Pharmacophore mapping, molecular docking, chemical synthesis of some novel pyrrolyl benzamide derivatives and evaluation of their inhibitory activity against enoyl-ACP reductase (InhA) and Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.bioorg.2018.08.035

In an effort to produce new lead antimycobacterial compounds, herein we have reported the synthesis of a sequence of new pyrrolyl benzamide derivatives. The new chemical entities were screened to target enoyl-ACP reductase enzyme, which is one of the key enzymes of M. tuberculosis that are involved in type II fatty acid biosynthetic pathway. Compound 3q exhibited H-bonding interactions with Tyr158, Thr196 and co-factor NAD⁺ that binds the active site of InhA. All the pyrrolyl benzamide compounds were evaluated as inhibitors of M. tuberculosis H₃₇Rv as well as inhibitors of InhA. Among them, few representative compounds were tested for mammalian cell toxicity on the human lung cancer cell-line (A549) and MV cell line that presented no cytotoxicity. Five of these compounds exhibited a good activity against InhA.

Full text of DOI:10.1016/j.bioorg.2018.08.035

Accepted Manuscript
Pharmacophore mapping, molecular docking, chemical synthesis of some novel
pyrrolyl benzamide derivatives and evaluation of their inhibitory activity against
enoyl-ACP reductase (InhA) and Mycobacterium tuberculosis
Shrinivas D. Joshi, Sheshagiri R. Dixit, Jeelan Basha, V.H. Kulkarni, Tejraj M.
Aminabhavi, Mallikarjuna N. Nadagouda, Christian Lherbet
PII:
S0045-2068(18)30686-2
DOI:
https://doi.org/10.1016/j.bioorg.2018.08.035
YBIOO 2492
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
10 July 2018
24 August 2018
27 August 2018
Please cite this article as: S.D. Joshi, S.R. Dixit, J. Basha, V.H. Kulkarni, T.M. Aminabhavi, M.N. Nadagouda, C.
Lherbet, Pharmacophore mapping, molecular docking, chemical synthesis of some novel pyrrolyl benzamide
derivatives and evaluation of their inhibitory activity against enoyl-ACP reductase (InhA) and Mycobacterium
tuberculosis, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.08.035
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Pharmacophore mapping, molecular docking, chemical synthesis of some novel pyrrolyl
benzamide derivatives and evaluation of their inhibitory activity against enoyl-ACP
reductase (InhA) and Mycobacterium tuberculosis
Shrinivas D. Joshi1*, Sheshagiri R. Dixit1, Jeelan Basha2, V.H. Kulkarni1, Tejraj M.
Aminabhavi1, Mallikarjuna N. Nadagouda1, Christian Lherbet3
1Novel Drug Design and Discovery Laboratory, Department of Pharmaceutical Chemistry,
S.E.T’s College of Pharmacy, Sangolli Rayanna Nagar, Dharwad, Karnataka, India
2Department of PG Studies and Research in Chemistry, Vijayanagar College, Hosapete,
Karnataka, India
3Universite de Toulouse, UPS, Laboratoire de Synthese et Physico-chimie de Molecules
d’Interet Biologique, LSPCMIB, 118 Roote de Narbonne, F-31062, Toulouse Cedex 9,
France.
Corresponding Author
Shrinivas D. Joshi, Novel Drug Design and Discovery Laboratory, Department of
Pharmaceutical Chemistry, Soniya Education Trust’s, College of Pharmacy, Sangolli
Rayanna Nagar, Dharwad- 580 002, India.
E-mail: shrinivasdj@rediffmail.com. Tel.: +91 9986151953; Fax; +91 836 2467190
1

Abstract:
In an effort to produce new lead antimycobacterial compounds, herein we have reported the
synthesis of a sequence of new pyrrolyl benzamide derivatives. The new chemical entities
were screened to target enoyl-ACP reductase enzyme, which is one of the key enzymes of M.
tuberculosis that are involved in type II fatty acid biosynthetic pathway. Compound 3q
exhibited H-bonding interactions with Tyr158, Thr196 and co-factor NAD+ that binds the
active site of InhA. All the pyrrolyl benzamide compounds were evaluated as inhibitors of M.
tuberculosis H37Rv as well as inhibitors of InhA. Among them, few representative
compounds were tested for mammalian cell toxicity on the human lung cancer cell-line
(A549) and MV cell line that presented no cytotoxicity. Five of these compounds exhibited a
good activity against InhA.
2

Keywords: Pyrroles, Tuberculosis, Enoyl-ACP reductase, InhA, GALAHAD.
3

1. Introduction
Tuberculosis (TB) is a disorder caused by various strains of mycobacteria such as
Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum,
Mycobacterium microti and Mycobacterium canetti. Among these, Mycobacterium
tuberculosis (M. tuberculosis) remains to be the major cause for TB, which is responsible for
worldwide infecting nearly 8 million and killing more than 2 million people every year. The
World Health Organization (WHO) has estimated that one-third of the world’s population is
infected by M. tuberculosis [1] of which India stands for one-fifth of the world. Therefore,
TB is a chronic communicable disease that not only attacks the lungs, but also affects other
vital parts of the body viz., spine, kidney, and brain if it is not treated appropriately in time.
Currently, there is an urgent need to develop new chemotherapeutics to combat TB,
especially multidrug and extensive drug resistivity. Even though innumerable new drugs have
appeared in the market, yet drug-resistance is a formidable issue. For this reason, the need to
develop novel anti-TB agents that are active against both acute and chronic growth phases of
mycobacterium in order to stop all types of drug resistant-TB is necessary. Recent literature
[2] has shown that most of the synthesized molecules were targeted to cell wall of
mycobacteria, mainly mycolic acid biosynthetic pathway alike to fatty acid synthase (FAS)
systems, such as FAS I and II. Mycolic acid is a mainstay constituent of mycobacterial cell
wall present in the FAS system of M. tuberculosis. InhA, the enoyl acyl carrier protein
reductase (ENR) from the M. tuberculosis, is the main enzyme for type II fatty acid synthesis
(FAS II), which catalyses the NADH-dependent reduction of 2-trans-enoyl-ACP (acyl carrier
protein) to give up NAD+ and reduced enoyl thioester-ACP substrate, which in turn, aids in
the synthesis of mycolic acid.
Pyrrole has been one of the important heterocycles that exist in plant (naturally occurring
chlorophyll) and animal kingdom (Hemin and vitamin-B12). Deidda et al. [3] reported for the
4

first time anti-TB activity of BM212, a derivative of pyrroles, shortly after that a number of
derivatives of BM212 have been synthesized [4]. Based on these findings, a sequence of
pyrrole compounds have been synthesized by Lupin, among them LL3858 is presently in pre-
clinical studies for the treatment of TB [5]; this suggests the significance of pyrrole based
analogs as anti-TB agents.
In efforts to universal fight against TB, from our laboratory several new chemical entities
have been designed and synthesized as possible inhibitors of InhA having pyrrole as the main
fragment (Fig. 2) [6-8] and these exhibited a considerable antitubercular activity. Keeping in
view of the biological and pharmacological potential of pyrroles and the importance of
peptide linkage, herein we have designed such new chemical entities comprising of both
pyrrole and peptide linkages as antitubercular and antibacterial agents.
Although there are many promising compounds with a significant anti-TB profile that act as
enoyl-ACP reductase enzyme inhibitors, but none of them are available in the market since
these are still in their early developmental stage. This has prompted us to carry out
comprehensive study on designing using pharmacophore mapping and synthesis of straight
enoyl-ACP reducatse inhibitors having pyrrolyl benzamide as the centre unit using in silico
methods that involve GALAHAD (pharmacophore based on an automated computational
alignment technique) as well as Surflex-docking studies to recognize the enoyl-ACP
reductase as a possible target for these pyrrolyl benzamide analogs. With these approaches,
we have established the quantitative pharmacophore mapping method, which is useful to
identify the structural and physicochemical needs for ligand binding and biological activity
[9] along with molecular modeling studies on pyrrolyl benzamide analogs as the potential
inhibitors of InhA and performed their antibacterial, antitubercular (Anti-TB) and cytotoxic
activities.
5

Hybridization approach was used here to construct the design perception for
novel anti-TB and antimicrobial derivatives. The derivatives of benzamide have exhibited
promising anti-TB activities, inhibiting the bacteria by blocking InhA [10]. Hence, novel
anti-TB agents are designed among benzamide and pyrrole moieties using the hybridization
approach. In continuation of our broad-based programm of research, we develop here to map
pharmacophore from the structure of 1-cyclohexyl-N-(3,5-dichlorophenyl)-5-oxopyrrolidine-
3-carboxamide, containing carboxamide H-bond acceptor and donor bridge between 5-
oxopyrrolidine and dichlorophenyl. In addition, it has three hydrophobic moieties viz., 5-
oxopyrrolidine, cyclohexyl and dichlorophenyl. Hence, 5-oxopyrrolidine and carboxamide
were mapped with a carboxamide linkage (-CO-NH-) that will support the interactions with
the hydroxyl group of the key residue Tyr158 of InhA [11] and at the same time, hydrophobic
moieties were mapped with o/m/p substituted phenyl derivatives and pyrrole ring as shown in
Fig. 1. In Fig. 2, few compounds reported before as the anti-TB agents, were considered for
the synthesis of new chemical entities following the Pall-Knorr pyrrole synthesis [12]. Up till
now, no such reports have appeared in the literature on these compounds and hence, our work
is innovative.
6

2. Computational Details.
2.1. General method
The crystal structure of M. tuberculosis enoyl reductase (InhA) complexed with 1-
cyclohexyl-N-(3,5-dichlorophenyl)-5-oxopyrrolidine-3-carboxamide (PDB ID 4TZK, 1.62 Å
X-ray resolution) was extracted from the Brookhaven Protein Database (PDB
http://www.rcsb.org/pdb). In the present situation, biopolymer and each molecule in the data
set was energetically minimized by employing the Tripos force field [13], Powell
optimization method [14], Amber7FF9902 (biopolymer) and MMFF94 (molecules) charges
(NB cut-off 9.0 and dielectric constant 4.0) with a convergence criterion set at 0.001 kcal/mol
Å. The pharmacophore models were generated and analyzed using GALAHAD (Genetic
Algorithm with Linear Assignment of Hypermolecular Alignment of Datasets) module. All
calculations were performed on a commercially available SYBYL-X 2.0 software package
(Tripos Associates, St. Louis, MO, USA) [15].
Modeling protein-ligand interactions is an important step in modern drug designing. If the
structure of a binding site is known, then docking studies will provide valuable insights into
such interactions and, in some favourable cases, one can identify the high-affinity ligands by
virtual high-throughput screening. The pharmacophore-based 3D searching also proved
particularly useful [16]. It is characterized in terms of subgroups of two, three or four
features, and arrangement of particular characteristics that are necessary for ligand binding as
well as spatial relationship between them; thus provides a fast and flexible tool for carrying
out these searches. The GALAHAD [17] was also utilized in the formulation of
pharmacophore hypothesis for the synthesized molecules, which is a unique method since it
does not require any template structure, but allows for immediate and effective generation of
partial-coverage models with multiple partial match constraints [18].
7

2.2. Alignment and pharmacophore generation
A total of 22 pyrrolyl benzamide anti-TB compounds were used as data set in the
identification of a feature-based pharmacophore and docking analysis. The alignment was
performed in two steps. In the first step, five compounds (3a, 3b, 3k, 3n and 3q labeled with
asterisks as shown in Table 1) were selected to carry out the pharmacophore hypothesis; the
genetic algorithm was used here to create conformers for all the molecules. The compounds
that were selected to generate pharmacophore hypothesis are highly active. All the selected
ligands were aligned flexibly by GALAHAD, completely independent of a template, with a
population size of 45 and a maximum generation value of 50 with molecular required the
hitting of 4. Twenty models were generated with default parameters and using all the
molecules in this stage to generate the flexible alignment that may lead to some features by
neglecting the interaction [19]. The biological activity of each compound was expressed as
minimal inhibitory concentration (MIC) against M. tuberculosis and the MIC values were
used in pharmacophore analysis.
Using the flexible alignment, GALAHAD produced a set of probable hypotheses (Table 3, 20
models). SPECIFICITY is a logarithmic indicator of the expected discrimination for each
query. The actual number hit is given in the N_HITS column and the values in FEATS
column indicate the total number of features in the model query. The next five columns are
model score components from the genetic algorithm. PARETO is a pareto rank of each
model, where all the models have a Pareto rank of zero. This means none of the models are
superior to any other when using all the four criteria in columns 5-8 (ENERGY, STERICS,
H_BOND, and MOL_QRY). ENERGY calculates the total energy of the model; STERICS is
a steric overlap for the model; H_BOND, the pharmacophoric concordance and MOL_QRY
is an agreement between the query tuplet and pharmacophoric tuplets for the ligands as a
8

group. The last four columns are the scores for individual ligands within each model. Thus, in
this case, every cell in each of the last four columns contains a list of five values.
In the second step, for rigid alignment of the remaining molecules in data set, we need to
select one best template model and the selection of the model from obtained 20 models in the
first stage based on the model needed to “hit” all the 5 active molecules. The model needed to
have high sterics with low energy and pharmacophoric features. We have constructed a
scatter plot (ENERGY vs STERICS vs MOL_QRY) to visualize the Pareto surface and
selected the best pharmacophore model (Fig. 3). Considering the ENERGY, STERICS and
MOL_QRY criteria, the best model is shown in the graph, where the ENERGY is reasonably
low and STERICS score is high. Among the considered models, MODEL_14 (represented
with a black circle in Fig. 3) has the optimal position because it fulfils all the three criteria
and has better Specificity, N_hits and Feats values [17,20,21].
Finally, the associated pharmacophore (MODEL_14) was used as a template to align all the
molecules using GALAHAD’s Align to template procedure (Table 4). Here, model fitness
evaluation starts by applying the corresponding torsions to the specified base configuration of
each ligand. Pharmacophore and steric bitmaps were created for each ligand, and
(compressed) the count vectors were then generated for the ensemble. Next, the post-
processing step in GALAHAD involves taking the genetic algorithm results and producing
the final models along with their alignments, scoring of the models, and displaying their rank.
In GALAHAD, the needed frame of reference is generated via post processing using
hypermolecular alignment program LAMDA (Linear Assignment for Molecular Dataset
Alignment) [19].
2.3. Surflex-docking
In order to explore the interaction and illustrate accurate binding model for the active site of
ENR with ligands, molecular docking was performed using Surflex-dock module of another
9

advanced version of SYBYL package (X 2.0). This docking approach aligns the ligand to a
“protomol” (called also idealized ligand) in the active site of the target. Surflex-Dock that
adopted an empirical scoring function and a patented search engine [22,23] was employed for
molecular docking study of the training set as well as the test set molecules into the active
site of the crystal structure of ENR catalytic core.
10

3. Results and discussion
3.1. Chemistry
All the new molecules were synthesized as per Scheme 1. The Pall Knorr pyrrole
synthesis involves the reaction of amine with diketone viz., 2,5-dimethoxytetrahydrofuran,
which is the common method for the synthesis of pyrrole ring and hence, 4-(1H-pyrrol-1-
yl)benzoic acid (2) was obtained by constructing pyrrole ring using amino group present at
the 4th position of 4-aminobenzoic acid (1) in the presence of dried glacial acetic acid.
Further, the titled compounds (3a-q) were synthesized by reacting the substituted aromatic
amines with 4-(1H-pyrrol-1-yl)benzoic acid (2) using peptide forming agents such as 2-(1H-
benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HBTU) and diisopropyl
ethylamine (DIEA). All the new compounds were purified by column chromatography and
their structures were confirmed with spectral data.
FTIR spectra of the compounds exhibited absorption bands at 3357-3140 cm-1 due to
secondary amine stretching, while the characteristic carbonyl stretches have appeared around
1
1663-1605 cm-1, confirming the formation of compounds 3a-q. The H NMR spectra
exhibited typical triplet signal for two protons of pyrrole at C3 and C4 positions with the δ
values of 5.75-6.42 ppm and aromatic proton signals (doublet, doublet of doublet, doublet of
triplet, triplet, triplet of doublet and multiplet) between δ 6.33 and 8.16 ppm. In the 13C NMR
spectra, C=O carbon resonance frequency was observed around δ of 164.25-168.01 ppm,
while the remaining aromatic carbons appeared around δ of 44.20-160.95 ppm. In addition,
structures of all the compounds were established by EI-MS (Electron Impact Ionization)
spectra that showed the molecular ion [M+], while all the spectroscopic measurements
confirmed the structures and their high purity.
11

3.2. Antitubercular and cytotoxicity studies
Antimycobacterial activity of the compounds was carried out with M. tuberculosis (Table 1).
INH and TCL were used as standard drugs for inhibitory activity against M. tuberculosis.
Most test derivatives exhibited quite moderate to good anti-TB activity. The investigation of
anti-TB activity has shown excellent anti-TB activity at the MIC values for all compounds
ranging from 3.125 to 25 µg/ml. In particular, compounds 3a, 3i, 3l, 3n and 3q exhibited
MIC value of 3.125 µg/ml. Some therapeutic properties are necessary to be considered if
antimycobacterial compound should have the potential to be a drug. Toxicity is one of these
significant criteria. Therefore, we have carried out the potential toxicity of five selected
pyrrolyl benzamide analogs (3a, 3i, 3l, 3n and 3q) towards mammalian Vero cell-lines and
A549 (lung adenocarcinoma) cell-lines up to concentrations of 62.5 µg/mL. These compounds
exhibited a reasonable cytotoxicity compared to standard INH (see Table 2).
3.3 Enzyme inhibition studies
Ten selected compounds were further tested for in vitro enzyme inhibition activity against
InhA from M. tuberculosis at 50 µM (and/or at 5 µM) (Table 1) using triclosan as a standard
drug, which showed a total inhibition of InhA enzyme. Out of ten compounds tested, five
compounds exhibited similar activities against InhA i.e., 99% inhibition at 50 µM.
3.4. Bacterial growth inhibition experiments with Gram positive and Gram negative bacteria
Among all the synthesized molecules, five compounds were selected for antibacterial activity
against both Gram positive and Gram negative bacterial strains and the results are presented
in Table 2. All the molecules show promising antibacterial activities in the MIC values of
3.12-12.5 µg/mL. For this study, ciprofloxacin was used as the control drug for antibacterial
activity. In general, all the compounds have demonstrated better activity against Gram
positive bacteria S. aureas than the Gram negative bacteria E. coli.
12

3.5. Pharmacophore results
Sum of 20 GALAHAD models were derived using five active molecules with the MIC values
of 3.12-6.25 µg/mL against M. tuberculosis. All the models showed Pareto rank 0, which
means no one model was superior to any other (Table 3). Among the 20 models tested,
MODEL_14 was chosen which exhibited the superior steric values, features, hits and sensibly
lower energy than the others. Therefore, MODEL_14 was considered for the final rigid
alignment of the left over compounds. The final MODEL_14 obtained from GALAHAD
displayed in Figure 4A-B is comprised of 5 substructures, one conformer for each molecule
from the selected active 5 molecules as the training set. This contains hydrophobes centered
on benzene and pyrrole rings, but the acceptor atom centers on oxygen atom of CONH
linkage. The pharmacophore model evidently shows the significance of hydrophobic phenyl
and pyrrole rings with polar oxygen to show the anti-TB activity.
3.6 Molecular docking results
To explore the mechanism of anti-TB activity and exhaustive intermolecular interactions
between the synthesized compounds, molecular docking studies were performed on the
crystal structure of M. tuberculosis enoyl reductase (InhA) complexed with 1-cyclohexyl-N-
(3,5-dichlorophenyl)-5-oxopyrrolidine-3-carboxamide (PDB ID 4TZK, 1.62 Å X-ray
resolution) using the surflex-dock programme of sybyl-X 2.0 software. Due to the fact that
larger level of resistance associated with INH isolates against InhA, docking studies were
done on InhA complex with 1-cyclohexyl-N-(3,5-dichlorophenyl)-5-oxopyrrolidine-3-
carboxamide, which indicates the presence of drug-receptor interactions.
Docking studies with Surflex-Dock demonstrated that almost all the synthesized molecules
have engaged the same binding sites as those of pyrrolidine carboxamide (Fig. 5A-B).
The 4TZK exhibited two H-bonding interactions, the oxygen of carbonyl group on
pyrrolidine makes H-bonds with that of OH of the active site of Tyr158 (2.41 Å) and NAD+
13

ribose (1.82 Å) (Fig. 6A-C). The compound 3q also binds in the same pattern i.e., each
oxygen atom of nitro group has made two H-bonding interactions, one with amino acid
Try158 (2.07, Å) and another with co-factor NAD+ (2.27 Å) (Fig. 7A-C). Figure 8A-B
represents the hydrophobic and hydrophilic amino acids encircled to the studied compounds
3a and 3q.
The compounds exhibited consensus score in the range of 6.20-2.80, signifying the necessity
of all forces of interaction between ligands and the InhA. Charge and van der Waals
interactions between the protein and ligands varied from -139.80 to -72.27. The Helmholtz
free energies of interactions for protein-ligands atom pairs that ranged between -68.16 and -
19.91 and its H-bonding, complex (ligand-protein), and internal (ligand-ligand) energies
range from -265.28 to -145.13, while those values ranging from -40.66 to -24.78 indicate the
ligands due to the presence of H-bonding, lipophilic contact, and rotational entropy. These
scores designate that the compounds preferentially bind to the InhA in comparison with
reference 4TZK (Table 5).
14

4. Experimental section
Melting points were determined using the Shital-digital programmable melting point
apparatus and are uncorrected. FTIR spectra in KBr pellets were recorded on a Bruker FTIR
1
13
spectrophotometer. The H and C NMR spectra were recorded on a Bruker AVANCE II at
400 and 100/75 MHz, respectively; chemical shifts are expressed in parts per million (δ ppm)
relative to TMS. Abbreviations used to describe the peak patterns are: (b) broad, (s) singlet,
(d) doublet, (t) triplet, (q) quartet, (dd) doublet of doublet, (brs) broad singlet, (q) quinet and
(m) multiplet.
Mass spectra (MS) were recorded using a JEOL GCMATE II GC-Mass spectrometer and
Schimadzu QP 20105 GC-Mass spectrometer. Analytical thin-layer chromatography (TLC)
was performed on precoated TLC sheets of silica gel 60 F254 (Merck, Darmstadt, Germany)
visualized by long- and short-wavelength UV lamps. Chromatographic purifications were
performed on Merck aluminium oxide (70-230 mesh) and Merck silica gel (70-230 mesh).
4.1 Synthesis of N-substituted-4-(1H-pyrrol-1-yl)benzamide derivatives (3a-q)
The 4-(1H-pyrrol-1-yl)benzoic acid (2) (0.0018 mol) and substituted aromatic amines (0.0019
mol) were dissolved in 50 mL of dry dimethyl formamide. HBTU (0.87 g, 0.0023 mol) and
DIEA (0.93 mL, 0.0053 mol) were then added to the above mixture and stirred for 24-30 h at
room temperature. The reaction was quenched by adding sodium chloride solution and
mixture was extracted with ethyl acetate (3 x 50 mL). The combined ethyl acetate layer was
washed with 1 N HCl and with saturated sodium bicarbonate, followed by sodium chloride.
The organic layer was dried over anhydrous sodium sulphate and was concentrated using a
rotary flash evaporator. Thus obtained residue was dried and purified by column
chromatography using petroleum ether:ethyl acetate (6:4) mixture as the eluent to afford the
compounds (3a-q).
4.1.1. N-(4-Chlorophenyl)-4-(1H-pyrrol-1-yl)benzamide (3a):
15

(Yield 58 %). mp 188-190 oC; FTIR (KBr): 3327 (-NH), 3097 (Ar-H), 1610 (C=O), 1242 (C-
O-C) cm-1; 1H NMR (400 MHz, DMSO-d6) δ ppm: 10.31 (s, 1H, NH), 8.07 (d, 2H, J = 8 Hz,
bridgingph-C3, C5-H), 7.84 (d, 2H, J = 8 Hz, bridgingph-C2, C6-H), 7.71 (d, 2H, chloroph- C2,
C6-H), 7.40 (s, 2H, chloroph-C3, C5-H), 7.35 (d, 2H, J = 12 Hz, pyrrole-C2, C5-H), 6.30 (t,
13
2H, pyrrole-C3, C4-H); C NMR (75 MHz, DMSO-d6) δ ppm: 111.62, 111.88, 117.21,
117.45, 119.22, 119.87, 123.92, 126.26, 127.70, 127.88, 127.92, 129.66, 130.23, 131.52,
142.46, 166.22; MS (EI): m/z = found 296.88 [M+]; calcd. 296.75. Anal. Calcd. For
C17H13ClN2O: C, 68.81; H, 4.42; N, 9.44. Found: C, 68.78; H, 4.41; N, 9.40.
4.1.2. N-(4-Bromo-2,6-dichlorophenyl)-4-(1H-pyrrol-1-yl)benzamide (3b):
(Yield 55 %). mp 78-80 oC; FTIR (KBr): 3300 (-NH), 3082 (Ar-H), 1611 (C=O), 1211 (C-O-
1
C) cm-1; H NMR (400 MHz, DMSO-d6) δ ppm: 8.58 (s, 1H, NH), 7.51 (d, 2H, J = 4 Hz,
bridgingph-C3, C5-H), 7.41 (t, 4H, bridgingph-C2, C6-H and ph- C3, C5-H), 7.08-7.10 (m, 2H,
13
pyrrole-C2, C5-H), 6.36 (t, 2H, pyrrole-C3, C4-H). C NMR (75 MHz, DMSO-d6) δ ppm:
107.96, 111.22, 120.03, 130.25, 130.27, 130.28, 130.30, 130.31, 139.45, 166.20; MS (EI):
m/z = found 410.10 [M+]; calcd. 410.09. Anal. Calcd. For C17H11BrCl2N2O: C, 49.80; H,
2.71; N, 6.80. Found: C, 49.79; H, 2.70; N, 6.83.
4.1.3. N-(3,5-Dichlorophenyl)-4-(1H-pyrrol-1-yl)benzamide (3c):
(Yield 77 %). mp 160-162 oC; FTIR (KBr): 3260 (-NH), 3072 (Ar-H), 1606 (C=O), 1227 (C-
O-C) cm-1; 1H NMR (400 MHz, DMSO-d6) δ ppm: 7.99 (s, 1H, NH), 7.95 (d, 2H, J = 8 Hz,
bridgingph-C3, C5-H), 7.66 (s, 2H, bridgingph-C2, C6-H), 7.52 (d, 2H, J = 8 Hz, chloroph-C2,
C6-H), 7.28 (s, 1H, chloroph-C4-H), 7.18 (t, 2H, pyrrole-C2, C5-H), 6.42 (t, 2H, pyrrole-C3,
C4-H); 13C NMR (75 MHz, DMSO-d6) δ ppm: 110.25, 111.62, 117.35, 117.78, 118.56,
119.58, 1243.77, 127.36, 127.78, 127.88, 127.96, 129.52, 130.55, 131.44, 143.28, 167.20;
MS (EI): m/z = found 331.55 [M+]; calcd. 331.20. Anal. Calcd. For C17H12Cl2N2O: C, 61.65;
H, 3.65; N, 8.46. Found: C, 61.66; H, 3.64; N, 8.44.
16

4.1.4. N-Phenyl-4-(1H-pyrrol-1-yl)benzamide (3d):
(Yield 68 %). mp 216-218 oC; FTIR (KBr): 3237 (-NH), 3043 (Ar-H), 1644 (C=O), 1250 (C-
O-C) cm-1; 1H NMR (400 MHz, DMSO-d6) δ ppm: 9.37 (s, 1H, NH), 7.92 (d, 2H, J = 8 Hz,
bridgingph-C3, C5-H), 7.49 (d, 2H, J = 12 Hz, bridgingph-C2, C6-H), 7.15-7.27 (m, 6H,
pyrrole-C2, C5-H and ph-C2, C3, C5, C6-H), 6.95 (t, 1H, ph-C4-H), 6.40 (t, 2H, pyrrole-C3, C4-
H). 13C NMR (75 MHz, DMSO-d6) δ ppm: 111.56, 120.32, 127.91, 148.28, 149.01, 168.01;
MS (EI): m/z = found 262.38 [M+]; calcd. 262.31. Anal. Calcd. For C17H14N2O: C, 77.84; H,
5.38; N, 10.68. Found: C, 77.60; H, 5.39; N, 10.66.
4.1.5. N-(2-Chlorophenyl)-4-(1H-pyrrol-1-yl)benzamide (3e):
(Yield 52%). mp 158-160 oC; FTIR (KBr): 3351 (-NH), 2977 (Ar-H), 1605 (C=O), 1276 (C-
1
O-C) cm-1. H NMR (400 MHz, DMSO-d6) δ ppm: 8.23 (s, 1H, NH), 8.11-8.13 (t, 2H,
bridgingph-C3, C5-H), 6.65-7.60 (m, 8H, bridgingph-C2, C6-H, chloroph-C3, C4, C5, C6-H and
13
pyrrole-C2, C5-H), 6.37-6.40 (t, 2H, pyrrole-C3, C4-H). C NMR (75 MHz, DMSO-d6) δ
ppm: 111.89, 119.22, 119.51, 127.57, 129.76, 143.33, 162.42, 165.83; MS (EI): m/z = found
296.58 [M+]; calcd. 296.75. Anal. Calcd. For C17H13ClN2O: C, 68.81; H, 4.42; N, 9.44.
Found: C, 68.88; H, 4.40; N, 9.48.
4.1.6. 3-Chlorophenyl)-4-(1H-pyrrol-1-yl)benzamide (3f):
(Yield 52 %). mp 178-180 oC; FTIR (KBr): 3325 (-NH), 2974 (Ar-H), 1656 (C=O), 1253 (C-
O-C) cm-1; 1H NMR (400 MHz, DMSO-d6) δ ppm: 9.82 (s, 1H, NH), 7.54 (d, 2H, J = 12 Hz,
bridgingph-C3, C5-H), 6.73-7.29 (m, 7H, bridgingph-C2, C6-H, chloroph-C2, C5, C6-H and
pyrrole-C2, C5-H), 6.28-6.33 (m, 1H, chloroph-C4-H), 5.75-5.79 (t, 2H, pyrrole-C3, C4-H).
13C NMR (75 MHz, DMSO-d6) δ ppm: 111.59, 118.95, 119.46, 127.36, 129.11, 129.59,
130.86, 132.44, 136.81, 142.49, 166.19; MS (EI): m/z = found 296.88 [M+]; calcd. 296.75.
Anal. Calcd. For C17H13ClN2O: C, 68.81; H, 4.42; N, 9.44. Found: C, 68.88; H, 4.40; N, 9.45.
4.1.7. N-(4-Hydroxyphenyl)-4-(1H-pyrrol-1-yl)benzamide (3g):
17

(Yield 58%). mp 202-204 oC; FTIR (KBr): 3333 (-NH), 2919 (Ar-H), 1610 (C=O), 1255 (C-
O-C) cm-1; 1H NMR (400 MHz, DMSO-d6) δ ppm: 9.95 (s, 1H, -OH), 9.17 (s, 1H, NH), 8.05
(s, 1H, bridgingph-C3, C5-H), 7.55 (s, 4H, bridgingph-C2, C6-H and pyrrole-C2, C5-H), 7.29
(s, 2H, hydroxyph-C2, C6-H), 6.77 (s, 2H, hydroxyph-C3, C5-H), 6.29 (t, 2H, pyrrole-C3, C4-
13
H); C NMR (75 MHz, DMSO-d6) δ ppm: 111.23, 119.45, 119.76, 127.55, 129.23, 129.69,
131.22, 132.88, 136.88, 143.25, 167.49; MS (EI): m/z = found 278.44 [M+]; calcd. 278.31.
Anal. Calcd. For C17H14N2O2: C, 73.37; H, 5.07; N, 10.07. Found: C, 73.10; H, 5.06; N,
10.06.
4.1.8. N-(2-Fluorophenyl)-4-(1H-pyrrol-1-yl)benzamide (3h):
(Yield 59%). mp 80-82 oC; FTIR (KBr): 3302 (-NH), 2929 (Ar-H), 1605 (C=O), 1274 (C-O-
C) cm-1; 1H NMR (400 MHz, DMSO-d6) δ ppm: 8.36-8.43 (m, 1H, NH), 7.07-8.13 (m, 10H,
bridgingph-C2, C3, C5, C6-H, fluoroph-C3, C4, C5, C6-H and pyrrole-C2, C5-H), 6.34-6.39 (t,
2H, pyrrole-C3, C4-H). 13C NMR (75 MHz, DMSO-d6) δ ppm: 111.71, 111.95, 116.14,
116.41, 119.08, 119.47, 124.78, 126.23, 127.50, 127.69, 127.97, 129.98, 130.57, 131.34,
142.77, 165.10; MS (EI): m/z = found 280.35 [M+]; calcd. 280.30. Anal. Calcd. For
C17H13FN2O: C, 72.85; H, 4.67; N, 9.99. Found: C, 72.44; H, 4.66; N, 9.97.
4.1.9. N-(3-Fluorophenyl)-4-(1H-pyrrol-1-yl)benzamide (3i):
(Yield 76%). mp 86-88 oC; FTIR (KBr): 3140 (-NH), 2975 (Ar-H), 1606 (C=O), 1189 (C-O-
1
C) cm-1; H NMR (400 MHz, DMSO-d6) δ ppm: 9.89 (s, 1H, NH), 8.08 (d, 2H, J = 8 Hz,
bridgingph-C3, C5-H), 7.90 (d, 2H, J = 8 Hz, fluoroph-C2-H), 7.44-7.67 (m, 4H, bridgingph-
C2, C6-H and fluoroph- C5, C6-H), 7.27-7.37 (m, 3H, pyrrole-C2, C5-H and fluoroph-C4-H),
13
6.26-6.29 (t, 2H, pyrrole-C3, C4-H). C NMR (75 MHz, DMSO-d6) δ ppm: 107.70, 110.69,
111.36, 111.92, 116.53, 118.89, 119.13, 119.46, 123.41, 127.96, 129.94, 130.72, 131.34,
141.45, 142.76, 160.95, 165.43; MS (EI): m/z = found 280.39 [M+]; calcd. 280.30. Anal.
Calcd. For C17H13FN2O: C, 72.85; H, 4.67; N, 9.99. Found: C, 72.96; H, 4.68; N, 9.97.
18

4.1.10. N-(4-Methoxyphenyl)-4-(1H-pyrrol-1-yl)benzamide (3j):
(Yield 78 %). mp 198-200 oC; FTIR (KBr): 3330 (-NH), 2954 (Ar-H), 1645 (C=O), 1246 (C-
1
O-C) cm-1; H NMR (400 MHz, DMSO-d6) δ ppm: 10.10 (s, 1H, NH), 8.16 (dd, 2H, J =
4Hz, 4Hz, bridgingph-C3, C5-H), 7.44-7.67 (m, 4H, bridgingph-C2, C6-H and methoxyph-C2,
C6-H) 7.31 (s, 2H, pyrrole-C2, C5-H), 6.69-6.91 (m, 2H, methoxyph-C3, C5-H), 6.28 (t, 2H,
13
pyrrole-C3, C4-H). C NMR (75 MHz, DMSO-d6) δ ppm: 55.67, 111.62, 114.23, 118.87,
119.48, 122.58, 129.73, 131.71, 132.65, 142.47, 164.25; MS (EI): m/z = found 292.55 [M+];
calcd. 292.34. Anal. Calcd. For C18H16N2O2: C, 73.95; H, 5.52; N, 9.58. Found: C, 73.85; H,
5.50; N, 9.57.
4.1.11. N-(2-Bromophenyl)-4-(1H-pyrrol-1-yl)benzamide (3k):
o
(Yield 76%). mp 98-100 C; FTIR (KBr): 3139 (-NH), 2926 (Ar-H), 1607 (C=O), 1295 (C-
O-C) cm-1; MS (EI): m/z = found 341.42 [M+]; calcd. 341.21. Anal. Calcd. For C17H13BrN2O:
C, 59.54; H, 3.82; N, 8.20. Found: C, 59.84; H, 3.84; N, 8.21.
4.1.12. N-(2,3-Dichlorophenyl)-4-(1H-pyrrol-1-yl)benzamide (3l):
(Yield 52%). mp 138-140 oC; FTIR (KBr): 3357 (-NH), 2926 (Ar-H), 1605 (C=O), 1278 (C-
O-C) cm-1; MS (EI): m/z = found 331.25 [M+]; calcd. 331.20. Anal. Calcd. For
C17H12Cl2N2O: C, 61.65; H, 3.65; N, 8.46. Found: C, 61.46; H, 3.64; N, 8.45.
4.1.13. N-Benzyl-4-(1H-pyrrol-1-yl)benzamide (3m):
(Yield 49%). mp 148-150 oC; FTIR (KBr): 3333 (-NH), 2923 (Ar-H), 1636 (C=O), 1245 (C-
O-C) cm-1; 1H NMR (400 MHz, DMSO-d6) δ ppm: 8.99 (t, 1H, NH), 8.02 (d, 2H, J = 8 Hz,
bridgingph-C3, C5-H), 7.55 (d, 2H, J = 4 Hz, bridgingph-C2, C6-H), 7.18-7.35 (m, 7H, ph-C2,
C3, C4, C5, C6-H and pyrrole-C2, C5-H), 6.28 (t, 2H, pyrrole-C3, C4-H), 4.54 (t, 2H, -CH2). 13C
NMR (75 MHz, DMSO-d6) δ ppm: 44.20, 111.32, 119.05, 119.67, 127.66, 128.63, 128.81,
128.83, 131.13, 138.17, 143.03, 166.50; MS (EI): m/z = found 276.38 [M+]; calcd. 276.34.
Anal. Calcd. For C18H16N2O: C, 78.24; H, 5.84; N, 10.14. Found: C, 78.29; H, 5.82; N,
19

10.12.
4.1.14. N-(2-Aminophenyl)-4-(1H-pyrrol-1-yl)benzamide (3n):
o
(Yield 50%). mp 183-185 C; FTIR (KBr): 3337 (-NH2), 3293 (-NH), 2923 (Ar-H), 1642
1
(C=O), 1279 (C-O-C) cm-1; H NMR (400 MHz, DMSO-d6) δ ppm: 9.71 (s, 1H, NH), 8.11
(d, 2H, J = 8 Hz, bridgingph-C3, C5-H), 7.70 (d, 2H, J = 4 Hz, bridgingph-C2, C6-H), 7.19-
7.67 (m, 4H, aminoph-C4, C6-H and pyrrole-C2, C5-H), 6.82 (d, 1H, J = 8 Hz, aminoph-C3-
H), 6.64 (t, 1H, aminoph-C5-H), 6.30 (t, 2H, pyrrole-C3, C4-H). 13C NMR (75 MHz, DMSO-
d6) δ ppm: 111.03, 118.98, 119.00, 119.88, 120.32, 127.91, 128.76, 148.28, 149.01, 163.25;
MS (EI): m/z = found 277.36 [M+]; calcd. 277.33. Anal. Calcd. For C17H15N3O: C, 73.63; H,
5.45; N, 15.15. Found: C, 73.15; H, 5.44; N, 15.02.
4.1.15. N-(3-Methoxyphenyl)-4-(1H-pyrrol-1-yl)benzamide (3o):
(Yield 69%). mp 123-125 oC; FTIR (KBr): 3348 (-NH), 2945 (Ar-H), 1663 (C=O), 1204 (C-
O-C) cm-1; 1H NMR (400 MHz, DMSO-d6) δ ppm: 10.21 (s, 1H, NH), 8.09 (d, 2H, J = 8.68
Hz, bridgingph-C3, C5-H), 7.66 (d, 2H, J = 6.6 Hz, bridgingph-C2, C6-H), 7.51 (t, 1H,
methoxyph-C6-H) 7.36 (t, 2H, pyrrole-C2, C5-H), 7.19-7.25 (m, 2H, methoxyph-C2, C5-
13
H),6.63-6.67 (m, 1H, methoxyph-C4-H), 6.31 (t, 2H, pyrrole-C3, C4-H); C NMR (75 MHz,
DMSO-d6) δ ppm: 55.48, 109.68, 111.94, 113.14, 119.47, 121.61, 123.43, 128.72, 129.86,
131.56, 132.69, 140.77, 142.61, 159.92, 165.29; MS (EI): m/z = found 292.87 [M+]; calcd.
292.34. Anal. Calcd. For C18H16N2O2: C, 73.95; H, 5.52; N, 9.58. Found: C, 73.88; H, 5.53;
N, 9.56.
4.1.16. N-(2-Chlorobenzyl)-4-(1H-pyrrol-1-yl)benzamide (3p):
(Yield 71%). mp 148-150 oC; FTIR (KBr): 3264 (-NH), 2920 (Ar-H), 1632 (C=O), 1276 (C-
1
O-C) cm-1; H NMR (400 MHz, DMSO-d6) δ ppm: 9.02 (s, 1H, NH), 8.04 (d, 2H, J = 8.72
Hz, bridgingph-C3, C5-H), 7.66 (d, 2H, J = 8.68 Hz, bridgingph-C2, C6-H), 7.26-7.43 (m, 6H,
2-Cl-benz- C3, C4, C5, C6-H and pyrrole-C2, C5-H), 6.30 (t, 2H, pyrrole-C3, C4-H), 4.59 (d,
20

2H, J = 5.8 Hz,CH2); 13C NMR (75 MHz, DMSO-d6) δ ppm: 56.26, 109.26, 111.44, 119.23,
119.48, 121.88, 123.52, 128.62, 129.78, 131.88, 132.59, 140.45, 142.61, 158.72, 166.88; MS
(EI): m/z = found 310.74 [M+]; calcd. 310.78. Anal. Calcd. For C18H15ClN2O: C, 69.57; H,
4.87; N, 9.01. Found: C, 69.54; H, 4.86; N, 9.00.
4.1.17. N-(2-Nitrophenyl)-4-(1H-pyrrol-1-yl)benzamide (3q):
(Yield 63%). mp 68-70 oC; FTIR (KBr): 3348 (-NH), 2923 (Ar-H), 1630 (C=O), 1241 (C-O-
C) cm-1; 1H NMR (400 MHz, DMSO-d6) δ ppm: 9.73 (s, 1H, NH), 8.10-8.12 (p, 2H, NO2Ph-
C3, C6-H), 7.33-7.37 (m, 2H, NO2Ph-C4, C5-H), 6.79. 6.81 (dd, 2H, J = 1.12 Hz, 1.12 Hz,
bridgingph-C3, C5-H), 6.68-6.72 (m, 2H, bridgingph-C2, C6-H), 6.05 (s, 4H, pyrrole-C2, C3,
13
C4, C5-H); C NMR (75 MHz, DMSO-d6) δ ppm: 111.29, 111.93, 116.30, 119.03, 119.15,
119.47, 119.99, 123.47, 125.82, 127.60, 127.96, 130.84, 131.51, 136.16, 143.50, 146.60,
167.22; MS (EI): m/z = found 307.54 [M+]; calcd. 307.31. Anal. Calcd. For C17H13N3O3: C,
66.64; H, 4.26; N, 13.67. Found: C, 66.56; H, 4.27; N, 13.66.
21

5.
Biological activity
5.1. In vitro evaluation of antitubercular studies
All the compounds were tested for inhibition of M. tuberculosis strain H37Rv using
Microplate Alamar Blue Assay (MABA) method described earlier [24]. The 96 wells plate
received 100 mL of Middlebrook 7H9 broth and serial dilution of compounds were made
directly on the plate with the drug concentrations of 0.2, 0.4, 0.8, 1.6, 3.125, 6.25,12.5, 25, 50
and 100 µg/mL. Plates were covered and sealed with parafilm and incubated at 37oC for 5
days. Then, 25 mL of freshly prepared 1:1 mixture of almar blue reagent and 10% Tween 80
was added to the plate and incubated for 24 h. A blue color in the well was interpreted as no
bacterial growth, while pink color was scored as the growth. The MIC value was defined as
the lowest drug concentration, which prevented the color change from blue to pink. Table 1
reveals the anti-TB activity data, expressed in MIC.
5.2. In vitro evaluation of antibacterial activity
MIC determination of the tested compounds was investigated by side-by-side comparison
with ciprofloxacin against Gram-positive (S. aureus) and Gram-negative bacteria (E. coli) by
the broth microdilution method [25, 26]. Serial dilutions of the test compounds and reference
drugs were prepared in Mueller-Hinton agar. Drugs (10 mg) were dissolved in
dimethylsulfoxide (DMSO, 1 mL). Further progressive dilutions with the molten Mueller-
Hinton agar were performed to obtain the required concentrations of 0.2, 0.4, 0.8, 1.6, 3.125,
6.25, 12.5, 25, 50 and 100 µg/mL. The tubes were inoculated with 105 cfu/mL (colony
forming unit/mL) and incubated at 37oC for 18 h. MIC was the lowest concentration of the
tested compound that yielded no visible growth on the plate. To ensure that solvent did not
affect the bacterial growth, the control was performed with the test medium supplemented
with DMSO using the same dilutions as used in the experiments, but DMSO did not show
22

any effect on the microorganisms in the concentration range studied. Table 2 reveals the
antibacterial activity expressed in terms of MIC.
5.3. MTT-based cytotoxicity activity
Cellular conversion of MTT [3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyl-tetrazolium bromide]
into a formazan product [27] was performed to evaluate cytotoxic activity (IC50) of some of
the compounds against A549 (lung adenocarcinoma) cell-line up to concentrations of 50
mg/mL using Promega Cell Titer 96 non-radioactive cell proliferation assay [28] using
cisplatin as the positive control. The IC50 values given in Table 2 are the averages ± SEM of
three independent experiments.
5.4. Enzyme inhibition studies
5.4.1. InhA expression and purification
The production and purification of InHA-6xHis protein from a protease-deficient strain
of E. coli (BL21) transformed with pHAT5/InhA plasmid were performed as follows. A 1 mL
of bacteria was grown in 100 mL of Lysogeny broth (LB) medium containing ampicillin (100
µg/mL) and 2% glucose at 37°C. After 4 h, the solution was re-diluted in 1 L of the same
medium and re-grown at 37°C. After attaining the proper concentration (OD595 = 0.6 - 0.8),
the culture was centrifuged at 3300 g factor for 10 min at 4oC and bacteria were suspended in
LB medium containing ampicillin (100 µg/mL). The His6-tagged InhA protein expression
was induced for overnight incubation in 1 mM isopropyl-β-D-galactopyranoside (IPTG) at
20°C. Cells were harvested by centrifugation at 6000 g for 30 min at 4°C. Dry pellet was kept
at -80°C for several months and purification was done with Ni-NTA Agarose from QIAGEN
following the manufacturer’s protocol. The purified recombinant protein was applied to PD-
10 desalting columns (GE Healthcare, Piscataway, NJ) equilibrated with PIPES 30 mM pH
6.8, 150 mM NaCl to remove imidazole. Samples were analyzed using SDS-PAGE and
23

Coomassie blue staining and then stored at 4°C for a short-term storage at -80°C with 20%
glycerin for long-term storage [29].
5.4.2. InhA activity inhibition
Triclosan and NADH were obtained from Sigma-Aldrich. Stock solutions of the selected
compounds were prepared in DMSO such that the final concentration of this co-solvent was
constant at 5% (v/v) in the final volume of 1 mL for all kinetic reactions. Kinetic assays were
performed using trans-2-dodecenoyl-coenzyme A (DDCoA) and wild type InhA as
previously described [30]. Briefly, reactions were performed at 25°C in an aqueous buffer (30
mM PIPES and 150 mM NaCl pH 6.8) containing 250 µM cofactor (NADH), 50 µM
substrate (DDCoA) and the test compound (at 50 µM). Reactions were initiated by the
addition of InhA (100 nM final) and NADH oxidation was monitored at a fixed wavelength
of 340 nm. Inhibitory activity of each derivative was expressed as % inhibition of InhA
activity (initial velocity of the reaction) with respect to control reaction without the inhibitor.
These results are shown in Table 1.
24

6. Conclusions
The pyrrolyl benzamide derivatives (3a-q) synthesized were found to be effective InhA
inhibitors. These molecules were screened for anti-TB activity against M. tuberculosis and
antibacterial activities against S. aureus and E. coli. The in vitro anti-TB results displayed
that molecules 3a, 3i, 3l, 3n and 3q are highly active showing the MIC value of 3.125 µg/mL
compared to molecules in the developed series. No noticeable cytotoxicity towards
mammalian vero cell-lines was observed, demonstrating their nontoxic behavior.
Furthermore, compounds 3a, 3i, 3l, 3n and 3q displayed excellent InhA inhibition activities.
The optimized pharmacophore model (MODEL_14) developed exhibited good statistical
parameters during the validation process. The pharmacophore delineates the characteristic
features that are common to peptide linkage ligands active against M. tuberculosis. The
corresponding features on peptide linkage and aromatic acceptor provide the necessary
interactions with the binding pockets. Molecular docking studies were carried out to improve
the reliability and accuracy of the model and for better understanding of the drug-receptor
interaction. It was shown that amino acid Try158 and NAD+ co-factor are essential for H-
bonding interaction, while amino acids (ILE122, PHE97, ALA201, VAL203, GLY96,
ALA198, MET199, ILE215, ILE95, LEU197, PHE149, GLY119, MET198) for hydrophobic
interaction that are vital for inhibiting protein. As per molecular simulation studies, these
molecules are bound primarily with the substrate binding site of InhA. The anti-TB results of
these molecules are fully supported by in silico molecular docking calculations. The
synthesized molecules may be useful as the lead compounds for developing InhA inhibitors.
25

Acknowledgements
The authors acknowledge the financial support from Rajiv Gandhi University of Health
Sciences, Bangalore, India (Order No. RGU: Adv. Res.: Proposal-P-161: 2015-16 dated 06.
01. 2016). We thank Dr. H. V. Dambal, President, S. E. T’s College of Pharmacy, Dharwad,
India for their support and Dr. K. G. Bhat of Maratha Mandal's Dental College, Hospital and
Research Centre, Belgaum, India for providing anti-TB activity and the Director, SAIF,
Panjab University, Chandigarh, Panjab, India have provided some of the NMR and mass
spectral data. The authors also appreciate the technical assistance from Mr. Imran Khan, Mr.
Shashank K R, and Mr. Umesh K.
26

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