synthesis and biological evaluation of thiazole...

Synthesis and Biological Evaluation of Thiazole,

Oxadiazole and Indole Derivatives

Hayat Ullah

DEPARTMENT OF CHEMISTRY

HAZARA UNIVERSITY

MANSEHRA

2018

Synthesis and Biological Evaluation of Thiazole,

Oxadiazole and Indole Derivatives

By

Hayat Ullah

A dissertation submitted in Partial fulfillment of the

requirement for the degree of

Doctor of Philosophy

in

Chemistry

DEPARTMENT OF CHEMISTRY

HAZARA UNIVERSITY

MANSEHRA

2018

In the name of ALLAH ALMIGHTTY

Most mmerciful, Most Beneficent, Most

Courteous, Most sympathetic ,

Whose Help and Guidance

I always solicit at every step, at every

moment and he unto whom wisdom is

given.

He truly hath relieved abundant good.

ALQURAN

(2:269)

i

Synthesis and Biological Evaluation of Thiazole, Oxadiazole and Indole Derivatives

CERTIFICATE

Certified that the work contained in this dissertation is carried out by Mr. Hayat Ullah

under our supervision from the Department of Chemistry, Hazara University

Mansehra, Pakistan and approved as to style and content.

Dr. Fazal Rahim Dr. Mohsan Nawaz Supervisor Co-Supervisor Department of Chemistry Department of Chemistry Hazara University Hazara University Mansehra, KP, Pakistan Mansehra, KP, Pakistan

Dr. Mohsan Nawaz Chairman, Department of Chemistry Hazara University, Mansehra, KP, Pakistan

ii

Author’s Declaration I Mr. Hayat Ullah hereby state that my PhD thesis titled “Synthesis and Biological

Evaluation of Thiazole, Oxadiazole and Indole Derivatives” is my own work and has

not been submitted previously by me for taking any degree from this University

“Hazara University Mansehra” or anywhere else in the country/world.

At any time if my statement is found to be incorrect even after my Graduate the

university has the right to withdraw my PhD degree.

Signature: ___________________ Author‟s Name: Hayat Ullah Date----------------------------

iii

Plagiarism Undertaking

I solemnly declare that research work presented in the thesis titled “Synthesis and

Biological Evaluation of Thiazole, Oxadiazole and Indole Derivatives” is solely my

research work with no significant contribution from any other person. Small

contribution/help wherever taken has been duly acknowledged and that complete

thesis has been written by me.

I understand the zero tolerance policy of the HEC and university “Hazara University

Mansehra” towards plagiarism.

Therefore I as Author of the above titled thesis declare that no portion of my thesis has

been plagiarized and any material used as reference is properly referred/cited.

I undertake that if I am found guilty of any formal plagiarism in the above titled thesis

even after award of PhD degree, the University reserves the rights to withdraw/revoke

my PhD degree and that HEC and the University has right to publish my name on the

HEC/University Website on which names of students are placed who submitted

plagiarized thesis.

Author Signature:___________________ Name: Hayat Ullah

iv

Certificate of Approval

This is to certify that the research work presented in this thesis, entitled “Synthesis and

Biological Evaluation of Thiazole, Oxadiazole and Indole Derivatives” was

conducted by Mr. Hayat Ullah under the supervision of Dr. Fazal Rahim.

No part of this thesis has been submitted anywhere else for any other degree. This

thesis is submitted to the Hazara University, Mansehra in partial fulfillment of the

requirements for the degree of Doctor of Philosophy in field of Chemistry, Department

of Chemistry University of Hazara University, Mansehra.

Student Name: Hayat Ullah Signature: -------------------

Examination Committee:

a) External Examiner 1: Signature: --------------------

Prof. Dr. Muhammad Ashraf

Department of chemistry

Baghdad Campus, Islamia

University Bahawalpur

External Examiner 2: Signature: ----------------------

Dr. Tariq Mehmood

Associate Professor

Department of Chemistry

CIIT, Abbottabad

b) Internal Examiner 1: Name

Dr. Fazal Rahim Signature: ----------------------

Assistant Professor

Department of Chemistry

Hazara University,

Mansehra

Supervisor Name: Dr. Fazal Rahim Signature: -----------------------

Name of Chairman: Dr. Mohsan Nawaz Signature: -----------------------

v

Dedicated To

My parents, Uncles, brothers and sisters in law and

also to my beloved wife who’s Encouragement,

Guidance, Support and continues prayers made me

strong enough to face all the challenges of my life

in future.

vi

ACKNOWLEDGEMENTS

First and foremost, I am thankful to Almighty Allah, the most Beneficent and the most

Merciful, who enabled me to complete my Ph.D. research work. All respects for his last

Holy Prophet (peace be upon him), who‟s teaching inspired me to widen my thoughts

and deliberate upon things deeply.

I wish to express my gratitude to a number of people who were involved in this work,

in one or another way. In this regard, first of all I am thankful to Dr. Muhammad Taha

for helping me in this thesis without his support and suggestion this document would

never be completed.

I have great pleasure in expressing my deep sense of gratitude to my supervisor, Dr.

Fazal Rahim for his valuable suggestions, expert guidance and active co-operation to

produce this dissertation. Without him this work would not be materialized in its

present form. I greatly appreciate his ever-ready helping attitude, which was a constant

motivating factor for me to complete this study.

I am also whole-heartedly thankful to my co-supervisor Dr. Mohsan Nawaz, Chairman,

Department of Chemistry, Hazara University Mansehra, who guided me in my research

work. I am especially thankful to him for his constant care and encouragement.

Thanks also go to all the faculty members of the Department that I had the privilege to

learn from as a Ph.D. student, especially Dr. Hameed Ullah Wazir, Dr. Obaid Ur

Rehman Abid, Dr. Ali Bahadar and Dr. Wajid Rehman.

I am also thankful to Dr. Zain-Ul-Wahab, Chairman, Department of conservation

Studies, Hazara University Mansehra, for providing us a laboratory, where I carried out

all of my research work.

I am also very grateful to Dr. Syed Adnan Ali shah and Dr. Syahrul Imran, Atta-ur-

Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA (UiTM),

Puncak Alam Campus, Bandar Puncak Alam, Selangor, Malaysia, for his kind co-

operation in solving biological analysis and characterizations of my compounds.

vii

I would like to express my sincere appreciation and thanks to my colleagues, Mr. Fahad

Khan, Mr. Imad Uddin, Mr. Muhammad Tariq Javid, Mr. Khalid Zaman and all my

junior colleagues.

I am also thankful to all technical and non-technical staff members of the department

for their help and kindness, especially Mr. Hafeez Ur Rehman and Mr. Chanzeb for

good co-operation during my research work.

I express my feelings of love and affection to my sympathetic and loving sons

Ayaan Wazir and Arsalan Wazir.

I am thankful to my friends specially Faiza Younis, Fazal Surhab Gul, Mehran, Hammid

Ali, Fayaz Ahmad, Muhammad Sulaiman, Kamran Farooqi, Inam Khan, Syed Noor Ul

Islam and Misbah.

I am also thankful to my cousins specially Fawad Ahmed, Hakim Ullah, Dr. Rafi Ullah,

Dr. Asmat Ullah, Zakir Ullah, Zabeeh Ullah, Waseem Ahmed and Zahid Ullah.

I also appreciate my dearest Uncle Mr. Zafar Ali Wazir who supported me at every

stage. Their prayers and invaluable patronage are the most important assets of my life.

Words are confined and inefficacious to express my huge indebtedness for my gracious

parents and my beloved brothers (Shahid Ullah Wazir, Sanat Ullah Wazir and Asad

Ullah Wazir) for their good wishes, love, prayers, moral support and very useful co-

operation.

May ALLAH bless all of above mentioned individuals with His kindness, and make their life

successful in this world and here after.

Hayat Ullah

March, 2018

viii

LIST OF PUBLICATIONS

1. Hayat Ullah, Fazal Rahim, Muhammad Taha, Imad Uddin, Abdul Wadood,

Syed Adnan Ali Shah, Rai Khalid Farooq, Mohsan Nawaz, Zainul Wahab, Khalid

Mohammed Khan. Synthesis, Molecular docking study and in vitro Thymidine

Phosphorylase Inhibitory Potential of Oxadiazole Derivatives. Bioorganic

Chemistry. 78 (2018) 58–67.

2. Muhammad Taha, Hayat Ullah, Laode Muhammad Ramadhan Al Muqarrabun,

Muhammad Naseem Khan, Fazal Rahim, Norizan Ahmat, Muhammad Tariq

Javid, Muhammad Ali, Khalid Mohammed Khan. Bisindolylmethane

thiosemicarbazides as potential inhibitors of urease: Synthesis and molecular

modeling studies. Bioorganic & Medicinal Chemistry. 26 (2018) 152-160.

3. Muhammad Taha, Hayat Ullah, Laode Muhammad Ramadhan Al Muqarrabun,

Muhammad Naseem Khan, Fazal Rahim, Norizan Ahmat, Muhammad Ali,

Shahnaz Perveen. Synthesis of bis-indolylmethanes as new potential inhibitors of

β-glucuronidase and their molecular docking studies. European Journal of

Medicinal Chemistry. 143 (2018) 1757-1767.

4. Muhammad Taha, Syed Adnan Ali Shah, Syahrul Imran, Muhammad Afifi,

Sridevi Chigurupati, Manikandan Selvaraj, Fazal Rahim, Hayat Ullah, Khalid

Zaman, Shantini Vijayabalan. Synthesis and in vitro study of benzofuran

hydrazone derivatives as novel alpha-amylase inhibitor. Bioorganic Chemistry.

75 (2017) 78-85.

5. Muhammad Taha, Muhammad Tariq Javid, Syahrul Imran, Manikandan

Selvaraj, Sridevi Chigurupati, Hayat Ullah, Fazal Rahim, Fahad Khan, Jahidul

Islam Mohammad, Khalid Mohammed Khan. Synthesis and study of the α-

amylase inhibitory potential of thiadiazole quinoline derivatives. Bioorganic

Chemistry. 74 (2017) 179-186.

6. Muhammad Taha, Fazal Rahim, Syahrul Imran, Nor Hadiani Ismail, Hayat

Ullah, Manikandan Selvaraj, Muhammad Tariq Javid, Uzma Salar, Muhammad

ix

Ali, Khalid Mohammed Khan. Synthesis, α-glucosidase inhibitory activity and in

silico study of tris-indole hybrid scaffold with oxadiazole ring: As potential leads

for the management of type-II diabetes mellitus. Bioorganic Chemistry. 74 (2017)

30-40.

7. Muhammad Taha, Syahrul Imran, Nor Hadiani Ismail, Manikandan Selvaraj,

Fazal Rahim, Sridevi Chigurupati, Hayat Ullah, Fahad Khan, Uzma Salar,

Muhammad Tariq Javid, Shantini Vijayabalan, Khalid Zaman, Khalid

Mohammed Khan. Biology-oriented drug synthesis (BIODS) of 2-(2-methyl-5-

nitro-1Himidazol-1-yl)ethyl aryl ether derivatives, in vitro α-amylase inhibitory

activity and in silico studies. Bioorganic Chemistry. 74 (2017) 1-9.

8. Tayyaba Noreen, Muhammad Taha, Syahrul Imran, Sridevi Chigurpati, Fazal

Rahim, Manikandan Selvaraj, Nor Hadiani Ismail, Jahidul Islam Mohammad,

Hayat Ullah, Muhammad Tariq javid, Faisal Nawaz, Maryam Irshad,

Muhammad Ali. Synthesis of Alpha Amylase Inhibitors Based on Privileged

Indole Scaffold. Bioorganic Chemistry. 72 (2017) 248-255.

9. Fazal Rahim, Samreen, Hayat Ullah, Muhammad Imran Fakhri, Uzma Salar,

Shahnaz Perveen, Khalid Mohammed Khan and M. Iqbal Choudhary. Anti-

Leishmanial Activities of Synthetic Biscoumarins. Journal of the Chemical Society

of Pakistan. 39 (2017) 79-82.

10. Khalid Mohammed Khan, Huma Rasheed, Bibi Fatima, Muhammad Hayat,

Fazal Rahim , Hayat Ullah, Abdul Hameed, Muhammad Taha, Affan Tahir and

Shahnaz Perveen. Anti-Cancer Potential of Benzophenone-Bis-Schiff bases on

Human Pancreatic Cancer Cell Line, Journal of the Chemical Society of Pakistan.

38 (2016) 954-958.

11. Khalid Mohammed Khan, Mohammad A. Mesaik, Omer M. Abdalla, Fazal

Rahim, Samreen Soomro, Sobia A. Halim, Ghulam Mustafa, Nida Ambreen, A.

Shukralla Khalid, Muhammad Taha, Shahnaz Perveen, Muhammad Tanveer

Alam, Zaheer Ul-Haq, Hayat Ullah, Zia Ur Rehman, Rafat Ali Siddiqui and

Wolfgang Voelter. The immunomodulation potential of the synthetic derivatives

x

of benzothiazoles: Implications in immune system disorders through in vitro and

in silico studies. Bioorganic Chemistry. 64 (2016) 21-28.

12. Fazal Rahim, Muhammad Ali, Shifa Ullah, Umer Rashid, Hayat Ullah,

Muhammad Taha, Muhammad Tariq Javed, Wajid Rehman, Obaid-Ur-Rehman

Abid, Aftab Ahmad Khan, Muhammad Bilal. Development of bis-

Thiobarbiturates as Successful Urease Inhibitors and their Molecular Modeling

Studies, Chinese Chemical Letters. 27 (2016) 693-697.

13. Umer Rashid, Fazal Rahim, Muhammad Taha, Muhammad Arshad, Hayat

Ullah, Tariq Mahmood, Muhammad Ali. Synthesis of 2-Acylated and Sulfonated

4-hydroxycoumarins: In vitro Urease Inhibition and Molecular Docking Studies,

Bioorganic Chemistry. 66 (2016) 111-116.

14. Muhammad Taha, Sadia Sultan, Herizal Ali Nuzar, Fazal Rahim, Syahrul Imran,

Nor Hadiani Ismail, Humera Naz, Hayat Ullah. Synthesis and biological

evaluation of novel N-arylidenequinoline-3-carbohydrazides as potent β-

glucuronidase inhibitors, Bioorganic & Medicinal Chemistry. 24 (2016) 3696-

3704.

15. Fazal Rahim, Hayat Ullah, Muhammad Taha, Abdul Wadood, Muhammad

Tariq Javed, Wajid Rehman, Mohsan Nawaz, Muhammad Ashraf, Muhammad

Ali, Muhammad Sajid, Farman Ali, Muhammad Naseem Khan, Khalid

Mohammed Khan. Synthesis and in vitro Acetylcholinesterase and

Butyrylcholinesterase Inhibitory Potential of Hydrazide based Schiff Bases,

Bioorganic Chemistry. 68 (2016) 30-40.

16. Muhammad Taha, Nor Hadiani Ismail, Syahrul Imran, Fazal Rahim, Abdul

Wadood, Huma Khan, Hayat Ullah, Uzma Salar, Khalid Mohammad Khan.

Synthesis, β-Glucuronidase Inhibition and Molecular Docking Studies of Hybrid

Bisindole-Thiosemicarbazides Analogs, Bioorganic Chemistry. 68 (2016) 56-63.

17. Farman Ali, Hayat Ullah, Zarshad Ali, Fazal Rahim, Fahad Khan and Zia Ur

Rehman. Polymer-clay Nanocomposites, Preparations and Current Applications:

A Review. Current Nanomaterials. 1(2): (2016) 83-95.

xi

18. Fazal Rahim, Khadim Ullah, Hayat Ullah, Abdul Wadood, Muhammad Taha,

Ashfaq Ur Rehman, Imad Uddin, Muhammad Ashraf, Ayesha Shaukat, Wajid

Rehman, Shafqat Hussain, Khalid Mohammad Khan. Triazinoindole analogs as

potent inhibitors of α-glucosidase: Synthesis, biological evaluation and molecular

docking studies. Bioorganic Chemistry 58 (2015) 81–87.

19. Fazal Rahim, Fazal Malik, Hayat Ullah, Abdul Wadood, Fahad Khan,

Muhammad Tariq Javid, Muhammad Taha, Wajid Rehman, Ashfaq Ur Rehman,

Khalid Mohammad Khan. Isatin based Schiff bases as inhibitors of α-glucosidase:

Synthesis, characterization, in vitro evaluation and molecular docking studies.

Bioorganic Chemistry 60 (2015) 42–48.

20. Fazal Rahim, Hayat Ullah, Muhammad Tariq Javid, Abdul Wadood,

Muhammad Taha, Muhammad Ashraf, Ayesha Shaukat, Muhammad Junaid,

Shafqat Hussain, Wajid Rehman, Rasheed Mehmood, Muhammad Sajid,

Muhammad Naseem Khan, Khalid Mohammad Khan. Synthesis, in vitro

evaluation and molecular docking studies of thiazole derivatives as new

inhibitors of α-glucosidase, Bioorganic Chemistry 62 (2015) 15–21.

21. Fazal Rahim, Muhammad Tariq Javed, Hayat Ullah, Abdul Wadood,

Muhammad Taha, Muhammad Ashraf, Qurat-ul-Aine, Muhammad Anas Khan,

Fahad Khan, Salma Mirza, Khalid Mohammad Khan. Synthesis, Molecular

Docking, Acetylcholinesterase and Butyrylcholinesterase Inhibitory Potential of

Thiazole Analogs as New Inhibitors for Alzheimer Disease, Bioorganic

Chemistry 62 (2015) 106–116.

22. Fazal Rahim, Khalid Zaman, Hayat Ullah, Muhammad Taha, Abdul Wadood,

Muhammad Tariq Javed, Wajid Rehman, Muhammad Ashraf, Reaz Uddin, Imad

Uddin, Humna Asghar, Aftab Ahmad Khan, Khalid M. Khan. Synthesis of 4-

Thiazolidinone Analogs as Potent in Vitro Anti-urease Agents. Bioorganic

Chemistry 63 (2015) 123–131.

xii

Table of Contents

Acknowledgment ………………………………………………………………………… VI

List of Publication ……………………………………………………………………….. Viii

Abstract …………………………………………………………………………………… xxxi

Background of the Research……………………..…………………………………..….. Xxxiii

CHAPTER-01 1

1. General Introduction 1

1.1 Introduction to Heterocycles 1

1.2 Introduction to thiazole 3

1.3 Biological importance of thiazole 5

1.3.1 Anti-microbial agent 5

1.3.2 Anti-tubercular Agents 10

1.3.3 Anti-viral Agents 11

1.3.4 Anti-cancer Agents 13

1.3.5 Anti-convulsant Agents 17

1.3.6 Anti-inflammatory agents 19

1.3.7 Anti-oxidant agents 21

1.3.8 Anti-diabetic agents 22

1.3.9 Anti-Alzheimer‟s Agents 23

1.4 Previous synthetic approaches towards thiazole 24

1.4.1 Synthesis of thiazole analogs by Hantzsch method 24

1.4.2 Thiazole analogs synthesis by acylation process 25

1.4.3 Synthesis of amino-thiazole analogs: 26

1.4.4 Synthesis of Allylic thiazoles analogs: 27

1.5 Results and discussion 28

1.5.1 Chemistry 28

1.5.3 Molecular docking 31

1.5.4 Docking Study 32

xiii

1.6 Conclusion 38

1.7 Material and methods 39

1.7.1 General procedure for synthesis of thiazole 39

1.7.1.1 2-((2,5-dimethylthiazol-4-yl)methyl)-5-(4-nitrophenyl)-1,3,4-oxadiazole (102)

39


39

1.7.1.3 2-(2,4-dichlorophenyl)-5-((2,5-dimethylthiazol-4-yl)methyl)-1,3,4-oxadiazole

(104) 40

1.7.1.4 2-(4-(benzyloxy)phenyl)-5-((2,5-dimethylthiazol-4-yl)methyl)-1,3,4-oxadiazole

(105) 40

1.7.1.5 4-(5-((2,5-dimethylthiazol-4-yl)methyl)-1,3,4-oxadiazol-2-yl)-N,N-

dimethylaniline (106) 40

1.7.1.6 2,4-dichloro-6-(5-((2,5-dimethylthiazol-4-yl)methyl)-1,3,4-oxadiazol-2-

yl)phenol (107) 41

1.7.1.7 2-(5-bromo-2-methoxyphenyl)-5-((2,5-dimethylthiazol-4-yl)methyl)-1,3,4-

oxadiazole (108) 41

1.7.1.8 2-(4-chlorophenyl)-5-((2,5-dimethylthiazol-4-yl)methyl)-1,3,4-oxadiazole (109)

41

1.7.1.9 2-(2,3-dimethoxyphenyl)-5-((2,5-dimethylthiazol-4-yl)methyl)-1,3,4-oxadiazole

(110) 42

1.7.1.10 2-(5-((2,5-dimethylthiazol-4-yl)methyl)-1,3,4-oxadiazol-2-yl)aniline (111) 42

1.7.1.11 2-chloro-4-(5-((2,5-dimethylthiazol-4-yl)methyl)-1,3,4-oxadiazol-2-yl)phenol

(112) 42

1.7.1.12 2-(5-((2,5-dimethylthiazol-4-yl)methyl)-1,3,4-oxadiazol-2-yl)-4-fluorophenol

(113) 43

1.7.1.13 2-(5-((2,5-dimethylthiazol-4-yl)methyl)-1,3,4-oxadiazol-2-yl)benzonitrile (114)

43

xiv


43


44

1.7.1.16 2-(anthracen-9-yl)-5-((2,5-dimethylthiazol-4-yl)methyl)-1,3,4-oxadiazole (117)

44

REFERENCES 45

CHAPTER-02 51

2.1 Oxadiazole 51

2.2 Pharmacological activity of 1,3,4-oxadiazoles 52

2.2.1 Anti-microbial activity 52

2.2.2 Anti-convulsant activity 56

2.2.3 Anti-inflammatory activity: 57

2.2.4 Analgesic activity 58

2.2.5 Anti-tumor activity 59

2.2.6 Anti-viral activity 60

2.2.7 Anti-hypertensive activity 62

2.3 Previous approaches toward synthesis of oxadiazole analogs 63

2.3.1 Synthesis of 5-argio-1, 3, 4-Oxadiazole analogs 63

2.3.2 Synthesis of 1,3,4-Oxadiazole-2-amines analogs 63

2.3.3 Synthesis of 5-substituted 1, 3, 4-Oxadiazole-2-thiol analogs 64

2.3.4 Synthesis of Nafion catalyzed 1, 3, 4-oxadiazole analogs 64

2.3.5 Synthesis of 1,3,4-oxadiazole through regioselective cyclization processes 65

2.4 Research background 66


2.5.1 Chemistry 67

2.5.2 In vitro thymidine phosphorylase inhibitory potential: 69


2.5.4 Docking study 73

xv

2.6 Conclusion 84

2.7 Material and Methods 85

2.7.1 Synthetic procedure for 4-cyanobenzohydrazide (61) 85

2.7.2 Synthetic procedure for methyl (E)-4-((2-(4-cyanobenzoyl)hydrazono)methyl)

benzoate (62) 85

2.7.3 Synthetic procedure for methyl 4-(5-(4-cyanophenyl)-1,3,4-oxadiazole-2-

yl)benzoate (63) 85

2.7.4 Synthetic procedure for 4-(5-(4-cyanophenyl)-1,3,4-oxadiazole-2-

yl)benzohydrazide (64) 86

2.7.5 General procedure for the synthesis of oxadiazole derivatives (65-80) 86

2.7.5.1 (E)-4-(5-(4-cyanophenyl)-1,3,4-thiadiazol-2-yl)-N'-(2,4-dichlorobenzylidene)

benzohydrazide (65) 86

2.7.5.2 (E)-N'-((4-chlorocyclohexa-1,3-dien-1-yl)methylene)-4-(5-(4-cyanophenyl)-

1,3,4-oxadiazol-2-yl)benzohydrazide (66) 87

2.7.5.3 (E)-N'-(4-(benzyloxy)benzylidene)-4-(5-(4-cyanophenyl)-1,3,4-oxadiazol-2-

yl)benzohydrazide (67) 87

2.7.5.4 (E)-N'-(5-bromo-2-methoxybenzylidene)-4-(5-(4-cyanophenyl)-1,3,4-

oxadiazol-2-yl)benzohydrazide (68)

87

2.7.5.5 (E)-N'-(3-chloro-4-hydroxybenzylidene)-4-(5-(4-cyanophenyl)-1,3,4-


88

2.7.5.6 (E)-4-(5-(4-cyanophenyl)-1,3,4-oxadiazol-2-yl)-N'-(4-

(dimethylamino)benzylidene) benzohydrazide (70)

88

2.7.5.7 (E)-N'-(4-(benzyloxy)-3-methoxybenzylidene)-4-(5-(4-cyanophenyl)-1,3,4-

oxadiazol-2-yl)benzohydrazide (71) 89

2.7.5.8 (E)-N'-(anthracen-9-ylmethylene)-4-(5-(4-cyanophenyl)-1,3,4-oxadiazol-2-yl)


xvi

2.7.5.9 (E)-4-(5-(4-cyanophenyl)-1,3,4-oxadiazol-2-yl)-N'-((2-hydroxynaphthalen-1-

yl)methylene)benzohydrazide (73) 90

2.7.5.10 (E)-4-(5-(4-cyanophenyl)-1,3,4-oxadiazol-2-yl)-N'-(4-hydroxybenzylidene)



hydroxybenzylidene)benzohydrazide (75) 90

2.7.5.12 (E)-4-(5-(4-cyanophenyl)-1,3,4-oxadiazol-2-yl)-N'-(3-hydroxy-4-

methoxybenzylidene) benzohydrazides (76)

91

2.7.5.13 (E)-4-(5-(4-cyanophenyl)-1,3,4-oxadiazol-2-yl)-N'-(4-hydroxy-3,5-dimethoxy

benzylidene)benzohydrazide (77) 91


methoxybenzylidene) benzohydrazide (78)

92


nitrobenzylidene)benzohydrazide (79) 92

2.7.5.16 (E)-N'-(2-cyanobenzylidene)-4-(5-(4-cyanophenyl)-1,3,4-oxadiazol-2-yl)

benzohydrazides (80) 92

REFERENCES 94

CHAPTER-03 99

3.1 Indole 99

3.2 Biological Importance of Indole 100

3.2.1 Anti-microbial activity 101

3.2.2 Anti-inflammatory activity 102

3.2.3 Anti-tumor activity 104

3.2.4 Hypocholestrolemic activity 105

3.2.5 Anti-cancer activity 105

3.2.6 Anti-oxidant activity 107

3.2.7 Anti-diabetic activity 108

xvii

3.2.8 Anti-parkinsonian activity 109

3.2.9 Anti-viral activity 109

3.3 Previous approaches towards indoles synthesis 110

3.4 Introduction to bis-indole derivatives 112

3.5 Previous approaches towards bis-(indolyl)methanes synthesis 114


3.6.1 Synthesis of bis-indolylmethane based Schiff base derivatives 117

3.6.2 In vitro β-Glucuronidase inhibitory Potential: 120

3.6.3 Molecular Docking 121

3.6.4 Docking Studies 122

3.7 Material and Methods 128

3.7.1 Synthesis of dimethyl-3,3'-(p-tolylmethylene)bis(1H-indole-5-carboxylate) 128

3.7.2 Synthesis of 3,3'-(p-tolylmethylene)bis(1H-indole-5-carbohydrazide) 128

3.7.3 Synthesis of the library of bis-indolylmethanes based Schiff base derivatives (71-

102) 128

3.7.3.1 3'-(p-Tolylmethylene)bis(N'-((E)-4-hydroxybenzylidene)-1H-indole-5-

carbohydrazide) (71) 129

3.7.3.2 3'3'-(p-Tolylmethylene)bis(N'-((E)-2,4-dihydroxybenzylidene)-1H-indole-5-


3.7.3.3 3,3'-(p-Tolylmethylene)bis(N'-((E)-3-nitrobenzylidene)-1H-indole-5-


3.7.3.4 3,3'-(p-Tolylmethylene)bis(N'-((E)-4-methoxybenzylidene)-1H-indole-5-


3.7.3.5 3,3'-(p-Tolylmethylene)bis(N'-((1E,2E)-3-phenylallylidene)-1H-indole-5-


3.7.3.6 3,3'-(p-Tolylmethylene)bis(N'-((E)-3-hydroxybenzylidene)-1H-indole-5-


3.7.3.7 3,3'-(p-Tolylmethylene)bis(N'-((E)-2,3-dihydroxybenzylidene)-1H-indole-5-


xviii

3.7.3.8 3,3'-(p-Tolylmethylene)bis(N'-((E)-benzylidene)-1H-indole-5-carbohydrazide)

(78) 132





3.7.3.11 3,3'-(p-Tolylmethylene)bis(N'-((E)-2,4,5-trihydroxybenzylidene)-1H-indole-5-




3.7.3.13 3,3'-(p-Tolylmethylene)bis(N'-((E)-2-hydroxy-4-methoxybenzylidene)-1H-

indole-5-carbohydrazide) (83) 135


indole-5-carbohydrazide) (84)

135



136

3.7.3.16 3,3'-(p-Tolylmethylene)bis(N'-((E)-3-hydroxy-2-iodo-4-methoxybenzylidene)-

1H-indole-5-carbohydrazide) (86) 136





3.7.3.19 3,3'-(p-Tolylmethylene)bis(N'-((E)-2-methylbenzylidene)-1H-indole-5-




xix



3.7.3.22 3,3'-(p-Tolylmethylene)bis(N'-((E)-2-chlorobenzylidene)-1H-indole-5-






3.7.3.25 3,3'-(p-Tolylmethylene)bis(N'-((E)-2,4-dichlorobenzylidene)-1H-indole-5-


3.7.3.26 3,3'-(p-Tolylmethylene)bis(N'-((E)-2-fluorobenzylidene)-1H-indole-5-






3.7.3.29 3,3'-(p-Tolylmethylene)bis(N'-((E)-3-bromo-4-fluorobenzylidene)-1H-indole-5-

carbohydrazide) (99)

143

3.7.3.30 3,3'-(p-Tolylmethylene)bis(N'-((E)-pyridin-3-ylmethylene)-1H-indole-5-






3.8 Synthesis of bis-indolylmethane thiosemicarbazides analogs 145

3.8.1 In vitro Urease Inhibition Study 147

3.8.2 Molecular Docking Studies 152

3.8.3 Docking Studies 153

xx

3.9. Material and Methods 159

3.9.1 Synthesis of dimethyl-3,3'-(p-tolylmethylene)bis(1H-indole-5-carboxylate) 159

3.9.2 Synthesis of 3,3'-(p-tolylmethylene)bis(1H-indole-5-carbohydrazide) 159

3.9.3 Synthesis of the library of new bis-indolylmethanes thiosemicarbazide

derivatives (103-120) 159

3.9.3.1 2,2'-(3,3'-(p-tolylmethylene)bis(1H-indole-3,5-diyl-5-carbonyl))bis(N-(4-

bromophenyl)hydrazine-1-carbothioamide) (103) 159






fluorophenyl)hydrazine-1-carbothioamide) (106) 161






chlorophenyl)hydrazine-1-carbothioamide) (109) 163


chlorophenyl)hydrazine-1-carbothioamide) (110) 163

3.9.3.9 2,2'-(3,3'-(p-tolylmethylene)bis(1H-indole-3,5-diyl-5-carbonyl))bis(N-(3,4-

dichlorophenyl)hydrazine-1-carbothioamide) (111) 164

3.9.3.10 2,2'-(3,3'-(p-tolylmethylene)bis(1H-indole-3,5-diyl-5-carbonyl))bis(N-(o-

tolyl)hydrazine-1-carbothioamide) (112) 164

3.9.3.11 2,2'-(3,3'-(p-tolylmethylene)bis(1H-indole-3,5-diyl-5-carbonyl))bis(N-(m-


3.9.3.12 2,2'-(3,3'-(p-tolylmethylene)bis(1H-indole-3,5-diyl-5-carbonyl))bis(N-(p-


xxi


methoxyphenyl)hydrazine-1-carbothioamide) (115) 166


methoxyphenyl)hydrazine-1-carbothioamide) (116) 166


nitrophenyl)hydrazine-1-carbothioamide) (117) 167


nitrophenyl)hydrazine-1-carbothioamide) (118) 168


(trifluoromethyl)phenyl)hydrazine-1-carbothioamide) (119) 168

3.9.3.18 N-((1s,3s)-adamantan-1-yl)-2-(3-((5-(2-(((3s,5s,7s)-adamantan-1-

yl)carbamothioyl)hydrazine-1-carbonyl)-1H-indol-3-yl)(p-tolyl)methyl)-1H-indole-5-

carbonyl)hydrazine-1-carbothioamide (120) 169

3.10 Synthesis of tris-indole analogs 170

3.10.1 α-Glucosidase inhibitory activity 171

3.10.2 Structure-activity relationship (SAR) 172


3.10.4 Docking studies 177

3.11 Material and methods 183

3.11.1 Synthesis of bis-indole ester derivatives 183

3.11.2 Synthesis of bis-indole hydrazide derivatives 183

3.11.3 Synthesis of trisindole-oxadiazole derivatives 183

3.11.3.1 3,3'-((4-(5-(1-Methyl-1H-indol-3-yl)-1,3,4-oxadiazol-2-

yl)phenyl)methylene)bis(1H-indole-5-carbonitrile) (126) 184

3.11.3.2 3,3'-((4-(5-(1H-Indol-3-yl)-1,3,4-oxadiazol-2-yl)phenyl)methylene)bis(1H-

indole-5-carbonitrile) (127) 184


yl)phenyl)methylene)bis(1H-indole-5-carbonitrile) (128) 185

xxii

3.11.3.4 2-(4-(Bis(1-methyl-1H-indol-3-yl)methyl)phenyl)-5-(1-methyl-1H-indol-3-yl)-

1,3,4-oxadiazole (129) 185


1,3,4-oxadiazole (130) 186

3.11.3.6 2-(4-(Bis(1-methyl-1H-indol-3-yl)methyl)phenyl)-5-(1H-indol-3-yl)-1,3,4-

oxadiazole (131) 187

3.11.3.7 2-(4-(Bis(2-phenyl-1H-indol-3-yl)methyl)phenyl)-5-(1H-indol-3-yl)-1,3,4-


3.11.3.8 2-(4-(Bis(2-phenyl-1H-indol-3-yl)methyl)phenyl)-5-(1-methyl-1H-indol-3-yl)-

1,3,4-oxadiazole (133) 188



3.11.3.10 2-(4-(Di(1H-indol-3-yl)methyl)phenyl)-5-(1-methyl-1H-indol-3-yl)-1,3,4-


3.11.3.11 2-(4-(Di(1H-indol-3-yl)methyl)phenyl)-5-(1H-indol-3-yl)-1,3,4-oxadiazole

(136) 190



3.11.3.13 2-(4-(Bis(1,2-dimethyl-1H-indol-3-yl)methyl)phenyl)-5-(1-methyl-1H-indol-3-

yl)-1,3,4-oxadiazole (138) 191


yl)-1,3,4-oxadiazole (139) 191

3.11.3.15 2-(4-(Bis(1,2-dimethyl-1H-indol-3-yl)methyl)phenyl)-5-(1H-indol-3-yl)-1,3,4-


3.11.3.16 2-(4-(Bis(5-chloro-1H-indol-3-yl)methyl)phenyl)-5-(1-methyl-1H-indol-3-yl)-

1,3,4-oxadiazole (141) 192


1,3,4-oxadiazole (142) 193

xxiii

3.11.3.18 2-(4-(Bis(5-chloro-1H-indol-3-yl)methyl)phenyl)-5-(1H-indol-3-yl)-1,3,4-


3.11.3.19 2-(4-(Bis(5-bromo-1H-indol-3-yl)methyl)phenyl)-5-(1-methyl-1H-indol-3-yl)-

1,3,4-oxadiazole (144) 194


1,3,4-oxadiazole (145) 195

3.11.3.21 2-(4-(Bis(5-bromo-1H-indol-3-yl)methyl)phenyl)-5-(1H-indol-3-yl)-1,3,4-


3.12 Ligand preparation 196

3.13 Rapid pharmacokinetic predictions of tris-indole series 196

3.14 Predicted pharmacokinetic for tris-indole derivatives 196

3.15 Conclusion 198

REFERENCES 200

CHAPTER-04 208

4. Procedures for Various Biological Assays 208

4.1 Assay Protocol of Thymidine phosphorylase 208

4.2 Urease Assay protocol 209

4.3 β-Glucuronidase assay 209

4.4 α-Glucosidase inhibition assay 210

REFERENCES 211

xxiv

List of Figures

Figure-1.1: Thiazole basic skeleton ............................................................................................ 3

Figure-1.2: Thiazole resonating structures ............................................................................... 3

Figure-1.3: Examples of thiazole bearing drugs ...................................................................... 5

Figure-1.4: Thiazole containing analogs as anti-microbial agents ........................................ 6

Figure-1.5: Thiazole containing analogs as anti-microbial agents ........................................ 7

Figure-1.6: Thiazole containing analogs as anti-bacterial agents .......................................... 8

Figure-1.7: Thiazole containing analogs as anti-bacterial agents .......................................... 9

Figure-1.8: Thiazole containing analogs as anti-fungal agents ............................................. 9

Figure-1.9: Thiazole containing analogs as anti-tubercular agents .................................... 11

Figure-1.10: Thiazole containing analogs as anti-viral agents ............................................. 12

Figure-1.11: Thiazole containing analogs as anti-viral agents ............................................. 13

Figure-1.12: Thiazole containing analogs as anti-cancer agents ......................................... 15



Figure-1.15: Thiazole containing analogs as anti-convulsant agents ................................. 18

Figure-1.16: Thiazole containing analogs as anti-convulsant agents ................................. 19

Figure-1.17: Thiazole containing analogs as anti-inflammatory agents ............................ 20

Figure-1.18: Thiazole containing analogs as anti-oxidant agents ....................................... 22

Figure-1.19: Thiazole containing analogs as anti-diabetic agents ....................................... 23

Figure-1.20: Thiazole containing analogs as anti-Alzheimer‟s agents ............................... 24

Figure-1.21: Structure of the potent analog 106 ..................................................................... 30

xxv

Figure-1.22: Comparison of structure activity relationship between 102 and 103 ........... 31

Figure-1.23: Comparison of structure activity relationship between 114, 115 and 116 ... 31

Figure-1.24: Docking conformations of compounds a) analog 106 and b) analog 103 on

thymidine phosphorylase enzyme .......................................................................................... 33

Figure-1.25: Correlation graph for IC50 values and docking predicted activity. .............. 34

Figure-2.1: Basic skeleton of oxadiazole ................................................................................. 51

Figure-2.2: Isomers of oxadiazole ............................................................................................ 51

Figure-2.3: Structure of Raltegravir (5) and Zibotentan (6) ................................................. 52

Figure-2.4: Oxadiazole containing analogs as anti-microbial agents ................................. 53



Figure-2.7: Oxadiazole containing analogs as anti-convulsant agents .............................. 57

Figure-2.8: Oxadiazole containing analogs as antiinflammatory agents ........................... 58

Figure-2.9: Oxadiazole containing analogs as analgesic agents .......................................... 59

Figure-2.10: Oxadiazole containing analogs as antitumor agents ...................................... 60

Figure-2.11: Basic structures of raltegravir (39) and its derivative (40) ............................. 61

Figure-2.12: 1,3,4-oxadiazole analogs having HIV-1 activity .............................................. 61

Figure-2.13: 1,3,4-Oxadiazole analogs having HIV and hepatitis C virus activity ........... 62

Figure-2.14: Oxadiazole containing analogs as anti-hypertensive agents ......................... 63

Figure-2.15: Rational of the current study ……………………………………………………...66

Figure-2.16: Comparison of structure activity relationship between analogs 76 and 78 . 70


xxvi


Figure-2.19: Docking conformations of compounds on thymidine phosphorylase

enzyme. (a) 3D binding mode of compound 77 as inhibitor of thymidine phosphorylase

enzyme. (b) 3D binding mode of compound 78 (c) 3D binding mode of compound 65 (d)

3D binding mode of compound 76 in binding cavity of thymidine phosphorylase

enzyme. Ligands are shown green color. ............................................................................... 74

Figure-2.20: Correlation graph for IC50 values and docking predicted activity. .............. 75

Figure-3.1: Basic skeleton of indole ......................................................................................... 99

Figure-3.2: Indole containing analogs as anti-microbial agents ........................................ 102

Figure-3.3: Indole containing analogs as anti-inflammatory agents ................................ 104

Figure-3.4: Indole containing analogs as anti-tumor agents .............................................. 105

Figure-3.5: Indole containing analogs as hypocholestrolemic agent ................................ 105

Figure-3.6: Indole containing analogs as anti-cancer agents ............................................. 107

Figure-3.7: Indole containing analogs as anti-oxidants agents ......................................... 107

Figure-3.8: Indole containing analog as anti-diabetic agent .............................................. 109

Figure-3.9: Indole containing analog as anti-parkinsonian agent ..................................... 109

Figure-3.10: Indole containing analog as anti-viral agent .................................................. 110

Figure-3.11: Structure of biologically active indirubin ....................................................... 112

Figure-3.12: Structure of biologically active dragmacidin ................................................. 113

Figure-3.13: Structure of biologically active bis-(indolyl)methanes complex with (Gd3+).

..................................................................................................................................................... 113

Figure-3.14: Structures of biologically active bis-indole derivatives ................................ 114

xxvii

Figure-3.15: Structure of Human-β-glucuronidase (PDB ID: 1BHG) and its active site

(red sphere and zoomed in).................................................................................................... 123

Figure-3.16: Models of the interaction of 81 (a) and 82 (b) with the binding site of

Human-β-glucuronidase generated by poseview. Dashed lines indicate hydrogen bond

interactions and a green line indicating hydrophobic interactions. ................................. 124

Figure-3.17: (a) Two-dimensional scheme of the interactions between 77 and Human β-

glucuronidase; (b) and interactions between 72 and Human β-glucuronidase generated

by Ligplot+. Only more important residues for binding are shown................................. 126

Figure-3.18: Stereoview of simulated docking poses of compounds (a) 81; (b) 77; (c) 82

and (d) 72 to Human-β-glucuronidase. Compounds 81, 77, 82 and 72 are shown as stick

models with carbon colored in bright yellow; nitrogen colored in bright blue and

oxygen atoms colored in dark red respectively. Important parts of the enzyme for

interaction were shown as a stick model colored in dark red. .......................................... 127

Figure-3.19: Comparison of structure activity relationship between compounds 111, 109

and 110 ....................................................................................................................................... 149


and 108 ....................................................................................................................................... 150


and 114 ....................................................................................................................................... 151

Figure-3.22: Comparison of structure activity relationship between compounds 117 and

118............................................................................................................................................... 152

xxviii

Figure-3.23: Urease from Jack bean (Ribbon form in light grey color), active site of urease

(enclosed in green color ring) and zoomed in docked conformation of compound 111

(yellow sticks along with Ni atoms in red color) ................................................................ 154

Figure-3.24: Docked conformation of synthesized compounds with the side chain

residues, Ligands shown in yellow color, side chain amino acids in dark orange color,

and distances in Angstrom colored in white; a) Interactions of compound 111 (most

active); b) Interactions of compound 119 (second most active); c) Interactions of

compound 118 (activity in-between) and d) Interactions of Compound 120 (least active

among series). ........................................................................................................................... 158

Figure-3.25: Structure-activity relationship of compounds 135-137 ................................. 173


Figure-3.27: Structure-activity relationship of compounds 19-21 ..................................... 174

Figure-3.28: Structure-activity relationship of compounds 129-131 and 138-140 ........... 175



Figure-3.31: Comparison of interaction between tris-indole series and acarbose in the

binding site of α-glucosidase. a) The active and moderately active tris-indole

compounds are circled in red dotted line, while the least active tris-indole compounds

are circled in blue dotted line. Acarbose binding mode is shown in blue stick. b) Shows

the binding mode comparison of acarbose in blue color stick with compound 141 in

olive color stick. ........................................................................................................................ 179

xxix

Figure-3.32: Graphical illustration of predicted binding mode of tris-indole series in the

binding site of α-glucuronidase. a) compound 141 (olive green color) b) compound 142

(magenta color), c) compound 143 (gray color), d) compound 144 (brown color). Key

residues are only shown and the hydrogen bond interactions are represented by yellow

dashed lines. Compounds are shown in stick and key amino acids in line. ................... 182

xxx

List of Tables

Table-1.1: Different substituents and thymidine phosphorylase activity of thiazole

analogs (103-118) ........................................................................................................................ 28

Table-1.2: Docking scores and report of predicted interactions of docked conformations

....................................................................................................................................................... 34

Table-2.1: Oxadiazole derivatives and their thymidine phosphorylase activity (65-80) . 68


....................................................................................................................................................... 75

Table-3.1: Different substituents of bis-indolylmethane analogs and their β-

glucuronidase activity (71-102) .............................................................................................. 118

Table-3.2: Different substituents and urease activity of the synthesized analogs (103-120)

..................................................................................................................................................... 146

Table-3.3: α-Glucosidase inhibitory activity of synthetic compounds (126-146) 171

Table-3.4: Pharmacokinetic predictions and Glide g score 197

xxxi

Abstract

This dissertation has been divided into four chapters and each chapter has its own

numbering of compounds and references.

General introduction related to the importance of natural products and natural product

based drugs, drug designing and value of lead molecules in drug designing. This

research work describes synthesis and bioactivities of different class of heterocycles

such as thiazole, oxadiazole and indole analogs in search of important therapeutic

agents. During this research study, a variety of thiazole, oxadiazole and indole analogs

were synthesized and screened for enzyme inhibition studies (thymidine

phosphorylase, α-glucosidase, urease and β-glucuronidase activities). The results

obtained from this study are encouraging which are discussed separately in the

forthcoming chapters 1, 2 and 3.

In the first chapter, thiazole analogs are described. In the thiazole series, sixteen analogs

(1-16) were synthesized and evaluated for thymidine phosphorylase inhibitory

potential. Out of sixteen analogs, nine analogs such as analogs 1, 2, 3, 5, 6, 8, 10, 13 and

15 showed good phosphorylase inhibitory potentials when compared with the standard

7-Deazaxanthine. The remaining seven analogs showed moderate to good activity.

In 2nd chapter, oxadiazole analogs are described. Sixteen analogs (1-16) of oxadiazole

were synthesized and evaluated for thymidine phosphorylase activity. Out of sixteen

analogs, fourteen analogs such as analogs 1-7 and 9-15 showed excellent thymidine

phosphorylase inhibitory potentials which is many folds better than the standard 7-

Deazaxanthine. Analog 16 showed good inhibitory potential while analog 8 remain

inactive among the series.

In chapter third, three series of indole are described. In first series, We have synthesized

thirty-two (32) bis-indolylmethane analogs (1-32) with varying degree of β-

glucuronidase inhibition potential ranging in between 0.10 ± 0.01 to 48.50 ± 1.10 μM

when compared with the standard drug D-saccharic acid 1,4-lactone (IC50 value 48.30 ±

xxxii

1.20 μM). All of thirty-two analogs 1-32, showed outstanding β-glucuronidase inhibitory

potentials.

In second series, eighteen derivatives (1-18) of bis-indolylmethane thiosemicarbazide

were synthesized and evaluated for their urease inhibitory potential. These derivatives

displayed varying degree of inhibition in the range of 0.14 ± 0.01 to 18.50 ± 0.90 μM

when compared with the standard inhibitor thiourea having an IC50 value 21.25 ± 0.90

μM. All derivatives showed outstanding urease inhibitory potentials which are many

folds better than the standard thiourea.

In third series, twenty one analogs of tris-indole hybrid scaffold with oxadiazole ring (1-

21) were synthesized and evaluated for α-glucosidase inhibitory potential. All

compounds displayed superior α-glucosidase inhibitory activities having IC50 value in

the range of 2.00 ± 0.01-292.40 ± 3.16 μM as compared to standard acarbose (IC50 =

895.09 ± 2.04 µM).

In chapter four, procedures for different biological assay are described.

xxxiii

Background of the Research

Our research group is continuously in struggle to identify new heterocyclic scaffold as

lead candidate. We mainly focus on enzyme inhibitors, because a lot of enzyme

inhibitors are currently used as drug in market against various diseases. The designing

of compounds was mainly based on previous literature relevant to these classes of

compounds which we have selected for our study, by keeping in view the basic

scaffold, structure modification and position of substituents. We have selected the

various test of enzyme inhibition mainly focuses on previous literature by keeping in

view the binding mode of enzymes.

We have design the compound for this study by keeping in view the biological

importance of these classes of compounds, structure modifications and substitution

pattern on compounds. After experimental we have obtained outstanding results for

various enzyme inhibitions which support our previous hypothesis. In order to explain

the inhibitory potential of our design compounds, we have perform molecular docking

in order to explain why some compounds are more active and why some are less active

or inactive.

1

CHAPTER-01

Synthesis of thiazole derivatives and their biological activity

Summary

………………………………………………………………………………………………………..

In this chapter we describe the synthesis of thiazole derivatives and study of their

thymidine phosphorylase with molecular docking.

………………………………………………………………………………………………………..

1. General Introduction

1.1 Introduction to Heterocycles

Those carbocyclic molecules which hold one or more atoms other than carbon such as

sulfur, nitrogen or oxygen within a ring structure are called heterocycles.

Insertion of sulfur, nitrogen, oxygen or an atom of a correlated element into a carbocyclic

ring as an alternate of a carbon atom give rise to a heterocyclic compounds. Commonly

dispersed heterocycles may be either aromatic or aliphatic; it is shown that nearly half of

the known organic molecules are heterocyclic; few of them are very significant for human

being. Heterocyclic molecule have important role in chemistry. In fact, they are essential

to life as we know purine and pyrimidine bases are exist in DNA and indoles and

pyrolidine moiety present in tryptophan and proline correspondingly. Heterocyclic

compound have many biological activities, some show greater activity as pesticides, co-

polymers, anti-oxidant, ligands or numerous synthetic intermediate. Numerous natural

drugs such as emetine, quinine, codeine, theophylline, atropine and reserpine are

heterocycle. Practically all synthetic drugs such as sumatriptane, methotrexate, anti-

pyrine, captopril, diazepam and metronidazole are also heterocycle. Heterocycles are

very significant for numerous industries such as solvent, photographic developer, in

2

rubber industry as vulcanization accelerators and dyestuff. Inside the human body, most

of heterocycle also take part in chemical reactions. Moreover, all biological progressions

are also chemical in nature.

The main purpose of this thesis is synthesis of selected heterocyclic molecules and

screens their biological significant in search of essential therapeutic agents and lead

molecules. These classes are:

1. Thiazole analogs

2. Oxadiazole analogs

3. Indole analogs

3

1.2 Introduction to thiazole

Thiazole is five membered rings having nitrogen and sulfur atoms. They are often

called 1,3-azoles while its 1,2 analogs are called iso-thiazole.

Figure-1.1: Thiazole basic skeleton

The boiling point of thiazole is 116-118 °C and it is clear to pale yellow liquid. It is

sparingly soluble in H2O and its specific gravity is 1.2 [1]. Due to localization of lone

pair from sulfur it is aromatic in nature [2]. Thiazole shows the following resonating

structures.

Figure-1.2: Thiazole resonating structures

Substituents greatly affect the thiazole ring and the change in acidity or basicity

strength arises certainly. With the introduction of electron donating group like is

positioned at C-4, C-5 and C-2, or of the thiazole ring than the nucleophilicity and

basicity increases. Placing CH3 at C-2 of thiazole ring than maximum substituent effect

is detected that might be due to the closeness of CH3 to the protonation centre and

being between two heteroatoms. The nucleophilicity or basicity of the thiazole ring is

decreases with the introduction of strong electron withdrawing as NO2 group is placed

on it. When NO2 group is present at C-4 than the biggest decrease occur in the basicity

4

[3]. In material sciences, thiazole analog is used due to varied importance. Numerous

heterocyclic derivatives are accomplished as conducting polymers, nonlinear optical

polymers and high potent polymers containing thiazole moiety in their basic structure.

The tensile strength of the molecule increases when thiazole is constructed in certain

polymers basic structure [4-6]. Complexes of bi-nuclear and multi-nuclear are made

with transition metal ions [7]. Due to exclusive shape and structure of the thiazole

compounds, complexes of transition metal are dynamic and due to its co-ordination,

some compounds act as anti-tumor agent [8]. Formamide thiazole is used as multi-

dentate ligands establishing bridging with metal atoms [9, 10].

Thiazole is very significant heterocyclic ring in various pharmacologically active

analogs that make it one of the widely studied heterocycle [11]. In various drugs,

thiazole play significant role. Examples of drugs which bear thiazole moiety are

tiazofurin 2 and dasatinib 3 (anti-neoplastic) [12], nitazoxanide 4 (anti-parasitic agent)

[13], ravuconazole 5 (anti-fungal) [14], fanetizole 6, fentiazac 7 and meloxicam 8 (anti-

inflammatory) [15], nizatidine 9 (anti-ulcer) [16], thiamethoxam 10 (insecticide) [17] and

ritonavir 11 (anti-HIV) [18]. Thiazole moiety is an important five membered heterocycle

which utilized numerous roles in optimization and lead identification, containing

bioisosteric and pharmacophoric elements. Drug which contain thiazole moiety can be

determinant for its pharmaco-kinetic and physio-chemical properties.

5

Figure-1.3: Examples of thiazole bearing drugs

1.3 Biological importance of thiazole

1.3.1 Anti-microbial agent

Worldwide the conflict of disease causing bacteria towards available drugs has been

reported. For that reason, the researchers have been motivated towards improvement of

new anti-microbial agents with unique target [19]. Compounds bearing different

6

thiazole exhibit auspicious anti-microbial activities. Various compounds comprising

thiazole moiety as novel anti-microbial agents are described in the following

paragraphs. Karegoudar et al., synthesized (phenyllidenehydrazino)-1,3-thiazole analog

12 by the reaction of Phenacyl bromide with 2,3,5-tri-chloro benzaldehyde

thiosemicarbazide. Analogs 13 and 14 was afforded by condensing di-chloroacetone or

Phenacyl bromide with 2,3,5-tri-chlorobenzen carbothioamide. Anti-microbial liability

testing designated that various analogs exhibited good antibacterial activity. Also

analogs with 3-biphenyl and pyridyl and 4-mercaptopyrazolpyrimidine showed the

greatest anti-fungal activity [20]. Some schiff bases of 2,4-disubstituted thiazole 15-17

were prepared by cyclisation of substituted phenacyl bromide with thiosemicarbazone.

All the analogs were screened for their anti-microbial against six bacterial strains and

four fungi strains. Imine analogs generally exhibited somewhat greater anti-fungal and

anti-bacterial inhibitory potential than cyclopentamine and cyclohexaimine analogs

[21].

Figure-1.4: Thiazole containing analogs as anti-microbial agents

7

Boondock and his co-workers prepared various schiff bases of 4-thiazolidinone 18 and

19, thiazole 20, thiazole [5,4-d]pyrimidine 21 and thiazoline thione 22 as anti-microbial

agents and screened against two strains of fungi (Asperrgillus niger and Aspeergillus

oryzaee), three bacterial strains ( Bacillus subtilis, Bacillus magaterium, Escherichia

coli). Thiazole [5,4-d]pyrimidine, thiazoline and thiazole analogs exhibited excellent

inhibitory potential while thiazoldinones exhibited almost same inhibitory potential as

compared to the standard drugs fluconazole, ampicillin and chloramphenicol [22].

Figure-1.5: Thiazole containing analogs as anti-microbial agents

Vijish et al., synthesized with the help of Vilsmayer-Hacck reaction of suitable

semicarbazones, disubstituted thiazole schiff bases 23 and 24 comprising pyrazole ring

[23]. Anti-bacterial activity of all compounds was checked against P. aeruginosa, E. coli,

S. aureus and B. subtilis. Analogs with 2,4-dichlorophenyl and 2,5-dichlorothiophen

substituents attached to pyrazole moiety exhibited important anti-bacterial inhibitory

potential against all tested micro-organisms [24]. Holla and his co-workers prepared 2-

substituted-4-(2,4-dichloro-5-florophenyl)thiazole 25-27 by the reaction of 2,4-dichloro-

5-florophenacyl bromide with thiosemicarbazone or substituted thiourea and evaluated

8

against anti-bacterial activity. Result showed that all the synthesized analogs exhibited

weak to excellent activities [25].

Figure-1.6: Thiazole containing analogs as anti-bacterial agents

Zhu and his co-workers synthesized schiff base comprising thiazole template 28 and

found that it is a new anti-bacterial agent and an excellent inhibitor of FabH. From

docking studies and biological screening it was found that these analog not only

excellent inhibitor against E.coli FabH but also exhibited promising inhibitory potential

against other five strains of bacteria [26]. Dixit et al., prepared quinazolinone comprising

thiazole analog 29 and evaluated against anti-fungal and anti-bacterial activities.

Results exhibited that analogs showed similar activity to the standard drugs [27].

Desai et al., prepared 1,3,4-oxadiazole containing thiazole moiety 30 and screened for

anti-bacterial activity. Analog having floro or nitro moiety at para position of phenyl

rings exhibited many fold greater anti-bacterial activity than the standard drug

chloramphenicol. On the other side, analog with para methoxy group of phenyl ring

found potent anti-fungal than the standard drug ketoconazole [28].

9

Mohammad et al., synthesized phenyl-thiazole analogs 31-33 and evaluated against

different strains of anti-bacteria and found that all these analogs were active against

vancomycin- and methicillin-resistant staphylococcus aureus [29].

Figure-1.7: Thiazole containing analogs as anti-bacterial agents

Bizzarri and his co-workers synthesized 2,4-disubstituted thiazole schiff bases 34 and

evaluated against twenty pathogenic candida spp for their anti-fungal inhibitory

potential. The most prominent activities were shown by the derivatives which contain

thiophene, coumarin, naphthalene, pyridine analogs against candida glabrata and

candida albicans that were most potent than the standard drugs clotrimazole and

fluconazole [30].

Figure-1.8: Thiazole containing analogs as anti-fungal agents

10

1.3.2 Anti-tubercular Agents

Currently the rates of mycobacterium tuberculosis infections are growing very rapid

because of HIV pandemic and poverty. The main problems are the disorganization of

conservative abti-tubercular medications and multi-drugs resistant to Mycobacterium

tuberculosis [31-33]. Various thiazoles containing analogs exhibited encouraging anti-

tubercular activities.

Shiradkarand et al., prepared 1,2,4-triazole based thiazole analog 35 and screened for

anti-tubercular inhibitory potential against mycobacterium tuberculosis. In the first

level of screening all the analogs were found active while two analogs comprising

benzamido and acetamido at carbon-2 of thiazoles and 3-nitro-benzamido group at N

position of triazole moiety were found potent anti-tubercular agent [34]. Makam et al.,

prepared pyridine-2-yl based thiazole analog 36 and evaluated against mycobacterium

tuberculosis H37Rv and found that it exhibited potent inhibitory potential [35].

Jeankumar et al., testified various piperidine based phenyl-thiazole analog 37 for

mycobacterium DNA gyrase enzyme inhibitors. Results showed that such type of

analog exhibited encouraging inhibitory potential against mycobacterium tuberculosis

DNA gyrase [36]. Jeong et al., synthesized thiazole 38 and thiazolidinone 39 analogs and

screened against mycobacterium tuberculosis H37Rv. Among the testified analogs para-

floro and methoxy phenyl analogs of thiazole moiety and para-chloro and bromo-

phenyl derivatives of thiazolidinone showed many fold better result than the standard

drug rifampicin [37].

11

Figure-1.9: Thiazole containing analogs as anti-tubercular agents

1.3.3 Anti-viral Agents

Annually countless people are died from viral infections which are deliberated one of

the most hazardous and public diseases. Moreover, current advances in the field of anti-

viral medicines, there is alternative requirements to determine efficient and effective

medicines so far. In this way various thiazole containing analogs were prepared and

screened against anti-viral agents and several of them are described below.

Numerous 4,5-dihydropyrazole analogs of thiazole were prepared and evaluated them

for anti-viral infections against various type of virusis by cytopathicity test. Results

showed that all analogs not show any important inhibition. But thiazolone analog 40

was found the most potent against vesicular stomatitis virus [38]. Many thiazolyl-

oxazole analogs were prepared and evaluated them for their inhibitory potential against

12

influenza A virus. Among the prepared analogs, only two analogs 41 and 42 showed

weak inhibitory potential because they containing free carboxylic acid [39].

Liu et al., prepared some thiazolidine substituted derivatives and screened them against

neuramindase of influenza virus. Results showed that various analogs have weak to

good inhibitory potentials. Predominantly, methoxy-phenolic analog 43 which

comprising N-(2-aminoacetyl)- moiety was found the excellent inhibitor of NA which is

many fold better than the standard drug oseltamviir [40]. Li et al., prepared and

evaluated against flavi-virus envelop proteins in order to determined anti-viral agents.

Flavi-virus anvelop proteins show significant part in the assembly of virus, contagion

host cells and morphogenesis. N-(3,4-dichlorobenzyl)- analog 44 among the testified

analogs were found potent against the said viruses [41].

Figure-1.10: Thiazole containing analogs as anti-viral agents

Stachulski and his co-workers prepared numerous 2-hydroxyaryl-N-(thiazole-2-yl)

amides 45 and tested against anti-viral inhibitory potential. Results showed that these

13

analogs were found active and selective inhibitore of hepatitis B replication. These

derivatives were deliberated as a new compound with wide range of anti-infective

agents [42]. Cureli and his co-workers prepared thiazole comprising oxalamide analog

as anti-HIV-1 agent. From the structure activity relationship, it was concluded that

hydroxyethyl- and hydroxymethyl-thiazole analogs 46 and 47 showed potent inhibitory

potential against the HIV-1 [43].

Figure-1.11: Thiazole containing analogs as anti-viral agents

1.3.4 Anti-cancer Agents

Now-a-days, in world one of the most popular reason of death is cancer and

widespread efforts had been made to discover new anti-cancer agent. Presently

available anti-cancer medicines have toxicity, drug interaction effect and resistance, due

to which immediately a new anti-cancer drug is needed [44, 45].

Aliabad and his co-workers prepared 2-phenylthiazole-4-carboxamide 48 and screened

against three types of human cancer cells line. From the results, it was concluded that

analog with 2-methoxy substituent showed greater inhibitory potential against T47D

and HT-29 while analog with 4-methoxy substituent exhibited greatest inhibitory

potential against caco-2 cell [46]. Farani et al., prepared 2-phenylthiazole-4-carboxamide

14

49 and evaluated for human cancer cells line. From the toxicity results it was conclude

that analogs which have 4-nitro and 3-chloro employed the greatest inhibitory potential

towards Hep-G2 and SKNMC cell [47]. Zaharia et al., synthesized 4-toluenesulfonyl-

hydrazinothiazole analog 50 and testified against hepatocarcinoma and prostate cancer

cell line. From the results it was concluded that when phenyl, methyl and acetyl

substituents attached to 4-toluenesulfhonyl-hydrazinothiazole and N,N-

diacetylhydrazinothiazole analog with CH3 substituent showed important anti-cancer

inhibitory potential [48]. Numerous analogs of 2-anilino-4-aryl-1,3-thiazole 51 were

synthesized and screened as valosin-containing protein inhibitors. From results it was

determined that 2-anilino-4-(4-chlorophenyl)-1,3-thiazole analog in which R1= 4-NH2

and 4-OH found the most potent one [49]. Wilson et al., prepared carboxamidine analog

52 and studied as urokinase inhibitor. The greater effectiveness of this analog may be

due to the phenoxy group [50].

2-alkylthio-4-(2,3,4-trimethoxyphenyl)-5-arylthiazole 53 were prepared and examined

against MCF-7, AGS and HT-29 for their proliferation inhibitory potential. 2-benzylthio

analog comprising 4-chloro, 4-floro and 4-nitro substituents perceived good summary

of cytotoxicity [51].

15

Figure-1.12: Thiazole containing analogs as anti-cancer agents

Emami et al., prepared 3-(trimethoxyphenyl)-2(3H)-thiazole thiones 54 and screened

against combretastine A-4. Cytotoxic activity screening of analog exhibited that analog

containing 4-methyl group had greatest inhibitory potential against all types of cells line

deprived important toxicity towards non-tumor cell [52]. Series of diarylthiazole-2-(3H)-

ones analog 55 and their imine analog 56 were prepared and screened as tubuline

polymeriization inhibitor against different types of human cancer cell line comprising

human breast cancer cell, human colon adenocarcinoma, human pancreatic cancer cells,

human liver cancer cell. The 3,4,5-trimethoxyphenyl analog containing R1 = 3-NH2,

MeO, R2 = 3,4,5-(MeO)3] and R3 = Cl or H exhibited excellent cytotoxicity activity

against non-solid human CEM cell lines [53]. Shi et al., described anti-cancer inhibitory

16

potential of arylaminothiazole analog 57. MTT experiment showed that analogs with

3,4-Cl2, 2-CH3 or CH3 substituents at R1 and 3-MeO, H,3,4,5-(MeO)3 at R2 exhibited

greatest anti-cancer inhibitory potential [54].


Bis-2-(2-hydroxyphenyl)-thiazole-4-carboxamides 58 and thio-carboxamides 59 were

prepared and evaluated for anti-cancer inhibitory potential against several of cell lines.

Results showed that methyl ester of thio-carboxamide derivative exhibited good anti-

proliferative/anti-cancer inhibitory potential than the reference drug deferoxamine [55].

Shafiee et al., prepared azolidinones analog 60 comprising hydantion or rhodanine

thiazoldine-2,4-dione skeletons in search of new chemotherapeutic agents. MTT

experiment exposed that thiazolidine-2,4-dione analog was found the most potent

against lymphoblastic leukemia cell [56].

17


1.3.5 Anti-convulsant Agents

Epilepsy is a crucial disease described by unusual and too much release of neurons [57].

Anti-convulsant medicines which commercially exist can controls the attacks in only

less than 70% of patients. Adverse effects such as ataxia, anemia, headache and

ineffectiveness of medicines exhibit the necessity for exploration of new anti-epileptic

medicines with lower toxicity and more selectivity [58, 59].

Siddique and his co-workers prepared various 3-(1, 3-thiazole-2-yl-amino)-4,5-di-hydro-

1H-1,2,4-triazole-5-thiones analog 61 and evaluated for anti-convulsant inhibitory

potential. Among the testified analogs, 3-[4-(4-bromo-phenyl)-1, 3-thiazole-2-yl-amino]-

4-(2-methyl-phenyl)-4,5-dihydro-1H-1,2,4-triuazole-5-thiones exhibited outstanding

inhibitory potential in both models. The analog also exhibited complex lipophilicity and

minor neurotoxicity, consequently smaller duration of action and a greater protective

18

index was estimated [60]. Emami et al., prepared a series of substituted azoles 62 (a, b)

and evaluated against anti-convulsant activity. Both analogs exhibited excellent anti-

convulsant activity. The SAR also showed good defense [61]. Rahman et al., prepared

coumarine based analogs holding thiazolone, thiazoles and thiazolidin-4-one and

screened for their anti-convulsant inhibitory potential against seizures by comparing

with the standard drug phenobarbital. Results show that thiazole analog 63a and

thiazolone-5-carboxylic acid ethyl aster 63b were the most potent derivatives.

Thiazolone analogs were most potent than thiazolidinone analog and this may be

attributed due to methyl and carboxylic acids esters in thiazolone unit which increase

inhibition [62]

Figure-1.15: Thiazole containing analogs as anti-convulsant agents

19

1.3.6 Anti-inflammatory agents

Auto-immune illness, elimination of transplanted organs and allergy causes

inflammation. Kulkarni et al., described 64 comprising cabostyril and coumarin with

anti-inflammatory potential. The derivatives have been testifying for their in vivo anti-

inflammatory and analgesic activities by comparing to the standard drug phenyl

butazone [63]. New series of 3-(4-chlorophenyl)-2-(4-methyl-2-(methyl-amino)thiazol-5-

yl)quinazolin-4-(3H)-one analogs 65 were prepared and screened for their anti-

inflammatory inhibitory potential. The testified analog exhibited favorable inhibitory

potential for inflammation [64]. Kumar et al., prepared and screened 3-thiazolyl

quinazolin-4-ones 66 against analgesic, ulcer-genic and anti-inflammatory inhibitory

potentials. They described 2-chloro phenyl analog at a dosage of 25 and 50 mg/kg

showed anti-inflammatory inhibitory potential equal to the standard drug phenyl

butazone while at a dosage of 100 mg/kg showed superior anti-inflammatory

inhibitory potential that the standard drug [65].

Figure-1.16: Thiazole containing analogs as anti-inflammatory agents

Aggerwal et al., prepared coumarin-thiazoles analogs 67 (a, b) and screened against

anti-inflammatory activity. All the testify analogs exhibited fast acting anti-

20

inflammatory inhibitory potentials. Furthermore 3,5-bis-tri-fluoromethyl analogs

possess greatest anti-inflammatory inhibitory potential. The presence of tri-floromethyl

moiety in pyrazoline ring improved the anti-inflammatory inhibitory potential [66].

Helal et al., planned, prepared various series of morpholino-phenylthiazoles and

screened against anti-inflammatory activity. Among these analogs, thiazole-2-amine 68

exhibited excellent inhibition of carrageenan induce inflammation in rat paw that was

analogous to the standard indomethacin [67]. Various N-(4-aryl-1,3-thiazole-2-yl)

acetamides 69 were prepared and screened against anti-inflammatory activity. Results

showed that analogs with phenyl and ortho- methoxy phenyl showed greater IL-1β

inhibitory potential related to the standard drug piroxicam. Moreover, analogs with

ortho or para-floro substituents showed superior MCP-1 inhibitory potential as

compared to the standard drug pioglitazone [68].

Figure-1.17: Thiazole containing analogs as anti-inflammatory agents

21

1.3.7 Anti-oxidant agents

Yadla et al., using DPPH procedure to study the anti-oxidant inhibitory potential of

thiazole based thiazolidinone analogs. Their report showed that para-bromo analog 70

exhibited excellent anti-oxidant potential as compared to the standard drug ascorbic

acid [69]. In order to discover new anti-oxidant agents, some pyrazoline analogs were

prepared for free radical scavenging and xanthine oxidase inhibitory potentials. Among

the prepared analogs, analog 71 comprising 2-(benzyl-thio)-5-chloro-thiophen-3-yl

group show greatest free radical scavenging and xanthine oxidase inhibitory potentials

when compared to the standard drug ascorbic acid and allopurinol [70]. To discover

new therapeutic for neurodegenerative illnesses, kim et al., planned unique anti-

oxidants 72-74 comprising N-t-butyl-N-hydroxyl-amino-phenyl group. N-t-butyl-N-

hydroxyl-amino-phenyl skeleton might have potent anti-oxidant inhibitory potential.

[71]. some series of thiazole modified analogs 75 and 76 comprising hydrazine

imidazolyl group was screened against anti-oxidant activity. Among the prepared

analogs, 4-florobenzylidene-thiazolidin-4-one analog was the most active analog [72].

22

Figure-1.18: Thiazole containing analogs as anti-oxidant agents

1.3.8 Anti-diabetic agents

Diabetes is a metabolic syndrome and has affected 6.4% of world population by insulin

resistance and dyslipidemia [73, 74].

Different analogs were designed to the treatment of diabetes and some of them possess

good potential. Controlling high level of insulin and triglycerides are associated with

enzyme stearoyl-CoA desaturase-1 (SCD1). Compounds which inhibit SCD1 are the

better option for the treatment of diabetes [75]. Numerous thiazole carboxamide

derivatives were planned, prepared and tested as SCD1 inhibitors. The substituted

amide analog 77 showed the greatest effectiveness in the whole cell assay against SCD1

[76]. Kang et al., designated benzyl thiazole analogs 78 as sodium glucose co-transporter

23

inhibitors. Result showed that nearly all analogs exhibited good inhibitory potential.

But 4-ethyl benzyl-5-chloro-thiazole analog showed the most active activity [77].

Figure-1.19: Thiazole containing analogs as anti-diabetic agents

1.3.9 Anti-Alzheimer’s Agents

Alzheimer‟s is associated with neuro-degeneration, abnormal behaviors and memory

loss. Raza et al., have been planned 3-thiozolo-coumarinyl Schiff base analogs 79 (a, b)

as anti-Alzheimer‟s agents. All the newly prepared thiazole analogs were active cheap

inhibitors of AChE and BuChE. Analog having bromo group on phenyl ring was the

most active compound against AChE while on the other hand analog having benzyloxy

group at 2-postion of the phenyl ring exhibited superior inhibition against BuChE [78].

Thiazole diamide analogs 80 were screened as γ-secretase inhibitors to depressed

Alzheimer‟s development. Results showed that the long side chain at 5-position of

thiazole ring had a vital role in whole cell effectiveness. Nearly all analogs showed

potent γ-secretase inhibitory potential [79]. Shiradkar et al., designated the preparation

of tri-azolylthiazole analogs as new cyclic dependent kinase for the cure of Alzheimer‟s

disease. The inhibitory potential of the analogs were screened in scintillation proximity

assay (SPA). The two 4-chlorobenzamide analogs 81 comprising chloro-acetylamino or

24

acetyl-amino substituents on 2-position of the thiazole ring possess the greatest

potential [80].

Figure-1.20: Thiazole containing analogs as anti-Alzheimer’s agents

1.4 Previous synthetic approaches towards thiazole

1.4.1 Synthesis of thiazole analogs by Hantzsch method

Thionicotinamide (82) was treated with chloro acetone, 3-chloroacetylacetone, p-

chloroacetyl-acetanilide and 3-bromo-acetylcoumarin using Et3N in ethanol to give us

2-(3-pyridyl) thiazole analogs (83-86) [81].

25

Scheme-1.1: Synthesis of thiazole analogs by Hantzsch method

1.4.2 Thiazole analogs synthesis by acylation process

Thiourea was treated with Phenacyl bromide (87a, b) in ethanol to give us 4-(4-

bromophenyl)thiazole-2-amine and 4-phenylthiazole-2-amine analogs (88a, b)

respectively, which was then acylated with naphthoic acid and phenyl acetic acid in

CH2Cl2, EDC in the presence of HOBt to give us final analogs (89a-h, 89j-k, 90a-h, 90j-

k). Moreover derivatives (88 a, b) were treated with benzoyl chloride or 4-

morpholincarbonyl chloride in pyridine to give us analogs (89i, 89l, 90i and 90l) [82].

26

Scheme-1.2: Synthesis of thiazole analogs by acylation process

1.4.3 Synthesis of amino-thiazole analogs:

1-(4-nitrophenyl)ethan-1-one 91 was treated with bromine in chloroform to give us 2-

bromo-1-(4-nitrophenyl)ethan-1-one 92, which was then treated with thiourea in

ethanol to give us cyclic product 2-amino-5-(4-nitrophenyl)thiazole 93. Lastly, the

analog 93 was reduced to diamine using Pd/C to give us the final product 2-(4-

aminophenyl) thiazol-5-amine 94 [83].

27

Scheme-1.3: Synthesis of amino-thiazoles analogs

1.4.4 Synthesis of Allylic thiazoles analogs:

Aromatic amine 95 was treated with allyl isothiocyanates 96 which give us N-allyl-N՜-

substituted thiourea (97 a-h), which was then treated with Phenacyl bromide in the

presence of Et3N to give us allyl-3H-thiazole (98 a-h) [84].

Scheme-1.4: Synthesis of allylic thiazole derivatives

28

1.5 Results and discussion

1.5.1 Chemistry

Methyl 2-(2,5-dimethylthiazol-4-yl)acetate (99) was mixed with excess of hydrazine

hydrate in methanol and refluxed for 4 hrs to give 2-(2,5-dimethylthiazol-4-

yl)acetohydrazide (100). Intermediate (100) was then treated with different substituted

aldehyde in methanol to give thiazole bearing Schiff bases (101), which was then

subjected through oxidative cyclization using phenyliododiacetate (PhI(OAc)2) in

dichloromethane to form thiazole bearing oxadiazole analogs (102-117).

Scheme-1.5: Synthesis of thiazole derivatives (102-117)

29

Table-1.1: Different substituents and thymidine phosphorylase activity of thiazole

analogs (102-117)

S. No R IC50 Values S. No R IC50 Values

102

37.70 ± 0.01 110

47.10 ± 0.01

103

33.80 ± 0.01 111

34.10 ± 0.01

104

35.30 ± 0.01 112

39.70 ± 0.02

105

54.10 ± 0.02 113

40.10 ± 0.02

106

32.40 ± 0.01 114

37.90 ± 0.01

107

37.10 ± 0.05 115

39.10 ± 0.01

108

42.20 ± 0.05 116

38.30 ± 0.03

30

109

36.10 ± 0.03 117

51.10 ± 0.01

St Drug 7-Deazaxanthined 38.68 ± 1.12 μM

1.5.2 Biological activity

We have synthesized sixteen thiazole bearing oxadiazole analogs (102-117) and

evaluated for thymidine phosphorylase inhibitory potential. All analogs showed a

varied degree of thymidine phosphorylase inhibitory potential with IC50 values ranging

between 32.40 ± 0.01 to 54.10 ± 0.02 μM when compared with standard drug 7-

Deazaxanthine (IC50 = 38.68 ± 1.12 μM). The structure activity relationship was mainly

based upon by bring about difference of substituent on phenyl ring.

The most potent analog among the series is analog 106 (IC50 = 32.40 ± 0.01 μM) having

di-methyl amine group on phenyl ring. The greater potential of this analog is mainly

seems to be due to di-methyl amine group which is electron donating group.

Figure-1.21: Structure of the potent analog 106

If we compare analog 102 (IC50 = 37.70 ± 0.01 μM) with analog 103 (IC50 = 33.80 ± 0.01

μM), both the analogs have nitro group on phenyl ring but position of nitro group is

31

different. The difference in the activity may be due to different position of substituents

on phenyl ring.

Figure-1.22: Comparison of structure activity relationship between 102 and 103

By comparing analog 114 (IC50 = 37.90 ± 0.01 μM) with analog 115 (IC50 = 39.10 ± 0.01

μM) and analog 116 (IC50 = 38.30 ± 0.03 μM), all three analogs have cyano group on

phenyl ring but position of substituent is different. The difference in activity is may be

due to different position of substituents on phenyl ring.

Figure-1.23: Comparison of structure activity relationship between 114, 115 and 116

Electron donating group (EDG) and electron withdrawing group (EWG) greatly affects

potential while positions and numbers of substituents also play a vital role in increasing

the potential. Molecular docking study was carried out to find the potent molecule.

1.5.3 Molecular docking

Interaction between inhibitor and target is best explored by molecular docking study.

To explore the interaction between inhibitor and active site of thymidine

32

phosphorylase, MOE-Dock program was used. Protein Databank (PDB) was applied to

retrieve the 3D structure. The synthesized compounds were docked in the enzyme

active sites by default parmeters i-e Placement: Triangle Matcher, Rescoring 1: London

dG, Refinement: Force field, Rescoring 2: London dG. Ten conformations were recorded

for each ligand and the best one was selected based on docking score [85].

1.5.4 Docking Study

The docking results of thiazole series with the thymidine phosphorylase enzyme have

given good information about the nature of the binding mode that was excellent

correlated with the experimental results. Docking study observed that best confirmation

of all compounds well accommodate inside active site of enzyme and involved different

interactions with enzyme i.e., His 85, Tyr 168, Arg 171, Ile 183, Ser 186, Lys 190, Phe 210

and Met 211 etc. docking score and interactions are listed in the Table-1.2. Compound

106 with docking scores -13.5480 show good potential against thymidine phosphorylase

enzyme. This indicates the best fitness of compound inside the enzyme cavity.

Compound 103 showed good inhibitory potential. Compound 106 showed some

interation with enzyme in different pattern His 85, Tyr 168, Ar 171, Ile 183, Ser 186, Lys

190, Phe 210 and Met 211 (Figure-1.24a). Ser 186 formed H-bond with the -NH of the

2,5-dimethylthiazole moiety while Arg 171 made H-bond with the nitrogen atom of the

2,5-dimethyl-1,3,4-oxadiazole moiety of the compound. Met 211 was observed making

H-donor interaction with the N, N-dimethyl aniline moiety of the ligand. His 85, Tyr

168, Ile 183, Lys 190 and Phe 210 active residues formed H-pi linkages with the

compound as shown in Figure-1.24a. The electron cloud system of the compound 106

33

greatly affects the potency of the compound. In case of compound 103, His 85 residue

formed polar interaction with the “O” atoms of the nitro group of the analog. Arg 171

and Ser 186 residues formed H-acceptor and H-donor interactions with the nitrogen

atoms of the 1,3,4-oxadiazole and 2,4,5-trimethylthiazole moieties of the compound

respectively. Tyr 168 and Phe 210 formed H-pi contacts with the carbon atoms and

benzene ring of the compound as shown in Figure-1.24b. The best potential of the

analog is might be due to the electron withdrawing effect of nitro group. Good

relationship was perceived between active site and docking scores.

Figure-1.24: Docking conformations of compounds a) analog 106 and b) analog 103 on

thymidine phosphorylase enzyme

34

Figure-1.25: Correlation graph for IC50 values and docking predicted activity


S.NO Docking

Scores

Interaction Report

102 -9.1298 Ligand Receptor Interaction Distance E

(kcal/mol)

N 3 OE1 GLN 156 (A) H-donor 2.67 -4.2

C 15 O SER 113 (A) H-donor 3.23 -0.5

N 20 NZ LYS 190 (A) H-acceptor 3.11 -1.0

O 35 NH1 ARG 171 (A) H-acceptor 2.85 -5.5


35

6-ring CG2 ILE 183 (A) pi-H 4.42 -0.5

103 -13.5240 N 3 O SER 186 (A) H-donor 1.71 -5.6

N 20 NH2 ARG 171 (A) H-acceptor 2.30 -1.0

O 35 NE2 HIS 85 (A) H-acceptor 2.44 -1.1

C 11 6-ring TYR 168 (A) H-pi 4.30 -0.3

C 15 6-ring TYR 168 (A) H-pi 3.62 -0.6

C 26 6-ring PHE 210 (A) H-pi 3.81 -0.3

104 -10.5803 S 1 O SER 113 (A) H-donor 3.26 -0.7

N 3 O SER 186 (A) H-donor 2.73 -7.4


C 15 6-ring TYR 168 (A) H-pi 3.96 -1.0

5-ring CG1 ILE 183 (A) pi-H 4.10 -0.5

5-ring CG2 ILE 183 (A) pi-H 3.99 -0.9

105 -6.8903 N 20 NH2 ARG 171 (A) H-acceptor 3.08 -4.4

C 29 6-ring PHE 210 (A) H-pi 4.19 -0.8

5-ring CA GLY 114 (A) pi-H 4.77 -1.0

106 -13.5480 N 3 O SER 186 (A) H-donor 1.75 -6.3

C 34 SD MET 211 (A) H-donor 3.64 -0.4


C 7 5-ring HIS 85 (A) H-pi 3.56 -0.1

C 15 6-ring TYR 168 (A) H-pi 3.61 -0.1

36

C 38 6-ring PHE 210 (A) H-pi 4.18 -0.1

6-ring CG2 ILE 183 (A) pi-H 4.51 -0.1

5-ring CE LYS 190 (A) pi-H 4.42 -0.2

107 -9.1003 N 19 CB SER 186 (A) H-acceptor 3.16 -1.1




O 31 NH1 ARG 171 (A) ionic 2.64 -7.4

O 31 NH2 ARG 171 (A) ionic 2.75 -6.4

5-ring CG2 ILE 183 (A) pi-H 4.09 -0.5

108 -7.9869 S 1 OG SER 186 (A) H-donor 4.36 -0.6

C 27 SD MET 211 (A) H-donor 3.21 -0.6

N 19 NE2 HIS 85 (A) H-acceptor 2.99 -2.6

6-ring 6-ring PHE 210 (A) pi-pi 3.69 -0.0

109 -9.4287 N 3 OG SER 186 (A) H-donor 2.79 -5.2

N 20 NE2 HIS 85 (A) H-acceptor 3.16 -4.4

5-ring 6-ring TYR 168 (A) pi-pi 3.76 -0.0

110 -7.5438 S 1 O SER 86 (A) H-donor 3.20 -1.0



111 -10.3428 S 1 O GLN 372 (A) H-donor 3.52 -0.6

37

N 3 OD1 ASP 172 (A) H-donor 2.66 -5.1

C 15 OD2 ASP 172 (A) H-donor 3.15 -0.5

N 33 O VAL 177 (A) H-donor 3.22 -1.4

N 33 OD1 ASP 178 (A) H-donor 2.86 -4.2

N 19 OH TYR 168 (A) H-acceptor 2.93 -0.6

N 20 OH TYR 168 (A) H-acceptor 3.16 -0.6

5-ring CD1 LEU 117 (A) pi-H 3.73 -1.0

5-ring CD2 LEU 117 (A) pi-H 3.50 -0.7


112 -8.2318 C 26 6-ring TYR 168 (A) H-pi 3.53 -0.6


113 -8.0013 5-ring N GLN 156 (A) pi-H 4.65 -0.7


114 -12.7621 N 34 NE2 HIS 85 (A) H-acceptor 2.72 -4.7

5-ring NZ LYS 190 (A) pi-cation 3.94 -8.6


115 -8.5409 N 3 O SER 113 (A) H-donor 2.91 -1.0

N 34 OG SER 95 (A) H-acceptor 2.77 -1.9

N 34 OG1 THR 123 (A) H-acceptor 3.0 -1.1

5-ring CE1 HIS 85 (A) pi-H 3.32 -1.0

6-ring CB SER 86 (A) pi-H 4.71 -0.6

38

116 -8.7865 N 19 NZ LYS 190 (A) H-acceptor 3.17 -7.2


117 -7.1290 S 1 O TYR 168 (A) H-donor 3.53 -1.1

C 7 O TYR 168 (A) H-donor 3.37 -0.8



Standard

7-

Deazaxanthine

-8.9439 N 1 OG SER 186 (A) H-donor 3.18 -2.3

O 7 NZ LYS 190 (A) H-acceptor 2.61 -10.4



6-ring NE2 HIS 85 (A) pi-H 4.64 -1.9

1.6 Conclusion

We have synthesized sixteen thiazole bearing oxadiazole analogs (102-117) and

evaluated for thymidine phosphorylase inhibitory potential. All analogs showed a

varied degree of thymidine phosphorylase inhibitory potential with IC50 values ranging

between 32.40 ± 0.01 to 54.10 ± 0.02 μM when compared with standard drug 7-

Deazaxanthine (IC50 = 38.68 ± 1.12 μM). Molecular docking study was carried out to

find potent molecule. The structure activity relationship was mainly based upon by

bring about difference of substituent on phenyl ring.

39

1.7 Material and methods

1.7.1 General procedure for synthesis of thiazole

Methyl 2-(2,5-dimethylthiazol-4-yl)acetate (5 mmol) was mixed with 20 mL of

hydrazine hydrate in methanol (15 mL) and refluxed for 4 hrs to give 2-(2,5-

dimethylthiazol-4-yl)acetohydrazide. Intermediate was then treated with different

substituted aldehydes (1 mmol) in methanol (15 mL) to give thiazole bearing Schiff

bases, which was then subjected through oxidative cyclization using

phenyliododiacetate (PhI(OAc)2) in dichloromethane to form thiazole bearing

oxadiazole analogs (102-117).


Yield: 86%. Yellow solid, m.p. 184-186 °C. 1HNMR (500 MHz, DMSO-d6): δ 8.39 (dd, J =

8.2, 1.3 Hz, 2H, H-3/5), 8.20 (dd, J = 8.0, 1.2 Hz, 2H, H-2/6), 3.79 (s, 2H, -CH2-), 2.75 (s,

3H, -CH3), 2.28 (s, 3H, -CH3). 13CNMR (125 MHz, DMSO-d6): δ 166.2, 164.3, 159.2, 150.2,

147.8, 132.1, 130.7, 130.7, 128.6, 128.6, 122.4, 27.7, 19.5, 10.7. HREI-MS: m/z calcd for

C14H12N4O3S, [M]+ 316.0630; Found: 316.0628.


Yield: 84%. Pale yellow solid, m.p. 190-192 °C. 1HNMR (500 MHz, DMSO-d6): δ 8.01 (dd,

J = 8.0, 1.3 Hz, 1H, H-6), 7.98 (dd, J = 8.3, 1.4 Hz, 1H, H-3), 7.88 (m, 1H, H-5), 7.70 (m,

1H, H-4), 3.78 (s, 2H, -CH2-), 2.74 (s, 3H, -CH3), 2.27 (s, 3H, -CH3). 13CNMR (125 MHz,

DMSO-d6): δ 166.2, 164.3, 159.2, 150.2, 146.7, 135.1, 131.5, 129.4, 128.3, 124.3, 122.3, 27.6,

19.4, 10.7. HREI-MS: m/z calcd for C14H12N4O3S, [M]+ 316.0630; Found: 316.0627.

40

1.7.1.3 2-(2,4-dichlorophenyl)-5-((2,5-dimethylthiazol-4-yl)methyl)-1,3,4-oxadiazole

(104)

Yield: 80%. Light white solid, m.p. 177-179 °C. 1HNMR (500 MHz, DMSO-d6): δ 7.67 (s,

1H, H-3), 7.62 (dd, J = 8.2, 8.1 Hz, 1H, H-6), 7.40 (dd, J = 8.4, 8.0 Hz, 1H, H-5), 3.76 (s, 2H,

-CH2-), 2.73 (s, 3H, -CH3), 2.25 (s, 3H, -CH3). 13CNMR (125 MHz, DMSO-d6): δ 166.2,

164.2, 159.2, 150.2, 135.5, 135.0, 133.4, 130.7, 130.2, 127.2, 122.4, 27.6, 19.3, 10.7. HREI-MS:

m/z calcd for C14H11Cl2N3OS, [M]+ 339.0000; Found: 339.0000.

1.7.1.4 2-(4-(benzyloxy)phenyl)-5-((2,5-dimethylthiazol-4-yl)methyl)-1,3,4-oxadiazole

(105)

Yield: 78%. Light yellow solid, m.p. 173-175 °C. 1HNMR (500 MHz, DMSO-d6): δ 8.00

(dd, J = 8.1, 1.0 Hz, 2H, H-2/6), 7.45 (dd, J = 8.0, 1.3 Hz, 2H, H-2՜/6՜), 7.38 (m, 2H, H-

3՜/5՜), 7.30 (m, 1H, H-4՜), 7.01 (dd, J = 7.8, 1.2 Hz, 2H, H-3/5), 5.14 (s, 2H, -CH2-O-), 3.74

(s, 2H, -CH2-), 2.72 (s, 3H, -CH3), 2.26 (s, 3H, -CH3). 13CNMR (125 MHz, DMSO-d6): δ

166.2, 164.3, 159.2, 158.9, 150.2, 136.6, 128.8, 128.8, 127.5, 127.0, 127.0, 122.2, 118.3, 115.7,

115.7, 114.7, 114.7, 70.6, 27.7, 19.5, 10.7. HREI-MS: m/z calcd for C21H19N3O2S, [M]+

377.1198; Found: 377.1195.

1.7.1.5 4-(5-((2,5-dimethylthiazol-4-yl)methyl)-1,3,4-oxadiazol-2-yl)-N,N-

dimethylaniline (106)

Yield: 80%. Yellowish solid, m.p. 170-172 °C. 1HNMR (500 MHz, DMSO-d6): δ 7.56 (dd, J

= 7.8, 1.2 Hz, 2H, H-2/6), 6.91 (dd, J = 7.7, 1.0 Hz, 2H, H-3/5), 3.75 (s, 2H, -CH2-), 3.01 (s,

6H, -CH3), 2.75 (s, 3H, -CH3), 2.22 (s, 3H, -CH3). 13CNMR (125 MHz, DMSO-d6): δ 166.2,

41

164.3, 159.2, 155.2, 150.2, 128.1, 128.1, 122.3, 115.3, 112.6, 112.6, 41.2, 41.2, 27.6, 19.4, 10.7.

HREI-MS: m/z calcd for C16H18N4OS, [M]+ 314.1201; Found: 314.1200.

1.7.1.6 2,4-dichloro-6-(5-((2,5-dimethylthiazol-4-yl)methyl)-1,3,4-oxadiazol-2-yl)phenol

(107)

Yield: 81%. White yellow solid, m.p. 163-165 °C. 1HNMR (500 MHz, DMSO-d6): δ 9.55 (s,

1H, -OH), 7.65 (d, J = 1.4 Hz, 1H, H-6), 7.40 (d, J = 1.3 Hz, 1H, H-4), 3.78 (s, 2H, -CH2-),

2.71 (s, 3H, -CH3), 2.29 (s, 3H, -CH3). 13CNMR (125 MHz, DMSO-d6): δ 166.2, 164.3, 159.2,

154.1, 150.2, 131.5, 127.3, 126.8, 126.0, 122.4, 114.8, 27.7, 19.5, 10.7. HREI-MS: m/z calcd

for C14H11Cl2N3O2S, [M]+ 354.9949; Found: 354.9947.

1.7.1.7 2-(5-bromo-2-methoxyphenyl)-5-((2,5-dimethylthiazol-4-yl)methyl)-1,3,4-

oxadiazole (108)

Yield: 75%. Greenish solid, m.p. 160-162 °C. 1HNMR (500 MHz, DMSO-d6): δ 7.43 (d, J =

1.5 Hz, 1H, H-6), 7.22 (dd, J = 8.1, 1.4 Hz, 1H, H-4), 6.80 (d, J = 8.3 Hz, 1H, H-3), 3.72 (s,

2H, -CH2-), 3.77 (s, 3H, -CH3), 2.69 (s, 3H, -CH3), 2.23 (s, 3H, -CH3). 13CNMR (125 MHz,

DMSO-d6): δ 166.2, 164.3, 159.2, 156.1, 150.2, 134.0, 132.3, 122.3, 117.0, 114.3, 112.5, 56.0,

27.4, 19.3, 10.7. HREI-MS: m/z calcd for C15H14BrN3O2S, [M]+ 378.9990; Found: 378.9987.

1.7.1.8 2-(4-chlorophenyl)-5-((2,5-dimethylthiazol-4-yl)methyl)-1,3,4-oxadiazole (109)

Yield: 78%. Light green solid, m.p. 145-147 °C. 1HNMR (500 MHz, DMSO-d6): δ 7.70 (dd,

J = 8.0, 1.0 Hz, 2H, H-2/6), 7.51 (dd, J = 8.2, 1.4 Hz, 2H, H-3/5), 3.80 (s, 2H, -CH2-), 2.75

(s, 3H, -CH3), 2.24 (s, 3H, -CH3). 13CNMR (125 MHz, DMSO-d6): δ 166.2, 164.3, 159.2,

150.2, 134.0, 129.0, 129.0, 128.6, 128.6, 124.0, 122.1, 27.3, 19.2, 10.7. HREI-MS: m/z calcd

for C14H12ClN3OS, [M]+ 305.0390; Found: 305.0388.

42

1.7.1.9 2-(2,3-dimethoxyphenyl)-5-((2,5-dimethylthiazol-4-yl)methyl)-1,3,4-oxadiazole

(110)

Yield: 82%. Light yellow solid, m.p. 154-156 °C. 1HNMR (500 MHz, DMSO-d6): δ 7.44 (d,

J = 8.3 Hz, 1H, H-6), 7.10 (dd, J = 8.1, 8.3 Hz, 1H, H-5), 7.02 (d, J = 8.0 Hz, 1H, H-4), 3.82

(s, 6H, -CH3), 3.73 (s, 2H, -CH2-), 2.67 (s, 3H, -CH3), 2.21 (s, 3H, -CH3). 13CNMR (125

MHz, DMSO-d6): δ 166.2, 164.3, 159.2, 152.0, 150.2, 148.1, 130.0, 122.2, 122.2, 115.1, 111.4,

60.5, 56.0, 27.6, 19.2, 10.7. HREI-MS: m/z calcd for C16H17N3O3S, [M]+ 331.0991; Found:

331.0990.

1.7.1.10 2-(5-((2,5-dimethylthiazol-4-yl)methyl)-1,3,4-oxadiazol-2-yl)aniline (111)

Yield: 81%. Light pale yellow solid, m.p. 166-168 °C. 1HNMR (500 MHz, DMSO-d6): δ

7.60 (d, J = 8.0 Hz, 1H, H-6), 7.25 (m, 1H, H-4), 6.97 (d, J = 7.9 Hz, 1H, H-3), 6.90 (m, 1H,

H-5), 5.77 (s, 2H, -NH2), 3.77 (s, 2H, -CH2-), 2.65 (s, 3H, -CH3), 2.19 (s, 3H, -CH3).

13CNMR (125 MHz, DMSO-d6): δ 166.2, 164.3, 159.2, 150.2, 145.0, 129.3, 128.2, 123.0,

122.4, 119.1, 116.4, 27.1, 19.1, 10.7. HREI-MS: m/z calcd for C14H14N4OS, [M]+ 286.0888;

Found: 286.0885.

1.7.1.11 2-chloro-4-(5-((2,5-dimethylthiazol-4-yl)methyl)-1,3,4-oxadiazol-2-yl)phenol

(112)

Yield: 76%. White yellow solid, m.p. 161-163 °C. 1HNMR (500 MHz, DMSO-d6): δ 9.90 (s,

1H, -OH), 7.77 (d, J = 1.5 Hz, 1H, H-2), 7.70 (dd, J = 8.5, 1.3 Hz, 1H, H-6), 6.92 (d, J = 8.4

Hz, 1H, H-5), 3.70 (s, 2H, -CH2-), 2.68 (s, 3H, -CH3), 2.17 (s, 3H, -CH3). 13CNMR (125

MHz, DMSO-d6): δ 166.2, 164.3, 159.2, 153.3, 150.2, 128.4, 124.5, 122.3, 120.0, 117.7, 114.3,

27.3, 19.2, 10.7. HREI-MS: m/z calcd for C14H12ClN3O2S, [M]+ 321.0339; Found: 321.0337.

43

1.7.1.12 2-(5-((2,5-dimethylthiazol-4-yl)methyl)-1,3,4-oxadiazol-2-yl)-4-fluorophenol

(113)

Yield: 84%. Light pale yellow solid, m.p. 174-176 °C. 1HNMR (500 MHz, DMSO-d6): δ

9.60 (s, 1H, -OH), 7.33 (d, J = 1.1 Hz, 1H, H-6), 7.10 (d, J = 7.8 Hz, 1H, H-4), 6.95 (d, J =

7.9 Hz, 1H, H-3), 3.73 (s, 2H, -CH2-), 2.64 (s, 3H, -CH3), 2.15 (s, 3H, -CH3). 13CNMR (125

MHz, DMSO-d6): δ 166.2, 164.3, 159.2, 154.4, 153.0, 150.2, 122.4, 118.0, 117.1, 116.4, 113.3,

27.1, 19.0, 10.7. HREI-MS: m/z calcd for C14H12FN3O2S, [M]+ 305.0634; Found: 305.0633.


Yield: 80%. Yellowish solid, m.p. 162-164 °C. 1HNMR (500 MHz, DMSO-d6): δ 7.92 (d, J

= 8.2 Hz, 1H, H-6), 7.75 (d, J = 8.4 Hz, 1H, H-3), 7.75 (m, 1H, H-5), 7.70 (m, 1H, H-4), 3.76

(s, 2H, -CH2-), 2.62 (s, 3H, -CH3), 2.15 (s, 3H, -CH3). 13CNMR (125 MHz, DMSO-d6): δ

166.2, 164.3, 159.2, 150.2, 140.0, 133.1, 132.2, 129.3, 128.0, 122.4, 117.0, 104.0, 27.6, 19.3,

10.7. HREI-MS: m/z calcd for C15H12N4OS, [M]+ 296.0732; Found: 296.0730.


Yield: 74%. Light yellow solid, m.p. 170-172 °C. 1HNMR (500 MHz, DMSO-d6): δ 8.30

(dd, J = 8.3, 1.4 Hz, 1H, H-6), 8.00 (d, J = 8.1 Hz, 1H, H-4), 7.83 (d, J = 1.3 Hz, 1H, H-2),

7.70 (dd, 8.5, 8.4 Hz, 1H, H-5), 3.77 (s, 2H, -CH2-), 2.73 (s, 3H, -CH3), 2.21 (s, 3H, -CH3).

13CNMR (125 MHz, DMSO-d6): δ 166.2, 164.3, 159.2, 150.2, 132.1, 131.7, 130.4, 129.8,

126.7, 122.2, 118.4, 113.0, 27.5, 19.4, 10.7. HREI-MS: m/z calcd for C15H12N4OS, [M]+

296.0732; Found: 296.0730.

44


Yield: 73%. Dark yellow solid, m.p. 148-150 °C. 1HNMR (500 MHz, DMSO-d6): δ 7.93

(dd, J = 8.1, 1.1 Hz, 2H, H-2/6), 7.81 (dd, J = 8.0, 1.4 Hz, 2H, H-3/5), 3.73 (s, 2H, -CH2-),

2.73 (s, 3H, -CH3), 2.20 (s, 3H, -CH3).

13CNMR (125 MHz, DMSO-d6): δ 166.2, 164.3, 159.2, 150.2, 132.3, 132.3, 130.1, 128.0,

128.0, 122.1, 118.2, 112.5, 27.6, 19.2, 10.7. HREI-MS: m/z calcd for C15H12N4OS, [M]+

296.0732; Found: 296.0730.

1.7.1.16 2-(anthracen-9-yl)-5-((2,5-dimethylthiazol-4-yl)methyl)-1,3,4-oxadiazole (117)

Yield: 76%. Light yellow solid, m.p. 171-173 °C. 1HNMR (500 MHz, DMSO-d6): δ 8.53 (s,

1H, H-6), 8.19 (d, J = 8.4 Hz, 2H, H-2/10), 8.00 (d, J = 8.3 Hz, 2H, H-5/7), 7.48 (dd, J =

8.0, 8.1 Hz, 2H, H-4/8), 7.44 (dd, J = 8.4, 8.2 Hz, 2H, H-3/9), 3.70 (s, 2H, -CH2-), 2.70 (s,

3H, -CH3), 2.22 (s, 3H, -CH3).

13CNMR (125 MHz, DMSO-d6): δ 166.2, 164.3, 159.2, 150.2, 134.0, 132.0, 132.0, 130.3,

130.3, 129.6, 128.0, 128.0, 125.3, 125.3, 125.3, 125.3, 124.0, 124.0, 122.3, 27.5, 19.5, 10.7.

HREI-MS: m/z calcd for C22H17N3OS, [M]+ 371.1092; Found: 371.1090.

45

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51

CHAPTER-02

Synthesis of oxadiazole derivatives and their biological activity

Summary

………………………………………………………………………………………………………..

In this chapter we describe the synthesis of oxadiazole derivatives and study of their

thymidine phosphorylase with molecular docking.

………………………………………………………………………………………………………..

2.1 Oxadiazole

Oxadiazole (1) is an aromatic five membered heterocyclic molecule having one oxygen

and two nitrogen atoms [1].

Figure-2.1: Basic skeleton of oxadiazole

There are three known isomers of oxadiazole such as 1,2,3-oxadiazole (2), 1,2,4-

oxadiazole (3) and 1,2,5-oxadiazole (4). But 1, 2, 4-oxadiazole and 1, 3, 4-oxadiazole are

famous and most commonly studied by researches because of their numerous

significant biological and chemical characteristics.

Figure-2.2: Isomers of oxadiazole

Among aromatic heterocyclic derivatives, oxadiazole has become a significant structure

design for progression of new and novel drugs. Derivatives comprising of oxadiazole

moiety have wide-range of biological activities including antidiabetic [2], anti-fungal

52

[3], antibacterial [4], anticonvulsant [5], anti-cancer [6], analgesic [7], antiviral [8] and

anti-inflammatory activities [9]. They attracted attention in chemistry because they are

used as bioisostere for esters, carboxamides and carboxylic acids. 1,3,4-Oxadiazole

analogs have the ability to go through numerous chemical reactions which made them

significant for molecular designing due to its privileged structure [10]. Two drugs that

possess oxadiazole moiety that recently used are: raltegravir® (5), an anti-retroviral

agent [11] and zibotentan® (6), an anti-cancer agent [12].

Figure-2.3: Structure of Raltegravir (5) and Zibotentan (6)

2.2 Pharmacological activity of 1,3,4-oxadiazoles

2.2.1 Anti-microbial activity

The current appearances of drug resistance when curing infective syndromes has

highlighted the need for fresh, harmless and well-organized anti-microbial drugs. Many

researchers have reported potent anti-microbial agent of derivatives comprising of

oxadiazole moiety.

Oliveira et al., testified anti-staphylococcal inhibitory potential of 1,3,4-oxadiazole

having 5-nitro-furan analog (7) against various strain of staphylococcus aureus. All

analogs exhibited effective anti-staphylococcal inhibitory potential which is many times

53

greater than the reference drug chloramphenicol [13]. New series of 5-((naphthalen-2-

yloxy)methyl)-1,3,4-oxadiazole-2-thiol (8) were prepared and screened against anti-

microbial inhibitory potential. All the derivatives were found potent against the various

strains such as C. albicans, C. parapsilosis, S. aureus, P. aeruginosa and E. coli [14].

Patel et al., confirmed the anti-bacterial inhibitory potential of novel series of

compounds comprising 1,3,4-Oxadizaole moiety against Gram positive bacteria and

Gram negative bacteria by using reference drug ampicillin. Two analogs such as analog

(9) and analog (10) correspondingly showed greater inhibitory potential than the

reference drug ampicillin [15].

Figure-2.4: Oxadiazole containing analogs as anti-microbial agents

2,5-disubstituted oxadiazole analogs (11) comprising acetyl moiety at position-3 of 1,3,4-

Oxadiazole moieties were prepared and evaluated for antifungal and anti-bacterial

activities against two strain of bacteria and two specie of fungi by using disks diffusion

54

methods. Fluconazole/ampicillin was used as reference inhibitor respectively. In

evaluation, all the analogs were equally active to the reference drug fluconazole and

ampicillin [16].

The anti-fungal and anti-bacterial activities of 2-(5-amine-1, 3, 4-oxadiazolyl)-4-bromo-

phenol (12) and 5-(3, 5-di-bromophenyl)-1, 3, 4-oxadiazol-2-amine (13) were evaluated

for anti-fungal and anti-bacterial activities against two strains of gram positive bacteria,

two strains of gram negative of bacteria and two specie of fungi. The assessment

exhibited inhibitory potential which were almost equal to the reference drug

griseofulvin and streptomycin respectively [17].

Sangshetti et. al., examined the anti-fungal inhibitory potential of di-substituted

oxadiazoles (14) which confined tri-azole moiety at 5-position of oxadiazole ring. The

analogs were testified against five species of fungi. For evaluation, fluconazole and

miconazole were used as a reference drugs. The analog comprising methyl sulfone

moiety bonded to nitrogen of piperidine moiety and OH/Cl moiety to phenyl ring

showed superior biological activity against some strains of fungi [18].

Analogs 15 and 16 respectively were many folds better than standard inhibitor furacin

when screened against P. aeruginosa and E. coli. Analogs 17 and 18 were twice as active

then reference drug fluconazole [19].

55


The analog 2-((naphthalen-2-yloxy)methyl)-5-(phenoxymethyl)-1,3,4-oxadiazole (19)

shows antimycobacterium inhibitory potential at a lowest inhibiitory concentretion [20].

Kumar et al., planned a new series of substituted-phenyl-1,3,4-Oxadiazole analog (20)

which exhibited anti-mycobacterial inhibitory potential against Mycobacterium

tuberculosis. The analog comprising chloro group showed highest results at a lowest

inhibitory concentration [21]. Yosheda et al., designated preparation and optimiization

of antimycobacterium inhibitory potential of Cephem analogs. Analog 21 showed anti-

mycobacterium inhibitory potential at a lowest inhibitory concentration [22].

Bakel et al., examined antitubercular inhibitory potential of a new series of 2, 5-di-

substituted oxadiazole analogs against M. tuberculosis. Analog 22 showed inhibitory

56

potential which is similar to the reference drug isoniazid while analog 23 showed many

fold better inhibitory potential against M. tuberculosis and INH resistant M. tuberculosis

than standard drug isoniazid [23, 24].


2.2.2 Anti-convulsant activity

Kashew et al., planned and prepared two new series of (E)-3-(5-(4-amino-phenyl)-1,3,4-

Oxadiazol-2-yl)-2-(4-fluorostyryl)quinazolinone analog (24), 5-(2-((4-

fluorobenzyl)thio)phenyl)-1,3,4-oxadiazol-2-amine analog (25) and screened for anti-

convulsant inhibitory potential. The authors claimed that by introducing NH2 at

position-2 of 1,3, 4-oxadiazole moiety and „F‟ at position-4 of benzyl-thio moiety

increases anti-convulsant inhibitory potential [25, 26].

Rajak et al., prepared semicarbazones comprising of 1,3,4-oxadiazole moiety 26 and

screened for anti-convulsant inhibitory potential against three models such as MES,

57

scPTZ and scSTY. Most of the analogs exhibited good inhibitory potential in all the

testified models [27].

Figure-2.7: Oxadiazole containing analogs as anti-convulsant agents

2.2.3 Anti-inflammatory activity:

Manjunatha et al., examined a new series of oxadiazole analogs 27 which comprise of

aryl-piperazine moiety at 3-position of oxadiazole ring for anti-inflammatory inhibitory

potential by using the method of paw edema induced by carrageenan with sodium

diclofenac as standard drug. These analogs having 3-Cl, 4-F, 4-Cl and 4-NO2 moieties

were found more potent than the standard drug while analogs with 2-OEt and 4-OMe

moieties exhibited low inhibitory potential [28].

Analog 28 was prepared and screened for antiinflammatory inhibitory potential by

using sodium diclofenac and fenbufen as reference drug through carrageenan induced

paw edema methods. The analogs comprising of 4-MeO, 4-F, 4-NO2 and 4-Cl moieties

showed equal potency to that of standard drugs and the analogs comprising of 3,4-

dimethoxy moiety was found most active than standard drugs [29]. 2-((3ϒ, 5ϒ, 7ϒ)-

Adamantan-1-yl)-5-(4-chloro-phenyl)-1,3,4-Oxadiazole analog (29) showed robust

dosage dependent activity.

58

Burbulene et al., examined 5-((2-amino-6-methyl-4-pyrimidinyl)thio)methyl)-1,3,4-

Oxadiazole-2(3H)-thione analog (30) for antiinflammatory inhibitory potential and

found this derivatives are most active then the standard drug [30].

Figure-2.8: Oxadiazole containing analogs as antiinflammatory agents

2.2.4 Analgesic activity

5-(2-((2,6-Dichlorophenyl)amino)benzyl)-N-(4-floro-phenyl)-1,3,4-oxadiazol-2-amine

analog (31) was screened for analgesic inhibitory potential and found most potent than

standard inhibitor sodium diclofenac [31]. The analog 32 having 2,4-di-chloro-phenyl

moiety present at position-2 of 1,3,4-oxadiazole moiety exhibited a maximum inhibitory

potential which is almost equal to the standard drug ibuprofen [32].

59

Figure-2.9: Oxadiazole containing analogs as analgesic agents

2.2.5 Anti-tumor activity

Savariz et al., prepared new Mannich bases and screened for in vitro anti-tumor

inhibitory potential. Among the planned analogs, analog 33 exhibited active inhibitory

potential against lung (NCI-460) and melanoma (UACC-62) cell line [33].

Liu et al., prepared a series of 2-(benzyl-thio)-5-aryl-oxadiazole analogs and testified for

anti-proliferative inhibitory potential. Analog 34 exhibited active biological activity [34].

Ouyang et al., and Tuma et al., prepared and screened numerous 1,3,4-oxadiazole

analogs to prevent polymeriization and slab the mitotic division of tumor cell. Analogs

35 and 36 showed active inhibitory potential. In vitro study of analog 35 specified that at

nano-concentration its disturbed mitotic division in tumor and breast cancer cells line

comprised multidrug resistant cell. In vivo study of analog 36 presented a desired

pharmacokinetics profile and was considerably most potent then standard inhibitor [35,

36].

New series of 1-(2-phenyl-5-(3,4,5-tri-methoxy-phenyl)-1,3,4-Oxadiazol-3-yl)ethan-1-one

(37) and 1-(5-(4-methoxyphenyl)-2-phenyl-1,3,4-oxadiazol-3-yl)ethanone (38) analogs

were prepared and screened for anti-proliferative activity. Results showed that this

60

class of analogs prevents polymeriization, proliferation of human and murine cancer

cell. Both analogs showed moderate potential when compared with combretastine [37].

Figure-2.10: Oxadiazole containing analogs as antitumor agents

2.2.6 Anti-viral activity

Us Food and Drug Administration on 16th October, 2007 accepted raltegravir (39) for

management of HIV-1 infections, in arrangement with other anti-retroviral agent in

cure experienced mature patient which give indication of viral replications and HIV

strain impervious to numerous anti-retroviral mediators [38].

Wang et al., prepared a new derivative of raltegravir from its parent structure only by

bringing upon changes in 5-hydroxyl group of pyrimidine ring and screened for anti-

HIV inhibitory potential which considerably improved their inhibitory potential.

61

Analog 40 was more active anti-HIV agents among all of prepared analogs and hence

appeared as novel and active anti-HIV agents [39].

Figure-2.11: Basic structures of raltegravir (39) and its derivative (40)

The inhibitory potentials of analogs 41 and 42 against HIV were resolute by using XTT

protocol. Analog 42 was more potent among testified analogs, creating 37, 43 and 100%

decrease in viral reproduction at concentrations of 10, 50 and 2µg/mL correspondingly.

Analog 41 showed less anti-viral replication potential. All the testified analogs were

non-cytotoxic [40].

Iqbal et al., testified inhibitory potential for analogs 43 and 44 against HIV which was

also resolute by using XTT protocol. Analog 43 in which R= Cl moiety was the most

potent among the testified analogs [41].

Figure-2.12: 1,3,4-oxadiazole analogs having HIV-1 activity

For curing HIV infection and AIDS, another protease inhibitor, indinavir is also used as

a part of anti-retroviral treatment. Kim et al., prepared 1,3,4-Oxadiazole containing

62

piperazin-2-carboxamide analog (45) and screened for protease inhibitory potential.

All synthesized analogs introverted protease inhibitory potential at molar concentration

[42]. Johns et al., testified anti-viral inhibitory potential for two new analogs such as 7-

(5-(4-floro-benzyl)-1,3,4-Oxadiazol-2-yl)-1,6-naphthyridinol analog (46) and

(2R,6S,13aR,14aR,16aS,E)-6-((tert-butoxy-carbonyl)amino)-2-((7-methoxy-2-(5-methyl-

1,3,4-Oxadiazol-2-yl)4-quinolinyl)oxy)-5,16-dioxo-16a-tetra-deca-hydro-cyclo-

propa[e]pyrrol [1,4]di-aza-cyclo-penta-decine-14a(5H)-carboxylic acid (47) exhibits

excellent inhibitory potential against hepatitis C virus [43, 44].

Figure-2.13: 1,3,4-Oxadiazole analogs having HIV and hepatitis C virus activity

2.2.7 Anti-hypertensive activity

The major reasons of morbidity and mortality are cardiovascular and hypertension.

Bankers et al., testified vasorelaxant effect of 4-(3-acetyl-5-(3-pyridinyl)-2,3-di-hydro-

1,3,4-Oxadiazol-2-yl)phenylacetate analog 48 in rats aortic ring causes hindering

through L-type calcium channel [45].

63

Figure-2.14: Oxadiazole containing analogs as anti-hypertensive agents

2.3 Previous approaches toward synthesis of oxadiazole analogs

2.3.1 Synthesis of 5-argio-1, 3, 4-Oxadiazole analogs

Acyl-thiosemicarbazide (49) was cyclized in NaOH, KI, H2O in the presence of

oxidizing agent 1,3-di-bromo-5,5-di-methyl hydantion to give 5-argio-1,3,4-Oxadiazole-

2-amine analogs (50). This process is useful for large scale preparation because the key

benefit of this process is that chemicals used are easily available, commercially

inexpensive and harmless to work [46].

Scheme-2.1: Synthesis of 5-argio-1, 3, 4-Oxadiazole-2-amine analogs

2.3.2 Synthesis of 1,3,4-Oxadiazole-2-amines analogs

2-(2-(naphthalene-2-yloxy)acetyl)-N-phenyl hydrazine carboxamide (51) was heated in

EtOH in the presence of NaOH and I2 to give the corresponding 1,3,4-Oxadiazole-2-

amines analogs (52) [47].

64

Scheme-2.2: Synthesis of 5-((naphthalen-2-yl-oxy)methyl)-N-phenyl-1,3,4-Oxadiazol-

2-amine analogs

2.3.3 Synthesis of 5-substituted 1, 3, 4-Oxadiazole-2-thiol analogs

Acyl-hydrazide (53) was mixed with CS2 in ethanol in the presence of KOH followed by

the acidification of mixture to give us 5-substituted 1,3,4-oxadiazole-2-thiol analogs (54)

[48].

Scheme-2.3: synthesis of 5-substituted 1, 3, 4-Oxadiazole-2-thiol analogs

2.3.4 Synthesis of Nafion catalyzed 1, 3, 4-oxadiazole analogs

1,3,4-oxadiazole (57) can be synthesized in excellent yield by condensation of

benzohydrazide (55) with tri-ethyl-ortho-alkanets (56) under microwave condition and

well catalyzed by phosphorus penta-sulfide in alumina and Nafion®NR50 [49].

Scheme-2.4: Synthesis of Nafion catalyzed 1,3,4-oxadiazole analogs

65

2.3.5 Synthesis of 1,3,4-oxadiazole through regioselective cyclization processes

Substituted amines 1,3,4-oxadiazole analogs (59) can be synthesized by regioselective

cyclization processes of thiosemicarbazide (58) intermediate with EDC.HCl in DMSO

[50].

Scheme-2.5: Synthesis of 1,3,4-oxadiazole analogs through regioselective cyclization

process

66

2.4 Research background

Our research group has reported the α-glucosidase inhibitory activity of oxindole based

oxadiazole analogs [51], α-glucosidase inhibitory of 5-aryl-2-(6‟-nitrobenzofuran-2‟-yl)-

1,3,4-oxadiazoles [52], as well as also reported the novel sulfonamides having

oxadiazole ring as potential class of β-glucuronidase inhibitors [53] (Figure-2.15).

Keeping in view the great biological potential of oxadiazole here in this study we

decided to synthesize oxadiazole derivatives and evaluation for their thymidine

phosphorylase inhibition potential.

Figure-2.15: Rational of the current study

67


2.5.1 Chemistry

Methyl 4-cyanobenzoate (60) was mixed with excess of hydrazine hydrate in methanol

and refluxed for 6 hrs to give us compound (61). Compound (61) was then treated with

methyl 4-formylbenzoate in methanol in the presence of catalytic amount of acetic acid

to afford hydrazone (62), which was then subjected through an oxidative cyclization

using phenyliododiacetate (PhI(OAc)2) in dichloromethane to form 1,3,4-oxadiazole

(63). The ester functional group of compound (63) was further converted into hydrazide

by treating with hydrazine hydrate to afford compound (64), which was then reacted

with various different substituted benzaldehydes in methanol to targeted 1,3,4-

oxadiazole analogs (65-80).

Scheme-2.6: Synthesis of oxadiazole derivatives (65-80)

68

Table-2.1: Oxadiazole derivatives and their thymidine phosphorylase activity (65-80)

S. No R IC50 values S. No R IC50 values

65

4.30 ± 0.10 73

29.20 ± 0.50

66

9.40 ± 0.20 74

8.10 ± 0.30

67

39.10 ± 0.80 75

17.30 ± 0.40

68

28.60 ± 0.40 76

4.60 ± 0.2

69

6.60 ± 0.20 77

1.10 ± 0.05

70

16.10 ± 0.20 78

2.40 ± 0.10

69

71

36.20 ± 0.90 79

17.50 ± 0.30

72

N.A 80

49.60 ± 1.30

7-Deazaxanthine 38.68 ± 1.12 μM

2.5.2 In vitro thymidine phosphorylase inhibitory potential

In continuous effort on enzyme inhibition [54]; sixteen derivatives of oxadiazole (65-80)

were synthesized and screened for thymidine phosphorylase inhibitory potential. These

derivatives displayed variable degree of inhibition in the range of 1.10 ± 0.05 to 49.60 ±

1.30 μM when compared with the standard inhibitor 7-Deazaxanthine having an IC50

value 38.68 ± 1.12 μM (Table-2.1). Derivatives 65-71 and 73-79 showed excellent

thymidine phosphorylase inhibitory potentials with IC50 values 4.30 ± 0.10, 9.40 ± 0.20,

39.10 ± 0.80, 28.60 ± 0.40, 6.60 ± 0.20, 16.10 ± 0.20, 36.20 ± 0.90, 29.20 ± 0.50, 8.10 ± 0.30,

17.30 ± 0.40, 4.60 ± 0.2, 1.10 ± 0.05, 2.40 ± 0.10 and 17.50 ± 0.30 μM respectively, which is

many folds better than the standard 7-Deazaxanthine. Derivative 80 showed good

inhibitory potential while derivative 72 was found inactive among the series. If we

compare the TP inhibition of our design compounds with other oxadiazole analogs as

reported by Shahzad et al., [55, 56], our compounds are superior. The SAR in previous

study was mainly based on substitution pattern on phenyl ring. Here in this study the

70

structural activity relationship (SAR) was mainly based upon by bring about different

substituents on phenyl ring.

The most active derivative among the series is compound 77 (IC50 = 1.10 ± 0.05 μM)

having two methoxy and one hydroxy substituents on the phenyl ring. The greater

potential of this compound is mainly seems to be due to the two methoxy groups which

is electron donating group.

If we compare derivative 76 (IC50 = 4.60 ± 0.2 µM) having one methoxy and one

hydroxy substituents with derivative 78 (IC50 = 2.40 ± 0.10 µM) also having one

methoxy and one hydroxy substituents on phenyl ring. In derivative 76 the methoxy

group is present at 4-position and hydroxy is present at 3-position and in derivative 78

the methoxy group is present at 3-position and hydroxy at 4-position on phenyl ring.

Both derivatives have the same methoxy and hydroxy groups but the position of the

hydroxy and methoxy groups are different on phenyl ring. Compound 78 was found to

be superior who showed that position of substituent also play role in this inhibition.

Figure-2.16: Comparison of structure activity relationship between analogs 76 and 78

71

All those analogs having chloro group on phenyl ring like analog 65 and 66 showed

greater potential among the series. The analog 65, a 2,4-dichloro analog (IC50 = 4.30 ±

0.10 µM) show greater potential as compare to 4-chloro analog 66 (IC50 = 9.40 ± 0.20

µM). This shows that increasing the number of chloro group correspondingly increase

the inhibitory potential.


If we compare derivative 74 having IC50 value 8.10 ± 0.30 μM with derivative 75 having

IC50 value 17.30 ± 0.40 μM, both the derivatives have hydroxy group on phenyl ring, but

the arrangement of hydroxy group is different in them which confirm that the

difference in position of substituents greatly affect the inhibitory potentials of the

derivatives.

72


To understand the binding interaction of the most active analogs molecular docking

study was performed.


The docking is a significant tool to explore the interactions between an inhibitor and the

target [57]. To find the binding interactions of these compounds in the active sites of the

thymidine phosphorylase, the MOE-Dock program (www.chemcomp.com) was used to

perform molecular docking. The 3D crystal structure of the thymidine phosphorylase

(4EAD) was retrieved from the Protein Databank (PDB). The synthesized compounds

were docked into the active site of the target enzyme in MOE (www.chemcomp.com) by

the default parameters i-e Placement: Triangle Matcher, Rescoring 1: London dG,

Refinement: Force field, Rescoring 2: London dG. For each ligand ten conformations

were generated and the top ranked conformation based on docking score was selected

for further studies in molecular docking. After the molecular docking, we analyzed the

best poses having polar, H-pi and pi-H interactions by Pymol software.

73

2.5.4 Docking study

The docking results of the oxadiazole series with the thymidine phosphorylase enzyme

have given good information about the nature of the binding mode that was excellent

correlated with the experimental results. From the docking calculation study, it was

observed that the top ranked conformations of almost all compounds were well

accommodated inside the active site of thymidine phosphorylase enzyme and were

involved in various type of interactions with the active site residues of thymidine

phosphorylase enzyme i.e., His 85, Arg 115, Thr 123, Tyr 168, Arg 171, Ile 183, Ser 186,

Lys 190 and Phe 210 etc. The detail of docking scores and interactions for all

compounds are listed in Table-2.2. The structural features observed in this group for

the active nature of compounds are the presence of electronegative groups like -OH,

halogen and Methoxy (MeO) groups. Among halogen Cl containing compounds were

found superior than Br supported by hydroxyl group. Figure-2.19 (a-d) displays the

interaction modes of some most active compounds among these docked conformations.

The confirmations obtained after docking showed good docking scores and

demonstrated healthy in-silico inhibition of the thymidine phosphorylase enzyme.

Overall a good correlation was observed between the docking study and biological

evaluation of active compounds.

74

Figure-2.19: Docking conformations of compounds on thymidine phosphorylase

enzyme. (a) 3D binding mode of compound 77 as inhibitor of thymidine

phosphorylase enzyme. (b) 3D binding mode of compound 78 (c) 3D binding mode

of compound 65 (d) 3D binding mode of compound 76 in binding cavity of

thymidine phosphorylase enzyme. Ligands are shown green color.

75

Figure-2.20: Correlation graph for IC50 values and docking predicted activity


Compounds Docking

scores (S)

Interaction Report

65

-12.9432

Ligand

N 28

N 31

5-ring

N 32

Receptor

OD1 TYR 168

(A)

N ARG 115

(A)

N ARG 115

(A)

NZ LYS 190

(A)

Interaction

H-donor

H-acceptor

pi-H

H-acceptor

Distance

3.03

2.63

3.76

2.22

E

(kcal/mol)

-0.2

-0.2

-0.2

-8.2

66 -11.4997 N 13

O 27

N 45

6-ring

NH2 ARG

171 (A)

NZ LYS 190

(A)

N MET

H-acceptor

H-acceptor

H-acceptor

pi-pi

3.28

2.42

3.55

3.59

-0.7

-2.6

-1.3

-0.0

76

211 (A)

6-ring TYR

168 (A)

67 -8.1102 C 1

C 24

N 28

N 30

N 59

6-ring

6-ring

6-ring

OD2 ASP 92

(A)

O ARG

115 (A)

O LEU 117

(A)

CE1 PHE

210 (A)

N SER 95

(A)

CA GLY 122

(A)

CB ALA

175 (A)

CZ PHE

210 (A)

H-donor

H-donor

H-donor

H-acceptor

H-acceptor

pi-H

pi-H

pi-H

3.04

3.41

2.91

4.26

3.89

4.80

4.60

4.09

-1.4

-0.3

-0.8

-0.1

-0.3

-0.1

-0.1

-0.7

68 -8.8854 C 37

O 27

N 49

N 49

6-ring

6-ring

5-ring

6-ring

6-ring

OD2 ASP

172 (A)

CB LEU

117 (A)

CG MET

111 (A)

N ILE 112

(A)

CA HIS 85

H-donor

H-acceptor

H-acceptor

H-acceptor

pi-H

pi-H

pi-H

pi-H

pi-H

2.78

3.61

4.14

3.91

4.79

4.33

4.22

4.51

4.05

-0.4

-0.1

-0.2

-0.4

-0.1

-0.4

-0.2

-0.3

-0.6

77

6-ring

6-ring

(A)

N SER 86

(A)

CB SER 86

(A)

CB SER 86

(A)

CA SER 113

(A)

CB THR

120 (A)

CG2 VAL

177 (A)

pi-H

pi-H

4.36

4.53

-0.1

-0.4

69 -11.7449 C 34

C 36

CL 42

O 43

N 12

N 13

6-ring

6-ring

6-ring

6-ring

5-ring

5-ring

5-ring

6-ring

OD2 ASP 83

(A)

SD MET

111 (A)

OG1 THR

123 (A)

OG SER 95

(A)

CA LYS 165

(A)

NZ LYS 190

(A)

CA HIS 85

(A)

N SER 86

H-donor

H-donor

H-donor

H-donor

H-acceptor

H-acceptor

pi-H

pi-H

pi-H

pi-H

pi-H

pi-H

pi-H

pi-H

3.75

3.57

3.40

2.67

3.36

3.92

4.23

3.74

4.43

3.88

4.16

4.24

3.41

3.12

-0.2

-0.1

-0.1

-2.0

-0.1

-0.1

-0.6

-0.5

-0.1

-0.1

-0.1

-0.5

-0.3

-0.1

78

(A)

CB SER 86

(A)

CA SER 113

(A)

CA LYS 165

(A)

CB TYR 168

(A)

CD2 TYR

168 (A)

CE LYS

190 (A)

70 -11.4853 C 36

N 12

N 53

C 34

6-ring

6-ring

5-ring

O TYR 168

(A)

N THR

123 (A)

N SER 95

(A)

6-ring TYR

168 (A)

OG SER

113 (A)

CD1 LEU

117 (A)

CA GLY

122 (A)

H-donor

H-acceptor

H-acceptor

H-pi

pi-H

pi-H

pi-H

3.81

2.71

2.69

3.87

4.32

4.20

3.32

-0.1

-3.3

-3.1

-0.1

-0.1

-0.8

-0.2

71 -8.2376 C 22 OD2 ASP H-donor 3.14 -0.4

79

C 43

C 58

N 63

6-ring

164 (A)

O SER 86

(A)

O SER

186 (A)

CD1 LEU

117 (A)

OH TYR

168 (A)

H-donor

H-donor

H-acceptor

pi-H

3.28

3.20

3.87

4.47

-0.4

-0.1

-0.2

-0.1

72 NA NA NA

73 -8.6509 C 43

N 52

N 52

O 49

6-ring

6-ring

6-ring

6-ring

OD2 ASP

172 (A)

CD LYS 191

(A)

NZ LYS

191 (A)

6-ring PHE

210 (A)

CD1 LEU

117 (A)

CA GLY

118 (A)

CA GLY

118 (A)

5-ring HIS

85 (A)

H-donor

H-acceptor

H-acceptor

H-pi

pi-H

pi-H

pi-H

pi-pi

3.76

2.75

3.26

4.50

4.60

4.88

4.12

3.68

-0.2

-0.6

-3.0

-0.2

-0.1

-0.2

-0.2

-0.0

74 -9.4593 O 43

O 27

O ARG

371 (A)

H-donor

H-acceptor

3.66

3.10

-0.1

-0.1

80

N 30

C 9

6-ring

6-ring

CB LEU

117 (A)

CD1 LEU

117 (A)

5-ring HIS

85 (A)

CB ILE

112 (A)

CA GLY

118 (A)

H-acceptor

H-pi

pi-H

pi-H

3.60

3.53

4.04

3.71

-0.1

-0.1

-0.1

-0.3

75 -9.6861 C 4

O 43

N 12

O 27

N 46

6-ring

6-ring

O ILE

112 (A)

O ARG

370 (A)

N THR

123 (A)

CE1 PHE

210 (A)

CB LYS 84

(A)

CA HIS

85 (A)

CA GLY

118 (A)

H-donor

H-donor

H-acceptor

H-acceptor

H-acceptor

pi-H

pi-H

3.19

3.43

3.88

3.97

2.85

4.83

4.12

-0.7

-0.2

-0.2

-0.1

-0.6

-0.3

-0.1

76 -12.5698 N 33

N 9

5-ring

O 27

NH1 ARG

171 (A)

NZ LYS

190 (A)

H-acceptor

H-acceptor

pi-cation

H-acceptor

2.79

2.21

3.26

3.43

-1.4

-0.2

-4.2

-3.2

81

6-ring NZ LYS

190 (A)

NE2 HIS

85 (A)

CG2 ILE

183 (A)

pi-H 4.40 -0.7

77 -14.2399 O 42

C 44

O 42

O 32

N 9

N 9

N 13

O 27

N 54

C 1

C 19

6-ring

NH TYR

168 (A)

O ILE

183 (A)

NH ARG

171 (A)

NH ARG

171 (A)

OG1 THR

123 (A)

NH THR

123 (A)

OG1 THR

123 (A)

NE2 HIS

85 (A)

CE1 PHE

210 (A)

6-ring PHE

210 (A)

5-ring HIS

85 (A)

H-donor

H-donor

H-donor

H-donor

H-acceptor

H-acceptor

H-acceptor

H-acceptor

H-acceptor

H-pi

H-pi

pi-H

2.15

3.52

2.01

2.01

1.91

2.01

2.17

1.93

4.27

4.66

4.46

4.79

-2.4

-0.2

-2.4

-0.2

-2.3

-2.3

-2.3

-3.2

-0.1

-0.1

-0.1

-0.1

82

CB TYR

168 (A)

78 -13.9108 C 1

C 9

N 12

N 13

N 12

O 42

O 27

O 27

6-ring

6-ring

6-ring

5-ring

5-ring

6-ring

OD1 ASP

178 (A)

O VAL

177 (A)

NH1 ARG

171 (A)

NH2 ARG

171 (A)

NH2 ARG

171 (A)

OH TYR

168 (A)

NE2 HIS

85 (A)

CE LYS

190 (A)

NE2 HIS

85 (A)

N ARG

115 (A)

CB ARG

115 (A)

CG1 ILE

183 (A)

CG2 ILE

183 (A)

H-donor

H-donor

H-acceptor

H-acceptor

H-acceptor

H-acceptor

H-acceptor

H-acceptor

pi-H

pi-H

pi-H

pi-H

pi-H

pi-H

3.50

3.16

2.10

2.06

2.51

3.01

2.81

3.53

4.78

3.87

3.83

4.12

3.75

4.27

-0.2

-0.3

-6.1

-6.2

-6.2

-5.2

-0.1

-0.2

-0.5

-0.1

-0.1

-0.1

-0.7

-0.1

83

CD1 ILE

183 (A)

79 -9.6860 C 4

C 19

C 22

C 24

C 34

6-ring

6-ring

6-ring

5-ring

O ILE

112 (A)

OH TYR

168 (A)

O LEU

117 (A)

OG1 THR

120 (A)

OD1 ASP

172 (A)

CB SER

86 (A)

CB LEU

117 (A)

CD1 LEU

117 (A)

CA GLY

122 (A)

H-donor

H-donor

H-donor

H-donor

H-donor

pi-H

pi-H

pi-H

pi-H

3.19

2.67

3.13

2.53

3.23

4.58

3.72

4.30

4.06

-0.5

-0.3

-0.1

-0.1

-0.3

-0.1

-0.1

-0.1

-0.6

80 -6.7812 C 36

C 38

O 27

N 46

N 46

N 46

6-ring

ND1 HIS

85 (A)

OE1 GLU

194 (A)

CB GLN

156 (A)

CB GLN

156 (A)

H-donor

H-donor

H-acceptor

H-acceptor

H-acceptor

H-acceptor

pi-H

2.80

2.80

2.93

2.74

2.49

3.07

4.70

-0.2

-0.7

-0.1

-0.1

-0.1

-0.2

-0.1

84

CG GLN

156 (A)

CD LYS

190 (A)

CA SER

113 (A)

Standard -8.0025 N 1

O 7

O 8

O 8

6-ring

OG SER

186 (A)

NZ LYS

190 (A)

NH1 ARG

171 (A)

NH2 ARG

171 (A)

NE2 HIS

85 (A)

H-donor

H-acceptor

H-acceptor

H-acceptor

pi-H

3.18

2.61

3.28

3.16

4.64

-2.3

-0.4

-2.3

-4.0

-1.9

2.6 Conclusion

In conclusion we have synthesized sixteen oxadiazole derivatives (1-16) and screened

against thymidine phosphorylase inhibitory potential. All derivatives exhibited a varied

degree of thymidine phosphorylase inhibition with IC50 values ranging between 1.10 ±

0.05 to 49.60 ± 1.30 μM when compared with standard drug 7-Deazaxanthine having an

IC50 value38.68 ± 1.12 μM. Our synthesized compounds are more active than the

previously reported oxadiazole analogs for the thymidine phosphorylase activity on the

basis of IC50 values. The SAR was mainly based on substitution pattern on phenyl ring

which is in consistent with previous reported oxadiazole analogs for the thymidine

85

phosphorylase activity. Molecular docking study was performed to understand the

binding interaction of the most active derivatives with enzyme active site.

2.7 Material and Methods

2.7.1 Synthetic procedure for 4-cyanobenzohydrazide (61)

Methyl 4-cyanobenzoate (20 mmol) was mixed with hydrazine hydrate (20 mL) in

methanol (15 mL). The mixture was refluxed for 6 hrs. Methanol was then evaporated

and the product formed was being washed with distilled water to remove excess of

hydrazine hydrate. The product formed was left to dry at room temperature.

2.7.2 Synthetic procedure for methyl (E)-4-((2-(4-cyanobenzoyl)hydrazono)methyl)

benzoate (62)

Compound 61 (15 mmol) was reacted and refluxed with methyl 4-formylbenzoate (15

mmol) in methanol (mL) in the presence of catalytic amount of acetic acid. The solvent

was evaporated and the residue (62) was washed with ether, filtered, dried and then

crystallized from ethanol.

2.7.3 Synthetic procedure for methyl 4-(5-(4-cyanophenyl)-1,3,4-oxadiazole-2-

yl)benzoate (63)

A mixture of compound 62 (12 mmol) and equivalent amount of PhI(OAc)2 was stirred

in dichloromethane (20 mL) at room temperature overnight. The solvent was

evaporated and the residue (63) was washed with ether, filtered, dried and then

crystallized from ethanol.

86

2.7.4 Synthetic procedure for 4-(5-(4-cyanophenyl)-1,3,4-oxadiazole-2-

yl)benzohydrazide (64)

Compound 63 (10 mmol) was mixed and refluxed with hydrazine hydrate (15 mL) in

methanol (15 mL). Methanol was then evaporated and the product formed was washed

with distilled water to remove excess of hydrazine hydrate. The product formed (64)

was left to dry at room temperature.

2.7.5 General procedure for the synthesis of oxadiazole derivatives (65-80)

Compound 64 (1 mmol) was treated and refluxed with different benzaldehyde (1 mmol)

in methanol (15 mL) to give us the resulting oxadiazole derivatives (65-80). The

resulting solid was filtered and recrystallized from methanol in good yields.

2.7.5.1 (E)-4-(5-(4-cyanophenyl)-1,3,4-thiadiazol-2-yl)-N'-(2,4-dichlorobenzylidene)

benzohydrazide (65)

Yield: 87%. Dark yellow solid, m.p. 230-232 °C. 1H-NMR (500 MHz, DMSO-d6): δ 11.50

(s, 1H, NH), 8.93 (s, 1H, CH=N), 8.05 (d, J = 7.9 Hz, 2H, Ar), 8.03 (d, J = 7.7 Hz, 1H, Ar),

7.91 (d, J = 7.5 Hz, 2H, Ar), 7.90 (d, J = 7.6 Hz, 2H, Ar), 7.80 (d, J = 7.3 Hz, 2H, Ar), 7.73

(s, 1H, Ar), 7.50 (d, J = 7.1 Hz, 1H, Ar), 13C-NMR (125 MHz, DMSO-d6): δ 164.8, 164.1,

163.0, 138.4, 132.4, 132.2, 132.0, 131.7, 131.3, 130.2, 130.1, 130.0, 129.8, 124.1, 129.3, 128.9,

128.7, 128.4, 127.5, 127.3, 126.0, 118.1, 112.2. HREI-MS: m/z calcd for C23H13Cl2N5O2 [M]+

461.0446, Found 461.0442.

87

2.7.5.2 (E)-N'-((4-chlorocyclohexa-1,3-dien-1-yl)methylene)-4-(5-(4-cyanophenyl)-1,3,4-


Yield: 82%. Yellow solid, m.p. 235-237 °C. 1H-NMR (500 MHz, DMSO-d6): δ 10.70 (s, 1H,

NH), 8.91 (s, 1H, CH=N), 8.11 (d, J = 7.9 Hz, 2H, Ar), 7.91 (d, J = 7.5 Hz, 2H, Ar), 7.90 (d,

J = 7.6 Hz, 2H, Ar), 7.80 (d, J = 7.3 Hz, 2H, Ar), 7.77 (d, J = 7.4 Hz, 2H, Ar), 7.44 (d, J = 7.3

Hz, 2H, Ar), 13C-NMR (125 MHz, DMSO-d6): δ 164.4, 164.2, 163.1, 144.3, 137.4, 136.2,

133.4, 132.4, 132.1, 130.8, 130.3, 130.1, 129.6, 129.1, 128.0, 127.9, 127.7, 127.1, 122.2, 118.3,

112.2, 34.1, 19.3. HREI-MS: m/z calcd for C23H16ClN5O2 [M]+ 427.0836, Found 427.0831.

2.7.5.3 (E)-N'-(4-(benzyloxy)benzylidene)-4-(5-(4-cyanophenyl)-1,3,4-oxadiazol-2-


Yield: 90%. Light yellow solid, m.p. 245-247 °C. 1H-NMR (500 MHz, DMSO-d6): δ 11.70



(d, J = 7.1 Hz, 2H, Ar), 7.37 (m, 2H, Ar), 7.30 (dd, J = 7.9, 3.5 Hz, 1H, Ar), 7.10 (d, J = 7.0

Hz, 2H, Ar), 5.13 (s, 2H, CH2), 13C-NMR (125 MHz, DMSO-d6): δ 164.2, 164.1, 163.4,

161.0, 146.3, 136.2, 133.8, 133.1, 132.4, 130.2, 130.0, 139.8, 139.6, 130.4, 129.2, 128.6, 128.4,

128.1, 127.9, 127.6, 127.4, 127.2, 126.9, 126.4,126.3, 118.4, 114.2, 114.0, 112.3, 70.3. HREI-

MS: m/z calcd for C30H21N5O3 [M]+ 499.1644, Found 499.1639.

2.7.5.4 (E)-N'-(5-bromo-2-methoxybenzylidene)-4-(5-(4-cyanophenyl)-1,3,4-oxadiazol-

2-yl)benzohydrazide (68)

Yield: 85%. Yellowish solid, m.p. 240-242 °C. 1H-NMR (500 MHz, DMSO-d6): δ 11.70 (s,

1H, NH), 8.92 (s, 1H, CH=N), 8.09 (d, J = 7.9 Hz, 2H, Ar), 7.91 (d, J = 7.5 Hz, 2H, Ar),

88

7.90 (d, J = 7.6 Hz, 2H, Ar), 7.90 (s, 1H, Ar), 7.80 (d, J = 7.3 Hz, 2H, Ar), 7.56 (d, J = 7.3

Hz, 1H, Ar), 6.94 (d, J = 6.5 Hz, 1H, Ar), 3.80 (s, 3H, CH3), 13C-NMR (125 MHz, DMSO-

d6): δ 164.1, 164.0, 163.0, 156.4, 145.0, 134.7, 132.5, 132.3, 132.2, 131.4, 130.8, 130.4, 130.0,

129.3, 128.9, 128.7, 127.4, 127.2, 119.8, 118.4, 113.2, 112.3, 110.2, 55.7. HREI-MS: m/z calcd

for C24H16BrN5O3 [M]+ 501.0437, Found 501.0431.

2.7.5.5 (E)-N'-(3-chloro-4-hydroxybenzylidene)-4-(5-(4-cyanophenyl)-1,3,4-oxadiazol-2-




7.93 (d, J = 7.6 Hz, 2H, Ar), 7.80 (d, J = 7.3 Hz, 2H, Ar), 7.72 (s, 1H, Ar), 7.52 (d, J = 7.3

Hz, 1H, Ar), 6.83 (d, J = 6.3 Hz, 1H, Ar), 13C-NMR (125 MHz, DMSO-d6): δ 164.3, 164.1,

163.0, 155.5, 146.6, 132.6, 132.4, 132.2, 130.8, 130.6, 130.4, 130.1, 129.3, 128.5, 128.3, 128.0,

127.5, 127.4, 127.1, 124.1, 118.4, 117.2, 112.4. HREI-MS: m/z calcd for C23H14ClN5O3 [M]+

443.0785, Found 443.0782.


(dimethylamino)benzylidene) benzohydrazide (70)




(d, J = 6.3 Hz, 2H, Ar), 3.00 (s, 6H, CH3), 13C-NMR (125 MHz, DMSO-d6): δ 164.2, 164.1,

163.0, 153.3, 146.4, 132.4, 132.2, 132.0, 130.8, 130.6, 130.2, 129.3, 128.8, 128.6, 128.2, 128.0,

89

127.4, 127.1, 123.0, 118.3, 112.3, 111.7, 111.4, 41.2, 41.0. HREI-MS: m/z calcd for

C25H20N6O2 [M]+ 436.1648, Found 436.1643.

2.7.5.7 (E)-N'-(4-(benzyloxy)-3-methoxybenzylidene)-4-(5-(4-cyanophenyl)-1,3,4-




7.90 (d, J = 7.6 Hz, 2H, Ar), 7.80 (d, J = 7.3 Hz, 2H, Ar), 7.54 (s, 1H, Ar), 7.44 (m, 2H, Ar),

7.41 (dd, J = 8.3, 3.3 Hz, 2H, Ar), 7.30 (m, 1H, Ar), 7.23 (d, J = 7.1 Hz, 1H, Ar), 6.96 (d, J =

6.3 Hz, 1H, Ar), 5.14 (s, 2H, CH2), 3.82 (s, 3H, CH3), 13C-NMR (125 MHz, DMSO-d6): δ

164.3, 164.5, 163.0, 149.5, 148.4, 146.0, 136.4, 132.4, 132.2, 132.1, 130.4, 130.2, 130.0, 129.2,

129.0, 128.7, 128.5, 128.3, 128.0, 127.9, 127.6, 127.3, 126.1, 126.0, 122.3, 118.3, 112.3, 111.3,

111.0, 71.3, 56.3. HREI-MS: m/z calcd for C31H23N5O4 [M]+ 529.1750, Found 529.1744.

2.7.5.8 (E)-N'-(anthracen-9-ylmethylene)-4-(5-(4-cyanophenyl)-1,3,4-oxadiazol-2-yl)

benzohydrazide (72)

Yield: 73%. Light white solid, m.p. 266-268 °C. 1H-NMR (500 MHz, DMSO-d6): δ 11.00 (s,

1H, NH), 8.70 (s, 1H, Ar), 8.32 (s, 1H, CH=N), 8.02 (d, J = 7.6 Hz, 2H, Ar), 8.02 (d, J = 7.9

Hz, 2H, Ar), 7.93 (d, J = 7.5 Hz, 2H, Ar), 7.92 (d, J = 7.6 Hz, 2H, Ar), 7.86 (d, J = 7.5 Hz,

2H, Ar), 7.80 (d, J = 7.3 Hz, 2H, Ar), 7.47 (m, 4H, Ar), 13C-NMR (125 MHz, DMSO-d6): δ

164.1, 164.0, 163.6, 143.0, 133.8, 132.2, 132.0, 131.9, 131.7, 130.5, 130.2, 130.0, 129.2, 129.0,

128.7, 128.5, 128.3, 128.0, 128.0, 127.9, 127.7, 127.4, 127.2, 126.6, 125.2, 125.0, 124.6, 124.2,


90

2.7.5.9 (E)-4-(5-(4-cyanophenyl)-1,3,4-oxadiazol-2-yl)-N'-((2-hydroxynaphthalen-1-

yl)methylene)benzohydrazide (73)

Yield: 81%. White yellow solid, m.p. 259-261 °C. 1H-NMR (500 MHz, DMSO-d6): δ 11.90



(d, J = 7.3 Hz, 2H, Ar), 7.80 (d, J = 7.5 Hz, 1H, Ar), 7.50 (dd, J = 8.6, 3.4 Hz, 1H, Ar), 7.34

(m, 1H, Ar), 7.15 (d, J = 7.1 Hz, 1H, Ar), 13C-NMR (125 MHz, DMSO-d6): δ 171.3, 164.1,

164.0, 163.0, 143.0, 133.8, 133.4, 132.5, 132.3, 132.0, 130.9, 130.7, 130.5, 129.3, 129.0, 128.9,

128.7, 128.5, 127.3, 127.1, 126.5, 123.4, 120.1, 118.4, 118.0, 112.1, 108.1. HREI-MS: m/z

calcd for C27H17N5O3 [M]+ 459.1331, Found 459.1325.


hydroxybenzylidene) benzohydrazide (74)




Hz, 2H, Ar), 13C-NMR (125 MHz, DMSO-d6): δ 164.1, 164.0, 163.5, 160.4, 146.4, 133.8,

132.4, 132.1, 131.6, 130.8, 130.6, 130.4, 129.8, 129.1, 128.8, 128.6, 127.4, 127.1, 126.1, 118.4,



hydroxybenzylidene)benzohydrazide (75)

Yield: 84%. Dark yellowish solid, m.p. 233-235 °C. 1H-NMR (500 MHz, DMSO-d6): δ

11.14 (s, 1H, NH), 8.43 (s, 1H, CH=N), 8.04 (d, J = 7.9 Hz, 2H, Ar), 7.91 (d, J = 7.5 Hz, 2H,

91

Ar), 7.90 (d, J = 7.6 Hz, 2H, Ar), 7.80 (d, J = 7.3 Hz, 2H, Ar), 7.30 (d, J = 7.0 Hz, 1H, Ar),

7.22 (s, 1H, Ar), 7.12 (dd, J = 8.1, 3.5 Hz, 1H, Ar), 6.90 (d, J = 6.4 Hz, 2H, Ar), 13C-NMR

(125 MHz, DMSO-d6): δ 165.0, 164.2, 163.0, 158.3, 146.5, 138.2, 132.4, 132.5, 132.4, 130.9,

130.7, 130.5, 130.1, 129.2, 128.6, 128.4, 127.3, 127.0, 121.6, 118.2, 118.0, 114.7, 112.4. HREI-



methoxybenzylidene) benzohydrazides (76)





d6): δ 164.2, 164.1, 163.0, 152.1, 147.0, 146.4, 132.2, 132.5, 132.3, 131.5, 130.2, 130.0, 130.0,

129.2, 128.8, 128.6, 127.4, 127.2, 122.4, 118.3, 115.6, 112.2, 112.1, 56.5. HREI-MS: m/z calcd

for C24H17N5O4 [M]+ 439.1281, Found 439.1276.

2.7.5.13 (E)-4-(5-(4-cyanophenyl)-1,3,4-oxadiazol-2-yl)-N'-(4-hydroxy-3,5-

dimethoxy benzylidene)benzohydrazide (77)

Yield: 83%. Greenish solid, m.p. 220-222 °C. 1H-NMR (500 MHz, DMSO-d6): δ 11.49 (s,

1H, NH), 8.43 (s, 1H, CH=N), 8.05 (d, J = 7.9 Hz, 2H, Ar), 7.93 (d, J = 7.5 Hz, 2H, Ar),

7.91 (d, J = 7.6 Hz, 2H, Ar), 7.80 (d, J = 7.3 Hz, 2H, Ar), 7.02 (s, 2H, Ar), 3.81 (s, 6H, CH3),

13C-NMR (125 MHz, DMSO-d6): δ 164.3, 164.1, 163.5, 148.0, 148.6, 146.4, 139.3, 132.4,

132.1, 132.0, 130.2, 130.0, 129.4, 129.0, 128.8, 128.6, 128.2, 127.3, 127.2, 118.2 , 112.6, 104.1,


92


methoxybenzylidene) benzohydrazide (78)

Yield: 89%. White yellow solid, m.p. 231-233 °C. 1H-NMR (500 MHz, DMSO-d6): δ 11.33




d6): δ 164.8, 164.2, 163.6, 152.2, 147.1, 146.5, 132.4, 132.2, 132.1, 131.4, 130.0, 130.0, 129.2,

129.0, 128.5, 128.1, 127.8, 127.4, 122.4, 118.2, 115.7, 112.4, 112.0, 56.3. HREI-MS: m/z calcd

for C24H17N5O4 [M]+ 439.1281, Found 439.1276.


nitrobenzylidene)benzohydrazide (79)




Hz, 2H, Ar), 7.67 (dd, J = 8.1, 2.7 Hz, 1H, Ar), 7.55 (dd, J = 8.1, 2.6 Hz, 1H, Ar), 13C-NMR

(125 MHz, DMSO-d6): δ 164.8, 164.4, 163.5, 147.6, 143.0, 134.6, 132.8, 132.6, 132.4, 131.7,

130.9, 130.6, 130.3, 130.0, 129.1, 128.9, 128.6, 128.4, 127.4, 127.1, 124.4, 118.8, 112.2. HREI-


2.7.5.16 (E)-N'-(2-cyanobenzylidene)-4-(5-(4-cyanophenyl)-1,3,4-oxadiazol-2-yl)

benzohydrazides (80)



93


(dd, J = 8.1, 2.7 Hz, 1H, Ar), 7.64 (dd, J = 8.1, 2.6 Hz, 1H, Ar), 7.52 (d, J = 7.1 Hz, 1H, Ar).

13C-NMR (125 MHz, DMSO-d6): δ 164.7, 164.2, 163.0, 143.0, 134.3, 133.4, 133.1, 132.9,

132.6, 132.4, 131.1, 130.6, 130.3, 130.0, 129.7, 129.2, 128.5, 128.1, 127.3, 127.2, 118.2, 115.3,

112.2, 111.5. HREI-MS: m/z calcd for C24H14N6O2 [M]+ 418.1178, Found 418.1173.

94

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CHAPTER-03

Synthesis of indole derivatives and their biological activities

Summary

………………………………………………………………………………………………………..

In this chapter we describe the synthesis of three series of indole derivatives and studies

of their β-glucuronidase, urease and α-glucosidase with molecular docking.

………………………………………………………………………………………………………..

3.1 Indole

Indole is a planner bi-cyclic compound in which benzene ring is bonded to pyrole ring.

According to Huckel‟s rules, indole is aromatic in nature having ten π-electrons. Indole

freely undergoes electrophilic substitution reaction analogous to benzene ring due to

delocalization of too much π-electron [1].

Figure-3.1: Basic skeleton of indole

Indole is very reactive in nature with strong acid due to weak basicity like pyrole. On

the basis of molecular orbital calculation, position-3 of indole has maximum electron

density and it is most reactive position for electrophilic substitution reaction [2].

Indole is found as solid in nature but it can be synthesized by bacteria through

biotransformation of tryptophan amino acid. It is present in high concentration in

flowers and also in perfume which give them flowery smell. The tough facial odor of

human face indicates its presence in human face. Adolf Von Baeyer synthesized indole

by the reduction of oxindole in the presence of zinc dust while he also predicted the

100

formula for indole in 1869 [3]. Synthesis, derivatives and applications of indole has been

reported by many researchers [4, 5]. Due to having great biological and pharmacological

applications, the indole skeleton has got more attention and is an attractive motif in

chemistry [6, 7]. This important skeleton is widely found in therapeutic drugs and

natural products because of its physiological significant [8-10]. Biologically potent

heterocyclic moieties in natural and synthetic alkaloids constitute the essential block of

indole [11, 12]. Indole is the important motif in medicinal chemistry [13]. The indole

nucleus is an important motif in many medicinal agents [14]. Synthesis of indole

derivatives and their application has been a subject of interest in many years. Indole

derivatives have been studied for variety of biological activities including anti-

inflammatory [15,16], anti-convulsant [17], anti-tumor [18], anti-microbial [19], anti-

bacterial [20, 21] and anti-fungal [22]. Indole is a significant constituent of perfume [23]

which displayed to defeat carcinogenicity and hepatotoxicity of numerous carcinogens

[24]. Numerous aroma compounds precursor has been synthesized from indole

derivatives [25,26]. Indole diterpene alkaloids show very good insecticidal activity and

found to be effective against tick and flea infestation in cats and dogs [27]. This

invertebrates specific glutamate gated chloride ion networks confirm no toxicity [28]. In

medicinal chemistry, indole moiety is widely used and is deliberated as most important

scaffold [29].

3.2 Biological Importance of Indole

The great biological importance of indole makes it one of central motif in medicinal

chemistry. Indole analogs are reported to possess numerous biological activities i.e.

101

anticancer, antidepressant, analgesic, anti-tubercular, insecticidal, antihypertensive,

antimicrobial, antiinflammatory, antioxidant, anti-viral and antidiabetic activities

[30,31].

3.2.1 Anti-microbial activity

Due to increase in drug resistance such as vancomycin etc. there is a vital need of novel

anti-fungal and anti-bacterial agents.

Deschenes et al., synthesized 6-amido-2-aryindoles (2) which act against histidine kinase

and help in motivating bacterial resistance [32]. Sivaprasad et al., synthesized a new

series of pyrazolyl-bisindoles (3) with antifungal activities [33]. Samosorn et al.,

synthesized 2-aryl-5-nitro-1H-indole (4) and studied their inhibitory potential against

NorA efflux pump of human pathogenic bacterium Staphylococcus aureus, accountable

for multi-drug resistance [34]. Hiari group reported the synthesis and antimicrobial

potential of 3-aryl(heteroaryl)indole (5) against Staphylococcus aureus and Escherichia coli

[35]. 3-(4-Tri-fluoromethyl-2-nitrophenyl) indole was found to be the most active analog

against Staphylococcus aureus and Escherichia coli. Leboho et. al., reported the synthesis of

2-aryl-5-methoxyindoles (6) and evaluated for antimicrobial activities against gram

positive micro-organism Bacillus cereus [36].

102

Figure-3.2: Indole containing analogs as anti-microbial agents

3.2.2 Anti-inflammatory activity

Cyclo-oxygenase (COX) involves in the bio-synthesis of prostaglandin and COX

inhibition provide relief from pain and inflammation symptom. Non-steroidal drugs

like aspirin and ibuprofen which exerts their effect causes the inhibition of COX. COX

found in two isozyme form such as COX-1 and COX-2. COX-1 is always active in

healthy condition while COX-2 is motivated in rheumatoid arthritis and is a target for

drugs to reduce pro-inflammatory prostaglandins production. COX-2 inhibitors are

used to decrease pain, inflammation and therefor have been produced as new anti-

inflammatory drugs. Hu et al., have evaluated 2-phenyl-3-sulfonylphenyl-indole (7) as a

selective and potent COX-2 inhibitor when related with celecoxib under celluar assay

studies [37]. Preethi et al., synthesized 3-pyrazolinyl-indole (8) and were found to be

the most active anti-inflammatory agent with fewer ulcerogenic aptitude and toxicity

103

[38]. Narayana et al., reported the synthesis and antiinflammatory inhibitory potential of

novel series of 2-(6-nitro-1H-indol-2-yl)-1,3,4-oxadiazole (9). The study suggests that

these compounds show potent antiinflammatory inhibitory potential [39]. Radwan et al.,

reported synthesis of 1H-indol-3-yl heterocycles (10) which show active anti-

inflammatory and analgesic activities [40]. Kuduk et al., synthesized 2-aryl-indole (11)

as inhibitor against acid sensing ion channel-3 (ASIC-3) (ASIC-3) which was supposed

to be involved in neurodegenerative and inflammation syndromes [41]. Dharmedra et

al., reported synthesis of sulfonyl-substituted-2-phenyl-1H-indole (12) and evaluated for

anti-inflammatory and anti-bacterial activities. Thirumurugan et al., reported 1H-indol-

3-yl-pyridine-3,5-dicarbonitrile analog (13) and showed active analgesic and anti-

inflammatory activities [42,43].

104

Figure-3.3: Indole containing analogs as anti-inflammatory agents

3.2.3 Anti-tumor activity

Hendricks et al., reported 3-(2-argio-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl)-1H-indol-

1-yl)propyl carbamimidothioate (14) which prevent protein kinase and therefor have

active anti-tumor activity [44]. Zhang et al., reported 2-amino-6-(1-methyl-1H-indol-3-

yl)-4-substituted phenylnicotinonitrile analog (15) exhibit most potent anti-tumor

inhibitory potential against numerous cell lines [45]. Zhang et al., developed a new

series of (E)-3-(4-(2-amino-3-(1H-indol-3-yl)propoxy)phenyl)-N-hydroxyacrylamide (16)

which were found to show histone deacetylase, anti-tumor and anti-proliferative

inhibitory potentials [46].

105

Figure-3.4: Indole containing analogs as anti-tumor agents

3.2.4 Hypocholestrolemic activity

Belimin et al., synthesized 1-(2-(1H-indol-3-yl)ethyl)-3-(morpholinomethyl)urea (17) as

acetyl-co-enzyme-A acetyltransferase inhibitor and their structural change directed to

the growth of new hypocholestrolemic agents [47].

Figure-3.5: Indole containing analogs as hypocholestrolemic agent

3.2.5 Anti-cancer activity

Medarde et al., reported 5-methoxy-3-(3,4,5-trimethoxyphenyl)-1H-arylindole (18) and

studied their cyctotoxic inhibitory potential against numerous cancer cell line [48].

Synthesis of indolopyrrolemaleimides (19) was carried out by Xu et. al., and screened

them for cytotoxicity inhibitory potential against numerous cancer cell line in human

[49]. Bromo-substituted derivatives were found to be the most potent among the series.

Kaufmann et al., synthesized 2-phenylindole-3-carbaldehydes (20) analogs as antitumor

106

agent. They found that these derivatives depress polymerization of tubulin which

inhibits cancer cell growth in breast [50]. Some other aryl indole substituted derivatives

have been synthesized that also inhibit the tubulin polymerization [51]. In 2008, CDK

inhibitors and anticancer agents were designed by Ulrich and company. These were

composed of 3-(4-(5-(1-(2-(dimethylamino)ethyl)-1H-indol-2-yl)pyridin-3-yl)phenoxy)-

N,N-dimethylpropan-1-amine and 1-(2-(dimethylamino)ethyl)-2-(5-(4-(3-

(dimethylamino)propoxy)phenyl)pyridin-3-yl)-1H-indol-5-ol analogs (21) [52]. Wu et al.,

designed and screened 3-aryl-indole analogs (22) for their anti-cancer inhibitory

potential [53]. Hong et al., prepared a new series of several tri-cyclic and tetra-cyclic

indole and studied their anti-cancer inhibitory potential. From the whole study, it was

concluded that compounds having methoxy and hydroxy group present in their

structure were found to be the most potent when studied in vitro against gastric

adenocarcinoma and human nasopharyngeal carcinoma [54]. 2-Amino-4-argio-6-(1H-

indol-3-yl)-4H-pyran-3,5-dicarbonitrile analogs (23) were synthesized by Vidhya

Lakshmi et. al., and studied their antioxidant and anticancer activity [55]. Some of these

analogs showed potent anticancer potential against numerous breast cancer cell lines

when compared with the standard inhibitor. 2-Argio-4-(1H-indol-3-yl)oxazole (24) were

synthesized by Dalip et al., under microwave condition in good yield. These

compounds have found to have better cytotoxicity against cancer cell line in human.

Dalip et al., also screened N-argio-5-methyl-1,3,4-thiadiazol-2-amine compound with

1H-indole (1:1) (25) for anti-cancer inhibitory potential against numerous breast cancer

cell lines [56, 57]. Meric et al., designed a new series of 3-((4-argiopiperazin-1-

107

yl)methyl)-1H-indole (26) which exhibited cytotoxicity against colon and human liver

cancer cell lines [58]. Mac-Donough et al., synthesized (2-(3-hydroxyphenyl)-6-methoxy-

1H-indol-3-yl)(substituted-phenyl)methanone (27) and studied their efficacy and

cytotoxicity for preventing tubulin polymeriization [59].

Figure-3.6: Indole containing analogs as anti-cancer agents

3.2.6 Anti-oxidant activity

Andreadou et al., synthesized new indoles analog having tri-azole moiety (28) in their

structures shows antioxidant properties. They have investigated their anti-ischemic

108

activity. Later Monica et. al., synthesized novel indole having tryptamine and

tryptophan analogs (29) and studied their anti-oxidant inhibitory potential. The study

suggests that these compounds showed different inhibitory potential and their potency

depend on the position of substituents of indole nucleus [60, 61]. A series of indol-3-

ylpyrimidine derivatives (30) were synthesized by Mosaad et al., and evaluate their

antioxidant and antibacterial potential. These analogs possess higher antioxidant

activity and antibacterial activity [62]. In 2013, DPPH radical scavenging inhibitory

potential was carried out for newly synthesized substituted 2-arylindoles (31). Study

suggests that compounds having fluorine in their structures have high potency when

compared with standard drug melatonin [63].

Figure-3.7: Indole containing analogs as anti-oxidants agent

3.2.7 Anti-diabetic activity

Several indole analogs were synthesized and have been screened for insulin and

glucose depressing effect. Analog (32) which have chloro benzyl group showed higher

inhibitory potential of PPARc agent. This indicates lower serum glucose and contributes

109

good anti-diabetic inhibitory potential. These analogs can be used a substitute remedy

for the management of type-II diabetes and other metabolic syndromes [64].

Figure-3.8: Indole containing analog as anti-diabetic agent

3.2.8 Anti-parkinsonian activity

Sunil et al., designed new 2-argio-5-methoxy-1H-indole analog (33) and studied their

anti-parkinsonian activity. Study showed that such type of analog exhibited higher

inhibitory potential [65].

Figure-3.9: Indole containing analog as anti-parkinsonian agent

3.2.9 Anti-viral activity

Abdel Gawad et al., designed (E)-4-((2-(1-(1H-indol-3-

yl)ethylidene)hydrazinyl)methyl)thiazole analog (34) and evaluated for anti-viral

activity. Results showed better activity [66].

110

Figure-3.10: Indole containing analog as anti-viral agent

3.3 Previous approaches towards indoles synthesis

Reaction of 2-bromotrifluoroacetanilide (35) and terminal alkyne (36) in DMF catalyzed

by CuI/L-proline lead to the development of resultant indoles (37) (Scheme-1) [67].

Scheme-3.1: CuI/L-proline-catalyzed synthesis of indoles

The most broad and applicable way to synthesize functionalized indoles 40 is the

reaction of chloroaniline 38 with ketones 39. This is the most efficient way to obtain

functionalized indole analogs (Scheme-2) [68].

Scheme-3.2: Pd-catalyzed synthesis of indole

Thermal rearrangement of 2-aryl-2H-azirines 41 in the presence of xylene lead to the

development of substituted indole-3-carbonitriles 42 (Scheme-3) [69].

111

Scheme-3.3: Synthesis of indole derivatives in the presence of xylene

Intra-molecular amination of amino alkenes 43 and allyl acetate 44 in the presence of

Rhodium catalyst [RuCl2(CO)3]2/dppp, N-methyl piperidine and potassium carbonate

leads to the synthesis of substituted indole derivatives 45 in good yield (Sheme-4) [70].

Scheme-3.4: Ruthenium catalyzed synthesis of indole analogs

2-Alkynylaniline 46 can be annulated by gold catalyst at room temperature to obtained

indole analog 47 in good yields. Ethanol and water were used as co-solvent (Scheme-5).

Furthermore; Synthesis of 3-bromo and 3-iodoindoles is reported to be gold catalyzed

reaction product of 2-alkynylanilines [71].

Scheme-3.5: Gold (III) catalyzed synthesis of indoles

112

3.4 Introduction to bis-indole derivatives

Bis(indolyl)methanes are formed from basic indole unit. Bis(indolyl)methanes are an

important heterocycle possess various biological and pharmacological activities and

play a role in the treatment of chronic fatigue, irritable bowel syndrome and

fibromyalgia [72, 73]. They are also accountable for advancement of beneficial estrogen

metabolism and yield apoptosis in human cancer cells [74]. Bis(indolyl)alkanes was

collected from numerous marine and terrestrial natural source and possess many

biological activities such as coronary dilatory properties, antibacterial activity and

genotoxicity [75]. The bis-indole alkaloid indirubin (48) sometimes exist in human urine

were among early cycline dependent kinasse inhibiitors [76, 77]. It can be used for

treating several diseases including chronic myelocytic leukemia [78]. They also target

aurora kinase, glycogen synthase kinases-3 [79, 80] and as a dioxin receptor [81].

Recently it was studied that several indirubin can delay phosphorrylation and

stimulation of transcription factors state, which leads to down-regulation of survival

factor like Mc-1 and thus cause induction of cells death [82].

Figure-3.11: Structure of biologically active indirubin

Dragmacidine 49 which is bis(indolyl)alkanes demonstrated an emergent class of

bioactive marine natural products derived from deep water sponges [83, 84]. The

113

dragmacidins derivatives having a piperazine linker hold antifungal, cytotoxic and

antiviral activities. In short, dragmacidin analogs are potent inhibitor of serinethreonine

protein phosphatases. Initial studies showed that dragmacidin derivatives are also a

selective inhibitor of PP1 versus PP2A [85].

Figure-3.12: Structure of biologically active dragmacidin

The radioactive metal ions (Gd3+) enclosed in complex (50) of bis-(indolyl)methanes are

convenient contrast agents for radio-imaging and visualization of different organs &

tissues [86].

Figure-3.13: Structure of biologically active bis-(indolyl) methanes complex with

(Gd3+)

114

Protozoa causes many diseases especially in tropical and subtropical region which

increases the mortality and morbidity rate of world death. Some bisindole alkaloids,

like macrocarpamine (51), macralstonine acetate and villastonine (52) possess significant

antiprotozoal activity against Plasmodium falciparum and Entamoeba histolytica [87, 88].

Figure-3.14: Structures of biologically active bis-indole derivatives

3.5 Previous approaches towards bis-(indolyl)methanes synthesis

Bis(indolyl)methanes analog 55 were designed by mixing of indole 53 with

benzaldehyde 54 using zeolite catalyst at room temperature (Scheme-6) [89].

Scheme-3.6: Zeolite mediated synthesis of bis-indole analogs

115

Bis(indolyl)methanes containing Sulfonyl moiety 58 have been synthesized by reacting

indole 57 with acetylene sulfone 56 using Cu(OTf)2 as catalyst in DCM at room

temperature (Scheme-7) [90].

Scheme-3.7: Copper catalyzed synthesis of bisindole derivatives

Bis-(indolyl)methanes 61 have been designed by mixing of indole 59 with aldehyde 60

in acetonitrile at room temperature in the presence of Zirconyl(IV) chloride (Scheme-

8) [91].

Scheme-3.8: Zirconyl (IV) chloride catalyzed synthesis of bis-indole analogs

Bis(indolyl)methanes 64 have been synthesized by the reaction of 2-Arylindole

derivatives 62 with different aldehydes 63 in the presence of glacial acetic acid as

catalyst (Scheme-9) [92].

116

Scheme-3.9: Synthesis of bisindole analogs through M.W.

Reaction of ortho-ethynyl-anilines (65) with substituted aldehydes in acetonitrile

catalyzed by gold (I) under nitrogen atmosphere to give bis-(indolyl)methanes 66 in a

good yield (Scheme-10) [93].

Scheme-3.10: Gold catalyzed synthesis of substituted bisindole analogs

117


3.6.1 Synthesis of bis-indolylmethane based Schiff base derivatives

Methyl 1H-indole-5-carboxylate (67), 20 mmol, and 4-methyl benzaldehyde (68), 10

mmol, were reacted in 50 mL of refluxing acetic acid for 3 hours. The reaction mixture

was poured into crushed ice. Crude bis-indolylmethane-bis-methyl ester (69) was

formed as solid which was filtered, washed with water to remove excess acetic acid,

and then dried. Intermediate (69) was converted into hydrazide by reacting it with 50

mL hydrazine hydrate in 50 mL MeOH. The mixture was refluxed for 6 hours and then

solvents were evaporated in vacuo. The crude product (70) was washed with water and

then dried to give 89.6% yield. The synthesis of novel bis-indolylmethane based Schiff

base derivatives (71-102) was accomplished by reacting different aldehydes with

hydrazide (70) in methanol.

Scheme-3.11: Synthesis of bis-indolylmethane based Schiff base derivatives (71-102)

118

Table-3.1: Different substituents of bis-indolylmethane analogs and their β-

glucuronidase activity (71-102)

Compd No. R IC50 ± SEMa Compd No. R IC50 ± SEMa

71

2.348±0.444 87

1.50 ± 0.1

72

0.30 ±0.04 88

48.50 ± 1.10

73

7.571 ± 0.5 89

22.20 ± 0.50

74

32.80 ± 0.7 90

33.50 ± 0.70

75

2.98± 0.10 91

12.50 ± 0.50

119

76

5.874±0.05 92

2.80 ± 0.1

77

0.1 0 ± 0.05 93

6.90 ± 0.3

78

42.75 ± 1.10 94

5.80 ± 0.20

79

1.14 ± 0.01 95

1.10 ±0.10

80

0.3 ± 0.01 96

1.20 ±0.1

81

0.10 ± 0.01 97

2.20 ±0.10

82

0.20 ± 0.01 98

2.8 ± 0.1

120

83

3.448± 0.10 99

15.8 ± 0.20

84

2.1 ± 0.10 100

32.90 ± 0.60

85

2.67 ± 0.10 101

23.4 ± 0.5

86

43.50 ± 1.05 102

13.10 ± 0.2

D-saccharic acid 1,4 lactone 48.30 ± 1.20 μM

3.6.2 In vitro β-Glucuronidase inhibitory Potential:

We have synthesized thirty-two bis-indolylmethane analogs (71-102) which shows

varying degree of β-glucuronidase inhibition potential ranging in between 0.10 ± 0.01 to

48.50 ± 1.10 μM when compared with the standard drug D-saccharic acid 1,4-lactone

(IC50 value 48.30 ± 1.20 μM). The structure activity relationship was mainly based upon

by bringing different substituents on phenyl part.

Analogs 77 and 81 with IC50 values of 0.10 ± 0.05 and 0.10 ± 0.01 μM were found to be

the most potent and powerful inhibitors among the series having di- and tri- hydroxy

121

substituents on the both sides phenyl rings. Similarly, other di- hydroxyl groups

containing analogs such as 72 (IC50 = 0.30 ± 0.04 μM), 79 (IC50 = 1.14 ± 0.01 μM), 80 (IC50

= 0.3 ± 0.01 μM) and with tri- hydroxyl substitutions such as in the compound 82 (IC50 =

0.20 ± 0.01 μM), the activity pattern in all these compounds is somewhat similar. Minute

differences of inhibition between these compounds are difficult to explain, however, the

obvious thing is that the position of substitutions slightly affects the potential of

inhibition.

If we compare analog 95 (IC50 = 1.10 ± 0.10 μM) having di-chloro substituent at ortho

and para positions on both side phenyl ring with analog 92 (IC50 = 2.80 ± 0.1μM), analog

93 (IC50 = 6.90 ± 0.3 μM) and analog 94 (IC50 = 5.80 ± 0.20 μM) having mono-chloro

substituent on both side phenyl ring but position of the substituents are different. The

slight difference in the activity of these analogs showed that the positions, as well as the

number of substituents greatly, affect the inhibition.

To understand the binding interaction of the most active analogs molecular docking

study was performed.

3.6.3 Molecular Docking

Molecular docking was carried out to study the interactions between the synthesized

compounds and the active site of the enzyme using Auto dock Vina [94]. Keeping in

view the previous molecular docking study importance [95] here in this study, receptor

was treated as rigid while ligands flexible with its active rotatable bonds ranging from 9

to 13 keeping the amide bond non-rotatable. Three-dimensional X-ray crystal structure

of Human-β-glucuronidase was downloaded from protein data bank

122

[www.rcsb.org/pdb] (PDB ID 1bhg). Heteroatoms and chain B were removed from the

dimer using Discovery Studio Visualizer [96] and was saved as pdb file. Prior to

autodocking the pdb file was converted into pdbqt format containing gasteiger charges,

polar hydrogen atoms and merged nonpolar hydrogen atoms using Autodock Tools

[97]. Two-dimensional structures of ligands were sketched and optimized using

Chembi3D Ultra (Version; 14.0.0.117) and were saved in pdb format. After that

gasteiger charges were computed for the entire ligand and „auto dock type‟ was

assigned to each atom using ADT. The search space was defined by constructing a grid

box of size 20 × 20 × 20 Å centered at X = 81.733, Y= 82.591 and Z = 89.436. These

parameters along with pdbqt files of receptor and ligand and with a number of modes

(20) were included in the configurational file. The docking results were analyzed using

Lig Plot+ [98], Poseview [99] and Discovery Studio Visualizer.

3.6.4 Docking Studies

In vitro inhibition potential studies of synthesized compounds against Human-β-

glucuronidase were supported by performing molecular docking studies. Molecular

docking has vast applications in drug finding and development. All the synthesized

compounds were docked against target enzyme and all of them show different

interactions with different residues of the active site. The active site of the protein was

selected based on targeting the key residues i.e. Glu451 and Glu540 [100]. X-ray crystal

structure of Human-β-glucuronidase (PDB ID: 1BHG) downloaded from PDB [101] and

its active site is shown in Figure-3.15.

123

Figure-3.15: Structure of Human-β-glucuronidase (PDB ID: 1BHG) and its active site

(red sphere and zoomed in).

First, interactions of the most active compound 81 (IC50 = 0.10 ± 0.01 µM) were analyzed

with the residues of the target protein. Graphical investigation of the lowest energy

pose illustrated that except one hydroxyl group at the phenyl groups all the remaining

five hydroxyl groups mediated hydrogen bond interactions with the side chain residues

of the active site. In these interactions, the residues acting as hydrogen bond donors

were Asn450, Glu540, Trp528 and Lys606 respectively. While, amino acids acting as

hydrogen bond acceptors were His385, Asn502 and Gln524. Also, another hydrogen

bond interaction was found between the amide nitrogen atom and the hydroxyl group

of Tyr508 acting as hydrogen bond donor. Other side chain residues involved in

hydrophobic contacts were Asp207, His385, Asn484, His509, Tyr508, Trp587 and Thr599

as shown in Figure-3.16 a.

124

Binding mode of compound 82, another active derivative (IC50 = 0.20 ± 0.01 µM)

showed that three hydrogen bond interactions occurred between the ligand and side

chain amino acids including Glu451. All the interacting amino acids acted as a

hydrogen bond acceptors. Carbonyl group of Val410 accepted hydrogen from NH

group of indole ring, the carboxyl group of Glu451 accepted hydrogen from NH of

amide group whereas, carbonyl oxygen of Asn484 accepted hydrogen from the

hydroxyl group attached at the ortho position of the phenyl ring. Other major residues

involved in hydrophobic interactions were Glu451 and Asp207 as shown in Figure-3.16

b.

Figure-3.16: Models of the interaction of 81 (a) and 82 (b) with the binding site of

Human-β-glucuronidase generated by poseview. Dashed lines indicate hydrogen

bond interactions and a green line indicating hydrophobic interactions.

Binding modes of another highly active compound 77 (IC50 = 0.10 ± 0.05 µM) showed

that the compound was involved in various interactions. Strong to medium hydrogen

bond interactions were found between the ligand and receptor residues. Residues

125

acting as hydrogen bond acceptors were His385, Asn484 and Tyr508 while, residues

acting as hydrogen bond donors were Asp207 and Asn450 respectively. Amino acids

involved in hydrophobic interactions with the ligands were Tyr205, Phe206, Glu451,

Asn502, Tyr504 and Trp587 as shown in Figure-3.17 a.

Compound 72 is the fourth most active compound (IC50 = 0.30 ± 0.04 µM) among the

synthesized derivatives. The predicted binding modes of this compound showed strong

to medium hydrogen bond interactions with the side chain amino acids, His385, Val410,

Glu451 and Trp528 were involved in making hydrogen bonds with the hydroxyl groups

attached to phenyl rings, while, Asn484, Tyr504 and His509 were involved in making

hydrogen bonds with carbonyl groups of amides. Also, another hydrogen bond

occurred between Asn484 and -NH- of amide group respectively. Other amino acids

involved in hydrophobic interactions were Phe206, Asn486, Asn502 and Tyr508 as

shown in Figure-3.17 b.

126

Figure-3.17: (a) Two-dimensional scheme of the interactions between 77 and Human

β-glucuronidase; (b) and interactions between 72 and Human β-glucuronidase

generated by Ligplot+. Only more important residues for binding are shown.

Along with two-dimensional analysis, the predicted binding modes of these selected

compounds with the active site of Human-β-D-glucoridase were also visualized in three-

dimensions. And the graphical analysis showed that these compounds fit well into the

active site of the protein as shown in Figure-3.18. Also, these studies showed that the

substituted phenyl groups specifically containing hydroxyl groups are generally

involved in hydrogen bond interactions and the indole groups of these synthesized

compounds are involved in hydrophobic interactions resulting in minimizing the free

energy of binding (FEB) score and making them able to fit well into the active site.

127

Figure-3.18: Stereoview of simulated docking poses of compounds (a) 81; (b) 77; (c) 82

and (d) 72 to Human-β-glucuronidase. Compounds 81, 77, 82 and 72 are shown as

stick models with carbon colored in bright yellow; nitrogen colored in bright blue

and oxygen atoms colored in dark red respectively. Important parts of the enzyme for

interaction were shown as a stick model colored in dark red.

128

3.7 Material and Methods

3.7.1 Synthesis of dimethyl-3,3'-(p-tolylmethylene)bis(1H-indole-5-carboxylate)

Two equimolar of methyl 1H-indole-5-carboxylate (67) (20 mmol) was treated with one

equimolar of 4-methyl benzaldehyde (68) (10 mmol) in 50 mL of acetic acid. The

mixture was refluxed at 200 °C for 3 hours. The reaction completion was monitored by

TLC. After completion of the reaction, the mixture was poured into crushed ice.

Derivative (69) formed was filtered, washed with water to remove excess acetic acid,

and then dried.

3.7.2 Synthesis of 3,3'-(p-tolylmethylene)bis(1H-indole-5-carbohydrazide)

The ester group on compound (69) was converted into hydrazide by reacting it with 50

mL hydrazine hydrate in 50 mL MeOH. The mixture was refluxed at 200 °C for 6 hours

and then rotavaped. The reaction completion was monitored by TLC. After completion

of the reaction, the product (70) was washed with water and then dried to give 89.6%

yield.

3.7.3 Synthesis of the library of bis-indolylmethanes based Schiff base derivatives

(71-102)

The synthesis of novel bis-indolylmethanes based schiff base derivatives was

accomplished by reacting 2 equimolar (0.25 mmol) of different aldehydes with

compound (70) (0.1 mmol) in 10 mL methanol.

129

3.7.3.1 3'-(p-Tolylmethylene)bis(N'-((E)-4-hydroxybenzylidene)-1H-indole-5-


Yield: 84%. Dark brown oily liquid. 1H NMR (500 MHz, DMSO-d6): δ 11.70 (s; 2H), 10.81

(s; 2H), 9.71 (s; 2H), 8.40 (s; 4H), 7.92 (d; J = 8.0 Hz, 2H), 7.69 (dd, J = 8.5, 1.5 Hz; 4H),

7.67 (d, J = 8.4 Hz; 2H), 7.16 (dd, J = 8.2, 1.1 Hz; 2H), 7.08 (dd, J = 8.6, 1.2 Hz; 2H), 6.88

(dd, J = 8.0, 1.3 Hz; 4H), 6.68 (s, 2H), 5.50 (s, 1H), 2.22 (s; 3H). 13C NMR (125 MHz,

DMSO-d6): δ 163.0, 163.0, 160.6, 160.6, 146.5, 146.5, 139.8, 139.8, 135.0, 135.3, 130.5, 130.5,

130.5, 130.5, 128.8, 128.8, 128.8, 128.8, 127.4, 127.4, 126.6, 126.6, 126.2, 126.2,123.2, 123.2,

119.3, 119.3, 116.1, 116.1,116.1, 116.1,112.3, 112.3, 111.3, 111.3, 111.2, 111.2, 54.5, 21.2.

HREI-MS: m/z Calcd for C40H32N6O4 [M]+660.2485; Found: 660.2480.

3.7.3.2 3'3'-(p-Tolylmethylene)bis(N'-((E)-2,4-dihydroxybenzylidene)-1H-indole-5-


Yield: 81%. Brownish oily liquid. 1H NMR (500 MHz, DMSO-d6): δ 11.71 (s; 2H), 11.69 (s;

2H), 10.83 (s; 2H), 10.13 (s; 2H), 8.83 (s; 2H), 8.39 (s; 2H), 7.94 (d, J= 8.0 Hz, 2H), 7.71 (d, J

= 8.4 Hz; 2H), 7.57 (d, J = 8.3 Hz; 2H), 7.54 (s, 2H), 6.36 (d, J = 8.2 Hz; 2H),7.17 (dd, J =

8.0, 1.2 Hz; 2H), 7.09 (dd, J = 8.1, 1.1 Hz; 2H), 6.67 (s, 2H), 5.46 (s, 1H), 2.24 (s; 3H). 13C

NMR (125 MHz, DMSO-d6): δ 163.0, 163.0, 162.3, 162.3, 162.0, 162.0, 146.2, 146.2, 139.7,

139.7, 135.0, 135.3, 133.6, 133.6, 128.7, 128.7, 128.7, 128.7, 127.3, 127.3, 126.6, 126.6, 123.2,

123.2, 119.3, 119.3, 112.0, 112.0, 111.1, 111.1, 111.0, 111.0, 111.0, 111.0, 108.5, 108.5, 103.6,

103.6, 54.5, 21.2. HREI-MS: m/z Calcd for C40H32N6O6 [M]+ 692.2383; Found: 692.2377.

130



Yield: 82%. Light grey sticky liquid. 1H NMR (500 MHz, DMSO-d6): δ 11.74 (s; 2H), 10.83

(s; 2H), 8.52 (s; 2H), 8.49 (s; 2H), 8.40 (s; 2H), 8.12 (d, J= 8.5 Hz, 4H), 7.95 (d, J = 8.7 Hz;

2H), 7.75 (dd, J = 8.0, 8.3 Hz; 2H), 7.63 (d, J = 8.6 Hz; 2H), 7.21 (dd, J = 8.4, 1.4 Hz; 2H),

7.11 (dd, J = 8.5, 1.5 Hz; 2H), 6.62 (s, 2H), 5.44 (s, 1H), 2.25 (s; 3H). 13C NMR (125 MHz,

DMSO-d6): δ 163.0, 163.0, 148.1, 146.5, 146.5, 139.8, 139.8, 135.2, 135.0, 134.4, 133.6, 132.3,

131.3, 129.5, 129.0, 129.0, 129.0, 128.5, 128.7, 128.7, 128.7, 128.6, 127.3, 127.3, 126.5, 126.5,

126.0, 123.2, 123.2, 121.5, 119.2, 119.2, 112.0, 112.0, 111.1, 111.1, 111.0, 111.0, 54.5, 21.2.

HREI-MS: m/z Calcd for C40H30N8O6 [M]+ 718.2288; Found: 718.2281.

3.7.3.4 3,3'-(p-Tolylmethylene)bis(N'-((E)-4-methoxybenzylidene)-1H-indole-5-


Yield: 80%. Dark grey sticky liquid. 1H NMR (500 MHz, DMSO-d6): δ 11.77 (s; 2H), 10.72

(s; 2H), 8.44 (s; 4H), 7.97 (d, J = 8.4 Hz; 2H), 7.73 (d, J = 8.3 Hz; 6H), 7.20 (dd, J = 8.6, 1.2

Hz; 2H), 7.17 (d, J = 8.3 Hz; 4H), 7.11 (dd, J = 8.8, 1.7 Hz; 2H), 6.75 (s, 2H), 5.55 (s, 1H),

3.85 (s; 6H), 2.27 (s; 3H). 13C NMR (125 MHz, DMSO-d6): δ 163.0, 163.0, 162.7, 162.7,

146.6, 146.6, 139.8, 139.8, 135.2, 135.0, 130.0, 130.0, 130.0, 130.0, 128.6, 128.6, 128.6, 128.6,

127.4, 127.4, 126.5, 126.5, 126.2, 126.2, 123.1, 123.1, 119.1, 119.1, 114.2, 114.2, 114.2,

114.2,112.0, 112.0, 111.1, 111.1, 111.0, 111.0, 55.7, 55.7, 54.4, 21.2. HREI-MS: m/z Calcd for

C42H36N6O4 [M]+ 688.2798; Found: 688.2794.

131

3.7.3.5 3,3'-(p-Tolylmethylene)bis(N'-((1E,2E)-3-phenylallylidene)-1H-indole-5-


Yield: 86%. Grey sticky liquid. 1H NMR (500 MHz, DMSO-d6): δ 10.92 (s; 2H), 10.84 (s;

2H), 8.48 (s; 2H), 7.98 (d, J = 6.7 Hz; 2H), 7.92 (d, J = 8.7 Hz; 2H), 7.66 (d, J = 8.6 Hz; 2H),

7.58 (d, J = 8.2; 4H), 7.44 (m; 4H), 7.40 (m; 2H), 7.27 (d, J = 6.9; 2H), 7.21 (dd, J = 8.9, 1.8

Hz; 2H), 7.11 (dd, J = 8.4, 1.9 Hz; 2H), 6.88 (d, J = 7.1 Hz; 2H), 6.76 (s, 2H), 5.42 (s, 1H),

2.28 (s; 3H). 13C NMR (125 MHz, DMSO-d6): δ 163.0, 163.0, 139.5, 139.5, 137.0, 137.0,

135.3, 135.1, 135.1, 135.0, 134.0, 134.0, 128.6, 128.6, 128.6, 128.6, 128.3, 128.3, 128.3, 128.3,

128.2, 128.2, 128.2, 128.2, 127.7, 127.7, 127.3, 127.3, 126.4, 126.4, 126.1, 126.1, 123.3, 123.3,

119.2, 119.2, 112.0, 112.0, 111.1, 111.1, 111.0, 111.0,54.4, 21.2. HREI-MS: m/z Calcd for

C44H36N6O2 [M]+ 680.2900; Found: 680.2897.



Yield: 88%. Light brown oily liquid. 1H NMR (500 MHz, DMSO-d6): δ 11.83 (s; 2H), 10.90

(s; 2H), 9.55 (s; 2H), 8.47 (s; 2H), 8.41 (s; 2H), 7.88 (d, J = 8.1 Hz; 2H), 7.75 (d, J = 8.2 Hz;

2H), 7.35 (d, J = 8.5 Hz; 2H), 7.29 (d, J = 1.9Hz; 2H), 7.20 (dd, J = 8.2, 8.0Hz; 2H), 7.17 (dd,

J = 8.3, 1.4 Hz; 2H), 7.12 (dd, J = 8.7, 1.7 Hz; 2H), 6.96 (d, J = 7.8 Hz; 2H), 6.76 (s, 2H), 5.53

(s, 1H), 2.29 (s; 3H). 13C NMR (125 MHz, DMSO-d6): δ 163.0, 163.0, 158.3, 158.3, 146.5,

146.5, 139.5, 139.5, 138.6, 138.6, 135.2, 135.0, 130.0, 130.0, 128.5, 128.5, 128.5, 128.5, 127.4,

127.4, 126.6, 126.6, 123.3, 123.3, 121.7,121.7, 119.3, 119.3, 118.0, 118.0, 114.7, 114.7, 112.0,

112.0, 111.1, 111.1, 111.0, 111.0, 54.4, 21.2. HREI-MS: m/z Calcd for C40H32N6O4 [M]+

660.2485; Found: 660.2482.

132



Yield: 78%. Brown oily liquid. 1H NMR (500 MHz, DMSO-d6): δ 13.82 (s; 2H), 11.84 (s;

2H), 10.90 (s; 2H), 9.60 (s; 2H), 8.88 (s; 2H), 8.48 (s; 2H), 7.96 (d, J = 8.8 Hz; 2H), 7.66 (d, J

= 8.7 Hz; 2H), 7.19 (dd, J = 8.9, 1.9 Hz; 2H), 7.13 (dd, J = 8.9, 1.8 Hz; 2H), 7.05 (d, J = 7.9

Hz; 2H), 6.78 (d, J = 7.5 Hz; 2H), 6.76 (dd, J = 7.5, 7.8 Hz; 2H), 6.69 (s, 2H), 5.58 (s, 1H),

2.29 (s; 3H). 13C NMR (125 MHz, DMSO-d6): δ 163.0, 163.0, 151.6, 151.6, 146.3, 146.3,

146.1, 146.1, 139.7, 139.7, 135.2, 135.0, 128.5, 128.5, 128.5, 128.5, 127.3, 127.3, 126.4, 126.4,

124.5, 124.5, 123.1, 123.1, 122.5, 122.5, 119.6, 119.6, 119.3, 119.3, 119.1, 119.1, 112.0, 112.0,

111.1, 111.1, 111.0, 111.0, 54.4, 21.2. HREI-MS: m/z Calcd for C40H32N6O6 [M]+ 692.2383;

Found: 692.2380.

3.7.3.8 3,3'-(p-Tolylmethylene)bis(N'-((E)-benzylidene)-1H-indole-5-carbohydrazide)

(78)

Yield: 79%. Light brown solid, m.p. 266-268 °C. 1H NMR (500 MHz, DMSO-d6): δ 11.79

(s; 2H), 10.80 (s; 2H), 8.40 (s; 4H), 7.96 (m; 4H), 7.93 (d, J = 8.9 Hz; 2H), 7.64 (d, J = 8.4 Hz;

2H), 7.56 (m; 6H), 7.15 (dd, J = 8.6, 1.5 Hz; 2H), 7.06 (dd, J = 8.4, 1.6 Hz; 2H), 6.62 (s, 2H),

5.44 (s, 1H), 2.21 (s; 3H). 13C NMR (125 MHz, DMSO-d6): δ 163.0, 163.0, 146.7, 146.7,

139.7, 139.7, 135.2, 135.0, 133.5, 133.5,131.1, 131.1, 129.0, 129.0,129.0, 129.0,128.7, 128.7,

128.7, 128.7, 128.6, 128.6, 128.6, 128.6, 127.2, 127.2, 126.3, 126.3, 123.1, 123.1, 119.2, 119.2,

112.0, 112.0, 111.1, 111.1, 111.0, 111.0, 54.4, 21.2. HREI-MS: m/z Calcd for C40H32N6O2

[M]+ 628.2587; Found: 628.2581.

133



Yield: 83%. Dark brown sticky substance. 1H NMR (500 MHz, DMSO-d6): δ 11.72 (s; 2H),

11.69 (s; 2H), 10.74 (s; 2H), 9.42 (s; 2H), 8.73 (s; 2H), 8.34 (s; 2H), 7.87 (d, J = 8.5 Hz; 2H),

7.63 (d, J = 8.8 Hz; 2H), 7.11 (dd, J = 8.2, 1.1 Hz; 2H), 7.06 (s; 2H), 7.02 (dd, J = 8.1, 1.3 Hz;

2H), 6.71 (d, J = 7.6 Hz; 2H), 6.63 (d, J = 7.8 Hz; 2H), 6.60 (s, 2H),5.41 (s, 1H), 2.22 (s; 3H).

13C NMR (125 MHz, DMSO-d6): δ 163.0, 163.0, 153.5, 153.5, 151.0, 151.0, 146.2, 146.2,

139.7, 139.7, 135.3, 135.0, 128.8, 128.8, 128.8, 128.8, 127.3, 127.3, 126.5, 126.5,123.1, 123.1,

120.3, 120.3, 119.8, 119.8, 119.4, 119.4, 119.2, 119.2, 116.1, 116.1, 112.0, 112.0, 111.1, 111.1,

111.0, 111.0, 54.4, 21.2. HREI-MS: m/z Calcd for C40H32N6O6 [M]+ 692.2383; Found:

692.2378.



Yield: 87%. Brown sticky substance. 1H NMR (500 MHz, DMSO-d6): δ 11.73 (s; 2H), 10.82


2H), 7.21 (d, J = 1.4 Hz; 2H), 7.19 (dd, J = 7.6, 0.9 Hz; 2H), 7.15 (dd, J = 7.9, 1.3 Hz; 2H),

7.09 (dd, J = 8.2, 1.2 Hz; 2H),6.74 (d, J = 7.3 Hz; 2H), 6.60 (s, 2H), 5.40 (s, 1H), 2.25 (s; 3H).

13C NMR (125 MHz, DMSO-d6): δ 163.0, 163.0, 149.4, 149.4,146.5, 146.5, 146.0, 146.0,

139.6, 139.6, 135.2,135.0, 131.2, 131.2, 128.7, 128.7, 128.7, 128.7, 127.2, 127.2, 126.5, 126.5,

123.1, 123.1,123.0, 123.0, 119.2, 119.2, 117.1, 117.1, 116.1, 116.1, 112.0, 112.0,111.1,

111.1,111.0, 111.0,54.4, 21.2. HREI-MS: m/z Calcd for C40H32N6O6 [M]+ 692.2383; Found:

692.2378.

134



Yield: 77%. Brown oily liquid. 1H NMR (500 MHz, DMSO-d6): δ 11.70 (s; 2H), 11.69 (s;

2H), 10.82 (s; 2H), 9.51 (s; 4H), 8.77 (s; 2H), 8.36 (s; 2H), 7.87 (d, J = 8.3 Hz; 2H), 7.65 (d, J

= 8.2 Hz; 2H), 7.12 (dd, J = 8.1, 1.5 Hz; 2H), 7.04 (dd, J = 8.3, 1.3 Hz; 2H), 6.90 (s, 2H),

6.64 (s, 2H), 6.14 (s, 2H), 5.47 (s, 1H), 2.18 (s; 3H). 13C NMR (125 MHz, DMSO-d6): δ

163.0, 163.0, 155.0, 155.0, 152.3, 152.3,146.1, 146.1, 139.7, 139.7, 138.6, 138.6, 135.2, 135.0,

128.8, 128.8, 128.8, 128.8,127.2, 127.2, 126.3, 126.3, 123.3, 123.3,119.1, 119.1, 117.4, 117.4,

112.4, 112.4, 112.0, 112.0,111.1, 111.1,111.0, 111.0,105.0, 105.0,54.4, 21.2. HREI-MS: m/z

Calcd for C40H32N6O8 [M]+ 724.2282; Found: 724.2274.




(s; 2H), 10.37 (s; 2H), 10.24 (s; 4H), 8.35 (s; 2H), 8.33 (s; 2H), 7.85 (d, J = 8.8 Hz; 2H), 7.61

(d, J = 8.5 Hz; 2H), 7.11 (dd, J = 8.2, 1.6 Hz; 2H), 7.07 (s; 4H),7.02 (dd, J = 8.4, 1.5 Hz; 2H),

6.67 (s, 2H), 5.42 (s, 1H), 2.17 (s; 3H). 13C NMR (125 MHz, DMSO-d6): δ 163.8, 163.8,

163.8, 163.8,163.4, 163.4, 163.0, 163.0, 143.1, 143.1,139.6, 139.6, 135.2, 135.0, 128.7, 128.7,

128.7, 128.7, 127.3, 127.3, 126.4, 126.4, 123.1, 123.1, 119.3, 119.3, 112.0, 112.0, 111.1, 111.1,

111.0, 111.0,106.1, 106.1, 96.1, 96.1, 96.1, 96.1, 54.4, 21.2. HREI-MS: m/z Calcd for

C40H32N6O8 [M]+ 724.2282; Found: 724.2274.

135



Yield: 85%. Light brown sticky substance. 1H NMR (500 MHz, DMSO-d6): δ 11.72 (s;

2H), 10.81 (s; 2H),10.23 (s; 2H), 8.83 (s; 2H), 8.41 (s; 2H), 7.94 (d, J = 8.3 Hz; 2H), 7.79 (d, J

= 8.5 Hz; 2H), 7.69 (d, J = 8.8 Hz; 2H), 7.18 (dd, J = 8.4, 1.7 Hz; 2H), 7.07 (dd, J = 8.1, 1.8

Hz; 2H), 6.72 (s, 2H), 6.57 (d, J = 7.8 Hz; 2H), 6.50 (s, 2H), 5.50 (s, 1H), 3.87 (s; 6H), 2.23

(s; 3H). 13C NMR (125 MHz, DMSO-d6): δ 164.2, 164.2, 163.0, 163.0, 162.0, 162.0, 146.1,

146.1, 139.7, 139.7, 135.2, 135.0, 133.3, 133.3, 128.7, 128.7, 128.7, 128.7,127.3, 127.3, 126.6,

126.6, 123.1, 123.1,119.1, 119.1, 112.0, 112.0, 111.1, 111.1, 111.0, 111.0, 110.7, 110.7, 107.1,


720.2696; Found:720.2691.

3.7.3.14 3,3'-(p-Tolylmethylene)bis(N'-((E)-2-hydroxy-5-methoxybenzylidene)-

1H-indole-5-carbohydrazide) (84)

Yield: 76%. Dark grey sticky substance. 1H NMR (500 MHz, DMSO-d6): δ 11.73 (s; 2H),

11.35 (s; 2H), 10.81 (s; 2H), 8.75 (s; 2H), 8.41 (s; 2H), 7.91 (d, J = 8.6 Hz; 2H), 7.70 (d, J =

8.5 Hz; 2H), 7.20 (s; 2H), 7.10 (dd, J = 8.9, 1.3 Hz; 2H), 7.02 (dd, J = 8.6, 1.4 Hz; 2H), 6.80

(d, J = 8.0 Hz; 4H), 6.60 (s, 2H), 5.50 (s, 1H), 3.75 (s; 6H), 2.23 (s; 3H). 13C NMR (125 MHz,

DMSO-d6): δ 163.0, 163.0, 153.2, 153.2, 153.1, 153.1, 146.1, 146.1, 139.7, 139.7, 135.2, 135.0,

128.7, 128.7, 128.7, 128.7, 127.2, 127.2, 126.6, 126.6, 123.1, 123.1, 119.2, 119.2, 119.3, 119.3,

118.1, 118.1, 117.1, 117.1, 113.3, 113.3, 112.0, 112.0, 111.1, 111.1, 111.0, 111.0, 55.6, 55.6,

54.4, 21.2.HREI-MS: m/z Calcd for C42H36N6O6 [M]+ 720.2696; Found: 720.2691.

136



Yield: 84%. Light grey oily liquid. 1H NMR (500 MHz, DMSO-d6): δ 11.71 (s; 2H), 10.76


2H), 7.37 (s; 2H), 7.16 (dd, J = 8.4, 1.6 Hz; 2H), 7.12 (d, J = 7.7 Hz; 2H), 7.01 (d, J = 8.1 Hz;

4H), 6.63 (s, 2H), 5.44 (s, 1H), 3.88 (s; 6H), 2.26 (s; 3H). 13C NMR (125 MHz, DMSO-d6): δ

163.0, 163.0,152.1, 152.1, 147.1, 147.1, 146.4, 146.4, 139.6, 139.6, 135.3, 135.0, 131.1, 131.1,

128.7, 128.7, 128.7, 128.7, 127.4, 127.4, 126.6, 126.6, 123.1, 123.1, 122.7, 122.7, 119.2, 119.2,

115.7, 115.7, 112.1, 112.1, 112.0, 112.0, 111.1, 111.1,111.0, 111.0, 56.0, 56.0, 54.4, 21.2.HREI-

MS: m/z Calcd for C42H36N6O6 [M]+ 720.2696; Found: 720.2691.

3.7.3.16 3,3'-(p-Tolylmethylene)bis(N'-((E)-3-hydroxy-2-iodo-4-

methoxybenzylidene)-1H-indole-5-carbohydrazide) (86)

Yield: 75%. Brownish oily liquid. 1H NMR (500 MHz, DMSO-d6): δ 11.60 (s; 2H), 10.72 (s;

2H), 9.55 (s; 2H), 8.35 (s; 2H), 8.33 (s; 2H), 7.85 (d, J = 9.0 Hz; 2H), 7.65 (d, J = 8.9 Hz; 2H),

7.19 (dd, J = 8.6, 1.2 Hz; 2H), 7.15 (d, J = 7.9 Hz; 2H), 7.07 (dd, J = 8.3, 1.1 Hz; 2H), 6.81

(d, J = 7.8 Hz; 2H), 6.61 (s, 2H), 5.41 (s, 1H), 3.85 (s; 6H), 2.27 (s; 3H). 13C NMR (125 MHz,

DMSO-d6): δ 163.0, 163.0, 155.2, 155.2, 151.7, 151.7, 143.1, 143.1,139.7, 139.7, 135.2, 135.0,

133.6, 133.6, 128.8, 128.8, 128.8, 128.8, 127.3, 127.3, 126.4, 126.4, 124.3, 124.3, 123.1, 123.1,

119.3, 119.3, 112.0, 112.0, 111.1, 111.1, 111.1, 111.1, 111.0, 111.0, 86.1, 86.1, 56.0, 56.0, 54.4,

21.2. HREI-MS: m/z Calcd for C42H34I2N6O6 [M]+ 972.0629; Found: 972.0623.

137



Yield: 79%. Dark grey sticky substance. 1H NMR (500 MHz, DMSO-d6): δ 11.58 (s; 2H),

11.03 (s; 2H), 10.71 (s; 2H), 8.75 (s; 2H), 8.31 (s; 2H), 7.83 (d, J = 8.5 Hz; 2H), 7.60 (d, J =

8.7 Hz; 2H), 7.49 (d, J = 8.0 Hz; 2H), 7.30 (m, 2H), 7.13 (dd, J = 8.3, 1.4 Hz; 2H), 7.09 (dd, J

= 8.0, 1.3 Hz; 2H), 6.97 (d, J = 7.9 Hz; 2H), 6.94 (m, 2H), 6.68 (s, 2H), 5.54 (s, 1H), 2.24 (s;

3H). 13C NMR (125 MHz, DMSO-d6): δ 163.0, 163.0, 157.0, 157.0,146.1, 146.1,139.6, 139.6,

135.2, 135.0, 132.1, 132.1,128.7, 128.7, 128.7, 128.7,127.3, 127.3, 127.3, 127.3, 126.6, 126.6,

123.1, 123.1,121.3, 121.3, 119.2, 119.2, 118.2, 118.2, 117.4, 117.4, 112.0, 112.0, 111.1,

111.1,111.0, 111.0, 54.4, 21.2. HREI-MS: m/z Calcd for C40H32N6O4 [M]+ 660.2485; Found:

660.2479.





2H), 7.91 (d, J = 8.6 Hz; 2H), 7.68 (m, 2H), 7.65 (d, J = 8.2 Hz; 2H),7.58 (m, 2H), 7.12 (dd, J

= 8.5, 1.7 Hz; 2H), 7.04 (dd, J = 8.5, 1.5 Hz; 2H), 6.64 (s, 2H), 5.48 (s, 1H), 2.18 (s; 3H). 13C

NMR (125 MHz, DMSO-d6): δ 163.0, 163.0, 147.7, 147.7, 143.1, 143.1, 139.6, 139.6, 135.2,

135.0, 134.5, 134.5, 131.6, 131.6,130.0, 130.0, 128.7, 128.7, 128.7, 128.7, 128.2, 128.2, 127.4,

127.4, 126.6, 126.6, 124.2, 124.2, 123.1, 123.1, 119.3, 119.3, 112.0, 112.0, 111.1, 111.1, 111.0,

111.0, 54.2, 21.2. HREI-MS: m/z Calcd for C40H30N8O6 [M]+ 718.2288; Found: 718.2285.

138



Yield: 82%. Brown solid, m.p. 284-286 °C. 1H NMR (500 MHz, DMSO-d6): δ 11.54 (s; 2H),

10.69 (s; 2H), 8.34 (s; 2H), 8.32 (s; 2H), 7.83 (d, J = 8.7 Hz; 2H), 7.73 (d, J = 8.1 Hz; 2H),

7.62 (d, J = 8.4 Hz; 2H), 7.25 (m, 6H), 7.09 (dd, J = 8.7, 1.4 Hz; 2H), 7.01 (dd, J = 8.3, 1.2

Hz; 2H), 6.58 (s, 2H), 5.41 (s, 1H), 2.45 (s, 6H), 2.15 (s, 3H). 13C NMR (125 MHz, DMSO-

d6): δ 163.0, 163.0, 143.1, 143.1,139.6, 139.6, 135.2, 135.2, 135.2, 135.0, 131.0, 131.0, 130.8,

130.8,129.1, 129.1, 128.8, 128.8, 128.8, 128.8,127.4, 127.4, 126.6, 126.6, 126.3, 126.3, 125.6,

125.6, 123.1, 123.1, 119.1, 119.1, 112.0, 112.0, 111.1, 111.1, 111.0, 111.0, 54.4, 21.2, 18.8, 18.8.




Yield: 82%. Light brownish solid, m.p. 258-260 °C. 1H NMR (500 MHz, DMSO-d6): δ

11.58 (s; 2H), 10.80 (s; 2H), 8.48 (s; 2H), 8.38 (s; 2H), 7.92 (d, J = 8.4 Hz; 2H), 7.75 (s, 2H),

7.67(d, J = 8.5 Hz; 4H), 7.46 (dd, J = 8.4, 8.1 Hz; 2H), 7.28 (d, J = 8.0 Hz; 4H), 7.14 (dd, J =

8.5, 1.1 Hz; 2H), 7.06 (dd, J = 8.5, 1.3 Hz; 2H), 6.66 (s, 2H), 5.49 (s, 1H), 2.43 (s, 6H), 2.20

(s, 3H). 13C NMR (125 MHz, DMSO-d6): δ 163.0, 163.0, 146.7, 146.7,139.7, 139.7, 138.3,

138.3, 135.2, 135.0, 133.5, 133.5, 131.2, 131.2, 129.3, 129.3, 128.6, 128.6, 128.6, 128.6,128.6,

128.6, 127.4, 127.4, 126.4, 126.4, 126.0, 126.0, 123.1, 123.1, 119.1, 119.1, 112.0, 112.0, 111.1,


656.2900; Found: 656.2898.

139



Yield: 83%. Dark brown solid, m.p. 271-273 °C. 1H NMR (500 MHz, DMSO-d6): δ 11.60

(s; 2H), 10.79 (s; 2H), 8.39 (s; 4H), 7.92 (d, J = 8.8 Hz; 4H), 7.91 (d, J = 8.9 Hz; 2H), 7.68(d,

J = 8.7 Hz; 2H),7.41 (d, J = 8.2 Hz; 4H), 7.13 (dd, J = 8.2, 1.3 Hz; 2H), 7.07 (dd, J = 8.3, 1.1

Hz; 2H), 6.67 (s, 2H), 5.51 (s, 1H), 2.44 (s, 6H), 2.14 (s, 3H). 13C NMR (125 MHz, DMSO-

d6): δ 163.0, 163.0, 146.7, 146.7,140.5, 140.5, 139.4, 139.4, 135.2, 135.0, 130.2, 130.2, 129.0,

129.0, 129.0, 129.0, 128.6, 128.6, 128.6, 128.6, 127.3, 127.3, 126.3, 126.3, 126.0, 126.0, 126.0,

126.0, 123.1, 123.1, 119.1, 119.1, 112.0, 112.0, 111.1, 111.1, 111.0, 111.0, 54.4, 21.2, 21.2, 21.2.




Yield: 81%. Grey oily liquid. 1H NMR (500 MHz, DMSO-d6): δ 11.61 (s; 2H), 10.80 (s; 2H),

8.97 (s; 2H), 8.38 (s; 2H), 7.94 (d, J = 8.7 Hz; 2H), 7.76 (d, J = 8.3 Hz; 2H), 7.69 (d, J = 8.2

Hz; 2H), 7.52 (m, 4H), 7.40 (m, 2H), 7.19 (dd, J = 8.4, 1.5 Hz; 2H), 7.10 (dd, J = 8.6, 1.3 Hz;

2H), 6.71 (s, 2H), 5.52 (s, 1H), 2.16 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ 163.0, 163.0,

138.5, 138.5, 139.6, 139.6, 135.2, 135.0, 134.4, 134.4, 133.6, 133.6, 132.1, 132.1,130.0, 130.0,

128.7, 128.7, 128.7, 128.7, 127.3, 127.3, 127.0, 127.0, 126.7, 126.7, 126.5, 126.5, 123.1, 123.1,

119.3, 119.3, 112.0, 112.0, 111.1, 111.1, 111.0, 111.0, 54.4, 21.2. HREI-MS: m/z Calcd for

C40H30Cl2N6O2 [M]+ 696.1807; Found: 696.1803.

140



Yield: 81%. Light grey sticky substance. 1H NMR (500 MHz, DMSO-d6): δ 11.62 (s; 2H),

10.81 (s; 2H), 8.48 (s; 2H), 8.39 (s; 2H), 7.95 (d, J = 8.6 Hz; 2H), 7.77 (s, 2H), 7.70(d, J = 8.4

Hz; 2H), 7.66 (d, J = 8.1 Hz; 2H),7.56 (d, J = 8.3 Hz; 2H), 7.50 (dd, J = 8.7, 8.5 Hz; 2H), 7.20

(dd, J = 8.7, 1.8 Hz; 2H), 7.11 (dd, J = 8.4, 1.6 Hz; 2H), 6.72 (s, 2H), 5.52 (s, 1H), 2.15 (s,

3H). 13C NMR (125 MHz, DMSO-d6): δ 163.0, 163.0, 146.7, 146.7, 139.8, 139.8, 135.2, 135.0,

135.0, 135.0, 134.3, 134.3, 131.0, 131.0, 130.0, 130.0,128.7, 128.7, 128.7, 128.7,127.4, 127.4,

127.1, 127.1, 127.0, 127.0, 126.6, 126.6, 123.1, 123.1, 119.3, 119.3, 112.0, 112.0, 111.1, 111.1,

111.0, 111.0, 54.4, 21.2. HREI-MS: m/z Calcd for C40H30Cl2N6O2 [M]+ 696.1807; Found:

696.1803.



Yield: 82%. Light grey oily liquid. 1H NMR (500 MHz, DMSO-d6): δ 11.63 (s; 2H), 10.86

(s; 2H), 8.42 (s; 4H), 7.98 (d, J = 8.8 Hz; 2H), 7.85 (d, J = 8.7 Hz; 4H), 7.70 (d, J = 8.6 Hz;


6.76 (s, 2H), 5.56 (s, 1H), 2.23 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ 163.0, 163.0,

146.6, 146.6,1397, 139.7, 1365, 136.5,135.2, 135.0, 131.7, 131.7, 130.5, 130.5, 130.5, 130.5,

128.8, 128.8, 128.8, 128.8, 128.8, 128.8, 128.8, 128.8, 127.4, 127.4, 126.5, 126.5, 123.2, 123.2,

119.2, 119.2, 112.0, 112.0, 111.1, 111.1,111.0, 111.0, 54.4, 21.2. HREI-MS: m/z Calcd for

C40H30Cl2N6O2 [M]+ 696.1807; Found: 696.1803.

141

3.7.3.25 3,3'-(p-Tolylmethylene)bis(N'-((E)-2,4-dichlorobenzylidene)-1H-indole-5-



(s; 2H), 8.97 (s; 2H), 8.44 (s; 2H), 8.01 (d, J = 9.0 Hz; 2H), 7.99 (d, J = 8.7 Hz; 2H), 7.71 (s;

2H), 7.70 (d, J = 8.2 Hz; 2H), 7.53 (d, J = 9.1 Hz; 2H), 7.26 (dd, J = 8.6, 1.4 Hz; 2H), 7.13

(dd, J = 8.7, 1.8 Hz; 2H), 6.77 (s, 2H), 5.59 (s, 1H), 2.25 (s, 3H). 13C NMR (125 MHz,

DMSO-d6): δ 163.0, 163.0, 139.6, 139.6, 138.5, 138.5,135.2, 135.0, 132.5, 132.5, 131.2, 131.2,

129.2, 129.2, 129.1, 129.1, 128.7, 128.7, 128.7, 128.7, 128.0, 128.0, 127.4, 127.4, 127.1, 127.1,

126.6, 126.6, 123.1, 123.1, 119.3, 119.3, 112.0, 112.0, 111.1, 111.1, 111.0, 111.0, 54.4, 21.2.

HREI-MS: m/z Calcd for C40H28Cl4N6O2 [M]+ 764.1028; Found: 764.1024.



Yield: 80%. Brown solid, m.p. 285-287 °C. 1H NMR (500 MHz, DMSO-d6): δ 11.69 (s; 2H),

10.89 (s; 2H), 8.45 (s; 2H), 8.39 (s; 2H), 7.89 (d, J = 8.8 Hz; 2H), 7.80 (d, J = 8.6 Hz; 2H),

7.75 (d, J = 8.5 Hz; 2H), 7.53 (m, 2H), 7.40 (dd, J = 8.2, 1.1 Hz; 2H), 7.30 (m, 2H), 7.21 (dd,

J = 8.3, 1.3 Hz; 2H), 7.14 (dd, J = 8.6, 1.6 Hz; 2H), 6.72 (s, 2H), 5.60 (s, 1H), 2.20 (s, 3H).

13C NMR (125 MHz, DMSO-d6): δ 163.0, 163.0, 159.5, 159.5, 143.1, 143.1, 139.6, 139.6,

135.2, 135.0, 132.3, 132.3, 130.7, 130.7, 128.7, 128.7, 128.7, 128.7, 127.2, 127.2, 126.4, 126.4,

124.2, 124.2, 123.1, 123.1, 119.3, 119.3, 118.0, 118.0, 115.5, 115.5, 112.0, 112.0, 111.1, 111.1,

111.0, 111.0, 54.4, 21.2. HREI-MS: m/z Calcd for C40H30F2N6O2 [M]+ 664.2398; Found:

664.2392.

142





2H), 7.66 (d, J = 8.4 Hz; 2H), 7.63 (m, 2H), 7.53 (m, 2H), 7.44 (dd, J = 8.0, 1.2 Hz; 2H), 7.13


3H). 13C NMR (125 MHz, DMSO-d6): δ 163.0, 163.0, 162.9, 162.9, 146.7, 146.7, 139.7, 139.7,

135.2, 135.2, 135.2, 135.0,130.3, 130.3, 128.8, 128.8, 128.8, 128.8, 127.4, 127.4, 126.6, 126.6,

124.7, 124.7, 123.1, 123.1, 119.1, 119.1, 117.7, 117.7, 114.1, 114.1, 112.0, 112.0, 111.1, 111.1,

111.0, 111.0, 54.4, 21.2. HREI-MS: m/z Calcd for C40H30F2N6O2 [M]+ 664.2398; Found:

664.2392.



Yield: 88%. Brownish solid, m.p. 270-272 °C. 1H NMR (500 MHz, DMSO-d6): δ 11.72 (s;

2H), 10.73 (s; 2H), 8.34 (s; 4H), 7.90 (d, J = 8.7 Hz; 2H), 7.77 (dd, J = 8.5, 1.9 Hz; 4H), 7.69

(d, J = 8.6 Hz; 2H), 7.30 (dd, J = 8.5, 8.2 Hz; 4H), 7.19 (dd, J = 8.2, 1.2 Hz; 2H), 7.09 (dd, J

= 8.5, 1.4 Hz; 2H), 6.67 (s, 2H), 5.49 (s, 1H), 2.17 (s, 3H). 13C NMR (125 MHz, DMSO-d6):

δ 165.0, 165.0, 163.0, 163.0, 146.6, 146.6, 139.8, 139.8, 135.2, 135.0, 130.7, 130.7, 130.7, 130.7,

129.2, 129.2, 128.7, 128.7, 128.7, 128.7, 127.4, 127.4, 126.6, 126.6, 123.1, 123.1, 119.3, 119.3,

115.5, 115.5,115.5, 115.5, 112.0, 112.0, 111.1, 111.1, 111.0, 111.0, 54.4, 21.2. HREI-MS: m/z

Calcd for C40H30F2N6O2 [M]+ 664.2398; Found: 664.2392.

143

3.7.3.29 3,3'-(p-Tolylmethylene)bis(N'-((E)-3-bromo-4-fluorobenzylidene)-1H-


Yield: 76%. Brown sticky substance. 1H NMR (500 MHz, DMSO-d6): δ 11.73 (s; 2H), 10.71

(s; 2H), 8.45 (s; 2H), 8.36 (s; 2H), 7.89 (d, J = 8.8 Hz; 2H), 7.87 (m, 2H), 7.84 (d, J = 8.3 Hz;


7.03 (dd, J = 8.3, 1.4 Hz; 2H), 6.62 (s, 2H), 5.47 (s, 1H), 2.16 (s, 3H). 13C NMR (125 MHz,

DMSO-d6): δ 167.6, 167.6, 163.0, 163.0, 146.6, 146.6, 139.7, 139.7, 135.2, 135.0, 134.0, 134.0,

131.4, 131.4,129.6, 129.6, 128.8, 128.8, 128.8, 128.8, 127.4, 127.4, 126.4, 126.4, 123.1, 123.1,

119.2, 119.2, 117.7, 117.7, 112.0, 112.0, 111.1, 111.1, 111.0, 111.0, 110.0, 110.0, 54.4, 21.2.

HREI-MS: m/z Calcd for C40H28Br2F2N6O2 [M]+ 820.0609; Found: 820.0601.



Yield: 89%. Dark brown oily liquid. 1H NMR (500 MHz, DMSO-d6): δ 10.89 (s; 2H), 10.77

(s; 2H), 9.07 (s; 2H), 8.72 (d, J = 8.6 Hz; 2H), 8.41 (d, J = 8.5 Hz; 2H), 8.36 (s; 2H), 8.33 (s;

2H), 7.84 (d, J = 8.5 Hz; 2H), 7.60 (d, J = 8.8 Hz; 2H), 7.43 (dd, J = 8.5, 8.7 Hz; 2H), 7.12


3H). 13C NMR (125 MHz, DMSO-d6): δ 163.0, 163.0, 151.8, 151.8,149.1, 149.1,143.2, 143.2,

139.7, 139.7, 135.2, 135.0, 133.6, 133.6, 130.3, 130.3, 128.6, 128.6, 128.6, 128.6, 127.2, 127.2,

126.6, 126.6, 123.7, 123.7, 123.1, 123.1, 119.3, 119.3, 112.0, 112.0, 111.1, 111.1, 111.0, 111.0,

54.4, 21.2. HREI-MS: m/z Calcd for C38H30N8O2 [M]+ 630.2492; Found: 630.2487.

144




(s; 2H), 8.61 (d, J = 8.9 Hz; 4H), 8.33 (s; 2H), 8.30 (s; 2H), 7.96 (d, J = 8.8 Hz; 4H), 7.93 (d, J


Hz; 2H), 6.72 (s, 2H), 5.56 (s, 1H), 2.27 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ 163.0,

163.0, 149.1, 149.1, 149.1, 149.1,146.6, 146.6,144.1, 144.1, 139.7, 139.7, 135.2, 135.0, 128.8,

128.8, 128.8, 128.8,127.4, 127.4, 126.6, 126.6, 123.1, 123.1, 120.3, 120.3, 120.3, 1203, 119.3,

119.3, 112.0, 112.0, 111.1, 111.1, 111.0, 111.0, 54.4, 21.2. HREI-MS: m/z Calcd for

C38H30N8O2 [M]+ 630.2492; Found: 630.2487.



Yield: 87%. Brownish sticky substance. 1H NMR (500 MHz, DMSO-d6): δ 10.83 (s; 2H),

10.73 (s; 2H), 8.53 (d, J = 9.2 Hz; 2H), 8.36 (s; 2H), 7.90 (s; 2H), 7.87 (d, J = 8.6 Hz; 2H),

7.82 (d, J = 8.9 Hz; 2H), 7.79 (m, 2H), 7.69 (d, J = 8.8 Hz; 2H), 7.40 (m, 2H), 7.17 (dd, J =

8.5, 1.5 Hz; 2H), 7.07 (dd, J = 8.1, 1.5 Hz; 2H), 6.67 (s, 2H), 5.47 (s, 1H), 2.17 (s, 3H). 13C

NMR (125 MHz, DMSO-d6): δ 163.0, 163.0, 153.5, 153.5, 149.0, 149.0, 144.6, 144.6,139.7,

139.7, 136.0, 136.0, 135.2, 135.0, 128.7, 128.7, 128.7, 128.7, 127.3, 1273, 126.5, 126.5, 126.0,

126.0, 123.1, 123.1, 120.1, 120.1,119.2, 119.2, 112.0, 112.0, 111.1 111.1, 111.0, 111.0, 54.4,

21.2. HREI-MS: m/z Calcd for C38H30N8O2 [M]+ 630.2492; Found: 630.2487.

145

3.8 Synthesis of bis-indolylmethane thiosemicarbazides analogs

Methyl 1H-indole-5-carboxylate (67) (20 mmol, 2 eq.) was treated with 4-methyl

benzaldehyde (68) (10 mmol, 1 eq.) in 50 mL of acetic acid. The mixture was refluxed for

3 hours. After completion as guided by TLC, the crude reaction mixture was poured

into crushed ice. Intermediate (69) formed so was filtered, washed with water to remove

excess of acetic acid and then dried. Intermediate (69) was then converted into

corresponding hydrazide by treating it with excess of hydrazine hydrate in refluxing

MeOH. The product (70) was obtained in excellent yield, washed with plenty of ether,

dried and was employed as such for next reaction. The synthesis of new bis-indole

bearing thiosemicarbazide derivatives was accomplished by reacting the hydrazide (70)

with different isothiocyanates in THF (Scheme-12).

Scheme-3.12: Synthesis of bis-indolylmethanes thiosemicarbazides 103-120

146

Table-3.2: Different substituents and urease activity of the synthesized analogs (103-

120)

Compd.

No

R IC50 ±SEMa Compd.

No

R IC50 ±SEMa

103

1.30 ±0.10 112

3.3 ± 0.1

104

1.80 ± 0.1 113

5.10 ± 0.1

105

2.50 ± 0.5 114

4.20 ± 0.1

106

0.80 ± 0.01 115

8.4 ± 0.20

107

0.98 ± 0.01 116

12.1 ± 0.30

147

108

1.10 ± 0.01 117

7.60 ± 0.20

109

1.20 ± 0.05 118

9.50 ± 0.5

110

2.75 ± 0.10 119

0.50 ± 0.01

111

0.14 ± 0.01 120

18.50 ± 0.90

Thiourea 21.25 0.90 μM

3.8.1 In vitro Urease Inhibition Study

Eighteen derivatives of bis-indolylmethane thiosemicarbazide (103-120) were

synthesized and evaluated for their urease inhibitory potential. These derivatives

displayed varying degree of inhibition in the range of 0.14 ± 0.01 to 18.50 ± 0.90 μM

when compared with the standard inhibitor thiourea having an IC50 value 21.25 ± 0.90

μM. All derivatives showed outstanding urease inhibitory potentials with IC50 values

1.30 ±0.10, 1.80 ± 0.1, 2.50 ± 0.5, 0.80 ± 0.01, 0.98 ± 0.01, 1.10 ± 0.01, 1.20 ± 0.05, 2.75 ±

0.10, 0.14 ± 0.01, 3.3 ± 0.1, 5.10 ± 0.1, 4.20 ± 0.1, 8.4 ± 0.20, 12.1 ± 0.30, 7.60 ± 0.20, 9.50 ±

148

0.5, 0.50 ± 0.01 and 18.50 ± 0.90 μM respectively, which is many folds better than the

standard thiourea. The structure activity relationship was mainly based upon by bring

about different substituents on phenyl ring.

The most active compound among the series was compound 111 (IC50 value 0.14 ± 0.01

μM) having two chloro substituents at meta- and para- positions of the phenyl ring. If we

compare compound 111 with compound 109 (IC50 value 1.20 ± 0.05µM) having one

chloro group at ortho position and compound 110 (IC50 value2.75 ± 0.10µM) also having

one chloro group at para position. All compounds have the same chloro group but the

position as well as number of the chloro group is different at the phenyl ring.

Compound 111 was found to be superior who showed that position as well as number

of substituent also play role in this inhibition (Figure-3.19).

149

Figure-3.19: Comparison of structure activity relationship between compounds 111,

109 and 110

If we compare compound 106 having IC50 value 0.80 ± 0.0 μM with compound 107

having IC50 value 0.98 ± 0.0μM and compound 108 with IC50 value 1.10 ± 0.0 μM, all

three compounds have fluoro group on phenyl ring, but the arrangement of fluoro

group is different in them which confirm that the difference in position of substituents

greatly affect the inhibitory potentials of the compounds (Figure-3.20).

150


107 and 108


having IC50 value 5.10 ± 0.1μM and compound 114 with IC50 value 4.20 ± 0.1 μM, all

three compounds have methyl group on phenyl ring, but the arrangement of methyl

group is different in them which confirm that the difference in position of substituents

greatly affect the inhibitory potentials of the compounds (Figure-3.21).

151


113 and 114


having IC50 value 9.50 ± 0.5μM, both the compounds have nitro group on phenyl ring,

but the arrangement of nitro group is different in them which confirm that the

difference in position of substituents greatly affect the inhibitory potentials of the

compounds (Figure-3.22).

152

Figure-3.22: Comparison of structure activity relationship between compounds 117

and 118

In this study, we observed that either electron withdrawing group (EWG) or electron

donating group (EDG) on phenyl ring showed potential but the slight difference in

potential was mainly affected by the position of the substituent as well as in some cases

the number of substituent also play a role. To understand the binding interaction of the

most active analogs molecular docking study was performed.

3.8.2 Molecular Docking Studies

The binding interactions of synthesized compounds in the active site of urease enzyme

from Jack bean were analyzed performing computational docking studies. X-ray crystal

structure of urease (PDB ID: 4H9M) from Jack bean urease was downloaded from

Protein Data Bank [www.rcsb.org/pdb]. Water molecules and acetohydroxamic acid

were removed using Discovery Studio Visualizer [96]. After that, gasteiger charges (q)

and atom types (t) were added to receptor using Autodock Tools [97] and was saved as

pdbqt file. Structures of synthesized ligands were sketched and their energies were

153

minimized using Chembio3D Ultra (Version; 14.0.0.117). Charges and atom types were

added to ligand molecules and were saved as pdbqt files using Openbabel (ver. 2.4.1)

[102]. Autodock Vina [94] software was used to operate docking calculations. Docking

parameters were set as follow: search box of size 35 × 35 × 35 Å centered at X = 21.546, Y

= -68.863 and Z = -22.802, 50 maximum number of binding modes, maximum energy

difference between best and worst binding mode was set 4 KCal/mol and

exhaustiveness of the global search was set 64 respectively. Docking results were

analyzed using Discovery Studio Visualizer.

3.8.3 Docking Studies

Molecular docking studies were carried out to support the in vitro studies of the

synthesized compounds. Enzyme selected for docking studies was downloaded from

RCSB Protein Data Bank (PDB ID; 4H9M). The size of the grid was selected keeping in

view the size of the ligands and the important residues of the enzyme including Ni901

and Ni902. The urease enzyme and the conformation adopted by the ligands in the

active site are shown in Figure-3.23.

154

Figure-3.23: Urease from Jack bean (Ribbon form in light grey color), active site of

urease (enclosed in green color ring) and zoomed in docked conformation of

compound 111 (yellow sticks along with Ni atoms in red color)

Compound 111 the most active compound (IC50 = 0.14 ± 0.01 µM) was analyzed for its

interactions with side chain amino acids in the active site of urease as shown in Figure-

3.24 a. Hydrogen bond interaction was found between NH of thiourea group next to

phenyl group and carbonyl oxygen of side chain Asp494 (2.17 Å). Interactions which

resulted from variable group (in this case m- p- dichlorophenyl) are metal-acceptor

interaction between Ni902 and chlorine atom present at para position with a bond

length of 3.22 Å, π-Alkyl interactions of His492 and His519 ring with both chlorines

present at para (4.65 Å with His492 and 4.43 Å with His519) and meta (4.63 Å with

His492 and 4.48 Å with His519) positions, between ring of His593 and chlorine atom at

meta position with distance of 5.06 Å and carbon-chlorine interaction (3.59 Å) between

meta chlorine of the second phenyl ring and Ala440. π-Alkyl (4.57 Å) and π-π Stacked

(3.98 Å) interactions were also spotted between phenyl ring of Phe605 and middle

155

phenyl ring of the compound. Other interactions involved in stabilizing the complex

were π-alkyl interaction; between methyl group of Ala636 and variable phenyl ring

(5.30 Å), chlorinated phenyl ring and carbon atoms of Ala440 (4.33 Å) and between

indole ring and middle phenyl ring of compound 111 with side chain Leu523

respectively.

Interactions between the second highly active compound 119 (IC50 = 0.50 ± 0.01 µM) and

active site of urease were analyzed as shown in Figure-3.24 b. Five hydrogen bond

interactions were found between the ligand and side chain amino acids. First, one

fluorine atom of trifluoromethyl group present at para position of variable phenyl ring

mediated hydrogen bond interaction with the NH group of His492 (2.74 Å). Second,

hydrogen bond interactions occurred between the NH of thiourea group and carbonyl

oxygen of Asp494 (2.14 Å). Two hydrogen bond interactions were found between

fluorine atoms of trifluoromethyl group and NH‟s of Arg439 with a distance of 2.56 Å

and 2.71 Å respectively. Last hydrogen bond with a bond distance of 2.08 Å was

established between NH of indole ring and carboxyl oxygen of side chain Glu493.

Interactions mediated by the fluorine atoms were as follow: metal acceptor; two fluorine

interactions between F‟s and both Nickle atoms i.e. Ni901 (2.97 Å) and Ni902 (2.87 Å),

interaction between carbonyl oxygen of Gly550 and two fluorine atoms (3.16 Å and 3.00

Å respectively), halogen (fluorine) interaction between fluorine atoms at the same

phenyl ring and carboxyl oxygens of Asp633 (3.17 Å and 3.18 Å) and two fluorine

nitrogen interactions between fluorine atoms and imidazole nitrogen‟s of side chain

156

His409 and His519 with a distance of 3.70 Å and 3.56 Å were found respectively. Other

interactions almost remained the same as those in the previous one (i.e. compound 111).

Compound 118 with IC50 value of 9.50 ± 0.5 µM (in-between highly and least active

compound) was analyzed for its interactions with the active site of urease enzyme

(Figure-3.24 c). It was found that the parent part (non-variable) was giving the same

interactions as found in active compounds but in addition the oxygen of nitro group at

variable phenyl ring mediated metal bond interactions with both Nickle atoms i.e.

Ni901 (2.46 Å) and Ni902 (2.65 Å) respectively. The NH of thiourea group next to

carbonyl group showed hydrogen bond interaction with the carbonyl oxygen of Asp294

(2.78 Å) and NH of the second thiourea group mediated hydrogen bond interaction

with the carbonyl oxygen of CME592 (non-standard protein residue) with the distance

of 2.13 Å. Hydrogen bond interaction was formed between NH of indole ring and nitro

group present at second phenyl ring and carboxyl oxygen of Glu493 (2.19 Å) and NH

group of Arg439 (2.41 Å) respectively. Other interactions found in this compound were

almost the same as that in highly active compounds. The reason which makes

compound 118 less active than compound 111 and 119 could be the absence of large

number of halogen interactions which were spotted in previously discussed

compounds.

Compound 120, the least active compound among the series (IC50 = 18.50 ± 0.90 µM)

showed four hydrogen bond interactions with the side chain amino acids;

i) Two hydrogen bond interactions between the carbonyl oxygen next to indole

ring and NH groups of Arg609 with the distance of 2.11 Å and 2.49 Å,

157

ii. single hydrogen bond interaction between NH of the thiourea group and

carbonyl oxygen of Asp494 (2.88 Å) and

iii. hydrogen bond interaction occurred between NH of indole ring and carbonyl

oxygen of side chain Arg439 with a distance of 2.54 Å.

Other interactions like π-π, π-Anion and π-Alkyl interactions were almost the same

as shown by the parent part of other highly active compounds (Figure-3.24 d). The

main reason which makes this compound the least active among the series could be

the presence of adamantane and absence of interactions of this compound with the

Nickle atoms and other interactions which were shown by the halogen atoms and

nitro groups present at variable phenyl rings of highly active compounds.

158

Figure-3.24: Docked conformation of synthesized compounds with the side chain

residues, Ligands shown in yellow color, side chain amino acids in dark orange color,

and distances in Angstrom colored in white; a) Interactions of compound 111 (most

active); b) Interactions of compound 119 (second most active); c) Interactions of

compound 118 (activity in-between) and d) Interactions of Compound 120 (least

active among series).

159

3.9. Material and Methods

3.9.1 Synthesis of dimethyl-3,3'-(p-tolylmethylene)bis(1H-indole-5-carboxylate)

2 equimolar of methyl 1H-indole-5-carboxylate (67) (20 mmol) was treated with 1

equimolar of 4-methyl benzaldehyde (68) (10.5 mmol) in 50 mL of acetic acid. The

mixture was refluxed at 200 °C for 3 hours. The reaction completion was monitored by

TLC. After completion of reaction, the mixture was poured into crushed ice. Derivative

(69) formed was filtered, washed with water to remove excess acetic acid, and then

dried.

3.9.2 Synthesis of 3,3'-(p-tolylmethylene)bis(1H-indole-5-carbohydrazide)

The ester group on compound (69) was converted into hydrazide by reacting it with 50

mL hydrazine hydrate in 50 mL MeOH. The mixture was refluxed at 200 °C for 6 hours

and then rotavaped. The reaction completion was monitored by TLC. After completion

of reaction, the product (70) was washed with water and then dried to give 89.6% yield.

3.9.3 Synthesis of the library of new bis-indolylmethanes thiosemicarbazide

derivatives (103-120)

The synthesis of new bis-indolylmethanes bearing thiourea derivatives was

accomplished by reacting 2 equimolar (0.25 mmol) of different isothiocyanates with

compound (70) (0.1 mmol) in THF.


bromophenyl)hydrazine-1-carbothioamide) (103)

Yield: 86%. Light brownish sticky substance. 1H NMR (500 MHz, DMSO-d6) δ 11.30 (s;

2H), 10.76 (s; 2H), 10.3 (s; 2H), 8.35 (s; 2H), 8.33 (s; 2H), 7.88 (d, J = 8.2 Hz, 2H), 7.64 (d, J

160

= 8.4 Hz; 2H), 7.53 (dd, J = 8.5, 1.5 Hz; 4H), 7.47 (dd, J = 8.6, 1.7 Hz; 4H), 7.11 (dd, J = 8.3,

1.2 Hz; 2H), 7.04 (dd, J = 8.2, 1.5 Hz; 2H), 6.63 (s, 2H), 5.46 (s, 1H), 2.17 (s; 3H). 13CNMR

(125 MHz, DMSO-d6): 181.0, 181.0, 164.5, 164.5, 139.7, 139.7, 137.4, 137.4, 135.2, 135.0,

131.8, 131.8, 131.8, 131.8, 131.5, 131.5, 131.5, 131.5, 128.7, 128.7, 128.7, 128.7, 127.3, 127.3,

126.6, 126.6, 123.1, 123.1, 122.5, 122.5, 119.2, 119.2, 112.0, 112.0, 111.1, 111.1, 111.0, 111.0,

54.4, 21.1. HREI-MS: m/z calcd for C40H32Br2N8O2S2, [M]+878.0456; Found:878.0453;

Anal. Calcd for C40H32Br2N8O2S2, C, 54.55; H, 3.66; N, 12.72; Found: C, 54.53; H, 3.64; N,

12.70.



Yield: 86%. Brownish sticky substance. 1H NMR (500 MHz, DMSO-d6) δ 11.29 (s; 2H),

10.75 (s; 2H), 10.2 (s; 2H), 8.34 (s; 2H), 8.32 (s; 2H), 7.87 (d, J = 8.3 Hz, 2H), 7.75 (s, 2H),

7.63 (d, J = 8.2 Hz; 2H), 7.45 (d, J = 8.5 Hz; 2H), 7.40 (dd, J = 8.7 Hz; 2H), 7.12 (dd, J = 8.5,

8.2 Hz; 2H), 7.10 (dd, J = 8.5, 1.6 Hz; 2H), 7.03 (dd, J = 8.4, 1.7 Hz; 2H), 6.62 (s, 2H), 5.45

(s, 1H), 2.16 (s; 3H). 13CNMR (125 MHz, DMSO-d6): 181.0, 181.0, 164.5, 164.5, 139.7,

139.7, 139.1, 139.1, 135.2, 135.0, 130.0, 130.0, 128.7, 128.7, 128.7, 128.7, 127.4, 127.4, 127.3,

127.3, 126.5, 126.5, 125.6, 125.6, 125.4, 125.4, 123.2, 123.2, 123.1, 123.1, 119.2, 119.2, 112.0,

112.0, 111.1, 111.1, 111.0, 111.0, 54.4, 21.1. HREI-MS: m/z calcd for C40H32Br2N8O2S2, [M]+;

878.0456; Found:878.0452; Anal. Calcd for C40H32Br2N8O2S2, C, 54.55; H, 3.66; N, 12.72;

Found: C, 54.54; H, 3.65; N, 12.71.

161



Yield: 86%. Light brown oily liquid. 1H NMR (500 MHz, DMSO-d6) δ 11.27 (s; 2H), 10.73

(s; 2H), 10.1 (s; 2H), 8.32 (s; 2H), 8.30 (s; 2H), 7.85 (d, J = 8.6 Hz, 2H), 7.73 (d, J = 8.7 Hz;

2H), 7.61 (d, J = 8.5 Hz; 2H), 7.59 (d, J = 8.8 Hz; 2H), 7.48 (m, 2H), 7.28 (m, 2H), 7.09 (dd,

J = 8.6, 1.8 Hz; 2H), 7.01 (dd, J = 8.5, 1.5 Hz; 2H), 6.60 (s, 2H), 5.43 (s, 1H), 2.14 (s; 3H).

13CNMR (125 MHz, DMSO-d6): 181.0, 181.0, 164.5, 164.5, 139.7, 139.7, 137.0, 137.0,

135.2, 135.0, 132.0, 132.0, 131.8, 131.8, 130.3, 130.3, 128.7, 128.7, 128.7, 128.7, 128.2, 128.2,

127.3, 127.3, 126.5, 126.5, 123.1, 123.1, 122.3, 122.3, 119.2, 119.2, 112.0, 112.0, 111.1, 111.1,

111.0, 111.0, 54.4, 21.1. HREI-MS: m/z calcd for C40H32Br2N8O2S2, [M]+; 878.0456;

Found:878.0450; Anal. Calcd for C40H32Br2N8O2S2, C, 54.55; H, 3.66; N, 12.72; Found: C,

54.51; H, 3.62; N, 12.68.


fluorophenyl)hydrazine-1-carbothioamide) (106)

Yield: 86%. Brownish solid, m.p. 240-242 °C. 1H NMR (500 MHz, DMSO-d6) δ 11.25 (s;


= 8.4 Hz; 2H), 7.59 (d, J = 8.3 Hz; 2H), 7.15 (m, 2H), 7.08 (dd, J = 8.7, 1.5 Hz; 2H), 7.05 (m,

2H), 7.01 (dd, J = 8.4, 1.6 Hz; 2H), 7.00 (m, 2H), 6.56 (s, 2H), 5.41 (s, 1H), 2.12 (s; 3H).

13CNMR (125 MHz, DMSO-d6): 181.0, 181.0, 164.5, 164.5, 163.0, 163.0, 139.7, 139.7,

135.2, 135.0, 130.1, 130.1, 129.3, 129.3, 128.7, 128.7, 128.7, 128.7, 128.0, 128.0, 127.3, 127.3,

126.5, 126.5, 123.1, 123.1, 120.1, 120.1, 119.2, 119.2, 115.7, 115.7, 112.0, 112.0, 111.1, 111.1,

111.0, 111.0, 54.4, 21.1. HREI-MS: m/z calcd for C40H32F2N8O2S2, [M]+; 758.2058;

162

Found:758.2056; Anal. Calcd for C40H32F2N8O2S2, C, 63.31; H, 4.25; N, 14.77; Found: C,

63.28; H, 4.22; N, 14.74.



Yield: 86%. Dark brown solid, m.p. 226-228 °C. 1H NMR (500 MHz, DMSO-d6) δ 11.22 (s;


= 8.4 Hz; 2H), 7.64 (d, J = 8.0 Hz; 2H), 7.39 (m, 2H), 7.26 (d, J = 8.1 Hz; 2H), 7.15 (dd, J =

8.6, 1.8 Hz; 2H), 7.07 (dd, J = 8.6, 1.2 Hz; 2H), 6.99 (m, 2H), 6.67 (s, 2H), 5.49 (s, 1H), 2.22

(s; 3H). 13CNMR (125 MHz, DMSO-d6): 181.0, 181.0, 164.5, 164.5,163.0, 163.0, 139.7,

139.7, 138.6, 138.6, 135.2, 135.0, 130.5, 130.5, 128.7, 128.7, 128.7, 128.7, 127.3, 127.3, 126.6,

126.6, 123.1, 123.1, 122.0, 122.0, 121.3, 121.3, 119.2, 119.2, 116.3, 116.3, 112.0, 112.0, 111.1,

111.1, 111.0, 111.0, 54.4, 21.1. HREI-MS: m/z calcd for C40H32F2N8O2S2, [M]+; 758.2058;


63.30; H, 4.23; N, 14.75.



Yield: 86%. Light brown solid, m.p. 232-234 °C. 1H NMR (500 MHz, DMSO-d6) δ 11.33 (s;


= 8.1 Hz; 2H), 7.51 (dd, J = 8.2, 1.3 Hz; 4H), 7.17 (dd, J = 8.3, 1.7 Hz; 2H), 7.14 (dd, J = 8.3,


(125 MHz, DMSO-d6): 181.0, 181.0, 164.5, 164.5, 163.0, 163.0, 139.7, 139.7, 135.2, 135.0,

134.0, 134.0, 131.1, 131.1, 131.1, 131.1, 128.7, 128.7, 128.7, 128.7, 127.3, 127.3, 126.5, 126.5,

163

123.1, 123.1, 119.2, 119.2, 115.7, 115.7, 115.7, 115.7, 112.0, 112.0, 111.1, 111.1, 111.0, 111.0,

54.4, 21.1. HREI-MS: m/z calcd for C40H32F2N8O2S2, [M]+; 758.2058; Found:758.2055;

Anal. Calcd for C40H32F2N8O2S2, C, 63.31; H, 4.25; N, 14.77; Found: C, 63.27; H, 4.20; N,

14.71.


chlorophenyl)hydrazine-1-carbothioamide) (109)

Yield: 86%. Brown oily liquid. 1H NMR (500 MHz, DMSO-d6) δ 11.35 (s; 2H), 10.82 (s;

2H), 10.10 (s; 2H), 8.42 (s; 2H), 8.39 (s; 2H), 7.96 (d, J = 8.4 Hz, 2H), 7.94 (d, J = 8.3 Hz,

2H), 7.72 (d, J = 8.4 Hz; 2H), 7.57 (d, J = 8.3 Hz; 2H), 7.44 (m, 2H), 7.29 (m, 2H), 7.19 (dd,

J = 8.5, 1.8 Hz; 2H), 7.12 (dd, J = 8.5, 1.4 Hz; 2H), 6.73 (s, 2H), 5.53 (s, 1H), 2.25 (s; 3H).

13CNMR (125 MHz, DMSO-d6): 181.0, 181.0, 164.5, 164.5, 139.7, 139.7, 135.6, 135.6,

135.2, 135.0, 133.8, 133.8, 131.3, 131.3, 130.1, 130.1, 130.1, 130.1, 128.7, 128.7, 128.7, 128.7,

127.3, 127.3, 126.6, 126.6, 124.2, 124.2, 123.1, 123.1, 119.2, 119.2, 112.0, 112.0, 111.1, 111.1,

111.0, 111.0, 54.4, 21.1. HREI-MS: m/z calcd for C40H32Cl2N8O2S2, [M]+ 790.1467;

Found:790.1465; Anal. Calcd for C40H32Cl2N8O2S2, C, 60.68; H, 4.07; N, 14.15; Found: C,

60.66; H, 4.05; N, 14.13.


chlorophenyl)hydrazine-1-carbothioamide) (110)

Yield: 86%. Dark brown sticky substance. 1H NMR (500 MHz, DMSO-d6) δ 11.38 (s; 2H),

10.86 (s; 2H), 10.12 (s; 2H), 8.43 (s; 2H), 8.40 (s; 2H), 7.97 (d, J = 8.6 Hz, 2H), 7.74 (d, J =

8.5 Hz; 2H), 7.64 (dd, J = 8.8, 1.8 Hz, 4H), 7.42 (dd, J = 8.7, 1.7 Hz; 4H), 7.21 (dd, J = 8.3,


164

(125 MHz, DMSO-d6): 181.0, 181.0, 164.5, 164.5, 139.7, 139.7, 136.5, 136.5, 135.2, 135.0,

133.4, 133.4, 131.0, 131.0, 131.0, 131.0, 129.0, 129.0, 129.0, 129.0, 128.7, 128.7, 128.7, 128.7,

127.3, 127.3, 126.5, 126.5, 123.1, 123.1, 119.2, 119.2, 112.0, 112.0, 111.1, 111.1, 111.0, 111.0,

54.4, 21.1. HREI-MS: m/z calcd for C40H32Cl2N8O2S2, [M]+ 790.1467; Found:790.1462;

Anal. Calcd for C40H32Cl2N8O2S2, C, 60.68; H, 4.07; N, 14.15; Found: C, 60.64; H, 4.03; N,

14.12.

3.9.3.9 2,2'-(3,3'-(p-tolylmethylene)bis(1H-indole-3,5-diyl-5-carbonyl))bis(N-(3,4-

dichlorophenyl)hydrazine-1-carbothioamide) (111)

Yield: 86%. Greyish oily liquid. 1H NMR (500 MHz, DMSO-d6) δ 11.39 (s; 2H), 10.88 (s;

2H), 10.14 (s; 2H), 8.46 (s; 2H), 8.42 (s; 2H), 7.99 (d, J = 8.4 Hz, 2H), 7.65 (s, 2H), 7.73 (d, J


Hz; 2H), 6.77 (s, 2H), 5.58 (s, 1H), 2.29 (s; 3H). 13CNMR (125 MHz, DMSO-d6): 181.0,

181.0, 164.5, 164.5, 139.7, 139.7, 136.5, 136.5, 135.2, 135.2, 135.2, 135.0, 131.1, 131.1, 129.2,

129.2, 129.0, 129.0, 128.7, 128.7, 128.7, 128.7, 127.3, 127.3, 126.5, 126.5, 123.1, 123.1, 121.0,

121.0, 119.2, 119.2, 112.0, 112.0, 111.1, 111.1, 111.0, 111.0, 54.4, 21.1. HREI-MS: m/z calcd

for C40H30Cl4N8O2S2, [M]+ 858.0687; Found:858.0684; Anal. Calcd for C40H30Cl4N8O2S2,

C, 55.82; H, 3.51; N, 13.02; Found: C, 55.80; H, 3.50; N, 13.00.

3.9.3.10 2,2'-(3,3'-(p-tolylmethylene)bis(1H-indole-3,5-diyl-5-carbonyl))bis(N-(o-

tolyl)hydrazine-1-carbothioamide) (112)

Yield: 86%. Light grey solid, m.p. 217-219 °C. 1H NMR (500 MHz, DMSO-d6) δ 10.82 (s;

4H), 10.11 (s; 2H), 8.47 (s; 2H), 8.42 (s; 2H), 7.97 (d, J = 8.6 Hz, 2H), 7.73 (d, J = 8.5 Hz;

2H), 7.28 (dd, J = 8.7, 1.5 Hz; 2H), 7.18 (m 2H), 7.15 (dd, J = 8.4, 1.4 Hz; 4H), 7.12 (m, 2H),

165

7.07 (dd, J = 8.2, 1.4 Hz; 2H), 6.78 (s, 2H), 5.60 (s, 1H), 2.30 (s; 9H). 13CNMR (125 MHz,

DMSO-d6): 181.0, 181.0, 164.5, 164.5, 139.7, 139.7, 136.4, 136.4, 135.8, 135.8, 135.2, 135.0,

130.7, 130.7, 129.4, 129.4, 128.7, 128.7, 128.7, 128.7, 127.3, 127.3, 127.1, 127.1, 126.5, 126.5,

126.1, 126.1, 123.1, 123.1, 119.2, 119.2, 112.0, 112.0, 111.1, 111.1, 111.0, 111.0, 54.4, 21.1,

17.7, 17.7. HREI-MS: m/z calcd for C42H38N8O2S2, [M]+750.2559; Found:750.2557; Anal.

Calcd for C42H38N8O2S2, C, 67.18; H, 5.10; N, 14.92; Found: C, 67.15; H, 5.08; N, 14.90.

3.9.3.11 2,2'-(3,3'-(p-tolylmethylene)bis(1H-indole-3,5-diyl-5-carbonyl))bis(N-(m-


Yield: 86%. Grey solid, m.p. 225-227 °C. 1H NMR (500 MHz, DMSO-d6) δ 11.30 (s; 2H),


8.7 Hz; 2H), 7.45 (s, 2H), 7.40 (d, J = 8.6 Hz; 2H), 7.19 (dd, J = 8.4, 8.1 Hz; 2H), 7.18 (dd, J

= 8.3, 1.5 Hz; 2H), 7.11 (dd, J = 8.5, 1.6 Hz; 2H), 7.02 (d, J = 8.1 Hz; 2H), 6.79 (s, 2H), 5.63

(s, 1H), 2.31 (s; 9H). 13CNMR (125 MHz, DMSO-d6): 181.0, 181.0, 164.5, 164.5, 139.7,

139.7, 138.6, 138.6, 137.2, 137.2, 135.2, 135.0, 128.7, 128.7, 128.7, 128.7, 128.7, 128.7, 127.3,

127.3, 126.6, 126.6, 125.2, 125.2, 125.1, 125.1, 123.4, 123.4, 123.1, 123.1, 119.2, 119.2, 112.0,

112.0, 111.1, 111.1, 111.0, 111.0, 54.4, 21.1, 21.1, 21.1. HREI-MS: m/z calcd for

C42H38N8O2S2, [M]+ 750.2559; Found:750.2554; Anal. Calcd for C42H38N8O2S2, C, 67.18; H,

5.10; N, 14.92; Found: C, 67.16; H, 5.07; N, 14.88.

3.9.3.12 2,2'-(3,3'-(p-tolylmethylene)bis(1H-indole-3,5-diyl-5-carbonyl))bis(N-(p-


Yield: 86%. Dark grey solid, m.p. 236-238 °C. 1H NMR (500 MHz, DMSO-d6) δ 11.42 (s;

2H), 10.89 (s; 2H), 10.15 (s; 2H), 8.50 (s; 2H), 8.47 (s; 2H), 8.03 (d, J = 8.5 Hz, 2H), 7.80 (d,

166

J = 8.6 Hz; 2H), 7.41 (dd, J = 8.5, 1.4 Hz; 8H), 7.22 (dd, J = 8.5, 1.4 Hz; 2H), 7.14 (dd, J =

8.3, 1.4 Hz; 2H), 6.73 (s, 2H), 5.61 (s, 1H), 2.36 (s, 6H), 2.31 (s; 3H). 13CNMR (125 MHz,

DMSO-d6): 181.0, 181.0, 164.5, 164.5, 139.7, 139.7, 137.0, 137.0, 135.4, 135.4, 135.2, 135.0,

129.1, 129.1, 129.1, 129.1, 128.7, 128.7, 128.7, 128.7, 127.3, 127.3, 126.5, 126.5, 126.2, 126.2,

126.2, 126.2, 123.1, 123.1, 119.2, 119.2, 112.0, 112.0, 111.1, 111.1, 111.0, 111.0, 54.4, 21.1,

21.1, 21.1. HREI-MS: m/z calcd for C42H38N8O2S2, [M]+ 750.2559; Found:750.2552; Anal.



methoxyphenyl)hydrazine-1-carbothioamide) (115)


(s; 2H), 8.52 (s; 2H), 8.47 (s; 2H), 8.05 (d, J = 8.7 Hz, 2H), 7.78 (d, J = 8.6 Hz, 2H), 7.73 (d, J

= 8.8 Hz; 2H), 7.22 (m, 2H), 7.19 (dd, J = 8.6, 1.7 Hz; 2H), 7.14 (m, 2H), 7.11 (dd, J = 8.1,

1.3 Hz; 4H), 6.81 (s, 2H), 5.63 (s, 1H), 3.88 (s, 6H), 2.33 (s; 3H). 13CNMR (125 MHz,

DMSO-d6): 181.0, 181.0, 164.5, 164.5, 154.4, 154.4, 139.7, 139.7, 135.2, 135.0, 128.7, 128.7,

128.7, 128.7, 128.5, 128.5, 127.3, 127.3, 126.6, 126.6, 125.3, 125.3, 125.1, 125.1, 123.1, 123.1,

121.2, 121.2, 119.2, 119.2, 112.8, 112.8, 112.0, 112.0, 111.1, 111.1, 111.0, 111.0, 55.7, 55.7,

54.4, 21.1. HREI-MS: m/z calcd for C42H38N8O4S2, [M]+ 782.2457; Found:782.2456; Anal.



methoxyphenyl)hydrazine-1-carbothioamide) (116)

Yield: 86%. Dark grey sticky substance. 1H NMR (500 MHz, DMSO-d6) δ 11.50 (s; 2H),


167

8.6 Hz; 2H), 7.27 (s, 2H), 7.22 (dd, J = 8.6, 8.8 Hz; 2H), 7.19 (d, J = 8.2 Hz; 2H), 7.17 (dd, J

= 8.4, 1.5 Hz; 2H), 7.13 (dd, J = 8.3, 1.5 Hz; 2H), 6.84 (d, J = 8.0 Hz; 2H), 6.81 (s, 2H), 5.71

(s, 1H), 3.78 (s, 6H), 2.34 (s; 3H). 13CNMR (125 MHz, DMSO-d6): 181.0, 181.0, 164.5,

164.5, 160.7, 160.7, 139.7, 139.7, 138.0, 138.0, 135.2, 135.0, 130.1, 130.1, 128.7, 128.7, 128.7,

128.7, 127.3, 127.3, 126.5, 126.5, 123.1, 123.1, 119.2, 119.2, 118.7, 118.7, 116.7, 116.7, 112.0,

112.0, 111.1, 111.1, 111.0, 111.0, 110.4, 110.4, 55.7, 55.7, 54.4, 21.1. HREI-MS: m/z calcd for

C42H38N8O4S2, [M]+ 782.2457; Found:782.2453; Anal. Calcd for C42H38N8O4S2, C, 64.43; H,

4.89; N, 14.31; Found: C, 64.40; H, 4.85; N, 14.30.


nitrophenyl)hydrazine-1-carbothioamide) (117)

Yield: 86%. Brownish solid, m.p. 246-248 °C. 1H NMR (500 MHz, DMSO-d6) δ 11.52 (s;

2H), 10.91 (s; 2H), 10.19 (s; 2H), 8.60 (s, 2H), 8.54 (s; 2H), 8.51 (s; 2H), 7.97 (d, J = 9.0 Hz,

2H), 7.95 (d, J = 8.7 Hz, 2H), 7.89 (d, J = 8.9 Hz, 2H), 7.75 (d, J = 8.4 Hz; 2H), 7.63 (dd, J =

8.8, 8.6 Hz, 2H), 7.25 (dd, J = 8.5, 1.6 Hz; 2H), 7.17 (dd, J = 8.2, 1.4 Hz; 2H), 6.80 (s, 2H),

5.67 (s, 1H), 2.35 (s; 3H). 13CNMR (125 MHz, DMSO-d6): 181.0, 181.0, 164.5, 164.5,

148.0, 148.0, 139.7, 139.7, 138.2, 138.2, 135.2, 135.0, 132.5, 132.5, 129.6, 129.6, 128.7, 128.7,

128.7, 128.7, 127.3, 127.3, 126.5, 126.5, 123.1, 123.1, 119.8, 119.8, 119.2, 119.2, 119.2, 119.2,

112.0, 112.0, 111.1, 111.1, 111.0, 111.0, 54.4, 21.1. HREI-MS: m/z calcd for C40H32N10O6S2,

[M]+ 812.1948; Found:812.1945; Anal. Calcd for C40H32N10O6S2, C, 59.10; H, 3.97; N,

17.23; Found: C, 59.09; H, 3.95; N, 17.21.

168


nitrophenyl)hydrazine-1-carbothioamide) (118)

Yield: 86%. Light brownish solid, m.p. 234-236 °C. 1H NMR (500 MHz, DMSO-d6) δ

11.97 (s; 2H), 10.84 (s; 2H), 10.18 (s; 2H), 8.58 (s; 2H), 8.54 (s; 2H), 8.18 (dd, J = 8.9, 1.8 Hz,

4H), 8.03 (d, J = 8.4 Hz, 2H), 7.75 (dd, J = 8.7, 1.7 Hz, 4H), 7.70 (d, J = 8.7 Hz; 2H), 7.26

(dd, J = 8.6, 1.3 Hz; 2H), 7.19 (dd, J = 8.4, 1.5 Hz; 2H), 6.82 (s, 2H), 5.73 (s, 1H), 2.36 (s;

3H). 13CNMR (125 MHz, DMSO-d6): 181.0, 181.0, 164.5, 164.5, 144.4, 144.4, 143.8, 143.8,

139.7, 139.7, 135.2, 135.0, 128.7, 1287, 128.7, 128.7, 127.3, 127.3, 126.5, 126.5, 124.7, 124.7,

124.7, 124.7, 124.1, 124.1, 124.1, 124.1, 123.1, 123.1, 119.2, 119.2, 112.0, 112.0, 111.1, 111.1,

111.0, 111.0, 54.4, 21.1. HREI-MS: m/z calcd for C40H32N10O6S2, [M]+ 812.1948;

Found:812.1942; Anal. Calcd for C40H32N10O6S2, C, 59.10; H, 3.97; N, 17.23; Found: C,

59.06; H, 3.93; N, 17.20.


(trifluoromethyl)phenyl)hydrazine-1-carbothioamide) (119)

Yield: 86%. Dark brown sticky substance. 1H NMR (500 MHz, DMSO-d6) δ 11.55 (s; 2H),


8.5 Hz; 2H), 7.64 (dd, J = 8.6, 1.7 Hz, 4H), 7.45 (dd, J = 8.5, 1.6 Hz, 4H), 7.30 (dd, J = 8.4,


(125 MHz, DMSO-d6): 181.0, 181.0, 164.5, 164.5, 141.6, 141.6, 139.7, 139.7, 135.2, 135.0,

132.4, 132.4, 128.7, 128.7, 128.7, 128.7, 127.3, 127.3, 126.7, 126.7, 126.7, 126.7, 126.5, 126.5,

125.3, 125.3, 125.3, 125.3, 124.0, 124.0, 123.1, 123.1, 119.2, 119.2, 112.0, 112.0, 111.1, 111.1,

111.0, 111.0, 54.4, 21.1. HREI-MS: m/z calcd for C42H32F6N8O2S2, [M]+ 858.1994;

169


58.71; H, 3.73; N, 13.02.

3.9.3.18N-((1s,3s)-adamantan-1-yl)-2-(3-((5-(2-(((3s,5s,7s)-adamantan-1-

yl)carbamothioyl)hydrazine-1-carbonyl)-1H-indol-3-yl)(p-tolyl)methyl)-1H-indole-5-

carbonyl)hydrazine-1-carbothioamide (120)


(s; 2H), 8.59 (s; 2H), 8.55 (s; 2H), 8.05 (d, J = 8.4 Hz, 2H), 7.85 (d, J = 8.2 Hz; 2H), 7.38 (s;

2H), 7.31 (dd, J = 8.2, 1.3 Hz; 2H), 7.25 (dd, J = 8.1, 1.4 Hz; 2H), 6.86 (s, 2H), 5.72 (s, 1H),

2.39 (s; 7H), 1.99 (s, 8H), 1.90 (s, 6H), 1.79 (s, 12H). 13CNMR (125 MHz, DMSO-d6):

184.1, 184.1, 164.5, 164.5, 139.7, 139.7, 135.2, 135.0, 128.7, 128.7, 128.7, 128.7, 127.3, 127.3,

126.5, 126.5, 123.1, 123.1, 119.2, 119.2, 112.0, 112.0, 111.1, 111.1, 111.0, 111.0, 54.4, 51.6,

51.6, 41.1, 41.1, 41.1, 41.1, 41.1, 41.1, 36.4, 36.4, 36.4, 36.4, 36.4, 36.4, 30.2, 30.2, 30.2, 30.2,

30.2, 30.2, 21.1. HREI-MS: m/z calcd for C48H54N8O2S2, [M]+838.3811; Found:838.3808;

Anal. Calcd for C48H54N8O2S2, C, 68.71; H, 6.49; N, 13.35; Found: C, 68.70; H, 6.46; N,

13.33.

170

3.10 Synthesis of tris-indole analogs

Indole (2 mmol) (121) was mixed with methyl 4-formyl benzoate (1mmol) (122) in acetic

acid and refluxed for 6 hours. Reaction completion was monitored by TLC. The

intermediate (1mmol) (123) obtained in the first step was further treated with hydrazine

hydrate (1mmol) in methanol and reflux for 6 hours. After reaction completion, the

indole based hydrazide (1mmol) (124) was obtained in good yield which was further

treated with substituted indole (1mmol) (125) in triethylamine as solvent in the presence

of phosphorus trichloride to get the desired product of tris-indole-oxadiazole hybrid

analogs (126-146). Different spectroscopic techniques such as EI-MS, 1HNMR and

13CNMR were used to determine the structure of all analogs.

Scheme-3.13: Synthesis of trisindole analogs (126-146)

171

3.10.1 α-Glucosidase inhibitory activity

All compounds displayed superior α-glucosidase inhibitory activities having IC50 value

in the range of 2.00 ± 0.01-292.40 ± 3.16 μM as compared to standard acarbose (IC50 =

895.09 ± 2.04 µM). All structural features of molecules such as indole ring, oxadiazole

ring and different substitutions “R” at indole ring are apparently played their role in the

inhibitory activity; however, variation in the inhibitory potential is attributed by the

type of substitutions and their respective positions.

Table-3.3: α-Glucosidase inhibitory activity of synthetic compounds (126-146)

Compound R1 R2 R3 R4 R5 IC50 (µM ± SEMa)

126 H H CN Me H 119.80 ± 1.9

127 H H CN H H 143.60 ± 2.05

128 H H CN H Me 150.50 ±2.17

129 Me H H Me H 71.80 ± 1.87

130 Me H H H Me 45.1 ± 1.7

131 Me H H H H 82.00 ± 1.84

132 H Ph H H H 292.40 ± 3.16

172

133 H Ph H Me H 248.50 ±3.05

134 H Ph H H Me 286.50 ± 3.15

135 H H H Me H 29.10 ± 0.76

136 H H H H H 19.80 ± 0.50

137 H H H H Me 43.60 ± 0.85

138 Me Me H Me H 22 ± 0.37

139 Me Me H H Me 31.80 ± 0.47

140 Me Me H H H 25.1 ± 0.40

141 H H Cl Me H 2.00 ± 0.01

142 H H Cl H Me 2.40 ± 0.01

143 H H Cl H H 3.50 ± 0.01

144 H H Br Me H 15 ± 0.15

145 H H Br H Me 17.6 ± 0.20

146 H H Br H H 19.10 ± 0.25

Standardb Acarboseb 895.09 ± 2.04

µM

SEMa (Standard error mean); Acarboseb (Standard inhibitor for α-Glucosidase inhibitory activity)

3.10.2 Structure-activity relationship (SAR)

Limited structure-activity relationship (SAR) was rationalized by looking at the

substitution pattern (type and their respective position of R1-R5) on indole moiety.

Compound 136 (IC50 = 19.80 ± 0.50 µM) with no substitutions on rings “A-C”, showed

forty-five fold enhanced inhibitory activity as compared to standard acarbose (IC50 =

173

895.09 ± 2.04 µM). Comparison of its activity with the structurally similar compound

135 (IC50 = 29.10 ± 0.76 µM) with an extra methyl group as R4 on ring “C”, showed

decreased activity. However, switching of methyl group from nitrogen to the next

carbon as R5 as in compound 137 (IC50 = 43.60 ± 0.85 µM), leads to further decline in the

activity (Figure-3.25).

Figure-3.25: Structure-activity relationship of compounds 135-137

Compounds 141-143 with the chloro substitutions as R3 showed most potent activity as

compared to the other analogs of the series. It means that chloro groups are strongly

interacting with the active site of enzyme. Amongst the chloro substituted derivatives,

compound 141 (IC50 = 2.00 ± 0.01 µM) with the methyl group as R4, was found to be

more than four hundred times better activity as compared to standard acarbose (IC50 =

895.09 ± 2.04 µM). Its positional isomer 142 (IC50 = 2.40 ± 0.01 µM) with methyl as R5,

showed almost similar activity. However, lack of methyl on ring “C”, leads to slight

decline in the activity. Slight enhanced activity of compounds 141 and 142 might be due

to extra hydrophobic interaction by the methyl group with the active site of enzyme

(Figure-3.26).

174


If, we compare the activity of chloro substituted compounds 141-143 with the bromo

substituted derivatives 144-146 a decreased activity was observed. The decreased

activity might be due to not strong interaction of bromo groups with the active site of

enzyme. Figure-3.27 displayed that compound 146 (IC50 = 19.10 ± 0.25 µM) which

doesn‟t have any methyl group on indole ring showed slightly less inhibitory activity as

compared to methyl substituted analogs 144 (IC50 = 15.00 ± 0.15 µM) and 145 (IC50 = 17.6

± 0.20 µM). It indicated that slight decreased activity of compound 146 might be due to

less hydrophobic interactions with the active site (Figure-3.27).


Amongst the methyl substituted compounds, compound 130 (IC50 = 45.1 ± 1.7 µM) with

methyl substitutions as R1 and R5, showed twenty times better activity than standard

acarbose (IC50 = 895.09 ± 2.04 µM). Its positional isomer 129 (IC50 = 71.80 ± 1.87 µM)

175

showed decreased inhibitory activity. Might be compound 129 attained the

conformation which is not very well fit into the active site. Another compound 131 (IC50

= 82.00 ± 1.84 µM) which lacks methyl on ring “C”, demonstrated further decreased

activity might be due to the less hydrophobic interactions. Figure-3.28 displayed that

dimethyl substitutions on rings “A” and “B” in compounds 138 (IC50 = 22.00 ± 0.37 µM),

139 (IC50 = 31.80 ± 0.47 µM), and 140, (IC50 = 25.1 ± 0.40 µM) demonstrated better

inhibitory activity as compared to mono-methyl substituted derivatives 129-131

(Figure-3.28).

Figure-3.28: Structure-activity relationship of compounds 129-131 and 138-140

In case of compounds with cyano substitutions as R3 on rings “A” and “B”, compound

126 (IC50 = 119.80 ± 1.9 µM) with methyl group as R4, found to be seven-fold more active

than standard acarbose (IC50 = 895.09 ± 2.04 µM). Lack of methyl group in compound

127 (IC50 = 143.60 ± 2.05 µM), showed decreased activity, might be due to the decreased

176

hydrophobic interactions with the active site. However, compound 128 (IC50 = 150.50 ±

2.17 µM) showed further decline in the activity, might be compound 128 attained the

conformation which is not fitted well into the active site (Figure-3.29).


Compounds with the phenyl substitutions on rings “A” and “B”, were found to be the

least active analogs but still they are more potent than standard. The comparatively less

activity might be due the steric hindrance created by the phenyl ring to bind with the

active site of enzyme (Figure-3.30).


In nut shell, the whole series was found to have superior inhibitory activity than the

standard acarbose. All structural features are contributed in the activity; however,

comparatively less activity for some derivatives is due to substitutions effects.

177

However, in order to rationalize the binding interactions of molecules into the active

site, molecular docking studies was conducted.


Docking studies were performed using Glide: A complete solution for ligand-receptor

docking in small molecule drug discovery suite [103]. Receptor grid generation was

done on the α-glucosidase protein structure (pdb id: 3top) were the grid box was set on

acarbose binding site as center with 12 Å radius. The standard precision (SP) mode was

chosen during the Glide docking process and Glide gscore was considered during

analysis. While, GOLD (Genetic Optimization for Ligand Docking) version 5.1 [104] was

used as docking validation tool. Gold scoring function was chosen as a fitness function.

The genetic algorithm (GA) with search efficiency of 100% was set. The specified

centroid was acarbose inhibition site on α-glucosidase protein structure (pdb id: 3top)

with a cavity of 12 A radius defined as active site. Results divergent by less than 1.5 Å

in ligand all atom RMSDs were clustered together. Best clusters and top rank scored

binding mode was analyzed in Pymol [105] and Maestro visualization.

3.10.4 Docking studies

The present molecular docking studies using Glide for twenty-one tris-indole

derivatives along with the substrate acarbose were carried out targeting the C-terminal

domain of the human intestinal α-glucosidase and the binding mode of each compound

was analyzed. On the basis of docked Glide g score and binding mode orientation, the

compounds interactions (hydrogen bonding, hydrophobic and π-π interactions) pattern

were analyzed and identified. From the docking studies, it was observed that all the

178

active tris-indole derivatives showed considerable binding interactions within the active

site (acarbose inhibition site) of α-glucosidase.

The binding modes of the compounds were clear enough to discriminate the difference

in the binding mode between active and least active compounds. The active and

moderately active compounds (compounds 141, 142, 143, 144, 146, 145, 136, 138, 140,

135, and 139) binding mode were all oriented more or less in similar fashion, while in

contrast, all the least active compounds (compound 129, 131, 126, 127, 128, 133, 134, and

132) binding mode were all oriented differently from the active ones as shown in

Figure-3.31 a.

The topmost four active compounds in this series, i.e. 141, 142, 143, and 144 bind

relatively stronger within the C-terminal domain of α-glucosidase. Comparison of the

binding mode of the most active compound 141 and the acarbose (substrate) show that

there is a close similarity in the binding mode as shown in Figure-3.31 b. While

compound 141 forms a better molecular interaction network with α-glucosidase with

high affinity (2.00 ± 0.01 µM), while acarbose exhibits fairly similar binding mode but

considerably lower affinity (856.45 ± 5.60 µM) towards α-glucosidase. It was hard to

determine any definite coherence between the Glide g score (Table-3.4) and IC50 value

of the tris-indole series but there is a stable binding mode of active compounds showing

high binding affinities with the C-terminal domain of α-glucosidase in comparison with

substrate acarbose. The binding mode of the top most four active compounds 141, 142,

143, and 144 are discussed in detail.

179

Figure-3.31: Comparison of interaction between tris-indole series and acarbose in the

binding site of α-glucosidase. a) The active and moderately active tris-indole

compounds are circled in red dotted line, while the least active tris-indole

compounds are circled in blue dotted line. Acarbose binding mode is shown in blue

stick. b) Shows the binding mode comparison of acarbose in blue color stick with

compound 141 in olive color stick.

Docking analysis of the binding mode of the most active compound 141 (IC50 = 2.00 ±

0.01 µM) shows that compound 141 adopts a binding mode that well fit the entire

furrow of the binding site of α-glucuronidase. One among the tris-indole indole ring

nitrogen forms hydrogen bond with the Gln1158, while the Pro1159 and Lys1164 form

hydrophobic interaction with the same ring. Furthermore, the second indole ring forms

hydrophobic interaction with side chain of Lys1460, Asp1370 and with aromatic indole

ring of Trp1369. Additionally, the centered oxadiazole group forms π-π stacking with

the phenyl ring of Phe1560 side chain. There is a pool of hydrophobic residues such as

180

Phe1559, Trp1523, Trp1418, Met1421, Ile1315, Ile1280, Trp1355 and Trp1251 forms

hydrophobic interaction with the methyl indole ring. Likewise, the other indole ring

forms hydrophobic interaction with side chain of Asp1526, Arg1501, Asp1420, Asp1279,

and Asp1157 (Figure-3.32 a).

The second most active in this series is compound 142 (IC50 = 2.40 ± 0.01 µM). One

among the tris-indole indole ring, which is chloro-indole ring forms π-π stacking with

the phenyl ring of Phe1560, while the nitrogen of the indole group forms hydrogen

bond with Asp1526. Subsequently, the Pro1159, Arg1510, Tyr1167, Phe1559, Met1421,

Trp1355, Tyr1251 forms hydrophobic interaction. The other chloro-indole ring forms

hydrophobic interaction with Ile1587 and Thr1586. Furthermore, the centered benzene

rings of compound 142 forms π-π stacking with Trp1369 and also form hydrophobic

interaction with side chain of Asp1157. Moreover, the methyl indole at the terminal end

forms hydrophobic interaction with series of residues such as Ser1426, Phe1427,

Asn1429, Lys1460, Val1428, Arg1156, Arg1455, Ser1452, and Ser1459 as in Figure-3.32 b.

The third most active compound in the series is compound 143 (IC50 = 3.50 ± 0.01 µM).

The tris-indole, indole ring nitrogen forms hydrogen bond with the Asp1420, while the

Arg1510 and Tyr1251 form π-π stacking with the indole ring. While the same forms

hydrophobic interaction with side chain of Trp1355, Asp1279, Ile1280, Trp1418, Ile1315,

Tyr1251, Trp1523, Met1421, Tyr1167, Phe1559, and Asp1526. Furthermore, the centered

oxadiazole and phenyl ring form π-π stacking with Phe1560. In the other end the chloro

indole ring forms π-π stacking with Trp1369. While the Leu1367, Gly1365, Gln1372,

181

Arg1377, Ile1587, Gly1588, Thr1586, Thr1589, and Gln1561 forms hydrophobic

interaction with the chloro indole ring as shown in Figure-3.32 c.

The binding mode analysis of compound 144 (IC50 = 15 ± 0.15 µM) shows that one

among the bromo-indole nitrogen forms hydrogen bond with Gln1158, while these two

indole rings form hydrophobic interaction with Lys1460, Trp1369, Asp1370, Gln1158,

Pro1159, and Lys1164. Likewise, the centered phenyl and oxadiazole ring form π-π

stacking with Phe1560 and while oxadiazole ring also form π-π stacking with Trp1355

and also forms hydrophobic interaction with Asp1157 side chain. While the methyl

indole ring form hydrophobic interaction with side chains of Ile1280, Tyr1251, Asp1279,

Ile1315, Trp1418, Asp1420, Trp1523, Arg1510, Asp1526, Phe1559, and Asp1526 as shown

in Figure-3.32 d.

182

Figure-3.32: Graphical illustration of predicted binding mode of tris-indole series in

the binding site of α-glucuronidase. a) Compound 141 (olive green color) b)

compound 142 (magenta color), c) compound 143 (gray color), d) compound 144

(brown color). Key residues are only shown and the hydrogen bond interactions are

represented by yellow dashed lines. Compounds are shown in stick and key amino

acids in line.

It is noteworthy that a detailed binding mode analysis of active compounds of the tris-

indole series in the active site of α-glucosidase is facilitated by the network of hydrogen

183

bonds with the side chain and backbone groups. In addition, the hydrophobic

interactions with the key residues in the active site also significantly stabilize the

inhibitors. Taking all these to consideration into account will enhance the designing of

the selective α-glucosidase inhibitors in the near future.

3.11 Material and methods

3.11.1 Synthesis of bis-indole ester derivatives

For synthesis of bis-indole ester derivatives, indole (2 mmol) was mixed with methyl 4-

formyl benzoate (1mmol) in acetic acid and refluxed for 6 hours. Reaction completion

was monitored by TLC. After reaction completion, the reaction mixture was poured in

cold water which gives precipitate, filtered and then washed with cold water to get

desire product in good yield [106-108].

3.11.2 Synthesis of bis-indole hydrazide derivatives

For synthesis of bis-indole hydrazide derivatives, bis-indole ester (1mmol) obtained in

first step was mixed with hydrazine hydrate (1mmol) in methanol and refluxed for 6

hours. The reaction completion was monitored by TLC. After completion of reaction,

the solvent from reaction mixture were evaporated and the solid residue was washed

and dried [109, 110].

3.11.3 Synthesis of trisindole-oxadiazole derivatives

For synthesis of tris-indole-oxadiazole derivatives, indole based hydrazide (1mmol)

obtained in second step was treated with substituted indole (1mmol) in triethylamine as

solvent in the presence of phosphorus trichloride to get the desired product of

trisindole-oxadiazole hybrid analogs. The reaction completion was monitored by TLC.

184

After completion of reaction, the solvent of reaction mixture was evaporated and the

crude product was washed and dried.


yl)phenyl)methylene)bis(1H-indole-5-carbonitrile) (126)

Yield: 87%. Brown solid, m.p. 232-234 °C. 1H NMR (500 MHz, DMSO-d6): δ 10.89 (s, 2-H,

H-1,1′), 8.19 (dd, J = 7.1, 1.2 Hz, 1-H, H-4′′,), 8.12 (dd, J = 7.5, 0.9 Hz, 2-H, H-3′′′, 5′′′), 7.94

(d, J = 1.2 Hz, 2-H, H-4/4′), 7.69 (d, J = 7.5 Hz, 2-H, H-7,7′), 7.60 (dd, J = 7.5, 0.9 Hz, 1-H,

H-7′′), 7.49 (d, J = 7.5 Hz, 2-H, H-6,6′), 7.45 (m, 1-H, H-6′′), 7.39 (dd, J = 7.8, 1.1 Hz, 2-H,

H-2′′′,6′′′), 7.35 (m, 1-H, H-5′′), 7.21 (s, 1-H, H-2′′,), 6.69 (s, 2-H, H-2,2′), 5.51 (s, 1-H, CH),

3.76 (s, 3-H, CH3). 13CNMR (125 MHz, DMSO-d6): δ 163.2, 154.5, 141.7, 138.6, 138.6,

137.2, 134.5, 128.1, 128.1, 128.1, 128.1, 127.5, 127.5, 126.9, 126.9, 125.8, 125.2, 124.5, 124.5,

122.2, 121.7, 121.2, 120.6, 120.6, 119.5, 119.5, 114.5, 114.5, 112.4, 112.4, 109.1, 107.3, 107.3,

99.2, 35.8, 34.5; HREI-MS: m/z Calcd for C36H23N7O [M]+ 569.1964; Found 569.1957;

Anal. Calcd for C36H23N7O, C, 75.91; H, 4.07; N, 17.21; Found C, 75.92; H, 4.05; N, 17.19.

3.11.3.2 3,3'-((4-(5-(1H-Indol-3-yl)-1,3,4-oxadiazol-2-yl)phenyl)methylene)bis(1H-

indole-5-carbonitrile) (127)


(s, 1-H, H-1′′), 10.81 (s, 2-H, H-1,1′), 8.64 (s, 1-H, H-2′′), 8.15 (dd, J = 7.1, 1.2 Hz, 1-H, H-

4′′), 8.00 (dd, J = 7.8, 0.7 Hz, 2-H, H-3′′′,5′′′), 7.92 (d, J = 1.2 Hz, 2-H, H-4,4′), 7.68 (d, J =

7.5 Hz, 2-H, H-7,7′), 7.60 (dd, J = 7.6, 0.7 Hz, 1-H, H-7′′), 7.47 (d, J = 7.4 Hz, 2-H, H-6,6′),

7.39 (dd, J = 7.7, 1.1 Hz, 2-H, H-2′′′,6′′′), 7.34 (m, 2-H, H-5′′,6′′), 6.66 (s, 2-H, H-2,2′), 5.49

(s, 1-H, CH). 13CNMR (125 MHz, DMSO-d6): δ 163.9, 153.8, 141.7, 139.4, 139.4, 136.2,

185

136.2, 133.5, 128.9, 128.6, 128.2, 128.2, 128.2, 128.2, 127.6, 127.6, 126.4, 124.7, 123.2, 121.9,

121.3, 120.9, 120.8, 120.8, 120.7, 112.7, 112.7, 112.3, 112.3, 109.6, 99.0, 35.4, 33.5, 32.8, 32.8;

HREI-MS: m/z Calcd for C35H21N7O [M]+ 555.1808; Found 555.1815; Anal. Calcd for

C35H21N7O, C, 75.66; H, 3.81; N, 17.65; Found C, 75.68; H, 3.83; N, 17.64.


yl)phenyl)methylene)bis(1H-indole-5-carbonitrile) (128)

Yield: 91%. Brownish solid, m.p. 245-247 °C. 1H NMR (500 MHz, DMSO-d6): δ 11.68 (s,

1-H, H-1′′), 10.81 (s, 2-H, H-1,1′), 8.05 (dd, J = 7., 0.9 Hz, 2-H, H-3′′′,5′′′), 7.95 (d, J = 1.1

Hz, 2-H, H-4,4′), 7.69 (d, J = 7.6 Hz, 2-H, H-7,7′), 7.67 (d, J = 7.1 Hz, 2-H, H-6,6′), 7.49 (dd,

J = 7.1, 1.2 Hz, 1-H, H-4′′), 7.38 (dd, J = 7.4, 1.0 Hz, 2-H, H-2′′′,6′′′), 7.30 (dd, J = 7.5, 1.2

Hz, 1-H, H-7′′), 7.02 (m, 2-H, H-5′′,6′′), 6.64 (s, 2-H, H-2,2′), 5.50 (s, 1-H, CH), 2.79 (s, 3-H,

CH3). 13CNMR (125 MHz, DMSO-d6): δ 164.2, 160.4, 142.6, 139.6, 137.7, 137.7, 137.2,

128.3, 128.3, 128.3, 128.3, 127.5, 127.5, 127.2, 126.8, 126.8, 125.4, 124.6, 124.6, 124.2, 122.5,

121.8, 121.8, 118.5, 118.5, 113.4, 113.4, 112.3, 112.3, 111.7, 106.1, 106.1, 95.9, 33.8, 13.1;




1,3,4-oxadiazole (129)

Yield: 90%. Dark brown sticky substance. 1H NMR (500 MHz, DMSO-d6): δ 8.19

(dd, J = 7.6, 0.9 Hz, 1-H, H-4′′), 8.03 (dd, J = 7.6, 1.0 Hz, 2-H, H-3′′′,5′′′), 7.89 (dd, J = 7.3,

1.0 Hz, 2-H, H-4,4′), 7.60 (d, J = 7.4 Hz, 3-H, H-7,7′, 7′′), 7.48 (m, 2-H, H-6,6′), 7.39 (m, 2-

H, H-5′′,6′′), 7.36 (dd, J = 7.7, 0.8 Hz, 2-H, H-2′′′,6′′′), 7.21 (s, 1-H, H-2′′), 6.86 (m, 2-H, H-

186

5,5′), 6.27 (s, 2-H, H-2,2′), 5.49 (s, 1-H, CH), 3.76 (s, 9-H, 3-CH3). 13CNMR (125 MHz,

DMSO-d6): δ 163.9, 153.5, 143.7, 139.4, 139.4, 136.2, 134.2, 129.2, 128.5, 128.5, 128.5, 128.5,

128.1, 128.1, 127.2, 127.2, 126.4, 124.7, 123.2, 122.3, 121.7, 120.9, 120.5, 120.5, 120.2, 120.2,

113.7, 113.7, 112.3, 112.3, 109.4, 98.6, 36.4, 34.5, 31.8, 31.8; HREI-MS: m/z Calcd for

C36H29N5O [M]+ 547.2372; Found 547.2379; Anal. Calcd for C36H29N5O, C, 78.95; H, 5.34;

N, 12.79; Found C, 78.97; H, 5.32; N, 12.81.



Yield: 88%. Brown yellow oily liquid. 1H NMR (500 MHz, DMSO-d6): δ 11.68 (s,

1-H, H-1′′), 8.02 (dd, J = 7.9, 0.7 Hz, 2-H, H-3′′′,5′′′), 7.88 (dd, J = 7.3, 1.0 Hz, 2-H, H-4,4′),

7.61 (d, J = 7.4 Hz, 2-H, H-7,7′), 7.49 (dd, J = 7.6, 0.6 Hz, 1-H, H-4′′), 7.45 (m, 2-H, H-6,6′),

7.39 (dd, J = 7.5, 1.1 Hz, 2-H, H-2′′′,6′′′), 7.34 (dd, J = 7.6, 0.9 Hz, 1-H, H-7′′), 7.02 (m, 1-H,

H-6′′), 6.99 (m, 1-H, H-5′′), 6.86 (m, 2-H, H-5,5′), 6.26 (s, 2-H, H-2,2′), 5.49 (s, 1-H, CH),

3.77 (s, 6-H, 2-CH3), 2.75 (s, 3-H, CH3). 13CNMR (125 MHz, DMSO-d6): δ 165.1, 160.4,

143.7, 140.4, 139.3, 139.3, 136.3, 128.9, 128.9, 128.5, 128.5, 128.2, 128.2, 127.4, 127.4, 126.8,

124.7, 124.1, 121.6, 120.9, 120.9, 120.6, 120.6, 120.6, 119.8, 119.8, 114.7, 114.7, 112.6, 112.6,

110.6, 93.7, 34.4, 31.8, 31.8, 15.1; HREI-MS: m/z Calcd for C36H29N5O [M]+ 547.2372;

Found 547.2363; Anal. Calcd for C36H29N5O, C, 78.95; H, 5.34; N, 12.79; Found C, 78.94;

H, 5.33; N, 12.81.

187

3.11.3.6 2-(4-(Bis(1-methyl-1H-indol-3-yl)methyl)phenyl)-5-(1H-indol-3-yl)-1,3,4-

oxadiazole (131)

Yield: 81%. Light brown grey sticky substance. 1H NMR (500 MHz, DMSO-d6): δ

12.40 (s, 1-H, H-1′′), 8.63 (s, 1-H, H-2′′), 8.17 (dd, J = 7.4, 1.0 Hz, 1-H, H-4′′), 8.00 (dd, J =

7.6, 0.9 Hz, 2-H, H-3′′′,5′′′), 7.89 (dd, J = 7.2, 1.3 Hz, 2-H, H-4,4′), 7.58 (d, J = 7.4 Hz, 3-H,

H-7,7′,7′′), 7.42 (m, 2-H, H-6,6′), 7.38 (dd, J = 7.7, 1.2 Hz, 2-H, H-2′′′,6′′′), 7.33 (m, 2-H, H-

6′′,5′′), 6.85 (m, 2-H, H-5,5′), 6.24 (s, 2-H, H-2,2′), 5.51 (s, 1-H, CH), 3.75 (s, 6-H, 2-CH3).

13CNMR (125 MHz, DMSO-d6): δ 163.7, 154.8, 141.7, 138.4, 138.4, 136.8, 135.3, 128.5,

128.5, 128.3, 128.3, 128.0, 128.0, 127.3, 127.3, 125.8, 124.3, 122.2, 121.6, 121.1, 120.9, 120.9,

120.6, 120.6, 120.3, 120.3, 113.7, 113.7, 112.8, 112.4, 112.4, 97.9, 35.1, 33.8, 33.8; HREI-MS:

m/z Calcd for C35H27N5O [M]+ 533.2216; Found 533.2221; Anal. Calcd for C35H27N5O, C,

78.78; H, 5.10; N, 13.12; Found C, 78.77; H, 5.08; N, 13.13.


oxadiazole (132)

Yield: 85%. Brownish sticky substance. 1H NMR (500 MHz, DMSO-d6): δ 12.40 (s,

1-H, H-1′′), 11.51 (s, 2-H, H-1,1′), 8.62 (s, 1-H, H-2′′), 8.13 (dd, J = 7.2, 0.8 Hz, 1-H, H-4′′),

8.04 (dd, J = 7.4, 1.0 Hz, 2-H, H-4,4′), 8.02 (dd, J = 7.6, 0.8 Hz, 2-H, H-3′′′,5′′′), 7.60 (d, J =

7.3 Hz, 3-H, H-7,7′,7′′), 7.51 (m, 2-H, H-4′′′′,4′′′′′), 7.46 (m, 8-H, H-2′′′′,3′′′′,5′′′′,6′′′′,

2′′′′′,3′′′′′,5′′′′′,6′′′′′), 7.39 (dd, J = 7.9, 0.9 Hz, 2-H, H-2′′′,6′′′), 7.32 (m, 2-H, H-6′′,5′′), 6.98 (m,

2-H, H-6,6′), 6.83 (m, 2-H, H-5,5′), 5.50 (s, 1-H, CH). 13CNMR (125 MHz, DMSO-d6): δ

163.4, 156.0, 137.9, 136.8, 136.8, 135.6, 135.3, 131.7, 131.7, 130.7, 130.7, 129.4, 129.4, 129.4,

129.4, 129.0, 129.0, 128.4, 128.4, 128.2, 128.2, 128.2, 128.2, 126.9, 126.9, 126.2, 126.2, 125.7,

188

124.8, 124.8, 124.1, 122.4, 121.6, 121.2, 120.3, 120.3, 120.3, 120.3, 112.5, 112.1, 112.1, 111.7,

111.7, 99.5, 37.6; HREI-MS: m/z Calcd for C45H31N5O [M]+ 657.2529; Found 657.2536;


3.11.3.8 2-(4-(Bis(2-phenyl-1H-indol-3-yl)methyl)phenyl)-5-(1-methyl-1H-indol-3-yl)-


Yield: 85%. Slight brown oily liquid. 1H NMR (500 MHz, DMSO-d6): δ 11.47 (s, 2-H, H-

1,1′), 8.18 (dd, J = 7.3, 1.2 Hz, 1-H, H-4′′), 8.04 (dd, J = 7.9, 0.8 Hz, 2-H, H-3′′′,5′′′), 8.02 (dd,

J = 7.3, 0.8 Hz, 2-H, H-4,4′), 7.59 (d, J = 7.6 Hz, 3-H, H-7,7′,7′′), 7.50 (m, 2-H, H-4′′′′,4′′′′′),

7.44 (m, 10-H, H-6′′,5′′,2′′′′,3′′′′,5′′′′,6′′′′, 2′′′′′,3′′′′′,5′′′′′,6′′′′′), 7.33 (dd, J = 7.7, 1.0 Hz, 2-H, H-

2′′′,6′′′), 7.20 (s, 1-H, H-2′′), 6.94 (m, 2-H, H-6,6′), 6.81 (m, 2-H, H-5,5′), 5.50 (s, 1-H, CH),


136.2, 134.5, 131.4, 131.4, 130.6, 130.6, 129.6, 129.6, 129.6, 129.6, 128.9, 128.9, 128.7, 128.7,

128.2, 128.2, 128.2, 128.2, 127.4, 127.4, 126.3, 126.3, 125.4, 124.8, 124.8, 124.1, 122.2, 121.6,

121.5, 120.2, 120.2, 120.2, 120.2, 112.3, 112.3, 110.8, 110.8, 109.4, 99.5, 39.3, 33.7; HREI-MS:

m/z Calcd for C46H33N5O [M]+ 671.2685; Found 671.2692; Anal. Calcd for C46H33N5O, C,

82.24; H, 4.95; N, 10.42; Found C, 82.22; H, 4.94; N, 10.43.


oxadiazole (134)

Yield: 92%. Brown yellow sticky substance. 1H NMR (500 MHz, DMSO-d6): δ

11.61 (s, 1-H, H-1′′), 11.48 (s, 2-H, H-1,1′), 8.06 (dd, J = 7.1, 0.8 Hz, 2-H, H-4,4′), 8.00 (dd,

J = 7.7, 0.9 Hz, 2-H, H-3′′′,5′′′), 7.56 (d, J = 7.5 Hz, 2-H, H-7,7′), 7.54 (m, 2-H, H-4′′′′,4′′′′′),

6.99 (m, 2-H, H-6′′,5′′), 7.46 (m, 8-H, H-2′′′′,3′′′′,5′′′′,6′′′′, 2′′′′′,3′′′′′,5′′′′′,6′′′′′), 7.42 (dd, J = 7.2,

189

1.2 Hz, 1-H, H-4′′), 7.36 (m, 1-H, H-7′′), 7.35 (dd, J = 7.9, 1.1 Hz, 2-H, H-2′′′,6′′′), 6.98 (m, 2-

H, H-6,6′), 6.78 (m, 2-H, H-5,5′), 5.46 (s, 1-H, CH), 2.87 (s, 3-H, CH3). 13CNMR (125 MHz,

DMSO-d6): δ 164.2, 162.4, 141.6, 137.9, 137.5, 137.5, 136.3, 131.4, 131.4, 130.7, 130.7, 129.1,

129.1, 129.1, 129.1, 129.0, 129.0, 128.7, 128.7, 128.4, 128.4, 128.4, 128.4, 127.8, 127.8, 127.0,

127.0, 126.2, 126.2, 124.5, 124.5, 123.8, 123.8, 121.3, 120.7, 119.8, 119.8, 119.6, 112.3, 112.3,

111.7, 111.7, 111.3, 94.9, 38.4, 15.3; HREI-MS: m/z Calcd for C46H33N5O [M]+ 671.8040;

Found 671.8033; Anal. Calcd for C46H33N5O, C, 82.24; H, 4.95; N, 10.42; Found C, 82.25;

H, 4.94; N, 10.40.


oxadiazole (135)

Yield: 89%. Light brown grey sticky substance. 1H NMR (500 MHz, DMSO-d6): δ 10.80

(s, 2-H, H-1,1′), 8.19 (dd, J = 7.1, 1.2 Hz, 1-H, H-4′′), 8.03 (dd, J = 7.7, 0.7 Hz, 2-H, H-

3′′′,5′′′), 7.64 (m, 2-H, H-6,6′), 7.60 (m, 1-H, H-7′′), 7.39 (m, 2-H, H-6′′,5′′), 7.33 (dd, J = 7.7,

1.1 Hz, 2-H, H-2′′′,6′′′), 7.31 (d, J = 7.6 Hz, 2-H, H-7,7′), 7.20 (s, 1-H, H-2′′), 7.08 (dd, J =

7.4, 0.8 Hz, 2-H, H-4,4′), 6.81 (m, 2-H, H-5,5′), 6.63 (s, 2-H, H-2,2′), 5.47 (s, 1-H, CH), 3.72

(s, 3-H, CH3). 13CNMR (125 MHz, DMSO-d6): δ 163.9, 153.5, 142.7, 137.2, 135.5, 128.1,

128.1, 127.8, 127.8, 127.2, 127.2, 127.2, 127.2, 126.2, 125.7, 122.2, 121.6, 121.5, 121.2, 121.2,

121.0, 121.0, 119.7, 119.7, 119.2, 119.2, 112.1, 112.1, 111.7, 111.7, 109.6, 99.0, 34.8, 33.5;



190

3.11.3.11 2-(4-(Di(1H-indol-3-yl)methyl)phenyl)-5-(1H-indol-3-yl)-1,3,4-oxadiazole

(136)

Yield: 87%. Dark brownish oily liquid. 1H NMR (500 MHz, DMSO-d6): δ 12.41 (s, 1-H, H-

1′′), 10.79 (s, 2-H, H-1,1′), 8.15 (dd, J = 7.0, 1.1 Hz, 1-H, H-4′′), 8.05 (s, 1-H, H-2′′), 8.04 (dd,

J = 7.9, 0.9 Hz, 2-H, H-3′′′,5′′′), 7.62 (m, 1-H, H-7′′), 7.34 (m, 2-H, H-6′′,5′′), 7.36 (dd, J = 7.6,

1.0 Hz, 2-H, H-7,7′), 7.31 (dd, J = 7.8, 1.1 Hz, 2-H, H-2′′′,6′′′), 7.12 (dd, J = 7.3, 0.8 Hz, 2-H,

H-4,4′), 6.98 (m, 2-H, H-6,6′), 6.86 (m, 2-H, H-5,5′), 6.64 (s, 2-H, H-2,2′), 5.48 (s, 1-H, CH).

13CNMR (125 MHz, DMSO-d6): δ 163.7, 155.1, 142.8, 136.5, 136.1, 128.4, 128.4, 127.5,

127.5, 127.2, 127.2, 127.2, 127.2, 126.7, 125.2, 122.7, 121.8, 121.4, 121.0, 121.0, 120.4, 120.4,

119.5, 119.5, 119.2, 119.2, 113.6, 112.3, 112.3, 111.5, 111.5, 98.6, 35.8; HREI-MS: m/z Calcd

for C33H23N5O [M]+ 505.1903; Found 505.1892; Anal. Calcd for C33H23N5O, C, 78.40; H,

4.59; N, 13.85; Found C, 78.38; H, 4.61; N, 13.86.


oxadiazole (137)

Yield: 80%. Slight brown grey solid, m.p. 222-224 °C. 1H NMR (500 MHz, DMSO-d6): δ

11.71 (s, 1-H, H-1′′), 10.82 (s, 2-H, H-1,1′), 8.03 (dd, J = 7.8, 0.9 Hz, 2-H, H-3′′′,5′′′), 7.49

(dd, J = 7.1, 1.2 Hz, 1-H, H-4′′), 7.36 (dd, J = 7.1, 1.1 Hz, 1-H, H-7′′), 7.35 (dd, J = 7.5, 1.1

Hz, 2-H, H-7,7′), 7.30 (dd, J = 7.8, 1.1 Hz, 2-H, H-2′′′,6′′′), 7.09 (dd, J = 7.1, 0.8 Hz, 2-H, H-

4,4′), 6.98 (m, 2-H, H-6′′,5′′), 6.93 (m, 2-H, H-6,6′), 6.81 (m, 2-H, H-5,5′), 6.60 (s, 2-H, H-

2,2′), 5.48 (s, 1-H, CH), 2.80 (s, 3-H, CH3). 13CNMR (125 MHz, DMSO-d6): δ 165.1, 161.3,

142.6, 139.6, 137.1, 128.3, 128.3, 127.8, 127.8, 127.5, 127.5, 127.5, 127.5, 127.0, 125.6, 124.0,

121.6, 121.3, 121.1, 120.9, 120.9, 120.5, 119.7, 119.7, 119.2, 119.2, 112.0, 112.0, 111.7, 111.7,

191

111.2, 93.6, 34.1, 14.5. HREI-MS: m/z Calcd for C34H25N5O [M]+ 519.2059; Found

519.2067; Anal. Calcd for C34H25N5O, C, 78.59; H, 4.85; N, 13.48; Found C, 78.61; H, 4.84;

N, 13.46.


yl)-1,3,4-oxadiazole (138)

Yield: 83%. Slight brown sticky substance. 1H NMR (500 MHz, DMSO-d6): δ 8.05 (dd, J =

7.7, 0.8 Hz, 2-H, H-3′′′,5′′′), 7.74 (dd, J = 7.2, 1.2 Hz, 1-H, H-4′′), 7.48 (m, 2-H, H-6,6′), 7.44

(dd, J = 7.3, 0.8 Hz, 2-H, H-4,4′), 7.41 (m, 2-H, H-6′′,5′′), 7.34 (dd, J = 7.9, 1.4 Hz, 2-H, H-

2′′′,6′′′), 7.29 (dd, J = 7.0, 1.0 Hz, 3-H, H-7,7′,7′′), 6.96 (m, 2-H, H-5,5′), 5.53 (s, 1-H, CH),

3.68 (s, 9-H, CH3), 2.63 (s, 3-H, CH3), 2.29 (s, 6-H, CH3). 13CNMR (125 MHz, DMSO-d6): δ

163.9, 153.5, 140.2, 140.2, 140.0, 140.0, 138.9, 137.2, 135.5, 129.0, 129.0, 127.8, 127.8, 127.5,

127.5, 127.0, 127.0, 126.2, 124.1, 122.4, 121.9, 121.5, 120.0, 120.0, 119.5, 119.5, 109.7, 109.1,

109.1, 103.6, 103.6, 99.1, 43.1, 34.5, 30.1, 30.1, 10.4, 10.4; HREI-MS: m/z Calcd for

C38H33N5O [M]+ 575.2685; Found 575.2692; Anal. Calcd for C38H33N5O, C, 79.28; H, 5.78;

N, 12.16; Found C, 79.26; H, 5.80; N, 12.17.


yl)-1,3,4-oxadiazole (139)

Yield: 79%. Dark grey sticky substance. 1H NMR (500 MHz, DMSO-d6): δ 11.70 (s, 1-H,

H-1′′), 8.04 (dd, J = 7.9, 0.8 Hz, 2-H, H-3′′′,5′′′), 7.49 (dd, J = 7.0, 1.5 Hz, 1-H, H-4′′), 7.47

(m, 2-H, H-6,6′), 7.39 (dd, J = 7.4, 1.0 Hz, 2-H, H-4,4′), 6.98 (m, 2-H, H-6′′,5′′), 7.36 (dd, J =

7.8, 1.2 Hz, 2-H, H-2′′′,6′′′), 7.33 (dd, J = 7.6, 0.9 Hz, 1-H, H-7′′), 7.28 (dd, J = 7.4, 1.0 Hz, 2-

H, H-7,7′), 6.95 (m, 2-H, H-5,5′), 5.51 (s, 1-H, CH), 3.69 (s, 6-H, CH3), 2.80 (s, 3-H, CH3),

192


139.6, 139.6, 137.5, 137.1, 129.0, 129.0, 127.9, 127.9, 127.7, 127.7, 127.0, 127.0, 127.0, 124.2,

124.2, 121.5, 120.8, 119.7, 119.7, 118.4, 118.4, 111.6, 109.2, 109.2, 102.6, 102.6, 93.9, 42.1,

31.6, 31.6, 13.1, 11.7, 11.7; HREI-MS: m/z Calcd for C38H33N5O [M]+ 575.2685; Found

575.2678; Anal. Calcd for C38H33N5O, C, 79.28; H, 5.78; N, 12.16; Found C, 79.28; H, 5.78;

N, 12.16.

3.11.3.15 2-(4-(Bis(1,2-dimethyl-1H-indol-3-yl)methyl)phenyl)-5-(1H-indol-3-yl)-1,3,4-

oxadiazole (140)

Yield: 90%. Brownish oily liquid. 1H NMR (500 MHz, DMSO-d6): δ 12.42 (s, 1-H, H-1′′),

8.62 (s, 1-H, H-2′′), 8.07 (dd, J = 7.8, 0.9 Hz, 2-H, H-3′′′,5′′′), 8.16 (dd, J = 7.1, 1.2 Hz, 1-H,

H-4′′), 7.45 (m, 2-H, H-6,6′), 7.42 (dd, J = 7.1, 0.8 Hz, 2-H, H-4,4′), 7.35 (m, 2-H, H-6′′,5′′),

7.32 (dd, J = 7.8, 1.1 Hz, 2-H, H-2′′′,6′′′), 7.33 (dd, J = 7.8, 0.9 Hz, 1-H, H-7′′), 7.24 (dd, J =

7.1, 1.1 Hz, 2-H, H-7,7′), 6.97 (m, 2-H, H-5,5′), 5.50 (s, 1-H, CH), 3.65 (s, 6-H, CH3), 2.25 (s,

6-H, CH3). 13CNMR (125 MHz, DMSO-d6): δ 164.2, 154.8, 140.2, 140.2, 140.0, 140.0, 138.9,

136.6, 136.3, 129.0, 129.0, 127.8, 127.8, 127.5, 127.5, 127.0, 127.0, 125.3, 124.1, 124.1, 122.4,

121.6, 121.5, 121.5, 120.0, 120.0, 119.8, 119.8, 112.6, 109.2, 109.2, 102.6, 102.6, 98.9, 42.1,

30.6, 30.6, 10.7, 10.7; HREI-MS: m/z Calcd for C37H31N5O [M]+ 561.2529; Found 561.2532;




Yield: 91%. Light brown solid, m.p. 247-249 °C. 1H NMR (500 MHz, DMSO-d6): δ

10.80 (s, 2-H, H-1,1′), 8.11 (dd, J = 7.2, 1.3 Hz, 1-H, H-4′′), 8.05 (dd, J = 7.8, 0.9 Hz, 2-H, H-

193

3′′′,5′′′), 7.71 (d, J = 1.2 Hz, 2-H, H-4,4′), 7.62 (dd, J = 7.3, 1.0 Hz, 1-H, H-7′′), 7.45 (d, J =

7.3, 2-H, H-7,7′), 7.39 (m, 2-H, H-6′′,5′′), 7.29 (dd, J = 7.7, 1.2 Hz, 2-H, H-2′′′,6′′′), 7.20 (s, 1-

H, H-2′′), 7.19 (d, J = 7.3 Hz, 2-H, H-6,6′), 6.68 (s, 2-H, H-2,2′), 5.49 (s, 1-H, CH), 3.72 (s, 3-

H, CH3). 13C NMR (125 MHz, DMSO-d6): δ 164.7, 154.5, 143.7, 138.2, 137.8, 137.8, 135.4,

128.1, 128.1, 127.4, 127.4, 127.2, 127.2, 126.2, 125.7, 124.1, 124.1, 122.6, 121.7, 121.4, 121.1,

121.1, 120.8, 120.8, 119.6, 119.6, 113.7, 113.7, 112.2, 112.2, 109.6, 99.0, 34.8, 33.5; HREI-MS:

m/z Calcd for C34H23Cl2N5O [M]+ 587.1280; Found 587.1287; Anal. Calcd for

C34H23Cl2N5O, C, 69.39; H, 3.94; N, 11.90; Found C, 69.40; H, 3.93; N, 11.88.



Yield: 82%. Dark brownish sticky substance. 1H NMR (500 MHz, DMSO-d6): δ 11.79 (s,

1-H, H-1′′), 10.79 (s, 2-H, H-1,1′), 8.07 (dd, J = 7.8, 1.0 Hz, 2-H, H-3′′′,5′′′), 7.71 (d, J = 1.1

Hz, 2-H, H-4,4′), 7.49 (dd, J = 7.2, 1.2 Hz, 1-H, H-4′′), 7.43 (d, J = 7.3, 2-H, H-7,7′), 7.34 (d,

J = 7.3 Hz, 1-H, H-7′′), 7.30 (dd, J = 7.7, 1.1 Hz, 2-H, H-2′′′,6′′′), 7.20 (d, J = 7.3 Hz, 2-H, H-

6,6′), 6.98 (m, 2-H, H-6′′,5′′), 6.66 (s, 2-H, H-2,2′), 5.45 (s, 1-H, CH), 2.79 (s, 3-H, CH3).

13CNMR (125 MHz, DMSO-d6): δ 165.2, 161.4, 142.7, 140.6, 137.3, 136.8, 136.8, 128.1,

128.1, 127.4, 127.4, 127.2, 127.2, 127.0, 125.7, 124.2, 124.2, 124.1, 121.5, 121.2, 121.2, 120.8,

120.8, 120.8, 119.6, 119.6, 113.4, 113.4, 112.2, 112.2, 111.6, 94.9, 34.6, 14.3; HREI-MS: m/z

Calcd for C34H23Cl2N5O [M]+ 587.1280; Found 587.1273; Anal. Calcd for C34H23Cl2N5O,

C, 69.39; H, 3.94; N, 11.90; Found C, 69.37; H, 3.92; N, 11.91.

194

3.11.3.18 2-(4-(Bis(5-chloro-1H-indol-3-yl)methyl)phenyl)-5-(1H-indol-3-yl)-1,3,4-

oxadiazole (143)

Yield: 87%. Grey sticky substance. 1H NMR (500 MHz, DMSO-d6): δ 12.43 (s, 1-H, H-1′′),

10.82 (s, 2-H, H-1,1′), 8.63 (s, 1-H, H-2′′), 8.18 (dd, J = 7.0, 1.1 Hz, 1-H, H-4′′), 8.05 (dd, J =

7.7, 0.9 Hz, 2-H, H-3′′′,5′′′), 7.67 (d, J = 1.3 Hz, 2-H, H-4,4′), 7.62 (dd, J = 7.2, 0.8 Hz, 1-H,

H-7′′), 7.40 (d, J = 7.2, 2-H, H-7,7′), 7.32 (dd, J = 7.4, 1.4 Hz, 2-H, H-2′′′,6′′′), 7.28 (m, 2-H,

H-6′′,5′′), 7.18 (d, J = 7.0 Hz, 2-H, H-6,6′), 6.63 (s, 2-H, H-2,2′), 5.47 (s, 1-H, CH). 13CNMR

(125 MHz, DMSO-d6): δ 163.9, 155.0, 142.7, 136.8, 136.8, 136.6, 136.3, 128.1, 128.1, 127.4,

127.4, 127.2, 127.2, 125.7, 125.3, 124.2, 124.2, 124.2, 121.6, 121.5, 121.2, 121.2, 120.8, 120.8,

119.6, 119.6, 113.7, 113.7, 112.6, 112.2, 112.2, 98.9, 34.8; HREI-MS: m/z Calcd for

C33H21Cl2N5O [M]+ 573.1123; Found 573.1128; Anal. Calcd for C33H21Cl2N5O, C, 69.00;

H, 3.68; N, 12.19; Found C, 67.99; H, 3.67; N, 12.20.



Yield: 86%. Light grey oily liquid. 1H NMR (500 MHz, DMSO-d6): δ 10.75 (s, 2-H,

H-1,1′), 8.13 (dd, J = 7.0, 1.2 Hz, 1-H, H-4′′), 8.00 (dd, J = 7.7, 1.1 Hz, 2-H, H-3′′′,5′′′), 7.88

(d, J = 1.0 Hz, 1-H, H-4′), 7.65 (d, J = 1.2 Hz, 1-H, H-4), 7.62 (dd, J = 7.2, 0.9 Hz, 1-H, H-

7′′), 7.40 (d, J = 7.2, 2-H, H-7,7′), 7.46 (m, 1-H, H-6′′), 7.36 (d, J = 7.0 Hz, 2-H, H-6,6′), 7.35

(dd, J = 7.8, 1.1 Hz, 2-H, H-2′′′,6′′′), 7.31 (m, 1-H, H-5′′), 7.24 (s, 1-H, H-2′′), 6.60 (s, 2-H, H-

2,2′), 5.44 (s, 1-H, CH), 3.77 (s, 3-H, CH3). 13CNMR (125 MHz, DMSO-d6): δ 163.8, 153.5,

142.7, 138.5, 138.5, 138.2, 136.5, 129.1, 129.1, 127.5, 127.5, 127.2, 127.2, 126.9, 126.7, 126.0,

126.0, 123.9, 123.9, 122.1, 121.8, 121.3, 120.9, 120.9, 116.4, 116.4, 116.9, 113.6, 112.2, 112.2,

195

109.4, 98.6, 33.8, 32.5; HREI-MS: m/z Calcd for C34H23Br2N5O [M]+ 675.0269; Found

675.0272; Anal. Calcd for C34H23Br2N5O, C, 60.29; H, 3.42; N, 10.34; Found C, 60.31; H,

3.43; N, 10.35.



Yield: 87%. Slight brown sticky substance. 1H NMR (500 MHz, DMSO-d6): δ 11.69

(s, 1-H, H-1′′), 10.73 (s, 2-H, H-1,1′), 8.05 (dd, J = 7.7, 0.9 Hz, 2-H, H-3′′′,5′′′), 7.89 (d, J =

1.3 Hz, 2-H, H-4,4′), 7.50 (dd, J = 7.1, 1.0 Hz, 1-H, H-4′′), 7.37 (dd, J = 7.2, 0.7 Hz, 1-H, H-

7′′), 7.35 (d, J = 7.2, 2-H, H-7,7′), 6.99 (m, 1-H, H-6′′), 7.40 (dd, J = 7.1, 0.9 Hz, 2-H, H-6,6′),

7.30 (dd, J = 7.8, 0.8 Hz, 2-H, H-2′′′,6′′′), 6.94 (m, 1-H, H-5′′), 6.62 (s, 2-H, H-2,2′), 5.41 (s, 1-

H, CH), 2.80 (s, 3-H, CH3). 13CNMR (125 MHz, DMSO-d6): δ 165.1, 162.7, 142.7, 141.4,

136.5, 136.5, 135.3, 128.1, 128.1, 127.4, 127.4, 127.2, 127.2, 127.2, 127.0, 125.7, 125.0, 125.0,

124.1, 123.7, 123.7, 121.5, 120.8, 120.8, 116.5, 116.5, 113.9, 113.9, 112.2, 112.2, 111.6, 93.9,

34.8, 14.1; HREI-MS: m/z Calcd for C34H23Br2N5O [M]+ 675.0269; Found 675.0274; Anal.

Calcd for C34H23Br2N5O, C, 60.29; H, 3.42; N, 10.34; Found C, 60.27; H, 3.41; N, 10.35.

3.11.3.21 2-(4-(Bis(5-bromo-1H-indol-3-yl)methyl)phenyl)-5-(1H-indol-3-yl)-1,3,4-

oxadiazole (146)

Yield: 89%. Light grey sticky substance. 1H NMR (500 MHz, DMSO-d6): δ 12.45 (s, 1-H,

H-1′′), 10.63 (s, 2-H, H-1,1′), 8.64 (s, 1-H, H-2′′), 8.19 (dd, J = 7.1, 1.2 Hz, 1-H, H-4′′), 8.04

(dd, J = 7.7, 1.1 Hz, 2-H, H-3′′′,5′′′), 7.82 (d, J = 1.2 Hz, 2-H, H-4,4′), 7.63 (dd, J = 7.2, 0.7

Hz, 1-H, H-7′′), 7.44 (dd, J = 7.0, 1.2 Hz, 2-H, H-6,6′), 7.39 (d, J = 7.2, 2-H, H-7,7′), 7.34 (m,

2-H, H-5′′,6′′), 7.28 (dd, J = 7.8, 0.6 Hz, 2-H, H-2′′′,6′′′), 6.60 (s, 2-H, H-2,2′), 5.54 (s, 1-H,

196

CH). 13CNMR (125 MHz, DMSO-d6): δ 163.7, 154.7, 141.6, 138.2, 137.5, 136.6, 136.3, 128.1,

128.1, 127.4, 127.4, 127.2, 127.2, 125.7, 125.3, 125.0, 125.0, 123.7, 123.7, 122.4, 121.6, 121.5,

120.8, 120.8, 116.5, 116.5, 113.9, 113.9, 112.6, 112.2, 112.2, 98.9, 34.8; HREI-MS: m/z Calcd

for C33H21Br2N5O [M]+ 661.0113; Found 661.0118; Anal. Calcd for C33H21Br2N5O, C,

59.75; H, 3.19; N, 10.56; Found C, 59.73; H, 3.18; N, 10.57.

3.12 Ligand preparation

All the twenty one tris-indole series 3D structures were sketched using the Schrodinger

Maestro [103] using build option and the structure were further optimized using

Ligprep application with OPLS_2005 force field, generation of possible states of pH 7.0

± 2.0. Generating possible tautomers, with all combinations of stereoisomer and with

lowest energy conformation.

3.13 Rapid pharmacokinetic predictions of tris-indole series

In silico pharmacokinetic properties and ADME were evaluated for the twenty-one tris-

indole derivatives using the QikProp program [111], implemented in Maestro [112].

Prior performing QikProp the compounds were neutralized using ligprep module of

Maestro. Particularly, four descriptors were taken into consideration for evaluation:

Lipinski‟s rule of five is a rule of thumb to assess drug likeness [113], the predicted

aqueous solubility (QP log S), the predicted octanol/water partition coefficient (QP

logP), and the predicted Caco-2 cell permeability (QPPcaco).

3.14 Predicted pharmacokinetic for tris-indole derivatives

The twenty-one tris-indole derivatives and their four pharmacokinetic descriptor

properties were analyzed using QikProp program, which consist of predicted Lipinski‟s

197

rule of five, the predicted octanol/water partition coefficient (QP logP), predicted

aqueous solubility (QP log S) and the predicted Caco-2 cell permeability (QPPcaco) and

their results are listed in Table-3.4.

Table-3.4: Pharmacokinetic predictions and Glide g score

Compounds

Lipinski’s

rule of

five[a]

QPlog P

o/w[b] QPlog S[c]

QP Pcaco[d]

[nms-1]

Glide g

score

126 2 15.173 -11.694 75.842 -4.211

127 2 16.981 -11.042 34.072 -4.777

128 2 16.682 -11.444 39.323 -3.36

129 2 8.585 -12.119 3667.623 -3.646

130 2 10.033 -11.821 2332.137 -5.059

131 2 10.331 -11.4 2019.61 -5.237

132 2 14.889 -14.422 998.531 -4.926

133 2 13.002 -14.894 1837.12 -4.994

134 2 14.889 -14.422 998.531 -4.926

135 2 11.832 -10.98 1081.895 -4.827

136 2 13.58 -10.52 596.275 -4.649

137 2 13.359 -10.812 687.786 -4.062

138 2 8.002 -12.417 3678.232 -5.346

139 2 9.448 -12.318 2340.223 -3.469

198

140 2 9.747 -12.022 2027.482 -5.213

141 2 11.426 -12.424 1082.243 -3.846

142 2 12.874 -12.251 688.411 -4.277

143 2 13.123 -11.957 595.995 -5.358

144 2 11.439 -14.293 1082.326 -4.342

145 2 12.883 -14.115 688.478 -5.609

146 2 13.141 -13.821 596.018 -5.216

[a] Predicted Rule of Five: range of recommended values is maximum number 4.

[b] Predicted octanol/water partition coefficient (QP log Po/w)

[c] Predicted aqueous solubility (QP log S): values less than -6 or greater than -1 are undesirable.

[d] Predicted apparent Caco-2 cell permeability (QP Pcaco): value < 25 is poor.

The desired molecular properties are important in the drug‟s pharmacokinetics in the

human bodies that are crucial for being an ideal drug candidate. All of the

pharmacokinetic parameters of twenty-one tris-indole derivatives are within the

acceptable range for drug-likeness.

3.15 Conclusion

In this chapter, we have synthesized two series of bis-indolylmethane namely bis-

indolylmethane based Schiff base and bisindolylmethane thiosemicarbazides and one

series of tris-indole-oxadiazole hybrid analogs.

A new series of bis-indolylmethanes based Schiff base derivatives was designed with

the aim to synthesize more potent β-glucuronidase inhibiting agents. In vitro β-

glucuronidase inhibition activity of these molecules helped in introducing some new β-

199

glucuronidase inhibitors. These derivatives have displayed excellent efficacy results as

compared to standard drug D-saccharic acid 1,4 lactone.

A series of bisindolylmethane thiosemicarbazides has been synthesized successfully

and evaluated for their in vitro urease inhibition potential. All analogs showed excellent

inhibitory potential.

A variety of synthetic tris-indole-oxadiazole hybrid analogs were evaluated for their α-

glucosidase inhibitory activity and found to be many folds better than standard drug

acarbose. In silico study was performed to identify the structural elements which

involved in the inhibitory activity and to rationalize the structure-activity relationship

(SAR). This study has identified a whole series of lead candidates which can serve for

the development of novel class of α-glucosidase inhibitors.

SAR studies were carried out to investigate the role of substitutions and nature of the

functional groups attached to the phenyl ring which exert imperative influence on the

inhibitory potential. The most probable binding modes of these derivatives with

enzyme‟s active sites were described through molecular docking.

200

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208

CHAPTER-04

Biological Assay Protocols

Summary

………………………………………………………………………………………………………..

In this chapter we describe biological assay protocol used for all derivatives.

………………………………………………………………………………………………………..

4. Procedures for Various Biological Assays

4.1 Assay Protocol of Thymidine phosphorylase

Thymidine phosphorylase/PD-ECGF (human and E. coli) activity was determined by

measuring the absorbance at 290 nm spectrophotometrically. The original method

described by Krenitsky and Bush by (1979) was modified [1, 2]. In brief, total reaction

mixture of 200 µL contained 145 µL of potassium phosphate buffer (pH 7.4, 50 mM), 30

µL of enzyme (human and E. coli) at concentration 0.05 and 0.002 U, respectively, were

incubated with 5 µL of test materials (0.5 mM) for10 min at 25 °C in microplate reader.

After incubation, pre-read at 290 nm was taken to deduce the absorbance of substrate

particles. Substrate “Thymidine” (20 µL, 1.5 mM), was dissolved in potassium

phosphate buffer, was immediately added to plate and continuously read after 10, 20,

and 30 min in microplate reader (spectra max, molecular devices, CA, USA). 7-

Deazaxanthine was used as positive control. All assays were performed in triplicate.

Source/ Supplier of the enzymes used:

Thymidine phosphorylase enzyme (from E. coli, EC Number 2.4.2.4), thymidine, and

potassium phosphate monobasic were purchased from Sigma Aldrich, USA.

209

4.2 Urease Assay protocol

The reaction mixtures, comprising 25 μL of enzyme (jack bean urease) solution and 55

μL of buffers containing 100 mM urea, were incubated with 5 μL of the test compounds

(0.5 mM concentration) at 30 °C for 15 min in 96-well plates. For the kinetics assessment

the urea concentrations were changed from 2-24 mM. Urease activity was determined

by measuring ammonia production using the indophenol method as described by

Weatherburn [3]. Briefly, 45 μL of phenol reagent (1% w/v phenol and 0.005% w/v

sodium nitroprussside) and, 70 μL of alkali reagent (0.5% w/v NaOH and 0.1% active

chloride NaOCl) were added to each well. The increasing absorbance at 630 nm was

measured after 50min, using a microplate reader (Molecular Device, USA). All reactions

were performed in triplicate in a final volume of 200 μL. The results (change in

absorbance per min) were processed by using SoftMaxPro software (molecular Device,

USA). The entire assays were performed at pH 6.8. Percentage inhibition was calculated

from the formula 100-(ODtest well/ODcontrol) ×100. Thiourea was used as the standard

inhibitor for urease.

4.3 β-Glucuronidase assay

Taha et al., describe the method for the β-glucuronidase activity by absorbance

measurement at 405 nm of p-nitrophenol formed substrate by spectrophotometric

method. The volume of the total reaction was found to be 250 µL. Reaction mixture

including 185 µL of 1M acetate buffer, 10 µL of enzyme solution and 5 µL of test

compound solution were incubated at 37 °C for 30 minutes. The plates were recorded at

405 nm (spectra Max plus 384) after the addition of nitrophenol solution. Triplates were

210

used for preforming the experiments [5]. Volume of reaction was increased and

concentration of compound was decreased to avoid precipitation. Addition of

detergents was not needed because the precipitation probability was less.

4.4 α-Glucosidase inhibition assay

Shibano et al describe the method for the enzyme inhibition with slight changes [6]. This

method was selected because of the solubility of the compound in DMSO with 95 μl of

50 mM phosphate buffer solution. 25 μl from compound stoke solution was diluted to

0.0625 U/ml with buffer and incubate for 10 minutes. 25 μl of 5 mM PNPG (dissolve 1.5

mg in 1 ml of phosphate buffer) and absorbance at time 0 min was measured for

reaction initiation. Incubation was carried out for 30 mints at 37 °C and the absorbance

was measured. 10 μl of DMSO was used fo negative control and acarbose was used for

positive control. The hydrolysis of the enzyme were recorded by monitoring the

amount of nitrophenol released by observation at 405 nm using microplate reader

(Spectrostar Nano BMG Labtech, Germany). The results were expressed as the mean ±

S.E.M of three determinations. The % inhibition was calculated using equation.

In the equation control sample are control of different absorbance at time t30 and

t0 respectively.

211

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