Design and Synthesis of 1,2,3/1,2,4-Triazole based Novel Heterocyclic

Compounds as Anti-tubercular Agents

THESIS

Submitted in partial fulfilment of the requirements for the degree of DOCTOR OF PHILOSOPHY

by AMAROJU SURESH ID No: 2012PHXF530H

Under the supervision of Prof. K.V.G. Chandra Sekhar

BITS Pilani Pilani | Dubai | Goa | Hyderabad

BIRLA INSTITUTE OF TECHNOLOGY & SCIENCE, PILANI 2018 BIRLA INSTITUTE OF TECHNOLOGY & SCIENCE, PILANI

CERTIFICATE

This is to certify that the thesis entitled “Design and Synthesis of 1,2,3/1,2,4-Triazole based

Novel Heterocyclic Compounds as Anti-tubercular Agents” submitted by AMAROJU

SURESH ID No: 2012PHXF530H for award of Ph. D. of the Institute embodies original work done by him under my supervision.

Signature of the Supervisor :

Name in capital letters : K.V.G. CHANDRA SEKHAR

Designation : Associate Professor

Date:

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Acknowledgements

It gives me great pleasure that I have an opportunity to place on record of long travelled path, the contributions of several people, some of whom were with me from the beginning, some who joined me at some stage during the journey, whose rally round kindness, love and blessings have brought me to this day. I wish to thank each and every one who have been instrumental in crystallising this thesis.

It gives me immense pleasure and pride to express my gratitude and respect for my teacher and guide Prof. K.V.G. Chandra Sekhar for his expert, inspiring guidance and valuable suggestions throughout the period of my work. I am indebted to him for enlightening me on the finer skills of dealing with synthetic problems. It would have been impossible to achieve this goal without his constant support and encouragement. I consider myself fortunate to be associated with him who gave a decisive turn and a significant boost to my career.

I gratefully acknowledge Head of the chemistry department, my DAC member Prof. Manab Chakravarty for his understanding, encouragement and personal attention which has provided good and smooth basis for my Ph.D. tenure. I also thank him for his valuable teaching of Heterocyclic chemistry during coursework.

I gratefully acknowledge my DAC member Prof. Anupam Bhattacharya for his understanding, encouragement and personal attention which have provided good and smooth basis for my Ph.D. tenure. I also thank him for his valuable teaching of Structure and reactivity of organic compounds during coursework and for providing me with all the necessary laboratory facilities and having helped me at various stages of my research work.

I am grateful to Prof. Souvik Bhattacharyya, Vice-Chancellor, BITS-Pilani.

I take this opportunity to thank Prof. G. Sundar, Director (Hyderabad campus) and Prof. V.S. Rao former Acting Vice-Chancellor (BITS) and Director (Hyderabad campus), for allowing me to carry out my doctoral research work in the institute.

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I am sincerely thankful to Prof. S.K. Verma, Dean, Academic Research Division, BITS-Pilani, Pilani and Prof. Vidya Rajesh, Associate Dean, Academic Research Division, BITS-Pilani, Hyderabad campus for their co-operation and encouragement at every stage of this research. During my research work, I have benefited from discussions with several people. Iam thankful from my bottom of my heart to DRC convenor Prof. Jayanthi Subbalaksmi and former HOD’s Prof. N. Rajesh, Prof. K. Sumithra and faculty members Dr. Balaji Gopalan, Prof. R. Krishnan of department of chemistry.

I am thankful to Prof. D. Sriram, Deparment of Pharmacy, for his help rendered in carrying out in vitro anti-tubercular activity, Pantothenate enzyme assay and cytotoxicity studies. I sincerely thank Prof. Augustynowicz-Kopeć Ewa, Microbiology Department, National Tuberculosis and Lung Diseases Research Institute, 01-138 Warsaw, Poland, for carrying out in vitro anti-tubercular activity studies.

I am sincerely thankful to Dr. S. Murugesan, Dr. Muthyala Murali Krishna Kumar, Dr. Mallika Alvala for docking studies.

I take this opportunity to sincerely acknowledge the Department of Biotechnology (DBT) and Department of Science & Technology (DST), Government of India, New Delhi, for providing financial assistance in the form of project fellowship. I would also thank BITS-Pilani, Hyderabad Campus for providing institute fellowship.

It gives me a golden opportunity to put on record my sincere gratitude to my labmates and friends Dr. K. Mahalakshmi Naidu, Dr. N. Suresh, Dr. H.N. Nagesh, P. Ravikiran, C. Surendar, S. Srinivas Rao, Dr. A. Mahesh, Dr. T. Vikramaditya T. Yadagiri, R. Santhosh, M. Sai Sudhakar, N. Ravikiran, T. Uday kumar and research scholars in chemistry and other departments for the time they had spent for me and making my stay at campus a memorable one. I take this opportunity to thank one and all who helped me directly or indirectly.

I would like to thank my parents Ramulu and Uppalamma and my brother Raju who have given their blessings for the great desire to see me succeed and get the highest degree in education. I

iii must specially thank my wife Viplava for the support and encouragement which helped me in keeping my morale high and son Akshith for giving joyful environment. I would like to do that by dedicating this thesis to my family.

I am truly grateful to my dear friends, Maragani Ramesh, A. Chiranjivi, Ch. Veeranna and G. Chandra mohan who have been my pillars of mental strength. Words are inadequate for expressing such feeling.

I express my thanks to our laboratory assistants, Mr. Ashok, Mrs. Shanta kumari and Mr. Sudhir.

My sincere thanks to Central Analytical Lab, staff and library of BITS-Pilani Hyderabad Campus staff for their excellent cooperation throughout my research work.

As much as my doctoral research work has been a personal pursuit, the story would not have been completed without the efforts & help from my co-workers, friends and well-wishers who have been an integral part of this saga for the last four years. My heartfelt thanks and deep sense of appreciation to all the people mentioned here and others whose names I might have omitted unwittingly.

Date: AMAROJU SURESH

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Table of contents

Contents Page No. Certificate i Acknowledgements ii Abstract x List of Tables xii List of Figures xiii Abbreviations xiv Chapter I: Introduction 1-53 1. Tuberculosis 1-4 1.1. Mycobacterium tuberculosis - the etiological agent of TB 5 1.2. Mycobacterium tuberculosis (MTB): An overview 5-6 1.3. Classification of mycobacteria 6-8

1.4. The Mycobacterium tuberculosis genome 8

1.5. The mycobacterial cell envelope 8-12 1.6. Tuberculosis: Drug resistance 12-13 1.6.1. Multidrug-resistant TB (MDR TB) 13-14 1.6.2. Extensively Drug-Resistant TB (XDR TB) 14 1.6.3. Totally drug-resistant TB (TDR TB) or extremely drug resistant TB 14-15 (XXDR TB)

1.6.4. Rifampicin-resistant TB (R TB 15

1.7. TB in HIV 15-16 1.8. Current treatment in TB 17 1.8.1. Treatment for latent-TB infection (LTBI) 17 1.8.2. Treatment for drug susceptible-TB 17-18 1.8.3. Treatment for drug resistant-TB 18-19

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Contents Page No. 1.9. Classification of anti-TB drugs 20 1.9.1. Cell wall synthesis inhibitors 20-22 1.9.2. Nucleic Acid Synthesis Inhibitors 22-24 1.9.3. Protein synthesis Inhibitors 24-25 1.9.4. Electron transport across membrane inhibitors 25-26 1.10. Current emerging pipeline new anti-TB drugs 27-28 1.10.1. Q203 28-29 1.10.2. Sutezolid 29 1.10.3. SQ109 30-31 1.10.4. Levofloxacin (LEV) 31-32 1.10.5. Delamanid 32-33 1.10.6. Pretomanid (PA-824) 33-34 1.10.7. Rifapentine 34 1.10.8. Moxifloxacin 34-35 1.10.9. Bedaquiline 35-37 1.11. Molecular Modification 37-38 1.11.1. Prodrug approach 38-39 1.11.2. Bioisosterism 39-43 1.11.3. Molecular hybridization (MH) 43-45 1.12. References 45-53 Chapter II: Objectives 54 Chapter III: Identification and development of 1-((1-(substituted)-1H-1,2,3- triazol-4-pyrazolo[4,3-c]pyridine-5(4H)-carboxamides as Mycobacterium 55-87 tuberculosis Pantothenate synthetase inhibitors

3.1. Introduction 55-58 3.1.1. Design and chemistry 58-59

3.2. Results and Discussion 59-60

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Contents Page No. 3.2.1. In vitro MTB screening 60-62 3.2.2. Pantothenate synthetase enzyme inhibition studies 63-64 3.2.3. Docking study 64-65 3.2.4. In vitro cytotoxicity studies 66 3.2.5. Single Crystal X-ray Crystallographic Structure of Compound 7g 66-68 3.3. Conclusion 69 3.4. Experimental 69 3.4.1. Materials and methods 69 3.4.2.Chemistry 69-83 3.4.3. Biological activity 83 3.4.3.1. MTB PS screening 83 3.4.3.2. In vitro MTB screening 83-84 3.4.3.3. In vitro cytotoxicity screening 84 3.4.4. Docking Study 84-85 3.5. References 85-87

Chapter IV: Design, synthesis of 9H-fluorenone based 1,2,3-triazole analogues as 88-138 Mycobacterium tuberculosis InhA inhibitors

4.1. Introduction 88-93 4.2. Results and Discussion 93 4.2.1. Chemistry 93-95 4.2.2. In vitro MTB screening 95-99 4.2.3. InhA enzyme Inhibition studies 99-100 4.2.4. Docking study 100-104 4.2.5. In vitro cytotoxicity studies 104 4.2.6. Single Crystal X-ray Crystallographic Structure of Compound 15a 105-106 4.2.6.1. Single Crystal X-ray Crystallographic Structure of Compound 15r 107-108 4.3. Conclusion 109

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Contents Page No. 4.4. Experimental section 109 4.4.1. Materials and methods 109 4.4.2. Chemistry 110-128 4.4.3. Biological activity 128 4.4.3.1. InhA activity inhibition 128 4.4.3.2. In vitro MTB screening 129 4.4.3.3. In vitro cytotoxicity screening 129 4.4.4. Docking Study 130 4.5. References 130-133 Chapter V: Design, synthesis and biological evaluation of new substituted 134-165 sulfonamide tetrazole derivatives as anti-tubercular agents 5.1. Introduction 134-138 5.2. Results and Discussion 138 5.2.1. Chemistry 138-140 5.2.2. Antimycobacterial activity 140-144 5.2.3. Cytotoxicity assay 144-145 5.3. Conclusion 146-145 5.4. Experimental section 146 5.4.1. Materials and methods 146 5.4.2. Chemistry 146-162

5.4.3. Anti-tubercular activity against MTB H37RV strain 162-163 5.4.4. CHO-K1 Cytotoxicity 163 5.5. References 163-165 Chapter VI: 6-chloro, 6,7-dichloro and 2-methyl-3-(((1-(substitutedphenyl)-1H-

1,2,3-triazol-4-yl)methoxy)carbonyl)quinoxaline 1,4-dioxide derivatives as anti- 166-191 tubercular agents 6.1. Introduction 166-169

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Contents Page No. 6.2. Results and Discussion 169 6.2.1. Chemistry 169-170 6.2.2. In vitro MTB screening 170-174 6.2.3. In vitro cytotoxicity studies 174 6.3. Conclusion 175 6.4. Experimental 175 6.4.1. Materials and methods 170 6.4.2. Chemistry 176-188 6.4.3. Biology 188 6.4.3.1. In vitro MTB screening 188 6.4.3.2. In vitro cytotoxicity screening 188-189 6.5. References 189-191 Chapter IX: Summary and Conclusion 192-194 Future perspectives 195 Appendix 196-198 List of publications 196-198 List of papers presented at conferences 198 Biography of supervisor 199 Biography of candidate 199

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Abstract

Mycobacterium tuberculosis, the fatal agent to humans is estimated to claim two million deaths annually. Even though the existing drugs are remarkable in controlling the disease to a certain extent, but they still suffer from several shortcomings. The drug discovery efforts are progressively becoming rational, focused at various enzymes and identification of appropriate targets is becoming a fundamental pre-requisite. In the present study, we paid attention on achieving promising anti-tubercular agents based on reported and promising anti-tubercular leads. Utilizing the medicinal chemistry tools of structure based drug design and molecular hybridization/scaffold hopping; we designed and synthesized the compounds. All synthesized novel compounds were characterized by spectral data (IR, NMR and MS), elemental analysis and few compounds were confirmed by single crystal XRD and screened for anti- mycobacterial activity.

In chapter 3, twenty six novel 1-((1-(substituted)-1H-1,2,3-triazol-4-yl)methyl)-N,3-diphenyl- 6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-carboxamide derivatives were synthesized and evaluated for their antimycobacterial activity against MTB H37Rv strain and pantothenate synthetase enzyme inhibition was also done. Among the synthesized compounds, 7d exhibited good activity with MTB MIC 24.72 μM and better MTB PS inhibition with IC50 1.01±0.32 μM. The most active compound 7d showed SI value 13.76.

In chapter 4, we designed 9H-fluorenone linked 1,2,3-triazoles based on reported InhA inhibitors of 9H-fluorenone analogues and anti-TB agents of 1,2,3-triazoles. In this chapter, fifty compounds were synthesized and evaluated for their antimycobacterial activity against three strains MTB H37Rv, MTB Spec. 192 and MTB Spec. 210. Among the synthesized compounds, 15 whose activities were ≤ 50 μg/mL were screened for the InhA activity. Amongst 15 compounds, 17f & 17p showed >73% of InhA inhibition at 50 μM. Compounds 17p showed good MTB activity with MIC 52.35 μM against MTB H37Rv. Most active compounds did not exhibit cytotoxicity against HEK 293 cell line at 50 μM.

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In chapter 5, a series of thirty six substituted sulfonamide tetrazole derivatives were designed based on the reported MTB inhibitors of 1,2,4, triazoles, tetrazole and sulfonamide analogues using molecular hybridization strategy. The compounds were synthesized over seven steps and evaluated for their anti-tubercular activity. Among the tested compounds, 26c emerged as a prospective candidate by inhibiting the MTB H37Rv strain at concentration 0.78 µg/mL. In addition, all the active compounds with MIC ≤ 6.25 μg/mL were subjected to cytotoxic studies against CHO-K1 cell lines at concentration 100 µM and the selectivity index values for most of the compounds is >12 indicating suitability of compounds in an endeavour to attain lead molecule for further drug development.

In chapter 6, 6-chloro, 6,7-dichloro and 2-methyl-3-(((1-(substitutedphenyl)-1H-1,2,3-triazol-4- yl)methoxy)carbonyl)quinoxaline 1,4-dioxide derivatives were designed by the approaching molecular hybridization based on the reported MTB inhibitors of 1,4-di-N-oxide-quinoxaline and 1,2,3 triazole analogues. Thirty one compounds were synthesized and evaluated for their anti- tubercular activity against three different strains MTB H37Rv, MTB Spec. 192 and MTB Spec. 210. Among the synthesized compounds, 36a and 37d showed excellent MTB MIC of 30.35, 47.60 μM and respectively. Cytotoxicity of 36a, 36e, 36i, 36l, 37b, 37c & 37d was determined against HEK 293 cell line. The most active compounds did not show toxic nature.

With new anti-TB agents desperately needed, we believe that the present class of triazole based inhibitors reported in this work would be an interesting potential lead to be worked for rational drug design against MTB from pharmaceutical point of view.

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List of Tables

Table No. Description Page No.

Table 1.1 Classification of mycobacteria according to the risk of infection 7 Table 1.2 Group name and mechanism of action of first and second line anti- 16 TB agents Table 1.3 Classic bioisosteres, classifications, their atoms and groups 39-40 Table 1.4 Non-classical bioisosteres classifications of their atoms and groups 40-41 Table 3.1 Result of antimycobacterial screening of title compounds 61-62 Table 3.2 Docking scores and MTB PS assay 63-64 Table 3.3 Cytotoxicity results of the active compounds 66 Table 3.4 Crystal data and structure refinement for 7g 67-68 Table 4.1 Antimycobacterial activities of compounds 12a-p, 15a-r & 17a-p 95-97 against MTB H37Rv, Spec. 192 and Spec. 210 in μM. Table 4.2 MTB InhA activity 100 Table 4.3 Docking scores 103-104 Table 4.4 Crystal data and structure refinement for 15a 105-106 Table 4.5 Crystal data and structure refinement for 15r 108-109 Table 5.1 Result of Antimycobacterial screening of title compounds 140-142

Table 5.2 IC50 (µg/mL) and selectivity index (SI) values of active compounds 144-145 Table 6.1 Result of Antimycobacterial screening of title compounds 171-172

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List of Figures

Figure No. Page No. Description Figure 1.1 Stages of Mycobacterium tuberculosis infection 1 Figure 1.2 First line anti-TB drugs 2 Figure 1.3 Second line anti-TB drugs 3 Morphological variations in MTB. (A) Thin section transmission electron micrograph of MTB (extracted from Figure 1.4 www.wadsworth.org/databank/mycotubr.htm); (B) Scanning 6 electron microscope shows shape variation in MTB at exponential phase of growth Figure 1.5 The mycobacterial cell wall 9 Figure 1.6 Fatty acid/mycolic acid biosynthesis in mycobacteria 11 Effectiveness and tolerability relation of first- line and second-line Figure 1.7 18 drugs used in TB treatment

Figure 1.8 Cell wall synthesis inhibitors 19 Figure 1.9 Mechanism of action of existing drugs/new anti-tubercular drugs in 26 development Figure 1.10 Various agents that are currently being investigated for TB therapy 27 Figure 1.11 Structure of anti-TB agents under preclinical development 28 Figure 1.12 Structure of anti-TB agents under phase-I & II clinical trials 31 Figure 1.13 Structure of anti-TB agents under phase-III clinical trials 37 Figure 1.14 FQ drugs were obtained using bioisosteric replacement 42 Figure 1.15 Chemical structure of linezolid bioisosteric derivatives 43 Figure 1.16 Different hybrid compounds obtained by molecular hybridization. 44 Figure 1.17 Molecular hybridization between FQ and INH 45 Figure 1.18 Quinoxaline-1,4-di-N-oxide molecular hybridization with INH 45 Figure 3.1 Pyrazole based drugs 55 Figure 3.2 Some of the pyrazole based anti-tubercular agents 56 Figure 3.3 Some of the triazole based anti-tubercular agents 57

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Figure No. Page No. Description Figure 3.4 Structures of literature reported MTB PS inhibitors 58 Figure 3.5 Design strategy of the title compounds 59 Figure 3.6 Superimposed view of co-crystallized ligand (green) with its X-ray 64 pose (red) in 3IUB Figure 3.7 Docked pose of compound 7d inside the 3IUB, showing two- 65 dimensional interactive diagram Figure 3.8 ORTEP diagram showing the X-ray crystal structure of the 67 compound 7g with a methanol solvent of crystallization Figure 4.1 Fluorenone containing natural products and semi synthetic 88 compounds Figure 4.2 InhA inhibitors 90 Figure 4.3 Anti-TB activity of triazole 92

Figure 4.4 Design strategy to achieve title compounds 93 Figure 4.5 Superimposed view of co-crystallized ligand in 1BVR 101 Figure 4.6 Docked pose of compound 17f inside the 1BVR, showing two- 102 dimensional interactive diagram Figure 4.7 Docked pose of compound 17p inside the 1BVR, showing two- 102 dimensional interactive diagram Figure 4.8 Cytotoxicity assay of 12p, 15e, 15f, 15g, 15q & 17p on HEK-293 104 cells Figure 4.9 ORTEP diagram showing the X-ray crystal structure of the 106 compound 15a Figure 4.10 ORTEP diagram showing the X-ray crystal structure of the 107 compound 15r Figure 5.1 Some of the1,2,4 triazole based anti-tubercular agents 135 Figure 5.2 Some of the tetrazole based anti-tubercular agents 136 Figure 5.3 Some of the sulfonamide based anti-tubercular agents 137 Figure 5.4 Design strategy to achieve title compounds 138 Figure 6.1 Some of the N-oxide based anti-tubercular agents 167 Figure 6.2 Some of the triazole based anti-tubercular agents 168 Figure 6.3 Design strategy of the title compounds 169 Figure 6.4 Cytotoxicity assay of 36, 36e, 36f, 36i, 36l, 37b , 37c & 37d on 174 HEK-293 cells Figure 7.1 Most active compounds structures of synthesized compounds 194

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List of Abbreviations

µg : Microgram µM : Micromolar 13C NMR : Carbon nuclear magnetic resonance 1H NMR : Proton nuclear magnetic resonance ACN : Acetonitrile ADH : Alanine dehydrogenase AMK : Amikacin ATP : Adenosine triphosphate BCG : Bacillus Calmettee Guerin t-BuOH : tert-butanol t-BuOK : Potassium tert-butoxide CAP : Capromycin CCDC : Cambridge Crystallographic Data Center

CDCl3 : Chloroform- deuterated CHO : Chinese hamster ovary

CHCl3 : Chloroform

CH2Cl2 : Dichloromethane CI : Confidence intervals

CO2 : Carbon dioxide CP : Ciprofloxacin

Cs2CO3 : Cesium carbonate CuBr : Copper(I) bromide CuCl : Copper(I) chloride CuI : Copper(I) iodide

CuSO4.5H2O : Copper sulphate pentahydrate d : Doublet DCM : Dichloromethane DCE : 1,2-Dichloroethane

xv dd : Doublet of doublet DIPEA : N,N-Diisopropylethylamine DMEDA : N,N1-Dimethylethylene diamine DMF : N,N-Dimethylformamide DMSO : Dimethylsulfoxide

DMSO-d6 : Dimethyl sulphoxide deuterated DNA : Deoxyribonucleic acid DOTS : Directly observed treatment short course DSF : Differential Scanning Fluorimeter EtOH : Ethanol EMB : Ethambutol EU : European Union EtOAc : Ethyl acetate FDA : Food and Drug Administration FQ : Fluoroquinolone G : Gram H : Hour HBr : Hydrobromic acid

H2SO4 : Sulfuric acid

H2O : Water HIV : Human immunodeficiency virus HRMS : High-resolution mass spectra HTS : High throughput screening

NH2OH.HCl : Hydroxylamine hydrochloride Hz : Hertz INH : Isoniazid

IC50 : Half Maximal Inhibitory Concentration IR : Infrared Spectroscopy J : Coupling constant KAN : Kanamycin

K2CO3 : Potassium carbonate

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KBr : Potassium bromide KI : Potassium iodide LCMS : Liquid chromatography–Mass Spectrometry LEV : Levofloxacin LJ medium : Lowenstein–Jensen medium m : Multiplet MA : Mycolicacid m.p. : Melting point MABA : Micro alamar blue assay MDR : Multi-drug resistant MIC : Minimum inhibitory concentration MOA : Mechanism of action MeOH : Methanol mg : Milligram MHz : Megahertz mmol : Millimolar MS : Mass spectrometry MTB : Mycobacterium tuberculosis MH : Molecular hybridization MTT assay : [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay MWI : Microwave irradiation

N2 : Nitrogen NADH : Nicotinamide Adenine Dinucleotide nm : Nanometer NAG-NAM : N-acetyl glucosamine – N-acetyl muramic acid

NaN3 : Sodium azide NaOH : Sodium hydroxide

Na2SO4 : Sodium sulfate NTZ : Nitazoxanide

O2 : Oxygen OADC : Oleic Albumin Dextrose Catalase

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PAS : Para aminosalicylic acid PDB : Protein Data Bank POA : Pyrazinoic acid ppm : Parts per million PPA : Polyphosphoric acid PS : Pantothenate synthetase PTSA : p-Toluenesulfonic acid PZA : Pyrazinamide RB flask : Round bottom flask RMP : Rifampicin RMSD : Root Mean Square deviation RNA : Ribonucleic acid rRNA : Ribosomal Ribonucleic acid RT : Room temperature s : Singlet SAR : Structure Activity Relationship SI : Selectivity index SM : Streptomycin t : Triplet TAACF : Tuberculosis antimicrobial acquisition and coordinating facility TB : Tuberculosis TBAB : Tetrabutyl ammonium bromide TDR : Totally drug-resistant TEA : Triethylamine TFA : Trifluoroacetic acid THF : Tetrahydrofuran TLC : Thin-layer chromatography TMEDA : Tetramethylethylene diamine TMS : Tetramethylsilane TMSI : Trimethylsulfoxonium iodide

TMSN3 : Trimethylsilyl azide

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Tt : Triplet of triplet UV : Ultra violet VS : Virtual Screening WHO : World Health Organization XDR : Extensively-drug resistant XP : Extra Precision

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Introduction

Chapter I

Introduction

Introduction

Introduction Chapter 1

1. Tuberculosis Tuberculosis (TB) remains a leading infectious killer in the world. One of the hallmarks of TB is its persistent phase of infection. One-third of the world's population is thought to be contaminated with the causative agent Mycobacterium tuberculosis (MTB) a gram positive bacterium, the causative agent of TB. The disease is transmitted via the respiratory route as highly infectious aerosol, whose exposure result ranges from immediate organism damage by the host’s immune system to infected individuals developing active primary TB [1]. It is the leading cause of morbidity and mortality among the infectious diseases. In immunocompetent individuals the initial acute infection is controlled by the immune system, and living bacteria are restricted in a peculiar localized pulmonary structure called granuloma. However, these patients have the risk of 10% to develop active form during their life even with the absence of any cause of immunosuppression (Figure 1.1) [2]. There the bacteria endures indefinitely in a latent non- virulent form, and gets reactivated whenever an immunosuppressive condition occurs [3]. If importance of a disease for mankind is measured from the number of fatalities which are due to it, then tuberculosis must be considered much more important than those most feared infectious diseases, like., cholera, plague, and the statistics have shown that 1/7 of all humans die of tuberculosis.

Figure 1.1: Stages of Mycobacterium tuberculosis infection.

1

Introduction

Even now TB continues to claim about 2 million deaths per each year (WHO report, 2015) [4] and results in vast mortality with a huge economic burden on undeveloped countries. The standard treatment recommended by World Health Organisation (WHO) contains four drugs to be administered for six months to treat drug sensitive TB [5]. Prolonged TB treatment results in poor patient fulfilment and severe side effects arising from some of the suggested drugs. During the past years most of the drugs developed for TB were ineffective due to the emergence and spread of resistant MTB strains to these front line drugs resulting in multidrug resistant (MDR), extremely drug resistance (XDR) and totally drug resistant (TDR) strains of MTB. MDR MTB is the one at least resistant to isoniazid and one of the antibiotics like rifampicin (RMP) is called as multi drug resistance strain. XDR MTB is the MDR strain which is resistant to fluoroquinolone (FQ) and an injectable aminoglycoside is termed as an extremely drug resistant strain. The strain of MTB which is resistant to all first line and second line licensed anti-tubercular drugs is defined as totally drug resistant stain (TDR MTB) [6]. The global emergence of these MDR, XDR and TDR TB strains makes to fail greatly the control and suppression of TB.

Figure 1.2: First line anti-TB drugs.

2

Introduction

Figure 1.3: Second line anti-TB drugs.

The World Health Organization (WHO) estimated that in 2015, about 9.6 million people developed TB and 1.5 million died from the disease (4,00,000 of whom were HIV-positive), with the huge majority of these from developing parts of the world [4]. An estimated 1.2 million (12%) of the 9.6 million people who developed TB in 2014 were HIV-positive. In 2014, an estimated 4,80,000 women died as a result of TB. From 2000 to 2014, 43 million lives were

3

Introduction saved through effective diagnosis and treatment. Out of 9.6 million people who developed TB in 2014, more than half (58%) were in the South-East Asia and Western Pacific regions. India and China alone accounted for 23% and 10% of total cases, respectively [4]. In 2016, 87% of latent TB cases took place inside the 30 excessive TB burden international locations. Seven nations accounted for 64% of the new TB cases: India, Indonesia, China, Philippines, Pakistan, Nigeria, and South Africa. Global development depends on advances in TB prevention and care in these countries. WHO estimates that there were 600 000 new cases with resistance to rifampicin – the handiest first-line drug – of which 490 000 had MDR-TB. The MDR-TB burden in large part falls on three countries – India, China and the Russian Federation – which together account for nearly half of the global cases. About 6.2% of MDR-TB instances had XDR-TB in 2016 [4]. India is one of the highest burdens of TB. According to WHO, It is estimated that about 40% of the Indian population is infected with TB bacteria, the vast majority of whom have latent TB rather than TB. An estimated incidence of around 2.79 million cases of TB were reported in 2016 for India including HIV TB patients. Totally, 1,78,00 males were effected. 1,47,00 MDR TB cases were identified in 2016 with including 84,000 new cases. A total of 4,23,000 people died due to TB [4]. The revised estimates are based on data from various sources including sub-national prevalence surveys and enhanced TB notification from the private sector. The Revised National Tuberculosis Control Programme notified 17.5 lakh TB patients in 2016 including both from public and private health sectors and 33,820 drug resistant TB patients are notified additionally. Major TB cases were identified in Uttar Pradesh (297,746), Maharashtra (195,139), Madhya pradesh (129,915), Gujarat (126,665), Rajasthan (106,756). In 2016 out of the total reported patients, almost one fifth of the patients were reported from the private sector. As shown below there was wide variation from state to state in respect of the proportionate reporting of TB patients from the two sectors. In Kerala the reporting was almost equal, whilst the number reported was nil in some of the northeast states [5]. This situation highlights the relative shortcomings of the current treatment strategies for TB and the limited effectiveness of public health systems; particularly in resource-poor countries where the main TB burden lies.

4

Introduction

1.1. Mycobacterium tuberculosis - the etiological agent of TB TB has plagued humans for thousands of years. It has been found in the skulls and spines of Egyptian mummies. Hippocrates who was ancient Greek physician noted that tuberculosis which at the time was called phthisis or consumption was the most widespread disease and fatal to almost everyone who became infected with it. During the 17th and 18th centuries, TB had made its way to Europe. MTB during this time was called the “White Plague" in Europe. In 1882, Robert Koch developed a staining technique which allowed him to see tubercle bacillus which identified the etiological agent. The disease was finally named tuberculosis in 1839 by J.L. Schonlein because of the numerous tubercles or holes formed in the lungs by the bacterium. Mycobacterium tuberculosis is an obligate intracellular pathogen which can survive up to decades in a phenotypically non-replicating state, primarily in hypoxic granulomas in the lung [7]. It has outstanding mechanisms to run away from elimination and a high degree of intrinsic resistance to most antibiotics, chemotherapeutic agents and immune eradication [8]. Mycolic acids are the hallmark of the cell envelope of MTB, which are long chain α-alkyl-β-hydroxy fatty acids, the major constituents of this protective layer, has been shown to be critical for the survival of MTB. One major problem for host defence mechanisms and therapeutic intervention is robust, mycolic acid-rich cell wall, which is unique among prokaryotes [9]. Mycolic acids are the primary constituent of the mycobacterial cell wall which contributes to the outer membrane permeability and integrity as well as virulence [10]. This contributes to the chronic nature of the disease, imposes long treatment regimens and represents a formidable obstacle for researchers [11].

1.2. Mycobacterium tuberculosis (MTB): An overview The causative agent of TB, Mycobacterium tuberculosis is typically a nonmotile, rod-shaped, non-spore forming, and aerobic bacteria, classified as acid-fast bacilli. Morphologies can be observed when grown on solid media and some species exist as curved rods or shorter coccibacilli on artificial media [12]. The rods are 2-4 µM in length and 0.2-0.5 µM in width. The distinguishing characteristics of MTB are its complex cell wall and its slow generation time. Escherichia coli can replicate in approximately 20 minutes but MTB replication times are 16-20 hours. In nature, the bacterium can grow only within the cells of a host organism, but MTB can be cultured in the laboratory [13].

5

Introduction

The MTB morphology is classified in two categories; i) which are frequently seen at exponential phase of growth that is rod, V, Y-shape, branched or buds (Figure 1.4A), and ii) those that are seen occasionally under stress or environmental conditions which are round, oval, ultravirus, spore like, and cell wall defiant or L-forms (Figure 1.4B) [14].

Figure 1.4: Morphological variations in MTB. (A) Thin section transmission electron micrograph of MTB (extracted from www.wadsworth.org/databank/mycotubr.htm); (B) Scanning electron microscope shows shape variation in MTB at exponential phase of growth [14].

1.3. Classification of mycobacteria The classification of mycobacteria started in 1896 when Lehmann and Neumann proposed for the first time the genus mycobacterium which included Mycobacterium tuberculosis and Mycobacterium leprae species [15]. DNA-based molecular taxonomy, mycobacteria are classified as grampositive bacteria due to genes high similarity with other gram-positive organisms, such as Bacillus [16]. Based on the mycobacteria growth rate genus is usually separated into two major groups: i) slow-growing species including M. tuberculosis, M. bovis and M. leprae; and ii) fast-growing species such as M. smegmatis. Based on their epidemiological features, mycobacterium includes: i) non-pathogenic or rarely pathogenic mycobacteria, ii) strictly pathogenic mycobacteria iii) potentially pathogenic mycobacteria [17].

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Introduction

Table 1.1: Classification of mycobacteria according to the risk of infection.

Rare pathogens Potential pathogens Strict pathogens

M. smegmatis M. aurum M. avium M. tuberculosis M. phlei M. chitae M. intracellulare M. bovis M. fallax M. duvalii M. chelonae M. africanum M. thermoresistibile M. gadium M. fortuitum M. ulcerans M. parafortuitum M. gilvum M. kansasii M. microti M. gastri M. komossense M. malmoense M. canetti M. triviale M. lepraemurium M. marinum M. caprae M. nonchromogenicum M. neoaurum M. scrofulaceum M. pinnipedii M. gordonae M. terrae M. simiae M. leprae M. flavescens M. vaccae M. szulgai M. farcinogenes M. agri M. xenopi M. senegalense M. aichiense M. asiaticum M. paratuberculosis M. paratuberculosis M. haemophilum M. porcinum M. chubuense M. shimoidei M. diernhoferi M. obuense M. pulveris M. rhodesiae M. tokaiense M. moriokaense M. poriferae

The pathogenic species the most relevant for human health are M. tuberculosis and M. leprae, the causative agents of two of the world’s oldest diseases, tuberculosis and leprosy, respectively [16]. TB causative agents M. canettii and M. africanum, were isolated from African patients. M. bovis demonstrates the broadest spectrum of host infection, affecting humans, domestic or wild bovines and goats. M. microti can also cause disease in immune compromised human patients [18-19] and M. pinnipedii infects seals [20]. M. kansasii, M. malmoense and M. xenopi represent pulmonary opportunists, while M. marinum is the skin pathogen infecting organism by entering through damaged or ulcerated skin. M. ulcerans is the causative organism of buruli (tropical) ulcer [21]. All of the species of MTB complex (MTBC), which includes M. tuberculosis, M. canettii, M. africanum, M. microti, M. bovis, M. caprae and M. pinnipedii, are known to cause TB in humans. The M. avium complex (MAC) comprises M. avium subspecies responsible for disease in birds, but also for disseminated disease in patients with AIDS, causing systemic infections late in the

7

Introduction progress of AIDS, cervical lymphadenitis, and chronic lung disease in immunocompetent or non- HIV patients [22]. The Mycobacterium fortuitum complex includes M. peregrinum, M. fortuitum, M. chelonae and M. abcessus which are regularly responsible for abscess formation in local injection or surgical wounds and can be related with pulmonary disease [23].

1.4. The Mycobacterium tuberculosis genome The genome of MTB was studied generally using the strain MTB H37Rv. The genome sequence of MTB is one of the first complete genomes to be sequenced, and was decoded in 1998 by Cole and co-workers [24]. It contains sequence of 4,411,529 bp and characteristically high guanine plus cytosine (G+C) content (65.5%). Genome analysis revealed an efficient DNA repair system with nearly 45 genes related to DNA repair mechanisms [25] and despite over 10,000 years of evolution, when 16 genetically diverse clinical strains were examined for conservation of 24 genes known to encode antigenic proteins, minimal variation was observed [26]. MTB H37Ra is the avirulent counterpart of virulent strain H37Rv and both strains are derived from their virulent parent strain H37, which was originally isolated from a 19 year-old male patient with chronic pulmonary tuberculosis by Edward R. Baldwin in 1905 [27]. H37Rv and its avirulent counterpart H37Ra strains have been widely used as reference strains for studying virulence and pathogenesis of MTB worldwide since 1940. H37Ra is used as an adjuvant to boost immunogenicity during immunization with BCG [28]. Several of the MTB genes discussed are attractive targets for healing intervention, either through drug development or through incorporation into vaccine strains. It is also previously obvious that a more complete understanding of the pathogenic strategies of this highly successful intracellular pathogen will elucidate novel features of macrophage defenses and the host immune response. In addition to this anticipated scientific dividend, the dividend of greatest immediate importance is the development of new drugs and vaccines against this deadly disease [29].

1.5. The mycobacterial cell envelope The mycobacterial cell wall is a complex structure required for cell growth, resistance to antibiotics and virulence [30]. It is an elaborate cell envelope comprised of several layers. Each of these layers display different chemical modifications and the architecture of the cell wall is

8

Introduction also molded through complex regulation. High molecular weights of lipids represent the complexity of the cell wall [31]. Unusual impermeable properties of MTB cell wall are thought to be advantageous for the bacilli in stressful conditions of osmotic shock [32] and the polymers, covalently linked with peptidoglycan and trehalose dimycolate, provide a thick layer involved in MTB resistance to antibiotics and the host defense mechanisms [33]. The mycobacterial cell wall consists of an inner layer and an outer layer that surround the plasma membrane. The outer compartment consists of both lipids and proteins (capsuleprotien and capsule sugars). The inner compartment has three distinct macromolecules of peptidoglycan (PG), arabinogalactan (AG), and mycolic acids (MA) covalently linked together to form an insoluble complex referred to as the essential core of the mycobacterial cell wall [34]. (Figure 1.5)

Figure 1.5: The mycobacterial cell wall.

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Introduction

The peptidoglycan, which forms the “backbone’ of the cell wall skeleton [35]. The PG is made of peptides and glycan strands and covered with the plasma membrane. The long glycan strand typically consists of repeating N-acetylglucosamines (NAGs) linked to N-acetylmuramic acid (NAM). These strands are cross linked by peptides bound to the lactyl group on NAMs from different glycan strands [34, 36]. These peptide chains normally consist of L-alanyl-D-iso- glutaminyl-meso-diaminopimelic acid (DAP) from one strand linked to the terminal D-alanine residue from L-alanyl-D-iso-glutaminyl meso-DAP-D-alanine from a different strand. Mycobacterial PG is heavily crosslinked; up to 80% of the PG contains ‘nontraditional’ 3→3 peptide crosslinks instead of the ‘traditional’ 4→3 crosslink [37]. The highly cross-linked glycan meshwork of PG that surrounds bacteria is the primary agent that maintains bacterial shape.

Arabinogalactan (AG) forms a mycolyl-arabinogalactan-peptidoglycan (mAGP) complex. This complex is comprised of AG moiety anchored into a PG layer unique to mycobacteria and esterified at the distal end by a dense layer of long chain mycolic acids [38]. AG lacks repeating units and is instead made up of a distinct structural motif: The entire AG structure is tethered to the PG at the C-6 position of the N-glycolylmuramic acid by a linker unit containing a diglycosylphosphoryl bridge, α-L-Rha-(1→3)-α-D-GlcNAc-(1→P), common among only Actinomycetes [39]. Arabinan is ligated with long carbon chain mycolic acids. It forms the characteristic thick waxy lipid coat of mycobacteria is responsible for the impermeability of the cell wall and virulence [40].

Mycolic acids are long-chain fatty acids, up to 90 carbon atoms long, that are α-branched and β- hydroxylated [40]. These lipids make up 60% of the dry weight of MTB compared to 10% in most other bacteria and they are bound to the AG by esterification of a terminal pentaarabinofuranosyl [41]. MA are heterogeneous with regard to chain length, number of double bonds, cyclopropyl groups and side groups (keto-, methoxyand epoxy-groups) [40].

The biosynthetic pathway of mycolic acid involves type I and type II fatty acid synthases, FAS-I and FAS-II respectively. FAS-I catalyses the de novo synthesis of fatty acids from acetyl-CoA. In contrast, FAS-II is similar to systems found in bacteria, apicomplexa parasites and plants, and is composed of four dissociable enzymes that act successively and repetitively to elongate the

10

Introduction growing acyl chain. Acyl-primers are continually activated via thioester linkage to the prosthetic group of coenzyme A (CoA) for FAS-I, or of an acyl carrier protein (ACP) for FAS-II (Figure 1.6) [42].

Figure 1.6: Fatty acid/mycolic acid biosynthesis in mycobacteria.

FAS-I is involved in the synthesis of C16 and C26. The C16 acyl-CoA product acts as a substrate for the synthesis of meromycolic acids by FAS-II, whereas the C26 fatty acid constitutes the α- branch of the final mycolic acid. MtFabH has been proposed to be the link between FAS-I and

FAS-II by converting C14-CoA generated by FAS-I to C16-AcpM, which is channelled into the FAS-II cycle. This latter comprises four enzymes which will act successively and repeatedly to ensure fatty acid elongation, ultimately leading to meromycolates (C56). These enzymes are the condensing enzymes KasA and KasB, the keto-reductase MabA, an unidentified dehydratase, and the enoyl-reductase InhA. Finally, the polyketide synthase Pks13 catalyses the condensation of the α-branch and the meromycolate to produce mycolic acids. Targets for the action of activated INH, ethionamide (ETH), triclosan (TRC), or thiolactomycin (TLM) are indicated. FAS-II enzymes are labelled in black except the condensing enzymes, which are indicated in red.

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Introduction

The relative contribution of FAS-I and FAS-II activities in fatty acid/mycolic acid biosynthesis is represented in green and purple respectively [42]. It’s interesting to note that mammals do not synthesize mycolic acids and thus compounds that antagonize the enzymes involved in mycolic acid biosynthesis are promising leads for developing novel anti-tubercular agents. Many efforts currently focus on revisiting and optimizing existing inhibitors of validated drug targets in FAS-II, particularly because it can remove much of the uncertainty surrounding new drug targets and in vivo clinical efficacy. Mycolic acids give rise to important characteristics, including resistance to chemical injury, resistance to dehydration, low permeability to hydrophobic antibiotics, virulence [43]. InhA encoded by the MTB gene InhA, catalyses the final enzymatic step in the elongation cycle of the FAS-II pathway [44]. It is a NADH-dependent reductase that exhibits specificity for long chain enoyl thioester substrates, and its reduction mechanism [45]. InhA, an enoyl-ACP reductase, involved in mycolic acid synthesis, is a well-known target for front-line anti-tubercular drugs [46] such as INH [47], and ETH [44], much interest has been devoted to deciphering the chemistry and biosynthesis of mycolic acids in the alarming context of the emergence of MDR, XDR, and TDR TB. Mycolic acids are processed and matured by a cascade of enzymes [48], which results in three distinct meromycolate variants: α- meroacids, methoxy-meroacids and keto-meroacids [30]. All three variants are required for full virulence during infection and have varying levels of saturation, cyclopropanation and oxygenation [42, 43].

1.6. Tuberculosis: Drug resistance A growing trend of drug-resistance in the TB disease is threatening the gains in global TB control. Despite the implementation and success of DOTS course, there is a steady increase in the number of patients infected with multi and extensively mycobacterial drug resistant strains [49]. Drug resistance in TB therapy is not an immediate past, as the strains of MTB that were resistant to streptomycin were experimental soon after its introduction for TB treatment in 1944 [50]. Nowadays resistance to all the available anti-tubercular drugs have been found in different constituents of the world. The most important factors causing drug resistance is incomplete and inadequate treatment methods and it emerges mostly where TB control

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Introduction programmes are weak [51]. TB intrinsically resistant to many antibiotics due to the low permeability of its mycolic acid-rich waxy cell envelope, the action of efflux pumps and the presence of chromosomally encoded resistance genes [52]. K.D. Green and S. Garneau-Tsodikova reported drug resistance are in MTB due to this three factors (i) mutations or modifications of the drug targets (RMP, EMB, kanamycin (KAN), amikacin (AMK), Capromycin (CAP), and the fluoroquinolones (FQ), (ii) the inability in prodrugs activation (INH, PZA and ETH) due to mutations which leads to a loss of function, and (iii) enzymatic inactivation of the drug (KAN) [53]. TB resistant classify as a) resistant to INH and RMP as MDR TB; b) Resistance of MDR TB strains to the FQ and aminoglycosides are classed as XDR TB; c) TDR TB has been used to describe strains found resistant to all available drugs, but there is not yet an agreed definition of TDR TB; (d) RMP-resistant TB (RR TB) is also known exist [54]. Expression of clinical efficacy in drug-sensitive TB is demanding, given high success rates for existing regimens, concerns about substituting an investigational agent for the most effective agents in a regimen and difficulties in determining the effect size of the components of a combination regimen. In difference, exploring efficacy of novel treatments in the setting of drug-resistant disease may experience with the activity and the safety of new agents in drug- resistant disease may provide a stage which their sign can diversify to include drug-sensitive disease [55].

1.6.1. Multidrug-resistant TB (MDR TB) MDR TB, also known as Vank's disease) is defined as TB where a bacterium is resistant were to least two of the most powerful first-line anti-TB drugs, INH and RMP. WHO in 2014 estimated that 300,000 had MDR TB. Globally in 2014; 123,000 patients with MDR TB or RR TB were actually notified. It was just 41% of the number of estimated cases of 300,000. There were also approximately 190,000 deaths from MDR TB. More than half of these patients were in India, China and the Russian Federation [4]. MDR TB is consequence of inappropriate use of essential anti-TB drugs. MDR TB results primarily from accumulation of mutations in individual drug target genes. The chance of resistance is very high for less effective anti-tubercular drugs such as ETH, CAP, thiacetazone, cycloserine, and viomycin; intermediate for drugs such as INH, SM, EMB, KAN, and PAS; and

13

Introduction lowest for RMP. In addition to accretion of mutations in the individual drug target genes, the permeability barrier imposed by the MTB cell wall can also involve to the development of low- level drug resistance. Studies addressing resistance to SM have found evidence of such a two- step mechanism for the development of drug resistance [56]. Treatment options are limited and expensive, recommended medicines are not always available, and patients experience many adverse effects from the drugs [4]. Treatment of MDR TB requires treatment with second-line drugs, usually four or more anti-TB drugs for a minimum of 6 months, and possibly extending for 18-24 months if RMP resistance has been identified in the specific strain of TB with which the patient has been infected [14]. In general, second-line drugs are less effective, more toxic and much more expensive than first-line drugs. Under ideal program conditions, MDR TB cure rates can approach 70% only [56].

1.6.2. Extensively Drug-Resistant TB (XDR TB) XDR TB (extensively drug resistant TB) is defined as strains resistant to at least RMP and INH. This is in addition to strains being resistant to one of the FQ, as well as resistant to at least one of the second line injectable TB drugs AMK, KAN or CAP, resulting in a longer treatment course for a minimum of 18-24 months, lower cure rates, and significantly increased healthcare costs [14]. Moreover, second-line therapeutic treatment requires strict patient monitoring, supervision, counseling, and support to prevent further drug resistance that could potentially render the disease untreatable [14]. XDR TB is of special concern for persons with HIV infection or other conditions that can weaken the immune system. These persons are more likely to develop TB once they are infected, and also have a higher risk of death once they develop TB.

1.6.3. Totally drug-resistant TB (TDR TB) or extremely drug resistant TB (XXDR TB) Researchers have recently identified the existence of the most dangerous form of TB strain reported till date. TDR TB refers to MTB clinical strains exhibiting in vitro resistance not only to RMP and INH, two of the main first line TB drugs, but strains that are also resistant to fluoroquinolone and to at least one of the second line injectable TB drugs (INH, RMP, SM, ethambutol, PZA, ETH, PAS, cycloserine, ofloxacin, AMK, ciprofloxacin, CAP, KAN). TDR TB, or XXDR TB, refers to strains that are resistant to all the first line drugs as well as all the

14

Introduction second line TB drugs. The presence of TDR TB was first observed in Italy in 2003, subsequently in Iran and India [57]. TDR TB is relatively poorly documented, as many countries do not test patient samples against a broad enough range of drugs to diagnose such a comprehensive array of resistance. The United Nations' Special Programme for Research and Training in Tropical Diseases has set up a TDR TB Specimen Bank to archive specimens of TDR TB [58]. TDR TB bacilli while designing new drugs and if it is so, whether the previously designed drugs could be effective? Last but not the least; as far as, there is no cure for TDR TB patient, hence it is not exaggeration to say that world is on danger of untreatable drug resistant TB strain. Therefore, if authorized health organization do not consider immediate action plan for such bacilli, then we may face a new outbreaks of untreatable TB [59]. Recently, Bedaquiline (TMC-207), Delamanid (OPC-67683) and Linezolid, three new drugs approved by the US-Food and Drug Administration and the European Medicines Agency, may offer therapeutic solutions for TDR TB. With more new anti-tubercular agents in the pipeline, there is hope of identifying drugs that may be bactericidal or bacteriostatic in TB treatment and challenge the TDR TB terminology [60].

1.6.4. Rifampicin-resistant TB (R TB): If bacteria are just resistant to RMP then it is called rifampicin-resistant TB. It is also with or without resistance to other drugs includes any resistance to rifampicin, whether mono-resistance, multidrug resistance, polydrug resistance or extensive drug resistance. Both MDR TB and XDR TB are forms of RR TB [61].

1.7. TB in HIV The risk of developing TB is estimated to be between 26 and 31 times greater in people living with HIV than among those without HIV infection. In 2014, in the new cases of TB, 1.2 million were people living with HIV and 0.4 million among people with HIV-positive died along with TB disease [4]. In patients with CD4 counts ≥50 cells/mm3 who present with clinical disease of major severity, as indicated by clinical evaluation (including low Karnofsky score, low body mass index, low hemoglobin, low albumin, organ system dysfunction, or extent of disease),

15

Introduction antiretroviral therapy should be initiated within 2 to 4 weeks of starting TB treatment. The strength of this recommendation varies on the basis of CD4 cell count. In HIV-infected patients with documented MDR and XDR in TB, antiretroviral therapy should be initiated within 2 to 4 weeks of confirmation of TB drug resistance and initiation of second-line TB therapy [62]. It must also be noted that several of the problems associated with TB, such as resistance and HIV co-infection are highly coupled. For example, due to the high abandon rate of treatment by co- infected patients, there is a higher emergence of drug resistant strains [63]. Such opportunistic infections have a disastrous effect on the mortality rate in infected patients.

1.8. Current treatment in TB Table 1.2: Group name and mechanism of action of first and second line anti-TB agents. Name of the group Drug* Mechanism of action Isoniazid Inhibition of Mycolic acid biosynthesis Rifampin Inhibition of RNA synthesis First line anti-TB drugs Disruption of electron transport across the Pyrazinamide membrane Ethambutol Arabinogalactone synthesis inhibitor Kanamycin Protein Synthesis Inhibitor Second line Injectable Amikacin Protein Synthesis Inhibitor anti-TB drugs Capreomycin Protein Synthesis Inhibitor Levofloxacin Inhibition of DNA gyrase Gatifloxacin Inhibition of DNA gyrase Second line Ofloxacin Inhibition of DNA gyrase Fluoroquinolones Ciprofloxacin Inhibition of DNA gyrase Moxifloxacin Inhibition of DNA gyrase Ethionamide Cell wall synthesis inhibitor

Second line (oral Prothionamide Cell wall synthesis inhibitor bacteriostatic) anti-TB Cycloserine Inhibition of peptidoglycan synthesis drugs p-Aminosalicylic acid Inhibition of folic acid and Iron metabolism Bedaquiline ATP synthetase Inhibitor *Drugs in bold letters are FDA-approved for use in TB therapy.

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Introduction

1.8.1. Treatment for latent-TB infection (LTBI) TB germs can live in our body without making you sick. This is called latent TB infection. People with latent TB infection do not have symptoms, and they cannot spread TB bacteria to others. LTBI testing is very mandatory for these people; close contacts of infectious TB patients, health care workers (particularly susceptible to TB exposure and infection) and frequent travellers abroad people [64]. For treating LTBI there are few regimens used based on the results of drug susceptibility testing [65]. INH nine months therapy but it is long treatment, RMP four months therapy but it is direct treatment and RMP–PZA two months therapy due to stern hepatic injury and death, this regimen was not recommended. CDC has recommended a 12-dose regimen; the regimen is a combination of INH and RMP doses under directly observed treatment. This 12-dose regimen is very effective which reduces the required treatment for LTBI from 270 daily doses over 9 months.

1.8.2. Treatment for drug susceptible-TB The existing TB treatment consists of isoniazid, ethambutol, RMP and PZA for two months followed by isoniazid and RMP for four months. This standard TB therapy is prolonged as patients have to take the drugs for six months and often leads to patient’s non-adherence. In these circumstances an incomplete treatment results in development of drug resistance. To confront this situation, WHO promoted a program known as “Directly Observed Treatment-Short course (DOTS)”, DOTS is effective in many controlled trials; few studies have evaluated its effectiveness under programmatic conditions [66]. In this type of treatment there is a direct observation by trained personnel on patients undergoing treatment. The DOTS therapy has established to be one of the most cost effective health interventions available today around the world. It has been proven to be one of the most efficient approaches to fight the global TB epidemics [4]. Effectiveness and tolerability relation of first- and second-line drugs used in TB treatment is depicted in figure 1.7.

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Introduction

Figure 1.7: Effectiveness and tolerability relation of first- line and second-line drugs used in TB treatment.

1.8.3. Treatment for drug resistant-TB Drug resistant TB can be cured with the suitable combination and reasonable use of available anti-tubercular drugs. The poly drug-resistant TB therapy of contagions involving (INH + EMB, INH + PZA, EMB + PZA, or INH + EMB + PZA) MTB strains entails careful clinical evaluation but can be managed with extended treatment up to 18 months by regimens containing first-line drugs, fluoroquinolones, KAN/AMK/CAP and some second-line agents. Treatment of MDR TB is lengthy, expensive, toxic, and associated with higher rates of clinical failure and disease relapse. For the treatment of MDR TB, WHO recommends the use of DOTS-Plus therapy, which includes drugs used in DOTS therapy plus second line TB drugs composition of MDR TB drug regimen; 1): a) Injectible second line drugs b) Later generation fluoroquinolone c) ETH or prothionamide (PTH) d) Cycloserine or terizidone; 2): Usage of at 4 drugs (it is unclear whether all patients with MDR TB/XDR TB should be treated with PZA); 3): Group five drugs to be used only if needed to sum up to at least four active drugs; 4): Healing for a total of 24 months with an intensive phase of 8 months; 5): Prolongation of duration of therapy should be considered based on success [67]. Bedaquiline and delamanid are recently approved by US FDA for the treatment of MDR TB in adults [4]. Bedaquiline and delamanid can be used for treatment of MDR TB in

18

Introduction adults when patients in serious or life-threatening conditions do not have an effective treatment regimen [68].

Drugs for treatment of XDR TB or TDR TB need to be selected stepwise on basis of safety and efficacy. New drugs (pretomanid, delamanid and bedaquiline) and novel regimens (PA- 824+Moxifloxacin+ PZA and NC-003) for curing drug resistant TB are now available. The new combination 3 (NC-003) clinical trials tested the bedaquiline + PTH + PZA (BpaZ) regimen, consisting of bedaquiline, PA-824, and PZA. The two-week study found that the PZA regimen killed more than 99% of TB bacteria over the course of 14 days, and that the treatment was safe. These novel chemospheres are reducing the treatment period and cost of therapy [69, 70]. If sufferers are untreated, the analysis for people laid low with drug-resistant TB is similar to the analysis for people with drug-touchy TB (10 yr case fatality charges of about 70%). The modern- day WHO-endorsed MDR TB routine has an approximate 50% treatment price, while the cure fee in endemic settings of drastically drug-resistant TB within the absence of drugs together with bedaquiline, delamanid and linezolid is about. Thus, TB (and drug-resistant TB specially) poses a grave risk to human fitness and great of existence. High-excellent patient care, regular with the International Standards for TB Care, is essential to make sure exact consequences and hold the pleasant of lifestyles. Unfortunately, international standards are frequently not met in lots of low- income, high-burden international locations, mainly within the personal fitness quarter, which is a primary company of health care in many countries with a excessive TB incidence. Poor best of care is, therefore, a key driver of TB mortality in excessive-burden nations, and might give an explanation for the persistently excessive TB incidence in some settings. Whereas country wide programmes are responsible to country wide and worldwide authorities regarding their implementation of proper standards of care, one of the finest demanding situations in TB manipulate continues to be enticing and regulating the non-public sector. Innovative public-non- public blend procedures are required to overcome this task, together with social franchising, insurance-based totally projects, middleman corporations and provider consolidation, with a heavy emphasis on the use of records and conversation technologies.

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Introduction

1.9. Classification of anti-TB drugs The available anti-TB drugs are classified based on their mechanism of action or inhibition. These are classified under following heads; i) cell wall synthesis inhibitors (INH, EMB, ETH and cycloserine), ii) nucleic acid synthesis inhibitors (rifampin and quinolones), iii) protein synthesis (SM, AMK, KAN & CAP), and iv) electron transport across the bacterial membrane or energy inhibitors. (PZA) [71].

1.9.1. Cell wall synthesis inhibitors Cell wall synthesis inhibitors such as INH, EMB, ETH, PTH and cycloserine are used for TB treatment.

Figure 1.8: Cell wall synthesis inhibitors. Isoniazid (INH) Isonicotinyl hydrazide or Isonicotinic acid hydrazide or INH was introduced in 1951 for the treatment of TB and it is more potent drug than streptomycin and p-aminosalicylic acid. It is a prodrug activated by ‘catalase peroxidase’ enzyme (KatG) and active against growing tubercle bacilli, but not active against nonreplicating bacilli. The primary target of inhibition is the cell wall mycolic acid synthesis pathway [50]. KatG links the isonicotinic acyl part to NADH resulting in an isonicotinic acyl-NADH complex. This complex binds efficiently to the InhA which is an enoyl-acyl carrier protein reductase, and blocks the natural enoyl-AcpM substrate and the action of fatty acid synthase. Consequently, the inhibition of synthesis of mycolic acids is terminated [50, 71].

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Introduction

Recent research shows that besides InhA it also attacks DfrA (‘dihyrofolate reductase’ involved in DNA synthesis) [50]. Since, its wide range of usage resistance to INH has been seen more repeatedly among clinical isolates of MTB infected patients. Resistance to INH occurs due to the mutations in KatG gene; as a result the ability of catalase peroxidase to activate INH prodrug reduces. The Hepatitis, lupus-like syndrome, peripheral neuropathy and drug-drug interactions are major adverse reactions of isoniazid [72]. Ethambutol (EMB) EMB ([(S,S)-2,2(ethylenediimino)di-1-butanol]) was discovered as anti-tubercular agent in 1961. EMB plays a pivotal role in the chemotherapy of drug-resistant TB, including MDR TB. It is a first line anti-tubercular drug. Ethambutol appears to target the cell wall of tubercle bacilli through interfering with arabinosyl transferases, encoded by the embCAB operon, comprised of three homologous genes, designated embC, embA, and embB, and involved in the biosynthesis of arabinogalactan and lipoarabinomannan, the key structural components of the mycobacterial cell wall. The proposed scenario of EMB action on MTB is that upon interaction with the EmbCAB proteins EMB inhibits the arabinan synthesis leading to lack of arabinan receptors for mycolic acids and accumulation of mycolic acid results in cell death [71, 73]. Resistance to EMB has repeatedly been associated with alterations in the embB gene, particularly in mutations in embCAB operon are responsible for resistance to EMB and are found in approximately 65% of clinical isolates of MTB resistant to EMB [71]. Some inconsistent reports revealed that one quarter of all EMB resistant MTB isolates do not harbour mutations in any of the above named genes, investigations suggesting further canvases are needed to explore possible mechanism of EMB resistance [72]. Ethionamide (ETH) and prothionamide (PTH) Thioamide drugs are Ethionamide (ETH) and Prothionamide (PTH), these are generally considered second-line drugs for treatment of TB and MDR TB. ETH is structurally related to INH and as well as a prodrug that is activated by the enzyme EtaA (a monooxygenase, also called EthA) and inhibits the same target InhA as INH of the mycolic acid synthesis pathway [44]. EtaA/EthA is a flavin adenosine dinucleotide (FAD) containing enzyme that oxidises ETH to the corresponding S-oxide. Similar to INH, ETH inhibits mycolic acid synthesis by binding to the enzyme InhA. EthA to activate ETH has been convincingly demonstrated, very limited data exist on the occurrence of EthA mutations in ETH-resistant MTB clinical isolated [73]. The

21

Introduction existence of partially cross-resistant phenotypes has long been known. Low-level INH-resistant strains frequently display low-level ETH resistance, while high-level INH-resistant strains typically remain ETH susceptible. The structural similarity and existence of cross-resistant phenotypes suggested that these two drugs share a common molecular target [71]. PTH shares almost identical structure and activity as ETH, where the R group in ETH is

C2H5 and the R group in PTH is C3H7. It is a better tolerated and less toxic drug than its predecessor of ETH [74]. The activated PTH form adducts with nicotinamide adenine dinucleotide (NAD), which is inhibitor of the InhA enzyme in MTB. Inhibition of InhA results in inhibition of mycolic acid biosynthesis and cell lysis. Mutations in the drug-activating enzyme EtaA/EthA and the target InhA cause resistance to ETA [75]. Cycloserine (CS) Cycloserine is a D-alanine analogue; it is classified as a second-line drug. CS is particularly used for healing MDR TB and XDR TB. CS interrupts peptidoglycan synthesis by inhibiting the enzymes D-alanine racemase (AlrA) and D-alanine:D-alanine ligase (Ddl) [76] which inhibits the synthesis of cell wall mycobacteria. Resistance to CS is due to over expression of AlrA and Ddl) [76].

1.9.2. Nucleic Acid Synthesis Inhibitors Rifampin and other rifamycins

The Rifamycins include Rifampin (RMP), Rifapentine, and Rifabutin. Of these, RMP is most commonly used, as first-line therapy for treatment of mycobacterial disease (including TB). The addition of rifampin to treatment regimens for TB reduces the duration of therapy needed for active disease from 12 to 6 months and reduces the duration of therapy needed for latent infection from 9 months to 2 to 3 months. RMP interfere with bacterial DNA-dependent RNA

22

Introduction polymerase and are potent bactericidal agents. RMP and its analogues kill actively multiplying extracellular organisms, intracellular mycobacteria, and semidormant mycobacteria in tissues [77]. RMP contain an aromatic nucleus linked on both sides by an aliphatic bridge so as to easily diffuse across the MTB cell membrane due to their lipophilic profile. These have ability to inhibit bacterial DNA-dependent RNA synthesis, due to high binding affinity with RNA polymerase [71]. Resistance to RMP is a result of mutations in the rpoB gene, which encodes the β-subunit of RNA polymerase [77]. Fluoroquinolones (FQ)

FQs were derived from quinine. Nalidixic acid, the first quinolone derivatives was introduced in 1962 by George Lesher et al., discovered as a by-product of chloroquine synthesis [78]. The fluoroquinolones (moxifloxacin, gatifloxacin, sparfloxacin, levofloxacin, ofloxacin, and ciprofloxacin), possess excellent activity against MTB and are used as second-line drugs in TB treatment [71]. Most FQs are being evaluated as potential anti-TB drugs, also for their proven potential to shorten TB treatment duration. Use of FQs is one of the major strategies for TB control. They are the class of antibiotics that have potent antimicrobial activity against a wide range of gram positive and gram negative organisms. The treatment of MDR TB relies upon a backbone of an injectable agent (KAN, CAP, AMK and a fluoroquinolone namely gatifloxacin, levofloxacin, moxifloxacin, or ofloxacin. The most active fluoroquinolones are: moxifloxacin = gatifloxacin > levofloxacin > ofloxacin [79]. In addition to the aforementioned fluoroquinolones, several clinical studies have investigated the efficacy of sparfloxacin and lomefloxacin.

23

Introduction sparfloxacin appears effective for treating MDR TB, the role for lomefloxacin in TB therapy is unclear. Gatifloxacin and moxifloxacin are under phase III clinical evaluation aiming at better TB treatment [79]. FQs inhibits DNA synthesis by targeting the DNA gyrase A and B subunits. It blocks the movement of replication works and transcription complexes in MTB [71]. Resistance to FQs in MTB is due to mutations in the conserved quinolone resistant determining region of gyrA and gyrB involved in the interaction between the drug and DNA gyrase [71]. In addition to the bacteria gaining resistance to the FQ class of drugs, a few serious side effects such as tendonitis and tendon rupture due to collagen damage, QTc interval prolongation by blocking voltage-gated potassium channels etc associated with these classes of drugs has limited their clinical use and future progress, necessitating research into development of new antibacterial agents that lack cross-resistance mediated by mutations in the bacterial targets [80].

1.9.3. Protein synthesis Inhibitors Streptomycin (SM)

Streptomycin is an antibiotic (antimycobacterial) drug, the first of a class of drugs called aminoglycosides to be discovered, and it was the first effective treatment for TB [71]. Streptomycin acts as inhibitor of protein synthesis by binding to the S12 protein of the 30S subunit of the bacterial ribosome and interfering with the binding of formyl-methionyl-tRNA to the 30S subunit of the ribosome [81]. These results in precarious ribosomal-mRNA complex, foremost to frameshift mutation and flawed protein synthesis and further to cell death. Mutations in rpsL and rrs are the major mechanism of SM resistance and it is exhibits toxic manifestations

24

Introduction on peripheral and central nervous system at higher doses and leads to hypersensitivity reactions [81]. Amikacin, Kanamycin and Capreomycin

The aminoglycosides KAN and AMK and the macrocyclic peptide CAP are key drugs for the treatment of MDR TB as second line drugs. These are protein synthesis inhibitors. These drugs bind to 16S rRNA in the 30S small ribosomal subunit and inhibit protein synthesis. The A1401G mutation has been assorted with AMK resistance in MTB, and change in their rrs genes. CAP is a macrocyclic polypeptide, like streptomycin and KAN it modifies the ribosomal structure at 16S RNA there by inhibiting protein MTB resistant to KAN and CAP has been associated with mutations in the rrs gene encoding 16S rRNA [82]. Mutations at 16S rRNA position 1400 are associated with high-level resistance to KAN and AMK. Cross-resistance may be observed between KAN and CAP but a recent study found little cross-resistance between KAN and AMK [71, 82].

1.9.4. Electron transport across membrane inhibitors

Pyrazinamide (PZA) or pyrazine-2-carboxamide is an important front-line drug for the treatment of TB; it is used in combination with additional drugs viz. INH and RMP for the treatment of MTB. It is still part of WHO suggested standard TB therapy. The use of PZA was first introduced in 1954 and was a great success as it resulted in shortening the duration of the TB

25

Introduction

therapy to current 6 months than initial 9 months [83]. PZA is a prodrug that stops the growth of MTB. PZA diffuses into the granuloma of MTB, where the enzyme pyrazinamidase converts PZA to the active form pyrazinoic acid. Under acidic conditions, the pyrazinoic acid that slowly leaks out converts to the protonated conjugate acid, which is thought to diffuse easily back into the bacilli and accumulate. The net effect is that more pyrazinoic acid accumulates inside the bacillus at acid pH than at neutral pH [83]. Pyrazinoic acid was thought to inhibit the enzyme fatty acid synthase (FAS) I, which is required by the bacterium to synthesise fatty acids. PZA and its analogues inhibited the activity of purified FAS I. Pyrazinoic acid binds to the ribosomal protein S1 (RpsA) and inhibits trans-translation. This may explain the ability of the drug to kill dormant mycobacteria [84]. The PZA resistance in MTB is due to mutations in the pncA, furthermore to PZA resistance associated with pncA, rpsA or panD mutations, it also described that can change pncA expression, altered PZA update, or dysregulated pyrazinoic acid efflux, which creates fault in the functioning of pyrazinamidase [83, 85].

Figure 1.9: Mechanism of action of existing drugs/new anti-tubercular drugs in development.

26

Introduction

1.10. Current emerging pipeline new anti-TB drugs Substantial progress has been made in last 40 years and a promising portfolio of new anti- tubercular drugs is on the horizon. Some have the potential to become the cornerstone of future TB treatment. There is now recognition that new drugs to treat TB are urgently required, specifically for use in shorter treatment regimens than are possible with the current agents and which can be employed to treat multidrug-resistant and latent disease. A variety of new initiatives have been created to tackle these objectives, the most recent of which is the establishment of the so-called Global Alliance for TB Drug Development [70, 86]. The Alliance, a public/private partnership in which WHO is a partner, is a not-for-profit venture that will accelerate the discovery and development of new drugs to fight TB using a virtual operating model to outsource projects. The current decade blossoms with a promising anti-TB drug pipeline, with various potential drugs targeting diverse MTB terminating sites in several stages of drug development.

Figure 1.10: Various agents that are currently being investigated for TB therapy.

27

Introduction

Eight drugs and combinations are in Phase I, Phase II or Phase III trials for the treatment of drug- susceptible, MDR TB or LTBI.

Figure 1.11: Structure of anti-TB agents under preclinical development.

1.10.1. Q203 Q203 is the first in new class of amide imidazopyridine compounds. The rapid inhibition of ATP synthesis at low concentration strongly suggests that the inhibition of cytochrome bc1 activity is the primary mode of action of Q203, which is a bacterial enzyme complex needed for respiration. Q203 causes a rapid depletion of intracellular ATP at an IC50 of 1.1 nM and interrupts ATP homeostasis in dormant Mtb at an IC50 of 10 nM. Both of these values are better

28

Introduction than bedaquiline's measures, and they explain Q203's excellent killing profile in chronic Mtb infection models. Q203 did not inhibit any P450 isoforms and was not a substrate or inhibitor of P-gP efflux, which indicated a low risk of drug–drug interactions. It is found that Q203, being a new chemical entity is able to inhibit MDR TB and XDR TB [87]. In phase I dose-escalation study to evaluate safety, tolerability and pharmacokinetics of single and multiple doses of Q203 in healthy volunteers started in March 2016. Though it should be noted that like bedaquiline, Q203 is a highly lipophilic drug, with very high serum protein binding. The Phase I clinical trial (clinicaltrials.gov identifier: NCT02530710) enrolling healthy patients is a dose-escalation study starting at 100 mg dosing that will be adjusted based on PK analysis.

1.10.2. Sutezolid Sutezolid (PNU-100480, PF-02341272) is an oxazolidinone antibiotic currently in development as a treatment for XDR TB. It has safety profile than linezolid. Its activity and pharmacokinetic data shows that sutezolid converts into sulfone and sulfoxide metabolites, the sulfoxide metabolite is more active and reaches four times higher in concentration than parent compound [88]. It inhibits protein synthesis by the ribosomal initiation complex [89]. Mouse model studies showed that addition of sutezolid to current first line TB drugs improved the bactericidal activity. It also gave better results when used in combination with moxifloxacin and PZA. Sutezolid was safe and well-‐tolerated at doses up to 1200 mg daily for up to 14 days, or 600 mg twice daily for up to 28 days. 4 A Phase 2 trial demonstrated that sutezolid has significant early bactericidal activity and may have clinical efficacy in humans in a larger Phase 2 trial. The addition of sutezolid to the standard TB treatment regimen leads to significantly improved efficacy. In vivo studies in the chronic mouse model of TB demonstrated that the addition of sutezolid to the standard TB regimen has the potential to significantly shorten treatment. It not only reduced the numbers of bacteria in the lungs more quickly, but also led to a relapse- free cure with a shorter duration of treatment. Pfizer recently completed a phase IIa, open-label, early bactericidal activity and whole-blood activity study. This study of adults with pulmonary DS-TB compared two experimental arms one with sutezolid twice daily at 600 mg, the other with sutezolid once daily at 1,200 mg with Rifafou. These outputs suggest that sutezolid has the potential to reduce the treatment duration in both drug susceptible and drug resistant TB [88].

29

Introduction

1.10.3. SQ109 SQ109 1,2-ethylenediamine, is an analogue of ethambutol. It was discoverednby screening a combinatorial library of more than 63,000 compounds with anwhole bacterium high throughput screen. Unlike EMB, SQ109 has different mechanism of action and belongs to the classes of cell wall inhibitors. The drug is active against both drug-susceptible and drug-resistant TB by targeting MmpL3 in MTB and specifically inhibiting the protein synthesis [90, 91]. In clinical trials study to determine safety, tolerability, pharmacokinetics and bacteriological effect of different doses of SQ109 alone and in combination with RMP was administered over 14 days. Several in vivo research in the chronic mouse version of TB the use of combinations of SQ109 and popular anti-TB tablets display both higher efficacy and shorter time to obtain the equal discount in MTB as preferred remedy with ethambutol. In studies in which SQ109 replaced EMB in the trendy first line treatment routine, no or few micro organism have been cultured from lungs of mice treated for 2 months, suggesting that SQ109 effects in a extra fast cure. It revealed no adverse drug-drug interactions, good activity against drug susceptible and drug resistant TB was observed. The in vitro bacterial mutation rate for SQ109 is very low which could limit the development of drug resistance to SQ109. In vitro, it has some synergic effects with bedaquiline and favourable interactions with sutezolid [92]. 82 unfavourable occasions, of which fifty six% were gastrointestinal occasions One affected person died during the 14 day comply with-up duration because of big hemoptysis. This turned into deemed unrelated to have a look at drug by way of the investigator. No different serious negative activities (SAEs). There had been no ECG- associated treatment discontinuations. There became no prolongation of QTcB or QTcF past 500ms, or an growth of more than 60ms in comparison to baseline. Safety/Tolerability of SQ109 in TB patients. It‘s principal facet effect is nausea, which is more reported within the 300mg dose There have been no systematic increases in QT in the SQ109 groups Steady kingdom seems to be reached at ~day 7; the induction of CYP2C19 through Rif can be conquer with 300mg SQ109 SQ109 had no bactericidal effect in humans over 14 days; RIF had a 1-log effect in human beings over 14 days. Mouse modeling statistics suggest that: - EBA statistics in people mimics that seen in mouse - SQ109 results are obvious the longer the drug is taken [92]. .

30

Introduction

Figure 1.12: Structure of anti-TB agents under phase-I & II clinical trials

1.10.4. Levofloxacin (LEV)

LEV is higher-generation fluoroquinolone antibiotic. It inhibits the two type II topoisomerase enzymes, namely DNA gyrase and topoisomerase IV. Topoisomerase IV is necessary to separate DNA that has been replicated (doubled) prior to bacterial cell division. With the DNA not being separated, the process is stopped, and the bacterium cannot divide. DNA gyrase, on the other hand, is responsible for supercoiling the DNA, so that it will fit in the newly formed cells. Both mechanisms amount to killing the bacterium. In this way, LEV acts as a bactericide [92]. LEV and moxifloxacin imposed no additional hepatotoxicity on patients with drug-induced liver injury (DILI) secondary to first-line anti-TB therapy. These two drugs could be safely prescribed while waiting for liver function normalization. No cases of DILI occurred among patients during the follow-up period. LEV and moxifloxacin were safe for long-term use [93]. Phase-II studies determined the LEV dose and exposure that achieves the greatest reduction in MTB burden with acceptable tolerability by studying 100 adults with smear- and culture- positive pulmonary MDR TB at sites in Peru and South Africa [93]. While its side outcomes are generally mild to slight, serious reactions to levofloxacin now and again occur.

31

Introduction

Prominent among those are side effects that became the challenge of a black box warning by means of the FDA in 2016. An FDA protection evaluation has shown that fluoroquinolones when used systemically (i.E. Pills, drugs, and injectable) are associated with disabling and probably permanent severe side outcomes that can occur together. These facet results can involve the tendons, muscle tissue, joints, nerves, and important anxious device. Such injuries, such as tendon rupture, have been determined up to 6 months after cessation of remedy; the elderly, transplant patients, and people with a present day or historic corticosteroid use are at elevated risk. A precise evaluate of threat factors for fluoroquinolone-related tendon rupture has been posted; superior age, concurrent treatment with corticosteroids, and better doses of fluoroquinolone appear like the maximum crucial chance factors. The U.S. Label for levofloxacin additionally includes a black field warning for the exacerbation of the signs of the neurological disorder myasthenia gravis.

1.10.5. Delamanid The Delamanid (OPC-67683) (Deltyba) is a nitroimidazo-oxazole derivative; it is a new anti-TB drug which exhibits potent in vitro and in vivo anti-TB activity against drug-susceptible and drug-resistant strains of MTB. The new drugs delamanid and bedaquiline are increasingly used to treat MDR-TB & XDR-TB. Its early bactericidal activity is approved in the EU and Japan for the treatment of MDR-TB, when administered in combination with an optimized background regimen [94]. Delamanid is currently being tested in a Phase III clinical trial, as an addition to an optimized background regimen (OBR) for the treatment of MDR-TB. The trial is comparison six months of treatment with delamanid plus the OBR with a placebo plus OBR. Delamanid was well tolerated, QT prolongation was more frequently reported in patients receiving delamanid against those receiving placebo. Based on the available evidence, WHO recommends the use if delamanid at the dose of 100 mg twice daily for 6 months, added to OBR in adults, when pharmacovigilance is in place and informed consent ensured. As a result delamanid has favorable safety profile compared to existing second-line drugs [4, 93, 94]. Delamanid primarily inhibit synthesis of methoxy-mycolic and keto-mycolic acid, which are components of the mycobacterial cell wall; unlike INH, the drug does not inhibit alpha- mycolic acid but it has no action against gram-negative or gram-positive bacteria and this is

32

Introduction clinically beneficial as its restriction prevent the generation of resistance. Delamanid is a prodrug that requires metabolic activation for anti-TB activity to be exerted. Reactive intermediates in the metabolic pathway of the bicyclic nitroimidazoles may provide additional mechanisms of action, including interruption of cellular respiration. Activation of delamanid is mediated via the mycobacterial F420 coenzyme system [94, 95]. The most common aspect consequences are nausea, vomiting and dizziness. These might also affect as many as a 3rd of all sufferers. There is likewise a critical aspect impact referred to as QT prolongation. QT prolongation is an alteration of the electrical pastime of the heart. It can cause a life-threatening abnormality of the heart rhythm. Anxiety, pins & needles, and shaking are other vital facet effects [94].

1.10.6. Pretomanid (PA-824) Pretomanid (PA-824) is an investigational anti-TB drug. PA-824 is a bicyclic nitroimidazole- identical molecule with a very complex mechanism of action. PA-824 was developed at Pathogenesis Corporation and later transferred to the TB Alliance, where it is currently undergoing Phase III clinical trials. New regimens based on nitroimidazole novel agents are required in order to shorten or abridge the treatment of both drug-susceptible and drug-resistant forms of TB [4]. Now it is in phase-II trials and has shown significant early bactericidal activity alone and in combination with the newly approved agent bedaquiline or with pyrazinamide and in phase-III trials with pyrazinamide and moxifloxacin. PA-824 also shows promise for MDR TB patients who are sensitive to the drugs in the regimen, reducing treatment from 2 years to 4 months and costing just a fraction of the current MDR TB treatment. Additionally, PaMZ regimen can be administered in a fixed dose for all patients, and will therefore be simpler for health systems to deliver and patients to use [93]. PA-824 is also a prodrug like INH and requires the activation of aromatic nitro group by F420- dependent mechanism. It inhibits both protein and lipid synthesis but does not affect nucleic acid synthesis. It undergoes nitro reduction producing highly reactive intermediates which then reacts with multiple targets inside the bacterial cell. PA-824 has been observed to kill bacteria in two distinct mechanisms: a) by interfering with the synthesis of ketomycolate which is an essential component of the mycobacterial cell wall, and b) by acting as a nitric oxide donor and causing respiratory poising [95]. Pretomanid recently was shown to be safe, well tolerated, and

33

Introduction efficacious at doses of 100–200 mg daily in a dose-ranging study among drug-sensitive, sputum smear positive, adult pulmonary TB patients.

1.10.7. Rifapentine Rifapentine (also known as cyclopentyl rifampicin and Priftin) is a medication recommended by the WHO as a first-line treatment for TB and approved by the U.S. FDA as a treatment for pulmonary TB in 1998 [4, 77]. Like other RMP, rifapentine inhibits bacterial DNA-dependent RNA polymerase. It kills TB bacteria by inhibiting bacterial RNA polymerase, which is the enzyme responsible for transcribing DNA into RNA (RNA is subsequently used to make bacterial proteins). By disrupting the bacterial RNA polymerase only, rifapentine eliminates TB bacteria while leaving human RNA polymerase unaffected. Rifapentine has a long half-life in serum and is therefore administered less frequently. Its half-life is 5 times that of RMP [71, 77]. Rifapentine should be given with isoniazid during the continuation phase of the treatment of drug-susceptible pulmonary TB after an intensive phase that consists of at least rifampin (or rifabutin), INH, PZA, and ethambutol administered for two months [77]. The 6-month regimen that included weekly administration of high-dose rifapentine and moxifloxacin was as effective as the control regimen. Rifapentine is presently in phase-III trials with moxifloxacin for the treatment of drug-susceptible TB [4]. Rifapentine was shown to be safe and effective in HIV negative patients, which was the basis for the current Centers for Disease Control and Prevention recommendation for using rifapentine and isoniazid in selected patients during the continuation phase of therapy. Common side effects consist of low neutrophil counts inside the blood, multiplied liver enzymes, and white blood cells inside the urine. Serious facet effects may additionally consist of liver issues or Clostridium difficile related diarrhea. It is uncertain if use all through being pregnant is safe. Rifapentine is inside the rifamycin circle of relatives of drugs and works with the aid of blockading DNA-structured RNA polymerase [77].

1.10.8. Moxifloxacin Moxifloxacin is a fourth-generation synthetic FQ antibacterial. Moxifloxacin is an important option for the treatment of MDR TB. A retrospective analysis showed that levofloxacin and moxifloxacin showed equivalent efficacy for treating MDR-TB. Moxifloxacin can be replaced with gatifloxacin, the FQ used in the earlier studies of shorter MDR TB regimens. Moxifloxacin

34

Introduction is a key component of the experimental arms of a trial that is intended both to shorten treatment from 6 to 4 months for patients with susceptible disease and to provide an all-oral 6-month treatment for patients with MDR-TB. This provides a significant advantage over the standard-of- care MDR-TB regimen that includes an injectable antibiotic for at least 3 months [71]. Rapid evaluation of moxifloxacin in TB study to determine whether the replacement of either INH or EMB with moxifloxacin would provide effective TB treatment in 4 months, as compared with the standard 6 months regimen. In phase-III clinical trials used with moxifloxacin with refapentine for the drug-susceptible TB. Pretomanid + Moxifloxacin + Pyrazinamide was tested in the Phase-IIa NC-001 trial, in which it killed TB bacteria faster when compared with the current TB regimen, as well as other experimental regimens over the first two weeks of treatment. It was subsequently tested in NC-002, in which it met its primary endpoint after eight weeks treatment. PaMZ has the potential to cure both TB and some forms of MDR TB in 4 months, drastically improving treatment [4, 71]. Moxifloxacin causes higher QT prolongation than LEV, knowing LEV has comparable efficacy is very useful in designing regimens with other QT-prolonging drugs, such as bedaquiline and delamanid. Moxifloxacin inhibiting DNA gyrase, a type II topoisomerase, and topoisomerase IV, enzymes necessary to separate bacterial DNA, thereby inhibiting cell replication [77, 90]. Rare but severe unfavorable outcomes that could occur due to moxifloxacin remedy consist of irreversible peripheral neuropathy, spontaneous tendon rupture and tendonitis, hepatitis, psychiatric consequences (hallucinations, melancholy), torsades de pointes, Stevens- Johnson syndrome and Clostridium difficile-related ailment and photosensitivity/phototoxicity reactions [71].

1.10.9. Bedaquiline Bedaquiline (trade name Sirturo, code names TMC207 and R207910) is new class of diarylquinoline drug. Although the drug is active against many different bacteria, it has been registered specifically for the treatment of MDR-TB. It was discovered by a team led by Koen Andries at Janssen Pharmaceutica. Bedaquiline was approved on 28th December 2012 by the US FDA, and a drug of novel class to be approved over 40 years for treatment of MDR TB as part of combination therapy for adults with pulmonary TB [95]. In February 2016 it was announced that bedaquiline is to be made available in India. The drug will be available as part of second line

35

Introduction treatment for patients suffering from MDR-TB and XDR-TB [95]. Phase-III trial to investigate the safety and efficacy of bedaquiline when used in combination shortened MDR TB regimens of 9 and 6 months duration. By the end of 2014, 43 countries reported having used bedaquiline to treat patients as part of efforts to expand access to treatment for MDR TB [4]. Bedaquiline have to now not be co-administered with different tablets that are strong inducers or inhibitors of CYP3A4, the hepatic enzyme responsible for oxidative metabolism of the drug. Co-management with rifampin, a strong CYP3A4 inducer, results in a 52% decrease within the AUC of the drug. This reduces the exposure of the body to the drug and reduces the antibacterial impact. Co- management with ketoconazole, a robust CYP3A4 inhibitor, outcomes in a 22% increase in the AUC, and potentially an increase in the price of adverse results experienced [95]. Bedaquiline specifically inhibits the mycobacterium ATP synthetase as compared to mitochondrial ATP synthetase. ATP synthase is a critical enzyme in the ATP synthesis of MTB. Binding of bedaquiline to the oligomeric and proteolipic subunit-c of mycobacterial ATP synthase leads to inhibition of ATP synthesis, which subsequently results in bacterial death [95]. Bedaquiline can affect the heart’s electrical activity causing prolongation of the QT interval, which could lead to an abnormal and potentially fatal heart rhythm. Accordingly, the FDA has approved bedaquiline as part of combination therapy to treat adults with MDR pulmonary TB when other alternatives are not available. The FDA also granted fast-track designation, priority review and orphan-product designation to bedaquiline [95]. Bedaquiline has been stated to disturb the characteristic of the heart and liver specifically. Interactions with different drugs, in particular lopinavir and efavirenz (used in the remedy of HIV), ketoconazole, as well as other capsules used inside the remedy of MDR-TB (eg moxifloxacin, clofazimine) may be expected. More deaths have been pronounced among sufferers taking bedaquiline for the duration of the research completed to research the drug, even though it is not clean whether or not this became due to the drug. For all these motives, it is critical that patients are closely monitored and that unfavourable activities are systematically stated (“energetic pharmacovigilance”), specially the ones which can be critical and existence-threatening. Clinical tracking of signs, performance of special assessments at appropriate periods, and engagement of the patient to document untoward effects of remedy to the appropriate pharmacovigilance organization are the cornerstones for the powerful control of side results in a timely style [95].

36

Introduction

Figure 1.13: Structure of anti-TB agents under phase-III clinical trials.

1.11. Limitation of current drugs A major adverse reaction of antituberculosis drugs, which results in discontinuation of that drug, has several implications. There may be considerable morbidity, even mortality, particularly with drug-induced hepatitis. More severe than side effects, life threatening, change in dosage of drug, discontinuation of drug, additional treatment or hospitalizations these are toxicities [96]. The length of therapy makes patient compliance difficult, most of the TB drugs available now a days are inactive against persist bacilli expect for RIF and PZA. However, there are still persist bacterial populations that are not killed by any available TB drugs. Based on these, there is a need to design drugs that are more than active against slowly growing or non-growing persistent bacilli [97].

1.12. Molecular Modification Molecular modification is chemical alteration of a known and previously characterized lead compound for the purpose of enhancing its usefulness as a drug. This may perhaps enhance its

37

Introduction specificity for a particular target site, increase its potency, improve its rate and extent of absorption, and modify to advantage its time course in the body, reduce its toxicity, change its physical or chemical properties to provide desired features. In molecular modification three approaches have been generally been explored: prodrug approach, bioisosterism and molecular hybridization [96].

1.12.1. Prodrug approach Prodrug definition was introduced by Albert in 1958 which define prodrug as “any compound that undergoes biotransformation prior to exhibiting its pharmacological effects” [97]. Then Haper in 1959 proposed the term as latentiation. Drug latentiation is the chemical modification of a biologically active compound to form a new compound, which in vivo will liberate the parent compound. A prodrug is a medication or compound that, after administration, is metabolized (i.e., converted within the body) into a pharmacologically active drug. Prodrugs are bioreversible derivatives of drug molecules which undergo chemical transformation or enzymatic conversion in vivo to release the active parent drug which shows desired pharmacologic effect. In both drug discovery and development, prodrugs have become an established tool for enhancing biopharmaceutical, physiochemical, or pharmacokinetic properties of therapeutic agents. The use of a prodrug is widely encouraged to optimize absorption, distribution, metabolism, and excretion (ADME) processes [97]. In general the prodrugs could be classified into two main classes: carrier prodrugs and bioprecursors. Carrier prodrugs are designed using labile linkage between a carrier group (ester or amide) and an active compound. These are classified as; Bipartate: if the drug is directly attached to the carrier group

Tripartate: if there is a linker group between the drug and the carrier moiety

Mutual prodrug: here two drugs are linked together and synergistic to each other

These types of prodrugs have greatly modified lipophilicity due to the attached carrier group. The active drug is released by hydrolytic cleavage either chemically or enzymatically.

38

Introduction

Advantages of carrier prodrugs are increasing absorption and chemical stability, injection site pain relief, elimination of unpleasant taste, decreasing metabolic inactivation etc. Bioprecursor prodrugs rely on oxidative or reductive activation reactions unlike the hydrolytic activation of carrier-linked prodrugs. They metabolize into a new compound that may itself be active or further metabolized to an active metabolite. Bioprecursor is metabolized by molecular modification into either an active form or into an intermediate that will be farther metabolized. Bioprecursor does contain any carrier group. Several examples of drugs available in the market used this strategy such as sulindac, acyclovir, losartan among others [98]. The prodrug is usually inactive or less active than parental drug.

1.12.2. Bioisosterism The notion of isosterism was introduced in 1919 by Langmuir. Compared the physical properties, chemical behavior and reactivity of various molecules possessing atoms or groups with the samenumber of valence electrons, i.e isoelectronic. Examples of various atoms and - molecules; C=O and N=N; CO2 and NO2; N=N=N and N=C=O [99]. Bioisosteres are substituents or groups with similar physical or chemical properties which produce broadly similar biological properties to a chemical compound. In biologically active molecule the replacement of an atom or group of atoms by another one presenting the same physicochemical properties [99]. In 1970, Burger classified and subdivided bioisosteres into two broad categories according to the degree of electronic and steric factors i.e., classic and non-classic. The classical bioisosteres are subdivided into: a) monovalent atoms or groups; b) divalent atoms or groups; c) trivalent atoms or groups; tetravalent atoms and e) ring equivalents (Table 1.3)

Table 1.3: Classic bioisosteres, classifications, their atoms and groups.

Monovalent Divalent Trivalent Tetravalent Ring equivalents Sulfadiazine in -OH, -NH2, -CH3, -OR -CH2- =CH- =C= Pyrimidine ring Sulfamethoxazole -F, -Cl, -Br, -I, -SH, -PH2 -O- =N- =Si= in

39

Introduction

Isoxazole ring Piroxicam + -SiCH3, -SR -S- =P- =N = Benzene ring Tenoxicam in + -Se- =As- =As = Thiophene ring

-Te- =Sb- =Sb+= =P+=

The non-classical bioisosteres do not obey the steric and electronic definition of classical isosteres. Further they do not have the same number of atoms of the substituent or moiety replaced. But retain the focus on providing similar sterics and electronic profile to the original functional group. Non-classical bioisosteres are much more dependent on the specific binding needs of the ligand in question and may substitute a linear functional group for a cyclic moiety, an alkyl group for a complex heteroatom moiety, or other changes that go far beyond a simple atom-for-atom switch. Non-classical bioisosteres we could cite: functional groups, noncyclic or cyclic and retroisosterism [100].

Table 1.4: Non-classical bioisosteres classifications of their atoms and groups.

-CO- -COOH -SO2NH2 -H -CONH- -COOR -CONH2 - -CO -SO H -PO(OH)NH -F -NHCO -CSNH 2 3 2 - ROCO- 2 - -SO -tetrazole -OH 2- catechol

-SO2NR- -SO2NHR -CH2OH - -CON- -SO2NH2 benzimi dazole -3-hydroxy -CH(CN) -NHCONH C H S - isoxazole 2 4 4

-2-hydroxy R-S-R -NHCSNH -C H N chromones 2 5 4

40

Introduction

(R-O-R’) =N- -C6H5 - -RN(CN)- -C(CN)=R’ NHC(CHNO2)NH2 -C4H4NH -NHC(CHCN)NH2 -halide

-CF3 -CN

-N(CN)2

-C(CN)3 Bioisosterism represents an approach used by the medicinal chemist for the rational modification of lead compounds into safer and more clinically effective agents. It has significant value in drug design and lead optimization process as it may enhance the desired biological or physical properties of a compound, reduce toxicity and also alter the metabolism of the lead. Bioisosteric replacement is not simple replacement with another isostere but they are firstly analyzed by structural, solubility and electronic parameters to obtain molecules having similar biological activity. Bioisosteresim applications applied in aminopyrine to prpopylphenazone; cholesterol to diazacholesterol; tolrestat to oxotolrestat; sulfadiazine to sulfamethoxazole; piroxicam to tenoxicam and nimesulide to flosulide [99, 100]. Bioisosterism strategy has been used to discovery new compounds to treat TB. One example is the class of FQs. The FQs derivatives gatifloxacin and moxifloxacin were derivatized scaffolds from the parent nalidixic acid using the bioisosterism as molecular modification. The uses of FQs occur mainly in patients with MDR TB. The most active quinolones for the treatment of TB are: ciprofloxacin, sparfloxacin, ofloxacin, moxifloxacin and LEV. Studies comparing the bactericidal activity of various FQs against MTB in the latent and exponential growth phases demonstrated that most promising drugs are moxifloxacin and LEV [100].

41

Introduction

Figure 1.14: FQ drugs were obtained using bioisosteric replacement.

Linezolid is oxazolidinone drug belonging to antibacterial agents. The drug has been evaluated in the treatment of MDR TB showing interesting results [100]. Based on this interesting result, some oxazolidinone biosisoteres have been developed. Sutezolid is an analogue of linezolid in clinical trial phase-II to be used in TB treatment. Other linezolid derivatives such as radezolid and torelozid are obtained by isosteric replacement [100].

The metronidazole scaffold was used to design PA-824 and OPC-67683 using bioisosterism and molecular hybridization, the second line drug ethambutol was used as scaffold to develop the compound SQ109 using bioisosterism and molecular hybridization [100].

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Figure 1.15: Chemical structure of linezolid bioisosteric derivatives

1.12.3. Molecular hybridization (MH) Molecular hybridization is a structural modification strategy useful in the design of new optimized ligands and prototypes with new molecular architectures composed of two or more known bioactive derivatives, through the adequate fusion of these sub-unities. The molecular hybridization strategy is particularly interesting for the development of new prototypes for the treatment of physicopathologies where treatment is restricted to few commercial drugs or in cases when bioactive compounds are discovered but present high toxicity or pharmacokinetic and pharmacodynamic restrictions [101]. Structural requirements, ligand-protein interaction mode, site ligandreceptor interactions and quantitative structure-activity relationships, which tends to become faster and more efficient the development of new drugs [102]. On the other hand, if the degree of template-hybrid homology is either low or inexistent, the discovery of new lead-compounds should be made by massive screening of the generated chemical library. The advantage of using MH is to activate different targets by a single molecule, thereby increasing therapeutic efficacy as well as to improve the bioavailability profile. MH strategy shows the drug A interacts only with the receptor A. The drug B interacts only with the receptor B. It is prohibitive the interaction between drug A and receptor B (and vice versa) is

43

Introduction prohibited but is possible to design compounds that can interact with both receptors contributing synergically for a desired effect [103].

Figure 1.16: Different hybrid compounds obtained by molecular hybridization.

The drug design of hybrid compound must judge three different conditions: a) the desired subunits are linked by a spacer agent; b) both subunits are linked without spacer agent and they are fused; c) the desired activities are merged in a new structure. These different conditions are in order to design a new drug [100, 102]. MH strategy has been used in TB drug discovery to increase the efficacy and reduce drug resistance. Imrarovský and co-workers combined through MH the scaffold of three anti-TB drugs: INH, PZA and ciprofloxacin. The novel compounds showed great activity against MTB [102].

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Figure 1.17: Molecular hybridization between FQ and INH.

Another example, Torres and co-workers reported the antitubercular activity from the new quinoxaline-1,4-di-N-oxide derivates obtained by molecular hybridization with the first line drug isoniazid [103].

Figure 1.18: Quinoxaline-1,4-di-N-oxide molecular hybridization with INH.

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[96] (a) M. Abdo Arbex, M. de Castro Lima Varella, H. Ribeiro de Siqueira, F. Augusto Fiúza de Mello, J Bras Pneumol. 2010, 36, 626; (b) D. Yee, C. Valiquette, M. Pelletier, I. Parisien, I. Rocher, D. Menzies, Am J Respir Crit Care Med, 2013, 167, 1472 [97] (a) E. Martins de Queiroz, M. Cecilia De-La-Torre-Ugarte-Guanilo, K. Ribeiro Ferreira, Maria Rita Bertolozzi, Rev. Latino-Am. Enfermagem, 2012 20, 369; (b) M. Pai, M. A. Behr, D. Dowdy, Keertan Dheda, M. Divangahi, C. C. Boehme, A. Ginsberg, S. Swaminathan, M. Spigelman, H. Getahun, D. Menzies, M. Raviglione, Nature Reviews Disease Primers, 2016, 2, 1607. [98] (a) J. L. Dossantos, L. A. Dutra de T. R. F. Melo, C. M. Dr. Chin, Pere-Joan Cardona (Ed.) (2012). InTech, (b) R. E. Notari, J. Pharm. Sci.,1973, 62, 865. [99] A. Albert, Nature., 1958, 182, 421. [100] (a) S. Supriya, S. Sheetal, S. Manik, IJPCBS., 2015, 5, 232; (b) A. T. A. Silva, L. F. Castro, R. V. C. Guido, M. C. Chung, Min. Rev. Med. Chem., 2005, 5, 893; (c) A. A. Sinkula, Annual Reports in Medicinal Chemistry., 1975, 10, 306. [101] (a) A. Burger, Pro. Drug Res., 1991, 37, 287; (b) L. Priyanka Gaikwad, S. Priyanka Gandhi, M. Deepali Jagdale, J. Vilasrao Kadam, Am. J. PharmTech Res., 2012, 2, 4. [102] (a) L. M. Lima, E. J. Barreiro, Curr. Med. Chem., 2005, 12, 23; (b) R. Cremades, J. C. Rodríguez, E. García-Pachón, A. Galiana, M. Ruiz-García, P. López, G. Royo, J. Antimicrob. Chemother., 2011, 66, 2281; (c) K. L. Leach, S. J. Brickner, M. C. P. F. Miller, Annals of the New York Academy Sciences., 2011, 1222, 49. [103] (a) C. Viegas-Junior, A. Danuello, V. da Silva Bolzani, E. J. Barreiro, C. A. Fraga, Curr. Med. Chem., 2007, 14, 1829; (b) L. A. Dutra, T. R. Ferreira de Melo, C. Man Chin, J. L. dos Santos, Int. Res. J. Pharm. Pharmacol., 2012, 2, 1. [104] C. Lazar, A. Kluczyk, T. Kiyota, Y. Konishi, J. Med. Chem., 2004, 47, 6973. [105] (a) A. Imramovský, P. Slovenko, J. Vins, M. Kocevar, J. Jampılek, Z. Reckova, J. Kaustova, Bioorg. Med. Chem., 2007, 15, 2551; (b) S. Ansizu, E. Moreno, B. Solano, R. Villar, A. Buerguete, E. Torres, S. Pérez-Silanes, L. Aladana, A. Monge, Bioorg. Med. Chem.,2010, 18, 2713; (c) E. Torres, E. Moreno, S. Ancizu, C. Barea, S. Galiano, I. Aldana, A. Monge, S. Pérez-Silanes, Bioorg. Med Chem. Lett., 2011, 21, 3699.

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Objectives

Chapter II

Objectives

Objectives

Objectives Chapter 2

The global pandemic of drug sensitive tuberculosis as well as the increasing threat from various drug resistant forms of TB drives the quest for newer, safer, more effective TB treatment options. A thorough review of the various literatures available enlightened the importance of pantothenate synthetase and InhA biosynthetic pathways in the lifecycle of Mycobacterium tuberculosis. The present study thus focused on utilizing the pharmaceutically underexploited pantothenate synthetase and InhA as potential target platforms for exploring newer anti tubercular agents that lack cross-resistance mediated by mutations in the bacterial targets.

The main objectives of the proposed work are:

1. To design novel anti-tubercular agents based on reported anti-tubercular leads by molecular hybridisation strategy and rational drug derivatization based on medicinal chemistry approach.

2. To synthesize the designed molecules by conventional methods, an environmental benign technique like, click chemistry method.

3. To undertake in vitro antimycobacterial screening of the synthesized compounds against Mycobacterium tuberculosis, pantothenate synthetase enzyme assay and InhA studies.

4. To perform the in vitro cytotoxicity studies of the synthesized compounds.

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Chapter III

Identification and development of 1-((1-(substituted)-1H-1,2,3-triazol-4- pyrazolo[4,3-c]pyridine-5(4H)-carboxamides as Mycobacterium tuberculosis Pantothenate synthetase inhibitors

Chapter 3

Chapter 3

Identification and development of 1-((1-(substituted)-1H-1,2,3-triazol-4- pyrazolo[4,3-c]pyridine-5(4H)-carboxamides as Mycobacterium tuberculosis Pantothenate synthetase inhibitors 3.1. Introduction Pyrazole is a five membered and two-nitrogen containing heterocyclic ring. Some of the pyrazole containing drugs like celecoxib, phenazone, fezolamine, apixaban, metamizole, rimonabant, phenylbutazone, lonazolac and many more are already in the market (Figure 3.1) [1]. Pyrazole or systems containing pyrazole fused with a six membered heterocycle have been extensively studied these derivatives are known to contain broad spectrum of pharmacological properties such as antifungal [2], antidiabetic [3], antitumor [4], antibacterial [5]. Pyrazoles play an essential role in biologically active compounds and therefore signify an interesting template for medicinal chemistry [6]. Many compounds have been synthesized as target structures by many researchers and were evaluated for their biological activities.

Figure 3.1: Pyrazole based drugs.

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Mamolo et al., reported 5-aryl-1-isonicotinoyl-3-(pyridin-2-yl)-4,5-dihydro-1H-pyrazole derivatives which exhibited an interesting in vitro antimycobacterial activity against MTB, with minimum inhibitory concentration (MIC) values ranging from 8 to 16 µg/mL [7]. Ravindra et al., reported 3-(4-chlorophenyl)-4-substituted pyrazole derivatives with MIC values ranging from 0.35 to 3.15 µg/mL against MTB H37Rv [8]. Chovatia et al., reported 1-acetyl-3,5-diphenyl-4,5- dihydro-(1H)-pyrazole derivatives which were screened against MTB H37Rv [9]. Series of N- phenyl-3-(4-fluorophenyl)-4-substituted pyrazole derivatives exhibited significant antimycobacterial activity with IC50 values ranging from 0.47 to 118.0 µM against MTB H37Rv [10]. Palanisamy et al., reported analogues of N,1-diphenyl-4,5-dihydro-1H- [1]benzothiepino[5,4-c]pyrazole-3-carboxamide and N,1-diphenyl-4,5-dihydro-1H- [1]benzothiepino[5,4-c]pyrazole-3-carboxamide-6,6-dioxides which were screened against MTB H37Rv [11] (Figure 3.2).

Figure 3.2: Some of the pyrazole based anti-tubercular agents.

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Triazoles have several medicinal applications [12]. It is quite obvious that, the favorable properties of 1,2,3-triazole ring like moderate dipole character, hydrogen bonding capability, rigidity and stability under in vivo conditions are responsible for their enhanced biological activities [13, 14]. For instance, a variety of 1H-1,2,3-triazole compounds have been known to exhibit antitubercular activity (Figure 3.3) [15-19].

Figure 3.3: Some of the triazole based anti-tubercular agents.

The biosynthetic pathway of pantothenate involves four steps catalyzed by panB, panC, panD, and panE genes [20]. The last step of pantothenate biosynthesis viz., the ATP-dependent condensation of D-pantoate and β-alanine to form pantothenate is catalyzed by PanC. Pantothenate is essential for several processes such as cell signaling, fatty acid metabolism, and synthesis of non-ribosomal peptides and polyketides [21]. The biosynthesis of pantothenate is essential for the growth of MTB. The pantothenate biosynthesis pathway is a latent drug target and hence is important for determined growth and virulence of MTB and PS is very good target for developing new therapeutics to treat TB [22, 23].

Till date many MTB PS inhibitors are reported (Figure 3.4), which include 5-tert-butyl-N- pyrazol-4-yl-4,5,6,7-tetrahydrobenzo[d]isoxazole-3-carboxamide derivatives [21], 3-phenyl- 4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine derivatives [24], 2,6-disubstituted 4,5,6,7-

57

Chapter 3 tetrahdrothieno[2,3-c]pyridine-3-carboxamide derivatives [25], nafronyl oxalate [26], N’-(1- naphthoyl)-2-methylimidazo[1,2-a]pyridine-3-carbohydrazide [27], and imidazo[2,1-b]thiazole and benzo[d]imidazo[2,1-b]thiazole derivatives [28].

Figure 3.4. Structures of literature reported MTB PS inhibitors.

3.1.1. Design and chemistry The molecular hybridization approach involves rational design of new ligands or the recognition of pharmacophoric sub-units in the molecular structure of two or more known bioactive derivatives which, through the adequate combination of these subunits, lead to the design of new hybrid architectures that maintain preselected characteristics of the original templates. In this work, we designed novel MTB inhibitors by hybridizing reported MTB PS inhibitor 1-benzoyl- N-(4-nitrophenyl)-3-phenyl-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-carboxamide [25] and 3-(4-((1-(4-bromo-3-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)methyl)piperazin-1- yl)benzo[d]isoxazole [29], anticipating a new lead in the development of novel MTB PS inhibitors with potential MTB MIC (Figure 3.5). 3-(4-((1-(4-bromo-3-(trifluoromethyl)phenyl)-

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1H-1,2,3-triazol-4-yl)methyl)piperazin-1-yl)benzo[d]isoxazole is our most active compound from our previous work and when screened for MTB PS it exhibited panC IC50 58.4 µM.

Figure 3.5: Design strategy of the title compounds.

3.2. Results and Discussion The designed molecules were synthesized in six steps (Scheme 3.1). Initially we prepared 1,3- dicarbonyl intermediate (2) using 1-Boc-4-piperidone (1), morpholine, p-toluenesulfonic acid (catalytic) and benzoyl chloride (stork enamine reaction conditions). Treatment of 2 with hydrazine hydrate yielded pyrazole ring (3) [30]. Compound 3 on reacting with propargyl bromide in the presence of Cs2CO3 formed N-alkyl product (4). Compound 4 was then deprotected using trifluoroacetic acid to yield compound (5). With weak base TEA, more nucleophilic amine of aliphatic ring reacted with phenylisocyanate yielding urea derivative (6). The free acetylene group was converted to various 1H-1,2,3-triazoles using different aromatic azides via click chemistry method [19]. The purity of compounds synthesized was checked by LC-MS and elemental analyses. Structures of the compounds were confirmed by spectral data. In 1H NMR and 13C NMR, the signals of the respective protons and carbons were verified on the basis of their chemical shifts, multiplicities, and coupling constants. The results of elemental analysis were within ± 0.05 of the theoretical values.

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Scheme 3.1: Synthetic protocol of titled compounds. Reagents and conditions: (i) (a) Morpholine (1.0 eq), PTSA (0.01 eq), toluene, 100 °C, 16 h;

(b) Benzoyl chloride (1.1 eq), TEA (2.0 eq), DCM, 0 °C - rt, 4 h. (ii) N2H4 (1.0 eq), EtOH, 0 °C - rt, 4 h (iii) Propargyl bromide (80% in toluene) (1.2 eq), Cs2CO3 (1.5 eq), DMF, rt, 16 h. (iv)

CF3COOH, DCM, rt, 16 h. (v) Phenyl isocyanate (1.3 eq), TEA (3.0 eq), DMF, rt, 4 h. (vi)

Substituted aromatic azides, CuSO4.5H2O (10 mol %), Sodium ascorbate (10 mol %), t H2O: BuOH (1:2), rt, 4 h.

3.2.1. In-vitro MTB screening All the synthesized compounds were tested for their capacity to inhibit the growth of MTB. In assay three different M. tuberculosis strains were used. One of them was reference strain M. tuberculosis H37Rv ATTC 25618 and the others were ‘wild’ strains isolated from tuberculosis patients [29, 31]. MTB strain spec. 210 was resistant to p-aminosalicylic acid (PAS), INH, ETB and RMP and another (Spec. 192) fully sensitive to the administrated tuberculostatics [31]. In this study three different strains were used for screening as we wanted to know the kind of activity synthesized compounds showed against the reference strain as well as against the strains isolated from TB patients. In this study the influence of the compound on the growth of mycobacteria at a certain concentration: 3.1, 6.2, 12.5, 25, 50 and 100 μg/mL were evaluated

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INH was used as reference drug. The in vitro antimycobacterial results of title compounds are arranged in Table 3.1 as MIC (µM) and the activity ranged from 24.72 - >200 µM.

Table 3.1: Result of Antimycobacterial screening of title compounds

MIC (µM) MIC (µM) MIC (µM) Entry R against against MTB against MTB MTB H37Rv Spec. 192 Spec. 210

7a Phenyl 105.14 105.14 105.14 7b 4-Methylphenyl 25.53 25.53 51.06 7c 4-Ethylphenyl 49.64 49.64 49.64 7d 4-Methoxyphenyl 24.72 24.72 24.72 7e 4-Fluorophenyl >200 >200 >200 7f 4-Chlorophenyl 196.08 196.08 196.08 7g 4-Bromophenyl 90.18 90.18 90.18 7h 4-Iodophenyl 166.26 166.26 166.26 7i 4-Nitrophenyl 192.10 192.10 192.10 7j 4-Trifluoromethylphenyl 91.98 91.98 91.98 7k 2-Chlorophenyl 196.08 196.08 196.08 7l 2-Fluorophenyl >200 >200 >200 7m 2-Bromophenyl 180.36 180.36 180.36 7n 2-Iodophenyl 166.26 166.26 166.26 7o 2-Nitrophenyl 192.10 192.10 192.10 7p 3-Chlorophenyl 98.04 98.04 98.04 7q 3-Methoxyphenyl 98.89 98.89 98.89 7r 2,4-dichlorophenyl 183.67 183.67 183.67 7s 3,4-dichlorophenyl 183.67 183.67 183.67 7t 3,5-dichlorophenyl 91.83 91.83 91.83 7u 3-Chloro-4-Fluorophenyl 189.40 189.40 189.40 7v 3,4-difluorophenyl 195.49 195.49 195.49 7w 3,4-dimethoxyphenyl 186.70 186.70 186.70

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4-bromo-3 7x 160.65 160.65 160.65 trifluoromethylphenyl 7y 3,4-dimethylphenyl 198.57 198.57 198.57 7z benzo[d][1,3]dioxole 192.47 192.47 192.47 INH - ≤22.60 ≤22.60 ≤91.14

Among the twenty six compounds screened, eight compounds (7b, 7c, 7d, 7g, 7j, 7p, 7q and 7t) showed activity against MTB with MIC ˂100 µM. Three compounds (7b, 7c, and 7d) inhibited MTB with MIC <50 µM. Compound 7d, (1-((1-(4-methoxyphenyl)-1H-1,2,3-triazol-4- yl)methyl)-N,3-diphenyl-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)carboxamide) was found to be the most active compound with in vitro MIC 24.72 µM. In addition, compounds 7b, 7c and 7d with MIC <50 µM were further subjected to toxicity studies on normal cell line to analyze the selectivity profile. Amongst synthesized derivatives, electron donating group containing substituent play major impact in exhibiting anti-TB activity. Structural activity relationship studies are explained based on activity of compound 7a. Structural changes at 4th position alter the activity. Compound 7a was inhibiting 99% growth of MTB H37Rv strain at 105.14 µM. In this series, introduction of electron donating groups at 4th position on phenyl ring increased the activity. Introduction of electron donating ethyl group (7c) increased the activity by two fold with MIC 49.64 µM. Presence of electron donating methyl group (7b) increased the activity by four folds with MIC 25.53 µM. With introduction of methoxy group (7d) activity increased by four folds (MIC 24.72 µM), with same methoxy group at meta position on phenyl ring (7q, MIC 98.89 µM) activity remained unaltered compared to compound 7a. Presence of electron withdrawing groups viz., F, Cl, Br and I resulted in decrease in activity compared to compounds 7b, 7c, and 7d. Even, presence of electron withdrawing groups at ortho or meta position decreased the activity. Introduction of electron donating methoxy group at meta and para position (7w, MIC 186.70 µM) yielded decrease in activity by two folds compared to compound 7a. In conclusion, presence of electron donating methoxy group at para position on phenyl ring enhanced the activity. Amongst the synthesized derivatives, 7d emerged to be the most active compound.

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3.2.2. Pantothenate synthetase enzyme inhibition studies PS enzyme inhibition studies are carried on synthesized compounds by estimating the amount of NAD+ produced [20, 28]. The NAD+ produced can be examined spectrophotometrically at 340 nm. In the initial screening at 50 μM, all compounds exhibited more than 50% inhibition against

MTB PS and their IC50’s were further determined. Most of the compounds showed good IC50 ranging from 0.91±0.32 to 8.97±0.05 µM (Table 3.2). Seven compounds (7b, 7d, 7h, 7p, 7r, 7s and 7v) inhibited MTB PS with IC50 ˂2.00 µM. Compounds 7d and 7s emerged as the most active compounds with IC50 1.01±0.32 and 0.91±0.32 µM respectively.

Table 3.2: Docking scores and MTB PS assay Compound In-silico In-vitro XP MTB PanC Entry Glide energy GScore IC50 µM Co-LIGAND -8.32 -78.66 -- 7a -7.19 -71.33 2.13±0.12 7b -5.69 -58.61 1.54±0.22 7c -6.44 -64.89 2.56±0.04 7d -8.19 -66.25 1.01±0.32 7e -6.61 -67.18 3.41±0.08 7f -3.25 -69.57 6.36±0.12 7g -5.24 -65.40 2.46±0.07 7h -7.61 -73.33 1.04±0.55 7i -3.80 -62.50 8.97±0.05 7j -4.70 -58.79 5.04±0.54 7k -4.62 -65.89 2.78±0.07 7l -4.46 -61.60 5.93±0.64 7m -6.31 -71.76 3.54±0.55 7n -5.24 -60.42 3.92±0.19 7o -6.33 -72.18 4.94±0.03 7p -7.69 -70.79 1.14±0.19 7q -4.25 -65.23 7.78±0.56

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7r -6.07 -67.82 1.29±0.14 7s -7.83 -69.26 0.91±0.32 7t -5.99 -63.35 4.76±0.02 7u -5.37 -63.08 6.43±0.12 7v -7.37 -64.55 1.56±0.11 7w -4.96 -64.16 8.21±0.03 7x -4.44 -57.95 6.54±0.21 7y -6.60 -56.87 6.91±0.07 7z -4.84 -73.86 8.39±0.14

3.2.3. Docking study All the final compounds were docked into the crystal structure of MTB PS protein (PDB ID: 3IUB) to know the exact binding pattern with the receptor. Validation of docking protocol revealed that, the value of RMSD obtained between experimental binding mode of co- crystallized ligand (as in X-ray) and its re-docked pose (Figure 3.6) was found to be 0.76, which suggested that, docking procedure could be relied on for further docking studies.

Figure 3.6: Superimposed view of co-crystallized ligand (green) with its X-ray pose (red) in 3IUB.

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Further, in the docking studies, molecules exhibited good binding energy in the range of -3.25 to -8.19 kcal/mol and exhibited good fitness with the MTB PS protein. Several compounds displayed hydrogen bonding interaction with HIE47, HIE44, Met40, Ser196, Tyr82 and Ser197, amino acid residues. One of the most active ligands, 1-((1-(4-methoxyphenyl)-1H-1,2,3-triazol- 4-yl)methyl)-N,3-diphenyl-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)carboxamide (7d) with

IC50 1.01±0.32 µM showed docking score of -8.19 kcal/mol. The active site in the hydrophobic pocket is within the vicinity of Leu146, Val187, Val142, Met195, Ala42, Leu50, Pro38, Ile168 and Met40 some polar amino acid residues HIE47, Ser196, Ser197 and Thr82 respectively. The ligand also exhibited hydrogen bonding interaction with Met40, Ser197, Val187 residues [28]. The binding pattern of 7d with MTB PS is shown in Figure 3.7.

Figure 3.7: Docked pose of compound 7d inside the 3IUB, showing two-dimensional interactive diagram.

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3.2.4. In vitro cytotoxicity studies The active compounds 7b, 7c and 7d were evaluated for Promega Cell Titer 96 non-radioactive cell proliferation assay to analyze the selectivity profile against mouse macrophage (RAW264.7) cell lines [32]. The IC50 values and selectivity index (SI) values are tabulated in Table 3.3. The most active compound (7d) showed SI value 13.76. The results imply that the compounds are suitable for further investigation in TB.

Table 3.3: Cytotoxicity results of the active compounds * a MIC (µM) in IC50 SI values Entry MTB H37Rv approximation IC50/MIC 7b 25.53 335.19 13.12 7c 49.64 370.12 7.45 7d 24.72 340.15 13.76 a Selectivity index; * units in µM

3.2.5. Single Crystal X-ray Crystallographic Structure of Compound 7g The suitable crystals of the compound 7g for X-ray crystallographic study were grown from methanol solution. The single crystal X-ray diffraction measurement of the molecule

(C28H24N7OBr.CH3OH) was done using Rigaku XtaLAB P200 diffractmeter using graphite monochromated Mo-Kα radiation (λ = 0.71073 Å) on 0.1mm x 0.1mm x 0.1mm pale yellow crystal. Data were collected and processed using CrysAlisPro (Rigaku Oxford Diffraction). The data were collected at a temperature of -180 ± 1 °C to a maximum 2θ value of 58.2°. Of the 19673 reflections collected, 6164 were unique (Rint = 0.0791) and equivalent reflections were merged. The diffraction data were refined and structure was solved using Crystal Structure 4.2.2 software program. The structure was solved by direct methods and expanded using Fourier techniques. The non-hydrogen atoms were refined anisotropically. Hydrogen atoms were refined using the riding model. The compound crystallized into a triclinic crystal system with P-1 space group. In a single unit cell four partially occupying molecules along with two methanol solvent of crystallization are observed with Z=2. The basic crystallographic data are shown in Table 3.4. The molecular structure of the compound with methanol solvent of crystallization is given as an ORTEP diagram in Figure 3.8. The part of the molecule containing p-bromophenyl ring is

66

Chapter 3 directly attached to triazole ring nitrogen. These two rings are not coplanar; the phenyl ring plane is deviated from the triazole plane by around 30°. The torsional angle between these two rings with selected bonds C5-C4-N1-N2 = 30.90 and C3-C4-N1-C7 = 31.71 degrees. The deviation is comparatively less in the other part of the molecule. Pyrazole ring plane with respect to the attached phenyl ring plane deviated by around 7.5°, the corresponding dihedral angle for the selected four bonds are N5-C12-C13-C18 = 7.54, C11-C12-C13-C14 = 7.34 degrees. Crystallographic data for the compound 7g is deposited to the Cambridge Crystallographic Data Center and corresponding deposition number is CCDC 1500428.

Figure 3.8: ORTEP diagram showing the X-ray crystal structure of the compound 7g with a methanol solvent of crystallization.

Table 3.4: Crystal data and structure refinement for 7g

Empirical Formula C28H24BrN7O. CH3OH Formula Weight 586.49 Crystal Color, Habit Light yellow Crystal Dimensions 0.1 mm x 0.1 mm x 0.1 mm Crystal System Triclinic

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Chapter 3

Lattice Type Primitive Lattice Parameters a = 7.8845(3) A° b = 11.9643(4) A° c = 14.9423(4) A° α = 102.268(2) A° β = 101.916(2) A° γ = 95.003(3) A° δ = 1334.95(8) A°3 Space Group P-1 (#2) Z value 2 3 Dcalc 1.459 g/cm

F000 604.00 µ(MoKα) 15.855 cm-1 Radiation Mo-Kα(λ = 0.71073 A°) Radiation monochromator Graphite Voltage, Current 50kV, 40mA Temperature -180.0 °C Maximum 2θ 58.2° Number of measured reflections 19673°

Number of Unique reflections 6164 (Rint = 0.0791) Number of parameters 380 Goodness-of-fit on F2 1.00 - 3 Δρmax,mix(e /A° ) 1.89, -0.78 Reflection/Parameter Ratio 16.22 Residuals: R1 (I>2.00σ(I)) 0.0479 Residuals: R (All reflections) 0.0538 Residuals: wR2 (All reflections) 0.1931 Crystal refinement CrystalStructure 4.2.2

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3.3. Conclusions In this work, we designed novel 1-((1-(substituted)-1H-1,2,3-triazol-4-yl)methyl)-N,3-diphenyl- 6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-carboxamide derivatives by molecular hybridization approach using reported MTB PS inhibitor and substituted 1H-1,2,3-triazole antitubercular compounds. Twenty six compounds were synthesized and well characterized. One of the compounds 7d showed better MTB PS inhibition and MTB MIC than one of the lead compound (1-benzoyl-N-(4-nitrophenyl)-3-phenyl-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine- 5(4H)-carboxamide. Thus, this 1-((1-(substituted)-1H-1,2,3-triazole scaffold could be further optimized to develop MTB PS specific agents. In conclusion, it has been shown that, the potency and low cytotoxicity of the title compounds make them suitable leads for synthesizing new compounds with better anti-tubercular activity.

3.4. Experimental 3.4.1. Materials and methods Chemicals and solvents were procured from commercial source. The solvents and reagents were of LR grade and if necessary purified before use. Thin-layer chromatography (TLC) was carried out on aluminium-supported silica gel plates (Merck 60 F254) with visualization of components by UV light (254 nm). Column chromatography was carried out on silica gel (Merck 100-200 mesh). 1H NMR and 13C NMR spectra were recorded at 400 MHz and 101 MHz respectively using a Bruker AV 400 spectrometer (Bruker CO., Switzerland) in CDCl3 and DMSO-d6 solution with tetramethylsilane as the internal standard and chemical shift values (δ) were given in ppm. Melting points were determined on an electro thermal melting point apparatus (Stuart- SMP30) in open capillary tubes and are uncorrected. Elemental analyses were performed by Elementar Analysensysteme GmbH vario MICRO cube CHN Analyzer. Mass spectra (ESI-MS) were recorded on Schimadzu MS/ESI mass spectrometer. Purity of all tested compounds were determined by LC-MS/MS on Schimadzu and was greater than 95%.

3.4.2. Chemistry tert-butyl 3-benzoyl-4-oxopiperidine-1-carboxylate (2) In a two neck 100 mL round-bottom flask equipped with a Dean-stark trap, a reflux condenser and an internal thermocouple, compound 1 (5.0 g, 23.15 mmol), toluene (50 mL), morpholine

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(2.1 mL, 23.15 mmol), and p-toluenesulfonic acid (catalytic) were added sequentially. The reaction mixture was refluxed under N2 atmosphere for 16 h. The solvent was evaporated and the crude reaction mixture was dissolved in DCM (40 mL) and then triethylamine (5.35 mL, 37.65 mmol) was added at 0 °C, under N2, benzoyl chloride (2.9 mL, 25.1 mmol) was added over 10 min, the ice bath was then removed and the reaction solution was stirred at room temperature for 4 h. Once completion of the reaction, as indicated by TLC, the reaction was quenched with

NaHCO3 solution and extracted with DCM. The organic layers were collected, washed with saturated brine solution, dried over anhydrous Na2SO4 and concentrated in vacuo. The resultant crude product was purified by column chromatography [ethyl acetate / hexane (10 - 15%)] to get the 1,3-dicarbonyl compound 2 (7.0 g, 92%) as a colorless liquid. ESI-MS found 304 (M+H)+. tert-butyl 3-phenyl-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-carboxylate (3) A stirred solution of tert-butyl 3-benzoyl-4-oxopiperidine-1-carboxylate (2) (7.6 g, 25.05 mmol) in ethanol was cooled to 0 °C and hydrazine hydrate (0.8 mL, 25.05 mmol) was added and stirred for 4h. Once completion of the reaction, as indicated by TLC the reaction mixture was concentrated under reduced pressure and the crude residue was purified by column chromatography to get compound 3 (6.9 g, 92%) as an off-white solid. ESI-MS found 300 + 1 (M+H) . H NMR (400 MHz, CDCl3) δ 9.48 (s, 1H), 7.58-7.19 (m, 5H), 4.82 (s, 2H), 3.91 (t, J = 13 7.6 Hz, 2H), 2.83 (t, J = 7.8 Hz, 2H), 1.34 (s, 9H); C NMR (100 MHz, CDCl3) 163.54, 146.99, 143.56, 136.64, 132.75, 129.31, 127.39, 117.45, 81.67, 43.91, 36.89, 31.49, 27.79; Anal. calcd for C17H21N3O2: (%) C, 68.20; H, 7.07; N, 14.04, Found: C, 68.24; H, 7.09; N, 14.13. tert-butyl-3-phenyl-1-(prop-2-yn-1-yl)-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)- carboxylate (4) A solution of tert-butyl 3-phenyl-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-carboxylate (3)

(5.0 g, 16.70 mmol) in DMF was cooled to 0 °C and Cs2CO3 (8.16 g, 25.05 mmol) and propargyl bromide (80% in toluene) (1.64 mL, 21.17 mmol) were added and allowed to reach room temperature and stirred for 16 h. Once completion of the reaction, as indicated by TLC, the reaction was quenched with cold water and extracted with diethyl ether. The organic layers were collected, washed with saturated brine solution, dried over anhydrous Na2SO4 and concentrated in vacuo. The resultant crude product was purified by column chromatography [ethyl acetate /

70

Chapter 3 hexane (20 - 30%)] to get the compound 4 (4.6 g, 83%) as a semisolid. ESI-MS found 338.15 + 1 (M+H) . H NMR (400 MHz, CDCl3) δ 7.60-7.23 (m, 5H), 4.80 (s, 2H), 4.56 (s, 2H), 3.92 (t, J = 13 7.4 Hz, 2H), 2.81 (t, J = 7.6 Hz, 2H), 2.72 (s, 1H), 1.31 (s, 9H); C NMR (100 MHz, CDCl3) 163.57, 147.64, 143.65, 136.76, 132.87, 129.38, 127.40, 117.51, 81.7, 77.96, 69.05, 43.91, 42.07,

36.77, 31.59, 27.90; Anal. calcd for C20H23N3O2: (%) C, 69.79; H, 6.96; N, 13.20, Found: C, 69.80; H, 7.01; N, 13.23.

3-phenyl-1-(prop-2-yn-1-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine (5) A solution of tert-butyl 3-phenyl-1-(prop-2-yn-1-yl)-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-

5(4H)-carboxylate (4) (4.0 g, 11.85 mmol) in CH2Cl2 was cooled to 0 °C and CF3COOH (4.5 mL, 59.27 mmol) was added drop wise and stirred at room temperature for 16 h. The reaction mixture was concentrated under reduced pressure and the crude residue was washed with hexane and diethyl ether to get compound 5 (2.6 g, 92%) as an off-white solid. ESI-MS showed 238.10 (M+H)+.

N,3-diphenyl-1-(prop-2-yn-1-yl)-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-carboxamide (6) Phenylisocyanate (1.3 mL, 10.95 mmol), was added to the stirred solution of Compound 5 (2.0 g,

8.42 mmol) and Et3N (3.5 mL, 25.26 mmol) in DMF at 0 °C under N2 atm, and stirred at room temperature for 4 h. Once completion of the reaction, as indicated by TLC, the reaction was quenched with cold water then solid was filtrated and washed with water and hexane to get the key intermediate 6 (2.7 g, 90%) as an off-white solid. ESI-MS showed 357.15 (M+H)+. 1H NMR

(400 MHz, DMSO-d6) δ 8.74 (s, 1H), 7.88 (d, J = 8.6 Hz, 2H), 7.78 (d, J = 8.8 Hz, 2H), 7.67 (d, J = 7.6 Hz, 2H), 7.72 – 7.64 (m, 2H), 7.28 – 7.18 (m, 2H), 5.47 (s, 2H), 4.71 (s, 2H), 3.86 (t, J = 13 7.6 Hz, 2H), 2.90 (t, J = 7.5 Hz, 2H), 2.69 (s, 1H); C NMR (100 MHz, CDCl3) 164.58, 155.57, 143.57, 139.51, 136.69, 132.85, 129.38, 128.92, 128.12, 127.40, 121.16, 117.52, 81.73, 77.94,

69.0, 44.01, 42.12, 23.94; Anal. calcd for C22H20N4O: (%) C, 74.14; H, 5.66; N, 15.72, Found: C, 74.16; H, 5.67; N, 15.73.

N,3-diphenyl-1-((1-phenyl-1H-1,2,3-triazol-4-yl)methyl)-6,7-dihydro-1H-pyrazolo[4,3- c]pyridine-5(4H)-carboxamide (7a-z)

71

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A solution of N,3-diphenyl-1-(prop-2-yn-1-yl)-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)- carboxamide (6) (0.20 g, 1.0 equiv.) is reacted with substituted phenyl azides (1.2 equiv.) in the presence of sodium ascorbate (0.01 equiv.), CuSO4.5H2O (0.02 equiv.) and t-BuOH: H2O (2:1), at rt for 4 h. Once completion of the reaction, as indicated by TLC, the reaction was quenched with cold water and extracted with DCM. The DCM layers were collected, washed with saturated brine solution, dried over anhydrous Na2SO4 and concentrated in vacuo. The resultant crude product was purified by column chromatography [MeOH / DCM (1 -3%)] to yield the title compounds 7a-z.

1 H NMR spectrum (400MHz, DMSO-d6) of compound 7a

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13 C NMR spectrum (101MHz, DMSO-d6) of compound 7a

N,3-diphenyl-1-((1-phenyl-1H-1,2,3-triazol-4-yl)methyl)-6,7-dihydro-1H-pyrazolo[4,3- c]pyridine-5(4H)-carboxamide (7a) -1 Off white solid (83%); m.p. 221-223 °C; IR (KBr) ʋmax / cm 3490, 3021, 2843, 1650, 1410, 1 1340, 1060. H NMR (400 MHz, DMSO-d6) δ 8.88 (s, 1H), 8.75 (s, 1H), 7.94 – 7.86 (m, 2H), 7.69 – 7.66 (m, 2H), 7.58 (dd, J = 8.6, 7.1 Hz, 2H), 7.50 – 7.42 (m, 5H), 7.38 – 7.28 (m, 1H), 7.28 – 7.15 (m, 2H), 6.94 (t, J = 7.2, 1.2 Hz, 1H), 5.49 (s, 2H), 4.72 (s, 2H), 3.83 (t, J = 5.8 Hz, 13 2H), 2.98 (t, J = 5.8 Hz, 2H). C NMR (101 MHz, DMSO-d6) δ 155.67, 145.55, 144.46, 140.85, 139.15, 136.98, 133.97, 130.32, 129.20, 129.14, 128.74, 127.78, 126.51, 122.38, 122.39, 120.61, 120.48, 112.00, 44.40, 42.17, 41.16, 40.59, 22.33. EI-MS m/z 476.20 (M+H)+; Anal. calcd for

C28H25N7O: (%) C, 70.72; H, 5.30; N, 20.62; Found: C, 70.74; H, 5.31; N, 20.63.

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N,3-diphenyl-1-((1-(p-tolyl)-1H-1,2,3-triazol-4-yl)methyl)-1,4,6,7-tetrahydro-5H-pyrazolo[4,3- c]pyridine-5-carboxamide (7b) -1 Light yellow solid (87%); m.p. 204-206 °C; (KBr) ʋmax / cm 3455, 3025, 2867, 1645, 1420, 1 1348, 1062. H NMR (400 MHz, DMSO-d6) δ 8.81 (s, 1H), 8.75 (s, 1H), 7.77 (d, J = 8.5 Hz, 2H), 7.67 (d, J = 7.2 Hz, 2H), 7.48 – 7.42 (m, 4H), 7.40 – 7.30 (m, 3H), 7.26 – 7.20 (m, 2H), 6.94 (t, J = 6.8 Hz, 1H), 5.47 (s, 2H), 4.71 (s, 2H), 3.83 (t, J = 5.6 Hz, 2H), 2.97 (t, J = 5.3 Hz, 13 2H), 2.36 (s, 3H). C NMR (101 MHz, DMSO-d6) δ 155.68, 145.54, 144.42, 140.91, 139.16, 138.77, 134.74, 133.95, 130.71, 129.21, 128.82, 127.78, 126.48, 122.38, 122.24, 120.49, 120.47, 111.99, 44.46, 42.18, 41.66, 22.29, 21.06. EI-MS m/z 490.23 (M+H)+; Anal. calcd for

C29H27N7O: (%) C, 71.15; H, 5.56; N, 20.03; Found: C, 71.16; H, 5.58; N, 20.06.

1-((1-(4-ethylphenyl)-1H-1,2,3-triazol-4-yl)methyl)-N,3-diphenyl-6,7-dihydro-1H-pyrazolo[4,3- c]pyridine-5(4H)-carboxamide (7c) -1 Off white solid (89%); m.p. 128-130 °C; IR (KBr) ʋmax / cm 3478, 3031, 2913, 1635, 1422, 1 1341, 1056. H NMR (400 MHz, DMSO-d6) δ 8.82 (s, 1H), 8.75 (s, 1H), 7.78 (d, J = 8.4 Hz, 2H), 7.68 (d, J = 7.3 Hz, 2H), 7.48 – 7.42 (m, 4H), 7.40 – 7.30 (m, 3H), 7.26 – 7.20 (m, 2H), 6.94 (t, J = 6.8 Hz, 1H), 5.47 (s, 2H), 4.71 (s, 2H), 3.83 (t, J = 5.6 Hz, 2H), 2.97 (t, J = 5.4 Hz, 13 2H), 2.76 (m, 2H), 1.36 (t, 3H). C NMR (101 MHz, DMSO-d6) δ 155.17, 145.09, 144.67, 144.21, 143.62, 140.38, 138.66, 133.47, 129.93, 128.66, 128.22, 127.25, 126.04, 121.89, 121.84, 121.77, 119.95, 114.78, 48.12, 44.19, 41.67, 28.23, 21.85, 14.45. EI-MS m/z 504.25 (M+H)+;

Anal. calcd for C30H29N7O: (%) C, 71.55; H, 5.80; N, 19.47; Found: C, 71.56; H, 5.82; N, 19.48.

1-((1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-N,3-diphenyl-6,7-dihydro-1H- pyrazolo[4,3-c]pyridine-5(4H)-carboxamide (7d) -1 Brown solid (90%); m.p. 115-117 °C; IR (KBr) ʋmax / cm 3442, 3027, 2832, 1645, 1424, 1365, 1 1034, 1020. H NMR (400 MHz, DMSO-d6) δ 8.76 (s, 1H), 8.73 (s, 1H), 7.81 – 7.78 (m, 2H), 7.68 – 7.64 (m, 2H), 7.47 – 7.41 (m, 4H), 7.35 – 7.31 (m, 1H), 7.25 – 7.21 (m, 2H), 7.11 (d, J = 9.1 Hz, 2H), 6.95 (d, J = 7.4 Hz, 1H), 5.46 (s, 2H), 4.70 (s, 2H), 3.83 (s, 3H), 3.82 (t, J = 7.1 Hz, 13 2H), 2.97 (t, J = 5.4 Hz, 2H). C NMR (101 MHz, DMSO-d6) δ 156.47, 153.18, 145.02, 143.72, 140.36, 138.63, 133.49, 129.91, 128.64, 128.24, 127.27, 126.01, 121.88, 121.86, 121.77, 119.98,

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Chapter 3

114.78, 111.48, 55.51, 48.12, 44.93, 41.67, 22.13. EI-MS m/z 506.20 (M+H)+; Anal. calcd for

C29H27N7O2: (%) C, 68.89; H, 5.38; N, 19.39; Found: C, 68.91; H, 5.39; N, 19.41.

1-((1-(4-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-N,3-diphenyl-6,7-dihydro-1H- pyrazolo[4,3-c]pyridine-5(4H)-carboxamide (7e) -1 Off white solid (82%); m.p. 158-160 °C; IR (KBr) ʋmax / cm 3490, 3021, 2843, 1655, 1410, 1 1340, 1120, 1060. H NMR (400 MHz, DMSO-d6) δ 8.85 (s, 1H), 8.74 (s, 1H), 8.00 – 7.90 (m, 2H), 7.71 – 7.63 (m, 2H), 7.50 – 7.29 (m, 7H), 7.28 – 7.17 (m, 2H), 6.94 (tt, J = 7.3, 1.2 Hz, 1H), 5.48 (s, 2H), 4.71 (s, 2H), 3.82 (t, J = 5.7 Hz, 2H), 2.97 (t, J = 5.4 Hz, 2H). 13C NMR (101 MHz,

DMSO-d6) δ 161.01, 155.21, 145.02, 143.72, 140.36, 138.63, 133.49, 129.91, 128.64, 128.24, 127.27, 126.01, 121.88, 121.86, 121.77, 119.98, 114.78, 111.48, 50.19, 48.93, 41.87, 24.23. EI- + MS m/z 494.20 (M+H) ; Anal. calcd for C28H24FN7O: (%) C, 68.14; H, 4.90; N, 19.87; Found: C, 68.16; H, 4.91; N, 19.89.

1-((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-N,3-diphenyl-1,4,6,7-tetrahydro-5H- pyrazolo[4,3-c]pyridine-5-carboxamide (7f) -1 White solid (89%); m.p. 145-147 °C; (KBr) ʋmax / cm 3428, 3027, 2834, 1647, 1412, 1343, 1 1043, 615. H NMR (400 MHz, DMSO-d6) δ 8.82 (s, 1H), 8.73 (s, 1H), 7.80 (d, J = 9.2 Hz, 2H), 7.66 (d, J = 8.5 Hz, 2H), 7.43 (t, J = 7.6 Hz, 4H), 7.37 (t, J = 7.5 Hz, 1H), 7.27 – 7.21 (m, 2H), 7.18 (d, J = 9.5 Hz, 2H), 6.91 (t, J = 7.8 Hz, 1H), 5.46 (s, 2H), 4.74 (s, 2H), 3.83 (t, J = 5.7 Hz, 13 2H), 2.96 (t, J = 5.2 Hz, 2H). C NMR (101 MHz, DMSO-d6) δ 155.28, 145.12, 143.92, 141.36, 138.63, 134.12, 133.39, 129.91, 128.64, 128.24, 127.27, 126.11, 121.88, 121.89, 121.77, 119.88, 114.78, 111.48, 52.19, 48.83, 41.67, 24.83. EI-MS m/z 510.20 (M+H)+; Anal. calcd for

C28H24ClN7O: (%) C, 65.94; H, 4.74; N, 19.23; Found: C, 65.16; H, 4.75; N, 19.89.

1-((1-(4-bromophenyl)-1H-1,2,3-triazol-4-yl)methyl)-N,3-diphenyl-6,7-dihydro-1H- pyrazolo[4,3-c] pyridine-5(4H)-carboxamide (7g) -1 Pale yellow solid (87%); m.p. 150-152 °C; IR (KBr) ʋmax / cm 3492, 3022, 2913, 1657, 1422, 1 1332, 1055, 680. H NMR (400 MHz, DMSO-d6) δ 8.90 (s, 1H), 8.75 (s, 1H), 7.88 (d, J = 8.6 Hz, 2H), 7.78 (d, J = 8.8 Hz, 2H), 7.67 (d, J = 7.6 Hz, 2H), 7.45 (dt, J = 7.8, 3.6 Hz, 4H), 7.33 (t, J = 7.4 Hz, 1H), 7.23 (t, J = 7.8 Hz, 2H), 6.94 (t, J = 7.4 Hz, 1H), 5.48 (s, 2H), 4.71 (s, 2H), 3.82

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Chapter 3

13 (t, J = 5.6 Hz, 2H), 2.96 (t, J = 6.0 Hz, 2H). C NMR (101 MHz, DMSO-d6) δ 155.66, 145.58, 144.65, 140.84, 139.15, 136.17, 133.95, 133.19, 129.14, 128.74, 127.79, 126.45, 122.51, 122.43, 122.38, 121.92, 120.42, 112.05, 44.33, 42.24, 41.24, 22.22. EI-MS m/z 554.12(M+H) +; 556.10 +2 (M+H) ; Anal. Calcd for C28H24BrN7O: (%) C, 60.66; H, 4.36; N, 17.68; Found: C, 60.68; H, 4.37; N, 17.69.

1-((1-(4-iodophenyl)-1H-1,2,3-triazol-4-yl)methyl)-N,3-diphenyl-6,7-dihydro-1H-pyrazolo[4,3- c]pyridine-5(4H)-carboxamide (7h) -1 Light brown solid (80%); m.p.147-149 °C; ; IR (KBr) ʋmax / cm 3485, 30311, 2905, 1658, 1402, 1 1043, 560. H NMR (400 MHz, DMSO-d6) δ 8.85 (s, 1H), 8.76 (s, 1H), 7.99 (d, J = 9.2 Hz, 2H), 7.76 (d, J = 8.5 Hz, 2H), 7.46 (t, J = 7.8 Hz, 4H), 7.35 (t, J = 7.5 Hz, 1H), 7.27 – 7.21 (m, 2H), 7.12 (d, J = 9.3 Hz, 2H), 6.92 (t, J = 7.8 Hz, 1H), 5.47 (s, 2H), 4.69 (s, 2H), 3.83 (t, J = 5.8 Hz, 13 2H), 2.97 (t, J = 5.4 Hz, 2H). C NMR (101 MHz, DMSO-d6) δ 155.48, 145.02, 144.23, 143.72, 140.36, 138.63, 129.91, 128.64, 128.24, 127.27, 126.01, 121.88, 121.86, 121.77, 119.98, 116.78, 115.48, 95.15, 45.23, 42.13, 41.67, 23.93. EI-MS m/z 602.12 (M+H)+; Anal. Calcd for

C28H24IN7O: (%) C, 55.92; H, 4.03; N, 16.30; Found: C, 55.94; H, 4.31; N, 16.32.

1-((1-(4-nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl)-N,3-diphenyl-6,7-dihydro-1H-pyrazolo[4,3- c]pyridine-5(4H)-carboxamide (7i) -1 Light yellow solid (81%); m.p. 144-146 °C; IR (KBr) ʋmax / cm 3505, 3031, 2912, 1675, 1532, 1 1372, 1027. H NMR (400 MHz, DMSO-d6) δ 9.08 (s, 1H), 8.74 (s, 1H), 8.48 – 8.40 (m, 2H), 8.28 – 8.19 (m, 2H), 7.71 – 7.63 (m, 2H), 7.50 – 7.39 (m, 4H), 7.40 – 7.27 (m, 1H), 7.28 – 7.18 (m, 2H), 6.94 (tt, J = 7.3, 1.2 Hz, 1H), 5.51 (s, 2H), 4.71 (s, 2H), 3.83 (t, J = 5.7 Hz, 2H), 2.97 (t, 13 J = 5.4 Hz, 2H). C NMR (101 MHz, DMSO-d6) δ 155.65, 147.17, 145.65, 145.10, 141.20, 140.83, 139.19, 133.92, 129.13, 128.74, 127.81, 126.50, 125.96, 122.89, 122.38, 121.14, 120.47, + 112.03, 44.29, 42.15, 41.13, 22.28. EI-MS m/z 521.19 (M+H) ; Anal. Calcd for C28H24N8O3: (%) C, 64.61; H, 4.65; N, 21.53; Found: C, 64.63; H, 4.66; N, 21.55.

N,3-diphenyl-1-((1-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)methyl)-6,7-dihydro-1H- pyrazolo[4,3-c]pyridine-5(4H)-carboxamide (7j)

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Chapter 3

-1 White solid (76%); m.p. 246-248 °C; IR (KBr) ʋmax / cm 3505, 3029, 2903, 1650, 1402, 1357, 1 1279, 1045. H NMR (400 MHz, DMSO-d6) δ 9.03 (s, 1H), 8.75 (s, 1H), 8.17 (d, J = 8.4 Hz, 2H), 7.97 (d, J = 8.5 Hz, 2H), 7.69 – 7.64 (m, 2H), 7.47 – 7.42 (m, 4H), 7.37 – 7.29 (m, 1H), 7.27 – 7.20 (m, 2H), 6.96 – 6.92 (m, 1H), 5.51 (s, 2H), 4.72 (s, 2H), 3.83 (t, J = 5.7 Hz, 2H), 2.97 13 (t, J = 6.7 Hz, 2H). C NMR (101 MHz, DMSO-d6) δ 155.66, 145.62, 144.86, 140.84, 139.75, 139.18, 133.94, 129.14, 128.73, 127.80, 127.60, 127.60, 126.51, 122.91, 122.71, 122.38, 121.06, 120.47, 112.02, 44.33, 42.16, 41.14, 22.30. EI-MS m/z 544.19 (M+H)+; Anal. Calcd for

C29H24F3N7O3: (%) C, 64.08; H, 4.45; N, 18.04; Found: C, 64.10; H, 4.46; N, 18.05.

1-((1-(2-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-N,3-diphenyl-6,7-dihydro-1H- pyrazolo[4,3-c]pyridine-5(4H)-carboxamide (7k) -1 Off white solid (88%); m.p. 137-139 °C; IR (KBr) ʋmax / cm 3523, 3032, 2923, 1675, 1445, , 1 1052, 602. H NMR (400 MHz, DMSO-d6) δ 8.87 (s, 1H), 8.75 (s, 1H), 7.81 (d, J = 9.2 Hz, 2H), 7.69 (d, J = 8.5 Hz, 2H), 7.40 (t, J = 7.6 Hz, 5H), 7.37 (t, J = 7.5 Hz, 1H), 7.27 – 7.21 (m, 1H), 7.19 (d, J = 9.5 Hz, 2H), 6.99 (t, J = 7.8 Hz, 1H), 5.42 (s, 2H), 4.64 (s, 2H), 3.84 (t, J = 5.7 Hz, 13 2H), 2.96 (t, J = 5.2 Hz, 2H). C NMR (101MHz, DMSO-d6) δ 155.28, 145.22, 142.92, 141.36, 138.53, 136.23, 134.12, 133.39, 129.91, 128.64, 128.24, 127.27, 126.11, 121.88, 121.89, 121.77, 120.21, 119.88, 114.78, 111.48, 52.19 48.83, 41.67, 24.83. EI-MS m/z 510.15 (M+H)+; EI-MS + m/z 510.15 (M+H) ; Anal. calcd for C28H24ClN7O: (%) C, 65.94; H, 4.74; N, 19.23; Found: C, 65.16; H, 4.75; N, 19.89.

1-((1-(2-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-N,3-diphenyl-6,7-dihydro-1H- pyrazolo[4,3-c]pyridine-5(4H)-carboxamide (7l) -1 White solid (91%); m.p. 156-158 °C; IR (KBr) ʋmax / cm 3576, 3031, 2925, 1665, 1421, 1330, 1 1050. H NMR (400 MHz, DMSO-d6) δ 8.76 (s, 1H), 8.68 (s, 1H), 7.83 (t, J = 7.8 Hz, 1H), 7.68 (d, J = 7.6 Hz, 2H), 7.64 – 7.51 (m, 2H), 7.51 – 7.38 (m, 5H), 7.33 (t, J = 7.4 Hz, 1H), 7.24 (t, J = 7.7 Hz, 2H), 6.95 (t, J = 7.4 Hz, 1H), 5.51 (s, 2H), 4.72 (s, 2H), 3.83 (t, J = 6.1 Hz, 2H), 2.98 13 (t, J = 6.3 Hz, 2H). C NMR (101 MHz, DMSO-d6) δ 155.67, 153.06, 145.56, 143.95, 140.85, 139.19, 133.96, 131.82, 129.16, 128.75, 127.80, 126.51, 125.99, 125.74, 125.13, 122.38, 120.48, 117.66, 117.47, 112.00, 44.20, 42.16, 41.16, 22.34. EI-MS m/z 494.20 (M+H)+; Anal. calcd for

C28H24FN7O: (%) C, 68.14; H, 4.90; N, 19.87; Found: C, 68.16; H, 4.91; N, 19.89.

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1-((1-(2-bromophenyl)-1H-1,2,3-triazol-4-yl)methyl)-N,3-diphenyl-6,7-dihydro-1H- pyrazolo[4,3-c]pyridine-5(4H)-carboxamide (7m) -1 White solid (90%); m.p. 119-121 °C; IR (KBr) ʋmax / cm 3490, 3021, 2843, 1650, 1410, 1340, 1 570. H NMR (400 MHz, DMSO-d6) δ 8.75 (s, 1H), 8.71 (s, 1H), 7.81(d, J = 9.1 Hz, 2H), 7.67 (d, J = 8.4 Hz, 2H), 7.44 (t, J = 7.9 Hz, 5H), 7.33 (t, J = 7.4 Hz, 1H), 7.26 – 7.20 (m, 1H), 7.11 (d, J = 9.1 Hz, 2H), 6.94 (t, J = 7.8 Hz, 1H), 5.46 (s, 2H), 4.73 (s, 2H), 3.82 (t, J = 5.6 Hz, 2H), 13 2.97 (t, J = 5.4 Hz, 2H). C NMR (101MHz, DMSO-d6) δ 155.21, 145.02, 144.23, 143.72, 140.36, 139.21, 138.63, 134.24, 133.49, 133.67, 129.91, 128.64, 128.24, 127.27, 126.01, 121.88, 121.86, 121.77, 120.08, 118.12, 48.12, 44.73, 41.67, 22.83 EI-MS m/z 554.12(M+H) +, 556.10 +2 (M+H) ; Anal. Calcd for C28H24BrN7O: (%) C, 60.66; H, 4.36; N, 17.68; Found: C, 60.68; H, 4.37; N, 17.69.

1-((1-(2-iodophenyl)-1H-1,2,3-triazol-4-yl)methyl)-N,3-diphenyl-6,7-dihydro-1H-pyrazolo[4,3- c]pyridine-5(4H)-carboxamide (7n) -1 White solid (87%); m.p. 188-120 °C; (KBr) ʋmax / cm 3512, 3025, 2843, 1650, 1408, 1344, 500. 1 H NMR (400 MHz, DMSO-d6) δ 8.79 (s, 1H), 8.72 (s, 1H), 7.99 (d, J = 9.2 Hz, 2H), 7.76 (d, J = 8.5 Hz, 2H), 7.46 (t, J = 7.8 Hz, 5H), 7.35 (t, J = 7.5 Hz, 2H), 7.19 (d, J = 9.3 Hz, 2H), 6.98 (t, J = 7.8 Hz, 1H), 5.47 (s, 2H), 4.70 (s, 2H), 3.82 (t, J = 5.6 Hz, 2H), 2.97 (t, J = 5.4 Hz, 2H). 13C

NMR (101 MHz, DMSO-d6) δ 155.46, 145.02, 144.23, 143.72, 140.36, 138.63, 130.23, 129.91, 128.64, 128.24, 127.61, 127.27, 126.01, 121.88, 121.86, 121.77, 119.98, 116.78, 115.48, 95.15, + 48.23, 44.83, 41.67, 22.93. EI-MS m/z 602.12 (M+H) ; Anal. Calcd for C28H24IN7O: (%) C, 55.92; H, 4.03; N, 16.30; Found: C, 55.94; H, 4.31; N, 16.32.

1-((1-(2-nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl)-N,3-diphenyl-6,7-dihydro-1H-pyrazolo[4,3- c]pyridine-5(4H)-carboxamide (7o) -1 Off white solid (86%); m.p. 118-120 °C; IR (KBr) ʋmax / cm 3505, 3027, 2846, 1655, 1525, 1 1410, 1360, 1025. H NMR (400 MHz, DMSO-d6) δ 9.07 (s, 1H), 8.75 (s, 1H), 8.49 – 8.41 (m, 2H), 8.28 – 8.20 (m, 2H), 7.73 – 7.62 (m, 2H), 7.50 – 7.39 (m, 4H), 7.40 – 7.27 (m, 1H), 7.28 – 7.18 (m, 2H), 6.94 (tt, J = 7.3, 1.2 Hz, 1H), 5.47 (s, 2H), 4.69 (s, 2H), 3.82 (t, J = 5.6 Hz, 2H), 13 2.97 (t, J = 5.4 Hz, 2H). C NMR (101 MHz, DMSO-d6) δ 155.18, 147.87, 147.15, 145.12,

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143.72, 141.36, 138.63, 135.23, 134.56, 133.49, 129.91, 128.64, 128.24, 127.27, 126.01, 121.88, 121.86, 121.77, 120.98, 112.03, 44.29, 42.15, 41.13, 22.28. EI-MS m/z 521.19 (M+H)+; Anal.

Calcd for C28H24N8O3: (%) C, 64.61; H, 4.65; N, 21.53; Found: C, 64.63; H, 4.66; N, 21.55.

1-((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-N,3-diphenyl-6,7-dihydro-1H- pyrazolo[4,3-c]pyridine-5(4H)-carboxamide (7p) -1 White solid (86%); m.p. 147-149 °C; IR (KBr) ʋmax / cm 3497, 3029, 2847, 1650, 1444, 1306, 1 1032, 753. H NMR (400 MHz, DMSO-d6) δ 8.80 (s, 1H), 8.75 (s, 1H), 7.89 (s, 1H), 7.81 (d, J = 9.2 Hz, 2H), 7.69 (d, J = 8.5 Hz, 2H), 7.40 (t, J = 7.6 Hz, 4H), 7.37 (t, J = 7.5 Hz, 1H), 7.27 – 7.21 (m, 1H), 7.19 (d, J = 9.5 Hz, 2H), 6.93 (t, J = 7.8 Hz, 1H), 5.42 (s, 2H), 4.64 (s, 2H), 3.84 (t, 13 J = 5.7 Hz, 2H), 2.96 (t, J = 5.2 Hz, 2H). C NMR (101 MHz, DMSO-d6) δ 155.38, 145.42, 142.82, 141.39, 138.55, 136.23, 134.32, 133.30, 129.96, 128.74, 128.29, 127.20, 126.51, 121.98, 121.80, 121.75, 121.34, 120.31, 119.78, 118.78, 45.29 42.80, 41.66, 22.73. EI-MS m/z 510.15 + (M+H) ; Anal. calcd for C28H24ClN7O: (%) C, 65.94; H, 4.74; N, 19.23; Found: C, 65.95; H, 4.76; N, 19.24.

1-((1-(3-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-N,3-diphenyl-6,7-dihydro-1H- pyrazolo[4,3-c]pyridine-5(4H)-carboxamide (7q) -1 Brown solid (79%); m.p. 135-136 °C; IR (KBr) ʋmax / cm 3502, 3023, 2875, 1657, 1454, 1350, 1 1090. H NMR (400 MHz, DMSO-d6) δ 8.78 (s, 1H), 8.75 (s, 1H), 7.80 (d, J = 9.1 Hz, 2H), 7.67 (d, J = 8.4 Hz, 2H), 7.47 – 7.41 (m, 4H), 7.34 (t, J = 7.4 Hz, 1H), 7.26 – 7.20 (m, 2H), 7.11 (d, J = 9.1 Hz, 2H), 6.94 (t, J = 7.8 Hz, 1H), 5.48 (s, 2H), 4.71 (s, 2H), 3.83 (t, J = 5.7 Hz, 2H), 2.96 13 (t, J = 5.5 Hz, 2H). C NMR (100 MHz, DMSO-d6) δ 156.25, 154.08, 145.22, 143.87, 141.36, 138.63, 133.49, 129.91, 128.64, 128.24, 127.27, 126.01, 125.32, 121.88, 121.76, 121.77, 120.14, 119.98, 114.78, 111.42, 53.51, 46.12, 44.73, 41.87, 22.21. EI-MS m/z 506.20 (M+H)+; Anal. calcd for C29H27N7O2: (%) C, 68.89; H, 5.38; N, 19.39; Found: C, 68.91; H, 5.39; N, 19.41.

1-((1-(2,4-dichlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-N,3-diphenyl-6,7-dihydro-1H- pyrazolo[4,3-c]pyridine-5(4H)-carboxamide (7r) -1 White solid (82%); m.p. 139-141 °C; IR (KBr) ʋmax / cm 3505, 3029, 2840, 1654, 1415, 1342, 1 1070, 740. H NMR (400 MHz, DMSO-d6) δ 8.77 (s, 1H), 8.73 (s, 1H), 7.80 (d, J = 9.2 Hz, 2H),

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7.66 (d, J = 8.5 Hz, 2H), 7.69 (s, 1H), 7.62 (d, J = 9.5 Hz, 2H), 7.50 (t, J = 7.6 Hz, 2H), 7.41 (t, J = 7.5 Hz, 3H), 6.91 (t, J = 7.8 Hz, 1H), 5.45 (s, 2H), 4.69 (s, 2H), 3.84 (t, J = 5.7 Hz, 2H), 2.96 13 (t, J = 5.2 Hz, 2H). C NMR (101 MHz, DMSO-d6) δ 155.31, 145.53, 144.16, 142.72, 141.48, 139.56, 136.29, 135.74, 133.40, 129.99, 129.24, 128.44, 127.25, 126.21, 123.58, 123.49, 121.57, 120.21, 119.88, 114.78, 46.21 44.63, 41.67, 22.23. EI-MS m/z 544.13 (M+H)+; Anal. calcd for

C28H23Cl2N7O: (%) C, 61.77; H, 4.26; N, 18.01; Found: C, 61.79; H, 4.27; N, 18.03.

1-((1-(3,4-dichlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-N,3-diphenyl-6,7-dihydro-1H- pyrazolo[4,3-c]pyridine-5(4H)-carboxamide (7s) -1 Off white solid (83%); m.p. 228-230 °C; IR (KBr) ʋmax / cm 3503, 3029, 2842, 1654, 1410, 1 1345, 1066, 745. H NMR (400 MHz, DMSO-d6) δ 8.78 (s, 1H), 8.74 (s, 1H), 7.81 (d, J = 9.2 Hz, 2H), 7.77 (s, 1H), 7.66 (d, J = 8.6 Hz, 2H), 7.62 (d, J = 9.4 Hz, 2H), 7.54 (t, J = 7.5 Hz, 2H), 7.44 (t, J = 7.4 Hz, 3H), 7.01 (t, J = 7.9 Hz, 1H), 5.43 (s, 2H), 4.63 (s, 2H), 3.82 (t, J = 5.7 Hz, 13 2H), 2.98 (t, J = 5.4 Hz, 2H). C NMR (101 MHz, DMSO-d6) δ 155.18, 146.41, 143.80, 141.43, 138.26, 136.49, 134.31, 133.41, 129.90, 128.52, 128.41, 127.43, 125.32, 121.78, 121.59, 121.47, 120.20, 119.82, 115.78, 111.78, 46.65, 46.24, 40.97, 22.73. EI-MS m/z 544.20 (M+H)+; Anal. calcd for C28H23Cl2N7O: (%) C, 61.77; H, 4.26; N, 18.01; Found: C, 61.79; H, 4.27; N, 18.03.

1-((1-(3,5-dichlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-N,3-diphenyl-6,7-dihydro-1H- pyrazolo[4,3-c]pyridine-5(4H)-carboxamide (7t) -1 White solid (85%); m.p. 139-141 °C; IR (KBr) ʋmax / cm 3505, 3027, 2846, 1650, 1411, 1345, 1 1030, 752. H NMR (400 MHz, DMSO-d6) δ 8.98 (s, 1H), 8.74 (s, 1H), 8.08 (d, J = 1.8 Hz, 2H), 7.78 – 7.64 (m, 3H), 7.45 (t, J = 7.4 Hz, 4H), 7.38 – 7.16 (m, 3H), 6.94 (t, J = 7.3 Hz, 1H), 5.49 (s, 2H), 4.71 (s, 2H), 3.83 (t, J = 5.8 Hz, 2H), 2.95 (t, J = 5.9 Hz, 2H13C NMR (101 MHz,

DMSO-d6) δ 155.64, 145.60, 144.79, 140.84, 139.15, 138.57, 135.63, 133.93, 129.13, 128.73, 127.80, 126.50, 122.83, 122.38, 120.47, 119.25, 112.02, 44.35, 42.16, 41.12, 22.26. EI-MS m/z + 544.13 (M+H) ; Anal. calcd for C28H23Cl2N7O: (%) C, 61.77; H, 4.26; N, 18.01; Found: C, 61.79; H, 4.27; N, 18.03.

1-((1-(3-chloro-4-fluorophenyl)-1H-1,2,3-triazol-4-yl) methyl)-N,3-diphenyl-6,7-dihydro-1H- pyrazolo[4,3-c]pyridine-5(4H)-carboxamide (7u)

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-1 White solid (77%); m.p. 240-242 °C; IR (KBr) ʋmax / cm 3505, 3022, 2840, 1654, 1410, 1345, 1 1178, 1020, 780. H NMR (400 MHz, DMSO-d6) δ 8.90 (s, 1H), 8.73 (s, 1H), 8.23 (dd, J = 6.4, 2.7 Hz, 1H), 8.01 – 7.92 (m, 1H), 7.71 – 7.62 (m, 3H), 7.49 – 7.39 (m, 4H), 7.38 – 7.28 (m, 1H), 7.28 – 7.18 (m, 2H), 6.98 – 6.89 (m, 1H), 5.48 (s, 2H), 4.70 (s, 2H), 3.82 (t, J = 5.8 Hz, 2H), 2.95 13 (t, J = 5.8 Hz, 2H). C NMR (101 MHz, DMSO-d6) δ 155.28, 145.09, 144.14, 140.34, 138.63, 133.46, 128.62, 128.22, 127.27, 126.00, 122.44, 122.30, 121.87, 120.95, 120.77, 120.58, 119.99, 118.13, 117.90, 111.51, 43.88, 41.67, 40.64, 21.79. EI-MS m/z 528.17 (M+H)+; Anal. calcd for

C28H23ClFN7O: (%) C, 63.70; H, 4.39; N, 18.57; Found: C, 63.72; H, 4.40; N, 18.59.

1-((1-(3,4-difluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-N,3-diphenyl-6,7-dihydro-1H- pyrazolo[4,3-c]pyridine-5(4H)-carboxamide (7v) -1 Off white solid (80%); m.p. 226-228 °C; IR (KBr) ʋmax / cm 3501, 3021, 2846, 1659, 1412, 1 1335, 1130, 1060. H NMR (400 MHz, DMSO-d6) δ 8.91 (s, 1H), 8.73 (s, 1H), 8.24 (dd, J = 6.5, 2.6 Hz, 1H), 8.01 – 7.91 (m, 1H), 7.70 – 7.62 (m, 3H), 7.46 – 7.40 (m, 4H), 7.35 (t, J = 7.4 Hz, 1H), 7.20 (t, J = 7.9 Hz, 2H), 7.04 (t, J = 7.3 Hz, 1H), 5.48 (s, 2H), 4.70 (s, 2H), ), 3.82 (t, J = 5.6 13 Hz, 2H), 2.95 (t, J = 5.4 Hz, 2H). C NMR (101 MHz, DMSO-d6) δ 155.21, 149.84, 149.21, 145.02, 143.72, 141.78, 140.36, 138.63, 133.49, 130.12, 129.91, 128.64, 128.24, 127.27, 126.01, 124.12, 121.88, 121.86, 121.77, 119.98, 45.22, 42.13, 40.81, 23.53. EI-MS m/z 528.17 (M+H)+;

Anal. calcd for C28H23F2N7O: (%) C, 65.74; H, 4.53; N, 19.17; Found: C, 65.76; H, 4.54; N, 19.19.

1-((1-(3,4-dimethoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-N,3-diphenyl-6,7-dihydro-1H- pyrazolo[4,3-c]pyridine-5(4H)-carboxamide (7w) -1 Brown solid (88%); m.p. 207-209 °C; IR (KBr) ʋmax / cm 3500, 3020, 2845, 1654, 1410, 1345, 1 1080. H NMR (400 MHz, DMSO-d6) δ 8.78 (s, 1H), 8.72 (s, 1H), 7.82 (d, J = 9.1 Hz, 2H), 7.67 (d, J = 8.4 Hz, 2H), 7.44 (t, J = 7.9 Hz, 4H), 7.33 (t, J = 7.4 Hz, 1H), 7.26 (m, 1H), 7.11 (d, J = 9.1 Hz, 2H), 6.94 (s, 1H), 5.46 (s, 2H), 4.72 (s, 2H), 3.84 (s, 6H), 3.82 (d, J = 7.2 Hz, 2H), 2.97 13 (t, J = 5.4 Hz, 2H). C NMR (101 MHz, DMSO-d6) δ 156.31, 153.21, 148.74, 139.76, 136.99, 135.44, 129.89, 129.54, 128.44, 127.45, 126.81, 123.98, 123.69, 121.77, 120.81, 119.88, 118.78, 114.25, 109.13, 101.21, 55.86, 46.90, 44.63, 40.32, 22.09. EI-MS m/z 536.20 (M+H)+; Anal. calcd for C30H29N7O3: (%) C, 67.27; H, 5.47; N, 18.31; Found: C, 67.29; H, 5.48; N, 18.33.

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1-((1-(4-bromo-3-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)methyl)-N,3-diphenyl-6,7- dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-carboxamide (7x) -1 White solid (79%); m.p. 221-223 °C; IR (KBr) ʋmax / cm 3500, 3022, 2854, 1660, 1405, 1343, 1 1119, 1070. H NMR (400 MHz, DMSO-d6) δ 8.91 (s, 1H), 8.73 (s, 1H), 8.24 (dd, J = 6.5, 2.6 Hz, 1H), 8.01 – 7.82 (m, 1H), 7.69 – 7.53 (m, 3H), 7.46 – 7.41 (m, 4H), 7.36 (t, J = 7.4 Hz, 1H), 7.21 (t, J = 7.9 Hz, 2H), 7.04 (t, J = 7.3 Hz, 1H), 5.48 (s, 2H), 4.70 (s, 2H), ), 3.82 (t, J = 5.6 Hz, 13 2H), 2.95 (t, J = 5.4 Hz, 2H). C NMR (101 MHz, DMSO-d6) δ 155.18, 145.02, 143.72, 140.36, 139.10, 138.63, 136.89, 136.12, 133.49, 130.23, 129.91, 128.64, 128.24, 127.27, 126.01, 121.88, 121.86, 121.77, 120.98, 120.01, 114.78, 48.01, 46.32, 44.83, 22.19. EI-MS m/z 622.20, 624.15 + (M+1, M+2) ; Anal. calcd for C30H23BrF3N7O: (%) C, 55.96; H, 3.72; N, 15.75; Found: C, 55.98; H, 3.74; N, 15.76.

1-((1-(3,4-dimethylphenyl)-1H-1,2,3-triazol-4-yl)methyl)-N,3-diphenyl-6,7-dihydro-1H- pyrazolo[4,3-c]pyridine-5(4H)-carboxamide (7y) -1 Off white solid (84%); m.p. 221-223 °C; IR (KBr) ʋmax / cm 3505, 3022, 2844, 1658, 1412, 1 1345, 1076. H NMR (400 MHz, DMSO-d6) δ 8.82 (s, 1H), 8.79 (s, 1H), 7.78 (d, J = 8.6 Hz, 2H), 7.66 (d, J = 7.3 Hz, 2H), 7.47 – 7.42 (m, 3H), 7.44 (s, 1H), 7.41 – 7.31 (m, 2H), 7.27 – 7.20 (m, 2H), 6.93 (t, J = 6.9 Hz, 1H), 5.47 (s, 2H), 4.72 (s, 2H), 3.82 (t, J = 5.7 Hz, 2H), 2.97 (t, J = 13 5.5 Hz, 2H), 2.36 (s, 6H). C NMR (101 MHz, DMSO-d6) δ 159.25, 155.18, 145.02, 143.72, 140.36, 138.63, 136.89, 136.12 133.49, 129.91, 128.64, 128.24, 127.27, 126.01, 121.88, 121.86, 121.77, 119.98, 114.78, 111.48, 45.51, 44.02, 41.57, 22.23, 20.92. EI-MS m/z 504.25 (M+H)+;

Anal. calcd for C30H29N7O: (%) C, 71.55; H, 5.80; N, 19.47; Found: C, 71.56; H, 5.82; N, 19.49.

1-((1-(benzo[d][1,3]dioxol-5-yl)-1H-1,2,3-triazol-4-yl)methyl)-N,3-diphenyl-6,7-dihydro-1H- pyrazolo[4,3-c]pyridine-5(4H)-carboxamide (7z) -1 Brown solid (78%); m.p. 168-170 °C; IR (KBr) ʋmax / cm 3504, 3025, 2840, 1655, 1416, 1345, 1 1080. H NMR (400 MHz, DMSO-d6) δ 8.77 (s, 1H), 8.72 (s, 1H), 7.81 (d, J = 9.1 Hz, 2H), 7.67 (d, J = 8.4 Hz, 2H), 7.44 (t, J = 7.9 Hz, 4H), 7.33 (t, J = 7.4 Hz, 1H), 7.26 (m, 1H), 7.11 (d, J = 9.1 Hz, 2H), 6.94 (s, 1H), 5.94 (s, 2H), 5.46 (s, 2H), 4.70 (s, 2H), 3.82 (d, J = 7.1 Hz, 2H), 2.97 13 (t, J = 5.4 Hz, 2H). C NMR (101 MHz, DMSO-d6) δ 156.31, 148.74, 147.53, 139.76, 136.99,

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135.44, 133.49, 129.89, 129.54, 128.44, 127.45, 126.81, 123.98, 123.69, 121.77, 120.81, 119.88, 114.78, 109.98, 101.21, 52.21, 48.63, 41.67, 24.73. EI-MS m/z 520.20 (M+H)+; Anal. calcd for

C29H25N7O3: (%) C, 67.05; H, 4.85; N, 18.87; Found: C, 67.06; H, 4.87; N, 18.89.

3.4.3. Biological activity 3.4.3.1 MTB PS screening The MTB panC gene (Rv3602c) encoding the pantothenate synthetase was cloned and changed into BL21 (DE3) cells and the protien was expressed.7, 15 For the assay, to each well of a 96-well plate, 60 μL of PS reaction mixture containing 0.4 mM NADH, 10 mM MgCl2, 5 mM β-alanine, 5 mM pantoic acid, 1 mM potassium phosphoenolpyruvate,10 mM ATP, and 20 μL of enzyme mixture consisting of 18 units/mL each of chicken muscle myokinase, rabbit muscle lactate dehydrogenase and rabbit muscle pyruvate kinase, diluted in 100 mM HEPES buffer were added. The reaction mixture and enzyme mixture were added to the plate to a final volume of 100 μL with 100 mM HEPES buffer (pH 7.8). Concentration of enzyme was determined based on the range finding experiments by varying the concentration of enzymes. Compound solutions were then added to the plates (from 50 μM to lower concentration) and the reaction began with the addition of 10 μL of 4.32 pM of MTB PS, diluted in buffer. The test plate was immediately moved to a microplate reader and the reduction of NADH was quantified at 340 nm. The reaction elements except MTB PS were mixed in the well and the background reaction was calculated; MTB PS was then added and the reaction kinetics was monitored. Reactions were carried out at 37 °C in a heat controlled Perkin Elmer Victor X3 Spectrophotometer. % inhibitions were calculated using following formula: 100 x [(1 - compound rate - background rate) / (full reaction rate - background rate)] [20, 28].

3.4.3.2. In vitro MTB screening The antimycobacterial activities of the compounds 7a-z were evaluated against MTB H37Rv strain and two “wild” strains extracted from tuberculosis patients: one strain is Spec. 210 resistant to PAS, INH, ETB and RMP and the other strain is Spec. 192 fully sensitive to the administrated anti-TB agents. In vitro anti-TB activity is performed by a classical test-tube method of successive dilution in Youmans’ modification of the Proskauer and Beck liquid medium containing 10% of bovine serum [31]. Bacterial respites were prepared from 14 days old

83

Chapter 3 cultures of gradually growing strains. Solutions of compounds in DMSO were tested. Stock solutions contained 10 mg of compounds in 1 mL. Dilutions (in geometric progression) were prepared in Youmans’ medium [31]. The medium is without compounds and containing INH as reference drug was used for comparison. Incubation was performed at 37 °C. The MIC values were determined as MIC inhibiting the growth of tested TB strains in relation to the probe with no tested compound. The influence of the compound on the growth of bacteria at concentrations of 3.12, 6.25, 12.5, 25, 50 and 100 μg/mL was evaluated.

3.4.3.3. In vitro cytotoxicity screening Compounds 7b, 7c and 7d were further tested for toxicity in a RAW 264.7 cell line at the concentration of 50 μM. After 72 h of exposure, viability was evaluated on the basis of cellular translation of MTT into a formazan product by the Promega Cell Titer 96 nonradioactive cell proliferation assay [32, 33].

3.4.4. Docking Study Docking studies of the title compounds (7a-z) was performed using Glide 5.9 (Extra Precision) running on maestro version 9.4, in order to investigate their in-silico biding affinity as well as their binding pattern with enzyme Pantothenate synthetase [34]. Enzyme used for the docking study was retrieved from RCSB Protein Data Bank (PDB ID: 3IUB) in complex with co- crystallised ligand (indole-2-carboxamide derivative). Selected protein consist of two chains asymmetric units (A and B), both consist of co-crystallized ligand, in the current study unit A was separated and used further for docking studies. Protein preparation wizard of Schr dinger suite was used for preparation of selected protein. Protein was pre-processed separately by deleting the substrate co-factor as well as the crystallographically observed water molecules (water without H bonds), followed by optimization of hydrogen bonds. After assigning charge and protonation position, finally energy was minimized with root mean square deviation (RMSD) value of 0.30 Å using optimized potentials for liquid simulations-2005 (OPLS-2005) force field [35]. Finally energy minimized protein and co-crystallized ligand was used to build energy grids using the default value of protein atom scaling (1.0 Å) within a cubic box of 14 Å dimensions, centered on the centroid of the X-ray ligand pose [36]. The structures of 7a-z were drawn using ChemSketch and converted to 3D structure with the help of 3D optimization tool.

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Using LigPrep 2.6 module, the drawn ligands were geometry optimized; partial atomic charges were computed using OPLS-2005 force field [37]. Finally, prepared ligands were docked with prepared protein using Glide 5.9 module, in extra precision mode (XP). The leading docked pose (with lowest Glide score value) found from Glide was analyzed. RMSD value was calculated between the experimental binding mode of co-crystallized ligand as in X-ray and re-docked pose to ensure accuracy and reliability of the docking procedure.

3.5. References [1] S. G. Küçükgüzel, S. Şenkardeş, Eur. J. Med. Chem., 2015, 97, 786. [2] P. P. Deohate, J. P. Deohate, B. N. Berad, Asian. J. Chem., 2004, 16, 255. [3] K. L. Kees, J. J. Fitzgerald, K. E. Steiner, J. F. Mattes, B. Mihan, T. Tosi, D. Moondoro, M. L. McCaleb, J. Med. Chem., 1996, 39, 3920. [4] (a) P. Diana, A. Carbone, P. Barraja, A. Martorana, O. Gia, L. DallaVia, G. Cirrincione, Bioorg. Med. Chem. Lett., 2007, 17, 6134; (b) H. J. Park, K. Lee, S. J. Park, B. Ahn, J. C. Lee, H. Y. Cho, K. I. Lee, Bioorg. Med. Chem. Lett., 2005, 15, 3307. [5] (a) N. Harikrishan, A. M. Isloor, K. Ananda, A. Obaid, H. K. Fun, New J. Chem., 2016, 40, 73; (b) N. K. Piyush, P. S. Shailesh and K. R. Dipak, New J. Chem., 2014, 38, 2902; (c) R. Manikannan, R. Venkatesan, S. Muthusubramanian, P. Yogeeswari, D. Sriram, Bioorg. Med. Chem. Lett., 2010, 20, 6920. [6] D. Raffa, B. Maggio, M. V. Raimondi, S. Cascioferro, F. Plescia, G. Cancemi, G. Daidone, Eur. J. Med. Chem., 2015, 97, 732. [7] M. G. Mamolo, D. Zampieri, V. Falagiani, L. Vio, E. Banfi, Il Farmaco, 2001, 56, 593. [8] R. B. Pathak, P. T. Chovatia, H. H. Parekh, Bioorg. Med. Chem. Lett., 2012, 22, 5129. [9] P. T. Chovatia, J. D. Akabari, P. K. Kachhadia, P. D. Zalavadia, H. S. Joshi, J. Serb. Chem. Soc., 2007, 71, 713. [10] R. C. Khunt, V. M. Khedkar, R. S. Chawda, N. A. Chauhan, A. R. Parikh, E. C. Coutinho, Bioorg. Med. Chem. Lett., 2012, 22, 666. [11] P. Palanisamy, S. Kumaresan, RSC. Adv., 2013, 3, 4704. [12] A. Suresh, N. Suresh, S. Misra, M. M. Krishna Kumar, K. V. G. Chandra Sekhar, ChemistrySelect, 2016, 1, 1705.

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[13] (a) M. Bhat, G.K. Nagarjuna, R. Kayarmar, S.K. Peethamber, R. Mohammed Shafeeulla, RSC Adv., 2016, 6, 59375; (b) H. C. Kolb, K. B. Sharpless, Drug. Discov. Today., 2003, 8, 1128. [14] D. Kumar, G. B. Khare, S. Kidwai, A. K. Tyagi, R. Singh, D. S. Rawat, Eur. J. Med. Chem., 2014, 81, 301. [15] S. R. Patpi, L. Pulipati, P. Yogeeswari, D. Sriram, N. Jain, B. Sridhar, R. Murthy, T. Anjana Devi, S. V. Kalivendi, S. Kantevari, J. Med. Chem., 2012, 55, 3911. [16] N. Boechat, V. F. Ferreira, S. B. Ferreira, M. L. G. Ferreira, F. C. da Silva, M. M. Bastos, M. S. Costa, M. S. Lourenço, A. C. Pinto, A. U. Krettli, A. C. Aguiar, B. M. Teixeira, N. V. da Silva, P. R. C. Martins, F. F. M. Bezerra, A. S. Camilo, G. P. da Silva, C. C. P. Costa, J. Med. Chem., 2011, 54, 5988. [17] S. Kim, S. Cho, T. Oh, P. Kim, Bioorg. Med. Chem. Lett., 2012, 22, 6844. [18] P. Shanmugavelan, S. Nagarajan, M. Sathishkumar, A. Ponnuswamy, P.Yogeeswari, D. Sriram, Bioorg. Med. Chem. Lett., 2011, 21, 7273. [19] H. N. Nagesh, K. Mahalakshmi Naidu, D. Harika Rao, J. P. Sridevi, D. Sriram, P. Yogeeswari, K. V. G. Chandra Sekhar, Bioorg. Med. Chem. Lett., 2013, 23, 6805. [20] R. Zheng, J. S. Blanchard, Biochemistry, 2001, 40, 12904. [21] S. Velaparthi, M. Brunsteiner, R. Uddin, B. Wan, S. G. Franzblau, P. A. Petukhov, J. Med. Chem. 2008, 51, 1999. [22] S. Wang, D. Eisenberg, Prot. Sci. 2003, 12, 1097. [23] Y. Yang, P. Gao, Y. Liu, X. Ji, M. Gan, Y. Guan, X. Hao, Z. Li, C. Xiao, Bioorg. Med. Chem. Lett., 2011, 21, 3943. [24] G. Samala, P. B. Devi, R. Nallangi, J. P. Sridevi, P. Yogeeswari, D. Sriram, Eur. J. Med. Chem., 2013, 69, 356. [25] G. Samala, P. B. Devi, R. Nallangi, J. P. Sridevi, P. Yogeeswari, D. Sriram, Bioorg. Med. Chem., 2014, 22, 1938. [26] E. L. White, K. Southworth, L. Ross, S. Cooley, R. B. Gill, M. I. Sosa, A. Manouvakhova, L. Rasmussen, C. Goulding, D. Eisenberg, T. M. Fletcher, J. Biomol. Screen., 2007, 12, 100. [27] G. Samala, R. Nallangi, P. B. Devi, S. Saxena, R. Yadav, J. P. Sridevi, P. Yogeeswari, D. Sriram, Bioorg. Med. Chem., 2014, 22, 4223.

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[28] G. Samala, P. B. Devi, S. Saxena, N. Meda, P. Yogeeswari, D. Sriram, Bioorg. Med. Chem., 2016, 24, 1298. [29] K. Mahalakshmi Naidu, S. Srinivasarao, N. Agnieszka, A. Ewa, M. M. Krishna Kumar, K. V. G. Chandra Sekhar, Bioorg. Med. Chem. Lett., 2016, 26, 2245. [30] X. M. Ye, A. W. Konradi, J. Smith, D. L. Aubele, A. W. Garofalo, J. Marugg, M. L. Neitzel, C. M. Semko, H. L. Sham, M. Sun, A. P. Truong, J. Wu, H. Zhang, E. Goldbach, J. M. Sauer, E. F. Brigham, M. Bova, G. S. Basi, Bioorg. Med. Chem. Lett., 2010, 20, 2195. [31] G.P. Youmans, A.S. Youmans, J. Bactriol., 1949, 58, 247. [32] P. B. Devi, G. Samala, J. P. Sridevi, S, Saxena, M, Alvala, E. G, Salina, D, Sriram, P. Yogeeswari, Chemmedchem, 2014, 9, 2538. [33] D. Gerlier, N. Thomasset, Immunol. Methods, 1986, 94, 57. [34] Glide, Schrödinger, LLC, New York, 2013 version 5.9. [35] W. L. Jorgensen, D. S. Maxwell, R. J. Tirado, J. Am. Chem. Soc., 1996, 118, 11225. [36] S. Chander, A. Penta, S. Murugesan, Journal of Pharmacy Research, 2014, 8, 552. [37] Lig-Prep, Schrödinger, LLC, New York, 2013 version 2.6.

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Chapter IV

Design, synthesis of 9H-fluorenone based 1,2,3-triazole analogues as Mycobacterium tuberculosis InhA inhibitors

Chapter 4

Chapter 4

Design, synthesis of 9H-fluorenone based 1,2,3-triazole analogues as Mycobacterium tuberculosis InhA inhibitors

4.1. Introduction Fluorenones contain a planar skeleton with fused aromatic rings and only one carbonyl prochiral center; these are normally used as photo-catalysts in organic synthesis. The fluorenone scaffold is widespread both in natural products and in industrial by-products. Over the last few years its derivatives have generated interest because of their use in various fields ranging from drugs to materials science. The fluorenone scaffold is found in natural biologically active (Kinamycins, Nakiterpiosin, Gracilamine, Dendroflorin, Gramniphenols D & E, and caulophine) and semisynthetic compounds [1, 2]. The representative fluorenone containing natural products are shown in Figure 4.1.

Figure 4.1: Fluorenone containing natural products and semi synthetic compounds.

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Dendroflorin containing fluorenone scaffold has antioxidant properties. Tilorone and 9- Fluorenylmethyl Polyglycosides are used for different viral diseases [3] and 2,7-disubstituted amidofluorenones[4], show anticancer properties. 9-Fluorenone-2-carboxylic acid is tubulin binders\ [2], 9-fluorenone carboxyhydroxyesters and amides are immunomodulators and anti- herpes simplex virus-2 agents [5]. 9-Hydroxyazafluorenes are thrombin inhibitors [6].

The fatty acid synthase arrangement of Mycobacterium tuberculosis contains unique signature fatty acid, the mycolic acid, which is a central constituent of the mycobacterial cell wall. Mycolic acid biosynthesis is carried out by several successive enzymatic cycles equivalent to two related but different Fatty Acid Synthase (FAS) systems, FAS I and II [7]. FAS II system is present in bacteria but is absent in humans. InhA protein (ENR, EC number: 1.3.1.9) is a key enzyme of FAS II and shows a NADH-dependent enoyl-ACP reductase activity. It has already been validated as the primary molecular target of the frontline anti-tubercular drug isoniazid [8]. It is a prodrug which upon activation by KatG, the mycobacterial catalase-peroxidase, forms adduct with NADH called NAD-INH [9]. The X-ray structure of the complex InhA isonicotinoyl moiety of this adducts shows that it occupies a hydrophobic pocket. Different research groups validated this cavity as a suitable site to increase inhibitors potency. Several approaches exist towards the design of new inhibitors for InhA [10].

Tilorone and doxorubicin inhibit primase DnaG from B. anthracis and MTB at the low micromolar range of IC50. Based on this Choi et al., modified fluorenone scaffold to various derivatives of C2 symmetry compounds that are similar to tilorone. They added different chain lengths and terminal groups and synthesized 9-fluorenone alkyl amines which exhibited antibacterial properties [1]. Genz-10850 (also called GEQ) (A) has been identified as a very promising inhibitor of InhA, after in vitro screening of a library of five million compounds [11]. Later, He et al., synthesized a series of GEQ derivatives with InhA inhibitory activities ranging from 0.09 to 2.04 μM [12]. Amongst these, (4-(9H-fluoren-9-yl)piperazin-1- yl)(phenyl)methanone (B) was the most active compound with IC50 0.09 μM. Unfortunately these molecules have poor in vitro activity against MTB (MIC ≥ 125 µM) because of their low absorptivity [12]. Matviiuk et al. reported 3-(9H-fluoren-9-yl)pyrrolidine-2,5-dione derivatives

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Chapter 4 with InhA inhibition at 50 μM ranging from 8 to ≥ 95% and they exhibited MIC in the range from 2 to ≥ 16 µg/mL against MTB H37Rv. Among these, C was the most active with InhA inhibition ≥ 95% at 50 μM. It also exhibited MTB MIC 19.6 μM against MTB H37Rv. Same group reported 3-heteryl substituted pyrrolidine-2,5-diones derivatives with InhA inhibition at 50 μM ranging from 9 to 56% and its exhibited MTB MIC ranged from 2.5 to 40 µg/mL. Amongst these, 1-benzyl-3-[4-(9H-fluoren-9-yl)-1-piperazinyl]-2,5-pyrrolidinedione (D) was the most active with the InhA inhibition of 56% at 50 μM and MTB MIC 2.5 µg/mL against MTB H37Rv [13]. Chollet et al., incorporated modifications in GEQ skeleton; piperazine was replaced with piperidine and other modifications include replacement of amide with sulfonyl, phosphonyl and phosphinamide groups. They also introduced 2- and 3-alkyl-substituted fluorenone derivatives as inhibitors of InhA with IC50 102 to 2690 nM and these derivatives exhibited MTB MIC ranging from 11 to 88.2 µM against MTB H37Rv. Among these series, (4-(3-(hexyloxy)-9H-fluoren-9- yl)piperazin-1-yl)(phenyl) methanone (E) bearing an additional hexyloxy chain on the fluorenone moiety demonstrated enhanced activity against InhA enzyme (IC50 up to 102 nM) as well as MTB growth (MIC 11 μM) [14]. Described InhA active compounds are depicted in Figure 4.2.

Figure 4.2: InhA inhibitors.

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Additionally, 1,2,3-triazole and its derivatives have attracted continued interest in the medicinal field owing to their varied biological activities such as anti-fungal [15], anti-bacterial [16], anti- allergic [17], anti-HIV, antiviral [18], anti-inflammatory, anesthetic [19], anti-cancer [20] and β - lactamase inhibition properties [21]. It is quite obvious that the favourable properties of 1,2,3- triazole ring similar to moderate dipole character, hydrogen bonding capability, and π stacking interactions, rigidity and stability under in vivo conditions are responsible for their easy binding with the biological targets and also improve their solubility in biological systems[22].

Kumar and co-workers reported the synthesis of triazole-isoniazid conjugates (F) and their in vitro evaluation as possible anti-TB agents against MTB H37Rv. The compounds exhibited potent activity against MTB strain with MIC values ranging from 0.195 to 1.56 μM [23]. Pullapati et al., reported synthesis of novel piperidine, piperazine, morpholine and thiomorpholine appended dibenzo[b,d]thiophene-1,2,3-triazoles (G) for in vitro activity against MTB H37Rv with MIC ranging from 0.78 to 1.56 μg/mL and these compounds showed lower cytotoxicity [24]. Boechat et al., reported (E)-N'-((1-(1-aryl)-1H-1,2,3-triazol-4- yl)methylene)isonicotinohydrazide derivatives (H) which exhibited activity with MIC values ranging from 0.62 to 1.5 μg/mL [25]. Yempala et al., published dibenzo[b,d]furan-1,2,3-triazole conjugates (I) with in vitro activity against MTB with MIC in the range of 0.78 to 50.0 μg/mL [26]. Goverdhan et al., reported novel-substituted 1,2,3-triazolylmethyl carbazoles (J) for in vitro antimycobacterial activity against MTB H37Rv with MIC values ranging from 6.25 to 50 μg/mL [27]. Menendez et al., reported 1,4-disubstituted triazoles and α-ketotriazole derivatives (K) these compounds with MIC values varied from 2 to 100 μg/mL against MTB H37Rv they also exhibited InhA inhibition from 10 to 58% at 50 μM [28]. The representative triazole derivatives are given in Figure 4.3.

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Figure 4.3: Anti-TB activity of triazole.

In our design, two key elements for InhA inhibition, i.e. the fluorenyl and the triazole moieties, were considered. The fluorenyl moiety that could act as an anchor [14] will occupy the same hydrophobic position than the long alkyl chain of the substrate. The triazole moiety could interact by hydrogen bonds with the hydroxyl group of the key residue Tyr158, essential for a good recognition. Furthermore, triazoles, synthesized in one step by “click” chemistry reaction, could bring diversity [23]. With this collective information and emphasizing on molecular hybridization approach we drew a synthetic stratagem to fit these imperative pharmacophoric groups into one distinct scaffold and synthesized N-((1-substituted phenyl-1H-1,2,3-triazol-4- yl)methyl)-9H-fluoren-9-amine, 4-(((9H-fluoren-9-yl)thio)methyl)-1-substituted phenyl-1H- 1,2,3-triazole, and 4-(((9H-fluoren-9-yl)sulfonyl)methyl)-1-substituted phenyl-1H-1,2,3-triazole analogues (Figure 4.4). Of note, only aryl groups were introduced on the triazole moiety of our designed target (Figure 4.4) because molecules bearing alkyl groups at this position were found to be less efficient as inhibitors against InhA enzyme [14].

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Figure 4.4: Design strategy to achieve title compounds.

4.2. Results and Discussion 4.2.1. Chemistry We synthesized N-((1-substituted phenyl-1H-1,2,3-triazol-4-yl)methyl)-9H-fluoren-9-amine, 4- (((9H-fluoren-9-yl)thio)methyl)-1-substituted phenyl-1H-1,2,3-triazole, and 4-(((9H-fluoren-9- yl)sulfonyl)methyl)-1-substituted phenyl-1H-1,2,3-triazole analogues as sketched in scheme 4.1 and scheme 4.2.

Scheme 4.1: Synthetic protocol to achieve the compound (12a-p)

Reagents and conditions: (i) NaBH4 (0.5 eq), MeOH, 0 ºC - rt, 4 h. (ii) PBr3 (1.2 eq), DCM, 0

ºC - rt, 4 h. (iii) Propargyl amine (1.2 eq), K2CO3 (2.0 eq), ACN, rt, 16 h. (iv) Substituted phenyl t- azides, CuSO4.5H2O (0.02 eq), Sodium ascorbate (0.01 eq), H2O: BuOH (1:2), rt, 4 h.

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In the scheme 4.1 we adopted reported procedure with slight modification to prepare 9H-fluoren- 9-ol (9), then the compound 9 was brominated with phosphorus tribromide to form compound

(10) [29]. Compound 10 on reacting with propargyl amine in the presence of K2CO3 formed N- alkyl product (11). The free acetylene group was converted to various N-((1-substituted phenyl- 1H-1,2,3-triazol-4-yl)methyl)-9H-fluoren-9-amines (12a-p) using different aromatic azides via click chemistry method [30].

Scheme 4.2: Synthetic protocol to achieve the compounds (15a-r) & (17a-p).

Reagents and conditions: (i) Lawesson's reagent (2.0 eq), toluene, 110 ºC, 16 h. (ii) Propargyl bromide (80% in toluene) (2.0 eq), TEA (3.0 eq), DCM, 16 h. (iii) Substituted aromatic azides, t- CuSO4.5H2O (10 mole %), Sodium ascorbate (5 mole %), H2O: BuOH (1:2), rt, 16 h. (iv) meta-

Chloroperoxybenzoic acid (2.0 eq), DCM, rt, 2h. (v) Substituted aromatic azides, CuSO4.5H2O t- (10 mol %), Sodium ascorbate (5 mole %), H2O: BuOH (1:2), rt, 16 h.

In the scheme 4.2 9H-fluorene-9-thiol (13) was obtained from compound 9 using lawesson's reagent in toluene at 110 °C for 16 h [31]. 13 on reacting with propargyl bromide in the presence of TEA formed (9H-fluoren-9-yl)(prop-2-yn-1-yl)sulfane (14). The compound 14 was converted to various 4-(((9H-fluoren-9-yl)thio)methyl)-1-substituted phenyl-1H-1,2,3-triazoles (15a-r) using different aromatic azides via click chemistry method [30]; further compound 14 on oxidation with meta-chloroperbenzoic acid in the presence of DCM at room temperature formed

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9-(prop-2-yn-1-ylsulfonyl)-9H-fluorene (16). Subsequently 16 was converted to various 4-(((9H- fluoren-9-yl) sulfonyl) methyl)-1-substituted phenyl-1H-1,2,3-triazole (17a-p) using different aromatic azides [30]. The purity of compounds synthesized was checked by LC-MS and elemental analyses. Structures of the compounds were confirmed by spectral data. In 1H NMR and 13C NMR, the signals of the respective protons and carbons were verified on the basis of their chemical shifts, multiplicities, and coupling constants. The results of elemental analysis were within ± 0.05 of the theoretical values.

4.2.2. In-vitro MTB screening All the synthesized compounds were tested for their capacity to inhibit the growth of MTB. In assay three different M. tuberculosis strains were used. One of them was reference strain M. tuberculosis H37Rv ATTC 25618 and the others were ‘wild’ strains isolated from tuberculosis patients [32]. MTB strain spec. 210 was resistant to p-aminosalicylic acid (PAS), INH, ETB and RMP and another Spec. 192 was fully sensitive to the administrated tuberculostatics [33]. In this study three different strains were used for screening as we wanted to know the kind of activity synthesized compounds showed against the reference strain as well as against the strains isolated from TB patients. The influence of the compound on the growth of mycobacteria at certain concentrations 3.1, 6.2, 12.5, 25, 50 and 100 μg/mL were evaluated. INH was used as reference drug. The in vitro antimycobacterial results of title compounds are arranged in Table 4.1 as MIC (μM) and the activity ranged from 52.35 ->250 μM.

Table 4.1: Antimycobacterial activities of compounds 12a-p, 15a-r & 17a-p against MTB H37Rv, Spec. 192 and Spec. 210 in μM. MIC (µg/mL) MIC (µg/mL) MIC (µg/mL) Entry Ar against MTB against MTB against MTB H37Rv Spec. 192 Spec. 210 12a Phenyl >295.49 (>100) >295.49 (>100) >295.49 (>100) 12b 4-Methylphenyl 141.87 (50) 141.87 (50) 141.87 (50) 12c 4-Ethylphenyl 136.44 (50) 136.44 (50) 136.44 (50) 12d 4-Methoxyphenyl 135.71 (50) 135.71 (50) >271.42 (>100)

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MIC (µg/mL) MIC (µg/mL) MIC (µg/mL) Entry Ar against MTB against MTB against MTB H37Rv Spec. 192 Spec. 210 12e 4-Fluorophenyl 280.58 (100) 280.58 (100) >280.58 (>100) 12f 4-Chlorophenyl 268.22 (100) 268.22 (100) 268.22 (100) 12g 4-Bromophenyl 239.63 (100) 239.63 (100) 239.63 (100) 12h 4-Trifluoromethylphenyl 123.30 (50) 123.30 (50) 123.30 (50) 12i 4-Nitrophenyl >260.82 (>100) >260.82 (>100) >260.82 (>100) 12j 2-Fluorophenyl 280.58 (100) 280.58 (100) 280.58 (100) 12k 2-Nitrophenyl 260.82 (>100) >260.82 (>100) >260.82 (>100) 12l 3,4-dimethylphenyl >272.88 (>100) >272.88 (>100) >272.88 (>100) 12m 3-Chloro,4-fluorophenyl >255.85 (>100) >255.85 (>100) >255.85 (>100) 12n 2,4-dichlorophenyl >245.51 (>100) >245.51 (>100) >245.51 (>100) 12o 3,5-dichlorophenyl >245.51 (>100) >245.51 (>100) >245.51 (>100) 12p 3,4,5-trimethoxyphenyl 58.34 (25) 58.34 (25) 58.34 (25) 15a Phenyl 140.66 (50) 140.66 (50) 140.66 (50) 15b 4-Methylphenyl 135.32 (50) 135.32 (50) 135.32 (50) 15c 4-Ethylphenyl 260.74 (100) 260.74 (100) >260.74 (>100) 15d 4-Methoxyphenyl 129.70 (50) 129.70 (50) >259.41 (>100) 15e 4-Fluorophenyl 66.94 (25) 66.94 (25) >267.77 (>100) 15f 4-Chlorophenyl 74.20 (25) 74.20 (25) >256.80 (>100) 15g 4-Bromophenyl 57.55 (25) 57.55 (25) 115.1 (50) 15h 4-Trifluoromethylphenyl >236.21 (>100) >236.21 (>100) >236.21 (>100) 15i 4-Nitrophenyl >249.71 (>100) >249.71 (>100) >249.71 (>100) 15j 2-Chlorophenyl >256.47 (>100) >256.47 (>100) >256.47 (>100) 15k 2-Nitrophenyl >249.71 (>100) >249.71 (>100) >249.71 (>100) 15l 3,4-dimethylphenyl >260.74 (>100) >260.74 (>100) >260.74 (>100) 15m 4-Fluoro, 2-nitrophenyl >238.98 (>100) >238.98 (>100) >238.98 (>100)

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MIC (µg/mL) MIC (µg/mL) MIC (µg/mL) Entry Ar against MTB against MTB against MTB H37Rv Spec. 192 Spec. 210 15n 2,4-dichlorophenyl >235.65 (>100) >235.65 (>100) >235.65 (>100) 15o 3,4-dichlorophenyl >235.65 (>100) >235.65 (>100) >235.65 (>100) 15p 3,5-dichlorophenyl 117.82 (50) 117.82 (50) >235.65 (>100) 15q 3,4,5-trimethoxyphenyl 56.11 (25) 56.11 (25) 56.11 (25) 15r 3-Chloro,4-fluorophenyl 245.61 (100) 245.61 (100) >245.61 (>100) 17a Phenyl >258.09 (>100) >258.09 (>100) 258.09 (>100) 17b 4-Methylphenyl 124.53 (50) 124.53 (50) 124.53 (50) 17c 4-Ethylphenyl 120.33 (50) 120.33 (50) 240.66 (>100) 17d 4-Methoxyphenyl 119.76 (50) 119.76 (50) 119.76 (50) 17e 4-Fluorophenyl 123.32 (50) 123.32 (50) 123.32 (50) 17f 4-Chlorophenyl 118.51 (50) 118.51 (50) 118.51 (50) 17g 4-Bromophenyl >214.43 (>100) >214.43 (>100) >214.43 (>100) 17h 4-Trifluoromethylphenyl 109.78 (50) 109.78 (50) 109.78 (50) 17i 4-Nitrophenyl >231.24 (>100) >231.24 (>100) >231.24 (>100) 17j 2-Nitrophenyl >231.24 (>100) >231.24 (>100) >231.24 (>100) 17k 2-Chlorophenyl 237.02 (100) 237.02 (100) 237.02 (100) 17l 3,4-dimethylphenyl 120.33 (50) 120.33 (50) 120.33 (>100) 17m 4-Fluoro, 2-nitrophenyl >222.00 (>100) >222.00 (>100) >222.00 (>100) 17n 2,4-dichlorophenyl >219.13 (>100) >219.13 (>100) >219.13 (>100) 17o 3,4-dichlorophenyl >219.13 (>100) >219.13 (>100) >219.13 (>100) 17p 3,4,5-trimethoxyphenyl 52.35 (25) 52.35 (25) 52.35 (25) INH - 22.59 (<3.1) 22.59 (<3.1) 91.14 (<12.5)

Among the three series of 9H-fluorenone analogues, totally fifty compounds were screened. Fifteen compounds (12b, 12c, 12d, 12h, 15a, 15b, 15d, 15p, 17b, 17c, 17d, 17e, 17f, 17h, & 17l) showed moderate activity with MIC ranging from 141.87 to 109.78 μM. Five compounds

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(12p, 15e, 15f, 15g, & 15q) showed good activity with MIC 74.20 to 56.11 μM. Compound 17p (4-(((9H-fluoren-9-yl)sulfonyl)methyl)-1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazole) was found to be the most active compound with in vitro MIC 52.35 μM.

Structure activity relationship (SAR) of N-((1-substituted phenyl-1H-1,2,3-triazol-4-yl)methyl)- 9H-fluoren-9-amines derivatives (12a-p) In this series, we screened sixteen compounds against the three different strains (MTB H37Rv, MTB spec. 192 & MTB spec. 210). We noticed that among electron withdrawing and electron donating substituents on the triazoles, electron donating group containing substituent show major impact in exhibiting anti-TB activity. SAR is explained based on activity of 12a. Compound 12a was inhibiting 99% growth of MTB strains at 295.49 μM. Introduction of electron donating group on the phenyl ring increased activity. Compounds 12b (MIC 141.87 μM), 12c (MIC 136.44 μM) and 12d (MTB 135.71 μM) with methyl, ethyl, methoxy groups increased the activity by two folds compared to 12a. Introduction of electron withdrawing groups viz., F, Cl, Br and NO2 at either second or fourth position in phenyl resulted in either decrease in th activity or the activity remained unaltered. Exception being electron withdrawing CF3 at 4 position with which the activity increased by two fold (12h, MIC 123.30 μM). Compound 12p with three electron donating methoxy groups emerged to be the most active compound among these sixteen derivatives with MIC 58.34 μM exhibiting fivefold increase in activity compared to 12a.

SAR of sulfide derivatives (15a-r) Among the eighteen sulfide derivates SAR is explained with respect to compound 15a (140.66 μM). Presence of electron donating groups at the 4th position impacted the activity. Interestingly, presence of electron withdrawing halogens like F, Cl, Br at the 4th position resulted in increase in activity by the two folds. But the presence of electron withdrawing at the ortho position decreased the activity by two folds. Among the dichloro substituted compounds, 15p was most active with MIC 117.82 μM similar to that of 15a. In this series, trimethoxy derivative 15q was most active with MIC 56.11 μM.

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SAR of sulfonyl derivatives (17a-p) Eighteen compounds based on sulfonyl were synthesized and screened for MTB. SAR is explained with respect to 18a which showed MIC 258.09 μM. Activity increased by more than two folds with electron dontaining 4-methyl, 4-ethyl and 4-methoxy (MIC 124.53, 120.33 and 119.76 μM with respectively). Among the halo derivatives, activity remained unaltered with bromo where as it increased by two folds with 4-flouro and 4-chloro with derivatives. Electron nd th withdrawing NO2 at 2 & 4 positions did not impact the activity with electron withdrawing disubstituted derivatives activity remained with unaltered (17m, 17n & 17o). Presence of electron donating dimethyl increased the activity by two folds (17l, MIC 120.33). Among this series, 17p with three methoxy groups emerged to be the most active compound (MIC 52.35 μM). Over all, we notice that sulfide derivatives exhibited better anti-TB activity followed by sulfonyl derivative and amines. Electron donating 3,4,5-trimethoxy derivates emerged to be the most active compound in all the series of compounds.

4.2.3. InhA enzyme Inhibition studies The compounds were tested for their capacity to inhibit the reduction of the substrate double bond by NADH in the presence of InhA. The assays were performed in triplicate in the presence of the substrate analogue 2-trans-dodecenoyl-CoA and the percentage of InhA inhibition was determined by measuring the conversion of the NADH cofactor to its oxidized form NAD+ by means of the decreasing of the absorbance at 340 nm [34]. The molecules were tested at 50 Μm, GEQ was used as reference and results are reported in Table 4.2. 15 compounds whose activities were ≤ 50 μg/mL were selected for screening the InhA activity. Among the amine derivatives (12a-p) the most active compound 12p (MIC 25 μg/mL) was selected for screening; among the sulfide derivatives except 15d all other compounds with MIC ≤ 50 μg/mL were selected. Among the sulfonyl derivatives except 17b and 17d remaining all other compounds with MIC ≤ 50 μg/mL were selected for screened. Investigations on these fifteen compounds indicated that among the -NH- group containing 9H- fluorene derivate, 12p exhibited only 7% inhibition. Presence of sulpur in the 9H-fluorene increased the InhA activity, as the results were moderate in this series of compounds. 15p showed the maximum InhA inhibition of 31%. The series of sulfonyl (-SO2-) compounds showed

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Chapter 4 relatively good inhibition. Compounds 17f and 17p were the most active with 74 and 73% inhibition. On the whole we notice that compounds with (O=S=O) exhibited the highest InhA inhibition which is in agreement with the already reported literature [14]. Table 4.2: MTB InhA activity Compound % of inhibition S. No Code at 50 μM of inhibitor 1 12p 7 2 15a 23 3 15b 24 4 15e 6 5 15f < 5 6 15g NI 7 15h 21 8 15p 31 9 15q 30 10 17c 27 11 17e NI 12 17f 74 13 17h 27 14 17l NI 15 17p 73 16 GEQ 88 NI = No inhibition 4.2.4. Docking study All the final compounds were docked into the crystal structure of InhA protein (PDB ID: 1BVR) to know the exact binding pattern with the receptor. Validation of docking protocol revealed that, the value of RMSD obtained between experimental binding mode of co-crystallized ligand (as in X-ray) and its re-docked pose (Figure 4.6) was found to be 0.76, which suggested that, docking procedure could be relied on for further docking studies.

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Figure 4.5: Superimposed view of co-crystallized ligand in 1BVR.

Further, in the docking studies, molecules exhibited good binding energy in the range of -5.65 to -10.36 kcal/mol and exhibited good fitness with the InhA protein. Several compounds displayed interactions with hydrophobic pockets MET103, ILE215, ALA22, ALA157, ALA198, LEU63, LEU218, PRO193, MET103, MET199, PHE41, PHE149, ILE194, ALA191, ILE95, ILE21, ILE122, VAL65, MET147, ILE95 and MET161, hydrogen bonding interaction with ILE194 amino acid residues. Ligand also exhibited π-π interactions with amino acids. Compound 4- (((9H-fluoren-9-yl)sulfonyl)methyl)-1-(4-chlorophenyl)-1H-1,2,3-triazole (17f) with 74% of inhibition at 50 μM showed docking score of -7.808 kcal/mol. This compound exhibits hydrophilic interaction with THR196 and SER94. The active site in the hydrophobic pocket is within the vicinity of MET103, ILE215, ALA157, LEU218, PRO193, MET199, PHE149, ILE194, ALA191, ILE21, MET147, ILE95 and MET161. 17f showed π-π interactions with PHE149 and TYR 158. One of the ligands, 4-(((9H-fluoren-9-yl)sulfonyl)methyl)-1-(3,4,5- trimethoxyphenyl)-1H-1,2,3-triazole (17p) with 73% of inhibition at 50 μM showed docking score of -8.298 kcal/mol. The active site in the hydrophobic pocket is within the vicinity of MET103, MET199, ALA198, ILE194, ILE21, ALA22, PHE41, VAL65, ILE122, PHE97, LE63, ILE15, ILE16 and ILE95 as well as some polar amino acid residues THR196, SER20, SER94, GLN66, THR39 and SER13 respectively. The ligand also exhibited π-π interactions with PHE41.

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These results correlate with the in vitro InhA and MTB screening. The binding pattern of 17f and 17p with InhA is shown in Figures 4.6 & 4.7.

Figure 4.6: Docked pose of compound 17f inside the 1BVR, showing two-dimensional interactive diagram.

Figure 4.7: Docked pose of compound 17p inside the 1BVR, showing two-dimensional interactive diagram.

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Table 4.3: Docking scores

Compound Docking XPG Glide glide score Score gscore emodel 1bvr.pdb.1_ligand(Standard) -6.812 -8.867 -8.867 -65.276 12a -8.287 -8.364 -8.364 -62.465 12b -8.215 -8.292 -8.292 -57.717 12c -8.452 -8.472 -8.472 -59.783 12d -8.701 -9.951 -9.951 -59.502 12e -7.881 -7.958 -7.958 -58.549 12f -7.376 -8.625 -8.625 -65.755 12g -8.064 -8.141 -8.141 -62.141 12h -8.872 -8.948 -8.948 -65.417 12i -6.06 -6.137 -6.137 -67.008 12j -8.639 -8.716 -8.716 -60.259 12k -5.515 -6.764 -6.764 -70.419 12l -8.734 -8.811 -8.811 -61.442 12m -8.67 -8.747 -8.747 -65.104 12n -6.763 -8.013 -8.013 -68.347 12o -8.834 -8.91 -8.91 -70.276 12p -8.543 -8.543 -8.543 -61.742 15a -8.282 -8.282 -8.282 -66.587 15b -8.265 -8.265 -8.265 -64.06 15c -7.995 -7.995 -7.995 -63.307 15d -6.551 -6.551 -6.551 -66.408 15e -7.699 -7.699 -7.699 -63.197 15f -8.54 -8.54 -8.54 -62.504 15g -7.935 -7.935 -7.935 -66.545 15h -9.421 -9.421 -9.421 -67.959 15i -5.655 -5.655 -5.655 -73.157 15j -8.1 -8.1 -8.1 -59.23 15k -8.308 -8.308 -8.308 -56.864 15l -8.777 -8.777 -8.777 -67.67 15m -6.695 -6.695 -6.695 -64.54 15n -8.205 -8.205 -8.205 -66.244 15o -8.324 -8.324 -8.324 -71.519 15p -9.233 -9.233 -9.233 -63.54 15q -10.365 -10.365 -10.365 -66.401 17a -8.572 -8.572 -8.572 -71.139

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17b -8.753 -8.753 -8.753 -69.495 17c -8.886 -8.886 -8.886 -67.672 17d -6.609 -6.609 -6.609 -69.43 17e -8.203 -8.203 -8.203 -71.884 17f -7.808 -7.808 -7.808 -66.086 17g -8.193 -8.193 -8.193 -71.343 17h -9.655 -9.655 -9.655 -72.269 17i -6.274 -6.274 -6.274 -71.62 17j -8.154 -8.154 -8.154 -71.356 17k -7.715 -7.715 -7.715 -74.456 17l -6.563 -6.563 -6.563 -71.969 17m -6.416 -6.416 -6.416 -71.38 17o -9.279 -9.279 -9.279 -78.853 -8.298 -8.298 -8.298 -68.615 17p

4.2.5. In vitro cytotoxicity studies Compounds with MTB MIC < 25 µg/mL were subjected to cytotoxicity studies against HEK 293 cell line. Cytotoxicity assay of 12p, 15e, 15f, 15g, 15q & 17p was determined. Cell viability was measured by in vitro MTT assay [35]. Cells were exposed to compounds for 24 hours at three concentrations 50µM, 25 µM and 10 µM (n=2). Data represent mean values of measurements ± s.d. (Figure 4.8). Data clearly indicate the active compounds were not toxic at even 50 µM.

Figure 4.8: Cytotoxicity assay of 12p, 15e, 15f, 15g, 15q & 17p on HEK-293 cells.

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4.2.6. Single Crystal X-ray Crystallographic Structure of Compound 15a The suitable crystals of the compound 15a for X-ray crystallographic study were grown from ethylacetate solution. The single crystal X-ray diffraction measurement of the molecule

(C22H17N3S) was done using Rigaku XtaLAB P200 diffract meter using graphite monochromated Mo-Kα radiation (λ = 0.71073 Å) on 0.2 mm x 0.15 mm x 0.1 mm pale yellow crystal. Data were collected and processed using CrysAlisPro (Rigaku Oxford Diffraction). The data were collected at a temperature of 20 ± 2 °C to a maximum 2θ value of 49.99°. Of the 10529 reflections collected, 2118 were unique (Rint = 0.0328) and equivalent reflections were merged. The diffraction data were refined and structure was solved using Olex 2 version 2.1, ShelXL software program. The structure was solved by direct methods and expanded using Fourier techniques. The non-hydrogen atoms were refined anisotropically. Hydrogen atoms were refined using the riding model. The compound crystallized into a monoclinic crystal system with P21/c space group. In a single unit cell four partially occupying molecules of crystallization are observed with Z=4. The basic crystallographic data are shown in Table 4.4. The molecular structure of the compound crystallization is given as an ORTEP diagram in Figure 4.8. Crystallographic data for the compound 15a is deposited to the Cambridge Crystallographic Data Center and corresponding deposition number is CCDC 1523811.

Table 4.4: Crystal data and structure refinement for 15a

Empirical Formula C22H17N3S Formula Weight 355.44 Crystal Color, Habit Light yellow Crystal Dimensions 0.2 mm × 0.15 mm × 0.1 mm Crystal System Monoclinic Lattice Type Primitive Lattice Parameters a = 13.509(2) A° b = 5.6467(8) A° c = 22.601(4) A° α = 90 A° β = 95.080(16) A° γ = 90 A°

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δ = 1717.3(5) A°3

Space Group P21/c Z value 4 3 Dcalc 1.375g/cm

F000 744.00 µ(MoKα) 19.99 cm-1 Radiation Mo-Kα(λ = 0.71073 A°) Radiation monochromator Graphite Voltage, Current 50kV, 40mA Temperature 19.5 °C Maximum 2θ 49.992° Number of measured reflections 10529°

Number of Unique reflections 2998 (Rint = 0.0328) Number of parameters 235 Goodness-of-fit on F2 1.071 - 3 Δρmax,mix(e /A° ) 0.35, -0.28

Residuals: R1 (I>2.00σ(I)) R1 = 0.0346, wR2 = 0.0931

Residuals: R (All reflections) R1 = 0.0388 Residuals: wR2 (All reflections) 0.0955 Crystal refinement Olex 2 version 2.1, ShelXL, ShelXL

Figure 4.9: ORTEP diagram showing the X-ray crystal structure of the compound 15a.

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4.2.5.1 Single Crystal X-ray Crystallographic Structure of Compound 15r The suitable crystals of the compound 15r for X-ray crystallographic study were grown from ethylacetate solution. The single crystal X-ray diffraction measurement of the molecule

(C22H15ClFN3S) was done using Rigaku XtaLAB P200 diffract meter using graphite monochromated Mo-Kα radiation (λ = 1.54184 Å) on 0.7 mm x 0.05 mm x 0.05 mm pale yellow crystal. Data were collected and processed using CrysAlisPro (Rigaku Oxford Diffraction). The data were collected at a temperature of -173 ± 2 °C to a maximum 2θ value of 133.144°. Of the 7975 reflections collected, 3213 were unique (Rint = 0.0122) and equivalent reflections were merged. The diffraction data were refined and structure was solved using Olex 2 version 2.1, ShelXL software program. The structure was solved by direct methods and expanded using Fourier techniques. The non-hydrogen atoms were refined anisotropically. Hydrogen atoms were refined using the riding model. The compound crystallized into a triclinic crystal system with P-1 space group. In a single unit cell four partially occupying molecules of crystallization are observed with Z=2. The basic crystallographic data are shown in Table 4.5. The molecular structure of the compound crystallization is given as an ORTEP diagram in Figure 4.9.

Figure 4.10: ORTEP diagram showing the X-ray crystal structure of the compound 15r.

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Table 4.5: Crystal data and structure refinement for 15r

Empirical Formula C22H15ClFN3S Formula Weight 407.88 Crystal Color, Habit Light yellow Crystal Dimensions 0.7 mm × 0.05 mm × 0.05 mm Crystal System Triclinic Lattice Type Primitive Lattice Parameters a = 6.7474(2) A° b = 7.4267(2) A° c = 18.8856(5) A° α = 86.538(2) A° β = 82.680(2) A° γ = 77.639(2) A° Volume/ A°3 δ = 916.37(4) Space Group P-1 Z value 2 3 Dcalc 1.478g/cm

F000 420.00 µ(MoKα) 31.02 cm-1 Radiation Cu-Kα(λ = 0.71073 A°) Radiation monochromator Graphite Voltage, Current 50kV, 40mA Temperature -173 °C Maximum 2θ 133.144° Number of measured reflections 7975°

Number of Unique reflections 3213 (Rint = 0.0122) Number of parameters 253 Goodness-of-fit on F2 1.049 - 3 Δρmax,mix(e /A° ) 0.35, -0.28

Residuals: R1 (I>2.00σ(I)) R1 = 0.0291, wR2 = 0.0757

Residuals: R (All reflections) R1 = 0.0293

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Residuals: wR2 (All reflections) 0.0759 Crystal refinement Olex 2 version 2.1, ShelXS, ShelXL

4.3. Conclusion In this chapter, we designed novel 9H-fluorenone analogues with three different series N-((1- substituted phenyl-1H-1,2,3-triazol-4-yl)methyl)-9H-fluoren-9-amine, N4-(((9H-fluoren-9- yl)thio)methyl)-1-substituted phenyl-1H-1,2,3-triazole & 4-(((9H-fluoren-9-yl) sulfonyl) methyl)-1-substituted phenyl-1H-1,2,3-triazole) by the molecular hybridization approach using reported MTB InhA inhibitors and substituted 1H-1,2,3-triazole antitubercular compounds. Fifty compounds were synthesized and characterized. One of the compounds 17p showed good MTB activity with MIC 52.35 μM. Out of fifty compounds studied InhA activity was studied for fifteen compounds. Amongst these compounds, 17f & 17p showed >73% of inhibition at 50 μM. Further, the most active compounds did not exhibit cytotoxicity against HEK 293 cell line for the most active compounds at 50 μM.

4.4. Experimental Section 4.4.1. Materials and methods Chemicals and solvents were procured from commercial source. The solvents and reagents were of LR grade and if necessary purified before use. Thin-layer chromatography (TLC) was carried out on aluminium-supported silica gel plates (Merck 60 F254) with visualization of components by UV light (254 nm). Column chromatography was carried out on silica gel (Merck 100-200 mesh). 1H NMR spectra and 13C NMR spectra were recorded at 400 MHz using a Bruker AV

400 spectrometer (Bruker CO., Switzerland) in CDCl3 and DMSO-d6 solution with tetramethylsilane as the internal standard, and chemical shift values (δ) were given in ppm. Melting points were determined on an electro thermal melting point apparatus (Stuart-SMP30) in open capillary tubes and are uncorrected. IR spectra were recorded with an FT-IR spectrophotometer (Jasco FTIR-4200). Elemental analyses were analyzed by Elementar Analysensysteme GmbH vario MICRO cube CHNS/O Analyzer. Mass spectra (ESI-MS) were recorded on Schimadzu MS/ESI mass spectrometer. Purity of all tested compounds was greater than 95%.

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4.4.2. Chemistry Synthesis of 9H-fluoren-9-ol (9) A solution of 9H-fluoren-9-one (10.0 g, 55.49 mmol ) in methanol was cooled to 0 °C add sodium borohydride (1.0 g, 27.74 mmol) was slowly added at 0 °C andallowed to reach room temperature andstirred the reaction mixture for 2 h. Once completion of the reaction, as indicated by TLC, the reaction was quenched with excess of methanol at 0 °C. Added excess of water and stirred for 30 minutes. White solid formed was filtered and was washed with excess of water. 9H-fluoren-9-ol (2) was dried in oven at 60 °C for 6 h. Yield (9.5 g, 93%). ESI-MS found m/z 183.07 (M+H)+; m.p. 153-154 ºC (reported m.p. 152-156 ºC).

Synthesis of 9-bromo-9H-fluorene (10) A solution of 9H-fluoren-9-ol (5.0 g, 27.43 mmol) in dichloromethane was cooled at 0 °C under the N2. Then PBr3 (3.12 mL, 32.92 mmol) was slowly added over 15 minutes at 0 °C to it. The mixture was kept at 0 °C for two hours and then saturated potassium bromide solution was slowly added under stirring until no bubble was generated. Water was added to reaction mixture and extracted three times with dichloromethane. The combined organic layers were washed with brine and dried over anhydrous sodium sulfate, filtered, and concentrated to provide a crude product which was purified by recrystalization from petroleum ether to afford pale yellow crystals yield:(6.2 g, 92%). ESI-MS found m/z 244.98 (M+H)+; 246.98 (M+H)+2; m.p. 104-105 ºC (reported m.p. 101-105 ºC).

Synthesis of N-(prop-2-yn-1-yl)-9H-fluoren-9-amine (11) A solution of 9-bromo-9H-fluorene (10) (5.0 g, 20.39 mmol) in ACN was cooled to 0 °C and

K2CO3 (5.63 g, 40.79 mmol) and propargylamine (1.56 mL, 24.47 mmol) were added and allowed to reach room temperature and stirred for 16 h. Once completion of the reaction, as indicated by TLC, the reaction was quenched with cold water and extracted with diethyl ether. The organic layers were collected, washed with saturated brine solution, dried over anhydrous

Na2SO4 and concentrated in vacuo. The resultant crude product was purified by column chromatography [ethyl acetate / hexane (15 - 25%)] to get the compound 11 (4.8 g, 85%) as a + +2 1 brown solid. ESI-MS found m/z 220.11 (M+H) ; 222.11 (M+H) . H NMR (400 MHz, CDCl3) δ 7.68 (d, J = 7.5 Hz, 2H), 7.51 (d, J = 8.1 Hz, 2H), 7.32 (t, J = 7.4 Hz, 2H), 7.25 (dd, J = 7.9, 5.9

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13 Hz, 2H), 5.03 (s, 1H), 3.31 (s, 2H), 2.61 (s, 1H); C NMR (101 MHz, CDCl3) δ 145.55, 141.08, 128.79, 127.91, 126.78, 126.18, 83.47, 70.17, 63.16, 37.56.

Synthesis of N-((1-substituted phenyl-1H-1,2,3-triazol-4-yl)methyl)-9H-fluoren-9-amine (12a- p) A solution of N-(prop-2-yn-1-yl)-9H-fluoren-9-amine (11) (0.30 g, 1.0 equiv.) is reacted with substituted phenyl azides (1.2 equiv.) in the presence of sodium ascorbate (0.01 equiv.), t- CuSO4.5H2O (0.02 equiv.) and BuOH:H2O (2:1), at room temperature for 4 h. Once completion of the reaction, as indicated by TLC, the reaction was quenched with cold water and extracted with DCM. The DCM layers were collected, washed with saturated brine solution, dried over anhydrous Na2SO4 and concentrated in vacuo. The resultant crude product was purified by column chromatography [MeOH / DCM (1 -3%)] to yield the title compounds 12a-p.

Synthesis of 9H-fluorene-9-thiol (13) To a solution of 9H-fluoren-9-ol (9) (5.0 g, 27.43 mmol) in toluene, Lawesson reagent (11.09 g,

27.43 mmol) was added. The reaction mixture was refluxed under N2 atmosphere for 16 h. Once completion of the reaction, as indicated by TLC, the reaction was quenched with cold water and extracted with diethyl ether. The organic layers were collected, washed with saturated brine solution, dried over anhydrous Na2SO4 and concentrated in vacuo. The resultant crude product was purified by column chromatography [ethyl acetate / hexane (5 - 7%)] to get the 9H-fluorene- 9-thiol (13) (4.0 g, 74%) as a light yellow solid. ESI-MS found m/z 199.06 (M+H)+; m.p. 105- 106 ºC (reported m.p. 103-107 ºC) .

Synthesis of (9H-fluoren-9-yl)(prop-2-yn-1-yl)sulfane (14) To a stirred solution of 9H-fluorene-9-thiol (4.0 g, 20.17 mmol) in dichloromethane (DCM), triethylamine (8.48 mL, 60.52 mmol) and propargyl bromide (80% in toluene) (3.0 mL, 40.34 mmol) were added. Reaction mixture was stirred at ambient temperature for 16 h. Reaction was monitored by TLC and water was added to reaction mixture once complete and was followed by extraction with ethyl acetate. Combined organic layers were collected and dried over dry sodium sulphate. Concentrated the organic layer and purified by column chromatography [ethyl acetate /

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Chapter 4 hexane (10 - 20%)]to get compound 14 (4.2 g, 87%) as light yellow solid. ESI-MS found m/z 237.08 (M+H)+.

Synthesis of 4-(((9H-fluoren-9-yl)thio)methyl)-1-substituted phenyl-1H-1,2,3-triazole (15a-r) To a stirred solution of compound 14 (1.0 mmol) and substituted phenyl azide (1.2 mmol) in t butanol:water (1:1) (4 mL), CuSO4.5H2O (10 mol %) (0.2 mmol) and sodium ascorbate (5 mol %) (0.2 mmol) were added and the reaction mixture was stirred at RT for 16 h. After completion of the reaction, as indicated by TLC, butanol was removed under reduced pressure. The residue was extracted with ethyl acetate (3 x10 mL) and combined organic layers were collected and washed with saturated brine solution, dried over anhydrous Na2SO4 and concentrated in vacuo to get the crude product. The product was further purified by column chromatography [ethyl acetate / hexane (35 - 40%)] to afford the title compounds 15a-r.

Synthesis of 9-(prop-2-yn-1-ylsulfonyl)-9H-fluorene (16)A stirred solution of compound 14 (3.0 g, 12.69 mmol) in dichloromethane was cooled to 0 °C and meta-Chloroperoxybenzoic acid (8.48 mL, 25.38 mmol) was added slowly at 0 °C. The reaction mixture was stirred at room temperature for 2 h. Reaction was monitored by TLC and water was added to reaction mixture once complete and was followed by extraction with dichloromethane. Combined organic layers were collected and washed NaHCO3 solutions. Concentrated the organic layer and purified by column chromatography [ethyl acetate / hexane (20 - 30%)]to get compound 9 (3.1 g, 91%) as light yellow solid. ESI-MS found m/z 269.07 (M+H)+.

Synthesis of 4-(((9H-fluoren-9-yl)sulfonyl)methyl)-1-substituted phenyl-1H-1,2,3-triazole (17a-p) To a stirred solution of compound 16 (1.0 mmol) and substituted phenyl azide (1.2 mmol) in t butanol:water (1:1) (4 mL), CuSO4.5H2O (1 mol %) (0.2 mmol) and sodium ascorbate (5 mol %) (0.2 mmol) were added and the reaction mixture was stirred at room temperature for 16 h. After completion of the reaction, as indicated by TLC, butanol was removed under reduced pressure. The residue was extracted with ethyl acetate (3 x10 mL) and combined organic layers were collected and washed with saturated brine solution, dried over anhydrous Na2SO4 and

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Chapter 4 concentrated in vacuo to get the crude product. The product was further purified by column chromatography [ethyl acetate / hexane (40 - 45%)] to afford the title compounds 17a-p.

N-((1-phenyl-1H-1,2,3-triazol-4-yl)methyl)-9H-fluoren-9-amine (12a) -1 Off white solid (82%); m.p. 167-168 ºC; IR (KBr) ʋmax / cm 3342, 3027, 2832, 1645, 1250, 990. 1H NMR (400 MHz, Chloroform-d) δ 7.65 (d, J = 7.5 Hz, 2H), 7.60 (d, J = 7.5 Hz, 2H), 7.58 (d, J = 8.3 Hz, 3H), 7.48 (d, J = 8.1 Hz, 2H), 7.32 (t, J = 7.4 Hz, 2H), 7.25 (dd, J = 7.9, 5.9 13 Hz, 4H), 5.00 (s, 1H), 3.65 (s, 2H). C NMR (101 MHz, CDCl3) δ 148.35, 142.92, 140.86, 133.10, 128.17,128.61, 127.45, 124.68, 122.28, 121.53, 119.62, 119.17, 64.21, 39.41. EI-MS m/z + 339.15 (M+H) ; Anal. calcd for C22H18N4: (%) C, 78.08; H, 5.36; N, 16.56; Found: C, 78.09; H, 5.37; N, 16.58.

N-((1-(p-tolyl)-1H-1,2,3-triazol-4-yl)methyl)-9H-fluoren-9-amine (12b) -1 Off white solid (79%); m.p. 160-161 ºC; IR (KBr) ʋmax / cm 3348, 3029, 2842, 1650, 1590, 1260, 860. 1H NMR (400 MHz, Chloroform-d) δ 7.65 (d, J = 7.4 Hz, 2H), 7.62 – 7.54 (m, 3H), 7.45 (d, J = 8.0 Hz, 2H), 7.31 (t, J = 7.5 Hz, 2H), 7.25 – 7.16 (m, 4H), 5.00 (s, 1H), 3.64 (s, 2H), 13 2.34 (s, 3H). C NMR (101 MHz, CDCl3) δ 141.84, 139.09, 134.92, 134.59, 134.36, 131.21, 130.11, 128.18, 127.18, 122.36, 120.82, 120.33, 71.05, 39.34, 21.25. EI-MS m/z 353.15 (M+H)+;

Anal. calcd for C23H20N4: (%) C, 78.38; H, 5.72; N, 15.90; Found: C, 78.39; H, 5.73; N, 15.91.

N-((1-(4-ethylphenyl)-1H-1,2,3-triazol-4-yl)methyl)-9H-fluoren-9-amine (12c) -1 Off white solid (86%); m.p. 167-168 ºC; IR (KBr) ʋmax / cm 3340, 3032, 2847, 1651, 1590, 1260, 891. 1H NMR (400 MHz, Chloroform-d) δ 7.65 (d, J = 7.5 Hz, 2H), 7.58 (d, J = 8.3 Hz, 3H), 7.48 (d, J = 8.1 Hz, 2H), 7.32 (t, J = 7.4 Hz, 2H), 7.25 (dd, J = 7.9, 5.9 Hz, 4H), 5.00 (s, 13 1H), 3.65 (s, 2H), 2.64 (q, J = 7.6 Hz, 2H), 1.20 (t, J = 7.6 Hz, 3H). C NMR (101 MHz, CDCl3) δ 142.54, 141.15, 139.72, 138.58, 130.15, 128.73, 127.63, 125.75, 120.20, 119.82, 119.80, 64.20, + 39.23, 22.46, 21.08. EI-MS m/z 367.15 (M+H) ; Anal. calcd for C24H22N4: (%) C, 78.66; H, 6.05; N, 15.29; Found: C, 78.67; H, 6.06; N, 15.30.

N-((1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-9H-fluoren-9-amine (12d)

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-1 White solid (75%); m.p. 148-149 ºC; IR (KBr) ʋmax / cm 3348, 3029, 2856, 1649, 1560, 1205, 1032, 864. 1H NMR (400 MHz, Chloroform-d) δ 7.65 (d, J = 7.5 Hz, 2H), 7.58 (d, J = 7.4 Hz, 2H), 7.54 – 7.44 (m, 3H), 7.32 (t, J = 7.4 Hz, 2H), 7.27 – 7.16 (m, 2H), 6.96 – 6.88 (m, 2H), 5.00 13 (s, 1H), 3.79 (s, 3H), 3.64 (s, 2H). C NMR (101 MHz, CDCl3) δ 159.66, 142.16, 141.06, 140.12, 129.12, 127.80, 126.35, 125.70, 122.32, 121.92, 120.83, 114.59, 75.24, 51.13, 40.20. EI- + MS m/z 369.15 (M+H) ; Anal. calcd for C23H20N4O: (%) C, 74.98; H, 5.47; N, 15.21; Found: C, 74.99; H, 5.48; N, 15.22.

N-((1-(4-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-9H-fluoren-9-amine (12e) -1 Pale yellow solid (80%); m.p. 126-127 ºC; IR (KBr) ʋmax / cm 3340, 3025, 2874, 1641, 1563, 1383, 1235, 890. 1H NMR (400 MHz, Chloroform-d) δ 7.65-7.54 (m, 5H), 7.37 (d, J = 8.4 Hz, 2H), 7.29 (t, J = 7.5 Hz, 2H), 7.24 (t, J = 7.8 Hz, 4H), 5.02 (s, 1H), 3.61 (s, 2H). 13C NMR (101

MHz, CDCl3) δ 161.59, 141.29, 140.12, 132.13, 129.88, 129.83, 129.43, 129.34, 129.01, 127.76, 125.67, 124.21, 121.55, 120.18, 65.89, 39.36. EI-MS m/z 357.15 (M+H)+; Anal. calcd for

C22H17FN4: (%) C, 74.14; H, 4.81; N, 15.72; Found: C, 74.15; H, 4.82; N, 15.73.

N-((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-9H-fluoren-9-amine (12f) -1 Off white solid (73%); m.p. 166-167 ºC; IR (KBr) ʋmax / cm 3347, 3021, 2875, 1644, 1560, 1235, 881, 653. 1H NMR (400 MHz, Chloroform-d) δ 7.67 (d, J = 7.4 Hz, 2H), 7.52 (d, J = 8.1 Hz, 3H), 7.48 (d, J = 8.2 Hz, 2H), 7.32 (t, J = 7.6 Hz, 2H), 7.27 (dd, J = 7.9, 5.8 Hz, 4H), 5.00 (s, 13 1H), 3.65 (s, 2H). C NMR (101 MHz, CDCl3) δ 141.18, 134.44, 129.88, 129.83, 129.61, 129.43, 129.34, 129.01, 127.74, 125.66, 125.29, 121.55, 120.14, 65.76, 38.38. EI-MS m/z 373.10 + +2 (M+H) ; 556.10 (M+H) ; Anal. calcd for C22H17Cl2N4: (%) C, 70.87; H, 4.60; N, 15.03; Found: C, 70.88; H, 4.61; N, 15.04.

N-((1-(4-bromophenyl)-1H-1,2,3-triazol-4-yl)methyl)-9H-fluoren-9-amine (12g) -1 Pale white solid (81%); m.p. 168-169 ºC; IR (KBr) ʋmax / cm 3415, 3021, 2871, 1644, 1560, 1235, 876, 560. 1H NMR (400 MHz, Chloroform-d) δ 7.62 (d, J = 7.5 Hz, 2H), 7.51 (d, J = 8.1 Hz, 3H), 7.46 (d, J = 8.2 Hz, 2H), 7.38 (t, J = 7.7 Hz, 2H), 7.29 (dd, J = 7.9, 5.8 Hz, 4H), 5.01 (s, 13 1H), 3.69 (s, 2H). C NMR (101 MHz, CDCl3) δ 148.35, 144.93, 140.84, 132.82, 128.27, 127.36, 124.98, 122.18, 121.83, 119.92, 119.67, 63.20, 39.40. EI-MS m/z 417.07 (M+H)+2;

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+ 419.07 (M+H) ; Anal. calcd for C22H17BrN4: (%) C, 63.32; H, 4.11; N, 13.43; Found: C, 63.34; H, 4.12; N, 13.44.

N-((1-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)methyl)-9H-fluoren-9-amine (12h) -1 Off white solid (72%); m.p.126-128 ºC; IR (KBr) ʋmax / cm 3419, 3029, 2901, 1657, 1509, 1267, 1355, 876, 631, 560. 1H NMR (400 MHz, Chloroform-d) δ 7.76 – 7.69 (m, 4H), 7.67 – 7.60 (m, 3H), 7.57 (d, J = 7.4 Hz, 2H), 7.32 (t, J = 7.4 Hz, 2H), 7.24 (t, J = 7.4 Hz, 2H), 5.00 (s, 13 1H), 3.65 (s, 2H). C NMR (101 MHz, CDCl3) δ 141.06, 139.31, 129.65, 128.78, 127.80, 127.63, 127.03, 125.43, 125.31, 124.89, 120.62, 120.28, 120.08, 77.22, 41.25. EI-MS m/z 407.15 + (M+H) ; Anal. calcd for C23H17F3N4: (%) C, 67.97; H, 4.22; N, 13.79; Found: C, 67.98; H, 4.23; N, 13.80.

N-((1-(4-nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl)-9H-fluoren-9-amine (12i) -1 Yellow solid (81%); m.p. 155-157 ºC; IR (KBr) ʋmax / cm 3420, 3021, 2911, 1632, 1530, 1280, 1355, 1020, 876. 1H NMR (400 MHz, Chloroform-d) δ 8.01 – 7.95 (m, 2H), 7.82 (d, J = 7.7 Hz, 2H), 7.78 (s, 1H), 7.58 – 7.49 (m, 4H), 7.35 (td, J = 7.6, 1.4 Hz, 2H), 7.30 (d, J = 8.3 Hz, 2H), 13 5.15 (s, 1H), 3.86 (s, 2H), C NMR (101 MHz, CDCl3) δ 147.89, 143.73, 141.84, 139.19, 134.97, 134.36, 128.71, 128.08, 127.16, 126.26, 123.29, 120.62, 69.45, 45.42. EI-MS m/z 384.10 + (M+H) ; Anal. calcd for C22H17N5O2: (%) C, 68.92; H, 4.47; N, 18.27; Found: C, 68.93; H, 4.48; N, 18.28.

N-((1-(2-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-9H-fluoren-9-amine (12j) -1 Pale yellow solid (71%); m.p. 123-124 ºC; IR (KBr) ʋmax / cm 3341, 3029, 2867, 1654, 1543, 1373, 1243, 895. 1H NMR (400 MHz, Chloroform-d) δ 7.66-7.51 (m, 5H), 7.38 (d, J = 8.4 Hz, 2H), 7.31 (t, J = 7.5 Hz, 2H), 7.25 (t, J = 7.8 Hz, 4H), 5.04 (s, 1H), 3.62 (s, 2H). 13C NMR (101

MHz, CDCl3) δ 162.59, 141.29, 140.12, 132.13, 129.88, 129.83, 129.43, 129.34, 129.01, 127.76, 125.67, 124.21, 121.55, 120.18, 65.89, 39.36. EI-MS m/z 357.15 (M+H)+; Anal. calcd for

C22H17FN4: (%) C, 74.14; H, 4.81; N, 15.72; Found: C, 74.15; H, 4.82; N, 15.73.

N-((1-(2-nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl)-9H-fluoren-9-amine (12k)

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-1 Yellow solid (81%); m.p. 153-154 ºC; IR (KBr) ʋmax / cm 3422, 3029, 2921, 1641, 1523, 1275, 1351, 1032, 881. 1H NMR (400 MHz, Chloroform-d) δ 7.82 (d, J = 2.4 Hz, 1H), 7.74 (dd, J = 7.5, 1.1 Hz, 2H), 7.65 (dd, J = 7.4, 1.1 Hz, 2H), 7.61 (d, J = 4.7 Hz, 1H), 7.58 (s, 1H), 7.53 (dd, J = 8.7, 2.4 Hz, 1H), 7.44 – 7.38 (m, 2H), 7.35 – 7.29 (m, 3H), 5.08 (s, 1H), 3.71 (s, 2H). 13C

NMR (101 MHz, CDCl3) δ 148.55, 144.84, 140.83, 136.05, 133.87, 132.62, 131.40, 128.31, 127.39, 124.99, 122.14, 119.95, 119.73, 119.32, 63.16, 39.26. EI-MS m/z 384.15 (M+H)+; Anal. calcd for C22H17N5O2: (%) C, 68.92; H, 4.47; N, 18.27; Found: C, 68.93; H, 4.48; N, 18.28.

N-((1-(3,4-dimethylphenyl)-1H-1,2,3-triazol-4-yl)methyl)-9H-fluoren-9-amine (12l) -1 Off white solid (77%); m.p. 106-108 ºC; IR (KBr) ʋmax / cm 3347, 3029, 2902, 1643, 1567, 1264, 866. 1H NMR (400 MHz, Chloroform-d) δ 7.66 (d, J = 7.4 Hz, 2H), 7.64 – 7.54 (m, 3H), 7.46 (d, J = 8.0 Hz, 2H), 7.37 (t, J = 7.5 Hz, 2H), 7.25 – 7.16 (m, 3H), 5.02 (s, 1H), 3.69 (s, 2H), 13 2.31 (s, 6H). C NMR (101 MHz, CDCl3) δ 141.72, 135.89, 133.99, 132.13, 131.56, 128.94, 127.17, 125.73, 121.43, 120.33, 119.88, 119.27, 76.24, 48.21, 18.19. EI-MS m/z 367.18 (M+H)+;

Anal. calcd for C24H22N4: (%) C, 78.66; H, 6.05; N, 15.29; Found: C, 78.67; H, 6.06; N, 15.30.

N-((1-(3-chloro-4-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-9H-fluoren-9-amine (12m) -1 White solid (86%); m.p. 153-154 ºC; IR (KBr) ʋmax / cm 3343, 3020, 2861, 1653, 1545, 1370, 1247, 891, 657. 1H NMR (400 MHz, Chloroform-d) δ 7.67 (t, J = 8.3 Hz, 3H), 7.56 (d, J = 7.4 Hz, 2H), 7.53 – 7.42 (m, 2H), 7.36 – 7.16 (m, 5H), 5.00 (s, 1H), 3.63 (s, 2H). 13C NMR (101

MHz, CDCl3) δ 159.7.21, 148.49, 144.90, 142.25, 140.84, 132.59, 128.28, 127.37, 126.12, 124.98, 122.97, 12071, 119.93, 116.91, 63.19, 39.31. EI-MS m/z 391.10 (M+H)+; Anal. calcd for

C22H16ClFN4: (%) C, 67.61; H, 4.13; N, 14.33; Found: C, 67.62; H, 4.14; N, 14.34.

N-((1-(2,4-dichlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-9H-fluoren-9-amine (12n) -1 White solid (80%); m.p.102-103 ºC; IR (KBr) ʋmax / cm 3347, 3020, 2889, 1657, 1549, 1247, 885, 659. 1H NMR (400 MHz, Chloroform-d) δ 7.66 (d, J = 7.4 Hz, 2H), 7.64 – 7.54 (m, 3H), 7.47 (d, J = 8.1 Hz, 2H), 7.37 (t, J = 7.5 Hz, 2H), 7.28 – 7.17 (m, 3H), 5.01 (s, 1H), 3.71 (s, 2H). 13 C NMR (101 MHz, CDCl3) δ 144.76, 141.72, 135.89, 133.99, 132.13, 131.56, 128.94, 127.17, 126.65, 125.73, 121.43, 120.33, 119.88, 119.27, 76.21, 47.29. EI-MS m/z 407.10 (M+H)+; Anal. calcd for C22H15ClN4: (%) C, 64.88; H, 3.96; N, 13.76; Found: C, 64.89; H, 3.97; N, 13.77.

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N-((1-(3,5-dichlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-9H-fluoren-9-amine (12o) -1 White solid (81%); m.p. 174-175 ºC; IR (KBr) ʋmax / cm 3337, 3021, 2879, 1649, 1549, 1247, 883, 657. 1H NMR (400 MHz, Chloroform-d) δ 7.69-7.66 (m, 3H), 7.56 – 7.54 (m, 2H), 7.48 (d, J = 8.2 Hz, 2H), 7.39 (t, J = 7.6 Hz, 2H), 7.29 – 7.18 (m, 3H), 5.01 (s, 1H), 3.70 (s, 2H). 13C

NMR (101 MHz, CDCl3) δ 141.32, 135.79, 133.99, 132.13, 131.46, 128.74, 127.27, 126.75, 123.53, 121.43, 119.88, 119.27, 76.21, 47.29. EI-MS m/z 407.10 (M+H)+; Anal. calcd for

C22H15ClN4: (%) C, 64.88; H, 3.96; N, 13.76; Found: C, 64.89; H, 3.97; N, 13.77.

N-((1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-9H-fluoren-9-amine (12p) -1 White solid (81%); m.p. 117-118 ºC; IR (KBr) ʋmax / cm 3327, 3029, 2879, 1641, 1541, 1241, 1022, 883. 1H NMR (400 MHz, Chloroform-d) δ 7.81 (d, J = 7.8 Hz, 2H), 7.80 (d, J = 6.8 Hz, 3H), 7.52 (t, J = 7.5 Hz, 2H), 7.41 (t, J = 7.7 Hz, 2H), 6.84 (s, 2H), 5.37 (s, 1H), 3.94 (s, 9H), 13 3.80 (s, 2H). C NMR (101 MHz, CDCl3) δ 153.85, 141.84, 138.36, 134.94, 134.73, 132.49, 130.05, 128.13, 127.18, 122.51, 120.66, 98.32, 76.97, 61.19, 56.42, 45.17. EI-MS m/z 429.15 + (M+H) ; Anal. calcd for C25H24N4O3: (%) C, 70.08; H, 5.65; N, 13.08; Found: C, 70.09; H, 5.66; N, 15.09.

N-((1-(3,4-dichlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-9H-fluoren-9-amine (12q) -1 White solid (85%); m.p. 156-157 ºC; IR (KBr) ʋmax / cm 3341, 3021, 2885, 1657, 1547, 1247, 886, 650. 1H NMR (400 MHz, Chloroform-d) δ 7.66 (d, J = 7.4 Hz, 2H), 7.64 – 7.54 (m, 3H), 7.47 (d, J = 8.1 Hz, 2H), 7.37 (t, J = 7.5 Hz, 2H), 7.28 – 7.17 (m, 3H), 5.01 (s, 1H), 3.71 (s, 2H). 13 C NMR (101 MHz, CDCl3) δ 141.73, 135.89, 133.80, 132.63, 131.56, 128.64, 127.77, 125.83, 121.93, 121.33, 119.91, 119.18, 76.24, 49.21. EI-MS m/z 408.08 (M+H)+; Anal. calcd for

C22H16Cl2N4: (%) C, 64.88; H, 3.96; N, 13.76; Found: C, 64.89; H, 3.97; N, 13.77.

4-(((9H-fluoren-9-yl)thio)methyl)-1-phenyl-1H-1,2,3-triazole (15a) -1 Off white solid (70%); m.p. 136-137 ºC; IR (KBr) ʋmax / cm 3031, 2913, 2595, 1649, 1542, 1237, 886. 1H NMR (400 MHz, Chloroform-d) δ 7.64 (dd, J = 12.7, 7.5 Hz, 4H), 7.48 (d, J = 7.9 Hz, 2H), 7.41 (t, J = 7.7 Hz, 2H), 7.27 (dq, J = 24.0, 8.4, 7.5 Hz, 5H), 6.96 (s, 1H), 4.93 (s, 1H), 13 3.25 (s, 2H). C NMR (101 MHz, CDCl3) δ 141.85, 134.98, 130.01, 129.71, 128.126, 128.27,

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127.16, 126.16, 122.26, 120.61, 120.49, 119.21, 49.28, 22.35. EI-MS m/z 356.12 (M+H)+; Anal. calcd for C22H17N3S: (%) C, 74.34; H, 4.82; N, 11.80; Found: C, 74.35; H, 4.83; N, 11.81.

4-(((9H-fluoren-9-yl)thio)methyl)-1-(p-tolyl)-1H-1,2,3-triazole (15b) -1 Off white solid (81%); m.p. 133-134 ºC; IR (KBr) ʋmax / cm 3025, 2925, 2547, 1577, 1235, 886. 1H NMR (400 MHz, Chloroform-d) δ 7.63 (dd, J = 12.4, 7.5 Hz, 4H), 7.37 – 7.33 (m, 2H), 7.29 (t, J = 7.5 Hz, 2H), 7.25 – 7.21 (m, 4H), 6.94 (s, 1H), 4.93 (s, 1H), 3.24 (s, 2H), 2.34 (s, 3H). 13C

NMR (101 MHz, CDCl3) δ 145.74, 144.19, 141.12, 138.45, 130.25, 128.43, 126.69, 125.59, 120.29, 119.85, 119.71, 49.26, 22.46, 20.12. EI-MS m/z 370.14 (M+H)+; Anal. calcd for

C23H29N3S: (%) C, 74.77; H, 5.19; N, 11.37; Found: C, 74.78; H, 5.20; N, 11.39.

4-(((9H-fluoren-9-yl)thio)methyl)-1-(4-ethylphenyl)-1H-1,2,3-triazole (15c) -1 Off white solid (72%); m.p. 136-137 ºC; IR (KBr) ʋmax / cm 3021, 2920, 2510, 1557, 1245, 896. 1H NMR (400 MHz, Chloroform-d) δ 7.67 (dd, J = 12.5, 7.3 Hz, 4H), 7.36 – 7.33 (m, 2H), 7.28 (t, J = 7.5 Hz, 2H), 7.25 – 7.21 (m, 4H), 6.96 (s, 1H), 4.94 (s, 1H), 3.24 (s, 2H), 2.67 (q, J = 7.8 13 Hz, 2H), 1.21 (t, J = 7.7 Hz, 3H). C NMR (101 MHz, CDCl3) δ 145.54, 144.15, 140.72, 138.55, 130.05, 128.13, 127.60, 125.70, 120.20, 119.82, 119.80, 59.23, 49.20, 22.46, 21.08. EI- + MS m/z 384.16 (M+H) ; Anal. calcd for C24H21N3S: (%) C, 75.16; H, 5.52; N, 10.96; Found: C, 75.18; H, 5.53; N, 10.97.

4-(((9H-fluoren-9-yl)thio)methyl)-1-(4-methoxyphenyl)-1H-1,2,3-triazole (15d) -1 Off white solid (71%); m.p. 116-117 ºC; IR (KBr) ʋmax / cm 3029, 2921, 2511, 1549, 1254, 1020, 884. 1H NMR (400 MHz, Chloroform-d) δ 7.64 (dd, J = 12.4, 7.5 Hz, 4H), 7.38 – 7.33 (m, 2H), 7.30 (t, J = 7.5 Hz, 2H), 7.25 – 7.19 (m, 4H), 6.94 (s, 1H), 4.94 (s, 1H), 3.69 (s, 3H), 3.25 13 (s, 2H). C NMR (101 MHz, CDCl3) δ 159.27, 144.16, 141.26, 140.72, 128.22, 127.60, 126.35, 125.70, 122.32, 121.92, 119.83, 114.59, 54.24, 49.20, 22.56. EI-MS m/z 386.05 (M+H)+; Anal. calcd for C23H29N3OS: (%) C, 71.66; H, 4.97; N, 10.91; Found: C, 71.78; H, 4.98; N, 10.92.

4-(((9H-fluoren-9-yl)thio)methyl)-1-(4-fluorophenyl)-1H-1,2,3-triazole (15e) -1 Off white solid (90%); m.p. 108-109 ºC; IR (KBr) ʋmax / cm 3026, 2921, 2517, 1549, 1322, 799. 1H NMR (400 MHz, Chloroform-d) δ 7.63 (dd, J = 12.5, 7.4 Hz, 4H), 7.37 (d, J = 8.1 Hz, 2H),

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7.29 (t, J = 7.4 Hz, 2H), 7.23 (t, J = 7.8 Hz, 4H), 6.95 (s, 1H), 4.92 (s, 1H), 3.24 (s, 2H). 13C

NMR (101 MHz, CDCl3) δ 164.27, 143.06, 141.26, 140.72, 128.22, 127.60, 126.71, 125.70, 122.35, 121.94, 119.73, 115.59, 49.22, 22.66. EI-MS m/z 374.10 (M+H)+; Anal. calcd for

C22H16FN3S: (%) C, 70.76; H, 4.32; N, 11.25; Found: C, 70.78; H, 4.33; N, 11.26.

4-(((9H-fluoren-9-yl)thio)methyl)-1-(4-chlorophenyl)-1H-1,2,3-triazole (15f) -1 Off white solid (75%); m.p. 140-142 ºC; IR (KBr) ʋmax / cm 3028, 2921, 2519, 1545, 799, 655. 1H NMR (400 MHz, Chloroform-d) δ 7.66 (dd, J = 12.3, 7.4 Hz, 4H), 7.38 – 7.32 (m, 2H), 7.30 (t, J = 7.6 Hz, 2H), 7.26 – 7.19 (m, 4H), 6.95 (s, 1H), 4.93 (s, 1H), 3.26 (s, 2H). 13C NMR (101

MHz, CDCl3) δ 142.13, 140.75, 138.55, 134.34, 131.05, 128.16, 127.60, 125.71, 120.10, 119.80, + 119.81, 49.24, 22.41. EI-MS m/z 390.05 (M+H) ; Anal. calcd for C22H16ClN3S: (%) C, 67.77; H, 4.14; N, 10.78; Found: C, 67.78; H, 4.16; N, 10.79.

4-(((9H-fluoren-9-yl)thio)methyl)-1-(4-bromophenyl)-1H-1,2,3-triazole (15g) -1 Off white solid (81%); m.p. 154-155 ºC; IR (KBr) ʋmax / cm 3031, 2926, 2535, 1533, 799, 585. 1H NMR (400 MHz, Chloroform-d) δ 7.61 (dd, J = 12.4, 7.5 Hz, 4H), 7.38 – 7.32 (m, 2H), 7.29 (t, J = 7.6 Hz, 2H), 7.25 – 7.19 (m, 4H), 6.95 (s, 1H), 4.94 (s, 1H), 3.27 (s, 2H). 13C NMR (101

MHz, CDCl3) δ 141.83, 140.65, 137.50, 134.44, 131.15, 128.76, 127.61, 125.81, 120.50, 119.81, 119.83, 49.25, 22.41. EI-MS m/z 434.05 (M+H)+; 436.05 (M+H)+2; Anal. calcd for

C22H16BrN3S: (%) C, 60.83; H, 3.71; N, 9.67; Found: C, 60.84; H, 4.72; N, 9.69.

4-(((9H-fluoren-9-yl)thio)methyl)-1-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazole (15h) -1 Pale yellow solid (89%); m.p. 128-129 ºC; IR (KBr) ʋmax / cm 3034, 2921, 2530, 1532, 1334, 872. 1H NMR (400 MHz, Chloroform-d) δ 7.70 – 7.65 (m, 3H), 7.65 – 7.60 (m, 4H), 7.29 (t, J = 7.4 Hz, 2H), 7.23 (d, J = 7.5 Hz, 2H), 6.89 (s, 1H), 4.94 (s, 1H), 3.23 (s, 2H). 13C NMR (101

MHz, CDCl3) δ 142.06, 140.73, 128.18, 127.66, 126.93, 126.91, 125.76, 124.24, 123.12, 120.16, + 119.84, 119.57, 99.99, 49.24, 22.26. EI-MS m/z 424.10 (M+H) ; Anal. calcd for C23H16F3N3S: (%) C, 65.24; H, 3.81; N, 9.92; Found: C, 65.25; H, 3.83; N, 9.93.

4-(((9H-fluoren-9-yl)thio)methyl)-1-(4-nitrophenyl)-1H-1,2,3-triazole (15i)

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-1 1 Yellow solid (77%); m.p. 167-168 ºC; IR (KBr) ʋmax / cm 3035, 2912, 2545, 1531, 872. H NMR (400 MHz, Chloroform-d) δ 7.94 (dd, J = 12.5, 7.6 Hz, 4H), 7.29 – 7.32 (m, 2H), 7.26 (t, J = 7.6 Hz, 2H), 7.21 – 7.12 (m, 4H), 6.95 (s, 1H), 4.94 (s, 1H), 3.27 (s, 2H). 13C NMR (101 MHz,

CDCl3) δ 141.83, 140.65, 137.50, 134.44, 131.15, 128.76, 127.61, 125.81, 120.50, 119.81, + 119.83, 49.25, 22.41. EI-MS m/z 401.11 (M+H) ; Anal. calcd for C22H16N4O2S: (%) C, 65.98; H, 4.03; N, 13.99; Found: C, 65.99; H, 4.05; N, 14.00.

4-(((9H-fluoren-9-yl)thio)methyl)-1-(2-chlorophenyl)-1H-1,2,3-triazole (15j) -1 Pale yellow solid (68%); m.p. 118-119 ºC; IR (KBr) ʋmax / cm 3029, 2923, 2519, 1535, 876, 665. 1H NMR (400 MHz, Chloroform-d) δ 7.69 (dd, J = 12.5, 7.5 Hz, 4H), 7.38 – 7.32 (m, 2H), 7.30 (t, J = 7.6 Hz, 2H), 7.25 – 7.19 (m, 4H), 6.96 (s, 1H), 4.91 (s, 1H), 3.24 (s, 2H). 13C NMR

(101 MHz, CDCl3) δ 141.13, 140.75, 138.55, 134.34, 132.55, 132.23, 131.05, 128.16, 127.60, 126.34, 125.71, 120.10, 119.80, 119.89, 49.29, 22.46. EI-MS m/z 390.05 (M+H)+; Anal. calcd for C22H16ClN3S: (%) C, 67.77; H, 4.14; N, 10.78; Found: C, 67.79; H, 4.15; N, 10.79.

4-(((9H-fluoren-9-yl)thio)methyl)-1-(2-nitrophenyl)-1H-1,2,3-triazole (15k) -1 1 Yellow solid (91%); m.p. 122-123 ºC; IR (KBr) ʋmax / cm 3032, 2919, 2541, 1535, 877. H NMR (400 MHz, Chloroform-d) δ 7.97 (dd, J = 12.5, 7.6 Hz, 4H), 7.36 – 7.32 (m, 2H), 7.29 (t, J = 7.5 Hz, 2H), 7.21 – 7.15 (m, 4H), 6.97 (s, 1H), 4.95 (s, 1H), 3.29 (s, 2H). 13C NMR (101 MHz,

CDCl3) δ 145,12, 142.13, 140.65, 137.50, 134.44, 131.15, 129.23, 128.76, 127.87, 127.61, 125.81, 120.51, 119.89, 119.89, 49.29, 22.49. EI-MS m/z 401.10 (M+H)+; Anal. calcd for

C22H16N4S: (%) C, 65.98; H, 4.03; N, 13.99; Found: C, 75.18; H, 5.53; N, 10.97.

4-(((9H-fluoren-9-yl)thio)methyl)-1-(3,4-dimethylphenyl)-1H-1,2,3-triazole (15l) -1 Pale yellow solid (72%); m.p. 119-120 ºC; IR (KBr) ʋmax / cm 3025, 2925, 2547, 1577, 1235, 886. 1H NMR (400 MHz, Chloroform-d) δ 7.62 (dd, J = 12.4, 7.5 Hz, 4H), 7.37 – 7.31 (m, 2H), 7.29 (t, J = 7.5 Hz, 2H), 7.25 – 7.21 (m, 3H), 6.94 (s, 1H), 4.95 (s, 1H), 3.25 (s, 2H), 2.36 (s, 13 6H). C NMR (101 MHz, CDCl3) δ 143.74, 142.19, 141.12, 138.45, 132.80, 130.25, 128.43, 126.69, 125.59, 124.21, 120.29, 119.85, 119.71, 49.26, 22.46, 20.09. EI-MS m/z 384.10 (M+H)+;

Anal. calcd for C24H21N3S: (%) C, 75.16; H, 5.52; N, 10.96; Found: C, 75.17; H, 5.53; N, 10.97.

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4-(((9H-fluoren-9-yl)thio)methyl)-1-(4-fluoro-2-nitrophenyl)-1H-1,2,3-triazole (15m) -1 Yellow solid (78%); m.p. 146-147 ºC; IR (KBr) ʋmax / cm 3029, 2921, 2547, 1545, 1332, 1239, 886. 1H NMR (400 MHz, Chloroform-d) δ 7.78 – 7.71 (m, 2H), 7.62 (dd, J = 12.5, 7.4 Hz, 4H), 7.29 (t, J = 7.4 Hz, 2H), 7.27 – 7.22 (m, 3H), 6.96 (s, 1H), 4.95 (s, 1H), 3.25 (s, 2H). 13C NMR

(101 MHz, CDCl3) δ 162.14, 148.15, 145.54, 144.15, 140.72, 138.55, 132.47, 130.05, 128.13, 127.60, 125.70, 123.54, 120.20, 119.82, 49.20, 22.46. EI-MS m/z 419.10 (M+H)+; Anal. calcd for C22H15FN4O2S: (%) C, 63.16; H, 3.61; N, 13.39; Found: C, 63.17; H, 3.62; N, 13.40.

4-(((9H-fluoren-9-yl)thio)methyl)-1-(2,4-dichlorophenyl)-1H-1,2,3-triazole (15n) -1 Off white solid (69%); m.p. 152-153 ºC; IR (KBr) ʋmax / cm 3022, 2930, 2817, 1634, 1447, 887, 560. 1H NMR (400 MHz, Chloroform-d) δ 7.79 – 7.67 (m, 4H), 7.56 (d, J = 2.1 Hz, 1H), 7.49 – 13 7.28 (m, 6H), 7.20 (d, J = 0.8 Hz, 1H), 5.01 (s, 1H), 3.36 (s, 2H). C NMR (101 MHz, CDCl3) δ 144.74, 142.19, 141.12, 138.45, 135.72, 132.80, 130.25, 128.43, 126.69, 125.59, 124.21, 120.29, + 119.85, 119.71, 49.26, 22.46. EI-MS m/z 424.04 (M+H) ; Anal. calcd for C22H15Cl2N3S: (%) C, 62.27; H, 3.56; N, 9.90; Found: C, 62.28; H, 3.59; N, 9.92.

4-(((9H-fluoren-9-yl)thio)methyl)-1-(3,4-dichlorophenyl)-1H-1,2,3-triazole (15o) -1 Off white solid (64%); m.p. 147-148 ºC; IR (KBr) ʋmax / cm 3028, 2930, 2817, 1634, 1447, 887, 560. 1H NMR (400 MHz, Chloroform-d) δ 7.78 – 7.66 (m, 4H), 7.57 (d, J = 2.1 Hz, 1H), 7.50 – 7.30 (m, 6H), 7.19 (d, J = 0.9 Hz, 1H), 5.04 (s, 1H), 3.36 (s, 2H). 13C NMR (101 MHz,

CDCl3) δ 142.10, 141.05, 135.72, 134.01, 132.89, 130.25, 128.43, 126.69, 125.59, 124.31, + 123.19, 119.95, 119.73, 49.29, 22.41. EI-MS m/z 424.04 (M+H) ; Anal. calcd for C22H15Cl2N3S: (%) C, 62.27; H, 3.56; N, 9.91; Found: C, 62.28; H, 3.57; N, 9.92.

4-(((9H-fluoren-9-yl)thio)methyl)-1-(3,5-dichlorophenyl)-1H-1,2,3-triazole (15p) -1 White solid (80%); m.p. 124-126 ºC; IR (KBr) ʋmax / cm 3024, 2921, 2817, 1532, 1435, 876, 573. 1H NMR (400 MHz, Chloroform-d) δ 7.78 – 7.66 (m, 4H), 7.57 (d, J = 2.1 Hz, 1H), 7.50 – 13 7.30 (m, 6H), 7.19 (d, J = 0.9 Hz, 1H), 5.04 (s, 1H), 3.36 (s, 2H). C NMR (101 MHz, CDCl3) δ 146.44, 144.14, 140.63, 138.14, 136.07, 128.31, 128.24, 127.67, 125.83, 119.90, 119.61, 118.58, + 49.44, 22.29. EI-MS m/z 424.04 (M+H) ; Anal. calcd for C22H15Cl2N3S: (%) C, 62.27; H, 3.56; N, 9.91; Found: C, 62.28; H, 3.57; N, 9.92.

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4-(((9H-fluoren-9-yl)thio)methyl)-1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazole (15q) -1 Off white solid (77%); m.p. 105-107 ºC; IR (KBr) ʋmax / cm 3031, 2921, 2532, 1549, 1236, 1025, 874. 1H NMR (400 MHz, Chloroform-d) δ 7.71 (d, J = 7.8 Hz, 2H), 7.67 (d, J = 6.8 Hz, 2H), 7.52 (t, J = 7.5 Hz, 2H), 7.41 (t, J = 7.7 Hz, 2H), 6.94 (s, 1H), 6.84 (s, 2H), 4.94 (s, 1H), 13 3.85 (s, 9H), 3.25 (s, 2H). C NMR (101 MHz, CDCl3) δ 152.27, 141.26, 139.56, 132.89, 128.22, 127.60, 126.35, 125.70, 122.89, 122.32, 119.83, 109.11, 56.02, 54.64, 49.29, 22.46. EI- + MS m/z 446.20 (M+H) ; Anal. calcd for C25H23N3O3S: (%) C, 67.41; H, 5.20; N, 9.43; Found: C, 67.42; H, 5.21; N, 9.44.

4-(((9H-fluoren-9-yl)thio)methyl)-1-(3-chloro-4-fluorophenyl)-1H-1,2,3-triazole (15r) -1 Brown solid (86%); m.p. 113-114 ºC; IR (KBr) ʋmax / cm 3037, 2964, 2556, 1535, 1342, 1269, 886, 654. 1H NMR (400 MHz, Chloroform-d) δ 7.78 – 7.66 (m, 4H), 7.57 (d, J = 2.1 Hz, 1H), 13 7.50 – 7.30 (m, 6H), 6.96 (s, 1H), 5.01 (s, 1H), 3.36 (s, 2H). C NMR (101 MHz, CDCl3) δ 158.99, 156.49, 144.06, 140.71, 133.37, 128.20, 127.64, 125.77, 122.68, 122.40, 122.21, 119.88, + 117.61, 117.38, 49.22, 22.23. EI-MS m/z 408.08 (M+H) ; Anal. calcd for C22H15ClFN3S: (%) C, 64.78; H, 3.72; N, 10.30; Found: C, 64.79; H, 3.73; N, 10.31.

4-(((9H-fluoren-9-yl)sulfonyl)methyl)-1-phenyl-1H-1,2,3-triazole (17a) -1 Off white solid (75%); m.p. 167-168 ºC; IR (KBr) ʋmax / cm 3037, 2964, 2556, 1535, 1269, 1187, 1050, 886. 1H NMR (400 MHz, Chloroform-d) δ 8.00 – 7.96 (m, 4H), 7.80 (d, J = 7.7 Hz, 2H), 7.76 (s, 1H), 7.52 – 7.47 (m, 3H), 7.39 (td, J = 7.5, 1.4 Hz, 2H), 7.35 (d, J = 8.2 Hz, 2H), 13 5.37 (s, 1H), 3.83 (s, 2H). C NMR (101 MHz, CDCl3) δ 141.85, 134.98, 130.01, 129.71, 128.126, 128.27, 127.16, 126.16, 122.26, 120.61, 120.49, 119.21, 70.08, 45.45. EI-MS m/z + 388.11 (M+H) ; Anal. calcd for C22H17N3O2S: (%) C, 68.20; H, 4.42; N, 10.85; Found: C, 68.21; H, 4.43; N, 10.86.

4-(((9H-fluoren-9-yl)sulfonyl)methyl)-1-(p-tolyl)-1H-1,2,3-triazole (17b) -1 Pale yellow solid (81%); m.p. 203-205 ºC; IR (KBr) ʋmax / cm 3029, 2962, 2566, 1539, 1279, 1186, 1051, 881. 1H NMR (400 MHz, Chloroform-d) δ 8.00 – 7.97 (m, 2H), 7.81 (d, J = 7.6 Hz, 2H), 7.77 (s, 1H), 7.53 – 7.48 (m, 4H), 7.37 (td, J = 7.5, 1.2 Hz, 2H), 7.31 (d, J = 8.2 Hz, 2H),

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13 5.35 (s, 1H), 3.86 (s, 2H), 2.44 (s, 3H). C NMR (101 MHz, CDCl3) δ 141.84, 139.09, 134.97, 134.79, 134.36, 130.21, 130.01, 128.08, 127.16, 122.26, 120.62, 120.37, 70.00, 45.42, 21.14. EI- + MS m/z 402.13 (M+H) ; Anal. calcd for C23H19N3O2S: (%) C, 68.81; H, 4.77; N, 10.47; Found: C, 68.82; H, 4.78; N, 10.48.

4-(((9H-fluoren-9-yl)sulfonyl)methyl)-1-(4-ethylphenyl)-1H-1,2,3-triazole (17c) -1 White solid (63%); m.p. 175-176 ºC; IR (KBr) ʋmax / cm 3033, 2956, 2534, 1541, 1265, 1165, 1057, 871. 1H NMR (400 MHz, Chloroform-d) δ 8.00 – 7.97 (m, 2H), 7.81 (d, J = 7.6 Hz, 2H), 7.77 (s, 1H), 7.53 – 7.48 (m, 4H), 7.37 (td, J = 7.5, 1.2 Hz, 2H), 7.31 (d, J = 8.2 Hz, 2H), 5.35 (s, 1H), 3.86 (s, 2H), 2.64 (q, J = 7.6 Hz, 2H), 2.44 (s, 3H), 1.20 (t, J = 7.6 Hz, 3H). 13C NMR (101

MHz, CDCl3) δ 145.37, 141.86, 134.99, 130.00, 129.03, 128.07, 127.16, 122.24, 120.60, 120.50, + 70.04, 45.47, 28.46, 15.39. EI-MS m/z 416.15 (M+H) ; Anal. calcd for C24H21N3O2S: (%) C, 69.17; H, 5.10; N, 10.11; Found: C, 69.18; H, 5.11; N, 10.12.

4-(((9H-fluoren-9-yl)sulfonyl)methyl)-1-(4-methoxyphenyl)-1H-1,2,3-triazole (17d) -1 Off white solid (74%); m.p. 186-187 ºC; IR (KBr) ʋmax / cm 3030, 2951, 2533, 1544, 1264, 1162, 1051, 1020, 889. 1H NMR (400 MHz, Chloroform-d) δ 7.99 – 7.91 (m, 2H), 7.79 (d, J = 7.5 Hz, 2H), 7.76 (s, 1H), 7.53 – 7.46 (m, 4H), 7.38 (td, J = 7.5, 1.3 Hz, 2H), 7.30 (d, J = 8.4 Hz, 13 2H), 5.36 (s, 1H), 3.87 (s, 2H), 3.66 (s, 3H). C NMR (101 MHz, CDCl3) δ 160.52, 141.85, 135.01, 134.72, 129.99, 128.06, 127.16, 126.52, 122.36, 122.11, 120.60, 114.73, 70.04, 55.63, + 45.46. EI-MS m/z 418.12 (M+H) ; Anal. calcd for C23H19N3O3S: (%) C, 66.17; H, 4.59; N, 10.07; Found: C, 66.18; H, 4.60; N, 10.08.

4-(((9H-fluoren-9-yl)sulfonyl)methyl)-1-(4-fluorophenyl)-1H-1,2,3-triazole (17e) -1 Pale yellow solid (76%); m.p. 209-210 ºC; IR (KBr) ʋmax / cm 3035, 2941, 2543, 1554, 1269, 1162, 1331, 1051, 881. 1H NMR (400 MHz, Chloroform-d) δ 7.98 – 7.91 (m, 2H), 7.79 (d, J = 7.5 Hz, 2H), 7.77 (s, 1H), 7.53 – 7.46 (m, 4H), 7.39 (td, J = 7.5, 1.3 Hz, 2H), 7.31 (d, J = 8.3 Hz, 13 2H), 5.35 (s, 1H), 3.81 (s, 2H). C NMR (101 MHz, CDCl3) δ 158.23, 141.83, 134.94, 134.38, 130.03, 129.92, 128.18, 127.16, 122.19, 121.71, 120.53, 99.98, 70.16, 45.44. EI-MS m/z 406.10 + (M+H) ; Anal. calcd for C22H16BrN3O2S: (%) C, 65.17; H, 3.98; N, 10.36; Found: C, 65.18; H, 3.99; N, 10.37.

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4-(((9H-fluoren-9-yl)sulfonyl)methyl)-1-(4-chlorophenyl)-1H-1,2,3-triazole (17f) -1 White solid (69%); m.p. 215-216 ºC; IR (KBr) ʋmax / cm 3031, 2942, 2547, 1557, 1269, 1162, 1331, 1051, 881, 651.1H NMR (400 MHz, Chloroform-d) δ 8.01 – 7.94 (m, 2H), 7.78 (d, J = 7.6 Hz, 2H), 7.75 (s, 1H), 7.53 – 7.46 (m, 4H), 7.38 (td, J = 7.5, 1.2 Hz, 2H), 7.31 (d, J = 8.4 Hz, 13 2H), 5.36 (s, 1H), 3.87 (s, 2H). C NMR (101 MHz, CDCl3) δ 141.83, 135.11, 134.95, 134.78, 130.03, 129.92, 128.08, 127.16, 122.15, 121.61, 120.63, 99.98, 70.12, 45.34. EI-MS m/z 422.08 + (M+H) ; Anal. calcd for C22H16ClN3O2S: (%) C, 62.63; H, 3.82; N, 9.96; Found: C, 62.64; H, 5.53; N, 9.97.

4-(((9H-fluoren-9-yl)sulfonyl)methyl)-1-(4-bromophenyl)-1H-1,2,3-triazole (17g) -1 Off white solid (81%); m.p. 225-226 ºC; IR (KBr) ʋmax / cm 3027, 2941, 2537, 1547, 1261, 1142, 1331, 1052, 881, 543. 1H NMR (400 MHz, Chloroform-d) δ 8.01 – 7.95 (m, 2H), 7.78 (d, J = 7.6 Hz, 2H), 7.75 (s, 1H), 7.53 – 7.46 (m, 4H), 7.38 (td, J = 7.5, 1.2 Hz, 2H), 7.31 (d, J = 8.4 13 Hz, 2H), 5.36 (s, 1H), 3.87 (s, 2H). C NMR (101 MHz, CDCl3) δ 141.83, 135.11, 134.97, 131.13, 129.97, 128.18, 127.16, 123,23, 122.15, 121.61, 120.63, 99.98, 70.12, 45.34. EI-MS m/z + +2 465.02 (M+H) ; 467.02 (M+H) ; Anal. calcd for C22H16BrN3O2S: (%) C, 56.66; H, 3.46; N, 9.01; Found: C, 56.67; H, 3.47; N, 9.02.

4-(((9H-fluoren-9-yl)sulfonyl)methyl)-1-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazole (17h) -1 Pale yellow solid (72%); mp.231-232 ºC; IR (KBr) ʋmax / cm 3025, 2940, 2537, 1547, 1341, 1146, 1331, 1052, 881. 1H NMR (400 MHz, Chloroform-d) δ 7.89 (d, J = 7.6 Hz, 2H), 7.76 (s, 1H), 7.72 (t, J = 5.7 Hz, 6H), 7.42 (t, J = 7.5 Hz, 2H), 7.28 (t, J = 7.6 Hz, 2H), 5.28 (s, 1H), 3.77 13 (s, 2H). C NMR (101 MHz, CDCl3) δ 141.82, 138.96, 135.60, 134.89, 131.08, 130.75, 130.07, 128.11, 127.17, 124.84, 122.17, 120.66, 120.38, 70.11, 45.23. EI-MS m/z 456.10 (M+H)+; Anal. calcd for C23H16F3N3O2S: (%) C, 60.65; H, 3.54; N, 9.23; Found: C, 60.66; H, 3.55; N, 9.24.

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1 H NMR spectrum (400MHz, CDCl3) of compound 17h

13 C NMR spectrum (101MHz, CDCl3) of compound 17h

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4-(((9H-fluoren-9-yl)sulfonyl)methyl)-1-(4-nitrophenyl)-1H-1,2,3-triazole (17i) -1 Yellow solid (91%); m.p. 246-247 ºC; IR (KBr) ʋmax / cm 3029, 2942, 2545, 1531, 1143, 872. 1H NMR (400 MHz, Chloroform-d) δ 8.12 (dd, J = 12.5, 7.6 Hz, 4H), 7.76 (s, 1H), 7.29 – 7.32 (m, 2H), 7.26 (t, J = 7.6 Hz, 2H), 7.21 – 7.15 (m, 4H), 5.04 (s, 1H), 3.67 (s, 2H). 13C NMR (101

MHz, CDCl3) δ 142.83, 141.65, 138.50, 135.44, 132.15, 128.76, 127.61, 125.81, 120.50, 119.81, + 119.73, 71.35, 46.01. EI-MS m/z 433.10 (M+H) ; Anal. calcd for C22H16N4O4S: (%) C, 61.10; H, 3.73; N, 12.96; Found: C, 61.11; H, 3.74; N, 12.97.

4-(((9H-fluoren-9-yl)sulfonyl)methyl)-1-(2-nitrophenyl)-1H-1,2,3-triazole (17j) -1 Yellow solid (77%); m.p. 186-187 ºC; IR (KBr) ʋmax / cm 3042, 2939, 2542, 1535, 1149, 877. 1H NMR (400 MHz, Chloroform-d) δ 7.97 (dd, J = 12.5, 7.6 Hz, 4H), 7.76 (s, 1H), 7.36 – 7.32 (m, 2H), 7.29 (t, J = 7.5 Hz, 2H), 7.21 – 7.15 (m, 4H), 5.05 (s, 1H), 3.59 (s, 2H). 13C NMR (101

MHz, CDCl3) δ 145,17, 142.16, 140.60, 137.51, 134.42, 132.15, 129.23, 128.76, 127.85, 127.61, + 125.81, 120.51, 119.89, 71.43, 46.19. EI-MS m/z 433.10 (M+H) ; Anal. calcd for C22H16N4O4S: (%) C, 61.10; H, 3.73; N, 12.96; Found: C, 61.11; H, 3.74; N, 12.97.

4-(((9H-fluoren-9-yl)sulfonyl)methyl)-1-(2-chlorophenyl)-1H-1,2,3-triazole (17k) -1 Off white solid (68%); m.p. 221-222 ºC; IR (KBr) ʋmax / cm 3039, 2934, 2542, 1535, 1149, 876, 567. 1H NMR (400 MHz, Chloroform-d) δ 7.98 (d, J = 7.7 Hz, 2H), 7.83 – 7.78 (m, 3H), 7.66 (d, J = 8.4 Hz, 2H), 7.51 (t, J = 7.5 Hz, 4H), 7.37 (t, J = 7.5 Hz, 2H), 5.36 (s, 1H), 3.85 (s, 2H). 13C

NMR (101 MHz, CDCl3) δ 141.82, 135.56, 135.28, 135.46, 134.92, 132.90, 130.05, 128.10, 127.16, 122.66, 122.11, 122.13, 121.82, 120.64, 70.08, 45.30. EI-MS m/z 422.08 (M+H)+; Anal. calcd for C22H16ClN3O2S: (%) C, 62.63; H, 3.83; N, 9.96; Found: C, 62.64; H, 3.84; N, 9.97.

4-(((9H-fluoren-9-yl)sulfonyl)methyl)-1-(3,4-dimethylphenyl)-1H-1,2,3-triazole (17l) -1 White solid (61%); m.p. 179-180 ºC; IR (KBr) ʋmax / cm 3023, 2921, 2547, 1547, 1151, 886, 563. 1H NMR (400 MHz, Chloroform-d) δ 7.89 (dd, J = 12.4, 7.5 Hz, 4H), 7.76 (s, 1H), 7.37 – 7.31 (m, 2H), 7.29 (t, J = 7.5 Hz, 2H), 7.25 – 7.21 (m, 3H), 5.15 (s, 1H), 3.81 (s, 2H), 2.36 (s, 13 6H). C NMR (101 MHz, CDCl3) δ 144.74, 142.19, 140.12, 138.42, 132.70, 130.21, 128.33, 126.62, 125.69, 124.20, 120.27, 119.85, 119.71, 70.45, 45.46, 20.09. EI-MS m/z 416.15 (M+H)+;

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Anal. calcd for C24H21N3O2S: (%) C, 69.37; H, 5.09; N, 10.11; Found: C, 69.38; H, 5.10; N, 10.12.

4-(((9H-fluoren-9-yl)sulfonyl)methyl)-1-(4-fluoro-2-nitrophenyl)-1H-1,2,3-triazole (17m) -1 Yellow solid (79%); m.p. 179-180 ºC; IR (KBr) ʋmax / cm 3021, 2921, 2547, 1545, 1332, 1239, 1145, 887. 1H NMR (400 MHz, Chloroform-d) δ 7.89 – 7.75 (m, 2H), 7.73 (s, 1H), 7.62 (dd, J = 12.4, 7.5 Hz, 4H), 7.32 (t, J = 7.4 Hz, 2H), 7.28 – 7.21 (m, 3H), 5.21 (s, 1H), 3.78 (s, 2H). 13C

NMR (101 MHz, CDCl3) δ 162.17, 148.35, 145.64, 144.19, 140.70, 138.57, 133.47, 131.05, 128.13, 127.61, 125.68, 123.54, 121.41, 119.82, 71.20, 46.46. EI-MS m/z 451.19 (M+H)+; Anal. calcd for C21H15FN4O4S: (%) C, 58.66; H, 3.36; N, 12.44; Found: C, 58.67; H, 3.37; N, 12.45.

4-(((9H-fluoren-9-yl)sulfonyl)methyl)-1-(2,4-dichlorophenyl)-1H-1,2,3-triazole (17n) -1 Off white solid (67%); m.p. 156-157 ºC; IR (KBr) ʋmax / cm 3027, 2931, 2819, 1524, 1132, 897, 562; 1H NMR (400 MHz, Chloroform-d) δ 7.89 – 7.81 (m, 4H), 7.76 (d, J = 2.1 Hz, 1H), 7.49 – 13 7.29 (m, 6H), 7.21 (d, J = 0.8 Hz, 1H), 5.27 (s, 1H), 3.66 (s, 2H). C NMR (101 MHz, CDCl3) δ 144.84, 142.39, 141.62, 138.75, 135.77, 132.81, 130.26, 128.47, 126.69, 125.59, 124.21, 120.26, + 119.81, 119.65, 77.26, 46.41. EI-MS m/z 456.04 (M+H) ; Anal. calcd for C22H15Cl2N3O2S: (%) C, 57.90; H, 3.31; N, 9.21; Found: C, 57.91; H, 3.32; N, 9.22.

4-(((9H-fluoren-9-yl)sulfonyl)methyl)-1-(3,4-dichlorophenyl)-1H-1,2,3-triazole (17o) -1 White solid (78%); m.p. 223-224 ºC; IR (KBr) ʋmax / cm 3026, 2931, 2817, 1534, 1136, 887, 567. 1H NMR (400 MHz, Chloroform-d) δ 7.98 – 7.95 (m, 4H), 7.70 (d, J = 2.1 Hz, 1H), 7.51 – 13 7.39 (m, 6H), 7.21 (d, J = 0.9 Hz, 1H), 5.34 (s, 1H), 3.69 (s, 2H). C NMR (101 MHz, CDCl3) δ 142.10, 141.05, 135.72, 134.01, 132.89, 130.25, 128.43, 126.69, 125.59, 124.31, 123.19, 119.95, 13 119.73, 49.29, 22.41. C NMR (101 MHz, CDCl3) δ 142.82, 136.58, 134.73, 134.11, 133.23, 131.56, 130.17, 128.81, 127.27, 122.33, 122.19, 120.76, 119.43, 76.19, 45.31. EI-MS m/z 456.04 + (M+H) ; Anal. calcd for C22H15Cl2N3O2S: (%) C, 57.90; H, 3.31; N, 9.21; Found: C, 57.91; H, 3.32; N, 9.22.

4-(((9H-fluoren-9-yl)sulfonyl)methyl)-1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazole (17p)

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-1 White solid (76%); m.p. 194-196 ºC; IR (KBr) ʋmax / cm 3027, 2939, 2814, 1540, 1130, 1024, 884, 569. 1H NMR (400 MHz, Chloroform-d) δ 7.99 (d, J = 7.7 Hz, 2H), 7.82 (d, J = 6.8 Hz, 3H), 7.53 (t, J = 7.5 Hz, 2H), 7.40 (t, J = 7.6 Hz, 2H), 6.84 (s, 2H), 5.37 (s, 1H), 3.94 (s, 6H), 13 3.90 (s, 3H), 3.84 (s, 2H). C NMR (101 MHz, CDCl3) δ 153.85, 141.84, 138.36, 134.94, 134.73, 132.49, 130.05, 128.13, 127.18, 122.51, 120.66, 98.32, 69.97, 61.09, 56.48, 45.07. EI- + MS m/z 478.19 (M+H) ; Anal. calcd for C25H23N3O5S: (%) C, 62.88; H, 4.85; N, 8.80; Found: C, 62.89; H, 4.86; N, 8.81.

4-(((9H-fluoren-9-yl)sulfonyl)methyl)-1-(3-nitrophenyl)-1H-1,2,3-triazole (17q) -1 Yellow solid (87%); m.p. 190-192 ºC; IR (KBr) ʋmax / cm 3039, 2939, 2540, 1536, 1147, 879. 1H NMR (400 MHz, Chloroform-d) δ 8.01 (dd, J = 12.5, 7.6 Hz, 4H), 7.79 (s, 1H), 7.40 – 7.36 (m, 2H), 7.29 (t, J = 7.5 Hz, 2H), 7.22 – 7.18 (m, 4H), 5.15 (s, 1H), 3.62 (s, 2H). 13C NMR (101

MHz, CDCl3) δ 148.86, 141.81, 137.29, 135.96, 134.95, 130.98, 130.10, 128.15, 127.22, 125.88, 123.39, 122.21, 120.69, 115.26, 70.24, 45.33. EI-MS m/z 433.10 (M+H)+; Anal. calcd for

C22H16N4O4S: (%) C, 61.10; H, 3.73; N, 12.96; Found: C, 61.11; H, 3.74; N, 12.97.

4.4.3. Biological activity 4.4.3.1. InhA activity inhibition. Triclosan and NADH were obtained from Sigma-Aldrich. Stock solutions of all compounds were prepared in DMSO such that the final concentration of this co-solvent was constant at 5% v/v in a final volume of 1 mL for all kinetic reactions. Kinetic assays were performed using trans-2-dodecenoyl-coenzyme A (DDCoA) and wild type InhA method.[34] Briefly, reactions were performed at 25 °C in an aqueous buffer (30 mM PIPES and 150 mM NaCl pH 6.8) containing additionally 250 μM cofactor (NADH), 50 μM substrate (DDCoA) and the tested compound (at 50 μM or 10 μM). Reactions were initiated by addition of InhA (100 nM final) and NADH oxidation was followed at 340 nm. The inhibitory activity of each derivative was expressed as the percentage inhibition of InhA activity (initial velocity of the reaction) with respect to the control reaction without inhibitor. Triclosan was used as a positive control. All activity assays were performed in triplicate.

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4.4.3.2. In vitro MTB screening The antimycobacterial activities of the compounds 12a-p, 15a-r & 17a-p were evaluated against MTB H37Rv strain and two “wild” strains extracted from tuberculosis patients: one strain is Spec. 210 resistant to PAS, INH, ETB and RMP and the other strain is Spec. 192 fully sensitive to the administrated anti-TB agents. In vitro anti-TB activity is performed by a classical test-tube method of successive dilution in Youmans’ modification of the Proskauer and Beck liquid medium containing 10% of bovine serum [32]. Bacterial respite was prepared from 14 days old cultures of gradually growing strains. Solutions of compounds in DMSO were tested. Stock solutions contained 10 mg of compounds in 1 mL. Dilutions (in geometric progression) were prepared in Youmans’ medium [33]. The medium is without compounds and containing INH as reference drug was used for comparison. Incubation was performed at 37 °C. The MIC values were determined as MIC inhibiting the growth of tested TB strains in relation to the probe with no tested compound. The influence of the compound on the growth of bacteria at concentrations of 3.12, 6.25, 12.5, 25, 50 and 100 μg/mL was evaluated.

4.4.3.3. In vitro cytotoxicity screening The human embryonic kidney cells (HEK-293) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Himedia Laboratories Pvt. Ltd., Mumbai, India), supplemented with 10% heat inactivated fetal bovine serum (Himedia Laboratories Pvt. Ltd., Mumbai, India) and 1 % of Antibiotic solution (10000 U Penicillin and 10 mg Streptomycin per ml, Himedia Laboratories

Pvt. Ltd., Mumbai, India). Cells were cultured at 37 °C in humidified atmosphere with 5% CO2. Stock solutions of compounds was prepared in DMSO at a concentration of 50 μM and stored.

Cytotoxicity screening of the synthesized compounds was determined using MTT assay [35]. 7.5×103 cells were seeded in 96 well plates and incubated overnight. Cells were treated with synthesized compounds at three concentrations (50µM, 25 µM & 10 µM) in duplicates and incubated for 24 hrs. 50 µL of 5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Himedia Laboratories Pvt. Ltd., Mumbai, India) was added and incubated for 4 hours. 150 µL of DMSO was added to dissolve formazan crystals and evaluated spectrophotometrically at 570 nm and 650 nm using Spectramax M4 (Molecular Devices, USA).

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4.4.4. Docking Study Docking studies of the title compounds (12a-p, 15a-r & 17a-p) was performed using Glide 5.9 (Extra Precision) running on maestro version 9.4, in order to investigate their binding pattern with enzyme InhA [36]. Enzyme used for the docking study was retrieved from RCSB Protein Data Bank (PDB ID: 1BVR) in complex with co-crystallised ligand (NICOTINAMIDE). Protein preparation wizard of Schr dinger suite was used for preparation of selected protein. Protein was pre-processed separately by deleting the substrate co-factor as well as the crystallographically observed water molecules (water without H bonds), followed by optimization of hydrogen bonds. After assigning charge and protonation position, finally energy was minimized with root mean square deviation (RMSD) value of 0.30 Å using optimized potentials for liquid simulations-2005 (OPLS-2005) force field [37]. Finally energy minimized protein and co- crystallized ligand was used to build energy grids using the default value of protein atom scaling (1.0 Å) within a cubic box of 14 Å dimensions, centered on the centroid of the X-ray ligand pose. The structures of 12a-p, 15a-r & 17a-p were drawn using ChemSketch and converted to 3D structure with the help of 3D optimization tool. Using LigPrep 2.6 module, the drawn ligands were geometry optimized; partial atomic charges were computed using OPLS-2005 force field [37]. Finally, prepared ligands were docked with prepared protein using Glide 5.9 module, in extra precision mode (XP). The leading docked pose (with lowest Glide score value) found from Glide was analyzed. RMSD value was calculated between the experimental binding mode of co- crystallized ligand as in X-ray and re-docked pose to ensure accuracy and reliability of the docking procedure.

4.5. References [1] (a) Y. Shi, S. Gao, Tetrahedron, 2016, 72, 1717; (b) S. Wang, B. Wen, N. Wang, J. Liu, L. He, Arch. Pharmacal Res., 2009, 32, 521; (b) S.R. Choi, M. A. Larson, S.H. Hinrichs, P. Narayanasamy, Bioorg. Med. Chem. Lett., 2016, 26, 1997. [2] F. Calogero, S. Borrelli, G. Speciale, M. S. Christodoulou, D. Cartelli, D. Ballinari, F. Sola, C. Albanese, A. Ciavolella, D. Passarella, G. Cappelletti, S. Pieraccini, M. Sironi, ChemPlusChem, 2013, 78, 663. [3] A. Tramice, A. Arena, A. De Gregorio, R. Ottanà, R. Maccari, B. Pavone, N. Arena, D. Iannello, M. G. Vigorita, A. Trincone, ChemMedChem, 2008, 9, 1419.

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Chapter V

Design, synthesis and biological evaluation of new substituted sulfonamide tetrazole derivatives as anti-tubercular agents

Chapter 5

Chapter 5

Design, synthesis and biological evaluation of new substituted sulfonamide tetrazole derivatives as anti-tubercular agents

5.1. Introduction 1,2,4 triazoles and their base derivatives correspond to an interesting class of compounds possessing a wide spectrum of biological activities. Number of 1,2,4 triazole containing ring systems exhibited anti-tubercular [1-3], antibacterial [4-6], anticancer [7, 8], antiviral [9, 10] and antifungal activities [11-13]. Based on the incorporation of various substitutents into 1,2,4 triazole ring their heterocyclic derivatives lead to compounds with enhanced biological activities. Thompson et al., reported (7S)-2-nitro-7-((4-(trifluoromethoxy)benzyl)oxy)-5a,6,7,8,9a,10- hexahydro-5H-pyrano[2,3-d][1,2,4]triazolo[1,5-a]pyridine derivatives which inhibited MTB H37RV strain with MIC ranging from 112-128 µM [14]. Krishna and co-workers published (E)- 4-(benzylideneamino)-3-(2-(2,6-dichlorophenylamino)benzyl)-1-(morpholinomethyl)-1H-1,2,4- triazole-5(4H)-thione derivatives which showed anti-TB activity ranging from 0.2-25 µM against MTB H37RV strain [15]. Klimesova’s group reported 1,2,4-triazole 3-benzylsulfanyl derivatives which exhibited MTB activity with MIC ranging from 32-250 μM. Pattan et al., reported 4-(3- mercapto-5- substituted 1H-1,2,4-triazol-4-yl)benzenesulfonamide compounds which inhibited MTB H37Rv strain at 25.0 µg/mL [16]. Suresh Kumar et al., reported 4-substituted- 5-(4- isopropylthiazol-2-yl)-4H-1,2,4-triazole-3-thiols which exhibited MIC ranging from 4-125 µg/mL against MTB H37RV [17]. Khanage et al., reported 6-(substitutedaryl)-4-(3,5-diphenyl- 1H-1,2,4-triazol-1-yl)-1,6-dihydropyrimidine-2-thiol derivatives which inhibited MTB H37Rv strain at 0.156 µg/mL [17]. 1,2,4 triazole based anti-TB agents are portrayed in figure 5.1.

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Figure 5.1: Some of the1,2,4 triazole based anti-tubercular agents.

Tetrazoles and its derivatives are known for biological activities such as antibacterial, anti-inflammatory, antifungal, antiviral, anti-TB, antinociceptive, hypoglycemic and anti-cancer [18]. The tetrazole ring is resistant to biological degradation and therefore used as isosteric substituents of various functional groups [19]. Indeed, substituted tetrazoles have similar pKa values to that of corresponding carboxylic acid [20]. Further, tetrazole analogues increase lipophilicity facilitating easier crossing of compounds across the plasma membrane [19]. Karabanovich’s group reported 1- and 2-alkyl-5-[(3,5-dinitrobenzyl)sulfanyl]-2H-tetrazole derivatives and their selenium bioisosteres with highest antimycobacterial activity, with MIC values 0.37-0.46 µg/mL against MTB CNCTC My 331/88 [21]. Chauhan and co-researchers reported a novel series of thiazolone piperazine tetrazole derivatives with MICs ranging from 1.56 -12.5 µg/mL [19]. Mohite et al., reported 3-chloro-4-(substituted phenyl)- 1-{[2-oxo-2-(5- phenyl-1H-tetrazol-1-yl) ethyl] amino} azetidin-2-one derivatives with MIC values ranging from 10.0-0.156 µg/mL against MTB H37Rv [18]. Tetrazole based anti-TB agents are depicted in figure 5.2.

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Figure 5.2: Some of the tetrazole based anti-tubercular agents.

Aryl sulfonamide derivatives display enhanced antibacterial and anti-TB activity. Lipophilicity is an important parameter related to membrane permeation of compounds in biological systems [22]. Thomas et al., reported 1-[1-(quinolin-4-yl)-1H-1,2,3-triazol-4- yl]methyl sulphonamides with MIC values 0.625-100 μg/mL [23]. Ranjith et al., reported N-(4- (4-chloro-1H-imidazol-1- yl)-3-methoxyphenyl)sulfonamide derivatives as anti-TB agents with MIC values ranging from 0.625-5.0 μg/mL againt MTB H37Rv [24]. Nagesh et al., reported series of 3-(4-(substitutedsulfonyl)piperazin-1-yl)benzo[d]isoxazole analogues with MIC ranging from 3.125 to ≥50 μg/mL [25] and novel 6-(piperazin-1-yl)phenanthridine amide and sulphonamide analogues exhibiting MIC between 1.56 to ≥50 μg/mL were reported [26]. Sulfonamide based anti-TB compounds are displayed in figure 5.3.

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Figure 5.3: Some of the sulfonamide based anti-tubercular agents.

With this collective information and confident by our recent anti-TB results emphasizing on molecular hybridization approach, we drew a synthetic stratagem to knit all these imperative pharmacophoric groups into one single scaffold and synthesized 2-(2,4-dihalophenyl)-1-(4-(1-( substitutedsulfonyl)-1H-tetrazol-5-yl)phenoxy)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol and 2-(2,4- dihalo)-1-(2-methoxy-4-(1-(substitutedsulfonyl)-1H-tetrazol-5-yl)phenoxy)-3-(1H-1,2,4-triazol- 1-yl)propan-2-ol analogues (Figure 5.4).

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Figure 5.4: Design strategy to achieve title compounds

5.2. Results and Discussion 5.2.1. Chemistry The designed molecules were synthesized in seven steps as sketched in Scheme 5.1. Initially, we prepared 2-chloro-1-(2,4-dihalophenyl)ethanone (19) from dihalobenzene (dichlorobenzene or diflourobezene), chloroacetylchloride, and AlCl3 via Friedel craft acylation. Treatment of 19 with 4-amino-4H-1,2,4-triazole in acetonitrile yielded 4-amino-1-(2-(2,4-dihalophenyl)-2-oxoethyl)- 4H-1,2,4-triazol-1-ium chloride salt (20). Compound 20 on deamination followed by 1.5N HCl,

NaNO2 yielded compound 21. Corey-Chaykovsky epoxidation of 21 using trimethylsulfoxonium

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Chapter 5 iodide and NaOH yielded epoxide 22 [27]. Compound 22 on treatment with 4- hydroxybenzonitrile/4-hydroxy-3-methoxybenzonitrile in the presence K2CO3 and TBAB resulted in compound 23 [28]. Conversion of cyano compound 23 to tetrazole 24 was carried out using trimethylsilyl azide and TBAB.3H2O in the sealed tube [29]. The title tetrazole containing substituted aryl sulfonyls (25a-i, 26a-i, 27a-i & 28a-i) were synthesized from 24 by varying substituted aryl sulfonylchlorides in the presence of TEA in dichloromethane.

Scheme 5.1: Synthetic protocol of tetrazole sulfonamides.

Reagents and conditions: (i) ClCH2COCl (1.2 eq), AlCl3 (2.0 eq), DCE, 60 ºC, 12 h. (ii) 4- amino-4H-1,2,4-triazole (1.2 eq), ACN, 80 ºC, 16 h. (iii) 1.5N HCl, NaNO2 (1.2 eq), 0 ºC, 1 h (iv) TMSI (1.2 eq), 20% NaOH (1.0 eq), toluene, 60 ºC, 12 h. (v) 4-hydroxybenzonitrile/4- hydroxy-3-methoxybenzonitrile (1.3 eq), K2CO3 (3.0 eq), TBAB (0.1 eq), EtOAc, 80 ºC, 12 h.

(vi) TMSN3 (1.2 eq), TBAB.3H2O (1.0 eq), 12 h. (vii) substituted aryl sulfonylchlorides (1.3 eq), TEA (3.0 eq), DCM, rt, 16 h.

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In this synthesis, cyano compound (23) to tetrazole compound (24) conversation was confirmed by disappearance of IR signal at 2224 cm-1 (cyano peak). Structures of 25a-i, 26a-i, 27a-i & 28a- i was further substantiated through 1H NMR and mass spectrometry. All the synthesized compounds displayed doublets of doublets in the range 4.36–4.95 ppm corresponding to their enantiotopic (–CH2–) protons, singlet in the range 3.65–3.87 ppm corresponding to –OCH3 proton, and protons of 1,2,4-triazole ring resonated in the range 7.8–8.2 and 8.3–8.5 ppm. Both analytical and spectral data (1H NMR, 13C NMR, IR and LCMS) of all the synthesized compounds confirmed the obtained structures.

5.2.2 Antimycobacterial activity All the synthesized compounds were tested for their ability to inhibit the growth of MTB H37RV by MABA [30-32]. Isoniazid, Rifampicin and Ethambutol were used as the positive drug controls. The antimycobacterial test results of synthesized compounds are represented in the Table 5.1 as minimum inhibitory concentration ranging from 0.78 to ≥25 µg/mL. Compounds with MIC ≤ 6.25 µg/mL were further subjected to cytotoxicity studies.

Table 5.1: Result of Antimycobacterial screening of title compounds

MIC in µM Entry X R Ar against MTB H37Rv (µg/mL) 25a F H Phenyl 5.78 (3.12)

25b F H 4-Methylphenyl 11.29 (6.25)

25c F H 4-Fluorophenyl 2.79 (1.56)

25d F H 4-Bromophenyl 5.04 (3.12)

25e F H 4-Methoxyphenyl 5.47 (3.12)

25f F H 4-Nitrophenyl 2.66 (1.56)

25g F H 4-ter-butylphenyl 10.49 (6.25)

25h F H 2-bromothiophene 4.99 (3.12)

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25i F H 4-trifluoromethoxyphenyl 10.02 (6.25)

26a Cl H Phenyl 43.67 (25)

26b Cl H 4-Methylphenyl 5.32 (3.12)

26c Cl H 4-Fluorophenyl 1.32 (0.78)

26d Cl H 4-Bromophenyl 9.59 (6.25)

26e Cl H 4-Methoxyphenyl 10.37 (6.25)

26f Cl H 4-Nitrophenyl 20.24 (12.5)

26g Cl H 4-ter-butylphenyl 2.48 (1.56)

26h Cl H 2-bromothiophene 19.01 (12.5)

26i Cl H 4-trifluoromethoxyphenyl 38.08 (25.0)

27a F OCH3 Phenyl 5.47 (3.12)

27b F OCH3 4-Methylphenyl 10.70 (6.25)

27c F OCH3 4-Fluorophenyl 10.63 (6.25)

27d F OCH3 4-Bromophenyl 4.81 (3.12)

27e F OCH3 4-Methoxyphenyl 2.60 (1.56)

27f F OCH3 4-Nitrophenyl 20.34 (12.5)

27g F OCH3 4-ter-butylphenyl 2.49 (1.56)

27h F OCH3 2-bromothiophene 2.38 (1.56)

27i F OCH3 4-trifluoromethoxyphenyl 4.77 (3.12)

28a Cl OCH3 Phenyl 41.49 (25)

28b Cl OCH3 4-Methylphenyl 5.06 (3.12)

28c Cl OCH3 4-Fluorophenyl 10.07 (6.25)

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28d Cl OCH3 4-Bromophenyl 2.28 (1.56)

28e Cl OCH3 4-Methoxyphenyl 9.88 (6.25)

28f Cl OCH3 4-Nitrophenyl 4.81 (3.12)

28g Cl OCH3 4-ter-butylphenyl 18.98 (12.5)

28h Cl OCH3 2-bromothiophene 2.26 (1.56)

28i Cl OCH3 4-trifluoromethoxyphenyl 9.10 (6.25)

Ethambutol - - - 7.63 (1.56)

Isoniazid - - - 0.036 (0.05)

Rifampicin - - - 0.12 (0.10)

Structure activity relationship of compounds 25a-i & 26a-i For structure activity relationship (SAR) study, among the nine compounds which contained 1,3 difluorobenzene skeleton, we varied it with 3 electron donating para substituted sulfonyl chlorides, 4 electron withdrawing para substituted sulfonylchlorides and one heterocyclic sulfonylchloride. SAR for both the series is explained with respect to unsubstituted compounds (25a and 26a).

Among the 1,3 difluorobezene derivatives, the unsubstituted benzene sulfonylchloride compound (25a) exhibited MTB MIC value of 3.12 μg/mL. Electron releasing methyl and tertiary butyl groups reduced (25b, 25g) the activity by two folds (MIC = 6.25 μg/mL) but the presence of electron releasing methoxy group in the para position did not impact the activity (25e, MIC = 3.12 μg/mL). Presence of electron withdrawing groups showed major impact on the activity. Nitro group in compound 25f at para position enhanced the activity by two folds (MIC =1.56

μg/mL), while the electron withdrawing -OCF3 decreased the activity by two folds (25i, MIC = 6.25 μg/mL). With the presence of electron withdrawing fluoro the activity was enhanced by two folds 25c (MIC =1.56 μg/mL); replacing with bromo group had no effect on the activity. With introduction of heterocyclic sulfonylchloride (2-bromothiophene) the activity (25h) remained unaltered (MIC = 3.12 μg/mL).

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Among the synthesized, 1,3 dichlorobenzene derivatives 26a-i, 26a, unsubstituted benzene sulfonyl chloride compound exhibited activity with MIC = 25 μg/mL. With the introduction of electron withdrawing groups at the para position on phenyl ring the activity increased. Presence of methyl group enhanced the activity by eight folds (26b, MIC = 3.12 μg/mL) but with presence of tertiary butyl group activity was enhanced by sixteen fold (26g, MIC = 1.56 μg/mL). Presence of the electron releasing methoxy increased the activity by four folds (26e, MIC = 6.25 μg/mL).

With electron withdrawing -OCF3 the activity remained unaltered (26i, MIC = 25 μg/mL). Presence of the withdrawing nitro increased the activity by two folds (26f, MIC = 12.5 μg/mL). Introduction of fluoro at para position drastically enhanced the activity by thirty two folds (26c, MIC = 0.78 μg/mL) but with the presence of bromo (26d, MIC = 6.25 μg/mL) activity increased four folds. With heterocyclic sulfonylchloride (2-bromothiophene) activity increased by two fold in 26h (MIC = 12.5 μg/mL).

SAR of compounds 27a-i & 28a-i For SAR study, among the nine compounds which contained 1,3 difluorobenzene skeleton, we varied it with 3 electron donating para substituted sulfonyl chlorides, 4 electron withdrawing para substituted sulfonylchlorides and one heterocyclic sulfonylchloride. SAR for both the series is explained with respect to unsubstituted compounds (27a and 28a). In these 1,3 difluorobezene derivatives, the unsubstituted benzene sulfonylchloride compound (27a) exhibited MTB MIC value of 3.12 μg/mL. Electron releasing methoxy and tertiary butyl groups enhanced (27e, 27g) the activity by two folds (MIC = 1.56 μg/mL) but the presence of methyl group in 27b at the para position led to decrease in the activity by two folds (MIC = 6.25 μg/mL). Electron withdrawing groups showed major impact on the activity. Presence of Nitro group in compound 27f at para position decreased the activity by four folds (MIC =12.5 μg/mL), when the electron withdrawing group -OCF3 was introduced no much change in the activity was observed. Presence of electron withdrawing fluoro decreased the activity by two folds in compound 27c (MIC = 6.25 μg/mL); replacing with bromo group had no effect on the activity spectrum. With introduction of heterocyclic sulfonylchloride (2-bromothiophene) the activity (27h) increased by two folds (MIC = 1.56 μg/mL).

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Among the synthesized 1,3 dichlorobenzene derivatives 28a-i, 28a, unsubstituted benzene sulfonyl chloride compound exhibited activity with MIC = 25 μg/mL. With the introduction of electron withdrawing groups at the para position on phenyl ring the activity increased.

Introduction of electron withdrawing –OCF3 exhibited four folds increase in activity (28i, MIC =

6.25 μg/mL). Hopping to -NO2 group improved the activity by eight folds (28f, MIC = 3.12 μg/mL). In this series, introduction of electron donating groups like tertiary butyl, methyl and methoxy resulted in increase of activity. With the introduction of tertiary butyl group activity increased by two folds in 28g (MIC = 12.5 μg/mL); varying with methyl group at the para position in 28b the activity increased by eight folds (MIC = 3.12 μg/mL). With –OCH3 at the para position (28e) the activity increased by four folds (MIC = 6.25 μg/mL). Introduction of fluoro at the para position in 28c led to increase in activity by four folds (MIC = 6.25 μg/mL), whereas changing to bromo at the para position the MTB activity was enhanced by sixteen folds in 28d (MIC = 1.56 μg/mL). With heterocyclic sulfonylchloride (2-bromothiophene) activity drastically increased by sixteen fold in 28h (MIC = 1.56 μg/mL).

5.2.3. Cytotoxicity Overall, anti-TB results indicate that the 1,3 difluorobenzene derivatives (25a-r) exhibited better anti-TB activity than the 1,3 dichlorobenzene derivatives (26a-r) as all twenty eight compounds exhibited anti-TB activity MIC ≤ 12.5 μg/mL. The compounds with MIC ≤ 6.25 μg/mL were subjected to in vitro cytotoxicity studies by MTT assay method against CHO-K1 cell lines at a concentration 100 µM [33]. The IC50 and selectivity index (SI) values are tabulated in Table 5.2 and the results imply the suitability of the compounds in further drug development for TB.

Table 5.2: IC50 (µg/mL) and selectivity index (SI) values of active compounds MIC (µg/mL) in IC (µg/mL) aSI values Entry 50 MTB H37Rv approximation IC50/MIC 25a 3.12 >150 >48 25b 6.25 >150 >24 25c 1.56 >150 >96 25d 3.12 >150 >48 25f 1.56 123.45 79 25g 6.25 82.55 13

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25h 3.12 >150 >48 25i 6.25 >150 >24 26b 3.12 >150 >48 26c 0.78 >150 >192 26d 6.25 96.69 15 26e 6.25 >150 >24 26g 1.56 >150 >96 27a 3.12 >150 >48 27b 6.25 >150 >24 27c 6.25 >150 >24 27d 3.12 38.12 12 27e 1.56 >150 >96 27g 1.56 >150 >96 27h 1.56 >150 >96 27i 3.12 58.12 18 28b 3.12 >150 >48 28c 6.25 >150 >24 28d 1.56 >150 >96 28e 6.25 >150 >24 28f 3.12 >150 >48 28h 1.56 >150 >96 28i 6.25 >150 >24 a Selectivity index 5.3. Conclusion Our preliminary anti-TB results encourage us to engineer the chemical structure of 1,2,4 triazole containing tetrazole with sulfonyl groups to generate essential pharmacophoric features that could lead to the synthesis of a promising candidate to develop anti-TB agents. We proved that incorporating sulfonyl group in the pharmacophore plays a pivotal role in the activity profile. In vitro anti tubercular screening results indicate that ten compounds 25a, 25d, 25e, 25h, 26b, 27a, 27d, 27i, 28b and 28f showed moderate activity (MIC = 3.12 µg/mL). Eight compounds 25c, 25f, 26g, 27e, 27g, 27h, 28d and 28h displayed good anti-TB activity (MIC = 1.56 µg/mL). Compound 26c exhibited excellent anti-TB activity (MIC = 0.78µg/mL). Most of the compounds did not show toxicity (SI value >13). Detailed in vivo studies of compounds 25a-i, 26a-i, 27a-i &

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28a-i further structural modification in 1,2,4 triazoles and tetrazoles need to be explored in future.

5.4. Experimental Section: 5.4.1. Materials and methods Chemicals and solvents were purchased from commercial sources and are analytically pure. Thin-layer chromatography (TLC) was carried out on aluminium-supported silica gel plates (Merck 60 F254) with visualization of components by UV light (254 nm). Column chromatography was carried out on silica gel (Merck 100-200 mesh). 1H and 13C NMR spectra were recorded at 300 MHz using a Bruker AV 300 spectrometer or 400 MHz using a Bruker AV

400 spectrometer (Bruker CO., Switzerland) in CDCl3 or DMSO-d6 solution with tetramethylsilane as the internal standard, and chemical shift values (δ) are given in ppm. IR spectra were recorded on a FT-IR spectrometer (Schimadzu) and peaks are reported in cm-1. Melting points were determined on an electro thermal melting point apparatus (Stuart-SMP30) in open capillary tubes and are uncorrected. Mass spectra (ESI-MS) were recorded on Schimadzu MS/ESI mass spectrometer.

5.4.2. Chemistry Representative procedure for the synthesis of compound 19 To a solution of 1,3-difluorobenzene/1,3-dichlorobenzene (1.0 eq) in 1,2-dichloroethane (DCE), anhydrous aluminum chloride (2.0 eq) was added at 25 to 30 °C and stirred for 30 min. The reaction mixture was then cooled to 0 °C and chloroacetyl chloride (1.2 eq) in DCE was added into it over a period of 30 min at 0 to 10 °C. The reaction mixture was then stirred at 60 °C for 12 h and diluted with DCE and poured into 5% hydrochloric acid (50 ml) at 0 to 5 °C. The product was extracted with DCE (2 ×50 ml) and the combined organic layer was washed with

5% aqueous NaHCO3 solution (20 ml), water (2 × 20 ml), brine (20 ml) and dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated under reduced pressure to yield the product 19, as yellow solid; yield (80%).

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Representative procedure for the synthesis of compound 20 To a solution of compound 19 (1.0 eq) in acetonitrile (ACN), 4-amino-4H-1,2,4-triazole (1.2 eq) was added and stirred for 16 h, at 80 ºC. The reaction mixture was then cooled to room temperature and filtered. The solid was washed with ethyl ether to afford the light yellow salt (20).

Representative procedure for the synthesis of compound 21 The compound 20 was dissolved in 1.5N hydrochloric acid. To this solution was obtained an aqueous solution of sodium nitrite (1.2 eq) was added drop wise and the reaction mixture was stirred for 1 h at 0 ºC to room temperature. Aqueous ammonia was used to adjust to neutral PH. The precipitated solid was filtered to afford 87% white product 21.

Representative procedure for the synthesis of compound 22 To a solution of 21 (1.0 eq) in toluene, was added trimethylsulfoxonium iodide (1.2 eq) followed by the addition of 20% sodium hydroxide solution. The reaction mixture was then heated at 60 °C for 12 h. After the reaction was over, it was diluted with toluene and poured into chilled water. The organic layer was washed with water, brine solution dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated under reduced pressure to give compound 22 as light brown oil; yield 64%.

Representative procedure for the synthesis of compound 23

To a solution of compound 22 (1.0 eq) in ethyl acetate, K2CO3 (3.0 eq) and tetra-butyl ammonium bromide (TBAB) (0.1 eq) were added. The reaction mixture was allowed to stir under reflux for 12 h under nitrogen atmosphere. It was then cooled to room temperature, diluted with water, extracted with ethyl acetate, dried over anhydrous Na2SO4, concentrated and purified by column chromatography using pet ether-ethyl acetate (40:60) as eluent. Yield; 82% (white solid).

Representative procedure for the synthesis of compound 24

To a screw capped vial equipped with a magnetic stirrer were added TBAB.3H2O (1.0 eq), compound 23 (1.0 eq) and TMSN3 (1.2 eq), and the resulting mixture was heated under vigorous

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Chapter 5 stirring at 85 ºC for 18 h. The crude reaction mixture was transferred into a separatory funnel with 20 mL of ethyl acetate, and TBAF was removed by washing the organic phase with a 1 M

HCl aqueous solution The organic layer was dried (Na2SO4) and concentrated under reduced pressure to furnish pure intermediate compound 24 as a white solid in 88% yield.

Representative procedure for the synthesis of compounds 25a-i, 26a-i, 27a-i & 28a-i 1-(4-(1H-tetrazol-5-yl)phenoxy)-2-(2,4-dihalophenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol/2- (2,4-dihalophenyl)-1-(2-methoxy-4-(1H-tetrazol-5-yl)phenoxy)-3-(1H-1,2,4-triazol-1-yl)propan- 2-ol (24) (1.0 eq) was dissolved in dichloromethane then triethylamine (3.0 eq), and substituted aryl sulfonylchloride (1.3 eq) were added. Resultant mixture was stirred at room temperature for 16 h. After the reaction was complete as indicated by TLC, compound was extracted using DCM. Combined organic layers were washed with saturated brine solution, dried over anhydrous sodium sulphate and evaporated in vacuo. The crude products were purified over silica gel column chromatography [MeOH / DCM (2 – 6%)] to afford required compounds 25a-i, 26a-i, 27a-i & 28a-i.

2-(2,4-difluorophenyl)-1-(4-(1-(phenylsulfonyl)-1H-tetrazol-5-yl)phenoxy)-3-(1H-1,2,4-triazol- 1-yl)propan-2-ol (25a) -1 White solid (85%); m.p. 133-135 ºC; IR (KBr) ʋmax / cm 3520, 3041, 2844, 1614, 1527, 1 1415,1362, 1152, 823. H NMR (300MHz, DMSO-d6) δ 8.39 (s, 1H), 7.83 (s, 1H), 6.98 – 7.80 (m, 12H), 6.45 (s, 1H), 4.72 (dd, J = 14.4, 28.4 Hz, 2H), 4.30 (dd, J = 13.2, 38.8 Hz, 2H). 13C

NMR (101 MHz, DMSO-d6) δ 161.03, 158.28, 154.35, 150.74, 145.28, 136.99, 133.70, 133.50, 133.11, 131.42, 131.38, 130.23, 129.97, 128.06, 127.05, 126.51, 121.72, 114.53, 75.09, 71.20. + EI-MS m/z 540 (M+H) ; Anal. Calcd for C24H19F2N7O4S: (%) C, 53.43; H, 3.55; N, 18.17; Found: C, 53.45; H, 3.56; N, 18.19.

2-(2,4-difluorophenyl)-1-(4-(1-tosyl-1H-tetrazol-5-yl)phenoxy)-3-(1H-1,2,4-triazol-1yl)propan- 2-ol (25b) -1 Pale yellow solid (77%); m.p. 141-143 ºC; IR (KBr) ʋmax / cm 3540, 3031, 2854, 1624, 1522, 1 1425, 1360, 1150, 820, 720. H NMR (300MHz, DMSO-d6) δ 8.39 (s, 1H), 7.83 (s, 1H), 6.98 – 7.80 (m, 11H), 6.45 (s, 1H), 4.72 (dd, J = 14.4, 28.4 Hz, 2H), 4.30 (dd, J = 13.2, 38.8 Hz, 2H),

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13 2.43 (s, 3H). C NMR (100 MHz, DMSO-d6) δ 161.03, 158.28, 154.35, 150.74, 145.28, 136.99, 133.70, 133.50, 133.11, 131.42, 131.38, 130.23, 129.97, 128.06, 127.05, 126.51, 121.72, 114.53, + 75.09, 71.20, 27.8. EI-MS m/z 554 (M+H) ; Anal. Calcd for C25H21F2N7O4S: (%) C, 53.24; H, 3.82; N, 17.71; Found: C, 53.25; H, 3.85; N, 17.73.

2-(2,4-difluorophenyl)-1-(4-(1-((4-fluorophenyl)sulfonyl)-1H-tetrazol-5-yl)phenoxy)-3-(1H- 1,2,4-triazol-1-yl)propan-2-ol (25c) -1 Pale yellow solid (98%); m.p. 130-132 ºC; IR (KBr) ʋmax / cm 3544, 3040, 2854, 1614, 1522, 1 1425, 1361, 1141, 1075, 818, 721. H NMR (300MHz, DMSO-d6) δ 8.39 (s, 1H), 7.83 (s, 1H), 7.00 – 7.80 (m, 11H), 6.49 (s, 1H), 4.82 (dd, J = 14.1, 27.4 Hz, 2H), 4.30 (dd, J = 13.2, 38.8 Hz, 13 2H). C NMR (100 MHz, DMSO- d6) δ 162.04, 161.03, 158.28, 150.74, 145.28, 136.99, 133.70, 133.50, 133.11, 131.42, 131.38, 130.23, 129.97, 128.06, 127.05, 126.51, 121.72, 114.53, 75.09, + 71.20. EI-MS: m/z 558 (M+H) ; Anal. Calcd for C24H18F3N7O4S: (%) C, 51.70; H, 3.25; N, 17.59; Found: C, 51.73; H, 3.26; N, 17.62.

1-(4-(1-((4-bromophenyl)sulfonyl)-1H-tetrazol-5-yl)phenoxy)-2-(2,4-difluorophenyl)-3-(1H- 1,2,4-triazol-1-yl)propan-2-ol (25d) -1 Pale yellow solid (78%); m.p. 126-128 ºC; (KBr) ʋmax / cm 3545, 3020, 2850, 1605, 1528, 1 1425, 1375, 1145, 825, 720, 512. H NMR (300 MHz, DMSO-d6) δ 8.39 (s, 1H), 7.82 (s, 1H), 6.92 – 7.98 (m, 11H), 6.51 (s, 1H), 4.95 (dd, J = 14.1, 27.4 Hz, 2H), 4.49 (dd, J = 13.2, 38.1 Hz, 13 2H). C NMR (100 MHz, DMSO-d6) δ 162.74, 161.73, 159.28, 150.74, 145.28, 136.99, 133.70, 133.50, 136.61, 131.38, 130.23, 129.97, 128.06, 127.05, 126.51, 125.56, 121.72, 114.53, 75.09, + +2 71.20. EI-MS: m/z 617 (M+H) , 619 (M+H) ; Anal. Calcd for C24H18BrF2N7O4S: (%) C, 46.61; H, 2.94; N, 15.85; Found: C, 46.63; H, 2.95; N, 15.86.

2-(2,4-difluorophenyl)-1-(4-(1-((4-methoxyphenyl)sulfonyl)-1H-tetrazol-5-yl)phenoxy)-3-(1H- 1,2,4-triazol-1-yl)propan-2-ol (25e) -1 Off white solid (77%); m.p. 130-131 ºC; IR (KBr) ʋmax / cm 3533, 3020, 2855, 1603, 1520, 1 1430, 1365, 1140, 1073, 835, 717. H NMR (300 MHz, DMSO-d6) δ. 8.32 (s, 1H), 7.82 (s, 1H), 6.92 – 7.98 (m, 11H), 6.48 (s, 1H), 4.93 (dd, J = 14.9, 26.4 Hz, 2H), 4.35 (dd, J = 13.2, 38.1 Hz, 13 2H), 3.76 (s, 3H). C NMR (100 MHz, DMSO-d6) δ 162.47, 161.99, 159.98, 154.77, 151.74,

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145.28, 139.45, 136.99, 133.50, 136.61, 131.38, 29.97, 128.06, 127.05, 126.51, 125.56, 121.72, + 114.53, 78.89, 72.10, 59.65. EI-MS: m/z 570 (M+H) ; Anal. Calcd for C25H21F2N7O5S: (%) C, 52.73; H, 3.72; N, 17.22; Found: C, 52.75; H, 3.75; N, 17.83.

2-(2,4-difluorophenyl)-1-(4-(1-((4-nitrophenyl)sulfonyl)-1H-tetrazol-5-yl)phenoxy)-3-(1H- 1,2,4-triazol-1-yl)propan-2-ol (25f) -1 Yellow solid (68%); m.p. 139-141 ºC; IR (KBr) ʋmax / cm 3542, 3023, 2845, 1601, 1510, 1430, 1 1365, 1259, 1145, 1070, 830, 715. H NMR (300 MHz, DMSO-d6) δ 8.49 (s, 1H), 8.12 (s, 1H), 6.82 – 8.25 (m, 11H), 6.51 (s, 1H), 4.95 (dd, J = 14.1, 27.4 Hz, 2H), 4.49 (dd, J = 13.2, 38.1 Hz, 13 2H). C NMR (100 MHz, DMSO-d6) δ 162.74, 161.73, 159.28, 154.35, 150.74, 145.28, 136.99, 133.70, 133.50, 136.61, 131.38, 130.23, 129.97, 128.06, 127.05, 126.51, 124.56, 121.72, 114.53, + 71.20. EI-MS m/z 585 (M+H) ; Anal. Calcd for C24H18F2N8O6S: (%) C, 49.33; H, 3.10; N, 19.17; Found: C, 49.35; H, 3.11; N, 19.19.

1-(4-(1-((4-(tert-butyl)phenyl)sulfonyl)-1H-tetrazol-5-yl)phenoxy)-2-(2,4-difluorophenyl)-3- (1H-1,2,4-triazol-1-yl)propan-2-ol (25g) -1 Off white solid (93%); m.p. 143-144 ºC; IR (KBr) ʋmax / cm 3540, 3020, 2843, 1606, 1516, 1 1430, 1365, 1145, 1075, 813, 722. H NMR (300 MHz, DMSO-d6) δ 8.33 (s, 1H), 7.98 (s, 1H), 6.98 – 7.80 (m, 11H), 6.45 (s, 1H), 4.72 (dd, J = 14.4, 28.4 Hz, 2H), 4.30 (dd, J = 13.2, 38.8 Hz, 13 2H), 1.33 (s, 9H). C NMR (100 MHz, DMSO-d6) δ 161.03, 158.48, 154.35, 150.74, 145.28, 136.99, 133.70, 133.50, 133.11, 131.42, 131.38, 130.23, 129.97, 128.06, 127.05, 126.51, 121.72, + 114.53, 75.09, 71.20, 35.12, 30.63. EI-MS m/z 596 (M+H) ; Anal. Calcd for C28H27F2N7O4S: (%) C, 56.46; H, 4.57; N, 16.47; Found: C, 56.47; H, 4.59; N, 16.49.

1-(4-(1-((5-bromothiophen-2-yl)sulfonyl)-1H-tetrazol-5-yl)phenoxy)-2-(2,4-difluorophenyl)-3- (1H-1,2,4-triazol-1-yl)propan-2-ol (25h) -1 Pale yellow solid (87%); m.p. 125-127 ºC; IR (KBr) ʋmax / cm 3543, 3022, 2845, 1678, 1510, 1 1432, 1360, 1145, 804, 721, 513. H NMR (300 MHz, DMSO-d6) δ 8.35 (s, 1H), 7.82 (s, 1H), 7.03 – 7.69 (m, 9H), 6.37 (s, 1H), 4.95 (dd, J = 14.1, 28.4 Hz, 2H), 4.35 (dd, J = 12.9, 39.1 Hz, 13 2H). C NMR (100 MHz, DMSO-d6) δ 163.74, 160.93, 159.48, 158.35, 151.84, 143.66, 136.99, 131.87, 131.37, 130.23, 129.55, 129.33, 128.69, 114.53, 111.98, 110.87,105.67, 98.87, 75.09,

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+1 +2 68.20. EI-MS m/z 623 (M+H) , 625 (M+H) ; Anal. Calcd for C22H16BrF2N7O4S2: (%) C, 42.32; H, 2.58; N, 15.70; Found: C, 42.34; H, 2.59; N, 15.71.

2-(2,4-difluorophenyl)-1-(1H-1,2,4-triazol-1-yl)-3-(4-(1-((4- (trifluoromethoxy)phenyl)sulfonyl)-1H-tetrazol-5-yl)phenoxy)propan-2-ol (25i) -1 Off white solid (76%); m.p. 140-142 ºC; IR (KBr) ʋmax / cm 3530, 3024, 2825, 1605, 1525, 1 1434, 1368, 1140, 1070, 803, 720. H NMR (300 MHz, DMSO-d6) δ 8.46 (s, 1H), 7.80 (s, 1H), 7.00 – 8.10 (m, 11H), 6.45 (s, 1H), 4.85 (dd, J = 14.7, 27.8 Hz, 2H), 4.37 (dd, J = 13.6, 9.1 Hz, 13 2H). C NMR (100 MHz, DMSO-d6) δ 161.54, 161.23, 158.56, 150.98, 146.38, 137.29, 133.80, 133.86, 133.70, 133.76, 131.87, 131.15, 130.45, 128.67, 127.24, 126.45, 125.87, 121.45, 114.53, + 72.20, 59.12. EI-MS m/z 623 (M+H) ; Anal. Calcd for C26H18F5N7O5S: (%) C, 48.16; H, 2.91; N, 15.72; Found: C, 48.17; H, 2.92; N, 15.73.

2-(2,4-dichlorophenyl)-1-(4-(1-(phenylsulfonyl)-1H-tetrazol-5-yl)phenoxy)-3-(1H-1,2,4- triazol-1-yl)propan-2-ol (26a) -1 White solid (88%); m.p. 120-121 ºC; IR (KBr) ʋmax / cm 3520, 3043, 2840, 1615, 1520, 1415, 1 1362, 1155, 805, 655. H NMR (300 MHz, DMSO-d6) δ 8.49 (s, 1H), 7.83 (s, 1H), 7.63 (s, 1H), 6.98 – 7.80 (d, 11H), 6.50 (s, 1H), 4.98 (dd, J = 14.4, 28.9 Hz, 2H), 4.41 (dd, J =13.3, 38.7 Hz, 13 2H). C NMR (100 MHz, DMSO-d6) δ 164.25, 161.03, 158.28, 154.35, 150.74, 145.28, 136.99, 133.70, 133.50, 131.42, 131.38, 130.23, 129.97, 128.06, 127.05, 126.51, 121.72, 114.53, 75.09, + 71.20. EI-MS m/z 572 (M+H) ; Anal. Calcd for C24H19Cl2N7O4S: (%) C, 50.36; H, 3.36; N, 17.13; Found: C, 50.37; H, 3.38; N, 17.38.

2-(2,4-dichlorophenyl)-1-(4-(1-tosyl-1H-tetrazol-5-yl)phenoxy)-3-(1H-1,2,4-triazol-1- yl)propan-2-ol (26b) -1 Brown solid (86%); m.p. 133-135 ºC; IR (KBr) ʋmax / cm 3545, 3035, 2855, 1624, 1520, 1425, 1 1360, 1150, 812, 722, 608. H NMR (300 MHz, DMSO-d6) δ 8.46 (s, 1H), 7.80 (s, 1H), 7.65 (s, 1H), 6.95 – 7.82 (d, 10H), 6.44 (s, 1H), 4.96 (dd, J = 14.7, 28.5 Hz, 2H), 4.44 (dd, J = 13.7, 38.9 13 Hz, 2H), 2.43 (s, 3H). C NMR (100 MHz, DMSO-d6) δ 161.23, 158.14, 154.55, 150.74, 145.26, 136.99, 133.79, 133.57, 131.62, 131.56, 130.87, 130.23, 129.97, 128.06, 127.05, 126.51,

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+ 121.72, 114.53, 75.09, 71.20, 27.8. EI-MS m/z 586 (M+H) ; Anal. Calcd for C25H21Cl2N7O4S: (%) C, 51.21; H, 3.61; N, 16.73; Found: C, 51.23; H, 3.62; N, 16.74.

2-(2,4-dichlorophenyl)-1-(4-(1-((4-fluorophenyl)sulfonyl)-1H-tetrazol-5-yl)phenoxy)-3-(1H- 1,2,4-triazol-1-yl)propan-2-ol (26c) -1 Off white solid (87%); m.p 133-135 ºC; IR (KBr) ʋmax / cm 3538, 3035, 2844, 1614, 1520, 1 1428, 1361, 1151, 1075, 820, 717, 601. H NMR (300 MHz, DMSO-d6) δ 8.46 (s, 1H), 7.78 (s, 1H), 7.65 (s, 1H), 6.98 – 7.82 (d, 10H), 6.50 (s, 1H), 4.89 (dd, J = 14.9, 28.4 Hz, 2H), 4.39 (dd, J 13 = 13.5, 38.9 Hz, 2H). C NMR (100 MHz, DMSO-d6) δ 164.23, 162.55, 158.99, 151.01, 150.34, 145.87, 137.10, 133.65, 133.99, 132.62, 131.67, 130.99, 129.67, 128.78, 127.58, 126.68, 121.92, + 114.63, 75.19, 71.20. EI-MS m/z 590 (M+H) ; Anal. Calcd for C24H18Cl2FN7O4S: (%) C, 48.82; H, 3.08; N, 16.61; Found: C, 48.83; H, 3.10; N, 16.62.

1-(4-(1-((4-bromophenyl)sulfonyl)-1H-tetrazol-5-yl)phenoxy)-2-(2,4-dichlorophenyl)-3-(1H- 1,2,4-triazol-1-yl)propan-2-ol (26d) -1 Pale yellow solid (88%); m.p. 127-129 ºC; IR (KBr) ʋmax / cm 3535, 3018, 2856, 1605, 1525, 1 1421, 1365, 1136, 815, 713, 608, 510. H NMR (300 MHz, DMSO-d6) δ 8.39 (s, 1H), 7.82 (s, 1H), 7.69 (s, 1H), 6.92 – 7.98 (d, 10H), 6.51 (s, 1H), 4.95 (dd, J = 14.1, 27.4 Hz, 2H), 4.49 (dd, J 13 = 13.2, 38.1 Hz, 2H). C NMR (100 MHz, DMSO-d6) δ 162.74, 161.73, 159.28, 150.74, 145.28, 136.99, 133.70, 133.50, 136.61, 131.38, 130.23, 129.97, 128.06, 127.05, 126.51, 125.56, 121.72, 114.53, 75.09, 71.20. EI-MS m/z 649 (M+H)+1, 651 (M+H)+2; Anal. Calcd for

C24H18BrCl2N7O4S: (%) C, 44.26; H, 2.79; N, 15.05; Found: C, 44.27; H, 2.80; N, 15.06.

2-(2,4-dichlorophenyl)-1-(4-(1-((4-methoxyphenyl)sulfonyl)-1H-tetrazol-5-yl)phenoxy)-3-(1H- 1,2,4-triazol-1-yl)propan-2-ol (26e) -1 Off white solid (70%); m.p. 130-132 ºC; IR (KBr) ʋmax / cm 3538, 3029, 2855, 1603, 1527, 1 1420, 1361, 1132, 812, 711, 610. H NMR (300 MHz, DMSO-d6) δ 8.32 (s, 1H), 7.82 (s, 1H), 6.99 – 7.98 (d, 10H), 6.48 (s, 1H), 4.93 (dd, J = 14.9, 26.4 Hz, 2H), 4.35 (dd, J = 13.2 Hz, 38.1 13 Hz, 2H), 3.76 (s, 3H). C NMR (100 MHz, DMSO-d6) δ 163.47, 162.99, 161.88, 159.98, 154.77, 151.74, 145.28, 139.45, 136.99, 133.50, 136.61, 131.38, 129.97, 128.06, 127.05, 126.51,

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125.56, 121.72, 114.53, 72.10, 59.65. EI-MS m/z 602 (M+H)+; Anal. Calcd for

C24H18BrCl2N7O4S: (%) C, 44.26; H, 2.79; N, 15.05; Found: C, 44.27; H, 2.80; N, 15.06.

2-(2,4-dichlorophenyl)-1-(4-(1-((4-nitrophenyl)sulfonyl)-1H-tetrazol-5-yl)phenoxy)-3-(1H- 1,2,4-triazol-1-yl)propan-2-ol (26f) -1 Yellow solid (68%); m.p. 134-136 ºC; IR (KBr) ʋmax / cm 3545, 3030, 2875, 1606, 1515, 1431, 1 1364, 1259, 1135, 1070, 815, 715, 610. H NMR (300 MHz, DMSO-d6) δ 8.49 (s, 1H), 8.12 (s, 1H), 7.80 (s, 1H), 6.82 – 8.25 (d, 10H), 6.49 (s, 1H), 4.93 (dd, J = 14.9, 27.6 Hz, 2H), 4.40 (dd, J 13 = 13.5, 38.2 Hz, 2H). C NMR (100 MHz, DMSO-d6) δ 162.94, 161.63, 159.28, 150.74, 145.38, 136.79, 133.80, 133.50, 136.61, 131.38, 130.23, 129.97, 128.16, 127.25, 126.55, 124.66, 122.72, + 115.53, 75.09, 72.20. EI-MS m/z 617 (M+H) ; Anal. Calcd for C24H18Cl2N8O6S: (%) C, 46.69; H, 2.94; N, 18.15; Found: C, 46.70; H, 2.95; N, 18.16.

1-(4-(1-((4-(tert-butyl)phenyl)sulfonyl)-1H-tetrazol-5-yl)phenoxy)-2-(2,4-dichlorophenyl)-3- (1H-1,2,4-triazol-1-yl)propan-2-ol (26g) -1 Pale yellow solid (76%); m.p. 123-125 ºC; IR (KBr) ʋmax / cm 3548, 3030, 2853, 1604, 1522, 1 1419, 1362, 1130, 811, 604. H NMR (300 MHz, DMSO-d6) δ 8.46 (s, 1H), 7.80 (s, 1H), 7.65 (s, 1H), 6.95 – 7.82 (d, 10H), 6.44 (s, 1H), 4.93 (dd, J = 14.6, 28.9 Hz, 2H), 4.47 (dd, J = 13.4, 38.2 13 Hz, 2H), 1.38 (s, 9H). C NMR (100 MHz, DMSO-d6) δ 164.23, 161.03, 158.28, 154.55, 150.74, 145.26, 136.99, 133.79, 133.57, 131.62, 131.56, 130.87, 129.02, 128.12, 127.45, 126.51, 121.72, 114.53, 75.09, 71.20, 35.12, 30.63. EI-MS m/z 628 (M+H)+; Anal. Calcd for

C28H27Cl2N7O4S: (%) C, 53.51; H, 4.33; N, 15.60; Found: C, 53.52; H, 4.34; N, 15.61.

1-(4-(1-((5-bromothiophen-2-yl)sulfonyl)-1H-tetrazol-5-yl)phenoxy)-2-(2,4-dichlorophenyl)-3- (1H-1,2,4-triazol-1-yl)propan-2-ol (26h) -1 Pale yellow solid (89%); m.p. 141-143 ºC; IR (KBr) ʋmax / cm 3544, 3026, 2845, 1670, 1510, 1 1431, 1360, 1145, 804, 720, 608, 509. H NMR (300 MHz, DMSO-d6) δ 8.35 (s, 1H), 7.82 (s, 1H), 7.76 (s, 1H), 7.06 – 7.79 (d, 8H), 6.49 (s, 1H), 4.95 (dd, J = 14.2, 29.1 Hz, 2H), 4.32 (dd, J 13 = 12.3, 39.4 Hz, 2H). C NMR (100 MHz, DMSO-d6) δ 163.99, 161.83, 159.48, 158.35, 151.84, 143.66, 136.99, 131.87, 131.37, 130.23, 129.55, 129.33, 128.69, 114.53, 111.98, 110.87,105.67,

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98.87, 75.19, 68.65. EI-MS m/z 655 (M+H)+1, 657 (M+H)+2; Anal. Calcd for

C22H16BrCl2N7O4S2: (%) C, 40.20; H, 2.45; N, 14.92; Found: C, 40.22; H, 2.46; N, 14.93.

2-(2,4-dichlorophenyl)-1-(1H-1,2,4-triazol-1-yl)-3-(4-(1-((4- (trifluoromethoxy)phenyl)sulfonyl)-1H-tetrazol-5-yl)phenoxy)propan-2-ol (26i) -1 Off White solid (85%); m.p. 144-145 ºC; IR (KBr) ʋmax / cm 3540, 3025, 2845, 1605, 1525, 1 1410, 1360, 1133, 812, 715, 610. H NMR (300 MHz, DMSO-d6) δ 8.35 (s, 1H), 7.80 (s, 1H), 7.69 (s, 1H), 6.98 – 7.82 (d, 10H), 6.49 (s, 1H), 4.79 (dd, J = 14.4, 28.9 Hz, 2H), 4.35 (dd, J = 13 13.9, 38.1 Hz, 2H) C NMR (100MHz, DMSO-d6) δ 164.34, 162.55, 158.99, 155.20, 150.34, 145.87, 137.10, 133.65, 133.99, 132.62, 131.67, 130.99, 129.67, 128.78, 127.58, 126.68, 121.92, + 114.63, 79.43, 75.19, 72.40. EI-MS m/z 656 (M+H) ; Anal. Calcd for C25H18Cl2F3N7O5S: (%) C, 45.74; H, 2.76; N, 14.94; Found: C, 45.76; H, 2.77; N, 14.95.

2-(2,4-difluorophenyl)-1-(2-methoxy-4-(1-(phenylsulfonyl)-1H-tetrazol-5-yl)phenoxy)-3-(1H- 1,2,4-triazol-1-yl)propan-2-ol (27a) -1 Off white solid (79%); m.p. 125-127 ºC; IR (KBr) ʋmax / cm 3520, 3041, 2844, 1614, 1527, 1 1415, 1362, 1152, 823. H NMR (400 MHz, DMSO-d6) δ 8.47 (s, 1H), 7.87 (s, 1H), 7.60 (s, 1H), 7.01 – 7.96 (m, 10H), 6.50 (s, 1H), 5.10 (dd, J = 14.9, 26.9 Hz, 2H), 4.49 (dd, J = 13.9, 38.3 Hz, 13 2H), 3.89 (s, 3H). C NMR (100 MHz, DMSO-d6) δ 166.78, 161.03, 158.28, 154.39, 150.74, 151.76, 145.28, 136.99, 133.70, 133.50, 131.38, 130.93, 129.56, 128.26, 127.77, 126.97, 121.72, + 114.53, 87.67,72.19, 69.11, 57.89. EI-MS m/z 570 (M+H) ; Anal. Calcd for C25H21F2N7O5S: (%) C, 52.72; H, 3.72; N, 17.22; Found: C, 52.73; H, 3.74; N, 17.23.

2-(2,4-difluorophenyl)-1-(2-methoxy-4-(1-tosyl-1H-tetrazol-5-yl)phenoxy)-3-(1H-1,2,4-triazol- 1-yl)propan-2-ol (27b) -1 Pale yellow solid (83%); m.p. 133-134 ºC; IR (KBr) ʋmax / cm 3540, 3031, 2854, 1624, 1522, 1 1425, 1360, 1150, 820, 720. H NMR (400MHz, DMSO-d6) δ 8.45 (s, 1H), 7.79 (s, 1H), 7.68 (s, 1H), 6.95 – 7.91 (m, 9H), 6.43 (s, 1H), 4.99 (dd, J = 14.9, 26.4 Hz, 2H), 4.52 (dd, J =13.5, 38.9 Hz, 1H), 4.33 (dd, J =12.7, 37.3 Hz, 1H), 3.90 (s, 3H), 2.48 (s, 3H). 13C NMR (100 MHz,

DMSO-d6) δ 164.67, 161.87, 158.45, 154.58, 150.87, 151.99, 145.67, 141.87, 136.67, 132.98, 133.67, 131.12, 130.43, 129.54, 128.65, 127.86, 126.99, 121.67, 114.85, 85.63, 72.77, 69.68,

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+ 58.89, 21.3. EI- MS m/z 584 (M+H) ; Anal. Calcd for C26H23F2N7O5S: (%) C, 53.51; H, 3.97; N, 16.80; Found: C, 53.53; H, 3.98; N, 16.82.

2-(2,4-difluorophenyl)-1-(4-(1-((4-fluorophenyl)sulfonyl)-1H-tetrazol-5-yl)-2- methoxyphenoxy)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (27c) -1 Pale yellow solid (82%); m.p. 117-118 ºC IR (KBr) ʋmax / cm 3544, 3040, 2854, 1614, 1522, 1 1425, 1361, 1141, 1075, 818, 721. H NMR (400 MHz, DMSO-d6) δ 8.39 (s, 1H), 7.83 (s, 1H), 7.00 – 7.80 (m, 9H), 6.91 (s, 1H), 6.49 (s, 1H), 4.79 (dd, J = 14.7, 27.4 Hz, 2H), 4.45 (dd, J = 13.3, 38.5 Hz, 1H), 4.28 (dd, J = 13.1, 37.8 Hz, 1H), 3.69 (s, 3H). 13C NMR (100 MHz, DMSO- d6) δ 168.12, 163.50, 161.23, 158.28, 154.67, 151.74, 145.28, 133.10, 133.54, 133.65, 131.66, 131.38, 130.23, 129.97, 128.06, 127.15, 126.76, 121.79, 114.56, 75.76, 72.88, 55.19, 53.89. EI- + MS m/z 588 (M+H) ; Anal. Calcd for C25H20F3N7O5S: (%) C, 51.11; H, 3.43; N, 16.69; Found: C, 51.12; H, 3.45; N, 16.70.

1-(4-(1-((4-bromophenyl)sulfonyl)-1H-tetrazol-5-yl)-2-methoxyphenoxy)-2-(2,4- difluorophenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (27d) -1 White solid (81%); m.p. 155-157 ºC; (KBr) ʋmax / cm 3545, 3020, 2855, 1600, 1528, 1425, 1 1374, 1145, 825, 720, 510. H NMR (400 MHz, DMSO-d6) δ 8.39 (s, 1H), 7.83 (s, 1H), 7.00 – 7.80 (m, 9H), 6.91 (s, 1H), 6.49 (s, 1H), 4.79 (dd, J = 14.7, 27.4 Hz, 2H), 4.45 (dd, J = 13.3, 38.5 13 Hz, 1H), 4.28 (dd, J = 13.1, 37.8 Hz, 1H), 3.69 (s, 3H). C NMR (100 MHz, DMSO-d6) δ 168.12, 163.50, 161.23, 158.28, 154.67, 151.74, 145.28, 133.10, 133.54, 133.65, 131.66, 131.38, 130.23, 129.97, 128.06, 127.15, 126.76, 121.79, 114.56, 75.76, 72.88, 55.19, 53.89. EI-MS m/z +1 +2 647 (M+H) , 649 (M+H) ; Anal. Calcd for C25H20BrF2N7O5S: (%) C, 46.31; H, 3.11; N, 15.12; Found: C, 46.32; H, 3.13; N, 15.13.

2-(2,4-difluorophenyl)-1-(2-methoxy-4-(1-((4-methoxyphenyl)sulfonyl)-1H-tetrazol-5- yl)phenoxy)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (27e) -1 Off white solid (83%); m.p. 129-131 ºC; IR (KBr) ʋmax / cm 3535, 3025, 2850, 1605, 1518, 1 1435, 1364, 1145, 1070, 830, 715. H NMR (400MHz, DMSO-d6) δ 8.19 (s, 1H), 7.90 (s, 1H), 6.91 (s, 1H), 6.99 – 7.97 (m, 9H), 4.87 (dd, J = 14.6, 26.7 Hz, 2H), 4.30 (dd, J =13.6, 37.9 Hz, 13 2H), 3.78 (s, 3H), 3.65 (s, 3H). C NMR (100 MHz, DMSO-d6) δ 163.45, 162.47, 161.88,

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159.98, 154.77, 151.74, 145.28, 139.45, 136.99, 133.50, 136.61, 131.38, 129.97, 128.06, 127.05, 126.51, 125.56, 121.72, 114.53, 83.15, 78.89, 72.10, 60.10, 58.90. EI-MS m/z 600 (M+H)+;

Anal. Calcd for C26H23F2N7O6S: (%) C, 52.08; H, 3.87; N, 16.35; Found: C, 52.12; H, 3.89; N, 16.36.

2-(2,4-difluorophenyl)-1-(2-methoxy-4-(1-((4-nitrophenyl)sulfonyl)-1H-tetrazol-5- yl)phenoxy)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (27f) -1 Yellow solid (71%); m.p. 150-151 ºC; ; IR (KBr) ʋmax / cm 3542, 3023, 2845, 1601, 1510, 1 1430, 1365, 1259, 1145, 1070, 830, 715. H NMR (400 MHz, CDCl3) δ 8.47 (s, 1H), 8.21 (s, 1H), 6.98 – 8.20 (m, 9H), 6.50 (s, 1H), 4.96 (dd, J = 14.3 Hz, 27.9 Hz, 2H), 4.50 (dd, J = 13.6 13 Hz, 38.6 Hz, 2H), 3.74 (s, 3H). C NMR (100 MHz, CDCl3) δ 163.64, 162.73, 159.43, 154.7, 150.87, 145.87, 136.92, 136.61, 133.66, 133.52, 131.38, 130.28, 129.92, 128.06, 127.55, 126.67, 124.99, 121.34, 114.88, 86.45,78.34, 72.01, 58.99 EI-MS m/z 615 (M+H)+; Anal. Calcd for

C25H20F2N8O7S: (%) C, 48.86; H, 3.28; N, 18.23; Found: C, 48.89; H, 3.29; N, 18.25.

1-(4-(1-((4-(tert-butyl)phenyl)sulfonyl)-1H-tetrazol-5-yl)-2-methoxyphenoxy)-2-(2,4- difluorophenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (27g) -1 Pale yellow solid (85%); m.p. 118-120 ºC IR (KBr) ʋmax / cm 3540, 3021, 2840, 1605, 1515, 1 1432, 1365, 1145, 1070, 810, 720. H NMR (400 MHz, DMSO-d6) δ 8.36 (s, 1H), 7.98 (s, 1H), 7.79 (s, 1H), 6.98 – 7.80 (m, 10H), 6.48 (s, 1H), 4.68 (dd, J = 14.4, 28.4 Hz, 2H), 4.34 (dd, J = 13 13.2, 38.8 Hz, 2H), 3.81 (s, 3H), 1.33 (s, 9H). C NMR (100 MHz, DMSO-d6) δ 161.03, 158.48, 154.35, 150.74, 145.28, 136.99, 133.70, 133.50, 133.11, 131.42, 131.38, 130.23, 129.97, 128.06, 127.05, 126.51, 121.72, 114.53,87.12, 75.09, 71.20, 65.43, 35.12, 30.63. EI-MS m/z 626 + (M+H) ; Anal. Calcd for C29H29F2N7O5S: (%) C, 55.67; H, 4.67; N, 15.67; Found: C, 55.69; H, 4.68; N, 15.69.

1-(4-(1-((5-bromothiophen-2-yl)sulfonyl)-1H-tetrazol-5-yl)-2-methoxyphenoxy)-2-(2,4- difluorophenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (27h) -1 Pale yellow solid (87%); m.p. 135-136 ºC IR (KBr) ʋmax / cm 3543, 3022, 2844, 1678, 1515, 1 1432, 1365, 1145, 805, 721, 515. H NMR (400 MHz, DMSO-d6) δ 8.44 (s, 1H), 7.88 (s, 1H), 7.68 (s, 1H), 7.13 – 7.80 (m, 7H), 6.45 (s, 1H), 4.95 (dd, J = 14.6, 28.9 Hz, 2H), 4.39 (dd, J =

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13 12.4, 39.0 Hz, 2H), 3.74 (s, 3H). C NMR (100 MHz, DMSO-d6) δ 165.32, 163.89, 162.23, 159.55, 158.43, 154.77, 151.67, 143.45, 136.87, 132.87, 131.87, 130.93, 129.85, 129.63, 128.79, 114.55, 111.57, 111.83, 105.67, 98.89, 75.59, 69.20, 59.80 EI-MS m/z 653 (M+H)+, 655 +2 (M+H) ; Anal. Calcd for C23H18BrF2N7O5S2: (%) C, 55.67; H, 4.67; N, 15.67; Found: C, 55.69; H, 4.68; N, 15.69.

2-(2,4-difluorophenyl)-1-(2-methoxy-4-(1-((4-(trifluoromethoxy)phenyl)sulfonyl)-1H-tetrazol- 5-yl)phenoxy)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (27i) -1 Off White solid (85%); m.p. 144-145 ºC; IR (KBr) ʋmax / cm 3530, 3024, 2825, 1605, 1525, 1 1434, 1368, 1140, 1070, 803, 720. H NMR (400 MHz, DMSO-d6) δ 8.35 (s, 1H), 7.80 (s, 1H), 7.69 (s, 1H), 6.98 – 7.82 (d, 9H), 6.49 (s, 1H), 4.68 (dd, J = 14.4, 28.4 Hz, 2H), 4.34 (dd, J = 13 13.2, 38.8 Hz, 2H), 3.74 (s, 3H). C NMR (100 MHz, DMSO-d6) δ 167.87, 162.55, 158.99, 155.20, 153.78, 150.34, 145.87, 137.10, 133.65, 133.99, 132.62, 131.67, 130.99, 129.67, 129.12, 128.78, 127.58, 126.68, 121.92, 114.63, 83.54, 75.19, 72.40, 56.78. EI-MS m/z 654 (M+H)+;

Anal. Calcd for C26H20F5N7O6S: (%) C, 47.78; H, 3.08; N, 15.00; Found: C, 47.79; H, 3.10; N, 15.01.

2-(2,4-dichlorophenyl)-1-(2-methoxy-4-(1-(phenylsulfonyl)-1H-tetrazol-5-yl)phenoxy)-3-(1H- 1,2,4-triazol-1-yl)propan-2-ol (28a) -1 Brown solid (78%); m.p. 156-158 ºC; IR (KBr) ʋmax / cm 3525, 3040, 2839, 1614, 1525, 1418, 1 1362, 1150, 803, 650. H NMR (400 MHz, DMSO-d6) δ 8.45 (s, 1H), 7.79 (s, 1H), 7.65 (s, 1H), 7.54 (s, 1H), 6.81 – 7.88 (d, 9H), 6.50 (s, 1H), 5.01 (dd, J = 14.0, 27.1 Hz, 1H), 4.81 (dd, J = 13.7, 26.9 Hz, 1H), 4.81 (dd, J = 14.1, 27.1 Hz, 1H), 4.51 (dd, J = 13.6, 38.8 Hz, 1H), 3.68 (s, 13 3H). C NMR (100 MHz, DMSO-d6) δ 168.32, 161.23, 159.39, 154.77, 151.94, 151.56, 145.47, 137.10, 133.68, 133.70, 131.54, 130.89, 129.76, 128.47, 127.87, 127.97, 124.76, 121.99, 115.53, + 82.76, 72.34, 69.16, 57.99. EI-MS m/z 602 (M+H) ; Anal. Calcd for C25H21Cl2N7O5S: (%) C, 49.84; H, 3.51; N, 16.27; Found: C, 49.86; H, 3.52; N, 16.28.

2-(2,4-dichlorophenyl)-1-(2-methoxy-4-(1-tosyl-1H-tetrazol-5-yl)phenoxy)-3-(1H-1,2,4-triazol- 1-yl)propan-2-ol (28b)

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-1 Off white solid (80%); m.p. 136-137 ºC; IR (KBr) ʋmax / cm 3545, 3030, 2854, 1624, 1522, 1 1425, 1360, 1151, 810, 722, 605. H NMR (400 MHz, DMSO-d6) δ 8.35 (s, 1H), 7.80 (s, 1H), 7.76 (s, 1H), 7.45 (s, 1H), 6.97 – 7.95 (d, 8H), 6.49 (s, 1H), 4.91 (dd, J = 14.6, 26.9 Hz, 2H), 13 4.49 (dd, J = 13.9, 37.9 Hz, 2H), 3.78 (s, 3H), 2.42 (s, 3H). C NMR (100 MHz, DMSO-d6) δ 166.78, 164.76, 162.77, 159.55, 154.76, 150.75, 151.34, 145.86, 136.97, 132.18, 133.97, 132.12, 130.73, 129.94, 128.55, 127.57, 126.86, 121.89, 115.85, 86.12, 72.67, 69.78, 58.79, 29.49. EI- + MS m/z 616 (M+H) ; Anal. Calcd for C26H23Cl2N7O5S: (%) C, 50.66; H, 3.76; N, 15.90; Found: C, 50.21; H, 3.77; N, 15.91.

2-(2,4-dichlorophenyl)-1-(4-(1-((4-fluorophenyl)sulfonyl)-1H-tetrazol-5-yl)-2- methoxyphenoxy)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (28c) -1 Pale yellow solid (82%); m.p. 151-153 ºC; IR (KBr) ʋmax / cm 3538, 3035, 2844, 1614, 1520, 1 1428, 1361, 1151, 1075, 820, 717, 601. H NMR (400 MHz, DMSO-d6) δ 8.46 (s, 1H), 7.89 (s, 1H), 7.66 (s, 1H),6.85 (s, 1H), 7.16 – 7.80 (d, 8H), 6.35 (s, 1H), 5.02 (dd, J = 14.8, 27.6 Hz, 1H), 4.87 (dd, J = 13.7, 38.5 Hz, 1H), 4.67 (dd, J = 13.3, 38.1 Hz, 1H), 4.52 (dd, J = 13.1, 37.9 Hz, 13 1H), 3.69 (s, 3H). C NMR (100 MHz, DMSO-d6) δ 163.60, 161.83, 158.58, 154.77, 151.94, 145.48, 138.80, 133.10, 133.54, 133.65, 131.66, 131.38, 130.23, 129.97, 128.06, 127.15, 126.76, 121.79, 114.56, 85.17, 75.76, 71.88, 57.89. EI-MS m/z 620 (M+H)+; Anal. Calcd for

C25H20Cl2FN7O5S: (%) C, 48.40; H, 3.25; N, 15.80; Found: C, 48.41; H, 3.27; N, 15.81.

1-(4-(1-((4-bromophenyl)sulfonyl)-1H-tetrazol-5-yl)-2-methoxyphenoxy)-2-(2,4- dichlorophenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (28d) -1 Off white solid (82%); m.p. 129-130 ºC; IR (KBr) ʋmax / cm 3535, 3019, 2855, 1601, 1525, 1 1421, 1364, 1135, 815, 710, 608, 508. H NMR (400 MHz, DMSO-d6) δ 8.49 (s, 1H), 7.96 (s, 1H), 7.86 (s, 1H), 6.79 (s, 1H), 7.61 (s, 1H), 7.16 – 7.80 (d, 8H), 6.45 (s, 1H), 5.18 (dd, J = 14.9, 28.0 Hz, 1H), 4.85 (dd, J = 13.85, 37.9 Hz, 1H), 4.66 (dd, J = 13.7, 37.4 Hz, 1H), 4.46 (dd, J = 13 13.1, 37.5 Hz, 1H), 3.76 (s, 3H). C NMR (100 MHz, DMSO-d6) δ 163.20, 161.63, 158.68, 154.97, 151.84, 145.58, 138.79, 133.18, 133.84, 133.75, 131.76, 131.48, 130.33, 129.87, 128.66, 127.45, 126.87, 121.80, 114.56, 87.21,75.76, 71.88, 57.89. EI-MS m/z 679 (M+H)+, 681 +2 (M+H) ; Anal. Calcd for C25H20BrCl2N7O5S: (%) C, 44.07; H, 2.96; N, 14.39; Found: C, 44.18; H, 2.97; N, 14.39.

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2-(2,4-dichlorophenyl)-1-(2-methoxy-4-(1-((4-methoxyphenyl)sulfonyl)-1H-tetrazol-5- yl)phenoxy)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (28e) -1 Pale yellow solid (82%); m.p. 144-146 ºC; IR (KBr) ʋmax / cm 3538, 3029, 2855, 1603, 1527, 1 1420, 1361, 1132, 812, 711, 610. H NMR (400 MHz, DMSO-d6) δ 8.39 (s, 1H), 7.79 (s, 1H), 7.66 (s, 1H), 6.85 (s, 1H), 7.12–7.90 (d, 8H), 6.45 (s, 1H), 5.12 (dd, J = 14.3 Hz, 27.1 Hz, 1H), 4.87 (dd, J = 13.72, 37.6 Hz, 2H), 4.67 (dd, J = 13.72, 37.4 Hz, 1H), 4.57 (dd, J = 13.62, 37.7 13 Hz, 1H), 3.91 (s, 3H), 3.70 (s, 3H). C NMR (100 MHz, DMSO-d6) δ 166.78, 163.10, 162.90, 161.89, 158.77, 154.87, 151.74, 145.88, 138.82, 133.40, 133.64, 133.15, 131.36, 131.88, 129.67, 128.36, 127.65, 126.46, 121.99, 114.66, 75.76, 71.88, 60.86, 57.89. EI-MS m/z 632 (M+H)+;

Anal. Calcd for C26H23Cl2N7O6S: (%) C, 49.37; H, 3.67; N, 15.50; Found: C, 49.38; H, 3.69; N, 15.51.

2-(2,4-dichlorophenyl)-1-(2-methoxy-4-(1-((4-nitrophenyl)sulfonyl)-1H-tetrazol-5- yl)phenoxy)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (28f) -1 Yellow solid (76%); m.p. 133-134 ºC; IR (KBr) ʋmax / cm 3545, 3030, 2875, 1606, 1515, 1431, 1 1364, 1259, 1135, 1070, 815, 714, 608. H NMR (400 MHz, DMSO-d6) δ 8.47 (s, 1H), 8.21 (s, 1H), 7.89 (s, 1H), 6.89 (s, 1H), 6.98 – 7.95 (d, 8H), 6.50 (s, 1H), 5.10 (dd, J = 14.3, 27.9 Hz, 1H), 4.81 (dd, J =13.6, 38.6 Hz, 1H), 4.65 (dd, J = 13.5, 38.3 Hz, 1H), 4.51 (dd, J = 13.4, 38.2 13 Hz, 1H), 3.74 (s, 3H). C NMR (100 MHz, DMSO-d6) δ 165.12, 163.64, 162.73, 159.43, 154.7, 150.87, 145.87, 136.92, 136.61, 133.66, 133.52, 131.38, 130.28, 129.92, 128.06, 127.55, 126.67, 124.99, 121.34, 114.88, 78.34, 72.01, 58.99. EI-MS m/z 647 (M+H)+; Anal. Calcd for

C25H20Cl2N8O7S: (%) C, 46.38; H, 3.11; N, 17.31; Found: C, 46.39; H, 3.12; N, 17.33.

1-(4-(1-((4-(tert-butyl)phenyl)sulfonyl)-1H-tetrazol-5-yl)phenoxy)-2-(2,4-dichlorophenyl)-3- (1H-1,2,4-triazol-1-yl)propan-2-ol (28g) -1 Pale yellow solid (71%); m.p. 120-121 ºC; IR (KBr) ʋmax / cm 3548, 3030, 2853, 1604, 1522, 1 1419, 1362, 1130, 811, 604. H NMR (400 MHz, DMSO-d6) δ 8.46 (s, 1H), 7.88 (s, 1H), 7.79 (s, 1H), 6.89 (s, 1H), 6.98 – 7.80 (d, 8H), 6.50 (s, 1H), 5.07 (dd, J = 14.8, 27.9 Hz, 1H), 4.84 (dd, J = 14.1, 38.5 Hz, 1H), 4.64 (dd, J = 14.0, 38.3 Hz, 1H), 4.49 (dd, J = 14.1, 38.5 Hz, 1H), 3.62 (s, 13 3H), 1.29 (s, 9H). C NMR (100 MHz, DMSO-d6) δ 161.13, 158.88, 154.65, 150.84, 147.12,

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145.88, 142.12, 137.01, 133.55, 133.78, 133.56, 131.89, 131.87, 130.93, 129.97, 128.76, 127.25, 126.61, 121.82, 114.73, 87.12, 75.19, 71.90, 35.82, 30.99. EI-MS m/z 658 (M+H)+; Anal. Calcd for C29H29Cl2N7O5S: (%) C, 52.89; H, 4.44; N, 14.89; Found: C, 52.91; H, 4.45; N, 14.90.

1 H NMR spectrum (400MHz, DMSO-d6) of compound 28g.

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13 C NMR spectrum (100 MHz, DMSO-d6) of compound 28g.

1-(4-(1-((5-bromothiophen-2-yl)sulfonyl)-1H-tetrazol-5-yl)-2-methoxyphenoxy)-2-(2,4- dichlorophenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (28h) -1 Pale yellow solid (83%); m.p. 119-121 ºC; IR (KBr) ʋmax / cm 3544, 3026, 2845, 1670, 1510, 1 1431, 1360, 1145, 804, 720, 608, 509. H NMR (400 MHz, DMSO-d6) δ 8.44 (s, 1H), 8.28 (s, 1H),7.78 (s,1H), 6.89 (s,1H), 7.13–7.91 (d, 6H), 6.47 (s, 1H), 5.01 (dd, J = 14.7 Hz, 28.9 Hz, 1H), 4.82 (dd, J = 12.9, 39.2 Hz, 2H), 4.66 (dd, J = 12.5, 39.0 Hz, 2H), 4.52 (dd, J = 12.9, 39.2 13 Hz, 1H), 3.64 (s, 3H). C NMR (100 MHz, DMSO-d6) δ 165.12, 163.89, 162.23, 159.55, 158.43, 151.67, 143.45, 136.87, 132.87, 131.87, 130.93, 129.85, 129.63, 128.79, 114.55, 111.57, 111.83, 105.67, 98.89, 86.12,75.59, 69.20, 59.80. EI-MS m/z 685 (M+H)+, 687 (M+H)+2; Anal.

Calcd for C23H18BrCl2N7O5S2: (%) C, 40.19; H, 2.64; N, 14.26; Found: C, 40.20; H, 2.65; N, 14.37.

2-(2,4-dichlorophenyl)-1-(2-methoxy-4-(1-((4-(trifluoromethoxy)phenyl)sulfonyl)-1H-tetrazol- 5-yl)phenoxy)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (28i) -1 Pale yellow solid (85%); m.p. 144-145 ºC; IR (KBr) ʋmax / cm 3540, 3021, 2843, 1603, 1520, 1 1410, 1361, 1130, 810, 715, 608. H NMR (400 MHz, DMSO-d6) δ 8.41(s, 1H), 7.80 (s, 1H),

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7.69 (s, 1H), 6.95 (s, 1H), 7.28 – 7.82 (d, 8H), 6.49 (s, 1H), 5.09 (dd, J = 14.4, 28.9 Hz, 1H), 4.85 (dd, J = 13.9, 37.1 Hz, 1H), 4.70 (dd, J = 14.9, 37.9 Hz, 1H), 4.49 (dd, J = 15.1, 37.4 Hz, 13 1H), 3.61 (s, 3H). C NMR (100 MHz, DMSO-d6) δ 166.75, 164.65, 162.55, 160.12, 158.99, 155.20, 150.34, 145.87, 141.43, 137.10, 133.65, 133.99, 132.62, 131.67, 130.99, 129.67, 128.78, 127.58, 126.68, 121.92, 114.63, 86.1, 75.19, 72.40. EI-MS m/z 686 (M+H)+; Anal. Calcd for

C27H22Cl2F3N7O7S: (%) C, 45.49; H, 2.94; N, 14.28; Found: C, 45.50; H, 2.95; N, 14.29.

5.4.2. Anti-tubercular activity against MTB H37RV strain The antimycobacterial activities of title compounds 25a-i, 26a-i, 27a-i and 28a-i were evaluated against MTB H37Rv (ATCC 27294) strain by using MABA [30-32]. Ethambutol, Isoniazid and Rifampcin are used as positive controls. Compound stock solutions were prepared in DMSO at a concentration of 100 µL and the final test concentrations ranged from 25 to 0.78 μg/mL. 200 mL of sterile deionized water was added to all outer-perimeter wells of sterile 96-well plates to minimize evaporation of the medium in the test wells during incubation. The wells in rows B to G in columns 3 to 11 received 100 μl of 7H9GC broth. 100 µL of 2 × drug solutions were added to the wells in rows B to G in columns 2 and 3. By using a multichannel pipette, 100 μl was transferred from column 3 to column 4, and the contents of the wells were mixed well. Identical serial 1:2 dilutions were continued through column 10 and 100 μl of excess medium was discarded from the wells in column 10. 100 µL of MTB inoculum was added to the wells in rows B to G in columns 2 to 10. 100 µL of medium to B11 and C11 (media control), 100 µL of MTB inoculum to D11 and E11 and 100 µL of MTB inoculum with 3-5% DMSO to F11 and G11 (solvent control) were added. The plates were sealed with parafilm and were incubated at 37°C for 5 days. 50 µL of a freshly prepared 1:1 mixture of 10 x Alamar Blue (Accumed International, Westlake, Ohio) reagent and 10% Tween 80 were added to well D11. The plates were reincubated at 37 °C for 24 h. If well D11 turned pink, the reagent mixture was added to all wells in the microplate (if the well remained blue, the reagent mixture would be added to another control well and the result would be read on the following day). The microplates were resealed with parafilm and were incubated for an additional 24 h at 37 °C, and the colors of all wells were recorded. A blue color in the well was interpreted as no growth, and a pink color was scored as growth. A few wells appeared violet after 24 h of incubation, but they invariably changed to pink after another day of incubation and thus were scored as growth (while the adjacent blue wells

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remained blue). The MIC was defined as the lowest drug concentration which prevented a color change from blue to pink [32].

5.4.3. CHO-K1 Cytotoxicity Standard MTT assay (Sigma Aldrich) was applied according to the manufacturer´s protocol on Chinese hamster ovary (CHO-K1) cell lines. The cells were cultured according to ECACC recommended conditions and seeded in a density of 8 × 103, 12 × 103 per well respectively for CHO-K1 cells. Cells were exposed to test compounds for 48 hours, then the medium was replaced for a medium containing 100 μM of MTT and cells were allowed to produce formazan for another approximately 2 h under observation. Then, medium with MTT was sucked out and crystals of formazan were dissolved in DMSO. Cell viability was assessed spectrophotometrically by the amount of formazan produced. Absorbance was measured at 570 nm with 650 nm reference wavelength [33].

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[25] H. N. Nagesh, K. Naidu, D. Harika Rao, J. P. Sridevi, D. Sriram, P. Yogeeswari, K. V. G. Chandra Sekhar, Bioorg. Med. Chem. Lett., 2013, 23, 6805. [26] K. Naidu, H. N. Nagesh, M. Singh, D. Sriram, P. Yogeeswari, K. V. G. Chandra Sekhar, Eur. J. Med. Chem., 2015, 92, 415. [27] S. Kae-shyang, C. Lie-Rong, L. Ching-Wie, J. W. China-Lin, U.S. Patent. 1998, 57102890. [28] H. B. Borate, S. P. Sawargave, S. P. Chavan, M. A. Chandavarkar, R. Iyer, A. Tawte, D. Rao, J. V. Deore, A. S. Kudale, P. S. Mahajan, G. S. Kangire, Bioorg. Med. Chem. Lett., 2011, 21, 4873. [29] D. Amantini, R. Beleggia, F. Fringuelli, F. Pizzo, L. Vaccaro, J. Org. Chem., 2004, 69, 2896. [30] H. B. Borate, S. P. Sawargave, S. P. Chavan, M. A. Chandavarkar, R. Iyer, A. Tawte, D. Rao, J. V. Deore, A. S. Kudale, P. S. Mahajan, G. S. Kangire, Bioorg. Med. Chem. Lett., 2011, 21, 4873. [31] D. Amantini, R. Beleggia, F. Fringuelli, F. Pizzo, L. Vaccaro, J. Org. Chem., 2004, 69, 2896. [32] L. A. Collins, S. G. Franzblau, Antimicrob. Agents Chemother., 1997, 41, 1004. [33] J. Zitko, B. Servusova, A. Janoutova, P Paterova, J. Mandikova, V. Gara j, M. Vejsova, J. Marek, M. Dolezal, Bioorg. Med. Chem., 2015, 23, 174.

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Chapter VI

6-chloro, 6,7-dichloro and 2-methyl-3-(((1-(substitutedphenyl)-1H-1,2,3- triazol-4-yl)methoxy)carbonyl)quinoxaline 1,4-dioxide derivatives as anti-tubercular agents

Chapter 6

Chapter 6

6-chloro, 6,7-dichloro and 2-methyl-3-(((1-(substitutedphenyl)-1H-1,2,3- triazol-4-yl)methoxy)carbonyl)quinoxaline 1,4-dioxide derivatives as anti-tubercular agents

6.1. Introduction Quinoxaline derivatives are a class of compounds that show very exciting biological properties and the importance in these compounds is growing within the field of medicinal chemistry [1]. Over the last three decades, many mono- and di-N-oxides and 2-oxo derivatives of this heterocyclic system have appeared on the scene and their biological activities have been reported. Oxidation of both nitrogens of the pyrazine ring to obtain quinoxaline-1,4-di-N-oxide dramatically increases the diversity of biological properties [2]. The quinoxaline 1,4-di-N-oxides were known as potent antibacterial agents since the 1940s. Quinoxaline-1,4-di-N-oxide derivatives even improve the biological results shown by their reduced analogues and are endowed with antiviral, anticancer, antibacterial and antiprotozoal activities [3]. Sainza et al., reported quinoxaline l,4-di-N-oxide derivatives with different substituents in 2, 3, 6 and 7 positions gave in order to obtain new hypoxia selective agents. Some of these products gave good results as antituberculosis agents [4]. Different 7-chloro-3-(para substituted) phenylaminoquinoxaline-2-carbonitrile 1,4-di-N-oxides have shown to possess MTB growth inhibition value of 99% [5]. 6,7-dichloro-2-ethoxycarbonyl-3-methylquinoxaline 1,4-di-N-oxide and 3-acetamide-6,7-dichloroquinoxaline-2-carbonitrile 1,4-di-N-oxide derivatives produced growth inhibition values of 100% [4, 6]. Jsco et al., reported 2-acetyl and 2-benzoyl-6(7)- substituted quinoxaline 1,4-di-N-oxide derivatives with MTB MIC ranging from 3.3 to 62.5 μM against MTB H37Rv. Same groups reported twenty nine new 6 (7)-substituted quinoxaline-2- carboxylate 1,4-dioxides with better activity MTB MIC ranging from 0.10 to >6.25 μg/mL [7]. Torres et al., reported 1,4-di-N-oxide-quinoxaline-2-ylmethylene isonicotinic acid hydrazide with MTB IC50 ranging from 0.58 to 90.84 μM against MTB H37Rv [8]. Twenty seven 2- acetylquinoxaline 1,4-di-N-oxide and seven 2-benzoylquinoxaline 1,4-di-N-oxide derivatives

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showed MTB IC50 ranging from 0.20 to 99.91 μg/mL against MTB H37Rv [9]. Pan et al., synthesized thirty one compounds of quinoxaline 1,4-di-N-oxides variously substituted at C-2 position and evaluated their antimycobacterial activity with MTB MIC ranging from 0.39 to 50 μg/mL [10]. Quinoxaline N-oxide based anti-TB agents are depicted in figure 6.1.

Recently some select analogues were found to be active against a panel of single-drug-resistant strains of MTB and in the TAACF macrophage model [9]. Two derivatives, compounds C and D were successful in vivo in a murine model of low dose aerosol infection. Moreover, these two compounds also showed activity against non-replicating (NRP) bacteria. If the bactericidal activity and activity on NRP bacteria in vitro translate to in vivo conditions, quinoxaline 1,4-di- N-oxides may lead to reduced therapy, since the presence of NRP bacteria is assumed to be major cause responsible for the prolonged nature of antitubercular therapy [11].

Figure 6.1: Some of the N-oxide based anti-tubercular agents

1,2,3-triazole and its derivatives have attracted continued interest in the medicinal chemistry field owing to their varied biological activities such as anti-bacterial, antiviral, antifungal, anti- allergic, anti-HIV, anticonvulsant, anti-inflammatory and β-lactamase inhibition properties [12]. It is quite evident that the favorable properties of 1,2,3-triazole ring viz., moderate dipole character, hydrogen bonding capability, rigidity and stability under in vivo conditions are

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Chapter 6 responsible for their enhanced biological activities [13, 14]. Triazole derivatives have also been shown to possess strong inhibitory activities in vitro and in vivo against MTB. These inhibit bacteria by blocking the biosynthesis of certain bacterial lipids. 1,2,3-triazole at all positions with varied substituents has produced potent anti-TB activity. Till date many triazole anti-TB agents were published here depicted in Figure 6.2 some triazole anti-TB agents [15].

Figure 6.2: Some of the triazole based anti-tubercular agents

Quinoxaline 1,4-di-N-oxide and 1,2,3-triazoles moieties are kwit in a single molecular scaffold and antitubercular activity was studied. It has been established that more efficacious antimicrobial compounds can be designed by joining two or more biologically active heterocyclic systems together in a single molecular framework. Based on this strategy we designed and synthesized the target compounds [14].

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Figure 6.3: Design strategy of the title compounds.

6.2. Results and Discussion 6.2.1. Chemistry The designed molecules were synthesized in five steps (Scheme 6.1). Initially we prepared N- oxide intermediate (31a-c) via azides compound (30a-c). Ethyl acetoacetate was treated with proprgyl alcohol in toluene at 110 °C to get transesterified compound 33. The free acetylene group of 33 was converted to various 1H-1,2,3-triazoles (34a-l) using different aromatic azides via click chemistry method [16]. Compound 34 on reacting with various N-Oxide intermediates (31a-c) in the presence of triethylamine formed quinoxaline 1,4-dioxide (35a-l, 36a-l & 37a-g). The purity of synthesized compounds was checked by LC-MS and elemental analyses. Structures of the compounds were confirmed by spectral data. In 1H NMR and 13C NMR, the signals of the respective protons and carbons were verified on the basis of their chemical shifts, multiplicities, and coupling constants. The results of elemental analysis were within ± 0.05 of the theoretical values.

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Scheme 6.1: Synthetic protocol of titled compounds.

Reagents and conditions: (i) NaNO2 (1.50 eq), NaN3 (1.50 eq), 6N HCl (8 wt/v), 0 °C, 2 h. (ii) toluene (30 wt/v), 110 °C, 24 h. (iii) Propargyl alcohol (10.0 eq), toluene, 24 h. (iv) Substituted t phenyl azides, CuSO4.5H2O (10 mol %), Sodium ascorbate (10 mol %), H2O: BuOH (1:2), rt, 16 h. (v) N-oxide intermediate (31a-c) (1.2 eq), Triethylamine, rt, 16 h.

6.2.2. In-vitro MTB screening All the synthesized compounds were tested for their capacity to inhibit the growth of MTB. In assay three different M. tuberculosis strains were used. One of them was reference strain M. tuberculosis H37Rv ATTC 25618 and the others were ‘wild’ strains isolated from tuberculosis patients [17, 18]. MTB strain spec. 210 was resistant to p-aminosalicylic acid (PAS), INH, ETB

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Chapter 6 and RMP and another (Spec. 192) fully sensitive to the administrated tuberculostatics [18]. In this study three different strains were used for screening as we wanted to know the kind of activity synthesized compounds showed against the reference strain as well as against the strains isolated from TB patients. In this study the influence of the compound on the growth of mycobacteria at a certain concentration: 3.1, 6.2, 12.5, 25, 50 and 100 μg/mL were evaluated INH was used as reference drug. The in vitro antimycobacterial results of title compounds are arranged in Table 6.1 as MIC (µM) and the activity ranged from 30.35- >252 µM.

Table 6.1: Result of Antimycobacterial screening of title compounds MIC in µM MIC in µM MIC in µM (µg/mL) (µg/mL) (µg/mL) Entry X Y Ar against MTB against MTB against MTB H37Rv Spec. 192 Spec. 210 35a H H phenyl 132.50 (50) 132.50 (50) 132.50 (50) 35b H H 4-Ethylphenyl 123.33 (50) 123.33 (50) 123.33 (50) 35c H H 4-Fluorophenyl >252.94 (>100) >252.94 (>100) >252.94 (>100) 35d H H 4-Chlorophenyl >242.84 (>100) >242.84 (>100) >242.84 (>100) 35e H H 4-Bromophenyl 109.59 (50) 109.59 (50) 109.59 (50) 35f H H 4-Nitrophenyl >236.77 (>100) >236.77 (>100) >236.77 (>100)

35g H H 2- Fluorophenyl 126.47 (50) 126.47 (50) 126.47 (50) 35h H H 2-Chlorophenyl 60.71 (25) 60.71 (25) 60.71 (25) 35i H H 2-Nitrophenyl 59.19 (25) 59.19 (25) 59.19 (25) 35j H H 3-Nitrophenyl 59.19 (25) 59.19 (25) 59.19 (25) 3-Trifluoromethyl 35k H H 224.54 (100) 224.54 (100) 112.27 (50) pheny 35l H H 3,5-dichlorophenyl 97.06 (50) 97.06 (50) 194.12 (100) 36a Cl H phenyl 30.35 (12.5) 30.35 (12.5) 30.35 (12.5) 36b Cl H 4-Ethylphenyl 56.83 (25) 56.83 (25) 56.83 (25) 36c Cl H 4-Fluorophenyl 58.16 (25) 58.16 (25) 58.16 (25)

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MIC in µM MIC in µM MIC in µM (µg/mL) (µg/mL) (µg/mL) Entry X Y Ar against MTB against MTB against MTB H37Rv Spec. 192 Spec. 210 36d Cl H 4-Chlorophenyl 56.02 (25) 56.02 (25) 56.02 (25) 36e Cl H 4-Bromophenyl 50.94 (25) 50.94 (25) >203.76 (>100) 36f Cl H 4-Nitrophenyl 54.72 (25) 54.72 (25) 54.72 (25) 36g Cl H 2-Fluorophenyl 58.16 (25) 58.16 (25) 58.16 (25) 36h Cl H 2-Chlorophenyl 56.02 (25) 56.02 (25) 56.02 (25) 36i Cl H 2-Nitrophenyl 54.72 (25) 54.72 (25) 109.44 (50) 36j Cl H 3-Nitrophenyl 109.45 (50) 109.45 (50) 109.45 (50) 3-Trifluoromethyl 36k Cl H 104.21 (50) 104.21 (50) 104.21 (50) phenyl 36l Cl H 3,5-dichlorophenyl 52.00 (25) 52.00 (25) 52.00 (25) 37a Cl Cl phenyl 112.04 (50) 112.04 (50) 112.04 (50) 37b Cl Cl 4-Fluorophenyl 53.85 (25) 53.85 (25) 53.85 (25) 37c Cl Cl 4-Chlorophenyl 52.00 (25) 52.00 (25) 52.00 (25) 37d Cl Cl 4-Bromophenyl 47.60 (25) 47.60 (25) 47.60 (25) 37e Cl Cl 2-Fluorophenyl 107.70 (50) 107.70 (50) 107.70 (50) 37f Cl Cl 2-Chlorophenyl 104.01 (50) 104.01 (50) 104.01 (50) 37g Cl Cl 3,5-dichlorophenyl 97.06 (50) 97.06 (50) 97.06 (50) INH - - - <22.59 (<3.1) <22.59 (<3.1) 91.15 (12.5)

Among the thirty one compounds screened , eight compounds ( 36a, 36e, 36f, 36i, 36l, 37b, 37c and 37d) showed activity against MTB with MIC <55.00 µM. Two compounds (36a & 37d) inhibited MTB with MIC <50.00 µM. Compound 36a (6-chloro-2-methyl-3-(((1-phenyl-1H- 1,2,3-triazol-4-yl)methoxy)carbonyl)quinoxaline 1,4-dioxide) was found to be the most active compound with MTB MIC 30.35 µM.

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SAR of compound 35a-l SAR is explained based on activity of compound 35a. Structural changes at ortho, meta & para positions alter the activity. Compound 35a was inhibiting growth of MTB H37Rv strain at 132.50 µM. In this series, introduction of electron donation ethyl group at the 4th position activity remained unaltered. Presence of electron withdrawing F and Cl on the phenyl ring at para position resulted in decease in activity by the two folds (35c, MIC >252.94 µM; 35d, MIC >242.84 µM) but the presence of bromo (35e) at the para position resulted in increase in activity by one fold with MIC 109.59 µM. Presence of electron withdrawing nitro group at para position decreased activity by two folds (35f, >236.77) but nitro at the ortho and meta position activity increased by two folds (35i, MIC 59.19 µM; 35j, MIC 59.19 µM). With introduction of fluoro group at ortho position activity remained unchanged compared with compound 35a but the presence of chloro (35h) at ortho position enhanced the activity by two folds with MIC 60.71 µM. Presence of two Cl (35l) at 3rd and 5th position resulted increase in activity by one fold with

MIC 97.06 µM. Introduction of withdrawing CF3 group (35k) at meta position activity decreased by two folds with MIC 224.54 µM.

SAR of compound 36a-l SAR is explained based on activity of compound 36a. Compound 36a was inhibiting growth of MTB H37Rv strain at 30.35 µM. With the presence of electron donating ethyl group at para position activity fell by two folds with MIC 56.83 µM. Introduction of electron withdrawing groups like viz., F, Cl & Br at artho, meta and para position with mono or disubstituted resulted in decrease in activity by two folds. Presence of electron withdrawing nitro group at para position (36f) decreased in activity two folds with MIC 54.72 µM but the introduction nitro group at meta position (36j) activity decreased by four folds with MIC 109.45

µM. Introduction of electro withdrawing CF3 group (36k) at meta position activity decreased by four folds with MIC 104.21 µM. All these results show that the insertion of a halogen moiety will increases the anti-tubercular activity.

SAR of compound 37a-g In this series, two Cl groups were introduced on the quinoxaline 1,4-dioxide frame but activity did not improve. SAR explained based on activity of compound 37a (MIC 112.04 µM).

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With introduction of electron withdrawing halogens like viz., F, Cl & Br at the para position on the phenyl, activity increased by two folds (37b, MIC 53.85 µM; 37c, MIC 52.00 µM & 37d, MIC 47.60 µM) but presence of F & Cl at the ortho position activity remained unchanged (37e, MIC 107.70 µM; 37f, MIC 104.01 µM). Introduction of chlorine at the 3rd and 5th position resulted in retention of activity.

Over all, we notice that 6-chloro-3-(((1-(substituted phenyl)-1H-1,2,3-triazol-4- yl)methoxy)carbonyl)-2-methylquinoxaline 1,4-dioxide derivatives (36a-l) exhibited better anti- TB activity followed by 2-(((1-(substituted phenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)-3- methylquinoxaline 1,4-dioxide derivative (35a-l) and 6,7-dichloro-2-(((1-(4-fluorophenyl)-1H- 1,2,3-triazol-4-yl)methoxy)carbonyl)-3-methylquinoxaline 1,4-dioxide derivatives (37a-g).

6.2.3. In vitro cytotoxicity studies Compounds with MTB MIC < 12.5 µg/mL were subjected to cytotoxicity studies against HEK 293 cell line. Cytotoxicity assay of 36a, 36e, 36f, 36i, 36l, 37b, 37c & 37d was determined. Cell viability was measured by in vitro MTT assay [19]. Cells were exposed to compounds for 24 hours at three concentrations 50 µM, 25 µM and 10 µM (n=2). Data represent mean values of measurements ± s.d. (Figure 6.4). Data clearly indicate the active compounds 36e, 36f, 36i, 36l & 37d were not toxic at even 50 µM. However, the compounds 36a and 37c were moderately toxic.

Figure 6.4: Cytotoxicity assay of 36a, 36e, 36f, 36i, 36l, 37b, 37c & 37d on HEK-293 cells.

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6.3. Conclusion In this chapter, quinoxaline 1,4-dioxide analogues synthesized with three different series; 2- methyl-3-(((substituted pheny)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)quinoxaline 1,4-dioxide derivatives, 6-chloro-3-(((1-(substituted phenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)-2- methylquinoxaline 1,4-dioxide derivatives and 6,7-dichloro-2-(((1-(4-fluorophenyl)-1H-1,2,3- triazol-4-yl)methoxy)carbonyl)-3-methylquinoxaline 1,4-dioxide derivatives by the molecular hybridization approach using reported 1,4-dioxides anti-TB agents and substituted 1H-1,2,3- triazol antitubercular agents. Amongst the synthesized compounds, 6-chloro-3-(((1- (substitutedphenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)-2-methylquinoxaline 1,4-dioxide derivatives showed good ant-TB results. One of the compound 36a showed excellent MTB activity with MIC 30.35 µM. Further, the most active compounds (36e, 36f, 36i, 36l & 37d) did not exhibit cytotoxicity against HEK 293 cell line for the most active compounds at 50 μM while 36a and 37c were moderately toxic.

6.4. Experimental 6.4.1. Materials and methods Chemicals and solvents were procured from commercial source. The solvents and reagents were of LR grade and if necessary purified before use. Thin-layer chromatography (TLC) was carried out on aluminium-supported silica gel plates (Merck 60 F254) with visualization of components by UV light (254 nm). Column chromatography was carried out on silica gel (Merck 100-200 mesh). 1H NMR and 13C NMR spectra were recorded at 400 MHz and 101 MHz respectively using a Bruker AV 400 spectrometer (Bruker CO., Switzerland) in CDCl3 and DMSO-d6 solution with tetramethylsilane as the internal standard and chemical shift values (δ) were given in ppm. Melting points were determined on an electro thermal melting point apparatus (Stuart- SMP30) in open capillary tubes and are uncorrected. Elemental analyses were performed by Elementar Analysensysteme GmbH vario MICRO cube CHN Analyzer. Mass spectra (ESI-MS) were recorded on Schimadzu MS/ESI mass spectrometer. Purity of all tested compounds were determined by LC-MS/MS on Schimadzu and was greater than 95%.

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6.4.2. Chemistry Representative procedure for the synthesis of compound (30a-c) A stirred solution of compound (29) (1.0 equiv.) in 6N HCl was cooled to 0 °C. The reaction mixture was stirred for 5 minutes and NaNO2 (1.50 equiv.) in water at 0 °C was slowly added and stirred for 5 minutes. This was followed by addition of NaN3 (1.50 equiv.) in water at 0 °C it was stirred for 2 h at 0 °C. Once completion of the reaction, as indicated by TLC, the reaction was quenched with cold water and extracted with ethyl acetate. The organic layers were collected, washed with saturated brine solution, dried over anhydrous Na2SO4 and concentrated in vacuo. The product was obtained as yellow solid (30a-c) yields (70-85%).

Representative procedure for the synthesis of compound (31a-c) A stirred solution of compound (30a-c) in toluene was heated at 110 °C for 24 h under argon. After completion of the reaction, as indicated by TLC, the reaction was concentrated in vacuo. The resultant crude product was purified by column chromatography [ethyl acetate / hexane (5 - 10%)] to get the compound 31a-c (75-85%) as yellow solid.

Representative procedure for the synthesis of compound (33) To solution of compound 32 (1.0 equiv) in toluene propargyl alcohol (5.0 equiv.) was added. The solution was heated at 110 °C for 12 h then another 5.0 equivalence of propargyl alcohol was added and continued for another 12 h at 110 °C. Once completion of the reaction, as indicated by TLC, the toluene was removed in vacuo. The crude product was purified by column chromatography [ethyl acetate / hexane (30 - 40%)] to get the compound 33 (79-90%) as yellow oil.

Representative procedure for the synthesis of compound (34a-l) A solution of compound 33 (1.0 equiv.) is reacted with substituted phenyl azides (1.2 equiv.) in the presence of sodium ascorbate (0.01 equiv.), CuSO4.5H2O (0.02 equiv.) and t-BuOH: H2O (2:1), at rt for 16 h. Once completion of the reaction, as indicated by TLC, the reaction was quenched with cold water and extracted with DCM. The DCM layers were collected, washed with saturated brine solution, dried over anhydrous Na2SO4 and concentrated in vacuo. The

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Chapter 6 resultant crude product was purified by column chromatography [ethyl acetate / hexane (30 - 40%)] to yield (35-65%) the title compounds 34a-l.

Representative procedure for the synthesis of compound (35a-l, 36a-l & 37a-h) To stirred suspension of an appropriate compound 31a-c (1.0 equiv.) compound 34a-l (1.2 equiv.) in triethylamine is added and stirred 16 hours at room temperature under nitrogen. Once completion of the reaction, as indicated by TLC, the triethylamine was removed under vacuum distillation. The remaining reaction mixture was dissolved in ethyl acetate and washed with water. The organic layer was separated, dried over Na2SO4 and concentrated under reduced pressure to give crude product. The resultant crude product was purified by column chromatography [ethyl acetate / hexane (30 - 40%)] to get compound (35a-l, 36a-l & 37a-h). Yield ranging from 45 to 85%.

2-methyl-3-(((1-phenyl-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)quinoxaline 1,4-dioxide (35a) -1 Yellow solid (47%); m.p. 188-189 °C; IR (KBr) ʋmax / cm 3017, 2895, 1663, 1339, 1020, 960. 1H NMR (400 MHz, Chloroform-d) δ 8.65 – 8.62 (m, 1H), 8.60 – 8.56 (m, 1H), 8.32 (s, 1H), 7.95 – 7.83 (m, 2H), 7.81 – 7.75 (m, 2H), 7.60 – 7.53 (m, 2H), 7.53 – 7.45 (m, 1H), 5.77 (s, 2H), 13 2.58 (s, 3H). C NMR (101 MHz, CDCl3) δ 159.34, 143.10, 139.01, 138.18, 137.85, 136.14, 135.28, 133.06, 132.22, 129.73, 129.42, 122.03, 121.99, 121.04, 119.04, 60.71, 14.68. EI-MS + m/z 378.12 (M+H) ; Anal. calcd for C19H15N5O4: (%) C, 60.48; H, 4.01; N, 18.56; Found: C, 60.49; H, 4.03; N, 18.58.

2-(((1-(4-ethylphenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)-3-methylquinoxaline 1,4- dioxide (35b) -1 Pale yellow solid (63%); m.p. 134-135 °C; IR (KBr) ʋmax / cm 3012, 2890, 1670, 1340, 1060, 975. 1H NMR (400 MHz, Chloroform-d) δ 8.65 – 8.60 (m, 1H), 8.57 (dd, J = 8.6, 1.5 Hz, 1H), 8.25 (s, 1H), 7.87 (dddd, J = 18.2, 8.5, 7.0, 1.5 Hz, 2H), 7.69 – 7.62 (m, 2H), 7.40 – 7.33 (m, 2H), 5.75 (s, 2H), 2.72 (t, J = 7.6 Hz, 2H), 2.56 (s, 3H), 1.29 (t, J = 7.6 Hz, 3H). 13C NMR (101

MHz, DMSO-d6) δ 159.90, 145.28, 142.09, 138.93, 138.14, 136.79, 134.88, 134.83, 133.32, 132.21, 129.63, 123.98, 120.72, 120.14, 120.13, 60.17, 28.15, 15.91, 14.54. EI-MS m/z 405.15

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+ (M+H) ; Anal. calcd for C21H19N5O4: (%) C, 62.23; H, 4.72; N, 17.27; Found: C, 62.24; H, 4.73; N, 17.28.

2-(((1-(4-fluorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)-3-methylquinoxaline 1,4- dioxide (35c) -1 Yellow solid (44%); m.p. 220-221 °C; (KBr) ʋmax / cm 3025, 2897, 1675, 1412, 1369, 1127, 1022. 972, 1H NMR (400 MHz, Chloroform-d) δ 8.68 – 8.61 (m, 1H), 8.61 – 8.55 (m, 1H), 8.29 (s, 1H), 7.90 (dddd, J = 18.4, 8.4, 7.0, 1.5 Hz, 2H), 7.81 – 7.72 (m, 2H), 7.29 (d, J = 1.9 Hz, 1H), 13 7.27 – 7.24 (m, 1H), 5.77 (s, 2H), 2.59 (s, 3H). C NMR (101 MHz, DMSO-d6) δ 159.89, 142.25, 138.94, 138.15, 136.80, 134.87, 133.34, 132.23, 124.35, 123.18, 123.09, 120.15, 117.41, + 117.18, 60.10, 14.55. EI-MS m/z 396.10 (M+H) ; Anal. calcd for C19H14FN5O4: (%) C, 57.72; H, 3.57; N, 17.71; Found: C, 57.73; H, 3.58; N, 17.72.

2-(((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)-3-methylquinoxaline 1,4 dioxide (35d) -1 Off white solid (57%); m.p. 221-222 °C; (KBr) ʋmax / cm 3075, 2867, 1675, 1412, 1369, 1020, 987, 765. 1H NMR (400 MHz, Chloroform-d) δ 8.69 – 8.62 (m, 2H), 8.31 (s, 1H), 7.99 – 7.86 13 (m, 2H), 7.70 – 7.26 (m, 4H), 5.76 (s, 2H), 2.57 (s, 3H). C NMR (101 MHz, DMSO-d6) δ 159.99, 144.25, 138.14, 138.75, 136.80, 134.87, 134.34, 133.34, 132.73, 125.55, 123.78, 123.19, + 121.19, 117.55, 117.28, 60.10, 14.59. EI-MS m/z 412.21 (M+H) Anal. calcd for C19H14ClN5O4: (%) C, 50.02; H, 3.09; N, 15.35; Found: C, 50.03; H, 3.10; N, 15.36.

2-(((1-(4-bromophenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)-3-methylquinoxaline 1,4- dioxide (35e) -1 Off white solid (49%); m.p. 204-205 °C; (KBr) ʋmax / cm 3092, 2867, 1685, 1412, 1373, 1024, 962, 653. 1H NMR (400 MHz, Chloroform-d) δ 8.66 – 8.60 (m, 2H), 8.33 (s, 1H), 7.92 – 7.87 13 (m, 2H), 7.70 – 7.28 (m, 4H), 5.77 (s, 2H), 2.59 (s, 3H). C NMR (101 MHz, CDCl3) δ 159.85, 142.51, 139.03, 138.07, 136.84, 135.78, 134.97, 133.03, 132.77, 131.62, 122.81, 122.12, 122.05, 120.31, 120.26, 60.27, 14.43. EI-MS m/z 457.21 (M+H)+2; 455.02 (M+H)+; Anal. calcd for

C19H14BrN5O4: (%) C, 50.02; H, 3.09; N, 15.35; Found: C, 50.03; H, 3.10; N, 15.36.

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2-methyl-3-(((1-(4-nitrophenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)quinoxaline 1,4- dioxide (35f) -1 Yellow solid (51%); m.p. 220-221 °C; (KBr) ʋmax / cm 3035, 2877, 1695, 1510, 1273, 1031, 987. 1H NMR (400 MHz, Chloroform-d) δ 8.67 – 8.61 (m, 1H), 8.57 (dd, J = 8.6, 1.5 Hz, 1H), 8.23 (dddd, J = 18.3, 8.5, 7.1, 1.6 Hz, 2H), 8.09 (s, 1H), 7.69 – 7.62 (m, 2H), 7.40 – 7.33 (m, 13 2H), 5.78 (s, 2H), 2.56 (s, 3H). C NMR (101 MHz, DMSO-d6) δ 159.97, 144.72, 142.87, 138.10, 138.74, 136.86, 135.12, 134.73, 134.15, 132.60, 130.96, 129.10, 129.49, 128.09, 127.11, + 126.09, 119.89, 60.39, 14.56. EI-MS m/z 423.11 (M+H) ; Anal. calcd for C19H14N6O6: (%) C, 54.03; H, 3.34; N, 19.90; Found: C, 54.04; H, 3.36; N, 19.91.

2-(((1-(2-fluorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)-3-methylquinoxaline 1,4- dioxide (35g) -1 Pale yellow solid (63%); m.p. 148-150 °C; (KBr) ʋmax / cm 3082, 2897, 1697, 1412, 1373, 1027, 972, 697. 1H NMR (400 MHz, Chloroform-d) δ 8.64 – 8.52 (m, 2H), 8.37 (d, J = 2.7 Hz, 1H), 8.01 – 7.80 (m, 3H), 7.47 (tdd, J = 8.2, 4.9, 1.8 Hz, 1H), 7.40 – 7.27 (m, 2H), 5.77 (s, 2H), 13 2.55 (s, 3H). C NMR (101 MHz, CDCl3) δ 159.76, 154.65, 152.15, 141.81, 139.06, 137.97, 136.81, 134.96, 132.72, 131.55, 130.63, 130.55, 125.49, 125.41, 125.35, 125.31, 125.04, 124.94, 120.31, 120.16, 117.20, 117.00, 60.14, 14.37. EI-MS m/z 396.10 (M+H)+; Anal. calcd for

C19H14FN5O4: (%) C, 57.73; H, 3.57; N, 17.72; Found: C, 57.74; H, 3.58; N, 17.73.

2-(((1-(2-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)-3-methylquinoxaline 1,4- dioxide (35h) -1 Off white solid (54%); m.p. 181-182 °C; (KBr) ʋmax / cm 3076, 2893, 1691, 1421, 1363, 1025, 966, 653. 1H NMR (400 MHz, Chloroform-d) δ 8.71 – 8.65 (m, 2H), 8.39 (d, J = 2.5 Hz, 1H), 8.11 – 7.90 (m, 3H), 7.40 (tdd, J = 8.4, 4.7, 1.8 Hz, 1H), 7.36 – 7.26 (m, 2H), 5.76 (s, 2H), 2.58 13 (s, 3H). C NMR (101 MHz, CDCl3) δ 158.86, 154.65, 150.15, 142.80, 138.16, 137.17, 136.31, 134.96, 132.72, 131.55, 130.63, 130.55, 125.49, 125.41, 125.35, 125.31, 125.44, 124.48, 120.14, 120.61, 117.11, 117.21, 60.44, 14.57. EI-MS m/z 412.07 (M+H)+; Anal. calcd for

C19H14ClN5O4: (%) C, 55.42; H, 3.43; N, 17.02; Found: C, 55.74; H, 3.44; N, 17.03.

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2-methyl-3-(((1-(2-nitrophenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)quinoxaline 1,4- dioxide (35i) -1 Yellow solid (48%); m.p. 197-198 °C; (KBr) ʋmax / cm 3096, 2913, 1693, 1427, 1363, 1033, 986. 1H NMR (400 MHz, Chloroform-d) δ 8.65 – 8.57 (m, 2H), 8.39 (d, J = 2.5 Hz, 1H), 8.11 – 7.94 (m, 3H), 7.57 (tdd, J = 8.4, 4.8, 1.6 Hz, 1H), 7.42 – 7.29 (m, 2H), 5.76 (s, 2H), 2.58 (s, 3H). 13 C NMR (101 MHz, DMSO-d6) δ 159.87, 144.52, 141.87, 139.00, 138.14, 136.76, 135.02, 134.83, 133.25, 132.20, 131.96, 129.80, 129.40, 128.29, 127.37, 126.09, 119.87, 59.91, 14.46. + EI-MS m/z 423.11 (M+H) ; Anal. calcd for C19H14N6O6: (%) C, 54.03; H, 3.34; N, 19.90; Found: C, 54.04; H, 3.36; N, 19.91.

2-methyl-3-(((1-(3-nitrophenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)quinoxaline1,4- dioxide (35j) -1 Yellow solid (65%); m.p. 186-187 °C; (KBr) ʋmax / cm 3032, 2943, 1695, 1424, 1360, 1023, 976. 1H NMR (400 MHz, Chloroform-d) δ 8.69 – 8.57 (m, 2H), 8.41 (d, J = 2.7 Hz, 1H), 8.32 (s, 1H), 8.14 – 7.93 (m, 2H), 7.57 (tdd, J = 8.6, 4.8, 1.8 Hz, 1H), 7.40 – 7.26 (m, 2H), 5.75 (s, 2H), 13 2.56 (s, 3H). C NMR (101 MHz, DMSO-d6) δ 159.77, 143.52, 140.97, 139.10, 138.24, 136.86, 135.12, 133.83, 133.27, 132.27, 131.96, 129.80, 129.40, 128.29, 127.37, 126.09, 119.87, 60.21, + 14.49. EI-MS m/z 423.11 (M+H) ; Anal. calcd for C19H14N6O6: (%) C, 54.03; H, 3.34; N, 19.90; Found: C, 54.04; H, 3.36; N, 19.91.

2-methyl-3-(((1-(3-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl) quinoxaline 1,4-dioxide (35k) -1 Pale yellow (40%); m.p. 193-194 °C; (KBr) ʋmax / cm 3042, 2963, 1689, 1420, 1361,1150, 1029, 973. 1H NMR (400 MHz, Chloroform-d) δ 8.69 – 8.62 (m, 2H), 8.44 (s, 1H), 8.19 (t, J = 1.9 Hz, 1H), 8.04 – 7.99 (m, 3H), 7.87 (dt, J = 7.4, 2.1 Hz, 1H), 7.60 (m, 1H), 5.77 (s, 2H), 2.56 13 (s, 3H). C NMR (101 MHz, CDCl3) δ 159.49, 142.58, 139.29, 138.78, 137.13, 136.61, 135.59, 133.63, 132.71, 132.38, 130.69, 127.32, 125.76, 123.64, 122.27, 121.92, 119.72, 117.60, 60.27, + 14.41. EI-MS m/z 446.11 (M+H) ; Anal. calcd for C20H14F3N5O4: (%) C, 53.95; H, 3.17; N, 15.73; Found: C, 53.96; H, 3.19; N, 15.74.

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2-(((1-(3,5-dichlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)-3-methylquinoxaline 1,4- dioxide (35l) -1 Pale yellow solid (52%); m.p. 205-206 °C; (KBr) ʋmax / cm 3076, 2893, 1691, 1421, 1363, 1025, 966, 653. 1H NMR (500 MHz, Chloroform-d) δ 8.63 (dd, J = 8.5, 1.5 Hz, 1H), 8.60 – 8.55 (m, 1H), 8.34 (s, 1H), 7.88 (dddd, J = 21.2, 8.4, 7.0, 1.5 Hz, 2H), 7.74 (d, J = 1.8 Hz, 2H), 7.50 – 13 7.42 (m, 1H), 5.75 (s, 2H), 2.58 (s, 3H). C NMR (101 MHz, CDCl3) δ 159.58, 142.74, 138.46, 137.79, 137.17, 137.03, 136.92, 136.54, 135.05, 134.64, 132.17, 129.55, 123.17, 121.82, 119.25, + 60.49, 14.52. EI-MS m/z 445.04 (M+H) ; Anal. calcd for C19H13Cl2N5O4: (%) C, 51.15; H, 2.94; N, 15.80; Found: C, 51.16; H, 2.95; N, 15.81.

6-chloro-2-methyl-3-(((1-phenyl-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)quinoxaline 1,4- dioxide (36a) -1 Off white solid (61%); m.p. 193-194 °C; (KBr) ʋmax / cm 3066, 2893, 1701, 1383, 1043, 966, 715. 1H NMR (400 MHz, Chloroform-d) δ 8.66 – 8.44 (m, 2H), 8.30 (d, J = 3.4 Hz, 1H), 7.79 (dd, J = 17.5, 8.4 Hz, 3H), 7.52 (dt, J = 32.0, 7.4 Hz, 3H), 5.75 (s, 2H), 2.55 (s, 3H). 13C NMR

(101 MHz, CDCl3) δ 159.48, 142.00, 139.31, 138.68, 137.15, 136.74, 136.58, 133.56, 132.48, 129.86, 129.12, 122.33, 121.91, 120.64, 119.74, 60.41, 14.39. EI-MS m/z 411.08 (M+H)+; Anal. calcd for C19H14ClN5O4: (%) C, 55.42; H, 3.44; N, 17.01; Found: C, 55.43; H, 3.45; N, 17.02.

1H NMR spectrum (400 MHz, Chloroform-d)) of compound 36a

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13C NMR spectrum (101 MHz, Chloroform-d)) of compound 36a

6-chloro-3-(((1-(4-ethylphenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)-2-methylquinoxaline 1,4-dioxide (36b) -1 Yellow solid (49%); m.p. 207-208 °C; (KBr) ʋmax / cm 3096, 2893, 1694, 1383, 1049, 986, 727. 1H NMR (500 MHz, Chloroform-d) δ 8.62 – 8.48 (m, 2H), 8.23 (d, J = 4.1 Hz, 1H), 7.81 (dd, J = 9.2, 2.2 Hz, 1H), 7.69 – 7.61 (m, 2H), 7.41 – 7.33 (m, 2H), 5.76 (s, 2H), 2.74 (q, J = 7.6 Hz, 2H), 13 2.56 (s, 3H), 1.29 (t, J = 7.6 Hz, 3H). C NMR (101 MHz, CDCl3) δ 158.23, 143.10, 139.33, 138.45, 137.56, 136.64, 136.18, 134.88, 132.18, 129.76, 128.22, 122.33, 121.22, 120.34, 119.55, + 60.34, 28.25, 15.87, 14.59. EI-MS m/z 439.11 (M+H) ; Anal. calcd for C21H18ClN5O4: (%) C, 57.34; H, 4.13; N, 15.92; Found: C, 57.35; H, 4.14; N, 15.93.

6-chloro-3-(((1-(4-fluorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)-2- methylquinoxaline 1,4-dioxide (36c)

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-1 Pale yellow solid (65%); m.p. 208-209 °C; (KBr) ʋmax / cm 3096, 2951, 1667, 1401, 1039, 976, 707. 1H NMR (500 MHz, Chloroform-d) δ 8.64 – 8.46 (m, 2H), 8.25 (d, J = 4.3 Hz, 1H), 7.81 (dd, J = 9.1, 2.3 Hz, 1H), 7.78 – 7.71 (m, 2H), 7.25 (d, J = 1.3 Hz, 2H), 5.75 (s, 2H), 2.55 (s, 13 3H). C NMR (101 MHz, CDCl3) δ 159.79, 143.25, 138.84, 138.35, 136.78, 135.07, 134.87, 133.76, 132.03, 124.87, 123.65, 123.19, 121.15, 117.43, 117.28, 60.21, 14.56. EI-MS m/z 430.06 + (M+H) ; Anal. calcd for C19H13ClFN5O4: (%) C, 53.11; H, 3.05; N, 16.30; Found: C, 53.12; H, 3.06; N, 16.31.

6-chloro-3-(((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)-2- methylquinoxaline 1,4-dioxide (36d) -1 Off white solid (55%); m.p. 222-223 °C; (KBr) ʋmax / cm 3084, 2981, 1677, 1411, 1022, 944, 737. 1H NMR (500 MHz, Chloroform-d) δ 8.69 – 8.48 (m, 2H), 8.33 (d, J = 4.1 Hz, 1H), 7.78 (dd, J = 9.2, 2.2 Hz, 1H), 7.66 – 7.60 (m, 2H), 7.49 – 7.36 (m, 2H), 5.74 (s, 2H), 2.57 (s, 3H). 13C

NMR (101 MHz, CDCl3) δ 158.73, 144.12, 139.73, 139.45, 137.56, 136.94, 135.18, 134.78, 134.76, 132.28, 129.66, 128.72, 122.33, 121.22, 119.95, 60.34, 14.59. EI-MS m/z 436.04 + (M+H) ; Anal. calcd for C19H13Cl2N5O4: (%) C, 51.15; H, 2.94; N, 15.69; Found: C, 51.16; H, 2.96; N, 15.70.

3-(((1-(4-bromophenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)-6-chloro-2- methylquinoxaline 1,4-dioxide (36e) -1 Pale yellow solid (59%); m.p. 222-223 °C; (KBr) ʋmax / cm 3079, 2971, 1687, 1421, 1036, 976, 798, 707. 1H NMR (500 MHz, Chloroform-d) δ 8.70 – 8.56 (m, 2H), 8.39 (d, J = 4.4 Hz, 1H), 7.79 (dd, J = 9.1, 2.2 Hz, 1H), 7.79 – 7.73 (m, 2H), 7.28 (d, J = 1.3 Hz, 2H), 5.78 (s, 2H), 2.59 13 (s, 3H). C NMR (101 MHz, DMSO-d6) δ 159.79, 143.25, 138.84, 138.35, 136.78, 135.07, 134.87, 133.76, 132.03, 124.87, 123.65, 123.19, 121.15, 117.43, 117.28, 60.21, 14.56. EI-MS +2 + m/z 490.98 (M+H) ; 488.96 (M+H) ; Anal. calcd for C19H13BrClN5O4: (%) C, 46.51; H, 2.68; N, 14.28; Found: C, 46.53; H, 2.69; N, 14.29.

6-chloro-2-methyl-3-(((1-(4-nitrophenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)quinoxaline 1,4-dioxide (36f)

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-1 Yellow solid (65%); m.p. 220-221 °C; (KBr) ʋmax / cm 3089, 2982, 1690, 1411, 1026, 954, 757. 1H NMR (500 MHz, Chloroform-d) δ 8.66 – 8.46 (m, 2H), 8.30 (d, J = 4.1 Hz, 1H), 7.79 (dd, J = 9.2, 2.2 Hz, 1H), 7.68 – 7.60 (m, 2H), 7.50 – 7.39 (m, 2H), 5.78 (s, 2H), 2.57 (s, 3H). 13C NMR

(101 MHz, CDCl3) δ 158.83, 144.92, 139.73, 139.45, 138.96, 136.94, 135.08, 134.78, 134.16, 133.78, 129.96, 128.82, 122.33, 121.22, 119.95, 60.74, 14.61. EI-MS m/z 457.08 (M+H)+; Anal. calcd for C19H13ClN6O6: (%) C, 49.96; H, 2.88; N, 18.41; Found: C, 49.97; H, 2.89; N, 18.42.

6-chloro-3-(((1-(2-fluorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)-2- methylquinoxaline 1,4-dioxide (36g) -1 Off white solid (46%); m.p. 195-196 °C; (KBr) ʋmax / cm 3078, 2972, 1693, 1421, 1145, 1026, 974, 787. 1H NMR (400 MHz, Chloroform-d) δ 8.64 – 8.47 (m, 2H), 8.36 (t, J = 2.3 Hz, 1H), 7.97 (td, J = 7.8, 1.7 Hz, 1H), 7.80 (ddd, J = 14.7, 9.2, 2.2 Hz, 1H), 7.49 (tdd, J = 8.1, 4.9, 1.7 13 Hz, 1H), 7.41 – 7.28 (m, 2H), 5.77 (s, 2H), 2.54 (s, 3H). C NMR (101 MHz, DMSO-d6) δ 159.64, 155.58, 153.09, 141.73, 139.42, 137.42, 137.02, 135.48, 133.54, 132.01, 127.40, 126.50, 126.07, 124.96, 122.58, 119.32, 117.54, 60.12, 14.48. EI-MS m/z 430.06 (M+H)+; Anal. calcd for C19H13ClFN5O4: (%) C, 53.11; H, 3.05; N, 16.30; Found: C, 53.12; H, 3.06; N, 16.31.

6-chloro-3-(((1-(2-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)-2- methylquinoxaline 1,4-dioxide (36h) -1 Pale yellow solid (67%); m.p. 176-177 °C; (KBr) ʋmax / cm 3079, 2979, 1697, 1434, 1026, 973, 776. 1H NMR (400 MHz, Chloroform-d) δ 8.64 – 8.47 (m, 2H), 8.36 (t, J = 2.3 Hz, 1H), 7.97 (td, J = 7.8, 1.7 Hz, 1H), 7.80 (ddd, J = 14.7, 9.2, 2.2 Hz, 1H), 7.49 (tdd, J = 8.1, 4.9, 1.7 Hz, 1H), 13 7.41 – 7.28 (m, 2H), 5.77 (s, 2H), 2.54 (s, 3H). C NMR (101 MHz, DMSO-d6) δ 159.70, 153.58, 150.09, 142.73, 139.02, 137.42, 137.12, 135.48, 133.54, 132.01, 127.40, 126.92, 126.77, 125.16, 122.78, 119.52, 117.64, 60.52, 14.68. EI-MS m/z 436.04 (M+H)+; Anal. calcd for

C19H13Cl2N5O4: (%) C, 51.15; H, 2.94; N, 15.69; Found: C, 51.16; H, 2.96; N, 15.70.

6-chloro-2-methyl-3-(((1-(2-nitrophenyl)-1H-1,2,3-triazol-4- yl)methoxy)carbonyl)quinoxaline 1,4-dioxide (36i) -1 Yellow solid (64%); m.p. 165-166 °C; (KBr) ʋmax / cm 3067, 2989, 1695, 1414, 1026, 975, 766. 1H NMR (400 MHz, Chloroform-d) δ 8.66 – 8.49 (m, 2H), 8.33 (t, J = 2.4 Hz, 1H), 7.95 (td, J =

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7.9, 1.8 Hz, 1H), 7.81 (ddd, J = 14.7, 9.2, 2.2 Hz, 1H), 7.50 (tdd, J = 8.1, 4.9, 1.6 Hz, 1H), 7.43 – 13 7.26 (m, 2H), 5.78 (s, 2H), 2.58 (s, 3H). C NMR (101 MHz, CDCl3) δ 159.71, 150.58, 148.19, 142.73, 139.11, 137.32, 137.02, 135.54, 134.44, 132.11, 127.66, 126.92, 126.87, 125.06, 122.78, + 119.59, 117.64, 60.58, 14.58. EI-MS m/z 457.08 (M+H) ; Anal. calcd for C19H13ClN6O6: (%) C, 49.96; H, 2.88; N, 18.41; Found: C, 49.97; H, 2.89; N, 18.42.

6-chloro-2-methyl-3-(((1-(3-nitrophenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)quinoxaline 1,4-dioxide (36j) -1 Yellow solid (62%); m.p. 205-206 °C; (KBr) ʋmax / cm 3097, 2988, 1685, 1422, 1031, 961, 771. 1H NMR (400 MHz, Chloroform-d) δ 8.64 – 8.42 (m, 2H), 8.30 (t, J = 2.4 Hz, 1H), 7.92 (td, J = 7.9, 1.8 Hz, 1H), 7.79 (ddd, J = 14.6, 9.3, 2.2 Hz, 1H), 7.44 – 7.25 (m, 3H), 5.77 (s, 2H), 2.56 (s, 13 3H). C NMR (101 MHz, CDCl3) δ 158.71, 151.59, 148.19, 142.73, 139.21, 137.42, 137.52, 136.57, 134.44, 132.11, 127.68, 126.22, 126.07, 125.16, 123.78, 119.79, 117.64, 60.58, 14.68. + EI-MS m/z 457.08 (M+H) ; Anal. calcd for C19H13ClN6O6: (%) C, 49.96; H, 2.88; N, 18.41; Found: C, 49.97; H, 2.89; N, 18.42.

6-chloro-2-methyl-3-(((1-(3-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4- yl)methoxy)carbonyl)quinoxaline 1,4-dioxide (36k) -1 Pale yellow solid (66%); m.p. 160-161 °C; (KBr) ʋmax / cm 3095, 2887, 1688, 1420, 1132, 1039, 971, 772. 1H NMR (400 MHz, Chloroform-d) δ 8.59 – 8.52 (m, 2H), 8.42 (s, 1H), 8.09 (t, J = 1.9 Hz, 1H), 8.01 (dt, J = 7.4, 2.0 Hz, 1H), 5.77 (s, 2H), 2.56 (s, 3H). 13C NMR (101 MHz,

CDCl3) δ 159.49, 142.58, 139.29, 138.78, 137.13, 136.61, 135.59, 133.63, 132.71, 132.38, 130.69, 127.32, 125.76, 123.64, 122.27, 121.92, 119.72, 117.60, 60.27, 14.41. EI-MS m/z 480.06 + (M+H) ; Anal. calcd for C20H13ClF3N5O4: (%) C, 50.08; H, 2.74; N, 14.61; Found: C, 50.09; H, 2.75; N, 14.62.

6-chloro-3-(((1-(3,5-dichlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)-2- methylquinoxaline 1,4-dioxide (36l) -1 Off white solid (55%); m.p. 197-198 °C; (KBr) ʋmax / cm 3079, 2891, 1691, 1420, 1364, 1029, 969, 663. 1H NMR (500 MHz, Chloroform-d) δ 8.66 (dd, J = 8.5, 1.5 Hz, 1H), 8.60 – 8.55 (m, 1H), 8.42 (s, 1H), 7.88 (m, 2H), 7.74 (d, J = 1.8 Hz, 1H), 7.50 (s, 1H), 5.78 (s, 2H), 2.56 (s, 3H).

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13 C NMR (101 MHz, CDCl3) δ 159.48, 142.64, 139.26, 138.79, 137.97, 137.13, 136.62, 136.41, 135.55, 133.64, 132.57, 129.05, 122.17, 121.92, 119.73, 119.05, 60.19, 14.42. EI-MS m/z 480.01 + (M+H) ; Anal. calcd for C19H12Cl3N5O4: (%) C, 47.48; H, 2.52; N, 14.58; Found: C, 47.49; H, 2.54; N, 14.59.

6,7-dichloro-2-methyl-3-(((1-phenyl-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)quinoxaline 1,4- dioxide (37a) -1 Off white solid (66%); m.p. 219-220 °C; (KBr) ʋmax / cm 3076, 2899, 1698, 1422, 1374, 1049, 989, 653. 1H NMR (400 MHz, Chloroform-d) δ 8.76 (s, 1H), 8.64 (s, 1H), 8.33 (s, 1H), 7.79 – 7.71 (m, 3H), 7.28 (d, J = 4.8 Hz, 2H), 5.78 (s, 2H), 2.59 (s, 3H). 13C NMR (101 MHz, DMSO- d6) δ 169.89, 166.77, 148.73, 134.66, 133.71, 131.76, 129.87, 129.65, 128.53, 127.02, 126.65, 124.34, 123.15, 123.06, 119.38, 60.29, 14.59. EI-MS m/z 446.04 (M+H)+; Anal. calcd for

C19H13Cl2N5O4: (%) C, 51.14; H, 2.95; N, 15.69; Found: C, 51.15; H, 2.96 N, 15.71.

6,7-dichloro-2-(((1-(4-fluorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)-3- methylquinoxaline 1,4-dioxide (37b) -1 Off white solid (64%); m.p. 223-224 °C; (KBr) ʋmax / cm 3091, 2889, 1697, 1432, 1374, 1165, 1040, 980, 673. 1H NMR (400 MHz, Chloroform-d) δ 8.74 (s, 1H), 8.68 (s, 1H), 8.26 (s, 1H), 7.81 – 7.70 (m, 2H), 7.29 (d, J = 4.9 Hz, 2H), 5.76 (s, 2H), 2.57 (s, 3H). 13C NMR (101 MHz,

DMSO-d6) δ 171.99, 167.78, 146.53, 134.94, 132.11, 131.66, 129.30, 129.12, 127.83, 127.12, 126.61, 124.34, 123.15, 123.06, 121.88, 117.38, 117.15, 60.22, 14.59. EI-MS m/z 464.04 + (M+H) ; Anal. calcd for C19H12Cl2FN5O4: (%) C, 49.16; H, 2.62; N, 15.09; Found: C, 49.17; H, 2.63 N, 15.11.

6,7-dichloro-2-(((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)-3- methylquinoxaline 1,4-dioxide (37c) -1 Pale yellow solid (63%); m.p. 213-214 °C; (KBr) ʋmax / cm 3089, 2980, 1687, 1401, 1042, 964, 750. 1H NMR (500 MHz, Chloroform-d) δ 8.72 (s, 1H), 8.66 (s, 1H), 8.28 (s, 1H), 8.30 (d, J = 4.1 Hz, 1H), 7.66 – 7.60 (m, 2H), 7.39 – 7.32 (m, 2H), 5.76 (s, 2H), 2.58 (s, 3H). 13C NMR (101

MHz, CDCl3) δ 160.73, 143.12, 139.93, 139.75, 137.66, 136.14, 135.88, 134.78, 133.76, 132.83,

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129.09, 128.92, 122.83, 121.42, 119.87, 60.44, 14.61. EI-MS m/z 480.01 (M+H)+; Anal. calcd for C19H12Cl3N5O4: (%) C, 47.47; H, 2.52; N, 14.58; Found: C, 47.49; H, 2.53 N, 14.59.

2-(((1-(4-bromophenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)-6,7-dichloro-3- methylquinoxaline 1,4-dioxide (37d) -1 Off white solid (54%); m.p. 214-215 °C; (KBr) ʋmax / cm 3099, 2983, 1688, 1451, 1046, 984, 779, 675. 1H NMR (500 MHz, Chloroform-d) δ 8.72 (s, 1H), 8.68 (s, 1H), 8.29 (s, 1H), 8.31 (d, J = 4.1 Hz, 1H), 7.68 – 7.62 (m, 2H), 7.41 – 7.36 (m, 2H), 5.78 (s, 2H), 2.56 (s, 3H). 13C NMR

(101 MHz, CDCl3) δ 160.79, 143.82, 139.90, 139.55, 138.86, 137.74, 135.18, 134.78, 133.76, 132.83, 129.19, 128.02, 122.83, 121.22, 119.87, 60.54, 14.68. EI-MS m/z 524.96 (M+H)+2; + 522.94 (M+H) ; Anal. calcd for C19H12BrCl2N5O4: (%) C, 43.46; H, 2.32; N, 13.34; Found: C, 43.47; H, 2.33 N, 13.35.

6,7-dichloro-2-(((1-(2-fluorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)-3- methylquinoxaline 1,4-dioxide (37e) -1 Off white solid (61%); m.p. 219-220 °C; (KBr) ʋmax / cm 3079, 2893, 1697, 1443, 1165, 1046, 980, 769, 675. 1H NMR (400 MHz, Chloroform-d) δ 8.74 (s, 1H), 8.68 (s, 1H), 8.36 (d, J = 2.7 Hz, 1H), 7.99 (td, J = 7.7, 1.7 Hz, 1H), 7.50 (tdd, J = 7.8, 4.9, 1.8 Hz, 1H), 7.43 – 7.28 (m, 2H), 13 5.78 (s, 2H), 2.55 (s, 3H). C NMR (101 MHz, DMSO-d6) δ 159.50, 141.69, 139.28, 137.42, 136.73, 136.20, 135.64, 132.57, 132.07, 131.99, 127.37, 126.51, 126.09, 121.90, 121.83, 117.74, + 117.55, 60.19, 14.55. EI-MS m/z 464.04 (M+H) ; Anal. calcd for C19H12Cl2FN5O4: (%) C, 49.16; H, 2.62; N, 15.09; Found: C, 49.17; H, 2.63 N, 15.11.

6,7-dichloro-2-(((1-(2-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)-3- methylquinoxaline 1,4-dioxide (37f) -1 Yellow solid (62%); m.p. 182-183 °C; (KBr) ʋmax / cm 3091, 2890, 1682, 1433, 1046, 984, 763, 675. 1H NMR (400 MHz, Chloroform-d) δ 8.76 (s, 1H), 8.66 (s, 1H), 8.38 (d, J = 2.8 Hz, 1H), 7.98 (m, 1H), 7.55 (tdd, J = 7.6, 4.9, 1.8 Hz, 1H), 7.43 – 7.29 (m, 2H), 5.78 (s, 2H), 2.56 (s, 3H). 13 C NMR (101 MHz, DMSO-d6) δ 160.50, 144.69, 139.78, 138.02, 136.83, 136.44, 135.04, 132.77, 132.17, 131.09, 128.37, 126.50, 125.19, 121.90, 121.03, 117.84, 117.55, 60.59, 14.69.

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+ EI-MS m/z 480.01 (M+H) ; Anal. calcd for C19H12Cl3N5O4: (%) C, 47.47; H, 2.52; N, 14.58; Found: C, 47.49; H, 2.53 N, 14.59.

6,7-dichloro-2-(((1-(3,5-dichlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)-3- methylquinoxaline 1,4-dioxide (37g) -1 Pale yellow solid (55%); m.p. 214-215 °C; (KBr) ʋmax / cm 3090, 2899, 1695, 1421, 1374, 1039, 968, 683. 1H NMR (500 MHz, Chloroform-d) δ 8.70 (s, 1H), 8.64 (s, 1H), 8.40 (s, 1H), 13 7.86 (m, 2H), 7.70 (d, J = 1.8 Hz, 1H), 5.75 (s, 2H), 2.58 (s, 3H). C NMR (101 MHz, CDCl3) δ 159.88, 142.64, 139.76, 138.99, 137.97, 136.93, 136.52, 136.01, 135.55, 134.04, 132.07, 129.05, 123.67, 121.92, 118.93, 119.15, 60.39, 14.59. EI-MS m/z 512.98 (M+H)+; Anal. calcd for

C19H11Cl4N5O4: (%) C, 44.31; H, 2.15; N, 13.61; Found: C, 44.32; H, 2.16 N, 13.62.

6.4.3. Biological activity 6.4.3.1. In vitro MTB screening The antimycobacterial activities of the compounds 35a-l, 36a-l & 37a-g were evaluated against MTB H37Rv strain and two “wild” strains extracted from tuberculosis patients: one strain is Spec. 210 resistant to PAS, INH, ETB and RMP and the other strain is Spec. 192 fully sensitive to the administrated anti-TB agents. In vitro anti-TB activity is performed by a classical test-tube method of successive dilution in Youmans’ modification of the Proskauer and Beck liquid medium containing 10% of bovine serum [18]. Bacterial respites were prepared from 14 days old cultures of gradually growing strains. Solutions of compounds in DMSO were tested. Stock solutions contained 10 mg of compounds in 1 mL. Dilutions (in geometric progression) were prepared in Youmans’ medium [18]. The medium is without compounds and containing INH as reference drug was used for comparison. Incubation was performed at 37 °C. The MIC values were determined as MIC inhibiting the growth of tested TB strains in relation to the probe with no tested compound. The influence of the compound on the growth of bacteria at concentrations of 3.12, 6.25, 12.5, 25, 50 and 100 μg/mL was evaluated.

6.4.3.2. In vitro cytotoxicity screening The human embryonic kidney cells (HEK-293) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Himedia Laboratories Pvt. Ltd., Mumbai, India), supplemented with 10%

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heat inactivated fetal bovine serum (Himedia Laboratories Pvt. Ltd., Mumbai, India) and 1 % of Antibiotic solution (10000 U Penicillin and 10 mg Streptomycin per ml, Himedia Laboratories

Pvt. Ltd., Mumbai, India). Cells were cultured at 37 °C in humidified atmosphere with 5% CO2. Stock solutions of compounds was prepared in DMSO at a concentration of 50 μM and stored.

Cytotoxicity screening of the synthesized compounds was determined using MTT assay [19]. 7.5×103 cells were seeded in 96 well plates and incubated overnight. Cells were treated with synthesized compounds at three concentrations (50µM, 25 µM & 10 µM) in duplicates and incubated for 24 hrs. 50 µL of 5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Himedia Laboratories Pvt. Ltd., Mumbai, India) was added and incubated for 4 hours. 150 µL of DMSO was added to dissolve formazan crystals and evaluated spectrophotometrically at 570 nm and 650 nm using Spectramax M4 (Molecular Devices, USA).

6.5. References [1] (a) G. W. H. Cheeseman, R. F. Cookson, in: A. Weissberger, E. C. Taylor (Eds.), The Chemisty of Heterocyclic Compounds, vol. 35, John Wiley and Sons, New York, 1979, pp. 1–27; (b) A.E.A. Porter, in: A.R. Katrizky, C.W. Rees (Eds.), Comprehensive Heterocyclic Chemistry, vol. 3, Pergamon Press, New York, 1984, p. 195. [2] A. Carta, G. Paglietti a, M. E. R. Nikookar, P. Sanna, L. Sechi, S. Zanetti, Eur. J. Med. Chem.,2002, 37, 355. [3] (a) E. Vicente, R. Villar, B. Solano, A. Burguete, S. Ancizu, S. Pérez-Silanes, I. Aldana, A. Monge, A. An. R. Acad. Nac. Farm., 2007, 73, 927; (b) G. Aguirre, H. Cerecetto, R. Di Maio, M. Gonzalez, M.E.M. Alfaro, A. Jaso, B. Zarranz, M. A. Ortega, I. Aldana, A. Monge- Vega, Bioorg. Med. Chem. Lett., 2004, 14, 3835; (c) C. Urquiola, M. Vieites, G. Aguirre, A. Marin, B. Solano, G. Arrambide, P. Noblia, M. L. Lavaggi, M.H. Torre, M. Gonzalez, A. Monge, D. Gambino, H. Cerecetto, Bioorg. Med. Chem., 2006, 14, 5503; (d) A. Carta, M. Loriga, G. Paglietti, A. Mattana, P. L. Fiori, P. Mollicotti, L. Sechi, S. Zanetti, Eur. J. Med. Chem., 2004, 39, 195; (e) B. Ganley, G. Chowdhury, J. Bhansali, J.S. Daniels, K.S. Gates, Bioorg. Med. Chem., 2001, 9, 2395. [4] Y. Sainza, M. E. Montoya, F. J. Martínez-Crespo, M. A. Ortega, A. L. de Ceráin, A. Monge, Arzneim.-Forsch./Drug Res., 1999, 49, 56.

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[5] M. A. Ortega, Y. Sainz, M. E. Montoya, et al., Die Pharm., 1999, 54, 24. [6] M. E. Montoya, Y. Sainz, M. A. Ortega, et al., Organizacio´n Farmace´utica Ibero- Latinoamericana (OFIL), 1998, 8, 36. [7] (a) A. Jaso, B. Zarranz, I. Aldana, A. Monge, Eur. J. Med. Chem., 2003, 38, 791; (b) A. Jaso, B. Zarranz, I. Aldana, A. Monge, J. Med. Chem., 2005, 48, 2019. [8] E. Torres, E. Moreno, S. Ancizu, C. Barea, S. Galiano, I. Aldana, A. Monge, S. Pérez- Silanes, Bioorg. Med. Chem. Lett., 2011, 21, 3699. [9] E. Moreno, S. Pérez-Silanes, S. Gouravaram, A. Macharam, S. Ancizu, E. Torres, I. Aldana, A. Monge, P. W. Crawford, Electrochimica Acta, 2011, 56, 3270. [10] Y. Pan, P. Li, S. Xie, Y. Tao, D. Chen, M. Dai, H. Hao, L. Huang, Y. Wang, L. Wang, Z. Liu, Z.Yuan, Bioorg. Med. Chem. Lett., 2016, 26, 4146.

[11] E. Vicente, R. Villar, S.Pérez-Silanes, I. Aldana, R. C. Goldman, A. Monge, Infectious Disorders – Drug Targets, 2011, 11, 196. [12] (a) W. Zhang, Z. Li, M. Zhou, F. Wu, X. Hou, H. Luo, H. Liu, X.Han, G. Yan, Z. Ding, R. Li, Bioorg. Med. Chem. Lett., 2014, 24, 799; (b) M. J. Giffin, H. Heaslet, A. Brik, Y. C. Lin, G. Cauvi, C. H. Wong, D. E. McRee, J. H. Elder, C. D. Stout, B. E. Torbett, J. Med. Chem., 2008, 51, 6263; (c) Z. C. Dai, Y. F. Chen, M. Zhang, S. K. Li, T. T. Yang, L. Shen, J. X. Wang, S. S. Qian, H. L. Zhu, Y. H. Ye, Org. Biomol. Chem., 2015, 13, 477; (d) D. R. Buckle, C. J. M. Rockell, H. Smith, B. A. Spicer, J. Med. Chem., 1986, 29, 2269. [13] H. C. Kolb, K. B. Sharpless, Drug Discov. Today., 2003, 8, 1128. [14] R. S. Keri, S. A. Patil, S. Budagumpi, B. M. Nagaraj, Chem Biol Drug Des., 2015, 86, 410. [15] (a) J. Xie, C. T. Seto, Bioorg. Med. Chem., 2007, 15, 458; (b) K. D. Thomas, A. V. Adhikari, I. H. Chowdhury, E. Sumesh, N. K. Pal, Eur. J. Med. Chem., 2011, 46, 2503; (c) L. Pulipati, P. Yogeeswari, D. Sriram, S. Kantevari, Bioorg. Med. Chem. Lett., 2016, 26, 2649; (d) H. N. Nagesh, K. Mahalakshmi Naidu, D. Harika Rao, J. P. Sridevi, D. Sriram, P. Yogeeswari, K. V. G. Chandra Sekhar, Bioorg. Med. Chem. Lett., 2013, 23, 6805; (e) N. Boechat, V. F. Ferreira, S. B. Ferreira, M. L. G. Ferreira, F. C. da Silva, M. M. Bastos, M. S. Costa, M. S. Lourenço, A. C. Pinto, A. U. Krettli, A. C. Aguiar, B. M. Teixeira, N. V. da Silva, P. R. C. Martins, F. F. M. Bezerra, A. S. Camilo, G. P. da Silva, C. C. P. Costa, J. Med. Chem., 2011, 54, 5988.

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[16] H. N. Nagesh, K. Mahalakshmi Naidu, D. Harika Rao, J. P. Sridevi, D. Sriram, P. Yogeeswari, K. V. G. Chandra Sekhar, Bioorg. Med. Chem. Lett., 2013, 23, 6805. [17] K. Mahalakshmi Naidu, S. Srinivasarao, N. Agnieszka, A. Ewa, M. M. Krishna Kumar, K. V. G. Chandra Sekhar, Bioorg. Med. Chem. Lett., 2016, 26, 2245. [18] G.P. Youmans, A.S. Youmans, J. Bactriol., 1949, 58, 247. [19] J. Van Meerloo, G. J. L. Kaspers, J. Cloos, Methods in Molecular Biology, 731, 237.

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Summary and Conclusion

Chapter VII

Summary and Conclusion

Summary and Conclusion

Summary and Conclusion Chapter 7

TB has become one of the most dangerous infectious diseases of the modern times with the epidemic of acquired immune deficiency syndrome (AIDS) in the 1980s. The emergence of drug resistant strains of MTB along with some other factors has resulted in multidrug-resistant (MDR), extensively drug-resistant (XDR), or more recently, totally drug-resistant (TDR-TB). This has rendered the presently available anti-tubercular drug regimen inadequate to address the many inherent and emerging challenges of treatment. These factors initiate the need for the development of newer, safer and more effective drugs which can reduce the TB treatment duration drastically. Hence we designed the compounds emphasizing molecular hybridisation approach to merit cost effective and reduced treatment time. Active core of existing antitubercular molecules were identified and made an attempt to tailor them in a single entity anticipating improved features. All compounds were designed with reported active core and varied with triazoles. All synthesized novel compounds were characterized by spectral data (IR, NMR and MS), elemental analysis and few compounds were confirmed by single crystal XRD. All compounds were evaluated for their antimycobacterial activity and most active compounds were evaluated for cytotoxicity studies in normal cell line.

In chapter 3, novel 1-((1-(substituted)-1H-1,2,3-triazol-4-yl)methyl)-N,3-diphenyl-6,7- dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-carboxamide derivatives designed and synthesized by molecular hybridization approach using reported MTB PS inhibitor and substituted 1H-1,2,3- triazole antitubercular compounds. Twenty six compounds were synthesized and evaluated for their antimycobacterial activity against MTB H37Rv, MTB Spec. 192 and MTB Spec. 210 strains and pantothenate synthetase enzyme studies was also carried out. Amongst, the synthesized compounds, 7c exhibited 99% inhibition of MTB H37Rv strain with MIC 49.64 µM. Compound 7b was significantly active against MTB with MIC 25.53 µM. Compound 7d exhibited good activity with MIC 24.72 µM. Seven compounds (7b, 7d, 7h, 7p, 7r, 7s & 7v) inhibited MTB PS with IC50 ˂2.00 µM. Compounds 7d and 7s emerged as the most active compounds with IC50 1.01±0.32 and 0.91±0.32 µM respectively. The active compounds 7b, 7c and 7d were evaluated for cytotoxicity (RAW264.7 cell line). These compounds showed low cytotoxicity.

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The anti-tubercular SAR profile suggests that tailoring methoxy and triazole group by means of appropriate substituent or functional group might provide an insight to obtain the lead compound.

In chapter 4, novel 9H-fluorenone analogues were synthesized with three modifications of these three groups -NH-, -S- and -SO2-. In scheme 4.1, N-((1-substituted phenyl-1H-1,2,3- triazol-4-yl)methyl)-9H-fluoren-9-amine analogues and in scheme 4.2 N-4-(((9H-fluoren-9- yl)thio)methyl)-1-substituted phenyl-1H-1,2,3-triazole & 4-(((9H-fluoren-9-yl) sulfonyl) methyl)-1-substituted phenyl-1H-1,2,3-triazole) analogues were synthesized, evaluated for their antimycobacterial activity against MTB. In this fifty compounds, N-4-(((9H-fluoren-9- yl)thio)methyl)-1-substituted phenyl-1H-1,2,3-triazole analogues showed good activity. 17p showed excellent MTB activity with MIC 52.35 μM. Out of fifty compounds synthesized, InhA activity was studied for fifteen compounds. Amongst these compounds, 17f & 17p showed >73% of inhibition at 50 μM. The most active compounds did not exhibit cytotoxicity against HEK 293 cell line at 50 μM.

From these schemes 4.1 & 4.2, preliminary anti-tubercular screening results drive us to engineer the chemical structure of 9H-fluorenone derivative to generate essential pharmacophoric features that could lead to the synthesis of a promising candidate to develop anti-tubercular agent. We discovered that incorporation of sulfonyl group on the 9H-fluorenone in the moiety plays a pivotal role in the activity profile.

In chapter 5, thirty six sulfonamide tetrazole and 1,2,4, triazole containing derivatives were synthesized and evaluated for their antimycobacterial activity. In vitro anti tubercular screening results indicate that ten compounds viz., 25a, 25d, 25e, 25h, 26b, 27a, 27d, 27i, 28b and 28f showed moderate activity (MIC = 3.12 µg/mL). Eight compounds, 25c, 25f, 26g, 27e, 27g, 27h, 28d and 28h displayed good anti-TB activity (MIC = 1.56 µg/mL). Compound 26c exhibited excellent anti-TB activity (MIC = 0.78µg/mL). Most of the compounds did not show toxicity when screened in CHO-K1 cell line (SI value >13).

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Summary and Conclusion

In chapter 6, thirty one, quinoxaline 1,4-dioxide tethered 1,2,3 triazole analogues were synthesized. In this chapter quinoxaline 1,4-dioxide was varied with chloro at 6,7 positions. .In vitro anti tubercular screening results indicate that 6-chloro-3-(((1-(substituted phenyl)-1H-1,2,3- triazol-4-yl)methoxy)carbonyl)-2-methylquinoxaline 1,4-dioxide derivatives showed good activity. Two compounds 36a and 37d displayed excellent anti-TB activity with respect MIC 30.35, 47.60 µM. Cytotoxicity assay of 36a, 36e, 36i, 36l, 37b, 37c & 37d was determined against HEK 293 cell line. These compounds did not exhibit cytotoxicity against HEK 293 cell line.

Figure 7.1: Most active compounds amongst the synthesized compounds

In conclusion, the most active compounds from the synthesized derivatives are depicted in figure 7.1. The class of compounds described here besets a collection of promising lead compounds for further optimization and development to yield best novel drugs aimed to combat ever-present and ever-increasing mycobacterial infections. The study also provides the basis for further chemical optimization of these potent inhibitors as potential anti-TB agents.

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Future perspectives

Future perspectives

 The present thesis described development of series of molecules as potential anti- tubercular agents, pantothenate synthetase enzyme inhibitors and InhA inhibitors. The molecules reported herein displayed considerable in-vitro enzyme inhibition and potency against Mycobacterium tuberculosis H37Rv strain. Although these results were encouraging further lead optimization can be carried out.

 The progression of any of the candidate molecules presented in this thesis along a drug development track would require a substantial investment in medicinal chemistry, preclinical and clinical studies.

 Extensive side effect profile of all the synthesized compounds may be studied. Sub- acute and acute toxicological screening of novel chemical entities has to be carried out.

 Extensive pharmacodynamic and pharmacokinetic studies of the safer compounds have to be undertaken in higher animal models.

 Based on the pharmacophore model proposed, various substituents which lead to activity proposed could be incorporated into the compounds synthesized and study further in various animal models.

Further, the viability, reduce the therapy period, cost effectiveness and reproducibility of synthesizing these compounds in big scale has to be attempted.

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Appendix

List of Publications

From thesis work:

1) A. Suresh, K. Mahalakshmi Naidu, S. Srinivasarao, N. Agnieszka, A. Ewa, S. Murugesan, S. Chander, R. Krishnan, K. V. G. Chandra Sekhar, Identification and development of pyrazolo[4,3-c]-pyridine carboxamides as Mycobacterium tuberculosis pantothenate synthetase inhibitors. New J. Chem., 2017, 41, 347.

2) A. Suresh, S. Srinivas, N. Agnieszka, A. Ewa, A. Mallika, C. Lherbet, K. V. G. Chandra Sekhar, Design, synthesis of 9H-fluorenone based 1,2,3-triazole analogues as Mycobacterium tuberculosis InhA inhibitors. Chemical Biology & Drug Design, DOI: 10.1111/cbdd.13127 (Accepted manuscript).

3) A. Suresh, N. Suresh, S. Misra, M. M. Krishna Kumar, K. V. G. Chandra Sekhar, Design, Synthesis and Biological Evaluation of New Substituted Sulfonamide Tetrazole Derivatives as Antitubercular Agents. Chemistry Select, 2016, 1, 1705.

4) A. Suresh, S. Srinivasarao, N. Agnieszka, A. Ewa, S. Murugesan, S. Chander, R. Krishnan, K. V. G. Chandra Sekhar, 6-chloro, 6,7-dichloro and 2-methyl-3-(((1- (substitutedphenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)quinoxaline 1,4-dioxide derivatives as anti-tubercular agents. (Manuscript under preparation)

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Other publications:

1) N. Suresh, C. Surendhar, A. Suresh, D. Battacharjee, B. Bhaskara Rao, N. Jain, A. Mallika, K. V. G. Chandra Sekhar, Design and synthesis of 4-morpholino-6-(1,2,3,6 tetrahydropyridin-4-yl)-N-(3,4,5-trimethoxyphenyl)-1,3,5-triazin-2-amine analogues as tubulin polymerization inhibitors. Bioorg. Med. Chem. Lett. 2017, 24, 3794.

2) H. N. Nagesh, A. Suresh, M. Nagarjuna Reddy, N. Suresh, J. Subbalakshmi, K. V. G. Chandra Sekhar, Multicomponent cascade reaction: Dual role of copper in the synthesis of 1,2,3-triazole tethered benzimidazo[1,2-a]quinoline and their photophysical studies. RSC Adv., 2016, 6, 15884.

3) H. N. Nagesh, A. Suresh, D. Sairam, P. Yogeeswari, K. V. G. Chandra Sekhar, Design, synthesis and antimycobacterial evaluation of 1-(4-(2-substitutedthiazol-4-yl)phenethyl)-4- (3-(4-substitutedpiperazin-1-yl)alkyl)piperazine hybrid analogues. Eur. J. Med. Chem., 2014, 84, 605.

4) K. Mahalakshmi Naidu, A. Suresh, J. Subbalakshmi, D. Sriram, P. Yogeeswari, P. Raghavaiah, K. V. G. Chandra Sekhar, Design, synthesis and antimycobacterial activity of various 3-(4-substituted sulfonylpiperazin-1-yl)benzo[d]isoxazole derivatives. Eur. J. Med. Chem., 2014, 87, 71.

5) K. V. G. Chandra Sekhar, V. S. Rao. T. V. N. V. Tara Sasank, H. N. Nagesh, N. Suresh, K. Mahalakshmi Naidu, A. Suresh, Synthesis of 3,5-diarylisoxazoles under solvent-free conditions using iodobenzene diacetate. Chin. Chem. Lett., 2013, 24, 1045.

6) N. Suresh, C. Surendhar, A. Suresh, G. Sridhar, A. Mallika, D. Battacharjee, N. Jain, K. V. G. Chandra Sekhar, Design, synthesis and biological evaluation of 2-(4-aminophenyl) benzothiazole analogues as antiproliferative agents. Bioorg. Med. Chem. Lett. (Manuscript under review).

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7) A. Suresh, N. Shashidhar, K. V. G. Chandra Sekhar, Pantothenate Synthetase: Novel Therapeutic Target for Tuberculosis Drug Discovery, Chemistry Select, (Under review).

8) H. N. Nagesh, S. Srinivas Rao, A. Suresh, K. V. G. Chandra Sekhar, One-pot synthesis of 4-substituted-1H-1,2,3-triazole employing sulfur to deprotect methylene nitrile group. (Manuscript under preparation).

Papers presented at Conferences

1) A. Suresh, K. Mahalakshmi Naidu, S. Srinivasrao, A. Ewa, M. Murali Krishna, K. V. G. Chandra Sekhar, Design, synthesis and anti-tubercular activity of various 3 (4- ((substituted-1H-1,2,3-triazol-4yl)methyl)piperazin-1yl)benzo[d]isoxazole derivatives. International conference on Nascent Developments in Chemical Science (NDCS-2015), BITS Pilani, Pilani campus, October 16-18th, 2015.

2) A. Suresh, K. Mahalakshmi Naidu, S. Srinivas Rao, K. V. G.Chandra Sekhar. Synthesis and characterization of new tetrazole derivatives as anti-tubercular agents. International Symposium on Bioorganic Chemistry (ISBOC-10), Indian Institute of Science Education and Research, Pune, January 11-15th, 2015.

3) National Symposium on Human Diseases at BITS-Pilani, Hyderabad Campus, Hyderabad, March 15-16th, 2014.

4) A. Suresh, H. N. Nagesh, N. Suresh, K. Mahalakshmi Naidu, D. Sriram, P. Yogeeswari, K. V. G. Chandra Sekhar, Synthesis and anti-tubercular activity of 6-(4- substitutedpiperazin-1-yl)phenanthridine analogues. International conference on Chemical Biology, Disease mechanisms and Therapeutics (IICB-2014), Indian Institute of Chemical Technology, Hyderabad, February 6-8th, 2014.

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Biography of Prof. K.V.G. Chandra Sekhar

Prof. K.V.G. Chandra Sekhar completed his B. Pharm (Hons.) in 1999 from BITS Pilani and after working a faculty in Gurukul vidyapeeth junior college, Hyderabad for two years, he re- joined BITS Pilani in 2001 as Teaching assistant and completed his M. Pharm in 2003. He then worked as Assistant Lecturer for one year and then as Lecturer up to 2008. He was awarded Ph. D in synthetic medicinal chemistry in 2008. From 2008 to 2014 he worked as Assistant professor and currently he is working as Associate professor since 2015. His areas of research interests are synthetic medicinal chemistry and drug design. As investigator, he successfully completed major research projects funded by DBT, DST and UGC. He has published over 27 research articles in well renowned international journals and presented around 36 papers in various conferences/symposia and workshops. Two students were awarded doctorate under his guidance and six of them are currently pursuing their Ph. D. He is a life member of association of pharmacy teachers of India, CRSI, Indian pharmacological society, Indian council of chemist, Indian association of chemistry teachers etc.

Biography of Mr. Amaroju Suresh

Mr. Amaroju Suresh completed his B. Sc (Botany, Zoology and Chemistry) in 2005 from University of Kakatiya. He completed his M. Sc (Organic chemistry) in 2007 from University of Kakatiya. He started his career as Trainee - Regulatory Affairs, Sri Krishna Pharmaceuticals Ltd (2007-12), Hyderabad, India. He was appointed as Junior Research Fellow in DBT project from December 2012–October 2016 in BITS Pilani, Hyderabad campus under the supervision of Prof. K.V.G. Chandra Sekhar. Later he was appointed as Senior Research Fellow in DST project from November 2016 to January 2017. He is currently working as Institute Research Fellow at BITS Pilani, Hyd. campus since February 2017. He has published six scientific papers in international journals and presented four papers at national and international conferences.

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