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
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
1.7.1.2 2-((2,5-dimethylthiazol-4-yl)methyl)-5-(2-nitrophenyl)-1,3,4-oxadiazole (103)
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
1.7.1.14 3-(5-((2,5-dimethylthiazol-4-yl)methyl)-1,3,4-oxadiazol-2-yl)benzonitrile (115)
43
1.7.1.15 4-(5-((2,5-dimethylthiazol-4-yl)methyl)-1,3,4-oxadiazol-2-yl)benzonitrile (116)
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 Results and discussion 67
2.5.1 Chemistry 67
2.5.2 In vitro thymidine phosphorylase inhibitory potential: 69
2.5.3 Molecular docking 72
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-
oxadiazol-2-yl)benzohydrazide (69)
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)
benzohydrazide (72) 89
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)
benzohydrazide (74) 90
2.7.5.11 (E)-4-(5-(4-cyanophenyl)-1,3,4-oxadiazol-2-yl)-N'-(3-
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
2.7.5.14 (E)-4-(5-(4-cyanophenyl)-1,3,4-oxadiazol-2-yl)-N'-(4-hydroxy-3-
methoxybenzylidene) benzohydrazide (78)
92
2.7.5.15 (E)-4-(5-(4-cyanophenyl)-1,3,4-oxadiazol-2-yl)-N'-(2-
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 Results and discussion 117
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-
carbohydrazide) (72) 129
3.7.3.3 3,3'-(p-Tolylmethylene)bis(N'-((E)-3-nitrobenzylidene)-1H-indole-5-
carbohydrazide) (73) 130
3.7.3.4 3,3'-(p-Tolylmethylene)bis(N'-((E)-4-methoxybenzylidene)-1H-indole-5-
carbohydrazide) (74) 130
3.7.3.5 3,3'-(p-Tolylmethylene)bis(N'-((1E,2E)-3-phenylallylidene)-1H-indole-5-
carbohydrazide) (75) 131
3.7.3.6 3,3'-(p-Tolylmethylene)bis(N'-((E)-3-hydroxybenzylidene)-1H-indole-5-
carbohydrazide) (76) 131
3.7.3.7 3,3'-(p-Tolylmethylene)bis(N'-((E)-2,3-dihydroxybenzylidene)-1H-indole-5-
carbohydrazide) (77) 132
xviii
3.7.3.8 3,3'-(p-Tolylmethylene)bis(N'-((E)-benzylidene)-1H-indole-5-carbohydrazide)
(78) 132
3.7.3.9 3,3'-(p-Tolylmethylene)bis(N'-((E)-2,5-dihydroxybenzylidene)-1H-indole-5-
carbohydrazide) (79) 133
3.7.3.10 3,3'-(p-Tolylmethylene)bis(N'-((E)-3,4-dihydroxybenzylidene)-1H-indole-5-
carbohydrazide) (80) 133
3.7.3.11 3,3'-(p-Tolylmethylene)bis(N'-((E)-2,4,5-trihydroxybenzylidene)-1H-indole-5-
carbohydrazide) (81) 134
3.7.3.12 3,3'-(p-Tolylmethylene)bis(N'-((E)-2,4,6-trihydroxybenzylidene)-1H-indole-5-
carbohydrazide) (82) 134
3.7.3.13 3,3'-(p-Tolylmethylene)bis(N'-((E)-2-hydroxy-4-methoxybenzylidene)-1H-
indole-5-carbohydrazide) (83) 135
3.7.3.14 3,3'-(p-Tolylmethylene)bis(N'-((E)-2-hydroxy-5-methoxybenzylidene)-1H-
indole-5-carbohydrazide) (84)
135
3.7.3.15 3,3'-(p-Tolylmethylene)bis(N'-((E)-3-hydroxy-4-methoxybenzylidene)-1H-
indole-5-carbohydrazide) (85)
136
3.7.3.16 3,3'-(p-Tolylmethylene)bis(N'-((E)-3-hydroxy-2-iodo-4-methoxybenzylidene)-
1H-indole-5-carbohydrazide) (86) 136
3.7.3.17 3,3'-(p-Tolylmethylene)bis(N'-((E)-2-hydroxybenzylidene)-1H-indole-5-
carbohydrazide) (87) 137
3.7.3.18 3,3'-(p-Tolylmethylene)bis(N'-((E)-2-nitrobenzylidene)-1H-indole-5-
carbohydrazide) (88) 137
3.7.3.19 3,3'-(p-Tolylmethylene)bis(N'-((E)-2-methylbenzylidene)-1H-indole-5-
carbohydrazide) (89) 138
3.7.3.20 3,3'-(p-Tolylmethylene)bis(N'-((E)-3-methylbenzylidene)-1H-indole-5-
carbohydrazide) (90) 138
xix
3.7.3.21 3,3'-(p-Tolylmethylene)bis(N'-((E)-4-methylbenzylidene)-1H-indole-5-
carbohydrazide) (91) 139
3.7.3.22 3,3'-(p-Tolylmethylene)bis(N'-((E)-2-chlorobenzylidene)-1H-indole-5-
carbohydrazide) (92) 139
3.7.3.23 3,3'-(p-Tolylmethylene)bis(N'-((E)-3-chlorobenzylidene)-1H-indole-5-
carbohydrazide) (93) 140
3.7.3.24 3,3'-(p-Tolylmethylene)bis(N'-((E)-4-chlorobenzylidene)-1H-indole-5-
carbohydrazide) (94) 140
3.7.3.25 3,3'-(p-Tolylmethylene)bis(N'-((E)-2,4-dichlorobenzylidene)-1H-indole-5-
carbohydrazide) (95) 141
3.7.3.26 3,3'-(p-Tolylmethylene)bis(N'-((E)-2-fluorobenzylidene)-1H-indole-5-
carbohydrazide) (96) 141
3.7.3.27 3,3'-(p-Tolylmethylene)bis(N'-((E)-3-fluorobenzylidene)-1H-indole-5-
carbohydrazide) (97) 142
3.7.3.28 3,3'-(p-Tolylmethylene)bis(N'-((E)-4-fluorobenzylidene)-1H-indole-5-
carbohydrazide) (98) 142
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-
carbohydrazide) (100) 143
3.7.3.31 3,3'-(p-Tolylmethylene)bis(N'-((E)-pyridin-4-ylmethylene)-1H-indole-5-
carbohydrazide) (101) 144
3.7.3.32 3,3'-(p-Tolylmethylene)bis(N'-((E)-pyridin-2-ylmethylene)-1H-indole-5-
carbohydrazide) (102) 144
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
3.9.3.2 2,2'-(3,3'-(p-tolylmethylene)bis(1H-indole-3,5-diyl-5-carbonyl))bis(N-(3-
bromophenyl)hydrazine-1-carbothioamide) (104) 160
3.9.3.3 2,2'-(3,3'-(p-tolylmethylene)bis(1H-indole-3,5-diyl-5-carbonyl))bis(N-(2-
bromophenyl)hydrazine-1-carbothioamide) (105) 161
3.9.3.4 2,2'-(3,3'-(p-tolylmethylene)bis(1H-indole-3,5-diyl-5-carbonyl))bis(N-(2-
fluorophenyl)hydrazine-1-carbothioamide) (106) 161
3.9.3.5 2,2'-(3,3'-(p-tolylmethylene)bis(1H-indole-3,5-diyl-5-carbonyl))bis(N-(3-
fluorophenyl)hydrazine-1-carbothioamide) (107) 162
3.9.3.6 2,2'-(3,3'-(p-tolylmethylene)bis(1H-indole-3,5-diyl-5-carbonyl))bis(N-(4-
fluorophenyl)hydrazine-1-carbothioamide) (108) 162
3.9.3.7 2,2'-(3,3'-(p-tolylmethylene)bis(1H-indole-3,5-diyl-5-carbonyl))bis(N-(2-
chlorophenyl)hydrazine-1-carbothioamide) (109) 163
3.9.3.8 2,2'-(3,3'-(p-tolylmethylene)bis(1H-indole-3,5-diyl-5-carbonyl))bis(N-(4-
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-
tolyl)hydrazine-1-carbothioamide) (113) 165
3.9.3.12 2,2'-(3,3'-(p-tolylmethylene)bis(1H-indole-3,5-diyl-5-carbonyl))bis(N-(p-
tolyl)hydrazine-1-carbothioamide) (114) 165
xxi
3.9.3.13 2,2'-(3,3'-(p-tolylmethylene)bis(1H-indole-3,5-diyl-5-carbonyl))bis(N-(2-
methoxyphenyl)hydrazine-1-carbothioamide) (115) 166
3.9.3.14 2,2'-(3,3'-(p-tolylmethylene)bis(1H-indole-3,5-diyl-5-carbonyl))bis(N-(3-
methoxyphenyl)hydrazine-1-carbothioamide) (116) 166
3.9.3.15 2,2'-(3,3'-(p-tolylmethylene)bis(1H-indole-3,5-diyl-5-carbonyl))bis(N-(3-
nitrophenyl)hydrazine-1-carbothioamide) (117) 167
3.9.3.16 2,2'-(3,3'-(p-tolylmethylene)bis(1H-indole-3,5-diyl-5-carbonyl))bis(N-(4-
nitrophenyl)hydrazine-1-carbothioamide) (118) 168
3.9.3.17 2,2'-(3,3'-(p-tolylmethylene)bis(1H-indole-3,5-diyl-5-carbonyl))bis(N-(4-
(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
3.10.3 Molecular docking 177
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
3.11.3.3 3,3'-((4-(5-(2-Methyl-1H-indol-3-yl)-1,3,4-oxadiazol-2-
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
3.11.3.5 2-(4-(Bis(1-methyl-1H-indol-3-yl)methyl)phenyl)-5-(2-methyl-1H-indol-3-yl)-
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-
oxadiazole (132) 187
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
3.11.3.9 2-(4-(Bis(2-phenyl-1H-indol-3-yl)methyl)phenyl)-5-(1H-indol-3-yl)-1,3,4-
oxadiazole (134) 188
3.11.3.10 2-(4-(Di(1H-indol-3-yl)methyl)phenyl)-5-(1-methyl-1H-indol-3-yl)-1,3,4-
oxadiazole (135) 189
3.11.3.11 2-(4-(Di(1H-indol-3-yl)methyl)phenyl)-5-(1H-indol-3-yl)-1,3,4-oxadiazole
(136) 190
3.11.3.12 2-(4-(Di(1H-indol-3-yl)methyl)phenyl)-5-(2-methyl-1H-indol-3-yl)-1,3,4-
oxadiazole (137) 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
3.11.3.14 2-(4-(Bis(1,2-dimethyl-1H-indol-3-yl)methyl)phenyl)-5-(2-methyl-1H-indol-3-
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-
oxadiazole (140) 192
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
3.11.3.17 2-(4-(Bis(5-chloro-1H-indol-3-yl)methyl)phenyl)-5-(2-methyl-1H-indol-3-yl)-
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-
oxadiazole (143) 194
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
3.11.3.20 2-(4-(Bis(5-bromo-1H-indol-3-yl)methyl)phenyl)-5-(2-methyl-1H-indol-3-yl)-
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-
oxadiazole (146) 195
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-1.13: Thiazole containing analogs as anti-cancer agents ......................................... 16
Figure-1.14: Thiazole containing analogs as anti-cancer agents ......................................... 17
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-2.5: Oxadiazole containing analogs as anti-microbial agents ................................. 55
Figure-2.6: Oxadiazole containing analogs as anti-microbial agents ................................. 56
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
Figure-2.17: Comparison of structure activity relationship between analogs 65 and 66 . 71
xxvi
Figure-2.18: Comparison of structure activity relationship between analogs 74 and 75 . 72
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
Figure-3.20: Comparison of structure activity relationship between compounds 106, 107
and 108 ....................................................................................................................................... 150
Figure-3.21: Comparison of structure activity relationship between compounds 112, 113
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-3.26: Structure-activity relationship of compounds 141-143 ................................. 174
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-3.29: Structure-activity relationship of compounds 126-128 ................................. 176
Figure-3.30: Structure-activity relationship of compounds 132-134 ................................. 176
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
Table-2.2: Docking scores and report of predicted interactions of docked conformations
....................................................................................................................................................... 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].
Figure-1.13: Thiazole containing analogs as anti-cancer agents
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
Figure-1.14: Thiazole containing analogs as anti-cancer agents
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
Table-1.2: Docking scores and report of predicted interactions of docked conformations
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
O 35 NH2 ARG 171 (A) H-acceptor 2.99 -1.1
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
N 19 NH2 ARG 171 (A) H-acceptor 2.90 -4.1
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
N 20 NH2 ARG 171 (A) H-acceptor 2.22 -3.8
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
N 20 NH2 ARG 171 (A) H-acceptor 2.77 -2.5
O 31 NH1 ARG 171 (A) H-acceptor 2.6 -1.8
O 31 NH2 ARG 171 (A) H-acceptor 2.75 -1.5
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
N 18 NH2 ARG 171 (A) H-acceptor 2.92 -1.2
5-ring 6-ring TYR 168 (A) pi-pi 3.65 -0.0
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
6-ring 6-ring PHE 210 (A) pi-pi 3.90 -0.0
112 -8.2318 C 26 6-ring TYR 168 (A) H-pi 3.53 -0.6
5-ring 6-ring PHE 210 (A) pi-pi 3.94 -0.0
113 -8.0013 5-ring N GLN 156 (A) pi-H 4.65 -0.7
5-ring 6-ring TYR 168 (A) pi-pi 3.82 -0.0
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
5-ring 6-ring TYR 168 (A) pi-pi 3.12 -0.0
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
5-ring 6-ring TYR 168 (A) pi-pi 3.41 -0.0
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
6-ring 6-ring PHE 210 (A) pi-pi 2.99 -0.0
6-ring 6-ring PHE 210 (A) pi-pi 2.76 -0.0
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
O 8 NH1 ARG 171 (A) H-acceptor 3.28 -2.3
O 8 NH2 ARG 171 (A) H-acceptor 3.16 -4.0
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).
1.7.1.1 2-((2,5-dimethylthiazol-4-yl)methyl)-5-(4-nitrophenyl)-1,3,4-oxadiazole (102)
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.
1.7.1.2 2-((2,5-dimethylthiazol-4-yl)methyl)-5-(2-nitrophenyl)-1,3,4-oxadiazole (103)
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.
1.7.1.13 2-(5-((2,5-dimethylthiazol-4-yl)methyl)-1,3,4-oxadiazol-2-yl)benzonitrile (114)
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.
1.7.1.14 3-(5-((2,5-dimethylthiazol-4-yl)methyl)-1,3,4-oxadiazol-2-yl)benzonitrile (115)
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
1.7.1.15 4-(5-((2,5-dimethylthiazol-4-yl)methyl)-1,3,4-oxadiazol-2-yl)benzonitrile (116)
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
References
1. Eicher, T.; Hauptmann, A. 3rd, completely revised and enlarged Edition, Weinheim:
Wiley-VCH Verlag GmbH, 2012.
2. Abele, E.; Abele, R.; Lukevics, E. New York: Springer, 2007.
3. Guray, T.; Ackkalp, E.; Yarlıgan, S. J. Mol. Graph. Model. 2007, 26, 154–165.
4. Ulrich, D. R. Polymer, 1987, 28, 533-542.
5. Roche, E. J.; Takahashi, T.; Thomas, E. L. ACS Symposium Series, 1980, 141, 303-313.
6. Odell, J. A.; Keller, A.; Atkins, E. D. T.; Metes, M. J. J. Mater. Sci. 1981, 16, 3309-3318.
7. Kabir, S. E.; Ahmed, F.; Das, A.; Hassan, M. R.; Lindeman, S. V.; GolzarHossain, G.
M.; Siddiquee, T. A.; Bennett, D. W. J. Organomet. Chem. 2007, 692, 4337–4345.
8. Bolos, C. A.; Chaviara, A. T.; Mourelatos, D.; Iakovidou, Z.; Mioglou, E.;
Chrysogelou, E.; Papageorgiou, A. Bioorg. Med. Chem. 2009, 17, 3142–3151.
9. Wu, Y.; Yeh, C.; Chan, Z.; Lin, C.; Chen, J.; Wang, J. J. Mol. Struct. 2008, 890, 48-56.
10. Ren, T. Coord. Chem. Rev. 1998, 175, 43–58.
11. Siddiqui, N.; Arshad, M. F.; Alam, M. S. Int. J. Pharm. Sci. Drug Res. 2009, 1, 136–143.
12. Das, J.; Chen, P.; Norris, D.; Padmanabha, R.; Lin, J.; Moquin, R. V.; Shen, Z.; Cook,
L. S.; Doweyko, A. M.; Wityak, J. C. J. Med. Chem. 2006, 49, 6819–32.
13. Fox, L. M.; Saravolatz, L. D. Clin. Infect. Dis. 2005, 40, 1173–1180.
14. Pasqualotto, A. C.; Goldani, L. Z. Curr. Opin. Investig. Drugs, 2010, 11, 165-74.
15. Lednicer, D.; Mitscher, G. I. Org. Chem. Drug Synth. 1990, 4, 95–97.
16. Knadler, M. P.; Callaghan, J. T.; Rubin, A. Drug Metab. Dispos. 1986, 14, 175–182.
17. Nauen, R.; Ebbinghaus, K. U.; Kaussmann, M. Pest. Biochem. Physiol. 2003, 76, 55–69.
46
18. De Souza, M. V. N.; De Almeida, M. V. Quim. Nova. 2003, 26, 366–372.
19. Grange, J. M.; Zumla, A. J. R. Soc, Health, 2002, 122, 78–81.
20. Karegoudar, P.; Karthikeyan, M. S.; Prasad, D. J.; Mahalinga, M.; Holla, B. S.;
Kumari, N. S. Eur. J. Med. Chem. 2008, 43, 261–267.
21. Bharti, S. K.; Nath, G.; Tilak, R.; Singh, S. K. Eur. J. Med. Chem. 2010, 45, 651–660.
22. El-Wahab, H. A.; El-Fattah, M. A.; El-Khalik, N. A.; Nassar, H. S.; Abdelall, M. M.
Prog. Org. Coat. 2014, 77, 1506–1511.
23. Vijesh, A. M.; Isloor, A. M.; Malladi, S. Eur. J. Med. Chem. 2010, 45, 5460–5464.
24. Baraldi, P. G.; Barbara, C.; Giampiero, S.; Romeo, R.; Giovanni, B.; Abdel, N. Z.;
Maria, J.; Pineda, D. L. Synthesis, 1997, 1140–1147.
25. Holla, B. S.; Malini, K. V.; Kumari, N. S. Eur. J. Med. Chem. 2003, 38, 313–318.
26. Lv, P. C.; Wang, K. R.; Yang, Y.; Mao, W. J.; Chen, J.; Xiong, J.; Zhu, H. L. Bioorg.
Med. Chem. Lett. 2009, 19, 6750–6754.
27. Jagani, C. L.; Sojitra, N. A.; Vanparia, S. F.; Patel, T. S.; Dixit, R. B.; Dixit, B.C. J. Saudi.
Chem. Soc. 2012, 16, 363–369.
28. Desai, N. C.; Bhatt, N.; Somani, H.; Trivedi, A. Eur. J. Med. Chem. 2013, 67, 54–59.
29. Mohammad, H.; Mayhoub, A. S.; Ghafoor, A.; Soofi, M.; Alajlouni, R. A.; Cushman,
M.; Seleem, M. N. J. Med. Chem. 2014, 57, 1609–1615.
30. Chimenti, F.; Bizzarri, B.; Bolasco, A.; Secci, D.; Granese, A.; Carradori, S.; Ascenzio,
M. D.; Lilli, D.; Rivanera, D. Eur. J. Med. Chem. 2011, 46, 378–382.
31. Ballell, L.; Field, R. A.; Duncan, K.; Young, R. J. Antimicrob. Agents Chemother. 2005,
49, 2153–2163.
47
32. Daele, I. V.; Calenbergh, S. Exp. Opin. Ther. Pat. 2005, 15, 131–140.
33. Zhang, Y.; Post-Martens, K.; Denkin, S. Drug Discov. Today, 2006, 11, 21–27.
34. Shiradkar, M. R.; Murahari, K. K.; Gangadasu, H. R.; Kaur, R.; Burange, P.; Ghogare,
J.; Mokalec, V.; Raut, M. Bioorg. Med. Chem. 2007, 15, 3997–4008.
35. Makam, P.; Kankanala, R.; Kannan, T. Eur. J. Med. Chem. 2013, 69, 564–576.
36. Jeankumar, V.U.; Renuka, J.; Santosh, P.; Jonnalagadda, V. S.; Sridevi, P.;
Suryadevara, P.; Sriram, P. Y. D. Eur. J. Med. Chem. 2013, 70, 143–153.
37. Aridoss, G.; Amirthaganesan, S.; Jeong, Y. T. Eur. J. Med.Chem. 2009, 44, 4199–4210.
38. El-Sabbagh, O. I.; Baraka, M. M.; Ibrahim, S. M.; Pannecouque, C.; Andrei, G.;
Snoeck, R.; Balzarini, J.; Rashad, A. A. Eur. J. Med. Chem. 2009, 44, 3746–3753.
39. Wang, W. L.; Yao, D. Y.; Gu, M.; Fan, M. Z.; Li, J. Y.; Xing, Y. C.; Nana, F. J. Bioorg.
Med. Chem. Lett. 2005, 15, 5284–5287.
40. Liu, Y.; Jing, F.; Xu, Y.; Li, M.; Xu, W. Bioorg. Med. Chem. 2011, 19, 2342–2348.
41. Li, Z.; Khaliq, M.; Kuhn, R.; Cushman, M. J. Med. Chem. 2008, 51, 4660–4671.
42. Stachulski, A. V.; Pidathala, C.; Row, E. C.; Sharma, R.; Berry, N. G.; Iqbal, M.;
Bentley, J.; Allman, S. A.; Edwards, G.; Korba, B. E.; Semple, J. E.; Rossignol, J. F. J.
Med. Chem. 2011, 54, 4119–4132.
43. Curreli, F.; Choudhury, S.; Pyatkin, I.; Zagorodnikov, V. P.; Bulay, A. K.; Altieri, A.;
Kwong, P. D.; Debnath, A. K. J. Med. Chem. 2012, 55, 4764–4775.
44. Moghaddam, G.; Ebrahimi, S. A.; Rahbar-Roshandel, N.; Foroumadi, A. Phytother.
Res. 2012, 26, 1023–1028.
48
45. Xiong, S.; Nguyen, B.; Jia, S.; Herich, J.; Guastella, J.; Reddy, S.; Tseng, B.; Drewe, J.;
Kasibhatla, S. J. Med. Chem. 2003, 46, 2474–2481.
46. Aliabadi, A.; Shamsa, F.; Ostad, S. N.; Emami, S.; Shafiee, A.; Davoodi, J.;
Foroumadi, A. Eur. J. Med. Chem. 2010, 45, 5384–5389.
47. Mohammadi-Farani, A.; Foroumadi, A.; Rezvani, M.; Aliabadi, A. Iran. J. Basic Med.
Sci. 2014, 17, 502–508.
48. Zaharia, V.; Ignat, A.; Palibrod, N.; Ngameni, B.; Kuete, V.; Fokunang, C. N.;
Moungang, M. L.; Ngadjui, B. T.; Eur. J. Med. Chem. 2010, 45, 5080–5085.
49. Bursavich, M. G.; Parker, D. P.; Willardsen, J. A.; Gao, Z. H.; Davis, T.; Ostanin, K.;
Cimbora, D. M.; Zhu, J. F.; Richards, B. Bioorg. Med. Chem. Lett. 2010, 20, 1677–1679.
50. Wilson, K. J.; Illig, C. R.; Subasinghe, N.; Hoffman, J. B.; Rudolph, M. J.; Soll, R.;
Zhang, M.; Lewandowski, F. A.; Zhou, Z.; Sharp, C.; Maguire, D.; Grasberger, B.;
DesJarlais, R. L.; Spurlino, J. Bioorg. Med. Chem. Lett. 2001, 11, 915–918.
51. Salehi, M.; Amini, M.; Ostad, S. N.; Riazi, G. H.; Assadieskandar, A.; Shafiei, B.;
Shafiee, A. Bioorg. Med. Chem. 2013, 21, 7648–7654.
52. Banimustafa, M.; Kheirollahi, A.; Safavi, M.; Kabudanian, S.; Aryapour, H.;
Foroumadi, A.; Emami, S. Eur. J. Med. Chem. 2013, 70, 692–702.
53. Liu, Z. Y.; Wang, Y. M.; Boykin, D.W. Bioorg. Med. Chem. Lett. 2009, 19, 5661–5664.
54. Shi, H. B.; Zhang, S. J.; Ge, Q. F.; Guo, D. W.; Cai, C. M.; Hua, W. X. Bioorg. Med.
Chem. Lett. 2010, 20, 6555–6559.
55. Rodriguez, L. D.; Gaboriau, F.; Rivault, F.; Schalk, I. J.; Lescoat, G.; Mislin, G. L. A.
Bioorg. Med. Chem. 2010, 18, 689–695.
49
56. Azizmohammadi, M.; Khoobi, M.; Ramazani, A.; Emami, S.; Zarrin, A.; Firuzi, O.;
Miri, R.; Shafiee, A. Eur. J. Med. Chem. 2013, 59, 15–22.
57. Jones, O. T. Eur. J. Pharmacol. 2002, 447, 211–225.
58. Perucca, E. Ther. Drug Monit. 2002, 24, 74–80.
59. Duncan, J. S. Br. J. Clin. Pharmacol. 2002, 53, 123–131.
60. Siddiqui, N.; Ahsan, W. Eur. J. Med. Chem. 2010, 45, 1536–1543.
61. Ahangar, N.; Ayati, A.; Alipour, E.; Pashapour, A.; Foroumadi, A.; Emami, S. Chem.
Biol. Drug Des. 2011, 78, 844–852.
62. Amin, K. M.; Al-Eryani, Y. A. Bioorg. Med. Chem. 2008, 16, 5377-5388.
63. Kalkhambkar, R. G.; Shivkumar, H.; Rao, N. Eur. J. Med. Chem. 2007, 42, 1272–1276.
64. Giri, R. S.; Thaker, H. M.; Giordano, T.; Williams, J.; Rogers, D.; Sudersanam, V.;
Vasu, K. K. Eur. J. Med. Chem. 2009, 44, 2184–2189.
65. Kumar, A.; Rajput, C. S.; Bhati, S. K. Bioorg. Med. Chem. 2007, 15, 3089–3096.
66. Aggarwal, R.; Kaushik, P.; Gupta, G. K. Eur. J. Med. Chem. 2013, 62, 508–514.
67. Helal, M. H.; Salem, M. A.; Aljahdali, M. Eur. J. Med. Chem. 2013, 65, 517–526.
68. Koppireddi, S.; Komsani, J. R.; Avula, S.; Pombala, S.; Vasamsetti, S.; Kotamraju, S.;
Yadla, R. Eur. J. Med. Chem. 2013, 66, 305–313.
69. Rajendran, P.; Nandakumar, N.; Rengarajan, T.; Palaniswami, R.; Gnanadhas, E. N.;
Lakshminarasaiah, U.; Gopas, J.; Nishigaki, I. Clin. Chim. Acta. 2014, 436, 332–347.
70. Mandawad, G. G.; Dawane, B. S.; Beedkar, S. D.; Khobragade, C. N.; Yemul, O. S.
Bioorg. Med. Chem. 2013, 21, 365–372.
71. Kim, K. M.; Kim, K. H.; Kang, C.; Shin, I. Bioorg. Med. Chem. Lett. 2003, 13, 2273–2275.
50
72. Wahab, B. F.; Awad, G. E. A.; Badria, F. A. Eur. J. Med. Chem. 2011, 46, 1505–1511.
73. Masakazu, F.; Tetsuya, H.; Naoko, I. Bioorg. Med. Chem. 2002, 10, 3113–3122.
74. William, P. C.; Jaswinder, K. S. FEBS Lett. 2008, 582, 117–131.
75. Attie, A. D.; Krauss, R. M.; Gray-Keller, M. P.; Brownlie, A.; Miyazaki, M.;
Stalenhoef, A. F.; Hayden, M. R.; Ntambi, J. M. J. Lipid Res. 2002, 43, 1899–1907.
76. Li, C. S.; Belair, L.; Guay, J.; Murgasva, R.; Sturkenboom, W.; Ramtohul, Y. K.;
Zhang, L.; Huang, Z. Bioorg. Med. Chem. Lett. 2009, 19, 5214–5217.
77. Kang, S. Y.; Song, K. S.; Lee, S. H.; Lee, J. Bioorg. Med. Chem. 2010, 18, 6069–6079.
78. Raza, R.; Saeed, A.; Arif, M.; Mahmood, S.; Muddassar, M.; Raza, A.; Iqbal, J. Chem.
Biol. Drug Des. 2012, 80, 605–615.
79. Chen, Y. L.; Cherry, K.; Corman, M. L.; Ebbinghaus, C. F.; Liston, D.; Martin, B. A.;
Oborskib, C. E.; Sahagan, B. G. Bioorg. Med. Chem. Lett. 2007, 17, 5518–5522.
80. Shiradkar, M. R.; Akula, K. C.; Dasari, V.; Baru, V.; Chiningiri, B.; Gandhid, S.;
Kaure, R. Bioorg. Med. Chem. 2007, 15, 2601–2610.
81. Sahu, S. K.; Mishra, A.; Behera, R. K. Ind. J. Heterocycl. Chem. 1996, 6, 91-96.
82. Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.; Belew, R. K.;
Olson, A. J. Comput. Chem. 1998, 19, 1660.
83. Mehdipour-Ataei, S.; Arabi, H.; Bahri-Laleh, N. Eur. Polym. J. 2006, 42, 2343.
84. Shih, M. H.; Ke, F. Y. Bioorg. Med. Chem. 2004, 12, 4633–4643.
85. Leach, A. R.; Shoichet, B. K.; Peishoff, C. J. Med. Chem. 2006, 49, 5851-5855.
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
Figure-2.5: Oxadiazole containing analogs as anti-microbial agents
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].
Figure-2.6: Oxadiazole containing analogs as anti-microbial agents
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 Results and discussion
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.
Figure-2.17: Comparison of structure activity relationship between analogs 65 and 66
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
Figure-2.18: Comparison of structure activity relationship between analogs 74 and 75
To understand the binding interaction of the most active analogs molecular docking
study was performed.
2.5.3 Molecular docking
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
Table-2.2: Docking scores and report of predicted interactions of docked conformations
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-
oxadiazol-2-yl)benzohydrazide (66)
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-
yl)benzohydrazide (67)
Yield: 90%. Light yellow solid, m.p. 245-247 °C. 1H-NMR (500 MHz, DMSO-d6): δ 11.70
(s, 1H, NH), 8.93 (s, 1H, CH=N), 8.03 (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), 7.83 (d, J = 7.6 Hz, 2H, Ar), 7.80 (d, J = 7.3 Hz, 2H, Ar), 7.45
(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-
yl)benzohydrazide (69)
Yield: 89%. Light yellow solid, m.p. 255-257 °C. 1H-NMR (500 MHz, DMSO-d6): δ 11.50
(s, 1H, NH), 8.45 (s, 1H, CH=N), 8.07 (d, J = 7.9 Hz, 2H, Ar), 7.93 (d, J = 7.5 Hz, 2H, Ar),
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.
2.7.5.6 (E)-4-(5-(4-cyanophenyl)-1,3,4-oxadiazol-2-yl)-N'-(4-
(dimethylamino)benzylidene) benzohydrazide (70)
Yield: 80%. Dark yellow solid, m.p. 250-252 °C. 1H-NMR (500 MHz, DMSO-d6): δ 11.45
(s, 1H, NH), 8.35 (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), 7.52 (d, J = 7.1 Hz, 2H, Ar), 6.80
(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-
oxadiazol-2-yl)benzohydrazide (71)
Yield: 78%. Dark yellow solid, m.p. 225-227 °C. 1H-NMR (500 MHz, DMSO-d6): δ 11.34
(s, 1H, NH), 8.45 (s, 1H, CH=N), 8.04 (d, J = 7.9 Hz, 2H, Ar), 7.92 (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), 7.54 (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,
123.6, 118.2, 112.1. HREI-MS: m/z calcd for C31H19N5O2 [M]+ 493.1539, Found 493.1535.
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
(s, 1H, NH), 9.00 (s, 1H, CH=N), 8.17 (d, J = 8.3 Hz, 1H, 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), 7.81 (d, J = 7.3 Hz, 1H, Ar), 7.80
(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.
2.7.5.10 (E)-4-(5-(4-cyanophenyl)-1,3,4-oxadiazol-2-yl)-N'-(4-
hydroxybenzylidene) benzohydrazide (74)
Yield: 71%. Yellow solid, m.p. 256-258 °C. 1H-NMR (500 MHz, DMSO-d6): δ 11.10 (s, 1H,
NH), 8.34 (s, 1H, CH=N), 8.03 (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), 7.62 (d, J = 7.3 Hz, 2H, Ar), 6.81 (d, J = 6.4
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,
116.8, 116.4, 112.1. HREI-MS: m/z calcd for C23H15N5O3 [M]+ 409.1175, Found 409.1171.
2.7.5.11 (E)-4-(5-(4-cyanophenyl)-1,3,4-oxadiazol-2-yl)-N'-(3-
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-
MS: m/z calcd for C23H15N5O3 [M]+ 409.1175, Found 409.1171.
2.7.5.12 (E)-4-(5-(4-cyanophenyl)-1,3,4-oxadiazol-2-yl)-N'-(3-hydroxy-4-
methoxybenzylidene) benzohydrazides (76)
Yield: 80%. Light yellow solid, m.p. 227-229 °C. 1H-NMR (500 MHz, DMSO-d6): δ 11.40
(s, 1H, NH), 8.44 (s, 1H, CH=N), 8.05 (d, J = 7.9 Hz, 2H, Ar), 7.93 (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), 7.41 (s, 1H, Ar), 7.08 (d, J = 7.0
Hz, 1H, Ar), 7.02 (d, J = 7.0 Hz, 1H, Ar), 3.82 (s, 3H, CH3), 13C-NMR (125 MHz, DMSO-
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,
104.0, 56.4, 56.0. HREI-MS: m/z calcd for C25H19N5O5 [M]+ 469.1386, Found 469.1380.
92
2.7.5.14 (E)-4-(5-(4-cyanophenyl)-1,3,4-oxadiazol-2-yl)-N'-(4-hydroxy-3-
methoxybenzylidene) benzohydrazide (78)
Yield: 89%. White yellow solid, m.p. 231-233 °C. 1H-NMR (500 MHz, DMSO-d6): δ 11.33
(s, 1H, NH), 8.43 (s, 1H, CH=N), 8.02 (d, J = 7.9 Hz, 2H, Ar), 7.92 (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), 7.34 (s, 1H, Ar), 7.31 (d, J = 7.1
Hz, 1H, Ar), 6.83 (d, J = 7.1 Hz, 1H, Ar), 3.80 (s, 3H, CH3), 13C-NMR (125 MHz, DMSO-
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.
2.7.5.15 (E)-4-(5-(4-cyanophenyl)-1,3,4-oxadiazol-2-yl)-N'-(2-
nitrobenzylidene)benzohydrazide (79)
Yield: 93%. Yellow solid, m.p. 243-245 °C. 1H-NMR (500 MHz, DMSO-d6): δ 11.30 (s, 1H,
NH), 8.53 (s, 1H, CH=N), 8.03 (d, J = 7.9 Hz, 1H, Ar), 8.01 (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), 7.90 (d, J = 7.7 Hz, 1H, Ar), 7.80 (d, J = 7.3
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-
MS: m/z calcd for C23H14N6O4 [M]+ 438.1077, Found 438.1070.
2.7.5.16 (E)-N'-(2-cyanobenzylidene)-4-(5-(4-cyanophenyl)-1,3,4-oxadiazol-2-yl)
benzohydrazides (80)
Yield: 88%. Dark yellow solid, m.p. 217-219 °C. 1H-NMR (500 MHz, DMSO-d6): δ 11.09
(s, 1H, NH), 8.32 (s, 1H, CH=N), 8.07 (d, J = 7.9 Hz, 2H, Ar), 7.96 (d, J = 7.7 Hz, 1H, Ar),
93
7.92 (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), 7.76
(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
References
1. Nagaraj, K. C.; Niranjan, M. S.; Kiran, S. Int. J. Pharm. Pharm. Sci. 2011, 3, 9–16.
2. Rajak, H.; Agarawal, A.; Parmar, P.; Thakur, B. S.; Veerasamy, R.; Sharma, P. C.;
Kharya, M. D. Bioorg. Med. Chem. Lett. 2011, 21, 5735–5738.
3. Onishi, T.; Iwashita, H.; Uno, Y.; Kunitomo, J.; Saitoh, M.; Kimura, E.; Fujita, H.;
Uchiyama, N.; Kori, M.; Takizawa, M. J. Neurochem. 2011, 119, 1330–1340.
4. Boström, J.; Hogner, A.; Llinàs, A.; Wellner, E.; Plowright, A. T. J. Med. Chem.
2012, 55, 1817–1830.
5. Katritzky, A. R.; Vedensky, V.; Rogovoy, B.; Steel, P. J. Arkivoc. 2002, 6, 82–90.
6. Savarino, A. Expert Opin. Investig. Drugs, 2006, 15, 1507–1522.
7. Kampmanna, T.; Yennamallia, R.; Campbella, P.; Stoermerc, M. J.; David, P.
Fairlie, D. P.; Kobea, B.; Young, P. R. Antivir. Res. 2009, 84, 234–241.
8. Gupta, V.; Kashaw, S. K.; Jatav, V.; Mishra, P. Med. Chem. Res. 2008, 17, 205–211.
9. James, N. D.; Growcott, J. W. Drugs Future, 2009, 34, 624–633.
10. Boström, J.; Hogner, A.; Llinàs, A.; Wellner, E.; Plowright, A. T. J. Med. Chem.
2012, 55, 1817–1830.
11. Savarino, A. Expert Opin. Investig. Drugs 2006, 15, 1507–1522.
12. James, N. D.; Growcott, J.W. Zibotentan. Drugs Future 2009, 34, 624–633.
13. Oliveira, C. S.; Lira, B. F.; Falcão-Silva, V. S.; Siqueira-Junior, J. P.; Barbosa-Filho,
J. M.; Athayde-Filho, P. F. Molecules, 2012, 17, 5095–5107.
14. Sahin, G.; Palaska, E.; Ekizoglu, M.; Ozalp, M. Il Farmaco. 2002, 57, 539–542.
15. Patel, N.B.; Patel, J. C. Sci. Pharm. 2010, 78, 171–193.
95
16. Fuloria, N. K.; Singh, V.; Shaharyar, M.; Ali, M. Molecules, 2009, 14, 1898–1903.
17. Kumar, S. J. Chil. Chem. Soc. 2010, 55, 126–129.
18. Sangshetti, J. N.; Shinde, D. B. Bioorg. Med. Chem. Lett. 2011, 21, 444–448.
19. Chandrakantha, B.; Shetty, P.; Nambiyar, V.; Isloor, N.; Isloor, A. M. Eur. J. Med.
Chem. 2010, 45, 1206–1210.
20. Yar, M. S.; Siddiqui, A. A.; Ali, M. A. J. Chin. Soc. 2007, 54, 5–8.
21. Kumar, G. V. S.; Rajendraprasad, Y.; Mallikarjuna, B. P.; Chandrashekar, S. M.;
Kistayya, C. Eur. J. Med. Chem. 2010, 45, 2063–2074.
22. Yoshida, Y.; Matsuda, K.; Sasaki, H.; Matsumoto, Y.; Matsumoto, S.; Tawara, S.;
Takasugi, H. Bioorg. Med. Chem. 2000, 8, 2317–2335.
23. Bakal, R. L.; Gattan, S. G. Eur. J. Med. Chem. 2012, 47, 278–282.
24. Alia, M. A.; Shaharyar, M. Bioorg. Med. Chem. Lett. 2007, 17, 3314–3316.
25. Kashaw, S. K.; Gupta, V.; Kashaw, V.; Mishra, P.; Stables, J. P.; Jain, N. K. Med.
Chem. Res. 2010, 19, 250–261.
26. Zarghi, A.; Tabatabai, S. A.; Faizi, M.; Ahadian, A.; Navabi, P.; Zanganeh, V.;
Shafiee, A. Bioorg. Chem. Lett. 2005, 15, 1863–1865.
27. Rajak, H.; Deshmukh, R.; Veerasamy, R.; Sharma, A. K.; Mishra, P.; Kharya, M.
D. Bioorg. Med. Chem. Lett. 2010, 20, 4168–4172.
28. Manjunatha, K.; Poojary, B.; Lobo, P. L.; Fernandes, J.; Kumari, N. S. Eur. J. Med.
Chem. 2010, 45, 5225–5233.
29. Husain, A.; Ahmad, A.; Ahuja, P. Eur. J. Med. Chem. 2009, 44, 3798–3804.
96
30. Burbuliene, M. M.; Jakubkiene, V.; Mekuskiene, G.; Udrenaite, E.; Smicius, R.;
Vainilavicius, P. Farmaco, 2004, 59, 767–774.
31. Amir, M.; Kumar, S. Acta Pharm. 2007, 57, 31–45.
32. Gilani, S. J.; Khan, S. A.; Siddiqui, N. Bioorg. Med. Chem. Lett. 2010, 20, 4762–4765.
33. Savariz, F. C.; Formagio, A. S. N.; Barbosa, V. A.; Foglio, M. A. Carvalho, J. E.;
Duarte, M. C. T.; Sarragiotto, M. H. J. Braz. Chem. Soc. 2010, 21, 288–298.
34. Liu, K.; Lu, X.; Zhang, H. J.; Zhu, H. L. Eur. J. Med. Chem. 2012, 47, 473–478.
35. Ouyang, X.; Piatnitski, E. L.; Pattaropong, V.; Chen, X.; He, H. Y.; Kiselyov, A. S.;
Velankar, A.; Labelle, M.; Smith, L. Bioorg. Med. Chem. Lett. 2006, 16, 1191–1196.
36. Tuma, M. C.; Malikzay, A.; Ouyang, X.; Surgulazde, D.; Fleming, J.; Mitelman, S.;
Camara, M.; Doody, J.; Chekler, E. L. P. Transl. Oncol. 2010, 3, 318–325.
37. Lee, L.; Robb, L. M.; Davis, R.; Mackay, H.; Chavda, S.; Babu, B.; O‟brien, E. L.;
Risinger, A. L.; Mooberry, S. L.; Lee, M. J. Med. Chem. 2010, 53, 325–334.
38. Temesgen, Z.; Siraj, D. S. Ther. Clin. Risk Manage. 2008, i, 493–500.
39. Wang, Z.; Wang, M.; Yao, X.; Li, Y.; Qiao, W.; Geng, Y.; Liu, Y.; Wang, Q. Eur. J.
Med. Chem. 2012, 50, 361–369.
40. El-Emam, A. A.; Al-Deeb, O.A.; Al-Omar, M.; Lehmann, J. Bioorg. Med. Chem.
2004, 12, 5107–5113.
41. Iqbal, R.; Zareef, M.; Ahmed, S.; Zaidi, J. H.; Arfan, M.; Shafique, M.; Al-masoudi,
N. A. J. Chin. Chem. Soc. 2006, 53, 689–696.
42. Kim, R. M.; Rouse, E. A.; Chapman, K. T.; Schleif, W. A.; Olsen, D. B.; Stahlhurt,
M.; Emini, E. A.; Tata, J. R. Bioorg. Med. Chem. Lett. 2004, 14, 4651–4654.
97
43. Johns, B.; Weatherhead, J. G.; Allen, S. H.; Thompson, J. B.; Garvey, E. P.; Foster,
S. A.; Jeffrey, J. L.; Miller, W. H. Bioorg. Med. Chem. Lett. 2009, 19, 1807–1810.
44. Llinàs-Brunet, M.; Bailey, M. D.; Bolger, G.; Brochu, C.; Faucher, A. M.; Ferland, J.
M.;Garneau, M.; Gorys, V.; Grand-Maître, C. J. Med. Chem. 2004, 47, 1605–1608.
45. Bankar, G. R.; Nandakumar, K.; Nayak, P. G.; Thakur, A.; Chamallamudi, M. R.;
Nampurath, G. K. Chem. Biol. Interact. 2009, 181, 377–382.
46. Rivera, N. R.; Balsells, J.; Hansen, K. B. Tetrahedron Lett. 2006, 47, 4889–4891.
47. El-Sayed, W. A.; Ali, O. M.; Abdel-Rahman, A. H. Chin. J. Chem. 2012, 30, 77–83.
48. Koparır, M.; Çetin, A.; Cansız, A. Molecules, 2005, 10, 475–480.
49. Polshettiwar, V.; Varma, R. S. Tetrahedron Lett. 2008, 49, 879–883.
50. Yang, S. J.; Lee, S. H.; Kwak, H. J.; Gong, Y. D. J. Org. Chem. 2013, 78, 438-444.
51. Taha, M.; Imran, S.; Rahim, F.; Wadood, A.; Khan, K. M. Bioorg. Chem. 2018, 76,
273-280.
52. Taha, M.; Ismail, N. H.; Imran, S.; Wadood, A.; Rahim, F.; Saad, S. M.; Khan, K.
M.; Nasir, A. Bioorg. Chem. 2016, 66, 117–123.
53. Taha, M.; Baharudin, M. S.; Ismail, N. H.; Selvaraj, M.; Salar, U.; Alkadi, K. A. A.;
Khan, K. M. Bioorg. Chem. 2016, 71, 86-96.
54. a) Taha, M.; Ismail, N. H.; Imran, S.; Selvaraj, M.; Rahim, F. RSC Adv. 2016, 6,
3003-3012; (b) Taha, M.; Ismail, N. H.; Imran, S.; Selvaraj, M.; Rahim, A.; Ali, M.;
Siddiqui, S.; Rahim, F.; Khan, K. M. Bioorg. Med. Chem. 2015, 23, 7394. (c) Taha,
M.; Ullah, H.; Muqarrabun, L. M. R. A.; Khan, M. N.; Rahim, F.; Ahmat, N.; Ali,
M.; Perveen, S. Eur. J. Med. Chem. 2018, 143, 1757-1767; (d) Taha, M.; Imran, S.;
98
Ismail, N. H.; Rahim, F.; Javed, M. T.; Khan, K. M.; Ali, M. Bioorg. Chem. 2017, 72,
323–332. (e) Taha, M.; Ismail, N. H.; Imran, S.; Wadood, A.; Rahim, F.;
Muqarrabin, L. M. R. A.; Ahmat, N.; Nasir, A.; Khan, F. Bioorg. Chem. 2016, 68, 15-
22. (f) Taha, M.; Sultan, S.; Nuzar, H. A.; Rahim, F.; Imran, S.; Naz, H.; Ismail, N.
H.; Ullah, H. Bioorg. Med. Chem. 2016, 24, 3696-3704. (g) Taha, M.; Ismail, N. H.;
Jamil, W.; Khan, K. M.; Salar, U.; Kashif, S. M.; Rahim, F.; Latif, Y. Med. Chem. Res.
2015, 24, 3166-3173. (h) Zawawi, N. K. N. A.; Taha, M.; Ahmat, N.; Wadood, A.;
Ismail, N. H.; Rahim, F.; Ali, M. Bioorg. Med. Chem. 2015, 23, 3119–3125. (I) Khan,
K. M.; Rahim, F.; Wadood, A.; Taha, M.; Khan, M.; Ambreen, N.; Choudhary, M.
I. Bioorg. Med. Chem. Lett. 2014, 24, 1825–1829.
55. Shahzad, S. A.; Yar, M.; Bajda, M.; Jadoon, B.; Khan, Z. A.; Naqvi, S. A. R.;
Shaikh, A. J.; Hayat, K. A.; Filipek, S. Bioorg. Med. Chem. 2014, 22, 1008–1015.
56. Shahzad, S. A.; Yar, M.; Bajda, M.; Shahzadi, L.; Khan, Z. A.; Naqvi, S. A.;
Mutahir, S.; Khan, K. M. Bioorg Chem. 2015, 60, 37-41.
57. Leach, A. R.; Shoichet, B. K.; Peishoff, C. J. Med. Chem. 2006, 49, 5851-5855.
99
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 Results and discussion
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-
carbohydrazide) (71)
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-
carbohydrazide) (72)
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
3.7.3.3 3,3'-(p-Tolylmethylene)bis(N'-((E)-3-nitrobenzylidene)-1H-indole-5-
carbohydrazide) (73)
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-
carbohydrazide) (74)
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-
carbohydrazide) (75)
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.
3.7.3.6 3,3'-(p-Tolylmethylene)bis(N'-((E)-3-hydroxybenzylidene)-1H-indole-5-
carbohydrazide) (76)
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
3.7.3.7 3,3'-(p-Tolylmethylene)bis(N'-((E)-2,3-dihydroxybenzylidene)-1H-indole-5-
carbohydrazide) (77)
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
3.7.3.9 3,3'-(p-Tolylmethylene)bis(N'-((E)-2,5-dihydroxybenzylidene)-1H-indole-5-
carbohydrazide) (79)
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.
3.7.3.10 3,3'-(p-Tolylmethylene)bis(N'-((E)-3,4-dihydroxybenzylidene)-1H-indole-5-
carbohydrazide) (80)
Yield: 87%. Brown sticky substance. 1H NMR (500 MHz, DMSO-d6): δ 11.73 (s; 2H), 10.82
(s; 2H), 9.46 (s; 4H), 8.44 (s; 2H), 8.34 (s; 2H), 7.87 (d, J = 8.8 Hz; 2H), 7.62 (d, J = 8.6 Hz;
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
3.7.3.11 3,3'-(p-Tolylmethylene)bis(N'-((E)-2,4,5-trihydroxybenzylidene)-1H-indole-5-
carbohydrazide) (81)
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.
3.7.3.12 3,3'-(p-Tolylmethylene)bis(N'-((E)-2,4,6-trihydroxybenzylidene)-1H-indole-5-
carbohydrazide) (82)
Yield: 81%. Light brown oily liquid. 1H NMR (500 MHz, DMSO-d6): δ 11.64 (s; 2H), 10.75
(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
3.7.3.13 3,3'-(p-Tolylmethylene)bis(N'-((E)-2-hydroxy-4-methoxybenzylidene)-1H-
indole-5-carbohydrazide) (83)
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,
107.1, 103.3, 103.3, 55.5, 55.5, 54.4, 21.2. HREI-MS: m/z Calcd for C42H36N6O6 [M]+
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
3.7.3.15 3,3'-(p-Tolylmethylene)bis(N'-((E)-3-hydroxy-4-methoxybenzylidene)-1H-
indole-5-carbohydrazide) (85)
Yield: 84%. Light grey oily liquid. 1H NMR (500 MHz, DMSO-d6): δ 11.71 (s; 2H), 10.76
(s; 2H), 9.30 (s; 2H), 8.43 (s; 2H), 8.33 (s; 2H), 7.86 (d, J = 8.8 Hz; 2H), 7.62 (d, J = 8.7 Hz;
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
3.7.3.17 3,3'-(p-Tolylmethylene)bis(N'-((E)-2-hydroxybenzylidene)-1H-indole-5-
carbohydrazide) (87)
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.
3.7.3.18 3,3'-(p-Tolylmethylene)bis(N'-((E)-2-nitrobenzylidene)-1H-indole-5-
carbohydrazide) (88)
Yield: 78%. Light brown solid, m.p. 273-275 °C. 1H NMR (500 MHz, DMSO-d6): δ 11.56
(s; 2H), 10.73 (s; 2H), 8.56 (s; 2H), 8.36 (s; 2H), 8.07 (d, J = 8.7 Hz; 2H), 7.94 (d, J = 8.9 Hz;
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
3.7.3.19 3,3'-(p-Tolylmethylene)bis(N'-((E)-2-methylbenzylidene)-1H-indole-5-
carbohydrazide) (89)
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.
HREI-MS: m/z Calcd for C42H36N6O2 [M]+ 656.2900; Found: 656.2898.
3.7.3.20 3,3'-(p-Tolylmethylene)bis(N'-((E)-3-methylbenzylidene)-1H-indole-5-
carbohydrazide) (90)
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,
111.1, 111.0, 111.0, 54.4, 21.2, 21.2, 21.2. HREI-MS: m/z Calcd for C42H36N6O2 [M]+
656.2900; Found: 656.2898.
139
3.7.3.21 3,3'-(p-Tolylmethylene)bis(N'-((E)-4-methylbenzylidene)-1H-indole-5-
carbohydrazide) (91)
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.
HREI-MS: m/z Calcd for C42H36N6O2 [M]+ 656.2900; Found: 656.2898.
3.7.3.22 3,3'-(p-Tolylmethylene)bis(N'-((E)-2-chlorobenzylidene)-1H-indole-5-
carbohydrazide) (92)
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
3.7.3.23 3,3'-(p-Tolylmethylene)bis(N'-((E)-3-chlorobenzylidene)-1H-indole-5-
carbohydrazide) (93)
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.
3.7.3.24 3,3'-(p-Tolylmethylene)bis(N'-((E)-4-chlorobenzylidene)-1H-indole-5-
carbohydrazide) (94)
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;
2H), 7.48 (d, J = 8.9 Hz; 4H), 7.21 (dd, J = 8.4, 1.5 Hz; 2H), 7.12 (dd, J = 8.6, 1.9 Hz; 2H),
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-
carbohydrazide) (95)
Yield: 80%. Light brown oily liquid. 1H NMR (500 MHz, DMSO-d6): δ 11.64 (s; 2H), 10.87
(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.
3.7.3.26 3,3'-(p-Tolylmethylene)bis(N'-((E)-2-fluorobenzylidene)-1H-indole-5-
carbohydrazide) (96)
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
3.7.3.27 3,3'-(p-Tolylmethylene)bis(N'-((E)-3-fluorobenzylidene)-1H-indole-5-
carbohydrazide) (97)
Yield: 79%. Light brown solid, m.p. 281-283 °C. 1H NMR (500 MHz, DMSO-d6): δ 11.67
(s; 2H), 10.78 (s; 2H), 8.47 (s; 2H), 8.37 (s; 2H), 7.90 (d, J = 8.5 Hz; 2H), 7.80 (d, J = 8.2 Hz;
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
(dd, J = 8.5, 1.4 Hz; 2H), 7.05 (dd, J = 8.4, 1.5 Hz; 2H), 6.65 (s, 2H), 5.48 (s, 1H), 2.19 (s,
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.
3.7.3.28 3,3'-(p-Tolylmethylene)bis(N'-((E)-4-fluorobenzylidene)-1H-indole-5-
carbohydrazide) (98)
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-
indole-5-carbohydrazide) (99)
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;
2H), 7.64 (d, J = 8.7 Hz; 2H), 7.23 (dd, J = 8.7, 8.4 Hz; 2H), 7.15 (dd, J = 8.1, 1.6 Hz; 2H),
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.
3.7.3.30 3,3'-(p-Tolylmethylene)bis(N'-((E)-pyridin-3-ylmethylene)-1H-indole-5-
carbohydrazide) (100)
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
(dd, J = 8.0, 1.3 Hz; 2H), 7.02 (dd, J = 8.2, 1.5 Hz; 2H), 6.59 (s, 2H), 5.41 (s, 1H), 2.23 (s,
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
3.7.3.31 3,3'-(p-Tolylmethylene)bis(N'-((E)-pyridin-4-ylmethylene)-1H-indole-5-
carbohydrazide) (101)
Yield: 89%. Light brown oily liquid. 1H NMR (500 MHz, DMSO-d6): δ 10.86 (s; 2H), 10.74
(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
= 8.7 Hz; 2H), 7.58 (d, J = 8.9 Hz; 2H), 7.20 (dd, J = 8.3, 1.6 Hz; 2H), 7.11 (dd, J = 8.4, 1.2
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.
3.7.3.32 3,3'-(p-Tolylmethylene)bis(N'-((E)-pyridin-2-ylmethylene)-1H-indole-5-
carbohydrazide) (102)
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
Figure-3.20: Comparison of structure activity relationship between compounds 106,
107 and 108
If we compare compound 112 having IC50 value 3.3 ± 0.1 μM with compound 113
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
Figure-3.21: Comparison of structure activity relationship between compounds 112,
113 and 114
If we compare compound 117 having IC50 value 7.60 ± 0.20 μM with compound 118
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.
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)
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.
3.9.3.2 2,2'-(3,3'-(p-tolylmethylene)bis(1H-indole-3,5-diyl-5-carbonyl))bis(N-(3-
bromophenyl)hydrazine-1-carbothioamide) (104)
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
3.9.3.3 2,2'-(3,3'-(p-tolylmethylene)bis(1H-indole-3,5-diyl-5-carbonyl))bis(N-(2-
bromophenyl)hydrazine-1-carbothioamide) (105)
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.
3.9.3.4 2,2'-(3,3'-(p-tolylmethylene)bis(1H-indole-3,5-diyl-5-carbonyl))bis(N-(2-
fluorophenyl)hydrazine-1-carbothioamide) (106)
Yield: 86%. Brownish solid, m.p. 240-242 °C. 1H NMR (500 MHz, DMSO-d6) δ 11.25 (s;
2H), 10.71 (s; 2H), 10.6 (s; 2H), 8.33 (s; 2H), 8.30 (s; 2H), 7.83 (d, J = 8.7 Hz, 2H), 7.81 (d, J
= 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.
3.9.3.5 2,2'-(3,3'-(p-tolylmethylene)bis(1H-indole-3,5-diyl-5-carbonyl))bis(N-(3-
fluorophenyl)hydrazine-1-carbothioamide) (107)
Yield: 86%. Dark brown solid, m.p. 226-228 °C. 1H NMR (500 MHz, DMSO-d6) δ 11.22 (s;
2H), 10.69 (s; 2H), 10.8 (s; 2H), 8.39 (s; 2H), 8.36 (s; 2H), 7.92 (d, J = 8.5 Hz, 2H), 7.68 (d, J
= 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;
Found:758.2054; Anal. Calcd for C40H32F2N8O2S2, C, 63.31; H, 4.25; N, 14.77; Found: C,
63.30; H, 4.23; N, 14.75.
3.9.3.6 2,2'-(3,3'-(p-tolylmethylene)bis(1H-indole-3,5-diyl-5-carbonyl))bis(N-(4-
fluorophenyl)hydrazine-1-carbothioamide) (108)
Yield: 86%. Light brown solid, m.p. 232-234 °C. 1H NMR (500 MHz, DMSO-d6) δ 11.33 (s;
2H), 10.80 (s; 2H), 10.9 (s; 2H), 8.40 (s; 2H), 8.38 (s; 2H), 7.94 (d, J = 8.2 Hz, 2H), 7.69 (d, J
= 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,
1.1 Hz; 4H), 7.08 (dd, J = 8.7, 1.5 Hz; 2H), 6.69 (s, 2H), 5.51 (s, 1H), 2.23 (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,
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.
3.9.3.7 2,2'-(3,3'-(p-tolylmethylene)bis(1H-indole-3,5-diyl-5-carbonyl))bis(N-(2-
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.
3.9.3.8 2,2'-(3,3'-(p-tolylmethylene)bis(1H-indole-3,5-diyl-5-carbonyl))bis(N-(4-
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,
1.5 Hz; 2H), 7.13 (dd, J = 8.2, 1.3 Hz; 2H), 6.74 (s, 2H), 5.55 (s, 1H), 2.27 (s; 3H). 13CNMR
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
= 8.7 Hz; 2H), 7.50 (d, J = 8.9 Hz; 4H), 7.23 (dd, J = 8.6, 1.6 Hz; 2H), 7.14 (dd, J = 8.4, 1.2
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-
tolyl)hydrazine-1-carbothioamide) (113)
Yield: 86%. Grey solid, m.p. 225-227 °C. 1H NMR (500 MHz, DMSO-d6) δ 11.30 (s; 2H),
10.83 (s; 2H), 10.15 (s; 2H), 8.47 (s; 2H), 8.44 (s; 2H), 8.00 (d, J = 8.8 Hz, 2H), 7.74 (d, J =
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-
tolyl)hydrazine-1-carbothioamide) (114)
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.
Calcd for C42H38N8O2S2, C, 67.18; H, 5.10; N, 14.92; Found: C, 67.13; H, 5.05; N, 14.91.
3.9.3.13 2,2'-(3,3'-(p-tolylmethylene)bis(1H-indole-3,5-diyl-5-carbonyl))bis(N-(2-
methoxyphenyl)hydrazine-1-carbothioamide) (115)
Yield: 86%. Light brown oily liquid. 1H NMR (500 MHz, DMSO-d6) δ 10.90 (s; 4H), 10.16
(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.
Calcd for C42H38N8O4S2, C, 64.43; H, 4.89; N, 14.31; Found: C, 64.41; H, 4.86; N, 14.28.
3.9.3.14 2,2'-(3,3'-(p-tolylmethylene)bis(1H-indole-3,5-diyl-5-carbonyl))bis(N-(3-
methoxyphenyl)hydrazine-1-carbothioamide) (116)
Yield: 86%. Dark grey sticky substance. 1H NMR (500 MHz, DMSO-d6) δ 11.50 (s; 2H),
10.90 (s; 2H), 10.18 (s; 2H), 8.52 (s; 2H), 8.49 (s; 2H), 8.07 (d, J = 8.8 Hz, 2H), 7.80 (d, J =
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.
3.9.3.15 2,2'-(3,3'-(p-tolylmethylene)bis(1H-indole-3,5-diyl-5-carbonyl))bis(N-(3-
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
3.9.3.16 2,2'-(3,3'-(p-tolylmethylene)bis(1H-indole-3,5-diyl-5-carbonyl))bis(N-(4-
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.
3.9.3.17 2,2'-(3,3'-(p-tolylmethylene)bis(1H-indole-3,5-diyl-5-carbonyl))bis(N-(4-
(trifluoromethyl)phenyl)hydrazine-1-carbothioamide) (119)
Yield: 86%. Dark brown sticky substance. 1H NMR (500 MHz, DMSO-d6) δ 11.55 (s; 2H),
10.91 (s; 2H), 10.20 (s; 2H), 8.60 (s; 2H), 8.56 (s; 2H), 8.08 (d, J = 8.6 Hz, 2H), 7.84 (d, J =
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,
1.5 Hz; 2H), 7.20 (dd, J = 8.2, 1.6 Hz; 2H), 6.85 (s, 2H), 5.73 (s, 1H), 2.38 (s; 3H). 13CNMR
(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
Found:858.1992; Anal. Calcd for C42H32F6N8O2S2, C, 58.73; H, 3.76; N, 13.05; Found: C,
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)
Yield: 86%. Light brown oily liquid. 1H NMR (500 MHz, DMSO-d6) δ 10.92 (s; 2H), 10.11
(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
Figure-3.26: Structure-activity relationship of compounds 141-143
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).
Figure-3.27: Structure-activity relationship of compounds 19-21
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).
Figure-3.29: Structure-activity relationship of compounds 126-128
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).
Figure-3.30: Structure-activity relationship of compounds 132-134
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.
3.10.3 Molecular docking
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.
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)
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)
Yield: 89%. Light brown solid, m.p. 239-241 °C. 1H NMR (500 MHz, DMSO-d6): δ 12.41
(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.
3.11.3.3 3,3'-((4-(5-(2-Methyl-1H-indol-3-yl)-1,3,4-oxadiazol-2-
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;
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, 4.09; N, 17.22.
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)
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.
3.11.3.5 2-(4-(Bis(1-methyl-1H-indol-3-yl)methyl)phenyl)-5-(2-methyl-1H-indol-3-yl)-
1,3,4-oxadiazole (130)
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.
3.11.3.7 2-(4-(Bis(2-phenyl-1H-indol-3-yl)methyl)phenyl)-5-(1H-indol-3-yl)-1,3,4-
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;
Anal. Calcd for C45H31N5O, C, 82.17; H, 4.75; N, 10.65; Found C, 82.15; H, 4.77; N, 10.66.
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)
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),
3.75 (s, 3-H, CH3). 13CNMR (125 MHz, DMSO-d6): δ 163.9, 152.5, 139.3, 138.4, 137.3,
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.
3.11.3.9 2-(4-(Bis(2-phenyl-1H-indol-3-yl)methyl)phenyl)-5-(1H-indol-3-yl)-1,3,4-
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.
3.11.3.10 2-(4-(Di(1H-indol-3-yl)methyl)phenyl)-5-(1-methyl-1H-indol-3-yl)-1,3,4-
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;
HREI-MS: m/z Calcd for C34H25N5O [M]+ 519.2059; Found 519.2065; Anal. Calcd for
C34H25N5O, C, 78.59; H, 4.85; N, 13.48; Found C, 78.57; H, 4.86; N, 13.49.
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.
3.11.3.12 2-(4-(Di(1H-indol-3-yl)methyl)phenyl)-5-(2-methyl-1H-indol-3-yl)-1,3,4-
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.
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)
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.
3.11.3.14 2-(4-(Bis(1,2-dimethyl-1H-indol-3-yl)methyl)phenyl)-5-(2-methyl-1H-indol-3-
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
2.29 (s, 6-H, CH3). 13CNMR (125 MHz, DMSO-d6): δ 165.2, 161.3, 140.4, 140.1, 140.1,
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;
Anal. Calcd for C37H31N5O, C, 79.12; H, 5.56; N, 12.47; Found C, 79.14; H, 5.57; N, 12.48.
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)
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.
3.11.3.17 2-(4-(Bis(5-chloro-1H-indol-3-yl)methyl)phenyl)-5-(2-methyl-1H-indol-3-yl)-
1,3,4-oxadiazole (142)
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.
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)
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.
3.11.3.20 2-(4-(Bis(5-bromo-1H-indol-3-yl)methyl)phenyl)-5-(2-methyl-1H-indol-3-yl)-
1,3,4-oxadiazole (145)
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
References
1. Lakhdar, S.; Westermaier, M.; Terrier, F.; Goumont, R.; Boubaker, T.; Ofial, A. R.;
Mayr, H. J. Org. Chem. 2006, 71, 9088-9095.
2. Alvarez-Builla, J.; Vaquero, J. J.; Barluenga, J. Wiley-VCH Verlag GmbH & Co.
KGaA, Germany. 2011, 5, 377.
3. Baeyer, A.; Emmerling, A. Chemische Berichte, 1869, 2, 679.
4. Cerighelli, R. Compt. Rend. 1924, 179, 1193.
5. Sack. J. Pharm. Weekblad. 1911, 48, 307
6. Sundberg, R. J. Indoles. London: Academic Press. 1996, 48, 113.
7. Joule, J. A.; Thomas. E. J. Ed. Georg Thieme: Stuttgart. 2000, 10, 361-652.
8. Gribble, G. W. J. Chem. Soc. Perkin Trans. 2000, 1, 1045-1075.
9. Hibino, S.; Choshi, T. Nat. Prod. Rep. 2002, 19, 148-180.
10. Joule, J. A.; Mills, K.; Smith, G. F. Stanley Thornes Ltd: Cheltenham. 1995.
11. Sundberg, R. J. Indoles. London: Academic Press. 1996, 113.
12. Kuethe, J. T.; Wong, A.; Qu, C.; Smitrovich, J.; Davis, I. W.; Hughes, D. L. J. Org.
Chem. 2005, 70, 2555-2567.
13. Ninomiya, I. J. Nat. Prod. 1992, 55, 541.
14. Smith, A. L.; Stevenson, L.; Swain, J.; Castro, J. L. Tetrahedron Lett. 1998, 39, 8317.
15. Dubey, P. K.; Kumar, T. V.; Kumar, K. A. Ind. J. Chem. 2006, 45, 2128-2132.
16. Sharma, R. Chem. Abstr. 2002, 136, 9.
17. Archana, P.; Rani, K.; Bajaj, V. K.; Srivastava, R.; Chandra, A. K. Arzneim
Forsch/Drug Res. 2003, 55, 31.
201
18. Zahran, M. A. H.; Ibrahim, A. M. J. Chem. Sci. 2009, 121, 455.
19. Sharma, P.; Kumar, A.; Pandey, P. Ind. J. Chem. 2006, 45, 2077.
20. Biradar, S. J.; Manjunath, Y. S. Ind. J. Chem. 2004, 43, 389.
21. Pardasani, T. R.; Pardasani, P.; Chaturvedi, V. Ind. J. Chem. 2001, 40, 1275.
22. Gadaginamath, S. G.; Kavali, R. R. Ind. J. Chem. 1999, 38, 156-159.
23. Phillipson, J. D.; Zenk, M. H. Academic Press Inc. New York. 1980, 295.
24. Hopp, M. L.; Matsumoto, M.; Lee, C.; Oyasu, R. Cancer Res. 1976, 36, 234.
25. Heonicke, K.; Simat, T. J.; Steinhart, H.; Kohler, H. J.; Schwab, A. J. Agric. Food
Chem. 2001, 49, 5494.
26. Hoenicke, K.; Borchert, O.; Simat, T. J. J. Agric. Food Chem. 2002, 50, 4303.
27. Cordell, G. A. Elsevier Academic Press. 2003, 60, 51, 163.
28. Ludmere, S. W.; Warren, V. A.; Williams, B. S.; Zheng, Y.; Hunt, D. C.; Ayer, M.
B.; Wallace, M. A.; Chaudhary, A. G.; Meimke, P. T. Biochem. 2002, 41, 6548-6560.
29. Cacchi, S.; Fabrizi, G. Chem. Rev. 2005, 105, 2873-2920.
30. Abele, E., Abele, R., Lukevics, E. Chem. Heterocycl. Compd. 2003, 39, 3-35.
31. Nagendra, K. K.; Neha, K.; Pankaj, A.; Naresh, K.; Chung, H. K.; Akhilesh, K. V.;
Eun, H. C. Molecules, 2013, 18, 6620-6662.
32. Deschenes, R. J.; Lin, H.; Ault, A. D.; Fassler, J. S.; Antimicrob. Agents Chemother.
1999, 43, 1700-1703.
33. Sivaprasad, G.; Perumal, P. T.; Prabavathy, V. R.; Mathivanan, N. Bioorg. Med.
Chem. Lett. 2006, 16, 6302–6305.
34. Samosorn, S.; Bremner, J. B.; Lewis, K. Bioorg. Med. Chem. 2006, 14, 857-865.
202
35. Hiari, Y. M. A.; Qaisi, A. M.; Abadelah, M. M.; Voelter, W. Monatshefte Fur
Chemie, 2006, 137, 243-248.
36. Leboho, T.C.; Michael, J. P.; Van Otterlo, W. A. L.; Van Vuuren, S. F.; De Koning,
C.B. Bioorg. Med. Chem. Lett. 2009, 19, 4948-4951.
37. Hu, W.; Guo, Z.; Chu, F.; Cheng, G. Bioorg. Med. Chem. 2003, 11, 5539–5544.
38. Preethi, R.; Srivastava, V. K.; Ashok, K. Eur. J. Med. Chem. 2004, 39, 449–452.
39. Narayana, B.; Ashalatha, B. V.; Vijaya, R. K. K.; Fernandes, J.; Sarojini, B.K.
Bioorg. Med. Chem. 2005, 13, 4638–4644.
40. Radwan, M. A. A.; Ragab, E. A.; Sabrya, N. M.; El-Shenawy, S. M. Bioorg. Med.
Chem. 2007, 15, 3832–3841.
41. Kuduk, S. D.; Chang, R. K.; Wai, J. M. C.; Di Marco, C. N.; Cofre, V.; Di- Pardo, R.
Hartman, G. D.; Bilodeau, M. T. Bioorg. Med. Chem.Lett. 2009, 19, 4059-4063.
42. Dharmendra, K.; Narendra, K.; Sandeep, K.; Tarun, S.; Singh, C. P. Int. J. Eng. Sci.
Tech. 2010, 2, 2553-2557.
43. Thirumurugan, P.; Mahalaxmi, S.; Perumal, P. T. J. Chem. Sci. 2010, 122, 819–832.
44. Hendricks, R. T.; Sheramn, D.; Broka, C. A. Bioorg. Med. Chem. Lett. 1995, 5, 67-72.
45. Zhang, F.; Zhao, Y.; Sun, L.; Ding, L.; Gu, Y.; Gong, P. Eur. J. Med. Chem. 2011, 46,
3149-3157.
46. Zhang, Y.; Yang, P.; Wang, X.; Xu, W. ACS Med. Chem. Lett. 2013, 4, 235–238.
47. Bellimin, R.; Decerprit, A.; Festal, D. Eur. J. Med. Chem. 1996, 31, 123-132.
48. Medarde, M.; Ramos, A. C.; Caballero, E.; Pelaez-Lamamie de Clairac, R.; Lopez,
J. L.; Gravalos, D. G.; Feliciano, A. S. Bioorg. Med. Chem. Lett. 1999, 9, 2303-2308.
203
49. Xu, G.; Guo, V.; Yang, B.; Hu, Y. Chem. Pharm. Bull. 2007, 55, 1302-1307.
50. Kaufmann, D.; Pojarova, M.; Vogel, S.; Liebl, R.; Gastpar, R.; Gross, D.; Nishino,
T.; Pfaller, T.; Von Angerer, E. Bioorg. Med. Chem. 2007, 15, 5122-5136.
51. Gastpeer, R.; Goldbrunner, M.; Angerer, E. V. J. Med. Chem. 1998, 41, 4965-4972.
52. Ulrich, J.; Nathalie, D.; Amelie, L.; Christian, B.; Cedric, L.; Jean-Michel, R.;
Olivier, L.; Jean-Yves, M.; Sylvain, R. Bioorg. Med. Chem. 2008, 16, 4932–4953.
53. Wu, Y. S.; Coumar, M. S.; Chang, J. Y.; Sun, H. Y.; Kuo, F. M.; Kuo, C. C.; Chen, Y.
J.; Chang, C. Y.; Hsiao, C. L.; Liou, J. P.; Chen, C. P.; Yao, H. T.; Chiang, Y. K.;
Tan, U. K.; Chen, C. T.; Chu, C. Y.; Wu, S. Y.; Yeh, T. K.; Lin, C. Y.; Hsieh, H. P. J.
Med. Chem. 2009, 52, 4941–4945.
54. Sharma, V.; Pradeep, K.; Devender, P. J. Heterocycl. Chem. 2010, 47, 491-502.
55. Vidhya, L. N.; Thirumurugan, P.; Noorulla, K. M.; Perumal, P. T. Bioorg. Med.
Chem. Lett. 2010, 20, 5054–5061.
56. Dalip, K. N.; Maruthi, K.; Swapna, S.; Emmanuel, J. O.; Kavita, S. Eur. J. Med.
Chem. 2010, 45, 1244–1249.
57. Dalip, K. N.; Maruthi, K.; Brett, N.; Kavita, S. Eur. J. Med. Chem. 2012, 55, 432-438.
58. Meric, K. A.; Mine, Y. Y.; Irem, D.; Rengul, C. A. Turk. J. Chem. 2012, 36, 515 – 525.
59. MacDonough, M. T.; Strecker, T. E.; Hamel, E.; Hall, J. J.; Chaplin, D. J.; Trawick,
M. L.; Pinney, K. G. Bioorg. Med. Chem. 2013, 21, 6831–6843.
60. Andreadou, I.; Tasouli, A.; Bofilis, E.; Chrysselis, M.; Rekka, E.; Tsantili-
Kakoulidou, A.; Iliodromitis, E.; Siatra, T.; Kremastinos, D. T. Chem. Pharm. Bull.
2002, 50, 165-168.
204
61. Monica, E. S.; Luisa, C. C.; Daniela, R.; Diana, C.; Marisa, F.; Ana, G.; Ferreira,
L.M.; Eduarda, F.; Manuel, M.; Marques, B. Eur. J. Med. Chem. 2010, 45, 4869-4878.
62. Mosaad, S. M.; Nagla, M. A. Med. Chem. Res. 2014, 23, 3374–3388.
63. Karaaslan, C.; Kadri, H.; Coban, T.; Suzen, S.; Westwell, A. D. Bioorg. Med. Chem.
Lett. 2013, 23, 2671-2674.
64. Li, Y. Y.; Wu, H. S.; Tang, L.; Feng, C. R.; Yu, H. Y.; Yang, Y. S.; Yang, B. J.
Pharmacol. Res. 2007, 56, 335-343.
65. Sunil, K.; Hemlata, K.; Saxena, K. K.; Monica, S.; Pinki, V.; Ashok, K. Indian J.
chem. B, 2010, 49, 1398-1405.
66. Abdel-Gawad, H.; Mohamed, H. A.; Dawood, K. M.; Abdel-Rahem, B. F. Chem.
Pharm. Bull. 2010, 58, 1529-1531.
67. Liu, F.; Ma, D. J. Org. Chem. 2007, 72, 4844-4850.
68. Nazare, M.; Schneider, C.; Lindenschmidt, A.; Will, D. W. Angewandte Chemie
International Edition, 2004, 43, 4526-4528.
69. Li, X.; Du, Y.; Liang, Z.; Li, X.; Pan, Y.; Zhao, K. Org. lett. 2009, 11, 2643-2646.
70. Kondo, T.; Okada, T.; Mitsudo, T. J. Am. Chem. Soc. 2002, 124, 186-187.
71. Arcadi, A.; Bianchi, G.; Marinelli, F. Synthesis, 2004, 610-618.
72. Bradfield, C. A.; Bjeldanes, L. F. J. Toxico. Environ. Health, 1987, 21, 311-323.
73. Dashwood, R. H.; Uyetake, L.; Fong, A. T.; Hendricks, J. D.; Bailey, G. S. Food and
Chem. Toxico. 1989, 27, 385-392.
74. Zeligs, M. A. J. Med. Food, 1998, 1, 67-82.
75. Chakrabarty, M.; Basak, R.; Harigaya, Y. Heterocycles, 2001, 55, 2431-2447.
205
76. Adachi, J.; Mori, Y.; Matsui, S.; Takigami, H.; Fujino, J.; Kitagawa, H.; Miller, C.
A.; Kato, T., Saeki, K.; Matsuda, T. J. Biolog. Chem. 2001, 276, 31475-31478.
77. Hoessel, R.; Leclerc, S.; Endicott, J. A.; Nobel, M. E. M.; Lawrie, A.; Tunnah, P.;
Leost, M.; Damiens, E.; Marie, D.; Marko, D. Nat. Cell Biol. 1999, 1, 60-67.
78. Xiao, Z.; Hao, Y.; Liu, B.; Qian, L. Leukemia and Lymphoma, 2002, 43, 1763-1768.
79. Leclerc, S.; Garnier, M.; Hoessel, R.; Marko, D.; Bibb, J. A.; Snyder, G. L.;
Greengard, P.; Biernat, J.; Mandelkow, E. M. J. Biolog. Chem. 2001, 276, 251-260.
80. Myrianthopoulos, V.; Magiatis, P.; Ferandin, Y.; Skaltsounis, A. L.; Meijer, L.;
Mikros, E. J. Med. Chem. 2007, 50, 4027-4037.
81. Kawanishi, M.; Sakamoto, M.; Kishi, K.; Yagi, T. Mutation Res. 2003, 540, 99-105.
82. Nam, S.; Buettner, R.; Turkson, J.; Kim, D.; Cheng, J. Q.; Merz, K. H.; Eisenbrand,
G. Proc. Nat. Acad. Sci. 2005, 102, 5998-6003.
83. Kohmoto, S.; Kashman, Y.; McConnell, O. J.; Rinehart Jr, K. L.; Wright, A. ;
Koehn, F. J. Org. Chem. 1988, 53, 3116-3118.
84. Wright, A. E.; Cross, S. S.; McCarthy, P. J. Org. Chem. 1992, 57, 4772-4775.
85. Capon, R. J.; Rooney, F.; Murray, L. M.; Collins, E.; Sim, A. T. R.; Rostas, J. A. P.;
Butler, M. S.; Carroll, A. R. J. Nat. Prod. 1998, 61, 660-662.
86. Gresens, E.; Ni, Y.; Verbruggen, A.; Marchal, G. US Patent 0053911A1, 2004.
87. Wright, C. W.; Phillipson, J. D. Phytotherapy Res. 1990, 4, 127-139.
88. Wright, C. W.; Allen, D.; Cai, Y.; Phillipson, J. D.; Said, I. M.; Kirby, G. C.;
Warhurst, D. C. Phytotherapy Res. 1992, 6, 121-124.
206
89. Karthik, M.; Tripathi, A. K.; Gupta, N. M.; Palanichamy, M.; Murugesan, V.
Catalysis Communications, 2004, 5, 371-375.
90. Mei, H. X.; Fa, D. X.; Gao, F. L.; Jin, H. Z. Tetrahedron Lett. 2010, 51, 1213-1215.
91. Nagawade, R. R.; Shinde, D. B. Acta Chimica Slovenica, 2006, 53, 210-213.
92. Zahran, M.; Abdin, Y.; Salama, H. ARKIVOC. 2008, 11, 256-265.
93. Praveen, C.; Wilson Perumal, P. T. Tetrahedron Lett. 2009, 50, 644-647.
94. Trott, O.; Olson, A. J. J. Comput. Chem. 2010, 31, 455–461.
95. Ugwu, D.I.; Okoro, U.C.; Ukoha, P.O.; Okafor, S.; Ibezim, A.; Kumar, N.M. Eur. J.
Med. Chem. 2017, 135, 349-369.
96. Dassault Systèms BIOVIA, Discovery Studio Modeling Environment, Release 4.5,
San Diego: Dassault Systèms, 2015.
97. Morris, G.M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew, R. K.; Goodsell, D.
S. J. Comput. Chem. 2009, 30, 2785–2791.
98. Wallace, A. C.; Laskowski, R.; Thornton, J. M. Protein engineering, 1995, 8, 127-134.
99. Jain, S.; Drendel, W. B.; Chen, Z. W.; Mathews, F. S.; Sly, W. S.; Grubb, J. H. Nat.
Struct. Biol. 1996, 3, 375.
100. Stierand, K.; Maaß, P. C.; Rarey, M. Bioinformatics, 2006, 22, 1710-1716.
101. Jain, S. Nat. Struc. Mol. Biol. 1996, 3, 375-381.
102. Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D.
M.; Meng, E. C.; Ferrin, T. E. J. Comput. Chem. 2004, 25, 1605-1612.
103. Schrödinger Release, Maestro, version 10.1, Schrödinger, LLC, NY. 2015.
104. Verdonk, M. L.; Cole, J. C.; Hartshorn, M. J.; Murray, C.W.; Taylor, R. D.
207
Proteins, 2003, 52, 609.
105. PyMOL, Molecular Graphics System L, NY, USA, PyMOL New York, US:
Schrodinger. 2010.
106. Imran, S.; Taha, M.; Ismail, N. H.; Khan, K. M.; Naz, F.; Hussain, M.;
Tauseef, S. Molecules, 2014, 19, 11722-11740.
107. Imran, S.; Taha, M.; Ismail, N. H.; Fayyaz, S.; Khan, K. M.; Choudhary, M.
I. Bioorg. Chem. 2015, 62, 83-93.
108. Imran, S.; Taha, M.; Ismail, N. H.; Fayyaz, S.; Khan, K. M.; Choudhary, M.
I. Bioorg. Chem. 2016, 68, 90-104.
109. Taha, M.; Ismail, N. H.; Imran, S.; Ali, M.; Jamil, W.; Uddin, N.; Kashif, S.
M. RSC Advances, 2016, 6, 3276-3289.
110. Taha, M.; Ismail, N. H.; Imran, S.; Rahim, F.; Wadood, A.; Khan, H.; Ullah,
H.; Salar, U.; Khan, K. M. Bioorg. Chem. 2016, 68, 56-63.
111. Duffy, E.M.; Jorgensen, W.L. J. Am. Chem. Soc. 2000, 122, 2878.
112. Maestro, Version 9.2, Schrodinger, LLC, New York, NY, USA. 2011.
113. Lipinski, C.A.; Lombardo, F.; Feeney, P. J. Adv. Drug Deliv. Rev. 1997, 23, 3.
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
References
1. Krenitsky, T. A. US Pat. 1979, 212, 1.
2. Taha, M.; Ismail, N. H.; Imran, S.; Rahim, F.; Wadood, A.; Al Muqarrabun, L. M.
R.; Khan, K. M.; Ghufran, M.; Ali, M. Bioorg. Chem. 2016, 68, 80-89.
3. Weatherburn, M. W. Anal. Chem. 1967, 39, 971.
4. Taha, M.; Ismail, N. H.; Imran, S.; Selvaraj, M.; Rahim, A.; Ali, M.; Siddiqui, S.;
Rahim, F.; Khan, K. M. Bioorg. Med. Chem. 2015, 23, 7394-7404.
5. a) Jamil, W.; Perveen, S.; Shah, S. A. A.; Taha, M.; Ismail, N. H.; Perveen, S.;
Ambreen, N.; Khan, K. M.; Choudhary, M. I. Molecules, 2014, 19, 8788-8802; b)
Khan, K. M.; Saad, S. M.; Shaikh, N. N.; Hussain, S.; Fakhri, M. I.; Perveen, S.;
Taha, M.; Choudhary, M. I. Bioorg. Med. Chem. 2014, 22, 3449-3454; c) Khan, K.
M.; Ambreen, N.; Taha, M.; Halim, S. A.; Naureen, S.; Rasheed, S.; Perveen, S.;
Ali, S.; Choudhary, M. I. J. Comput. Aided Mol. Des. 2014, 28, 577-585.
6. (a) Rahim, F.; Ullah, H.; Javid, M. T.; Wadood, A.; Taha, M.; Ashraf, M.; Shaukat,
A.; Junaid, M.; Hussain, S.; Rehman, W.; Mehmood, R.; Sajid, M.; Khan, M. N.;
Khan, K. M. Bioorg. Chem. 2015, 62, 15–21. (b) F. Rahim, K. Ullah, H. Ullah, A.
Wadood, M. Taha, A. Rehman, I. Uddin, M. Ashraf, A. Shaukat, W. Rehman, S.
Hussain, K. M. Khan. Bioorg. Chem. 58 (2015) 81–87. (c) S. Imran, M. Taha, N.H.
Ismail, S.M. Kashif, F. Rahim, W. Jamil, M. Hariono, M. Yusuf, H. Wahab. Euro. J.
Med. Chem. 105 (2015) 156-170.