Synthesis and antimycobacterial evaluation of 3a,4-dihydro-3H-indeno [1,2-c] pyrazole-2-carboxamide analogues

Article abstract of DOI:10.1016/j.ejmech.2011.09.035

In the present investigation, a series of 3a,4-dihydro-3H-indeno [1,2-c] pyrazole-2-carboxamide analogues were synthesized and were evaluated for antitubercular activity by two fold serial dilution technique. All the newly synthesized compounds showed low to good inhibitory activities against Mycobacterium tuberculosis H₃₇Rv and multi-drug resistant M. tuberculosis (MDR-TB). 3-(4-fluorophenyl)-N-(4-chlorophenyl)-6,7-dimethoxy-3a,4- dihydro-3H-indeno [1,2-c] pyrazole-2-carboxamide (4c) was found to be the most promising compound active against M. tuberculosis, H₃₇Rv and MDR-TB with minimum inhibitory concentrations 0.83 μM and 3.32 μM respectively.

Full text of DOI:10.1016/j.ejmech.2011.09.035

European Journal of Medicinal Chemistry 46 (2011) 5694e5697
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis and antimycobacterial evaluation of 3a,4-dihydro-3H-indeno [1,2-c]
pyrazole-2-carboxamide analogues
,
b
,
a
a
Mohamed Jawed Ahsana , Jeyabalan Govinda Samy , Habibullah Khalilullah ,
*
Mohamed Afroz Bakhtc, Mohd. Zaheen Hassand
a New Drug Discovery Research, Department of Medicinal Chemistry, Alwar Pharmacy College, Alwar, Rajasthan 301 030, India
b Department of Pharmaceutical Sciences, National Institute of Medical Sciences University, Jaipur 303 121, India
c Department of Pharmaceutical Chemistry, College of Pharmacy in Al-Kharj University, P.O. Box 11323, Saudi Arabia
d Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Jamia Hamdard University, New Delhi 110 062, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 July 2011
Received in revised form
14 September 2011
Accepted 20 September 2011
Available online 29 September 2011
In the present investigation,
a series of 3a,4-dihydro-3H-indeno [1,2-c] pyrazole-2-carboxamide
analogues were synthesized and were evaluated for antitubercular activity by two fold serial dilution
technique. All the newly synthesized compounds showed low to good inhibitory activities against
Mycobacterium tuberculosis H37Rv and multi-drug resistant M. tuberculosis (MDR-TB). 3-(4-fluorophenyl)-
N-(4-chlorophenyl)-6,7-dimethoxy-3a,4-dihydro-3H-indeno [1,2-c] pyrazole-2-carboxamide (4c) was
found to be the most promising compound active against M. tuberculosis, H37Rv and MDR-TB with
minimum inhibitory concentrations 0.83
m
M and 3.32
m
M respectively.
Keywords:
Pyrazolines
Ó 2011 Elsevier Masson SAS. All rights reserved.
Antitubercular agents
Mycobacterium tuberculosis
Multi-drug resistant tuberculosis
1. Introduction
persistent bacteria. Also the treatment of TB with the frontline
drugs is associated with severe side effects including hepatotox-
icity, ocular toxicity, thrombocytopoenia, neuropathy, rashes, fever,
drug induced hepatitis. Nowadays there is an apparent need for fast
acting drugs with less toxicity, which are capable to eliminate
infection within a few weeks.
Tuberculosis or TB is a dreadful life threatening disease caused
by the bacteria Mycobacterium tuberculosis and to a lesser degree by
Mycobacterium Bovis and Mycobacterium Africana. There are one-
third of world’s population which are currently infected with TB,
with about 9.4 million worldwide and 1.6e2.4 million cases in India
[1,2]. An estimated 1.7 million people died from TB in 2009 as per
WHO survey. There are estimated 1.3 million multi-drug/
extensively drug resistant tuberculosis (M/XDR-TB) cases which
will need to be treated between 2010 and 2015 [2]. The develop-
ment of multi-drug resistant tuberculosis (MDR-TB; resistant to
isoniazid and rifampicin) and extensively drug resistant tubercu-
losis (XDR-TB; resistant to quinolones and also to any one of
kanamycin, capreomycin or amikacin) are alarming for the
discovery of new drugs to reduce the potential hazards caused by
TB. The current strategies to cure the diseases are very complicated
as they take several months of chemotherapy to eliminate
The azole group of heterocyclic compounds possess significant
pharmacokinetic property, lipophilicity that influence the ability of
drug to reach the target by transmembrane diffusion and showed
promising activity against resistant TB by inhibiting the biosyn-
thesis of lipids [3,4]. Pyrazoline is an important class of heterocyclic
compounds, and were found to have various biological activities
including antiamoebic, anticonvulsant, antimicrobial, anti-
inflammatory, antiviral, antiarrhythmic, antidepressant, anti-
cancer, antidiabetic, and antitubercular, etc [5e13]. Earlier we have
reported the antimycobacterial activity of diketones and pyrazo-
lines [14,15]. The present investigation is the continuation of the
previous work. In this investigation we have focussed on the design
and synthesis of some novel 3a,4-dihydro-3H-indeno [1,2-c] pyr-
azole-2-carboxamide analogues based on the structure of the
known antitubercular agent, thiacetazone (Fig. 1) in which the “S”
atom of the thiacetazone was replaced with isosteric “O” atom in
the title compounds (4aer). All the synthesized compounds were
evaluated for their anti tubercular activity.
* Corresponding author. Alwar Pharmacy College, New Drug Discovery Research,
Department of Medicinal Chemistry, Alwar, Rajasthan 301 030, India. Tel.: þ91
9694087786; fax: þ91 144 5121120.
E-mail address: jawedpharma@gmail.com (M.J. Ahsan).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.09.035

M.J. Ahsan et al. / European Journal of Medicinal Chemistry 46 (2011) 5694e5697
5695
Isostericre placement of 'S' with 'O'
O
recrystallization with absolute ethanol. The purity of the
compounds was checked by TLC using eluant benzene:acetone
(9:1) and elemental analyses. Both the analytical and spectral data
(IR, 1H NMR and MS) of all the synthesized compounds were in full
accordance with the proposed structures.
Ar2
N
N
N
H
O
O
Ar1
Title compounds (4a-r)
3. Biology
S
All the compounds were screened for their in vitro anti-
mycobacterial activity against MTB and MDR-TB. The primary
screening was carried out by agar dilution method using double
dilution technique recommended by the National Committee for
Clinical Laboratory Standards [17]. Isoniazid was used as standard
drug. The observed data on the antimycobacterial activity of the
title compounds (4aer) and standard drug are given in Table 1.
All the active compounds were tested for cytotoxicity (IC50) in
N
NH2
N
H
O
H3C
N
H
Thiacetazone
Fig. 1. Design of the title compounds based on known antitubercular drug.
VERO cells at concentrations of 62.5
per reported method [18].
mg/mL or 10 times the MIC as
2. Chemistry
The 3a,4-dihydro-3H-indeno [1,2-c] pyrazole-2-carboxamide
analogues (4aer) described in this study are shown in Table 1
and the reaction sequence for the synthesis is summarised in
Scheme 1. In the initial step 5,6-dimethoxy-2,3-dihydro-1H-inden-
1-one (0.01 mol) and appropriate aromatic aldehydes (0.01 mol) in
diluted methanolic sodium hydroxide solution stirred at room
temperature (ClaiseneSchmidt condensation) giving the
2-substituted-5,6-dimethoxy-2,3-dihydro-1H-indene-1-one deriv-
atives (3aef). In the subsequent step 2-substituted-5,6-dimethoxy-
2,3-dihydro-1H-indene-1-one derivatives treated with appropriate
substituted phenyl semicarbazide/semicarbazide furnished the
titled compounds (4aer). The substituted phenyl semicarbazides
were synthesized as per reported method [16]. The yields of
the titled compounds were ranging from 62% to 88% after
4. Results and discussion
In the present work, a series of eighteen 3a,4-dihydro-3H-
indeno [1,2-c] pyrazole-2-carboxamide analogues were synthe-
sized. The synthesis of pyrazolines might be followed by cyclization
of semicarbazone. In general, the IR spectra of the compounds
afforded pyrazoline C]N stretching at 1501e1576 cmꢀ1, CeH
deformation at 1362e1464 cmꢀ1
1069e1189 cmꢀ1
3112e3481 cmꢀ1 and C]O stretching at 1675e1687 cmꢀ1 bands.
The spectra showed singlet at 2.34e2.92 ppm corresponding to
CH3; multiplet at 3.27e3.35 ppm corresponding to CH; a doublet
,
C2eN1 stretching at
,
and carbamoyl group NeH stretching at
d
d
at
d
3.43e3.49 ppm corresponding to CH2 group; a singlet at
Table 1
Physical constant, antimycobacterial activity and cytotoxicity of the title compounds (4aer).
O
O
2
Ar
N
N
N
N
NH
N
2
O
O
O
H
1
1
Ar
Ar
O
4m-4r
4a-4l
Compound
Ar1
Ar2
Yield (%)
Mp (ꢁC)
IC50
(
m
M)
MIC (
MTBa
mM)
Vero
MTBb
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
4-pyridinyl-
4-chlorophenyl-
4-chlorophenyl-
4-chlorophenyl-
4-chlorophenyl-
4-chlorophenyl-
4-chlorophenyl-
4-nitrophenyl-
4-nitrophenyl-
4-nitrophenyl-
4-nitrophenyl-
4-nitrophenyl-
4-nitrophenyl-
e
72
76
70
84
82
76
62
68
72
88
72
66
72
68
70
78
72
68
e
116
124
128
136
166
188
168
130
144
168
160
164
224
242
238
216
220
226
e
>139.2
NT
>133.6
NT
>139.4
NT
68.0
NT
94.8
NT
51.08
NT
NT
NT
6.96
13.92
0.83
9.8
6.97
10.46
6.80
12.67
3.16
12.05
6.81
12.78
11.79
10.75
8.44
10
>13.92
3.32
>9.8
10.46
2-chlorophenyl-
4-fluorophenyl-
3,4-dimethoxyphenyl-
Phenyl-
4-methoxyphenyl-
4-pyridinyl-
2-chlorophenyl-
4-fluorophenyl-
3,4-dimethoxyphenyl-
Phenyl-
4-methoxyphenyl-
4-pyridinyl-
2-chlorophenyl-
4-fluorophenyl-
3,4-dimethoxyphenyl-
Phenyl-
4-methoxyphenyl-
e
>10.46
>13.6
>12.67
6.32
>12.05
13.62
>12.78
>11.79
>10.75
6.62
>10.05
>11.83
>10.86
45.57
e
e
176.85
NT
NT
NT
>456
e
10.05
11.83
10.86
0.56
e
e
INH
e
NT ¼ not tested.
a
M. tuberculosis H37Rv.
MDR-TB.
b

5696
M.J. Ahsan et al. / European Journal of Medicinal Chemistry 46 (2011) 5694e5697
O
open tube capillary method and are uncorrected. Purity of the
O
CH OH/NaOH
3
O
O
O
O
compounds was checked on TLC plates (silica gel G) using eluants
benzeneeacetone (9:1), the spots were located under iodine
vapours or UV light. IR spectra were obtained on a Schimadzu 8201
PC, FT-IR spectrometer (KBr pellets). 1H NMR spectra were recorded
on a Bruker AC 300 MHz spectrometer using TMS as internal
standard in DMSO. Mass spectra were recorded on a Bruker Esquire
LCMS using ESI and elemental analyses were performed on Per-
kineElmer 2400 Elemental Analyzer.
1
+
Ar CHO
1
Ar
2a-f
1
3a-f
2
AcOH
Ar NHCONHNH
2
O
2
Ar
N
N
H
N
O
5.2. General method for the synthesis of 2-substituted-5,6-
dimethoxy-2,3-dihydro-1H-indene-1-one derivatives (3aef)
1
Ar
O
4a-r
5,6-dimethoxy-2,3-dihydro-1H-inden-1-one (0.001 mol) with
appropriate aldehyde (0.001 mol) in diluted methanolic sodium
hydroxide solution was stirred under room temperature for 4 h. The
resulting solution was allowed to stand overnight and then the
reaction mixture was poured into cold water and neutralized with
dilute HCl. The solid was filtered, dried and recrystallized with
ethanol furnished the 2-substituted-5,6-dimethoxy-2,3-dihydro-
1H-indene-1-one.
Scheme 1. Protocol for the synthesis.
d
3.79e3.83 ppm corresponding to OCH3 group; a doublet at
4.9e5.4 ppm corresponding to CH; multiplet at 6.51e8.38 ppm
corresponding to aromatic protons; broad singlet at
8.88e10.02 ppm corresponding to CONH2. The mass spectra of the
d
d
compounds revealed in each case, a peak corresponding to their
molecular ion peaks. The elemental analysis results were within
ꢂ0.4% of the theoretical values.
5.3. General method for the synthesis of 3a,4-dihydro-3H-indeno
[1,2-c] pyrazole-2-carboxamide analogues (4aer)
Among the eighteen synthesized compounds, six compounds
were found to be active with minimum inhibitory concentration of
To
a 2-substituted-5,6-dimethoxy-2,3-dihydro-1H-indene-1-
0.83e6.97
mM. The compound 4c was found to be active against
one (3aeg) (0.01 mol) and substituted phenyl semicarbazide
(0.01 mol) in 20 ml glacial acetic acid was refluxed for 12 h. The
excess of solvent was removed under reduced pressure and then
the reaction mixture was poured into the crushed ice. The solid
mass was filtered dried and recrystallized with ethanol furnished
the 3-substituted-N-aryl-6,7-dimethoxy-3a,4-dihydro-3H-indeno
[1,2-c] pyrazole-2-carboxamide.
MTB and MDR-TB at a MIC of 0.83 and 3.32
m
M respectively. When
compared with isoniazid (INH) the compound, 4c was less potent
against MTB, while 13.6 folds more active against MDR-TB. In the
title compounds (4aer), both the C3 aryl group and the N-aryl
group influenced the antitubercular activity. The 3-substituted
compounds with electron withdrawing groups such as 4-
flourophenyl, 4-pyridyl produced more inhibitory activity than 2-
chlorophenyl and the electron releasing groups such as 4-
methoxyphenyl, 3,4-dimethoxyphenyl showed less inhibitory
activity. The 4-chlorophenyl substitution on N-aryl group showed
maximum inhibitory activity than 4-nitrophenyl substitution or
when there was no substitution. The compound, 4i showed good
5.3.1. 3-(Pyridin-4-yl)-N-(4-chlorophenyl)-6,7-dimethoxy-3a,4-
dihydro-3H-indeno [1,2-c] pyrazole-2-carboxamide (4a)
IR: (KBr) cmꢀ1: 3334 (NH), 1680 (C]O), 1563 (C]N), 1110
(CeN). 1H NMR (300 MHz, DMSO-d6):
d 3.29e3.35 (1H, m, CH),
3.41e3.43 (2H, d, J ¼ 6.2 Hz, CH2), 3.81 (3H, s, OCH3), 3.83 (3H, s,
OCH3), 5.1 (1H, d, J ¼ 6.4 Hz, CH), 6.98e8.31 (10H, m, Ar), 8.89 (1H, s,
CONH); m/z ¼ 449 (Mþ1)þ.
inhibitory activity against MTB at MIC 3.12
compounds 4g, 4k, 4a and 4e showed moderate inhibitory activity
against MTB at MIC 6.80 Me6.97 M (Table 1).
All the active compounds were tested for cytotoxicity (IC50) in
VERO cells at concentrations of 62.5 g/mL or 10 times the MIC. The
mM, while the
m
m
5.3.2. 3-(4-Fluorophenyl)-N-(4-chlorophenyl)-6,7-dimethoxy-3a,4-
dihydro-3H-indeno [1,2-c] pyrazole-2-carboxamide (4c)
m
title compounds having N-aryl substitution with 4-nitrophenyl
group showed more toxicity than 4-chlorophenyl group. The
IR: (KBr) cmꢀ1: 3331 (NH), 1681 (C]O),1565 (C]N), 1144 (CeN)
787 (CeF). 1H NMR (300 MHz, DMSO-d6):
d 3.29e3.33 (1H, m, CH),
compounds showed IC50 ranging from 51.08 to >176.85
mM
3.41e3.44 (2H, d, J ¼ 6.3 Hz, CH2), 3.80 (3H, s, OCH3), 3.83 (3H, s,
OCH3), 5.1 (1H, d, J ¼ 6.5 Hz, CH), 6.98e8.29 (10H, m, Ar), 9.19 (1H, s,
CONH); m/z ¼ 466 (Mþ1)þ.
(Table 1). The compound 4c was non-toxic up to >133.6 M and
m
showed selective index (IC50/MIC) of more than 161 for MTB and
more than 81 for MDR-TB.
The synthesized novel series of pyrazoline were synthesized in
satisfactory yields. The antimycobacterial activity showed prom-
ising results. The SAR studies confirmed the compound 4c is the
potent lead compound for drug discovery with negligible cytotox-
icity. The pyrazoline derivatives discovered in this study may
provide valuable therapeutic intervention for the treatment of
tubercular disease.
5.3.3. 3-(3,4-Dimethoxyphenyl)-N-(4-chlorophenyl)-6,7-
dimethoxy-3a,4-dihydro-3H-indeno [1,2-c] pyrazole-2-
carboxamide (4d)
IR: (KBr) cmꢀ1: 3336 (NH), 1685 (C]O), 1565 (C]N), 1174
(CeN). 1H NMR (300 MHz, DMSO-d6):
d 3.29e3.33 (1H, m, CH),
3.40e3.44 (2H, d, J ¼ 6.7 Hz, CH2), 3.80 (6H, s, OCH3), 3.83 (6H, s,
OCH3), 5.2 (1H, d, J ¼ 6.4 Hz, CH), 6.78e8.19 (9H, m, Ar), 9.39 (1H, s,
CONH); m/z ¼ 439 (Mþ1)þ.
5. Experimental
5.3.4. 3-Phenyl-N-(4-chlorophenyl)-6,7-dimethoxy-3a,4-dihydro-
3H-indeno [1,2-c] pyrazole-2-carboxamide (4e)
5.1. Chemistry
IR: (KBr) cmꢀ1: 3331 (NH), 1681 (C]O), 1565 (C]N), 1144 (CeN).
The entire chemicals were supplied by E. Merck (Germany) and
S. D. Fine Chemicals (India). Melting points were determined by
1H NMR (300 MHz, DMSO-d6):
d
3.27e3.31 (1H, m, CH), 3.41e3.43
(2H, d, J ¼ 6.0 Hz, CH2), 3.81 (3H, s, OCH3), 3.83 (3H, s, OCH3), 5.1

M.J. Ahsan et al. / European Journal of Medicinal Chemistry 46 (2011) 5694e5697
5697
(1H, d, J ¼ 6.3 Hz, CH), 6.98e8.33 (11H, m, Ar), 9.49 (1H, s, CONH); m/
z ¼ 448 (Mþ1)þ.
All the active compounds were tested for cytotoxicity (IC50) in
VERO cells at concentrations of 62.5 g/mL or 10 times the MIC by
serial double dilution technique. After 72 h exposure, viability was
assessed on the basis of cellular conversion of MTT into a formazan
product using the Promega Cell Titer 96 Non-radioactive cell
proliferation method. Most of the active compounds were found to
m
5.3.5. 3-(Pyridin-4-yl)-N-(4-nitrophenyl)-6,7-dimethoxy-3a,4-
dihydro-3H-indeno [1,2-c] pyrazole-2-carboxamide (4g)
IR: (KBr) cmꢀ1: 3334 (NH), 1685 (C]O), 1560 (C]N), 1121
(CeN). 1H NMR (DMSO-d6) ppm: 3.29e3.34 (1H, m, CH), 3.41e3.43
(2H, d, J ¼ 6.8 Hz, CH2), 3.81 (3H, s, OCH3), 3.83 (3H, s, OCH3), 5.1
(1H, d, J ¼ 6.0 Hz, CH), 6.88e8.19 (10H, m, Ar), 10.19 (1H, s, CONH);
m/z ¼ 459 (Mþ1).
be non-toxic up to 62.5 mg/mL.
The molecular docking simulation for possible action on InhA
are currently under investigation. Also studies to acquire more
information about Quantitative Structure Activity Relationships
(QSAR) and MDR are in progress in our laboratory.
5.3.6. 3-(4-Fluorophenyl)-N-(4-nitrophenyl)-6,7-dimethoxy-3a,4-
dihydro-3H-indeno [1,2-c] pyrazole-2-carboxamide (4i)
IR: (KBr) cmꢀ1: 3331 (NH), 1680 (C]O), 1565 (C]N), 1145
(CeN), 786 (CeF). 1H NMR (DMSO-d6) ppm: 3.29e3.33 (1H, m, CH),
3.39e3.42 (2H, d, J ¼ 6.3 Hz, CH2), 3.81 (3H, s, OCH3), 3.83 (3H, s,
OCH3), 5.1 (1H, d, J ¼ 6.1 Hz, CH), 7.02e8.28 (10H, m, Ar), 9.09 (1H, s,
CONH); m/z ¼ 477 (Mþ1)þ.
Acknowledgements
Authors are thankful to the management people of Alwar
Pharmacy College, Alwar, Rajasthan, India for providing research
facilities. The authors are also thankful to Dr. K. P. Singh and Dr.
(Mrs.) Sobha Tomar, National Institute of Medical Sciences
University, Jaipur, Rajasthan, India for their guidance.
5.3.7. 3-Phenyl-N-(4-nitrophenyl)-6,7-dimethoxy-3a,4-dihydro-
3H-indeno [1,2-c] pyrazole-2-carboxamide (4k)
References
IR: (KBr) cmꢀ1: 3331 (NH), 1680 (C]O), 1561 (C]N), 1134
(CeN). 1H NMR (DMSO-d6) ppm: 3.23e3.25 (1H, m, CH), 3.39e3.40
(2H, d, J ¼ 6.2 Hz, CH2), 3.81 (3H, s, OCH3), 3.83 (3H, s, OCH3), 5.1
(1H, d, J ¼ 6.1 Hz, CH), 6.98e8.31 (11H, m, Ar), 9.59 (1H, s, CONH);
m/z ¼ 458 (Mþ1).
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