Development of new pyrazole hybrids as antitubercular agents: Synthesis, biological evaluation and molecular docking study

Article abstract of DOI:10.22159/ijpps.2017v9i11.20469

Objective: Synthesis of new 1, 3-diphenyl pyrazole derivatives 9(a-f) and 10(a-f) using molecular hybridization approach and evaluation of their antitubercular and cytotoxic studies. Methods: The structures of synthesized compounds were confirmed by 1H NMR, 13C NMR and mass spectra. The antitubercular activity of compounds and standard drugs were assessed against Mycobacterium tuberculosis using Microplate alamar blue assay (MABA). The cytotoxic activities were performed by Sulforhodamine B (SRB) assay. The molecular docking and in silico ADME prediction studies were also performed by using Schrodinger. Results: The results reveal that compounds 9c, 9d, 10c and 10d exhibited substantial antitubercular potential with MIC<20 μM. The cytotoxic studies revealed that active compounds (9c, 9d, 10c and 10d) are non-toxic to HeLa cancer cell lines with the selectivity index>10. The molecular docking study was performed to study the binding orientation and affinity of synthesized compounds for InhA enzyme. Conclusion: The study explored that 1, 3-diphenyl pyrazole hybrid coupled with well-known antitubercular drugs could be a potential lead for antitubercular agents. In silico molecular docking, study helps to identify their corresponding intermolecular ligand-protein interactions with target enzyme. Also, ADME prediction studies revealed that the compounds were in acceptable range to have good pharmacokinetic parameters.

Full text of DOI:10.22159/ijpps.2017v9i11.20469

International Journal of Pharmacy and Pharmaceutical Sciences
ISSN- 0975-1491
Vol 9, Issue 11, 2017
Original Article
DEVELOPMENT OF NEW PYRAZOLE HYBRIDS AS ANTITUBERCULAR AGENTS: SYNTHESIS,
BIOLOGICAL EVALUATION AND MOLECULAR DOCKING STUDY
SAMEER I. SHAIKH, ZAHID ZAHEER*, SANTOSH N. MOKALE, DEEPAK K. LOKWANI
Y. B. Chavan College of Pharmacy, Dr. Rafiq Zakaria Campus, Aurangabad 431001, (M. S.), India
Email: zahidzresearch2@gmail.com
Received: 03 Jun 2017 Revised and Accepted: 21 Sep 2017
ABSTRACT
Objective: Synthesis of new 1, 3-diphenyl pyrazole derivatives 9(a-f) and 10(a-f) using molecular hybridization approach and evaluation of their
antitubercular and cytotoxic studies.
Methods: The structures of synthesized compounds were confirmed by 1H NMR, 13C NMR and mass spectra. The antitubercular activity of
compounds and standard drugs were assessed against Mycobacterium tuberculosis using Microplate alamar blue assay (MABA). The cytotoxic
activities were performed by Sulforhodamine B (SRB) assay. The molecular docking and in silico ADME prediction studies were also performed by
using Schrodinger.
Results: The results reveal that compounds 9c, 9d, 10c and 10d exhibited substantial antitubercular potential with MIC<20 μM. The cytotoxic
studies revealed that active compounds (9c, 9d, 10c and 10d) are non-toxic to HeLa cancer cell lines with the selectivity index>10. The molecular
docking study was performed to study the binding orientation and affinity of synthesized compounds for InhA enzyme.
Conclusion: The study explored that 1, 3-diphenyl pyrazole hybrid coupled with well-known antitubercular drugs could be a potential lead for
antitubercular agents. In silico molecular docking, study helps to identify their corresponding intermolecular ligand-protein interactions with target
enzyme. Also, ADME prediction studies revealed that the compounds were in acceptable range to have good pharmacokinetic parameters.
Keywords: Pyrazole, Antitubercular activity, Cytotoxicity, Molecular docking
© 2017 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open-access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
DOI: http://dx.doi.org/10.22159/ijpps.2017v9i11.20469
for tuberculosis and its latent infections, it suffers from two
pitfalls as enzymatic acetylation of isoniazid by N-
acetyltransferase (NATs) and it’s associated liver toxicity. So,
blocking N-acetylation via chemical modification of N-terminal
of isoniazid makes it to be more effective and less hepatotoxic
than isoniazid [10]. Linezolid is recommended by the WHO to
treat drug-resistant tuberculosis [11]. The study also found that
it is potentially important for patients with XDR-TB. It acts by
inhibiting protein synthesis at an early stage of translation [12].
3-Fluoro-4-morpholinoaniline, a bioactive segment of linezolid
plays a very crucial role in the antitubercular activity. 3-Fluoro
group attached to phenyl ring enhances the potency while
morpholine ring improves pharmacokinetic and water solubility
of linezolid [13].
INTRODUCTION
Tuberculosis (TB) is a re-emerging global health threat caused by an
infectious bacillus called Mycobacterium tuberculosis and is the
second largest killer disease caused by a single infectious agent after
the human immunodeficiency virus (HIV) [1]. World Health
Organization (WHO) Global Tuberculosis Report 2016 suggested
that, in 2015, there were an estimated 10.4 million new tuberculosis
cases worldwide. People living with HIV accounted for 1.2 million
(11 %) of all new tuberculosis cases [2].
World Health Organization (WHO) promoted a comprehensive
tuberculosis management program known as DOTS (Directly
Observed Treatment, Shortcourse). DOTS therapy involves the use
of four first-line agents as isoniazid, ethambutol, pyrazinamide,
and rifampicin for six months [3].
The molecular hybridization approach is one of the most
valuable structural modification tools useful for the discovery of
ligands and prototypes. Recently, the emerging interest to
discover hybrid molecules resulting from the combination of
pharmacophoric moieties of different known lead compounds
Despite notable progress in antitubercular agents, multi-drug
resistance tuberculosis (MDR-TB), extremely drug resistance
tuberculosis (XDR-TB) and HIV co-infection are the major
hurdles in control of tuberculosis infection [4]. Therefore, the
discovery and development of new chemical entities with a novel
mechanism of action, safe and efficacious drugs, and shorter
duration of treatment are the desperate needs for infectious
diseases research programs. Pioneering scientists had reported
pyrazole and its derivatives for an extensive range of
pharmacological activities such as antipyretic, analgesic,
has been developed with
multifactorial diseases [14].
a new hope for the treatment of
In the light of above consideration, it seems rational to combine
the pharmacophoric potential of the two well-known
antitubercular agents like isoniazid and linezolid with one core
scaffolds, pyrazole (fig. 1). Finally, these hybridized molecules
were evaluated for antitubercular and cytotoxicity studies. The
molecular docking study was performed to determine the possible
mechanism of action of titled compounds by which they exert
antitubercular activity. The in silico molecular docking study helps
to identify their corresponding intermolecular ligand-protein
interactions with target enzyme to set a basis for mycobacterial
inhibition. In addition to this, absorption, distribution,
metabolism, and excretion (ADME) prediction studies were also
performed to explore the results.
antimicrobial,
anticancer,
antitubercular,
antiviral,
antihypertensive, antioxidant, antidepressant, and anxiolytic [5-7].
Rangappa S. Keri et al. reviewed and discussed the possible
structure-activity relationship of different types of pyrazole
scaffold for designing of better antitubercular agents [8].
Synthetic analogues of pyrazole are known to exhibit significant
antitubercular activity especially 1, 3-diphenyl pyrazole motif is
known to be a potent antitubercular agent (fig. 1) [9]. Even
though isoniazid (INH) has been the most widely used treatment

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Int J Pharm Pharm Sci, Vol 9, Issue 11, 50-56
Fig. 1: Pharmacophores derived from structurally related and well known antitubercular agents
MATERIALS AND METHODS
129.48, 130.93, 131.04, 131.22, 137.87, 137.87, 140.31, 150.63,
154.73, 164.03; m/z=370.25 (M+1) (% Mol. Wt.: 99.70).
Materials
N'-((3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)methyl)
isonicotinohydrazide (9b)
All the solvents and reagents were used as obtained from the
supplier or recrystallized/redistilled unless otherwise stated. The
progress of the reaction was monitored by thin layer
chromatography (TLC) using silica gel-G pre-coated aluminium
plates (Merck) and visualized under UV light. Melting points were
measured in open capillary tubes and uncorrected. The synthesized
compounds were characterized by spectral analysis like 1H NMR and
13C NMR and Mass spectroscopy. The nuclear magnetic resonance 1H
NMR and 13C NMR spectra were recorded with a Bruker DRX-400
MHz and Bruker DRX-100 MHz NMR spectrometer. Chemical shifts
(δ) are reported in parts per million (ppm). The mass spectra were
recorded under ESI mode on Waters Micromass equipment (model
Q-TOF micro).
Yellow solid; Yield=92%; m. p =185-187 ᵒC; Rf value=0.60; 1H NMR
(CDCl3, 400 MHz, δ ppm): 3.14 (s, 1H,-CH2-NH-), 3.90 (s, 2H,-CH2-),
7.43-7.83 (m, 9H, Ar-H), 7.56 (s, 1H,-CH-of pyrazole), 7.73 (s, 1H,-
NH-C=O), 7.89-8.79 (m, 4H, pyridine-H); [13]C NMR (CDCl3, 100
MHz, δ ppm): 49.63, 120.17, 121.05, 125.27, 126.55, 129.15, 129.48,
130.89, 131.02, 131.22, 134.00, 137.87, 140.31, 150.63, 154.73,
164.03; m/z=404.60 (M+1) (% Mol. Wt.: 99.63).
N'-((3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)methyl)
isonicotinohydrazide (9c)
Dark-yellow solid; Yield=90%; m. p =208-210 ᵒC; Rf value=0.54; 1H NMR
(CDCl3, 400 MHz, δ ppm): 2.87 (s, 1H,-CH2-NH-), 3.92 (s, 2H,-CH2-), 7.38-
7.84 (m, 9H, Ar-H), 7.51 (s, 1H,-CH-of pyrazole), 7.57 (s, 1H,-NH-C=O),
7.90-8.79 (m, 4H, pyridine-H); [13]C NMR (CDCl3, 100 MHz, δ ppm):
49.63, 116.69, 116.89, 120.17, 121.07, 125.27, 126.55, 128.41, 128.44,
129.48, 131.22, 132.32, 132.40, 137.87, 140.31, 150.63, 154.73, 162.94,
164.03, 165.46; m/z=388.53 (M+1) (% Mol. Wt.: 99.64).
General procedure for synthesis of N'-((3-(4-substituted
phenyl)-1-phenyl-1H-pyrazol-4-yl)
methylen)
isonicotino-
hydrazide 7(a-f) and N-((3-(4-substituted phenyl)-1-phenyl-1H-
pyrazol-4-yl) methyenel)-3-fluoro-4-morpholinoaniline 8(a-f)
A mixture of substituted 3-aryl-1-phenylpyrazol-4-carbaldehydes 4(a-
f) (1.0 mmol) and isoniazid
5 (1.0 mmol) or 3-fluoro-4-
N'-((3-(4-bromophenyl)-1-phenyl-1H-pyrazol-4-yl)methyl)
isonicotinohydrazide (9d)
morpholinoaniline 6 (1.0 mmol) was dissolved in absolute ethanol (20
ml) and heated under reflux for 6–8 h in the presence of glacial acetic
acid (2.0 mmol) as a catalyst. Then, the reaction mixture was poured in
ice-cold water and filtered under suction; the precipitate thus obtained
was washed with water and recrystallized from ethanol.
Brown solid; Yield=91%; m. p =292-294 ᵒC; Rf value=0.71; 1H NMR
(CDCl3, 400 MHz, δ ppm): 3.86 (s, 1H,-CH2-NH-), 3.91 (s, 2H,-CH2-),
7.44-7.64 (m, 9H, Ar-H), 7.56 (s, 1H,-CH-of pyrazole), 7.81-8.79 (m,
4H, pyridine-H), 8.32 (s, 1H,-NH-C=O); 13C NMR (CDCl3, 100 MHz, δ
ppm): 49.63, 120.17, 121.05, 123.85, 125.27, 126.55, 128.50, 129.48,
129.95, 130.84, 137.87, 140.31, 150.63, 154.73, 164.03; m/z=448.64
(M+1) (% Mol. Wt.: 99.52).
General procedure for synthesis of N'-((3-(4-substituted
phenyl)-1-phenyl-1H-pyrazol-4-yl)
hydrazide 9(a-f) and N-((3-(4-substitutedphenyl)-1-phenyl-1H-
pyrazol-4-yl) methyl)-3-fluoro-4-morpholinoaniline 10(a-f)
methyl)
isonicotino-
N'-((3-(4-nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)methyl)
isonicotinohydrazide (9e)
Corresponding imines 7(a-f) and 8(a-f) (0.01 mmol) were dissolved in
absolute methanol (10-15 ml) and placed in a two-necked flask fitted
with two glass stoppers and a reflux condenser. This solution was
warmed with continuous stirring. To this was added an equimolar
amount of solid sodium borohydride (NaBH4). The portion-wise
addition was made through one of the necks of the flask. If the reaction
became too vigorous, the flask was moved to room temperature. When
the initial reaction had subsided, the contents of the flask were
refluxed for 15 min and then cooled. The precipitate of secondary
amine thus formed was collected, washed with water, and dried. Then
the compounds were recrystallized using methanol.
Dark grey solid; Yield=82%; m. p =276-278 ᵒC; Rf value=0.55; 1H
NMR (CDCl3, 400 MHz, δ ppm): 3.90 (s, 1H,-CH2-NH-), 4.12 (s, 2H,-
CH2-), 7.44-7.50 (m, 9H, Ar-H), 7.51 (s, 1H,-CH-of pyrazole), 7.81-
8.79 (m, 4H, pyridine-H), 8.50 (s, 1H,-NH-C=O); [13]C NMR (CDCl3,
100 MHz, δ ppm): 49.63, 120.17, 121.05, 123.63, 125.27, 126.55,
127.40, 129.48, 131.22, 137.87, 140.31, 148.87, 150.63, 154.73,
164.03; m/z=415.34 (M+1) (% Mol. Wt.: 99.73).
N'-((3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)methyl)
isonicotinohydrazide (9f)
N'-((1,3-diphenyl-1H-pyrazol-4-yl)methyl)isonicotinohydrazide
(9a)
Yellow solid; Yield=85%; m. p =162-164 ᵒC; Rf value=0.63; 1H NMR
(CDCl3, 400 MHz, δ ppm): 2.87 (s, 1H,-CH2-NH-), 3.81 (s, 3H,-OCH3-of
phenyl), 3.90 (s, 2H,-CH2-), 7.02-7.49 (m, 9H, Ar-H), 7.50 (s, 1H,-CH-of
pyrazole), 7.76-8.79 (m, 4H, pyridine-H), 7.77 (s, 1H,-NH-C=O); [13]C
NMR (CDCl3, 100 MHz, δ ppm): 49.63, 55.39, 114.70, 120.17, 121.05,
125.27, 126.55, 129.48, 130.82, 131.22, 137.87, 140.31, 150.63,
154.73, 161.23, 164.03; m/z=400.4 (M+1) (% Mol. Wt.: 99.45).
Pale yellow solid; Yield=86%; m. p. =152-154 ᵒC; Rf value=0.56; 1H
NMR (CDCl3, 400 MHz, δ ppm): 2.98 (s, 1H,-CH2-NH-), 3.92 (s, 2H,-
CH2-), 7.52 (s, 1H,-CH-of Pyrazole), 7.47-7.83 (m, 10H, Ar-H), 7.72 (s,
1H,-NH-C=O), 7.90-8.79 (m, 4H, pyridine-H); 13C NMR (CDCl3, 100
MHz, δ ppm): 49.63, 120.17, 121.05, 125.27, 126.55, 128.53, 128.97,
51

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Int J Pharm Pharm Sci, Vol 9, Issue 11, 50-56
N-((1,3-diphenyl-1H-pyrazol-4-yl)methyl)-3-fluoro-4-
Biological activities
morpholinoaniline (10a)
Antitubercular activity
Brown solid; Yield=94%; m. p =226-228 ᵒC; Rf value=0.64;1H NMR
(CDCl3, 400 MHz, δ ppm): 2.93-2.96 (t, 4H,-CH2-of morpholine, J=7.2
Hz), 3.72-3.75 (t, 4H,-CH2-of morpholine, J=7.2 Hz), 4.64 (s, 2H,-CH2-
), 6.32 (s, 1H, NH), 6.15-7.92 (m, 13H, Ar-H); 7.49 (s, 1H,-CH-of
pyrazole), [13]C NMR (CDCl3, 100 MHz, δ ppm): 40.66, 51.10, 66.81,
102.60, 102.80, 113.73, 113.76, 120.17, 121.19, 121.27, 125.01,
126.55, 128.53, 128.97, 129.48, 130.93, 131.04, 131.17, 134.12,
134.32, 140.31, 144.01, 144.09, 154.73, 155.09, 157.61; MS:
m/z=429.8 (M+1) (% Mol. Wt.: 99.24).
The antitubercular activity of the synthesized compounds and
standard drugs were assessed against Mycobacterium tuberculosis
using Microplate alamar blue assay (MABA) [15]. This methodology is
non-toxic, uses a thermally stable reagent and shows good correlation
with proportional and BACTEC radiometric method. Briefly, 200 µl of
sterile deionized water was added to all outer perimeter wells of
sterile 96 wells plate to minimized evaporation of medium in the test
wells during incubation. The 96 wells plate received 100 µl of the
Middlebrook 7H9 broth (HiMedia, Mumbai) and serial dilution of
compounds was made directly on the plate. The final drug
concentrations tested were made. Plates were covered and sealed with
parafilm and incubated at 37 °C for five days. After this time, 25 µl of
freshly prepared 1:1 mixture of almar blue reagent and 10 % tween 80
was added to the plate and incubated for 24 h. The minimum
inhibitory concentration (MIC) was defined as the minimum
concentration of compound required to give complete inhibition of
bacterial growth. The antitubercular screening was performed in
triplet and the standard errors were all within 10 % of the mean.
N-((3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)methyl)-3-
fluoro-4-morpholinoaniline (10b)
Yellowish-brown solid; Yield=94%; m.
p =228-230 ᵒC; Rf
value=0.53;1H NMR (CDCl3, 400 MHz, δ ppm): 2.93-2.96 (t, 4H,-CH2-
of morpholine, J=7.2 Hz), 3.72-3.75 (t, 4H,-CH2-of morpholine, J=7.2
Hz), 4.56 (s, 2H,-CH2-), 5.89 (s, 1H, NH), 5.88-7.92 (m, 12H, Ar-H);
7.46 (s, 1H,-CH-of pyrazole); 13C NMR (CDCl3, 100 MHz, δ ppm):
40.66, 51.14, 66.81, 102.60, 102.80, 113.73, 113.76, 120.17, 121.19,
121.27, 125.01, 126.55, 129.15, 129.48, 130.89, 131.02, 131.17,
134.00, 134.12, 134.32, 140.31, 144.01, 144.09, 154.73, 155.09,
157.61.; MS: m/z=463.3 (M+1) (% Mol. Wt.: 99.84).
Cytotoxicity study
The active compounds 9c, 9d, 10c and 10d were evaluated for their
in vitro cytotoxic activity against HeLa human cancer cell line, by
Sulforhodamine B (SRB) assay according to the reported procedures
[16]. This assay gives Growth inhibition concentration (GI50) values
which were taken as the lowest concentration of the compound
killing 50 % of the cells.
3-Fluoro-N-((3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-
yl)methyl)-4-morpholinoaniline (10c)
Brown solid; Yield=89%; m. p =200-202 ᵒC; Rf value=0.62; 1H NMR
(CDCl3, 400 MHz, δ ppm): 2.93-2.96 (t, 4H,-CH2-of morpholine, J=7.2
Hz), 3.72-3.75 (t, 4H,-CH2-of morpholine, J=7.2 Hz), 4.60 (s, 2H,-CH2-
), 6.24 (s, 1H, NH), 6.12-7.92 (m, 12H, Ar-H); 7.46 (s, 1H,-CH-of
pyrazole); [13]C NMR (CDCl3, 100 MHz, δ ppm): 40.66, 51.14, 66.81,
102.60, 102.80, 113.73, 113.76, 116.69, 116.89, 120.17, 121.19,
125.01, 126.55, 128.41, 128.44, 129.48, 131.17, 132.32, 132.40,
134.12, 140.31, 144.01, 144.09, 154.73, 155.09, 157.61, MS:
m/z=447.3 (M+1) (% Mol. Wt.: 99.43).
Selectivity index
The selectivity index (SI) was calculated by dividing GI50 for human
cancer cell line (HeLa) by the MIC (μg/ml) for in vitro activity against
Mycobacterium tuberculosis. If the selectivity index (SI) is ≥ 10, the
compounds are processed further for drug development [17].
Computational studies
Molecular docking study
N-((3-(4-bromophenyl)-1-phenyl-1H-pyrazol-4-yl)methyl)-3-
fluoro-4-morpholinoaniline (10d)
Dark-brown solid; Yield=85%; m. p. =188-190 ᵒC; Rf value=0.43; 1H
NMR (CDCl3, 400 MHz, δ ppm): 2.93-2.96 (t, 4H,-CH2-of morpholine,
J=7.2 Hz), 3.72-3.75 (t, 4H,-CH2-of morpholine, J=7.2 Hz), 4.59 (s,
2H,-CH2-), 7.47 (s, 1H, NH), 7.45 (s, 1H,-CH-of pyrazole), 7.46-7.92
(m, 12H, Ar-H); [13]C NMR (CDCl3, 100 MHz, δ ppm): 40.66, 51.10,
66.81, 102.60, 102.80, 113.73, 113.76, 120.17, 121.19, 121.27,
123.85, 125.01, 126.55, 128.50, 129.48, 129.95, 130.84, 131.17,
134.12, 134.32, 140.31, 144.09, 154.73, 155.09, 157.61; MS:
m/z=507.7 (M+1) (% Mol. Wt.: 99.40).
The molecular docking studies were performed in Maestro 9.1 using
Glide (Schrodinger, LLC, New York, NY, 2015) [18]. This is an
interactive molecular graphics program for docking calculations, for
identification of the probable binding site of the biomolecules, and
for visualizing ligand-receptor interactions. All compounds were
built using Maestro build panel and optimized to lower energy
conformers using Ligprep v2.4 which uses an OPLS_2005 force field.
Epik v2.1was used to generate an ionized state of all compounds at
target pH 7.0±2.0. The coordinate for InhA enzyme (PDB: 2X23) [19]
were taken from RCSB Protein Data Bank and prepared for docking
using ‘protein preparation wizard’ in Maestro 9.1. Water molecules
in the structures were removed and termini were capped by adding
ACE and NMA residue. The bond orders and formal charges were
added for hetero groups and hydrogens were added to all atoms in
the structure. Side chains that are not close to the binding cavity and
do not participate in salt bridges were neutralized. After
preparation, the structure was refined to optimize the hydrogen
bond network using an OPLS_2005 force field. This helps in the
reorientation of side chain hydroxyl group. The minimization was
terminated when the energy converged or the RMSD reached a
maximum cutoff of 0.30 Ǻ. Grids were then defined around refined
structure by centring on ligand using default box size. The extra
precision (XP) docking mode for all compounds was performed on a
generated grid of protein structure. The final evaluation of ligand-
protein binding was done with glide score (docking score). The extra
precision (XP) docking mode for all compounds was performed on a
generated grid of protein structure. The final evaluation of ligand-
protein binding was done with glide score (docking score).
3-Fluoro-4-morpholino-N-((3-(4-nitrophenyl)-1-phenyl-1H-
pyrazol-4-yl)methyl)aniline (10e)
Dark-brown solid; Yield=85%; m. p =188-190 ᵒC; Rf value=0.56;1H
NMR (CDCl3, 400 MHz, δ ppm): 2.93-2.96 (t, 4H,-CH2-of morpholine,
J=7.2 Hz), 3.72-3.75 (t, 4H,-CH2-of morpholine, J=7.2 Hz), 4.59 (s,
2H,-CH2-), 6.15 (s, 1H, NH), 7.52 (s, 1H,-CH-of pyrazole), 5.98-8.24
(m, 12H, Ar-H); [13]C NMR (CDCl3, 100 MHz, δ ppm): 40.66, 51.10,
66.81, 102.60, 102.80, 113.73, 113.76, 120.17, 121.19, 121.27,
123.63, 125.01, 126.55, 127.40, 129.48, 131.17, 134.12, 134.32,
137.87, 140.31, 144.09, 148.87, 154.73, 155.09, 157.61; MS:
m/z=474.8 (M+1) (% Mol. Wt.: 99.65).
3-Fluoro-N-((3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-
yl)methyl)-4-morpholinoaniline (10f)
Pale yellow solid; Yield=90%; m. p =238-240 ᵒC; Rf value=0.70; 1H
NMR (CDCl3, 400 MHz, δ ppm): 2.93-2.96 (t, 4H,-CH2-of morpholine,
J=7.2 Hz), 3.72-3.75 (t, 4H,-CH2-of morpholine, J=7.2 Hz), 3.81 (s,
3H,-OCH3-of Phenyl), 4.65 (s, 2H,-CH2-), 6.40 (s, 1H, NH), 7.47 (s, 1H,-
CH-of pyrazole), 6.13-7.92 (m, 12H, Ar-H);13C NMR (CDCl3, 100 MHz,
δ ppm): 40.66, 51.14, 55.39, 66.81, 102.60, 102.80, 113.73, 113.76,
114.70, 120.17, 121.19, 121.27, 125.01, 125.75, 126.55, 129.48,
130.82, 131.17, 134.12, 134.32, 140.31, 144.09, 154.73, 155.09,
157.61, 161.23; MS: m/z=459.3 (M+1) (% Mol. Wt.: 99.41).
RESULTS AND DISCUSSION
Chemistry
The pyrazolyl derivatives 9(a-f) and 10(a-f) were synthesized as
presented in Scheme 1. Initially, 3-aryl-1-phenylpyrazole-4-
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Int J Pharm Pharm Sci, Vol 9, Issue 11, 50-56
carbaldehydes 4(a-f) were synthesized from phenylhydrazones 3(a-
f) by Vilsmeier-Haack formylation as reported by Raquib Alam et al.
[20]. Then 3-aryl-1-phenylpyrazole-4-carbaldehydes 4(a-f) were
condensed with isoniazid 5 and 3-fluoro-4-morpholinoaniline 6
which gives corresponding imines 7(a-f) and 8(a-f) respectively. The
last step involves reduction amination of imines 7(a-f) and 8(a-f)
using sodium borohydride (NaBH4) in methanol to afford
corresponding titled compounds 9(a-f) and 10(a-f).
Scheme 1: General procedure for synthesis of pyrazolyl derivatives 9(a-f) and 10(a-f); (i) EtOH, H2SO4, reflux; (ii) a. POCl3/DMF, 80 ᴼC; b.
NaHCO3/H2O; (iii) EtOH, AcOH, reflux; (iv) Methanol, NaBH4
Biological activity
compounds 9(a-f) and 10(a-f), the pyrazole-linezolid like conjugates
10(a-f) exhibited superior inhibitory activity than pyrazole-isoniazid
9(a-f) derivatives. The basic skeleton 1, 3-diphenyl pyrazole scaffold is
supposed to have significant antitubercular activity. Further, we
investigated the impact of substitution of p-position on the 3-phenyl of
pyrazole ring. The p-position on the 3-phenyl of pyrazole ring
substituted with electron withdrawing (Cl, F, Br, and NO2) or electron
donating groups (-OCH3) were investigated. In the pyrazole-isoniazid
9(a-f) derivatives, the introduction of electron withdrawing groups
like p-flouro 9c (MIC = 16.06 μM) and p-bromo 9d (MIC = 13.91 μM) on
3-phenyl of pyrazole leads to increase activity by two folds when
compared with electron donating group p-OCH3 9f (MIC 31.15 μM).
In vitro antitubercular activity
All pyrazole-isoniazid 9(a-f) and pyrazole-linezolid like conjugates
10(a-f) were tested for their in vitro antitubercular activity against
Mycobacterium tuberculosis H37Ra (ATCC 27294). The antitubercular
activity in terms of minimum inhibitory concentration (MIC) of titled
compounds and standard drugs were given in table 1. As evident from
the data, among the pyrazole-isoniazid 9(a-f) conjugates two
compounds 9c and 9d were found to be most active against
Mycobacterium tuberculosis H37Ra having MIC 16.06 μM and 13.91
μM, respectively. The compound 9f showed good antitubercular
activity with MIC 31.15 μM. Other compounds 9b (MIC = 61.70 μM)
and 9g (MIC = 60.07 μM) also showed moderate antitubercular
activity. Further, pyrazole-linezolid like conjugates 10(a-f) was found
to possess excellent potency against Mycobacterium tuberculosis
H37Ra. From this series, compound 10c and 10d exhibited excellent
antitubercular effect with MIC 13.94 μM and 12.30 μM, respectively.
However, rest of compounds 10b, 10e, 10a, and 10f were also found to
have moderate inhibitory effect against Mycobacterium tuberculosis. As
a result of this discussion, compound 10d (MIC = 12.30 μM) was found
to be most potent in comparison of both the series.
Other electron withdrawing groups like p-chloro 9b (MIC = 61.70 μM)
and p-nitro 9e (MIC = 60.07 μM) on 3-phenyl of pyrazole showed least
antitubercular activity. In pyrazole-linezolid like conjugates 10(a-f),
the presence of electron withdrawing flouro group on phenyl ring
largely influences the antitubercular activity (10c; MIC = 13.94 μM).
Along with this flouro group, the introduction of strong electron
withdrawing bromo group at p-position on the 3-phenyl of pyrazole
ring (10d; MIC
= 12.30 μM) leads to significant increase in
antitubercular effect. Introduction of least electron withdrawing
groups like p-chloro 10b (MIC = 26.92 μg/ml) and p-flouro 10e (MIC =
26.30 μM) also responsible for enhancement of antitubercular activity.
The compounds with unsubstituted phenyl ring and presence of
electron donating p-OCH3 group resulted in a decrease in activity (10a,
MIC = 134.70 μM; 10a, MIC = 116.22 μg/ml).
Structure-activity relationship (SAR)
Observing the results, we deduce the valuable data about structure-
activity correlations of the tested compounds. From the two series of
53

Zaheer et al.
Int J Pharm Pharm Sci, Vol 9, Issue 11, 50-56
Table 1: In vitro antitubercular activity, cytotoxicity study and selectivity index of compounds 9(a-f) and 10(a-f)
Entry
9a
9b
9c
9d
9e
9f
10a
10b
10c
10d
10e
R
H
Anti-TB MIC (μM)
134.70
61.70
16.06
13.91
60.07
31.15
116.22
26.92
13.94
12.30
26.30
108.64
0.87
9.41
Cytotoxicity HeLa GI50 (μM)
-
-
>256.96
>222.67
-
-
Selectivity Index
-
-
>16
>16
-
-
-
4-Cl
4-F
4-Br
4-NO2
4-OCH3
H
4-Cl
4-F
4-Br
4-NO2
4-OCH3
-
-
-
>223.11
>201.80
-
-
NA
NA
NA
0.004
>16
>16
-
10f
-
aIsoniazid
aCiprofloxacin
aRifampicin
bPaclitaxel
NA
NA
NA
NA
0.70
NA
aStandard antitubercular drug; standard anticancer drug; DPPH: 1,1-Dipheny-1-picrylhydrazyl; NA: Not applicable; NT: Not tested; Selectivity index
(S. I.) = (GI50/MIC); GI50 = concentration inhibits 50 % growth of cells. (Biological activities performed in the triplet. The results were with 10 % of
standard error mean, SEM)
In vitro cytotoxicity study
studies. Visual inspection of the minimum energy docked poses
revealed that these derivatives snuggly fitted into the binding pocket
of InhA enzyme (PDB: 2X23) making close contacts with the
surrounding residues. The synthesized compounds showed good
docking scores. A detailed per-residue interaction analysis between
the protein and the most active compound 10d only is elucidated for
the sake of brevity through which we can speculate regarding the
binding patterns in the cavity. The binding interaction of 10d is
presented in fig. 2. It showed multiple interactions with the residues
in the active site, however for visibility and clarity only selected
interacting residues are exhibited. The compound 10d helped in the
stabilization of compound by making hydrophobic interactions with
amino acid residues like ILE95, PHE97, ILE202, MET103, MERT161,
MET199, VAL203, LEU218, PRO193, PHE149, ILE194, ALA191,
ILE21 and MET147 of InhA enzyme. Strong binding of 10d with the
active site of InhA enzyme is also contributed by its position in the
pi-interaction in space of TYR158. On the basis of activity data and
docking result, it was found that the compound 10d has potential to
inhibit InhA enzyme and can be processed further to develop a lead
compound.
The safety profile of antitubercular active compounds with MIC<20
μM was assayed for in vitro cytotoxicity against HeLa cell line by
Sulforhodamine B (SRB) assay method. None of the synthesized
compounds 9c, 9d, 10c and 10d was cytotoxic at concentration up to
200 μM.
Selectivity index
Table 1 observations interpret that, the screened compounds 9c, 9d,
10c and 10d were shown selectivity index>10. This indicates that
the screened compounds were further investigated for future drug
development process.
Computational studies
Molecular docking study
In order to rationalize the observed antitubercular results and to get
insight into the inhibition pattern, interactions of synthesized
compounds were analyzed and depicted using molecular docking
Fig. 2: (A) 2D docking pose of compound 10d; (B) 3D docking pose of compound 10d
54

Zaheer et al.
Int J Pharm Pharm Sci, Vol 9, Issue 11, 50-56
candidate. Other parameters as solvent accessible surface area
(SASA; 709.441-829.983) and a number of rotatable bonds (#rotor;
<15; 3-5) also have a great influence on the oral bioavailability of the
drug molecules and found in acceptable range. Investigation of
intestinal absorption, permeation, and metabolism is also greatly
contributed to minimizing the clinical trial failures. Hence, predicted
Caco-2 cell permeability (QPPCaco), apparent MDCK cell
permeability (QPPMDCK), predicted aqueous solubility (QPlogS),
percent scale human oral absorption (%HOA), and number of likely
metabolic reactions (#metab) were also analyzed and displayed a
reliable results falling in the prescribed range as shown in table 2.
From all in silico prediction of physicochemical and pharmacokinetic
properties (ADME) study, these compounds could be developed as
an oral active drug, making them potentially capable for
antitubercular treatment.
Prediction of physicochemical and pharmacokinetic properties
All the synthesized compounds 9(a-f) and 10(a-f) were evaluated for
prediction of physicochemical and pharmacokinetic properties for
drug-likeliness using QikProp module of Schrodinger 9.0 [21]. As per
Lipinski’s rule-of-five, a molecule likely to be developed as an orally
active drug candidate should show no more than one violation of the
four parameters such as molecular weight (MW<500), hydrogen
bond donor (HBD; 0-6), hydrogen bond acceptor (HBA; 2-20), and
partition coefficient (QlogP; 1-5) [22]. Except for partition
coefficient (3.52-6.88), other physicochemical properties like
molecular weight (371.441-491.420), hydrogen bond donor (1-2)
and hydrogen bond acceptor (4.7-6.75) found the results within
their acceptable range. So, the synthesized compound has shown no
more than one violation and can be developed as a good oral drug
Table 2: Physicochemical properties for the titled compounds 9(a-f) and 10(a-f) predicted by QikProp
Entry MW
(<500)
HBD HBA
QlogPo/w SASA
#rotor No. of
(0-15) violations
(<1)
QPPCaco
(>500
great)
QPPMDCK QPlogS
% HOA
(>80%) (1-8)
#metab
(0-
6)
2
(2-
20)
6
(1-5)
(300-
(>500)
(-6.5-
0.5)
1000)
9a
371.441
405.886
389.431
450.337
416.438
401.467
430.524
464.969
448.514
491.420
475.521
460.550
4.22
4.69
4.43
4.76
3.52
4.98
6.30
6.79
6.52
6.88
5.59
6.36
709.411
732.779
718.268
738.357
754.269
829.983
742.260
766.793
748.262
775.253
782.232
779.035
4
4
4
4
5
5
3
3
3
3
4
4
0
0
0
0
0
0
1
1
1
1
1
1
857.23
879.61
879.46
879.10
99.065
1318.83
5954.59
5884.19
5910.85
5982.35
701.45
5750.95
418.833
1060.693
776.501
1134.407
40.65
667.204
5231.022
10000
9364.362
10000
518.474
5026.507
-5.576
-6.259
-5.932
-7.153
-6.09
-5.888
-7.447
-8.159
-7.818
-9.081
-7.965
-7.759
100
100
100
100
83.30
100
100
100
100
100
100
100
3
3
3
3
4
4
4
4
4
4
5
5
9b
9c
9d
9e
2
6
2
2
6
6
2
2
1
1
1
1
1
1
7
9f
6.75
4.7
4.7
4.7
4.7
5.7
5.45
10a
10b
10c
10d
10e
10f
MW-Molecular weight; HBD-Hydrogen bond donor; HBA-Hydrogen bond acceptor; QlogPo/w-Predicted octanol/water partition coefficient; SASA-
Solvent accessible surface area; #rotor-Number of rotatable bonds; QPPCaco-Possible CaCO cell permeability; QPPMDCK-Predicted apparent MDCK
cell permeability; QPlogS-Predicted aqueous solubility; % HOA-Percent human oral absorption; #metab-Number of likely metabolic reactions.
CONCLUSION
CONFLICT OF INTERESTS
In conclusion, some new structural hybrids of 1, 3-diphenyl
pyrazole bearing isoniazid and linezolid like moieties were
synthesized and investigated for their in vitro antitubercular
activity with an anticipation of generating new structural leads.
Four of the synthesized compounds (9c, 9d, 10c and 10d)
possessing electron withdrawing groups such as flouro and bromo
at para position were identified as the most potent antitubercular
agents. The potent antitubercular activity of the most potent
The authors declare that there is no conflict of interest
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AUTHORS’ CONTRIBUTION
Sameer I. Shaikh researched and wrote this overview. Zahid Zaheer
and Santosh N. Mokale provided guidance, critical review and
revision. Deepak K. Lokwani carried out all computational studies
(Molecular docking and in silico properties prediction). All authors’
read and approved the final version of this manuscript.
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