to my mummy, papa, sisters and brother; you all mean the...

To My Mummy, Papa, Sisters andBrother;

You All Mean the World to Me

Date:

Certificate

This is to certify that the work embodied in this thesis entitled

“Design, Synthesis, Biological Evaluation of Nitrogen and/or Sulphur

Containing Heterocyclic Compounds and Biosynthesis of Biologically

Active Alkaloids” has been carried out by Ms. Kanika under my

supervision and fulfills all the requirements for the award of the degree of

Doctor of Philosophy from University of Lucknow, Lucknow.

The work reported in this thesis is original and does not form a

basis for the award of any other degree of any other university.

(Prof. (Ms) Sudha Jain)

Certificate

This is to certify that all the regulations necessary for the

submission of Ph.D thesis of Ms. Kanika has been fully observed.

(Prof. R. N. Pathak)Head

Department of ChemistryUniversity of Lucknow

Lucknow.

Acknowledgements

i

Acknowledgements

To complete a multiyear task, like PhD is not possible without the help and guidance of

mentors, many seasoned experts, colleagues and other technical persons and my task was not an

exception. It is a moment of gratification and pride to look back with a sense of contentment at the

long traveled path, to be able to recapture some of the fine moments, to think of infinite number of

people, some who were with me from the beginning, some who joined me at different stages during this

journey, whose kindness, love and blessings has brought me to this day. I wish to thank each of them

from the bottom of my heart.

First and foremost I express my entire honour to “My Parents” without whose blessing this

task would not have been accomplished. I bow my head in utter humility and complete dedication from

within my heart. My vocabulary is not wide enough to put my sentiments in words to repay them for

what they have done to groom me in such a great fashion. Hats off to the Omnipresent, Omniscient

and Almighty God, the glorious fountain and continuous source of inspirations! I offer salutation to

the Omnipotent Lord “Krishna”.

Words are insufficient to record my deep sense of gratitude to my esteemed teacher, mentor

and guide Prof. (Ms) Sudha Jain, Professor, Department of Chemistry, University of Lucknow,

Lucknow for her constant inspiration with keen interest, parental affectionate behaviour and ever

vigilant guidance, without which this task could not have been achieved. I appreciated the freedom

she allowed, which permitted me to develop scientific thoughts and experimental skill independently.

Her sincere involvement in my work has triggered and nourished my intellectual maturity that I will

benefit from, for a long time to come. I would also like to express my indebtedness to Dr. Anil Kumar

Saxena, Ex-Chief Scientist, Division of Medicinal and Process Chemistry, CSIR-Central Drug

Research Institute (CSIR-CDRI), Lucknow, for his constant support, encouragement and supreme

guidance in my entire research work at CSIR-CDRI. Without their valuable supervision and guidance

this work would not be possible. The only way to thank them would be perhaps to strive to work

similarly in years ahead, and continue the chain succession.

I express my sincere thanks to Prof. S. B. Nimse, Vice Chancellor, University of Lucknow,

Dr. S. K .Puri, Director, CSIR-CDRI, Dr. T. K. Chakraborty, Ex-Director, CSIR-CDRI, Lucknow,

Prof. R. N. Pathak, Head, Department of Chemistry, University of Lucknow, Dr. B. Kundu, Chief

Scientist and Head, Medicinal and process chemistry Division for providing competent laboratory

facilities and infrastructures to carry out the work successfully.

Acknowledgements

ii

I also express my sincere gratitude to Dr. A. K. Srivastava, Senior Principal Scientist and

Head, Division of Biochemistry, Dr. Geetika. Bhatia, Chief Scientist, Division of Biochemistry, Dr.

Anil K. Gaikwad, Principal Scientist, Division of Biochemistry and Dr. Madhu Dixit, Division of

Pharmacology, CSIR-CDRI, Lucknow for providing Biological data of the synthesized targeted

compounds.

I am thankful to Dr. Abha Bishnoi, and all the teachers in Department of Chemistry,

University of Lucknow, who have delivered very fruitful lectures during pre-Ph.D course work. I am

also thankful Dr. V. L. Sharma, Dr. A. K. Sinha, Dr. Y. S. Prabhakar, Dr. R. Bhatta, Dr.

Namrata Rastogi Scientists, CSIR-CDRI for their moral support, suggestions and encouragements.

I wish to confer my sincere thanks to Mr. A. S. Kushwaha, Mr. Zahid Ali and Mr. D. N.

Vishwakarma for their valuable support throughout the work.

An attempt such as a Ph. D. is unattainable to achieve without the generous help and

support of friends and colleagues. I would like to take this opportunity to thank those whom I was

fortunate to know, work and form friendship with over the past few years.

I wish to express my sincere obligations to my seniors, Dr. Paras Nath Chaudhary, Dr. Anjuli

Natu, Vishal sir, Rajesh sir from University of Lucknow and Dr. Amit Gupta, Dr. Swati Gupta, Dr.

Imran Ahmd Khan, Dr. K.K. Roy, Dr. Shailendra Chaudhaery from CSIR-CDRI who inspired me to

carry out strategic research work with full dedication.

I take this opportunity to thank my lab mates Praveen, Ashok Sir in University of Lucknow

and Supriya Ma’am, Sugandha, Vishal, Shome sir, Azad, and my biologists Neha ma’am, Arun, Rohit,

Akanksha, Vaibhav, Neetu ma’am, Ravi in CSIR-CDRI for providing a great working environment

and for the help extended by them during my Ph.D. tenure.

Thanks to my never failing research colleagues and friends Anil, Shaheen, Suruchi, Ritesh and

research students from Department of Chemistry, for the stimulating companionship, timely

assistance. I am fortunate to have few peoples, who have always stood by me during my good as well

as bad time. I would like to thank my sister Parul and my best friend Amit from the bottom of my

heart to stay by my side throughout selflessly.

I am extremely thankful to Manju Bua, Badi Bua, Laxmi aunty, Baby Bua, Anju Bua,

Bhuri, Bulbul and Kinshu for their affectionate behavior and making me feel at home whenever I was

with them.

At this moment, I would like to thank and remember my late grandfather. I am sure my

achievement would have filled his heart with pride and happiness. I would like to put on record that

Acknowledgements

iii

the love, affection and support extended by my siblings Kopal and Aman along with my uncle Naresh

Mama cannot be ignored and deeply acknowledged herewith.

I would like to thank to SAIF division, CSIR-CDRI for providing spectral data of the

synthesized compounds. Finally, I take this opportunity to express my sincere thanks to Council of

Scientific and Industrial Research (CSIR), New Delhi for approving my research proposal for the

award of Direct-Senior Research Fellowship.

Lastly, I wish to offer my heartfelt thanks to all, whose names could not be included, but will

be fondly remembered.

Kanika

Table of ContentsTable of Contents

ACKNOWLEDGMENTS iPREFACE ivLIST OF ABBREVIATIONS vi

Chapter 1 SYNTHESIS, SAR AND DOCKING STUDIES OF THIAZOLE DERIVATIVES ASPROTEIN TYROSINE PHOSPHATASE INHIBITORS (PTP1B) 1-31

1.0 Diverse biological activities of Thiazole and Therapeutic Approach to Type 2Diabetes ………………………………………………………………………………….. 1

1.1 Biological Activities of Thiazole …………………………………………………........ 21.2 Diabetes ………………………………………………………………………………….. 121.3 Treatment Approaches ………………………………………………………………… 131.3.1 Protein Tyrosine Phosphatase 1B …………………………........................................ 14

PTP1B: Structure and Biology………………………………………………………...... 15Protein Tyrosine Phosphatase 1B in Diabetes……………………………………….. 17PTP1B Inhibitors……………………................................................................................ 18

Part 1ASYNTHESIS, STRUCTURE ACTIVITY RELATIONSHIP (SAR) AND DOCKINGSTUDIES OF SUBSTITUTED ARYL THIAZOLYL PHENYLSULFONAMIDES ASPOTENTIAL PROTEIN TYROSINE PHOSPHATASE 1B INHIBITORS

32-59

1A.1 Results and Discussion …………………………………………………………….. 331A.1.1 Chemistry ……………………………………………………………………………… 331A.1.2 In vitro and Structure Activity Relationship Study ……………………………… 361A.1.3 Docking Studies ……………………………………………………………………… 381A.1.4 In vivo Antihyperglycaemic activity………………………………………………… 411A.2 Conclusion………………………………………………….......................................... 421A.3 Experimental …………………………………………………………………………. 45

Part 1B

SYNTHESIS, STRUCTURE ACTIVITY RELATIONSHIP (SAR) AND DOCKINGSTUDIES OF SUBSTITUTED ARYL PHENYL THIAZOLYL PHENYLCARBOXAMIDE AS POTENTIAL PROTEIN TYROSINE PHOSPHATASE 1BINHIBITORS

60-100

1B.1 Result and Discussion ……………………………………………………………… 611B.1.1 Chemistry ……………………………………………………………………………… 611B.1.2 In vitro and Structure Activity and Relationship Study ………………………… 651B.1.3 Kinetics Measurements and Mechanism of Inhibition …………………………. 661B.1.4 Docking Studies ……………………………………………………………………… 681B.1.5 In vivo Biological Activity ……………………………………………………………. 681B.2 Conclusion………………………………………........................................................... 751B.3 Experimental …………………………………………………………………………… 76

References……………………………………………………………………............... 91

Table of ContentsChapter 2 DESIGN, SYNTHESIS AND BIOLOGICAL EVALUATION OF INDOLE-FUSED

HETEROCYCLIC DERIVATIVES101-115

2.1 Introduction……………………………………………………………………………..... 1012.2 Indole: Chemical and Biological Importance ……………………………………..... 1012.2.1 Bioactive Indoles Developed over the Past Few Years ………………………….. 103

Part 2ALIPID LOWERING OXOPROPANYL INDOLE HYDRAZONE DERIVATIVES WITHANTIOXIDANT, ANTIHYPERGLYCAEMIC AND IMPROVED LIPOPROTEINLIPASE ACTIVITIES

116-138

2A.1 Results and Discussion ……………………………………………………………….. 1172A.1.1 Chemistry…………………………………………………………………………………. 1172A.1.2 Biological activities ………………………………………………………………………… 1202A.2 Conclusion……………………………………………....................................................... 1272A.3 Experimental……………………………………………………………………………… 129

Part 2BSYNTHESIS, STRUCTURE ACTIVITY RELATIONSHIP (SAR) AND DOCKINGSTUDIES OF SUBSTITUTED ARYL PHENYL THIAZOLYL PHENYLCARBOXAMIDE AS POTENTIAL PROTEIN TYROSINE PHOSPHATASE 1BINHIBITORS

139-158

2B.1 Result and Discussion……………………………………………………………………… 1402B.1.1 Chemistry……………………………………………………………………………………… 1402B.1.2 In vivo antithrombotic activity…………………………………………………………….. 1442B.2 Conclusion……………………………………………………………………………………. 1452B.3 Experimental ………………………………………………………………………………… 146

References………………………………………………………………………………… 154

Chapter 3 ABERRANT BIOSYNTHESIS OF 8-BROMORETICULINE 159-198

3.1 Introduction…………………………………………………………………………………… 1593.2 Benzylisoquinoline Alkaloids……………………………………………………………... 1593.3 Biogenesis of Reticuline………………………………………………………………… 1613.4 Aberrant Biosynthesis……………………………………………………………............ 1643.5 Present Investigation………………………………………………………….................. 1643.6 Discussion……………………………………………………………………...................... 1773.7 Experimental………………………………………………………………………………... 179

References……………………………………………………………………………………. 196

Preface

iv

Preface

Chemistry of heterocycles is one of the most complex branches of organic

chemistry. Because of the diversity in the synthetic procedures, physiological and

industrial significance, heterocyclic chemistry has been and continues to be one of the

most active areas of organic chemistry.

Among various heterocycles, sulphur and nitrogen-containing heterocyclic

compounds have maintained the interest of researchers through decades of historical

development of organic synthesis. Thiazole is one of the most important class of

heterocyclic compounds found in many potent biologically active molecules such as

Sulfathiazole (antimicrobial drug), Ritonavir (antiretroviral drug), Abafungin (antifungal

drug). Thiazoles are also found in drugs which are used for the treatment of allergies,

hypertension, inflammation, schizophrenia, bacterial, HIVinfection and hypnotics.

Moreover, many thiazole derivatives are reported as antidiabetic agents in literature.

The Indole scaffold also represents one of the most important structural subunits

for the discovery of new drug candidates. At present, among the 200 most commercially

successful products in the world medicinal market contain the indole fragment in their

structure. Due to the wide structural variety of the biologically active indole derivatives

and their characteristically high degree of binding with various biological targets

substituted indoles for more than half a century remain "privileged structures" in the

creation of new potential drugs and some of the most popular subjects of investigations

in the synthetic chemistry of heterocyclic compounds.

Ever since the structures of alkaloids were elucidated, the bio-organic chemists

speculated the way plants build up these molecules. Biosynthesis in general terms is

used to the formation of any substance in a living organism. There are several

approaches to the study of biosynthesis. Most of the work on alkaloid biosynthesis has

been carried out by feeding of labelled putative precursors to alkaloid producing plants.

Very little is known about the potentiality of higher plants to carry out

transformation on organic molecules which they do not normally produce or contain. This

type of transformation where an ‘unnatural precursor’ is converted into an ‘unnatural

product’ in living system is called ‘aberrant biosynthesis’. The ability of higher plants to

carryout unnatural or aberrant synthesis is relatively less explored area of research. A

few reported examples of aberrant biosynthesis are conversion of 5-fluoronicotinic acid

to 5-fluoronicotine in the tobacco plant Nicotiana tabacum, N-Methyl-Δ’-piperidinium

Preface

v

chloride was converted into higher homologue of nicotine, racemic 2’-bromonor-

reticuline and 2’-nitronorreticuline into 2‘-bromopapaverine and 2’-nitropapaverine

respectively. These experiments suggest that many of the enzymes in plants are non-

specific and are able to catalyse the biosynthesis of unnatural alkaloids form unnatural

precursors. Since the unnatural product (analogue of biologically active molecule)

possesses a structural label in addition to radiolabel, these can be used in study of

metabolism.

Based on the above theme, in the thesis are presented the results of research

work on the two broad topics: (1) Design, synthesis and biological evaluation of nitrogen

and/or sulphur heterocycles. (2) Biosynthesis of biologically active alkaloid. The research

work embodied in the present thesis has been divided in three chapters.

The first chapter of the thesis gives an overview of diverse biological activities of

thiazole derivatives and Protein tyrosine phosphatise 1B (PTP1B) inhibitors as

antidiabetic agents. This chapter is further divided into two parts, Part A and Part B. Part

A deals with synthesis, SAR and docking studies of substituted aryl thiazolyl

phenylsulfonamides as potential Protein Tyrosine Phosphatase 1B Inhibitors. While Part

B deals with the substituted aryl phenylthiazolyl phenylcarboxamide as potential protein

tyrosine phosphatase 1B inhibitors.

The second chapter of the thesis deals with the indepth review on diverse

biological activities of Indole. This chapter is further divided in two parts, Part A and Part

B. Part A deals with the synthesis of oxopropanyl indole hydrazone derivatives which

were further evaluated for their antioxidant, antidyslipidemic antihyperglycaemic and

antiadepogenic activities. The part B describes the synthesis of indolyl triazole

derivatives which were evaluated for their antithrombotic activity.

The third chapter of the thesis describes the aberrant biosynthesis of

bromoreticulines in the leaves of intact living young plant of Litsea glutinosa var.

glabraria Roxb. plant.

List of Abbreviations

vi


3D - Three-Dimensionalaq - AqueousÅ - Angstromb.p. - Boiling pointbs - Broad singletCADD - Computer-Aided Drug Designd - Doubletdd - Double DoubletdL deciliterDCM - DichloromethaneDMF - DimethylformamideDMSO - DimethylsulfoxideEt - EthylFT - Fourier Transformg - GramH - Hydrophobic FieldHDL - High Density LipoproteinHz - HertzH-bond - Hydrogen Bondi-Bu - Isobutyli-Pro - IsopropylIR - Infra-red SpectroscopyJ - Coupling ConstantLDL - Low Density Lipoproteinm - MultipletM+ - Molecular Ion Peakm.p. - Melting PointmL - Milliliterµg - MicrogramµL - MicroliterµM - MicromolarmM - MilimolarmCi - MiliCurieMe - MethylMS - Mass SpectroscopyMW - Molecular WeightnM - NanomolarNMR - Nuclear Magnetic ResonanceORO - Oil Red OPDB - Protein Data BankPh - PhenylPHLA - Post Heparin Lipolytic ActivityPL - PhospholipidPro - PropylPTP1B - Protein tyrosine phosphatase 1BQSAR - Quantitative Structure-Activity Relationships - SingletSAR - Structure-Activity RelationshipSBDD - Structure-based Drug Design


vii

SD - Standard Deviationt - TripletTBARS - Thiobarbituric Acid Reactive SubstancesTC - Total CholesterolTG - TriglycerideTHF - TetrahydrofuranTLC - Thin Layer ChromatographyTMS - TetramethylsilaneVLDL - Very Low Density Lipoprotein

Chapter 1Chapter 1Chapter 1Chapter 1

SynthesisSynthesisSynthesisSynthesis,,,, SSSStructure tructure tructure tructure AAAActivity Relationship ctivity Relationship ctivity Relationship ctivity Relationship and Docking and Docking and Docking and Docking

Studies of Thiazole Derivatives as Protein Tyrosine Studies of Thiazole Derivatives as Protein Tyrosine Studies of Thiazole Derivatives as Protein Tyrosine Studies of Thiazole Derivatives as Protein Tyrosine

Phosphatase Inhibitors (PTP1B)Phosphatase Inhibitors (PTP1B)Phosphatase Inhibitors (PTP1B)Phosphatase Inhibitors (PTP1B)

Synthesis, SAR and Docking Studies of Thiazole Derivatives as

Protein Tyrosine Phosphatase Inhibitors (PTP1B)

Chapter 1

Page | 1

1.0 Diverse Biological Activities of Thiazole and Therapeutic

Approach to Type 2 Diabetes

Over a century, heterocycles have constituted one of the largest area of

research in organic chemistry. These have not only contributed to the development of

society from a biological and industrial point of view but also to the understanding of

life processes and to the efforts to improve the quality of life. Apart from the majority

of pharmaceuticals, biologically active agrochemicals and countless additives in

industrial applications are also heterocyclic in nature.

Almost 80% of the drugs in clinical use are based on heterocyclic

constitution.1 Many natural drugs2-4 such as vinblastine, vincristine, colchicine,

papaverine, theobromine, quinine, emetine, theophylline, atropine, procaine, codeine,

reserpine and morphine etc. are heterocycles. Synthetic heterocycles also have

widespread therapeutic uses such as antibacterial, antifungal, antimycobacterial,

trypanocidal, anti-HIV activity, antileishmanial agents, genotoxic, antitubercular,

antimalarial, herbicidal, analgesic, antiinflammatory, muscle relaxants,

anticonvulsant, anticancer, lipid peroxidation inhibitor, hypnotics, antidepressant,

antitumoral, anthelmintic and insecticidal agents.5-11

Among various heterocycles, sulphur and nitrogen containing heterocyclic

compounds have maintained the interest of researchers through decades of historical

development of organic synthesis.12 Nitrogen and sulphur organic aromatic

heterocycles are formally derived from aromatic carbocyclic ring with a heteroatom

taking the place of a ring carbon atom or a complete CH=CH group. The presence of

heteroatom results in significant changes in the cyclic molecular structure due to the

availability of unshared pair of electrons and the difference in electronegativity

between heteroatom and carbon. Therefore, nitrogen and sulphur heterocyclic

compounds display physicochemical characteristics and reactivity quite different from

the parent aromatic hydrocarbons.

Thiazole (1) is one class of heterocyclic compound that contains both nitrogen

and sulphur atoms in the 1 and 3-positions of an aromatic five membered ring

respectively.

S

N

Thiazole (1)

1

4

5 2

3



Chapter 1

Page | 2

1.1 Biological Activities of Thiazole

The thiazole moiety is present in many potent biologically active molecules

found in nature e.g., it exists in Thiamine (2) a coenzyme required for the oxidative

decarboxylation of keto acids,14 as well as in synthetic products with wide range of

pharmacological activities for instance, tetrahydrothiazole appears in the skeleton of

Penicillin (3) which is one of the first and still the most important of the broad

spectrum antibiotics.15

A large number of drugs are being used containing thiazole ring such as

Sulfathiazole (4) an antimicrobial drug, Ritonavir (5) an antiretroviral drug, Abafungin

(6) an antifungal drug with trade name Abasol cream, Tiazofurin (7) an antineoplastic

drug, Talipexole (8) an antiparkinsonian drug and Meloxicam (9) an antiinflammatory

drug.15

H2N

S

HN

S

N

OO

NH

O

ON

SHO

HN

O

NH

O

N

S

N

Sulfathiazole (4) Ritonavir (5)

O

S

N

HN

NHN

Abafungin (6)

O

HO

OHOH

NS

NH2

O

Tiazofurin (7)

N

N

S

NH2

Talipexole (8)

NS

ON

S

O O

OH

Meloxicam(9)



Chapter 1

Page | 3

It has been noticed continuously over the years that interesting biological

activities16,17 were associated with thiazole derivatives. The applications of thiazoles

were found in drug development for the treatment of allergies,18 hypertension,19

inflammation,20 schizophrenia,21 bacterial,22 HIV infections,23 hypnotics24 and for the

treatment of pain,25 as fibrinogen receptor antagonists with antithrombotic activity26

and as new inhibitors of bacterial DNA gyrase B.27

A number of thiazoles, however have been found to possess a variety of

biological activities. A few are listed in Table 1.

Table 1: Diverse biological activities of thiazole derivatives

S.No Thiazole Derivtaives Pharmacological Uses

1

N

N

S

N

H2N

S

(10)

Antitumor activity28

2


3


4




Chapter 1

Page | 4

5

N

N S

N

Cl

NH

N

S

O

Cl

O

(14)

Antiinflammatory

activity32

6

Antiinflammatory

activity33

7

Antiinflammatory

activity33

8

Antiinflammatory

activity34

9

Antiinflammatory

activity35



Chapter 1

Page | 5

10

Antimicrobial

activity36,37

11

Antimicrobial activity38

12

NS

CN

O

C6H5

O

C6H5

(21)


13

N

NS

S

NN NH

C6H5

Br (22)


14


15




Chapter 1

Page | 6

16


17


18


19


20

Antioxidant activity45

21


22




Chapter 1

Page | 7

23


24

Antitubercular activity49

25


26

SNH

O

N N

N

CH3

C6H5

C6H5 CH3(35)


27

O

N

S

OH2N

Cl

(36)

Antifungal activity51

28




Chapter 1

Page | 8

29


30

Anticonvulsant activity53

31


32


33


34

Phosphatidylinositol-3-

kinase (PI3K)

inhibitors56



Chapter 1

Page | 9

35


kinase (PI3K)

inhibitors57

36 N

O

S N

O

OH

Cl

Cl(45)


kinase (PI3K)

inhibitors58

37

Protein kinase

inhibitors59

38

Protein kinase

inhibitors60

39

Protein kinase

inhibitors61



Chapter 1

Page | 10

40

N

ON

S

O

NH

N

N

NH

(49)

Protein kinase

inhibitors62

41

AntiAlzheimers

activity63

42

AntiHIV activity64

43

AntiHIV activity65

44

AntiHIV activity66



Chapter 1

Page | 11

45

Antidiabetic activity67

46


47

Antidiabetic

activity(Glucokinase

activators) 68

48

Antidiabetic activity

(protein tyrosine

phosphatase

inhibitors)69

49


50




Chapter 1

Page | 12

51


52


1.2 Diabetes

Diabetes is a growing disease in epidemic proportions world-wide. It is the

root cause of several chronic and progressive diseases that adversely affect a

number of organs including nervous and vascular system.74 According to

International Diabetes Federation (IDF), diabetes is one of the major challenging

health problems of 21st century.75 India, China, and United States are expected to

have the largest number of diabetic people by 2030, and India has been considered

to be the diabetes time bomb.76

There are two major categories of diabetes mellitus:

1. Type I or juvenile-onset diabetes or insulin-dependent diabetes

mellitus (IDDM).

2. Type II or adult-onset diabetes or non-insulin-dependent diabetes

mellitus (NIDDM).

Type I diabetes is an autoimmune destruction of the pancreatic β-cells (that

synthesize and secrete insulin) leading to absolute insulin deficiency and the patient

is absolutely dependent upon exogenous insulin for survival. Approximately 5-10% of

the diabetic population accounts with this type of diabetes mellitus.

Type II diabetes (T2DM) is a complex metabolic disorder, which accounts for

90% of the cases of diabetes. It is characterized by high blood glucose in context of



Chapter 1

Page | 13

relative insulin deficiency, reduced insulin action and insulin resistance of glucose

transport in skeletal muscle and adipose tissue.77 If left untreated, then

hyperglycaemia may cause long-term microvascular and macrovascular

complications, such as nephropathy, neuropathy, retinopathy and atherosclerosis.78

Prospective studies have shown that defects in both insulin secretion and

insulin action at target tissues are independent risk factors for T2DM. Nonetheless

insulin resistance is often the initial recognizable feature of the disease. In early

stages of the disease, insulin resistance in peripheral tissues, such as muscles and

fat, is associated with a compensatory increase in insulin secretion by pancreatic β-

cells. The secreted insulin promotes glucose utilization in peripheral tissues and

decreases hepatic gluconeogenesis.

During fasting, insulin level progressively increases in a stepwise fashion until

the β-cells can no longer compensate for the increased insulin resistance, and β-cell

failure ensues. Subsequent loss of pancreatic β-cells and insulin secretion results in

levels of hyperglycaemia defined as T2DM.

1.3 Treatment Approaches

The corner stone of the treatment of diabetes is lifestyle modification through

increased physical activity and attention to food intake, particularly among the obese

in whom weight loss is the principal goal. When lifestyle modification does not result

in normalization of metabolic abnormalities, pharmacotherapy is required.

Seven categories of oral hypoglycaemic agents are available in the market for

the treatment of type 2 diabetes and they are classified according to the mechanism

of their action (Table 2). In current scenario, the treatment of type 2 diabetes has

been revolutionized with the advent of insulin sensitizers like Rosiglitazone and

Pioglitazone that not only ameliorate insulin resistance, thereby normalize elevated

blood glucose levels but are also associated with hepatotoxicity, weight gain and

edema.79,80 The alarming situation emphasized the need to discover new

antihyperglycaemic agents with reduced or no side effects.

The development pipeline for new oral therapeutic agents for type 2 diabetes

is encouraging and continues to expand.81 These intensive researches and

developmental efforts are in response to increasing prevalence of the disease and

related co-morbidities, realization by care givers that successful glycaemic control will

likely require combination therapy, a growing understanding of the pathophysiology

of the diseases and the identification and validation of new pharmacological targets.82

These targets include various receptors and enzymes which exhibit mechanisms of



Chapter 1

Page | 14

action distinct from current therapies, thus could be beneficial in the treatment of

diabetes without having any major side effects.

Some of the new and emerging approaches due to their promise for future

clinical success and different mechanisms of action from existing therapies are

Table 2: Drug therapies used for the treatment of Type II diabetes

Class of Drug Drug Role in Blood Glucose

Management

Sulfonyl Ureas

1st Generations Acetohexamide, Chlopromazine,

Tolbutamide, Tolazamide

Insulin Secretagogues

2nd Generations Glibenclamide, Gluburide,

Glipizide

Insulin Secretagogues

3rd Generations Glimepride Insulin Secretagogues

Meglitinides (glinides) Repaglinide, Nateglinide Insulin Secretagogues

Biguanides Metformin, Phenformin Insulin Sensitizers

Thiazolidinediones (PPAR γ

agonists; glitazones)

Rosiglitazones, Pioglitazones Insulin Sensitizers and

decrease insulin resistance

Gliptins ( DPPIV inhibitors) Sitagliptin, Saxagliptin Insulin Secretagogues

Glucagon like peptides-1 (GLP 1) Exenatide, Liraglutide Insulin Secretagogues

α-Glucosidase inhibitors Acarbose, Miglitol, Voglibose Inhibitors of glucose uptake

depicted in Table 3. Protein Tyrosine Phosphatase 1B (PTP1B) has recently

emerged as a promising molecular level legitimate therapeutic target in the effective

management of type 2 diabetes.

1.3.1 Protein Tyrosine Phosphatase 1B

Protein tyrosine phosphatases (PTPs) constitute a large family of signaling

enzymes that control several fundamental cellular functions via phosphorylation and

dephosphorylation reactions.83,84 Deregulation of these PTPs activity can lead to a

number of diseases including cancer and diabetes.85,86 Thus, PTPs are considered as

viable targets for the design of novel therapeutics that are capable of inhibiting or

modulating activities of these crucial enzymes. 87

Among various members of the PTP superfamily, PTP1B, an intracellular

enzyme causing negative regulation of insulin receptors (IR) as well as leptin

signaling system, has emerged as promising legitimate therapeutic target for the

treatment of NIDDM and obesity.88-90 Two landmark papers reported that PTP1B

deficient mice are more sensitive to insulin, have improved glycaemic control and are

resistant to diet induced obesity.91,92



Chapter 1

Page | 15

Table 3: New approaches for the management of Type II diabetes

Target Role in blood glucose management

Glycogen synthase kinase-3 (GSK-3) inhibitors Activation of glycogen synthase

Peroxisome Proliferator-Activated Receptors

(PPAR) α/γ dual agonist

Insulin sensitizers

Na+ glucose co-transporter (SGLT) inhibitors Inhibits renal glucose absorption from urine

Hepatic Glucose Output (HGO)

inhibitors

Insulin sensitizers and decrease insulin resistance

β3-Adrenoreceptor agonists Decrease food consumption

Retinoid X receptor Controls lipid and carbohydrate metabolism

Protein tyrosine phosphates 1B (PTP1B)

inhibitors

Prevents dephosphorylation of activated insulin

receptors.

Furthermore, treatment of diabetic mice with PTP1B antisense

oligonucleotides reduced the expression level of this enzyme and subsequently

normalized blood glucose levels and improved insulin sensitivity.93,94 A PTP1B

inhibitor may provide a novel strategy for the treatment of type II diabetes and

obesity. Recent studies have shown that PTP1B also plays a role in tumorigenesis.

As a result, PTP1B inhibitors represent attractive pharmaceutical agents for treating

type II diabetes, obesity and cancer.95,96

Despite of the continuous efforts, no PTP1B inhibitor has successfully

completed the clinical trials. It may be due to the poor cell permeability and

bioavailability of these inhibitors. However, efforts are made to discover orally active

and selective PTP1B inhibitors which may also be useful for probing signal

transduction pathways as well as for the treatment of diabetes and obesity.

PTP1B: Structure and Biology

All of the PTPases possess at least one catalytic domains of approximately

240 amino acids which contains the active site signature sequence (H/V)C(X)R(S/T)

motif and the general acid containing surface loop.97 PTP1B is a member of the class

I cysteine-based PTPs in the classical nonreceptor PTP (NR-PTP) subfamily. The

native protein consists of 435 amino acid residues. Residues 30-278 correspond to

the catalytic domain, while the 35 C-terminal residues target the enzyme to the

cytosolic face of the endoplasmic reticulum.

It was the first PTPase isolated in pure form from human placental tissue 98

and structure elucidated by tungstate on bound X-ray crystallography in 1994.99 The

main structural feature of PTP1B is the catalytic site containing the catalytic residue

Cys215, the WPD (tryptophan, proline and aspartic acid) loop and the secondary aryl

phosphate binding site (Fig. 1).100



Chapter 1

Page | 16

Active Site

The active site of PTP1B is defined by the 214-221 PTP signature motif

[Histidine(His)-Cysteine(Cys)-Serine(Ser)-Alanine(Ala)-Glycine(Gly)-Isoleucine(Ile)-

Gly-Arginine (Arg) in PTP1B], a loop of eight aminoacid residues that form a rigid,

cradle like structure that coordinates to the aryl phosphate moiety of the substrate.

This loop also contains the active site nucleophile Cys215. Aspartic acid (Asp181),

phenylalanine (Phe182), tyrosine (Tyr46), valine (Val49), lysine (Lys120) and

glutamine (Gln262) form the sides of the catalytic cleft and contribute to catalysis and

substrate recognition.

The WPD Loop

PTP1B contains a WPD (tryptophan, proline, aspartic acid) loop (amino acids

79–187) which helps PTP1B to undergo conformational changes associated with its

catalytic activity. This loop moves up to 12 Å to close down on the phenyl ring of the

substrate and maximize hydrophobic interactions. Asp181 moves into a position in

which it can act as a general acid to protonate the tyrosyl leaving group.

Figure 1: A ribbon view of the crystal structure of the protein tyrosine phosphatase 1B

(PTP1B) highlighting the main regions of the protein.



Chapter 1

Page | 17

Secondary Aryl Phosphate Binding Site

PTP1B contains a second aryl phosphate binding site adjacent to the active

site101 demarcated by Arg254 and Arg24 which is catalytically inactive and provides

weaker binding interactions compared with the primary site. It is not conserved

among the PTPases and has important implications in designing selective and

specific inhibitors.

YRD Motif

Targeting interactions between Arg47 and Asp48, which are close to the

active site, has allowed selectivity to be achieved over most other PTPs. Arg47 and

Asp48 form a charged region at the top of the binding pocket of PTP1B. This region

is known as the YRD motif. By forming a salt bridge between Asp48 and an inhibitor

that contains a basic nitrogen in that region, it has been shown that selectivity can be

achieved over six other PTPs.100

Protein Tyrosine Phosphatase 1B in Diabetes

Insulin, on binding to its receptor, induces the activation of the insulin-receptor

kinase (IRK) through phosphorylation (Fig. 2). Recruitment of the insulin-receptor

substrate (IRS) proteins induces activation of phosphatidylinositol-3-kinase (PI3K)

through binding the p85 subunit and activating the catalytic p110 subunit. PI3K

activation induces downstream effectors, such as phosphatidyl inositol dependent

kinase 1 (PDK1) and protein kinase B (PKB; also known as AKT), leading to the

translocation of glucose transporter 4 (GLUT4) and glucose uptake in muscle and

inactivation of glycogen-synthase kinase 3 (GSK3). The binding of leptin to its obese

receptor (ObR) leads to phosphorylation of Janus Kinase 2 (JAK2), activating the

JAK/signal transducer and activator of transcription (STAT) pathway and possibly the

PI3K pathway through less well-defined mechanisms. Activation of STAT3 through

JAK2 phosphorylation induces translocation of STAT3 to the nucleus. STAT3

induces gene responses that reduce transcription of acetyl coenzyme-A carboxylase

(ACC), reducing malonyl CoA and fatty acid synthesis, while increasing fatty acid

oxidation. Endoplasmic reticulum bound or cytosolic PTP1B dephosphorylates

membrane bound or endocytosed insulin receptors and leptin receptors, causing

their deactivation. Therefore, any changes in expression levels or activity of PTP1B

relative to the insulin receptor could affect insulin signaling and possibly contribute to

the insulin resistance observed in type II diabetes.101



Chapter 1

Page | 18

Figure 2: Regulation of the leptin and insulin signaling pathways by PTP1B.

PTP1B Inhibitors

A number of small molecule inhibitors for PTP1B have been identified with

potential application in the treatment of type II diabetes. The development of

selective PTP1B inhibitors would certainly help in clarifying the role of PTP1B in type

II diabetes. This will be a daunting challenge due to the large number of PTP family

members and the conservation of the PTP catalytic domain, especially in the case of

T-cell PTPase (TCPTP), a phosphatase implicated in regulating T-cell activation,102

which has the highest homology to PTP1B with 74% sequence identity to PTP1B in

the catalytic domain and 100% sequence identity in the catalytic site.

Selective inhibition of PTP1B over TCPTP is therefore highly desirable and

represents one of the most challenging aspects for drug discovery. Indeed, only a

few groups have addressed this issue in a structure-based fashion, despite a large

number of PTP1B inhibitors reported in the recent years.103,104 Progress in developing

selective PTP inhibitors is being made, although most inhibitors described till date

are nonselective. A large number of chemical scaffolds belonging to peptides,

peptide mimetics and non-peptide class have been explored as selective inhibitors,



Chapter 1

Page | 19

however only a few have shown potential for further development and are described

here.

(A) DFMP phosphotyrosyl (pTyr) Mimetics

Burke et al.105 in 1992 made a major breakthrough in designing PTP1B

inhibitors by preparing phosphate based and difluoromethylenephosphonate (DFMP)

analogs as a non hydrolyzable phosphotyrosine mimetic. Among them, the

compound 62 is the most potent and selective PTP1B inhibitor identified till date (Ki

2.4 nM) with 10 fold selectivity over the highly homologous TCPTP. Unfortunately,

the highly polar nature of the bisanionic DFMPs and peptide scaffold provided

compounds with poor physicochemical properties and thus poor cell permeability and

low oral bioavailability.

The development of non-peptide scaffolds incorporating the DFMP group has

been pursued by several pharmaceutical companies. Merck Frosst’s106-108 efforts

have led to promising PTP1B inhibitor classes such as arylketone (63), benzotriazole

(64), and naphthyl (65) with improved potency (IC50 5 nM) and selectivity (7 fold) of

the dual binding bisDFMP, containing benzotriazole (64) compared to initially

nonselective monoDFMP leads 63 (IC50 120 nM) and 65 (IC50 120 nM) were

designed on the basis of crystallographic data to bind both the catalytic site and non-

catalytic aryl phosphate binding site (B-site).

Affymax109 has identified a sulfonamide scaffold bearing the DFMP (66) (IC50

28 nM) and a novel ketophosphonate pTyr mimetic 67 (IC50 600 nM) as PTP1B

inhibitors. Compound 66 is nonselective over TCPTP, while selectivity of compound

67 has not been reported. The presence of a thiadiazole ring and a DFMP group is

sufficient to afford potent inhibitors of PTP1B, and even more potent inhibitors result

from inclusion of an additional oxyacetic group into the molecules. The compound 68

emerged as the most potent compound of the series with IC50 and Ki at

submicromolar level.110

(B) Carboxylic Acid pTyr Mimetic:

Replacement of the phosphorus based mimetics with carboxylate based pTyr

mimetic was sought to improve the membrane permeability of inhibitors bearing

these pharmacophores. Dicarboxylic acid containing O-malonyltyrosine (OMT)

tyrosine111 and o-carboxy-(O-carboxymethyl) tyrosine112 peptide derivatives were the

first carboxylic acid containing pTyr mimetics published. Since these early findings,

several research groups have made significant advances in peptide and non-peptide



Chapter 1

Page | 20

carboxylic acid derivatives as potent PTP1B inhibitors. A lack of membrane

permeability remains an issue for this class of compounds.112

The Abbott research group113,114 used a fragment based NMR approach to

identify novel PTP1B inhibitors. The 2-oxalylarylaminobenzoic acid derivative (69)

demonstrated enhanced potency (IC50 18 nM) and TCPTP selectivity (3.6 fold). The

diacid class of inhibitors displayed reduced membrane penetration and cellular

activity presumably due to their bis anionic nature. Replacement of the oxamic acid

moiety with monoacids (70) and (71) provided micromolar inhibitors of PTP1B with

greater than 20 fold selectivity over TCPTP.115 They also identified a novel isoxazole

carboxylic acid (72), as a micromolar inhibitor (Ki 2.1 µM) with good selectivity (>15

fold).116

The 2-oxalylaminobenzoic acid (OBA) containing pTyr mimetic was

discovered by researchers at Novo Nordisk.117 Optimization of a thiophene based



Chapter 1

Page | 21

scaffold bearing the OBA provided compound 73 as a potent PTP1B inhibitor. These

diacid compounds lacked cellular activity.

Kyorin Pharmaceuticals118 has reported inhibitors bearing novel

hydroxypropionic acid pTyr mimetic (74) with micromolar potency. The Wyeth

group119 identified a dicarboxylic acid containing thiophene scaffold that after

extensive optimization led to the potent inhibitor 75 (Ki 5 nM). Unfortunately, 75

lacked cellular activity, so an effort was made to eliminate or replace one or more of

the carboxylic acid substituent with a variety of functional groups, including tetrazole.

All derivatives suffered a loss of several orders of magnitude in potency except for

the 1,2,5-thiadiazolidin-3-one-1,1-dioxide (TDZ) pTyr (76), with a heterocyclic

replacement for the phenoxyacetic acid. The TDZ pTyr mimetic was originally

reported by an Astra Zeneca group, 120 and the details of this discovery will be

discussed in the following section.

(C) Thiazolidinediones

During last decade, a new class of drugs called "glitazones" such as

Rosiglitazone (RSG) and Pioglitazone were approved by the FDA for the treatment of

type 2 diabetes. These agents share a common molecular scaffold: 2,4-

thiazolidinediones (TZDs).Various studies have suggested that existing TZDs could

inhibit PTP1B. It was postulated that high fat feeding increases the levels of muscle

PTP1B in mice increasing the possibility that the antidiabetic effects of RSG involves

decreasing muscle PTP1B levels. The inhibitory effect of RSG on PTP1B activity is

more likely mediated by reducing PTP1B protein expression in skeletal muscle and

liver.120

Maccari and co-workers121 demonstrated TZD scaffolds containing arylidene

moiety at position 5 with significant inhibitory effects against human PTP1B (h-

PTP1B) enzymes. Benzoic acid could act as non-phosphorus containing pTyr mimic

therefore it was considered of interest to insert the p-methylbenzoic acid residue at

N-3 of the TZD scaffold. It has also been reported that carboxylic group mimics the

interactions of the phosphate group of pTyr with Arg221 in the PTP1B catalytic site

while the benzene ring might interact with Phe182 and Tyr46. Among the series

compound 77 proved to be most effective at micromolar level against h-PTP1B.

Further Maccari et al.122 performed optimization by introducing second

carboxyl group at benzyloxybenzylidene moiety of TZD scaffold. It is considered that

this second pTyr mimic group could enhance the affinity of these bidentate inhibitors

for the secondary non-catalytic pocket of PTP1B. Compound 78 proved to be the



Chapter 1

Page | 22

CO2H

CO2H

O

Et

NH

O

O

O O

OH

NHAc

(69), IC50 18nM

HO

NH

O

O

O O

OH

NHAc

O COOH

(70), Ki 9 M

NH

O

O

O O

OH

NHBoc

O COOH

OHN

HO

O OMe

O

N

ONH2

COOH

NH

S

HN

O

HN

HN

O

CO2HHOOC

(71), Ki 8.4 M (72), Ki 2.1 M (73), Ki 0.018 M

CO2H

HO CF3

N

S

Cl

S

OCO2H

HO2C

Br

NH

NS

OO

S

NH

NS

OO

N

S

HN O

O

O

(74), IC50 3.2 M (75), Ki 5 M (76), Ki 4.3 M

most potent PTP1B inhibitor with sub-micromolar IC50 value against h-PTP1B.

Molecular docking studies also indicated that this compound interact with amino acid

residues involved in the affinity and selectivity of inhibitors towards this enzyme.122

Liu et al.123 synthesized a biphenyl thiazolidinone library and found a series of

novel PTP1B inhibitors that exhibited submicromolar potency. Among these the

compounds, 79 was tested in an animal model for its efficacy as an antidiabetic

agent. Bhattarai et al.124 identified TZD scaffolds as PTP1B inhibitors by computer

aided drug design protocol involving virtual screening. A series of benzylidene-2, 4-

thiazolidinedione derivatives with substitutions on the phenyl ring at



Chapter 1

Page | 23

N

SO

O

HO2C

O

N

SO

O

HO2C

O

CO2H

HN S

O

O O

CF3

BrSNH

O

O

O

NS

N

CO2H

O

O

NS

N

CO2H

O

O

F3C

(77), IC50 1.6 M(78), IC50 0.24 M

(79), IC50 1.6 M(80), IC50 5.0 M

(81), IC50 1.1 M(82), IC50 1.4 M

the ortho or para positions of the thiazolidinedione (TZD) group were reported with

IC50 values in a low micromolar range. The compound 80 was the most potent

inhibitor of PTP1B with an IC50 value of 5.0 µM.

A further significant modification was reported by Ottana et al.125 where

replacement of carbonyl group at position 2 of the 2, 4-thiazolidinedione scaffold with

a phenylimino moiety resulted in the development of 5-arylidene-2-phenylimino-4-

thiazolidinones as potent inhibitors of h-PTP1B. Enhancement of the

inhibitor/enzyme affinity is attributed to the favorable interactions with residues at the

active site and the surrounding loops particularly with the WPD loop. Among the

series, the compound 81 was the most active with IC50 1.1 µM.

Recently, Ottana and colleagues126 demonstrated that the negatively charged

compound 82 inhibited PTP1B in vitro with an IC50 of 1.4 µM, and was able to

increase IR Tyr phosphorylation and glucose uptake in cultured myotubes.



Chapter 1

Page | 24

Aher et al.73 synthesized a series of thiazolidinone derivatives and evaluated

the inhibitory activity against PTP1B. Most of the compounds behaved as inhibitors of

PTP1B with the lowest IC50 of 1.4 µM observed with 62. In contrast to the in vitro

efficacy of 62 as a potent PTP1B inhibitor, but has no effect as an in vivo antiobesity

or hypoglycaemic agent.

(D) Isothiazolidinone derivatives:

Combs et al.127,128 reported the discovery of novel heterocyclic (S)-

isothiazolidinone [(S)-IZD] as phosphotyrosine (pTyr) mimetics using structure based

drug design approach. It was observed that incorporation of heterocycles into

dipeptides resulted in exceptionally potent, competitive and reversible inhibitors of

PTP1B. The isothiazolidinone heterocycle was chosen because it allowed the two

sulfonyl oxygens to effectively mimic the oxygens of the DFMP inhibitor, while the

carbonyl mimicked the carboxymethyl salicylic acid (CMS) inhibitor, carbonyl and the

ionized NH mimic the carboxylic anion or DFMP anion. The compound 83, which

contains a isothiazolidinone group, is also an excellent pTyr mimetic128 when

incorporated into a dipeptide structure, the isothiazolidinone containing inhibitor 83

has a Ki of 0.19 µM. Using the isothiazolidinone group as the pTyr mimetic, a peptide

based inhibitor, 84 (IC50 40 nM) was synthesized. This demonstrates the utility of the

isothiazolidinone to serve as a highly efficacious pTyr mimetic.129

Sparks et al.130 reported a unique combination of benzothiazole

benzimidazole (S)-isothiazolidinone derivatives through a peptidomimetic

modification of the peptidic (S')-IZD inhibitors. These derivatives are potent,

competitive and reversible inhibitors of PTP1B. X-ray co-crystal structure of PTP1B

with compound 85 (IC50 270 nm) demonstrated that the benzothiazole benzimidazole

forms bidentate H-bonds to Asp48, and the benzothiazole interacts with the surface

of the protein in a solvent exposed region towards the C-site.

The benzothiazole binds toward the C-site above Tyr46. The nitrogen lone

pair of the benzothiazole although forms a hydrogen bond to a water molecule but is

otherwise solvent exposed on the surface of the enzyme. Caco-2 permeability was

significantly enhanced for this series of non-peptidic benzothiazole benzimidazole

IZD inhibitors presumably due to the lower polar surface area (PSA).

Douty et al.131 further reported isosteric replacements of the benzimidazole

with imidazoles and imidazolines as potent inhibitors of PTP1B. Both heterocycles

maintained the critical hydrogen bonding interactions with Asp48 of PTP1B through

the nitrogen atoms which were found to be necessary for high afinity binding. Two



Chapter 1

Page | 25

highly potent PTP1B inhibitors 86 and 87 having IC50 value 32 and 22 nM

respectively interact with the B site.

Recently, novel sulfathiazole related compounds were designed as PTP1B

inhibitors based on a previously reported allosteric inhibitor 88 of PTP1B by Tang et

al.132 These compounds were synthesized and evaluated against human recombinant

PTP1B. The most active compound 89 showed IC50 value of 3.2 µM and kinetic

analysis indicated that it is a non-competitive inhibitor of PTP1B. Furthermore,

compound 88 demonstrated excellent selectivity to PTP1B over other PTPs. It also

displayed in vivo insulin sensitizing effect in the insulin resistant mice.

NH2

S NH

HN

O

O

O

OAcHN

O

HN

S NH

C5H11

HN

O

O

O

OHN

OO

O

N

HN

S NHO

O

O

(83), Ki 0.19 M (84), IC50 40 nM (85), IC50 270 nM

S

N

N

HN

S NHO

O

O

HN

S

O OF

O

HO2COH

N

HN

S NHO

O

O

HN

S

O OF

O

HO2COH

(86), IC50 32 nM (87), IC50 22 nM

S

NH

N

S

NHO

O SO

O

O

O

Br

OH

Br

S

NH

N

S

NHO

O

O

OCH3

O

(88), IC50 40 nM (89), IC50 3.2 M

(E) α-Ketocarboxylic acid derivatives:

To improve bioavailability, efforts were directed towards the replacement of

phosphate with the less charged, non-phosphorus containing functional groups. The

glycosyl α-ketocarboxylic acids were identified as promising sugar-based PTP1B

inhibitors for the first time with at least several fold selectivity over a panel of

homologous PTPs by Song et al.133



Chapter 1

Page | 26

In addition, docking study plausibly proposed a typical competitive inhibitory manner

of ketoacid (90) with the enzymatic target

(F) Isoxazole carboxylic acid analogues:

Isoxazole carboxylic acid analogues, oxamic acid mimics, were identified as

potent and selective PTP1B inhibitor from X-ray crystallographic study and NMR-

based screening. The compound 91 (Ki 148 µM) gave slightly weak inhibitory activity

against PTP1B and a poor selectivity against TCPTP. After tailored with a salicylic

motif, two derivatives 92 (Ki 5.7 µM) and 93 (Ki 6.9 µM) showed greater than 30 fold

selectivity over TCPTP, as well as inhibitory activity against LAR, CD45, Cdc25, and

SHP-2 at the concentration of 300 µM. Since novel isoxazole carboxylic acid based

PTP1B inhibitors gave potent inhibitory activity, selectivity and cell permeability,

some useful transformations of the linker and the substitution on the isoxazole ring

were conducted to get an improvement of interaction with active site in PTP1B.

Introduction of substituent in isoxazole as 4-amino (94) (Ki 2.1 µM) and 4-

hydroxymethyl (95) (Ki 0.92µM) provided more active and selective inhibitors over

TCPTP.134

NH

O

O

N

HOOC

CH

O

O

N

HOOC

RCOOMe

OH

(91) Ki 148 M (92) R=H, Ki 5.7 M

(93) R=F, Ki 6.9 M

CH

O

O

N

HOOC

COOMe

OH

R

(94) R=NH2, Ki 2.1 M

(95) R=CH2OH, Ki 0.92 M

4

Recently Basu et al.135 used heterocyclic carboxylic acid derivative as a pTyr

mimetic to synthesize compound 96 with improved cell permeability of PTP1B

inhibitors by reducing the number of negative charges. The compound 96 showed

high in vitro potency (Ki 0.3M) and acceptable pharmacokinetic properties. The

compound 96 was synthesized by replacement of highly negatively charged DFMP in



Chapter 1

Page | 27

Merck Frost’s106 compound with neutral isoxazole and addition of more rigid

oxadiazole to achieve better binding to PTP1B.

(G) Benzofuran/Benzothiophene derivatives:

Modeling studies using the X-ray crystal structure of hepatic PTP1B led to the

synthesis of the compound 97 and its structural modifications like the replacement of

the butyl group at position 2 of benzofuran with benzyl led to the most active

inhibitors of both the series which showed IC50 values in the range of 20-50 nM

against recombinant human PTP1B. The (2S)-2-[4'-(2-benzylbenzofuran-3-yl)-biphe-

-nyl-4-yloxy]-3-phenyl propionic acid trimethamine salt (97) emerged as the most

potent compound in in vivo studies with normalizing plasma glucose levels at the

25mg/kg dose (p.o.) and the 1 mg/kg dose (i.p.). It also demonstrated 10 to 100 fold

selectivity against other homologous PTPases and was least selective (10 fold) over

LAR.136

Ertiprotafib (98) a monocarboxylic acid benzothiophene phosphotyrosine

mimetic (Ki 384 nM) was developed by Wyeth laboratory137 as a drug for the

treatment of type II diabetes, but its further development was stopped in 2002 at

phase II clinical trials due to unsatisfactory clinical efficacy and the occurrence of

dose limiting side effects. Ertiprotafib has also been shown to be a PPAR modulator

and some of its biological effects may be linked to this activity.



Chapter 1

Page | 28

(H) Oxadiazolidinedione derivatives:

A large number of oxadiazolidinediones have been examined as PTP1B

inhibitors. The compounds 99-101 were the potent inhibitors with sub-micromolar

IC50 values. The meta-substituted oxadiazolidinedione (101, IC50 0.2 µM) has a

better in vitro profile when compared to 99 (IC50 0.3 µM) and 100 (IC50 0.4

µM).138

O

NO

NNH

O

O

O

F3C

O

NO

NNH

O

O

O

F3C

n-octyl

(100), IC50 0.4 M

O

NO

NNH

O

O

OF3C

n-octyl

(101), IC50 0.2 M

(99), IC50 0.3 M

(I) Pyridazine Derivatives:

A series of novel pyridazine derivatives were tested which showed a

moderate to strong inhibitory activity against PTP1B. The high throughput screening

(HTS) of these compound led to the novel reversible PTP1B inhibitor 102 (IC50 5.7

µM). It’s O-ethyl and O-butyl derivatives 103 and 104 respectively were among the

most active PTP1B inhibitors which were not selective over other PTPases.

However, several compounds in this series showed 6–10 fold selectivity against

PTP1B over TCPTP. Among them, the compound 105 (IC50 5.6 µM) showed a 20

fold selectivity for PTP1B over both LAR and TCPTP. The compounds 102 and 103

also prolonged the activated state of insulin receptor in an in vitro cellular assay and

showed increased effect on insulin receptor phosphorylation.139



Chapter 1

Page | 29

(J) Sulfamic acids and Sulfonamide derivatives:

Klopfenstein et al.140 in 2006 identified novel tetrahydroisoquinolinyl sulfamic

acids as PTP1B inhibitors by HTS. Among them, the compound 106 was developed

based on hits identified from a compound collection with an IC50 of 42.5 µM and four

fold selectivity compared with TCPTP.140 Incorporation of the second sulfamic acid in

107 resulted in 13 fold increase in potency. The second aryl sulfamic acid showed

good interaction with the Arg24 and Arg254. The compound 107 was screened

against a panel of 14 PTPases. High selectivity was observed against several other

therapeutically useful phosphatases. Unfortunately, selectivity over the closely

homologous TCPTP was poor.

Navarrete-Vazquez et al.141 synthesized a small library of arylsulfonylamino

benzothiazoles and evaluated these compounds for their PTP1B inhibitory activity.

The compound 108 is a rapid reversible (mixed type) inhibitor of PTP1B with IC50

values in the low micromolar range. The most active compound 108 was docked into

the crystal structure of PTP1B. Docking results indicated potential hydrogen bond

interactions between the nitro group in the compound and the catalytic amino acid

residues in Arg221 and Ser216.

Huang et al.142 discovered a novel series of trifluoromethyl sulfonyl and

trifluoromethyl sulfonamido compounds as PTP inhibitors, where compound 109 was

found to be the most active compound of the series and made good interactions with

both catalytic and non-catalytic site.

N

NH

O

O

O

NH

SO3H

(106), IC50 42.5 M

S

N

NH

SO

O

NO2

(108), IC50 19.5 M

O

NH

S

OO

OH

O

NH

HN

SHO

O

O

(107), IC50 4.8 M

O

O NHS CF3

O O

NH

S

F3C

OO

(109), IC50 11 M



Chapter 1

Page | 30

(K) Quinoline/Naphthalene-difluoromethylphosphonates:

Yongxin Han et al.143 reported a series of quinoline/naphthalene

difluoromethyl phosphonates as potent PTP1B inhibitors. Most of these compounds

bearing polar functionalities or large lipophilic residues did not show appreciable oral

bioavailability in rodents while small and less polar analogues displayed moderate to

good oral bioavailability.

(L) Vanadium Compounds:

For efficient inhibition of PTP1B, the inhibitors must contain non hydrolysable

phosphotyrosine (pTyr) mimetic, which will competitively occupy the high affinity

catalytic site of PTP1B. To achieve that, inhibitors should carry polar groups which

are negatively charged at physiological pH. Vanadate was among the first

phosphotyrosine mimetics used to inhibit PTP1B in order to potentiate insulin

signalling in the treatment of T2DM. Recently, several N3O-coordinated oxovanadium

(IV) complexes with mixed ligand including a tridentate salicylaldehyde anthranilic

acid derivative and a bidentate polypyridyl ligand were identified as PTP1B inhibitors

using structure based design.144 Compound 110 inhibited PTP1B with IC50 value of

42 nM which is over 2 fold lower than orthovanadate (IC50 90 nM). Molecular docking

studies suggest that the phenolate oxygen of the complex 110 is close to sulphur

atom of the Cys215 in the active site whereas vanadyl oxygen forms hydrogen bonds

with Arg221. Oxovanadium complexes showed 2 fold selectivity over TCPTP,

however further improvement of selectivity via a structure based design is necessary

for these agents to be of clinical benefit.

N

N

V

O

O

N

O

O

Br

(110)

PTP1B inhibitors have emerged a legitimate approach for the management of

diabetes mellitus and obesity i.e."diabesity". Researches during the past decade

have identified a number of PTP1B inhibitors as promising candidates for the

treatment of obesity and T2DM. Design of PTP1B inhibitors is complicated by the

strong homology between PTPs, requiring a high selectivity for PTP1B inhibitors over



Chapter 1

Page | 31

closely related PTPs. Furthermore, development of PTP1B inhibitors is challenging

because of the highly charged nature of the catalytic pocket of the enzyme.

A principal focus of the most research effort to date has centered on the

design of inhibitors which interact both with the catalytic as well as the secondary

binding pockets of PTP1B. The discovery of potent small molecule inhibitors that

incorporate thiazole containing TDZ and (S)-IZD pTyr mimetics has provided the

most potent pTyr mimetics class of PTP1B inhibitors reported to date.73,120-132

The future of successful PTP1B inhibitors suitable for clinical trials is likely the

bidentate inhibitors, interacting with both the catalytic and secondary sites of PTP1B

and carrying side chain modifications for improved bioavailability. The wide variety of

information available till date indicates that this search is worthwhile and identification

of a hit would have a marked impact in combating global epidemic of diabesity.

Taken as a whole, these advancements in the field of phosphatase drug

discovery have provided direction to medicinal chemists pursuing these challenging

drug targets and hope that a pharmacological inhibitor of PTP1B suitable for human

clinical trials may emerge.

Thus, thiazole is considered a suitable pharmacophore in the medicinal

chemistry research and different modifications on thiazole moiety has displayed a

wide range of valuable biological activities. Therefore, analyzing the biological

importance of the thiazole moiety, we decided to choose the thiazole nucleus as the

core unit for constructing our target molecules. Moreover, many thiazole derivatives

are reported as antidiabetic agents in literature, 67-73 as shown in Table 1 and also

considering diabetes as the major human health concern, it was thought to

synthesize thiazole derived molecules and antidiabetic screening.

Further, considering the importance of thiazole as PTP1B inhibitors in

diabetes, the synthesis, structure activity relationship and docking studies of

substituted aryl thiazolyl phenylsulfonamides and substituted aryl phenylthiazolyl

phenylcarboxamide as potential Protein Tyrosine Phosphatase 1B Inhibitors were

carried out and screened for PTP1B inhibitory and antidiabetic activity. The results

are reported in following parts two:

Part 1A: Synthesis, Structure Activity Relationship (SAR) and Docking Studies of

Substituted Aryl thiazolyl phenylsulfonamides as potential Protein Tyrosine

Phosphatase 1B Inhibitors.

Part 1B: Synthesis, Structure Activity Relationship (SAR) and Docking Studies of

Substituted Aryl phenylthiazolyl phenylcarboxamide as potential Protein Tyrosine

Phosphatase 1B Inhibitors.

Synthesis, Structure Activity Relationship (SAR) and Docking

Studies of Substituted Aryl thiazolyl phenylsulfonamides as

potential Protein Tyrosine Phosphatase 1B Inhibitors

Part 1A

Page | 32

Part 1A: Synthesis, Structure Activity Relationship (SAR) and Docking Studies of Substituted Aryl thiazolyl phenyl sulfonamides as potential Protein Tyrosine Phosphatase 1B Inhibitors

Protein tyrosine phosphatase 1B (PTP1B) is a typical member of protein

tyrosine phosphatase superfamily and its inhibition results both in increased insulin

sensitivity and resistance to obesity with no abnormalities in growth, fertility or other

pathogenetic effects,91,92 hence PTP1B is considered to be an excellent target for the

treatment of type 2 diabetes and obesity.86,100 Efforts are being made to discover

orally active and selective PTP1B inhibitors that could be useful for probing signal

transduction pathways as well as for the treatment of diabetes and obesity.142

Several active PTP1B inhibitors reported in recent years include

difluoromethylene phosphonate (DFMP) pTyr mimetics (62),145 arylketone DFMP

(63),106 benzotriazole DFMP (64),107 naphthyl DFMP (75),108 dicarboxylic acid

containing O-malonyltyrosine (OMT) (71)111 and o-carboxy-(O-carboxymethyl)-

tyrosine peptide (70),112 benzoic acid derivatives (73),117,133 hydroxypropionic acid

pTyr mimetics (74),118 dicarboxylic acid containing thiophene scaffolds (75),119

thiadiazolidinedione (TDZ) and thiazolidinediones scaffolds (82),144 unsaturated

isothiazolidin-3-(2H)-one-1,1-dioxide and saturated isothiazolidin-3-one-1,1-dioxide

(IZD) pTyr mimetics,128 sulfonamideimidazoles (86,87),131 aryl trifluoromethyl

sulfones, aryl trifluoromethyl sulfonamide pTyr mimetics (109),142 aryl sulfamic acids

(107)140 and 2-arylsulfonylaminobenzothiazoles (108).141

Most of the inhibitors containing multiple charged phosphate mimicking

components cannot be developed into effective drugs because of their low cell

permeability and bioavailability. In view of this and considering the present PTP1B

inhibitors with different scaffolds containing thiazole moiety, a series of twenty eight

novel aryl thiazolyl phenylsulfonamides were synthesized and screened for PTP1B

inhibitory activity (Table 4). The design of these molecules were based on the

observation that the sulfonyl moiety in different scaffolds such as aryl trifluoromethyl

sulfone and aryl trifluoromethyl sulfonamide pTyr mimetics142 and arylsulfamic acid 140

interacts with the catalytic A site (Ile221, Ala217, Gln262), the non-catalytic B site

(Arg254, Arg24, Tyr20) and the C site (Arg47, Asp48, Phe182). In addition

sulfonamides are known to function as good hydrogen acceptors in biological

systems.




Part 1A

Page | 33

1A.1 Results and Discussion

Using the fragment based approach, twenty eight new compounds containing

a sulfonyl moiety were designed, synthesized and evaluated for their in vitro PTP1B

inhibitory and in vivo antidiabetic (STZ model) activities. Since the synthesized

compounds show promising PTP1B inhibitory activity, docking simulations of

selected compounds were also performed to analyze the potential binding mode of

these compounds. The results of these studies are presented herein.

1A.1.1 Chemistry

All the new compounds 114a-114i, 115a-115k and 116a-116h were

synthesized from the cyclized intermediates (113a-113c) according to literature

procedure.146 The 4-fluoroaniline (111a), on reaction with benzoyl isothiocyanate in

dry benzene, resulted in the formation of N-(4-fluorophenylcarbamothioyl)benzamide

(111d), which upon alkaline hydrolysis gave 1-(4-fluorophenyl)thiourea (112a).

Condensation of the phenylthiourea (112a) with 2-bromoethylamine in presence of

hydrogen bromide afforded N-(4-fluorophenyl)-4,5-dihydrothiazol-2-amine (113a),

which on reaction with 1-naphthalenebenzenesulfonylchlorides gave the desired

compounds N-(4,5-Dihydrothiazol-2-yl)-N-(4-fluorophenyl)napthalene-1-sulfonamide

(114a) (Scheme 1).

The key intermediate 113b and 113c were synthesized from 111b and 111c

respectively, as shown in Scheme 1. All other final compounds 114b-114i, 115a-

115k and 116a-116h were synthesized by the same procedure as described above

for the synthesis of 114a from key intermediates 113a, 113b and 113c respectively

(Table 1). The compound 114b-114i were synthesized by using 113a and 2,5-

dichlorobenzenesulphonylchloride, 4-nitrobenzenesulphonylchloride. 2,4,6-tri-

isopropylbenzenesulphonylchloride, 2-naphthalenesulphonylchloride, 2-trifluorometh-

-ylbenzenesulphonylchloride, 2-thiophenesulphonylchloride, 4-methoxybenzenesulp-

-honylchloride and 4-fluorobenzenesulphonylchloride respectively. The compound

115a-115k were synthesized in the similar manner using 113b and 1-naphthalenesu-

-lphonylchloride, 4-methoxybenzenesulphonylchloride, 3-trifluoromethylsulphonylchl-

-oride, 2-thiophenesulphonylchloride, 2,4,6-triisopropylbenzenesulphonylchloride, 2-

nitrobenzenesulphonylchloride, 4-nitro benzenesulphonylchloride, 2-naphthalenesul-

-phonylchloride, benzene sulphonylchloride, 2,5-dichlorobenzenesulphonylchloride

and 4-fluorobenzenesulphonylchloride respectively. The compound 116a-116h were

synthesized by using 113c and 4-methoxybenzenesulphonylchloride, 2-trifluorometh-




Part 1A

Page | 34

-ylbenzenesulphonylchloride, 4-nitrobenzenesulphonylchloride, 2,5-dichlorobenzene-

-sulphonylchloride, 2,4,6-triisopropylbenzenesulphonylchloride, 4-fluorobenzenesulp-

-honylchloride, 2-naphthalenebenzenesulphonylchloride and p-toluenesulphonylchlo-

-ride respectively.

All the final synthesized compounds 114a-114i, 115a-115k, 116a-116h and

their intermediates were characterized by spectroscopic techniques. All the spectra

were in agreement with the assigned structures. The compound 112a showed a

sharp singlet at δ 8.30 ppm for the NH2 group of thiourea which was found absent

when the compound 112a was cyclized to dihydrothiazolyl amine (113a). In the

infrared (IR) spectra the NH vibrations of secondary amine in the key intermediate

113a in the region 3456 cm-1 was also found absent in the compound N-(4,5-

Dihydrothiazol-2-yl)-N-(4-fluorophenyl)napthalene-1-sulfonamide (114a) and a sharp

peak for sulphonamide group in region 1352 cm-1 was observed confirming the

attachment of sulfonyl group to the secondary amine in 113a. Further, the 1H-NMR

spectra of key intermediate compound 113a showed two triplets that belong to CH2

protons of the thiazolyl groups in position 4 and 5 in the region δ 3.35 and δ 3.88

ppm. These peaks were shifted between δ 3.29- 4.49 respectively in the final phenyl

sulphonamide derivative (114a). Finally, the structure of 114a was confirmed by its

mass spectrometry which showed molecular ion peak (M+1) at 387.

i ii

iii

iv

112a-c

113a-c

NH2

N N

S

S O

NH NH

S O

NH NH2

S

NH N

S

R3

O

Scheme 1. Reagents and conditions (i) C6H5CONCS, dry benzene (ii) 10 % NaOH (iii)

Br(CH2) NH2, HBr, alcohol, distilled water (iv) R3SO2Cl, TEA, dry acetone.

111a, R=H, R1= F, R2=H

111b, R=OMe, R1= H, R2=OMe

111c, R=CF3, R1= H, R2=H

R

R1

R

R1

R

R1

R

R1

R2 R2 R2

R2

R

R2

R1

111d, R=H, R1= F, R2=H

111e, R=OMe, R1= H, R2=OMe

111f, R=CF3, R1= H, R2=H

112a, R=H, R1= F, R2=H


112c, R=CF3, R1= H, R2=H

111d-f111a-c

113a, R=H, R1= F, R2=H


113c, R=CF3, R1= H, R2=H

114a-i,115a-k,116a-h

114a, R= H, R1= F, R2= H, R3= 1-naphthyl

4

5

4

5




Part 1A

Page | 35

Similarly, all other compounds 2,5-Dichloro-N-(4,5-dihydrothiazol-2-yl)-N-(4-

fluorophenyl)benzenesulfonamide (114b), N-(4,5-Dihydrothiazol-2-yl)-N-(4-fluorophe-

-enyl)-4-nitrobenzenesulfonamide (114c), N-(4,5-Dihydrothiazol-2-yl)-N-(4-fluoroph-

-enyl)-2,4,6-triisopropylbenzenesulfonamide (114d), N-(4,5-Dihydrothiazol-2yl)-N-(4-

fluorophenyl)napthalene-2-sulfonamide (114e), N-(4,5-Dihydrothiazol-2-yl)-N-(4-fluo-

-rophenyl)-2-(trifluoromethyl)benzenesulfonamide (114f), N-(4,5-Dihydrothiazol-2-yl)-

N-(4-fluorophenyl)thiophene-2-sulfonamide (114g), N-(4,5-Dihydrothiazol-2-yl)-N-(4-

fluorophenyl)-4-methoxybenzenesulfonamide (114h), N-(4,5-Dihydrothiazol-2-yl)-4-fl-

-uoro-N-(4-fluorophenyl)benzenesulfonamide (114i), N-(4,5-Dihydrothiazol-2-yl)-N-

(2,5-dimethoxyphenyl)naphthalene-1-sulfonamide (115a), N-(4,5-Dihydrothiazol-2-yl)

-N-(2,5-dimethoxyphenyl)-4-methoxybenzenesulfonamide (115b), N-(4,5-Dihydrothi-

-azol-2-yl)-N-(2,5-dimethoxyphenyl)-2-(trifluoromethyl)benzenesulfonamide (115c),

N-(4,5-Dihydrothiazol-2-yl)-N-(2,5-dimethoxyphenyl)thiophene-2-sulfonamide (115d),

N-(4,5-Dihydrothiazol-2-yl)-N-(2,5-dimethoxyphenyl)-2,4,6-triisopropylbenzenesulfon-

-amide (115e), N-(4,5-Dihydrothiazol-2-yl)-N-(2,5-dimethoxyphenyl)-2-nitrobenzenes-

-ulfonamide (115f), N-(4,5-Dihydrothiazol-2-yl)-N-(2,5-dimethoxyphenyl)-4-nitrobenz-

-ensulfonamide (115g), N-(4,5-Dihydrothiazol-2-yl)-N-(2,5-dimethoxyphenyl)naphth-

alene-2-sulfonamide (115h), N-(4,5-Dihydrothiazol-2-yl)-N-(2,5-dimethoxyphenyl)be-

-nzenesulfonamide (115i), 2,5-Dichloro-N-(4,5-dihydrothiazol-2-yl)-N-(2,5-dimethoxy-

-phenyl)benzenesulfonamide (115j), N-(4,5-Dihydrothiazol-2-yl)-N-(2,5-dimethoxyph-

-enyl)-4-fluorobenzenesulfonamide (115k), N-(4,5-Dihydrothiazol-2-yl)-4-methoxy-N-

(2-(trifluoromethyl)phenyl)benzenesulfonamide (116a), N-(4,5-Dihydrothiazol-2-yl)-2-

(trifluoromethyl)-N-(2-(trifluoromethyl)phenyl)benzenesulfonamide (116b) N-(4,5-

Dihydrothiazol-2-yl)-4-nitro-N-(2(trifluoromethyl)phenyl)benzenesulfonamide (116c),

2,5-Dichloro-N-(4,5-dihydrothiazol-2-yl)-N-(2-(trifluoromethyl)phenyl)benzenesulfona-

-mide (116d), (4,5-Dihydrothiazol-2-yl)-2,4,6-triisopropyl-N-(2-(trifluoromethyl)pheny-

-l)benzenesulfonamide (116e), N-(4,5-Dihydrothiazol-2-yl)-4-fluoro-N-(2-(trifluoromet

hyl)phenyl)benzenesulfonamide (116f), N-(4,5-Dihydrothiazol-2-yl)-N-(2-(trifluoromet

hyl)phenyl)naphthalene-2-sulfonamide (116g) and N-(4,5-Dihydrothiazol-2-yl)-4-met-

-hyl-N-(2-(trifluoromethyl)phenyl)benzenesulfonamide (116h) were characterized on

the basis of corresponding spectral data. The spectral data are reported in

experimental part of this chapter.




Part 1A

Page | 36

1A.1.2 In vitro and Structure Activity Relationship Study

In order to fully explore the structure activity relationships associated with the

PTP1B inhibitors, we sought to probe hydrophobic, steric and electronic

requirements by efficiently and systematically installing R1 and R2 substituents on the

core aryl thiazolyl phenylsulfonamide scaffold. A series of twenty eight substituted

aryl thiazolyl phenylsulfonamides were synthesized and evaluated against PTP1B

using a colorimetric, non-radioactive PTP1B assay (Table 4).

Among these, the most active compound 116a with R as trifluoromethyl, R1,

R2 as H and 4-methoxyphenyl group at R3 showed good in vitro inhibitory activity with

73% inhibition at 10 µm (IC50 1.08 µm). Replacement of the trifluoromethyl group at R

position with H, R1 position by fluoro group resulted in compound 114h, with slight

decreased activity (IC50 2.96 µm). Insertion of R, R2=OMe, R1=H, and a 4-

methoxyphenyl group at R3 to give compound 115b demonstrated a further decrease

in activity, exhibiting only 48.5% inhibition of PTP1B in vitro.

Among the series of compounds 116a-116h prepared keeping same

substituents for R=H, R1=fluoro and R2=H respectively and altering only R3 with a

2,5-dichlorophenyl in compound 116d, a 4-fluorophenyl in compound 116f and a p-

toluene group in compound 116h showed 50% inhibition against PTP1B at 10 µm.

However, replacement of the trifluoromethyl group at R with OMe and insertion of

OMe group at R2 position and different substituents at R3 position gave compounds

115a-115k, which resulted in good PTP1B inhibitory activity (Table 4). Among these

compounds, the compound 115k with 4-fluorophenyl ring at the R3 position showed

almost 69% inhibition against PTP1B (IC50 1.46 µm). Assessment of these

compounds indicated that derivatives with an ortho substituent at the R3 position

showed potent inhibition. In order to support this hypothesis, compounds substituted

at the R3 position with a 2-thiophene (115d), 2,4,6-isopropylphenyl (115e), 2-

nitrophenyl (115f), 2-naphthyl (115h) or a 2,5-dichlorophenyl (115j) group were

synthesized and evaluated. Interestingly, most of these compounds showed more

than 50% PTP1B inhibition at 10 µm and the compound 115f was the second most

active derivative of the series with 72.2% inhibition (IC50=1.25 µm). Replacement of

the R=H and R1, R2=OMe in these compounds by R=H, R1=F, R2= H group led to

compounds 114a–114i with decreased activity, with the exception of compounds

114c and 114h, which showed 66.3% and 59.48% PTP1B inhibition respectively,

compared with 2,5-dimethoxy analogues 115g and 115b with 46.41% and 48.5%




Part 1A

Page | 37

Table 4. In vitro PTP1B enzyme inhibitory and in vivo antihyperglycaemic activity in STZ model for compound (114-116)

Compd.

R

R1

R2

R3

% inhibition at 10 µM

% lowering on Blood glucose

levels (100 mg/kg) 0-5h 0-24h

114a H F H 1-Naphthyl 7.60% - -

114b H F H 2,5-diClPh 2.9 - -

114c H F H 4-NO2Ph 66.3% (1.7)a 20.6** 18.8*

114d H F H 2,4,6 tri-isoPrPh 56.9% (9.09) a 11.1 6.43

114e H F H 2-Naphthyl 51.28% (9.65) a 13.0 11.43

114f H F H 2-CF3Ph 3.9 - -

114g H F H C4H4S 48.20 17.3* 9.15

114h H F H 4-MeOPh 59.48 (2.96) a 12.2 14.5

114i H F H 4-FluoroPh 43.2 - -

115a OMe H OMe 1-Naphthyl 39.1 - -

115b OMe H OMe 4-MeOPh 48.5 19.4** 18.0*

115c OMe H OMe 3-CF3Ph 12.4 (Maximum inhibition 13% at 1.0

µM)b

- -

115d OMe H OMe C4H4S 59.9 (3.99) a 13.4 11.7

115e OMe H OMe 2,4,6 tri-isoPrPh 52.82 (7.26) a 8.55 7.80

115f OMe H OMe 2-NO2Ph 72.2 (1.25) a 19.1** 17.5*

115g OMe H OMe 4-NO2Ph 46.41 - -

115h OMe H OMe 2-Naphthyl 55.5(6.59) a 13.0 11.7

115i OMe H OMe Phenyl 46.66 - -

115j OMe H OMe 2,5-diCl-Ph 43.24(Maximum inhibition 44% at

0.3µM)b

- -

115k OMe H OMe 4-FluoroPh 69(1.46) a 19.3** 20.3**

116a CF3 H H 4-MeOPh 73.6(1.08) a 15.5 15.4

116b CF3 H H 2-CF3Ph 38.8 - -

116c CF3 H H 4-NO2Ph 31.5 - -

116d CF3 H H 2,5-diClPh 50.2(9.93) a 18.3* 14.7

116e CF3 H H 2,4,6-tri-isoPrPh 49.7 10.1 8.68

116f CF3 H H 4-FPh 50(10.1) a 20.7** 20.5**

116g CF3 H H 2-Naphthyl 44.6(Maximum inhibition 46% at 1.0 µM)

b

- -

116h CF3 H H 4-MePh 50.76(4.72) a 16.3* 21.0**

Suramin - - 60(9.5) a - -

Metformin - - - 20.9** 21.3**

Values are mean ± SEM, n=6, p< 0.05 * p<0.01**, aIC50 value in µM,

b the compound showed the same inhibition

upto 10 µM




Part 1A

Page | 38

inhibition, respectively. All the other analogues 114a, 114b, 114d-114i, along with

derivatives 115c and 116b exhibited decreased inhibitory activities.

1A.1.3 Docking Studies

Docking studies were performed to identify correct poses of ligands in the

binding pocket of a protein and to predict the affinity between the ligand and the

protein. Molecular docking has made immense contribution to drug discovery for

many years as compared to the fast and successful method of 3D pharmacophore

modeling through virtual screening.147,148 Computational docking of a small molecule

to a biological target involves efficient sampling of possible poses of the former in

the specified binding pocket of the latter in order to identify the optimal binding

geometry, as measured by a user-defined fitness or scoring function. During

computational docking, a pose is typically generated, scored and compared to the

previous pose(s). The current pose is then accepted or rejected on the basis of its

score. If the current pose is rejected then a new pose is generated and the search

process iterates to an end point. Reliable rank ordering of the ligands based on their

docked scores appears to be even more challenging than searching the conformation

and orientation space.149,150A trend has been to employ consensus scoring (apply a

number of scoring functions to the same docked pose identified by docking) to

eliminate false positives.149,151 Over the last few years, extensive efforts have been

directed towards developing efficient docking methods and scoring functions as tools

for the identification of lead compounds.

The complexity of computational docking increases in the following order:

• rigid body docking- where both the receptor and small molecule are

treated as rigid

• flexible ligand docking- where the receptor is held rigid but the ligand is

treated as flexible and

• flexible docking- where both receptor and ligand flexibility is

considered.149

A reliable docking algorithm should exhaustively search all the possible

binding modes between the ligand and the receptor. However, this is impractical due

to the huge size of the search space. Therefore, in practice the computational

expense is limited by applying constraints, restraints and approximations to reduce

the dimensionality of the problem in an attempt to locate the global minimum as




Part 1A

Page | 39

efficiently as possible. As a consequence of the huge conformational space available

to protein structures, receptor flexibility is not often considered in many docking

algorithms, however some partial flexibility (side chains) have been incorporated

recently in some docking algorithms e.g. GOLD152, AUTODOCK153, FlexX154,

GLIDE155, INDUCEDFIT,156 AFFINITY157 etc.

1A.1.3.1 Application of above techniques in the present study

The aim of molecular modeling is often the discovery of new lead candidates.

Successful use of docking studies to understand the structure activity relationships

and to elucidate the essential structural requirements for molecules acting on the

same receptor/enzyme has been reported in literature.158,159 Thus, in order to

increase the understanding of the role of physicochemical and structural features

important for activity, docking studies have been carried out on the PTP1B inhibitors.

In the present work, we have evaluated the twenty eight synthesized

substituted aryl thiazolyl phenylsulfonamides 114a-114i, 115a-115k and 116a-116h

by docking the compounds in the PTP1B binding site using the GLIDE 5.6 module of

the Schrodinger software package, considering the X-ray high resolution crystal

structure of PTP1B catalytic domain taken from the protein data bank (PDB:

2ZMM).160,161 All the ligands were prepared with the LigPrep 2.3 application using an

OPLS 2005 force field. The protein structure was prepared with the protein

preparation wizard available in the Schrodinger software package and used for grid

generation (active site of the protein). Compounds 114a-114i, 115a-115k and 116a-

116h were docked into the prepared grid using the automated flexible ligand docking

in the extra precision (XP) mode.

From the docking results, the formation of hydrogen bonds and hydrophobic

interactions with the active site are predicted to be the most crucial factors affecting

the inhibitory potency of these compounds. The predicted binding pose of the most

active compound 116a suggested that the observed improved potency arises due to

the extensive hydrophobic interactions predicted to be formed with the side chains of

Tyr46, Phe182, Ala217, Ile219, Gln262 and Val49 (Fig. 3 a-c). The π–π interaction

predicted to form between the 4-methoxy phenyl ring of 116a and Phe182 and the

predicted water molecule (325 and 600) mediated hydrogen bond interactions

involving amino acid residues Ser216, Arg221, Gly220 with the oxygen atom of the 4-

methoxyphenyl group of 116a anchoring the compound in the active site.




Part 1A

Page | 40

Furthermore, the trifluoromethylphenyl moiety of 116a was predicted to occupy the

hydrophobic cleft formed by residues Asp48 and Val49 resulting its improved activity.

The ligand binding analysis of one of the least active analogues 114a

predicted hydrophobic interactions between the phenyl thiazolyl moiety and residues

Phe182 and Ile219 and between the naphthyl moiety and Val49, but it lacked the

anchoring interactions predicted to be formed between the active analogues and the

binding site of PTP1B explaining its decreased activity (Fig. 3 d-f).

Figure 3: (a) Binding pose analysis of the compound 116a with PTP1B, (b) Schematic 2D plots of

binding site observed for 116a and PTP1B (PDB ID:2ZMM) (c) binding site cavity of PTP1B with 116a,

(d) Binding pose analysis of the compound 114a with PTP1B, (e) Schematic 2D plots of binding site

observed for 114a and PTP1B (PDB ID: 2ZMM) (f) Binding site cavity of PTP1B with 114a

In order to confirm that the occupancy of hydrophobic cleft formed by Asp48

and Val49 is thought to be responsible for the better activity of 116a, the docking

studies on the analogues of 116a in which the 2-trifluomethylphenyl moiety was

replaced by a 4-fluorophenyl (114h) and 2,5-dimethoxyphenyl moiety (115b)

respectively, were performed. The 2,5-dimethoxyphenyl moiety in compound (115b)

failed to occupy the hydrophobic cleft formed by Val49 and Asp48 explaining its

reduced activity, however, the 4-fluorophenyl moiety in compound 114h had slight

contact with the residue Asp48 which might explain good activity of this compound

(Fig. 4).




Part 1A

Page | 41

Figure 4: Schematic 2D plots of binding site observed for 114h (A), 115b (B), 116a (C) and PTP1B

(PDB ID: 2ZMM)

The amino acid residues close to the active site (Arg47 and Asp48) have

been found to impart selectivity towards PTP1B over most other PTPs,162,163 which is

due to the Asp Asn at position 48 in other PTPs. Asp48 is close to the active site

and plays an important role in positioning the ligand for binding in the active site, as

judged by B-factors in the published PTP1B structures.164–165

1A.1.4 In vivo Antihyperglycaemic activity

Compounds that exhibited potent in vitro inhibition of PTP1B (>48% at 10 µm;

Table 4) were next evaluated in a streptozotocin induced (STZ) rat model of

diabetes. Table 5, 6 and 7 depict the effects of test compounds (100 mg/kg b.w.) and

Metformin (100 mg/kg b.w.) on streptozotocin induced diabetic rats.

Among these sixteen compounds 114c-114e, 114g, 114h, 115b, 115d-115f,

115h, 115k, 116a, 116d-116f and 116h, the compounds 114c, 115b, 115f, 115k and

116f showed significant lowering in around 20.6(p< 0.01), 19.4(p< 0.01), 19.1(p<

0.01), 19.3(p< 0.01) and 20.7(p< 0.01) % during 5h and 18.8(p< 0.05), 18.0(p< 0.05),

17.5(p< 0.05), 20.3(p< 0.01) and 20.5(p< 0.01) % during 24h respectively whereas

the compound 114g, 116a, 116d and 116h showed mild activity in the tune of

17.3(p>0.05), 15.5, 18.3(p>0.05), and 16.3(p>0.05) after 5h and 9.15, 15.4, 14.7 and

21.0(p>0.01) % activity after 24h intervals respectively (Fig. 5). The standard drug

Metformin treated group at the dose level of 100 mg/kg showed a blood glucose

lowering effect of 20.9 % (p < 0.01) and 21.3 % (p < 0.01) after 5h and 24h intervals

respectively (Fig. 6). The most active compound 116f showed comparable activity to

the reference antidiabetic drug Metformin, at the same dose.

Since no difference was found in food consumption between the sham treated

control groups compared to any of the experimental group during 24h, the observed




Part 1A

Page | 42

Figure 5. Antihyperglycaemic activity of test compounds in low dosed Streptozotocin induced diabetic

rats. Statistical analysis was made by Dunnet test (Prism Software 3).166

blood glucose lowering effect is significant. The rest of the compounds viz. 114d,

114e, 114h, 115d, 115e, 115h and 116e did not cause any significant lowering on

the blood glucose level of streptozotocin induced diabetic rats at 100 mg/kg dose

level.

0 50 100 150 200 250 3000

100

200

300

400

500

600Vehicle

115 e

114 e

1420 1440

114 g

114 d

Metformin

t/min

Blo

od

Glu

co

se/m

gd

l-1

Figure 6. Effect of test compounds (100 mg/kg) and standard antidiabetic drug Metformin (100 mg/kg)

on blood glucose level of low dosed Streptozotocin induced diabetic rats.

1A.2 Conclusion

Among the synthesized twenty eight novel aryl thiazolyl phenylsulfonamide

compounds synthesized 114a-114i, 115a-115k and 116a-116h, sixteen compounds

exhibited greater than 48% inhibition of PTP1B in vitro. Docking studies were

performed on the most active and one of the least active compounds 116a and 114a

respectively to gain insight into the molecular interactions with the binding site. The

compound 116a formed good interactions with the active site amino acid residues

Gln262 and Ala217 as well as with C-site amino acid residues Asp48 and Phe182. In

contrast, the docking results for inactive compound 114a lacked interactions with

Tyr46, Ala217, Gln262 and Val49 and the water mediated hydrogen bond, which

were predicted to be formed with the active analogue. Interestingly, the most active

compound 116a was suggested to interact with Asp48, a residue specific to PTP1B,

0 50 100 150 200 250 3000

100

200

300

400

500

600

1420 1440

Control

116e

115h

115d

t/min

Blo

od

Glu

co

se/m

gd

l-1




Part 1A

Page | 43

Table 5 : Effect of the test compounds (100 mg/kg) on the blood glucose levels of Streptozotocin (STZ)

induced diabetic rats at various time intervals (mean ±S.E.M.)

Blood glucose profile (mg/dL)

Groups

Pre treatment Post treatment

% Reduction

compared

to control

0

min

30

min

60

min

90

min

120

min

180

min

240

min

300

min

1400

min

5h

24h

Sham

control

323

±3.0

403.1

±2.7

459.1

±11.9

493

±7.8

465

±5.6

447

±6.4

435.6

±8.5

415.1

±5.4

552.1

±16.2

-- --

114d 322.8

±3.9

376

±13.5

377.6

±15.9

365.6

±14.8

336.6

±14.3

335.3

±7.8

336

±3.1

349.1

±7.3

463.1

±18.8

11.1 6.43

114e 323.6

±1.4

386.8

±13.2

406.6

±12.8

392.3

±14.5

389.3

±12.2

390.1

±5.5

380.6

±6.7

351.6

±2.5

518.8

±16.2

13.0 11.3

114g 322.6

±0.8

385

±18.4

351.3

±15.7

385.3

±6.1

342

±11.1

345.5

±9.2

382.6

±7.2

380.1

±5.1

501.5

±13.0

17.3 9.15

115e 321.5

±1.6

385.1

±12.2

368.8

±8.2

379.5

±13.7

391.8

±17.4

414.8

±17.6

430.6

±17.3

431.1

±12.7

488.1

±14.2

8.55 7.80

Metformin 322.8

±3.9

376

±13.5

377.6

±15.9

365.6

±14.8

336.6

±14.3

335.3

±7.8

336

±3.1

349.1

±7.3

409.8

±35.0

20.9** 21.3**

Values are mean ± SEM, n=6 *p<0.05 and **p<0.01 vs. Control

Table 6: Effect of the test compounds (100 mg/kg) on the blood glucose levels of Streptozotocin

(STZ) induced diabetic rats at various time intervals (mean ±S.E.M.)


Groups


%Reduction

compared

to control

0

min

30

min

60

min

90

min

120

min

180

min

240

min

300

min

1400

min

5h

24h

Sham

control

368

±4.6

492.0

±3.8

487.0

±4.9

475.2

±5.7

467.8

±5.4

435.0

±5.7

385.6

±2.8

363.0

±6.0

551.6

±19.8

-- --

114h 367.2

±20.9

446.0

±10.4

428.6

±7.2

389.6

±7.0

374.8

±6.98

359.8

±4.5

363.4

±7.2

340.0

±21.1

425.2

±34.2

12.2 14.5

115f 367.2

±14.0

456.2

±8.3

442.2

±11.0

402.0

±10.0

362.8

±15.4

322.4

±15.5

279.2

±14.4

275.6

±10.2

487.2

±24.7

19.1* 17.5*

116 a 367.8

±6.12

464.8

±5.58

448.2

±8.87

414.0

±4.24

383.2

±3.42

346.0

±7.70

310.4

±8.14

279.2

±7.87

491.6

±13.2

15.5 15.4

116 h 367.4

±10.8

451.6

±17.3

406.6

±11.1

384.2

±6.67

360.8

±9.33

342.2

±6.70

319.2

±1.74

329.2

±1.90

367.2

±24.4

16.3* 21.0**


indicating potential selectivity for PTP1B over other PTPs.

Out of the sixteen compounds exhibiting greater than 48% inhibition against

PTP1B in vitro, nine analogues 114c, 114g, 115b, 115f, 115k, 116a, 116d, 116f and

116h showed good in vivo activity in the STZ rat model of diabetes and the

compounds 116f and 114c showed comparable in vivo activity to Metformin. Thus,




Part 1A

Page | 44

these studies may be useful in the development of novel PTP1B inhibitors with

promising selectivity and improved pharmacological properties.

Table 7: Effect of the test compounds (100 mg/kg) on the blood glucose levels of Streptozotocin (STZ) induced diabetic rats at various time intervals (mean ±S.E.M.)


Groups


% Reduction compared

to control

0 min

30 min

60 min

90 min

120 min

180 min

240 min

300 min

1400 min

5h

24h

Vehicle 326.4

±17.3

417.4

±17.8

436.0

±17.1

422.8

±10.8

413.8

±17.7

409.8

±22.5

398.8

±18.7

385.0

±17.2

468.0

±22.4

-- --

114c 318.0

±12.8

361.0

±16.5

348.6

±20.5

337.8

±14.7

333.4

±13.1

311.8

±9.96

297.4

±12.9

309.2

±19.3

389.6

±7.06

20.6** 18.8*

115b 326.2

±14.8

364.0

±2.42

362.2

±8.19

349.4

±7.41

337.4

±8.95

322.6

±9.53

306.0

±7.88

279.8

±9.20

416.8

±7.47

19.4** 18.0*

115d 324.6

±14.1

354.6

±16.1

430.0

±16.3

445.4

±18.2

397.6

±17.7

339.2

±16.1

293.8

±11.1

258.6

±8.33

440.4

±21.0

13.4 11.7

115h 325.8

±11.6

381.2

±4.53

389.2

±17.0

379.0

±19.5

373.4

±11.4

348.0

±11.9

326.4

±10.4

308.2

±13.3

443.4

±14.3

13.0 11.7

115k 325.0

±14.0

366.6

±16.3

352.6

±10.3

371.4

±17.0

376.0

±19.6

329.6

±15.5

279.4

±10.3

249.8

±7.17

417.0

±15.7

19.3** 20.3**

116d 323.6

±14.9

363.8

±19.1

361.2

±16.3

345.2

±9.15

343.2

±11.1

316.8

±10.9

315.0

±9.90

316.0

±16.8

415.8

±13.9

18.3* 14.7

116e 329.6

±6.9

406.6

±14.0

409.2

±23.8

383.2

±13.6

370.0

±11.5

365.0

±5.7

343.4

±7.9

319.8

±2.5

462.4

±12.5

10.1 8.68

116f 320.6

±8.26

377.8

±16.3

356.6

±19.5

325.6

±17.7

337.6

±17.7

312.6

±14.4

302.8

±14.7

277.2

±17.0

393.6

±6.70

20.7** 20.5**





Part 1A

Page | 45

1A.3 Experimental

Melting points were determined on an electrical heated m. p. apparatus /using

silicon oil bath. Reactions were monitored by thin layer chromatography on self-made

plates of silica gel G (Merck, India) or 0.25mm ready-made plates of silica gel 60F

254, (Merck, Darmstadt, Germany). Column chromatography was performed on silica

gel (Merck, 60 to 120mesh). Infrared spectra (IR) were recorded on Perkin-FTIR

model PC spectrophotometer with frequency of absorptions reported in wave

numbers. Mass were recorded on JEOL spectrometer with fragmentation pattern

reported as values, 1H NMR was recorded on Bruker spectrometer with a

multinuclear inverse probe head with gradient at room temperature (298 K) using

CDCl3 as solvent and tetramethylsilane (TMS) as internal standard. Chemical shifts

were described in parts per million (ppm) relative to TMS (0.00 ppm) using scale and

coupling constants were reported in hertz (Hz).

Preparation of N-(4-fluorophenylcarbamothioyl)benzamide (111a)

A solution of benzoyl isothiocyanate (3.2 g, 0.02 mol) in dry benzene (20 mL)

was added dropwise to a stirred solution of 4-fluoroaniline (2.2 g, 0.02 mol) in dry

benzene (30 mL) at 200C over 10 min. Stirring continued at 200C for 2h, concentrated

under reduced pressure, filtered and washed with a small amount of cold dry

benzene and dried to give 111a.

Yield: 70 %; mp 1290C (lit.167 mp 128-129.50C); IR (KBr cm-1): 772, 846, 1263, 1518,

1604, 1664, 2371, 3253, 3410; 1H NMR (CDCl3): δ 7.60 (d, J = 6.9 Hz, 2H), 7.69 (d, J

= 6.87 Hz, 1H), 7.93 (d, J = 6.87 Hz, 2H), 8.07 (d, J = 8.16 Hz, 2H), 8.30(d, J = 8.16

Hz, 2H), 9.21 (s, 1H), 13.11 (s,1H); MS (ESI+): m/z = 275 (M+H) +

Similarly, other compounds 111b-111c of this series were prepared from

corresponding substituted anilines.

N-(2,5-dimethoxyphenylcarbamothioyl)benzamide (111b)

The compound was prepared by similar method as described for the synthesis of

111a using benzoyl isothiocyanate (3.2g) and 2,5-dimethoxy aniline (3.62g).

Yield: 72 %; mp 1360C (lit.167 mp 1380C); IR (KBr cm-1): 770, 840, 1267, 1514, 1604,

1662, 2370, 3413, 3557; 1H NMR (CDCl3): δ 6.60- 6.25 (m, 3H), 7.70 (d, J = 6.87 Hz,

1H), 8.11 (d, J = 8.16 Hz, 2H), 8.25(d, J = 8.16 Hz, 2H), 9.68 (s, 1H), 12.06 (s,1H);

MS (ESI+): m/z = 317 (M+H) +




Part 1A

Page | 46

N-(2-(trifluoromethyl)phenylcarbamothioyl)benzamide (111c)

The compound was prepared by similar method as 111a using benzoyl

isothiocyanate (3.2g) and 2-trifluoromethyl aniline (3.22g).

Yield: 71%; mp 1320C; IR (KBr cm-1): 774, 846, 1265, 1517, 1609, 1665, 2371,

3412, 3587; 1H NMR (CDCl3): δ 7.65 -7.43 (m, 4H), 7.73 (d, J = 6.87 Hz, 1H), 8.16

(d, J = 8.16 Hz, 2H), 8.25(d, J = 8.16 Hz, 2H), 9.11 (s, 1H), 12.21 (s,1H); MS (ESI+):

m/z = 325 (M+H) +

Preparation of 1-(4-fluorophenyl)thiourea (112a).

The compound 111a (3.78 g, 0.014 mol) was added in one portion to a

stirring 5 % aqueous NaOH (20 mL) solution at 90 0C and stirring cotinued for further

20 min. The hot reaction mixture was filtered and the filtrate obtained cooled,

acidified with 15 % aqueous HCl, pH adjusted to ~8.0 with aq. ammonia to remove

benzoic acid. The solid obtained was filtered, washed with water and dried to give

112a.

Yield: 95 %; mp 930C; IR (KBr cm-1): 707, 812, 1212, 1536, 1625, 1689, 2363,

3339(NH2), 3437(NH); 1H NMR (CDCl3): δ 6.17 (s, 2H), 7.13-7.65 (m, 3H), 8.13(d, J

= 7.2 Hz, 1H), 8.3 (s, NH); MS (ESI+): m/z = 171 (M+H) +

Similarly, other compounds 112b-112c of this series were prepared.

1-(2,5-Dimethoxyphenyl) thiourea (112b)


112a using 111b (4.42g).

Yield :75 %; mp 1480C; IR (KBr cm-1): 711, 820, 1221, 1518, 1624, 1692, 2364,

3343(NH2), 3457(NH); 1H NMR (CDCl3): δ 3.80 (d, J = 15 Hz, 6H), 6.30 (s, 1H), 6.7-

6.9 (m, 2H), 7.8 (s, 1H), 8.0 (s, NH); MS (ESI+): m/z = 213 (M+H) +

1-(2-(Trifluoromethyl)) thiourea (112c)


112a using 111c (4.53g).




Part 1A

Page | 47

Yield: 90 %; mp 1700C; IR (KBr cm-1: 763, 928, 1216, 1536, 1621, 1689, 2366,

3345(NH2), 3428(NH); 1H NMR (CDCl3): δ 7.73-7.53 (m, 4H), 8.05(d, J = 7.02 Hz,

2H), 8.92 (s, NH); MS (ESI+): m/z = 221 (M+H) +

Preparation of N-(4-fluorophenyl)-4,5-dihydrothiazol-2-amine (113a)

A solution of 2-bromoethylamine hydrobromide (2.05 g, 0.01 mol) in 5.0 mL

water was added to a solution of 112a (1.70 g, 0.01 mol) in ethanol (40 mL) and the

reaction mixture refluxed under stirring overnight. Ethanol was distilled off under

reduced pressure and the reaction mixture basified with dilute aq. ammonia solution.

The product 113a was filtered, washed with water, dried and recrystallized with

methanol.

Yield: 60 %; mp 1340C; IR (KBr cm-1): 770, 1216, 1636, 3023, 3456(NH); 1H NMR

(CDCl3): δ 3.35 (t, J = 7 Hz, 2H,-CH2-CH2-), 3.88 (t, J = 7 Hz, 2H, -CH2-CH2-), 6.78-

7.19 (m, 3H), 8.03 (d, J = 8.7 Hz, 1H), 9.3 (s, 1H); MS (ESI+): m/z = 197 (M+H)+.

Similarly, other compounds 113b-113c of this series were prepared.

N-(2,5-Dimethoxyphenyl)-4,5-dihydrothiazol-2-amine (113b)


113a using 2-bromoethylamine hydrobromide (2.05 g) and 112b (2.12g).


(CDCl3): δ 3.33 (t, J = 7 Hz, 2H, -CH2-CH2-), 3.76 (s,1H), 3.81(s,3H) 4 .11 (t, J = 7

Hz, 2H, -CH2-CH2-), 6.48-6-44 (m,1H), 7.25 (s,1H), 7.83 (s, 1H); MS (ESI+): m/z =

239 (M+H)+.

N-(2-(Trifluoromethyl))-4,5-dihydrothiazol-2-amine (113c)


113a using 2-bromoethylamine hydrobromide (2.05 g) and 112c (2.2g).


(CDCl3): δ 3.09 (t, J = 7 Hz, 2H, -CH2-CH2-), 3.47 (t, J = 7 Hz, 2H, -CH2-CH2-), 7.50 -

7.01 (m,4H); MS (ESI+): m/z = 247 (M+H)+.

N-(4,5-Dihydrothiazol-2-yl)-N-(4-fluorophenyl)napthalene-1-sulfonamide (114a)




Part 1A

Page | 48

To a stirred solution of 113a (1.97g, 0.01 mol) in dry acetone (10mL) was

added triethylamine (2.08 mL, 0.015 mol) and 1-naphthalene sulphonyl chloride (2.25

g, 0.01 mol) under dry conditions. The mixture was stirred at room temperature for 12

h. The solvent was evaporated in vacuo and water added to the residue followed by

extraction with ethyl acetate (4x20mL). The organic layer was dried over anhyd.

sodium sulphate. The solvent was evaporated under reduced pressure and the

residue scratched with hexane to afford compound 114a.

Yield: 77.2 % ; mp 170oC; IR (KBr cm-1): 767, 1093, 1352(S=O), 1594, 2369, 3006;

1H NMR (CDCl3, 300MHz): δ 3.24 (t, J = 6.0 Hz, 2H, -CH2-CH2-), 4.49 (t, J = 6.0 Hz,

2H, -CH2-CH2-), 6.42-6.47 (m, 2H), 6.85 (t, J = 9.0 Hz, 2H), 7.58-7.67 (m, 2H), 7.71

(d, J = 5.5 Hz, 1H), 7.99 (d, J = 6.0 Hz, 1H), 8.14 (d, J = 6.0 Hz, 1H), 8.53 (d, J = 6.0

Hz, 1H); MS (ESI+): m/z = 387 (M+H) +; HRMS (ESI+) calcd. for C19H16FN2O2S2+ H

387.0637, found 387.0636.

2,5-Dichloro-N-(4,5-dihydrothiazol-2-yl)-N-(4-fluorophenyl)benzenesulfonamide

(114b)


114a using 113a (1.97 g) and 2,5-dichlorobenzenesulfonyl chloride (2.43g).

Yield: 71.2 %; mp 145oC; IR (KBr cm-1): 760, 1100, 1350(S=O), 1648, 2362, 3026; 1H

NMR (CDCl3, 300MHz): δ 3.32 (t, J = 6.6 Hz, 2H, -CH2-CH2-), 4.46 (t, J = 6.6 Hz, 2H,

-CH2-CH2-), 6.54-6.58 (m, 2H), 6.89-6.95 (m, 2H), 7.46-7.52 (m, 1H), 7.53-7.55 (m,

1H), 8.30 (d, J = 2.3 Hz, 1H); MS (ESI+): m/z = 405 (M+H)+, HRMS (ESI+) calcd. for

C15H12F1N2O2S2+ H 404.97013, found 404.96924.

N-(4,5-Dihydrothiazol-2-yl)-N-(4-fluorophenyl)-4-nitrobenzenesulfonamide

(114c)


114a using 113a (1.97 g) and 4-nitrobenzenesulfonyl chloride (2.20g).

Yield: 47.6 %; mp 208oC; IR (KBr cm-1): 671, 762, 1216, 1353(S=O), 1637, 2359,

3021; 1H NMR (CDCl3, 300MHz): δ 3.29 (t, J = 6.6 Hz, 2H, -CH2-CH2-), 4.27 (t, J =

6.6 Hz, 2H, -CH2-CH2-), 6.66–6.71 ( m, 2H), 6.97 (t, J = 8.7 Hz, 2H), 8.29 (d, J = 8.8

Hz, 2H), 8.41 (d, J = 8.8 Hz, 2H); MS (ESI+): m/z = 384 (M+H) +; HRMS (ESI+) calcd.

for C15H13F1N3O4S2+ H 382.03315, found 382.03201.




Part 1A

Page | 49

N-(4,5-Dihydrothiazol-2-yl)-N-(4-fluorophenyl)-2,4,6-triisopropylbenzenesulfona-

-mide (114d)

The compound was prepared by similar method as as described for the synthesis of

114a using 113a (1.97 g) and 2,4,6-triisopropylbenzenesulfonyl chloride (3.02g).

Yield: 47 %; mp 120oC; IR (KBr cm-1): 670, 761, 1094, 1356(S=O), 1596, 2963,

3019; 1H NMR (CDCl3, 200MHz): δ 1.23-1.29 (m, 18H), 2.88-2.97 (m, 3H), 4.14 (t, J

=6.6 Hz, 2H, -CH2-CH2-), 4.33 (t, J = 6.6 Hz, 2H, -CH2-CH2-), 6.46 (t, J =4.8 Hz, 2H),

6.86 (t, J = 8.4 Hz, 2H), 7.14 (s, 2H); MS (ESI+): m/z = 463 (M+H)+, HRMS (ESI+)

calcd. for C24H31F1N2O2S2+ H 463.1889, found 463.1891.

N-(4,5-Dihydrothiazol-2-yl)-N-(4-fluorophenyl)napthalene-2-sulfonamide (114e)


114a using 113a (1.97 g) and 2-naphthalenesulfonylchloride (2.25g).



-CH2-CH2-), 6.65-6.58 (m, 2H), 6.85-6.94 (m, 2H), 7.65-7.69 (m, 2H), 7.92-8.04 (m,

2H), 8.68 (s, 1H); MS (ESI+): m/z = 384 (M+H) +; HRMS (ESI+) calcd. for

C19H15FN2O2S2 + H 386.0559, found 386.0565.

N-(4,5-Dihydrothiazol-2-yl)-N-(4-fluorophenyl)-2-(trifluoromethyl)benzenesulfon-

-amide (114f)


114a using 113a (1.97 g) and 2-trifluoromethylbenzenesulfonyl chloride (2.44g).



6.6 Hz, 2H, -CH2-CH2-), 6.51-6.57 (m, 2H), 6.89 (t, J = 7.5 Hz, 2H), 7.23 (d, J = 6.9

Hz, 1H), 7.68-7.76 (m, 1H), 7.89 (t, J = 4.3 Hz, 1H), 8.54 (t, J = 5.7 Hz, 1H); MS

(ESI+): m/z = 405 (M+H) +; HRMS (ESI+) calcd. for C16H12F4N2O2S2 + H 405.0315,

found 405.0318.

N-(4,5-Dihydrothiazol-2-yl)-N-(4-fluorophenyl)thiophene-2-sulfonamide(114g)


114a using 113a (1.97 g) and 2- thiophenesulfonylchloride (1.81g).




Part 1A

Page | 50



6.6 Hz, 2H, -CH2-CH2-), 7.02-7.15 (m, 5H), 7.70-7.79 (m, 2H); MS (ESI+): m/z = 342

(M+H) +; HRMS (ESI+) calcd. for C13H11FN2O2S3 + H 342.9967, found 343.0128

N-(4,5-Dihydrothiazol-2-yl)-N-(4-fluorophenyl)-4-methoxybenzenesulfonamide

(114h)


114a using 113a (1.97 g) and 4-methoxybenzenesulfonylchloride (2.06g).

Yield: 50 % ; mp 165oC; IR (KBr cm-1): 672, 763, 1163, 1356(S=O), 1636, 2365,

3067; 1H NMR (CDCl3, 200MHz): δ 3.25 (t, J = 6.6 Hz, 2H, -CH2-CH2-), 3.9 (s,

3H,OCH3), 4.15 (t, J = 6.6 Hz, 2H, -CH2-CH2-), 6.91-7.06 (m, 2H), 7.08-7.18 (m, 2H),

7.23 (d, J = 6.9 Hz, 1H), 7.21-7.28 (m, 2H), 7.89-7.91 (m, 2H); MS (ESI+): m/z = 367

(M+H) +; HRMS (ESI+) calcd. for C16H15FN2O3S2 + H 367.0586, found 367.05721.

N-(4,5-Dihydrothiazol-2-yl)-4-fluoro-N-(4-fluorophenyl)benzenesulfonamide

(114i)


114a using 113a (1.97 g) and 4-fluorobenzenesulfonylchloride (1.94g).

Yield : 46.8 %; mp 176oC; IR (KBr cm-1): 722, 1079, 1350(S=O), 1526, 1627, 2362,

2925, 3021; 1H NMR (CDCl3, 300MHz): δ 3.24 (t, J = 6.6 Hz, 2H, -CH2-CH2-), 4.23 (t,

J = 6.6 Hz, 2H, -CH2-CH2-), 6.69-6.72 (m, 2H), 6.94-7.00 (m, 2H), 7.21-7.28 (m, 2H),

8.11-8.14 (m, 2H); MS (ESI+): m/z = 355 (M+H) +; HRMS (ESI+) calcd. for

C15H12F2N2O2S2 + H 355.03718, found 355.03865.

(4,5-Dihydrothiazol-2-yl)-N-(2,5-dimethoxyphenyl)naphthalene-1-sulfonamide

(115a)


114a using 113b (2.39g) and 1-naphthalenesulphonylchloride (2.25 g).

Yield: 74.4 %; mp=116oC; IR (KBr cm-1): 767, 1116, 1356(S=O), 1632, 2367, 3003;

1H NMR (CDCl3, 200MHz): δ 3.19 (t, J = 6.6 Hz, 2H, -CH2-CH2- ), 3.30 (s, 3H,OCH3),

3.65 (s, 3H, OCH3), 4.49 (t, J = 6.6 Hz, 2H, -CH2-CH2-), 6.06 (d, J = 3.0 Hz, 1H), 6.5

(dd, J = 3.0 Hz, J = 8.8 Hz, 1H), 6.67 (d, J = 9.0 Hz, 1H), 7.51-7.69 (m, 3H), 7.97 (d,

J = 7.6 Hz, 1H), 8.10 (d, J = 8.2 Hz, 1H), 8.54 (d, J = 7.4 Hz, 1H), 8.66 (d, J = 8.2 Hz,




Part 1A

Page | 51

1H); MS (ESI+): m/z = 333 (M+H) +; HRMS (ESI+) calcd. for C21H21N2O4S2+ H

429.09427, found 429.09235.

N-(4,5-Dihydrothiazol-2-yl)-N-(2,5-dimethoxyphenyl)-4-methoxybenzenesulfona-

-mide (115b)


114a using 113b (2.39g) and 4-methoxybenzenesulphonylchloride (2.06 g).

Yield: 71.0 %; mp=128oC; IR (KBr cm-1): 571, 827, 1011, 1160, 1365(S=O), 1633,

2368, 3048; 1H NMR (CDCl3, 200MHz): δ 3.16 (t, J = 6.6 Hz, 2H, -CH2-CH2-), 3.61 (s,

3H, OCH3), 3.88 (s, 3H, OCH3), 4.19 (t, J = 6.6 Hz, 3H, -CH2-CH2-), 6.30 (d, J = 2.9

Hz, 1H), 6.56 (dd, J = 3.0 Hz, J = 8.9 Hz, 1H), 6.77 (d, J = 8.9 Hz, 1H), 6.98 (d, J =

8.9 Hz, 2H); MS (ESI+): m/z = 409 (M+H) +; HRMS (ESI+) calcd. for C18H21N2O5S2+

H 409.08919, found 409.08717.

N-(4,5-Dihydrothiazol-2-yl)-N-(2,5-dimethoxyphenyl)-2-(trifluoromethyl)benzene

sulfonamide (115c)

The compound was prepared by similar method as 114a using 113b (2.39g) and 2-

trifluoromethylbenzenesulphonylchloride (2.43 g).


NMR (CDCl3, 300MHz): δ 3.24 (t, J = 6.6 Hz, 2H, -CH2-CH2-), 3.48 (s, 3H, OCH3),

3.71 (s, 3H, OCH3), 4.38 (t, J = 6.6 Hz, 2H-CH2-CH2-), 6.16 (d, J = 3.0 Hz, 1H), 6.47-

6.51 (dd, J = 3.0 Hz, J = 8.9 Hz, 1H), 6.68 (d, J = 8.6 Hz, 1H), 7.71 (t, J = 4.0 Hz,

2H), 7.89 (t, J = 3.71 Hz, 1H), 8.61 (t, J = 5.52 , 1H); MS (ESI+): m/z = 447 (M+H) +;

HRMS (ESI+) calcd. for C18H17F3N2O4S2+ H 447.0805, found 447.0827.

N-(4,5-Dihydrothiazol-2-yl)-N-(2,5-dimethoxyphenyl) thiophene-2-sulfonamide

(115d)


thiophenesulphonylchloride (1.81g).




(dd, J = 3.0 Hz, J = 8.8 Hz, 1H), 6.80 (d, J = 8.86 Hz, 1H), 7.13-7.17 (m, 1H), 7.67-




Part 1A

Page | 52

7.71 (m, 1H), 8.01-8.04 (m, 1H); MS (ESI+): m/z = 385 (M+H) +; HRMS (ESI+) calcd.

for C15H16N2O4S3+ H 385.0276 found 385.0289.

N-(4,5-Dihydrothiazol-2-yl)-N-(2,5-dimethoxyphenyl)-2,4,6-triisopropylbenzene

sulfonamide (115e)

The compound was prepared by similar method as 114a using 113b (2.39g) and

2,4,6-triisopropylbenzenesulfonylchloride (3.02g).

Yield: 64.9 %; mp 145oC; IR (KBr cm-1): 761, 1166, 1365(S=O), 1492, 1637, 2961,

3027;1H NMR (CDCl3, 300MHz): δ 1.24 (t, J = 6.6 Hz, 18 H), 2.84-2.93 (m, 1H), 3.19

(s, 3H, OCH3), 3.23 (t, J = 6.6 Hz, 2H, -CH2-CH2-), 3.70 (s, 3H, OCH3), 4.14-4.23 (m,

2H, -CH2-CH2-), 4.33 (t, J = 6.6 Hz, 2H), 6.18 (d, J = 2.9 Hz, 1H), 6.45 (d, J = 2.9 Hz,

1H), 6.48 (d, J = 2.9 Hz, 1H), 6.69 (d, J = 8.79 Hz, 1H), 7.71 (s, 2H); MS (ESI+): m/z

= 405 (M+H) +; HRMS (ESI+) calcd. for C26H36N2O4S2+ H 505.2116 found 505.2207.

N-(4,5-Dihydrothiazol-2-yl)-N-(2,5-dimethoxyphenyl)-2-nitrobenzene sulfonami-

-de (115f)

The compound was prepared by similar method as 114a using 113b (2.39g) and 2 -

nitrobenzenesulfonylchloride (2.20g).


3020; 1H NMR (CDCl3, 300MHz): δ 3.32 (t, J = 6.6 Hz, 2H, -CH2-CH2-), 3.50 (s, 3H,

OCH3), 3.70 (s, 3H, OCH3), 4.38 (t, J = 6.6 Hz, 2H, -CH2-CH2-), 6.21 (d, J = 2.97 Hz,

1H), 6.52 (dd, J = 3.0 Hz, J = 8.8 Hz, 1H), 6.70 (d, J = 8.8 Hz, 1H), 7.65-7.74 (m,

3H), 8.51 (t, J = 6.9 Hz, 1H); MS (ESI+): m/z = 424 (M+H) +; HRMS (ESI+) calcd. for

C17H17N3O6S2+ H 423.0612, found 423.0609.

N-(4,5-Dihydrothiazol-2-yl)-N-(2,5-dimethoxyphenyl)-4-nitrobenzenesulfon ami-

-de (115g)

The compound was prepared by similar method as 114a using 113b (2.39g) and 4 -

nitrobenzenesulfonylchloride (2.20g).




(d, J = 2.8 Hz,1H), 6.77 (d, J = 8.8 Hz, 2H), 8.33 (d, J = 5.34 Hz, 2H), 8.38 (s, 1H);




Part 1A

Page | 53

MS (ESI+): m/z = 424 (M+H) +; HRMS (ESI+) calcd. for C17H18N3O6S2+ H 424.06370,

found 424.06206.

N-(4,5-Dihydrothiazol-2-yl)-N-(2,5-dimethoxyphenyl)naphthalene-2-sulfonamide

(115h)


naphthalenesulphonylchloride (2.25 g).

Yield: 52 %; mp 155oC; IR (KBr cm-1): 654, 1225, 1357(S=O), 1661, 2365, 3032; 1H


3.76 (s, 3H, OCH3), 4.31 ( t, J = 6.6 Hz, 2H, -CH2-CH2-), 6.22 (d, J = 3.0 Hz, 1H),

6.50 (d, J = 3.0 Hz, 1H), 6.53 (d, J = 3.0 Hz, 1H), 7.59-7.70 (m, 2H), 7.92-8.04 (m,

2H), 8.08-8.12 (m, 1H), 8.75 (s, 1H); MS (ESI+): m/z = 429 (M+H)+; HRMS (ESI+)

calcd. for C21H21N2O4S2+ H 429.0942, found 429.09182.

N-(4,5-Dihydrothiazol-2-yl)-N-(2,5-dimethoxyphenyl)benzenesulfonamide (115i)

The compound was prepared by similar method as 114a using 113b (2.39g) and

benzenesulphonylchloride (1.75 g).

Yield: 91.0 %; mp 85oC; IR (KBr cm-1): 762, 1222, 1345 (S=O), 1626, 2358, 3072,

3059; 1H NMR (CDCl3, 300MHz): δ 3.19 (t, J = 6.6 Hz, 2H, -CH2-CH2-), 3.60 (s, 3H,

OCH3), 3.72 (s, 3H, OCH3), 4.23 (t, J = 6.6 Hz, 2H, -CH2-CH2-), 6.28 (d, J =1.8 Hz,

1H), 6.56-6.59 (m, 1H),6.78 (d, J = 8.7 Hz, 1H), 7.52-7.66 (m, 3H), 8.16 (d, J = 7.8

Hz, 2H); MS (ESI+): m/z = 379 (M+H) +; HRMS (ESI+) calcd. for C17H19N2O4S2+H

379.07862, found 379.08061.

2,5-Dichloro-N-(4,5-dihydrothiazol-2-yl)-N-(2,5-dimethoxyphenyl)benzene-

sulfonamide (115j)

The compound was prepared by similar method as 114a using 113b (2.39g) and 2,5-

dichlorobenzenesulphonylchloride (2.43 g).


NMR (CDCl3, 200MHz): δ 3.28 (t, J = 6.6 Hz, 2H, -CH2-CH2-), 3.48 (s, 3H), 3.76 (s,

3H,OCH3), 4.47 (t, J = 6.7 Hz, 2H, -CH2-CH2-), 6.19 (d, J = 2.97 Hz, 1H), 6.6 (dd, J =

8.8 Hz, J = 33.2 Hz, 1H),7.35-7.42 (m, 2H), 8.19 (d, J = 1.9 Hz, 1H); MS (ESI+): m/z

= 447 (M+H)+ HRMS (ESI+) calcd. for C17H16Cl2N2O4S2 + H 445.9929, found

445.9952.




Part 1A

Page | 54

N-(4,5-Dihydrothiazol-2-yl)-N-(2,5-dimethoxyphenyl)-4-fluorobenzenesulfonami-

-de (115k)


fluorobenzenesulphonylchloride (1.94 g).

Yield: 81.4 %; mp 104oC; IR (KBr cm-1): 832, 1359(S=O), 1498, 1643, 2948, 3004;

1H NMR (CDCl3, 200MHz): δ 3.20 (t, J = 6.5 Hz, 2H, -CH2-CH2-), 3.61 (s, 3H,OCH3),

3.72 (s, 3H,OCH3), 4.21 (t, J = 5.6 Hz, 2H, -CH2-CH2-), 6.31 (dd, J = 1.1 Hz, J = 2.8

Hz, 1H), 6.59 (d, J = 3.0 Hz, 1H), 6.77 (d, J = 8.9 Hz, 1H), 7.16 -7.25 (m, 2H), 8.15-

8.22 (m, 2H); MS (ESI+): m/z = 396 (M+H) +; HRMS (ESI+) calcd. for

C17H17F1N2O4S2+ H 397.06315, found 397.06081.

N-(4,5-Dihydrothiazol-2-yl)-4-methoxy-N-(2-(trifluoromethyl)phenyl)benzenesulf

-onamide(116a)

The compound was prepared by similar method as 114a using 113c (2.47g) and 4-

methoxybenzenesulphonylchloride (2.06g).

Yield: 53.1 %; mp 126oC; IR (KBr cm-1): 680, 761, 1165, 1320(S=O), 1644, 2365; 1H

NMR (CDCl3, 200MHz): δ 3.21 (t, J = 6.6 Hz, 2H, -CH2-CH2-), 3.88 (s, 3H,OCH3),

4.25 (t, J = 6.6 Hz, 2H, -CH2-CH2-), 6.84 (d, J = 7.6 Hz, 1H), 6.96 (d, J = 2.0 Hz, 2H),

7.13 (t, J = 7.8 Hz, 1H), 7.40 (d, J = 7.6 Hz, 1H), 7.55 (d, J = 7.8 Hz, 1H), 7.98 (d, J =

2.2 Hz, 2H); MS (ESI+): m/z = 417 (M+H)+; HRMS (ESI+) calcd. for

C17H16F3N2O3S2+ H 417.05544, found 417.05096.

N-(4,5-Dihydrothiazol-2-yl)-2-(trifluoromethyl)-N-(2-(trifluoromethyl) phenyl)

benzenesulfonamide (116b)


trifluoromethylbenzenesulphonylchloride (2.43 g).

Yield : 44.17 %; mp 118oC; IR (KBr cm-1): 766, 1175, 1322(S=O), 1639, 3053; 1H


-CH2-CH2-), 6.85 (d, J = 7.9 Hz, 1H), 7.11 (t, J = 7.6 Hz, 1H), 7.36-7.50 (m, 1H), 7.66

(d, J = 1.5 Hz, 1H), 7.71 (t, J = 2.8 Hz, 1H), 7.90 (t, J = 4.4 Hz, 1H), 8.47 (t, J = 4.4

Hz, 1H); MS (ESI+): m/z = 454 (M+H) +; HRMS (ESI+) calcd. for C17H13F6N2O2S2+ H

455.03226 found 455.03404.




Part 1A

Page | 55

N-(4,5-Dihydrothiazol-2-yl)-4-nitro-N-(2 (trifluoromethyl)phenyl)benzenesulfona-

-mide (116c)


nitrobenzenesulphonylchloride (2.20 g).



6.6 Hz, 2H, -CH2-CH2-), 6.86 (d, J = 7.97 Hz, 1H), 7.16 (t, J = 7.7 Hz, 1H), 7.40-7.57

(m, 1H), 7.60-7.77 (m, 1H), 8.24 (d, J = 8.9 Hz, 2H), 8.36 (d, J = 8.75 Hz, 2H); MS

(ESI+): m/z = 432 (M+H) +; HRMS (ESI+) calcd. for C16H12F3N3O4S2+ H 432.03315,

found 432.03201.

2,5- Dichloro-N-(4,5-dihydrothiazol-2-yl)-N-(2-(trifluoromethyl)phenyl)benzene-

sulfonamide (116d)

The compound was prepared by similar method as 114a using 113c (2.47g) and 2,5-

dichlorobenzenesulphonylchloride (2.43 g).



6.6 Hz, 2H, -CH2-CH2-), 6.81 (d, J = 6.0 Hz, 1H), 7.06 (d, J = 9.0 Hz, 1H), 7.32-7.46

(m, 4H), 8.15 (s, 1H); MS (ESI+): m/z = 454 (M+H) ++; HRMS (ESI+) calcd. for

C16H11Cl2F3N2O2S2+ H 454.9591, found 454.9720.

N-(4,5-Dihydrothiazol-2-yl)-2,4,6-triisopropyl-N-(2-(trifluoromethyl)phenyl)benz-

-enesulfonamide (116e)

The compound was prepared by similar method as 114a using 113c (2.47g) and

2,4,6-triisopropylbenzenesulphonylchloride (3.02 g).


2966; 1H NMR (CDCl3, 300MHz): δ 1.21 (d, J = 6.8 Hz, 12H), 1.27 (d, J = 6.9 Hz,

6H), 2.86-2.95 (m, 1H), 3.27 (t, J = 6.7 Hz, 2H, -CH2-CH2-), 4.03-4.12 (m, 2H), 4.35

(t, J = 6.7 Hz, 2H, -CH2-CH2-), 6.81 (d, J = 7.89 Hz, 1H), 7.08 (d, J = 7.6 Hz, 1H),

7.12 (s, 2H), 7.39 (t, J = 7.6 Hz, 1H), 7.46 (d, J = 7.74 Hz, 1H); MS (ESI+): m/z = 512

(M+H) +; HRMS (ESI+) calcd. for C25H31F3N2O2S2+ H 513.1857, found 513.1853.

N-(4,5-Dihydrothiazol-2-yl)-4-fluoro-N-(2-(trifluoromethyl)phenyl)benzenesulfon-

-amide (116f)




Part 1A

Page | 56


fluorobenzenesulphonylchloride (1.94 g).

Yield: 80 %; mp 127oC; IR (KBr cm-1): 761, 1136, 1320(S=O), 1637, 3047; 1H NMR

(CDCl3, 200MHz): δ 3.25 (t, J = 6.6 Hz, 2H, -CH2-CH2-), 4.28 (t, J = 7.6 Hz, 2H, -CH2-

CH2-), 6.86 (d, J = 7.9, 2H), 7.24-7.11 (m, 1H), 7.43 (t, J = 7.6 Hz, 2H), 7.55 (d, J =

7.78 Hz, 1H), 8.05-8.12 (m, 2H); MS (ESI+): m/z = 405 (M+H) +; HRMS (ESI+) calcd.

for C16H13F4N2O2S2 + H 405.0354, found 405.0308.

N-(4,5-Dihydrothiazol-2-yl)-N-(2-(trifluoromethyl)phenyl)naphthalene-2-sulfona-

-mide (116g)


naphthalenesulphonylchloride (2.25 g).

Yield : 53 %; mp 152oC; IR (KBr cm-1): 774, 1114, 1353(S=O), 1646, 2364, 3031; 1H


-CH2-CH2-), 6.84 (d, J = 7.9 Hz, 1H), 7.14 (t, J = 7.7 Hz, 1H), 7.41 (t, J = 7.9 Hz, 1H),

7.53 (d, J = 7.8 Hz, 1H), 7.59-7.64 (m, 2H), 7.64-7.71 (m, 4H), 7.80 (s, 1H); MS

(ESI+): m/z = 437 (M+H) +; HRMS (ESI+) calcd. for C20H16F3N2O2S2+ H 437.06053,

found 437.06005.

N-(4,5-Dihydrothiazol-2-yl)-4-methyl-N-(2-(trifluoromethyl)phenyl)benzenesulfo-

-namide (116h)


methylbenzenesulphonylchloride (1.89 g).

Yield : 49 %; mp 145oC; IR (KBr cm-1): 680, 762, 1103, 1318(S=O), 1637, 2363,

2934, 3047; 1H NMR (CDCl3, 200MHz) : δ 2.49 (s, 3H), 3.25 (t, J = 6.6 Hz, 2H, -CH2-

CH2-), 4.27 (t, J =6.6 Hz, 2H, -CH2-CH2-), 6.86 (d, J = 8.1 Hz, 1H), 7.14 (t, J = 7.5 Hz,

1H), 7.32 (d, J = 8.4 Hz, 2H), 7.45 (s, 1H), 7.56 (d, J =7.8 Hz, 1H), 7.94 (d, J = 8.4

Hz, 2H); MS (ESI+): m/z = 401 (M+H) +; HRMS (ESI+) calcd. for C17H15F3N2O2S2+ H

401.0527, found 401.0540.

1A.3.1 Biological Assay

1A.3.1.1 In vitro assay




Part 1A

Page | 57

All the synthesized compounds were evaluated for in vitro antihyperglycaemic

activity against protein tyrosine phosphatase 1B using colorimetric, non-radioactive

PTP1B tyrosine phosphatase drug discovery kit -BML-AK 822 from Enzo Life

Sciences, USA. PTP1B enzyme inhibitory activity of compounds were evaluated

using human recombinant PTP1B enzyme provided in the kit at five different

concentration i.e. 0.3 µM, 1.0 µM, 3.0 µM, 5.0 µM and 10 µM concentration taking

suramin as a control and IC50 was calculated for the compounds showing >50%

inhibition at 10µM concentration.

Other components of the kit include substrate (IR5 insulin receptor residues),

biomol red (phosphate determining reagent), assay buffer, suramin (PTP1B inhibitor)

and calibration standards. Assay was done according to the Kit manufacturer’s

protocol, in brief the reaction was carried out in 96 well flat bottomed microtiter plate

by the addition of assay buffer, solution of test compounds and diluted PTP1B

enzyme. Enzyme reaction was initiated by addition of 50µL of warmed 2x substrate

then incubated the plate at 300ºC for 30min. After incubation for 30 min. Reaction

was terminated by addition of 25 µL of biomol red reagent and mixed thoroughly by

repeated pipetting. Test compounds were dissolved in dimethyl sulfoxide (DMSO)

and solution of 100µM concentration was prepared of which 10 µL solution was

added in each reaction well to achieve final concentration of 10 µM in reaction

mixture. Volumes and dilution of other component were made accordingly as

instructed in the manual provided in the kit. The 50 µL “reaction” may consist either

of PTP1B phosphatase acting on the phosphopeptide substrate or free phosphate.

The detection of free phosphate released is based on classic Malachite green

assay.168 After adding biomol red to reaction wells after 30 min of incubation as

described earlier the plate was incubated for another 20 min to develop the colour.

Absorbance was recorded at 620 nm on a microplate reader. The percentage

inhibition of PTP1B enzyme by test compounds was calculated based on activity in

the control tube (without inhibitor) taking as 100 % from three independent set of

experiments. The concentration of dimethyl sulfoxide (DMSO) in the test well (1.0 %)

had no demonstrable effect on PTP1B enzyme activity.

1A.3.1.2 In vivo study

Experimental Animals

Male albino rats of Sprague Dawley strain of 8 to 10 weeks of age and 160 ±

20 g of body weight were procured from the animal colony of the Institute. Research

on animals was conducted in accordance with the guidelines of the Committee for




Part 1A

Page | 58

the Purpose of Control and Supervision of Experiments on Animals (CPCSEA)

formed by the Government of India in 1964. The institute has taken permission from

the animal ethical committee for the work (No. 129/07/Biochem/IAEC). Rats were

always placed in the group of 5 to 6 in polypropylene cages. The following norms

were always followed for animal room environment: temperature 23 ± 2°C; humidity

50-60%; light 300 lux at floor level with regular 12 h light cycle; noise level 50 decibel;

ventilation 10-15 air changes per hour. The animals had free access to pellet diet and

tap water unless stated otherwise.

Preparation of test samples and the standard antidiabetic drug:

The test compounds and standard antidiabetic drug Metformin were prepared

in 1.0 % freshly prepared Gum acacia.169

Evaluation of test compounds for antihyperglycaemic activity in Streptozotocin

(STZ)-induced diabetic rats:

Induction of diabetes in rats:

Streptozotocin (STZ) is a broad spectrum antibiotic and selected to induce

experimental diabetes because of its greater selectivity of β-cells, lower mortality and

relatively longer half life (15 min) of STZ in the body. A solution of STZ (60 mg/kg) in

100 mM citrate buffer, pH 4.5 was prepared and calculated amount of the fresh

solution dosed to overnight fasted rats intraperitoneally. Two days later baseline

blood glucose was drawn from tail vein and glucose levels determined by glucostrips

(Roche) to confirm the induction of diabetes.

Animal Modelling, Grouping and Treatment

Assessment of antihyperglycaemic effect by measuring fall in blood glucose

level on Streptozotocin treated diabetic rats

Rats having hyperglycaemia of the range of 270 and 450 mg/dL were

considered as diabetic, selected and divided into groups of five animals each. One

group used for normal control receives only vehicle (gumacacia) and this group was

considered as diabetic control. The blood glucose measured at this time was termed

the baseline (0 min) blood glucose. Rats of experimental groups were orally

administered suspension of the desired test samples (made in 1.0% gum acacia) at

desired dose levels and the biguanide derivative Metformin was used as standard

antidiabetic drug and was always given at a dose of 100 mg/kg body weight orally to

the experimental group. After 30 min of drug treatment, blood glucose level was

again measured with glucometer. The blood glucose assessment were collected

from tail vein just prior administration of test sample i.e. 0, 30, 60, 90, 120, 180, 240,




Part 1A

Page | 59

300 and 1440 min post test sample administration. After 300 min the STZ treated

animals were allowed to feed over night to overcome drug induced hypoglycaemia.

The animals were fed ad lib during 5 to 24h of experiments. The average fall in AUC

(area under curve) in experimental group compared to control group was termed as

% antihyperglycaemic activity. Statistical analysis was done by Dunnett’s test.166

Statistical analysis:

Quantitative glucose tolerance of each animal was calculated by area under

curve (AUC) method using Prism Software. Comparing the AUC of experimental and

control groups determined the percentage antihyperglycaemic activity. Statistical

comparison was made by Dunnett’s test.166 Results were expressed as mean ± SEM.

Synthesis, SAR and Docking Studies of Substituted Aryl

phenylthiazolyl phenylcarboxamide as potential Protein Tyrosine

Phosphatase 1B Inhibitors

Part 1B

Page | 60

Part 1B: Synthesis, Structure Activity Relationship (SAR) and Docking Studies of Substituted Aryl Phenylthiazolyl Phenyl carboxamide as Potential Protein Tyrosine Phosphatase 1B Inhibitors

Protein tyrosine phosphatase 1B (PTP1B) is been considered a tried and

tested molecular target for the development of novel insulin-sensitizer agents

addressing both T2DM and obesity.100,149,170 Due to the highly conserved and

charged nature of the PTP active site identification of selective, safe and orally

available small molecule, PTP inhibitors has been a challenge. However, the

presence of unique features in the loops bordering the catalytic site of each PTP can

be used for the structure based design of selective inhibitors. A secondary,

noncatalytic, arylphosphate binding site located close to the PTP1B active site is not

present in all PTPs, thus providing a structural basis for the targeted design of

selective bidentate inhibitors that can simultaneously occupy both the active site and

the nearby noncatalytic site.1, 3,171-172

In Part 1A the design of novel substituted aryl thiazolyl phenylsulphonamides

as nonphosphorous small molecule inhibitors of PTP1B using fragment based

approach has been disclosed, where the compound 116a (IC50 1.08µM) has been

described as the most active PTP1B inhibitor. Docking studies also revealed that this

molecule showed all the relevant interactions essential for PTP1B inhibition.

However, many carboxamide derivatives are also known as potent PTP1B inhibitors

in literature. 172-176 Inspired by the key structure of N-phenyl-4,5-dihydrothiazol-2-

amine in part 1A, it appeared of interest to replace the sulfonyl moiety in the

compound 116a by benzoyl group (region II) affording compound 117 and observe its

impact on the PTP1B inhibitory activity (Fig. 7). Interestingly, the compound 117

showed moderate activity with 50.5% PTP1B inhibiton although less active as

compared to compound 116a (73.6 % PTP1B inhibition) with sulfonyl moiety. As

many carboxamide core are reported as PTP1B inhibitors in literature,172-176 further

structural modification were done on compound 117 (50% PTP1B inhibition) at

Region I, II and III to improve PTP1B inhibitory activity.

Dihydrothiazolyl moiety (Region I) in compound 117 was substituted by

thiazolyl group and substituted phenyl groups were introduced to its C5 position to




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Figure 7: Optimization of compound 117

investigate if the phenyl ring may provide more extension of the molecule from the

active site towards the site C ( Arg47, Asp48, Phe182) of PTP1B enzyme. Further

studies were centered on systemic substitution by different groups around region II

and III and subsequently identified a series of substituted thiazolyl-N-phenyl-

benzamide derivatives as PTP1B inhibitors with good PTP1B activity and improved in

vivo efficacy. The results of these studies are presented herein.

1B.1 Result and Discussion

1B.1.1 Chemistry

The compound 117 was synthesized using 2-trifluoromethyl aniline (111c)

according to the procedure mentioned in Scheme 1 of Part 1A. Rest of the new

compounds 131-154 were synthesized from the cyclized intermediates 123-130

according to literature procedure.177 Phenylcarbamothioyl benzamides (111d-111e

and 118b) and thioureas (112a-112b and 119) were synthesized according to the

procedure mentioned in Part 1A (Table 8). Condensation of the 1-(4-

fluorophenyl)thiourea (112a) with 2-bromo-4’-methylacetophenone (120) in THF at

room temperature for 30 min resulted in the formation of suspension, which was

filtered and dried to afford N-(4-fluorophenyl)-4-p-tolylthiazol-2-amine (123), which on

reaction with 4-chlorobenzoyl chloride gave the desired compound 4-chloro-N-(4-

fluorophenyl)-N-(4-p-tolylthiazol-2-yl)benzamide (131) (Scheme 2). All the other final

compounds 131-154 were synthesized according to the same procedure described

above from key intermediates 123-130 (Table 8 and 9).




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Table 8 : Key intermediates synthesized (123-130)

Compd No. R R1 R2 R3 n

123 H Fluoro H Me 0

124 H Fluoro H OMe 0

125 H Fluoro H H 0

126 OMe H OMe Me 0

127 OMe H OMe OMe 0

128 OMe H OMe H 0

129 H OMe H Me 2

130 H OMe H OMe 2

The compound 132 and 133 were synthesized using 123 and,

hydrocinnamoyl chloride and phenyl acetylchloride respectively. The compounds

134-136 were synthesized using 124 and 4-chlorobenzoylchloride, hydrocinnamoyl

chloride and phenylacetylchloride respectively. The compounds 137-139 were

synthesized using 125 and 4-chlorobenzoylchloride, hydrocinnamoyl chloride and

phenylacetylchloride respectively.




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Table 9: In vitro PTP1B enzyme inhibitory and in vivo antihyperglycaemic activity in STZ model for compound 117,131-154

Compd.

No

R

R1

R2

R3

R4

n

% inhibition at 10 µM

a

Blood glucose levels

b

0-5h 0-24h

117 CF3 H H - 4-OMePh - 48 - -

131 H F H Me 4-ClPh 0 35.2 - -

132 H F H Me CH2CH2Ph 0 33.0 - -

133 H F H Me CH2Ph 0 60.3 (8.0) 14.1 23.2

134 H F H OMe 4-ClPh 0 56.9 (8.8) - -

135 H F H OMe CH2CH2Ph 0 74.7 (7.0) - -

136 H F H OMe CH2Ph 0 9.8 - -

137 H F H H 4-ClPh 0 37.2 - -

138 H F H H CH2CH2Ph 0 69.0 (6.9) 2.44 11.7

139 H F H H CH2Ph 0 24.7 - -

140 OMe H OMe Me 4-ClPh 0 26.5 - -

141 OMe H OMe Me CH2CH2Ph 0 56.2 (8.6) - -

142 OMe H OMe Me CH2Ph 0 63.6 (7.7) 11.4 15.8

143 OMe H OMe OMe 4-ClPh 0 64.1 (7.55) 13.4 17.8

144 OMe H OMe OMe CH2CH2Ph 0 64.3 (7.34) 10.6 9.56

145 OMe H OMe OMe CH2Ph 0 75.0 (6.3) 17.6 25.1*

146 OMe H OMe H 4-ClPh 0 71.3 (7.33) 2.48 6.78

147 OMe H OMe H CH2CH2Ph 0 52.7 (8.6) - -

148 OMe H OMe H CH2Ph 0 57.2 (7.94) 18.3 16.6

149 H OMe H Me 4-ClPh 2 9.8 - -

150 H OMe H Me CH2CH2Ph 2 24.6 - -

151 H OMe H Me CH2Ph 2 21.6 - -

152 H OMe H OMe 4-ClPh 2 14.6 - -

153 H OMe H OMe CH2CH2Ph 2 69.9 (6.9) 6.10 13.2

154 H OMe H OMe CH2Ph 2 76.6 (5.8) 22.8* 23.7**

Suramin - - - - - - 67.3 - -

Sodiumorthovanadate - - - - - -- 24.3* 30.1**

Values are mean ± SEM of six values, p< 0.05 * p<0.01**, a

(IC50 in µM), b

% lowering at the dose of 100 mg/kg)

The compounds 140-142 were synthesized using 126 and 4-chlorobenzoyl

chloride, hydrocinnamoylchloride and phenylacetylchloride respectively. The compo-

-unds 143-145 were synthesized using 127 and 4-chlorobenzoylchloride, hydrocinna-




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-moylchloride and phenylacetylchloride respectively. The compounds 146-148 were

synthesized using 128 and 4-chlorobenzoylchloride, hydrocinnamoyl chloride and

phenylacetylchloride respectively. The compounds 149-151 were synthesized using

129 and 4-chlorobenzoylchloride, hydrocinnamoyl chloride and phenylacetylchloride

respectively. The compounds 152-154 were synthesized using 130 and 4-chloro

benzoylchloride, hydrocinnamoylchloride and phenylacetylchloride respectively.

All the final compounds 131-154 were identified by their detailed spectral

studies such as IR, 1H NMR, 13C NMR and Mass spectroscopy as discussed below.

The cyclization of 112a to 123 was confirmed by the 1H NMR spectra as the NH2

peak at δ 8.30 ppm in 112a was found absent in 123. Moreover, the compound 123

showed singlet peak at δ 3.81 ppm for the methyl group attached to phenyl at C5

position of thiazolyl ring. The singlet at δ 6.66 ppm for NH group in compound 123

was found absent in the final carboxamide derivative (131). The IR spectrum of final

carboxamide derivative (131) also showed characteristic absorption band in the

region 1656 cm-1 characteristic for the keto group confirming the attachment of

benzoyl group to the compound 123. Its 13C NMR spectrum also supported the

structure which showed peaks at δ 21.2 (CH3), 107.4 (S-CH) and displayed

characteristic carbonyl carbon signal at δ 158.5 (NH-CO). The final support for the

formation of product came from its mass spectrum, which showed M+1 molecular ion

peak at 422.

Similarly, all other final compounds N-(4-fluorophenyl)-3-phenyl-N-(4-p-

tolylthiazol-2-yl)propanamide (132), N-(4-fluorophenyl)-2-phenyl-N-(4-p-tolylthiazol-2-

yl)acetamide (133), 4-chloro-N-(4-fluorophenyl)-N-(4-(4-methoxyphenyl)thiazol-2-yl)-

-benzamide (134), N-(4-fluorophenyl)-N-(4-(4-methoxyphenyl)thiazol-2-yl)-3-phenyl-

-propanamide (135), N-(4-fluorophenyl)-N-(4-(4-methoxyphenyl)thiazol-2-yl)-2-pheny-

-l acetamide (136), 4-chloro-N-(4-fluorophenyl)-N-(4-phenylthiazol-2-yl)benzamide

(137), N-(4-fluorophenyl)-3-phenyl-N-(4-phenylthiazol-2-yl)propanamide (138), N-(4-

fluorophenyl)-2-phenyl-N-(4-phenylthiazol-2-yl)acetamide (139), 4-chloro-N-(2,5-dim-

-ethoxyphenyl)-N-(4-p-tolylthiazol-2-yl)benzamide (140), N-(2,5-dimethoxyphenyl)-3-

phenyl-N-(4-p-tolylthiazol-2-yl)propanamide (141), N-(2,5-dimethoxyphenyl)-2-pheny-

-l-N-(4-p-tolylthiazol-2-yl)acetamide (142), 4-chloro-N-(2,5-dimethoxyphenyl)-N-(4-(4-

methoxyphenyl)thiazol-2-yl)benzamide (143), N-(2,5-dimethoxyphenyl)-N-(4-(4-meth-

-oxyphenyl)thiazol-2-yl)-3-phenylpropanamide (144), N-(2,5-dimethoxyphenyl)-N-(4-(-




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-4-methoxyphenyl)thiazol-2-yl)-2-phenylacetamide (145), 4-chloro-N-(2,5-dimethoxy-

-phenyl)-N-(4-phenylthiazol-2-yl)benzamide (146), N-(2,5-dimethoxyphenyl)-3-phen- -

yl-N-(4-phenylthiazol-2-yl)propanamide (147), N-(2,5-dimethoxyphenyl)-2-phenyl-N-

(4-phenylthiazol-2-yl)acetamide (148), 4-chloro-N-(4-methoxyphenethyl)-N-(4-p-tolylt-

-hiazol-2-yl)benzamide (149), N-(4-methoxyphenethyl)-3-phenyl-N-(4-p-tolylthiazol-2-

yl)propanamide (150), N-(4-methoxyphenethyl)-2-phenyl-N-(4-p-tolylthiazol-2-yl)acet-

-amide (151), 4-chloro-N-(4-methoxyphenethyl)-N-(4-(4-methoxyphenyl)thiazol-2-yl)-

-benzamide (152), N-(4-methoxyphenethyl)-N-(4-(4-methoxyphenyl)thiazol-2-yl)-3-p-

-henylpropanamide (153) and N-(4-methoxyphenethyl)-N-(4-(4-methoxyphenyl)thiaz-

-ol-2-yl)-2-phenylacetamide (154) were characterized on the basis of spectral data.

The spectral data are reported in experimental part.

1B.1.2 In vitro and Structure Activity and Relationship Study

A set of twenty five substituted phenylthiazolyl-N-phenylbenzamide derivative

(117, 131-154) with different functionalities at R, R1, R2, R3 and R4 were synthesized

and evaluated against PTP1B. The effect of the synthesized compounds on PTP1B

was studied using colorimetric, non-radioactive PTP1B drug discovery kit-BMLAK

822 from Enzo Life Sciences, USA. The assay was done according to the Kit protocol

and first all the compounds were evaluated for PTP1B inhibition at 10 µM

concentration. Further compounds showing more than 57% inhibition against PTP1B

were evaluated at five different concentrations to calculate their IC50 value. Suramin

was used as a reference standard.

The antidiabetic activity including PTP1B inhibitory activities of the thiazolyl-

N-phenylcarboxamide derivatives are presented in Table 9. It was observed from the

results that the systemic substitution at R, R1, R2, R3 and R4 positions influenced the

biological activity of the synthesized analogues. Initial SAR studies were focused on

C5 position of thiazolyl ring (R3) to explore if binding with the non catalytic binding

site of PTP1B could be enhanced. Therefore, as a starting point -Me group was

inserted at R3 position and was kept constant to see the effect of R, R1, R2 and R4

substituent on the PTP1B inhibition. Compound 133 having R, R2 as H, R1 as fluoro,

R3 as Me and R4 as CH2Ph showed 60.3% PTP1B inhibition. Replacement of R, R2

with OMe, R1 as H and keeping R4 as CH2Ph in compound 142 showed slight

increase in the activity (63% PTP1B inhibition). However, on substituting 2,5-OMe (R,

R2=OMe, R1=H) by 4-OMePhCH2CH2 (R, R2=H, R1=OMe, n=2) and R4 as CH2Ph in




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compound 151 led to great decrease in the activity. Further, replacement of R4 group

in compound 133 by 4-ClPh to give compound 131(35% PTP1B inhibition) and -

CH2CH2Ph to give compound 132 (33% inhibition) led to reduced activity as

compared to compound 133. Similar trends were observed in analogous compounds

140, 141 with 2,5-OMe (R, R2=OMe, R1=H) and 150 with 4-OMePhCH2CH2 (R,

R2=H, R1=OMe, n=2) substitutions.

To further explore the SAR Me group at R3 position in the above compounds

were replaced by OMe group. In general, the compounds with OMe at R3 position

were more active in comparison to compounds with Me group except the compound

136 and 152 which showed less than 15% PTP1B inhibition. The compound 154 (R,

R2=H, R1, R3=OMe, n=2, R4=CH2Ph) was found to be the most active compound of

the series with 76.6% PTP1B inhibition. Replacement of R, R1 and R2 in compound

154 by R, R2, R3=OMe, R1=H and R4 as CH2Ph gave the compound 145 as the

second most active compound of the series with 75% PTP1B inhibition. However,

insertion of F group at R1 in place of H and R, R2=H in place of OMe led to least

activity in compound 136 (9.8% PTP1B inhibition). Further, the replacement of

CH2Ph group at R4 position by CH2CH2Ph led to slight reduction in the activity in the

compound 153 (R, R2=H, R1=OMe, n=2, R3= OMe, R4=CH2CH2Ph, 69.9% PTP1B

inhibition) and 144 (R, R2=OMe, R1=H, R3=OMe, 64.3% PTP1B inhibition). In

contrast to compound 153 and 144, compound 135 (R, R2=H, R1=F, R3=OMe and

R4=CH2CH2Ph) showed increased activity with 74.7% PTP1B inhibition. Incorporation

of 4-ClPh group in place of CH2CH2Ph at R4 position in compound 137 (R, R2=H,

R1=F, R3=OMe) showed decrease in activity with 56.9% PTP1B inhibition. Similar

trend was observed in case of compound 146 (R, R2=OMe, R1=H, R3=OMe, 64.1%

PTP1B inhibition) but in compound 152 (R, R2=H, R1=OMe, n=2, R3=OMe) which

showed highly reduced activity with 14.6% PTP1B inhibition in comparison to its

analogous compound. Removal of the 4-substituent in R3 position and keeping it as

H resulted in further decrease in the PTP1B inhibitory activity in comparison to their

analogues. The compound 138 and 147 showed slight increase in activity in

comparison to counter analogues with 69% and 71% PTP1B inhibition respectively.

1B.1.3 Kinetics Measurements and Mechanism of Inhibition

The compound 154 inhibits the PTP1B, a tyrosine phosphatase enzyme

having an important role in insuln signalling. Kinetic study was done to determine the




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type of inhibition of the compound by using PTP1B tyrosine phosphate drug

discovery kit BML AK 822 from Enzo life science USA, as used previously to

determine the % inhibition of PTP1B by the test compounds. For determination of

type of inhibition the activity assay was performed at different concentration of 0µM,

5 µM, 10µM and 20µM of the most active compound 154 with varying concentration

of substrate IR5 (sequence from the insulin receptor ß subunit domain provided in

the kit) from 40 to 140 µM and drawn the Lineweaver-Burk double reciprocal plot

(Fig. 8). The Plot shows that intercept of all lines obtained with 0µM, 5µM, 10µM and

20µM compound concentration converging at y-axis (1/Vmax ), whereas slope and x-

axis intercept (-1/Km ) vary with inhibitor concentration which suggest that inhibitor

follows a competitive inhibition kinetics therefore, compound 154 is a competitive

inhibitor of PTP1B.

Figure 8: Competitive inhibitory profile of compound 154

From this plot Km for the subsrate IR5 was calculated and found to be 60.1

µM. The Ki value was determined by plotting the slope values vs [I]. The resulting

secondary plot or "replot" have a yaxis intercept of Km/Vmax and a slope of Km/Vmax Ki.

The value of Ki was calculated from intercept on x-axis of this replot and found 8.59

µM.




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1B.1.4 Docking Studies

To elucidate the essential structural requirements for the active molecule

acting on the PTP1B enzyme, docking studies were done on the most active

compound 154 on the PTP1B enzyme active site to gain an insight and

understanding of the important interactions with the crucial amino acid residues. The

binding pose analysis of the most active compound 154 revealed that 4-

methoxyphenyl group at the C5 position of the thiazolyl ring binds well with the active

site of PTP1B and shows good interaction with Ser216, Phe182, Arg221. It also

shows water molecule (325 and 600) mediated hydrogen bond interactions with

amino acid residue Asp181. These water molecules (325 and 600) mediated H-

bonds also play a key role in the binding mode of the co-crystallized ligand (PDB ID-

2ZMM; 161 Fig. 9). Tyr46 was anticipated to establish aromatic stacking interactions

with the thiazolyl scaffold which are commonly observed in many known

PTP1B/inhibitor complexes.113,119,178,179 An additional interaction through hydrogen

bond between Tyr46 and the CH2 of phenyl acetyl group of compound 154 was also

possible.

The amino acid residues close to the active site (Arg47 and Asp48; Site C)

have been demonstrated to be residues for addressing selectivity because of the

point mutation between PTP1B/TCPTP and other phosphatases such as LAR

(Asn48) and SHP-2 (Asn48). 162,163 The phenyl acetyl group in the compound 154

showed potential to form hydrogen bond interaction with Asp 48 and was in close

contact to Arg47. Thus the compound 154 may provide selectivity for PTP1B over

other PTPs. Because of these multiple interactions, compound 154 positioned itself

effectively at the enzyme active site to serve as a potent inhibitor.

1B.1.5 In vivo Biological Activity

(a) Streptozotocin Rat Model Study

Based on the high PTP1B inhibitory activity in vitro, a limited number of

compounds with more than 57% PTP1B inhibition were evaluated in vivo in

Streptozotocin induced rats model (STZ). Table 9 depicts the effect of submitted ten

compounds 133, 138, 142-146, 148, 153, 154 and standard compound Sodium

orthovanadate on decline in blood glucose level of streptozotocin treated diabetic




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rats. Sodium orthovanadate was taken as positive control. It is evident from the

results that out of ten compounds tested only one compound i.e. compound 154

Figure 9: A) Pose attained by compound 154 (pink) and co-crystallized ligand of PDB Id:

2ZMM (green) in the active site of PTP1B; B) Binding site cavity of PTP1B with 154; C)

Schematic 2D plots of binding site observed for 154 and PTP1B (PDB ID:2ZMM); D) Binding

pose surface view of the compound 154 with PTP1B.

showed significant decline in blood glucose levels on streptozotocin induced diabetic

rats where the decline in blood glucose level was around 22.8 % (p<0.05) and 23.7

% (p<0.01) during 0-5h and 0-24h, respectively (Fig. 10A and B). Standard drug

sodium orthovanadate demonstrated maximum decline in blood glucose levels to the

tune of 24.3 % (p<0.05) during 0-5h and 30.1 % (p<0.01) during 0-24h, post

treatment respectively on STZ-induced diabetic rats at 100 mg/kg oral dose.

Compound 133, 138, 142-146, 148 and 153 though showed decline in blood glucose

to the tunes of 14.1 %, 2.44 %, 11.4 %, 13.4 %, 10.6 %, 17.6 %, 2.48 %, 18.3 % and

6.10 %, during 0- 5h and 23.2 %, 11.7 %, 15.8 %, 17.8 %, 9.56%, 25.1 % (p<0.05),

3.78 %, 16.6 %, and 13.2 % during 0-24h, respectively on STZ-induced diabetic rats




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at 100 mg/kg oral dose level, however the effect was not found statistically significant

in any of the case except compound 154 during 0-24 h where the decline in blood

glucose was observed around 23.7 %. (Fig.10)

Figure 10: A and B) Effect of compound 133, 138, 142-146, 148 and Standard compound

Sodium orthovanadate on blood glucose levels of the streptozotocin treated diabetic rats at

various time intervals, C and D) Area under curve (AUC) at 5h for the test compounds, E and

F) Area under curve (AUC) at 24h for the test compounds.




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(b) db/db Mouse Model Study

Effect on hyperglycaemia

Inspired by the findings the most potent compound 154 was further evaluated

for antihyperglycaemic and antidyslipidemic activities in C57BL/KsJ-db/db mice

(Table 10). Antihyperglycaemic activity of the most active compound 154 was carried

out in vivo using db/db mice by observing overall glucose lowering effect and also

improvement on oral glucose tolerance at 30mg/kg for the period of 15 days.

Pioglitazone was taken as positive control. It is evident from the Fig. 11A that

compound 154 exerted its effect on blood glucose from day 5 while the significant

effect was observed from day 7 and persisted till the end of the experiment, whereas,

Pioglitazone (at the dose of 10mg/kg) significantly declined the random blood

glucose from day 6 which persisted up to the end of the experiment as compared to

vehicle treated control group.

Table 10: Antihyperglycaemic and antidyslipidemic activity in db/db mice( % efficacy 15 days)

Compd no Antihyperglycaemic Antidyslipidemic Insulin resistant reversal

OGTT(15 days)

PTT ITT TG CHOL HDL Fasting blood glucose

Serum insulin level

HOMA index

154 36.6 25.7 39.2 18.5 11.5 27.3 44.5 39.4 66.6

Pioglitazone 52.9 35.8 62.4 12.6 9.17 11.1 64.7 46.1 70.3

Effect on oral glucose tolerance

To observe the effect of drug on glucose tolerance an OGTT was conducted

on day 14 during the course of dosing in overnight fasted mice. After 14 days of

consecutive dosing an oral glucose tolerance test was carried out and the results

showed that inhibition of PTP1B by compound 154 effectively resisted the rise in

postprandial hyperglycaemia during 30 and 60 min post glucose load and

significantly enhanced the glucose clearance from blood at 60, 90 and 120 time

points (Fig. 11B) and an overall improvement of 36.6% (p<0.01) on glucose disposal

was calculated by AUC analysis (0-120min) with no observed hyperglycaemia,

whereas, reference drug Pioglitazone showed an improvement of 52.9% (p<0.01) on

glucose AUC as shown in Fig. 11C. The fasting baseline blood glucose value at 0

time point was also found lower in compound 154 treated groups compared to




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vehicle treated control group at corresponding time point because of

antihyperglycaemic effect. It is evident from the graph that compound 154 and

Pioglitazone effectively declined the rise in postprandial hyperglycaemia induced by

glucose load of 3g/kg.

Figure 11: Antihyperglycaemic profile and insulin resistance reversal profile of db/db mice.

(A) Random blood glucose, (B) Oral glucose tolerance, (C) Glucose AUC (OGTT), (D) ITT,

(E) AUC (ITT), (F) PTT, (G) AUC (PTT), (H) FBG, (I) serum Insulin (J) HOMA-index.

To assess the whole body insulin sensitivity, insulin tolerance test (ITT) was

performed in vehicle treated, compound 154 and Pioglitazone treated mice. It was




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found that compound 154 significantly improved the insulin sensitivity at 15 and 30

min after a bolus of 0.75 unit of human insulin as showed in Fig. 11D and area under

the curve (AUC) analysis also showed a 39.2% improvement in insulin tolerance (Fig.

11E) as compared to vehicle treated control group, whereas reference drug

Pioglitazone improved the insulin sensitivity by 62.4% (p<0.01). Further to assess the

effect of compound 154 on hepatic insulin sensitivity, pyruvate tolerance test was

performed by intraperitoneally administrating pyruvate, a major gluconeogenic

substrate. The results of pyruvate tolerance test showed that compound 154

significantly increased the insulin sensitivity and effectively resisted the rise in blood

glucose (Fig. 11F) caused by a bolus of pyruvate injection as compared to vehicle

treated control group. AUC analysis indicated an improvement of 25.7 and 35.8%

(p<0.01) (Fig. 11G) on pyruvate tolerance by compound 154 and reference drug

Pioglitazone, respectively as compared to vehicle treated control group.

Effect on fasting blood glucose, serum insulin level, HOMA-index.

Insulin resistance is one of the characteristic features of db/db mice.

Decreased insulin sensitivity leads to hyperglycaemia, hyperinsulinemea. Treatment

with compound 154, near normalizes the fasting blood glucose by 44.5% (p<0.01)

and restores the altered insulin level by 39.4% (p<0.01) in treated diabetic mice as

compared to vehicle treated control group as shown in Fig. 11H and 11I.

Homeostatic model assessment (HOMA) is a method used to quantify insulin

resistance that is quantified on the basis of fasting blood glucose and fasting serum

insulin level, treatment with compound 154 significantly improves the insulin

resistance state by improving the HOMA-index by 66.6% (p<0.01) (Fig. 11J).

Antidyslipidemic activity

Type 2 diabetes is known to be associated with several adverse

cardiovascular risk factors including obesity, hypertension and serum lipid

abnormalities, characterised mainly by elevated serum total triglycerides and low

levels of high density lipoprotein (HDL) cholesterol. Therefore, the most active

compound of the series compound 154 was further tested for serum lipid profile.

Treatment of compound 154 restores the altered serum lipid profile as evident from

the Fig.12 (A, B and C). The compound 154 significantly lowers the triglyceride and

serum cholesterol level by 18.5% and 11.5 % (p<0.05) respectively, with significant

increase in serum HDL-cholesterol by 27.3.8% (p<0.05) in comparison to the




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standard drug Pioglitazone which significantly declines the serum triglyceride and

cholesterol level by 12.6% (p<0.05)and 9.7% respectively.

Excessive consumption of food intake and body weight is responsible for the

development of obesity and it can be directly linked to type 2 diabetes. Further

observation on body weight during the antidiabetic activity in db/db mice study

resulted in decrease of body weight in mice during the treatment of compound 154

(Fig. 12D) .

Figure 12: Effect of compound 154 on serum lipid profile of db/db mice, A) Triglyceride; B)

Cholesterol; C) HDL-c; D) Body weight.

Western blot analysis of p-IRSI in skeletal muscle of db/db mice

Further, the inhibition of PTP1B by compound 154 was evident by western

blot analysis as showed in Fig.13A. Compound 154 effectively inhibited the PTP1B

which is evident with increase of approximately 2.34 fold increase level of p-IRS-1

and 1.95 fold increase in level of p-Akt, a downstream receptor of insulin signaling,

while reference drug Rosiglitazone exhibited an increase of 3.21 and 2.52 folds in p-

IRS1 and p-Akt respectively, as compared to vehicle treated control group. (Fig.13 B

and C). The activation of IRS1 and Akt by insulin results in clearance of circulating

blood glucose which is evident with the significant increase in protein level of Glut4 in

skeletal muscle. In the present study, it was observed that the inhibition of PTP1B

with compound 154 resulted in improved insulin signaling and glucose homeostasis

with an increase of approximately 2.1 fold in Glut4 (Fig. 13D).




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Figure 13: Inhibition of PTP1B by compound 154 improved insulin signaling in skeletal

muscle of db/db mice. A) Western blot analysis of p-IRSI, p-Akt and Glut4 in skeletal muscle

of db/db mice, 40 µg of protein was resolved on SDS-PAGE; B) Evaluation for levels of p-

IRS1; C) Evaluation for levels of p-Akt; D) Evaluation for levels of Glut4. The experiments

were repeated three times and values are means ± SEM of three independent experiments.

The blot shown were representatives of the indicated groups and the densitometric analyses

of the same are given below. *p< 0.05, **p < 0.01

1B.2 Conclusion

A series of aryl phenylthiazolyl phenylcarboxamide derivatives were

synthesized and evaluated against PTP1B enzyme. Among the twenty five

synthesized compounds six compounds showed good PTP1B inhibitory activity

namely 135 (IC50=7.0 µM), 138 (IC50=6.9 µM), 145 (IC50=6.3 µM), 146 (IC50=7.33

µM), 153 (IC50=6.9 µM) and 154 (IC50=5.8 µM). The docking studies on the most

active compound 154 provided rational explanation to its higher potency as

compared to the corresponding derivative 117. The compound 154 interacts well with

the active site residues Arg221, Asp181 and Gln262. It also showed good interaction

with Arg47 and Asp48 which may attribute to its selectivity over highly homologous

phosphtase TCPTP. Further, the compound 154 also showed promising

antihyperglycaemic, antidyslipidemic and insulin resistant reversal activities in vivo, in

STZ model and db/db mice model. Thus, these studies may be helpful in developing

novel PTP1B inhibitors with improved pharmacological properties.




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1B.3 Experimental


silicon oil bath. Reactions were monitored by thin layer chromatography on self-made

plates of silica gel G (Merck, India) or 0.25mm ready-made plates of silica gel 60F

254, (Merck, Darmstadt, Germany). Column chromatography was performed on silica

gel (Merck, 60 to 120mesh). Infrared spectra (IR) were recorded on Perkin- FTIR

model PC spectrophotometer with frequency of absorptions reported in wave

numbers. Mass were recorded on JEOL spectrometer with fragmentation pattern

reported as values, 1H NMR was recorded on Bruker spectrometer with a



were described in parts per million (ppm) relative to TMS (0.00 ppm) using scale and


N-(4-fluorophenylcarbamothioyl)benzamide (111a) and N-(2,5dimethoxyphenyl

carbamothioyl)benzamide (111b)

These compounds were prepared in a similar manner as discussed in chapter1 Part

1A.

N-(4-methoxyphenethylcarbamothioyl)benzamide (118b)


111a using benzoylisothiocyanate (3.2g) and 4-methoxyphenethyl amine (3.02g).

Yield 83%; mp 115-117oC; IR (KBr cm-1): 774, 856, 1270, 1516, 1600, 1666, 2368,

3411, 3547; 1H NMR (CDCl3 ): δ 10.76 (bs,1H), 9.00(bs,1H), 7.83-7.86(m, 2H), 7.61-

7.67(m,1H), 7.50-7.55(m, 2H), 7.20(m, 2H), 6.87-6.92(m,2H), 3.94-3.98(m,2H),

3.82(s,3H), 3.00(t, J= 6Hz, 2H); MS(ESI+): m/z 315 (M+H)+

1-(4-fluorophenyl)thiourea (112a) and 1-(2,5-dimethoxyphenyl)thiourea (112b)

These compounds were prepared in a similar manner as discussed in chapter1 Part

1A.




Part 1B

Page | 77

1-(4-methoxyphenethyl)thiourea (119)


112a using 118a (4.41g).

Yield 79%; mp 161oC; IR (KBr cm-1): 710, 822, 1220, 1516, 1634, 1695, 2364, 3346,

3450; 1H NMR (CDCl3): δ 2.87(t, j= 6Hz, 2H), 3.39-3.42 (m,2H), 3.77 (bs, 1H),

3.82(s,3H), 6.87-6.90(m, 2H), 7.17-7.20 (m,2H), 7.45 (s,1H); MS(ESI+): m/z 211

(M+H)+.

N-(4-fluorophenyl)-4-p-tolylthiazol-2-amine (123)

The reaction of the equimolar (0.01 mol) amounts of the particular thiourea

(112a, 1.71g) and substituted 4-methyl-2-bromoacetophenone (120, 2.13g) in THF at

room temperature for 30 min resulted in the formation of a suspension, which was

filtered and dried to yield the key intermediate 123 in high yield.

Yield 87% mp 202oC; IR (KBr cm-1): 759, 1103, 1216, 1406, 1510, 1596, 1614, 2402,

3021, 3428; 1H NMR (CDCl3): δ 2.40 (s, 3H,CH3), 6.6 (s, NH), 7.15-7.18 (m, 2H),

7.20-7.28 (m, 3H), 7.31-7.39 (m, 2H), 7.40-7.41(m,2H); MS(ESI+): m/z 285 (M+H)+

The other compounds 124-130 were synthesized by the above procedure using

corresponding substituted thiourea 112(a-b), 119 and 2-bromoacetophenones (120-

122)

N-(4-fluorophenyl)-4-(4-methoxyphenyl)thiazol-2-amine (124)

The compound 124 was prepared by similar method as described for the synthesis of

123 using 121 (2.27g) and 112a (1.71g).

Yield 93%; mp 100oC; IR (KBr cm-1): 760, 1030, 1216, 1409, 1509, 1620, 2401,

3021, 3430; 1H NMR (CDCl3): δ 3.86 (s, 3H, CH3), 6.6 (s, NH), 6.96-7.03 (m, 2H),

7.17-7.22 (m, 2H), 7.44-7.48 (m, 2H), 7.67-7.70(m,2H), 7.96-7.99(m,1H); MS(ESI+):

m/z 301 (M+H)+

N-(4-fluorophenyl)-4-phenylthiazol-2-amine (125)


123 using 122 (1.97g) and 112 (1.71g).




Part 1B

Page | 78

Yield 92%; mp 168oC; IR (KBr cm-1): 760.77, 1216, 1512, 1612, 1692, 2401, 3021,

3430; 1H NMR (CDCl3): δ 6.67(s, NH), 7.17-7.25(m,2H), 7.34-7.45 (m,2H), 7.50-

7.55(m,3H), 7.78-7.83(m,2H); MS(ESI+): m/z 271 (M+H)+

N-(2,5-dimethoxyphenyl)-4-p-tolylthiazol-2-amine (126)


123 using 120 (2.13g) and 112b (2.13g).


3021, 3423; 1H NMR (CDCl3): δ 2.37 (s, 3H, CH3), 3.80(s, 6H, 2xOCH3), 6.6 (s, NH),

6.79-6.82 (m, 1H), 6.93-6.98 (m, 2H), 7.26-7.29 (m, 2H), 7.71-7.74(m,2H), 11.13

(s,1H); MS(ESI+): m/z 327 (M+H)+

N-(2,5-dimethoxyphenyl)-4-(4-methoxyphenyl)thiazol-2-amine (127)


123 using 121 (2.27g) and 112b (2.13g).

Yield 87%; mp 104oC; IR (KBr cm-1): 1031, 1581, 2365, 2859, 3050, 3450; 1H NMR

(CDCl3): δ 3.86 (s, 6H, 2xOCH3), 3.89 (s, 3H, OCH3), 6.49- 6.53 (s, NH), 6.74 (s,

1H), 6.82-6.85 (m, 1H), 6.95-6.97 (m, 1H), 7.83-7.85 (m,3H), 7.94 (s,1H); MS(ESI+):

m/z 343 (M + 1)+.

N-(2,5-dimethoxyphenyl)-4-phenylthiazol-2-amine (128)


123 using 122 (1.97g) and 112b (2.13g).


3431; 1H NMR (CDCl3): δ 3.83 (s,3H, OCH3), 3.92 (s,3H, OCH3), 6.70 (s, NH), 6.84-

6.85 (m,1H), 6.95-6.98 (m,1H), 7.06-7.07(m,1H), 7.47-7.51 (m,3H), 7.85-7.09 (m,2H);

MS(ESI+): m/z 313 (M+H)+

N-(4-methoxyphenethyl)-4-p-tolylthiazol-2-amine (129)


123 using 120 (2.13g) and 119 (2.11g).




Part 1B

Page | 79


2021, 3429; 1H NMR (CDCl3): δ 2.42(s, 3H, CH3), 3.08(t, J= 6Hz, 2H, CH2), 3.84(s,

3H, OCH3), 4.46 (m,2H, CH2), 6.66(NH), 6.90-6.93(m, 2H), 7.14-7.19(m,5H), 7.83-

7.86(m,2H); MS(ESI+): m/z 325 (M + 1)+.

N-(4-methoxyphenethyl)-4-(4-methoxyphenyl)thiazol-2-amine (130)


123 using 121 (2.27g) and 119 (2.11g).


3020, 3414; 1H NMR (CDCl3): δ 2.94 (t, J= 6Hz, 2H, CH3), 3.54 (m, 2H, CH2), 3.82

(s,3H, OCH3), 3.85 (s, 3H, OCH3), 5.53 (bs, NH), 6.57 (s, 1H), 6.87 -6.95 (m, 4H),

7.16-7.18( m, 2H), 7.73-7.77(m, 2H); MS(ESI+): m/z 341 (M + 1)+.

N-(4,5-dihydrothiazol-2-yl)-4-methoxy-N-(2-(trifluoromethyl)phenyl)benzamide

(117)


114a using 113c (2.47g) and 4-methoxybenzoylchloride (1.70g).

Yield: 47%; mp 118oC; IR (KBr cm-1): 670, 761, 1094, 1356, 1650, 2963, 3419; 1H

NMR (CDCl3, 200MHz): δ 3.81(s, 3H, OCH3), 4.14 (t, J =6.6 Hz, 2H, -CH2-CH2-), 4.33

(t, J = 6.6 Hz, 2H, -CH2-CH2-), 7. 16-7.18(m, 2H), 7.45-7.50 (m, 2H), 7.71-7.98 (m,

4H); MS(ESI+): m/z 381 (M+H) +.

Preparation of 4-chloro-N-(4-fluorophenyl)-N-(4-p-tolylthiazol-2-yl)benzamide

(131)

To a stirred solution of 123 (0.284g, 0.001 mol) in dry THF (10mL) was added

potassium carbonate (0.068g, 0.0005 mol) and 4-chlorobenzoyl chloride (0.154mL,

0.0012 mol) under dry conditions. The mixture was refluxed for 8h. The volatiles were

evaporated and water added to the residue followed by extraction with ethyl acetate.

The organic layer was dried over sodium sulphate. The solvent was evaporated and

scratched with hexane to afford compound 131.

The other compounds were prepared in the same manner as 131 using the

respective thiazol-2-amines and benzoylchlorides.




Part 1B

Page | 80

Yield: 82%; mp 166oC; IR (KBr cm-1): 759, 1093, 1216, 1317, 1504, 1656, 3020,

3430; 1H NMR (300 MHz, CDCl3): δ 2.35 (s, 3H, CH3), 7.06-7.16 (m, 4H), 7.22-7.30

(m, 7H), 7.51-7.54 (m, 2H); 13C NMR (CDCl3, 75 MHz) δ 21.2, 107.4(-SCH-), 114.0,

114.3, 123.2, 127.0, 127.2, 128.5, 128.8, 129.1, 129.2, 130.0, 131.7, 132.5, 134.1,

137.7, 150.2, 158.5(CONH), 159.4, 170.5; MS(ESI+): m/z (M+1)+ 422.

N-(4-fluorophenyl)-3-phenyl-N-(4-p-tolylthiazol-2-yl)propanamide (132)

The compound was prepared by similar method as described for the synthesis of 131

using 123 (0.284 g) and hydrocinnomoylchloride (0.178 mL).

Yield 78%; mp 170oC; IR (KBr cm-1): 759, 1064, 1218, 1312, 1504.8, 1677, 3021,

3429; 1H NMR (300 MHz, CDCl3): δ 2.34 (s, 3H, CH3), 2.59 (t, 2H, J = 15 Hz, CH2),

3.05 (t, 2H, J = 15 Hz, CH2), 7.11-7.52 (m, 12H), 7.52-7.55 (m, 2H); 13C NMR (CDCl3,

75 MHz) δ 21.1, 31.1, 37.3, 107.7(-SCH-), 116.6, 116.9, 125.7, 126.3, 128.4, 128.5,

129.1, 129.2, 130.7, 130.8, 131.7, 131.5, 140.3, 150.2, 162.9(CONH), 171.6;

MS(ESI+): m/z (M+1)+ 417.

N-(4-fluorophenyl)-2-phenyl-N-(4-p-tolylthiazol-2-yl)acetamide (133)


using 123 (0.284 g) and phenylacetylchloride (0.159 mL).

Yield 80%; mp 112oC; IR (KBr cm-1): 758.4, 1034, 1222, 1301, 1509.8, 1674.5, 3018;

1H NMR (300 MHz, CDCl3): δ 2.34 (s, 3H, CH3), 3.84 (s, 2H, CH2), 7.21-7.42 (m,

12H), 7.52-7.55 (m, 2H); 13C NMR (CDCl3, 75 MHz) δ 21.2, 41.4, 107.9(-SCH-),,

114.0, 116.7, 125.4, 125.8, 129.2, 130.2, 131.1, 131.2, 135.5, 137.5, 149.4,

158.7(CONH), 160.9, 164.2, 170.7; MS(ESI+): m/z (M+1)+ 403.

4-chloro-N-(4-fluorophenyl)-N-(4-(4-methoxyphenyl)thiazol-2-yl)benzamide

(134)


using 124 (0.301 g) and 4-chlorobenzoylchloride (0.154mL).

Yield 80%; mp 200oC; IR (KBr cm-1): 757.15, 1023, 1239, 1277, 1499.2, 1691, 3018,

3408; 1H NMR (300 MHz, CDCl3): δ 3.90 (s, 3H, OCH3), 6.98(d, 2H, J = 9 Hz), 7.22-

7.33 (m, 2H), 7.45-7.46(d, 2H, J = 6 Hz), 7.96(d,2H, J = 9Hz), 8.10(d, 2H, J = 9 Hz);




Part 1B

Page | 81

13C NMR (CDCl3, 75 MHz) δ 55.8, 105.0(-SCH-),, 114.8, 115.7, 123.2, 125.3, 128.9,

130.1, 132.5, 134.1, 137.7, 150.2, 156.3(CONH), 160.6, 162.9, 173.2; MS(ESI+): m/z

(M+1)+ 439.

N-(4-fluorophenyl)-N-(4-(4-methoxyphenyl)thiazol-2-yl)-3-phenylpropanamide

(135)



Yield 74%; mp 159oC; IR (KBr cm-1): 760, 1032, 1221, 1304, 1498., 1673, 3019,

3429; 1H NMR (300 MHz, CDCl3): δ 2.59 (t, 2H, J = 9Hz), 3.04 (t, 2H, J = 9Hz ), 3.80

(s,3H, OCH3), 6.85 (d,2H, J = 9 Hz), 7.09-7.28 (m, 12H), 7.56 (d, 2H, J = 9Hz); 13C

NMR (CDCl3, 75 MHz) δ31.1, 37.3, 55.2, 106.7(-SCH-), 113.9, 116.6, 116.9, 126.3,

127.14, 127.4, 128.4, 130.7, 130.8, 135.6, 140.3, 149.2, 159.3(CONH), 164.1, 171.6;

MS(ESI+): m/z (M+1)+ 433.

N-(4-fluorophenyl)-N-(4-(4-methoxyphenyl)thiazol-2-yl)-2-phenylacetamide (136)



Yield 65%; mp 110oC; IR (KBr cm-1): 755.4, 1030, 1249, 1306, 1498.8, 1676.5, 3028,

3412; 1H NMR (300 MHz, CDCl3): δ 3.69 (s, 2H), 3.80 (s, 3H OCH3), 6.83-7.86 (m,

2H), 7.08-7.30 (m, 12H), 7.52-7.58(m,2H); 13C NMR (CDCl3, 75 MHz) δ 37.5, 55.8,

113.4, 115.7, 120.1, 123.2, 125.3, 127.6, 128.5, 129.2, 135.1, 150.2, 160.6,

163.7(CONH), 173.2; MS(ESI+): m/z (M+1)+ 419.

4-chloro-N-(4-fluorophenyl)-N-(4-phenylthiazol-2-yl)benzamide (137)



Yield 55%; mp 161oC; IR (KBr cm-1): 761, 1029, 1217.7, 1307, 1504, 1672, 3020,

3412; 1H NMR (300 MHz, CDCl3): δ 7.07-7.12(m, 3H), 7.23-7.34(m, 5H), 7.69-

7.72(m,4H), 8.03-8.06(m,2H); 13C NMR (CDCl3, 75 MHz) δ 109.2(-SCH-), 115.9,

116.1, 125.7, 127.8, 128.2, 128.4, 129.4, 130.6, 131.0, 131.2, 138.7, 150.2,

156.3(CONH), 162.9, 167.4; MS(ESI+): m/z (M+1)+ 409.




Part 1B

Page | 82

N-(4-fluorophenyl)-3-phenyl-N-(4-phenylthiazol-2-yl)propanamide (138)




3430; 1H NMR (300 MHz, CDCl3): δ 2.62 (t, 2H, J= 9Hz), 3.57 (t, 2H, J= 9Hz), 7.18-

7.30(m, 13H), 7.35-7.40(m,2H); 13C NMR (CDCl3, 75 MHz) δ 31.1, 37.3, 108.6(-SCH-

), 116.6, 116.9, 125.9, 126.4, 127.8, 128.4, 128.5, 128.6, 130.8, 134.4, 135.5, 140.3,

149.4, 160.0 (CONH), 160.9, 164.2, 171.7; MS(ESI+): m/z (M+1)+ 389.

N-(4-fluorophenyl)-2-phenyl-N-(4-phenylthiazol-2-yl)acetamide (139)



Yield 72%; mp 172oC; IR (KBr cm-1): 761, 1029, 1217.7, 1307, 1504, 1672, 3020,

3412; 1H NMR (300 MHz, CDCl3): δ 3.67(m,2H), 7.02-7.07(m, 2H), 7.19-

7.27(m,11H), 7.62-7.64(m,2H); 13C NMR (CDCl3, 75 MHz) δ42.3, 108.7, 116.5,

116.8, 125.8, 127.2, 127.8, 128.5, 128.6, 129.1, 131.1, 131.2, 133.4, 134.3, 135.4,

149.4, 160.1(CONH), 161.0, 164.3, 170.4; MS(ESI+): m/z (M+1)+ 403.

4-chloro-N-(2,5-dimethoxyphenyl)-N-(4-p-tolylthiazol-2-yl)benzamide (140)




3415;1H NMR (300 MHz, CDCl3): δ 2.32 (s, 3H, CH3), 3.57 (s, 3H, OCH3), 3.76 (s,3H,

OCH3), 7.1-8.0(m,12H); 13C NMR (CDCl3, 75 MHz) δ 21.3, 55.8, 105.7(-SCH-),

110.5, 125.7, 129.1, 129.3, 129.7, 130.1, 131.7, 134.1, 135.5, 150.2, 150.7, 153.1,

156.3(CONH), 173.2; MS(ESI+): m/z (M+1)+ 465.

N-(2,5-dimethoxyphenyl)-3-phenyl-N-(4-p-tolylthiazol-2-yl)propanamide (141)






Part 1B

Page | 83


3401; 1H NMR (300 MHz, CDCl3): δ 2.33 (s,3H, CH3), 2.60- 2.62(m, 2H, CH2), 3.02-

3.04 (m, 2H), 3.6 (s,3H, OCH3), 3.80 (s,3H, OCH3), 6.83-7.2 (m,11H), 7.09-7.28(m,

12H), 7.55(d, 2H, J = 6Hz); 13C NMR (CDCl3, 75 MHz) δ21.9, 30.7, 36.4,55.9, 56.4,

107.5(-SCH-), 113.4, 115.7, 116.0, 125.8, 126.1, 128.41, 128.45, 128.8, 129.1,

132.0, 137.3, 140.7, 149.3, 149.5, 153.9(CONH), 172.1; MS(ESI+): m/z (M+1)+ 459.

N-(2,5-dimethoxyphenyl)-2-phenyl-N-(4-p-tolylthiazol-2-yl)acetamide (142)




3407; 1H NMR (300 MHz, CDCl3): δ 1.59 (s, 2H), 2.30 (s,3H), 3.68(s, 3H, OCH3),

3.75 (s, 3H, OCH3), 6.80-6.81 (m, 1H), 7.02-7.28 (m, 10H), 7.54-7.56 (m,2H); 13C

NMR (CDCl3, 75 MHz) δ21.21, 41.7, 55.8, 56.3, 105.2(-SCH-), 105.7, 113.3, 116.12,

116.19, 128.3, 129.35, 134.0, 135.6, 138.4, 150.0, 154.1(CONH), 165.0, 172.1;

MS(ESI+): m/z (M+1)+ 445

4-chloro-N-(2,5-dimethoxyphenyl)-N-(4-(4-methoxyphenyl)thiazol-2-yl)benzami-

-de (143)




3409; 1H NMR (300 MHz, CDCl3): δ 3.59 (s, 3H, OCH3), 3.78 (s, 3H, OCH3), 3.81 (s,

3H, OCH3), 6.76-7.28(m,8H), 7.39-7.42(m, 2H), 7.64-7.67(m,2H); 13C NMR (CDCl3,

75 MHz) δ55.2, 55.9, 56.0, 107.2(-SCH-), 113.6, 113.8, 115.5, 116.6, 127.2, 127.6,

127.9, 129.5, 133.3, 136.4, 149.0, 149.2, 153.4, 159.3(CONH), 159.7, 169.0;

MS(ESI+): m/z (M+1)+ 481.

N-(2,5-dimethoxyphenyl)-N-(4-(4-methoxyphenyl)thiazol-2-yl)-3-phenylpropana-

-mide (144)






Part 1B

Page | 84


3421; 1H NMR (300 MHz, CDCl3): δ 2.62-2.64 (m, 2H), 3.02-3.07 (m, 2H), 3.73 (s,3H,

OCH3), 3.80(s.6H, 2x OCH3), 6.83-7.86 (m,3H), 7.00-7.27(m, 8H), 7.59-7.61(m,2H);

13C NMR (CDCl3, 75 MHz) δ 30.78, 36.40, 55.27, 55.93, 56.43, 106.58(-SCH-),

113.43, 113.83, 115.69, 116.08, 126.16, 127.2, 128.41, 128.45, 128.83, 146.80,

149.02, 149.5, 153.9, 159.22(CONH), 172.16; MS(ESI+): m/z (M+1)+ 475.

N-(2,5-dimethoxyphenyl)-N-(4-(4-methoxyphenyl)thiazol-2-yl)-2-phenylacetami-

-de (145)



Yield 72%; mp 135oC; IR (KBr cm-1): 759, 1037, 1217, 1316, 1498.3, 1671.15, 3018,

3409; 1H NMR (300 MHz, CDCl3): δ 3.57 (m,2H), 3.68 (s,3H, OCH3), 3.76 (s,3H,

OCH3), 3.80 (s,3H, OCH3), 6.80-6.86 (m,3H), 6.99-7.08 (m,5H), 7.29-7.31 (m,3H),

7.57-7.62 (m,2H); 13C NMR (CDCl3, 75 MHz) δ41.7, 55.2, 55.8, 56.2, 106.7(-SCH-),

113.3, 113.8, 116.1, 116.2, 126.9, 127.2, 127.8, 128.4, 128.7, 129.3, 134.0, 149.0,

149.5, 153.7, 159.2(CONH), 170.9; MS(ESI+): m/z (M+1)+ 461.

4-chloro-N-(2,5-dimethoxyphenyl)-N-(4-phenylthiazol-2-yl)benzamide (146)




3437; 1H NMR (300 MHz, CDCl3): δ 3.6 (s, 3H, OCH3), 3.79 (s,3H, OCH3), 6.78-

6.81(m, 1H), 6.89-6.93(m,1H); 7.02-7.03(m,1H), 7.21-7.43(m, 8H), 7.72-7.74( m,2H);

13C NMR (CDCl3, 75 MHz) δ55.9, 56.0, 109.0(-SCH-), 115.6, 116.6, 125.9, 127.7,

127.9, 128.5, 129.5, 129.52, 133.2, 134.6, 136.5, 149.0, 149.5, 153.4, 159.8(CONH),

169.0; MS(ESI+): m/z (M+1)+ 451.

N-(2,5-dimethoxyphenyl)-3-phenyl-N-(4-phenylthiazol-2-yl)propanamide (147)






Part 1B

Page | 85


3424;1H NMR (300 MHz, CDCl3): δ 2.58-2.59 ( m, 2H), 3.02 (m, 2H), 3.68 (s, 3H,

OCH3), 3.77(s,3H, OCH3), 6.80-6.81(m, 1H), 6.98-6.99(m,1H); 7.10-7.12(m,1H),

7.16-7.30( m,9H), 7.63-7.65(m,2H); 13C NMR (CDCl3, 75 MHz) δ30.7, 36.4, 55.9,

56.4, 108.3(-SCH-), 113.3, 115.7, 116.0, 125.9, 126.1, 127.5, 128.4, 128.47, 128.69,

134.7, 140.7, 149.2, 153.8(CONH), 173.1; MS(ESI+): m/z (M+1)+ 445.

N-(2,5-dimethoxyphenyl)-2-phenyl-N-(4-phenylthiazol-2-yl)acetamide (148)




3427; 1H NMR (300 MHz, CDCl3): δ 3.68 (s, 3H, OCH3), 3.76(s, 3H, OCH3), 6.82-

6.84(m, 1H), 6.99-7.04(m,3H); 7.05-7.30(m,8H), 7.65-7.68( m,2H); 13C NMR (CDCl3,

75 MHz) δ 37.5, 55.6, 55.8, 105, 105.7(-SCH-), 110.5, 127.5, 127.8, 128.7, 129.2,

129.6, 133.0, 135.6, 138.1, 150.2, 156.7(CONH), 163.7, 172.2; MS(ESI+): m/z

(M+1)+ 431.

4-chloro-N-(4-methoxyphenethyl)-N-(4-p-tolylthiazol-2-yl)benzamide (149)




3429; 1H NMR (300 MHz, CDCl3): δ 2.40( s, 3H, CH3), 3.03 (t, 2H, J= 6Hz), 3.78 (s,

3H, OCH3), 4.40(t,2H, J= 6Hz), 6.76-6.79(m,2H), 6.86-6.89(m,2H); 7.14-7.17( m,2H),

7.23-7.27(s,3H), 7.35-7.38(m,2H), 7.82-7.85(m,2H); 13C NMR (CDCl3, 75 MHz)

δ21.3, 33.5, 51.6, 55.3, 108.6(-SCH-),114.0, 125.9, 128.3, 128.7, 129.4, 130.0,

131.9, 133.2, 136.4, 137.8, 149.8, 158.4(CONH), 169.7; MS(ESI+): m/z (M+1)+ 463.

N-(4-methoxyphenethyl)-3-phenyl-N-(4-p-tolylthiazol-2-yl)propanamide (150)




3021, 3410; 1H NMR (300 MHz, CDCl3): δ 2.34 ( s,3H, CH3), δ 2.62 (t, 2H, J= 6Hz),




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Page | 86

2.96 ( t,2H, J= 6Hz), 3.08 ( t,2H,J= 6Hz), 3.78 (s, 3H, OCH3), 4.40 (m,2H), 6.86-6.88

(m,2H); 6.96-6.99 (m,2H), 7.15-7.17 ( m,4H), 7.40-7.48 (m,4H), 7.63-7.69 (m,2H);

13C NMR (CDCl3, 75 MHz) δ21.3, 33.4, 38.6, 47.1, 55.2, 105.0(-SCH-), 114.1, 125.7,

125.9, 127.7, 128.2, 128.7, 129.9, 130.0, 131.7, 139.5, 150.2, 158.4(CONH), 172.8,

179.6; MS(ESI+): m/z (M+1)+ 457.

N-(4-methoxyphenethyl)-2-phenyl-N-(4-p-tolylthiazol-2-yl)acetamide (151)




3398; 1H NMR (300 MHz, CDCl3): δ 2.42 (s,3H, CH3), 3.08 (t, J= 6Hz, 2H), 3.72

(s,2H), 3.84 (s,3H, OCH3), 4.46 (m,2H), 6.90-6.93 (m,2H), 7.14-7.19 (m,5H), 7.33-

7.88(m,5H),7.83-7.86(m,2H); 13C NMR (CDCl3, 75 MHz) δ21.2, 33.5, 41.5, 50.5,

55.3, 105.2(-SCH-), 114.3, 125.9, 127.2, 128.8, 129.1, 129.3, 130.0, 130.4, 137.6,

150.2, 158.6(CONH), 170.5; MS(ESI+): m/z (M+1)+ 443.

4-chloro-N-(4-methoxyphenethyl)-N-(4-(4-methoxyphenyl)thiazol-2-yl)benzamid-

-e (152)




3435; 1H NMR (300 MHz, CDCl3): δ 3.03 ( t,2H, J= 6Hz), 3.78 (s, 3H, OCH3), 3.86 (s,

3H, OCH3), 4.39 (t,2H, J= 6Hz), 6.76-6.79 (m,2H), 6.86-6.88 (m,2H); 6.96-6.99

(m,2H), 7.15-7.17 ( m,3H), 7.35-7.38 (m,2H), 7.86-7.89 (m,2H); 13C NMR (CDCl3, 75

MHz) δ33.5, 51.6, 55.3, 107.6(-SCH-), 114.1, 127.3, 127.6, 128.3, 130.0, 133.2,

136.4, 150.2, 158.2(CONH), 159.3, 170; MS(ESI+): m/z (M+1)+ 465.

N-(4-methoxyphenethyl)-N-(4-(4-methoxyphenyl)thiazol-2-yl)-3-phenylpropana-

-mide (153)






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3402; 1H NMR (300 MHz, CDCl3): δ 2.62 (t, 2H, J= 6Hz), 2.96 ( t,2H, J= 6Hz), 3.08 (

t,2H,J= 6Hz), 3.78 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 4.40 (m,2H), 6.86-6.89 (m,2H);

6.97-7.00 (m,2H), 7.11-7.16 ( m,4H), 7.23-7.34 (m,4H), 7.87-7.90 (m,2H); 13C NMR

(CDCl3, 75 MHz) δ30.8, 33.5, 36.2, 49.3, 55.2, 55.3, 105.2(-SCH-), 114.0, 114.2,

126.3, 127.2, 127.8, 128.3, 128.5, 130.0, 130.4, 140.6, 158.5 (CONH), 159.4, 171.6;

MS(ESI+): m/z (M+1)+ 473.

N-(4-methoxyphenethyl)-N-(4-(4-methoxyphenyl)thiazol-2-yl)-2-phenylacetamid-

-e (154)


using 130 (0.341 g) and phenylacetylchloride (0.159 ml).

Yield 69%; mp =164oC; IR (KBr cm-1): 757, 1032, 1216, 1391, 1492, 1659, 3018,

3412; 1H NMR (300 MHz, CDCl3): δ 3.10 ( t,2H, J= 7.05 Hz), 3.85 (s, 3H), 3.92

(s,3H), 4.47 (m,2H), 6.93 (d, 2H, J=8.43 Hz), 6.96 (d,2H,J=8.67 Hz); 7.10-7.12

(m,1H), 7.13-7.18 ( m,4H), 7.30-7.37 (m,4H), 7.91 (d, 2H, J= 8.64 Hz); 13C NMR

(CDCl3, 75 MHz) δ33.5, 41.5, 50.5, 55.3, 55.36, 105.2(-SCH-), 114.0, 114.3, 124.2,

128.8, 129.1, 130.0, 130.4, 135.6, 150.2, 158.6(CONH), 159.4, 166.4; MS(ESI+): m/z

(M+1)+ 445.

1B.3.2 Biological Testing

In vitro assay : Same as mentioned in Chapter1 Part 1A.

In vivo asaay:

Stereptozotocin Study:

Chemicals and Reagents: Sodium orthovanadate and Streptozotocin were

purchased from Sigma Aldrich Co., USA, one touch glucometer (Accu-Check sensor)

and glucostrips purchased from Roche Diagnostics India Ltd.

Preparation of dosage of active drug and Synthetic compound:

Sodium orthovanadate: Sodium orthovanadate was in microcrystalline form and

freely soluble in water. The dosage was prepared in solution from using sterilized

water that, each 0.1 mL of solution contained sodium orthovanadate at dose of 100

mg/kg body weight since sodium orthovanadate is effective in such dose.




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Test Samples: The synthetic compounds were dissolved in 1% gum acacia to

prepare the solution according to the dose of 100 mg/kg body weight.

Evaluation of test compounds for antihyperglycaemic activity in Streptozotocin

(STZ)-induced diabetic rats and assessment of antihyperglycaemic effect by

measuring fall in blood glucose level on Streptozotocin treated diabetic rats:

Same as mentioned in Chapter1 Part 1A

db/db mice study

Male C57BL/Ks strain of mouse (db/db mouse) 10-12 weeks of age and

around 40 ±3 g of body weight were procured from the animal colony of the Institute.

The animals were housed four or five in a polypropylene cage in the animal house.

The following norms were always followed for animal room environment: temperature

23 ± 2°C; humidity 50-60%; light 300 lux at floor level with regular 12h light cycle;

noise level 50 decibel; ventilation 10-15 air changes per hour. After randomization

into groups, the mice were acclimatized for 2-3 days in the new environment before

initiation of experiment. Standard pellets were used as a basal diet during the

experimental period.

Antihyperglycaemic and Antidyslipidemic activity assessment in C57BL/Ks

strain of mouse (db/db mouse)

Experimental Design

The animals were allocated into groups of 5 animals in each. Prior to start of

the sample feeding, a vehicle training period was followed from day -3 to day 0

during which all the animals were given vehicle ( 1% gum acacia) at a dose volume

of 10 mL/kg body weight. At day 0 the animals having blood group level between 350

to 500 mg/dL were selected and divided into three groups containing 5 animals in

each. One group was considered as control group while the other group was

treatment group. The experimental group was given suspensions of compound 154

and Pioglitazone at 30.0 and 10.0mg/kg body weight dose respectively. The control

group was given an equal amount of vehicle. All the animals had free access to fresh

water and normal diet. Random blood glucose of each mouse was checked daily at

10.00 pm. On day 10 and day 15 oral glucose tolerance (OGTT) test was performed

to study the effect of compound on glucose tolerance. Blood has been withdrawn

from the retroorbital plexus of mice eye for the estimation of lipid profile on DIALAB




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Page | 89

DTN-410-K and insulin level by CALBIOTECH Insulin ELISA Kit. Body weight of each

animal was measured on alternate day for studying the effect of test sample on body

weight. The skeletal muscle from each mouse were quickly excised at the end of

experiment under light anesthesia and frozen at -80oC until further use.

Oral glucose and intraperitoneal insulin and pyruvate tolerance test

Oral glucose and pyruvate tolerance test was performed on 12h fasted mice

while insulin tolerance test was performed on 4h fasted mice. For OGTT mice were

administered glucose 3.0 g/kg by gavages whereas for ITT and PTT, insulin

(0.8U/kg) and pyruvate (2g/kg) was injected intraperitoneally, respectively. Blood

samples were obtained via tail nick at 0, 30, 60, 90 and 120 min during OGTT and

PTT while at 0, 15, 30, 60, 90 and 120 time points in ITT. Glucose was measured

with the One Touch Ultra glucometer (Accu-Chek Sensor, Roche Diagnostics).

Western blot analysis

Collected tissues and cells were homogenized into PBS containing 1% NP40,

5 mM EDTA, phosphatase inhibitors and protease inhibitors cocktail ( Ripalysis

buffer). Samples were homogenized and incubated on ice for 15 min. Sample is then

stored at – 80ºC and thawed at 37ºC in waterbath. Sample was then centrifuged at

16000 rpm at 4ºC. Then supernatant was taken and quantified by Bradford assay. 40

µg protein (supernatant) of each incubation was resolved on SDS-PAGE, transferred

to nitrocellulose membranes and probed with p-Akt and p-IRS1, Glut4 (Cell

Signaling, MA, USA) and β-actin (Santa Cruz) was taken as the loading control.

Immunoreactive bands were visualized by Enhanced Chemiluminescence according

to manufacturer’s instructions (GE Healthcare, UK).

Densitometry analysis

Protein expression was evaluated by densitometric analysis performed with

Alpha DigiDoc 1201 software (Alpha Innotech Corporation, CA, USA).180 The same

size rectangle box was drawn surrounding each band and the intensity of each was

analyzed by the program after subtraction of the background intensity.

Statistical analysis

The homeostatic model assessment (HOMA) was used to calculate relative insulin

resistance as follows: [Fasting blood glucose (mg/dL) × Fasting serum insulin




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Page | 90

(µLU/mL)]/405. All statistical calculations were performed using Graph-Pad Prism

version 3.02 for Windows (GraphPad Software). Statistical analysis was carried out

by Students t test.181 Data was expressed as mean +SE. The criterion for statistical

analysis was significant (*p<0.05), more significant (**p<0.01), highly significant

(**p<0.01) and not significant (ns). The results are reported as mean values ± SEM.




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Design, Synthesis and Biological Evaluation of IndoleDesign, Synthesis and Biological Evaluation of IndoleDesign, Synthesis and Biological Evaluation of IndoleDesign, Synthesis and Biological Evaluation of Indole----

Fused Heterocyclic DerivativesFused Heterocyclic DerivativesFused Heterocyclic DerivativesFused Heterocyclic Derivatives

Design, Synthesis and Biological Evaluation of Indole-Fused

Heterocyclic Derivatives

Chapter 2

Page | 101

2.1 Introduction

Heterocyclic chemistry is one of the most valuable sources of novel

compounds with diverse biological activities, mainly because of the unique ability of

the resulting compounds to mimic the structure of peptides and to bind reversibly to

proteins.1,2 To medicinal chemists, the true utility of heterocyclic structures is the

ability to synthesize one library based on one core scaffold and to screen it against a

variety of different receptors, yielding several active compounds.

The Indole (1) scaffold probably represents one of the most important

structural subunits for the discovery of new drug candidates. It is an aromatic

heterocyclic organic compound which has a bicyclic structure, consisting of a six-

membered benzene ring fused to a five-membered nitrogen-containing pyrrole ring.

2.2 Indole: Chemical and Biological Importance

Indole is a popular component of fragrances and the precursor to many

pharmaceuticals. Compounds that contain an indole ring are called indoles. The

Tryptophan (2) is an essential amino acid and as such is a constituent of most

proteins, it also serves as a biosynthetic precursor for a wide variety of tryptamine-

indole, and 2,3-dihydroindole containing secondary metabolites.3 In animals,

Serotonin (5-hydroxytryptamine) (3) is a very important neurotransmitter in the CNS

and also in the cardiovascular and gastrointestinal systems. Biochemically derived

from tryptophan, serotonin is primarily found in the gastrointestinal tract, platelets and

in the central nervous system of humans and animals. It is a well known contributor

to feelings of well-being therefore, it is also known as a happiness hormone. The

structurally similar hormone Melatonin (4) is thought to control the diurnal rhythm of

physiological functions. It is produced in the brain by the pineal gland, from the amino

acid tryptophan. It has a significant role in the protection of nuclear and mitochondrial

DNA. Study and classification of 5-hydroxytryptamine receptors has resulted in the

design and synthesis of highly selective medicines such as Sumatriptan4 (5) for the

treatment of migraine, Ondansetron5 (6) for the suppression of the nausea and

vomiting caused by cancer chemotherapy and radiotherapy and Alosetron6 (7) for the

treatment of irritable bowel syndrome.



Chapter 2

Page | 102

Since Indoles are also found in abundance in biologically active compounds

such as pharmaceuticals, agrochemicals and alkaloids, indole derivatives have

therefore, captured the attention of organic synthetic chemists. Acute chronic

inflammation and different type of arthritis are the inflammatory disorders which are a

big blow to humanity and continual search for newer nonsteroidal antiinflammatory

agents is the only way to fortify against this awful threat. The discovery of

Indomethacin7 (8) as a successful agent for clinical treatment of antiinflammatory

disorders has led to the exploration of indole moiety to obtain better antiinflammatory

agents.

Some indole alkaloids exert considerable pharmacological activity but quite

different effects may be obtained even from alkaloids of one genus e.g., the

Strychnos alkaloids, Strychnine acts powerfully causing muscle contraction, while the

Toxiferines is a muscle relaxant. Of the clinically useful alkaloids, three groups are

notable:

(a) the Ergot alkaloids—Ergometrine8 (9) with its direct action on the

contraction of uterine muscle, Ergotamine8 (10) for migraine relief and

the modified alkaloid, Bromocriptine,9 (11) which suppresses lactation

and has some application for the treatment of mammary carcinoma

(b) the Rauvolfia alkaloid Reserpine,10 (12) the forerunner of the

tranquillisers,

(c) the dimeric anticancer alkaloids of Catharanthus Vinblastine (13) and

Vincristine (14).11



Chapter 2

Page | 103

2.2.1 Bioactive Indoles Developed over the Past Few Years

The incorporation of indole nucleus, a biologically accepted pharmacophore

in medicinal compounds, has made it versatile heterocycle possessing wide

spectrum of biological activities. (Fig. 1)



Chapter 2

Page | 104

Figure 1: Diverse biological activities of Indole

Given below is a brief account of various indole ring containing derivatives

and their associated biological activities developed in past few years.

Table: 2.1 Diverse Biological activities of Indole derivatives

S.No Structure Biological Activity

1

Antiinflammatory

12

2

Antiinflammatory13

Indole

Anti

inflammatory Antifungal

Antibacterial

Antioxidant

Antidiabetic

Insecticidal

Opoid

AntagonistAnti-

tubercular

Thrombin

Catalytic

Antiviral

&

Anti-HIV

Anti-

histaminic

Antiarrythmic

Anti-

hypertensive

Anticancer



Chapter 2

Page | 105

3

Antiinflammatory14

4

Antiinflammatory

15

5

Antiinflammatory

16

6

Antibacterial

17

7

NH

O

HN

H

NH2.HCl

CO2H(21)

Antibacterial

18

8

Antiviral

19,

Antiarrythmic19



Chapter 2

Page | 106

9

Antiviral

20

10

Antiviral

21

11

NH

O

N

N

O

F

(25)

AntiHIV

22

12

Antifungal

23

13.

Antifungal

24

14

Analgesic

25



Chapter 2

Page | 107

15

Analgesic

19

16

Analgesic

25

17

Analgesic

3

18

Anticonsulvant

26

19

Anticonsulvant

27

20

Anticonsulvant

28



Chapter 2

Page | 108

21

Anticancer

29

22

Anticancer

30

23

Anticancer

31

24

Anticancer

32

25

Anticancer

33



Chapter 2

Page | 109

26

Anticancer

34

27

Anticancer

35

28

Anticancer

36

29

Anticancer

37

30

Anticancer

38

31

Anticancer

39



Chapter 2

Page | 110

32

Anticancer

40

33

Antitumor

41

34

Antitumor

42

35

Antioxidant

43

36

Antioxidant

44



Chapter 2

Page | 111

37

Antioxidant

45

38

Antidiabetic

45

39

Antidiabetic

46

41

Antidiabetic47

42

Antiobesity and CNS disorder

48

43

CNS disorder

49



Chapter 2

Page | 112

44

Antiarrythmic

50

45.

Hypocholesterolemic agent

51

46

Antiarrythmic

19

47

Antiischemic

52

48

ACAT hypocholesterolemic agent

53



Chapter 2

Page | 113

49

Antithrombotic54

50

Antidyslipidemic

55

51

Antidyslipidemic

56

52

Antithrombotic

57

53

Antithrombotic

58

54

Antithrombotic

59



Chapter 2

Page | 114

55

Antithrombotic60

56

Antioxidant61

57

Antidiabetic

62,

Antiatherosclerotic62

58

Antiatherosclerotic

63,64

59

Antiatherosclerotic65

Indole derivatives have therefore, captured the attention of organic synthetic

chemists. They represent a very important class of molecules that play a major role in

cell biology and are potential naturally occurring products. Due to the diverse and

versatile biological properties of indole derivatives, they are of great interest to the

research community. Significant analgesic, antiinflammatory, antipyretic and

antitumor activity is displayed by some effective substituted indole derivative which



Chapter 2

Page | 115

are presently leading drug in the market. Some modified indole are found to be

effective as antihypertensive, antidepressant, opoid antagonist and antiemetic

agents, whereas some of the derivatives of indole are found to show the

antiasthmatic, antiarrhythmic, antithrombotic, antidyslipidemic, antiobesity,

antiatherosclerotic and antiviral activity.

Keeping in mind the versatile use of indole derivatives in pharmaceuticals, we

decided to use indole nucleus as the basic core unit for constructing oxopropanyl

indole and indolyl triazole derivatives and evaluate for various biological activities.

The present study has been divided in the following two parts:

Part 2A. Lipid Lowering Oxopropanyl Indole Hydrazone Derivatives with Antioxidant,

Antihyperglycaemic and Improved Lipoprotein Lipase Activities

Part 2B. Synthesis and Biological Evaluation of Antithrombotic Activity of Indole

Based Triazole Derivatives.

Lipid Lowering Oxopropanyl Indole Hydrazone Derivatives with

Antioxidant, Antihyperglycaemic and Improved Lipoprotein Lipase

Activities

Part 2A

Page | 116

Part 2A: Lipid Lowering Oxopropanyl Indole Hydrazone

Derivatives with Antioxidant, Antihyperglycaemic and

Improved Lipoprotein Lipase Activities

Atherosclerosis, the major contributor to the cardiovascular diseases (CVDs),

is the largest cause of mortality around the World. According to WHO, by 2030 more

than 23 million people will die annually from CVDs.66 It is a multifactorial disease.

Some group of diseases (commonly called metabolic syndrome) like obesity,

diabetes mellitus, impaired glucose tolerance, insulin resistance, dyslipidemia (high

LDL cholesterol, low HDL cholesterol and high triglyceride levels in blood) and

increased blood pressure raise the risk of cardiovascular disease.67

The involvement of oxidative stress in combination with hyperlipidemia in the

pathogenesis and development of atherosclerosis as well as type 2 diabetes has

been reported.68 There is growing evidence that hydroxyl free radicals are involved

as major factor causing peroxidative damage to lipoproteins present in the blood,

which are responsible for the onset and advancement of atherosclerosis.69 Moreover,

in hyperglycaemic patients, the occurance of several non enzymatic glycosylations is

accompanied by glucose oxidation catalyzed by Cu2+ and Fe2+ resulting in the

formation of O2- and •OH radicals which further accelerates the risk of cardiac

diseases in dyslipidemic patients.70

Despite of intensive therapy with high dose ‘‘statins’’ to lower LDL-C levels,

there is significant level of cardiovascular event in patients with established coronary

artery disease making way for new strategies for reducing the residual cardiovascular

risk.71 Moreover, antidyslipidemic statins have several side effects like myostitis,

arthralgia, gastrointestinal upset and elevated liver function tests. Raising levels of

HDL-C provides an important strategy for addressing the residual cardiovascular risk

attributing to its ability to remove cellular cholesterol, as well as its antiinflammatory,

antioxidant and antithrombotic properties, which act in concert to improve endothelial

function and inhibit atherosclerosis.72 Fibrates represent one such class of

compounds used for the treatment of hyperlipidemia but require higher doses for

significant effects.73 The combination therapy is also used for these two classes but

have met with serious safety concerns. Therefore, different classes of lipid lowering

agent along with antioxidant activity and antidiabetic activity will be able to protect

endothelial and myocardial function and thus may serve as a good antiatherosclerotic

agent.



Activities

Part 2A

Page | 117

Lipoprotein lipase (LPL) is a rate limiting enzyme which is responsible for the

hydrolysis of triglyceride rich lipoproteins viz. chylomicrons and very low density

lipoprotein (VLDL) present in the bloodstream. Increased LPL activity is associated

with decreased level of triglycerides (TG) and increased high density lipoprotein

(HDL) and thus have protective effect against atherosclerosis.74 Several studies have

been carried out showing LPL as antiatherogenic enzyme and is been identified as

potential therapeutic target for correcting dyslipidemia and reduction of CVDs. Thus,

LPL activators can potentially help battle CVD residual risk in the future.75-77

Hydrazones as well as carboxamides represent an important class of

compounds found in versatile building blocks for the preparation of

pharmaceuticals.78,79 Moreover, these classes of compounds have been clinically

used as therapeutic options in the treatment of diabetes, obesity, metabolic

syndrome (dyslipidemia) and CVDs. Additionally, it is known that many amino acids

have potential antioxidant activity. Several tryptophan derivatives function as a free

radical scavengers and antioxidants.80, 81 Considering this background, it appeared of

interest to synthesize a series of oxopropanylindole hydrazone derivatives and

evaluate them against antioxidant, antidyslipidemic and antihyperglycaemic activities.

Thus the synthesis and biological (antioxidant, antidyslipidemic,

antiadepogenic and antihyperglycaemic) activities of a series of twelve substituted

oxopropanylindole hydrazone derivatives with different functionalities at R1, R2, R3

and R4 are presented herein.

2A.1 Results and Discussion

2A.1.1 Chemistry

The synthesis of final oxopropanylindole hydrazone derivatives (87a-87d,

88a-88d, 89a-89d) is outlined in Scheme 1. (Table 2) The starting material DL-

tryptophan methyl ester (73) was synthesized by the reported procedure82 which on

reaction benzoylchloride (74) in the presence of triethylamine afforded DL- N-benzoyl

tryptophanmethylester (77). The ester 77 on refluxing with hydrazine hydrate in

ethanol gave the key intermediate 80. Condensation of key intermediate 80 with 4-

chlorobenzaldehyde (83) gave the desired compound N-(1-(2-(4-chlorobenzylidene)-

-hydrazinyl)-3-(1H-indol-3-yl)-1-oxopropan-2-yl)-benzamide (87a) (Scheme 1). The

key intermediate 81 and 82 were synthesized from 78 and 79 respectively, as shown

in Scheme 1. All other final compounds 87b-87d, 88a-88d and 89a-89d were



Activities

Part 2A

Page | 118

synthesized by the same procedure as described above for from key intermediates

77, 78 and 79 respectively (Table 2).

The compounds 87b-87d were synthesized by using 77 and 4-methoxy

benzaldehyde, 4-methylbenzaldehyde and 3,4,5-methoxybenzaldehyde respectively.

The compounds 88a-88d were synthesized by using 78 and 4-chlorobenzaldehyde,

4-methoxybenzaldehyde, 4-methylbenzaldehyde and 3,4,5-methoxybenzaldehyde

respectively. The compounds 89a-89d were synthesized by using 79 and 4-

chlorobenzaldehyde, 4-methoxybenzaldehyde, 4-methylbenzaldehyde and 3,4,5-

methoxybenzaldehyde respectively.



Activities

Part 2A

Page | 119

All the intermediates along with the final products were fully characterized by

their detailed spectral analysis. However, we have taken N-(1-(2-(4-

chlorobenzylidene)-hydrazinyl)-3-(1H-indol-3-yl)-1-oxopropan-2-yl)benzamide (87a)

as the reference compound for characterization. We first protected the amino group

of 73 with benzoyl chloride (74) to obtain DL- N-benzoyl tryptophan methyl ester (77).

The structure of 77 was confirmed by its IR spectrum which showed two

characteristic bands at 1625 cm-1 and 1748 cm-1 for the benzoyl and ester carbonyl

group respectively. In the compound 80 the IR band for the ester group shifted to

1661 cm-1 indicating the formation of amidic bond. Further, absorption band at 3311

and 3206 cm-1 showed presence of NH stretching which was also confirmed by sharp

singlet at δ 3.92 and δ 9.06 for the NH protons in its 1H NMR spectrum. The final

compound 87a exhibited characteristic absorption at 1532 cm-1 for >C=N stretching

in its IR spectrum which indicated the formation of Schiff’s base. The absence of a

singlet at δ3.92 for hydrazide NH2 in compound 80 also confirmed the conversion of

hydrazide to hydrazone (87a). Further, absorption band at 3341 cm-1 showed the

presence of NH stretching which was also confirmed by the presence of singlet at δ

8.53, δ 9.75 and δ 11.01 for the NH protons in its 1H NMR spectrum.

Table 2: Synthesized oxopropanylindole hydrazone derivatives 86a-86d, 87a-87d and 88a-88d

Compd. No R1 R2 R3 R4

87a Ph H Cl H

87b Ph H OMe H

87c Ph H CH3 H

87d Ph OMe OMe OMe

88a CH2Ph H Cl H

88b CH2Ph H OMe H

88c CH2Ph H CH3 H

88d CH2Ph OMe OMe OMe

89a CH2CH2Ph H Cl H

89b CH2CH2Ph H OMe H

89c CH2CH2Ph H CH3 H

89d CH2CH2Ph OMe OMe OMe

The final support of the formation of 87a came from its mass spectral data

that showed (M+1)+ peak at 445 corresponding to its molecular weight. Thus on the

basis of above mentioned spectral data the compound was characterized as N-(1-(2-

-(4-chlorobenzylidene)-hydrazinyl)-3-(1H-indol-3-yl)-1-oxopropan-2-yl)benzamide

(87a). Similarly, all the synthesized compounds 80b-80d, 81a-81d and 82a-82d N-(3-

(1H-indol-3-yl)-1-(2-(4-methoxybenzylidene)hydrazinyl)-1-oxopropan-2-yl)benzamide



Activities

Part 2A

Page | 120

(87b), N-(3-(1H-indol-3-yl)-1-(2-(4-methylbenzylidene)hydrazinyl)-1-oxopropan-2-yl)-

-benzamide (87c), N-(3-(1H-indol-3-yl)-1-oxo-1-(2-(3,4,5-trimethoxybenzylidene)- -

hydrazinyl)propan-2-yl)benzamide (87d), N-(1-(2-(4-chlorobenzylidene)hydrazinyl)-3

-(1H-indol-3-yl)-1-oxopropan-2-yl)-2-phenylacetamide (88a), N-(3-(1H-indol-3-yl)-1-

(2-(4-methoxybenzylidene)hydrazinyl)-1-oxopropan-2-yl)-2-phenylacetamide (88b), N

-(3-(1H-indol-3-yl)-1-(2-(4-methylbenzylidene)hydrazinyl)-1-oxopropan-2-yl)-2-phenyl-

-acetamide (88c), N-(3-(1H-indol-3-yl)-1-oxo-1-(2-(3,4,5-trimethoxybenzylidene)hyd-

-razinyl)propan-2-yl)-2-phenylacetamide (88d), N-(1-(2-(4-chlorobenzylidene)hydraz-

-inyl)-3-(1H-indol-3-yl)-1-oxopropan-2-yl)-3-phenylpropanamide (89a), N-(3-(1H-indol

-3-yl)-1-(2-(4-methoxybenzylidene)hydrazinyl)-1-oxopropan-2-yl)-3-phenylpropanami-

-de (89b), N-(3-(1H-indol-3-yl)-1-(2-(4-methylbenzylidene)hydrazinyl)-1-oxopropan-2-

yl)-3-phenylpropanamide (89c), N-(3-(1H-indol-3-yl)-1-oxo-1-(2-(3,4,5-trimethoxybe-

-nzylidene)hydrazinyl)propan-2-yl)-3-phenylpropanamide (89d) were characterized

and the spectral data are recorded in experimental part.

2A.1.2 Biological Activities

Antioxidant activity

Recent studies have demonstrated that the generation of large quantities of

reactive oxygen species can cause activation of lipid peroxidation, protein

modification, which leads to cardiovascular diseases (CVDs).83 All the twelve

compounds 80b-80d, 81a-81d and 82a-82d were screened for their antioxidant

activity using earlier reported method.83 The scavenging potential of compounds at

200 µg/mL against formation of O2- and ●OH in non enzymatic systems was studied

(Table 3). Further, the effect of compounds on lipid peroxidation in microsomes was

also studied. Among the twelve compounds three compounds 88c, 89b and 89d

showed significant decrease in superoxide anions (33%, 27%, and 25%) as

compared to the standard drug Allopurinol which showed 43% inhibition of

superoxide anions. The compounds 88c, 89b and 89d also reduced hydroxyl radicals

by 35%, 29%, and 23% respectively in comparison to mannitol which showed 47%

inhibition in hydroxyl radicals. Furthermore, the effect of these compounds on the non

enzymatic peroxidation of microsomal membrane lipids was also studied The

compounds 88c, 89b and 89d at 200 µg/mL reduced the microsomal lipid

peroxidation by 35%, 26%, and 23% respectively as compared to α-tocopherol with

50% inhibiton.



Activities

Part 2A

Page | 121

Table 3. Effect of compounds 87-89 on generation of superoxide anions, hydroxyl radicals and lipid-

peroxidation

Series of

compounds

Dose

(µg/ml)

Superoxide anions (%)

(n mol formazone

formed/minute)

Hydroxyl radicals

(%) (n mol MDA

formed/h/mg

protein)

Lipid peroxidation

(%) (n mol MDA

formed/h/mg

protein)

87a 200 -21** -20*** -21**

87b 200 -16* -18* -15*

87c 200 -11* -10* -12*

87d 200 -20** -20** -22**

88a 200 -17* -19* -19*

88b 200 -11* -13* -14*

88c 200 -33*** -35*** -34***

88d 200 -14* -12* -13*

89a 200 -17* -15* -19*

89b 200 -27*** -29*** -26***

89c 200 -14* -17* -15*

89d 200 -25*** -23*** -23***

Standards 200 -43*** (Allopurinol) -47*** (Mannitol) -50*** (α-

tocopherol)

Each value is mean±SD of six values, *P < 0.05; **P < 0.01; ***P < 0.001 experimental values

compared with control values. NOTE: NS (non significant)

LDL oxidation activity

The cascade of cellular processes can lead to the formation of fatty streaks

and ultimately atherosclerotic lesions in the arterial wall as it is triggered by oxidative

modification of LDL, so the compounds 88c, 89b and 89d were also examined for

their antioxidant activities on human LDL oxidation induced by cupric ions (CuSO4).

Aerobic oxidation of LDL even in the absence of metal ions caused formation

of thiobarbituric acid reactive substances (TBARS) (nmol MDA/mg protein), which

were greatly increased by 10 to 15 folds in the presence of Cu+2. Addition of

compounds 88c, 89b and 89d at 200 µg/mL concentrations in above reaction mixture

attenuated LDL oxidation by 32%, 24%, and 17% respectively with decreased

plasma MDA levels in normolipidemic rats (Fig. 2).


Antioxidant, Antihyperglyc

In vivo antidyslipidemic activity

The in vivo anti

in triton induced hyperlipidemic model based on the reduction of experimentally

induced elevated plasma lipid

Figure 2: Compounds 88c

normal donor blood sample. Data expressed as mean

to treated values with Control

Experimental hyperlipidemia was successfully

WR 1339 administration, with an increase in plasma total cho

phospholipids (PL, 3.25 fold) and

control.

All the substituted oxopropanylindole hydrazone derivatives particularly

89b and 89d significantly improved blood lipid profile in

rats. The compounds

total cholesterol (TC, 26% and 24%), plasmalipid (PL, 27% and 23%,) and

triglyceride (TG, 25% and 23%) level in triton induced hyperlipidemic rats

analysis of hyperlipidemic serum of triton administered rats showed significant

increase in the level of VLDL

compared to control rats. Treatment with compounds

decreased the levels of VLDL

induced hyperlipidemic rats (Fig. 3

Gemfibrozil (used as antidyslipidemic drug), at the same dose reduced plasma

by 33%, PL by 31% and

post heparin lipolytic activity (

(88c) and (89d) also showed



Activities

In vivo antidyslipidemic activity

antidyslipidemic activity of these derivatives were


induced elevated plasma lipid84-86 (Table 4).

88c, 89b, and 89d reduced the LDL-oxidation at dose of 200µg/m

sample. Data expressed as mean±S.D. *P < 0.05; **P < 0.01 compared

to treated values with Control

Experimental hyperlipidemia was successfully established 24

WR 1339 administration, with an increase in plasma total cholesterol

3.25 fold) and triglyceride levels (TG, 2.67 fold) as

All the substituted oxopropanylindole hydrazone derivatives particularly

significantly improved blood lipid profile in triton induced hyp

rats. The compounds 88c and 89d (100 mg/kg b.w) improved the serum lipoproteins


25% and 23%) level in triton induced hyperlipidemic rats


increase in the level of VLDL-TG and LDL-TG followed by decrease in HDL

compared to control rats. Treatment with compounds 88c and

levels of VLDL-TG and LDL-TG and increased HDL

uced hyperlipidemic rats (Fig. 3). Under the same experimental conditions,

emfibrozil (used as antidyslipidemic drug), at the same dose reduced plasma

by 33%, PL by 31% and TG by 35 %. Triton treated rats caused inhibition of plasma

post heparin lipolytic activity (PHLA) (38.54 fold) as compared to control. Compound

also showed enhancement of post heparin lipolytic activity (PHLA)


emic and Improved Lipoprotein Lipase

Activities

Part 2A

Page | 122

derivatives were also evaluated


oxidation at dose of 200µg/mL in

±S.D. *P < 0.05; **P < 0.01 compared

established 24h after Triton

lesterol (TC, 3.10 fold),

2.67 fold) as compared to

All the substituted oxopropanylindole hydrazone derivatives particularly 88c,

triton induced hypolipidemic

(100 mg/kg b.w) improved the serum lipoproteins


25% and 23%) level in triton induced hyperlipidemic rats. The


TG followed by decrease in HDL-TG as

and 89d significantly

TG and increased HDL-TG in triton

). Under the same experimental conditions,

emfibrozil (used as antidyslipidemic drug), at the same dose reduced plasma TC

ton treated rats caused inhibition of plasma

(38.54 fold) as compared to control. Compound

lipolytic activity (PHLA)



Activities

Part 2A

Page | 123

Table 4: Percentage (%) change of plasma lipids with the treatment of compounds in Triton-induced

hyperlipidemic rats at dose of 100 mg/kg body weight.

Animal Groups

L I P I D S P R O F I L E PHLA

( nmol of free

fatty acids

formed/h/mL of

plasma)

TC

(mg/dL)

PL

(mg/dL)

TG

(mg/dL)

Control 7 8 .5 3 ±5.13 8 2 .6 4 ±4.71 79.43±6.08 18.86±1.37

Triton 244.27±16.84 c

(+3.110 Fold)

269.23±19.82 c

(+3.257 Fold)

239.38±16.38 c

(+3.013 Fold)

11.59±0.93 c

(-38.54 Fold)

Triton+87a 214.95±17.32*

(-12%)

242.30±17.83*

(-10%)

213.04±17.83*

(-11)

11.93±0.79 NS

(+9%)

Triton+87b 227.17±12.87 NS

(-7%)

255.76±22.31 NS

(-5%)

220.22±15.39 NS

(-8)

2.51±0.96 NS

(+3%)

Triton+878c 224.72±18.69 NS

(-8%)

253.07±19.47 NS

(-6%)

220.22±18.46 NS

(-8)

12.63±0.66 NS

(+3%)

Triton+87d 210.07±14.63*

(-14%)

231.53±18.72*

(-14%)

208.26±16.26*

(-13)

13.67±0.51 NS

(+8%)

Triton+88a 222.28±29.35 NS

(-9%)

247.69±15.82 NS

(-8%)

217.83±13.29 NS

(-9)

11.93±0.71 NS

(+3%)

Triton+88b 219.84±17.26*

(-10%)

239.61±13.67*

(-11%)

208.26±18.61*

(-13)

12.05±0.73 NS

(+4%)

Triton+88c 180.75±13.49***

(-26%)

196.53±16.11***

(-27%)

179.53±13.73***

(-25)

11.93±0.52*

(+15%)

Triton+89d 217.40±14.83*

(-11%)

242.30±15.48*

(-10%)

210.65±12.96*

(-12)

13.32±1.2 NS

(+4%)

Triton+89a 212.51±18.27*

(-13%)

242.30±17.91*

(-10%)

210.65±14.52*

(-12)

12.28±0.88 NS

(+6%)

Triton+89b 202.74±15.91*

(-17%)

226.15±14.26*

(-16%)

203.47±18.21*

(-15)

12.74±0.91*

(+10%)

Triton+89c 217.40±16.62*

(-11%)

242.30±21.68*

(-10%)

215.44±17.33*

(-10)

12.16±0.63 NS

(+5%)

Triton+89d 185.64±11.63***

(-24%)

199.23±16.19***

(-26%)

184.32±11.85***

(-23)

13.21±1.11*

(+14%)

Triton+Gemfibrozil 163.66±11.38***

(-33%)

185.76±11.66***

(-31%)

155.59±10.72***

(-35%)

13.67±0.51*

(+18%)

Each parameter represents pooled data from 6 rats/group and values are expressed as mean ± S.D. c is

P < 0.001, Triton treated group compared with control group and *P < 0.05; **P < 0.01; ***P < 0.001

Triton plus compounds groups compared with Triton treated group only. NOTE: NS (non significant)

and F (Fold change over control group). Units are amg/dL; nmol of free fatty acids formed/h/mL of

plasma.



by 15% and 14% respectively, as compared to G

reversal activity of this enzyme.Among all the test compounds, the compounds

89b and 80d showed good antioxida

showed moderate antioxidant activity.

All the compounds were also subjected to antidyslipidemic activity, where

compounds 88c and 8

general, the analysis of structure activity relationship indicated that although the

compounds 87a and

antioxidant activity, the lipid lowering activity of these compounds were reduced as

compared to analogues

position. Moreover, it was also observed that compounds with electron donating

substituent at R3 position were more active than the compounds with electron

withdrawing group.

Figure 3: Effect of compounds

hyperlipidemic rat. Compound

VLDL-TG and LDLTG followed by increase in HDL

rats. Each parameter represents pooled data from 6 rats/group and values are expressed as

mean±S.D. ***P < 0.001 between control and triton,

between triton versus treated rat groups. Note: Gemfibrozil (100 mg/kg) has

standard drug.

Lipoprotein lipase activity of active compounds

Lipoprotein lipase (LPL) is a major enzyme in overall lipid metabolism and

transport, being responsible for hydrolysis of triglycerides present in circulating

lipoproteins. The potential antiatherogenic role of LPL has been derived from several

studies.75,76 Increased LPL activity positively correlate



Activities

respectively, as compared to Gemfibrozil which showed 18% of

reversal activity of this enzyme.Among all the test compounds, the compounds

showed good antioxidant activity, whereas compounds

showed moderate antioxidant activity.


89d improved blood lipid profile and enhanced PHLA activity. In

the analysis of structure activity relationship indicated that although the

and 87d having phenyl ring at R1 position showed moderate


compared to analogues having CH2Ph 88a-88d) and CH2CH2Ph


position were more active than the compounds with electron

Effect of compounds 88c and 89d on lipoprotein triglyceride level in triton induced

hyperlipidemic rat. Compounds 88c and 89d at dose of 100mg/kg attenuate the level of

TG and LDLTG followed by increase in HDL-TG level in triton induced hyperlipidemic


mean±S.D. ***P < 0.001 between control and triton, aP < 0.05;

bP < 0.001;

s treated rat groups. Note: Gemfibrozil (100 mg/kg) has

Lipoprotein lipase activity of active compounds



tential antiatherogenic role of LPL has been derived from several

Increased LPL activity positively correlates with HDL



Activities

Part 2A

Page | 124

emfibrozil which showed 18% of

reversal activity of this enzyme.Among all the test compounds, the compounds 88c,

nt activity, whereas compounds 87a and 87d


improved blood lipid profile and enhanced PHLA activity. In

the analysis of structure activity relationship indicated that although the

position showed moderate


Ph 89a-89d) at R1


position were more active than the compounds with electron

on lipoprotein triglyceride level in triton induced

at dose of 100mg/kg attenuate the level of

TG level in triton induced hyperlipidemic


P < 0.001; cP < 0.001

s treated rat groups. Note: Gemfibrozil (100 mg/kg) has been taken as



tential antiatherogenic role of LPL has been derived from several

with HDL-C levels and also



increase the clearance of atherogenic TG

increased LPL activity has led to increased

atherosclerosis in rats. Moreover, decrease of LPL activity in adipose tissue is

considered to be responsible for the hypertriglyceridaemia which is also associated

with insulin resistance and type

88d were further evaluated for lipoprotein lipase activity (LPLA) in triton induced

hyperlipidemic rats (Fig.

activity in liver. The compounds

similar to standard drug G

oxopropanylindole hydrazone derivatives

Figure 4: Effect of compound

hyperlipidemic rat. Each parameter represents pooled data from 6 rats/group and values are

expressed as mean±S.D. ***P < 0.001; *P < 0.05 between control and triton, triton plus

compound treated rats groups,

Note: Gemfibrozil (100 mg/kg)

Antiadipogenic activity

Excessive accumulation of adipose mass tissue not only contributes to

obesity but also increase

The active compounds of the series

preadipocytes. To investigate the anti

cells were treated with MDI and simultaneously with

Adipogenesis was assessed by ORO staining of lipid droplets. ORO staining results



Activities

increase the clearance of atherogenic TG-rich lipoproteins. Studies have shown

ty has led to increased levels of HDL-C and inhibition of



th insulin resistance and type II diabetes. The effect of active compounds

were further evaluated for lipoprotein lipase activity (LPLA) in triton induced

idemic rats (Fig. 4). Administration of triton in rats markedly decreases

The compounds 88c and 89d significantly increased LPL act

similar to standard drug Gemfibrozil explaining the mode of action of these

oxopropanylindole hydrazone derivatives.

Effect of compound 88c and 89d on lipoprotein lipase activity in triton

Each parameter represents pooled data from 6 rats/group and values are


compound treated rats groups, aP < 0.05;

bP < 0.01 between triton verses treated rat groups.

Note: Gemfibrozil (100 mg/kg) has been taken as standard drug.

Antiadipogenic activity


obesity but also increases the risk of cardiovascular disease and type 2 diabetes.

The active compounds of the series 88c and 89d were also evaluated on 3T3

To investigate the antiadipogenic effect of compound

cells were treated with MDI and simultaneously with 88c and




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rich lipoproteins. Studies have shown that

C and inhibition of



active compounds 88c and

were further evaluated for lipoprotein lipase activity (LPLA) in triton induced

on in rats markedly decreases LPL

significantly increased LPL activity

emfibrozil explaining the mode of action of these

on lipoprotein lipase activity in triton induced

Each parameter represents pooled data from 6 rats/group and values are


es treated rat groups.


the risk of cardiovascular disease and type 2 diabetes.

were also evaluated on 3T3-L1

adipogenic effect of compounds 88c and 89d,

and 89d at 20µM.




showed that compound

at 20µM as observed microscopically (Fig. 5

Figuer 5: (A) Reduced lipid accumulation leading to fewer lipid cells by compound

89d on MDI-induced adipogenesis in 3T3

compound 88c and 89d

with Oil Red O solution. Data are the mean±S.D., n=3 and p<0.001 when compared with MDI

induced control group.

MDI-induced greater lipid accumulation was reduced approximately 44% and 31% in

the cells treated w

spectrophotmetrically (Fig. 5

In vivo Antihyperglyc

Taking into consideration the promising antioxidant, antidyslipidemic and

antiadepogenic activity of compound

tested for their antidiabetic activity in streptozotocin induced diabetic rats. Fig.

6b shows the effect of compounds

glucose level on streptozotocin

Metformin was taken as positive control.

It is evident from the results that

singnificant decline in blood glucose levels

However, compound 88

Compounds 88c, 89d

in blood glucose to the tune of 20.5 % (p<0.01) 16.7 % (P<0.05) and 23.3 % (p<0.01)



Activities

showed that compound 88c and 89d likely reduces MDI-mediated lipid accumulation

microscopically (Fig. 5A).

Reduced lipid accumulation leading to fewer lipid cells by compound

induced adipogenesis in 3T3-L1 preadipocytes. (B) Reduction in adipogenesis by

d in differentiation of 3T3-L1 preadipocytes as indicated by staining


induced greater lipid accumulation was reduced approximately 44% and 31% in

the cells treated with compound 88c and 89d at 20µM, as measur

spectrophotmetrically (Fig. 5B).

In vivo Antihyperglycaemic activity


antiadepogenic activity of compound 88c and 89d, these compounds were further

tested for their antidiabetic activity in streptozotocin induced diabetic rats. Fig.

the effect of compounds 88c, 89d and standard drug on decline in blood

glucose level on streptozotocin induced diabetic rats at various time intervals.

was taken as positive control.

It is evident from the results that both compounds 88c

singnificant decline in blood glucose levels on streptozotocin induced

88c was found to be the most active compound of the series.

d and standard drug Metformin demonstrated maximum decline




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mediated lipid accumulation

Reduced lipid accumulation leading to fewer lipid cells by compound 88c and

) Reduction in adipogenesis by

L1 preadipocytes as indicated by staining


induced greater lipid accumulation was reduced approximately 44% and 31% in

at 20µM, as measured


, these compounds were further

tested for their antidiabetic activity in streptozotocin induced diabetic rats. Fig. 6a and

and standard drug on decline in blood

at various time intervals.

c and 89d showed

induced diabetic rats.

most active compound of the series.

etformin demonstrated maximum decline




at 5h and 24.3 % (p<0.01), 18.2 % (p<0.

on the STZ-induced diabetic rats at 100 mg/kg oral dose.

Figure 6: Effect of test compounds on blood glucose profile of streptozotocin

rats.Statistical analysis was made by Dunnet test

2A.2 Conclusion

A series of twelve

evaluated for the antioxidant, antidyslipidemic activity in an acute triton induced

hyperlipidemic model. Among the

two compounds 88c and

anions and hydroxyl radicals along with enhanced protection against lipid

peroxidation. Both the compounds

against LDL oxidation with decresed MDA levels in normolipidemic rats. The

compounds also improved blood lipid profile reducing the TC, PL and TG levels by

23-27% and also showed enhancement of post heparin lipolytic activity (

14-15% as compared to G

enzyme. The active compounds also improved lipoprotein lipase activity explaining



Activities

at 5h and 24.3 % (p<0.01), 18.2 % (p<0.01) and 29.5 % (p<0.01) at 24h respectively

induced diabetic rats at 100 mg/kg oral dose.

Effect of test compounds on blood glucose profile of streptozotocin

nalysis was made by Dunnet test86

(Prism Software 3)

series of twelve substituted hydrazone derivatives were synthesized and


hyperlipidemic model. Among the twelve compounds 87a-87d, 88a

and 89d showed good antioxidant activity reducing superoxide


peroxidation. Both the compounds 88c and 89d also showed effective protection

xidation with decresed MDA levels in normolipidemic rats. The


27% and also showed enhancement of post heparin lipolytic activity (

15% as compared to Gemfibrozil which showed 18% of reversal activity of this




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and 29.5 % (p<0.01) at 24h respectively

Effect of test compounds on blood glucose profile of streptozotocin-treated diabetic

substituted hydrazone derivatives were synthesized and


a-88d and 89a-89d

showed good antioxidant activity reducing superoxide


also showed effective protection

xidation with decresed MDA levels in normolipidemic rats. The


27% and also showed enhancement of post heparin lipolytic activity (PHLA) by

which showed 18% of reversal activity of this




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their mode of action. The active compounds of the series 88c and 89d also showed

antiadepogenic and antihyperglycaemic activity, where compound 88c was found to

be the most active compound with 44% reduction in lipid accumulation and the

blood glucose by 20.5% at 5h and 24.3% at 24h in comparison to standard drug

Metformin. Thus these compounds with balanced activities may be useful in battling

CVDs risk in the future.



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2A.3 Experimental


silicon oil bath. Reactions were monitored by thin layer chromatography on selfmade

plates of silica gel G (Merck, India) or 0.25mm readymade plates of silica gel 60F

254, (E.Merck, Darmstadt, Germany). Column chromatography was performed on

silica gel (Merck, 60 to 120mesh). Infrared spectra (IR) were recorded on Perkin-

FTIR model PC spectrophotometer with frequency of absorptions reported in wave

numbers. MS were recorded on JEOL spectrometer with fragmentation pattern

reported as values. 1H NMR was recorded on Bruker spectrometer with a



were described in parts per million (ppm) relative to TMS (0.00 ppm) using scale, and


dl-Tryptophanmethylester hydrochloride (73)

Thionyl chloride (7mL, 0.01mol) was added drop wise to a stirred suspension

of dl-tryptophan (10g, 0.05mol) in dry methanol (45 mL) at -10°C during 30 min and

stirring continued for 6h during which temperature was gradually raised from -10°C

to room temperature (25°C). The reaction mixture upon filteration under suction

afforded the cream colored product tryptophan methyl ester which was washed with

ether (10mL) and dried.

Yield: 92%; mp 220-223°C; IR (KBr cm-1): 603, 731, 1034,1225, 1285, 1352, 1443,

1499, 1580, 1748 (COOCH3), 2021, 3292; 1HNMR (200 MHz, CDCl3+ DMSO-d6): δ

3.45 (d, J=5.90, 2H), 3.75 (s, 3H), 4.17 (m, 1H), 7.07(m, 2H), 7.28(s, 1H), 7.41(d,

J=7.72, 1H), 7.52(d, J=7.54, 1H), 8.60 (brs, 1H), 10.76(s,1H); MS(ESI+): m/z 218

(M+).

dl- N-benzoyltryptophanmethylester (77)

Benzoyl chloride (74) (1.52mL, 0.012mol) in dry THF (5mL) was added to a stirred

solution of dl tryptophanmethylester (73) (2.18gm, 0.01mol) and dry triethylamine

(1.66 mL, 0.012mol) in dry THF (8mL) during 15 min and was allowed to stir for 2h at

room temperature. The reaction mixture was concentrated under vacuum the residue

triturated with water (20 mL) to get 77, which was crystallized with methanol.



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Yield: 92%; mp 111°C; IR (KBr cm-1): 603, 731, 1330,1370, 1450, 1580, 1625

(CONH), 1745 (COOCH3), 2951, 3060, 3292, 3410; 1HNMR (300 MHz, CDCl3+

DMSO-d6): δ 3.21 (m,1H), 3.46 (m, 1H), 3.68(s,3H), 4.81 (m, 1H), 7.11-7.18 (m, 3H),

7.28(s, 1H), 7.61-7.67(m, 3H), 7.97(m, 2H), 9.57 (s, 1H), 10.85 (s,1H); MS(ESI+):

m/z 323 (M+1)+.

Compounds 78–79 were synthesized in the same manner.

Methyl-3-(1H-indol-3-yl)-2-(2-phenylacetamido)propanoate (78)


77 using 73 (2.18g) and phenylacetylchloride (75) (1.32 mL).



DMSO-d6): δ 2.96-3.17 (m,2H), 3.25 (s,2H), 3.66 (s,3H), 4.05-4.10 (m,1H), 6.90-7.34

(m,9H), 7.63(s,1H), 8.95(s,1H), 10.12(s,1H); MS(ESI+): m/z 337 (M+1)+.

Methyl-3-(1H-indol-3-yl)-2-(3-phenylpropanamido) propanoate (79)


77 using 73 (2.18g) and hydrocinnomoylchloride (76) (1.48 mL).



DMSO-d6): δ 2.32-2.34 (m, 2H) 2.73-2.76 (m, 2H), 3.17-3.20 (m, 2H), 3.58 (s, 3H),

4.85-4.90(m, 1H), 6.56-6.85 (m, 5H), 7.03-7.39 (m, 5H), 7.45 (s, 1H), 8.40 (s, 1H);

MS(ESI+): m/z 351 (M+1)+.

(dl)-N-(1-hydrazinyl-3-(1H-indol-3-yl)-1-oxopropan-2-yl)benzamide (80)

A solution of 77 (1.61 g, 0.005 mol) in ethanol (20 mL) containing 85% hydrazine

hydrate (10 mL) was refluxed for 6h. The reaction mixture was concentrated under

vacuum to give white solid which was triturated with water and dried to give

compound 80, crystallized with methanol.

Yield: 70%; mp 225-228°C; IR (KBr cm-1): 613, 741, 1330, 1356, 1443, 1580, 1625

(CONH), 1661 (CONHNH2), 2950, 3060, 3206 (NH), 3311 (NH); 1HNMR (300 MHz,

CDCl3+ DMSO-d6): δ 2.91-2.96 (m,1H), 3.09-3.16 (m, 1H), 3.92 (s, 2H, NH2), 4.03-



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4.08 ( m, 1H), 6.87-7.34 (m, 8H), 7.85 (m, 2H), 8.34(s, 1H), 9.06(s, NH), 10.23(s,

1H); MS(ESI+): m/z 323 (M+1)+.

Compounds 81–82 were synthesized in the same manner.

N-(1-hydrazinyl-3-(1H-indol-3-yl)-1-oxopropan-2-yl)-2-phenylacetamide (81)


80 using 78 (1.68 g) and hydrazine (10mL).

Yield: 65%; mp 181°C; IR (KBr cm-1): 612, 736, 1335, 1360, 1440, 1560, 1628


CDCl3+ DMSO-d6): δ 2.90-2.94 (m,1H), 3.05-3.10 (m, 1H), 3.23 (s,2H), 3.91 (s, 2H,

NH2), 4.04-4.08 ( m, 1H), 7.05-7.24 (m, 3H), 7.35-7.70 (m, 2H), 8.31(s, 1H), 9.01(s,

NH), 10.19(s, 1H); MS(ESI+): m/z 337 (M+1)+.

N-(1-hydrazinyl-3-(1H-indol-3-yl)-1-oxopropan-2-yl)-3-phenylpropanamide (82)


80 using 79 (1.75 g) and hydrazine (10mL).

Yield: 68%; mp 191°C; IR (KBr cm-1): 613, 741, 1335, 1360, 1455, 1570, 1630


CDCl3+ DMSO-d6): δ 3.16-3.20 (m, 2H), 3.90 (s, 2H, NH2), 4.02-4.07 ( m, 1H), 7.12-

7.20 (m, 3H), 7.30-7.85 (m, 7H), 8.32 (s, 1H), 9.03 (s, NH), 10.14 (s, 1H);

MS(ESI+): m/z 351 (M+1)+.

N-(1-(2-(4-chlorobenzylidene)hydrazinyl)-3-(1H-indol-3-yl)-1-oxopropan-2-

yl)benzamide (87a)

Equimolar quantities of hydrazide 80 (0.323 g, 0.001mol) and the 4-

chlorobenzaldehyde (83) (0.140g, 0.001 mol) (existing in the liquid or solid state

under normal conditions) were mixed in DMSO. The mixtures was stirred at room

temperature for 4h to give white solid compound 87a.

Yield: 70%; mp 132oC; IR (KBr cm-1): 754, 1215, 1484, 1532 (>C=N), 1625, 1664,

2998, 3341 (NH); 1H NMR (300 MHz, CDCl3): δ 3.01-3.16 (m, 1H), 4.72-4.78 (m, 1H),

5.58-5.61(m,1H), 6.70-7.67 (m, 14H), 7.82 (s, 1H), 8.53 (s, NH), 9.75 (s, NH), 11.01

(s, NH); ; ESI-Ms : m/z (M+1)+ 445; Anal. Calcd. for C25H21ClN4O2: C, 67.49; H, 4.76;

N, 12.59; found: C, 67.18; H, 4.41; N, 12.34 %



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N-(3-(1H-indol-3-yl)-1-(2-(4-methoxybenzylidene)hydrazinyl)-1-oxopropan-2-yl)

benzamide (87b)

The compound 87b was prepared by similar method as described for the synthesis of

87a using 80 (0.323 g) and 4-methoxybenzaldehyde (84) (0.136 g).

Yield: 72%; mp 200oC; IR (KBr cm-1): 759, 1216, 1456, 1527(>C=N), 1625, 1661,

2928, ,3385 (NH); 1H NMR (300 MHz, CDCl3): δ 3.40-3.45 (m,1H), 3.89 (s, 3H), 5.01-

5.03 (m, 1H), 5.86-5.89 (m,1H), 6.88-7.91 (m, 14H), 7.98 (s, 1H), 8.60 (s, NH), 9.78

(s, NH), 10.91 (s, NH); ESI-Ms : m/z (M+1)+ 441; Anal. Calcd. for C26H24N4O3 : C,

70.89; H, 5.49; N, 12.72; Found: C, 70.68; H, 5.41; N, 12.84 %.

N-(3-(1H-indol-3-yl)-1-(2-(4-methylbenzylidene)hydrazinyl)-1-oxopropan-2-

yl)benzamide (87c)

The compound 87c was prepared by similar method as described for the synthesis of

87a using 80 (0.323 g) and 4-methylbenzaldehyde (85) (0.120 g).

Yield: 69%; mp 190oC; IR (KBr cm-1): 767, 1218, 1456, 1517 (>C=N), 1632,1665,

2928, ,3375 (NH); 1H NMR (300 MHz, CDCl3): δ 2.84(s,3H), 3.36-3.40 (m,1H), 4.98-

5.03 (m, 1H), 5.82-5.86(m,1H), 70.8-8.11 (m, 14H), 7.90 (s, 1H), 8.56 (s, NH), 10.18


73.56; H, 5.70; N, 13.20; Found: C, 73.48; H, 5.46; N, 13.04 %.

N-(3-(1H-indol-3-yl)-1-oxo-1-(2-(3,4,5-trimethoxybenzylidene)hydrazinyl)propan-

-2-yl)benzamide (87d)

The compound 87d was prepared by similar method as described for the synthesis of

87a using 80 (0.323 g) and 3,4,5 –trimethoxybenzaldehyde (86) (0.196 g).

Yield: 75%; mp 120 oC; IR (KBr cm-1): 760, 1217, 1460, 1525 (>C=N), 1632,1676,

3397 (NH); 1H NMR (300 MHz, CDCl3): δ 3.43-3.48 (m,1H), 3.89 (s, 9H), 5.04-5.07

(m, 1H), 5.93-5.96 (m,1H), 6.94-7.89 (m, 12H), 7.94 (s, 1H), 8.59 (s, NH), 9.59 (s,

NH), 10.94 (s, NH); ESI-Ms : m/z (M+1)+ 501; Anal. Calcd. for C28H28N4O5 : C, 67.19;

H, 5.64; N, 11.10; found: C, 66.48; H, 5.56; N, 11.04 %.

N-(1-(2-(4-chlorobenzylidene)hydrazinyl)-3-(1H-indol-3-yl)-1-oxopropan-2-yl)-2-

phenylacetamide (88a)



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The compound 88a was prepared by similar method as described for the synthesis of

87a using 81 (0.336 g) and 4-chlorobenzaldehyde (83) (0.140 g).

Yield: 72%; mp 170 oC; IR (KBr cm-1): 758, 1217, 1486, 1520 (>C=N),1651, 3401

(NH); 1H NMR (300 MHz, CDCl3): δ 2.58 (s,2H), 3.51-3.57 (m,1H), 4.77-4.79 (m, 1H),

5.64-5.66 (m, 1H) 6.89-7.89 (m, 14H), 8.02 (s, 1H), 8.61 (s, NH), 9.93 (s, NH), 11.13

(s, NH); ESI-Ms : m/z (M+1)+ 459; Anal. Calcd. for C28H28N4O5: C, 68.04; H, 5.05; N,

7.72; found: C, 67.88; H, 4.96; N, 7.44 %.

N-(3-(1H-indol-3-yl)-1-(2-(4-methoxybenzylidene)hydrazinyl)-1-oxopropan-2-yl)-

2-phenylacetamide (88b)




(NH); 1H NMR (300 MHz, CDCl3): δ 2.60 (s,2H), 3.01-3.29 (m, 1H), 3.82 (s, 3H),

4.80-4.83 (m, 1H), 5.65-5.70 (m,1H), 6.64-7.84 (m, 14H), 7.92 (s, 1H), 8.58 (s, NH),

9.85 (s, NH), 10.91 (s, NH); ESI-Ms : m/z (M+1)+ 455; Anal. Calcd. for C27H26N4O3: C,

71.35; H, 5.77; N, 12.33; found: C, 70.88; H, 5.66; N, 12.13 %.

N-(3-(1H-indol-3-yl)-1-(2-(4-methylbenzylidene)hydrazinyl)-1-oxopropan-2-yl)-2-

phenylacetamide (88c)



Yield: 75%; mp 180oC; IR (KBr cm-1): 760, 1215, 1421, 1521(>C=N),1643, 3406

(NH); 1H NMR (300 MHz, CDCl3): δ 2.64 (s, 2H), 3.13( s,3H), 3.54-3.57 (m,1H), 4.81-

4.83 (m, 1H), 5.69-5.70 (m,1H), 6.91-7.52 (m, 12H), 7.55-7.89 (m,2H), 7.99 (s, 1H),

8.59 (s, NH), 10.13 (s, NH), 11.13 (s, NH); ESI-Ms : m/z (M+1)+ 439; Anal. Calcd. for

C27H26N4O2: C, 73.95; H, 5.98; N, 12.78; Found: C, 73.83; H, 5.72; N, 12.53 %.

N-(3-(1H-indol-3-yl)-1-oxo-1-(2-(3,4,5-trimethoxybenzylidene)hydrazinyl)propan-

2-yl)-2-phenylacetamide (88d)


87a using 81 (0.336 g) and 3,4,5-trimethoxybenzaldehyde (86) (0.196 g).



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(NH); 1H NMR (300 MHz, CDCl3): δ 2.58 (s, 2H), 3.54-3.57 (m,1H), 3.86(s,9H), 4.91-

4.93 (m, 1H), 5.67-5.69 (m,1H), 7.11-7.52 (m, 12H), 8.19 (s, 1H), 8.69 (s, NH), 10.11


67.69; H, 5.88; N, 10.89; found: C, 67.56; H, 5.85; N, 10.23 %.

N-(1-(2-(4-chlorobenzylidene)hydrazinyl)-3-(1H-indol-3-yl)-1-oxopropan-2-yl)-3-

phenylpropanamide (89a)


87a using 82 (0.350 g) and 4-chlorobenzaldehyde (83) (0.140 g).


(NH); 1H NMR (300 MHz, CDCl3): δ 2.47-2.49 (m, 2H), 2.83-2.86( m,2H), 3.15-3.17

(m,1H), 4.81-4.83 (m,1H), 5.54-5.63 (m, 1H), 6.91-7.64 (m, 14H), 7.93 (s, 1H), 8.13

(s, NH), 10.48 (s, NH), 11.28 (s, NH); ESI-Ms : m/z (M+1)+ 473; Anal. Calcd. for

C27H25ClN4O2: C, 68.56; H, 5.33; N, 11.85; found: C, 68.16; H, 5.05; N, 11.53 %.

N-(3-(1H-indol-3-yl)-1-(2-(4-methoxybenzylidene)hydrazinyl)-1-oxopropan-2-yl)-

3-phenylpropanamide (89b)



Yield: 73%; mp 197 oC; IR (KBr cm-1): 759, 1216, 1423, 1508 (>C=N),1653, 3341,

3403 (NH); 1H NMR (300 MHz, CDCl3): δ 2.44-2.52 (m, 2H), 2.80-2.85( m,2H), 3.11-

3.14 (m,1H), 3.83 (s,3H), 4.80-4.82 (m,1H), 5.64-5.72 (m, 1H), 7.11-7.84 (m, 14H),

7.95 (s, 1H), 8.15 (s, NH), 10.50 (s, NH), 11.18 (s, NH); ESI-Ms : m/z (M+1)+ 469;

Anal. Calcd. for C28H28N4O3: C, 71.78; H, 6.02; N, 11.96; found: C, 71.56; H, 6.05; N,

11.87 %.

N-(3-(1H-indol-3-yl)-1-(2-(4-methylbenzylidene)hydrazinyl)-1-oxopropan-2-yl)-3-

phenylpropanamide (89c)



Yield: 68%; mp 198oC; IR (KBr cm-1): 759, 1215, 1424, 1508 (>C=N),1637, 3380

(NH); 1H NMR (300 MHz, CDCl3): δ 2.50-2.53 (m, 2H), 2.63 (s, 3H), 2.85-2.90 (

m,2H), 3.18-3.21 (m,1H), 4.77-4.79 (m,1H), 5.69-5.71 (m, 1H), 6.91-7.79 (m, 14H),



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12.17 %.

N-(3-(1H-indol-3-yl)-1-oxo-1-(2-(3,4,5-trimethoxybenzylidene)hydrazinyl)propa- -

n-2-yl)-3-phenylpropanamide (89d)




(NH);1H NMR (300 MHz, CDCl3): δ 2.45-2.50 (m, 2H), 2.83-2.86( m,2H), 3.15-

3.18(m,1H), 3.83 (s, 9H), 4.71-4.76 (m,1H), 5.63-5.65 (m,1H), 6.95-7.87 (m, 12H),



10.57 %.

2A.3.1 Biological Assay

Materials and Methods

Animal used

Rats (Charles Foster strain, male, adult, body wt 200-225 g) were kept in a room

with controlled temperature at 25-26ºC, humidity 60- 80% and 12/12h light/dark cycle

(light on from 8.00 AM to 8:00 PM) under hygienic conditions. Animals were

acclimatized for one week before starting the experiment. The animals had free

access to the normal diet and water.

Antioxidant activity

Super oxide anions were generated enzymatically by xanthine (160 mM),

xanthine oxidase (0.04 U), and nitroblue tetrazolium (320 µM) in absence and

presence of the test compounds listed in Scheme 1 at concentrations 200 µg/mL in

100 mM phosphate buffer (pH 8.2). Compounds were sonicated well in phosphate

buffer before use. The reaction mixtures were incubated at 37ºC and after 30 min the

reaction was stopped by adding 0.5 mL glacial acetic acid. The amount of formazone

formed was calculated spectrophotometrically. In another set of experiments, the

effect of compounds on the generation of hydroxyl radical was also studied by non

enzymatic reactants. Briefly, •OH were generated in a non enzymatic system

comprising deoxyribose (2.8 mM), FeSO4.7H2O (2 mM), sodium ascorbate (2.0 mM)

and H2O2 (2.8 mM) in 50 mM KH2PO4 buffer (pH 7.4) to a final volume of 2.5 mL. The



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above reaction mixtures in the absence or presence of test compounds were

incubated at 37ºC for 90 min The test compounds were also studied for their

inhibitory action against microsomal lipid peroxidation in vitro by non enzymatic

inducer. Reference tubes and reagents blanks were also run simultaneously.

Malondialdehyde (MDA) contents in both experimental and reference tubes were

estimated spectrophotometrically by thiobarbituric acid.87 Alloprinol, Mannitol and α-

tocopherol were used as standard drugs for superoxide, hydroxylations and

microsomal lipid peroxidation.

LDL oxidation

Serum was separated from the blood of normolipemic donors who were fasted

overnight and fractionated into very lowdensity lipoprotein (VLDL), low density

lipoprotein (LDL) and high density lipoprotein (HDL) by ultracentrifugation.88 The

lipoproteins preparations were dialysed against 150 mM NaCl containing EDTA (0.02

% w/v) in presence of N2 gas in cold. The purity of LDL was checked on

polyacrylamide gel electrophoresis. LDL (0.71 mg) and CuCl2.2H2O (10 µM) in the

absence or presence of the test compounds in 50 mM phosphate buffer saline (pH

7.4) to a final volume of 1.5 mL, was incubated at 37ºC for 16h. The level of lipid

peroxides in unoxidized LDL, oxidized LDL with Cu++ in the absence or presence of

test compounds at dose of 200 µg/mL were assayed as thiobarbituric acid reactive

substances (TBARS). Briefly, the reaction mixture contained 0.5ml SDS (8% w/v),

0.5 mL glacial acetic acid, 1.5 mL TBA (0.8% w/v) was heated in a boiling water bath

for 1h. After cooling up to room temperature optical density of reaction mixture was

read at 532 nm with respective reagent blank. The level of lipid peroxide as nmol of

Malondialdehyde formed was calculated by taking absorption coefficient of MDA as

1.78X105 cm-1 M-1 mg protein.

Antidyslipidemic activity in triton induced hyperlipidemic rat model

Rats were divided into different groups- control, triton treated and triton plus

compounds treated groups, every group containing six rats each. In the acute

experiment of 18h, hyperlipidemia was developed by administration of triton WR-

1339 (Sigma Chemical Company, St. Louis, MO, USA) at a dose of 400 mg/kg, b.w.

intraperitoneally (i.p) to animals of all the treated groups with extraction, fractionation

and the test compounds were macerated with gum acacia, suspended in water and

fed simultaneously with triton at a dose of 100 mg/kg p.o. to the animals of treated

groups, diet was withdrawn. Animals of control and triton group without treatment



Activities

Part 2A

Page | 137

with extraction, fractionation and the test compounds were given same amount of

gum acacia suspension (vehicle). After 18h of treatment the animals were

anaesthetized with thiopentone solution (50 mg/kg b.w.) prepared in normal saline

and 1mL blood was withdrawn from retro-orbital sinus using glass capillary in EDTA

coated tubes (3.0 mg/mL blood). The blood was centrifuged at 2500 x g for 10 min at

4ºC and plasma was separated. Plasma was diluted with normal saline (ratio of 1:3)

and used for analysis of total cholesterol (TC), phospholipid (PL) and triglyceride

(TG) by standard enzymatic methods. Using Beckmann auto analyzer and standard

kit purchase from Merck Company and post heparin lipolytic activity (PHLA) was

assayed using spectrophotometer.88

Lipoprotein measurement

Plasma was fractionated into very low density lipoprotein (VLDL), low density

lipoprotein (LDL) and high density by standard procedures reported earlier.89

Lipoprotein lipase activity in liver of triton induced hyperlipidemic rats

Liver was homogenized (10%, w/v) in cold 100 mM lipoprotein (HDL) by poly

anionic precipitation methods. Lipoproteins were analyzed for their triglyceride (TG)

level phosphate buffer pH 7.2 and used for the assay of total lipolytic activity of

lipoprotein lipase (LPL).90

Cell culture and adipogenic differentiation

3T3-L1 mouse embryo fibroblasts cell line was obtained from the American

Type Culture Collection. Cells were cultured in a humidified atmosphere at 37ºC and

5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) heat

inactivated fetal bovine serum and antibiotic penicillin and streptomycin. For

adipogenesis induction 50000 cells were seeded in 24 multi-well plates. After 2 days,

when cells achieved near complete confluence, culture media was replaced with

adipogenesis media I (containing Insulin 5 µg/mL, IBMX 0.5 mM and Dexamethason-

-e 250 nm in culture medium). This media was then replaced after 72h with

adipogenesis media II (Insulin 5 µg/mL in DMEM with 10% FBS). After replacement

of this media, cells were then maintained next 2 days in 10% FBS containing DMEM

medium. Lipid globules in the adipogenic cells starts forming from day 4 onwards

after treatment and fully developed adipocytes were observed after day 8 of

adipogenesis treatment. More than 80% cells do have lipid globules at this stage.

Triglyceride assay and Oil Red O staining

To study effect of compound on adipogenic differentiation, cells were

differentiated as mentioned in above protocol along with compound at 20µM



Activities

Part 2A

Page | 138

concentrations. Fully differentiated 3T3-L1 (with or without compound) adipocytes

were rinsed in phosphate buffered saline (pH 7.4). The adipocytes lipid globules were

stained with Oil Red O (0.36% in 60% isopropanol) for 20 min Unstained Oil Red O

was removed by rinsing wells twice with phosphate buffer saline. After complete

removal of PBS, finally, 100% isopropanol was used to extract the dye from the cells

and extracted dye absorbance was measured at 492 nm.

Antihyperglycaemic activity

Male albino rats of Sprague-Dawley strain (8 to 10 weeks of age, body weight

160 ± 20 g) were selected for this study. Streptozotocin (Sigma, USA) was dissolved

in 100 mM citrate buffer pH 4.5 and calculated amount of the fresh solution was

injected to overnight fasted rats (60 mg/kg) intraperitoneally. Fasting blood glucose

was checked 48h later by glucometer by using glucostrips and animals showing

blood glucose values over 270 mg/dL were selected and divided into groups of five

animals each. Rats of experimental groups were administered suspension of

standard drug and the desired test samples orally (made in 1.0% gum acacia) at a

dose of 100 mg/kg body weight. Animals of control group were given an equal

amount of 1.0 % gum acacia. The blood glucose level of each animal was

determined just before the administration of standard drug and test samples (0 min)

and thereafter at 30, 60, 90, 120, 180, 240, 300 and 1440 min Food but not water

was withdrawn from the cages during 0 to 300 min The average lowering in blood

glucose level between 0 to 300 min and 0 to 1440 min was calculated by plotting the

blood glucose level on y-axis and time on x-axis and determining the area under

curve (AUC). Comparing the AUC of experimental group with that to control group

determined the percent lowering of blood glucose level during the period. Statistical

analysis was made by Dunnett’s test (Prism Software).86

Statistical evaluation

All results are presented as the means ±S.D. of results from three independent

experiments. Groups were analyzed via t-tests (two-sided) or ANOVA for

experiments with more than two subgroups. Probability values of p < 0.05 were

considered to be statistically significant.

Synthesis and biological evaluation of antithrombotic activity of

Indole based triazole derivatives

Chapter 2B

Page | 139

2B. Synthesis and Biological Evaluation of Antithrombotic

Activity of Indole Based Triazole Derivatives

The cardiovascular system disorder related diseases are the prime cause of

mortality and morbidity in today’s world leading towards major socio-economic

consequences. Thrombotic events are leading causes of cardiovascular associated

death in the developed world.91,92 Intravascular thrombosis leads to many acute

cardiovascular diseases.93 Controlled activation of blood coagulation, that is

haemostasis is necessary to prevent the blood loss after fatal injury, whereas,

uncontrolled activation could lead to vascular and arterial thrombosis, which in turn

could result into various serious thromboembolic diseases.

The current therapies include the use of antiplatelet agents, Aspirin,

Ticlopidine, Clopidogrel and anticoagulants such as Heparin and Warfarin. However,

these drugs do not work in acute conditions and require extended periods of closely

monitored therapy mainly because of the high risk of bleeding. 94-96

On the other hand, a number of drug-class-specific adverse reactions include,

for instance, Aspirin related gastrointestinal ulcers and bleeding, thrombotic

thrombocytopenic purpura that may be associated to thienopyridines,97 Heparin

induced thrombocytopenia, Warfarin induced skin necrosis etc.98 Moreover, Warfarin

has a narrow therapeutic window and its activity is affected by diet and genetic

makeup, so requiring continuous monitoring for accurate dosing. Hence, the search

for more potent and safer new chemical entities for antiplatelet/antithrombotic activity

is of current interest.

Indole derivatives are class of privileged structure with wide range of

biological activities and many indole containing derivatives are known to possess

antithrombotic activity.57-60 Pharmacologically, β-carbolines and tetrahydro-β-

carbolines are important rings in indole alkaloids with a wide spectrum of

pharmacological actions.97-100 Harmalol, Harmaline, Norharmane, Harmol, Harmine

and Harmane belonging to β -carboline, were only capable of inhibiting the platelet

aggregation induced by collagen and had more than 130 µM of IC50 value.99

Moreover, triazoles moiety is a substructure of a number of biologically active

compounds101 and coupling of triazoles with other biologically active heterocycles is

expected to show an increased spectrum of activities. It appeared therefore, of

interest to synthesize indolyltriazole derivatives and test them for their antithrombotic

activity.



Chapter 2B

Page | 140

Hence, a number of substituted indolyl triazole derivatives were synthesized

and evaluated for their antithrombotic in vivo activity in mice. The two compounds

with 40% of antithrombotic activity were further optimized to get better results. The

details of the study are reported herein.

2B.1 Results and Discussion

2B.1.1 Chemistry

The synthesis of final compounds indolyl triaozoles derivatives 94a-94c, 95a-

95c, 96a-96c, 100a-100c, 106 and 107 is outlined in Scheme 2, 3 and 4 respectively.

The key intermediates hydrazides 80, 82, and 92 required for the synthesis of

substituted indolyl triazole derivatves were synthesized according to the procedure

mentioned in Chapter 2A. The hydrazide 80 synthesized was further cyclocondensed

with 4-methoxy benzaldehyde (84) in the presence of ammonium acetate and acetic



Chapter 2B

Page | 141

acid to afford the corresponding final compound 94a (Scheme 2).

All other final compounds 94b-94c, 95a-95c and 96a-96c were synthesized by

the same procedure as described above from key intermediates 80, 82 and 92

respectively (Table 5). The compounds 94b-94c were synthesized by using 80 and

3,4,5-trimethoxy benzaldehyde (86) and isopropyl benzaldehyde (93) respectively.

The compounds 95a-95c were synthesized by using 82 and 4-methoxy

benzaldehyde (84), 3,4,5-trimethoxybenzaldehyde (86) and isopropylbenzaldehyde

(93) respectively. The compounds 96a-96c were synthesized by using 92 and 4-

methoxybenzaldehyde (84), 3,4,5-trimethoxybenzaldehyde (86) and isopropyl

benzaldehyde (93) respectively.

The starting material dl-tryptophan methyl ester (73) was synthesized as

mentioned in Chapter 2A which on reaction with 4-methoxysulphonylchloride (97) in

the presence of triethylamine afforded methyl-3-(1H-indol-3-yl)-2-(4-methoxyphenyl

sulfonamido)propanoate (98). The ester 98 on refluxing with hydrazine hydrate in

ethanol gave the key intermediate N-(1-hydrazinyl-3-(1H-indol-3-yl)-1-oxopropan-2-

yl)-4-methoxybenzenesulfonamide (99). Condensation of 99 with 4-methoxyben-

-zaldehyde (84) in the presence of ammonium acetate and acetic acid afforded the

corresponding final compound 100a (Scheme 3).



Chapter 2B

Page | 142

The compound 100b was synthesized by using 99 and 3,4,5-trimethoxy

benzaldehyde (86). The compound 100c was synthesized by using 99 and 4-

isopropylbenzaldehyde 93.

The intermediate (RS) methyl 1,2,3,4-tetrahydro-9H-pyrido (3,4-b)-indole-3-

carboxylate (101) was prepared by cyclization of methyl ester of tryptophan 73 with

formaldehyde via Pictet Spengler reaction.102 The intermediate obtained by

cyclization with formaldehyde, followed by basification, on reaction with benzoyl

chloride (74) yielded methyl-2-benzoyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-

carboxylate (102). The compound 102 on refluxing with hydrazine hydrate in

ethanol gave the key intermediate 2-benzoyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-

b]indole-3-carbohydrazide (104) which was further cyclocondensed with 4-methoxy

benzaldehyde (84) in the presence of ammonium acetate and acetic acid to obtain

corresponding target compound (3-(5-(4-methoxyphenyl)-4H-1,2,4-triazol-3-yl)-3,4-

dihydro-1H-pyrido[3,4-b]indol-2(9H)-yl)(phenyl)methanone (106) (Scheme 4).

The compound 107 was also synthesized in a similar manner as described

for the synthesis of 106 using 2-(3-phenylpropanoyl)-2,3,4,9-tetrahydro-1H-

pyrido[3,4-b]indole-3-carbohydrazide (105) and 3,4,5-trimethoxy benzaldehyde (86)

(Table 5).



Chapter 2B

Page | 143

All the final indolyltriazole 94a-94c, 95a-95c, 96a-96c, 100a-100c, 106 and

107 have been fully characterized by their IR, 1H NMR and Mass spectrum. The

compound N-(2-(1H-indol-3-yl)-1-(5-(4-isopropylphenyl)-4H-1,2,4-triazol-3-yl)ethyl)

benzamide (94c) was taken as reference here for the discussion of spectral

characterization. The reaction of 92 with isopropyl benzaldehyde (93) in 1:1 ratio in

acetic acid and ammonium acetate yielded compound 94c.

The characteristic absorption band in IR spectrum at 3311 cm-1 and 3206cm-1

for NH2 stretching of amino group of hydrazide (92) was found absent in 94c

indicating that hydrazide moiety has reacted with aldehyde group and formed triazole

ring in the compound 94c. The singlet at δ 2.51 for hydrazide NH2 in compound 92

was found absent in the compound 94c, also confirming the conversion of hydrazide

to triazole (94c).

Its mass spectrum showed (M+1)+ peak in ESI MS at m/z 450. Its IR

spectrum showed absorption band at 3244.6 cm-1 and 3377.7 cm-1 due to the

presence of NH stretching which was also confirmed by the presence of broad singlet

at δ 9.35 and δ10.80 for the NH protons in its1H NMR spectrum.

The compound 107 in Scheme 4 was also characterized in a similar manner.

The cyclization of dl tryptophan ester (73) to β-carboline ring was confirmed by the

presence of two multiplets in the range δ 4.57-4.98 for the two CH2 protons at

position 1 in the piperidine ring of β-carboline ring. A multiplet for the four benzylic

proton in the hydrocinnomoyl group was also observed in the range δ 2.75-3.01. A

singlet for nine protons for three methoxy group was seen at δ 3.89 confirming the

coupling of 3,4,5-trimethoxybenzaldehyde with the the hydrazide to form triazole ring.

The IR spectrum also showed a peak at 1687 cm-1 confirming the presence of amide

group in the compound 107. Further, its mass spectrum showed (M+1) + peak at m/z

450.

Similarly, all other compounds N-(2-(1H-indol-3-yl)-1-(5-(3,4,5-

trimethoxyphenyl)-4H-1,2,4-triazol-3-yl)ethyl)benzamide (94b), N-(2-(1H-indol-3-yl)-1-

(5-(4-isopropylphenyl)-4H-1,2,4-triazol-3-yl)ethyl)benzamide (94c), N-(2-(1H-indol-3-

yl)-1-(5-(4-methoxyphenyl)-4H-1,2,4-triazol-3-yl)ethyl)-3-phenylpropanamide (95a),

N-(2-(1H-indol-3-yl)-1-(5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazol-3-yl)ethyl)-3-phe-

-nylpropanamide (95b), N-(2-(1H-indol-3-yl)-1-(5-(4-isopropylphenyl)-4H-1,2,4-triazol-

3-yl)ethyl)-3-phenylpropanamide (95c), N-(2-(1H-indol-3-yl)-1-(5-(4-methoxyphenyl)-

4H-1,2,4-triazol-3-yl)ethyl)-2,4-dichlorobenzamide (96a), N-(2-(1H-indol-3-yl)-1-(5-

(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazol-3-yl)ethyl)-2,4-dichloro benzamide (96b), N-

(2-(1H-indol-3-yl)-1-(5-(4-isopropylphenyl)-4H-1,2,4-triazol-3-yl)ethyl)-2,4-dichlorobe-



Chapter 2B

Page | 144

-nzamide (96c), N-(2-(1H-indol-3-yl)-1-(5-(4-methoxyphenyl)-4H-1,2,4-triazol-3-yl)et-

-hyl)-4-methoxybenzenesulfonamide (100a), N-(2-(1H-indol-3-yl)-1-(5-(3,4,5-trimeth-

-oxyphenyl)-4H-1,2,4-triazol-3-yl)ethyl)-4-methoxybenzenesulfonamide (100b), N-(2-

(1H-indol-3-yl)-1-(5-(4-isopropylphenyl)-4H-1,2,4-triazol-3-yl)ethyl)-4-methoxyben- -

zenesulfonamide (100c), and (3-(5-(4-Methoxyphenyl)-4H-1,2,4-triazol-3-yl)-3,4-

dihydro-1H-pyrido[3,4-b]indol-2(9H)-yl)(phenyl)methanone (106) were characterized

on the basis of corresponding spectral data. The spectral data are reported in

experimental part of this chapter.

2B.1.2 In vivo Antithrombotic activity

All the compounds synthesized were tested for their antithrombotic activity103

as shown in Table 5. Out of all fourteen compounds 94a-94c, 95a-95c, 96a-96c,

100a-100c, 106 and 107 evaluated for in vivo antithrombotic activity, two compounds

94a and 95b showed 40% activity. Further optimization of the compound with 40%

antithrombotic activity 94a and 95b led to compound 106 and 107, with improved

activity with 50% protection against pulmonary thromboembolism. The other ten

compounds 94b, 94c, 95a, 95c, 96a-96c and 100a-100c exhibited activity between

10-30%.

Table 5: Antithrombotic activity of the test compounds (94a-94c, 95a-95c, 96a-96c, 100a-100c, 106

and 107)

S. No R1 R2 R3 R4 Antithrombotic activity (%

protection)

94a Phenyl H OMe H 40

94b Phenyl OMe OMe OMe 30

94c Phenyl H Isopropyl H 10

95a CH2CH2Ph H OMe H 10

95b CH2CH2Ph OMe OMe OMe 40

95c CH2CH2Ph H Isopropyl H 20

96a 2,4-diClPh H OMe H 30

96b 2,4-diClPh OMe OMe OMe 10

96c 2,4-diClPh H Isopropyl H 20

100a 4-OMePh H OMe H 10

100b 4-OMePh OMe OMe OMe 20

100c 4-OMePh H Isopropyl H 10

106 Phenyl H OMe H 50

107 CH2CH2Ph OMe OMe OMe 50

Aspirin - - - - 37



Chapter 2B

Page | 145

2B.2 Conclusion

In conclusion, a series of fourteen indolyltriazole derivatives were synthesized

and evaluated for antithrombotic activity. Out of fourteen compounds 94a-94c, 95a-

95c, 96a-96c, 100a-100c, 106 and 107, two compounds 94a and 95b showed 40%

of antithrombotic activity, which were further optimized to their cyclized analogue 106

and 107. These two analogues 106 and 107 interestingly showed improved

protection of about 50% against pulmonary thromboembolism. The current study led

to the development of indole based antithrombotic compounds and offers a promising

opportunity to design and develop a more potent and active series of antithrombotic

agents.



Chapter 2B

Page | 146

2B.3 Experimental


silicon oil bath. Reactions were monitored by thin layer chromatography on selfmade

plates of silica gel G (Merck, India) or 0.25mm readymade plates of silica gel 60F

254, (E.Merck, Darmstadt, Germany). Column chromatography was performed on

silica gel (Merck, 60 to 120mesh). Infrared spectra (IR) were recorded on Perkin-

FTIR model PC spectrophotometer with frequency of absorptions reported in wave

numbers. MS were recorded on JEOL spectrometer with fragmentation pattern

reported as values. 1H NMR was recorded on Bruker spectrometer with a



were described in parts per million (ppm) relative to TMS (0.00 ppm) using scale, and


Synthesis of dl-tryptophanmethylesterhydrochloride (73), dl-N-benzoyltrypt-

-ophanmethylester (77) and Methyl-3-(1H-indol-3-yl)-2-(3-phenylpropanamido)

propanoate (79)

Same as mentioned in Chapter2-Part 2A.

Methyl-2-(2,4-dichlorobenzamido)-3-(1H-indol-3-yl)propanoate (91)


77 using 73 (2.18g) and 2,4-dichlorobenzoylchloride (90) (1.66 mL).

Yield: 88%; mp 132oC; IR (KBr cm-1): 744.6, 834.3, 878.9, 1108, 1639.3 (CONH),

1741.5 (COOCH3), 2361, 3071, 3460.5; 1H NMR (CDCl3, 200MHz): δ 3.65-

3.71(m,1H), 3.81(s,6H), 3.98-4.01(m,1H), 5.02-5.04(m,1H), 6.60-7.96(m,13H),

8,53(s,1H), 10.41(s,1H), 11.03(s,1H); MS (ESI+): m/z = 392 (M+H) +.

Methyl 3-(1H-indol-3-yl)-2-(4-methoxyphenylsulfonamido) propanoate (99)


77 using 73 (2.18 g) and 4-methoxybenzenesulfonylchloride (98) (2.46 g).

Yield: 89%; mp 125oC; IR (KBr cm-1): 670, 761, 1037, 1160 (SO2), 1216, 1360 (SO2),

1738(COOCH3), 2361, 3020, 3454; 1H NMR (CDCl3, 200MHz): δ 3.65-3.71(m,1H),

3.81(s,6H), 3.98-4.01(m,1H), 5.02-5.04(m,1H), 6.60-7.96(m,13H), 8,53(s,1H),

10.41(s,1H), 11.03(s,1H); MS (ESI+): m/z = 389 (M+H) +.



Chapter 2B

Page | 147

Synthesis of (dl)-N-(1-hydrazinyl-3-(1H-indol-3-yl)-1-oxopropan-2-yl)benzamide

(80) and N-(1-hydrazinyl-3-(1H-indol-3-yl)-1-oxopropan-2-yl)-3-phenylpropan-

-amide (82)

Same as mentioned in chapter 2-Part 2A

2,4-dichloro-N-(1-hydrazinyl-3-(1H-indol-3-yl)-1-oxopropan-2-yl)benzamide (92)


80 using 91 (1.95 g) and hydrazine hydrate (10mL).

Yield: 65%; mp 205oC; IR (KBr cm-1): 745.3, 838.4, 879.3, 1105.2, 1639.5 (CONH),

1687(CONHNH2), 2361, 3076, 3206 (NH), 3311 (NH2); 1H NMR (CDCl3, 200MHz): δ

2.51(s,2H, NH2), 3.10-3.24(m,2H,), 4.73-4.74(m,1H), 6.96-7.03(m,2H), 7.15(s,1H),

7.30(s,1H), 7.60-7.63(m,2H), 7.76-7.83(m,1H), 8.03-8.06(m,1H), 8.70(bs, 1NH),

9.36(bs,1NH), 10.67(bs, 1NH); MS (ESI+): m/z = 391 (M+H) +.

N-(1-hydrazinyl-3-(1H-indol-3-yl)-1-oxopropan-2-yl)-4-methoxybenzenesulfona-

-mide (100)


80 using 99 (1.94 g) and hydrazine hydrate (10mL).

Yield: 59%; mp 140oC; IR (KBr cm-1): 675, 759.8, 1043.8, 1165.9, 1215, 1356.9,

1684 (CONHNH2), 2361,3020, 3210 (NH), 3309 (NH2); 1H NMR (CDCl3, 200MHz): δ

2.51(s,2H, NH2), 2.87-2.95(m,2H), 3.86(s,3H), 3.93-3.98(m,1H), 6.85-6.90(m,1H),

6.97-7.02(m,1H), 7.06(s,1H), 7.20-7.22(m,1H), 7.29-7.34(m,1H), 7.37-7.38(m,1H),

7.40-7.41(m,2H), 7.61(s,NH), 9.24(s,NH), 10.70(s,NH); MS (ESI+): m/z = 389

(M+H)+.

(RS)-methyl-1,2,3,4,-tetrahydro-9H-pyrido[3,4-b]indole-3-carboxylate (101)

Formaldehyde (38 weight % solution in water, 7 mL) was added to a solution

of dl-tryptophan methylester hydrochloride (73) (11g, 0.43 mol) in aq. methanol (77

mL; ratio 10:1) during 30 min. The reaction mixture was stirred for 4h at room

temperature, concentrated and cooled to give (RS)-methyl-1,2,3,4,-tetrahydro-9H-

pyrido(3,4-b)indole-3-carboxylate hydrochloride which was basified with 10 % aq

solution of Na2CO3 to give 101.

Yield: 57%; mp 150°C; IR (KBr cm-1): 738, 820, 866, 1004, 1056, 1128, 1190, 1234,

1302, 1346, 1442, 1504, 1590, 1626, 1740 (COOCH3), 1916, 2312, 2752, 2938,



Chapter 2B

Page | 148

3058, 3150, 3324;1H NMR (200 MHz, CDCl3): δ 2.83-2.96 (m, 2H), 3.16 (m, 1H),

3.80 (s, 4H), 4.13(bs, 2H), 7.06-7.19 (m, 2H), 7.30 (d, J=7.10, 1H), 7.47 (d, J=8.18,

1H), 7.83 (brs, 1H); MS(FAB): m/z 230(M+).

Methyl-2-benzoyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylate (102)


77 using 101 (2.30 g) and benzoylchloride (74) (1.52 mL).

Yield: 87%; mp 200°C; IR (KBr cm-1): 744.6, 1034.1, 1108.6, 1415.0, 1699.4

(CONH), 1741.6 (COOCH3), 2301, 2949.5, 3071, 3379; 1H NMR (CDCl3, 200MHz): δ

3.12-3.24(m,1H), 3.48-3.58(m,1H), 3.72(s,3H), 4.60-4.66(m,1H), 4.84-4.96(m,1H),

5.40-5.45(m,1H), 7.12-7.17(m,4H), 7.40-7.71(m,3H), 7.65-7.71(m,2H), 8.07(s,1H);

MS (ESI+): m/z = 334 (M+H) +.

Methyl-2-(3-phenylpropanoyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-

carboxylate (103)


77 using 101 (2.30 g) and hydrocinnomoylchloride (76) (1.48 mL).


(CONH), 1744.6 (COOCH3), 2363, 3058.4, 3267, 3461.5; 1H NMR (CDCl3, 200MHz):

δ 2.74-3.30(m,4H), 3.36-3.42(m,1H), 3.81(s,3H), 4.49-4.76(m,1H), 4.96-5.22(m,1H),

5.66-5.76(m,1H), 6.76-6.86(m,1H), 7.07-7.53(m,7H), 7.84-8.00(m,1H), 8.42(s,1H);

MS (ESI+): m/z = 363 (M+H) +.

2-Benzoyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carbohydrazide (104)


using 102 (1.67 g) and hydrazine hydrate (10mL).


(CONH), 1670.9 (CONHNH2), 2363.4, 3058.2, 3207.9 (NH), 3317 (NH2), 3461.6; 1H

NMR (CDCl3, 200MHz): δ 2.51(s,NH2), 3.12-3.24(m,1H), 3.48-3.58(m,1H), 4.60-

4.66(m,1H), 4.84-4.96(m,1H), 5.40-5.45(m,1H), 7.12-7.17(m,4H), 7.40-7.71(m,3H),

7.65-7.71(m,2H), 8.07(s,1H) 9.10(s,1H); MS (ESI+): m/z = 335 (M+H) +.

2-(3-Phenylpropanoyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carbohydra-

-zide (105)



Chapter 2B

Page | 149


using 103 (1.81 g) and hydrazine hydrate (10mL).

Yield: 62%; mp 130°C; IR (KBr cm-1): 741.9, 1010.3, 1621.5(CONH),

1675.4(CONHNH2), 1246.6, 2371.1, 2981.8, 3146.3 (NH), 3317.7 (NH2), 3418.7; 1H

NMR (CDCl3, 200MHz): δ 2.51(s,NH2), 2.74-3.30(m,4H), 3.36-3.42(m,1H), 4.49-

4.76(m,1H), 4.96-5.22(m,1H), 5.66-5.76(m,1H), 6.76-6.86(m,1H), 7.07-7.53(m,7H),

7.84-8.00(m,1H), 8.42(s,1H), 9.32(s,1H); MS (ESI+): m/z = 363 (M+H) +.

N-(2-(1H-indol-3-yl)-1-(5-(4-methoxyphenyl)-4H-1,2,4-triazol-3-yl)ethyl)benzami-

-de (94a)

A mixture of key intermediate 80 (0.323g, 0.001 mol) and 4-methoxy

benzaldehyde (84) (0.136 g), 0.001 mol) in 1:1 ratio was dissolved in glacial acetic

acid in a 50 mL round bottom flask. To the mixture, ammonium acetate was added.

The reaction mixture was stirred for a period of 4h at room temperature. The

progress of the reaction was monitored by TLC. After completion of the reaction, the

mixture was poured into ice cold water and neutralized with ammonia. The

precipitated product was filtered, washed with water to obtain the desired product

94a.

Yield: 75%; mp 210oC; IR (KBr cm-1): 760.1, 1028.7, 1168.8, 1421.8, 1512.9, 1629.3,

1666.2, 2979.8, 3020.8, 3285.3 (NH), 3387.5 (NH); 1H NMR (CDCl3, 200MHz): δ

3.44 (m, 1H), 3.86( s, 3H), 4.95-4.97 (m, 1H), 5.81-5.83(m, 1H),6.88-8.07(m, 14H),

8.59( s, 1H), 10.09(bs, NH), 11.09(bs, NH); MS (ESI+): m/z = 438 (M+H) +.

N-(2-(1H-indol-3-yl)-1-(5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazol-3-yl)ethyl)ben-

-zamide (94b)




1585.3, 1676.2, 2964.8, 3021.5, 3279.5 (NH), 3387 (NH); 1H NMR (CDCl3, 200MHz):

δ 2.28-2.30 (m, 1H), 3.76( s, 3H), 3.85(s, 6H), 4.86-4.88 (m, 1H), 5.74-5.75(m,

1H),6.97-8.17(m, 12H), 10.69(bs, NH), 11.44(bs, NH); MS (ESI+): m/z = 498 (M+H) +.

N-(2-(1H-indol-3-yl)-1-(5-(4-isopropylphenyl)-4H-1,2,4-triazol-3-yl)ethyl)benzami-

-de (94c)



Chapter 2B

Page | 150


94a using 80 (0.323 g) and 4-isopropylbenzaldehyde (93) (0.148g).


1671.8, 2974.6, 3084.7, 3244.6 (NH), 3377.7b(NH); 1H NMR (CDCl3, 200MHz): δ

1.23-1.26 (m, 6H), 2.88-2.94( m, 1H), 3.39-3.41(m, 1H), 4.99-5.01 (m, 1H), 5.84-

5.86(m, 1H),6.96-7.95(m, 14H), 9.35( s, NH), 10.80(bs, NH); MS (ESI+): m/z = 450

(M+H) +.

N-(2-(1H-indol-3-yl)-1-(5-(4-methoxyphenyl)-4H-1,2,4-triazol-3-yl)ethyl)-3-phenyl

propanamide (95a)


94a using 82 (0.350 g) and 4-methoxybenzaldehyde 84 (0.136 g).

Yield: 72%; mp 165oC; IR (KBr cm-1): 761.6, 1031.6, 1216.9, 1426.6, 1510.2, 1656.6

(CONH), 2929.9, 3025.7, 3342.0 (NH), 3437.1 (NH); 1H NMR (CDCl3, 200MHz): δ

1.23 (t, J= 6Hz, 6H), 2.42 (t, J= 6Hz, 2H), 2.70-2.76 (m,2H), 2.90-2.95(m,1H), 3.15-

3.20 (m,1H), 4.63-4.66(m,1H), 5.50-5.53(m,1H), 6.89-7.29(m,10H), 7.51-7.59(m,2H),

7.98-8.13(m,2H), 10.64(bs,NH), 11.24(bs,NH) ; MS (ESI+): m/z = 466 (M+H) +.

N-(2-(1H-indol-3-yl)-1-(5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazol-3-yl)ethyl)-3-

phenylpropanamide (95b)



Yield: 74%; mp 185oC; IR (KBr cm-1): 761.6, 1043.11, 1216.33, 1420.6, 1507.4,

1664.6 (CONH), 3020.7, 3376.0 (NH), 3426.1 (NH); 1H NMR (CDCl3, 200MHz): δ

2.45-2.47(m,2H), 2.76-2.79 (m,1H), 3.10-3.25(m,1H), 3.76 ( s,6H), 3.82(s,1H), 4.69-

4.72(m,1H), 5.61-5.63(m,1H), 6.78-7.76(m,12H), 8.47(s, 1H), 9.25(bs, NH),

10.57(bs,NH); MS (ESI+): m/z = 526 (M+H) +.

N-(2-(1H-indol-3-yl)-1-(5-(4-isopropylphenyl)-4H-1,2,4-triazol-3-yl)ethyl)-3-phenyl

propanamide (95c)




(CONH), 2961.9, 3079.7, 3314.5 (NH), 3396.3 (NH); 1H NMR (CDCl3, 200MHz): δ



Chapter 2B

Page | 151

1.23 (t, J= 6Hz, 6H), 2.42 (t, J= 6Hz, 2H), 2.70-2.76 (m,2H), 2.90-2.95(m,1H), 3.15-

3.20 (m,1H), 4.63-4.66(m,1H), 5.50-5.53(m,1H), 6.89-7.29(m,10H), 7.51-7.59(m,2H),

7.98-8.13(m,2H), 10.64(bs,NH), 11.24(bs,NH) ; MS (ESI+): m/z = 478 (M+H) +.

N-(2-(1H-indol-3-yl)-1-(5-(4-methoxyphenyl)-4H-1,2,4-triazol-3-yl)ethyl)-2,4-dichl-

-orobenzamide (96a)




(CONH), 3066.7, 3291.1 (NH), 3397.4 (NH); 1H NMR (CDCl3, 200MHz): δ 3.79-

3.80(m,1H), 3.82(s,3H), 4.80-4.82(m,1H), 5.65-5.66(m,1H), 6.93-7.05(m,4H), 7.22-

7.31(m,1H), 7.62-7.79(m,5H), 8.0-8.13(m,1H), 8.74-8.93(m, 1H), 10.67(bs,NH),

11.30(bs,NH); MS (ESI+): m/z = 506 (M+H) +.

N-(2-(1H-indol-3-yl)-1-(5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazol-3-yl)ethyl)-2,4-

dichlorobenzamide (96b)




1652.6 (CONH), 3211.7, 3351.0 (NH), 3493.1 (NH); 1H NMR (CDCl3, 200MHz): δ

3.79-3.80(m,1H), 3.82(s,9H), 4.80-4.82(m,1H), 5.65-5.66(m,1H), 6.93-6.97(m,2H),

7.12-7.61(m,5H), 7.79-8.09(m,3H), 8.74 (s,1H), 10.67(bs,NH), 11.30(bs,NH); MS

(ESI+): m/z = 566 (M+H)+.

N-(2-(1H-indol-3-yl)-1-(5-(4-isopropylphenyl)-4H-1,2,4-triazol-3-yl)ethyl)-2,4-

dichlorobenzamide (96c)




(CONH), 3066.7, 3291.1 (NH), 3397.4 (NH); 1H NMR (CDCl3, 200MHz): δ 1.22-

1.25(m,6H), 2.90-2.95(m,1H), 3.15-3.17(m,1H), 4.78-4.81( m,1H), 5.64-5.66(m,1H),

6.92-8.13(m,13H), 10.61(bs, NH), 11.33(bs, NH); MS (ESI+): m/z = 518 (M+H) +.

N-(2-(1H-indol-3-yl)-1-(5-(4-methoxyphenyl)-4H-1,2,4-triazol-3-yl)ethyl)-4-metho-

-xybenzenesulfonamide (100a)



Chapter 2B

Page | 152

The compound 100a was prepared by similar method as described for the synthesis

of 94a using 99 (0.388 g) and 4-methoxybenzaldehyde (84) (0.136 g).

Yield: 78%; mp 175oC; IR (KBr cm-1): 759.7, 1028.07, 1162 (SO2), 1253.02, 1360.8

(SO2), 1420.37, 1459.36, 3017.7, 3291.1 (NH), 3396.4 (NH); 1H NMR (CDCl3,

200MHz): δ 3.65-3.71(m,1H), 3.81(s,6H), 3.98-4.01(m,1H), 5.02-5.04(m,1H), 6.60-

7.96(m,13H), 8,53(s,1H), 9.41(bs,NH), 10.03(bs,NH); MS (ESI+): m/z = 504 (M+H) +.

N-(2-(1H-indol-3-yl)-1-(5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazol-3-yl)ethyl)-4-

methoxybenzenesulfonamide (100b)

The compound 100b was prepared by similar method as described for the synthesis

of 94a using 99 (0.388 g) and 3,4,5-trimethoxybenzaldehyde (86) (0.196 g).


(SO2), 1412.7, 1459.0, 3066.7, 3293.1, 3387.4; 1H NMR (CDCl3, 200MHz): δ 3.74-

3.77(m,1H), 3.82(s,9H), 4.80-4.82(m,1H), 5.63-5.66(m,1H), 6.83-6.90(m,2H), 7.12-

7.71(m,9H), 7.69(s,NH), 9.63(bs,NH), 10.30(bs,NH); MS (ESI+): m/z = 564 (M+H) +.

N-(2-(1H-indol-3-yl)-1-(5-(4-isopropylphenyl)-4H-1,2,4-triazol-3-yl)ethyl)-4-meth-

-oxybenzenesulfonamide (100c)

The compound 100c was prepared by similar method as described for the synthesis

of 94a using 99 (0.388 g) and 4-isopropylbenzaldehyde (93) (0.148g).


(SO2), 1419.37, 1459.36, 3017.7, 3290.1, 3393.4; 1H NMR (CDCl3, 200MHz): 1.20-

1.24(m,6H), 2.90-2.94(m,1H), 3.12-3.14(m,1H), 4.73-4.76( m,1H), 5.60-5.64(m,1H),

6.95-7.20(m, 5H), 7,36-7.70(m, 6H), 7.78(s,1H), 8.09-8.13(m,2H), 9.61(bs, NH),

10.33(bs, NH); MS (ESI+): m/z = 516 (M+H) +.

(3-(5-(4-Methoxyphenyl)-4H-1,2,4-triazol-3-yl)-3,4-dihydro-1H-pyrido[3,4-b]indol-

2(9H)-yl)(phenyl)methanone (106)




1669.95 (CONH), 3021.7, 3291.1, 3396.4; 1H NMR (CDCl3, 200MHz): δ 3.10-

3.24(m,2H), 4.53-4.57(m,1H, 1-CH2), 5.03-5.08(m,1H, 1-CH2), 5.49-5.54(m,1H),

7.10-8.14(m,13H), 8.70(bs, 1NH), 9.36(bs,1NH); MS (ESI+): m/z = 450 (M+H) +.



Chapter 2B

Page | 153

3-Phenyl-1-(3-(5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazol-3-yl)-3,4-dihydro-1H-

pyrido[3,4-b]indol-2(9H)-yl)propan-1-one (107)




1687.18 (CONH), 3020.8, 3205.6, 3380.0; 1H NMR (CDCl3, 200MHz): δ 2.75-

3.01(m, 4H, CH2CH2Ph), 3.19-3.45(m,1H), 3.75-3.83(m,1H), 3.86(s, 9H), 4.57-

4.72(m,1H, 1-CH2), 4.95-4.98(m,1H, 1CH2), 5.76-5.78(m,1H), 6.83-6.88(m,2H), 7.04-

7.56(m, 8H), 7.89-8.05(m,1H), 8.52(bs,1H), 9.83(bs,1H); MS (ESI+): m/z = 538

(M+H) +.

2B.3.1 Biological testing

Swiss mice (20–25 g, from CDRI animal colony) were used in a group of at

least 10 animals each. Thrombosis was induced by infusion of a mixture of 15 mg

collagen and 5 mg adrenaline in a volume of 100 mL into the tail vein of each mouse.

This resulted either death or hind limb paralysis of 100% animals. Pulmonary

thromboembolism was induced by following a method of Diminno and Silver.103 Each

of the test compounds 94a-94c, 95a-95c, 96a-96c, 100a-100c, 106 and 107

(30µΜ/Kg) and vehicle were separately administered orally 60 min prior to the

thrombotic challenge. A mixture of collagen (150µg/mL) and adrenaline (50µg/mL)

was administered by the rapid intravenous injection into the tail vein to induce hind

limb paralysis or death. A group of 10 animals were used to evaluate the effect of test

compound, while 5 mice were used to assess effect of a standard drug, aspirin and a

group of vehicle treated (n=5) mice was also used in each experiment.

% Protection was expressed as:

(P control –P test)x100

P control

Ptest is the number of animals paralyzed/dead in the test compound treated group,

and Pcontrol is the number of animals paralyzed/dead in vehicle treated group.



Chapter 2B

Page | 154

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Aberrant Biosynthesis of Bromoreticulines Chapter 3

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Aberrant Biosynthesis of Aberrant Biosynthesis of Aberrant Biosynthesis of Aberrant Biosynthesis of BromoreticulineBromoreticulineBromoreticulineBromoreticulinessss


Page | 159

3.1 Introduction

For thousands of years, plants have been used for their medicinal properties

to treat diseases and to promote health and wellbeing.1 Plants produce a vast array

of secondary metabolites in response to biotic or abiotic interactions with their

environment, which impart flavor, color and fragrance and confer protection through a

variety of antimicrobial, pesticidal and pharmacological properties.2

Today, most of the pharmaceuticals derived from natural sources have been

obtained from terrestrial organisms. In India, traditional medicine was documented in

the Ayurveda in around 900 B.C.3 Because of their sessile way of life, plants have

developed a rich arsenal of chemicals, encompassing more than 200,000 known

compounds.4

About 20 % of plant species accumulate alkaloids which are one of the most

important groups of natural products, i.e. compounds that are synthesised by the

secondary metabolism of living organisms. These molecules play important roles in

the species that synthesize them but are not essential for life, unlike products of

primary metabolism, e.g. nucleosides, amino acids, carbohydrates or lipids.5,6 They

are derived from biologically active metabolic degradation products of excess amino

acids (or amino acid intermediates).6

Although rare in mammals, alkaloids are particularly abundant in higher

plants, insects, amphibians and fungi. Within these species, alkaloids are

synthesised to act as poisons or anaesthetics for predators, or even as mediators of

ecological interactions. In any case, their purpose is to increase chances of survival.

3.2 Benzylisoquinoline alkaloids

The benzylisoquinoline alkaloids (BIAs) show an unusually rich variety of

structural types.7,8 They comprise a large and diverse group of nitrogen-containing

secondary metabolites with about 2,500 compounds identified in plants. As

secondary or specialized metabolites, BIAs are not essential for normal growth and

development but appear to function in the defense of plants against herbivores and

pathogens.9,10

Benzylisoquinoline is the structural backbone of many classes of alkaloids

with a wide variety of structures e.g. cularine, proaporphine, aporphine, protopine,

protoberberine, benzophenanthridine, pavine, morphinanedienone and bisbenzyl

isoquinolines alkaloids7,8 (Fig. 1).


Page | 160

Many benzylisoquinoline alkaloids have been reported to show therapeutic

properties.9-11 The BIAs morphine and codeine are potent analgesics that have been

exploited for thousands of years and are produced exclusively in the opium poppy

(Papaver somniferum).12 Other BIAs used as pharmaceuticals include sanguinarine

and berberine13 (antimicrobial), noscapine4 (antitussive and potentially

antineoplastic), papaverine14 (vasodilator) and (+)-tubocurarine15 (muscle relaxant).

Thebaine is a metabolic precursor to morphine and codeine and is used for the

synthesis of analgesics such as oxycodone, naltrexone and buprenorphine.11 The

aporphine type alkaloid magnoflorine has been reported to protect HDL during

oxidant stress to prevent the development of atherosclerotic disease and to inhibit

human lymphoblastoid cell-killing by HIV-1.16-18 A report has also stated the

antimicrobial agent berberine had cholesterol lowering activity.19

Most of these compounds are not feasible targets for de novo chemical

synthesis owing primarily to the occurrence of multiple chiral centers, therefore,

plants remain the only commercial sources for many pharmaceutical alkaloids.11

Figure 1: Benzylisoquinoline derived alkaloids

The naturally occuring tetraoxygenated 1-benzyl tetrahydroisoquinoline

alkaloids of interest are reticuline (1) isolated from Papaver somniferum and other

plants, protosinomenine (2) isolated from Erythrina lithosperma and nororientaline (3)

isolated from Argemone platyceras.7,8 Racemic reticuline functions as a dopamine

blocking agent in the CNS20 and also stimulates hair growth.21


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Reticuline (1) is a branch-point intermediate in the biosynthesis of many types

of benzylisoquinoline alkaloids and also a nonnarcotic alkaloid of pharmaceutical

significance that is useful in the development of antimalarial and anticancer drugs. 22

Its substitution pattern suits it for intramolecular coupling reactions leading to a pleth-

-ora of alkaloid types, e.g. morphinans,23 protoberberines,24 benophenanthridines,25

phthalideisoquinolines 26 and protopine.27,28

Despite this storied use and palpable presence in the current pharmacopeia,

relatively little is known about the biosynthesis, regulation and transport of these

molecules. In the past years, there has been a substantial increase in our knowledge

of the structure elucidation and biosynthesis of alkaloids. As a result of this extensive

research of the elucidation of structures of alkaloids and their unprecedented

structural diversity has led to a great number of biosynthetic hypotheses, some of

which were found correct by radiotracer experiments.

3.3 Biogenesis of Reticuline

Winterstein and Trier29 suggested that two molecules of 3,4-dihydroxy

phenylalanine (DOPA) (6) might be modified in the plant as follows:

Decarboxylation of one molecule of DOPA (6) could give rise to 3,4-

dihydroxyphenethylamine (dopamine, 7) whereas oxidative deamination of second

molecule of DOPA (6) could give rise to 3,4-dihydroxyphenylacetaldehyde (8a) via.

3,4-dihydroxyphenylpyruvicacid (8) (Scheme 1). Condensation of 7 and 8a could

then form norlaudanosoline (16a) recognized as a potential precursor for more

complex isoquinoline alkaloids29. Later on this hypothesis was subsequently

elaborated, extended and refined mainly by Robinson. 30,31

The dual role of (L)-tyrosine (4) in the biosynthesis of 1-benzyltetrahydroisoq-

-uinoline alkaloids was established. It was suggested that (L)-tyrosine (4) gave rise to


Page | 162

two different aromatic units dopamine (7) and 4-hydroxyphenylacetaldehyde (11) as

follows:

Tyrosine (4) by decarboxylation formed tyramine (5) which by ortho

hydroxylation could form dopamine (7). On the other hand tyrosine (4) could give rise

to 4-hydroxyphenylpyruvicacid (10) by transamination. Further decarboxylation of 10

could form 4-hydroxyphenylacetaldehyde (11). Alternatively, dopamine (7) may

undergo condensation with 3,4-dihydroxyphenylpyruvicacid (8) derived from the

transamination of DOPA (6). Condensation of dopamine (7) with 8 could form the

norlaudanosoline-1-carboxylic acid (16b) intermediate.32,33 Thus, in the biosynthetic

pathway, two molecules of tyrosine (4) are used, only the phenylethylamine fragment

i.e. the upper part of the tetrahydroisoquinoline ring system is formed via DOPA (6)

and the remaining lower part carbons are derived via 4-hydroxyphenylpyruvicacid

(10) and 4-hydroxyphenylacetaldehyde (11).34 Stadler et al.34 ruled out the

intermediacy of norlaudanosoline-1-carboxylicacid (16b) in the biosynthesis of

reticuline. It was further established that the product from the mannich like reaction of

7 and 11 is the trihydroxylated 1-benzylisoquinoline alkaloid norcoclaurine (12)

formed stereospecifically as (S)-enantiomer. The biosynthetic pathway studies in

Annona reticulate34 plant demonstrated a common biogenetic origin of trihydroxylated


Page | 163

1-benzylisoquinoline of coclaurine type (13) and tetraoxygenated bases of reticuline

(1) type. It was established by Zenk et al.35 that the trihydroxylated intermediate

norcoclaurine (12) was formed by the stereospecific condensation of dopamine (7)

and 4-hydroxyphenylacetaldehyde (11). Norcoclaurine (12) thus was considered to

be the first true 1-benzylisoquinoline intermediate in the biosynthesis of reticuline (1)

and reticuline derived alkaloids.

(S)-Norcoclaurine (12) by the action of S-adenosylmethionine (SAM) was

converted into (S)-coclaurine (13) which by N-methylation gave rise to (S)-N-

methylcoclaurine (14). Subsequent hydroxylation and O-methylation of 14 in the

benzylic half gave rise to reticuline (1).36 Alternatively (S)-coclaurine (13) by 6-O-

methylation could give rise to 6-O-methylnorlaudanosoline (15) which by subsequent

4-O-methylation could form norreticuline (17). Finally, N-methylation of 17 could form


Page | 164

reticuline (1). In another possibility (S)-norcoclaurine (12) by 3’-hydroxylation could

form (S)-norlaudanosoline (16a) intermediate which could undergo either 6-O-

methylation to form 15 or 4’-O-methylation to form 4’-O-methylnorlaudanosoline (18).

Norreticuline (17) could then be formed either by 4’-O-methylation of 15 or by 6-O-

methylation of 18. Finally N-methylation of 17 could form reticuline (1).

3.4 Aberrant Biosynthesis

The enzymes present in a plant normally convert a normal precursor into a

normal product. Generally the enzymes are substrate specific, however the enzymes

present in some micro-organism are not strictly substrate specific and are capable of

converting abnormal precursor into abnormal product. This aspect of biosynthesis is

called aberrant biosynthesis.

Gorman et al.37 reported that an antibiotic pyrrolnitrin-1 is biosynthesized from

tryptophan by a strain of Pseudomonas aurefaciens. Feeding of 6-fluorotryptophan

and 7-methyltryptophan to the same micro-organism demonstrated the formation of

4’-fluoropyrrolnitrin and 3’-methyl-3’-dechloropyrrolnitrin respectively.37

Another aspect of aberrant biosynthesis is the conversion of abnormal

precursors into normal products. There are also reports available where abnormal

precursors have been converted by micro-organism into normal products.38 Although

aberrant biosynthesis is common in micro-organism, however, this aspect of

biosynthsis is less investigated in higher plants.39-44

3.5 Present Aim

Study of aberrant biosynthesis has great advantages as:

(i) Analogues of biologically active complex natural products can be prepared

using the same enzyme system as is used for the conversion of abnormal

precursor into normal product.

(ii) Since the unnatural product (analogue of biologically active molecule)

possesses a structural label in addition to radiolabel, these can be used in

study of metabolism.

Since reticuline (1) is the potential intermediate of various class of alkaloids

viz. morphinanes, protoberberines, aporphines, pavine, phthalidisoquinolines,

bisbenzylisoquinolines etc., it was, therefore, thought worthwhile to explore the


Page | 165

potentiality of higher plants by undertaking the aberrant biosynthesis of 8-

bromoreticuline (19), 2’-bromoreticuline (20) and 5-bromoreticuline (21) in Litsea

glutinosa (Lour.) C.B. (Roxb.) var. glabraria Hook (Lauraceae) plant and the results

of this study are presented herein.

3.5.1 Biogenesis of 8-Bromoreticuline (19)

5-Bromo-3,4-dihydroxyphenethylamine (22) could undergo condensation

with 4-hydroxyphenylacetaldehyde (11) to give 8-bromonorcoclaurine (23) which

could give rise to 8-bromococlaurine (24) by 6-O-methylation. (Scheme-2)

C-3'-hydroxylation of 24 in benzylic half could give 8-bromo-6-

methoxynorlaudanosoline (25). Further specific 4’-O-methylation of 25 could give 8-

bromonorreticuline (26) which could be converted finally into 8-bromoreticuline (19)

by subsequent N-methylation.


Page | 166

Alternatively 23 first could undergo C-3’-hydroxylation to form 8-

bromonorlaudanosoline (27) which either by specific 6-O-methylation could give 25

or by specific 4’-O-methylation could give 8-bromo-4’-O-methylnorlaudanosoline (28).

6-O-Methylation followed by N-methylation of 28 could form 8-bromoreticuline (19) as

shown in Scheme 2.

3.5.2 Biogenesis of 2’-Bromoreticuline

Dopamine (7) could undergo condensation with 2-bromo-4-hydroxy

phenylacetaldehyde (29) to give 2’-bromonorcoclaurine (30). 2’-Bromoreticuline (20)

could then be formed from 30 by the same reaction sequence as described for the

formation of 8-bromoreticuline (19) from 23 involving the intermediacy of either 31,

32 and 33 or 34, 35 through 33 (Scheme 3).

NH2HO

HO

HOH

O

29

7NH

HO

HO

HO

+

NH

HO

MeO

HO

NH

HO

HO

HO

OH

30

34

NH

HO

MeO

HO

OH

32

5

8

31

1'

2'3'

5

NH

MeO

HO

HO

OH

35

NH

MeO

MeO

HO

OH

NMe

MeO

MeO

HO

OH33

20

BrBr Br

BrBrBr

BrBr

Scheme 3: Biogenesis of 2'-Bromoreticuline


Page | 167

3.5.3 Biogenesis of 5-Bromoreticuline

2-Bromo-3,4-dihydroxyphenethylamine (36) could undergo condensation with

4-hydroxyphenylacetaldehyde (11) to form 5-bromonorcoclaurine (37). By the same

reaction sequence as shown for the formation of 8-bromoreticuline (19) from 8-

bromonorcoclaurine (23), 5-bromonorcoclaurine (37) could be converted into 5-

bromoreticuline (21) via the intermediacy of either 38, 39 and 40 or 41, 42 through 40

(Scheme 4).

NH2HO

HO

HOH

O

11

36NH

HO

HO

HO

+

NH

HO

MeO

HO

NH

HO

HO

HO

OH

37

41

NH

HO

MeO

HO

OH

39

5

8

38

1'

2'3'

5

NH

MeO

HO

HO

OH

42

NH

MeO

MeO

HO

OH

NMe

MeO

MeO

HO

OH40

21

Br

Br Br

BrBrBr

BrBr

Scheme 4: Biogenesis of 5-Bromoreticuline


Page | 168

3.5.4 Syntheses of precursors

Synthesis of Substituted Bromobenzaldehydes (44a-44d)

Bromination of 4-hydroxy-3-methoxybenzaldehyde (43a) in glacial acetic acid

furnished 5-bromo-4-hydroxy-3-methoxybenzaldehyde (44a) (with 90% yield)

however, the 2-bromo derivative of 43a was obtained by the sequential acetylation,

nitration, reduction and diazotization respectively as follows: 4-Hydroxy-3-methoxy

benzaldehyde (43a) on acetylation gave 43b, which on treatment with concentrated

nitric acid afforded 4-acetoxy-3-methoxy-2-nitrobenzaIdehyde (43c) (80% yield)

(Scheme 5). 2-Amino-4-hydroxy-3-methoxybenzaIdehyde (43d) was obtained by

treatment of 43c with ferrous hydroxide. Compound 43d afforded 2-bromo-4-

hydroxy-3-methoxybenzaldehyde (44b) with hydrobromic acid and sodium nitrite

(Scheme 5).

MeO

HO

CHO CHOMeO

HO

43a 44a

Br

(i)

MeO

AcO

CHO MeO

AcO

CHO

NO2

MeO

HO

CHO

NH2

MeO

HO

CHO

Br

(iii)

(ii)

(iv)

(v)

Scheme 5: (i) Br2, gl. AcOH (ii) Ac2O/pyridine (iii) Conc.HNO3 (iv) Fe(OH)2 (v) HBr, NaNO2

43b 43c 43d

44b

3-Hydroxy-4-methoxybenzaldehyde (43e) when brominated in acetic acid

furnished the 2-bromoderivative 44c, (53% yield) which was further demethylated to

give 2-bromo-3,4-dihydroxybenzaldehyde (44d) in good yield (Scheme 6).


Page | 169

Synthesis of substituted phenethylamine (9, 36, 49a and 49b)

Benzylation of the aldehydes 43a, 44a, 44b and 44d in presence of

potassium carbonate and sodium iodide gave the benzylated products 45a-45d

respectively (~85% yield). The nitrostyrene 46a-46d was obtained by nitroaldol

condensation of the benzylated aldehydes 45a-45d with nitromethane (75% yield)

(Scheme 7).


Page | 170

The nitrostyrenes 46a-46c were then treated with sodium borohydride to

afford the nitroethane derivatives 47a-47c (53% yield) which on reduction with Raney

nickel followed by hydrogenolysis afforded the substituted phenethylamines 49a, 49b

and 36, respectively, whereas 9 was obtained by LiAlH4 reduction of 46d (Scheme

7).

Synthesis of substituted phenyl acetylchlorides (54a-54d)

Sodium borohydride reduction of the aldehydes 43e, 44c, 43f and 43g

separately gave the corresponding alcohols 50a-50d. The benzyl chlorides 51a-51d

were prepared by treatment of the corresponding benzyl alcohols 50a-50d with

thionyl chloride. The chlorides 51a-51d were separately treated with potassium

cyanide to afford the corresponding benzyl nitriles 52a-52d (75% yield) which on

alkaline hydrolysis yielded phenyl acetic acids 53a-53d respectively. The phenyl

acetic acid 53a-53d, were then converted to the corresponding acid chlorides 54a-

54d by treatment with thionyl chloride (Scheme 8).

CHO

R3

R2 CH2OH

R3

R2(i) CH2Cl

R3

R2

CH2CN

R3

R2CH2COOH

R3

R2CH2COCl

R3

R2

(iii)

(iv)

(v)(iii)

Scheme 8: (i) C6H5CH2Cl, K2CO3, KI, Ethanol, reflux (ii) NaBH4, THF:Methanol(1:1)

(iii) SOCl2, Benzene (iv) KCN (v) (CH2OH)2,KOH

R1 R1 R1

R1R1R1

R1 R2 R343e, H OH OMe

44c, Br OH OMe

43f, H H OH

43g, H OH OH

(ii)

R1 R2 R350a, H OBn OMe

50b, Br OBn OMe

50c, H H OBn

50d, H OBn OBn


51b, Br OBn OMe

51c, H H OBn

51d, H OBn OBn


52b, Br OBn OMe

52c, H H OBn

52d, H OBn OBn


53b, Br OBn OMe

53c, H H OBn

53d, H OBn OBn


54b, Br OBn OMe

54c, H H OBn

54d, H OBn OBn


Page | 171

Synthesis of substituted 1-benzyltetrahydroisoquinolines precursors (24-26,

33, 35 and 58)

The acid chlorides 54a-54d on condensation with appropriate amines 7, 9,

49a and 49b afforded the suitably substituted amides 55a-55f (~75% yield). Bischler-

Napieralski cyclization of the amide 55a-55f gave the corresponding dihydroisoquino-

-line 56a-56f hydrochloride (~80% yield) (Scheme 9). Sodium borohydride reduction

of the dihydroisoquinoline 56a-56f in ice cooled methanol furnished the

corresponding 1,2,3,4-tetrahydroisoquinoline 57a-57f. Hydrogenolysis of the benzyl

ethers afforded the desired bromo-1-benzyltetrahydroisoquinoline 24, 25, 26, 35, 33

and 58 respectively (Scheme 9).

NH

R2

BnO

R6

R5

R4

O

N

R3

R2

BnO

NH

R3

R2

BnO

R5R6

NH

R3

R2

HO

R5R6

(iii)

Scheme 9: Reagents (i) POCl3/C6H6 (ii) NaBH4/MeOH (iii) EtOH/HCl

R3

(i)

(ii)

R1

R1 R2 R3 R4 R5 R655a H OMe Br H H OBn

55b H OMe Br H OBn OBn

55c H OMe Br H OBn OMe

55d H OBn H Br OBn OMe

55e H OMe H Br OBn OMe

55f Br OMe H H OBn OMe


56b H OM Br H OBn OBn





R1

R4

R5R6

R1

R4R4

R1

R1 R2 R3 R4 R5 R624 H OMe Br H H OH

25 H OMe Br H OH OH

26 H OMe Br H OH OMe

35 H OH H Br OH OMe

33 H OMe H Br OH OMe

58 Br OMe H H OH OMe


57b H OM Br H OBn OBn






Page | 172

Treatment of bromo-1-benzyltetrahydroisoquinoline 57c, 57e and 57f with

formic acid and formaldehyde gave the corresponding N-methyl-1,2,3,4-tetrahydro

isoquinolines 59a-59c (~70% yield). Debenzylation of 59a-59c afforded the

corresponding 1-benzyltetrahydroisoquinolines (19-21) (~71% yield) (Scheme 10).

NH

R2

MeO

BnO

MeO

(i)

NMe

R2

MeO

BnO

MeO

NMe

R2

MeO

HO

MeO

(ii)

Scheme 10: Reagents (i) HCOOH, HCHO (ii) EtOH, HCl

R1

R3

OBn

R1

R3

OBn

R3

OH

R1

R1 R2 R357c H Br H

57e H H Br

57f Br H H

R1 R2 R319 H Br H

20 H H Br

21 Br H H

R1 R2 R359a H Br H

59b H H Br

59c Br H H

3.5.5 Labelling of precursors

Acid catalyzed exchange reactions45 were used to prepare tritium labelled

compounds. To a mixture of thionyl chloride and tritiated water, was added (±) 8-

bromoreticuline (19). The mixture was heated under nitrogen (sealed tube) for 110 h

at 100°C. Work up in the usual manner afforded (±)-[aryl-3H]-8-bromoreticuline (19).

(±)-[Aryl-3H]-3,4-dihydroxyphenethylamine (7), (±)-5-bromo-[aryl-3H]-4-hydrox-

-y-3-methoxyphenethylamine (49a), (±)-8-bromo-[aryl-3H]-coclaurine (24), (±)-8-

bromo-[aryl-3H]-6-O-methylnorlaudanosoline (25), (±)-2'-bromo-[aryl-3H]-4'-O-methyl-

-norlaudanosoline (35), 2’-bromo-[aryl-3H]-norreticuline (33) and 2-bromo-[aryl-3H]-

3,4-dihydroxyphenethylamine (36) were prepared by the same method as described

above. (±)-8-Bromo-[3-14C]norreticuline (26) was prepared by total synthesis.

3.5.6 Feeding experiments

To check whether the plant was actively biosynthesizing reticuline or not, (L)-

[U-14C]-tyrosine (4) (experiment 1) was initially fed to young cut branches of L.

glutinosa var. glabraria Roxb. plant. After 5 days the biosynthetic reticuline was

isolated and crystallized to constant activity. The radiochemical purity of the isolated


Page | 173

base was established by dilution technique. The results of various feeding

experiments are recorded in Table 1.

To test the biosynthesis of 8-bromoreticuline (19, abnormal product) from 3,4-

dihydroxyphenethylamine (7, normal precursor), (±)-[aryl-3H]-3,4-dihydroxyphenethyl

amine (7) (experiment 2), was fed to young cut branches of L. glutinosa var. glabraria

Roxb. plant. After 5 days the biosynthetic 8-bromoreticuline (19) was isolated as

usual and crystallized to constant activity. The incorporation of 7 into 19 is recorded

in Table 1.

To check the possibility of condensation of 5-bromo-4-hydroxy-3-

methoxyphenethyl amine (49a) with 4-hydroxyphenylacetaldehyde (11) to

biosynthesize 8-bromoreticuline (19), (±)-5-bromo-[aryl-3H]-4-hydroxy-3-

methoxyphenethylamine (49a) (experiment 3), was fed to young cut branches of L.

glutinosa var. glabraria Roxb. plant. Biosynthetic 8-bromoreticuline (19) was isolated

after 5 days by dilution method and crystallized to constant activity. The incorporation

of labelled 49a into 19 is recorded in Table 1.

To test the biosynthesis of 8-bromoreticuline (19) from 8-bromococlaurine

(24) and 8-bromo-6-O-methylnorlaudanosoline (25), (±)-8-bromo-[aryl-3H]coclaurine

(24) (experiment 4) and (±)-8-bromo-[aryl-3H]-6-O-methylnorlaudanosoline (25)

(experiment 5) were separately fed to young cut branches of L. glutinosa var. glabria

Roxb. plant. After 5 days biosynthetic 8-bromoreticuline (19) was isolated in both

experiments by usual method and crystallized to constant activity. The radiochemical

purity of the isolated base was established by reverse dilution technique. The

incorporations of labelled 24 and 25 into 19 are recorded in Table 1.

To check whether 4’-O-methylation of norlaudanosoline (16a) can occur prior

to 6-O-methylation, 2’-bromo-[aryl-3H]-4’-O-methylnorlaudanosoline (35) (experiment

6) was fed to young cut branches of L. glutinosa var. glabraria Roxb. plant.

Biosynthetic 2’-bromoreticuline (20) was isolated after 5 days by usual method and

crystallized to constant activity. The incorporation of labelled 35 into 20 is recorded in

Table 1.

(±)-2’-Bromo-[aryl-3H]-norreticuline (33) (experiment 7) was then fed to young

cut branches of L. glutinosa var. glabraria Roxb. plant. After 5 days biosynthetic 2’-

bromoreticuline (20) was isolated by usual workup and crystallized to constant

activity. The incorporation of labelled 33 into 20 is given in Table 1.


Page | 174

To test the intact incorporation of 8-bromonorreticuline (26) and

regiospecificity of label in the biosynthetic 8-bromoreticuline (19), (±)-8-bromo-[3-

14C]-norreticuline (26) (experiment 8) was fed to young cut branches of L. glutinosa

var. glabraria Roxb. plant. After 5 days biosynthetic 8-bromoreticuline (19) was

isolated as usual and crystallized to constant activity. The incorporation of 26 into 19

is recorded in Table 1.

To confirm that the conversion of abnormal precursor 2-bromo-3,4-

dihydroxyphenethylamine (36) into abnormal product 5-bromoreticuline (21) follows

the same enzymatic sequence as for the conversion of normal precursor 3,4-

dihydroxyphenethylamine (dopamine, 7) into reticuline (1), (±)-2-bromo-[aryl-3H]-3,4-

dihydroxyphenethylamine (36) (experiment 9) was fed to young cut branches of L.

glutinosa var. glabraria Roxb. plant. After 5 days biosynthetic 5-bromoreticuline (21)

was isolated as usual and crystallized to constant activity. The radiochemical purity of

the isolated base was established by dilution technique. The incorporation of 36 into

21 is recorded in Table 1.


Page | 175

Table 1: Tracer experiments on L. glutinosa var. glabraria Roxb. plant

Experiment Precursor fed Incorporation (%) into

Reticuline (1) 8-Bromoreticuline (19) 2’-Bromoreticuline (20) 5-Bromoreticuline (21)

1 L-[U-14

C] Tyrosine (4) 0.04 - - -

2 (±)-[Aryl-3H]-3,4-dihydroxyphenethyl

amine (7)

-

0.0003

-

-

3 (±)-5-Bromo-[aryl-3H]-4-hydroxy-3-m-

-ethoxyphenethylamine (49a)

-

0.0002

-

-

4 (±)-8-Bromo-[aryl-3H]-coclaurine (24) - 1.02 - -

5 (±)-8-Bromo-[aryl-3H]-6-O-methylnorl-

-audanosoline (25)

-

1.50

-

-

6 (±)-2’-Bromo-[aryl-3H]-4’-O-methylno-

-rlaudanosoline (35)

- - 0.0004 -

7 (±)-2’-Bromo-[aryl-3H]-norreticuline

(33)

- - 1.1 -

8 (±)-8-Bromo-[3-14

C]-norreticuline (26) - 3.80 - -

9 (±)-2-Bromo-[aryl-3H]-3,4–dihydroxy-

-phenethylamine (36)

0.0001 - - 0.06


Page | 176

3.5.7 Degradations

Treatment of radioactive (±)-8-bromo[3-14C]-reticuline (19) derived from the

feeding of 8-bromo-[3-14C]-norreticuline (26) (experiment 8) with diazomethane gave

8-bromolaudanosine (60).

Debromination of 60 with lithium aluminium hydride gave laudanosine (61)

which was converted into laudanosine methiodide (62) with methyl iodide. The

methiodide (62) was passed through freshly generated amberlite IR-410 resin (OH-


Page | 177

form) to give the corresponding methohydroxide (62a). Hofmann degradation of 62a

furnished a mixture of t rans and cis Iaudanosine methines (63a) and (63b).

Catalytic hydrogenation of the mixture of 63a and 63b gave dihydrolaudanosine

methine (64) with practically no loss of radioactivity. A second Hofmann degradation

of 64 via the intermediacy of methohydroxide (65a) yielded 1-(4,5-dimethoxy-2-

vinylphenyl)-2-(3',4'-dimethoxyphenyl)ethane (66). Treatment of 66 with osmium

tetroxide furnished formaldehyde which was trapped as formaldehyde dimethone

derivative (67) (95% of the original activity) (Scheme 11).

3.6 Discussion

Initial feeding of (L)-[U-14C]-tyrosine (experiment 1) demonstrated that the

enzyme system responsible for the biosynthesis of reticuline (1) was active at the

time of feeding experiments. Incorporation results of (L)-[U-14C]-tyrosine (4)

(experiment 1) in parallel with (±)-[aryl-3H]-3,4-dihydroxyphenylethylamine (7)

(experiment 2), (±)-5-bromo-[aryl-3H]-4-hydroxy-3-methoxyphenylethylamine (49a)

(experiment 3), (±)-8-bromo-[aryl-3H]-coclaurine (24) (experiment 4), (±)-8-bromo-

[aryl-3H]-6-O-methylnorlaudanosoline (25) (experiment 5), (±)-2’-bromo-[aryl-3H]-4’O-

methylnorlaudanosoline (35) (experiment 6), (±)-2’-bromo-[aryl-3H]-norreticuline (33)

(experiment 7), (±)-8-bromo-[3-14C]-norreticuline (26) (experiment 8) and (±)-2-bromo

-[aryl-3H]-3,4-dihydroxyphenylethylamine (36) (experiment 9) in 8-bromoreticuline

(19), 2’-bromoreticuline (20) and 5-bromoreticuline (21) in L.glutinosa var. glabraria

(Roxb.) are recorded in Table 1.

Feeding of (±)-[aryl-3H]-3,4-dihydroxyphenylethylamine (7) and (±)-5-bromo-

[aryl-3H]-4-hydroxy-3-methoxyphenylethylamine (49a) in parallel with (L)-[U-14C]-

tyrosine revealed that both 7 and 49a are poorly metabolised by L.glutinosa var.

glabraria plant to form 8-bromoreticuline (19). The incorporation results thus

demonstrated that the enzyme system present in L. glutinosa var. glabraria Roxb.

plant is unable to synthesize a normal (natural) precursor 7 into abnormal (unnatural)

product 19 and also supported that 3,4-dihydroxyphenethylamine (7) undergoes

condensation or in other words O-methylation of dihydroxyphenylethylamine takes

place after condensation with 4-hydroxyphenylacetaldehyde (11) as established

earlier in normal biosynthesis of reticuline.

Feeding with (±)-8-bromo-[aryl-3H]-coclaurine (24) and (±)-8-bromo-

[aryl-3H]-6-O-methylnorlaudanosoline (25) (experiments 4 and 5 respectively)


Page | 178

showed that both 24 and 25 are efficient precursors of 8-bromoreticuline (19) in L.

glutinosa var. glabraria (Roxb.) plant.

(±)-2’-Bromo-[aryl-3H]-4’-O-methylnorlaudanosoline (35) was poorly

metabolised by the enzyme system present in the plant L. glutinosa var. glabraria

Roxb. plant (experiment 6) to form 2’-bromoreticuline (20).

The foregoing feeding experiments thus suggested that the biosynthesis of

abnormal product 8-bromoreticuline (19) from abnormal precursors 24 and 25 in L.

glutinosa var. glabraria (Roxb.) plant follows the same pathway as demonstrated in

normal biosynthesis i.e. 6-O-methylation takes precedence over 4’-O-methylation in

the biosynthesis of reticuline.

(±)-2’-Bromo-[aryl-3H]-norreticuline (33) was efficiently incorporated into

2’-bromoreticuline (20) (experiment 7), thus establishing the intermediacy of

bromonorreticuline in the biosynthesis of bromoreticuline.

All the foregoing experiments thus established that 8-bromonorreticuline

(26) is the penultimate precursor of 8-bromoreticuline (19) however, the precursor

used was labeled with tritium in aromatic ring only which are vulnerable to exchange.

The intact incorporation of label in 8-bromoreticuline (19) was therefore,

demonstrated by efficient incorporation of (±)-8-bromo-[3-14C]-norreticuline (26)

(experiment 8) into 8-bromoreticuline (19). The regiospecificity of label in 8-

bromoreticuline (19) was established by specific degradation of the biosynthetic 19

derived from the feeding of 8-bromo-[3-14C]-norreticuline (26) (Scheme 11). The

formaldehyde dimethone so formed was found to have 95% of original radioactivity.

Efficient incorporation of (±)-2-bromo-[aryl-3H]-3,4-dihydroxyphenylethyl

amine (36) into 5-bromoreticuline (21) indirectly supported that the condensation of

2-bromo-3,4-dihydroxyphenylethylamine and 4-hydroxyphenylacetaldehyde follows

the same sequence utilizing the same enzyme system responsible for the formation

of reticuline and are not substrate specific. Further negligible incorporaton of (±)-2-

bromo-[aryl-3H]-3,4-dihydroxyphenylethylamine (36) (experiment 9) into reticuline

demonstrated that the abnormal precursor cannot be converted into normal product

by the enzymes present in L. glutinosa var. glabraria (Roxb.) plant.

The foregoing feeding experiments strongly demonstrated that the

enzymes present in L. glutinosa var. glabraria (Roxb.) plant are: (i) Not substrate

specific, (ii) Capable of synthesizing abnormal products from abnormal precursors;

(iii) Cannot synthesize abnormal product from normal precursors; (iv) Cannot

synthesize normal product from abnormal precursors.


Page | 179

3.7 Experimental

Counting method

Packard Model 3330 Tri-carb Liquid Scintillation Spectrometer was used for

measurement of 14C and 3H activities. Samples after dissolution in methanol or

dimethylsulphoxide (0.5 mL) were counted in scintillation fluid (8.0 mL). Relative

efficiencies were obtained by counting [2-14C]- and [1,2-3H2]-hexadecane standards.

Dopamine41 (7), 2-bromo-3-benzyloxy-4-methoxyphenylacetylchloride44 (54b),

(±)-2’-bromonorreticuline41 (33), (±)-2’-bromoreticuline41 (20), (±)-5-bromoreticuline44

(21) were prepared by standard literature procedure.

Synthesis of Precursors

Synthesis of 5-bromo-4-hydroxy-3-methoxyphenethylamine (49a)

5-Bromo-4-hydroxy-3-methoxybenzaldehyde (44a)

To a stirred mixture of 4-hydroxy-3-methoxybenzaldehyde (vanillin) (43a)

(15g) and NaOAc (5g) in glacial AcOH (35 mL), a solution of bromine (6.15 mL) in

AcOH (30 mL) was added at 10°C during 1h and the stirring continued for another 1h

at room temperature. The resulting mixture was poured in ice-water (300mL)

containing sodium metabisulphite (3g). The precipitated 5-bromovanillin (44a) thus

obtained was filtered and dried. Yield 91%; mp 162-163°C (Lit.46164°C).

4-Benzyloxy-5-bromo-3-methoxy benzaldehyde (45a)

A mixture of 44a (20g), K2CO3 (20g), NaI (500mg), EtOH (300mL) and benzyl

chloride (20mL) was heated under reflux for 4h. It was filtered and the solution from

the filtrate removed. The residue was extracted with EtOAc (3x100 mL), 10%

aqueous NaOH (3x50mL), H2O (3x50mL), dried (anhyd.Na2SO4) and the solvent

removed to give 45a. Yield 87%; mp 45-46°C (Lit.47 45.5-46°C).

4-Benzyloxy-5-bromo-3-methoxy-ω-nitrostyrene (46a)

A mixture of 45a (20g), CH3COONH4 (8g), glacial AcOH (80 mL) and MeNO2

(20mL) was refluxed for 2h. The excess of solvent and reagent was removed in

vacuo and the residue treated with H2O. The precipitate thus obtained was filtered,

washed with H2O, MeOH and dried to afford the nitrostyrene 46a. Yield 63%; mp

127-28°C (Lit.48 129-131.5°C).


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4-Benzyloxy-5-bromo-3-methoxyphenyl-ω-nitroethane (47a)

To an ice cooled stirred solution of 46a (14g) in MeOH (280mL), NaBH4 (2.8g)

was added in portions and the stirring continued for 2h at room temperature. The

excess of solvent was removed in vacuo and the residue extracted with CHCl3

(3x100mL), washed with H2O (3x50mL), dried (anhyd.Na2SO4) and the solvent

removed to give the product, which was chromatographed over a column of silica-

gel. Elution (TLC monitored) with C6H6-hexane (1:1) furnished 47a. Yield 53%; mp

62°C (Lit.49 62°C).

4-Benzyloxy-5-bromo-3-methoxyphenethyl amine (48a)

To an ice cooled, stirred solution of 47a (7g) in EtOH (170 mL) and

NH2.NH2.H2O (7mL), Raney-Ni (7g) was added in portions and the mixture stirred for

1h at ambient temperature. The resulting mixture was filtered and the solvent from

the filtrate removed in vacuo. The residue, thus obtained, was extracted with CHCl3

(3x50mL), washed with H2O (2x25mL), dried (anhyd.Na2SO4) and the solvent

removed to afford an oil, which was purified to afford the amine 48a HCl. Yield 68%;

mp 155-157°C (Lit.48 155-157°C).

5-Bromo-4-hydroxy-3-methoxyphenethyl amine (49a)

A mixture of 48a (100mg), EtOH (10mL) and 12N HCl (10 mL) was heated

under reflux for 3h. The excess of solvent and reagent was removed in vacuo to

afford 5-bromo-4-hydroxy-3-methoxyphenethylamine 49a as oil. Yield 68%.

Synthesis of 8-bromococlaurine (24)

4-Benzyloxybenzyl alcohol (50c)

A solution of 4-hydroxybenzaldehyde (43f) (10.0g) was benzylated and

treated with NaBH4 (2.8g) at 0oC temperature in THF:MeOH (1:1). The resulting

mixture was concentrated in vacuo. The residue diluted with water (4×20 mL) and

extracted with Et2O (4×50 mL). The ether extract was washed with water (4×30 mL),

dried (anhyd. Na2SO4) and concentrated to furnish the alcohol 50c. Yield 70%; mp

86-87°C (Lit.50 84-86°C).

4-Benzyloxybenzyl chloride (51c)

SOCl2 (8.0 mL) was added drop wise to a solution of 50c (8.0g) in dry

benzene (15 mL). The resulting mixture was stirred for 4h and then worked up as

usual to give the chloride 51c. Yield 73%; mp 74-75°C (Lit.51 75-76°C).


Page | 181

4-Benzyloxyphenylacetonitrile (52c)

To a stirred solution of KCN (3.0g) in dry DMSO (20 mL) was added a

solution of 51c (7.5g) in dry DMSO (75 mL) and the stirring continued overnight. The

resulting mixture was worked up to furnish the nitrile 52c as oil. Yield 72%.

4-Benzyloxyphenylacetic acid (53c)

A mixture of the nitrile 52c (6.5 g), (CH2OH)2 (120 mL), KOH (3.0g) and water

(25 mL) was refluxed for 18h. The resulting mixture was worked up to yield the acid

53c. Yield 68%; mp 121-122°C (Lit.52 120-121°C).

4-Benzyloxyphenylacetylchloride (54c)

A mixture of the acid 53c (6.0g), dry benzene (100 mL) and SOCl2 (6.0 mL)

was stirred at room temperature for 2h and then refluxed for 1h. Solvent and excess

of SOCl2 were removed in vacuo to afford the acid chloride 54c as oil. Yield 74%.

N-(4-Benzyloxy-5-bromo-3-methoxyphenethyl)-4-benzyloxyphenylacetamide

(55a)

To a stirred mixture of amine 48a HCl (7g) in 4N NaOH (200mL) and C6H6

(400mL) was added a solution of acid chloride 54c (10g) in dry C6H6 (400mL). After

20h the C6H6 layer was separated, washed with H2O (3x50mL) dried (anhyd.

Na2SO4) and the solvent removed to give 55a. Yield 67%; mp 145°C (Lit.53 145-

146°C).

1-(4-Benzyloxybenzyl)-7-benzyloxy-8-bromo-6-methoxy-3,4-dihydroisoquinolin-

-e (56a)

A mixture of 55a (10g), dry C6H6 (100 mL) and freshly distilled POCl3 (20 mL)

was heated on water bath for 2h. The excess of solvent and reagent from the

resulting mixture were removed in vacuo to give 56a HCl. Yield 95%; mp 159°C (Lit53

160°C).

1-(4-Benzyloxybenzyl)-7-benzyloxy-8-bromo-6-methoxy-1,2,3,4-tetrahydro

isoquinoline (57a)

To an ice cooled and stirred solution of 56a HCl (9.2g) in MeOH (350mL) was

added NaBH4 (5g) portion wise. The stirring continued for 2h at room temperature.

The excess of solvent from the resulting mixture was removed in vacuo. Usual work

up gave crude product which was chromatographed over a column of silica gel.

Elution (TLC monitored) of the column with CHCl3: MeOH (99:1) gave 57a as an oil.

Yield 76%.


Page | 182

1-(4-hydroxybenzyl)-8-bromo-7-hydroxy-6-methoxy-1,2,3,4-tetrahydroisoquinol-

-ine [(±)-8-bromococlaurine] (24)

A mixture of 57a (4g), EtOH (150mL) and 12N HCl (150mL) was heated

under reflux for 3h. Excess of solvent and reagent from the resulting mixture was

removed in vacuo. The product thus obtained was basified with 10%. NH3 and the

free base extracted with EtOAc (3x50 mL), washed with H2O, dried (anhyd.Na2SO4).

The solvent was removed in vacuo to afford 24. Yield 91%; Base HCl mp185°C (Lit.53

195°C).

Synthesis of (±)-8-bromo-6-O-methylnorlaudanosoline (25)

3,4-Dibenzyloxybenzyl alcohol (50d)

A solution of 3,4-dihydroxybenzaldehyde (43g) (30 g) was benzylated and

treated with NaBH4 (10.8 g) at 0oC in THF: MeOH (1:1). The resulting mixture was

concentrated in vacuo and worked up as usual to afford the alcohol 50d. Yield 65%;

mp 70oC (Lit.54 70oC).

3,4-Dibenzyloxybenzyl chloride (51d)

SOCl2 (24 mL) was added drop wise to an ice cooled solution of 50d (24 g) in

dry benzene (120 mL) during 3h. The resulting mixture was stirred at room

temperature for 1h and worked up to yield the chloride 51d. Yield 68%; mp 39-40oC

(Lit.54 40oC).

3,4-Dibenzyloxyphenylacetonitrile (52d)

To a stirred solution of KCN (17 g) in dry DMSO (160 mL) was added drop

wise a solution of 51d (20 g) in dry DMSO (100 mL) and stirring continued overnight.

The resulting mixture was worked up as usual to afford the nitrile 52d. Yield 72%;

mp 70oC (Lit.54 70oC).

3,4-Dibenzyloxyphenylacetic acid (53d)

A mixture of 52d (12 g), (CH2OH)2 (260 mL), KOH (9.0 g) and H2O (80 mL)

was refluxed for 18h. It was then worked up to afford the acid 53d. Yield 74%; mp

106oC (Lit.54 106oC).

3,4-Dibenzyloxyphenylacetylchloride (54d)

The acid 53d (10.0 g) in dry benzene (100 mL) was treated with SOCl2 (12

mL) at room temperature for 2h and then refluxed for 1h. The solvent and excess of

SOCl2 were removed in vacuo to yield the acid chloride 54d as oil. Yield 60%.


Page | 183

N-(4-Benzyloxy-5-bromo-3-methoxyphenethyl)-3-benzyloxy-4-methoxyphenyl

acetamide (55b)

To a sitrred mixture of 48a HCl (7g), 4N NaOH (200mL) and C6H6 (100mL)

was added a solution of the acid chloride 54d (11.0g) in dry C6H6 (400 mL). The

mixture was stirred for 3h and worked up as usual to afford the amide 55b (10.5g)

Yield 68% mp 110°C; IR(KBr cm-1): 3320 (NH), 1630 (C=O); 1HMR(CDCl3): δ 2.52 (t,

J=6.5 Hz, 2H, CH2CH2NH), 3.25 (m, 4H, CH2NHCO, CH2CONH), 3.65 (s, 3H, OCH3),

4.81, 4.9 and 4.95 (s each, 6H, 3xOCH2Ph), 6.5-6.9 (m, 4H, Ar-H), 7.1-7.5 (m, 15H,

Ar-H); MS: m/z 665 and 667 (M+ 79Br and 81Br), 349 (M+ 317).

1-(3,4-Dibenzyloxybenzyl)-7-benzyloxy-8-bromo-6-methoxy-3,4-dihydroisoquin-

-oline (56b)

A mixture of 55b (10g), dry C6H6 (100mL) and freshly distilled POCl3 (20mL)

was refluxed for 2h. The excess of solvent and reagent from the reacting mixture

were removed in vacuo to give the dihydroisoquinoline 56b HCl as an oil. Yield 90%;

IR (KBr cm-1):1630 (C=N ); MS (ESI+): m/z 647 and 649 (M+ 79Br and 81Br).

1-(3,4-Dibenzyloxybenzyl)-7-benzyloxy-8-bromo-6-methoxy-1,2,3,4-tetrahydro-

-isoquinoline (57b)

To a stirred solution of 56b HCl (5g) in MeOH (250 mL) was added NaBH4

(2.5g) portion wise. The resulting mixture was worked up as before to afford the

tetrahydroisoquinoline 57b as an oil. Yield 90%.

1-(3,4-Dihydroxybenzyl)-8-bromo-7-hydroxy-6-methoxy-1,2,3,4-tetrahydro

isoquinoline (25)

A mixture of 57b HCl (4g), 12N HCl (125 mL) and EtOH (125 mL) was heated

under reflux for 3h. The excess of solvent and reagent from the resulting mixture

were removed in vacuo to afford 25 HCl. Yield 74%; mp 250° (dec); 1H NMR (CDCl3+

DMSO-d6): δ 3.75 (s, 3H, OCH3), 6.9-7.56 (m, 4H, Ar-H); MS(ESI+): m/z 379 and

381 (M+ 79Br and 81Br)

Synthesis of (+)-2'-bromo-4'-O-methylnorlaudanosoline (35)

N-(3,4-Dibenzyloxyphenethyl)-3-benyloxy-2-bromo-4-methoxy-phenylacetamide

(55d)

To a stirred mixture of 7 HCl (7g), 4N NaOH (150mL) and C6H6 (70mL) was

added a solution of 54b (10g) in dry C6H6 (250mL). The mixture was stirred for 3h

and worked up as described earlier to afford the amide 55d. Yield 59%; mp 110°C;


Page | 184

IR(KBr cm-1): 3320 (NH), 1080 (C=O); 1HNMR(CDCl3): δ 2.52 (t, J=6.5Hz, 2H,

CH2CH2NH), 3.25 (t, J=6.5Hz, 2H, CH2NHCO), 3.50 (s, 2H, CH2CONH), 3.72 (s, 3H,

OCH3), 4.80, 4.95 (s, 6H, 3xOCH2Ph) 6.5-6.71 (m, 4H, Ar-H), 7.1-7.5 (m, 15H, Ar-H).

1-(3-Benzyloxy-2-bromo-4-methoxybenyl)-6,7-dibenzoloxy-3,4-dihydroisoquino-

-line (56d)

A mixture of 55d (8g), dry C6H6 (70 mL) and POCl3 (15 mL) was heated for

2h. The excess of solvent and reagent from the resulting mixture were removed in

vacuo to afford the dihydroisoquinoline 56d HCl. Yield 84%; mp 212°C; IR(KBr):

1630 ( C=N ); MS (ESI+): m/z 649 and 647 (M+ 81Br and 79Br).

1-(3-Benzyloxy-2-bromo-4-methoxybenzyl)-6,7-dibenzyloxy-1,2,3,4-tetrahydro

isoquinoline (57d)

To an ice cooled and stirred solution of 56d HCl (5.0 g) in MeOH (200 mL)

was added NaBH4 (3.0g) portionwise. The resulting mixture was worked up as

described earlier, the product thus obtained was purified as 57d HCl. Yield 70%; mp

190°C; IR(KBr cm-1): 3450 (NH); 1H NMR(CDCl3): δ 3.80 (s, 3H, OCH3), 4.7 and 4.95

(s, 6H, 3xOCH2Ph), 6.58 (s, 3H, Ar-H), 7.0-7.5 (m, 15H, Ar-H).

1-(2-Bromo-3-hydroxy-4-methoxybenzyl)-6,7-dihydroxy-1,2,3,4-tetrahydroisoqu-

-inoline (35)

A mixture of 57d HCl (3.0g), EtOH (125mL) and12N HCl (125 mL) was

refluxed for 3h and worked up as described earlier to afford 35 HCl. Yield 85%; mp

225°C; IR(KBr cm-1) : 3500 (OH); 1H NMR(CDCl3+ DMSO-d6): δ 3.81(s, 3H, OCH3),

6.5-7.3 (m, 4H, Ar-H); MS (ESI+): m/z 379 and 381 (M+ 79Br and 81Br).

Synthesis of 2-bromo-3,4-dihydroxyphenethylamine (36)

2-Bromo-3,4-dihydroxybenzaldehyde (44d)

2-bromoisovanillin (43e) (1.5g) in acetic acid (50ml) and 48% hydrobromic

acid (50mL) was heated under reflux for 30 min. The solution was concentrated in

vacuo EtOAc (100ml) was added to the residue and it was washed with water

(2x15mL), dried (anhyd .Na2SO4). The solvent was concentrated in vacuo to afford 2-

bromo-3,4-dihydroxybenzaldehyde (44d). Yield 58%; mp 175°C (Lit.49 179-181°C).

2-Bromo-3,4-dibenzyloxy benzaldehyde (45c)

A mixture of 44d (0.5g), K2CO3 (0.43g), NaI (11mg), benzyl chloride

(0.53mL) and ethanol (30 mL) was heated under reflux for 4h. It was filtered and the

solvent from the filtrate removed. The residue was extracted with EtOAc (3x25 mL),


Page | 185

washed with 10% aqueous NaOH (3x15 mL), H2O (3x15 mL), dried (anhyd. Na2SO4)

and the solvent removed to give 2-bromo-3,4-dibenzyloxy benzaldehyde (45c) as

viscous solid. Yield 50%; mp 93oC (Lit.49 90-91oC).

2-Bromo-3,4-dibenzyloxynitrostyrene (46c)

To the compound 45c (400 mg) was added nitromethane (0.5 mL), glacial

acetic acid (1.5mL) and CH3COONH4 (200 mg). The reaction mixture was refluxed

for 2h. Excess of solvent and reagent were removed in vacuo and the residue treated

with H2O. The precipitate thus obtained was filtered, washed with H2O (50 mL),

MeOH (50 mL) and dried to afford the nitrostyrene 46c as an oil. It was used without

purification. Yield 60%.

2-Bromo-3,4-dibenzyloxyphenyl)-ω-nitroethane (47c)

To an ice cooled and stirred solution of 46c (250mg) in MeOH (25 mL),

NaBH4 (50 mg) was added in portions and the stirring continued for 2h at room

temperature. Usual work up afforded the nitroethane 47c. It was used without further

purification. Yield 56%; mp 94oC (Lit.50 95oC)

2-Bromo-3,4-dibenzyloxyphenethylamine (48c)

To an ice cooled, stirred solution of 47c (200 mg) in EtOH (25 mL) and

NH2.NH2.H2O(2 mL), Ra-Ni (1g) was added in portions and the reaction mixture

stirred for 1h at ambient temperature. The resulting mixture was filtered and the

solvent removed to afford an oil, which was purified as the amine 48c HCl. Yield

57%; MS (ESI+): m/z 413 and 411 (M+ 79Br, 81Br) 320 (M+CH2C6H5)

2-Bromo-3,4-dihydroxy phenethylamine (36)

To 2-bromo-3,4-dibenzyloxyphenethylamine (48c) (50mg) was added

absolute ethanol (15mL) and 12N HCl. The reaction mixture was refluxed for 2h and

the solvent concentrated in vacuo to afford 2-bromo-3,4-dihydroxyphenethylamine

(36) as an oil.Yield 90%; MS (ESI+): m/z 233 and 234 (M+ 81Br and 79Br).

Synthesis of (±) 8-bromoreticuline (19)

3-Benzyloxy-4-methoxybenzylalcohol (50a)

A stirred solution of 3-hydroxy-4-methoxybenzaldehyde (43e) (12.0g) was

benzylated and treated with NaBH4 (4.8g) portionwise at 0-4°C in MeOH (150mL)

and C6H6 (15mL). The resulting mixture was further stirred at room temperature for

2h. Excess of solvent removed under reduced pressure, diluted the residue obtained

with H2O (20mL) and extracted with CHCl3 (6x30mL). The combined CHCl3 layer


Page | 186

washed with H2O (5x15mL), dried (anhyd.Na2SO4) and solvent removed to afford the

alcohol 50a. Yield 75%; mp 71-72°C (Lit49. 70-71°C).

3-Benzyloxy-4-methoxybenzylchloride (51a)

To a stirred solution of alcohol 50a (10.0g) in dry C6H6 (100 mL), was added

dry SOCl2 (10mL) dropwise at 0-5°C. The resulting reaction mixture was further

stirred at room temperature for 3h. Excess of solvent and reagent was concenterated

in vacuo to afford the chloride 51a as oil, which was used as such in subsequent

reaction without any purification. Yield 70%.

3-Benzyloxy-4-methoxyphenylacetonitrile (52a)

To a stirred suspension of KCN (5.0g) in dry DMSO (50mL) was added 51a

(9.0g) in dry DMSO (100mL). The resulting mixture was further stirred for 3h and left

overnight. Diluted the mixture with H2O and extracted with EtOAc (6x35mL). The

combined EtOAc layer was washed with H2O (5x30mL), dried (anhyd. Na2SO4) and

solvent removed to afford the nitrile 52a Yield 69%; mp 80-81°C (Lit.55, 79.5-80.5°C).

3-Benzyloxy-4-methoxyphenylacetic acid (53a)

A mixture of nitrile 52a (8.0g), (CH2OH)2 (150 mL), KOH (4.0g) and H2O (40

mL) was refluxed for 18h. The resulting mixture was cooled, extracted with Et2O (5x

30mL). The aqueous layer left was acidified with 10% HCl and again extracted with

CHCl3 (6x30mL). Both the organic layers washed with H2O (4x20 mL), dried (anhyd.

Na2SO4) and concentrated under reduced pressure to afford the acid 53a. Yield 72%;

mp 122-124°C (Lit.49,55 125-126°C).

3-Benzyloxy-4-methoxyphenylacetylchloride (54a)

To a stirred solution of acid 53a (6.0g) in dry C6H6 (l00mL) was added dry

SOCl2 (6mL) at 0-5°C.The resulting mixture was further stirred at room temperature

for 3h. Concentrated in vacuo to afford the acid chloride 54a as oil. Yield 66%.

N-(4-Benzyloxy-5-bromo-3-methoxyphenethyl)-3-benzyloxy-4-methoxyphenyl

acetamide (55c)

To a stirred mixture of 48a HCl (7g), 4N NaOH (200mL) and C6H6 (100mL)

was added a solution of acid chloride (54a) (10.5g) in dry C6H6 (400mL) for 3h and

the resulting mixture worked up as usual to afford the amide 55c. Yield 68%; mp

110°C (Lit49 110°C).


Page | 187

1-(3-Benzyloxy-4-methoxybenzyl)-7-benzyloxy-8-bromo-6-methoxy-3,4-dihydro

isoquinoline (56c)

A mixture of 55c (10g), dry C6H6 (100 mL) and freshly distilled POCl3 (20mL)

was heated on water bath for 2h. The excess of solvent and reagent was removed in

vacuo to give dihydroisoquinoline 56c HCl as an oil which was used without

purification. Yield 73%.

1-(3-Benzyloxy-4-methoxybenzyl)-7-benzyloxy-8-bromo-6-methoxy-1,2,3,4-tetra

hydroisoquinoline (57c)

To an ice cooled and stirred solution of 56c HCl (5.0 g) in MeOH (200 mL)

was added NaBH4 (3.0g) portion wise. The resulting mixture was worked up as

described earlier, the product thus obtained was purified as 57c HCl. Yield 70% mp

190°C (Lit.55 192oC).

1-(3-Benzyloxy-4-methoxybenzyl)-8-bromo-7-benzyloxy-6-methoxy-1,2,3,4-

tetetrahydro-2-methylisoquinoline (59a)

A mixture of 1-(3-(benzyloxy)- 4-methoxybenzyl)-7-(benzyloxy)-8-bromo-6-

methoxy-1,2,3,4-tetrahydroisoquinoline (57c) (10g), HCO2H (98%, 150mL) and

HCHO (37-41%, 150 mL) was refluxed for 2h excess of HCO2H and HCHO were

removed in vacuo, the residue was diluted with H2O (50 mL) and basified with

Na2CO3. The liberated base was extracted with CHCl3 (5×40mL), washed with H2O

(4×15mL), dried (anhyd. Na2SO4) and solvent removed to furnish the isoquinoline

59a as oil.

1-(3-Hydroxy-4-methoxybenzyl)-8-bromo-7-hydroxy-6-methoxy-1,2,3,4-tetrahyd-

-ro-2methylisoquinoline (19)

A mixture of 59a (2g), EtOH (160mL) and 12N HCl (60mL) was heated under

reflux 3h and worked up to give (19). Yield 72%; mp133°C (Lit49.133-134°).

LABELLING OF PRECURSORS

(±)-[Aryl-3H]-3,4-dihydroxyphenethylamine (7)

A mixture of 7 (100mg), tritiated H2O (0.2 mL; activity, 200 mCi) and SOCl2

(0.1mL) gas heated in a sealed tube (nitrogen atmosphere) on a steam bath for 96 h.

The resulting mixture was cooled, diluted with H2O (5mL), basified with Na2CO3 and

the liberated base extracted with CHCl3-MeOH (3:1) (3x15mL), washed with H2O

(3x10mL), dried (anhy Na2SO4) and solvent removed to furnish (±)-[aryl-3H]-3,4-

dihydroxyphenethylamine (7) (48mg)


Page | 188

Specific activity = 0.104 mCi/mg

(±)-5-Bromo-[aryl-3H]-4-hydroxy-3-methoxyphenethylamine (49a)

T2O (0.2mL, activity 200 mCi) and SOCl2 (0.1mL) were added to 49a (100

mg) and the reaction mixture was similarly heated as described above. Usual work

up gave (±)-5-bromo-[aryl-3H]-4-hydroxy-3-methoxyphenethylamine (49a) (58mg).


(±)-8-Bromo-[aryl-3H]coclaurine (24)

A mixture of 24 (100 mg), T2O (0.2mL, activity 200mCi) and SOCl2 (0.1mL)

was heated in a sealed tube for 96 h. Usual work up afforded (±)-8-bromo-[aryl-

3H]coclaurine (24) (55mg).


(±)-8-Bromo-[aryl-3H]-6-O-methylnorlaudanosoline (25)

A mixture of 25 (100mg), T2O (0.2mL, activity 200mCi) and SOCl2 (0.1mL)

was heated on a steam bath for 96h. Usual work up afforded (±)-8-bromo-[aryl-3H]-6-

O-methylnorlaudanosoline (25) (45mg).


8-Bromo-[3-14C]norreticuline (26) was prepared by total synthesis using K14CN

reported in the literature procedure.55


(±)-2’-Bromo-[aryl-3H]-4’-O-methylnorlaudanosoline (35) was prepared as



(±)-2’-Bromo-[aryl-3H]-norreticuline (33) was prepared as reported in the literature

procedure.56


(±)-2-Bromo-[aryl-3H]-3,4-dihydroxyphenethylamine (36) was prepared as




Page | 189

Feeding Experiments

Feeding of (L)-(U-14C]-tyrosine (experiment 1)

Freshly cut young branches (7 nos.) of Litsea glutinosa var. glabraria Roxb.

plant were dipped into an aqueous solution (1mL) of (L)-(U-14C]-tyrosine (activity=

0.1mCi) and allowed to take up precursors. When uptake was complete, the twigs

were dipped into H2O, kept alive for 5 days and harvested. The plant material (130 g

wet wt.) was macerated in ethanol (350mL) with inactive 8-bromoreticuline (60 mg)

and left overnight. The ethanolic solution was decanted and the residue percolated

with fresh ethanol (5x 200mL).

The combined ethanolic percolate was concentrated under reduced pressure.

The green viscous mass, so obtained, was extracted with 10%HCl and the acidic

solution defatted with petroleum ether (5x20mL), basified with Na2CO3 (pH 8). The

precipitated bases were extracted with CHCl3 (5x40mL). The combined CHCl3 layer

was washed with H2O, dried (anhyd. Na2SO4) and solvent removed. The residue was

purified by TLC over SiO2 gel (solvent: CHCl3:MeOH, 98:2). 8-bromoreticuline was

eluted with CHCl3-MeOH (80:20), fractions mixed (tlc control), evaporated and

crystallized from MeOH to constant activity.

Specific activity 7.4 x 102dis/min/mg

Specific activity 3.01 x 105dis/min/mmol

Incorporation (%) 0.04

Feeding of (±)-[aryl-3H]-3,4-dihydroxyphenethylamine (experiment 2)

(±)-[Aryl-3H]-3,4-dihydroxyphenethylamine (activity = 0.104 mCi) in H2O (1mL)

was fed to young cut branches (6 nos.) of L. glutinosa var. glabraria Roxb. plant and

kept alive for 5 days. Harvested plant material (120g wet wt.) was macerated in

ethanol (250mL) with inactive 8-bromoreticuline (55.5 mg) and left overnight.

Biosynthetic 8-bromoreticuline were isolated as in the previous experiment and

crystallised from MeOH to constant activity.

Specific activity 1.248 x 103 dis/min/mg

Specific activity 5.08 x 105 dis/min/mmol


Molar Activity (original) dis/min/mmol

Feeding of (±)-5-bromo-[aryl-3H]-4-hydroxy-3-methoxyphenethylamine (experi-

-ment 3)


Page | 190

(±)-5-Bromo-[aryl-3H]-4-hydroxy-3-methoxyphenethylamine (activity =0.083

mCi) in H2O (1mL) was fed to young cut branches (6 nos.) of L. glutinosa var.

glabraria Roxb. plant and kept alive for 5 days. Harvested plant material (120 g wet

wt.) was macerated in ethanol (250mL) with inactive 8-bromoreticuline (59.2 mg) and

left overnight. Biosynthetic 8-bromoreticuline were isolated as in the previous

experiment and crystallised from MeOH to constant activity.

Specific activity 6.22 dis/min/mg



Biosynthetic 8-bromoreticuline (3.2 mg) was diluted with inactive 8-

bromoreticuline ( ) and crystallised to constant activity.


Molar Activity 4.26 x 103 dis/min/mmol


(original)

Feeding of (±)-8-bromo-[aryl-3H]coclaurine (experiment 4)

Young cut branches (7 nos.) of L. glutinosa var. glabraria Roxb. plant were

dipped into a solution of (±)-8-Bromo-[aryl-3H]coclaurine (activity =0.0346 mCi) in

H2O (1mL) containing tartaric acid (20mg) the twigs were kept alive for 5 days, then

harvested (125 g wet wt.). The plant material was macerated in ethanol (280mL) with

inactive 8-bromoreticuline (71.2 mg). Reisolated the biosynthetic 8-bromoreticuline

as described earlier and crystallised from MeOH to constant activity.




Labelled 8-bromoreticuline (2.8 mg) was diluted with inactive 8-

bromoreticuline (14 mg) respectively and crystallised.




(original)


Page | 191

Feeding of (±)-8-bromo-[aryl-3H]-6-O-methylnorlaudanosoline (experiment 5)

A solution of (±)-8-bromo-[aryl-3H]-6-O-methylnorlaudanosoline (0.02 mCi) in

H2O (1 mL containing 0.2 mL DMSO) was fed to young cut branches (6 nos.) of L.

glutinosa var. glabraria Roxb. plant the twigs were kept alive for 5 days. The

harvested plant material (120 g wet wt.) was macerated with inactive 8-

bromoreticuline (75.2 mg) in ethanol (300mL) and left overnight. 8-bromoreticuline

was isolated in the usual manner and crystallised from MeOH to constant activity.




The above biosynthetic 8-bromoreticuline (2.62 mg) was diluted with inactive

8-bromoreticuline (18.2 mg) respectively and crystallised.




(original)

Feeding of (±)-2'-bromo-[aryl-3H]-4'-O-methylnorlaudanosoline (experiment 6)

A solution of (±)-2'-bromo-[aryl-3H]-4'-O-methylnorlaudanosoline (activity

=0.0384 mCi) in H2O (1 mL containing 0.2 mL DMSO) was fed to young cut branches

(6 nos.) of L. glutinosa var. glabraria Roxb. plant the twigs were kept alive for 5 days.

The harvested plant material (120 g wet wt.) was macerated with inactive 8-






The above biosynthetic 8-bromoreticuline (2.6 mg) was diluted with inactive 8-

bromo reticuline (14.2 mg) respectively and crystallised.




(original)


Page | 192

Feeding of (±)-2’-Bromo-[aryl-3H]-norreticuline (experiment 7)

A solution of (±)-2’-bromo-[aryl-3H]-norreticuline (activity =0.078 mCi) in H2O

(1 mL containing 0.2 mL DMSO) was fed to young cut branches (6 nos.) of L.

glutinosa var. glabraria Roxb. plant. The twigs were kept alive for 5 days. The

harvested plant material (120 g wet wt.) was macerated with inactive 2’-

bromoreticuline (69.2 mg) in ethanol (300mL) and left overnight. 2’-bromoreticuline



Molar activity 1.12 x 108 dis/min/mmol


The above biosynthetic 2’-bromoreticuline (1.92 mg) were diluted with inactive

2’-bromoreticuline (18.2 mg) respectively and crystallised.




(original)

Feeding of (±)-8-Bromo-[3-14C]-norreticuline (experiment 8)

A solution of (±)-8-bromo-[3-14C]-norreticuline hydrochloride (activity =0.3818

mCi) in H2O (1 mL containing 0.2 mL DMSO) was fed to young twigs (7 nos.) of L.

glutinosa var. glabraria Roxb. plant. The plant material was harvested, macerated in

ethanol (280mL) with inactive 8-bromoreticuline (64.6 mg). After 18h it was worked

up in the usual way to furnish radioactive 8-bromoreticuline, crystallised from MeOH

to constant





bromoreticuline (20.6 mg) respectively and crystallised.




(original)

Feeding of (±)-2-Bromo-[aryl-3H]-3,4-dihydroxyphenethylamine (experiment 9)


Page | 193

A solution of (±)-2-bromo-[aryl-3H]-3,4-dihydroxyphenethylamine (activity

=0.032 mCi) in H2O (1 mL containing 0.2 mL DMSO) was fed to young cut branches

(6 nos.) of L. glutinosa var. glabraria Roxb. plant the twigs were kept alive for 5 days.

The harvested plant material (120 g wet wt.) was macerated with inactive 5-







bromoreticuline (15.2 mg) respectively and crystallised.




(original)

Degradation Experiment

Degradation of the radioactive-8-bromo-[3-14C]-reticuline (19) derived from

incubation of 8-bromo-[3-14C]norreticuline (26)

To a solution of labelled 8-bromo-[3-14C]-reticuline (19) (100mg) (molar

activity =1.88 x 108 dis/min/mmol) derived from 8-Bromo-[3-14C]-norreticuline (26)

was added an excess of ethereal diazomethane. After 20h the resulting mixture was

worked up to give the crude product 60 (100 mg). Compound 60 (100 mg) in THF

(25mL) was added to LAH (150 mg) in THF (100mL) and refluxed at 55oC for 5h. The

excess of LAH was decomposed with EtOAc (120mL) and 20% NaOH solution

(15mL). The resulting mixture was filtered, the solvent removed. The residue was

extracted with CHCl3-MeOH (9:1) (3x15mL), dried (anhyd. Na2SO4) and solvent

removed. The crude product, thus obtained was chromatographed on a column of

neutral Al2O3 (5.0 g). Elution of the column with C6H6: Hexane (3:1) afforded

radioactive laudanosine 61 (0.82 mg) .Crystallized from CH2Cl2-pet.ether, m p.114-

15oC (Lit55. 114-15°C).


Molar activity 1.78 x108 dis/min/mmol

Radioactive laudanosine (65 mg) was diluted with inactive laudanosine (125

mg) and crystallized to constant activity


Page | 194


Molar activity 5.71 x 107dis/min/mmol


(original)

The preceding (±)-[3-14C] laudanosine (61) (150 mg) in MeOH (4 mL) was

refluxed with MeI (2 mL) for 2h to give radioactive laudanosine methiodide 62 (175

mg) m.p.210-212°C (Lit.57212-213°C).

Specific activity 3.45 x 105dis/min/mg

Molar activity 1.72 x108 dis/min/mmol

A solution of the radioactive (±)-laudanosine methiodide (62) (160 mg) in

MeOH was passed through a column of freshly generated amberlite IR-410 anion-

exchange resin (OH- form) (8.0g) and the process was recycled five times. The

column was finally washed with MeOH (100mL). The solvent from the methanolic

solution was removed in vacuo to afford radioactive methohydroxide 62a. The

methohydroxide in MeOH (10 mL) and KOH (3.5 g) was refluxed for 2h and worked

up to give a mixture of trans and cis laudanosine methines 63a and 63b (100 mg).

A mixture of the radioactive 63a and 63b (98 mg) was hydrogenated in the

presence of PtO2 (30 mg) to give radioactive methine (78 mg) (64).

The radioactive methine (64) (75 mg) in MeOH (2.5 mL) was refluxed with

MeI (1.5 mL) for 3h to give the radioactive methine methiodide (65) (90 mg) mp 196-

197°C (Lit.24 197°C).

Specific activity 4.4 x 105dis/min/mg.


The foregoing radioactive dihydrolaudanosine methine methiodide (65) (85

mg) in MeOH (25 mL) was treated with amberlite IR-410 anion exchange resin (OH-

form) (6.0 g) to give the corresponding methohydroxide which was refluxed with KOH

(3.0 g) for 2h. The resulting mixture was worked up to afford 1-(4,5-dimethoxy-2

vinylphenyl)-2-(3,4-dimethoxyphenyl)ethane (66) (40 mg) which was crystallized from

EtOH as plates, mp 73-74°C (Lit.24 74°C).

OsO4 was added to radioactive 1-(4,5-dimethoxy-2-vinylphenyl)-2-(3,4-

dimethoxyphenyl)ethane (66) (38 mg) in t-BuOH (4mL) and H2O (3.5mL). To the

orange complex so obtained, NaIO4 (45 mg) was added. After 5h the resulting

mixture was treated with saturated solution of As2O3 (20mL) and extracted with Et2O.

The aqueous solution was adjusted to pH 10 with K2CO3, dimedone (120 mg) added


Page | 195

to the reaction mixture and pH adjusted to 6. The resulting mixture was worked up

after 12h in the usual way to give formaldehyde dimedone (67), crystallized from

MeOH-ether to constant activity. mp 188oC (Lit.24 188°C).



The formaldehyde dimethone has 95% of the original molar radioactivity


Page | 196

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