Synthesis, antimycobacterial screening and ligand-based molecular docking studies on novel pyrrole derivatives bearing pyrazoline, isoxazole and phenyl thiourea moieties

Article abstract of DOI:10.1016/j.ejmech.2015.10.047

We report 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 Mycobacterium tuberculsosi

Full text of DOI:10.1016/j.ejmech.2015.10.047

Accepted Manuscript
Synthesis, antimycobacterial screening and ligand-based molecular docking studies
on novel pyrrole derivatives bearing pyrazoline, isoxazole and phenyl thiourea
moieties
Shrinivas D. Joshi, Sheshagiri R. Dixit, M.N. Kirankumar, Tejraj M. Aminabhavi,
K.V.S.N. Raju, Ramanuj Narayan, Christian Lherbet, Kap Seung Yang
PII:
S0223-5234(15)30329-9
10.1016/j.ejmech.2015.10.047
EJMECH 8182
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 September 2015
Revised Date: 22 October 2015
Accepted Date: 28 October 2015
Please cite this article as: S.D. Joshi, S.R. Dixit, M.N. Kirankumar, T.M. Aminabhavi, K.V.S.N. Raju, R.
Narayan, C. Lherbet, K.S. Yang, Synthesis, antimycobacterial screening and ligand-based molecular
docking studies on novel pyrrole derivatives bearing pyrazoline, isoxazole and phenyl thiourea moieties,
European Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.10.047.
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ACCEPTED MANUSCRIPT
Synthesis, antimycobacterial screening and ligand-based molecular docking studies on
novel pyrrole derivatives bearing pyrazoline, isoxazole and phenyl thiourea moieties
Shrinivas D. Joshi, Sheshagiri R. Dixit, Kirankumar M. N, Tejraj M. Aminabhavi, K.
V. S. N. Raju, Ramanuj Narayan, Christian Lherbet, Kap Seung Yang
Antitubercular activity of novel series of pyrrolyl chalcone, pyrazline, isoxazole and phenyl
urea derivatives was analyzed. Molecular modeling constructed using Surflex-Dock study
using enoyl ACP reductase from Mycobacterium tuberculosis.

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Synthesis, antimycobacterial screening and ligand-based molecular docking studies on
novel pyrrole derivatives bearing pyrazoline, isoxazole and phenyl thiourea moieties
Shrinivas D. Joshi*a, Sheshagiri R. Dixita, Kirankumar M. N.a, Tejraj M. Aminabhavia,
K. V. S. N. Rajub**, Ramanuj Narayanb, Christian Lherbetc, Kap Seung Yangd
aNovel Drug Design and Discovery Laboratory, Department of Pharmaceutical Chemistry,
S.E.T’s College of Pharmacy, Sangolli Rayanna Nagar, Dharwad-580 002, India.
bDivision of Polymers and Functional Materials CSIR-Indian Institute of Chemical
Technology, Hyderabad 500 607, India.
cUniversite 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
dDepartment of Polymer and Fiber System Engineering, Chonnam National University, 300
Yongbong-Dong, Bukgu, Gwangju, 500 757, Korea.
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
** Authors dedicate this work on the eve of retirement of Dr. K. V. S. N. Raju after 37 years
of service at IICT, Hyderabad
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Abstract:
We report 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 toxicity using human lung cancer cell-line (A549) and were nontoxic. Some
compounds exhibited inhibition activities against InhA.
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Keywords: Surflex docking, Pyrrolyl chalcones, Pyrrolyl isoxazoles, Pyrrolyl pyrazolines,
Anti-tubercular activity, Cytotoxicity activity, Enzyme inhibition studies.
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1. Introduction
Tuberculosis (TB) is a chronic disorder caused by five closely related mycobacteria such as
Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum,
Mycobacterium microti and Mycobacterium canetti. Among these, Mycobacterium
tuberculosis (M. tuberculosis), is an infection caused by slow-growing bacteria in parts of the
body having high level of blood and oxygen is often found in lungs, called pulmonary TB.
The disease also spreads to other parts of the body, called as extra-pulmonary TB that may be
latent or active. In other case, treatment of the active TB is more complex due to multi-drug
resistance (MDR-TB), extensive-drug resistance (XRD-TB) and HIV infection. The MDR-
TB is a type of TB which occurs once MTB strain turns resistant to the most efficient anti-TB
drugs i.e. rifampin and isoniazid. In 2013, 0.45 million people developed MDR-TB
worldwide and there were 0.21 million deaths resulting from MDR-TB. XDR-TB occurs
when MTB strain is resistant to at least isoniazid and rifampin in addition to being resistant to
one of the fluoroquinolones, as well as resistant to at least one of the second line injectable
drugs i.e. amikacin, kanamycin or capreomycin. XDR-TB was found worldwide in 100
countries by the end of 2013. About, 9% of MDR-TB cases lead to XDR-TB which is related
with higher mortality rate than MDR-TB. World Health Organization (WHO) estimated 9.0
million people with cases of TB in 2013 of which 1.5 million died and 360,000 of them were
affected with HIV-positive. TB is a foremost public health problem in India. India accounts
for one-fifth of the world TB incident cases. Every year about 2 million people in India
develop TB, of which around 0.87 million are infectious cases. Furthermore, it is estimated
that yearly around 330,000 Indians die due to TB. The MTB generally attacks the lungs,
spine, kidney, and brain. Therefore, if TB is not treated properly, it can be severe and fatal.
Over the past decades, several anti-tubercular (anti-TB) drugs have been developed see Fig.
1, but drug-resistance issue has not been solved. There is thus a tremendous need to develop
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new anti-TB drugs that are active against both acute and chronic growth phases of
mycobacterium to stop all forms of drug resistant-TB [1, 2]. In this perspective, many studies
have been made on targeting the cell wall of mycobacteria.
Mycolic acid biosynthesis has been carried out [2] by numerous successive enzymatic cycles
equivalent to Fatty Acid Synthase (FAS) systems viz., FAS I and II. Mycolic acid is a unique
signature fatty acid, which is a core constituent of the mycobacterial cell wall present in fatty
acid synthase system of M. tuberculosis. InhA, the enoyl acyl carrier protein reductase (ENR)
from M. tuberculosis is the key enzyme for type II fatty acid synthesis (FAS II), which
catalyses NADH-dependent reduction of 2-trans-enoyl-ACP (acyl carrier protein) to yield
NAD+ and reduced enoyl thioester-ACP substrate, which in turn, helps the synthesis of
mycolic acid.
Chalcone is a central core for many important biological compounds that are synthesized by
aldol condensation reaction between substituted aryl ketones and aromatic aldehydes in the
presence of sodium hydroxide as a catalyst. These undergo variety of chemical reactions to
produce innumerable heterocyclic compounds that are used as intermediates to prepare drugs
with therapeutic value. Literature reveals that chalcone derivatives from natural and synthetic
analogs exhibit diverse pharmacological activities such as anti-TB, anti-inflammatory, anti-
cancer, anti-neoplastic, anti-bacterial, anti-fungal, anti-malarial, anti-viral, anti-allergic and
estrogenic [3-9]. On the other hand, isoxazole derivatives constitute a class of nitrogen and
oxygen containing five membered heterocyclic compounds that are the important class of
heterocyclic pharmaceuticals due to their wide spectrum of biological activities, including
potent and selective antagonism of NMDA receptor [10], anti-HIV activity [11], anti-
tuberculosis, anti-bacterial, antibiotic, anti-fungal, anti-cancer, ulcerogenic activities and also
used as COX-2 inhibitors and anti-inflammatory drugs [12-16].
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Pyrazolines have been widely used as anti-tubercular [17], anti-bacterial [18] and anti-cancer
[19] agents, as these have a broad spectrum antimicrobial activity and hence, can be explored
further. The most prominent compounds featuring pyrazoline nucleus are econazole,
ampyrone, phenazone and propylphenzone see Fig. 2a. Some of the reported pyrazoline
skeletals which exhibit anti-tubercular activity are shown in Fig. 2b [20-23].
Pyrrole 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. First isolated in 1857 from
the products of bone pyrolysis, it showed biological activities that are characteristic of
haemoglobin [24], Earlier, pyrrole derivatives have shown in vitro anti-tubercular activity
[25, 26] and recently, much of the research was carried out on anti-TB drug design using
pyrroles as templates for synthesis [27, 28], including molecular modeling along with the
laboratory investigations [29-31]. Biava et al [32] reported several 1,5-diarylpyrrole
derivatives with a very good activity against MTB (BM 212). Based on the work of Deidda
et al. [33], Lupin developed a series of pyrrole compounds, of which LL3858 is currently in
pre-clinical trials for the treatment of TB [34], suggesting the importance of pyrrole
derivatives as the anti-TB agents. This prompted us to undertake detailed investigation on the
design and synthesis of new pyrrole derivatives useful as anti-TB agents.
In our previous studies [35, 36], we have synthesized the potential inhibitors of InhA bearing
pyrrole as a central core with different pharmacophores in a single molecular framework
along with 2D and 3D-QSAR studies. In this work, we have undertaken to develop new
chemical entities containing pyrrole as the core that inhibits enoyl ACP reductase enzyme
along with their in vitro anti-bacterial and anti-TB activities. Fig. 2 represents some of the
marketed drugs that were considered in the synthesis of new derivatives following the Paal-
Knorr pyrrole synthesis. Fig. 3 indicates the design concept used to describe our framework
by combining molecular docking and classification techniques to understand the structural
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characteristics affecting the binding of pyrrolylchalcones, pyrazolines and isoxazoles with
enoyl ACP reductase receptor. Molecular docking studies have been used to correlate in
silico results with in vitro analysis to find the ENR as a potential target of pyrrolylchalcone,
pyrazoline and isoxazole derivatives.
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2. Molecular modeling/docking studies
The 3D structures were generated using SYBYL package (Tripos Associates, St. Louis, MO,
USA) [37]. The geometry optimization was done with the help of standard Tripos force field
[38] using a distance dependent-dielectric function, energy gradient of 0.001 kcal/ mol and
MMFF94 as the electrostatics. Conformational analyses of all the 61 compounds were
performed using a repeated molecular dynamics-based simulated annealing approach as
implemented in Sybyl-X 2.0. The molecule was heated up to 1000 K within 2000 fs, held at
this temperature for 2000 fs and annealed to 0 K for 10,000 fs using an exponential annealing
function. By employing this procedure, 100 conformations were sampled out during 100
cycles to account the conformational flexibility to find the most likely conformations
occurring most often in the resulting pool. All conformations were minimized with Tripos
force field and atomic charges were calculated using MMFF94 (Merck Molecular Force
Field) method.
2.1. Molecular docking using Surflex-Dock
Molecular docking was used to clarify the binding mode of the compounds to provide
straightforward information for further structural optimization. Surflex-Dock that adopted an
empirical scoring function and a patented searching engine [39, 40] was employed for
molecular docking. 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). During the docking process, water molecules and all the
ligands in the crystal structures were removed (except co-factor NAD+). The polar hydrogens
as well as united atom Amber7 FF99 were assigned for the protein PDB ID 4TZK. Then,
ligand-based mode was adopted to generate the “protomol”, leaving the threshold and bloat
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parameters at their default values of 0.50 and 0 Å; all the inhibitors were docked within the
prepared protein.
The mode of interaction of the relative ligand in the crystal structure against 4TZK PDB was
used as a standard docked model. The maximum number of poses per ligand was set to 20
with no constraints to perform the molecular docking. The docking complex was assumed to
represent the ligand-receptor interactions, which was selected based on three criteria: (i)
docking score of the pose possessed the highest docking score, (ii) its orientation of aromatic
rings of the ligand oriented into the active site in a similar manner with the cocrystallized
ligands orientation, and (iii) preservation of the two key interactions viz., H- bonds with
Tyr158 and Co-factor NAD+. For comparative analysis of the designed molecules, D_score
[41], PMF_score [42], G_score [43] and Chem_score [44] were estimated using the C-Score
module of the Sybyl-X 2.0.
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3. Results and discussion
3.1. Chemistry
All the compounds were synthesized as per steps outlined in Schemes 1, 2 and 3. The Paal-
Knorr reaction was performed to synthesize (4-pyrrol-1-yl)acetophenone (2) by condensing
4-amino acetophenone (1) with 2,5-dimethoxytetrahydrofuran. The required key
intermediates viz., chalcones (3a-r) were obtained by Claisen-Schmidt condensation of (4-
pyrrol-1-yl)acetophenone (2) with the substituted aldehydes in the presence of sodium
hydroxide catalyst in ethanol. Chalcones (3a-r) were treated with hydrazine hydrate and
glacial acetic acid in a solvent free condition to obtain the corresponding N-acetyl pyrazolines
(4a-s). The reaction of chalcones with hydroxylamine hydrochloride and sodium acetate in
the presence of glacial acetic acid led to the synthesis of 5-(4-(1H-pyrrol-1yl)phenyl)-3-
substituted phenylisoxazoles (5a-r) as per schemes 1 and 2.
o-Phenylenediamine (6) reacted with acetonyl acetone (7) to afford 2-(2,5-dimethyl-1H-
pyrrol-1-yl)aniline (8). Next, different phenylisothiocyanates were reacted with intermediate
8 in dry chloroform to get the final desired 1-(2-(2,5-dimethyl-1H-pyrrol-1-yl)phenyl)-3-
(substituted phenyl)thioureas (9a-k) with good yields (Scheme 3).
1
13
All the synthesized compounds were characterized by FTIR, H NMR, C NMR and mass
spectroscopy. FTIR spectrum of the compound 3e, as a representative of chalcone series,
showed an absorption band at 1657 cm-1 associated with carbonyl group from the α, β-
1
unsaturated fragment. In the H NMR spectrum of compound 3e, a singlet at δ 3.74 ppm
indicates the presence of methoxy group, two doublets at δ 7.98 and 8.00 ppm are assigned to
Hα and Hβ, respectively for vinylic system. Protons of pyrrole moiety resonated as two triplets
13
at δ 6.29 and 7.07 ppm, respectively. The C NMR spectrum of compound 3e showed the
signal at δ 188.92 ppm corresponding to carbonyl group of α, β-unsaturated fragment. The
signal corresponding to methoxy group appears at δ 55.45 ppm. All the other aromatic
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carbons resonated in the expected region of δ 111.55-144.74 ppm. The molecular ion peak at
m/z 288.13 (100%) (EI-MS) confirmed the formation of the desired product.
FTIR spectrum of pyrazoline (Compound 4g) showed absorption bands at 1661 (C=O) and
1609 cm-1 (C=N). Formation of pyrazoline was further confirmed by 1H NMR, wherein CH2
protons of pyrazoline ring resonated as a pair of doublets of doublets at δ 3.17 ppm (Ha) and δ
3.79 ppm (Hb) with JAB = 22.2-29.5 Hz. The –CH(Hx) proton appeared as a doublet of
doublets at δ 5.60 ppm due to vicinal coupling with two magnetically non-equivalent protons
of methylene group at position 4 of the pyrazoline ring with a JAX value of 16.44 Hz. A
singlet at δ 2.42 ppm was assigned to methyl protons of acetyl group (-COCH3). The 13C
NMR data of the compound 4g supported the structure via COCH3 and COCH3 resonances
appearing at δ 168.86 and 21.92 ppm, respectively. Furthermore, peaks observed at δ 152.94,
59.35 and 42.24 ppm are due to C1, C4 and C5 of pyrazoline, respectively. The mass spectrum
of 4g showed a molecular ion peak at m/z 347 (80%), which confirmed its molecular weight.
The 1H NMR spectrum of 5e showed a doublet at δ 6.72 ppm (J = 16 Hz) due to C4 proton of
isoxazole. The structure of 5e was further confirmed by 13C NMR, which displayed signals at
δ 159.29, 110.54 and 156.22 ppm due to C3, C4 and C5 carbons of isoxazole ring,
respectively. All the other carbons resonated in the expected regions. The mass spectrum of
5e exhibited molecular ion peak at m/z 317 (100%) confirming its molecular weight.
FTIR spectrum of 9c displayed stretching bands at 3304 and 3170 cm-1 due to NH group. The
1H NMR spectrum of this sample displayed a singlet at δ 1.81 ppm due to methyl protons of
pyrrole, while a singlet at δ 5.81 ppm was due to C3 and C4 protons of pyrrole. Two singlets
at δ 8.56 and 8.28 ppm were assigned to two NH protons. The 13C NMR data of 9c also
supported the structure, which displayed the peaks at δ 12.38 ppm due to two methyl carbons
of pyrrole. The C=S resonance appeared at δ 178.45 ppm. The mass spectrum of 9c showed a
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molecular ion peak at m/z 339 (80%), which confirmed its molecular weight. Various
compounds synthesized with their data are indicated in the experimental section.
3.2. Antitubercular and antibacterial activities
The anti-TB activity of the compounds was studied with M. tuberculosis (see Table 1). The
preliminary anti-TB screening revealed that majority of compounds showed quite moderate
to good activity. The activities of 3a-r, 4a-q, 5a-o and 9a-k are expressed in terms of
minimum inhibitory concentration (MIC) values using ethambutol and rifampicin as the
reference drugs. In the first series of compounds, 4d, 4e, 4m, 4p, 4q and 5m showed better
activity with the inhibition of mycobacterium at MIC of 6.25 µg/mL, while those from the
third series of compounds 9b and 9d showed the highest activity with MIC value of 0.8
µg/mL. A good anti-TB activity is due to the presence of pharmacologically active hetero-
aryl groups such as pyrazoline, isoxazole and aromatic ring with -NH-CS-NH-linkage
attached to pyrrole ring. The pyrazoline derivatives displayed relatively higher inhibitory
activity compared to chalcones and isoxazoles. It is encouraging to observe that compounds
9b and 9d showed very good anti-TB activities against MTB strain (MIC=0.8 µg/mL), while
9c showed the promising activity (MIC=1.6 µg/mL).
Antibacterial activity was also carried out for all the compounds against both Gram positive
bacteria (S. aureus) and Gram negative bacteria (E. Coli). The antimicrobial screening data
revealed that all the compounds showed moderate to significant microbial inhibition. Among
all the compounds tested, compounds 9a-k showed excellent activity with the MIC values of
0.2-1.6 µg/mL. Compounds 3a-r, 4a-q, 5a-o exhibited good to moderate antimicrobial
activity with the MIC values of 6.25-100 µg/mL.
Among the compounds tested (Table 1), compounds 9d and 9b bearing para-trifluoro and
ortho-fluoro substitution respectively, on the aryl ring gave the best MIC. Clearly the fluoro
substitution at the aryl ring, which is more electro-negative than other halogens like Cl and
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Br, is important for activity compared to other molecules in the series. The presence of chloro
or bromo substitution at 3rd or 4th position on the aryl ring shows comparatively less activity.
The lack of inhibition for these compounds on InhA in contrast with low MIC suggests that
the target is not InhA.
The presence of trifluoro substitution at 4th position of aryl ring resulted in low MIC values.
Compound 9j bearing chloro substitution exhibit lower MIC (6.25 µg/mL) by comparison
with compound 9d, which is a non-chloro derivative (MIC 0.8 µg/mL). Therefore we believe
that some of these derivatives might have biological target other than InhA and a mode of
action different than triclosan thereby making them relevant candidates for further drug
design in MDR-TB research. In general, these compounds are not only active on InhA but
also on M. tuberculosis H37Rv strain.
3.3.MTT-based cytotoxicity studies
Certain therapeutic properties are to be identified to show the antimycobacterial potential of a
drug. Toxicity is one of these criteria. Hence, we have investigated the potential toxicity of
eight selected pyrrolyl derivatives (3d, 3n, 4m, 4e, 5m, 5e, 9b and 9d) towards A549 (lung
adenocarcinoma) cell-lines up to concentrations of 62.5 µg/mL. These compounds showed a
moderate cytotoxicity compared to cisplatin (Table 2). Specifically, the most potent
compounds viz., 4m and 5m exhibited a good safety profile as their IC50 value was 39.6
µmol/L against the A549 cell-line.
3.4.Enzyme inhibition studies
By considering in vitro antimycobacterial studies, we have selected four compounds for in
vitro enzyme inhibition activity against InhA from the M. tuberculosis at 50 µM by applying
the commonly used approach. Triclosan was tested first at the same concentration and
showed complete inhibition of InhA at 50 µM. The results are shown in Table 3.
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The compounds bearing chlorine atom (compound 9h) on the aromatic ring are very week (or
not) inhibitors of InhA. In fact it doesn’t showed any inhibition at 50 µM. but the 2,4-
dichloro substituted analogue 9j shows a 13% inhibition at 50 µM. Based on previous
reported research’s, we hypothesized that substitution of highly electro-negative groups
mimic the InhA substrate might provide compounds with higher affinity. The introduction of
lipophilic groups like bulkier groups or halogen might facilitate more hydrophobic interaction
in the pocket. The result shows an increase in the inhibitory activity (Table 3). Compound 9d
bearing trifluoro substitution by in comparison with 9j presents 100% inhibition at 50 µM.
3.5.Molecular docking studies
To investigate the mechanism of anti-TB activity and detailed 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. On the basis of
greater level of resistance associated with INH isolates against InhA, docking studies were
performed on InhA complex with 1-cyclohexyl-N-(3,5-dichlorophenyl)-5-oxopyrrolidine-3-
carboxamide, which indicates the presence of drug-receptor interactions. All the 61 inhibitors
were docked into the active site of ENR as shown in Fig 4A and 4B. The predicted binding
energies of the compounds are listed in Table 4.
The interaction of compound 4q with the enzyme depicted in Fig. 6A and 6B shows that
oxygen of methoxy group at the 4th position of phenyl ring attached to pyrazoline moiety of
compound 4q has one H-bonding interaction with the hydrogen of MET98 (-OCH3 ------ H-
MET98). As depicted in Fig. 7A and 7B, the fluorine atom of CF3 group of compound 9d
exhibited H-bonding interaction with the hydrogen of MET98 (C-F ---- H-MET98; 2.49 Å).
In Fig. 8A and 8B, the oxygen and nitrogen of isoxazole ring (compound 5g) makes two H-
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bonding interactions with the hydrogen of MET98 (O ----- H-MET98; 2.49 Å and N ----- H-
MET98; 1.90 Å), while fluorine atom makes an H-bonding interaction with GLN100 (F ------
HE-GLN100; 2.40 Å). On the other hand, hydrophobic (Ile105, Leu207, Met103, Trp160,
Ala206, Met161, Met98, Pro99, Ala157, Ala211, Pro156, Ala201, Ile202, Val203, Ile215,
Leu218, Leu217, Phe97, Ile122, Val65, Ile16, Ile95, Ala198, Leu197, Met199, Phe149,
Pro193, Ile194, Ala191, Ile21, Met147, Trp222, Trp230) and hydrophilic (Asp148, Ser94,
Gly192, Asp150, Gly14, Lys165, Ser20, Ser19, Thr196, Arg195, Glu219, Gly96, Thr162,
Ser123, Gly119, Asp64, Tyr158, Asn159, Thr196, Arg195, Glu219, Gln216, Ser200, Gln214,
Gly205, Gly104, Gln100, Gly102, Asn159, Tyr158, Gly119) amino acid residues are
surrounded to the representative compound 4q are depicted in the Figure 9A and 9B.
All the compounds showed consensus score in the range 7.51-1.60, indicating the summary
of all forces of interaction between ligands and the InhA. Charge and van der Waals
interactions between protein and ligands varied from -75.28 to -180.16. The Helmholtz free
energies of interactions for protein ligands atom pairs range between -16.05 and 88.56
However, its H-bonding, complex (ligand-protein), and internal (ligandeligand) energies
range from -113.69 to -323.98, while those values -26.14 to -49.79 indicate the ligands due to
H-bonding, lipophilic contact, and rotational entropy, as well as intercept terms. These scores
indicate that molecules preferentially bind to InhA in comparison to the reference 4TZK
ligand (Table 4). In general, it was observed that -OCH3 and C=O groups make the H-bond
with a substrate binding site and presence of electron donating or withdrawing substitution on
the aromatic ring attached to pyrazoline/isoxazole moiety may favours the activity, while
those of pyrrole, pyrazoline, isoxazole and phenylthiourea moieties help to occupy or
penetrate the molecule at the active sites.
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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. The 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 the 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. General procedure for the synthesis of 4-(1-H-pyrrol-1-yl)-acetophenone (2) 45
A mixture of 2,5-dimethoxytetrahydrofuran (4.23 g, 0.032 mol) and 4-aminoacetophenone
(4.05 g, 0.030 mol) taken in glacial acetic acid (12 mL) was refluxed for 1 h, poured into ice
cold water and basified with NaHCO3 solution. The solid separated was washed with water,
dried and recrystallized from ethanol.
4.2.General procedure for the synthesis of 1-(4-(1H-pyrrol-1yl)phenyl)-3-substitutedprop-2-
en-1-ones (3a-r)
To a mixture of 4-(1-H-pyrrol-1-yl)-acetophenone (0.01 mol) and substituted aldehydes
(0.01 mol) in ethanol (20 mL), a solution of sodium hydroxide (40%, 8 mL) was added
slowly. The mixture was stirred for 24-30 h, poured into ice-cold water, and neutralized with
hydrochloric acid. The solid separated was filtered off, washed with water, dried and purified
16

ACCEPTED MANUSCRIPT
by column chromatography on silica gel with ethyl acetate/petroleum ether (6:4) as the
eluent.
4.2.1.1-(4-(1H-pyrrol-1yl)phenyl)-3-phenylprop-2-en-1-one (3a)
(Yield 70%). mp 255-257 oC; FTIR (KBr): 1653.95 (C=O) cm-1; 1H NMR (400 MHz, CDCl3)
δ ppm: 6.40 (t, 2H, pyrrole-C3, C4-H), 7.19 (t, 2H, pyrrole-C2, C5-H), 7.40-7.44 (m, 3H,
phenyl-C3, C4, C5-H), 7.49-7.53 (m, 2H, phenyl-C2, C6-H), 7.67 (q, 2H, bridging phenyl-C2,
C6-H), 7.86 (d, 2H, J =16 Hz, bridging phenyl-C3, C5-H), 8.09-8.13 (m, 2H, -CH=CH-); MS
(ESI): m/z = found 273.12 [M+]; calcd. 273.12. Anal. Calcd. For C19H15NO: C, 83.49; H,
5.53; N, 5.12. Found: C, 84.60; H, 6.32; N, 6.33.
4.2.2. 1-(4-(1H-pyrrol-1yl)phenyl)-3-(4-chlorophenyl)prop-2-en-1-one (3b)
(Yield 70%). mp 208-210 oC; FTIR (KBr): 1655.83 (C=O) cm-1; 1H NMR (400 MHz, CDCl3)
δ ppm: 6.34 (dd, 2H, pyrrole-C3, C4-H), 7.39 (dd, 2H, pyrrole-C2, C5-H), 7.42-7.95 (m, 8H, 4-
chlorophenyl-C2, C3, C4, C6-H and bridging phenyl-C2, C3, C5, C6-H), 8.23 (d, 2H, J =8 Hz, -
CH=CH-); MS (ESI): m/z = found 308.08 [M+ + 1]; calcd. 307.08. Anal. Calcd. For
C19H14ClNO: C, 74.15; H, 4.58; N, 4.55. Found: C, 75.75; H, 5.32; N, 5.33.
4.2.3. 1-(4-(1H-pyrrol-1yl)phenyl)-3-(2-chlorophenyl)prop-2-en-1-one (3c)
(Yield 65%). mp 103-105 oC; FTIR (KBr): 1656.02 (C=O) cm-1; 1H NMR (400 MHz, CDCl3)
δ ppm: 6.41 (dd, 2H, pyrrole-C3, C4-H), 7.16-7.20 (m, 2H, pyrrole-C2, C5-H), 7.30-7.37 (m,
2H, 2-chlorophenyl-C4, C5-H), 7.43-8.13 (m, 6H, 2-chlorophenyl-C3, C6-H and bridging
phenyl-C2, C3, C5, C6-H), 8.22 (d, 2H, J =16 Hz, -CH=CH-); 13C NMR (100 MHz, CDCl3) δ
ppm:111.62, 119, 119.44, 124.40, 127.07, 127.79, 130.31, 130.49, 131.20, 133.21, 134.65,
135.50, 140.62, 143.94, 188.80; MS (ESI): m/z = found 308.08 [M+ + 1]; calcd. 307.08. Anal.
Calcd. For C19H14ClNO: C, 74.15; H, 4.58; N, 4.55. Found: C, 75.75; H, 5.32; N, 5.33.
4.2.4. 1-(4-(1H-pyrrol-1yl)phenyl)-3-p-tolylprop-2-en-1-one (3d)
17

ACCEPTED MANUSCRIPT
o
1
(Yield 70%). mp 188-190 C; FTIR (KBr): 1656.63 (C=O) cm-1; H NMR (400 MHz,
CDCl3) δ ppm: 2.39 (s, 3H, -CH3), 6.40 (t, 2H, pyrrole-C3, C4-H), 7.19 (t, 2H, 4-
methylphenyl-C3, C5-H), 7.25 (t, 2H, pyrrole-C2, C5-H), 7.48-7.84 (m, 6H, 4-methylphenyl-
C2, C6-H and bridging phenyl-C2, C3, C5, C6-H), 8.09-8.12 (m, 2H, -CH=CH-); 13C NMR
(100 MHz, CDCl3) δ ppm: 21.58, 111.59, 119.07, 119.48, 120.62, 128.55, 129.76, 130.35,
132.14, 135.16, 141.21, 143.81, 144.99, 188.99; MS (ESI): m/z = found 288.13 [M+ + 1];
calcd. 287.13. Anal. Calcd. For C20H17NO: C, 83.59; H, 5.96; N, 4.87. Found: C, 84.75; H,
6.32; N, 6.33.
4.2.5.1-(4-(1H-pyrrol-1yl)phenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (3e)
o
1
(Yield 80%). mp 168-170 C; FTIR (KBr): 1657.47 (C=O) cm-1; H NMR (400 MHz,
CDCl3) δ ppm: 3.74 (s, 3H, -OCH3), 6.29 (t, 2H, pyrrole-C3, C4-H), 6.84 (d, 2H, J =8 Hz, 4-
methoxyphenyl-C3, C5-H), 7.07 (t, 2H, pyrrole-C2, C5-H), 7.30-7.72 (m, 6H, 4-
methoxyphenyl-C2, C6-H and bridging phenyl-C2, C3, C5, C6-H), 8.00 (dd, 2H, -CH=CH-);
13C NMR (100 MHz, CDCl3) δ ppm: 55.45, 111.55, 111.47, 119.47, 127.61, 130.28, 130.30,
135.31, 143.72, 144.74, 161.76, 188.92; MS (ESI): m/z = found 304.13 [M+ + 1]; calcd.
303.13. Anal. Calcd. For C20H17NO2: C, 79.19; H, 5.65; N, 4.62. Found: C, 80.75; H, 6.32;
N, 6.33.
4.2.6.1-(4-(1H-pyrrol-1yl)phenyl)-3-(3-bromophenyl)prop-2-en-1-one (3f)
o
1
(Yield 60%). mp 158-160 C; FTIR (KBr): 1657.46 (C=O) cm-1; H NMR (400 MHz,
CDCl3) δ ppm: 6.41-6.44 (m, 2H, pyrrole-C3, C4-H), 7.18-7.84 (m, 10H, pyrrole-C2, C5-H;
bridging phenyl-C2, C3, C5, C6 and 3-bromophenyl-C2, C4, C5, C6-H), 8.12-8.16 (m, 2H, -
CH=CH-); 13C NMR (100 MHz, CDCl3) δ ppm: 111.66, 119.44, 122.74, 123.08, 127.29,
130.40, 130.47, 130.78, 133.27, 134.59, 136.94, 142.93, 144, 188.35; MS (ESI): m/z = found
352.03 [M+ + 1]; calcd. 351.03. Anal. Calcd. For C19H14BrNO: C, 64.79; H, 4.01; N, 3.98.
Found: C, 66.15; H, 5.32; N, 5.33.
18

ACCEPTED MANUSCRIPT
4.2.7.1-(4-(1H-pyrrol-1yl)phenyl)-3-(4-fluorophenyl)prop-2-en-1-one (3g)
o
1
(Yield 70%). mp 198-200 C; FTIR (KBr): 1654.24 (C=O) cm-1; H NMR (400 MHz,
CDCl3) δ ppm: 6.31 (t, 2H, pyrrole-C3, C4-H), 7.22 (q, 2H, 4-fluorophenyl-C3, C5-H), 7.43
(t, 2H, pyrrole-C2, C5-H), 7.71 (t, 2H, 4-fluorophenyl-C2, C6-H), 7.74-7.91 (m, 4H, bridging
13
phenyl-C2, C3, C5, C6-H), 8.22 (d, 2H, J=8Hz, -CH=CH-); C NMR (100 MHz, CDCl3) δ
ppm: 111.62, 116.01, 116.30, 119.01, 119.45, 121.28, 130.31, 130.34, 130.43, 131.11,
134.84, 143.55, 143.91, 162.42, 188.68; MS (ESI): m/z = found 292.11 [M+ + 1]; calcd.
291.11. Anal. Calcd. For C19H14FNO: C, 78.33; H, 4.84; N, 4.81. Found: C, 79.75; H, 5.32;
N, 5.33.
4.2.8.1-(4-(1H-pyrrol-1yl)phenyl)-3-(2-bromophenyl)prop-2-en-1-one (3h)
(Yield 60%). mp 214-216 oC; FTIR (KBr): 1656.29 (C=O) cm-1; 1H NMR (400 MHz, CDCl3)
δ ppm: 6.38-6.41 (m, 2H, pyrrole-C3, C4-H), 7.15-7.20 (m, 2H, pyrrole-C2, C5-H), 7.24-8.17
(m, 10H, 2-bromophenyl C3, C4, C5, C6-H; bridging phenyl-C2, C3, C5, C6-H and -CH=CH-);
13C NMR (100 MHz, CDCl3) δ ppm: 111.62, 119, 119.42, 124.66, 125.89, 127.70, 127.87,
130.51, 131.35, 133.56, 134.60, 135.01, 143.19, 143.94, 188.82; MS (ESI): m/z = found
352.03 [M+ + 1]; calcd. 351.03. Anal. Calcd. For C19H14BrNO: C, 64.79; H, 4.01; N, 3.98.
Found: C, 66.15; H, 5.32; N, 5.33.
4.2.9. 1-(4-(1H-pyrrol-1yl)phenyl)-3-(3,4-dimethoxyphenyl)prop-2-en-1-one (3i)
o
1
(Yield 78%). mp 183-185 C; FTIR (KBr): 1657.02 (C=O) cm-1; H NMR (400 MHz,
CDCl3) δ ppm: 3.94 (s, 3H, -OCH3), 3.96 (s, 3H, -OCH3), 6.41 (t, 2H, pyrrole-C3, C4-H),
6.92 (d, 1H, J= 8 Hz, methoxyphenyl-C5-H), 7.17-7.20 (m, 2H, methoxyphenyl-C2, C6-H),
7.24-7.27 (m, 2H, pyrrole-C2, C5-H), 7.50-7.53 (d, 2H, bridging phenyl-C2, C6-H), 7.82 (d,
2H, J= 16 Hz, phenyl-C3, C5-H), 8.09-8.13 (m, 2H, -CH=CH-); 13C NMR (100 MHz,
CDCl3) δ ppm: 55.93, 55.96, 110.04, 111.07, 111.50, 119.38, 119.48, 123.18, 127.76,
130.23, 135.18, 143.65, 144.98, 149.20, 151.44, 188.86; MS (ESI): m/z = found 334.14 [M+
19

ACCEPTED MANUSCRIPT
+ 1]; calcd. 333.14. Anal. Calcd. For C21H19NO3: C, 75.66; H, 5.74; N, 4.20. Found: C,
76.75; H, 6.32; N, 5.33.
4.2.10. 1-(4-(1H-pyrrol-1yl)phenyl)-3-(2,4-dichlorophenyl)prop-2-en-1-one (3j)
o
1
(Yield 70%). mp 192-194 C; FTIR (KBr): 1655.78 (C=O) cm-1; H NMR (400 MHz,
CDCl3) δ ppm: 6.41 (dd, 2H, pyrrole-C3, C4-H), 7.20 (dd, 2H, pyrrole-C2, C5-H), 7.32 (dd,
1H, chlorophenyl-C5-H), 7.47-7.53 (m, 4H, chlorophenyl-C3, C6-H and bridging phenyl-C2,
C6-H), 7.71 (d, 2H, J= 8 Hz, phenyl-C3, C5-H), 8.08-8.12 (m, 2H, -CH=CH); 13C NMR (100
MHz, CDCl3) δ ppm: 111.69, 118.97, 119.42, 124.56, 127.54, 128.48, 130.14, 130.47,
131.78, 134.46, 136.07, 136.49, 139.29, 144.03, 188.42; MS (ESI): m/z = found 342.04 [M+
+ 1]; calcd. 341.04. Anal. Calcd. For C19H13Cl2NO: C, 66.68; H, 3.83; N, 4.09. Found: C,
67.75; H, 4.32; N, 5.33.
4.2.11. 1-(4-(1H-pyrrol-1yl)phenyl)-3-(2,3-dichlorophenyl)prop-2-en-1-one (3k)
o
1
(Yield 67%). mp 157-159 C; FTIR (KBr): 1651.62 (C=O) cm-1; H NMR (400 MHz,
CDCl3) δ ppm: 6.39-6.41 (m, 2H, pyrrole-C3, C4-H), 7.20 (t, 2H, pyrrole-C2, C5-H), 7.29 (t,
1H, chlorophenyl-C5-H), 7.46-7.54 (m, 3H, chlorophenyl-C6-H and bridging phenyl-C2, C6-
H), 7.67 (dd, 1H, chlorophenyl-C4-H), 8.09-8.21 (m, 4H, bridging phenyl-C3, C5-H and -
CH=CH-); 13C NMR (100 MHz, CDCl3) δ ppm: 111.70, 118.99, 119.44, 125.47, 125.89,
127.38, 130.02, 130.52, 131.65, 134.40, 135.60, 140.49, 144.06, 188.48; MS (ESI): m/z =
found 342.04 [M+ + 1]; calcd. 341.04. Anal. Calcd. For C19H13Cl2NO: C, 66.68; H, 3.83; N,
4.09. Found: C, 67.75; H, 4.32; N, 5.33.
4.2.12. 1-(4-(1H-pyrrol-1yl)phenyl)-3-(2,6-dichlorophenyl)prop-2-en-1-one (3l)
(Yield 67%). mp 136-140 oC; FTIR (KBr): 1662.12 (C=O) cm-1; 1H NMR (400 MHz, CDCl3)
δ ppm: 6.41 (t, 2H, pyrrole-C3, C4-H), 7.20 (t, 2H, pyrrole-C2, C5-H), 7.24 (d, 1H, J=8 Hz,
chlorophenyl-C4-H), 7.41 (d, 2H, J= 8 Hz, chlorophenyl-C3, C5-H), 7.50-7.53 (m, 2H,
bridging phenyl-C2, C6-H), 7.71 (d, J=16 Hz, 1H, bridging phenyl-C3-H), 7.91 (d, 1H, J=16
20

ACCEPTED MANUSCRIPT
Hz, bridging phenyl-C5-H), 8.13 (m, 2H, -CH=CH); 13C NMR (100 MHz, CDCl3) δ ppm:
111.67, 118.99, 119.45, 128.86, 130.61, 134.40, 135.19, 137.80, 144.06, 188.57; MS (ESI):
m/z = found 342.04 [M+ + 1]; calcd. 341.04. Anal. Calcd. For C19H13Cl2NO: C, 66.68; H,
3.83; N, 4.09. Found: C, 67.75; H, 4.32; N, 5.33.
4.2.13. 1-(4-(1H-pyrrol-1yl)phenyl)-3-(4-isopropylphenyl)prop-2-en-1-one (3m)
(Yield 70%). mp 196-198 oC; FTIR (KBr): 1655.40 (C=O) cm-1; 1H NMR (400 MHz, CDCl3)
δ ppm: 1.28 (t, 6H, -CH(CH3)2), 2.97 (t, 1H, -CH(CH3)2), 6.40 (t, 2H, pyrrole-C3, C4-H), 7.19
(t, 2H, pyrrole-C2, C5-H), 7.30 (d, 2H, J= 8 Hz, 4-isopropylphenyl-C3, C5-H), 7.49-7.85 (m,
6H, 4-isopropylphenyl-C2, C6-H and bridging phenyl-C2, C3, C5, C6-H), 8.09-8.12 (m, 2H, -
CH=CH-); 13C NMR (100 MHz, CDCl3) δ ppm: 23.75, 34.12, 111.53, 119.01, 119.43,
120.66, 127.10, 128.62, 130.91, 132.47, 135.11, 143.75, 144.96, 152.05, 188.98; MS (ESI):
m/z = found 316.17 [M+ + 1]; calcd. 315.41. Anal. Calcd. For C22H21NO: C, 83.78; H, 6.71;
N, 4.44. Found: C, 84.75; H, 7.32; N, 5.33.
4.2.14. 1-(4-(1H-pyrrol-1yl)phenyl)-3-(2,4-dimethoxyphenyl)prop-2-en-1-one (3n)
(Yield 75%). mp 108-110 oC; FTIR (KBr): 1655.59 (C=O) cm-1; 1H NMR (400 MHz, CDCl3)
δ ppm: 3.86 (s, 3H, -OCH3), 3.91 (s, 3H, -OCH3), 6.40 (t, 2H, pyrrole-C3, C4-H), 6.49 (d, 1H,
J= 4 Hz, methoxyphenyl-C3-H), 6.56 (dd, 1H, methoxyphenyl-C5-H), 7.19 (t, 2H, pyrrole-C2,
C5-H), 7.48-7.59 (m, 4H, bridging phenyl-C2, C3, C5, C6-H), 8.05-8.11 (m, 3H,
methoxyphenyl-C6-H and -CH=CH-); 13C NMR (100 MHz, CDCl3) δ ppm: 55.48, 55.54,
98.43, 105.40, 111.37, 117.07, 119.03, 119.39, 119.94, 130.21, 131.05, 135.65, 140.58,
143.46, 160.43, 163.06, 189.56; MS (ESI): m/z = found 333 [M+]; calcd. 333.14. Anal. Calcd.
For C21H19NO3: C, 75.66; H, 5.74; N, 4.20. Found: C, 76.75; H, 6.32; N, 5.33.
4.2.15. 1-(4-(1H-pyrrol-1yl)phenyl)-3-(3-methoxyphenyl)prop-2-en-1-one (3o)
o
1
(Yield 85%). mp 130-132 C; FTIR (KBr): 1660.54 (C=O) cm-1; H NMR (400 MHz,
CDCl3) δ ppm: 3.85 (s, 3H, -OCH3), 6.40 (dd, 2H, pyrrole-C3, C4-H), 6.95-6.98 (m, 1H, 3-
21

ACCEPTED MANUSCRIPT
methoxyphenyl-C4-H), 7.15-7.81 (m, 9H, pyrrole-C2, C5-H; 3-methoxyphenyl-C2, C5, C6-H
13
and bridging phenyl-C2, C3, C5, C6-H), 8.08-8.12 (m, 2H, -CH=CH-); C NMR (100 MHz,
CDCl3) δ ppm: 55.32, 111.57, 113.48, 119, 119.42, 121.07, 121.87, 129.95, 130.35, 134.89,
136.19, 143.84, 144.73, 159.92, 188.84; MS (ESI): m/z = found 303 [M+]; calcd. 303.13.
Anal. Calcd. For C20H17NO2: C, 79.19; H, 5.65; N, 4.62. Found: C, 80.75; H, 6.32; N, 6.33.
4.2.16. 1-(4-(1H-pyrrol-1yl)phenyl)-3-(3-phenoxyphenyl)prop-2-en-1-one (3p)
(Yield 70%). mp 126-128 oC; FTIR (KBr): 1655.59 (C=O), 1249.88 (C-O-C) cm-1; 1H NMR
(400 MHz, CDCl3) δ ppm: 6.40 (t, 2H, pyrrole-C3, C4-H), 6.94-7.52 (m, 13H, pyrrole-C2, C5-
H; phenoxyphenyl-C2, C4, C5, C6, C9, C10, C11, C12, C13-H and bridging phenyl-C2, C6-H),
7.79 (d, 2H, J= 12 Hz, phenyl-C3, C5-H), 8.01-8.10 (q, 2H, -CH=CH-); 13C NMR (100 MHz,
CDCl3) δ ppm: 111.61, 117.07, 118.10, 119.02, 119.43, 120.82, 122.24, 123.53, 123.68,
129.67, 129.90, 130.90, 134.76, 136.66, 143.91, 144.10, 157.60, 157.82, 188.76; MS (ESI):
m/z = found 365 [M+]; calcd. 365.14. Anal. Calcd. For C25H19NO2: C, 82.17; H, 5.24; N,
3.83. Found: C, 83.75; H, 6.32; N, 5.33.
4.2.17. 1-(4-(1H-pyrrol-1yl)phenyl)-3-(2-hydroxyphenyl)prop-2-en-1-one (3q)
o
1
(Yield 50%). mp 174-176 C; FTIR (KBr): 3425.61 (-OH), 1645.39 (C=O) cm-1; H NMR
(400 MHz, CDCl3) δ ppm: 6.29 (t, 2H, pyrrole-C3, C4-H), 6.83 (t, 1H, hydroxyphenyl-C3-H),
6.91 (t, 1H, hydroxyphenyl-C5-H), 7.15-7.19 (m, 1H, hydroxyphenyl-C4-H), 7.28 (t, 2H,
pyrrole-C2, C5-H), 7.56 -7.58 (m, 2H, hydroxyphenyl-C6-H; phenyl-CH=CH-), 7.61-7.63 (m,
1H, phenyl-CH=CH-), 7.75 (d, 1H, J= 16 Hz, bridging phenyl-C2-H), 8.03 (d, 1H, J= 16 Hz,
bridging phenyl-C6-H), 8.10 (q, 2H, bridging phenyl-C3, C5-H), 9.99 (s, 1H, -OH); 13C NMR
(100 MHz, CDCl3) δ ppm: 111.55, 116.65, 119.01, 119.39, 120.86, 122.28, 127.84, 129.63,
130.48, 131.83, 131.97, 140.91, 143.80, 155.88, 190.10; MS (ESI): m/z = found 289 [M+];
calcd. 289.11. Anal. Calcd. For C25H19NO2: C, 78.87; H, 5.23; N, 4.84. Found: C, 80.75; H,
6.32; N, 6.33.
22

ACCEPTED MANUSCRIPT
4.2.18. 1-(4-(1H-pyrrol-1yl)phenyl)-3-(3-furan-2-yl)prop-2-en-1-one (3r)
(Yield 80%). mp 177-179 oC; FTIR (KBr): 1656.40 (C=O) cm-1; 1H NMR (400 MHz, CDCl3)
δ ppm: 6.40 (t, 2H, pyrrole-C3, C4-H), 6.53 (q, 1H, furan-C4-H), 6.74 (d, 2H, IJ= 4 Hz, furan-
C3-H), 7.18 (t, 2H, pyrrole-C2, C5-H), 7.45-7.54 (m, 4H, bridging phenyl-C2, C3, C5, C6-H),
7.64 (d, 1H, J= 16 Hz, furan-C5-H), 8.10-8.14 (m, 2H, -CH=CH-); 13C NMR (100 MHz,
CDCl3) δ ppm: 111.53, 112.70, 119, 119.42, 130.25, 130.63, 134.91, 143.80, 144.95, 151.63,
188.16; MS (ESI): m/z = found 264.10 [M+ + 1]; calcd. 263.09. Anal. Calcd. For C17H13NO2:
C, 77.55; H, 4.98; N, 5.32. Found: C, 79.75; H, 5.32; N, 6.33.
4.3.General procedure for the synthesis of 1-(3-(4-(1H-pyrrol-1-yl)phenyl)-5-
substitutedphenyl-4,5-dihydro-1H-pyrazol-1-yl)ethanones (4a-p)
A mixture of chalcones (3a-r) (0.67 mmol), hydrazine hydrate 99% (0.87 mmol) and dried
acetic acid (2 mL) was heated under reflux for 7 h. The reaction was monitored using TLC.
After cooling to ambient temperature, the reaction mixture was neutralized with strong
ammonia solution. The separated solid was filtered, washed with water, dried and purified by
column chromatography on silica gel with ethyl acetate/petroleum ether (6:4) as eluent to
afford the corresponding pyrazole derivatives in high purity with good yields.
4.3.1. 1-(3-(4-(1H-pyrrol-1-yl)phenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)ethanone (4a)
o
1
(Yield 70%). mp 156-158 C; FTIR (KBr): 1677.81 (C=O), 1564.80 (C=N) cm-1; H NMR
(400 MHz, CDCl3) δ ppm: 2.43 (s, 3H, -COCH3), 3.19 (dd, 1H, pyrazoline-C4-Ha), 3.79 (dd,
1H, pyrazoline-C4-Hb), 5.63 (dd, 1H, pyrazoline-C5-Hx), 6.39 (t, 2H, pyrrole-C3, C4-H), 7.14
(t, 2H, pyrrole-C2, C5-H), 7.22-7.27 (m, 3H, phenyl-C2, C4, C6-H), 7.34 (d, 2H, J= 8 Hz,
phenyl-C3, C5-H), 7.42-7.45 (m, 2H, bridging phenyl-C2, C6-H), 7.77-7.80 (m, 2H, bridging
phenyl-C3, C5-H); MS (ESI): m/z = found 330.16 [M+ + 1]; calcd. 329.15. Anal. Calcd. For
C21H19N3O: C, 76.57; H, 5.81; N, 12.76. Found: C, 77.75; H, 7.32; N, 14.33.
23

ACCEPTED MANUSCRIPT
4.3.2. 1-(3-(4-(1H-pyrrol-1-yl)phenyl)-5-(4-chlorophenyl)-4,5-dihydro-1H-pyrazol-1-
yl)ethanone (4b)
o
1
(Yield 60%). mp 162-164 C; FTIR (KBr): 1657.95 (C=O), 1606.57 (C=N) cm-1; H NMR
(400 MHz, CDCl3) δ ppm: 2.42 (s, 3H, -COCH3), 3.16 (dd, 1H, pyrazoline-C4-Ha), 3.80 (dd,
1H, pyrazoline-C4-Hb), 5.59 (dd, 1H, pyrazoline-C5-Hx), 6.39 (t, 2H, pyrrole-C3, C4-H), 7.13-
7.30 (m, 6H, chlorophenyl-C2, C3, C5, C6-H and pyrrole-C2, C5-H), 7.46 (d, 2H, J= 12 Hz,
bridging phenyl-C2, C6-H), 7.79 (d, 2H, J= 8 Hz, bridging phenyl-C3, C5-H).
4.3.3. 1-(3-(4-(1H-pyrrol-1-yl)phenyl)-5-(2-chlorophenyl)-4,5-dihydro-1H-pyrazol-1-
yl)ethanone (4c)
o
1
(Yield 60%). mp 166-168 C; FTIR (KBr): 1661.98 (C=O), 1608.24 (C=N) cm-1; H NMR
(400 MHz, CDCl3) δ ppm: 2.49 (s, 3H, -COCH3), 3.09 (dd, 1H, pyrazoline-C4-Ha), 3.89 (dd,
1H, pyrazoline-C4-Hb), 5.95 (dd, 1H, pyrazoline-C5-Hx), 6.38 (t, 2H, pyrrole-C3, C4-H), 7.05-
7.08 (m, 1H, chlorophenyl-C4-H), 7.13 (t, 2H, pyrrole-C2, C5-H), 7.18-7.23 (m, 2H,
chlorophenyl-C5, C6-H), 7.39-7.44 (m, 3H, bridging phenyl-C2, C6-H and chlorophenyl-C3-
13
H), 7.76-7.79 (m, 2H, phenyl-C3, C5-H); C NMR (100 MHz, CDCl3) δ ppm: 21.87, 41.39,
57.77, 111.17, 119.97, 125.82, 127.25, 127.96, 128.28, 128.79, 130.02, 131.68, 138.32,
141.94, 153.45, 168.87; MS (ESI): m/z = found 363 [M+]; calcd. 363.11. Anal. Calcd. For
C21H18ClN3O: C, 69.32; H, 4.99; N, 11.55. Found: C, 70.75; H, 5.32; N, 13.33.
4.3.4. 1-(3-(4-(1H-pyrrol-1-yl)phenyl)-5-p-tolyl-4,5-dihydro-1H-pyrazol-1-yl)ethanone (4d)
o
1
(Yield 60%). mp 128-130 C; FTIR (KBr): 1678.83 (C=O), 1564.33 (C=N) cm-1; H NMR
(400 MHz, CDCl3) δ ppm: 2.30 (s, 3H, -COCH3), 2.42 (s, 3H, -CH3), 3.18 (dd, 1H,
pyrazoline-C4-Ha), 3.78 (dd, 1H, pyrazoline-C4-Hb), 5.59 (dd, 1H, pyrazoline-C5-Hx), 6.39 (t,
2H, pyrrole-C3, C4-H), 7.11-7.14 (m, 4H, methylphenyl-C2, C3, C5, C6-H), 7.26 (s, 2H,
pyrrole-C2, C5-H), 7.45 (t, 2H, bridging phenyl-C2, C6-H), 7.77-7.80 (m, 2H, bridging
24

ACCEPTED MANUSCRIPT
phenyl-C3, C5-H); MS (ESI): m/z = found 344.17 [M+ + 1]; calcd. 343.17. Anal. Calcd. For
C22H21N3O: C, 76.94; H, 6.61; N, 12.24. Found: C, 78.75; H, 7.32; N, 14.33.
4.3.5. 1-(3-(4-(1H-pyrrol-1-yl)phenyl)-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-
yl)ethanone (4e)
o
1
(Yield 60%). mp 124-125 C; FTIR (KBr): 1658.31 (C=O), 1608.60 (C=N) cm-1; H NMR
(400 MHz, CDCl3) δ ppm: 2.41 (s, 3H, -COCH3), 3.19 (dd, 1H, pyrazoline-C4-Ha), 3.74 (t,
1H, pyrazoline-C4-Hb), 3.77 (s, 3H, -OCH3), 5.58 (dd, 1H, pyrazoline-C5-Hx), 6.39 (t, 2H,
pyrrole-C3, C4-H), 6.83-6.86 (m, 2H, methoxyphenyl-C3, C5-H), 7.13-7.19 (m, 4H, pyrrole-
C2, C5-H and methoxyphenyl-C2, C6-H), 7.45 (dd, 2H, bridging phenyl-C2, C6-H), 7.78-7.80
(m, 2H, bridging phenyl-C3, C5-H); 13C NMR (100 MHz, CDCl3) δ ppm: 21.95, 42.22, 55.24,
59.48, 111.14, 114.20, 120, 126.87, 127.92, 133.94, 141.83, 153.03, 158.99, 168.78; MS
(ESI): m/z = found 359 [M+]; calcd. 359.16. Anal. Calcd. For C22H21N3O2: C, 73.52; H, 5.89;
N, 11.69. Found: C, 71.75; H, 6.32; N, 12.33.
4.3.6. 1-(3-(4-(1H-pyrrol-1-yl)phenyl)-5-(3-bromophenyl)-4,5-dihydro-1H-pyrazol-1-
yl)ethanone (4f)
o
1
(Yield 75%). mp 120-122 C; FTIR (KBr): 1660.58 (C=O), 1570.62 (C=N) cm-1; H NMR
(400 MHz, CDCl3) δ ppm: 2.42 (s, 3H, -COCH3), 3.17 (dd, 1H, pyrazoline-C4-Ha), 3.80 (dd,
1H, pyrazoline-C4-Hb), 5.58 (dd, 1H, pyrazoline-C5-Hx), 6.39 (t, 2H, pyrrole-C3, C4-H), 7.13-
7.48 (m, 8H, pyrrole-C2, C5-H, bridging phenyl-C2, C6-H and bromophenyl-C2, C4, C5, C6-H),
13
7.77-7.79 (m, 2H, bridging phenyl-C3, C5-H); C NMR (100 MHz, CDCl3) δ ppm: 21.30,
42.24, 59.46, 111.22, 120.03, 123, 124.31, 128.01, 128.18, 128.57, 130.51, 130.87, 142,
143.95, 152.92, 168.93; MS (ESI): m/z = found 407 [M+], 409 [M+ + 2]; calcd. 407.06. Anal.
Calcd. For C21H18BrN3O: C, 61.78; H, 4.44; N, 10.29. Found: C, 62.75; H, 5.02; N, 12.33.
4.3.7. 1-(3-(4-(1H-pyrrol-1-yl)phenyl)-5-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-
yl)ethanone (4g)
25

ACCEPTED MANUSCRIPT
o
1
(Yield 75%). mp 172-174 C; FTIR (KBr): 1661.21 (C=O), 1568.89 (C=N) cm-1; H NMR
(400 MHz, CDCl3) δ ppm: 2.42 (s, 3H, -COCH3), 3.17 (dd, 1H, pyrazoline-C4-Ha), 3.79 (m,
1H, pyrazoline-C4-Hb), 5.60 (dd, 1H, pyrazoline-C5-Hx), 6.39 (t, 2H, pyrrole-C3, C4-H), 6.98-
7.03 (m, 2H, fluorophenyl-C3, C5-H), 7.14 (t, 4H, pyrrole-C2, C5-H), 7.19-7.23 (m, 2H,
fluorophenyl-C2, C6-H), 7.43-7.46 (m, 2H, bridging phenyl-C2, C6-H), 7.80 (dd, 2H, bridging
13
phenyl-C3, C5-H); C NMR (100 MHz, CDCl3) δ ppm: 21.92, 42.24, 59.35, 111.21, 115.62,
115.91, 120.02, 127.29, 127.41, 127.96, 137.52, 137.56, 141.94, 152.94, 160.50, 168.86; MS
(ESI): m/z = found 347 [M+]; calcd. 347.14. Anal. Calcd. For C21H18FN3O: C, 72.61; H, 5.22;
N, 12.10. Found: C, 73.75; H, 6.32; N, 13.33.
4.3.8. 1-(3-(4-(1H-pyrrol-1-yl)phenyl)-5-(2-bromophenyl)-4,5-dihydro-1H-pyrazol-1-
yl)ethanone (4h)
(Yield 75%). mp 160-162 oC; FTIR (KBr): 1671.47 (C=O), 1604.39 (C=N) cm-1.
4.3.9. 1-(3-(4-(1H-pyrrol-1-yl)phenyl)-5-(3,4-dimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-
yl)ethanone (4i)
o
1
(Yield 60%). mp 180-182 C; FTIR (KBr): 1660.91 (C=O), 1608.20 (C=N) cm-1; H NMR
(400 MHz, CDCl3) δ ppm: 2.43 (s, 3H, -COCH3), 3.20 (dd, 1H, pyrazoline-C4-Ha), 3.78 (dd,
1H, pyrazoline-C4-Hb), 3.87 (d, 6H, -2OCH3), 5.58 (dd, 1H, pyrazoline-C5-Hx), 6.39 (t, 2H,
pyrrole-C3, C4-H), 6.77-6.82 (m, 3H, methoxyphenyl-C2, C5, C6-H), 7.15 (t, 2H, pyrrole-C2,
C5-H) 7.46 (d, 2H, J= 8 Hz, bridging phenyl-C2, C6-H), 7.81 (d, 2H, J= 8 Hz, bridging
phenyl-C3, C5-H); MS (ESI): m/z = found 389 [M+]; calcd. 389.17. Anal. Calcd. For
C23H23N3O3: C, 70.93; H, 5.95; N, 10.79. Found: C, 72.75; H, 7.32; N, 12.33.
4.3.10. 1-(3-(4-(1H-pyrrol-1-yl)phenyl)-5-(2,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-1-
yl)ethanone (4j)
o
1
(Yield 75%). mp 118-120 C; FTIR (KBr): 1662.25 (C=O), 1561.83 (C=N) cm-1; H NMR
(400 MHz, CDCl3) δ ppm: 2.48 (s, 3H, -COCH3), 3.06 (dd, 1H, pyrazoline-C4-Ha), 3.88 (dd,
26

ACCEPTED MANUSCRIPT
1H, pyrazoline-C4-Hb), 5.88 (dd, 1H, pyrazoline-C5-Hx), 6.38 (t, 2H, pyrrole-C3, C4-H), 7.02
(d, 1H, J= 8 Hz, chlorophenyl-C6-H), 7.13 (t, 4H, pyrrole-C2, C5-H), 7.20 (dd, 1H,
chlorophenyl-C5-H), 7.42-7.78 (m, 5H, chlorophenyl-C3-H and bridging phenyl-C2, C3, C5,
C6-H); 13C NMR (100 MHz, CDCl3) δ ppm: 21.85, 41.23, 57.40, 111.23, 118.95, 119.97,
126.96, 127.97, 129.82, 132.39, 133.89, 137.06, 142.02, 153.34, 168.91; MS (ESI): m/z =
found 398 [M+ + 1]; calcd. 397.07. Anal. Calcd. For C21H17Cl2N3O: C, 63.33; H, 4.30; N,
10.55. Found: C, 65.15; H, 5.32; N, 11.33.
4.3.11. 1-(3-(4-(1H-pyrrol-1-yl)phenyl)-5-(2,3-dichlorophenyl)-4,5-dihydro-1H-pyrazol-1-
yl)ethanone (4k)
o
1
(Yield 60%). mp 144-146 C; FTIR (KBr): 1662.17 (C=O), 1607.52 (C=N) cm-1; H NMR
(400 MHz, CDCl3) δ ppm: 2.49 (s, 3H, -COCH3), 3.07 (dd, 1H, pyrazoline-C4-Ha), 3.91 (dd,
1H, pyrazoline-C4-Hb), 5.95 (dd, 1H, pyrazoline-C5-Hx), 6.38 (t, 2H, pyrrole-C3, C4-H), 6.99
(d, 1H, J= 8 Hz, chlorophenyl-C6-H), 7.12-7.39 (m, 4H, pyrrole-C2, C5-H and chlorophenyl-
C4, C5-H), 7.44 (d, 2H, J= 8 Hz, bridging phenyl-C2, C6-H), 7.78 (d, 2H, J= 8 Hz, bridging
13
phenyl-C3, C5-H); C NMR (100 MHz, CDCl3) δ ppm: 21.87, 41.39, 57.77, 111.24, 119.98,
123.93, 127.72, 127.99, 128.09, 129.54, 129.99, 133.74, 140.65, 142.03, 153.40, 168.90; MS
(ESI): m/z = found 397 [M+]; calcd. 397.07. Anal. Calcd. For C21H17Cl2N3O: C, 63.33; H,
4.30; N, 10.55. Found: C, 64.75; H, 5.32; N, 12.33.
4.3.12. 1-(3-(4-(1H-pyrrol-1-yl)phenyl)-5-(2,6-dichlorophenyl)-4,5-dihydro-1H-pyrazol-1-
yl)ethanone (4l)
o
1
(Yield 60%). mp 148-150 C; FTIR (KBr): 1664.34 (C=O), 1608.92 (C=N) cm-1; H NMR
(400 MHz, CDCl3) δ ppm: 2.37 (s, 3H, -COCH3), 3.34 (dd, 1H, pyrazoline-C4-Ha), 3.74 (dd,
1H, pyrazoline-C4-Hb), 6.27 (dd, 1H, pyrazoline-C5-Hx), 6.39 (t, 2H, pyrrole-C3, C4-H), 7.12-
7.37 (m, 5H, pyrrole-C2, C5-H, bridging phenyl-C2, C6-H and chlorophenyl-C4-H), 7.44-7.46
(m, 2H, chlorophenyl-C3, C5-H), 7.79-7.81 (m, 2H, bridging phenyl-C3, C5-H).
27

ACCEPTED MANUSCRIPT
4.3.13. 1-(3-(4-(1H-pyrrol-1-yl)phenyl)-5-(4-isopropylphenyl)-4,5-dihydro-1H-pyrazol-1-
yl)ethanone (4m)
o
1
(Yield 60%). mp 170-172 C; FTIR (KBr): 1662.05 (C=O), 1526.30 (C=N) cm-1; H NMR
(400 MHz, CDCl3) δ ppm: 1.22 (dd, 6H, -CH(CH3)2), 2.43 (s, 3H, -COCH3), 2.83-2.89 (p,
1H, -CH(CH3)2), 3.20 (dd, 1H, pyrazoline-C4-Ha), 3.77 (dd, 1H, pyrazoline-C4-Hb), 5.61 (dd,
1H, pyrazoline-C5-Hx), 6.38 (t, 2H, pyrrole-C3, C4-H), 7.13-7.19 (m, 6H, pyrrole-C2, C5-H
and isopropylphenyl-C2, C3, C5, C6-H), 7.46 (dd, 2H, bridging phenyl-C2, C6-H), 7.80 (t, 2H,
phenyl-C3, C5-H); 13C NMR (100 MHz, CDCl3) δ ppm: 21.94, 23.88, 23.90, 33.73, 42.28,
59.75, 111.14, 120, 125.44, 126.93, 127.94, 128.53, 139, 141.84, 148.22, 153.17, 168.85; MS
(ESI): m/z = found 371 [M+]; calcd. 371.20. Anal. Calcd. For C22H21N3O2: C, 77.60; H, 6.78;
N, 11.31. Found: C, 79.75; H, 8.32; N, 13.33.
4.3.14. 1-(3-(4-(1H-pyrrol-1-yl)phenyl)-5-(3-phenoxyphenyl)-4,5-dihydro-1H-pyrazol-1-
yl)ethanone (4n)
(Yield 60%). mp 194-196 oC; FTIR (KBr): 1671.47 (C=O), 1604.39 (C=N) cm-1.
4.3.15. 1-(3-(4-(1H-pyrrol-1-yl)phenyl)-5-(furan-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone
(4o)
o
(Yield 60%). mp 194-196 C; FTIR (KBr): 1675.54 (C=O), 1608.08 (C-O), 1591.43 (C=N)
1
cm-1; H NMR (400 MHz, CDCl3) δ ppm: 2.40 (s, 3H, -COCH3), 3.48 (dd, 1H, pyrazoline-
C4-Ha), 3.63 (dd, 1H, pyrazoline-C4-Hb), 5.73 (dd, 1H, pyrazoline-C5-Hx), 6.31-6.34 (m, 2H,
furan-C3, C4-H), 6.39 (t, 2H, pyrrole-C3, C4-H), 7.15 (t, 2H, pyrrole-C2, C5-H) 7.30 (d, 1H,
furan-C5-H), 7.46 (d, 2H, J= 8 Hz, bridging phenyl-C2, C6-H), 7.82 (d, 2H, J= 8 Hz, bridging
13
phenyl-C3, C5-H); C NMR (100 MHz, CDCl3) δ ppm: 21.95, 38.18, 53.37, 107.61, 110.53,
111.16, 120.02, 127.96, 128.39, 141.90, 141.96, 151.96, 153.20, 168.94; MS (ESI): m/z =
found 319 [M+]; calcd. 319.13. Anal. Calcd. For C22H21N3O2: C, 71.46; H, 5.37; N, 13.16.
Found: C, 72.75; H, 6.32; N, 15.33.
28