Discovery of rimonabant and its potential analogues as anti-TB drug candidates

Article abstract of DOI:10.1007/s00044-015-1346-4

Rimonabant and its analogues have been synthesized in moderate to good yields using a simple synthetic route. All the newly synthesized compounds were subjected to in vitro screening against M. tuberculosis and M. smegmatis. The most potent analogue JMG-14 exhibits MIC value of 3.13 compared to 3.25 and 50 μ/ml for ethambutol and pyrazinamide, respectively. The molecular docking reveals that pyrazole ring, number and position of halogen atoms play a crucial role in deciding interactions with MTCYP-121. These findings open up a new avenue in the search of potent anti-TB drugs with rimonabant and its novel analogue JMG-14 as lead molecules.

Full text of DOI:10.1007/s00044-015-1346-4

Med Chem Res
DOI 10.1007/s00044-015-1346-4
ORIGINAL RESEARCH
Discovery of Rimonabant and its potential analogues as anti-TB
drug candidates
•
J. M. Gajbhiye N. A. More Manoj D. Patil R. Ummanni
•
•
•
•
S. S. Kotapalli P. Yogeeswari D. Sriram V. H. Masand
•
•
Received: 25 September 2014 / Accepted: 19 February 2015
Ó Springer Science+Business Media New York 2015
Abstract Rimonabant and its analogues have been syn-
thesized in moderate to good yields using a simple syn-
thetic route. All the newly synthesized compounds were
subjected to in vitro screening against M. tuberculosis and
M. smegmatis. The most potent analogue JMG-14 exhibits
MIC value of 3.13 compared to 3.25 and 50 lg/ml for
ethambutol and pyrazinamide, respectively. The molecular
docking reveals that pyrazole ring, number and position of
halogen atoms play a crucial role in deciding interactions
with MTCYP-121. These findings open up a new avenue in
the search of potent anti-TB drugs with rimonabant and its
novel analogue JMG-14 as lead molecules.
Graphical Abstract
Keywords Rimonabant ꢀ Diaryl pyrazoles ꢀ
Tuberculosis ꢀ Mycobacterium tuberculosis ꢀ H37Rv ꢀ
MTCYP-121
J. M. Gajbhiye (&) ꢀ N. A. More ꢀ M. D. Patil
Division of Organic Chemistry, CSIR-National Chemical
Laboratory, Pune 411008, India
e-mail: jm.gajbhiye@ncl.res.in
Introduction
R. Ummanni ꢀ S. S. Kotapalli
Centre for Chemical Biology, Indian Institute of Chemical
Technology, Hyderabad 500607, India
Tuberculosis (TB) is among the deadly diseases afflicting
mankind, and it still remains a major public health burden in
many developing countries (Corbett et al., 2003). Ap-
proximately one-third of the world’s population is still under
the ill influence of TB. In 2011, nearly 9 million people
around the world suffered from TB (WHO 2013), with 1.4
million deaths worldwide. Recent reports reveal that every
year 0.2 million from 0.7 million HIV patients die due to TB,
P. Yogeeswari ꢀ D. Sriram
Tuberculosis Drug Discovery Laboratory, Pharmacy Group,
Birla Institute of Technology and Science, Pilani, Hyderabad
Campus, Hyderabad 500078, India
V. H. Masand
Department of Chemistry, VidyaBharati College, Camp,
Amravati 444602, India
123

Med Chem Res
and thus, TB is a leading killer in HIV-infected patients
(Nunn et al., 2005; Masand et al., 2012; Shukla et al., 2014).
Emerging resistance to the marketed anti-tuberculosis drugs
is a major concern that threatens progress made in TB care
and control worldwide (Shukla et al., 2014). In most of the
cases, drug resistance (Chiang and Schaaf, 2010) arises due
to the improper use of antibiotics in chemotherapy, admin-
istration of improper treatment regimens and failure to en-
sure that patient completes the whole course of the treatment
(Elzinga et al., 2004; Kumar et al., 2013). To combat the
continuous rise in multidrug-resistant TB, scientific efforts
are concentrated on inventing new agents for the treatment of
TB. Despite the efforts executed in laboratories and hospi-
tals, TB is nevertheless a major challenge to medicinal
chemists.
Developing a new drug is a long, expensive and tedious
process, often consuming several years and resources. In
addition, the regulatory approval process following the drug
discovery/design involves several tests and trials. Even after
completing the whole process of drug development, certain
drugs such as celecoxib, sibutramine, rimonabant, though
approved by the regulatory agencies, were withdrawn from
the market due to the appearance of the side effects. At the
current stage, it would be a highly beneficial and attractive
strategy to identify a drug candidate, which has been ap-
proved as a drug by regulatory authorities, effective for the
treatment of TB. This would enable a quicker introduction
of the drug into the market for TB treatment. In the recent
years, azoles viz. pyrazoles, imidazoles and triazoles have
attracted the attention of researchers. Some clinically used
drugs such as clotrimazole, fluconazole and voriconazole,
which contain azole moiety, can be used to treat generalized
systemic mycoses. Literature survey reveals that selected
azole drugs possess potent anti-mycobacterial activity in the
nanomolar range (Kumar et al., 2013; Menozzi et al., 2004)
(Fig. 1).
Fig. 1 Azole and pyrazole derivatives possessing anti-tubercular
activity
N
HN
O
N
N
l
C
l
C
It has been established (McLean et al., 2002; Hudson
et al., 2012) that many azole compounds possessing potent
anti-tubercular activity bind to MT-CYP51 and MT-
CYP121, proteins with crucial role in metabolism of M. tu-
berculosis, possessing high affinity and retard the growth of
Mycobacterium bovis and Mycobacterium smegmatis, the
two mycobacterial species which closely resemble M. tu-
berculosis. In recent years, cytochrome P450 (CYP121) has
gained popularity as an attractive target for designing potent
azole-based drugs against M. tuberculosis, a plausible reason
is that MT-CYP121, which has unique catalytic action, binds
commercially available azole drugs with a higher affinity
than MT-CYP51 (Menozzi et al., 2004). In addition, it has no
equivalent enzyme in humans. MT-CYP121 is necessary for
bacterial viability since it catalyzes an unusual in-
tramolecular carbon–carbon coupling reaction (Dumas
et al., 2013). The high affinity of the azole derivatives such as
l
C
Fig. 2 2D-structure of rimonabant
clotrimazole for MT-CYP121 is attributed to their bulky,
polycyclic structure, enhancing favorable interactions with
the hydrophobic residues in the largely non-polar active site
of the enzyme. Moreover, in many studies, the presence of
halogen attached to aromatic moiety has been found to
escalate antifungal and anti-mycobacterial activities (Dumas
et al., 2013; Sundaramurthi et al., 2011; Belin et al., 2009).
Rimonabant (1) (Fig. 2), a pyrazole carboxylic acid deriva-
tive discovered by Sanofi-Synthelabo in 1994 as the first
potent, orally active, selective CB1 cannabinoid receptor
antagonist and sold under several trade names as an anorectic
123

Med Chem Res
anti-obesity drug. Rimonabant and its analogues were also
shown to have promising activities and also proved safe to
human (Lange and Kruse, 2005). It was recently withdrawn
(Kang and Park, 2012) from the market following post-
marketing surveillance studies, which confirmed a risk of
depressive disorders among users. However, modification or
elimination of the side chain attached to carboxamide
(Jagerovic et al., 2008) which is responsible for crossing the
blood–brain barrier might reduce the side effects. There are
several important anti-TB drug candidates such as PA-824
and BM212, which are structurally reminiscent to ri-
monabant 1, and hence, we were curious to study its anti-TB
properties. All these observations prompted us to develop
new rimonabant analogues that may prove effective against
multidrug-resistant strains of the tuberculosis pathogen.
amidation was executed to afford the target compounds 1,
4, 5, 6, 10, 14, 15, 16, 17, 18 and 19 in moderate to good
yields (Schemes 2, 3, 4). The structures of newly synthe-
sized compounds were established on the basis of spectral
and physicochemical analyses.
Results and discussion
Our initial study involved screening of rimonabant 1 against
M. smegmatis. Interestingly, it exhibited a MIC value of
13.56 compared to 16 lg/ml for isoniazid. Therefore, its 18
analogues were synthesized (Lan et al., 1999; Kotagiri et al.,
2007), and few of them were screened against M. smegmatis.
They showed moderate to high activity (Table 1). Encour-
aged by the results, rimonabant and all the synthesized
analogues were then screened for Mycobacterium tubercu-
losis (virulent strain H37Rv) in vitro (Moore et al., 1999).
The details of the screening data are shown in Table 1.
The precursor of rimonabant acid 2 showed improved
MTB activity and ester 3 retains the activity as compared to
rimonabant. Similarly, the analogues 2, 4, 7, 11 and 15–18
exhibited enhanced activity, and the analogues 3, 5, 6, 8, 10,
12 & 19 were also found to be active. Most importantly, the
analogues 9 and 13 showed very good MTB activity. The
analogue JMG-14 came out to be a promising lead with
highest activity. Compared with one of the first-line anti-TB
drug ethambutol (MIC 3.25 lg/ml), the analogue JMG-14
was found to be equally active. When compared to pyr-
azinamide (MIC 50.0 lg/ml), all the 18 analogues were
found to be more potent. The analogue 13 also emerged as
another lead molecule, which can be explored further.
Chemistry
The synthesis of compounds 2, 3, 7, 8, 9, 11, 12, and 13
was carried out as outlined in the Scheme 1. 4-Chloro-
propiophenone (A) on condensation with diethyl oxalate in
the presence of LiHMDS gave lithium salt of ethyl 4-(4-
chlorophenyl)-3-methyl-4-oxydo-2-oxobuten-3-oate (B).
This on condensing with 2, 4-dichlorophenyl hydrazine
hydrochloride and phenyl hydrazine hydrochloride in
ethanol gave hydrazones, which on refluxing in acetic acid
for 24 h yielded esters 3 and 8, respectively. Subsequent
reaction of B with suitable hydrazines in 50 % sulfuric acid
and ethanol at 79 °C for 14 h yielded desired acids (2, 7, 9,
11, 12 and 13). These acids on subsequent treatment with
thionyl chloride afforded acid chlorides. Finally, the
Li
CO2H
O
O
O
a
b
CO2Et
N
N
X
X
X
R1
B: X= Cl
C: X= H
A
2: X=Cl, R1=2,4-dichlorophenyl
7: X=Cl, R1=phenyl
X=H or Cl
c
9: X=H, R1=2,4-dichlorophenyl
11: X=Cl, R1=H
O
N
12: X=Cl, R1=2-chlorophenyl
13: X=Cl, R1=2,4-difluorophenyl
O
N
X
R1
3: X=Cl, R1=2,4-dichlorophenyl
8: X=Cl, R1=phenyl
Scheme 1 Reagents and conditions: (a) LiHMDS, diethyl oxalate, methyl cyclohexane, 15–25 °C, 6th; (b) R1-NH-NH2ꢀHCl, 50 % aq. H2SO4,
EtOH, 79 °C, 14th; (c) EtOH, rt, 20 h, then AcOH, reflux, 24 h
123

Med Chem Res
O
N
CO2H
R2
a
b
N
N
N
X
X
R1
R1
HN
N
1: X=Cl, R1=2,4-dichlorophenyl, R2=
4: X=Cl, R1=2,4-dichlorophenyl, R2=
O
O
N
N
NH
N
HN
NH
O
5: X=Cl, R1=2,4-dichlorophenyl, R2=
6: X=Cl, R1=2,4-dichlorophenyl, R2=
Cl
N
N
O
CH3
17
N
O
N
N
NH
O
10: X=H, R1=2,4-dichlorophenyl, R2=
14: X=Cl, R1=phenyl, R2=
N
HN
N
NH
N
Cl
HN
N
Cl
15: X=Cl, R1=2-chlorophenyl, R2=
HN
16: X=Cl, R1=2,4-difluorophenyl, R2=
18: X=H, R1=2,4-dichlorophenyl, R2=
19
Cl
N
N
CH3
HN
N
Scheme 2 Reagents and conditions: (a) thionyl chloride. DMF (cat.) toluene. 100 °C, 4 h (b) Et3N sutiable amide. 0°-rt. 12 h
Scheme 3 Reagents and
conditions: (a) LiHMDS,
diethyl oxalate, methyl
cyclohexane, 15–25 °C, 6h;
(b) NH2OHꢀHCl, 50% aq.
H2SO4, EtOH, 79 °C, 14h;
(c) thionyl chloride, DMF (cat),
toluene, 100 °C, 4th; (d) Et3N,
1-aminopiperidine, 0°-rt, 12 h
Li
O
O
O
a
CO2Et
Cl
Cl
B
A
b
O
O
COOH
NH
N
N
O
c
N
d
Cl
17
Cl
17a
123

Med Chem Res
Scheme 4 Reagents and
conditions: (a) LiHMDS,
diethyl oxalate, methyl
Li
O
O
O
cyclohexane, 15–25 °C, 6h;
(b) 2,4-dichlorophenylhy-
drazine. HCl, 50% aq. H2SO4,
EtOH, 79 °C, 14 h; (c) thionyl
chloride, DMF (cat), toluene,
100 °C, 4 h; (d) Et3N,
a
CO2Et
Cl
Cl
C
A'
1-aminopiperidine, 0°-rt, 12 h
b
N
COOH
O
NH
N
N
c
N
Cl
N
Cl
Cl
d
Cl
Cl
19a
Cl
19
Table 1 In vitro anti-mycobacterial activity of rimonabant (1) and synthesized analogues
S. No.
CLogPb
% inhibition
M. Smegmatis
MIC (lg/ml)
S. No.
CLogPb
% inhibition
M. Smegmatis
MIC (lg/ml)
M. TB
M. TBa
1
6.471
6.121
7.083
5.532
5.666
5.335
4.681
5.234
5.407
4.818
3.035
5.404
99.33 MIC 13.56 lg/ml
25
13
4.981
5.031
5.754
4.526
3.342
5.756
6.841
NT
6.25
3.13
12.5
12.5
12.5
12.5
25
2
22.84
14.07
19.21
14.50
NT
12.5
25
14
99.70
NT
3
15
4
12.5
25
16
NT
5
17
NT
6
[25
12.5
25
18
32.37
NT
7
17.70
22.70
15.73
1.362
NT
19
8
Isoniazid
Rifampicin
Ethambutol
Pyrazinamide
16 lg/ml
2 lg/ml
NT
0.05
0.1
9
6.25
25
10
11
12
3.25
50.0
12.5
25
NT
NT
NT not tested
a
Mycobacterium tuberculosis H37Rv
b
Calculated LogP using ChemDraw 12
Based on the analogue 9, a simple replacement of chlorine
by hydrogen might provide a more active analogue of 13.
The main purpose of this study was to introduce
modification on the side chain on carboxamide moiety of
rimonabant and its analogues to develop new potential
anti-TB agents. In the present work, diverse functional
groups were introduced on the side chain of the carbox-
amide moiety of rimonabant to identify structure–activity
relationships. It appears that the piperidinyl side chain is
not must for high activity and derivatives with free acids
are better candidates for further evaluation. Variation of
phenyl ring attached to nitrogen (of pyrazole moiety)
shows prominent effect on the activity, and this obser-
vation is further vindicated by the docking analysis. The
substitution on the ring or its complete removal as well as
replacement of that nitrogen with oxygen shows positive
effect. Alteration on the p-chlorophenyl ring present on
central pyrazole also influences the bioactivity. The
methyl group seems to be important for the activity, since
the analogue without methyl shows diminished activity.
123

Med Chem Res
Fig. 3 Docking pose for
a rimonabant (compound 1) and
b most active compound 14
Docking analysis
Thr-77, Val-82, Val-83, Asn-85, Met-86, Ala-167, Ph-168,
Thr-229, Ala-233, Ser-237, Gln-385 and Arg-386. The cat-
alytic site is located on top of the distal side of the heme,
where oxygen binds, near to I-helix (Dumas et al., 2013;
Sundaramurthi et al., 2011; Belin et al., 2009).
Molecular docking analysis was performed to determine the
structural features that steer the biological profile of Ri-
monabant and its novel analogues. As stated earlier (Dumas
et al., 2013; Sundaramurthi et al., 2011; Belin et al., 2009),
the mechanism of action for anti-TB activity involves in-
teraction of pyrazoles with MTCYP-121; therefore, mole-
cular docking analysis was performed to determine the
structural features that govern the anti-TB of present series
molecules. The docking analysis was performed for all the
compounds, but for the sake of convenience, we represent the
docking poses for compounds 1 and 14 as representatives.
The active site of MT-CYP121, a heme-based enzyme that
transfers a single oxygen atom to the substrate, comprises of
According to the results of our docking experiments (see
Fig. 3), the prominent interactions between compound 1
and the receptor are van der Waals, H-bonding and hy-
drophobic in nature. The compound 1 could adopt bent ‘T’
shape (or weird propeller shape) while interacting with the
3
˚
cytochrome catalytic site (1350 A in size) orienting chloro
atom attached to benzene ring 1 toward the iron atom of the
heme group with the heterocyclic rings (pyrazole and
piperidine) pointing toward polar residues. Binding of
compound 1 involves numerous van der Waals contacts
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Fig. 4 Summary of SAR and molecular docking analysis for anti-TB activity of rimonabant and its analogues
with the hydrophobic side chains of Leu-76, Val-78, Val-
83, Ala-167 and Phe-168. There are water-mediated
H-bond network with the N (non-substituted) of pyrazole
ring and with N (amide group). Furthermore, a strong
H-bond between Arg-72 with N (piperidine ring) enhances
the anchoring of compound 1 with CYP-121. The presence
of pyrazole moiety in close proximity to Gln-385 indicates
that it plays a crucial role in inhibiting the normal func-
tioning of CYP-121.
do not coordinate with the heme iron, as is typically ob-
served for the azole antifungal CYP inhibitors (Hudson
et al., 2012). Our docking experiment also concurs with this.
˚
The N (d-)/N–H (d?) with a distance of 2.20 A (depicted in
Fig. 3), acting as polar pharmacophore, are responsible for
establishing the polar contacts with the receptor.
The molecular docking and structure–activity relation-
ships (SAR) have been summarized in Fig. 4.
Further studies being carried out in our laboratory to
explore the mechanism of action of this scaffold and
evaluation of these compounds for anti-bactericidal activity
as well as activity under alternate growth conditions such
as non-replicating growth conditions.
Compound 14, like compound 1, adopts bent ‘T’ shape
while interacting with CYP-121, but interacts with more
number of residues of the active site than the compound 1.
The ring 2 is closer to heme moiety with ring 1 pointing
toward hydrophobic residues Val-78, Phe-168, Trp- 182 and
Val-228. Another remarkable difference in interaction pat-
tern of compounds 1 and 14 is due to the involvement of one
and two water molecules, respectively, in enhancing the
polar interactions with the receptor. The binding between the
receptor and the substrate is enhanced by the water-mediated
H-bonding between Thr-77 and the oxygen atom of amide
group as well between Arg-72 and N (piperidine moiety).
A plausible reason for bent ‘T’ shape for compounds 1
and 14 is the presence of bulky Ser-237 in close proximity to
phenyl ring attached to N of pyrazole moiety. Another rea-
son could be the presence of unusually close other I-helix
residues above the heme. Compounds 1 and 14 have adopted
exactly opposite conformations while interacting with the
receptor, and this could be attributed to the steric repulsion
exerted by bulkier -Cl atoms present as substituents on the
phenyl ring. Hudson et al. recently reported that the azoles
Pharmacology
Anti-mycobacterial activity assay
Anti-mycobacterial activity of the synthesized compounds
was performed against Mycobacterium smegmatis MC2155
strain using growth inhibition assay by agar dilution fol-
lowed by turbidimetry method. The assay was semi-
throughput and conducted in a 96-well plate (sterile). For
M. tuberculosis, H37Rv strain was utilized by performing a
growth inhibition assay from fresh Middlebrook 7H11 agar
slants. This method is similar to that recommended by the
National Committee for Clinical Laboratory Standards
(NCCLS, 1995) for the determination of minimum in-
hibitory concentration (MIC) in duplicate.
123

Med Chem Res
precipitate was obtained. The precipitate so obtained was
filtered and washed with ethanol (10 ml) and dried under
vacuum to give a pale yellow solid (1.1 g), which was
dissolved in acetic acid (10 ml) and refluxed for 24 h. On
completion of the reaction (TLC), the reaction mixture was
poured into cold water (20 ml) and then extracted with
ethyl acetate (3 9 15 ml). The combined extracts were
washed with water, saturated sodium bicarbonate solution,
brine and then dried under vacuum to give the crude
product. It was then purified by column chromatography on
silica gel using ethyl acetate/petroleum ether (1:9) as elu-
tion system to give the ester 3 (Kotagiri et al., 2007),
(670 mg, 45 %,) pale yellow solid.mp 128–129 °C;1H
NMR (CDCl3, 200 MHz): d 7.39 (d, J = 2.02 Hz, 1H),
7.34 (d, J = 1.37 Hz, 1H), 7.33–7.28 (m, 3H), 7.08 (d,
J = 8.59 Hz, 2H), 4.51–4.41 (q, J = 7.08 Hz, 2H), 2.34 (s,
3H), 1.43 (t, J = 7.08 Hz, 3H); 13C NMR (CDCl3,
100 MHz): d 162.7, 142.9, 136.0, 135.9, 135.0, 133.0,
130.9, 130.7, 130.1, 128.9, 127.7, 127.0, 119.1, 60.9, 14.4,
and 9.6.HRMS-ESI (m/z) Calcd. C19H15O2N2Cl3 ? H)?:
409.0272 found: 409.0265.
Conclusion
For the first time, it has been shown that rimonabant could
be a lead molecule for finding more potent anti-TB drug
candidates. In the present work, JMG-14 is the most potent
analogue of rimonabant, emerging as a suitable lead
molecule, which can be further optimized using the SAR
and molecular docking analyses. Currently, research work
is in progress on the development of more rational ana-
logues and their anti-TB.
Experimental
Chemistry
All the reagents and the solvents were used as received
from commercial sources unless and otherwise stated. All
the experiments were carried out under an atmosphere of
nitrogen in round-bottom flask. Pre-coated plates (silica gel
60 PF254, 0.25 mm or 0.5 mm) were utilized for thin-layer
chromatography (TLC). Column chromatographic purifi-
cations were carried out on flash silica gel (100–200 mesh)
using either petroleum ether and ethyl acetate or dichlor-
omethane and methanol as eluents. Melting points (mp) are
uncorrected and were measured on an electrothermal
melting point apparatus. The IR spectra were recorded on
an FT-IR spectrometer. The 1H and 13C NMR spectra were
recorded on 200/400/500 MHz and 50/100/125 MHz NMR
spectrometer, respectively, in CDCl3/DMSO-d6. Mass
spectra were taken on LC–MS (ESI) mass spectrometer.
HRMS were scanned at NCL, Pune.
Preparation of 1-(5-(4-Chlorophenyl)-4-methyl-1-phenyl-
1H-pyrazol-3-yl)propan-1-one (8) Synthesis of 8 was ac-
complished according to the procedure applied for 3. Pale
1
orange solid; 595 mg, 48 %; mp 121–123 °C; H NMR
(CDCl3, 400 MHz): d 7.31–7.36 (m, 5H), 7.24–7.27 (m, 2H),
7.10 (d, J = 8.6 Hz, 2H), 4.48 (q, J = 14.14 Hz, 2H), 2.33
(s, 3H), 1.46 (t, J = 7.07 Hz, 3H); 13C NMR (CDCl3,
100 MHz): d 163.0, 142.3, 140.9, 139.3, 134.7, 131.3, 128.9,
128.0, 127.9, 127.0, 125.4, 120.0, 60.8, 14.4, 9.7; HRMS-
ESI (m/z) Calcd. C19H17O2N2ClNa: 363.0871 found:
363.0868.
Preparation of Lithium salt of Ethyl 4-(4-chlorophenyl)-3-
methyl-4-oxydo-2-oxobuten-3-oate (B) Methylcyclohex-
ane (30 ml) and lithium hexamethyldisilazane (30 ml,
31.7 mmol) were introduced in a round-bottom flask under
nitrogen atmosphere and cooled to 15–25 °C. A solution of
4-chloropropiophenone (A, 5.0 g, 29.65 mmol) in
methylcyclohexane (12.5 ml) was slowly added with con-
tinuous stirring for 2.5 h. To this reaction mixture, diethyl
oxalate (4.78 g, 32.6 mmol) was added with stirring. The
reaction progress was monitored by TLC; the solid ob-
tained was filtered, washed with methylcyclohexane
(30 ml) and dried under vacuum (30 min) to afford the
lithium salt (Kotagiri et al., 2007) (Cream yellow solid,
5.7 g, 71.9 %).
Preparation of 5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-
4-methyl-1H-pyrazole-3-carboxylic acid (2) A mixture of
lithium salt of ethyl 4-(4-chlorophenyl)-3-methyl-4-oxydo-
2-oxobuten-3-oate (B, 1.0 g, 3.6 mmol), ethanol (25 ml),
2,4-dichlorophenyl hydrazine hydrochloride (0.777 g,
3.6 mmol), and 50 % sulfuric acid (10 ml) was refluxed for
6 h. After the reaction was complete (TLC), ethanol was
removed under reduced pressure, and again, a second in-
stallment of 50 % sulfuric acid (20 ml) was added, fol-
lowed by refluxing for 8 h. The reaction mixture was
cooled to room temperature (35 °C) and was poured onto
crushed ice, stirred for 15 min, filtered and washed with
water (20 ml). The wet solid so obtained was stirred with
water (30 ml), and the pH was adjusted to [10 with 20 %
dil. NaOH. This aqueous layer was washed with petroleum
ether. The aqueous layer was separated, cooled to 0 °C, and
pH was adjusted to 2.0 by concentrated hydrochloric acid.
Solid so obtained was filtered, washed with water (100 ml)
and dried to afford 2 (Kotagiri et al., 2007), (0.923 mg,
65 %), Off white solid, mp 208–209 °C; 1H NMR (CDCl3,
Preparation of ethyl 5-(4-Chlorophenyl)-1-(2,4-dichlor-
ophenyl)-4-methyl-1H-pyrazole-3-carboxylate (3) To a
continuously stirring solution of lithium salt B (1.0 g,
3.64 mmol) in 15 ml of ethanol was added 2,4-dichlor-
ophenyl hydrazine hydrochloride (0.777 g, 3.64 mmol) at
room temperature. The shaking was carried for 20 h till
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Med Chem Res
400 MHz): d 7.44–7.41 (m, 1H), 7.35 (d, J = 1.89 Hz,
1H), 7.33–7.28 (m, 3H), 7.09 (d, J = 8.47 Hz, 2H), 2.36 (s,
3H); 13C NMR (CDCl3, 100 MHz): d 166.4, 143.3, 136.2,
135.6, 135.1, 133.5, 130.8, 130.5, 129.8, 128.9, 127.8,
127.7, 126.7, 119.6, 9.6; HRMS-ESI (m/z) Calcd.
C17H11O2N2Cl3Na: 402.9778 found: 402.9781.
6.72 (s, 1H), 2.25 (s, 3H); 13C NMR (CDCl3, 125 MHz): d
161.7, 157.5, 155.5, 143.1, 135.0, 130.7, 130.0, 128.9, 127.0,
123.7, 119.5, 112.1, 111.9, 104.8, 9.6; HRMS-ESI (m/z)
Calcd. C17H11O2N2ClF2Na: 371.0369 found: 371.0369.
Procedure for the synthesis of 5-(4-Chlorophenyl)-1-(2,4-
dichlorophenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-
3-carboxamide 1 To a stirred solution of 2 (382 mg;
1.0 mmol) in toluene (10 ml), one drop of dimethyl for-
mamide was added. The reaction mixture was cooled to
0 °C and thionyl chloride (140 mg: 1.2 mmol) in 2 ml
toluene was added dropwise for the period of 2 min at the
same temperature. The reaction mixture was allowed to
attain room temperature and heated at 100 °C for 4 h.
Excess of thionyl chloride and toluene was distilled off
under reduced pressure. In another flask under nitrogen
atmosphere was introduced 1-aminopiperidine (100 mg;
1.0 mmol) and triethyl amine (101 mg; 1.0 mmol) in
5.0 ml dichloromethane. The flask was cooled to 0 °C. To
this was added a cooled solution of acid chloride dropwise
at the same temperature. The resulting reaction mixture
was allowed to attain room temperature, and then, it was
stirred for 12 h. After completion of the reaction (mon-
itored by TLC), the reaction mixture was diluted with
water (10 ml) and organic layer was separated, washed
with water (2 9 5 ml) and brine solution (5 ml), dried over
Na2SO4 and concentrated in vacuo. The residue was puri-
fied by column chromatography over silica gel (ethyl ac-
etate/petroleum ether 1:9 (v/v)) afforded pure product.
Preparation of 5-(4-Chlorophenyl)-4-methyl-1-phenyl-1H-
pyrazole-3-carboxylic acid (7) Compound 7 was synthe-
sized by the procedure applied for 2. Pale brown solid:
719 mg, 58 %; mp 203–204 °C; 1H NMR (CDCl3,
200 MHz): d 7.46 (s, 1H), 7.32–7.38 (m, 5H), 7.30–7.20
(m, 2H), 7.13 (d, J = 6.36 Hz, 2H), 2.35 (s,3H); 13C NMR
(CDCl3, 400 MHz): d 165.9, 141.3, 140.9, 139.0, 134.9,
131.3, 129.0, 128.2, 127.6, 125.4, 125.1, 120.5, 9.6;
HRMS-ESI (m/z) Calcd. C17H13O2N2ClNa: 335.0571
found: 335.0566.
Preparation of 1-(2,4-dichlorophenyl)-4-methyl-5-phenyl-
1H-pyrazole-3-carboxylic acid (9) Compound 9 was
synthesized by the procedure applied to 2. Cream solid:
865 mg, 59 %; mp 188–189 °C; 1H NMR (CDCl3,
400 MHz): d 7.46 (s, 1H), 7.16–7.27 (m, 5H), 7.08 (s, 2H),
6.02 (s, 1H), 2.17 (s, 3H); 13C NMR (CDCl3, 100 MHz): d
167.2, 145.6, 136.0, 135.1, 132.2, 131.2, 130.8, 129.6, 129.1,
128.5, 128.3, 128.2, 127.8, 118.0, 9.7; HRMS-ESI (m/z)
Calcd. C17H12O2N2Cl2Na: 369.0168 found: 369.0164.
Preparation of 5-(4-chlorophenyl)-4-methyl-1H-pyrazole-
3-carboxylic acid (11) Compound 11 was synthesized by
the procedure applied to 2. Cream solid; 387 mg, 45 %; mp
White solid: 292 mg, 63 %; mp 182–183 °C;1H NMR
(CDCl3, 400 MHz): d 7.63 (s,1H), 7.41–7.36 (m, 1H),
7.30–7.26 (m, 3H), 7.04 (d, J = 8.28 Hz, 2H), 2.93–2.78
(m, 4H), 2.35 (s, 3H), 1.78–1.70 (m, 4H), 1.48–1.37 (m,
2H);13C NMR (CDCl3, 100 MHz): d 159.9, 144.4, 142.9,
136.0, 135.9, 134.9, 132.9, 130.8, 130.6, 130.3, 128.9,
127.9, 127.2, 118.2, 57.0, 25.4, 23.3, 9.3; HRMS-ESI (m/z)
Calcd. C22H22ON3Cl3 ? H)?: 463.0854 found:463.0853.
1
288–290 °C; H NMR (DMSO-d6, 500 MHz): d 13.54 (s,
1H), 7.89, (d, J = 8.24 Hz, 2H), 7.77 (d, J = 8.24 Hz,
2H), 2.61 (s, 3H); 13C NMR (DMSO-d6, 125 MHz): d
162.4, 132.7, 130.9, 129.4, 128.9, 128.5, 117.0, 60.5, 9.9;
HRMS-ESI (m/z) Calcd. C11H9O2N2ClNa: 259.0245
found: 259.0243.
Preparation of 1-(2-chlorophenyl)-5-(4-chlorophenyl)-4-
methyl-1H-pyrazole-3-carboxylic acid (12) Compound
12 was synthesized by the procedure applied to 2. Pale
N0-(5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-
1H-pyrazole-3-carbonyl) isonicotinohydrazide (4) Com-
pound 4 was synthesized according to the procedure ap-
plied to 1. Off white solid: 499 mg, 59 %; mp 237–239 °C;
1H NMR (CDCl3, 400 MHz): d 10.33 (s,1H), 9.44 (s, 1H),
8.71 (s, 1H), 7.74 (s, 2H), 7.41 (s, 1H), 7.30–7.32 (m, 5H),
7.05 (d, J = 8.53 Hz, 2H), 2.17 (s, 3H); 13C NMR (CDCl3,
100 MHz): d 163.5, 161.2, 150.2, 143.3, 142.4, 136.2,
135.6, 135.2, 132.7, 130.7, 130.5, 130.3, 128.9, 128.8,
127.9, 126.7, 118.4, 30.9, 9.2; HRMS-ESI (m/z) C23H16-
O2N5Cl3 ? H)?: Calcd. 500.0442 found: 500.0443.
1
yellow solid: 656 mg, 52 %; mp 204–206 °C; H NMR
(CDCl3, 200 MHz): d 7.33–7.42 (m, 4H), 7.26 (d,
J = 6.41 Hz, 2H), 7.04 (d, J = 7.79 Hz, 2 H), 2.32 (s,
3H);13C NMR (CDCl3, 100 MHz): d 165.6, 143.3, 141.7,
136.9, 134.9, 131.9, 130.9, 130.3, 130.0, 129.8, 128.8
127.5, 126.9, 119.4, 9.6; HRMS-ESI (m/z) Calcd.
C17H12O2N2Cl2Na: 369.0168 found: 369.0168.
Preparation of 5-(4-chlorophenyl)-1-(2,4-difluorophenyl)-
4-methyl-1H-pyrazole-3-carboxylic acid (13) Compound
13 was synthesized by the procedure applied to 2. Pale brown
(5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-
pyrazol-3-yl)(4-methylpiperazin-1-yl) methanone (5) Com-
pound 5 was synthesized according to the procedure ap-
plied to 1. Brown solid; 306 mg, 50 %; mp 111–113 °C;
1
solid; 781 mg, 62 %; mp 196–198 °C; H NMR (CDCl3,
500 MHz): d 9.31 (bs, 1H), 7.44 (s, 1H), 7.27 (d,
J = 8.46 Hz, 2H), 7.03 (d, J = 8.46 Hz, 2H), 6.87 (s, 1H),
123

Med Chem Res
1H NMR (CDCl3, 400 MHz): d 7.48 (s, 1H), 7.31–7.34 (m,
3H), 7.20 (d, J = 8.54 Hz, 1H), 7.10 (d, J = 8.28 Hz, 2H),
3.99–3.80 (m, 4H), 2.61–2.45 (m, 4H), 2.37 (s, 3H), 2.24
(s, 3H); 13C NMR (CDCl3, 100 MHz): d 163.2, 146.4,
141.9, 135.9, 135.7, 134.8, 133.0, 130.6, 128.9, 127.8,
127.3, 116.8, 55.5, 54.7, 47.1, 45.9, 41.9, 9.0; HRMS-ESI
(m/z) C22H21ON4Cl3 ? H) ? :Calcd. 463.0854 found:
463.0862.
100 MHz): d 160.3, 143.4, 141.1, 136.5, 135.0, 131.5,
130.9, 130.8, 130.3, 129.8, 128.8, 127.7, 126.6, 118.3,
55.7, 23.8, 21.1, 9.2; HRMS-ESI (m/z) C22H22ON4-
Cl2 ? H)?: Calcd. 429.1243 found: 429.1246.
(5-(4-chlorophenyl)-1-(2,4-difluorophenyl)-4-methyl-1H-pyra-
zol-3-yl)(4-methylpiperazin-1-yl) methanone (16) Com-
pound 16 was synthesized according to the procedure
applied to 1. White solid; 518 mg, 84 %; mp 174–176 °C;
1H NMR (CDCl3, 200 MHz): d 7.30–7.24 (m, 1H),
7.24–7.20 (m, 2H), 7.06–6.96 (m, 2H), 6.91–6.69 (m, 2H),
3.91–3.63 (m, 4H), 2.56–2.34 (m, 4H), 2.29 (s, 3H), 2.12
(s, 3H); 13C NMR (CDCl3, 50 MHz): d 163.2, 146.6,
141.9, 134.8, 130.5, 129.7, 128.8, 127.4, 124.1, 116.8,
112.1, 111.6, 105.0, 104.5, 55.5, 54.7, 47.0, 45.9, 41.8, 8.9;
(5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-
pyrazol-3-yl) (morpholino) methanone (6) Compound 6
was synthesized according to the procedure applied to 1.
Cream yellow solid: 590 mg, 63 %; mp 170–172 °C;1H
NMR (CDCl3, 500 MHz): d 7.47 (d, J = 2 Hz, 1H), 7.33 (d,
J = 8.54 Hz, 2H), 7.27–7.30 (m, 1H), 7.19 (d, J = 8.55 Hz,
1H), 7.09 (d, J = 8.54 Hz, 2H), 3.85–3.92 (m, 4H),
3.75–3.83 (m, 4H), 2.25 (s, 3H); 13C NMR (CDCl3,
125 MHz): d 163.2, 146.0, 142.1, 135.9, 135.8, 134.9, 133.0,
130.6, 130.5, 130.3, 128.9, 127.8, 127.3, 117.2, 67.3, 66.9,
47.8, 42.6, 9.1; HRMS-ESI (m/z) C21H18O2N3Cl3Na:Calcd.
472.0357 found: 472.0355.
HRMS-ESI
(m/z)
C22H21ON4ClF2 ? H)?: Calcd.
431.1445 found: 431.1447.
5-(4-chlorophenyl)-4-methyl-N-(piperidin-1-yl) isoxazole-
3-carboxamide (17) Compound 17 was synthesized ac-
cording to the procedure applied to 1. White solid: 240 mg,
1
45 %; mp 103–104 °C; H NMR (CDCl3, 500 MHz): d
N0-(1-(2,4-dichlorophenyl)-4-methyl-5-phenyl-1H-pyrazole-
3-carbonyl)isonicotinohydrazide (10) Compound 10 was
synthesized according to the procedure applied to 1. White
7.63–7.64 (d, J = 8.24 Hz, 1H), 7.57–7.58 (d,
J = 8.24 Hz, 1H), 7.47–7.49 (d, J = 8.24 Hz, 2H),
2.99–2.81 (m, 4H), 2.45 (s, 3H), 1.85–1.71 (m, 4H),
1.53–1.41 (m, 2H); 13C NMR (CDCl3, 125 MHz): d 165.8,
163.0, 157.3, 156.4, 136.1, 129.2, 126.0, 111.7, 56.9, 25.2,
23.1, 8.3; HRMS-ESI (m/z) C16H18O2N3Cl ? H)?: Calcd.
320.1160 found: 320.1161.
1
solid: 273 mg, 68 %; mp 167–168 °C; H NMR (CDCl3,
400 MHz): d 10.28 (bs, 1H), 9.43 (s, 1H), 8.71 (bs, 1H),
7.75 (s, 1H), 7.41 (d, J = 1.47, 2H), 7.28–7.34 (m, 5H),
7.14 (d, J = 4.40, 2H), 7.01–7.04 (m, 1H) 2.33 (s,3H); 13
C
NMR (CDCl3, 100 MHz): d 163.4, 161.2, 150.3, 144.4,
142.3, 138.8, 135.9, 135.8, 132.8, 130.5, 130.2, 129.5,
128.9, 128.6, 128.2, 127.8, 121.2, 118.3, 9.3; HRMS-ESI
(m/z) C23H17O2N5Cl2 ? H)?: Calcd. 466.0832 found:
463.0830.
1-(2,4-dichlorophenyl)-4-methyl-5-phenyl-N-(piperidin-1-
yl)-1H-pyrazole-3-carboxamide (18) Compound 18 was
synthesized according to the procedure applied to 1. Pale
brown solid: 288 mg, 61 %; mp 172–173 °C; 1H NMR
(CDCl3, 400 MHz): d 7.72 (s, 1H), 7.41 (s, 1H), 7.27–7.32
(m, 5H), 7.11 (s, 2H), 3.05–2.75 (m, 4H), 2.38 (s, 3H),
1.91–1.65 (m, 4H), 1.54–1.34 (m, 2H); 13C NMR (CDCl3,
100 MHz): d 160.1, 144.2, 144.0, 136.1, 135.7, 133.0,
130.6, 130.2, 129.5, 128.6, 128.6, 128.4, 127.7, 117.9,
57.0, 25.3, 23.2, 9.3; HRMS-ESI (m/z) C22H22ON4-
Cl2 ? H)?: Calcd. 429.1243 found: 429.1244.
5-(4-chlorophenyl)-4-methyl-1-phenyl-N-(piperidin-1-yl)-
1H-pyrazole-3-carboxamide (14) Compound 14 was
synthesized according to the procedure applied to 1. White
1
solid; 580 mg, 65 %; mp 201–202 °C; H NMR (CDCl3,
400 MHz): d 7.77 (bs, 1H), 7.33 (d, J = 7.27 Hz, 5H),
7.21 (d, J = 7.78 Hz, 2H), 7.08 (d, J = 8.28 Hz, 2H),
2.98–2.79 (m, 4H), 2.35 (s, 3H), 1.85–1.68 (m, 4H),
1.52–1.38 (m, 2H); 13C NMR (CDCl3, 100 MHz): d 160.1,
143.5, 140.9, 139.3, 134.6, 131.2, 129.0, 128.9, 128.0,
127.9, 125.0, 119.1, 57.1, 25.4, 23.3, 9.3; HRMS-ESI (m/z)
C22H23ON4Cl ? H)?: Calcd. 395.1633 found: 395.1631.
5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-N-(piperidin-1-yl)-
1H-pyrazole-3-carboxamide (19) Compound 19 was syn-
thesized from the corresponding acid according to the
procedure applied to 1. Cream solid: 199 mg, 53 %; mp
139–140 °C; 1H NMR (CDCl3, 400 MHz): d 7.61 (d,
J = 2.29 Hz, 1H), 7.57 (s, 1H), 7.55 (d, J = 2.29 Hz, 1H),
7.53 (d, J = 1.83 Hz, 1H), 7.42 (d, J = 9.15 Hz, 2H), 7.26
(d, 2H), 7.23 (s, 1H), 3.35–3.19 (m, 4H), 2.01–1.95 (m,
4H), 1.70–1.59 (m, 2H); 13C NMR (CDCl3, 100 MHz): d
158.8, 146.8, 145.6, 136.3, 135.7, 135.1, 132.8, 130.5,
130.4, 129.1, 129.0, 128.2, 127.3, 107.5, 56.8, 24.9, 22.7;
HRMS-ESI (m/z) C21H19ON4Cl3 ? H)?: Calcd. 449.0697
found: 449.0701.
1-(2-chlorophenyl)-5-(4-chlorophenyl)-4-methyl-N-(piperid-
in-1-yl)-1H-pyrazole-3-carboxamide (15) Compound 15
was synthesized according to the procedure applied to 1.
1
Yellow solid: 207 mg, 42 %; mp 248–250 °C; H NMR
(CDCl3, 200 MHz): d 7.69 (bs, 1H), 7.37–7.30 (m,
1H),7.29–7.24 (m, 2H), 7.23–7.15 (m, 3H), 6.98 (d,
J = 8.71 Hz, 2H), 2.95–2.72 (m, 4H), 2.30 (s,3H),
1.78–1.61 (m, 4H), 1.45–1.29 (m, 2H); 13C NMR (CDCl3,
123

Med Chem Res
Anti-mycobacterial activity assay (Abreu et al., 2012;
Stigliani et al., 2012; Handoko et al., 2012; Tiwari
et al., 2012; NCCLS, 1995)
In vitro anti-mycobacterial activity against M. tubercu-
losis H37Rv (MTB) (Abreu et al., 2012; Stigliani et al.,
2012; Handoko et al., 2012; Tiwari et al., 2012; NCCLS,
1995).
Isolated single colonies of M. smegmatis MC2155 (ATCC
14468) grown on 7H10 agar plate were developed over-
night in Middlebrook 7H9 medium (0.47 % Middlebrook
7H9 broth base, 10 % ADS, 0.2 % glycerol and 0.1 %
Tween 80) to mid-exponential phase at 37 °C. When the
OD of this culture reached approximately 0.8, a secondary
culture was inoculated in 5 ml Middlebrook 7H9 medium.
The secondary culture was incubated overnight and al-
lowed to grow at 37 °C to early log phase (OD600 & 0.3).
For the anti-mycobacterial assay, 98 ll of 1:1000-folds
dilution of the secondary culture was dispensed into
96-well microtiter plate per well along with 2 ll of test
compound in triplicate, and 240 ll of sterile water was
added to each well of the peripheral rows of 96-well plate
to minimize media evaporation during assay incubation.
The final concentration of the test compound in each well
was 30 lM. Bacterial growth was assessed after 32 h of
incubation by measuring turbidity at 600 nm OD600 values
using TECAN Infinite 200 PROTM (Tecan Instruments,
Switzerland). Depending upon the percentage of growth,
the percentage of inhibition was calculated at a standard
concentration of 30 lM. Isoniazid and rifampicin were
included in every assay plate as positive controls of growth
inhibition using stock solutions of INH (10 mg/ml,
HiMedia) and rifampicin (10 mg/ml, HiMedia) to achieve
the final concentration of 16 lg/ml for INH and 2 lg/ml
for rifampicin. Additional controls DMSO (solvent without
compound) and medium without inoculums were included
in all the assay plates avoiding intra-assay variability. The
results were analyzed as the percentage of growth
inhibition.
MTB H37Rv strain was obtained from National Institute
for Research in Tuberculosis (NIRT), Chennai. Tenfold
serial dilutions of each test compound were prepared and
incorporated into Middlebrook 7H11 agar medium with
OADC growth supplement. Inoculation of M. tuberculosis
H37Rv ATCC 27294 (MTB) was prepared from fresh
Middlebrook 7H11 slants with OADC growth supplement
and was adjusted to 1 mg/ml (wet weight) in Tween 80
(0.05 %) saline diluted to 10–2 to achieve a concentration
of *107 cfu/ml (culture forming units). Then, a bacterial
suspension of 5 ll was spotted in 7H11 agar tubes having
tenfold serial dilutions of compounds per ml. Cultures were
then incubated at 37 °C for 4 weeks. The MIC values of
the synthesized compounds along with the standard drugs
for comparison are tabulated in Table 1.
Experimental methodology for molecular docking
analysis
For molecular docking, the structures were drawn using
ChemDraw 12 followed by MMFF94 optimization using
the default settings. The crystallographic coordinates of
CYP121 (pdb id: 2IJ7) from M. tuberculosis were obtained
from protein data bank (www.rcsb.org). For CYP121 of M.
tuberculosis, we opted for pdb-2IJ7 for docking, because it
˚
has been crystallized with fairly good resolution (1.90 A)
with fluconazole as a ligand. For docking analysis, the
standard procedure mentioned in literature (Abreu et al.,
2012; Stigliani et al., 2012; Handoko et al., 2012; Tiwari
et al., 2012; Masand et al., 2013) was followed using
AutoDock 4.2, running under Linux Ubuntu 12.04.
Acknowledgments JMG gratefully acknowledges CSIR-OSDD,
CSIR-ORIGIN and DST-SERB, for the financial support.
MIC Assay (Abreu et al., 2012; Stigliani et al., 2012;
Handoko et al., 2012; Tiwari et al., 2012; NCCLS,
1995)
After the compounds were screened for percentage inhibi-
tion, the promising compounds were further screened to
obtain the MIC values. Minimum inhibitory concentration
(MIC) is the concentration of compound which inhibits the
90 % growth of bacteria under optimum conditions. The
growth inhibition assays were carried out in the same ana-
logy as explained above using various concentrations of the
test compounds prepared by serial dilutions 100, 50, 25, 12.5
and 6.25 lM to obtain the final concentrations of 46.37,
23.18, 11.59, 5.79 and 2.89 lg/ml, respectively. From the
rate of inhibition bacterial growth, theascertained MIC of the
compound was calculated. The MIC value of the test com-
pound 1 (rimonabant) is 13.56 lg/ml (29.24 lM 1.47).
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