Pyrazole clubbed triazolo[1,5-a]pyrimidine hybrids as an anti-tubercular agents: Synthesis, in vitro screening and molecular docking study

Article abstract of DOI:10.1016/j.bmc.2015.11.018

A series of novel pyrazole linked triazolo-pyrimidine hybrids were synthesized and evaluated for their anti-tuberculosis activity against M.tb H37Rv strain. Some of the screened entities rendered promising anti-tb activity (MIC: 0.39 μg/mL) and were found non toxic against Vero cells (IC₅₀: ≥20 μg/mL). Further, the docking study against wild type InhA enzyme of Mycobacterium tuberculosis using Glide reproduced the most active inhibitors (J21 and J27) with lowest binding energies and highest Glide XP scores demonstrating efficient binding to the active pocket. Additionally, the enzyme inhibition assay and ADME prediction of the active proved to be an attest to the possibility of developing compound J27 as a potent anti-tubercular lead.

Full text of DOI:10.1016/j.bmc.2015.11.018

Accepted Manuscript
Pyrazole clubbed triazolo[1,5-a]pyrimidine hybrids as an anti-tubercular agents:
Synthesis, in vitro screening and molecular docking study
Jaimin D. Bhatt, Chaitanya J. Chudasama, Kanuprasad D. Patel
PII:
S0968-0896(15)30143-7
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.11.018
BMC 12666
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
11 September 2015
6 November 2015
16 November 2015
Please cite this article as: Bhatt, J.D., Chudasama, C.J., Patel, K.D., Pyrazole clubbed triazolo[1,5-a]pyrimidine
hybrids as an anti-tubercular agents: Synthesis, in vitro screening and molecular docking study, Bioorganic &
Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.11.018
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Pyrazole clubbed triazolo[1,5-a]pyrimidine
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docking study
Leave this area blank for abstract info.
Jaimin D. Bhatt1, Chaitanya J. Chudasama2, Kanuprasad D. Patel1
1Chemistry Department, V. P. & R. P. T. P. Science College, Affiliated to Sardar Patel University, Vallabh
Vidyanagar-388 120, Gujarat, India.
2Department of Biochemistry, Shree Alpesh N. Patel P. G. Institute, Affiliated to Sardar Patel University,
Anand-388001, Gujarat, India.
1

Bioorganic & Medicinal Chemistry
Pyrazole clubbed triazolo[1,5-a]pyrimidine hybrids as an anti-tubercular agents:
Synthesis, in vitro screening and molecular docking study
Jaimin D. Bhatt a, Chaitanya J. Chudasama b, Kanuprasad D. Patela
aChemistry Department, V. P. & R. P. T. P. Science College, Affiliated to Sardar Patel University, Vallabh Vidyanagar-388120, Gujarat, India.
bDepartment of Biochemistry, Shree Alpesh N. Patel P. G. Institute, Affiliated to Sardar Patel University, Anand-388001, Gujarat, India.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A series of novel pyrazole linked triazolo-pyrimidine hybrids were synthesized and evaluated for
their anti-tuberculosis activity against M.tb H37Rv strain. Some of the screened entities rendered
promising anti-tb activity (MIC: 0.39 µg/mL) and were found non toxic against Vero cells (IC50:
≥20 µg/mL). Further, the docking study against wild type InhA enzyme of M. tuberculosis using
Glide reproduced the most active inhibitors (J21 & J27) with lowest binding energies and
highest Glide XP scores demonstrating efficient binding to the active pocket. Additionally, the
enzyme inhibition assay and ADME prediction of the active proved to be an attest to the
possibility of developing compound J27 as a potent anti-tubercular lead.
Available online
Keywords:
Triazolopyrimidine
Diaryl pyrazole aldehydes
Glide
2009 Elsevier Ltd. All rights reserved.
Enzyme inhibition assay
ADME
1. Introduction
eliciting the ability to prevail against the chemical injuries.5
Protein enoyl acyl carrier protein (ACP) reductase (InhA) plays a
Tuberculosis (Tb) which is mainly caused by Mycobacterium
Tuberculosis (M.tb), is still throned a devastating threat across
the globe.1 Despite of having acute antitubercular chemotherapy,
over 2 million new cases are reported every year.2 The drug
resistance surveillance data indicated 0.5 million new cases of
multidrug resistance Tb (MDR-Tb) during the year 2013.3 The
increasing multidrug resistance among the pathogens has urged
the urgency for development of new agents that can shorter the
lengthy Tb therapy as well as inhibits the targets involved in
persistency or dormancy.4
catalytic role in final step of fatty acid elongation pathway and is
also a target for the activated form of Isoniazide (INH), a half
century old drug.6
Heterocyclic and fused heterocyclic compounds are ubiquitous
and plays vital role in metabolism of all living cells.7 A class of
fused heterocyclic entity is bridge headed nitrogen containing
triazolopyrimidine derivatives, resembling to purine has
remarkable therapeutic and pharmacological applications.8 They
are functional therapeutics, especially for the treatment and
prevention of cardiovascular diseases, also known to posses
smooth muscles cell growth inhibiting efficacy, are efficient
analgesics and anti inflammatory agents.9 Another class of
heterocyclic entities, pyrazole derivative occurs in many drugs
and synthetic products. Synthetic analogues of pyrazole are
known to show phenomenal antitubercular activity especially 1,3-
diphenyl pyrazole motif is known to be potent antituberculosis
agent.10,11 The carboxamide side chain has shown enhancement in
the pharmacological output of pyrimidine as parent motif11 and
further it was noted that replacement of benzene by pyridine ring
augmented fungicidal, antibacterial and anticancer activities.12a
Moreover, the pyrazole and carboxamide derivatives are reported
to owe potent InhA direct inhibitory activity both in vitro and in
vivo.12b
On vantage point of drug discovery and medicinal chemistry,
choice of the proteins involved in regulatory functions as an
inhibitory target is a crux of matter in drug development regime.
In the case of Mycobacterium, FAS-II co-exists with the type I
(FAS-I) pathway, a situation that is unique to this genus. Here
FAS-I is responsible for de novo synthesis leading to C16–C26
fatty acids, which are used for production of phospholipids and as
primers for complex lipids. The FAS-II system here in extends
these fatty acids up to C56 making the long chain precursors
which are required for the synthesis of cell wall-associated
mycolic acids that are specific to Mycobacteria.5 The cell wall of
M.tb comprises of mycolic acids covalently linked to
arabinogalactam, which provide complex mycobacterial envelope
———
 Corresponding author. Tel.: +91 2692-230011; fax: +91-2692-235207; e-mail: drkdpatel64@yahoo.co.in (KD Patel);
jaiminbhatt1488@gmail.com (JD Bhatt)
2

Scrutinizing the above implications, we here in developed
hybrid 1-3 diarylpyrazole ligated triazolopyrimidines possessing
pyridyl carboxamide moieties. The developed hybrid molecules
were screened for their antituberculosis activity against H37Rv
strain of M. tuberculosis. Furthermore, docking studies and
ADME prediction were carried out computationally to get insight
of the structural parameters leading to activity.
screening compounds J3, J15, J21, J25 and J27 inhibited M. tb
in range of 90-100% as summarized in Table 1. Further, the
secondary screening results represented that two compounds (J3
& J15) inhibited the mycobacterium strain at MIC 3.13 µg/mL
and 1.56 µg/mL, while the three compounds (J21, J25 & J27)
inhibited M. tb at MIC lower than 1 µg/mL. These results
revealed that the compounds demonstrate moderate activity as
compared to Isoniazide but good activity against M. tb as
compared to Ethambutol and Rifampicin.
2. Result and Discussion
2.1. Chemistry
Table 1: In-vitro anti-tuberculosis activity and cytotoxicity of
triazolo-pyrimidine hybrids
The conjectured target triazolopyrimidine derivatives (J1-J30)
were obtained in single step by multicomponent amalgamation
of pyridin-2-yl-3-oxobutanamide derivatives, 1,3-diphenyl-1H-
pyrazole-4-carbaldehyde derivatives and amino-triazole (1.5
equivalent) in appropriate quantity using dimethylformamide as
reaction medium under refluxing condition as delineated in
Scheme 1. The one pot reaction methodology adopted to obtain
the final hybrid 7-(1,3-diphenyl-pyrazol-4-yl)-5-methyl-N-
Compound
% Inhibition
MIC µg/mL
R1
R2
R3
(at 6.25 µg /mL)
J1
J2
72
66
95
45
42
54
56
49
64
55
46
66
84
68
96
74
68
78
84
79
98
81
76
84
99
86
99
84
82
88
99
99
99
N.D.
N.D.
H
H
Me
H
CH3 Me
J3
3.13 ±0.313
N.D.
H
Br
H
Me
J4
H
i-Pr
J5
N.D.
H
CH3 i-Pr
J6
N.D.
H
Br
H
i-Pr
J7
N.D.
Me
Me
Me
Me
Me
Me
NO2
Me
J8
N.D.
CH3 Me
J9
N.D.
Br
H
Me
J10
J11
J12
J13
J14
J15
J16
J17
J18
J19
J20
J21
J22
J23
J24
J25
J26
J27
J28
J29
J30
INH
Rifampicin
Ethambutol
N.D.
i-Pr
N.D.
CH3 i-Pr
(pyridin-2-yl)-4,7-dihydro[1,2,4]
triazolo[1,5-a]pyrimidine-6-
N.D.
Br
H
i-Pr
carboxamide in good yields.
N.D.
Me
Scheme 1: Synthesis of triazolopyrimidines J1-J30; Reagent
and Conditions: DMF, reflux
N.D.
NO2 CH3 Me
1.56 ± 0.083
N.D.
NO2 Br
NO2
Me
The formation of triazolopyrimidines was confirmed by
observing the characteristic spectral data of the synthesized
compounds J1-J30 which were fully in agreement with their
proposed structures. FT-IR results also indicates the
conformation of the synthesized compound by peculiar band of
amide as well as amine at 3419-3372 cm-1, also a band at ~1566
cm-1 indicates C=N stretching of triazole ring and a band at 1697-
1657 cm-1 corresponds to C=O stretching of amide linkage. Two
medium bands observed at 2978-2950 and 2941-2899 cm-1
corresponds to asymmetrical and symmetrical stretching of
methyl group. All the entitled motifs showed a singlet of
asymmetric CH proton in 1H NMR spectrum between 6.20-5.94 δ
ppm, a singlet for the methine proton of triazole ring at ~7.65 δ
ppm, singlets for amino as well as amide group protons at 10.12-
9.95 and 10.63-10.39 δ ppm, respectively. A singlet was
observed at 2.17-2.08 corresponding to methyl group at C2
position of triazolopyrimidine ring, characteristic multiplet and
two singlets were observed at ~3.9, 1.24-1.07 and 1.16-1.06
H
i-Pr
N.D.
NO2 CH3 i-Pr
N.D.
NO2 Br
i-Pr
N.D.
Cl
Cl
Cl
Cl
Cl
Cl
F
H
Me
N.D.
CH3 Me
0.78 ±0.039
N.D.
Br
H
Me
i-Pr
N.D.
CH3 i-Pr
N.D.
Br
H
i-Pr
0.78 ±0.039
N.D.
Me
F
CH3 Me
0.39 ± 0.019
N.D.
F
Br
H
Me
F
i-Pr
respectively
corresponding
to
isopropyl
group
of
N.D.
F
CH3 i-Pr
triazolopyrimidine ring. The aromatic ring protons and J value
were found to be in accordance with substitution pattern on
phenyl ring. All the final products were also confirmed by
performing ESI-MS and observing the molecular ion peak.
N.D.
F
--
Br
--
i-Pr
--
0.3
--
--
--
0.5
--
--
--
3.125
2.2. In-vitro antituberculosis activity
ND denotes Not determined, Minimum inhibitory
concentration (MIC) against H37Rv strain of M. tuberculosis
(µg/mL).
The synthesized compounds J1-J30 were evaluated for their
anti-tb potency against M.tb H37Rv strain (MTCC 300) using LJ
medium and broth dilution technique. Preliminary screening was
carried out at 6.25 µg/mL concentration and the compounds
exhibiting more than or equal to 90% inhibition in the initial
screen were retested at and below 6.25 µg/mL using twofold
dilution to determine the actual MIC. In the preliminary
The substitutional pattern at three positions R1, R2 and R3 of
the hybrid molecules were studied for their effect on anti-
tuberculosis efficacy. It emerged out that substitution at position
R1 of aryl ring of pyrazole demonstrated maximum effect on the
3

activity pattern of the hybrid molecule. The electron withdrawing
(J15, J21, J25 & J27), whereas methyl group at this position
group (–F, –Cl, –NO2) enhanced the activity of the compounds
Figure 1: The binding model of docked compound J21 with enoyl ACP reductase (InhA) enzyme. The enzyme is displayed as cartoon, while the compounds as
stick model. (a) 2D model enumerating the interactions of the ligand J21 with the enzyme (b) 3D model of compound J21 in the binding pocket of InhA.
Figure 2: The binding model of docked compound J27 with enoyl ACP reductase (InhA) enzyme. The enzyme is displayed as cartoon, while the compounds as
stick model. (a) 2D model enumerating the interactions of the ligand J27 with the enzyme (b) 3D model of compound J27 in the binding pocket of InhA.
greatly reduced the potency of the compounds. Comparably
fluorine substituted entities were found to be most potent owing
to high electronegativity and hydrophobicity (J25 & J27). The
substitutional changes at para position of pyridyl ring of
carboxamide side arm also influence the activity pattern, the
presence of –Br group greatly increases the potency of the
compounds as compared to methyl substituted and unsubstituted
pyridyl ring. The position R3 substituted with methyl group
demonstrated better activity as compared to isopropyl substituted
motifs, which clearly indicates the effect of increase in chain
length and branching leading to downfall of potency.
Table 2: Glide XP score, binding energies and enzyme
inhibition assay of triazolo-pyrimidine hybrids
IC50
Enzyme
Inhibition
CC50
VERO
cells
Binding
energy
RMSa
Å
S.I.*
Glide
XP
score
Compound
(kcal/mol)
(µg/mL)
(µg/mL)
2.739
2.271
1.866
1.474
1.509
0.85 ±
0.042
J3
-7.47
-7.46
-9.07
-7.17
-9.07
-44.89
25
29.41
89.28
0.28 ±
0.014
J15
J21
J25
J27
-56.60
25
20
20
20
0.11 ±
0.005
-49.48
181.81
125.00
181.81
2.3. Docking Study
0.16 ±
0.008
Molecular docking studies were performed for the active
compounds (J3, J15, J21, J25 and J27) against the active site of
InhA enzyme (PDB Id: 2B35) structure. The results of the
docking study with the docking scores, RMS deviation and
energies are as summarized in Table 2. The results clearly
demonstrate the significant binding affinities with docking
energies ranging from -44.89 kcal mol-1 to -56.60 kcal mol-1 and
the RMS deviation values were observed to fall in range of 2-3 Å
which can be considered to be acceptable value of deviation.
-46.59
0.11 ±
0.005
-46.29
RMSa: The deviation was calculated considering the Triclosan
as reference molecule. Measurement of cytotoxicity in VERO
cells: 50% inhibitory concentrations or cytotoxic concentrations
(µg/mL), * Selectivity index (in vitro): CC50 of VERO cells/ IC50
of enzyme inhibition.
4

Table 3: Prediction of Lipinski’s rule of 5
Compound
mol_MW
donorHB
accptHB
(<10)
QPlogPo/w
(<5)
PSA
Å
#rotor
‘N’ of
(<500 amu)
(<5)
violations
(70-200)
106.253
150.573
105.642
105.671
97.995
(<2)
J3
529.395
574.392
563.84
1
1
1
1
1
4.5
5.5
4.5
4.5
4.5
6.305
5.673
6.704
6.152
6.636
2
3
2
2
2
2
3
2
1
2
J15
J21
J25
J27
468.489
547.385
MW: Molecular Weight; donorHB: Hydrogen Bond Donor; accptHB: Hydrogen Bond Acceptor; QPlogPo/w: Partition Coefficient; PSA: Polar Surface Area;
#rotor: Rotatable Bonds; ‘N’ of violations: Number of Lipinski Rule Violations.
Table 4: ADME prediction of triazolo-pyrimidine hybrids
Compound
Percent Human Oral Absorption*
QPPCaco*
(<25 poor, >500 great)
693.605
QPlogBB*
(-3.0 – 1.5)
-0.649
QPlogKhsa*
(-1.5 – 1.5)
1.50
QPPMDCK*
(<25 poor >500 great)
885.563
QPlogS*
(-6.5 –5)
-8.52
(>80% -high & <25% - poor)
J3
87.425
57.316
90.073
100
J15
J21
J25
J27
103.077
-1.773
1.50
113.023
-8.936
-9.194
-8.032
-9.01
712.72
-0.467
1.60
2249.327
760.583
-0.653
1.399
665.543
93.707
1016.463
-0.377
1.50
2433.214
*Calculated using QikProp v 3.5. Range/recommended values calculated for 95% known drugs.
The Figures 1 and 2 illustrates the binding poses as well as
interactions of the compounds J21 and J27 with highest docking
score. The compound J21 with binding energy -49.48 kcal mol-1
with their permissible ranges are delineated in Table 4. The
optimum value of rotatable bonds (<15) and polar surface area
(7-200 Å) holds a great importance on the oral bioavailability of
the drug molecules.13 The active test hybrid triazolopyrimidine
derivatives demonstrated results of the descriptors to be in the
prescribed range thus owing good bioavailability. Intestinal
absorption or permeation is also one of the important factor to be
studied in concern with the absorption of the drug molecule,
which was further confirmed by predicted Caco-2 cell
permeability (QPPCaco), used as model for gut- blood barrier.14
Caco-2 cell permeability prediction of the test compounds
indicates excellent results predicting good intestinal absorption.
Further, the test results for QPlogkhsa descriptor of Quickprop
indicating the predicted values of human serum albumin binding
indicated that test molecules were found to fall in the permissible
range (-1.5 to 1.5). Also, the Quickprop descriptor for
blood/brain partition coefficient QPlogBB showed reliable
prediction for all the test compounds and reference drugs. The
cell permeability of the blood brain barrier mimic MDCK cells
(QPPMDCK) also displayed reliable results falling in the
prescribed range. The aqueous solubility parameter (QPlogS) of
the test entities was assessed and the compounds were found to
be out laid from the permissible range (-6.5-0.5) indicating slight
poor solubility of the test molecules which should be further
rectified by utilizing solubility enhancers.
and
considerably
high
XP
glide
score
-9.07, binds with InhA active site forming a hydrogen bond
between NH of pyrimidines ring and oxygen of carboxylate
group of Gly14 at a distance of 2.17 Å. Further, π-π stacking was
observed between the two phenyl rings linked to pyrazole ring
with phenyl ring of Phe97 and amine group of Arg43 respectively
(Figure 1). Proceedingly, the compound J27 having low binding
energy -46.29 kcal mol-1 and high XP glide score -9.07, actively
interact with the binding pocket of the enzyme structure in the
similar pattern as compound J21 leading to hydrogen bonding
between –NH of pyrimidines ring and oxygen of carboxylate
group of Gly14 at a distance of 2.28 Å. Additionally, π-π
stacking was observed between the two phenyl rings linked to
pyrazole ring with phenyl ring of Phe97 and amine group of
Arg43 as summarized in figure 2. The other active compounds
(J3, J15 and J25) similarly binds to the active binding pocket of
InhA by forming significant hydrogen bonding as well as π-π
interaction and van der Waals interaction.
2.4. Pharmacokinetic properties prediction
Different pharmacokinetic parameters of the synthesized
compounds showing good antitubercular activity were calculated
using ADME predictions by Quickprop v4.3. The compounds
were preliminarily screened considering the basic parameters of
Lipinski’s rule of 5. Table 3 shows the results obtained from
Quickprop with their permissible range. In general an orally
active compound should not have more than 2 violations of the
Lipinski rule. The active test compounds in present study were
not found violating the rule more than the maximum permissible
limits and thus proving their drug likeness properties.
2.5. InhA inhibition assay & Cytotoxicity
The active molecules with lower MIC values and good
docking score were further evaluated for InhA inhibitory
potency. The results obtained are delineated as IC50 values of
enzyme inhibition in Table 2. The compounds J21, J25 as well as
J27 demonstrated good inhibitory potency among screened
compounds with IC50 value of 0.11, 0.16 and 0.11 µg/mL
respectively. This indicates a good agreement between docking
experiment and InhA inhibitory activity of the potent entities
concluding that the anti-tuberculosis activity of the compounds is
due to their enzyme inhibitory potency. The active entities
The optimum values of the descriptors, polar surface area and
rotatable bonds also have great influence on the oral
bioavailability of the drug molecules. The important parameters
5

obtained after preliminary screening were further tested for their
cytotoxicity on Vero cell using MTT assay and the results were
reproduced as 50% growth inhibitory concentration values or
cytotoxicity concentration (CC50) which are as displayed in Table
2. The CC50 values of the screened compounds emerged no sign
of toxicity up to a concentration of 25 µg/mL thus owing a non
toxic nature. The S.I. value indicates that the anti-tb potency of
the compounds is not attributed due to non-specific activity and
cytotoxicity as well.
A mixture of the appropriate aldehyde 1 (0.01 mol),
aminoazole 2 (0.015 mol) and appropriate acetoacetanilides 3
(0.01 mol) were refluxed in 0.4 mL of DMF15 for specific time
interval as visualized by TLC. After cooling, acetone (~10 mL)
was added. The reaction mixture was allowed to stand overnight
and then filtered to give the solid triazolopyrimidine products J1-
J30, which were crystallized from ethanol and subsequently dried
in air.
3. Conclusion
4.2. In Vitro antitubercular activity
Summing up here with, we report the synthesis, anti-
tuberculosis activity, docking study, enzyme inhibition study and
ADME prediction of novel pyrazole clubbed triazolo[1,5
a]pyrimidine hybrids. The in vitro study of the hybrids yielded
five potent entities (J3, J15, J21, J25 and J27) owing their
potency in micro molar range and further exerting no cytotoxicity
against Vero cells. Eventually, docking study of the active entries
against InhA enzyme active site was performed to study binding
pattern of the active motifs. Compounds J21 and J27 were found
to show good binding affinity with the active binding site of InhA
enzyme with lowest binding energies and comparably high Glide
XP scores. Further, the results of enzyme inhibition assay and
ADME prediction study provides a confirmation to consider the
active molecules as lead targets for further progress in drug
discovery process.
All the synthesized compounds were examined for
antituberculosis activity against Mycobacterium tuberculosis
H37Rv (MTCC 300) strain using Lowenstein-Jensen medium
method.16 Isoniazide, Ethambutol and Rifampicin was used as the
standard drug. The dilutions of standard drugs were prepared in
DMSO to get different concentrations of 0.1-6.25 µg/mL. Stock
solutions of synthesized compounds were prepared in DMSO.
Further dilutions were prepared in DMSO to get different
concentrations 1.0 mL of each concentration was used for the
study, to this 9.0 mL of Lowenstein-Jensen medium was added.
A sweep from M. tuberculosis H37RV strain culture was
discharged with the help of Nichrome wire loop having 3 mm
external diameter into a vial containing 4 mL of sterile distilled
water. The vial was shaken for 5 min. The suspension was
inoculated on the surface test compounds containing Lowenstein-
Jensen medium. Further test media was incubated for 4 weeks at
37 °C. Readings were taken after incubation period of 4 weeks.
Further the activity was determined by measuring the OD at 600
nm followed by the calculating the percentage inhibition.
4. Experimental
4.1. General Chemistry
All chemicals and solvents were obtained from commercial
suppliers and were used without further purification. Melting
points were determined on an electro thermal melting point
apparatus (Buchi BM530) in open capillary tubes and are
uncorrected. All reactions were monitored by thin-layer
chromatography (TLC on alluminium plates coated with silica
gel 60 F254, 0.25 mm thickness, E. Merck, Mumbai-India) and
detection of the components were measured under UV light or
explore in Iodine chamber. IR spectra for the compounds were
recorded using FTIR Perkin Elmer Spectrum 100 spectrometer as
KBr pellets with absorption in cm−1. 1H and 13C NMR
measurements were carried out on Avance-II 400 Bruker NMR
spectrometer. Chemical shifts are expressed in δ (ppm) relative to
tetramethylsilane. Elemental analysis was performed on a
Thermo Scientific (FLASH 2000) elemental analyzer. Mass
analysis was performed on Waters Micromass Q-Tof Micro,
mass spectrometer.
% Inhibition =
4.3. Cell viability by MTT assay
The determination of the cytotoxicity of the synthesized
compounds was determined by MTT assay against Vero cell
using Promega CellTiter 96 Non-radioactive Cell Proliferation
Assay (Promega, Madison, WI, USA). The cellular viability was
assessed on basis of conversion of MTT into formazan by the
Vero cells after 72 hours of incubation at 37 °C. Further the
activity was determined by measuring the absorbance at 540 nm
followed by the calculating the percentage cell viability.17
Percentage cell viability =
Where, Ao = Absorbance of cells treated with 0.1% DMSO
medium, At = Absorbance of cells treated with various
concentration of the samples.
4.1.1. Typical procedure for synthesis of 1,3-
diphenyl-1-H-pyrazole-4-carbaldehyde derivatives
and pyridin-2-yl-3-oxobutanamide derivatives
Each treatment was performed in triplicate and the 50%
inhibitory concentration (IC50) of each compound was obtained
using the Graph Pad Prism program, version 3 (San Diego, CA).
The divergent substituted acetoacetanilide derivatives
retaining electron donating and electron withdrawing group were
synthesized by refluxing corresponding substituted 2-
aminopyridine
with
ethylacetoacetate
and
4.4. Computational studies and ADMET prediction method
methylisobutyrylacetate respectively under microwave irradiation
(Scheme S2).11 Proceedingly, 1,3-diarylpyrazole-4-carbaldehyde
derivatives were prepared following a two step protocol
involving treatment of corresponding hydrazones formed from
various substituted acetophenones and phenylhydrazine by excess
Vilsmeier reagent (Scheme S1) as reported by Yadlapalli et al.11
The molecular structures of all the compounds were drawn
using ChemBioDraw Ultra 14.0 (www.cambridgesoft.com).
These structures were then imported into Maestro implemented in
Schrödinger, further energy of the 3D structures was minimized
using Ligprep 3.3 module. The crystal structure of InhA (PDB
ID: 2B35) was extracted from protein data bank (www.rcsb.org).
The refining of the protein structure and addition of hydrogen
atoms to the structure was accomplished utilizing Protein
Preparation Wizard (Maestro 10.1 Schrödinger, LLC, New York,
NY, 2015). The binding site in the protein molecules was
4.1.2. Typical procedure for synthesis of 7-(1,3-
diphenyl-1H-pyrazol-4-yl)-5-methyl-N-(pyridin-2-
yl)-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidine-6-
carboxamide (J1) and related compounds (J2-J30)
6

predicted using Sitemap 3.4 wizard and further the center of the
grid was defined, which was generated using Glide 6.6
(Schrödinger, LLC, New York, NY, 2015) with default settings
for all parameters. The grid size was kept sufficiently high so as
to include the atoms participating in the interaction and then all
the compounds were docked against the grid of prepared
receptors (mutant InhA) using Glide in XP (Extra Precession)
mode18 with other default setting for scoring function.
Scheme S1-S2, Table S1, Experimental data and Figure S1-S7
are available in supplementary information.
References and notes
1.
2.
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scourge to a curable disease-journey over a millennium. The
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Snider, D.; Raviglione, M.; Kochi, A.; Bloom, B., Tuberculosis:
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The pharmacokinetic profile of the test compounds showing
good antitubercular activity were predicted by using programs
Qikprop v4.3 (Schrödinger, Inc., New York, NY, 2015). The
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calculation of pharmacokinetic parameters by QikProp v4.3. The
program QikProp v4.3, utilizes the method of Jorgensen19 to
3.
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4.5. Enzyme Inhibition Assay
Spectrophotometric method was utilized to assay the
inhibition of Wild type enoyl-ACP reductase (InhA) procured by
over expression of gene cloned in pET-28a(+) vector transformed
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with the oxidation of NADH to NAD+, which is monitored at
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activity of the enzyme at various concentrations of these
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corresponds to the concentration of the compound that inhibited
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Acknowledgments
We articulate our appreciation to authorities of V. P. & R. P.
T. P. Science College, Sardar Patel University, Vallabh
Vidyanagar (Gujarat-India) for providing infrastructural &
chemical facilities. We forward our gratefulness to DST-Delhi
for providing financial aid to the author Jaimin D. Bhatt in form
of INSPIRE fellowship (IF120757). The support of Dr. Raghu R.
and the team Schrodinger (Bangalore) for Maestro‐2015‐1 is
greatly lauded. We are thankful to the Director SAIF, Chandigarh
for spectroscopic analysis facility. The support of Director,
SICART, Vallabh Vidyanagar is appreciated for elemental
analysis and IR spectroscopy.
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8