CHAPTER II

LITERATURE REVIEW

Docking is now routine in virtual screening or lead optimization for drug

screening and design. The number of papers related to docking has dramatically
increased over the past decade. However, there are many issues to consider when
undertaking a docking study. Frequent problems or issues arise, such as the wrong
binding site of the target protein, screening using an unsuitable small-molecule
database, the choice of docking pose, high dock score but failed in molecular dynamics
(MD) simulation, and lack of clarity over whether the compound is an inhibitor or
agonist. These problems should be cause for caution and concern before performing
docking. Some papers show comprehensive biochemistry experiments but only a simple
docking figure. This procedure presents some evidence to show that the docking might
be questionable, despite a high score. In some cases, the accuracy of docking can even
change from 0% to 92.66%.

2.1 DOCKING FOR STRUCTURE-BASED DRUG DESIGN

Since its beginnings in the 1960s, docking, along with the tremendous
developments in physics, chemistry, informational technology, biochemistry, and
computers, has become a powerful tool and an essential technique, not only in drug
screening but also in protein–protein interactions and the behaviour of nanomaterials.
The current field of computer-aided drug design (CADD) is dominated by technologies
used to dock small molecules into macromolecules, particularly protein targets, and its
use is increasing year by year. In modern CADD, structure-based drug design is
essential (Warren, et al., 2012; Merz, et al., 2010; Zheng, et al., 2013; Pei, et al., 2014)
and most big pharmaceutical companies have this department. Many commercial drugs
are directly designed from CADD method (Sliwoski, et al., 2014). Protein–ligand or
protein–protein docking is a computational technology to predict the orientation of a
ligand when it is bound to a protein receptor or enzyme. In most cases, one can choose
the best ‘binding affinity’ to be the potent ligand for further biochemistry experiments
and development. Because docking is simple and the equipment requirement is low (it
even works well on a personal computer), docking related papers have sharply increased
over the past decade. It can provide a critical survey of the field, pointing out the

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strengths and weaknesses of the current family of docking protocols. Careful evaluation
shows that accuracy is a major problem with docking studies, because if the docking is
not approached with precision then these papers will be of little value (Friesner, et al.,
2004; Hou, et al., 2013; Lee and Sun, 2007). Questionable docking results can be found,
even in high-profile journals. There are frequent problems such as an inaccurate binding
site of the target protein, screening using an unsuitable small-molecule database, the
choice of docking pose, high dock score (binding affinity) but failed in MD simulation,
lack of clarity over whether the compound is an inhibitor or agonist, or the docking
results are inconsistent with bioassays. Although some papers declare docking results
with a high accuracy by comparing the ligand pose before and after docking, here I
present some evidence that the docking might be still questionable. In some cases the
accuracy of docking can even change from 0% to 92.66%.

2.2 DOCKING ALGORITHMS AND PROGRAMS

The original concept of docking comes from the concept of ‘lock and key’ of
rational drug design, but the precise algorithms used to fit the ‘key’ (the ligand) into the
‘lock’ (the receptor protein) vary across programs. The latest developments in docking
programs, the docking web server, screens software, and screen web server are
available, from which we can see that the number of new algorithms has been
increasing in recent years and that the most commonly used docking programs are
Autodock (Goodsell and Olson, 1990) and GOLD (Jones, et al., 1995). This does not
mean that Autodock or GOLD are more accurate than other docking programs, they are
merely more popular and well known. Nowadays, a new algorithm to predict protein
structure for docking, Rosetta (http://boinc.bakerlab.org/), has also been highly
evaluated. Although they vary, the different algorithms of each docking program must
strike a balance between speed and accuracy. The algorithms for docking also vary by
differences in scoring functions. Binding affinity is usually considered to be a priority in
the evaluation of the best candidate for virtual screening. There are several docking
programs for a user to choose from based on his or her particular requirements. At
present, docking algorithms emphasize different aspects of structure-based drug design
(SBDD), such as fragment-based drug design(Chen and Shoichet, 2009; Schmidt, et al.,
2013; Pérot, et al., 2010), flexible docking (Rosenfeld, et al., 1995), docking in water,
solvation, and specific pH (Tsai, et al., 2011; Chang, et al., 2011). For example, if we

21
need to screen more than 10000 compound from a database, then flexible docking may
be not a good choice unless we have a very powerful and high-speed computer. By
contrast, if we need to dock only a few compounds in the specific protein binding site,
at a specific pH, water, or solvation, then the flexible docking program might be a good
choice. In our experience, translating drug discovery from CADD to clinical trials is
difficult. For example, some top potent hits screened by docking from the TCM
database failed in the final bioactivity test (Tou and Chen, 2014; Li, et al., 2014; Tou, et
al., 2013; Yang, et al., 2011; Chang, et al., 2011; Tsou, et al., 2012; Chen, et al.,
2012). However, if a hit comes first from a bioassay – for example, if it was screened
from high-throughput screening (HTS) by ‘automation-friendly’ robotics, data
processing, and control software in vitro – then it is easier to move to in vivo.
Explaining the underlying biological mechanism is easier and more reasonable (Tou, et
al., 2013). However, given the rise of docking studies, it is important to conduct them
with care. The following considers some important considerations that developers
should keep in mind when undertaking docking analyses.

Program Name Novel features

Docking software

Autodock Freeopen-source EA-based docking software. Flexible ligand.

Flexible protein side chains. Maintained by the Molecular Graphics
Laboratory, Scripps Research Institute, La Jolla.

DOCK Anchor-and-grow based docking program. Free for academic use.

Flexible ligand. Flexible protein. Maintained by the Soichet group
at the University of California San Francisco (UCSF).

GOLD GA-based docking program. Flexible ligand. Partial flexibility for

protein. Product of collaboration between the University of
Sheffield, GlaxoSmithKline, and the Cambridge Crystallographic
Data Centre (CCDC).

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Glide Exhaustive search-based docking program. Exists in extra precision
(XP), standard precision (SP) and virtual high-throughput
screening modes. Ligand and protein flexible. Provided by
Schro¨dinger.

SCIGRESS Desktop/server molecular modelling software suite employing

linear scaling semi-empirical quantum methods for protein
optimization and ligand docking. Developed and distributed by
Fujitsu.

GlamDock Docking program based on a Monte-Carlo with minimization

(basin-hopping) search in a hybrid interaction matching/internal
coordinate search space. Part of the Chilsuite. Open for general
research.

GEMDOCK Program for computing a ligand conformation and orientation

(Generic evolutionary relative to the active site of target protein.

method for molecular

docking)

iGEMDOCK Graphic environment for the docking, virtual screening, and post-
screening analysis. Free for non-commercial researches. For
Windows and Linux.

HomDock Program for similarity-based docking, based on a combination of

the ligand-based GMA molecular Alignment tool and the docking
tool GlamDock. Part of the Chilsuite. Open for general research.

ICM Docking program based on pseudo-Brownian sampling and local

minimization. Ligand and protein flexible. Provided by MolSoft.

FlexX, Flex-Ensemble Incremental build-based docking program. Flexible ligand. Protein

(FlexE) flexibility through ensemble of protein structure. Provided by
BioSolveIT.

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Fleksy Program for flexible and induced fit docking using receptor
ensemble (constructed using backbone-dependent rotamer library)
to describe protein flexibility. Provided by the Centre for
Molecular and Biomolecular Informatics, Radboud University,
Nijmegen

FITTED Suite of programs to dock flexible ligands into flexible proteins.

(Flexibility induced This software relies on a genetic algorithm to account for flexibility
of the two molecules and location of water molecules, and on a
through targeted
novel application of a switching function to retain or displace water
evolutionary
molecules and to form potential covalent bonds (covalent docking)
description)
with the protein side chains. Part of the Molecular FORECASTER
package and FITTED Suite. Free for an academic site license
(excluding cluster).

VLifeDock Multiple approaches for protein–ligand docking. Provides three

docking approaches: grid-based docking, GA docking, and VLife’s
own GRIP docking program. Several scoring functions can be
used:

PLP score, XC score, and Steric + Electrostatic score. Available for

Linux and Windows. Provided by VLife. (www.vlifesciences.com)

ParaDockS Free open-source program for docking small, drug-like molecules

(Parallel docking suite) to a rigid receptor employing either the knowledge-based potential
PMF04 or the empirical energy function p-Score.

MolegroVirtual Docker Protein–ligand docking program with support for displaceable

waters, induced-fit-docking, user- defined constraints, molecular
alignment, ligand-based screening, and KNIME workflow
integration. Provided by Molegro (http://www.scientific software
solutions.com/product.php?productid=17625)

eHiTS Exhaustive search-based docking program. Provided by

SimBioSys.

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DAIM–SEED–FFLD Free open-source fragment-based docking suite. The docking is
realized in three steps. DAIM (decomposition and identification of
molecules) decomposes the molecules into molecular fragments
that are docked using SEED (program for docking libraries of
fragments with salvation energy evaluation). Finally, the molecules
are reconstructed ‘in situ’ from the docked fragments using the
FFLD program (program for fragment-based flexible ligand
docking). Developed and maintained by the Computational
Structural Biology of ETH, Zurich, Switzerland.

Autodock Vina MC-based docking software. Free for academic use. Flexible
ligand. Flexible protein side chains. Maintained by the Molecular
Graphics Laboratory, The Scripps Research Institute, La Jolla.

VinaMPI Massively parallel message-passing interface (MPI) program based

on the multi-threaded virtual docking program Autodock Vina.
Free and open-source. Provided by the University of Tennessee.

FlipDock GA-based docking program using FlexTree data structures to

represent a protein–ligand complex. Free for academic use.
Flexible ligand. Flexible protein. Developed by the Department of
Molecular Biology at the Scripps Research Institute, La Jolla.

FRED FRED performs a systematic, exhaustive, non-stochastic

examination of all possible poses within the protein active site,
filters for shape complementarity and pharmacophore features
before selecting and optimizing poses using the Chemgauss4
scoring function. Provided by Open Eye scientific software.

HYBRID Docking program similar to FRED, except that it uses the chemical
Gaussian overlay (CGO) ligand based scoring function. Provided
by Open Eye scientific software.

POSIT Ligand-guided pose prediction. POSIT uses bound ligand

information to improve pose prediction. Using a combination of
several approaches, including structure generation, shape

25
 alignment, and flexible fitting, it produces a predicted pose whose
accuracy depends on similarity measures to known ligand poses.
As such, it produces a reliability estimate for each predicted pose.
In addition, if provided with a selection of receptors from a
crystallographic series, POSIT will automatically determine which
receptor is best suited for pose prediction. Provided by Open Eye
scientific software.

Rosetta Ligand Monte Carlo minimization procedure in which the rigid body
position and orientation of the small molecule and the protein side
chain conformations are optimized simultaneously. Free for
academic and non-profit users.

Surflex-Dock Docking program based on an idealized active site ligand (a

protomol), used as a target to generate putative poses of molecules
or molecular fragments, which are scored using the Hammer head
scoring function. Distributed by Tripos.

CDocker CHARMm-based docking program. Random ligand conformations

are generated by MD and the positions of the ligands are optimized
in the binding site using rigid body rotations followed by simulated
annealing. Provided by Accelrys.

LigandFit CHARMm-based docking program. Ligand conformations

generated using Monte Carlo techniques are initially docked into
an active site based on shape, followed by further CHARM
minimization. Provided by Accelrys.

MOE Suite of medicinal chemistry tools including ligand–receptor

docking, protein/ligand interaction diagrams, contact statistics,
electrostatic and interaction maps, LigX (ligand optimization
inpocket), ligand and structure-based scaffold replacement,
multiple molecule flexible alignment, conformation generation,
analysis, and clustering, active site detection and visualization,
multi-fragment search, ligand- and structure-based query editor,

26
 high-throughput conformation generation, pharmacophore search.
Distributed by Chemical Computing Group.

LeadFinder Program for molecular docking, virtual screening, and quantitative

evaluation of ligand binding and biological activity. Distributed by
Moltech. for Windows and linux.

YASARA Structure Adds support for small-molecule docking to YASARA

View/Model/Dynamics using Autodock and Fleksy. provided by
YASARA (http://www.yasara.org/).

GalaxyDock Protein–ligand docking program that allows flexibility of pre-

selected side chains of ligand. Developed by the Computational
Biology Lab, Department of Chemistry, Seoul National University.

MS-Dock Free multiple-conformation generator and rigid docking protocol

for multi-step virtual ligand screening.

FINDSITE-LHM Homology modelling approach to flexible ligand docking. It uses a

collection of common molecule substructures derived from
evolutionarily related templates as the reference compounds in
similarity-based ligand-binding pose prediction. It also provides a
simple scoring function to rank the docked compounds. Freely
available to all academic users and not-for-profit institutions.
Provided by the Skolnick Research Group.

BetaDock Molecular docking simulation software based on the theory of

Beta-complex.

ADAM Automated docking tool. Can be used for vHTS. Distributed by

IMMD (http://www.immd.co.jp/en/product_2.html).

hint! (Hydropathic Estimates LogP for modeled molecules or data files; numerically
interactions). and graphically evaluates binding of drugs or inhibitors into protein
structures and scores DOCK orientations, constructs hydropathic
(LOCK and KEY) complementarity maps that can be used to
predict a substrate from a known receptor or protein structure or to

27
 propose the hydropathic structure from known agonists or
antagonists, and evaluates/predicts the effects of site-directed
mutagenesis on protein structure and stability.

DockVision Docking package including Monte Carlo, genetic algorithm, and

database screening docking algorithms.

PLANTS Docking algorithm based on a class of stochastic optimization

(Protein-ligand ANT algorithms termed ant colony optimization (ACO). In the case of
protein–ligand docking, an artificial ant colony is employed to find
system)
a minimum energy conformation of the ligand in the binding site.
These ants are used to mimic the behaviour of real ants and mark
low-energy ligand conformations with pheromone trails. The
artificial pheromone trail information is modified in subsequent
iterations to generate low-energy conformations with a higher
probability. Developed by Konstanz University.

EADock Hybrid evolutionary docking algorithm with two fitness functions,

in combination with a sophisticated management of the diversity.
EADock is interfaced with the CHARMM package for energy
calculations and coordinate handling.

EUDOC Program for the identification of drug interaction sites in

macromolecules and drug leads from chemical databases.

FLOG Rigid body docking program using databases of pre-generated

conformations. Developed by the Merck research laboratories.

Hammerhead Automatic, fast fragment-based docking procedure for flexible

ligands, with an empirically tuned scoring function and an
automatic method for identifying and characterizing the binding
site on a protein.

ISE-Dock Docking program based on the iterative stochastic elimination

(ISE) algorithm.

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ASEDock Docking program based on a shape-similarity assessment between
a concave portion (i.e., concavity) on a protein and the ligand.
Developed by Yoka Systems.

HADDOCK An approach that makes use of biochemical and/or biophysical

(High ambiguity interaction data such as chemical shift perturbation data resulting
from NMR titration experiments, mutagenesis data or
driven
bioinformatic predictions. First developed from protein–protein
biomolecular docking)
docking, it can also be applied to protein–ligand docking.
Developed and maintained by the Bijvoet Center for Biomolecular
Research, The Netherlands.

Computer-aided drug- PyMOL plugins provide a graphical user interface incorporating

design platform individual academic packages designed for protein preparation

using PyMOL (AMBER package and Reduce), molecular mechanics applications

(AMBER package), and docking and scoring (AutoDock Vina and
SLIDE).

Autodock Vina plugin Allows defining binding sites and export to Autodock and VINA
for PyMOL input files, performing receptor and ligand preparation
automatically, starting docking runs with Autodock or VINA from
within the plugin, viewing grid maps generated by autogrid in
PyMOL, handling multiple ligands and setting up virtual
screenings, and setting up docking runs with flexible side chains.

GriDock Virtual screening front-end for AutoDock 4. GriDock was designed

to perform the molecular dockings of a large number of ligands
stored in a single database (SDF or Zip format) in the lowest
possible time. It takes full advantage of all local and remote CPUs
through the MPICH2 technology, balancing the computational load
between processors/grid nodes. Provided by the Drug Design
Laboratory of the University of Milano.

29
DockoMatic GUI application that is intended to ease and automate the creation
and management of Auto Dock jobs for high-throughput screening
of ligand/receptor interactions.

BDT Graphic front-end application which controls the conditions of

Auto Grid and Auto Dock runs. Maintained by the University at
Rovira i Virgili.

2.3 DOCKING STUDIES RELATED TO THIS WORK

Ulahannan, et al., (2015), revealed that FT-IR and FT-Raman spectra of 2-[(E)-
2-phenylethenyl] quinoline-5-carboxylic acid were recorded and obtained and analyzed.
The vibrational wave numbers were computed using DFT quantum chemical
calculations. The geometrical parameters (SDD) of the title compound are in agreement
with that of similar derivatives. Stability of the molecule arising from the hyper
conjugative interactions, charge delocalization has been analyzed using natural bond
orbital analysis. From the natural and Mulliken charges, it can be concluded that
electrophilic substitution of the quinoline scaffold is more preferred than nucleophilic
substitution. From the MEP map it is evident that the negative regions are mainly
localized over the carbonyl group and are possible sites for electrophilic attack. The title
compound forms a stable complex with PknB as is evident from the binding affinity
values and the molecular docking study suggests that the compound might exhibit
inhibitory activity against PknB.
Loza Mejía and Salazar (2015), showed that the triterpenes and sterols are good
candidates for the development of anti-inflammatory drugs and use in chemoprevention
or chemotherapy of cancer via the interaction with therapeutic targets related to
inflammation, such as COX-1 and -2; LOX-5; MPO, PLA2 and i-NOS. In this study,
we use molecular docking to evaluate the potential binding of a database of selected
sterol and triterpenoid compounds with several skeletons against enzymes related to
inflammation to propose structural requirements beneficial for anti-inflammatory
activity that can be used for the design of more potent and selective anti-inflammatory
and antitumor drugs. Our results suggest that the substitution pattern is important and

30
that there is an important relationship between the class of sterol or triterpenoid skeleton
and enzyme binding.
Dipankar Das, et al., (2015), reported that the 4-(Z)-((2-Hydroxy-4-oxopent-2-
en-3-yl)diazenyl)-N-(5-methylisoxazol-3-yl)benzene sulfonamide (HL) and its
nickel(II) complex [Ni(L)2(H2O)4] have been characterized by spectroscopic and single
crystal X-ray diffraction measurements. Time dependent DFT computations have been
used to explain the electronic spectra of the compounds. The interaction of CT DNA
with [Ni(L)2(H2O)4] (Kb, 12.20 x 105M-1) is stronger than with HL (Kb, 6.09 x 105M-1).
The antimicrobial activity of HL and the Ni(II) complex has been examined against
Bacillus subtilis (ATCC 6633; IC50 : 63.72 µg/ml (HL) and 81.49 µg/ml [Ni(L)2(H2O)4]
and Escherichia coli (ATCC 8739; IC50: 77.25 µg/ml (HL) and 78.28 µg/ml
[Ni(L)2(H2O)4]. The in silico test of HL with DHPS protein from E. coli helps in
understanding the drug metabolism and has explained the drug–molecule interaction.
Abdelkader and Rania (2015), revealed that formulating hydrophobic drugs in
micro emulsions starts with screening the solubility of the active pharmaceutical
ingredients in different oils and thereby selecting the best candidate according to its
solubilising power. We hypothesise that in silico methods such as molecular dynamics
to simulate the oils domains together with docking of the investigated drug(s) on these
simulated domains can offer extremely valuable tools saving researches long
experimentation time in the laboratories and incalculable efforts exerted in developing
sensitive and accurate methods of analysing drugs in oils.
Joshi, et al., (2016), reported here the synthesis, antibacterial and antitubercular
evaluation of 61 novel pyrrolyl derivatives bearing pyrazoline, isoxazole and phenyl
thiourea moieties. Molecular docking was carried out on enoyl ACP reductase from M.
tuberculsosis using Surflex-Dock, which is one of the key enzymes involved in type II
fatty acid biosynthetic pathway of M. tuberculosis, an attractive target for designing
novel antitubercular agents. Docking analysis of the crystal structure of ENR performed
using Surflex-Dock in Sybyl-X 2.0 software indicates the occupation of substituted
pyrrolyl derivatives into hydrophobic pocket of InhA enzyme. Compounds 9b and 9d
exhibited the highest antitubercular activity almost close to isoniazid (0.4 µg/mL) with a
MIC value of 0.8 µg/mL. All other compounds showed the good activity with a MIC
value of 6.25-100 µg/mL. The compounds were further tested for mammalian cell

31
toxicity using human lung cancer cell-line (A549) and were nontoxic. Some compounds
exhibited inhibition activities against InhA.
Sashidhara, et al., (2015), reported that the search for safe and efficacious
antifilarials, a series of novel chalcone-benzothiazole hybrids have been synthesized and
evaluated for their Brugia malayi thymidylate kinase (BmTMK) enzyme inhibition
activity. Their selectivity towards BmTMK was studied and compared to the human
TMK (HsTMK) by an in silico method. Out of seventeen derivatives, compounds 34
and 42 showed higher interactions with the BmTMK active site. MolDock docking
model revealed the interactions of these two derivatives and the results corroborated
well with their in vitro antifilarial activities. This study suggests that these hybrids are
selective towards the BmTMK enzyme and may serve as potential therapeutic agents
against filariasis.
Noorullah, et al., (2015), had reported the six amino acid derived N-
glycoconjugates of D-glucose were synthesized, characterized and tested for
antibacterial activity against G(+)ve (Bacillus cereus) as well as G(-)ve µ(Escherichia
coli and Klebsiella pneumoniae) bacterial strains. All the tested compounds exhibited
moderate to good antibacterial activity against these bacterial strains. The results were
compared with the antibacterial activity of standard drug Chloramphenicol, where
results of A5 (Tryptophan derived glycoconjugates) against Escherichia coli and A4
(Isoleucine derived glycoconjugates) against Klebsiella pneumoniae bacterial strains are
comparable with the standard drug molecule. In silico docking studies were also
performed in order to understand the mode of action and binding interactions of these
molecules. The docking studies revealed that, occupation of compound A5 at the ATP
binding site of subunit GyrB (DNA gyrase, PDB ID: 3TTZ) via hydrophobic and
hydrogen bonding interactions may be the reason for its significant in vitro antibacterial
activity.
Zawawi, et al., (2015), has showed the Biscoumarin analogs 1–18 have been
synthesized, characterized by EI-MS and 1H NMR and evaluated for g-glucosidase
inhibitory potential. All compounds showed variety of g-glucosidase inhibitory potential
ranging in between 13.5 ± 0.39 and 104.62 ± 0.3 µM when compared with standard
acarbose having IC50 value 774.5 ± 1.94 µM. The binding interactions of the most
active analogs were confirmed through molecular docking. The compounds showed

32
very good interactions with enzyme. All synthesized compounds 1–18 are new. Our
synthesized compounds can further be studied to developed lead compounds.
Shruthy and Shakkeela (2014), revealed that the major objective of present study
was to synthesize a series of substituted schiff base derivatives of 4-phenyl thiazoles
designed as anticancer and anti-inflammatory agents using computational methods. The
thiazole derivatives for synthesis were selected based on docking studies performed on
active site of protein tyrosine kinase (PDB:1T46) for anticancer activity and protein
kinase (PDB:3DPK) for anti-inflammatory activity using Schrödinger 9.3 software.
Molecules with better docking score was subjected to analysis for cytotoxic activity by
in vitro MTT assay on cervical cancer HeLa cell lines and anti-inflammatory activity.
Among the five thiazole derivatives, CSB2: (4-(4-Methoxyphenyl)-n-3-phenylprop-2-
en-1-ylidene]-1,3-thiazol-2-amine) was found to have highest docking score and the
same exhibited maximum in vitro cytotoxic and anti-inflammatory activity. Thiazole
schiff bases derivatives showed good anti-inflammatory and cytotoxic activity as
predicted using molecular docking on respective receptors.
Meenakshi, et al., (2014), reported that the plant Acanthophora spicifera, the red
algae which is used as fresh vegetables in some parts of Vietnam and the Philippines
was subjected to different solvent extraction (Hexane, Ethylacetate and Ethanol) and the
ethanol extract of the Acanthophora spicifera was screened against the following strains
Escherichia coli, Proteus vulgaris, Staphylococcus aureus, Streptococcus pyogenes,
Pseudomonas aeuroginosa, Bacillus subtilis and also subjected to the in vitro cytotoxic
assay. From the data obtained it was evident that the extract showed significant increase
in the death rate of Ehrlich Ascites carcinoma cell lines. At the dose level of 100µg/ml,
36.46% of cell death was noticed. With GC-MS (Gas chromatography Mass
spectrometry) technique, 42 compounds were identified. Five of the compounds were
docked with the BCL-2 protein by using Auto Dock software. The compound Phenol, 3-
methyl-5(1-methylethyl)-, methylcarbamate showed least binding energy of -9.3. In this
study, it is confirmed that the Acanthophora spicifera has significant anticancer activity.
Krishna, et al., (2013), reported that Prodigiosin and cycloprodigiosin are
tripyrrole red pigmented compounds with medical importance for their anticancer
property. In the present investigation, molecular docking studies were performed for
both prodigiosin and cycloprodigiosins to evaluate the in silico anti-inflammatory

33
activity against Cyclo-oxygenase-2 (COX-2) protein as model compound and the data
compared with rofecoxib and celcoxid. Cycloprodigiosin showed higher initial
potential, initial RMS gradient and potential energy values compared to prodigiosin.
Analysis of COX-2 protein and ligand binding revealed that cycloprodigiosin interacted
with COX-2 protein amino acid residues of Tyr324, Phe487 and Arg89 while prodigiosin
interaction was observed with two amino acids i.e. Leu321 and Tyr324. The
computational ligand binding interaction suggested > 45% higher fitness score value for
prodigiosin to that of cycloprodigiosin with COX-2 protein while the standard
compounds rofecoxib and celecoxib revealed fitness score of 44 and 62, respectively.
The prodigiosin ligand revealed the best fitness score compared with the standard drug
rofecoxib suggesting the prodigiosin could be effective as the potential inhibitor
compound against COX-2 protein and can be evaluated as anti-inflammatory drug
molecule using clinical trials.

2.4 ANTICANCER ACTIVITY

Tumor is a biomedically complex gathering of diseases including cell change,

deregulation of apoptosis, expansion, invasion, angiogenesis and metastasis. The
creating learning of disease science recommends that controlling cytotoxic medication
treatment at high measurements is not generally suitable. A potential point of interest of
phytochemicals and different mixes got from regular items is that they may act through
various cell-flagging pathways and diminish the advancement of resistance by disease
cells. New medicates with milder reactions are required urgently to supplant and
enhance existing prescription and to give new avenues to treating malignancy that
oppose treatment with current medications. A lot of data is currently accessible
demonstrating that few characteristic items are supplied with intense anticancer
movement. It has been seen that most regular items with anticancer movement go about
as solid cell reinforcements and/or alter the action of one or more protein kinases
included in cell cycle control.

2.4.1 Survey for anticancer activity

The World Cancer Research Foundation 2007, report estimates that 35% of the
growth rate worldwide could be alluded to way of lifestyle factors, for example,
nourishment, foodstuff and physical movement. Expanding evidence demonstrates that
devouring natural nourishments like organic products, vegetables, spices and nuts could
34
ensure against disease. Studies show that people who expend more organic product,
vegetables and spices are associated with lower rate of malignancy (Bertuccio, et al.,
2009; Bravi, et al., 2009; Buiatti, et al., 1989; Zaid, et al., 2010). Malignancy is a
nonexclusive term for an extensive gathering of ailments that can influence any a part of
the body. Different terms utilized are harmful tumors and neoplasms. One
characterizing highlight of growth is the fast production of strange cells that become
past their regular limits and which can then attack bordering parts of the body and
spread to different organs. This methodology is alluded to as metastasis, which is the
significant reason for death from malignancy. Tumor is in charge of numerous death
(1in 8) around the world. The universal tumor weight multiplied somewhere around
1975 and 2000 and is situated to twofold again by 2020 and almost triple by 2030.
There were around 12 million new disease cases and 7 million tumor death worldwide
in 2008,with 20-26 million new cases and 13-17 million death anticipated for 2030.

2.4.2 Effective management, treatment for cancer

Accordingly, the requirement for an effective management, treatment and cure

of growth is without a doubt pivotal. The control of malignancy, one of the main
reasons for death around the world, may advantage from the potential that lives in
option treatments. Traditional treatments cause genuine reactions and, best case
scenario, only augment the persistent's lifespan by a couple of years. Better growth
medications with milder reactions are urgently required. There is in this way the need to
use elective ideas or ways to deal with the counteractive action of tumor (Reddy, et al.,
2003). An integrative methodology for dealing with a patient with growth ought to
focus on the various biochemical and physiological pathways that support tumor
development and minimize ordinary tissue poisonous quality. Interestingly, both
research center analyses and clinical trials have shown that when joined with
chemotherapy, herbal medications could raise the adequacy level and lower poisonous
responses. These certainties raised the possibility of the blend of natural drug and
chemotherapy (Ruan, et al., 2006). Higher plants have made vital commitments in the
range of tumor treatment. Early sample incorporates antileukaemic alkaloids,
vinblastine and vincristine, which were both gotten from the Madagascan periwinkle
(Catharanthus roseus syn. Vinca roseus). Other disease helpful specialists incorporate
taxol, homoharringtonine and a few subordinates of camplothein (Iwu, et al.,

35
1999).Cancer is an ailment that is regularly described by too little apoptosis. Apoptosis
or customized cell death, is a typical segment of the development and health of
multicellular organisms. Cells die on in response to a variety of stimuli and among
apoptosis they do as such in a controlled regulated fashion. Under ordinary
circumstances harmed cells will experience apoptosis, at the same time on account of
disease cells changes may have happened that keep cells from experiencing apoptosis.
In these cases there is no look out for the cell multiplication and hence the illness can
advance to the development of tumors. By and large these tumors can be hard to kill the
same number of malignancy medications depend on harming the cells with radiation or
chemicals and mutation in the apoptotic pathway frequently deliver cells that are
impervious to this sort of attack. Apoptosis can be incited in cells under in vitro
conditions by various ways. One of the established system is introduction of thymocytes
to glucocorticoids. Different approaches incorporate DNA damage either by irradiation,
introduction to medications that inhibit topoisomerase, withdrawal of growth factors
from growth media, cell cycle perturbation, exposure to inhibitors/activators of kinases
or phosphatases, obstruction with Ca2+ homeostasis, over interpretation of
p[53],members of Ced-3/ICE and numerous more (Saran, 2000). Tumors are brought on
by various hereditary alterations. Mutation in oncogenes or tumor suppressor genes
stand for the primary genetic lesions their enactment and inactivation, correspondingly,
trigger carcinogenesis. Countless mutational events and modified programme of gene
expression not withstanding, set in when primary tumors advance to their last
threatening state (Patel and Sinha, 2001).When devastating diseases, for example,
growth strike, alternative therapies are regularly looked for which utilize less dangerous
and unpleasant treatments than current chemotherapy and radiation medications (Colic
and Pavelic, 2000). A lot of data is presently accessible demonstrating that few regular
items are enriched with potent anticancer action. It is in fact hard to envision the
possible biochemical mechanism of the anticancer activity of natural products.
Numerous analysts have recently tested the activity of such items and the possible
mechanism of their anticancer activity (Hesketh, 1997; Chinery, et al., 1997a; Chinery,
et al., 1997b; Bai Fulu, et al., 1998; Kuzumaki, et al., 1998; Sadzuka, et al., 1998). A
typical movement noted for most of such items is that they go about as powerful
antioxidants and free radical scavengers (Colic and Pavelic, 2000). Plants are incapable

36
to damage caused by active oxygen and hence create various antioxidant defence
system bringing about formation of various potent antioxidants. In basic words "Cancer
prevention agents are a sort of complex compounds found in our diet that proceed as a
defensive shield for our body against certain grievous enemies (diseases, for example,
blood vessel and cardiovascular diseases joint inflammation, cataracts) furthermore
premature ageing alongside a few chronic diseases." Plants contain certain chemicals,
for example, like carotenoids, flavonoids, biflavonoids, phenols, phytosterols and so on
that have antioxidative properties. Since responsive oxygen radicals assume an essential
part in carcinogenesis and other human diseases states, antioxidants in plants have
gotten impressive consideration as tumor chemopreventive operators (Lee, et al., 1998).
The strongest anticancer activity has been exhibited by natural compounds with
multifunctional activity. For example, antioxidants, which likewise ties to and adjust the
activity of protein kinases included in signal transduction cascades show both cytostatic
and cytotoxic action towards tumor cells. Different activities, for example, angiogenesis
inhibition, nitric oxide synthase hindrance and prooxidants generation have likewise
been observed (Colic and Pavelic, 2000).

2.4.3 Screening methods of anticancer activity In vitro methods

2.4.3.1 Trypan blue dye exclusion technique

Trypan blue is a blue colour acid dye, contains two group of azo functional
chromophores. This dye can't penetrate the cell wall of cells grown in culture medium.
It is used to estimate the number of feasible cells present in the residents (Phillips and
Terryberry, 1957).

2.4.3.2 Sulphorodamine B assay

Sulphorodamine B (SRB) is an intense pink aminoxanthine derivative dye

contains two sulfonic functional groups. In acidic condition this dye merge to amino
acid (basic) group in trichloro acetic acid, the permanent cells to give a perceptive index
of protein (cellular) content (Skehan, et al., 1989; Skehan, et al., 1990).

2.4.3.3 Microculture tetrazolium (MTT) assay

The 3(4,5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) is a

yellow colour water soluble dye reduce by succinate dehydrogenase in viable cells, into

37
an insoluble, formazan which is measure the viability of the cells by colorimetrically
(Mosmann, 1983 and Wilson, 2000).

2.4.3.4 XTT assay

In order to measure the proliferation response, The (2,3-bis[2-methoxy-4-nitro-

5-sulfophenyl]- 2H-tetrazolium-5-carboxyanilide (XTT) salt is a yellow colour salt to
form an orange colour formazan, which is measure the viable cells by
spectrophotometrically (Economou, et al., 2008).

2.4.3.5 LDH (Lactic dehydrogenase) Assay

Analyzing NADH reduction through the pyruvate lactate transformation. The

percentage of Lactic dehydrogenase (LDH) released is measured by
spectrophotometrically in the culture medium (Russo, et al., 2006).

2.5 MOLECULAR DOCKING STUDIES USED FOR ANTICANCER ACTIVITY

Subhani, et al., 2015, reported that the MDR1, a protein commonly involved in
drug transport, has been linked to multi drug resistance and disease progression in
cancers such as non-small cell lung cancer. Hence, targeting this protein is essential for
improving drug design and preventing adverse drug-drug interactions. The aim of the
study was to examine chemotherapeutic drug binding to MDR1 and the interactions
therein. We have used Schrodinger suite 2014, to perform homology modelling of
human MDR1 based on Mouse MDR1, followed by Induced Fit Docking with
Paclitaxel, Docetaxel, Gemcitabine, Carboplatin and Cisplatin drugs. Finally, we
evaluated drug binding affinities using Prime/MMGBSA and using these scores we
compared the affinities of combination therapies against MDR1. Analysis of the
docking results showed Paclitaxel > Docetaxel > Gemcitabine > Carboplatin > Cisplatin
as the order of binding affinities, with Paclitaxel having the best docking score. The
combination drug binding affinity analysis showed Paclitaxel + Gemcitabine to have the
best docking score and hence, efficacy. Through our investigation we have identified
the residues Gln 195 and Gln 946 to be more frequently involved in drug binding
interactions with MDR1. Our results suggest that, Paclitaxel or combination of
Paclitaxel + Gemcitabine could serve as a suitable therapy against MDR1 in NSCLC
patients. Thus, our study provides new insight into the possible repurposing of
chemotherapeutic drugs in targeting elevated MDR1 levels in NSCLC patients, thereby

38
ensuring better overall outcome. Further our study highlights the use of in silico
methodologies in understanding drug binding to protein targets and its relevance to
advancing lung cancer therapy.
Penthala, et al., (2015), performed a series of heterocyclic combretastatin
analogues have been synthesized and evaluated for their anticancer activity against a
panel of 60 human cancer cell lines. The most potent compounds were two 3,4,5-
trimethoxy phenyl analogues containing either an (Z)-indol-2-yl (8)or(Z)-
benzo[b]furan-2-yl (12) moiety; these compounds exhibited GI50 values of <10 nM
against 74% and 70%, respectively, of the human cancer cell lines in the 60-cell panel.
Compounds 8, and 12 and two previously reported compounds in the same structural
class, i.e. 29 and 31, also showed potent anti-leukemic activity against leukemia MV4-
11 cell lines with LD50 values = 44 nM, 47 nM, 18 nM, and 180 nM, respectively. From
the NCI anticancer screening results and the data from the in vitro toxicity screening on
cultured AML cells, seven compounds: 8, 12, 21, 23, 25, 29 and 31 were screened for
their in vitro inhibitory activity on tubulin polymerization in MV4-11 AML cells; at 50
nM, 8 and 29 inhibited polymerization of tubulin by >50%. The binding modes of the
three most active compounds (8, 12 and 29) to tubulin were also investigated utilizing
molecular docking studies. All three molecules were observed to bind in the same
hydrophobic pocket at the interface of α- and β-tubulin that is occupied by colchicine,
and were stabilized by van der Waals’ interactions with surrounding tubulin residues.
The results from the tubulin polymerization and molecular docking studies indicate that
compounds 8 and 29 are the most potent anti-leukemic compounds in this structural
class, and are considered lead compounds for further development as anti-leukemic
drugs.
Subhani and Jamil (2015), reported that the CYP3A4, a ‘‘heme’’ containing
isoform, abundantly found in the liver, gastro-intestinal tract, lungs and renal cells, also
known as drug metabolising enzyme (DME) may be responsible for the disease
progression in cancers such as lung cancer. Hence, we have targeted this protein for
improving drug selection and in preventing adverse reactions. The aim of this study was
to examine chemotherapeutic drug binding to CYP3A4 and the interactions therein. We
have used Schrodinger suite 2014, to perform molecular docking of human CYP3A4,
by Induced Fit Docking using gemcitabine, cisplatin, carboplatin, docetaxel and

39
paclitaxel drugs. We evaluated drug-binding affinities using Prime/MMGBSA and
using these scores we compared the affinities of combination therapies against
CYP3A4. Analysis of the docking results showed gemcitabine > carboplatin > cisplatin
as the order of binding affinities, with gemcitabine having the best docking score.
Interestingly, docetaxel and paclitaxel did not bind to CYP3A4 1B. The combination
drug-binding affinity analysis showed gemcitabine + carboplatin to have the best
docking score and hence, efficacy. This investigation has identified the residue Arg 105
to be more frequently involved in drug binding to CYP3A4. Our results suggest that
gemcitabine or combination of gemcitabine + carboplatin could serve as an excellent
therapy against CYP3A4 in NSCLC patients. Thus, our study depicts binding of
chemotherapeutic drugs to CYP3A4 and has identified the residues that may be targeted
for therapy in NSCLC patients.
Agarwal, et al., (2015), reported that the recent developments in the target based
cancer therapies have identified HSF1 as a novel non oncogenic drug target. The
present study delineates the design and molecular docking evaluation of Rohinitib
(RHT)- Cantharidin (CLA) based novel HSF1 inhibitors for target-based cancer
therapy. Here, we exploited the pharmacophoric features of both the parent ligands for
the design of novel hybrid HSF1 inhibitors. The RHT-CLA ligands were designed and
characterized for ADME/Tox features, interaction with HSF1 DNA binding domain and
their pharmacophoric features essential for interaction. From the results, amino acid
residues Ala17, Phe61, His63, Asn65, Ser68, Arg71 and Gln72 were found crucial for
HSF1 interaction with the Heat shock elements (HSE). The hybrid ligands had better
affinity towards the HSF1 DNA binding domain, in comparison to RHT or CLA and
interacted with most of the active site residues. Additionally, the HSF1-ligand complex
had a reduced affinity towards HSE in comparison to native HSF1. Based on the results,
ligand RC15 and RC17 were non carcinogenic, non-mutagenic, completely
biodegradable under aerobic conditions, had better affinity for HSF1 (1.132 and 1.129
folds increase respectively) and diminished the interaction of HSF1 with HSE (1.203
and 1.239 folds decrease respectively). The simulation analysis also suggested that the
ligands formed a stable complex with HSF1, restraining the movement of active site
residues. In conclusion, RHT-CLA hybrid ligands can be used as a potential inhibitor of
HSF1 for non-oncogene target based cancer therapy.

40
 Arul and Sunisha (2014), revealed to design, synthesize and in vitro anticancer
and antitubercular evaluation of some new isatin derivatives containing azetidinone.
Novel azetidinone containing isatin derivatives were designed by using various
softwares such as ACD Lab ChemSketch 12.0, Marvin Sketch, Molinspiration, PASS
and Schrodinger. The designed molecules having required physico-chemical properties,
drug likeness and obeying Lipinski’s rule of five were selected for the synthesis. These
compounds were synthesized by conventional and microwave assisted synthetic
methods through a series of three steps. The synthesized compounds were subjected to
TLC, melting point determination, IR, 1HNMR and Mass spectroscopic studies. The in
vitro anticancer activity of selected compounds was evaluated against MCF7 and L929
by MTT assay method. The antitubercular activity of selected compounds was
evaluated by REMA method. Ten derivatives (AZ-2, AZ-3, AZ-5, AZ-8, AZ-9, AZ-10,
AZ-12, AZ-13, AZ-14 and AZ-15) were selected for synthesis with the help of in silico
modelling. In the synthesis, the microwave method took minimum reaction time and
gave maximum yield comparing with conventional method. All the synthesized
compounds showed characteristic peak in IR, 1HNMR and Mass spectroscopic studies.
Based on the Schrodinger Glide XP score, the compounds AZ-13, AZ-5 and AZ-3 were
selected for in vitro anticancer and AZ-9, AZ-14 and AZ-3 were selected for
antitubercular evaluation. The compound AZ-13 showed significant anticancer activity
particularly against L929 cell line and the compound AZ-9 showed significant
antitubercular activity comparing with other selected compounds.
Pingaew, et al., (2014), had synthesized a new series of chalcone-coumarin
derivatives (9-19) linked by the 1,2,3-triazole ring were synthesized through the
azide/alkyne dipolar cycloaddition. Hybrid molecules were evaluated for their cytotoxic
activity against four cancer cell lines (e.g., HuCCA-1, HepG2, A549 and MOLT-3) and
antimalarial activity towards Plasmodium falciparum. Most of the synthesized hybrids,
except for analogs 10 and 16, displayed cytotoxicity against MOLT-3 cell line without
affecting normal cells. Analogs (10, 11, 16 and 18) exhibited higher inhibitory efficacy
than the control drug, etoposide, in HepG2 cells. Significantly, the high cytotoxic
potency of the hybrid 11 was shown to be non-toxic to normal cells. Interestingly, the
chalcone- coumarin 18 was the most potent antimalarial compound affording IC50 value
of 1.60 mM. Molecular docking suggested that the cytotoxicity of reported hybrids

41
could be possibly due to their dual inhibition of β-tubulins at GTP and colchicine
binding sites, respectively. Furthermore, falcipain-2 was identified to be a plausible
target site of the hybrids given their antimalarial potency.
Kaur, et al., (2014), had synthesized some novel Ospemifene derived analogs
and their evaluation as anti-breast cancer agents against MCF-7 (ER-positive) and
MDA-MB-231 (ER-negative) human breast cancer cell lines are described. Few of
these analogs for instance, compounds 6, 7 and 8 are shown to be more effective than
recent Selective Estrogen Receptor Modulators (SERMs) i.e. Ospemifene and
Tamoxifen, against these cell lines. Compound 8 was relatively more cytotoxic to MCF-
7 cells similar to Ospemifene and Tamoxifen, while most potent compounds 6 and 7
were equally effective in inhibiting growth of both ER-positive and ER-negative cell
lines. The observed activity profiles were further supported by the docking studies
performed against estrogen receptors (ERα and ERβ). Compounds 6, 7 and 8 exhibited
stronger binding affinities with both ERα and ERβ compared to Ospemifene and
Tamoxifen.
Chengyuan Liang, et al., (2014), showed that the Topoisomerase I inhibitors
have been developed for a variety of clinical applications. In the present study, we
report the antiproliferative activity of some sulphonic acid esters derived from
evodiamine as well as 5-methylene evodiamine, for the purpose of improving
therapeutic benefits of evodiamine. The synthesized compounds 3a-3g and 5a-5g were
evaluated for their in vitro cytotoxicity against A549, HepG-2, U251, HeLa and MCF-7
human carcinoma cell lines by MTT assay. Compound 3e exhibited potent anti-tumor
activities on all cell lines (IC50 ranged between 2.68 µM and 18.42 µM). Moreover, 3e
was found to be able to inhibit substantially the tumor growth on the HepS-bearing mice
at a dose of 80 mg/kg. Subsequently, preliminary structure-activity relationship was
explored based on the biological data, which could provide guidance for designing new
analogues of evodiamine. Finally, molecular modelling studies of sulfonic evodiamine
esters revealed that they could form hydrogen-bonding and hydrophobic interactions
with several amino acid residues of topoisomerase I, resembling the binding format
between camptothecin and topoisomerase I.
Ahmed, et al., (2014), reported that the series of imidazo[2,1-b]thiazoles bearing
pyrazole moieties 4-6(a-c) was synthesized through the reaction of 6-

42
hydrazinylimidazo[2,1-b]thiazoles 3aec with different b-dicarbonyl compounds. Eleven
compounds were screened at the National Cancer Institute (NCI), USA for anticancer
activity at a single dose (10mM). The in vitro anticancer evaluation revealed that
compounds 2a and 4-6(a) exhibited increased potency towards CNS SNB-75 and Renal
UO-31 cancer cell lines. Compare analyses showed strong to considerable correlations
with rapamycin (mTOR inhibitor). The results of assessment of toxicities, drug likeness,
and drug score profiles of compounds 2a and 4-6(a) are promising. Some of the target
compounds showed good docking scores with potential anticancer targets, chosen based
on pharmacophore mapping of the established derivatives.

Megha Sahu and Nerkar (2014), reported that the main objective of this research
was to in silico screen, synthesize, characterize and in vitro evaluate some quinazolin-
eone derivatives as dihydrofolate reductase (DHFR) inhibitors for anticancer activity.
The present study reports a new series of Quinazoline and quinazolinone as potent
inhibitors of human DHFR for anticancer activity. In silico screening of compound was
performed on Vlife MDS 4.3 software and ADME studies were performed on
PreADMET online software for prioritization of molecules for actual synthesis and in
vitro evaluation. Prioritized molecules were synthesized by known reactions.
Characterizations of derivatives were done by 1HNMR, 13CNMR, IR and melting point.
In vitro anticancer activity was performed at ACTREC, Navi Mumbai, India for the
prioritized molecules on ten human cell-lines. Molecules were prioritized with
comparable docking score as compared with Methotrexate used as standard in docking.
ADMET parameters such as HIA, Caco 2 cell permeability, MDCK and PPB were
considered for prioritization. Prioritized synthesized molecules complied with
spectroscopic assignments. Further prioritized molecules were evaluated for in vitro
anticancer activity .Molecules showed potent anticancer activity. Prioritized compounds
BrQSB1, BrAQ3, BAQ23 were highly active on A549, HeLa, SK-OV-3, KB, HCT15,
SiHa, MCF7, DU145 at a concentration of < 10 µg/ml. These can further serve as
templates for development of anticancer agent.
El Ansary, et al., (2014), showed that the synthesis of some new 3-alkyl-
7-hydroxy-4-methyl-8-substituted-1H-benzopyran-2-ones, 6-alkyl-7-methyl-2-
substituted amino-5H-pyrano[6,5-e]benzoxazol-5-ones, 7-alkyl-8-methyl-3-substituted-

43
2,6-dihydropyrano[6,5-f]-1,4-benzoxazin-6-ones, 7,8-disubstituted-3-ethyl-4-methyl-
1H-benzopyran-2-ones and 3-alkyl-4-methyl-7-substituted-1H-benzopyran-2-ones were
described. Fourteen compounds were selected by National Cancer Institute (NCI),
Bethesda, and evaluated for their in vitro anticancer activity in the full NCI 60 cell lines
panel assay by a single dose test. Compounds 4a, 18a, 18b and 23a were found to be
broad-spectrum antitumors showing effectiveness toward numerous cell lines that
belong to different tumor subpanels. Furthermore, docking studies were undertaken to
gain insight into the possible binding mode of these compounds with the binding site of
the casein kinase II (CK2) enzyme which is involved in cell survival and proliferation
through a number of downstream effectors.
Adriana, et al., (2014), showed that the present study evaluated the carvacrol
(CARV) effect on hyperalgesia and nociception induced by sarcoma 180 (S180) in
mice. Carvacrol treatment (12.5–50 mg/kg s.c.) once daily for 15 days was started 24 h
after injection of the sarcoma cells in the hind paw (s.c.). Mice were evaluated for
mechanical sensitivity (von Frey), spontaneous and palpation-induced nociception, limb
use and tumor growth on alternate days. CARV effects on the central nervous system
were evaluated through immunofluorescence for Fos protein. Molecular docking studies
also were performed to evaluate intermolecular interactions of the carvacrol and
muscimol, as ligands of interleukin-10 and GABAA receptors. CARV was able to
significantly reduce mechanical hyperalgesia and spontaneous and palpation induced
nociception, improve use paw, decrease the number of positively marked neurons in
lumbar spinal cord and activate periaqueductal gray, nucleus raphe magnus and locus
coeruleus. CARV also caused significant decreased tumor growth. Docking studies
showed favorable interaction overlay of the CARV with IL-10 and GABAA.Together,
these results demonstrated that CARV may be an interesting option for the development
of new analgesic drugs for the management of cancer pain.
Baskaran and Ramachandran (2012), reported that the cancer can be described
as the uncontrolled growth of abnormal cells. Lung cancer is one of the commonest
malignant neoplasms all over the world. Oncogenic fusion genes consisting of EML4
and anaplastic lymphoma kinase (ALK) are present in non-small-cell lung cancers,
representing 2 to 7 of such tumors. ALK proteins play a vital role in deactivating the
apoptosis process in cancer disease. Some of the most commonly used non-small-cell

44
lung cancers % drugs are Crizotinib, Sunitinib malate, Tandutinib etc..., Non-small-cell
lung cancer Cells need anaplastic lymphoma kinase (ALK) to cell growth and
proliferation the role of ALK in malignant proliferation and as a valid drug target. These
drugs mainly work against the effects of ALK on these cells. The Protein- Ligand
interaction plays a significant role in structural based drug designing. In our research
work we have taken the Human anaplastic lymphoma kinase (ALK) and the
commercially available drugs against non-small-cell lung cancer. The ALK was docked
to the above said drugs and the energy value obtained as follows Crizotinib (-9.86),
Sunitinib malate (-8.26), Tandutinib (-8.05) using the Argus Lab docking software.
Depending on the energy values we have chosen the best two drugs they are Crizotinib
(-9.86) and Sunitinib malate (-8.26). We tried to improve the binding efficiency and
steric compatibility of the two drugs namely Crizotinib (-9.86) and Sunitinib malate(-
8.26). Several modifications were made to the probable functional groups which were
interacting with the receptor molecule. Analogs of this drug molecule were prepared
using ACD ChemSketch and docked using Argus Lab docking software. Crizotinib
Analog 2 and Sunitinib malate analog 1 were detected with significant energy values
and probable lead molecules. The Modified drugs was sketched using Chemsketch were
found to be better than the conventional drugs available. Further from this work we can
improve the steric compatibility and then ADME/T properties of the Analogs can be
analyzed using in silico ADME/T tools available.
Adinarayana, et al., (2012), revealed that the computational tools have been
widely utilized to design potent leads against specific targets as a part of drug discovery
process. Moreover, virtual screening has been reported as an efficient concept in
filtering actives from a huge library of compounds. In this paper, a computational
approach employing virtual screening of Drug Bank database was implemented to dock
1400 drugs against caspase-3 to evaluate the binding affinities of ligands with the
receptor. Docking analysis with Molegro Virtual Docker revealed top 10 drugs and
hence consensus re-scoring was employed to obtain true hits. Classes were generated
using rank-sum technique and the top five hits (Olmesartan, Verteporfin, Saprisartan,
Atorvastatin and Lapatinib) were tested experimentally to determine the inhibitory
effects on growth of HeLa cells in vitro. It was observed that proliferation of HeLa cells
could be significantly inhibited by Atorvastatin in a concentration dependent manner.

45
 Dandawate, et al., (2012), revealed that the novel plumbagin hydrazonates were
prepared, structurally characterized and evaluated for anti-proliferative activity against
estrogen receptor-positive MCF-7 and triple negative MDA-MB-231 and MDA-MB-
468 breast cancer cell lines which exhibited superior inhibitory activity than parent
plumbagin compound. Molecular docking studies indicated that hydroxyl groups on
plumbagin and hydrazonate side chain favor additional hydrogen bonding interactions
with amino acid residues in p50-subunit of NF-κB protein and these compounds
inhibited NF-κB expression which may be responsible for the enhanced anti-
proliferative activity. These compounds were found to be more effective against triple
negative breast cancer cells and might serve as a starting point for building future
strategies against triple negative breast cancers which are known for their increased
drug resistance and poor prognosis of breast cancer patients.
Ismail, et al., (2010), reported Novel series of 7-substituted-benzopyran-2-ones
was synthesized by incorporating heterocyclic rings as oxadiazole, triazole, pyrazole or
pyrazolin-5-one to benzopyran-2-one nucleus at p-7 via methylene-oxy or acetoxy
linker. In vitro anticancer activity was evaluated for these hybrids; twelve compounds
were selected by National Cancer Institute for anticancer screening. Among them,
compound 9a exhibited broad spectrum antitumor activity showing full panel median
growth inhibition (GI50) = 5.46 mM. According to docking results using Molsoft ICM
3.4-8c program, the target compounds may act through inhibition of topoismerase 1,
where camptothecin is used as ligand.
Pérez-Montoto, et al., (2009), reported a new class of invariants for MD
trajectories based on the spectral moments πκ(L) of the Markov matrix associated to
lattice network-like (LN) graph representations of Molecular Dynamics (MD)
trajectories. The procedure embeds the MD energy profiles on a 2D Cartesian
coordinates system using simple heuristic rules. At the same time, we associate the LN
with a Markov matrix that describes the probabilities of passing from one state to other
in the new 2D space. We construct this type of LNs for 422 MD trajectories obtained in
DNA–drug docking experiments of 57 furocoumarins. The combined use of psoralens +
ultraviolet light (UVA) radiation is known as PUVA therapy. PUVA is effective in the
treatment of skin diseases such as psoriasis and mycosis fungoides. PUVA is also useful
to treat human platelet (PTL) concentrates in order to eliminate Leishmania spp. and

46
Trypanosoma cruzi. Both are parasites that cause Leishmaniosis (a dangerous skin and
visceral disease) and Chagas disease, respectively; and may circulate in blood products
collected from infected donors. We included in this study both lineal (psoralens) and
angular (angelicins) furocoumarins. In the study, we grouped the LNs on two sets; set1:
DNA–drug complex MD trajectories for active compounds and set2: MD trajectories of
nonactive compounds or no-optimal MD trajectories of active compounds. We
calculated the respective πκ (L) values for all these LNs and used them as inputs to train
a new classifier that discriminate set1 from set2 cases. In training series the model
correctly classifies 79 out of 80 (specificity = 98.75%) set1 and 226 out of 238
(Sensitivity = 94.96%) set2 trajectories. In independent validation series the model
correctly classifies 26 out of 26 (specificity = 100%) set1 and 75 out of 78 (sensitivity =
96.15%) set2 trajectories. We propose this new model as a scoring function to guide
DNA-docking studies in the drug design of new coumarins for anticancer or
antiparasitic PUVA therapy.
Kumar, et al., (2008), revealed that this study documents the analysis of a
hitherto unreported dynamic behaviour of androgen receptor (AR), a member of the
nuclear receptor super family. Employing GFP-tagged AR, we observed agonist-
mediated docking of AR onto the mitotic chromatin during all the stages of mitosis.
When bound to therapeutic drugs with intrinsically absolute or partial agonistic
properties, AR concomitantly associated with the mitotic chromatin. Conversely, pure
antagonists known to bind and subsequently translocate unliganded AR from cytoplasm
to nuclear compartment did not provoke such association. The agonist-mediated
docking of AR could not be competed with other transcription factors that constitutively
preoccupied the chromosomal docking sites. Amongst the previously reported proteins,
AR is first example of a transcription factor whose response on mitotic chromatin
platform can be modulated in a ligand-specific manner. However, data from live cell
imaging revealed that coactivators of agonist-activated receptor that are recruited into
“nuclear foci” of interphase chromatin are dislodged from the mitotic chromatin during
cell division. This implies that in absence of critical co-activators, AR transverses
mitotic phase in transcriptionally silenced state. Finally, results indicate that ligand-
mediated dynamic relationship of nuclear receptors with mitotic chromatin can be
effectively exploited to study, analyze and authenticate therapeutic ligands.

47
2.6 ANTI TUBERCULOSIS DRUGS

Tuberculosis (TB) is a contagious infectious disease caused by species

belonging to the Mycobacterium tuberculosis complex. At present, it is a re-emerging
disease, due to co-infection with the Human Immunodeficiency Virus (HIV), but also
to global bacterial resistance, and lack of adequate treatment in some places in the
world. Approximately one third of the world’s population is infected with M.
tuberculosis, and out of these people, about 1.1 million people die every year of TB
(World Health Organization, 2011), making this disease the main cause of bacterial
infectious death in adolescents and adults all around the world. In 2010 there was an
estimation of 8.8 million incident cases and 12.0 million prevalent cases of TB
worldwide. M. tuberculosis drug-resistant isolates have appeared giving origin to
multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains. XDR-TB has
been identified in every continent of the planet. By 2010, the World Health
Organization (WHO) was notified of the existence of 53.018 cases of multi-drug
resistant TB (MDR-TB) worldwide; figure that only represents 18% of the TB-MDR
estimated cases among reported pulmonary TB cases around the world (World Health
Organization, 2011). Currently, there is global alarm since the infection with these
strains is cured only in 66% of MDR cases and in 60% of the XDR cases (Mitnick, et
al., 2008).
More than sixty years ago, the introduction of the first anti-TB drugs for the
treatment of TB (streptomycin (STR), p-aminosalicylic acid (PAS), isoniazid (INH) and
then later ethambutol (EMB) and rifampicin (RIF)) gave optimism to the medical
community, and it was believed that the disease would be completely eradicated soon.
After a 30-year halt of anti-TB drug Research & Development pipeline, the Global
Alliance for TB Drug Development (TB Alliance) started to fill the gap between the
existing chemotherapeutics and the clinical need. Despite the efforts carried out with
candidates in clinical trials such as PA-824 and bedaquiline, there is an urgent need of
in-depth medicinal chemistry discovery studies for assuring enough leads and
candidates feeding the pipeline within the next decade (Casenghi, et al., 2007).
Emerging chemical entities must shorten the time of treatment, be potent and safe while
effective facing resistant strains and non-replicative, latent forms, and not interfere in
the antiretroviral therapy (Sacks & Behrman, 2008). In this review, we explore why we

48
require to work continuously on the development of novel antiTB agents, the stages
necessary for the development of new anti-TB agents, breakthroughs in the discovery of
new active principles and targets, the preclinical and clinical development of drugs, as
well as the new approaches for the search of anti-TB active principles.
Tuberculosis (TB) is a disease of antiquity which is thought to have evolved
sometime between the seventh and sixth millennia BC (Manchester, 1984).Current
estimates suggest that one third of the world’s population are infected resulting in some
2 million deaths per year (Dye, et al., 1999).The introduction of the first drugs for TB
treatment some 50 years ago - streptomycin, para-aminosalicylic acid, isoniazid - led to
optimism that the disease could be controlled if not eradicated (Centers for Disease
Control and Prevention, 1999). These medicaments, coupled with generally increasing
standards of health care, caused a rapid decline of tuberculosis in many industrialized
countries which produced a climate of indifference to the need for fresh drugs. As a
result of this apathy and the perception by the pharmaceutical industry that such agents
would be unlikely to generate a suitable return on investment, few new drugs have been
introduced in the last 30 years (Weil, 1994).However, since the 1980’s the disease has
been undergoing a resurgence driven by a variety of changes in social, medical and
economic factors. Thus, a dramatic increase in immuno-suppressed individuals due
mainly to AIDS (but also to cancer chemotherapy and organ-transplant practices),
coupled with increasing urbanization and poverty in developing countries, has
compromised primary health care structures and led to large increases in TB incidence
(Snider, 1994).Concomitant with the resurgence of TB has been the occurrence of
multidrug-resistant disease which has exposed the frailties of the current drug
armamentarium. There is now recognition that new drugs to treat TB are urgently
required, specifically for use in shorter treatment regimens than are possible with the
current agents and which can be employed to treat multidrug-resistant and latent
disease. A variety of new initiatives have been created to tackle these objectives, the
most recent of which is the establishment of the so-called Global Alliance for TB Drug
Development (International Union Against Tuberculosis and Lung Disease, 2000).The
Alliance, a public/private partnership in which WHO is a partner, is a not-for-profit
venture that will accelerate the discovery and development of new drugs to fight TB
using a virtual operating model to outsource projects. As a first step to identifying

49
suitable candidate molecules, information in the public domain on anti-TB compounds
was reviewed at a meeting held in Geneva, 56 December 2000, under the joint auspices
of the UNDP/World Bank/WHO Special Programme for Research and Training in
Tropical Diseases and the Global Alliance for TB Drug Development.
The information in this report was initially presented at the meeting in a draft
document prepared by Dr Toshiko Imamura (WHO; financially assisted by the
Rockefeller Foundation). This contained data extracted mainly from the public domain
via Medline, R&D Focus, Pharma-Transfer, World Drug Alert (Accord), Investigational
Drugs weekly highlights, a Boston Consulting Group presentation to the Global
Alliance for TB Drug Development (working session, 11 May 2000), and various
pharmaceutical company web-sites. In some instances non-confidential information was
also obtained direct from representatives of companies involved in the development of
several of the compounds cited. As a result of the review, additional material was added
by the delegates and used to produce the ensuing report (Rattan, et al., 1998).

2.7 COMPOUNDS WITH ANTI-TB ACTIVITY

As in the original draft document, this report presents chemical, biological, and
in some cases, clinical data for each of the candidate molecules and uses this to conduct
a SWOT (Strength, Weakness, Opportunity, Threat) analysis. The compounds are
grouped according to their perceived state of development for the TB indication -
discovery research, preclinical development and clinical development. It should be
noted that for many of the compounds, particularly those in the discovery and
preclinical phases, there were insufficient data to enable accurate assessments of their
anti-TB potential to be made. Nevertheless, despite this paucity of information, it was
possible to conclude which molecules should be progressed further to generate the
required data to facilitate go/no go recommendations.
Discovery
In assessing the compounds discussed below, particular attention was paid to the
following properties:
Drug like:
• Molecular structure compatible with ADME (absorption, distribution,
metabolism and excretion)
Requirements

50
 • Avoid metabolically fragile molecules, high molecular weight, very polar, and
highly lipophilic compounds
• Avoid compounds containing known toxocophores and chemically reactive
groups.
Novelty:
• At target site, avoid cross resistance with existing drugs
• Structure makes patent protection feasible and may also avoid cross resistance
with existing drugs even if directed to the same target protein.
In vitro data:
• Activity v M. Tuberculosis strains
• Activity v mammalian cells
• Bacteriostatic v bactericidal activity.
In vivo data:
• Efficacy
• ADME
• Toxicity.
Potential cost of goods:
• Ease of synthesis; achiral preferred to chiral molecules
• Availability of starting materials
• Lack of hazardous intermediates.
Many of the above parameters were not known with certainty, but sufficient
details were usually available to permit a judgment to be made.
Ashtekar, et al., 1991 reported that the DL-S-n-(3-(4-acetyl)-2-oxo-5-
oxazolidynyl methyl) acetamide (DuP-721) is an orally active representative of the
oxazolidinone series of antimicrobials. At concentrations ranging from 1.5 to 4µg/ml,
DuP-721 inhibited equally the strains of Mycobacterium tuberculosis susceptible and
resistant to conventional antituberculosis drugs. DuP-721 inhibited M. gordonae and M.
fortuitum at 3.9µg/ml, M. kansasii at 1.95, and M. scrofulaceum at 15.6 µg/ml. It was
not active against M. avium and M. intracellulare at concentrations of 250µg/ml. The
inhibition of the metabolism of M. tuberculosis as indicated by the liquid scintillation
radiometric method was 56% at fourfold the minimum inhibitory concentration (MIC)
of DuP-721 that compared well to that of the fourfold MIC concentrations of rifampicin

51
and isoniazid. The in vitro activity of DuP-721 was not affected by reducing the pH
from 6.8 to 5.5. In mice infected with M. tuberculosis, the 50% effective dose (ED50)
for DuP-721 was 13.2mg/kg when administered daily beginning 4 h post infection for
17 days. The ED50 was 71.8mg/kg when DuP-721 was administered only on days 11
and 12 post infection. A 100% survival rate was obtained at 50 and 160mg/kg when
DuP-721 was administered daily for 17 days, and only on days 11 and 12 after the
infection, respectively. The increase in the survival time by DuP-721 at 100 mg/kg (8
fold the ED50 dose) when administered daily for 17 days beginning 4h after infection
was inferior to that by eightfold the ED50 dose of rifampicin and isoniazid administered
on days 11 and 12 post infection.
Galina, et al., (2014), reported that this work, a new class of highly potent
antituberculosis agents, 1-substituted-5-[(3,5-dinitrobenzyl) sulfanyl]-1H-tetrazoles and
their oxa and selanyl analogs, is described. The minimal inhibitory concentration (MIC)
values reached 1mM (0.36-0.44 µg/mL) against Mycobacterium tuberculosis CNCTC
My 331/88 and 0.25-1µM against six multidrug-resistant clinically isolated strains of M.
tuberculosis. The antimycobacterial effects of these compounds were highly specific
because they were ineffective against all eight bacterial strains and eight fungal strains
studied. Furthermore, these compounds exhibited low in vitro toxicity in four
mammalian cell lines (IC50> 30 µM). We also examined the structure activity
relationships of the compounds, particularly the effects on antimycobacterial activity of
the number and position of the nitro groups, the linker between tetrazole and benzyl
moieties, and the tetrazole itself. Relatively high variability of substituent R1 on the
tetrazole in the absence of negative effects on antimycobacterial activity allows further
structural optimization with respect to toxicity and the ADME properties of the 1-
substituted-5-[(3,5-dinitrobenzyl)sulfanyl]-1H-tetrazoles lead compounds.
Variam, et al., (2015), revealed that the Gyrase ATPase domain, the
pharmaceutical under exploited segment of DNA gyrase, the sole Type II topoisomerase
present in Mycobacterium tuberculosis represents an attractive target for antitubercular
drug discovery. Here we report, the development of a novel series of MTB DNA gyrase
B inhibitor identified through a medium throughput screening (MTS) of BITS in-house
chemical library (3000 compounds). The MTS hit was further remodeled by chemical
synthesis to identify the most potent analogue N-(4-Fluorobenzyl)-1-(5-nitrothiazol-2-

52
yl)piperidin-4amine exhibiting an in vitro gyrB inhibitory IC50 of 0.15 µM. The series
also demonstrated well correlating gyrase super coiling activity and in vitro anti-
mycobacterial potency against MTB H37Rv strain. Furthermore the compounds
displayed good safety profile in their subsequent cytotoxicity and hERG toxicity
evaluations, to be worked out from a pharmaceutical point of view as potential
antitubercular agents.
Richard, et al., (2015), reported that the development of new drugs is required to
control human tuberculosis (TB). This study examined whether drug hypersensitive
mutants could be used to reveal novel aspects of the mechanism of action of a TB drug.
A transposon mutant collection with an estimated 1.1-fold genome coverage (7680
mutants) was constructed in Mycobacterium smegmatis and screened in high-throughput
against isoniazid. Hypersensitive transposants with mutations in genes known to
influence the mode of action of isoniazid were isolated. To further investigate the role
of one of these genes, nudC, the corresponding mutant was tested for sensitivity towards
isoniazid structural analogues. Over expression of nudC, as well as inhA which encodes
a known target of isoniazid, increased M. smegmatis resistance to isoniazid, but failed to
increase resistance to three of the analogues, NSC27607, NSC33759, and NSC40350. In
contrast, over expression of katG resulted in increased sensitivity to each of the
isoniazid analogues tested including NSC27607, NSC33759, and NSC40350. This
provides evidence that the latter isoniazid analogues are activated by KatG in a NudC-
independent manner and that InhA may not be their primary target. In summary,
characterisation of drug hypersensitive mutants detected genes involved in the mode of
action of isoniazid. Furthermore, it identified isoniazid analogues which are resilient to
both InhA- and NudC dependent mechanisms of resistance.
Sanchaita, et al., (2015), revealed that the InhA is an attractive target to combat
tuberculosis (TB), which is targeted by many pro-drugs (isoniazid, etc.) and drugs such
as triclosan. However, triclosan is less useful as an antitubercular drug due to its low
bioavailability and therefore, in order to overcome this difficulty, many derivatives of
triclosan were prepared. Here, we have combined various computational techniques to
virtually screen out four potential triclosan derivatives. Molecular docking methods
have been employed to screen out 32 out of 62 triclosan derivatives considering the
mode of binding and the top re-rank scores. A comparative study on the chemical

53
properties of triclosan and some of its derivatives has been performed using density
functional theory (DFT) calculations. DFT based global reactivity descriptors (GRD),
such as hardness, chemical potential, chemical softness, electrophilicity index, Fukui
function, and local philicity calculated at the optimized geometries were used to
investigate the usefulness of these descriptors for understanding there active nature and
sites of the molecules. QSAR equations were built using these descriptors considering
these 32 compounds. Four common compounds showing the best correlation and the
best docking scores were considered for the ADMET property calculations and their
dynamical movements have been studied using molecular dynamics simulations. This
results showed that these four compounds are chemically more active than triclosan and
have the potential to inhibit the Mycobacterium tuberculosis enoyl acyl carrier protein
reductase. This work shows that combination of different computational techniques may
help to screen out potential drug candidates from a list of possible ones.
Ze Qi, et al., (2006), reported that the Pyranocoumarin compounds were
identified to embody a novel and unique pharmacophore for anti-TB activity. A
systematic approach was taken to investigate the structural characteristics. Focused
libraries of compounds were synthesized and evaluated for their anti-TB activity in
primary screening assays. Compounds shown to be active were further determined for
MIC and MBC values. Three of the four bactericidal compounds were amino
derivatives, with MIC values of 16 µg/mL and respective MBC values of 32, 32, and 64
µg/mL.
Dakshina, et al., (2015), revealed that the various classes of natural products and
synthetic compounds were tested against reference strains and clinical multidrug
resistant isolates of Mycobacterium tuberculosis. Vermelhotin (19), a natural tetramic
acid from fungi, was the most active toward clinical MDR TB isolates (MIC
1.5e12.5µg/mL). Synthetic compounds (i.e. benzoxazocines, coumarins, chromenes,
and pyrrolodiquinoline derivatives) were prepared by green chemistry approaches.
Under microwave irradiation, a one-pot synthesis of pyrrolodiquinoline5-
methylpyrrolo[1,2-a:3,4-c']diquinolin-5-ium iodide was achieved by homocoupling of
1-methylquinolinium iodide; the structure of 85 was confirmed by single-crystal X-ray
analysis. Compound 5-methylpyrrolo [1,2-a:3,4-c']diquinolin-5-ium iodide and its
derivative 5-methyl-5,6-dihydropyrrolo[1,2-a:3,4-c']diquinoline exhibited potent

54
antitubercular activity (MIC 0.3-6.2µg/mL) against clinical MDR TB isolates, and they
displayed weak cytotoxicity toward normal cell line. The scaffold of 5-
methylpyrrolo[1,2-a:3,4-c']diquinolin-5-ium iodide and 5-methyl-5,6-dihydropyrrolo
[1,2-a:3,4-c']diquinoline is potential for antimycobacterial activity.
Riffat, et al., (2013), revealed that the Tuberculosis (TB) is one of the leading
infectious causes of death due to single infectious agent after HIV/AIDS. Rifampicin,
Isoniazid (INH), Ethambutol, Pyrazinamide and/or their combinations are extensively
prescribed to treat TB. Despite several therapeutic implications, these drugs also
produce several toxic effects at cellular level. MTT assay and Ames test were adopted
in this study for the determination of cytotoxic and mutagenic potential of these anti-TB
drugs. Among all tested drugs, cytotoxic potential of RIF was strongest with highly
significant decline (p < 0.001) in cell numbers at the concentration of 250µg/ml with
LC50 at 325µg/ml, while significant decline (p < 0.01) in cell count was observed in
INH treated group at the concentration 500µg/ml with LC50 at 1000µg/ml. Moreover,
combination RIPE demonstrated significant reduction (p < 0.01) in cell number at the
concentration of 25–500–500–500g/ml with LC50 at 60–1200–1200–1200µg/ml. It is
apparent from the data that almost all drugs represented identical mutagenic pattern i.e.,
more significant results were achieved in TA100 with metabolic activation (+S9). RIF
proved to be highly mutagenic of all tested drugs with significant mutagenicity (p <
0.01) at 0.0525g/plate against TA98 strain with S9. The combination RIPE exhibited
highly significant mutagenic activity (p<0.01) at concentration 0.125–3–3–3g/plate
without S9, while addition of S9 resulted in similar activity at lower doses, i.e., 0.0525–
1–1–1µg/plate. It was concluded from the data that all anti-TB drugs possess significant
cytotoxic and mutagenic potential, especially in combination, making TB patient more
vulnerable to cytotoxic and mutagenic effects of anti-TB drugs, which could produce
further health complications in TB patients.
Anisha, et al., (2013), revealed that the range of polyvinylpyrrolidone–
polycaprolactone diblock copolymers with varying chain lengths were synthesized by
Atom Transfer Radical Polymerisation (ATRP) using bromopolycaprolactone as
macroinitiator and copper(I) bromide/bipyridine catalytic system. The copolymers self-
assembled in solution into core–shell micelles with sizes varying from150 to 205 nm
and critical micelle concentration of the order of 10-5to 10-6M. Front line anti-

55
tuberculosis drugs Rifampicin (RIF), Pyrazinamide (PZA) and Isoniazid (INH) were
successfully encapsulated within the micelle hydrophobic core singly or in dual
combination. The effect of length of hydrophobic and hydrophilic segments on drug
loading, micelle size and drug release was investigated. Determination of binding
constants showed that RIF binds more strongly to the micelle core than PZA and INH,
leading to highest drug loading content. All drugs were released in vitro (PBS solution
at 37˚C) in a sustained manner with zero-order kinetics and followed the order INH >
PZA > RIF.

2.8 ANTI INFLAMMATORY DRUGS

Drugs for the relief of pain, fever, and inflammation have been known for
centuries, and the history of anti-inflammatory drugs is an impressive example of
successful drug development. Many of these agents have their origin in extracts or
decoctions of plants such as willow leaves or bark. Already the Assyrians have used the
extract of willow leaves for the reduction of painful conditions, and the ancient
Egyptians have treated joint pain with decoctions of myrtle and willow leaves. In
modern age, the extraction of salicin from willow barks by Johann Andreas Buchner in
1828 followed by the isolation and synthesis of salicylic acid by Felix Hoffmann in
1897, and finally the acetylation of salicylic acid and the birth of acetylsalicylic acid
(Aspirin®) were milestones in pharmaceutical history (Jack, 1997).Today a broad
spectrum of anti-inflammatory drugs is available, which can be divided by their
chemical structure into biologicals and small molecules, which include steroidal and
non-steroidal agents. Despite the growing influence of biologicals especially in the
autoimmune and cancer market, there is still a strong need for potent and safe small
molecules. Apparently, small molecules are less expensive compared to biologicals and
can usually be administered orally. The last vast reviews on anti-inflammatory drugs
focused on the enormous range and type of new anti-inflammatory agents and the
discovery of new therapeutic targets (Rainsford, 2007) as well as the current status of
anti-inflammatory agents, with focus on anti-cytokine therapy as new anti-inflammatory
strategy (Dinarello, 2010). The focus of the present review shall lie on small molecules
in clinical trials for chronic inflammatory diseases, belonging to the field of immune-
mediated inflammatory diseases (IMIDs), and especially to the field of auto immune
diseases.

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 The cellular and molecular mechanisms of an acute inflammation are widely
understood (Medzhitov, 2008). In general, tissue-resident macrophages become
activated by exogenous or endogenous inducers, which cause the release of
inflammatory mediators. These inflammatory mediators are responsible for endothelial
activation and recruitment of further effector cells to the site of inflammation. The
effector cells then release their cytotoxic contents, such as reactive oxygen species
(ROS) or certain types of proteases, which neutralize the cause of inflammation but at
the same time damage surrounding tissue. This general mechanism of an acute
inflammation is usually self-limiting through the initiation of the pro-resolution phase
that prevents further recruitment and activity of effector cells and resolves the
inflammatory response. However, if the acute inflammatory response fails to resolve the
inflammation, chronification might follow. In contrast to an acute inflammation, the
pathomechanisms of chronic inflammation are much less understood.
In particular, the triggers of chronic inflammation are not comparable to those of
an acute inflammation, which is mostly caused by tissue injury or infections. Moreover,
in chronic inflammation, the activation of the inflammatory cascade is often caused by a
malfunction of the affected tissue and associated with a homeostatic imbalance in the
physiological processes of inflammation. The challenge to review the stage of
development of anti-inflammatory drugs in clinical trials is the versatile appearance of
inflammation in many diseases. Search terms such as “inflammation” or “anti-
inflammatory drugs” in clinical databases would therefore have led to unsatisfactory
results. For many conditions, e.g., in infectious diseases, a causal (anti-infective)
therapy is available and anti-inflammatory agents are predominantly used in chronic
inflammation. We will therefore focus on immune-mediated inflammatory diseases,
especially autoimmune diseases, which include multiple sclerosis, chronic obstructive
pulmonary disease (COPD), inflammatory bowel diseases (IBDs), psoriasis, and
rheumatoid arthritis (RA).
To systematically review clinically evaluated compounds with anti-
inflammatory properties, we explored the database of the U.S. National Institute of
Health, listed on clinicaltrials.gov, which we screened for the keywords rheumatoid
arthritis, multiple sclerosis, psoriasis, chronic obstructive pulmonary disease, and
inflammatory bowel diseases. The results were then filtered for small molecules vs.

57
biologicals, and the molecular targets of all small molecules that are in clinical trials for
one of the five mentioned diseases were evaluated for their anti-inflammatory
properties. Interestingly, only a small number of the listed clinical trials were performed
with small molecules with anti-inflammatory properties (about 9–17%). Besides these,
many clinical trials have been performed with biological in RA and IBD, while
phototherapy has a high significance in psoriasis. For MS and COPD, there are
predominantly small molecules under investigation. However, clinical trials are
dominated by neuroprotective agents and vitamin (especially vitamin D)
supplementation for MS and by β2 adrenoceptor agonists as well as anticholinergic
agents for COPD.
Rajanarendar, et al., (2015), revealed that the synthesis of novel 6-
methylisoxazolo[5,4-d]isoxazol-3-yl-aryl-methanones 5 has been achieved via nitro-
nitrite rearrangement by utilizing vinylogous nitroaldol adducts as synthons under mild
conditions. Furthermore, the new series of compounds 5a–i were assessed for molecular
properties prediction, drug-likeness by Molinspiration (Molinspiration, 2008) &
MolSoft (MolSoft, 2007) softwares, lipophilicity and solubility parameters using
ALOGPS 2.1 program. The new series of compounds 5a–i were screened for their anti-
inflammatory activity.

58
 Figure 2.1: classification of anti-inflammatory drugs
Rahul, et al., (2016), had searched novel, safe and effective anti-inflammatory
agents has remained an evolving research enquiry in the mainstream of inflammatory
disorders. In the present investigation series of thiazoles bearing pyrazole as a possible

59
pharmacophore were synthesized and assessed for their anti-inflammatory activity using
in vitro and in vivo methods. In order to decipher the possible anti-inflammatory
mechanism of action of the synthesized compounds, cyclooxygenase I and II (COX-I
and COX-II) inhibition assays were also carried out. The results obtained clearly focus
the significance of compounds 1-(4-(4-Chlorophenyl) thiazol-2-yl)-4-((1,3-diphenyl-1H
pyrazol-4-yl)methylene)-3-methyl-1H-pyrazol-5(4H)-one,1-(4-(4-Chlorophenyl)
thiazol-2-yl)-4-((3-(4-methoxyphenyl)1-phenyl-1H-pyrazol-4-yl) methylene)-3-methyl-
1H-pyrazol-5(4H)one and 3-Methyl-4-((1,3-diphenyl-1H-pyrazol-4-yl) methylene)-1-
(4phenylthiazol-2-yl)-1H-pyrazol-5(4H)-one as selective COX-II inhibitors. Moreover,
compound 1-(4-(4-Chlorophenyl) thiazol-2-yl)-4-((3-(4-methoxyphenyl)1-phenyl-1H-
pyrazol-4-yl)methylene)-3-methyl-1H-pyrazol-5(4H)one was also identified as a lead
molecule for inhibition of the carrageenin induced rat paw edema in animal model
studies. Molecular docking results revealed significant interactions of the test
compounds with the active site of COX-II, which perhaps can be explored for design
and development of novel COX-II selective anti-inflammatory agents.
Santhisudha, et al., (2016), reported that a series of bioactive bis(indolyl)
methanes are synthesized by one-pot green reaction of indole with various substituted
aldehydes by microwave irradiation under solvent free conditions. The antibacterial
activity against Staphylococcus aureus and anti-inflammatory activity of the
synthesized bis (indolyl) methanes are evaluated in vitro and compared to standard
drugs tetracycline and diclofenac, respectively. The majority of the compounds showed
good antibacterial and anti-inflammatory activity. Interestingly, 4 compounds exhibited
much higher anti-inflammatory activity than the standard diclofenac drug and thus
qualify for clinical trials to be used as an anti-inflammatory compound.
Felix, et al., 2010 revealed that the synthesis and in vivo anti-inflammatory
activity of a series of pseudopteros in analogues are presented. Synthetic tricyclic
catechol aglycons with different substitution patterns were monofucosylated or -
xylosylated. Anti-inflammatory activity was conserved over a wide range of structural
modifications. The most active synthetic compound (3R*,9aR*)-4-Hydroxy-3,3-
dimethyl-2,3,7,8,9,9a-hexahydro-1H-phenalen-5-yl6-deoxya-L-galactopyranoside
reduced phorbol myristate acetate (PMA)-induced inflammation in the mouse ear by
72% at 50µg/ear. This corresponds to 80% of the activity of natural pseudopterosin A.

60
 Karim, et al., 2016 revealed that the Abstract The oleanolic acid (I), a natural
pentacyclic triterpenoid, was quantitatively isolated from pomace olive (Oleaeuropaea
L.) under ultra-sonication conditions (6.8g (3.4 mg/g DW)).Two series of oleanolic
acid-1-phenyl-1H-[1,2,3]triazol-4-ylmethylester 6a–g and new oleanolic acid-1-phenyl-
1H-[1,2,3]triazol-5-ylmethylester 7a–g congeners have been designed and synthesized
in an attempt to develop potent anticancer and anti-inflammatory agents. A facile and
regiospecific synthesis of 1,2,3-triazoles catalyzed by Cu(I) (CuAAC) or Ru(II)
(RuAAC) and conducted under microwave conditions of oleanolic acid–alkyne
derivative 5 with various aromatic azides 2 afforded a series of 1,4- and 1,5-triazolyl
derivatives, respectively. Their structures were confirmed by using1HNMR, 13
CNMR,
NOESY and HRMS analysis. Most of the compounds were evaluated for their
anticancer and anti-inflammatory activities. Oleanolic acid I exhibited promising
anticancer activity against murine breast (EMT-6) and human colon (SW480) cancer
cells. Its derivatives (1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl(3b)-3-
hydroxyolean-12-en-28-oate and (1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl-
(3b)3-hydroxyolean-12-en-28-oate (1,4-regioisomers) and (1-(4-methoxyphenyl)-1H-
1,2,3-triazol-5-yl)methyl(3b)-3-hydroxyolean-12-en-28-oate(1,5-regioisomer) were
found to be anticancer agents. On the other hand, only (1-(4-methoxyphenyl)-1H-1,2,3-
triazol-4-yl)methyl(3b)-3-hydroxyolean-12-en-28-oate displays anti-inflammatory
activity.
Kharkar, et al., (2015), reported that the Computational drug repositioning is a
powerful tool to guide the experimental drug repositioning campaigns. Both structure-
based (e.g., reverse docking) and ligand-based (e.g., 3D pharmacophore) approaches
can be used for the generation of repurposing hypotheses. In an attempt to discover
novel anti-inflammatory agents, computational repurposing of approved small molecule
drugs was undertaken using diclofenac (nonselective cyclooxygenase (COX) inhibitor),
celecoxib (selective COX-2 inhibitor) and a potent microsomal prostaglandin E
synthase – 1 (mPGES-1) inhibitor as query molecules for shape-and electrostatics-based
virtual screening. Several approved drugs (other than anti-inflammatory) were amongst
the top 5% of the hits. These hits (approved drugs) may serve as starting points for
clinical repositioning (anti-inflammatory indication) or as lead structures in drug
discovery programs.

61
 Khaled, et al., (2016), reported that the two series of new thiazolidin-4-one
derivatives 4a–c and 8a–e were designed and prepared. All the synthesized compounds
were evaluated for their in vitro COX-2 selectivity and anti-inflammatory activity in
vivo. Compounds 3-(4-Aminosulfonylphenylamino)-2-(4-chlorophenyl)-5methyl-4-
thiazolidinone and 3-(4-Aminosulfonylphenylamino)-2-(4-fluorophenyl)-5methyl-4-
thiazolidinone showed the best overall in vitro COX-2 selectivity (selectivity indexes of
4.56 and 5.68 respectively) and in vivo activities (edema inhibition % = 61.8 and 67
after 3 h, respectively) in comparison with the reference drug celecoxib (S.I. = 7.29,
edema inhibition % = 60 after 3 h). In addition, 3-(4-Aminosulfonylphenylamino)-2-(4-
chlorophenyl)-5methyl-4-thiazolidinone and -(4-Aminosulfonylphenylamino)-2-(4-
fluorophenyl)-5methyl-4-thiazolidinone were evaluated for their mean effective anti-
inflammatory doses (ED50= 27.7 and18.1µmol/kg respectively, celecoxib ED50 = 28.2
µmol/kg) and ulcerogenic liability (reduction in ulcerogenic potential) versus celecoxib
= 85%, 92% respectively. Molecular docking studies were performed and the results
were in agreement with that obtained from the in vitro COX inhibition assays.
Angela, et al., (2016), revealed that the series of substituted 1,5-diarylpyrrole-3-
alkoxyethyl ethers were previously synthesized and the potential anti-inflammatory and
antinociceptive activities of these compounds were evaluated in vivo. The compounds
displayed a very good activity against both carrageenan-induced hyperalgesia and
oedema in the rat paw test. Therefore, in a very preliminary test, compounds (8a, b)
showed anti-proliferative activity in the HaCaT (aneuploid immortal keratinocyte from
adult human skin) cell models. On these basis, this research continued with the
synthesis of fluorinated derivatives (8c,d, 9b-d, and 10b-d) with the aim of improving
the pharmacokinetic profile of the previous active compounds. Substitution of a
hydrogen atom by a fluorine atom may change the conformational preferences of the
molecules due to stereo electronic effects and also fluorine atom may be able to exert
the metabolic obstruction reducing the “first-pass effect”. Compound para-fluoro
derivative exhibited a prominent in vivo anti-inflammatory and antinociceptive
activities, in addition its antiproliferative power in an in vitro model of human skin
cancer is herein described.
Yasodakrishna, et al., (2016), revealed that an efficient three-component
protocol is described for the synthesis of benzo [6,7] cyclohepta [1,2-b] pyridine

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derivatives using b-chloroacroleins, 1,3-dicarbonyls and ammonium acetate under
catalyst free conditions by using ethanol as reaction media. The mild reaction
conditions, operational simplicity and high yields are the advantages of this protocol
and the broad scope of this one-pot reaction makes this procedure promising for
practical usages. All the final compounds were screened for anti-inflammatory activity.
Among the compounds tested, the compounds methyl-2,9,10-trimethyl-6,7-dihydro-5H-
benzo[6,7]cyclohepta[1,2-b] pyridine -3-carboxylate (5a), 5b, 5c, 5d, 5f, and 5k
exhibited significant inhibition of IL-1b and MCP-1 secretion as a measure of anti-
inflammatory activity.
Mayela, et al., (2015), revealed that this study evaluated the potential anti-
inflammatory effects of natural phthalides, isolated from Ligusticum porteri, and of
semi-synthetic phthalides. Anti-inflammatory activity was investigated in two mouse
models; one with ear edema, induced with 12-O-tetradecanoylphorbol-13-acetate, and
the other with paw edema, induced with carrageenan. The effect on the RAW 264.7
stimulated with lipopolysaccharide cells was evaluated and after application of 12-O-
tetradecanoylphorbol-13-acetate, the activity of myeloperoxidase was assessed to serve
as an index of leukocytes infiltration together with the histological evaluations. We also
assessed the inhibition of cyclooxygenases 1 and 2 in vitro. Our results demonstrated
that administration of semi-synthetic phthalides significantly inhibited the ear edema
induced by 12-O-tetradecanoylphorbol-13-acetate, and reduced the paw edema caused
by carrageenan. The anti-inflammatory activity of phthalides could, in part, be
explained by the reduction in myeloperoxidase activity and the infiltration of
leukocytes. The semi-synthetic phthalides also inhibited the production of oxide nitric
in RAW cells.

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Aim and Objectives