SOLID-PHASE SYNTHESIS OF PURINE DERIVATIVES

FU HAN

(M.Sc., FUDAN UNIVERSITY)

A THESIS SUBMITTED

FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

DEPARTMENT OF CHEMISTRY

NATIONAL UNIVERSITY OF SINGAPORE

2006

ACKNOWLEDGEMENTS

A very special thank you to my supervisor, Dr. Lam Yulin for her guidance,

encouragement and patience, which have been a tremendous help for me throughout the

entire course of my Ph.D. study. She showed me her wide knowledge and stimulating

suggestions during many hours of discussions we had. And most of all she gave me

untiring help during my difficult moments.

I would like to gratefully acknowledge the support of Dr. Teresa Tan in Dept. of

Biochem., who gave me the chance to do the biological test in her lab. My special thanks

to Yang Fei for her help on biological experiments. And I also want to thank Dr. Go Mei

Lin and Leng Zhijin for their help on microwave-assistant reactions.

I also wish to thank all my group members, Madam Liang Eping, Kong Hah Hoe, Mark

Tan Kheng Chuan, Makam Shantha Kumar Raghavendra, He Rongjun, Gao Yongnian,

and Soh Chai Hoon⎯for all the help and interesting hints. Their support has been great.

I want to express my gratitude to Han Yanhui and Peggy Ler, for their help with

performing NMR spectra analyses. And thanks to Wong Lai Kwai and Lai Hui Ngee,

who have helped me with mass spectral analyses.

I am deeply indebted to my husband, whose patience and understanding I am very

thankful for. My deepest gratitude is reserved for my parents for their long-distance

support and love.

I would like to thank National University of Singapore for awarding me a research

scholarship to pursue my doctorate degree.

i

TABLE OF CONTENTS

TABLE OF CONTENTS i

SUMMARY iv

LIST OF TABLES vi

LIST OF FIGURES vii

LIST OF ABBREVIATIONS ix

LIST OF PUBLICATIONS xiv

CHAPTER 1: INTRODUCTION 1

1.1 Solid-phase synthesis (SPS) 1

1.1.1 Solid supports 2

1.1.2 Linkers 3

1.1.3 Reaction monitoring in solid-phase synthesis 13

1.1.4 Solid-phase synthetic libraries---from peptides to small organic molecules 13

1.2 Solid-phase synthesis of purine 15

1.2.1. SPS of purines based on halogenated/aminated purine 16

1.2.2. SPS of purine based on purine ring construction 24

1.3 Purpose of the research work in this thesis 27

1.4 References 28

CHAPTER 2: DESIGN, SYNTHESIS AND BIOLOGICAL EVALUATION OF 2,9-

DISUBSTITUTED-6-OXOPURINES AS INHIBITORS OF MULTIDRUG

RESISTANCE PROTEIN (MRP4/ABCC4) 31

ii

2.1 Introduction 31

2.1.1 Importance of purine 31

2.1.2 Multidrug resistance protein (MRP) 32

2.2 Outline of our synthetic strategy 33

2.3 Results and discussions 34

2.3.1 Solution-phase study 34

2.3.2 Solid-phase Study 42

2.3.3 Biological evaluation of 2,9-disubstituted-6-oxopurines as MRP4 inhibitor 51

2.4 Conclusions 55

2.5 Experimental 55

2.6 References 74

CHAPTER 3: TRACELESS SOLID-PHASE SYNTHESIS OF 1,7-DISUBSTITUTED

PURINES 77

3.1 Introduction 77

3.2 Outline of our strategy 78

3.3 Results and discussions 79

3.3.1 Solution-phase synthesis study 79

3.3.2 Solid-phase study 88

iii

3.4 Conclusion 91

3.5 Experimental 92

3.6 References 104

CHAPTER 4: TRACELESS SOLID-PHASE SYNTHESIS OF VARIOUS

SUBSTITUTED PURINES FROM p-BENZYLOXYBENZYLAMINE (BOBA) RESIN

105

4.1 Introduction 105

4.2 Outline of our strategy 106

4.3 Results and discussions 107

4.3.1 Solution-phase study towards 1,7,8-trisubstituted purines 107

4.3.2 Solution-phase study of other various substituted purines 117

4.3.3 Solid-phase study 131

4.4 Conclusions 137

4.5 Experimental 139

4.6 References 163

APPENDIX A: X ray crystal data 164

APPENDIX B: Spectral analyses 176

iv

SUMMARY

This thesis reports the development of novel methodologies for the solid-phase synthesis

of purine derivatives.

The first project involves the solid-phase synthesis of 2,9-disubstituted-6-oxopurines

using Wang resin. The synthetic strategy involves loading 6-chloropurine scaffolds

directly onto the solid support via an ether linker. Following this, combinatorial

modifications include Mitsunobu alkylation at the N9 position, amination or Sonogashira

coupling at the C2 position, bromination and subsequent alkylation at the C8 position

were carried out. Then resin was eventually cleaved and 2,9-disubstituted-6-oxopurines

were released. A small library of purine derivatives was prepared and overall yields

obtained were 24-70%. The effects of these compounds on multidrug resistance protein 4

(MRP4/ABCC4) facilitated bimane-GS efflux were examined. Compounds 2-16 and 2-

25d were active in inhibiting MRP4 mediated efflux of the bimane-glutathione conjugate.

In addition, both compounds were also able to reverse MRP4 mediated resistance to the

anti-cancer drug 6-thioguanine.

The second project focuses on the investigation of the regioselective solid-phase

synthesis of N7-substituted purine using REM resin. The synthetic strategy was devised

to anchor the REM resin at N9 of 6-chloropurine via Michael addition, leaving N7 as the

steric priority for alkylation. Subsequent hydrolysis of 6-chloride was carried out

followed by alkylation at N1. The resin bound N1-substituted purine was then

quaternized at N7 with different alkylation agents. The 1,7-disubstituted-6-oxopurine

derivatives were released from the resin via Hofmann elimination. With this method, a

library of 15 1,7-disubstituted-6-oxopurines was synthesized in high purity and good

v

yields. This study gives the first example of a highly regioselective solid-phase synthesis

of 1,7-disubstituted-6-oxopurine derivatives.

The third project centers on widening the solid-phase synthesis of purines based on the

purine ring construction strategy. The synthetic strategy was designed to load the 5-

amino-4,6-dichloropyrimidine onto BOBA resin via an amine linker to construct the

diamine key intermediate for elaboration to various substituted purines. After cyclization,

the N7 position possesses the steric priority to be alkylated. This, in turn, resulted in a

regioselective N7 alkylation being achieved. At the end of the reaction, the BOBA linker

was easily cleaved and the target purines were released. During this study, we have also

extended the use of the key intermediate polymer supported diamine for other solid-phase

synthesis including 1,7,8-trisubstituted purines, 8-unsubstituted purines, 8-azapurines and

[i]-condensed purines.

In all these three projects, solid-phase-oriented synthesis in solution was examined to

establish the requisite solid-phase reaction conditions.

vi

LIST OF TABLES

Table 1.1 Acid labile solid-phase linkers

5

Table 2.1

Synthesis of compound 2-4a 36

Table 2.2

Effects of 2-16 and 2-25d on bimane-GS efflux 52

Table 2.3

Viability of M and V following exposure to 2-16 and 2-25d 53

Table 2.4

IC50 for 6TG in the presence of the purine derivatives 54

Table 2.5

Effects of inhibitors on MRP4-mediated efflux of bimane-GS 54

Table 3.1

Synthesis of compound 3-2 80

Table 3.2

Solution-phase synthesis of compound 3-5 86

Table 4.1

Synthesis of compound 4-2a 109

Table 4.2

Various ring closure conditions applied on 4-9 112

Table 4.3

Cyclization with aldehyde 114

Table 4.4

Different hydrolysis conditions of 4-13a 120

vii

LIST OF FIGURES

Figure1.1

Illustration of a solid-phase synthesis 1

Figure 1.2

Structure of Wang resin 4

Figure 1.3

Silyl linkers for traceless SPS 9

Figure 1.4

Purine structure and numbering 16

Figure 2.1

Structures of hypoxanthine and guanine 31

Figure 2.2

X ray crystal structure of 2-3 35

Figure 2.3

X ray crystal structure of compound 2-4b 38

Figure 2.4

Library of 6-oxopurine derivatives 45

Figure 2.5

X ray Crystal Structure of 2-23a 46

Figure 2.6

X ray Crystal Structure of 2-23b 46

Figure 2.7

X ray Crystal Structure of 2-23d 47

Figure 2.8

NOESY spectrum of 2-25a 49

Figure 2.9

NOESY spectrum of 2-26 50

Figure 2.10

Structure of compound 2-6 58

Figure 2.11

Structure of compound 2-22e 67

Figure 3.1

9-H and 7-H purine 77

Figure 3.2

NOESY spectrum of compound 3-4 83

Figure 3.3

NOESY spectrum of compound 3-4a 84

Figure 3.4

NOESY spectrum of compound 3-4b 85

Figure 3.5

Library of 1,7-disubstituted-6-oxopurine 90

Figure 3.6

X-ray crystal structure of 3-6f 91

viii

Figure 4.1

Structure of 9-(4-(benzyloxy)benzyl)-6-chloro-8-hexyl-9H-purine (4-12)

114

Figure 4.2

X ray crystal structure of 4-13a 119

Figure 4.3

X ray crystal structure of 4-18a 122

Figure 4.4

Structures of side products 4-25 and 4-26 125

Figure 4.5

NOESY of compound 4-21a 126

Figure 4.6

Two possible pathways of the cyclization of the pendant alcohol

127

Figure 4.7

NOESY of compound 4-21b 129

Figure 4.8

NOESY of compound 4-23 130

Figure 4.9

X ray crystal structure of compound 4-6i 133

Figure 4.10

X ray crystal structure of compound 4-17b 135

Figure 4.11

X ray crystal structure of compound 4-17c 135

Figure 4.12

Library of various substituted purines 138

ix

LIST OF ABBREVIATIONS

δ chemical shift in ppm

AcOH acetic acid

aq aqueous

bimane-GS bimane-glutathione

Bn benzyl

nBu n-butyl

BOBA resin p-benzyloxybenzylamine resin

Bpoc 2-(biphenyl)-isopropyloxycarbonyl

BuOH n-butanol

calcd calculated

CAN ceric ammonium nitrate

CDK cyclin-dependent kinase

d doublet

DABCO 1,4-diazabicyclo-[2.2.2]octane

DBU diazabicyclo[5.4.0]undec-11-ene

DEPT distortionless enhancement of polarization transfer

dd doublet of doublets

DiAD diisopropyl azodicarboxylate

dt doublet of triplets

DCM dichloromethane

DHP 3,4-dihydro-[ 2H ]-pyran

x

DiEA N,N-diisopropylethylamine

DMA N,N-dimethylacetamide

DMEM Dulbecco’s modified eagle medium

DMF N,N-dimethylformamide

DMSO dimethylsulfoxide

DVB divinylbenzene

equiv. equivalent

EI electron ionization

ESI electrospray ionization

Et2O diethyl ether

EtOAc ethyl acetate

Fmoc 9-fluorenylmethoxycarbonyl

FTIR fourier trasform infrared spectroscopy

HAL hypersensitive acid labile

HBSS Hank’s balanced salt solution

HFIP hexafluoroisopropanol

HMDS hexamethyldisilazane

HRMAS high resolution magic angle spinning spectroscopy

HRMS high resolution mass spectroscopy

IC50 half maximal inhibitory concentration

J coupling constant

LiTMP lithium 2,2,6,6-tetramethylpiperidine

m multiplet

xi

MCB monochlorobimane

mCPBA 3-chloroperoxybenzoic acid

MRP multidrug resistance protein

MS mass spectroscopy

MsCl methanesulfonyl chloride

MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo

phenyl)-2H-tetrazolium

MW microwave irradiation

NBS N-bromosuccinimide

ND not determined

NIS N-iodosuccinimide

NMP N-methyl-2-pyrrolidone

NMR nuclear magnetic resonance

NOESY nuclear overhauser enhancement spectroscopy

Nu nucleophile

o/n overnight

PAL 5-(4-(g-Fmoc)aminomethyl-3,5-dimethoxyphenoxy) valeric acid

Pd (dppe) Cl2 dichloro [1,2-bis(diphenylphosphino)ethane] palladium(II)

Pd (dppf) Cl2 dichloro[1,1’-ferrocenylbis(diphenyl-phosphine)] palladium(II)

PEG poly(ethylene glycol)

PES phenazine ethosulfate

ph phenyl

PNP p-nitrophenol

xii

PS polystyrene

q quartet

REM regeneratable resin linker initially functionalized via a Michael reaction

rt room temperature

s singlet

SASRIN super-acid-sensitive resin

SD standard deviation

SDS sodium dodecyl sulfate

SEM silylethoxy methyl

t triplet

TEA triethylamine

temp. temperature

TFA trifluoroacetic acid

TFE trifluoroethanol

6TG 6-thioguanine

THF tetrahydrofuran

THP tetrahydropyran

TLC thin layer chromatography

TMS tetramethylsilane

TsOH p-methylbenzenesulfonic acid

xiii

LIST OF PUBLICATIONS

1. Traceless solid-phase synthesis of 1,7,8-trisubstituted purines. Han Fu and Yulin

Lam. In preparation.

2. Design, Synthesis and Biological Evaluation of Novel Purine Analogs as

Inhibitors of Multidrug Resistance Protein 4 (MRP4/ABCC4). Theresa M. C. Tan,

Fei Yang, Han Fu, Makam S. Raghavendra and Yulin Lam. Submitted to Journal

of Combinatorial Chemistry.

3. Traceless Solid-Phase Synthesis of N1,N7-Disubstituted Purines. Han Fu and

Yulin Lam. Journal of Combinatorial Chemistry 2005 7(5) 734-738.

CONFERENCE PAPER

1. Traceless Solid-Phase Synthesis of N1,N7-Disubstituted Purines. Han Fu and Yulin Lam.

Pacifichem 2005, Honolulu, Hawaii, USA, December 15-20, 2005.

2. Solid-phase Synthesis of 6-Oxopurine Derivatives. Han Fu and Yulin Lam.

Singapore International Chemical Conference 3, 2003, Singapore, December 15-17,

2005.

1

CHAPTER 1: Introduction

1.1 Solid-phase synthesis (SPS)

Solid-phase synthesis (SPS) is a methodology whereby the reactions take place on the

molecule attached to an insoluble material referred to as a solid support. Such a SPS is

composed of a polymer bead (generally cross-linked, insoluble, polymeric material inert

to the conditions of synthesis) and a linker (a bifunctional chemical moiety that joins the

polymer and the molecule to be synthesized). A building block is firstly coupled to the

solid support via the functionality present on the solid support. Several modification steps

can be performed to achieve the solid support bound final molecule and eventually it is

cleaved from the solid support (Figure 1.1).

Figure1.1 Illustration of a solid-phase synthesis

This concept of solid-phase synthesis was first raised by R. B. Merrifield in 1963 for

efficient peptides synthesis.[1] Later this methodology was expanded to synthesize other

bio-oligomers such as oligonucleotides and oligosaccharides.[2] Today it is a powerful

method for the synthesis of small molecules with biological importance. The main

advantage of SPS is the convenience of purification. Only simple filtration is needed for

2

the purification because compounds are bound to the solid support. The filtration and

washing steps can easily remove the excess reagents used. Other benefits include the

possibility of using excess reagents to force the chemical reaction to completion and the

ease of straightforward automation. However solid-phase synthesis needs large amount of

reagents and solvents, and, depending on the synthetic strategy, extra attachment and

cleavage steps are at times also required. In addition, it is also more difficult to monitor

the progress of a solid-phase reaction because the substrate and product are attached to

the solid support which reduces spectral resolution.

1.1.1 Solid supports

Solid support is an insoluble material to which molecules may be attached (via a linker).

This insoluble material allows the full separation of the solid support from excess

reagents, soluble by-products, or solvents by filtration. Many solid supports have been

developed for SPS of organic molecules. This includes cross-linked organic polymer,

linear organic polymer, dendrimers and inorganic supports.[3] The most frequently used

solid supports are 1) hydrophobic polystyrene resin; 2) hybrid hydrophilic polystyrene

resin; and 3) macroporous non-swelling resin.

The hydrophobic polystyrene resin is normally cross-linked with 1-2% divinylbenzene

(DVB). This cross-linking increases the mechanical stability, diffusion and swelling

property of the resin. Swelling is an essential property of resin in SPS because it

manifests an internal flexibility of the polymer backbone that can move to maximize the

available functionality. It also allows free diffusion of solvents and reagents into the

beads. The swollen resin beads thus have an enlarged surface area to obtain optimum

3

reaction efficiency. The sizes of polystyrene beads commonly used in SPS are between

90-200 μm. These resins are fairly cheap and easily functionalized with high loading.

Thus it is the most extensively used resin in SPS.

Hybrid hydrophilic polystyrene resin is a grafted polystyrene resin with hydrophilic

monofunctional or bifunctional polyethylene glycol (PEG) chains.[4] The monofunctional

PEG grafted polystyrene resin is commonly named Tentagel (TG) and bifunctional PEG

grafted polystyrene resin is called Argogel (AG). This kind of resin shows better swelling

property in aqueous solution and has less mechanical stability compared to hydrophobic

polystyrene resin. However, these resins are very expensive and have lower loading value

which limits their use in SPS.

Macroporous non-swelling resin contains macroscopic pores embedded inside their rigid

structure which does not give any swelling. The rigid structure increases the mechanical

stability. This type of solid support is usually applied for automated oligonucleotide and

peptide synthesis.

1.1.2 Linkers

Linker is another vital component for SPS and it is a molecular moiety connecting the

solid support and the compound to be prepared. This molecular moiety is tethered to the

solid support and contains a reactive functional group which is ready for the attachment

of the first reactant (Figure 1.1). In fact most resins today are named to indicate the linker

grafted onto them. For example Wang resin is named to define the p-alkoxybenzyl

alcohol linker grafted on polystyrene resin (Figure 1.2).

4

O

OHPS resin

linker linking functional group

Figure 1.2 Structure of Wang resin

An ideal linker has to meet some criteria. It should be stable enough to tolerate all the

reaction conditions. On the other hand it is supposed to be sensitive enough to be cleaved

after the reaction is completed.

Many linkers used in SPS can be categorized according to their cleavage condition,[5]

such as acid labile linkers, base labile linkers, photo labile linkers, metal-assisted cleaved

linkers, oxidative/reductive cleaved linkers, cyclatively cleaved linkers, safety-catch

linkers, traceless linkers and multifunctional linkers, etc. However, it is obvious that

some linkers are attributed to more than one family. The most frequently used families of

linkers such as the acid labile linkers, base labile linkers, photo labile linkers, safety-catch

linkers, traceless linkers and cyclatively cleaved linkers are described below.

1.1.2.1 Acid labile linkers

This is the most widely used class of solid-phase linker. Its popularity may be attributed

to the ease of reaction–––cleavage of the acid labile linker and the deprotection of

protecting groups on the resin bound compound sometimes can be achieved in a single

step. Many historically important resins (Merrifield, Wang, Sasrin, Rink resins) have

linkers that are cleaved under acidic conditions. The acids used are normally TFA, HBr,

HF, acetic acid etc. The commonly used acid labile resins and their cleavage conditions

are listed in Table 1.1. [5c]

5

Table 1.1 Acid labile solid-phase linkers

Name of linker Structure Cleavage conditionsa

Wang O

OH

TFA/DCM=1/1, rt,

30 min

SASRIN O

OH

OMe

1 % TFA/DCM, rt

HAL (X=O)

PAL (X=NH) NH

O

O

XH

OMe

OMe

4

0.1 % TFA/DCM, rt,

5 min or

TFA/PhOH=95/5, rt, 2 h

Rink Acid

(X=O)

Rink amide

(X=NH) O

XH

OMe

OMe

0.2 % TFA/DCM, rt,

3 min or 50% TFA/DCM,

rt, 15 min,

THP O

O

TFA/H2O=95/5, rt

Wang halide O

X

TFA/H2O=95/5, rt

Indole NH

N

O

CHO

TFA/DCM=1/1, rt,

30 min

Trityl chloride Cl

Cl

0.5% TFA/DCM or

AcOH/TFE/DCM=1/1/8

or HFIP/DCM=1/4

a) Cleavage conditions depend on particular compound prepared

6

1.1.2.2 Base/nucleophile labile linkers

Although not as popular as the acid labile linker, the nucleophile labile linker has also

been developed to some extent. The cleavage mechanism normally involves β-

elimination, hydrolysis, hydrazinolysis or aminolysis. The main advantage of this linker

is its ability to introduce diversity in the cleavage step. Scheme 1.1 gives an example of a

nucleophilic cleavage. p-Thiophenol linker prepared from methyl amine linker was first

anchored at chloropyridazine and then after combinatorial modification, the final product

3,6-disubstituted pyridazine was released from the solid support by treatment with

primary or secondary amine.[6]

NH

O

SH

NNCl Cl

NH

O

SNN

R

NHR1R2

90oC, 24 h

NNRN

R2

R1

Scheme 1.1 Nucleophilic cleavage of p-thiophenol linker

1.1.2.3 Photo labile linkers

Photo labile linkers use a photon source to cleave the bond between the final compound

and solid support. The target molecule is released into solution and no additional step is

needed to remove the cleavage reagent. Photolytic conditions can be mild and selective.

Only compounds with specific structures can be cleaved photochemically. This kind of

linker usually contains an o-nitrobenzyl group which can be cleaved by a 350 nm light.

An example of a photo cleavage reaction is shown in Scheme 1.2.[7]

7

NH

O

NO2

Br

RCOOHNH

O

NO2

O

O

R'

hv, 350 nm

MeOH, rt, 24 h

R'COOH

Scheme1.2 Photo cleavage of o-nitrobenzyl bromide linker

1.1.2.4 Safety-catch linkers

Safety-catch linker is the linker that is only labile after activation which increases the

lability of the linker to the cleavage conditions. Thus this kind of linker involves two-step

cleavage: activation and cleavage. This linker makes the linkage moiety completely

stable enough to a wide range of reaction conditions during the synthesis unless it is

activated. An example of a safety-catch linker, called the Kenner sulfonamide-based

linker, is shown in Scheme 1.3.[8] This linker was initially coupled with carboxylic acid.

After synthetic elaboration, treatment of diazomethane activated the linker which was

subsequently cleaved with a nucleophile such as NH3, hydrazine or NaOH to release the

amide, hydrazide or carboxylic acid respectively.

SNH2

O O RCOOHS

NH

O O O

R'

SN

OO O

R'

nucleophile O

R'Nu

CH2N2

(activation) (cleavage)

Scheme 1.3 Kenner sulfonamide-based safety-catch linker

1.1.2.5 Traceless linkers

A major drawback to traditional linker is that after cleavage, a specific functional group

through which the compound was attached to the solid support would be left on the target

molecule, e.g. carboxylic acids and amides in peptide synthesis. The presence of these

appendages is acceptable if the target molecule embodies these functionalities. However,

8

complications may arise if these vestigial functionalities are redundant and affect the

activities of the compounds. To address this issue, traceless linkers were developed. A

traceless linker does not leave a residual functional group after cleavage. It normally

creates a C-C or C-H bond at the site of cleavage. Traceless linkers are so called because

an examination of the final compound reveals no trace of the anchoring point. However,

some traceless linkers are also known as multifunctional linkers when cleavage causes an

introduction of a new functionality at the linkage site by either nucleophilic or

electrophilic substitutions.[9] Presently development of traceless linkers is a major area of

interest in SPS.[9, 10] Some commonly used traceless linkers are illustrated below.

1.1.2.5.1 Silyl-based traceless linker

The first and most widely explored traceless linker is the silyl-based linker. It was first

reported by Ellman in 1995 and now it is called Ellman silyl linker.[11] Silicon attached to

a phenyl group can be cleaved by either acids or a fluoride ion, leaving hydrogen on the

aromatic ring (Scheme 1.4). In this case traceless cleavage gave a C-H bond at the

linkage site.

NHBpoc

SnMe3

Si

N

R3O

R2

R1

HF N

R3O

R2

R1

H

NH

O

O

OSi

Ellman silyl linker

Scheme 1.4 Traceless SPS of benzodiazepines using silyl linker

9

Figure 1.3 gives the structures of other silyl-based linkers towards traceless SPS.

O

O Si

R

Veber silyl linker

OSi

R

Showalter silyl linker Figure 1.3 Silyl linkers for traceless SPS

1.1.2.5.2 Sulfur-based traceless linker

The first sulfur traceless linker was the aryl sulfide linker developed by Suto in 1997.[12]

The aryl sulfide linker was activated by oxidation with mCPBA to form a sulfone linker

which was then cleaved with primary or secondary amine to give 2-aminopyrimidines

(Scheme 1.5). This linker is also a typical example for safety-catch linker because the

oxidation can be considered as an activation of the linker for the final cleavage.

Meanwhile it is also regarded as a multifunctional linker because the final cleavage

introduces various amino groups at the linkage site.

S N

N

R1

R2 mCPBA, DCM, rt

oxidation

S N

N

R1

R2

O O

HNR3R4

N

N

R1

R2

NR3

R4

Scheme 1.5 Traceless cleavage of aryl sulfide-based linker

Besides aryl sulfide-based traceless linker, alkyl sulfide linker was also reported for

traceless SPS of biarylmethane through Pd-catalyzed release of resin bound

benzylsulfonium (Scheme 1.6).[13]

10

OSH

SX

ArB(OH)2

Pd(dppf)Cl2K2CO3

XAr

Scheme 1.6 Traceless SPS of biarylmethane

Sulfone linker is another well-developed sulfur-based traceless linker. This linker

provides tethers robust to various chemical transformations.[14] Sodium benzenesulfinate

is an example of a traceless sulfone linker. After combinatorial modifications,

imidazo[1,2-a]puridin-2-yl-enones were released by treatment of resin with base (Scheme

1.7).

SO2Na S

O

O

N

N

R1

R2N

N

R1

R2

OR3

R3O

base

Scheme 1.7 Traceless cleavage of sulfone linker

1.1.2.5.3 Selenium-based traceless linkers

Selenium and sulfur share similar properties. However, the use of selenium reagents is

often preferable to sulfur because not only the oxidation of selenides proceeds more

quickly than that of sulfides but s C-Se bond is weaker than the C-S bond.[15] Selenium

has proven to be a useful element for traceless SPS and many studies on selenium-based

traceless linker have been reported.[16] An example is given in Scheme 1.8. The selenium

linker was obtained form Merrifield resin first. After elaboration to resin bound

substituted benzopyran, oxidative deselenylation released the final product in high

yield.[16a]

11

R1

R2

R3

R4

OH

Se

Br R2

R3

R4

O

R1

Se

R6

R7

R8

O

R5

elaboration R6

R7

R8

O

R5

Se

R6

R7

R8

O

R5

Se

O

oxidation

Scheme 1.8 Traceless SPS of 2,2-dimethylbenzopyran

1.1.2.5.4 Nitrogen-based traceless linker

The first nitrogen-based traceless linker was developed by Komogawa in 1983 as a

sulfonylhydrazone linker.[17] Sulfonylhydrazone linker was easily prepared from sulfonyl

hydrazine resin. After being treated with ketone or aldehyde, the modified resin then was

cleaved either under reduction condition to generate alkane or under basic condition to

offer alkene.

S

HN NH2

OO

S

NH

N

O O

PhPh

reducing reagentPh Ph

Ph Ph

base

Scheme 1.9 Sulfonylhydrazone traceless linker

Amongst the various nitrogen-based traceless linkers, the triazene linker is the most well

studied and frequently used.[10a, 18] Triazene linker, such as T1 linker, has shown the

12

versatility of diazonium type anchoring and its suitability for traceless cleavage. T1

triazene traceless linker was first developed for SPS of arene. The triazene linker was

prepared from diazotization of secondary amine resin and aniline. After chemical

modification such as Heck reaction, the triazene linker was cleaved with HCl or H3PO2 in

dichloroacetic acid.

NH

Ph

N Ph

NN

Heck reactionN Ph

NN

or HCl

CO2Bu

Br CO2Bu

H3PO2

Scheme 1.10 Traceless SPS of arene via T1 linker.

1.1.2.6 Cyclative cleavage strategy

Cyclative cleavage is also an often-used strategy and has begun to play an increasingly

important role in SPS. It produces the intramolecular cyclization of resin bound

intermediate and releases the final cyclized product from the solid support. The

advantage of cyclative cleavage is the ability to generate the final cyclized product in

high purity since any uncyclized side products still remain on the solid support. However

this kind of cleavage is restricted to substrates that contain the structural requirements for

ring closure. An example of cyclative cleavage is shown in Scheme 1.11. The REM

linker was first treated with primary amine followed by the reaction with isocyanate to

yield β-ureido ester as the precursor for ring closure. Treatment of β-ureido ester under

acidic condition gave a direct formation of the final product with concomitant cleavage

from the solid support.[19]

13

O

O

O

O

N NHR2

O

R1N

N

OO

R1

R2

HCl

toluene

Scheme 1.11 Traceless SPS of 1,3-disubstituted-5,6-dihydropyrimidine-2,4-diones

1.1.3 Reaction monitoring in solid-phase synthesis

Although the nature of solid-phase reaction makes it ‘blind’ to some extent and its

reaction monitoring is not as easy as in solution, there are still some methods available

for solid-phase reaction monitoring. These analytical methods can be classified as off-

beads method and on-beads methods. In off-beads method, the resin-bound intermediate

is cleaved off the resin and characterized by classical analytical techniques. This method

is accurate but time-consuming and sometimes the reagents used for cleavage may cause

contamination. In on-beads method, the characterizations are carried out directly on the

resin-bound compounds. Compared with off-beads method, on-beads method is rapid and

more straightforward. The frequently used on-beads methods are on-beads IR, gel phase

NMR, HRMAS-NMR and MS. Despite these methods, solid-phase reaction monitoring is

still a big challenge because the substrate and product are attached to the solid support

which could reduce spectral resolution and new methods and techniques are still required.

1.1.4 Solid-phase synthetic libraries---from peptides to small organic molecules

In 1963 Merrifield realized the efficient synthesis of L-leucyl-L-alanylglycyl-L-valine on

solid support.[1] In order to extend the peptide chain, the deprotection, neutralization and

coupling steps were repeated for each of the subsequent amino acid until the desired

14

sequence was assembled. Finally, the completed peptide was deprotected and cleaved

from the solid support. Due to its speed and simplicity, this technique eventually led to

the rapid development of solid-phase peptide synthesis. Although peptide library is the

most exploited oligomeric molecule generated by SPS, this synthetic methodology was

also extended to the preparation of other biologically important oligomeric molecules

such as oligonucleotides and oligosaccharides.[2] SPS was used to prepare only oligomers

for almost three decades until in 1992 Ellman published their convenient and high yield

synthesis of a library of ten 1,4-benzodiazepine analogs.[20] This was a turning point in

the history of SPS. From then on, the center of SPS was directed at small organic

molecules because many of these molecules were potential lead compounds for drug

discovery. During a drug exploration process, a large number of libraries of organic

molecules are needed for lead discovery and lead optimization and SPS technique can be

used to provide a large collection of small organic molecules expediently. However SPS

of small organic molecules is more challenging. Unlike oligomers, the reaction

conditions for the synthesis of small molecules are more versatile. It requires the solid

support and linker to be stable under the various reaction conditions. To date many small

organic molecule libraries have been prepared as bioactive templates using SPS

technique.

It is worth noting that, during SPS of small organic molecules, after designing the solid-

phase synthetic route, a solution-phase synthesis validation is usually examined to

establish the requisite solid-phase reaction conditions. This validation of the planned

synthetic route is necessary to carry out successful corresponding reactions on the solid

phase. Normally the solution-phase synthetic route must provide all the intermediates and

15

target compound with good yield and high purity before it is transferred onto the solid

support. Meanwhile because of different nature of solid-phase reactions from solution-

phase ones, some modifications of the reaction conditions are still necessary to achieve

better results in solid-phase reactions. For example sometime a specific co-solvent should

be added to the solid-phase reaction to allow the resin to achieve better swelling property.

1.2 Solid-phase synthesis of purine

The purine ring is a critical structural element in biology because of its potential as a

target nucleotide-binding protein and its important role in numerous cellular processes. A

large number of purine syntheses have been developed in solution.[21] Ever since Gray

discovered the trisubstituted purine as cyclin-dependent kinase (CDK) inhibitors, [22] SPS

of purines have been developed to cater to the demand of purine derivatives with higher

diversity. SPS has proved to be an effective and convenient technique to generate purine

library. From 1990’s various methods for the SPS of purine derivatives have been

reported. These methodologies generally involve two main strategies. In the first strategy,

a halogenated/aminated purine is usually used. Modification on such purine ring can

generate purine libraries. The second strategy is based on the construction of the purine

ring. This is achieved via the synthesis of substituted pyrimidine ring followed by closure

of the imidazole ring or through the generation of the imidazole ring first followed by

cyclocondensation of the pyrimidine ring.

16

1.2.1. SPS of purines based on halogenated/aminated purine

This strategy involves loading the purine scaffold bearing halo- or amino- functional

groups directly onto the solid support. After modification at various positions, the

substituted purine is released. Although it looks there are seven positions on the purine

ring which can be used as points of attachment to the solid support, only three of them are

commonly employed. They are the N9, C2 and C6 positions.

N

N NH

N1

2

3

4

56 7

8

9

Figure 1.4 Purine structure and numbering

1.2.1.1 Purine scaffold connected to solid support at N9

In 1996 Norman and co-workers devised a strategy to load the modified purine onto the

aminoalkyl solid support.[23] 2-amino-6-chloropurine was firstly alkylated at N9 to form

2-amino-6-chloro-9-(2-hydroxyethyl)purine, which was then treated with dihydropyran to

generate the hydroxyethyl-THP linker. After loading on the aminoalkyl solid support,

alkylation at the amino group on C2 exocyclic nitrogen and amination on C6 position

were carried out. This was followed by cleavage from the support giving 2,6-

diaminopurine alcohols as final products (Scheme 1.12). However this work has obvious

limitations as only amino substituents could be introduced at the C2 and C6 positions. In

addition, the substituent at N9 is invariable because ethyl alcohol was always obtained

after cleavage.

17

N

N NH

N

Cl

H2N

N

N N

N

Cl

H2N

OH

N

N N

N

Cl

H2NO O

O

OEt

N

N N

N

Cl

H2NO O

O

NH

N

N N

N

NHR2

R1HNO O

O

NH

N

N N

N

NHR2

R1HN

OH

Scheme 1.12

In 1997 Nugiel reported another similar SPS of C2 and C6-focused purines (Scheme

1.13).[24] This method was inspired by the purine tetrahydropyranyl (THP) protection.

THP linker was first generated from Merrifield resin and attached to the N9 position of

2,6-dichloropurine. Different amination conditions were required for reactions at the C6

and C2 positions–––displacement at C6 was carried out first with 5 equiv. of amine and 5

equiv. of TEA at 80oC for 3 h while the amination at C2 had to be carried out at elevated

temperature and using amine as the reaction solvent. This is because the C6-chloro

position was the more reactive site and with an amino group at C6, the amination at C2

became more difficult. Finally 2,6-diaminopurine was easily released by treating the resin

with mild acid. This procedure could be considered as a traceless cleavage. However,

further modification at N9 had to be conducted in solution.

18

Cl OO

N

N N

N

Cl

ClO

O

N

N N

N

NR1

R2NO

O

N

N NH

N

NHR1

R2HNN

N N

N

NHR1

R2HNMe

N

N NH

NCl

Cl

Scheme 1.13

In 2001 Brill and co-workers reported the SPS of 2,6,8-trisubstituted purine (Scheme

1.14). In his strategy, the resin bound purine was prepared by treating activated Rink acid

resin with 2,6-dichloropurine. After which the introduction of amino substituents was

accomplished sequentially by displacement of chlorides. Alkylation at C2 with boric acid

could also be performed successfully. Incidentally this work presented an effective

method for the bromination at C8 position, which provided the possibility of further

modifications at this position.[25]

OH OTFAN

N NH

NCl

Cl N

N N

N

Cl

Cl

N

N N

N

NHR1

R2

N

N N

N

NHR1

R2Br

activation

N

N N

N

NHR1

R2R3 N

N NH

N

NHR1

R2R3

Br2-2,6-lutidine complex

Rink acid resin

Scheme 1.14

19

1.2.1.2 Purine scaffold connected to solid support at C2

In this strategy the purine scaffold was usually attached to the solid support through an

amine linker.

In 1996 glycinamide linker was applied to SPS of 2-(acylamino)-6-aminopurines

(Scheme 1.15).[23] Starting with 2-amino-6-chloropurine, the purine core was attached to

the Rink amide resin at the C2 position. This was followed by the combinatorial acylation

at the exocyclic nitrogen and displacement of C6-chloro by primary or secondary amines.

Since an exocyclic nitrogen was required for the attachment to the solid support, the

diversity at C2 was limited. Furthermore, functionalization at N9 had to be performed in

solution before the purine framework was put onto solid support. Hence this method is

restricted to synthesize of C6-focused purine library.

N

N NH

N

Cl

H2N

N

N N

N

Cl

NH

O

PNPO

Me

NH2 N

N N

N

Cl

NH

O Me

NH

N

N N

N

Cl

N

O Me

NH

O

R1

N

N N

N

NHR2

N

O Me

NH2

O

R1

Rink amide resin

Scheme 1.15

To circumvent the limitation that one substituent is held invariant in order to anchor the

purine to the solid support, Ding and co-workers reported a traceless SPS of 2,6,9-

trisubstituted purines (Scheme 1.16).[26] Starting with 2-fluoro-6-phenylsulfenylpurine,

Mitsunobu reaction was carried out in solution to introduce the first point of diversity at

20

the N9 position. At the same time, primary amines were coupled with the 4-formyl-3,5-

dimethoxyphenoxymethyl-functionalized resin by reductive amination in order to

generate a PAL linker. The modified purine scaffold was then loaded onto the solid

support via the C2 position. The C6-thioether was then oxidatively-activated to a sulfone

so that C6 amination could be achieved. Although the PAL linker allowed traceless

cleavage, only amino substituents could be introduced at the C2 and C6 positions and

only a secondary amino at C2 could be obtained. Besides these drawbacks, since

oxidation to convert 6-thioether to sulfone had to be performed for further substitution,

substituents sensitive to the oxidative conditions could not be introduced in the first two

combinatorial modification steps. In addition, C6 displacement of sulfone was restricted

to primary amines and cyclic secondary amines.

N

N NH

N

SPh

F

N

N N

N

SPh

F

R1

N

N N

N

SPh

N

R1R2

N

N N

N

SPh

N

R1R2

O

O

N

N N

N

NHR3

N

R1R2

N

N N

N

NHR3

HN

R1R2

O

OMe

OMeHN R2

PAL resin

Scheme 1.16

1.2.1.3 Purine scaffold connected to solid support at C6

This is the most common strategy used in the SPS of purine derivatives. In this strategy,

the purine scaffold was attached to a solid support through either an amine linker or a

thioether linker.

21

1.2.1.3.1 Purine scaffold connected to solid support at C6 via amine linker

PAL linker can be attached to purine via the C2 position (as shown in Scheme 1.16) or at

C6 position (Scheme 1.17).[27]. In this report 4-formyl-3,5-dimethoxyphenoxy methyl-

functionalized resin was coupled with the 2-fluoro-6-(4-aminobenzylamino)purine core

via a reductive amination in the presence of sodium triacetoxyborohydride. Following

this, Mitsunobu reaction at N9 and displacement of C2-fluoro with amines were easily

achieved. Finally cleavage with TFA yielded 2,9-substituted purines. It is obvious that

the disadvantage of this method is that one potential combinatorial site is lost. To

circumvent this problem, a modified SPS based on PAL linker was reported by Schultz

and co-workers.[28]

N

N NH

N

HN

F

NH2

NH

O

O CHO

OMe

OMe

4N

N NH

N

HN

F

NH

N

N N

N

HN

F

NH

R1

N

N N

N

HN

N

NH

R1

N

N N

N

HN

N

NH2

R1R2

R3

R2

R3

Scheme 1.17

This modified synthetic route (Scheme 1.18) commenced with the coupling of 4-formyl-

3,5-dimethoxyphenoxymethyl-functionalized resin with a series of primary amines via

reductive amination. This was followed by loading the 6-chloro-2-fluoro-9-SEM purine

core onto the solid support via PAL linker. The purpose of introducing 9-trimethyl

22

silylethoxy methyl (SEM) to the purine core before loading onto the solid support was to

increase the electrophilicity of the purine ring so that a resin capture at C6 could be

performed. Subsequent deprotection of the SEM group, Mitsunobu alkylation at N9 and

amination at C2 position could be carried out. Since the purine was attached to the solid

support at C6 via an amine linker, this implied that only secondary amine can be

introduced at the C6 position upon cleavage.

NH

O

O CHO

OMe

OMe

4 NH

O

O NH

OMe

OMe

4R1

reductiveamination

N

N N

NCl

FO SiMe3

NH

N

N N

N

NR2

NR1

N

N N

N

F

O SiMe3

NR1

N

N N

N

FR2

R1

R3

R4

1)deprotection

2) Mitsunobu

Scheme 1.18

In 2001 a similar method was reported by Dorff and co-workers.[29] However, instead of

using a PAL linker, the purine core was attached to the solid support via an indole linker

(Scheme 1.19).

From the synthetic routes described above, it is observed that 2,6-dihalopurine is the most

frequently used purine core to be loaded onto a solid support. Nucleophilic substitution of

the C2-halogen with amine is a common way to introduce diversity at the C2 position.

However this displacement usually requires harsh reaction conditions and long reaction

time. Thus in 2002 Austin et. al. reported a microwave assisted SPS of 2,6,9-

trisubstituted purine.[30] The synthetic route is similar to the method shown in Scheme

23

1.18. PAL linker was also employed to connect the purine core and solid support.

Microwave irradiation was applied during amination at the C2 position.

NH

N

O

CHOreductive amination

NH

N

O

HN R1N

N NH

N

Cl

F

N

N NH

N

N

F

R1

N

N N

N

HN

N

R1

R3

R2

Me

indole resin

N

N N

N

N

F

R1

Me

Scheme 1.19

1.2.1.3.2 Purine scaffold connected to solid support at C6 via thioether linker

In 2001 Brun and co-workers published the SPS of 2,6,9-trisubstituted purine using

Merrifield-SH resin (Scheme 1.20).[31a] The resin was anchored onto the C6 position of 2-

chloro-2-iodo-9-isopropylpurine. This was followed by substitution at the C2 position

with primary and secondary amines. After oxidative activation of the thioether to sulfone,

both amination and cleavage from the resin was carried out at the C6 position. In order to

expand the scope of substituents at C2, the same group, in 2002, reported the solid-phase

alkylation at C2 using palladium catalyzed cross coupling reactions such as Suzuki and

Sonogashira coupling.[31b] This method expands the diversity at C2 position because both

amino and alkyl substituents can now be introduced.

24

N

N N

N

Cl

I

SH N

N N

N

S

I

N

N N

N

S

R1

N

N N

N

S

R1

O O

N

N N

N

NHR2

R1

R1=amino or alkyl

Scheme 1.20

In 2002 a similar method was also reported by the Schultz group (Scheme 1.21).[32]

Instead of 6-chloro-2-iodo-9-alkylpurine, 6-chloro-2-fluoro-9-alkylpurine was used as the

initial purine scaffold. Amination at C2 was then applied followed by oxidation at C6 and

substitution of sulfone with amines.

N

N NH

N

Cl

F

N

N N

N

Cl

FR1

SHN

N N

N

S

FR1

N

N N

N

S

NR1

R2

R3

N

N N

N

S

R1

O O

N

N N

N

N

R1

R4 R5

N

R2

R3N

R2

R3

Mitsunobu

Scheme 1.21

1.2.2. SPS of purine based on purine ring construction

SPS of purine library based on loading halogenated/aminated purine directly onto solid

support can easily provide purine derivatives with diversity on C2, C6 and N9 positions.

However, SPS of purine library based on imidazole ring formation has an important

25

advantage of providing the possibility of introducing C8 substituents more easily,

although it is less straightforward than the first method. This strategy usually commences

with the attachment of substituted pyrimidine on the solid support followed by functional

group generation, combinatorial modification and ring closure.

In 2000 Lucrezia et al reported the first SPS of purine from pyrimidine.[33] In this method

(Scheme 1.22), Rink amide resin was attached to 4,6-dichloro-5-nitropyrimidine via the

C6 position of the pyrimidine ring. Subsequent amination at C4 and reduction of nitro

group at C5 provided the precursor for closure of the imidazole ring. Elaboration to

purine was achieved by cyclization with isothiocyanate, formamide or aldehyde.

Although this work gave only 8,9-disubstituted purines, it provides an avenue for

cyclization to be carried out on solid phase to generate purines with C8 diversity.

However, with the Rink amide resin, only adenine analogs could be prepared as cleavage

resulted in the generation of a NH2 group. Moreover the overall yield is only ~7%

indicating an average yield of just 59% for each step.

N

N

Cl

NO2

Cl

NH2N

N

NO2

Cl

NH

N

N

NH2

NHR1

NH

N

N

NH

N

N

R1

R2

N

N

NH2

N

N

R1

R2

Scheme 1.22

In 2002, the SPS of 2-(6-(benzylamino)-9-methyl-9H-purin-2-yl-amino)ethanol

(olomoucine) based on purine imidazole ring formation was also reported (Scheme

1.23).[34] Argogel MB-CHO resin was coupled with benzylamine by reductive amination.

The resin bound secondary amine was then attached to 4,6-dichloro-2-(methylthio)-5-

26

nitropyrimidine. After substituting the C4-chloro with methylamine, the C2-thiomethyl

was oxidized to sulfone followed by substitution with protected ethanolamine. The nitro

group was reduced and the imidazole ring was closed with trimethyl orthoformate.

Subsequent deprotection and cleavage provided olomoucine. Although this report offered

only olomoucine one compound, it provides an opportunity to generate purine library

with higher diversity by varying of the individual amines used in the displacement steps

and orthoesters used in during the cyclization step.

O OMe

CHO

O OMe

NH Ph

N

N

N

MeS

NO2

NH

Bn

Me

N

NMeS

NO2

Cl

Cl

N

N

N

NH

NH2

NH

Bn

MeTBDPSO

N

N

N

NH

N

N

Bn

Me

TBDPSO

N

N

HN

NH

N

N

Bn

Me

HO

olomoucine

Scheme 1.23

In 2005 He reported the SPS of 1,3-substituted xanthines.[35] This study gave the first

example of SPS of purines obtained by generating both the imidazole and pyrimidine ring

on solid support (Scheme 1.24). Starting from glycine ethyl ester, PS-MB-CHO resin

bound substituted imidazole ring was first constructed. After treatment with various

isocyanates, the pyrimidine ring was closed under basic condition. Additional diversity at

N3 was introduced by N-alkylation and acidic cleavage gave the desired purines.

27

OO

OMe

ON

N

O

OEt

NH2

OMe

N

NH

N

N

X

O

R1O

OMe

ON

N

O

OEt

NH

OMe

NHR1

X

N

N N

HN

X

O

R1

R2

X=O or S

Scheme 1.24

1.3 Purpose of the research work in this thesis

With the solid-phase synthetic methods described in Section 1.2, various substituted

purine derivatives have been prepared. However, those methods mostly focus on

adenines (6-aminopurines) synthesis. The SPS of 6-oxopurines is unexplored. Therefore

one of the purposes of our research is to develop novel methodologies to traceless SPS of

6-oxopurine derivatives.

As described in Section 1.2, the SPS of purines has concentrated on the synthesis of N9

substituted purine derivatives. In recent years, there has been much interest in the N7

regioisomers due to its unique biological activities. Hence the second goal of this

research is to investigate the regioselective SPS of N7-substituted purine derivatives.

Finally, the extent to which reported SPS of purines could be applied to generate more

diversity in purine is limited. For this reason, our research aims to widen the existing SPS

protocol for purine so as to enable one to prepare purine derivatives with higher diversity.

28

1.4 References

[1] Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149-2154.

[2] (a) Letsinger, R. L.; Mahadevan, V. J. Am. Chem. Soc. 1966, 88, 5319-5324.

(b) Frechet, J. M.; Schuerch, C. J. Am. Chem. Soc. 1971, 93, 492-496.

[3] Obrecht, D.; Villalgordo, J. M. Tetrahedron Organic Chemistry Series Volume

17, Elsevier Science Ltd. 1998

[4] (a) Shepperd, R. C. J. Chem. Br. 1983, 19, 402-414. (b) Small, P. W.;

Sherrington, D.C. J. Chem. Soc. Chem. Commun. 1989, 1589-1591.

[5] (a) Guillier, F.; Orain, D.; Bradley, M. Chem. Rev. 2000, 100, 2091-2157. (b)

Lazny, R.; Michalak, M. Wiadomosci Chemiczne 2003, 57, 11-12. (c) Seneci,

P. Solid-phase Synthesis And Combinatorial Technologies, John Wiley & Sons,

Inc. 2000.

[6] Parot, I.; Werinuth, C. G.; Hibert, M. Tetrahedron Lett. 1999, 7975-7978.

[7] Rich, D. H.; Gurwara, S. K. J. Am. Chem. Soc. 1975, 97, 1575-1579.

[8] Backes, B. J.; Eliman, J. A. J. J. Am. Chem. Soc. 1975, 116, 11171-11172.

[9] Knepper, K.; Gil, C.; Brase S. Highlights In Bioorganic Chemistry 2003, 449-

484.

[10] (a) Brase, S.; Dahmen, S. Chem. Eur. J. 2000, 6, 1899-1905. (b) Blaney, P.;

Grigg, R.; Sridharan, V. Chem. Rev. 2002, 102, 2607-2624. (c) Phoon, C. W.;

Sim, M. M. Curr. Org. Chem. 2002, 6, 937-964.

[11] Plunkkett, M. J.; Ellman, J. A. J. Org. Chem. 1995, 60, 6006-6007.

[12] Gayo, L. M.; Suto, M. J. Tetrahedron Lett. 1997, 38, 211.

29

[13] Vanier, C.; Lorge, F.; Wagner, A.; Mioskowaski. C. Angew. Chem. Int. Ed.

2000, 39, 1679-1683.

[14] Chen, Y.; Lam, Y.; Lai, Y. H. Org. Lett. 2002, 4, 3935-3937.

[15] Nicolaou, K. C.; Petasis, N. A. Selenium in Natural Product Synthesis CIS Inc:

Philadelphia, PA, 1984.

[16] (a) Nicolaou, K. C.; Pfefferkorn, J. A.; Roecker, A. J.; Cao, G. Q.; Barluenga,

S.; Mitchell, H. J. J. Am. Chem. Soc. 2000, 122, 9939-9953. (b) Ruhland, T.;

Anderson, K.; Pederson, H. J. Org. Chem. 1998, 63, 9204-9211. (c) Nicolaou,

K. C.; Pastor, J.; Barluenga, S.; Winssinger, N. J. Chem. Soc., Chem. Commun.

1998, 18, 1947-1948. (d) Nicolaou, K. C.; Roecker, A. J.; Pfefferkorn, J. A.;

Cao, G. Q. J. Am. Chem. Soc. 2000, 122, 2966-2967.

[17] Kamogawa, H.; Kanzawa, A.; Kadoya, M.; Naito, T.; Nanasawa, M. M. Bull.

Chem. Soc. Jpn. 1983, 56, 762-765.

[18] Brase, S.; Enders, D.; Kobberling, J.; Avemarie, F. Angew. Chem. Int. Ed.

1998, 37, 3413-3415.

[19] Kolodziej, S. A.; Hamper, B. C. Tetrahedron Lett. 1996, 37, 5277-5280.

[20] Bunin, B. A.; Ellman, J.A. J. Am. Chem. Soc. 1992, 114, 10997-10998.

[21] Bork, J. T.; Lee, J. W.; Chang, Y-T. QSAR Comb. Sci. 2004, 23, 245-260.

[22] Gray, N. S.; Wodicka, L.; Thunnissen, A-M. W. H.; Norman, T. C.; Kwon, S.;

Espinoza, F. H.; Morgan, D. O.; Barnes, G.; LeClerc, S.; Meijer, L.; Kim, S-H.;

Lockhart, D. J.; Schultz, P. G. Science 1998, 281, 533-538.

[23] Norman, T. C.; Gray, N. S.; Koh, J. T. and Schultz, P. G. J. Am. Chem. Soc.

1996, 118, 7430-7431.

30

[24] Nugiel, D. A.; Cornelius, L. A. M.; Corbett, J. W. J. Org. Chem. 1997, 62, 201-

203.

[25] (a) Brill W. K. D. and Toniolo C. R. Tetrahedron Lett. 2001, 42, 6279-6282. (b)

Brill W. K. D.; Toniolo C. R. Tetrahedron Lett. 2001, 42, 6515-6518.

[26] Ding, S; Gray, N. S.; Ding, Q and Schultz, P. G. J. Org. Chem. 2001, 66, 8273-

8276.

[27] Gray, N. S.; Kwon, S.; Schultz, P. G. Tetrahedron Lett. 1997, 38, 1161-1164.

[28] Chang, Y. T.; Gray, N. S.; Rosania, G. R.; Sutherlin,D. P.; Kwon, S.; Norman,

T. C.; Sarohia, R.; Leost, M.; Meijer, L. and Schultz, P. G. Chemistry and

Biology 1999, 6, 361-375.

[29] Dorff, P. H. and Garigipati, R. S. Tetrahedron Lett. 2001, 42, 2771-2773.

[30] Austin, R. E.; Okonya, J. F.; Bond, D. R. S.; Al-Obeidi, F. Tetrahedron Lett.

2002, 43, 6169-6171.

[31] (a) Brun, V.; Legraverend, M.; Grierson, D. S. Tetrahedron Lett. 2001, 42

8165-8167. (b) Brun, V.; Legraverend, M.; Grierson, D. S. Tetrahedron 2002,

58, 7911-7923.

[32] Ding, S.; Gray, N. S.; Ding, Q.; Wu, X. and Schultz P. G. J. Comb. Chem.

2002, 4, 183-186.

[33] Lucrezia, R. D.; Gilbert, I. H. and Floyd, C. D. J. Comb. Chem. 2000, 2, 249-

253.

[34] Hammarstrom, L. G. J.; Meyer, M. E.; Smith, D. B.; Talamas, F. X.

Tetrahedron Lett. 2003, 44, 8361-8363.

[35] He, R. J.; Lam, Y. L. J. Comb. Chem. 2005, 7, 916-920.

31

CHAPTER 2: Design, Synthesis and Biological Evaluation of 2,9-

Disubstituted-6-oxopurines as Inhibitors of Multidrug Resistance

Protein (MRP4/ABCC4)

2.1 Introduction

2.1.1 Importance of purine

Purine ring is a critical structural element in biology because it is the component of

nucleoside and nucleotide. Amongst the various purine derivatives, hypoxanthine and

guanine (Figure 2.1) have found applications in a variety of therapeutics[1] and are known

to (i) modulate multidrug resistance[2]; (ii) represent important anti-infective, anti-herpes

and antitumor agents; and (iii) act as selective agonists and antagonists of specific

receptors in cardiovascular or central nervous system complaints.[1] It is therefore

reasonable to expect that combinatorial libraries of 6-oxopurine derivatives may provide

inhibitors of these processes and act as useful biological probes or lead molecules for

drug development efforts.

N

NHN

N

OH

N

NHNH2N

HN

O

hypoxanthine guanine

Figure 2.1 Structures of hypoxanthine and guanine

32

2.1.2 Multidrug resistance protein (MRP)

The ATP-binding cassette (ABC) transporters are membrane proteins that facilitate the

transport of a diverse variety of molecules. In humans, there are 48 ABC transporters

belonging to 6 different subfamilies.[3] Members of the human ABC superfamily have

been implicated in multidrug resistance in chemotherapy. To date, P-glycoprotein

(Pgp/MDR1/ABCB1) is the best studied in terms of its role in multidrug resistance.[4]

Other human ABC transporters that are implicated in drug resistance include the breast

cancer resistance protein (BCRP/ABCG2) and the multidrug resistance protein 1

(MRP1/ABCC1).[5]

The ABCC subfamily has 12 members of which 9 are MRP proteins (MRP1-6/ABCC1-6

and MRP7-9/ABCC10-12).[6] Although most ABC proteins contain two transmembrane

domains and two nucleotide binding domains, some MRP proteins also contain a third

transmembrane domain at the N-terminal.[6] These include MRP1-3, MRP6 and MRP7.

In contrast, MRP4, MRP5, MRP8 and MRP9 all have the typical two transmembrane

domains and two nucleotide binding domains topology.

MRP1 was cloned in 1992 and studies with cell lines overexpressing MRP1 showed that

MRP1 is able to confer resistance to anthracyclines, vinca alkaloids,

epipodophyllotoxins, camptothecins and methotrexate.[7] However, to date, the

expression of MRP1 in clinical samples has not been systematically examined and the

contribution of MRP1 to resistance to the anti-cancer drugs in humans is still not well

established.[5a] Similar to the observations for MRP1, overexpression of MRP4 and

MRP5 too can confer resistance to therapeutic agents. However, unlike MRP1, MRP4

and MRP5 confer resistance to nucleoside and nucleobase analogs which are used

33

therapeutically as anti-cancer or anti-viral agents. These include compounds such as 6-

mercaptopurine, 6-thioguanine (6TG), 9-(2-phosphonylmethoxyethyl)adenine (PMEA)

and azidothymidine (AZT).[8] Overexpression of MRP4 also results in resistance to

camptothecins.[9] In addition to therapeutic agents, MRP4 is also able to transport various

endogenous molecules including cAMP and cGMP as well as conjugated steroids, bile

acids, prostaglandins and glutathione.[8, 10]

To date, various compounds have been shown to inhibit the activities of MRP4. These

include probenecid, sulfinpyrazone, indomethacin, dipyridamole and compounds

containing the purine (eg. 6TG and PMEA).[10d, 11]

2.2 Outline of our synthetic strategy

In this project, we have designed a general method for the traceless SPS of 2,9-

disubstituted-6-oxopurine derivatives (Scheme 2.1). The strategy involves the direct

loading of the purine scaffold onto the solid support. This was achieved through a resin

capture-release chemistry whereby the purine was initially captured on the Wang resin at

the 6-position through a C6-O bond. After various combinatorial modifications on the

purine moiety, the product was released by debenzylative cleavage of the C6-O bond.

N

N NH

N

Cl

I I

N

N NH

N

O

HN

N N

N

O

R2

R1

OH

Scheme 2.1 Overall strategy

34

2.3 Results and discussions

2.3.1 Solution-phase study

Prior to the solid-phase synthesis, preliminary solution-phase studies were carried out to

survey the requisite reaction conditions and establish the modifications required for solid-

phase organic synthesis.

2.3.1.1 Resin capture-release chemistry in solution

Ph O-Na+N

N NH

N

N

N

+ Cl-

DMF

Ph O

N

N NH

N

ROH, PPh3, DiAD

Ph O

N

N N

N

R

THF

30% TFA/CH2Cl2

O

HN

N N

N

nBu

Cl

N

N NH

NDABCO

DMF

2-1

2-2 2-3

2-4a R=nBu2-4b R=iPr

2-5

Scheme 2.2 Simulation of resin capture-release chemistry in solution

2.3.1.1.1 Synthesis of 6-(benzyloxy)-9H-purine (2-3)

We first examined the resin capture-release chemistry (Scheme 2.2). Benzyl alcohol was

used to mimic the structure of Wang resin in solution-phase study. According to the

literature, the chloride at the 6-position can be easily replaced by benzyl thiol.[12]

35

However sulfur is better nucleophile than oxygen. Hence we needed to convert the 6-

chloride in compound 2-1 into a better leaving group. A standard method is to convert the

6-chloride into a 6-ammonio species.[13] This is commonly achieved through the reaction

of 6-chloropurine with trimethylamine. However, the latter reagent being very volatile

and toxic is difficult to handle, especially on a large scale. In addition, alkylation on the

methyl group of trimethylammonio will be an undesirable competitive reaction during the

subsequent displacement reaction. Hence in our synthesis, 1,4-diazabicyclo-[2.2.2]octane

(DABCO) was used instead of trimethylamine.[14] Treatment of 2-1 with DABCO gave

the DABCO-purine salt 2-2 which was reacted with sodium benzyloxide in DMF at room

temperature to give 6-benzyloxypurine 2-3 in 91% yield. Attempts to carry out the

reaction at 100oC resulted in a series of side-products and 2-3 was isolated in a much

lower yield (22%). The X ray crystal structure of 2-3 is shown in Figure 2.2.

Figure 2.2 X ray crystal structure of 2-3

36

2.3.1.1.2 Solution-phase N9 alkylation

According to the literature, distribution between N9 and N7 alkylated products is strongly

dependent on the substituents at the 6-position of purine, the choice of the base used and

the reaction temperature.[15] Thus, to establish a regioselective N9 alkylation, various

conditions were examined (Table 2.1).

Table 2.1 Synthesis of compound 2-4a

Reaction conditions Ratio of N9:N7a

1 nBuBr/LiH or NaH, 80°C, 6 h 1:1

2 nBuBr/LiH, rt, 4 h 3:2

3 PhSO3nBub/K2CO3/18-crown-6, rt, o/n 1:1

4 ROH/DiAD/PPh3, rt, o/n 1:0 a) Determined by 1HNMR(C2H integral value). b) Prepared from benzenesulfonyl chloride and 1-

butanol.

Kjellberg and coworker have reported the regioselective N9-alkylation of 6-alkoxy-9H-

purine with LiH or NaH. [15] However our attempts to alkylate 2-3 with 1-bromobutane in

the presence of LiH or NaH provided the N7 and N9 alkylated regioisomers in a 1:1 ratio

(Entry 1). Reaction at lower temperature favored the N9 isomer and the N9 and N7

alkylated regioisomers were obtained in a 3:2 ratio (Entry 2). Treatment of 2-3 and

PhSO3nBu in the presence of K2CO3 and 18-crown-6 also gave poor selectivity and the

N7:N9 isomers were obtained in a 1:1 ratio. To selectively alkylate the N9-position, we

therefore proceeded to explore the Mitsunobu reaction. Reaction of 2-3 with 1-butanol

under Mitsunobu condition gave exclusively the N9 isomer 2-4a (determined by

1HNMR) in 68% yield. The mechanism of the Mitsunobu reaction is depicted in Scheme

2.3.

37

N NOO

OOPPh3

N NOO

OO+PPh3

PPh3

N

N NH

NOPh

OR

NHN

OOOO

Ph3P=ON

N N

N

OPh

R2-4

H OR

NHN

OOOO

+PPh3

+

+

N

N N

N

OPh

PPh3 OR

HN

HN

OOOO

+

+

2-3

RO

Scheme 2.3 Mechanism of Mitsunobu reaction

When 2-propanol was used for N9 Mitsunobu alkylation, 6-(benzyloxy)-9-isopropyl-9H-

purine (2-4b) was obtained exclusively. Figure 2.3 shows the X ray crystal structure of 2-

4b.

2.3.1.1.3 Synthesis of 9-butyl-1H-purin-6(9H)-one (2-5)

After the N9 alkylation, the benzyl group can be easily removed by catalytic

hydrogenation or acid hydrolysis. Since the conditions for catalytic hydrogenation could

not be conveniently adapted onto the solid-phase format, we proceeded to investigate the

removal of the benzyl group using acid hydrolysis. In our study 2-benzyloxy-9-

38

butylpurine 2-4 was hydrolyzed in 30% TFA/DCM or 1 M HCl in methanol to afford 9-

butyl-1H-purin-6(9H)-one 2-5. IR spectrum of compound 2-5 showed a C=O stretch at

1692 cm-1 which could be attributed to the cyclic amide carbonyl group.

Figure 2.3 X ray crystal structure of compound 2-4b

2.3.1.2 Solution-phase study towards 2,9-disubstituted-6-oxopurines

The above study in solution phase demonstrated the efficiency of our resin capture-

release strategy for the synthesis of 6-oxo-purine derivatives. To achieve combinatorial

modification at the C2 position, 6-chloro-2-iodopurine 2-9 was prepared as the starting

39

halogenated purine scaffold. The solution-phase synthesis of 2,9-disubstituted-6-

oxopurines from 2-9 is summarized in Scheme 2.5.

N

N NH

N

N

N

I

+ Cl-

N

N NH

N

I

OPh

N

N N

N

I

OPh

nBu

N

N N

N

N

OPh

nBuO

HN

N N

N

N

O

nBuO

nBuOH, PPh3, DiAD

Morpholine, NPr3

TFA,CH2Cl2

N

N NH

N

Cl

I

2-92-10 2-11

2-12 2-132-14

DMF NaH

PhCH2OH

THF

DMA

2-15 2-16

N

N N

N

OPh

nBuO

HN

N N

N

O

nBuO

TFA,CH2Cl2

4-Methoxyphenylboronic acidPd(PPh3)4, DiEA

DABCO

Scheme 2.5 Overall solution-phase study

2.3.1.2.1 Synthesis of 6-chloro-2-iodopurine (2-9) as starting material

6-Chloro-2-iodo-purine (2-9) was obtained from 6-chloropurine (2-1) via protection of

N9 with DHP, lithiation-mediated stannyl migration, subsequent iodination and final

deprotection (Scheme 2.4).[16]

40

N

N NH

N

Cl

N

N N

N

Cl

THP

N

N N

N

Cl

THP

Bu3Sn

N

N N

N

Cl

THP

I

N

N NH

N

Cl

I

2-1 2-6 2-7

2-8 2-9

DHP, TsOH.H2O

CH3CN

1) LiTMP

2) nBu3SnCl

I2, THF TFA,CH2Cl2

Scheme 2.4 Preparation of 6-chloro-2-iodopurine (2-9)

2.3.1.2.2 Synthesis of 6-(benzyloxy)-9-butyl-2-iodo-9H-purine (2-12)

6-benzyloxy-2-iodopurine (2-11) was obtained using the same procedure as the synthesis

of 6-benzyloxy-purine (2-3). It was noted that dihalogenated purines have different

reactivities at the C2 and C6 positions. Earlier studies have shown that nucleophilic

reactions with 2-iodo (or bromo- or chloro)-6-chloropurines would occur preferably at

the C6 position instead of the C2 position.[17] Hence treatment of 6-chloro-2-iodopurine

with DABCO/PhCH2OH/NaH provided regioselectively 2-11. Reaction of 2-11 under

Mitsunobu condition resulted in the key intermediate 2-12 which facilitated further

modifications on purine ring. In this project, we have examined the modifications by 1)

amination via nucleophilic aromatic substitution, and 2) arylation via Suzuki coupling.

2.3.1.2.3 Synthesis of 6-(benzyloxy)-9-butyl-2-morpholino-9H-purine (2-13)

Halogens on purines can be readily replaced by various nucleophiles. Since the halogen

at the C2 position has a lower reactivity than those at the C6 position, harsher reaction

41

condition was required for the substitution of C2-I by an amine. Treatment of 2-12 with

morpholine at 120°C overnight in the presence of tripropylamine gave 2-13 in 83% yield.

2.3.1.2.4 Synthesis of 6-(benzyloxy)-9-butyl-2-(4-methoxyphenyl)-9H-purine (2-15)

The C2 aryl substituted 2-15 can be formed via Suzuki cross coupling of 2-12 and 4-

methoxyphenylboronic acid. Suzuki reaction is the coupling of an aryl or vinyl boronic

acid with an aryl or vinyl halide using a palladium catalyst. Compound 2-12 was coupled

with 4-methoxyphenylboronic acid in the presence of tetrakis(triphenylphosphine)

palladium(0) via Suzuki cross coupling procedure to give 2-15 in 94% yield. The

mechanism of this reaction involves oxidative addition, transmetalation, and reductive

elimination steps and is illustrated in Scheme 2.6.

Pd(0)

Pd(II)Pd(II)Ar1 Ar2

Ar1I

B(OH)2Ar2 B(OH)2Ar2

Ar2Ar1

IAr1

activation

base

base

oxidative additionreductive elimination

B(OH)2

base

+I-transmetallation

Scheme 2.6 Mechanism of Suzuki cross coupling

2.3.1.3 Study of bromination at C8 position in solution

In order to provide the possibility of modification at the C8-position, different

halogenation conditions (Br2/DMF, NBS/DMF, NBS/THF and NIS/DMF) were examined

but none provided the desired 8-halopurine. While Br2-2,6-lutidine[18] complex succeeded

in generating 8-bromopurine, 2-17 was obtained in 44% yield (Scheme 2.7).

42

Br2-lutidineN

N N

N

N

OPh

nBuO

BrHN

N N

N

N

O

nBuO

TFA,CH2Cl2Br

2-17 2-18

N

N N

N

N

OPh

nBuO2-13

Scheme 2.7 Bromination of 2-13 at C8 position

After the N9 and C2 modifications, compounds 2-13, 2-15 and 2-17 were then

hydrolyzed with 30% TFA/CH2Cl2 to give the respective substituted 6-oxopurines.

2.3.2 Solid-phase Study

2.3.2.1 Solid-phase synthesis of 2,9-disubstituted-6-oxopurine

Having corroborated the approach in solution, we proceeded to apply the method to SPS.

Scheme 2.8 summarizes our solid-phase process. The starting purine scaffolds, either 6-

chloropurine (2-1) or 6-chloro-2-iodo-purine (2-9), were first loaded on the Wang resin.

The resin bound purine 2-19 was N9-alkylated under Mitsunobu condition. The

alkylation of resin bound purine can be monitored by High-Resolution Magic Angle

Spinning (HRMAS) NMR for the appearance of peaks corresponding to the alkyl group.

After obtaining the resin bound key intermediate 2-20, further modifications were carried

out in accordance to the solution-phase study.

C2-amino groups were introduced through the reaction of resin 2-20b with a variety of

primary and secondary amines in DMA at 120˚C. Meanwhile key intermediate 2-20 was

also readily cleaved with acid to obtain compound 2-23.

43

OH

DMF

O

N

N NH

N

X

R1OH, PPh3, DiAD

O

N

N N

N

X

R1

THF

R2NH2, NPr3

O

N

N N

N

R2HN

R1

TFA/CH2Cl2

O

HN

N N

N

R2HN

R1

N

N NH

N

N

N

X

+ Cl-N

N NH

N

Cl

X

O

N

N N

N

R1

R3

Alkyne, PdCl2(dppe)CuI, DiEA, DMF

TFA/CH2Cl2

O

HN

N N

N

R1

R3

2-1 X=H2-9 X=I

2-19

2-20a X=H2-20b X=I

2-212-22

2-24 2-25

DMA

TFA/CH2Cl2O

HN

N N

N

R1

2-23 X=H, I

X

2-2 X=H2-10 X=I

Scheme 2.8 Solid-phase studies

However the established solution-phase C2-alkylation via Suzuki cross coupling gave a

less clean reaction on solid phase. This may be attributed to the low solubility of the

Pd(PPh3)4 catalyst in the reaction solvent thus preventing the reaction from being carried

out efficiently on solid phase. Hence we proceeded to examine the C2-alkylation using

Sonogashira coupling. This coupling of terminal alkynes with aryl or vinyl halides was

performed with a palladium catalyst, a copper (I) co-catalyst, and an amine as base under

44

anhydrous and anaerobic conditions. Resin 2-20b was reacted with different alkynes in

the presence of [1,2-bis(diphenylphosphino)ethane] dichloro palladium(II), copper(I)

iodide and DiEA to give 2-alkylated compound 2-24. The target molecule 2-25 was

finally efficiently cleaved from the solid support with 30% TFA in CH2Cl2. The

mechanism of this reaction is illustrated in Scheme 2.9.

oxidative additionreductive elimination

transmetallation

(dppe)Pd( II)Cl2

RC CH R3N, CuIRC CR

(dppe)Pd(0)ArX

(dppe)PdAr

X

RC CH R3N, CuI

R3N+HX-

(dppe)PdAr

C CR

ArC CR

Scheme 2.9 Mechanism of Sonogashira coupling

Thus an efficient and novel SPS of 6-oxopurine derivatives using Wang resin as solid

support was developed. The synthetic strategy was designed to load the purine scaffold

directly onto the solid support. 6-Chloropurine was captured onto Wang resin at C6

position via an ether linker. After modifications at N9 and C2, the resin was cleaved,

resulting in 2,9-disubsittued-6-oxopurine derivatives.

To illustrate the versatility of this chemistry, a small library of C2 and N9 focused 6-

oxopurine derivatives was synthesized. The structures are illustrated in Figure 2.4. The

overall yields obtained were 24-70% (purities of >95% by NMR), indicating an average

yield of 70% for each step of the SPS.

45

HN

N N

N

O

O

N

HN

N N

N

O

HN

PhN

N N

N

O

NnBu

O

HN

HN

N N

N

O

HN

Ph

HN

N N

N

O

nBu

O

HN

N N

N

O

N

Ph

HN

N N

N

O

HN

N N

N

O

NH

N

HN

N N

N

O

OH

HN

N N

N

O

HN

InBu

HN

N N

N

O

HN

N N

N

O

I

OH

HN

N N

N

O

HN

N N

N

O

2-23a 70% 2-23c 40%

2-22a 28% 2-22c 29%

2-22g 32%2-22e 26% 2-25a 33%

2-22d 29%

2-25b 28%

2-22f 24%

2-23b 47% 2-23d 28%

2-22b 24%

2-25c 24%

HN

N N

N

O

2-25d 28%

Figure 2.4 Library of 6-oxopurine derivatives

The crystal structures of 2-23a, 2-23b and 2-23d are shown in Figure 2.5, Figure 2.6 and

Figure 2.7 respectively.

46

Figure 2.5 X ray Crystal Structure of 2-23a

Figure 2.6 X ray Crystal Structure of 2-23b

47

Figure 2.7 X ray Crystal Structure of 2-23d

2.3.2.2 Solid-phase bromination and further C8 modification study

Although bromination of 6-benzyloxy-9-nbutyl-2-morpholin-4-yl-9H-purine 2-13 can be

carried out in solution, on solid phase, it gave a very complex product mixture upon

cleavage (Scheme 2.10).

O

N

N N

N

R2HN

R1

2-21

O

N

N N

N

R2HN

R1

Br

Br2-lutidine

Scheme 2.10 Bromination of resin 2-21

However we found that bromination of resin bound 9-nbutylpurine 2-20a showed a

positive result (Scheme 2.11). Treatment of 2-20a with bromine-lutidine complex

48

followed by Sonogashira coupling with 3-methyl-pent-1-yn-3-ol and cleavage yielded 8-

alkynyl-9-nbutyl-6-oxo-purine 2-26 in 19 % overall yield.

O

N

N N

N

C4H9

O

N

N N

N

C4H9

Br

O

N

N N

N

C4H9

R

O

HN

N N

N

C4H9

R

R=HO

2-20a 2-26

Scheme 2.11 Bromination of resin 2-20a

The bromination site on the purine ring could be identified from the position of the

alkyne group introduced during Sonogashira coupling. In order to determine this position

in 2-26 (R=3-hydroxy-3-methyl-pent-1-ynyl), NOESY experiment was carried out and

compared with the NOESY spectrum of 2-25a which was obtained via 6-chloro-2-

iodopurine (2-9) on solid phase. The interaction between C8H and N9CH2 was observed

in the NOESY spectrum of 2-25a (Figure 2.8). No such interaction was observed

between C2H and N9CH2 in 2-26 which indicates that the alkyne substituent in 2-26 is at

the C8 position (Figure 2.9).

49

HN

N N

N

O

OH

H

2-25a

H

Figure 2.8 NOESY spectrum of 2-25a

50

HN

N N

N

O

HOH

2-26

Figure 2.9 NOESY spectrum of 2-26

51

2.3.3 Biological evaluation of 2,9-disubstituted-6-oxopurines as MRP4 inhibitor

2.3.3.1 Effects of 2,9-disubstituted-6-oxopurines on bimane-GS efflux

MRP4 is able to mediate the transport of conjugated molecules. These include molecules

conjugated with glucuronate or with glutathione.[10d,11b] It has been reported that cells

overexpressing MRP4 is able to mediate the efflux of the fluorescent bimane-glutathione

(bimane-GS) adduct.[10d] Thus we first examined the effects of 2,9-disubstituted-6-

oxopurines on MRP4-mediated bimane-GS efflux. This was carried out using HepG2

cells stably overexpressing MRP4 (denoted as MRP4/HepG2). Cells stably transfected

with the empty vector (denoted as V/HepG2) served as the control. Similar to

observations in an earlier study, MRP4/HepG2 cells were able to facilitate the efflux of

bimane-GS at a significantly highly rate than that of V/HepG2.

Of the screened 2,9-disubstituted-6-oxopurines, only 2-16 and 2-25d were able to

influence the bimane-GS transport ability of MRP4 at 100 μM. The presence of 25 μM of

2-16 led to significant reduction in MRP4-mediated transport (Table 2.2). The efflux was

reduced from 7.7 nmol/mg protein to 2.9 nmol/mg protein. Significant inhibition of

MRP4-mediated transport was also observed in the presence of 25 μM of 2-25d although

the degree of inhibition was much less than that observed for 2-16. At 25 μM both 2-16

and 2-25d did not affect efflux from the control V/HepG2 cells. In addition, the effects of

both compounds were specific to the efflux process as there were no differences in the

total bimane-GS synthesis between cells that were exposed to 2-16 or 2-25d and those

that were not. Significant inhibition of MRP4-mediated transport was also observed at 50

μM of 2-16 and 2-25d but at this concentration, there was also significant reduction in

the efflux from the control V/HepG2 cells.

52

Table 2.2 Effects of 2-16 and 2-25d on bimane-GS efflux

Concentration of compound

Total bimane-GS synthesized in V/HepG2 (top) and MRP4/HepG2 (bottom) (nmol/mg protein)

1Bimane efflux from V/HepG2 cells (nmol/mg protein)

2Bimane efflux from MRP4/HepG2 cells (nmol/mg protein)

Efflux mediated by MRP4 (2-1) (nmol/mg protein)

0 μM (control)

48.2 + 1.1 / 48.5 + 1.7

14.6 + 0.7

22.3 + 1.0

7.7 + 0.5

25 μM

50.2 + 3.5 / 48.3 + 3.4

14.3 + 1.0 17.1 + 0.6a 2.9 + 0.8a

2-16 50 μM

50.0 + 3.7 / 49.5 + 2.5

11.8 + 1.3a 16.0 + 1.4a 4.3 + 1.3a

0 μM (control)

50.5 + 2.4 / 49.2 + 2.8

15.6 + 0.6 23.3 + 0.9 7.7 + 0.4

25 μM

49.7 + 2.4 / 50.0 + 3.8

14.3 + 0.8 19.7 + 0.9a 5.4 + 0.9a

2-25d 50μM

51.0 + 2.0 / 50.2 + 1.8

13.1 + 1.1a 19.2 + 0.6a 6.1 + 0.7a

a) Compared with the corresponding untreated cells, p<0.05, ANOVA analysis.

2.3.3.2 Effects of 2,9-disubstituted-6-oxopurines on 6TG resistance

Given the fact that MRP4 has the ability to mediate resistance to nucleoside and

nucleobase analogs, and also to transport the phosphorylated metabolites of

mercaptopurines,[11, 19] it is highly possible that the expression of MRP4 may play a role

in the pharmacokinetics of resistance to these therapeutic molecules. Thus one of the

aims of this study was to examine if it were possible to reverse the MRP4-mediated

resistance to 6TG. The approach in this study was to use a series of 2,9-disubstituted-6-

oxopurines which are not cytotoxic to the cells. Given the fact that 2-16 and 2-25d were

53

active in inhibiting MRP4 mediated bimane-GS efflux, the effects of these analogs on

resistance to 6TG were also investigated.

MRP4/HepG2 cells (M) and V/HepG2 cells (V) were first treated with 0-100 μM of these

compounds for 48 h to examine the effects on cell proliferation. As shown in Table 2.3,

the IC50 for each of these compounds was >100 μM. There was no effect on cell growth

and proliferation when the cells were exposed to 10 μM of compound 2-16 and 25 μM of

compounds 2-25d for 48 h. Thus these concentrations were used for the next series of

experiments.

Table 2.3 Viability of M and V following exposure to 2-16 and 2-25da

Concentration 0 μM 10 μM 25 μM 50 μM 100 μM

M 100% 94% ± 1.0% 87% ± 0.5% 77% ± 0.3% 76% ± 0.5% 2-16 V 100% 93% ± 0.1% 90% ± 4.6% 78% ± 2.2% 68% ± 3.0%

M 100% ND 96% ± 0.3% 86% ± 0.4% 84% ± 0.9%2-25d

V 100% ND 97% ± 2.3% 83% ± 0.8% 77% ± 2.3% a) The cells were exposed to compound 2-16 and 2-25d at the concentrations indicated for 48 h. 20 μL

of MTS reagent was then added and cells were incubated at 37ºC for 1 h. The absorbance at 490 nm was measured. Each concentration was carried out in triplicate. Data are expressed as mean ±S.D. from three independent experiments.

The IC50 values for 6TG for the MRP4/HepG2 cells were significantly reduced in the

presence of both compounds as shown in Table 2.4. The concentrations used to achieve

this reversal of resistance ranged from 10 μM to 25 μM. At these concentrations, the

compounds had no adverse effects on cell proliferation and viability (Table 2.3). It is also

obvious that the purine analog inhibitors did not affect the uptake or metabolism of 6TG

as the IC50 values in the control V/HepG2 cells were not affected by the presence of these

analogs (Table 2.4).

54

Table 2.4 IC50 for 6TG in the presence of the purine derivatives

IC50 (μM) 1MRP4/HepG2 2V/HepG2 Fold resistance (1/2)

10 μM 2-16 18.5±0.3a 13.0±1.1 1.4

25 μM 2-25d 29.4±0.9a 15.3±1.0 1.9

Control 37.1±3.8 13.9±0.6 2.7

a) Compared with the corresponding untreated cells, p<0.05, ANOVA analysis.

MRP4 can confer resistance to nucleoside-based drugs such as 6TG. In the earlier study,

it was observed that 6TG is capable of inhibiting MRP4-mediated efflux of bimane-

GS[10d]. In this study, two 2,9-disubstituted-6-oxopurines, 2-16 and 2-25d, were found to

inhibit MRP4-mediated efflux of bimane-GS. This inhibition was solely on the efflux as

the synthesis of bimane-GS was not affected by the compounds. A comparison of the

effectiveness of these inhibitors with 6TG revealed that 2-16 was a better inhibitor than

6TG (Table 2.5).

Table 2.5 Effects of inhibitors on MRP4-mediated efflux of bimane-GS

Inhibitor Percent of control (%)

None 100a

100 μM 6TG 56±9b

25 μM 2-16 37±9c

25 μM 2-25d 70±14c a) Export in the absence of inhibitor was designated as 100%. b) Data for 6TG was

previously published by Bai et al., 2004.[10d] c) Compared with export in the absent of an inhibitor, p<0.05, ANOVA analysis.

55

2.4 Conclusions

The strategy for the SPS of 2,9-disubstituted-6-oxopurines was described. Key steps in

the synthesis include: (i) attachment of the 6-chloropurine scaffold onto the Wang resin,

(ii) Mitsunobu alkylation at the N9 position, (iii) amination or alkylation at the C2

position, (iv) bromination and alkylation at the C8 position, and (v) traceless product

release by debenzylation. Since a variety of reagents can be used in steps (ii), (iii) and

(iv), the overall strategy appears applicable for library generation. With this strategy a

small library of 2,9-disubstituted-6-oxopurines was synthesized in good yield and high

purity. The effects of these compounds on multidrug resistance protein 4

(MRP4/ABCC4) facilitated efflux was examined. Compounds 2-16 and 2-25d were

active in inhibiting MRP4 mediated efflux of the bimane-glutathione conjugate. In

addition, both compounds were also able to reverse MRP4 mediated resistance to the

anti-cancer drug 6-thioguanine.

2.5 Experimental

General Procedures. All chemicals were obtained from commercial suppliers and used

without purification. Column chromatography was performed with silica gel (Merck,

230-400 mesh). NMR spectra (1H and 13C) were recorded using Bruker DPX300 or

AMX500, and chemical shifts are expressed in parts per million related to internal TMS.

Mass spectrometry was performed on VG Micromass 7035 spectrometer under EI or ESI.

56

2.5.1 Synthesis of 6-(benzyloxy)-9H-purine (2-3) 0

6-Chloropurine (2-1) (0.46 g, 3.00 mmol) and DABCO (1.85 g, 16.50 mmol) were

dissolved in DMF (5 mL). The reaction mixture was stirred at room temperature for 3.5

h. Meanwhile benzyl alcohol was added to a suspension of NaH in anhydrous DMF and

the reaction was stirred at room temperature for 1 h. The DABCO-purine mixture was

then added to sodium benzyloxide and the reaction mixture was stirred at room

temperature for 48 h. After which, the reaction mixture was cooled in an ice water bath

and neutralized with 5% HCl to pH7. The aqueous layer was extracted with ethyl acetate.

The combined organic layer was washed with brine, dried with MgSO4 and purified by

silica gel column chromatography (CH3OH/CH2Cl2=1/19) to give 2-3 as a white solid

(0.43 g, 94.3%). 1HNMR (CDCl3, 300 Hz) δ(ppm): 5.70 (s, 2H, PhCH2), 7.32-7.55 (m,

5H, ArH), 8.10 (s, 1H, C8H), 8.61 (s, 1H, C2H). 13CNMR (CD3OD, 300 Hz) δ(ppm):

68.88, 117.86, 128.49, 128.62, 128.71, 136.74, 143.13, 152.21, 155.81, 159.62.

HRMS(EI) Calcd for C12H10N4O 226.0855; found 226.0853.

2.5.2 Synthesis of 6-(benzyloxy)-9-butyl-9H-purine (2-4a)

6-Benzyloxy-9H-purine (2-3) (0.75 g, 3.30 mmol), 1-butanol (0.42 g, 5.61 mmol) and

PPh3 (1.56 g, 5.95 mmol) were dissolved in THF (20 mL). DiAD (1.00 g, 4.95 mmol)

was then added dropwise under ice water bath temperature. The reaction was stirred at

room temperature overnight. The solvent was evaporated and the residue was dissolved in

ethyl acetate and water. The aqueous layer was extracted with ethyl acetate. The

combined organic layer was washed with brine, dried over MgSO4 and purified by silica

gel column chromatography (EtOAc/hexane=1/2) to give 2-4a as a white solid (0.63 g,

68%). 1HNMR (CDCl3, 300 Hz) δ(ppm): 0.97 (t, J=7.3 Hz, 3H, CH3), 1.31-1.44 (m, 2H,

57

CH2), 1.84-1.94 (m, 2H, CH2), 4.22-4.27 (t, J=7.3 Hz, 2H, N9CH2), 5.68 (s, 2H, PhCH2),

7.31-7.40 (m, 5H, ArH), 7.90 (s, 1H, C8H), 8.55 (s, 1H, C2H). 13CNMR (CDCl3, 500 Hz)

δ(ppm): 14.13, 20.51, 32.69, 44.56, 69.02, 122.20, 128.75, 128.98, 129.09, 136.89,

142.80, 152.56, 153.00, 161.21. HRMS(EI) Calcd for C16H18N4O 282.1481; found

282.1481.

2.5.3 Synthesis of 9-butyl-1H-purin-6(9H)-one (2-5)

To solution of 2-4 (88.70 mg, 0.31 mmol) in CH2Cl2 (4 mL) was added TFA (2 mL). The

reaction was stirred at room temperature for 4 h. The solvent was removed and the

residue was dissolved in ethyl acetate and NaHCO3 (aq.). The aqueous layer was

extracted with ethyl acetate and the combined organic layer was washed with brine, dried

with MgSO4 and purified by silica gel column chromatography (CH3OH/CH2Cl2=1/10) to

give 2-5 as a white solid (51.30 mg, 85%). 1HNMR (CD3OD, 300 Hz) δ(ppm): 0.97 (t,

J=7.3 Hz, 3H, CH3), 1.29-1.42 (m, CH2), 1.82-1.92 (m, CH2), 4.25 (t, J=7.1 Hz, 2H,

N9CH2), 8.03 (s, 1H, C8H), 8.06 (s, 1H, C2H). 13CNMR (CD3OD, 500 Hz) δ(ppm):

19.19, 26.15, 38.66, 50.36, 130.55, 147.52, 151.84, 155.68, 164.43. HRMS(EI) Calcd for

C9H12N4O 192.1011; found 192.1012.

2.5.4 Synthesis of 6-chloro-2-iodopurine

2.5.4.1 6-Chloro-9-(tetrahydropyran-2-yl)-9H-purine (2-6)

6-Chloro-9H-purine (2-1) (1.86 g, 12.00 mmol), p-toluenesulfonic acid (cat.) and DHP

were dissolved in CH3CN (40 mL). The mixture was stirred at 55°C overnight. The

solvent was evaporated and the residue was dissolved in ethyl acetate and ammonium

58

hydroxide. The aqueous layer was extracted with ethyl acetate and the combined organic

layer was washed with brine, dried with MgSO4 and purified by silica gel column

chromatography (EtOAc:Hexane = 1:2) to give 2-6 as a white solid (2.53 g, 88%).

1HNMR (CDCl3, 300 Hz) δ(ppm): 1.65-2.20 (m, 6H, H2’+H3’+H4’), 3.75-3.84 (td, J=2.8

Hz, J=11.2 Hz, 1H, H5’), 4.17-4.22 (m, 1H, H5’), 5.80 (dd, J=2.4 Hz, J=10.1 Hz, 1H,

H1’), 8.34 (s, 1H, C8H), 8.76 (s, 1H, C2H). 13CNMR (CDCl3, 300 Hz) δ(ppm): 21.93,

24.10, 30.91, 68.10, 81.82, 130.92, 142.77, 150.01, 150.36, 151.17. MS(EI): m/z 238.1.

N

N N

N

Cl

O1'

2'

3'

4'

5'

8

2

Figure 2.10 Structure of compound 2-6

2.5.4.2 6-Chloro-9-(tetrahydropyran-2-yl)-2-tributylstannanyl-9H-purine (2-7)

6-Chloro-9-(tetrahydropyran-2-yl)-9H-purine, 2-6, (2.61 g, 10.93 mmol) in THF (8 mL)

was added dropwise to a THF solution of LiTMP (54.64 mmol, 5 equiv.) under dry

nitrogen atmosphere, while maintaining the reaction temperature below -70°C. After

stirring for 5 min, nBu3SnCl (8.89 g, 27.32 mmol, 2.5 equiv.) was added and stirring was

continued for an additional 1 h. The reaction was quenched with aqueous NH4Cl. The

aqueous layer was extracted with ethyl acetate and the combined organic layer was

washed with brine, dried with MgSO4 and purified by silica gel column chromatography

(EtOAc:Hexane = 1:5-1:2) to give 2-7 as a pale yellow oil (5.59 g, 97%). 1HNMR

(CDCl3, 300 Hz) δ(ppm): 0.90 (t, J=7.3 Hz, 9H, 3CH3), 1.18-1.20 (t, J=8.0 Hz, 6H,

59

3SnCH2), 1.23-1.42 (m, 6H, 3CH2CH3), 1.57-1.69 (m, 12H, H2’, 3’, 4’+3CH2Et), 3.78 (td,

J=3.5 Hz, J=10.8 Hz, 1H, H5’), 4.17-4.21 (m, 1H, H5’), 5.78 (dd, J= 2.8 Hz, J=10.1 Hz ,

1H, H1’), 8.21 (s, 1H, H8). MS(ESI, M+H): 528.9.

2.5.4.3 6-Chloro-2-iodo-9-(tetrahydropyran-2-yl)-9H-purine (2-8)

A solution of 2-7 (5.59 g, 10.6 mmol) and iodine (3.23 g, 12.72 mmol) in THF (60 mL)

was stirred at room temperature for 1 h. The reaction mixture was diluted with 5%

aqueous Na2S2O3. The aqueous layer was extracted with ethyl acetate and the combined

organic layer was washed with brine, dried with MgSO4 and purified by silica gel column

chromatography (EtOAc:Hexane = 1:2) to give 2-8 as a pale yellow oil (2.83 g, 73%).

1HNMR (CDCl3, 300 Hz) δ(ppm): 1.65-2.18 (m, 6H, H2’, 3’, 4’), 3.78 (td, J=3.3 Hz, J=11.1

Hz, 1H, H5’), 4.16-4.20 (m, 1H, H5’), 5.78 (dd, J=2.8 Hz, J=10.1 Hz, 1H, H1’), 8.24 (s,

1H, C8H). 13CNMR (CDCl3, 500 Hz) δ(ppm): 151.58, 150.36, 143.01, 131.59, 116.52,

82.37, 68.88, 32.06, 24.70, 22.47. HRMS(EI) Calcd for C10ClH10IN4O 363.9588; found

363.9586.

2.5.4.4 6-Chloro-2-iodo-9H-purine (2-9)

A solution of 2-8 (1.57 g, 4.32 mmol) and TFA (13 mL) in CH2Cl2 (50 mL) was stirred at

room temperature for 1 h. The volatile material was evaporated on the rotavapor and the

residue obtained was dissolved in ethyl acetate and saturated aqueous NaHCO3. The

aqueous layer was extracted with ethyl acetate and the combined organic layer was

washed with brine, dried with MgSO4 and purified by silica gel column chromatography

(EtOAc:Hexane = 1:1-2:1) to give 2-9 as a white solid (0.88 g, 73%). 1HNMR (CD3OD,

60

300 Hz) δ(ppm): 8.50 (s, 1H, C8H). 13CNMR (CD3OD, 500 Hz) δ(ppm): 157.81, 149.74,

148.28, 131.12, 118.24. HRMS(EI) Calcd for C5ClH2IN4 279.9013; found 279.9000.

2.5.5 Synthesis of 6-(benzyloxy)-2-iodo-9H-purine (2-11)

Compound 2-11 was prepared from 2-9 as described in Section 2.5.1. 1HNMR (DMSO-

d6, 500 Hz) δ(ppm): 5.56 (s, 2H, PhCH2), 7.37-7.55 (m, 5H, ArH), 8.34 (s, 1H, C8H).

13CNMR (CDCl3, 500 Hz) δ(ppm): 68.09, 116.97, 127.82, 127.91, 128.19, 128.69,

132.25, 135.18, 142.69, 156.95. HRMS(EI) Calcd for C12H9IN4O 351.9821; found

351.9820.

2.5.6 Synthesis of 6-(benzyloxy)-9-butyl-2-iodo-9H-purine (2-12)

Compound 2-12 was prepared from 2-11 as described in Section 2.5.2. 1HNMR (CDCl3,

500 Hz) δ(ppm): 0.96 (t, 3H, J=7.2 Hz, CH3), 1.31-1.38 (m, 2H, CH2), 1.83-1.89 (m, 2H,

CH2), 4.19 (t, 2H, J=7.2 Hz, N9CH2), 5.62 (s, 2H, PhCH2), 7.31-7.56 (m, 5H, ArH), 7.78

(s, 1H, C8H). 13CNMR (CDCl3, 500 Hz) δ(ppm): 13.44, 19.78, 31.89, 43.95, 69.38,

117.23, 128.44 (x2), 128.93 (x2), 135.55, 141.92, 153.09, 159.25. HRMS(EI) Calcd for

C16H17ION4 408.0447; found 408.0451.

2.5.7 Synthesis of 6-(benzyloxy)-9-butyl-2-morpholino-9H-purine (2-13)

A solution of 2-12 (0.1 g, 0.245 mmol), morpholine (0.064g, 0.735 mmol) and

tripropylamine (0.105 g, 0.733 mmol) in DMF (7 mL) was stirred at 120°C under

nitrogen for 24 h. After evaporating all the volatile materials, the residue was dissolved in

CH2Cl2 and water. The aqueous layer was extracted with CH2Cl2. The combined organic

61

layer was washed with brine, dried with MgSO4 and purified by silica gel column

chromatography (EtOAc:Hexane = 1:1) to give 2-13 as a white solid (0.073 g, 83%).

1HNMR (CDCl3, 500 Hz) δ(ppm): 0.95 (t, 3H, J=7.4 Hz, CH3), 1.30-1.79 (m, 2H, CH2),

1.79-1.85 (m, 2H, CH2), 3.76-3.81 (m+m, 4H+4H, 2CH2N+2CH2O), 4.06 (t, 2H, J=7.2

Hz, N9CH2), 5.56 (s, 2H, PhCH2), 7.29-7.50 (m, 5H, ArH), 7.58 (s, 1H, C8H). 13CNMR

(CDCl3, 500 Hz) δ(ppm): 13.47, 19.75, 31.79, 43.02, 45.07, 66.84, 67.73, 114.81, 127.83,

127.99, 128.37, 154.35, 158.50, 160.26. HRMS(EI) Calcd for C20H25N5O2 367.2008;

found: 367.2009.

2.5.8 Synthesis of 9-butyl-2-morpholino-1H-purin-6(9H)-one (2-14)

9-Butyl-2-morpholino-1H-purin-6(9H)-one (2-14) was prepared from 2-13 as described

in Section 2.5.3. 1HNMR (CDCl3, 500 Hz) δ(ppm): 0.95 (t, J=7.3 Hz, 3H, CH3), 1.31-

1.38 (m, 2H, CH2), 1.77-1.83 (m, 2H, CH2), 3.76-3.85 (m+m, 4H+4H, 2CH2N +2CH2O),

4.01 (t, J=7.3 Hz, 2H, CH2N9), 7.51 (s, 1H, C8H), 11.51 (s, 1H, NH). 13CNMR (CDCl3,

500 Hz) δ(ppm): 13.47, 19.70, 31.95, 43.07, 45.61, 66.43, 117.09, 138.10, 151.47,

152.51, 159.29. HRMS(EI) Calcd for C13H19N5O2 277.1539; found:277.1543.

2.5.9 Synthesis of 6-(benzyloxy)-9-butyl-2-(4-methoxyphenyl)-9H-purine (2-15)

K2CO3 (61.00 mg, 0.44 mmol), Pd(PPh3)4 (17.00 mg, 0.0145 mmol), and 4-

methoxyphenylboric acid (74.00 mg, 0.44 mmol) were added to the solution of 2-12

(120.00 mg, 0.29 mmol) in DMF. The reaction mixture was stirred in the dark at 100oC

for 48 h and then evaporated to dryness and chromatographed on a silica gel column

(EtOAc:Hexane = 1:2) to give 2-15 as a pale yellow solid (107.00 mg, 94%). 1HNMR

62

(CDCl3, 500 Hz) δ(ppm): 0.96 (t, J=7.4 Hz, 3H, CH3), 1.32-1.40 (m, 2H, CH2), 1.85-1.95

(m, 2H, CH2), 3.86 (s, 3H, OCH3), 4.24 (t, J=7.2 Hz, 2H, N9CH2), 5.77 (s, 2H, PhCH2),

6.98-8.48 (m, 9H, ArH), 7.83 (s, 1H, C8H). 13CNMR (CDCl3, 500 Hz) δ(ppm): 13.39,

19.73, 31.90, 43.43, 55.25, 67.92, 113.60, 119.67, 127.87, 128.24, 128.31, 129.68,

130.75, 136.69, 141.83, 153.34, 158.15, 159.88, 161.30. HRMS(EI) Calcd for

C23H24N4O2 388.1899; found: 388.1893.

2.5.10 Synthesis of 9-butyl-2-(4-methoxyphenyl)-1H-purin-6(9H)-one (2-16)

2-16 was prepared from 2-15 using the method described in Section 2.5.3. 1HNMR

(CDCl3, 500 Hz) δ(ppm): 0.99 (t, J=7.4 Hz, 3H, CH3), 1.01-1.46 (m, 2H, CH2), 1.86-1.96

(m, 2H, CH2), 3.89 (s, 3H, OCH3), 4.22 (t, J=7.2 Hz, 2H, N9CH2), 7.06-8.27 (m, 4H,

ArH), 7.77 (s, 1H, C8H) 11.98 (s, 1H, NH). 13CNMR (CDCl3, 500 Hz) δ(ppm): 13.44,

19.76, 32.18, 43.63, 55.47, 114.46, 122.40, 124.55, 129.34, 139.89, 150.03, 153.13,

159.18, 162.39. HRMS(EI) Calcd for C16H18N4O2 298.1430; found: 298.1432.

2.5.11 Synthesis of 8-bromo-9-butyl-2-morpholino-1H-purin-6(9H)-one (2-18)

2.5.11.1 Preparation of Br2-lutidine complex

Br2 (1.54 mL, 29.26 mmol) was added dropwise to 2,6-lutidine at 0°C in 40 min and the

reaction mixture was stirred at 0°C for 2 h. Then the precipitate was filtered, washed with

n-pentane, dissolved in 1,2-dichloroethane (10 mL) and re-precipitated by adding n-

pentane (100 mL). The precipitated complex was then filtered and dried in vacuum in the

dark.

63

Brominating reagent: Br2-lutidine complex (0.27 g) was added to a mixture of 2,6-

lutidine (0.11 g) and NMP (10 mL).

2.5.12 Synthesis of 6-(benzyloxy)-8-bromo-9-butyl-2-morpholino-9H-purine (2-17)

from compound 2-13

To 2-13 (20.00 mg, 0.05 mmol) was added the brominating reagent (10 mL). The reaction

mixture was stirred at room temperature for 6 h. After which, the reaction mixture was

dissolved in ethyl acetate and brine. The aqueous layer was extracted with ethyl acetate

and the combined organic layer was washed with brine, dried with MgSO4 and purified

by silica gel column chromatography (EtOAc:Hexane = 1:4) to give 2-17 (10.70 mg,

44%). 1HNMR (CDCl3, 300 Hz) δ(ppm): 0.94 (t, J=7.3 Hz, 3H, CH3), 1.25-1.47 (m, 2H,

CH2), 1.72-1.82 (m, 2H, CH2), 3.77 (m, 8H, 2CH2N+2CH2O), 4.07 (t, J=7.1 Hz, 2H,

CH2N9), 5.53 (s, 2H, PhCH2), 7.29-7.49 (m, 5H, ArH). HRMS(EI) Calcd for

C20H24BrN5O2 445.1113; found: 445.1109, 447.1098.

2.5.13 Synthesis of 8-bromo-9-butyl-2-morpholino-1H-purin-6(9H)-one (2-18)

A solution of 2-17 (11.00 mg, 0.05 mmol) and TFA (0.5 mL) in CH2Cl2 (2 mL) was

stirred at room temperature for 4 h. After evaporating all the volatile materials, the

residue was neutralized with saturated aqueous NaHCO3. The reaction mixture was

concentrated under reduced pressure and the residue obtained was purified by silica gel

column chromatography (CH3OH:CH2Cl2 = 1:20) of to give 2-18 as a white solid (6.80

mg, 80%). 1HNMR (CDCl3, 300 Hz) δ(ppm): 0.96 (t, J=7.2 Hz, 3H, CH3), 1.25-1.38 (m,

64

2H, CH2), 1.70-1.80 (m, 2H, CH2), 3.73-3.84 (m+m, 4H+4H, 2CH2N+2CH2O), 4.02 (t,

J=7.2 Hz, 2H, CH2N9). HRMS(EI) Calcd for C13H18BrO2N5 355.0644; found: 355.0633.

2.5.14 Preparation of resin bound 2-19

2-1 or 2-9 (0.39 g, 0.395 mmol) and DABCO (0.86 g, 7.67 mmol) were dissolved in

DMF (10 mL). The reaction mixture was stirred rapidly at room temperature for 4 h. A

suspension of Wang resin (1.15 mmol/g, 0.60 g) in DMF was treated with 5 equiv. of

sodium hydride at room temperature for 4 h. The DABCO-purine mixture was then added

and the suspension was shaken at room temperature for 48 h. The resin was filtered,

washed successively with DMF, H2O, EtOH, CH2Cl2 and Et2O and dried in a vacuum

oven at 40°C.

2.5.15 General procedure for the N9 Mitsunobu alkylation of resin bound purine (2-

20)

Resin 2-19 was swollen in THF at room temperature for 30 min. Alcohol (1.5 equiv.) and

PPh3 (1.7 equiv.) were added and then DiAD (1.8 equiv.) was added dropwise under ice

water bath temperature. The mixture was allowed to shake at room temperature

overnight. The resin was then filtered, washed successively with THF, EtOH, CH2Cl2 and

Et2O and dried in a vacuum oven at 40°C.

2.5.16 General procedure for the C2 amination of resin bound purine (2-21)

Resin 2-20b was swollen in DMA at room temperature for 30 min. The respective amine

(10 equiv.) and tripropylamine (3 equiv.) were added and the mixture was then stirred

65

slowly at 80°C under nitrogen for 24 h, filtered, washed successively with DMF, H2O,

EtOH, CH2Cl2 and Et2O and dried in.

2.5.17 General procedure for the C2-C bond formation via Sonogashira coupling of resin

bound purine (2-24)

Resin 2-20b was swollen in DMA at room temperature for 30 min. To the suspension was

added dichloro(1,2-bis(diphenylphosphino)ethane)-palladium(II) (1.1 equiv.), DiEA (30

equiv.), CuI (2.2 equiv.) and alkyne (20 equiv.). The mixture was stirred slowly at 100°C

in the dark for 48 h and then filtered. The resin was washed successively with DMF, H2O,

EtOH, CH2Cl2 and Et2O and dried in a vacuum oven at 40°C.

2.5.18 Bromination of resin bound 9-nbutyl-purine (2-20a)

Loaded resin 2-20a was swollen in NMP for 30 min. Then brominating reagent (prepared

as described in Section 2.5.11.1) (10 mL) was added and the mixture was shaken at room

temperature in the dark for 7 h. Then the solution was drained. This process was repeated

for 4 times and the resin was washed with DMF, H2O, EtOH, CH2Cl2 and Et2O and dried

in a vacuum oven at 40°C.

2.5.19 General procedure for the cleavage of resin bound purine

A suspension of resin in 30% TFA/CH2Cl2 was shaken at room temperature for 5 h. The

resin was removed by filtration and washed with MeOH and CH2Cl2. The filtrate and

washings were combined and neutralized with saturated aqueous NaHCO3. The mixture

was then concentrated under reduced pressure and the residue obtained was purified by

66

silica gel column chromatography (CH3OH:CH2Cl2 = 1:15-1:10). to give the final

compound.

2-22a: 9-Butyl-2-morpholino-1H-purin-6(9H)-one

1HNMR (CDCl3, 500 Hz) δ(ppm): 0.95 (t, J=7.3 Hz, 3H, CH3), 1.31-1.38 (m, 2H, CH2),

1.77-1.83 (m, 2H, CH2), 3.76-3.85 (m+m, 4H+4H, 2CH2N+2CH2O), 4.01 (t, J=7.2 Hz,

2H, CH2N9), 7.51 (s, 1H, C8H), 11.51 (s, 1H, NH). 13CNMR (CDCl3, 500 Hz) δ(ppm):

13.47, 19.70, 31.95, 43.07, 45.61, 66.43, 117.09, 138.10, 151.47, 152.51, 159.29.

HRMS(EI) Calcd for C13H19N5O2 277.1539; found: 277.1543.

2-22b: 9-Isopropyl-2-morpholino-1H-purin-6(9H)-one

1HNMR (CDCl3, 300 Hz) δ(ppm): 1.53 (d, J=7.0 Hz, 6H, 2CH3), 3.43-3.82 (m+m,

4H+4H, 2CH2N+2CH2O), 4.57-4.66 (m, 1H, CHN9), 7.60 (s, 1H, C8H). 13CNMR

(CDCl3, 300 Hz) δ(ppm): 22.50, 45.58, 46.71, 66.38, 115.48, 135.77, 152.32, 159.39.

HRMS(EI) Calcd for C12H17N5O2 263.1382; found: 263.1361.

2-22c: 9-Benzyl-2-morpholino-1H-purin-6(9H)-one

1HNMR (CDCl3, 300 Hz) δ(ppm): 3.78-3.79 (m+m, 4H+4H, 2CH2N+2CH2O), 5.19 (s,

2H, PhCH2), 7.33-7.56 (m, 5H, ArH), 7.56 (s, 1H, C8H). 13CNMR (CDCl3, 300 Hz)

δ(ppm): 45.55, 46.98, 66.34, 116.42, 127.72, 128.28, 128.95, 132.97, 135.74, 137.98,

152.68, 159.18. HRMS(EI) Calcd for C16H17N5O2 311.1382; found: 311.1380.

67

2-22d: 9-Butyl-2-(2-(piperidin-1-yl)ethylamino)-1H-purin-6(9H)-one

1HNMR (CD3OD, 300 Hz) δ(ppm): 0.99 (t, J=7.3 Hz, 3H, CH3), 1.31-1.43 (m, 2H,

CH3CH2), 1.79-1.96 (m, 8H, CH3CH2CH2+H3’+H4’+H5’), 3.57 (t, J=5.4 Hz, 2H, CH2N1’),

3.75 (t, J=5.4 Hz, 3H, CH2NHC2), 4.07 (t, J=7.2 Hz, 2H, N9CH2), 7.75 (s, 1H, C8H).

13CNMR (CD3OD, 300 Hz) δ(ppm): 13.89, 20.86, 22.96, 24.06, 32.89, 36.85, 44.53,

54.12, 55.97, 117.43, 139.45, 151.99, 153.47, 158.86. HRMS(ESI, M+H) Calcd for

C16H27N6O 319.2246; found 319.2254.

2-22e: 9-Benzyl-2-(2-(piperidin-1-yl)ethylamino)-1H-purin-6(9H)-one

1HNMR (CD3OD, 300 Hz) δ(ppm): 1.62 (m, 2H, H4’), 1.80-1.84 (m, 4H, H3’+H 5’), 3.30-

3.32 (m, 2H, NHCH2), 3.35-3.36 (m, 4H, H2’+H6’), 3.69 (t, J=5.8 Hz, 2H, N1’CH2), 5.27

(s, 2H, PhCH2), 7.29-7.34 (m, 5H, ArH), 7.81 (s, 1H, C8H). 13CNMR (CD3OD, 300 Hz)

δ(ppm): 23.37, 24.50, 24.58, 54.57, 56.76, 117.41, 128.81, 129.17, 129.94, 137.76,

139.71, 152.52, 154.02 HRMS(ESI, M+H) Calcd for C19H25N6O 353.2090; found

353.2091.

HN

N N

N

O

HN

PhN1'

2'

3'

4'

5'

6'

Figure 2.11 Structure of compound 2-22e

68

2-22f: 9-Butyl-2-(butylamino)-1H-purin-6(9H)-one

1HNMR (CDCl3, 300 Hz) δ(ppm): 0.94-1.03 (m, 6H, 2CH3), 1.38-1.52 (m, 4H,

2CH3CH2), 1.61-1.70 (m, 2H, CH2CH2NH), 1.91-2.01 (m, 2H, CH2CH2N9), 3.40-3.47

(m, 2H, CH2NH), 4.22 (t, J=7.2 Hz, 2H, CH2N9), 7.74 (s, 1H, C8H), 10.29, 13.08 (s+s,

2H, 2NH). 13CNMR (CD3OD, 300 Hz) δ(ppm): 13.34, 13.73, 19.57, 20.04, 30.96 (x2),

41.09, 45.28, 119.33, 133.05, 138.62, 150.61, 154.89. HRMS(EI) Calcd for C13H21N5O

263.1746; found 263.1743.

2-22g: 9-Benzyl-2-(butylamino)-1H-purin-6(9H)-one

1HNMR (DMSO-d6, 300 Hz) δ(ppm): 0.89 (t, J=7.3 Hz, 3H, CH3), 1.28-1.38 (m, 2H,

CH2), 1.44-1.54 (m, 2H, CH2), 3.24-3.32 (m, 2H, NHCH2), 5.16 (s, 2H, PhCH2), 7.33-

7.34 (m, 5H, ArH), 7.79 (s, 1H, C8H). 13CNMR (CDCl3, 500 Hz) δ(ppm): 13.16, 18.98,

30.38, 38.16, 45.52, 115.89, 127.17, 127.32, 128.05, 136.66, 136.73, 150.40, 152.27,

156.52. HRMS(EI) Calcd for C16H19N5O 297.1590; found: 297.1591.

2-23a: 9-Butyl-1H-purin-6(9H)-one

1HNMR (CD3OD, 300 Hz) δ(ppm): 0.97 (t, J=7.3 Hz, 3H, CH3), 1.29-1.42 (m, CH2),

1.82-1.92 (m, 2H, CH2), 4.25 (t, J=7.2 Hz, 2H, CH2N9), 8.03 (s, 1H, C8H), 8.06 (s, 1H,

C2H). 13CNMR (CD3OD, 500 Hz) δ(ppm): 19.19, 26.15, 38.66, 50.36, 130.55, 147.52,

151.84, 155.68, 164.43. HRMS(EI) Calcd for C9H12N4O 192.1011; found 192.1012.

69

2-23b: 9-Isopropyl-1H-purin-6(9H)-one

1HNMR (CD3OD, 300 Hz) δ(ppm): 1.61 (d, J=6.6 Hz, 6H, CH3+CH3), 8.03 (s, 1H,

C8H), 8.15 (s, 1H, C2H). 13CNMR (CD3OD, 300 Hz) δ(ppm): 21.87, 124.64, 139.05,

145.32, 148.95, 158.25. HRMS(EI) Calcd for C8H10N4O 178.0855; found 178.0855.

2-23c: 9-Butyl-2-iodo-1H-purin-6(9H)-one

1HNMR (CD3OD, 300 Hz) δ(ppm): 0.98 (t, J=7.2 Hz, 3H, CH3), 1.29-1.43 (m, 2H,

CH2CH3), 1.82-1.90 (m, 2H, CH2CH2N9), 4.24 (t, J=7.2 Hz, 2H, CH2N9), 8.33 (s, 1H,

C8H). 13CNMR (CD3OD, 500 Hz) δ(ppm): 13.74, 20.67, 32.98, 45.59, 108.88, 122.73,

141.39, 149.75, 157.85. HRMS(EI) Calcd for C9H11IN4O 317.9978; found: 317.9981.

2-23d: 2-Iodo-9-isopropyl-1H-purin-6(9H)-one

1HNMR (CD3OD, 500 Hz) δ(ppm): 1.59 (d, J=6.47 Hz, 6H, 2CH3), 4.75-4.79 (m, 1H,

CH), 8.08 (s, 1H, C8H). 13CNMR (CDCl3, 500 Hz) δ(ppm): 22.69, 48.82, 106.92, 124.95,

139.55, 149.66, 159.07. HRMS(ESI, M+H) Calcd for C8H10IN4O 304.9899; found:

304.9893.

2-25a: 9-Butyl-2-(3-hydroxy-3-methylpent-1-ynyl)-1H-purin-6(9H)-one

1HNMR (CD3OD, 300 Hz) δ(ppm): 0.94 (t, 3H, J=7.3 Hz, CH3CH2CH2), 1.08 (t, J=7.5

Hz, 3H, CH2CH3C), 1.11-1.37 (m, 2H, CH3CH2CH2), 1.54 (s, 3H, CCH3), 1.76-1.88 (m,

4H, CH3CH2C+NCH2CH2), 4.20 (t, J=7.3 Hz, 3H, N9CH2), 8.09 (s, 1H, C8H). 13CNMR

(CD3OD, 300 Hz) δ(ppm): 9.15, 13.81, 20.72, 28.72, 33.23, 37.03, 44.98, 69.29, 76.81,

70

97.63, 125.16, 139.76, 142.79, 149.86, 158.80. HRMS(ESI, M+H) Calcd for C15H21N4O2

289.1665; found 289.1661.

2-25b: 9-Benzyl-2-(3-hydroxy-3-methylpent-1-ynyl)-1H-purin-6(9H)-one

1HNMR (CD3OD, 300 Hz) δ(ppm): 1.09 (t, J=7.3 Hz, 3H, CH3CH2C), 1.54 (s, 3H,

CH3C), 1.76-1.86 (m, 2H, CH3CH2C), 5.39 (s, 2H, N9CH2), 7.28-7.34 (m, 5H, ArH), 8.11

(s, 1H, C8H). 13CNMR (CD3OD, 300 Hz) δ(ppm): 9.13, 28.70, 37.04, 69.32, 76.85,

97.75, 125.19, 128.77 (x2), 129.32, 129.99 (x2), 137.33, 140.11, 142.72, 158.86.

HRMS(ESI, M+H) Calcd for C18H18N4NaO2 345.1327; found 345.1322.

2-25c: 9-Benzyl-2-(hept-1-ynyl)-1H-purin-6(9H)-one

1HNMR (CDCl3, 300 Hz) δ(ppm): 0.89 (t, J=7.2 Hz, 3H, CH3), 1.25-1.66 (m, 6H, 3CH2),

2.42 (t, J=7.2 Hz, 2H, CCH2), 5.59 (s, 2H, N9CH2), 7.36 (s, 5H, ArH), 8.10 (s, 1H, C8H).

13C NMR (CD3OD) δ 13.87, 19.16, 22.09, 27.45, 31.03, 50.95, 74.19, 94.98, 128.12,

128.18, 128.74(×2), 129.15(×2), 135.27, 138.14, 154.81. HRMS(EI) Calcd for

C19H20N4O 320.1637; found 320.1633.

2-25d: 9-Butyl-2-(hept-1-ynyl)-1H-purin-6(9H)-one

1H NMR (CD3OD) δ 0.87-0.98(m, 6H, 2CH3), 1.28-1.38 (m, 6H, 3CH2), 1.60-1.71 (m,

4H, 2CH2), 1.82-1.92 (m, 6H, 3CH2), 2.49 (t, J=7.1 Hz, 2H, CCH2), 4.39 (t, J=7.3 Hz,

2H, N9CH2), 8.17 (s, 1H, C8H). 13C NMR (CD3OD) δ 13.84, 14.24, 19.72, 20.51, 23.22,

28.67, 30.74, 32.19, 34.41, 74.98, 95.41, 116.64, 130.69, 140.27, 145.67, 156.21.

HRMS(EI) Calcd for C16H22N4O 286.1794; found 286.1788.

71

2-26: 9-Butyl-8-(3-hydroxy-3-methylpent-1-ynyl)-1H-purin-6(9H)-one

1HNMR (CD3OD, 300 Hz) δ(ppm): 0.97 (t, J=7.3 Hz, 3H, CH3CH2CH2), 1.12 (t, J=7.5

Hz, 3H, CH3CH2C), 1.34-1.41 (m, CH3CH2CH2), 1.57 (s, 3H, CCH3), 1.78-1.89 (m, 4H,

CH3CH2C+NCH2CH2), 4.29 (t, J=7.3 Hz, 3H, N9CH2), 8.06 (s, 1H, C8H). 13CNMR

(CD3OD, 300 Hz) δ(ppm): 9.33, 13.95, 20.83, 28.99, 32.89, 37.16, 44.97, 69.48, 101.56,

124.99, 131.50, 132.26, 135.46, 147.27, 158.35. HRMS(EI, M+) Calcd for C15H20N4O2

288.1586; found 288.1586.

2.5.20 Biological evaluation of 2,9-disubstituted-6-oxopurines as MRP4 inhibitor

2.5.20.1 Cell lines and culture conditions

HepG2 cells stably expressing the human MRP4 protein were previously described [20].

The MRP4 clone used in this study was MRP4/HepG2. The blasticidine clone, V/HepG2

which was transfected with the pcDNA6 vector was included as the control. Cells were

routinely grown in complete medium consisting of Dulbecco’s Modified Eagle Medium

(DMEM), 1 mM sodium pyruvate, 2 mM glutamine, 0.1 mM non-essential amino acids,

100 units/mL penicillin, 100 μg/mL streptomycin, 10% fetal bovine serum and 0.25

μg/mL blasticidin. The cells were grown at 37oC in a humidified atmosphere of 95% air

and 5% CO2.

2.5.20.2 Bimane-glutathione efflux

The measurement of bimane-GS synthesis and efflux from MRP4/HepG2 and V/HepG2

cells was carried out as previously described.[10d] In brief, cells were seeded in triplicate

72

at a density of 6 x 105 cells per well into 6-well plates and incubated at 37ºC for 24 h. The

cells were then incubated with 1 mL DMEM medium containing 100 μM

monochlorobimane (MCB) at 10oC for 60 min with different concentrations of the purine

derivatives. Controls consist of cells incubated with DMSO, which was used to dissolve

the compounds. After pretreatment with MCB, the plates were placed on ice, the medium

was removed and the cells were washed with cold Hank’s balanced salt solution (HBSS),

without glucose twice. The cells were then incubated with 0.6 mL HBSS containing 5.6

mM glucose and different concentrations of synthesized compounds at 37oC for 5 min.

0.2 mL of the incubation buffer and 0.2 mL of the cell lysate (in 0.2% sodium dodecyl

sulfate, SDS) were collected. The bimane-GS content in the sample was measured by

determining the fluorescence intensity at an excitation wavelength of 385 nm and an

emission wavelength of 478 nm in a Gemini XS microplate spectrofluorometer from

Molecular Devices Corp, USA. A series of bimane-GS standards was used to generate a

calibration curve for quantifying the amount of bimane-GS. The protein determination

was carried out using the Bio-Rad Protein Dye with bovine serum albumin dissolved in

0.2% SDS as the standard.

2.5.20.3 Effects of the purine derivatives on 6TG resistance

Cells were plated in triplicate at the density of 4 x 103 per well in 96-well tissue culture

plate. After 24 h of incubation at 37ºC, the cells were treated with 6TG or the purine

derivative or both. Control cells were incubated either with medium containing 0.1%

DMSO or in medium only. 48 h later, 20 μL of 3-(4,5-dimethylthiazol-2-yl)-5-(3-

carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium /phenazine ethosulfate

73

(MTS/PES) reagent (Promega) was added to each well. After incubation at 37ºC for 60

min, absorbance was measured at 490 nm. The data were used to calculate the 50%

growth inhibitory concentration (IC50). At least five 6TG concentrations were used to

determine the IC50 value.

74

2.6 References

[1] (a) Veseley, J,; Havlicek, L.; Strnad, M.; Blow, J.J.; Donella-Deana, A.; Pinna,

L.; Letham, D.S.; Kato, J.-Y.; Detivaud, L.; Leclerc, S.; Meijer, L. Eur. J.

Biochem. 1994, 224, 771-786. (b) De Azevedo, W. F.; Leclerc, S.; Meijer, L.

Havlicek, L.; Strnad, M.; Kim, S.H. Eur. J. Biochem. 1997, 243, 518-526. (c)

Legraverend, M.; Ludwig, O.; Bisagni, E.; Leclerc, S.; Meijer, L.; Giocanti,

N.; Sadri, R and Favaudon, V. Bioorg. Med. Chem. 1999, 7, 1281-1293. (d)

Sielecki, T. M.; Boylan, J. F.; Benfield, P. A. and Trainor, G. L. J. Med. Chem.

2000, 43, 1-18.

[2] Dhainaut, A.; Regnier, G,; Tizot, A.;Pierre, A.; Leonce, S.; Guilbaud, N.;

Kraus-Berthier, L.; Atassi, G. J. Med .Chem. 1996, 39, 4099-4108.

[3] Dean, M.; Annilo, T. Annu. Rev. Genomics Hum. Genet. 2005, 6, 123-142.

[4] Gottesman, M.; Ling, V. FEBS Lett. 2006, 580, 998-1009.

[5] (a) Polgar, O.; Bates, S. Biochem. Soc. Trans. 2005, 33, 241-245. (b) Haimeur,

A.; Conseil, G.; Deeley, R.; Cole, S. Curr. Drug Metab. 2004, 5, 21-53.

[6] Kruh, G.; Belinsky, M. Oncogene 2003, 22, 7537-7552.

[7] Deeley, R.; Cole, S. FEBS Lett. 2006, 580, 1103-1111.

[8] Ritter, C.; Jedlitschky, G.; Meyer zu Schwabedissen, H.; Grube, M.; Kock, K.;

Kroemer, H. Drug Metab. Rev. 2005, 37, 253-278.

[9] Tian, Q.; Zhang, J.; Tan, T.; Chan, E.; Duan, W.; Chan, S.; Boelsterli, U.; Ho,

P.; Yang, H.; Bian, J.; Huang, M.; Zhu, Y.; Xiong, W.; Li, X.; Zhou, S.

Pharm. Res. 2005, 22, 1837-1853.

75

[10] (a) Zelcer, N.; Reid, G.; Wielinga, P.; Kuil, A.; van der Heijden, I.; Schuetz, J.;

Borst, P. Biochem. J. 2003, 371, 361-367. (b) Rius, M.; Hummel-Eisenbeiss, J.;

Hofmann, A.; Keppler, D. Am. J. Physiol. Gastrointest. Liver Physiol. 2006,

290, G64064-9. (c) Reid, G.; Wielinga, P.; Zelcer, N.; van der Heijden, I.; Kuil,

A.; de Haas, M.; Wijnholds, J.; Borst, P. Proc. Natl. Acad. Sci. U. S. A. 2003,

100, 9244-9249. (d) Bai, J.; Lai, L.; Yeo, H.; Goh, B.; Tan, T. Int. J. Biochem.

Cell Biol. 2004, 36, 247-257.

[11] (a) Reid, G.; Wielinga, P.; Zelcer, N.; De Haas, M.; van Deemter, L.;

Wijnholds, J.; Balzarini, J.; Borst, P. Mol. Pharmacol. 2003, 63, 1094-1103. (b)

Chen, Z.; Lee, K.; Kruh, G.; J. Biol. Chem. 2001, 276, 33747-33754. (c)

Adachi, M.; Sampath, J.; Lan, L.; Sun, D.; Hargrove, P.; Flatley, R.; Tatum, A.;

Edwards, M.; Wezeman, M.; Matherly, L.; Drake, R.; Schuetz, J. J. Biol.

Chem. 2002, 277, 38998-39004.

[12] Brun, V.; Legraverend, M.; Grierson, D. S. Tetrahedron 2002, 58, 7911-7923.

[13] Kiburis, J.; Lister, J. H. J. Chem. Soc. C 1971, 1587-1589.

[14] Lembicz, N. K.; Grant, S.; Clegg, W.; Griffin, R. J.; Heath, S. L.; Golding, B.

T. J. Chem. Soc. Perkin Trans.1 1997, 185-186.

[15] Kjellberg, J.; Liljenberg, M. Tetrahedron Lett. 1989, 27, 877-880.

[16] Kato, K.; Hayakawa, H.; Tanaka, H.; Kumamoto, H.; Shindoh, S.; Shuto, S.;

Miyasaka, T. J. Org. Chem. 1997, 62, 6833-6841.

[17] Bork, J. T.; Lee, J. W.; Chang, Y-T. QSAR Comb. Sci. 2004, 23, 245-260.

[18] Brill, W. K-D.; Riva-Toniolo, C. Tetrahedron Lett. 2001, 42, 6279-6282.

76

[19] (a) Schuetz, J.; Connelly, M.; Sun, D.; Paibir, S.; Flynn, P.; Srinivas, R.;

Kumar, A.; Fridland, A. Nat. Med. 1999, 5, 1048-1051. (b) Lai, L.; Tan, T.

Biochem. J. 2002, 361, 497-503. (c) Wielinga, P.; Reid, G.; Challa, E.; van der

Heijden, I.; van Deemter, L.; de Haas, M.; Mol, C.; Kuil, A.; Groeneveld, E.;

Schuetz, J.; Brouwer, C.; de Abreu, R.; Wijnholds, J.; Beijnen, J.; Borst, P.

Mol. Pharmacol. 2002, 62, 1321-1331.

[20] Tan, L. L. T. Biochem. J. 2002, 361, 497-503.

77

CHAPTER 3: Traceless Solid-phase Synthesis of 1,7-Disubstituted

Purines

3.1 Introduction

N

N NH

N

9H-purine 7H-purine

1

2

3

4

56

7

8

9

N

N N

HN

1

2

3

4

56

7

8

9

Figure 3.1

Although N9-substituted purines are the natural nucleosides, in recent years, there has

been much interest in the N7 regioisomers as these isomers have found applications as

antiviral agents.[1] N7 guanines are also important DNA adducts formed as a result of

exposure to electrophiles.[2] Consequently, N7-alkylguanines are the main types of DNA

adducts excreted in urine and are therefore important markers for the development of

diagnostic methods to detect and quantitate specific types of DNA damages.[3] Studies of

1,7-disubstituted guanines have shown that they are capable of inhibiting telomerase

activity and possess the ability to enhance the efficacy of other chemotherapeutic agents

in the treatment of cancer.[2] Earlier studies have shown that under kinetic control,

alkylation of purines gives predominantly the N9 isomer.[4] Hence selective N7 alkylation

of purine is important for achieving high regioselectivity.[5] Some strategies in the

literature were known to achieve regioselective N7 alkylation in solution phase.[1,6]

78

However, to our knowledge there has been no prior reports on the solid-phase selective

N7 alkylation.

3.2 Outline of our strategy

We herein describe a traceless solid-phase route for the synthesis of 1,7-disubstituted

purines. The linkage strategy involves the use of the REM resin which has been widely

used in the synthesis of tertiary amines.[7] It generally involves 1) coupling the starting

secondary amine to REM linker via Michael addition; 2) further alkylation of the

attached amine; 3) quaternization of the resultant amine and 4) Hofmann elimination to

release the target products (Scheme 3.1).

OH

Cl

O

DiEA/DCMO

O

O

O

N

HNR1R2

O

O

N+

R3X

DMF

DMF

R1

R2

R1

R2

R3DiEA/DCM

NR1

R2

R3

Scheme 3.1 REM resin cycle

Our synthetic route was designed such that the linker was anchored at the N9 position of

the purine scaffold, leaving the N7 position as the steric priority to be alkylated to form a

quaternary salt, thus allowing a highly regioselective N7 synthesis to be achieved

(Scheme 3.2).

79

O

OX-

N

N

O

N

N

R1R2

N

N

O

O

N

N

O

R1 R2

X= I orBr Scheme 3.2 Overall strategy

3.3 Results and discussions

3.3.1 Solution-phase synthesis study

Prior to the solid-phase synthesis, preliminary solution-phase studies were carried out to

survey the requisite reaction conditions and establish the modifications required for solid-

phase synthesis. The solution-phase synthetic route is summarized in Scheme 3.3.

N

NHN

N

Cl

N

N

Cl

O

N

N

OPh

O

O

Ph

N

N

X

O

HN

N

OPh

N

N

O

O

N

N

OPh

C4H9

N

N

O

O

N

N

OPh

C4H9

C4H9

I-

N

N

O

N

N

C4H9

C4H9

nBuI, DBU

DMF, rt

3-1DiEA, DMF

3-2

3-3a X=O3-3b X=S 3-4

3-5

+

3-6a

NH3/MeOH

70oC

nBuI, DMF

OHPh

acryloyl chloride

DiEA

Scheme 3.3 Solution-phase study

80

3.3.1.1 Synthesis of benzyl acrylate (3-1):

In the first step, benzyl acrylate (3-1) was chosen to mimic the structure of the REM

resin. 3-1 was easily synthesized according to the standard procedure for the preparation

of REM resin from the Wang resin.[7]

3.3.1.2 Synthesis of benzyl 3-(6-chloro-9H-purin-9-yl)propanoate (3-2)

Treatment of 3-1 with 6-chloropurine, in the presence of base, yielded 3-2 via Michael

addition. Table 3.1 shows the various reaction conditions examined for this reaction.

Table 3.1 Synthesis of compound 3-2

Reaction condition Yield of 3-2 (%)

1 No base/DMF -

2 LiH/DMF -

3 NaOCH3/DMF, rt -a

4 K2CO3/DMF, rt <10

5 K2CO3/DMF/18-crown-6 60

6 DiEA/DMF, rt 74

7 DiEA/DMF, 70°C 60b a) 3-(6-chloro-purin-9-yl)-propionic acid was obtained. b) N7 alkylated regioisomer was also

observed.

LiH gave no product at either room temperature or elevated temperatures, whilst

NaOCH3 gave 3-(6-chloro-purin-9-yl)-propionic acid, the ester hydrolysis product. The

results of Entry 2 and 3 indicated both LiH and NaOCH3 are too strong bases for this

Michael addition. Thus weaker bases should be inspected. Although reaction with

K2CO3/DMF gave very low yield (<10%), it provided 3-2 in 60% yield when 18-crown-6

was applied as a phase transfer catalyst (Entry 5). Therefore K2CO3/DMF/18-crown-6

was satisfactory for solution-phase synthesis. However, it may not be an appropriate

81

system for the solid-phase synthesis because K2CO3 is only sparingly soluble in DMF.

Since our aim is to develop a procedure for solid-phase synthesis, it is necessary to

choose a base that would dissolve well in a solvent which as good resin swelling effect.

Further investigation provided DiEA/DMF which gave 3-2 in 74% yield when the

reaction was carried out at room temperature (Entry 6). However, the yields were lower

at higher temperatures and the N7 alkylated regioisomer was observed at temperatures

above 70oC (Entry 7). These results suggest that DiEA/DMF/room temperature is the best

reaction condition for solid-phase synthesis.

3.3.1.3 Synthesis of benzyl 3-(6-oxo-1,6-dihydropurin-9-yl)propanoate (3-3a)

We next examined the hydrolysis of 3-2. Hydrolysis of chloride to hydroxyl group can be

carried out under both basic and acidic condition. Since compound 3-2 contains an ester

bond, acidic condition was chosen. Both TFA and formic acid were examined. When

50% TFA (aq.) was used, the reaction was completed after stirring at room temperature

for 24 h and 3-3a was obtained in 30% yield. This low yield may be due to the partial

hydrolysis of the ester bond since TFA is a strong acid. When 85% formic acid (aq.) was

used, the reaction was completed after reflux 3 h and 3-3a was obtained in 96% yield.

The IR spectrum of 3-3a showed C=O stretch at 1724 cm-1 and 1692 cm-1 which could be

attributed to the ester carbonyl and cyclic amide carbonyl groups respectively (Scheme

3.4).

82

N

N N

N

OO

Ph

Cl

N

N N

N

OO

Ph

OH

HN

N N

N

OO

Ph

O

1692cm-1

1724cm-1

85%HCOOH/H2O

3-23-3a

Scheme 3.4 Hydrolysis of compound 3-2 with formic acid

The synthesis of benzyl 3-(6-thioxo-1,6-dihydropurin-9-yl)propanoate 3-3b was also

studied. 3-2 reacted readily with thiourea in ethanol to give 3-3b as a pale yellow solid in

85% yield.

3.3.1.4 Synthesis of benzyl 3-(1-butyl-6-oxo-1,6-dihydropurin-9-yl)propanoate (3-4)

N1-alkylation of 3-3a was examined with butyl bromide as the alkylation reagent. The

reaction could be carried out with either DBU/DMF at room temperature or K2CO3/DMF

at 90oC to provide 3-4 in over 90% yields. The structure of 3-4 was confirmed by

NOESY experiments which clearly showed the CH2N1/HC2 interaction (Figure 3.2).

However, when NaOEt was used as base, product 3-4 was isolated in only 37% yield. A

side product 3-4a isolated during N1 alkylation gives NMR and MS data which are

similar to compound 3-4. However the NOESY spectrum of compound 3-4a shows no

CH2N1/HC2 interaction which suggests that the side product 3-4a could be the O-

alkylated product (Figure 3.3).

83

N

N N

N

OO

OH

H

H

3-4

Figure 3.2 NOESY spectrum of compound 3-4

84

N

N N

N

OO

O

H

3-4a

H

H

Figure 3.3 NOESY spectrum of compound 3-4a

85

Analogous alkylation of compound 3-3b gave only one product. NOESY experiment of

this product, however, showed it was the S-alkylated product 3-4b (Figure 3.4).

N

N N

N

OO

S

H

H

H

3-4b

Figure 3.4 NOESY spectrum of compound 3-4b

86

3.3.1.5 Synthesis of 3-(1,7-dibutyl-6-oxo-1,6-dihydro-purin-9-yl)-propionic acid

phenyl ester salt (3-5)

We next proceeded to N7 alkylation to form a quaternary salt. A number of

quaternization conditions were experimented (Table 3.2) and nBuI/ 70oC / 18 h provided

the most favorable condition. Since the quaternization process occurred rather slowly, a

large excess (20 equiv.) of the alkylation reagent was used to ensure complete conversion

to 3-5.

Table 3.2 Solution-phase synthesis of compound 3-5

Reaction conditiona Yield of compound 3-5(%)

1 nBuBr, 50°C, 48 h 52

2 nBuBr, NaI, 50°C, 24 h 71

3 nBuI, 50°C, 18 h 73

4 nBuI, 70°C, 18 h 100 a) Equivalence of alkylating reagents was 20 equiv..

When 1-bromobutane was used as an alkylating reagent and the reaction mixture was

heated at 50°C, only partial quaternization was observed even after 2 days. TLC showed

the existence of starting material and the quaternary salt was isolated in 52% yield.

Addition of NaI as a catalyst shortened the reaction time to 24 h, enabled complete

consumption of the starting material and provided the quaternized product in 71% yield

(Entry 2). Quaternization with 1-iodobutane gave similar results except for a shorter

reaction time (18 h). However when the reaction temperature was raised to 70°C,

quaternization of 3-4 with 1-iodobutane proceeded smoothly to give 3-5 in quantitative

yield (Entry 4).

87

Quaternization resulted in a purinium salt with an electron-deficient imidazole ring.

Consequently, the chemical shift of C8H was shifted to a lower field at 10.63 ppm

(compared to the chemical shift of C8H before quaternizaiton which was at 7.78 ppm).

3.3.1.6 Synthesis of 1,7-dibutyl-1,7-dihydro-purin-6-one (3-6a)

Subsequent treatment of 3-5 with TEA or DiEA was expected to give the final product 3-

6a via Hoffmann elimination. However the yields obtained were very low (<20%) and

the major product isolated was the imidazolium ring-opened compound 3-7 which was

characterized using NMR and MS. The proposed mechanism for the formation of 3-7 is

shown in Scheme 3.5.

NH

N

O

O

N

N

OBn

C4H9

C4H9

H

3-7

ON

N N

N

OOBn

O

C4H9

C4H9

3-5

X-N

N N

N

OOBn

O

C4H9

C4H9

H

OH

Scheme 3.5 Formation of side product 3-7 in solution-phase cleavage step

The formation of 3-7 may be attributed to the electron-deficient C8 which causes the

imidazolium structure to be unstable even under mild basic conditions.[8] To effect the

formation of 3-6a, ammonia in methanol, a milder base, was chosen and upon stirring for

12 h, compound 3-6a was obtained in 72% yield. The mechanism for the Hoffmann

elimination of 3-5 is illustrated in Scheme 3.6.

88

N

N N

N

OO

Ph

C4H9

HN

N N

N

C4H9

OO

Ph+

B

OOC4H9

C4H9

3-5 3-6a3-1

Scheme 3.6 Hoffmann elimination of compound 3-5

Thus a solution-phase study oriented to solid-phase synthesis was successfully

established. All the intermediates and final compound were obtained in good yield and

high purity.

3.3.2 Solid-phase study

N

NHN

N

Cl

N

N

Cl

O

N

N

O

OHCl

O

O

O

85% HCOOH/H2O

X-

N

N

O

N

N

R1R2

N

N

O

O

N

N

O

R1R2

DMF

DiEA, DMF3-8

3-9

3-6

N

N

O

O

HN

N

O3-10

N

N

O

O

N

N

O3-11

R1 R2X/DMF

3-12

X = I or Br

NH3/MeOH

R1Br, DBU

DMF

Scheme 3.7 Solid-phase study

89

With the solution-phase synthetic pathway well established, the solid-phase synthesis was

evaluated. As outlined in Scheme 3.7, REM resin 3-8 was developed by treating the

Wang resin with acryloyl chloride in the presence of DiEA. The formation of 3-8 was

amenable to KBr FTIR monitoring (i.e. appearance of carbonyl stretch at 1724 cm-1). 3-8

was then reacted with 6-chloropurine to give 3-9 which was subsequently hydrolyzed

with formic acid/H2O/DMF. Compared with the solution-phase reaction condition, DMF

was added as a co-solvent in this hydrolysis step to facilitate resin swelling. This is

because in the solution-phase hydrolysis, compound 3-2 easily dissolved in 85% aqueous

formic acid and therefore the reaction mixture is homogeneous. However solid-phase

hydrolysis is a heterogeneous reaction and resin swellability is important. In this case

DMF not only gave good swelling of the Wang resin but was also inert to the hydrolysis

reaction. The resin-bound hydrolyzed intermediate 3-10 was subsequently alkylated at the

N1 position under basic condition followed by quaternization at N7 with different alkyl

halides. When a bromide was used, the addition of sodium iodide as catalyst to the

quaternization mixture improved the efficiency of the solid-phase alkylation. Our study

also showed that lowering the temperature from 70oC to 60oC during quaternization gave

less side products upon cleavage. The final product 3-6 was finally released from the

resin by an overnight treatment with 2 M ammonia in methanol.

To illustrate the general applicability of this synthetic procedure, a small library of 15

compounds (3-6a ~3-6o) was prepared on solid-phase (Figure 3.5). The overall yields

obtained were 13-27% (purities of >95% by NMR), indicating an average yield of at least

70% for each step of the reaction.

90

N

N N

N

O

C7H15

N

N

O

N

N

C4H9

C4H9

N

N N

N

O

Ph

N

N N

N

O Ph

N

N N

N

O Ph

C7H15

N

N N

N

C4H9O

N

N N

N

PhO

N

N N

N

OC4H9

N

N N

N

O Ph

3-6a 18% 3-6b 17% 3-6c 15%

3-6d 20% 3-6e 19% 3-6f 18%

3-6g 14% 3-6h 16% 3-6i 18%

3-6j 13% 3-6k 13% 3-6l 27%

3-6m 20% 3-6n 15% 3-6o 16%

N

N

O

N

N

C4H9

N

N

O

N

N

C6H5

C4H9

N

N

O

N

N

C4H9

C7H15

N

N

O

N

N

C4H9

Ph

N

N

O

N

N

C4H9

N

N N

N

O C4H9

Figure 3.5 Library of 1,7-disubstituted-6-oxopurine

91

Figure 3.6 X-ray crystal structure of 3-6f

Figure 3.6 depicts the X-ray crystal structure and defines the atomic numbering of

compound 3-6f. It gives direct evidence that regioselective alkylation had occurred at the

N7 position rather than the N9 position.

3.4 Conclusion

In summary, a novel and facile solid-phase synthetic procedure has been developed for

the synthesis of N1, N7-disubstituted-purine using REM resin. Key steps in the synthetic

strategy involve (i) coupling of 6-chloropurine to the REM resin (Michael addition), (ii)

hydrolysis, (iii) N1-alkylation, (iv) quaternization and (v) product release through

92

Hofmann elimination. This study gave the first example of a highly regioselective solid-

phase synthesis of 1, 7-disubstituted purine derivatives. Using this method a library of

1,7-disubstituted-6-oxopurine was successfully synthesized.

3.5 Experimental

General Procedures. 1HNMR and 13CNMR spectra were measured at 298K on a Bruker

DPX 300 Fourier Transform spectrometer. Chemical shifts were reported in δ (ppm),

relative to the internal standard of tetramethylsilane (TMS). All Infra-red (IR) spectra

were recorded on a Bio-Rad FTS 165 spectrometer. Mass spectra were performed on VG

Micromass 7035 spectrometer under electron impact (EI). All chemical reagents were

obtained from commercial suppliers and used without further purification. Analytical

TLC was carried out on pre-coated plates (Merck silica gel 60, F254) and visualized with

UV light. Flash column chromatography was performed with silica (Merck, 70-230

mesh).

3.5.1 Synthesis of benzyl acrylate (3-1)

DiEA (4.79 g, 37.04 mmol) and acryloyl chloride (3.35 g, 37.04 mmol) were added to a

solution of benzyl alcohol (0.50 g, 4.63 mmol) in CH2Cl2 (30 mL). The reaction mixture

was stirred at room temperature for 4 h and then concentrated to dryness. The brown

solid obtained was dissolved in EtOAc and water. The aqueous layer was extracted with

EtOAc and the combined organic layer obtained was washed with brine, dried with

MgSO4, concentrated to dryness and purified by column chromatography (EtOAc:hexane

= 1:2). 3-1 was obtained as a colorless oil (0.69 g, 92%). 1HNMR (CDCl3): δ(ppm) 5.17

93

(s, 2H, PhCH2), 5.79 (dd, J=1.2 Hz, J=10.4 Hz, 1H, CHCH2), 6.14 (dd, J=10.5 Hz,

J=17.3 Hz, 1H, CH), 6.42 (dd, J=1.2 Hz, J=17.3 Hz, 1H, CHCH2), 7.27-7.35 (m, 5H,

ArH). 13CNMR (CDCl3): δ(ppm) 66.08, 128.04 (x2), 128.15, 128.37, 130.81, 135.75,

165.72. HRMS(EI): Calcd for C10H10O2 162.0681; found: 162.0680.

3.5.2 Synthesis of benzyl 3-(6-chloro-9H-purin-9-yl)propanoate (3-2)

Compound 3-1 (0.16 g, 1.0 mmol) and DiEA (0.14 g, 1.1 mmol) were added to a solution

of 6-chloropurine (0.17 g, 1.1 mmol) in DMF (2 mL). The reaction mixture was stirred at

room temperature for 48 h and then extracted with EtOAc and water. The combined

organic layer was washed with brine, dried with MgSO4, concentrated to dryness and

purified by column chromatography (EtOAc:hexane = 1:2) to give 3-2 as a white solid

(0.26 g, 74%). 1HNMR (CDCl3): δ(ppm) 3.03 (t, J=6.0 Hz, 2H, N9CH2CH2), 4.60 (t, 2H,

J=6.2 Hz, N9CH2), 5.10 (s, 2H, PhCH2), 7.24-7.36 (m, 5H, ArH), 8.21 (s, 1H, C8H), 8.71

(s, 1H, C2H). 13CNMR (CDCl3): δ(ppm) 33.69, 39.89, 67.01, 128.28, 128.53 (x2),

131.52, 134.90, 145.90, 150.89, 151.56, 151.76, 170.30. HRMS(EI): Calcd for

C15H13ClN4O2 316.0727; found: 316.0723.

3.5.3 Synthesis of benzyl 3-(6-oxo-1,6-dihydropurin-9-yl)propanoate (3-3a)

Compound 3-2 (0.44 g, 1.4 mmol) was dissolved in 80% formic acid (13 mL) and the

reaction mixture was stirred at 70oC for 3 h and then evaporated to dryness. The white

solid obtained was extracted with EtOAc and water. The combined organic layer was

washed with brine, dried with MgSO4, concentrated to dryness and purified by column

chromatography (MeOH:CH2Cl2 = 1:8) to provide 3-3a as a white solid (0.40 g, 96%).

94

1HNMR (DMSO-d6): δ(ppm) 3.02 (t, J=6.8 Hz, 2H, N9CH2CH2), 4.40 (t, J=6.8 Hz, 2H,

N9CH2), 5.07 (s, 2H, PhCH2), 7.26-7.35 (m, 5H, ArH), 8.02 (s, 1H, C8H), 8.04 (s, 1H,

C2H), 12.26 (s, 1H, N1H). 13CNMR (CDCl3): δ(ppm) 33.27, 38.83, 65.34, 123.44,

127.46, 127.57, 127.89, 135.30, 139.81, 145.03, 147.82, 156.14, 169.86. HRMS(EI):

Calcd for C15H14N4O3 298.1066; found: 298.1079.

3.5.4 Synthesis of benzyl 3-(6-thioxo-1,6-dihydropurin-9-yl)propanoate (3-3b)

Compound 3-2 (0.429 g, 1.36 mmol) was dissolved in absolute ethanol followed by

addition of thiourea (0.83 g, 10.86 mmol). The reaction mixture was stirred under reflux

for 3 h and then cooled under ice water bath until solid precipitated. The solid was

filtered and washed with cold ethanol to give compound 3-3b as a pale yellow solid (0.36

g, 85%). 1HNMR (DMSO-d6, 300 Hz) δ(ppm): 3.04 (t, 2H, J=7.0 Hz, N9CH2CH2), 4.43

(t, J=7.0 Hz, 2H N9CH2), 5.06 (s, 2H, PhCH2), 7.25-7.35 (m, 5H, ArH), 8.16 (s, 1H,

C8H), 8.25 (s, 1H, C2H). 13CNMR (DMSO-d6, 300 Hz) δ(ppm): 33.04, 38.82, 65.37,

127.46, 127.57, 127.88, 134.45, 135.25, 142.57, 143.58, 144.36, 169.81, 175.27.

HRMS(EI): Calcd for C15H14N4O2S 314.0837; found: 314.0836.

3.5.5 Synthesis of benzyl 3-(1-butyl-6-oxo-1,6-dihydropurin-9-yl)propanoate (3-4)

1-Butyl bromide (47.40 mg, 0.346 mmol) and DBU (52.70 mg, 34.6 mmol) were added

to the solution of 3-3 (86 mg, 0.29 mmol) in DMF (2 mL). The reaction mixture was

stirred at room temperature overnight and then extracted with EtOAc and brine. The

combined organic layer was washed with brine, dried with MgSO4, concentrated to

dryness and purified by column chromatography (EtOAC:hexane = 1:1 followed by

95

MeOH:CH2Cl2 = 1:10) to provide 3-4 as a pale yellow oil (92.90 mg, 91%). 1HNMR

(CDCl3): δ(ppm) 0.89 (t, J=7.3 Hz, 3H, CH3), 1.26-1.39 (m, 2H, CH2CH3), 1.65-1.75 (m,

2H, CH2CH2CH3), 2.90 (t, J=6.3 Hz, 2H, N9CH2CH2), 3.99 (t, J=7.3 Hz, 2H, N1CH2),

4.42 (t, J=6.4 Hz, 2H, N9CH2), 5.05 (s, 2H, PhCH2), 7.22-7.36 (m, 5H, ArH), 7.78 (s,

1H, C8H), 7.99 (s, 1H, C2H). 13CNMR (CDCl3): δ(ppm) 13.44, 19.58, 31.62, 34.25,

39.41, 46.52, 66.78, 124.21, 128.21, 128.33, 128.44, 134.96, 140.25, 146.86, 147.33,

156.26, 170.19. HRMS(EI): Calcd for C19H22N4O3 354.1692; found: 354.1675.

Benzyl 3-(6-butoxy-9H-purin-9-yl)propanoate (3-4a): 1HNMR (CDCl3): δ(ppm) 0.97

(t, J=7.3 Hz, 3H, CH3), 1.49-1.56 (m, 2H, CH2CH3), 1.87-1.92 (m, 2H, CH2CH2CH3),

2.99 (t, J=6.3 Hz, 2H, COCH2), 4.54 (t, J=7.3 Hz, 2H, OCH2), 4.58 (t, J=6.4 Hz, 2H,

N9CH2), 5.08 (s, 2H, PhCH2), 7.25-7.34 (m, 5H, ArH), 7.95 (s, 1H, C8H), 8.49 (s, 1H,

C2H). 13CNMR (CDCl3): δ(ppm) 13.76, 19.12, 30.84, 34.09, 39.55, 66.96, 67.06, 109.40,

121.45, 128.38, 128.51, 128.60, 135.09, 142.58, 152.03, 161.02, 170.54.

Benzyl 3-(6-(butylthio)-9H-purin-9-yl)propanoate (3-4b): 1HNMR (CDCl3): δ(ppm)

0.91 (t, J=7.3 Hz, 3H, CH3), 1.40-1.45 (m, 2H, CH2CH3), 1.47-1.70 (m, 2H,

CH2CH2CH3), 2.94 (t, J=6.1 Hz, 2H, COCH2), 3.34 (t, J=7.3 Hz, 2H, SCH2), 4.49 (t,

J=6.3 Hz, 2H, N9CH2), 5.04 (s, 2H, PhCH2), 7.20-7.29 (m, 5H, ArH), 7.97 (s, 1H, C8H),

8.63 (s, 1H, C2H). 13CNMR (CDCl3): δ(ppm) 13.49, 21.77, 28.19, 31.30, 33.82, 39.30,

66.76, 109.40, 128.15, 128.30, 128.41, 131.16, 134.94, 143.01, 147.96, 151.58, 161.36,

170.34. HRMS(EI): Calcd for C19H22N4O2S 370.1463 found: 370.1457.

96

3.5.6 Synthesis of 3-(1,7-dibutyl-6-oxo-1,6-dihydro-purin-9-yl)-propionic acid phenyl

ester salt (3-5)

1-Iodobutane (2.94 g, 16 mmol) was added to the solution of 3-4 (0.28 g, 0.8 mmol) in

DMF (7 mL). The reaction mixture was stirred at 70oC for 24 h and then evaporated to

dryness. The residue was purified by column chromatography (MeOH:CH2Cl2 = 1:15) to

give 3-5 as a yellow solid (0.43 g, 100%). 1HNMR (CDCl3): δ(ppm) 0.97 (t, J=7.3 Hz,

6H, CH3), 1.37-1.47 (m, 4H, CH2), 1.70-1.80 (m, 2H, CH2), 1.94-2.05 (m, 2H, CH2), 3.24

(t, J=6.4 Hz, 2H, CH2), 4.07 (t, J=7.5 Hz, 2H, CH2), 4.61 (t, J=7.5 Hz, 2H, CH2), 4.84 (t,

J=6.4 Hz, 2H, CH2), 5.09 (s, 2H, PhCH2), 7.31-7.32 (m, 5H, ArH), 8.23 (s, 1H, C2H),

10.63 (s, 1H, C8H). 13CNMR (CDCl3): δ(ppm) 13.34, 13.44, 19.41, 19.70, 31.44, 32.07,

33.15, 42.37, 47.52, 50.13, 67.08, 114.04, 128.42, 128.46, 128.54, 135.12, 141.46,

146.52, 150.83, 152.05, 169.84. HRMS(EI): Calcd for C23H31N4O3 411.2396; found:

411.2391.

3.5.7 Synthesis of 1,7-dibutyl-1,7-dihydro-purin-6-one (3-6a)

3-5 (0.12 g, 0.23 mmol) was dissolved in the solution of 2 M ammonia in methanol (2.3

mL). Reaction was stirred at room temperature for 24 h and then evaporated to dryness.

The residue was purified by column chromatography (CH3CN:CH2Cl2 =1:1 followed by

MeOH:CH2Cl2 = 1:15) to give 3-6a as a white solid (41.00 mg, 72%).

Benzyl 3-(1-butyl-5-(N-butylformamido)-6-oxo-1,6-dihydropyrimidin-4-ylamino)

propanoate (3-7): 1HNMR (CDCl3): δ(ppm) 0.83-0.95 (m, 6H, 2CH3), 1.23-1.72 (m, 8H,

4CH2), 2.62 (t, 2H, COCH2), 3.30-3.88 (m, 6H, 2CH2+NHCH2), 5.09 (s, 2H, PhCH2),

97

5.73 (t, 1H, NH), 7.30-7.31(m, 5H, ArH), 7.82 (s, 1H, C8H), 7.85 (s, 1H, C2H). MS(EI):

m/z 277(M+), 428.2, 400.3, 91.1, 265.1.

3.5.8 General procedure for the preparation of the REM resin (3-8)

Wang resin (loading 1.47 mmol/g) was swollen in CH2Cl2. DiEA (8 equiv.) and acryloyl

chloride (8 equiv.) were added and the reaction mixture was shaken at room temperature

for 4 h. After which, the mixture was filtered and the resin washed sequentially with

DMF (20 mL x 2), H2O (20 mL x 2), EtOH (20 mL x 2), CH2Cl2 (20 mL x 2) and Et2O

(20 mL x 2), and dried overnight in a vacuum oven at 40°C to afford resin 3-8.

3.5.9 General procedure for the preparation of resin bound benzyl 3-(6-chloro-9H-

purin-9-yl)propanoate (3-9)

3-8 was swollen in DMF and 6-chloropurine (2 equiv.) and DiEA (2 equiv.) were added.

The reaction mixture was shaken at room temperature for 48 h and then filtered and

washed with DMF (20 mL x 2), EtOH (20 mL x 2) and CH2Cl2 (20 mL x 2) and dried in

vacuum. This procedure was repeated once to afford resin 3-9.

3.5.10 General procedure for the preparation of resin bound benzyl 3-(6-oxo-1,6-

dihydropurin-9-yl)propanoate (3-10)

3-9 was swollen in DMF for 30 min. 80% formic acid was added and the reaction

mixture was stirred at 70°C for 4 h. Then the resin was filtered and washed with DMF

(20 mL x 2), EtOH (20 mL x 2) and CH2Cl2 (20 mL x 2) and dried in vacuum at 40°C to

afford resin 3-10.

98

3.5.11 General procedure for the preparation of resin bound benzyl 3-(1-

substituted-6-oxo-1,6-dihydro-purin-9-yl)propanoate (3-11)

3-10 was swollen in DMF for 30 min and 1-bromobutane (1.5 equiv.) and DBU (2

equiv.) were added. After shaking at room temperature overnight, the resin was filtered

and washed with DMF (20 mL x 2), EtOH (20 mL x 2) and CH2Cl2 (20 mL x 2) and

dried in vacuum.

3.5.12 General procedure for the preparation of resin bound benzyl 3-(1,7-

disubstituted-6-oxo-1,6-dihydro-purin-9-yl)propanoate (3-12)

3-11 was swollen in DMF for 30 min. 1-Iodobutane (20 equiv.) was added and the

mixture was stirred slowly at 50oC for 24 h. After which, the resin was filtered and

washed with DMF (20 mL x 2), H2O (20 mL x 2), EtOH (20 mL x 2) and CH2Cl2 (20 mL

x 2) and dried in vacuum.

3.5.13 General procedure for the preparation of 1,7-disubstituted-6-oxopurine (3-6)

Resin 3-12 was swollen in DCM for 30 min. 2 M ammonia in methanol (20 equiv.) was

added and the mixture was shaken at room temperature for 24 h. The resin was filtered

and washed with methanol (20 mL x 2) and CH2Cl2 (20 mL x 2). The washings were

combined with the filtrate and concentrated to dryness and purified by column

chromatography.

3-6a: 1,7-Dibutyl-1,7-dihydro-purin-6-one

1HNMR (CDCl3): δ(ppm) 0.90-0.96 (m, 6H, 2CH3), 1.29-1.41 (m, 4H, CH2), 1.68-1.78

(m, 2H, CH2), 1.80-1.90 (m, 2H, CH2), 3.99 (t, J=7.3 Hz, 2H, CH2), 4.35 (t, J=7.3 Hz,

99

2H, CH2), 7.80 (s, 1H, C8H), 7.98 (s, 1H, C2H). 13CNMR (CDCl3): δ(ppm) 13.42, 13.52,

19.50, 19.76, 31.82, 33.31, 46.26, 47.14, 115.10, 143.14, 146.31, 154.20, 156.95.

HRMS(EI): Calcd for C13H20N4O 248.1637; found: 248.1636.

3-6b: 7-Allyl-1-butyl-1,7-dihydro-purin-6-one

1HNMR (CDCl3): δ(ppm) 0.92 (t, J=7.3 Hz, 3H, CH3), 1.29-1.42 (m, 2H, CH2), 1.66-1.76

(m, 2H, CH2), 3.97 (t, J=7.3 Hz, 2H, N1CH2), 5.01 (d, J=5.6 Hz, 2H, N7CH2), 5.21 (dd,

J=10.5 Hz, J=23.0 Hz, 2H, CH2), 5.99-6.10 (m, 1H, CH), 7.82 (s, 1H, C8H), 7.97 (s, 1H,

C2H). 13CNMR (CDCl3): δ(ppm) 13.48, 19.69, 31.75, 46.18, 49.12, 118.94, 132.41,

139.59, 143.01, 146.41, 154.19, 156.76. HRMS(EI): Calcd for C12H16N4O 232.1324;

found: 232.1320.

3-6c: 7-Benzyl-1-butyl-1,7-dihydro-purin-6-one

1HNMR (CDCl3): δ(ppm) 0.95 (t, J=7.3 Hz, 3H, CH3), 1.32-1.44 (m, 4H, CH2), 1.68-1.78

(m, 2H, CH2), 3.99 (t, J=7.3 Hz, 2H, N1CH2), 5.59 (s, 2H, PhCH2), 7.29-7.36 (m, 5H,

ArH), 7.83 (s, 1H, C8H), 7.98 (s, 1H, C2H). 13CNMR (CDCl3): δ(ppm) 13.53, 19.76,

31.83, 46.25, 50.53, 115.11, 127.90, 128.45, 129.00, 135.66, 143.16, 146.48, 154.37,

156.87. HRMS(EI): Calcd for C16H18N4O 282.1481; found: 282.1478.

3-6d: 7-Allyl-1-heptyl-1,7-dihydro-purin-6-one

1HNMR (CDCl3): δ(ppm) 0.84 (t, J=7.3 Hz, 3H, CH3), 1.22-1.31 (m, 8H, 4CH2), 1.71-

1.76 (m, 2H, CH2), 3.98 (t, J=7.3 Hz, 2H, N1CH2), 5.04 (dd, J=1.4 Hz, J=5.9 Hz, 2H,

N7CH2), 5.24 (dd, J=10.1 Hz, J=20.2 Hz, 2H, CHCH2), 5.99-6.11 (m, 1H, CH), 7.96 (s,

100

1H, C8H), 8.00 (s, 1H, C2H). 13CNMR (CDCl3): δ 13.90, 22.41, 26.46, 28.70, 29.76,

31.53, 46.56, 49.30, 119.19, 132.30, 142.89, 146.69, 152.21, 154.13, 156.28. HRMS(EI):

Calcd for C15H22N4O: 274.1794; found: 274.1781.

3-6e: 7-Butyl-1-heptyl-1,7-dihydro-purin-6-one

1HNMR (CDCl3): δ(ppm) 0.86 (t, J=6.6 Hz, 3H, CH3), 0.95 (t, J=7.3 Hz, 3H, CH3), 1.26-

1.39 (m, 10H, 5CH2), 1.73-1.92 (m, 4H, 2CH2), 1.80-1.90 (m, 2H, CH2), 4.00 (t, J=7.5

Hz, 2H, N1CH2), 4.38 (t, J=7.3 Hz, 2H, N7CH2), 7.90 (s, 1H, C8H), 8.00 (s, 1H, C2H).

13CNMR (CDCl3): δ(ppm) 13.45, 13.94, 19.55, 22.47, 26.54, 28.77, 29.85, 31.60, 33.33,

46.61, 47.30, 115.16, 143.06, 146.52, 154.18, 156.64. HRMS(EI): Calcd for C16H26N4O

290.2107; found: 290.2100.

3-6f: 7-Benzyl-1-heptyl-1,7-dihydro-purin-6-one

1HNMR (CDCl3): δ(ppm) 0.88 (t, J=6.8 Hz, 3H, CH3), 1.25-1.35 (m, 8H, 4CH2), 1.75-

1.79 (m, 2H, CH2), 4.01 (t, J=7.5 Hz, 2H, N1CH2), 5.64 (s, 2H, PhCH2), 7.35-7.36 (m,

5H, ArH), 8.02 (s, 1H, C8H), 8.05 (s, 1H, C2H). 13CNMR (CDCl3): δ(ppm) 13.99, 22.51,

26.55, 28.80, 29.88, 31.63, 46.72, 50.91, 128.13 (x2), 128.70, 129.14 (x2), 142.77,

147.03. HRMS(EI): Calcd for C19H24N4O 324.1950; found: 324.1947.

3-6g: 1-Benzyl-7-butyl-1,7-dihydro-purin-6-one

1HNMR (CDCl3): δ(ppm) 0.87 (t, J=7.5 Hz, 3H, CH3), 1.23-1.33 (m, 2H, CH2), 1.75-

1.85(m, 2H, CH2), 4.30 (t, J=7.3 Hz, 2H, N7CH2), 5.14 (s, 2H, PhCH2), 7.21-7.26 (m,

5H, ArH), 7.78 (s, 1H, C8H), 8.07 (s, 1H, C2H). 13CNMR (CDCl3): δ(ppm) 13.24, 19.27,

101

33.04, 46.92, 48.77, 114.87, 127.57, 127.93, 128.69, 135.71, 143.13, 146.24, 153.94,

156.71. HRMS(EI): Calcd for C16H18N4O 282.1481; found: 282.1479.

3-6h: 7-Allyl-1-benzyl-1,7-dihydro-purin-6-one

1HNMR (CDCl3): δ(ppm) 5.04 (dd, J=1.4 Hz, J=5.9 Hz, 2H, N7CH2), 5.19 (dd, J=1.0

Hz, J=10.5 Hz, 2H, CHCH2), 5.16 (s, 2H, PhCH2), 5.93-6.06 (m, 1H, CH), 7.21-7.26 (m,

5H, ArH), 7.81 (s, 1H, C8H), 8.08 (s, 1H, C2H). 13CNMR (CDCl3): δ(ppm) 48.79, 48.96,

114.78, 118.81, 127.58, 127.96, 128.70, 132.23, 135.65, 143.03, 146.35, 153.98, 156.59.

HRMS(EI): Calcd for C15H14N4O 266.1168; found: 266.1164.

3-6i: 1-Allyl-7-butyl-1,7-dihydro-purin-6-one

1HNMR (CDCl3): δ(ppm) 0.83 (t, J=7.3 Hz, 3H, CH3), 1.20-1.33 (m, 2H, CH2), 1.71-1.81

(m, 2H,CH2), 4.28 (t, J=7.2 Hz, 2H, N7CH2), 4.55 (d, J=6.0 Hz, 2H, N1CH2), 5.13 (dd,

J=10.4 Hz, J=13.7 Hz, 2H, CH2), 5.83-5.92 (m, 1H CH), 7.77 (s, 1H, C8H), 7.92 (s, 1H,

C2H). 13CNMR (CDCl3): δ(ppm) 13.16, 19.21, 33.01, 46.89, 47.46, 114.70, 118.32,

131.88, 143.11, 146.01, 153.66, 156.67. HRMS(EI): Calcd for C12H16N4O 232.1324;

found: 232.1321.

3-6j: 7-Benzyl-1-isopropyl-1,7-dihydro-purin-6-one

1HNMR (CDCl3): δ(ppm) 1.47 (d, J= 6.9 Hz, 6H, 2CH3), 5.15-5.24 (m, 1H, N1CH), 5.60

(s, 2H, PhCH2), 7.30-7.35 (m, 5H, ArH), 7.85 (s, 1H, C8H), 8.09 (s, 1H, C2H). 13CNMR

(CDCl3): δ(ppm) 22.46 (x2), 45.51, 50.53, 127.97, 128.50, 129.06, 135.68, 143.28,

143.61, 154.22, 156.34. HRMS(EI): Calcd for C15H16N4O 268.1324; found: 268.1356.

102

3-6k: 7-Butyl-1-isopropyl-1,7-dihydro-purin-6-one

1HNMR (CDCl3): δ(ppm) 0.92 (t, J=7.3 Hz, 3H, CH3), 1.29-1.36 (m, 2H, CH2), 1.45 (d,

J=6.9 Hz, 6H, 2CH3), 1.79-1.89 (m, 2H, CH2), 4.35 (t, J=7.3 Hz, 2H, N7CH2), 5.10-5.24

(m, 1H, N1CH), 7.82 (s, 1H, C8H), 8.06 (s, 1H, C2H). 13CNMR (CDCl3): δ(ppm) 13.39,

19.49, 22.40, 33.28, 45.43, 47.10, 114.78, 143.19, 143.41, 153.96, 156.31. HRMS(EI):

Calcd for C12H18N4O 234.1481; found: 234.1475.

3-6l: 7-Butyl-1-prop-2-ynyl-1,7-dihydro-purin-6-one

1HNMR (CDCl3): δ(ppm) 0.82 (t, J=7.3 Hz, 3H, CH3), 1.19-1.27 (m, 2H, CH2), 1.71-1.81

(m, 2H, CH2), 2.43-2.45 (m, 1H, CH), 4.27 (t, J=7.3 Hz, 2H, N7CH2), 4.74 (dd, J=1.0

Hz, 2.8 Hz, 2H, N1CH2), 7.77 (s, 1H, C8H), 8.19 (s, 1H, C2H). 13CNMR (CDCl3):

δ(ppm) 13.19, 19.23, 32.98, 34.51, 46.95, 74.70, 114.45, 139.85, 143.27, 145.18, 153.22,

156.74. HRMS(EI): Calcd for C12H14N4O 230.1168; found: 232.1162.

3-6m: 7-Benzyl-1-prop-2-ynyl-1,7-dihydro-purin-6-one

1HNMR (CDCl3): δ(ppm) 2.46-2.48 (m, 1H, CH), 4.77 (d, J=2.4 Hz, 2H, N1CH2), 5.55

(s, 2H, PhCH2), 7.27-7.31 (m, 5H, ArH), 7.86 (s, 1H, C8H), 8.24 (s, 1H, C2H). 13CNMR

(CDCl3): δ(ppm) 34.62, 50.48, 74.97, 114.63, 127.80 (x2), 128.39, 128.91, 135.44,

143.40, 145.39, 153.53, 156.86. HRMS(EI): Calcd for C15H12N4O 264.1011; found:

264.1011.

3-6n: 7-Butyl-1-methyl-1,7-dihydro-purin-6-one

103

1HNMR (CDCl3): δ(ppm) 0.84 (t, J=7.3 Hz, 3H, CH3), 1.20-1.28 (m, 2H, CH2), 1.72-1.82

(m, 2H, CH2), 3.52 (s, 3H, N1CH3), 4.28 (t, J=7.1 Hz, 2H, N7CH2), 7.75 (s, 1H, C8H),

7.97 (s, 1H, C2H). 13CNMR (CDCl3): δ(ppm) 13.24, 19.28, 33.09, 33.40, 46.96, 114.76,

143.06, 146.46, 154.39, 156.94. HRMS(EI): Calcd for C10H14N4O 206.1168; found:

206.1167.

3-6o: 7-Benzyl-1-methyl-1,7-dihydro-purin-6-one

1HNMR (CDCl3): δ(ppm) 3.57 (s, 3H, N1CH3), 5.57 (s, 2H, PhCH2), 7.30 (m, 5H, ArH),

7.85 (s, 1H, C8H), 8.01 (s, 1H, C2H). 13CNMR (CDCl3): δ(ppm) 33.51, 50.51, 114.96,

127.82, 128.42, 128.96, 135.65, 143.19, 146.69, 154.70, 157.07. HRMS(EI): Calcd for

C13H12N4O 240.1011; found: 240.1013.

104

3.6 References

[1] (a) Naesens, L.; Lenaerts, L.; Andrei, G.; Snoeck, R.; Van Beers, D.; Holy, A.;

Balzarini, J.; De Clercq, E. Antimicrob.Agents Chemother. 2005, 49, 1010-

1016. (b) Neyts, J.; Balzarini, J.; Andrei, G.; Zhu, C.; Snoeck, R.;

Zimmermann, A.; Mertens, T.; Karlssen, A.; De Clercq, E. Mol. Pharmacol.

1998, 53, 157-165. (c) Hakimelahi, G. H.; Ly, T. W.; Moosavi-Movahedi, A.

A.; Jain, M. L.; Zakerinia, M.; Davari, H.; Mei, H.-C.; Sambaiah, T.;

Moshfegh, A. A.; Hakimelahi, S. J. Med. Chem. 2001, 44, 3710-3720. (d)

Jähne, G.; Kroha, H.; Müller, A.; Helsberg, M.; Winkler, I.; Gross, G.; Scholl,

T. Angew. Chem. Int. Ed. Engl. 1994, 33, 562-563.

[2] (a) Gates, K. S.; Nooner, T.; Dutta, S. Chem. Res. Toxicol. 2004, 17, 839-856.

(b) Novák, J.; Linhart, I.; Dvořákavá, H. Eur. J. Org. Chem. 2004, 2738-2746.

[3] Shuker, D. E. G.; Farmer, P. B. Chem. Res. Toxicol. 1992, 5, 450-460.

[4] Kjellberg, J.; Jphansson, N. G. Nucleosides Nucleotides 1989, 8, 225-256.

[5] Bargiotti, A.; Ermoli, A.; Menichincheri, M.; Vanotti, E.; Bonomini, L.; Fretta,

A. US Patent 0,138,212, 2004; SciFinder Scholar AN 2002:868932.

[6] (a) Pappo, D. Kashman, Y. Tetrahedron 2003, 59, 6493-6501. (b) Hocková, D.;

Buděšínský, M.; Marek, R.; Marek, J.; Holý, A. Eur. J. Org. Chem. 1999,

2675-2682.

[7] Brown, A. R.; Rees, D. C.; Rankovic, A. Morphy, J. R. J. Am. Chem. Soc.

1997, 119, 3288-3295.

[8] Saito, T.; Inoue, I.; Fujii, T. Chem. Pharm. Bull. 1990, 38, 1536-1547.

105

CHAPTER 4: Traceless Solid-Phase Synthesis of Various Substituted

Purines From p-Benzyloxybenzylamine (BOBA) Resin

4.1 Introduction

Combinatorial technologies encompass numerous strategies to prepare and screen

collections or ‘libraries’ of structurally related molecules. In SPS strategies, a substrate is

attached to a functionalized polymer. These functionalized polymers are prepared using

copolymerization processes that incorporate the functionalized monomer, or by direct

derivatization of the polymer itself. The selection of an appropriate linkage is critical to

the success of any SPS. The primary function of the linker is to covalently attach the

initial substrate to the polymer support. However a major drawback to the traditional

linkers used in SPS is that, upon cleavage, it leaves a functional group on the final

compound. The presence of these appendages is fine if the compounds require these

functionalities. However complications may arise if these appendages are redundant and

affect the activities of the compounds. To overcome this drawback, traceless linkers

which leave the target compound with no “memory” of the SPS was developed and has

become a major area of interest in SPS.[1]

The purine moiety is a principle component in nucleic acids and substrates of many

regulatory, biosynthetic and signal transduction proteins including kinases, DNA and

RNA polymerases, G proteins, and purine biosynthetic and metabolic enzymes.[2] Their

importance in biology has generated much interest and over the years various solid-phase

synthetic methodologies have been studied. These methodologies generally involve two

106

main strategies. In the first strategy, a halogenated/aminated purine is used. Modification

on the purine ring can generate substituents at the 2-, 6- and 9- positions. The second

strategy is based on the synthesis of substituted pyrimidine ring followed by the closure

of the imidazole ring. Although this strategy is less straightforward than the first, it has an

important advantage of providing the possibility to introduce C8 substituents more easily.

However the difficult reaction condition for cyclization in solid-phase synthesis could

have limited the usage of this strategy as only one paper using this strategy has thus far

been reported.[3] In this paper, 4,6-dichloro-5-nitropyrimidine was coupled to Rink amide

resin. Subsequent amination at C4 and reduction of nitro group at C5 provided the

precursor for closure of the imidazole ring (Chapter 1, Scheme 1.22). Limitations of this

reported method are (i) the substitution on C6 is invariable because an NH2 group is

always left after the cleavage of the Rink amide resin and (ii) low yield (overall yield was

7% indicating an average yield of 59% for each step). In order to introduce greater

diversity on the purine moiety, a new linker and methodology should be developed.

4.2 Outline of our strategy

Herein we present our results on the development of a traceless solid-phase synthesis of

various substituted purines starting from 5-amino-4,6-dichloropyrimidine and p-

benzyloxybenzylamine (BOBA) resin 4-27. 5-Amino-4,6-dichloropyrimidine was

coupled to BOBA resin to form a resin bound diamine 4-28 as the key intermediate for

elaboration to various substituted purines. After cyclization, the N7 position possessed

the steric priority to be alkylated. This, in turn, resulted in a regioselective N7 alkylation

being achieved. At the end of the reaction, the BOBA linker was easily cleaved and the

107

target purines were released (Scheme 4.1). During this study, we have also extended the

use of 4-28 to other SPS.

O

NH2

N

N

Cl

NH2

NH

O

N

N

O

N

N

R1

R2

R3

N

N

O

N

N

R1

R2

N

N

O

N

NH

N

R

N

N

N

N

NH

X

4-22 X= N4-24 X=C

4-6

4-17

4-35

4-27 4-28

Scheme 4.1 Overall strategy

4.3 Results and discussions

4.3.1 Solution-phase study towards 1,7,8-trisubstituted purines

The initial solution-phase validation used (4-(benzyloxy)phenyl)methanamine (4-1a) to

mimic the p-benzyloxybenzylamine (BOBA) resin. The solution-phase synthetic route is

depicted in Scheme 4.2.

108

NH2

BnO

N

N

ClNH2

ClN

N

Cl

NH2

NH

OBn

1)C6H13CHO/HOAc/THF HN

N

O

N

N

OBn

C6H13

DBU/DMF

N

N

O

N

N

OBn

C6H13

nBuI/DMF

N

N

O

N

N

C6H13

C4H9N

N

O

N

N

OBn

C6H13

C4H9

I-

2)HCOOH/H2O

H2SO4/PhMe/DCM

propargyl bromide

or CAN/CH3CN/H2O

4-1a 4-2a

4-3 4-4

4-5 4-6a

Ph X1) 4-hydroxybenzamide

2) LiAlH4 or BH3X=Br or Cl

Scheme 4.2 Solution-phase study

4.3.1.1 Synthesis of (4-(benzyloxy)phenyl)methanamine (4-1a)

Compound 4-1a was synthesized by first treating benzyl bromide or benzyl chloride with

4-hydroxybenzamide in the presence of NaOH in DMSO. Benzyl bromide gave a much

higher yield than benzyl chloride (80% vs. 25%). After which, reduction was carried out

by using BH3/THF (4 equiv.) under reflux or LiAlH4/THF (4 equiv.) at room temperature

to give 4-1a in 88% yield. Compound 4-1a was not stable and turned from white to

yellow when exposed to air.

109

4.3.1.2 Synthesis of N4-(4-(benzyloxy)benzyl)-6-chloropyrimidine-4,5-diamine (4-2a)

The pyrimidine core 5-amino-4,6-dichloropyrimidine was reacted with (4-

(benzyloxy)phenyl)methanamine (4-1a) through a SNAr displacement. The reaction was

studied using different bases and solvents and the results are summarized in Table 4.1.

Table 4.1 Synthesis of compound 4-2a

Base Reaction conditions Yield of 4-2a (%)

1 - Reflux in BuOH overnight -

2 NaHCO3 Reflux in THF for 2 h 18

3 NaHCO3 Reflux in BuOH for 24 h 66

4 DiEA Stirred at 100oC in DMF for 5 h 25

5 DiEA Reflux in BuOH for 24 h 88

It is evident that the presence of a base is important for the SNAr displacement. Without

base, formation of compound 4-2a was not observed. At the same time, the reaction

solvent was also a crucial entity. Table 4.1 shows that the reaction proceeded more

favorably in the presence of a protic solvent than an aprotic solvent, such as DMF or

THF. Since DiEA had a better solubility than NaHCO3 in BuOH (Entry 5), it resulted in a

higher yield and was used as the base for our SPS.

4.3.1.3 Studies of cyclization of N4-(4-(benzyloxy)benzyl)-6-chloropyrimidine-4,5-

diamine (4-2a)

Different methods for cyclization of 4-2a were studied. That included cyclization with

orthoester, cyclization with acid choride and cyclization with aldehyde.

110

4.3.1.3.1 Cyclization with triethyl orthoacetate

Various solution-phase cyclization of 4,5-diaminopyrimidine with orthoesters have been

reported earlier.[4] However treatment of diamine (4-2a) with triethyl orthoacetate in the

presence of TsOH or HCl at room temperature for 3 h resulted in a pale yellow oil (99%

yield) which upon analysis by 1HNMR, 13CNMR, Dept135 and accurate MS showed that

this oil was not the desired cyclized product 4-8 but could be the intermediate ethyl N-4-

(4-(benzyloxy)benzylamino)-6-chloropyrimidin-5-ylacetimidate (4-7) (Scheme 4.3).

N

N

Cl

NH2

NH

OBn

OEt

OEt

OEt

TsOH or HClN

N

Cl

N

NH

OBn

OEt

4-2a 4-7

N

N

Cl

N

N

OBn4-8

Scheme 4.3 Cyclizaiton with triethyl orthoacetate

A possible mechanism for the reaction of diamine (4-2a) with triethyl orthoacetate is

shown in Scheme 4.4.

N

N

Cl

NH2

NH

OBn

OEt

OEt

OEt

N

N

Cl

N+

NH

OBn

OEtH H OEt

N

N

Cl

NH

NH

OBn

HOEtOEt

N

N

Cl

N

NH

OBn

OEt

4-2a

4-7

N

N

Cl

N

N

OBn

N

N

Cl

N

N

OBn4-8

-H+ OEt

Scheme 4.4 Mechanism of reaction of compound 4-2a with triethyl orthoacetate

111

In order to obtain the cyclized compound 4-8, the reaction was attempted at room

temperature for 24 h and then at elevated temperature (100oC). However these reaction

conditions did not provide 4-8. Then Lewis acid was added to promote the cyclization.

The isolated intermediate compound 4-7 was stirred with ferric chloride in anhydrous

ethanol. After 1 h, TLC showed the disappearance of the starting material and the

formation of two new products. Analysis of both products revealed that the first

compound (major product) isolated was 4-2a whilst the second compound was the

desired cyclized product 4-8 (46%). Since 4-2a was the major product, a one-pot reaction

of compound 4-2a with triethyl orthoacetate in the presence of ferric chloride was carried

out. However, it generated a lot of side products which gave the procedure no preparative

utility.

4.3.1.3.2 Cyclization with benzoyl choride

N

N

Cl

NH2

NH

OBn

Ph

O

Cl

DiEA N

N

Cl

HN

NH

OBn

O

Ph N

N

X

N

N

OBn

Ph

4-2a 4-94-10 X=Cl

4-11 X=OH

Scheme 4.5 Cyclization with benzoyl chloride

A less straightforward two-step cyclization method was also examined.[5] As shown in

Scheme 4.5, starting diamine 4-2a was first reacted with benzoyl chloride to give

112

compound 4-9 in near quantitative yield. Different ring closure conditions applied on 4-9

were examined (Table 4.2).

Table 4.2 Various ring closure conditions applied on 4-9

Ring closure conditions Products

1 HMDS/(NH4)2SO4, reflux o/n 4-9 and 4-10

2 10% KOH (aq.)/CH3CN, reflux o/n 4-9, 4-10 and 4-11

3 10% NaOH (aq.)/CH3OH, 60oC, o/n 4-9 and 4-11

4 10% NaOH (aq.)/DMA, 100oC, o/n 4-9

From Table 4.2 it is clear that all the examined conditions failed to provide complete

disappearance of the starting material 4-9. However, reaction with 10% KOH

(aq.)/CH3CN under reflux condition (Entry 2) gave both 6-chloro-cyclized product 4-10

and 6-hydroxy-cyclized product 4-11. This implied that cyclization and hydrolysis of 6-

chloro had occurred during the one-pot reaction. Replacing acetonitrile with methanol

(Entry 3) resulted only in the formation of 6-hydroxy-cyclized product 4-11. Though the

desired product was formed using 10% NaOH (aq.)/CH3OH, the yield obtained was low

(20%) and thus we had to press on in the search for a better cyclization method.

4.3.1.3.3 Cyclization with aldehydes

Condensation of diaminopyrimidine with an aldehyde is a classic method to generate

purine imidazole ring.[3,6] With the intention of seeking a high yielding and

straightforward reaction condition for solid-phase synthesis, this cyclization method was

studied (Table 4.3). Treatment of diamine 4-2a with heptan-1-al (5 equiv.) in the

presence of acetic acid (40 equiv.) under reflux condition gave complete disappearance of

the starting material 4-2a. After that reaction mixture was evaporated to dryness and

113

treated with ferric chloride at room temperature for 3 h, 6-hydroxy-cyclized product 4-3

was isolated in 51% yield (Entry 1). This result indicated that hydrolysis had occurred

during the reaction and this may be attributed to the presence of acetic acid. Overnight

treatment with ferric chloride (Entry 2) further increased the yield of 4-3 (69%).

However, when 1,4-dioxane was used as solvent[6b] and treatment with ferric chloride

was carried out at 100oC, it generated a very dirty reaction mixture and no product was

isolated (Entry 3). Since the reaction was made more complex by ferric chloride, we

decided to study this reaction in a stepwise manner. First, we examined the treatment of

diamine 4-2a with heptan-1-al. With disappearance of the starting material, there were

two products formed in a 1:1 ratio (Entry 4). NMR and MS identification showed that the

first product (colourless oil) was the 6-chloro-cyclized product, 9-(4-(benzyloxy)benzyl)-

6-chloro-8-hexyl-9H-purine (4-12), whilst the second product (pale yellow solid) was the

6-hydroxy-cyclized product 4-3. This result showed that cyclization was already

completed even without treatment of ferric chloride and hydrolysis had already taken

place. This inspired us to perform the cyclization and hydrolysis in one pot without ferric

chloride. We also tried to raise the ratio of acetic acid from 40 equiv. to 80 equiv.. But

this did not improve the yields of compounds 4-3 (Entry 5). At the same time, reaction

with formic acid, a stronger acid than acetic acid, was also examined. However neither 4-

3 nor 4-12 was obtained and the starting diamine 4-2a was recovered (Entry 6). Finally 4-

2a was treated with heptan-1-al and acetic acid in THF overnight and then evaporated to

dryness. The obtained residue was then stirred with 85% formic acid at 70°C for another

3 h which provided the desired product 4-3 in 81% overall yield (Entry 7).

114

Table 4.3 Cyclization with aldehyde

Reaction conditions Yield of product 4-3 (%)

1 1) AcOH (40 equiv.)/MeOH reflux o/n 2) FeCl3/EtOH, rt, 3 h 51

2 1) AcOH (40 equiv.)/MeOH reflux o/n 2) FeCl3/EtOH, rt, o/n 69

3 1) AcOH (40 equiv.)/MeOH reflux o/n 2) FeCl3/dioxane, reflux, o/n -

4 AcOH (40 equiv.)/MeOH reflux o/n 53a

5 AcOH (80 equiv.)/MeOH reflux 24 h 50a

6 HCOOH(80 equiv.)/MeOH reflux 5 h -

7 1) AcOH (80 equiv.)/THF reflux o/n 2) 85% HCOOH (aq.) 81

a) Compound 4-12 was also isolated.

N

N

Cl

N

N

OBn

C6H13

Figure 4.1 Structure of 9-(4-(benzyloxy)benzyl)-6-chloro-8-hexyl-9H-purine (4-12)

The mechanism of this reaction is depicted in Scheme 4.6. During the reaction the

iminium ion was formed first followed by oxidative cyclization.

115

N

N

ClNH2

NH

OBn

R

O

HN

N

ClN

NH

OBn

O

R

N

N

ClNH

N

OBn

R

4-2a

N

N

ClHN

NH

OBn

OH

R

H+

N

N

Cl HN

NH

OBn

OH2

R

N

N

ClHN

NH

OBn

R N

N

Cl

OBn

RN

N

4-12

-H+ N

N

ClN

NH

OBn

R

H

H

Scheme 4.6 Mechanism of cyclization with aldehyde

4.3.1.4 Synthesis of 9-(4-(benzyloxy)benzyl)-8-hexyl-1-(prop-2-ynyl)-1H-purin-

6(9H)-one (4-4)

Next N1 alkylation was easily achieved using propargyl bromide as alkylating reagent

(1.2 equiv.) in the presence of DBU (1.2 equiv.) as base. The alkylation was initially

carried out according to the reaction condition described in Chapter 3 (Section 3.3.1.4).

However after reacting at room temperature overnight, TLC showed the existence of

starting material and product 4-4 was isolated in 60% yield. We subsequently increased

the amount of alkylating reagent and DBU to 4 equiv.. This provided complete

consumption of the starting material and product 4-4 was obtained in 98% yield.

116

4.3.1.5. Synthesis of 9-(4-(benzyloxy)benzyl)-7-butyl-8-hexyl-1-(prop-2-ynyl)-1H-

purin-6(9H)-one salt (4-5)

Quarternization of 4-4 with 1-iodobutane was carried out initially with 20 equiv. of 1-

iodobutane according to the reaction condition described in Chapter 3 (Section 3.3.1.5).

However after stirring at 70°C in DMF for 48 h, TLC showed the existence of starting 4-

4. Increasing the reaction temperature to 90oC resulted in a very dirty reaction mixture.

Hence, we proceeded to increase the equivalence of 1-iodobutane to 40 equiv.. Under this

reaction condition, the quarternization reaction was completed after stirring at 70°C

overnight and gave product 4-5 in 93% yield. This result suggested that quaternization

was more sluggish with substitution at the C8 position.

4.3.1.6 Synthesis of 7-butyl-8-hexyl-1-(prop-2-ynyl)-1H-purin-6(7H)-one (4-6a)

Having accomplished the quarternization, we proceeded with the N9-debenzylation. This

reaction was carried out to mimic the cleavage of the solid support to achieve a traceless

character. Initial attempts to carry out the N9-benzylation with TFA/DCM did not provide

the desired 4-6a. Subsequently, we treated 4-5 with 10 equiv. of 96% H2SO4 at room

temperature in the presence of toluene and provided the final product 4-6a in 95% yield.

The debenzylation proceeded via carbonium formation.[7] In this reaction, toluene was

used as both a solvent and a carbonium trap which led the reaction to completion

(Scheme 4.7). It is worth noting that in order to obtain a neutral final product, it is

necessary to neutralize the reaction mixture with saturated NaHCO3 at the end of the

reaction.

117

4-5+H N

N

O

NH

OBn

C6H13

C4H9

N

N

O

N

N

C6H13

C4H9

H B

N

N

O

N

N

C6H13

C4H9

4-6a

BnO CH2

toluene

CH3

CH2 OBn

+

N

Scheme 4.7 Mechanism of cleavage with sulfuric acid/toluene

Another cleavage method was also investigated. CAN/CH3CN/H2O is a classic method

for oxidative debenzylation.[8] With this method, the 4-(benzyloxy)benzyl group was

cleaved via benzylic oxidation (Scheme 4.8). Treatment of compound 4-5 with 4 equiv.

CAN in CH3CN/H2O (4:1) also gave the final compound 4-6a in high yield.

4-5N

N

O

N

N

C6H13

R

C4H9

4-6a

N

N

O

N

N

OBn

C6H13

R

C4H9

CAN H2O N

N

O

N

N

OBn

C6H13

R

C4H9

HO

R=prop-2-ynyl

Scheme 4.8 Mechanism of cleavage with CAN/CH3CN/H2O

4.3.2 Solution-phase study of other various substituted purines

We were interested to expand the chemistry at the C8 position of purine. As described

above, 4-2a could react with orthoesters, acid chloride or aldehydes to generate 8-

substituted purine. Besides this, other utilizations of this key diamine 4-2a were also

118

investigated. The following sections describe the use of 4-2a for the preparation of 1,7-

disubstituted purines, 8-azapurines and [i]-condensed purines.

4.3.2.1 Solution-phase synthesis of 1,7-disubstituted purines (4-17a)

Starting from N4-(4-(benzyloxy)benzyl)-6-chloropyrimidine-4,5-diamine (4-2a) 1,7-

disubstituted purine were prepared as shown in Scheme 4.9.

N

N

Cl

N

N

OBn4-13a

4-2aHC(OMe)3

HCl (Cat.)

N

N

O

N

N

OBn

C4H9

N

N

O

N

N

OBn

C4H9

C4H9

N

N

O

N

N

C4H9

C4H9

85% HCOOH BuBr/DBUHN

N

O

N

N

OBn

H2SO4

Toluene

BuI

4-14

4-15 4-16 4-17a

Scheme 4.9 Synthesis of 1,7-dibutyl-1H-purin-6(7H)-one (4-17a)

In this case cyclization of imidazole was achieved using either orthoformate or

formamide. Compared to the cyclization of 4-2a with triethyl orthoacetate (section

4.3.1.3.1), cyclization of 4-2a with trimethyl orthoformate proceeded more readily.

Compound 4-2a reacted smoothly with trimethyl orthoformate in the presence of acid to

give 9-(4-(benzyloxy)benzyl)-6-chloro-9H-purine (4-13a). 1HNMR of 4-13a showed the

presence of the purine C8H peak indicating the formation of the imidazole ring. The X

ray crystal structure of 4-13a is shown in Figure 4.2.

119

Figure 4.2 X ray crystal structure of 4-13a

On the other hand, cyclization with formamide was less mild. In order to facilitate

cyclization, compound 4-2a had to react with formamide at 160oC for 24 h. Analysis of

the product showed that the compound obtained was 4-14. Although the reaction was

quite straightforward, the overall yield obtained for two steps was low (29%). This

together with the harsh reaction condition made it impracticable for solid-phase synthesis.

We next proceeded to study the hydrolysis reaction. Unlike REM linker (Chapter 3) that

is base labile, BOBA linker is more stable under basic condition. Thus both acidic and

basic hydrolysis conditions were explored. The results are listed in Table 4.4.

120

Table 4.4 Different hydrolysis conditions of 4-13a

Hydrolysis conditions Yield of 4-14 (%)

1 1 M NaOH, reflux in CH3CN for 3 h 11

2 2 M NaOH, reflux in CH3CN for 3 h 59

3 2 M NaOH, stirred in DMF at 80oC for 3 h 76

4 2 M NaOH, reflux in CH3CN for 1 h 75

5 1.5 M HCl, reflux in CH3CN for 1.5 h 90

6 TFA/H2O(2:1), rt, 48 h 41

7 85% HCOOH (aq), 70oC, 1 h 92

According to Table 4.4 basic hydrolysis provided lower yields than acidic hydrolysis.

This could be attributed to side product formed. We observed that whenever hydrolysis

was performed under basic condition, a side product was isolated. NMR and MS of this

product suggested that it was the ring-open product 4-2a. This implied that the imidazole

ring in 4-13a was not stable under refluxing basic conditions. It is worth noting that

neutralization was always required after base hydrolysis because compound 4-14 could

be deprotonated to form a salt which is soluble in the aqueous layer during extraction

workup. Amongst the various reactions listed in Table 4.4, 85% formic acid gave the best

result (Entry 7). IR spectrum of 4-14 showed the cyclic amide C=O stretch at 1680 cm-1

and phenyl alkyl ether C-O stretch at 1247 cm-1 and 1019 cm-1.

Having obtained 4-14, we proceeded with the N1 alkylation, N7 quaternization and N9

debenzylation according to the methods described in Sections 4.3.1.4~4.3.1.6 to obtain

1,7-dibutyl-1H-purin-6(7H)-one (4-17a) (Scheme 4.9).

121

4.3.2.2 Preliminary solution-phase synthesis of 8-azapurine

Although the solution-phase synthesis of 8-azapurines via the cyclization of 4,5-

diaminopyrimidine with sodium nitrite had been reported earlier, [9] there are no earlier

reports on the application of this reaction on solid phase. Since purine is prone to

substantial changes in reactivity due to substituents effect, we decided to study the

cyclization of 4-2a and subsequent hydrolysis in solution before adapting the reaction

onto solid phase (Scheme 4.10).

N

N

Cl

N

N

OBn4-18a

4-2aNaNO2

N 85% HCOOH HN

N

O

N

N

OBn

N

4-19

HOAc

Scheme 4.10 Solution-phase synthesis of 8-azapurine

Cyclization with sodium nitrite was first examined in THF. Starting diamine 4-2a was

dissolved in THF and 50% HOAc (aq.) followed by the addition of sodium nitrite. The

reaction mixture was stirred at room temperature for 30 min and product 4-18a was

isolated in 43% yield. When DCM was used as the reaction solvent, a heterogeneous

reaction mixture was obtained but with vigorous stirring at room temperature for 30 min,

product 4-18a was obtained in 96% yield. The X ray crystal structure of 4-18a is shown

in Figure 4.3. Hydrolysis of 4-18a could be achieved with established method described

in Section 4.3.2.1.

122

Since N1 alkylation and N9 debenzylation conditions were well examined in Section

4.3.1.4 and Section 4.3.1.6, further study of N1 alkylation and N9 debenzylation of 4-19

was not performed.

Figure 4.3 X ray crystal structure of 4-18a

4.3.2.3 Solution-phase synthesis of [i]-condensed purines

Studies showed [i]-condensed purines were effective for reducing side effects of

xanthines.[10a] However there are very few reports on the synthesis of purine derivatives

condensed with other heterocyclic rings to generate [i]-condensed purines [10] and, to our

knowledge, no study on solid-phase synthesis of [i]-condensed purines have been

reported. In this section of our research, commercially available 4-methoxybenzyl amine

was chosen to mimic BOBA resin instead of (4-(benzyloxy)phenyl)methanamine (4-1a)

123

due to the laborious preparation and unstable nature of the latter. The procedure described

for the syntheses of 4-2a and 4-18a were adopted for the preparation of the key

intermediate diamine 4-2b and 4-18b respectively. 4-18b was subsequently reacted with

ethanolamine in THF to give 4-20a in almost quantitative yield (Scheme 4.11).

NH2CH2CH2OH

N

N

NH

N

N

O

x

OH

N

N

NN

N

O

x

N

N

N

N

N

O

C4H9

N

N

N

N

N

C4H9

O

NH2

N

N

Cl

NH2

ClN

N

Cl

NH2

NH

O

N

N

Cl

N

N

O

X

BuI

N

N

NN

NH

N

SOCl2/DMF

MW 180oC 20min

DiEA, BuOH

4-2b 4-18b X=N4-13b X=CH

4-21a X=N4-21b X=CH

4-23

4-24

4-20a X=N4-20b X=CH

4-22

Scheme 4.11 Solution-phase synthesis of [i]-condensed purines

An earlier report had described the synthesis of heterocycle-condensed purines from 6-

hydroxyethylaminopurine and MsCl/TEA, SOCl2/DCM, or POCl3.[10] Applying these

reaction conditions to our system, various studies on the cyclization of 4-20a were

performed (Table 4.5).

124

Table 4.5 Studies on the cyclization of 4-20a

Cyclization conditions Solvent Temp. and Time Yield of 4-

21a (%)

1 MsCl(2 equiv.), TEA(2 equiv.) DCM rt, 6 h 59

2 MsCl(2 equiv.), TEA(2 equiv.) DCM rt, o/n 66a

3 MsCl(5 equiv.), TEA(5 equiv.) DCM rt, o/n 43

4 MsCl(2 equiv), TEA(2 equiv.) DMF rt, 6 h 52

5 MsCl(2 equiv.), TEA(2 equiv.) DMF 50oC, 6 h -

6 SOCl2(90 equiv.) DCM rt, 6 h -b

7 SOCl2(90 equiv.) CHCl3 reflux, 4 h -b

8 SOCl2(90 equiv.) CHCl3 reflux, o/n 60c

9 SOCl2(3 equiv.) CHCl3 reflux, o/n 36c

10 SOCl2(1.5 equiv.) CHCl3 MW 80oC, 10 min 30

11 SOCl2(1.5 equiv.) DMF MW 180oC, 10 min 85 a) Side product 4-25was isolated. b) Starting 4-20a was recovered. c) Side product 4-26 was isolated.

Entries 1-5 showed that increasing the quantities of MsCl and TEA gave lower yield

(Entry 3) whilst higher reaction temperature gave more complex reaction mixture and

practically no cyclized product (Entry 5). The best result obtained with MsCl/TEA is

shown in Entry 2. However the intermediate, 2-(3-(4-methoxybenzyl)-3H-

[1,2,3]triazolo[4,5-d]pyrimidin-7-ylamino)ethyl methanesulfonate (4-25), was isolated

along with the desired 4-21a (Figure 4.4).

125

N

N

NH

N

N

O

N

Cl

N

N

NH

N

N

O

N

O S

O

O

4-25 4-26

Figure 4.4 Structures of side products 4-25 and 4-26

Entries 6-9 summarize the cyclization with thionyl chloride. It shows that larger excess of

SOCl2 and longer reaction time gave better result (Entry 8). However there was always a

side product isolated. This was identified as 3-(4-methoxybenzyl)-N-(2-chloroethyl)-3H-

[1,2,3]triazolo[4,5-d]pyrimidin-7-amine (4-26) (Figure 4.4).

Cyclization under microwave conditions are given in Entries 10-11. When CHCl3 was

used as the reaction solvent and 4-20a was treated with 1.5 equiv. SOCl2 at 80oC for 10

min under microwave condition, 4-21a was obtained in 30% yield (Entry 10). This low

yield may be attributed to the poor solubility of starting 4-20a in CHCl3. Replacing

CHCl3 with DMF permitted higher reaction temperature to be used and product 4-21a

was obtained in 85% yield (Entry 11). NOESY spectrum of 4-21a clearly showed the

interaction between N1CH2 and C2H which indicates the successful formation of the

third ring. 4-21a was then subjected to debenzylation to obtain product 4-22 (Figure 4.5).

126

N

N

N

N

N

O

N

H

H

4-21a

Figure 4.5 NOESY of compound 4-21a

For compound 4-13b, the first attempt to generate the third ring involves a one-pot

reaction between 4-13b and 3-bromopropylamine hydrobromide followed by cyclization

(Scheme 4.12). Unfortunately this reaction failed to provide complete disappearance of

127

the starting material and gave a very complicated reaction mixture. Pretreatment of 3-

bromopropylamine hydrobromide with Amberlyst A21 resin (weak base) gave similar

results.

N

N

NN

N

O

4-13b

4-21b

Br NH2

N

N

NH

N

N

O

Br

HBr

Scheme 4.12 Initial attempt to 4-21b

Next, we proceeded to investigate the stepwise formation of the third ring for compound

4-13b. As in the formation of 4-21a from 4-18b, 4-13b was reacted first with

ethanolamine in THF to give 4-20b in high yield (96%) (Scheme 4.11). Subsequent

formation of the third ring was carried out according to the reaction condition described

in Table 4.5 Entry 11. However, unlike 4-20a, 4-20b appeared to undergo cyclization via

two possible pathways as shown in Figure 4.6.

N

N

NH

N

N

O

OHN

N

NN

N

O

N

N

N

N

O

HN

4-20b

4-21b

Figure 4.6 Two possible pathways of the cyclization of the pendant alcohol

128

Earlier studies have shown that the pendant alcohol always cyclizes at the N1 position

because of (i) the greater nucleophilicity of N1 compared to N7 and (ii) the greater

probability of forming a five-member ring.[10c] In order to obtain experimental evidence

for the cyclization direction, NOESY experiment of 4-21b was carried out (Figure 4.7).

The NOESY data clearly showed the interaction between C2H and N1CH2, which

confirmed the cyclization direction of the third ring.

To expand the diversity of the library, compound 4-21b was subjected to quaternization

with BuI in DMF. After stirring with 10 equiv. BuI at 70oC overnight, compound 4-21b

was completely consumed and the product was obtained as a yellow solid. NMR and MS

data confirmed that this yellow solid was the alkylated product of 4-21b. In order to

determine the position of the butyl group, NOESY experiment was carried out (Figure

4.8). The NOESY spectrum showed the interaction between Ha and Hb which indicated

the direction of cyclization of the ring fused to purine. The NOESY data also showed

interaction between Hc and Hd and the lack of interaction between Hd and He which

gave evidence that the butyl group is located at C6N rather than N7 position. Thus the

quaternization product is 4-23. Subsequent debenzylation of 4-23 can be achieved using

either sulfuric acid or CAN to offer final product 4-24.

129

N

N

NN

N

O

H

H

4-21b

Figure 4.7 NOESY of compound 4-21b

130

N

N

NN

N

O

Ha

Hb

HcHd

He

4-23

Figure 4.8 NOESY of compound 4-23

131

4.3.3 Solid-phase study

4.3.3.1 Preparation of BOBA resin

Having validated the approach to synthesize 1,7,8-trisubstituted purines in solution, we

proceeded to adapt the strategy on solid support. p-Benzyloxybenzylamine (BOBA) resin

(4-27) was firstly prepared in two steps using reported procedure[11]: Bromo Merrifield

resin (2.3 mmol/g) was reacted with 4-hydroxybenzamide in the presence of NaOH

followed by reduction with 1 M borane in THF under reflux overnight (Scheme 4.13).

Br

CNH2HO

O

NaOH

O

NH2

OO

NH2

4-27

Scheme 4.13 Preparation of BOBA resin from bromomethylated polystyrene resin

Since the borane solution was difficult to handle and harsh reaction condition was needed

for the reaction to proceed, we attempted a simple and milder method to prepare the

BOBA resin (Scheme 4.14). Since Mitsunobu reaction is known to convert an alcohol to

a primary amine, the Wang resin (1.6 mmol/g) was chosen as the starting resin and was

firstly treated with PPh3, DiAD and phthalimide, followed by hydrazinolysis with 1 M

hydrazine in THF. IR spectrum of the resin showed the appearance of C=O stretch at

1770 cm-1 and 1714 cm-1 and the disappearance of these stretches after hydrazinolysis.

Meanwhile loading of BOBA resin calculated from the loading of Fmoc release UV

assay[12] showed almost quantitatively conversion from the starting Wang resin to the

BOBA resin (1.55 mmol/g).

132

4.3.3.2 Solid-phase synthesis of 1,7,8-trisubstituted purines

O

N

O

ON

N

ClNH2

Cl

N

N

ClNH2

NH

O

OH

O

NH2

1. R1CHO/HOAc/THF HN

N

O

N

N

O

R1 R2X/DBU/DMF N

N

ON

N

O

R1R2

R3X N

N

O

N

N

O

R1R2

R3

N

N

ON

NR1

R2

R3

H2SO4/PhMe/DCM

4-29 4-30

4-6

H2NNH2/THFPPh3/DiAD

DiEA/BuOH/DMA

4-28

2. HCOOH/H2O

phthalimide

4-27

4-31

Scheme 4.14 Solid-phase synthesis of 1,7,8-trisubstituted purines

The prepared BOBA resin (4-27) was then subjected to SNAr displacement with 5-amino-

4,6-dichloropyrimidine at elevated temperature as established in solution-phase study

(Scheme 4.14). However in the solid-phase reaction, DMA was required as a co-solvent

to ensure good swelling of the resin (DMF was also examined as a possible co-solvent

but it decomposed at the high temperature under basic condition). Formation of 1,7,8-

trisubstituted purines was then performed by cyclization of resin 4-28 with various

aldehydes in the presence of acetic acid according to our solution-phase study. Resin 4-28

was stirred with various aldehydes in THF under reflux followed by hydrolysis with 85%

formic acid to offer polymer supported 9-(4-(benzyloxy)benzyl)-8-hexyl-1-(prop-2-ynyl)-

133

1H-purin-6(9H)-one (4-29). Subsequent N1 alkylation introduced the second point of

diversity followed by quaternization at N7 with different alkyl halides. For the

quaternization reaction, when an alkyl bromide was used, the addition of sodium iodide

to the quaternization mixture improved the efficiency of the alkylation. The final product

4-6 was then released from the solid support by treatment with 96% sulfuric acid at room

temperature for 3 h in the presence of toluene. In this step CH2Cl2 was required as a co-

solvent to achieve good swelling of the resin. Using this strategy compounds 4-6a~4-6i

were prepared in 8-12% overall yields (Figure 4.12), indicating an average yield of at

least 73% for each step of the reaction. The solid-phase cyclization chemistry with

aldehydes has been validated for both aliphatic and aromatic aldehydes. With the

aromatic aldehydes, it was observed that having an electron-donating group on the para

position, such as 4-methoxybenzaldehyde, gave better result whilst the presence of an

electron-withdrawing group on the para position, e.g. 4-nitrobenzaldehyde, did not give

the desired product. X-ray crystal analysis of 4-6i confirmed the structure obtained.

Figure 4.9 X ray crystal structure of compound 4-6i

134

4.3.3.3 Solid-phase synthesis of simple 1,7-disubstituted purines and 8-azapurines

As shown from our solution-phase study, 1,7-disubstituted purines and 8-azapurines can

be prepared from diamine 4-2a through different routes. We now proceeded to adapt the

methodology for SPS. Resin bound diamine 4-28 was employed as a key intermediate for

the SPS of various purines.

HN

N

ON

N

O

XN

N

ON

N

O

X

R1

N

N

ON

NH

N

R1N

N

ClN

N

O

XH2SO4

N

N

ON

N

O

R1R2

N

N

ON

N

R1R2

4-28R1X/DBU

4-32a X=CH4-32b X=N 4-33

4-35

4-364-17

4-34a X=CH4-34b X=N

HCOOH

R2X

Scheme 4.15 Solid-phase synthesis of simple purines and 8-azapurines

Formation of simple 1,7-disubstituted purines by cyclization of the key intermediate resin

bound diamine 4-28 was first studied (Scheme 4.15). Treatment of resin 4-28 with

trimethyl orthoformate facilitated cyclization to offer 4-32a. Subsequent hydrolysis of the

6-choride, N1 alkylation and regioselective N7 alkylation can also be accomplished

according to the method described in solution-phase study. With this strategy, compounds

4-17a~4-17d were prepared in ~18% overall yields which implied that an average 81%

yield was obtained for each step of the reaction (Figure 4.12). X-ray crystal analyses of 4-

17b (Figure 4.10) and 4-17c (Figure 4.11) were performed and confirmed their exact

structures.

135

Figure 4.10 X ray crystal structure of compound 4-17b

Figure 4.11 X ray crystal structure of compound 4-17c

136

Then the SPS of 8-azapurines was studied. Reaction of resin 4-28 with sodium nitrite in

the presence of 50% HOAc in DCM easily gave resin bound 8-azapurine 4-32b. Similar

hydrolysis and N1 alkylation can also be performed to give 4-35 which was directly

cleaved from the resin with 96% H2SO4 or CAN/CH3CN/H2O. (Scheme 4.15) With this

synthetic route, compounds 4-35a~4-35d were prepared on solid-phase in high yields

(Figure 4.12). This study gave the first example of SPS of 8-azapurines.

4.3.3.4 Solid-phase synthesis of [i]-condensed purine

Additionally, [i]-condensed purine can be prepared from the key intermediate resin bound

diamine 4-28 (Scheme 4.16).

NH2CH2CH2OHN

N

NHN

N

O

X

OH

N

N

NN

N

O

X N

N

NN

NH

N

N

N

NN

N

O

N

N

NN

N

N

N

ClN

N

O

XSOCl2/DMF

4-32a X=CH4-32b X=N

4-22

4-39 4-24

MW 180oC 20min

4-38a X=CH4-38b X=N

4-37a X=CH4-37b X=N

C4H9 C4H9

Scheme 4.16 Solid-phase synthesis of [i]-condensed purine

Resin 4-32 was obtained from 4-28 using the same methods as described above. Resin 4-

32 was subsequently treated with ethanolamine to give resin 4-37 which in turn reacted

with SOCl2 in DMF under microwave condition to form the third five-member ring. The

137

final [i]-condensed purine was obtained through cleavage with H2SO4/toluene. Obtained

4-38a could be treated with 1-iodobutane to enable quarternizaiton at the C6N position.

Using this strategy, compounds 4-22 and 4-24 were prepared in 28% overall yield. This is

the first reported solid-phase synthesis of [i]-condensed purines.

4.4 Conclusions

In conclusion, we have demonstrated both the solution-phase study and the traceless

solid-phase synthesis of various substituted purines with BOBA resin. The target purines

were obtained in high purity and yields. This solid-phase approach offers various

advantages - 1) key intermediate resin bound diamine 4-28 can provide more

combinatorial modification on the purine C8 position; 2) regioselective N7 alkylation can

be achieved; 3) the first SPS of 8-azapurines and [i]-condensed purines have been

developed; and 4) the BOBA linker allows for traceless cleavage.

138

N

N

O

N

NC6H13

C4H9

N

N

O

N

NC6H13

Ph

Ph

N

N

O

N

NC6H13

Ph

PhN

N N

N

O C4H9

N

N N

N

O

C4H9

Ph

PhN

N N

N

O C4H9

O

N

N N

N

O

OPh N

N N

N

O C4H9

Ph

N

N N

N

O

C4H9

Ph

N

N N

N

O

C4H9

Ph

N

N N

N

O Ph

N

N N

N

NC4H9

N

N NH

NN

N

N

N N

N

O C4H9

4-17d 16%

4-6g 12%

4-6f 11%

4-6c 9%

4-6d 11%

4-6b 8%4-6a 9%

4-6e 11%

4-6h 8% 4-6i 9%

4-17b 18% 4-17c 19%

4-22 28% 4-24 28%

N

N N

N

O

C4H9

C4H9

4-17a 17%

N

N NH

N

N

O

N

N NH

N

N

O

Ph

N

N NH

N

N

O

N

N NH

NN

O

C4H9

4-35d 34%4-35c 24%

4-35b 25%4-35a 36%

Figure 4.12 Library of various substituted purines

139

4.5 Experimental

General Procedures. 1HNMR and 13CNMR spectra were measured at 298K on a Bruker

DPX 300 or AMX500 Fourier Transform spectrometer. Chemical shifts were reported in

δ (ppm), relative to the internal standard of tetramethylsilane (TMS). All Infra-red (IR)

spectra were recorded on a Bio-Rad FTS 165 spectrometer. Mass spectra were performed

on VG Micromass 7035 spectrometer under electron impact (EI). All chemical reagents

were obtained from commercial suppliers and used without further purification.

Analytical TLC was carried out on pre-coated plates (Merck silica gel 60, F254) and

visualized with UV light. Flash column chromatography was performed with silica

(Merck, 70-230 mesh). Microwave reaction was performed on InitiatorTM microwave

synthesizer.

4.5.1 Synthesis of (4-(benzyloxy)phenyl)methanamine (4-1a)

4.5.1.1 Synthesis of 4-(benzyloxy)benzamide

NaOH (1.50 g, 37.5 mmol) was added to a solution of 4-hydroxybenzamide (3.09 g, 22.5

mmol) in DMSO (40 mL). The mixture was stirred at 90°C for 1 h before benzyl bromide

(2.57 g, 15 mmol) was added. The reaction was then stirred at 90°C for an additional 3 h

and extracted with EtOAc and water. The combined organic layer was washed with brine,

dried with MgSO4, concentrated to dryness and purified by column chromatography

(EtOAc:hexane = 2:1) to give 4-(benzyloxy)benzamide as a white solid (3.00g, 87%).

1HNMR (DMSO-d6, 300 Hz) δ(ppm): 5.16 (s, 2H, PhCH2), 7.05 (m, 2H, ArH), 7.33-7.47

(m, 5H, ArH), 7.84 (m, 2H, ArH). 13CNMR (DMSO-d6, 300 Hz) δ(ppm): 68.76, 113.64,

140

136.18, 127.13, 127.32, 127.85, 128.73, 136.16, 160.06, 166.79. HRMS(EI) Calcd for

C14H13NO2 227.0946; found: 227.0946.

4.5.1.2 Synthesis of (4-(benzyloxy)phenyl)methanamine (4-1a)

To a solution of borane (0.60 mmol) in THF (1 mL) was added 4-(benzyloxy)benzamide

(67.00 mg, 0.30 mmol) in THF (2 mL). The reaction mixture was stirred under reflux

overnight and then concentrated to dryness. The white solid obtained was dissolved in

EtOAc and washed with water. The aqueous layer was then extracted with EtOAc and the

combined organic layer was washed with brine, dried with MgSO4, concentrated to

dryness and purified by column chromatography (EtOAc 100% followed by

MeOH:CH2Cl2=1:8) to give 4-1a as a white solid (55.00 mg, 88%). 1HNMR (MeOD, 300

Hz) δ(ppm): 4.43 (s, 4H, 2CH2), 6.49-6.97 (m, 9H, ArH). 13CNMR (DMSO-d6, 300 Hz)

δ(ppm): 45.59, 70.99, 116.11, 128.50, 128.81, 129.45, 130.07, 132.32, 138.69, 159.53.

MS(EI): m/z 213.1.

4.5.2 Synthesis of N4-(4-(benzyloxy)benzyl)-6-chloropyrimidine-4,5-diamine (4-2a)

and N4-(4-methoxybenzyl)-6-chloropyrimidine-4,5-diamine (4-2b)

A solution of compound 4-1a or 4-methoxybenzyl amine, 5-amino-4,6-

dichloropyrimidine (1.5 equiv.) and DiEA (3 equiv.) in 1-butanol was stirred under reflux

for 24 h and then evaporated to dryness. The residue obtained was extracted with EtOAc

and water. The combined organic layer was washed with brine, dried with MgSO4,

concentrated to dryness and purified by column chromatography (EtOAc:hexane=1:4) to

give 4-2a and 4-2b in 75% and 100% yield respectively.

141

4-2a: 1HNMR (CDCl3, 300 Hz) δ(ppm): 3.62 (s, 2H, NH2), 4.57 (d, J=5.2 Hz, 2H,

NHCH2), 5.03 (s, 2H, OCH2), 5.45 (s, 1H, NH), 6.90-7.43 (m, 9H, ArH), 8.03 (s, 1H,

C2H). 13CNMR (DMSO-d6, 300 Hz) δ(ppm): 45.00, 69.96, 114.94, 121.96, 127.34,

127.93, 128.52, 129.23, 130.36, 136.76, 142.00, 149.09, 154.32, 158.18. HRMS(EI)

Calcd for C18H17N4OCl 340.1091 found: 340.1087.

4-2b: 1HNMR (DMSO-d6, 300 Hz) δ(ppm): 2.85(s, 3H, CH3), 3.68 (d, J=5.2 Hz, 2H,

NHCH2), 4.21 (s, 2H, PhCH2), 5.45 (s, 1H, NH), 6.00-6.40 (m, 4H, ArH), 8.03 (s, 1H,

C2H). 13CNMR (DMSO-d6, 300 Hz) δ(ppm): 43.17, 54.54, 113.21, 123.03, 128.32,

130.75, 136.38, 145.11, 151.26, 157.81. HRMS(EI) Calcd for C12H13N4OCl 264.0778;

found: 264.0776.

4.5.3 Synthesis of 9-(4-(benzyloxy)benzyl)-8-hexyl-1H-purin-6(9H)-one (4-3) and 9-

(4-(benzyloxy)benzyl)-6-chloro-8-hexyl-9H-purine (4-12)

Acetic acid (0.5 mL) was added to a solution of 4-2a (66.00 mg, 0.2 mmol) and heptan-1-

al (0.11 mL, 0.8 mmol) in THF (2 mL). The reaction mixture was stirred under reflux for

24 h and then concentrated to dryness. The residue was dissolved in 85% formic acid (2.1

mL), stirred at 70°C for 3 h and then evaporated to dryness and chromatographed on

silica gel column using EtOAc:hexane=1:1 followed by EtOH:CH2Cl2=1:10 as eluent to

give 4-3 as pale yellow solid (65.00 mg, 81%). 4-3: 1HNMR (DMSO-d6, 300 Hz)

δ(ppm): 0.83 (t, J=6.6 Hz, 3H, CH3), 1.19-1.59 (m, 8H, 4CH2), 2.67 (t, J=7.7 Hz, 2H,

C8CH2), 5.05 (s, 2H, N9CH2), 5.28 (s, 2H, OCH2), 6.96 (m, 2H, ArH), 7.11-7.42 (m, 9H,

ArH), 8.02 (s, 1H, C2H), 12.25 (s, 1H, N1H). 13CNMR (DMSO-d6, 300 Hz) δ(ppm):

13.29, 21.34, 25.81, 26.04, 27.60, 30.34, 73.94, 68.68, 114.44, 122.13, 127.03, 127.23,

142

127.73, 127.82, 128.23, 136.38, 144.49, 148.60, 150.54, 155.71, 157.27. HRMS(EI)

Calcd for C25H28N4O2 416.2212; found: 416.2200.

9-(4-(Benzyloxy)benzyl)-6-chloro-8-hexyl-9H-purine (4-12): 1HNMR (CDCl3, 300 Hz)

δ(ppm): 0.86 (t, J=6.6 Hz, 3H, CH3), 1.25-1.37 (m, 6H, 3CH2), 1.70-1.77 (m, 2H, CH2),

2.83 (t, J=7.7 Hz, 2H, C8CH2), 5.01 (s, 2H, N9CH2), 5.37 (s, 2H, OCH2), 6.89-7.39 (m,

9H, ArH), 8.69 (s, 1H, C2H). 13CNMR (CDCl3, 300 Hz) δ(ppm): 13.89, 22.33, 27.10,

28.20, 28.97, 31.26, 45.79, 70.01, 115.33, 127.29, 127.36, 127.96, 128.43, 128.51,

130.86, 136.53, 148.92, 151.14, 153.23, 158.74, 158.86. HRMS(EI) Calcd for

C25H27ClN4O 434.1873; found: 434.1873.

4.5.4 Synthesis of 9-(4-(benzyloxy)benzyl)-8-hexyl-1-(prop-2-ynyl)-1H-purin-6(9H)-

one (4-4)

Propargyl bromide (0.14 g, 1.2 mmol) and DBU (0.18 g, 1.2 mmol) were added to a

solution of 4-3 (0.11 g, 0.3 mmol) in DMF (2 mL). The reaction mixture was stirred at

room temperature overnight and then extracted with EtOAc and brine. The combined

organic layer was washed with brine, dried with MgSO4, concentrated to dryness and

purified by column chromatography (EtOAc:hexane = 1:1 followed by acetone:hexane=

1:4) to provide 4-4 as yellow oil (0.13 g, 98%). 1HNMR (CDCl3, 300 Hz) δ(ppm): 0.84

(t, J=6.4 Hz, 3H, CH3), 1.22-1.76 (m, 8H, 4CH2), 2.49 (t, J=2.4 Hz, 1H, CH), 2.68 (t,

J=7.8 Hz, 2H, C8CH2), 4.86 (d, J=2.0 Hz, 2H, N1CH2), 5.01 (s, 2H, N9CH2), 5.24 (s,

2H, OCH2), 6.89 (m, 2H, ArH), 7.05 (m, 2H, ArH), 7.30-7.39 (m, 5H, ArH), 8.25 (s, 1H,

C2H). 13CNMR (CDCl3, 300 Hz) δ(ppm): 13.93, 22.40, 27.05, 27.72, 28.92, 31.35,

143

34.87, 45.39, 70.02, 75.38, 76.58, 115.24, 127.33, 127.94 (x2), 127.98, 128.21, 128.53,

136.63, 145.17, 148.46, 152.88, 155.65, 158.57. HRMS(EI) Calcd for C28H30N4O2

454.2369; found: 454.2360.

4.5.5 Synthesis of 9-(4-(benzyloxy)benzyl)-7-butyl-8-hexyl-1-(prop-2-ynyl)-8,9-

dihydro-1H-purin-6(7H)-one salt (4-5)

1-Iodobutane (0.8 mL, 7 mmol) was added to a solution of 4-4 (0.08 g, 0.18 mmol) in

DMF (2 mL). The reaction mixture was stirred at 70oC for 24 h and then evaporated to

dryness. The residue was purified by column chromatography (ethanol:CH2Cl2 = 1:15) to

give 4-5 (0.11 g, 93%). 1HNMR (CDCl3, 300 Hz) δ(ppm): 0.77 (t, J=7.0 Hz, 3H, CH3),

0.92 (t, J=7.3 Hz, 3H, CH3), 1.04-1.94 (m, 12H, 6CH2), 2.53 (t, J=2.4 Hz, 1H, CH), 3.22

(t, J=8.0 Hz, 2H, C8CH2), 4.36 (t, J=8.0 Hz, 2H, N7CH2), 4.91 (d, J=2.4 Hz, 2H,

N1CH2), 4.97 (s, 2H, N9CH2), 5.64 (s, 2H, OCH2), 6.88 (m, 2H, ArH), 7.21 (m, 2H,

ArH), 7.24-7.32 (m, 5H, ArH), 8.69 (s, 1H, C2H). 13CNMR (CDCl3, 300 Hz) δ(ppm):

13.32, 13.82, 19.83, 22.14, 26.81, 27.37, 29.01, 30.91, 32.50, 36.27, 48.27, 48.86, 70.07,

75.42, 76.45, 113.36, 115.57, 125.55, 127.32, 128.07, 128.56, 129.51, 136.31, 146.74,

150.27, 151.36, 153.43, 159.26. HRMS(ESI) Calcd for C32H39N4O2 511.3073; found:

511.3070.

4.5.6 Synthesis of 7-butyl-8-hexyl-1-(prop-2-ynyl)-1H-purin-6(7H)-one (4-6a)

Method A: 96% Sulfuric acid (0.5 mL) and toluene (0.5 mL) were added to a solution of

4-5 in dichloromethane (1 mL). The reaction mixture was stirred vigorously at room

temperature for 3 h, neutralized with saturated NaHCO3 to pH8 and then extracted with

144

EtOAc and water. The combined organic layer was washed with brine, dried with

MgSO4, concentrated to dryness and purified by column chromatography (EtOAc:hexane

= 1:1 followed by acetone:hexane= 1:2) to provide 4-6a as yellow oil (0.13 g, 95%).

1HNMR (CDCl3, 300 Hz) δ(ppm): 0.87 (t, J=7.0 Hz, 3H, CH3), 0.90 (t, J=7.4 Hz, 3H,

CH3), 1.29-1.91 (m, 12H, 6CH2), 2.46 (t, J=2.6 Hz, 1H, CH), 2.78 (t, J=7.8 Hz, 2H,

C8CH2), 4.33 (t, J=7.4 Hz, 2H, N7CH2), 4.79 (d, J=2.8 Hz, 2H, N1CH2), 8.23 (s, 1H,

C2H). 13CNMR (CDCl3, 300 Hz) δ(ppm): 13.63, 13.95, 19.75, 22.45, 27.04, 27.56,

29.03, 31.43, 33.48, 34.63, 45.25, 74.82, 76.58, 114.70, 114.93, 147.38, 153.19, 156.34.

HRMS(EI) Calcd for C18H26N4O 314.2107; found: 314.2103.

Method B: CAN (4 equiv.) was added protionwise to a stirred solution of compound 4-5

and in acetonitrile/H2O (4:1). The reaction mixture was stirred rapidly at room

temperature for 8 h and then quenched with NaHCO3 (aq.). The quenched mixture was

stirred vigorously for 10 min before extraction with EtOAc and brine. The combined

organic layer was washed with brine, dried with MgSO4, concentrated to dryness and

purified by column chromatography (EtOAc:hexane = 1:1 followed by acetone:hexane=

1:2) to provide 4-6a.

4.5.7 Synthesis of ethyl N4-(4-(benzyloxy)benzylamino)-6-chloropyrimidin-5-yl-

acetimidate (4-7)

Catalytic amount of TsOH or HCl was added to the solution of 4-2a (0.12 g, 0.34 mmol)

in triethyl orthoacetate (4 mL). The reaction mixture was stirred at room temperature for

3 h, concentrated to dryness and purified by silica gel column chromatography

(EtOAc:Hexane = 1:1) to give 4-7 as a pale yellow oil (0.24 g, 99%). 1HNMR (CDCl3,

145

300 Hz) δ(ppm): 1.33 (t, J=7.0 Hz, 3H, CH3), 1.86 (s, 3H, CH3), 4.27-4.29 (m, 2H,

OCH2), 4.61 (d, J=5.6 Hz, 2H, NHCH2), 5.05 (s, 2H, CH2), 6.93-7.43 (m, 9H, ArH), 8.19

(s, 1H, C2H). 13CNMR (CDCl3, 300 Hz) δ(ppm): 13.92, 17.62, 44.41, 62.67, 69.87,

114.91, 123.83, 127.26, 127.82, 128.43, 128.72, 130.55, 136.73, 145.49, 152.41, 156.42,

158.10, 166.89. HRMS(EI) Calcd for C22H23ClN4O2 410.1510; found: 410.1497.

4.5.8 Synthesis of 9-(4-(benzyloxy)benzyl)-6-chloro-8-methyl-9H-purine (4-8)

A solution of ferric chloride (0.05 g, 0.31 mmol) in ethanol (1 mL) was added to a

solution of 4-7 (0.07 g, 0.18 mmol) in ethanol (3 mL). The reaction mixture was stirred

under reflux condition for 1 h then concentrated to dryness and purified by silica gel

column chromatography (EtOAc:Hexane = 1:1) to give 4-8 as a white solid (0.03 g,

46%). 1HNMR (CDCl3, 300 Hz) δ(ppm): 2.60 (s, 3H, CH3), 5.03 (s, 2H, CH2), 5.36 (s,

2H, CH2), 6.87-8.06 (m, 9H, ArH), 8.71 (s, 1H, C2H). 13CNMR (CDCl3, 300 Hz)

δ(ppm): 14.78, 46.04, 77.06, 114.99, 115.41, 127.01, 127.37, 127.97, 128.04, 128.57,

128.65, 129.33, 136.52, 149.49, 151.32, 155.23. HRMS(EI) Calcd for C20H17ClN4O

364.1091; found: 364.1091.

4.5.9 Synthesis of N4-(4-(benzyloxy)benzylamino)-6-chloropyrimidin-5-yl)benzamide

(4-9)

A solution of 4-2a (138 mg, 0.41 mmol), benzoyl chloride (63 mg, 0.45 mmol) and DiEA

(78 mg, 0.61 mmol) in THF was stirred at room temperature for 7 h and then

concentrated to dryness and purified by silica gel column chromatography

(EtOAc:Hexane = 1:4-1:2) to give 4-9 (163 mg, 91%). 1HNMR (CDCl3, 300 Hz) δ(ppm):

146

4.63 (d, J=5.2 Hz, 2H, C4NH), 5.01 (s, 2H, CH2), 5.36 (s, 2H, CH2), 6.89-7.88 (m, 14H,

ArH), 7.99 (s, 1H, C2H). 13CNMR (CDCl3, 300 Hz) δ(ppm): 44.71, 69.95, 113.17,

114.97, 127.36, 127.49, 127.89, 128.50, 128.81, 128.85, 130.24, 132.49, 132.71, 136.82,

154.17, 155.71, 158.12, 158.74, 166.44. HRMS(EI) Calcd for C25H21ClN4O2 444.1353;

found: 444.1357.

4.5.10 Synthesis of 9-(4-(benzyloxy)benzyl)-6-chloro-8-phenyl-9H-purine (4-10) and

9-(4-(benzyloxy)benzyl)-8-phenyl-1H-purin-6(9H)-one (4-11)

A mixture of 4-9 and 10% KOH in CH3CN was stirred under reflux for overnight and

then was neutralized to pH7-8, concentrated to dryness and purified by silica gel column

chromatography (EtOAc:Hexane = 1:1-Acetone:Hexane=1:1) to give 4-10 and 4-11 in

ratio of 1:1.

4-10: 1HNMR (CDCl3, 300 Hz) δ(ppm): 5.01 (s, 2H, CH2), 5.50 (s, 2H, CH2), 6.85-7.70

(m, 14H, ArH), 8.78 (s, 1H, C2H). MS(EI): m/z 426.0.

4-11: 1HNMR (MeOD, 300 Hz) δ(ppm): 5.02 (s, 2H, CH2), 5.46 (s, 2H, CH2), 6.85-7.62

(m, 14H, ArH), 8.25(s, 1H, C2H). MS(EI): m/z 407.0.

4.5.11 Synthesis of 9-(4-(benzyloxy)benzyl)-6-chloro-9H-purine (4-13a) and 9-(4-

methoxybenzyl)-6-chloro-9H-purine (4-13b)

Compounds 4-13a and 4-13b were prepared from 4-2a and 4-2b correspondingly using

the method described in Section 4.5.7 (trimethyl orthoformate was used instead of triethyl

orthoacetate) in 91% and 90% yield respectively.

147

4-13a: 1HNMR (CDCl3, 300 Hz) δ(ppm): 5.01 (s, 2H, OCH2), 5.34 (s, 1H, CH2), 6.92-

7.36 (m, 9H, ArH), 8.07 (s, 1H, C8H), 8.75 (s, 1H, C2H). 13CNMR (CDCl3, 300 Hz)

δ(ppm): 47.28, 69.91, 115.36, 126.66, 127.22, 127.90, 128.44, 129.40, 131.39, 136.38,

144.81, 150.79, 151.64, 151.88, 158.95. MS(EI): m/z 350.0.

4-13b: 1HNMR (CDCl3, 300 Hz) δ(ppm): 3.62 (s, 3H, CH3), 5.26 (s, 1H, CH2), 6.69-7.15

(m, 4H, ArH), 8.06 (s, 1H, C8H), 8.59 (s, 1H, C2H). 13CNMR (CDCl3, 300 Hz) δ(ppm):

46.91, 54.77, 113.94, 126.09, 129.02, 130.93, 144.75, 150.10, 151.27, 151.37, 159.28.

HRMS(EI) Calcd for C13H11N4OCl 274.0621; found: 274.0613.

4.5.12 Synthesis of 9-(4-(benzyloxy)benzyl)-1H-purin-6(9H)-one (4-14)

A solution of 4-13a in 85% formic acid was stirred at 70oC for 1 h and then concentrated

to dryness. The residue obtained was dissolved in EtOAc and brine. The aqueous layer

was extracted with EtOAc and the combined organic layer was washed with brine, dried

with MgSO4 and purified by silica gel column chromatography (MeOH:DCM = 1:15) to

give 4-14 as a white solid in 92% yield. 1HNMR (DMSO-d6, 300 Hz) δ(ppm): 5.07 (s,

2H, OCH2), 5.28 (s, 1H, CH2), 6.96-7.40 (m, 9H, ArH), 8.03 (s, 1H, C8H), 8.16 (s, 1H,

C2H). 13CNMR (DMSO-d6, 300 Hz) δ(ppm): 46.06, 69.19, 114.94, 123.98, 127.62,

127.81, 128.40, 128.99, 129.12, 136.93, 140.13, 145.68, 148.22, 156.64, 157.97. MS(EI):

m/z 332.0.

4.5.13 Synthesis of 9-(4-(benzyloxy)benzyl)-1-butyl-1H-purin-6(9H)-one (4-15)

Compound 4-15 was prepared from 4-14 using the method described in Section 4.5.4 in

89% yield. 1HNMR (CDCl3, 300 Hz) δ(ppm): 0.94 (t, 3H, J=7.4 Hz, CH3), 1.33-1.71 (m,

148

2H, CH2), 1.74-1.77 (m, 2H, CH2), 4.03 (t, 3H, J=7.4 Hz, N1CH2), 5.03 (s, 2H, OCH2),

5.21 (s, 1H, CH2), 6.91-7.34 (m, 9H, ArH), 7.59 (s, 1H, C8H), 7.95 (s, 1H, C2H).

13CNMR (CDCl3, 300 Hz) δ(ppm): 13.51, 19.71, 31.77, 46.61, 46.90, 70.00, 115.31,

124.33, 127.30, 127.51, 127.95, 128.51, 129.19, 136.57, 139.60, 146.98, 147.60, 156.52,

158.83. HRMS(EI) Calcd for C23H24N4O2 388.1899; found: 388.1893.

4.5.14 Synthesis of 9-(4-(benzyloxy)benzyl)-1,7-dibutylpurin-6-one salt (4-16)

Compound 4-16 was prepared from 4-15 using the method described in Section 4.5.5 in

92% yield. 1HNMR (CDCl3, 300 Hz) δ(ppm): 0.91-0.97 (m, 6H, 2CH3), 1.35-1.43 (m,

4H, 2CH2), 1.71-1.75 (m, 2H, CH2), 1.96-2.01 (m, 2H, CH2), 4.10 (t, 3H, J=7.5 Hz,

CH2), 4.10 (t, 3H, J=7.6 Hz, CH2), 4.98 (s, 2H, OCH2), 5.64 (s, 1H, CH2), 6.91-7.34 (m,

9H, ArH), 8.43 (s, 1H, C2H), 10.87 (s, 1H, C8H). 13CNMR (CDCl3, 300 Hz) δ(ppm):

13.23, 13.37, 19.32, 19.57, 47.38, 49.11, 49.96, 69.86, 114.02, 115.39, 124.96, 127.26

(x2), 127.89, 128.42 (x2), 131.07, 136.33, 139.97, 146.16, 151.18, 151.99, 159.40.

HRMS(ESI) Calcd for C27H33N4O2 445.2604; found: 445.2590.

4.5.15 Synthesis of 1,7-dibutyl-1H-purin-6(7H)-one (4-17a)

Compound 4-17a was prepared from 4-16 using the method described in Section 4.5.6 in

96% yield. 1HNMR (CDCl3, 300 Hz) δ(ppm): 0.92-0.98 (m, 6H, 2CH3), 1.31-1.43 (m,

4H, 2CH2), 1.70-1.92 (m, 4H, 2CH2), 4.00 (t, 3H, J=7.3 Hz, CH2), 4.40 (t, 3H, J=7.3 Hz,

CH2), 7.81 (s, 1H, C8H), 7.99 (s, 1H, C2H). 13CNMR (CDCl3, 300 Hz) δ(ppm): 13.44,

13.55, 19.54, 19.81, 31.86, 33.34, 46.32, 47.21, 115.41, 143.15, 146.38, 154.25, 156.94.

HRMS(ESI) Calcd for C13H20N4O 248.1637; found: 248.1637.

149

4.5.16 Synthesis of 3-(4-(benzyloxy)benzyl)-7-chloro-3H-[1,2,3]triazolo[4,5-d]

pyrimidine (4-18a) and 3-(4-methoxybenzyl)-7-chloro-3H-[1,2,3]triazolo[4,5-d]

pyrimidine (4-18b)

Sodium nitrite (4.7 equiv.) was added to a solution of 4-2a or 4-2b in DCM and 50%

HOAc (aq). The reaction mixture was stirred vigorously at room temperature for 30 min

and then concentrated to dryness and purified by silica gel column chromatography

(EtOAc:Hexane = 1:2) to give 4-18a or 4-18b in 96% and 94% yield respectively.

4-18a: 1HNMR (CDCl3, 300 Hz) δ(ppm): 5.01 (s, 2H, CH2), 5.80 (s, 2H, CH2), 6.91-7.44

(m, 9H, ArH), 8.91 (s, 1H, C2H). 13CNMR (CDCl3, 300 Hz) δ(ppm): 50.86, 69.85,

115.14, 126.09, 127.26, 127.92, 128.46, 130.00, 133.99, 136.41, 149.42, 153.91, 155.34,

159.02. HRMS(EI) Calcd for C18H14N5OCl 351.0887; found: 351.0890.

4-18b: 1HNMR (CDCl3, 300 Hz) δ(ppm): 3.77 (s, 3H, CH3), 5.82 (s, 2H, CH2), 6.85-7.45

(m, 4H, ArH), 8.92 (s, 1H, C2H). 13CNMR (CDCl3, 300 Hz) δ(ppm): 50.66, 54.97,

113.98, 125.67, 129.75, 133.75, 149.24, 153.57, 155.13, 159.59. HRMS(EI) Calcd for

C12H10N5OCl 275.0574; found: 275.0579.

4.5.17 Synthesis of 3-(4-(benzyloxy)benzyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7

(6H) -one (4-19)

Compound 4-19 was prepared from 4-18 using the method described in Section 4.5.12 in

92% yield. 1HNMR (DMSO-d6, 300 Hz) δ(ppm): 5.07 (s, 2H, CH2), 5.67 (s, 2H, CH2),

6.97-7.41 (m, 9H, ArH), 8.26 (s, 1H, C2H). 13CNMR (DMSO-d6, 300 Hz) δ(ppm): 49.39,

150

69.19, 115.00, 127.54, 127.66, 127.85, 128.16, 128.43, 129.50, 129.67, 148.38, 149.83,

155.28, 158.21. HRMS(EI) Calcd for C18H15N5O2 333.1226; found: 333.1229.

4.5.18 Synthesis of 2-(3-(4-methoxybenzyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-

ylamino)ethanol (4-20a) and 2-(9-(4-methoxybenzyl)-9H-purin-6-ylamino)ethanol

(4-20b)

A solution of 4-18b or 4-13b and ethanolamine (3 equiv.) in THF was stirred at 0oC for 3

h and then concentrated to dryness and purified by silica gel column chromatography

(EtOAc:Hexane = 1:2) to give 4-20a or 4-20b in 100% and 96% yield respectively.

4-20a: 1HNMR (DMSO-d6, 300 Hz) δ(ppm): 3.60-3.62 (m, 4H, 2CH2), 3.71 (s, 3H,

CH3), 5.67 (s, 2H, CH2), 6.88-7.31 (m, 5H, ArH), 8.39 (s, 1H, C2H). 13CNMR (CDCl3,

300 Hz) δ(ppm): 50.86, 69.85, 115.14, 126.09, 127.26 (x2), 127.92, 128.46 (x2), 130.00,

133.99, 136.41, 149.42, 153.91, 155.34, 159.02. HRMS(EI) Calcd for C14H16N6O

300.1335; found: 300.1339.

4-20b: 1HNMR (CDCl3, 300 Hz) δ(ppm): 3.66-3.82 (m, 7H, CH3+2CH2), 5.13 (s, 2H,

CH2), 6.76-7.18 (m, 4H, ArH), 7.61 (s, 1H, C8H), 8.27 (s, 1H, C2H). 13CNMR (CDCl3,

300 Hz) δ(ppm): 43.40, 46.41, 54.98, 61.25, 114.06, 118.97, 127.22, 129.04, 139.19,

148.65, 152.75, 154.69, 159.29. HRMS(EI) Calcd for C15H17N5O 299.1382; found:

299.1379.

151

4.5.19 Synthesis of 4-21a and 4-21b

4.5.19.1 Synthesis of 4-21a and 4-21b

A solution of 4-20a or 4-20b and thionyl chloride (1.5 equiv.) in DMF was stirred at

180oC under microwave irradiation for 20 min. After evaporating all the volatile

materials, the residue was dissolved in CH2Cl2 and saturated Na2CO3. The aqueous layer

was extracted with CH2Cl2. The combined organic layer was washed with brine, dried

with MgSO4 and purified by silica gel column chromatography (Acetone:DCM=1:1-

EtOH:DCM= 1:10) to give 4-21a (83%) and 4-21b (88%).

4-21a: 1HNMR (DMSO-d6, 300 Hz) δ(ppm): 3.72 (s, 3H, CH3), 3.98 (t, J=9.5 Hz, 2H,

CH2), 4.10 (t, J=9.2 Hz, 2H, CH2), 5.51 (s, 2H, CH2), 6.81-7.33 (m, 4H, ArH), 7.77 (s,

1H, C2H). 13CNMR (CDCl3, 300 Hz) δ(ppm): 45.89, 49.93, 54.33, 55.13, 113.99, 125.99,

126.71, 129.55, 146.19, 147.99, 156.62, 159.48. HRMS(EI) Calcd for C14H14N6O

282.1229; found: 282.1240.

4-21b: 1HNMR (DMSO-d6, 300 Hz) δ(ppm): 3.71 (s, 3H, CH3), 3.88 (t, J=9.1 Hz, 2H,

CH2), 4.10 (t, J=9.6 Hz, 2H, CH2), 5.20 (s, 2H, CH2), 6.87-7.26 (m, 4H, ArH), 8.02 (s,

1H, C8H), 8.06 (s, 1H, C2H). 13CNMR (CDCl3, 300 Hz) δ(ppm): 43.40, 46.41, 54.98,

61.25, 114.06, 118.97, 127.22, 129.04, 139.19, 148.65, 152.75, 154.69, 159.29.

HRMS(EI) Calcd for C15H15N5O 281.1277; found: 281.1287.

4.5.19.2 Data of side products 4-25 and 4-26

2-(3-(4-Methoxybenzyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-ylamino)ethyl methane

sulfonate (4-25): 1HNMR (DMSO-d6, 300 Hz) δ(ppm): 2.89 (s, 3H, CH3), 3.29-3.35 (m,

2H, NHCH2), 4.11 (m, 2H, OCH2), 5.68 (s, 2H, PhCH2), 6.90-7.34 (m, 4H, ArH), 7.32

152

(br, 1H, NH), 8.46 (s, 1H, C2H). 13CNMR (DMSO-d6, 300 Hz) δ(ppm): 40.34, 41.39,

47.47, 50.22, 56.00, 113.65, 126.73, 128.66, 129.08, 147.30, 152.17, 154.37, 158.68.

HRMS(EI) Calcd for C15H18N6O4S 378.1110; found: 378.1113.

3-(4-Methoxybenzyl)-N-(2-chloroethyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-amine

(4-26): 1HNMR (DMSO-d6, 300 Hz) δ(ppm): 3.71 (s, 3H, OCH3), 3.83-3.85 (m, 4H,

2CH2), 5.70 (s, 2H, PhCH2), 6.89-7.33 (m, 4H, ArH), 8.43 (s, 1H, C2H), 9.13 (br, 1H,

NH). HRMS(EI) Calcd for C14H15N6OCl 318.0996; found: 318.0992.

4.5.20 9-Butyl-4-(4-methoxybenzyl)-tetrahydro-imidazo[2,1-i]purine salt (4-23)

Compound 4-23 was prepared using the method described in Section 4.5.5 in 84% yield.

1HNMR (CDCl3, 300 Hz) δ(ppm): 0.94 (t, J=7.3 Hz, 3H, CH3), 1.38-1.45 (m, 2H, CH2),

1.74-1.81 (m, 2H, CH2), 3.77 (s, 3H, CH3), 4.09 (t, J=7.3 Hz, 2H, N7CH2), 4.33 (t, J=9.8

Hz, 2H, CH2), 5.27 (t, J=9.6 Hz, 2H, CH2), 5.34 (s, 2H, CH2), 6.85-7.30 (m, 4H, ArH),

8.00 (s, 1H, C8H), 8.89 (s, 1H, C2H). 13CNMR (CDCl3, 300 Hz) δ(ppm): 12.81, 18.91,

28.25, 46.43, 46.76, 47.90, 48.15, 54.67, 113.56, 115.36, 125.78, 129.08, 143.27, 143.29,

148.78, 149.51, 158.86. HRMS(ESI) Calcd for C19H24N5O 338.1981; found: 338.1986.

4.5.21 Tetrahydro-imidazo[1,2-c][1,2,3]triazolo[4,5-e]pyrimidine (4-22) and 9-butyl-

tetrahydro-imidazo[2,1-i]purine (4-24)

Compounds 4-22 and 4-24 were prepared according to the procedure given in Section

4.5.6.

153

4-22: 1HNMR (DMSO-d6, 300 Hz) δ(ppm): 4.17 (t, J=9.4 Hz, 2H, CH2), 4.69 (t, J=9.5

Hz, 2H, CH2), 8.81 (s, 1H, C2H). 13CNMR (MeOD, 300 Hz) δ(ppm): 45.32 (x2), 114.84,

148.25, 148.53, 159.58. HRMS(EI) Calcd for C6H6N6 162.0654; found: 162.0651.

4-24: 1HNMR (MeOD, 300 Hz) δ(ppm): 1.00 (t, J=7.3 Hz, 3H, CH3), 1.44-1.54 (m, 2H,

CH2), 1.79-1.86 (m, 2H, CH2), 4.11-4.22 (m, 4H, 2CH2), 4.80 (t, J=7.3 Hz, 2H, N1CH2),

8.35 (s, 1H, C8H), 8.51 (s, 1H, C2H). 13CNMR (MeOD, 300 Hz) δ(ppm): 12.99, 19.66,

29.28, 47.04, 47.84, 48.41, 117.07, 144.52, 145.45, 151.08, 152.81. HRMS(EI) Calcd for

C11H15N5 217.1327; found: 217.1324.

4.5.22 Preparation of BOBA resin (4-27)

DiAD (3 equiv.) was added dropwise to a mixture of Wang resin (loading 1.6 mmol/g),

phthalimide (3 equiv.) and triphenylphosphine (3 equiv.) in THF under ice water bath

temperature. The reaction mixture was shaken at room temperature overnight and the

resin was filtered and washed sequentially with DMF (20 mL x 2), H2O (20 mL x 2),

EtOH (20 mL x 2), CH2Cl2 (20 mL x 2) and ether (20 mL x 2), and dried overnight in a

vacuum oven at 40°C. The dried resin was then subjected to hydrazinolysis using a

solution of hydrazine (15 equiv.) in THF at room temperature overnight. After which, the

resin was washed sequentially with DMF (20 mL x 2), H2O (20 mL x 2), EtOH (20 mL x

2), CH2Cl2 (20 mL x 2) and ether (20 mL x 2), and dried overnight in a vacuum oven at

40°C.

154

4.5.23 Synthesis of resin bound N4-(4-(benzyloxy)benzyl)-6-chloropyrimidine-4,5-

diamine (4-28)

Resin 4-27 was swollen in DMA for 30 min and 1-butanol, 5-amino-4,6-dichloro-

pyrimidine and DiEA were added. The reaction mixture was stirred slowly at 140°C for

24 h. After which, the resin was filtered and washed sequentially with DMF (20 mL x 2),

H2O (20 mL x 2), EtOH (20 mL x 2), CH2Cl2 (20 mL x 2) and ether (20 mL x 2), and

dried overnight in a vacuum oven at 40°C.

4.5.24 General solid-phase cyclization of 4-28 with aldehydes

Resin 4-28 was swollen in THF for 30 min and acetic acid and the appropriate aldehydes

(8 equiv.) were added. The reaction mixture was then slowly stirred under reflux

condition for 24 h. After which, the resin was filtered and treated with 85% formic acid

and DMF and stirred at 70°C for 3 h.

4.5.25 General procedure for solid-phase N1 alkylation

The resin 4-29 was swollen in DMF for 30 min and the respective alkylating reagents (3

equiv.) and DBU (3 equiv.) were added. After shaking at room temperature overnight, the

resin was filtered and washed with DMF (20 mL x 2), EtOH (20 mL x 2) and CH2Cl2 (20

mL x 2) and dried in vacuum.

4.5.26 General procedure for solid-phase N7 quaternization

Resin 4-30 was swollen in DMF for 30 min. The respective quarternization reagent (40 or

50 equiv.) was added and the mixture was stirred slowly at 70oC for 36 h. After which,

155

the resin was filtered and washed with DMF (20 mL x 2), H2O (20 mL x 2), EtOH (20

mL x 2) and CH2Cl2 (20 mL x 2) and dried in vacuum.

4.5.27 Synthesis of resin bound 9-(4-(benzyloxy)benzyl)-6-chloro-9H-purine (4-32a):

Resin 4-28 was swollen in DMF for 30 min and then trimethyl orthoformate (50 equiv.)

was added. The reaction mixture was then cooled in an ice water bath and HCl (cat.) was

added. The reaction mixture was then shaken at room temperature overnight. After

which, the resin was filtered and washed with DMF (20 mL x 2), H2O (20 mL x 2), EtOH

(20 mL x 2) and CH2Cl2 (20 mL x 2) and dried in vacuum.

4.5.28 Synthesis of resin bound 3-(4-(benzyloxy)benzyl)-7-chloro-3H-[1,2,3] triazolo

[4,5-d]pyrimidine (4-32b)

Resin 4-28 was swollen in DCM for 30 min and 50% aqueous acetic acid and NaNO2 (2

equiv.) were added. After shaking vigorously at room temperature for 30 min, the resin

was filtered and washed with EtOH (20 mL x 2) and CH2Cl2 (20 mL x 2) and dried in

vacuum.

4.5.29 Solid-phase synthesis of 4-37 via amination at C6 with ethanolamine

The loaded resin 4-32 was swollen in THF for 30 min and then ethanolamine was added.

The reaction mixture was stirred slowly at 60oC for 4 h. After which, the resin was

filtered and washed sequentially with EtOH (20 mL x 2), CH2Cl2 (20 mL x 2) and Et2O

(20 mL x 2), and dried overnight in a vacuum oven at 40°C.

156

4.5.30 Solid-phase synthesis of 4-38 with thionyl chloride

Resin 4-37 was swollen in DMF for 30 min and then SOCl2 (2 equiv.) was added. The

reaction mixture was stirred at 180oC under microwave irradiation for 20 min. After

which, the resin was filtered and washed sequentially with EtOH (20 mL x 2), saturated

NaHCO3 (20 mL x 2), H2O (20 mL x 2), EtOH (20 mL x 2), CH2Cl2 (20 mL x 2) and

ether (20 mL x 2), and dried overnight in a vacuum oven at 40°C.

4.5.31 General cleavage procedure

Resin was swollen in DCM for 30 min. Toluene and 96% sulfuric acid were added and

the reaction mixture was shaken at room temperature for 4 h. The resin was then filtered

and washed with EtOAc. The filtrate was neutralized with saturated NaHCO3 and

extracted with EtOAc and water. The combined organic layer was washed with brine,

dried with MgSO4, concentrated to dryness and purified by column chromatography.

4-6a: 7-Butyl-8-hexyl-1-(prop-2-ynyl)-1H-purin-6(7H)-one

1HNMR (CDCl3, 300 Hz) δ(ppm): 0.87 (t, J=7.0 Hz, 3H, CH3), 0.90 (t, J=7.4 Hz, 3H,

CH3), 1.29-1.91 (m, 12H, 6CH2), 2.46 (t, J=2.6 Hz, 1H, CH), 2.78 (t, J=7.8 Hz, 2H,

C8CH2), 4.33 (t, J=7.4 Hz, 2H, N7CH2), 4.79 (d, J=2.8 Hz, 2H, N1CH2), 8.23 (s, 1H,

C2H). 13CNMR (CDCl3, 300 Hz) δ(ppm): 13.63, 13.95, 19.75, 22.45, 27.04, 27.56,

29.03, 31.43, 33.48, 34.63, 45.25, 74.82, 76.58, 114.70, 114.93, 147.38, 153.19, 156.34.

HRMS(EI) Calcd for C18H26N4O 314.2107; found: 314.2103.

157

4-6b: 1,7-Dibenzyl-8-hexyl-1H-purin-6(7H)-one

1HNMR (CDCl3, 300 Hz) δ(ppm): 0.84 (t, J=6.6 Hz, 3H, CH3), 1.24-1.31 (m, 6H, 3CH2),

1.71-1.75 (m, 2H, CH2), 2.71 (t, J=7.7 Hz, 2H, N7CH2), 5.2 (s, N1CH2), 5.66 (s, 1H,

N7CH2), 7.12-7.34 (m, 10H, ArH), 8.09 (s, 1H, C2H). 13CNMR (CDCl3, 300 Hz) δ(ppm):

13.96, 22.40, 27.22, 27.26, 28.92, 31.36, 48.37, 49.03, 115.25, 126.72 (x2), 127.84,

127.92, 128.22 (x2), 128.90, 128.97, 135.96, 136.25, 146.19, 154.10, 156.05, 156.98.

HRMS(EI) Calcd for C25H28N4O 400.2263; found: 400.2257.

4-6c: 1-Allyl-7-benzyl-8-hexyl-1H-purin-6(7H)-one

1HNMR (CDCl3, 300 Hz) δ(ppm): 0.84 (t, J=6.6 Hz, 3H, CH3), 1.24-1.34 (m, 6H, 3CH2),

1.71-1.76 (m, 2H, CH2), 2.72 (t, J=7.7 Hz, 2H, C8CH2), 4.63 (d, J=5.6 Hz, N1CH2), 5.24

(m, 2H, CH2), 5.65 (s, 2H, N7CH2), 5.93-6.02 (m, 1H, CH), 7.11-7.33 (m, 5H, ArH), 8.00

(s, 1H, C2H). 13CNMR (CDCl3, 300 Hz) δ(ppm): 13.96, 22.40, 27.22, 27.26, 28.92,

31.36, 48.37, 49.03, 126.72, 127.84, 127.92, 128.22, 128.90, 128.97, 135.96, 136.25,

146.19, 154.10, 156.05, 156.98. HRMS(EI) Calcd for C21H26N4O 350.2107; found:

350.2111.

4-6d: 1-Allyl-7-butyl-8-phenyl-1H-purin-6(7H)-one

1HNMR (CDCl3, 300 Hz) δ(ppm): 0.82 (t, J=7.5 Hz, 3H, CH3), 1.19-1.27 (m, 2H, CH2),

1.73-1.81 (m, 2H, CH2), 4.43 (t, J=7.5 Hz, 2H, N7CH2), 4.65 (d, J=6.0 Hz, N1CH2), 5.23

(m, 2H, CHCH2), 5.93-6.03 (m, 1H, CH), 7.48-7.69 (m, 5H, ArH), 8.01 (s, 1H, C2H).

13CNMR (CDCl3, 300 Hz) δ(ppm): 13.37, 19.47, 33.57, 46.34, 47.80, 116.00, 118.53,

158

128.72, 129.21, 129.27, 130.17, 132.15, 146.39, 153.88, 154.13, 156.35. HRMS(EI)

Calcd for C18H20N4O 308.1637; found: 308.1637.

4-6e: 7-Benzyl-1-butyl-8-phenyl-1H-purin-6(7H)-one

1HNMR (CDCl3, 300 Hz) δ(ppm): 0.95 (t, J=7.3 Hz, 3H, CH3), 1.32-1.42 (m, 2H, CH2),

1.69-1.79 (m, 2H, CH2), 4.00 (t, J=7.3 Hz, 2H, N1CH2), 5.74 (s, 2H, N7CH2), 6.99-7.64

(m, 10H, 2ArH), 8.05 (s, 1H, C2H). 13CNMR (CDCl3, 300 Hz) δ(ppm): 13.57, 19.76,

31.87, 46.34, 49.54, 126.33, 127.72, 127.81, 128.23, 128.77, 128.96, 129.37, 130.35,

136.95, 146.80, 154.19, 154.58, 156.36. HRMS(EI) Calcd for C22H22N4O 358.1794;

found: 308.1788.

4-6f: 7-Butyl-8-(4-methoxyphenyl)-1-(prop-2-ynyl)-1H-purin-6(7H)-one

1HNMR (CDCl3, 300 Hz) δ(ppm): 0.86 (t, J=7.3 Hz, 3H, CH3), 1.24-1.31 (m, 2H, CH2),

1.77-1.84 (m, 2H, CH2), 2.49 (t, J=2.6 Hz, 2H, N1CH2), 3.88 (s, 3H, OCH3), 4.44 (t,

J=7.7 Hz, N7CH2), 4.83 (d, J=2.6 Hz, CH), 7.02 (m, 2H, ArH), 7.64(m, 2H, ArH), 8.30

(s, 1H, C2H). 13CNMR (CDCl3, 300 Hz) δ(ppm): 13.47, 19.59, 33.58, 34.83, 46.48,

55.39, 74.99, 76.70, 114.26, 121.36, 130.79, 145.48, 153.40, 154.38, 156.42, 161.14.

HRMS(EI) Calcd for C19H20N4O2 336.1586; found: 336.1588.

4-6g: 7-Allyl-1-benzyl-8-(4-methoxyphenyl)-1H-purin-6(7H)-one

1HNMR (CDCl3, 300 Hz) δ(ppm): 3.88 (s, 3H, CH3), 5.04 (m, 2H, N7CH2), 5.17 (dd,

J=13.7 Hz, J=81.2 Hz, CHCH2), 5.23 (s, 2H, N1CH2), 6.09-6.20 (m, 1H, CH), 7.02-7.23

(m, 2H, ArH), 7.34-7.36 (m, 5H, ArH), 7.74 (m, 2H, ArH), 8.16 (s, 1H, C2H). 13CNMR

159

(CDCl3, 300 Hz) δ(ppm): 48.47, 49.03, 55.29, 114.11, 115.98, 116.98, 120.95, 127.79,

128.14, 128.89, 130.69, 133.68, 135.89, 146.62, 153.92, 154.29, 156.20, 161.19.

HRMS(EI) Calcd for C22H20N4O2 372.1586; found: 372.1578.

4-6h: 7-Butyl-1-(prop-2-ynyl)-8-styryl-1H-purin-6(7H)-one

1HNMR (CDCl3, 300 Hz) δ(ppm): 0.97 (t, J=7.3 Hz, 3H, CH3), 1.35-1.48 (m, 2H, CH2),

1.79-1.89 (m, 2H, CH2), 2.49 (t, J=2.4 Hz, 1H, CH), 4.53 (t, J=7.3 Hz, 2H, N7CH2), 4.81

(d, J=2.8 Hz, 2H, N1CH2), 6.95 (d, J=15.7 Hz, 1H, CH), 7.36-7.60 (m, 5H, ArH), 8.04

(d, J=15.7 Hz, 1H, CH), 8.29 (s, 1H, C2H). 13CNMR (CDCl3, 300 Hz) δ(ppm): 13.66,

19.72 (x2), 33.67 (x2), 34.78, 45.09, 75.07, 111.31, 127.47, 128.91 (x2), 129.56, 135.49,

145.67, 151.62, 153.17, 156.34. HRMS(EI) Calcd for C20H20N4O 332.1637; found:

332.1621.

4-6i: 7-Allyl-1-butyl-8-styryl-1H-purin-6(7H)-one

1HNMR (CDCl3, 300 Hz) δ(ppm): 0.96 (t, J=7.3 Hz, 3H, CH3), 1.36-1.44 (m, 2H, CH2),

1.73-1.78 (m, 2H, CH2), 3.99 (t, J=7.4 Hz, 2H, N1CH2), 5.05-5.28 (m, 2H, CH2), 5.23 (d,

J=5.0 Hz, N7CH2), 5.99-6.10 (m, 1H, CH), 6.92 (d, J=15.8 Hz, 1H, CH), 7.36-7.57 (m,

5H, ArH), 7.99 (d, J=15.7 Hz, 1H, CH), 8.04 (s, 1H, C2H). 13CNMR (CDCl3, 300 Hz)

δ(ppm): 13.56, 19.79, 31.85, 46.29, 46.92, 111.63, 117.53, 127.41, 128.39, 128.83,

129.44, 132.68, 135.50, 139.38, 146.71, 151.71, 153.91, 156.24. HRMS(EI) Calcd for

C20H22N4O 334.1794; found: 334.1777.

160

4-17b: 7-Benzyl-1-(prop-2-ynyl)-1H-purin-6(7H)-one

1HNMR (CDCl3, 300 Hz) δ(ppm): 2.48 (t, J=2.4 Hz, 1H, CH3), 4.80 (d, J=2.8 Hz, 2H,

N1CH2), 5.58 (s, 2H, N7CH2), 7.32-7.36 (m, 5H, ArH), 7.87 (s, 1H, C8H), 8.27 (s, 1H,

C2H). 13CNMR (CDCl3, 300 Hz) δ(ppm): 34.75, 50.62, 75.13, 114.73, 127.91, 128.55

(x2), 129.04, 135.41, 143.45, 145.46, 153.63, 156.92. HRMS(EI) Calcd for C15H12N4O

264.1.11; found: 264.1.11.

4-17c: 7-Butyl-1-(prop-2-ynyl)-1H-purin-6(7H)-one

1HNMR (CDCl3, 300 Hz) δ(ppm): 0.89 (t, J=7.3 Hz, 3H, CH3), 1.25-1.35 (m, 2H, CH2),

1.77-1.86 (m, 2H, CH2), 2.46 (dt, J=0.7 Hz, J=2.4 Hz, 1H, CH3), 4.32 (t, J=7.3 Hz, 2H,

N1CH2), 4.77 (dd, J=0.7 Hz, J=2.6 Hz, 2H, N1CH2), 7.81 (s, 1H, C8H), 8.23(s, 1H,

C2H). 13CNMR (CDCl3, 300 Hz) δ(ppm): 12.90, 18.95, 32.70, 34.22, 46.71, 74.48,

76.07, 114.45, 142.95, 144.87, 152.94, 156.40. HRMS(EI) Calcd for C12H14N4O

230.1168, found: 230.1162.

4-17d: 7-Benzyl-1-butyl-1H-purin-6(7H)-one

1HNMR (CDCl3, 300 Hz) δ(ppm): 0.95 (t, J=7.3 Hz, 3H, CH3), 1.35-1.43 (m, 2H, CH2),

1.69-1.79 (m, 2H, CH2), 4.00 (t, J=7.5 Hz, 2H, N1CH2), 5.60 (s, 2H, N7CH2), 7.30-7.34

(m, 5H, ArH), 7.84 (s, 1H, C8H), 7.99 (s, 1H, C2H). 13CNMR (CDCl3, 300 Hz) δ(ppm):

13.57, 19.77, 31.84, 46.30, 50.55, 127.92, 128.48, 129.02, 135.64, 143.17, 143.29,

146.51, 154.38, 156.86. HRMS(EI) Calcd for C16H18N4O 282.1481; found: 282.1467.

161

4-35a: 6-(Prop-2-ynyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7(6H)-one

1HNMR (CDCl3, 300 Hz) δ(ppm): 3.42 (t, J=2.5 Hz, 1H, CH), 4.85 (d, J=2.6 Hz, 2H,

N1CH2), 8.55 (s, 1H, C2H). 13CNMR (CDCl3, 300 Hz) δ(ppm): 34.49, 75.38, 77.94,

126.91, 149.86, 151.95, 153.73. HRMS(EI) Calcd for C7H7N5O 175.0494; found:

175.0494.

4-35b: 6-Benzyl-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7(6H)-one

1HNMR (DMSO-d6, 300 Hz) δ(ppm): 5.22 (s, 2H, CH2), 7.29-7.34 (m, 5H, ArH), 8.59 (s,

1H, C2H). 13CNMR (DMSO-d6, 300 Hz) δ(ppm): 47.85, 127.05, 127.17 (x2), 128.14,

136.44, 149.76, 152.74, 154.61. HRMS(EI) Calcd for C11H9N5O 227.0807; found:

227.0805.

4-35c: 6-Allyl-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7(6H)-one

1HNMR (DMSO-d6, 300 Hz) δ(ppm): 4.64 (d, J=5.2 Hz, 2H, N1CH2), 5.13 (m, 2H,

CH2), 5.92-6.05 (m, 1H, CH), 8.40 (s, 1H, C2H). 13CNMR (DMSO-d6, 300 Hz) δ(ppm):

46.70, 116.95, 126.97, 132.69, 149.99, 152.36, 154.23. HRMS(EI) Calcd for C7H7N5O

177.0651, found: 177.0646.

4-35d: 6-Butyl-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7(6H)-one

1HNMR (MeOD, 300 Hz) δ(ppm): 0.95 (t, J=7.3 Hz, 3H, CH3), 1.32-1.45 (m, 2H, CH2),

1.69-1.79 (m, 2H, CH2), 4.09 (t, J=7.5 Hz, 2H, N1CH2), 8.38 (s, 1H, C2H). 13CNMR

162

(MeOD, 300 Hz) δ(ppm): 13.94, 20.75, 32.60, 47.51, 128.75, 152.23, 153.80, 156.91.

HRMS(EI) Calcd for C8H11N5O 193.0964; found: 193.0956.

163

4.6 References

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165

Appendix A

1. X ray crystal data for 2-3 165

2. X ray crystal data for 2-4b 166

3. X ray crystal data for 2-23a 167

4. X ray crystal data for 2-23b 168

5. X ray crystal data for 2-23d 169

6. X ray crystal data for 3-6f 170

7. X ray crystal data for 4-6i 171

8. X ray crystal data for 4-13a 172

9. X ray crystal data for 4-18a 173

10. X ray crystal data for 4-17b 174

11. X ray crystal data for 4-17c 175

166

Table 1. Crystal data and structure refinement for 2-3.

Identification code 2-3

Empirical formula C12 H10 N4 O

Formula weight 226.24

Temperature 223(2) K

Wavelength 0.71073 Å

Crystal system Orthorhombic

Space group P2(1)2(1)2(1)

Unit cell dimensions a = 8.3887(9) Å α= 90°.

b = 9.8874(10) Å β= 90°.

c = 12.8009(13) Å γ = 90°.

Volume 1061.74(19) Å3

Z 4

Density (calculated) 1.415 Mg/m3

Absorption coefficient 0.096 mm-1

F(000) 472

Crystal size 0.47 x 0.26 x 0.26 mm3

Theta range for data collection 2.60 to 27.49°.

Index ranges -10<=h<=10, -10<=k<=12, -16<=l<=16

Reflections collected 7565

Independent reflections 2437 [R(int) = 0.0395]

Completeness to theta = 27.49° 100.0 %

Absorption correction SADABS

Max. and min. transmission 0.9754 and 0.9562

Refinement method Full-matrix least-squares on F2

Data / restraints / parameters 2437 / 0 / 162

Goodness-of-fit on F2 0.965

Final R indices [I>2sigma(I)] R1 = 0.0450, wR2 = 0.0845

R indices (all data) R1 = 0.0566, wR2 = 0.0887

Absolute structure parameter 0.00

Largest diff. peak and hole 0.197 and -0.199 e.Å-3

167

Table 1. Crystal data and structure refinement for 2-4b.

Identification code 2-4b

Empirical formula C15 H16 N4 O

Formula weight 268.32

Temperature 295(2) K

Wavelength 0.71073 Å

Crystal system Triclinic

Space group P-1

Unit cell dimensions a = 5.380(5) Å α= 99.932(16)°.

b = 10.617(10) Å β= 90.153(18)°.

c = 12.547(11) Å γ = 92.275(18)°.

Volume 705.4(11) Å3

Z 2

Density (calculated) 1.263 Mg/m3

Absorption coefficient 0.083 mm-1

F(000) 284

Crystal size 0.52 x 0.34 x 0.34 mm3

Theta range for data collection 1.65 to 25.00°.

Index ranges -6<=h<=6, -12<=k<=12, -14<=l<=14

Reflections collected 7522

Independent reflections 2491 [R(int) = 0.0322]

Completeness to theta = 25.00° 100.0 %

Absorption correction Sadabs, (Sheldrick 2001)

Max. and min. transmission 0.9723 and 0.9580

Refinement method Full-matrix least-squares on F2

Data / restraints / parameters 2491 / 0 / 183

Goodness-of-fit on F2 1.087

Final R indices [I>2sigma(I)] R1 = 0.0627, wR2 = 0.1578

R indices (all data) R1 = 0.0787, wR2 = 0.1674

Largest diff. peak and hole 0.253 and -0.151 e.Å-3

168

Table 1. Crystal data and structure refinement for 2-23a.

Identification code 2-23a

Empirical formula C9 H12 N4 O

Formula weight 192.23

Temperature 293(2) K

Wavelength 0.71073 Å

Crystal system Monoclinic

Space group P2(1)/c

Unit cell dimensions a = 9.686(13) Å α= 90°.

b = 9.044(11) Å β= 95.32(17)°.

c = 11.075(12) Å γ = 90°.

Volume 966(2) Å3

Z 4

Density (calculated) 1.322 Mg/m3

Absorption coefficient 0.092 mm-1

F(000) 408

Crystal size 0.60 x 0.38 x 0.05 mm3

Theta range for data collection 2.11 to 24.99°.

Index ranges -11<=h<=11, -8<=k<=10, -11<=l<=13

Reflections collected 4138

Independent reflections 1679 [R(int) = 0.0672]

Completeness to theta = 24.99° 98.5 %

Absorption correction SADABS

Max. and min. transmission 0.9954 and 0.9469

Refinement method Full-matrix least-squares on F2

Data / restraints / parameters 1679 / 12 / 132

Goodness-of-fit on F2 1.072

Final R indices [I>2sigma(I)] R1 = 0.0925, wR2 = 0.2523

R indices (all data) R1 = 0.1204, wR2 = 0.2726

Largest diff. peak and hole 0.455 and -0.367 e.Å-3

169

Table 1. Crystal data and structure refinement for 2-23b.

Identification code 2-23b

Empirical formula C8 H12 N4 O2

Formula weight 196.22

Temperature 223(2) K

Wavelength 0.71073 Å

Crystal system Monoclinic

Space group P2(1)/n

Unit cell dimensions a = 9.6078(7) Å α= 90°.

b = 8.9381(6) Å β= 113.3110(10)°.

c = 11.8443(8) Å γ = 90°.

Volume 934.11(11) Å3

Z 4

Density (calculated) 1.395 Mg/m3

Absorption coefficient 0.104 mm-1

F(000) 416

Crystal size 0.52 x 0.52 x 0.26 mm3

Theta range for data collection 2.33 to 27.50°.

Index ranges -12<=h<=12, -11<=k<=11, -9<=l<=15

Reflections collected 6469

Independent reflections 2145 [R(int) = 0.0184]

Completeness to theta = 27.50° 100.0 %

Absorption correction None

Max. and min. transmission 0.9735 and 0.9479

Refinement method Full-matrix least-squares on F2

Data / restraints / parameters 2145 / 0 / 141

Goodness-of-fit on F2 1.083

Final R indices [I>2sigma(I)] R1 = 0.0378, wR2 = 0.0997

R indices (all data) R1 = 0.0440, wR2 = 0.1028

Largest diff. peak and hole 0.300 and -0.193 e.Å-3

170

Table 1. Crystal data and structure refinement for 2-23d.

Identification code 2-23d

Empirical formula C8 H9 I N4 O

Formula weight 304.09

Temperature 496(2) K

Wavelength 0.71073 Å

Crystal system Triclinic

Space group P-1

Unit cell dimensions a = 7.7879(4) Å α= 85.3740(10)°.

b = 8.1761(4) Å β= 70.7910(10)°.

c = 9.1395(4) Å γ = 62.0530(10)°.

Volume 483.58(4) Å3

Z 2

Density (calculated) 2.088 Mg/m3

Absorption coefficient 3.283 mm-1

F(000) 292

Crystal size 0.26 x 0.13 x 0.13 mm3

Theta range for data collection 2.37 to 27.50°.

Index ranges -10<=h<=10, -10<=k<=10, -11<=l<=11

Reflections collected 6365

Independent reflections 2219 [R(int) = 0.0184]

Completeness to theta = 27.50° 99.9 %

Absorption correction None

Max. and min. transmission 0.6749 and 0.4823

Refinement method Full-matrix least-squares on F2

Data / restraints / parameters 2219 / 0 / 133

Goodness-of-fit on F2 1.073

Final R indices [I>2sigma(I)] R1 = 0.0175, wR2 = 0.0428

R indices (all data) R1 = 0.0180, wR2 = 0.0430

Largest diff. peak and hole 0.606 and -0.620 e.Å-3

171

Table 1. Crystal data and structure refinement for 3-6f.

Identification code 3-6f

Empirical formula C19 H26 N4 O2

Formula weight 342.44

Temperature 223(2) K

Wavelength 0.71073 Å

Crystal system Monoclinic

Space group P2(1)/c

Unit cell dimensions a = 17.164(3) Å α= 90°.

b = 8.4372(14) Å β= 99.752(4)°.

c = 13.333(2) Å γ = 90°.

Volume 1902.9(6) Å3

Z 4

Density (calculated) 1.195 Mg/m3

Absorption coefficient 0.079 mm-1

F(000) 736

Crystal size 0.78 x 0.65 x 0.26 mm3

Theta range for data collection 2.41 to 23.29°.

Index ranges -14<=h<=19, -9<=k<=8, -14<=l<=11

Reflections collected 9008

Independent reflections 2736 [R(int) = 0.0274]

Completeness to theta = 23.29° 99.7 %

Absorption correction Sadabs

Refinement method Full-matrix least-squares on F2

Data / restraints / parameters 2736 / 26 / 270

Goodness-of-fit on F2 1.045

Final R indices [I>2sigma(I)] R1 = 0.0465, wR2 = 0.1256

R indices (all data) R1 = 0.0591, wR2 = 0.1345

Largest diff. peak and hole 0.249 and -0.154 e.Å-3

172

Table 1. Crystal data and structure refinement for 4-6i.

Identification code 4-6i

Empirical formula C20 H22 N4 O

Formula weight 334.42

Temperature 223(2) K

Wavelength 0.71073 Å

Crystal system Monoclinic

Space group P2(1)/c

Unit cell dimensions a = 10.9860(5) Å α= 90°.

b = 20.6191(9) Å β= 90.5090(10)°.

c = 16.1222(6) Å γ = 90°.

Volume 3651.9(3) Å3

Z 8

Density (calculated) 1.216 Mg/m3

Absorption coefficient 0.078 mm-1

F(000) 1424

Crystal size 0.23 x 0.13 x 0.10 mm3

Theta range for data collection 1.60 to 27.50°.

Index ranges -14<=h<=11, -26<=k<=26, -20<=l<=18

Reflections collected 26060

Independent reflections 8397 [R(int) = 0.0771]

Completeness to theta = 27.50° 100.0 %

Absorption correction None

Max. and min. transmission 0.9920 and 0.9821

Refinement method Full-matrix least-squares on F2

Data / restraints / parameters 8397 / 0 / 453

Goodness-of-fit on F2 1.019

Final R indices [I>2sigma(I)] R1 = 0.0802, wR2 = 0.1413

R indices (all data) R1 = 0.1558, wR2 = 0.1679

Largest diff. peak and hole 0.234 and -0.185 e.Å-3

173

Table 1. Crystal data and structure refinement for 4-13a.

Identification code 4-13a

Empirical formula C21.71 H17.14 Cl1.14 N4.57 O1.14

Formula weight 400.91

Temperature 223(2) K

Wavelength 0.71073 Å

Crystal system Triclinic

Space group P-1

Unit cell dimensions a = 9.7872(8) Å α= 100.106(2)°.

b = 17.9940(15) Å β= 91.111(2)°.

c = 19.4196(16) Å γ = 97.268(2)°.

Volume 3336.9(5) Å3

Z 7

Density (calculated) 1.397 Mg/m3

Absorption coefficient 0.244 mm-1

F(000) 1456

Crystal size 0.39 x 0.24 x 0.23 mm3

Theta range for data collection 1.07 to 27.50°.

Index ranges -12<=h<=12, -23<=k<=23, -25<=l<=25

Reflections collected 43958

Independent reflections 15307 [R(int) = 0.0614]

Completeness to theta = 27.50° 99.9 %

Absorption correction None

Refinement method Full-matrix least-squares on F2

Data / restraints / parameters 15307 / 0 / 901

Goodness-of-fit on F2 1.077

Final R indices [I>2sigma(I)] R1 = 0.0862, wR2 = 0.1611

R indices (all data) R1 = 0.1526, wR2 = 0.1899

Largest diff. peak and hole 0.420 and -0.265 e.Å-3

174

Table 1. Crystal data and structure refinement for 4-18a.

Identification code 4-18a

Empirical formula C18 H14 Cl N5 O

Formula weight 351.79

Temperature 223(2) K

Wavelength 0.71073 Å

Crystal system Monoclinic

Space group P2(1)/c

Unit cell dimensions a = 7.479(3) Å α= 90°.

b = 20.043(10) Å β= 99.58(5)°.

c = 11.195(7) Å γ = 90°.

Volume 1654.6(15) Å3

Z 4

Density (calculated) 1.412 Mg/m3

Absorption coefficient 0.248 mm-1

F(000) 728

Crystal size 0.40 x 0.30 x 0.28 mm3

Theta range for data collection 2.03 to 27.50°.

Index ranges -9<=h<=9, -26<=k<=19, -13<=l<=14

Reflections collected 11703

Independent reflections 3802 [R(int) = 0.0214]

Completeness to theta = 27.50° 100.0 %

Absorption correction Sadabs(Sheldrick,2001)

Max. and min. transmission 0.9339 and 0.9074

Refinement method Full-matrix least-squares on F2

Data / restraints / parameters 3802 / 0 / 226

Goodness-of-fit on F2 1.069

Final R indices [I>2sigma(I)] R1 = 0.0527, wR2 = 0.1243

R indices (all data) R1 = 0.0607, wR2 = 0.1297

Largest diff. peak and hole 0.472 and -0.392 e.Å-3

175

Table 1. Crystal data and structure refinement for 4-17b.

Identification code 4-17b

Empirical formula C15 H12 N4 O

Formula weight 264.29

Temperature 223(2) K

Wavelength 0.71073 Å

Crystal system Orthorhombic

Space group P2(1)2(1)2(1)

Unit cell dimensions a = 8.1795(6) Å α= 90°.

b = 8.7638(6) Å β= 90°.

c = 18.5052(12) Å γ = 90°.

Volume 1326.52(16) Å3

Z 4

Density (calculated) 1.323 Mg/m3

Absorption coefficient 0.088 mm-1

F(000) 552

Crystal size 0.52 x 0.39 x 0.26 mm3

Theta range for data collection 2.20 to 27.50°.

Index ranges -10<=h<=10, -11<=k<=11, -18<=l<=24

Reflections collected 9573

Independent reflections 3053 [R(int) = 0.0533]

Completeness to theta = 27.50° 100.0 %

Absorption correction None

Max. and min. transmission 0.9776 and 0.9558

Refinement method Full-matrix least-squares on F2

Data / restraints / parameters 3053 / 0 / 181

Goodness-of-fit on F2 1.037

Final R indices [I>2sigma(I)] R1 = 0.0534, wR2 = 0.1005

R indices (all data) R1 = 0.0787, wR2 = 0.1095

Absolute structure parameter 1(2)

Largest diff. peak and hole 0.184 and -0.149 e.Å-3

176

Table 1. Crystal data and structure refinement for 4-17c.

Identification code 4-17c

Empirical formula C24 H25 N8 O3

Formula weight 473.52

Temperature 223(2) K

Wavelength 0.71073 Å

Crystal system Monoclinic

Space group C2/c

Unit cell dimensions a = 20.7072(14) Å α= 90°.

b = 10.7727(7) Å β= 102.957(2)°.

c = 11.6071(8) Å γ = 90°.

Volume 2523.3(3) Å3

Z 4

Density (calculated) 1.246 Mg/m3

Absorption coefficient 0.087 mm-1

F(000) 996

Crystal size 0.39 x 0.31 x 0.31 mm3

Theta range for data collection 2.02 to 27.45°.

Index ranges -26<=h<=26, -13<=k<=11, -15<=l<=13

Reflections collected 8634

Independent reflections 2876 [R(int) = 0.0294]

Completeness to theta = 27.45° 99.7 %

Absorption correction None

Max. and min. transmission 0.9735 and 0.9670

Refinement method Full-matrix least-squares on F2

Data / restraints / parameters 2876 / 2 / 170

Goodness-of-fit on F2 1.058

Final R indices [I>2sigma(I)] R1 = 0.0649, wR2 = 0.1829

R indices (all data) R1 = 0.0765, wR2 = 0.1940

Largest diff. peak and hole 0.666 and -0.198 e.Å-3

177

Appendix B

1. Spectrum 2.1 1HNMR spectrum of compound 2-13 178

2. Spectrum 2.2 13CNMR spectrum of compound 2-13 178

3. Spectrum 2.3 1HNMR spectrum of compound 2-16 179

4. Spectrum 2.4 13CNMR spectrum of compound 2-16 179

5. Spectrum 2.5 1HNMR spectrum of compound 2-23d 180

6. Spectrum 2.6 13CNMR spectrum of compound 2-23d 180

7. Spectrum 2.7 1HNMR spectrum of compound 2-25a 181

8. Spectrum 2.8 13CNMR spectrum of compound 2-25a 181

9. Spectrum 2.9 1HNMR spectrum of compound 2-26 182

10. Spectrum 2.10 13CNMR spectrum of compound 2-26 182

11. Spectrum 3.1 1HNMR spectrum of compound 3-5 183

12. Spectrum 3.2 13CNMR spectrum of compound 3-5 183

13. Spectrum 3.3 1HNMR spectrum of compound 3-6d 184

14. Spectrum 3.4 13CNMR spectrum of compound 3-6d 184

15. Spectrum 3.5 1HNMR spectrum of compound 3-6l 185

16. Spectrum 3.6 13CNMR spectrum of compound 3-6l 185

17. Spectrum 3.7 1HNMR spectrum of compound 3-6o 186

18. Spectrum 3.8 13CNMR spectrum of compound 3-6o 186

19. Spectrum 4.1 1HNMR spectrum of compound 4-5 187

20. Spectrum 4.2 13CNMR spectrum of compound 4-5 187

21. Spectrum 4.3 1HNMR spectrum of compound 4-6g 188

178

22. Spectrum 4.4 13CNMR spectrum of compound 4-6g 188

23. Spectrum 4.5 1HNMR spectrum of compound 4-7 189

24. Spectrum 4.6 13CNMR spectrum of compound 4-7 189

25. Spectrum 4.7 1HNMR spectrum of compound 4-8 190

26. Spectrum 4.8 13CNMR spectrum of compound 4-8 190

27. Spectrum 4.9 1HNMR spectrum of compound 4-16 191

28. Spectrum 4.10 13CNMR spectrum of compound 4-16 191

29. Spectrum 4.11 1HNMR spectrum of compound 4-17d 192

30. Spectrum 4.12 13CNMR spectrum of compound 4-17d 192

31. Spectrum 4.13 1HNMR spectrum of compound 4-22 193

32. Spectrum 4.14 13CNMR spectrum of compound 4-22 193

33. Spectrum 4.15 1HNMR spectrum of compound 4-24 194

34. Spectrum 4.16 13CNMR spectrum of compound 4-24 194

35. Spectrum 4.17 1HNMR spectrum of compound 4-35d 195

36. Spectrum 4.18 13CNMR spectrum of compound 4-35d 195

179

1.0

00

0

1.7

90

9

2.5

64

9

2.1

11

7

2.1

63

7

8.2

52

7

2.1

51

8

2.3

15

4

3.0

76

9

Inte

gra

l

7.5

76

27

.49

85

7.4

84

77

.35

25

7.3

38

67

.33

58

7.3

23

87

.29

98

7.2

85

97

.26

00

5.5

64

4

4.0

69

54

.05

56

4.0

40

83

.80

23

3.7

99

53

.79

40

3.7

92

13

.78

66

3.7

76

43

.77

08

3.7

69

03

.76

25

3.7

60

73

.75

61

1.8

52

51

.83

77

1.8

34

01

.82

29

1.8

19

21

.80

81

1.7

93

31

.37

72

1.3

62

51

.34

77

1.3

31

91

.31

72

1.3

02

40

.96

49

0.9

50

10

.93

53

(ppm)

0.00.40.81.21.62.02.42.83.23.64.04.44.85.25.66.06.46.87.27.68.08.48.8

Spectrum 2.1 1HNMR of 2-13

16

0.2

63

31

58

.50

04

15

4.3

53

6

13

9.3

72

5

13

6.9

03

0

12

8.3

31

21

27

.99

57

12

7.8

31

6

11

4.8

13

2

77

.25

69

77

.00

00

76

.75

02

67

.72

87

66

.83

65

45

.06

79

43

.02

66

31

.79

26

19

.75

20

13

.47

12

(ppm)

5101520253035404550556065707580859095100105110115120125130135140145150155160165

Spectrum 2.2 13CNMR of 2-13

180

1.0

16

6

2.0

33

5

1.0

00

0

2.0

58

8

1.9

02

9

3.0

75

1

2.4

70

9

2.0

28

3

3.0

00

8

Inte

gra

l

11

.97

73

8.2

67

58

.23

85

7.7

67

2

7.2

60

07

.09

40

7.0

63

8

4.2

43

24

.22

00

4.1

95

6

3.8

88

0

1.9

55

41

.93

10

1.9

06

61

.88

23

1.8

57

9

1.4

33

11

.40

87

1.3

83

11

.35

88

1.3

34

41

.00

94

0.9

85

00

.96

06

(ppm)

0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.011.512.012.5

Spectrum 2.3 1HNMR of 2-16

16

2.3

87

2

15

9.1

84

8

15

3.1

38

6

15

0.0

37

6

13

9.8

93

1

12

9.3

35

6

12

4.5

59

41

22

.40

11

11

4.4

69

4

77

.42

85

77

.00

00

76

.57

93

55

.47

99

43

.63

68

32

.19

88

19

.76

36

13

.45

25

(ppm)

5101520253035404550556065707580859095100105110115120125130135140145150155160165170

Spectrum 2.4 13CNMR of 2-16

181

1.0

00

0

0.9

46

1

6.2

89

0

Inte

gra

l

8.0

83

3

4.8

28

14

.80

96

4.8

05

94

.79

29

4.7

79

14

.76

52

3.3

13

73

.31

00

3.3

07

2

1.5

90

41

.57

74

(ppm)

0.40.81.21.62.02.42.83.23.64.04.44.85.25.66.06.46.87.27.68.08.48.8

Spectrum 2.5 1HNMR of 2-23d

15

9.0

70

6

14

9.6

56

5

13

9.5

50

2

12

4.9

47

4

10

6.9

18

7

49

.51

39

49

.34

26

49

.17

13

49

.00

00

48

.82

87

48

.65

74

48

.48

61

22

.68

50

(ppm)

5101520253035404550556065707580859095100105110115120125130135140145150155160165

Spectrum 2.6 13CNMR of 2-23d

182

1.0

00

0

2.3

47

8

4.4

65

3

2.4

27

6

3.2

70

2

3.3

97

0

Inte

gra

l

8.0

94

6

4.8

43

3

4.2

39

84

.21

54

4.1

91

0

3.3

20

43

.31

58

3.3

10

03

.30

42

3.2

99

6

1.8

99

71

.87

53

1.8

50

91

.84

40

1.8

25

41

.81

84

1.7

99

91

.79

29

1.7

74

31

.76

85

1.4

15

71

.39

01

1.3

64

61

.33

91

1.3

14

71

.29

03

1.1

29

01

.10

46

1.0

79

00

.99

08

0.9

66

50

.94

21

(ppm)

0.00.40.81.21.62.02.42.83.23.64.04.44.85.25.66.06.46.87.27.68.08.48.8

Spectrum 2.7 1HNMR of 2-25a

15

8.9

38

3

14

9.8

92

3

14

2.8

33

2

13

9.8

64

6

12

5.2

24

4

97

.60

35

76

.87

02

69

.34

36

49

.85

71

49

.56

88

49

.28

83

49

.00

00

48

.71

95

48

.43

12

48

.15

07

45

.01

07

37

.07

90

33

.26

11

28

.72

65

20

.75

58

13

.81

35

9.1

46

4

(ppm)

0102030405060708090100110120130140150160

Spectrum 2.8 13CNMR of 2-25a

183

1.0

00

0

1.9

66

4

4.3

14

5

2.0

13

2

2.7

64

2

2.9

60

0

Inte

gra

l

8.0

60

9

4.8

56

1

4.3

18

74

.29

43

4.2

69

9

3.3

15

83

.31

00

3.3

04

2

1.8

88

11

.86

37

1.8

40

51

.82

77

1.8

15

01

.80

34

1.7

90

61

.77

90

1.4

11

01

.38

66

1.3

61

11

.33

56

1.1

45

21

.12

08

1.0

95

30

.99

66

0.9

72

30

.94

67

(ppm)

0.40.81.21.62.02.42.83.23.64.04.44.85.25.66.06.46.87.27.68.08.48.8

Spectrum 2.9 1HNMR of 2-26

158.3

461

147.2

666

124.9

906

101.5

616

69.4

839

49.8

493

49.5

688

49.2

883

49.0

000

48.7

195

48.4

312

48.1

507

44.9

718

37.1

647

32.8

949

28.9

914

20.8

337

13.9

460

9.3

256

(ppm)

5101520253035404550556065707580859095100105110115120125130135140145150155160165

Spectrum 2.10 13CNMR of 2-26

184

0.8

89

7

0.8

72

5

4.5

14

3

1.8

77

8

1.8

26

1

1.8

11

0

1.9

70

1

1.8

08

4

2.3

01

2

2.2

60

9

4.3

34

1

6.0

00

0

Inte

gra

l

10

.63

08

8.2

31

5

7.3

18

07

.30

64

7.2

60

0

5.0

87

14

.86

19

4.8

41

04

.81

90

4.6

40

24

.61

47

4.5

90

3

4.0

92

34

.06

80

4.0

42

4

3.2

62

43

.24

15

3.2

19

4

2.0

45

92

.02

27

1.9

97

21

.97

16

1.9

46

11

.80

45

1.7

80

11

.75

46

1.7

30

21

.70

47

1.4

67

91

.44

35

1.4

19

11

.39

36

1.3

69

20

.99

89

0.9

74

60

.95

02

(ppm)

0.00.61.21.82.43.03.64.24.85.46.06.67.27.88.49.09.610.210.811.4

Spectrum 3.1 1HNMR of 3-5

169.6

176

151.9

387

151.1

050

146.3

133

140.9

605

134.9

221

128.3

305

128.2

136

113.8

227

77.4

207

77.0

000

76.5

715

66.8

399

49.8

544

47.2

598

42.1

564

32.9

312

31.8

482

31.2

483

19.4

831

19.1

948

13.2

966

13.1

720

(ppm)

5101520253035404550556065707580859095100105110115120125130135140145150155160165170175

Spectrum 3.2 13CNMR of 3-5

185

1.0

00

0

1.0

77

5

1.0

92

6

2.2

68

1

2.1

34

9

2.1

93

2

2.2

66

4

9.0

25

7

3.2

25

9

Inte

gra

l

8.0

04

07

.95

64

7.2

60

0

6.1

15

56

.09

58

6.0

80

76

.07

60

6.0

61

06

.03

89

6.0

23

86

.01

92

6.0

05

25

.98

55

5.2

98

35

.29

60

5.2

64

75

.26

12

5.2

31

05

.22

87

5.1

74

15

.17

18

5.0

51

15

.04

65

5.0

31

45

.02

79

4.0

02

93

.97

86

3.9

54

2

1.7

59

21

.73

49

1.7

10

5

1.3

14

71

.30

07

1.2

92

61

.23

81

1.2

21

80

.85

97

0.8

37

60

.81

44

(ppm)

0.00.40.81.21.62.02.42.83.23.64.04.44.85.25.66.06.46.87.27.68.08.48.89.2

Spectrum 3.3 1HNMR of 3-6d

15

6.2

78

61

54

.12

81

15

2.2

11

4

14

6.6

87

2

14

2.8

92

8

13

2.2

96

3

11

9.1

91

0

77

.42

07

77

.00

00

76

.57

15

49

.30

12

46

.55

86

31

.52

87

29

.76

01

28

.70

04

26

.46

43

22

.41

27

13

.90

44

(ppm)

5101520253035404550556065707580859095100105110115120125130135140145150155160165

Spectrum 3.4 13CNMR of 3-6d

186

1.0

00

0

1.0

32

5

5.2

34

4

2.2

96

1

2.1

51

1

0.9

85

4

Inte

gra

l

8.2

40

8

7.8

61

3

7.2

97

17

.28

55

7.2

73

97

.26

00

5.5

52

5

4.7

72

54

.76

44

2.4

76

62

.46

84

2.4

60

3

(ppm)

0.40.81.21.62.02.42.83.23.64.04.44.85.25.66.06.46.87.27.68.08.48.89.2

Spectrum 3.5 1HNMR of 3-6l

15

6.7

38

3

15

3.2

16

5

14

5.1

83

51

43

.27

46

11

4.4

46

0

77

.42

85

77

.00

00

76

.57

93

76

.49

36

74

.70

15

46

.94

82

34

.50

51

32

.97

80

19

.22

60

13

.18

76

(ppm)

5101520253035404550556065707580859095100105110115120125130135140145150155160165170175

Spectrum 3.6 13CNMR of 3-6l

187

1.0

00

0

0.9

56

6

4.8

57

6

1.9

98

1

2.9

16

1

Inte

gra

l

8.0

07

5

7.8

49

7

7.3

04

17

.27

97

5.5

74

6

3.5

73

5(ppm)

0.40.81.21.62.02.42.83.23.64.04.44.85.25.66.06.46.87.27.68.08.48.8

Spectrum 3.7 1HNMR of 3-6o

15

7.0

65

51

54

.70

47

14

6.6

87

2

14

3.1

88

9

13

5.6

54

5

12

8.9

61

61

28

.41

62

12

7.8

16

2

11

4.9

60

2

77

.42

85

77

.00

00

76

.57

93

50

.50

89

33

.50

78

(ppm)

5101520253035404550556065707580859095100105110115120125130135140145150155160165

Spectrum 3.8 13CNMR of 3-6o

188

1.0

000

7.3

295

2.3

233

2.0

568

2.2

087

2.0

826

2.1

230

2.1

073

1.0

114

2.3

643

2.4

129

8.3

204

3.0

208

3.2

027

Inte

gra

l

8.7

388

7.3

703

7.3

645

7.3

505

7.3

250

7.3

169

7.2

995

7.2

704

7.2

600

6.9

431

6.9

141

5.6

953

5.0

174

4.9

640

4.9

559

4.4

382

4.4

115

4.3

848

3.3

030

3.2

763

3.2

496

2.5

845

2.5

764

2.5

683

2.0

204

1.9

960

1.9

716

1.9

461

1.5

143

1.4

899

1.4

656

1.4

400

1.2

601

1.2

369

1.1

614

1.1

394

1.1

266

0.9

989

0.9

746

0.9

502

0.8

480

0.8

260

0.8

016

(ppm)0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5

Spectrum 4.1 1HNMR of 4-5

159.2

484

153.4

188

151.3

527

150.2

753

146.7

259

136.2

991

129.5

103

128.5

584

128.0

639

127.3

187

125.5

403

115.5

562

113.3

499

77.4

206

77.0

000

76.5

794

76.4

318

75.4

208

70.0

709

48.8

410

48.2

507

36.2

743

32.4

888

30.9

096

29.0

132

27.3

676

26.7

994

22.1

358

19.8

261

13.8

194

13.3

176

(ppm)102030405060708090100110120130140150160

Spectrum 4.2 13CNMR of 4-5

189

1.0

00

0

2.0

64

8

5.2

93

6

2.0

94

5

0.9

53

8

1.0

76

1

1.9

95

1

2.9

21

1

3.0

32

8

Inte

gra

l

8.1

16

8

7.7

19

17

.71

25

7.6

90

1

7.3

21

97

.31

10

7.3

00

57

.26

00

7.0

00

36

.97

13

6.1

72

56

.15

72

6.1

39

16

.12

27

6.1

15

56

.10

68

6.1

00

26

.08

27

6.0

65

16

.04

98

5.2

77

95

.24

34

5.1

96

85

.07

68

5.0

69

15

.06

15

5.0

29

74

.97

27

3.8

41

4(ppm)

0.40.81.21.62.02.42.83.23.64.04.44.85.25.66.06.46.87.27.68.08.48.8

Spectrum 4.3 1HNMR of 4-6g

16

1.1

89

1

15

6.2

00

81

54

.28

96

15

3.9

20

6

14

6.6

15

2

13

5.8

93

31

33

.67

95

13

0.6

90

91

28

.89

04

12

8.1

37

71

27

.79

09

12

0.9

50

4

11

6.9

80

4

11

4.1

09

9

77

.42

06

77

.00

00

76

.57

20

55

.29

04

49

.03

29

48

.47

21

(ppm)

5101520253035404550556065707580859095100105110115120125130135140145150155160165170

Spectrum 4.4 13CNMR of 4-6g

190

1.0

00

0

5.3

35

3

2.2

43

1

2.1

70

0

1.0

46

6

2.1

59

2

2.1

16

7

1.7

19

9

3.2

12

8

3.4

92

5

Inte

gra

l

8.1

88

6

7.4

33

07

.40

86

7.3

74

97

.35

05

7.3

34

37

.24

72

7.2

18

2

6.9

60

56

.93

15

5.3

30

85

.31

34

5.2

94

9

5.0

49

9

4.6

22

84

.60

42

4.2

87

34

.26

53

1.8

59

1

1.3

55

31

.33

21

1.3

08

9

(ppm)

1.21.62.02.42.83.23.64.04.44.85.25.66.06.46.87.27.68.08.48.89.29.610.0

Spectrum 4.5 1HNMR of 4-7

16

6.8

90

6

15

8.1

01

81

56

.41

88

15

2.4

06

2

14

5.4

87

4

13

6.7

29

7

13

0.5

51

01

28

.72

00

12

8.4

31

71

27

.82

40

12

7.2

55

21

23

.82

70

11

4.9

13

5

77

.42

07

77

.00

00

76

.57

15

69

.87

08

62

.67

14

44

.40

81

17

.62

09

13

.92

00

(ppm)

5101520253035404550556065707580859095100105110115120125130135140145150155160165170175

Spectrum 4.6 13CNMR of 4-7

191

1.0

00

0

5.7

00

4

2.3

02

1

2.3

29

9

2.3

84

2

2.4

29

5

3.2

82

9

Inte

gra

l

8.7

12

1

8.0

95

78

.08

99

7.4

01

67

.38

07

7.3

65

67

.33

89

7.2

63

57

.25

88

7.2

39

17

.21

82

7.2

11

37

.14

74

7.1

21

96

.93

15

6.9

28

06

.90

37

6.8

74

6

5.3

63

3

5.0

27

9

2.6

04

3

(ppm)

0.40.81.21.62.02.42.83.23.64.04.44.85.25.66.06.46.87.27.68.08.48.8

Spectrum 4.7 1HNMR of 4-8

15

8.8

10

8

15

5.2

34

5

15

1.3

15

41

49

.49

22

14

8.9

31

2

13

6.5

19

31

29

.32

78

12

8.6

49

91

28

.57

20

12

8.0

42

21

27

.97

20

12

7.3

72

11

27

.00

59

11

5.4

12

11

14

.99

14

77

.42

07

77

.00

00

76

.57

15

70

.05

00

46

.04

43

14

.78

48

(ppm)

5101520253035404550556065707580859095100105110115120125130135140145150155160165

Spectrum 4.8 13CNMR of 4-8

192

1.0

000

1.0

322

2.0

858

5.3

223

2.1

977

2.0

311

2.2

170

2.0

694

2.0

875

2.1

828

2.1

904

4.4

604

6.2

724

Inte

gra

l

10.8

746

8.4

300

7.6

547

7.6

257

7.3

633

7.3

413

7.3

204

7.2

937

7.2

856

7.2

763

7.2

635

6.9

385

6.9

095

5.6

419

4.9

850

4.6

356

4.6

100

4.5

845

4.1

260

4.1

016

4.0

761

2.0

320

2.0

065

1.9

821

1.9

566

1.9

310

1.7

790

1.7

546

1.7

291

1.7

059

1.6

792

1.4

331

1.4

226

1.4

075

1.3

983

1.3

832

1.3

588

1.3

483

0.9

665

0.9

583

0.9

421

0.9

340

0.9

177

0.9

096

(ppm)

0.40.81.21.62.02.42.83.23.64.04.44.85.25.66.06.46.87.27.68.08.48.89.29.610.010.410.811.211.6

Spectrum 4.9 1HNMR of 4-16

15

9.3

95

2

15

1.9

85

51

51

.18

29

14

6.1

57

4

13

9.9

71

0

13

6.3

32

3

13

1.0

73

11

28

.41

62

12

7.8

86

31

27

.26

30

12

4.9

56

7

11

5.3

88

81

14

.01

75

77

.42

07

77

.00

00

76

.57

15

69

.86

30

49

.96

35

49

.10

64

47

.38

45

32

.13

65

31

.34

96

19

.56

88

19

.31

95

13

.37

45

13

.23

43

(ppm)

5101520253035404550556065707580859095100105110115120125130135140145150155160165

Spectrum 4.10 13CNMR of 4-16

193

1.0

00

0

1.1

90

6

8.5

28

7

2.5

95

6

2.0

73

4

2.1

30

2

2.3

93

8

3.1

65

3

Inte

gra

l

8.0

87

07

.97

80

7.8

39

4

7.3

02

57

.26

91

5.5

79

0

5.1

82

9

4.0

06

13

.98

20

3.9

56

8

1.7

73

61

.74

89

1.7

23

71

.71

55

1.6

98

51

.67

33

1.4

30

11

.40

54

1.3

80

81

.35

50

1.3

29

81

.31

40

1.3

06

30

.95

89

0.9

34

30

.91

02

(ppm)

0.00.40.81.21.62.02.42.83.23.64.04.44.85.25.66.06.46.87.27.68.08.48.8

Spectrum 4.11 1HNMR of 4-17d

0.8

10

4

1.1

14

2

7.5

31

7

2.7

68

6

1.7

99

9

1.8

44

5

2.0

10

2

3.0

00

0

Inte

gra

l

8.0

93

47

.99

13

7.8

43

9

7.3

41

37

.32

27

7.2

98

37

.26

00

5.5

96

7

5.1

99

7

4.0

25

04

.00

06

3.9

75

1

1.7

92

91

.76

85

1.7

43

01

.71

86

1.6

93

1

1.4

50

51

.42

61

1.4

01

71

.37

50

1.3

50

61

.33

44

1.3

26

30

.97

92

0.9

54

80

.93

05

(ppm)

0.40.81.21.62.02.42.83.23.64.04.44.85.25.66.06.46.87.27.68.08.48.8

Spectrum 4.12 13CNMR of 4-17d

194

1.0

000

2.1

135

2.4

392

Inte

gra

l

8.8

052

4.7

228

4.6

926

4.6

578

4.2

086

4.1

738

4.1

436

3.8

627

2.5

000

(ppm)0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.5

Spectrum 4.13 1HNMR of 4-22

Spectrum 4.14 13CNMR of 4-22

159.

5846

148.

5305

148.

2501

114.

8445

49.8

486

49.5

682

49.2

804

49.0

000

48.7

122

48.4

318

48.1

440

45.3

178

(ppm)

5101520253035404550556065707580859095100105110115120125130135140145150155160165

195

Spectrum 4.15 1HNMR of 4-24

15

2.8

10

51

51

.08

37

14

5.4

53

41

44

.51

62

11

7.0

73

0

49

.85

60

49

.56

82

49

.42

06

49

.28

78

49

.00

00

48

.86

72

48

.71

96

48

.43

18

48

.15

14

48

.05

55

30

.29

38

20

.67

13

13

.99

31

(ppm)

5101520253035404550556065707580859095100105110115120125130135140145150155160165

Spectrum 4.16 13CNMR of 4-24

1.0

000

1.0

174

2.2

944

4.1

473

2.0

975

2.0

917

3.0

902

Inte

gra

l

8.5

148

8.3

511

4.8

805

4.8

248

4.7

946

4.7

609

4.2

165

4.1

840

4.1

620

4.1

388

4.1

132

3.3

204

3.3

146

3.3

100

3.3

042

3.2

996

1.8

637

1.8

393

1.8

138

1.7

894

1.7

639

1.5

410

1.5

167

1.4

911

1.4

656

1.4

412

1.4

168

1.0

338

1.0

094

0.9

850

(ppm)0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.0

196

1.0

00

0

2.0

27

4

2.0

50

7

2.0

44

6

2.9

87

7

Inte

gra

l

8.3

76

6

5.0

97

5

4.1

13

24

.08

89

4.0

63

3

3.3

10

0

1.7

92

91

.76

85

1.7

43

01

.73

49

1.7

17

51

.69

19

1.4

21

51

.39

71

1.3

71

61

.34

60

0.9

76

90

.95

25

0.9

28

2

(ppm)

0.40.81.21.62.02.42.83.23.64.04.44.85.25.66.06.46.87.27.68.08.48.89.29.6

Spectrum 4.17 1HNMR of 4-35d

15

6.9

05

9

15

3.7

99

21

52

.22

75

12

8.7

46

9

49

.56

82

49

.28

04

49

.00

00

48

.71

96

47

.50

94

32

.60

34

20

.74

51

13

.94

15

(ppm)

5101520253035404550556065707580859095100105110115120125130135140145150155160165

Spectrum 4.18 13CNMR of 4-35d