DESIGN, SYNTHESIS, CHARACTERIZATION AND
BIOLOGICAL EVALUATION OF NOVEL HETEROCYCLIC
DERIVATIVES AS ANTI-TUBUERCULAR AGENTS
THE TAMILNADU Dr. M.G.R. MEDICAL UNIVERSITY
In partial fulfilment for the award of the degree of
DOCTOR OF PHILOSOPHY IN
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.
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
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
It is privileged to thank Dr. Muthuswamy, Mpharm., Ph.D., Collage of
Pharmacy, Madras Medical Collage, Chennai for his valuable suggestions and
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,
I am also indebted to Issac Christian Moses., M.D. Dean, Madras Medical
College, Chennai, for providing the necessary requisites and facilities for my
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
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
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
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
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
LIST OF TABLES
Table No Title of Table Page No
1 2D and energy minimized 3d structures of the selected
2 Docking results of the selected 35 analogues 59
3 Residue interaction pattern for the synthesized
compounds against target enzyme Fab D
4 In-silico ADME properties of the selected 35 ligand
5 In-silico toxicity assessment result of the 35 ligand
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
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
11 Lung CFU lung values in animals treated with test
samples and standard controls.
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
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
15 Energy minimized 3D structure of prepared protein Fab
D (PDB ID-2QC3)
16 Docked possess of all the 35 ligands at the active site of
target enzyme Fab D
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
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
CHAPTER TITLE PAGE. No
1 INTRODUCTION 1
` 1.1. History of tuberculosis 2
1.2. Tuberculosis Epidemiology : Prevalence ,
1.3. Pathogenesis and immune response: the
interactions between MTB and the host cell.
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
3.2 Literature review based on In-silico approach 25
` 3.3 Literature review based on Chemistry 26
3.4 Conclusion 36
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
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
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
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
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
Figure2: Mycobacterium tuberculosis208
Synonym: Tubercle bacillus Koch 1882.
Mycobacterial cell wall:
Genome: The genome sequence of strain of Mtb, H37Rv has been determined and
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
1.2 Tuberculosis Epidemiology: prevalence, airborne transmission
MTB or TB is epidemic in any developing countries. Because of the
Figure 5: Virulence life cycle of mycobacterium tuberculosis
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
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
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
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
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.
Figure 10: Tuberculosis-drugs-and-actions: (Source: www.google.com)
Figure 12: The traditional regimen for TB: (Source: www.google.com)
Figure 13: Mechanism of action of current TB Drugs:
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
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
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
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
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
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
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
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
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
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
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
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
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
AIM AND OBJECTIVES
The aim of present research is to develop novel Anti-tubercular molecules,
which inhibit target enzymes, Fab D (Malonyl CoA - acyl carrier protein
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
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
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
3.1 Literature review based on Pharmacology, Epidemiology, Prevalence
and WHO data:
1. World Health Organization. ”The Sixteenth Global Report on
2. Takayama K et al., 2(2005) explained about on reestablishment of
3. Zhang Y., 3(2005) evaluated on emergence of drug resistance of
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
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
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
10. Roberts CA, et al, 13(2003) a global view on a re-emerging disease
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
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
27. Ernst JD37 (2012) Reviewed about The immunological life cycle of
28. Shaler CR et al38 (2013) Discussed on contribution of the granuloma
to the dissemination, persistence and transmission of Mycobacterium
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
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
33. Van Crevel R, et al 44 (2002) reviewed on Innate immunity to
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.
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
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
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
73) Radiszewskiet al.220detailed condensation reaction of diketones
MW at 240W
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
77) Bharadwajet al225(2010) the condensation of different under microwave
oven. The structures reported in better yield as compared to conventional
78) Raghavendraet al 226 (2011) a series of imidazole quinoline analogs were
synthesized by condensation of substituted imidazoleand substituted
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
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
81) Nalageet al221 (2010) described an efficient procedure imidazole.
84) Ermolat’ev et al 231 (2006), A green chemistry for the synthesis of
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
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
NH N H
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
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 N O
95) Sajeevan Gaikwad et al., (2012) reported new thiazolidinone derivatives
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.
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
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
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
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,
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
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
All the compounds will be subjected to in-vitro anti-tubercular
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
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
In-house chemical library containing more than 2400 molecules
based on heterocyclic nucleus such as “Chalcones”, “3,
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
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®)
The binding sites (Active sites) were analyzed by “Sitemap” v-2.6
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
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.
5.2.1 Molecular Docking
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®)
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
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
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
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 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
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
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,
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
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
The ligand molecules selected for the synthesis are
Chalcones containing [(furan-2-yl) moiety and (4-hydroxy phenyl)
1,3,4 Oxadiazole derivatives
Pyrazol-1-yl (pyridin-4-yl) methanone derivatives
Thiazolo pyrimidin-3(5H)-one, and Oxazolo Pyrimidin-3(5H)-one
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
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
2D Structure 3D Structure
6 Q O
7 AFDC O
12 ATRIM O O O O O C H3 CH3 CH3 13 B1B2 O H O O O
O O C H3 O CH3 16 2C THIOU REA O NH N H S