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Design, synthesis, and biological evaluation of novel therapeutics for cancer

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Design, synthesis, and biological evaluation of novel therapeutics for cancer

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Content DESIGN, SYNTHESIS, AND BIOLOGICAL EVALUATION OF NOVEL THERAPEUTICS FOR CANCER by Marcos A. Sainz A Dissertation Presented to the FACULTY OF THE USC GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (CHEMISTRY) December 2014 Copyright 2014 Marcos A. Sainz ii Dedication To Mom, Dad, and Alex iii Acknowledgements I would like to thank my advisor, Professor Nicos Petasis for his guidance, encouragement, and patience. Thank you for teaching me how to approach problems independently and in new ways, and for allowing me the freedom to explore my ideas in the laboratory, no matter how far- fetched they may have seemed. The problem-solving skills I learned in your group are truly invaluable. I would like to additionally thank my qualifying committee members Professors G.K. Surya Prakash, Travis Williams, Matthew Pratt, and Dr. Amir Goldkorn. A very special thanks to my thesis committee member Professor Stan Louie for outstanding work as a collaborator as well as the many helpful discussions we shared regarding science and life. A huge thank you goes out to all past and present member of the Petasis group for making the lab a great place to work for the past few years: Dr. Kevin Gaffney, for always challenging me to go the extra mile and run one more experiment, Dr. Kalyan Nagulapalli Ventkata for his words of wisdom, Dr. Jamie Jarusiewicz for helping me out when I started my lab work, Steve Glynn for being a great friend and co-worker, Caitlin DeAngelo for all of the help in the lab, as well as Dr. Jeremy Winkler, Dr. Min Zhu, Nikita Vlasenko Dr. Anne-Marie Finaldi, Charles Arden, Dr. Alex Butkevich, Dr. Rong Yang, and Dave Rosenberg. Additionally, I would like thank Yuan Ding for her constant love and support. I thank the staff of the Loker Hydrocarbon Institute and the Department of Chemistry at USC: Carole Phillips, Jessy May, David Hunter, Dr. Robert Anizfeld, Michele Dea, Magnolia Benitez, and Katie McKissick. Lastly, I would like to thank my friends and family for their unwavering encouragement and support; without whom this would not have been possible. iv Table of Contents Dedication ii Acknowledgements iii List of Tables vi List of Figures vii List of Schemes x Abstract xi Chapter 1. Design, Synthesis and Evaluation of dUTPase Inhibitors. 1 1.1. Introduction. The role of deoxyuridine triphosphatase (dUTPase) as a therapeutic target. 1 1.2. dUTPase inhibitor design and synthesis 4 1.2.1. Virtual screening and fragment-based design 4 1.2.2. Synthesis of dUTPase inhibitors 7 1.2.3. Biological screening and activity of dUTPase inhibitors 15 1.3. Conclusion 22 1.4. Experimental 22 1.4.1. Structure-based design 22 1.4.2. Synthetic chemistry 23 1.4.3. Biological screening 71 1.5. Chapter 1 references 72 Chapter 2. Chemical Investigation of the covalent interaction between bortezomib and epigallocatechin gallate (EGCG). 76 2.1. Introduction 76 2.2. Biological screening of drug-drug interactions of bortezomib 77 2.2.1. EGCG and related polyphenolic compounds 77 2.2.2. Other polyphenols 78 2.3. NMR experiments 81 2.4. Conclusion 83 2.5. Experimental 83 2.5.1. Biological screening 83 2.5.2. NMR screening and compound synthesis 88 2.6. Chapter 2 references 90 v Chapter 3. Efforts towards the synthesis of a novel class of bi-functional peptide nucleic acids (PNA) 91 3.1. Introduction 91 3.2. Synthesis of nucleic acid base derivatives 93 3.3. Synthesis of PNA backbone 96 3.4. Monomer assembly 99 3.5. Solid phase peptide synthesis (SPPS) of PNAs 100 3.6. Conclusion 110 3.7. Experimental 110 3.8. Chapter 3 references 121 Chapter 4. Biological screening of MEF2 inhibitors for the treatment of cancer 124 4.1. Introduction 124 4.2. Design of MEF2 inhibitors 126 4.3. Biological screening and structure-activity relationship of MEF2 inhibitors 128 4.4. Conclusion 132 4.5. Experimental 133 4.6. Chapter 4 references 143 Bibliography 145 Appendix: Selected spectra 153 vi List of Tables Table 1.1 MTT viability assay data for first analog class of dUTPase 18 inhibitors Table 1.2 MTT viability assay data for second analog class of dUTPase Inhibitors 19 Table 1.3 MTT viability assay data for third analog class of dUTPase inhibitors 20 Table 4.1 Activity of MEF2-HDAC inhibitors at 10µM in luciferase assay 128 Table 4.2 Activity of MEF2-HDAC inhibitors in HCT-116 and RPMI8226 cells 129 Table 4.3 Activity of reverse amide MEF2-HDAC inhibitors in HCT-116 and RPMI8226 cells 131 Table 4.4 Activity of benzamide MEF2-HDAC inhibitors in HCT-116 and RPMI8226 cells 132 vii List of Figures Figure 1.1 Function of dUTPase and thymidylate synthase (TS) 1 Figure 1.2 Role of dUTPase in cancer 3 Figure 1.3 Structures of literature-reported dUTPase inhibitors 4 Figure 1.4 Mechanism of dUTP hydrolysis 5 Figure 1.5 Crystal structure of dUTPase and modeling of the active site 5 Figure 1.6 Fragment screening of dUTPase using ZINC Database fragments 6 Figure 1.7 Generation of toluoylimidazole screening hit and fragment-linking strategy 7 Figure 1.8 Graphical representation of novel dUTPase inhibitor design 8 Figure 1.9 Mechanism of MTT viability assay 16 Figure 1.10 Structure of literature-reported, non-selective dUTPase inhibitors 21 Figure 1.11 Cell viability assays of dUTPase inhibitors in HCT-116 human colorectal carcinoma cells 21 Figure 2.1 Influence of EGCG on RPMI8226 and LN229 cell viability, in combination with 20 nM of bortezomib 77 Figure 2.2 Structural breakdown of EGCG fragments. 78 Figure 2.3 Influence of EGCG and related fragments on RPMI8226 and LN229 cell viability, in combination with 20 nM of bortezomib 78 Figure 2.4 Influence of substituted phenols on RPMI8226 and LN229 cell viability, in combination with 20 nM of bortezomib 79 Figure 2.5 Influence of substituted catechols on RPMI8226 and LN229 cell viability, in combination with 20 nM of bortezomib 80 Figure 2.6 Compiled cell viability assay data of polyphenol influence on the antitumor activity of BZM in RPMI8226 and LN229 cell lines. 81 viii Figure 2.7 Representation of the reversible covalent interaction between BZM and EGCG 81 Figure 2.8 11 B NMR screening of bortezomib in the presence of EGCG and related fragments 82 Figure 2.9 Compiled graphs of RPMI8226 cell viability data of all polyphenols 84 Figure 2.10 Compiled graphs of LN229 cell viability data of all polyphenols 85 Figure 2.11 96-well plates of RPMI8226 and LN229 cells in the presence of EGCG and ascorbic acid. 86 Figure 2.12 96-well plates of RPMI8226 and LN229 cells in the presence of NCAT and ISO 86 Figure 2.13 96-well plates of RPMI8226 and LN229 cells in the presence of PYRO and CAT 87 Figure 2.14 96-well plates of RPMI8226 and LN229 cells in the presence of RES and PHE 87 Figure 2.15 96-well plates of RPMI8226 and LN229 cells in the presence of DHBA 88 Figure 3.1 Mechanism of telomerase activity 91 Figure 3.2 Design strategy of diPNA 11-mer 92 Figure 3.3 Retrosynthetic strategy of diPNA 11-mer 93 Figure 3.4 Synthetic strategy for the synthesis of the PNA monomer backbone 97 Figure 3.5 General reaction mechanism of the Kaiser test 101 Figure 3.6 Reaction setup for solid phase peptide synthesis of 11-mer PNA 102 Figure 3.7 Deprotection of Fmoc-PAL-PEG-PS resin, followed by attachment of C-G monomer (3.27c) via amide coupling to yield PNA monomer 3.28 102 Figure 3.8 Capping, deprotection, and amide coupling of 3.28 with A-A monomer 3.27a to yield Fmoc dimer 3.29 103 ix Figure 3.9 Capping and deprotection of 3.29 followed by Amide coupling of 3.29 with A-U monomer 3.27b to yield Fmoc trimer 3.30 103 Figure 3.10 Yield quantification using UV-Vis analysis of the 4-MP/dibenzo- fulvene adduct formed after Fmoc deprotection 105 Figure 3.11 Coupling of AEEA spacer 3.35 to deprotected PAL-PEG-PS resin 105 Figure 3.12 Structures of Fmoc-PAL-PEG-PS and NovaPEG rink amide resin LL 106 Figure 3.13 Coupling of AEEA spacer 3.35 to deprotected NovaPEG rink amide resin 106 Figure 3.14 Coupling of NovaPEG resin-spacer with A-A monomer 3.27a 107 Figure 3.15 Coupling reaction of 3.36 with A-U monomer 3.27b 107 Figure 3.16 Coupling of NovaPEG resin with C-G monomer 3.27c 108 Figure 3.17 Coupling of 3.38 with C-G monomer 3.27c to give dimer 3.39 109 Figure 3.18 Coupling of 3.39 with A-U monomer 3.27b to give trimer 3.40 109 Figure 4.1 Role of HATs and HDACs in gene expression 124 Figure 4.2 Reported HDAC inhibitors 125 Figure 4.3 Structural comparison of binding sites for HDAC9 and BML-210 on MEF2 126 Figure 4.4 Effect of MEF2 inhibitors on HCT116 cell viability 129 Figure 4.5 Crystallographic understanding of analog activity and design 130 Figure 4.6 Scaffold change of MEF2 inhibitors by reversing the amide bond between the A region and the hydrophobic linker. 130 x List of Schemes Scheme 1.1 Synthesis of Amides 1.3a-b and 1.8a-b 9 Scheme 1.2 Synthesis of sulfonamide 1.11 9 Scheme 1.3 Synthesis of alkyl benzimidazoles 1.14a-b 10 Scheme 1.4 Preparation of substituted phenylenediamines 1.17a and 1.17b 11 Scheme 1.5 Synthesis of substituted benzimidazoles 1.18a-b and 1.19a-k 11 Scheme 1.6 Synthesis of aminobenzimidazole 23 12 Scheme 1.7 Synthesis of cyanobenzimidazole 1.29 13 Scheme 1.8 Synthesis of methyl ester 1.34 13 Scheme 1.9 Preparation of dUTPase inhibitor 1.38 using a piperidine linker 14 Scheme 1.10 Synthesis of thiophene analogs 1.47a-c and 1.48a-c 15 Scheme 3.1 Synthesis of N-Boc-protected cytosine 3.3 94 Scheme 3.2 Synthesis of Bis-N-Boc-protected adenine 3.6 95 Scheme 3.3 Synthesis of Boc-protected guanine 3.9 96 Scheme 3.4 Synthesis of uracil acetic acid 3.10 96 Scheme 3.5 Synthesis of PNA backbone diamine intermediate 3.21 98 Scheme 3.6 Synthesis of monomers 3.27a-c 100 Scheme 4.1 Synthesis of MEF2 inhibitors 4.3a-c, f, 4.4a-b, and 4.5a-b 127 xi Abstract This dissertation details my efforts towards the design, synthesis and biological evaluation of novel, small molecule anti-cancer agents. This was accomplished with a joint collaboration with the USC School of Pharmacy. Chapter 1 describes the computational fragment-based design, synthesis, and biological validation of a series of novel inhibitors of the enzyme dUTPase as potential anti-cancer agents. Chapter 2 reports the investigation into the chemical interaction of green tea extract epigallocatechin gallate (EGCG) with the anticancer drug bortezomib (BZM, Velcade™). This work described focuses mainly on the phenotypic assays used to screen the interaction of EGCG with BZM and other biologically relevant polyphenol compounds. Chapter 3 provides a description of the development of a novel class of peptide nucleic acids (PNAs). This includes a rational design of the PNA oligomer, the synthesis of the nucleic acid monomer units, as well as efforts to synthesize the PNA using solid phase peptide synthesis. Chapter 4 reviews the structure-activity relationship of a series of small molecules capable of disrupting the protein-protein interaction between transcription factor MEF2 and class IIa HDACs. The work described herein focuses on the SAR developed through in vitro cellular assay screening. 1 Chapter 1: Design, Synthesis and Evaluation of dUTPase Inhibitors 1.1 Introduction. The role of dUTPase as a therapeutic target. According to the National Cancer Institute, approximately 1 in 20 Americans will be diagnosed with colorectal cancer (CRC) in their lifetime, including over 130,000 new cases in 2014 alone 1 . Anticancer drugs targeting DNA synthesis and repair are among the most widely prescribed cancer therapeutics in the world. For several decades, medicines such as 5- fluorouracil (5-FU) have been the standard treatment for a wide variety of cancers through reversible inhibition of thymidylate synthase (TS), an enzyme that catalyzes the methylation of deoxyuridine monophosphate (dUMP) (Figure 1.1.A) and is responsible for DNA synthesis, repair, and cell division 2-5 . Inhibition of TS reduces DNA incorporation of 2’-deoxythymidine 5’-triphosphate (dTTP) and, while increasing levels of deoxyuridine triphosphate (dUTP) 6-7 , causing misincorporation of uracil into DNA during replication and repair by DNA polymerase, resulting in cell death. However, many patients display a lack of response to 5-FU-based therapies due to intrinsic or acquired resistance to the drug. Figure 1.1. (A) Methylation of deoxyuridine monophosphate (dUMP) by thymidylate synthase (TS); (B) hydrolysis of deoxyuridine triphosphate (dUTP) by dUTPase to yield dUMP and pyrophosphate. Deoxyuridine triphosphatase (dUTPase) is an ubiquitous enzyme that catalyzes the hydrolysis of the α,β-pyrophosphate bond of dUTP to furnish dUMP (Figure 1.1.B), which serves as a substrate for TS in normal thymidylate metabolism and DNA synthesis 8 (Figure 1.2.A). dUTPase is responsible maintaining DNA integrity by regulating dUTP/dTTP ratios; lack of the enzyme leads to abnormally high uracil concentrations, which would overload the DNA base-excision repair pathway, resulting in DNA double-strand breakage and cell death 8 . It has been shown that dUTPase is over-expressed in several 5-FU-resistant tumors, which serves 2 to overcome TS inhibition by regulating uracil levels in DNA 8-12 . This is accomplished by preventing the mis-incorporation of dUTP into DNA while concomitantly increasing concentrations of dUMP substrate (Figure 1.2.B). 3 dUTP dUDP dUMP dUTPase TS TS inhibition Repair AP Sites UDG B dUTP dUDP dUMP dUTPase TS TS inhibition Repair AP Sites UDG C dUTPase inhibition Figure 1.2. Role of dUTPase in cancer. (A) Normal thymidylate metabolism; (B) Thymidylate metabolism in response to TS inhibition, resulting in overexpression of dUTPase ; (C) Therapeutic strategy of combining a TS inhibitor with a dUTPase inhibitor, resulting in enhanced cell death. Altogether, these findings demonstrate that dUTPase inhibition may enhance the efficacy of TS inhibitors and ameliorate resistance when used as a combinational therapy (Figure 1.2.C). Over the last 20 years, several dUTPase inhibitors have been reported in the literature. Many of 4 these make use of a non-hydrolyzable dUTP analog by replacing the oxygen atom comprising the α,β-pyrophosphate bond of dUTP 13-14 (Figure 1.3.A and Figure 1.3.B). However, many of these compounds suffer from poor solubility and cell permeability owing to their inherent polarity. Other compounds, including O-tritylated dUTP derivatives 15-16 (Figure 1.3.C), have shown high efficacy against other forms of dUTPase such as Plasmonium falciparum, while exerting much weaker activity against human dUTPase. Figure 1.3. Structures of literature-reported dUTPase inhibitors. 1.2. dUTPase Inhibitor Design, Synthesis and Biological Evaluation 1.2.1 Design of dUTPase Inhibitors Prior to the design of our inhibitors, we needed a mechanistic understanding of the dUTPase enzyme. Human dUTPase is an ubiquitous, homotrimeric enzyme with three active sites that selectively converts dUTP to dUMP and pyrophosphate via hydrolysis of the α,β- pyrophosphate bond. Hydrolysis is initiated by the coordination of a Mg 2+ ion cofactor to the three phosphate groups of dUTP, followed by selective attack of a catalytic water molecule at the α-pyrophosphate bond (Figure 1.4) 17 . Binding of dUTP is mediated by a flexible C-terminal segment, which opens to accommodate the substrate and subsequently closes upon binding to allow for hydrolysis to take place. After undergoing hydrolysis, the C-terminus reopens, and dUTP and pyrophosphate are released from the active site , allowing for entry of another dUTP molecule and cycle repetition 18-19 . Due to the rapid kinetics of this reaction (k cat = 7 molecules of dUTP per second 20 ), attempts to co-crystallize the ES-complex have been unsuccessful. Alternatively, a dUTP mimetic, α,β-imido-dUTP, was used to obtain a crystal structure (PDB:2HQU, Figure 1.5A) 21 . This mimetic replaced the α,β-pyrophosphate oxygen atom with nitrogen, preventing substrate hydrolysis to reveal other key interactions within the active site. 5 Figure 1.4. Mechanism of dUTP hydrolysis 17 . C D A B Figure 1.5. (A) Crystal structure of human dUTPase 19 . (B) Computational model of α,β-imido-dUTP bound in the dUTPase active site. (C) Binding interactions of α,β-imido-dUTP. (D) Structural breakdown of dUTPase active site. Using our knowledge of the dUTPase structure and mechanism, efforts were made to design novel structural types of dUTPase inhibitors using a rational, structure-based approach. To initiate the design of dUTPase inhibitors, my colleague Kevin Gaffney and I used an in silico fragment screen to generate ideas of potential molecular motifs and binding interactions. These 6 fragments were docked into a computational model of the crystal structure of human dUTPase, which was accessed via the Protein Data Bank (PDB:2HQU, Figure 1.5.A) 21 . In this crystal structure, the aforementioned non-hydrolyzable α,β-imido-dUTP is bound into the active site of dUTPase(Figure 1.5.B). The active site was divided into three regions based on the native substrate: (a) the uracil-binding site (U), the sugar group (S), and the phosphate-binding site (P) (Figure 1.5.D). The main objective of our design was to investigate the binding interactions made by dUTP and imido-dUTP (Figure 1.5.C) within the active site and to identify functional groups and moieties that are capable of these interactions. These groups, or fragments, can then serve as a blueprint for our inhibitor design. Using Schrodinger’s Glide, a group of approximately 350,000 molecular fragments (MW ≤ 250 daltons, logP ≤ 3.5, # rotatable bonds ≤ 5) from the ZINC Database were virtually screened in a human dUTPase crystal structure (PDB:3ARA) 22-26 . Initially, the docked fragments were biased towards the uracil binding site due to its high H-bonding density (Figure 1.6A), and as a result, the screen yielded mostly nucleobase analogs (uracil, thymine, cytosine, Figure 1.6B). This was problematic in part because our aim was to identify fragments that could bind in different regions of the active site in order to construct a design of an inhibitor that could occupy the phosphate-binding site as well as the uracil-binding site. Figure 1.6. (a) Overlay of initial screen of ZINC Database fragments into the crystal structure of human dUTPase (PDB:3ARA), which were biased towards the uracil-binding site; (b) identification of carbocyclic analog of 5’-aminouradine, a screening hit resulting from the high H-bonding density of the uracil-binding site. In order to overcome the inherent bias of fragment binding into the uracil-binding site of dUTPase, we employed an in silico alkylation of the backbone NH of G99 and G110 of the 7 uracil binding site, blocking these interactions and allowing for docking into the sugar- and phosphate-binding sites (Figure 1.7A). The virtual screen was then re-performed using the same group of fragments, which resulted in an array of docking hits in the sugar- and phosphate- binding sites (Figure 1.7B). A manual analysis of the screening results biasing for lipophilic molecules and hydrogen bonding to D102 yielded a toluoylimidazole fragment (Figure 1.7C). An overlay of the two fragments (Figure 1.6D) shows the methyl group of the toluoyl fragment in the same position as the 1’ carbon of dUTP, or the analogous position on the uridine analog, and inspired a fragment linking strategy (Figure 1.7E). From these initial hits, I designed a focused library of uracil-containing compounds with varying linker (alkyl, aryl, thiophenyl, piperidinyl), orientation, and N-H bond donors (amides, sulfonamides, N-heterocycles) while simultaneously considering the synthetic accessibility of these designs (Figure 1.7F). Due to its accessibility and resemblance to the screening hits, a library of uracil-based compounds containing a 2-substituted benzimidazole group was considered for scaffold development. Figure 1.7. (a) In silico alkylation of the backbone NH of G99 and G110 of the uracil-binding site of dUTPase; (b) blocking the uracil-binding site resulted in fragment binding in other regions of the active site; (c) toluoylimidazole fragment screening hit; (d) overlay of initial carbocyclic uridine fragment (aqua) with toluoylimidazole (purple); (e) fragment-linking strategy and (f) binding interactions of representative molecules generated from in silico screening. 1.2.2 Synthesis of dUTPase Inhibitors Using the results gathered from a manual analysis of the screening results, a focused library of uracil-containing compounds was designed utilizing a fragment-linking strategy. 8 These compounds combined the strong binding affinity of uracil and an N-H bond donor using a molecular linker (Figure 1.8). The linkers were varied greatly (alkyl, aryl, thiophenyl, piperidinyl) in order to probe size, distance, and orientation of the molecule with regard to the active site. Various N-H bond donors (amides, sulfonamides, N-heterocycles) were incorporated into the molecules to evaluate hydrogen bonding to D102. Selected analogs were prepared jointly with my colleague Kevin Gaffney. Figure 1.8. Graphical representation of a novel class of dUTPase inhibitors. Analogs combined the uracil moiety of the native substrate dUTP, along with an N-H donor and a molecular linker. Due to the synthetic tractability and access to commercially available starting materials, a series of alkyl and aryl amides were synthesized (Scheme 1.1). Uracil alkylation of commercially available bromoalkyl and benzyl bromide esters 1.1a-b and 1.4 followed by saponification with lithium hydroxide monohydrate afforded carboxylic acids 1.2a-b and 1.5a-c. These carboxylic acids were then coupled with commercial anilines using the peptide coupling reagent O-(7-azabenzotriazol-1-yl)-N,N,N’N’-tetramethyluroniumhexafluorophosphate (HATU) to furnish amides 1.3a-b and 1.8a-b. The chain length of the alkyl benzamides was selected based on molecular models which indicated the proximity of the N-H donor to the uracil fragment. Initially, there were concerns that the uracil alkylation would result in alkylation at the N-3 position of the uracil ring rather than at the N-1 position. To test this hypothesis, alkylations were performed with uracil and (TMS) 2 uracil 16,27 , which resulted in identical NMR spectra. Thus, alkylations were generally carried out in DMSO in an open flask at room temperature in the presence of cesium carbonate due to ease of preparation. 9 Scheme 1.1. Synthesis of Amides 1.3a-b and 1.8a-b. Reagents and conditions: (a) Uracil, Cs 2 CO 3 , DMSO, room temperature, 2.5-3 h; (b) LiOH·H 2 O, THF/H 2 O/EtOH, room temperature, 2.5-3 h; (c) o- Phenylenediamine, HATU, DIPEA, DMF, room temperature, 3 h. Due to the availability of commercially available benzenesulfonyl chloride 1.9, sulfonamide 1.11 was synthesized in two steps via nucleophilic displacement reactions. (Scheme 1.2). Scheme 1.2. Synthesis of sulfonamide 1.11. Reagents and conditions: (a) Benzylamine, DIPEA, Et 2 O, O o C to room temperature, 2 h; (b) 1. Uracil, TMSCl, HMDS, reflux, 2 h, 2. 1.10, TBAI, CH 2 Cl 2 , room temperature, 96 h. 10 Based off of the toluoylimidazole fragment hit from in silico screening, a series of alkyl and aryl benzimidazoles was synthesized (Scheme 1.3 and Scheme 1.4). Initial efforts to synthesize the benzimidazoles in one pot from reaction of the parent carboxylic acid with phenylenediamines were unsuccessful, despite several reported methods in the literature. Instead, amide coupling of the carboxylic acid or acyl chloride was carried out, followed by AcOH-catalyzed cyclization to yield benzimidazoles 1.14a-b, 1.18a-b and 1.19a-f. In the case of compounds 1.14a-b, Boc-protected o-phenylenediamine was acylated with commercially available acid chlorides, followed by uracil alkylation and an acid-catalyzed cyclization (Figure 1.11). This multistep approach allowed for chromatographic purification of intermediates prior to cyclization, which was imperative given the inherent polarity of the benzimidazole group. The final products were typically purified via recrystallization. Scheme 1.3. Synthesis of alkyl benzimidazoles 1.14a-b. Reagents and conditions: (a) Tert-butyl (2- aminophenyl)carbamate, DIPEA, CH 2 Cl 2 , O o C to room temperature, 14 h; (b) Uracil, Cs 2 CO 3 , DMSO, room temperature, O/N; (c) AcOH, 120-130 o C, O/N. Substituted alkyl benzimidazoles 1.18a-b were synthesized in order access a hydrophobic pocket within the phosphate-binding region of the dUTPase active site. 28 The substituted phenylenediamine precursors were synthesized via O-alkylation of the hydroxynitroanilines 1.15a and 1.15b to give 1.16a-b, which were then reduced to alkoxyphenylenediamines 1.17a-b via Pd-mediated hydrogenation (Figure 1.12). 29-30 These phenylenediamines were then subjected to HATU-catalyzed amide coupling with carboxylic acid 1.2b, followed by AcOH- mediated cyclization to yield 1.18a-b. 31-32 The corresponding meta- and para-substituted aryl benzimidazoles were synthesized in a similar fashion via amide coupling with carboxylic acids 1.5a-c to yield 1.19a-k (Scheme 1.5). This strategy allowed for a quick generation of compounds using parallel synthesis. 11 Scheme 1.4. Preparation of substituted phenylenediamines 1.17a and 1.17b. Reagents and conditions: (a) (Bromomethyl)cyclopropane, K 2 CO 3 , KI, MeCN, 70 o C, 12-13 h; (b) H 2 , Pd/C, MeOH, room temperature, 2-2.5 h. Scheme 1.5. Synthesis of substituted benzimidazoles 1.18a-b and 1.19a-k. Reagents and conditions: (a) 17a or 17b, HATU, DIPEA, DMF, room temperature, O/N; (b) AcOH or AcOH/HCl, 100-120 o C, 10-72 h; (c) Uracil or thymine, Cs 2 CO 3 , DMSO, room temperature, 3-16 h; (d) LiOH·H 2 O, THF/H 2 O/EtOH or THF/H 2 O, room temperature, 16 h. 12 Other substituents were implemented using a more linear approach. The three-step synthesis of aminobenzimidazole 1.23 allowed for incorporation of an additional N-H bond donor (Scheme 1.6). Cyanobenzimidazole 1.29 was synthesized in five steps from 1.4a, which included the synthesis and alkylation of 3-benzoyluracil 33 (1.27) and a one-pot condensation/cyclization from benzaldehyde 1.28 and the parent diamine (Scheme 1.7). Curiously, this was the only instance where this approach was successful. Compound 1.34 was generated from amide coupling/cyclization of Boc-protected diamine 1.33, which was synthesized from commercially available benzoic acid 1.30 for both ease of purification and regioselectivity of the aforementioned amide coupling reaction (Scheme 1.8). Scheme 1.6. Synthesis of aminobenzimidazole 23. Reagents and conditions: (a) 1. Uracil, TMSCl, HMDS, reflux, 2 h, 2. 20, TBAI, CH 2 Cl 2 , room temperature, 96 h; (b) H 2 , Pd/C, room temperature, 2.5 h; (c) 2-chlorobenzimidazole, KH 2 PO 4 , n-BuOH, 90 o C, 12 h. 13 Scheme 1.7. Synthesis of cyanobenzimidazole 1.29. Reagents and conditions: (a) DIBAL/hexanes, CH 2 Cl 2 , -78 o C to room temperature, 19 h; (b) MnO 2 , CH 2 Cl 2 , room temperature, 15 h; (c) BzCl, pyridine, MeCN, room temperature, 26 h; (d) 1.27, Cs 2 CO 3 , MeCN, room temperature, 13 h; (e) 3,4- Diaminobenzonitrile, Na 2 S 2 O 5 , DMF, 100-110 o C, 14 h; (f) NH 4 OH, room temperature, 14 h. Scheme 1.8. Synthesis of methyl ester 1.34. Reagents and conditions: (a) H 2 SO 4 , MeOH, reflux, 16 h; (b) NaH, THF, Boc 2 O, 0 o C to room temperature, 10 h; (c) H 2 , Pd/C, EtOAc/MeOH (1:1), room temperature, 13 h; (d) 1.5a, DIPEA, HATU, DMF, 14 h; (e) AcOH, 100 o C, 14 h. 14 The aforementioned meta- and para-substituted aryl benzimidazoles were synthesized in order to probe the orientation of the benzimidazole moiety in the phosphate-binding region of the dUTPase active site. While the flexible alkyl analogues 1.3a-b, 1.14a-b and 1.18a-b were used to probe the optimal distance between the uracil and N-H bond donors, the rigid aryl derivatives 1.19a-k were designed with the premise of investigating molecular orientation and selectivity. Additionally, other linkers such as piperidine and thiophene were investigated with the goal of optimizing orientation and molecular geometry. Iodomethylpiperidine 1.36 was synthesized via iodination 34 from commercially-available 1-Boc-4-hydroxymethylpiperidine (1.35), which was then alkylated with 3-benzoyluracil (1.27), followed by Boc-deprotection and nucleophilic displacement to yield piperidinyl benzimidazole 1.38 (Scheme 1.9). Scheme 1.9. Preparation of dUTPase inhibitor 1.38 using a piperidine linker. Reagents and conditions: (a) PPh 3 , imidazole, I 2 , THF, O o C to room temperature, 3 h; (b) BzCl, pyridine, MeCN, room temperature, 26 h; (c) 1.27, Cs 2 CO 3 , DMF, room temperature, 43 h; (d) TFA, CH 2 Cl 2 , room temperature, 23 h; (e) 2-chlorobenzimidazole, DIPEA, DMF, 135 o C, 9.5 h. Thiophene-based analogs were synthesized in a similar fashion to benzene derivatives 1.19a-e. Commercially available 2,5-thiophenedicarboxylic acid 1.39 was converted to diester 1.40 via Fischer esterification, which was reduced with lithium borohydride to yield monoester 1.41 and subsequently brominated to furnish bromoester 1.42. Using the alkylation, saponification, and amide-coupling/cyclization conditions established for the benzylic analogs, compounds 1.47a-c and 1.48a-c were quickly synthesized using both uracil and thymine as the nucleic base (Scheme 1.10). 15 Scheme 1.10. Synthesis of thiophene analogs 1.47a-c and 1.48a-c. Reagents and conditions: (a) H 2 SO 4 , MeOH, reflux, 21 h; (b) LiBH 4 , THF, 0 o C to room temperature, 4 h; (c) PPh 3 , imidazole, Br 2 , room temperature, 1 h; (d) Uracil or thymine, Cs 2 CO 3 , DMSO, room temperature, 1-37 h; (e) LiOH·H 2 O, NaOH, EtOH/H 2 O or MeOH/H 2 O, room temperature, 6-72 h; (f) o-phenylenediamine, HATU, DIPEA, DMF, room temperature, 8-24 h; (g) AcOH, 100-110 o C, 13-48 h. 1.2.3 Biological Screening and Activity of dUTPase Inhibitors Due to the emergence of phenotypic screening as a means of ameliorating clinical attrition rates in drug discovery, compound efficacy was evaluated using an MTT cell viability assay in collaboration with Professor Stan G. Louie (USC School of Pharmacy). It has been shown that cells were sensitized to 5-fluorodeoxyuridine (FdUrd) in vitro when dUTPase expression was silenced with siRNA. 35 Taiho Pharmaceutical, which is currently developing a dUTPase inhibitor for the treatment of resistant cancers, expanded this work by combining FUDR with a dUTPase inhibitor in an MTT assay to evaluate its effect on cell viability. 28 16 Following the synthesis of our focused library, these molecules were screened in an MTT assay for their effects on HeLa and HCT-116 cell viability, and the results allowed for the selection of the best N-H donor and the best two linkers for further optimization. Molecular linkers were evaluated on the basis of length, size, and conformation, while N-H donors were initially synthesized on the basis of the aforementioned docking results. Cells were grown, seeded in 96-well plates, treated with compound + FdUrd (1 µM) at various concentrations, and incubated for 72 hours. The wells were then treated with methylthiazolyldiphenyl-tetrazolium bromide (MTT), which is converted by mitochondrial reductase in living, metabolically active cells to yield 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (Figure 1.9). 36 The amount of MTT formazan produced in each well is directly proportional to the amount of viable cells 37 and is quantified by absorbance at 490 nm using a spectrophotometric plate reader. Figure 1.9. Cleavage of methylthiazolyldiphenyl-tetrazolium bromide (MTT) by mitochondrial reductase yields 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT formazan). MTT formazan is quantified using a spectrophotometric plate reader at a wavelength of 490 nm. The MTT assay data for the initial class of analogs are summarized in Table 1.1 for the human colorectal cancer HCT-116 and human cervical cancer HeLa, respectively. The general activity trend was as follows: aryl benzimidazole > alkylamides > arylsulfonamide > alkylbenzimidazole. With respect to the benzyl linker, it was shown that benzimidazole substitution at the para position (compound 1.19b) resulted in increased potency in both cell lines compared to meta substitution (compound 1.19a). For this reason, para-substituted 1.19d was synthesized, and it was revealed that the addition of a cyclopropylmethyl ether substituent resulted in increased efficacy across both cell lines. This finding bolstered our predictions that having an alkyl substituent on the benzimidazole ring would increase potency due to its higher clogP (cell permeability) and via access to a hydrophobic pocket in the phosphate-binding region 17 of the dUTPase active site. This strategy proved effective in increasing the efficacy of alkylbenzimidazoles (compounds 1.18a-b) as well, though none were as potent as substituted arylbenzimidazole 1.19d. 18 Compound R Linker N-H Donor EC50 (µM), HCT116 EC50 (µM), HeLa 1.3a H 160 67 1.3b H >160 38 1.11 H 108 143 1.14a H >160 >160 1.14b H 103 160 1.18a H 75 68 1.18b H 53 33 1.19a H 135 73 1.19b H 75 30 1.19d H 19 25 1.23 H >160 78 Table 1.1. MTT viability assay data for first analog class of dUTPase inhibitors. Growth inhibition of human colorectal cancer HCT-116 and human cervical cancer HeLa cells was evaluated in response to 1 µM FdUrd and dUTPase inhibitors. 19 Inspired by the results of aryl-linked, cyclopropylmethyl ether 1.19d, we sought to optimize the arylbenzimidazole scaffold through the synthesis and biological evaluation of other substituted benzimidazole analogs (Table 1.2). It was predicted that functionalization of the aryl ring of the N-H donor to access a hydrophobic pocket proximal to the phosphate binding site would result in an increase in potency. We were very interested in incorporating fluorine into our inhibitors due to the propensity of fluorine groups to participate in electrostatic interactions, enhance metabolic stability and increase half-life of several known drugs 38 . As expected, trifluoromethyl benzimidazole 1.19f and trifluoromethoxy 39 benzimidazole 1.19g exhibited very good potency against both HCT-116 and HeLa cell lines. Although arylnitriles are generally very robust and not easily metabolized 40 , compound 1.25 did not decrease cell viability compared to the fluorine-containing analogs. Methyl ester 1.30 was synthesized to explore the possibility of using a prodrug moiety to increase cellular uptake 41 , though it did not have any significant effect on cell viability in either cell line. Compound R Linker N-H Donor EC50 (µM), HCT116 EC50 (µM), HeLa 19f H 35 30 19g H 22 30 25 H 80 90 30 H 140 >160 Table 1.2. MTT viability assay data for second analog class of dUTPase inhibitors. Growth inhibition of human colorectal cancer HCT-116 and human cervical cancer HeLa cells was evaluated in response to 1 µM FdUrd and dUTPase inhibitors. Fluorine-containing compounds 1.19f and 1.19g exhibited increased potency against HCT-116 and HeLa compared with nitrile 1.25 and methyl ester 1.30. 20 After evaluating the combined MTT data, a series of analogs were synthesized using a 2,5-disubstituted thiophene linker along with –CF 3 , -OCF 3 , and unsubstituted benzimidazoles as the N-H donor. These compounds were subjected to MTT assay screening to assess possible replacement of the benzene linker (Table 1.3). Overall, the substituted thiophene- benzimidazoles 1.47a and 1.47b exhibited comparable potency with 1.19f and 1.19g, with the – OCF 3 analogs having the greatest effect against cell viability. Compound R Linker N-H Donor EC50 (µM), HCT116 EC50 (µM), HeLa 47a H >100 155 47b H 45 38 47c H 20 15 Table 1.3. MTT viability assay data for third analog class of dUTPase inhibitors, which incorporated a thiophene linker. Growth inhibition of human colorectal cancer HCT-116 and human cervical cancer HeLa cells was evaluated in response to 1 µM FdUrd and dUTPase inhibitors. While the MTT data alluded to potential anti-proliferative effects of these compounds, a series of literature-reported in vitro enzymatic assays using a reported 5’-tritylated nucleoside dUTPase inhibitor 16 as a control to directly detect dUTPase inhibition were wildly unsuccessful. Around that same time, a “selective” dUTPase inhibitor from Taiho Pharmaceuticals 28 was revealed to be a dual dUTPase/DPD (dihydropyrimidine dehydrogenase) inhibitor 42 (Figure 1.10). 21 Figure 1.10. Structure of literature-reported dUTPase inhibitors, which include a dual dUTPase/DPD inhibitor from Taiho Pharmaceuticals (left) and a 5’-tritylated nucleoside analog (right). In order to address the possibility of off-target cytotoxic effects, thymine derivatives of each compound were synthesized and screened. It was found that replacing the uracil moiety of 1.19f for thymine (compound 1.19i) dramatically decreased the growth-inhibiting capacity. Those compounds that retained MTT activity as thymine derivatives were deprioritized as part of their activity was dUTPase-independent. Interestingly, the thymine analog of the control compound was more potent than the parent compound, suggesting that it may be acting on more than one target (Figure 1.11). Figure 1.11. Cell viability assays of dUTPase inhibitors in HCT-116 human colorectal carcinoma cells. (A) When the uracil moiety (blue) was replaced with thymine (orange), the potency of compound 1.19f significantly decreased, as opposed to a previously published 5’-tritylated nucleoside dUTPase inhibitor (B), which was used as a control. These preliminary results suggest that the potency of these compounds can be attributed to selective inhibition of dUTPase, therefore providing an interesting starting point for further optimization. Most recently, a group of 5-fluorouracil analogs were synthesized (1.19j-k) to address the possibility of increasing potency via the release of 5-fluorouracil after the compound is metabolized. These compounds are to be screened in cellular and enzymatic assays in the near future. 22 1.3 Conclusion We have demonstrated the design and synthesis of a novel class of anticancer agents. Design of the inhibitors was done through molecular modeling of the active site of the dUTPase enzyme based on its crystal structure. Several inhibitors were synthesized that used the inherent binding affnity of uracil while incorporating an N-H donor. By inhibiting dUTPase, increasing levels of uracil are incorporated into DNA, overloading its base-excision epair capacity and resulting in apoptosis. Addition of dUTPase inhibitors to existing TS inhibitors such as 5-FU increases the anticancer effect by elevating dUTP levels and inducing cell death in cancer cells in 5-FU resistant tumors. 1.4 Experimental 1.4.1 Structure-based Design All molecular modeling, virtual screening and computational chemistry was carried out as described previously. The dUTPase crystal structure PDB:3ARA was chosen for its uniform active sites and high resolution. The protein was prepared for docking using Schrodinger’s Protein Preparation Wizard tool to add hydrogens, correct bond orders, delete non-essential waters, predict side-chain protonation states, tautomers, and polar hydrogen orientations, and minimize the energy of the protein structure. The receptor grid was prepared as a 20-Å box centered around G110, A45, R85, and Q131. The ZINC Database was sorted for subsets by property and 354,309 “Clean Fragments” (MW≤ 250, logP≤ 3.5, # rotatable bonds≤ 5) were downloaded as .sdf files in Linux. These molecules were prepared for docking with LigPrep using the OPLS_2005 force field to generate protonation states between pH 5-9 using Epik. Initial studies showed identical results for all three active sites, so the active site between chain A and B that would be covered by the C-terminus of chain C was chosen for the remaining docking studies. The fragments were initially docked and scored using the HTVS setting in Glide 5.8 and then the top docking were redocked using the SP setting. The U-site was blocked manually in Maestro 9.3 by converting the –NH of the G99 and G110 to –NC with the “Set element” feature in the Build toolbar, hydrogens were added to the carbon with the “Add 23 hydrogens” feature of the Edit toolbar. A cycle of converting one of the hydrogens to a carbon, adding hydrogens, and adjusting the bond angles using the “Quick Torsion” tool in the “Adjust” feature in the Edit toolbar was repeated until the chain from G99 was connected to G110 with the “Draw structure” feature of the Build toolbar. A grid file was set up using the same parameters as above and docked with the fragments using the same HTVS to SP protocol. The top docking fragments for the U- and P-sites were overlay in the Maestro 9.3 window and the proposed linking strategies were drawn in ChemDraw, saved as .sdf file, prepared with LigPrep, docked into the original grid using the SP setting, and selected for synthesis based on GlideScore. 1.4.2. Synthetic Chemistry All reactions, unless noted otherwise, were conducted using commercially available solvents and reagents as received, without additional preparation or purification, in ordinary glassware. 1H, 13C, and 19F spectra were recorded on Mercury 400, Varian 400-MR (400 MHz), Varian VNMRS-500 (500 MHz) 2-channel, or Varian VNMRS-600 (600 MHz) 3-channel NMR spectrometers, using residual 1H or 13C signals of deuterated solvents as internal standards. Silica gel (60 Å, 40-63 μm; Sorbent Technologies) was used as a sorbent for flash column chromatography. Automated flash chromatography was performed on Isolera One flash purification system (Biotage), default fraction volume – 14 mL. 6-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)hexanoic acid (1.2a): To a stirred solution of uracil (2.01 g, 17.934 mmol) in DMSO (40 mL) was added Cs 2 CO 3 (5.42 g, 17.927 mmol). After stirring for 10 minutes at room temperature, the slurry was charged dropwise with ethyl 6-bromohexanoate (1.60 mL, 8.992 mmol) and was allowed to stir at room temperature for 3 hours. The reaction mixture was diluted with EtOAc (60 mL) and washed with brine (60 mL). The aqueous layer was extracted with EtOAc (3 x 20 mL), and the combined organic layers were washed with brine (30 mL), dried over Na 2 SO 4 and concentrated in vacuo onto Celite. The residue was purified by automated column chromatography (75-85% 24 EtOAc/hexanes) to afford ethyl 6-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)hexanoate as a white solid (1.50 g, 62% yield). 1 H NMR (400 MHz, CDCl 3 ) δ 9.39 (s, 1H), 7.15 (d, J = 8.1 Hz, 1H), 5.69 (d, J = 7.8 Hz, 1H), 4.11 (q, J = 7.1 Hz, 2H), 3.71 (t, J = 6.9 Hz, 2H), 2.29 (t, J = 7.0 Hz, 2H), 1.67 (m, 4H), 1.36 (m, 2H), 1.23 (t, J = 7.1 Hz, 3H). 13 C NMR (100 MHz, CDCl 3 ) δ 173.50, 164.00, 150.97, 144.55, 102.25, 60.48, 48.74, 34.06, 28.81, 25.95, 24.43, 14.35. To a stirred solution of ethyl 6-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)hexanoate (0.817 g, 3.045 mmol) in 1:1:1 THF/H 2 O/EtOH (9 mL) was added lithium hydroxide monohydrate (0.35 g, 8.341 mmol, and the mixture was allowed to stir at room temperature for 3 hours. After evaporating the solvents, the residue was charged with water (15 mL), and the mixture was adjusted to pH 2 with 6N HCl. The resulting precipitate was collected by filtration and washed with H 2 O and EtOAc to yield 1.2a as a white powder (0.400 g, 55% yield). 1 H NMR (400 MHz, DMSO-d 6 ) δ 12.04 (s, 1H), 11.20 (s, 1H), 7.67 (d, J = 7.9 Hz, 1H), 5.52 (d, J = 7.7 Hz, 1H), 3.63 (t, J = 7.2 Hz, 2H), 2.20 (t, J = 7.3 Hz, 2H), 1.53 (m, 4H), 1.24 (m, 2H). 13 C NMR (100 MHz, DMSO-d 6 ) δ 174.74, 164.16, 151.33, 146.18, 101.15, 47.67, 33.94, 28.59, 25.73, 24.50. 7-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)heptanoic acid (1.2b): Cs 2 CO 3 (5.100 g, 16.868 mmol) was added to a stirred solution of uracil (1.891 g, 16.868 mmol) in DMSO (40 mL), and the suspension was stirred at room temperature for 10 minutes. Ethyl 7- bromoheptanoate (1.64 mL, 8.434 mmol) was added dropwise over 5 minutes, and the resulting white slurry was allowed to stir for 2.5 hours at room temperature. The reaction mixture was charged with H 2 O and extracted with EtOAc (4 x 40 mL). The organic layers were combined, washed with brine, dried over Na 2 SO 4 , and concentrated in vacuo onto Celite. The residue was purified by automated column chromatography (89-100% EtOAc/hexanes) to yield ethyl 7-(2,4- dioxo-3,4-dihydropyrimidin-1(2H)-yl)heptanoate as a white solid (1.407g, 62% yield). 1 H NMR (400 MHz, CDCl 3 ) δ 9.07 (brs, 1H), 7.14 (d, J = 7.9 Hz, 1H), 5.69 (d, J = 7.8 Hz, 1H), 4.11 (q, J = 7.2 Hz, 2H), 3.71 (t, J = 7.4 Hz, 2H), 2.28 (t, 2H), 1.78 – 1.54 (m, 4H), 1.44 – 1.31 (m, 4H), 1.24 (td, J = 7.2, 1.2 Hz, 3H). 13 C NMR (150 MHz, CDCl 3 ) δ 173.72, 163.79, 150.88, 144.51, 102.24, 60.41, 48.90, 34.23, 28.96, 28.70, 26.19, 24.79, 14.38. 25 To a stirred solution of 7-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)heptanoate (1.407 g, 5.244 mmol) in 1:1:1 THF/H 2 O/EtOH (12 mL) was added lithium hydroxide monohydrate (0.572 g, 13.634 mmol), and the resulting slurry was allowed to stir at room temperature for 3 hours. The reaction mixture was concentrated under reduced pressure, and the resulting residue was triturated with 6N HCl. The precipitate was filtered, washed with H 2 O, CH 2 Cl 2 and dried to yield 1.2b a white solid (0.470g, 37% yield). 1 H NMR (400 MHz, DMSO-d 6 ) δ 11.97 (s, 1H), 11.19 (s, 1H), 7.64 (d, J = 7.8 Hz, 1H), 5.52 (d, J = 7.8 Hz, 0H), 3.63 (t, J = 7.3 Hz, 2H), 2.18 (t, J = 7.3 Hz, 2H), 1.81 – 1.41 (m, 4H), 1.39 – 1.13 (m, 4H). 13 C NMR (100 MHz, DMSO-d 6 ) δ 174.84, 164.14, 151.32, 146.11, 101.16, 40.37, 40.16, 33.96, 28.71, 28.53, 25.94, 24.77. 6-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-N-phenylhexanamide (1.3a): To a stirred solution of 6-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)hexanoic acid (1.2a, 0.192 g, 0.849 mmol) in DMF (10 mL) was added DIPEA (0.3 mL, 1.697 mmol) and HATU (0.323 g, 0.849 mmol). After stirring for five minutes, aniline (0.079 g, 0.849 mmol) was added, and the resulting solution was allowed to stir at room temperature for 3 hours. The solvent was evaporated, and the residue was dissolved in EtOAc, washed three times with brine, dried over Na 2 SO 4 and concentrated in vacuo onto Celite. The residue was purified by automated column chromatography (EtOAc) to yield 1.3a as a light-orange solid (0.119 g, 46% yield). 1 H NMR (400 MHz, CD 3 OD) δ 8.07 (d, J = 7.8 Hz, 1H), 8.03 (dd, J = 8.7, 1.1 Hz, 2H), 7.79 (dd, J = 8.5, 7.5 Hz, 2H), 7.65 – 7.54 (m, 1H), 6.12 (d, J = 7.8 Hz, 1H), 4.28 – 4.22 (m, 2H), 2.88 (t, J = 7.4 Hz, 2H), 2.36 – 2.15 (m, 4H), 2.01 – 1.84 (m, 2H). 13 C NMR (100 MHz, CD 3 OD) δ 174.32, 166.73, 152.79, 147.34, 139.82, 129.75, 125.13, 121.26, 102.13, 38.87, 37.62, 29.61, 26.93, 26.31. MS (ESI) m/z 302.2 [M+H] + ; 300.1 [M+H] - . 26 7-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-N-phenylheptanamide (1.3b): To a stirred solution of 7-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)heptanoic acid (0.195 g, 0.805 mmol) in DMF (10 mL) was added DIPEA (0.28 mL, 1.610 mmol) and HATU (0.306 g, 0.805 mmol). After stirring for five minutes, aniline (0.075 g, 0.805 mmol) was added, and the resulting solution was allowed to stir at room temperature for 3 hours. The solvent was evaporated, and the residue was dissolved in EtOAc, washed three times with brine, dried over Na 2 SO 4 and concentrated in vacuo onto Celite. The residue was purified by automated column chromatography (EtOAc) to yield 1.3b as a light-brown solid (0.198 g, 78% yield). 1 H NMR (400 MHz, CD 3 OD) δ 8.05 (m, 3H), 7.88 – 7.70 (m, 1H), 7.57 (t, J = 7.4 Hz, 2H), 6.13 (d, J = 7.8 Hz, 1H), 4.37 – 4.11 (m, 2H), 2.87 (t, J = 7.4 Hz, 2H), 2.20 (h, J = 7.1 Hz, 4H), 2.02 – 1.82 (m, 4H). 13 C NMR (100 MHz, CD 3 OD) δ 174.47, 166.71, 152.77, 147.28, 139.88, 129.75, 125.08, 121.21, 102.13, 38.87, 37.77, 29.80, 29.78, 27.15, 26.65. MS (ESI) m/z 316.2 [M+H] + ; 314.2 [M+H] - . 4-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)benzoic acid (1.5a): Cesium carbonate (3.959g, 13.096 mmol) was added to a stirred solution of uracil (1.468g, 13.096 mmol) in DMSO (30 mL), and the suspension was stirred at room temperature for 1 hour. A solution of methyl 4-bromomethyl)benzoate (1.4a, 1.000g, 4.365 mmol) in DMSO (4 mL) was added dropwise, and the mixture was allowed to stir overnight at room temperature. The reaction mixture was poured into H 2 O (40 mL) and extracted with EtOAc (4 x 30 mL). The 27 combined organic layers were washed with brine (30 mL), dried over Na 2 SO 4 and concentrated in vacuo onto Celite. The residue was purified by automated column chromatography (80-100% EtOAc/hexanes) to yield methyl 4-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)benzoate as a white powder (0.469g, 41% yield). 1 H NMR (600 MHz, DMSO-d 6 ) δ 11.36 (s, 1H), 7.95 (d, J = 8.0 Hz, 2H), 7.78 (d, J = 7.9 Hz, 1H), 7.41 (d, J = 8.1 Hz, 2H), 5.62 (d, J = 7.8 Hz, 1H), 4.96 (s, 2H), 3.84 (s, 3H). 13 C NMR (150 MHz, DMSO-d 6 ) δ 165.91, 163.64, 150.98, 145.63, 142.32, 129.50, 128.84, 127.44, 101.52, 52.14, 50.11. To a stirred solution of methyl 4-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)benzoate (0.450g, 1.729 mmol) in 1:1 THF/MeOH (40 mL) was added lithium hydroxide monohydrate (0.189g, 4.496 mmol), and the resulting mixture was stirred at room temperature for 19 hours, followed by a second addition of LiOH (0.189g, 4.496 mmol) and H 2 O (7 mL). After stirring overnight, TLC revealed completion of the reaction. The reaction mixture was concentrated under reduced pressure, and the residue was charged with H 2 O (20 mL) and acidified to pH 1 with 2N HCl. The resulting precipitate was filtered, washed with H 2 O and hexanes, and dried to afford 1.5a as a white powder (0.343g, 81% yield). 1 H NMR (400 MHz, DMSO-d6) δ 12.95 (s, 1H), 11.35 (s, 1H), 7.93 (d, J = 6.8 Hz, 2H), 7.78 (d, J = 7.8 Hz, 1H), 7.38 (d, J = 6.8 Hz, 2H), 5.62 (d, J = 7.9 Hz, 1H), 4.95 (s, 2H). 13 C NMR (100 MHz, DMSO-d6) δ 166.98, 163.66, 151.00, 145.65, 141.82, 130.04, 129.66, 127.30, 101.50, 50.12. 4-((5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)benzoic acid (1.6a): To a stirred solution of thymine (3.303g, 26.193 mmol) in DMSO (90 mL) was added Cs 2 CO 3 (7.919g, 26.193 mmol), and the suspension was stirred at room temperature for 20 minutes. The resulting thick slurry was charged dropwise with a solution of methyl 4-(bromomethyl) benzoate in DMSO (22 mL) over a period of 20 minutes. The resulting bright yellow-orange slurry was stirred at room temperature for 11 hours. The reaction mixture was poured in ice-cold H 2 O (150 mL) and extracted with EtOAc (4 x 60 mL). The organic layers were combined, washed with brine (50 mL), dried over Na 2 SO 4 , and concentrated in vacuo onto Celite. The residue was purified by automated column chromatography (70-85% EtOAc/hexanes) to yield a white solid, 28 which was triturated with EtOAc (30 mL), filtered and dried to give methyl 4-((5-methyl-2,4- dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)benzoate as a white powder 0.906 g, 38% yield). 1 H NMR (400 MHz, DMSO-d 6 ) δ 11.35 (s, 1H), 7.95 (d, J = 8.6 Hz, 2H), 7.65 (d, J = 1.3 Hz, 1H), 7.41 (d, J = 8.3 Hz, 2H), 4.92 (s, 2H), 3.84 (s, 3H), 1.76 (s, 3H). 13 C NMR (101 MHz, DMSO-d 6 ) δ 165.92, 164.24, 150.98, 142.48, 141.29, 129.49, 128.81, 127.47, 109.17, 52.14, 49.90, 11.95. To a stirred suspension of methyl 4-((5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)- yl)methyl)benzoate (0.902 g, 3.289 mmol) in 1:1 MeOH/H 2 O (30 mL) was added 2N NaOH (13.2 mL), and the resulting homogeneous solution was stirred at room temperature for 3 hours. Methanol was removed under reduced pressure, and the remaining aqueous solution was acidified to pH 1 with concentrated HCl (aq.), resulting in precipitation. The precipitate was collected by vacuum filtration, washed with H 2 O and EtOAc, and dried to yield 1.6a as a white solid (0.694 g, 81% yield). 1 H NMR (400 MHz, DMSO-d 6 ) δ 12.94 (s, 1H), 11.35 (s, 1H), 7.92 (d, J = 8.2 Hz, 2H), 7.64 (s, 1H), 7.38 (d, J = 8.1 Hz, 2H), 4.91 (s, 2H), 1.76 (s, 3H). 13 C NMR (101 MHz, DMSO-d 6 ) δ 166.99, 164.25, 151.00, 141.99, 141.30, 130.00, 129.65, 127.32, 109.16, 49.90, 11.96. 4-((5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)benzoic acid (1.7a): A 500 mL flask containing a stirred solution of 5-fluorouracil (8.517 g, 65.482 mmol) in DMSO (170 mL) was charged with K 2 CO 3 (9.050 g, 65.482 mmol), and the resulting suspension was stirred at room temperature for 45 minutes. The suspension was then charged dropwise with a solution of methyl 4-(bromomethyl)benzoate (5.000 g, 21.827 mmol) in DMSO (32 mL) over a period of 1 hour. The resulting light-yellow solution was allowed to stir at room temperature for 21 hours. The solidified reaction mixture was cooled in an ice bath, charged with H 2 O (400 mL), and extracted with EtOAc (4 x 150 mL). The organic layers were combined, washed with brine (150 mL) and concentrated under reduced pressure. The resulting white semisolid was treated with a 29 solution of 1:1 EtOAc/hexanes (250 mL) and was stirred at room temperature for 40 minutes. The precipitate was collected by filtration, washed with water and 1:1 EtOAc/hexanes, and dried in a 70 0 C vacuum oven to yield methyl 4-((5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)- yl)methyl)benzoate as a white solid (1.183 g, 19% yield). 1 H NMR (400 MHz, DMSO-d 6 ) δ 11.88 (s, 1H), 8.24 (d, J = 6.7 Hz, 1H), 7.95 (d, J = 8.6 Hz, 2H), 7.45 (d, J = 8.7 Hz, 2H), 4.91 (s, 2H), 3.85 (s, 3H). 13 C NMR (101 MHz, DMSO-d 6 ) δ 165.92, 157.58, 157.33, 149.65, 141.94, 141.00, 138.72, 130.24, 129.91, 129.46, 128.89, 127.50, 52.16, 50.46. 19 F NMR (376 MHz, DMSO-d 6 ) δ -168.95, -168.94. MS (ESI) m/z 279.1 [M+H] + ; 277.1 [M-H] - . A 250 mL flask containing a stirred suspension of methyl 4-((5-fluoro-2,4-dioxo-3,4- dihydropyrimidin-1(2H)-yl)methyl)benzoate (1.173 g, 4.216 mmol) in 1:1 MeOH/H 2 O (50 mL) was charged dropwise with 2N NaOH (16.86 mL). The resulting colorless solution was allowed to stir at room temperature for 24 hours. The reaction mixture was concentrated under reduced pressure to remove methanol. The remaining aqueous solution was acidified to pH 0-1 with 12N HCl, resulting in precipitation. The precipitate was collected by filtration, washed with water and dried in a 70 0 C vacuum oven to yield 1.7a as a white solid (0.981 g, 88% yield). 1 H NMR (400 MHz, DMSO-d 6 ) δ 12.96 (s, 1H), 11.88 (d, J = 5.1 Hz, 1H), 8.23 (d, J = 6.7 Hz, 1H), 7.93 (d, J = 8.5 Hz, 2H), 7.42 (d, J = 8.6 Hz, 2H), 4.90 (s, 2H). 13 C NMR (101 MHz, DMSO-d 6 ) δ 167.01, 157.60, 157.34, 149.67, 141.44, 141.01, 138.73, 130.25, 130.08, 129.92, 129.63, 127.37, 50.48. 19 F NMR (376 MHz, DMSO-d 6 ) δ -168.95. MS (ESI) m/z 263.1 [M-H] - . 4-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)-N-phenylbenzamide (1.8a): HATU (0.309 g, 0.812 mmol) was added to a vial containing a stirred solution of 1.5a (0.200 g, 0.812 mmol) and DIPEA (0.28 mL, 1.625 mmol) in DMF (4 mL). After stirring at room temperature for a period of 10 minutes, aniline (0.08 mL, 0.894 mmol) was added, and the resulting yellow solution was allowed to stir at room temperature for an additional 70 hours. The 30 reaction mixture was subsequently treated with saturated NaHCO 3 (10 mL) and extracted with EtOAc (4 x 5 mL). The cloudy organic layers were combined, washed with H 2 O (5 mL) and filtered. The recovered solid was washed with EtOAc and H 2 O and dried to yield 1.8a as a white powder (0.174 g, 67% yield). 1 H NMR (400 MHz, DMSO-d 6 ) δ 11.35 (s, 1H), 10.22 (s, 1H), 7.93 (d, J = 7.7 Hz, 2H), 7.80 (d, J = 7.9 Hz, 1H), 7.76 (d, J = 8.0 Hz, 2H), 7.43 (d, J = 8.0 Hz, 2H), 7.40 – 7.30 (m, 2H), 7.10 (t, J = 7.6 Hz, 1H), 5.63 (d, J = 7.9 Hz, 1H), 4.96 (s, 2H). 13 C NMR (DMSO-d 6 ) δ 165.22, 163.66, 151.00, 145.62, 140.37, 139.10, 134.35, 128.60, 128.01, 127.28, 123.66, 120.30, 101.47, 50.11. 4-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)-N-(4- (trifluoromethyl)phenyl)benzamide (1.8b): HATU (0.309 g, 0.812 mmol) was added to a vial containing a stirred solution of 1.5a (0.200 g, 0.812 mmol) and DIPEA (0.28 mL, 1.625 mmol) in DMF (4 mL). After stirring at room temperature for a period of 10 minutes,4-(trifluoromethyl)aniline (0.11 mL, 0.894 mmol) was added dropwise, and the mixture was allowed to stir at room temperature for an additional 38 hours. The reaction mixture was charged with brine (10 mL) and cooled in an ice bath. The resulting precipitate was collected by vacuum filtration, washed with copious amounts of water and dried to yield 1.8b as an off-white solid (0.160 g, 51% yield). 1 H NMR (400 MHz, DMSO-d 6 ) δ 11.36 (s, 1H), 10.56 (s, 1H), 8.00 (d, J = 8.5 Hz, 2H), 7.95 (d, J = 8.0 Hz, 2H), 7.80 (d, J = 7.9 Hz, 1H), 7.72 (d, J = 8.5 Hz, 2H), 7.45 (d, J = 7.9 Hz, 2H), 5.63 (d, J = 7.8 Hz, 1H), 4.97 (s, 2H). 13 C NMR (101 MHz, DMSO-d 6 ) δ 165.74, 163.66, 151.00, 147.94, 145.62, 142.77, 140.81, 133.85, 132.08, 128.18, 127.35, 125.90, 120.06, 101.49, 50.13. MS (ESI) m/z 390.0 [M+H] + . 31 N-benzyl-4-(bromomethyl)benzenesulfonamide (1.10): To an oven-dried flask containing an ice-cold solution of 4-(bromomethyl)benzenesulfonyl chloride (7, 1.190 g, 4.400mmol) in anhydrous Et 2 O (2 mL) was added DIPEA (0.782 mL, 4.500 mmol) followed by benzylamine (0.483 mL, 4.420 mmol), and the reaction was warmed to room temperature. After stirring for 2 hours, the reaction mixture was concentrated in vacuo, and purified by automated column chromatography (20% EtOAc/hexanes) to yield 1.10 (0.227 g, 15% yield). 1 H NMR (400 MHz, CD 3 OD) δ 7.79 (dd, J = 11.2, 8.5 Hz, 2H), 7.56 (dd, J = 8.6, 2.1 Hz, 2H), 7.26 – 7.16 (m, 5H), 4.70 (s, 1H), 4.61 (s, 1H), 4.07 (s, 2H). N-benzyl-4-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)benzenesulfonamide (1.11): A suspension of uracil (0.074 g, 0.66mmol), chlorotrimethylsilane (0.17 mL, 1.34mmol), and hexamethyldizsilazane (1.38 mL, 6.6mmol) was heated at refluxing temperature for 2 hours. A solution of N-benzyl-4-(bromomethyl)benzenesulfonamide (1.8, 0.227 g, 0.670 mmol) and tetrabutylammonium iodide (0.016 g, 0.04mmol) in anhydrous CH 2 Cl 2 (1.4 mL) was added to the uracil solution and stirred at room temperature for 96hrs. H 2 O was then added and the precipitate was filtered to yield 1.11 as an off-white solid (0.079 g, 3% yield). 1 H NMR (400 MHz, CD3OD) δ 8.08 (d, J = 8.5 Hz, 2H), 7.97 (d, J = 7.9 Hz, 1H), 7.75 (d, J = 8.6 Hz, 2H), 7.52 – 7.43 (m, 5H), 6.02 (d, J = 7.8 Hz, 1H), 5.29 (s, 2H), 4.37 (s, 2H). 13 C NMR (101 MHz, CD 3 OD) δ 146.99, 142.24, 142.02, 138.47, 129.38, 129.36, 128.90, 128.53, 128.41, 102.90, 51.87, 47.90. LC-MS: calcd. [M+H] + 372.09 (100.0%), 373.10 (19.8%), & 374.09 (4.7%); found 372.1 (100%), 373.1 (21.42%), & 374.1 (7.31%). MS (ESI) m/z 372.1 [M+H] + ; 370.1 [M+H] - . 32 tert-butyl (2-(6-bromohexanamido)phenyl)carbamate (1.13a): Tert-butyl (2-aminophenyl)carbamate (1.00g, 4.802 mmol) was charged to an oven-dried RBF and purged with argon. Dry CH 2 Cl 2 (10 mL) was added, and the solution was cooled in an ice bath and charged dropwise with DIEA (1.67 mL, 9.603 mmol) and 6-bromohexanoyl chloride (1.12a, 740 µL, 4.802 mmol). After stirring at 0 0 C for 10 minutes, the solution was allowed to stir overnight at room temperature under argon atmosphere for 14 hours. The solvent was evaporated and the resulting residue was dissolved in EtOAc and washed with H 2 O. The aqueous layer was extracted an additional 4x with EtOAc, and the organic layers were combined, dried over Na 2 SO 4 , and concentrated in vacuo onto Celite. The residue was purified by automated column chromatography (28-30% EtOAc/hexanes) to yield 1.13a as a colorless syrup (1.697g, 92% yield). 1 H NMR (400 MHz, CDCl 3 ) δ 8.14 (s, 1H), 7.46 (d, J = 9.0 Hz, 1H), 7.35 (d, J = 9.1 Hz, 1H), 7.19 – 7.11 (m, 2H), 6.85 (s, 1H), 3.42 (t, J = 6.7 Hz, 2H), 2.37 (t, J = 7.4 Hz, 2H), 2.02 – 1.82 (m, 2H), 1.82 – 1.66 (m, 2H), 1.52 (s, 9H). 13 C NMR (100 MHz, CDCl 3 ) δ 171.93, 154.39, 130.65, 130.25, 126.31, 125.68, 124.65, 81.17, 37.12, 33.66, 32.56, 28.45, 27.84, 24.88. tert-butyl (2-(7-bromoheptanamido)phenyl)carbamate (1.13b): An oven-dried RBF was charged with 7-bromoheptanoic acid (0.580 g, 2.775 mmol) and purged several times with argon. Dry CH 2 Cl 2 (5 mL) was added, and the resulting solution was charged dropwise with oxalyl chloride (940 µL, 11.102 mmol) and anhydrous DMF (1 drop). After stirring at room temperature for 2 hours, the solution was concentrated under reduced pressure to yield acid chloride 1.12b, and the residue was dissolved in anhydrous CH 2 Cl 2 (5 mL) and added dropwise under argon to a stirred solution of tert-butyl (2-aminophenyl)carbamate (578 mg, 2.775 mmol) and DIEA (970 µL, 5.551 mmol) in CH 2 Cl 2 (5 mL) at 0 o C. After 10 minutes, the 33 solution was allowed to stir at room temperature overnight. The reaction mixture was subsequently evaporated, and the residue was diluted with EtOAc and washed with H 2 O. The aqueous layer was extracted an additional 3 times with EtOAc. The organic layers were combined, washed with sat. NaCl, dried over Na 2 SO 4 and concentrated in vacuo onto Celite. The residue was purified by automated column chromatography (30% EtOAc/hexanes) to yield 1.13b as a pale-yellow oil (0.942g, 85% yield). 1 H NMR (400 MHz, CDCl 3 ) δ 8.16 (s, 1H), 7.43 (d, J = 7.3 Hz, 1H), 7.35 (d, J = 7.2 Hz, 1H), 7.21 – 7.07 (m, 1H), 6.91 (s, 1H), 3.41 (t, J = 7.7 Hz, 2H), 2.40 – 2.30 (m, 1H), 1.94 – 1.81 (m, 2H), 1.80 – 1.68 (m, 2H), 1.52 (s, 9H), 1.49 – 1.33 (m, 4H). 13 C NMR (100 MHz, CDCl 3 ) δ 172.22, 154.36, 130.74, 130.19, 126.27, 125.56, 124.62, 81.06, 37.20, 33.90, 32.65, 28.44, 28.39, 27.96, 25.57. 1-(5-(1H-benzo[d]imidazol-2-yl)pentyl)pyrimidine-2,4(1H,3H)-dione (1.14a): Cesium carbonate (2.638g, 8.726 mmol) was added to a stirred solution of uracil (0.978g, 8.726 mmol) in DMSO (20 mL), and the suspension was stirred at room temperature for 20 minutes. A solution of 1.11a (1.681g, 4.363 mmol) in DMSO (4 mL) was added dropwise, and the mixture was allowed to stir overnight at room temperature. The mixture was treated with brine and extracted with EtOAc. The organic layer was washed with H 2 O and brine, dried over Na 2 SO 4 , and concentrated in vacuo onto Celite. The residue was purified by automated column chromatography (3-5% MeOH/CH 2 Cl 2 ) to afford tert-butyl (2-(6-(2,4-dioxo-3,4- dihydropyrimidin-1(2H)-yl)hexanamido)phenyl)carbamate product as a white foam (0.456g, 25% yield). 1 H NMR (400 MHz, CDCl 3 ) δ 8.32 (brs, 1H), 7.54 (brs, 1H), 7.47 – 7.34 (m, 1H), 7.22 – 7.03 (m, 4H), 5.67 (d, J = 7.9 Hz, 1H), 3.72 (t, J = 7.3 Hz, 2H), 2.37 (t, J = 7.3 Hz, 2H), 1.74 – 1.70 (m,, 4H), 1.51 (s, 9H), 1.46 – 1.36 (m, 2H). 13 C NMR (100 MHz, CDCl 3 ) δ 171.96, 163.78, 154.44, 144.64, 125.62, 102.50, 48.62, 36.81, 31.73, 28.46, 25.73, 24.80. 34 A flask containing tert-butyl (2-(6-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)- yl)hexanamido)phenyl)carbamate (0.443g, 1.064 mmol) was charged with 13:2 AcOH/HCl (15 mL), and the resulting colorless solution was heated at refluxing temperature (130 o C) overnight. The solution was subsequently cooled and concentrated under reduced pressure. The residue was neutralized with saturated NaHCO 3 , resulting in a sticky solid. Trituration with EtOAc/hexanes, followed by filtration and washing with copious amounts of H 2 O, EtOAc, and hexanes yielded 1.14a as a light-brown solid (0.066g, 21% yield). 1 H NMR (400 MHz, DMSO-d 6 ) δ 12.13 (s, 1H), 11.19 (s, 1H), 7.63 (d, J = 7.6 Hz, 1H), 7.56 – 7.36 (m, 2H), 7.11 – 7.09 (m, 2H), 5.50 (d, J = 7.8 Hz, 1H), 3.64 (t, J = 7.3 Hz, 2H), 2.79 (t, J = 7.4 Hz, 2H), 1.94 – 1.73 (m, 2H), 1.83 – 1.75 (m, 2H), 1.35 – 1.28 (m, 2H). 13 C NMR (100 MHz, DMSO-d 6 ) δ 164.14, 155.33, 151.32, 146.11, 141.05, 121.17, 118.46, 101.15, 47.71, 28.76, 28.60, 27.49, 25.83. 1-(6-(1H-benzo[d]imidazol-2-yl)hexyl)pyrimidine-2,4(1H,3H)-dione (1.14b): Cs 2 CO 3 1.375g (4.548 mmol) was added to a stirred solution of uracil (0.510g, 4.548 mmol) in DMSO (10 mL). The resulting suspension was stirred for 5 minutes before being charged dropwise with a solution of 1.13b (0.908g, 2.274 mmol) in DMSO (2 mL). The resulting pale yellow mixture was allowed to stir overnight at room temperature. The reaction mixture was subsequently charged with brine and extracted with EtOAc. The organic layer was washed with H 2 O and brine, dried over Na 2 SO 4 and concentrated in vacuo onto Celite. The residue was purified by automated column chromatography (3-4% MeOH/CH 2 Cl 2 ) to yield tert-butyl (2-(7- (2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)heptanamido)phenyl)carbamate as a white foam (0.469g, 48% yield). 1 H NMR (400 MHz, CDCl 3 ) δ 9.49 (brs, 1H), 8.32 (brs, 1H), 7.48 (brs, 1H), 7.40 (d, J = 9.2 Hz, 1H), 7.22 – 7.03 (m, 4H), 5.67 (d, J = 2.8 Hz, 1H), 3.70 (t, J = 3.6 Hz, 2H), 2.36 (t, J = 7.4 Hz, 2H), 1.81 – 1.63 (m, 4H), 1.50 (s, 9H), 1.46 – 1.30 (m, 4H). 13 C NMR (100 MHz, CDCl 3 ) δ 172.28, 163.91, 154.39, 151.29, 130.70, 130.18, 126.15, 125.45, 124.62, 102.44, 48.55, 37.00, 28.92, 28.45, 28.41, 25.90, 25.40. A flask containing tert-butyl (2-(7-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)- yl)heptanamido)phenyl)carbamate (0.432g, 1.003 mmol) was charged with a 11:1 mixture of 35 HCl/AcOH (12 mL), and the resulting solution was heated at refluxing temperature (120 ○ C) for 19 hours. Afterwards, the solution was cooled to room temperature and concentrated under reduced pressure. The residue was neutralized with saturated NaHCO 3 and diluted with EtOAc. After several minutes a precipitate was formed, filtered and washed with copious amounts of H 2 O and EtOAc to afford 1.14b as a light-brown powder (0.266g, 85% yield). 1 H NMR (400 MHz, DMSO-d 6 ) δ 12.13 (s, 1H), 11.20 (s, 1H), 7.53 – 7.36 (m, 2H), 7.09 (dd, J = 6.1, 3.1 Hz, 2H), 5.52 (d, J = 7.7 Hz, 1H), 3.63 (t, J = 7.2 Hz, 2H), 2.79 (t, J = 7.5 Hz, 2H), 1.87 – 1.64 (m, 2H), 1.67 – 1.49 (m, 2H), 1.41 – 1.18 (m, 4H). 13 C NMR (100 MHz, DMSO-d 6 ) δ 164.14, 155.45, 151.33, 146.10, 121.43, 101.16, 47.81, 40.58, 40.37, 40.21, 40.16, 39.95, 39.74, 39.54, 39.33, 28.83, 28.74, 28.66, 27.80, 25.96. 4-(cyclopropylmethoxy)-2-nitroaniline (1.16a): To a solution of 4-amino-3-nitrophenol (1.15a, 2.00g, 12.977 mmol) in CH 3 CN (30 mL) was added K 2 CO 3 (3.587g, 25.953 mmol), (bromomethyl)cyclopropane (1.89 mL, 19.645 mmol), and KI (0.215g, 1.298 mmol). The mixture was stirred at 70 o C for 13 hours. The reaction mixture was poured into H 2 O (100 mL) and extracted with EtOAc (3 x 125 mL). The organic layers were combined, washed with brine, dried over Na 2 SO 4 , and concentrated in vacuo onto Celite. The residue was purified by flash column chromatography (15% EtOAc/hexanes). The relevant fractions were combined and concentrated under reduced pressure to yield 1.16a as a bright red- orange solid (2.814g, 81% yield). 1 H NMR (400 MHz, CDCl 3 ) δ 7.52 (d, J = 3.0 Hz, 1H), 7.10 (ddd, J = 9.1, 2.9, 1.4 Hz, 1H), 6.76 (d, J = 9.1 Hz, 1H), 5.02 (brs, 2H), 3.77 (d, J = 7.0 Hz, 2H), 1.44 – 1.12 (m, 1H), 0.65 (d, J = 8.2 Hz, 2H), 0.35 (d, J = 4.9 Hz, 2H). 13 C NMR (100 MHz, CDCl 3 ) δ 150.33, 139.83, 127.38, 127.37, 120.23, 120.20, 107.50, 73.77, 10.26, 3.35. 36 2-(cyclopropylmethoxy)-6-nitroaniline (1.16b): To a stirred suspension of 2-amino-3-nitrophenol in CH 3 CN (30 mL) was added (bromomethyl)cyclopropane (1.15b, 2.000g, 12.977 mmol), K 2 CO 3 (3.587g, 25.953 mmol), and KI (0.215g, 1,298 mmol), and the resulting dark-red mixture was stirred at 70 o C for 12 hours. The reaction mixture was subsequently cooled to room temperature and concentrated in vacuo onto Celite. The residue was purified by silica gel flash column chromatography (10% EtOAc/hexanes) to yield 1.16b as an orange solid (2.478g, 92% yield). 1 H NMR (400 MHz, CDCl 3 ) δ 7.79 – 7.66 (m, 1H), 6.84 (d, J = 7.8 Hz, 1H), 6.57 (t, 1H), 6.48 (s, 2H), 3.87 (d, J = 7.0 Hz, 2H), 1.45 – 1.11 (m, 1H), 0.70 – 0.66 (m, 2H), 0.39 – 0.36 (m, 2H). 13 C NMR (101 MHz, CDCl 3 ) δ 147.68, 137.43, 131.85, 117.46, 114.69, 114.63, 74.31, 10.30, 3.47. 4-(cyclopropylmethoxy)benzene-1,2-diamine (17a): A flask containing a solution of 1.16a (1.00g, 4.803 mmol) in methanol (30 mL) was purged several times with argon. Palladium on carbon (5 wt%, 0.200g) was added, and the flask was quickly re-purged with argon, and then several times with hydrogen. The mixture was allowed to stir at room temperature under a hydrogen atmosphere for 2 hours. The reaction mixture was subsequently filtered over a pad of Celite, and the cake was washed with copious amounts of methanol. The filtrate was concentrated under reduced pressure to yield 1.17a as a deep purple solid (0.828g, 97% yield). 1 H NMR (600 MHz, CDCl 3 ) δ 6.61 (d, J = 8.3 Hz, 1H), 6.33 (d, J = 2.7 Hz, 1H), 6.25 (d, J = 5.8 Hz, 1H), 3.70 (d, J = 6.9 Hz, 2H), 3.50 (brs, 2H), 3.07 (brs, 2H), 1.39 – 1.13 (m, 1H), 0.60 (d, J = 8.9 Hz, 2H), 0.31 (d, J = 5.1 Hz, 2H). 13 C NMR (150 MHz, CDCl 3 ) δ 154.09, 137.09, 127.50, 118.38, 105.23, 103.96, 73.37, 10.56, 3.25. 37 3-(cyclopropylmethoxy)benzene-1,2-diamine (17b): A flask containing a solution of 1.16b (0.500 g, 2.401 mmol) in methanol (25 mL) was purged several times with nitrogen and charged with Pd/C (5 wt %, 0.100 g). The flask was back-filled several times more with nitrogen, followed by H 2 gas. The mixture was subsequently allowed to stir at room temperature under H 2 atmosphere. After 2.5 hours, TLC revealed completion of the reaction. The reaction mixture was passed through a syringe filter to remove Pd/C, and the filtrate was concentrated under reduced pressure to yield 1.17b as a red-brown flaky solid (0.417 g, 97% yield). 1 H NMR (400 MHz, CDCl 3 ) δ 6.64 (td, J = 8.0, 1.8 Hz, 1H), 6.38 (ddt, J = 8.0, 6.2, 1.5 Hz, 2H), 3.82 (d, J = 6.9 Hz, 2H), 3.52 (s, 4H), 1.33-1.23 (m, 1H), 0.80 – 0.54 (m, 2H), 0.36-0.32 (m, 2H). 13 C NMR (101 MHz, CDCl 3 ) δ 148.12, 135.52, 124.09, 119.30, 109.84, 104.07, 73.56, 10.64, 3.27. 1-(6-(6-(cyclopropylmethoxy)-1H-benzo[d]imidazol-2-yl)hexyl)pyrimidine-2,4(1H,3H)- dione (1.18a): To a stirred solution of 1.2b (0.404g, 1.683 mmol) and DIPEA (0.59 mL, 3.366 mmol) in DMF (7 mL) was added HATU (0.640g, 1.683 mmol). After stirring for 10 minutes, the solution was charged with 1.17a (0.300g, 1.683 mmol), and the resulting purple solution was allowed to stir overnight at room temperature. The reaction mixture was concentrated under reduced pressure. The residue was taken in EtOAc, washed twice with saturated NaHCO 3 and once with brine, dried over Na 2 SO 4 and concentrated in vacuo onto Celite. The residue was purified by automated column chromatography (5% MeOH/CH 2 Cl 2 ) to yield N-(2-amino-4- (cyclopropylmethoxy)phenyl)-7-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)heptanamide as a light-brown foam (0.128g, 19% yield). 1 H NMR (400 MHz, CDCl 3 ) δ 8.65 (s, 1H), 7.24 (s, 1H), 38 7.15 (d, J = 7.9 Hz, 1H), 6.99 (d, J = 8.3 Hz, 1H), 6.38 – 6.25 (m, 2H), 5.68 (d, J = 8.0 Hz, 1H), 3.77 – 3.65 (m, 4H), 2.38 (t, J = 7.4 Hz, 2H), 1.78 – 1.69 (m, 4H), 1.45 – 1.38 (m, 2H), 1.28 – 1.23 (m, 2H), 0.88 – 0.86 (m, 1H), 0.69 – 0.56 (m, 2H), 0.46 – 0.20 (m, 2H). 13 C NMR (100 MHz, CDCl 3 ) δ 172.09, 165.81, 159.64, 153.90, 145.89, 144.52, 127.00, 117.58, 106.13, 104.23, 102.37, 77.48, 76.84, 73.05, 48.68, 36.62, 29.61, 28.62, 26.02, 25.56, 10.41, 3.32. Glacial acetic acid (6 mL) was added to a flask containing N-(2-amino-4- (cyclopropylmethoxy)phenyl)-7-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)heptanamide (0.120g, 0.300 mmol), and the resulting brown solution was heated and stirred at 100 o C for 11 hours. The reaction mixture was concentrated under reduced pressure, and the resulting orange- red oil was treated with saturated NaHCO 3 (5 mL) and extracted with EtOAc (7 mL). The aqueous layer was washed with EtOAc (3 x 4 mL), and the combined organic washes were dried over Na 2 SO 4 and concentrated in vacuo onto Celite. The residue was purified by automated column chromatography (4-5% MeOH/CH 2 Cl 2 ) to yield 1.18a as a light-brown foam (0.091g, 79% yield). 1 H NMR (400 MHz, DMSO-d 6 ) δ 11.95 (s, 1H), 11.20 (s, 1H), 7.62 (d, J = 7.5 Hz, 1H), 7.30 (d, J = 8.6 Hz, 1H), 6.92 (s, 1H), 6.72 (d, J = 9.0 Hz, 1H), 5.52 (d, J = 7.9 Hz, 1H), 3.79 (d, J = 6.9 Hz, 2H), 3.63 (t, J = 7.2 Hz, 2H), 2.73 (t, J = 7.5 Hz, 2H), 1.76 – 1.69 (m, 2H), 1.60 – 1.52 (m, 2H), 1.34 – 1.21 (m, 4H), 0.87 – 0.83 (m, 1H), 0.58 – 0.54 (m, 2H), 0.33 – 0.30 (m, 2H). 13 C NMR (100 MHz, DMSO-d 6 ) δ 163.70, 154.34, 150.89, 145.66, 110.64, 100.72, 72.50, 47.37, 28.38, 28.31, 28.21, 27.41, 25.52, 10.33, 3.08. MS (ESI) m/z 383.2 [M+H] + ; 381.2 [M+H] - . 1-(6-(7-(cyclopropylmethoxy)-1H-benzo[d]imidazol-2-yl)hexyl)pyrimidine-2,4(1H,3H)- dione (1.18b): To a stirred solution of 1.2b (0.425 g, 1.769 mmol) and DIPEA (0.612 mL, 3.514 mmol) in DMF (10 mL) was added HATU (0.669 g, 1.760 mmol). After stirring for 10 minutes, the solution was charged with 1.17b (0.313 g, 1.756 mmol) and was stirred at room temperature for 14 hours. 39 The reaction mixture was subsequently concentrated in vacuo onto Celite, and the residue was purified by automated column chromatography (10% MeOH/CH 2 Cl 2 ) to yield N-(2-amino-3- (cyclopropylmethoxy)phenyl)-7-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)heptanamide as a tan foam (0.499 g, 57% yield). N-(2-amino-3-(cyclopropylmethoxy)phenyl)-7-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)- yl)heptanamide (0.230g, 0.574 mmol) was dissolved in glacial acetic acid (10 mL), and the resulting solution was stirred at 100 o C for 10 hours. The reaction mixture was subsequently concentrated under reduced pressure, neutralized with saturated NaHCO 3 , and concentrated once more. The residue was treated with water, sonicated, and filtered to yield 1.18b as a purple solid (0.078g, 35%). 1 H NMR (400 MHz, DMSO-d6) δ 12.16 (s, 1H), 11.19 (s, 1H), 7.63 (d, J = 7.8 Hz, 1H), 7.03 – 6.95 (m, 2H), 6.61 (d, J = 7.6 Hz, 1H), 5.52 (d, J = 7.8 Hz, 1H), 3.98 (d, J = 6.9 Hz, 2H), 3.63 (t, J = 7.2 Hz, 2H), 2.76 (t, J = 7.5 Hz, 2H), 1.78 – 1.71 (m, 2H), 1.60 – 1.53 (m, 2H), 1.36 – 1.24 (m, 4H), 0.87 – 0.81 (m, 1H), 0.61 – 0.57 (m, 2H), 0.38 – 0.34 (m, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 163.71, 150.90, 145.68, 103.64, 100.73, 72.54, 47.38, 30.95, 28.31, 28.22, 27.57, 25.51, 10.41, 3.27. MS (ESI) m/z 383.2 [M+H] + ; 381.2 [M-H] - . 3-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)benzoic acid (1.5b): An oven-dried flask was charged with uracil (1.160 g, 10.349 mmol), methyl 3- (bromomethyl)benzoate (1.4b, 2.360 g, 10.303 mmol) and Cs 2 CO 3 (3.110 g, 10.287 mmol) and was subsequently purged with nitrogen gas. DMSO (10 mL) was added, and the mixture was stirred at room temperature for 3 hours. The mixture was subsequently dissolved in EtOAc (30 mL) and washed with brine (2 x 10 mL) and 2N aq. NaOH (2 x 10 mL). The aqueous layer was acidified with 6N HCl, extracted with EtOAc, dried with MgSO 4 , filtered through cotton, and concentrated in vacuo to yield methyl 3-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)- yl)methyl)benzoate (0.680 g, 25% yield). 1 H NMR (400 MHz, CD3OD) δ 7.97 (d, J = 11.4 Hz, 2H), 7.68 (d, J = 7.9 Hz, 1H), 7.61 – 7.57 (m, 1H), 7.49 (t, J = 7.7 Hz, 1H), 5.68 (d, J = 7.9 Hz, 40 1H), 4.99 (s, 2H), 3.90 (s, 3H). 13 C NMR (101 MHz, CD 3 OD) δ 168.06, 166.55, 152.85, 146.94, 138.31, 133.59, 132.00, 130.23, 130.19, 129.77, 102.82, 52.74, 51.96. To a stirring solution of methyl 3-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)benzoate (0.680 g, 2.613 mmol) in 1:1 H 2 O/THF (20 mL) was added lithium hydroxide monohydrate (0.436, 10.391 mmol). After stirring at room temperature for 16 hours, the reaction mixture was concentrated under reduced pressure, and the reside was dissolved in H 2 O, acidified to pH 3 with 6N HCl upon which a white precipitate formed. The precipitate was collected by vacuum filtration, washed with H 2 O, and dried to yield 1.5b as a white powder (0.158 g, 25% yield). 1 H NMR (400 MHz, DMSO-d 6 ) δ 13.04 (s, 1H), 11.35 (s, 1H), 7.89 – 7.85 (m, 2H), 7.81 (d, J = 7.9 Hz, 1H), 7.55 (d, J = 7.7 Hz, 1H), 7.52 – 7.47 (m, 1H), 5.62 (dd, J = 7.8, 2.2 Hz, 1H), 4.94 (s, 2H). 13 C NMR (101 MHz, DMSO-d 6 ) δ 167.45, 164.05, 151.44, 146.06, 137.84, 132.37, 129.41, 129.01, 128.63, 101.89, 50.48. 1-(3-(1H-benzo[d]imidazol-2-yl)benzyl)pyrimidine-2,4(1H,3H)-dione (1.19a): To a stirring solution of 1.5b (0.158 g, 0.642 mmol) and DIPEA (0.144 mL, 0.827 mmol) in DMF (4 mL) was added HATU (0.315 g, 0.829 mmol). After stirring for 10 minutes, the solution was charged with o-phenylenediamine (0.090 g, 0.832 mmol), and was stirred at room temperature for an additional 16 hours. The reaction mixture was subsequently concentrated under reduced pressure, and the residue was dissolved in EtOAc and extracted with 2N NaOH. The aqueous layer was then neutralized with 6N HCl, extracted with EtOAc, the organic layer was then dried with MgSO 4 , concentrated in vacuo onto Celite. The residue was purified by silica gel flash chromatography (5-8% MeOH/CH 2 Cl 2 ) to yield N-(2-aminophenyl)-3-((2,4- dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)benzamide (0.093 g, 43% yield). 1 H NMR (400 MHz, CD3OD) δ 7.94 (d, J = 8.9 Hz, 2H), 7.69 (d, J = 7.9 Hz, 1H), 7.54 (dt, J = 15.1, 7.7 Hz, 2H), 7.18 (dd, J = 7.9, 1.3 Hz, 1H), 7.12 – 7.04 (m, 1H), 6.90 (dd, J = 8.0, 1.2 Hz, 1H), 6.77 (td, J = 7.7, 1.3 Hz, 1H), 5.69 (d, J = 7.9 Hz, 1H), 5.02 (s, 2H). 13 C NMR (101 MHz, CD 3 OD) δ 165.14, 145.52, 136.77, 134.86, 130.88, 128.79, 127.18, 127.08, 126.90, 126.21, 118.19, 117.26, 101.41, 50.68. 41 N-(2-aminophenyl)-3-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)benzamide (0.093 g, 0.277 mmol) was dissolved in glacial acetic acid (10 mL) and 12N HCl (1 mL) and stirred at 120°C for 72 hours. The reaction mixture was subsequently concentrated under reduced pressure, and the residue was dissolved in a minimal amount of MeOH and triturated with EtOAc to yield 1.19a as a light-brown powder (0.068 g, 77% yield). 1 H NMR (400 MHz, CD3OD) δ 8.11 (d, J = 9.0 Hz, 2H), 7.84 (dd, J = 6.2, 3.1 Hz, 2H), 7.77 (t, J = 7.2 Hz, 3H), 7.64 (dd, J = 6.2, 3.1 Hz, 2H), 5.74 (d, J = 7.9 Hz, 1H), 5.12 (s, 2H). 13 C NMR (101 MHz, CD 3 OD) δ 147.01, 140.10, 134.19, 133.23, 131.68, 128.62, 128.25, 127.89, 124.80, 115.00, 103.10, 52.11. MS (ESI) m/z 319.1 [M+H] + . 1-(4-(1H-benzo[d]imidazol-2-yl)benzyl)pyrimidine-2,4(1H,3H)-dione (1.19b): To a solution of 1.5b (0.158 g, 0.642 mmol) and DIPEA (0.144 mL, 0.827 mmol) in DMSO (4 mL) was added HATU (0.315 g, 0.829 mmol). After stirring for 10 minutes, the solution was charged with o-phenylenediamine (0.090 g, 0.832 mmol), and was stirred at room temperature for an additional 240 hours. The reaction mixture was subsequently diluted in EtOAc (20 mL) and washed with brine (3 x 5 mL). The organic layer was then dried with MgSO 4 and concentrated in vacuo onto Celite. The residue was purified by silica gel flash chromatography (2-5% MeOH/CH 2 Cl 2 ) to yield N-(2-aminophenyl)-4-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)- yl)methyl)benzamide (0.114 g, 56% yield). N-(2-aminophenyl)-4-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)benzamide (0.114 g, 0.339 mmol) was dissolved in 10mL of glacial acetic acid and 1mL of 12N HCl and stirred at 120°C for 14 hours. The reaction mixture was subsequently concentrated in vacuo. The resulting residue was dissolved in a minimal amount of MeOH and treated with EtOAc. The precipitate was recovered by vacuum filtration and dried to yield 1.19b as a light-brown powder (0.081g, 70% yield). 42 1 H NMR (400 MHz, DMSO-d 6 ) δ 11.38 (s, 1H), 8.29 (d, J = 8.4 Hz, 2H), 7.89 – 7.77 (m, 3H), 7.61 (d, J = 8.4 Hz, 2H), 7.53 (dd, J = 6.1, 3.1 Hz, 2H), 5.65 (dd, J = 7.9, 2.2 Hz, 1H), 5.01 (s, 1H). 13 C NMR (101 MHz, DMSO-d 6 ) δ 163.72, 151.05, 148.86, 145.67, 141.92, 128.30, 128.14, 125.45, 123.49, 114.19, 109.56, 101.62, 50.23. MS (ESI) m/z 319.1 [M+H] + . 1-(4-(1H-benzo[d]imidazol-2-yl)benzyl)-5-methylpyrimidine-2,4(1H,3H)-dione (1.19c): To a stirred solution of 1.6a (0.300 g, 1.153 mmol) and DIPEA (0.37 mL, 2.097 mmol) in DMF (4 mL) was added HATU (0.439 g, 1.153 mmol), and the resulting yellow solution was stirred at room temperature for 10 minutes. The solution was then charged with o-phenylenediamine in one portion and was allowed to stir at room temperature for 14 hours. The reaction mixture was subsequently partitioned between EtOAc (5 mL) and saturated NaHCO 3 (10 mL). The aqueous layer was back-extracted with EtOAc (3 x 5 mL), and the combined organic layers were washed with brine (5 mL) and concentrated in vacuo onto Celite. The residue was purified by automated column chromatography (90% EtOAc/hexanes) to yield N-(2-aminophenyl)-4-((5-methyl-2,4- dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)benzamide as a yellow solid (0.162g, 44% yield). N-(2-aminophenyl)-4-((5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)benzamide (0.145 g, 0.414) was added glacial acetic acid (10 mL), and was stirred at 100 o C for 6 hours. The reaction mixture was subsequently concentrated under reduced pressure. The residue was treated with H 2 O (10 mL) and sonicated. The resulting precipitate was collected by vacuum filtration and dried to yield 1.19c as a light-brown solid (0.060 g, 43% yield). 1 H NMR (400 MHz, DMSO-d 6 ) δ 11.36 (s, 1H), 8.16 (d, J = 8.0 Hz, 2H), 7.68 (s, 1H), 7.63 – 7.55 (m, 2H), 7.46 (d, J = 8.0 Hz, 2H), 7.21 (d, J = 2.8 Hz, 2H), 4.92 (s, 2H), 1.77 (s, 3H). 13 C NMR (101 MHz, DMSO-d 6 ) δ 164.29, 151.06, 150.80, 141.32, 138.84, 129.30, 127.97, 126.75, 122.25, 109.16, 49.94, 11.99. 43 MS (ESI) m/z 333.1 [M+H] + . 1-(4-(6-(cyclopropylmethoxy)-1H-benzo[d]imidazol-2-yl)benzyl)pyrimidine-2,4(1H,3H)- dione (19d): To a stirred solution of 1.5a (0.335g, 1.361 mmol) and DIPEA (0.47 mL, 2.721 mmol) in DMF (15 mL) was added HATU (0.517g, 1.361 mmol). After stirring for 10 minutes, the solution was charged with 1.17a (0.242g, 1.361 mmol), and the resulting dark solution was stirred for 12 hours at room temperature. The reaction mixture was concentrated under reduced pressure, and the oily residue was partitioned between EtOAc and NaHCO 3 , resulting in an emulsion and subsequent precipitation. The precipitate was collected by filtration and washed with copious amounts of EtOAc and H 2 O to yield N-(2-amino-4-(cyclopropylmethoxy)phenyl)-4-((2,4-dioxo- 3,4-dihydropyrimidin-1(2H)-yl)methyl)benzamide as a brown powder (0.241g, 44% yield). 1 H NMR (400 MHz, DMSO-d6) δ 11.35 (s, 1H), 9.50 (s, 1H), 7.95 (d, J = 7.9 Hz, 2H), 7.79 (d, J = 7.8 Hz, 1H), 7.39 (d, J = 8.0 Hz, 2H), 6.98 (d, J = 8.6 Hz, 1H), 6.33 (d, J = 2.4 Hz, 1H), 6.16 (d, J = 8.5 Hz, 1H), 5.62 (d, J = 7.7 Hz, 1H), 4.94 (s, 2H), 4.88 (brs, 2H), 3.72 (d, J = 6.9 Hz, 2H), 1.23 – 1.15 (m, 1H), 0.57 – 0.53 (m, 2H), 0.32 – 0.28 (m, 2H). 13 C NMR (100 MHz, DMSO-d6) δ 165.03, 163.65, 157.57, 150.99, 145.61, 144.53, 140.03, 128.06, 127.81, 127.18, 116.33, 102.55, 101.44, 101.34, 71.75, 50.11, 10.25, 3.06. Glacial acetic acid (10 mL) was added to a flask containing N-(2-amino-4- (cyclopropylmethoxy)phenyl)-4-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)benzamide, and the mixture was stirred at 100 o C for 12 hours. The reaction mixture was subsequently concentrated under reduced pressure, and the residue was neutralized with saturated NaHCO 3 . The resulting precipitate was collected by filtration and washed with copious amounts of saturated NaHCO 3 , water, and EtOAc, and dried to yield 1.19d as a light-purple solid (0.161 g, 72% yield). 1 H NMR (400 MHz, DMSO-d6) δ 12.73 (s, 1H), 11.36 (s, 1H), 8.11 (d, J = 8.1 Hz, 2H), 7.80 (d, J = 7.9 Hz, 1H), 7.43 (d, J = 8.2 Hz, 3H), 7.03 (s, 1H), 6.83 (d, J = 8.6 Hz, 1H), 5.63 (d, J = 7.8 44 Hz, 1H), 4.94 (s, 2H), 3.84 (d, J = 6.9 Hz, 2H), 1.28 – 1.21 (m, 1H), 0.58 (d, J = 7.5 Hz, 2H), 0.34 (d, J = 5.0 Hz, 2H). 13 C NMR (100 MHz, DMSO-d6) δ 163.70, 151.06, 145.65, 138.14, 129.73, 127.91, 126.37, 101.47, 72.49, 50.13, 10.30, 3.13. MS (ESI) m/z 389.1 [M+H] + . MS (ESI) m/z 387.1 [M-H] - . 1-(4-(7-(cyclopropylmethoxy)-1H-benzo[d]imidazol-2-yl)benzyl)pyrimidine-2,4(1H,3H)- dione (1.19e): To a solution of 1.5a (0.551 g, 2.238 mmol) and DIPEA (0.779 mL, 4.472 mmol) in DMF (10 mL) was added HATU (0.851 g, 2.238 mmol). After stirring for 10 minutes, the solution was charged with 1.17b (0.400 g, 2.244 mmol) and stirred at room temperature for an additional 3 hours. The reaction mixture was diluted with EtOAc and concentrated in vacuo onto Celite. The residue was purified by automated column chromatography (100% EtOAc) to yield N-(2-amino- 6-(cyclopropylmethoxy)phenyl)-4-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)- yl)methyl)benzamide as a brown foam (0.880 g, 96% yield). N-(2-amino-6-(cyclopropylmethoxy)phenyl)-4-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)- yl)methyl)benzamide (0.230g, 0.566 mmol) was dissolved in glacial acetic acid (10 mL), and the resulting solution was stirred at 100 o C for 10 hours. The reaction mixture was subsequently concentrated under reduced pressure, neutralized with saturated NaHCO 3 , and concentrated once more. The residue was treated with water, sonicated, and filtered to yield 1.19e as a brown solid (0.069g, 31%). 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.20 (d, J = 8.0 Hz, 2H), 7.80 (d, J = 7.8 Hz, 1H), 7.43 (d, J = 8.1 Hz, 2H), 7.14 (d, J = 8.0 Hz, 1H), 7.07 (t, J = 7.8 Hz, 1H), 6.68 (d, J = 7.8 Hz, 1H), 5.62 (d, J 45 = 7.8 Hz, 1H), 4.94 (s, 2H), 4.04 (d, J = 7.0 Hz, 2H), 0.87 – 0.81 (m, 1H), 0.64 – 0.59 (m, 2H), 0.40 – 0.38 (m, 2H). 13 C NMR (101 MHz, DMSO-d 6 ) δ 163.98, 151.25, 145.58, 138.24, 129.75, 127.83, 126.76, 122.82, 104.16, 101.49, 72.72, 50.14, 10.40, 3.38. 1-(4-(5-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)benzyl)pyrimidine-2,4(1H,3H)-dione (19f): To a solution of 1.5a (0.250 g, 1.015 mmol) and DIPEA (0.353 mL, 2.027 mmol) in DMF (3 mL) was added HATU (0.420 g, 1.105 mmol). After stirring for 10 minutes, the solution was charged with 4-(trifluoromethyl)benzene-1,2-diamine (0.162 g, 0.920 mmol), and was stirred at room temperature for an additional 16 hours. The reaction mixture was subsequently diluted in EtOAc (20 mL) and washed with brine (3 x 5 mL). The organic layer was then dried with MgSO 4 and concentrated in vacuo onto Celite. The residue was purified by automated column chromatography (60% acetone/hexanes, 1% MeOH) to yield N-(2-amino-4- (trifluoromethyl)phenyl)-4-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)benzamide as a tan powder (0.297 g, 80% yield). N-(2-amino-4-(trifluoromethyl)phenyl)-4-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)- yl)methyl)benzamide (0.297 g, 0.735 mmol) was dissolved in glacial acetic acid (10 mL) and stirred at 120 o C for 16 hours. The reaction mixture was concentrated under reduced pressure. The resulting residue was dissolved in a minimal amount of MeOH and treated with EtOAc. The precipitate was recovered by vacuum filtration and dried to yield 1.19f as an off-white powder (0.227 g, 80% yield). 1 H NMR (400 MHz, DMSO-d 6 ) δ 13.35 (s, 1H), 11.36 (s, 1H), 8.19 (d, J = 8.3 Hz, 2H), 7.95 (s, 1H), 7.82 (d, J = 7.8 Hz, 1H), 7.77 (s, 1H), 7.64 – 7.41 (m, 3H), 5.64 (d, J = 7.9 Hz, 1H), 4.97 (s, 2H). MS (ESI) m/z 385.1 [M-H] - . MS (ESI) m/z 387.1 [M+H] + . 46 1-(4-(5-(trifluoromethoxy)-1H-benzo[d]imidazol-2-yl)benzyl)pyrimidine-2,4(1H,3H)-dione (1.19g): A flask containing a solution of 2-nitro-4-(trifluoromethoxy)aniline (0.500 g, 2.251 mmol) in methanol (20 mL) was purged several times with nitrogen and subsequently charged with Pd/C (5 wt %, 0.100 g). The flask was repurged with nitrogen and then purged several times with hydrogen gas. The mixture was allowed to stir at room temperature for 17 hours under a hydrogen atmosphere. The reaction mixture was filtered over a pad of Celite and washed with copious amounts of EtOAc. The combined filtrate was concentrated in vacuo onto Celite. The residue was purified by automated column chromatography (33-43% EtOAc/hexanes) to yield 4- (trifluoromethoxy)benzene-1,2-diamine as a red oil (0.398 g, 92% yield). A solution of 1.5a (0.295 g, 1.298 mmol) and DIPEA (0.362 mL, 2.078 mmol) in DMF (3 mL) was added HATU (0.514 g, 1.352 mmol). After stirring for 10 minutes, the solution was charged with a solution of 4-(trifluoromethoxy)benzene-1,2-diamine (0.199 g, 1.035 mmol) in DMF (1 mL), and was stirred at room temperature for an additional 16 hours. The reaction mixture was subsequently concentrated under reduced pressure and the residue was partitioned between saturated NaHCO 3 and EtOAc. The organic layer was washed with brine and concentrated in vacuo onto Celite. The residue was purified by automated column chromatography (50% acetone/hexanes, 1% MeOH) to yield N-(2-amino-4-(trifluoromethoxy)phenyl)-4-((2,4-dioxo- 3,4-dihydropyrimidin-1(2H)-yl)methyl)benzamide as a light-brown powder (0.086 g, 20% yield). N-(2-amino-4-(trifluoromethoxy)phenyl)-4-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)- yl)methyl)benzamide (0.086 g, 0.205 mmol) was dissolved in glacial acetic acid (6 mL) and stirred at 120 o C for 16 hours. The reaction mixture was concentrated under reduced pressure and the residue was stirred in saturated NaHCO 3 (10 mL) for 30 minutes. The resulting solid was filtered, washed with hexanes, H 2 O, and acetone to yield an off-white powder, which was washed further with MeOH to yield 1.19g as an off-white solid (0.063 g, 77% yield). 47 1 H NMR (400 MHz, DMSO-d 6 ) δ 13.19 (s, 1H), 11.36 (s, 1H), 8.16 (d, J = 8.3 Hz, 2H), 7.81 (d, J = 7.8 Hz, 1H), 7.65 (s, 2H), 7.48 (d, J = 8.4 Hz, 2H), 7.20 (d, J = 9.0 Hz, 1H), 5.63 (d, J = 7.9 Hz, 1H), 4.96 (s, 2H). 13 C NMR (101 MHz, DMSO-d 6 ) δ 163.80, 153.87, 151.13, 145.64, 143.47, 143.45, 140.82, 138.72, 138.36, 129.74, 127.92, 126.89, 120.39 (q, J = 254.8 Hz), 115.37, 108.51, 101.50, 50.15. 19 F NMR (376 MHz, DMSO-d 6 ) δ -56.98. MS (ESI) m/z 403.1 [M+H] + . MS (ESI) m/z 401.1 [M-H] - . 5-methyl-1-(4-(5-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)benzyl)pyrimidine- 2,4(1H,3H)-dione (1.19h): To a stirred solution of 1.6a (0.300 g, 1.153 mmol) and DIPEA (0.37 mL, 2.097 mmol) in DMF (4 mL) was added HATU (0.439 g, 1.153 mmol), and the resulting yellow solution was stirred at room temperature for 10 minutes. The solution was then charged with 3,4- diaminobenzotrifluoride (0.185 g, 1.049 mmol) in one portion, and the resulting brown solution was stirred at room temperature for an additional 3 hours. The reaction mixture was partitioned between EtOAc (5 mL) and saturated NaHCO 3 (10 mL). The aqueous layer was extracted with EtOAc (3 x 5 mL), and the combined organic layers were washed with brine (5 mL), dried over Na 2 SO 4 and concentrated in vacuo onto Celite. The residue was purified by automated column chromatography (80-90% EtOAc/hexanes) to yield N-(2-amino-4-(trifluoromethyl)phenyl)-4- ((5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)benzamide as a light-brown solid (0.104 g). N-(2-amino-4-(trifluoromethyl)phenyl)-4-((5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)- yl)methyl)benzamide (0.098 g, 0.234 mmol) was dissolved in glacial acetic acid (10 mL) and was stirred at 100 o C for 12 hours. The reaction mixture was concentrated under reduced pressure, and the residue was treated with water (20 mL) and neutralized to pH 6-7 with dropwise addition of 2N NaOH (aq.). The resulting precipitate/aqueous mixture was washed 48 with EtOAc (3 x 5 mL). The organic layers were removed and the resulting aqueous layer was filtered to yield 1.19h as an off-white solid (0.048 g, 12% yield over two steps). 1 H NMR (400 MHz, DMSO-d 6 ) δ 13.36 (s, 1H), 11.36 (s, 1H), 8.19 (d, J = 8.0 Hz, 2H), 8.02 (s, 1H), 7.83 (s, 1H), 7.68 (s, 1H), 7.51 (m, 3H), 4.93 (s, 2H), 1.78 (s, 3H). 13 C NMR (101 MHz, DMSO-d 6 ) δ 164.28, 151.05, 141.31, 128.74, 128.04, 127.08, 109.16, 49.93, 11.98. MS (ESI) m/z 401.0 [M+H] + . 5-methyl-1-(4-(5-(trifluoromethoxy)-1H-benzo[d]imidazol-2-yl)benzyl)pyrimidine- 2,4(1H,3H)-dione (1.19i): To a stirred solution of 1.6a (0.293 g, 1.127 mmol) and DIPEA (0.39 mL, 2.254 mmol) in DMF (2 mL) was added HATU (0.428 g, 1.127 mmol), and the resulting yellow solution was stirred at room temperature for 5 minutes. The solution was then charged with a solution of 4-(trifluoromethoxy)benzene-1,2-diamine (0.866 g, 4.507 mmol) in DMF (2.5 mL). The resulting dark solution was stirred at room temperature for 19 hours. The reaction mixture was partitioned between EtOAc (20 mL) and saturated NaHCO 3 (50 mL). The organic layer was collected, and the aqueous layer was back-extracted with EtOAc (3 x 20 mL). The organic layers were combined, washed with brine (30 mL), dried over Na 2 SO 4 and concentrated in vacuo onto Celite. The residue was purified by automated column chromatography (83-85% EtOAc/hexanes) to yield N-(2-amino-4-(trifluoromethoxy)phenyl)-4-((5-methyl-2,4-dioxo-3,4- dihydropyrimidin-1(2H)-yl)methyl)benzamide as a light yellow-brown solid (0.281 g). MS (ESI) m/z 435.1 [M+H] + . The solid was taken in glacial acetic acid (10 mL), and the reaction mixture was stirred at 100 o C for 15 hours. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The residue was treated with saturated NaHCO 3 (10 mL) and sonicated for 5 minutes, resulting in precipitation. The solid was collected by filtration, washed with water and dried in a vacuum oven to yield 1.19i as a light-brown solid (0.239 g, 51% over two steps). 49 1 H NMR (500 MHz, DMSO-d 6 ) δ 13.20 (s, 1H), 11.36 (s, 1H), 8.16 (d, J = 8.2 Hz, 2H), 7.68 – 7.59 (m, 3H), 7.47 (d, J = 8.1 Hz, 2H), 7.20 (d, J = 7.7 Hz, 1H), 4.92 (s, 2H), 1.77 (s, 3H). 13 C NMR (125 MHz, DMSO-d 6 ) δ 164.29, 153.04, 151.06, 143.74, 141.32, 139.26, 128.94, 128.01, 126.89, 121.38, 119.35, 109.17, 49.93, 11.99. 19 F NMR (470 MHz, DMSO-d 6 ) δ -57.00. MS (ESI) m/z 417.1 [M+H] + ; 415.1 [M-H] - . 5-fluoro-1-(4-(5-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)benzyl)pyrimidine- 2,4(1H,3H)-dione (1.19j): An 8-dram vial containing a stirred solution of 1.6c (0.300 g, 1.135 mmol) and DIPEA (0.40 mL, 2.271 mmol) in DMF (4 mL) was charged with HATU (0.432 g, 1.135 mmol), and the resulting yellow solution was stirred at room temperature for 5 minutes. The mixture was then charged with 3,4-diaminobenzotrifluoride (0.800 g, 4.542 mmol) and allowed to stir at room temperature for 48 hours. The reaction mixture was partitioned between EtOAc (5 mL) and saturated NaHCO 3 (10 mL). The organic layer was collected, and the aqueous layer was extracted with additional EtOAc (3 x 5 mL). The organic layers were combined, washed with brine (5 mL), and concentrated in vacuo onto Celite. The residue was purified twice by automated column chromatography (70% EtOAc/hexanes) to yield N-(2- amino-4-(trifluoromethyl)phenyl)-4-((5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)- yl)methyl)benzamide as a yellow solid (0.215 g, 45% yield). MS (ESI) m/z 421.1 [M-H] - . N-(2-amino-4-(trifluoromethyl)phenyl)-4-((5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)- yl)methyl)benzamide (0.215 g, 0.509 mmol) was loaded into a 50 mL flask, taken in glacial acetic acid (5 mL), and stirred at 100 0 C for 20 hours. The reaction mixture was concentrated under reduced pressure, and the residue was treated with saturated NaHCO 3 (10 mL) and sonicated for 15 minutes, resulting in precipitation. The precipitate was collected by filtration, washed with copious amounts of water, and dried in a 70 0 C vacuum oven to yield 1.19j as a yellow-orange solid (0.151 g, 73% yield). 1 H NMR (400 MHz, DMSO-d 6 ) δ 13.36 (s, 1H), 11.89 (s, 1H), 8.27 (d, J = 6.7 Hz, 1H), 8.19 (d, J = 8.3 Hz, 2H), 8.03 (s, 1H), 7.73 (d, J = 8.4 Hz, 1H), 7.52 (d, J = 8.3 Hz, 2H), 4.92 (s, 2H). 50 19 F NMR (376 MHz, DMSO-d 6 ) δ -58.76, -168.85. MS (ESI) m/z 405.1 [M+H] + ; 403.1 [M-H] - . 5-Fluoro-1-(4-(5-(trifluoromethoxy)-1H-benzo[d]imidazol-2-yl)benzyl)pyrimidine- 2,4(1H,3H)-dione (1.19k): A 50 mL flask containing a stirred solution of 1.6c (0.314 g, 1.189 mmol) and DIPEA (0.41 mL) in DMF (4 mL) was charged with HATU (0.452 g, 1.189 mmol), and the resulting yellow solution was allowed to stir at room temperature for 5 minutes. The mixture was then charged with a solution of 4-(trifluoromethoxy)benzene-1,2-diamine (0.914 g, 4.757 mmol) in DMF (4 mL) and allowed to stir at room temperature for 18 hours. The reaction mixture was subsequently partitioned between EtOAc (10 mL) and saturated NaHCO 3 (20 mL). The organic layer was collected, and the aqueous layer was extracted with additional EtOAc (3 x 5 mL). The organic layers were combined, washed with brine (20 mL) and concentrated in vacuo onto Celite. The residue was purified by automated column chromatography (70% EtOAc/hexanes) to yield N-(2-amino-4-(trifluoromethoxy)phenyl)-4-((5-fluoro-2,4-dioxo-3,4- dihydropyrimidin-1(2H)-yl)methyl)benzamide as a yellow-brown solid (0.345 g, 66% yield). MS (ESI) m/z 439.1 [M+H] + ; 437.1 [M-H] - . N-(2-amino-4-(trifluoromethoxy)phenyl)-4-((5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)- yl)methyl)benzamide (0.345 g, 0.787 mmol) was loaded into a 50 mL flask, taken in glacial acetic acid (5 mL), and stirred at 100 0 C for 21 hours. The reaction mixture was concentrated under reduced pressure, and the residue was treated with saturated NaHCO 3 (15 mL) and sonicated for 20 minutes, resulting in precipitation. The precipitate was recovered by filtration, washed with water and dried in a 70 0 C vacuum oven to yield 1.19k as a light-brown solid (0.288 g, 87% yield). 1 H NMR (500 MHz, DMSO-d 6 ) δ 13.21 (s, 1H), 11.87 (s, 1H), 8.26 (d, J = 6.6 Hz, 1H), 8.16 (d, J = 8.3 Hz, 2H), 7.77 – 7.57 (m, 2H), 7.50 (d, J = 8.1 Hz, 2H), 7.22 – 7.17 (m, 1H), 4.91 (s, 2H). 13 C NMR (126 MHz, DMSO-d 6 ) δ 157.61, 157.40, 153.01, 149.72, 143.71, 140.80, 138.97, 138.70, 130.22, 129.95, 129.02, 128.03, 126.85, 121.37, 119.34, 50.50. 19 F NMR (470 MHz, DMSO-d 6 ) δ -57.00, -168.94. 51 MS (ESI) m/z 421.1 [M+H] + ; 419.1 [M-H] - . 1-(3-nitrobenzyl)pyrimidine-2,4(1H,3H)-dione (1.21): Uracil (5.188g, 46.290 mmol) was charged to an oven-dried RBF and purged with argon. Hexamethyldisilazane (96.52 mL, 462.899 mmol) and TMSCl (11.75 mL, 92.580 mmol) were added under argon, and the suspension was heated at refluxing temperature for 3 hours under an argon atmosphere. The resulting solution was subsequently cooled to room temperature and concentrated under reduced pressure, and the reaction vessel was repurged several times with argon. A solution of 3-nitrobenzyl bromide (1.20, 10.000g, 46.290 mmol) and N- tetrabutylammonium iodide (1.094g, 2.963 mmol) in CH 2 Cl 2 (60 mL) was added, and the mixture was allowed to stir at room temperature for 4 days under an argon atmosphere. The reaction mixture was poured into water (300 mL), and the resulting precipitate was filtered and washed with copious amounts of water and EtOAc to yield 1.21 as a white powder (8.400g, 73% yield). 1 H NMR (400 MHz, DMSO-d6) δ 11.37 (s, 1H), 8.20 – 8.16 (m, 2H), 7.85 (d, J = 7.9 Hz, 1H), 7.77 (d, J = 7.4 Hz, 1H), 7.69 – 7.65 (m, 1H), 5.64 (d, J = 7.8 Hz, 1H), 5.01 (s, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 163.61, 151.03, 147.85, 145.50, 139.05, 134.27, 130.25, 122.67, 122.37, 101.65, 49.79. 52 1-(3-aminobenzyl)pyrimidine-2,4(1H,3H)-dione (1.22): To a stirred solution of 1.21 (3.000g, 12.135 mmol) in methanol (150 mL) was added palladium on carbon (5 wt %, 0.600g), and the reaction vessel was quickly evacuated and backfilled several times with argon. The vessel was then backfilled several times with hydrogen gas, and the mixture was subsequently allowed to stir for 2.5 hours at room temperature under a hydrogen atmosphere. The reaction was filtered over a pad of Celite to remove Pd/C, and the filtrate was concentrated in vacuo onto Celite. The residue was purified by automated column chromatography (4-5% MeOH/CH 2 Cl 2 ) to yield 1.22 as an off-white crystalline powder (2.068g, 78% yield). 1 H NMR (400 MHz, DMSO-d6) δ 11.29 (s, 1H), 7.64 (d, J = 7.8 Hz, 1H), 6.98 (t, J = 7.7 Hz, 1H), 6.47 (d, J = 8.3 Hz, 1H), 6.44 – 6.36 (m, 2H), 5.58 (d, J = 7.8 Hz, 1H), 5.12 (s, 2H), 4.72 (s, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 163.71, 151.00, 149.00, 145.66, 137.41, 129.15, 114.49, 113.14, 112.21, 101.17, 50.21. 1-(3-((1H-benzo[d]imidazol-2-yl)amino)benzyl)pyrimidine-2,4(1H,3H)-dione (1.23): To a vial containing 1.22 (0.300g, 1.381 mmol), 2-chlorobenzimidazole (0.203g, 1.328 mmol), and KH 2 PO 4 (0.188g, 1.381 mmol) was added n-BuOH (3 mL), and the mixture was stirred at 90 o C for 12 hours, at which point an off-white slurry had formed. The mixture was cooled to room temperature and filtered. The solid was washed with n-BuOH, water, and copious amounts of methanol. Upon drying, 1.23 was recovered as an off-white solid (0.212g, 46% yield). 53 1 H NMR (400 MHz, DMSO-d6) δ 11.35 (s, 1H), 9.74 (s, 1H), 7.83 – 7.70 (m, 2H), 7.56 (s, 1H), 7.38 – 7.25 (m, 3H), 7.01 (dd, J = 5.8, 3.2 Hz, 2H), 6.84 (d, J = 7.6 Hz, 1H), 5.63 (d, J = 7.8 Hz, 1H), 4.89 (s, 2H). 13 C NMR (100 MHz, DMSO-d6) δ 163.77, 151.07, 150.20, 145.79, 140.92, 137.61, 129.23, 120.33, 119.56, 116.53, 115.87, 101.36, 50.24. MS-52-287 (4-(bromomethyl)phenyl)methanol (1.24): An oven-dried flask was charged with 1.4a (5.000 g, 21.827 mmol) and purged several times with N 2 . Dry CH 2 Cl 2 (200 mL) was added, and the solution was cooled to -78 o C and charged with diisobutylaluminum hydride (1.0M in hexanes, 54.56 mL) over a period of 10 minutes. The resulting colorless solution was allowed to warm to room temperature and was stirred for an additional 19 hours. The reaction was quenched with 10% aqueous sodium potassium tartrate (8 mL). The resulting thick slurry was diluted with CH 2 Cl 2 (100 mL) and washed with H 2 O (50 mL) and brine (50 mL). The organic layers were filtered, and the filtrate was washed with additional brine (50 mL), dried over Na 2 SO 4 and concentrated under reduced pressure to yield 1.24 as a white solid (3.481 g, 88%). 1 H NMR (400 MHz, CDCl 3 ) δ 7.40 (d, J = 8.3 Hz, 2H), 7.35 (d, J = 8.4 Hz, 2H), 4.70 (s, 1H), 4.50 (s, 2H). 13 C NMR (101 MHz, CDCl 3 ) δ 141.32, 137.31, 129.42, 127.48, 65.08, 33.38. 4-(bromomethyl)benzaldehyde (1.25): Manganese (IV) dioxide (19.931 g, 229.245 mmol) was charged to a stirring solution of 1.24 (3.841 g, 19.104 mmol) in CH 2 Cl 2 (50 mL). The mixture was allowed to stir at room temperature for 15 hours. The reaction mixture was subsequently filtered over a pad of Celite, and the filter cake was washed with copious amounts of CH 2 Cl 2 . The filtrate was concentrated 54 in vacuo onto Celite, and the residue was purified by automated column chromatography (9-12% EtOAc/hexanes) to yield 1.25 as a white solid (2.866 g, 75% yield). 1 H NMR (600 MHz, CDCl 3 ) δ 10.08 (s, 1H), 7.86 (d, J = 6.0 Hz, 2H), 7.56 (d, J = 6.0 Hz, 2H), 4.52 (s, 2H). 13 C NMR (150 MHz, CDCl 3 ) δ 191.63, 144.39, 136.29, 130.31, 129.82, 32.10. 3-benzoylpyrimidine-2,4(1H,3H)-dione (1.27): To an oven-dried flask containing a slurry of uracil (1.26, 2.241g, 19.986 mmol) in anhydrous CH 3 CN (20 mL) was added dry pyridine (8 mL). Benzoyl chloride (5.2 mL, 44.798 mmol) was added dropwise, and the resulting homogeneous mixture was allowed to stir at room temperature for 26 hours under an argon atmosphere. The mixture was concentrated under reduced pressure, and the resulting yellow solid was partitioned between CH 2 Cl 2 (110 mL) and H 2 O (100 mL). The organic layer was dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure. The residue was taken up in a mixture of dioxane (40 mL) and 0.5 M aq. K 2 CO 3 (20 mL) and stirred at room temperature for 40 minutes. The mixture was acidified with glacial acetic acid (pH 5), and the mixture was concentrated to give a solid residue. The solid was treated with aq. NaHCO 3 (100 mL) and stirred for 1 hour at room temperature, and filtered. The resulting solid was washed with cold water, and dried. The residue was stirred in hot acetone and filtered to yield 1.27 as a white powder (2.032g, 47% yield). 1 H NMR (400 MHz, DMSO-d6) δ 11.59 (s, 1H), 7.95 (d, J = 8.4 Hz, 2H), 7.78 (t, J = 7.4 Hz, 1H), 7.66 (d, J = 7.4 Hz, 1H), 7.60 (t, J = 7.2 Hz, 2H), 5.74 (d, J = 7.2 Hz, 1H). 13 C NMR (100 MHz, DMSO-d6) δ 169.98, 162.89, 150.03, 143.30, 135.38, 131.30, 130.17, 129.47, 100.05. 55 4-((3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)benzaldehyde (1.28): An oven-dried fask was charged with 1.27 (0.790 g, 3.654 mmol), 1.25 (0.800 g, 4.019 mmol) and CsCO 3 (2.209 g, 7.308 mmol), and was subsequently purged several times with N 2 . Dry acetonitrile (50 mL) was added under N 2 , and the resulting white suspension was allowed to stir at room temperature for 13 hours under N 2 atmosphere. The reaction mixture was charged with saturated NH 4 Cl (60 mL) and extracted with EtOAc (3 x 50 mL). The organic layers were combined, dried over Na 2 SO 4 , and concentrated in vacuo onto Celite. The residue was purified by automated column chromatography (75-80% EtOAc/hexanes) to yield 1.28 as a white solid (0.789 g, 65% yield). 1 H NMR (400 MHz, DMSO-d 6 ) δ 10.01 (s, 1H), 8.07 (d, J = 8.0 Hz, 1H), 8.02 – 7.90 (m, 4H), 7.78 (t, J = 7.4 Hz, 1H), 7.59 (dd, J = 20.4, 8.0 Hz, 4H), 5.95 (d, J = 8.0 Hz, 1H), 5.07 (s, 2H). 13 C NMR (101 MHz, DMSO-d 6 ) δ 192.70, 169.45, 162.23, 149.61, 146.70, 142.91, 135.63, 135.53, 131.01, 130.27, 129.91, 129.51, 127.92, 101.28, 51.04. 2-(4-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)phenyl)-1H-benzo[d]imidazole-5- carbonitrile (1.29): A vial containing a solution of 1.28 (0.100 g, 0.299 mmol) and 3,4-diaminobenzonitrile in DMF (3 mL) was charged with sodium metabisulfite. The resulting yellow-orange mixture was allowed to stir at 100-110 o C for 14 hours. The mixture was cooled to room temperature, charged with a few drops of 29% NH 4 OH, and stirred at room temperature for an additional 14 hours. The reaction mixture was concentrated under reduced pressure to remove DMF. The residue 56 was triturated with H 2 O, filtered, washed with methanol, H 2 O and hexanes, and dried in a vacuum oven overnight to yield 1.29 as a pale-yellow/brown solid (0.045 g, 44% yield). 1 H NMR (400 MHz, DMSO-d 6 ) δ 11.36 (s, 1H), 8.20 (d, J = 8.3 Hz, 2H), 8.15 (s, 1H), 7.81 (d, J = 7.9 Hz, 1H), 7.74 (s, 1H), 7.60 (dd, J = 8.3, 1.6 Hz, 1H), 7.49 (d, J = 8.3 Hz, 2H) , 5.64 (dd, J = 7.8, 2.3 Hz, 1H), 4.97 (s, 2H). 13 C NMR (101 MHz, , DMSO-d 6 ) δ 163.68, 151.04, 145.65, 139.57, 128.49, 128.02, 127.19, 119.99, 109.53, 104.00, 101.51, 50.14. MS (ESI) m/z 343.9 [M+H] + . methyl 4-amino-3-nitrobenzoate (1.31): To a flask containing a stirred suspension of 4-amino-3-nitrobenzoic acid (1.30, 2.000 g, 10.981 mmol) in methanol (30 mL) was added 18 M H 2 SO 4 (4 drops), and the mixture was heated at refluxing temperature for 16 hours. The reaction mixture was subsequently cooled to room temperature, and the precipitate was collected by vacuum filtration, washed with cold methanol, and dried to yield 1.31 as a bright-yellow powder (1.513 g, 70% yield). 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.55 (d, J = 2.1 Hz, 1H), 7.98 (s, 2H), 7.86 (dd, J = 9.0, 2.1 Hz, 1H), 7.06 (d, J = 8.9 Hz, 1H), 3.81 (s, 3H). 13 C NMR (101 MHz, DMSO-d 6 ) δ 164.88, 148.85, 134.80, 129.60, 128.04, 119.39, 116.13, 51.94. MS (ESI) m/z 197.0 [M+H] + . methyl 4-((tert-butoxycarbonyl)amino)-3-nitrobenzoate (1.32): 57 An oven-dried flask was purged several times with N 2 and charged with dry THF (15 mL). After cooling to 0 o C, the flask was charged with NaH (60% dispersion in mineral oil, 0.918 g), and the flask was back-filled with N 2 several times more. The suspension was charged in three portions with a suspension of 1.31 (1.500 g, 7.647 mmol) in dry THF (30 mL). The resulting dark mixture was stirred under nitrogen at 0 o C for 10 minutes, and then at room temperature for 1 hour. Afterwards the mixture was charged with Boc 2 O (2.003 g, 9.176 mmol) and stirred at room temperature for 10 hours. The reaction mixture was then poured into cold saturated NH 4 Cl (100 mL) and extracted with EtOAc (3 x 50 mL). The organic layers were combined, washed with brine (20 mL), dried over Na 2 SO 4 and concentrated in vacuo onto Celite. The residue was purified by automated column chromatography (3-7% EtOAc/hexanes) to yield 1.32 as a light- yellow solid (1.601 g, 71% yield). 1 H NMR (400 MHz, CDCl 3 ) δ 9.88 (s, 1H), 8.87 (s, 1H), 8.69 (d, J = 9.1 Hz, 1H), 8.22 (d, J = 8.5 Hz, 1H), 3.94 (s, 3H), 1.55 (s, 9H). 13 C NMR (101 MHz, CDCl 3 ) δ 165.00, 151.86, 139.62, 136.33, 135.29, 127.91, 123.76, 120.31, 82.77, 52.67, 28.29. MS (ESI) m/z 295.1 [M-H] - . methyl 3-amino-4-((tert-butoxycarbonyl)amino)benzoate (1.33): A flask containing a stirred solution of 1.32 (1.590 g, 5.367 mmol) in 1:1 EtOAc/MeOH (50 mL) was purged several times with N 2 and charged with Pd/C (5 wt%, 0.318 g). The flask was back- filled with N 2 and then several times with hydrogen gas. The resulting mixture was allowed to stir at room temperature under a H 2 atmosphere for 13 hours. The reaction mixture was subsequently filtered over a pad of Celite, and the filter cake was washed with copious amounts of EtOAc and MeOH. The filtrate was concentrated under reduced pressure to yield 1.33 as a white solid (1.294 g, 91% yield). 1 H NMR (400 MHz, CDCl 3 ) δ 7.70 – 7.45 (m, 3H), 6.59 (s, 1H), 3.88 (s, 3H), 1.52 (s, 9H). 13 C NMR (101 MHz, CDCl 3 ) δ 166.99, 153.17, 137.34, 130.96, 126.46, 122.35, 121.91, 119.58, 52.14, 28.44. MS (ESI) m/z 265.1 [M-H] - . 58 methyl 2-(4-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)phenyl)-1H- benzo[d]imidazole-5-carboxylate (1.34): To a stirring solution of 1.5a (0.500 g, 2.031 mmol) and DIPEA (0.707 mL, 4.061 mmol) in DMF (25 mL) was added HATU (0.772 g, 2.031 mmol). After stirring for 10 minutes, the solution was charged with 1.33 (0.595 g, 2.234 mmol), and was stirred at room temperature for an additional 14 hours. The reaction mixture was poured into saturated NH 4 Cl (50 mL) and extracted with EtOAc (4 x 30 mL). The organic layers were combined, dried over Na 2 SO 4 and concentrated in vacuo onto Celite. The residue was purified by silica gel flash chromatography (80% EtOAc/hexanes) to yield methyl 4-((tert-butoxycarbonyl)amino)-3-(4-((2,4-dioxo-3,4- dihydropyrimidin-1(2H)-yl)methyl)benzamido)benzoate as an off-white foam (0.815 g, 81% yield). MS (ESI) m/z 493.2 [M-H] - . Glacial acetic acid (5 mL) was added to a flask containing methyl 4-((tert- butoxycarbonyl)amino)-3-(4-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)- yl)methyl)benzamido)benzoate (0.100 g, 0.202 mmol), and the solution was stirred at 100 o C for 14 hours. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The residue was triturated twice with EtOAc and dried under high vacuum to yield 1.34 as an off-white powder (0.020 g, 26% yield). 1 H NMR (400 MHz, DMSO-d 6 ) δ 13.28 (s, 1H), 11.36 (s, 1H), 8.18 (d, J = 8.1 Hz, 3H), 7.85 (d, J = 8.2 Hz, 1H), 7.82 (d, J = 7.9 Hz, 1H), 7.68 (s, 1H), 7.49 (d, J = 8.1 Hz, 2H), 5.64 (dd, J = 8.0, 1.8 Hz, 1H), 4.96 (s, 2H), 3.88 (s, 3H). MS (ESI) m/z 375.1 [M-H] - . MS (ESI) m/z 377.0 [M+H] + . 59 tert-butyl 4-(iodomethyl)piperidine-1-carboxylate (1.36): An oven-dried flask was charged with 1-Boc-4-hydroxymethylpiperidine (1.35, 1.000g, 4.645 mmol), imidazole (0.379g, 5.574 mmol), and triphenylphosphine (1.464g, 5.574 mmol), and was backfilled several times with argon. Dry THF (4 mL) was added, and the resulting colorless solution was cooled to 0 o C in an ice bath. A solution of iodine (1.415g, 5.574 mmol) in dry THF (6 mL) was added dropwise over 5 minutes. After a few minutes, the resulting brown mixture was allowed to warm to room temperature. After stirring at room temperature for 3 hours, the reaction mixture was diluted with 10% EtOAc/hexanes, and the precipitate was filtered over a pad of Celite. The filter cake was washed with copious amounts of 20% EtOAc/hexanes, and the filtrate was concentrated in vacuo onto Celite. The residue was purified by silica gel flash column chromatography (5-10% EtOAc/hexanes) to yield 1.36 as a colorless oil (1.301g, 86% yield). 1 H NMR (400 MHz, CDCl 3 ) δ 4.14 – 4.07 (m, 2H), 3.09 (d, J = 6.2 Hz, 2H), 2.71 – 2.64 (m, 2H), 1.82 (d, J = 13.1 Hz, 1H), 1.45 (s, 9H), 1.18 – 1.08 (m, 2H). 13 C NMR (100 MHz, CDCl 3 ) δ 154.80, 79.65, 43.70, 38.80, 32.72, 28.58, 13.62. 1-(piperidin-4-ylmethyl)pyrimidine-2,4(1H,3H)-dione (1.37): To a solution of 1.27 (0.300g, 1.388 mmol) in DMF (8 mL) was added Cs 2 CO 3 (0.839g, 2.775 mmol). After stirring for 20 minutes, the resulting bright yellow mixture was charged with a solution of 1.36 (0.866g, 2.663 mmol) in DMF (3 mL) and was allowed to stir at room temperature for 43 hours. The reaction mixture was diluted with EtOAc and washed with 60 saturated NH 4 Cl. The aqueous layer was back-extracted 3x with EtOAc, and the combined organic layers were washed with brine, dried over Na 2 SO 4 , and concentrated in vacuo onto Celite. The residue was purified by automated column chromatography(66-70% EtOAc/hexanes) to yield tert-butyl 4-((3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)- yl)methyl)piperidine-1-carboxylate as a pale-yellow oil, which became a colorless foam upon vigorous drying (0.546g, 95% yield). 1 H NMR (400 MHz, CDCl 3 ) δ 7.93 – 7.91 (m, 2H), 7.66 (t, J = 7.5 Hz, 1H), 7.50 (t, J = 7.6 Hz, 2H), 7.19 (d, J = 8.0 Hz, 1H), 5.80 (d, J = 7.9 Hz, 1H), 4.16 – 4.12 (m, 2H), 3.63 (d, J = 7.1 Hz, 2H), 2.72 – 2.65 (m, 2H), 2.00 – 1.92 (m, 1H), 1.68 – 1.64 (m, 2H), 1.45 (s, 9H), 1.25 – 1.13 (m, 2H). 13 C NMR (100 MHz, CDCl 3 ) δ 168.80, 162.40, 154.77, 150.04, 144.59, 135.27, 131.59, 130.53, 129.33, 102.09, 79.82, 54.89, 43.41, 35.90, 29.63, 28.56. To a stirred solution of tert-butyl 4-((3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)- yl)methyl)piperidine-1-carboxylate (0.539g, 1.307 mmol) in CH 2 Cl 2 (5 mL) was added TFA (5 mL), and the solution was allowed to stir at room temperature for 23 hours. The reaction mixture was concentrated under reduced pressure. The residue was neutralized with saturated NaHCO 3 , concentrated in vacuo onto Celite, and purified by automated column chromatography (20% MeOH/CH 2 Cl 2 ). The relevant fractions were concentrated under reduced pressure to afford 1.37 as an orange oil (0.153g, 56% yield). 1 H NMR (400 MHz, CD 3 OD) δ 7.57 (d, J = 7.8 Hz, 1H), 5.67 (d, J = 7.8 Hz, 1H), 3.71 (d, J = 7.2 Hz, 2H), 3.41 (d, J = 13.2 Hz, 2H), 3.00 – 2.93 (m, 2H), 2.16 – 2.07 (m, 1H), 1.92 – 1.88 (m, 2H), 1.54 – 1.44 (m, 2H). 13 C NMR (100 MHz, CD 3 OD) δ 166.57, 153.02, 147.33, 102.39, 53.62, 44.70, 34.85, 27.30. 1-((1-(1H-benzo[d]imidazol-2-yl)piperidin-4-yl)methyl)pyrimidine-2,4(1H,3H)-dione (1.38): A microwave tube containing a solution of 1.33 (0.040g, 0.191 mmol) and 2- chlorobenzimidazole (0.022 g, 0.143 mmol) in DMF (1 mL) was charged with DIPEA (8 µL), 61 and the mixture was stirred at 135 o C for 9.5 hours. The reaction mixture was cooled to room temperature and concentrated in vacuo onto Celite. The residue was purified by automated column chromatography (6% MeOH/CH 2 Cl 2 ) to yield 1.38 as a colorless gum (0.005g, 33% yield). 1 H NMR (400 MHz, CD 3 OD) δ 7.57 (d, J = 7.8 Hz, 1H), 7.24 – 7.21 (m, 2H), 7.03 – 6.96 (m, 2H), 5.66 (d, J = 7.8 Hz, 1H), 4.15 – 4.06 (m, 2H), 3.68 (d, J = 7.3 Hz, 2H), 3.09 – 2.98 (m, 2H), 2.12 – 2.04 (m, 1H), 1.77 (d, J = 11.5 Hz, 2H), 1.45 – 1.34 (m, 2H). MS (ESI) m/z 326.2 [M+H] + . MS (ESI) m/z 324.1 [M-H] - . dimethyl thiophene-2,5-dicarboxylate (1.40) Thiophene-2,5-dicarboxylic acid (1.39, 6.886 g, 40 mmol) was charged to an oven-dried flask and purged with argon. Anhydrous methanol (40 mL) and concentrated sulfuric acid (1 mL) were added, and the resulting white slurry was stirred at refluxing temperature for 21 hours. After cooling to room temperature, the mixture was cooled in an ice bath and filtered. The recovered solid was washed with methanol to yield 1.40 as a white powder (6.873 g, 86% yield). 1 H NMR (400 MHz, DMSO-d 6 ) δ 7.82 (s, 2H), 3.86 (s, 6H). 13 C NMR (101 MHz, DMSO-d 6 ) δ 161.22, 138.09, 133.75, 52.81. methyl 5-(hydroxymethyl)thiophene-2-carboxylate (1.41) An ice-cold suspension of 1.38 (1.00 g, 5 mmol) in anhydrous THF (14 mL) was charged dropwise with LiBH 4 (2.0 M in THF, 1.40 mL), and the mixture was stirred at room temperature for 4 hours. The resulting solution was concentrated under reduced pressure, treated with water 62 (40 mL) and extracted with EtOAc (1 x 75 mL, 1 x 50 mL). The organic layers were combined, washed with brine (10 mL), dried over Na 2 SO 4 and concentrated in vacuo onto Celite. The residue was purified by automated column chromatography (30% EtOAc/hexanes) to yield 1.41 as a colorless oil (0.390 g, 45% yield). 1 H NMR (400 MHz, CDCl 3 ) δ 7.66 (d, J = 3.7 Hz, 1H), 6.97 (d, J = 3.8 Hz, 1H), 4.84 (d, J = 0.9 Hz, 2H), 3.87 (s, 3H). 13 C NMR (101 MHz, CDCl 3 ) δ 162.85, 151.95, 133.68, 132.85, 125.44, 60.34, 52.32. methyl 5-(bromomethyl)thiophene-2-carboxylate (1.42) To an oven-dried flask containing a stirred solution of imidazole (0.228 g, 3.345 mmol) and PPh 3 (0.879 g, 3.345 mmol) in anhydrous CH 2 Cl 2 (9 mL) was added bromine (0.17 mL, 3.345 mmol). After stirring at room temperature for 5 minutes, the solution was charged dropwise with a solution of 1.41 (0.384 g, 2.230 mmol) in anhydrous CH 2 Cl 2 (3 mL). The resulting orange suspension was stirred at room temperature under an argon atmosphere for 1 hour, after which time TLC indicated completion of the reaction. The reaction mixture was directly concentrated in vacuo onto Celite, and the residue was purified by automated column chromatography (5-11% EtOAc/hexanes). The relevant fractions were combined and concentrated under reduced pressure to yield 1.42 as a colorless oil (0.437g, 83% yield). 1 H NMR (400 MHz, CDCl 3 ) δ 7.63 (d, J = 3.8 Hz, 1H), 7.09 (d, J = 3.8 Hz, 1H), 4.67 (s, 2H), 3.88 (s, 3H). 13 C NMR (101 MHz, CDCl 3 ) δ 162.42, 147.53, 134.36, 133.55, 128.55, 52.45, 25.59. 63 methyl 5-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)thiophene-2-carboxylate (1.43): Cesium carbonate (1.109 g, 3.667 mmol) was added to a flask containing a stirred solution of uracil (1.26, 0.411 g, 3.667 mmol) in DMSO (10 mL). After stirring for 15 minutes, the suspension was charged with a solution of 1.42 (0.431 g, 1.833 mmol) in DMSO (2 mL) and was stirred at room temperature for 1 hour. The reaction mixture was subsequently charged with H 2 O (20 mL) and extracted with EtOAc (3 x 30 mL). The organic layers were combined, washed with brine (20 mL), dried over Na 2 SO 4 and concentrated in vacuo onto Celite. The residue was purified by automated column chromatography (70-80% EtOAc/hexanes) to yield 1.43 a white solid (0.197 g, 40% yield). 1 H NMR (400 MHz, DMSO-d 6 ) δ 11.40 (s, 1H), 7.81 (d, J = 7.9 Hz, 1H), 7.69 (d, J = 3.8 Hz, 1H), 7.20 (d, J = 3.7 Hz, 1H), 5.62 (d, J = 7.9 Hz, 1H), 5.07 (s, 2H), 3.80 (s, 3H). 13 C NMR (101 MHz, DMSO-d 6 ) δ 163.51, 161.62, 150.75, 146.58, 144.88, 133.53, 132.55, 128.18, 101.76, 52.25, 45.50. methyl 5-((5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)thiophene-2- carboxylate (1.44) To a stirred solution of thymine (6.070 g, 48.133 mmol) in DMSO (300 mL) was added Cs 2 CO 3 (14.552 g, 48.133 mmol) in one portion, and the mixture was stirred at room temperature for 25 minutes to give a thick white slurry. A solution of methyl 5-(bromomethyl)thiophene-2- carboxylate (2.829 g, 12.033 mmol) in DMSO (30 mL) was added dropwise over a period of 1.5 hours, and the resulting red-orange mixture was stirred at room temperature for 37 hours. The reaction mixture was poured into cold saturated NH 4 Cl (300 mL) and extracted with EtOAc (4 x 64 150 mL). The organic layers were combined, washed with brine (100 mL), dried over Na 2 SO 4 and concentrated in vacuo onto Celite. The residue was purified by automated column chromatography (80% EtOAc/hexanes), and the relevant fractions were combined and concentrated under reduced pressure to yield 1.44 a light-orange solid (1.195 g, 35% yield). 1 H NMR (400 MHz, DMSO-d 6 ) δ 11.40 (s, 1H), 8.08 – 7.45 (m, 2H), 7.19 (d, J = 3.8 Hz, 1H), 5.03 (s, 2H), 3.79 (s, 3H), 1.75 (s, 3H). 13 C NMR (101 MHz, DMSO-d 6 ) δ 164.13, 161.64, 150.73, 146.78, 140.51, 133.52, 132.51, 128.10, 109.47, 52.24, 45.34, 11.96. 5-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)thiophene-2-carboxylic acid (1.45) To a stirred solution of 1.43 (0.850 g, 3.192 mmol) in absolute EtOH (100 mL) was added 2N aq. NaOH (4.79 mL, 9.577 mmol) and H 2 O (10 mL), and the resulting solution was stirred at room temperature for 4.5 hours, after which time only partial hydrolysis was observed. Approximately 20 mL of EtOH was removed by rotary evaporation, and additional portions of 2N aq. NaOH (4.79 mL) and H 2 O (10 mL) were added. After stirring for an additional 2 hours, consumption of the starting material was observed. The reaction mixture was concentrated under reduced pressure to remove the remaining ethanol, and the resulting aqueous solution was acidified to pH 1 using 2N aq. HCl. The resulting precipitate was collected by vacuum filtration, washed with cold H 2 O and Et 2 O and dried to yield 1.45 as a white solid (0.659 g, 82% yield). 1 H NMR (400 MHz, DMSO-d 6 ) δ 13.09 (brs, 1H), 11.39 (s, 1H), 7.80 (d, J = 7.9 Hz, 1H), 7.60 (d, J = 3.7 Hz, 1H), 7.16 (d, J = 3.8 Hz, 1H), 5.62 (d, J = 7.8 Hz, 1H), 5.06 (s, 2H). 13 C NMR (101 MHz, DMSO-d 6 ) δ 163.54, 162.64, 150.75, 145.92, 144.92, 134.54, 133.01, 128.05, 101.73, 45.52. 65 5-((5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)thiophene-2-carboxylic acid (1.46) A flask containing a stirred slurry of 1.44 (1.184 g, 4.224 mmol) in 1:1 MeOH/H 2 O (50 mL) was charged dropwise with 2N aq. NaOH (16.9 mL, 33.792 mmol), and the resulting solution was stirred at room temperature for 3 days. The reaction mixture was subsequently concentrated under reduced pressure to remove methanol, and the remaining aqueous solution was acidified to pH 1 with concentrated HCl and extracted with EtOAc (4 x 20 mL). The organic layers were combined, dried over Na 2 SO 4 and concentrated under reduced pressure to yield 1.46 as an off- white solid (1.106 g, 98% yield). 1 H NMR (400 MHz, DMSO-d 6 ) δ 11.39 (s, 1H), 7.67 (s, 1H), 7.59 (d, J = 3.1 Hz, 1H), 7.16 (d, J = 3.6 Hz, 1H), 5.01 (s, 2H), 1.75 (s, 3H). 13 C NMR (101 MHz, DMSO-d 6 ) δ 164.15, 162.66, 150.73, 146.11, 140.55, 134.52, 132.99, 127.97, 109.43, 45.36, 11.97. 1-((5-(1H-benzo[d]imidazol-2-yl)thiophen-2-yl)methyl)pyrimidine-2,4(1H,3H)-dione (1.47a) To a stirred solution of 1.45 (0.100 g, 0.396 mmol) and DIPEA (0.14 mL, 0.793 mmol) in DMF (3 mL) was added HATU (0.151 g, 0.396 mmol). After stirring at room temperature for 10 minutes, the solution was charged with tert-butyl (2-aminophenyl)carbamate (0.091 g, 0.436 mmol) and was allowed to stir at room temperature for an additional 17 hours. The reaction mixture was charged with brine (20 mL) and extracted with EtOAc (4 x 5 mL). The organic layers were combined, dried over Na 2 SO 4 and concentrated in vacuo onto Celite. The residue was purified by automated column chromatography (2-3% MeOH/CH 2 Cl 2 ) to yield tert-butyl (2- 66 (5-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)thiophene-2- carboxamido)phenyl)carbamate as a light-orange paste (0.200 g) with residual DMF. A solution of tert-butyl (2-(5-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)thiophene-2- carboxamido)phenyl)carbamate (0.170 g, 0.384 mmol) was dissolved in glacial acetic acid (10 mL) and stirred at 100 o C for 13 hours, after which time LCMS indicated an incomplete reaction. The reaction mixture was charged with five drops of concentrated HCl and stirred for an additional 8 hours at 100 o C, resulting in consumption of the starting material. The reaction mixture was allowed to cool to room temperature and concentrated under reduced pressure to afford a light-brown solid, which was triturated with EtOAc and dried in a vacuum oven at 75 o C to yield 1.47a as a light-brown powder (0.113 g, 88% yield over two steps). 1 H NMR (400 MHz, DMSO-d 6 ) δ 11.46 (s, 1H), 8.73 (s, 1H), 8.16 (d, J = 3.7 Hz, 1H), 7.89 (d, J = 7.8 Hz, 1H), 7.70 (dd, J = 6.0, 3.1 Hz, 2H), 7.44 (dd, J = 6.0, 3.1 Hz, 2H), 7.38 (d, J = 3.7 Hz, 1H), 5.65 (d, J = 7.4 Hz, 1H), 5.17 (s, 2H). 13 C NMR (101 MHz, DMSO-d 6 ) δ 163.55, 150.84, 145.61, 144.92, 144.28, 133.54, 131.64, 128.90, 124.95, 113.99, 101.82, 45.50. MS (ESI) m/z 323.1 [M-H] - . MS (ESI) m/z 325.0 [M+H] + . 1-((5-(5-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)thiophen-2-yl)methyl)pyrimidine- 2,4(1H,3H)-dione (1.47b) To a stirred solution of 1.45 (0.250 g, 0.991 mmol) and DIPEA (0.31 mL, 1.802 mmol) in DMF (4 mL) was added HATU (0.377 g, 0.991 mmol). After stirring at room temperature for 10 minutes, the solution was charged with 3,4-diaminobenzotrifluoride (0.159 g, 0.901 mmol) and allowed to stir at room temperature for an additional 14.5 hours. The reaction mixture was treated with saturated NaHCO 3 (10 mL) and extracted with EtOAc (4 x 5 mL). The organic layers were combined, washed with brine (5 mL), dried over Na 2 SO 4 and concentrated in vacuo 67 onto Celite. The residue was purified by automated column chromatography (EtOAc) to yield N-(2-amino-4-(trifluoromethyl)phenyl)-5-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)- yl)methyl)thiophene-2-carboxamide (0.360g, 97% yield). MS (ESI) m/z 409.0 [M-H] - ; MS (ESI) m/z 411.0 [M+H] + . Glacial acetic acid (10 mL) was added to a flask containing N-(2-amino-4- (trifluoromethyl)phenyl)-5-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)thiophene-2- carboxamide (0.360 g, 0.877 mmol), and the resulting orange solution was stirred at 100 o C for 2 days. The reaction mixture was allowed to cool to room temperature and concentrated under reduced pressure. The residue was treated with water and the mixture was basified to pH 12 with 2N aq. NaOH. The resulting precipitate was extracted with EtOAc (2 x 30 mL), and the combined organic layers were concentrated in vacuo onto Celite. The residue was purified by automated column chromatography (2-4% MeOH/CH 2 Cl 2 ) to yield 1.47b an off-white solid (0.146 g, 42% yield). 1 H NMR (400 MHz, DMSO-d 6 ) δ 11.45 (s, 1H), 7.88 (s, 1H), 7.84 (d, J = 7.9 Hz, 1H), 7.76 (d, J = 3.7 Hz, 1H), 7.72 (d, J = 8.4 Hz, 1H), 7.50 (d, J = 8.1 Hz, 0H), 7.24 (d, J = 3.7 Hz, 1H), 5.64 (d, J = 7.9 Hz, 1H), 5.10 (s, 1H). 13 C NMR (101 MHz, DMSO-d 6 ) δ 163.60, 150.82, 149.31, 144.97, 142.42, 133.03, 128.41, 127.46, 126.37, 123.25, 122.93, 122.61, 118.97, 109.57, 101.74, 45.47. 1-((5-(5-(trifluoromethoxy)-1H-benzo[d]imidazol-2-yl)thiophen-2-yl)methyl)pyrimidine- 2,4(1H,3H)-dione (1.47c) To a stirred solution of 1.45 (0.279 g, 1.105 mmol) and DIPEA (0.35 mL, 2.009 mmol) in DMF (3 mL) was added HATU (0.420 g, 1.105 mmol). After stirring at room temperature for 10 minutes, the solution was charged with a solution of 4-(trifluoromethoxy)benzene-1,2-diamine (0.193 g, 1.004 mmol) in DMF (2 mL) and allowed to stir at room temperature for an additional 13 hours. The reaction mixture was diluted with EtOAc (5 mL) and washed with saturated NaHCO 3 (10 mL). The aqueous layer was extracted with additional EtOAc (3 x 5 mL), and the organic layers were combined, washed with brine (5 mL), and concentrated in vacuo onto Celite. The residue was purified by automated column chromatography (80% EtOAc/hexanes) to yield 68 N-(2-amino-4-(trifluoromethoxy)phenyl)-5-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)- yl)methyl)thiophene-2-carboxamide as a brown solid (0.147 g, 34% yield). Glacial acetic acid (10 mL) was charged to a flask containing N-(2-amino-4- (trifluoromethoxy)phenyl)-5-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)thiophene-2- carboxamide (0.140 g, 0.328 mmol), and the solution was stirred at 100 o C for 22 hours. The reaction mixture was allowed to cool to room temperature and was concentrated under reduced pressure. The residue was neutralized with water and 2N aq. NaOH and was subsequently sonicated. The resulting precipitate was collected by vacuum filtration, washed with water and dried to yield 1.47c as a light-brown solid (0.117 g, 87% yield). 1 H NMR (400 MHz, DMSO-d 6 ) δ 13.23 (s, 1H), 11.41 (s, 1H), 7.84 (d, J = 7.9 Hz, 1H), 7.72 (d, J = 3.7 Hz, 1H), 7.61 (d, J = 8.7 Hz, 1H), 7.53 (s, 1H), 7.23 (d, J = 3.7 Hz, 1H), 7.18 (d, J = 8.1 Hz, 1H), 5.64 (d, J = 7.9 Hz, 1H), 5.09 (s, 2H). 13 C NMR (101 MHz, DMSO-d 6 ) δ 163.56, 150.79, 144.94, 142.01, 133.21, 128.31, 127.00, 101.70, 45.43. MS (ESI) m/z 409.1 [M+H] + . MS (ESI) m/z 407.1 [M-H] - . 1-((5-(1H-benzo[d]imidazol-2-yl)thiophen-2-yl)methyl)-5-methylpyrimidine-2,4(1H,3H)- dione (1.48a): To a stirred solution of 1.46 (0.300 g, 1.127 mmol) and DIPEA (0.39 mL, 2.253 mmol) in DMF (5 mL) was added HATU (0.428 g, 1.127 mmol), and the mixture was stirred for 10 minutes at room temperature. The solution was subsequently charged with o-phenylenediamine (0.122 g, 1.127 mmol) and was stirred at room temperature for 24 hours. The reaction mixture was charged with sat. NaHCO 3 (10 mL), resulting in precipitation. The aqueous suspension was washed with EtOAc (4 x 5 mL) and filtered. The recovered solid was washed with H 2 O and EtOAc and dried to yield N-(2-aminophenyl)-5-((5-methyl-2,4-dioxo-3,4-dihydropyrimidin- 69 1(2H)-yl)methyl)thiophene-2-carboxamide as an off-white solid (0.199 g, 49% yield). 1 H NMR (400 MHz, DMSO-d 6 ) δ 11.34 (s, 1H), 9.67 (s, 1H), 7.83 (d, J = 3.8 Hz, 1H), 7.67 (s, 1H), 7.17 (d, J = 3.7 Hz, 1H), 7.09 (d, J = 7.8 Hz, 1H), 6.97 (t, J = 7.6 Hz, 1H), 6.76 (d, J = 8.0 Hz, 1H), 6.58 (t, J = 7.6 Hz, 1H), 5.02 (s, 2H), 4.89 (s, 2H), 1.76 (s, 3H). 13 C NMR (101 MHz, DMSO- d 6 ) δ 159.73, 158.96, 150.76, 144.14, 143.37, 140.51, 139.87, 128.80, 127.76, 126.92, 126.75, 116.19, 116.03, 109.37, 45.28, 11.99. N-(2-aminophenyl)-5-((5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)thiophene-2- carboxamide (0.196 g, 0.550 mmol) was dissolved in glacial acetic acid (10 mL) and stirred at 115 o C for 13 hours. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The residue was treated with water, sonicated, filtered and dried to yield 1.48a as a tan solid (0.103 g, 55% yield). 1 H NMR (400 MHz, DMSO-d 6 ) δ 12.93 (s, 1H), 11.41 (s, 1H), 7.70 (s, 1H), 7.67 (d, J = 3.8 Hz, 1H), 7.57 (s, 1H), 7.49 (s, 1H), 7.19 (m, 3H), 5.05 (s, 2H), 1.77 (s, 3H). 13 C NMR (101 MHz, DMSO-d 6 ) δ 164.19, 150.77, 146.69, 141.54, 140.60, 133.87, 128.12, 126.27, 122.69, 121.80, 118.56, 111.18, 109.38, 45.27, 11.99. MS (ESI) m/z 339.0 [M+H] + . 5-methyl-1-((5-(5-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)thiophen-2- yl)methyl)pyrimidine-2,4(1H,3H)-dione (1.48b) HATU (0.428 g, 1.127 mmol) was charged to a vial containing a stirred solution of 1.46 (0.300 g, 1.127 mmol) and DIPEA (0.39 mL) in DMF (5 mL), and the mixture was stirred at room temperature for 10 minutes. The mixture was subsequently charged with 3,4- diaminobenzotrifluoride (0.198 g, 1.127 mmol) in one portion and stirred for 19 hours at room temperature. The reaction mixture was partitioned between EtOAc (5 mL) and sat. NaHCO 3 (10 mL). The aqueous layer was back-extracted with EtOAc (3 x 5 mL), and the combined organic layers were washed with brine (5 mL) and concentrated in vacuo onto Celite. The residue was purified by automated column chromatography (87-90% EtOAc/hexanes) to yield N-(2-amino-4- 70 (trifluoromethyl)phenyl)-5-((5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)- yl)methyl)thiophene-2-carboxamide an off-white solid (0.408g). Glacial acetic acid (10 mL) was added to a flask containing N-(2-amino-4- (trifluoromethyl)phenyl)-5-((5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)- yl)methyl)thiophene-2-carboxamide (0.401 g, 0.945 mmol), and the mixture was stirred at 100 o C for 18 hours. The reaction mixture was allowed to cool to room temperature and was concentrated under reduced pressure. The residue was treated with water (10 mL), sonicated, filtered and dried to yield 1.48b a light-brown solid (0.322 g, 71% yield over two steps) 1 H NMR (400 MHz, DMSO-d 6 ) δ 11.41 (s, 1H), 7.88 (s, 1H), 7.83 – 7.63 (m, 3H), 7.51 (d, J = 8.5 Hz, 1H), 7.24 (d, J = 3.7 Hz, 1H), 5.06 (s, 2H), 1.77 (s, 3H). 13 C NMR (101 MHz, DMSO-d 6 ) δ 164.18, 150.78, 149.23, 149.10, 142.67, 140.59, 132.86, 128.29, 127.47, 126.37, 122.68, 118.97, 109.42, 45.28, 11.99. MS (ESI) m/z 407.0 [M+H] + . 5-methyl-1-((5-(6-(trifluoromethoxy)-1H-benzo[d]imidazol-2-yl)thiophen-2- yl)methyl)pyrimidine-2,4(1H,3H)-dione (1.48c) HATU (0.428 g, 1.127 mmol) was charged to a vial containing a stirred solution of 1.46 (0.300 g, 1.127 mmol) and DIPEA (0.39 mL) in DMF (4 mL), and the mixture was stirred at room temperature for 10 minutes. The mixture was subsequently charged with a solution of 4- (trifluoromethoxy)benzene-1,2-diamine (0.216 g, 1.127 mmol) in DMF (1 mL) and stirred at room temperature for an additional 8.5 hours. The reaction mixture was subsequently partitioned between EtOAc (5 mL) and sat. NaHCO 3 (10 mL), and the aqueous layer was back-extracted with EtOAc (3 x 5 mL). The combined organic layers were washed with brine (5 mL) and concentrated in vacuo onto Celite. The residue was purified by automated column chromatography (77-80% EtOAc/hexanes) to yield N-(2-amino-4-(trifluoromethoxy)phenyl)-5- ((5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)thiophene-2-carboxamide as a brown solid (0.193 g). 71 Glacial acetic acid (10 mL) was added to a flask containing N-(2-amino-4- (trifluoromethoxy)phenyl)-5-((5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)- yl)methyl)thiophene-2-carboxamide (0.174 g, 0.395 mmol), and the mixture was stirred at 115 o C for 27 hours. The reaction mixture was concentrated under reduced pressure, and the residue was treated with water and sonicated for 2 minutes. The resulting precipitate was recovered by vacuum filtration, washed with water and dried to yield 1.48c as a tan powder (0.130 g. 30% yield over two steps). 1 H NMR (400 MHz, DMSO-d 6 ) δ 11.41 (s, 1H), 7.71 (d, J = 4.5 Hz, 2H), 7.62 (d, J = 8.7 Hz, 1H), 7.54 (s, 1H), 7.21 (dd, J = 16.4, 6.4 Hz, 2H), 5.06 (s, 2H), 1.77 (s, 3H). 13 C NMR (101 MHz, DMSO-d 6 ) δ 164.19, 150.78, 148.71, 143.82, 142.42, 140.59, 132.89, 128.26, 127.19, 121.60, 119.07, 116.06, 109.42, 45.29, 11.99. MS (ESI) m/z 423.1 [M+H] + . 1.4.3 Biological Screening Cell lines and culturing Human colorectal carcinoma cell line HCT-116 (ATCC® CCL-247™) and human cervical cell line HeLa (ATCC® CCL-2™) were obtained from ATCC (Manassas, VA). Cells were propogated in either Dulbeccco’s modified Eagle’s medium (Cellgro, Herndon, VA) or folate- free RPMI-1640 (Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin (Gemini Bio-Products, West Sacramento, CA). All cell lines were grown in a humidified incubator at 37 o C and a 5% CO 2 atmosphere. MTT Viability Assay HCT-116 cells (5000 cells/well) were seeded in 96-well plates in culture medium (50 µL/well) and incubated for 24 hours in a humidified atmosphere of 37 o C and a 5% CO 2 . Compounds were diluted with culture medium to various concentrations and fed to the culture. Cells were then incubated for 72 hours and were subsequently treated with MTT (10 µL/well, 5 mg/mL in phosphate buffered saline) and incubated for an additional 4 hours. Lysis buffer (10% SDS/0.1% HCl in deionized water, 100 µL/well) was added to each well, and the cells were incubated at room temperature for 17 hours in darkness. 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Chem. 2010, 53, 7902-7917. 75 (41) Rautio, J.; Kumpulainen, H.; Heimbach, T.; Oliyai, R.; Oh, D.; Järvinen, T.; Savolainen, J. Nat. Rev. Drug Discov. 2008, 7, 255-270. (42) Saito, K.; Nagashima, H.; Noguchi, K.; Yoshisue, K.; Yokogawa, T.; Matsushima, E.; Tahara, T.; Takagi, S. Cancer Chemother. Pharmacol. 2014, 73, 577-583. 76 Chapter 2: Chemical Investigation of the Covalent Interaction Between Bortezomib (BZM) and Epigallocatachin Gallate (EGCG) 2.1. Introduction Bortezomib (BZM) is a 26S proteosome inhibitor approved for the treatment of multiple myeloma and mantle cell lymphoma. It is sold commercially as Velcade™ and has achieved blockbuster drug status with global sales of over $1 billlion 1 . As the first FDA-approved boron- containing drug, BZM sparked renewed interest in the development of boron-containing drugs for a wide array of diseases ranging from cancer to anti-bacterial treatment. The boronic acid moiety of BZM is crucial for its activity, enabling the compound to bind to a catalytic threonine residue in the active site of the 26S proteasome. In recent years, there has been increasing evidence of potential drug-drug interactions between BZM and naturally-occurring polyphenols, resulting in the amelioration of antitumor activity. In 2009, Golden and co-workers demonstrated that epigallocatechin gallate (EGCG), a polyphenolic compound naturally found in green tea and related supplements, negated the antiproliferative effects of BZM in the multiple myeloma cell line RPMI8226 and in the glioblastoma cell line LN229 in a concentration-dependent manner 2 . This finding was of great interest, since green tea is the fourth most commonly used nutritional supplement 3 and has long been touted for its chemopreventative properties in against a variety of cancers 4 . Furthermore, EGCG belongs to a class of dietary polyphenols, which make up the largest fraction of antioxidant in our diet 5-6 . Using 1 H and 13 C NMR experiments, it was shown that BZM indeed forms a complex with EGCG. Therefore, it was hypothesized that a covalent interaction between BZM and EGCG was responsible for the inactivation of BZM across multiple cell lines. Additionally, it was speculated that the covalent interaction was occurring at the boronic acid group of BZM. However, due to the myriad of functional groups of EGCG, the binding mechanism and kinetics were still unknown. We sought to investigate and characterize the interaction between BZM and EGCG using various chemical and biological methods. Using my familiarity with MTT viability assays, I sought to phenotypically screen a variety of EGCG fragments and other polyphenols in RPMI8226 and LN229 with the goal of providing insight to the nature of the BZM-EGCG interaction. By coupling the phenotypic screening data with spectroscopic techniques, it was proposed that BZM-EGCG binding could be thoroughly characterized, which in turn could 77 provide a method for screening drug-drug interactions with BZM and other boronic acid- containing compounds. 2.2. Biological Screening of Drug-Drug Interactions of Bortezomib 2.2.1. EGCG and Related Polyphenolic Compounds Building off of the work by Golden and co-workers, we sought to evaluate the drug-drug interactions of bortezomib in vitro using an MTT viability assay. We sought to extend the study of the antitumor activity of BZM in combination with a variety of structurally similar and relevant polyphenols in RPMI8226 and LN229 cells. The antitumor tumor activity was evaluated using concentration escalation (0-40 µM) of the polyphenols in the absence or presence of 20 nM using an MTT assay. At 20 nM BZM, no proliferation of RPMI8226 was detected, while 80% inhibition of LN229 was observed after 48 hours of treatment. As reported previously, increasing concentrations of EGCG was able to mitigated the cytotoxic effects of BZM, with up to 82% RPMI8226 (Figure 2.1) and 100% LN229 cell viability observed when 40 µM EGCG was employed. Figure 2.1. (A) Influence of EGCG on RPMI8226 cell viability. Percentage cell viability of RPMI/8226 cells was determined via MTT assay after 48 hours of treatment with 0-40 µM EGCG in the presence of 20 nM BZM. (B) Cell viability of LN229 cells. Percentage cell viability of RPMI/8226 cells was determined via MTT assay after 48 hours of treatment with 0-40 µM EGCG in the presence or absence of 20 nM BZM. Based on the polyphenolic structure of EGCG, our first initiative was to examine the various fragments of EGCG to determine their influence on the efficacy of BZM (Figure 2.2). Isopropyl 3,4,5-trihydroxybenzoate (ISO) and pyrogallol (PYRO) exhibited similar inhibition of BZM-mediated antitumor activity in both cell lines at 40 µM (Figure 2.3A-B). Thus, the greater 78 binding affinity of BZM-pyrogallol is reflected by the RPMI8226 cytoprotective effect of pyrogallol relative to its isopropyl ester counterpart. Although the opposite effect was observed in glioblastoma cells, this can be attributed in part to the greater inherent cytotoxicity of pyrogallol in LN229 relative to RPMI8226 (see Experimental). As expected, the 1,3-diol resorcinol (RES) had no effect on viability in either cell line using the same parameters. Figure 2.2. Structural breakdown of EGCG fragments. Figure 2.3. Influence of EGCG and fragments on (A) RPMI8226 and (B) LN229 cell viability. Percentage cell viability was determined via MTT assay after 48 hours of treatment with 0-40 µM phenols in the presence of 20 nM BZM. 2.2.2. Other Polyphenols In order to explore the underlying effects of BZM-phenol binding, we widened our screen to include other relevant and electronically-variable phenols. We were particularly interested in the effect of the number and position of phenol groups on cell viability. The polyphenols PYRO, catechol (CAT), phenol (PHE), and resveratrol (RSV) were compared (Figure 2.4). 79 Figure 2.4. (A) Structures of phenols differing in the number and position of hydroxyl groups; (B) Influence of phenol number and position on RPMI8226 and (C) LN229 cell viability. Percentage cell viability was determined via MTT assay after 48 hours of treatment with 0-40 µM phenols in the presence of 20 nM BZM. The polyphenols PYRO and CAT, which contain two or more vicinal hydroxyl groups, exhibited the greatest effect on cell viability in both cell lines when combined with 20 nM BZM. On the other hand, RSV and PHE, which contain either a single or non-vicinal hydroxyl group, showed no effect on cell viability in either cell line when combined with BZM. This validates earlier theory that a polyphenol must contain two or more adjacent hydroxyl groups (i.e. a catechol group) in order to efficiently produce the boronic ester and therefore enhance cell viability. These data corroborate earlier evidence of the interactions between boronic acids and diols 7-9 , particularly with polyphenols 10-11 . Next, we investigated the effects of electron-withdrawing substitutents on catechol reactivity. It has been documented that BZM reacts with the nucleophilic oxygen lone pair of Thr1Oγ of the 20S proteasome in accordance to the Lewis hard-soft-acid-base principle. Taking this into consideration an expected outcome was that a stronger electron-withdrawing group on a polyphenol would afford decreased production of the boronic ester. It was hypothesized that the inductive effects on the aromatic ring would decrease the ability of the hydroxyl group to donate electrons decreasing its potency as a lewis base species due to the electron withdrawing substituent. This was partially supported by MTT assay screening, as 4-nitrocatechol (NCAT) 80 and 3,4-dihydroxybenzoic acid (DHBA) had a lower effect on the antitumor activity of BZM in both cell lines than CAT or EGCG (Figure 2.5). However, while it was expected that the stronger electron-withdrawing group of NCAT would result in significantly less cell viability rescue than DHBA, this was not consistently observed in cellular assays. This may be attributed to the intrinsic cytotoxic effects of some catechols, which stems from oxidation and formation of quinones that bind to protein thiols in DNA 12-13 . Additionally, these effects have been observed in cell culture 14 , which reveals a fundamental limitation of these assays to evaluate such interactions. Thus, although electron-withdrawing groups generally appeared to hamper complexation of catechols to BZM, a clear trend could not be concluded to MTT assays alone. Figure 2.5. (A) Structures of catechols differing in electronic substituents; (B) Influence of phenol number and position on RPMI8226 and (C) LN229 cell viability. Percentage cell viability was determined via MTT assay after 48 hours of treatment with 0-40 µM phenols in the presence of 20 nM BZM. After compiling the data, it was apparent that only phenols containing vicinal diols were effective in rescuing cell viability in the presence of 20 nM BZM (Figure 2.6). As expected, PYRO and ISO were very effective in negating the antitumor effects of BZM in a dose- dependent manner. However, discerning the degree of BZM inactivation between the two phenols was inconclusive by phenotypic screening, as the data were not consistent through both 81 cell lines. Since electronegative/inductive effects were also inconsistent across cell lines, it was impossible to establish a clear trend based on phenotypic screening alone. Figure 2.6. Compiled cell viability assay data of polyphenol influence on the antitumor activity of BZM in (A) RPMI8226 and (B) LN229 cell lines. 2.3. NMR Experiments The findings of the MTT screen were validated by 19 F and 11 B NMR experiments, which were primarily carried out by my colleague, Steve Glynn. These experiments verified that the amelioration of BZM activity in cancer cells was due to a complex formed between the boronic acid moiety of BZM and the vicinal diol groups of EGCG (Figure 2.7). Although the work by Golden and co-workers provided evidence of a covalent EGCG-BZM interaction through the use of 1 H and 13 C NMR, the complex could not be fully characterized, which led us to speculate which functional group(s) were responsible for this interaction. Figure 2.7. Schematic of the reversible covalent interaction between BZM and EGCG. 82 Using 11 B NMR, it was shown that BZM complexes with EGCG to form a boronate adduct (Figure 2.8A). Increasing the concentration of EGCG resulted in the emergence of a peak corresponding to the boronate adduct with concomitant disappearance of the peak corresponding to the BZM boronic acid. When this screen was applied to the EGCG fragments, a similar effect was observed (Figure 2.8B). While PYRO and ISO effectively formed the boronic ester reaching 88% and 85%, respectively, at 4:1 ratio to BZM, RES showed a decreased capacity with the majority of the BZM remaining as a free boronic acid. This effect reflected the results observed in the MTT assay screen, as only phenols with vicinal diol groups had a pronounced effect on the rescue of cell viability. The trends in polyphenol electronegativity were also consistent with the MTT assay data, as electron-withdrawing groups promoted complexation with the BZM boronic acid due to the increased acidity of the polyphenol (see Experimental). Figure 2.8. (A) 11 B NMR screening of BZM and EGCG in molar ratios of 1:0, 1:0.5, 1:1, 1:2, and 1:4 BZM:EGCG. The free boronic acid of BZM has a broad peak at 28ppm this peak is shifted to a boronic ester peak at 19.62ppm. (B) 11 B NMR screening of BZM and EGCG fragments in molar ratios of 1:0, 1:0.5, 1:1, 1:2, and 1:4 BZM:phenol. 83 2.4. Conclusion We have fully characterized the chemical interaction of BZM with the green tea extract EGCG. Through the validation and utilization of 11 B NMR, we were able to quantify the interaction and determine its equilibrium coefficient. The application of MTT viability assays in combination with these spectroscopic techniques allowed for the development of trends that could be applied to the entire class of polyphenols including dietary and biologically relevant polyphenols such as resveratrol. These results of NMR screening were validated in biological experiments highlighting the inactivation of bortezomibs cell death properties in glioblastoma (LN229) and multiple myeloma (RPMI8226) cell lines thus indicating the formation of an adduct. The combination of these trends and the evaluation of the BZM/EGCG interaction allowed us to design a model outlining the chemical properties of EGCG that allow it to be a potent inhibitor of BZM apoptosis activity. Furthermore, these methods may provide a simple and effective way to screen drug-drug and food-drug interactions of boron-containing drugs. 2.5. Experimental 2.5.1. Biological Screening Human multiple myeloma cell line RPMI/8226 (ATCC® CCL-155™) and glioblastoma cell line LN229 (ATCC® CRL-2611™) were obtained from ATCC (Manassas, VA). MM cells were propagated in RPMI-1640 (Cellgro, Herndon, VA). Glioblastoma cells were propagated in Dulbecco’s modified Eagle’s medium (Cellgro, Herndon, VA). All cells were grown with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin (Gemini Bio-Products, West Sacramento, CA) in a humidified incubator at 37 o C and a 5% CO 2 atmosphere. MTT assays were performed in 96-well plates with the use of 2.5 x 10 4 cells per well for multiple myeloma cells and 3.0 x 10 3 cells per well for glioblastoma cells. Cells in culture medium (50 µL/well) were seeded in 96-well plates and incubated for 24 hours in a humidified atmosphere of 37 o C and a 5% CO 2 . Compounds were diluted with culture medium to various concentrations and fed to the culture. Cells were then incubated for 48 hours and were subsequently treated with MTT (10 µL/well, 5 mg/mL in phosphate buffered saline) and incubated for an additional 4 hours. Lysis buffer (10% SDS/0.1% HCl in deionized water, 100 84 µL/well) was added to each well, and the cells were incubated at room temperature for 17 hours in darkness. The absorbance at 490 nm was measured by a microplate reader. In individual experiments, each condition was set up in triplicate. EGCG 0 10 20 30 40 0 20 40 60 80 100 EGCG + 20 nM BZM EGCG Alone Concentration ( M) % Viability (+)-Catechin 0 10 20 30 40 0 20 40 60 80 100 CATN + 20 nM BZM CATN Alone Concentration ( M) % Viability Isopropyl 3,4,5-trihydroxybenzoate 0 10 20 30 40 0 20 40 60 80 100 ISO + 20 nM BZM ISO Alone Concentration ( M) % Viability Pyrogallol 0 10 20 30 40 0 20 40 60 80 100 PYR + 20 nM BZM PYR Alone Concentration ( M) % Viability 4-Nitrocatechol 0 10 20 30 40 0 20 40 60 80 100 NCAT + 20 nM BZM NCAT Alone Concentration ( M) % Viability Catechol 0 10 20 30 40 0 20 40 60 80 100 CAT + 20 nM BZM CAT Alone Concentration ( M) % Viability 4-Fluorocatechol 0 10 20 30 40 0 20 40 60 80 100 FCAT + 20 nM BZM FCAT Alone Concentration ( M) % Viability 3,4-Dihydroxybenzoic acid 0 10 20 30 40 0 20 40 60 80 100 DHBA + 20 nM BZM DHBA Alone Concentration ( M) % Viability Phenol 0 10 20 30 40 0 20 40 60 80 100 PHE + 20 nM BZM PHE Alone Concentration ( M) % Viability Resorcinol 0 10 20 30 40 0 20 40 60 80 100 RES + 20 nM BZM RES Alone Concentration ( M) % Viability Resveratrol 0 10 20 30 40 0 20 40 60 80 100 RSV + 20 nM BZM RSV Alone Concentration ( M) % Viability A B C D E F G H I J K Fig.2.9. Cell viability of RPMI 8226 cells in the presence of phenols (0-40 µM). Cells were seeded in a 96-well plate at 2.5 x 10 4 cells/well and treated with 1,2-diphenols (0-40 µM). Following 48-hour incubation at 37 o C, cell viability was determined by MTT assay. Compounds were tested alone (red) and with 20 nM bortezomib (BZM, blue). A = EGCG; B = (+)-catechin (CATN); C = isopropyl 3,4,5- trihydroxybenzoate (ISO); D = pyrogallol (PYRO); E =4-nitrocatechol (NCAT); F = catechol (CAT); G = 4-fluorocatechol (FCAT); H = 3,4-dihydroxybenzoic acid (DHBA); I = phenol (PHE); J = resorcinol (RES); K = resveratrol (RSV). 85 EGCG 0 10 20 30 40 0 20 40 60 80 100 EGCG Alone EGCG + 20 nM BZM Concentration ( M) % Viability (+)-Catechin 0 10 20 30 40 0 20 40 60 80 100 120 CATN + 20 nM BZM CATN Alone Concentration ( M) % Viability Isopropyl 3,4,5-trihydroxybenzoate 0 10 20 30 40 0 20 40 60 80 100 ISO Alone ISO + 20 nM BZM Concentration ( M) % Viability Pyrogallol 0 10 20 30 40 0 20 40 60 80 100 PYR Alone PYR + 20 nM BZM Concentration ( M) % Viability 4-Nitrocatechol 0 10 20 30 40 0 20 40 60 80 100 NCAT Alone NCAT + 20 nM BZM Concentration ( M) % Viability Catechol 0 10 20 30 40 0 20 40 60 80 100 CAT Alone CAT + 20 nM BZM Concentration ( M) % Viability 4-Fluorocatechol 0 10 20 30 40 0 20 40 60 80 100 120 FCAT + 20 nM BZM FCAT Alone Concentration ( M) % Viability 3,4-Dihydroxybenzoic acid 0 10 20 30 40 0 20 40 60 80 100 DHBA Alone DHBA + 20 nM BZM Concentration ( M) % Viability Phenol 0 10 20 30 40 0 20 40 60 80 100 PHE Alone PHE + 20 nM BZM Concentration ( M) % Viability Resorcinol 0 10 20 30 40 0 20 40 60 80 100 RES Alone RES + 20 nM BZM Concentration ( M) % Viability Resveratrol 0 10 20 30 40 0 20 40 60 80 100 120 RSV + 20 nM BZM RSV Alone Concentration ( M) % Viability A B C D E F G H I J K Fig.2.10. Cell viability of LN229 cells in the presence of phenols (0-40 µM). Cells were seeded in a 96- well plate at 3.0 x 10 3 cells/well and treated with 1,2-diphenols (0-40 µM). Following 48-hour incubation at 37 o C, cell viability was determined by MTT assay. Compounds were tested alone (red) and with 20 nM bortezomib (BZM, blue). A = EGCG; B = (+)-catechin (CATN); C = isopropyl 3,4,5- trihydroxybenzoate (ISO); D = pyrogallol (PYRO); E =4-nitrocatechol (NCAT); F = catechol (CAT); G = 86 4-fluorocatechol (FCAT); H = 3,4-dihydroxybenzoic acid (DHBA); I = phenol (PHE); J = resorcinol (RES); K = resveratrol (RSV). Fig.2.11. Cell viability of (A) RPMI8226 and (B) LN229 cells in the presence of EGCG (top) and ascorbic acid (bottom). Compounds were tested alone (left half of plate) and with 20 nM bortezomib (right half of plate) at escalating concentrations of phenols (0-40 µM, left to right). Fig.2.12. Cell viability of (A) RPMI8226 and (B) LN229 cells in the presence of NCAT (top) and ISO (bottom). Compounds were tested alone (left half of plate) and with 20 nM bortezomib (right half of plate) at escalating concentrations of phenols (0-40 µM, left to right). 87 Fig.2.13. Cell viability of (A) RPMI8226 and (B) LN229 cells in the presence of PYRO (top) and CAT (bottom). Compounds were tested alone (left half of plate) and with 20 nM bortezomib (right half of plate) at escalating concentrations of phenols (0-40 µM, left to right). Fig.2.14. Cell viability of (A) RPMI8226 and (B) LN229 cells in the presence of RES (top) and PHE (bottom). Compounds were tested alone (left half of plate) and with 20 nM bortezomib (right half of plate) at escalating concentrations of phenols (0-40 µM, left to right). 88 Fig.2.15. Cell viability of (A) RPMI8226 and (B) LN229 cells in the presence of DHBA (top). Compounds were tested alone (left half of plate) and with 20 nM bortezomib (right half of plate) at escalating concentrations of phenols (0-40 µM, left to right). 2.5.2. NMR Screening and Compound Synthesis Materials All commercially available products were used without further purification steps. Bortezomib was purchased from LC laboratories as a free base. Epigallocatechin gallate, pyrogallol, gallic acid monohydrate, resorcinol, 1,2-dihydroxybenzene, phenol, 3,4- dihydroxybenzoic acid, 4-nitrocatechol, resveratrol, and (-)-epicatechin were all purchased from Sigma-Aldrich. 4-fluorocatechol was purchased from Combi-Blocks. NMR Spectroscopy NMR spectra were recorded on a Varian 500 2-channel NMR spectrometer with an Agilent OneNMR probe ( 1 H- 19 F/ 31 P- 15 N 5 mm PFG OneNMR probe) and a Varian 400 2- channe; NMR spectrometer with an Agilent OneNMR probe ( 1 H- 19 F/ 31 P- 15 N 5 mm PFG OneNMR probe). Chemical shifts (δ) are expressed in parts per million (ppm). All the experiments were performed at 25°C. All samples were diluted to 1mL in an 80% CD 3 CN, 20% D 2 O solvent system. For all samples 2.80mg of bortezomib (7.3×10 -3 mmols) and adequate quantity of phenol was added by mass and spun for 15 minutes at room temperature. All spectra were processed with MestReNova v8.1.2 software. 89 Isopropyl 3,4,5-trihydroxybenzoate (ISO) Gallic acid monohydrate (0.3 grams, 1.8 mmols), 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide (0.341 grams, 2.2 mmols) (0.022 grams, 0.18 mmols) 4- dimethylaminopyridine in 5 mL of dry CH 2 Cl 2 stirred at 0°C. Isopropyl alcohol (0.132 grams, 2.2 mmols) was added to the mixture and stirred for 30 minutes at 0°C then allowed to reach room temperature and stirred for another 48 hours. The reaction mixture was quenched with sodium bicarbonate and extracted with ethyl acetate to yield pure Isopropyl 3,4,5- trihydroxybenzoate as a tan powder (61%). 90 2.6 Chapter 2 References (1) Côté, A.; Keating, B. Value in Health, 2012, 15, 1185–1191. (2) Golden, E. B.; Lam, P. Y.; Kardosh, A.; Gaffney, K.J.; Cadenas, E.; Louie, S.G.; Petasis, N.A.; Chen, T.C.; Schönthal, A.H. Blood, 2009, 113, 5927–5937. (3) Sarma, D.N.; Barret, M.L.; Chavez, M.L.; Gardiner, P.; Ko, R; Mahady, G.B.; Marles, R.J.; Pellicore, L.S.; Giancaspro, G.I.; Low Dog, T. Drug Saf. 2008, 31, 469-484. (4) Zaveri, N.T. Life Sci. 2006, 78, 2073-2080. (5) Seeram, N.P.; Henning, S.M.; Niu, Y.; Lee, R.; Schueller, H.S.; Hever, D. J. Agric. Food Chem. 2006, 54, 1599-1603. (6) Scalbert, A.; Johnson, I.T.; Saltmarsh, M. Am. J. Clin. Nutr. 2005, 81, 215S-7S. (7) Mulla, H.R.; Agard, M.J.; Basu, A. Bioorg. Med. Chem. Lett. 2004, 14, 25-27. (8) Yang, W.; Gao, X.; Want, B. Med. Res. Rev. 2003, 23, 346-368. (9) Springsteen, G.; Wang, B. Tetrahedron 2002, 58, 5291-5300. (10) Kin, T.Y.; Park, J.; Oh, B.; Min, H.J.; Jeong, T.-S.; Lee, J.H.; Suh, C.; Cheong, J.-W.; Kim, H.J.; Yoon, S.-S.; Park, S.B.; Lee, D.S.; the Korean Multiple Myeloma Working Party (KKMMWP). Brit. J. Haematol. 2009, 146, 270-281. (11) Yan, J.; Springsteen, G.; Deeter, S.; Wang, B. Tetrahedron 2004, 60, 11205-11209. (12) Passi, S.; Picardo, M.; Nazzaro-Porro, M. Biochem. J. 1987, 245, 537-542. (13) Wright, J.S.; Shadnia, H. Chem. Res. Toxicol. 2008, 21, 1426-1431. (14) Nemeikaitė-Čėnienė, A.; Imbrasaitė, A.; Sergedienė, E.; Čėnas, N. Arch. Biochem. Biophys. 2005, 441, 182-190. 91 Chapter 3: Efforts towards the synthesis of a novel class of bi-functional peptide nucleic acids (PNAs) 3.1 Introduction Telomerase is a reverse transcriptase ribonucleoprotein that synthesizes specific DNA sequence repeats at the 3’ end of DNA strands at the end of eukaryotic chromosomes, known as telomeric DNA 1-4 . During DNA replication, DNA polymerases are unable to fully replicate the end of a duplex DNA, and as a result, telomeres shorten with each replication cycle, which ultimately leads to cell arrest or death 5-8 . By continuously adding sequence repeats to the chromosome ends of terminal DNA, telomerase allows cells to divide beyond their programmed limit. In 1985, Carol Greider and Elizabeth Blackburn discovered telomerase 1 as a therapeutic target for cancer, for which they were awarded the Nobel Prize in Physiology or Medicine in 2009. Telomerase is expressed in over 90% of human cancer cells 9-10 , while absent in most somatic tissues, providing researchers a major therapeutic advantage in comparison with other cancer targets. Figure 3.1. 11-12 Mechanism of telomerase activity. Our goal was to design and synthesize an inhibitor that directly inhibits telomerase activity. In order to do so, we needed an understanding of the mechanism of action by which 92 telomerase is activated. The telomerase enzyme consists of an RNA template, a substrate anchor site and a catalytic binding/alignment site (Figure 3.1) 11-12 . The telomere end selectively binds to the complementary template via base-pairing interaction, followed by elongation of six nucleotides (GGTTAG) to the telomere, which is then shifted back by six nucleotides via translocation to allow for another catalytic cycle. Recently, Geron Corporation has developed a first-in-class telomerase inhibitor (GRN163) that works by binding directly to the RNA template, preventing binding of the telomere and subsequent elongation 13-14 . GRN163 (imetelstat) is a lipid-conjugated 13-mer that contains a thiophosphoramidate backbone that achieves selective binding by incorporating nucleobases complementary to the RNA template sequence 15 . During the time that GRN163 was entering clinical trials, we developed a scheme that expands on the template antagonist strategy. We adopted a strategy based on peptide nucleic acids (PNAs) due to the growing amount of interest in PNA molecules as antisense agents 16 . Like DNA, PNA is a nucleic acid analog that participates in Watson-Crick base pairing, but replaces the deoxyribose backbone with a peptide chain 17 . With this in mind, we sought to take the unique approach of designing and synthesizing a double-sided, or diPNA analog. This molecule would have the distinct advantage of binding to the telomerase template as well as the telomere end (Figure 3.2). Since the sequence of the template and telomere are known, we can fine tune the sequence of our PNA to complement both sides simultaneously. We hypothesized that dual binding could increase selectivity and potency, as well as providing other mechanistic insights. In the end, we conceived a design of a PNA oligomer with 11 double-sided units (11- mer) to overlap with the template while attempting to capture additional interactions with the telomere. Figure 3.2. Design strategy of diPNA 11-mer. The double-sided PNA selectively binds to both the telomere end of DNA in addition to the telomerase template, increasing potency and selectivity. 93 With a general design in hand, our next goal was to design a retrosynthetic scheme for the diPNA 11-mer (Figure 3.3). Due to the inherent polarity of the molecule and the high difficulty for purification, it was decided that the oligomer be synthesized from double-sided PNA monomers using solid phase peptide synthesis (SPPS). Each PNA monomer could be synthesized convergently from pre-functionalized nucleic acid bases and an amino acid backbone via amide coupling. The backbone would therefore need to be able to accommodate two amide couplings selectively in a stepwise manner while avoiding unwanted side reactions from the nucleobases. In order to accomplish this, an exhaustive, orthogonal protecting group strategy was required. Figure 3.3. Retrosynthetic strategy of diPNA 11-mer. A convergent synthesis of a PNA backbone and pre-functionalized nucleobases allows for the regioselective synthesis of diPNA monomers, which could then be elongated using solid phase peptide synthesis (SPPS). 3.2 Synthesis of Nucleic Acid Base Derivatives With a convergent synthetic strategy in mind, we sought out to synthesize functionalized nucleic acid bases (adenine, cytosine, uracil, and guanine), which could then be sequentially joined with the complementary backbone piece via amide coupling to construct various monomers. Thus, each nucleobase was appended with a methylene carbonyl group and, with the 94 exception of uracil, a Boc protecting group. To our delight, Robert Hudson and co-workers 18 had done extensive work on PNA oligomers using a similar nucleobase functionalization strategy for adenine, cytosine and guanine. This enabled rapid synthesis and scale-up of these nucleobases. Our foray into the nucleobase synthesis commenced with the synthesis of N-Boc- protected cytosine 3.3 (Scheme 3.1). Commercially available cytosine was selectively alkylated at the N-1 position with ethyl bromoacetate to yield 3.1 in moderate to good yield. This reaction takes advantage of the lower pKa of the secondary amine (4.4), which is more susceptible to deprotonation by sodium hydride and thus undergoes alkylation more readily than the primary amine (pKa 12.2) 19 . Due to the low reactivity of the primary amine, Boc-protection using di- tert-butyl dicarbonate was unsuccessful. To circumvent this issue, 1,1’-carbonyldiimidazole (CDI) was used to generate a reactive carbonyl intermediate, which subsequently underwent nucleophilic attack with tert-butanol to furnish the Boc-protected cytosine ester 3.2. Finally, saponification using 2N aqueous sodium hydroxide quickly yielded 3.3 in near-quantitative yield. This route was particularly advantageous due to its amenability for multi-gram synthesis and the fact that no chromatography was required at any stage. Scheme 3.1. Synthesis of N-Boc-protected cytosine 3.3. Reagents and conditions: (a) 1. NaH, DMF, 0 0 C to room temperature, 2 h, 2. Ethyl bromoacetate, room temperature, 11 h; (b) 1. CDI, DMF, room temperature, 2 h, 2. t-BuOH, 65 0 C, 20 min; (c) 2N NaOH (aq.), dioxane/H 2 O, 0 0 C to room temperature, 20 min. This chemistry was then successfully applied to the synthesis of the Bis-N-Boc-protected adenine 3.6 (Scheme 3.2). Starting from cheaply available adenine, selective N-alkylation was carried out with ethyl bromoacetate in the presence of sodium hydride to yield 3.4, owing to the lower pKa (4.1) of the 9-H proton 19 . Unlike cytosine, the mild acidity of the adenine primary amine (pKa 9.80) facilitated Bis-N-Boc-protection using mild conditions to afford 3.5 in good 95 yield. Carboxylic acid 3.6 was then obtained using saponification conditions described for the synthesis of 3.3. With the exception of 3.5, all adenine compounds were obtained in multi-gram scale without the need for chromatography. Scheme 3.2. Synthesis of Bis-N-Boc-protected adenine 3.6. Reagents and conditions: (a) 1. NaH, DMF, room temperature, 2 h, 2. Ethyl bromoacetate, room temperature, 2 h; (b) Boc 2 O, DMAP, THF, room temperature, 14 h; (c) 2.5M NaOH (aq.), dioxane/H 2 O, 0 0 C to room temperature, 10 min. Due to the multiple reactive sites on guanine 20 , it was not used a starting material. Instead, commercially available 2-amino-6-chloropurine was employed for the synthesis of compound 3.9 (Scheme 3.3). By “protecting” the lactam group of guanine with a chloro group, the reactivity of the purine core could be manipulated easier. Due to the deactivation of the exocyclic amine by the purine ring, alkylation of 2-amino-6-chloropurine was selective at the 9- position to yield benzyl ester 3.7. In order to protect the exocyclic amine, triphosgene was employed as a carbonyl surrogate, followed by nucleophilic attack by tert-butanol to afford 3.8. Nucleophilic displacement of the chlorine atom of 3.8 by 3-hydroxypropionitrile in the presence of sodium hydride, followed by β-hydride elimination 21 , afforded guanine 3.9 as the final product. Unlike the other nucleobases, uracil did not require a protecting group, and the methylene carboxylic acid was readily synthesized in one step from chloroacetic acid using a known procedure 22 (Scheme 3.4). 96 Scheme 3.3. Synthesis of Boc-protected guanine 3.9. Reagents and conditions: (a) 1. K 2 CO 3 , DMF, 80- 85 0 C, 30 min, 2. Benzyl bromoacetate, 0 0 C, 3 h, then room temperature, 9.5 h; (b) 1. Triphosgene, THF, DIPEA, 0 0 C, 1.5 h, 2. t-BuOH, room temperature, 12 h; (c) 1. NaH, THF, -78 0 C, 2. 3- hydroxypropionitrile, THF, 0 0 C, 2.5 h, 3. 3.8, THF, room temperature, 2 h, 4. 2N NaOH (aq.), room temperature, 5 min. Scheme 3.4. Synthesis of uracil acetic acid 3.10 22 . Reagents and conditions: (a) 1. Chloroacetic acid, KOH, H 2 O, reflux, 1 h, 2. Concentrated HCl (l), room temperature, 15 h. 3.3 Synthesis of the PNA backbone Although construction of the nucleic acid base derivatives was relatively straightforward using established chemical transformations, the synthesis of the PNA backbone proved extremely challenging. In addition to selecting an amino acid backbone with the optimal size and orientation to accommodate attachment of two nucleobases per unit, the backbone required several reactive sites that could be accessed for nucleobase installation as well as chain elongation. Consequently, an orthogonal protecting group strategy was needed in order to selective append each group in a stepwise fashion. After much consideration and experimentation, L-asparagine was selected as a monomeric chiral amino acid feedstock (Figure 3.4). Using a series of synthetic steps, L-asparagine could be functionalized into a diamine intermediate that could accommodate the appendage of two nucleobase derivatives, followed by Fmoc-protection to allow for selective chain/resin installation and subsequent chain elongation 97 via solid-phase peptide synthesis (SPPS). In order to synthesize adequate quantities of the three target monomers for SPPS, a robust and scalable route was required for synthesis of the monomer backbone. Figure 3.4. Synthetic strategy for the synthesis of the PNA monomer backbone. With a building block strategy in hand, I set out to synthesize the PNA backbone (Scheme 3.5) with the help of my colleagues, Dr. Kalyan Nagulapalli and Dr. Rong Yang. Using cheap and commercially available L-asparagine ($0.77/g from Sigma-Aldrich), compound 3.12 was synthesized via simple tert-butyloxycarbonyl (Boc) protection. The primary amide was converted to an amine by a Hofmann rearrangement using phenyliodine diacetate (PIDA) to yield pure 3.13 in good yield 23 . Subsequent carbobenzyloxy (Cbz) protection was carried out in aqueous solution using commercially available Cbz-OSu to give 3.14. At this point, the first three steps of the synthesis could be carried out with inexpensive reagents in mild conditions (aqueous solvents, low temperatures) on decagram scale without the need for chromatography. Compound 3.14 was then converted to Weinreb amide 24 3.15 via amide coupling with N,O- dimethylhydroxylamine hydrochloride using N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC●HCl) as a catalyst. Because of the stability of the Weinreb amide and the Cbz group under acidic conditions, compound 3.16 was obtained readily upon treatment with trifluoroacetic acid (TFA). Due to the fact that nearly all of the functionalized nucleic acid bases were Boc- protected, coupled with the strategy of elongating the PNA chain after attaching said nucleic bases, we needed to incorporate yet another protecting group that was orthogonal to Boc, Cbz, and the Weinreb amide. After scouring the literature, we found the 2- (trimethylsilyl)ethoxycarbonyl (Teoc) protecting group to be a viable option; it has great stability in the presence of reducing agents and basic conditions, and can be cleaved in the presence of TBAF, which does not affect stability of the Boc-protected nucleobases. Although the surrogate 98 for the Teoc protecting group, Teoc-OSu (3.18), was not cheaply available, it could be synthesized in a two-step, one pot reaction 25 . Hence, 2-TMS-ethanol (3.17) was reacted with triphosgene to create a reactive carbonyl intermediate, and was subsequently treated with N- hydroxysuccinimide (HOSu) to yield 3.18 in multi-gram quantities. Unlike Teoc-Cl, Teoc-OSu could still retain its reactivity under aqueous conditions, which facilitated handling and use in our strategy. Consequently, 3.18 was reacted with primary amine 3.16 at room temperature under aqueous conditions to yield several grams of Teoc-protected 3.19. At this junction, Dr. Yang and Dr. Nagulapalli took the material forward to optimize reaction conditions for synthesis of diamine intermediate 3.21. Treatment of Weinreb amide 3.19 with lithium aluminum hydride (LiAlH 4 ) quickly furnished aldehyde 3.20, which, after purification, was subjected to reductive amination with glycine methyl ester hydrochloride to yield 3.21. Scheme 3.5. Synthesis of PNA backbone diamine intermediate 3.21. Reagents and conditions: (a) Boc anhydride, Na 2 CO 3 , dioxane/H 2 O (1:1), room temperature, 7 h; (b) PIDA, EtOAc/MeCN/H 2 O (2:2:1), 0 o C for 20 min, then room temperature for 16 h; (c) Cbz-OSu, 5% Na 2 CO 3 , MeCN, 0 o C to room 99 temperature, 15 h; (d) 1. EDC●HCl, N,O-dimethylhydroxylamine hydrochloride, NMM, -78 o C, 2. 3.14, CH 2 Cl 2 , -78 o C to room temperature, 14 h; (e) TFA, CH 2 Cl 2 , room temperature, 8.5 h; (f) 1. triphosgene, Et 3 N, CH 2 Cl 2 , -78 o C to 0 o C, 5 min, then room temperature for 1 h, 2. HOSu, MeCN, -78 o C to room temperature, 16 h; (g) 3.16, MeCN, 5% NaHCO 3 , room temperature, 11 h; (h) LiAlH 4 , THF, -78 o C to 5 o C, 10 min; (i) glycine methyl ester hydrochloride, NaBH(OAc) 3 , Et 3 N, CH 2 Cl 2 , -78 o C to room temperature, 1.5 h. 3.4 Monomer assembly After completing the synthesis of the backbone and nucleobases, we devised a strategy to combine them in a site-selective manner in order to synthesize the three monomeric units for PNA construction. At this point in the project, I was charged with the task of scaling up additional quantities of nucleobases and key intermediates for the PNA backbone, while Dr. Nagulapalli and Dr. Yang worked to find and optimize reaction conditions for assembly of the monomers. The general reaction scheme is outlined in Scheme 3.6. Initially, there were several problems with the scheme that required a great deal of troubleshooting. The secondary amine of the PNA backbone 3.21 showed low activity under standard coupling conditions. To overcome the inherently low reactivity, free acids 3.3 and 3.6 were activated with HOSu prior to treatment with 3.21 to generate 3.22 in good to excellent yield. Standard Cbz-deprotection via hydrogenation in the presence of Pd/C (5 wt%) afforded 3.23. The second nucleobase was then attached through HOSu-mediated amide coupling to yield 3.24. Methyl ester saponification was accomplished using either NaOH or LiOH to furnish free acid 3.25. The deprotection of the Teoc group using a solution of TBAF in THF was initially very slow; it was found that adjusting the pH to ~10 and elevating the reaction temperature (~50 o C) led to shorter reaction times and better yields of 3.26. Because 3.26 contained both a free primary amine and a free acid, there were great difficulties in its purification and isolation. To circumvent this issue, the pH of the reaction mixture containing 3.26 was adjusted to ~8.5 and immediately taken forward to the final step. Treatment with Fmoc-OSu (along with reaction monitoring by LC/MS) yielded monomers 3.27a-c, which could be purified using silica with a small particle size (<30 microns) along with acidified (0.2% acetic acid) DCM and MeOH. 100 Scheme 3.6. Synthesis of monomers 3.27a-c. 3.5 Solid Phase Peptide Synthesis (SPPS) of PNA Oligomers Once the laborious task of synthesizing di-functionalized PNA monomers was completed, the challenge of linking them strategically using solid phase peptide synthesis (SPPS) 26 immediately followed. The idea of using SPPS to synthesize the target 11-mer PNA arose due to the inherent polarity of the monomers, each of which difficult to purify. Our strategy was to use amide coupling conditions to attach the first monomer of the sequence, 3.27c (C-G), to a polyethylene glycol (PEG) based resin due to its inherent compatibility in polar solvents 27 . Because the reaction takes place on the surface of the resin, any unreacted starting materials and byproducts could then be easily discarded by filtration. Using the Fmoc strategy, the Fmoc group of the monomer could then be selectively deprotected under mild basic conditions 28 , followed by amide coupling to the second monomer. The coupling cycles were to be repeated until the eleven monomeric units were coupled. Initial efforts made use of the colorimetric Kaiser test 29 , which utilizes ninhydrin to detect the presence of primary amines following Fmoc-deprotection of a monomeric unit following the amide coupling step. Upon heating in the presence of ninhydrin, the primary amine-containing 101 resin beads will typically turn a bluish color due to the conversion of ninhydrin to an imino compound (Figure 3.5). Figure 3.5. General reaction mechanism of the Kaiser test. Fmoc-PAL-PEG-PS resin (PAL-PEG-PS = peptide amide linker – poly(ethylene glycol)- polystyrene) was selected as the SPPS resin due to the increased flexibility and solubility of the PEG/PS backbone chain as well as the chemical stability of the Fmoc-protected amine prior to use. Reactions were performed in a glass vessel fitted with a stopcock; a steady stream of bubbling nitrogen gas served to mix the resin with the reaction mixture (Figure 3.6). Solid phase synthesis of the 11-mer commenced with Fmoc-deprotection of the resin, which was complete in 15 minutes (positive Kaiser test) using a solution of 25% 4-methylpiperidine (4-MP) in DMF. After draining the reaction vessel and washing the beads thoroughly, the resin was immediately reacted with a preactivated solution of 3.27c, HBTU, HOBt, and N- methylmorpholine (NMM) in DMF (Figure 3.7). A subsequent negative Kaiser test suggested completion of the reaction. Amide coupling reactions were “capped” with acetic anhydride, which serves to acetylate any unreacted primary amines that were not detected by the Kaiser test. 102 Figure 3.6. Reaction setup for solid phase peptide synthesis of 11-mer PNA. (a) A 10-mL glass vessel containing the resin and reaction mixture was connected to a nitrogen inlet; (b) The reaction components are allowed to mix under bubbling nitrogen. Figure 3.7. Deprotection of Fmoc-PAL-PEG-PS resin, followed by attachment of C-G monomer (3.27c) via amide coupling to yield PNA monomer 3.28. After capping the first amide coupling reaction, 3.28 underwent Fmoc deprotection and subsequent amide coupling with A-A monomer 3.27a to yield dimer 3.29 (Figure 3.8). Both reactions appeared to proceed smoothly via Kaiser test analysis. However, after repeating the 103 coupling cycle with A-U monomer 3.27b, the subsequent Fmoc deprotection did not proceed to completion despite several attempts (Figure 3.9). Figure 3.8. 1. Capping of first coupling reaction with acetic anhydride; 2. Deprotection of Fmoc-3.28; 3. Amide coupling of 3.28 with A-A monomer 3.27a to yield Fmoc dimer 3.29. Figure 3.9. 1. Capping of second coupling reaction with acetic anhydride; 2. Deprotection of Fmoc- 3.29; 3. Amide coupling of 3.29 with A-U monomer 3.27b to yield Fmoc trimer 3.30; 4. Capping of third coupling reaction with acetic anhydride; 5. Unsuccessful deprotection of Fmoc-3.30. In order to investigate the problems with the Fmoc-deprotection of 3.30, the conditions underlying the previous steps were scrutinized, and as a result, several modifications were made 104 to the protocol. Initially, an excess of resin (relative to the Fmoc-protected monomer) was used in the first coupling step, which yielded large amounts of unreacted (and eventually capped) resin and a low yield, effectively diminishing the yields of the subsequent steps. Furthermore, short reaction times and choice of catalysts were known to have a dramatic effect on yield; the reaction time of the initial coupling step was increased, and the HBTU/HOBt catalyst system was replaced by the more reactive HATU. In order to save precious monomer building blocks, these conditions were first applied to the synthesis of a phenylalanine 5-mer using inexpensive Fmoc-Phe-OH, which proved successful. With these new conditions in hand, we set out to conduct further testing by homocoupling A-A monomer 3.27a, due to its availability and easier synthetic tractability. Although the Kaiser test had suggested successful completion of six consecutive coupling cycles, it could not be verified by mass spectrometry. Furthermore, after attempting to apply these conditions to the synthesis of the target 11-mer, the same problems arose with the deprotection of trimer 3.30. It was postulated that the synthetic problems may arise due to increasing aggregation of the monomeric units with the polymeric resin chain. As a result, a PNA spacer strategy was attempted wherein a linker was inserted between the resin and the C-G monomer of 3.28. It was predicted that the length and flexibility of the linker would increase coupling reaction efficiency while decreasing the probability of intramolecular aggregation. We sought to use a known PNA linker 30 that could be synthesized in four steps from commercially available reagents (Scheme 3.7). Scheme 3.7. Synthesis of PNA spacer 3.35 30 . Reagents and conditions: (a) BnBr, K 2 CO 3 , MeCN, 50 o C, 16 h; (b) 1. NaH, THF, 0 o C, 5 min, 2. Bromoacetic acid, reflux, 16 h; (c) H 2 , Pd/C, MeOH, room temperature, 24 h; (d) Fmoc-OSu, K 2 CO 3 , acetone, room temperature, 16 h. 105 Additionally, we sought to address the issue of evaluating the efficiency of each coupling reaction, as the Kaiser test proved unreliable towards our molecules. In order to quantify the efficiency of the coupling step, reactions were evaluated after the subsequent Fmoc deprotection step. We used ultraviolet-visible spectroscopy (UV-Vis) to quantify the amount of 4- methylpiperidine/dibenzofulvene adduct (λ max = 301 nm) formed after Fmoc deprotection 31-32 . By comparing the absorbance of the reaction mixture during each Fmoc deprotection, the coupling reaction yield can indirectly be quantified and evaluated. Thus, the absorbance value yielded from the initial resin Fmoc deprotection served as the positive control, or 100% yield (Figure 3.10). Figure 3.10. Yield quantification using UV-Vis analysis of the 4-MP/dibenzofulvene adduct formed after Fmoc deprotection. Applying this method to the linker-coupling step, it was found that the yield was roughly 18% (Figure 3.11). This was particularly puzzling since none of the PNA monomers had been added at this point, while at the same time the coupling reagents and conditions had seemingly been improved. Figure 3.11. Coupling of AEEA spacer 3.35 to deprotected PAL-PEG-PS resin. The reaction yield was analyzed by UV-Vis spectroscopy and compared with the absorbance value of the resin deprotection reaction. 106 After ruling out coupling agents and monomer-resin aggregation, our focus was turned to the PNA resin itself. The Fmoc-PAL-PEG-PS resin was replaced with another PEG-based resin, NovaPEG rink amide resin LL (Figure 3.12). In order to promote completion of coupling reactions, each coupling step was carried out twice. Interestingly, the absorbance (A = 1.79) of the linker-coupling step was higher than observed when carried out with the NovaPEG rink amide resin (Figure 3.13), suggesting that the combination of the resin and the repeated coupling cycle contribute to higher yield. Figure 3.12. Structures of Fmoc-PAL-PEG-PS (left) and NovaPEG rink amide resin LL (right). Figure 3.13. Coupling of AEEA spacer 3.35 to deprotected NovaPEG rink amide resin. The reaction yield was analyzed by UV-Vis spectroscopy and compared with the absorbance value of the resin deprotection reaction. Once the coupling of the AEEA spacer was confirmed by UV-Vis analysis, the product was taken forward to evaluate the effect of the resin/linker/conditions on PNA monomer reaction yield. When the NovaPEG resin-bound AEEA linker was coupled with A-A monomer 3.27a, UV-Vis analysis of the subsequent deprotection step revealed a 76.5% yield (3.36), a vast improvement over the PAL-PEG-PS resin (Figure 3.14). 107 Figure 3.14. Coupling of NovaPEG resin-spacer with A-A monomer 3.27a. Although the first PNA monomer coupling step proceeded with good yield, we observed a significant decrease in yield after just one additional coupling step. The resin-linker-(A-A) monomer 3.36 was coupled with A-U monomer 3.27b in a similar fashion to afford dimer 3.37, but yielded only a 55.5% yield (Figure 3.15). Furthermore, attempts to install a second A-U monomer onto the N-terminal chain of 3.37 were unsuccessful, and LC/MS analysis of a TFA- cleaved sample was inconclusive. Attempts to repeat this synthesis starting with C-G monomer (3.27c) in place of A-A was also unsuccessful, as the stock of AEEA linker had decomposed by this time. Figure 3.15. Coupling reaction of 3.36 with A-U monomer 3.27b. 108 With our stock of AEEA linker decomposed, we attempted to react NovaPEG rink amide resin directly with PNA monomers using coupling conditions that had been developed thus far. Direct coupling of the resin free amine with G-C monomer 3.27c resulted in a yield similar to that observed when the AEEA spacer was used (Figure 3.16). Subsequent coupling with a second unit of 3.27c gave the G-C dimer 3.39 in 74.1% yield (Figure 3.17), a significant improvement from the previous campaign. However, when a third coupling was attempted using A-U monomer 3.27b, trimer 3.40 was obtained in only 9.3% yield (Figure 3.18). Figure 3.16. Coupling of NovaPEG resin with C-G monomer 3.27c. 109 Figure 3.17. Coupling of 3.38 with C-G monomer 3.27c to give dimer 3.39. Figure 3.18. Coupling of 3.39 with A-U monomer 3.27b to give trimer 3.40. Despite the increased coupling yields through the first two steps, there appeared to be an inherent problem with the synthesis of the diPNA trimer. This may be traced back to our initial hypothesis that intramolecular aggregation 33 increases as more monomeric units are added to the 110 growing PNA chain. Although many attempts to resolve this issue have been futile, the conditions and methods developed thus far have provided an ample starting point for diPNA synthesis. 3.6 Conclusion We have demonstrated the design and synthesis of a novel class of diPNA monomers. These monomers were constructed from an L-asparagine-based peptide backbone and pre- functionalized nucleic acid bases, with the purpose of synthesizing a diPNA oligomer that can bind to both the RNA template of telomerase as well as the complementary telomere. By binding to both sites, it is postulated that these molecules will exert enhanced anticancer effects. While the final sequence of the diPNA synthesis using solid phase peptide synthesis has so far been unsuccessful, synthetic and analytical techniques developed herein have provided a strong basis for further development. 3.7 Experimental All reactions, unless noted otherwise, were conducted using commercially available solvents and reagents as received, without additional preparation or purification, in ordinary glassware. 1 H and 13 C spectra were recorded on Mercury 400, Varian 400-MR (400 MHz), Varian VNMRS- 500 (500 MHz) 2-channel, or Varian VNMRS-600 (600 MHz) 3-channel NMR spectrometers, using residual 1H or 13C signals of deuterated solvents as internal standards. Silica gel (60 Å, 40-63 μm; Sorbent Technologies) was used as a sorbent for flash column chromatography. Automated flash chromatography was performed on Isolera One flash purification system (Biotage), default fraction volume – 14 mL. Ethyl 2-(4-amino-2-oxopyrimidin-1(2H)-yl)acetate (1): An oven-dried 250 mL flask was charged with cytosine (5.000 g, 45.005 mmol) and purged several times with argon. Dry DMF (100 mL) was added, and the resulting slurry was cooled in an ice bath. Sodium hydride (60% 111 dispersion in mineral oil, 1.800 g, 45.005 mmol) was added in one portion, and the flask was quickly sealed, evacuated by vacuum, and back-filled several times with argon. The suspension was stirred at room temperature for 2 hours under argon atmosphere. Ethyl bromoacetate (4.99 mL, 45.005 mmol) was added dropwise over 2 hours. The resulting dark orange solution was allowed to stir for 11 hours at room temperature under an argon atmosphere. The reaction mixture was concentrated under reduced pressure to remove DMF. The residue was treated with H 2 O (100 mL) and filtered. The recovered solid was washed with copious amounts of water and suction-dried to yield 3.1 as an off-white/light-pink powder (3.719 g, 42% yield). 1 H NMR (400 MHz, DMSO-d 6 ) δ 7.54 (d, J = 7.2 Hz, 1H), 7.11 (d, J = 31.1 Hz, 2H), 5.67 (d, J = 7.2 Hz, 1H), 4.42 (s, 2H), 4.11 (q, J = 7.1 Hz, 2H), 1.19 (t, J = 7.1 Hz, 3H). 13 C NMR (101 MHz, DMSO-d 6 ) δ 168.73, 166.34, 155.70, 146.29, 93.47, 60.74, 49.92, 14.04. MS (ESI) m/z 198.0 [M+H] + ; 220.0 [M+Na] - . Ethyl 2-(4-((tert-butoxycarbonyl)amino)-2-oxopyrimidin-1(2H)-yl)acetate (3.2): An oven- dried 250 mL flask was charged with 3.1 (3.719 g, 18.860 mmol) and purged several times with argon. Dry DMF (63 mL) was added, and after stirring for a few minutes, the slurry was charged with 1,1’-carbonyldiimidazole (4.893 g, 30.176 mmol) in a glovebag. The mixture was allowed to stir at room temperature under an argon atmosphere for 2 hours. The mixture was then charged with t-BuOH (12.6 mL, 132.020 mmol) and the mixture was stirred at 65 0 C for 20 minutes. The reaction was quenched with methanol (2 mL), and the mixture was concentrated under reduced pressure. The resulting oily residue was co-evaporated with methanol and water, producing an off-white solid residue. This residue was treated with ethyl ether, filtered and washed with additional amounts of ethyl ether. Suction-drying afforded 3.2 as a white solid (3.992 g, 71% yield). 1 H NMR (400 MHz, DMSO-d 6 ) δ 10.36 (s, 1H), 8.00 (d, J = 7.4 Hz, 1H), 7.00 (d, J = 7.1 Hz, 1H), 4.59 (s, 2H), 4.14 (q, J = 7.1 Hz, 2H), 1.46 (s, 9H), 1.20 (t, J = 7.1 Hz, 3H). 13 C NMR (101 MHz, DMSO-d 6 ) δ 167.97, 163.55, 149.94, 135.11, 94.19, 81.00, 61.06, 50.52, 27.77, 14.01. 112 MS (ESI) m/z 298.0 [M+H] + ; 296.1 [M-H] - . 2-(4-((tert-butoxycarbonyl)amino)-2-oxopyrimidin-1(2H)-yl)acetic acid (3.3): A 500 mL flask containing a stirred solution of 3.2 (3.254 g, 10.945 mmol) in dioxane (140 mL) and water (42 mL) was cooled in an ice bath and charged dropwise with 2N NaOH (42.6 mL, 85.205 mmol) over 4 minutes. The ice bath was subsequently removed and the solution was allowed to stir at room temperature for 20 minutes. The reaction mixture was transferred to a separatory funnel containing 1M KHSO 4 (250 mL) and EtOAc (250 mL). The organic layer was collected and aqueous layer was extracted with EtOAc (4 x 200 mL). The organic layers were combined, dried over Na 2 SO 4 , and concentrated under reduced pressure to afford 3.3 as a shiny white solid (2.641 g, 90% yield). 1 H NMR (400 MHz, DMSO-d 6 ) δ 10.30 (s, 1H), 7.97 (d, J = 7.3 Hz, 1H), 6.96 (d, J = 7.3 Hz, 1H), 4.49 (s, 2H), 1.44 (s, 9H). 13 C NMR (101 MHz, DMSO-d 6 ) δ 169.38, 163.42, 155.07, 152.12, 150.03, 94.04, 80.96, 50.45, 27.78. MS (ESI) m/z 270.0 [M+H] + ; 268.1 [M-H] - . Ethyl 2-(6-amino-9H-purin-9-yl)acetate (3.4): An oven-dried 250 mL flask was charged with adenine (5.000 g, 37.001 mmol) and purged several times with argon. Dry DMF (75 mL) was added under an argon atmosphere, and the suspension was stirred briefly. Sodium hydride (60% dispersion in mineral oil, 1.687 g, 42.182 mmol) was added, and the flask was quickly resealed and purged several times with argon. The resulting thick white slurry was allowed to stir for 2 hours at room temperature under an argon atmosphere. Ethyl bromoacetate (8.21 mL, 74.003 mmol) was added dropwise under argon over a period of 3 hours, and the resulting brown suspension was allowed to stir at room temperature for an additional 2 hours. The reaction 113 mixture was thoroughly concentrated under reduced pressure to remove DMF, and the residual thick, dark-orange syrup was treated and mixed vigorously with H 2 O (50 mL), resulting in precipitation. The solid was collected by filtration, washed with copious amounts of H 2 O and dried to yield 3.4 as an off-white powder (5.241 g, 64% yield). 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.12 (d, J = 6.9 Hz, 2H), 7.26 (s, 2H), 5.06 (s, 2H), 4.16 (q, J = 7.1 Hz, 2H), 1.21 (t, J = 7.1 Hz, 3H). 13 C NMR (101 MHz, DMSO-d 6 ) δ 167.95, 155.95, 152.62, 149.69, 141.22, 118.23, 61.33, 43.90, 14.00. MS (ESI) m/z 222.1 [M+H] + . Ethyl [N 6 ,N 6 -Bis(tert-butoxycarbonyl)adenine-9-yl]acetate (3.5): A 250 mL flask containing a stirred suspension of 3.4 (2.000 g, 9.041 mmol) in THF (50 mL) was charged with 4- (dimethylamino)pyridine (3.313 g, 27.122 mmol) and di-tert-butyl dicarbonate (5.919 g, 27.122 mmol). The resulting brown suspension was allowed to stir at room temperature for 14 hours. The reaction mixture was subsequently concentrated in vacuo onto Celite. The residue was purified by silica gel flash column chromatography (1:1 EtOAc/hexanes) to yield 3.5 as a sticky yellow foam (2.005 g, 53% yield). 1 H NMR (400 MHz, CDCl 3 ) δ 8.86 (s, 1H), 8.16 (s, 1H), 5.05 (s, 2H), 4.27 (q, J = 7.1 Hz, 2H), 1.44 (s, 18H), 1.29 (t, J = 7.2 Hz, 3H). 13 C NMR (101 MHz, CDCl 3 ) δ 166.77, 153.51, 152.44, 150.61, 150.42, 145.06, 83.94, 62.65, 44.53, 27.92, 14.21. [N6,N6-Bis(tert-butoxycarbonyl)adenin-9-yl]acetic acid (3.6): A 250 mL flask containing an ice cold solution of 3.5 (3.940 g, 9.349 mmol) in dioxane (75 mL) and H 2 O (30 mL) was charged 114 dropwise with aqueous NaOH (2.5 M, 28.4 mL) over 2-3 minutes. The ice bath was removed and the solution was allowed to stir at room temperature for 10 minutes. The reaction mixture was poured into a separatory funnel containing aq. KHSO 4 (1M, 150 mL) and EtOAc (150 mL). The organic layer was collected, and the aqueous layer was extracted with additional EtOAc (3 x 100 mL). The organic layers were combined, dried over Na 2 SO 4 , and concentrated under reduced pressure to yield a yellow oil, which was co-evaporated twice with CH 2 Cl 2 /hexanes and dried under high vacuum to yield 3.6 as a yellow crunchy foam (3.701 g, 100% yield). 1 H NMR (400 MHz, CDCl 3 ) δ 8.88 (s, 1H), 8.39 (s, 1H), 5.09 (s, 2H), 1.42 (s, 18H). 13 C NMR (101 MHz, CDCl 3 ) δ 169.11, 153.35, 152.48, 150.26, 150.20, 146.23, 127.65, 84.34, 44.54, 27.91. Ethyl 2-(2-amino-6-chloro-9H-purin-9-yl)acetate (3.7): A two-necked 100 mL flask containing a suspension of 6-chloro-2-aminopurine (3.000 g, 17.692 mmol) in DMF (30 mL) was charged with K 2 CO 3 (3.668 g, 26.538 mmol) and the mixture was heated to 80-85 0 C for 30 minutes. The resulting yellow mixture was cooled to 0 0 C and charged dropwise with benzyl bromoacetate (3.08 mL, 19.461 mmol) over a period of 30 minutes. After the addition was complete, the mixture was stirred at 0 0 C for 3 hours and then at room temperature for 9.5 hours. The reaction mixture was filtered and washed with DMF (10 mL). The filtrate was poured into a vigorously stirred solution of 1N HCl (70 mL), which resulted in immediate precipitation and a mild exotherm. After stirring for 2 hours, the solid was collected by filtration, washed with H 2 O (2 x 50 mL) and dried to yield 3.7 as an off-white powder (4.575 g, 81% yield). 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.12 (s, 1H), 7.47 – 7.29 (m, 5H), 6.99 (s, 2H), 5.20 (s, 2H), 5.06 (s, 2H). 13 C NMR (101 MHz, DMSO-d 6 ) δ 167.59, 159.93, 154.24, 149.44, 143.42, 135.30, 128.49, 128.29, 128.08, 122.88, 66.75, 44.04. MS (ESI) m/z 319.9 [M+H] + . 115 2-(2-((tert-butoxycarbonyl)amino)-6-oxo-1H-purin-9(6H)-yl)acetic acid (3.9): An oven- dried 100 mL flask was charged with 3.7 (1.500 g, 4.722 mmol) and purged several times with argon. Dry THF (20 mL) was added under argon and the solution was cooled in an ice bath. Triphosgene (0.505 g, 1.700 mmol) was added, and the flask was quickly sealed and repurged with argon. The resulting yellow solution was stirred at 0 0 C for 1 hour, and was subsequently charged dropwise with N,N-diisopropylethylamine (1.81 mL, 10.389 mmol). After stirring for an additional 30 minutes at 0 0 C, t-BuOH (0.54 mL, 5.667 mmol) was added in one portion, and the resulting orange suspension was allowed to stir overnight at room temperature under an argon atmosphere for 12 hours. The reaction mixture was concentrated under reduced pressure, and the resulting orange oil was dissolved in CH 2 Cl 2 (50 mL), washed with 10% aq. citric acid (15 mL) and 5% aq. NaHCO 3 (10 mL), dried over Na 2 SO 4 , and concentrated under reduced pressure to yield benzyl 2-(2-((tert-butoxycarbonyl)amino)-6-chloro-9H-purin-9-yl)acetate (3.8) as an orange foam ([M-H] - = 416.1), which was carried to the next step without further purification. An oven-dried 100 mL flask was purged several times with argon and charged with dry THF (30 mL). Sodium hydride (60% dispersion in mineral oil, 0.944 g, 23.609 mmol) was added, and the mixture was purged again with argon and cooled to -78 0 C. The slurry was charged dropwise with 3-hydroxypropionitrile (1.61 mL, 23.609 mmol), and the mixture was stirred at 0 0 C for 2.5 hours. A solution of 3.5 in dry THF (6 mL) was added dropwise, and the resulting mixture was allowed to stir at room temperature under an argon atmosphere for 2 hours. The reaction mixture was charged with a few drops of 2N NaOH (to hydrolyze residual unreacted benzyl ester), stirred for a few minutes, and concentrated under reduced pressure. The residue was redissolved in H 2 O (30 mL) and acidified with 20% aq. citric acid. Attempts to recover the product via extraction with EtOAc were unsuccessful. The aqueous layer was charged with a small aliquot of EtOAc and acidified with concentrated HCl, resulting in precipitation. The organic layer was collected, concentrated, triturated with ethyl ether, and filtered to yield 3.9 as a white solid (0.333 g, 23% yield). 1 H NMR (400 MHz, DMSO-d 6 ) δ 11.39 (s, 1H), 11.12 (s, 1H), 7.91 (s, 1H), 4.85 (s, 2H), 1.48 (s, 9H). 13 C NMR (101 MHz, DMSO-d 6 ) δ 169.05, 155.06, 153.78, 149.37, 147.68, 140.06, 119.08, 82.49, 27.77. 116 MS (ESI) m/z 310.0 [M+H] + ; 308.1 [M-H] - . 2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetic acid (3.10): A 250 mL flask was charged with uracil (3.000 g, 26.767 mmol), potassium hydroxide (6.596 g, 117.559 mmol) and chloroacetic acid (4.513 g, 47.752 mmol). The flask was submerged in an ice bath, and water was added (100 mL), which resulted in a large exotherm. The resulting solution was heated at refluxing temperature for 1 hour. The reaction mixture was allowed to cool to room temperature and was acidified to pH 2 with concentrated HCl, resulting in precipitation. After the slurry had stirred at room temperature for 15 hours, it was cooled to 0 0 C, stirred for an additional 30 minutes, and filtered. The filter cake was washed with cold H 2 O and dried to yield 3.10 as a white solid (2.735 g, 60% yield). 1 H NMR (400 MHz, DMSO-d 6 ) δ 13.12 (s, 1H), 11.33 (s, 1H), 7.61 (d, J = 7.9 Hz, 1H), 5.59 (dd, J = 7.8, 2.2 Hz, 1H), 4.41 (s, 2H). 13 C NMR (101 MHz, DMSO-d 6 ) δ 169.56, 163.79, 151.01, 146.06, 100.87, 48.59. MS (ESI) m/z 171.1 [M+H] + ; 193.1 [M+Na] + ; 169.1 [M-H] - . (R)-4-amino-2-((tert-butoxycarbonyl)amino)-4-oxobutanoic acid (3.12): To a stirred suspension of L-asparagine (3.11, 10.0 g, 75.689 mmol) and Na 2 CO 3 (8.00 g, 75.748 mmol) in 1:1 H 2 O/dioxane (300 mL) was added di-tert-butyl dicarbonate (20.0 g, 91.638 mmol) in two portions. The resulting mixture was allowed to stir at room temperature for 7 hours. The reaction mixture was concentrated under reduced pressure to remove dioxane, and the remaining aqueous solution was acidified to pH 2 by addition of 12N HCl, resulting in precipitation. After cooling the flask in an ice bath, the precipitate was collected by filtration, washed with copious amounts of water, and dried to yield 3.12 as a white crystalline powder (16.353 g, 67% yield). 117 1 H NMR (400 MHz, DMSO-d 6 ) δ 12.51 (s, 1H), 7.31 (s, 1H), 6.90 (s, 1H), 6.87 (d, J = 8.4 Hz, 1H), 4.23 (m, 1H), 2.48 – 2.32 (m, 2H), 1.37 (s, 9H). 13 C NMR (101 MHz, DMSO-d 6 ) δ 173.41, 171.41, 155.21, 78.14, 50.24, 36.74, 28.22. MS (ESI) m/z 255.1 [M+Na] + ; 231.2 [M-H] - . (R)-3-amino-2-((tert-butoxycarbonyl)amino)propanoic acid (3.13): To a stirred suspension of 3.12 (15.0 g, 64.591 mmol) in 2:2:1 EtOAc/MeCN/H 2 O (180 mL) was added (diacetoxyiodo)benzene (PIDA, 24.066 g, 77.509 mmol). After stirring for 30 minutes at 0 0 C, the pale-yellow solution was allowed to stir at room temperature for 16 hours, resulting in precipitation. After cooling the flask in an ice bath, the precipitate was collected by filtration, washed with EtOAc and dried to yield 3.13 as a white powder (7.94 g, 60% yield.) 1 H NMR (400 MHz, CD 3 OD) δ 4.05 (t, J = 6.5 Hz, 1H), 3.24 – 2.95 (m, 2H), 1.46 (s, 9H). 13 C NMR (101 MHz, CD 3 OD) δ 174.76, 80.91, 53.89, 43.14, 28.66. MS (ESI) m/z 205.1 [M+H] + ; 203.1 [M-H] - . (R)-3-(((benzyloxy)carbonyl)amino)-2-((tert-butoxycarbonyl)amino)propanoic acid (3.14): To an ice-cold mixture of 3.13 (11.869 g, 58.119 mmol) in MeCN (132 mL) and 5% aq. NaHCO 3 (161 mL) was added Cbz-OSu (17.381 g, 69.742 mmol) in two portions. After stirring at 0 0 C for 3 minutes, the mixture was allowed to stir at room temperature for 15 hours. The reaction mixture was subsequently concentrated under reduced pressure to remove MeCN. The remaining aqueous solution was acidified to pH 3-4 by addition of 2N HCl, resulting in precipitation of a white solid. The residue was extracted with EtOAc (4 x 150 mL), and the 118 organic extracts were combined, dried over Na 2 SO 4 and concentrated under reduced pressure to yield 3.14 as a sticky white foam (14.035 g, 71% yield). 1 H NMR (400 MHz, CDCl 3 ) δ 7.39 – 7.28 (m, 5H), 5.77 (s, 1H), 5.52 (s, 1H), 5.07 (s, 2H), 4.32 (s, 1H), 3.60 (d, J = 7.0 Hz, 2H), 1.43 (s, 9H). 13 C NMR (101 MHz, CDCl 3 ) δ 173.66, 171.33, 156.45, 136.20, 128.68, 128.66, 128.35, 128.21, 80.93, 67.36, 54.68, 42.78, 28.41. MS (ESI) m/z 361.1 [M+Na] + ; 337.1 [M-H] - . (R)-benzyl tert-butyl (3-(methoxy(methyl)amino)-3-oxopropane-1,2-diyl)dicarbamate (3.15): An oven-dried flask was purged several times with argon and charged with 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride (12.410 g, 64.735 mmol) and N,O- dimethylhydroxylamine hydrochloride (6.314 g, 64.735 mmol) under nitrogen in a glovebag. The flask was sealed and purged several times with argon, charged with N-methylmorpholine (8.21 mL, 74.694 mmol) and cooled to -78 0 C. A solution of 3.14 (16.849 g, 49.796 mmol) in anhydrous CH 2 Cl 2 (125 mL) was added via cannula under argon, and the resulting pale-yellow solution was allowed to stir under argon atmosphere at room temperature for 14 hours. The reaction mixture was subsequently washed with saturated NH 4 Cl (125 mL) and H 2 O (125 mL), and the organic layer was collected. The aqueous layers were combined, acidified to pH 4 with 5% aq. citric acid, and extracted with CH 2 Cl 2 (150 mL). The organic layers were combined, washed with saturated NaHCO 3 (50 mL) and brine (50 mL), dried over Na 2 SO 4 , and concentrated under reduced pressure. The residue was purified by automated column chromatography (43% EtOAc/hexanes), and the relevant fractions were combined and concentrated under reduced pressure to yield a colorless foam, which upon standing solidified to give 3.15 as a white solid (11.727 g, 62% yield). 1 H NMR (400 MHz, CDCl 3 ) δ 7.40 – 7.29 (m, 5H), 5.51 (s, 1H), 5.19 (s, 1H), 5.08 (s, 2H), 4.77 (s, 1H), 3.77 (s, 3H), 3.59 – 3.45 (m, 3H), 3.19 (s, 3H), 1.43 (s, 9H). 13 C NMR (101 MHz, CDCl 3 ) δ 171.20, 156.64, 155.62, 136.59, 130.03, 128.60, 128.19, 80.16, 66.93, 61.80, 51.03, 43.13, 32.47, 28.45. MS (ESI) m/z 404.1 [M+Na] + ; 426.2 [M+HCOO] - . 119 2,5-dioxopyrrolidin-1-yl (2-(trimethylsilyl)ethyl) carbonate (Teoc-OSu, 3.18): An oven- dried flask was charged with triphosgene (5.00 g, 16.849 mmol) and purged several times with argon. Anhydrous CH 2 Cl 2 (50 mL) was added under argon, and the solution was cooled to - 78 0 C, followed by addition of 2-(trimethylsilyl)ethanol (3.17, 7.25 mL, 50.548 mmol) and triethylamine (7.05 mL, 50.548 mmol). The resulting white slurry was allowed to warm to 0 0 C for 5 minutes and then warmed to room temperature for 1 hour. The mixture was then cooled to -78 0 C and charged with a solution of N-hydroxysuccinimide (7.563 g, 65.712 mmol) and triethylamine (7.05 mL, 50.548 mmol) in acetonitrile (100 mL). The resulting mixture was stirred in a melting ice bath and allowed to warm to room temperature. After stirring for 16 hours, the reaction mixture was poured into water (400 mL) and extracted with ethyl ether (3 x 150 mL). The organic layers were combined, washed with 1N HCl (150 mL) and H 2 O (2 x 150 mL), dried over Na 2 SO 4 and concentrated under reduced pressure to yield Teoc-OSu (3.18) as a white powder (5.071 g, 39% yield). 1 H NMR (400 MHz, CDCl 3 ) δ 5.01 – 4.03 (m, 2H), 2.83 (s, 4H), 1.54 – 0.87 (m, 2H), 0.07 (s, 9H). 13 C NMR (101 MHz, CDCl 3 ) δ 168.88, 151.63, 70.75, 25.62, 17.66, -1.45. MS (ESI) m/z 260.0 [M+H] + . (R)-benzyl (2-(trimethylsilyl)ethyl) (3-(methoxy(methyl)amino)-3-oxopropane-1,2- diyl)dicarbamate (3.19): Trifluoroacetic acid (4 mL) was added dropwise to a solution of 3.15 (4.00 g, 10.487 mmol) in CH 2 Cl 2 (4 mL) with stirring. The resulting pale yellow solution was allowed to stir at room temperature for 8.5 hours. The reaction mixture was subsequently concentrated under reduced pressure. The residue was treated with saturated NaHCO 3 and solid Na 2 CO 3 until the mixture reached pH 9, followed by extraction with EtOAc (4 x 30 mL). The organic layers were combined, dried over Na 2 SO 4 and concentrated under reduced pressure to yield (R)-benzyl (2-amino-3-(methoxy(methyl)amino)-3-oxopropyl)carbamate (3.16) a pale yellow oil (2.870 g, 10.202 mmol, 97% yield). MS (ESI) m/z 282.0 [M+H] + . 120 The entire product was dissolved in acetonitrile (20 mL) and 5% aq. NaHCO 3 (24 mL) and charged with Teoc-OSu (3.18, 3.175 g, 12.243 mmol) in two portions with stirring. The resulting colorless mixture was stirred at room temperature for 11 hours. The reaction mixture was concentrated under reduced pressure to remove MeCN, and the resulting aqueous mixture was extracted with EtOAc (4 x 30 mL). The organic layers were combined, washed with saturated NH 4 Cl (2 x 20 mL) and concentrated in vacuo onto Celite. The residue was purified by silica gel flash column chromatography (1:1 EtOAc/hexanes). The relevant fractions were combined and concentrated under reduced pressure to yield 3.19 as a colorless syrup (3.337 g, 77% yield). 1 H NMR (400 MHz, CDCl 3 ) δ 7.43 – 7.27 (m, 5H), 5.64 (s, 1H), 5.25 – 5.02 (m, 3H), 4.81 (s, 1H), 4.25 – 4.06 (m, 2H), 3.77 (s, 3H), 3.53 – 3.56 (m, 2H), 3.19 (s, 3H), 1.09 – 0.87 (m, 2H), 0.03 (s, 9H). 13 C NMR (101 MHz, CDCl 3 ) δ 156.68, 130.04, 128.62, 128.22, 67.00, 63.70, 61.81, 51.48, 43.00, 32.48, 25.56, 17.83, -1.35. MS (ESI) m/z 448.0 [M+Na] + ; 470.0 [M+HCOO] - . 121 3.8 Chapter 3 References (1) Greider, C.W. & Blackburn, E.H. Cell 1985, 51, 405-413. (2) Greider C.W. & Blackburn, E.H. Nature 1989, 337, 331-337. (3) Liu, D.; O’Connor, M.S.; Qin, J. & Songyang, Z. J. Biol. Chem. 2004, 279, 51338-51342. (4) de Lange, T.; Genes Dev. 2005, 19, 2100-2110. (5) Harley, C.B.; Futcher, A.B.; Greider, C.W. Nature 1990, 345, 458-460. (6) Hastie, N.D.; Dempster, M.; Dunlop, M.G.; Thompson, A.M.; Green, D.K.; Allshire, R.C. Nature 1990, 346, 866-868. 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Many efforts to combat these diseases target proteins involved in the underlying pathways of epigenetic regulation, which control gene expression without modifying the DNA sequence. These changes are controlled by a class of proteins known as transcription factors, which modify gene expression through a number of processes, including methylation, ubiquitinylation, phosphorylation, or acetylation of the nucleosomal histone tail 4-6 . Histone acetyltransferases (HATs) are enzymes that cause dissociation of DNA from histone protein, and are responsible for regulating a number of pathways 6 . DNA-histone dissociation enables gene transcription/translation by allowing transcription factors to access the free DNA 7 . This process is reversed by histone deacetylases (HDACs), responsible for deacetylating the lysine residues on histone, resulting in gene silencing (Figure 4.1). 7 Histone deacetylation has been implicated in several cancers, including leukemia, 8 making HDACs an attractive therapeutic target. Figure 4.1. Role of HATs and HDACs in gene expression. As a result, a lot of HDAC inhibitors have been developed in recent years. In 2006, Vorinostat (SAHA, Zolinza ® , Merck) became the first FDA-approved HDAC inhibitor, approved for advanced cutaneous T-cell lymphoma 9 . In addition, several other compounds, including LBH-589 (Novartis AG), CI-994 (Pfizer), BML-210, and MS-275 have been or are undergoing clinical trials (Figure 4.2) 10-13 . 125 Figure 4.2. Reported HDAC inhibitors There are 17 human HDACs that have been implicated in gene silencing, and these are categorized into four classes (class I: HDACs 1,2,3, and 8; class II: HDACs 4, 5, 6, 7, 9, and 10; class III: sirtuins 1-7, class IV: HDAC 11). Many of the small molecule HDAC inhibitors in development exhibit a myriad of side effects 14 due to pan-HDAC inhibition 18 and the resulting complex cellular responses, raising the importance of isoform selectivity. Among the family of HDACs are the class IIa HDACs (HDACs 4, 5, 6, 7, 9, and 10), which, while catalytically inactive, act as structural proteins 15 and are involved with binding to transcription factors prior to histone deacetylation 16 as a result of their unique amino acid sequence. Additionally, class IIa HDACs have been shown to be disregulated or mutated in lymphoma and leukemia 17-19 . Although many of the HDAC inhibitors in development bind to an array of HDACs, many exhibit dimished activity for the class IIa HDACs. With this in mind, we sought to develop compounds specifically targeting class IIa HDACs by inhibiting the interaction of the class IIa HDACs with an associated transcription factor, myocyte enhancer factor-2 (MEF2). It has been shown that a hydrophobic groove exists on the surface of the MADS/MEF2 domain of MEF2 20 and crystallographic data demonstrated MEF2 was capable of binding class IIa HDACs (Figure 4.3.A) 21 . Previous studies from our group, in collaboration with Dr. Lin Chen, showed that HDAC inhibitor BML-210 was capable of disrupting HDAC9-MEF2 binding (Figure 4.3.B) 16 . With crystallographic data in hand, we sought to develop small molecules capable of binding to the hydrophobic groove of MEF2 and potentially block the interaction of MEF2 with class IIa HDACs, which in turn would influence MEF2-dependent gene expression. 126 Figure 4.3 16 : Structural comparison of binding sites for HDAC9 and BML-210 on MEF2. (a) X-ray crystal structure of the α-helix of HDAC9 (purple helix) bound to MEF2 (grey surface) (PDB:1TQE); (b) X-ray crystal structure of small molecule BML-210 (yellow) bound to MEF2 in the same cleft as the α- helix of HDAC9 (PDB:3MU6). 4.2. Design of MEF2 Inhibitors A series of molecules based on the BML-210 scaffold (Table 4.1) were previously synthesized by my colleague Kevin Gaffney to explore their ability to disrupt the interaction between MEF2 and class IIa HDAC4 were synthesized according to Scheme 4.1. These compounds consisted of an aliphatic linker with two capping regions A and B. The analogs were prepared via two routes starting with hydrocarbon dicarboxylic acids adipic or pimelic acids (4.1a-b). The dicarboxylic acids 4.1a-b were either heated as neat mixture of with a variety of anilines provided or reacted with HBTU and DIPEA then reacted with the anilines to generate monocarboxylic acids 4.2a-f. The resulting monocarboxylic acids 4.2a-f were then coupled to either anilines, phenylenediamines, or tert-butyl (2-aminophenyl)carbamate in the presence of HBTU and DIPEA to give 4.3a-f. The use of tert-butyl (2-aminophenyl)carbamate necessitated a final deprotection using trifluoroacetic acid to provide analogs 4.4a-b. Two further analogs of 4.3b were synthesized to explore the importance of the ortho-anilidine moiety. The N,N- dimethyl analog 4.5a was synthesized by reductive amination of 4.3b with paraformaldehyde. Formylated compound 4.5b was synthesized, in poor yield, from the reaction 4.3b with formic acid and sodium formate, in poor yield. B A 127 Scheme 4.1. Synthesis of MEF2 inhibitors 4.3a-c, f, 4.4a-b, and 4.5a-b. Reagents and conditions: (a) HBTU, DIPEA, DMSO, tert-butyl (2-aminophenyl)carbamate or ArNH 2 , 140 o C, 24 h, 26-34% ; (b) HBTU, DIPEA, DMSO or DMF, tert-butyl (2-aminophenyl)carbamate or ArNH 2 , rt, 3-24 h, 6-68%; (c) CF 3 COOH, CH 2 Cl 2 , 0° to rt, 3 h, 18-79%; (d) HCOONa, HCOOH, rt, 18 h, 9%, or paraformaldehyde, NaBH 4 , CF 3 COOH, THF, rt, 24 h, 38%. The analogs were tested in the previously described mammalian two hybrid luciferase assay 16 which directly detects the disruption of the HDAC-MEF2 protein-protein interaction. The percent inhibition of the interaction was recorded at 10µM for each compound. 128 Table 4.1 16 . Activity of MEF2-HDAC inhibitors at 10µM in luciferase assay. Entry Structure % Inhibition Entry Structure % Inhibition 4.3a 66 4.4a 80 4.3b 98 4.4b 96 4.3c 4.3f 92 95 4.5a 4.5b <50 <50 _______________________________________________________________________ 4.3. Biological Screening and Structure-Activity Relationship of MEF2 Inhibitors The compounds were tested in the human colorectal carcinoma cell line HCT-116 and the multiple myeloma cell line RPMI8226 to examine their effects on cell proliferation (Figure 4.4). The anti-proliferation activity was evaluated using concentration escalation (0-160 µM) in an MTT assay. At 10 µM 4.3b, 92% inhibition of HCT-116 was observed after 72 hours of treatment. It was observed that having a primary ortho-aniline functionality in region B was critical for cytotoxicity, as compounds 4.5a and 4.5b exerted much weaker anti-proliferative effects despite sharing an identical linker length and A region to that of 4.3b. Similarly, changing the length of the linker greatly influenced biological activity, as shortening the six- carbon linker of 4.3b by one carbon atom (4.3a) resulted in a significant loss of cytotoxicity. In general, it was found that substitution in the meta-position of the Region A phenyl ring provided good inhibitory activity, with the methyl and trifluoromethyl groups exhibiting the most activity. With the exception of 4.5a-b, the compounds exhibited good activity in RPMI8226 cells. Overall these results are consistent with crystallographic studies (Figure 4.5) that reveal a 129 channel which could accommodate bulkier substituents at the meta-position of Region B. Altogether, these results (Table 4.2) validate the trends observed in the aforementioned luciferase assay (Table 4.1). 0 20 40 60 80 100 120 140 160 0 10 20 30 40 50 60 70 80 90 100 4.3b 4.4a 4.5c 4.3f 4.5a 4.5b 4.3a 4.4b Concentration ( M) % Viability, HCT-116 Figure 4.4. Effect of MEF2 inhibitors on HCT116 cell viability. Percentage cell viability of HCT116 cells was determined via MTT assay after 72 hours of treatment with 0-160 µM compounds. Table 4.2. Activity of MEF2-HDAC inhibitors in HCT-116 and RPMI8226 cells. Compound R Linker Length R’ HCT-116 EC 50 (µM) RPMI-8226 EC 50 (µM) 4.3a 3-CF 3 4 2-NH 2 73 18 4.3b 3-CF 3 5 2-NH 2 6 5 4.3c 3-F 5 2-NH 2 13 6 4.3f 3-CH 3 5 2-NH 2 5 5 4.4a 4-F 5 2-NH 2 10 6 4.4b 3-Br 5 2-NH 2 15 17 4.5a 3-CF 3 5 2-N(CH 3 ) 2 63 46 4.5b 3-CF 3 5 2-NH(CHO) >100 >100 130 Figure 4.5. Crystallographic understanding of analog activity and design. (a) Structural explanation of the activity ortho<meta<para activity trend. While the ortho positions make direct contact with the protein preventing the addition of substituents, the blue arrow shows a small gap between L67A and D63A for para substituents and the red arrow shows a larger channel between D63A and T70B to accommodate meta substituents. These assays were then used to evaluate a second class of MEF2 inhibitors that were synthesized by my colleague Jamie Jarusiewicz. These inhibitors made use of a reverse amide bond in region A, which was predicted to increase potency through an increase in binding affinity (Figure 4.6). Figure 4.6. Scaffold change of MEF2 inhibitors by reversing the amide bond between the A region and the hydrophobic linker. After a large library of compounds had been synthesized and tested in the aforementioned luciferase assay, I was tasked with evaluating the efficacy of a collection of representative compounds in an attempt to gain insight on the structure-activity relationship (SAR) of these compounds across multiple cell lines (Table 4.3). Many of these included heterocyclic derivatives, such as indoles, that showed increased activity in luciferase assays, presumably due to the extended region of hydrophobicity being accommodated by the hydrophobic groove of 131 MEF2. Although these analogs were generally more potent than the first-generation compounds in both cell lines, the increase in activity was only marginal. Table 4.3. Activity of reverse amide MEF2-HDAC inhibitors in HCT-116 and RPMI8226 cells. Compound R Linker Length R’ HCT-116 EC 50 (µM) RPMI-8226 EC 50 (µM) 4.6 6 H 2.5 2.35 4.7 5 H 2 2.6 4.8 5 H 7 6.3 4.9 5 H 1.6 1.8 4.10 5 H 3.12 3.6 4.11 5 H 6 4 4.12 5 H 11 18 4.13 5 F 10 1.25 Additionally, selected benzamide derivatives were investigated based on the structures of MS-275 and CI-994 (Table 3.4). These compounds had comparable activity in HCT-116 and RPMI8226, suggesting that substitution of the alkyl linker with a benzene ring does not result in increase of potency. This corroborates the results from the first-generation compounds as well as 132 the crystallographic data for BML-210, which shows that a flexible linker promotes inhibition of MEF2:HDAC binding. Although these second-generation compounds did not display a vast increase in activity against cancer cells, their activity is comparable to those of MS-275 and CI- 994. Furthermore, the antitumor activity of MS-275 and CI-994 is likely influenced by its pan- HDAC inhibitory activity, whereas the compounds described herein have been shown to exert selective inhibition of class II HDACs. Table 4.4. Activity of benzamide MEF2-HDAC inhibitors in HCT-116 and RPMI8226 cells. Compounds were tested alongside known HDAC inhibitors MS-275 and CI-994, respectively. Compound R HCT-116 EC 50 (µM) RPMI-8226 EC 50 (µM) MS-275 1.6 0.7 CI-994 6.25 N/A 4.14 4.5 3.35 4.15 2.5 2.3 4.4. Conclusion We have designed, synthesized, and evaluated a series of compounds for their potential ability to function as modulators of epigenetic activity through inhibition of the interaction of class IIa HDACs and the associated transcription factor MEF2. Many of these novel analogs exhibit comparable inhibition with the parent analog BML-210. In particular, we have determined that the presence of an aliphatic linker connecting an ortho-aniline capping region (region A) and a meta-substituted aromatic capping region (region B) is important for biological activity in HCT-116 and RPMI8226 cells, whereas a wide range of functionality located on the distal side of the molecule, particularly with substitution at the meta position, is amenable to 133 inhibitory activity. Incorporation of a reverse-amide linker and a heterocyclic moiety in region B resulted in a slight increase in potency. The inhibition of cell proliferation by the compounds described is comparable with pan-HDAC inhibitors MS-275 and CI-994. It is theorized that the isoform selectivity of these compounds may provide better pharmacokinetic properties and thus serve as a basis for further development. 4.5. Experimental Cell lines and culturing Human colorectal carcinoma cell line HCT-116 (ATCC® CCL-247™) and the multiple myeloma cell line RPMI/8226 (ATCC® CCL-155™) were obtained from ATCC (Manassas, VA). RPMI/8226 cells were propagated in RPMI-1640 (Cellgro, Herndon, VA). HCT-116 cells were propogated in Dulbeccco’s modified Eagle’s medium (Cellgro, Herndon, VA) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin (Gemini Bio-Products, West Sacramento, CA). All cell lines were grown in a humidified incubator at 37 o C and a 5% CO 2 atmosphere. MTT cell viability assay RPMI/8226 cells (2.5 x 10 3 cells/well) and HCT-116 cells (5000 cells/well) were seeded in 96-well plates in culture medium (50 µL/well) and incubated for 24 hours in a humidified atmosphere of 37 o C and a 5% CO 2 . Compounds were diluted with culture medium to various concentrations and fed to the culture. 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Abstract (if available)

Abstract This dissertation details my efforts towards the design, synthesis and biological evaluation of novel, small molecule anti-cancer agents. This was accomplished with a joint collaboration with the USC School of Pharmacy. ❧ Chapter 1 describes the computational fragment-based design, synthesis, and biological validation of a series of novel inhibitors of the enzyme dUTPase as potential anti-cancer agents. ❧ Chapter 2 reports the investigation into the chemical interaction of green tea extract epigallocatechin gallate (EGCG) with the anticancer drug bortezomib (BZM, Velcade™). This work described focuses mainly on the phenotypic assays used to screen the interaction of EGCG with BZM and other biologically relevant polyphenol compounds. ❧ Chapter 3 provides a description of the development of a novel class of peptide nucleic acids (PNAs). This includes a rational design of the PNA oligomer, the synthesis of the nucleic acid monomer units, as well as efforts to synthesize the PNA using solid phase peptide synthesis. ❧ Chapter 4 reviews the structure-activity relationship of a series of small molecules capable of disrupting the protein-protein interaction between transcription factor MEF2 and class IIa HDACs. The work described herein focuses on the SAR developed through in vitro cellular assay screening.

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Creator Sainz, Marcos A. (author)

Core Title Design, synthesis, and biological evaluation of novel therapeutics for cancer

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Chemistry

Publication Date 11/17/2015

Defense Date 08/15/2014

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag bortezomib,dUTPase,medicinal chemistry,MEF2,OAI-PMH Harvest,organic chemistry,PNA,synthesis

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Petasis, Nicos A. (committee chair), Louie, Stan G. (committee member), Prakash, G. K. Surya (committee member)

Creator Email msainz@usc.edu,sainz.marcos@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-518515

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Legacy Identifier etd-SainzMarco-3091.pdf

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Document Type Dissertation

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Rights Sainz, Marcos A.

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Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

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