Synthesis of 2,4,6-Substituted Pyrrolo[2,3-D]Pyrimidines As Potential Anticancer Agents Si Yang

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SYNTHESIS OF 2,4,6-SUBSTITUTED PYRROLO[2,3-d] PYRIMIDINES AS

POTENTIAL ANTICANCER AGENTS

A Thesis

Submitted to the Graduate School of Pharmaceutical Sciences

Duquesne University

In partial fulfillment of the requirements for

the degree of Master of Science

Si Yang

December 2017

Si Yang

2017

SYNTHESIS OF 2,4,6-SUBSTITUTED PYRROLO[2,3-d] PYRIMIDINES AS

POTENTIAL ANTICANCER AGENTS

Si Yang

Approved November 16th, 2017

______Aleem Gangjee Marc W. Harrold Professor of Medicinal Chemistry Professor of Medicinal Chemistry (Committee Chair) (Committee Member)

______Patrick Flaherty Assistant Professor of Medicinal Chemistry (Committee Member)

iii

ABSTRACT

SYNTHESIS OF 2,4,6-SUBSTITUTED PYRROLO[2,3-d] PYRIMIDINES AS

POTENTIAL ANTICANCER AGENTS

Si Yang

December 2017

Thesis supervised by Professor Aleem Gangjee

More and more people are suffering from cancers. Scientists have been putting so much effort to wish find a solution which can defeat tumor cells. As analogs of folic acid which is essential for human being life, antifolates are currently clinically used for cancer chemotherapy.

Under the guidance of Professor Aleem Gangjee, I had been focusing on synthesizing a series of pyrrolo[2,3-d]pyrimidines to investigate its inhibitory potency against tumor cells over normal tissues during my Master degree in Duquesne University.

This thesis will focus on the introduction of the background and work have been done in the areas of antifolates development, such as folate function, its three uptake mechanisms inside human cells, antifolates’ role in chemotherapy, et. al. In addition, the

Structure-Activity-Relationship design rationale for the series of antifolates will also be

discussed. Nevertheless, the details of synthesizing these pyrrolo[2,3-d]pyrimidines as potential antifolates have been described, including chemistry reviews on the pyrrolo[2,3- d]pyrimidine scaffold, and the challenges encountered and the solutions how to solve or improve in order to achieve better yield.

In this study, sixty new compounds have been synthesized and ten antifolates have been sent for biological evaluation.

DEDICATION

Dedicated To My Family For Their Love And Support

ACKNOWLEDGEMENT

I would like to thank all the people who have helped me to make this work done.

Especially, I’ll give my most appreciation to Dr. Aleem Gangjee, my supervisor. Without his help, support and guidance, I could not have made this thesis possible. Not only his enthusiastic passion on scientific research that set a good example for me, but also his support both financially and in spirit. I would like to thank all my thesis committee members: Drs. Marc W. Harrold, Patrick Flaherty, and Aleem Gangjee for their academic and knowledge support. Also, I want to acknowledge Dr Larry H. Matherly at the Barbara

Ann Karmanos Cancer Institute, Wayne State University School of Medicine for his cooperation on research. I would like to express my sincere appreciation to Dr. James

Drennen for his thoughtfulness and kindness during my master degree at Duquesne

University. As a Dean, he tried his best to help me. I wish to thank Nancy Hosni and Jackie

Farrer for their help at all time. In the end, I want to give my great appreciation to my family for their love and support.

vii

TABLE OF CONTENTS

Page

Abstract ...... iv

Dedication ...... vi

Acknowledgement ...... vii

List of Tables ...... ix

List of Figures ...... x

List of Schemes ...... xii

List of Abbreviations ...... xv

I. Biochemical Review ...... 1

II. Chemical Review ...... 45

III. Statement of The Problem ...... 67

IV. Chemical Discussion ...... 79

V. Summary ...... 95

VI. Experimental ...... 96

Bibliography ...... 127

Appendix 1 ...... 159

Appendix 2 ...... 168

Appendix 3 ...... 174

viii

LIST OF TABLES

Page

Table 1 Pyrimidines as GARFTase Inhibito ...... 42

Table 2 IC50’s (in nM) for 6-substituted pyrrrolo[2,3-d]pyrimidine thienoyl antifolates

3-4, and analogs with fused aromatic side chain 135-138 and Classical

Antifolates in hRFC, hPCFT, and FR-Expressing Cell Lines ...... 165

Table 2 IC50’s (in nM) for 6-substituted pyrrrolo[2,3-d]pyrimidine phenyl antifolates 2

and analogs with naphthoic ring as side chain 139, 140 and Classical

Antifolates in hRFC, hPCFT, and FR-Expressing Cell Lines ...... 166

Table 4 IC50’s (in nM) for 6-substituted pyrrrolo[2,3-d]pyrimidine thienoyl antifolates

3-4, and analogs with amino acid modification 141-144 and Classical

Antifolates in hRFC, hPCFT, and FR-Expressing Cell Lines ...... 167

LIST OF FIGURES

Page

Figure 1 Structure of folic acid ...... 1

Figure 2 Structure of classical folates ...... 2

Figure 3 Cellular folate metabolism in the cytosol and mitochondria ...... 4

Figure 4 Representative examples of classical antifolates (and their principal

target(s)) ...... 6

Figure 5 Representative examples of nonclassical antifolates (and their principal

target(s)) ...... 7

Figure 6 Homeostasis of folates and cellular accumulation of antifolates ...... 9

Figure 7 Human RFC topology model...... 10

Figure 8 Three-dimensional (3D) homology models of human RFC ...... 12

Figure 9 Model for trafficking of human folate receptors ...... 18

Figure 10 Structures of folate receptors depicting states of biological trafficking ...... 19

Figure 11 Conformational changes in the hFR ligand binding pocket during states of

biological trafficking ...... 20

Figure 12 Crystal structure of FR bound to folic acid ...... 21

Figure 13 Structural and biochemical analysis of FRa–folic acid interactions ...... 23

Figure 14 Interaction map of folic acid with ligand-binding-pocket residues ...... 25

Figure 15 Schematic structure of human PCFT membrane topology...... 30

Figure 16 De novo Purine Biosynthesis ...... 33

Figure 17 Proposed mechanism for GARFTase ...... 37

Figure 18 Crystal structure (MOE 2008.10) of hGARFTase in a binary complex with

inhibitor 10-CF3CO-DDACTHF at pH 7 (PDB ID 1NJS) ...... 38

Figure 19 The structure of classical 2-amino-4-oxo-6-substituted pyrrolo[2,3- d]

pyrimidine antifolate 135 ...... 67

Figure 20 The structures of ONX0801 ...... 69

Figure 21 Calculation of the total distance from the C6 of the scaffold to the C1’ of the

side chain thioenyl ring of 3. (MOE 2011.10) ...... 71

Figure 22 Calculation of the total distance from the C6 of the scaffold to the C1’ of the

side chain thioenyl ring of 4. (MOE 2011.10) ...... 71

Figure 23 Calculation of the total distance from the C6 of the scaffold to the C7a’ of the

benzo[b]thioenyl ring of 135. (MOE 2011.10) ...... 72

Figure 24 The structures of classical 2-amino-4-oxo-6-substituted pyrrolo[2,3- d]

pyrimidine antifolates 135-138 ...... 73

Figure 25 The structures of classical 2-amino-4-oxo-6-substituted pyrrolo[2,3- d]

pyrimidine antifolates 139 and 140...... 74

Figure 26 The structures of classical 2-amino-4-oxo-6-substituted pyrrolo[2,3- d]

pyrimidine antifolates 141-144 ...... 78

Figure 27 Molecular modeling study of compound 135 with FRα...... 174

Figure 28 Molecular modeling study of compound 3 with FRα...... 175

Figure 29 Molecular modeling study of compound 139 with FRα...... 176

LIST OF SCHEMES

Page

Scheme 1 Synthesis of pyrrolo[2,3-d]pyrimidines 8 ...... 46

Scheme 2 Synthesis of pyrrolo[2,3-d]pyrimidine 11 ...... 46

Scheme 3 Synthesis of pyrrolo[2,3-d]pyrimidines 15 ...... 47

Scheme 4 Synthesis of pyrrolo[2,3-d]pyrimidines 20 and 21 ...... 48

Scheme 5 Synthesis of pyrrolo[2,3-d]pyrimidines 25 ...... 49

Scheme 6 Synthesis of pyrrolo[2,3-d]pyrimidines 29 ...... 49

Scheme 7 Synthesis of pyrrolo[2,3-d]pyrimidine 32 ...... 50

Scheme 8 Synthesis of pyrrolo[2,3-d]pyrimidines 34,-36 ...... 50

Scheme 9 Synthesis of pyrrolo[2,3-d]pyrimidines 40 ...... 51

Scheme 10 Synthesis of pyrrolo[2,3-d]pyrimidines 43 ...... 51

Scheme 11 Synthesis of pyrrolo[2,3-d]pyrimidines 47 and 49...... 52

Scheme 12 Synthesis of pyrrolo[2,3-d]pyrimidines 52s ...... 53

Scheme 13 Synthesis of pyrrolo[2,3-d]pyrimidines 55s ...... 54

Scheme 14 Synthesis of pyrrolo[2,3-d]pyrimidines 60 and 61 ...... 54

Scheme 15 Synthesis of pyrrolo[2,3-d]pyrimidines 65 and 67 and furo[2,3-d]

pyrimidines 66 ...... 55

Scheme 16 Synthesis of pyrrolo[2,3-d]pyrimidines 71 and 72 ...... 56

Scheme 17 Synthesis of pyrrolo[2,3-d]pyrimidines 76 ...... 57

Scheme 18 Synthesis of pyrrolo[2,3-d]pyrimidines 79-80 ...... 57

Scheme 19 Synthesis of pyrrolo[2,3-d]pyrimidines 83 ...... 58

Scheme 20 Synthesis of pyrrolo[2,3-d]pyrimidines 86 ...... 58

xii

Scheme 21 Synthesis of pyrrolo[2,3-d]pyrimidines 89 ...... 59

Scheme 22 Synthesis of pyrrolo[2,3-d]pyrimidines 92 ...... 59

Scheme 23 Synthesis of pyrrolo[2,3-d]pyrimidine 97 ...... 60

Scheme 24 Synthesis of pyrrolo[2,3-d]pyrimidines 103 ...... 61

Scheme 25 Synthesis of pyrrolo[2,3-d]pyrimidines 106 ...... 62

Scheme 26 Synthesis of pyrrolo[2,3-d]pyrimidines 108 ...... 62

Scheme 27 Synthesis of pyrrolo[2,3-d]pyrimidines 111 ...... 63

Scheme 28 Synthesis of 5,6-disubstitutedpyrrolo[2,3-d]pyrimidine 114 ...... 63

Scheme 29 Synthesis of pyrrolo[2,3-d]pyrimidine 118 ...... 64

Scheme 30 Synthesis of pyrrolo[2,3-d]pyrimidine 121 ...... 64

Scheme 31 Synthesis of pyrrolo[2,3-d]pyrimidine 124 ...... 65

Scheme 32 Synthesis of pyrrolo[2,3-d]pyrimidine 129 ...... 66

Scheme 33 Synthesis of pyrrolo[2,3-d]pyrimidine 134 ...... 66

Scheme 34 Synthesis of classical 2-amino-4-oxo-6-substituted-pyrrolo[2,3- d]

pyrimidines 135 and 136 ...... 80

Scheme 35 Synthesis of classical 2-amino-4-oxo-6-substituted-pyrrolo[2,3- d]

pyrimidine 137 ...... 83

Scheme 36 Synthesis of classical 2-amino-4-oxo-6-substituted-pyrrolo[2,3- d]

pyrimidine 138 ...... 85

Scheme 37 Synthesis of classical 2-amino-4-oxo-6-substituted-pyrrolo[2,3- d]

pyrimidine 139 ...... 87

Scheme 38 Synthesis of classical 2-amino-4-oxo-6-substituted-pyrrolo[2,3- d]

pyrimidine 140 ...... 89

xiii

Scheme 39 Synthesis of pteroic acid 204 ...... 90

Scheme 40 Synthesis of classical 2-amino-4-oxo-6-substituted-pyrrolo[2,3- d]

pyrimidine 142 ...... 92

Scheme 41 Synthesis of classical 2-amino-4-oxo-6-substituted-pyrrolo[2,3- d]

pyrimidine 144 ...... 92

Scheme 42 Synthesis of classical 2-amino-4-oxo-6-substituted-pyrrolo[2,3- d]

pyrimidine 141 ...... 93

Scheme 43 Synthesis of classical 2-amino-4-oxo-6-substituted-pyrrolo[2,3- d]

pyrimidine 143 ...... 94

xiv

LIST OF ABBREVIATIONS

ATP Adenosine-5’-triphosphate

DHFR Dihydrofolate reductase

TS Thymidylate Synthase

DMF N,N-dimethyl formamide

DMSO Dimethyl sulfoxide

DNA Deoxyribonucleic acid

GDP Guanosine Diphosphate

GTP Guanosine triphosphate dATP 2’-Deoxyadenosine-5’-triphosphate dGTP 2’-Deoxyguanosine-5’-triphosphate dTDP 2’-Deoxythymidine-5’-diphosphate dTMP 2’-Deoxythymidine-5’-monophosphate dTTP 2’-Deoxythymidine-5’-triphosphate dUMP 2’-Deoxyuridine-5’-monophosphate dUTP 2’-Deoxyuridine-5’-triphosphate

E. coli Escherichia coli

EGFR Epidermal growth factor receptor

FA Folic Acid

DHF 7,8-Dihydrofolate

THF 5,6,7,8-Tetrahydrofolate

5-CH3-THF 5-methyl-Tetrahydrofolate

GlpT Glycerol-3-phosphate antiporter

FGFR Fibroblast Growth Factor Receptor

FPGH Folylpolyglutamate Hydrolase

FPGS Folyl Poly--glutamate Synthetase

MFS Major facilitator super family

RFC Reduced Folate Carriers

PCFT Proton-Coupled Folate Transportor

FR Folate Receptor

GAR Glycinamide Ribosyl-5-phosphate

GARFTase Glycinamide Ribonucleotide Formyl Transferase

AICAR Aminoimidazole-4-carboxamide ribosyl-5-phosphate

AICARFTase Aminoimidazole-4-carboxamide ribosyl-5-phosphate

Formyl Transferase

MS methionine synthase

AdoMet S-adenosylmethionine

MFT mitochondrial folate transporter

MTSES 2-Sulfonatoethyl methanethiosulfonate

IMP Inositol Monophosphate

TMD Transmembrane domain

MRP Multidrug Resistance Protein

MTX Methotrexate

PMX Pemetrexed

RTX Raltitrexed

xvi

LMTX Lometrexol

AG-2037 Pelitrexol

10-formyl-TDAF 10-formyl-5,8,10-trideazafolic acid

10-CF3CO-DDACTHF 10-(Trifluoroacetyl)-5,10-dideaza-acyclic-5,6,7,8-

tetrahydrofolic acid

PDDF N10-propargyl-5,8-dideazafolate

FdUMP 5-Fluoro-2’-deoxyuridine-5’-monophosphate

AG337 Nolatrexed

TMP Trimethoprim

PTX Piritrexim

TMQ Trimetrexate

NADPH Nicotinamide Adenine Dinucleotide Phosphate

NCI National Cancer Institute

NMR Nuclear Magnetic Resonance

NRTI Nucleoside reverse transcriptase inhibitors

Piv Pivaloyl (trimethyl acetyl)

RNA Ribonucleic Acid

TMP Trimethoprim

TMQ Trimetrexate

Trp Tryptophan

Val Valine

Arg Arginine

Lys Lysine

xvii

Asn Asparagine

Tyr Tyrosine

Ser Serine

Ile Isoleucine

Ala Alanine

Cys Cysteine

Asp Aspartate

His Histidine

Gly Glycine

Leu Leucine

Glu Glutamate

Pro Proline

xviii

I. BIOCHEMICAL REVIEW

1. Folate Biology

Figure 1 Structure of folic acid

1.1 Folic acid structure

Folates (folic acid in Figure 1), as essential B9 vitamins, are found to be crucial one- carbon donors in many biological pathways in eukaryotic cells, notably the conversion of homocysteine to methionine and de novo nucleotide synthesis.1-5 Since human cannot synthesize folates de novo, it is essential to absorb the cofactors from foods, like liver and dark green leafy vegetables.7 In the 1940s, folic acid was firstly developed for the treatment of anemia. 6 Since then, it has been treated as a natural folate supplement because folic acid can be transformed to functional metabolites via dihydrofolate reductase (DHFR). 1,10

The chemical structure of folic acid consists of three parts: i) hetero-bicyclic pteridine ring; ii) p-aminobenzoic acid (PABA); iii) glutamic acid moiety (Figure 1). The

5 pteridine ring reduction in folic acid may occur at the 7,8-H2 and 5,6,7,8-H4. Also, N or

N10 or both may be methylated; Polyglutamylation may occur on the glutamate moiety via

γ-peptide bonds, which is mediated by folylpolyglutamate synthase.

Folates are found in two forms: i) oxidized form; ii) reduced forms. Folic acid is its oxidized form. Reduced folates, as naturally occurring forms in human beings, include partially reduced form 7,8-dihydrofolate (DHF) and the reduced 5,6,7,8-tetrahydrofolate

(THF). Among the reduced species, the primary circulating one is 5-methyl-THF (5-CH3-

THF).

Figure 2. Structure of classical folates

1.2 Folates update system

Naturally occurring reduced folates are anionic in physiological pH since they are water-soluble members of the vitamin B class. Thus, sophisticated uptake systems are evolved to facilitate cellular update of folates in mammalian cells.4,8

The major cellular transport systems include: i) Reduced folate carrier (RFC), an anionic transport carrier that is the major uptake route to transport folates into cells at physiological pH.4,7,9 ii) Folate receptors (FRs) are membrane glycoproteins with high folate-binding affinity including FRα, FRβ and FRγ. FRs transport folates into cells via

endocytosis at neutral or modest acidic pH.2,11 iii) Proton-coupled folate transporter

(PCFT), a member of solute carrier, transports folates optimally in acidic pH and has limited distribution in humans.8,9

1.3 Cellular folates metabolism

After folates are transported inside mammalian cells via the carriers, folate metabolism is found to be separated into two subcellular compartments in eukaryotes: i) cytosol, ii) mitochondria.10

i) In Cytosol

In cytosols, reduced folates play important roles to facilitate folate-dependent enzymes in multiple one-carbon transfer reactions.

5 10 5,10-Methylene tetrahydrofolate (N ,N -CH2-FH4), a one-carbon provider, serves as the essential cofactor for the conversion of 2’-deoxyuridine monophosphate (dUMP) to

2’-deoxythymidine monophosphate (dTMP) mediated by thymidylate synthase (TS). DHF, as the byproduct for the reductive methylation reaction by TS, is recycled to THF through dihydrofolate reductase (DHFR) to maintain THF pool in cytosol.

The reduced folate, 10-CHO-THF is an essential cofactor for the last two steps in the generation of purine intermediate inosine 5’-monophosphate (IMP). The first reaction is the formation of the purine imidazole ring by the transfer of one carbon from 10-CHO-

THF via the enzyme glycineamide ribonucleotide formyltransferase (GARFTase); 10-

CHO-THF also serves as the one-carbon provider in the latter transportation, mediated by

5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFTase).

Figure 3. Cellular folate metabolism in the cytosol and mitochondria.9

Note: TS: Thymidylate Synthase; DHFR: Dihydrofolate Reductase; SHMT: Serine

Hydroxymethyltransferase; GARFTase: Glycinamide-ribonucleotide Formyl Transferase;

AICARFTase: Amino-imidazole-carboxamide-ribonuleotide Formyl Transferase; AICAR:

Aminoimidazole-4-carboxamide ribosyl-5-phosphate; dTMP: 2’-Deoxythymidylate 5’- monophosphate; dUMP: 2’-Deoxyuridylate-5’-monophosphate; dTMP: 2’-

Deoxythymidylate 5’-triphosphate; GAR: Glycinamide Ribosyl-5-phosphate; DNA:

Deoxyribonucleotide; IMP: Inositol monophosphate.

Another cellular reduced folate cofactor, 5-methyl-THF (5-CH3-THF) facilitates methionine synthase (MS) to synthesize methionine from homocysteine by providing a methyl group, along with vitamin B12. The formation of S-adenosylmethionine (AdoMet) through conjugating ATP and methionine, which is a widely used one-carbon donor in

multiple methylation reactions, such as methylation reaction of neurotransmitters, phospholipids, RNA, cytosine nucleotide residues within CpG islands in DNA, as well as proteins including histones.12

ii) In Mitochondria

Mitochondria is known as a major folate warehouse in mammalian cells accumulating as much as 40% of total cellular folates.12,16 Folates in the cytosol are transported into the mitochondria via mitochondrial folate transporter (MFT/SLC25A32) with the monoglutamate form, which is THF.13-15 Without exchange from mitochondria to cytosol, some one-carbon providers including glycine, serine and formate are commuted between cytosol and mitochondria.10

Formate, as the methyl group donor for the cytoplasmic reactions and biosynthesis of mitochondrial glycine, is mainly derived from THF in mitochondria (Figure 3). 13-15

1.3 Folate polyglutamylation

Folates are transported inside cells via the three uptake carriers as a monoglutamate form, containing a single L-glutamic acid. Polyglutamylation occurs to afford folypoly-γ- glutamate by attaching multiple L-glutamic acids via γ-peptide bonds. The whole process is catalyzed by folypoly-γ-glutamate synthetase (FPGS).

Polyglutamylation is known to facilitate better folate uptake in human tissue. It is mainly summarized in three respects: i) help to increase folates retention in cytosol; ii) beneficial to increasing folate cofactors’ retention in mitochondria; iii) help to bind to folate-dependent enzymes in case of insufficient folate sources. 17-19

Folate polyglutamate can also be hydrolyzed back to its monoglutamate form. The hydrolysis reaction is mediated by folypolyglutamate hydrolase (FPGH), which is found in the lysosomes.20 Either endo or exopeptidase FPGH can be based on species type and has not been studied as well as FPGS.21

Figure 4. Representatives of classical antifolates (and their principal target(s)).

1.4 Therapy of Antifolates

Antifolates are categorized into two types, classical and nonclassical, based on their transport mechanism and polyglutamation.

A. Classical antifolates

The classical antifolates contains an L-glutamate moiety such as methotrexate

(MTX), pemetrexed (PMX, LY231514, Alimta), raltitrexed (RTX, ZD1694, Tomudex),

PDDF (N10-propargyl-5,8-dideazafolate) (Figure 4), of which the structures are akin to the

folates and their metabolites, such that they can be transported into cells by the RFC, FRs, and PCFT, followed by polyglutamation by FPGS.18

Figure 5. Representatives of non-classical antifolates (and their principal target(s)).

B. Non-classical antifolates

The non-classical antifolates do not have the L-glutamate moiety but lipophilic side chains, which are proposed to be uptaken inside cells by passive and/or facilitative diffusion.10-11. To overcome drug resistance and improve targeted treatment of infections,

Non-classical antifolates have been developed to associate with classical antifolate, such as nolatrexed (AG337, Thymitaq), AG331, pyrimethamine, trimethoprim (TMP), piritrexim (PTX) and trimetrexate (TMQ) (Figure 5).

2. Cellular folate transporters

A. Reduced Folate Receptor (RFC)

RFC, ubiquitously expressed in epithelia and nonepithelial cells,22,23 is reported to be the major transport system for folates in mammalian cells and tissues (Figure 6).9, 25,26

Exhibiting preferences for reduced folates, the carrier binds substrates with a high affinity in a low capacity.27,28 Loss of RFC expression may cause physiological and developmental problems due to folate deficiency.29,30

Beside its physiological role, RFC plays an important role in the pharmacology of folates because it is considered the major transport system for antifolates, such as methotrexate (MTX), pemetrexed, and raltitrexed for chemotherapy.9, 30 Antifolate resistance may be observed with loss of RFC expression or mutation in the RFC protein in tumor cells, which leads to incomplete intracellular target inhibition and low level polyglutamylation of antifolate substrates. 9, 24, 31

1. Structure of RFC

Since murine RFC was first cloned in mid 1990s,26, 32 studies on its physiologic and pharmacologic importance have been extensively studied.9, 29, 33 Understating the protein structure is a prerequisite for determining the mechanism of membrane transport.1

RFC is a mammalian member of the major facilitator super family (MFS). MFS of transporters includes a large group of carriers, catalyzing a variety substrates’ uptake, such as amino acids, neurotransmitters, sugars, vitamins, nucleosides and organic phosphate.34

MFS proteins usually have 400-600 amino acids and comprise a symmetrical structural motif with two halves, each of which has six transmembrane-spanning -helices linked by a large hydrophilic loop, and cytoplasmic N- and C- termini.1 Structures of the bacteria

MFS proteins, lactose/proton symporter (LacY)34 and inorganic phosphate/glycerol-3- phosphate antiporter (GlpT),35 are well studied via X-ray crystallography. In both proteins, large hydrophilic cavities are found as substrate-binding sites, which are composed of helices-I, -II, -IV and -V of the N-termini, and helices-VII, -VIII, -X and -XI of the C-

termini. Helices-III, -VI, -IX and -XII, embedded in the lipid bilayer, are not found to be directly involved in substrate binding. 34-35

Figure 6. Homeostasis of folates and cellular accumulation of antifolates.9

Note: Influx and efflux transporters that transport (anti)folates. Once inside the cell,

(anti)folates undergo polyglutamylation in the cytosol and mitochondria, whereas the counteracting process of hydrolysis occurs in the lysosome.9

However, structural information for the mammalian MFS transporter human RFC

(hRFC) is limited since it is difficult to isolate sufficient quantities of purified proteins for crystallization for X-ray diffraction studies.42 State-of-the-art molecular biology and biochemistry methods have been used to characterize structural determinants of hRFC transport that impact its physiology and pharmacology, which includes its membrane

topology, N-glycosylation, functionally and structurally important binding domains and amino acids, and, more recently, tertiary and quaternary structures.32,36-42

Figure 7. Human RFC topology model.42

Note: A topology model is shown for human RFC, with 12 TMDs, internal N- and C- termini, and a loop domain connecting TMD6 and 7. The structurally and functionally important amino acids, as described in the text, are shown as red circles. A conserved stretch of amino acids (Lys204–Arg214) in the TMD6/TMD7 loop domain, which is important for transport activity, is shown as yellow circles. N-glycosylation occurs at

Asn58, which is labeled as a red triangle.42

RFC has 12 transmembrane domains (TMDs) with N- and C- termini oriented intracellular27,42-45 and a large intracellular loop between TMD6 and TMD7.27,44 Human

RFC includes 591 amino acids and is N-glycosylated at Asn58 in the loop domain connected by TMD1 and TMD2 (Figure 7).25,42 High conservation is observed in RFC from

diverse species with substantial conservation within the TMDs and the lowest homology in the intracellular loop domain connecting TMD6 and TMD7, and also in the N- and C- termini.25,42

Growing evidence has risen that the quaternary structure involving higher order oligomers is essential for structure and function of membrane transporters.47-48 Recent studies showed compelling evidence that hRFC, like many other MFS proteins, exists as a homo-oligomer.29,50 Although their co-folding forms oligomers, each hRFC monomer provides a single translocation pathway for substrates and is functionally independent of other monomers.29 Thus, hRFC monomer is identified as operational minimal functional unit for membrane transport by this system that mediates cellular uptake of folate substrates.29 However, homo-oligomers composed of hRFC monomers seems to be particular critical for cellular trafficking from the endoplasmic reticulum (ER) to plasma membranes. A dominant-negative phenotype, produced by co-expressing wild-type (WT) hRFC and inactive hRFC mutant (S138C) with impaired intracellular trafficking, led to profoundly decreased surface expression of WT hRFCs.29 This implies that oligomerization of RFC may be crucial in functional coupling between monomers by impacting plasma membrane stabilities of transporters or by modulating protein/protein interactions in the plasma membrane.29,42,51-52 Further investigation of oligomeric RFC, such as structural and regulatory determinants of oligomer formation, is important to better understand the physiological and pharmacologic role of RFC.42

Figure 8. Three-dimensional (3D) homology models of human RFC.42

Note: A 3D model for human RFC is presented, based on structure alignments between

RFC and LacY/GlpT and experimental data. (A) A side view of the RFC for which the extended C-terminal segment is truncated at Lys479. TMD1, TMD2, TMD4, and TMD5 of the N-terminal region and TMD7, TMD8, TMD10, and TMD11 of the C-terminal region are involved in formation of the hydrophilic binding site for anionic folates (colored sky

blue). TMD3, TMD6, TMD9, and TMD12 are buried in the lipid bilayer and do not directly participate in substrate binding (colored green). Panel (A) also depicts key amino acids

(shown in assorted colors) that may contribute to the binding pocket for anionic folate substrates, as described in the text. (B) A cytosolic view of only the TMD segments of the human RFC molecule so that the order of helix packing can be seen easily. TMD coloring is the same as described in (A). (C) Enhanced view of the hypothetical substrate-binding site comprising the same key amino acids depicted in (A), including Lys411, Ser313,

Tyr281, and Arg373, as described in the text. Other residues that may contribute to the substrate-binding pocket are also shown and include Arg133, Ile134, Ala135, Tyr136, and

Ser138. The physical distances between the αcarboxyl groups of Lys411, Ser313, Tyr281, and Arg373 are given in angstroms.42

2. Structure-Activity Relationship (SAR) of RFC

In studies of the SAR of RFC, researchers have been seeking to identify domains and/or residues, as well as TMD helix packing, that contribute to binding and/or translocation of (anti)folate substrates.42

A three-dimensional structural model for the hRFC monomer is generated from homology modelling from the crystal structures of bacterial RFC proteins and results of biochemical studies (Figure 8). The structure model includes TMDs 1, 2, 4, 5, 7, 8, 10 and

11 as components of an aqueous membrane-spanning translocation pathway flanked by

TMDs 3, 6, 9 and 12.53

In contrast to many other nutrient transporters, integrity of the membrane-spanning hydrophobic backbone is critical for export of the polypeptide from the ER to the cell surface rather than the cytoplasmic N- or C- termini of the protein.42, 54 Truncation of the

cytoplasmic N- or C- or both tails of hRFC did not affect the plasma membrane targeting of these constructs. Although the NH2- and COOH- cytoplasmic tails are not essential for cell surface localization, these regions play accessory roles in modulating the efficiency of expression.54 The linker domain connecting TMD6 and TMD7 in RFC is quite different across various species except for highly conserved segment Lys204-Arg214. Deleting segments (49 or 60 amino acids; positions 215–263 and 204–263, respectively) of the

TMD6/TMD7 linker from human RFC abolished transport activity.55 The transport activity was not abolished when hRFC was expressed as a half molecule TMD1-6 and TMD7-12, respectively.56 Above all, it indicate that neither the N- or C- cytoplasmic tails nor the

TMD6/TMD7 linker is critical for folate substrate binding and membrane translocation.

The TMD6-TMD7 loop domain is mainly to provide appropriate spacing between the

TMD1–6 and TMD7–12 segments for optimal carrier function.42

Scanning cysteine accessibility method (SCAM) with a functional “Cys-less” hRFC is used, in which the 11 cysteine residues were replaced with serines,43 in order to identify domains that form the putative substrate-binding pocket/translocation pathway.57,58 Studies on the patterns of transport inhibition by 2-sulfonatoethyl methanethiosulfonate (MTSES) and protection from

MTSES inhibition by leucovorin identified that the hydrophilic cavity composed of TMD4,

5, 7, 8, 10, and 11 is responsible for translocation of (anti)folates. 53,57-58

In the cysteine-scanning studies for hRFC, ten inactivating cysteine substitutions suggested the functional or structural importance of residues including Arg133, Ile134,

Ala135, Tyr136 and Ser138 in TMD4, Tyr281 in TMD7, Ser313 in TMD8, Arg373 in

TMD10, and Lys411 in TMD11. TMD helices including these resides comprise the hRFC

substrate-binding pocket.53,58 Therefore, individual replacement of Arg373, Tyr281 or

Ser313 in hRFC with cysteine caused to almost total loss of the transport function.53,58 The same result was found in the aliphatic substitutions of Arg373 although transport activity was preserved with lysine replacement.26 Lys411 in TMD11 was found to be the primary target for electrophilic attack by N-hydroxysuccinimide-activated MTX ester and can interact with (anti)folate substrate primarily through an ionic association with -carboxyl group.26 However, this interaction is not necessary for transport activity since not only the

-carboxyl group is expandable but high-affinity reversible binding of substrates to the carrier was shown with its replacement by an uncharged hydrogen or methyl group in a series of furo[2,3-d]pyrimidine antifolates, as long as the -carboxylate is kept intact.26

Systematic site-directed mutagenesis for Ser313, and cysteine-insertion mutagenesis and homobifunctional cross-linking between TMD8 and juxtaposed TMD5 are used to investigate the functional significance of TMD8 and Ser313 in RFC membrane transport.42,53 Substantial differences were observed between structurally diverse RFC

(anti)folate substrates in their binding to RFCs with mutated Ser313, and in inducing conformational changes involving the proximal end of TMD8, determined by protein cross-linking. Therefore, TMD8 and Ser313 play an essential role in binding and/or membrane translocation of (anti)folate substrates.42,53

Based on the above studies, a hypothetical model for binding (anti)folate substrates to hRFC is proposed: i) interactions between the pteridine ring of MTX and Tyr281 and

Ser313. Tyr281 is juxtaposed to pteridine ring of MTX through - interactions; Although a hydrogen bond is depicted between Ser313 and the 4-amino group of MTX, this must not be obligatory since replacement of Ser313 with alanine does not abolish the transport

functionality; ii) An ionic association between Arg373 and the -carboxylate of

(anti)folates26 is suggested; iii) Lys411 interacts with the γ-carboxyl group of MTX, and is not necessary for binding and transport activity; iv) Ser313, flanking 2-sulfonatoethyl methanethiosulfonate (MTSES) reactive positions 311 and 314 in the proximal end of

TMD8 lines the aqueous transmembrane pathway for hRFC.26

3. Transport mechanism of RFC

Folates are hydrophilic dianions since two glutamate carboxyl groups are fully ionized at physiological pH. Although it generates only small transmembrane chemical gradients, RFC is actually considered to produce a substantial electrochemical potential difference for cellular folate substrates’ uptake within the context of the membrane potential.36 It was found a unique energy source for this uphill process, in which RFC function is not related to ATP hydrolysis and is independent to Na+ and H+.59,60 However,

RFC-mediated transport exhibits high sensibility to the transmembrane anion gradient, especially the organic phosphate gradient.59,60 In ATP-dependent reactions, organic phosphates are largely synthesized and highly retained within cells. Driving force for RFC- mediated uphill cellular folate uptake is caused by the asymmetrical distribution of organic phosphates across cell membranes.60

B. Folate Receptors (FRs)

Folate receptors, a group of cysteine-rich cell-surface glycoproteins, mediate cellular folate uptake through endocytosis with high folate-substrate binding affinity

(Figure 6).9,61 Three isoforms of FRs have been identified and cloned to date, including

FR- from KB cells, CaCo-2 cells, and placenta; FR- from placenta; and FR- from malignant hematopoietic cells.14

1. Structure of FRs

In contrast to the MFS protein RFC, the isoforms FR and FR are glycosylphosphatidylinositol-anchored (GPI), whereas FR isoform is a soluble protein constitutively secreted by lymphoid cells.62-66 Genes, located on the long arm of chromosome 11 q11.3-q13.5, have been identified to encode functional folate receptor isoforms FR, FR and FR respectively in human beings.2,3,67-74 FR gene is regulated by two promoter regions, and resides upstream to exon 1, named P1; whereas the second is P4, located upstream of exon 4.75,76 FRβ expression is regulated by retinoid receptors and upregulated by all-trans retinoic acid, particularly in combination with histone deacetylase inhibitors.77, 78 And FR is secreted is because it lacks a signal sequence for

GPI anchor attachment.79-81 The three isoforms of FRs share 68-79% identical homology and consist of 229 to 236 amino acids with two N-glycosylation sites for FR and , three for FR.82

2. Transport mechanism of FRs

Cellular (anti)folate uptake by GPI anchored FRs is through receptor-mediated endocytosis. 2,82-84 To delineate discrete structural conformations representative of key stages in the endocytic trafficking of FRs and propose models for pH-dependent conformational changes, a series of six unique hFR structures that outline three distinct states relevant to the endocytic trafficking of the receptor and four structures in complex

Figure 9. Model for trafficking of human folate receptors.87

Note: (A) Schematic of ligand transport via endocytosis of hFR is depicted with three biological trafficking states. At the cell surface, the receptor at neutral to slightly basic pH is in an apo-FR conformation competent to bind ligand (state I). On ligand binding, structural transitions occur and lead to complex formation (state II). After endocytosis, ligand release occurs in the mildly acidic microenvironment of the recycling endosome.

After ligand release and under acidic conditions, the receptor likely adopts a third distinct conformation (state III) before recycling to the cell surface.87 with (anti)folate ligands2,85,86 were established (Figure 9).87 Five of the six unique structures were identified for hFRβ at pH 7.4–8.2, whereas the sixth structure was from hFRα crystallized at pH 5.5.3 To understand the molecular details of folate uptake, Dann et al.87 purified proteins via heterologous expression in eukaryotic host cells and subsequently used these properly folded glycoproteins to solve structures of hFRs in three states relevant to endocytic transport.

Figure 10. Structures of folate receptors depicting states of biological trafficking.87

Note: Cartoon models representing proposed states I, II, and III (A–C) of folate transport are represented by apo-hFRβ and the hFRβ/FOL complex at near neutral pH, and apo- hFRα at acidic pH, respectively. (A) Cartoon model of the apo-hFRβ structure is shown with conserved disulfides colored orange. (B) A cartoon depiction of the hFRβ/FOL complex shows the position of the ligand binding pocket with residues that interact with folate shown as sticks. (C) The apo-hFRα model illustrates global conformational differences in the structure of apo-hFR at pH 5.5 relative to the same at near neutral pH (cf.

A and C). (D) Conformational differences between the three trafficking states are highlighted. Four regions of the folate receptors that undergo significant conformational changes are numbered with arrows to indicate the general direction of movements and colored as seen in A–C. Variable regions in each individual model are emphasized with darker shading of the same color.87

State I. the first structure of apo-hFRβ represents the conformation of the receptor at the cell surface before association with folate (Figure 10A, Figure 11A), which was crystallized under physiological conditions (pH 7.4) with a resolution of 1.8 Å. By investigating crystal structure of apo-hFRβ, it has been proposed that i) The majority of apo-hFRβ lacks defined secondary structure, with ∼30% of residues in six α-helices and less than 8% in four short β-strands (Figure 9, Figure 10A); ii) Eight disulfide bonds

Figure 11. Conformational changes in the hFR ligand binding pocket during states of biological trafficking.87

Note: Residues that interact with folate in the complex structure are modeled to highlight the movements of conformationally variable loops (darker shading) between the open state at neutral pH (A), the folate complex (B), and the closed state at acidic pH (C). Polar interactions in the folate complex are designated with dashed lines. Tyr-76, which forms hydrophobic interactions benzoyl moiety of the folate ligand, is removed for clarity.87 stabilize the fold by tethering loops and secondary elements; the most prominent feature is iii) A ∼10 × 15 Å cleft with a depth of ∼20 Å serves as the binding site for ligands and that the receptor is in an open conformation for interacting with folates.87

Figure 12. Crystal structure of FR bound to folic acid.61

Note: a. Two views of the complex, with FR in green, folic acid in grey, NAG in orange and the disulphide bonds depicted as yellow sticks. The N and C termini are labelled. b

Ribbon diagram of FR, with folic acid and NAG in green stick presentations, overlaid with the semi-transparent receptor surface. c. Charge distribution surface of FR with a close-up view of the ligand binding pocket entrance. Folic acid carbon atoms are coloured grey, nitrogen atoms blue, and oxygen atoms red. A colour-code bar (bottom) shows an electrostatic scale from 23 to 13 eV.61

State II. To clarify structural determinants for ligand binding and to deduce conformational changes in the receptor when interacting with (anti)folates, the second

structure hFRβ/folate complex was crystallized at neutral to basic pH values (7.0–8.5), representing the general conformation of the receptor at the cell surface after binding to a ligand (Figures 9, Figure 10B, Figure 11B). It is identified that i) The pterin ring of folate is positioned deep within the aforementioned cleft; ii) the 4-aminobenozyl linker extends through the central region of the cleft; iii) and the glutamic tail is partially exposed to solvent with the γ-carboxylate protruding from the binding cleft (Figure 10B, Figure

11B).87

State III. The third structure, structure of apo-hFRα has been determined at pH 5.5 in three distinct crystal forms from three protein constructs to resolutions of 1.55, 1.8, and

2.2 Å, with Rfree values of 19.9, 26.9, and 19.9%, respectively, representing the endosomal conformation of the receptor after folate has been released (Figures 9, Figure 10C, Figure

11C). Endocytosis leads to folate internalization mediated by FRs and wherein is occurred folate release. This release has been proposed to depend on pH switch in the hFR/folate complex as the micro-enviroment of the endosome acidifies after folate is transported by the encapsulated endosome. It has been observed that i) folates in the three crystal forms are statistically identical and that ii) several conformational changes take place on loops that surround the binding cleft on acidification.87

With the exploration of the structures representing three biological trafficking states of hFRs, some conclusions were drawn: i) hFRs are poised in an open conformation at the cell surface and undergo minor rearrangements to make specific interactions with folates;3,87,88 ii) After ligand release under acidic conditions, a major reorganization occurs to hFRs wherein the folate binding site is occluded through the movements of four surface loops; iii) A key residue, Arg125, anchors

Figure 13. Structural and biochemical analysis of FRa–folic acid interactions.61

Note: a, The sA-weighted 2Fo–Fc electron density map for folic acid, shown as a grey mesh. b, The internal charge distribution surface of the binding pocket is shown using the same colour code as in Fig. 1c, with folic acid shown in stick presentation. c, Folic acid- binding network with close-ups of the folic acid head and tail groups. Residues that line the binding pocket are shown in green and folic acid is shown in grey. Hydrogen bonds are indicated by dashed lines.61 the inhibitory loop in the binding site that is stabilized by a series of ionic interactions mediated by a His54, preventing the pterin ring of folate from entering the binding cleft of the receptor; iv) pH of the receptor environment plays a major role in the structural

rearrangements with the folate binding cleft poised in an open conformation at neutral pH and occluded at acidic pH in the endosome3,87,88 v) pH-dependent changes are indicated in the affinity for folate and less changes for antifolates.87

3. Structure-activity relationship (SAR) of FRs

To elucidate structural determinants for ligand binding of FRs, hFR was expressed as a secreted IgG Fc fusion protein (FR–Fc) in HEK293 cells by deleting its carboxy- terminal glycophosphatidylinositol anchor.61 The deglycosylated FR–Fc had a folic acid- binding affinity similar to the fully glycosylated protein and is crystalized in a resolution of 2.8A˚.61

Based on its crystal structure, FR has an overall globular structure, composed of four long -helices, two short a-helices, four short β-strands (β1–β4) and many loop regions. Eight disulfide bonds formed by 16 conserved cysteine residues greatly stabilize the tertiary structure. With three predicted N-glycosylation sites, clear electron density for

N-acetylglucosamine (NAG) is observed for Asn47 and Asn139, and partial electron density for Asn179.61

Helices 1, 2, 3 and 5 tied together by four disulfide bridges (Cys35–Cys83,

Cys44–Cys87, Cys74–Cys124 and Cys117–Cys167) build up the core domain. The long and open folate-binding pocket of FR is formed by 1, 2 and 3 in the back; the amino- terminal loop, β1 and β2 in the bottom; the 1–2 and 3–4 loops in the left and top; and

4, 5, β4 and β3 in the right (Figure 12, Figure 13b).61

Figure 14. Interaction map of folic acid with ligand-binding-pocket residues.61

Note: The folic acid chemical structure is shown in magenta, pocket residues in black and hydrogen bonds as green dashed lines with bond distances.61

Pteridine ring. Folic acid with the pterin scaffold is oriented into an extended groove of

FR in the direction roughly perpendicular to the plane formed by helices 1, 2 and 3.

The interactions around the pteroate moiety contain both hydrogen bonds and hydrophobic interactions: 1) the pterin ring is stacked between the parallel side chains of Tyr85 and

Trp171, and capped by Tyr175; 2) the hydrophilic pterin ring N and O atoms form a series of hydrogen bonds with receptor residues. Specifically, the pterin N1 and N2 atoms form strong hydrogen bonds with the side-chain carboxyl group of Asp81, the N3 and O4 atoms with the Ser174 hydroxyl group, the O4 atom forms two hydrogen bonds with the guanidinium groups of Arg103 and Arg106, and the N5 atom forms one hydrogen bond

(Figure 13c, Figure 14).61

The folic acid aminobenzoate is stabilized by hydrophobic interactions with Tyr60,

Trp102 and Trp134, which line the middle of the long ligand binding pocket (Figure 14).

Extensive interactions are also observed for the glutamate group, which engages six hydrogen bonds, contributed by the side chains of Trp102, Lys136 and Trp140, as well as by backbone interactions with His135, Gly137 and Trp138 (Figure 13c, Figure 14). Most residues involved in ligand binding are identical among different subtypes of FR regardless of their origins, indicating that the observed folate-binding interactions are probably conserved in all three different receptor subtypes. In addition, the most physiologically prevalent folate, 5-M THF, can be easily docked into the FR ligand-binding pocket in a mode very similar to that of folic acid, suggesting that the fundamental mechanism of folate recognition is conserved.61

To validate the structural observations, a series of ligand binding affinities of FRα mutants have been examined that have alanine mutations in the key folate-contacting residues. Mutation on Trp171 abolished the expression of the receptor, suggesting that this residue is critical for protein stability. Compared with that of wt FRα binding to [3H]-folic acid with a Kd of ~0.19 nM, replacement of Asp81 decreased affinity by more than one order of magnitude. The result indicates that this interaction is a key contributor to high- affinity ligand binding, which is consistent with the strong interaction of the aspartate carboxyl oxygens with the pterin N1 and N2 nitrogens. However, mutations of Tyr175,

Lys136 and Arg106 have little effect on folic acid binding.61

Above all, a structural rationale of designing FRs-targeted antifolates is identified based on structural and mutational analyses: i) the pterin group or its equivalent is absolutely required to anchor folates in the binding pocket of the receptor; ii) glutamate

group is required for conjugation with drugs and imaging reagents without adversely influencing receptor and ligand interactions.61

4. FRs distribution in human body

In contrast to RFC, which is ubiquitously expressed in normal tissues, FR isoforms are not evenly distributed among the tissues and cell types.

FRα primarily limits its expression to epithelial cells and is upregulated in malignant tissues, derived from the same cell types including choroid plexus, proximal kidney tubules, fallopian tube, uterus, epididymis, submandibular salivary, bronchial gland, acinar cells of the breast, type I and type II pneumocytes in the lung, and trophoblasts of the placenta.89-91 Expression of FRα in proximal renal tubule cells is at the apical (lumial) surface; whereas in retinal pigment epithlium, it is expressed on the basolateral membrane.89,92

FRβ is expressed in latter stages of normal myelopoiesis and in placenta, spleen, and thymus.72,93 Functional FRβ is found in myeloid leukemia and in activated macrophages associated with inflammation and malignant tumor.62,94 Therefore, FRβ is potentially useful as a marker for myeloid leukemia, for chronic inflammatory diseases such as rheumatoid arthritis, and for tumor-associated macrophages.77,94-96

Expression of FRγ has been identified in normal and malignant hematopoietic cells, as well as in carcinomas of the ovary, endometrium, and cervix.63,76,82

5. FRs and antifolate chemotherapy

In apical cells of normal tissues, FRs are not involved in the circulation, The FRs serve as markers for diseased cells in cancers and inflammatory disease, and the

development of three distinct types of FR-targeted therapeutics based on antibodies, folate- conjugates, and antifolates are currently being pursued.11,111,112 Monoclonal antibodies against hFRα and hFRβ could promote clearance of FR positive cells by the immune system, folate-conjugates aim to deliver cytotoxic cargo or imaging agents to FR-positive cells, and FR-targeted antifolates would potentially eliminate cytotoxic side effects of current antifolates that are non-selectively delivered to normal cell via RFC.103-110 These therapies have enormous potential for the treatment of cancer and inflammatory disease.

Elevated FRα expression has been considered a potential negative prognostic factor for chemotherapy resistance for at least breast, ovarian, and endometrial cancers.98 Also, the expression of FRα on the apical surface of most normal cells is very low. Such that, the difference in expression makes FRα a very attractive therapeutic target for novel anticancer agents that would have limited toxicity on normal tissues.99,100

Approximately 90% of epithelial ovarian cancers express FRα, and its expression is associated with parameters of biological aggressiveness;63,65,81,86 indeed, the highest FRα expression level is correlated with poorly differentiated tumors.81,95 Furthermore, the selective upregulation of FRα on tumors compared with normal tissue suggests FRα as a therapeutic target in epithelial ovarian cancer.101

C. Proton-coupled folate transporter (PCFT)

PCFT, a new folate transporter was identified in 2006,113 belongs to the superfamily of solute carriers, but is functionally distinct from the RFC (Figure 6).9 PCFT functions optimally at acidic rather than neutral pH. Besides that, it exhibits differences in its specificities for particular (anti)folate substrates and tissue distribution compared with

RFC.114,115

A distinguishing characteristic of PCFT is identified as acidic optimum (pH 5–

5.5).113,115 PCFT transport decreases dramatically as the pH increases from pH 5.5. and abolishes transport above pH 7 even though the extent of residual transport varies for different substrates.114-117

1. Structure of PCFT

PCFT consists of 459 amino acids with an approximate MW of ~50kDa and is located on chromosome 17q11.2. Like other MSF proteins, PCFT has 12 TMDs with the N- and C- termini oriented into the cytoplasm (Figure 15). This is supported by immunofluorescence staining of hemagglutinin (HA)-tagged PCFT constructs and by SCAM with MTSEA (2- aminoethyl methanethiosulfonate)-biotin.118 Two N-glycosylation sites (Asn58 and Asn68) have been identified in the loop domain between TMD1 and TMD2 of PCFT(Figure 15).118

The integrity of these sites is not essential for transport function since mutation on either of the asparagine residues to glutamine did not abolish PCFT expression and function.118

However, about 40% decreased transport activity of that for wt PCFT was observed with the replacement both Asn58 and Asn68 to glutamine. Deleting the C-terminus of human

PCFT at position 449 had no effect on apical membrane targeting or transporter in Madin-

Darby canine kidney and Caco-2 cells.118

With employment of a series studies including molecular characterization of human

PCFT mutants that result in loss of function in hereditary folate malabsorption (HFM) cases, and systematic mutagenesis of these HFM mutants, a group of structurally and functionally important amino acids in human PCFT have been identified.

Figure 15. Schematic structure of human PCFT membrane topology.42

Note: A topology model is shown for human PCFT, with 12 TMDs and internal N- and C- termini. Structurally or functionally important amino acids, as determined from published mutagenesis studies and in patients with hereditary folate malabsorption (HFM), are shown as red circles. GXXXG putative oligomerization motifs are shown as yellow circles

(Phe157 and Gly158 in the G155XXXG159 motif are shown as red circles because they are also structurally and functionally important). N-glycosylation occurs at Asn58 and

Asn68 (shown as red triangles).42

His247 is predicted to interact with Ser172 to limit substrate access to the folate- binding pocket such that transport substrates selectively, which is localized to the loop region separating TMD6 and TMD7, lie in the cytoplasmic opening of the water-filled translocation pathway (Figure 15).118 His281, oriented to the extracellular region in TMD7, is believed to play an important role in PCFT protonation with increased substrate binding to the carrier .118

PCFT has a highly conserved segment DXXGRR (Figure 15) which links TMD2 and TMD3 (residues 109–114) and includes a β-turn.115,119,120 Key residues in this segment, including Asp109 and Arg113, that are essential for substrate binding and/or translocation because transport function is totally abolished with both conservative and nonconservative replacement.42,119,120

A series of mutagenesis studies identified a group of residues to be functionally important in human PCFT, including Leu161 in TMD4, Glu232 in TMD6, and Ile304 in

TMD8, Gly189 and Gly192 (TMD5).116,121 The following experimental results further support the prediction: loss of transport was associated with i) a reduced rate of carrier translocation in Glu232 Gly mutant or ii) lower substrate affinities in Ile304Phe and

Leu161Arg mutants. In later studies by Shin et. al122 in 2012, although it was probably not directly involved in substrate binding, Pro425 was predicted to be essential for selectively transporting substrates by controlling PCFT conformation. It was shown that mutating

Pro425 to Arg in the loop junction flanking TMD12 eliminated binding of MTX and other substrates to PCFT, but preserved binding of PMX.122 Given its location, Pro425 is proposed to regulate the conformational change that selectively inhibits (anti)folate substrate binding rather than directly participate in the substrate binding.

2. Transport mechanism of PCFT

Folates are bivalent anionic with two carboxyl groups ionized at physiological pH, which indicates that more than two protons must be transported with each folate molecule to explain the net positive charge of the PCFT–folate–proton complex.113,116,123,124 PCFT is identified as a folate–proton symporter in which the downhill flow of protons is coupled to the uphill flow of folates into cells. In the absence of a transmembrane pH gradient,

interestingly, PCFT can still function well. Membrane potentials is the power that drives the folate substrate transport,113 similar to the divalent metal-ion transporters. At low pH,

PCFT showed channel-like activities (i.e., protons can flow independent of folates).121

Removing extracellular Na+, K+, Ca2+, Mg2+, or Cl− or changing membrane potential does not impact PCFT activity,113 which is consistent with an electroneutral mode of PCFT transport. But the inwardly directed proton gradient is required for PCFT- mediated folate transport based on the following observations: (i) Reduced transport by

PCFT was observed if the transmembrane proton gradient was dissipated by a proton ionophore in Xenopus oocytes and a K+/H+-exchanging ionophore in HEK293 cells.113 (ii)

A transvesicular pH gradient led to increased unidirectional folate transport and substantial transmembrane folate concentration gradients in rabbit jejunum, from the low pH to the high pH compartment, consistent with a proton-coupled process. (iii) PCFT transport was inhibited with treatment of HeLa cells with nitrate or bisulfite, which abolished the pH gradient.121 Finally, (iv) Proton coupling was confirmed by cellular acidification accompanied folate transport into Xenopus oocytes.113

3. PCFT distribution in human body

Within the intestine, apical brush border membrane of the proximal jejunum and duodenum is identified as the site with the highest PCFT expression; whereas other segments of the intestine and colon levels showed remarkably decreased level of PCFT expression.113 PCFT is also expressed in other normal tissues including the kidney, placenta, spleen, the sinusoidal membrane of the liver, the basolateral membrane of the choroid plexus, and the retinal pigment epithelium.113,114,121 Beside normal tissues, solid tumors are another major sites of PCFT expression, such as breast, lung, ovarian, and lung

human tumor cell lines, while human leukemia cell lines exhibited a very low level expression of PCFT.114

Figure 16. De novo Purine Biosynthesis.127,130 (Note: the circle indicates the carbon in imidazole ring by transferring one carbon from N10-formyl THF; the number associated with the circle indicates the step of the formyl-transfer reaction in the whole purine biosynthesis)

4. PCFT and antifolate chemotherapy

PCFT transports folates most effectively at pH 5-5.5, exhibiting an acidic pH optimum. Above pH 7, PCFT has a limiting capability of folate transport, although specific transport efficiencies with increasing pH vary for particular (anti)folate molecules, largely reflecting differences in substrate binding.203 The interstitial pH of solid tumors is often acidic,227-228 which is favor of PCFT transport over RFC and FRs. High levels of human

PCFT (hPCFT) transcripts were observed in a broad range of human tumors226 and a prominent low-pH transport route was identified in 29 of 32 solid human tumor cell lines.210 The discovery indicates that hPCFT is a promising chemotherapy target for it transports cellular (anti)folates more efficiently in solid tumor cells over normal tissues due to solid tumors’ acidic microenvironment.

2. Intracellular Target ------Glycinamide-ribonucleotide transformylase

(GARFTase)

10 The third reaction (Figure 16), transfer of one formyl group from N -CHO-FH4 to the primary side-chain amino group of glycinamide ribonucleotide (GAR) to yield formylglycinamide ribonucleotide (FGAR) and tetrahydrofolate, is catalyzed by

GARFTase. As the key step of de novo purine biosynthesis, the reaction is the first one of two folate-dependent formyl transfers. The FGAR formed is further converted to amino- imidazolecarboxamide ribosyl-5-phosphate (AICAR). Then, Amino- imidazolecarboxamide ribosyl-5-phosphate formyl transferase (AICARFTase) converts

AICAR to formyl-amino-imidazolecarboxamide ribosyl-5-phosphate (FAICAR), and ultimately leads to the biosynthesis of inosinic acid (IMP) by incorporating C-8, which is a purine precursor that can be further metabolized to guanosine and adenosine nucleotides.127

Since lometrexol (LMTX) (Table 1) was evaluated for clinical trials by the Eli Lilly

Corporation in 1985, unceasing interest was afforded to GARFTase, because LMTX inhibits the catalytic mechanism of the formyl group transfer,128,129,131-133 and hence the synthesis of DNA precursor purines,130 and its potential as an important intracellular target for chemotherapeutic drug design.109,117

1. Structure of GARFTase

First discovered and partially characterized from pigeon liver in a pioneering investigation by Warren and Buchanan,135 the structure of GARFTase (EC 2.1.2.2) was then intensively studied by employing crystallography technique.

In the Escherichia coli (E. coli), the crystal structure of GARFTase was established at pH 3.5 (1.8 Å),151 pH 6.75 (2.8 and 3.0 Å),142 and pH 7.5 (1.9 Å)137 and at neutral pH binding with GAR and 5-deazatetrahydrofolate (2.5 Å),132 GAR and 10-formyl-5,8,10- trideazafolic acid (2.1 Å),133 and two multi-substrate adduct inhibitors (1.96 and 1.6

Å).131,138 Later, the crystal structure of hGARFTase was established at pH 4.2 (1.7 Å), at pH 8.5 (2 Å). At pH 8.5 binding with the substrate β-GAR (2.2 Å)134 and at pH 7 in the complex with the substrate 10-trifluoroacetyl-5,10-dideaza-acyclic-5,6,7,8-tetrahydrofolic

139 138 acid (10-CF3CO-DDACTHF) (Table 1) (2 Å) and a series of folate inhibitors.

With a comparison of the structures of hGARFTase and E. coli GARFTase, a series of key points have been identified: i) In contrast to the E. coli GARFTase dimer below pH

6.8, a monomer hGARFTase was observed at a wide range of pH values; ii) In E. coli

GARFTase, the active site loop helix composed of residues 110-131 was observed a pH dependent order-disorder transition, while that of hGARFTase had a highly conserved conformation under all pH ranges from pH 4 to pH 9; iii) Compared with the same

conformation adopted by the substrate-binding pocket in E. coli GARFTase from pH 3.5 to pH 8, changes from an open to an occluded conformation occured with the loop of residues 8-14 in the hGARFTase under a wide range of pH conditions, to prohibit substrate binding. iv) In the unliganded hGARFTase, conformations of the folate-binding loop are different from those previously identified for the E. coli GARFTase.134

2. Catalytic Mechanism of GARFTase

GARFTase catalyzes the biosynthesis of FGAR by transferring the formyl group

10 from N -CHO-FH4 with the byproduct tetrahydrofolate. A sequential mechanism is suggested through kinetic studies on the GARFTase of E. coli,128 human140 and murine.141

It has been proposed that the GAR amino group directly attacks the formyl carbon of N10-

CHO-FH4, and a tetrahedral intermediate is formed. It indicates that proton transfers are required to form this intermediate and to break down to the final product FGAR. Although it has not been experimentally verified, an “anchored” water molecule has been proposed responsible for the critical proton transfer between GAR and the folate cofactor, instead of the invariant amino acids of the active site.142

The mechanism for the formyl transfer mediated by GARFTase is shown in Figure

10 17. i) In the first step, when the folate cofactor N -CHO-FH4 binds to the active site of

GARFTase, two hydrogen bonds are formed between the formyl group of folate and the hydrogens of Asn106 and the protonated imidazolium group of His108. In addition, a salt bridge is formed between Asp144 and the imidazolium of His108 in the binding domain ii)

In the second step, a tetrahedral intermediate is formed by the nucleophilic attack of GAR’s free amino group to the activated formyl group of folate. iii) In the third reaction, a catalytic

Figure 17. Proposed mechanism for GARFTase.129 water molecule is proposed to mediate the proton transfer from GAR to the N10 of folate.

Then the product FGAR is formed through the collapse of the tetrahedral intermediate, in which the carboxylate of Asp144 facilitates to position this catalytic water molecule by a hydrogen bond.129

Figure 18. Crystal structure (MOE 2008.10) of hGARFTase in a binary complex with inhibitor 10-CF3CO-DDACTHF at pH 7 (PDB ID 1NJS).

3. Catalytic Binding domain of GARFTase

To elucidate structural determinants of hGARFTase binding domain, intensive work has been carried out. The X-ray crystal structure (Figure 18) of hGARFTase with the

139 co-substrate analog inhibitor 10-CF3CO-DDACTHF was established by Zhang et al. at pH 7 with a resolution of 1.98 Å (PDB entry 1NJS). The substrate binding domain of hGARFTase is located at the interface between the N-terminal and the C-terminal mononucleotide binding domain, which consists of three parts: i) the pteridine ring binding cleft; ii) the benzoyl-glutamate region; and iii) the formyl transfer region.

Pteridine Binding Cleft.

The quinazoline ring of 10-formyl-5,8,10-trideazafolic acid (10-formyl-TDAF) in the E. coli GARFTase complex (PDB entry 1C2T) binds to the diaminopyrimidinone ring

of 10-CF3CO-DDACTHF (Table 1) in a similar fashion in the active binding pocket, which is a long cleft buried deep in the hGARFTase.133,139 The aliphatic hydrocarbon bridge, connecting the diaminopyrimidinone ring, exhibits more flexibility to conformationally adjust itself to the binding site, such that it allows optimization of the gem-diol group interactions with the protein. Because the hydrocarbon bridge is longer than the fused benzene ring in 10-formyl-TDAF.

All the key interactions with the quinazoline ring of 10-formyl-TDAF are also conserved with the diaminopyrimidinone ring, while additional critical hydrogen bond interactions with hGARFTase are observed. The deep binding cavity is encircled by several hydrophobic residues that hold the diaminopyrimidinone ring, and are composed of Leu85,

Ile91, Leu92, Phe96, and Val97 that reside on one side and the folate-binding loop with residues 141−146 at the other side.133,139

Eight key hydrogen bonds are observed between the diaminopyrimidinone ring and the main chain amides and carbonyls of residues Arg90, Leu92, Ala140, Glu141, and

Asp144, along with two “fixed” water molecules (W18 and W70). N2 of the diaminopyrimidinone is oriented within the hydrogen bonding range (3.1 Å) of the Glu141 backbone carbonyl oxygen through 15° tilt of the diaminopyrimidinone ring of 10-CF3CO-

DDACTHF relative to the quinazoline ring of 10-formyl-TDAF. The N8 of the folate pteridine ring has been proposed to be of importance in targeting and interacting with the folate-binding domain. In the E. coli GARFTase complex, the quinazoline ring of 10- formyl-TDAF replaces the N8 of the folate pteridine ring with a carbon without abolishing the binding interaction with GARFTase. It indicates that the N8 more likely contributes to substrate recognition by the folate transport system and/or FPGS rather than binding. In

contrast, the N8 is preserved in the diaminopyrimidinone ring of 10-CF3CO-DDACTHF

(Table 1), in which the hydrogen bond interactions are observed between this amino nitrogen and the carbonyl oxygen of Arg90 (2.8 Å) and an anchored water molecule W70

(2.7 Å).133,139

Benzoyl-glutamate region

By exploring the crystal structure of hGARFTase in the binary complex with 10-

CF3CO-DDACTHF, the p-aminobenzoate moiety, oriented in the middle of the side chains of Ile91 and Ser118, inserts deep into a hydrophobic cavity. The electron density of the carbonyl group is found to be in the same plane as the phenyl ring.139

Approximately perpendicular to the p-aminobenzoate plane, the glutamate tail is also parallel to the hydrocarbon bridge of the diaminopyrimidinone ring in 10-CF3CO-

DDACTHF. In contrast to its poorly-ordered structure in E. coli GARFTase complex, the solvent-exposed glutamate tail shows highly conservative-oriented structure in the binary complex of hGARFTase with 10-CF3CO-DDACTHF. Key interactions are observed and provide the evidence to support the well-ordered glutamate tail structure in hGARFTase: i)

The glutamate α-carboxyl group interacts with the Arg64 through a salt bridge (2.7 Å), such that it orients the γ-carboxylate outwards to the solvent. ii) Another H-bond interaction is observed between the Ile91 backbone amide and the α-glutamate carboxyl group (2.8 Å), which may restrict the orientation of the glutamate moiety in the binding pocket.139

Formyl Transfer Region and the Gem-Diol Structure.

In the formyl transfer region, several key interactions are identified for the high

139 binding affinity of hGARFTase with substrate 10-CF3CO-DDACTHF. The ketone

group is proposed to be hydrated as a gem-diol since strong electron density is observed next to the ketone oxygen, similar as that observed in the complex of the E. coli GARFTase with two substrates 10-formyl-TDAF and β-GAR(PDB ID 1C2T). The gem-diol intermediate interacts with the formyl transfer region mainly through hydrogen bonds, in which Asp144 and His108 play an essential role in the formyl transfer reaction with the intermediate. Several key hydrogen-bond interaction are observed between both hydroxyl groups of the gem-diol intermediate and carboxylate group of Asp144 (2.5 and 2.7 Å), N3 in the imidazole ring of His108 [OA1 (2.7 Å) and OA2 (3.1 Å)], and a potential hydrogen bond is proposed between one of the hydroxyl group [OA2 (3.0 Å)] with the backbone carbonyl oxygen of Gly117.139

4. GARFTase targeted chemotherapy

Since purine nucleotides are essential precursors for RNA and DNA biosynthesis,

GARFTase-targeted cancer chemotherapy has been proposed, which inhibits de novo purine biosynthesis.76-78 Not only does it interferes with DNA replication, but also inhibits purine production in tumor cells and disturbs the cellular energy balance via ATP depletion.

GARFTase-targeted cancer chemotherapy has been supported by the evidence that

GARFTase was inhibited by a potent antitumor agent 5,10-dideazatetrahydrofolate, such that de novo purine biosynthesis was also inhibited.143 However, these analogs were found to be too toxic for clinical use.218

In contrast to normal cells that can rely solely on the salvage pathway for purines, many types of cancer cells are strongly dependent on the de novo purine biosynthetic pathway due to an impaired purine salvage pathway.136,144 Some have been shown to have lost the capacity for purine salvage in the course of genomic deletions responsible for cell

Structure Compound Biological Activity Ref.

Lometrexol 220 (DDATHF) hGARFTase (Ki) 6 nM (LMTX)

AG-2037 222 (pelitrexol) hGARFTase (Ki) 0.5 nM

10-CF3CO- rhGARFTase (Ki) 15 nM DDACTHF 139 R = CF3 CCRF-CEM (IC50) 16 nM

1 n = 3 hGARFTase 2.44 µM KB 1.7 nM IGROV1 2.2 nM 207 2 n = 4 hGARFTase 0.15 µM KB 1.9 nM IGROV1 3.6 nM

3 n = 3 hGARFTase 0.06 µM KB 0.55 nM IGROV1 0.97 nM 117 4 n = 4 hGARFTase 3.31 µM KB 3.17 nM

IGROV1 176 nM Table 1. Pyrimidines as GARFTase Inhibitors

transformation.144 The 9p21 chromosomal deletion, as one of the most prevalent oncogenic

transformation steps in many tumors, leads to the loss of the tumor suppressor genes

CDKN2A and CDKN2B which encode proteins that bind Hdm2, regulating the tumor suppressor p53 and the accompanying loss of the nearby MTAP gene. Being a key purine salvage enzyme, loss of Methythioadenosine phosphorylase (MTAP) renders that the tumor cells completely dependent on de novo synthesis (versus salvage) of purines. Tumors with such 9p21 chromosomal deletions are uniquely sensitive to purine biosynthesis inhibitors, while normal cells are not. Such chromosomal deletion is found in many of the most refractory tumors, including 70% of all gliomas, 50% of all T-cell leukemias, 40% of all lung cancers and 47% of all pancreatic cancers.145,146 Thus, inhibitors of de novo purine biosynthesis are able to target sensitive tumor cells selectively over normal cells, including

GARFTase inhibitors.145,146

Lometrexol. Lometrexol (LMTX, Table 1) is the first GARFTase inhibitor to enter clinical trials, synthysized by Taylor and coworker in 1985 with the term of the 6-(R)- isomer of 5,10-dideazatetrahydrofolic acid (DDATHF).220 Since then, GARFTase and the purine de novo biosynthetic pathway have been established as viable targets for chemotherapy. LMTX exhibits poor inhibition against both DHFR and TS, but is a potent

220 inhibitor against GARFTase (Ki = 6 nM) and a substrate for FPGS. The diastereomers were separated and found to be equipotent as inhibitors of cell growth and as substrates for

FPGS.220 The (6R)-diastereomer was chosen for further development for it corresponds to the configuration found in natural tetrahydrofolates. Severe and cumulative myelosuppression and mucositis were observed among patients with treatment of LMTX in phase I clinical trials. The cumulative toxicity of LMTX is thought to be lack of cellular uptake selectivity of FRs over RFC, resulting in increased cellular levels.212,213

AG-2037. AG-2037 (Table 1) was designed from the X-ray structure of the

GARFTase. AG-2037 (pelitrexol) is currently in phase I clinical development (Table 1).221

Compared with LMTX, the configuration at C-6 is reversed and the thiophene is methylated at the 4-position of AG-2037. As a potent inhibitor of GARFTase (Ki = 0.5 nM), AG-2037 exhibits significant antiproliferative effects against tumor cells in vitro and in vivo.222 Several phase I studies have been completed that indicate that AG-2037 is well tolerated and its maximum tolerated dose and schedule for phase II studies have been determined.223-224

139 10-CF3CO-DDACTHF. Boger and coworkers reported the synthesis and evaluation of 10-(Trifluoroacetyl)-5,10-dideaza-acyclic-5,6,7,8-tetrahydrofolic acid (10-

CF3CO-DDACTHF, Table 1). Being a potent inhibitor of tumor cell proliferation, 10-

CF3CO-DDACTHF exhibits inhibition against CCRF-CEM cells with an IC50 of 16 nM, which represents a 10-fold improvement over LMTX. 10-CF3CO-DDACTHF specifically inhibits recombinant hGARFTase (Ki = 15 nM), and is stable without displaying competitive oxidative deacylation.139

Pyrrolo[2,3-d]pyrimidines. A series of novel 6-substituted classical pyrrolo[2,3- d]pyrimidines were reported by Gangjee et al. recently117,207 as cytotoxic antifolates with varying lengths of the carbon bridge region (Table 1 shows only the three- and four-carbon bridge analogs). These antifolates are characterized by selective FRα and FRβ transport over RFC transport. The three- and four-carbon bridge analogs were the most active for both series toward FR-expressing human tumors (KB and IGROV1). Potent inhibition of

GARFTase, which is the first folate-dependent reaction in de novo purine nucleotide biosynthesis, resulted in the cytotoxicity.109,207 The three-carbon pyrrolo[2,3-d]pyrimidine

derivative, 1 (Table 1), was subsequently reported to also be a substrate for PCFT, thus providing another means of tumor targeting.225

In an attempt to identify antifolates with poor transport by the ubiquitously expressed RFC relative to the other major (anti)folate transport systems with GARFTase as the intracellular target, Gangjee and coworkers117 reported a series of pyrrolo[2,3- d]pyrimidines to afford compounds 3 and 4 by isosterically replacing the benzoyl ring

(such as 2) with a thienoyl ring (Table 1). The four-carbon analog 4 of this series showed remarkable inhibitory potency against tumor cell proliferation, resulting from FR- and

PCFT-mediated cellular uptake over RFC, and inhibition of GARFTase. Compared with the most potent of the previously published four-atom pyrrolo[2,3-d]pyrimidine 2 with a benzoyl ring in the side chain 207, compound 4 is much more potent in vitro (Table 1).

Significant in vivo antitumor activity was observed for compound 4 with severe combined immunodeficient (SCID) mice bearing both early and more importantly advanced stage

KB tumors.117

II. CHEMICAL REVIEW

The chemistry related to the present work will be reviewed and includes synthetic approaches to the synthesis of pyrrolo[2,3-d]pyrimidines.

Pyrrolo[2,3-d]pyrimidines

A large body of literature exists for the synthesis of pyrrolo[2,3-d]pyrimidines because of their application as deazapurine analogs. The synthetic strategies to this ring system have three broad classifications from:

1. From pyrimidines

2. From pyrroles

3. From triazines

4. From others

1. From pyrimidines

Scheme 1. Synthesis of pyrrolo[2,3-d]pyrimidines 8.147

Shaik Karamthulla et al.147 first reported the synthesis of pyrrolo[2,3-d]pyrimidine

8 (Scheme 1) by a three-component reaction of arylglyoxal monohydrate (1 mmol) 5, 6- amino-1,3-dimethyluracil (1 mmol) 6 and thiols 7 (1 mmol) at 130 °C for 20 min in acetic acid under microwave heating conditions without any catalyst or promoter. A good yield was obtained for a novel series of products.

Scheme 2. Synthesis of pyrrolo[2,3-d]pyrimidine 11.147

Shaik Karamthulla et al.147 also reported the synthesis of pyrrolo[2,3-d]pyrimidine

11 (Scheme 2) by the three-component reaction of phenylglyoxal monohydrate 5, 6-amino-

1,3-dimethyluracil 9 and malononitrile 10 to examine the replacement of thiol with a C-

Scheme 3. Synthesis of pyrrolo[2,3-d]pyrimidine 15.148 nucleophile malononitrile. By exploring a series of reagents, the three-component reaction in ethanol under microwave heating conditions without any catalyst or promoter obtained good yields of a novel series of products with pyrrolo[2,3-d]pyrimidinedione core.

Wang et al.148 reported a general, mild and environmentally benign method for the synthesis of pyrrolo[2,3-d]pyrimidine-6-carboxamide 15 (Scheme 3) through two routes by replacing Pd with Cu catalyst to perform more efficiently and more friendly to the environment . The two routes are modified by referencing Besong’s method149 and

Calienni’s method.150

The synthetic route 1, based on Besong’s method, started with the reaction of 5- bromo-2,4-dichloropyrimidine and cyclopentylamine in N,N-diisopropylethylamine to yield 12 (Scheme 3), and then Cu/6-methylpicolinic acid catalyzed Sonogashira

Scheme 4. Synthesis of pyrrolo[2,3-d]pyrimidines 20 and 21.152 reaction/cyclization of 12 with 3,3-diethoxy-propyne to afford 13 in 85% yield. The hydrolysis of 13 with hydrochloric acid generated aldehyde. Further oxidation of aldehyde with oxone gave the corresponding acid 15.

In route 2, based on Calienni’s method, the N-arylation reaction of cyclopentylamine with 5-bromo-2,4-dichloropyrimidine in N,N-diisopropylethylamine yielded 12 (Scheme 3), and then Cu/6-methylpicolinic acid catalyzed Sonogashira reaction/cyclization of 12 with propargyl alcohol gave the compound 14 in 60% yield, after the oxidation of the alcohol 14 using stoichiometric NaClO2, catalytic TEMPO, and catalytic NaClO to afford the corresponding acid 15.151

Mishra et al.152 reported the synthesis of pyrrolo[2,3-d]pyrimidines 20 (Scheme 4) and 21 (Scheme 4) by the three-component reaction of phenylglyoxal monohydrate 16 and

pyrimidines 18 and 19 on a 0.5 mmol scale with acetic acid as solvent and promoter under microwave heating at 130 °C for 5 min.

Scheme 5. Synthesis of pyrrolo[2,3-d]pyrimidines 25.153

Quiroga, J. et al.153 reported a three component one-pot reaction that the condensation of 2,4,6-triaminopyrimidine 22 (Scheme 5) with dimedone 23 and arylglyoxal 24 in ethanol with acetic acid as the catalyst produced an unexpected cyclization affording pyrrolo[2,3-d]pyrimidines 25 in moderate yields.

Scheme 6. Synthesis of pyrrolo[2,3-d]pyrimidines 29.154

Naidu and Bhuyan154 reported the synthetic details of a novel series of 5-arylamino- pyrrolo[2,3-d]pyrimidine derivatives 29 (Scheme 6), which were synthesized via a

microwave-assisted three-component reaction of N,N-dimethyl-6-aminouracil 26, aryl glyoxal monohydrates 27 and aryl amines 28. In the reaction process, acetic acid is identified to act as the Brønsted acid promoter as well as solvent.153-154

Scheme 7. Synthesis of pyrrolo[2,3-d]pyrimidine 32.155,156

Rodriguez et al.156 reported the synthesis of pyrrolo[2,3-d]pyrimidine, 32 (Scheme

7) by coupling bromo- (or iodo-) aniline 30 with an alkyne through the Sonogashira

Scheme 8. Synthesis of pyrrolo[2,3-d]pyrimidines 34,-36. 155-158 reaction, giving a satisfactory yield of 31. This precursor was then treated with a strong base in NMP at room temperature, giving good yields of pyrrolo[2,3-d]pyrimidine 32 through a 5-endo-dig cyclization.156

Similarly, cyclization of 6-chloropyrimidines, 33 (Scheme 8) using cesium carbonate under microwave conditions resulted in pyrrolo[2,3-d]pyrimidines 34 in excellent yields.156

Scheme 9. Synthesis of pyrrolo[2,3-d]pyrimidines 40. 154,159

Prieur et al.157-158 reported the synthesis of pyrrolo[2,3-d]pyrimidines 35 and 36

(Scheme 8), for which the one-pot procedure with alkylamines, hydrazine, or anilines and potassium tert-butoxide resulted in the corresponding 6-substituted pyrrolo[2,3- d]pyrimidines 35 and 36 in moderate (for hydrazine and anilines) to good (for alkylamines) yields.157-158

Scheme 10. Synthesis of pyrrolo[2,3-d]pyrimidines 43.155,160

Rad-Moghadam and Azimi159 developed a sequential tandem protocol for facile preparation of pyrrolo[2,3-d]pyrimidine derivatives 40 (Scheme 9) in one-pot via a cycloaddition reaction between 6-amino-uracils 37, isatins 38 and acetophenones 39 in ethanol at reflux.154,159

Ghahremanzadeh et al.160 reported the synthesis of pyrrolo[2,3-d]pyrimidine derivatives 43 (Scheme 10), which can be prepared by condensation reaction of 6-amino- uracils 41 and isatins 42 in aqueous media.155,160

Scheme 11. Synthesis of pyrrolo[2,3-d]pyrimidines 47 and 49.155,161,162

El Kaim et al.161,162 reported the synthesis of pyrrolo[2,3-d]pyrimidines 47 (Scheme

11) and 49 by an Ugi−Smiles four-component reaction with pyrimidinols 44 afforded

intermediates 45 in low to moderate yields. The subsequent Sonogashira coupling and treatment with a suitable base produced the desired pyrrolo[2,3-d]pyrimidines 47 and 49 in good yields. When aromatic aldehydes were used, the Ugi adducts and Sonogashira products could be isolated or the entire process could be performed in a one-pot setup.161,162

Paul et al.163,164 reported a new method for the synthesis of 5-arylpyrrolo[2,3- d]pyrimidine-2,4-diones 52a (Scheme 12) and 52b by reacting 6-aminouracil 50a with nitroalkenes 51 using PEG−SO3H as polymer-supported catalyst.

Scheme 12. Synthesis of pyrrolo[2,3-d]pyrimidines 52s.155,163,164

The highest yields of the pyrrolopyrimidinones were obtained with nitroolefins 52b bearing an electron-donating group on the aromatic ring. The nitroolefins could also be synthesized in situ by reacting uracils 50b with aromatic aldehydes in the presence of

155,163,164 nitromethane, using CuFe2O4 as catalyst.

Scheme 13. Synthesis of pyrrolo[2,3-d]pyrimidines 55s.155,165

Gibson et al.165 reported the synthesis of pyrrolo[2,3-d]pyrimidines 55a (Scheme

13) and 55b were synthesized in low yields from diarylethanone 54 with pyrimidines 53a and 53b, respectively.165,166

Scheme 14. Synthesis of pyrrolo[2,3-d]pyrimidines 60 and 61.155, 167,168

Trzoss et al.168 reported the synthesis of two pyrrolo[2,3-d]pyrimidines 60 (Scheme

14) and 61, which were synthesized via the dehydration of masked 2-oxo-alkylsubstituted aminopyrimidines 59. Reaction of methyl cyanoacetate 56 with bromobutanone 57 and

subsequent acetal formation afforded compound 58 in good yield. Pyrimidinones 59 were also obtained in good yields through the treatment with either urea or thiourea. Acid- catalyzed ring-closure of the thio-analogue 59 gave the pyrrolo[2,3-d]pyrimidines 60 in excellent yield. Similar treatment of the oxo-analogue and subsequent activation with

167,168 POCl3 afforded the other pyrrolo[2,3-d]pyrimidines 61.

Scheme 15. Synthesis of pyrrolo[2,3-d]pyrimidines 65 and 67 and furo[2,3-d]pyrimidines

66.169

Secrist and Liu169 reported synthetic details of 2,6-diamino-4-hydroxypyrimidine

62 (Scheme 15) with various -halo aldehydes and ketones. Two modes of cyclization are reported that produce either the pyrrolo[2,3-d]pyrimidine or the furo[2,3-d]pyrimidine or both during the cyclization. In the reaction with chloroacetone 63, both the furo[2,3- d]pyrimidine 66 and the pyrrolo[2,3-d]pyrimidine 65 were observed; whereas only the

pyrrolo[2,3-d]pyrimidine 67 was observed in the reaction with 64. Therefore, the authors suggest that a critical electron density is necessary at the C5 of the pyrimidine ring since it is the C5 that reacts with the -carbon atom of the -halo aldehydes or ketones to afford the exclusive form of pyrrolo[2,3-d]pyrimidines.

Scheme 16. Synthesis of pyrrolo[2,3-d]pyrimidines 71 and 72.155, 170

Davoll170 reported the synthesis of a series of pyrrolo[2,3-d]pyrimidine derivatives including 71 (Scheme 16). The reaction starts with the cyclization of the acetals ethyl(2,2- diethoxyethyl)acetate 68 with thiourea (or guanidine, urea) to afford 69. Then cyclization of pyrimidine 5-acetone onto the neighboring amino group is mediated by an acid to afford pyrrolo[2,3-d]pyrimidine 70. With large amount of Raney-Nickel reagents, desulfurization afforded pyrrolo[2,3-d]pyrimidine 71.

Scheme 17. Synthesis of pyrrolo[2,3-d]pyrimidine 76.171-173

Noell et al.172,173 reported an in-house process to synthesize pyrrolo[2,3- d]pyrimidines 76 (Scheme 17) by employing desulfurization of 75 as the final step. This was prompted by a lack of an efficient method for the preparation of the cyanoacetate 68

(Scheme 16).172,173 Sulfurized pyrrolopyrimidine 75 was in turn obtained by cyclization of chloro dimethoxypropane with pyrimidine 74, which was synthesized via methylation of

73 by Me2SO4.

Scheme 18. Synthesis of pyrrolo[2,3-d]pyrimidines 79-80.174

Cheung and co-workers174 reported the synthesis of pyrrolo[2,3-d]pyrimidines 79-

80 (Scheme 18) by employing the intramolecular cyclization of pyrimidine 78. The synthesis started from 77, which was obtained by the treatment of trihalogenated pyrimidine with ammonia. Further Stille coupling afforded the vinyl ether 78. Dissolution in HCl at reflux, in the presence or absence of trimethoxyaniline, furnished the key cyclized pyrrolo[2,3-d]pyrimidines 79-80 in excellent yields.174

Scheme 19. Synthesis of pyrrolo[2,3-d]pyrimidines 83.175

Janeba et al.175 reported the synthesis of pyrrolo[2,3-d]pyrimidines 83 (Scheme 19) through two different methods. A rare example of heteroatom exchange mediated synthesis was shown with the treatment of furo pyrimidine 81 with hot methanolic ammonia to give

83a (R = H) in good yield. Meanwhile, 6-alkyl-substituted derivatives were synthesized from iodouracil 82 through a one-pot procedure of Sonogashira coupling and subsequent ammonia addition.

Scheme 20. Synthesis of pyrrolo[2,3-d]pyrimidine 86.176

Esteban-Gamboa et al.176 reported the synthesis of pyrrolo[2,3-d]pyrimidines 85

(Scheme 20). Starting material monosubstituted urea 84 was condensed with ethyl cyanoacetate to yield 85, then pyrrolo[2,3-d]-pyrimidinone 86 was obtained by treating 85 with chloroacetaldehyde.

Scheme 21. Synthesis of pyrrolo[2,3-d]pyrimidines 89.177-179

Hammond et al.177 and Hornillo-Araujo178-179 reported the methods of synthesizing pyrrolo[2,3-d]pyrimidines 89 (Scheme 21). The synthesis started from pyrimidinol 87 which was condensed with nitroalkenes in good yield. Then, reduction of the nitro group and acidic treatment led to bicyclic heterocycles 89 via intermolecular cyclization.

Scheme 22. Synthesis of pyrrolo[2,3-d]pyrimidines 92.180

Ko´kail and coworkers180 reported a new synthetic route of providing convenient access to pyrrolo[2,3-d]pyrimidines 92 (Scheme 22).Treatment of the easily available ethyl

2-(4-chloropyrimidin-5-yl)acetate derivatives 90 (R = H, Me, Ph, SMe) with alcoholic ammonia afforded the corresponding 2-(4-aminopyrimidin-5-yl)acetamides 91, which were cyclized to target compounds 92 (R = H, Me, Ph, SMe).

Scheme 23. Synthesis of pyrrolo[2,3-d]pyrimidine 97.181

Wang and co-workers181 reported the synthesis of pyrrolo[2,3-d]pyrimidine 97 as described in Scheme 23. Compound 94 was prepared by reacting 4,6-dihydroxylpyrimidne

93 with freshly prepared Vilsmeier–Haack reagent.182 4,6-Dichloropyrimidine-5- carbaldehyde 94 underwent SNAr reaction with sarcosine benzylester in the presence of a base and afforded 95 in moderate yield. Intermediate 95 was then cyclized to form pyrrolidine ring 96 by treatment with triethylamine in acetonitrile at reflux for 8 h.

Dehydration of 96 with thionyl chloride (SOCl2) and pyridine in dichloromethane at room temperature provided 97 in good yield.

Mieczkowski and co-worker183 reported the synthesis of pyrrolo[2,3-d]pyrimidines

103 (Scheme 24). The reaction was conducted starting from uracil 98 protected by acetyl groups. Then iodouracil 99 was obtained through the iodination reaction by using I2 in the presence of cerium ammonium nitrate. Sonogashira coupling of alkynes afforded 5-alkynyl derivatives 99 as the main products and bicyclic furo[2,3-d]pyrimidines 101 as minor products. Then, treatment of compound 101 with methanolic ammonia at room temperature led to the fast removal of the acetyl groups followed by rather slow conversion of the

deprotected furo[2,3-d]pyrimidin-2(3H)-one nucleosides 102 to pyrrolo[2,3-d] pyrimidins

103.184,185

Scheme 24. Synthesis of pyrrolo[2,3-d]pyrimidines 103.183-185

2. From pyrroles

Mizar and Myrboh186 reported the synthesis of pyrrolo[2,3-d]pyrimidines 106

(Scheme 25) by a Biginelli-type reaction between five-membered tertiary amides 104, acetophenones 105, and urea or guanidine using KF/alumina as solid support that furnished bi- or tricyclic heterocycles 106 in good yields.155, 186

Scheme 25. Synthesis of pyrrolo[2,3-d]pyrimidines 106.155, 186

Kaspersen and coworkers187 reported the synthesis of pyrrolo[2,3-d]pyrimidines

108 (Scheme 26) by condensation of pyrroles 107 with formamide that resulted in pyrrolo[2,3-d]pyrimidines 108 in good yields, which followed classical pyrrolo[2,3- d]pyrimidine chemistry.155,287

Scheme 26. Synthesis of pyrrolo[2,3-d]pyrimidines 108.155,287

Pittala and coworkers188 reported the synthesis of pyrrolo[2,3-d]pyrimidines 111

(Scheme 27) started from trisubstituted pyrroles 109, which were easily condensed with 2- chloroethyl isocyanate that afforded ureas 110 in excellent yields. Treatment with 1-(2-R2- phenyl)piperazines (PPz’s) elongated the chain prior to final pyrimidine cyclization by

KOH in MeOH, which afforded target compounds 111 in good yields.155,188

Scheme 27. Synthesis of pyrrolo[2,3-d]pyrimidines 111.155,188

Tolman and coworkers189 reported the synthesis of pyrrolo[2,3-d]pyrimidine 114

(Scheme 28) by cyclization of 2-amino-5-bromo-3,4-dicyanopyrrole 113 with formamidine acetate. Ramasamy and coworkers190 have also utilized the pyrrole 113 to form the pyrrolo[2,3-d]pyrimidine ring system for use in nucleoside synthesis. The versatility of the pyrrole 113 prompted Swayze et al.191 to synthesize it in an efficient one- step reaction from tetracyanoethylene 112. On controlled addition of HBr in acetic acid

112 undergoes an intramolecular self-condensation to afford 113.

Scheme 28. Synthesis of 5,6-disubstitutedpyrrolo[2,3-d]pyrimidine 114.155, 189-191

Scheme 29. Synthesis of pyrrolo[2,3-d]pyrimidine 118.193

Girgis and Joergensen193 reported the synthesis of pyrrolo[2,3-d]pyrimidine 118

(Scheme 29). For example, pyrrole 116 was prepared from acetamidoacetone 115.192

However, the direct conversion of 116 to 118 using known procedures afforded poor yields.

Alternatively, treating 116 with formic acid in the presence of acetic anhydride gave the

N-formyl pyrrole 117 in good yield. However, conversion of 117 to the 118 by heating with concentrated phosphoric acid or phosphorus pentaoxide gave only trace amounts of

118.192-194

Scheme 30. Synthesis of pyrrolo[2,3-d]pyrimidine 121.195

Cirrincione and coworkers195 reported the synthesis of pyrrolo[2,3-d]pyrimidine

121 (Scheme 30). When 2-amino-4-methyl-1H-pyrrole-3-carboxamide (120) was prepared from 119 using similar conditions, it underwent annulation with ethyl formate to 121 in

about 80% yield. However, the yield of 120 was low, and the pyrrole was found to be unstable.195

3. From triazines

Scheme 31. Synthesis of pyrrolo[2,3-d]pyrimidine 124.196

Dang and Gomez-Galeno196 reported the synthesis of pyrrolo[2,3-d]pyrimidine 124

(Scheme 31). Various 1,3,5-triazines 122 underwent a nonconcerted inverse electron- demand Diels−Alder reaction with N-alkylated 2- aminopyrroles 123. The cycloadduct intermediate first eliminates a nitrile through retro-Diels−Alder. This nitrile subsequently reacts with the free amino group of the pyrrole, forming an amidinium ion intermediate, which by elimination produces the aromatic pyrrolo[2,3-d]pyrimidines 124 in good yields.

Several alkyl, benzyl, and thioether chains were prepared. The presence of electron- withdrawing substituents such as esters and trifluoromethyl groups enhance the reactivity of the triazine, allowing the reaction to occur at room temperature, while heating was necessary in their absence.197-200

4. From others

Scheme 32. Synthesis of pyrrolo[2,3-d]pyrimidine 129.201

cyanoacetamide 127 resulted in the formation of highly functionalized pyrroles 128.

Ring closure with oxalyl chloride afforded 7-deazaxanthines 129 in good overall yields.

Scheme 33. Synthesis of pyrrolo[2,3-d]pyrimidine 134.202

Modugu and co-workers202 reported the synthesis of pyrrolo[2,3-d]pyrimidine 134

(Scheme 33). Reaction of isoxazole 130 with phenacyl bromide 131 afforded 132 in good yields. Pyrroles 134 were then obtained by treatment with malononitrile, and condensation with thiourea gave the final pyrrolo[2,3-d]pyrimidine-2-thiones 134 in good yields.

III. STATEMENT OF THE PROBLEM

1. (S)-2-(6-(2-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6- yl)ethyl)benzo[b]thiophene-2-carboxamido)pentanedioic acid (135) as GARFTase inhibitor with selectivity for FRs and/or PCFT over RFC.

Figure 19 The structure of classical 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine antifolate 135.

One of the critical structural characteristics of classical antifolates is the glutamic acid moiety, of which the two carboxylic acids exist as anions at physiologic pH. As a consequence, they do not cross cell membranes by passive diffusion. So, specific transport systems are required for folate uptake inside the cells. The three major uptake systems including RFC, FRs and PCFT are shared by reduced folates and antifolates.4,5,9 RFC, as a

type II transmembrane protein, is the principle transporter for folates in mammalian cells and tissues at biologically physiologic pH.4,5,9 FRs are glycosylphosphatidylinositol- anchored proteins that bind folates with a high affinity and mediate folate transport via endocytosis.82,11,19 Finally, PCFT transports folates at acidic pH optimum in mammalian cells and tissues.113

RFC has ubiquitous expression in tissues and tumors.9 Its lack of selectivity for tumor cells over normal tissues causes the dose-limiting toxicity. However, FRs are promising targets for targeted antifolate chemotherapy, considering their more restricted pattern of tissue distribution and function. In the normal tissues, for example, FRα is expressed in the apical side of the membranes in the kidney, choroid plexus, and placenta, whereas FRβ is expressed in placenta, spleen, and thymus.11 In terms of the function in normal cells, FRα expressed in the apical side is inaccessible to folates and antifolates, due to, no circulation,2 whereas FRβ expressed in normal hematopoietic cells is incapable of binding to folate ligand.11 Nevertheless, FRα is overexpressed in a number of carcinomas including up to 90% of ovarian cancers, whereas FRβ is overexpressed in a broad range of myeloid leukemia cells in chronic myelogenous leukemia and in acute myelogenous leukemia.11 The selectivity for tumor cells over normal tissues of FRs indicates its importance for targeted antifolate chemotherapy.

PCFT is expressed in normal tissues such as the small intestine, colon, liver and kidney and functions optimally at acidic pH around 5-5.5. Above pH 7, no folate uptake by PCFT is observed.116,211 Thus, in tissues like liver and kidney, PCFT is unable to transport folates at physiologic pH (pH = 7.4), whereas at the acidic pH of small intestines, it indeed mediates folate absorption. However, the toxicity of chemotherapeutic agents to

the small intestine could be circumvented via iv administration. The distribution pattern of human PCFT (hPCFT) in tumors has not been systematically studied, however, compatible with the low-pH microenvironment of solid tumors, PCFT is the prominent mechanism for folate uptake in 29 of 32 human solid tumor cell lines.210 Therefore, PCFT is another promising means for targeted antifolate chemotherapy transport.

The FR-targeted chemotherapy has always been a hot topic in the treatment for cancer. The current strategies of targeting FRs include folate-conjugated cytotoxins, liposomes, radionuclides and cytotoxic antifolates.204-206 The clinically used classical antifolates (including RTX, PMX and LMX) have dose limiting toxicity toward normal cells, since these cytotoxic antifolates are not only transported by FRs but also by the ubiquitously expressed RFC.109,207 This could be a possible reason for the severe myelosuppression observed in Phase 1 clinical study of LMTX (Table 1).212,213

Figure 20 The structures of ONX0801.208,209

One of the strategies for FRs-targeted chemotherapy is employing prodrug conjugates.82,103,214,215 Cytotoxins like mitomycin C18 are covalently linked to folates or pteroates and are selectively cleaved to release the cytotoxic drugs upon internalization.

Even though these folate conjugates are not likely substrates of RFC, inefficient cleavage and limited release of the cytotoxic moiety could result in a decreased antitumor

activity.214,215 The decreased selectivity and increased toxicity to normal tissues could be caused by the premature cleavage of the prodrug conjugates prior to the tumor internalization. Moreover, the released free folic acids by the conjugates could serve as nutrients for tumor cells, which would also compromise antitumor activities. However, if a FR-targeted compound was itself cytotoxic without any RFC activity, targeted antifolate chemotherapy could ensue. Fortunately, the idea has been proved to be successful by a recently described series of cyclopenta[g]quinazoline antifolates,208,209 which potently inhibit thymidylate synthase (TS) and is selectively transported by FRs over RFC. When

ONX0801 (BGC945, Figure 20),208,209 was tested in mice, no systemic toxicity was observed including weight loss and macroscopic signs of toxicity to major organs.209 All these results demonstrate that the strategy of specifically targeting FRs is highly practical.

Gangjee and coworkers117,207,216 recently described a series of 6-substituted classical pyrrolo[2,3-d]pyrimidines as cytotoxic antifolates with varying lengths of the carbon bridge region (Table 1). These compounds were characterized as potent GARFTase inhibitors with selective transport via FRs and PCFT over RFC. In the series, the four- carbon bridge analog 4 was not a substrate of RFC,117 whereas the three-carbon bridge 3 exhibited a modest inhibitory activity (IC50 = 101 nM) against the RFC-expressing PC43-

10 cell line.216 In terms of the antitumor potency, however, the situation was reversed.

Compound 3 with a 3-carbon side chain showed the highest inhibitory potency toward KB and IGROV1 human tumor cell lines, whereas 4 with a 4-carbon side chain give slightly less potent activity toward these tumor cells than 3, but was transported selectively by FRs and PCFT over RFC. The major difference between 3 and 4 are the chain length of the carbon bridge and orientation of thienyl ring, indicating the distance between the bicyclic

scaffold and the glutamate moiety and/or orientation of thienyl ring might play an important role on both the selectivity and the inhibitory potency. Thus, it was postulated that the optimum potency and transport selectivity would probably be achieved if the bridge chain length of compounds was between 3- and 4-carbon and/or restricting the orientation

Figure 21 Calculation of the total distance from the C6 of the scaffold to the C1’ of the side chain thioenyl ring of 3. (MOE 2011.10)229

Figure 22 Calculation of the total distance from the C6 of the scaffold to the C1’ of the side chain thioenyl ring of 4. (MOE 2011.10)229

Figure 23 Calculation of the total distance from the C6 of the scaffold to the C7a’ of the benzo[b]thioenyl ring of 135. (MOE 2011.10)229 of side chain ring between that of compound 3 and compound 4. (See Table 2 in Appendix

Therefore, in order to investigate the optimum chain length and/or side chain orientation for the GARFTase inhibitory potency and the selectivity for FRs and PCFT over RFC, 135 with a 2,6-substituted benzo[b]thiophene ring was designed. Based on the result calculated by MOE (2011.10),229 the total distance from the C6 of the scaffold to the

C5’ of the benzo[b]thiophene ring of 135 is 5.94 Å (Figure 23), which is greater than that of compound 3 (4.52 Å) and slightly smaller almost the same as compound 4 (5.96 Å)

(Figure 21, 22). In contrast to lead compounds 3 and 4 with freely rotatable single bonds,

135 with the benzo[b]thiophene ring shows a more restricted conformation, of which the bicyclic ring restricts two carbon bonds to rotate but still remains some conformation flexibility with two single carbon bonds. Compound 135 is proposed to exhibit not only a similar or possible more potent GARFTase inhibitory activity than 3, but also the desired selectivity for FRs and/or PCFT over RFC with regard to the targeted antifolate chemotherapy.

2. (S)-2-(6-(2-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)ethyl)-2- naphthamido)pentanedioic acid (136), (S)-2-(5-(2-(2-amino-4-oxo-4,7-dihydro-3H- pyrrolo[2,3-d]pyrimidin-6-yl)ethyl)benzo[b]thiophene-2-carboxamido)pentanedioic acid

(137) and (S)-2-(6-(3-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6- yl)propyl)benzo[b]thiophene-2-carboxamido)pentanedioic acid (138) as GARFTase inhibitors with potential selectivity for FRs and/or PCFT over RFC.

As described above, it was envisioned that the distance between the bicyclic scaffold and the glutamate moiety was of importance to both transport selectivity and

Figure 24 The structures of classical 2-amino-4-oxo-6-substituted pyrrolo[2,3- d]pyrimidine antifolates 135-138.

antitumor activity. So, 135 was designed with a distance between three carbons and four carbons by restricting the conformation with the benzo[b]thiophene ring as shown in Figure

24. Even though its inhibitory activity against KB cell lines was ten-fold less potent (IC50

216 117 = 2.38 nM) than its lead compounds 3 (IC50 = 0.25 nM) and 4 (IC50 = 0.2 nM), it still partially met our expectation. Compound 135 with the restricted benzo[b]thiophene ring in the side chain was selectively transported into human CHO cell lines by FRs over PCFT and RFC. Compared with its lead compounds 3 and 4, compound 135 was a pure FR substrate and lost the inhibitory activity toward CHO cell lines expressing PCFT. One of the obvious structural differences between 135 and lead compounds are the fused aromatic

Figure 25 The structures of classical 2-amino-4-oxo-6-substituted pyrrolo[2,3- d]pyrimidine antifolates 139 and 140. ring system in the side chain. Based on the pure inhibitory activity toward FRs of 135, it is suggested that employing the extended aromatic ring system in the side chain might be favorable to differentiate FRs from other two transport systems. According to the crystal structures of FRs,61,87 it was proposed that the aromatic side chain of 135 perpendicularly

penetrate into the hydrophobic binding cleft of FRs. The selectivity of 135 for FRs over

RFC/PFCT is probably due to the strong π-π interactions between the fused aromatic side chain of the ligand and the parallel side chains of Tyr85 and Trp171, and capped by Tyr175 in the FR biding pocket.61

To further explore the SAR of the targeted specificity for FR and GARFTase inhibition, we designed a novel series of classical 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidines with a bicyclic aromatic system in the side chain (Figure 24).

The relative position of the 2-carbon bridge domain and L-glutamate moiety was considered and therefore, the 2,5-substituted regioisomer 137 (Figure 24) was designed. In addition, to further determine the importance of the distance between the scaffold and the glutamate moiety with respect to the selectivity toward FRs and the cytotoxic activity, a homologated analog 138 (Figure 24) was also designed with a 3-carbon bridge.

Gangjee et al.207 recently reported that the 4-atom chain benzoyl analog 2 (Table 1) was six-fold less potent than the corresponding 4-carbon thioenyl analog 4 (Table 1) as an inhibitor of KB and IGROV1 tumor cells in culture and of the target enzyme GARFTase both in vitro and in vivo. These results indicate that the side chain thioenyl ring exhibits higher inhibitory potency to GARFTase than the benzoyl ring in pyrrolo[2,3-d]pyrimidine scaffold (compare 2 and 4). This might be due to the extra hydrogen bond interaction between the S atom of the thiophene ring in the ligand and the enzyme. To further determine the importance of the hydrogen-bond interaction of the S atom, compound 136 was designed with the bioisosteric replacement of the S atom in 138 with the C-C double bond.

Since the C-C double bond is slightly shorter than the S atom, this bioisosteric replacement would decrease somewhat the side chain ring size of 135, and also alter

(increase) the angle between the carbon bridge and the L-glutamate moiety. In addition, the replacement of the S atom with the C-C double bond is an efficient means of probing the importance of the hydrogen bond interaction between the ligand and the target enzyme and/or transporter(s).

3. (S)-2-(4-(4-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)butyl)-1- naphthamido)pentanedioic acid (139) (Figure 25) and (S)-2-(4-(4-(2-amino-4-oxo-4,7- dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)but-1-yn-1-yl)-1-naphthamido)pentanedioic acid (140) (Figure 25) as GARFTase inhibitors with selectivity for FRs and/or PCFT over

RFC.

Gangjee et al.207 reported (S)-2-({5-[4-(2-amino-4-oxo-4,7-dihydro-3H- pyrrolo[2,3-d]-pyrimidin-6-yl)-butyl]-phenyl-2-carbonyl}-amino)-pentanedioic acid, 2

(Figure 25), as a potent inhibitor against KB and IGROV1 human tumor cells that express

RFC, FRα and PCFT. Compound 2 was selectively transported by FRs and PCFT over

RFC, and inhibited GARFTase as the intracellular target. Thus, compound 2 is an excellent lead analog for further structure optimization for inhibition of GARFTase and selective transport by FRs and/or hPCFT over RFC.

Structure-based drug design for FR targeted classical antifolates hasn’t been attempted until the crystal structure of FRα was recently reported by Chen et al.61 From the crystal structure of FRα, amino acids with aromatic side chains including Trp102,

Trp140 and Try60 exist at the cross-section of the binding pocket. Based on this result, we envisioned that higher inhibitory activities toward FRα and GARFTase might be achieved

if we employed an enlarged aromatic system in the side chain to replace the phenyl ring of

2. Thus, a series of analogs with extended aromatic side chain were designed by Dr.

Gangjee to probe the importance of the fused aromatic ring system in the side chain with respect to the FRα specificity and the cytotoxic activity. In the thesis, only the analog 139 with naphthyl ring in the side chain (Figure 25) was developed for the bioassay evaluation.

All the other analogs were developed by other coworkers. The unsaturated isomer 140

(Figure 19), as the synthetic intermediate of 139, was also evaluated for a bioassay.

4. (S)-2-(5-(4-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6- yl)butyl)thiophene-2-carboxamido)hexanedioic acid (141) (Figure 22), 4-(5-(4-(2-amino-

4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)butyl)thiophene-2- carboxamido)butanoic acid (142) (Figure 22), (S)-2-(5-(4-(2-amino-4-oxo-4,7-dihydro-

3H-pyrrolo[2,3-d]pyrimidin-6-yl)butyl)-N-methylthiophene-2-carboxamido)pentanedioic acid (143) (Figure 22) as a GARFTase inhibitors with selectivity for FRs and/or PCFT over

RFC, and (S)-2-(5-(4-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6- yl)butyl)thiophene-2-carboxamido)succinic acid (144) (Figure 22).

In a recent publication, Gangjee et al.217 reported a series of 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl analogues based on compound 3 with replacement of L- glutamate by different amino acids. The result established that amino acid modifications are well tolerated by FRα. It indicates that L-glutamate is not essential for binding and cellular uptake by FRs. However, L-glutamate is required for PCFT transport and α- carboxyl shows more importance than γ-carboxyl for substrate binding to hPCFT

To further explore the role of the terminal L-glutamate in antitumor selectivity and inhibitory potency against tumors, a potent GARFTase inhibitor with selective transport by FRs and PCFTs compound 4 has been chosen to go with the similar modification on L- glutamate moiety. So, a series of analogs were developed, including 141 with adipic acid,

142 with 4-amino butanoic acid, 143 with aspartic acid, 144 with N-methyl L-glutamate.

(Figure 26)

Figure 26 The structure of classical 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine antifolates 141-144. were designed and synthesized. They are proposed to determine the different roles of the

α- and γ-carboxylic acid on the FR binding affinity and the inhibitory activity against

GARFTase.

No structural modification on the amide bond at the R3 position (Figure 26) has been reported. It was of interest to investigate the importance of the amide hydrogen of the glutamate moiety to the FR binding affinity and the GARFTase inhibitory activity. Thus, compound 144 (Figure 26) was designed with a methyl group at the R3 position. Moreover, the methyl group would prevent the in vivo metabolism given that it is possible for the amide bond of the glutamate moiety to be hydrolyzed in vivo. In addition, the methyl group could serve as a probe to determine the impact of its hydrophobic effect on the FR binding affinity.

IV. CHEMICAL DISCUSSION

1. The synthesis of (S)-2-(6-(2-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-

6-yl)ethyl)benzo[b]thiophene-2-carboxamido)pentanedioic acid (135) and (S)-2-(6-(2-(2- amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)ethyl)-2- naphthamido)pentanedioic acid (136) as bioisosteric analogs.

The target compounds 135 and 136 were abtained via a ten-step synthesis employing the α-bromomethylketone condensation with the commercially available pyrimidine as the key step as outlined in Scheme 34.

The synthesis started with the palladium-catalyzed Sonogashira coupling of the propargyl alcohol, with methyl 6-bromobenzo[b]thiophene-2-carboxylate 145, and methyl

6-bromo-2-naphthoate 146, respectively, afforded propynyl alcohols 147 and 148 (82% and 87%). Subsequent hydrogenation of 147 and 148 in the presence of the 10% Pd/C catalyst in MeOH overnight yielded the saturated alcohols 149 and 150. PCC and periodic

acid were employed to give the oxidized carboxylic acids 151 and 152 (81% and 86%), which were converted to the acid chlorides 153 and 154 and immediately reacted with diazomethane followed by 48% HBr to achieve the desired α–bromomethyl ketones 157

Scheme 34a Synthesis of classical 2-amino-4-oxo-6-substituted-pyrrolo[2,3-d]pyrimidines

135 and 136.

a Conditions: (a) propargyl alcohol, CuI, PdCl2(PPh3)2, PPh3, Et3N, CH3CN, microwave,

100 C, 1 h; (b) 10% Pd/C, H2, 60 psi, MeOH, overnight; (c) PCC, H5IO6, CH3CN, 0 C

~RT; (d) oxalyl chloride, CH2Cl2, reflux, 1 h; (e) diazomethane, Et2O, RT, 1h; (f) HBr, 70-

80 C, 2h; (g) 2,6-diamino-3H-pyrimidin-4-one 177, DMF, RT, 3 days; (h) i. 1N NaOH,

RT, 12h; ii. 1N HCl; (i) N-methylmorpholine, 2-chloro-4,6-dimethoxy-1,3,5-triazine, L- glutamate diethyl ester hydrochloride, DMF, RT, 12h. and 158. Condensations of 2,6-diamino-3H-pyrimidin-4-one 117, with 157 and 158 at room temperature for 3 days afforded the 2-amino-4-oxo-6-substituted-pyrrolo[2,3- d]pyrimdines 159 and 160 (48% and 52%). Hydrolysis of 159 and 160 achieved the key pteroic acids 161 and 162. Subsequent peptide coupling with L-glutamate diethyl ester using N-methylmorpholine and 2-chloro-4,6-dimethoxy-1,3,5-triazine as the activating agents afforded the diesters 163 and 164. Final saponification of the diesters gave the desired compounds 135 and 136

Synthetic challenges and solutions:

a) For the Sonogashira coupling, instead of using the catalyst tetrakis-

(triphenylphosphine) palladium (0) Pd(PPh3)4, the bis(triphenylphosphine)palladium(II) dichloride Pd(PPh3)2Cl2 with double the amount of the ligand triphenylphosphine PPh3 were employed to increase the yield from 70% up to 90%. Another reason for the improved yield could be that the palladium (II) catalyst Pd(PPh3)2Cl2 is more stable and has a longer shelf life than that of the palladium(0) catalyst Pd(PPh3)4.

b) To achieve complete transformation from the unsaturated alcohol to the saturated alcohol as shown in the second step in the Scheme 36, the loading amount of the 10% Pd/C catalyst is essential. Generally, the catalyst loading amount is 20% with respect to the unsaturated starting material. However, the best loading quantity of 10% Pd/C is 70% for the bicyclic unsaturated alcohol in this study.

c) For the hydrogenation reaction in the second step (Scheme 34), the pressure, as another key element, plays an important role in the complete transformation. At 50 psi,

only around 10% of the unsaturated starting materials were hydrogenated. When the reaction time was extended from overnight to 3 days, no increased hydrogenation rate was observed. At 55 psi, around 40% of propynl alcohols were transformed to starting materials were hydrogenated. At 60 psi, both propynl alcohols were completely transformed to saturated ones after overnight.

4) For the oxidization reaction in the third step as outlined in the Scheme 34, instead of the harsh Jones reagents, much milder reagents of PCC and H5IO6 were employed to oxidize the primary alcohols, affording the carboxylic acids. It is critical to keep the reaction temperature at 0 C before PCC and H5IO6 are added. Generally, the yield of this oxidation is pretty high, usually up to 80%.

The most important step for the synthesis of 135 and 136 is the complete transformation of the hydrogenation reaction, otherwise it was very difficult to separate the propargyl alcohols, propenyl alcohols and propyl alcohols.

2. The synthesis of (S)-2-(5-(2-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-

6-yl)ethyl)benzo[b]thiophene-2-carboxamido)pentanedioic acid (137) as a regioisomer of compound 135.

The target compound 137 was obtained via a ten-step synthesis in Scheme 35, which is similar to the synthetic procedure as described for the compounds 135 and 136.

The synthesis started with the palladium-catalyzed Sonogashira coupling of the propargyl alcohol 165 (Scheme 35), with methyl 5-bromobenzo[b]thiophene-2- carboxylate 167 to afford the propynyl alcohol 168 (84%). Subsequent hydrogenation of

168 in the presence of the 10% Pd/C catalyst in MeOH overnight yielded the saturated

propyl alcohol 169 via a complete transformation. PCC and periodic acid were employed to give the oxidized carboxylic acid 170 (83%), which was converted to the acid chloride

171 and immediately reacted with diazomethane followed by 48% HBr to achieve the desired α–bromomethyl ketone 173. Condensation of 2,6-diamino-3H-pyrimidin-4-one

177, with 173 was at room temperature for 3 days afforded the 2-amino-4-oxo-6- substituted-pyrrolo[2,3-d]pyrimdine 174 (50%). Hydrolysis of 174 achieved the key pteroic acid 175. Subsequent peptide coupling with L-glutamate diethyl ester using N- methylmorphoilne and 2-chloro-4,6-dimethoxy-1,3,5-triazine as the activating agents afforded the diester 176. Final saponification of the diester gave the desired compounds

137.

Scheme 35a Synthesis of classical 2-amino-4-oxo-6-substituted-pyrrolo[2,3-d]pyrimidine

137.

a Conditions: (a) propargyl alcohol, CuI, PdCl2(PPh3)2, PPh3, Et3N, CH3CN, microwave,

100 C, 1h; (b) 10% Pd/C, H2, 60 psi, MeOH, overnight; (c)PCC, H5IO6, CH3CN, 0 C

~RT; (d) oxalyl chloride, CH2Cl2, reflux, 1h; (e) diazomethane, Et2O, RT, 1h; (f) HBr, 70-

80 C, 2h; (g) 177, DMF, RT, 3 days; (h) i. 1N NaOH, RT, 12h; ii. 1N HCl; (i) N- methylmorpholine, 2-chloro-4,6-dimethoxy-1,3,5-triazine, L-glutamate diethyl ester hydrochloride, DMF, RT, 12h.

Synthetic challenges and solutions in this study:

1) Similarly, the palladium (II) catalyst Pd(PPh3)2Cl2 with double amount of the ligand PPh3 were employed instead of the palladium (0) catalyst Pd(PPh3)4 for Sonogashira coupling reaction.

2) For the second step reaction as outlined in Scheme 35, the best reaction condition for the complete transformation of the hydrogenation reaction is 60% loading amount of

10% Pd/C, 60 psi and overnight.

3) For the third step reaction as outlined in Scheme 35, PCC and periodic acid were employed to give a high yield (83%) of the carboxylic acid under a mild reaction condition, compared with Jones oxidation conditions.

3. The synthesis of (S)-2-(6-(3-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-

6-yl)propyl)benzo[b]thiophene-2-carboxamido)pentanedioic acid 138 as a homologated compound of 135.

The target compound 138 was afforded via a ten-step synthesis employing the α- bromomethylketone condensation with the commercially available pyrimidine as the key step as outlined in Scheme 36.

Scheme 36a Synthesis of classical 2-amino-4-oxo-6-substituted-pyrrolo[2,3-d]pyrimidine

138. a Conditions: (a) but-3-yn-1-ol, CuI, PdCl2(PPh3)2, PPh3, Et3N, CH3CN, microwave, 100

C, 1h; (b) 10% Pd/C, H2, 60 psi, MeOH, overnight; (c)PCC, H5IO6, CH3CN, 0 C ~RT;

(d) oxalyl chloride, CH2Cl2, reflux, 1h; (e) diazomethane, Et2O, RT, 1h; (f) HBr, 70-80 C,

2h; (g) 117, DMF, RT, 3 days; (h) i. 1N NaOH, RT, 12h; ii. 1N HCl; (i) N- methylmorpholine, 2-chloro-4,6-dimethoxy-1,3,5-triazine, L-glutamate diethyl ester hydrochloride, DMF, RT, 12h.

The synthesis started with the palladium-catalyzed Sonogashira coupling of the commercially available methyl 6-bromobenzo[b]thiophene-2-carboxylate 145, with but-3- yn-1-ol 166, afforded the butynyl alcohol 178 (81%). Subsequent hydrogenation of 179 in

the presence of the 10% Pd/C catalyst in MeOH for overnight, achieved the saturated alcohol 179 via a complete transformation. PCC and periodic acid were employed to give the oxidized carboxylic acid 180 (85%), which was converted to the acid chloride 181 and immediately reacted with the diazomethane followed by 48% HBr to achieve the desired

α–bromomethyl ketone 183. Condensation of 2,6-diamino-3H-pyrimidin-4-one 177, with

183 was at room temperature for 3 days afforded the 2-amino-4-oxo-6-substituted- pyrrolo[2,3-d]pyrimdine 184 (50%). Hydrolysis of 184 achieved the key pteroic acid 185.

Subsequent peptide coupling with L-glutamate diethyl ester using N-methylmorphoilne and

2-chloro-4,6-dimethoxy-1,3,5-triazine as the activating agents afforded the diester 186.

Final saponification of the diester gave the desired compound 138.

Synthetic challenges and solutions:

1) Similarly, the palladium (II) catalyst Pd(PPh3)2Cl2 with double amount of the ligand PPh3 were employed instead of the palladium (0) catalyst Pd(PPh3)4 for Sonogashira coupling reaction. The yield was 81%.

2) For the second step reaction as outlined in Scheme 36, the best reaction condition for the complete transformation of the hydrogenation reaction is 60% loading amount of

10% Pd/C, 62 psi and overnight with 92% of the yield.

3) For the third step reaction as outlined in Scheme 36, PCC and periodic acid were employed to give a high yield (85%) of the carboxylic acid under a mild reaction condition, compared with Jones oxidation reagents.

4. The synthesis of (S)-2-(6-(3-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-

6-yl)propyl)benzo[b]thiophene-2-carboxamido)pentanedioic acid 139.

The target compound 139 was obtained via a nine-step synthesis employing the α- bromomethylketone condensation with the commercially available pyrimidine as the key step as outlined in Scheme 37.

Scheme 37a Synthesis of classical 2-amino-4-oxo-6-substituted-pyrrolo[2,3-d]pyrimidine

139. a Conditions: (a) methyl 4-bromo-1-naphthoate, CuI, PdCl2(PPh3)2, PPh3, Et3N, CH3CN,

microwave, 100 C, 1 h; (b)PCC, H5IO6, CH3CN, 0 C ~RT; (c) oxalyl chloride, CH2Cl2, reflux, 1h; (d) diazomethane, Et2O, RT, 1h; (e) HBr, 70-80 C, 2h; (f) 117, DMF, RT, 3

days; (g) N-methylmorpholine, 2-chloro-4,6-dimethoxy-1,3,5-triazine, L-glutamate diethyl ester hydrochloride, DMF, RT, 12h (h) i. 1N NaOH, RT, 12h; ii. 1N HCl; (i) 10% Pd/C,

H2, 60 psi, MeOH, overnight.

The synthesis started with the palladium-catalyzed Sonogashira coupling of the commercially available methyl 4-bromo-1-naphthoate, with pent-4-yn-1-ol 187, afforded the pentynyl alcohol 188 (79%). PCC and periodic acid were employed to give the oxidized carboxylic acid 189 (87%), which was converted to the acid chloride 190 and immediately reated with diazomethane followed by 48% HBr to achieve the desired α–bromomethyl ketone 192. Condensation of 2,6-diamino-3H-pyrimidin-4-one 177, with 192 was at room temperature for 3 days afforded the 2-amino-4-oxo-6-substituted-pyrrolo[2,3-d]pyrimdine

193(45%). Hydrolysis of 193 achieved the key pteroic acid 194. Subsequent peptide coupling with L-glutamate diethyl ester using N-methylmorphoilne and 2-chloro-4,6- dimethoxy-1,3,5-triazine as the activating agents afforded the diester 195. Saturated diester

196 was achieved via the Pd-catallyzed hydrogenation (63%). Final saponification of the diester gave the desired compound 139.

Synthetic challenges and solutions:

Based on the previous work done with the oxidation of primary alcohols in the second step, the free carboxylic acids could be obtained without any byproduct by employing 2 mol% of pyridinium chlorochromate (PCC) and 2 equivalents of periodic acid

(H5IO6) in acetonitrile for 2 h. However, under the same reaction condition, compared with the desired carboxylic acid, a byproduct with a higher Rf value was observed on the TLC plate. The byproduct did not disappear when the reaction was run overnight. Subsequently, a new batch of the oxidation reaction was run with 3 mol% of pyridinium chlorochromate

(PCC) and 3 equivalents of periodic acid (H5IO6) for 2 h. From the TLC, the byproduct was not observed and only the desired free acid was observed.

5. The synthesis of (S)-2-(4-(4-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-

6-yl)but-1-yn-1-yl)-1-naphthamido)pentanedioic acid (140)

Scheme 38a Synthesis of classical 2-amino-4-oxo-6-substituted-pyrrolo[2,3-d]pyrimidine

140.

aConditions: (a) i. 1N NaOH, RT, 6 h; ii. 1N HCl.

As shown in Scheme 38, the direct hydrolysis of the intermediate 195 afforded the target compound 140.

6. The synthesis of 4-(5-(4-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6- yl)butyl)thiophene-2-carboxamido)butanoic acid (142), (S)-2-(5-(4-(2-amino-4-oxo-4,7- dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)butyl)thiophene-2-carboxamido)succinic acid

(144), (S)-2-(5-(4-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6- yl)butyl)thiophene-2-carboxamido)hexanedioic acid (141) and (S)-2-(5-(4-(2-amino-4- oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)butyl)-N-methylthiophene-2- carboxamido)pentanedioic acid (143) as glutamate derivatives of compound 4.

The target compounds 141-144 were obtained via a ten-step synthesis from the commercially available methyl 5-bromo-thiophene-2-carboxylate using a peptide coupling

of the key intermediate pteroic acid and the corresponding amino acids. In this study, the palladium(II) catalyst Pd(PPh3)2Cl2 was employed in the Sonogashira coupling reaction instead of Pd(PPh3)4, together with double amount of the ligand PPh3 to achieve higher yield.

Scheme 39a Synthesis of pteroic acid 204. a Conditions: (a) 5-bromo-thiophene-2-carboxylic acid methyl ester, CuI, PdCl2(PPh3)2,

PPh3, Et3N, CH3CN, microwave, 100 C, 1h; (b) 10% Pd/C, H2, 55 psi, MeOH, overnight;

(c)PCC, H5IO6, CH3CN, 0 C ~RT; (d) oxalyl chloride, CH2Cl2, reflux, 1h; (e) diazomethane, Et2O, RT, 1h; (f) HBr, 70-80 C, 2h; (g) 177, DMF, RT, 3 days; (h) i. 1N

NaOH, RT, 12h; ii. 1N HCl.

The synthesis of pteroic acid 204 (Scheme 39) started from the commercially available pent-4-yl-1-ol 187. Sonogashira coupling of 187 with 5-bromo-thiophene-2- carboxylic acid methyl ester, afforded thiophenyl alcohol 197 in 89% yield. Instead of using the catalyst tetrakis-(triphenylphosphine) palladium (0) (Pd(PPh3)4), the bis(triphenylphosphine)palladium(II) dichloride Pd(PPh3)2Cl2 with twice the amount of the ligand triphenylphosphine PPh3 were employed to increase the yield from 70% up to 90%.

The following step is the Pd-catallyzed hydrogenation, which afforded the saturated alcohol 198 in quantitative yield. PCC and periodic acid were employed to give the oxidized carboxylic acid 199 (85%), which was converted to the acid chloride 200 and immediately reated with diazomethane followed by 48% HBr to achieve the desired α– bromomethyl ketone 202. Condensation of 2,6-diamino-3H-pyrimidin-4-one, 177, with

202 was at room temperature for 3 days afforded the 2-amino-4-oxo-6-substituted- pyrrolo[2,3-d]pyrimdine 202(51%). Hydrolysis of 203 achieved the desired pteroic acid

204.

Synthetic problems and improvements:

2) For the third step reaction as outlined in Scheme 39, PCC and periodic acid were employed to give a high yield (85%) of the carboxylic acid under a mild reaction condition, compared with the harsh Jones oxidation conditions.

Scheme 40a Synthesis of classical 2-amino-4-oxo-6-substituted-pyrrolo[2,3-d]pyrimidines

142. a Conditions: (a) i. SOCl2, MeOH, RT, 10 h; ii. NaHCO3. (b) N-methylmorpholine, 2- chloro-4,6-dimethoxy-1,3,5-triazine, ethyl 4-aminobutyrate hydrochloride, DMF, RT,

12h (c) i. 1N NaOH, RT, 12h; ii. 1N HCl.

With the key pteroic acid 204 in hand, compound 207 (Scheme 40) was synthesized by a peptide coupling of the pteroic acid 204 (Scheme 40) with ethyl 4-aminobutyrate

Scheme 41a Synthesis of classical 2-amino-4-oxo-6-substituted-pyrrolo[2,3-d]pyrimidines

144.

aConditions: (a) N-methylmorpholine, 2-chloro-4,6-dimethoxy-1,3,5-triazine, L-aspartate diethyl ester hydrochloride, DMF, RT, 12h (b) i. 1N NaOH, RT, 12 h; ii. 1N HCl. hydrochloride 206 using N-methylmorpholine and 2-chloro-4,6-dimethoxy-1,3,5-triazine as the activating agents in 49% yield. Final saponification of the diester with 1N NaOH gave the target compound 142.

Scheme 42a Synthesis of classical 2-amino-4-oxo-6-substituted-pyrrolo[2,3-d]pyrimidines

141. a Conditions: (a) i. SOCl2, MeOH, RT, 10 h; ii. NaHCO3. (b) N-methylmorpholine, 2- chloro-4,6-dimethoxy-1,3,5-triazine, L-2-aminoadipic acid dimethyl ester, DMF, RT, 12h

Similarly, compound 208 (Scheme 41) was synthesized by a peptide coupling of the pteroic acid 204 (Scheme 42) with L-aspartate diethyl ester hydrochloride using N- methylmorphoilne and 2-chloro-4,6-dimethoxy-1,3,5-triazine as the activating agents in 51% yield. Final saponification of the diester with 1N NaOH gave the target compound 144.

Scheme 43a Synthesis of classical 2-amino-4-oxo-6-substituted-pyrrolo[2,3-d]pyrimidines

143. a Conditions: (a) i. SOCl2, MeOH, RT, 10 h; ii. NaHCO3. (b) N-methylmorpholine, 2- chloro-4,6-dimethoxy-1,3,5-triazine, L-2-aminoadipic acid dimethyl ester, DMF, RT, 12h

Followed by the same procedure, the target compounds 141 (Scheme 42) and 143

(Scheme 43) were synthesized by the peptide coupling of the pteroic acid 204 with L-2- aminoadipic acid dimethyl ester and N-methyl-L-glutamate dimethyl ester, respectively, followed by the hydrolysis with 1N NaOH as shown in Scheme 42 and Scheme 43. In addition, L-2-aminoadipic acid dimethyl ester and N-methyl- L-glutamate dimethyl ester were not commercially available and synthesized from the carboxylic acids 209 and 212 via the esterification reaction as shown in Scheme 42 and Scheme 43, respectively.

V. SUMMARY

The design and synthesis of classical pyrrolo[2,3-d]pyrimidines as potential antifolates have been described. As a part of this study, a total of sixty new compounds have been synthesized and characterized. Of these, ten antifolate compounds were submitted for biological evaluation.

The target compounds synthesized as part of this study are as follows:

Ten Classical Antifolates for bio-evaluation:

1. (S)-2-(6-(2-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-

yl)ethyl)benzo[b]thiophene-2-carboxamido)pentanedioic acid (135).

2. (S)-2-(6-(2-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-

yl)ethyl)-2-naphthamido)pentanedioic acid (136).

3. (S)-2-(5-(2-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-

yl)ethyl)benzo[b]thiophene-2-carboxamido)pentanedioic acid (137) .

4. (S)-2-(6-(3-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-

yl)propyl)benzo[b]thiophene-2-carboxamido)pentanedioic acid (138).

5. (S)-2-(4-(4-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-

yl)butyl)-1-naphthamido)pentanedioic acid (139).

6. (S)-2-(4-(4-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-

yl)but-1-yn-1-yl)-1-naphthamido)pentanedioic acid (140).

7. (S)-2-(5-(4-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-

yl)butyl)thiophene-2-carboxamido)hexanedioic acid (141).

8. 4-(5-(4-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-

yl)butyl)thiophene-2-carboxamido)butanoic acid (142).

9. (S)-2-(5-(4-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-

yl)butyl)-N-methylthiophene-2-carboxamido)pentanedioic acid (143).

10. (S)-2-(5-(4-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-

yl)butyl)thiophene-2-carboxamido)succinic acid (144).

VI. EXPERIMENTAL

All evaporations were carried out in vacuum with a rotary evaporator. Analytical samples were dried in vacuo (0.2 mmHg) in a CHEM-DRY drying apparatus over P2O5 at 60 °C.

Melting points were determined on a MEL-TEMP II melting point apparatus with FLUKE

51 K/J electronic thermometer and are uncorrected. Nuclear magnetic resonance spectra for proton (1H NMR) were recorded on Bruker Avance II 400 (400 MHz) and 500 (500

MHz) spectrometer. 1H NMR analysis was performed with the software Mestrenova by

Mestrelab Research, Inc. is used for. The chemical shift values are expressed in ppm (parts per million) relative to tetramethylsilane as an internal standard: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad singlet. Thin-layer chromatography (TLC) was performed on Whatman Sil G/UV254 silica gel plates with a fluorescent indicator, and the spots were visualized under 254 and 366 nM illumination. Proportions of solvents used for

TLC are by volume. Column chromatography was performed on a 230-400 mesh silica gel

(Fisher, Somerville, NJ) column. The amount (weight) of silica gel for column

chromatography was in the range of 50-100 times the amount (weight) of the crude compounds being separated. Columns were dry-packed unless specified otherwise.

Elemental analyses were performed by Atlantic Microlab, Inc., Norcross, GA. Element compositions are within ± 0.4% of the calculated values. Fractional moles of water or organic solvents frequently found in some analytical samples of antifolates could not be prevented despite 24-48 h of drying in vacuo and were confirmed where possible by their presence in the 1H NMR spectra. High-resolution mass spectrometry (HRMS) was performed on a Waters Q-TOF (API-US) by Department of Chemistry, University of

Pittsburgh, Pittsburgh, PA. All solvents and chemicals were purchased from Aldrich

Chemical Co. and Fisher Scientific and were used as received.

General procedure for the synthesis of compounds 147, 148 and 168.

To a 20-mL vial for microwave reaction, was added a mixture of Bis(triphenylphosphine)palladium(II) dichloride (112 mg, 0.16 mmol), triphenylphosphine (84 mg, 0.32 mmol), triethylamine (1.01 g, 10 mmol), 145, 146 and 167 (271 mg, 1 mmol) and anhydrous acetonitrile (10 mL). To the stirred mixture, were added copper(I) iodide (61 mg, 0.32 mmol), and prop-2-yn-1-ol, 165 (84 mg, 1.5 mmol), and the vial was sealed and put into the microwave reactor at 100 °C for 1 h. Silica gel (0.5 g) was added and the solvent was evaporated under reduced pressure. The resulting plug was loaded on to a silica gel column (3.5 × 12 cm) and eluted with hexane followed by 20% EtOAc in hexane. The desired fraction (TLC) was collected and the solvent was evaporated under reduced pressure to afford 147, 148 and 168.

methyl 6-(3-hydroxyprop-1-yn-1-yl)benzo[b]thiophene-2-carboxylate (147):

Compound 147 was prepared using the general method described for the preparation of

147-148, 168, from 145 (270 mg, 1 mmol) to give 202 mg (82%) of 147 as a yellow powder.

1 mp 113.9-115.1 °C; TLC Rf 0.60 (hexane/EtOAc 1:1); H NMR (DMSO-d6):  3.90 (s, 3H,

COOCH3), 4.34-4.36 (d, 2 H, CH2, J = 6 Hz), 5.41-5.44 (t, 1 H, OH, J = 6 Hz, exch), 7.48-

7.50 (m, 1 H, Ar), 8.01-8.03 (d, 1 H, Ar, J = 8.4 Hz), 8.21 (s, 1 H, Ar), 8.23 (s, 1 H, Ar).

Anal. (C13H10O3S · 0.49 CH3CN · 0.24 CH3COOH) Cal. C: 61.86, H: 4.46, S: 11.43. Found

C: 61.87, H: 4.12, S: 11.33. methyl 6-(3-hydroxyprop-1-yn-1-yl)-2-naphthoate (148): Compound 148 was prepared using the general method described for the preparation of 147, 148 and 168, from 146 (264

mg, 1 mmol) to give 209 mg (87%) of 148 as a light-yellow oil. TLC Rf 0.60

1 (hexane/EtOAc 1:1); H NMR (DMSO-d6):  3.92 (s, 3H, COOCH3), 4.37-4.39 (d, 2 H,

CH2, J = 6 Hz), 5.46-5.49 (t, 1 H, OH, J = 6 Hz, exch), 7.50-7.52 (m, 1 H, Ar), 7.86 (s, 1

H, Ar), 8.02 (s, 2 H, Ar), 8.11-8.13 (d, 1 H, Ar, J = 8.4 Hz), 8.66 (s, 1 H, Ar). The reaction was carried over to the next step without further characterization. methyl 5-(3-hydroxyprop-1-yn-1-yl)benzo[b]thiophene-2-carboxylate (168):

Compound 168 was prepared using the general method described for the preparation of

147, 148 and 168, from 167 (270 mg, 1 mmol) to give 207 mg (84%) of 168 as a yellow

1 powder. mp 115.2-116.8 °C; TLC Rf 0.59 (hexane/EtOAc 1:1); H NMR (DMSO-d6): 

3.90 (s, 3 H, COOCH3), 4.34-4.35 (d, 2 H, CH2, J = 6 Hz), 5.38-5.41 (t, 1 H, OH, J = 6

Hz, exch), 7.53-7.55 (m, 1 H, Ar), 8.09-8.11 (d, 1 H, Ar, J = 8.4 Hz), 8.13 (s, 1 H, Ar), 8.22

(s, 1 H, Ar). Anal. (C13H10O3S · 0.90 CH3CN) Cal. C: 62.76, H: 4.52, S: 11.32. Found C:

62.72, H: 4.14, S: 11.23.

General procedure for the synthesis of compounds 149, 150 and 169.

To a Parr flask was added 147, 148 and 168 (492 mg, 2 mmol), 10% palladium on activated carbon (344 mg), and MeOH (100 mL). Hydrogenation was carried out at 62 psi of H2 overnight. The reaction mixture was filtered through Celite®, washed with MeOH (100 mL). The solvent was evaporated under reduced pressure. The resulting plug was loaded on to a silica gel column (3.5 × 12 cm) and eluted with hexane followed by 25% EtOAc in hexane. The desired fraction (TLC) was collected and the solvent was evaporated under reduced pressure to give 149, 150 and 169. methyl 6-(3-hydroxypropyl)benzo[b]thiophene-2-carboxylate (149): Compound 149 was prepared using the general method described for the preparation of 149, 150 and 169, from 147 (492 mg, 2 mmol) to give 465 mg (93%) of 149 as a light yellow powder. mp

1 134.8-135.6 °C; TLC Rf 0.51 (hexane/EtOAc 1:1); H NMR (DMSO-d6):  1.75-1.82 (m,

2 H, CH2), 2.74-2.78 (t, 2 H, CH2, J = 7.2 Hz), 3.89 (s, 3 H, COOCH3), 4.52-4.55 (t, 1 H,

OH, J = 5.2 Hz, exch), 7.33-7.35 (m, 1 H, Ar), 7.88 (s, 1 H, Ar), 7.93-7.95 (d, 1 H, Ar, J =

8.4 Hz), 8.17 (s, 1 H, Ar). Anal. (C13H14O3S) Cal. C: 62.38, H: 5.64, S: 12.81. Found C:

62.16, H: 5.86, S: 12.81. methyl 6-(3-hydroxypropyl)-2-naphthoate (150): Compound 150 was prepared using the general method described for the preparation of 149, 150 and 169, from 237 (480 mg,

2 mmol) to give 442 mg (92%) of 150 as a light-yellow oil. TLC Rf 0.52 (hexane/EtOAc

1 1:1); H NMR (DMSO-d6):  1.79-1.86 (m, 2 H, CH2), 2.80-2.84 (t, 2 H, CH2, J = 7.6 Hz),

3.43-3.48 (q, 2 H, CH2, J = 7.4 Hz), 3.91 (s, 3 H, COOCH3), 4.55-4.57 (t, 1 H, OH, J =

5.2 Hz, exch), 7.50-7.52 (m, 1 H, Ar), 7.80 (s, 1 H, Ar), 7.96 (s, 2 H, Ar), 8.05-8.07 (d, 1

H, Ar, J = 8.4 Hz), 8.60 (s, 1 H, Ar). The reaction was carried over to the next step without further characterization. methyl 5-(3-hydroxypropyl)benzo[b]thiophene-2-carboxylate (169): Compound 169 was prepared using the general method described for the preparation of 149, 150 and 169, from 168 (438 mg, 2 mmol) to give 465 mg (89%) of 169 as a light yellow powder. mp

1 125.7-127.1 °C; TLC Rf 0.51 (hexane/EtOAc 1:1); H NMR (DMSO-d6):  1.75-1.81 (m,

2 H, CH2), 2.74-2.77 (t, 2 H, CH2, J = 7.2 Hz), 3.90 (s, 3 H, COOCH3), 4.52-4.55 (t, 1 H,

OH , J = 5.2 Hz, exch), 7.41-7.43 (m, 1 H, Ar), 7.85 (s, 1 H, Ar), 7.97-7.99 (d, 1 H, Ar, J =

7.2 Hz), 8.17 (s, 1 H, Ar). Anal. (C13H14O3S) Cal. C: 62.38, H: 5.64, S: 12.81. Found C:

62.39, H: 5.80, S: 12.55.

General procedure for the synthesis of compounds 151, 152 and 170.

To acetonitrile (20 mL) was added periodic acid (502 mg, 2.2 mmol). The mixture was stirred vigorously at 0 °C for 30 min. 149, 150 and 169 (250 mg, 1 mmol) in acetonitrile

(5 mL) was then added followed by addition of PCC (4.32 mg, 0.02 mmol) in acetonitrile

(5 mL). The mixture was stirred in an ice bath for additional 2 h, and allowed to warm to the room temperature. The solvent was evaporated under reduced pressure to afford a residue. The resulting residue was then diluted with EtOAc (100 mL) and washed with brine, sat. aq NaHSO3 solution, and brine, respectively, dried over anhydrous Na2SO4.

Silica gel (5 g) was added and the solvent was evaporated under reduced pressure. The resulting plug was loaded on to a silica gel column (3.5 × 12 cm) and eluted with hexane followed by 25% EtOAc in hexane. The desired fraction (TLC) was collected and the

100

solvent was evaporated under reduced pressure to afford 151, 152 and 170.

3-(2-(methoxycarbonyl)benzo[b]thiophen-6-yl)propanoic acid (151): Compound 151 was prepared using the general method described for the preparation of 151, 152 and 170, from 149 (250 mg, 1 mmol) to give 214 mg (81%) of 151 as a light yellow powder. mp

1 274.9-275.4 °C; TLC Rf 0.42 (hexane/EtOAc 1:1); H NMR (DMSO-d6):  2.61-2.64 (t, 2

H, CH2, J = 7.6 Hz), 2.95-2.99 (t, 2 H, CH2, J = 7.6 Hz), 3.89 (s, 3 H, COOCH3), 7.37-7.39

(m, 1 H, Ar), 7.91 (s, 1 H, Ar), 7.93-7.95 (d, 1 H, Ar, J = 8.4 Hz), 8.17 (s, 1 H, Ar), 12.20

(br, 1 H, COOH, exch). Anal. (C13H12O4S) Cal. C: 59.08, H: 4.58, S: 12.13. Found C: 59.22,

H: 4.61, S: 11.98.

3-(6-(methoxycarbonyl)naphthalen-2-yl)propanoic acid (152): Compound 152 was prepared using the general method described for the preparation of 151, 152 and 170, from

150 (244 mg, 1 mmol) to give 222 mg (86%) of 152 as a light yellow powder. mp 127.8-

1 127.9 °C; TLC Rf 0.44 (hexane/EtOAc 1:1); H NMR (DMSO-d6):  2.65-2.68 (t, 2 H, CH2,

J = 7.2 Hz), 3.00-3.04 (t, 2 H, CH2, J = 7.2 Hz), 3.91 (s, 3 H, COOCH3), 7.53-7.55 (d, 1 H,

Ar, J = 8.4 Hz), 7.83 (s, 1 H, Ar), 7.96 (s, 2 H, Ar), 8.05-8.07 (d, 1 H, Ar, J = 8.4 Hz), 8.60

(s, 1 H, Ar), 12.22 (br, 1 H, COOH, exch). Anal. (C15H14O4 · 0.19 H2O) Cal. C: 68.84, H:

5.54. Found C: 68.85, H: 5.63.

3-(2-(methoxycarbonyl)benzo[b]thiophen-5-yl)propanoic acid (170): Compound 170 was prepared using the general method described for the preparation of 151, 152 and 170, from 169 (250 mg, 1 mmol) to give 219 mg (83%) of 170 as a light yellow powder. mp

1 140.8-141.3 °C; TLC Rf 0.41 (hexane/EtOAc 1:1); H NMR (DMSO-d6):  2.59-2.62 (t, 2

H, CH2, J = 7.2 Hz), 2.93-2.97 (t, 2 H, CH2, J = 7.2 Hz), 3.89 (s, 3 H, COOCH3), 7.44-7.46

101

(d, 1 H, Ar, J = 8.0 Hz), 7.87 (s, 1 H, Ar), 7.97-7.99 (d, 1 H, Ar, J = 8.4 Hz), 8.16 (s, 1 H,

Ar), 12.13 (br, 1 H, COOH, exch). Anal. (C13H12O4S) Cal. C: 59.08, H: 4.58, S: 12.13.

Found C: 59.16, H: 4.59, S: 12.19.

General procedure for the synthesis of compounds 159-160,174.

To 151, 152 and 170 (660 mg, 2.5 mmol) in a 100 mL flask was added oxalyl chloride (1.9 g, 15 mmol) and anhydrous CH2Cl2 (20 mL). The resulting solution was refluxed for 1 h and then cooled to room temperature. After evaporating the solvent under reduced pressure, the residue was dissolved in 20 mL of Et2O. The resulting solution was added dropwise to an ice-cooled diazomethane (generated in situ from 8 g of diazald with a Aldrich Mini

Diazald Apparatus) in an ice bath over 10 min. The resulting mixture was allowed to stand for 30 min and then stirred for an additional 1 h. To this solution was added 48% HBr (10 mL). The resulting mixture was refluxed for 1.5 h. After cooling to room temperature, the organic layer was separated and the aqueous layer was extracted with Et2O (2 × 50 mL).

The combined organic layer and Et2O extract was washed with two portions of 10%

Na2CO3 solution and dried over anhydrous Na2SO4. Evaporation of the solvent afforded

646 mg (95%) of 157, 158 and 173 as light yellow oil. TLC Rf 0.81 (hexane/EtOAc 1:1).

To the solution of 157, 158 and 173 in anhydrous DMF (15 mL) was added 2,6-diamino-

3H-pyrimidin-4-one, 177 (378 mg, 3 mmol). The resulting mixture was stirred under N2 at room temperature for 3 days. Silica gel (0.8 g) was then added and the solvent was evaporated under reduced pressure. The resulting plug was loaded on to a silica gel column

(1.5 × 12 cm) and eluted with CHCl3 followed by 3% MeOH in CHCl3 and then 5% MeOH in CHCl3. The desired fraction (TLC) was collected and the solvent was evaporated under reduced pressure to afford 159, 160 and 174.

102

3-(2-(methoxycarbonyl)benzo[b]thiophen-5-yl)propanoic acid (159 ester): Compound

159 was prepared using the general method described for the preparation of 159, 160 and

174, from 151 (660 mg, 2.5 mmol) to give 442 mg (48%) of 159 as a light yellow semisolid.

1 TLC Rf 0.48 (CHCl3/MeOH 5:1); H NMR (DMSO-d6):  2.85-2.89 (t, 2 H, CH2, J = 7.2

Hz), 3.04-3.08 (t, 2 H, CH2, J = 7.2 Hz), 3.89 (s, 3 H, COOCH3), 5.88 (s, 1 H, CH), 6.00

(s, 2 H, 2-NH2, exch), 7.35-7.37 (d, 1 H, Ar, J = 8.4 Hz), 7.90 (s, 1 H, Ar), 7.91-7.93 (d, 1

H, Ar, J = 8.4 Hz), 8.09 (s, 1 H, Ar), 10.17 (s, 1 H, 3-NH, exch), 10.94 (s, 1 H, 3-NH, exch).

The reaction was carried over to the next step without further characterization.

3-(2-(methoxycarbonyl)benzo[b]thiophen-5-yl)propanoic acid (160 ester): Compound

160 was prepared using the general method described for the preparation of 159, 160 and

174, from 152 (645 mg, 2.5 mmol) to give 470 mg (52%) of 160 as a yellow semisolid.

1 TLC Rf 0.48 (CHCl3/MeOH 5:1); H NMR (DMSO-d6):  2.90-2.94 (t, 2 H, CH2, J = 7.2

Hz), 3.10-3.14 (t, 2 H, CH2, J = 7.2 Hz), 3.90 (s, 3 H, COOCH3), 5.90 (s, 1 H, CH), 6.04

(s, 2 H, 2-NH2, exch), 7.56-7.58 (m, 1 H, Ar), 7.84 (s, 1 H, Ar), 7.96 (s, 2 H, Ar), 8.04-8.06

(d, 1 H, Ar, J = 8.4 Hz), 8.57 (s, 1 H, Ar), 10.17 (s, 1 H, 3-NH, exch), 10.97 (s, 1 H, 3-NH, exch). The reaction was carried over to the next step without further characterization.

3-(2-(methoxycarbonyl)benzo[b]thiophen-5-yl)propanoic acid (174 ester): Compound

174 was prepared using the general method described for the preparation of 159, 160 and

174, from 170 (660 mg, 2.5 mmol) to give 460 mg (50%) of 174 as a grey semisolid. TLC

1 Rf 0.48 (CHCl3/MeOH 5:1); H NMR (DMSO-d6):  2.83-2.87 (t, 2 H, CH2, J = 7.2 Hz),

3.01-3.05 (t, 2 H, CH2, J = 7.2 Hz), 3.88 (s, 3 H, COOCH3), 5.88 (s, 1 H, CH), 5.99 (s, 2 H,

2-NH2, exch), 7.40-7.43 (m, 1 H, Ar), 7.87 (s, 1 H, Ar), 7.96-7.98 (d, 1 H, Ar, J = 8.4 Hz),

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8.16 (s, 1 H, Ar), 10.16 (s, 1 H, 3-NH, exch), 10.94 (s, 1 H, 3-NH, exch). The reaction was carried over to the next step without further characterization.

General procedure for the synthesis of compounds 161, 162 and 175.

To a solution of 159, 160 and 174 (368 mg, 1.0 mmol) in MeOH (10 mL) was added 1 N

NaOH (10 mL) and the mixture was stirred under N2 at room temperature for 16 h. TLC showed the disappearance of the starting material (Rf 0.51) and one major spot at the origin

(CHCl3/MeOH 5:1). The reaction mixture was evaporated to dryness under reduced pressure. The residue was dissolved in water (10 mL), the resulting solution was cooled in an ice bath, and the pH was adjusted to 3-4 with dropwise addition of 1 N HCl. The resulting suspension was frozen in a dry ice-acetone bath, thawed to 4-5 °C in the refrigerator, and filtered. The residue was washed with a small amount of cold water and dried in vacuum using P2O5 to afford 161, 162 and 175.

6-(2-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6- yl)ethyl)benzo[b]thiophene-2-carboxylic acid (161): Compound 161 was prepared using the general method described for the preparation of 161, 162 and 175, from 159 (368 mg,

1.0 mmol) to give 336 mg (95%) of 161 as a light yellow powder. mp > 300 °C; TLC Rf

1 0.1 (CHCl3/MeOH 10:1); H NMR (DMSO-d6):  2.85-2.89 (t, 2 H, CH2, J = 7.2 Hz), 3.03-

3.07 (t, 2 H, CH2, J = 7.2 Hz), 5.88 (s, 1 H, CH), 6.00 (s, 2 H, 2-NH2, exch), 7.33-7.35 (d,

1 H, Ar, J = 8.4 Hz), 7.89 (s, 1 H, Ar), 7.90-7.92 (d, 1 H, Ar, J = 8.4 Hz), 8.06 (s, 1 H, Ar),

10.16 (s, 1 H, 3-NH, exch), 10.93 (s, 1 H, 3-NH, exch), 13.35 (br, 1 H, COOH, exch). Anal.

(C17H14N4O3S · 1.23 H2O · 0.10 HCl) Cal. C: 53.70, H: 4.39, N: 14.73, S: 8.43. Found C:

53.69, H: 4.39, N: 14.86, S: 8.24.

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6-(2-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)ethyl)-2- naphthoic acid (162): Compound 162 was prepared using the general method described for the preparation of 161, 162 and 175, from 160 (362 mg, 1.0 mmol) to give 327 mg

(94%) of 162 as a light yellow powder. mp 240.9-241.6 °C; TLC Rf 0.1 (CHCl3/MeOH

1 10:1); H NMR (DMSO-d6):  2.89-2.93 (t, 2 H, CH2, J = 7.2 Hz), 3.09-3.13 (t, 2 H, CH2,

J = 7.2 Hz), 5.89 (s, 1 H, CH), 6.00 (s, 2 H, 2-NH2, exch), 7.50-7.52 (m, 1 H, Ar), 7.82 (s,

1 H, Ar), 7.94 (s, 2 H, Ar), 8.02-8.04 (d, 1 H, Ar, J = 8.4 Hz), 8.55 (s, 1 H, Ar), 10.16 (s, 1

H, 3-NH, exch), 10.96 (s, 1 H, 3-NH, exch), 13.03 (br, 1 H, COOH, exch). Anal.

(C19H16N4O3 · 0.67 CH3OH · 0.64 HCl) Cal. C: 60.11, H: 4.95, N: 14.25. Found C: 60.12,

H: 4.93, N: 14.23, Cl: 1.33.

5-(2-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6- yl)ethyl)benzo[b]thiophene-2-carboxylic acid (175): Compound 175 was prepared using the general method described for the preparation of 161, 162 and 175, from 174 (368 mg,

1.0 mmol) to give 340 mg (96%) of 175 as a light yellow powder. mp 153.7-154.2 °C; TLC

1 Rf 0.1 (CHCl3/MeOH 10:1); H NMR (DMSO-d6):  2.83-2.87 (t, 2 H, CH2, J = 7.6 Hz),

3.00-3.04 (t, 2 H, CH2, J = 7.6 Hz), 5.89 (s, 1 H, CH), 6.03 (s, 2 H, 2-NH2, exch), 7.34-7.36

(m, 1 H, Ar), 7.80 (s, 1 H, Ar), 7.89-7.91 (d, 1 H, Ar, J = 8.4 Hz), 7.92 (s, 1 H, Ar), 10.22

(s, 1 H, 3-NH, exch), 10.93 (s, 1 H, 3-NH, exch), 13.32 (br, 1 H, COOH, exch). Anal.

(C17H14N4O3S · 1.59 H2O) Cal. C: 53.31, H: 4.52, N: 14.63, S: 8.37. Found C: 53.35, H:

4.31, N: 14.36, S: 8.38.

General procedure for the synthesis of target compounds 135-137.

To a solution of 161, 162 and 175. (354 mg, 1.0 mmol) in anhydrous DMF (10 mL) was

105

added N-methylmorpholine (202 mg, 2.0 mmol) and 2-chloro-4,6-dimethoxy-1,3,5- triazine (352 mg, 2.0 mmol). The resulting mixture was stirred at room temperature for 2 h. To this mixture was added N-methylmorpholine (202 mg, 2.0 mmol) and L-glutamate diethyl ester hydrochloride (480 mg, 2.0 mmol). The reaction mixture was stirred for an additional 4 h at room temperature. Silica gel (700 mg) was then added and the solvent was evaporated under reduced pressure. The resulting plug was loaded on to a silica gel column

(1.5 × 15 cm) and with 5% CHCl3 in MeOH as the eluent. Fractions that showed the desired spot (TLC) were pooled and the solvent evaporated to dryness to afford a residue. To this residue was added MeOH (10 mL) and 1 N NaOH (10 mL) and the mixture was stirred under N2 at room temperature for 16 h. TLC showed the disappearance of the starting material (Rf 0.38) and one major spot at the origin (CHCl3/MeOH 5:1). The reaction mixture was then evaporated to dryness under reduced pressure. The residue was dissolved in water (10 mL), the resulting solution was cooled in an ice bath, and the pH was adjusted to 3-4 with dropwise addition of 1 N HCl. The resulting suspension was frozen in a dry ice-acetone bath, thawed to 4-5 °C in the refrigerator, and filtered. The residue was washed with a small amount of cold water and dried in vacuum using P2O5 to afford 135-137.

(S)-2-(6-(2-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6- yl)ethyl)benzo[b]thiophene-2-carboxamido)pentanedioic acid (135): Compound 135 was prepared using the general method described for the preparation of 135-137, from 161

(354 mg, 1.0 mmol) to give 290 mg (60%) of 135 as a light yellow powder. mp 274.3-

1 275.1 °C; TLC Rf 0.10 (CHCl3/MeOH 5:1); H NMR (DMSO-d6):  1.91-2.00 (m, 1 H,

CH2), 2.07-2.15 (m, 1 H, CH2), 2.36-2.40 (t, 2 H, CH2, J = 7.2 Hz), 2.84-2.88 (t, 2 H, CH2,

J = 7.2 Hz), 3.02-3.06 (t, 2 H, CH2, J = 7.2 Hz), 4.37-4.42 (m, 1 H, CH), 5.89 (s, 1 H, CH),

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6.00 (s, 2 H, 2-NH2, exch), 7.31-7.33 (d, 1 H, Ar, J = 8.4 Hz), 7.85-7.87 (m, 2 H, Ar), 8.17

(s, 1 H, Ar), 8.88-8.90 (d, 1 H, CONH, J = 8.0 Hz, exch), 10.15 (s, 1 H, 3-NH, exch), 10.92

(s, 1 H, 3-NH, exch), 12.47 (br, 1 H, COOH, exch). Anal. (C22H21N5O6S · 0.90

CH3OH · 0.67 HCl) Cal. C: 51.24, H: 4.74, N: 13.05, S: 5.97. Found C: 51.20, H: 4.91, N:

13.07, S: 6.23.

(S)-2-(6-(2-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)ethyl)-2- naphthamido)pentanedioic acid (136): Compound 136 was prepared using the general method described for the preparation of 135-137, from 162 (348 mg, 1.0 mmol) to give

281 mg (59%) of 136 as a light yellow powder. mp 218.7-219.1 °C; TLC Rf 0.18

1 (CHCl3/MeOH 5:1); H NMR (DMSO-d6):  1.95-2.04 (m, 1 H, CH2), 2.08-2.16 (m, 1 H,

CH2), 2.38-2.42 (t, 2 H, CH2, J = 7.2 Hz), 2.89-2.93 (t, 2 H, CH2, J = 7.2 Hz), 3.08-3.12 (t,

2 H, CH2, J = 7.2 Hz), 4.43-4.48 (m, 1 H, CH), 5.89 (s, 1 H, CH), 5.90 (s, 2 H, 2-NH2, exch),

7.49-7.52 (m, 1 H, Ar), 7.80 (s, 1 H, Ar), 7.92 (s, 1 H, Ar), 7.95-7.97 (d, 1 H, Ar, J = 8.4

Hz), 8.45 (s, 1 H, Ar), 8.71-8.73 (d, 1 H, CONH, J = 7.6 Hz, exch), 10.15 (s, 1 H, 3-NH, exch), 10.95 (s, 1 H, 3-NH, exch), 12.47 (br, 1 H, COOH, exch). Anal. (C24H23N5O6 · 0.74

CF3COOH · 1.52 H2O) Cal. C: 51.97, H: 4.58, N: 11.90, F: 8.43. Found C: 52.06, H: 4.76,

N: 11.61, F: 8.10.

(S)-2-(5-(2-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6- yl)ethyl)benzo[b]thiophene-2-carboxamido)pentanedioic acid (137): Compound 137 was prepared using the general method described for the preparation of 135-137, from 175

(354 mg, 1.0 mmol) to give 280 mg (58%) of 137 as a light yellow powder. mp 257.6-

1 258.9 °C; TLC Rf 0.12 (CHCl3/MeOH 5:1); H NMR (DMSO-d6):  1.91-2.00 (m, 1 H,

107

CH2), 2.05-2.14 (m, 1 H, CH2), 2.36-2.40 (t, 2 H, CH2, J = 7.2 Hz), 2.84-2.88 (t, 2 H, CH2,

J = 7.2 Hz), 3.02-3.06 (t, 2 H, CH2, J = 7.2 Hz), 4.37-4.42 (m, 1 H, CH), 5.88 (s, 1 H, CH),

6.00 (s, 2 H, 2-NH2, exch), 7.34-7.36 (m, 2 H, Ar), 7.77(s, 1 H, Ar), 7.91-7.93 (d, 1 H, Ar,

J = 8.4 Hz), 8.14 (s, 1 H, Ar), 8.86-8.88 (d, 1 H, CONH, J = 7.6 Hz, exch), 10.17 (s, 1 H,

3-NH, exch), 10.93 (s, 1 H, 3-NH, exch), 12.52 (br, 1 H, COOH, exch). Anal.

(C22H21N5O6S · 0.99 CH3OH · 1.01 HCl) Cal. C: 50.02, H: 4.74, N: 12.69, S: 5.81. Found

C: 50.02, H: 4.74, N: 12.67, S: 5.84. methyl 6-(4-hydroxybut-1-yn-1-yl)benzo[b]thiophene-2-carboxylate (178): To a 20 mL vial for microwave reaction, was added a mixture of Bis(triphenyl-phosphine)palladium(II) dichloride (112 mg, 0.16 mmol), triphenylphosphine (84 mg, 0.32 mmol), triethylamine

(1.01 g, 10 mmol), 145 (270 mg, 1.0 mmol) and anhydrous acetonitrile (10 mL). To the stirred mixture, were added copper(I) iodide (61 mg, 0.32 mmol), and but-3-yn-1-ol, 166

(105 mg, 1.5 mmol), and the vial was sealed and put into the microwave reactor at 100 °C for 1 h. Silica gel (0.5 g) was added and the solvent was evaporated under reduced pressure.

The resulting plug was loaded on to a silica gel column (3.5 × 12 cm) and eluted with hexane followed by 20% EtOAc in hexane. The desired fraction (TLC) was collected and the solvent was evaporated under reduced pressure to afford 211 mg (81%) of 178 as an

1 orange powder. mp 107.5-108.6 °C; TLC Rf 0.41 (hexane/EtOAc 1:1); H NMR (DMSO- d6): 2.59-2.62 (t, 2 H, CH2, J = 6.8 Hz), 3.59-3.64 (q, 2 H, CH2, J = 7.2 Hz), 3.90 (s, 3 H,

COOCH3), 4.94-4.97 (t, 1 H, OH, J = 5.6 Hz, exch), 7.45-7.47 (m, 1 H, Ar), 7.98-8.00 (d,

1 H, Ar, J = 8.4 Hz), 8.16 (s, 1 H, Ar), 8.21 (s, 1 H, Ar). The reaction was carried over to the next step without further characterization.

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methyl 6-(4-hydroxybutyl)benzo[b]thiophene-2-carboxylate (179) To a Parr flask were added 178 (792 mg, 3.0 mmol), 10% palladium on activated carbon (554 mg), and MeOH

(35 mL). Hydrogenation was carried out at 60 psi of H2 for overnight. The reaction mixture was filtered through Celite®, washed with MeOH (100 mL) and concentrated under reduced

1 pressure to give 729 mg (92%) of 179 as yellow oil. TLC Rf 0.42 (hexane/EtOAc 1:1); H

NMR (DMSO-d6):  1.41-1.49 (m, 2 H, CH2), 1.61-1.70 (m, 2 H, CH2), 2.71-2.75 (t, 2 H,

CH2, J = 7.2 Hz), 3.47-3.61 (t, 2 H, CH2, J = 7.2 Hz), 3.88 (s, 3 H, COOCH3), 4.39-4.41 (t,

1 H, OH, J = 5.6 Hz, exch), 7.33-7.35 (d, 1 H, Ar, J = 8.0 Hz), 7.88 (s, 1 H, Ar), 7.93-7.95

(d, 1 H, Ar, J = 8.0 Hz), 8.17 (s, 1 H, Ar). Anal. (C14H16O3S · 0.22 H2O) Cal. C: 62.67, H:

6.18, S: 11.95. Found C: 62.71, H: 6.23, S: 11.76.

4-(2-(methoxycarbonyl)benzo[b]thiophen-6-yl)butanoic acid (180): To acetonitrile (20 mL) was added periodic acid (502 mg, 2.2 mmol). The mixture was stirred vigorously at

0 °C for 30 min. 179 (264 mg, 1 mmol) in acetonitrile (5 mL) was then added followed by addition of PCC (4.32 mg, 0.02 mmol) in acetonitrile (5 mL). The mixture was stirred in an ice bath for additional 2 h, and allowed to warm to the room temperature. The solvent was evaporated under reduced pressure to afford a residue. The resulting residue was then diluted with EtOAc (100 mL) and washed with brine, sat. aq NaHSO3 solution, and brine, respectively, dried over anhydrous Na2SO4. Silica gel (5 g) was added and the solvent was evaporated under reduced pressure. The resulting plug was loaded on to a silica gel column

(3.5 × 12 cm) and eluted with hexane followed by 25% EtOAc in hexane. The desired fraction (TLC) was collected and the solvent was evaporated under reduced pressure to

afford 228 mg (85%) of 180 as orange solid. mp 73.9-74.8 °C; TLC Rf 0.46 (hexane/EtOAc

1 1:1); H NMR (DMSO-d6):  1.83-1.90 (m, 2 H, CH2), 2.23-2.27 (t, 2 H, CH2, J = 7.2 Hz),

109

2.72-2.76 (t, 2 H, CH2, J = 7.2 Hz), 3.89 (s, 3 H, COOCH3), 7.33-7.35 (d, 1 H, Ar, J = 8.0

Hz), 7.89 (s, 1 H, Ar), 7.94-7.96 (d, 1 H, Ar, J = 8.4 Hz), 8.17 (s, 1 H, Ar), 12.11 (br, 1 H,

COOH, exch),. Anal. (C14H14O4S · 0.32 H2O) Cal. C: 59.20, H: 5.19, S: 11.29. Found C:

59.25, H: 4.98, S: 10.91. methyl 6-(3-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6- yl)propyl)benzo[b]thiophene-2-carboxylate (184): To 180 (670 mg, 2.5 mmol) in a 100 mL flask was added oxalyl chloride (1.9 g, 15 mmol) and anhydrous CH2Cl2 (20 mL). The resulting solution was refluxed for 1 h and then cooled to room temperature. After evaporating the solvent under reduced pressure, the residue was dissolved in 20 mL of Et2O.

The resulting solution was added dropwise to an ice-cooled diazomethane (generated in situ from 8 g of diazald by using Aldrich Mini Diazald Apparatus) in an ice bath over 10 min. The resulting mixture was allowed to stand for 30 min and then stirred for an additional 1 h. To this solution was added 48% HBr (10 mL). The resulting mixture was refluxed for 1.5 h. After cooling to room temperature, the organic layer was separated and the aqueous layer was extracted with Et2O (2 × 50 mL). The combined organic layer and

Et2O extract was washed with two portions of 10% Na2CO3 solution and dried over anhydrous Na2SO4. Evaporation of the solvent afforded 797 mg (90%) of 183 as yellow

oil. TLC Rf 0.82 (hexane/EtOAc 1:1). To the solution of 183 in anhydrous DMF (15 mL) was added 2,6-diamino-3H-pyrimidin-4-one, 177 (378 mg, 3 mmol). The resulting mixture was stirred under N2 at room temperature for 3 days. Silica gel (0.8 g) was then added and the solvent was evaporated under reduced pressure. The resulting plug was loaded on to a silica gel column (1.5 × 12 cm) and eluted with CHCl3 followed by 3% MeOH in CHCl3 and then 5% MeOH in CHCl3. The desired fraction (TLC) was collected and the solvent

110

was evaporated under reduced pressure to afford 477 mg (50%) of 184 as dark dark orange

1 semisolid. TLC Rf 0.43 (CHCl3/MeOH 5:1); H NMR (DMSO-d6):  1.95-2.03 (m, 2 H,

CH2), 2.36-2.40 (t, 2 H, CH2, J = 7.6 Hz), 2.76-2.80 (t, 2 H, CH2, J = 7.6 Hz), 3.88 (s, 3 H,

COOCH3), 5.93 (s, 1 H, CH), 6.03 (s, 2 H, 2-NH2, exch), 7.34-7.37 (m, 1 H, Ar), 7.89 (s, 1

H, Ar), 7.93-7.95 (d, 1 H, Ar, J = 8.0 Hz), 8.09 (s, 1 H, Ar), 10.20 (s, 1 H, 3-NH, exch),

10.87 (s, 1 H, 3-NH, exch). The reaction was carried over to the next step without further characterization.

6-(3-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6- yl)propyl)benzo[b]thiophene-2-carboxylic acid (185): To a solution of 184 (382 mg, 1.0 mmol) in MeOH (10 mL) was added 1 N NaOH (10 mL) and the mixture was stirred under

N2 at room temperature for 16 h. TLC showed the disappearance of the starting material

(Rf 0.43) and one major spot at the origin (CHCl3/MeOH 5:1). The reaction mixture was evaporated to dryness under reduced pressure. The residue was dissolved in water (10 mL), the resulting solution was cooled in an ice bath, and the pH was adjusted to 3-4 with dropwise addition of 1 N HCl. The resulting suspension was frozen in a dry ice-acetone bath, thawed to 4-5 °C in the refrigerator, and filtered. The residue was washed with a small amount of cold water and dried in vacuum using P2O5 to afford 342 mg (93%) of 185 as

1 dark dark brown powder. mp > 300 °C; TLC Rf 0.12 (CHCl3/MeOH 5:1); H NMR

(DMSO-d6):  1.92-2.00 (m, 2 H, CH2), 2.33-2.37 (t, 2 H, CH2, J = 7.6 Hz), 2.73-2.77 (t, 2

H, CH2, J = 7.6 Hz), 5.92 (s, 1 H, CH), 6.02 (s, 2 H, 2-NH2, exch), 7.31-7.34 (m, 1 H, Ar),

7.87 (s, 1 H, Ar), 7.91-7.93 (d, 1 H, Ar, J = 8.0 Hz), 8.07 (s, 1 H, Ar), 10.19 (s, 1 H, 3-NH, exch), 10.86 (s, 1 H, 3-NH, exch), 13.34 (br, 1 H, COOH, exch). Anal. (C18H16N4O3S · 0.49

HCl) Cal. C: 55.98, H: 4.30, N: 14.51, S: 8.30. Found C: 56.10, H: 4.49, N: 14.11, S: 8.11.

111

(S)-2-(6-(3-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6- yl)propyl)benzo[b]thiophene-2-carboxamido)pentanedioic acid (138): To a solution of

185 (368 mg, 1.0 mmol) in anhydrous DMF (10 mL) was added N-methylmorpholine (202 mg, 2.0 mmol) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (352 mg, 2.0 mmol). The resulting mixture was stirred at room temperature for 2 h. To this mixture was added N- methylmorpholine (202 mg, 2.0 mmol) and L-glutamate diethyl ester hydrochloride (480 mg, 2.0 mmol). The reaction mixture was stirred for an additional 4 h at room temperature.

Silica gel (800 mg) was then added and the solvent was evaporated under reduced pressure.

The resulting plug was loaded on to a silica gel column (1.5 × 15 cm) and with 5% CHCl3 in MeOH as the eluent. Fractions that showed the desired spot (TLC) were pooled and the solvent evaporated to dryness to afford a residue. To this residue was added MeOH (10 mL) and 1 N NaOH (10 mL) and the mixture was stirred under N2 at room temperature for 16 h. TLC showed the disappearance of the starting material (Rf 0.35) and one major spot at the origin (CHCl3/MeOH 5:1). The reaction mixture was then evaporated to dryness under reduced pressure. The residue was dissolved in water (10 mL), the resulting solution was cooled in an ice bath, and the pH was adjusted to 3-4 with dropwise addition of 1 N HCl.

The resulting suspension was frozen in a dry ice-acetone bath, thawed to 4-5 °C in the refrigerator, and filtered. The residue was washed with a small amount of cold water and dried in vacuum using P2O5 to afford 138 as dark dark brown powder. mp 234.8-236.3 °C;

1 TLC Rf 0.11 (CHCl3/MeOH 5:1); H NMR (DMSO-d6):  1.93-2.00 (m, 2 H, CH2), 2.04-

2.09 (m, 1 H, CH2), 2.11-2.16 (m, 1 H, CH2), 2.36-2.40 (t, 2 H, CH2, J = 7.2 Hz), 2.84-2.88

(t, 2 H, CH2, J = 7.2 Hz), 3.03-3.07 (t, 2 H, CH2, J = 7.2 Hz), 4.37-4.42 (m, 1 H, CH), 5.93

(s, 1 H, CH), 6.03 (s, 2 H, 2-NH2, exch), 7.32-7.35 (m, 1 H, Ar), 7.88 (s, 1 H, Ar), 7.92-

112

7.94 (d, 1 H, Ar, J = 8.0 Hz), 8.09 (s, 1 H, Ar), 8.89-8.91 (d, 1 H, CONH, J = 8.0 Hz, exch),

10.19 (s, 1 H, 3-NH, exch), 10.87 (s, 1 H, 3-NH, exch), 12.50 (br, 1 H, COOH, exch). Anal.

(C23H23N5O6S · 0.86 H2O) Cal. C, 53.85; H, 4.86; N, 13.65; O, 21.39; S, 6.25. Found C:

53.97, H: 4.89, N: 13.29, S: 5.92. methyl 4-(5-hydroxypent-1-yn-1-yl)-1-naphthoate (188): To a 20-mL vial for microwave reaction, was added a mixture of Bis(triphenyl-phosphine)palladium(II) dichloride (112 mg, 0.16 mmol), triphenylphosphine (84 mg, 0.32 mmol), triethylamine

(1.01 g, 10 mmol), 217 (264 mg, 1.0 mmol) and anhydrous acetonitrile (10 mL). To the stirred mixture, were added copper(I) iodide (61 mg, 0.32 mmol), and pent-4-yn-1-ol, 187

(126 mg, 1.5 mmol), and the vial was sealed and put into the microwave reactor at 100 °C for 1 h. Silica gel (0.5 g) was added and the solvent was evaporated under reduced pressure.

1 yellow powder. mp 115.6-117.1 °C; TLC Rf 0.45 (hexane/EtOAc 1:1); H NMR (DMSO- d6):  1.78-1.87 (m, 2 H, CH2), 2.66-2.69 (t, 2 H, CH2, J = 7.2 Hz), 3.58-3.63 (q, 2 H, CH2,

J = 6.4 Hz), 3.94 (s, 3 H, COOCH3), 4.63-4.51 (t, 1 H, OH, J = 5.2 Hz, exch), 7.71-7.73

(m, 3 H, Ar), 8.07-8.09 (d, 1 H, Ar, J = 7.6 Hz), 8.36-8.39 (m, 1 H, Ar), 8.78-8.81 (m, 1 H,

Ar). Anal. (C17H16O3) Cal. C: 76.10, H: 6.01. Found C: 75.83, H: 6.12.

5-(4-(methoxycarbonyl)naphthalen-1-yl)pent-4-ynoic acid (189): To acetonitrile (20 mL) was added periodic acid (502 mg, 2.2 mmol). The mixture was stirred vigorously at

0 °C for 30 min. 188 (268 mg, 1 mmol) in acetonitrile (5 mL) was then added followed by addition of PCC (4.32 mg, 0.02 mmol) in acetonitrile (5 mL). The mixture was stirred in

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an ice bath for additional 2 h, and allowed to warm to the room temperature. The solvent was evaporated under reduced pressure to afford a residue. The resulting residue was then diluted with EtOAc (100 mL) and washed with brine, sat. aq NaHSO3 solution, and brine, respectively, dried over anhydrous Na2SO4. Silica gel (5 g) was added and the solvent was evaporated under reduced pressure. The resulting plug was loaded on to a silica gel column

(3.5 × 12 cm) and eluted with hexane followed by 25% EtOAc in hexane. The desired fraction (TLC) was collected and the solvent was evaporated under reduced pressure to

afford 245 mg (87%) of 189 as light-yellow powder. mp 89.6-91.3 °C; TLC Rf 0.41

1 (hexane/EtOAc 1:1); H NMR (DMSO-d6):  2.64-2.68 (t, 2 H, CH2, J = 7.2 Hz), 2.82-2.85

(t, 2 H, CH2, J = 6.4 Hz), 3.94 (s, 3 H, COOCH3), 7.70-7.75 (m, 3 H, Ar), 8.08-8.10 (d, 1

H, Ar, J = 7.6 Hz), 8.38-8.40 (m, 1 H, Ar), 8.77-8.80 (m, 1H, Ar), 12.45 (br, 1 H, COOH, exch). Anal. (C17H14O4 · 0.24 CH3CN) Cal. C: 71.86, H: 5.08. Found C: 71.87, H: 5.14. methyl 6-(3-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6- yl)propyl)benzo[b]thiophene-2-carboxylate (193): To 189 (705 mg, 2.5 mmol) in a 100 mL flask was added oxalyl chloride (1.9 g, 15 mmol) and anhydrous CH2Cl2 (20 mL). The resulting solution was refluxed for 1 h and then cooled to room temperature. After evaporating the solvent under reduced pressure, the residue was dissolved in 20 mL of Et2O.

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Et2O extract was washed with two portions of 10% Na2CO3 solution and dried over anhydrous Na2SO4. Evaporation of the solvent afforded 814 mg (91%) of 192 as yellow

oil. TLC Rf 0.83 (hexane/EtOAc 1:1). To the solution of 192 in anhydrous DMF (15 mL) was added 2,6-diamino-3H-pyrimidin-4-one, 177 (378 mg, 3 mmol). The resulting mixture was stirred under N2 at room temperature for 3 days. Silica gel (0.8 g) was then added and the solvent was evaporated under reduced pressure. The resulting plug was loaded on to a silica gel column (1.5 × 12 cm) and eluted with CHCl3 followed by 3% MeOH in CHCl3 and then 5% MeOH in CHCl3. The desired fraction (TLC) was collected and the solvent was evaporated under reduced pressure to afford 434 mg (45%) of 193 as yellow semisolid.

1 TLC Rf 0.42 (CHCl3/MeOH 5:1); H NMR (DMSO-d6):  2.90-2.92 (t, 2 H, CH2, J = 5.2

Hz), 2.95-2.97 (q, 2 H, CH2, J = 5.2 Hz), 3.94 (s, 3 H, COOCH3), 6.02 (s, 1 H, CH), 6.12

(s, 2 H, 2-NH2, exch), 7.76-7.78 (t, 1 H, Ar, J = 5.6 Hz), 7.68-7.69 (d, 1 H, Ar, J = 6.0 Hz),

7.70 (s, 1 H, Ar), 8.06-8.08 (d, 1 H, Ar, J = 6.0 Hz), 8.17-8.19 (d, 1 H, Ar, , J = 6.8 Hz),

8.75-8.77 (d, 1 H, Ar, , J = 7.2 Hz), 10.20 (s, 1 H, 3-NH, exch), 10.97 (s, 1 H, 3-NH, exch).

The reaction was carried over to the next step without further characterization.

4-(4-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)but-1-yn-1-yl)-1- naphthoic acid (194): To a solution of 193 (386 mg, 1.0 mmol) in MeOH (10 mL) was added 1 N NaOH (10 mL) and the mixture was stirred under N2 at room temperature for

16 h. TLC showed the disappearance of the starting material (Rf 0.42) and one major spot at the origin (CHCl3/MeOH 5:1). The reaction mixture was evaporated to dryness under reduced pressure. The residue was dissolved in water (10 mL), the resulting solution was cooled in an ice bath, and the pH was adjusted to 3-4 with dropwise addition of 1 N HCl.

The resulting suspension was frozen in a dry ice-acetone bath, thawed to 4-5 °C in the

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refrigerator, and filtered. The residue was washed with a small amount of cold water and dried in vacuum using P2O5 to afford 331 mg (89%) of 194 as yellow powder. mp 163.5-

1 164.9 °C; TLC Rf 0.12 (CHCl3/MeOH 5:1); H NMR (DMSO-d6):  2.89-2.96 (m, 4 H,

CH2CH2), 6.02 (s, 1 H, CH), 6.11 (s, 2 H, 2-NH2, exch), 7.53-7.56 (t, 1 H, Ar, J = 6.0 Hz),

7.64-7.68 (m, 2 H, Ar), 8.06-8.07 (d, 1 H, Ar, J = 6.0 Hz), 8.16-8.17 (d, 1 H, Ar, J = 6.0

Hz), 8.86-8.88 (d, 1 H, Ar, , J = 7.2 Hz), 10.20 (s, 1 H, 3-NH, exch), 10.98 (s, 1 H, 3-NH, exch), 13.22 (br, 1 H, COOH, exch). Anal. (C21H16N4O3 · 1.14 CH3OH) Cal. C: 65.02, H:

5.07, N: 13.70. Found C: 64.89, H: 4.74, N: 13.77.

(S)-diethyl 2-(4-(4-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)but-

1-yn-1-yl)-1-naphthamido)pentanedioate (195): To a solution of 194 (372 mg, 1.0 mmol) in anhydrous DMF (10 mL) was added N-methylmorpholine (202 mg, 2.0 mmol) and 2- chloro-4,6-dimethoxy-1,3,5-triazine (352 mg, 2.0 mmol). The resulting mixture was stirred at room temperature for 2 h. To this mixture was added N-methylmorpholine (202 mg, 2.0 mmol) and L-glutamate diethyl ester hydrochloride (480 mg, 2.0 mmol). The reaction mixture was stirred for an additional 4 h at room temperature. Silica gel (800 mg) was then added and the solvent was evaporated under reduced pressure. The resulting plug was loaded on to a silica gel column (1.5 × 15 cm) and with 5% CHCl3 in MeOH as the eluent.

Fractions that showed the desired spot (TLC) were pooled and the solvent evaporated to

dryness to afford 256 mg (46%) of 195 as orange semisolid. TLC Rf 0.42 (CHCl3/MeOH

1 5:1); H NMR (DMSO-d6):  1.19-1.24 (m, 5 H, OCH2CH3), 1.22-1.27 (m, 5 H, OCH2CH3),

1.91-2.19 (m, 2 H, CH2), 2.93-2.95 (t, 2 H, CH2, J = 7.2 Hz), 4.17-4.19 (t, 2 H, CH2, J = 7.2

Hz), 4.20-4.23 (m, 2 H, CH2), 4.48-4.53 (m, 1 H, α-CH), 6.07 (s, 2 H, 2-NH2, exch), 6.11

(s, 1 H, CH), 7.52-7.62 (m, 3 H, Ar), 7.65-7.67 (d, 1 H, Ar, J = 7.2 Hz), 8.12-8.14 (d, 1 H,

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Ar, J = 7.2 Hz), 8.20-8.22 (d, 1 H, Ar, J = 7.2 Hz), 8.98-9.00 (d, 1 H, CONH, J = 7.2 Hz, exch), 10.23 (s, 1 H, 3-NH, exch), 10.98 (s, 1 H, 3-NH, exch). The reaction was carried over to the next step without further characterization.

(S)-diethyl 2-(4-(4-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)but-

1-yn-1-yl)-1-naphthamido)pentanedioate (196): To a Parr flask were added 195 (557 mg,

1.0 mmol), 10% palladium on activated carbon (390 mg), and MeOH (35 mL).

Hydrogenation was carried out at 62 psi of H2 for overnight. The reaction mixture was filtered through Celite®, washed with MeOH (100 mL) and concentrated under reduced

1 pressure to give 353 mg (63%) of 196 as yellow oil. TLC Rf 0.40 (CHCl3/MeOH 5:1). H

NMR (DMSO-d6):  1.18-1.29 (m, 6 H, 2 COOCH2-CH3), 1.91-2.00 (m, 1 H, β-CH2), 2.03-

2.11 (m, 1 H, β-CH2), 2.90-2.95 (m, 4 H, 2 CH2), 4.14-4.23 (m, 4 H, 2 COO-CH2), 4.48-

4.53 (m, 1 H, α-CH), 6.07 (s, 1 H, C5-CH), 6.11 (s, 2 H, 2-NH2, exch), 7.54-7.56 (d, 2 H,

Ar, J = 7.2 Hz), 7.58-7.62 (m, 1 H, Ar), 7.65-7.67 (d, 2 H, Ar, J = 7.6 Hz), 8.12-8.14 (d, 2

H, Ar, J = 8.0 Hz), 8.20-8.22(d, 2 H, Ar, J = 8.4 Hz), 8.98-9.00 (d, 1 H, NH-CO, J = 7.2

Hz) 10.23 (s, 1 H, 3-NH, exch), 10.98 (s, 1 H, 7-NH, exch). The reaction was carried over to the next step without further characterization.

(S)-2-(4-(4-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)butyl)-1- naphthamido)pentanedioic acid (139): To a solution of 196 (280 mg, 0.5 mmol) in

MeOH (10 mL) was added 1 N NaOH (10 mL) and the mixture was stirred under N2 at room temperature for 16 h. TLC showed the disappearance of the starting material (Rf 0.40) and one major spot at the origin (CHCl3/MeOH 5:1). The reaction mixture was evaporated to dryness under reduced pressure. The residue was dissolved in water (10 mL), the resulting solution was cooled in an ice bath, and the pH was adjusted to 3-4 with dropwise

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addition of 1 N HCl. The resulting suspension was frozen in a dry ice-acetone bath, thawed to 4-5 °C in the refrigerator, and filtered. The residue was washed with a small amount of cold water and dried in vacuum using P2O5 to afford 240 mg (95%) of 139 as light yellow

1 powder. mp 224.8-226.3 °C; TLC Rf 0.11 (CHCl3/MeOH 5:1); H NMR (DMSO-d6): 

1.67-1.73 (m, 4 H, 2 CH2), 1.87-1.96 (m, 1 H, β-CH2), 2.06-2.14 (m, 1 H, β-CH2), 2.39-

2.42 (t, 2 H, CH2, J = 7.2 Hz), 3.08-3.12 (m, 2 H, γ-CH2), 4.43-4.50 (m, 1H, α-CH), 5.87

(s, 1 H, C5-CH), 5.96 (s, 2 H, 2-NH2, exch), 7.39-7.41 (d, 1 H, Ar, J = 7.2 Hz), 7.51-7.57

(m, 3 H, Ar), 8.10-8.12 (m, 1 H, Ar), 8.25-8.28 (m, 1 H, Ar), 8.72-8.74 (d, 1 H, CO-NH, J

= 7.6 Hz), 10.13 (s, 1 H, 3-NH, exch), 10.81 (s, 1 H, 7-NH, exch), 12.45 (br, 2 H, 2 COOH).

Anal. (C26H27N5O6 · 1.26 H2O) Cal. C: 59.57, H: 4.91, N: 13.36. Found C: 59.14, H: 5.37,

N: 13.05.

(S)-2-(4-(4-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)but-1-yn-1- yl)-1-naphthamido)pentanedioic acid (140): To a solution of 195 (56 mg, 0.1 mmol) in

MeOH (10 mL) was added 1 N NaOH (5 mL) and the mixture was stirred under N2 at room temperature for 16 h. TLC showed the disappearance of the starting material (Rf 0.39) and one major spot at the origin (CHCl3/MeOH 5:1). The reaction mixture was evaporated to dryness under reduced pressure. The residue was dissolved in water (10 mL), the resulting solution was cooled in an ice bath, and the pH was adjusted to 3-4 with dropwise addition of 1 N HCl. The resulting suspension was frozen in a dry ice-acetone bath, thawed to 4-

5 °C in the refrigerator, and filtered. The residue was washed with a small amount of cold water and dried in vacuum using P2O5 to afford 34 mg (68%) of 140 as light-yellow powder.

1 mp 210.6-211.9 °C; TLC Rf 0.15 (CHCl3/MeOH 5:1); H NMR (DMSO-d6):  1.86-1.96

(m, 1 H, CH2), 2.04-2.13 (m, 1 H, CH2), 2.38-2.31 (t, 2 H, CH2, J = 7.2 Hz), 4.43-4.49 (m,

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1 H, α-CH), 6.03 (s, 1 H, CH), 6.11 (s, 2 H, 2-NH2, exch), 7.51-7.61 (m, 3 H, Ar), 7.64-

7.66 (d, 1 H, Ar, J = 7.6 Hz), 8.12-8.14 (d, 1 H, Ar, J = 8.4 Hz), 8.23-8.25 (d, 1 H, Ar, J =

8.4 Hz), 8.80-8.81 (d, 1 H, CONH, J = 7.6 Hz, exch), 10.21 (s, 1 H, 3-NH, exch), 10.97 (s,

1 H, 3-NH, exch), 12.56 (br, 1 H, COOH, exch). Anal. (C26H23N5O6 · 0.23 (C2 H5) 2O · 0.96

HCl) Cal. C: 58.42, H: 4.78, N: 12.65. Found C: 58.43, H: 4.75, N: 12.63. methyl 5-(5-hydroxypent-1-yn-1-yl)thiophene-2-carboxylate (197): To a 20 mL vial for microwave reaction, was added a mixture of Bis(triphenyl-phosphine)palladium(II) dichloride (112 mg, 0.16 mmol), triphenylphosphine (84 mg, 0.32 mmol), triethylamine

(1.01 g, 10 mmol), 216 (221 mg, 1.0 mmol) and anhydrous acetonitrile (10 mL). To the stirred mixture, were added copper(I) iodide (61 mg, 0.32 mmol), and pent-4-yn-1-ol, 187

(126 mg, 1.5 mmol), and the vial was sealed and put into the microwave reactor at 100 °C for 1 h. Silica gel (0.5 g) was added and the solvent was evaporated under reduced pressure.

1 yellow powder. mp 71.9-73.1 °C; TLC Rf 0.52 (hexane/EtOAc 1:1); H NMR (DMSO-d6):

1.65-1.72 (m, 1 H, CH2), 2.51-2.55 (t, 2 H, CH2, J = 6.0 Hz), 3.50-3.51(q, 2 H, CH2, J = 6.0

Hz), 3.82 (s, 3 H, COOCH3), 4.57-4.60 (q, 1 H, OH, J = 5.2 Hz, exch), 7.27-7.28 (d, 1 H,

Ar, J = 4.0 Hz), 7.70-7.71 (d, 1 H, Ar, J = 4.0 Hz). The reaction was carried over to the next step without further characterization. methyl 5-(5-hydroxypentyl)thiophene-2-carboxylate (198): To a Parr flask were added

197 (224 mg, 1.0 mmol), 10% palladium on activated carbon (67 mg), and MeOH (20 mL).

Hydrogenation was carried out at 55 psi of H2 for overnight. The reaction mixture was

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filtered through Celite, washed with MeOH (100 mL) and concentrated under reduced

1 pressure to give 217 mg (95%) of 198 as orange oil. TLC Rf 0.46 (CHCl3/MeOH 5:1); H

NMR (CDCl3):  1.44-1.51 (m, 2 H, CH2), 1.60-1.67 (m, 2 H, CH2), 1.72-1.79 (m, 2 H,

CH2), 2.85-2.89 (t, 2 H, CH2, J = 12.0 Hz), 3.66-3.69 (t, 2 H, CH2, J = 12.0 Hz), 3.88 (s, 3

H, COOCH3), 6.80-6.81 (d, 1 H, Ar, J = 4.0 Hz), 7.65-7.66 (d, 1 H, Ar, J = 4.0 Hz).

5-(5-(methoxycarbonyl)thiophen-2-yl)pentanoic acid (199): To acetonitrile (20 mL) was added periodic acid (502 mg, 2.2 mmol). The mixture was stirred vigorously at 0 °C for 30 min. 198 (228 mg, 1 mmol) in acetonitrile (5 mL) was then added followed by addition of PCC (4.32 mg, 0.02 mmol) in acetonitrile (5 mL). The mixture was stirred in an ice bath for additional 2 h, and allowed to warm to the room temperature. The solvent was evaporated under reduced pressure to afford a residue. The resulting residue was then diluted with EtOAc (100 mL) and washed with brine, sat. aq NaHSO3 solution, and brine, respectively, dried over anhydrous Na2SO4. Silica gel (5 g) was added and the solvent was evaporated under reduced pressure. The resulting plug was loaded on to a silica gel column

(3.5 × 12 cm) and eluted with hexane followed by 25% EtOAc in hexane. The desired fraction (TLC) was collected and the solvent was evaporated under reduced pressure to

1 afford 206 mg (85%) of 199 as light-yellow oil. TLC Rf 0.41 (hexane/EtOAc 1:1); H NMR

(CDCl3):  1.62-1.81 (m, 4 H, CH2CH2), 2.40-2.43 (t, 2 H, CH2, J = 12.0 Hz), 2.87-2.90 (t,

2 H, CH2, J = 12.0 Hz), 3.88 (s, 3 H, COOCH3), 6.81-6.82 (d, 1 H, Ar, J = 4.0 Hz), 7.65-

7.66 (d, 1 H, Ar, J = 4.0 Hz). The reaction was carried over to the next step without further characterization.

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methyl 5-(4-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6- yl)butyl)thiophene-2-carboxylate (203): To 199 (605 mg, 2.5 mmol) in a 100 mL flask was added oxalyl chloride (1.9 g, 15 mmol) and anhydrous CH2Cl2 (20 mL). The resulting solution was refluxed for 1 h and then cooled to room temperature. After evaporating the solvent under reduced pressure, the residue was dissolved in 20 mL of Et2O. The resulting solution was added dropwise to an ice-cooled diazomethane (generated in situ from 8 g of diazald by using Aldrich Mini Diazald Apparatus) in an ice bath over 10 min. The resulting mixture was allowed to stand for 30 min and then stirred for an additional 1 h. To this solution was added 48% HBr (10 mL). The resulting mixture was refluxed for 1.5 h. After cooling to room temperature, the organic layer was separated and the aqueous layer was extracted with Et2O (2 × 50 mL). The combined organic layer and Et2O extract was washed with two portions of 10% Na2CO3 solution and dried over anhydrous Na2SO4. Evaporation

of the solvent afforded 739 mg (93%) of 202 as orange oil. TLC Rf 0.85 (hexane/EtOAc

1:1). To the solution of 202 in anhydrous DMF (15 mL) was added 2,6-diamino-3H- pyrimidin-4-one, 177 (378 mg, 3 mmol). The resulting mixture was stirred under N2 at room temperature for 3 days. Silica gel (0.8 g) was then added and the solvent was evaporated under reduced pressure. The resulting plug was loaded on to a silica gel column

(1.5 × 12 cm) and eluted with CHCl3 followed by 3% MeOH in CHCl3 and then 5% MeOH in CHCl3. The desired fraction (TLC) was collected and the solvent was evaporated under

reduced pressure to afford 441 mg (51%) of 203 as dark yellow semisolid. TLC Rf 0.43

1 (CHCl3/MeOH 5:1); H NMR (DMSO-d6):  1.62-1.65 (m, 4 H, 2 CH2), 2.83-2.85 (t, 2 H,

CH2, J = 7.2 Hz), 3.87 (s, 3 H, COOCH3), 5.88 (s, 1 H, C5-CH), 5.98 (s, 2 H, 2-NH2, exch),

6.92-6.93 (d, 1 H, Ar, J = 3.6 Hz), 7.56-7.57 (d, 1 H, Ar, J = 3.6 Hz), 10.15 (s, 1 H, 3-NH,

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exch), 10.82 (s, 1 H, 7-NH, exch). The reaction was carried over to the next step without further characterization.

5-(4-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)butyl)thiophene-

2-carboxylic acid (204): To a solution of 203 (346 mg, 1.0 mmol) in MeOH (10 mL) was added 1 N NaOH (10 mL) and the mixture was stirred under N2 at room temperature for

16 h. TLC showed the disappearance of the starting material (Rf 0.43) and one major spot at the origin (CHCl3/MeOH 5:1). The reaction mixture was evaporated to dryness under reduced pressure. The residue was dissolved in water (10 mL), the resulting solution was cooled in an ice bath, and the pH was adjusted to 3-4 with dropwise addition of 1 N HCl.

1 159.8 °C; TLC Rf 0.25 (CHCl3/MeOH 5:1); H NMR (DMSO-d6):  1.62-1.65 (m, 4 H, 2

CH2), 2.83-2.86 (t, 2 H, CH2, J = 7.0 Hz), 5.87 (s, 1 H, C5-CH), 5.97 (s, 2 H, 2-NH2, exch),

6.91-6.92 (d, 1 H, Ar, J = 3.5 Hz), 7.55-7.56 (d, 1 H, Ar, J = 3.5 Hz), 10.14 (s, 1 H, 3-NH, exch), 10.81 (s, 1 H, 7-NH, exch), 12.80 (br, 1 H, COOH, exch). Anal.

(C15H16N4O3S · 0.92 H2O) Cal. C: 51.62, H: 5.15, N: 16.05, S: 9.19. Found C: 51.67, H:

4.98, N: 16.00, S: 8.86.

General procedure for the synthesis of compounds 206, 210, 213.

To a solution of 209, 212, 205 (322 mg, 2 mmol) in MeOH (15 mL) was added dropwise of SOCl2 (476 mg, 4 mmol) at 0 °C. The reacting mixture was warmed to room temperature and stirred for 10 h. The resulting solution was concentrated under reduced pressure and

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diluted in fresh MeOH (10 mL). To the solution was added solid NaHCO3 to neutralize to pH 7 and filtered the precipitation. The resulting solution was evaporated to dryness to afford a residue. To this residue was added CH2Cl2 (10 mL) to precipitate the salts NaCl and NaHCO3 and filtered. The resulting solvent was evaporated under reduced pressure to afford 206, 210, 213. methyl 4-aminobutanoate (206): Compound 206 was prepared using the general method described for the preparation of 206, 210, 213, from 205 (206 mg, 2 mmol) to give 306 mg

1 (79%) of 206 as a colorless oil. H NMR (DMSO-d6):  1.78-1.84 (m, 2 H, CH2), 2.43-

2.46 (t, 2 H, CH2, J = 7.5 Hz), 2.79-2.81 (t, 2 H, CH2, J = 7.5 Hz), 3.61 (s, 3 H, COOCH3),

7.94 (br, 3 H, NH3Cl, exch). The reaction was carried over to the next step without further characterization.

(S)-dimethyl 2-aminohexanedioate (210): Compound 210 was prepared using the general method described for the preparation of 206, 210, 213, from 209 (322 mg, 2 mmol) to give

1 306 mg (81%) of 210 as a colorless oil. H NMR (DMSO-d6):  1.52-1.61 (m, 1 H, β-CH2),

1.63-1.72 (m, 1 H, β-CH2), 1.78-1.84 (m, 2 H, γ-CH2), 2.34-2.38 (t, 2 H, CH2, J = 7.2 Hz),

3.60 (s, 3 H, COOCH3), 3.76 (s, 3 H, COOCH3), 4.04-4.07 (t, 1 H, α-CH, J = 6.4 Hz), 8.53

(br, 3 H, NH3Cl , exch). The reaction was carried over to the next step without further characterization.

(S)-dimethyl 2-aminohexanedioate (213): Compound 213 was prepared using the general method described for the preparation of 206, 210, 213, from 212 (322 mg, 2 mmol) to give

1 283 mg (75%) of 213 as a colorless oil. H NMR (DMSO-d6):  2.41-2.47 84 (m, 2 H,

CH2), 2.56-2.64 (m, 1 H, β-CH2), 2.70-2.78 (m, 1 H, β-CH2), 2.83 (br, 3 H, NH-CH3), 3.71

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(s, 3 H, COOCH3), 3.87 (s, 3 H, COOCH3), 3.96-4.03 (m, 1 H, α-CH), 9.86 (br, 1 H, NH, exch), 10.30 (br, 1 H, HCl, exch). The reaction was carried over to the next step without further characterization.

General procedure for the synthesis of target compounds 141-144.

To a solution of 204 (66.4 mg, 0.2 mmol) in anhydrous DMF (10 mL) was added N- methylmorpholine (40.4 mg, 0.4 mmol) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (70.4 mg, 0.4 mmol). The resulting mixture was stirred at room temperature for 2 h. To this mixture was added N-methylmorpholine (40.4 mg, 0.4 mmol) and 205, 209, 212, 219 (96 mg, 0.4 mmol). The reaction mixture was stirred for an additional 4 h at room temperature.

Silica gel (200 mg) was then added and the solvent was evaporated under reduced pressure.

The resulting plug was loaded on to a silica gel column (1.5 × 12 cm) and with 5% CHCl3 in MeOH as the eluent. Fractions that showed the desired spot (TLC) were pooled and the solvent was evaporated to dryness to afford a residue. To this residue was added MeOH

(10 mL) and 1 N NaOH (10 mL) and the mixture was stirred under N2 at room temperature for 16 h. TLC showed the disappearance of the starting material (Rf 0.40) and one major spot at the origin (CHCl3/MeOH 5:1). The reaction mixture was then evaporated to dryness under reduced pressure to afford 207, 208, 211, 214. The residue was dissolved in water

(10 mL), the resulting solution was cooled in an ice bath, and the pH was adjusted to 3-4 with dropwise addition of 1 N HCl. The resulting suspension was frozen in a dry ice- acetone bath, thawed to 4-5 °C in the refrigerator, and filtered. The residue was washed with a small amount of cold water and dried in vacuum using P2O5 to afford the target compounds 141-144.

124

(S)-dimethyl 2-(5-(4-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6- yl)butyl)thiophene-2-carboxamido)hexanedioate (211): Compound 211 was prepared using the general method described for the preparation of 207, 208, 211, 214, from 204

(66.4 mg, 0.2 mmol) to give 39 mg (47% over two steps) of 211 as a brown syrup. TLC Rf

1 0.13 (CHCl3/MeOH 5:1); H NMR (DMSO-d6):  1.56-1.66 (m, 5 H, β-CH2, 2 CH2), 1.74-

1.84 (m, 1 H, β-CH2), 2.32-2.36 (t, 2 H, CH2, J = 7.2 Hz), 2.80-2.83 (t, 2 H, CH2, J = 7.2

Hz), 3.03-3.13 (m, 2 H, CH2), 3.58 (s, 3 H, COOCH3), 3.69 (s, 3 H, COOCH3), 4.34-4.39

(m, 1 H, α-CH)， 5.86 (s, 1 H, C5-CH), 6.03 (s, 2 H, 2-NH2, exch), 6.88-6.89 (d, 1 H, Ar,

J = 3.6 Hz), 7.70-7.71 (d, 1 H, Ar, J = 4.0 Hz), 8.65-8.67 (d, 1 H, CO-NH, J = 7.6 Hz, exch), 10.19 (s, 1 H, 3-NH, exch), 10.81 (s, 1 H, 7-NH, exch). The reaction was carried over to the next step without further characterization.

(S)-2-(5-(4-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6- yl)butyl)thiophene-2-carboxamido)hexanedioic acid (141): Compound 141 was prepared using the general method described for the preparation of 141-144, from 210 (75.6 mg, 0.4 mmol) to give 47 mg (50% over two steps) of 141 as a light grey powder. mp

1 247.5-248.8 °C; TLC Rf 0.12 (CHCl3/MeOH 5:1); H NMR (DMSO-d6):  1.59-1.65 (m,

4 H, 2 CH2), 1.70-1.76 (m, 1 H, β- CH2), 1.78-1.86 (m, 1 H, β- CH2), 2.22-2.25 (t, 2 H,

CH2, J = 7.2 Hz), 2.80-2.83 (t, 2 H, CH2, J = 6.4 Hz), 4.26-4.31 (m, 1 H, α-CH), 5.86 (s, 2

H, 2-NH2, exch), 5.97 (s, 1 H, Ar), 6.87-6.88 (d, 1 H, Ar, J = 3.6 Hz), 7.69-7.70 (d, 1 H,

Ar, J = 3.6 Hz), 8.47-8.48 (d, 1 H, CO-NH, J = 7.6 Hz, exch), 10.14 (s, 1 H, 3-NH, exch),

10.81 (s, 1 H, 7-NH, exch), 12.39 (br, 1 H, 2 COOH, exch). Anal. (C21H25N5O6S · 1.10

H2O) Cal. C: 50.93, H: 5.53, N: 14.14, S: 6.47. Found C: 50.96, H: 5.30, N: 13.94, S: 6.42.

125

4-(5-(4-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6- yl)butyl)thiophene-2-carboxamido)butanoic acid (142): Compound 142 was prepared using the general method described for the preparation of 141-144, from 206 (67.2 mg, 0.4 mmol) to give 39 mg (47% over two steps) of 142 as a light grey powder. mp 217.9-

1 219.4 °C; TLC Rf 0.13 (CHCl3/MeOH 5:1); H NMR (DMSO-d6):  1.60-1.65 (m, 4 H, 2

CH2), 1.70-1.73 (t, 2 H, CH2, J = 6.4 Hz), 2.23-2.26 (t, 2 H, CH2, J = 6.4 Hz), 2.79-2.82 (t,

2 H, CH2, J = 6.8 Hz), 2.20-2.23 (t, 2 H, CH2, J = 7.2 Hz), 5.87 (s, 1 H, C5-CH), 5.97 (s, 2

H, 2-NH2, exch), 6.86 (s, 1 H, Ar), 7.54 (s, 1 H, Ar), 8.36-8.38 (d, 1 H, CO-NH, J = 6.0

Hz, exch), 10.13 (s, 1 H, 3-NH, exch), 10.81 (s, 1 H, 7-NH, exch), 12.08 (br, 1 H, COOH, exch). Anal. (C19H23N5O4S · 1.16 CH3COCH3 · 0.25 HCl) Cal. C: 54.65, H: 6.16, N: 14.18,

S: 6.49. Found C: 54.67, H: 5.98, N: 14.18, S: 6.40.

(S)-2-(5-(4-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)butyl)-N- methylthiophene-2-carboxamido)pentanedioic acid (143): Compound 143 was prepared using the general method described for the preparation of 141-144, from 213

(113.4 mg, 0.6 mmol) to give 37 mg (39% over two steps) of 143 as a light brown powder.

1 mp 238.4-240.1 °C; TLC Rf 0.12 (CHCl3/MeOH 5:1); H NMR (DMSO-d6):  1.60-1.67

(m, 4 H, 2 CH2), 1.93-2.02 (m, 1 H, β- CH2), 2.14-2.29 (m, 1 H, β- CH2, 2 H, CH2), 2.80-

2.84 (m, 3 H, N-CH3), 3.09-3.18 29 (m, 2 H, CH2), 4.82-4.88 (m, 1 H, α-CH), 5.86 (s, 2 H,

2-NH2, exch), 5.98 (s, 1 H, Ar), 6.87-6.92 (m, 1 H, Ar), 7.41-7.55 (m, 1 H, Ar), 10.15 (s, 1

H, 3-NH, exch), 10.81 (s, 1 H, 7-NH, exch), 12.66 (br, 2 H, 2 COOH, exch). Anal.

(C21H25N5O6S · 0.12 H2O · 0.11 CH3SO3H) Cal. C: 51.91, H: 5.30, N: 14.33, S: 7.31.

Found C: 51.80, H: 5.12, N: 14.63, S: 7.31.

126

(S)-2-(5-(4-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6- yl)butyl)thiophene-2-carboxamido)succinic acid (144): Compound 144 was prepared using the general method described for the preparation of 141-144, from 219 (79.2 mg, 0.4 mmol) to give 43 mg (48% over two steps) of 144 as a light grey powder. mp 221.3-

1 222.9 °C; TLC Rf 0.11 (CHCl3/MeOH 5:1); H NMR (DMSO-d6):  1.59-1.66 (m, 4 H, 2

CH2), 2.56-2.63 (m, 2 H, β- CH2), 2.77-2.81 (t, 2 H, CH2, J = 6.4 Hz), 4.59-4.65 (m, 1 H,

α-CH), 5.86 (s, 2 H, 2-NH2, exch), 5.97 (s, 1 H, Ar), 6.87-6.88 (d, 1 H, Ar, J = 3.6 Hz),

7.60-7.61 (d, 1 H, Ar, J = 4.0 Hz), 8.55-8.57 (d, 1 H, CO-NH, J = 6.8 Hz, exch), 10.13 (s,

1 H, 3-NH, exch), 10.81 (s, 1 H, 7-NH, exch), 12.82 (br, 1 H, COOH, exch). Anal.

(C19H21N5O6S · 2.17 H2O) C: 46.90, H: 5.25, N: 14.39, S: 6.59. Found C: 46.83, H: 4.88,

N: 14.56, S: 6.39.

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APPENDIX 1

The biological evaluations of the analogs listed in the following tables were performed by Dr. Larry H. Matherly (Developmental Therapeutics Program, Barbara Ann

Karmanos Cancer Institute and the Cancer Biology Program and Department of

Pharmacology, Wayne State University School of Medicine) against GARFTase, RFC- expressing PC43-10 cells, FRα-expressing RT16 cells, FRβ-expressing D4 cells and hPCFT-expressing R2/hPCFT4 cells; Dr. Roy L. Kisliuk (Department of Biochemistry,

Tufts University School of Medicine) against rhTS, rhDHFR, E. coli TS and E. coli DHFR;

Dr. Sherry F. Queener (Department of Pharmacology and Toxicology, Indiana University

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School of Medicine) against rlDHFR, P. carinii DHFR, T. gondii DHFR, and M. avium

DHFR; Dr. Michael Ihnat (Department of Cell Biology, University of Oklahoma Health

Science Center) against various kinase (VEGFR-1, VEGFR-2 and EGFR), A431 cytotoxicity and CAM assay (as described below).

Cell Lines and Assays of Antitumor Drug Activities. RFC- and FRR-null

MTXRIIOuaR2-4 (R2) CHO cells were gifts from Dr. Wayne Flintoff (University of

Western Ontario) and were cultured in R-minimal essential medium (MEM) supplemented with 10% bovine calf serum (Invitrogen, Carlsbad, CA), penicillin- streptomycin solution and L-glutamine at 37 °C with 5% CO2. PC43-10 cells are R2 cells transfected with hRFC.

RT16 cells are R2 cells transfected with human FRα, and D4 cells are R2 cells transfected with human FRβ. R2/hPCFT4 cells were prepared by transfection of R2 cells with a hPCFT cDNA, epitope tagged at the C-terminus with Myc-His6 (hPCFTMyc-His6) and cloned in pCDNA3.1. All the R2 transfected cells (PC43- 10, RT16, D4, R2/hPCFT4) were routinely cultured in R-MEM plus 1.5 mg/mL G418. Prior to the cytotoxicity assays (see below),

RT16 and D4 cells were cultured in complete folate-free RPMI1640 (without added folate) for 3 days. KB human cervical cancer cells were purchased from the American Type

Culture Collection (Manassas, VA), whereas IGROV1 ovarian carcinoma cells were a gift of Dr. Manohar Ratnam (Medical University of Ohio). Cells were routinely cultured in folate-free RPMI1640 medium, supplemented with 10% fetal bovine serum, penicillin- streptomycin solution, and 2 mM L-glutamine at 37 °C with 5% CO2. For growth inhibition assays, cells (CHO, KB, or IGROV1) were plated in 96 well dishes (∼2500-5000 cells/well, total volume of 200 µL medium) with a broad range of antifolate concentrations. The medium was RPMI1640 (contains 2.3 µM folic acid) with 10% dialyzed serum and

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antibiotics for experiments with R2 and PC43-10 cells. For RT16, D4, KB, and IGROV1 cells, the cells were cultured in folate-free RPMI media with 10% dialyzed fetal bovine serum (Invitrogen) and antibiotics supplemented with 2 nM LCV. The requirement for FR mediated drug uptake in these assays was established in a parallel incubation including 200 nM folic acid. For R2/hPCFT4 cells, the medium was folate-free RPMI1640 (pH 7.2) containing 25 nM LCV, supplemented with 10% dialyzed fetal bovine serum (Invitrogen) and antibiotics. Cells were routinely incubated for up to 96 h, and metabolically active cells

(a measure of cell viability) were assayed with Cell Titer-blue cell viability assay (Promega,

Madison, WI), with fluorescence measured (590 nm emission, 560 nm excitation) using a fluorescence plate reader. Raw data were exported from Softmax Pro software to an Excel spreadsheet for analysis and determinations of IC50s, corresponding to the drug concentrations that result in 50% loss of cell growth. For some of the in vitro growth inhibition studies, the inhibitory effects of the antifolate inhibitors on de novo thymidylate biosynthesis (i.e., TS) and de novo purine biosynthesis (GARFTase and AICARFTase) were tested by coincubations with thymidine (10 µM) and adenosine (60 µM), respectively.

For de novo purine biosynthesis, additional protection experiments used AICA (320 µM) as a means of distinguishing inhibitory effects at GARFTase from those at AICARFTase.

For assays of colony formation in the presence of the antifolate drugs, KB cells were harvested and diluted, and 200 cells were plated into 60mmdishes in folate-free RPMI1640 medium supplemented with 2 nM LCV, 10% dialyzed fetal bovine serum, penicillin- streptomycin, and 2 mM L-glutamine in the presence of antifolate drugs. The dishes were incubated at 37 °C with 5% CO2 for 10-14 days. At the end of the incubations, the dishes were rinsed with Dulbecco’s phosphate-buffered saline (DPBS), 5%trichloroacetic acid,

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and borate buffer (10 mM, pH 8.8), followed by 30 min incubation in 1% methylene blue in the borate buffer. The dishes were rinsed with the borate buffer, and colonies were counted for calculating percent colony-forming efficiency normalized to control.

FR Binding Assay. [3H]Folic acid binding was used to assess levels of surface FRs.

Briefly, cells (e.g., RT16 or D4; ∼1.6 × 106) were rinsed twice with Dulbecco’s phosphate- buffered saline (DPBS) followed by two washes in acidic buffer (10 mM sodium acetate,

150 mM NaCl, pH 3.5) to remove FR-bound folates. Cells were washed twice with ice- cold HEPES-buffered saline (20 mM HEPES, 140 mM NaCl, 5 mM KCl, 2 mM MgCl2, 5 mM glucose, pH 7.4; HBS), then incubated in HBS with [3H]folic acid (50 nM, specific activity 0.5 Ci/mmol) in the presence and absence of a range of concentrations of unlabeled folic acid or antifolate for 15 min at 0 °C. The dishes were rinsed three times with ice-cold

HBS, after which the cells were solubilized with 0.5 N sodium hydroxide and aliquots measured for radioactivity and protein contents. Protein concentrations were measured with Folin phenol reagent. Bound [3H]folic acid was calculated as pmol/mg protein.

Relative binding affinities for assorted folate/antifolate substrates were calculated as the inverse molar ratios of unlabeled ligands required to inhibit [3H]folic acid binding by 50%.

By definition, the relative affinity of folic acid is 1.

Transport Assays. For transport assays, R2/hPCFT4, PC43- 10, and R2(VC) CHO cells grown as monolayers were used to seed spinner flasks. For experiments to determine the inhibitions of transport by antifolate substrates, cells were collected and washed with

DPBS and resuspended in 2 mL of physiologic Hank’s balanced salts solution (HBSS) for

PC43-10 cells and in HBS adjusted to pH 7.2 or 6.8 or 4-morpholinepropanesulfonic acid

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(MES)-buffered saline (20 mM MES, 140 mM NaCl, 5 mM KCl, 2 mM MgCl2, and 5 mM glucose) adjusted to pH 6.5, 6.0, or 5.5 for R2/hPCFT4 cells. In either case, uptakes of

[3H]MTX (0.5 µM) were measured over 2 min at 37 °C in the presence and absence of unlabeled antifolates (10 µM). Uptakes of [3H]MTX were quenched with ice-cold DPBS.

Cells were washed with icecold DPBS (3×) and solubilized with 0.5 N NaOH. Levels of intracellular radioactivity were expressed as pmol/mg protein, calculated from direct measurements of radioactivity and protein contents of cell homogenates. Protein concentrations were measured with Folin phenol reagent. Percent MTX transport inhibition was calculated by comparing level of [3H]MTX uptake in the presence and absence of the inhibitors. Kinetic constants (Kt, Vmax) and Kis were calculated from Lineweaver-Burke and

Dixon plots, respectively.

In Vitro GARFTase Enzyme Inhibition Assay. Purified recombinant mouse

GARFTase enzyme was a gift from Dr. Richard Moran (Virginia Commonwealth

University, Richmond, VA). Briefly, enzyme activity was assayed spectrophotometrically at 37 °C using GARFTase (0.75 nM), α,β-GAR (11 µM), and coenzyme 10-formyl-5,8- dideazafolic acid (10 µM) in HEPES buffer (75 mM, pH 7.5) with or without antifolate inhibitor (10-30 000 nM). The absorbance of the reaction product, 5,8-dideazafolic acid, was monitored at 295 nM over the first minute as a measure of the initial rate of enzyme activity. IC50s were calculated as the concentrations of inhibitors that resulted in a 50% decrease in the initial velocity of the GARFTase reaction.

In Situ GARFT Enzyme Inhibition Assay. Incorporation of [14C]glycine into

[14C]FGAR, as an in situ measure of endogenous GARFTase activity, was described by

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Beardsley et al. and modified by Deng et al. For these experiments, KB cells were seeded in 4 mL of complete folate-free RPMI1640 plus 2 nM LCVin 60 mm dishes at a density of

2 × 106 cells per dish. On the next day, the medium was replaced with 2 mL of fresh complete folate-free RPMI1640 plus 2 nM LCV (without supplementing glutamine).

Azaserine (4 µM final concentration) was added in the presence and absence of the antifolate inhibitors (0.1, 1, 10, 100, or 1000 nM). After 30 min, L-glutamine (final concentration, 2 mM) and [14C]glycine (tracer amounts; final specific activity 0.1 mCi/L) were added. Incubations were at 37 °C for 15 h, at which time cells were washed (one-time) with ice-cold folate-free RPMI1640 plus serum. Cell pellets were dissolved in 2mL of 5% trichloroacetic acid at 0 °C. Cell debris was removed by centrifugation (the cell protein contents in the pellets were measured), and the supernatants were extracted twice with 2 mL of ice-cold ether. The aqueous layer was passed through a 1 cm column of AG1 × 8

(chloride form), 100-200 mesh (Bio-Rad), washed with 10 mL of 0.5 N formic acid and then 10 mL of 4 N formic acid, and finally eluted with 8 mL of 1 N HCl. The elutants were collected and determined for radioactivity. The accumulation of radioactive FGAR was calculated as pmol per mg protein over a range of inhibitor concentrations. IC50s were calculated as the concentrations of inhibitors that resulted in a 50% decrease in FGAR synthesis.

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Table 2. IC50’s (in nM) for 6-Substituted Pyrrrolo[2,3-d]pyrimidine thienoyl antifolates 3-4, and analogs with fused aromatic side chain

135-138 and Classical Antifolates in hRFC, hPCFT, and FR-Expressing Cell Linesa

hRFC hFRα hFRβ hPCFT hRFC/ FRα/hPCFT hRFC/ FRα/hPCFT Antifolate RT16 D4 KB IGROV1 PC43-10 R2 RT16 D4 R2/hPCFT4 R2(VC) KB IGROV1 (+FA) (+FA) (+FA) (+FA) 3 101.0(16.6) 273.5(49.1) 0.31(0.14) >1000 0.17(0.03) >1000 3.34(0.26) 288(12) 0.26(0.03) >1000 0.55(0.10) >1000 4 >1000 >1000 1.82(0.28) >1000 0.57(0.09) >1000 43.4(4.1) >1000 0.55(0.10) >1000 0.97(0.12) >1000 135 >1000 >1000 7.24 b 53.81 b >1000 b 1.58 >1000 b b 136 >1000 >1000 9.09 b 11.04 b >1000 b 6.94 >1000 b b 137 >1000 >1000 8.51 b 0.96 b >1000 b 6.73 >1000 b b 138 >1000 >1000 9.09 b 11.04 b >1000 b 6.94 >1000 b b MTX 12(1.1) 216(8.7) 114(31) 461(62) 106(11) 211(43) 120.5(16.8) >1000 6.0(0.6) 20(2.4) 21(3.4) 22(2.1) PMX 138(13) 894(93) 42(9) 388(68) 60(8) 254(78) 13.2(2.4) 974.0(18.1) 68(12) 327(103) 102(25) 200(18)

165 RTX 6.3(1.3) >1000 15(5) >1000 22(10) 746(138) 99.5(11.4) >1000 5.9(2.2) 22(5) 12.6(3.3) 20(4.3)

LMTX 12(2.3) >1000 12(8) 188(41) 2.6(1.0) 275(101) 38.0(5.3) >1000 1.2(0.6) 31(7) 3.1(0.9) 16(6) aGrowth inhibition assays were performed for CHO sublines engineered to express hRFC (PC43-10), FR (RT16, D4), or hPCFT (R2/hPCFT4), for comparison with transporter null [R2, R2(VC)] CHO cells, and for the KB and IGROV1 tumor sublines (expressing hRFC, FRR, and hPCFT), as described in the Experimental Section. For the FR experiments, growth inhibition assays were performed in the presence and the absence of 200 nM folic acid (FA). The data shown are mean values from 2-10 experiments (plus/minus SEM in parentheses). Results are presented as IC50 values, corresponding to the concentrations that inhibit growth by 50% relative to cells incubated without drug. b Not determined. c Not active.

Table 3. IC50’s (in nM) for 6-Substituted Pyrrrolo[2,3-d]pyrimidine phenyl antifolates 2 and analogs with naphthoic ring as side chain

139, 140 and Classical Antifolates in hRFC, hPCFT, and FR-Expressing Cell Linesa

hRFC hFRα hFRβ hPCFT hRFC/ FRα/hPCFT hRFC/ FRα/hPCFT Antifolate RT16 D4 KB IGROV1 PC43-10 R2 RT16 D4 R2/hPCFT4 R2(VC) KB IGROV1 (+FA) (+FA) (+FA) (+FA) 2 >1000 >1000 6.3(1.6) >1000 10(2) >1000 213(28) >1000 1.7(0.4) >1000 b b 139 >1000 >1000 26.41 b 119 b >1000 b 16.3 >1000 b b 140 >1000 >1000 7.18 b 59.4 b >1000 b >1000 b b b MTX 12(1.1) 216(8.7) 114(31) 461(62) 106(11) 211(43) 120.5(16.8) >1000 6.0(0.6) 20(2.4) 21(3.4) 22(2.1) PMX 138(13) 894(93) 42(9) 388(68) 60(8) 254(78) 13.2(2.4) 974.0(18.1) 68(12) 327(103) 102(25) 200(18) RTX 6.3(1.3) >1000 15(5) >1000 22(10) 746(138) 99.5(11.4) >1000 5.9(2.2) 22(5) 12.6(3.3) 20(4.3) LMTX 12(2.3) >1000 12(8) 188(41) 2.6(1.0) 275(101) 38.0(5.3) >1000 1.2(0.6) 31(7) 3.1(0.9) 16(6) a

166 Growth inhibition assays were performed for CHO sublines engineered to express hRFC (PC43-10), FR (RT16, D4), or hPCFT (R2/hPCFT4), for comparison with transporter null [R2, R2(VC)] CHO cells, and for the KB and IGROV1 tumor sublines (expressing hRFC, FRR, and hPCFT), as described in the Experimental Section. For the FR experiments, growth inhibition assays were performed in the presence and the absence of 200 nM folic acid (FA). The data shown are mean values from 2-10 experiments (plus/minus SEM in parentheses). Results are presented as IC50 values, corresponding to the concentrations that inhibit growth by 50% relative to cells incubated without drug. b Not determined. c Not active.

Table 4. IC50’s (in nM) for 6-Substituted Pyrrrolo[2,3-d]pyrimidine thienoyl antifolates 3-4, and analogs with amino acid modification 141-144 and Classical Antifolates in hRFC, hPCFT, and FR-Expressing Cell Linesa hRFC hFRα hFRβ hPCFT hRFC/ FRα/hPCFT hRFC/ FRα/hPCFT Antifolate RT16 D4 KB IGROV1 PC43-10 R2 RT16 D4 R2/hPCFT4 R2(VC) KB IGROV1 (+FA) (+FA) (+FA) (+FA) 3 101.0(16.6) 273.5(49.1) 0.31(0.14) >1000 0.17(0.03) >1000 3.34(0.26) 288(12) 0.26(0.03) >1000 0.55(0.10) >1000 4 >1000 >1000 1.82(0.28) >1000 0.57(0.09) >1000 43.4(4.1) >1000 0.55(0.10) >1000 0.97(0.12) >1000 141 >1000 >1000 7.5 b b b >1000 b 1.21 >1000 b b 142 >1000 >1000 21 b 106 b >1000 b >1000 b b b 143 b b b b b b b b b b b b 144 >1000 >1000 11.91 b 11.9 b >1000 b >1000 b b b MTX 12(1.1) 216(8.7) 114(31) 461(62) 106(11) 211(43) 120.5(16.8) >1000 6.0(0.6) 20(2.4) 21(3.4) 22(2.1) PMX 138(13) 894(93) 42(9) 388(68) 60(8) 254(78) 13.2(2.4) 974.0(18.1) 68(12) 327(103) 102(25) 200(18) RTX 6.3(1.3) >1000 15(5) >1000 22(10) 746(138) 99.5(11.4) >1000 5.9(2.2) 22(5) 12.6(3.3) 20(4.3)

167 LMTX 12(2.3) >1000 12(8) 188(41) 2.6(1.0) 275(101) 38.0(5.3) >1000 1.2(0.6) 31(7) 3.1(0.9) 16(6) a Growth inhibition assays were performed for CHO sublines engineered to express hRFC (PC43-10), FR (RT16, D4), or hPCFT (R2/hPCFT4), for comparison with transporter null [R2, R2(VC)] CHO cells, and for the KB and IGROV1 tumor sublines (expressing hRFC, FRR, and hPCFT), as described in the Experimental Section. For the FR experiments, growth inhibition assays were performed in the presence and the absence of 200 nM folic acid (FA). The data shown are mean values from 2-10 experiments (plus/minus SEM in parentheses). Results are presented as IC50 values, corresponding to the concentrations that inhibit growth by 50% relative to cells incubated without drug. b Not determined. c Not active.

APPENDIX 2

1. SAR of 2-amino-4-oxo-6-substituted-pyrrolo[2,3-d]pyrimidines with fused aromatic side chains

135-138 in whole cell assay as GARFTase inhibitors with selectivity for FRs over RFC and PCFT

(carbon bridge optimization and conformation restriction).

Figure 24 The structures of classical 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine antifolates 135-138

As shown in Table 2, 2-amino-4-oxo-6-substituted-pyrrolo[2,3-d]pyrimidines 135-138 with fused aromatic ring in the side chain are potent GARFTase inhibitors selectively transported by FRs over RFC and PCFT. Even though their inhibitory activities against KB tumor cell lines

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were less potent (IC50 value of 1.6-7 nM for KB human CHO cell lines) than their lead compounds

3 (IC50 = 0.26 nM for KB human CHO cell lines) and 4 (IC50 = 0.55 nM for KB human CHO cell lines), they still partially met our expectation. Compared with lead compounds 3 and 4, all the analogs with fused aromatic side chain are selective FR substrates without the inhibitory activity against PCFT. The big difference between the two lead compunds and the series of analogs is the fused aromatic ring system in the side chain. The result established that employing the extended aromatic ring system in the side chain might be favorable to differentiate FRs from other two transport systems. Also, compared with the freely rotatable single bond side chains of lead compounds 3 and 4, the fused aromatic ring in the side chain restrict the conformation of this series of analogs, to some extent tighten the bridge between the bicyclic scaffold and the glutamate moiety. Different antitumor activity of these analogs in the bioassay indicates that the different restricted confirmation may influence the binding mode between the antifolates and GARFTase, such that impact their antitumor activty, which needs further SAR study on the fused ring system in the side chain. Among the analogs, compound 135 with the 2, 6-substituted benzo[b]thiophene bridge is the most potent toward GARFTase (IC50 of 1.58 nM for KB human CHO cell lines) and cells expressing FRs (IC50 of 7.24 nM for FRα-expressing RT16 cells) but is modest potent toward

FRβ (IC50 of 53.81 nM for FRβ-expressing D4 cells) without the inhibitory activity against RFC and PCFT (IC50 > 1000 nM for hRFC expressing PC43-10 cells and hPCFT-expressing

R2/hPCFT4 cells). The less potent inhibitory activity against GARFTase of 135 is probably because that it’s only transported by FRs whereas lead compounds 3 and 4 are transported by FRs and PCFT, which might be favorable to a higher intracellular concentration of antifolates, such that more of the antifolate will inhibit the intracellular target GARFTase. The envision that the length of the bridge domain between the bycyclic scaffold and the glutamate moiety is important

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for binding and transport, with optimal activity for the 3- and 4-carbon bridge analogs needs further exploration with development of more analogs.

2. SAR of 2-amino-4-oxo-6-substituted-pyrrolo[2,3-d]pyrimidines with a naphthyl ring as side chain in whole cell assay as GARFTase inhibitors with selectivity for FRs over RFC and PCFT

(fused aromatic ring).

Figure 25 The structures of classical 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine antifolates 139 and 140.

The analog 139 with naphythyl ring in the side chain was evaluated for the bioassay and the result is shown in Table 3. The lead compound 2 with phenyl ring in the side chain is potent

GARFTase inhibitor selectively transported by FRs and PCFT over RFC (IC50 = 1.7 nM toward

KB human CHO cell lines); whereas the analog 139 is a selective FRs substrate and a less potent

GARFTase inhibitor, exhibiting around 10-fold less potent inhibitory activity against KB tumor cell line (IC50 = 16.3 nM). As discussed above, the fused aromatic ring system in the side chain

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may contribute to the selectivity for FRs over RFC and PCFT. Based on the crystal structure of

FRs, strong π-π interaction between the fused aromatic ring system in the side chain and the side chain of amino acids in the long biding cleft in FRs may help to explain the selective transport via

FRs over RFC and PCFT. To furthure confirm the envision, the molecular docking of FRα and the analog 139 was evaluated by MOE (2016) (Figure 29). The regioisomer 140, as the synthetic intermediate of 139, was also sent for bioassay. The result established that it is a selective FRs substrate but not a GARFTase inhibitor. Dr. Gangjee et al.217 recently reported that the docking mode of antifolates with FRs and with GARFTase is in an upside-down opposite way. The analog

140 with too restricted confirmation may abandon its biding to GARFTase (IC50 >1000 nM against

KB CHO cell line) and result in the loss of inhibitory activity toward GARFTase; whereas it inhibits toward FRs quite well (IC50 = 7.18 nM for FRα-expressing RT16 cells, IC50 of 59.4 nM for FRβ-expressing D4 cells). Compared with the lead compound 2, 139 is a less potent GARFTase inhibitor. It may because that 2 is transported by both FRs and PCFT into tumor cells while 139 is only transported by FRs with 4-fold less potent inhibitory activity (IC50 = 26.41 nM against FRα- expressing CHO cell line) than lead compound 2 (IC50 = 6.3 nM against FRα-expressing CHO cell line).

3. SAR of 2-amino-4-oxo-6-substituted-pyrrolo[2,3-d]pyrimidine thienoyl analogs with variations on L-glutamic acid in whole cell assay as GARFTase inhibitors with selectivity for FRs over RFC and PCFT (amino acid modification).

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Figure 26 The structures of classical 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine antifolates 141-144

In Table 4, a series of 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl analogues based on compound 4 with replacement of L-glutamate by different amino acids were evaluated against human CHO cell lines expressing RFC, FRs, and PCFT and all the three transport system. The result established that amino acid modifications are well tolerated by FRα and modest tolerated by

FRβ. However, variations on the terminal L-glutamate moiety led to the total lost of inhibitory potency against PCFT-expressing human CHO cell line for all the analogs. So, it indicates L- glutamate is not essential for biding and cellular uptake by FRs whereas it is required for PCFT transport. Analogs 142-144 with variations on α-carboxylic acid totally lost the inhibitory activity against KB tumor cell line (IC50 > 1000); whereas the analog 141 with the γ-carboxylic acid modified is still potent GARFTase inhibitor against KB tumor cell line with an IC50 value of 1.21

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nM. According to the result, it indicates that α-carboxyl shows more importance than γ-carboxyl for substrate binding to intracellular target GARFTase.

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APPENDIX 3

Figure 27 Molecular modeling study of compound 135 with FRα.

Note: Docked pose of 135 (brown) in human FRα (PDB 4LRH).61 Interacting amino acids in the binding pocket are depicted as gray lines (labeled), while noninteracting amino acids in the binding pocket are depicted as gray lines (unlabeled).

Figure 27 shows the docked pose of 135 in the human FRα (PDB 4LRH)61 binding site.

Compound 135 binds in the folate binding cleft of FRα. The 2-NH2 and 4-oxo moieties of 135 interact with the same amino acids as the corresponding groups of folic acid,61 with the 2-NH2 interacting with Asp81 and the 4-oxo forming hydrogen bonds with the side chain hydroxyl of

Ser174 and the side chain NH of Arg103. The pyrrolo[2,3-d]pyrimidine scaffold is sandwiched between the side chains of Tyr60 and Trp171, similar to that seen with the pteroyl ring of folic

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acid in its bound conformation.61 The L-glutamate moiety of 135 is oriented similarly to the corresponding glutamate in folic acid.61 The α-carboxylic acid of 135 forms a network of hydrogen bonds involving the backbone NH of Gly137 (labeled) and the side chain NH of Trp140. The γ- carboxylic acid interacts with the side chain NH groups of Gln100 and Trp102, as is seen with the corresponding glutamate portion of folic acid in the crystal structure.61 The benzo[b]thiophene ring of 135 forms hydrophobic interactions with Trp140 and His135, probably - interactions. The hydrophobic fused aromatic side chain of 135 may also form hydrophobic interactions with Tyr60,

Phe62, Trp102, Trp134. The docking score of 135 was -50.02 kJ/mol, compared with that for folic acid of −44.67 kJ/ mol. Ligand was drawn using MOE 2016, docked using general settings on

LeadIT 2.1.6. RMSD of the native ligand is 0.81 Å.

Figure 28 Molecular modeling study of compound 3 with FRα.

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Note: Docked pose of 3 (orange) in human FRα (PDB 4LRH).61, 217 Interacting amino acids in the binding pocket are depicted as gray cylinders (labeled), while noninteracting amino acids in the binding pocket are depicted as green lines (unlabeled).

Figure 28 shows the docked pose of 3 in the human FRα (PDB 4LRH)61 binding site retains most of the docking interactions between the ligand and FRα (PDB 4LRH), as seen with the docked pose of 135. The major docking difference between 135 and 3 is the hydrophobic interactions between the side chain bridge and the side chain of amino acids. The thiophene ring of 3 forms hydrophobic interactions with Trp140.217 The hydrophobic 3-C linker of 3 forms nonspecific hydrophobic interactions with Tyr60, Phe62, Trp102, Trp134, and His135. The hydrophobic

Figure 29 Molecular modeling study of compound 139 with FRα.

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Note: Docked pose of 139 (light blue) in human FRα (PDB 4LRH).61 Interacting amino acids in the binding pocket are depicted as gray lines (labeled), while noninteracting amino acids in the binding pocket are depicted as gray lines (unlabeled). benzo[b]thiophene ring of 135 also forms hydrophobic interactions with these amino acids. But these hydrophobic interactions are probably - interactions, much stonger than the nonspecific hydrophobic ones. The docking score of 3 was −40.35 kJ/mol, compared with that for 135 of

−50.02 kJ/ mol.

The docked pose of 139 in human FRα retain the docking interactions of the pyrrolo[2,3- d]pyrimidine scaffold and the aromatic side chain, as seen with the docked pose of 3 (Figure 29).

Variations were observed on the docked orientation of the naphythyl ring of 139 in the side chain and on hydrogen bonds between the L-glutamate moiety and the side chain of amino acids, which could explain, in part, the differences observed in binding of naphythyl ring analogue 139 to FRα from 3. The docking score of 139 was -50.004 kJ/mol, compared with that for 3 of −40.35 kJ/mol.

Ligand was drawn using MOE 2016, docked using general settings on LeadIT 2.1.6. RMSD of native ligand is 0.81 Å.

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