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理學博士學位請求論文

A Dissertation for Doctor of Philosophy

아이속사졸린 유도체들의 PTP1B 억제 및 비만 억제효과

와 4,4'-메틸렌비스(5-(퓨란-2-일)-2-하이드록시벤조산)의 생

물학적 평가

Isoxazolone Derivatives as PTP1B Inhibitors with Anti-

Obesity Effects and the Biological Evaluation of 4,4'-

Methylenebis(5-(furan-2-yl)-2-hydroxybenzoic acid).

2011 년 8 월

仁荷大學校大學院化學科 (化學專攻)

Department of Chemistry, Graduate School

Inha University

Bhooshan Kafle

理學博士學位請求論文

A Dissertation for Doctor of Philosophy

아이속사졸린 유도체들의 PTP1B 억제 및 비만 억제효과

와 4,4'-메틸렌비스(5-(퓨란-2-일)-2-하이드록시벤조산)의 생

물학적 평가

Isoxazolone Derivatives as PTP1B Inhibitors with Anti-

Obesity Effects and the Biological Evaluation of 4,4'-

Methylenebis(5-(furan-2-yl)-2-hydroxybenzoic acid).

2011 년 8 월

指導敎授趙亨鎭

이 論文을 博士學位論文으로 提出함

仁荷大學校大學院化學科 (化學專攻)

Department of Chemistry, Graduate School

Inha University

Bhooshan Kafle

Dedication

There is elixir in knowledge.

This dissertation is dedicated to my father late Netra Prashad Kafle

(Masters in Sanskrit grammar, Sampurnananda University, Varanasi, India, a

high school teacher of Sanskrit grammar). He started his formal education

when he was 22. He bunked several lunch and dinner in his student life due

to lack of money but did not surrender with poverty and continued his

education. He was a very good teacher. Even in vacation he was surrounded

with students at home. He rarely enjoyed his vacation. He was physically

disabled. But mentally, he was abnormally strong.

Buwa (father), the god did not let you to wait till my graduation but I hope

your soul is able to see this dissertation.

i

ACKNOWLEDGEMENTS

I would like to extend my heartfelt gratitude to Prof. Dr. Hyeongjin Cho, who trained,

encouraged and challenged me throughout my Ph.D. He never accepted less than my best

efforts. In fact, whatever the results I obtained during my Ph.D. and written in this

dissertation are creations of Professor Hyeongjin Cho. Beside the science he also trained me

for the ultimate condition and made me ultimate survivor. Where ever I will that doesn’t

matter. I will strive to imply the knowledge that I learned with him. Thank you Prof. Cho.

I would like to acknowledge to my thesis reviewers, Prof. Lee Keun Hyeung, Prof. Soh Jae-

Won Prof. Choi Eunmi, and Prof. Kim Taehyun for constructive and excellent advices after

detailed review of this dissertation.

I am deeply indebted to my senior Dr. Bharat Raj Bhattarai who introduced me in this lab

and trained me to play with chemicals, enzymes, bacteria and animals. I always missed him

during every hard time that I faced due to social and other circumstances.

I owe my sincerest thanks to Dr. Suja Shreshtha, Dr. Chuda Raj Lohani, Lok Nath Neupane,

Mandira Neupane, Dr Ranjan KC, Dr Suravi Baral, Dr Devi Basnet, Prabha Basnet, Dr Shila

Maskey, Dr Mrigendra Bir Karmacharya, Binika Hada, Sher Bahadur Rawal, Basanti Rawal,

Thakur Prasad Subedi, Deegendra Khadka, Dr Nilkanth G.Aher, Dr Thirupati, Dr Vivek

Thapa, Lee Jun and other Nepalese and Indian friends.

A special thanks to my Korean friends Kim Joung Min, Choi Unchal, Yang Mi Hwa, Jang

Su Jang , Mrs. You, Ms Lee, Miso Park, Mr lee and other Korean friends.

Most especially to my mother Na Kareshwari Kafle, my son Aagam Kafle, my brother

Alankar Kafle and my wife Anita Kafle, my brother in law & sisters Durga & Bani Guragain,

Bhim & Benu Khatiwada and Om & Karuna Wasti, my father in law and mother in law

Kashinath & Mina Baral. Words alone cannot express what I owe them for their

encouragement and whose patient love enabled me to complete this dissertation.

I want to acknowledge all the professors and staffs of Inha University for everything, Jung–

Seok International Scholarship, Brain Korea (BK 21) program and KRF of the Ministry of

Education, Government of Korea for providing financial supports during my doctoral

studies and researches. I am indebted to Institute of Engineering, Tribhuban University for

providing study leave.

Finally, no one is perfect and alone in this world; this graduate dissertation was not possible

without a supporting cast. It’s impossible to mention everyone’s name, so if you are missing,

please forgive me. It is not intentional.

ii

CONTENTS

Acknowledgement i

List of figures ix

List of tables xii

List of schemes xiii

List of abbreviations xiv

Abstract

In English xvii

In Korean xviii

TABLE OF CONTENTS

1. General Introduction 1

1.1 Phosphorylation and dephosphorylation 1

1.2 Phosphatases 1

1.3 Importance of phosphatase 2

1.4 Protein Tyrosin Phosphatase 1B 3

1.5 Landmarks in PTP1B research 3

1.6 General mechanism of dephosphorylation of substrate proteins by

PTP1B 5

1.7 Validation of PTP1B as a therapeutic target 6

1.8 Some important class of PTP1B inhibitors found in literature 7

1.8.1 Vanadate (VO4-3

) as a non-specific phosphatase inhibitor 7

1.8.2 DFMP pTyr mimetics 8

1.8.3 Nonhydrolyzable pTyr mimetics 9

1.8.3.1 Nonhydrolyzable DFMP pTyr mimetics 9

iii

1.8.3.2 Carboxylic acid pTyr mimetics 11

1.8.3.3 TDZ pTyr mimetics 13

1.8.3.4 Novel pTyr mimetics 15

1.9 PTP1B inhibitors reported from our lab 16

1.10 Present work 23

1.11 Objectives of this study 24

2.

Synthesis and Biological Evaluation of Isoxazol-5(4H)-one

Derivatives as a New Family of PTP1B Inhibitors with Anti-

Obesity Effects

25

2.1 Introduction 25

2.2 Result and discussion 27

2.2.1 Synthesis of lead compound (Z)-4-(2-(2-chlorobenzyloxy)

benzylidene)-3-phenylisoxazol-5(4H)-one (L4)

28

2.2.2 Synthesis of a small library of compounds L1-L6 29

2.2.2.1 Synthesis of aldehyde precursors (A & K-O) for small library

compounds 29

2.2.2.2 Precursors for small library compounds 29

2.2.2.3 Synthesis of small library compounds 30

2.2.2.4 Small library compounds 31

2.2.3 In vitro results of small library compounds 31

2.2.4 Chemical library synthesis 32

2.2.4.1 Synthesis of seven isoxazol-5(4H)-one precursors for chemical

library synthesis

32

iv

2.2.4.2 Synthesis of aldehyde precursor (A-F and H) for chemical library

synthesis 33

2.2.4.3 Synthesis of aldehydes precursors (G) for chemical library synthesis 34

2.2.4.4 Commercially available aldehydes precursor (I and J) for Chemical

Library Synthesis

35

2.2.4.5 Chemical library synthesis 37

2.2.4.6 Solubility determination of precursors for chemical library synthesis 37

2.2.4.7 Synthesis chemical library compounds 38

2.2.4.8 Nature of chemical library compounds 39

2.2.5 In vitro results of library compounds 42

2.2.5.1 Inhibitory potency of library compounds against PTP1B 42

2.2.5.2 Selectivity of most potent compound C3 against other PTPs 43

2.2.5.3 Enzyme kinetic experiments 44

2.2.6 Animal experiment 46

2.2.6.1 Toxicity test of compounds C3 on mice 46

2.2.6.1.1 Animal and diet selection for toxicity 46

2.2.6.1.2 Feeding 46

2.2.6.1.3 Drug dose, gavages preparation and administration 47

2.2.6.1.4 Effect of compounds C3 on (SIC: ICR) mice 47

2.2.6.1.5 Carcass of the mice at 6 days of C3 treatment 49

2.2.6.1.6 Conclusion 51

2.2.6.2 Effect of compounds C3 on diet induced diabetic mice 51

2.2.6.2.1 External appearance and body weight gain of mice after obesity

development period

51

v

2.2.6.2.2 Body weight at 4 weeks of C3 treatment 52

2.2.6.2.3 Body weight gain during 4 weeks of C3 treatment 53

2.2.6.2.4 Food intake 54

2.2.6.2.5 Feed efficiency 56

2.2.6.2.6 Intraperitoneal glucose tolerance test (IPGTT) 56

2.2.6.2.7 Carcass and liver appearance at 4 weeks C3 treatment 58

2.2.6.2.8 Liver, kidneys and lungs weights 59

2.2.6.2.9 Epididymal and retroperitoneal fat pad weights 60

2.2.6.2.10 Blood analysis 61

2.2.7 Chemical sub-library I compounds 63

2.2.7.1 Synthesized chemical sub-library I compounds 64

2.2.7.2 Inhibitory potency of chemical sub-library I compounds against

PTP1B

65

2.2.8 Chemical sub-library II 65

2.2.8.1 Chemical sub-library II compounds 65

2.2.8.2 Inhibitory potency of chemical sub-library II compounds against

PTP1B

66

2.3 Experimental section 68

2.3.1 Materials and methods for chemical synthesis 68

2.3.2 Synthesis of library compound precursors 69

2.3.2.1 Synthesis of aldehyde precursors 69

2.3.2.1.1 General procedure for the preparation of (A – E, H and K-O) 69

2.3.2.1.2 Procedure for the preparation of (F) 69

vi

2.3.2.1.3 Procedure for the preparation of (G) 69

2.3.2.2 Synthesis of isoxazol-5(4H)-one precursors 72

2.3.2.2.1 General procedures for synthesis of β-hydroxy esters 72

2.3.2.2.2 General procedures for synthesis of β-keto esters 75

2.3.2.2.3 General procedure for the synthesis of isoxazol-5(4H)-one 78

2.3.2.3 Library synthesis procedure: 81

2.3.3 In vitro studies 87

2.3.3.1 Materials 87

2.3.3.2 IC50 determination: 87

2.3.3.3 LB plot analysis 88

2.3.4 In vivo studies 89

2.3.4.1 Toxicity test of compounds C3 on mice 89


2.3.4.1.2 Feeding 89


2.3.4.2 Biological effect of C3 in diet-induced obese mice: 90

2.3.4.2.1 Animals and diets 90

2.3.4.2.2 Feeding 90

2.3.4.2.3 Drug administration 90

2.3.4.2.4 Body weight and food intake 91

2.3.4.2.5 Intraperitoneal glucose tolerance test 91

2.3.4.2.6 Blood and tissue collection 91

vii

2.3.4.2.7 Triglyceride, total cholesterol and free fatty acid analysis 92

2.3.4.2.8 Statistical analysis 92

2.4 Summary and Conclusions 93

3. Biological Evaluation of 4,4'-Methylenebis(5-(furan-2-yl)-2-

hydroxybenzoic acid) 95

3.1 Introduction 95

3.2 Result and discussion 98

3.2.1 Synthesis of difuranyl methylenedisalicylic acid 98

3.2.2 Animal experiment 99

3.2.2.1 Toxicity test of compounds SA37 on mice 99


3.2.2.1.2 Feeding 99


3.2.2.1.4 Effect of compounds SA37 on SLC: ICR Mice 100

3.2.2.1.5 Body weight after 6 days of SA37 treatment 101

3.2.2.1.6 Carcass of the mice at 6 days of SA37 treatment 101

3.2.2.1.7 Conclusion 103

3.2.2.2 Effect of compounds SA37 on diet induced diabetic mice 103

3.2.2.2.1 External appearance and body weight gain of mice after obesity

development period

104

3.2.2.2.2 Body weight after 4 weeks of SA37 treatment 105

3.2.2.2.3 Body weight gain during 4 weeks of SA37 treatment 107

viii

3.2.2.2.4 Food intake 108

3.2.2.2.5 Feed efficiency 109

3.2.2.2.6 Intraperitoneal glucose tolerance test (IPGTT) 110

3.2.2.2.7 Carcass appearance at 4 weeks of SA37 treatment 111

3.2.2.2.8 Liver, kidneys and lungs weights 112

3.2.2.2.9 Epididymal and retroperitoneal fat pad weights 113

3.2.2.2.10 Blood analysis 114

3.3 Experimental 115

3.3.1 Materials and methods for chemical synthesis 115

3.3.2 Synthesis of chemical compounds 116

3.3.3 Materials and methods for animal experiment 119

3.3.3.1 Animals and diets 119

3.3.3.2 Feeding 119

3.3.3.3 Drug administration 119

3.3.3.4 Body weight and food intake 120

3.3.3.5 Intraperitoneal glucose tolerance test 120

3.3.3.6 Blood and tissue collection 120

3.3.3.7 Triglyceride, total cholesterol and free fatty acid analysis 121

3.3.3.8 Statistical analysis 122

3.4 Summary and conclusions 122

ix

Figure List of figures Page

1-1 Protein phosphorylation and dephosphorylation 1

1-2 Catalytic mechanism of PTP1B 5

1-3 Bis(maltolato)oxovanadium(IV) compound 8

1-4 Peptide ligand with two DFMP 9

1-5 Nonhydrolyzable DFMP pTyr mimetics 10

1-6 Carboxylic acid pTyr mimetics 11

1-7 TDZ pTyr mimetics 13

1-8 Some novel pTyr mimetics 15

1-9 Some potent PTP1B inhibitors reported from our lab 18

1-10 Novel PTP1B inhibitors identified from virtual screenin 19

1-11 Two most potent PTP1B inhibitors from TZD-series 20

1-12 Most potent PTP1B inhibitors from barbituric acid-serirs (52) and

analogue compound of virtual hit 42 (53)

21

2-1 Some PTP1B ihibitors 27

2-2 Precursors for small library compounds 30

2-3 Small library compounds 31

2-4 Commercially available aldehydes precursor for Chemical Library

Synthesis

35

2-5 Precursors for chemical library synthesis 36

2-6 Solubility of precursors for chemical library compounds 38

2-7 Precipitation of chemical library compounds at room temperature 40

x

2-8 Chemical library compounds 41

2-9 Lineweaver burk plot analysis for PTP1B catalyzed reaction in presence

of C3

45

2-10 Physical appearance of control mouse and C3 treated mouse 48

2-11 Carcass of the mice at 6 days of C3 treatment 49

2-12 Liver and other organs appearance at 6 days of C3 treatment 50

2-13 Appearance of mice after obesity development period 52

2-14 Appearance of mice drug feeding period 53

2-15 Body weight gain during 4 weeks C3 treatment 54

2-16 Cumulative food intake during 4 weeks of C3 treatment 55

2-17 Feed efficiency in mice 56

2-18 Intraperitoneal glucose tolerance test (IPGTT) 57

2-19 Carcass of Mice after C3 treatment 58

2-20 Effect of C3 in Liver, Kidneys and Lungs Weights after 4 Weeks of

Treatment

59

2-21 Effect of C3 in Epididymal and Retroperitoneal Fat Pad Weights after 4

Weeks of Treatment

60

2-22 Effect of C3 in Serum Concentration of (A) Total Cholesterol and

Triglyceride; and (B) NEFA Levels after 4 Weeks of Treatment

62

2-23 Chemical Sub Library I Compounds 64

2-24 Chemical Sub Library II Compounds 66

3-1 Some MDSA derivatives 96

3-2 Physical appearance of HFD-control and SA37-treated mouse 100

3-3 Carcass of the mice after 6 days of SA37 treatment 102

xi

3-4 The appearance of organs after 6 days of SA37 treatment 103

3-5 Appearance of mice after 8 weeks of obesity development period 105

3-6 Appearance of mice after drug feeding period 106

3-7 Body weight gain during 4 weeks of SA37-treatment 107

3-8 Cumulative food intake during 4 weeks of SA37 treatment 108

3-9 Feed efficiency in mice 109

3-10 Intraperitoneal glucose tolerance test (IPGTT) 110

3-11 Carcass of HFD control and SA37-treated mice 111

3-12 Effect of SA37 in liver, kidneys and lungs weights after 4 weeks of

treatment

112

3-13 Effect of SA37 in epididymal and retroperitoneal fat pad weights after 4

weeks of treatment

113

3-14 Effect of SA37 in serum concentration of total cholesterol and

triglyceride; and NEFA levels

114

xii

Table List of tables Page

1-1 IC50 or Ki

of carboxylic acid pTyr mimetics 12

1-2 IC50 or Ki of TDZ pTyr mimetics 14

1-3 IC50 or Ki of some novel pTyr mimetics 16

2-1 Inhibitory effect of small library compounds against PTP1B 32

2-2 Nature of chemical library compounds 40

2-3 Percentage of inhibition by 10 µM solution of the library compounds A1-

J7 against PTP 1B 42

2-4 Inhibitory potency of compounds A3, B3, C2, C3, C5, C8 against PTP1B 43

2-5 Inhibitory effect of compound C3 on various PTPs 44

2-6 Body weight after 6 days of C3 treatment 48

2-7 Body weight gain during obesity development Period 52

2-8 Body weight after 4 weeks of C3 treatment 53

2-9 Inhibitory potency of chemical sub library I compounds against PTP1B 65

2-10 Inhibitory potency of chemical sub library II compounds against PTP1B 67

2-11 IC50 of chemical sub-library II compounds against PTP1B 68

3-1 IC50 values of disalicylic acid derivatives against PTP1B and IKK β 97

3-2 Body weight after 6 days of SA37 treatment 101

3-3 Body weight gain during 8 weeks of obesity development period 104

3-4 Body weight after 4 weeks of SA37 treatment 106

xiii

Scheme List of scheme Page

2-1 Synthesis of (Z) 4(2(2 chlorobenzyloxy)benzylidene) 3 phenylisoxazol-

5(4H)-one (Lead Compound)

28

2-2 Synthesis of aldehydes precursors for small librarycompounds 29

2-3 Synthesis of small library compounds 30

2-4 Synthesis of substituted 3 phenyliisoxazol-5(4H)-one derivative 33

2-5 Synthesis of aldehydes precursors for chemical library synthesis 34

2-6 Synthesis of aldehydes precursors (G) for chemical library synthesis 35

2-7 Chemical library synthesis 37

2-8 Synthesis of substituted 3 phenylisoxazol-5(4H)-one derivative 63

2-9 Hydrolysis of C7 to C3ix 64

3-1 Synthesis of difuranyl methylenedisalicylic acid 98

xiv

List of abbreviations

ACS Acyl-CoA synthetase

ACOD Acyl-CoA oxidase

ATP Adenosine triphosphate

BSA Bovine serum albumin

BW Body Weight

13 C NMR Carbon-13 nuclear magnetic resonance

Chemical shift

DAG Diacylglycerol

DMSO Dimethyl sulfoxide

DTT Dithiothreitol

E.coli Escherechia coli

EDTA Ethylenediaminetetraacetic acid

FFA Free fatty acids

kg; g; mg Kilogram; gram; milligram

HFD High fat diet

1H NMR Proton nuclear magnetic resonance

Hepes (N-[2-hydroxyethyl]piperazine-N’-[ethanesulfonic acid]

IC50

Half-maximal inhibitory concentration

IκB Inhibitor of NFκB

IKK IκB kinase

IL-1 β Interleukin -1 β

xv

IPGTT Intraperitoneal glucose tolerance test

IR Insulin receptor

IRS Insulin receptor substrate

JAK2 Janus kinase 2

LFD Low fat diet

mL; L Milliliter; microliter

M; mM; M Molar; millimolar; micromolar

MHz Megahertz

mmol Millimole

NEFA Non-esterified free fatty acids

nm Nanometer

NFκB Nuclear factor κB

pNPP p-Nitrophenyl phosphate

PTP Protein tyrosine phosphatase

PTP1B Protein tyrosine phosphatase 1B

PTK Protein tyrosine kinase

pTyr Phosphotyrosyl

SHP-1 Src homology 2 (SH2) domain-containing PTPase

TC-PTP T-cell protein tyrosine phosphatase

T-Cho Total Cholesterol

TG Triglyceride

TLC Thin-layer chromatography

xvi

TNF- α Tumor necrosis factor-α yeast protein tyrosine posphatase 1

VHR Vaccinia H1-related phosphatase

TZD 2, 4- Thiazolidinediones

YPTP1 Yeast Protein tyrosine phosphatase

xvii

Abstract

In developing inhibitors of therapeutic target enzymes, significant time and effort are

committed to the preparation of large numbers of compounds. In an effort to develop a

potent inhibitor of PTP1B as an anti-obesity and/or anti-diabetic agent, we constructed an

isoxazolone chemical library using a simplified procedure that circumvents tedious work up

and purification steps. The 10 7 isoxazolone derivatives were synthesized by coupling the

two halves of the target compounds. When mixed and heated in test tubes, the precursors

produced the reaction products as precipitates. After brief washing, the products were pure

enough to be used for enzymatic experiments. With the precursors for the coupling reactions

prepared, the 10 7 library compounds could be prepared in a day using the present

protocol. The library compounds thus obtained were examined for their inhibitory activities

against PTP1B. Among them, compound C3 was the most potent inhibitor of PTP1B with

an IC50 of 2.3 μM. The in vivo effect of C3 was also examined in an obesity-prone mouse

strain. Diet-induced obese/diabetic mice were divided into two groups and each group was

fed high fat diet (HFD) or HFD + C3 for 4 weeks. The group of C3-fed mice gained

significantly less weight compared to the HFD-fed control group during the 4 weeks of the

drug feeding period. In contrast to the anti-obesity effect of C3, no difference was observed

in the glycemic control of the HFD and HFD + C3 mice groups.

xviii

요 약

의약개발시 표적 표적효소에 대한 억제제 개발과정에서 다수의 화합물들을

합성하는데 상당한 시간과 노력이 소모된다. 비만 및 당뇨 치료제로 사용될 수

있는 PTP1B 억제제 개발을 위하여 옥사졸론 화합물 라이브러리를 제작하였는데

반응 후 처리와 정제과정을 생략할 수 있는 방법을 사용하였다. 표적화합물들의

반쪽에 해당하는 두 개의 전구체들을 축합하여 10 7 옥사졸론 유도체들을

합성하였는데, 전구체들을 시험관 내에서 혼합 가열하면 생성물이 침전형태로

얻어졌다. 약간의 용매로 세척하는 것만으로 효소 실험에 사용하기에 충분한

순도의 화합물을 얻을 수 있었다. 축합반응을 위한 전구체들이 준비된 상태에서

10 7 라이브러리를 하루 동안에 제작할 수 있었다. 라이브러리 화합물들에

대하여 PTP1B 억제활성을 측정한 결과 가장 강력한 억제제는 화합물 C3 로 IC50

값은 2.3 μM 이었다. 비만이 되기 쉬운 생쥐 모델을 이용하여 화합물 C3 의 생체

내 효과를 실험하였다. 생쥐들에게 고지방식을 먹여 비만/당뇨를 유발한 뒤

생쥐들을 두 그룹으로 나누고 각각 고지방식 또는 고지방식 + C3 를 4 주간

먹였다. 화합물 C3 투여 그룹은 고지방식만 먹은 그룹에 비해 체중증가가

현저하게 감소하였다. 화합물 C3 에 의한 혈당조절효과는 관찰되지 않았다.

1

CHAPTER I

1. General Introduction

1.1. Phosphorylation and dephosphorylation

Kinases and phosphatases are counteracting partners in regulating signaling

responses. The kinases control the amplitude of a signaling response, and

phosphatases play a role in controlling the rate and duration of the response.1

The diversity and complexity of the PTP and PTK are comparable. Similar to

PTK, PTP are equally important in controlling the signaling pathways in

both negative and positive manner. Furthermore, biochemical and genetic

studies indicate PTP play a key role in the regulation of many physiological

processes in various mammalian tissues and cells.2,3

Figure 1-1. Protein phosphorylation and dephosphorylation

1.2. Phosphatases

One hundred and seven PTP genes were found in human genome, and 81 of

them encode active phosphatase.4 All the PTPs are characterized by a

conserved active site sequence (H/V)C(X)5R(S/T), term as PTP signature

motif, in which the cysteine residue functions as a nucleophile and is

essential for catalysis.5 PTPs can be divided into three subclasses: dual-

2

specificity PTPs (DSP-PTPs), low molecular weight PTPs (LMW-PTPs) and

classical tyrosine-specific PTPs.

Dual specificity PTPs (DSP-PTPs) are capable of dephosphorylating serine

and threonine residues as well as tyrosine residues. LMW-PTPs constitute a

family of 18 kDa enzymes with specificity primarily towards

phosphotyrosine.

The classical tyrosine-specific PTPs can be further divided in two

subfamilies: receptor-like PTPs and cytosolic PTPs. The receptor-like PTP

contain a transmembrane domain, an extracellular receptor like domain and

one or two PTP catalytic intracellular domains. The catalytic activities of

PTP reside in membrane-proximal (D1) PTP domain except in PTPα6, while

the membrane-distal domain (D2) is almost inactive and is considered to

play a regulatory role.

Cytosolic PTPs contain a single catalytic domain and flanking regions with

putative roles in the regulation of catalytic activity, protein-protein

interactions and subcellular targeting.7 PTP1B is an example of cytosolic

PTPs, that contains a carboxy-terminal endoplasmic reticulum-targeting

domain. The most prominent substrates for PTP1B is the insulin receptor (IR)

and insulin receptor substrate (IRS) proteins in skeletal muscle and liver.8,9

1.3. Importance of phosphatases

About 4% of the ‘druggable genome’ is thought to be phosphatases.10

Some

of the validated phosphatases as a drug target are PTP1B, 8,11,12,13,14,15

SHP-1, SHP-2,16,17

CD45,18

PTP-α, PTP-ε, Prl-3, Cdc25,19,20

PTP-β (VE-

PTP), PTP-ε,21

PTP-σ, GLEPP-1(PTP-oc), and PTP-ε.22,23

Among PTPs,

PTP1B is the most exploited PTP, and recently validated as a therapeutic

target for obesity and diabetes.

3

1.4. Protein Tyrosin Phosphatase 1B

PTP1B is the first PTP to be purified from human placental tissue.24

PTP1B

is widely expressed cytosolic enzyme and is primarily localized in the

endoplasmic reticulum (ER) via a cleavable proline-rich C-terminal

segment.25

PTP1B is a 50 kD PTP that consists of 435 amino acids.

Residues 30-278 comprise catalytic domain and 35 carboxyl terminal

residues target enzyme to the cytosolic face of the endoplasmic reticulum.26

Most of the structural features of PTP1B such as P-loop (His-Cys-Ser-Ala-

Gly-Ile-Gly-Arg) and WPD loop are common in other PTPs.27

The active

site of PTP1B is defined by a loop of eight amino acid residues (214-221)

and are important for catalysis. The cysteine residue (Cys215) is critical for

catalytic activity. Second phosphotyrosine binding site has been discovered

adjacent to the catalytic site.28

Identification of the second phosphotyrosine

binding site provided an additional opportunity for the design of more potent

and specific inhibitors.28

1.5. Landmarks in PTP1B research29

(Yip S. C. et al, 2010):

1988 - Purification and characterization of PTP1B from human placenta.30,31

1990-Cloning of hPTP1B cDNA, Microinjection in Xenopus laevis

oocytes.32,33,34,27

1992 - ER targeting domain identified, the effect of PTP1B on Neu- and V-

src transformed fibroblasts.25

1993 - Serine phosphorylation of PTP1B, Calpain-induced cleavage of the

PTP1B C-terminal tail.35,36

1994 - Crystal structure of hPTP1B (1–321 at 2.8 A°), PTP1B over-

4

expression in human breast & ovarian cancers.27

1995 - Osmotic loading of neutralizing antibody.37

1996 - Characterization of SH3 domain-containing PTP1B binding partners.

38

1997 - Development of substrate-trapping mutant: PTP1B-D181A.39

1998 - Suppression of transformation.40

1999 - Ptp1b knockout mice generated.8

2000 - Positive role in c-Src activation in cancer cells.41

2001 - ROS regulation of PTP1B identified TYK2 and JAK2 as substrates.

42,43

2002 - PTP1B attenuates leptin signaling, FRET imaging of receptor

dephosphorylation.

2003 - Structure of oxidized PTP1B.44

2004 - Positive role in Ras signaling, Muscle-specific Ptp1b knockout mice

generated.45,46,47

2005 - Crystal structure of non-catalytic binding to IR.48

2006-Brain-specific Ptp1b knockout mice generated.8

2007 - SUMO regulation of PTP1B, MMTV-Erbb2; Ptp1b-/-

mouse models

generated.49,50,51

2008 - Quantitative SILAC proteomics of PTP1B substrates.52

2009 - Src activation in ErbB2-mediated transformation in 3D culture, Liver-

specific Ptp1b knockout mice generated.53,54

5

1.6. General mechanism of dephosphorylation of substrate proteins by

PTP1B

The phosphotyrosine of substrate proteins binds deeply in the catalytic site of

phosphate-binding loop (P-loop). The binding of phosphotyrosine are

mediated by residues 214-221 (His, Cys, Ser, Ala, Gly, Ile, Gly, Arg) of

PTP1B. The WPD (tryptophan, proline, aspartic acid) loop closes down onto

the substrate and Asp181 acts as a general acid catalyst. The phosphate is

cleaved from phosphotryosine residue, allowing dephoshorylated substrate to

diffuse from the active site and water to replace it. General base catalyzed

hydrolysis of the resultant phosphocysteine by Asp181 regenerates the active

form of phosphatase and completes the catalytic cycle.55

O

Enzyme

P

O

-O

-O

pTyr substrate

Asp

OHO

+ HO

Hydrolysed substrate

S PCys

O

O-O

-

HO

Ser

OH

H

Asp

O-

O

O

GlnH2N

Thiophosphoryl enzymeintermediate

Enzyme

+ P-O-O

OH

O

215

181

222

181

262

Cys S-

215

Cys S P

O

O-

-O

215

Cys S-

215

A

B

HO

Ser 222

HO

Ser 222

HO

Ser 222

Asp

OHO

181

Asp

O-

O

181

Thiophosphoryl enzymeintermediate

Figure 1-2. Catalytic mechanism of PTP1B56

6

Entire PTPs share a common catalytic mechanism. (A) Formation of

thiophosphoryl enzyme intermediate: thiolate anion from the active site

cysteine attacks phosphate of the substrate and forms a thiophosphoryl

enzyme intermediate. Expulsion of dephosphorylated substrate is aided by

the general acid (Asp) of the enzyme.

(B) Hydrolysis of thiophosphoryl enzyme intermediate: A water molecule

activated by the general base (Asp) attacks the intermediate. The glutamine-

262 positions the attacking water molecule. The hydrogen bond with the

serine-222 compensates the negative charge developed on the S atom of the

active site cysteine.

1.7. Validation of PTP1B as a therapeutic target

The validation of PTP1B as therapeutic target for diabetes and obesity are

supported by growing number of evidences collected from several

biochemical, pharmacological and genetic studies. Two research groups

independently reported that PTP1B knockout mice displayed a phenotype

strongly suggestive of a role in insulin and leptin signaling.8,11

The PTP1B

deficient mice showed enhanced insulin sensitivity, lower plasma glucose

and insulin levels, and resistance to weight gain compared to control mice

with high fat diets. The PTP1B deficient mice also exhibited normal

development and longevity. In contrast, TC-PTP deficient mice died at 3-5

weeks of age. The death of TC-PTP knockout mice was due to impaired B

cell and T cell functions. Even though, TC-PTP is the most homologous

phosphatase to PTP1B.57

Several studies on antisense oligionucleotide,

overexpression in vitro, human single nucleotide polymorphisms, and

observations of mutations within the human PTP1B gene sequence were

7

conducted to further validate the role of PTP1B as a therapeutic target.58,59

Xie, L. et al. reported that small-molecule inhibitors of PTP1B can work

synergistically with insulin to increase insulin signaling and augment insulin-

stimulated glucose uptake.60

In another approach, pretreatment of leptin-

resistant rats with a potent and selective PTP1B inhibitor results in a marked

improvement in leptin-dependent suppression of food intake.61

On the basis

of these evidences, PTP1B is considered as one of the validated biological

targets for non-insulin dependent diabetes and obesity.

1.8. Some important PTP1B inhibitors found in literature

Among PTPs, PTP1B is the most exploited phosphatase as a drug target.

Several screenings and structural studies were conducted to develop specific

PTP1B inhibitors. Almost all the research groups faced two major hurdles in

the development of potent and selective PTP1B inhibitors as a drug, 1)

highly conserved active site and 2) the regulation of multiple signaling

pathways by a single PTP as well as the regulation of single pathway by

diverse PTPs. To overcome aforementioned obstacles, different research

groups gleaned more biological roles and structural features of PTPs. On the

basis of available information, several research groups designed synthesized

and reported several inhibitors that target PTP1B.

1.8.1. Vanadate (VO4-3

) as a non-specific phosphatase inhibitor

In proper oxidation state, Vanadium is highly similar to phosphate.62

It can

enter into tight covalent complexes within the PTP catalytic site, and can

serve as an effective PTP inhibitor.63,64

Vanadate (VO4-3

) was found to be a

non-specific phosphatase inhibitor acting as insulin mimics.65 , 66

Bis(maltolato)oxovanadium(IV) compound 1, has been reported as a potent

8

PTP1B inhibitor with a blood glucose lowering effect in diabetic animal

models.67

Figure 1-3. Bis(maltolato)oxovanadium(IV) compound

1.8.2. DFMP pTyr mimetics

The pTyr moiety binds deeply into the phosphatase active site. It is

responsible for majority of binding energy to PTP1B. Thus, several small

molecule PTP1B inhibitors with non-hydrolyzable or poorly hydrolyzable

pTyr mimics were reported. Zhang (1997) reported a highly potent (PTP1B

Ki = 2.4 nM) and selective (TC-PTP 10-fold selectivity) peptide ligand 2

with two DFMP groups.28

Unfortunately, the highly polar nature of the bis-

anionic DFMPs and peptide scaffold provided compounds with poor

physiochemical properties i.e. poor cell permeability and low oral

bioavailability.

9

Figure 1-4. Peptide ligand with two DFMP

1.8.3. Nonhydrolyzable pTyr mimetics

As peptide backbone is not good for the development of effective drugs, the

focus has mainly been on the development of non-peptide low molecular

weight inhibitors. Combs A.P. (2010) classified PTP1B inhibitor into four

classes and are summarized hereafter.55

1.8.3.1. Nonhydrolyzable DFMP pTyr mimetics (Combs A.P., 2010)

10

Figure 1-5. Nonhydrolyzable DFMP pTyr mimetics

Several approaches of non-peptide scaffolds with DFMP group have been

trailed by various pharmaceutical companies. Merk Frosst reported three

promising PTP1B inhibitor classes: arylketone DFMP 3 (IC50 = 120 nM),

benzotriazole DFMP 4 (IC50 = 5 nM), and naphthyl DFMP 5 (IC50 = 120

nM).68,69 ,70

These inhibitor classes demonstrated modest degree of cell

permeability and oral bioavailability in rodents. However bis-anionic DFMP

demonstrated less desirable pharmacokinetics in higher species. Compounds

3 and 5 demonstrated an antidiabetic effect when tested in an oral glucose

tolerance test (oGTT) in diet induced obese (DIO) mice. A sulfonamide

scaffold bearing the DFMP 6 (IC50 = 28 nM) and a novel ketophosphonate

pTyr mimetic 7 (IC50 = 600 nM) as PTP1B inhibitors were identified.71

However, selectivity report for compound 6 and 7 do not exist.

11

1.8.3.2. Carboxylic acid pTyr mimetics

Figure 1-6. Carboxylic acid pTyr mimetics

12

Table 1-1. IC50 or Ki

of carboxylic acid pTyr mimetics

Compound

No

IC50 or Ki in µM PTP1B

Selectivity

with respect

to TC-PTP

Research

Group PTP1B TC-PTP

8 0.018 0.065

3.6 Abbott

72

9 9.0 182

20 Abbott

73

10 8.4 ˃200 ˃23 Abbott

52

11 2.1 ˃30 ˃15 Abbott

74

12 0.081 NA NA

Novo

Nordisk75

13 3.2 NA NA Kyorin

Pharma76

14 0.016 NA NA Inst. of

Pharm. Dis.77

15 0.004 0.005

1 Wyeth

78

Highly potent dicarboxylic acid containing PTP1B inhibitors were reported

by different group. But these inhibitors have poor membrane permeability.

When one of the carboxylic acid moieties was eliminated to improve the

13

membrane permeability, the resulting compounds were found to be 100-1000

fold less potent or converted in to extremely hydrophobic compounds that

may result in nonspecific inhibition.

1.8.3.3. TDZ pTyr mimetics

Figure 1-7. TDZ pTyr mimetics

14

Table 1-2. IC50 or Ki of TDZ pTyr mimetics

Compound

No

IC50 or Ki in µM PTP1B

Selectivity

with respect

to TC-PTP

Research

Group PTP1B TC-PTP

16 1600 NA NA AstraZeneca79

17 2.5 NA NA AstraZeneca58

18 0.14 0.15 1 Incyte80

19 0.032 0.025 0.8 Incyte81

20 0.27 0.25 1 Incyte82

21 0.023 0.025 1 Incyte83

22 4.3 NA NA Incyte

84

23 0.104 NA NA Novartis85





15

TDZ and (S)-IZD five-membered heterocyclic pTyr mimetics were reported

by four independent research groups. These small molecule inhibitors that

incorporate the TDZ and (S)-IZD pTyr mimetics were the most potent pTyr

mimetics class of PTP1B inhibitors with better membrane permeability then

carboxylic acid and DFMP containing inhibitors. But, still lack sufficient

cellular penetration for in vivo efficacy and oral bioavailability.

1.8.3.4. Novel pTyr mimetics

Figure 1-8. Some Novel pTyr mimetics

16

Table 1-3. IC50 or Ki of some pTyr mimetics

Compound

No

IC50 in µM PTP1B

Selectivity

with respect

to TC-PTP

Research

Group PTP1B TC-PTP

28 2.5 NA NA Sugen89

29 4.8 9.4 1.9 Proct. &

Gamble90

30 0.07 Na NA Proct. &

Gamble91

31 8 NA NA Taylor et al92

32 4.1 5.3 1.3 Tremblay et

al93

The sulfamic acid pTyr mimetic obtained from high throughput screening are

low molecular weight compounds. On optimization these compounds shows

extremely good inhibitory potency against PTP1B, but the membrane

permeability of compounds containing this pharmacophore were not found in

literature.

1.9. PTP1B inhibitors reported from our lab

As an effort to develop potent and selective PTP1B inhibitors, our lab

17

reported a novel series, formylchromone derivatives,94

disalicylic acid

derivatives56

and pyrogallol derivatives95

.

The most potent compound from formylchromone derivatives 33 with IC50 =

1µM, was irreversible inhibitor of PTP1B with good selectivity among

several PTPases including TC-PTP.

MDSA derivatives were synthesized and examined for the inhibitory activity

against diverse PTPs. Among those, three MDSA derivatives 34, 35 and 36

demonstrated about 10-fold selectivity over TC-PTP and two of the

compounds, 34 and 36 inhibited PTP1B reversibly. Oral administration of

compound 34 and 36 to DIO mice significantly reduced body weight gain

compared with that of the untreated HF control group without suppressing

food intake.56

Our lab further modified disalicylic acid compounds to 37 (IC50 = 5 µM) and

38 (IC50 = 0.5 µM) improved PTP1B inhibitory effect. Compound 37

showed glucose level lowering effect and improved glucose tolerance in

HFD-induced diabetic mice.96

Non-phosphorous small molecule derivatives of pyrogallol with one

carboxyllic acid group showed low IC50 value upto 2 µM and Ki value of 1.1

µM against PTP1B. The most potent compound 39 of pyrogallol series was

7-fold selective for PTP1B than TC-PTP. Oral administration the most potent

compound 39 of this series exhibited glucose lowering effect and improved

glucose tolerance in DIO mice without any overt toxicity.97

18

Figure 1-9. Some potent PTP1B inhibitors reported from our lab

In order to find potent inhibitors of PTP1B, we screened a drug library called

‘The Prestwick Chemical Library® ’

that contains 1120 small molecules, 90%

of which are drugs and 10% are bioactive alkaloids or related compound.98

This library contains a limited number of highly diverse drug molecules for

which bioavailability and toxicity studies have already been performed and

proven safety in humans. Thus, the initial hits of this library can be used as a

starting point for drug optimization in medicinal chemistry.26

We identified 9

novel inhibitors of PTP1B by applying a computer-aided drug design

protocol involving the structure-based virtual screening with docking

simulations under consideration of the effects of ligand solvation in the

scoring function. These inhibitors are structurally diverse and reveal potency

with IC50 values ranging from 10 to 50 µM.

19

Figure 1-10. Novel PTP1B inhibitors identified from virtual screening

20

Compound 45, was found to be a competitive inhibitor of PTP1B. Several

variations in substitutions in ortho-, meta- and para- directions from the

TZD moiety were synthesized and evaluated for their PTP1B inhibitory

activity. Most of the synthesized compounds exhibited low µM IC50 values

against PTP1B. Two of the most potent compounds of different series,

compound 50 and 51 were tested for their in vivo antihyperglycemic and

antiobesity effects.

Figure 1-11. Two most potent PTP1B inhibitors from TZD-series

Treatment of two of the most potent compounds 50 and 51 of that series to

the HFD-induced diabetic mice controlled the HFD-induced body weight

gain and exhibited an improved glucose tolerance effect. The serum

triglyceride, total cholesterol and free fatty acids levels were found

significantly lowered.

Compound 46 was found as a non-competitive inhibitor. We designed and

synthesized a series of barbituric acid derivative to investigate the potency,

mode of inhibition, steady-state kinetic experiments of PTP1B and YPTP1

were performed for compound 52. The mode of inhibition was determined

21

by the Lineweaver-Burk plot analysis of the results of the kinetic

experiments. The compound 52 inhibited PTP1B and YPTP1 in a

noncompetitive and a mixed-type noncompetitive fashion, respectively.

These results indicate binding site for the compound 52 is different from that

for the substrate. The noncompetitive inhibition of PTP1B might be

explained by the possible binding of the barbituric acid moiety in the

secondary aryl phosphate-binding site, present near the active site of PTP1B.

Figure 1-12. Most potent PTP1B inhibitors from barbituric acid-serirs (52)

and analogue compound of virtual hit 42 (53)

On the other hand, the mixed-type noncompetitive inhibition of YPTP1 by

52 could hardly be explained on the same basis, because no aryl phosphate-

binding site distinct from the active site has been identified on YPTP1.

Nondiscriminative inhibition of the three structurally diverse PTPs by 52

also implicates the possible similarity of the inhibitor binding sites on this

PTPs. Explanation of these apparently conflicting observations and

considerations requires additional studies including X-ray crystallographic

22

structure determination of the enzyme-inhibitor complex. Enzyme-inhibitor

interaction yet to be explained at the molecular level, the barbiturate moiety

was proven to be a promising scaffold for the design of PTP inhibitors.

To improve the inhibitory potency of barbituric acid derivatives we modified

the structure of compound 46 and di-substituted barbituric acid compounds

were synthesized. The work is in progress in our lab.

We synthesized compound 53 as an analogue of compound 42. The

inhibitory potency and kinetic study of the compound 53 revealed that it’s a

promising competitive inhibitor of PTP1B. Further modification of

compound 42 is in progress.

23

1.10. Present work

PTP1B is considered as one of the validated biological targets for non-

insulin dependent diabetes and obesity. Targeting PTP1B for drug discovery

is challenging because of the highly conserved and positively charged active-

site pocket. Potent and selective PTP1B inhibitors, with minimum charge

density, good pharmacokinetics and pharmacodynamics can be considered as

a drug.

Our present work is based on compound 44 (a compound with isoxazol-

5(4H)-one moiety). The compound was obtained from virtual screening.

24

1.11. Objectives of this study

Numerous researches have been performed for the development of selective

PTP1B inhibitors possessing beneficial effects for diabetes and obesity.

However, clinically usable drugs have not been developed yet. Aimed for the

development of PTP1B inhibitors with therapeutic effects, this study focused

on the synthesis and the biological evaluation of isoxazol-5(4H)-one

derivatives. Main objectives of the present work are summarized below.

1. To synthesize a chemical library with a isoxazol-5(4H)-one moiety as

a common scaffold.

2. To evaluate their inhibitory potency against PTP1B and the selectivity

profile of the most potent compound against various PTPases.

3. To study the inhibition pattern of a most potent compound by kinetic

experiments.

4. To examine the in vivo effect of the most potent PTP1B inhibitor in a

diet-induced obese/diabetic mouse model system.

25

CHAPTER – II

2. Synthesis and Biological Evaluation of Isoxazol-5(4H)-one Derivatives

as a New Family of PTP1B Inhibitors with Anti-Obesity Effects

2.1. Introduction

The rapid increase in the prevalence of obesity throughout the world poses a

serious health threat in modern society.99,100,101

Obesity enhances the risk of

associated morbidities, including diabetes, hypertension, dyslipidemia,

ischaemic heart disease, and even cancer.102

Existing therapies for obesity

are limited in number and effectiveness. Only two drugs, orlistat and

sibutramine, are currently approved for the long-term treatment of obesity.

These drugs have limitations in their use due to adverse side effects or

limited efficacy.103

Another drug, rimonabant, recently developed as an

appetite suppressant was approved in 2006 in the European Union. However,

rimonabant was found to increase the risk of adverse psychiatric effects,

including serious depression and suicide, and its use was suspended in

2008.104

To circumvent the problems of the current drugs, numerous studies have

been devoted to the identification of novel therapeutic targets and significant

progress has been made.105,106,107

Among these progresses, inhibition of

PTP1B was recognized as a promising therapeutic strategy for the

management of both obesity and diabetes.108

Genetic ablation of PTP1B in

mice suppressed weight gain and maintained insulin sensitivity upon feeding

a high fat diet (HFD). Wild-type mice, on the other hand, gained weight and

became insulin resistant. Furthermore, PTP1B-depleted mice were healthy,

without worrisome traits such as enhancement of mitogenic signaling.

26

Inhibition or reduction of the cellular abundance of PTP1B in mice also

resulted in increased sensitivity to leptin and insulin, and has been shown to

exhibit a protective effect against diet-induced obesity.8

Numerous active site-directed PTP1B inhibitors have been reported, many of

them with pharmacophores that mimic phosphotyrosine residues.109 , 110

Among those, Ertiprotafib (Figure 2-1) had progressed to clinical trials but it

was discontinued in the second phase due to unwanted side effects and

insufficient efficacy.111

A noncompetitive PTP1B inhibitor, trodusquemine

(Figure 2-1), has recently proceeded to the early stages of clinical trials with

promising preclinical results as both an appetite suppressant and a

hypoglycemic and hypocholestrolemic agent.112

First-in-class drug targeting

PTP1B is yet to be developed and intensive research is underway to develop

a potent and selective PTP1B inhibitor with anti-obesity and/or

hypoglycemic effect.

In an effort to find potent inhibitors of PTP1B, we previously performed a

virtual screening with docking simulations and identified nine compounds

with half-maximal inhibitory concentrations (IC50) lower than 50 μM toward

PTP1B. Among those, compound L4 (Figure 2-1) with an IC50 value 22 μM

was chosen as a lead compound for this study to develop potent PTP1B

inhibitors.

27

Figure-2-1: Some PTP1B inhibitors.

2.2. Result and discussion

To obtain a large number of drug-like molecules is one of the preliminary but

an important step in a drug discovery process. An efficient synthetic strategy

is required for the synthesis of large number of molecules with minimum

time and efforts. Diverse techniques have been developed to construct

chemical libraries such as polymer-supported synthesis, multi component

one pot condensation, and combinatorial synthesis. In the construction of a

chemical library, it would be better if a single reaction step is available with

a simple work-up and purification procedure.

With the construction of a chemical library in mind, compound L4 with IC50

22 μM against PTP1B is selected as lead compound. On kinetic study, the

compound was found to be a competitive inhibitor of PTP1B. We designed

28

and synthesized a structurally diverse chemical library with an isoxazol-

5(4H)-one moiety as a common scaffold.

2.2.1. Synthesis of lead compound (Z)-4-(2-(2-

chlorobenzyloxy)benzylidene)-3-phenylisoxazol-5(4H)-one (L4)

The precursor, 3-phenylisoxazol-5(4H)-one, was synthesized from

benzaldehyde via β-hydoxy ester and β-keto ester. And to prepare aldehyde

precursor 2-(2-chlorobenzyloxy)benzaldehyde commercially available 2-

hydroxy benzaldehydes were subjected to benzylation with the 1-chloro-2-

(chloromethyl)benzene in presence of K2CO3. Coupling 3-phenyl isoxazol-

5(4H)-one with 2-(2-chlorobenzyloxy)benzaldehyde, we got the lead

compound.(Scheme2-1).

Scheme 2-1. Synthesis of lead compound (Z)-4-(2-(2-

chlorobenzyloxy)benzylidene)-3-phenylisoxazol-5(4H)-one: Reagents and

conditions: (a) Zn/NH4Cl, rt, 1 h; (b) MnO2/DCM, rt, 24 h, (c) NH2OH·HCl,

pyridine, EtOH, 65 ºC, (d) K2CO3, DMF, 90 ºC, (e) isopropanol, 65 ºC, 4 h.

The synthetic strategy for the synthesis of lead compound was used for the

29

preparation of a small library of compounds L1-L6.

2.2.2. Synthesis of a small library of compounds L1-L6

2.2.2.1. Synthesis of aldehyde precursors (A & K-O) for small library

compounds

To prepare the ortho or meta or para-substituted derivatives A & K-O,

commercially available 2 or 3 or 4-hydroxybenzaldehydes were subjected to

benzylation with appropriate benzylchlorides in presence of K2CO3. (Scheme

2-2)

Scheme 2-2: Synthesis of aldehyde precursors (A & K-O) for small library

compounds: Reagents and Conditions: (a) K2CO3, DMF, 90 ºC, 3-6 h

2.2.2.2. Precursors for small library compounds

Six aldehyde precursors and 3-phenylisoxazol-5(4H)-one derivatives were

30

synthesized to set up the reaction conditions for chemical library synthesis

and to find the preliminary inhibitory potency trend.

Figure 2-2: Precursors for small library compounds.

2.2.2.3. Synthesis of small library compounds

To check the potency trend of isoxazol-5(4H)-one derivatives against PTP1B,

we synthesized two series of test compounds, L1-L3 and L4-L6, (Fig. 2-3)

bearing a benzyloxy or a chlorobenzyloxy substituent at the ortho, meta, and

para positions respectively on the ring A. Scheme 2-3.

Scheme 2-3. Synthesis of small library compounds: Reagents and

31

Conditions: (a) isopropanol, 65 ºC, 4 h

2.2.2.4. Small library compounds

Figure 2-3: Small library compounds

2.2.3. In vitro results of small library compounds

With the construction of a chemical library in mind, we synthesized two

series of test compounds, L1-L3 and L4-L6, bearing a benzyloxy or a

chlorobenzyloxy substituent at the ortho, meta, and para positions

respectively on the ring A of the 3,4-diarylisoxazol-5-one derivatives. These

32

compounds, when examined for their inhibitory potency against the enzyme

activity of PTP1B, exhibited different levels of potency in enzyme inhibition

with IC50 values ranging from 15 to 102 μM (Table 2-1).

Table 2-1. Inhibitory effect of small library compounds against PTP1B

Compd. Mol. Wt IC50(µM)a Compd. Mol. Wt IC50 (µM)

a

L1 355.39 102±10 L4 389.83 22±2

L2 355.39 71±4 L5 389.83 25±1

L3 355.39 43±3 L6 389.83 15±1

[a] Data expressed as means ± standard deviations of two experiments. The

kinetic data are analyzed using the GraFit 5.0 program (Erithacus Software).

Notably, the assay result revealed that para-isomers L3 and L6 were the

most potent within the two groups, L1-L3 and L4-L6. Based on this

observation, a chemical library was designed with structural variations on the

A and B rings of this series of compounds, with the substituent on the A ring

being fixed at the para position.

2.2.4. Chemical library synthesis

Ten aldehyde precursors and seven isoxazol-5(4H)-one precursors were

synthesized as synthetic scheme 2-4, 2-5 and 2-6.

2.2.4.1. Synthesis of seven isoxazol-5(4H)-one precursors for chemical

library synthesis

To prepare β-hydroxy ester derivative, mixture of appropriate commercially

33

available aldehyde and ethyl bromoacetate were ground with Zn/NH4Cl. The

reactions were carried out in solvent free condition for liquid aldehydes.

Minimum volume of THF (Technical Grade) was used for the solid

aldehydes. The β-hydroxy ester derivatives were oxidized to β-keto esters by

MnO2 catalyzed oxidation reaction. On pyridine catalyzed cyclisation, β-keto

esters converted into corresponding substituted 3-phenylisoxazol-5(4H)-one.

(Scheme 2-4).

Scheme 2-4: Synthesis of substituted 3-phenylisoxazol-5(4H)-one

derivative: Reagents and conditions: (a) Zn/NH4Cl, rt, 5 min – 2 h; (b)

MnO2/DCM, rt, 24 h, (c) NH2OH·HCl, pyridine, EtOH, 65 ºC.

2.2.4.2. Synthesis of aldehyde precursor (A-F and H) for chemical

library synthesis

4-hydroxybenzaldehydes were subjected to benzylation with the appropriate

34

benzylchlorides in presence of K2CO3. To prepare the p-tosyl derivative F

commercially available 4-hydroxybenzaldehydes were subjected to

tosylation with p-toluenesulfonyl chloride in presence of DIEPA. (Scheme 2-

5)

Scheme2-5: Synthesis of aldehyde precursors for chemical library Synthesis:

Reagents and conditions: (a) K2CO3, DMF, 90 ºC, 3-6 h (b) DIEPA, DMF, 0

ºC to rt.

2.2.4.3. Synthesis of aldehyde precursor (G) for chemical library

synthesis

To convert the benzonitrile to tetrazol, a solution of 4-(4-formylphenoxy)

35

benzonitrile, NaN3 and Et3N·HCl in toluene was refluxed for 6 h.

Scheme 2-6: Synthesis of aldehyde precursor (G) for chemical library

synthesis: Reagents and conditions: (a) NaN3, Et3N·HCl, 4-(4-

formylphenoxy) benzonitrile, and toluene, reflux for 6 h.

2.2.4.4. Commercially available aldehydes precursor (I and J) for

Chemical Library Synthesis

4-(5-pyrimidinyl)benzaldehyde (I) and 5-(3-chloro-4-methoxyphenyl)-2-

furaldehyde (J) were Purchased from Aldrich.

Figure 2-4: Commercially available aldehyde precursors (I and J) for

Chemical Library Synthesis:

36

Figure 2-5: Precursors for chemical library synthesis

37

2.2.4.5. Chemical library synthesis

In vitro data obtained from small chemical library hints that the para

substituent on the A ring might be better for further investigations. We

designed and synthesized seventeen precursors. Eight aldehyde precursors

(A–H) were synthesized. Two aldehyde precursors I and J were purchased

from Aldrich. Seven substituted 3-phenylisoxazol-5(4H)-one (1-7)

derivatives were synthesized. Table 2-5.

The aldehyde precursors were coupled with isoxazol-5(4H)-one precursors

by catalyst free Knoevenagel type condensation reaction.

Scheme 2-7. Chemical library synthesis: Reagents and Conditions: (a)

isopropanol, 65 ºC, and 4 h.

2.2.4.6. Solubility determination of precursors for chemical library

synthesis

The solubility of the aldehydes and isoxazol-5(4H)-one precursors was

determined by dissolving 0.05 mmol of aldehydes (A–J) and substituted 3-

phenylisoxazol-5(4H)-one (1–7) in a minimum volume of reaction solvent

isopropanol at reaction temperature 65 ºC. (Fig 2-6)

38

Figure 2-6: Solubility of precursors for chemical library compounds

2.2.4.7. Synthesis chemical library compounds

The strategy for the library synthesis is shown in (Scheme 2-7). The

condensation reaction of (A-J) and (1-7) was accomplished without catalyst

by heating the reaction mixture at 65 ºC. In this study, all the condensation

reactions proceeded in the absence of piperidine or any other catalysts.

Furthermore, the product precipitated on cooling of the reaction mixture,

which eliminated work up process and saved the time and effort for the

isolation of the desired product. This feature is one of the conspicuous

virtues in the library synthesis for the present study.

We developed a protocol that uses test tubes as reaction vessels and

39

eliminates the aqueous work up step to minimize the time and effort required

for the preparation of a chemical library of 70 compounds,. Briefly, 70 test

tubes (10 mL capacity) were set in a 10 × 7 array. Solutions of benzaldehyde

derivatives (A–J) and isoxazol-5(4H)-one derivatives (1–7) (0.05 mmole

each) in isopropanol were placed in glass test tubes in 10 × 7 combinations.

Following heating for 4 h at 65 ºC in a dry heating block and cooling to room

temperature, the reaction products had separated out as precipitates in most

of the reaction tubes. In some cases where no precipitate had formed, the

addition of hexane (0.5 mL) and cooling to -50 ºC effectively induced

precipitation. Removal of the solvent and washing the precipitate with

isopropanol (0.5 mL) afforded a solid product which showed essentially a

single spot in thin layer chromatography (TLC) analysis. Even though the

products can be further purified by recrystallization or column

chromatography, the precipitates were used as such for enzyme experiments.

2.2.4.8. Nature of chemical library compounds

After four hour reaction at 65 ºC, the reaction mixtures were cooled to room

temperature. Most of the reaction products were obtained as precipitate as

shown in Figure below.

40

Table 2-2: Nature of chemical library compounds

P = The product precipitate on cooling to room temperature

S = The product is soluble in reaction solvent even on cooling to room

temperature. Precipitation induced by adding hexane and cooling at -50 ºC.

Figure 2-7: Precipitation of chemical library compounds at room

temperature

41

Figure 2-8: Chemical library compounds

42

2.2.5. In vitro results of library compounds

2.2.5.1. Inhibitory potency of library compounds against PTP1B

Crude products were harvested from chemical library synthesis. 100 µM

solution of each product were prepared and assayed against PTP1B. The

final concentration of assayed solution was 10 µM.

Table 2-3. Percentage of Inhibition by 10 µM solution of the library

compounds (A1-J7) against PTP 1B

1 2 3 4 5 6 8

A 27.8 76.4 97.8 13.5 64.2 16.7 26.7

B 47.0 38.0 100.0 42.2 52.2 20.6 42.7

C 67.1 98.3 97.8 86.1 94.4 78.3 94.4

D 21.4 1.7 27.4 NI NI NI NI

E NI 35.1 57.0 0.0 7.8 0.5 NI

F 10.7 33.5 85.5 17.6 NI 36.1 27.1

G NI 27.3 34.6 87.4 25.0 31.5 27.1

H NI NI 38.5 NI NI NI NI

I NI NI 18.4 NI NI NI NI

J 33.9 63.2 83.8 NI 70.6 4.1 4.0

43

NI = No Inhibition

Eighteen compounds out of seventy showed >50% inhibition for PTP1B

enzyme activity. Six compounds inhibited the PTP1B activity by >90%.

Compounds at column 3 and row C exhibited higher potency compared to

other columns and rows. IC50 values are shown as numbers for the

compounds that inhibit >90% PTP1B enzyme activity.

Table 2-4. Inhibitory potency of compounds A3, B3, C2, C3, C5, C8 against

PTP1B


a

A3 457.83 2.5±1.3 C3 507.38 2.3±0.0

B3 457.83 3.9±1.1 C5 516.47 4.2±0.0

C2 518.28 2.4±0.1 C7 497.42 7.2±0.5

aData expressed as the mean standard deviations of two experiments.

2.2.5.2. Selectivity of most potent compound C3 against other PTPs

The inhibitory activity of C3 against other PTPs was tested in order to

examine the PTP1B selectivity of C3, the results of which are summarized in

Table 2-5. Compound C3 demonstrated a 10-fold greater selectivity over

TC-PTP, the most homologous with PTP1B among the human PTPs, and a

2.9-fold greater selectivity over the catalytic domain of SHP-1 (SHP-1cat).

Compound C3 showed a 12-fold greater selectivity for PTP1B versus VHR,

a dual specificity PTP. YPTP1, a yeast PTP, was also tested to compare the

PTPs of different origin, and it exhibited 4.6-fold selectivity.

44

Table 2-5. Inhibitory effect of compound C3 on various PTPs

Compound PTP1B TCPTP SHP1-cat YPTP1 VHR

C3 2.3 ± 0 24 ± 3 6.7 ± 1 11 ± 0 27 ± 1

IC50 (μM), Values are means ± standard deviations of two experiments. The

kinetic data were analyzed using the GraFit 5.0 program (Erithacus

Software).

2.2.5.3. Enzyme kinetic experiments

To investigate the mode of inhibition of C3, the enzyme activity of PTP1B

was determined with varying the concentration of the substrate, p-

nitrophenyl phosphate (pNPP). Lineweaver-Burk plot analysis of the results

by plotting the reciprocal of the enzyme reaction rates versus 1/[pNPP]

revealed the competitive pattern of inhibition, which indicates that C3 binds

to the active site of the enzyme.

45

Figure 2-9: Lineweaver-Burk Plot Analysis for PTP1B-catalyzed Reaction

in Presence of C3: Hydrolysis of pNPP by PTP1B was measured in absence

(○), or presence of 10 μM (●), 20 μM (□) and 30 μM (■) C3.

We successfully developed a simple and efficient library synthesis protocol

for the condensation of aldehydes and isoxazol-5(4H)-one derivatives. The

Knoevenagel condensation was accomplished in the absence of catalyst. By

employing this library synthesis protocol, a number of isoxazol-5(4H)-one

molecule were synthesized and assayed without purification against enzymes.

The IC50 value obtained from crude products and purified products were

similar. The crude products were TLC pure and all the NMR data of the

46

randomly selected compounds were essentially same before and after

purification. These result distinctly verified our library synthesis protocol.

The in vitro result revealed that, the compound C3 was the best inhibitor

among the 70 library compounds, exhibiting the lowest IC50 value of 2.3 μM

against PTP1B.

2.2.6. Animal experiment

2.2.6.1. Toxicity test of compounds C3 on mice

The most potent PTP1B inhibitor C3 was selected for the evaluation of in

vivo efficacy as anti-obesity and/or hypoglycemic agent, to avoid the risk of

animal death. The toxic effect of C3 was tested with 2 male, 5-week-old (30-

35 g) (SIC: ICR) mice. Two mice were kept as DMSO control.

2.2.6.1.1. Animal and diet selection for toxicity

Male, 4-week-old (18-20 g) (SIC: ICR) mice were purchased from Japan

SLC, OHHARA breeding branch and individually caged at 25 2 C. The

12 h light/dark cycle was maintained and food and water were supplied ad

libitum.

The experimental diets were obtained from Research Diets (New Brunswick,

NJ). Low fat diet (LFD, D12450B) contained 10% calories from fat, 70%

calories from carbohydrate and 20% calories from protein. All the foods

were low fat diet and in pellet form.

2.2.6.1.2. Feeding

All six mice (SIC: ICR) were provided with low fat diet during 7 days of

acclimatization period. Then they were weighed and divided in to two groups.

47

During the toxicity testing period (6 days), all the mice were deprived of

food for 6 hours and of water for 2 hours before the drug administration.

After 1 hour of drug administration, food and water were supplied ad libitum.

2.2.6.1.3. Drug dose, gavages preparation and administration

Drug dose for toxicity test was fixed as 400mg of C3/kg body weight of

mouse for 6 days. To maintain this drug dose, 120 mg of C3 was dissolved

in 3 mL of DMSO. The DMSO solution of C3 was administrated by needle

(shaft length 2 inch, ball diameter 21/4

mm, curved) as, 10 µL/gm body

weight of mouse i.e. 1 % of body weight of mouse.

2.2.6.1.4. Effect of compounds C3 on (SIC: ICR) mice

The mice were found highly stressed for ~6 h of drug administration but

slowly normalized. The external appearance perineum was found normal the

next day of the drug administration.

48

Figure 2-10: Physical appearance of control mouse and C3-treated mouse.

Table 2-6. Body weight after 6 days of C3 treatment:

During 6 days of toxicity testing period, the body weight gain was 4 g for the

DMSO control group, compared to 2 g for the C3-treated group.

Initial Weight (g) Final Weight (g)

DMSO Control 34.1± 0.3 38.10±1.9

DMSO + C3 31.65±0.6 33.73±1.5

49

2.2.6.1.5. Carcass of the mice at 6 days of C3 treatment

Figure 2-11: Carcass of the mice after 6 days of C3-treatment

At the last day of experiment, overnight fasted mice were anaesthetized with

physical dislocation and the mice were dissected to observe the organs. All

the organs were found normal on visual inspection except the liver which

was abnormally brown spotted, in C3- treated mouse.

50

Figure 2-12: Liver and other organs appearance after 6 days of C3-treatment

51

2.2.6.1.6. Conclusion: Even though the toxicity test was done in small group

of normal mice the result revealed that the mice can survive with C3. The

brown spots were seen in liver may be the side effect of overdose.

2.2.6.2. Effect of compound C3 on diet induced diabetic mice

C3 is the most potent inhibitor of PTP1B among the chemical library

compound. We tested the selectivity and nature of inhibition of compound

C3. The compound C3 was found ten times selective with TC-PTP and

competitive inhibitor. The toxicity test of C3 was performed and found to be

safe for animal experiment. With the reference of these in vitro and in vivo

results, C3 was chosen for determining their antiobesity or

antihyperglycemic effect in HFD-induced diabetic mice (C57BL/6J Jms Slc

male). After 1-week of acclimatization, the mice were divided into three

groups; two groups of which were fed HFD for further 8 weeks to develop

the HFD induced diabetes/obesity and the another one group was provided

with LFD to serve as a lean control group. After 8 weeks on HFD, one of the

groups was continued on HFD an obese control group whereas the other

group was provided with HFD + C3 for further 4 weeks.

2.2.6.2.1. External appearance and body weight gain of mice after

obesity development period:

During the obesity development period, body weight gain was 10 g for the

HFD-fed mouse groups, compared to 5 g for the LFD-fed group. The LFD-

fed lean control mice gained much lower body weight compared to the HFD-

fed mice and the two groups were clearly different in external appearances.

(Figure 2-13 and Table 2-7).

52

Table 2-7. Body weight gain during obesity development Period


HFD-Fed Groups LFD-Fed Groups

18.00 ±0.12 28.47 ±0.44 23.70 ± 0.29

Figure 2-13: Appearance of mice after obesity development period

2.2.6.2.2. Body weight after 4-weeks of C3 Treatment

No significant difference in body weights between HFD-fed control group

53

and C3-treated group was observed during and after 4 weeks of the

compounds treatment period (Table 2-8).

Table 2-8. Body weight after 4 weeks of C3 treatment:


HFD 28.55 ± 1.32 36.05 ±1.56

HFD + C3 28.18 ±0.81 34.1 ±1.14

LFD 23.70 ± 0.29 26.26 ±0.41

2.2.6.2.3. Body weight gain during 4-weeks of C3 treatment

Figure 2-14: Appearance of mice after drug feeding period

54

Figure 2-15. Body weight gain during 4 weeks C3 treatment

C3 (0.1% w/w) in HFD were mixed well and provided with food. Each

points represents the mean value ± SEM; n = 8/group. Mice provided with

LFD served as a reference group of lean control. Significance was calculated

by One-way ANOVA, where, ** represents p < 0.05.

There was significant difference in body weight gain between HFD control

group and C3-treated group mice beginning on day 12 and remaining for the

most of the duration of the study. (Figure 2-15).

2.2.6.2.4. Food intake

There was a slight difference in food intake in C3-treated mice group and

55

LFD control mice group as compare to the HFD control mice group. But,

there was no significantly difference in cumulative food intake between HFD

control and C3-treated mice groups during 4 weeks of treatment period.

(Figure 2-16.)

Figure 2-16: Cumulative food intake during 4 weeks of C3 treatment

C3 (0.1% w/w) in HFD were mixed well and provided with food. Each bar

represents the cumulative mean value ± SEM; n = 8/group

There was no significantly difference in cumulative food intake between

HFD control and C3-treated mice groups during 4 weeks of treatment period.

(Figure 2-16.)

56

2.2.6.2.5. Feed Efficiency

The ratio of weight gain to calories consumed, were described in terms of

feed efficiency. And the body weight gains per calories consumed were

calculated for the HFD control, C3-treated and LFD control groups. (Figure

2-17). Feed efficiency of C3-treated group was significantly lower compared

to HFD control group. This result revealed that, the body weight gain in C3-

treated group was due to high energy expenditure.

Figure 2-17. Feed efficiency in mice: Significance was calculated by One-

way ANOVA, where, ** represents p < 0.05.

2.2.6.2.6. Intraperitoneal Glucose Tolerance Test (IPGTT)

At the end of 4 weeks of C3-feeding period, fasting blood glucose level of

57

all the groups was measured i.e. 0 min (Figure 2-18). Then extra glucose

was loaded by i.p. injection.

Figure 2-18. Intraperitoneal glucose tolerance test (IPGTT)

Mice were fasted for 8 hrs starting from the beginning of light cycle. Just

before the intraperitoneal injection of glucose (1 g/kg BW), the blood was

withdrawn from the tip of the tail and base line glucose level was measured.

Then, after injection of the glucose, blood glucose levels were measured at

given times. All values are the mean values ± SEM; n= 8/group. Significance

was calculated by One-way ANOVA, where, ** represents p < 0.05.

At the end of the 4 weeks of drug-feeding period, the fasting glucose level of

the test compounds-fed groups was similar as the HFD control group

58

(Figure 2-18, 0 min) After injection of extra glucose, there were no any

significantly faster decrease in blood glucose concentration in C3-treated

group compared to the HFD control group.

2.2.6.2.7. Carcass appearance at 4-wks of C3 treatment

No any visual differences were seen in organs of the C3-treated mice and

HFD control mice. The fat pad deposits in HFD control groups were seen

significantly higher compared to HFD + C3 group after dissection of mice at

the end of experiment (Figure 2-19).

Figure 2-19. Carcass of mice after C3 treatment

There was no visual difference in liver of C3-treated mice and control group.

59

2.2.6.2.8. Liver, kidneys and lungs weights

Figure 2-20. Effect of C3 in liver, kidneys and lungs weights after 4 weeks

of treatment: C3 (0.1% w/w) in HFD were mixed well and provided with

food. All values are the mean values ± SEM; n = 8/group. Mice provided

with LFD served as a reference group of lean control. Significance was

calculated by One-way ANOVA.

The liver weights of C3-treated group of mice were significantly higher

compared to HFD and LFD control groups. But there was no significant

difference in lung and kidney weights between HFD control and C3-treated

mice groups.

60

2.2.6.2.9. Epididymal and retroperitoneal fat pad weights

Figure 2-21. Effect of C3 in epididymal and retroperitoneal fat pad weights

after 4 weeks of treatment

C3 (0.1% w/w) in HFD were mixed well and provided with food. All values

are the mean values ± SEM; n = 8/group. Mice provided with LFD served as

a reference group of lean control. Significance was calculated by One-way

ANOVA, where, ** represents p < 0.05.

There was no significant difference in Epididymal fat pad deposition in C3-

treated mice. But there was significant difference in retroperitoneal fat pad

deposition in C3 treated mice compared to HFD control (Figure 2-21).

61

2.2.6.2.10. Blood analysis

62

Figure 2-22. Effect of C3 in serum concentration of (a) total cholesterol and

triglyceride; and (b) nefa levels after 4 weeks of treatment

C3 (0.1% w/w) in HFD were mixed well and provided with food. Data

presented are the mean values ± SEM; n = 8/group. Mice provided with LFD

served as a reference group of lean control. Significance was calculated by

One-way ANOVA, where, ** represents p < 0.05.

No significant difference of total cholesterol, triglyceride and non-esterified

free fatty acids were found in serum of the C3-treated mice compared to

HFD control mice when tested after overnight deprivation of the food

(Figure 2-22).

63

2.2.7. Chemical sub-library I compounds

Compound C3 exhibited highest inhibitory potency against PTP1B among

the chemical library compounds. We fixed compound C as an aldehyde

precursor. Eight new isoxazol-5(4H)-one precursors were synthesized.

Chemical sub-library I compounds were synthesized by using same synthetic

strategy as library synthesis. And the library compound C7 was hydrolyzed

to get C3ix compound. Their inhibitory potency was tested against PTP1B.

Scheme 2-8. Synthesis of substituted 3-phenyl isoxazol-5(4H)-one

derivatives. Reagents and conditions: (a) Zn/NH4Cl, rt, 1h; (b) MnO2/DCM,

rt, 24 h, (c) Acetone, Johns’ reagent (d) NH2OH·HCl, pyridine, EtOH, 65

ºC

64

Scheme 2-9. Hydrolysis of C7 to C3ix: Reagents and conditions: (a) 1,4-

dioxane, 1M, NaOH, 100 ºC.

2.2.7.1. Synthesized chemical sub-library I compounds

Figure 2-23. Chemical sub-library I compounds

65

2.2.7.2. Inhibitory potency of chemical sub-library I compounds against

PTP1B:

Table 2-9. Inhibitory potency of Chemical sub library I compounds against

PTP1B


a

C3i 523.38 3.2±0.7 C3vi 455.38 12.5±4.0

C3ii 523.38 2.7±0.0 C3vii 609.57 1.5±0.2

C3iii

C3iv

C3v

541.37

541.37

541.37

4.0±0.1

2.3±0.3

6.8±0.6

C3viii

C3ix

599.48

483.39

0.7±0.1

18.6±1.1


Compound C3vii and C3viii exhibited higher potency compared to other

compound.

2.2.8. Chemical sub-library II

2.2.8.1. Chemical sub-library II compounds

To cross check the potency trend against PTP1B. Isoxazol-5(4H)-one

precursor 3vii and 3viii were selected to couple with aldehyde precursors of

library synthesis and their inhibitory potency was tested against PTP1B.

66

Figure 2-24. Chemical sub-library II compounds

2.2.8.2. Inhibitory potency of chemical sub-library II compounds against

PTP1B

Percentage of Inhibition of chemical sub-library compounds A3vii-J3vii and

67

A3viii- J3viii against PTP 1B by 10µM solution.

Compd. Mol. Wt Inhibition% Compd. Mol. Wt Inhibition%

A3vii 560.02 98 A3viii 549.92 100

B3vii 560.02 97 B3viii 549.92 100

D3vii 526.56 71 D3viii 516.47 75

E3vii 570.57 80 E3viii 560.48 88

F3vii 589.64 78 F3viii 579.54 89

G3vii 579.58 74 G3viii 569.49 70

H3vii 533.59 83 H3viii 523.50 87

I3vii 497.52 53 I3viii 487.43 63

J3vii 549.98 87 J3viii 539.89 99

Table 2-10. Inhibitory potency of Chemical sub library II compounds against

PTP1B : IC50 values are shown for the compounds that inhibit >90% PTP1B

enzyme activity.

68

Table 2-11. IC50 of chemical sub-library II compounds against PTP1B

Compd. Mol. Wt IC50(µM)a Compd. Mol. Wt IC50(µM)

a

A3vii 560.02 3.3±0.4 A3viii 549.92 1.1±0.0

B3vii 560.02 4.0±1.3 B3viii 549.92 1.6±0.1

J3viii 539.89 1.3±0.1


2.3. Experimental section

2.3.1. Materials and methods for chemical synthesis

1H and

13C NMR spectra were recorded on a Varian Inova 400 (400 MHz) or

Varian Vnmrs 400 (400 MHz) spectrometer using CDCl3 or DMSO-d6 as a

solvent. Chemical shifts are reported in unit of parts per million (ppm) from

tetramethylsilane with the solvent resonance as the internal standard (CHCl3:

δ = 7.26 ppm for 1H, CDCl3: δ = 77.0 ppm for

13C). Data are reported as

follows: chemical shift, multiplicity (s; singlet, d; doublet, t; triplet, q;

quartet, m; multiplet, br; broad), coupling constants (Hz) and integration. All

reactions were monitored by thin-layer chromatography using E. Merck

silica gel plates (60F-254) pre-coated plates (0.25 mm). TLC visualization

was done with UV light and/or 5% ethanolic p-anisaldehyde. IR spectra were

recorded on a Bruker Vertex 80V spectrophotometer. All reagents were

purchased from Aldrich (St. Louis, U.S.A.), Sigma (St. Louis, U.S.A.), or

TCI (Tokyo, Japan) and used as received.

69

2.3.2. Synthesis of library compound precursors

2.3.2.1. Synthesis of aldehyde precursors

2.3.2.1.1. General Procedure for the preparation of (A – E, H and K-O)

A solution of appropriate hydroxybenzaldehyde (5.0 mmol), appropriate

benzylchlorides and K2CO3 (12 mmol) in dry DMF (10 mL) was heated to

90ºC. After 3 – 6 h, the reaction mixture was cooled to r.t. and added into a

mixture of 0.1 M HCl (60 mL) and EtOAc (30 mL). The aqueous layer was

separated and extracted with EtOAc (30 mL × 2). Combined organic layers

were washed with H2O (20 mL × 3) and saturated NaCl (20 mL), dried in

(anh. Na2SO4), filtered and concentrated. The crude product was purified by

column chromatography on silica gel using Hexane/EtOAc mixture as a

solvent system.

2.3.2.1.2. Procedure for the preparation of (F)

4-methylbenzene-1-sulfonyl chloride (5.5 mmol) and DIEPA (6.5 mmol)

were used instead of benzylchlorides and K2CO3 and the reaction was carried

out at 0 ºC to room temperature.

2.3.2.1.3. Procedure for the preparation of (G)

A solution of 4-(4-formylphenoxy) benzonitrile (0.447 g, 2.0 mmol), NaN3

(0.390 g, 6.0 mmol,) and Et3N·HCl (0.823 g 6.0 mmol) in toluene (3 mL)

was refluxed for 6 h. The reaction mixture was cooled to rt. and poured into

1 M HCl (25 mL). It was extracted with EtOAc (30 mL × 3). The organic

layers were combined, washed with 1 M HCl (10 mL × 2) and saturated

NaCl (20 mL) successively, dried in anh. Na2SO4, filtered, and concentrated.

The crude product was used as such for next reaction.

70

4-(2-chlorobenzyloxy)benzaldehyde (A): White solid, 92% yield; Rf = 0.46

(hexane/ethyl acetate, 4:1); m.p. 54-55 ºC; 1H NMR (200 MHz, CDCl3): δ

5.31 (s, 2H), 7.15 (d, J = 8.8 Hz, 2H), 7.31-7.37 (m, 2H), 7.45- 7.61 (m, 2H),

7.91 (d, J = 8.8 Hz, 2H), 9.95 (s, 1H, CHO).

4-(4-chlorobenzyloxy)benzaldehyde (B): White solid, 89% yield; Rf = 0.35

(hexane/ethyl acetate, 4:1); m.p. 73-74 ºC; 1H NMR (200 MHz, CDCl3): δ

5.16 (s, 2H), 7.1 (d, J = 8.8 Hz, 2H), 7.42 (brs, 4H), 7.89 (d, J = 8.8 Hz, 2H),

9.93 (s, 1H, CHO).

4-(4-(trifluoromethoxy)benzyloxy)benzaldehyde (C): White solid, 96%

yield; Rf = 0.46 (hexane/ethyl acetate, 2:1); m.p. 45-46 ºC; 1H NMR (200

MHz, CDCl3): δ 5.20 (s, 2H), 7.12 (d, J = 8.8 Hz, 2H), 7.31 (d, J = 8.0 Hz,

2H), 7.52 (d, J = 8.0 Hz, 2H), 7.91 (d, J = 8.8 Hz, 2H), 9.95 (s, 1H, CHO).

4-(pyridin-3-ylmethoxy)benzaldehyde (D): Light Yellow solid, 55% yield;

Rf = 0.25 (hexane/ethyl acetate, 1:4); m.p. 71-72 ºC; 1H NMR (200 MHz,

CDCl3): δ 5.20 (s, 2H), 7.12 (d, J = 7.6 Hz, 2H), 7.36-7.42 (m, 1H), 7.81-

7.92 (m, 3H), 8.65 (d, J = 4.6 Hz, 1H), 8.74 (brs, 1H), 9.93 ppm (s, 1H,

CHO).

4-(4-nitrobenzyloxy)benzaldehyde (E): Light Yellow solid, 53% yield; Rf =

0.18 (hexane/ethyl acetate, 4:1); m.p. 110-111 ºC; 1H NMR (400 MHz,

CDCl3): δ 5.27 (s, 2H), 7.09 (d, J = 8.8 Hz, 2H), 7.62 (d, J = 8.4 Hz, 2H),

7.87 (d, J = 8.8 Hz, 2H), 8.27 (d, J = 8.4 Hz, 2H), 9.90 ppm (s, 1H, CHO).

4-Tosyloxy-benzaldehyde (F): White solid, 94% yield; m.p. 73-74 ºC; Rf =

0.33 (hexane/ethyl acetate, 4:1); 1H NMR (200 MHz, CDCl3): δ 2.50 (s, 3H),

7.21 (d, J = 8.6 Hz, 2H), 7.37 (d, J = 7.8 Hz, 2H), 7.76 (d, J = 7.8 Hz, 2H),

7.88 (d, J = 8.6 Hz, 2H), 10.01 ppm (s, 1H, CHO).

71

4-(4-(1H-tetrazol-5-yl)phenoxy)benzaldehyde (G): Cream color solid 93%

yield; Rf = 0.14 (ethyl acetate/MeOH, 19:1); m.p. 179-180 ºC; 1H NMR (400

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