synthesized via general procedure I using 2.19b (30 mg, 0.13 mmol), which yielded a white solid (11 mg, 23%); mp 295–296 °C dec, with a purity 95% 1 determined by qNMR. H NMR (400 MHz, DMSO-d6) δ 10.99 (s, 1H), 8.61 (s, 1H), 7.11 (dt, J = 8.6, 4.6 Hz, 1H), 6.85 (d, J = 8.1 Hz, 1H), 6.79 (d, J = 4.5 Hz, 2H), 5.36 (s, 1H), 4.47 (d, J = 11.4 Hz, 1H), 4.07 (m, 3H), 3.96–3.77 (m, 2H), 2.18 (s, 3H), 2.02–1.81 (m, 4H), 1.17 (t, J = 6.9 Hz, 3H).

138 4-Ethyl-6,14-dimethyl-5,10,11,16b-tetrahydro-1H,9H- benzo[9',10'][1,5]dioxecino[8',7':4,5]pyrido[2,3-d]pyrimidine-1,3,7(2H,4H)-trione (2.20c).

O O

O NH O N N O H

The title compound was synthesized via general procedure I using 2.19c (61 mg, 0.24 mmol), which yielded a white solid (16 mg, 18%); mp 311–312 °C dec; with a purity 94% 1 determined by qNMR. H NMR (400 MHz, DMSO-d6) δ 10.89 (s, 1H), 8.37 (s, 1H), 6.77– 6.82 (m, 2H), 6.67 (d, J = 7.9 Hz, 1H), 5.34 (s, 1H), 4.71 (m, 1H), 4.52–4.42 (m, 1H), 4.17– 3.95 (m, 4H), 2.21 (s, 3H), 2.11 (s, 3H), 1.96–1.86 (m, 1H), 1.80–1.70 (m, 1H), 1.17 (t, J = 6.9 Hz, 3H).

4-Ethyl-6,15-dimethyl-5,9,10,11,12,17b-hexahydro-1H- benzo[10',11'][1,6]dioxacycloundecino[9',8':4,5]pyrido[2,3-d]pyrimidine- 1,3,7(2H,4H)-trione (2.20d).

O O O NH O O N N H The title compound was synthesized via general procedure I using 2.19d (30 mg, 0.12 mmol), which yielded a white solid (14 mg, 30%); mp 300–301 °C dec; with a purity 92% 1 determined by qNMR. H NMR (400 MHz, DMSO-d6) δ 10.97 (s, 1H), 8.57 (s, 1H), 6.69– 6.62 (m, 2H), 6.59 (d, J = 7.8 Hz, 1H), 5.30 (s, 1H), 4.46 (d, J = 11.4 Hz, 1H), 4.06 (ddd, J = 41.4, 14.3, 7.1 Hz, 3H), 3.96–3.77 (m, 2H), 2.22 (s, 2H), 2.17 (s, 3H), 1.96–1.81 (m, 5H), 1.17 (t, J = 6.9 Hz, 3H).

139 3-Ethyl-1-methyl-3,6b,13,14,20,21-hexahydro-4H,12H- dibenzo[f,m]pyrimido[5',4':5,6]pyrido[3,4-k][1,5]dioxa[9]azacyclotetradecine- 4,6,22(2H,5H)-trione (2.26).

O O O O O O N NH AcOH H + N NH H O O H2N N O 110 ˚C N N O H O The title compound was synthesized via general procedure I using 2.25 (100 mg, 0.28 mmol), which yielded a white solid (25 mg, 18%); mp 249–250 °C dec; with a purity 94% 1 determined by qNMR. H NMR (400 MHz, DMSO-d6) δ 10.82 (s, 1H), 8.36 (s, 1H), 8.18 (dd, J = 8.4, 3.9 Hz, 1H), 7.26 (td, J = 7.8, 1.7 Hz, 1H), 7.20–7.11 (m, 4H), 7.08 (ddd, J = 8.0, 3.1, 1.1 Hz, 2H), 6.95 (td, J = 7.4, 1.3 Hz, 1H), 6.86 (td, J = 7.4, 1.0 Hz, 1H), 4.98 (s, 1H), 4.62 (ddd, J = 10.4, 8.2, 2.4 Hz, 1H), 4.52 (dd, J = 13.5, 8.3 Hz, 1H), 4.42–4.23 (m, 3H), 4.13–3.87 (m, 2H), 2.40 (s, 4H), 2.31–2.20 (m, 1H), 1.12 (t, J = 6.9 Hz, 3H).

4.4. Bifunctional Molecules: PROTACs 2-(2,6-Dioxopiperidin-3-yl)-4-hydroxyisoindoline-1,3-dione (2.33).

Based on a literature procedue,127 3-hydroxyphthalic anhydride (1.0 g, 6.1 mmol) and 3- aminoperidine-2,6-dione hydrochloride (1.0 g, 6.1 mmol) were mixed in toluene (50 mL) in a round-bottom flask. After charging TEA (0.9 mL, 6.7 mmol), the resulting mixture was heated to reflux for 12 h using a Dean-Stark trap apparatus. After cooling to rt, the solvent was evaporated under reduced pressure. The residue was re-dissolved in ethyl acetate and the organic phase was washed with brine, dried over anhydrous MgSO4, and concentrated under reduced pressure, and purified by flash column chromatography (40 gram RediSep Gold silica gel column, DCM + 2.5% methanol) to yield the desired product 1 2.33 as a slightly yellow solid (0.9 g, 47% yield). H NMR (400 MHz, DMSO-d6) δ 11.20 (s, 1H), 11.09 (s, 1H), 7.66 (t, J = 7.8 Hz, 1H), 7.33 (d, J = 7.2 Hz, 1H), 7.26 (d, J = 8.4

140 Hz, 1H), 5.08 (dd, J = 12.9, 5.4 Hz, 1H), 2.95–2.83 (m, 1H), 2.64–2.53 (m, 2H), 2.09–2.01 (m, 1H).

2-(2,6-Dioxopiperidin-3-yl)-4-((7-hydroxyheptyl)oxy)isoindoline-1,3-dione (2.34).

OH O O 7-bromoheptan-1-ol, HO O O O NH KI, KHCO3 NH N O N O DMF O O To a solution of 2.33 (0.44 g, 1.6 mmol) in DMF (10 mL), KI (26 mg, 0.16 mmol),

NaHCO3 (0.27 g, 3.2 mmol), and 7-bromo-1-heptanol (0.38 g, 1.9 mmol) were added sequentially. The mixture was heated to 60 °C and stirred for 12 h. After cooling to rt, the solution was poured into water and extracted with ethyl acetate. The organic phase was washed with brine, dried over anhydrous MgSO, concentrated under reduced pressure, and purified by flash column chromatography (24 gram RediSep Gold silica gel column, DCM + 3% methanol) to yield the desired product 2.34 as a pale yellow solid (0.38 g, 61% yield). 1 H NMR (400 MHz, DMSO-d6) δ 11.11 (s, 1H), 7.81 (t, J = 7.9 Hz, 1H), 7.52 (d, J = 8.5 Hz, 1H), 7.45 (d, J = 7.2 Hz, 1H), 5.09 (dd, J = 12.9, 5.3 Hz, 1H), 4.33 (t, J = 5.1 Hz, 1H), 4.21 (t, J = 6.4 Hz, 2H), 3.39 (q, J = 6.1 Hz, 2H), 2.96–2.81 (m, 1H), 2.65–2.53 (m, 2H), 2.08–1.99 (m, 1H), 1.82–1.71 (m, 2H), 1.52–1.39 (m, 4H), 1.37–1.27 (m, 4H).

7-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)heptyl Methanesulfonate (2.35A).

O S O O O HO O O O O O NH MsCl, TEA NH N O N O DCM, 0 oC O O Intermediate 2.34 (0.30 g, 0.77 mmol) and triethylamine (0.12 mL, 1.5 mmol) were dissolved in DCM (15 mL) and cooled to 0 °C. Methanesulfonyl chloride (0.32 mL, 2.3 mmol) was added dropwise into the system. After 2 h, the mixture was diluted with ethyl acetate, washed with brine, and dried over anhydrous MgSO4. The crude compound was purified by flash column chromatography (12 gram RediSep Gold silica gel column, DCM + 2% methanol) to yield the desired product 2.35A as a pale yellow solid (0.27 g, 74%

141 1 yield). H NMR (400 MHz, DMSO-d6) δ 11.11 (s, 1H), 7.81 (t, J = 7.6 Hz, 1H), 7.52 (d, J = 8.5 Hz, 1H), 7.45 (d, J = 7.2 Hz, 1H), 5.09 (dd, J = 12.9, 5.4 Hz, 1H), 4.26–4.15 (m, 4H), 3.16 (d, J = 1.0 Hz, 3H), 2.96–2.83 (m, 1H), 2.64–2.54 (m, 2H), 2.10–1.99 (m, 1H), 1.82– 1.73 (m, 2H), 1.72–1.63 (m, 2H), 1.52–1.44 (m, 2H), 1.42–1.35 (m, 4H).

4-((7-Azidoheptyl)oxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (2.35).

O S N3 O O O O O O O O NH NH NaN3 N O N O DMF, 70 oC O O Intermediate 2.35A (0.27 g, 0.57 mmol) was dissolved in anhydrous DMF (10 mL) and sodium azide (0.65 g, 10 mmol) was added. The mixture was stirred at 45 ºC for 16 h. After cooling to rt, the solvent was removed under reduced pressure. The residue was re- dissolved with methanol (10 mL), loaded on to silica gel, and purified by flash column chromatography (12 gram RediSep Gold silica gel column, DCM + 2% methanol) to yield the desired product 2.35 as a pale-yellow solid (94 mg, 42% yield). 1H NMR (400 MHz,

DMSO-d6) δ 11.11 (s, 1H), 7.82 (t, J = 7.9 Hz, 1H), 7.52 (d, J = 8.5 Hz, 1H), 7.45 (d, J = 7.2 Hz, 1H), 5.09 (dd, J = 12.9, 5.4 Hz, 1H), 4.22 (t, J = 6.4 Hz, 2H), 3.37–3.28 (m, 2H), 2.95–2.83 (m, 1H), 2.64–2.54 (m, 2H), 2.08–1.99 (m, 1H), 1.77 (p, J = 6.7 Hz, 2H), 1.60– 1.51 (m, 2H), 1.50–1.43 (m, 2H), 1.42–1.30 (m, 4H).

2-Chloro-N-(prop-2-yn-1-yl)acetamide.

O O K2CO3 Cl Cl + H2N N Cl H THF, 0 oC 2-Chloro-N-(prop-2-yn-1-yl)acetamide was synthesized based on a reported procedure.196 A solution of propargylamine (0.58 mL, 9.0 mmol) and triethylamine (1.3 mL, 9.0 mmol) in dry THF (20 mL) was cooled to 0 ºC (ice-bath) under N2. Chloroacetyl chloride (0.72 mL, 9.0 mmol) was added slowly through a septum to the solution which was kept at 0 ºC for 45 min and then stirred at rt for 2 h. The solvent was removed under reduced pressure and the residue was dissolved in DCM (100 mL). The resulting solution was washed sequentially with 10% HCl solution (50 mL) and water (50 mL); the aqueous layers were 142 then reextracted with DCM (100 mL). The organic layers were combined, dried over anhydrous MgSO4 and evaporated under reduced pressure to afford target molecule as an 1 off-white solid (0.89 g, 75%). H NMR (400 MHz, CDCl3) δ 6.77 (s, 1H), 4.14 (dd, J = 5.5, 2.5 Hz, 2H), 4.10 (s, 2H), 2.31 (t, J = 2.6 Hz, 1H).

4.4.1. General Procedure J

O Cl OH K CO , NaI O HN N + 2 3 H DMF, reflux CHO CHO O To a solution of the hydroxybenzaldehyde (1 equiv) in acetonitrile (50 mL) was added NaI

(0.1 equiv) and K2CO3 (1.1 equiv). The reaction mixture was refluxed for 45 min. Then chloroacetamide (1 equiv) was added dropwise and the final mixture was refluxed overnight. The reaction mixture was diluted with water and DCM. The layers were separated and the aqueous layer was extracted twice with DCM. The combined organic layers were washed twice with water, dried over anhydrous MgSO4, filtered and concentrated. The obtained oil was purified by flash chromatography (40 gram RediSep Gold silica gel column, 0–50% ethyl acetate in hexanes).

2-(3-Formylphenoxy)-N-(prop-2-yn-1-yl)acetamide (2.27a).

O O N H

CHO The title compound was synthesized via general procedure J using 3-hydroxybenzaldehyde 1 (2.5 g, 20 mmol), which yielded a colorless oil (2.5 g, 56%). H NMR (400 MHz, CDCl3) δ 10.02 (d, J = 0.8 Hz, 1H), 7.61–7.51 (m, 2H), 7.47–7.43 (m, 1H), 7.27–7.22 (m, 1H), 6.79 (s, 1H), 4.60 (s, 2H), 4.20 (dd, J = 5.6, 2.5 Hz, 2H), 2.37–2.24 (m, 1H).

143 2-(2-Formylphenoxy)-N-(prop-2-yn-1-yl)acetamide (2.27b).

CHO O O N H

The title compound was synthesized via general procedure J using 3-hydroxybenzaldehyde 1 (2.5 g, 20 mmol), which yielded as a colorless oil (2.8 g, 63%). H NMR (400 MHz, CDCl3) δ 10.20 (s, 1H), 7.95 (s, 1H), 7.81 (dd, J = 7.7, 1.7 Hz, 1H), 7.63 (td, J = 7.8, 1.7 Hz, 1H), 7.23 (t, J = 7.5 Hz, 1H), 6.96 (d, J = 8.3 Hz, 1H), 4.64 (s, 2H), 4.23 (dd, J = 5.6, 2.5 Hz, 2H), 2.32 (t, J = 2.6 Hz, 1H).

4.4.2. General Procedure K

O HN O O O O O HN NH O O AcOH, reflux O NH + + OBn H2N N O O BnO CHO O N N O H The benzaldehyde intermediate (1 equiv), 2.2 (1 equiv), and ethyl 4-(benzyloxy)-3- oxobutanoate (1 equiv) were added into a 20 mL vial followed by the addition of AcOH (10 mL). The mixture was heated to reflux overnight, during which the reaction turned into a clear light brown solution. After cooling to rt, the solution was poured into a saturated

NaHCO3 solution (50 mL) portionwise, stirred for 0.5 h to neutralize the acid, and extracted with ethyl acetate. The organic phase was washed with brine, dried over anhydrous MgSO4, and concentrated under reduced pressure. The residue was purified by flash column chromatography (24 gram RediSep Gold silica gel column, DCM + 2.5% methanol) to yield a yellow solid.

144 Ethyl 7-((Benzyloxy)methyl)-1-ethyl-2,4-dioxo-5-(3-(2-oxo-2-(prop-2-yn-1- ylamino)ethoxy)phenyl)-1,2,3,4,5,8-hexahydropyrido[2,3-d]pyrimidine-6- carboxylate (2.28a).

N H O

O O

O NH BnO N N O H

The title compound was synthesized via general procedure K using 2.27a (2.5 g, 12 mmol), 1 which yielded a yellow solid (1.1 g, 16%). H NMR (400 MHz, DMSO-d6) δ 11.03 (s, 1H), 8.55 (d, J = 7.1 Hz, 2H), 7.44–7.28 (m, 5H), 7.16 (t, J = 7.9 Hz, 1H), 6.84 (d, J = 7.7 Hz, 1H), 6.80 (s, 1H), 6.72 (dd, J = 8.5, 2.4 Hz, 1H), 5.01 (d, J = 13.9 Hz, 1H), 4.93 (s, 1H), 4.66–4.55 (m, 3H), 4.42 (s, 2H), 4.05 (q, J = 7.2 Hz, 3H), 3.99–3.84 (m, 3H), 3.10 (q, J = 2.1 Hz, 1H), 1.18–1.13 (m, 3H), 1.09 (t, J = 7.0 Hz, 3H).

Ethyl 7-((Benzyloxy)methyl)-1-ethyl-2,4-dioxo-5-(2-(2-oxo-2-(prop-2-yn-1- ylamino)ethoxy)phenyl)-1,2,3,4,5,8-hexahydropyrido[2,3-d]pyrimidine-6- carboxylate (2.28b).

NH O O O NH O N N O H BnO The title compound was synthesized via general procedure K using 2.27b (2.8 g, 13 mmol), 1 which yielded a yellow solid (2.1 g, 29%). H NMR (400 MHz, DMSO-d6) δ 11.04 (s, 1H), 8.76 (t, J = 5.9 Hz, 1H), 8.43 (s, 1H), 7.42–7.28 (m, 5H), 7.20–7.15 (m, 1H), 7.11 (t, J = 8.4 Hz, 1H), 6.87 (t, J = 7.4 Hz, 1H), 6.82 (d, J = 8.3 Hz, 1H), 5.37 (s, 1H), 4.96 (d, J = 14.1 Hz, 1H), 4.65–4.58 (m, 4H), 4.49 (d, J = 14.9 Hz, 1H), 4.04 (q, J = 7.3 Hz, 2H), 3.95 (t, J = 6.8 Hz, 3H), 3.92–3.80 (m, 1H), 3.07 (d, J = 2.5 Hz, 1H), 1.11 (t, J = 7.1 Hz, 3H), 1.06 (t, J = 7.1 Hz, 3H).

145 4.4.3. General Procedure L

O HN O HN O O BBr O O O 3 O O NH DCM, - 78 oC O NH BnO HO N N O N N O H H

Removal of the benzyl ether was conducted according to a modified procedure from the literature.3 The substrate (1 equiv) was dissolved in dry DCM (25 mL) in a two-necked round-bottomed flask (100 mL). The resulting yellow solution was cooled to –78 °C before the addition of BBr3 (1 M solution, 3 equiv) dropwise. The solution was stirred for an additional 0.5 h after the removal of the dry ice bath. After the total consumption of the starting material monitored by TLC, the reaction was quenched by the addition of methanol and a saturated NaHCO3 solution. The mixture was extracted with ethyl acetate and the organic phase was washed with brine, dried over anhydrous MgSO4, and concentrated under reduced pressure. The residue was then purified by flash column chromatography (24 gram RediSep Gold silica gel column, DCM + 2.5% methanol) to yield the title compound as a white solid.

Ethyl 1-Ethyl-7-(hydroxymethyl)-2,4-dioxo-5-(3-(2-oxo-2-(prop-2-yn-1- ylamino)ethoxy)phenyl)-1,2,3,4,5,8-hexahydropyrido[2,3-d]pyrimidine-6- carboxylate (2.29a).

N H O

O O

O NH HO N N O H

The title compound was synthesized via general procedure L using 2.28a (1.1 g, 1.8 mmol), 1 which yielded a yellow solid (0.50 g, 56%). H NMR (400 MHz, DMSO-d6) δ 11.03 (s, 1H), 8.56 (t, J = 5.7 Hz, 1H), 8.42 (s, 1H), 7.16 (t, J = 7.9 Hz, 1H), 6.85 (d, J = 7.7 Hz, 1H), 6.80 (t, J = 1.9 Hz, 1H), 6.71 (dd, J = 8.2, 2.5 Hz, 1H), 6.05–5.98 (m, 1H), 4.92 (s, 1H), 4.82 (dd, J = 16.3, 4.9 Hz, 1H), 4.59 (dd, J = 16.3, 5.2 Hz, 1H), 4.42 (s, 2H), 4.05 (qd,

146 J = 7.4, 3.0 Hz, 3H), 3.98–3.85 (m, 3H), 3.10 (d, J = 2.5 Hz, 1H), 1.19 (dt, J = 14.4, 7.0 Hz, 6H).

Ethyl 1-Ethyl-7-(hydroxymethyl)-2,4-dioxo-5-(2-(2-oxo-2-(prop-2-yn-1- ylamino)ethoxy)phenyl)-1,2,3,4,5,8-hexahydropyrido[2,3-d]pyrimidine-6- carboxylate (2.29b).

NH O O O NH O N N O H HO The title compound was synthesized via general procedure L using 2.28b (2.1 g, 3.7 mmol), 1 which yielded a yellow solid (0.90 g, 49%). H NMR (400 MHz, DMSO-d6) δ 11.04 (s, 1H), 8.79 (t, J = 5.8 Hz, 1H), 8.36 (s, 1H), 7.18 (dd, J = 7.6, 1.8 Hz, 1H), 7.10 (td, J = 7.8, 7.4, 1.6 Hz, 1H), 6.87 (t, J = 7.4 Hz, 1H), 6.81 (d, J = 8.3 Hz, 1H), 5.99 (t, J = 5.5 Hz, 1H), 5.35 (s, 1H), 4.79 (dd, J = 16.3, 5.2 Hz, 1H), 4.69–4.55 (m, 2H), 4.49 (d, J = 14.9 Hz, 1H), 4.08–3.80 (m, 6H), 3.06 (t, J = 2.6 Hz, 1H), 1.22 (t, J = 7.1 Hz, 3H), 1.07 (t, J = 7.1 Hz, 3H).

4.4.4. General Procedure M

O HN O HN

O O Imidazole, SO2Cl2 O O O O O NH DMF, 0 oC O NH HO Cl N N O N N O H H

4 Based on a reported method, a solution of SO2Cl2 (2 equiv) in DMF (10 mL) was added dropwise over 5 min to a stirred, ice-cooled solution of the hydroxyl intermediate (1 equiv) and imidazole (2.6 equiv) in DMF. After the removal of the ice bath, the mixture was stirred at rt for 0.5 h, diluted with ethyl acetate, washed with water, and dried over anhydrous

MgSO4. The solvent was removed under reduced pressure and the residue was purified by flash column chromatography (24 gram RediSep Gold silica gel column, DCM + 2.5% methanol) to yield the title compound as a light-yellow solid.

147 Ethyl 7-(Chloromethyl)-1-ethyl-2,4-dioxo-5-(3-(2-oxo-2-(prop-2-yn-1- ylamino)ethoxy)phenyl)-1,2,3,4,5,8-hexahydropyrido[2,3-d]pyrimidine-6- carboxylate (2.30a).

N H O

O O

O NH Cl N N O H

The title compound was synthesized via general procedure M using 2.29a (0.50 g, 1.0 1 mmol), which yielded a pale-yellow solid (0.39 g, 76%). H NMR (400 MHz, DMSO-d6) δ 11.07 (d, J = 4.1 Hz, 1H), 9.07 (s, 1H), 8.56 (t, J = 5.8 Hz, 1H), 7.17 (td, J = 7.9, 2.7 Hz, 1H), 6.85 (d, J = 7.8 Hz, 1H), 6.80 (d, J = 2.3 Hz, 1H), 6.73 (dd, J = 8.2, 2.4 Hz, 1H), 5.22 (d, J = 11.0 Hz, 1H), 4.94 (d, J = 5.8 Hz, 1H), 4.74 (d, J = 11.0 Hz, 1H), 4.43 (d, J = 2.8 Hz, 2H), 4.09 (dq, J = 13.9, 7.0 Hz, 3H), 3.99–3.86 (m, 3H), 3.13–3.07 (m, 1H), 1.23–1.08 (m, 6H).

Ethyl 7-(Chloromethyl)-1-ethyl-2,4-dioxo-5-(2-(2-oxo-2-(prop-2-yn-1- ylamino)ethoxy)phenyl)-1,2,3,4,5,8-hexahydropyrido[2,3-d]pyrimidine-6- carboxylate (2.30b).

NH O O O NH O N N O H Cl The title compound was synthesized via general procedure M using 2.29b (0.90 g, 1.8 1 mmol), which yielded a pale-yellow solid (0.20 g, 22%). H NMR (400 MHz, DMSO-d6) δ 11.08 (d, J = 6.9 Hz, 1H), 8.96 (s, 1H), 8.76 (td, J = 11.2, 5.6 Hz, 1H), 7.20–7.08 (m, 2H), 6.89 (td, J = 7.4, 4.2 Hz, 1H), 6.82 (d, J = 8.2 Hz, 1H), 5.39 (s, 1H), 5.17 (d, J = 10.8 Hz, 1H), 4.77 (d, J = 11.0 Hz, 1H), 4.63 (d, J = 15.0 Hz, 1H), 4.49 (dd, J = 15.1, 2.0 Hz, 1H), 4.04 (ddt, J = 38.6, 14.1, 7.0 Hz, 5H), 3.88–3.81 (m, 1H), 3.07 (t, J = 2.5 Hz, 1H), 1.17 (q, J = 7.4 Hz, 3H), 1.14–1.06 (m, 3H).

148 4.4.5. General Procedure N

O O RNH2 O O NH NH O R N N N O N N O H H Cl The chloromethyl intermediate (1 equiv) and the primary amine (1.1 equiv) were added into a dry microwave tube (2 mL) containing ethanol (1 mL). The mixture was heated to 120 °C for 30 sec in a microwave reactor. The mixture was purified by flash column chromatography (4 gram RediSep Gold silica gel column, DCM + 10% methanol) to yield the desired compound.

2-(3-(1-Ethyl-7-(4-hydroxyphenethyl)-2,4,6-trioxo-2,3,4,5,6,7,8,9-octahydro-1H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidin-5-yl)phenoxy)-N-(prop-2-yn-1-yl)acetamide (2.31a).

N H O

O O NH

HO N N O H

The title compound was synthesized via general procedure N using 2.30a (0.39 g, 0.78 mmol), which yielded a yellow solid (211 mg, 49%), with a purity 97% determined by 1 UPLC. H NMR (400 MHz, DMSO-d6) δ 10.42 (s, 1H), 9.18 (s, 1H), 8.80 (s, 1H), 8.55 (t, J = 5.7 Hz, 1H), 7.16 (t, J = 7.9 Hz, 1H), 6.90 (d, J = 6.7 Hz, 2H), 6.83 (d, J = 8.0 Hz, 2H), 6.73 (dd, J = 8.2, 2.4 Hz, 1H), 6.60 (d, J = 8.0 Hz, 2H), 5.17 (d, J = 16.5 Hz, 1H), 5.03 (d, J = 16.5 Hz, 1H), 4.91 (s, 1H), 4.44 (s, 2H), 4.03 (q, J = 6.9 Hz, 2H), 3.91 (dd, J = 5.8, 2.5 Hz, 2H), 3.43–3.37 (m, 2H), 3.10 (d, J = 2.5 Hz, 1H), 2.70–2.56 (m, 2H), 1.12 (d, J = 6.8 13 Hz, 3H). C NMR (100 MHz, DMSO-d6) δ 175.0, 172.8, 167.6, 162.1, 157.5, 155.7, 155.6, 151.0, 148.3, 129.6, 128.8, 128.3, 120.3, 115.1, 114.3, 111.5, 93.2, 89.4, 81.0, 75.1, 72.8, 66.8, 43.4, 35.7, 34.4, 32.8, 27.7, 14.0.

149 2-(2-(1-Ethyl-7-(4-hydroxyphenethyl)-2,4,6-trioxo-2,3,4,5,6,7,8,9-octahydro-1H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidin-5-yl)phenoxy)-N-(prop-2-yn-1-yl)acetamide (2.31b).

NH O O O NH N HO N N O H

The title compound was synthesized via general procedure N using 2.30b (0.12 g, 0.23 1 mmol), which yielded a yellow solid (27 mg, 21%). H NMR (400 MHz, DMSO-d6) δ 10.43 (s, 1H), 9.16 (s, 1H), 8.95 (s, 1H), 8.36 (d, J = 6.1 Hz, 1H), 7.15 (t, J = 7.5 Hz, 1H), 7.01–6.91 (m, 2H), 6.82 (d, J = 8.4 Hz, 1H), 6.76 (d, J = 8.0 Hz, 2H), 6.56 (d, J = 8.0 Hz, 2H), 5.22 (s, 1H), 4.99 (s, 2H), 4.59 (s, 2H), 4.11–4.00 (m, 4H), 3.49 (d, J = 7.6 Hz, 2H), 3.21 (d, J = 2.7 Hz, 1H), 2.62 (d, J = 6.9 Hz, 2H), 1.14 (t, J = 6.9 Hz, 3H).

1-Ethyl-7-(4-(prop-2-yn-1-yloxy)phenethyl)-5-(p-tolyl)-5,7,8,9-tetrahydro-1H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidine-2,4,6(3H)-trione (2.32).

O O NH N O N N O H The title compound was synthesized via general procedure N using 2.4 (56 mg, 0.13 mmol), 1 which yielded a yellow solid (13 mg, 19%). H NMR (400 MHz, DMSO-d6) δ 10.39 (s, 1H), 8.77 (d, J = 5.9 Hz, 1H), 7.15 (d, J = 7.8 Hz, 2H), 7.04 (d, J = 7.9 Hz, 2H), 6.93 (d, J = 8.2 Hz, 2H), 6.80 (d, J = 8.2 Hz, 2H), 5.16 (d, J = 16.5 Hz, 1H), 5.02 (d, J = 16.5 Hz, 1H), 4.87 (s, 1H), 4.75 (d, J = 2.4 Hz, 2H), 4.02 (q, J = 6.9 Hz, 2H), 3.55 (t, J = 2.3 Hz, 1H), 3.50–3.35 (m, 2H), 2.76–2.64 (m, 2H), 2.26 (s, 3H), 1.10 (t, J = 6.9 Hz, 3H). 13C NMR (100 MHz, DMSO) δ 175.1, 172.8, 162.1, 155.7, 155.4, 151.0, 143.7, 134.8, 131.0, 129.7, 128.4, 127.2, 114.6, 93.4, 89.9, 79.4, 78.1, 75.1, 55.3, 43.2, 35.7, 34.3, 32.4, 20.6, 14.0.

150 4.4.6. General Procedure O (Click Reaction)

The thalidomide azide (2.35) (1 equiv) and the dihydropyridine alkyne (2.31a, 2.31b and 2.32) (1 equiv) were added into a scintillation vial (20 mL) and DMF (5 mL) was added to dissolve the substrates. Sodium ascorbate (0.2 equiv) and copper sulfate pentahydrate (0.1 equiv) were added to another scintillation vial (20 mL). After addition of water (5 mL), the resulting dark brown solution was transferred to the reaction system via syringe. The mixture was allowed to stir at rt overnight. The reaction solution then was poured into water and extracted with ethyl acetate. The organic phase was washed with brine, and then dried over anhydrous MgSO4. The residue was purified by flash column chromatography (4 gram RediSep Gold silica gel column, DCM + 5% methanol) to obtain the desired product as a yellow solid.

N-((1-(7-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)heptyl)-1H-1,2,3- triazol-4-yl)methyl)-2-(3-(1-ethyl-7-(4-hydroxyphenethyl)-2,4,6-trioxo- 2,3,4,5,6,7,8,9-octahydro-1H-pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidin-5- yl)phenoxy)acetamide (2.36a).

O O N N HN O N NH O N O O O

O O NH N HO N N O H The title compound was synthesized via general procedure O using 2.31a (30 mg, 0.054 mmol), which yielded a yellow solid (10 mg, 19%); mp 169–170 °C dec, with a purity 91% 1 determined by UPLC. H NMR (400 MHz, DMSO-d6) δ 11.10 (s, 1H), 10.42 (s, 1H), 9.18 (s, 1H), 8.80 (s, 1H), 8.58 (t, J = 5.9 Hz, 1H), 7.85 (s, 1H), 7.81 (t, J = 7.9 Hz, 1H), 7.50 (d, J = 8.6 Hz, 1H), 7.44 (d, J = 7.2 Hz, 1H), 7.15 (t, J = 7.9 Hz, 1H), 6.95–6.86 (m, 2H), 6.82 (d, J = 8.1 Hz, 3H), 6.73 (dd, J = 8.3, 2.4 Hz, 1H), 6.59 (d, J = 8.0 Hz, 2H), 5.20–4.98 151 (m, 3H), 4.90 (s, 1H), 4.46 (s, 2H), 4.37 (dd, J = 5.9, 3.1 Hz, 2H), 4.30 (t, J = 7.1 Hz, 2H), 4.19 (t, J = 6.4 Hz, 2H), 4.13–3.98 (m, 2H), 3.45–3.26 (m, 2H), 3.18 (d, J = 5.3 Hz, 1H), 2.89 (ddd, J = 17.4, 13.5, 5.4 Hz, 1H), 2.66 (dd, J = 22.0, 5.0 Hz, 2H), 2.03 (dd, J = 11.2, 5.8 Hz, 1H), 1.77 (dp, J = 14.2, 6.8 Hz, 4H), 1.52–1.32 (m, 4H), 1.26 (d, J = 8.2 Hz, 2H), 1.11 (t, J = 6.9 Hz, 3H).

N-((1-(7-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)heptyl)-1H-1,2,3- triazol-4-yl)methyl)-2-(2-(1-ethyl-7-(4-hydroxyphenethyl)-2,4,6-trioxo- 2,3,4,5,6,7,8,9-octahydro-1H-pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidin-5- yl)phenoxy)acetamide (2.36b)

HN O O N O O N N O N NH O O O NH N HO N N O H The title compound was synthesized via general procedure O using 2.31b (27 mg, 0.049 mmol), which yielded a yellow solid (15 mg, 32%); mp 185–186 °C dec, with a purity 96% 1 determined by UPLC. H NMR (400 MHz, DMSO-d6) δ 11.11 (s, 1H), 10.44 (s, 1H), 9.16 (s, 1H), 8.98 (t, J = 5.7 Hz, 1H), 8.38 (t, J = 6.2 Hz, 1H), 7.92 (s, 1H), 7.81 (t, J = 7.9 Hz, 1H), 7.50 (d, J = 8.6 Hz, 1H), 7.44 (d, J = 7.2 Hz, 1H), 7.14 (t, J = 7.6 Hz, 1H), 6.96 (dt, J = 14.7, 7.3 Hz, 2H), 6.81 (d, J = 8.2 Hz, 1H), 6.71 (d, J = 8.0 Hz, 2H), 6.53 (d, J = 7.9 Hz, 2H), 5.23 (s, 1H), 5.08 (dd, J = 12.7, 5.4 Hz, 1H), 4.98 (s, 2H), 4.61 (s, 2H), 4.46 (d, J = 5.5 Hz, 2H), 4.31 (t, J = 7.1 Hz, 2H), 4.18 (t, J = 6.4 Hz, 2H), 4.07 (dt, J = 14.1, 6.3 Hz, 3H), 3.47 (d, J = 7.3 Hz, 2H), 3.31 (d, J = 5.1 Hz, 1H), 3.18 (d, J = 5.3 Hz, 1H), 2.95–2.82 (m, 1H), 2.60 (dt, J = 13.0, 6.1 Hz, 3H), 2.03 (dd, J = 11.5, 5.8 Hz, 1H), 1.75 (dt, J = 15.2, 7.3 Hz, 5H), 1.48–1.31 (m, 2H), 1.25 (d, J = 6.5 Hz, 1H), 1.14 (t, J = 6.9 Hz, 3H).

152 7-(4-((1-(7-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)heptyl)-1H- 1,2,3-triazol-4-yl)methoxy)phenethyl)-1-ethyl-5-(p-tolyl)-5,7,8,9-tetrahydro-1H- pyrrolo[3',4':5,6]pyrido[2,3-d]pyrimidine-2,4,6(3H)-trione (2.36c).

O O NH O N O O N N N O N H O N N O NH O The title compound was synthesized via general procedure O using 2.32 (30 mg, 0.060 mmol), which yielded a yellow solid (22 mg, 33%); mp 196–197 °C dec, with a purity 92% 1 determined by UPLC. H NMR (400 MHz, DMSO-d6) δ 11.11 (s, 1H), 10.39 (s, 1H), 8.75 (s, 1H), 8.21 (s, 1H), 7.81 (t, J = 7.9 Hz, 1H), 7.51 (d, J = 8.6 Hz, 1H), 7.44 (d, J = 7.4 Hz, 1H), 7.13 (d, J = 7.7 Hz, 2H), 7.03 (d, J = 7.8 Hz, 2H), 6.94 (d, J = 8.2 Hz, 2H), 6.85 (d, J = 8.2 Hz, 2H), 5.23–4.98 (m, 5H), 4.87 (s, 1H), 4.37 (t, J = 7.1 Hz, 2H), 4.20 (t, J = 6.3 Hz, 2H), 4.11–3.96 (m, 3H), 3.42 (d, J = 6.5 Hz, 2H), 2.97–2.81 (m, 1H), 2.75–2.63 (m, 2H), 2.24 (s, 3H), 2.02 (d, J = 17.1 Hz, 2H), 1.83 (p, J = 7.1 Hz, 2H), 1.74 (q, J = 6.9 Hz, 2H), 1.40 (dq, J = 29.9, 7.2, 6.8 Hz, 3H), 1.26 (t, J = 7.4 Hz, 2H), 1.18 (t, J = 7.2 Hz, 1H), 1.10 (t, J = 6.9 Hz, 3H).

4.5. Bifunctional Molecules: Bivalent Analog 6-Chloro-3-methoxy- [1,2,4]triazolo[4,3-b]pyridazine (2.37).

N N N N Cl Based on a reported method,97 3-chloro-6-hydrazinylpyridazine (1.0 g, 6.9 mmol) was suspended in DME (20 mL) and treated with tetramethoxymethane (1.5 mL, 11 mmol) and the resulting mixture was stirred at 90 °C for 24 h. The DME was evaporated and the residue was dissolved in 5% methanol in DCM and filtered through a silica plug. The filtrate was evaporated to dryness and then taken up in MTBE and slurred for 1 h. The solid was filtered and dried under vacuum to afford the target molecule (1.2 g, 91 %) as a cream

153 1 colored powder. H NMR (400 MHz, DMSO-d6) δ 8.24 (dd, J = 9.7, 1.9 Hz, 1H), 7.33 (dd, J = 9.7, 1.8 Hz, 1H), 4.26 (d, J = 1.8 Hz, 3H).

1-(3-Methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-ol (2.38).

N N N N N OH 4-Hydroxypiperidine (0.36 g, 3.6 mmol) was added to 6-chloro-3-methoxy- [1,2,4]triazolo[4,3-b]pyridazine (0.22 g, 1.2 mmol) and DIPEA (0.4 mL, 2.4 mmol) in ethanol (10 mL). The resulting solution was stirred at 55 °C for 18 h. The crude product was purified by flash silica chromatography (12 gram RediSep Gold silica gel column, DCM + 10% methanol) to afford the target molecule (140 mg, 47%). 1H NMR (400 MHz,

Methanol-d4) δ 7.72 (d, J = 10.3 Hz, 1H), 7.28 (d, J = 10.3 Hz, 1H), 4.28 (s, 3H), 4.09– 3.99 (m, 2H), 3.90 (dq, J = 8.8, 4.3 Hz, 1H), 3.82–3.72 (m, 2H), 3.28 (t, J = 3.5 Hz, 1H), 2.01–1.94 (m, 2H), 1.65–1.54 (m, 2H).

2-(4-(1-Ethyl-2,4,6-trioxo-1,2,3,4,5,6,8,9-octahydrofuro[3',4':5,6]pyrido[2,3- d]pyrimidin-5-yl)phenyl)acetic acid (2.40).

O O NH O N N O H

Tetramic acid (120 mg, 0.10 mmol), 2.1 (155 mg, 0.10 mmol), and methyl(p- formylphenyl)acetate (178 mg, 0.10 mmol) were dispensed in acetic acid (15 mL). The mixture was heated to reflux for 12 h. After the completion of the reaction, acetic acid was removed under reduced vacuum. The residue was re-dissolved in ethyl acetate and washed with saturated NaHCO3 solution, brine, and dried over anhydrous MgSO4. The ester intermediate was purified via a flash chromatography (4 gram RediSep Gold silica gel 1 column, DCM + 5% methanol). H NMR (400 MHz, DMSO-d6) δ 11.09 (s, 1H), 10.16 (s, 1H), 7.19 (d, J = 7.9 Hz, 2H), 7.14 (d, J = 7.9 Hz, 2H), 4.96 (d, J = 16.6 Hz, 1H), 4.88 (d,

154 J = 16.6 Hz, 1H), 4.66 (s, 1H), 3.93 (dq, J = 18.5, 7.5 Hz, 2H), 3.60 (s, 5H), 1.20 (t, J = 7.0

Hz, 3H). The ester precursor was then dissolved in THF/H2O (1:1). LiOH (168 mg, 0.70 mmol) was added to the solution and stirred at rt overnight. After consumption of the ester precursor, monitored by TLC, ethyl acetate and water were added. The aqueous layer was kept and acidified using 1M HCl solution to pH 1-2. The pale-yellow solid was collected through filtration and dried under high vacuum to afford the free acid (199 mg, 52% over 1 two steps). H NMR (400 MHz, DMSO-d6) δ 12.26 (s, 1H), 11.09 (s, 1H), 10.16 (s, 1H), 7.18 (d, J = 8.2 Hz, 2H), 7.13 (d, J = 8.2 Hz, 2H), 4.92 (q, J = 16.6 Hz, 2H), 4.66 (s, 1H), 3.95 (pt, J = 14.5, 7.1 Hz, 2H), 3.49 (s, 2H), 1.20 (t, J = 7.0 Hz, 3H).

1-(3-Methoxy-[1,2,4]triazolo[4,3-a]pyridin-6-yl)piperidin-4-yl 2-(4-(1-Ethyl-2,4,6- trioxo-1,2,3,4,5,6,8,9-octahydrofuro[3',4':5,6]pyrido[2,3-d]pyrimidin-5- yl)phenyl)acetate (2.41).

O HN O O N O NH

N O N O O N N N The acid 2.40 (38 mg, 0.10 mmol), HATU (42 mg, 0.11 mmol), and DIPEA (70 uM, 0.40 mmol) were dissolved in DMF (5 mL) and yielded a dark-yellow solution after stirring for 0.5 h. 2.37 (25 mg, 0.10 mmol) was then added to the solution and the mixture was stirred for 72 h. The reaction was poured into water and extracted with ethyl acetate. The organic phase was washed with brine, dried over anhydrous MgSO4, and concentrated under reduced pressure. The residue was purified by flash column chromatography (4 gram RediSep Gold silica gel column, DCM + 10% methanol), which yielded the target molecule 1 as white wax (7.8 mg, 13%). H NMR (400 MHz, DMSO-d6) δ 11.09 (s, 1H), 10.16 (s, 1H), 7.87 (d, J = 10.2 Hz, 1H), 7.27 (d, J = 10.2 Hz, 1H), 7.19 (d, J = 8.0 Hz, 2H), 7.14 (d, J = 8.1 Hz, 2H), 5.01–4.79 (m, 2H), 4.66 (s, 1H), 4.18 (s, 3H), 3.95 (h, J = 7.6 Hz, 2H), 3.79 (d, J = 13.6 Hz, 2H), 3.60 (s, 2H), 3.43–3.32 (m, 3H), 1.92 (d, J = 17.9 Hz, 2H), 1.69– 1.55 (m, 2H), 1.18 (t, J = 7.2 Hz, 3H).

155 4.6. Analog Affinity Determinations 4.6.1. AlphaScreen BRD4-1 and BRDT-1 AlphaScreen assays were performed with minimal modifications from the manufacturer’s protocol (PerkinElmer, USA). All reagents were diluted in 50 mM HEPES, 150 mM NaCl, 0.1% w/v BSA, 0.01% w/v Tween 20, pH 7.5 and allowed to equilibrate to rt prior to addition to plates. After the addition of Alpha beads to the master solutions, all subsequent steps were performed under low light conditions. A 2x solution of components with final concentrations of His-BRD4 or His-BRDT at 40 nM, Ni-coated Acceptor Bead at 25 µg/ml, and 20 nM biotinylated-JQ1(S) was added in 10 µL to 384-well plates (AlphaPlate-384, PerkinElmer, USA). Plates were spun down at 150 g, after which 100 nL of compound in DMSO from stock plates were added by pin transfer using a Janus Workstation (PerkinElmer, USA). The streptavidin-coated donor beads (25 µg/ml final) were added in the same manner as the previous solution, in a 2x solution of 10 µL volume. Following this addition, plates were sealed with foil to prevent light exposure and evaporation. The plates were spun down again at 150g. Plates were incubated at rt for 1 h and then read on an Envision 2104 (PerkinElmer, USA) using the manufacturer’s protocol. All analogs were tested once in duplicates.

4.6.2. FP Assay The FP assays for BRDT-1 and BRD4-1 were conducted following the published protocol.122 All affinity experiments were carried out in assay buffer comprised of 150 mM NaCl, 3 mM DTT, 4 mM CHAPS, 50 mM sodium phosphate, pH 7.4 containing 0.374% DMSO using 384-well plates (Corning 4511). Inhibitory potencies were determined in dose-response experiments at eight concentrations in duplicate using 3-fold dilutions. Analogs dissolved in DMSO (Sigma Aldrich, Saint Louis, MO) were first added to the appropriate wells using an Echo 550 contactless liquid handler (Labcyte). BET proteins prepared in assay buffer were then added to provide final concentrations (50 nM BRDT-1 and 50 nM BRD4-1). Finally, the fluorescent probe (CAS: 2283344-22-7), diluted into assay buffer, was added to each well (final concentration 7.5 nM). The plates were sealed

156 and incubated at rt for 60 min. Then FP measurements were performed on a CLARIOstar (BMG Labtech) plate reader (482-16 nm excitation, 530-40 nm emission, 504 nm dichroic).

4.7. Focused Library Syntheses Ethyl 2-Chloronicotinate.

Based on a reported procedure,197 2-chloropyridine-3-carboxylic acid (10 g, 63 mmol) was charged into a two necked round-bottom flask (250 mL) with a reflux condenser. After the addition of ethanol (150 mL), the resulting mixture was vigorously stirred. Concentrated

H2SO4 (5 mL) was then added dropwise via a syringe. The reaction mixture was heated to reflux for 4 h. Upon the completion of the reaction, monitored by TLC, the mixture was cooled to rt before the removal of ethanol under reduced pressure. The residue was dissolved in ethyl acetate and washed with saturated NaHCO3 solution, brine, and dried over MgSO4. The residue was purified by flash column chromatography (40 gram RediSep normal phase silica gel column, 0-30% ethyl acetate in hexanes), which gave 10 gram of 1 the title compound as oil (85%). H NMR (400 MHz, CDCl3) δ 8.49 (dd, J = 4.8, 2.0 Hz, 1H), 8.13 (dd, J = 7.7, 2.0 Hz, 1H), 7.31 (dd, J = 7.7, 4.8 Hz, 1H), 4.40 (q, J = 7.1 Hz, 2H), 1.38 (t, J = 7.1 Hz, 3H).

4.7.1. General Procedure for the Synthesis of the Acid Intermediates

Ethyl 2-chloronicotinate (1.0 equiv) and substituted phenol (1.1 equiv) were charged into a 20 mL scintillation vial. DMF (5 mL) was added as the solvent before the addition of

K2CO3 (2.0 equiv). The vial was flushed with nitrogen gas and sealed heated to 90-120°C for 12 h. After cooling to rt, the mixture was poured into water and extracted with ethyl acetate. The combined organic layer was washed with water, brine, and dried over

157 anhydrous MgSO4. The residue was purified by flash column chromatography on silica gel (24 gram RediSep normal phase silica gel column, 0-50% ethyl acetate in hexanes) to give the corresponding ethyl 2-arylnicotinate. The ester intermediate (1.0 equiv) was dissolved in mixture of water and 1,4-dioxane (1:1). After the addition of NaOH (2.0 equiv), the reaction mixture was stirred at rt for 3 h. After completion of the reaction, monitored by TLC, the mixture was poured into water and its pH was adjusted to 1-2 using concentrated HCl. The acid precipitate that formed was collected by filtration, washing with cold water and drying under reduced pressure.

Desired Nicotinic Acid Intermediates 2-(4-Propionylphenoxy)nicotinic Acid.

COOH

N O

The title compound was synthesized following the general procedure for the synthesis of the acid intermediate, using ethyl 2-chloronicotinate (1.0 g, 5.4 mmol) and 1-(4- hydroxyphenyl)propan-1-one (0.89 g, 5.9 mmol), as a white solid (0.56 g, 76% over two steps). 1H NMR (400 MHz, DMSO-d6) δ 13.31 (s, 1H), 8.37–8.26 (m, 2H), 8.04–7.97 (m, 2H), 7.31 (dd, J = 7.5, 4.8 Hz, 1H), 7.24–7.17 (m, 2H), 3.04 (q, J = 7.1 Hz, 2H), 1.09 (t, J = 7.2 Hz, 3H).

2-([1,1'-Biphenyl]-4-yloxy)nicotinic Acid.

COOH

N O

The title compound was synthesized following the general procedure for the synthesis of the acid intermediate, using ethyl 2-chloronicotinate (1.0 g, 5.4 mmol) and [1,1'-biphenyl]-

158 4-ol (1.0 g, 5.9 mmol), as a white solid (0.71 g, 45% over two steps). 1H NMR (400 MHz,

DMSO-d6) δ 13.25 (s, 1H), 8.31–8.23 (m, 2H), 7.68 (dd, J = 8.7, 7.0 Hz, 4H), 7.47 (t, J = 7.6 Hz, 2H), 7.37 (d, J = 7.4 Hz, 1H), 7.25 (dd, J = 7.5, 4.9 Hz, 1H), 7.22–7.16 (m, 2H).

2-([1,1'-Biphenyl]-2-yloxy)nicotinic Acid.

COOH

N O

The title compound was synthesized following the general procedure for the synthesis of the acid intermediate starting from ethyl 2-chloronicotinate (1.0 g, 5.4 mmol) and [1,1'- biphenyl]-2-ol (1.0 g, 5.9 mmol) as a white solid (0.43 g, 27% over two steps). 1H NMR

(400 MHz, DMSO-d6) δ 13.15 (s, 1H), 8.14 (d, J = 6.2 Hz, 2H), 7.54–7.46 (m, 3H), 7.41 (td, J = 7.7, 1.8 Hz, 1H), 7.35–7.19 (m, 4H), 7.15 (d, J = 8.0 Hz, 1H), 7.09 (dd, J = 7.4, 5.0 Hz, 1H).

2-(4-Benzoylphenoxy)nicotinic Acid.

COOH

N O

The title compound was synthesized following the general procedure for the synthesis of the acid intermediate, using ethyl 2-chloronicotinate (1.0 g, 5.4 mmol) and (4- hydroxyphenyl)(phenyl)methanone (1.2 g, 5.9 mmol), as a white solid (0.56 g, 33% over 1 two steps). H NMR (400 MHz, DMSO-d6) δ 13.34 (s, 1H), 8.36 (dd, J = 4.8, 2.0 Hz, 1H), 8.31 (dd, J = 7.6, 2.0 Hz, 1H), 7.84–7.78 (m, 2H), 7.78–7.73 (m, 2H), 7.68 (t, J = 7.4 Hz, 1H), 7.57 (t, J = 7.6 Hz, 2H), 7.33 (dd, J = 7.6, 4.8 Hz, 1H), 7.29–7.22 (m, 2H).

159 2-(4-Acetylphenoxy)nicotinic Acid.

COOH

N O

O The title compound was synthesized following the general procedure for the synthesis of the acid intermediate, using ethyl 2-chloronicotinate (1.0 g, 5.4 mmol) and 1-(4- hydroxyphenyl)ethan-1-one (0.80 g, 5.9 mmol), as a white solid (0.83 g, 60% over two 1 steps). H NMR (400 MHz, DMSO-d6) δ 13.31 (s, 1H), 8.31 (ddd, J = 9.6, 6.2, 2.0 Hz, 2H), 8.04–7.97 (m, 2H), 7.31 (dd, J = 7.6, 4.8 Hz, 1H), 7.24–7.17 (m, 2H), 2.58 (s, 3H).

2-(3-Cyanophenoxy)nicotinic Acid. COOH

N O

CN The title compound was synthesized following the general procedure for the synthesis of the acid intermediate, using ethyl 2-chloronicotinate (1.0 g, 5.4 mmol) and 3- hydroxybenzonitrile (0.70 g, 5.9 mmol), as a white solid (0.77 g, 59% over two steps). 1H

NMR (400 MHz, DMSO-d6) δ 13.29 (s, 1H), 8.30 (d, J = 6.3 Hz, 2H), 7.68 (d, J = 3.0 Hz, 2H), 7.62 (t, J = 8.1 Hz, 1H), 7.49 (dd, J = 8.2, 2.3 Hz, 1H), 7.32–7.25 (m, 1H).

2-([1,1'-Biphenyl]-3-yloxy)nicotinic Acid.

COOH

N O

The title compound was synthesized following the general procedure for the synthesis of the acid intermediate, using ethyl 2-chloronicotinate (1.0 g, 5.4 mmol) and [1,1'-biphenyl]- 3-ol (1.0 g, 5.9 mmol), as a white solid (0.47 g, 30% over two steps). 1H NMR (400 MHz,

DMSO-d6) δ 13.24 (s, 1H), 8.31–8.25 (m, 2H), 7.70–7.65 (m, 2H), 7.54–7.49 (m, 2H), 7.46 160 (dd, J = 8.3, 6.8 Hz, 2H), 7.40–7.36 (m, 2H), 7.24 (dd, J = 7.5, 4.9 Hz, 1H), 7.10 (dt, J = 7.1, 2.1 Hz, 1H).

2-(3-Acetylphenoxy)nicotinic Acid.

COOH

N O

O The title compound was synthesized following the general procedure for the synthesis of the acid intermediate, using ethyl 2-chloronicotinate (1.0 g, 5.4 mmol) and 1-(3- hydroxyphenyl)ethan-1-one (0.80 g, 5.9 mmol), as a white solid (0.62 g, 45% over two 1 steps). H NMR (400 MHz, DMSO-d6) δ 13.27 (s, 1H), 8.30–8.24 (m, 2H), 7.81 (d, J = 7.7 Hz, 1H), 7.62 (t, J = 2.0 Hz, 1H), 7.57 (t, J = 7.9 Hz, 1H), 7.40 (dd, J = 7.9, 2.4 Hz, 1H), 7.26 (dd, J = 6.8, 5.6 Hz, 1H), 2.58 (s, 3H).

2-(4-Chlorophenoxy)nicotinic Acid.

COOH

N O

Cl The title compound was synthesized following the general procedure for the synthesis of the acid intermediate, using ethyl 2-chloronicotinate (1.0 g, 5.4 mmol) and 4-chlorophenol (0.76 g, 5.9 mmol), as a white solid (0.33 g, 25% over two steps). 1H NMR (400 MHz,

DMSO-d6) δ 13.26 (s, 1H), 8.31–8.23 (m, 2H), 7.49–7.41 (m, 2H), 7.25 (dd, J = 7.5, 4.9 Hz, 1H), 7.16–7.11 (m, 2H).

161 2-(2-Chloro-5-fluorophenoxy)nicotinic Acid.

COOH

N O Cl

F The title compound was synthesized following the general procedure for the synthesis of the acid intermediate, using ethyl 2-chloronicotinate (1.0 g, 5.4 mmol) and 3,4- difluorophenol (0.86 g, 5.9 mmol), as a white solid (0.43 g, 30% over two steps). 1H NMR

(400 MHz, DMSO-d6) δ 13.30 (s, 1H), 8.30 (dd, J = 7.6, 1.9 Hz, 1H), 8.26 (dd, J = 4.9, 1.9 Hz, 1H), 7.62 (dd, J = 9.0, 5.8 Hz, 1H), 7.33 (dd, J = 9.3, 3.0 Hz, 1H), 7.27 (dd, J = 7.5, 4.9 Hz, 1H), 7.19 (td, J = 8.5, 3.0 Hz, 1H).

2-(4-Fluoro-3-(trifluoromethyl)phenoxy)nicotinic Acid.

COOH

N O

CF3 F The title compound was synthesized following the general procedure for the synthesis of the acid intermediate, using ethyl 2-chloronicotinate (1.0 g, 5.4 mmol) and 4-fluoro-3- (trifluoromethyl)phenol (1.1 g, 5.9 mmol), as a white solid (0.40 g, 25% over two steps). 1 H NMR (400 MHz, DMSO-d6) δ 13.30 (s, 1H), 8.29 (d, J = 6.2 Hz, 2H), 7.63–7.51 (m, 3H), 7.31–7.24 (m, 1H).

2-(2-Chloro-5-(trifluoromethyl)phenoxy)nicotinic Acid.

COOH

N O Cl

F3C The title compound was synthesized following the general procedure for the synthesis of the acid intermediate, using ethyl 2-chloronicotinate (1.0 g, 5.4 mmol) and 2-chloro-5- (trifluoromethyl)phenol (1.1 g, 5.9 mmol), as a white solid (0.60 g, 35% over two steps). 1 H NMR (400 MHz, DMSO-d6) δ 13.34 (s, 1H), 8.32 (dd, J = 7.5, 2.0 Hz, 1H), 8.26 (dd, J 162 = 4.9, 2.0 Hz, 1H), 7.84 (d, J = 8.4 Hz, 1H), 7.78 (d, J = 2.1 Hz, 1H), 7.67 (dd, J = 8.5, 2.1 Hz, 1H), 7.28 (dd, J = 7.5, 4.9 Hz, 1H).

2-(3,4-Difluorophenoxy)nicotinic Acid.

COOH

N O

F F The title compound was synthesized following the general procedure for the synthesis of the acid intermediate, using ethyl 2-chloronicotinate (1.0 g, 5.4 mmol) and 2-chloro-5- fluorophenol (0.77 g, 5.9 mmol), as a white solid (0.28 g, 21% over two steps). 1H NMR

(400 MHz, DMSO-d6) δ 13.34 (s, 1H), 8.30–8.24 (m, 2H), 7.47 (q, J = 9.5 Hz, 1H), 7.36 (ddd, J = 11.7, 6.9, 2.8 Hz, 1H), 7.26 (dd, J = 7.5, 4.9 Hz, 1H), 6.99 (dd, J = 8.9, 4.1 Hz, 1H).

2-(2-Chloro-3-(trifluoromethyl)phenoxy)nicotinic Acid.

COOH

N O Cl

CF3 The title compound was synthesized following the general procedure for the synthesis of the acid intermediate, using ethyl 2-chloronicotinate (1.0 g, 5.4 mmol) and 2-chloro-3- (trifluoromethyl)phenol (1.2 g, 5.9 mmol), as a white solid (0.61 g, 36% over two steps). 1 H NMR (400 MHz, DMSO-d6) δ 13.37 (s, 1H), 8.33 (dd, J = 7.6, 1.9 Hz, 1H), 8.26 (dd, J = 4.9, 1.9 Hz, 1H), 7.77 (dd, J = 7.4, 1.9 Hz, 1H), 7.68–7.59 (m, 2H), 7.28 (dd, J = 7.5, 4.8 Hz, 1H).

163 2-(4-Fluoro-3-methylphenoxy)nicotinic Acid.

COOH

N O

F The title compound was synthesized following the general procedure for the synthesis of the acid intermediate, using ethyl 2-chloronicotinate (1.0 g, 5.4 mmol) and 4-fluoro-3- methylphenol (0.74 g, 5.9 mmol), as a white solid (0.55 g, 41% over two steps). 1H NMR

(400 MHz, DMSO-d6) δ 13.23 (s, 1H), 8.28–8.21 (m, 2H), 7.21 (dd, J = 7.5, 4.8 Hz, 1H), 7.15 (t, J = 9.1 Hz, 1H), 7.05 (dd, J = 6.6, 2.9 Hz, 1H), 6.95 (dt, J = 8.3, 3.7 Hz, 1H), 2.23 (d, J = 1.9 Hz, 3H).

2-(3-(Trifluoromethyl)phenoxy)nicotinic Acid.

COOH

N O

CF3 The title compound was synthesized following the general procedure for the synthesis of the acid intermediate, using ethyl 2-chloronicotinate (1.0 g, 5.4 mmol) and 3- (trifluoromethyl)phenol (0.96 g, 5.9 mmol), as a white solid (0.56 g, 37% over two steps). 1 H NMR (400 MHz, DMSO-d6) δ 13.31 (s, 1H), 8.33–8.26 (m, 2H), 7.66 (t, J = 7.9 Hz, 1H), 7.58 (d, J = 7.9 Hz, 1H), 7.50 (d, J = 1.8 Hz, 1H), 7.44 (dd, J = 8.1, 2.2 Hz, 1H), 7.28 (dd, J = 7.3, 5.1 Hz, 1H).

2-(2,3,5-Trichlorophenoxy)nicotinic Acid.

COOH

N O Cl

Cl Cl The title compound was synthesized following the general procedure for the synthesis of the acid intermediate, using ethyl 2-chloronicotinate (1.0 g, 5.4 mmol) and 2,3,5- trichlorophenol (1.2 g, 5.9 mmol), as a white solid (0.48 g, 28% over two steps). 1H NMR 164 (400 MHz, DMSO-d6) δ 13.37 (s, 1H), 8.33 (dd, J = 7.5, 1.9 Hz, 1H), 8.28 (dd, J = 4.9, 1.9 Hz, 1H), 7.79 (d, J = 2.4 Hz, 1H), 7.58 (d, J = 2.4 Hz, 1H), 7.30 (dd, J = 7.5, 4.8 Hz, 1H).

2-(4-Acetyl-3-chlorophenoxy)nicotinic Acid.

O OH

N O

Cl O The title compound was synthesized following the general procedure for the synthesis of the acid intermediate, using ethyl 2-chloronicotinate (1.0 g, 5.4 mmol) and 1-(2-chloro-4- hydroxyphenyl)ethan-1-one (1.0 g, 5.9 mmol), as a white solid (0.75 g, 48% over two 1 steps). H NMR (400 MHz, DMSO-d6) δ 13.36 (s, 1H), 8.33 (dd, J = 7.6, 2.0 Hz, 1H), 8.26 (dd, J = 4.9, 2.0 Hz, 1H), 8.11 (d, J = 2.1 Hz, 1H), 7.99–7.94 (m, 1H), 7.42 (d, J = 8.5 Hz, 1H), 7.30 (dd, J = 7.6, 4.8 Hz, 1H), 2.62 (s, 3H).

4.7.2. General Procedure for the Amide Coupling Reaction The carboxylic acid (1.0 equiv) and HATU (1.3 equiv) were charged into a 10 mL scintillation vial. DCM (5 mL) was added to generate a cloudy mixture. After the addition of DIPEA (2.0 equiv), the reaction mixture was stirred for 0.5 h, which turned into a clear solution. The aniline or tetrahydroquinoline (1.2 equiv) was then added and the reaction mixture was stirred overnight. Upon the completion of reaction, monitored by TLC, the mixture was poured in water, and extracted with ethyl acetate twice. The combined organic layer was washed with brine and dried over anhydrous MgSO4. The residue was purified by flash column chromatography on silica gel (12 gram RediSep normal phase silica gel column, 0-75% ethyl acetate in hexanes) to give the desired compounds.

165 Desired Nicotinamide Analogs N-Methyl-2-(4-propionylphenoxy)-N-(o-tolyl)nicotinamide.

O N

N O

The title compound was synthesized following the general procedure for the coupling reaction using 2-(4-propionylphenoxy)nicotinic acid (100 mg, 0.370 mmol) and N-methyl- o-toluidine (53.8 mg, 0.444 mmol) as colorless wax (117 mg, 85%). 1H NMR (400 MHz,

CDCl3) δ 8.02–7.96 (m, 3H), 7.74 (dd, J = 7.4, 2.0 Hz, 1H), 7.17–7.09 (m, 2H), 7.09–7.00 (m, 2H), 6.98–6.89 (m, 3H), 3.41 (s, 3H), 3.00 (q, J = 7.2 Hz, 2H), 2.23 (s, 3H), 1.24 (t, J = 7.2 Hz, 3H).

1-(4-((3-(1,2,3,4-Tetrahydroquinoline-1-carbonyl)pyridin-2-yl)oxy)phenyl)propan-1- one.

O N

N O

The title compound was synthesized following the general procedure for the coupling reaction using 2-(4-propionylphenoxy)nicotinic acid (100 mg, 0.370 mmol) and 1,2,3,4- tetrahydroquinoline (59.1 mg, 0.444 mmol) as colorless wax (92 mg, 69%). 1H NMR (400

MHz, CDCl3) δ 8.16–8.00 (m, 2H), 7.86 (d, J = 8.2 Hz, 2H), 7.12 (dd, J = 7.4, 4.9 Hz, 1H), 7.03 (q, J = 8.9, 8.1 Hz, 2H), 6.88 (s, 1H), 6.57 (s, 1H), 6.49 (s, 2H), 4.78–4.29 (m, 1H), 3.56 (s, 1H), 2.95 (q, J = 7.3 Hz, 2H), 2.62 (s, 2H), 1.91 (s, 2H), 1.21 (t, J = 7.2 Hz, 3H).

166 2-(4-Propionylphenoxy)-N-(o-tolyl)nicotinamide.

O N H N O

The title compound was synthesized following the general procedure for the coupling reaction using 2-(4-propionylphenoxy)nicotinic acid (100 mg, 0.370 mmol) and 2- methylaniline (47.5 mg, 0.444 mmol) as colorless wax (176 mg, 99%). 1H NMR (400

MHz, CDCl3) δ 9.66 (s, 1H), 8.78 (dd, J = 7.7, 2.0 Hz, 1H), 8.33–8.26 (m, 2H), 8.17–8.10 (m, 2H), 7.35–7.28 (m, 4H), 7.21 (d, J = 7.3 Hz, 1H), 7.11 (td, J = 7.5, 1.3 Hz, 1H), 3.05 (q, J = 7.2 Hz, 2H), 2.28 (s, 3H), 1.27 (t, J = 7.2 Hz, 3H).

N-Ethyl-N-phenyl-2-(4-propionylphenoxy)nicotinamide.

O N

N O

The title compound was synthesized following the general procedure for the coupling reaction using 2-(4-propionylphenoxy)nicotinic acid (100 mg, 0.370 mmol) and N- ethylaniline (53.8 mg, 0.444 mmol) as colorless wax (113 mg, 82%). 1H NMR (400 MHz,

CDCl3) δ 8.00–7.92 (m, 3H), 7.74 (dd, J = 7.4, 2.0 Hz, 1H), 7.20 (dt, J = 5.0, 2.7 Hz, 3H), 7.12–7.05 (m, 2H), 6.94 (dd, J = 7.4, 4.9 Hz, 1H), 6.92–6.86 (m, 2H), 4.01 (q, J = 7.1 Hz, 2H), 2.99 (q, J = 7.2 Hz, 2H), 1.27 (d, J = 7.1 Hz, 3H), 1.22 (d, J = 7.2 Hz, 3H).

167 N-(2-Fluorophenyl)-N-methyl-2-(4-propionylphenoxy)nicotinamide.

O N F N O

O The title compound was synthesized following the general procedure for the coupling reaction using 2-(4-propionylphenoxy)nicotinic acid (100 mg, 0.370 mmol) and 2-fluoro- N-methylaniline (55.6 mg, 0.444 mmol) as colorless wax (97 mg, 73%). 1H NMR (400

MHz, CDCl3) δ 8.00 (dd, J = 4.9, 2.0 Hz, 1H), 7.97 (d, J = 8.7 Hz, 2H), 7.81 (dt, J = 7.5, 1.4 Hz, 1H), 7.25–7.11 (m, 2H), 7.03–6.94 (m, 3H), 6.94–6.89 (m, 2H), 3.46 (s, 3H), 2.99 (q, J = 7.2 Hz, 2H), 1.22 (t, J = 7.2 Hz, 3H).

N-Methyl-N-phenyl-2-(4-propionylphenoxy)nicotinamide.

O N

N O

The title compound was synthesized following the general procedure for the coupling reaction using 2-(4-propionylphenoxy)nicotinic acid (100 mg, 0.370 mmol) and N- methylaniline (47.6 mg, 0.444 mmol) as colorless wax (82 mg, 59%). 1H NMR (400 MHz,

CDCl3) δ 7.99 (dd, J = 4.9, 1.9 Hz, 1H), 7.94 (d, J = 8.6 Hz, 2H), 7.78 (dd, J = 7.4, 2.0 Hz, 1H), 7.20 (h, J = 3.9 Hz, 3H), 7.07 (dd, J = 7.3, 2.4 Hz, 2H), 6.96 (dd, J = 7.4, 4.9 Hz, 1H), 6.85–6.78 (m, 2H), 3.52 (s, 3H), 2.98 (q, J = 7.2 Hz, 2H), 1.22 (t, J = 7.2 Hz, 3H).

168 2-([1,1'-Biphenyl]-4-yloxy)-N-methyl-N-(o-tolyl)nicotinamide.

O N

N O

The title compound was synthesized following the general procedure for the coupling reaction using 2-([1,1'-biphenyl]-4-yloxy)nicotinic acid (100 mg, 0.340 mmol) and N- methyl-o-toluidine (49.4 mg, 0.408 mmol) as colorless wax (78 mg, 57%). 1H NMR (400

MHz, CDCl3) δ 7.99 (dd, J = 4.9, 2.0 Hz, 1H), 7.72 (dd, J = 7.4, 1.9 Hz, 1H), 7.61–7.54 (m, 4H), 7.47–7.40 (m, 2H), 7.36–7.34 (m, 1H), 7.16–7.10 (m, 3H), 7.06 (ddd, J = 8.3, 5.9, 2.8 Hz, 1H), 6.96 – 6.92 (m, 2H), 6.89 (dd, J = 7.4, 4.9 Hz, 1H), 3.42 (s, 3H), 2.28 (s, 3H).

(2-([1,1'-Biphenyl]-4-yloxy)pyridin-3-yl)(3,4-dihydroquinolin-1(2H)-yl)methanone.

O N

N O

The title compound was synthesized following the general procedure for the coupling reaction using 2-([1,1'-biphenyl]-4-yloxy)nicotinic acid (100 mg, 0.340 mmol) and 1,2,3,4- tetrahydroquinoline (54.3 mg ,0.408 mmol) as colorless wax (69 mg, 52%). 1H NMR (400

MHz, CDCl3) δ 8.13 (s, 1H), 8.04 (d, J = 7.3 Hz, 1H), 7.59–7.53 (m, 2H), 7.50–7.40 (m, 4H), 7.36–7.31 (m, 1H), 7.14–7.04 (m, 3H), 6.90 (s, 1H), 6.61 (s, 1H), 6.50 (s, 2H), 4.46 (d, J = 117.1 Hz, 1H), 3.47 (s, 1H), 2.70 (s, 2H), 2.33–2.14 (m, 1H), 2.00–1.76 (m, 1H).

169 2-([1,1'-Biphenyl]-4-yloxy)-N-(o-tolyl)nicotinamide.

O N H N O

The title compound was synthesized following the general procedure for the coupling reaction using 2-([1,1'-biphenyl]-4-yloxy)nicotinic acid (100 mg, 0.340 mmol) and o- toluidine (43.7 mg ,0.408 mmol) as colorless wax (79 mg, 57%). 1H NMR (400 MHz,

CDCl3) δ 9.86 (s, 1H), 8.78 (dd, J = 7.6, 2.1 Hz, 1H), 8.32 (dt, J = 5.9, 1.3 Hz, 2H), 7.73– 7.68 (m, 2H), 7.66–7.61 (m, 2H), 7.51–7.45 (m, 2H), 7.42–7.35 (m, 1H), 7.34–7.29 (m, 3H), 7.27–7.24 (m, 1H), 7.22 (d, J = 7.5 Hz, 1H), 7.10 (td, J = 7.5, 1.3 Hz, 1H), 2.33 (s, 3H).

2-([1,1'-Biphenyl]-4-yloxy)-N-ethyl-N-phenylnicotinamide.

O N

N O

The title compound was synthesized following the general procedure for the coupling reaction using 2-([1,1'-biphenyl]-4-yloxy)nicotinic acid (100 mg, 0.340 mmol) and N- ethylaniline (49.4 mg, 0.408 mmol) as colorless wax (96 mg, 71%). 1H NMR (400 MHz,

CDCl3) δ 8.00 (dd, J = 5.0, 2.0 Hz, 1H), 7.73 (dd, J = 7.5, 1.9 Hz, 1H), 7.62–7.53 (m, 4H), 7.45 (dd, J = 8.4, 6.8 Hz, 2H), 7.38–7.31 (m, 1H), 7.23 (td, J = 7.1, 6.6, 3.7 Hz, 3H), 7.18– 7.11 (m, 2H), 6.96–6.85 (m, 3H), 4.04 (q, J = 7.2 Hz, 2H), 1.27 (t, J = 7.1 Hz, 3H).

170 2-([1,1'-Biphenyl]-4-yloxy)-N-(2-fluorophenyl)-N-methylnicotinamide.

O N F N O

The title compound was synthesized following the general procedure for the coupling reaction using 2-([1,1'-biphenyl]-4-yloxy)nicotinic acid (100 mg, 0.340 mmol) and 2- fluoro-N-methylaniline (51.1 mg, 0.408 mmol) as colorless wax (43 mg, 31%). 1H NMR

(400 MHz, CDCl3) δ 8.02 (dd, J = 4.9, 2.0 Hz, 1H), 7.81 (dt, J = 7.4, 1.6 Hz, 1H), 7.61– 7.54 (m, 4H), 7.44 (t, J = 7.6 Hz, 2H), 7.38–7.32 (m, 1H), 7.26–7.19 (m, 2H), 7.07–6.97 (m, 2H), 6.97–6.90 (m, 3H), 3.49 (s, 3H).

(2-([1,1'-Biphenyl]-4-yloxy)pyridin-3-yl)(6-methyl-3,4-dihydroquinolin-1(2H)- yl)methanone.

O N

N O

The title compound was synthesized following the general procedure for the coupling reaction using 2-([1,1'-biphenyl]-4-yloxy)nicotinic acid (100 mg, 0.340 mmol) and 6- methyl-1,2,3,4-tetrahydroquinoline (60.1 mg, 0.408 mmol) as colorless wax (90 mg, 62%). 1 H NMR (400 MHz, CDCl3) δ 8.13 (s, 1H), 8.03 (s, 1H), 7.55 (d, J = 7.6 Hz, 2H), 7.48– 7.40 (m, 4H), 7.37–7.32 (m, 1H), 7.12–7.06 (m, 1H), 6.89 (s, 1H), 6.69 (s, 1H), 6.51 (d, J = 10.0 Hz, 3H), 4.59 (s, 1H), 3.43 (s, 1H), 2.64 (s, 2H), 2.28 (s, 4H), 1.93 (s, 1H).

171 2-([1,1'-Biphenyl]-4-yloxy)-N-methyl-N-phenylnicotinamide.

O N

N O

The title compound was synthesized following the general procedure for the coupling reaction using 2-([1,1'-biphenyl]-4-yloxy)nicotinic acid (100 mg, 0.340 mmol) and N- methylaniline (43.7 mg, 0.408 mmol) as colorless wax (116 mg, 89%). 1H NMR (400 MHz,

CDCl3) δ 8.00 (dd, J = 4.9, 2.3 Hz, 1H), 7.80–7.70 (m, 1H), 7.54 (dd, J = 11.0, 7.9 Hz, 4H), 7.42 (t, J = 7.6 Hz, 2H), 7.36–7.29 (m, 1H), 7.21 (p, J = 7.6, 6.5 Hz, 3H), 7.12 (d, J = 7.3 Hz, 2H), 6.91 (dd, J = 7.5, 4.8 Hz, 1H), 6.83 (d, J = 8.1 Hz, 2H), 3.53 (s, 3H).

2-([1,1'-Biphenyl]-2-yloxy)-N-methyl-N-(o-tolyl)nicotinamide.

O N

N O

The title compound was synthesized following the general procedure for the coupling reaction using 2-([1,1'-biphenyl]-2-yloxy)nicotinic acid (100 mg, 0.340 mmol) and N- methyl-o-toluidine (49.4 mg, 0.408 mmol) as colorless wax (100 mg, 74%). 1H NMR (400

MHz, CDCl3) δ 7.93 (dd, J = 5.0, 1.9 Hz, 1H), 7.53–7.48 (m, 2H), 7.46–7.43 (m, 1H), 7.38–7.34 (m, 2H), 7.30 (d, J = 2.0 Hz, 1H), 7.27 (dd, J = 9.2, 1.9 Hz, 2H), 7.23 (dq, J = 4.5, 2.6, 2.1 Hz, 1H), 7.10–7.04 (m, 2H), 6.91 (td, J = 7.2, 6.4, 2.4 Hz, 1H), 6.82 (dd, J = 7.7, 1.2 Hz, 1H), 6.79–6.73 (m, 2H), 3.35 (s, 3H), 2.17 (s, 3H).

172 2-([1,1'-Biphenyl]-2-yloxy)-N-(o-tolyl)nicotinamide.

O N H N O

The title compound was synthesized following the general procedure for the coupling reaction using 2-([1,1'-biphenyl]-2-yloxy)nicotinic acid (100 mg, 0.340 mmol) and o- toluidine (43.7 mg, 0.408 mmol) as colorless wax (86mg, 68%). 1H NMR (400 MHz,

CDCl3) δ 9.44 (s, 1H), 8.63 (d, J = 7.6 Hz, 1H), 8.29–8.20 (m, 1H), 8.11 (d, J = 8.1 Hz, 1H), 7.56–7.37 (m, 3H), 7.33 (d, J = 5.9 Hz, 2H), 7.25–7.19 (m, 5H), 7.18–7.07 (m, 2H), 7.04 (t, J = 7.4 Hz, 1H), 1.94 (s, 3H).

2-([1,1'-Biphenyl]-2-yloxy)-N-ethyl-N-phenylnicotinamide.

N O

The title compound was synthesized following the general procedure for the coupling reaction using 2-([1,1'-biphenyl]-2-yloxy)nicotinic acid (100 mg, 0.340 mmol) and N- ethylalinine (49.4 mg, 0.408 mmol) as colorless wax (116 mg, 86%). 1H NMR (400 MHz,

CDCl3) δ 7.90 (dd, J = 4.9, 2.0 Hz, 1H), 7.55–7.49 (m, 2H), 7.45 (ddd, J = 9.4, 7.4, 4.0 Hz, 2H), 7.36 (t, J = 7.5 Hz, 2H), 7.33–7.28 (m, 2H), 7.26–7.23 (m, 1H), 7.16–7.06 (m, 3H), 6.92 (dd, J = 6.6, 2.8 Hz, 2H), 6.82–6.70 (m, 2H), 3.95 (q, J = 7.2 Hz, 2H), 1.21 (t, J = 7.2 Hz, 3H).

173 2-(4-Benzoylphenoxy)-N-methyl-N-(o-tolyl)nicotinamide.

O N

N O

The title compound was synthesized following the general procedure for the coupling reaction using 2-(4-benzoylphenoxy)nicotinic acid (100 mg, 0.310 mmol) and N-methyl- o-toluidine (45.1 mg, 0.372 mmol) as colorless wax (22 mg, 15%). 1H NMR (400 MHz,

CDCl3) δ 8.01 (dd, J = 4.9, 2.0 Hz, 1H), 7.87–7.81 (m, 4H), 7.76 (dd, J = 7.5, 1.9 Hz, 1H), 7.63–7.57 (m, 1H), 7.50 (t, J = 7.7 Hz, 2H), 7.17–7.09 (m, 2H), 7.05 (ddd, J = 8.2, 6.6, 3.8 Hz, 2H), 6.98–6.91 (m, 3H), 3.42 (s, 3H), 2.25 (s, 3H).

(2-(4-Benzoylphenoxy)pyridin-3-yl)(3,4-dihydroquinolin-1(2H)-yl)methanone.

O N

N O

The title compound was synthesized following the general procedure for the coupling reaction using 2-(4-benzoylphenoxy)nicotinic acid (100 mg, 0.310 mmol) and 1,2,3,4- tetrahydroquinoline (49.5 mg, 0.372 mmol) as colorless wax (97 mg, 65%). 1H NMR (400

MHz, CDCl3) δ 8.13 (d, J = 4.5 Hz, 1H), 8.10–8.01 (m, 1H), 7.81–7.75 (m, 2H), 7.72 (d, J = 8.2 Hz, 2H), 7.61–7.55 (m, 1H), 7.47 (dd, J = 8.3, 6.9 Hz, 2H), 7.14 (dd, J = 7.4, 4.9 Hz, 1H), 7.01 (d, J = 5.1 Hz, 2H), 6.87 (s, 1H), 6.62 (s, 1H), 6.52 (s, 2H), 4.57 (s, 1H), 3.45 (s, 1H), 2.63 (s, 2H), 2.36–2.07 (m, 1H), 2.02–1.78 (m, 1H).

174 2-(4-Benzoylphenoxy)-N-(o-tolyl)nicotinamide.

O N H N O

The title compound was synthesized following the general procedure for the coupling reaction using 2-(4-benzoylphenoxy)nicotinic acid (100 mg, 0.310 mmol) and o-toluidine 1 (39.9 mg, 0.372 mmol) as colorless wax (92 mg, 66%). H NMR (400 MHz, CDCl3) δ 9.66 (s, 1H), 8.78 (dd, J = 7.7, 2.1 Hz, 1H), 8.33–8.26 (m, 2H), 8.01–7.94 (m, 2H), 7.85 (dt, J = 7.0, 1.5 Hz, 2H), 7.65–7.58 (m, 1H), 7.57–7.49 (m, 2H), 7.36–7.27 (m, 4H), 7.21 (d, J = 7.5 Hz, 1H), 7.10 (td, J = 7.5, 1.4 Hz, 1H), 2.29 (s, 3H).

2-(4-Benzoylphenoxy)-N-ethyl-N-phenylnicotinamide.

O N

N O

The title compound was synthesized following the general procedure for the coupling reaction using 2-(4-benzoylphenoxy)nicotinic acid (100 mg, 0.310 mmol) and N- ethylaniline (45.1 mg, 0.372 mmol) as colorless wax (108 mg, 74%). 1H NMR (400 MHz,

CDCl3) δ 8.00 (dd, J = 4.9, 1.9 Hz, 1H), 7.82 (t, J = 8.0 Hz, 4H), 7.75 (dd, J = 7.4, 2.0 Hz, 1H), 7.62–7.56 (m, 1H), 7.48 (dd, J = 8.3, 6.9 Hz, 2H), 7.23–7.15 (m, 3H), 7.08 (dd, J = 7.5, 2.1 Hz, 2H), 6.96 (dd, J = 7.4, 5.0 Hz, 1H), 6.94–6.89 (m, 2H), 4.02 (q, J = 7.1 Hz, 2H), 1.24 (t, J = 7.1 Hz, 3H).

175 2-(4-Benzoylphenoxy)-N-(2-fluorophenyl)-N-methylnicotinamide.

O N F N O

The title compound was synthesized following the general procedure for the coupling reaction using 2-(4-benzoylphenoxy)nicotinic acid (100 mg, 0.310 mmol) and 2-fluoro-N- methylaniline (46.6 mg, 0.372 mmol) as colorless wax (41 mg, 28%). 1H NMR (400 MHz,

CDCl3) δ 8.03 (dd, J = 4.9, 2.0 Hz, 1H), 7.82 (ddt, J = 9.5, 8.4, 1.8 Hz, 5H), 7.63–7.55 (m, 1H), 7.49 (dd, J = 8.3, 7.0 Hz, 2H), 7.24–7.11 (m, 2H), 6.97 (ddq, J = 12.8, 9.4, 2.8, 2.1 Hz, 5H), 3.47 (s, 3H).

(2-(4-Benzoylphenoxy)pyridin-3-yl)(6-methyl-3,4-dihydroquinolin-1(2H)- yl)methanone.

O N

N O

The title compound was synthesized following the general procedure for the coupling reaction using 2-(4-benzoylphenoxy)nicotinic acid (100 mg, 0.310 mmol) and 6-methyl- 1,2,3,4-tetrahydroquinoline (54.8 mg, 0.372 mmol) as colorless wax (115 mg, 74%). 1H

NMR (400 MHz, CDCl3) δ 8.09 (dd, J = 26.8, 6.0 Hz, 2H), 7.80–7.75 (m, 2H), 7.71 (d, J = 8.2 Hz, 2H), 7.62–7.54 (m, 1H), 7.48 (dd, J = 8.2, 6.9 Hz, 2H), 7.14 (dd, J = 7.4, 4.9 Hz, 1H), 6.79 (s, 1H), 6.54 (td, J = 45.0, 40.9, 8.1 Hz, 4H), 4.56 (s, 1H), 3.83–3.09 (m, 1H), 2.60 (d, J = 21.1 Hz, 2H), 2.33–2.07 (m, 4H), 2.02–1.61 (m, 1H).

176 2-(4-Benzoylphenoxy)-N-methyl-N-phenylnicotinamide.

O N

N O

The title compound was synthesized following the general procedure for the coupling reaction using 2-(4-benzoylphenoxy)nicotinic acid (100 mg, 0.310 mmol) and N- methylaniline (39.9 mg, 0.372 mmol) as colorless wax (103 mg, 74%). 1H NMR (400 MHz,

CDCl3) δ 8.03 (dd, J = 4.9, 2.0 Hz, 1H), 7.80 (dq, J = 7.1, 2.6, 1.7 Hz, 5H), 7.61–7.55 (m, 1H), 7.48 (dd, J = 8.3, 7.0 Hz, 2H), 7.24–7.13 (m, 3H), 7.13–7.05 (m, 2H), 6.98 (dd, J = 7.5, 4.9 Hz, 1H), 6.85 (d, J = 8.3 Hz, 2H), 3.53 (s, 3H).

2-(4-Acetylphenoxy)-N-methyl-N-(o-tolyl)nicotinamide.

O N

N O

O The title compound was synthesized following the general procedure for the coupling reaction using 2-(4-acetylphenoxy)nicotinic acid (100 mg, 0.390 mmol) and N-methyl-o- toluidine (56.7 mg, 0.468 mmol) as colorless wax (73 mg, 52%). 1H NMR (400 MHz,

CDCl3) δ 7.98–7.94 (m, 3H), 7.73 (dd, J = 7.4, 1.9 Hz, 1H), 7.15–7.08 (m, 2H), 7.07–6.99 (m, 2H), 6.95–6.89 (m, 3H), 3.40 (s, 3H), 2.59 (s, 3H), 2.23 (s, 3H).

177 1-(4-((3-(1,2,3,4-Tetrahydroquinoline-1-carbonyl)pyridin-2-yl)oxy)phenyl)ethan-1- one.

O N

N O

O The title compound was synthesized following the general procedure for the coupling reaction using 2-(4-acetylphenoxy)nicotinic acid (100 mg, 0.390 mmol) and 1,2,3,4- tetrahydroquinoline (62.3 mg, 0.468 mmol) as colorless wax (120 mg, 83%). 1H NMR (400

MHz, CDCl3) δ 8.20–7.98 (m, 2H), 7.85 (d, J = 8.2 Hz, 2H), 7.18–6.81 (m, 4H), 6.75–6.23 (m, 3H), 4.56 (s, 1H), 3.46 (s, 1H), 2.56 (s, 5H), 2.34–2.10 (m, 1H), 1.87–1.61 (m, 1H).

2-(4-Acetylphenoxy)-N-(o-tolyl)nicotinamide.

O N H N O

O The title compound was synthesized following the general procedure for the coupling reaction using 2-(4-acetylphenoxy)nicotinic acid (100 mg, 0.390 mmol) and o-toluidine 1 (50.1 mg, 0.468 mmol) as colorless wax (84 mg, 62%). H NMR (400 MHz, CDCl3) δ 9.63 (s, 1H), 8.77 (dd, J = 7.5, 2.1 Hz, 1H), 8.33–8.22 (m, 2H), 8.10 (d, J = 8.3 Hz, 2H), 7.39– 7.27 (m, 4H), 7.20 (d, J = 7.5 Hz, 1H), 7.09 (t, J = 7.5 Hz, 1H), 2.64 (s, 3H), 2.27 (s, 3H).

178 2-(4-Acetylphenoxy)-N-ethyl-N-phenylnicotinamide.

O N

N O

O The title compound was synthesized following the general procedure for the coupling reaction using 2-(4-acetylphenoxy)nicotinic acid (100 mg, 0.390 mmol) and N-ethylaniline 1 (56.7 mg, 0.468 mmol) as colorless wax (134 mg, 96%). H NMR (400 MHz, CDCl3) δ 8.00–7.92 (m, 3H), 7.74 (dd, J = 7.4, 1.9 Hz, 1H), 7.19 (dt, J = 5.0, 2.6 Hz, 3H), 7.11–7.05 (m, 2H), 6.94 (dd, J = 7.4, 4.9 Hz, 1H), 6.92–6.83 (m, 2H), 4.01 (q, J = 7.1 Hz, 2H), 2.59 (s, 3H), 1.24 (t, J = 7.1 Hz, 3H).

2-(4-Acetylphenoxy)-N-(2-fluorophenyl)-N-methylnicotinamide.

O N F N O

O The title compound was synthesized following the general procedure for the coupling reaction using 2-(4-acetylphenoxy)nicotinic acid (100 mg, 0.390 mmol) and 2-fluoro-N- methylaniline (58.6 mg, 0.468 mmol) as colorless wax (110 mg, 77%). 1H NMR (400 MHz,

CDCl3) δ 8.00 (dd, J = 4.9, 1.9 Hz, 1H), 7.98–7.92 (m, 2H), 7.81 (ddd, J = 7.4, 1.9, 1.0 Hz, 1H), 7.21 (tdd, J = 7.9, 4.9, 1.7 Hz, 1H), 7.17–7.11 (m, 1H), 7.02–6.94 (m, 3H), 6.94–6.90 (m, 2H), 3.46 (s, 3H), 2.59 (s, 3H).

179 1-(4-((3-(6-Methyl-1,2,3,4-tetrahydroquinoline-1-carbonyl)pyridin-2- yl)oxy)phenyl)ethan-1-one.

O N

N O

O The title compound was synthesized following the general procedure for the coupling reaction using 2-(4-acetylphenoxy)nicotinic acid (100 mg, 0.390 mmol) and 6-methyl- 1,2,3,4-tetrahydroquinoline (68.9 mg, 0.468 mmol) as colorless wax (135 mg, 90%). 1H

NMR (400 MHz, CDCl3) δ 8.13–8.08 (m, 1H), 8.06 (s, 1H), 7.84 (d, J = 8.2 Hz, 2H), 7.12 (dd, J = 7.5, 4.8 Hz, 1H), 6.78 (s, 1H), 6.67 (d, J = 8.0 Hz, 1H), 6.47 (dd, J = 30.3, 8.1 Hz, 3H), 4.42 (d, J = 101.2 Hz, 1H), 3.41 (s, 1H), 2.56 (s, 5H), 2.32–2.07 (m, 4H), 1.86 (d, J = 10.8 Hz, 1H).

3-((3-(1,2,3,4-Tetrahydroquinoline-1-carbonyl)pyridin-2-yl)oxy)benzonitrile.

O N

N O

CN The title compound was synthesized following the general procedure for the coupling reaction using 2-(3-cyanophenoxy)nicotinic acid (100 mg, 0.420 mmol) and 1,2,3,4- tetrahydroquinoline (67.1 mg, 0.504 mmol) as colorless wax (95 mg, 65%). 1H NMR (400

MHz, CDCl3) δ 8.18–8.02 (m, 2H), 7.43–7.30 (m, 2H), 7.15 (dd, J = 7.3, 5.1 Hz, 1H), 7.08 (td, J = 7.5, 1.2 Hz, 1H), 7.01 (d, J = 7.5 Hz, 1H), 6.90 (s, 1H), 6.78 (s, 1H), 6.50 (d, J = 42.3 Hz, 2H), 4.75–4.29 (m, 1H), 3.83–3.13 (m, 1H), 2.61 (s, 2H), 2.37–2.05 (m, 1H), 1.92 (s, 1H).

180 2-(3-Cyanophenoxy)-N-(o-tolyl)nicotinamide.

O N H N O

CN The title compound was synthesized following the general procedure for the coupling reaction using 2-(3-cyanophenoxy)nicotinic acid (100 mg, 0.420 mmol) and o-toluidine 1 (54.0 mg, 0.504 mmol) as colorless wax (66 mg, 48%). H NMR (400 MHz, CDCl3) δ 9.53 (s, 1H), 8.78 (dd, J = 7.6, 2.0 Hz, 1H), 8.30 –8.23 (m, 2H), 7.66–7.59 (m, 2H), 7.58–7.55 (m, 1H), 7.49 (dt, J = 7.0, 2.3 Hz, 1H), 7.31 (dd, J = 7.7, 4.8 Hz, 2H), 7.23 (d, J = 7.5 Hz, 1H), 7.15–7.09 (m, 1H), 2.29 (s, 3H).

2-(3-Cyanophenoxy)-N-ethyl-N-phenylnicotinamide.

O N

N O

CN The title compound was synthesized following the general procedure for the coupling reaction using 2-(3-cyanophenoxy)nicotinic acid (100 mg, 0.420 mmol) and N-ethylaniline 1 (61.1 mg, 0.504 mmol) as colorless wax (69 mg, 48%). H NMR (400 MHz, CDCl3) δ 7.96 (dd, J = 4.9, 1.9 Hz, 1H), 7.77 (dd, J = 7.4, 1.9 Hz, 1H), 7.48–7.41 (m, 2H), 7.25–7.18 (m, 3H), 7.10 (dt, J = 7.0, 2.4 Hz, 1H), 7.08–7.02 (m, 2H), 7.00–6.95 (m, 2H), 4.02 (q, J = 7.1 Hz, 2H), 1.24 (t, J = 7.1 Hz, 3H).

181 2-(3-Cyanophenoxy)-N-(2-fluorophenyl)-N-methylnicotinamide.

O N F N O

CN The title compound was synthesized following the general procedure for the coupling reaction using 2-(3-cyanophenoxy)nicotinic acid (100 mg, 0.420 mmol) and 2-fluoro-N- methylaniline (63.1 mg, 0.504 mmol) as colorless wax (131 mg, 91%). 1H NMR (400 MHz,

CDCl3) δ 7.99 (dd, J = 4.9, 2.0 Hz, 1H), 7.82 (ddd, J = 7.5, 2.0, 1.1 Hz, 1H), 7.48–7.41 (m, 2H), 7.27–7.20 (m, 1H), 7.18–7.09 (m, 2H), 7.07–6.93 (m, 4H), 3.47 (s, 3H).

3-((3-(6-Methyl-1,2,3,4-tetrahydroquinoline-1-carbonyl)pyridin-2- yl)oxy)benzonitrile.

O N

N O

CN The title compound was synthesized following the general procedure for the coupling reaction using 2-(3-cyanophenoxy)nicotinic acid (100 mg, 0.420 mmol) and 6-methyl- 1,2,3,4-tetrahydroquinoline (74.2 mg, 0.504 mmol) as colorless wax (72 mg, 46%). 1H

NMR (400 MHz, CDCl3) δ 8.09 (s, 2H), 7.43–7.30 (m, 2H), 7.15 (dd, J = 7.3, 5.1 Hz, 1H), 6.85 (d, J = 8.6 Hz, 1H), 6.81 (s, 1H), 6.69 (d, J = 8.1 Hz, 1H), 6.41 (d, J = 8.2 Hz, 1H), 6.36 (s, 1H), 4.57 (s, 1H), 3.39 (s, 1H), 2.54 (s, 2H), 2.29 (s, 3H), 2.25–2.06 (m, 2H).

2-(3-Cyanophenoxy)-N-methyl-N-phenylnicotinamide.

O N

N O

CN The title compound was synthesized following the general procedure for the coupling reaction using 2-(3-cyanophenoxy)nicotinic acid (100 mg, 0.420 mmol) and N- 182 methylaniline (54.0 mg, 0.504 mmol) as colorless wax (73 mg, 53%). 1H NMR (400 MHz,

CDCl3) δ 7.99 (dd, J = 5.1, 2.1 Hz, 1H), 7.81 (dd, J = 7.4, 2.1 Hz, 1H), 7.48–7.38 (m, 2H), 7.24–7.14 (m, 3H), 7.03 (ddd, J = 19.3, 6.7, 3.6 Hz, 4H), 6.87 (s, 1H), 3.53 (s, 3H).

2-([1,1'-Biphenyl]-3-yloxy)-N-methyl-N-(o-tolyl)nicotinamide.

O N

N O

The title compound was synthesized following the general procedure for the coupling reaction using 2-([1,1'-biphenyl]-3-yloxy)nicotinic acid (100 mg, 0.340 mmol) and N- methyl-o-toluidine (49.4 mg, 0.408 mmol) as colorless wax (97 mg, 71%). 1H NMR (400

MHz, CDCl3) δ 7.98 (dd, J = 5.0, 1.9 Hz, 1H), 7.73 (dd, J = 7.4, 1.9 Hz, 1H), 7.60–7.56 (m, 2H), 7.43 (dd, J = 7.3, 3.8 Hz, 4H), 7.39–7.34 (m, 1H), 7.15–7.09 (m, 4H), 7.07–7.02 (m, 1H), 6.91–6.83 (m, 2H), 3.43 (s, 3H), 2.28 (s, 3H).

(2-([1,1'-Biphenyl]-3-yloxy)pyridin-3-yl)(3,4-dihydroquinolin-1(2H)-yl)methanone.

O N

N O

The title compound was synthesized following the general procedure for the coupling reaction using 2-([1,1'-biphenyl]-3-yloxy)nicotinic acid (100 mg, 0.340 mmol) and 1,2,3,4- tetrahydroquinoline (54.3 mg, 0.408 mmol) as colorless wax (60 mg, 43%). 1H NMR (400

MHz, CDCl3) δ 8.11 (s, 1H), 8.04 (d, J = 7.3 Hz, 1H), 7.57–7.46 (m, 2H), 7.46–7.40 (m, 2H), 7.40–7.28 (m, 3H), 7.08 (t, J = 6.1 Hz, 2H), 7.02 (t, J = 7.4 Hz, 1H), 6.89 (s, 1H), 6.74–6.55 (m, 2H), 6.47 (s, 1H), 4.60 (s, 1H), 3.58 (d, J = 87.1 Hz, 1H), 2.70 (s, 2H), 2.38– 2.10 (m, 1H), 2.00–1.76 (m, 1H).

183 2-([1,1'-Biphenyl]-3-yloxy)-N-(o-tolyl)nicotinamide.

O N H N O

The title compound was synthesized following the general procedure for the coupling reaction using 2-([1,1'-biphenyl]-3-yloxy)nicotinic acid (100 mg, 0.340 mmol) and o- toluidine (43.7 mg, 0.408 mmol) as colorless wax (64 mg, 49%). 1H NMR (400 MHz,

CDCl3) δ 9.88 (s, 1H), 8.78 (dd, J = 7.6, 2.1 Hz, 1H), 8.36–8.27 (m, 2H), 7.65–7.60 (m, 2H), 7.59–7.55 (m, 2H), 7.49–7.43 (m, 3H), 7.42–7.36 (m, 1H), 7.33–7.28 (m, 1H), 7.27– 7.23 (m, 1H), 7.23–7.18 (m, 2H), 7.10 (td, J = 7.5, 1.3 Hz, 1H), 2.33 (s, 3H).

2-([1,1'-Biphenyl]-3-yloxy)-N-ethyl-N-phenylnicotinamide.

O N

N O

The title compound was synthesized following the general procedure for the coupling reaction using 2-([1,1'-biphenyl]-3-yloxy)nicotinic acid (100 mg, 0.340 mmol) and o- toluidine (49.4 mg, 0.408 mmol) as colorless wax (70 mg, 52%). 1H NMR (400 MHz,

CDCl3) δ 7.98 (dd, J = 5.1, 1.9 Hz, 1H), 7.73 (dd, J = 7.4, 1.9 Hz, 1H), 7.60–7.54 (m, 2H), 7.48–7.38 (m, 4H), 7.38–7.32 (m, 1H), 7.26–7.11 (m, 5H), 7.06 (t, J = 1.6 Hz, 1H), 6.94– 6.87 (m, 1H), 6.82 (dt, J = 6.5, 2.6 Hz, 1H), 4.04 (q, J = 7.1 Hz, 2H), 1.28 (t, J = 7.2 Hz, 3H).

184 2-([1,1'-Biphenyl]-3-yloxy)-N-(2-fluorophenyl)-N-methylnicotinamide.

O N F N O

The title compound was synthesized following the general procedure for the coupling reaction using 2-([1,1'-biphenyl]-3-yloxy)nicotinic acid (100 mg, 0.340 mmol) and o- toluidine (51.1 mg, 0.408 mmol) as colorless wax (29 mg, 21%). 1H NMR (400 MHz,

CDCl3) δ 8.01 (dd, J = 4.9, 2.0 Hz, 1H), 7.81 (dt, J = 7.5, 1.6 Hz, 1H), 7.60–7.54 (m, 2H), 7.47–7.40 (m, 4H), 7.40–7.33 (m, 1H), 7.26–7.17 (m, 2H), 7.11–7.09 (m, 1H), 7.05–6.97 (m, 2H), 6.92 (dd, J = 7.4, 4.9 Hz, 1H), 6.84 (dt, J = 6.2, 2.5 Hz, 1H), 3.49 (s, 3H).

(2-([1,1'-Biphenyl]-3-yloxy)pyridin-3-yl)(6-methyl-3,4-dihydroquinolin-1(2H)- yl)methanone.

O N

N O

The title compound was synthesized following the general procedure for the coupling reaction using 2-([1,1'-biphenyl]-3-yloxy)nicotinic acid (100 mg, 0.340 mmol) and o- toluidine (60.1 mg, 0.408 mmol) as colorless wax (120 mg, 83%). 1H NMR (400 MHz,

CDCl3) δ 8.09 (s, 1H), 8.02 (d, J = 7.7 Hz, 1H), 7.48 (d, J = 7.6 Hz, 2H), 7.41 (t, J = 7.7 Hz, 2H), 7.37–7.27 (m, 3H), 7.06 (dd, J = 7.5, 4.8 Hz, 1H), 6.85 (s, 1H), 6.65 (d, J = 8.1 Hz, 1H), 6.50 (d, J = 24.0 Hz, 3H), 4.59 (s, 1H), 3.39 (s, 1H), 2.58 (d, J = 31.5 Hz, 2H), 2.13 (s, 4H), 1.95–1.79 (m, 1H).

185 2-([1,1'-Biphenyl]-3-yloxy)-N-methyl-N-phenylnicotinamide.

O N

N O

CDCl3) δ 8.03–7.96 (m, 1H), 7.80–7.73 (m, 1H), 7.54 (d, J = 7.6 Hz, 2H), 7.47–7.30 (m, 5H), 7.24–7.09 (m, 5H), 6.97 (s, 1H), 6.95–6.89 (m, 1H), 6.74 (d, J = 7.0 Hz, 1H), 3.53 (s, 3H).

2-(3-Acetylphenoxy)-N-methyl-N-(o-tolyl)nicotinamide.

O N

N O

The title compound was synthesized following the general procedure for the coupling reaction using 2-(3-acetylphenoxy)nicotinic acid (100 mg, 0.390 mmol) and N-methyl-o- toluidine (56.7 mg, 0.468 mmol) as colorless wax (114 mg, 81%). 1H NMR (400 MHz,

CDCl3) δ 7.93 (dd, J = 4.9, 2.0 Hz, 1H), 7.79 (dt, J = 7.8, 1.3 Hz, 1H), 7.73 (dd, J = 7.4, 2.0 Hz, 1H), 7.44 (t, J = 7.9 Hz, 1H), 7.40 (t, J = 2.0 Hz, 1H), 7.18–7.11 (m, 2H), 7.10– 7.02 (m, 3H), 6.90 (dd, J = 7.4, 4.9 Hz, 1H), 3.41 (s, 3H), 2.57 (s, 3H), 2.24 (s, 3H).

186 1-(3-((3-(1,2,3,4-Tetrahydroquinoline-1-carbonyl)pyridin-2-yl)oxy)phenyl)ethan-1- one.

O N

N O

The title compound was synthesized following the general procedure for the coupling reaction using 2-(3-acetylphenoxy)nicotinic acid (100 mg, 0.390 mmol) and 1,2,3,4- tetrahydroquinoline (62.3 mg, 0.468 mmol) as colorless wax (141 mg, 97%). 1H NMR (400

MHz, CDCl3) δ 8.12–7.97 (m, 2H), 7.72 (d, J = 7.7 Hz, 1H), 7.34 (t, J = 8.0 Hz, 1H), 7.14– 7.02 (m, 3H), 6.93 (d, J = 20.8 Hz, 2H), 6.62 (d, J = 29.6 Hz, 2H), 4.56 (s, 1H), 3.46 (s, 1H), 2.64 (s, 2H), 2.52 (s, 3H), 2.32–2.10 (m, 1H), 1.98–1.77 (m, 1H).

2-(3-Acetylphenoxy)-N-(o-tolyl)nicotinamide.

O N H N O

The title compound was synthesized following the general procedure for the coupling reaction using 2-(3-acetylphenoxy)nicotinic acid (100 mg, 0.390 mmol) and o-toluidine 1 (50.1 mg, 0.468 mmol) as colorless wax (84 mg, 63%). H NMR (400 MHz, CDCl3) δ 9.71 (s, 1H), 8.76 (dd, J = 7.7, 2.0 Hz, 1H), 8.30–8.26 (m, 1H), 8.25 (dd, J = 4.8, 2.0 Hz, 1H), 7.91 (dt, J = 7.7, 1.3 Hz, 1H), 7.82 (t, J = 2.0 Hz, 1H), 7.60 (t, J = 7.9 Hz, 1H), 7.44 (ddd, J = 8.1, 2.6, 1.0 Hz, 1H), 7.32–7.27 (m, 1H), 7.25 (d, J = 4.9 Hz, 1H), 7.19 (d, J = 7.3 Hz, 1H), 7.09 (td, J = 7.4, 1.2 Hz, 1H), 2.63 (s, 3H), 2.28 (s, 3H).

187 2-(3-Acetylphenoxy)-N-ethyl-N-phenylnicotinamide.

O N

N O

The title compound was synthesized following the general procedure for the coupling reaction using 2-(3-acetylphenoxy)nicotinic acid (100 mg, 0.390 mmol) and o-toluidine 1 (56.7 mg, 0.468 mmol) as colorless wax (140 mg, 99%). H NMR (400 MHz, CDCl3) δ 7.94 (dd, J = 5.0, 1.9 Hz, 1H), 7.77 (dt, J = 7.8, 1.3 Hz, 1H), 7.73 (dd, J = 7.4, 1.9 Hz, 1H), 7.43 (t, J = 7.9 Hz, 1H), 7.38 (t, J = 2.0 Hz, 1H), 7.23 (d, J = 6.9 Hz, 3H), 7.11 (dd, J = 7.6, 2.0 Hz, 2H), 7.07–7.00 (m, 1H), 6.91 (dd, J = 7.4, 4.9 Hz, 1H), 4.02 (q, J = 7.1 Hz, 2H), 2.56 (s, 3H), 1.24 (t, J = 7.1 Hz, 3H).

2-(3-Acetylphenoxy)-N-(2-fluorophenyl)-N-methylnicotinamide.

O N F N O

The title compound was synthesized following the general procedure for the coupling reaction using 2-(3-acetylphenoxy)nicotinic acid (100 mg, 0.390 mmol) and 2-fluoro-N- methylaniline (58.6 mg, 0.468 mmol) as colorless wax (119 mg, 84%). 1H NMR (400 MHz,

CDCl3) δ 7.97 (dd, J = 4.9, 2.0 Hz, 1H), 7.79 (ddt, J = 10.6, 7.8, 1.5 Hz, 2H), 7.44 (t, J = 7.9 Hz, 1H), 7.40 (t, J = 2.1 Hz, 1H), 7.26–7.15 (m, 2H), 7.08 (ddd, J = 8.1, 2.5, 1.0 Hz, 1H), 7.06–6.97 (m, 2H), 6.97–6.90 (m, 1H), 3.47 (s, 3H), 2.57 (s, 3H).

188 1-(3-((3-(6-Methyl-1,2,3,4-tetrahydroquinoline-1-carbonyl)pyridin-2- yl)oxy)phenyl)ethan-1-one.

O N

N O

The title compound was synthesized following the general procedure for the coupling reaction using 2-(3-acetylphenoxy)nicotinic acid (100 mg, 0.390 mmol) and 6-methyl- 1,2,3,4-tetrahydroquinoline (68.9 mg, 0.468 mmol) as colorless wax (128 mg, 85%). 1H

NMR (400 MHz, CDCl3) δ 8.05 (dd, J = 11.1, 6.3 Hz, 2H), 7.71 (d, J = 7.7 Hz, 1H), 7.33 (t, J = 7.9 Hz, 1H), 7.09 (dd, J = 7.4, 4.9 Hz, 1H), 7.00 (s, 1H), 6.85 (s, 1H), 6.68 (d, J = 7.8 Hz, 2H), 6.45 (d, J = 8.1 Hz, 1H), 4.56 (s, 1H), 3.60–3.19 (m, 1H), 2.52 (s, 5H), 2.34– 2.06 (m, 4H), 2.00–1.67 (m, 1H).

2-(3-Acetylphenoxy)-N-methyl-N-phenylnicotinamide.

O N

N O

The title compound was synthesized following the general procedure for the coupling reaction using 2-(3-acetylphenoxy)nicotinic acid (100 mg, 0.390 mmol) and N- methylaniline (50.1 mg, 0.468 mmol) as colorless wax (108 mg, 80%). 1H NMR (400 MHz,

CDCl3) δ 7.97 (dd, J = 5.1, 2.1 Hz, 1H), 7.77 (td, J = 6.0, 5.5, 2.8 Hz, 2H), 7.42 (t, J = 7.9 Hz, 1H), 7.31 (t, J = 2.0 Hz, 1H), 7.22 (d, J = 7.1 Hz, 3H), 7.14–7.06 (m, 2H), 6.96 (ddd, J = 12.9, 7.8, 3.7 Hz, 2H), 3.53 (s, 3H), 2.56 (s, 3H).

189 2-(4-Chlorophenoxy)-N-methyl-N-(o-tolyl)nicotinamide.

O N

N O

Cl The title compound was synthesized following the general procedure for the coupling reaction using 2-(4-chlorophenoxy)nicotinic acid (100 mg, 0.400 mmol) and N-methyl-o- toluidine (58.2 mg, 0.480 mmol) as colorless wax (59 mg, 42%). 1H NMR (400 MHz,

CDCl3) δ 7.95 (dd, J = 4.9, 1.9 Hz, 1H), 7.72 (dd, J = 7.4, 2.0 Hz, 1H), 7.33–7.28 (m, 2H), 7.16–7.10 (m, 2H), 7.08–7.01 (m, 2H), 6.89 (dd, J = 7.4, 4.9 Hz, 1H), 6.81–6.75 (m, 2H), 3.41 (s, 3H), 2.24 (s, 3H).

(2-(4-Chlorophenoxy)pyridin-3-yl)(3,4-dihydroquinolin-1(2H)-yl)methanone.

O N

N O

Cl The title compound was synthesized following the general procedure for the coupling reaction using 2-(4-chlorophenoxy)nicotinic acid (100 mg, 0.400 mmol) and 1,2,3,4- tetrahydroquinoline (63.9 mg, 0.480 mmol) as colorless wax (120 mg, 82%). 1H NMR (400

MHz, CDCl3) δ 8.06 (d, J = 19.0 Hz, 2H), 7.19 (d, J = 8.3 Hz, 2H), 7.08 (dt, J = 10.3, 5.9 Hz, 3H), 6.88 (s, 1H), 6.57 (s, 1H), 6.34 (s, 2H), 4.57 (s, 1H), 3.46 (s, 1H), 2.66 (s, 2H), 2.40–2.08 (m, 1H), 1.92 (s, 1H).

190 2-(4-Chlorophenoxy)-N-(o-tolyl)nicotinamide.

O N H N O

Cl The title compound was synthesized following the general procedure for the coupling reaction using 2-(4-chlorophenoxy)nicotinic acid (100 mg, 0.400 mmol) and o-toluidine 1 (51.4 mg, 0.480 mmol) as colorless wax (86 mg, 63%). H NMR (400 MHz, CDCl3) δ 9.72 (s, 1H), 8.76 (dd, J = 7.7, 2.0 Hz, 1H), 8.34–8.26 (m, 2H), 7.49–7.44 (m, 2H), 7.31 (d, J = 7.3 Hz, 1H), 7.27–7.24 (m, 1H), 7.24–7.16 (m, 3H), 7.11 (td, J = 7.5, 1.3 Hz, 1H), 2.29 (s, 3H).

2-(4-Chlorophenoxy)-N-ethyl-N-phenylnicotinamide.

O N

N O

Cl The title compound was synthesized following the general procedure for the coupling reaction using 2-(4-chlorophenoxy)nicotinic acid (100 mg, 0.400 mmol) and N- ethylaniline (58.2 mg, 0.480 mmol) as colorless wax (89 mg, 63%). 1H NMR (400 MHz,

CDCl3) δ 7.95 (dd, J = 5.1, 2.1 Hz, 1H), 7.72 (dd, J = 7.2, 2.1 Hz, 1H), 7.29 (d, J = 8.5 Hz, 2H), 7.21 (d, J = 7.3 Hz, 3H), 7.14–7.05 (m, 2H), 6.90 (dd, J = 7.4, 4.9 Hz, 1H), 6.80–6.71 (m, 2H), 4.01 (q, J = 7.2 Hz, 2H), 1.24 (d, J = 6.7 Hz, 3H).

191 2-(4-Chlorophenoxy)-N-(2-fluorophenyl)-N-methylnicotinamide.

O N F N O

Cl The title compound was synthesized following the general procedure for the coupling reaction using 2-(4-chlorophenoxy)nicotinic acid (100 mg, 0.400 mmol) and 2-fluoro-N- methylaniline (60.1 mg, 0.480 mmol) as colorless wax (77 mg, 54%). 1H NMR (400 MHz,

CDCl3) δ 7.98 (dd, J = 5.0, 2.0 Hz, 1H), 7.79 (dt, J = 7.6, 1.6 Hz, 1H), 7.33–7. 29 (m, 2H), 7.25–7.13 (m, 2H), 7.04–6.96 (m, 2H), 6.93 (dd, J = 7.4, 5.0 Hz, 1H), 6.82–6.75 (m, 2H), 3.47 (s, 3H).

(2-(4-Chlorophenoxy)pyridin-3-yl)(6-methyl-3,4-dihydroquinolin-1(2H)- yl)methanone.

O N

N O

Cl The title compound was synthesized following the general procedure for the coupling reaction using 2-(4-chlorophenoxy)nicotinic acid (100 mg, 0.400 mmol) and 6-methyl- 1,2,3,4-tetrahydroquinoline (70.6 mg, 0.480 mmol) as colorless wax (103 mg, 68%). 1H

NMR (400 MHz, CDCl3) δ 8.12–8.05 (m, 1H), 8.01 (d, J = 7.3 Hz, 1H), 7.17 (d, J = 8.2 Hz, 2H), 7.07 (dd, J = 7.4, 4.8 Hz, 1H), 6.85 (s, 1H), 6.65 (d, J = 7.8 Hz, 1H), 6.42 (d, J = 8.1 Hz, 1H), 6.34 (d, J = 8.2 Hz, 2H), 4.42 (s, 1H), 3.40 (s, 1H), 2.57 (d, J = 17.5 Hz, 2H), 2.25–2.08 (m, 4H), 1.87 (s, 1H).

192 2-(2-Chloro-5-fluorophenoxy)-N-methyl-N-(o-tolyl)nicotinamide.

O N

N O Cl

F The title compound was synthesized following the general procedure for the coupling reaction using 2-(2-chloro-5-fluorophenoxy)nicotinic acid (100 mg, 0.370 mmol) and N- methyl-o-toluidine (53.8 mg, 0.444 mmol) as colorless wax (142 mg, 99%). 1H NMR (400

MHz, CDCl3) δ 7.95 (dd, J = 4.9, 1.9 Hz, 1H), 7.66 (dd, J = 7.4, 1.9 Hz, 1H), 7.40 (dd, J = 8.9, 5.8 Hz, 1H), 7.18–7.14 (m, 3H), 7.05 (ddd, J = 7.7, 5.2, 3.9 Hz, 1H), 6.93–6.86 (m, 2H), 6.60 (dd, J = 9.0, 2.9 Hz, 1H), 3.42 (s, 3H), 2.30 (s, 3H).

(2-(2-Chloro-5-fluorophenoxy)pyridin-3-yl)(3,4-dihydroquinolin-1(2H)- yl)methanone.

O N

N O Cl

F The title compound was synthesized following the general procedure for the coupling reaction using 2-(2-chloro-5-fluorophenoxy)nicotinic acid (100 mg, 0.370 mmol) and 1,2,3,4-tetrahydroquinoline (59.1 mg, 0.444 mmol) as colorless wax (120 mg, 84%). 1H

NMR (400 MHz, CDCl3) δ 8.10 (s, 1H), 8.01 (s, 1H), 7.33 (t, J = 7.5 Hz, 1H), 7.13–7.05 (m, 3H), 6.86 (d, J = 26.5 Hz, 2H), 6.61 (s, 1H), 5.83 (s, 1H), 4.41 (d, J = 67.6 Hz, 1H), 3.58 (s, 1H), 2.71 (s, 2H), 1.99 (d, J = 81.3 Hz, 2H).

193 2-(2-Chloro-5-fluorophenoxy)-N-(o-tolyl)nicotinamide.

O N H N O Cl

F The title compound was synthesized following the general procedure for the coupling reaction using 2-(2-chloro-5-fluorophenoxy)nicotinic acid (100 mg, 0.370 mmol) and o- toluidine (47.6 mg, 0.444 mmol) as colorless wax (98 mg, 74%). 1H NMR (400 MHz,

CDCl3) δ 9.53 (s, 1H), 8.75 (dd, J = 7.6, 2.0 Hz, 1H), 8.28–8.21 (m, 2H), 7.51 (dd, J = 8.9, 5.6 Hz, 1H), 7.33–7.26 (m, 2H), 7.22 (d, J = 7.3 Hz, 1H), 7.15–7.09 (m, 2H), 7.06 (ddd, J = 8.9, 7.7, 2.9 Hz, 1H), 2.30 (s, 3H).

2-(2-Chloro-5-fluorophenoxy)-N-ethyl-N-phenylnicotinamide.

O N

N O Cl

F The title compound was synthesized following the general procedure for the coupling reaction using 2-(2-chloro-5-fluorophenoxy)nicotinic acid (100 mg, 0.370 mmol) and N- ethylaniline (53.8 mg, 0.444 mmol) as colorless wax (108 mg, 78%). 1H NMR (400 MHz,

CDCl3) δ 7.95 (dd, J = 4.9, 1.9 Hz, 1H), 7.64 (dd, J = 7.4, 1.9 Hz, 1H), 7.39 (dd, J = 8.9, 5.7 Hz, 1H), 7.21 (dt, J = 17.2, 4.3 Hz, 5H), 6.94–6.85 (m, 2H), 6.61 (dd, J = 9.1, 2.9 Hz, 1H), 4.03 (q, J = 7.1 Hz, 2H), 1.26 (t, J = 7.2 Hz, 3H).

194 2-(2-Chloro-5-fluorophenoxy)-N-(2-fluorophenyl)-N-methylnicotinamide.

O N F N O Cl

F The title compound was synthesized following the general procedure for the coupling reaction using 2-(2-chloro-5-fluorophenoxy)nicotinic acid (100 mg, 0.370 mmol) and 2- fluoro-N-methylaniline (55.6 mg, 0.444 mmol) as colorless wax (35 mg, 25%). 1H NMR

(400 MHz, CDCl3) δ 7.98 (dd, J = 4.9, 1.9 Hz, 1H), 7.74 (dt, J = 7.6, 1.6 Hz, 1H), 7.40 (dd, J = 8.9, 5.7 Hz, 1H), 7.34–7.28 (m, 1H), 7.26–7.18 (m, 1H), 7.06–6.99 (m, 2H), 6.96–6.87 (m, 2H), 6.66 (dd, J = 9.0, 2.9 Hz, 1H), 3.48 (s, 3H).

(2-(2-Chloro-5-fluorophenoxy)pyridin-3-yl)(6-methyl-3,4-dihydroquinolin-1(2H)- yl)methanone.

O N

N O Cl

F The title compound was synthesized following the general procedure for the coupling reaction using 2-(2-chloro-5-fluorophenoxy)nicotinic acid (100 mg, 0.370 mmol) and 6- methyl-1,2,3,4-tetrahydroquinoline (65.4 mg, 0.444 mmol) as colorless wax (153 mg, 1 99%). H NMR (400 MHz, CDCl3) δ 8.11 (s, 1H), 8.02 (s, 1H), 7.32 (s, 1H), 7.12 (dd, J = 7.4, 4.9 Hz, 1H), 6.84 (d, J = 19.1 Hz, 2H), 6.68 (s, 1H), 6.47 (s, 1H), 5.74 (d, J = 9.1 Hz, 1H), 4.52 (s, 1H), 3.49 (s, 1H), 2.63 (s, 2H), 2.37–2.12 (m, 4H), 1.94 (s, 1H).

195 2-(2-Chloro-5-fluorophenoxy)-N-methyl-N-phenylnicotinamide.

O N

N O Cl

F The title compound was synthesized following the general procedure for the coupling reaction using 2-(2-chloro-5-fluorophenoxy)nicotinic acid (100 mg, 0.370 mmol) and N- methylaniline (47.6 mg, 0.444 mmol) as colorless wax (114 mg, 85%). 1H NMR (400 MHz,

CDCl3) δ 8.02–7.95 (m, 1H), 7.72–7.64 (m, 1H), 7.39 (dd, J = 8.9, 5.7 Hz, 1H), 7.20 (td, J = 11.0, 8.9, 4.5 Hz, 5H), 6.90 (ddd, J = 20.0, 7.9, 3.9 Hz, 2H), 6.52 (dd, J = 9.1, 2.9 Hz, 1H), 3.55 (s, 3H).

2-(4-Fluoro-3-(trifluoromethyl)phenoxy)-N-methyl-N-(o-tolyl)nicotinamide.

O N

N O

(400 MHz, CDCl3) δ 7.96 (dd, J = 4.9, 2.0 Hz, 1H), 7.77 (dd, J = 7.4, 1.9 Hz, 1H), 7.19 (d, J = 9.2 Hz, 1H), 7.16–7.10 (m, 2H), 7.04 (dq, J = 5.0, 2.5, 1.9 Hz, 3H), 6.96 (td, J = 8.2, 7.4, 5.4 Hz, 2H), 3.42 (s, 3H), 2.23 (s, 3H).

196 (3,4-Dihydroquinolin-1(2H)-yl)(2-(4-fluoro-3-(trifluoromethyl)phenoxy)pyridin-3- yl)methanone.

O N

N O

NMR (400 MHz, CDCl3) δ 8.08 (d, J = 5.6 Hz, 2H), 7.14 (dd, J = 7.2, 5.2 Hz, 1H), 7.06 (d, J = 4.9 Hz, 3H), 6.89 (s, 1H), 6.68 (s, 1H), 6.50 (d, J = 44.8 Hz, 2H), 4.64 (d, J = 47.8 Hz, 1H), 3.46 (s, 1H), 2.65 (s, 2H), 2.20 (s, 1H), 1.93 (s, 1H).

2-(4-Fluoro-3-(trifluoromethyl)phenoxy)-N-(o-tolyl)nicotinamide.

O N H N O

CDCl3) δ 9.57 (s, 1H), 8.77 (dd, J = 7.6, 2.0 Hz, 1H), 8.30–8.23 (m, 2H), 7.50 (dd, J = 5.8, 2.9 Hz, 1H), 7.43 (dt, J = 7.2, 3.6 Hz, 1H), 7.37–7.28 (m, 3H), 7.23 (d, J = 7.2 Hz, 1H), 7.12 (td, J = 7.5, 1.3 Hz, 1H), 2.30 (s, 3H).

197 N-Ethyl-2-(4-fluoro-3-(trifluoromethyl)phenoxy)-N-phenylnicotinamide.

O N

N O

MHz, CDCl3) δ 7.96 (dd, J = 4.9, 1.9 Hz, 1H), 7.78 (dd, J = 7.4, 2.0 Hz, 1H), 7.22 (dd, J = 5.1, 2.0 Hz, 3H), 7.16 (t, J = 9.3 Hz, 1H), 7.11–7.06 (m, 2H), 6.98 (ddd, J = 12.5, 7.3, 4.1 Hz, 2H), 6.92 (dd, J = 5.9, 2.9 Hz, 1H), 4.03 (q, J = 7.1 Hz, 2H), 1.28–1.25 (m, 3H).

2-(4-Fluoro-3-(trifluoromethyl)phenoxy)-N-(2-fluorophenyl)-N-methylnicotinamide.

O N F N O

NMR (400 MHz, CDCl3) δ 7.90 (dd, J = 5.0, 1.9 Hz, 1H), 7.75 (dt, J = 7.6, 1.5 Hz, 1H), 7.18–7.11 (m, 1H), 7.11–7.03 (m, 2H), 7.01–6.94 (m, 1H), 6.93 (s, 1H), 6.92–6.87 (m, 3H), 3.40 (s, 3H).

198 (2-(4-Fluoro-3-(trifluoromethyl)phenoxy)pyridin-3-yl)(6-methyl-3,4- dihydroquinolin-1(2H)-yl)methanone.

O N

N O

F3C F The title compound was synthesized following the general procedure for the coupling reaction using 2-(4-fluoro-3-(trifluoromethyl)phenoxy)nicotinic acid (100 mg, 0.330 mmol) and 6-methyl-1,2,3,4-tetrahydroquinoline (58.3 mg, 0.396 mmol) as colorless wax (101 1 mg, 71%). H NMR (400 MHz, CDCl3) δ 8.08 (d, J = 5.9 Hz, 2H), 7.13 (dd, J = 7.1, 5.2 Hz, 1H), 7.08 (t, J = 9.4 Hz, 1H), 6.88 (s, 1H), 6.75 (s, 1H), 6.69 (d, J = 8.1 Hz, 1H), 6.43 (s, 2H), 4.58 (s, 1H), 3.41 (s, 1H), 2.64 (s, 2H), 2.34-2.07 (m, 4H), 1.90 (s, 1H).

2-(4-Fluoro-3-(trifluoromethyl)phenoxy)-N-methyl-N-phenylnicotinamide.

O N

N O

(400 MHz, CDCl3) δ 7.99 (dd, J = 5.0, 1.9 Hz, 1H), 7.83 (dd, J = 7.4, 1.9 Hz, 1H), 7.21 (dd, J = 5.4, 2.0 Hz, 3H), 7.14 (t, J = 9.3 Hz, 1H), 7.11–7.05 (m, 2H), 7.00 (dd, J = 7.4, 4.9 Hz, 1H), 6.94 (dt, J = 8.1, 3.3 Hz, 1H), 6.83 (dd, J = 6.0, 2.9 Hz, 1H), 3.54 (s, 3H).

199 2-(2-Chloro-5-(trifluoromethyl)phenoxy)-N-methyl-N-(o-tolyl)nicotinamide.

O N

N O Cl

F3C The title compound was synthesized following the general procedure for the coupling reaction using 2-(2-chloro-5-(trifluoromethyl)phenoxy)nicotinic acid (100 mg, 0.310 mmol) and N-methyl-o-toluidine (45.1 mg, 0.372 mmol) as colorless wax (120 mg, 90%). 1 H NMR (400 MHz, CDCl3) δ 7.93 (dd, J = 4.9, 2.0 Hz, 1H), 7.71 (dd, J = 7.4, 1.9 Hz, 1H), 7.56 (d, J = 8.5 Hz, 1H), 7.40 (dd, J = 8.4, 2.0 Hz, 1H), 7.13 (td, J = 5.8, 5.3, 2.8 Hz, 3H), 7.03 (ddd, J = 8.3, 6.3, 2.4 Hz, 1H), 6.98 (d, J = 2.0 Hz, 1H), 6.92 (dd, J = 7.4, 4.9 Hz, 1H), 3.42 (s, 3H), 2.27 (s, 3H).

(2-(2-Chloro-5-(trifluoromethyl)phenoxy)pyridin-3-yl)(3,4-dihydroquinolin-1(2H)- yl)methanone.

O N

N O Cl

F3C The title compound was synthesized following the general procedure for the coupling reaction using 2-(2-chloro-5-(trifluoromethyl)phenoxy)nicotinic acid (100 mg, 0.310 mmol) and 1,2,3,4-tetrahydroquinoline (49.5 mg, 0.372 mmol) as colorless wax (118 mg, 1 87%). H NMR (400 MHz, CDCl3) δ 8.07 (d, J = 5.2 Hz, 2H), 7.49 (d, J = 8.4 Hz, 1H), 7.33 (d, J = 8.4 Hz, 1H), 7.13 (dd, J = 7.3, 5.0 Hz, 1H), 7.05 (d, J = 4.7 Hz, 2H), 6.90 (s, 1H), 6.60 (s, 1H), 6.20 (s, 1H), 4.38 (s, 1H), 3.47 (s, 1H), 2.68 (s, 2H), 2.21 (s, 1H), 1.97– 1.85 (m, 1H).

200 2-(2-Chloro-5-(trifluoromethyl)phenoxy)-N-(o-tolyl)nicotinamide.

O N H N O Cl

(400 MHz, CDCl3) δ 9.50 (s, 1H), 8.77 (dd, J = 7.6, 2.0 Hz, 1H), 8.27–8.20 (m, 2H), 7.69 (d, J = 8.4 Hz, 1H), 7.65–7.55 (m, 2H), 7.33–7.28 (m, 2H), 7.25–7.20 (m, 1H), 7.12 (td, J = 7.5, 1.3 Hz, 1H), 2.30 (s, 3H).

2-(2-Chloro-5-(trifluoromethyl)phenoxy)-N-ethyl-N-phenylnicotinamide.

O N

N O Cl

NMR (400 MHz, CDCl3) δ 7.93 (dd, J = 5.1, 1.9 Hz, 1H), 7.70 (dd, J = 7.5, 1.9 Hz, 1H), 7.55 (d, J = 8.4 Hz, 1H), 7.42–7.35 (m, 1H), 7.26–7.18 (m, 3H), 7.15 (dd, J = 8.0, 1.8 Hz, 2H), 6.99–6.96 (m, 1H), 6.93 (dd, J = 7.4, 4.9 Hz, 1H), 4.03 (q, J = 7.1 Hz, 2H), 1.28 (d, J = 2.1 Hz, 3H).

201 2-(2-Chloro-5-(trifluoromethyl)phenoxy)-N-(2-fluorophenyl)-N-methylnicotinamide.

O N F N O Cl

F3C The title compound was synthesized following the general procedure for the coupling reaction using 2-(2-chloro-5-(trifluoromethyl)phenoxy)nicotinic acid (100 mg, 0.310 mmol) and 2-floro-N-methylaniline (46.6 mg, 0.372 mmol) as colorless wax (88 mg, 66%). 1 H NMR (400 MHz, CDCl3) δ 7.96 (dd, J = 4.9, 1.9 Hz, 1H), 7.78 (dt, J = 7.4, 1.4 Hz, 1H), 7.57 (d, J = 8.4 Hz, 1H), 7.41 (dd, J = 8.4, 2.0 Hz, 1H), 7.30–7.19 (m, 2H), 7.06–6.99 (m, 3H), 6.99–6.93 (m, 1H), 3.48 (s, 3H).

(2-(2-Chloro-5-(trifluoromethyl)phenoxy)pyridin-3-yl)(6-methyl-3,4- dihydroquinolin-1(2H)-yl)methanone.

O N

N O Cl

F3C The title compound was synthesized following the general procedure for the coupling reaction using 2-(2-chloro-5-(trifluoromethyl)phenoxy)nicotinic acid (100 mg, 0.310 mmol) and 6-methyl-1,2,3,4-tetrahydroquinoline (54.8 mg, 0.372 mmol) as colorless wax 1 (124 mg, 88%). H NMR (400 MHz, CDCl3) δ 8.05 (d, J = 5.8 Hz, 2H), 7.49 (d, J = 8.4 Hz, 1H), 7.33 (d, J = 8.4 Hz, 1H), 7.12 (dd, J = 7.2, 5.2 Hz, 1H), 6.89 (s, 1H), 6.68 (d, J = 7.9 Hz, 1H), 6.45 (d, J = 8.2 Hz, 1H), 6.22 (s, 1H), 4.87–4.28 (m, 1H), 3.85–3.16 (m, 1H), 2.62 (s, 2H), 2.36–2.07 (m, 4H), 2.09–1.88 (m, 1H).

202 2-(2-Chloro-5-(trifluoromethyl)phenoxy)-N-methyl-N-phenylnicotinamide.

O N

N O Cl

NMR (400 MHz, CDCl3) δ 8.00–7.93 (m, 1H), 7.75 (dd, J = 7.2, 2.0 Hz, 1H), 7.55 (d, J = 8.4 Hz, 1H), 7.45–7.36 (m, 1H), 7.26–7.09 (m, 5H), 6.96 (dd, J = 7.4, 4.9 Hz, 1H), 6.91– 6.82 (m, 1H), 3.55 (s, 3H).

2-(3,4-Difluorophenoxy)-N-methyl-N-(o-tolyl)nicotinamide.

O N

N O

F F The title compound was synthesized following the general procedure for the coupling reaction using 2-(3,4-difluorophenoxy)nicotinic acid (100 mg, 0.400 mmol) and N-methyl- o-toluidine (58.2 mg, 0.480 mmol) as colorless wax (147 mg, 98%). 1H NMR (400 MHz,

CDCl3) δ 7.95 (dd, J = 4.9, 1.9 Hz, 1H), 7.72 (dd, J = 7.4, 2.0 Hz, 1H), 7.16–7.10 (m, 3H), 7.05–7.02 (m, 2H), 6.91 (dd, J = 7.4, 4.9 Hz, 1H), 6.68–6.58 (m, 2H), 3.40 (s, 3H), 2.24 (s, 3H).

203 (2-(3,4-Difluorophenoxy)pyridin-3-yl)(3,4-dihydroquinolin-1(2H)-yl)methanone.

O N

N O

F F The title compound was synthesized following the general procedure for the coupling reaction using 2-(3,4-difluorophenoxy)nicotinic acid (100 mg, 0.400 mmol) and 1,2,3,4- tetrahydroquinoline (63.9 mg, 0.480 mmol) as colorless wax (135 mg, 92%). 1H NMR (400

MHz, CDCl3) δ 8.07 (d, J = 16.6 Hz, 2H), 7.13–6.95 (m, 4H), 6.87 (s, 1H), 6.54 (s, 1H), 6.17 (d, J = 34.6 Hz, 2H), 4.56 (s, 1H), 3.45 (s, 1H), 2.66 (s, 2H), 2.35–2.07 (m, 1H), 2.02– 1.73 (m, 1H).

2-(3,4-Difluorophenoxy)-N-(o-tolyl)nicotinamide.

O N H N O

F F The title compound was synthesized following the general procedure for the coupling reaction using 2-(3,4-difluorophenoxy)nicotinic acid (100 mg, 0.400 mmol) and o- toluidine (51.4 mg, 0.480 mmol) as colorless wax (89 mg, 66%). 1H NMR (400 MHz,

CDCl3) δ 9.60 (s, 1H), 8.75 (dd, J = 7.7, 2.0 Hz, 1H), 8.29–8.23 (m, 2H), 7.29 (dd, J = 8.6, 5.9 Hz, 2H), 7.24 (d, J = 5.6 Hz, 1H), 7.21 (d, J = 7.6 Hz, 1H), 7.15–7.07 (m, 2H), 7.00– 6.95 (m, 1H), 2.28 (s, 3H).

204 2-(3,4-Difluorophenoxy)-N-ethyl-N-phenylnicotinamide.

O N

N O

F F The title compound was synthesized following the general procedure for the coupling reaction using 2-(3,4-difluorophenoxy)nicotinic acid (100 mg, 0.400 mmol) and N- ethylaniline (58.2 mg, 0.480 mmol) as colorless wax (140 mg, 99%). 1H NMR (400 MHz,

CDCl3) δ 7.95 (dd, J = 4.9, 1.9 Hz, 1H), 7.73 (dd, J = 7.4, 1.9 Hz, 1H), 7.26–7.18 (m, 3H), 7.15–7.05 (m, 3H), 6.93 (dd, J = 7.4, 4.9 Hz, 1H), 6.65–6.55 (m, 2H), 4.01 (q, J = 7.1 Hz, 2H), 1.24 (t, J = 7.1 Hz, 3H).

2-(3,4-Difluorophenoxy)-N-(2-fluorophenyl)-N-methylnicotinamide.

O N F N O

F F The title compound was synthesized following the general procedure for the coupling reaction using 2-(3,4-difluorophenoxy)nicotinic acid (100 mg, 0.400 mmol) and 2-fluoro- N-methylaniline (60.1 mg, 0.480 mmol) as colorless wax (131 mg, 92%). 1H NMR (400

MHz, CDCl3) δ 7.98 (dd, J = 5.0, 1.9 Hz, 1H), 7.79 (dt, J = 7.4, 1.5 Hz, 1H), 7.22 (tdd, J = 7.8, 4.9, 1.7 Hz, 1H), 7.19–7.07 (m, 2H), 7.05–6.97 (m, 2H), 6.97–6.92 (m, 1H), 6.70– 6.59 (m, 2H), 3.46 (s, 3H).

205 (2-(3,4-Difluorophenoxy)pyridin-3-yl)(6-methyl-3,4-dihydroquinolin-1(2H)- yl)methanone.

O N

N O

F F The title compound was synthesized following the general procedure for the coupling reaction using 2-(3,4-difluorophenoxy)nicotinic acid (100 mg, 0.400 mmol) and 6-methyl- 1,2,3,4-tetrahydroquinoline (70.7 mg, 0.480 mmol) as colorless wax (88 mg, 58%). 1H

NMR (400 MHz, CDCl3) δ 8.09 (s, 1H), 8.04 (d, J = 7.4 Hz, 1H), 7.10 (dd, J = 7.4, 4.9 Hz, 1H), 7.01 (d, J = 9.4 Hz, 1H), 6.86 (s, 1H), 6.67 (d, J = 8.0 Hz, 1H), 6.40 (d, J = 8.1 Hz, 1H), 6.29 (s, 1H), 6.01 (s, 1H), 4.56 (s, 1H), 3.40 (s, 1H), 2.59 (s, 2H), 2.20–2.12 (m, 4H), 1.97 (s, 1H).

2-(3,4-Difluorophenoxy)-N-methyl-N-phenylnicotinamide.

O N

N O

F F The title compound was synthesized following the general procedure for the coupling reaction using 2-(3,4-difluorophenoxy)nicotinic acid (100 mg, 0.400 mmol) and N- methylaniline (51.4 mg, 0.480 mmol) as colorless wax (99 mg, 73%). 1H NMR (400 MHz,

CDCl3) δ 7.98 (d, J = 4.9 Hz, 1H), 7.80–7.74 (m, 1H), 7.21 (d, J = 7.1 Hz, 3H), 7.12–7.03 (m, 3H), 6.95 (dd, J = 7.4, 5.0 Hz, 1H), 6.58–6.48 (m, 2H), 3.53 (s, 3H).

206 2-(2-Chloro-3-(trifluoromethyl)phenoxy)-N-methyl-N-(o-tolyl)nicotinamide.

O N

N O Cl

F3C The title compound was synthesized following the general procedure for the coupling reaction using 2-(2-chloro-3-(trifluoromethyl)phenoxy)nicotinic acid (100 mg, 0.310 mmol) and N-methyl-o-toluidine (45.1 mg, 0.372 mmol) as colorless wax (84 mg, 63%). 1 H NMR (400 MHz, CDCl3) δ 7.92 (dd, J = 4.9, 1.9 Hz, 1H), 7.65 (dd, J = 7.4, 1.9 Hz, 1H), 7.57 (dd, J = 7.9, 1.5 Hz, 1H), 7.34 (t, J = 8.1 Hz, 1H), 7.19 (d, J = 7.6 Hz, 1H), 7.16– 7.13 (m, 2H), 7.08–7.01 (m, 2H), 6.89 (dd, J = 7.4, 4.9 Hz, 1H), 3.42 (s, 3H), 2.31 (s, 3H).

(2-(2-Chloro-3-(trifluoromethyl)phenoxy)pyridin-3-yl)(3,4-dihydroquinolin-1(2H)- yl)methanone.

O N

N O Cl

F3C The title compound was synthesized following the general procedure for the coupling reaction using 2-(2-chloro-3-(trifluoromethyl)phenoxy)nicotinic acid (100 mg, 0.310 mmol) and 1,2,3,4-tetrahydroquinoline (45.1 mg, 0.372 mmol) as colorless wax (75 mg, 1 55%). H NMR (400 MHz, CDCl3) δ 8.06 (s, 1H), 8.00 (s, 1H), 7.54–7.46 (m, 1H), 7.20 (s, 1H), 7.16–7.01 (m, 3H), 6.89 (s, 1H), 6.63 (s, 1H), 6.30 (s, 1H), 4.70–4.22 (m, 1H), 3.51 (d, J = 78.3 Hz, 1H), 2.70 (s, 2H), 2.26–2.07 (m, 1H), 1.92–1.69 (m, 1H).

207 2-(2-Chloro-3-(trifluoromethyl)phenoxy)-N-(o-tolyl)nicotinamide.

O N H N O Cl

(400 MHz, CDCl3) δ 9.52 (s, 1H), 8.76 (dd, J = 7.6, 2.0 Hz, 1H), 8.24–8.19 (m, 2H), 7.72 (q, J = 4.5 Hz, 1H), 7.53 (d, J = 4.6 Hz, 2H), 7.33–7.27 (m, 2H), 7.25–7.21 (m, 1H), 7.16– 7.10 (m, 1H), 2.30 (s, 3H).

2-(2-Chloro-3-(trifluoromethyl)phenoxy)-N-ethyl-N-phenylnicotinamide.

O N

N O Cl

(400 MHz, CDCl3) δ 7.92 (dd, J = 5.1, 1.9 Hz, 1H), 7.64 (dd, J = 7.4, 1.9 Hz, 1H), 7.56 (d, J = 7.8 Hz, 1H), 7.33 (t, J = 8.0 Hz, 1H), 7.26–7.17 (m, 5H), 7.05 (d, J = 8.1 Hz, 1H), 6.90 (dd, J = 7.4, 4.9 Hz, 1H), 4.03 (q, J = 7.1 Hz, 2H), 1.28 (t, J = 7.1 Hz, 3H).

208 (2-(2-Chloro-3-(trifluoromethyl)phenoxy)pyridin-3-yl)(6-methyl-3,4- dihydroquinolin-1(2H)-yl)methanone.

O N

N O Cl

NMR (400 MHz, CDCl3) δ 8.07 (s, 1H), 8.00 (s, 1H), 7.50 (d, J = 7.8 Hz, 1H), 7.18 (s, 1H), 7.12 (dd, J = 7.4, 5.0 Hz, 1H), 6.86 (s, 1H), 6.68 (s, 1H), 6.51 (s, 1H), 6.34 (s, 1H), 4.46 (s, 1H), 3.63 (s, 1H), 2.64 (s, 2H), 2.26 (s, 4H), 2.00 (s, 1H).

2-(4-Fluoro-3-methylphenoxy)-N-methyl-N-(o-tolyl)nicotinamide.

O N

N O

F The title compound was synthesized following the general procedure for the coupling reaction using 2-(4-fluoro-3-methylphenoxy)nicotinic acid (100 mg, 0.400 mmol) and N- methyl-o-toluidine (58.2 mg, 0.480 mmol) as colorless wax (29 mg, 21%). 1H NMR (400

MHz, CDCl3) δ 7.94 (dd, J = 4.9, 2.0 Hz, 1H), 7.69 (dd, J = 7.4, 1.9 Hz, 1H), 7.15–7.11 (m, 2H), 7.09–7.01 (m, 2H), 6.96 (t, J = 8.9 Hz, 1H), 6.86 (dd, J = 7.4, 4.9 Hz, 1H), 6.62 (td, J = 8.7, 3.5 Hz, 2H), 3.40 (s, 3H), 2.24 (d, J = 3.0 Hz, 6H).

209 (3,4-Dihydroquinolin-1(2H)-yl)(2-(4-fluoro-3-methylphenoxy)pyridin-3- yl)methanone.

O N

N O

F The title compound was synthesized following the general procedure for the coupling reaction using 2-(4-fluoro-3-methylphenoxy)nicotinic acid (100 mg, 0.400 mmol) and 1,2,3,4-tetrahydroquinoline (63.9 mg, 0.480 mmol) as colorless wax (51 mg, 35%). 1H

NMR (400 MHz, CDCl3) δ 8.08 (d, J = 4.8 Hz, 1H), 8.00 (d, J = 7.3 Hz, 1H), 7.11–7.02 (m, 3H), 6.96–6.79 (m, 2H), 6.57 (s, 1H), 6.15 (d, J = 27.2 Hz, 2H), 4.57 (s, 1H), 3.44 (s, 1H), 2.65 (d, J = 12.0 Hz, 2H), 2.31–2.07 (m, 4H), 2.00–1.77 (m, 1H).

2-(4-Fluoro-3-methylphenoxy)-N-(o-tolyl)nicotinamide.

O N H N O

F The title compound was synthesized following the general procedure for the coupling reaction using 2-(4-fluoro-3-methylphenoxy)nicotinic acid (100 mg, 0.400 mmol) and o- toluidine (51.4 mg, 0.480 mmol) as colorless wax (47 mg, 35%). 1H NMR (400 MHz,

CDCl3) δ 9.79 (s, 1H), 8.74 (dd, J = 7.6, 2.0 Hz, 1H), 8.33–8.24 (m, 2H), 7.31–7.27 (m, 1H), 7.21 (td, J = 8.0, 5.7 Hz, 2H), 7.13–7.03 (m, 3H), 7.00 (dt, J = 8.6, 3.6 Hz, 1H), 2.32 (d, J = 2.0 Hz, 3H), 2.27 (s, 3H).

210 N-Ethyl-2-(4-fluoro-3-methylphenoxy)-N-phenylnicotinamide.

O N

N O

F The title compound was synthesized following the general procedure for the coupling reaction using 2-(4-fluoro-3-methylphenoxy)nicotinic acid (100 mg, 0.400 mmol) and N- ethylaniline (58.2 mg, 0.480 mmol) as colorless wax (82 mg, 58%). 1H NMR (400 MHz,

CDCl3) δ 7.95 (dd, J = 5.0, 1.9 Hz, 1H), 7.69 (dd, J = 7.4, 2.0 Hz, 1H), 7.21 (q, J = 7.2, 6.7 Hz, 3H), 7.14–7.08 (m, 2H), 6.94 (t, J = 8.9 Hz, 1H), 6.87 (dd, J = 7.4, 4.9 Hz, 1H), 6.59 (ddt, J = 11.9, 7.5, 3.1 Hz, 2H), 4.01 (q, J = 7.1 Hz, 2H), 2.23 (s, 3H), 1.23 (t, J = 7.1 Hz, 3H).

2-(4-Fluoro-3-methylphenoxy)-N-(2-fluorophenyl)-N-methylnicotinamide.

O N F N O

F The title compound was synthesized following the general procedure for the coupling reaction using 2-(4-fluoro-3-methylphenoxy)nicotinic acid (100 mg, 0.400 mmol) and 2- fluoro-N-methylaniline (60.1 mg, 0.480 mmol) as colorless wax (52 mg, 38%). 1H NMR

(400 MHz, CDCl3) δ 7.98 (dd, J = 5.0, 1.9 Hz, 1H), 7.77 (dt, J = 7.4, 1.5 Hz, 1H), 7.25- 7.17 (m, 2H), 7.05–6.95 (m, 3H), 6.95–6.87 (m, 1H), 6.66 (dd, J = 6.5, 2.9 Hz, 1H), 6.60 (dt, J = 7.8, 3.6 Hz, 1H), 3.47 (s, 3H), 2.24 (s, 3H).

211 (2-(4-Fluoro-3-methylphenoxy)pyridin-3-yl)(6-methyl-3,4-dihydroquinolin-1(2H)- yl)methanone.

O N

N O

F The title compound was synthesized following the general procedure for the coupling reaction using 2-(4-fluoro-3-methylphenoxy)nicotinic acid (100 mg, 0.400 mmol) and 6- methyl-1,2,3,4-tetrahydroquinoline (70.7 mg, 0.480 mmol) as colorless wax (45 mg, 29%). 1 H NMR (400 MHz, CDCl3) δ 8.09 (s, 1H), 8.01 (d, J = 7.3 Hz, 1H), 7.06 (dd, J = 7.3, 4.9 Hz, 1H), 6.88 (d, J = 14.0 Hz, 2H), 6.68 (d, J = 8.2 Hz, 1H), 6.45 (d, J = 8.2 Hz, 1H), 6.25 (s, 1H), 6.09 (s, 1H), 4.57 (s, 1H), 3.41 (s, 1H), 2.61 (s, 2H), 2.23 (d, J = 44.6 Hz, 7H), 1.90 (s, 1H).

2-(4-Fluoro-3-methylphenoxy)-N-(2-fluorophenyl)-N-methylnicotinamide.

O N

N O

F The title compound was synthesized following the general procedure for the coupling reaction using 2-(4-fluoro-3-methylphenoxy)nicotinic acid (100 mg, 0.400 mmol) and N- methylaniline (51.4 mg, 0.480 mmol) as colorless wax (73 mg, 54%). 1H NMR (400 MHz,

CDCl3) δ 7.97 (dd, J = 5.1, 2.0 Hz, 1H), 7.74 (dd, J = 7.4, 1.9 Hz, 1H), 7.21 (q, J = 7.2 Hz, 3H), 7.09 (d, J = 7.2 Hz, 2H), 6.96–6.86 (m, 2H), 6.52 (d, J = 6.2 Hz, 2H), 3.52 (s, 3H), 2.21 (s, 3H).

212 N-Methyl-N-(o-tolyl)-2-(3-(trifluoromethyl)phenoxy)nicotinamide.

O N

N O

MHz, CDCl3) δ 7.96 (dd, J = 4.9, 2.0 Hz, 1H), 7.76 (dd, J = 7.4, 2.0 Hz, 1H), 7.51–7.43 (m, 2H), 7.15 (ddd, J = 8.3, 6.1, 2.4 Hz, 1H), 7.11 (d, J = 1.4 Hz, 1H), 7.10–7.02 (m, 3H), 7.01 (d, J = 2.0 Hz, 1H), 6.94 (dd, J = 7.4, 4.9 Hz, 1H), 3.41 (s, 3H), 2.23 (s, 3H).

(3,4-Dihydroquinolin-1(2H)-yl)(2-(3-(trifluoromethyl)phenoxy)pyridin-3- yl)methanone.

O N

N O

NMR (400 MHz, CDCl3) δ 8.08 (t, J = 7.2 Hz, 2H), 7.37 (d, J = 4.2 Hz, 2H), 7.13 (dd, J = 7.3, 5.0 Hz, 1H), 7.04 (t, J = 5.0 Hz, 2H), 6.89 (s, 1H), 6.72 (s, 1H), 6.53 (d, J = 29.5 Hz, 2H), 4.81–4.30 (m, 1H), 3.45 (s, 1H), 2.63 (s, 2H), 2.38–2.08 (m, 1H), 2.03–1.79 (m, 1H).

213 N-(o-Tolyl)-2-(3-(trifluoromethyl)phenoxy)nicotinamide.

O N H N O

CDCl3) δ 9.65 (s, 1H), 8.78 (dd, J = 7.6, 2.0 Hz, 1H), 8.31–8.25 (m, 2H), 7.67–7.58 (m, 2H), 7.52 (s, 1H), 7.47–7.42 (m, 1H), 7.31 (td, J = 6.4, 5.6, 2.2 Hz, 2H), 7.22 (d, J = 8.0 Hz, 1H), 7.11 (td, J = 7.4, 1.2 Hz, 1H), 2.29 (s, 3H).

N-Ethyl-N-phenyl-2-(3-(trifluoromethyl)phenoxy)nicotinamide.

O N

N O

CDCl3) δ 7.97 (dd, J = 5.0, 1.9 Hz, 1H), 7.77 (dd, J = 7.4, 1.9 Hz, 1H), 7.49–7.40 (m, 2H), 7.21 (dd, J = 5.1, 1.9 Hz, 3H), 7.08 (dd, J = 6.7, 2.9 Hz, 2H), 7.04 (dd, J = 7.3, 2.0 Hz, 1H), 7.00–6.92 (m, 2H), 4.03 (q, J = 7.1 Hz, 2H), 1.27 (t, J = 7.1 Hz, 3H).

214 N-(2-Fluorophenyl)-N-methyl-2-(3-(trifluoromethyl)phenoxy)nicotinamide.

O N F N O

(400 MHz, CDCl3) δ 8.00 (dd, J = 4.9, 1.9 Hz, 1H), 7.84 (dt, J = 7.4, 1.4 Hz, 1H), 7.50– 7.42 (m, 2H), 7.26–7.19 (m, 1H), 7.15 (t, J = 7.6 Hz, 1H), 7.09 (dt, J = 7.5, 2.1 Hz, 1H), 7.04–6.94 (m, 4H), 3.48 (s, 3H).

(6-Methyl-3,4-dihydroquinolin-1(2H)-yl)(2-(3-(trifluoromethyl)phenoxy)pyridin-3- yl)methanone.

O N

N O

CF3 The title compound was synthesized following the general procedure for the coupling reaction using 2-(3-(trifluoromethyl)phenoxy)nicotinic acid (100 mg, 0.350 mmol) and 6- methyl-1,2,3,4-tetrahydroquinoline (61.8 mg, 0.420 mmol) as colorless wax (68 mg, 47%). 1 H NMR (400 MHz, CDCl3) δ 7.99 (t, J = 6.3 Hz, 2H), 7.29 (d, J = 4.6 Hz, 2H), 7.04 (dd, J = 7.3, 5.0 Hz, 1H), 6.76 (s, 1H), 6.70 (s, 1H), 6.60 (d, J = 8.1 Hz, 1H), 6.43–6.32 (m, 2H), 4.51 (s, 1H), 3.32 (s, 1H), 2.68–2.35 (m, 2H), 2.24–2.01 (s, 4H), 1.90–1.73 (m, 1H).

215 N-Methyl-N-phenyl-2-(3-(trifluoromethyl)phenoxy)nicotinamide.

O N

N O

CDCl3) δ 7.98 (dd, J = 5.0, 2.0 Hz, 1H), 7.80 (dd, J = 7.5, 1.9 Hz, 1H), 7.42 (d, J = 7.0 Hz, 2H), 7.19 (dd, J = 5.3, 2.0 Hz, 3H), 7.11–7.04 (m, 2H), 7.01–6.93 (m, 2H), 6.87 (s, 1H), 3.53 (s, 3H).

N-Methyl-N-(o-tolyl)-2-(2,4,5-trichlorophenoxy)nicotinamide.

O N

N O Cl

MHz, CDCl3) δ 7.95 (dd, J = 4.9, 1.9 Hz, 1H), 7.68 (dd, J = 7.4, 1.9 Hz, 1H), 7.55 (s, 1H), 7.17–7.11 (m, 3H), 7.05 (ddd, J = 8.4, 5.6, 3.2 Hz, 1H), 6.92 (dd, J = 7.4, 4.9 Hz, 1H), 6.87 (s, 1H), 3.42 (s, 3H), 2.29 (s, 3H).

216 (3,4-Dihydroquinolin-1(2H)-yl)(2-(2,4,5-trichlorophenoxy)pyridin-3-yl)methanone.

O N

N O Cl

NMR (400 MHz, CDCl3) δ 8.07 (dd, J = 23.2, 5.7 Hz, 2H), 7.48 (s, 1H), 7.14 (dd, J = 7.4, 4.9 Hz, 1H), 7.09 (d, J = 4.7 Hz, 2H), 6.91 (s, 1H), 6.59 (s, 1H), 6.02 (s, 1H), 4.52 (s, 1H), 3.57 (s, 1H), 2.67 (s, 2H), 2.16 (s, 1H), 1.94 (s, 1H).

N-Ethyl-N-phenyl-2-(2,4,5-trichlorophenoxy)nicotinamide.

O N

N O Cl

CDCl3) δ 7.96 (dd, J = 5.0, 1.9 Hz, 1H), 7.67 (dd, J = 7.4, 1.9 Hz, 1H), 7.55 (s, 1H), 7.23 (d, J = 7.6 Hz, 3H), 7.18–7.12 (m, 2H), 6.93 (dd, J = 7.4, 4.9 Hz, 1H), 6.87 (s, 1H), 4.03 (q, J = 7.1 Hz, 2H), 1.26 (t, J = 7.1 Hz, 3H).

217 N-(2-Fluorophenyl)-N-methyl-2-(2,4,5-trichlorophenoxy)nicotinamide.

O N F N O Cl

(400 MHz, CDCl3) δ 7.98 (dd, J = 4.9, 1.9 Hz, 1H), 7.76 (dt, J = 7.6, 1.4 Hz, 1H), 7.55 (s, 1H), 7.24 (ddd, J = 12.4, 7.7, 1.9 Hz, 2H), 7.03 (t, J = 7.8 Hz, 2H), 6.98–6.92 (m, 2H), 3.48 (s, 3H).

(6-Methyl-3,4-dihydroquinolin-1(2H)-yl)(2-(2,4,5-trichlorophenoxy)pyridin-3- yl)methanone.

O N

N O Cl

Cl Cl The title compound was synthesized following the general procedure for the coupling reaction using 2-(3-(trifluoromethyl)phenoxy)nicotinic acid (100 mg, 0.310 mmol) and 6- methyl-1,2,3,4-tetrahydroquinoline (54.8 mg, 0.372 mmol) as colorless wax (128 mg, 1 90%). H NMR (400 MHz, CDCl3) δ 8.08 (t, J = 7.9 Hz, 2H), 7.48 (s, 1H), 7.14 (dd, J = 7.4, 5.0 Hz, 1H), 6.91 (s, 1H), 6.70 (d, J = 8.1 Hz, 1H), 6.44 (d, J = 8.1 Hz, 1H), 5.93 (s, 1H), 4.55 (s, 1H), 3.45 (s, 1H), 2.63 (s, 2H), 2.30–2.04 (m, 4H), 1.999–1.88 (m, 1H).

218 N-Methyl-N-phenyl-2-(2,4,5-trichlorophenoxy)nicotinamide.

O N

N O Cl

CDCl3) δ 8.01–7.95 (m, 1H), 7.75–7.69 (m, 1H), 7.54 (s, 1H), 7.23 (d, J = 7.9 Hz, 3H), 7.15 (d, J = 7.3 Hz, 2H), 6.96 (dd, J = 7.4, 4.9 Hz, 1H), 6.76 (s, 1H), 3.55 (s, 3H).

2-(4-Acetyl-3-chlorophenoxy)-N-(o-tolyl)nicotinamide.

O N H N O

Cl O The title compound was synthesized following the general procedure for the coupling reaction using 2-(4-acetyl-3-chlorophenoxy)nicotinic acid (100 mg, 0.340 mmol) and o- toluidine (43.7 mg, 0.408 mmol) as colorless wax (117 mg, 86%). 1H NMR (400 MHz,

CDCl3) δ 9.51 (s, 1H), 8.76 (dd, J = 7.6, 2.0 Hz, 1H), 8.27–8.21 (m, 2H), 8.16 (d, J = 2.1 Hz, 1H), 8.01 (dd, J = 8.4, 2.1 Hz, 1H), 7.43 (d, J = 8.4 Hz, 1H), 7.33–7.27 (m, 2H), 7.24– 7.19 (m, 1H), 7.11 (td, J = 7.5, 1.3 Hz, 1H), 2.65 (s, 3H), 2.29 (s, 3H).

219 2-(4-Acetyl-3-chlorophenoxy)-N-ethyl-N-phenylnicotinamide.

O N

N O

Cl O The title compound was synthesized following the general procedure for the coupling reaction using 2-(4-acetyl-3-chlorophenoxy)nicotinic acid (100 mg, 0.340 mmol) and N- ethylaniline (49.4 mg, 0.408 mmol) as colorless wax (134 mg, 96%). 1H NMR (400 MHz,

CDCl3) δ 8.07 (d, J = 2.1 Hz, 1H), 7.95 (dd, J = 4.9, 1.9 Hz, 1H), 7.83 (dd, J = 8.5, 2.1 Hz, 1H), 7.64 (dd, J = 7.5, 1.9 Hz, 1H), 7.21 (q, J = 7.5, 6.9 Hz, 5H), 6.97–6.89 (m, 2H), 4.03 (q, J = 7.1 Hz, 2H), 2.60 (s, 3H), 1.27 (t, J = 7.1 Hz, 3H).

2-(4-Acetyl-3-chlorophenoxy)-N-(2-fluorophenyl)-N-methylnicotinamide.

O N F N O

Cl O The title compound was synthesized following the general procedure for the coupling reaction using 2-(4-acetyl-3-chlorophenoxy)nicotinic acid (100 mg, 0.340 mmol) and 2- fluoro-N-methylaniline (51.1 mg, 0.408 mmol) as colorless wax (110 mg, 77%). 1H NMR

(400 MHz, CDCl3) δ 8.07 (d, J = 2.1 Hz, 1H), 7.97 (dd, J = 5.0, 1.9 Hz, 1H), 7.85 (dd, J = 8.5, 2.1 Hz, 1H), 7.75 (dt, J = 7.3, 1.6 Hz, 1H), 7.35–7.29 (m, 1H), 7.26–7.17 (m, 1H), 7.07–6.98 (m, 3H), 6.94 (dd, J = 7.4, 4.9 Hz, 1H), 3.49 (s, 3H), 2.61 (s, 3H).

220 4.8. TGR5 cAMP Assay LANCE Ultra cAMP Kit (PerkinElmer, USA) was utilized to determine the cAMP production inside CHO-K1 cells after the incubation.

4.8.1. Buffer Preparation The IBMX solution, stimulation buffer, Eu-cAMP tracer solution, and Ulight-anti-cAMP solution were prepared following the LANCE Ultra cAMP Assay Development Guidelines. The cell lysate buffer (15 mL) was prepared as indicated in the table below. The pH value was adjusted to 7.4 with NaOH (0.1N). Reagent Volume 50mM HEPES (1M stock) 750 µL

10mM final CaCl2 22.05 mg 0.35% final Triton X-100 52.5 µL (100% stock) Distilled Water 14.2 mL

4.8.2. Cell Plate Preparation After the removal of culture media, CHO-K1 cells (cAMP Hunter™, DiscoverX) were rinsed with PBS (5 mL) and aspirated. Then AssayComplete™ Cell Plating Reagent (0.5 mL, DiscoverX) was added into the T75 flask, which was placed in the incubator for 5 min. The same cell dissociation solution (3 mL) was charged and the entire solution was transferred into a conical tube. The cell density then was determined by Countess cell counter (Invitrogen) before cells being reconstituted to appropriate concentrations with stimulation buffer. The prepared cell suspension (10 µL) was transferred to the appropriate wells of 384 white walled microplate by Echo550 (Labcyte), which gave a final concentration 1000 cell per well. The plate was centrifuged at 500 rpm for 30 sec for the next step. 4.8.3. Agonism Assay

To the prepared cell plate, positive control (GPBAR-A, Emax: 3 µM and EC80: 100 nM), negative control (DMSO) and compound to be tested (10 and 100 µM) were

221 dispensed (10 nL) respectively by Echo550. After the incubation at 37 °C for 30 min, the plate was cooled on ice immediately to prevent further signaling. The stimulation buffer was removed and ice-cold lysate buffer (20 µL) was added sequentially. The plate then was agitated at 600 rpm for 15-30 min at 4 °C. Of the original 20 µL of cell lysate in lysate buffer, 10 µL was transferred from each well to a new plate and spun down at 1000 rpm for 1 min. Eu-cAMP tracer solution (5 µL) and Ulight-anti-cAMP solution (5 µL) then were added into each well for another one-hour incubation at 25 °C, resulting in a 2x dilution of the original cell lysate concentrations. The measurements of wells were performed on LJL Analyst with UV380 mirror (320 nm excitation and 615/665 nm emissions).

4.8.4. Antagonism Assay

GPBAR-A at 3 µM and 35 nM were utilized as positive controls of Emax and EC80, whereas DMSO was the negative control. Both test compounds (10 and 100 µM) and

GPBAR-A (EC80, 35 nM) were added to each well. Under the same conditions, the cells were incubated, lysed, and treated with detecting solutions. The data was collected with LJL Analyst with UV380 mirror (320 nm excitation and 615/665 nm emission).

4.8.5. Data Analysis Data from the LJL was corrected in Excel using the following equation recommended by kit creator, Perkin Elmer: ((665 Read -665 Blank)) * 615 Max/ 615 Read). 665 Read = 665 nm raw emission value. 665 Blank = Just buffer, no cells or components. 615 Max = No cAMP, no cells, just buffer and kit components. 615 Read =

615 nm raw emission value. The GPBAR-A Emax (3 µM) and Low (DMSO) controls on the plate were used to calculate a % of control value for each concentration tested, also done in Excel.

A cAMP standard curve was run on every plate. Graph Pad Prism was used to determine the IC10 and IC90 for each cAMP standard curve since between IC10 and IC90, or between 10% and 90% inhibition, is considered the working dynamic range. Values that 222 fell outside of the working dynamic range determined by the 10-90% inhibition were considered unreliable for determining cAMP production and were eliminated. Where fmole of cAMP produced was estimated, it was done using the standard curve parameters for that plate.

223 References

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