Synthesis, biological evaluation and in silico molecular modeling of pyrrolyl benzohydrazide derivatives as enoyl ACP reductase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.11.032

In efforts to develop lead anti-TB compounds, a novel series of 19 pyrrolyl benzohydrazides were synthesized and screened to target enoyl-ACP reductase enzyme, which is one of the important enzymes involved in type II fatty acid biosynthetic pathway of M.

Full text of DOI:10.1016/j.ejmech.2016.11.032

Accepted Manuscript
Synthesis, biological evaluation and in silico molecular modeling of pyrrolyl
benzohydrazide derivatives as enoyl ACP reductase inhibitors
Shrinivas D. Joshi, Sheshagiri R. Dixit, Venkatarao H. Kulkarni, Christian Lherbet,
Mallikarjuna N. Nadagouda, Tejraj M. Aminabhavi
PII:
S0223-5234(16)30968-0
10.1016/j.ejmech.2016.11.032
EJMECH 9065
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 18 August 2016
Revised Date: 9 November 2016
Accepted Date: 13 November 2016
Please cite this article as: S.D. Joshi, S.R. Dixit, V.H. Kulkarni, C. Lherbet, M.N. Nadagouda, T.M.
Aminabhavi, Synthesis, biological evaluation and in silico molecular modeling of pyrrolyl benzohydrazide
derivatives as enoyl ACP reductase inhibitors, European Journal of Medicinal Chemistry (2016), doi:
10.1016/j.ejmech.2016.11.032.
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ACCEPTED MANUSCRIPT
Synthesis, biological evaluation and in silico molecular modeling of pyrrolyl benzamide
derivatives as enoyl ACP reductase inhibitors
Shrinivas D. Joshi*a, Sheshagiri R. Dixita, Venkatarao H. Kulkarnia, Christian
Lherbetb, c, Mallikarjun N. Nadagoudaa, Tejraj M. Aminabhavia
Antitubercular activity of novel series of pyrrolyl benzamide derivatives was analyzed.
Pharmacophore mapping constructed using GALAHAD and Molecular modeling studied
using Surflex-Dock on enoyl ACP reductase from Mycobacterium tuberculosis.

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Synthesis, biological evaluation and in silico molecular modeling of pyrrolyl
benzohydrazide derivatives as enoyl ACP reductase inhibitors
Shrinivas D. Joshi*a, Sheshagiri R. Dixita, Venkatarao H. Kulkarnia, Christian
Lherbetb, c, Mallikarjuna N. Nadagoudaa, Tejraj M. Aminabhavia
aNovel Drug Design and Discovery Laboratory, Department of Pharmaceutical Chemistry,
S.E.T’s College of Pharmacy, Sangolli Rayanna Nagar, Dharwad-580 002, India.
bUniversite 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
cITAV-USR3505, Université de Toulouse, CNRS, UPS, F-31106 Toulouse, France.
Corresponding Author
Shrinivas D. Joshi, Novel Drug Design and Discovery Laboratory, Department of
Pharmaceutical Chemistry, S.E.T’s College of Pharmacy, Sangolli Rayanna Nagar, Dharwad-
580 002, India.
E-mail: shrinivasdj@rediffmail.com. Tel.: +91 9986151953; Fax; +91 836 2467190
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Abstract:
In efforts to develop lead anti-TB compounds, a novel series of 19 pyrrolyl benzohydrazides
were synthesized and screened to target enoyl-ACP reductase enzyme, which is one of the
important enzymes involved in type II fatty acid biosynthetic pathway of M. tuberculosis.
Pharmacophores were constructed using GALAHAD to generate alignment of data sets and
calculated by Pareto ranking. The pharmacophore features were then filtered by Surflex-dock
study using enoyl ACP reductase from M. tuberculosis. Compounds 5b and 5d showed H-
bonding interactions with Tyr158, Thr196 and co-factor NAD+ that fitted well within the
binding pocket of InhA. All the synthesized compounds were screened for preliminary
antibacterial activities against Gram-positive S. aureus and Gram-negative E. coli and M.
tuberculosis H37Rv to evaluate their antitubercular activities. Some representative compounds
were further tested for mammalian cell toxicity using human lung cancer cell-line (A549) that
was found to be nontoxic. These compounds exhibited moderate inhibition activities against
InhA.
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Keywords: GALAHAD, Pharmacophore hypothesis, Surflex docking, Pyrrolyl
benzohydrazide derivatives, Antitubercular activity, Enzyme inhibition studies.
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1. Introduction
Tuberculosis (TB), a leading disorder, caused by Mycobacterium tuberculosis (M.
tuberculosis), is the second leading disease causing the death along with HIV [1]. According
to WHO 2015 report, nearly six million new cases of TB have been reported, of which about
63% of 9.6 million people were found to be affected with TB, suggesting that worldwide
approximately 37% of new cases have not been reported or not diagnosed; about 480,000
cases of multidrug-resistant (MDR-TB) were seen of which 25% cases were diagnosed and
reported [2]. The current common treatment method for TB prescribes more than 40 years old
drug. Despite established high efficacy in clinical trials [3], standardized short-course
chemotherapy (SCC) of active drug-susceptible TB requires a direct supervision to ensure
good adherence to prevent drug resistance, but drugs that are active against resistant-TB are
less potent, more toxic, and are to be taken for longer time.
The existence of virtually untreatable extensive drug-resistant TB (XDR-TB) has been a new
for controlling TB worldwide. In addition, treatment of patients who are co-infected with
HIV is more problematic due to drug-drug interactions [4]. Over the past decades, even
though several anti-tuberculosis (anti-TB) drugs have been developed, drug-resistance issue
has not yet been resolved. There is thus a tremendous need to develop new anti-TB drugs
having shorter and simpler regimens, well tolerated, effective against drug-susceptible and
drug-resistant TB, appropriate for combined HIV-TB treatment. In this direction, studies have
been made on targeting the cell wall of Mycobacteria [5].
The core member of mycobacterial cell wall is mycolic acid, whose biosynthesis is inhibited
by INH-NAD adduct in MTB by affecting InhA, an enoyl-ACP reductase enzyme of the type
II fatty acid synthesis (FAS-II) system, which catalyses the last step of fatty acid elongation
cycle [5]. Among them, fatty acid biosynthesis I (FabI) constitutes a single isoform in major
pathogens such as Staphylococcus aureus [6], Escherichia coli [7], and MTB [8]. The clinical
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success of InhA inhibitor isoniazid [9] and numerous reports of FabI inhibitors [10]
involving naphthyridinones [11], diazaborines [12], 4-pyridones [13], triclosan and its
analogues [14, 15] have validated this target as one of the most attractive of the FAS-II
pathways. This enzyme is recognized and validated as an important drug target in MTB, since
its homolog in human is absent.
In our previous studies, we have worked on pyrrole moiety, as it is an important heterocycle
of the plant and animal kingdom as a subunit of chlorophyll in plant cells. Hemin and vitamin
B12 in animal cells, and these derivatives have shown significant antimycobacterial activities
[16-18]. Keeping in view of the biological and medicinal properties of pyrroles and the
potentiality of peptide linkage, we have undertaken to design such molecules that contain
both pyrrole and peptide linkages to treat against the resistant bacterial species.
Even though some novel compounds possess anti-TB activity by inhibiting enoyl-ACP
reductase enzyme, but none of them are available in the market as these are still in their
preliminary research phase. This prompted us to undertake detailed investigations on
pharmacophore mapping, design and synthesis of direct enoyl-ACP reductase inhibitors
involving pyrrolyl benzohydrazide as a core structure using computational methods that
include GALAHAD (pharmacophore based on an automated computational alignment
technique) and Surflex-docking studies to identify enoyl-ACP reductase as a potential target
for these pyrrolyl benzohydrazide derivatives. Using these tools, we have demonstrated
quantitative pharmacophore mapping technique that is effective to identify physicochemical
and structural requirements for ligand binding and biological activity [19] along with
molecular docking investigations on N’-substituted benzoyl-4-(1H-pyrrol-1-yl)benzo
hydrazide derivatives as possible inhibitors of InhA and evaluated their possible antibacterial,
antitubercular and cytotoxic activities.
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Hybridization strategy was used to build our design concept for new antimicrobial and anti-
TB compounds. Benzohydrazide derivatives have shown interesting anti-TB and
antimicrobial activities, inhibiting the bacteria by blocking lipid biosynthesis [5]. Therefore,
new anti-TB agents are designed between benzohydrazide and pyrrole moieties utilizing
hybridization strategy. In continuation of our research work, herein we try to map
pharmacophore from the structure of 1-cyclohexyl-5-oxo-N-phenylpyrrolidine-3-
carboxamide, which contains carboxamide H-bond acceptor and donor bridge between 5-
oxopyrrolidine and phenyl as well as contain three hydrophobic moieties viz., 5-
oxopyrrolidine, cyclohexyl and phenyl. Therefore, 5-oxopyrrolidine and carboxamide were
mapped with a carboxamide linkage (-CO-NH-NH-CO) that will strengthen interactions with
the hydroxyl group of the key residue Tyr158 of InhA [20], while hydrophobic moieties were
mapped with o/m/p substituted phenyl derivatives and benzopyrrole ring as shown in Fig.1.
In Fig. 2, represents some of the drugs that are under clinical or preclinical trials that were
considered for the synthesis of new derivatives following the Pall-Knorr pyrrole synthesis
[21]. However, no previous literature studies have been made on these molecules and hence,
the present study is a novel approach.
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2. Computational Methods
All the calculations were performed using the commercially available SYBYL-X 2.0
software packages (Tripos Associates, St. Louis, MO, USA) [22].
2.1. Preparation of ligands
All the 3D structures were constructed using the CONCORD program built in SYBYL-X 2.0
molecular modeling software package (Tripos Inc, St. Louis, MO) followed by geometry
optimisation using the distance dependent dielectric function (Tripos molecular mechanics
force field) [23] by the Powell method with a convergence criterion of 0.001 kcal/mol Å and
a maximum iteration of 1000. Partial atomic charges were calculated by MMFF94 method
and minimized structures were used as initial conformations for GALAHAD as well as for
Surflex docking studies.
2.2.GALAHAD
In order to grow a pharmacophore model for InhA inhibitors, the GALAHAD program [24]
was employed. This utilizes a series of compounds with a common biological activity as an
input parameter and then genetic algorithm was used to flexibly align these compounds on
the basis of shared pharmacophoric features that are situated relative to each other in
coordinate spaces as points surrounded by a sphere of tolerance. Each sphere represents the
region in space that should be occupied by a certain chemical functionality capable of the
kind of interaction specified by the feature type. This method describes spatial relationship
between structural elements corresponding to the biological activity in the form of a series of
3D pharmacophore models; these models can then assist in the design and discovery of new
molecules, which incorporate the desired bioactivity.
The GALAHAD program was run using the four compounds (compounds 5c, 5d, 5l and 5s)
to carry out pharmacophore hypothesis, and conformers were generated using genetic
algorithm for all the molecules. Twenty models were found using a population size of 30 with
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the maximum generations was set to 60 with the value of molecular required hitting of 3
(suggested from data). Using more than four molecules led to a reduction in the quality of
pharmacophore while using a single molecule, we could induce nonspecific feature set.
Pharmacophore hypothesis calculates SPECIFICITY as a logarithmic indicator of the
expected discrimination for each query. The actual number of hit is given in N_HITS column.
The values in FEATS column indicate total number of features in the model. The next five
columns are model scores components from the genetic algorithm. PARETO is a pareto rank
of each model [25], wherein all models have a Pareto rank of 0. This means none of the
models is superior to any other when using all the four criteria in columns 5-8 (ENERGY,
STERICS, H_BOND, and MOL_QRY). ENERGY calculates 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 group.
One best template model needs to be selected out of the twenty models for rigid alignment of
the remaining molecules in data set. The selection of best model was based on the model
having high sterics with low energy and pharmacophoric features and the model needed to
“hit” all the active molecules. Among the considered models, MODEL_13 has the optimal
position because it fulfils the three criteria and has better Specificity, N_hits and Feats values
[26-28]. Finally all the molecules were aligned to predict various parameters using the best
selected template model (MODEL_13). (Fig. 3)
2.3.Preparation of protein
The crystal structure of M. tuberculosis enoyl reductase (InhA) complexed with 1-
cyclohexyl-5-oxo-N-phenylpyrrolidine-3-carboxamide, refined with new ligand restraints
(PDB code: 4U0J; 1.62 Å X-ray resolution) was retrieved from the Protein Data Bank
(http://www.rcsb.org/pdb/). The structure was prepared as per the following procedures:
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water molecules and co-crystallized ligand were removed from the crystal structure,
hydrogen atoms were added to protein by the biopolymer module implemented in the
SYBYL software, and assigning the Amber7 FF99 atom charges to the protein atoms. The
resultant structure was finally converted from the PDB format to MOL2 and used for
molecular docking experiments.
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3. Results and discussion
3.1. Chemistry
All the compounds were synthesized as per the Scheme 1. The Pall-Knorr pyrrole synthesis
involves the reaction of diketone like 2,5-dimethoxy tetrahydrofuran with amine, which is
among the most conventional method for the synthesis of heterocyclic pyrrole ring. Hence,
ethyl 4-(1H-pyrrol-1-yl)benzoate (2) was obtained by constructing pyrrole ring utilizing the
amino group at the 4th position of aromatic ring in the presence of dried glacial acetic acid.
Thus obtained ester was converted to the corresponding hydrazide (3) by hydrazinolysis
using hydrazine hydrate. The peptide forming agents such as 2-(1H-benzotriazole-1-yl)-
1,1,3,3-tetramethyl uroniumhexa fluorophosphate (HBTU) and diisopropyl ethylamine
(DIEA) were used in the reaction of substituted benzoic acid (4a-s) and ethyl 4-(1H-pyrrol-1-
yl)benzohydrazide (3), which resulted in the formation of title compounds, N’-substituted
benzoyl-4-(1H-pyrrol-1-yl)benzohydrazide derivatives (5a-s).
FTIR spectra showed the presence of -NH (2o amine) and carbonyl group as strong bands in
the region of 3402.44-3186.02 cm-1 and 1731.62-1519.67 cm-1, respectively. In the 1H NMR
spectra resonating signals of 2o amine (-NH) group, protons are seen as two singlets in the
range of δ 10.26-11.05 ppm. The pyrrole protons at the 3-H and 4-H positions appeared as a
singlet/triplet/multiplet from δ 6.28-6.32 ppm and those protons at the 2-H and 5-H positions
resonated as singlet/multiplet from δ 7.16-7.72 ppm. The 13C NMR spectra exhibited
characteristic signals of carbonyl carbon atom in the range of δ 163.12-168.54 ppm. The mass
spectra showed a precise molecular ion peak data for the respective compounds.
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3.2. Antitubercular and antibacterial activities
Anti-TB activity was determined for compounds 5(a-s) against M. tuberculosis strain H37Rv
using a Microplate Alamar Blue assay (MABA) method using ethambutol and rifampicin as
reference drugs. See results presented in Table 1. The preliminary anti-TB screening revealed
that majority of compounds showed a moderate to good activity. The activities of compounds
5(a-s) are expressed in terms of minimum inhibitory concentration (MIC) values.
Compounds, 5b, 5d, 5m, 5r and 5s showed the highest activity with a MIC value of 3.125
µg/mL, while compounds 5e and 5n showed better activities with a MIC value of 6.25
µg/mL. The good anti-TB activity may be due to the presence of pharmacologically active
amide linkage (CO-NH-NH-CO) and aromatic ring. The compounds with electron
withdrawing substitutions have shown better inhibitory activity compared to other
derivatives.
Antibacterial activity was also carried out against both Gram positive bacteria (S. aureus) and
Gram negative bacteria (E. coli) for all the compounds, that showed moderate to significant
microbial inhibition. Among all the compounds tested, Compounds 5a-s exhibited good to
moderate antimicrobial activity with the MIC values of 3.125-50 µg/mL.
3.3.MTT-based cytotoxicity studies
Based on in vitro antimycobacterial studies, we have selected 10 compounds for cytotoxicity
studies against human lung cancer cell-line A549 (Table 2), suggesting that the most active
molecules are quite safe to human lung cell-line. The tested compounds showed a moderate
cytotoxicity compared to cisplatin (Table 2). Specifically, the active compounds viz., 5b and
5d exhibited a good safety profile with a IC50 values of 40.2 µmol/L against the A549 cell-
line.
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3.4.Enzyme inhibition studies
Among the 10 compounds selected for cytotoxicity study were further tested for in vitro
enzyme inhibition activity against InhA from M. tuberculosis at 50 µM (Table 3) using
triclosan as a reference showed complete inhibition of InhA enzyme.
The compounds bearing halogen atoms (5b, 5d and 5e) viz., -Cl, Br, and F on aromatic ring
showed a good activity profile with % inhibition of 38%, 36% and 35%, respectively at 50
µM. Based on our previous study [18], we hypothesized that substitution of electro-negative
group might help to show higher affinity to mimic InhA substrate and introduction of
lipophilic bulkier groups or halogen might have facilitated hydrophobic interaction. Finally,
lack of inhibition of InhA in contrast with the low MICs clearly show that the target of these
compounds is not only InhA.
3.5.Pharmacophore results
Pharmacophore models of the representative molecules with high activities were generated
using GALAHAD. Twenty pharmacophore models were generated by GALAHAD. Table 4
lists the parameters of each model, while Fig. 3 shows the alignments of ligands obtained
using GALAHAD. Best models exhibit a high value of SPECIFICITY. MODEL_05,
MODEL_06, MODEL_13, MODEL_14, MODEL_15, MODEL_16, and MODEL_19
showed high SPECIFICITY values of 3.779, 3.779, 3.776, 3.777, 3.779, 3.778 and 4.004,
respectively. But these models were having lesser value of pharmacophore ‘hit’ or sterics.
The N_HITS column shows the number of ligands the pharmacophore ‘hit’ in the training
set, and many of the models matched 4 ligands in the dataset. The value in the FEATS
column indicates the total number of features possessed by each model. The values in
PARETO column indicate whether a model was superior to other based on the four criteria of
energy, sterics, H_bond, and MOL_QRY. All the models showed Pareto rank 0, which means
none of the model was superior to others. Amongst the 20 models, MODEL_13 was selected,
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which showed better steric values, features, hits and reasonably low energy than others.
Hence, MODEL_13 was considered for final rigid alignment of the remaining compounds.
Most pyrrolyl benzohydrazide analogs evaluated in this study are relatively active and the
variance in activities in this dataset is quite low (Table 5). However, dataset did not allow
further studies like 3D QSAR, since only 19 compounds were available in our database,
which were structurally related. The in vitro activity with MIC have ranged from 3.125 to 50
µg/mL.
3.6.Molecular docking results
After successful reproduction of crystallographic binding mode by the Surflex-Dock, all the
pyrrolyl benzohydrazide analogues were docked into the active site of InhA and the results of
surflex docking are depicted in Table 6. Here, our aim was to examine that all the docked
analogues will bind into the active site of InhA, in a similar way as those of crystallographic
binding modes. Docking studies with Surflex-Dock showed that compounds 5b and 5d
occupy the same binding sites as those of pyrrolidine carboxamide (Fig. 4A, B and C). The
pyrrolidine carboxamide exhibited two H-bonding interactions, the oxygen of carbonyl group
on pyrrolidine makes H-bonding interaction with active site amino acid Tyr158 and co-factor
NAD+. Our attention was focused on the most characteristic receptor-ligand interactions of
the most active analogue 5b. As predicted by Surflex-Dock binding mode, compound 5b
exhibited a similar type of orientation and conformation as that of crystallographic ligand
pyrrolidine carboxamide (Fig. 5). As depicted in Fig. 6A, the oxygen atom of carbonyl group
present at the 4th postion of bridging phenyl interact with the amino acid Tyr158 (2.09 Å) and
co-factor NAD+ (1.99 Å), while oxygen atom of carbonyl group present on the aromatic ring
exhibits H-bond interaction with amino acid Thr196 (2.12 Å). The compound 5d binds in the
same pattern as that of crystallographic ligand pyrrolidine carboxamide. Here, oxygen atom
of peptide linkage makes three H-bonds, two with amino acid Tyr158 (2.14 Å) and another
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with co-factor NAD+ (1.97 Å), yet another one with the amino acid Thr196 (2.10 Å) (Fig.
7A). The docking scores viz., C-score, Crash, Polar, D_Score, PMF_Score, G_Score and
Chem Score from the Surflex dock are given in Table 6. Fourteen molecules were observed
with better scores than the re-docked pyrrolidine carboxamide ligand. But compound 5b (C-
score 9.68, MIC 3.125 µg/mL) showed the highest docking score compared to compound 5d
(C-score 9.43, MIC 3.125 µg/mL). The hydrophobic and hydrophilic amino acids surrounded
to pyrrole and bridiging phenyl rings (Pro156, Tyr158, Asn159, Glu219, Trp160, Met161,
Thr162, Pro193, Ile 194, Arg195), peptide bridge (Ser20, Ile21, Ser123, Trp160, Met161),
and aromatic ring (Ile16, Ser19, Phe97, Met98, Pro99, Thr196) with the representative
compound 5b is depicted in Fig. 8(A and B).
3.7.Pharmacophore map correlation with the binding site
The docked conformation was correlated with the recognized pharmacophoric features; thus a
correlation between binding site and pharmacophoric features was observed. Interactions
identified by the docking program were quite consistent with the outcome of pharmacophore
modeling. This reveals that amino acid Tyr158 and co-factor NAD+ of the receptor protein
interacts with H-bond acceptor atoms (pyrrolidine C=O---H and peptide linkage C=O----H).
That of hydrophobic moieties (phenyl, pyrrolidine, pyrrole) help the molecule for penetration,
whereas negative values of crash score indicates the extent of penetration of a compound.
Figure 7b represents hydrophobic amino acids surrounded to the molecule.
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4. Experimental section
Melting points were determined using Shital-digital programmable melting point apparatus
and are uncorrected. FTIR spectra in KBr pellets were recorded on a Bruker FTIR
spectrophotometer. The 1H and 13C 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, and (m) multiplet.
Mass spectra (MS) were recorded in a JEOL GCMATE II GC-Mass spectrometer and
Schimadzu QP 20105 GC-Mass spectrometer. Elemental analysis data (performed on Leco
Tru Spec CHNS Analyzer) for C, H, and N were all within + 0.4% of the theoretical values.
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 ethyl 4-(1-H-pyrrol-1-yl)benzoate (2)
Compound 2 was synthesized by refluxing a mixture of ethyl 4-aminobenzoate (1) (2 g, 0.1
mol) in dried acetic acid (15 mL) and 2,5-dimethoxytetrahydrofuran (1.9 mL, 0.12 mol) for
45 min at 150-160 oC. This reaction mixture was poured onto crushed ice, neutralized by the
addition of saturated sodium bicarbonate, the solid separated was filtered and recrystallized
from ethanol [29].
4.2. Synthesis of 4-pyrrol-1-yl benzoic acid hydrazide (3)
Compound 3 was synthesized by refluxing a mixture of ethyl 4-pyrrol-1-yl benzoate (2) (3.22
g, 0.015 mol) with hydrazine hydrate (10 mL) in absolute ethanol (10 mL) for 3 h. The
reaction mixture was cooled and crystalline mass obtained was recrystallized from ethanol
[29].
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4.3. General procedure for the synthesis of N’-substituted benzoyl-4-(1H-pyrrol-1-
yl)benzohydrazide derivatives (5a-s)
4-(1H-pyrrol-1-yl)benzohydrazide (3) (0.0018 mol) and substituted benzoic acid (4a-s)
(0.0019 mol) were dissolved in 50 mL of dry DMF. HBTU (0.87 g, 0.0023 mol) and DIEA
(0.93 mL, 0.0053 mol) were then added 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 1N 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 by eluting with
petroleum ether-ethyl acetate (6:4) mixture to afford the compounds 5(a-s). The purity was
established by single spot on the TLC plates.
4.2.1. N'-benzoyl-4-(1H-pyrrol-1-yl)benzohydrazide (5a)
(Yield 85%). mp 230-232 oC; FTIR (KBr): 3244.13 (N-H), 1683.13, 1645.52 (C=O) cm-1; 1H
NMR (400 MHz, DMSO-d6) δ ppm: 6.31 (t, 2H, pyrrole-C3, C4-H), 7.39 (t, 2H, pyrrole-C2,
C5-H), 7.51 (t, 2H, benzoyl-C3, C5-H), 7.58 (t, 1H, benzoyl-C4-H), 7.67 (d, 2H, J =8 Hz
benzoyl-C2, C6-H), 7.97 (d, 2H, J =8 Hz, bridging phenyl-C2, C6-H), 8.07 (d, 2H, bridging
phenyl-C3, C5-H), 10.49, 10.52 (s, 2H, NH); 13C NMR (100 MHz, DMSO-d6) δ ppm: 112.22,
119.88, 128.67, 129.25, 130.45, 132.66, 133.72, 143.70, 166.49, 167.45; MS (ESI): m/z =
found 305 [M+]; calcd. 305.34. Anal. Calcd. For C18H15N3O2: C, 70.81; H, 4.95; N, 13.76.
Found: C, 71.86; H, 5.32; N, 15.33.
4.2.2. N'-(4-(1H-pyrrol-1-yl)benzoyl)-4-bromobenzohydrazide (5b)
o
(Yield 70%). mp 255-257 C; FTIR (KBr): 3254.63 (N-H), 1681.79, 1643.51 (C=O) cm-1;
1H NMR (400 MHz, DMSO-d6) δ ppm: 6.33 (t, 2H, pyrrole-C3, C4-H), 7.24 (t, 2H, pyrrole-
C2, C5-H), 7.48-7.64 (m, 4H, bromophenyl-C3, C5-H & bridiging phenyl-C2, C6-H), 7.87-
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7.93 (m, 2H, bromophenyl- C2, C6-H), 8.07 (d, 2H, J =8 Hz bridging phenyl-C3, C5-H).
13
10.29, 10.57 (s, 2H, NH); C NMR (100 MHz, DMSO-d6) δ ppm: 111.39, 119.11, 125.47,
128.12, 129.43, 129.61, 131.47, 131.71, 142.56, 158.80, 164.93; MS (ESI): m/z = found 385
[M+ + 1]; calcd. 384.23. Anal. Calcd. For C18H14BrN3O2: C, 56.27; H, 3.67; N, 10.94.
Found: C, 57.86; H, 4.42; N, 12.33.
4.2.3. N'-(4-(1H-pyrrol-1-yl)benzoyl)-4-nitrobenzohydrazide (5c)
o
(Yield 70%). mp 250-252 C; FTIR (KBr): 3199.95 (N-H), 1693.27, 1641.07 (C=O) cm-1;
1H NMR (400 MHz, DMSO-d6) δ ppm: 6.33 (s, 2H, pyrrole-C3, C4-H), 7.28 (s, 2H, pyrrole-
C2, C5-H), 7.58 (d, 2H, J =8.44 Hz, bridging phenyl-C2, C6-H), 8.10 (d, 2H, J =8.52 Hz,
bridging phenyl-C3, C5-H), 8.23 (d, 2H, J =6.8 Hz, nitrophenyl-C2, C6-H), 8.32 (d, 2H, J
13
=8.24 Hz, nitrophenyl-C3, C5-H), 10.87 (s, 2H, NH); C NMR (100 MHz, DMSO-d6) δ
ppm: 111.44, 119.09, 123.50, 129.23, 129.65, 139.80, 148.92, 163.12; MS (ESI): m/z =
found 350.09 [M+]; calcd. 350.33. Anal. Calcd. For C18H14N4O4: C, 61.71; H, 4.03; N,
15.99. Found: C, 62.86; H, 4.42; N, 16.30.
4.2.4. N'-(4-(1H-pyrrol-1-yl)benzoyl)-4-chlorobenzohydrazide (5d)
o
(Yield 75%). mp 248-250 C; FTIR (KBr): 3250.38 (N-H), 1682.21, 1643.42 (C=O) cm-1;
1H NMR (400 MHz, DMSO-d6) δ ppm: 6.33 (t, 2H, pyrrole-C3, C4-H), 7.25 (t, 2H, pyrrole-
C2, C5-H), 7.38-7.50 (m, 2H, chlorophenyl-C2, C6-H), 7.96-8.01 (m, 4H, bridging phenyl-C2,
C6-H & chlorophenyl-C3, C5-H), 8.09 (d, 2H, J =8 Hz, bridging phenyl-C3, C5-H), 10.53 (s,
2H, NH); MS (ESI): m/z = found 339 [M+]; calcd. 339.78. Anal. Calcd. For C18H14ClN3O2:
C, 63.63; H, 4.15; N, 12.37. Found: C, 65.20; H, 4.42; N, 13.40.
4.2.5. N'-(4-(1H-pyrrol-1-yl)benzoyl)-4-fluorobenzohydrazide (5e)
o
(Yield 70%). mp 260-262 C; FTIR (KBr): 3238.92 (N-H), 1682.97, 1644.37 (C=O) cm-1;
1H NMR (400 MHz, DMSO-d6) δ ppm: 6.33 (d, 2H, J =1.52 Hz, pyrrole-C3, C4-H), 7.17 (t,
2H, pyrrole-C2, C5-H), 7.23 (d, 2H, J =1.48 Hz, fluorophenyl-C3, C5-H), 7.53 (d, 2H, J=8
17

ACCEPTED MANUSCRIPT
Hz, bridging phenyl-C2, C6-H), 8.03-8.09 (m, 4H, bridging phenyl-C3, C5-H & fluorophenyl-
13
C2, C6-H), 10.35 (s, 2H, NH); C NMR (100 MHz, DMSO-d6) δ ppm: 111.37, 115.46,
119.06, 129.55, 129.77, 142.77, 162.97, 165.28, 165.42; MS (ESI): m/z = found 323 [M+];
calcd. 323.32. Anal. Calcd. For C18H14FN3O2: C, 66.87; H, 4.36; N, 13.00. Found: C, 67.10;
H, 5.02; N, 14.50.
4.2.6. N'-(4-(1H-pyrrol-1-yl)benzoyl)-4-hydroxybenzohydrazide (5f)
(Yield 60%). mp 270-272 oC; FTIR (KBr): 3418.02 (O-H), 3220.95 (N-H), 1731.62, 1647.75
1
(C=O) cm-1; H NMR (400 MHz, DMSO-d6) δ ppm: 6.28-6.31 (m, 2H, pyrrole-C3, C4-H),
6.70 (d, 2H, J =4 Hz, hydroxyphenyl-C3, C5-H), 7.31-7.33 (m, 2H, pyrrole-C2, C5-H), 7.55-
7.66 (m, 4H, hydroxyphenyl-C2, C6-H and bridging phenyl-C2, C6-H), 7.94-8.00 (m, 2H,
bridging phenyl-C3, C5-H), 9.98 (s, 1H, -OH), 10.72 (s, 2H, NH); MS (ESI): m/z = found
320.10 [M+-1]; calcd. 321.33. Anal. Calcd. For C18H15N3O3: C, 67.28; H, 4.71; N, 13.08.
Found: C, 68.10; H, 5.02; N, 14.50.
4.2.7. N'-(4-(1H-pyrrol-1-yl)benzoyl)-4-methoxybenzohydrazide (5g)
(Yield 70%). mp 253-255 oC; FTIR (KBr): 3254.87 (N-H), 1680.81, 1642.89 (C=O) cm-1; 1H
NMR (400 MHz, DMSO-d6) δ ppm: 3.85 (s, 3H, -OCH3), 6.31 (t, 2H, pyrrole-C3, C4-H), 7.03
(d, 2H, J=12 Hz, methoxyphenyl-C3, C5-H), 7.43 (t, 2H, pyrrole-C2, C5-H), 7.70 (d, 2H, J
=12 Hz, methoxyphenyl-C2, C6-H), 7.95 (d, 2H, J=8 Hz, bridging phenyl-C2, C6-H), 8.05 (d,
2H, J=8 Hz, bridging phenyl-C3, C5-H), 10.35, 10.46 (s, 2H, NH); MS (ESI): m/z = found
336 [M+ + 1]; calcd. 335.36. Anal. Calcd. For C19H17N3O3: C, 68.05; H, 5.11; N, 12.53.
Found: C, 69.10; H, 5.62; N, 13.50.
4.2.8. N'-(4-(1H-pyrrol-1-yl)benzoyl)-3-acetylbenzohydrazide (5h)
(Yield 70%). mp 265-267 oC; FTIR (KBr): 3291.91 (N-H), 1681.05, 1642.99 (C=O) cm-1; 1H
NMR (400 MHz, DMSO-d6) δ ppm: 2.64 (s, 3H, -OCH3), 6.30 (m, 2H, pyrrole-C3, C4-H),
7.12-8.52 (m, 10H, acetylphenyl-C2, C3, C5, C6-H, bridging phenyl-C2, C3, C5, C6-H and
18

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pyrrole-C2, C5-H), 11.05, 10.98 (s, 2H, NH); 13C NMR (100 MHz, DMSO-d6) δ ppm: 26.71,
111.02, 118.77, 126.97, 128, 128.84, 129.69, 130.68, 131.65, 134.86, 136.63, 141.62, 164.88,
165.01, 197.41; MS (ESI): m/z = found 346 [M+-1]; calcd. 347.37. Anal. Calcd. For
C20H17N3O3: C, 69.15; H, 4.93; N, 12.10. Found: C, 70.10; H, 5.62; N, 13.50.
4.2.9. N'-(4-(1H-pyrrol-1-yl)benzoyl)-3-methylbenzohydrazide (5i)
o
(Yield 70%). mp 201-203 C; FTIR (KBr): 3239.22 (N-H), 1684.08, 1644.91 (C=O) cm-1;
1H NMR (400 MHz, DMSO-d6) δ ppm: 2.40 (s, 3H, -OCH3), 6.31 (m, 2H, pyrrole-C3, C4-
H), 7.33-7.38 (m, 4H, methylphenyl-C4, C5-H and pyrrole-C2, C5-H), 7.58-7.79 (m, 4H,
methylphenyl-C2, C6-H and bridging phenyl-C2, C6-H), 8.07 (d, 2H, J =8 Hz, bridging
phenyl-C3, C5-H), 10.52 (s, 2H, NH); 13C NMR (100 MHz, DMSO-d6) δ ppm: 20.94,
111.13, 118.86, 124.55, 128.04, 128.24, 128.82, 129.42, 132.28, 132.57, 134.78, 137.69,
142.29, 165.03, 166.05; MS (ESI): m/z = found 320 [M++1]; calcd. 319. Anal. Calcd. For
C19H17N3O2: C, 71.46; H, 5.37; N, 13.16. Found: C, 73.10; H, 5.62; N, 14.08.
4.2.10. N'-(4-(1H-pyrrol-1-yl)benzoyl)-2-methylbenzohydrazide (5j)
(Yield 70%). mp 203-205 oC; FTIR (KBr): 3204.70 (N-H), 1689.67, 1636.91 (C=O) cm-1; 1H
NMR (400 MHz, DMSO-d6) δ ppm: 2.47 (s, 3H, -CH3), 6.31 (m, 2H, pyrrole-C3, C4-H),
7.18-7.72 (m, 8H, bridging phenyl-C2, C5-H, methylphenyl-C3, C4, C5, C6-H and pyrrole-C2,
13
C5-H), 8.07 (d, 2H, J =8 Hz, bridging phenyl-C3, C5-H), 10.47 (s, 2H, NH); C NMR (100
MHz, DMSO-d6) δ ppm: 19.34, 111.23, 118.87, 127.36, 128.78, 129.23, 129.77, 130.47,
130.61, 134.73, 135.94, 142.26, 164.84, 168.54; MS (ESI): m/z = found 320 [M++1]; calcd.
319. Anal. Calcd. For C19H17N3O2: C, 71.46; H, 5.37; N, 13.16. Found: C, 73.10; H, 5.62; N,
14.08.
4.2.11. N'-(4-(1H-pyrrol-1-yl)benzoyl)-4-aminobenzohydrazide (5k)
(Yield 70%). mp 118-120 oC; FTIR (KBr): 3230.48 (N-H), 1637.28, 1607.67 (C=O) cm-1; 1H
NMR (400 MHz, DMSO-d6) δ ppm: 5.62 (s, 2H, -NH2), 6.28-6.31 (m, 2H, pyrrole-C3, C4-H),
19

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6.53-6.61 (m, 2H, aminophenyl-C3, C5-H), 7.14-8.27 (m, 8H, aminophenyl-C2, C6-H,
bridging phenyl-C2, C3, C5, C6-H and pyrrole-C2, C5-H), 10.01, 10.51 (s, 2H, NH); MS (ESI):
m/z = found 322 [M++2]; calcd. 320. Anal. Calcd. For C18H16N4O2: C, 67.49; H, 5.03; N,
17.49. Found: C, 68.10; H, 5.62; N, 18.08.
4.2.12. N'-(4-(1H-pyrrol-1-yl)benzoyl)-2-aminobenzohydrazide (5l)
(Yield 70%). mp 205-207 oC; FTIR (KBr): 3270.18 (N-H), 1645.26, 1610.68 (C=O) cm-1; 1H
NMR (400 MHz, DMSO-d6) δ ppm: 6.28-6.32 (m, 2H, pyrrole-C3, C4-H), 6.36 (s, 2H, NH2),
6.56 (q, 1H, aminophenyl-C3-H), 6.75 (q, 1H, aminophenyl-C5-H), 7.04 (q, 1H, aminophenyl-
C4-H), 7.37 (t, 2H, pyrrol-C2, C5-H), 7.51-7.70 (m, 3H, bridging phenyl-C2, C6-H,
aminophenyl-C6-H), 8.03 (d, 2H, J =8 Hz, bridging phenyl-C3, C5-H), 10.47 (s, 2H, NH); 13C
NMR (100 MHz, DMSO-d6) δ ppm: 111.30, 116.40, 118.45, 118.88, 118.93, 128.19, 128.93,
129.15, 129.53, 132.19, 142.22, 149.86, 165.13, 168.33; MS (ESI): m/z = found 321 [M++1];
calcd. 320. Anal. Calcd. For C18H16N4O2: C, 67.49; H, 5.03; N, 17.49. Found: C, 68.10; H,
5.62; N, 18.08.
4.2.13. N'-(4-(1H-pyrrol-1-yl)benzoyl)-2-chlorobenzohydrazide (5m)
(Yield 75%). mp 210-212 oC; FTIR (KBr): 3206.28 (N-H), 1698.47, 1636.51 (C=O) cm-1; 1H
NMR (400 MHz, DMSO-d6) δ ppm: 6.31 (m, 2H, pyrrole-C3, C4-H), 7.40-7.72 (m, 8H,
bridging phenyl-C2, C6-H, chlorophenyl-C3, C4, C5, C6-H and pyrrole-C2, C5-H), 8.07 (d, 2H,
13
J =8 Hz, bridging phenyl-C3, C5-H), 10.67 (s, 2H, NH); C NMR (100 MHz, DMSO-d6) δ
ppm: 111.15, 118.66, 126.66, 128.17, 129.01, 129.66, 130.12, 130.97, 131.45, 134.31,
134.74, 142.20, 165.25, 165.48; MS (ESI): m/z = found 338 [M+]; calcd. 339.78. Anal. Calcd.
For C18H14ClN3O2: C, 63.63; H, 4.15; N, 12.37. Found: C, 65.10; H, 5.62; N, 14.78.
4.2.14. N'-(4-(1H-pyrrol-1-yl)benzoyl)-3-chlorobenzohydrazide (5n)
(Yield 78%). mp 212-214 oC; FTIR (KBr): 3317.95 (N-H), 1641.37, 1609.82 (C=O) cm-1; 1H
NMR (400 MHz, DMSO-d6) δ ppm: 6.30 (t, 2H, pyrrole-C3, C4-H), 7.34-7.36 (m, 2H,
20

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pyrrole-C2, C5-H), 7.40-7.51 (m, 2H, chlorophenyl-C4, C5-H), 7.59-7.67 (m, 2H,
chlorophenyl-C2, C6-H), 7.86-8.04 (m, 4H, bridging phenyl-C2, C3, C5, C6-H), 10.65 (s, 2H,
NH); 13C NMR (100 MHz, DMSO-d6) δ ppm: 111.25, 118.83, 126.92, 128.01, 128.20,
129.22, 129.78, 130.58, 131.75, 134.63, 142.33, 164.73, 165.77; MS (ESI): m/z = found 339
[M+-2]; calcd. 339. Anal. Calcd. For C18H14ClN3O2: C, 63.63; H, 4.15; N, 12.37. Found: C,
65.10; H, 5.62; N, 14.78.
4.2.15. N'-(4-(1H-pyrrol-1-yl)benzoyl)-4-methylbenzohydrazide (5o)
(Yield 80%). mp 218-220 oC; FTIR (KBr): 3244.13 (N-H), 1683.75, 1643.99 (C=O) cm-1; 1H
NMR (400 MHz, DMSO-d6) δ ppm: 2.39 (s, 3H, -CH3), 6.31 (q, 2H, pyrrole-C3, C4-H), 7.42-
7.45 (m, 2H, pyrrole-C2, C5-H), 7.67-7.99 (m, 6H, bridging phenyl-C2, C6-H and
methylphenyl-C2, C3, C5, C6-H), 8.05 (d, 2H, J =8 Hz, bridging phenyl-C3, C5-H), 10.43,
13
10.51 (s, 2H, NH); C NMR (100 MHz, DMSO-d6) δ ppm: 111.07, 118.77, 127.48, 128.82,
128.86, 129.18, 129.72, 141.66, 142.29, 165.10, 165.86; MS (ESI): m/z = found 317 [M+-2];
calcd. 319. Anal. Calcd. For C19H17N3O2: C, 71.46; H, 5.37; N, 13.16. Found: C, 73.10; H,
5.62; N, 14.08.
4.2.16. N'-(4-(1H-pyrrol-1-yl)benzoyl)-3-aminobenzohydrazide (5p)
(Yield 75%). mp 208-210 oC; FTIR (KBr): 3369.53 (NH2), 3215.25 (N-H), 1687.00, 1638.53
(C=O) cm-1; 1H NMR (400 MHz, DMSO-d6) δ ppm: 5.10 (s, 2H, -NH2), 6.31 (q, 2H, pyrrole-
C3, C4-H), 6.78 (m, 1H, aminophenyl-C4), 7.10-7.16 (m, 3H, pyrrole-C2, C5-H and
aminophenyl-C5), 7.37 (s, 2H, aminophenyl-C2, C6-H), 7.64 (d, 2H, J= 8 Hz, bridging
phenyl-C2, C6-H), 8.04-8.16 (q, 2H, bridging phenyl-C3, C5-H), 10.26 (s, 2H, NH); 13C NMR
(100 MHz, DMSO-d6) δ ppm: 110.99, 113.01, 117.01, 118.44, 118.70, 128.63, 129.12,
129.29, 133.52, 142.11, 148.44, 164.88, 166.41; MS (ESI): m/z = found 322 [M++2]; calcd.
320. Anal. Calcd. For C18H16N4O2: C, 67.49; H, 5.03; N, 17.49. Found: C, 68.10; H, 5.62; N,
18.08.
21

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4.2.17. N'-(4-(1H-pyrrol-1-yl)benzoyl)-3-nitrobenzohydrazide (5q)
(Yield 65%). mp 249-250 oC; FTIR (KBr): 3402.44 (N-H), 1681.35, 1643.65 (C=O) cm-1; 1H
NMR (400 MHz, DMSO-d6) δ ppm: 6.29-6.34 (m, 2H, pyrrole-C3, C4-H), 7.33-7.38 (m, 2H,
pyrrole-C2, C5-H), 7.77-8.44 (m, 7H, nitrophenyl-C4, C5, C6-H and bridging phenyl-C2, C3,
13
C5, C6-H), 8.85 (t, 1H, nitrophenyl-C2-H), 10.69 (s, 2H, NH); C NMR (100 MHz, DMSO-
d6) δ ppm: 111.34, 118.78, 124.88, 126.54, 128.42, 129.11, 130.11, 131.04, 133.96, 141.08,
147.76, 148.19, 163.82, 164.99; MS (ESI): m/z = found 350 [M+]; calcd. 350. Anal. Calcd.
For C18H14N4O4: C, 61.71; H, 4.03; N, 15.99. Found: C, 63.05; H, 4.62; N, 17.38.
4.2.18. N'-(4-(1H-pyrrol-1-yl)benzoyl)-2-bromobenzohydrazide (5r)
(Yield 65%). mp 225-227 oC; FTIR (KBr): 3207.58 (N-H), 1696.38, 1635.73 (C=O) cm-1; 1H
NMR (400 MHz, DMSO-d6) δ ppm: 6.28-6.31 (m, 2H, pyrrole-C3, C4-H), 7.32-7.69 (m, 8H,
pyrrole-C2, C5-H, bromophenyl-C2, C3, C5, C6-H and bridging phenyl-C2, C6-H), 8.08 (d, 2H,
J= 8 Hz, bridging phenyl-C3, C5-H), 10.42, 10.76 (s, 2H, NH); 13C NMR (100 MHz, DMSO-
d6) δ ppm: 111.37, 118.81, 119.40, 127.97, 128.26, 128.62, 129.44, 131.39, 132.85, 136.73,
142.33, 164.77, 166.60; MS (ESI): m/z = found 385 [M+]; calcd. 384.23. Anal. Calcd. For
C18H14BrN3O2: C, 56.27; H, 3.67; N, 10.94. Found: C, 58.05; H, 4.06; N, 12.38.
4.2.19. N'-(4-(1H-pyrrol-1-yl)benzoyl)-2,4-dichlorobenzohydrazide (5s)
(Yield 70%). mp 192-194 oC; FTIR (KBr): 3186.02 (N-H), 1606.01, 1519.67 (C=O) cm-1; 1H
NMR (400 MHz, DMSO-d6) δ ppm: 6.31 (p, 2H, pyrrole-C3, C4-H), 7.38 (t, 2H, pyrrole-C2,
C5-H), 7.46-7.66 (m, 5H, chlorophenyl-C3, C5, C6-H and bridging phenyl-C2, C6-H), 8.08 (d,
2H, J= 8Hz, bridging phenyl-C3, C5-H), 10.69 (s, 2H, NH); 13C NMR (100 MHz, DMSO-d6)
δ ppm: 111.11, 118.45, 118.79, 127.18, 128.50, 129.24, 129.38, 130.68, 131.85, 133.06,
135.45, 142.36, 164.76, 164.93; MS (ESI): m/z = found 372 [M+-2]; calcd. 374. Anal. Calcd.
For C18H13Cl2N3O2: C, 57.77; H, 3.50; N, 11.23. Found: C, 59.05; H, 3.82; N, 13.38.
22

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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 as described earlier [30]. 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 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 norfloxacin and ciprofloxacin against Gram-positive (S. aureus) and Gram-negative
bacteria (E. coli) by the broth microdilution method [31, 32]. 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 had
no effect on bacterial growth, a control was performed with the test medium supplemented
with DMSO at the same dilutions as used in the experiments and DMSO had no effect on the
23

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microorganisms in the concentration ranges studied. Table 1 reveals antibacterial activity
(MIC values) data.
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 [33] was used 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 [34] with cisplatin as the
positive control. The IC50 values are the averages ± SEM of three independent experiments,
which are presented in Table 2.
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. 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 a proper concentration (OD595 = 0.6 - 0.8), the
culture was centrifuged at 3300 g for 10 min at 4oC and the bacteria were suspended in LB
medium containing ampicillin (100 µg/mL). 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. The dry pellet was kept at -80°C for several
months and purification was performed with Ni-NTA Agarose from QIAGEN as described
by 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
24

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Coomassie blue staining and then stored at 4°C for a short-term storage at -80 °C with 20%
glycerin for long-term storage [35].
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 [36]. 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 followed 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 inhibitor. These
results are shown in Table 3.
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Conclusions
The novel compounds viz., N’-substituted benzoyl-4-(1H-pyrrol-1-yl)benzohydrazide
derivatives (5a-s) were synthesized that were found to possess good antibacterial activity, but
showed moderate InhA inhibition. These compounds were further explored for anti-TB and
antibacterial activity, against M. tuberculosis. The in vitro study suggests that compounds 5b,
5d, 5m, 5r and 5s are the most active with a MIC value of 3.125 µg/mL than the others in the
series. No noticeable cytotoxicity towards human lung cancer cell-line was observed,
indicating their non-toxicity Furthermore, compounds 5a-j have shown moderate InhA
inhibition. The moderate InhA inhibition in the micromolar range compared to good MIC
values were observed, which suggests that InhA enzyme is not the sole target.
The optimized pharmacophore model (MODEL_13) was developed that showed good
statistical parameters in the validation process. The pharmacophore delineates characteristic
features that are common to peptide linkage ligands active against M. tuberculosis. The
corresponding features on the peptide linkage and aromatic acceptor provide the necessary
interactions with the binding pockets. Molecular docking studies were performed to improve
the reliability and accuracy of the model. It was shown that amino acid (Tyr158, Thr196) and
NAD+ co-factor are essential for H-bonding interaction, while amino acids (Ala164, Ile95,
Gly94, Gly96, Met161, Ile21, Ala154, Met155, Pro193, Trp160, Phe97, Ile194, Pro156,
Ala157, Met103, Ile16, Pro99) are good for hydrophobic interaction, crucial for the inhibition
of protein. As per molecular modeling studies, these compounds are bound mainly in the
substrate binding site of InhA. Anti TB activity of these compounds is supported by the in
silico molecular docking calculations. These compounds show potential to be lead InhA
inhibitors.
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Acknowledgements
The authors acknowledge financial support from the Council of Scientific and Industrial
Research, New Delhi, India (Letter No. 02(0139)/13/EMR-II dated- 12/04/2013). We thank
Dr. H. V. Dambal, President, S. E. T’s College of Pharmacy, Dharwad, India for his support
and Dr. K. G. Bhat of Maratha Mandal's Dental College, Hospital and Research Centre,
Belgaum, India for providing anti-TB and cytotoxic activities. Director, SAIF Panjab
University, Chandigarh, Punjab, India has provided some of the NMR and mass spectral data.
The authors also appreciate the technical assistance from Mr. Manjunath Bangalore.
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