Design, Synthesis, Characterization and Biological Evaluation of Novel Heterocyclic Derivatives as Anti-Tubuercular Agents

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DESIGN, SYNTHESIS, CHARACTERIZATION AND

BIOLOGICAL EVALUATION OF NOVEL HETEROCYCLIC

DERIVATIVES AS ANTI-TUBUERCULAR AGENTS

THESIS

Submitted To

THE TAMILNADU Dr. M.G.R. MEDICAL UNIVERSITY

In partial fulfilment for the award of the degree of

DOCTOR OF PHILOSOPHY IN

PHARMACY

By

P.R.SURYA

Under the Guidance of

Dr. A. JERAD SURESH ., M.Pharm., Ph.D.,M.B.A.,

COLLEGE OF PHARMACY

MADRAS MEDICAL COLLEGE, PARK TOWN, CHENNAI – 600 003.

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ACKNOWLEDGEMENT

My deepest gratitude, love and prayers go to my family for their constant

love, care, trust and unconditional support throughout my life. I am indebted to my

dearest dad Mr. P. N. Ramakrishnan (Retd. Sub Postmaster) and my ever-loving

mom Mrs. P.K Thankam (Retd. Teacher) for their everlasting love. They are who

considered education is the best & most precious wealth of my life and introduced

me to this world of education and knowledge and who made this thesis possible.

They taught me that nothing is impossible this world and hard work is the key to

success. I love you dad and love you mom more than my breath. Hold my hands

forever in rain and shine throughout my life and take me in to right path. The most

beautiful thing in this world is to see my parents smiling, and the next best thing is

to know that I am the reason behind that smile.

I would like to remember and thank my sweetest Grandmother M. Lakshmi

kuttyAmma (Retd. Teacher) who filled utmost self-confidence in me and taught

about importance of knowledge and independence. I really miss you

Ammamma…you were so dear to me. I love you a lot. I always cherish those days

you spent with us.

This thesis has been kept on track and been seen through to completion with

the immense support and encouragement of my eldest brothers Mr. P.R. Harish

Babu & Mr. P.R. Rathnakumar.During the most difficult times of my education,

research and when writing this thesis, they gave me the moral support and the

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I would like to thank my dearest teacher, my advisor, Dr .A. Jerad Suresh,

M. Pharm., PhD., M.B.A., Principal, Professor & Head, Department of

Pharmaceutical Chemistry, College of Pharmacy, Madras Medical Collage, Chennai,

for his supervision, advice, and guidance from the very early stage of this

researchas well as giving me remarkable suggestions and experiences throughout my

research work. His continuous energy and passion in research has motivated me. His

perfection in research and time management will always inspire me. In addition, he

was always accessible and willing to help students including me with my research.

He was always there in my difficult times and gave me the moral support, valuable

suggestions, blessings, and the freedom I needed to move on. His constructive

guidance during the course of research helped me to present this thesis today. I

could not have imagined having a better advisor and mentor for my Ph.D. study. The

honesty, justice, perfection and goodness which he had filled in me will extend in

my whole life. My teacher is my role model who taught all that is necessary to

develop my knowledge and improve my responsibility.

I would like to express my sincere gratitude to my doctoral advisory

committee members Dr. Aruna, M. pharm., Ph.D., JDME pharmacy, Director of

Medical Education, Kilpauk , Chennai and Dr. N. Jayashree, M. pharm., Ph.D.,

Professor, Department of Pharmacognosy, Madras Medical College, Chennai for

their valuable suggestions, inspirations and timely advice during my research work.

I wish to convey my thanks to Dr. V. Niraimathi, M. pharm , Ph.D.,

Professor & Head,Department of Pharmaceutical Chemistry, Collage of Pharmacy,

Madurai Medical college, for her involvement , interest and encouragement in the

research work , the associated experience broadened my perspective on the practical

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It is privileged to thank Dr. Muthuswamy, Mpharm., Ph.D., Collage of

Pharmacy, Madras Medical Collage, Chennai for his valuable suggestions and

encouragements.

I am also indebted to Dr .V. Kanagasabai , M.D., and Dr .R. Vimala,

Former Dean, madras medical college, for allowing me to carry out my research as

a full-time research scholar in the College of Pharmacy ,Madras Medical College,

Chennai.

I am also indebted to Issac Christian Moses., M.D. Dean, Madras Medical

College, Chennai, for providing the necessary requisites and facilities for my

research work.

I am greatly thankful to prof. Dr. T.K Ravi, Principal, Sri Ramakrishna

Institute of Paramedical Sciences, Coimbatore, for his valuable suggestions and

encouragement to bring my admission to PhD.

Hereby I remember and thank all my Teachers throughout my way of

education for their blessings, love and care.

I would like to express my sincere gratitude to Dr. Sathish, M.Pharm.,

Ph.D., Dr. Priyadarshini, M.pharm., Ph.D., Dr. Sunitha M.Pharm., Ph.D.,

Mrs. Saraswathy, M.pharm., (Ph.D). Department of pharmaceutical chemistry for

the timely help during my thesis work.

I am greatly obligated to Dr. Sreenivelan, BVSc., chief Veterinarian, and

Mr. Kandaswamy, in charge, Central animal house, Madras Medical College, for

providing important research materials and constructive ideas in order to do the

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I also express my acknowledgment to Mr. Deshpande, and Mr. Maneesh,

Deshpande laboratories, Bhopal for conducting in-vivo study for my samples and

reports at the right time.

I would like to express my thanks to Mr. Vadivelan, M.pharm, PhD.,

Principal Scientist GVK Bioscience Pvt Ltd , Chennai, for his guidance and

suggestions towards the drug discovery informatics & training and computational

work in my thesis. I deeply admire his valuable help and encouragement to complete

my thesis work successfully.

I wish to express my thanks to Mr. Madheesan, Mr. Venkitesh,Vijay

Kumar and Vinoth Vijayan Scientific assistants NMR, GC-Mass Analyzers, and

IR analyzer, VIT, Vellore.

I convey my thanks to Dr. Kishore Bhatt, Professor and Head, Department

of Microbiology, Maratha Mandal’s Institute of Dental Science and Research

Institute, Belgaum, Karnataka and Mr. Sunil for helping me to carry out the

biological evaluation and speedy reports in right time.

I also express my thanks to Mr. Siva Kumar Infra-red Spectroscopy analyst

of college of Pharmacy, Madras Medical College, and the entire non-teaching staffs

for their constant help and co-operation during my research work.

All my lab buddies including my seniors Mr. K.M. Noorulla, and

Mrs. Devi Umesh, Ms. Velankanni, Mr. Ravi kumar, Ms.Narayani,

Ms. Menaka, Ms. Bharathi, Mr. Dinesh, Mr. Madhuraj and Ms. Leela at the

laboratory made it a convivial place to work. They had inspired me in research and

life through our interactions and with memorable moments which I always like to

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I owe a great deal of debt and whole hearted thanks to my eldest sisters

Sachin, Santhi and Bindu, for their love, care and prayer which raised me to

achieve my dreams. I also owe my most sincere gratitude to my brother in laws

Mr. Vasudevan, Mr. Harikrishnan and Mr. Pramodkumar for my moral strength,

Source of inspiration, and positive attitude towards life now and then. I also thankful

to buddies Indu, Vishnu and Surya for joining my hands to have funs and

memorable moments. All of them were my courage and back bone in completing the

project successfully and I thank all of them from the bottom of my heart.

I would like to express my everlasting love and thanks to the personality,

none other than one and only Super Star Padamavibhushan Mr. Rajnikanth, who

inspired me, a lot. I thank my dearest Rajni uncle for his blessings and for finding

his valuable time to meet me during the tight schedule of shoot. The down to earth

simplicity, humbleness, hard work and goodness in him made me speechless and

filled happiness in me. I deeply esteem and encouraged to become a good human

being. I love you Rajni uncle. Be there always for us.

I wish to thank one and only my dearest Rahul, who is my respect, my love,

and treasure of my life. Love you for everything. Wish to walk long ways with

broken silence. Just be there.

I thank my Lakshmi & shoes, my little kittens, who made me to smile now

and then.

I am very sorry, and it would not be fair if I do not regret for all those

innocent mice, which lost their lives for the sake for my in-vivo study without which

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Lastly, and most importantly, I wish to thank my own Lord Krishna, who is

always there for me, who listens me, who speak with me, sing with me , walk with

me and wipe away my tears whenever I am sad. My trust in God, in the power of

Almighty will always lead me to overcome the strange roads with utmost good faith

and self-confidence. Lord Krishna is always with me, and my music goes to his feet

as prayers. Krishna, when you know me, why should I afraid, you are within me, in

my heart and thoughts.

To anyone that may I have forgotten. I apologize. Thank you as well.

I dedicate this thesis to my ever loving Parents.

With love and prayers

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LIST OF TABLES

Table No Title of Table Page No

1 2D and energy minimized 3d structures of the selected

molecules 48

2 Docking results of the selected 35 analogues 59

3 Residue interaction pattern for the synthesized

compounds against target enzyme Fab D

62

4 In-silico ADME properties of the selected 35 ligand

molecules

85

5 In-silico toxicity assessment result of the 35 ligand

molecules

87

6 List of synthesised compounds with the IUPAC name 110

7 Mol. wet., Mol. formula, colour, solubility & Melting

point, percentage yield of the synthesized compounds

117

8 TLC Profile of the Synthesized compounds 121

9 Solubility data of the synthesized compounds 123

10 Ranking of the compounds based on the in-vitro anti

mycobacterial activity.

136

11 Lung CFU lung values in animals treated with test

samples and standard controls.

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LIST OF FIGURES

Figure No. Title of the figures Page No

1 Mycobacterial infection 2

2 Mycobacterium tuberculosis 3

3 Cell wall of mycobacterium tuberculosis 3

4 mycobacterium tuberculosis H37Rv 4

5 Virulence life cycle of mycobacterium tuberculosis 5

6 Estimated TB incidence rates, 2013 6

7 The pathogesis of

tuberculosis-adapted from Canadian tuberculosis standards

7

8 Patho physiology of Tuberculosis 7

9 First-Line Treatment of TB for Drug-Sensitive TB 8

10 Tuberculosis-drugs-and-actions 9

11 MDR TB Treatments 9

12 The traditional regimen for TB 10

13 Mechanism of action of current TB Drugs 10

14 Unprocessed 3D structure of prepared protein Fab D

(PDB ID-2QC3)

57

15 Energy minimized 3D structure of prepared protein Fab

D (PDB ID-2QC3)

58

16 Docked possess of all the 35 ligands at the active site of

target enzyme Fab D

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Figure No. Title of the figures Page No

17 Ligand interaction diagrams of all the ligand molecules

against active site of target enzyme Fab D (Protein Data

bank-2QC3)

66

18 Screen shot of in-silico toxicity assessment results 88

19 ORTEP DIAGRAM OF COMPOUND “C” 127

20. Lung CFU in animals treated with test samples and

standard controls, N = 3, error bars represent standard

deviation

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CONTENTS

CHAPTER TITLE PAGE. No

1 INTRODUCTION 1

` 1.1. History of tuberculosis 2

1.2. Tuberculosis Epidemiology : Prevalence ,

Airborne transmission

4

1.3. Pathogenesis and immune response: the

interactions between MTB and the host cell.

6

1.4. Drug discovery 11

1.5. Medicinal chemistry 12

1.6. In- silico Screening Approach 12

1.7. Biological target 16

1.8. Significance of heterocyclic compounds 18

2 AIM AND OBJECTIVE 20

2.1 Aim 20

2.2 Objective 20

3 LITERATURE REVIEW 21

3.1 Literature review based on Pharmacology,

Epidemiology, Prevalence and WHO data

21

3.2 Literature review based on In-silico approach 25

` 3.3 Literature review based on Chemistry 26

3.4 Conclusion 36

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4.1 Scope of the study 37

4.2 Plan of work - work flow 37

5 IN SILICO APPROACH 40

5.1 Materials 40

5.2 Experimental 41

5.3 Result and discussion Materials 46

6 CHEMISTRY 100

6.1 Materials 100

6.2 Experimental 102

6.3 Physical properties of the synthesised compounds 110

6.4 Result and discussions 124

7 IN-VITRO ANTIMYCOBACTERIAL ASSAY 128

7.1 Materials 128

7.2 Experimental 128

7.3 Result and discussions 129

8 INVIVO EFFICACY 138

8.1 Acute toxicity study 138

8.2 In-vivo antimycobacterial activity 141

9 SUMMARY AND CONCLUSION 145

9.1 Summary 145

9.2 Conclusion 148

10 IMPACT OF THE STUDY 150

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ANNEXTURES

S.NO ANNEXTURES Page No.

1 Plagiarism Screen Shot i

2 Plagiarism Screen Shot ii

3 Animal ethical Clearance Approval Letter iii

4 Patent Filing iv

5 Patent Filing v

6 Publications vi - xii

7 Presented Posters xiv & xvi

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CHAPTER 1

INTRODUCTION

Tuberculosis (TB), the disease caused by Mycobacterium tuberculosis

(Mtb),a leading cause of death killing approximately 5000 people per day

throughout the World’s is more common among men than women2.Today, the

inadequate drug compliance, the appearance of multiple-drug resistant strains, and

the HIV/ AIDS epidemics are some factors that have led to the resurgence in TB.

Drug resistance develops following inadequate compliance and HIV/AIDS patients

with weakened immune system are extremely susceptible to Mycobacterium

Tuberculosis and the expected cause of death2. Mycobacterium tuberculosis (Mtb)

has been aggravated by human immuno-deficiency virus (HIV) and their calamitous

synergism, since both are destructive together than individually. Approximately

70-80% of HIV infected patients are co-infected with Mycobacterium tuberculosis

(Mtb), as a result of which 60-70% of HIV positive patients develop active

TB3.Today, TB has become a disaster to the world due to the increasing emergence

of “multi-drug resistant tuberculosis” (MDR-TB), “extremely drug resistant

tuberculosis” (XDR-TB) and “totally drug resistant tuberculosis” (TDR-TB). Further

along with this, the HIV pandemic threatens disease control3.

The upsurge in the progress or spread in the drug resistant tuberculosis along

with HIV has the potential to influence TB care schemes. This highlights the need to

develop new and more effective anti-tb drugs.

The main theme of the present thesis is the exploration of new tactics in the

field of modern drug discovery for the development of new drugs, efficient of

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introduction to the history, epidemiology and pathogenesis of tuberculosis, aspects

of modern drug discovery and their applications in the medicinal chemistry are

given in the succeeding sections.

1.1 History of tuberculosis

TB is spread through the air when people who are affected with Pulmonary

TB, expel bacteria, by sneezing or coughing. Mycobacterium Tuberculosis is an

ancient menace.

Figure 1: Mycobacterial infection203

Till 18th century the disease was a mystery. Fortunately speculations came to

an end with announcement of German microbiologist Robert Koch that he had

identified the bacillus and gave the name mycobacterium tuberculosis.

The Bacillus - Calmette Guerin (BCG) a vaccine for tuberculosis, in 1908,

developed by two French scientists, Calmette and Guerin and the introduction of

specific anti-tuberculosis drugs in 1943, extended the hope to control the most fatal

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Figure2: Mycobacterium tuberculosis208

Synonym: Tubercle bacillus Koch 1882.

Mycobacterial cell wall:

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Genome: The genome sequence of strain of Mtb, H37Rv has been determined and

analyzed.

Figure: 4: mycobacterium tuberculosis H37Rv218

In 2013 there was an estimated 9 million new cases18and 2 million associated

deaths19 occurred in developing countries. Furthermore, tuberculosis treatment has

disadvantages such as substantial toxicity, patient noncompliance and long lasting

treatment periods which results in drug resistance, MDR-TB, XDR-TB and more

recently totally drug resistant tuberculosis (TDR-TB) mounts a challenge to

tuberculosis control.

1.2 Tuberculosis Epidemiology: prevalence, airborne transmission

MTB or TB is epidemic in any developing countries. Because of the

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Figure 5: Virulence life cycle of mycobacterium tuberculosis

(Source: www.google.com)

Across the globe, tuberculosis (TB) remains a great concern, due to the rise

in the estimated new cases of TB and related 2 million death cases. Malnutrition,

poverty and drug resistance are the main causes for a rapid increase in TB cases.

As per World Health Organization (WHO) report, “One third of the world’s

population has been infected with TB”6. A majority of deaths reported, in

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Figure 6: Estimated TB incidence rates, 2013

There were an estimated twelve million prevalent cases of TB in 2012. By

20thcentuary, the prevalence rate had fallen thirty seven percentages globally since

1990.

Tuberculosis (TB) cases in 2006 in showed that poverty, urbanization,

population densities are directly correlated.

1.3 Pathogenesis and Immune Response: The interactions between MTB and

the host cell

Once the bacterium Mycobacterium tuberculosis (MTB) is inhaled via

droplets, spread through direct contact. In alveoli, bacteria get surrounded by

macrophages, the most abundant immune effectors cells present in alveolar spaces

42, 43, 44

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Figure 7: The pathogesis of tuberculosis - adapted from Canadian tuberculosis standards:

Figure 8: Patho physiology of Tuberculosis: (Source: www.google.com)

The high incidence of TB is mainly due to overcrowding and

malnourishment due to the ease at which the infection can be transferred. The

emergence of multidrug resistant (MDR), extremely drug resistant (XDR-TB) and

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disease. Lack of patient compliance, delay in diagnosis, drug resistance further

complicates the situation.25, 47. Further Co-infection with Mtb and HIV

synergistically influence each other progress, rendering the host vulnerable to death.

About 80% of HIV infected patients are co infected with Mtb and as a result 70% of

HIV positive patients develop active TB.

Thus, TB is a major global health threat; in order to reduce the emergence of

drug resistance and to shorten the duration of therapy and we must improve the

existing treatment regimen with discovery of newer classes of anti-tubercular agents

to control the spread of TB.

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Figure 10: Tuberculosis-drugs-and-actions: (Source: www.google.com)

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Figure 12: The traditional regimen for TB: (Source: www.google.com)

Figure 13: Mechanism of action of current TB Drugs:

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Schematic diagrams of First-Line Treatment of Tuberculosis for

Drug-Sensitive TB (Figure 9), Tuberculosis-drugs-and-actions (Figure 10), MDR TB

Treatments (Figure 11), the traditional regimen for TB (Figure 12) and Mechanism

of action of current TB drugs (Figure 13). Source: www.google.com

1.4 Drug Discovery

In this era, drug discovery has developed into an interdisciplinary scientific

field integrating diverse disciplines of biology, chemistry, mathematics and

computers72. Any novel chemical entity with potential therapeutic value is

extensively studied for its safety and efficacy before it is marketed for public use.

This multi-stage process is generally referred as “Drug Discovery Pipeline” or

“Development Chain” 73. All the initial stages of the pipeline phenomenon i.e.

Identification and validation of the drug target, lead discovery and lead identification

is collectively represented by the term “Drug Discovery”.

The modern drug discovery and drug development shows an imperative role

in transforming a molecule from laboratory into a drug candidate. The Drug

discovery process can be usually split into two sections56.

1) Identification and optimization of lead molecules to improve their

selectivity towards the target including their toxicity profile56.

2) Development of a relevant biological system to test the compounds

in-vitro and in-vivo models to expedite the drug discovery process

and to enhance the screening efficiency and success rate56.

Drug discovery and development is an intricate, extended and an expensive

process since the safety, efficacy and other issues are mandatory. Mostly, it takes

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screening stage to final FDA approval and has a huge failure rate at each step of the

developmental process. To identify this issue, there are numerous new techniques

are available for instance molecular docking and QSAR analysis. Despite of such

modernization and progression in research and development, the number of new

chemical entities reaching the market has reduced distinctly, giving an impression

that, choice of the appropriate molecules for synthesis turn into one of the most

challenging tasks56.

New methodologies which pave way for faster development of promising

biologically active molecules.

1.5 Medicinal Chemistry

Medicinal chemistry deals with the interphase of organic chemistry and

biochemistry, genetics, molecular biology, pharmacology, pharmacokinetics, and

toxicology on one side, and chemistry-based disciplines for instance physical

chemistry, crystallography, spectroscopy, and computer-based techniques of

stimulation, data analysis and data visualization on the other side.74

“Medicinal chemistry concerns the discovery, the development, the

identification and the explanation of the mode of action of biologically active

compounds at the molecular levels”. Medicinal chemistry is as well concerned with

the “study, identification and the synthesis of the metabolic products of the drugs

and related compounds”59, 60.

1.6. In-silico screening approach

Over the past decade, the practice of computerized models to predict sequels

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biology. It ultimately assists a better understanding and prediction of chronic human

diseases pathogenesis and eventually facilitates to design better and more rational

approaches for developing and analyzing new drug candidates.

In over-all, Rational Drug Design (RDD) is “the indigenous process of

discovering new molecules based on the knowledge of the biological target”. Hence

the fundamental concept of drug design consists of “design of small molecules that

are complementary in shape and charge to the bio molecular target to which they

interact and therefore will bind to it”.

Computer Aided Drug Discovery and Development is being utilized in early

stages of DD process that comprises hit identification, lead selection and

optimization 70. Past three decades have witnessed the development of therapeutic

small molecules solely based on Computer-aided drug discovery/design methods 71.

In the post genomic era, Computer-Aided Drug Design (CADD) has found

significant applications in almost all stages in the drug discovery pipeline56. CADD

computational tools and software’s are used to stimulate the drug-receptor

interactions. In traditional based approach, drugs were discovered by the means of

trial and error methodologies making research and development process more time

consuming and expensive. Computational drug discovery aids scientists to get

insight into the drug receptor interaction and also aids to reduce the time and cost61.

Drug Discovery and Development process is a highly complex phenomenon.

Involvement of computers and related technologies has significantly improved

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RDD methods fall in to two different categories.

Ligand- based (Pharmacophore Modeling)

Structure based (Molecular Docking)

Ligand based drug designs(LBDD):

LBDD depend on the perception of other molecules that bind to the

biological enzyme target of interest. These and other molecules may be used to

derive a pharmacophore model which describes the minimum required structural

characteristics for a molecule to possess, thus as to bind to the target. Conversely,

quantitative study (QSAR) in which a correlation between calculated properties of

molecules and their related biological activities may be derived. QSAR relationship

is used to predict the activity of new analogues.

Structure based drug design (SBDD):

SBDD depend on emphasizing the three dimensional structure of the target

obtained through methods such as X-ray crystallography or NMR spectroscopy. On

the other hand, diverse automated computational procedures may be used to

contemplate the molecular target for which drugs are contemporarily designed.

Structure-based drug design is considered as one of the most innovative and

powerful approaches in drug design69.

1.6.1 Molecular Docking

Molecular docking is the technique which envisions the “preferred

orientation of one molecule to a second when bound to each other to form a stable

complex in three dimensional spaces.” The function of the protein can be inferred.

Accordingly the results of the docking are exceptionally valuable in finding drugs

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Docking Principle

The two main components that are vital for docking studies are:

Secondary structure of our protein of interest.

Library of ligands from suitable data base.

Docking tools are based on the search of algorithm and the scoring function.

A search algorithm finds the best docking pose measured by the scoring function. A

scoring function differentiates correct docking poses from incorrect ones.

The protein and ligand structures need preparation before docking to achieve

the best docking results63-65.

1.6.2 In-silico Toxicity Risk Assessment

Toxicity is accountable for 20-40% of drug failures. Commercially In-silico

tools are accessible and can be used for predicting potential toxicity issues; they are

typically classified in to two groups. The first approaches uses “expert systems that

develops models on the basis of abstracting and codifying information from human

and the scientific literature sources”. The next approach relies on “generating the

descriptors of chemical structure and statistical analysis of relations ships between

those descriptors and the toxicological end point.”

1.6.3 In-silico ADME predictions

In pharmaceutical research some new drug failures occur in the clinical trial

phase owing to absorption, distribution, metabolism and excretion (ADME)

properties. ADME prediction is an exceptionally challenging area as many of the

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1.7 Biological Target

Mycobacterium tuberculosis, the cause of tuberculosis, is a devastating

human pathogen. There is an alarming increase in cases of TB caused by

drug-resistant strains and to the co-infection with the HIV. The re-emergence of

tuberculosis (TB) as a global health crisis over the last few decades emphasizes the

need for discovery of new therapeutic drugs acting on the new targets against this

disease.

There are several biosynthetic target enzymes that are crucial for the survival

of the mycobacterium and are considered as potential drug targets.

The identification of 451 high-confidence targets of mycobacterium

tuberculosis studied by passing the whole mycobacterium tuberculosis proteome

into several filters / analysis as “ network analysis of the protein-protein interactome

(molecular interaction network), flux balance analysis of the reactome

(Mathematical method for simulating metabolism in genome-scale reconstruction of

metabolic networks), experimentally derived phenotype essentiality data (genes that

are indispensible for the survival of an organism), sequence analysis. (process of

subjecting a DNA, RNA or peptide sequence to any of a wide range of analytical

methods to recognize its features, function, structure, or evolution) and a structural

assessment of targetability (obtainability of crystal structure of the proteins so as to

increase the targetability)”.1

From those 451 high confidences target of Mycobacterium tuberculosis, for

crucial targets which pass the major filter s of the above study were chosen for the

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The target enzymes are as follows.

Fab H Lipid biosynthesis

Fab D (MCAT)

EmbC (arabinosylindolyl acetyl inositol synthase) Cell Wall biosynthesis

Glf (UDP–GALP MUTASE)

Mycobacterium tuberculosis Fab D (Malonyl CoA - acyl carrier protein

transacylase):

MCAT (MCAT) is an essential enzyme in the biosynthesis of fatty acids in

Mtb. This enzyme catalyzes the transacylation of malonate from malonyl CoA to

activated holo-ACP, to generate malonyl-ACP which is an elongation substrate in

fatty acid biosynthesis. It is a critical step in mycobacterial FASII for its viability

and pathogenicity. Thus the discovery of molecules specifically inhibit mtFab D

may lead to the development of new therapeutic anti tuberculosis agents.

Mycobacterium tuberculosis Fab H (3-oxoacyl-(acyl carrier protein) synthase):

Fab H catalyzes a two-step reaction that initiates the pathway of fatty acid

biosynthesis in bacteria.

Mycobacterium tuberculosis EmbC (arabinosylindolyl acetyl inositol synthase):

In mtb, EmbC is an essential gene under normal growth conditions.

MtbEmbC is a membrane protein, which involves in the biosynthesis of the

mycobacterial cell wall arabinan. Mtb EmbC encodes arabinosyl transferase, which

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Mycobacterium tuberculosis Glf (UDP–GALP MUTASE):

UDP Galactopyranose mutase is involved in Lipopolysacchride biosynthesis,

in the conversion of UDP galacto pyranose into UDP galacto furanose through 2

keto inter mediate.

1.8 Significance of Heterocyclic Compounds

Heterocyclic structures are always a part in the field of research and

development in organic chemistry. Utmost all the therapeutic molecules consist of

heterocyclic structures. Heterocyclic ring system encompasses the core of the active

moiety as pharmacophore. For a period of decade’s heterocyclic therapeutic agents

plays a pivotal role in chemotherapy. It is a major building block of carbohydrates,

vitamins, alkaloids and nucleic acids which indicates the profound influence of

heterocyclic structure on the physiological / functional activity. Therefore, the drug

design will involve docking study of some interesting heterocyclic ligands with

biological protein target of interest.

Heterocyclic ring containing nitrogen, sulfur, and oxygen, which shown to

have various important medicinal properties. Among heterocyclic molecules,

“Chalcones” containing [(furan-2-yl) moiety or (hydroxy phenyl) moiety], “3,

4-dihydropyrimidine-2(1H)-thione”, “3,4-dihydropyrimidine- 2(1H) one”, “thiazolo

pyrimidin-3(5H)-one”, “OxazoloPyrimidin-3(5H)-one”, “2, 4, 6 triaryl-1

H-imidazole”, “Pyridine-4-Carbohydrazide”,Oxadiazole,“Pyrazolines” and “Iso

Nicotino Hydrazide” and their fused ring systems were revealed to have several

significant biological activities such as anti-bacterial, anti-fungal, anti-viral, diuretic,

tuberculostatic, anti-HIV, anti-cancer, anticonvulsant, anti-inflammatory and

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pharmaceutically very important, since they have been reported to various

properties. The derivatives of imidazole, pyrazole and oxadiazole are the scaffolds

present in many standard drugs and it is recognized to boost the pharmacological

activity.

In view of the above facts, and statements the research attempt has been

undertaken to frame the strategy as per the protocol of drug discovery to design and

synthesize new chemical entities of diverse heterocyclic scaffolds like “Chalcones”,

“3, 4-dihydropyrimidine-2(1H)-thione”, “3,4-dihydropyrimidine- 2(1H)-one”,

“thiazolo pyrimidin-3(5H)-one”,“Oxazolo Pyrimidin-3(5H)-one”, “2, 4,

6triaryl-1H-imidazole”, “Pyridine Carbohydrazide” , “Oxadiazole”, “Pyrazolines” and “Iso

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CHAPTER 2

AIM AND OBJECTIVES

2.1 AIM:

The aim of present research is to develop novel Anti-tubercular molecules,

which inhibit target enzymes, Fab D (Malonyl CoA - acyl carrier protein

transacylase).

Thus the research study directly aims to design and synthesize some

heterocyclic analogues such as “Chalcones” containing [(furan-2-yl) moiety or

(4-hydroxy phenyl) moiety],“2, 4, 6 triaryl-1H-imidazole”,

“Pyridine-4-Carbohydrazide” , “Oxadiazole”, “Pyrazolines” and “IsoNicotinoHydrazide” and

some fused ring systems like “3, 4-dihydropyrimidine-2(1H)-thione”,

“3,4-dihydropyrimidine- 2(1H)-one”, “thiazolo pyrimidin-3(5H)-one”, “Oxazolo

Pyrimidin-3(5H)-one” which will prove to be effective against Mycobacterium

Tuberculosis.

2.2 OBJECTIVE:

The objective of this research study is to Design, Synthesize, and

Characterize Heterocyclic derivatives of some medicinally important

compounds and to evaluate its biological activity against Mycobacterium

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CHAPTER-3

REVIEW OF LITERATURE

The aim of the literature review is to establish a broad knowledge, based

upon the various in-silico approaches in the modern drug discovery. The literature

survey is also aimed to understand the significance of chemistry and biology in the

drug discovery of anti-tubercular agents. Thus, an extensive literature survey was

carried out based on in-silico approaches, Chemistry aspect and on Pharmacological

aspect.

3.1 Literature review based on Pharmacology, Epidemiology, Prevalence

and WHO data:

1. World Health Organization. ”The Sixteenth Global Report on

Tuberculosis-2011”.1

2. Takayama K et al., 2(2005) explained about on reestablishment of

tuberculosis.

3. Zhang Y., 3(2005) evaluated on emergence of drug resistance of

tuberculosis.

4. Hudson A et al., 4 explained on increase in TB incidence due to

poverty and urbanization.

5. Keshavajee .S et al.5, (2012) discussed on Tuberculosis and about the

drug resistance as well as about the history of modern medicine.

6. World health organization c 1948. (2014)It deals with the

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7. Daniel TM., 7et al. (2006) reported the history of tuberculosis from

ancient times 18th and 19th century. Reviewed on The modern era of

tuberculosis treatment and the discovery of streptomycin and

isoniazid.

8. Davies P.D.O, 9(1999) detailed on a brief history of tuberculosis, the

development of BCG Vaccine, and introduction of Anti-TB drugs.

9. Evans CC12 et al., (1994) historical background. Clinical

tuberculosis.

10. Roberts CA, et al, 13(2003) a global view on a re-emerging disease

tuberculosis.

11. M. S. Jawahar, 14(2004) explained about current trends in

chemotherapy of tuberculosis.

12. Ashish Kaushal15, et al.; (2012) review on recent advances in

chemotherapy of tuberculosis

13. Nature reviews. 16 (2013) provides proof on MDR TB, XDR TB

AND TDR TB.

14. Tomioka H et al.,19(2006) discussed about the development of

anti-tuberculant drugs: current status and future prospects

15. Fogel N., 21(2015) reviewed on the history of tuberculosis, its

epidemiology, transmission, pathogenesis, and its treatment control.

Concluded about the importance of complete understanding of

pathological immune responses and interactions in TB in the

development of drugs and vaccines.

16. Ruth McNerney et al., 22(2012) reported Needs, Challenges, Recent

Advances and opportunities in tuberculosis diagnostics and

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17. Godman L, Schafer AI.23 (2011) Tuberculosis: disease overview.

24TH ed. St. Louis (MO): Saunders Elsevier.

18. Cruz- Knight W24 et al. (2013). Tuberculosis: An overview. Prim

Care; 40(3): 743-56

19. Anderson P et al 25(2014) reviewed on Tuberculosis vaccine and

Trends in immunology.

20. De Martino M et al. 26 (2014) Detailed about reflections in the

immunology of tuberculosis.

21. “Improved data reveals higher global burden of tuberculosis”.

WHO.int.22 October (2014).31

22. GBD (2013) Mortality and causes of death collaborators., “ Global,

regional and national age-sex specific all cause and cause-specific

mortality for 240 causes of death,1990-2013: a systematic analysis

for the Global Burden of Disease Study 2013” Lancet.201432

23. Keshavjee S, et al 33(2012) discussed on Tuberculosis, drug,

resistance, and the history of modern medicine.

24. World Health Organization. c1948. Tuberculosis fact sheet. In:

Geneva (Switzerland): WHO global TB Programme. (2014)34

25. Comas I, et al. 35(2014).The past and future of tuberculosis research.

26. De Martino M et al, 36(2014) Reflections on the immunology of

tuberculosis.

27. Ernst JD37 (2012) Reviewed about The immunological life cycle of

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28. Shaler CR et al38 (2013) Discussed on contribution of the granuloma

to the dissemination, persistence and transmission of Mycobacterium

Tuberculosis.

29. HossainMM et al39 (2014) discussed on Pattern recognition receptors

and cytokines in Mycobacterium tuberculosis infection.

30. Chao MC et al, 40(2014) discussed on the role of dorman cystate in

tuberculosis.

31. Frieden TR, et al 42(2003) discussed about Tuberculosis.

32. Korf JE et al.43 (2006) explained about Macrophage reprogramming

by mycolic acid and promotion of a tolerogenic response in

experimental asthma.

33. Van Crevel R, et al 44 (2002) reviewed on Innate immunity to

Mycobacterium tuberculosis.

34. Nicod LP 45(2007) immunology of tuberculosis.

35. Mason RJ, 46 et al. (2010) tuberculosis In: Murray JF, Nadel JA,

Editors. Murray and Nadel.

36. Mi Yan Shutao Ma.47(2012)discussed about “Recent advances in the

research of heterocyclic compounds as anti-tubercular agents

37. Gutierrez-Lugo mt48 et al (2008). Noted on Natural products, small

molecules and genetics in tuberculosis drug development.

38. Tretter EM50 et al (2012) detailed on Mechanisms for defining super

coiling set point of DNA Gyrase.

39. Gutierrez-Lugo51 et al. (2008) Discussed on Natural Products, Small

Molecules, and Genetics in Tuberculosis Drug Development.

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41. Rivers, E. C53. et al. (2008) discussed about new anti-tuberculosis

drugs in clinical trials and their novel mechanisms of action.

42. Lombardino JGH55 et al. (2004) given the detailed information about

the role of the medicinal chemist in drug discovery-then and now.

43. Stratmann, H.G.56 (2010) Mentioned on Bad Medicine: When

Medical Research Goes Wrong.

44. Peter Imming.57 (2015) Conversed on Medicinal Chemistry:

Explained about the Definition and objectives, drug discovery phases

and classification of drugs.

45. Wermuth CG58etal (1998) Glossary of terms used in medicinal

chemistry. IUPAC Recommendations

46. Burger A59 et al(1990)Comprehensive medicinal chemistry.

47. Kavitha CV75 et al., (2006)details on Synthesis of new bioactive

venlaxine analogues: novel thiazoldin-4-ones as anti-microbial.

3.2 Literature review based on In-silico approach:

48. Lengauer T et al. 61(1996) computational methods for bimolecular

docking.

49. Jain AN.62(2006) Nattered on “Scoring Function For Protein-ligand

docking” and also about Current Protein And Peptide Science

50. Lensink MF63 et al. (2007) “Docking and scoring protein complexes:

Proteins: Structures, Function and Bioinformatics.

51. Robertson TA, 64(2007) talked on “An all atom, distance dependent

scoring function for the prediction of protein-DNA interactions from

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52. Roncaglioni A. et al. 65(2013), Enunciated about In Silico methods to

predict drug toxicity. Current Opinion in Pharmacology.

53. Lipinski CA 66(2001). Review details on advanced drug delivery.

54. Pieffet G 67(2005). Voiced on The application of molecular dynamics

simulation techniques and free energy. Groningen

55. Baldi A.68 (2010) Overviewed about Computational approaches for

drug design and discovery.

56. Suryawanshi SB69 et al., 69 (2013). Said about Computer aided drug

discovery and development-an important need of the hour..

57. Sliwoski G et al 70, (2014) computational methods in drug discovery.

58. Herrling PL 71(2005) stated about the drug discovery process.

3.3 Literature review based on Chemistry:

72. Debus et al. 219 (1858), Debus Synthesis of imidazole by using diketone

molecule.

73) Radiszewskiet al.220detailed condensation reaction of diketones

O

O

+ 2NH3

H O

MW at 240W

(40)

74) Qasimet al 221(2011) synthesis of 2- phenylimidazo [4, 5-f] [1, 10]

Phenanthroline derivatives, by reacting dicarbonyl compound and p

-substituted benzaldehyde. This is a type of acid catalyzed reaction with

excellent yields in a neutral ionic liquid, 1-methyl-3-heptyl imidazolium

tetrafluoroborate [(HeMIM) BF4], under solvent free and microwave assisted

conditions. Microwave reaction accompanies all the merits of microwave

reactions like easy workup, better yield, and environment friendly reaction.

75) Pathanet al223 (2006) reported the reaction of alkyl cyanide

76) Ermolatet al224 (2009) explained the synthesis of microwave assisted

hydrazinolysis.

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77) Bharadwajet al225(2010) the condensation of different under microwave

oven. The structures reported in better yield as compared to conventional

methods.

280W

280W

280 W

78) Raghavendraet al 226 (2011) a series of imidazole quinoline analogs were

synthesized by condensation of substituted imidazoleand substituted

(42)

79) Frank et al227 (2007) synthesis of substituted oxadiazoles containing the

nitroimidazole moiety by microwave-assisted as well as conventional

method was carried out and reported for antibacterial, antifungal and

anti-inflammatory activity.

80) Safari et al221 (2010) discussed about an efficient catalyst for an improved

and rapid synthesis of 2,4,5-trisubstituted imidazoles by a three-component,

one-pot condensation of benzil, aryl aldehydes and ammonium acetate in

good yields under solvent-free conditions using microwave irradiation. The

reactions in conventional heating conditions were compared with the

microwave-assisted reactions.

81) Nalageet al221 (2010) described an efficient procedure imidazole.

84) Ermolat’ev et al 231 (2006), A green chemistry for the synthesis of

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85) Sohet al 232 (2008) reviewed on- microwave-assisted synthetic reaction

86) Sparks et al223 (2004) synthesis of Triaryl-imidazoles in moderate to good

yields via by upon microwave irradiation.

87) Lupsori et al234 (1956) A series of imidazole derivatives synthesis by

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88) Jays et al 235(2011) synthesis of Isatin derivatives by agar diffusion method.

89) Sun J, et al., 236 (2013) Screening of novel compounds by a rapid and

effective approach for the discovery of potential chemical agents.

90) R.V.Sidhaye et al., “The synthesis of substituted oxadiazole and pyrazole

derivatives and screening anti-mycobacterial activity.”

91) Arora et al., 238(1990) 1, 3, 4oxadiazoles also find applications as

antiparkinian drugs. Initiated the preparation of 4(5-aryl-1, 3, 4-oxadiazole-yl

methyl)-1-phenyl piperazines.

N

N O

NH N H

R

92) Radha R et al., 239 (1990) Synthesized 5-(benzothiazol-2-yl-thiomethyl) 1, 3,

4-oxadiazole from mercaptobenzothiazole which showed moderate

anti-bacterial and anti-inflammatory activities. Some novel 1,3,4 oxadiazoles

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N S N N O NH R

93) Kalluraya B et al., 240(1995) Synthesized various 5- substituted 1, 3,

4-oxadiazole-2-hydrazines by reaction of oxadiazole with

5-substitued-2-furfural gave the hydrazine. These compounds are active against gram

positive and gram negative bacteria.

O N N O Ar -NH NH2

94) Chaudary BR et al.,241(1995) Anti tubercular activity of synthesized

derivatives of oxadiazoles.

N H

S R

C H3

N N O

N H

R

95) Sajeevan Gaikwad et al., (2012) reported new thiazolidinone derivatives

(46)

O CH3 O

+

O O R O NH N H S R O N N S O R EtOH/NaOH EtOH/HCL Ac2O/AcOH Monochloroacetic acid

96) Savita R. S et al., (2014), reported Synthesis, and evaluation of

anti-tubercular and analgesic activity of some novel pyrazolopyrimidine and

pyrazolopyridine derivatives. R O CH3

+

N NH NH2 O C H3 N CH3 NH EtOH/GAA/Reflux

97) K. Ilangoetal, (2010) reported synthesisof novel drugs which reported for

with anti-tubercular activity.

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O H OH O H COOC2H5 N2H4H2O/C2H5OH REFLUX 4hr O H OH O H CONHNH2 ArCHO/C2H5OH Reflux 6hrs N NH O H O H O H O R C H3

98) Shashikant Petal, (2013) Synthesis, antimicrobial and anti-tubercular

activity of some novel 1, 3, 4-oxadiazol-2-thiol derivatives.

Ar H O

+

N NH O

NH2 O NHN CH

3 Ar POCl3 Ar-COOH N O N N O Ar Ar NH3

(48)

99) Sadaf Jamal G et al.,(2011), Anti-microbial evaluation of oxadiazole derivatives. N N H NH2 O RCOOH POCl3 N O N N R

100) B.C. Revanasiddappa et al. (2010), reported synthesis of some novel 1, 3,

5-trisubstituted pyrazolines and biological evaluation of synthesized

compounds. O R + CH3 O R R O

H2N

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3.4 CONCLUSION

Based on literature survey, various in-silico approaches have been studied in

a detailed manner, which includes virtual screening like molecular docking studies,

docking with multiple targets, pharmacophore modeling, homology modeling

importance of insilico toxicity assessment and insilico ADME predictions.

In furtherance, a comprehensive survey based on the importance of the

heterocyclic in drug discovery was examined. The various synthetic strategies,

including the challenges involved in the synthesis of heterocyclic were studied.

In pharmacological aspects the importance of drug discovery against

tuberculosis was viewed. Further different types of critical targets available for

mycobacterium tuberculosis were studied. Finally different types of vitro and

in-vivo screening methods accessible for the anti-tuberculosis activity was studied.

Overall from the literature survey, concluded and framed the research

working order to identify some critical lead molecules against Mycobacterium

tuberculosis and their molecular docking studies synthesis characterization and

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CHAPTER 4

SCOPE AND PLAN OF WORK

4.1 Scope of the study:

The scope of the study directly focuses the study of ten scaffolds and design

and synthesis, characterization and biological evaluation for anti-tubercular activity

from these scaffolds. Design and Synthesis of selected novel heterocyclic molecules

such as “Chalcones”,“2, 4, 6 triaryl-1H-imidazole”, “Pyridine-4-Carbohydrazide” ,

“Oxadiazole”, “Pyrazolines” and “IsoNicotinoHydrazide” and some fused ring

systems like “3, 4-dihydropyrimidine-2(1H)-thione”,

2(1H)-one”, “thiazolo pyrimidin-3(5H)-one”, “Oxazolo Pyrimidin-3(5H)-one” and

their Characterization, acute toxicity study and biological evaluation including both

in-vitro and in-vivo activity against Mycobacterium tuberculosis.

4.2 Plan of work - Work Flow:

Based up on the review of literature, small heterocyclic analogues as

“Chalcones”, “2, 4, 6 triaryl-1H-imidazole”,

“Pyridine-4-Carbohydrazide” , “Oxadiazole”, “Pyrazolines” and

“IsoNicotinoHydrazide” and some fused ring systems like “3,

4-dihydropyrimidine-2(1H)-thione”, “3,4-dihydropyrimidine-

2(1H)-one”, “thiazolo pyrimidin-3(5H)-2(1H)-one”, “Oxazolo

Pyrimidin-3(5H)-one” were selected for computational design.

From an in-house chemical library comprising more than 2400

sketched molecules, preliminary docking will be conducted against

the pathophysiological target of Mycobacterium tuberculosis using

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Based on the preliminary docking results, top 100 molecules with

diverse heterocyclic nucleus will be chosen and further subjected to

an advanced docking against the patho physiological target of

Mycobacterium tuberculosis. In advanced docking ‘XP-extra

precision mode for the elimination of false positives by means of vast

sampling and advanced scoring, resulting in even higher enrichment.

Those 100 molecules will also be subjected to in-silico toxicity

assessment and in-silico Absorption, Distribution, Metabolism and

Excretion (ADME) prediction.

On the basis of XP mode docking results, silico toxicity data,

in-silico ADME data and synthetic feasibility, top 35 molecules will be

selected for synthesis.

Purification of the synthesized compounds will be carried out by

recrystallization, repeated recrystallization or column

chromatographic techniques to attain the expected purity.

The purity of the synthesized compounds will be confirmed by sharp

melting points and Thin Layer Chromatography (TLC).

Characterization of the synthesized compounds will be done by

spectral studies like GC-MASS Spectrometry, 13C-NMR

Spectroscopy, 1H-NMR spectroscopy, and single X-ray

crystallographic study.

All the compounds will be subjected to in-vitro anti-tubercular

activity.

Based up on the in-vitro results, synthesized compounds will be

further docked against multiple pathophysiological target enzymes of

Mycobacterium tuberculosis so as to validate the software and to

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and to find out the plausible mechanism by which the compounds

would have exhibited the activity.

All the synthesized compounds which exhibit promising in-vitro anti

mycobacterial activity will be subjected to acute toxicity studies to

find out the toxicity induced mortality and other behavioral changes.

Based up on the acute toxicity results, in-vitro anti-tubercular activity

rankings by the support of multiple molecular docking studies, top-3

compounds will be chosen and subjected to in-vivo anti-tubercular

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CHAPTER 5

IN-SILICO APPROACH

5.1 Materials

In-house chemical library containing more than 2400 molecules

based on heterocyclic nucleus such as “Chalcones”, “3,

4-dihydropyrimidine-2(1H)-thione”, “3,4-dihydropyrimidine-

2(1H)-one”, “thiazolo pyrimidin-3(5H)-2(1H)-one”, “Oxazolo

Pyrimidin-3(5H)-one”, “imidazole”, “Pyridine-4-Carbohydrazide” , “1,3,4

Oxadiazole”, and “ Pyrazol-1-yl (pyridin-4-yl) methanone”

“Nicotinohydrazide” was created by sketching the molecules using

chemdraw® , ultra, version 8.0, April 23, 2003 Cambridge soft

Cooperation , USA.

The target enzyme, “mtFab D, Malonyl CoA - acyl carrier protein

transacylase” an essential Malonyl-CoA: AcpMTransacylase

(MCAT). In Mycobacterium tuberculosis, it is one of the key

enzymes involved in the Lipid Biosynthesis FAS II for the production

of mycolic acids, which is critical for the survival and growth of

Mycobacterium tuberculosis. Mycolic acids form the pathogen’s

defensive layer. This target enzyme was selected from the in-silico

target identification pipeline for Mycobacterium tuberculosis which

comprises a total of 451high confidence targets. The crystal structure

of the enzyme was downloaded from the protein data bank. (“An

information Portal to Biological Macromolecular Structures”), (PDB

id – 2QC3).

Other Mycobacterium tuberculosis target enzymes used in the study

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1HZP and 2QNY), Arabinosylindolyl acetyl inositol synthase - Embc

(PDB id – 3PTY), UDP GALP Mutase Glf(PDB id – 1VOJ), and L,

D Transpeptidase 2 (PDB id – 3VAE)were also downloaded from

Protein Data Bank.

All the ligands were prepared by means of

“LigPrep®”v-2.5(Schrodinger®) software to generate the lowenergy 3D

conformers of the ligands.

The target protein was prepared using “protein preparation

wizard”from the workflows of “Maestro” v-9.3.515 (Schrodinger®)

platform.

The binding sites (Active sites) were analyzed by “Sitemap” v-2.6

(Schrodinger®) software.

Molecular docking was performed by “GLIDE®”V-5.8 (“Grid-Based

Ligand Docking with Energies”)(Schrodinger®) software.

In-silicoADME properties were predicted using “QikProp®” V-3.5

(Schrodinger®) software.

In-Silicotoxicity Assessment carried out using OSIRIS® Online Tool.

All the computational works expecting In-silico toxicity assessments

carried out by “Maestro” v-9.3.515 (Schrodinger®) platform.

5.2 Experimental

5.2.1 Molecular Docking

Ligand Preparation:

All the ligands from in-house chemical library were built using “Maestro

build panel”. They were prepared by means of “Lig Prep” v-2.5 (Schrodinger®)

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field gave the corresponding low energy 3d conformers of the ligands.” The ligand

preparation involves the following tasks.

Addition of Hydrogen atoms

Neutralization of charged groups, later generation of ionization and

tautomeric states with Epik.

Generation of stereo isomers, particularly if sterochemical

information is missing.

Generation of low-low energy ring conformations

Removal of any badly prepared structures.

Optimization of the geometrics.

As a whole, “ Lig Prep converts simple 2D structure to 3D structures by

including tautomeric, stereo chemical, and ionization variations, with energy

minimization and flexible filters to generate fully customized ligand libraries that are

optimized for additional computational analysis”.102

Protein preparation:

The crystal structure of target enzyme mtFabD (MCAT) from

Mycobacterium tuberculosis was downloaded from the Protein Data Bank (PDB id –

2QC3) and was prepared using “Protein Preparation Wizard” panel from the

workflows of Maestro.

In this course of action, the downloaded protein was processed in several

aspects so as to make the protein perfect for docking. Initially, the protein was

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chains and loops were also reoriented and corrected. Then all the water molecules

were removed with exception of water molecules which were co-ordinated to metal

atoms. Hydrogen atoms were added and the geometry of all the hetero groups was

corrected. Further optimization of the hydrogen bond network was carried out using

hydrogen bonds Assignment tool. Finally, with the default constraints (0.3 A° OF

RMSD and the OPLS_2005 force field) energy minimization was carried out using

Restrained Minimization tool.

Binding site analysis:

The active binding sites were searched using Sitemap Version 2.6

(Schrodinger®). It allocates numerical descriptors to evaluate the predicted binding

sites by a series of parameters such as size, tightness, hydrophobic/hydrophilic

character and possibilities of hydrogen bonding. These measurements help prioritize

possible binding sites.

In this task, the prepared protein was subjected to binding site analysis with

the default parameter settings to identify top-ranked potential receptor binding sites.

Based on the Site-Score, one receptor binding site was chosen for docking.

Receptor Grid Generation:

The prepared protein with its top ranked binding site was placed in the

workspace and the receptor grid generation was done by using the Receptor Grid

Generation panel. The grid was defined and represented by adjusting the size and

position of the active site so as to accommodate the ligands for docking using the

default box size (20×20×20A 3). Rotatable hydroxyl groups in the active site were

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Molecular docking:

Molecular docking is a process to find the best pose from the series of poses

of ligand binding to the active site of the protein using a scoring method. The

GLIDE software (version 5.8) provided by Schrodinger®, LLC suite offers three

docking procedures viz., High throughput virtual screening (HTVS), standard

precision (SP) docking and Extra precision (XP) docking.

Glide® docking:

Glide docking was executed by the ligand Docking panel. Primarily the

precision was set to Standard precision (SP) mode to rank the top hundred ligands

from two thousand four hundred in house library ligands, which involves diverse

heterocyclic molecules based on Chalcones containing[(furan-2-yl) moiety

and(4-hydroxy phenyl) moiety], ; “imidazole”, “Pyridine-4-Carbohydrazide” , “1,3,4

Oxadiazole”, and “Pyrazol-1-yl (pyridin-4-yl) methanone” and some fused ring

systems like “3, 4-dihydropyrimidine-2(1H)-thione”,

2(1H)-one”, “thiazolo pyrimidin-3(5H)-one”,and “Oxazolo Pyrimidin-3(5H)-one”.

For this, all the two thousand four hundred prepared ligands were generated as a

single LigPrep out file. The docking task was performed by specifying the receptor

grid base (in the receptor grid tool) and Lig-prep out file (in the ligands to be docked

tool). With the other default parameter settings the glide docking in Standard

precision (SP) Mode was performed. The best docking pose for every ligand was

visualized by importing pose viewer files after docking. Based up on the glide score,

top hundred molecules were selected.

Further, docking for those 100 molecules was performed with the same

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selected entries. Docking precision mode was set to extra precision (XP) mode.

While setting extra precision (XP) mode, writing XP descriptors information was

checked in the docking tool. The G-score and the energy minimized docking poses

for all the ligands were analyzed.

5.2.2 In-Silico toxicity Assessment

In-silico toxicity Assessment for those 100 molecules resulted from Standard

Precision (SP) mode were predicted by using OSIRIS®, a JAVA based online tool.

The tool predicts toxicity related parameters such as mutagenicity, tumorigenicity,

skin irritancy and the effects on reproduction. The prediction is based on the

fragment contribution group present in the structure of the molecule.

On assessing the OSIRIS® property Explorer, which is a JAVA applet,

allows us to draw chemical structures.

5.2.3 In-silico ADME predictions:

The ADMET properties of those 100 molecules were performed using

QikProp® program. The software program predicts the physically significant

descriptors and pharmaceutically relevant properties such as octanol/water log P, log

S for aqueous solubility, log BBB for blood /brain barrier, number of primary

metabolites, CNS activity, percentage human oral consumption in GI, log Khsa for

serum protein binding and log IC 50 for HERG K+ channel blockage, log Kp for

predicted skin permeability, Lipinski’s rule of five violations and Jorgenson’s rule of

three violations”. For this task, those top 100 LigPrep ligand molecules which was

resulted from Standard Precision (SP) mode docking, were selected from the Project

Table panel as selected entries and were given as a source in QikProp panel. The

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5.3 Results and Discussions

The energy minimized 3D structures of 2400 ligand molecules were docked

against “mtFab D” (Malonyl CoA - acyl carrier protein transacylase) (PDB ID-

2QC3) in a standard precision mode (SP). The docking score of all the ligand

molecules were found to be in the range of -3.85 to -8.48 kcal/mol. Based on the

docking score top 100 molecules with diverse heterocyclic nucleus were taken for

extra precision (XP) docking against the same target enzyme (PDB ID-2QC3)

The best docking pose of all the 100 ligand molecules were analyzed and

various XP descriptors were reviewed. The docking scores/ G-scores for 100 ligand

molecules in XP mode docking ranges from -4.6 to -8.48 kcal/mol.

In-Silico ADME property predictions of those 100 ligand molecules were

analyzed for any violations in the range of critical parameters such as “CNS activity

(CNS)”, total solvent accessible surface area (SASA), octanol/ water partition

co-efficient (QPlogPo/w), IC50 value for blockage of HERG K+ channels

(QPlogHERG), brain blood partition co-efficient (QPlogBB), binding human serum

albumin Lipinski’s rule of five “(Rule of Five)”. Some of the ligand molecules

showed violations, while the majority of the ligand molecules were found to be

within the range of recommended values for 95% of known drugs.

The in-silico toxicity assessment of those 100 ligand molecules gave better

insights in the selection of the molecules for the synthesis. More than one third of

the ligand molecules were found to be mutagenic in in-silico toxicity assessment.

Some ligand molecules were found to have mutagenic and tumeriogenic risk alerts,

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and tumorigenic, skin irritancy and reproductive effect which were considered to be

highly toxic based upon in-silico approach.

All those 100 ligand molecules were analyzed for their synthetic feasibility

based up on their synthetic pathway, chemicals and reagents, green techniques and

compound stability.

On considerations from the above in-silico approaches (molecular docking,

in-silico toxicity assessment, in-silico ADME predictions) and synthetic feasibility,

35 ligand molecules with diverse heterocyclic nucleus were selected for the

synthesis.

The ligand molecules selected for the synthesis are

Chalcones containing [(furan-2-yl) moiety and (4-hydroxy phenyl)

moiety],

Imidazole derivatives

Pyridine-4-Carbohydrazide derivatives

1,3,4 Oxadiazole derivatives

Pyrazol-1-yl (pyridin-4-yl) methanone derivatives

Dihydropyrimidine derivatives

Thiazolo pyrimidin-3(5H)-one, and Oxazolo Pyrimidin-3(5H)-one

derivatives

The 2D and energy minimized 3D structures of the selected molecules are

shown in Table 1. All the Synthesized analogues were recorded as alphabetically or

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U, NA, ATRIM, KSBA, HTPC, JA, IAF, B1B2, AFDC, PW, 2UTHIOU,

2ATHIO, 2UUREA, 2CUREA, 2CTHIOU, 2I/AF UREA, RG 6, RG 7, INHAF,

INHAP, SOS1, SOS2, NHD 1, NHD2, NHD4, CSP, 3rd PDT etc.

Table 1: 2D and energy minimized 3D structures of the selected molecules

Sl. No

Ligand code

2D Structure 3D Structure

1 C

O O

2 F

O H

O

O H

3 G/VA

O O

O

(62)

4 H/LA

O

O

5

M4F O

O

O CH3

O

6 Q O

O

O H

OH

OH

7 AFDC O

O

Cl

Cl

8 U

O

OH

(63)

9 X

O

O

Br

10 Y

O O

S

11 NA

OH

O

O

12 ATRIM O O O O O C H3 CH3 CH3 13 B1B2 O H O O O

(64)

14 KSBA

OH

O O

C

H3 CH3

15

IAF O

O O C H3 O CH3 16 2C THIOU REA O NH N H S

Figure

Table No Title of Table

Table No.

Title of Table . View in document p.8
Figure No. Title of the figures

Figure No.

Title of the figures . View in document p.10
Figure 1: Mycobacterial infection203

Figure 1.

Mycobacterial infection203. View in document p.15
Figure 3: Cell wall of mycobacterium tuberculosis  (source: www.tuberculosis.com, ref 2)

Figure 3.

Cell wall of mycobacterium tuberculosis source www tuberculosis com ref 2 . View in document p.16
Figure: 4: mycobacterium tuberculosis H37Rv218
Figure 4 mycobacterium tuberculosis H37Rv218. View in document p.17
Figure 5: Virulence life cycle of mycobacterium tuberculosis

Figure 5.

Virulence life cycle of mycobacterium tuberculosis . View in document p.18
Figure 6: Estimated TB incidence rates, 2013

Figure 6.

Estimated TB incidence rates 2013 . View in document p.19
Figure 8: Patho physiology of Tuberculosis: (Source: www.google.com)

Figure 8.

Patho physiology of Tuberculosis Source www google com . View in document p.20
Figure 7: The pathogesis  of  tuberculosis tuberculosis  standards:

Figure 7.

The pathogesis of tuberculosis tuberculosis standards . View in document p.20
Figure 9: First-Line Treatment of TB for Drug-Sensitive TB:  (Source: www.google.com)

Figure 9.

First Line Treatment of TB for Drug Sensitive TB Source www google com . View in document p.21
Figure 11: MDR TB Treatments: (Source: www.google.com)

Figure 11.

MDR TB Treatments Source www google com . View in document p.22
Figure 10: Tuberculosis-drugs-and-actions: (Source: www.google.com)

Figure 10.

Tuberculosis drugs and actions Source www google com . View in document p.22
Figure 12: The traditional regimen for TB: (Source: www.google.com)

Figure 12.

The traditional regimen for TB Source www google com . View in document p.23
Figure 13: Mechanism of action of current TB Drugs:

Figure 13.

Mechanism of action of current TB Drugs . View in document p.23
Table 1: 2D and energy minimized 3D structures of the selected molecules
Table 1 2D and energy minimized 3D structures of the selected molecules. View in document p.61
Figure 14: Unprocessed 3D structure of prepared protein Fab D (PDB ID-2QC3)

Figure 14.

Unprocessed 3D structure of prepared protein Fab D PDB ID 2QC3 . View in document p.70
Figure 16: Docked possess of all the 35 ligands at the active site of target enzyme Fab D

Figure 16.

Docked possess of all the 35 ligands at the active site of target enzyme Fab D . View in document p.71
Figure 15: Energy minimized 3D structure of prepared protein Fab D (PDB ID-2QC3)

Figure 15.

Energy minimized 3D structure of prepared protein Fab D PDB ID 2QC3 . View in document p.71
Table 2: Docking results of the selected 35 analogues

Table 2.

Docking results of the selected 35 analogues . View in document p.72
Table 3: Residue interaction pattern for the synthesized compounds against target enzyme Fab D

Table 3.

Residue interaction pattern for the synthesized compounds against target enzyme Fab D . View in document p.75
Figure 17: Ligand interaction diagrams of all the 35 ligand molecules

Figure 17.

Ligand interaction diagrams of all the 35 ligand molecules . View in document p.96
Table 5: In-silico toxicity assessment results of the 35 ligand molecules

Table 5.

In silico toxicity assessment results of the 35 ligand molecules . View in document p.100
Figure 18: Screen shot of in-silico toxicity assessment results

Figure 18.

Screen shot of in silico toxicity assessment results . View in document p.112
Table 6: List of synthesized compounds with their IUPAC NAME:

Table 6.

List of synthesized compounds with their IUPAC NAME . View in document p.123
Table 7: Molecular Weight, Molecular Formula, Color, Solubility, Melting

Table 7.

Molecular Weight Molecular Formula Color Solubility Melting . View in document p.130
Table 8: TLC Profile of the Synthesized Compounds:

Table 8.

TLC Profile of the Synthesized Compounds . View in document p.134
Figure 19: ORTEP Diagram Of Compound “C”

Figure 19.

ORTEP Diagram Of Compound C . View in document p.140
Table 10: Ranking of the compounds based on the in-vitro anti-mycobacterial activity

Table 10.

Ranking of the compounds based on the in vitro anti mycobacterial activity . View in document p.149
Table 11: Lung CFU values in animals treated with test samples and

Table 11.

Lung CFU values in animals treated with test samples and . View in document p.156
Figure 20: Lung CFU in animals treated with test samples and standard controls, N = 3, error bars represent standard deviation

Figure 20.

Lung CFU in animals treated with test samples and standard controls N 3 error bars represent standard deviation . View in document p.157

References

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