Synthesis of Novel 6-Substituted and 5-Substituted Pyrrolo ...

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Synthesis of Novel 6-Substituted and 5-SubstitutedPyrrolo[2,3-D] Pyrimidine Antifolates as TargetedAnticancer TherapiesXinxin LiDuquesne University

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SYNTHESIS OF NOVEL 6-SUBSTITUTED AND 5-SUBSTITUTED PYRROLO[2,3-d]

PYRIMIDINE ANTIFOLATES AS TARGETED ANTICANCER THERAPIES

A Dissertation

Submitted to the Graduate School of Pharmaceutical Sciences

Duquesne University

In partial fulfillment of the requirements for

the degree of Master of Science

By

Xinxin Li

December 2018

Copyright by

Xinxin Li

2018

iii



By

Xinxin Li Approved November 05, 2018 ________________________________ Aleem Gangjee Distinguished Professor, School of Pharmacy (Committee Chair)

________________________________ Marc W. Harrold Professor, School of Pharmacy (Committee Member)

________________________________ Kevin Tidgewell Assistant Professor, School of Pharmacy (Committee Member)

iv

ABSTRACT



By

Xinxin Li

December 2018

Dissertation supervised by Professor Aleem Gangjee, Ph. D.

The dissertation will give an introduction, background and current research progress in the

areas of antifolates and chemotherapy of anticancer. The design and synthesis of classical 6-

substituted pyrrolo[2,3-d]pyrimidines and 5-substituted pyrrolo[2,3-d]pyrimidines as potential

antifolates have been described. The design variations include: methylated thiophene regioisomers,

fluorinated phenyl regioisomers, thionyl regioisomers on the side chain of pyrrolo[2,3-

d]pyrimidines. As a part of this study, a series of new compounds have been synthesized and

characterized. Of these, ten final compounds were submitted for biological evaluation.

v

DEDICATION

Dedicated To My Family For Their Love And Support

vi

ACKNOWLEDGEMENT

I would like to thank all of those who have contributed to this work. Particularly, I am most

grateful to Professor Dr. Aleem Gangjee, my supervisor, for his help and support which made this

dissertation possible. I am indebted to him not just for his scientific guidance but also for his

encouragement, day after day, financial support and his friendship.

I would like to thank the members of my dissertation committee: Dr. Marc W. Harrold,

and Dr. Kevin Tidgewell for their scientific and emotional support. I wish to thank Dr. Larry H.

Matherly at Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine

for evaluating all the compounds for enzyme inhibitory activity and antitumor activity. I wish to

express my sincere appreciation to Nancy Hosni and Jackie Farrer in the office of pharmacy school

for their help and assistance. My thanks go to all my colleagues in the Graduate School of

Pharmaceutical Sciences for their help and advice which made my stay at Duquesne University a

most pleasant experience. I would like to thank the Graduate School of Pharmaceutical Sciences

for the financial support provided to me.

Finally, I would like to express my love and gratitude to my whole family and my friends

for their understanding, supporting and endless love.

vii

TABLE OF CONTENTS

Page

Abstract .............................................................................................................................. iv

Dedication ............................................................................................................................v

Acknowledgement ............................................................................................................. vi

List of Tables ................................................................................................................... viii

List of Figures .................................................................................................................... ix

List of Schemes .................................................................................................................. xi

List of Abbreviations ....................................................................................................... xiv

I. Biochemical Review .........................................................................................................1

II. Chemical Review ..........................................................................................................45

III. Statement of Problem ...................................................................................................72

IV. Chemical Discussion ...................................................................................................88

V. Summary .....................................................................................................................101

VI. Experimental ..............................................................................................................103

Bibliography ....................................................................................................................144

Appendix 1 .......................................................................................................................183

viii

LIST OF TABLES

Page

Table 1 5,6,7,8-Tetrahydropyrido[2,3-d]pyrimidines and 7,8-dihydropyrimido[5,4-

b][1,4]thiazines ............................................................................................................29

Table 2 Pyrimidines as GARFTase inhibitors ...........................................................................31

Table 3 Pyrrolo[2,3-d]pyrimidines and thieno[2,3-d]pyrimidines as GARFTase

inhibitors .......................................................................................................................35

Table 4 IC50’s (in nM) for 6-Substituted Pyrrrolo[2,3-d]pyrimidine Antifolates 3, 4 and

Classical Antifolates in hRFC, hPCFT, and FR-Expressing Cell lines .........................36

Table 5 IC50’s (in nM) for 2-amino-4-oxo-6-substituted straight chain pyrrolo[2,3-d]

pyrimidine Antifolates 7, 8 and Classical Antifolates in hRFC, hPCFT, and FR-

Expressing Cell Line ......................................................................................................38

ix

LIST OF FIGURES

Page

Figure 1 Structure of folic acid illustrating the three principle moieties of the

molecule…………………………………………………………………………...…..1

Figure 2 Folates and their roles in the biosynthesis of nucleic acid precursors and

amino acids……………………………………………………………………………4

Figure 3 TS catalyzed biosynthesis of dTMP from dUMP……………………………………..6

Figure 4 De novo synthesis of Purines…………………………………………………………..9

Figure 5 Representative examples of classical antifolates (and their principal

targets)………………………………………………………………………………..11

Figure 6 Representative examples of nonclassical antifolates (and their principal

targets)………………………………………………………………………………..12

Figure 7 hFRα-scaffold buried insides and glutamic acid is solvent exposed…………………16

Figure 8 hFRα-Interaction of folic acid with ligand-binding-pocket residues………………...17

Figure 9 Endocytosis of FRs…………………………………………………………….…….18

Figure 10 Human PCFT structure…………………………………………………………….…20

Figure 11 Proposed mechanism for GARFTase……………………………………………..….24

Figure 12 Crystal structure (MOE 2014) of hGARTase in a binary complex with inhibitor

10-CF3CO-DDACTHF at pH 7…………………………………………………...….25

Figure 13 Crystal structure of hGARTFase……………………………………………………..28

Figure 14 A proposed mechanism of AICARFTase…………………………………………….40

Figure 15 AICARFTase inhibitors………………………………………………………………41

x

Figure 16 The structures of classical 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]-pyrimidine

antifolates (7,8 and156-159)…………………………………… ……………………….73

Figure 17 Superimposition of docked poses of 7, 156 and157 in FRα………………………….77

Figure 18 Superimposition of docked poses of 7, 156 and157 in GARFTase…………………..78

Figure 19 Superimposition of docked poses of 8,158 and159 in FRα…………………………..80

Figure 20 Superimposition of docked poses of 8, 158 and159 in GARFTase ……………..…...81

Figure 21 6-substituted pyrrolo[2,3-d]pyrimidine with three atom chain length having nitrogen

bridge and fluorinated benzoyl regioisomers compounds design……………………….82

Figure 22 Superimposition of docked poses of 161, 162 and 163 in FR…………………..…

Figure 23 Superimposition of docked poses of 161, 162 and 163 in GARFTase……………….85

Figure 24 5-substituted pyrrolo[2,3-d]pyrimidine antifolates with three bridge carbons and

fluorinated benzoyl ring compound 167 as antitumor agents…………………………..86

Figure 25 5-substituted pyrrolo[2,3-d]pyrimidine antifolates with two- to- four bridge carbons

and thienoyl regioisomers in the side chain as antitumor agents……………………….87

Figure 26 Structure of 5-bromo-3-methylthiophene-2-carboxylate 175 and 5-bromo-4-

methylthiophene-2-carboxylate 176……………………………………………………..88

xi

LIST OF SCHEMES

Page

Scheme 1 Synthesis of pyrrolo[2,3-d]pyrimidines 11 and 13…………………………………46

Scheme 2 Synthesis of pyrrolo[2,3-d]pyrimidine 15…………………………………………..46

Scheme 3 Synthesis of pyrrolo[2,3-d] pyrimidines 20………………………………………...47

Scheme 4 Synthesis of furo[2,3-d]pyrimidines 22 and pyrrolo[2,3-d]pyrimidines 23………...47

Scheme 5 Synthesis of pyrrolo[2,3-d] pyrimidines 27………………………………………...48

Scheme 6 Synthesis of pyrrolo[2,3-d]pyrimidines 29 and furo[2,3-d]pyrimidines 30.………..49

Scheme 7 Synthesis of pyrrolo[2,3-d]pyrimidines 34…………………………………………50

Scheme 8 Synthesis of 5,6-disubstituted pyrrolo[2,3-d]pyrimidine 39………………………..50

Scheme 9 Synthesis of 2-amino-4-methyl pyrrolo[2,3-d]pyrimidine 45.……………………...51

Scheme 10 Synthesis of 2-(2-amino-4-oxo-4,7-dihydro-1H-pyrrolo[2,3-d] pyrimidin-6-

yl)acetic acid 47…………………………………………………………………….52


yl)acetic acid 49.……………………………………………………………………52

Scheme 12 Synthesis of pyrrolo[2,3-d]pyrimidine 54…………………………………………..53

Scheme 13 Synthesis of pyrrolo[2,3-d]pyrimidine 59 from 6-amino-5-pyrimidylacetalde-hydes

58…………………………………………………………………………………………54

Scheme14 Synthesis of PMX from α-bromo aldehyde 64……………………………………....54

Scheme 15 Synthesis of pyrrolo[2,3-d]pyrimidines 71…………………………………………55

Scheme 16 Synthesis of 4-methyl pyrrolo[2,3-d]pyrimidines 74……………………………….56

Scheme 17 Synthesis of 2,4-dimethyl pyrrolo[2,3-d]pyrimidines 80…………………………...56

Scheme 18 Synthesis of pyrrolo[2,3-d]pyrimidines 84………………………………………....57

xii

Scheme 19 Synthesis of pyrrolo[2,3-d]pyrimidines via Fischer indole cyclization…………….58

Scheme 20 Synthesis of pyrrolo[2,3-d]pyrimidines 94…………………...….……....................58

Scheme 21 Synthesis of 4-amino-5-cyanopyrrolo[2,3-d]pyrimidine 100.……………………...59

Scheme 22 Synthesis of 4-amino-5-substituted pyrrolo[2,3-d]pyrimidines 105……………….60

Scheme 23 Synthesis of 5,6-disubstitutedpyrrolo[2,3-d]pyrimidine 107……………………….61

Scheme 24 Synthesis of 2,5,6-trimethyl pyrrolo[2,3-d]pyrimidine 111………………………...61

Scheme 25 Synthesis of 2,5,-dimethyl-N7-substitutedpyrrolo[2,3-d]pyrimidine 118…………..62

Scheme 26 Synthesis of pyrrolo[2,3-d]pyrimidine 123.………………………………………...63

Scheme 27 Synthesis of N7-substituted analogs of PMX 131.………………………………….64

Scheme 28 Synthesis of 2,5-dimethylpyrrolo[2,3-d]pyrimidine 136.…………………………..65

Scheme29 Synthesis of 5-substituted 2-des-4-oxo-pyrrolo[2,3-d]pyrimidine 139……………...65

Scheme30 Synthesis of 2,5,6-trisubstitutedpyrrolo[2,3-d]pyrimidines 143…………………….66

Scheme 31 A general transformation of Sonogashira coupling…………………………………66

Scheme 32 Mechanism of Sonogashira cross-coupling…………………………………………67

Scheme 33 Synthesis of pyrrolo[2,3-d]pyrimidin-2-yl)-N-pivaloylpivalamide 146.…………...68

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

Sonogashira coupling…………………………………………………………………….68

Scheme 35 General transformation of Heck coupling………………………………………….69

Scheme 36 Heck coupling to synthesis aldehyde 152 and 153………………………………...69

Scheme 37 A proposed mechanism of Heck coupling to synthesis aldehyde………………….70

Scheme 38 Improved Heck coupling to synthesis aldehyde 152………………………………71

Scheme 39 Heck coupling with thiophenyl bromide 155………………………………………72

xiii

Scheme 40 Synthesis of classical three carbon atoms side chain methylated thiophene2-amino-4-

oxo-6-substitutedpyrrolo[2,3-d]pyrimidine isomers 156,157……………………...........89

Scheme 41 Synthesis of classical four carbon atoms side chain methylated thiophene2-amino-4-

oxo-6-substitutedpyrrolo[2,3-d]pyrimidine isomers 158,159……………………………93

Scheme 42 Retro synthetic route A………………………………………………………….......95

Scheme43 Condensation of 223, 228 and 214………………………………………………......96

Scheme 44 Synthesis of classical 6-substituted pyrrolo[2,3-d]pyrimidine with three atom chain

length having nitrogen bridge and fluorinated benzoyl regioisomers162,163…………..97

Scheme45 synthetic route A of 167……………………………………………………………..98

Scheme 46 Synthesis of classical 5-substituted pyrrolo[2,3-d]pyrimidine antifolates with three

bridge carbons and fluorinated benzoyl167…………………………………………......99

Scheme 47 Synthesis of classical 5-substituted pyrrolo[2,3-d]pyrimidine antifolates 172-

174………………………………………………………………………………..…….100

xiv

LIST OF ABBREVIATIONS

AICA Aminoimidazole-4-carboxamide

AICAR Aminoimidazole-4-carboxamide ribosyl-5-phosphate

AICARFTase Aminoimidazole-4-carboxamide ribosyl-5-phosphate Formyl

Transferase

AIDS Acquired Immunodeficiency Syndrome

ALL Acute Lymphoblastic Leukemia

AML Acute Myelogenous Leukemia

AMP Adenosine monophosphate

AMPK AMP-activated protein kinase

AMT Aminopterin

ARDS Acute respiratory distress syndrome

Arg Arginine

ATP Adenosine-5’-triphosphate

CAM

CML

Chorioallantoic membrane

Chronic myelogenous leukemia

CNS Central nervous system

DHFR Dihydrofolate reductase

DMAP 4-Dimethylamino pyridine

DMAI Disseminated Mycobacterium avium intracellulare

DMF N,N-dimethyl formamide

DMSO Dimethyl sulfoxide

xv

DNA Deoxyribonucleic acid

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

EDX 10-Ethyl-l0-deaza aminopterin

EGF Epidermal Growth Factor

EGFR Epidermal growth factor receptor

FA

fAICAR

fGAR

Folic Acid

Formyl-aminoimidazolecarboxamide ribosyl-5-phosphate

Formyl-glycinamide ribosyl-5-phosphate

FGFR Fibroblast Growth Factor Receptor

FH2 7,8-Dihydrofolate

FH4 5,6,7,8-Tetrahydrofolate

FPGH Folylpolyglutamate Hydrolase

FPGS Folyl Poly--glutamate Synthetase

FR Folate Receptor

FdUMP

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

xvi

5-FU 5- Fluorouracil

GAR Glycinamide Ribosyl-5-phosphate

GARFTase Glycinamide Ribonucleotide Formyl Transferase

GDP Guanosine Diphosphate

GPI Glycosylphosphatidylinositol

GTP Guanosine triphosphate

HAART highly active anti-retroviral therapy

IFN Interferon

IGFR Insulin-like Growth Factor Receptor

IMP Inositol Monophosphate

IR Insulin Receptor

KDR Kinase Insert Domain Receptor

L. casei Lactobacillus casei

LV Leucovorin

MAC Mycobacterium Avium Complex

M. avium Mycobacterium Avium

MAI Mycobacterium avium intracellulare

M. intracellulare Mycobacterium intracellulare

MRP Multidrug Resistance Protein

MTHFR Methylene Tetrahydrofolate Reductase

MTX Methotrexate

NADPH Nicotinamide Adenine Dinucleotide Phosphate

NCI National Cancer Institute

xvii

NMR Nuclear Magnetic Resonance

NRTI Nucleoside reverse transcriptase inhibitors

NSCLC Non-small cell lung cancer

PABA Para-amino Benzoic Acid

P. carinii Pneumocystis carinii

PCFT Proton coupled folate transporter

PCP Pneumocystis carinii Pneumonia

PDB Protein Data Bank

PDDF N10-propargyl-5,8-dideazafolate

PDGFR Platelet-Derived Growth Factor Receptor

Pgp P-glycoprotein

Piv

PMX

Pivaloyl (trimethyl acetyl)

Pemetrexed

P. jirovecii Pneumocystis jirovecii

PteGlu Pteroylglutamic Acid

PTX Piritrexim

Rh Recombinant human

rl Rat liver

RNA Ribonucleic Acid

RFC Reduced Folate Carriers

RTK Receptor Tyrosine Kinase

RTX Raltitrexed

SHMT Serine Hydroxymethyl Transferase

xviii

THF Tetrahydrofolate

T. gondii Toxoplasma gondii

TLC Thin Layer Chromatography

TMP Trimethoprim

TMQ Trimetrexate

TNP-351 N-[4-[3-(2,4-Diamino-7H-pyrrolo[2,3-d]pyrimidin-5-

yl)propyl]benzoyl-L-glutamic acid.

Trp Tryptophan

TS Thymidylate Synthase

Val Valine

VEGF Vascular Endothelial Growth Factor

VEGFR Vascular Endothelial Growth Factor Receptor

- 1 -

BIOCHEMICAL REVIEW

Figure 1 Structure of folic acid illustrating the three principle moieties of the molecule.

Folic acid (FA) (Figure 1), a water-soluble vitamin of the B-complex group, was first

isolated by Mitchell and coworkers1 from spinach leaves in 1941. The structure of FA consists of

three principle moieties: a hetero-bicyclic pteridine (scaffold), a p-aminobenzoic acid (PABA) and

a glutamic acid (Figure 1). The FA structure probably does not occur in nature as such, but it can

be considered as the parent compound of a group of naturally occurring folates, which belong to

the B9 family. Folate refers to the various reduced derivatives of folic acid which is naturally found

in food. There are several differences between FA and folates: 1) The reduction states of the

pteridine ring (oxidized, 7,8-H2, and 5,6,7,8-H4) could occur in folates. 2) In folate, one carbon

unite can be attached to N5 or N10 or both. 3) Glutamate residues (poly-γ-glutamates) of folates may

be attached to the glutamate moiety by γ-peptide bonds to give folate polyglutamates.

Folates are anionic hydrophilic molecules at physiologic pH and because of that they can

not cross biological membranes by diffusion.2 As a result, mammalian cells have evolved several

sophisticated membrane transport systems for facilitating cellular uptake of folate cofactors. There

are three major uptake systems for the membrane transport of folates and antifolates across

- 2 -

biological membranes.These include the reduced folate carrier (RFC),2 the folate receptors (FRs)

α and β,3and the proton-coupled folate transporter (PCFT).2-4 RFC is expressed ubiquitously and

is recognized to be the major transport system for folates in mammalian cells and tissues at

physiologic pH.2-4 FRs α and β are glycosylphosphatidylinositol-anchored proteins that transport

folates by receptor-mediated endocytosis.5-6 Finally, PCFT is a proton-folate symporter that

functions optimally at acidic pH by coupling the flow of protons down an electrochemical

concentration gradient to the uptake of folates into cells.7-9 The reduced form of folic acid including

5-methyltetrahydrofolate (N5-CH3-FH4) and10-formyl tetrahydrofolate (N10-CHO-FH4) are

actively taken up into the cell via the RFC system. However, folic acid has a higher affinity for

FRs, which has a higher affinity for the oxidized form of the folate cofactor than the reduced form.

Folic acid plays an important role in one-carbon transfers system, so it is essential for

deoxyribonucleic acid (DNA) synthesis, repair and cell replication.2,3,10 Folate antagonist or

antifolates with structures similar to folic acid are considered cytotoxic drugs. These drugs inhibit

one or more biosynthetic steps that required folate coenzymes. Antifolates represent an important

class of antimetabolites and are used as chemotherapeutic agents for cancer.11-15

1. FOLATE METABOLISM

The mammalian folic acid cycle is a highly complex and crucial process for transferring

one-carbon unite to amino acids, nucleotides and other biomolecules. In mammals, folic acid

cannot be synthesized and must be obtained through the diet, primarily fruits and vegetables.1 Folic

acid is the starting material for tetrahydrofolic acid (tetrahydrofolate, THF), which is the

fundamental molecule in folic acid cycle. Folic acid exists in several oxidative states, each of them

is critical for the entire biosynthesis of nucleic acid precursors and amino acids. Intracellular

- 3 -

reduction of the pteridine ring in folic acid is enzymatically reduced by nicotinamide adenine

dinucleotide phosphate (NADPH) - specific dihydrofolate reductase (DHFR) and leads to the

formation of 7,8-dihydrofolate (FH2) followed by 5,6,7,8-tetrahydrofolate (FH4) (Figure 2).7 FH4

functions as the coenzyme in the utilization of single-carbon units and is the central component of

folate metabolism. Tetrahydrofolate is subsequently processed through a variety of oxidative

enzymatic modifications that function as one-carbon donors. It is one of the critical components

in the synthesis of amino acids (glycine, serine, methionine, and histidine), nucleic acid (purine

nucleotide and the 5-methyl group of thymine) and the formation of formylmethionyl-tRNA.

(Figure 2). FH4 can carry various single carbon units, including methyl, methylene, and formyl

groups. These single carbon units can be oxidized to methanol, formaldehyde, and formic acid,

respectively. Subsequently these one-carbon units can be attached to the N5- and/or N10- positions

of FH4 to form 5-methyltetrahydrofolate (N5-CH3-FH4), 5,10-methylenetetrahydrofolate (N5,N10-

CH2-FH4), 5-formyltetrahydro-folate (N5-CHO-FH4),10-formyltetrahydrofolate (N10-CHO-FH4),

and 5-formiminotetra-hydrofolate (N5-CH=NH-FH4) respectively (Figure 2).

There are numbers of enzymes involve in folate-dependent reactions, using different

derivatives of FH4 in one-carbon transfer reactions. Such as: (i) 5,10-methylene tetrahydrofolate

(N5,N10-CH2-FH4) provides a carbon for the synthesis of thymidylate from deoxyuridylate

mediated by thymidylate synthase (TS), an initial step in the synthesis of precursors for DNA. (ii)

N10-CHO-FH4 could provide two carbons for the synthesis of the purine ring in reactions mediated

by glycinamideribonucleotide formyl transferase (GARFTase) and amino-imidazole-

carboxamideribonuleotide formyl transferase (AICARFTase). (iii) N5-CH3-FH4 provides the

methyl group for the vitamin B12 dependent synthesis of methionine from homocysteine mediated

by methionine synthetase, which is followed by the synthesis of S-adenosylmethionine (Figure 2).

- 4 -

Figure 2 Folates and their roles in the biosynthesis of nucleic acid precursors and amino acids.

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

Hydroxymethyltransferase; MTHFD: Methylenetetrahydrofolate Dehydrogenase; MTHFR:

Methylenetetrahydrofolate Reductase; GARFTase: Glycinamide-ribonucleotide Formyl

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

- 5 -

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.

TS can utilize the FH4 cofactor N5, N10-CH2- FH4, that acts as the source of the methyl group

as well as the reductant by concerted transfer of its methylene moiety and the 6-hydrogen atom in

the form of hydride to form the 5-methyl group of dTMP (Figure 3). This transformation represents

Figure 3 TS catalyzed biosynthesis of dTMP from dUMP.

- 6 -

the only de novo source of dTMP in dividing cells; hence, inhibition of TS, in the absence of

salvage, could lead to “thymineless death”.16

“Thymineless death” is observed in mammalian cells when severe deoxythymidine-

triphosphate (dTTP) depletion takes place due to methotrexate (MTX) or 5-fluorouracil (5-FU)

treatment.17 The dTTP depleted mammalian cells irreversible lose colony forming ability and

undergo cell death.16 The phenomenon of thymineless death is underlied in the mechanism of

several antibacterial, antimalarial and anticancer agents, such as sulfamethoxazole, trimethoprim,

methotrexate and fluorouracil16, 18-20. In 1954, Barner and Cohen21,22 discovered this phenomenon

that thymine requiring mutants of the E. coli bacterium lost viability when grown in a medium

lacking thymine but containing other essential nutrients. Subsequently, this discovery led to the

development of theories to explain the mechanism of action of several pyrimidine analogs that

targeted thymine metabolism in bacteria and tumor cells.23 The phenomenon was commonly

attributed to "unbalanced growth" wherein cells continued fundamental processes of RNA

transcription, protein synthesis and metabolism in the absence of DNA replication.22 This effect is

unusual in that the deprivation of many other nutritional requirements such as amino acids or

vitamins has a biostatic, but not lethal effect.24-25 Studies on numerous tumor cells have indicated

that thymine starvation has both direct and indirect effects. The direct effects involve both single-

and double-strand DNA breaks.17 Single-stand DNA breaks may be repaired effectively, but

double-strand DNA break will lead to cell death. Depletion of dTTP in mammalian cells induces

apoptosis, although the mechanism underlying this process remains to be elucidated.26-28

Methotrexate and 5-FU cause a decrease in dTTP levels and a concomitant increase in dUTP,

which is incorporated into DNA. This leads to extensive DNA damage as a result of the active

- 7 -

process of excision repair at the many uracil-containing sites in DNA, and thus triggers a DNA-

damage-induced apoptosis.17

During the TS catalyzed reaction, N5,N10-CH2-FH4 is oxidized to FH2 and converted back

to FH4 by dihydrofolate reductase (DHFR) which maintains the intracellular reduced folate pool.

Thus, inhibition of DHFR leads to a partial depletion of the intracellular reduced folate pool which

consequently limits cell growth.29 Both human TS and human DHFR are essential enzymes for

cell growth and hence both continue to represent attractive targets for chemotherapeutic agents.30,31

β-Glycinamide-ribonucleotide transformylase(GARFTase) and aminoimidazole-

carboxamide ribosyl-5-phosphate formyl transferase (AICARFTase) are the other two enssential

folate related enzymes in the de novo biosynthesis of purine nucleotides (Figure 4). These two

enzymes utilize the cofactor, N10-CHO-FH4 to transfer one carbon units.32 The C-8 carbon and C-

2 carbon of purine nucleotides are comprised of these “one carbon units”. GARFTase catalyzes

the third in the series of ten reactions required for purine synthesis (Figure 4), the conversion of

glycinamide ribosyl-5-phosphate (GAR) to formyl-glycinamide ribosyl-5-phosphate (fGAR),

utilizing N10-formyl-FH4. In mammals, GARFTase occurs as one enzyme in a trifunctional protein,

which catalyzes the second and the fifth steps of this pathway in addition to the third step. Or

fGAR formed is converted further to aminoimidazolecarboxamide ribosyl-5-phosphate (AICAR).

AICARFTase is responsible for the catalysis of the last two steps in de novo biosynthesis of purine.

AICARFTase utilizes N10-CHO-FH4 and converts AICAR to formyl-aminoimidazolecarboxamide

ribosyl-5-phosphate (fAICAR). The fAICAR continues along the purine biosynthetic pathway and

leads to the formation of inosine-5’-monophosphate (IMP), the precursor of adenosine-5’-

triphosphate (ATP) and guanosine-5’-triphosphate (GTP) necessary for ribonucleic acid (RNA)

synthesis and of 2’-deoxyadenosine-5’-triphosphate (dATP) and 2’-deoxyguanosine-5’-

- 8 -

triphosphate (dGTP) necessary for DNA synthesis and 2’-deoxyguanosine-5’-triphosphate (dGTP)

necessary for DNA synthesis.30

Figure 4 De novo synthesis of Purines.

As mentioned above, mammalian cells have three main folate transport system. Once

transported inside the cell, the cofactors are converted to the poly-γ-glutamyl species by the

- 9 -

enzyme folylpoly-γ-glutamate synthetase (FPGS), which adds glutamic acid residues to the

gamma carboxylic acid via amide bonds. Usually 4-8 glutamate residues are added to the γ-

carboxylic acid group of the cofactor or reduced folate. The polyglutamylated folates usually have

higher binding affinity to some folate dependent enzymes (e.g. TS) and have increased intracellular

retention time, because of their polyanionic nature.33,34

Due to the crucial role in the biosynthesis of nucleic acid precursors, folates and their

metabolism have been recognized as attractive targets for cancer chemotherapy.35-41 Based on the

transport mechanism and the ability to undergo polyglutamylation, antifolates are classified into

two types: classical antifolates and nonclassical antifolate.42 Classical antifolates contain an intact

L-glutamate side chain, while nonclassical antifolates contain a lipophilic side chain.43,44As shown

in Figure 5, representatives of classical antifolates include methotrexate (MTX), aminopterin,

edatrexate, PDDF (N10-propargyl-5,8-dideazafolate), raltitrexed (RTX, ZD1694, Tomudex),

pemetrexed (PMX, LY231514, Alimta), GW1843U89 and plevitrexed (ZD9331). These analogs

closely resemble the structure of endogenous folates and its metabolites. They are actively taken

up into cells by RFC, FR and/or PCFT systems present on the cell surface.33,38 The nonclassical

antifolates are represented by structures shown in Figure 6:

nolatrexed, (thymitaq), pyrimethamine, trimethoprim (TMP), piritrexim (PTX) and trimetrexate

(TMQ). Nonclassical antifolates are not taken up by the folate active transport systems (RFC, FR

and PCFT) and are presumably taken up by passive and/or facilitated diffusion.33,38

- 10 -

Figure 5 Representative examples of classical antifolates and their principal target enzyme.

- 11 -

Figure 6 Representative examples of nonclassical antifolates and their principal target.

2. MEMBRANE TRANSPORT OF FOLATES

As mentioned above, due to the lack of de novo biosynthetic enzymes in mammals for

folates, folate cofactors must be obtained from dietary resources. Because folates are hydrophilic

anionic molecules and cannot passive diffuse across biological membranes, several sophisticated

membrane transport systems have evolved to facilitate membrane translocation of these essential

cofactors. The three main transport systems include RFC, FR, and PCFT. Each of these plays a

unique role in mediating folate transport across epithelia and into tissues.’

- 12 -

2.1. The Reduced Folate Carrier (RFC)

RFC is the major transport system of folates in mammalian cells. It is ubiquitously

expressed in mammalian cells and tissues. In addition, RFC is the major transporter of antifolate

drugs used for cancer chemotherapy such as MTX, PMX, and RTX (Figure 5), and that the

effectiveness of these agents is closely linked to levels and activity of this transport system in both

tumors and normal tissues.45-47

2.1.1. Structure of RFC

In 1968, the functional properties of RFC were first report in murine leukemia cells by

Goldman and coworkers48. RFC (SLC19A1) belongs to the major facilitator superfamily (MFS)

of transporters. It is characterized by its anion exchange property. Human RFC (hRFC) is

comprised of 591 amino acids and is 64-66% conserved with rodent RFCs. RFC is an integral

membrane protein characterized by 12 transmembrane domains (TMDs) and cytoplasmic-oriented

N- and C-termini.49-52 hRFC is N-glycosylated at an N-glycosylation consensus site (Asn58) in the

first extracellular loop (EL) connecting TMD1 and TMD2.53A large loop domain that connects

TMD6 and TMD7 is poorly conserved between species and can be replaced altogether by a

nonhomologous segment from the SLC19A2 carrier.54 When separate TMD1-TMD6 and TMD7-

TMD12 RFC half-molecules are co-expressed in human RFC-null cells, they are targeted to the

cell plasma membrane surface and restore RFC transport activity.

2.1.2. RFC distribution

RFC is the primary transport system for folates cofactors in mammalian cells and tissues.

It can transport folates into cells of peripheral tissues from blood.55 In human tissues, the highest

level of hRFC transcript were detected in liver and placenta.56 The appreciable levels of hRFC

- 13 -

were recorded in kidney, leukocytes, lung, bone marrow, intestine, and portions of the central

nervous system (CNS) and brain.56 In other tissues such as the basolateral membrane of the renal

tubule epithelium, the apical brush border membrane of the small intestine and colon, hepatocyte

membranes, the apical surface of the choroid plexus, hRFC were also detected. RFC is essential

for tissue development since targeting both RFC alleles is embryonic lethal.57 In addition, at least

in some tissues (e.g., small intestine), mouse RFC is responsive to dietary folates supply such that

increased RFC transcripts and proteins were detected under conditions of dietary folate

deficiency.58

2.1.3. Transport mechanism of RFC

RFC, a bidirectional anion transporter, can transport reduced folates via counter-transport

with organic anions.3 5-methyl THF is the physiologic substrate of RFC.As an integral

transmembrane protein, RFC has a high affinity (~50-100-fold) for reduced folates (Ki ~1-5 µM)

and a low affinity for FA (Ki ~200 µM). Transport by RFC is characterized by a neutral pH

optimum and significantly decreased transport activity in acidic environment when pH is below

7.3,4,8.

RFC can generate small transmembrane chemical gradient. However, due to two glutamate

carboxyl groups of folates that are fully ionized at physiological pH, folates are negatively charged.

If considered with the within the context of the membrane potential, RFC produces a substantial

electrochemical potential difference for folates across cell membranes.49 The energy source for

this uphill process is unique. RFC function is not directly linked to ATP hydrolysis and it is neither

Na+ nor H+ dependent.59,60 Instead, RFC-mediated transport is highly sensitive to the

transmembrane anion gradient, especially, the organic phosphate gradient.59,60 Organic phosphates

are highly concentrated in cells where they are synthesized by ATP-dependent reactions and are

- 14 -

largely retained. Their resulting asymmetrical distribution across cell membranes provides the

driving force for RFC-mediated uphill transport of folates into cells.60

2.1.4. RFC in antifolate chemotherapy

MTX, RTX, and PMX, classical antifolates (Figure 5), are all actively transported into

mammalian cells by RFC as the major transporter.47,61 Membrane transport of antifolates such as

MTX is critical to antitumor activity since this provides sufficient intracellular drug concentration

to sustain maximal inhibition of enzyme targets (e.g. DHFR and TS) and for synthesis of

polyglutamate derivatives required for high affinity binding to some intracellular

enzymes and for sustained drug effects as plasma antifolate level decline. However, the loss of

active transport of MTX in drug resistance has been documented as early as 1962 in MTX resistant

L5178 murine leukemia cells.62 Since that time, impaired transport has emerged as one of the

dominant modes of tumor resistance to classical antifolate inhibitors such as MTX.63,64

Impaired transport that results in a loss of sensitivity to standard doses of antifolate should

be circumvented to some extent by increasing extracellular concentrations of drug. This increase

forces the drug into tumor cells expressing mutated or low levels of RFC, and involves alternate

uptake routes or passive diffusion, to a sufficient extent to inhibit intracellular enzymes and/or to

support polyglutamate synthesis. However, RFC is ubiquitously expressed in mammalian cells.

Therefore, the elevated extracellular antifolate concentration always results in dose-limiting

toxicity due to the lack of selectivity of uptake into the tumor cells compared to normal cells.

2.2 The folate receptors (FRs)

The FRs is a family of proteins with high affinity folate binding and represents another

mode of folate uptake into mammalian cells. They bind folic acid, reduced folates, many anti-

folates conjugates with high (low nanomolar) affinities.

- 15 -

2.2.1. Structure of FRs

The FRs are encoded by three distinct genes, designated α, β and γ localized to

chromosome 11q13.3-q13.5.65 FRs α, β and γ are highly homologous proteins (68-79% identical

amino acid sequence) and contain from 229 to 236 amino acids with two (β, γ) or three (α) N-

glycosylation sites.66 In contrast to RFC, FRα and FRβ are anchored in plasma membranes

via a glycosyl phosphatidylinositol (GPI) anchor, while FRγ is a secretory protein of unknown

function due to the lack of a signal sequence for GPI anchor attachment.6

Recently, Melcher and co-workers66reported the first crystal structure of FRα with folic

acid at 2.8 Å resolution. FRα has a globular structure stabilized by eight disulpfide bonds and

contains a deep open folate-binding pocket comprised of residues that are conserved in all receptor

subtypes. The pteroate moiety of folate is buried inside the receptor, while its glutamate moiety is

solvent-exposed and sticks out of the pocket entrance, allowing it to be conjugated to drugs without

adversely affecting FRα binding.67 (Figure7, 8).

Figure 7. hFRα- folate scaffold buried inside and glutamic acid is solvent exposed. (MOE 2014, PDB ID

4LRH).67

- 16 -

Figure 8. hFRα- interaction of folic acid with ligand-binding-pocket residues. (MOE 2014, PDB

ID 4LRH).67

2.2.2. FRs distribution

FRα is predominantly expressed on the apical (luminal) surface of polarized mammalian

epithelial cells, particularly in the proximal tubule cells of the kidney choroid plexus, retina, uterus

and placenta.68,69 Because FRα is predominantly expressed on the apical surface, it is not in contact

with the circulating folate. The unusual polarized expression of FRα appears to protect normal

tissues from FR-targeted cytotoxic agents in the circulation.70 FRβ is expressed in placenta, spleen,

thymus and in CD34+ monocytes and hematopoeitic cells.71-73 In normal bone marrow and

peripheral blood cells, expression of FRβ is restricted to myelomonocytic lineage (e.g. mature

neutrophils) and was reported to be nonfunctional.74 hFRγ is secreted at low levels from lymphoid

cells in the spleen, thymus, and bone marrow.74

FRα has been reported to be overexpress in malignant tissues, such as non-mucinous

adenocarcinomas of the ovary, uterus and cervix, and ependymal brain tumors.6 FRα levels

positively correlate with tumor stages and grades.75 FRβ malignant expression has been reported

- 17 -

to involve a substantial fraction of chronic myelogenous leukemia (CML) and acute myelogenous

leukemia (AML) cells, but not Acute Lymphoblastic Leukemia (ALL).75-77 Both FRα and FRβ in

malignant tissues seem to be functional, prompting the use of folic acid and pteroyl moieties for

tumor targeting of toxins, liposomes, imaging and cytotoxic agents.70,77,78

2.2.3. Transport mechanism of FRs

Figure 9. Endocytosis of FRs.

Folate internalization by membrane-associated FRs involves receptor-mediated

endocytosis.3,4,6,8 The process of internalization by FRs is initiated when a folate molecule binds

to a folate receptor on the cell surface. This is followed by invagination of the plasma membrane

at that site and the formation of vesicle (endosome) that migrates along microtubules in the

- 18 -

cytoplasm to the perinuclear endosomal compartment where it is acidified to a pH of 6.0-6.5. The

decrease in pH results in a conformational change of the complex which leads to dissociation of

the folate from folate receptor complex.79 The folate ligand is then exported into the cytoplasm by

a process requiring a trans-endosomal pH gradient.80-83 The endosome then migrates to the cell

surface and is fused with the cell membrane, the same FRs recycle. (Figure 9)

2.2.4. FRs and antifolate chemotherapy

Many reports have shown that FRα is overexpressed in solid tumors such as ovarian, lung

and breast carcinomas, including up to 90% of ovarian cancers.76 In addition, FRα expression has

been associated with higher stage, grade and differentiation status of ovarian tumors. However, in

normal tissues, FRα is reported to be inaccessible to the circulation.5 FRβ is expressed in a wide

range of myeloid leukemia cells, but FRβ is unable to bind folate, when it is in normal

hematopoetic cells.75 Thus, the high affinity FRs offer a potential means of selective tumor

targeting, given their restricted pattern of tissue expression and function. Folate-conjugated

cytotoxins, liposomes, radionuclides, or cytotoxic antifolates have all been used to target

FRs.5,33,84,85 However, for most folate-based therapeutics such as classical antifolates (including

MTX, RTX and PMX), tumor selectivity is lost, since substrates are shared between FRs and the

ubiquitously expressed RFC.

2.3. The proton-coupled folate transporter (PCFT)

In 2006, PCFT a new folate carrier (SLC46A1) and two other members of this family

(SLC46A2 and SLC46A3) were discovered.86,87 PCFT belongs to the superfamily of solute

transporter family. The gene that encodes human PCFT (SLC46A1) is located on chromosome

17q11.2. PCFT as a high-affinity folate transporter is a proton-folate symporter that functions

- 19 -

optimally at acidic pH 5.5 by coupling the flow of protons down an electrochemical concentration

gradient to the uptake of folates into cells.49,87 PCFT is a high-affinity folate transporter with a low

acidic pH optimum. Its primary role appears to be intestinal absorption of dietary folates and plays

a major role in in vivo folate homeostasis.88-89

2.3.1. Structure of PCFT

Hydropathy analyses predict PCFT is comprised of 459 amino acids with a molecular mass

of 49.8 kDa. It is predicted to include 12 TMDs with N- and C-termini oriented to the cytoplasm

(Figure 10). This structure has been validated by immunofluorescence analysis of hemagglutin

(HA)-tagged hPCFT molecules.90,91 The loop domain between the first and second TMDs must be

extracellular because the two putative N-glycosylation consensus sites in this region are

glycosylated. N-glycosylation does not appear to be required for either PCFT trafficking or

function.92

Figure 10 Human PCFT structure.48

2.3.2. PCFT expression patterns in human tissue

- 20 -

PCFT is expressed in many normal tissues including small intestine, colon, liver, kidney,

placenta, retina and brain. Within the intestine, high PCFT levels are found in the in apical brush-

border membranes in the proximal jejunum and duodenum. In addition, PCFT is also found in the

choroid plexus.92

2.3.3. Transport mechanism of PCFT

Folate transport mediated by human, mouse and rat PCFTs is electrogenic,

indicating that there is a net translocation of positive charges as each folate molecule is

transported.7,93-95 PCFT functions as a folate-proton symporter: the downhill flow of

protons via PCFT is coupled with the uphill flow of folates into cells. A trans vesicular pH

gradient results in increased unidirectional folate transport and substantial transmembrane

folate concentration gradients from the low-pH to the high-pH compartment, which is

consistent with a proton-coupled process.96 Acidic pH optimum is a distinguishing characteristic

of PCFT. For PCFT, transport is maximal at pH 5-5.5. As the pH increases above pH 5.5, transport

decreases dramatically; above pH 7, activity is not detectable.4,8 This leads to an important cancer

chemotherapy hypothesis that selective transport via PCFT could afford targeted therapy access of

solid tumors expressing PCFT. The pH of solid tumors is often acidic,94,97 which favors PCFT

transport. However, the role of hPCFT in antifolate activity and tumor selectivity is still under

investigation, transport of classical antifolates by PCFT has been described previously.95

2.3.4. PCFT and antifolate chemotherapy

The expression of RFC in both normal and tumor cells presents a potential obstacle to

antitumor selectivity. Further, loss of RFC results in resistance to MTX and RTX, yet PTX

cytotoxic activity can be preserved or even increased if PCFT is present.98 For agents such as PMX

- 21 -

that are transported by both RFC and PCFT, loss of tumor selectivity could be due to RFC transport

in normal tissues. Thus, there is a compelling rationale for developing cytotoxic antifolates that

are substrates for transporters other than RFC with limited expression and/or transport in normal

tissues compared with tumors. This rationale provided the impetus to develop targeted agents that

are selectively transported into tumors by FRs and PCFT over RFC, with potent intracellular

targets inhibition.48 Recently, Gangjee and coworkers99-101reported novel 6-substituted classical

pyrrolo[2,3-d]pyrimidine and thieno[2,3-d]pyrimidine as cytotoxic antifolates with varying

lengths of the carbon bridge region that are characterized by selective FR and/or PCFT transport

over RFC transport.

3. Glycinamide-ribonucleotide transformylase (GARTFase)

Glycinamide ribonucleotide transformylase (GARTFase) is one of the most important

trifunctional enzymes involved in purine synthesis. GARFTase (EC 2.1.2.2) was first discovered

and partially characterized from pigeon liver in pioneering investigations by Warren and

Buchanan.102 GARFTase catalyzes the transfer of the formyl group from N10-CHO-FH4 to the

primary, side-chain amino group of glycinamide ribonucleotide (GAR) to yield

formylglycinamide ribonucleotide (FGAR) and tetrahydrofolate (Figure 4), ultimately resulting in

the incorporation of C-8 into inosinic acid (IMP). This reaction is the third step and the first of two

folate-dependent formyl transfers in the de novo purine biosynthetic pathway. Purine nucleotides

play a critical role as precursors to RNA and DNA. Therefore, inhibition of de novo purine

biosynthesis might be a viable approach for cancer chemotherapy.103,104 This suggestion was

confirmed when it was demonstrated that 5,10-dideazatetrahydrofolate, a potent experimental

antitumor agent, has, as its mechanism of action, the inhibition of GARFTase and, consequently,

of de novo purine biosynthesis.105 GARFTase is of mechanistic interest for the ease with which it

- 22 -

catalyzes the formyl transfer,106,107of biological interest for its role in the synthesis of DNA

precursor purines.108 it is also structural interest for the delineation of key mechanistic features of

its catalytic reaction,109-112 and of medicinal interest as an important target for chemotherapeutic

drug design.113-123

3.1. Structure of GARFTase

Recently, The structure of the Escherichia coli (E. coli) GARFTase has been solved at pH

3.5 (1.8 Å),124 pH 6.75 (2.8 and 3.0 Å),125and pH 7.5 (1.9 Å)124 and at neutral pH in complexes

with GAR and 5-deazatetrahydrofolate (2.5 Å),110 GAR and 10-formyl-5,8,10- trideazafolic acid

(2.1 Å),111 and two multi-substrate adduct inhibitors (1.96 and 1.6 Å).109 The hGARFTase domain

is now readily available through cloning and overexpression.126,127 The hGARFTase is located at

the C-terminus of a trifunctional enzyme with a molecular mass of more than 110 kDa, which is

also responsible for catalyzing the second (glycinamide ribonucleotide synthetase) and fifth

(aminoimidazole ribonucleotide synthetase) reactions of de novo purine biosynthesis.128 The

crystal structure of hGARFTase has been reported at pH 4.2 (1.7 Å), at pH 8.5 (2 Å), at pH 8.5 in

the binary complex with the substrate β-GAR (2.2 Å)112 and at pH 7 in a binary complex with the

co-substrate analog inhibitor 10-trifluoroacetyl-5,10-dideaza-acyclic-5,6,7,8- tetrahydrofolic acid

(10-CF3CO-DDACTHF) (2 Å)129 and a series of other folate inhibitors.119

3.2. Catalytic Mechanism of GARFTase

Kinetic studies of the E. coli enzyme106, the human enzyme126 and the murine

enzyme130 suggest a sequential mechanism in which the formyl group is transferred by a direct

nucleophilic attack of the GAR amino group on the formyl carbon of the co-substrate, leading to

the formation of a tetrahedral intermediate. Formation of this intermediate and its collapse to

- 23 -

product require proton transfers. It has been proposed that a “fixed” water molecule, rather than

the invariant amino acids in the active site,131 mediates the required proton transfer between the

substrate and cofactor, but this has not been verified experimentally.

Figure 11 Proposed mechanism for GARFTase.132

A proposed mechanism for GARFTase is shown in Figure 11.132 As the cofactor

binds to the active site, Asp144 forms a salt bridge to the imidazolium of His108 and the

formyl group is positioned to form hydrogen bonds to Asn106 and the protonated

imidazolium group of His108. The free base form of the amino group of GAR then attacks the

- 24 -

activated formyl group to form a tetrahedral intermediate. A proton transfer from GAR to the N10

of folate is mediated by a catalytic water molecule, followed by breakdown of the tetrahedral

intermediate to form products. The positioning of this water molecule may be assisted by a

hydrogen bond to the carboxylate of Asp144. (For clarity, Asp144 is shown twice in Figure 11; it

spans the N1 of His108 to the bound H2O molecule.)

3.3. Binding of Inhibitors

Zhang et al.129 reported a 1.98 Å X-ray crystal structure (Figure 12) of hGARTFase in a

binary complex with the co-substrate analog inhibitor 10-CF3CO-DDACTHF (Table 2) at pH 7

(PDB entry 1NJS). The cofactor binding pocket of GARTFase is located at the interface between

the N-terminal mononucleotide binding domain and the C-terminal half of the structure. The

binding site for the folate cofactor moiety consists of three parts: the pteridine binding cleft, the

benzoylglutamate region, and the formyl transfer region.

Figure 12 Crystal structure (MOE 2014) of hGARTase in a binary complex with inhibitor 10-

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

Pteridine Binding Cleft. The diaminopyrimidinone ring is deeply buried in the active site

cleft and occupies the same location as the quinazoline ring of 10-formyl-5,8,10-trideazafolic acid

- 25 -

(10-formyl-TDAF) in the E. coli GARFTase complex (PDB entry 1C2T). The connecting stem

from the diaminopyrimidinone ring, composed of single carbon bonds, is longer than its

counterpart in 10-formyl-TDAF, due to the removal of the fused benzene ring, which makes it

more flexible when adapting to the binding site to optimize the gem-diol interactions with the

protein. The diaminopyrimidinone ring of 10-CF3CO-DDACTHF is tilted about 15° relative to the

quinazoline ring of 10-formyl-TDAF, which places N2 within hydrogen bonding range

(3.1 Å) of the backbone carbonyl oxygen of Glu141. The diaminopyrimidinone ring conserves all

of the key interactions that were previously observed with the quinazoline ring of 10-formyl-TDAF

and provides additional key hydrogen bonds with the enzyme. Several hydrophobic residues

encircle a deep cavity holding the heterocycle. The hydrophobic pocket consists of Leu85, Ile91,

Leu92, Phe96, and Val97 lining one end and the folate-binding loop of residues 141-146 at the

other. The diaminopyrimidinone ring makes six hydrogen bonds to the main chain amides and

carbonyls of Arg90, Leu92, Ala140, Glu141, and Asp144, and two hydrogen bonds to ordered

waters (W18 and W70). In the quinazoline ring of 10-formyl-TDAF, N8 of the folate pteridine

ring is replaced with a carbon. This nitrogen has been proposed to play a key role in the recognition

and interaction with folate-binding enzymes and forms one end of an H-bond

donor-acceptor-donor array. While its replacement with carbon does not preclude the

binding to GARFTase, its presence appears to contribute to substrate recognition by the

folate transport system and/or FPGS. The diaminopyrimidinone ring of 10-CF3CO- DDACTHF,

however, preserves this nitrogen (N8).

Glutamate Tail. In the 10-CF3CO-DDACTHF complex, the p-aminobenzoate

moiety locates in a hydrophobic pocket sandwiched between the side chains of Ile91

and Ser118. The electron density of the carbonyl group is well-defined and in the same

- 26 -

plane as the phenyl ring. The glutamate tail is oriented almost perpendicular to the p-

aminobenzoate plane and parallel to the aliphatic stem of the diaminopyrimidinone ring. In the

complex structure of 10-CF3CO-DDACTHF with human GARFTase, the glutamate moiety is

solvent-exposed, but exhibits a remarkably well-ordered structure, in contrast to its flexibility in

E. coli GARFTase complex structures. A salt bridge (2.7 Å) is formed between the glutamate α-

carboxylate and Arg64 so that the γ-carboxylate points to the solvent. An additional interaction

observed here includes a hydrogen bond between the Ile91 backbone amide and the α-glutamate

carboxylate (2.8 Å).

Formyl Transfer Region and the Gem-Diol Structure. Key interactions for tight binding of

inhibitor 10-CF3CO-DDACTHF to GARFTase are found in the formyl transfer region. Strong

density next to the ketone oxygen indicates that the ketone is hydrated to a gem-diol, similar to the

10-formyl-TDAF and β-GAR complex with the E.coli GARFTase (PDB ID 1C2T). The gem-diol

forms extensive interactions with the formyl transfer region, especially with Asp144 and His108,

two essential residues in the formyl transfer reaction. The Asp144 carboxylate hydrogen bonds

bind (2.5 and 2.7 Å) to each of the hydroxyl groups of the gem-diol. N3 in the imidazole ring of

His108 also forms hydrogen bonds with both hydroxyls of the gem-diol [OA1 (2.7 Å) and OA2

(3.1 Å)]. Moreover, OA2 also makes a potential hydrogen bond (3.0 Å) with the backbone

carbonyl oxygen of Gly117.

Recently X-ray crystal structure of hGARTFase with pemetrexed and 6-substituted

pyrrolo[2,3-d] pyrimidine antifolate (Figure 13) and β-GAR substrate were determined.133 Based

on the crystallographic models antifolates form six hydrogen bond interactions with hGARFTase

peptide backbone atoms via the pyrrolo[2,3-d] pyrimidine moiety. In addition, two hydrogen bonds

- 27 -

with the peptide backbone, a bidentate polar interaction with Arg871, and three long distance

charge–charge interactions with Arg897 and Lys844 are mediated via the glutamyl tail.

Figure 13 Crystal structure of hGARTFase. (PDB: 4ZZ1)133

3.4. GARFTase Inhibitors

Three types of GARFTase inhibitors are reviewed. These are listed in Table 1, Table 2 and

Table 3 below:

3.4.1. 5,6,7,8-Tetrahydropyrido[2,3-d]pyrimidines and 7,8-dihydropyrimido[5,4-

b][1,4]thiazines (Table 1)

3.4.2. Pyrimidines (Table 2)

3.4.3. Pyrrolo[2,3-d]pyrimidines and thieno[2,3-d]pyrimidines (Table 3)

- 28 -

3.4.1. 5,6,7,8-Tetrahydropyrido[2,3-d]pyrimidines and 7,8-dihydropyrimido[5,4-b][1,4]thiazines (Table 1)

Lometrexol. In 1985 Taylor and coworker139 synthesized the first GARFTase inhibitor to

enter clinical trials, the 6-(R)-isomer of 5, 10-dideazatetrahydrofolic acid (DDATHF), termed

lometrexol (LMTX, Table 1). This discovery established GARFTase and the purine de novo

biosynthetic pathway as viable targets for antineoplastic intervention. LMTX is a poor inhibitor of

both DHFR and TS, but a potent inhibitor of GARFTase (Ki = 6 nM) and a substrate for FPGS.140

The diastereomers were separated and found to be equipotent as inhibitors of cell growth and as

substrates for FPGS.134 The (6R)-diastereomer, which corresponds to the configuration found in

natural tetrahydrofolates, was chosen for further development. Patients treated with LMTX in

Structure Compound Biological Activity

Ref.

Lometrexol (DDATHF)

hGARFTase (Ki) 6 nM

134

LY-222306 (6-R,S)

hGARFTase (Ki) 0.77 nM

CCRF-CEM (IC50) 27 nM

135

LY-254155 (6-R,S)

LY-309887 (6-R)

hGARFTase (Ki) 2.1 nM


136

AG-2034 hGARFTase (Ki)

28 nM CCRF-CEM (IC50) 2.9 nM

137

AG-2037 (pelitrexol)

hGARFTase (Ki)

0.5 nM

138

- 29 -

phase I clinical trials developed severe and cumulative myelosuppression and mucositis. The

cumulative toxicity of LMTX is thought to be due in part to its ability to be

transported by both the RFC and FRs, resulting in increased cellular levels and a lack of selectivity

for tumor cells.141,142

(2S)-2-[[5-[2-[(6R)-2-amino-4-oxo-5,6,7,8-tetrahydro-1H-pyrido[2,3-d]pyrimidin-6-

yl]ethyl]thiophene-2-carbonyl]amino]pentanedioic acid (LY309887). Unexpected observations of

delayed cumulative toxicity with LMTX led to a search for a second-generation antimetabolite

with a more favorable toxicological, biochemical and pharmacological profile. In 1994, Habeck et

al.135 reported a novel class of classical antifolates that replaced the 1’,4’-phenyl group of LMTX

with a 2’,5’-furan (LY222306) and a 2’,5’-thiophene (LY254155) (Table 2). Both LY222306 and

LY254155 (mixtures of diastereomers) were found to be potent inhibitors of CCRF-CEM cell

growth (IC50 = 27 and 2.3 nM, respectively) and tight binders of human GARFTase (Ki = 0.77 and

2.1 nM, respectively). Further evaluation of LY254155 was restarted in 1996 after the compound

was resolved into its two diastereomers.136,143 LY309887 (6R-2’,5’-thienyl-5,10-

dideazatetrahydrofolic acid, Table 1) was more potent than LMTX at inhibiting tumor growth in

the murine C3H mammary tumor model and several tumor xenografts. LY309887 displayed a 9-

fold greater inhibitory potency against GARFTase compared to LMTX. In preclinical models,

LY309887 was more active than LMTX against two pancreatic xenografts and in the human LX-

1 lung carcinoma model. In two preliminary reports of phase I studies with LY309887, all patients

received concurrent folic acid but still suffered from cumulative toxicity, suggesting that, like

LMTX, cumulative toxicity remains a property of LY309887.144,145

AG-2034 and AG-2037. AG-2034 (Table 1) was designed from the X-ray structure of the

GARFTase domain of the human trifunctional enzyme. The only difference between AG-2034 and

- 30 -

LY-309887 is the 5-sulfur in the pteridine ring. AG-2034 inhibits hGARFTase (Ki = 28 nM) and

is a substrate for rat liver FPGS, with similar efficacy as LMTX, while AG-2034 is a more effective

ligand for FRs than LMTX. AG-2034 inhibited cell growth (IC50 = 4.0 and 2.9 nM, respectively,

against L1210 and CCRF-CEM cells) and exhibited in vivo antitumor activity against lung

carcinomas, lymphosarcomas, mammary and colon adenocarcinomas and melanoma. In phase I

clinical studies, AG-2034 displayed much less cumulative myelosuppression than LMTX, but like

LMTX did result in mucositis and diarrhea.146 AG-2037 (pelitrexol) is currently in phase I clinical

development (Table 1).147 The configuration at C-6 is reversed compared to LMTX and the

thiophene is methylated at the 4-position of AG-2037. AG-2037 is a potent inhibitor of GARFTase

(Ki = 0.5 nM) and exhibits significant antiproliferative effects against tumor cells in vitro and in

vivo.148 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.149,150

4.2. Pyrimidines (Table 2)

Structure Compound Biological Activity Ref.

7-DM-DDATHF X = CH2

5-DACTHF

X = NH

CCRF-CEM (IC50) 0.13 µM

hog liver GARTFase (IC50)

2.6 µM

151

152

Structure Compound Biological Activity Ref.

- 31 -

1 X = CH

2 X = N

hog liver GARTFase (IC50)

2.06 µM hog liver

GARTFase (IC50) 0.14 µM

153

154

10-CHO-DDACTHF

R = H

10-CF3CO-DDACTHF

R = CF3

hGARTFase (Ki) 14 nM


rhGARTFase (Ki)

15 nM CCRF-CEM (IC50)

16 nM

122

129

(10R)-CH3S-DDACTHF

(10S)-CH3S-DDACTHF

rhGARTFase (Ki) 210 nM


rhGARFTase (Ki)

180 nM CCRF-CEM (IC50)

50 nM

155

155

α-CH2-10-CF3CO-DDACTHF R1=COONH2 R2=COOH γ-CH2-10-CF3CO-DDACTHF R1=COOH R2=COONH2 γ -Tetrazole-10-CF3CO-DDACTHF R1=COOH R2=tetrazole

hGARFTase (Ki) 4.8 µM

CCRF-CEM (IC50) inactive

hGARFTase (Ki) 56 nM


hGARFTase (Ki) 130 nM


156

156

157

7-DM-DDATHF. During the development and evaluation of LMTX, open-chain

analogs of LMTX were synthesized and evaluated.151,152 These analogs did not possess a

- 32 -

stereocenter and were therefore less challenging to synthesize. 7-DesmethyleneDDATHF (7-DM-

DDATHF) (Table 2) is approximately 8-fold less potent than LMTX

against human leukemia CCRF-CEM cells (IC50 = 130 nM vs. 16 nM) and only 3-fold

less active against GARFTase isolated from murine leukemia L1210 cells. 7-DMDDATHF is also

a substrate for FPGS and its polyglutamated conjugates are more potent

inhibitors of GARFTase than the parent compound. Removal of the annulated

tetrahydropyridine ring of LMTX had minimal effect on binding to GARFTase. A similar

compound 5-DACTHF (Table 2) was reported by Kelley and coworkers152 in 1990. 5-

DACTHF showed an IC50 of 2.6 μM against GARFTase isolated from hog liver, and

slight inhibition of AICARFTase (IC50 = 200 μM in L1210 cells). Further SAR studies

afforded the thienyl and thiazolyl analogs of 5-DACTHF.153 The thiophene analog 1 (Table 2) was

equal in activity to 5-DACTHF in controlling MCF7 breast cancer cell growth, while thiazole 2

(Table 2) was 9-fold more active than 5-DACTHF and 4-times more active than LMTX.

Compound 2 and LMTX have very similar activity against hog liver GARFTase.

10-CF3CO-DDACTHF. Boger and coworkers122 reported the synthesis and

evaluation of 10-formyl-5,10-dideaza-acyclic-5,6,7,8-tetrahydrofolic acid (10-CHODDACTHF,

Table 2), an acyclic analog of LMTX bearing a nontransferable C-10 formyl

group. 10-CHO-DDACTHF inhibited cell growth (IC50 = 60 nM, CCRF-CEM) and

hGARFTase (Ki = 14 nM), but suffered from instability due to a facile oxidative

deformylation. Continued development of such GARFTase inhibitors was accomplished

by combining the 2,4-diaminopyrimidone core of 10-CHO-DDACTHF with a more

stable trifluoromethyl ketone moiety.13010-(Trifluoroacetyl)-5,10-dideaza-acyclic-

5,6,7,8-tetrahydrofolic acid (10-CF3CO-DDACTHF, Table 3) is a potent inhibitor of

- 33 -

tumor cell proliferation, with an IC50 of 16 nM against CCRF-CEM cells, which

represents a 10-fold improvement over LMTX. 10-CF3CO-DDACTHF specifically

inhibits recombinant hGARFTase (Ki = 15 nM), and is stable, displaying no competitive

oxidative deacylation.

10-CH3S-DDACTHF. Further exploration of analogs containing alternative tetrahedral

intermediate mimics continued with the synthesis and evaluation of 10-CH3SDDACTHF (Table

2).158 10-CH3S-DDACTHF proved to be potent, exhibiting an IC50 of 100 nM against CCRF-CEM

cells. A follow-up asymmetric synthesis of (10R)- and (10S)- diastereomers of 10-CH3S-

DDACTHF was recently reported in 2008.158 Both diastereomers are potent and selective

inhibitors of rhGARFTase (Ki = 210 and 180 nM, respectively, for 10R and 10S) and effective

inhibitors of cell growth (IC50 = 80 and 50 nM, respectively, against CCRF-CEM cells), which is

dependent on intracellular polyglutamation by FPGS but not transport by RFC.

γ-CONH2-10-CF3CO-DDACTHF and γ-tetrazole-10-CF3CO-DDACTHF.A further area

of examination in these efforts was the glutamic acid portion of 10-CF3CO-DDACTHF. As stated

previously, folates and many antifolates are polyglutamylated by FPGS after entering cells. Most

FPGS, including human FPGS, attach additional glutamates to the γ-carboxylic acid of folates and

antifolates. Polyglutamation of folates and antifolates increases their affinity for some enzymes

(e.g. TS and GARFTase) and makes them less susceptible to cellular efflux, providing a long-lived

cellular supply of the molecules. However, this long-term enhanced intracellular accumulation of

antifolates, due to their polyglutamation, contributes to their cumulative toxicity. Therefore, the

strategy that preventing polyglutamylation, while maintaining potent enzyme binding, is

considered a therapeutic asset. γ-CONH2-10-CF3CO-DDACTHF (Table 2) exhibited potent

inhibitory activity against rhGARFTase (Ki = 56 nM) and purine sensitive cytotoxic activity (IC50

- 34 -

= 300 nM, CCRF-CEM), whereas α-CONH2-10-CF3CO-DDACTHF showed decreased affinity

(Ki = 4.8 μM) and was inactive in cellular functional assays.155 Another potent

nonpolyglutamatable inhibitor γ-tetrazole-10-CF3CO-DDACTHF (Table 2) exhibited purine-

sensitive cytotoxic activity (IC50 = 40 nM) and was a selective inhibitor of hGARFTase (Ki = 130

nM).156 As anticipated, both γ-CONH2-10-CF3CO-DDACTHF and γ-tetrazole-10-CF3CO-

DDACTHF are not dependent on FPGS for acitivty, and hence not susceptible to resistance

mediated by FPGS.

4.3. Pyrrolo[2,3-d]pyrimidines and thieno[2,3-d]pyrimidines (Table 3)

Structure Compound Biological Activity (IC50) Ref.

HN

N NH

O

H2N

NH

O COOH

COOH

n

n = 1-6

3 n = 3 4 n = 4

hGARFTase 18 nM KB 1.7 nM IGROV1 2.2 nM hGARFTase 6.8 nM KB 1.9 nM IGROV1 3.6 nM

96

HN

N S

O

H2N

NH

O COOH

COOH

n

n = 2-8

5 n = 3 6 n = 4

hGARFTase 13.8 nM 5 KB 23 nM IGROV1 4.7 nM hGARFTase 13.3 nM 63 KB 4.9 nM IGROV1 5.9 nM

7 n = 3 8 n = 4

hGARFTase 0.69 nM KB 0.26 nM IGROV1 0.55 nM hGARFTase 1.96 nM KB 0.55 nM IGROV1 0.97 nM

1. SAR of 2-amino-4-oxo-6-substituted-pyrrolo[2,3-d]pyrimidines with para substituted benzoyl

- 35 -

aromatic rings as side chain in whole cell assay as GARTFase inhibitors with selectivity for FRs

and/or PCFT over RFC.96

Table 4. IC50’s (in nM) for 6-Substituted Pyrrrolo[2,3-d]pyrimidine Antifolates 3,4 and classical

Antifolates in hRFC, hPCFT, and FR-Expressing Cell Lines. (+F (folic acid) = 200 nM, plus/minus

SEM in parentheses, ND not determined).96

Gangjee and coworkers5,63,95, recently described novel 6-substituted classical pyrrolo[2,3-

d]pyrimidine and thieno[2,3-d]pyrimidine as cytotoxic antifolates with varying lengths of the

carbon bridge region (Table 3), that are characterized by selective FR and PCFT transport over

RFC transport. For every series, the three- and four-carbon bridge analogs were the most active

toward FR-expressing human tumors (KB and IGROV1) and the cytotoxicity was primarily due

to potent inhibition of GARFTase, although for the thieno[2,3-d]pyrimidine antifolates, a

secondary target, most likely AICARFTase, was also implied at higher concentration.

Engineered CHO (Chinese Hamster Ovary) Cell Line (IC50S in nM)

hRFC NIL hFRα hFRβ hPCFT NIL

PC43-10 R2 RT16 RT16+F D4 D4+F R2/hPCFT4 R2(VC)

3 304 448 4.1 >1000 5.6 >1000 23 >1000

4 >1000 >1000 6.3 >1000 10 >1000 213 >1000

PMX 138(13) 894 (93) 42 (9) 388 (68) 60 (8) 254 (78) 13.2 (2.4) 974.0 (18.1)

MTX 12 (1.1) 216 (8.7) 114 (31) 461(62) 106 (11) 211 (43) 120.5 (16.8) >1000

RTX 6.3(1.3) >1000 15(5) >1000 22 (10) 746 (138) 99.5 (11.4) >1000

- 36 -

The 6-substituted classical pyrrolo[2,3-d]pyrimidine with a para substituted benzoyl side

chain compound 3 and 4 were highly potent against tumor cells (3, IC50s of 1.7 nM for KB and

2.2 nM IGROV1 and 4, IC50s of 1.9 nM for KB and 3.6 nM IGROV1) . These compounds are

highly selective towards FRs and PCFT over RFC (Table 4). The selectivity of compound 3 is

slightly compromised by its RFC transport, however compound 4 has excellent selectivity for FRs

and PCFT over RFC. These in vitro cytotoxicity results establish that the growth inhibitory effects

of 3 and 4 are dependent on their cellular accumulation via FRs rather than RFC and that there is

no apparent difference in the activity of these drugs toward cells that express the FRα isoform from

cells that express FRβ.

2. SAR of 2-amino-4-oxo-6-substituted pyrrrolo[2,3-d]pyrimidines with para substituted thieno

rings as side chain in whole cell assay as GARTFase inhibitors with selectivity for FRs and/or

PCFT over RFC.5

The 6-substituted classical thieno[2,3-d]pyrimidines with a para substituted thieno side

chain 7 and 8 were highly potent against tumor cells (Table 5) . These compounds showed high

selective towards FRs over RFC and PCFT (Table 5). These compounds do not use RFC for

transport into the cell.

Table 5. IC50’s (in nM) for 6-Substituted pyrrrolo[2,3-d]pyrimidines Antifolates 7, 8 and

Classical Antifolates in hRFC, hPCFT, and FR-Expressing Cell Lines. (+F (folic acid) = 200 nM,

plus/minus SEM in parentheses, ND not determined)5

Table 5.

Engineered CHO (Chinese Hamster Ovary) Cell Line (IC50S in nM)

- 37 -

4. Aminoimidazole-4-carboxamide ribonucleotide transformylase (AICARFTase)

AICARFTase catalyzes the penultimate reaction in the de novo purine biosynthetic

pathway, producing formyl-AICAR (FAICAR) from AICAR using N10-formyltetrahydrofolate

(N10-CHOFH4) as the formyl donor. It exists as one domain of a bifunctional enzyme that also

contains IMP cyclohydrolase (IMP CHase) that catalyzes the final reaction of the pathway (Figure

4). Because of the large demand for purines by cancer cells, AICARFTase has been targeted by

antifolate inhibitors in chemotherapy along with GARFTase.160-161

4.1. Structure of AICARFTase

AICARFTase is highly conserved from E. coli to human but has no sequence homology

with other folate-dependent enzymes, such as GARFTase.162 Thus, folate based inhibitors of

GARFTase do not usually inhibit AICARFTase because of different interactions within the two

active sites.163 For example, lometrexol potently inhibits GARFTase but not AICARFTase.164

hRFC NIL hFRα hFRβ hPCFT NIL

PC43-10 R2 RT16 RT16+F D4 D4+F R2/hPCFT4 R2(VC)

7 101 273 0.31 >1000 0.17 >1000 3.34 >1000

8 >1000 >1000 1.82 >1000 0.57 >1000 43.4 >1000

PMX 138(13) 894 (93)

42 (9) 388 (68) 60 (8) 254 (78)

13.2 (2.4) 974.0 (18.1)

MTX 12(1.1)

216 (8.7)

114 (31) 461(62) 106 (11)

211 (43)

120.5 (16.8) >1000

RTX 6.3(1.3) >1000 15(5) >1000 22

(10) 746

(138) 99.5 (11.4) >1000

- 38 -

Wilson and coworkers161,163,165-167have previously reported several crystals

structures of human and avian ATIC (the bifunctional enzyme, aminoimidazole-4-

carboxamide ribonucleotide (AICAR) transformylase (AICARFTase)/IMP

cyclohydrolase (IMPCH)) in both unliganded and complexed forms. The ATIC forms an

intertwined symmetrical homodimer (each monomer being composed of 393 residues)

with the IMPCH domain at the N terminus (residues 1–199) and the AICARFTase

domain at the C terminus (residues 200-593). The IMPCH active sites are contained

within each monomer of the dimer, but the AICARFTase active site is located at the

dimer interface with key active-site residues being contributed from both monomers.

4.2. AICARFTase catalytic mechanism

The catalytic mechanism of AICARFTase was evaluated with pH dependent kinetics, site-

directed mutagenesis, and quantum chemical calculations.168-170 The results indicated that the

amide-assisted mechanism is concerted such that the proton transfers from the 5-amino group to

the formamide are simultaneous with nucleophilic attack by the 5-amino group. Because this

process does not lead to a kinetically stable intermediate, the intramolecular proton transfer from

the 5-amino group through the 4-carboxamide to the formamide proceeds via the same transition

state. Collectively, these experimental and theoretical analyses lead to a proposed mechanism for

AICARFTase catalysis shown in Figure 14.

- 39 -

Figure 14 A proposed mechanism of AICARFTase.170

In this proposed mechanism, Lys266, with the aid of His267, acts as a general acid catalyst

to interact with the N3 of the imidazole ring of AICAR. His267 aids Lys266 to be in the right

position for a hydrogen-bonding interaction. The 4-carboxamide mediates proton shuttling from

the 5-amino to 4-carboxamide and then to the nitrogen of the leaving group with simultaneous

nucleophilic attack of the 5-amino group on the formyl group of folate cofactor. The first proton

transfer is facilitated by the intramolecular hydrogen bond in the ground-state AICAR and the

protonated 4-carboxamide species is only present transiently. The theoretical results suggest that

protonation of the nitrogen of the leaving group is critical in the aminolysis reaction between

AICAR and the formyl group rather than the nucleophilic attack on the carbonyl group. The central

role assigned to proton transfer compensates for the weak nucleophilicity of the AICAR 5-amino

group.

4.3. AICARFTase inhibitors

Burroughs Wellcome (Research Triangle Park, NC) had designed and synthesized two

antifolates that are specific inhibitors (nM) for human AICARFTase, as compared with other folate

dependent enzymes, GARFTase, DHFR, and TS.162 These compounds are both

sulfamido-bridged 5,8-dideazafolate analogs identified as BW1540U88UD (BW1540)

- 40 -

and BW2315U89UC (BW2315) (Fig. 15) that differ only in the disposition of the imido

and sulfonyl groups within the bridge region. BW1540 and BW2315 have approximate Ki

values against human ATIC of 8 and 6 nM, respectively, whereas the Ki values against

GARFTase, DHFR and TS are within the micromolar range, except for BW1540, which

showed low nanomolar inhibition against DHFR. BW1540 and BW2315 were co-crystallized at

2.55 and 2.60 Å with human ATIC in the presence of substrate AICAR to elucidate their

mechanism of inhibition. It was found both of compounds bound to the AICARFTase active site

of ATIC and the sulfonyl groups dominate inhibitor binding and orientation through

interaction with the proposed oxyanion hole. Moreover, interaction of the sulfonyl oxygens with

the oxyanion hole of the AICARFTase active site suggests that it is the driving force behind the

high affinity for these sulfonyl-containing antifolates (The Km of 10-f-THF is 100 µM170, whereas

the Ki values for BW1540 and BW2315 are 8 and 6 nM, respectively).162 Thus, these two

antifolates bind more strongly to the AICARFTase active site, by at least 1000-fold, than the

natural co-factor N10-formyltetrahydrofolate.

Figure 15 AICARFTase inhibitors.162

- 41 -

Figure 15 AICARFTase inhibitors.162

b. NSC37173 and NSC30171

NSC30171 had nanomolar inhibition (Ki = 154 nM, IC50 = 600 nM), and NSC37173 (IC50

= 4.1 µM) against human AICARFTase.171 The human AICARFTase /BW1540 complex (PDB

1P4R) was selected as the docking template. NSC30171 was selected via NSC37173 similarity

search. Docking with and without AICAR substrate in the active site strongly indicates that this

nonfolate competes with the AICAR substrate for the AICAR binding site. Key interactions with

the AICARFTase active site residues appear to be the electrostatic and H-bonding interactions

between its sulfate and the enzyme “oxyanion hole”, and the aromatic stacking of the NSC30171

naphthalene ring with the Phe590 benzyl ring.

c. TNP-351

TNP-351, characterized by a pyrrolo[2,3-d]pyrimidine ring, exhibits potent antitumor

activities against mammalian solid tumors.172 The mechanism of action of TNP-351 was evaluated

using methotrexate-resistant CCRF-CEM human lymphoblastic leukemia cell lines as well as

- 42 -

partially purified FPGS, AICARFTase, and GARFTase from parent CCRF-CEM cells. TNP-351

was found to significantly inhibit the growth of L1210 and CCRF-CEM cells in culture, with the

doses effective against 50% of the cells (ED50 values) being 0.79 and 2.7 nM, respectively. The

methotrexate-resistant CCRFCEM cell line, which has an impaired methotrexate transport,

showed less resistance to TNP-351 than to methotrexate. Inhibitory activities of TNP-351 and its

polyglutamatesGn (n=1–6) for AICARFTase were found to be significantly enhanced with

increasing glutamyl chain length (inhibition constants (Ki): G1, 52 μM; G6, 0.07 μM). Neither TNP-

351 nor its polyglutamates were very potent AICARFTase inhibitors.

d. Pemetrexed (PMX, Alimta)

Pemetrexed (PMX) is currently in widespread clinical use as the first line therapy for

mesothelioma and non–small cell lung cancer (NSCLC) and is also currently being evaluated for

the treatment of a variety of other solid tumors in the US.173 Thymidylate synthase (TS) was

identified originally as the primary intercellular target174 of PMX. However, it is possible start that

PMX is a multitargeted antifolate that inhibits DHFR, GARFTase and AICARFTase in addition

to TS.175 However, TS Iis the pricipal target.

7. Multiple targeted antifolates in combinational anticancer chemotherapy

It has been of interest not only to design potent antifolates against specific

enzymes of DHFR, TS, GARFTase and AICARFTase but also to design and synthesize

single agents that have potent multiple inhibitory activities against these enzymes.176, 177

This strategy is particularly promising in anticancer chemotherapy against the multiple

drug resistant cancers.178 Such a single agent could act at more than one active site and

provide “combination chemotherapy”. The benefits of single agents are the following: 1)

- 43 -

circumvent the pharmacokinetic problems of multiple agents, 2) avoid of drug-drug interactions,

3) used at lower doses to alleviate toxicity, 4) devoid of overlapping toxicities, and most

importantly delay or prevent cancer drug resistance.178,179 Other advantages of such single agents

are cost reduction and patient compliance. In addition, the clinical success of pemetrexed

provided a good example of multiple targeted antifolates used in combinational anticancer

chemotherapy.179

- 44 -

II. CHEMICAL REVIEW

The chemistry related to the present work is reviewed and includes synthetic approaches to the

following 5-substituted, 6-substituted pyrrolo[2,3-d]pyrimidines and relevant reactions:

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

2. Sonogashira coupling in antifolate synthesis

3. Heck coupling reaction in one-step aldehyde synthesis

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

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 for synthesis from:

1.1 Pyrimidine precursors

1.2 Pyrrole precursors

1.3 Furan precursors

1.1. From pyrimidine precursors

- 45 -

Scheme 1 Synthesis of pyrrolo[2,3-d]pyrimidines 11and 13.

Noell and Robins180 first reported the synthesis of pyrrolo[2,3-d]pyrimidines 11 and 13 by

the reaction of chloroacetaldehyde 10 with 2-amino-6-alkylamino-4-hydroxypyrimidines 9 and

6-amino-1,3-dimethyluracil 12 respectively (Scheme 1).

Scheme 2 Synthesis of pyrrolo[2,3-d]pyrimidine 15

Noell and Robins180 also reported the synthesis of pyrrolo[2,3-d]pyrimidine 15 from 2-

methylthio-6-amino-4-pyrimidone 14 and chloroacetaldehyde 10 (Scheme 2)

- 46 -

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

In 1998, Gibson et al.181 reported a synthetic approach to prepare 2-amino-4-

oxopyrrolo[2,3-d]pyrimidines 20 (Scheme 3). In this method, 2,6-diamino-4-hydroxypyrimidine

16 was reacted with a biselectrophile, an oxime 17, in the presence of a weak base (sodium

carbonate, sodium acetate, or triethylamine) to afford the C-5 alkylated pyrimidine derivatives 19.

No side products resulting from substitution at any other position were isolated. Cyclization of

pyrimidine derivatives 19 at 120 °C under acid-catalyzed transoximation with benzaldehyde or

acetaldehyde afforded substituted pyrrolo[2,3-d]pyrimidines 20.

Scheme 4 Synthesis of furo[2,3-d]pyrimidines 22 and pyrrolo[2,3-d]pyrimidines 23.

- 47 -

In 1973, Fumio et al.182 reported that the reaction of 6-amino-1,3-dimethyluracil 12 with

phenacyl bromides 21 in DMF afforded 1,3-dimethyl-6-phenylpyrrolo[2,3-d]pyrimidines 23 in 69-

74% yields (Scheme 4). The reaction of 12 and 21 in acetic acid afforded the isomeric 1,3-

dimethyl-5-phenylfuro[2,3-d]pyrimidine-2,4(1H,3H)-diones 22 as a byproduct in addition to 23.

Scheme 5 Synthesis of pyrrolo[2,3-d] pyrimidines 27.

Davoll and coworker183 reported the synthesis of various pyrrolo[2,3-d]pyrimidines 27

(Scheme 5) by an acid mediated cyclization of suitable pyrimidine 5-acetone or acetaldehyde

side-chains 26 onto the neighboring amino group. The pyrimidines were in turn obtained from

the acetals ethyl(2,2-diethoxyethyl)acetate 24, or 2,2-diethoxyethylmalonodinitrile, or their ketal

derivatives respectively and cyclization with guanidine, urea or thiourea.

- 48 -

Scheme 6 Synthesis of pyrrolo[2,3-d]pyrimidines 29 and furo[2,3-d]pyrimidines 30.

Secrist and Liu184 provided a detailed study of the reaction of 2,6-diamino-4-

hydroxypyrimidine 16 with various -halo aldehydes and ketones (Scheme 6). They reported that

the cyclization occurred via two different route to produce either the pyrrolo[2,3-d]pyrimidine

and/or the furo[2,3-d]pyrimidine. Thus -halo ketones, chloroacetone 10 and 3-bromo-2-butanone

28, afforded both the furo[2,3-d]pyrimidine 30 and the pyrrolo[2,3-d]pyrimidine 29, whereas 16

afforded only the pyrrolo[2,3-d]pyrimidine 30 on reaction with 28. It was concluded that a critical

electron density is necessary at the C5 of the pyrimidine nucleus for it to react with the -carbon

atom of the -halo aldehydes or ketones to afford exclusively pyrrolo[2,3-d]pyrimidines.

- 49 -

Scheme 7 Synthesis of pyrrolo[2,3-d]pyrimidines 34.

Seela and Luepke185 first reported the synthesis of 6-substituted 2-amino-3,7-

dihydropyrrolo[2,3-d]pyrimidin-4-one 34 from 1,3-dioxolane-2-propanoic acid 33, α-cyano-2-

methyl-ethyl ester 31 and guanidine 32 (Scheme 7).

Gibson et al.186 also reported the synthesis of 5,6-disubstituted-pyrrolo[2,3-d]pyrimidine.

2-Chloro-2-cyanoethanal 36 was reacted with 35 to afford exclusively the 7-cyano-7-deazaguanine

37. Substituents at C-5 and C-6 were obtained by brominating the primary deazaguanine 37 with

N-bromosuccinimide in DMF to afford the 7-cyano-8-bromo derivative 39. These transformations

establish methods for the synthesis of a wide range of 5,6-disubstituted pyrrolo[2,3-d]pyrimidine.

Scheme 8. Synthesis of 5,6-disubstituted pyrrolo[2,3-d]pyrimidine 39.

- 50 -

Scheme 9 Synthesis of 2-amino-4-methyl pyrrolo[2,3-d]pyrimidine 45.

In 2000, Gangjee, et al.,187 reported the synthesis of 2-amino-4-methylpyrrolo[2,3-

d]pyrimidine 45 (Scheme 9) via a novel ring closure method. The synthesis started with

condensation of 2-acetylbutyrolactone 40 with guanidine carbonate 32 to afford the substituted

pyrimidine 41. Chlorination with POCl3 provided the dichloro compound 42 which was in turn

condensed with benzylamine to afford 2-amino-4-methyl-7-(N-benzyl)piperidinyl[2,3-

d]pyrimidine 43. Oxidative aromatization of this compound with MnO2 provided the N7-

benzylated compound 44. Compound 45 was obtained following debenzylation of 44 with metallic

sodium in ammonia.

In 2001, Gangjee, et al.,188 reported the synthesis of 2-(2-amino-4-oxo- 4,7-dihydro-1H-

pyrrolo[2,3-d]pyrimidin-6-yl)acetic acid 47 from the condensation of 2,6-diaminopyrimidin-

4(1H)-one 16 with ethyl 4-chloro-3-oxobutanoate 46 in the presence of sodium acetate (Scheme

10).

In another report of 2001, Gangjee, et al., also reported the synthesis of 2-amino-4-oxo-

4,7-dihydro-1H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile 49 (Scheme 11) from the condensation

- 51 -

of compound 16 with 2-chloro-3-oxopropanenitrile 48 under basic conditions.189


yl)acetic acid 47.

Scheme 11 Synthesis of 2-amino-4-oxo-4,7-dihydro-1H-pyrrolo[2,3-d]pyrimidine-5- carbonitrile

49.

In 1991, Miwa and coworkers190 reported a series of 2,4-diamino-5-arylalkyl substituted

classical pyrrolo[2,3-d]pyrimidine antifolates. In this synthetic approach (Scheme 12), the ring

system was constructed by the spontaneous lactam formation of the tert-butyl ester of (2,4,6-

triamino-pyrimidin-5-yl)-acetic acid which was in turn generated in situ by the condensation of

guanidine 32 and the malonodinitrile derivative 51. The resulting lactam 52 was subjected to

borane reduction to afford the pyrrolo[2,3-d] pyrimidine intermediate 54 along with its 5,6-dihydro

analog 53, which were separated by flash chromatography in 45% and 46% yields, respectively.

- 52 -

Scheme 12 Synthesis of pyrrolo[2,3-d]pyrimidine 54.

Taylor et al.191reported a novel synthesis of PMX (Scheme 13) which involved the

spontaneous cyclization of 6-amino-5-pyrimidylacetaldehydes 58 which in turn was generated

utilizing the Nef reaction with compound 58 onto the adjacent amino group. 2,6-Diamino-4-oxo-

pyrimidine, 16 was known to form Michael adducts at its unsubstituted C-5 position.192-196 The

synthesis of 58 thus involved a Michael addition reaction between the Michael acceptor 56 and

compound 16. The Michael acceptor 56 was in turn synthesized in three steps that involved a

palladium-catalyzed cross-coupling197 between methyl 4-iodobenzoate 56 and allyl alcohol, aldol

condensation with nitromethane followed by dehydration with methanesulfonyl chloride in the

presence of triethylamine.198 As expected, the condensation between 56 and 16 afforded the nitro

derivative 57 in high yield. Compound 58 was converted to 4-[2-(2-amino-4-oxo-7H-pyrrolo[2,3-

d]pyrimidin-5-yl]ethylbenzoic acid 59 by the Nef reaction in a one-pot, five-step procedure.

Conversion of acid 59 to PMX involved standard peptide coupling with diethyl L-glutamate using

2-chloro-4,6-dimethoxy-1,3,5-triazine as the coupling agent in presence of N-methylmorpholine

and final saponification.

- 53 -

Scheme 13 Synthesis of pyrrolo[2,3-d]pyrimidine 59 from 6-amino-5-pyrimidylacetalde-hydes 58.

Scheme 14 Synthesis of PMX from α-bromo aldehyde 64.

Barnett and coworkers also reported199 in 1999 a practical synthesis of PMX (Scheme 14)

which involved the cyclization of 2-bromo-4-arylbutanal 63 with 2,4-diamino-6-oxo-pyrimidine

16 regioselectively provided 5-substituted pyrrolo[2,3-d]pyrimidine 64 as shown in Scheme 14.

The key intermediate α-bromo aldehyde 63 was synthesized from aryl bromide 60 and alkyne 61

- 54 -

with Sonogashira coupling followed by reduction of the triple bond, oxidation to aldehyde and

corresponding bromination of the aldehyde 62.


Legraverend and coworkers200 reported the synthesis of pyrrolo[2,3-d]pyrimidines 71 from

2-amino-4,6-dichloro-5-(2,2-diethoxyethyl)pyrimidine 68 (Scheme 15). Thus, 5-allyl-2-amino-

4,6-dihydroxypyrimidine 65, prepared by the reaction of guanidine hydrochloride with diethyl

allylmalonate, was converted to the 4,6-dichloro derivative 66 by treatment with POCl3, and

diethylaniline in the presence of PCl5. The (2-amino-4,6-dichloropyrimidin-5-yl)acetaldehyde 67

was obtained from 66 by ozonolysis of the allyl group (Scheme 15). The diethylacetal derivative

69 of 68 was prepared by using standard methods. The acetal 69 was cyclized to 2-amino-4-chloro-

7-alkyl-7H-pyrrolo-[2,3-d]pyrimidine 70 by treatment with dilute aqueous HCl at room

temperature. Compound 71 was then obtained by hydrolysis of the 4-chloro group of 70 using 1

N HCl at 100 °C.

- 55 -

Kondo et al.201 reported the synthesis of 4-methyl pyrrolo[2,3-d]pyrimidine 74 via a

palladium(0) catalyzed cross-coupling of terminal acetylenes 73 with N-(5-halo-4-

pyrimidinyl)methane sulfonamides 72 (Scheme 16).

Scheme 16 Synthesis of 4-methyl pyrrolo[2,3-d]pyrimidines 74.

In the same report, Kondo and coworkers201 also reported the synthesis of 2,4-dimethyl

pyrrolo[2,3-d]pyrimidine 80 via a photoinduced or thermal cyclization of 4-azidopyrimidines 79

containing an olefinic functionality at the 5-position (Scheme 17). Intermediates 79 were obtained

by a palladium catalyzed cross-coupling between the 5-iodopyrimidine 75 and appropriate

stannanes 76, followed by nucleophilic displacement of the 4-chloro in pyrimidine 78 in the

presence of sodium azide.

Scheme 17 Synthesis of 2,4-dimethyl pyrrolo[2,3-d]pyrimidines 80

Sakamoto and coworkers202 reported in 1993 the synthesis of pyrrolo[2,3-d]pyrimidines 84

by utilizing an intramolecular cyclization of protected 5-acetaldehyde pyrimidines 83 (Scheme 18).

Compounds 83 were synthesized by palladium (0) catalyzed coupling of the appropriate 2,4-

disubstituted-5-bromo-6-acetamido pyrimidine 81 with (Z)-1-ethoxy-2-(tributylstannyl)ethane 82.

- 56 -

The same methodology was also applicable for the synthesis of pyrrolo[3,2-d]pyrimidines, except

that, 5-acetylamino-4-iodopyrimidine was used as the starting material.


Wright, et al.203 reported acid-catalyzed (Fisher indole cyclization) cyclization of 6-

(phenylhydrazino) uracils 86 to 9H-pyrimido[4,5-b]indole-2,4-diones 85. Several authors have

employed the Fischer indole cyclization of 4-pyrimidinylhydrazones 87 (Scheme 19) to afford the

pyrrolo[2,3-d]pyrimidine ring system 88.204-208The applicability of the Fischer-indole cyclization

to the synthesis of pyrrolo[2,3-d]pyrimidines, however is limited by the high reaction temperatures

and the steric constraints for the [3,3] sigmatropic rearrangement involved in the mechanism.

Scheme 19. Synthesis of pyrrolo[2,3-d]pyrimidines via Fischer indole cyclization.

Gangjee, et al.209 reported the synthesis of 2-amino-4-methylpyrrolo[2,3-d]pyrimidine.

- 57 -

Compound 89 (Scheme 20) and guanidine carbonate were refluxed with absolute ethanol in the

presence of triethylamine or sodium methoxide to afford 90. Compound 90 was converted to 91

by refluxing with phosphorus oxychloride. Condensation of benzylamine with 91 in the presence

of triethylamine under reflux in n-BuOH afforded the bicyclic compound 92. Compound 92 was

oxides to aromatic compound 93 using MnO2. Sodium in liquid ammonium at -78 °C afforded 94.

1.2 From Pyrroles precursors

in 1965, Taylor, et al.210 reported the synthesis of 4-amino-5-cyano-pyrrolo[2,3-

d]pyrimidine 100 (Scheme 21) starting from the tetracyanoethylene 95 via 2-mercapto-3,4-

dicyano-5-aminopyrrole 97.211 Reaction of 98 with triethylorthoformate followed by ammonia

results in the formation of a formamidine intermediate which then cyclizes to 99. The mercapto

group of 99 could be removed by Raney nickel (RaNi) to afford 100. Formamidine acetate 98 also

condensed with 97 to give 99 but in lower yields.

Scheme 20. Synthesis of pyrrolo[2,3-d]pyrimidines 94

- 58 -

Scheme 21 Synthesis of 4-amino-5-cyanopyrrolo[2,3-d]pyrimidine 100.

Scheme 22 Synthesis of 4-amino-5-substituted pyrrolo[2,3-d]pyrimidines 105

In the same report, Taylor, et al.210 synthesized 4-amino-5-methyl-pyrrolo[2,3-

d]pyrimidine 105 (Scheme 22) from 2-amino-3-cyano-5-substituted pyrroles 104 which in turn

were obtained from malonodinitrile 103 and the appropriate -aminoketones 101. Treatment of

pyrrole 104 with triethylorthoformate followed by ammonia resulted in the imidine intermediates

104 which underwent cyclization with sodium methoxide in pyridine to afford the pyrrolo[2,3-

- 59 -

d]pyrimidine 105. The fact that pyrroles 103, did not cyclize to pyrrolo[2,3-d]pyrimidine 105 in

ethanolic ammonia was attributed to the absence of the second nitrile group in the pyrrole 103.

Tolman and coworkers211 reported the synthesis of pyrrolo[2,3-d]pyrimidine 107 by

cyclization of 2-amino-5-bromo-3,4-dicyanopyrrole 106 with formamidine acetate (Scheme 23).

Ramasamy and coworkers212 have also utilized the pyrrole 106 to form the pyrrolo[2,3-

d]pyrimidine ring system for use in nucleoside synthesis. The versatility of the pyrrole 106

prompted Swayze et al213 to synthesize it in an efficient one-step reaction from tetracyanoethylene

95. On controlled addition of HBr in acetic acid 95 undergoes an intramolecular self-condensation

to afford 106.

Scheme 23 Synthesis of 5,6-disubstitutedpyrrolo[2,3-d]pyrimidine 107

Scheme 24 Synthesis of 2,5,6-trimethyl pyrrolo[2,3-d]pyrimidine 111

Eger and coworkers214,215reported the synthesis of 2,5,6-trimethylpyrrolo[2,3-d]pyrimidine

111 (Scheme 24) from 1-(1-phenylethyl)-2-amino-3-cyano-4,5-dimethylpyrrole 110 by heating

- 60 -

with a mixture of acetonitrile and sodium methoxide. The pyrrole 110 was in turn obtained by

cyclocondensation of 3-hydroxy-2-butanone 108, 1-phenyl-ethylamine 109 and malonodinitrile

102.

Chen, et al.216 reported an efficient synthesis of pyrrolo[2,3-d]pyrimidine 118 (Scheme 25).

Acetone 112 was condensed with malonodinitrile 102 to afford 113. Bromination of 113 using

NBS and benzoyl peroxide in chloroform afforded 114. Cyclization of 114 with aryl amine 115

afforded the substituted pyrrole intermediate 116. Compound 116 was then elaborated to the

pyrrolo[2,3-d]pyrimidine 118.

Scheme 25 Synthesis of 2,5,-dimethyl-N7-substitutedpyrrolo[2,3-d]pyrimidine 118.

Barnett, et al.217 reported the synthesis of a 2-amino-4-oxo-5,6-dihydropyrrolo[2,3-

d]pyrimidine 123 via a guanidine cyclization of a preformed 3-carbethoxy-2-thiopyrrolidine

- 61 -

intermediate 121 as the key step (Scheme 26). This intermediate was in turn prepared in several

steps from 4-propionaldehyde benzoic acid tert-butyl ester 120. Compound 122 was oxidized to

the pyrrolo[2,3-d]pyrimidine intermediate 123, 123 which was then elaborated to PMX in several

steps.

Scheme 26 Synthesis of pyrrolo[2,3-d]pyrimidine 123.

Taylor and coworkers218 reported the synthesis of a pyrrolo[2,3-d]pyrimidine analog of

PMX by a novel route (Scheme 27). A manganic triacetate dihydrate mediated radical cyclization

of racemic methyl N-crotyl-N-[1-(3,4-phenyl)-eth-1-yl)malonamide 128, afforded a

diastereomeric mixture of the 3-carbomethoxy-2-pyrrolidinone 129. Compound 128 was in turn

obtained by alkylation of racemic 1-(3,4-dimethoxy-phenyl)-ethylamine 124 with crotyl bromide

132 followed by a DMAP catalyzed acylation with methyl malonyl chloride 127. The

pyrrolidinone 129 was converted to the thiolactam 130 with P2S5 followed by cyclocondensation

with guanidine to afford the N7-protected 5,6-dihydro-5-allyl-pyrrolo[2,3-d]pyrimidine131.

- 62 -

Palladium catalyzed cross-coupling with diethyl-4-iodobenzoyl-L-glutamate fortuitously afforded

the ethano-bridged derivatives, and not the expected vinyl-bridged derivatives, by double bond

migration. This compound was then elaborated to analogs of PMX.

124 126

BrH3COOC COCl

DMAP/ Et3N

N

CH3

O

O

H3CO

N

CH3

N

CH3

HN

N

O

H2N

128

129 130 131

P2S5 Guanidine

H3COOCH3

NH2H3C

H3COOCH3

NHH3C

H3COOCH3

H3COOCH3

H3COOCH3

125 127

O

OH3CO

N

H3CO OCH3

CH3

S

OH3CO

Scheme 27 Synthesis of N7-substituted analogs of PMX 131.

Pedersen and coworkers219 reported the synthesis of 2,5-dimethyl pyrrolo[2,3-d]pyrimidine

136 (Scheme 28) from 2-acetylamino-3-cyano-4-methylpyrrole 135 by heating with 85%

phosphoric acid. The pyrrole 135 was in turn obtained by acetylation of 2-amino-3-cyano-4-

methylpyrrole 134 using acetic anhydride. Cyclocondensation of 132 or 133 with malonodinitrile

102 using sodium hydroxide afforded the precursor pyrrole 134.220,221

- 63 -

Scheme 28 Synthesis of 2,5-dimethylpyrrolo[2,3-d]pyrimidine 136.

In 2005, Bookser and coworkers222 reported the synthesis of pyrrolo[2,3-

d]pyrimidine 137 (Scheme 29) via the condensation between substituted pyrrole 138 and

triethylorthoformate 139 under acidic conditions.

Scheme 29 Synthesis of 5-substituted 2-des-4-oxo-pyrrolo[2,3-d]pyrimidine 139.

1.3 From Furans precursors

A general method to 2,5,6-trisubstituted-4-amino-pyrrolo[2,3-d]pyrimidines 143 was

reported by Taylor and coworkers223 (Scheme 30). Condensation of appropriate -hydroxyketones

140 with malonodinitrile 102 afforded the corresponding 2-amino-3-cyanofurans 141 which on

- 64 -

cyclization with amidines 142 afforded the corresponding pyrrolo[2,3-d]pyrimidines 143 by an

unexpected ring transformation/ring annulation sequence.

Scheme 30 Synthesis of 2,5,6-trisubstitutedpyrrolo[2,3-d]pyrimidines 143.

2. Sonogashira coupling in antifolate synthesis

Scheme 31 A general transformation of Sonogashira coupling.224, 225

In 1975, Sonogashira and coworkers226 reported the synthesis of symmetrically substituted

alkynes via a coupling reaction between acetylene gas and aryl iodides or vinyl bromides in the

presence of catalytic amounts of Pd(PPh3)Cl2 and CuI under mild conditions (Scheme 31). Thus,

the copper-palladium catalyzed coupling of terminal alkynes with aryl and vinyl halides to give

enynes is named the Sonogashira crosscoupling. Typically, two catalysts, a zerovalent palladium

complex and a halide salt of copper (I), are necessary for the reaction. Copper (I) salts, such as

copper (I) iodide, react with the terminal alkyne and produce a copper (I) acetylide, which acts as

an activated species to increase the rate of the coupling reactions.224 However, the copper-free

- 65 -

Sonogashira coupling of aryl iodides with terminal acetylenes has been developed recently.227-229

The Sonogashira coupling reaction also requires a base to neutralize the hydrogen halide produced

as the byproduct of this coupling reaction. The reactivity order of the aryl and vinyl halides is as:

I ≈ OTf > Br >> Cl.224, 225

Sonogashira coupling is believed to involve oxidative addition-reductive

elimination pathway (Scheme 32), although the mechanism is not clearly understood.

Scheme 32 Mechanism of Sonogashira cross-coupling.230

Gangjee and coworkers reported231 in 2007 the synthesis of N-(7-benzyl-4-methyl-5-

(phenylethynyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-N-pivaloylpivalamide 146 from N-(7-benzyl-

5-iodo-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-N-pivaloylpivalamide 144 and phenyl-

acetylene 145 via a Sonogashira cross-coupling in the presence of tetrakis (triphenylphosphine)

palladium(0) and CuI as catalysts in dichloromethane (Scheme 33).

- 66 -

Scheme 33 Synthesis of pyrrolo[2,3-d]pyrimidin-2-yl)-N-pivaloylpivalamide 146.

In 2010, Wang, et al.101 reported the synthesis of classical 2-amino-4-

oxo-6-substituted-pyrrolo[2,3-d]-pyrimidines 147a-c (Scheme 34) from terminal alkynes

149a-c and thiophenyl bromide 148 via a Sonogashira cross-coupling in the presence of

tetrakis(triphenylphosphine)palladium(0) and CuI as catalysts in DMF.

Scheme34 Synthesis of classical 2-amino-4-oxo-6-substituted-pyrrolo[2,3-d]-pyrimidines by

Sonogashira coupling.

- 67 -

3. Heck coupling reaction in one-step aldehyde synthesis

Scheme 35 General transformation of Heck coupling

The Heck coupling reaction (also called the Mizoroki-Heck reaction) is the

chemical reaction of an aryl or vinyl halide (or triflate) with an alkene and a base and

palladium catalyst to form a substituted alkene.231,232 Together with the other palladium catalyzed

cross-coupling reactions, this reaction is of great importance in modern organic

synthesis as reviewed.233,234 Richard F. Heck was awarded the 2010 Nobel Prize in

Chemistry for the discovery and development of this reaction. A General transformation

of Heck coupling is shown in Scheme 35.

Scheme 36 Heck coupling to synthesis aldehyde 152 and 153.

In 1968, Heck disclosed the formation of 3-aryl aldehydes and ketones by the reaction of

primary and secondary allylic alcohols with aryl palladium complexes prepared in situ from

arylmercuric chlorides or acetates and either an equimolecular amount of a palladium (II) salt, or

a catalytic amount of this salt with an equimolecular amount of copper (II) chloride to regenerate

the palladium after each reaction cycle.235 The teams of Heck and Chalk reported in 1976

- 68 -

simultaneously, but independently, a strong improvement in these couplings by disclosing that

such compounds are also obtained using aryl iodides or bromides and, furthermore, with a catalytic

amount of a palladium catalyst (Scheme 36). 236, As shown in Scheme 36, this reaction was carried

out with catalytic amount of palladium acetate at 100 C in MeCN for 0.5 hour to afford aldehydes

152 with good yield (60%) from phenyl iodide 150 and allyl alcohol 151. 236,237 This kind of Heck

coupling reactions with unsaturated alcohols has been widely used to synthesize aldehydes from

aryl halides in one step reaction.

A possible mechanism of this Heck coupling to synthesize aldehydes from phenyl iodide

and allyl alcohol was proposed in Scheme 37.

Scheme 37 A proposed mechanism of Heck coupling to synthesis aldehyde

- 69 -

In 1989, Larock and coworkers239 reported an improved Heck coupling to synthesis

aldehyde 152 with much better yield (90%) and lower temperature (70 C). (Scheme 38). This

reaction between phenyl iodide 150 and allyl alcohol 151 is carried out successfully with assistance

of palladium acetate as the key catalyst, Bu4NCl as the phase transfer catalyst and lithium acetate

as the base. The easy availability of the reactants and catalysts plused the excellent yield and mild

condition made this reaction very attractive to synthesize aldehydes as versatile intermediates.239-

241 However, the applications of this condition to heterocycle (such as thiophene and furan) halides

other than phenyl halides were not reported, which highly limited the application of this reaction.

239

Scheme 38 Improved Heck coupling to synthesis aldehyde 152

In 1977 Yoshida and coworkers reported242,243 a improved Heck coupling with thiophenyl

bromides as shown in Scheme 39. 2-Bromothiophene 154 was alkylated in the 2 position by

reaction with allylic alcohol 151 in the presence of Pd(OAc)2, NaI, NaHCO3 at 90 C under Ar2

for 4 h gave 76% 3-(thiophen-2-yl)propanal 155. Although this is a successful example of Heck

coupling with thiophenyl bromides, the drawbacks of this reaction are obvious: Only thiophenyl

bromide without any substitutions on the thiophenyl ring was demonstrated; the reaction tolerance

of different functional groups (e.g. –COOMe, -C=O) on the thiophene ring is not known; the

reaction condition is pretty hash: high temperature (90 C) and argon protection needed. Due to its

potential use in synthesis of classical antifolates and analogs such as RTX

- 70 -

and PMX,244 the mild conditioned and wide functional group tolerant Heck coupling of thiophenyl

halides with allyl alcohols to afford aldehydes in one step is highly attractive.

Scheme 39 Heck coupling with thiophenyl bromide 155

- 71 -

III. STATEMENT OF THE PROBLEM

1. Methylated (S)-2-({5-[4-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)-

alkyl]-thiophene-2-carbonyl}-amino)-pentanedioic acids regio-isomers (156 -159) as

GARFTase inhibitors with selectivity for FRs and/or PCFT over RFC.

Figure 16 The structures of classical 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine

antifolates (7,8 and 156-159).

Classical antifolates are all anions at physiologic pH, their ability to cross cell membranes

by passive diffusion is limited, so specific transporters are required for folates. There are three

major uptake systems involved in the transport of reduced folates system, all of which are shared

by antifolates.2-4 Reduced folate carrier is the major transport system for folates in mammalian

cells and tissues at physiologic pH.2-4 Folate receptor α and Folate receptor β are

glycosylphosphatidylinositol-anchored proteins that transport folates by receptor-mediated

endocytosis.5, 6 Finally, proton coupled folate transporter functions optimally at acidic pH.7, 8

- 72 -

Classical antifolates continue to serve a key role in the therapy of cancer as well as for

other diseases.245 Clinically relevant antifolates (Figure 1) such as methotrexate (MTX),

pemetrexed (PMX), and raltitrexed (RTX) are all substrates for the ubiquitously expressed reduced

folate carrier (RFC), which is the major folate transport system in tissues and tumors. RFC levels

and activities are important determinants of drug efficacy, and loss of RFC is a common mode of

antifolate drug resistance.62 However, RFC is also expressed in normal tissues and it is likely to

be a determinant of dose-limiting drug toxicity. Along with RFC, other folate membrane

transporters are recognized as important for physiologic functions in relation to in vivo folate

homeostasis and likely contribute to assorted clinical manifestations of folate deficiency. For

instance, FRα is expressed in epithelial tissues such as the renal tubules and cerebral

microvasculature where it is important in folate reabsorption and folate transport across the

bloodbrain barrier, respectively.6 A folate-proton symporter, PCFT is expressed in the upper small

intestine (e.g., jejunum) where it represents the primary mode of dietary folate absorption, and its

loss is causal in the autosomal recessive folate-deficient condition termed hereditary folate

malabsorption.4

Interestingly, these non-RFC transport mechanisms have also attracted attention for their

potentials in chemotherapy drug targeting, particularly for cancer, since FRs are expressed in a

subset of malignancies such as ovarian and endometrial cancers,65 and PCFT is present at high

levels in assorted solid tumors (including lung cancers, ovarian cancer, and hepatomas).246 Further,

PCFT is maximally active at pHs approximating those attained in the solid tumor

microenvironment.63 In response to the patterns of expression and function of FRs and PCFT in

tumors and normal tissues, new cytotoxic agents are being developed, by virtue of their

specificities for FRs and/or PCFT over RFC for selective tumor targeting.

- 73 -

Gangjee et al.101 recently reported 6-substituted classical pyrrolo[2,3-d]pyrimidines with

varying lengths of the carbon bridge region as cytotoxic tumor-targeted antifolates (Table 3).

Pyrrolo[2,3-d]pyrimidines and thieno[2,3-d]pyrimidines (Table 3)

Structure Compound Biological Activity (IC50) Ref.

HN

N NH

O

H2N

NH

O COOH

COOH

n

n = 1-6

3 n = 3 4 n = 4

hGARFTase 18 nM KB 1.7 nM IGROV1 2.2 nM hGARFTase 6.8 nM KB 1.9 nM IGROV1 3.6 nM

96

HN

N S

O

H2N

NH

O COOH

COOH

n

n = 2-8

5 n = 3 6 n = 4

hGARFTase 13.8 nM 5 KB 23 nM IGROV1 4.7 nM hGARFTase 13.3 nM 63 KB 4.9 nM IGROV1 5.9 nM

7 n = 3 8 n = 4

hGARFTase 0.69 nM KB 0.26 nM IGROV1 0.55 nM hGARFTase 1.96 nM KB 0.55 nM IGROV1 0.97 nM

These compounds were characterized by selective transport via FRα, FRβ and PCFT over

RFC. Three- and four-carbon bridge analogs were most active toward FR-expressing human

tumors and cytotoxicity was primarily due to the potent inhibition of GARFTase, the first folate-

dependent reaction in de novo purine nucleotide biosynthesis. The three-carbon pyrrolo[2,3-

d]pyrimidine derivative, 7 was subsequently reported122 to also be a substrate for PCFT, thus

providing an additional selective means of tumor targeting. Further structural optimization of

- 74 -

compounds such as 7, for improved drug efficacy due to selective cellular uptake by non-RFC

transport mechanisms, and as a sbustrate for folylpolyglutamate synthase for polyglutamylation

and binding to GARFTase to inhibit purine nucleotide biosynthesis, requires a systematic variation

of individual structural features. For example, although compound 7 is highly potent, its selective

tumor-targeting via FRs and PCFT is lower than compound 8 (Table 3).

Thus, it is of interest to further explore the structure−activity relationships (SAR) for this

series to determine if absolute selectivity over RFC along with high potency for FRα, FRβ, and/or

PCFT expressing cells over RFC would be possible. In this study, we consider the SAR for cellular

uptake and inhibition of cell proliferation for the pyrrolo[2,3-d]pyrimidine scaffold of 7 and 8 in

which the thienoyl ring was replaced by methylated thienoyl regioisomers. Our goal was to explore

the SAR for novel 6-substituted pyrrolo[2,3-d]pyrimidine methylated thienoyl antifolates related

to 7 and 8 (Figure 16), the most potent compounds of our series for inhibiting proliferation of FRα-

and PCFT-expressing cells with substantially reduced inhibitory effects toward RFC expressing

cells. Thus, 6-substituted pyrrolo[2,3-d]pyrimidine classical antifolates with methylated thienoyl

regioisomers in the side chain 156-159 (Figure 20) were designed and synthesized to evaluate the

effect of methylation in the side chain on transport by RFC, FR and PCFT and as inhibitors of

proliferation of human KB tumor cells (IC50) via inhibition of GARFTase. Methylated thienoyl

regioisomers analogues could provide metabolic stability and perhaps alter the conformation of

the molecule to allow FRα, FRβ, PCFT, and GARFTase and/or AICARFTase for better binding at

the required active sites.

1.1 Molecular Modeling Studies

- 75 -

Figure 17. (A) Superimposition of docked poses of 7(red) and 156 (green) in FRα (PDB:

5IZQ).12

Figure 17. (B) Superimposition of docked poses of 7(green) and 157 (red) in FRα (PDB:

5IZQ).12

Modeled using MOE 2014.08.

A

B

- 76 -

Figure 18. (A) Superimposition of docked poses of 7 (green) and 156 (red) in GARFTase (PDB:

4ZZ1).12

Figure 18. (B) Superimposition of docked poses of 7 (green) and 157 (red) in GARFTase (PDB:

4ZZ1).14 Modeled using MOE 2014.08.13

Figures 17 and 18 show the docked poses of methylated thienoyl regioisomers 156 and 157

in FRα (PDB: 5IZQ) and GARFTase (PDB ID: 4ZZ1), respectively. In FRα and GARFTase, 156

A

B

- 77 -

maintains the interaction patterns of the bicyclic scaffold and L-glutamate side chain moiety as 7.

The meta-methyl group in compound 157 decreases the distance (6.94 Å) between the scaffold

and the L-glutamate side chain moiety, when compareing with that of 7 (8.15 Å), resulting in the

loss of the interaction pattern of the L-glutamate side chain. In GARFTase, compound 157 with

the methyl group shifts away from the scaffold binding region, owing to the change in its linker

conformation (due to the steric clash with the methyl group). This results in a loss of the interaction

pattern of the bicyclic scaffold, when compared to the parent 7. Though the molecular modeling

of 157 predicts a lower activity for FRα, modeling in RFC and PCFT is not currently possible due

to an absence of crystal structures. Thus, it was of interest to determine the effects of methylation

on the thiophene ring to RFC and PCFT activity.

Figures 17 and 18 show the docked poses of methylated thienoyl regioisomers 156 and 157

in FRα (PDB: 5IZQ) and GARFTase (PDB ID: 4ZZ1), respectively. In FRα and GARFTase, 156

maintains the interaction patterns of the bicyclic scaffold and L-glutamate side chain moiety as 7.

The meta-methyl group in compound 157 decreases the distance (6.94 Å) between the scaffold

and the L-glutamate side chain moiety when compared with that of 7 (8.15 Å), resulting in the loss

of the interaction pattern of the L-glutamate side chain. In GARFTase, compound 157 with the

methyl group shifts away from the scaffold binding region, owing to the change in its linker

conformation (due to the steric clash with the methyl group). This results in a loss of the interaction

pattern of the bicyclic scaffold, when compared to the parent 7. Though the molecular modeling

of 157 predicts a lower activity for FRα, modeling in RFC and PCFT is not currently possible due

to an abance of crystal structures. Thus, it was of interest to determine the effects of methylation

on the thiophene ring to RFC and PCFT activity.

Figures 19 and 20 show the docked poses of methylated thienoyl regioisomers 158 and

- 78 -

159 in FRα (PDB: 5IZQ) and GARFTase (PDB ID: 4ZZ1), respectively. In FRα, 158, 159 retain

the interaction patterns of the bicyclic scaffold but loss of the interaction patterns of the L-

glutamate side chain moiety as 8. However, in GARFTase, both 158 and 159 overlap with 8 and

maintain the interaction patterns of the bicyclic scaffold and L-glutamate side chain moiety as 8.

Figure 19. (A) Superimposition of docked poses of 8 (yellow) and 158 (purple) in FRα (PDB:

5IZQ).12

Figure 19. (B) Superimposition of docked poses of 8 (yellow) and 159 (blue) in FRα (PDB:

5IZQ).

B

A

- 79 -

Figure 20. (A) Superimposition of docked poses of 8 (red) and 158 (green) in GARFTase (PDB:

4ZZ1).12

Figure 20. (B) Superimposition of docked poses of 8 (red) and 159 (yellow) in GARFTase (PDB:

4ZZ1).14 Modeled using MOE 2014.08.13

B

A

- 80 -

2. 6-substituted pyrrolo[2,3-d]pyrimidine with three atom chain length having a nitrogen in the

bridge and fluorinated benzoyl regioisomers as GARTFase inhibitors with selectivity for FRs

and/or PCFT over RFC.

Figure 21 6-substituted pyrrolo[2,3-d]pyrimidine with three atom chain length having nitrogen

bridge and fluorinated benzoyl regioisomers compounds design.

Gangjee et al.96 previously described novel 6-substituted pyrrolo[2,3-d]pyrimidine benzoyl

antifolates with selective cellular uptake by FRs and/or PCFT over RFC, resulting in potent

inhibitory activity toward human tumor cells.96 The most active analog of this series, 3 included

a 3-carbon bridge (Table 3), showed high level activity toward KB human tumors (IC50 = 1.7 nM)

expressing FRα and PCFT. Compound 4 has a 4-carbon atom bridge and showed better selectivity

than 3 for both FR and PCFT over RFC.63

- 81 -

The natural substrate for GARFTase, N10-formyl tetrahydrofolate (10-CHOTHF, Figure 3),

is a 6-substituted pteridine with a –CH2-N-2-atom bridge. The N10 a formyl (CHO) moiety forms

a hydroxylated, tetrahedral intermediate prior to transfer of the formyl group.129 Thus, replacement

of the benzylic CH2 of the 6-substituted pyrrolo[2,3-d] pyrimidine 3 (Table 3) with a NH would

afford “mimics” of the natural substrate side chain with a three-atom rather than two-atom bridge.

Gangjee et al.247 synthesized the 6-substituted three-atom bridged NH function as potent inhibitors

of human GARFTase rather than as substrates. The X-ray crystal structures of 161 248with FRα

and GARFTase showed that the bound conformations of 161 were different and required flexibility

for attachment to both FRα and GARFTase. Compound 161 showed high potency against FRα and

GARFTase, but its potency and selective tumor- targeting via PCFT is lower than 3.

Recently, Gangjee et al.249report that for 160 (Figure 21), fluorine incorporation on the

aromatic group adjacent to the L-glutamate amide had better selectivity, as well as potency, toward

FR- and PCFT-expressing CHO and KB cells than its des-fluorine compound 4 (Figure 21). We

proposed that fluorine substitution could improve the selectivity in antifolates through electrostatic

molecular interactions, such as those involving fluorine hydrogen bonding and dipole interactions.

Thus, it is of interest to further explore the SAR for this series to determine if fluorinated benzoyl

regioisomers 162 and 163 (Figure 21) could have absolute selectivity along with high potency for

FRα, FRβ, and/or PCFT over RFC.

2.1 Molecular Modeling

The docked poses of 162 (Figure 22A, 23A) in FRα and GARFTase retain the hydrogen

bonding and hydrophobic interactions seen in the docked pose of the lead compound 161. In

addition, the fluorine meta to the L-glutamate side chain in 162 facilitated the interaction with the

polar hydrophobic amino acid Phe 62 and results increased potency toward against FRα-expressing

- 82 -

cells. Comparing to the parent 162, fluorine ortho to the L-glutamate side chain in 163 (Figure 22B,

23B)results in a loss of interaction patterns of the L-glutamate region in FRα and GARFTase,

which causes a lower inhibitory activity toward cells expressing FRα. Alought the molecular

modeling predicts lower activity for FRα, modeling in RFC and PCFT is not currently possible

due to lack of crystal structures. Thus, it was of interest to determine the effects of fluorine on the

benzyl ring in relation to RFC, FRs and PCFT anti-proliferative activities.

Figure 22. (A) Superimposition of docked poses of 161 (red) and 162 (green) in FR(PDB:

5IZQ).247

Figure 22. (B) Superimposition of docked poses of 161 (red) and 163 (green) in FR(PDB:

5IZQ).247

Modeled using MOE 2014.08.12

B

A

- 83 -

Figure 23. (A) Superimposition of docked poses of 161 (green) and 162 (red) in GARFTase (PDB:

4ZZ1).247

Figure 23. (B) Superimposition of docked poses of AGF161 (green) and 163 (red) in GARFTase

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

fluorobenzoyl)-L-glutamic acid 167 as GARFTase inhibitors with potential selectivity for

FRs and/or PCFT over RFC

B

A

- 84 -

Figure 24 5-substituted pyrrolo[2,3-d]pyrimidine antifolates with three bridge carbons and

fluorinated benzoyl ring compound 167 as antitumor agent.

As mentioned above, pemetrexed (PMX) is clinically used for the treatment of

mesothelioma and non–small cell lung cancer (NSCLC) and is currently being evaluated for the

treatment of a variety of other solid tumors in the US.172 More recently, PMX has been approved

in combination with cisplatin as a first-line treatment for patients with locally advanced or

metastatic NSCLC other than squamous cell histology.174 PMX is a multitargeted antifolate that is

protected to several folate metabolism enzymes: TS, DHFR, AICARFTase and GARFTase.174

However, its potentional target is TS. PMX suffers from dose-limiting toxicity due to its transport

by RFC which is ubiquitously expressed in normal cells.248 To determine if altering the length of

the side chain of PMX would maintain the multitarget attributes of PMX and perhaps provide

selectivity for FR over RFC, Gangjee et al.249synthesized and evaluated the one to six carbon chain

homologs of PMX. Compound 164 and 166 (Figure 24) the 4- and 3- carbon chain homologs of

PMX, respectively have better KB cell inhibitiory potency than PMX. Compound 164 inhibits KB

cells at an IC50 of 3.6 nM, about 20-fold greater than PMX. However, 164 and 166 have poor

- 85 -

PCFT inhibition potency and poor KB tumor cell inhibitiory potency. Recently, Gangjee et al.247

reported that for 165 (Figure 24), with a fluorine in the benzene adjacent to the L-glutamate amide

had better potency for PCFT and FRs, as well as better selectivity, than its des-fluoro 164 (Figure

24). Based on this result, we designed and synthesized the fluorinated benzoyl analog 167 to

evaluate the effect of fluorine in the side chain on transport by RFC, FR and PCFT, and as

inhibitors of human KB tumor cells (IC50).

4. 5-Substituted pyrrolo[2,3-d]pyrimidine with two- to- four- bridge carbons and thienoyl

regioisomers 172-174 in the side chain as GARFTase inhibitors with selectivity for FRs

and/or PCFT over RFC.

Figure 25 5-substituted pyrrolo[2,3-d]pyrimidine antifolates with two- to- four- bridge carbons

and thienoyl regioisomers in the side chain as antitumor agents.

Gangjee and coworkers101 recently reported a series of 6-substituted pyrrolo[2,3-

d]pyrimidine thienoyl antifolates (Figure 25). The best characterized of this series, compound 8

included a 4- carbon bridge and was selectively transported into cells by FRs and PCFT (but not

RFC) whereupon it inhibited de novo purine biosynthesis at the level of GARFTase. Compound 8

- 86 -

was highly active toward both KB (IC50 = 3.17 nm) and IGROV1 (IC50 = 176 nm) tumors. In vivo

in SCID mice with KB tumor xenograft, 8 was highly active against both early and advanced stage

tumors. Based on the 5-substituted pyrrolo[2,3-d]pyrimidine structure of PMX and our results with

the 6-substituted compound 8, it was of interest to synthesize 5-substituted pyrrolo[2,3-

d]pyrimidine thienoyl analogs with 2 to 4- carbon atoms in the bridge (169-171) as hybrid

molecules of PMX and 8. These analogs were expected to afford the multi-targeted attributes of

PMX, while preserving FRα and/or PCFT specificity of the 6-substituted analogs previously

reported. However, 171 showed less activity against KB tumor cell, FRs and PCFT than 8.

Knowing that compound 863 is an excellent lead analog for further structure optimization for

inhibition of GARFTase and as for transport selectivity by FRs and/or hPCFT over RFC.

Compound 168 was designed to probe the importance of the regio-positions of the 4-carbon bridge

and the L-glutamate moiety on the side chain thienoyl ring of 8. The biological result showed that

168 had 10-fold more FRα activity than 8. Based on this result, compounds 172-174 were designed

to improve the potency and selectivity of 5-substituted pyrrolo[2,3-d]pyrimidine thienoyl

antifolates against FRs, PCFT and KB tumor cells.

IV. CHEMICAL DISCUSSION

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

pyrimidin-6-yl)-methyl]-thiophene-2-carbonyl}-amino)-pentanedioic acid isomers 156 and

157.

- 87 -

S Br

Xb

S

X

MeO

O

MeO

O

OH

S

X

MeO

O

OH

S

X

MeO

O

OH

Od

S

X

MeO

O

ClO

S

X

MeO

O

O N2

S

X

MeO

O

OBr

HN

N NH

H2N

O

a

ce

fg

S

YCOOR1

HN

N NH

H2N

O S

Y O

NHCOOR2

COOR2

189 X = H, Y= CH3, R1 = CH3 (41%) over 4 steps190 X = CH3, Y = H, R1 = CH3 (30%) over 4 steps

191 X = H, Y= CH3, R1 = H (95%)192 X = CH3, Y = H, R1 = H (97%)

193 X = H, Y= CH3, R2 = C2H5 (53%)194 X = CH3, Y = H, R2 = C2H5 (64%)

i

175 X = H, Y= CH3176 X = CH3, Y = H

Y

177 X = H, Y= CH3 (73%)178 X = CH3, Y = H (71%)

Y Y

179 X = H, Y= CH3 (84%)180 X = CH3, Y = H (88%)

181 X = H, Y= CH3 (94%)182 X = CH3, Y = H (83%)

Y Y

183 X = H, Y= CH3184 X = CH3, Y = H

185 X = H, Y= CH3186 X = CH3, Y = H

Y

187 X = H, Y= CH3188 X = CH3, Y = H

Y

X

h

156 X = H, Y= CH3, R2 = H (75%)157 X = CH3, Y = H, R2 = H (76%)

h

X

Scheme 40a Synthesis of classical three carbon atoms side chain methylated thiophene 2-amino-

4-oxo-6-substitutedpyrrolo[2,3-d]pyrimidine isomers 156,157.

a Conditions: (a) but-3-yn-ol, CuI, PdCl2, PPh3, Et3N, CH3CN, microwave,100 0C,10 min;

(b)10% Pd/C, H2, 55 psi, MeOH, overnight; (c) H5IO6, pyridinium chlorochromate, 0 0C, 3h; (d)

oxalyl chloride, CH2Cl2, reflux, 1h; (e) diazomethane, Et2O, RT, 1h; (f) HBr, reflux, 2h; (g) 2,6-

diamino-3H-pyrimidin-4-one, DMF, RT, 3 days; (h) i. 1 N NaOH, RT, overnight; ii. 1 N HCl; (i)

- 88 -

N-methylmorpholine, 2-chloro-4,6-dimethoxy-1,3,5-triazine, L-glutamate diethyl ester

hydrochloride, DMF, RT, overnight.

The synthesis of target compound 156 and 157 followed a modification of a reported

procedure101 as shown in Scheme 40. A palladium-catalyzed Sonogashira coupling of 5-bromo-3-

methylthiophene-2-carboxylate 175 and 5-bromo-4-methylthiophene-2-carboxylate 176, with but-

3-yn-1-ol respectively, afforded butynyl alcohols 177, 178 (73%, 71%) respectively, which were

catalytically hydrogenated to give the saturated alcohols 179, 180 respectively in quantitative yield.

Subsequent oxidation of 179 and 180 using Jones reagent afforded the carboxylic acids 181, 182

(94%, 83%) respectively, which were converted to the acid chlorides 183 and 184 and immediately

reacted with diazomethane followed by 48% HBr to give the desired α-bromomethyl ketones 187

and 188. Condensation of 2,6-diamino-3H-pyrimidin-4-one, with 187, 188 at room temperature

for 3 days afforded the 2-amino-4-oxo-6-substituted-pyrrolo[2,3-d]pyrimidines 189, 190 (41%,

30%) respectively. Hydrolysis of 189 and 190 afforded the corresponding free acids 191, 192 (95%,

97%). Subsequent coupling with L-glutamate diethyl ester using 2-chloro-4,6-dimethoxy-1,3,5-

triazine as the activating agent afforded the diesters 193, 194. Final saponification of the diesters

gave the desired compounds 156 and157.

In this study, one of the most important and difficult challenge is to characterize the

stuructures of these two regioisomers, compound156 and 157. Although the starting materials for

synthesis 156 and 157 are commercially available, it is still necessary to distinguish between

regioisomer 5-bromo-3-methylthiophene-2-carboxylate 175 and 5-bromo-4-methylthiophene-2-

carboxylate 176 (Figure 26).

- 89 -

Figure 26 Structure of 5-bromo-3-methylthiophene-2-carboxylate 175 and 5-bromo-4-

methylthiophene-2-carboxylate 176

Compound 175 and 176 have two electongeative founctional group are attached to the same

position of thiophene ring. These two electron withdrawing groups, bromine and carboxylic ester,

can affect chemical shift of the adjacent proton to be desheilded. The higher electonegativity

difference between H and its surrounding atoms results a higher chemical shift. This is because

the higher the electronegativity difference between H and another atom, the lower the electron

density is around the H (proton), and thus the more deshielding the proton experiences. In fact, the

electronegativity of the carboxylic ester is greater than bromine. Therefore, the proton ortho to the

bromine should have a lower chemical shift than the proton ortho to the carboxylic ester. The 1H

NMR data showed that the chemical shift of C3-H (δ 6.83 ppm) of 175 is less than the chemical

shif of the C3-H (δ 7.56 ppm) of 176. The chemical shift of the methyl group of 175 is greater than

the methyl group of 176. Based on this difference in 1H NMR chemical shifts in the thiophene ring,

the structure of 175 and 176 can be assigned.

In this study, the current synthetic method was optimized and simplified via synthetic steps,

separation procedure, shorte the reaction time and increased yield. Firstly, optimization the

reaction conditions of the palladium-catalyzed Sonogashira reaction. Without changing the

reaction regents, a microwave reaction replaced the high temperature reflux reaction (80 0C, 6-8h)

afforoded alcohols 177, 178 (73%, 71%) respectively. Comparing with the reflux condition,

microwave reaction shortened the Sonogashira reaction from 8 hours to 10 minutes. Instead of

- 90 -

generation multiple spots on TLC, this microwave reaction generated one clear spot. Therefore,

this microwave reaction significantly simplified the separation step and save cost of separation. In

addition, the microwave reacrion increased the yield from 54%, 58% to 73%, 71% respectively.

Since microwave reaction took 10 minutes to complete the Sonogashira coupling, it could be a

potential methodology for scale-up synthesis.

In the alcohol oxidation step, different reaction regents were selected to increase the yield

and shorton the reaction time. Instead of chromium trioxide as the oxidation regents (CrO3, H2SO4,

24h, rt), periodic acid with pyridinium chlorochromate (H5IO4, PCC, 3h, rt) was used to convert

the alcohol to the carboxylic acids 181, 182 (94%, 83%) respectively. This optimization shortened

the reaction time more than 21 hours and doubled the yield.

For the synthesis of the key intermediate α-bromomethyl ketones 187 and 188 step, fresh

diazomethane was used to convert acid chlorides 183 and 184 into the haloketones. Comparing

with the equivalent reagent trimethylsilyldiazomethane, freshly produced diazomethane works

much faster to generate the desired haloketone.The only limition of diazomethane is that it is

hazardous on an industrial scale without special precautions. However, it is a popular methylating

agent in the laboratory. It is universally used as a solution in diethyl ether and kept at lower

temperature.

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

d]pyrimidin-6-yl)butyl)thiophene-2-carbonyl)-L-glutamic acid isomers 158 and 159.

- 91 -

Scheme 41a Synthesis of classical four carbon atoms side chain methylated thiophene2-amino-4-

oxo-6-substituted pyrrolo[2,3-d]pyrimidine isomers 158,159.

a Conditions: (a) pent-4-yn-ol, CuI, PdCl2, PPh3, Et3N, CH3CN, microwave,100 0C,

10 min; (b)10%Pd/C, H2, 55psi, MeOH, overnight; (c) H5IO6, pyridinium chlorochromate, 0 0C,

3h; (d) oxalyl chloride, CH2Cl2, reflux, 1h; (e) diazomethane, Et2O, RT, 1h; (f) HBr, reflux, 2h;

(g) 2,6-diamino-3H-pyrimidin-4-one, DMF, RT, 3days; (h) i. 1N NaOH, RT, overnight; ii. 1 N

- 92 -

HCl; (i) N-methylmorpholine, 2-chloro-4,6-dimethoxy-1,3,5-triazine, L-glutamate diethyl ester

hydrochloride, DMF, RT, overnight.


procedure101as shown in Scheme 41. A palladium-catalyzed Sonogashira coupling of methyl 5-

bromo-3-methylthiophene-2-carboxylate 175 and methyl 5-bromo-4-methylthiophene-2-

carboxylate 176, with pent-4-yn-ol, afforded butynyl alcohols 195, 196 (72%, 78%) respectively,

which was catalytically hydrogenated to give the saturated alcohol 197, 198 in quantitative yield.

Subsequent oxidation of 197 and 198 using Jones reagent afforded the carboxylic acid 199, 200

(80%, 80%) respectively, which was converted to the acid chloride 201 and 202 and immediately

reacted with diazomethane followed by 48% HBr to give the desired α-bromomethylketone203

and 204 respectively. Condensation of 2,6-diamino-3H-pyrimidin-4-one, with 203, 204 at room

temperature for 3 days afforded the 2-amino-4-oxo-6-substituted-pyrrolo[2,3-d]pyrimidines 205,

206 (38%, 33%) respectively. Hydrolysis of 205, 206 afforded the corresponding free acid 207,

208 (81%, 87%) respectively. Subsequent coupling with L-glutamate diethyl ester using 2-chloro-

4,6-dimethoxy-1,3,5-triazine as the activating agent afforded the diesters 209, 210 respectively.

Final saponification of the diesters gave the desired compound 158 and 159.

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

fluorobenzoyl)-L-glutamic acid isomers 162 and 163.

The retro synthetic route A was proposed as shown in Scheme 42. From route A (Scheme

42), it was anticipatied that nucleophilic substitution of 217 and 218 would afford the ketone 216.

Ketone 216 could then can be converted to the α-bromoketone compound 215. Sequential

- 93 -

condensation with 2,6-diamino-3H-pyrimidin-4-one 214, hydrolysis, L-glutamate peptide

coupling, and saponification would afford the desired compound 213.

Scheme 42. Retro synthetic route A.

Synthesis of compounds 225 and 226 were attempted (Scheme 43). Compound 219 and

220 were reacted with the α, β- unsaturated methyl ketone 217 to afford 221 and 222 respectivly.

Compound 221, 222 reacted with 33% HBr in acetic acid and bromine afforded the α-bromoketo

compounds 223, 224 respectivly. Condensation of 223, 224 with compound 214 failed to provide

compounds 225, 226. TLC of the reaction shows multiple spots at Rf = 0.5 (5:1, Chloroform:

Methanol), separation of these spot could not be achieved. This could be because of the instability

of the α-bromomehtyl ketone in the presence of a secondary or tertiary nitrogen. Protection of the

nitrogen was considered as the next strategy to obtain the target compounds.

- 94 -

Scheme 43. Condensation recation between 223, 224 and 214.


procedure291as shown in Scheme 44. Commercially available methyl 4-amino-3- fluorobenzoate

219 and methyl 4-amino-2-fluorobenzoate 220 were reacted with methyl vinyl ketone through a

Michael addition to afford the β-keto amines 221 and 222 respectively. Selective bromination of

the α-carbon of 221 and 222 under acidic condition using 33% HBr in acetic acid/Br2 afforded the

α-bromoketone 223 and 224. Trifluroacetic anhydride was converted without protection of the α-

bromoketones 223 and 224. Subsequently, the protected amine was reacted with 2,6-diamino-3H-

pyrimidin-4-one in DMF at room temperature for 3 days to afford the 2-amino-4-oxo-6-

substituted-pyrrolo[2,3-d] pyrimidines 227 and 228. Hydrolysis of 227 and 228 gave the

corresponding free acids 229 and 230 respectively. Subsequent coupling with L-glutamate di- tert-

butyl ester using 2-chloro-4,6-dimethoxy-1,3,5-triazine as the activating agent afforded the di-tert-

butyl esters 231 and 232 respectively. Deprotection of di-tert-butyl ester using trifluroacetic acid

afforded the corresponding final compounds 162 and 163 respectively.

- 95 -

Scheme 44a Synthesis of classical 6-substituted pyrrolo[2,3-d]pyrimidine with a three atom chain

length with a nitrogen atom and fluorinated benzoyl regioisomers 162 and 163.

a Conditions: (a) but-3-en-2-one, ethanol, reflux, 5 h; (b) 33% HBr in CH3COOH, Br2, rt,

2.5 h; (c) (CF3CO)2O, rt, overnight; (d) 2,6-diaminopyrimidin-4(3H)-one, DMF, rt, 3 d; (e) 1N

NaOH, rt, 10 h; (f) N-methylmorpholine, 2-chloro-4,6-dimethoxy-1,3,5-triazine, L-glutamate

diethyl ester hydrochloride, DMF, RT, overnight; (g) CF3COOH, CH2Cl2, rt, 2 h

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

yl)propyl)-2-fluorobenzoyl)-L-glutamic acid 167.

- 96 -

Scheme 45. Synthetic route A of 167.

The retro synthetic route A was proposed as shown in Scheme 45. The proposed synthesis

followed a modification of a reported procedure.101A palladium-catalyzed Sonogashira coupling

of methyl 4-bromo-3-fluorobenzoate 233 with but-3-yn-1-ol 234, afforded alcohol 235, which was

catalytically hydrogenated to give the saturated alcohol 236. Subsequent oxidation of 236 afforded

the aldehyde 237. However, over three steps, the total yield of the aldehyde was less than 20%.

Starting from the Sonogashira coupling to afford aldehyde 237, usually takes three steps and more

than three days. To improve the yield and simplify the reaction, the synthesis of the aldehyde 237

was optimized by designing a Heck coupling instead of the Sonogashira coupling. Another

methodology optimization in the synthesis of 167 is to optimize the synthsis step of the key

intermediate α-bromoaldehyde 238. Bromine and 1,4-dioxane as a mild brominating agent was

used to convert the aldehyde 237 into the α-bromoaldehyde 238 (82%). Compared with other

brominating agents, such as 5,5-dibromo-2,2-dimethyl-1,3-dioxane-4,6-dione, this

- 97 -

dioxanedibromide shortened the bromination reaction from 36 hours to 3 hours and increased the

yield from 30% to 82%.

The synthesis of target compound 167 followed a modidification of a reported

procedure290as shown in Scheme 46. A palladium-catalyzed Heck coupling reaction of 233 with

pent-4-en-1-ol 234 in DMF 85 0C afforded the aldehyde 237. Reaction of 237 with bromine in

dioxane at 0 0C afforded the corresponding α-bromoaldehyde 238. The α-bromoaldehyde 238

reacted with 2,6-diamino-3H-pyrimidin-4-one in DMF at 45 0C in the presence of sodium acetate

to afford 5-substituted-pyrrolo[2,3-d]pyrimidine 239. Subsequent hydrolysis of 239 with 1 N

NaOH followed by coupling with L-glutamate dimethyl ester using N-methyl morpholine and 2,4-

dimethoxy-6-chlorotriazine as the activating agents afforded the diester 241. Final saponification

of the diester with 1 N NaOH gave the target compound 167.

- 98 -

Scheme 46a Synthesis of classical 5-substituted pyrrolo[2,3-d]pyrimidine antifolates with three

bridge carbons and fluorinated benzoyl 167.

a Conditions: (a) pent-4-en-1-ol, Pd(OAc)2, n-Bu4NCl, LiOAc, LiCl, ACN, 85 0C, 4h; (b)

Br2, Dioxane, 3h, 0C; (c) 2,6-diamino-3H-pyrimidin-4-one, CH3COONa, H2O/MeOH (1:1), 45

0C, 4h; (d) i. 1N NaOH, RT, overnight; ii. 1 N HCl; (e) N-methylmorpholine, 2-chloro-4,6-

dimethoxy-1,3,5-triazine, L-glutamate diethyl ester hydrochloride, DMF, RT, overnight. (d) i. 1N

NaOH, RT, overnight; ii. 1 N HCl;

5. The synthesis of (S)-5-substituted pyrrolo[2,3-d]pyrimidine with two- to- four bridge carbons

and thienoyl regioisomers 172-174.

- 99 -

Scheme 47a Synthesis of classical 5-substituted pyrrolo[2,3-d]pyrimidine antifolates 172-174.

a Conditions: (a) 3-buten-1-ol, 5-hexen-1-ol, pent-4-en-1-ol, Pd(OAc)2, n-Bu4NCl, LiOAc,

LiCl, ACN, 85 0C, 4h; (b) 5,5-dibromo-2,2-dimethyl-1,3-dioxane-4,6-dione, 2N HCl, 24 h, RT;

(c) 2,6-diamino-3H-pyrimidin-4-one, CH3COONa, H2O/MeOH (1:1), 45 0C, 4h; (d) i. 1N NaOH,

RT, overnight; ii. 1 N HCl; (e) N-methylmorpholine, 2-chloro-4,6-dimethoxy-1,3,5-triazine, L-

glutamate diethyl ester hydrochloride, DMF, RT, overnight. (d) i. 1N NaOH, RT, overnight; ii. 1

N HCl;

The synthesis of target compounds 172-174 followed a modidication of a reported

procedure290 as shown in Scheme 47. A palladium-catalyzed Heck coupling reaction of methyl 4-

bromothiophene-2-carboxylate 242 with 3-buten-1-ol, 5-hexen-1-ol, pent-4-en-1-ol in DMF 85 0C

afforded the aldehydes 243-245 respectively. Compounds 243-245 reacted with 5,5- dibromo-2,2-

dimethyl-4,6-dioxo-1,3-dioxane at room temperature afforded the corresponding α-bromo

- 100 -

aldehydes 246-248. Subsequently, the α-bromoaldehydes 246-248 were eacted with 2,6-diamino-

3H-pyrimidin-4-one in DMF at 45 0C in the presence of sodium acetate to afford 5-substituted-

pyrrolo[2,3-d] pyrimidines 249-251. Hydrolysis 249-251 with 1 N NaOH followed by coupling

with L-glutamate dimethyl ester using N-methyl morpholine and 2,4-dimethoxy-6-chlorotriaine as

the activating agents afforded the diester 255-257. Final saponification of the diester with 1 N

NaOH gave the target compounds 172-174.

- 101 -

V. SUMMARY

The design and synthesis of classical 6-substituted pyrrolo[2,3-d]pyrimidines and 5-

substituted pyrrolo[2,3-d]pyrimidines as potential antifolates have been described. As a part of

this study, a series of new compounds have been synthesized and characterized. Of these, over

ten compounds were submitted for biological evaluation.

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

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

yl)propyl)-3-methylthiophene-2-carbonyl)-L-glutamic acid 156

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

yl)propyl)-4-methylthiophene-2-carbonyl)-L-glutamic acid 157

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

yl)butyl)-3-methylthiophene-2-carbonyl)-L-glutamic acid 158

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

yl)butyl)thiophene-2-carbonyl)-L-glutamic acid 159

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

yl)ethyl)amino)-3-fluorobenzoyl)-L-glutamic acid 162

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

yl)ethyl)amino)-2-fluorobenzoyl)-L-glutamic acid 163


yl)propyl)-2-fluorobenzoyl)-L-glutamic acid 167


yl)ethyl)thiophene-2-carbonyl)-L-glutamic acid 172

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

yl)propyl)thiophene-2-carbonyl)-L-glutamic acid 173

- 102 -


yl)butyl)thiophene-2-carbonyl)-L-glutamic acid 174

During this study, a synthetic procedure for synthesizing 6-substituted pyrrolo[2,3-

d]pyrimidines was successfully exploited. In addition a synthetic method leading to novel 5-

substituted pyrrolo[2,3-d]pyrimidines was accomplished. The classic analog 157 showed excellent

potent activities for FRα, FRβ and PCFT, and high selectivity for FRα, FRβ and PCFT over RFC.

Compound 157 further demonstrated potent inhibition of tumor cells in culture (IC50 = 0.3 nM

toward KB cells).

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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. 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. All solvents

and chemicals were purchased from Aldrich Chemical Co. and Fisher Scientific and were used as

received.

- 104 -

Methyl 5-(4-hydroxybut-1-yn-1-yl)-3-methylthiophene-2-carboxylate 177.

To a 20-mL vial for microwave reaction, was added a mixture of palladium chloride (71

mg, 0.4 mmol), triphenylphosphine (131 mg, 0.4 mmol), copper iodide (304 mg, 1.6 mmol),

triethylamine (10.1 g, 100 mmol), methyl 5-bromo-4-methylthiophene-2-carboxylate 175 (2.35 g,

10 mmol) and anhydrous acetonitrile (10 mL). To the stirred mixture, was added but-3-yn-ol (1.05

g, 10.5 mmol), and the vial was sealed and placed into the microwave reactor at 100 0C for 10 min.

Then, silica gel (5 g) was added, and the solvent was evaporated to afford a plug under reduced

pressure. The resulting plug was loaded on to a silica gel column (3.5 × 12 cm) and eluted with

hexane followed by 40% EtOAc in hexane. The desired fraction (TLC) were pooled and

evaporated to afford 177 (1.63 g), yield 73% as yellow oil. TLC Rf = 0.37 (hexane/ EtOAc, 1:1); 1

H NMR (400 MHz, DMSO-d6) δ 2.43 (s, 3H, ArCH3), 2.60-2.62 (t, 2H, CH2CH2OH, J = 6.4 Hz),

3.57-3.59 (t, 2H, CH2CH2OH, J = 6.4 Hz), 3.79 (s, 3H, OCH3), 4.96-4.98 (t, 1H, OH, exch, J =

5.6 Hz), 7.16 (s, 1H, Ar).

Methyl 5-(4-hydroxybut-1-yn-1-yl)-4-methylthiophene-2-carboxylate 178.




10 mmol) and anhydrous acetonitrile (10 mL). To the stirred mixture, was added but-3-yn-ol (1.05

g, 10.5 mmol), and the vial was sealed and placed into the microwave reactor at 100 0C for 10 min.





- 105 -

H NMR (400 MHz, DMSO-d6) δ 2.23 (s, 3H, ArCH3), 2.63-2.66 (t, 2H, CH2CH2OH, J = 6.4 Hz),

3.57-3.60 (t, 2H, CH2CH2OH, J = 6.4 Hz), 3.80 (s, 3H, OCH3), 4.96-4.98 (t, 1H, OH, exch, J =

5.6 Hz), 7.63 (s, 1H, Ar).

Methyl 5-(4-hydroxybutyl)-3-methylthiophene-2-carboxylate 179

To a parr flask was added 177 (1.20 g, 7.14 mmol), 10% palladium on active carbon (50%

w/w), and MeOH (25 mL). Hydrogenation was carried out at 55 psi of H2 for overnight. The

reaction mixture was filtered through Celite, washed with MeOH (100 mL) and concentrated under

reduced pressure to give 179 (1.01 g,) yield 84% as yellow oil. TLC Rf = 0.35 (hexane/ EtOAc,

1:1); 1 H NMR (400 MHz, DMSO-d6) δ 1.44-1.49 (m, 2H, ArCH2CH2), 1.60-1.65 (m, 2H,

CH2CH2OH), 2.42 (s, 3H, ArCH3), 2.75-2.78 (t, 2H, ArCH2, J = 7.3 Hz), 3.40-3.42 (t, 2H, CH2OH,

J = 6.0 Hz), 3.75 (s, 3H, OCH3), 4.42-4.43 (t, 1H, OH, exch, J = 4.8 Hz), 6.83 (s, 1H, Ar).

Methyl 5-(4-hydroxybutyl)-4-methylthiophene-2-carboxylate 180

To a parr flask was added 178 (2.24 g, 10 mmol), 10% palladium on active carbon (50%



reduced pressure to give 180 (2.0 g,) yield 88% as yellow oil. TLC Rf = 0.35 (hexane/ EtOAc, 1:1);

1 H NMR (400 MHz, DMSO-d6) δ 1.45-1.50 (m, 2H, ArCH2CH2), 1.59-1.63 (m, 2H, CH2CH2OH),

2.13 (s, 3H, ArCH3), 2.72-2.76 (t, 2H, ArCH2CH2, J = 7.6 Hz), 3.39-3.42 (t, 2H, CH2OH, J = 6.0

Hz), 3.77 (s, 3H, OCH3), 4.42-4.44 (t, 1H, OH, exch, J = 5.2 Hz), 7.54 (s, 1H, Ar).

4-(5-(methoxycarbonyl)-3-methylthiophen-2-yl) butanoic acid 181

To MeCN (40 mL) was added H5IO6 (2.20 g, 9.65 mmol) and the mixture was stirred

vigorously at room temperature for 15 min. 179 (1 g, 4.38 mmol) was then added in iced-water

bath followed by addition of pyridinium chlorochromate (19 mg, 2 mol%) in MeCN (10 mL) and

- 106 -

the reaction mixture was stirred for 3 h. The reaction mixture was then diluted with EtOAc (100

mL) and washed with brine-water (1:1), sat. aq NaHSO3 solution, and brine, respectively, dried

over anhydrous Na2SO4 and concentrated to give the clean carboxylic acid 181 (1.0 g) yield 94%

as yellow oil. TLC Rf = 0.56 (hexane/ EtOAc, 1:1). 1 H NMR (400 MHz, DMSO-d6) δ 1.81-1.85

(m, 2H, ArCH2CH2), 2.25-2.29 (t, 2H, CH2COOH, J = 7.6 Hz), 2.43 (s, 3H, ArCH3), 2.77-2.80 (t,

2H, ArCH2, J = 7.6 Hz), 3.76 (s, 3H, OCH3), 6.84 (s, 1H, Ar), 12.15 (br, 1H, COOH, exch).

4-(5-(methoxycarbonyl)-3-methylthiophen-2-yl) butanoic acid 182


vigorously at room temperature for 15 min. 180 (673 mg, 2.95 mmol) was then added in iced-

water bath followed by addition of pyridinium chlorochromate (12.7 mg, 2 mol%) in MeCN (10

mL) and the reaction mixture was stirred for 3 h. The reaction mixture was then diluted with EtOAc

(100 mL) and washed with brine-water (1:1), sat. aq NaHSO3 solution, and brine, respectively,

dried over anhydrous Na2SO4 and concentrated to give the clean carboxylic acid 182 (590 mg)

yield 83% as yellow oil. TLC Rf = 0.55 (hexane/ EtOAc, 1:1). Mp 113.10C. 1 H NMR (400 MHz,

DMSO-d6) δ 1.79-1.82 (m, 2H, ArCH2CH2), 2.27-2.30 (m, 2H, CH2COOH), 2.13 (s, 3H, ArCH3),

2.74-2.76 (t, 2H, ArCH2, J = 7.6 Hz), 3.78 (s, 3H, OCH3), 7.55 (s, 1H, Ar), 12.14 (br, 1H, COOH,

exch).

Methyl 5-(3-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)propyl)-3-


To 181 (0.65 g, 2.69 mmol) in a 100 mL flask was added oxalyl chloride (2.0 g, 15.9 mmol)

and anhydrous CH2Cl2 (15 mL). The resulting solution was refluxed for 1 h and then cooled to the

room temperature. After evaporating the solvent under reduced pressure, the residue was dissolved

in 15 mL Et2O. The resulting solution was added dropwise to an ice-cooled diazomethane

- 107 -

(generated in stiu from 4.1 g of diazald by using Aldrich Mini Diazald Apparatus) in an ice bath

for 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 (6 mL). The resulting mixture was refluxed

for 1.5 h. After cooling to room temperature, the organic layer was separated, and aqueous layer

extracted with Et2O (3 × 50 mL). The organic layer was washed with 10% Na2CO3 solution and

dried over Na2SO4. To this residue anhydrous DMF (15 mL) was added 2,6-diamino-3H-

pyrimidin-4-one (338 mg, 2.68 mmol). The resulting mixture was stirred under N2 at room

temperature for 3 days. Silica gel (1.0 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 and then CHCl3 followed by 5% MeOH in CHCl3. The desired

fraction (TLC) was collected, and the solvent was evaporated under reduced pressure to

afford 189 (380 mg) yield 41% as yellow power. TLC Rf = 0.66 (CHCl3/MeOH, 6:1). Mp 189.3

0C. 1 H NMR (400 MHz, DMSO-d6) δ 1.90-1.96 (m, 2H, ArCH2CH2CH2Ar), 2.43 (s, 3H, CH3),

2.53-2.55 (t, 2H, ArCH2CH2CH2Ar, J = 7.6 Hz), 2.76-2.79 (t, 2H, ArCH2CH2CH2Ar, J = 7.6 Hz),

3.76 (s, 3H, COOCH3), 5.90 (s, 1H, C5-CH), 5.99 (s, 2H, 2-NH2, exch), 6.85 (s, 1H, Ar), 10.16 (s,

1H, 3-NH, exch), 10.85 (s, 1H, 7-NH, exch).

Methyl5-(3-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)propyl)-3-


To 182 (580 mg, 2.4 mmol) in a 100 mL flask was added oxalyl chloride (1.8 g, 14.4 mmol)



in 15 mL Et2O. The resulting solution was added dropwise to an ice-cooled diazomethane

(generated in stiu from 10 g of diazald by using Aldrich Mini Diazald Apparatus) in an ice bath

- 108 -





dried over Na2SO4. To this residue anhydrous DMF (15 mL) was added 2,6-diamino-3H-



reduced pressure. The resulting plug was loaded on to a silica gel column (1.5 × 12 cm) and eluted

with CHCl3 and then CHCl3 followed by 5% MeOH in CHCl3. The desired fraction (TLC) was

collected, and the solvent was evaporated under reduced pressure to afford 190 (250 mg) yield

30% as yellow power. TLC Rf = 0.63 (CHCl3/MeOH, 6:1). Mp 181.4 0C. 1 H NMR (400 MHz,

DMSO-d6) δ 1.88-1.92 (m, 2H, ArCH2CH2CH2Ar), 2.56-2.54 (t, 2H, ArCH2CH2CH2Ar, J = 7.6

Hz), 2.11 (s, 3H, ArCH3), 2.76-2.74 (t, 2H, ArCH2CH2CH2Ar, J = 7.6 Hz), 3.78 (s, 3H, OCH3),

5.90 (s, 1H, C5-CH), 6.00 (s, 2H, 2-NH2, exch), 7.66 (s, 1H, Ar), 10.17 (s, 1H, 3-NH, exch), 10.86

(s, 1H, 7-NH, exch).

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

methylthiophene-2-carboxylic acid 191

To a solution of 189 (370 mg, 1.06 mmol) in MeOH (15 mL) was added 1 N NaOH (10

mL) and the mixture was stirred under N2 at room temperature for overnight. TLC showed the

disappearance of the starting material and one major spot at the origin. The reaction mixture was

evaporated to dryness under reduced pressure. The residue was dissolved in water (10 mL). The

resulting solution was cooled in ice bath and 1 N HCl was added by dropwise to adjust pH to 3-4.

The resulting suspension was stored in a 4-5 0C refrigerator, and flitered. The residue was washed

- 109 -

with 20-40 mL cold water and dried in dried in vacuum using P2O5 to afford 191 (343 mg) yield

97% as yellow power. Mp 188.40C. 1 H NMR (400 MHz, DMSO-d6) δ 1.90-1.93 (m, 2H,

ArCH2CH2CH2Ar), 2.40 (s, 3H, ArCH3), 2.55-2.57 (t, 2H, ArCH2CH2CH2Ar, J = 7.6 Hz), 2.74-

2.77 (t, 2H, ArCH2CH2CH2Ar, J = 7.6 Hz), 5.90 (s, 1H, C5-CH), 6.01 (s, 2H, 2-NH2, exch), 6.80

(s, 1H, Ar), 10.17 (s, 1H, 3-NH, exch), 10.85 (s, 1H, 7-NH, exch), 12.74 (br, 1H, COOH, exch).

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


To a solution of 190 (346 mg, 1 mmol) in MeOH (15 mL) was added 1 N NaOH (10 mL)

and the mixture was stirred under N2 at room temperature for overnight. TLC showed the






95% as yellow power. Mp 181.4 0C. 1 H NMR (400 MHz, DMSO-d6) δ 1.87-1.90 (m, 2H,

ArCH2CH2CH2Ar), 2.55-2.57 (t, 2H, ArCH2CH2CH2Ar, J = 7.6 Hz), 2.13 (s, 3H, ArCH3), 2.72-

2.75 (t, 2H, ArCH2CH2CH2Ar, J = 7.6 Hz), 5.90 (s, 1H, C5-CH), 5.99 (s, 2H, 2-NH2, exch), 7.46

(s, 1H, Ar), 10.16 (s, 1H, 3-NH, exch), 10.86 (s, 1H, 7-NH, exch).

Diethyl (5-(3-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d] pyrimidin-6-yl)propyl)-4-

methylthiophene-2-carbonyl)glutamate 193

To a solution of 191 (330 mg, 1.0 mmol) in anhydrous DMF (10 mL) was added N-

methylmorpholine (180 mg, 1.8 mmol) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (314 mg, 1.8

mmol). The resulting mixtures were stirred at room temperature for 2 h. To this

- 110 -

mixture were added N-methylmorpholine (180 mg, 1.8 mmol) and L-glutamate diethyl ester

hydrochloride (360 mg, 1.5 mmol). The reaction mixture was stirred for overnight at room

temperature and then evaporated to dryness under reduced pressure. The residue was dissolved in

the minimum amount of CHCl3/MeOH (10:1) and chromatographed on a silica gel column (2 ×

15 cm) with 5% MeOH in CHCl3 as the eluent. Fractions that showed the desired spot (TLC) were

pooled, and the solvent evaporated to dryness to afford 193 (274 mg) yield 53 % as yellow power.

TLC Rf = 0.42 (CHCl3/ MeOH, 6:1); Mp 143.8 0C. 1 H NMR (400 MHz, DMSO-d6) δ 1.10-1.15

(m, 6H, COOCH2CH3), 1.82-1.84 (m, 2H, β-CH2), 1.87-1.93 (m, 2H, ArCH2CH2CH2Ar), 2.03 (s,

3H, CH3), 2.35-2.37 (t, 2H, J = 7.2 Hz, γ-CH2), 2.49-2.52 (t, 2H, ArCH2CH2CH2Ar, J = 7.6 Hz),

2.68-2.71 (t, 2H, ArCH2CH2CH2Ar, J = 7.6 Hz), 4.97-4.05 (m, 4H, COOCH2CH3), 4.30-4.34 (m,

1H, α-CH), 5.90 (s, 1H, C5-CH), 6.02 (s, 2H, 2-NH2, exch), 7.60 (s, 1H, Ar), 8.57-8.56 (d, 1H, J =

4.0 Hz, CONH, exch), 10.18(s, 1H, 3-NH, exch), 10.86 (s, 1H, 7-NH, exch).

Diethyl (5-(3-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d] pyrimidin-6-yl)propyl)-4-

methylthiophene-2-carbonyl)glutamate 194


methylmorpholine (230 mg, 2.28 mmol) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (400mg, 2.28

mmol). The resulting mixtures were stirred at room temperature for 2 h. To this






pooled, and the solvent evaporated to dryness to afford 194 (417 mg) yield 64% as yellow power.

- 111 -


(m, 6H, COOCH2CH3), 1.82-1.84 (m, 2H, β-CH2), 1.87-1.93 (m, 2H, ArCH2CH2CH2Ar), 2.03 (s,

3H, ArCH3), 2.35-2.37 (t, 2H, γ-CH2, J = 7.2 Hz), 2.40-2.44 (t, 2H, ArCH2CH2CH2Ar, J = 7.6 Hz),

2.70-2.74 (t, 2H, ArCH2CH2CH2Ar, J = 7.6 Hz), 4.04-4.11 (m, 4H, COOCH2CH3), 4.30-4.34 (m,

1H, α-CH), 5.90 (s, 1H, C5-CH), 5.98 (s, 2H, 2-NH2, exch), 7.60 (s, 1H, Ar), 8.55-8.57 (d, 1H,

CONH, exch, J = 4.0 Hz,), 10.15(s, 1H, 3-NH, exch), 10.86 (s, 1H, 7-NH, exch).

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

methylthiophene-2-carbonyl)glutamic acid 156

To a solution of 193 (90 mg, 0.17 mmol) in MeOH (20 mL) was added 1 N NaOH (10 mL).

The mixture was then stirred under N2 at room temperature overnight. TLC showed the

disappearance of the starting material and one major spot at the origin (CHCl3/ MeOH, 6:1). The

reaction mixture was then evaporated to dryness under reduced pressure. The residue was

dissolved in water (15 mL), the resulting solution was cooled in an ice bath, and the pH was

adjusted to 3-4 with dropwise addition of 1 N acetic acid. The resulting suspension was stored in

a 4-5 0C refrigerator and filtered. The residue was washed with 30-40 mL cold water and dried in

vacuum using P2O5 to afford 60 mg (76%) of 156 as a yellow powder. Mp 163.5 0C. 1 H NMR

(400 MHz, DMSO-d6) δ 1.90-2.08 (m, 4H, β-CH2), 2.32-2.34 (t, 2H, J = 7.2 Hz, γ-CH2), 2.36 (s,

3H, ArCH3), 2.54-2.59 (t, 2H, ArCH2CH2CH2Ar, J = 7.6 Hz), 2.71-2.73 (t, 2H, ArCH2CH2CH2Ar,

J = 7.6 Hz), 4.29-4.33 (m, 1H, α-CH), 5.91 (s, 1H, C5-CH), 5.99 (s, 2H, 2-NH2, exch), 6.74 (s, 1H,

Ar), 7.98-8.00 (d, 1H, J = 6.6 Hz, CONH, exch), 10.17 (s, 1H, 3-NH, exch), 10.85 (s, 1H, 7-NH,

exch). Anal. (C20H23N5O6S. 2.5573 H2O) C, H, N, S.

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

methylthiophene-2-carbonyl)glutamic acid 157

- 112 -






adjusted to 3-4 with dropwise addition of 1 N HCl. The resulting suspension was stored in a 4-5

0C refrigerator and filtered. The residue was washed with 30-40 mL cold water and dried in

vacuum using P2O5 to afford 105 mg (75%) of 157 as a yellow powder. Mp 167.8 0C. 1 H NMR

(400 MHz, DMSO-d6) δ 1.89-1.93 (m, 4H, β-CH2), 2.09 (s, 3H, ArCH3), 2.30-2.34 (t, 2H, γ-CH2,

J = 7.2 Hz), 2.54-2.58 (t, 2H, ArCH2CH2CH2Ar, J = 7.6 Hz), 2.71-2.73 (t, 2H, ArCH2CH2CH2Ar,

J = 7.6 Hz), 4.29-4.32 (m, 1H, α-CH), 5.89 (s, 1H, C5-CH), 5.97 (s, 2H, 2-NH2, exch), 7.58 (s, 1H,

Ar), 8.41-8.42 (d, 1H, CONH, exch, J = 8.0 Hz), 10.14(s, 1H, 3-NH, exch), 10.84 (s, 1H, 7-NH,

exch), 12.42 (br, 2H, COOH, exch). Anal. (C20H23N5O6S. 2.5573 H2O) C, H, N, S.

Methyl 5-(5-hydroxypent-1-yn-1-yl)-3-methylthiophene-2-carboxylate 195




10 mmol) anhydrous acetonitrile (10 mL). To the stirred mixture, was added pent-4-yn-ol (0.88 g,

10.5 mmol), and the vial was sealed and placed into the microwave reactor at 100 0C for 10 min.




evaporated to afford 195 (1.7 g), yield 71.4 % as yellow oil. TLC Rf = 0.57 (hexane/ EtOAc, 1:1);

- 113 -

1 H NMR (400 MHz, DMSO-d6) δ 1.64-1.71 (m, 2H, CH2CH2CH2OH), 2.43 (s, 3H, ArCH3), 2.51-

2.54 (t, 2H, CH2CH2CH2OH, J = 7.1 Hz), 3.47-3.51 (t, 2H, CH2CH2CH2OH, J = 6.2 Hz), 3.78 (s,

3H, COOCH3), 4.57 (s, 1H, OH, exch), 7.15 (s, 1H, Ar-H).

Methyl 5-(5-hydroxypent-1-yn-1-yl)-4-methylthiophene-2-carboxylate 196




10 mmol) and anhydrous acetonitrile (10 mL). To the stirred mixture, was added pent-4-yn-ol

(0.88 g, 10.5 mmol), and the vial was sealed and placed into the microwave reactor at 100 0C for

10 min. Then, silica gel (5 g) was added, and the solvent was evaporated to afford a plug under


with hexane followed by 40% EtOAc in hexane. The desired fraction (TLC) were pooled and


H NMR (400 MHz, DMSO-d6) δ 1.68-1.73 (m, 2H, CH2CH2CH2OH), 2.24 (s, 3H, ArCH3), 2.54-

2.58 (t, 2H, CH2CH2CH2OH, J = 7.2 Hz), 3.50-3.53 (t, 2H, CH2CH2CH2OH, J = 6.2 Hz), 3.80 (s,

3H, OCH3), 4.58 (Br, 1H, OH, exch), 7.64 (s, 1H, Ar-H).

Methyl 5-(5-hydroxypentyl)-3-methylthiophene-2-carboxylate 197

To a parr flask was added 195 (2.4 g, 9.67 mmol), 10% palladium on active carbon (50%



reduced pressure to give 197 g (1.90 g,) yield 82% as green oil. TLC Rf = 0.37 (hexane/ EtOAc,

1:1); 1 H NMR (400 MHz, DMSO-d6) δ 1.31-1.33 (m, 2H, ArCH2CH2CH2), 1.42-1.45 (m, 2H,

CH2CH2 OH), 1.59-1.63 (m, 2H, ArCH2CH2CH2), 2.42 (s, 3H, ArCH3), 2.74-2.78 (t, 2H, ArCH2,

- 114 -

J = 7.5 Hz), 3.35-3.38 (t, 2H, CH2OH, J = 6.0 Hz), 3.75 (s, 3H, OCH3), 4.35-4.37 (t, 1H, OH, exch,

J = 4.8 Hz), 6.82 (s, 1H, Ar-H).

Methyl 5-(5-hydroxypentyl)-4-methylthiophene-2-carboxylate 198

To a parr flask was added 196(600 mg, 2.5 mmol), 10% palladium on active carbon (50%



reduced pressure to give 198 (485 mg,) yield 80% as yellow oil. TLC Rf = 0.53 (hexane/ EtOAc,

1:1); 1 H NMR (400 MHz, DMSO-d6) δ 1.35-1.37 (m, 2H, Ar CH2CH2CH2CH2CH2OH), 1.43-

1.46 (m, 2H, ArCH2CH2CH2CH2CH2OH), 1.57-1.60 (m, 2H, ArCH2CH2CH2CH2CH2OH), 2.12

(s, 3H, ArCH3), 2.72-2.75 (t, 2H, ArCH2CH2, J = 7.5 Hz), 3.37-3.39 (t, 2H, CH2OH, J = 5.9 Hz),

3.78 (s, 3H, COOOCH3), 4.37-4.39 (t, 1H, OH, J = 5.0 Hz exch), 7.54 (s, 1H, Ar-H).

5-(5-(methoxycarbonyl)-4-methylthiophen-2-yl)-pentanoic acid 199


vigorously at room temperature for 15 min. 197 (1.90 g, 7.85 mmol) was then added in iced-water

bath followed by addition of pyridinium chlorochromate (34 mg, 2 mol%) in MeCN (10 mL) and

the reaction mixture was stirred for 3 h. The reaction mixture was then diluted with EtOAc (100

mL) and washed with brine-water (1:1), sat. aq NaHSO3 solution, and brine, respectively, dried

over anhydrous Na2SO4 and concentrated to give the clean carboxylic acid 199 (1.6 g) yield 80%

as yellow oil. TLC Rf = 0.56 (hexane/ EtOAc, 1:1). 1 H NMR (400 MHz, DMSO-d6) δ 1.51-1.62

(m, 4H, ArCH2CH2CH2CH2Ar), 2.22-2.26 (t, 2H, CH2COOH, J = 7.2 Hz), 2.42 (s, 3H, ArCH3),

2.75-2.79 (t, 2H, ArCH2, J = 7.3 Hz), 3.75 (s, 3H, OCH3), 6.83(s, 1H, Ar-H), 12.15 (br, 1H, COOH,

exch).

5-(5-(methoxycarbonyl)-3-methylthiophen-2-yl) pentanoic acid 200

- 115 -


vigorously at room temperature for 15 min. 198 (2.5 g, 10.3 mmol) was then added in iced-water

bath followed by addition of pyridinium chlorochromate (44.7 mg, 2 mol%) in MeCN (10 mL)

and the reaction mixture was stirred for 3 h. The reaction mixture was then diluted with EtOAc

(100 mL) and washed with brine-water (1:1), sat. aq NaHSO3 solution, and brine, respectively,

dried over anhydrous Na2SO4 and concentrated to give the clean carboxylic acid 200 (2.1 g) yield

80% as yellow oil. TLC Rf = 0.55 (hexane/ EtOAc, 1:1). 1 H NMR (400 MHz, DMSO-d6) δ 1.56-

1.61 (m, 4H, ArCH2CH2CH2CH2COOH), 2.13 (s, 3H, ArCH3), 2.24-2.27 (t, 2H, CH2COOH, J =

5.9 Hz), 2.74-2.77 (t, 2H, ArCH2, J = 6.9 Hz), 3.78 (s, 3H, COOCH3), 7.55 (s, 1H, Ar-H), 12.05

(br, 1H, COOH, exch).

Methyl 5-(4-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)butyl)-3-


To 199 (1.60 g, 6.25 mmol) in a 100 mL flask was added oxalyl chloride (4.85 g, 38.2

mmol) and anhydrous CH2Cl2 (15 mL). The resulting solution was refluxed for 1 h and then cooled

to the room temperature. After evaporating the solvent under reduced pressure, the residue was

dissolved in 15 of mL Et2O. The resulting solution was added dropwise to an ice-cooled

diazomethane (generated in stiu from 9.3 g of diazald by using Aldrich Mini Diazald Apparatus)

in an ice bath for 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 (13 mL). The resulting mixture

was refluxed for 1.5 h. After cooling to room temperature, the organic layer was separated, and

aqueous layer extracted with Et2O (3 × 50 mL). The organic layer was washed with 10% Na2CO3

solution and dried over Na2SO4. To this residue in anhydrous DMF (15 mL) residue and 2,6-

diamino-3H-pyrimidin-4-one (788 mg, 5.08 mmol) was added The resulting mixture was stirred

- 116 -

under N2 at room temperature for 3 days. Silica gel (1.0 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 and then CHCl3 followed by 5% MeOH in CHCl3. The desired

fraction (TLC) was collected, and the solvent was evaporated under reduced pressure to afford 207

(855 mg) yield 38% as yellow oil. TLC Rf = 0.50 (CHCl3/MeOH, 6:1). 1 H NMR (400 MHz,

DMSO-d6) δ 1.58-1.68 (m, 4H, ArCH2CH2CH2CH2Ar), 2.38-2.41 (t, 2H, ArCH2CH2CH2CH2Ar,

J = 9.3 Hz), 2.41 (s, 3H, ArCH3), 2.76-2.80 (t, 2H, ArCH2CH2CH2CH2Ar, J = 9.3 Hz), 3.75 (s,

3H, COOCH3), 5.86 (s, 1H, C5-CH), 5.99 (s, 2H, 2-NH2, exch), 6.81 (s, 1H, Ar-H), 10.16 (s, 1H,

3-NH, exch), 10.82 (s, 1H, 7-NH, exch).

Methyl 5-(4-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)butyl)-4-


To 200 (2.13 g, 8.3 mmol) in a 100 mL flask was added oxalyl chloride (4.7 g, 37.6 mmol)



in 15 of mL Et2O. The resulting solution was added dropwise to an ice-cooled diazomethane

(generated in stiu from 10 g of diazald by using Aldrich Mini Diazald Apparatus) in an ice bath





dried over Na2SO4. To this residue in anhydrous DMF (15 mL) was added 2,6-diamino-3H-



- 117 -


with CHCl3 and then CHCl3 followed by 5% MeOH in CHCl3. The desired fraction (TLC) was

collected, and the solvent was evaporated under reduced pressure to afford 208 (1. 01 g) yield 34%

as yellow power. TLC Rf = 0.53 (CHCl3/MeOH, 6:1). 1 H NMR (400 MHz, DMSO-d6) δ 1.57-

1.64 (m, 4H, ArCH2CH2 CH2CH2Ar), 2.12 (s, 3H, ArCH3), 2.51-2.54 (t, 2H, ArCH2CH2CH2Ar, J

= 6.9 Hz), 2.73-2.76 (t, 2H, ArCH2CH2CH2Ar, J = 6.9 Hz), 3.80 (s, 3H, COOCH3), 5.87 (s, 1H,

C5-CH), 6.06 (s, 2H, 2-NH2, exch), 7.45 (s, 1H, Ar-H), 10.21 (s, 1H, 3-NH, exch), 10.85 (s, 1H,

7-NH, exch).

5-(4-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)butyl)-3-ethylthiophene-

2-carboxylic acid 209

To a solution of 207 (360 mg, 1.0 mmol) in MeOH (15 mL) was added 1 N NaOH (10 mL)







81% as yellow oil. 1 H NMR (400 MHz, DMSO-d6) δ 1.53-1.63 (m, 4H, ArCH2CH2CH2CH2Ar),

2.40 (s, 3H, ArCH3), 2.47-2.51 (t, 2H, ArCH2CH2CH2CH2Ar, J = 7.6 Hz), 2.74-2.77 (t, 2H,

ArCH2CH2CH2CH2Ar, J = 7.6 Hz), 5.86 (s, 1H, C5-CH), 6.00 (s, 2H, 2-NH2, exch), 6.77(s, 1H,

Ar-H), 10.16 (s, 1H, 3-NH, exch), 10.81 (s, 1H, 7-NH, exch), 12.63 (br, 1H, COOH, exch).

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


- 118 -







with 20-40 mL cold water and dried in vacuum using P2O5 to afford 210 (330 mg) yield 87% as

yellow power. Mp 179.5 0C. 1 H NMR (400 MHz, DMSO-d6) δ 1.57-1.64 (m, 4H, ArCH2CH2

CH2CH2Ar), 2.12 (s, 3H, ArCH3), 2.51-2.54 (t, 2H, ArCH2CH2CH2Ar, J = 6.9 Hz), 2.73-2.76 (t,

2H, ArCH2CH2CH2Ar, J = 6.9 Hz), 5.87 (s, 1H, C5-CH), 6.06 (s, 2H, 2-NH2, exch), 7.45 (s, 1H,

Ar-H), 10.21 (s, 1H, 3-NH, exch), 10.85 (s, 1H, 7-NH, exch).

Diethyl (5-(4-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)butyl)-3-

methylthiophene-2-carbonyl)-L-glutamate 211



mmol). The resulting mixture was stirred at room temperature for 2 h. To this






pooled, and the solvent evaporated to dryness to afford 211 (260 mg) yield 53 % as yellow oil.

TLC Rf = 0.42 (CHCl3/ MeOH, 6:1). 1 H NMR (400 MHz, DMSO-d6) δ 1.15-1.21(m, 6H,

- 119 -

COOCH2CH3), 1.88-1.97 (m, 2H, β-CH2), 1.60-1.65 (m, 2H, ArCH2CH2CH2CH2Ar), 2.34 (s, 3H,

ArCH3), 2.38-2.42 (t, 2H, J = 7.2 Hz, γ-CH2), 2.49-2.52 (t, 2H, ArCH2CH2CH2CH2Ar, J = 7.6 Hz),

2.75-2.78 (t, 2H, ArCH2CH2CH2CH2Ar, J = 7.6 Hz), 4.06-4.11 (m, 4H, COOCH2CH3), 4.33-4.35

(m, 1H, α-CH), 5.87 (s, 1H, C5-CH), 5.99 (s, 2H, 2-NH2, exch), 6.70 (s, 1H, Ar-H), 8.13-8.15 (d,

1H, J = 4.0 Hz, CONH, exch), 10.18(s, 1H, 3-NH, exch), 10.86 (s, 1H, 7-NH, exch).

Diethyl (5-(4-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)butyl)-4-

methylthiophene-2-carbonyl)-L-glutamate 212


methylmorpholine (263 mg, 2.61 mmol) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (452mg,

2.61mmol). The resulting mixture was stirred at room temperature for 2 h. To this








(m, 6H, COOCH2CH3), 1.58-1.63 (m, 2H, β-CH2), 1.93-1.98 (m, 4H, ArCH2CH2CH2CH2Ar), 2.15

(s, 3H, ArCH3), 2.35-2.38 (t, 2H, γ-CH2, J = 7.2 Hz), 2.38-2.43 (t, 2H, ArCH2CH2CH2Ar, J = 7.6

Hz), 2.70-2.74 (t, 2H, ArCH2CH2 CH2CH2Ar, J = 7.6 Hz), 4.04-4.11 (m, 4H, COOCH2CH3), 4.30-

4.34 (m, 1H, α-CH), 5.86 (s, 1H, C5-CH), 5.97 (s, 2H, 2-NH2, exch), 7.58 (s, 1H, Ar), 8.55-8.57

(d, 1H, CONH, exch, J = 4.0 Hz,), 10.14(s, 1H, 3-NH, exch), 10.81 (s, 1H, 7-NH, exch).

- 120 -

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

methylthiophene-2-carbonyl)-L-glutamic acid 158


mL). The mixture was then stirred under N2 at room temperature overnight. TLC showed the




adjusted to 3-4 with dropwise addition of 1 N acetic acid. The resulting suspension was

stored in a 4-5 0C refrigerator and filtered. The residue was washed with 30-40 mL cold water and

dried in vacuum using P2O5 to afford 140 mg (81%) of 158 as a yellow powder.Mp 130.8 0C. 1 H

NMR (400 MHz, DMSO-d6) δ 1.60-2.62 (m, 4H, ArCH2CH2CH2CH2Ar), 1.88-1.96 (m, 2H, β-

CH2), 2.31-2.34 (t, 2H, J = 7.2 Hz, γ-CH2), 2.35 (s, 3H, ArCH3), 2.51-2.54 (t, 2H,

ArCH2CH2CH2CH2Ar, J = 7.6 Hz), 2.75-2.78 (t, 2H, ArCH2CH2CH2CH2Ar, J = 7.6 Hz), 4.28-

4.33 (m, 1H, α-CH), 5.86 (s, 1H, C5-CH), 5.98 (s, 2H, 2-NH2, exch), 6.74 (s, 1H, Ar), 7.97-7.99

(d, 1H, J = 6.6 Hz, CONH, exch), 10.14 (s, 1H, 3-NH, exch), 10.82 (s, 1H, 7-NH, exch), 12.25 (br,

2H, COOH, exch). Anal. (C21H25N5O6S. 0.9246 H2O. 0.7945CH3CO2H) C, H, N, S.

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

methylthiophene-2-carbonyl)-L-glutamic acid 159






- 121 -

adjusted to 3-4 with dropwise addition of 1 N HCl. The resulting suspension was


dried in vacuum using P2O5 to afford 87 mg (74%) of 159 as a yellow powder. Mp 190.2 0C. 1 H

NMR (400 MHz, DMSO-d6) δ 1.56-1.65 (m, 4H, ArCH2CH2CH2Ar), 1.87-1.93 (m, 2H, β-CH2),

1.99-2.09 (m, 2H, CH2), 2.12 (s, 3H, ArCH3), 2.35-2.32 (t, 2H, γ-CH2, J = 7.4 Hz), 2.54-2.71-2.75

(t, 2H, ArCH2CH2CH2CH2Ar, J = 7.2 Hz), 4.29-4.32 (m, 1H, α-CH), 5.87 (s, 1H, C5-CH), 5.98 (s,

2H, 2-NH2, exch), 7.58 (s, 1H, Ar-H), 8.43-8.45 (d, 1H, CONH, exch, J = 7.7 Hz), 10.15(s, 1H, 3-

NH, exch), 10.82 (s, 1H, 7-NH, exch), 12.42 (br, 2H, COOH, exch). Anal. (C20H23N5O6S. 1.2735

H2O. 0.8361 CH3CO2H) C, H, N, S.

Methyl 3-fluoro-4-((3-oxobutyl) amino) benzoate 221

To a solution of methyl 4-amino-3-fluorobenzoate 219 (10.14 g, 60 mmol) in in anhydrous

absolute ethanol (70 mL), methyl vinyl ketone (4.2 g, 60 mmol) was added. The reaction mixture

was stirred under reflux for 4.5 hours, and the resultant mixture was cooled to 0 °C. The

precipitated crystalline product was collected and washed with precooled ethanol and dried to

afford 221, yield 35 % as yellow power; Mp 98 °C; TLC Rf = 0.6 (hexane/ EtOAc, 1:1); 1 H NMR

(400 MHz, DMSO-d6) δ 2.21 (s, 3H, CH2COCH3), 2.80-2.83(t, 2H, CH2CH2O, J = 6.1 Hz), 3.51-

3.55 (t, 2H, CH2CH2O, J = 6.3 Hz), 3.88 (s, 3H, OCH3), 5.54 (bs, Ar-NH, exch), 6.65-6.70 (t, 1H,

Ar-CH, J = 8.4 Hz), 7.62-7.65 (d, 1H, Ar-CH, J = 7.0 Hz), 7.74-7.76 (d, 1H, Ar-CH, J = 7.0 Hz).

Methyl 2-fluoro-4-((3-oxobutyl)amino)benzoate 222

To a solution of methyl 4-amino-3-fluorobenzoate 220 (9.15 g, 50 mmol) in in anhydrous

absolute ethanol (10 mL), methyl vinyl ketone (3.4 g, 50 mmol) was added. The reaction mixture

was stirred under reflux for 4.5 hours, and the resultant mixture was cooled to 0 °C. The

precipitated crystalline product was collected and washed with precooled ethanol and dried to

- 122 -

afford 222 (6.9 g), yield 54 % as yellow power; Mp 86.8 0C. TLC Rf = 0.5 (hexane/ EtOAc, 1:1);

1 H NMR (400 MHz, DMSO-d6) δ 2.21 (s, 3H, CH2COCH3), 2.78-2.80 (t, 2H, CH2CH2O,

J = 5.9 Hz), 3.45-3.48 (t, 2H, CH2CH2O, J = 5.9 Hz), 3.88 (s, 3H, OCH3), 5.54 (bs, Ar-NH, exch),

6.27-6.30 (d, 1H, Ar-CH, J = 13.6 Hz), 6.37-6.39 (d, 1H, Ar-CH, J = 10.7 Hz), 7.78-7.80 (t, 1H,

Ar-CH, J = 8.5 Hz).

Methyl 4-((4-bromo-3-oxobutyl)amino)-3-fluorobenzoate 223

A suspension of 221 (8 g, 33.5 mmol) in 21 mL of 33% hydrogen bromide-glacial acetic

acid solution was treated with 2.6 mL of bromine in 5.24 mL of glacial acetic acid and stirred for

2.5 h at 25 °C. Ether (100 mL) was added to the reaction mixture with swirling until separation of

the syrupy hydrobromide was complete. The ether was decanted, and any ether remaining was

evaporated from the syrup along with excess hydrogen bromide. After further drying by

evaporation (bath 35 °C) of a solution in an equal volume of dichloromethane, the syrup

weighed 8.0 g. The syrup was then stirred with cold water and filtered to afford yellow solid.

Yellow solid obtained was then washed with water (3 × 20 mL) and dissolved in dichloromethane.

The solution was dried over sodium sulfate. Removal of solvent under reduced

pressure at room temperature gave a beige colored semisolid. The crude product was thoroughly

triturated with precooled ether, and the solid product was collected by filtration to afford 223 (4.2

g, yield 43%). 223 wasn’t stable. Used directly for the next step without purification.

Methyl 4-((4-bromo-3-oxobutyl)amino)-2-fluorobenzoate 224

A suspension of 222 (7 g, 29.0 mmol) in 18 mL of 33% hydrogen bromide-glacial acetic

acid solution was treated with 2.2 mL of bromine in 4.4 mL of glacial acetic acid and stirred for

2.5 h at 25 °C. Ether (100 mL) was added to the reaction mixture with swirling until separation of

the syrupy hydrobromide was complete. The ether was decanted, and any ether remaining was

- 123 -

evaporated from the syrup along with excess hydrogen bromide. After further drying by

evaporation (bath 35 °C) of a solution in an equal volume of dichloromethane, the syrup

weighed 3.3 g. The syrup was then stirred with cold water and filtered to afford yellow solid.

Yellow solid obtained was then washed with water (3 × 20 mL) and dissolved in dichloromethane.

The solution was dried over sodium sulfate. Removal of solvent under reduced

pressure at room temperature gave a beige colored semisolid. The crude product was thoroughly

triturated with precooled ether, and the solid product was collected by filtration to afford 224 (2.7

g) yield 33 % as yellow oil. 224 wasn’t stable. Used directly for the next step without purification.

Methyl 4-(N-(2-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)ethyl)-2,2,2-

trifluoroacetamido)-3-fluorobenzoate 227

A suspension of α-bromoketone 223 (3.15 g, 10 mmol) in trifluoroacetic anhydride (30 mL)

was stirred for overnight at room temperature and then evaporated to dryness under reduced

pressure to remove the excess trifluoroacetic anhydride. The residue was dissolved in

dichloromethane (30 mL). The reaction solution was washed with cold 2% HCl (100 mL), 5%

NaHCO3 (100 mL) and extraction with cold water (100 mL) and dried over Na2SO4. After

evaporation of solvent, the residue was dried in vacuo using P2O5 to afford amine protected α-

bromoketone 225 (3.1 g). 225 was then added to a suspension of 2,6-diaminopyrimidin-4-one (1.26

g, 10 mmol) in anhydrous DMF (25 mL). The resulting mixture was stirred under anhydrous

condition at room temperature for 3 days. After evaporation of the solvent under reduced pressure,

methanol (10 mL) was added followed by silica gel (5 g). The resulting plug was loaded on to a

silica gel column (3.5 cm × 12 cm) and eluted with CHCl3 followed by 5% MeOH in CHCl3 and

then 8% MeOH in CHCl3. The desired fraction (TLC) were pooled and evaporated to afford 227

(1.5 g), yield 34% as red power. Mp : 213 0C TLC Rf = 0.23 (CHCl3/ MeOH, 6:1); 1 H NMR (400

- 124 -

MHz, DMSO-d6) δ 2.76-2.80 (t, 2H, CH2CH2N, J = 7.3 Hz), 3.89 (s, 3H, OCH3), 4.09-4.12 (t, 2H,

CH2CH2N, J = 7.3 Hz), 5.96 (s, 2H, 2-NH2, exch), 6.02 (s, 1H, C5-CH), 7.51-7.55 (t, 1H, Ar-CH,

J = 7.9 Hz), 7.82-7.84 (d, 1H, Ar-CH, J = 8.3 Hz), 7.90-7.93 (d, 1H, Ar-CH, J = 10.2 Hz), 10.18

(s, 1H, 3-NH, exch), 10.83 (s, 1H, 7-NH, exch).

Methyl 4-(N-(2-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)ethyl)-2,2,2-

trifluoroacetamido)-2-fluorobenzoate 228

A suspension of α-bromoketone 224 (3.0 g, 10 mmol) in trifluoroacetic anhydride (30 mL)

was stirred for overnight at room temperature and then evaporated to dryness under reduced

pressure to remove the excess trifluoroacetic anhydride. The residue was dissolved in

dichloromethane (30 mL). The reaction solution was washed with cold 2% HCI (100 mL), 5%

NaHCO3 (100 mL) and extraction with cold water (100 mL) and dried over Na2SO4. After

evaporation of solvent, the residue was dried in vacuo using P2O5 to afford amine protected α-

bromoketone 226 (3.20 g, 10 mmol). 226 was then added to a suspension of 2,6-diaminopyrimidin-

4-one (1.26 g, 10 mmol) in anhydrous DMF (25 mL). The resulting mixture was stirred under

anhydrous condition at room temperature for 3 days. After evaporation of the solvent under

reduced pressure, methanol (10 mL) was added followed by silica gel (5 g). The resulting plug

was loaded on to a silica gel column (3.5 cm × 12 cm) and eluted with CHCl3 followed by 5%

MeOH in CHCl3 and then 8% MeOH in CHCl3. The desired fraction (TLC) were pooled and

evaporated to afford 228 (1.59g), yield 43 % as brown power. Mp 178.4 0C. TLC Rf = 0.23 (CHCl3/

MeOH, 6:1); 1 H NMR (400 MHz, DMSO-d6) δ 2.75-2.78 (t, 2H, CH2CH2N, J = 7.3 Hz), 3.89 (s,

3H, OCH3), 3.97-4.01 (t, 2H, CH2CH2N, J = 7.3 Hz), 5.95 (s, 1H, C5-CH), 6.02 (s, 2H, 2-NH2,

exch), 7.26-7.28 (d, 1H, Ar-CH, J = 10.1 Hz), 7.34-7.37 (d, 1H, Ar-CH, J = 10.1 Hz), 7.93-7.96

(t, 1H, Ar-CH, J = 8.1 Hz), 10.20 (s, 1H, 3-NH, exch), 10.85 (s, 1H, 7-NH, exch).

- 125 -

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

fluorobenzoic acid 229








86 % as yellow power. Mp 204.4 0C. 1 H NMR (400 MHz, DMSO-d6) δ 2.76-2.80 (t, 2H,

CH2CH2N, J = 7.3 Hz), 3.37-3.40 (t, 2H, CH2CH2N, J = 7.3 Hz), 5.99 (s, 2H, 2-NH2, exch), 6.02

(s, 1H, C5-CH), 6.34 (s, 1H, NH), 6.82-6.87 (t, 1H, Ar-CH, J = 8.5 Hz), 7.45-7.49 (d, 1H, Ar-CH,

J = 12.6 Hz), 7.59-7.62 (d, 1H, Ar-CH, J = 8.2 Hz), 10.20 (s, 1H, 3-NH, exch), 10.96 (s, 1H, 7-

NH, exch).


fluorobenzoic acid 230





resulting solution was cooled in ice bath and 1 N HCl was added by dropwise to adjust pH to 3-

4. The resulting suspension was stored in a 4-5 0C refrigerator, and flitered. The residue was

washed with 20-40 mL cold water and dried in dried in vacuum using P2O5 to afford 230 (264

- 126 -

mg) yield 84 % as brown power. Mp 213.3 0C. 1 H NMR (400 MHz, DMSO-d6) δ 2.74-2.77 (t,

2H, CH2CH2N, J = 7.2 Hz), 3.30-3.33 (t, 2H, CH2CH2N, J = 7.3 Hz), 6.03 (s, 2H, 1H, C5-CH),

6.12 (s, 2H, 2-NH2, exch), 6.34 (s, 1H, NH), 6.35-6.40 (d, 1H, Ar-CH, J = 14.5 Hz), 6.45-6.47

(d, 1H, Ar-CH, J = 10.6 Hz), 7.54-7.62 (t, 1H, Ar-CH, J = 8.8 Hz), 10.69 (s, 1H, 3-NH, exch),

11.26 (s, 1H, 7-NH, exch).

Di-tert-butyl (4-((2-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-

yl)ethyl)amino)-3-fluorobenzoyl)-L-glutamate 231




mixture were added N-methylmorpholine (483 mg, 4.8 mmol) and L-glutamate di-tert-butyl

hydrochloride (1.04 g, 3.53 mmol). The reaction mixture was stirred for overnight at room





TLC Rf = 0.63 (CHCl3/ MeOH, 6:1); Mp 202.3 0C. 1 H NMR (400 MHz, DMSO-d6) δ 1.39 (s, 9H,

C(CH3)3), 1.40 (s, 9H, C(CH3)3), 1.87 -2.04 (m, 2H, β-CH2), 2.30-2.34 (t, 2H, γ-CH2, J = 7.5 Hz),

2.77-2.81 (t, 2H, CH2CH2N, J = 7.5 Hz), 3.36-3.40 (t, 2H, CH2CH2N, J = 7.5 Hz), 4.22-4.31 (m,

1H, α-CH), 6.00 (s, 1H, NH), 6.02 (s, 2H, 2-NH2, exch), 6.08 (s, 1H, C5-CH), 6.82-6.87 (t, 1H,

Ar-CH, J = 8.6 Hz), 7.57-7.63(d, 2H, Ar-CH, J = 10.3 Hz), 8.26-8.28 (d, 1H, CONH, exch, J =

7.2 Hz), 10.19 (s, 1H, 3-NH, exch), 10.96 (s, 1H, 7-NH, exch).

- 127 -

Di-tert-butyl (4-((2-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-

yl)ethyl)amino)-2-fluorobenzoyl)-L-glutamate 232




mixture were added N-methylmorpholine (182 mg, 1.8 mmol) and L-glutamate di-tert-butyl

hydrochloride (442mg, 1.5 mmol). The reaction mixture was stirred for overnight at room




pooled, and the solvent evaporated to dryness to afford 232 (245 mg) yield 43% as brown power.

TLC Rf = 0.78 (CHCl3/ MeOH, 6:1); Mp 158.9 0C. 1 H NMR (400 MHz, DMSO-d6) δ 1.38 (s, 9H,

C(CH3)3), 1.40 (s, 9H, C(CH3)3), 1.86 -2.01 (m, 2H, β-CH2), 2.29-2.32 (t, 2H, γ-CH2, J = 7.5 Hz),


1H, α-CH), 5.99 (s, 1H, C5-CH), 6.01 (s, 2H, 2-NH2, exch), 6.39-6.42 (d, 1H, Ar-CH, J = 8.6 Hz),

6.45-6.49 (d, 1H, Ar-CH, J = 8.6 Hz), 6.61 (s, 1H, NH), 7.44-7.50 (t, 1H, Ar-CH, J = 8.8 Hz),

7.74-7.78 (t, 1H, CONH, exch, J = 7.2 Hz), 10.27 (s, 1H, 3-NH, exch), 11.20 (s, 1H, 7-NH, exch).


fluorobenzoyl)-L-glutamic acid 162

To a solution of 231 (109 mg, 0.19 mmol) in MeOH (10 mL) was added trifluoroacetic

acid (2 mL). The mixture was then stirred under N2 at room temperature for 2 h. TLC showed the



- 128 -

dissolved in 1 N NaOH, 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 stored in a 4-5 0C

refrigerator and filtered. The residue was washed with 10-20 mL cold water and dried in vacuum

using P2O5 to afford 60 mg (68%) of 162 as a yellow powder. Mp 222.9 0C. 1 H NMR (400 MHz,

DMSO-d6) δ 1.91 -2.04 (m, 2H, β-CH2), 2.32-2.36 (t, 2H, γ-CH2, J = 7.5 Hz), 2.77-2.82 (t, 2H,

CH2CH2N, J = 7.5 Hz), 3.36-3.40 (t, 2H, CH2CH2N, J = 7.5 Hz), 4.37-4.34 (m, 1H, α-CH), 6.00

(s, 1H, NH), 6.02 (s, 1H, C5-CH), 6.19 (s, 2H, 2-NH2, exch), 6.81-6.87 (t, 1H, Ar-CH, J = 8.6 Hz),

7.57-7.63(d, 2H, Ar-CH, J = 10.3 Hz), 8.29-8.31 (d, 1H, CONH, exch, J = 7.2 Hz), 10.33 (s, 1H,

3-NH, exch), 11.04 (s, 1H, 7-NH, exch). Anal. (C20H21FN6O6. 1.40447 H2O. 0.0582CF3COOH)

C, H, N, F.



To a solution of 232 (100 mg, 0.17 mmol) in MeOH (10 mL) was added trifluoroacetic

acid (2 mL). The mixture was then stirred under N2 at room temperature for 2 h. TLC showed the



dissolved in 1 N NaOH, 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


dried in vacuum using P2O5 to afford 60 mg (76%) of 163 as a yellow powder. Mp 187.3 0C. 1 H

NMR (400 MHz, DMSO-d6) δ1.86 -2.01 (m, 2H, β-CH2), 2.30-2.33 (t, 2H, γ-CH2, J = 7.3 Hz),


1H, α-CH), 6.00 (s, 1H, C5-CH), 6.14 (s, 2H, 2-NH2, exch), 6.38-6.42 (d, 1H, Ar-CH, J = 8.6 Hz),

- 129 -

6.48-6.50 (d, 1H, Ar-CH, J = 8.6 Hz), 6.61 (s, 1H, NH), 7.47-7.51 (t, 1H, Ar-CH, J = 8.8 Hz),

7.74-7.78 (t, 1H, CONH, exch, J = 7.2 Hz), 10.28 (s, 1H, 3-NH, exch), 11.00 (s, 1H, 7-NH, exch).

Anal. (C20H21FN6O6. 1.6967 H2O) C, H, N, F.

Methyl 3-fluoro-4-(5-oxopentyl) benzoate 237

To a solution of methyl 4-bromo-3-fluorobenzoate 233 (4.66 g, 20 mmol) in 20mL

anhydrous DMF was added pent-4-en-1-ol (2.06 g, 24 mmol), LiCl (840 mg, 20 mmol), LiOAc

(3.4 g, 50 mmol), n-Bu4NCl (3.36g, 10 mmol), Pd(OAc)2 (240 mg, 1.2 mmol) and the mixture was

stirred at 85 0C for 3 hours. Then, silica gel (5 g) was added, and the solvent was evaporated to

afford a plug 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)

were pooled and evaporated to afford 237 (2.38 g), yield 50 % as yellow oil. TLC Rf = 0.50

(hexane/ EtOAc, 1:1); 1 H NMR (400 MHz, DMSO-d6) δ 1.54-1.59 (m, 4H, ArCH2CH2CH2), 2.44-

2.47 (t, 2H, CH2CHO, J = 7.0 Hz), 2.62-2.65 (t, 2H, ArCH2CH2, J = 7.3 Hz), 3.82 (s, 3H, OCH3),

7.13-7.19 (t, 2H, Ar, J = 10.9 Hz), 7.77-7.81 (t, 1H, Ar, J = 7.8 Hz), 9.63-9.65 (s, 1H, CHO, J =

5.6 Hz).

Methyl 4-(4-bromo-5-oxopentyl)-3-fluorobenzoate 238

To a solution of aldehyde 237 (2.38 g, 10 mmol) in 10 mL dichloromethane was added the

Br2(0.66 g, 7.7 mmol), dioxane in dichloromethane solution dropwise and the mixture was stirred

at ice bath for 3 hours. TLC (hexane/ EtOAc, 1:1) showed the disappearance of the starting material

(Rf = 0.50) and formation of one major spot at Rf = 0.40 (hexane/ EtOAc, 1:1). The reaction solution

was washed with 5% NaHCO3 solution and extraction with H2O and dried over Na2SO4. After

evaporation of solvent the residue was dried in vacuo using P2O5 to afford 238 (3g). 238 wasn’t

stable. Used directly for the next step without purification.

- 130 -

Methyl 4-(3-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)propyl)-2-

fluorobenzoate 239

To a solution of 2,6-diamino-3H-pyrimidin-4-one 214 (1.25g, 10 mmol) and sodium

acetate (1.7 g, 20 mmol) in water (10 mL) and methanol (10 mL) was added α-bromo aldehyde

238 (3.17, 10 mmol). The reaction mixture was stirred at 45 0C for 3 hours. TLC showed the

disappearance of starting materials and the formation of one major spot at Rf = 0.73 (CHCl3/ MeOH,

6:1) After evaporation of solvent, CH3OH was added followed by silica gel (3 g). Evaporation of

the solvent afforded a plug, which was loaded onto a silica gel column (3.5 cm × 15 cm) and eluted

with CHCl3 followed by 8% MeOH in CHCl3. The desired fraction (TLC) were pooled and

evaporated to afford 239 (2.01g), yield 61 % as green power. Mp : 171.4 0C TLC Rf = 0.73 (CHCl3/

MeOH, 6:1); 1 H NMR (400 MHz, DMSO-d6) δ 1.91-1.95 (m, 2H, ArCH2CH2CH2), 2.56-2.59 (t,

2H, ArCH2CH2CH2Ar, J = 7.0 Hz), 2.64-2.68 (t, 2H, ArCH2CH2CH2Ar J = 7.3 Hz), 3.82 (s, 3H,

OCH3), 6.00 (s, 2H, 2-NH2, exch), 6.38 (s, 1H, C6-CH), 7.15-7.21 (t, 2H, Ar, J = 10.9 Hz), 7.77-

7.81 (t, 1H, Ar, J = 7.8 Hz), 10.14 (s, 1H, 3-NH, exch), 10.66 (s, 1H, 7-NH, exch).

4-(3-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)propyl)-2-fluorobenzoic

acid 240








- 131 -

81 % as green powder. Mp 202.0 0C. 1 H NMR (400 MHz, DMSO-d6) δ 1.91-1.96 (m, 2H,

ArCH2CH2CH2), 2.54-2.57 (t, 2H, ArCH2CH2CH2Ar, J = 7.6 Hz), 2.62-2.65 (t, 2H,

ArCH2CH2CH2Ar J = 7.4 Hz), 6.00 (s, 2H, 2-NH2, exch), 6.46 (s, 1H, C6-CH), 7.12-7.17 (t, 2H,

Ar, J = 7.9 Hz), 7.75-7.79 (t, 1H, Ar, J = 8.0 Hz), 10.67 (s, 1H, 3-NH, exch), 10.95 (s, 1H, 7-NH,

exch), 12.48 (br, 1H, COOH, exch)

Dimethyl (4-(3-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)propyl)-2-

fluorobenzoyl)-L-glutamate 241









pooled, and the solvent evaporated to dryness to afford 241 (131 mg) yield 46 % as yellow powder.


(m, 2H, ArCH2), 2.07-2.14 (m, 2H, β-CH2), 2.44-2.51 (t, 2H, γ-CH2, J = 7.2 Hz), 2.56-2.59 (t, 2H,

ArCH2CH2CH2Ar, J = 7.6 Hz), 2.64-2.67 (t, 2H, ArCH2CH2CH2Ar J = 7.5 Hz), 3.59 (s, 3H,

COOCH3, exch), 3.66 (s, 3H, COOCH3, exch), 4.43-4.48 (m, 1H, α-CH), 5.99 (s, 2H, 2-NH2, exch),

6.38 (s, 1H, C6-CH), 7.12-7.17 (t, 2H, Ar, J = 10.1 Hz), 7.50-7.54 (t, 1H, Ar, J = 8.0 Hz), 8.60-

8.62 (d, 1H, CONH, exch, J = 7.5 Hz,), 10.13 (s, 1H, 3-NH, exch), 10.66 (s, 1H, 7-NH, exch).

- 132 -

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









vacuum using P2O5 to afford 60 mg (77%) of 167 as a green powder. Mp 150.6 0C. 1 H NMR (400

MHz, DMSO-d6) δ 1.87-1.93 (m, 2H, ArCH2), 2.04-2.07 (m, 2H, β-CH2), 2.33-2.37 (t, 2H, γ-CH2,

J = 7.2 Hz), 2.56-2.59 (t, 2H, ArCH2CH2CH2Ar, J = 7.5 Hz), 2.64-2.67 (t, 2H, ArCH2CH2CH2Ar

J = 7.6 Hz), 4.37-4.41 (m, 1H, α-CH), 5.98 (s, 2H, 2-NH2, exch), 6.37 (s, 1H, C6-CH), 7.12-7.16

(t, 2H, Ar, J = 9.9 Hz), 7.51-7.54 (t, 1H, Ar, J = 7.5 Hz), 8.42-8.43 (d, 1H, CONH, exch, J = 6.8

Hz), 10.13(s, 1H, 3-NH, exch), 10.65 (s, 1H, 7-NH, exch), 12.48 (br, 2H, COOH, exch). Anal.

(C21H22FN5O6. 0.7908 CH3OH. 0.1795 HCl) C, H, N, F,Cl.

Methyl 4-(4-oxobutyl)thiophene-2-carboxylate 243

To a solution of methyl 4-bromothiophene-2-carboxylate 242 (4.42 g, 20 mmol) in 20mL

anhydrous DMF was added 3-buten-1-ol (1.75 g, 24 mmol), LiCl (840 mg, 20 mmol), LiOAc (3.4

g, 50 mmol), n-Bu4NCl (3.36g, 10 mmol), Pd(OAc)2 (240 mg, 1.2 mmol) and the mixture was




- 133 -

were pooled and evaporated to afford 243 (2.35 g), yield 55% as yellow oil. TLC Rf = 0.6 (hexane/

EtOAc, 1:1); 1 H NMR (400 MHz, DMSO-d6) δ 1.82-1.86 (m, 2H, ArCH2CH2CH2CHO), 2.42-

2.45 (t, 2H, ArCH2CH2CHO, J = 3.0 Hz), 2.58-2.60 (t, 2H, ArCH2CH2CH2CHO, J = 3.3 Hz), 3.81

(s, 3H, OCH3), 7.61 (s, 1H, Ar), 7.70 (s, 1H, Ar), 9.64-9.66 (t, 1H, CHO, J = 1.5 Hz).

Methyl 4-(5-oxopentyl)thiophene-2-carboxylate 244


anhydrous DMF was added 4-penten-1-ol (2.06 g, 24 mmol), LiCl (840 mg, 20 mmol), LiOAc

(3.4 g, 50 mmol), n-Bu4NCl (3.36g, 10 mmol), Pd(OAc)2 (240 mg, 1.2 mmol) and the mixture was




were pooled and evaporated to afford 244 (1.60 g), yield 35 % as yellow oil. TLC Rf = 0.6 (hexane/

EtOAc, 1:1); 1 H NMR (400 MHz, DMSO-d6) δ 1.50-1.60 (m, 4H, ArCH2CH2CH2CH2CHO),

2.46-2.48 (t, 2H, ArCH2CH2CH2CH2CHO, J = 3.0 Hz), 2.58-2.60 (t, 2H, ArCH2CH2CH2CH2CHO,

J = 7.2 Hz), 3.80 (s, 3H, OCH3), 7.58 (s, 1H, Ar), 7.68 (s, 1H, Ar), 9.64-9.66 (t, 1H, CHO, J = 1.5

Hz).

Methyl 4-(6-oxohexyl)thiophene-2-carboxylate 245


anhydrous DMF was added 5-hexen-1-ol (2.4 g, 24 mmol), LiCl (840 mg, 20 mmol), LiOAc (3.4

g, 50 mmol), n-Bu4NCl (3.36g, 10 mmol), Pd(OAc)2 (240 mg, 1.2 mmol) and the mixture was




- 134 -

were pooled and evaporated to afford 245 (2.16 g), yield 45 % as yellow oil. TLC Rf = 0.5 (hexane/

EtOAc, 1:1); 1 H NMR (400 MHz, DMSO-d6) δ 1.25-1.28 (m, 2H, ArCH2CH2CH2 CH2CH2CHO),

1.50-1.61 (m, 4H, ArCH2CH2CH2CH2CH2CHO), 2.41-2.45 (t, 2H, ArCH2CH2CH2CH2CH2CHO,

J = 7.4 Hz), 2.57-2.60 (t, 2H, ArCH2CH2, J = 7.6 Hz), 3.80 (s, 3H, OCH3), 7.58 (s, 1H, Ar), 7.68

(s, 1H, Ar), 9.65-9.66 (t, 1H, CHO, J = 1.6 Hz).

Methyl 4-(3-bromo-4-oxobutyl)thiophene-2-carboxylate 246

To a solution of aldehyde 243 (1.0 g, 4.72 mmol) in 10 mL of anhydrous Et2O was added

5,5-dibromo-2,2-dimethyl-1,3-dioxane-4,6-dione (0.71 g, 2.36 mmol) and 2N HCl (245 µL, 0.23

mmol) in Et2O solution and the mixture was stirred at room temperature for 24 hours. TLC

(hexane/ EtOAc, 1:1) showed the disappearance of the starting material (Rf = 0.60) and formation

of one major spot at Rf = 0.8 (hexane/ EtOAc, 1:1). The reaction solution was washed with 5%

NaHCO3 solution and extraction with H2O and dried over Na2SO4. After evaporation of solvent

the residue was dried in vacuo using P2O5 to afford 246 (1.26 g). Yield 91% as yellow oil. 246

wasn’t stable. Used directly for the next step without purification. 1 H NMR (400 MHz, DMSO-

d6) δ 2.72-2.74 (t, 2H, ArCH2CH2, J = 3.3 Hz), 2.79-2.81 (t, 2H, ArCH2CH2CHBr, J = 3.0 Hz),

3.81 (s, 3H, OCH3), 4.64-4.66 (m, 1H, CHBr), 7.67 (s, 1H, Ar), 7.74 (s, 1H, Ar), 9.42 (s, 1H,

CHO).

Methyl 4-(4-bromo-5-oxopentyl) thiophene-2-carboxylate 247

To a solution of aldehyde 244 (726 mg, 3.37 mmol) in 10 mL of anhydrous Et2O was added

5,5-dibromo-2,2-dimethyl-1,3-dioxane-4,6-dione (505 mg, 1.68 mmol) and 2N HCl (174 µL, 0.17




- 135 -


the residue was dried in vacuo using P2O5 to afford 247 (700 mg). 247 wasn’t stable. Used directly

for the next step without purification. 1 H NMR (400 MHz, DMSO-d6) δ1.50-1.60 (m, 2H,

ArCH2CH2), 2.72-2.74 (t, 2H, ArCH2CH2, J = 3.3 Hz), 2.79-2.81 (t, 2H, ArCH2CH2CH2CHBr, J

= 3.0 Hz), 3.81 (s, 3H, OCH3), 4.73-4.76 (m, 1H, CHBr), 7.61 (d, 1H, Ar), 7.69 (s, 1H, Ar), 9.42

(s, 1H, CHO).

Methyl 4-(5-bromo-6-oxohexyl)thiophene-2-carboxylate 248

To a solution of aldehyde 245 (2.4 g, 1.0 mmol) in 10 mL of anhydrous Et2O was added

5,5-dibromo-2,2-dimethyl-1,3-dioxane-4,6-dione (600 mg, 2 mmol) and 2N HCl (210 µL, 0.2





the residue was dried in vacuo using P2O5 to afford 248 (700 mg) yield 25 % as yellow oil. 248

wasn’t stable. Used directly for the next step without purification.

Methyl 4-(2-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl)thiophene-

2-carboxylate 249

To a solution of 2,6-diamino-3H-pyrimidin-4-one 214 (534 mg, 4.3 mmol) and sodium

acetate (740 mg, 9.0 mmol) in water (10 mL) and methanol (10 mL) was added α-bromo aldehyde

246 (1.26, 4.3 mmol). The reaction mixture was stirred at 45 0C for 3 hours. TLC showed the




- 136 -

with CHCl3 followed by 10 % MeOH in CHCl3. The desired fraction (TLC) were pooled and

evaporated to afford 249 (861mg), yield 63% as red power. Mp: 124.1 0C. TLC Rf = 0.5 (CHCl3/

MeOH, 6:1); 1 H NMR (400 MHz, DMSO-d6) δ 2.84-2.87 (t, 2H, ArCH2CH2Ar, J = 8.2 Hz),

2.92-2.95 (t, 2H, ArCH2CH2Ar, J = 8.2 Hz), 3.80 (s, 3H, OCH3), 6.00 (s, 2H, 2-NH2, exch), 6.33

(s, 1H, C6-CH), 7.54 (s, 1H, Ar), 7.65 (s, 1H, Ar), 10.15 (s, 1H, 3-NH, exch), 10.64 (s, 1H, 7-NH,

exch).

Methyl 4-(3-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-l)propyl)thiophene-

2-carboxylate 250

To a solution of 2,6-diamino-3H-pyrimidin-4-one 214 (1.03 g, 8.2 mmol) and sodium

acetate (1.4 g, 17.0 mmol) in water (10 mL) and methanol (10 mL) was added α-bromo aldehyde

247 (2.6 g, 8.5 mmol). The reaction mixture was stirred at 45 0C for 3 hours. TLC showed the




with CHCl3 followed by 10% MeOH in CHCl3. The desired fraction (TLC) were pooled and

evaporated to afford 250 (861mg), yield 63% as red power. Mp : 186.2 0C TLC Rf = 0.73 (CHCl3/

MeOH, 6:1); 1 H NMR (400 MHz, DMSO-d6) δ 1.92-1.95(m, 2H, ArCH2 CH2CH2Ar), 2.56-2.63

(m, 4H, ArCH2), 3.80 (s, 3H, OCH3), 6.00 (s, 2H, 2-NH2, exch), 6.37 (s, 1H, C6-CH), 7.60 (s, 1H,

Ar), 7.67 (s, 1H, Ar), 10.11 (s, 1H, 3-NH, exch), 10.64 (s, 1H, 7-NH, exch).

Methyl 4-(4-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)butyl)thiophene-

2-carboxylate 251

To a solution of 2,6-diamino-3H-pyrimidin-4-one 214 (1.15 g, 8.8 mmol) and sodium

acetate (1.56 g, 18.3 mmol) in water (10 mL) and methanol (10 mL) was added α-bromo aldehyde

- 137 -

248 (2.9 g, 9.14 mmol). The reaction mixture was stirred at 45 0C for 3 hours. TLC showed the




with CHCl3 followed by 10 % MeOH in CHCl3. The desired fraction (TLC) were pooled and

evaporated to afford 251 (2.12 g), yield 67% as red power. Mp 164.4 0C. TLC Rf = 0.5 (CHCl3/

MeOH, 6:1); 1 H NMR (400 MHz, DMSO-d6) δ 1.58-1.59(m, 4H, ArCH2CH2 CH2CH2Ar), 2.51-

2.61 (m, 4H, ArCH2), 3.80 (s, 3H, OCH3), 5.98 (s, 2H, 2-NH2, exch), 6.33 (s, 1H, C6-CH), 7.56 (s,

1H, Ar), 7.65 (s, 1H, Ar), 10.12 (s, 1H, 3-NH, exch), 10.62 (s, 1H, 7-NH, exch).

4-(2-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl)thiophene-2-

carboxylic acid 252

To a solution of 249 (318mg, 1.0 mmol) in MeOH (15 mL) was added 1 N NaOH (10 mL)





The resulting suspension was stored in a 4-5 0C refrigerator, and flited. The residue was washed


84% as red power. Mp 183.9 0C. 1 H NMR (400 MHz, DMSO-d6) δ 2.73-2.91 (m, 4H,

ArCH2CH2Ar), 6.00 (s, 2H, 2-NH2, exch), 6.34 (s, 1H, C6-CH), 7.43 (s, 1H, Ar), 7.56 (s, 1H, Ar),

10.11(s, 1H, 3-NH, exch), 10.65 (s, 1H, 7-NH, exch), 12.88 (br, 1H, COOH, exch)

4-(3-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)propyl)thiophene-2-

carboxylic acid 253

- 138 -








92 % as blue powder. Mp 167.9 0C. 1 H NMR (400 MHz, DMSO-d6) δ 1.89-1.92 (m, 2H, ArCH2

CH2CH2Ar), 2.57-2.65 (m, 4H, ArCH2), 5.99 (s, 2H, 2-NH2, exch), 6.36 (s, 1H, C6-CH), 7.45 (s,

1H, Ar), 7.65 (s, 1H, Ar), 10.11(s, 1H, 3-NH, exch), 10.65 (s, 1H, 7-NH, exch), 12.88 (br, 1H,

COOH, exch).


carboxylic acid 254








87% as red powder. Mp 153.2 0C. 1 H NMR (400 MHz, DMSO-d6) δ 1.52-1.64 (m, 4H, ArCH2

CH2CH2Ar), 2.55-2.64 (m, 4H, ArCH2), 6.13 (s, 2H, 2-NH2, exch), 6.35 (s, 1H, C6-CH), 7.49 (s,

- 139 -

1H, Ar), 7.56 (s, 1H, Ar), 10.23 (s, 1H, 3-NH, exch), 10.67 (s, 1H, 7-NH, exch), 12.88 (br, 1H,

COOH, exch).

Dimethyl (4-(2-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl)

thiophene-2-carbonyl)glutamate 255


methylmorpholine (137 mg, 1.35 mmol) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (241mg, 1.35







pooled, and the solvent evaporated to dryness to afford 255 (172 mg) yield 50% as red powder.


(m, 2H, β-CH2), 2.44-2.47 (t, 2H, γ-CH2, J = 7.2 Hz), 2.85-2.90 (m, 4H, ArCH2CH2Ar), 3.59 (s,

3H, COOCH3, exch), 3.65 (s, 3H, COOCH3, exch), 4.39-4.47 (m, 1H, α-CH), 6.06 (s, 2H, 2-NH2,

exch), 6.37 (s, 1H, C6-CH), 7.39 (s, 1H, Ar), 7.80 (s, 1H, Ar), 8.74-8.76 (d, 1H, CONH, exch, J =

7.5 Hz,), 10.21 (s, 1H, 3-NH, exch), 10.66 (s, 1H, 7-NH, exch).


carboxylic acid 257




- 140 -






pooled, and the solvent evaporated to dryness to afford 256 (320 mg) yield 64 % as red power. 256

is not very pure.

4-(4-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)butyl)thiophene-2-

carboxylic acid 257









pooled, and the solvent evaporated to dryness to afford 257 (320 mg) yield 63% as red powder.

(S)- (4-(2-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl)thiophene-2-

carbonyl)glutamic acid 172




- 141 -





vacuum using P2O5 to afford 172 82 mg yield 84% as red powder. Mp 169.0 0C. 1 H NMR (400

MHz, DMSO-d6) δ 1.84-2.16 (m, 2H, β-CH2), 2.28-2.34 (t, 2H, γ-CH2, J = 8.2 Hz), 2.83-2.95 (m,

4H, ArCH2CH2Ar), 4.29-4.41 (m, 1H, α-CH), 6.00 (s, 2H, 2-NH2, exch), 6.37 (s, 1H, C6-CH), 7.37

(s, 1H, Ar), 7.81 (s, 1H, Ar), 8.62-8.59 (d, 1H, CONH, exch, J = 10.8 Hz), 10.14 (s, 1H, 3-NH,

exch), 10.65 (s, 1H, 7-NH, exch), 12.48 (br, 2H, COOH, exch). Anal. (C18H19N5O6S. 0.596

CH3OH. 0.6232 HCl) C, H, N, S.

(S)- 4-(3-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)propyl)thiophene-

2-carbonyl)-L-glutamic acid 173








vacuum using P2O5 to afford 184 mg (83%) of 173 as a red powder. Mp 239.8 0C. 1 H NMR (400

MHz, DMSO-d6) δ 1.88 -1.94 (m, 2H, ArCH2CH2CH2), 2.04 -2.11 (m, 2H, β-CH2), 2.28-2.39 (t,

2H, γ-CH2, J = 8.2 Hz), 2.59-2.63 (m, 4H, ArCH2), 4.30-4.37 (m, 1H, α-CH), 5.99 (s, 2H, 2-NH2,

exch), 6.38 (s, 1H, C6-CH), 7.42 (s, 1H, Ar), 7.76 (s, 1H, Ar), 8.56-8.60 (d, 1H, CONH, exch, J =

- 142 -

10.8 Hz), 10.12 (s, 1H, 3-NH, exch), 10.66 (s, 1H, 7-NH, exch), 12.62 (br, 2H, COOH, exch).

Anal. (C19H21N5O6S. 2.2443 H2O) C, H, N, S.

(S)- 4-(4-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)butyl)thiophene-2-

carbonyl)-L-glutamic acid 174








vacuum using P2O5 to afford 190 mg (80%) of 174 as a green powder. Mp 146.1 0C. 1 H NMR

(400 MHz, DMSO-d6) δ 1.53 -1.59 (m, 2H, ArCH2CH2CH2), 1.88 -1.93 (m, 2H, ArCH2CH2CH2),

2.02 -2.07 (m, 2H, β-CH2), 2.30-2.35 (t, 2H, γ-CH2, J = 8.2 Hz), 2.56-2.65 (m, 4H, ArCH2), 4.30-

4.35 (m, 1H, α-CH), 5.98 (s, 2H, 2-NH2, exch), 6.38 (s, 1H, C6-CH), 7.39 (s, 1H, Ar), 7.75 (s, 1H,

Ar), 8.55-8.56 (d, 1H, CONH, exch, J = 10.8 Hz), 10.12 (s, 1H, 3-NH, exch), 10.62 (s, 1H, 7-NH,

exch), 12.62 (br, 2H, COOH, exch). Anal. (C20H23N5O6S. 0.333 CHCl3) C, H, N, S.

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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.

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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 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

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(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. 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, 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

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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 (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

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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 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

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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 1. IC50s (in nM) for 6-substituted pyrrolo[2,3-d]pyrimidine antifolates and classical antifolates in RFC-, PCFT-, and FR-expressing cell lines. Growth inhibition assays were performed for CHO sublines engineered to express human RFC (PC43-10), FRα (RT16), FRβ (D4), or PCFT (R2/PCFT4), for comparison with transporter-null [R2, R2(VC)] CHO cells and for the KB human tumor subline (expresses RFC, FRα, and PCFT), 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 3-10 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.

Comp

RFC FRα FRβ PCFT RFC/FRα/PCFT

PC43-10 R2 RT16 RT16 (+FA) D4

D4 (+FA) R2/PCFT4 R2(VC) KB

KB (+FA)

7 101.0(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

8 >1000 >1000 1.82(0.28) >1000 0.57(0.09 >1000 43.4(4.1) >1000 0.55(0.10) >1000 163 88.2 >1000 0.68 >1000 0.19 >1000 2.07 >1000 0.3 >1000 164 101.7 >1000 2.16 >1000 0.95 >1000 35.7 >1000 1.38 >1000 165 >1000 >1000 2.99 >1000 2.98 >1000 132 >1000 0.98 >1000 166 >1000 >1000 9.81 >1000 3.81 >1000 140 >1000 5.32 >1000 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)

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)

RTX 6.3(1.3) >1000 15(5) >1000 22(10) 746(138) 99.5(11.4) >1000 5.9(2.2) 22(5)

- 184 -

Table 2. IC50s (in nM) for 6-substituted pyrrrolo[2,3-d]pyrimidine benzoyl antifolates with heteroatom replacements, and classical antifolates in RFC-, PCFT- and FR-expressing cell lines. Proliferation assays were performed for CHO sublines engineered to express human RFC (PC43-10), FRα (RT16), FRβ (D4) or PCFT (R2/PCFT4), transporter-null [R2, R2(VC)] CHO cells,29, 46-48 and KB human tumor cells (express RFC, FRα, and PCFT). For the experiments measuring FR-mediated effects, assays were performed in the presence or absence of 200 nM folic acid (FA) (results are shown only for KB cells). Results are presented as IC50 values, corresponding to the concentrations that inhibit growth by 50% relative to cells incubated without drug. The data are mean values from 5-16 experiments (+/- standard errors in parentheses). Results are also summarized for KB cells for the protective effects of adenosine (60 µM), thymidine (10 µM), or 5-aminoimidazole-4-carboxamide (320 µM). Methods are summarized in the Experimental Section. Undefined abbreviations: Ade, adenosine; AICA, 5-aminoimidazole-4-carboxamide; FA, folic acid; ND, not determined;

Com


PC43-10 R2 RT16 D4 R2/PCFT4 R2(VC) KB KB (+FA) KB +

Ade/Thd/AICA

4 >1000 >1000 6.3 (1.6) >1000 213 (28) >1000 1.9 (0.7) >1000 Ade/AICA 160 62(12) 140(27) 1.12 (0.37) 3.87 (0.14) 3.82 (0.27) ND ND ND ND 161 510(90) >1000 3.04 (0.71) 0.62 (0.20) 87.4(9.9) >1000 0.32(0.05) 666(46) Ade/AICA 162 >1000 >1000 1.3 (0.8) 0.1 (0.02) 245 (102) >1000 0.21(0.04) ND ND 173 808 (81) >1000 4.1(1.4) 0.62 (0.04) 125 (15) >1000 3.81(0.92) ND ND

MTX 12(1.1) 114(31) 114 (31) 106 (11) 121(17) >1000 6.00(0.60) 20(2.4) Thd/Ade PMX 138(13) 42(9) 42 (9) 60 (8) 13.2(2.4) 974 (18) 68(12) 327(103) Thd/Ade RTX 6.3(1.3) 15(5) 15 (5) 22 (10) 99.5(11.4) >1000 5.90(2.20) 22(5) Thd

LMTX 12(2.3) 12(8) 12 (8) 2.6 (1.0) 38.0(5.3) >1000 1.20(0.60) 31(7) Ade/ AICA

- 185 -

Table 3. IC50s (in nM) for 5-substituted pyrrrolo[2,3-d]pyrimidine, and classical antifolates in RFC-, PCFT- and FR-expressing cell lines. Proliferation assays were performed for CHO sublines engineered to express human RFC (PC43-10), FRα (RT16), FRβ (D4) or PCFT (R2/PCFT4), transporter-null [R2, R2(VC)] CHO cells,29, 46-48 and KB human tumor cells (express RFC, FRα, and PCFT). For the experiments measuring FR-mediated effects, assays were performed in the presence or absence of 200 nM folic acid (FA) (results are shown only for KB cells). Results are presented as IC50 values, corresponding to the concentrations that inhibit growth by 50% relative to cells incubated without drug. The data are mean values from 5-16 experiments (+/- standard errors in parentheses). Results are also summarized for KB cells for the protective effects of adenosine (60 µM), thymidine (10 µM), or 5-aminoimidazole-4-carboxamide (320 µM). Methods are summarized in the Experimental Section. Undefined abbreviations: Ade, adenosine; AICA, 5-aminoimidazole-4-carboxamide; FA, folic acid; ND, not determined;

Com

RFC FRα PCFT RFC/FRα/PCFT

PC43-10 R2 RT16 RT16 (+FA)

R2/PCFT4 R2(VC) KB KB (+FA) KB +

Ade/Thd/AICA

164 56.6(5.8) >1000 8.6 (2.1) >1000 840(90) >1000 12.7 (5.4) ND Ade 165 >1000 >1000 0.58 >1000 75.4 ND 0.59 ND ND 166 68.8 (21.2) >1000 72.0 (27.1) >1000 329 (61) >1000 49.5 (13.2) 533(233) Ade 167 151 >1000 228 >1000 59 >1000 32.5 ND ND

MTX 12(1.1) 114(31) 114 (31) 106 (11) 121 (17) >1000 6.00(0.60) 20(2.4) Thd/Ade PMX 138(13) 42(9) 42 (9) 60 (8) 13.2(2.4) 974(18) 68(12) 327(103) Thd/Ade RTX 6.3(1.3) 15(5) 15 (5) 22 (10) 99.5(11.4) >1000 5.90(2.20) 22(5) Thd

LMTX 12(2.3) 12(8) 12 (8) 2.6 (1.0) 38.0(5.3) >1000 1.20(0.60) 31(7) Ade/ AICA

- 186 -

Table 4. IC50s (in nM) for 5-substituted pyrrrolo[2,3-d]pyrimidine, and classical antifolates in RFC-, PCFT- and FR-expressing cell lines. Proliferation assays were performed for CHO sublines engineered to express human RFC (PC43-10), FRα (RT16), FRβ (D4) or PCFT (R2/PCFT4), transporter-null [R2, R2(VC)] CHO cells,29, 46-48 and KB human tumor cells (express RFC, FRα, and PCFT). For the experiments measuring FR-mediated effects, assays were performed in the presence or absence of 200 nM folic acid (FA) (results are shown only for KB cells). Results are presented as IC50 values, corresponding to the concentrations that inhibit growth by 50% relative to cells incubated without drug. The data are mean values from 5-16 experiments (+/- standard errors in parentheses). Results are also summarized for KB cells for the protective effects of adenosine (60 µM), thymidine (10 µM), or 5-aminoimidazole-4-carboxamide (320 µM). Methods are summarized in the Experimental Section. Undefined abbreviations: Ade, adenosine; AICA, 5-aminoimidazole-4-carboxamide; FA, folic acid; ND, not determined;

Comp


PC43-10 R2 RT16 RT16 (+FA) D4

D4 (+FA) R2/PCFT4 R2(VC) KB

KB (+FA)

8 >1000 >1000 1.82(0.28) >1000 0.57(0.09 >1000 43.4(4.1) >1000 0.55(0.10) >1000 168 >1000 >1000 2.54(0.52) >1000 ND ND 41.6 (13.1) >1000 0.17(0.02) >1000 169 59.8(11.1) >1000 550 (50) ND ND ND 80.2(5.5) >1000 875 (125) ND

170 116.0(22.5

) >1000 109 (44) ND ND

ND 312 (90) >1000 211 (58) >1000

171 38.3(6.6) >1000 49.3(11.5) ND ND ND 141(40) >1000 66.0(14.4) >1000 172 ND ND ND ND ND ND ND ND ND ND 173 >1000 >1000 >1000 ND ND ND >1000 >1000 320 ND 174 >1000 >1000 >1000 ND ND ND 125 >1000 260 ND

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)

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

) RTX 6.3(1.3) >1000 15(5) >1000 22(10) 746(138) 99.5(11.4) >1000 5.9(2.2) 22(5)

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Synthesis of Novel 6-Substituted and 5-Substituted Pyrrolo ...

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