POMA analyses as new efficient bioinformatics' platform to predict and optimise bioactivity of synthesized 3a,4-dihydro-3H-indeno[1,2-c]pyrazole-2- carboxamide/carbothioamide analogues

Article abstract of DOI:10.1016/j.bmcl.2012.09.108

A series of 43, 3a,4-dihydro-3H-indeno[1,2-c]pyrazole-2-carboxamide/ carbothioamide analogues (D01-D43) were analysed using Petra, Osiris, Molinspiration and ALOGPS (POMA) to identify pharmacophore, toxicity prediction, lipophilicity and bioactivity. All the compounds were evaluated for anti-HIV activity. 3-(4-Chlorophenyl)-N-(4-fluorophenyl)-6,7-dimethoxy-3a,4-dihydro-3H- indeno[1,2-c]pyrazole-2-carboxamide (D07) was found to be the most active with IC₅₀ > 4.83 μM and CC₅₀ 4.83 μM. 3-(4-Fluorophenyl)-6,7-dimethoxy-3a,4-dihydro-3H-indeno[1,2-c] pyrazole-2-carbothioamide (D41) was found to be the most active compound against bacterial strains with MIC of 4 μg/ml, comparable to the standard drug ciprofloxacin while 3-(4-methoxyphenyl)-6,7-dimethoxy-3a,4-dihydro-3H-indeno[1, 2-c]pyrazole-2-carboxamide (D38) was found to be the most active compound against fungal strains with MIC 2-4 μg/ml, however less active than standard fluconazole. Toxicities prediction by Osiris were well supported and experimentally verified with exception of some compounds. In anticonvulsant screening, 3-(4-fluorophenyl)-N-(4-chlorophenyl)-6,7-dimethoxy-3a,4-dihydro-3H- indeno[1,2-c]pyrazole-2-carboxamide (D09) showed maximum activity showing 100% (4/4, 0.25-0.5 h) and 75% (3/4, 1.0 h) protection against minimal clonic seizure test without any toxicity.

Full text of DOI:10.1016/j.bmcl.2012.09.108

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7029–7035
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
POMA analyses as new efficient bioinformatics’ platform to predict and optimise
bioactivity of synthesized 3a,4-dihydro-3H-indeno[1,2-c]pyrazole-2-carboxam-
ide/carbothioamide analogues
Mohamed Jawed Ahsan a,b, , Jeyabalan Govindasamy a, Habibullah Khalilullah a, Govind Mohan b,
⇑
James P. Stables c, Christophe Pannecouque d, Eric De Clercq d
a New Drug Discovery Research, Department of Medicinal Chemistry, Alwar Pharmacy College, Alwar, Rajasthan 301 030, India
b Department of Pharmaceutical Chemistry, Nims Institute of Pharmacy, Nims University, Jaipur, Rajasthan 303 121, India
c Preclinical Pharmacology Section, Epilepsy Branch, National Institute of Health, Bethesda, MD 20892-9020, USA
d Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 July 2012
Revised 4 September 2012
Accepted 28 September 2012
Available online 6 October 2012
A
series of 43, 3a,4-dihydro-3H-indeno[1,2-c]pyrazole-2-carboxamide/carbothioamide analogues
(D01–D43) were analysed using Petra, Osiris, Molinspiration and ALOGPS (POMA) to identify pharmaco-
phore, toxicity prediction, lipophilicity and bioactivity. All the compounds were evaluated for anti-HIV
activity. 3-(4-Chlorophenyl)-N-(4-fluorophenyl)-6,7-dimethoxy-3a,4-dihydro-3H-indeno[1,2-c]pyrazole-
2-carboxamide (D07) was found to be the most active with IC50 > 4.83
phenyl)-6,7-dimethoxy-3a,4-dihydro-3H-indeno[1,2-c]pyrazole-2-carbothioamide (D41) was found to
be the most active compound against bacterial strains with MIC of 4 g/ml, comparable to the standard
drug ciprofloxacin while 3-(4-methoxyphenyl)-6,7-dimethoxy-3a,4-dihydro-3H-indeno[1,2-c]pyrazole-
2-carboxamide (D38) was found to be the most active compound against fungal strains with MIC
2–4 lg/ml, however less active than standard fluconazole. Toxicities prediction by Osiris were well
lM and CC50 4.83 lM. 3-(4-Fluoro-
Keywords:
Pyrazolines
Tautomerism
Petra/Osiris/Molinspiration/ALOGPS 2.1
program
l
supported and experimentally verified with exception of some compounds. In anticonvulsant screening,
3-(4-fluorophenyl)-N-(4-chlorophenyl)-6,7-dimethoxy-3a,4-dihydro-3H-indeno[1,2-c]pyrazole-2-
carboxamide (D09) showed maximum activity showing 100% (4/4, 0.25–0.5 h) and 75% (3/4, 1.0 h)
protection against minimal clonic seizure test without any toxicity.
Anti-HIV
Antibacterial
Antifungal
Anticonvulsant
Ó 2012 Elsevier Ltd. All rights reserved.
Now a day’s millions of new compounds are synthesized all
over the world and many of them entered pharmacological screen-
ing. The medicinal chemists are making intensive effort to synthe-
size new compounds and explore the old one available in order to
increase their biological activity, and this effort needs the use of
enormous chemicals, efforts and funding. Sometimes it may lead
to waste of efforts, chemicals and funds if we found the unenthu-
siastic results. Today we have various computational methods
available which one can use before synthesis of actual compounds
to reduce the excess waste of chemicals, precious time and efforts.
The chemistry of pyrazoline derivatives are much more ex-
plored today because of their interesting structural properties
and their biological potentials as antitubercular, anticonvulsant,
antimicrobial, anti-inflammatory, anti-HIV, anticancer and many
more.1–8 Earlier we have reported the antitubercular and anticon-
vulsant activities of some novel pyrazoline derivatives.9–12 The
present letter is the continuation of the previous work and we have
focused here on the bioinformatics of the synthesized compounds
with the help of computational method viz. Petra, Osiris, Molinspi-
ration and ALOGPS and correlate the biological activities, and to
identify the pharmacophores for antiviral and antibacterial activi-
ties. Pyrazoline exhibits tautomerism, isomerism and opening/
closing of pyrazoline nucleus and showed interesting structural
and spectroscopic properties. We reported herein 43 pyrazoline
derivatives. It was believed that pyrazoline can exist in two major
δ-
δ -
O
H
N
δ+
2
O
δ-
N
Ar
NH
2
Ar
NH
N
O
O
N
O
O
1
1
Ar
Ar
II
I
Antiviral
Antibacterial
⇑
Corresponding author. Tel.: +91 969 4087786; fax: +91 144 5121120.
E-mail address: jawedpharma@gmail.com (M.J. Ahsan).
Figure 1. Potential antibacterial/antiviral pharmacophores sites of compounds
(D01–D43).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.09.108

7030
M. J. Ahsan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7029–7035
CH
O
tautomeric form keto-imine and keto-enamine, although keto-
CH
O
3
O
3
O
CH OH/NaOH
3
imine form is predominant in the solid state. Hadda et al. investi-
gated the potential pharmacophores for antiviral and antibacterial
activity and verified them further with Petra/Osiris/Molinspiration
(POM) analyses.13 On the basis of their finding we can conclude
that pyrazoline derivatives we reported here also contain a poten-
tial pharmacophores for antiviral and antibacterial activities and
this is shown in Figure 1. For antibacterial activity the compounds
posses (XdÀÀYd+) pharmacophore site (II) and also it was hypothe-
sised that the difference in charges between two heteroatoms of
the same dipolar pharmacophoric site may facilitate the inhibition
of bacteria more than virus. And for antiviral activity the com-
pounds posses (XdÀÀYd-) pharmacophore site (I).13
1
+
Ar CHO
H C
1
H C
3
3
O
Ar
O
B01-B06
A1
C01-C06
2
Ar NHCXNHNH
AcOH
2
X
D01-D38; X = O
D39-D43; X = S
2
Ar
CH
3
N
N
N
O
H
1
Ar
H C
3
O
The protocol for the synthesis of 3a,4-dihydro-3H-indeno[1,2-
c]pyrazole-2-carboxamide/carbothioamide analogues (D01–D43)
is summarised in Scheme 1. In the initial step 5,6-dimethoxy-2,
D01-D43
Scheme 1. Protocol for the synthesis.
Table 1
Calculated partition coefficients and solubilities score of the 3a,4-dihydro-3H-indeno[1,2-c]pyrazole-2-carboxamide/carbothioamide analogues (D01–D43)
S
O
2
2
Ar
Ar
N
N
N
H
N
H
N
N
O
O
O
O
1
1
Ar
Ar
D39-D43
D01-D38
Ar2
Compd.
Ar1
ALOGPS
KoW WIN
XLOGP2
D01
D02
D03
D04
D05
D06
D07
D08
D09
D10
D11
D12
D13
D14
D15
D16
D17
D18
D19
D20
D21
D22
D23
D24
D25
D26
D27
D28
D29
D30
D31
D32
D33
D34
D35
D36
D37
D38
D39
D40
D41
D42
D43
4-Pyridinyl–
4-Fluorophenyl–
4-Fluorophenyl–
4-Fluorophenyl–
4-Fluorophenyl–
4-Fluorophenyl–
4-Fluorophenyl–
4-Chlorophenyl–
4-Chlorophenyl–
4-Chlorophenyl–
4-Chlorophenyl–
4-Chlorophenyl–
4-Bromophenyl–
4-Bromophenyl–
4-Bromophenyl–
4-Bromophenyl–
4-Bromophenyl–
4-Bromophenyl–
3-Chloro-4-fluoro–
3-Chloro-4-fluoro–
3-Chloro-4-fluoro–
3-Chloro-4-fluoro–
3-Chloro-4-fluoro–
2,4-Dimethylphenyl–
2,4-Dimethylphenyl–
2,4-Dimethylphenyl–
2,4-Dimethylphenyl–
2,4-Dimethylphenyl–
2,6-Dimethylphenyl–
2,6-Dimethylphenyl–
2,6-Dimethylphenyl–
2,6-Dimethylphenyl–
2,6-Dimethylphenyl–
H–
H–
H–
H–
H–
H–
H–
H–
H–
H–
H–
À4.44 (15.83 mg/l)
À4.84 (6.78 mg/l)
À5.16 (3.08 mg/l)
À5.11 (3.82 mg/l)
À5.01 (4.25 mg/l)
À5.11 (3.60 mg/l)
À4.65 (10.10 mg/l)
À5.62 (1.16 mg/l)
À5.37 (1.98 mg/l)
À5.32 (2.42 mg/l)
À5.22 (2.69 mg/l)
À4.90 (6.17 mg/l)
À5.58 (1.38 mg/l)
À5.37 (2.19 mg/l)
À5.29 (2.81 mg/l)
À5.24 (2.81 mg/l)
À5.28 (2.73 mg/l)
À4.85 (6.58 mg/l)
À5.79 (0.80 mg/l)
À5.55 (1.35 mg/l)
À5.50 (1.65 mg/l)
À5.40 (1.84 mg/l)
À4.76 (7.66 mg/l)
À5.71 (0.92 mg/l)
À5.56 (1.28 mg/l)
À5.45 (1.79 mg/l)
À5.41 (1.71 mg/l)
À5.72 (0.92 mg/l)
À5.45 (1.63 mg/l)
À5.42 (1.90 mg/l)
À5.39 (1.81 mg/l)
À5.41 (1.81 mg/l)
À3.39 (0.14 mg/l)
À4.31 (18.14 mg/l)
À3.86 (49.16 mg/l)
À3.85 (56.54 mg/l)
À3.72 (64.43 mg/l)
À3.79 (59.39 mg/l)
À4.40 (13.99 mg/l)
À5.08 (3.22 mg/l)
À4.86 (5.11 mg/l)
À4.80 (6.13 mg/l)
À4.72 (6.77 mg/l)
4.87
6.70
6.26
5.70
6.06
6.14
5.31
7.14
6.70
6.14
6.50
5.56
7.39
6.95
6.39
6.75
6.83
5.51
7.34
6.90
6.35
6.70
5.76
7.59
7.15
6.59
6.95
7.59
7.15
6.59
6.95
7.03
2.40
4.24
3.79
3.24
3.59
3.67
3.35
5.18
4.74
4.18
4.54
3.14
5.01
4.55
3.95
4.39
4.31
3.60
5.48
5.01
4.42
4.85
3.78
5.65
5.19
4.59
5.03
4.94
3.76
5.64
5.18
4.58
5.01
3.64
5.52
5.06
4.46
4.89
5.31
4.84
4.25
4.68
4.60
0.90
2.77
2.31
1.71
2.15
2.06
1.15
3.02
2.56
1.96
2.40
2-Chlorophenyl–
4-Fluorophenyl–
3,4-Dimethoxyphenyl–
Phenyl–
4-Methoxyphenyl–
4-Pyridinyl–
2-Chlorophenyl–
4-Fluorophenyl–
3,4-Dimethoxyphenyl–
Phenyl–
4-Pyridinyl–
2-Chlorophenyl–
4-Fluorophenyl–
3,4-Dimethoxyphenyl–
Phenyl–
4-Methoxyphenyl–
4-Pyridinyl–
2-Chlorophenyl–
4-Fluorophenyl–
3,4-Dimethoxyphenyl–
Phenyl–
4-Pyridinyl–
2-Chlorophenyl–
4-Fluorophenyl–
3,4-Dimethoxyphenyl–
Phenyl–
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–

M. J. Ahsan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7029–7035
7031
3-dihydro-1H-inden-1-one (0.1 mol) (A1) and appropriate aro-
matic aldehydes (0.1 mol) (B01–B06) in diluted methanolic so-
dium hydroxide solution stirred at room temperature giving the
2-substituted-5,6-dimethoxy-2,3-dihydro-1H-indene-1-one deriv-
atives (C01–C06). In the subsequent step 2-substituted-5,6-dime-
thoxy-2,3-dihydro-1H-indene-1-one derivatives were treated
with appropriate semicarbazides furnished the titled compounds
(D01–D43). The yields of the titled compounds were ranging from
62% to 88% after recrystallization with absolute ethanol.9–12 All the
title compounds (D01–D43) were subjected to Petra, Osiris, Molin-
spiration and ALOGPS (POMA) analyses.
Petra is a program package used for the calculation of physio-
chemical properties of organic compounds, have been developed
over the last 20 years in the research group of Professor. J. Gasteiger.
On the basis of finding based on Petra analyses by Hadda et al., we
can conclude that pyrazoline derivatives we reported here also
contain a potential pharmacophores for antiviral and antibacterial
activities.13 The ALOGPS method is part of the ALOGPS 2.1 program
used to predict lipophilicity and aqueous solubility of
compounds.14 The Log Kow (Kow-WIN) program estimates the
log octanol/water partition coefficient (logP) of organic chemicals
and drugs using an atom/fragment contribution method and the
XLOGP2 is an atom additive method applying corrections. Com-
puted partition coefficients for drugs studied varied between 0.90
and 5.64 (XLOGP2 method) and between 2.40 and 7.59
(KoW-WIN method) is presented in Table 1. The XLOGP2 method
best supported for most of the compounds on the basis of lipophil-
icity (65) to consider an oral drug/lead. Also the absorption of a
drug is usually very low if the calculated solubility is
<0.0001 mg/L.15 As per the criterion compounds of the series
(D01–D43) almost fulfill the requirement of solubility (ALOGPS).
Good intestinal absorption, reduced molecular flexibility (mea-
sured by the number of rotatable bonds), low polar surface area
or total hydrogen bond count (sum of donors and acceptors), are
important predictors of good oral bioavailability.16,17 Membrane
permeability and bioavailability is always associated with some
basic molecular descriptors such as logP (partition coefficient),
molecular weight (MW), or hydrogen bond acceptors and donors
counts in a molecule. Number of rotatable bonds is important for
conformational changes of molecules under study and ultimately
for the binding with receptors or channels. It is revealed that for
passing oral bioavailability criteria, number of rotatable bond
should be 610.16 The compounds in this series (D01–D43) in gen-
eral possess high number of rotatable bonds (3–6) and therefore,
Table 2
Pharmacokinetic parameters important for good oral bioavailability of 3a,4-dihydro-3H-indeno[1,2-c]pyrazole-2-carboxamide/carbothioamide analogues (D01–D43)
Compd.
%ABS
Volume (A3)
TPSA (A2)
NROTB
HBA
HBD
LogP
MW
Lipinski’s violations
Rule
D01
D02
D03
D04
D05
D06
D07
D08
D09
D10
D11
D12
D13
D14
D15
D16
D17
D18
D19
D20
D21
D22
D23
D24
D25
D26
D27
D28
D29
D30
D31
D32
D33
D34
D35
D36
D37
D38
D39
D40
D41
D42
D43
—
—
—
—
4
4
4
6
4
5
4
4
4
6
4
4
4
4
6
4
5
4
4
4
6
4
4
4
4
6
4
4
4
6
4
5
3
3
3
5
3
4
4
4
4
6
4
<10
5
4
4
6
4
5
5
4
4
6
4
5
4
4
6
4
5
5
4
4
6
4
5
4
4
6
4
4
4
6
4
5
5
4
4
6
4
5
5
4
4
6
4
<5
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
65
<500
432
465
449
491
431
461
448
482
465
508
447
492
526
510
552
492
522
467
500
484
525
465
442
475
459
501
441
475
459
501
441
471
338
372
355
397
337
367
354
387
371
413
453
61
0
0
0
0
0
0
0
1
0
1
0
0
2
2
1
0
1
0
1
1
1
0
0
1
0
1
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
89.50
90.69
90.69
85.13
90.69
87.79
86.47
90.69
90.69
85.13
90.69
87.43
90.69
90.69
85.13
90.69
87.79
86.34
89.53
89.59
85.13
90.69
87.61
90.87
90.87
85.31
90.87
90.91
90.91
85.36
90.91
88.02
84.04
87.29
87.29
81.74
87.29
84.40
89.10
92.35
92.35
86.80
92.35
406.12
426.78
416.74
474.27
410.82
442.67
417.40
439.08
482.02
485.55
422.10
422.06
442.72
432.68
490.21
426.76
458.61
429.95
456.66
440.56
490.42
426.98
441.97
463.65
452.59
510.11
446.67
463.59
452.53
510.06
446.61
478.46
327.97
349.65
338.59
396.12
332.67
364.52
340.03
361.71
350.65
408.18
344.73
56.53
53.10
53.10
69.19
53.10
61.48
62.53
53.10
53.10
69.19
53.10
62.53
53.10
53.10
69.19
53.10
61.48
65.70
56.44
56.27
69.19
53.10
62.00
52.57
52.57
68.67
52.57
52.46
52.46
68.55
52.46
60.84
72.36
62.93
62.93
79.03
62.93
71.32
57.70
48.27
48.27
64.37
48.27
3.02
5.02
4.29
3.91
4.17
4.23
3.59
5.77
4.86
4.47
4.74
3.79
6.01
5.06
4.68
4.94
5.00
3.94
6.07
5.21
4.70
4.96
3.67
5.86
4.94
4.56
4.82
5.10
4.19
3.80
4.07
4.12
0.59
2.77
1.86
1.48
1.74
1.79
0.96
3.15
2.23
1.85
2.11
% ABS, percentage of absorption; TPSA, topological polar surface area; NROTB, number of rotatable bonds; MW, molecular weight; LogP, logarithm of compound partition
coefficient between n-octanol and water; HBD, number of hydrogen bond donors; HBA, number of hydrogen bond acceptors.

7032
M. J. Ahsan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7029–7035
exhibit conformational flexibility. Molecular polar surface area
(TPSA) is a sum of surfaces of polar atoms (usually oxygen, nitrogen
and attached hydrogen) in a molecule is a very useful parameter
for the prediction of drug transport properties. TPSA and volume
is inversely proportional to %ABS. TPSA correlate well the passive
molecular transport through membranes and, therefore, allows
prediction of transport properties of drugs in the intestines and
blood–brain barrier crossing.15 TPSA was used to calculate the
percentage of absorption (%ABS) according to the equation:
%ABS = 109 0.345 Â TPSA, as reported.18 From all these parame-
ters, it can be observed that all the title compounds (D01–D43)
exhibited a great %ABS ranging from 81.74 to 92.35%. A computa-
tional study for prediction of ADME properties of all molecules
performed is presented in Table 2. The number of rotatable bonds
(NROTB) and Lipinski’s rule of five were also calculated.19 The rule
states that most molecules with good membrane permeability have
logP 6 5, molecular weight 6500, number of hydrogen bond
acceptors 610 (4–6), and number of hydrogen bond donors 65
(1–2). This rule is widely used as a filter for drug-like properties.
Furthermore, none of the compounds violated Lipinski’s parame-
ters except compounds D13 and D14, making them potentially
promising agents for biological activities. Prediction of toxicity
risk and bioactivity of 3a,4-dihydro-3H-indeno[1,2-c]pyrazole-2-
carboxamide/carbothioamide analogues were studied by Osiris
and Molinspiration software respectively available online.20,21 The
prediction results of compounds (D01–D43) are given in Table 3.
Osiris calculations reveal that the compounds have low toxicity
profile (except for compounds D23–D27) and Molinspiration
predicted efficient binding with the receptors and ion channels.
All the compounds were non toxic in VERO cells at concentration
of 62.5 l
g/ml as reported earlier.9–11 These were well supported
by Osiris calculation.
Among the 43 pyrazoline analogues (D01–D43), 8 compounds
(D07, D34, D40, D02, D29, D24, D13 and D19) showed good anti-
Table 3
Prediction of toxicity risk and bioactivity of 3a,4-dihydro-3H-indeno[1,2-c]pyrazole-2-carboxamide/carbothioamide analogues (D01–D43)
Compd.
Prediction of toxicity risks by Osiris
Prediction of bioactivity by Molinspiration
MUT
TUMO
IRRI
REP
GPCR
ICM
KI
NRL
PI
EI
D01
D02
D03
D04
D05
D06
D07
D08
D09
D10
D11
D12
D13
D14
D15
D16
D17
D18
D19
D20
D21
D22
D23
D24
D25
D26
D27
D28
D29
D30
D31
D32
D33
D34
D35
D36
D37
D38
D39
D40
D41
D42
D43
Cip.
Flu.
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
+
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
+
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À0.00
À0.05
À0.04
À0.04
À0.04
À0.05
À0.01
À0.06
À0.04
À0.05
À0.05
À0.09
À0.13
À0.11
À0.11
À0.12
À0.13
À0.02
À0.07
À0.05
À0.05
À0.05
À0.06
À0.11
À0.09
À0.09
À0.09
À0.10
À0.08
À0.08
À0.09
À0.09
À0.00
À0.07
À0.04
À0.05
À0.05
À0.06
À0.29
À0.35
À0.32
À0.30
À0.34
À0.12
À0.04
0.41
À0.11
À0.19
À0.16
À0.18
À0.16
À0.17
À0.10
À0.18
À0.16
À0.18
À0.16
À0.16
À0.23
À0.21
À0.23
À0.21
À0.21
À0.12
À0.19
À0.17
À0.22
À0.17
À0.17
À0.24
À0.22
À0.26
À0.21
À0.22
À0.20
À0.24
À0.19
À0.21
À0.16
À0.25
À0.23
À0.20
À0.23
À0.23
À0.49
À0.58
À0.55
À0.49
À0.56
À0.04
À0.01
À0.08
À0.06
À0.25
À0.16
À0.15
À0.16
À0.17
À0.10
À0.28
À0.17
À0.18
À0.20
À0.12
À0.31
À0.19
À0.20
À0.22
À0.22
À0.01
À0.19
À0.10
À0.10
À0.11
À0.13
À0.30
À0.19
À0.20
À0.22
À0.29
À0.18
À0.19
À0.21
À0.21
À0.04
À0.28
À0.14
À0.15
À0.17
À0.18
À0.11
À0.35
À0.20
À0.21
À0.24
À0.07
À0.09
À0.15
À0.16
À0.13
À0.13
À0.11
À0.14
À0.11
À0.19
À0.15
À0.15
À0.14
À0.17
À0.26
À0.23
À0.22
À0.21
À0.24
À0.21
À0.15
À0.12
À0.12
À0.11
À0.13
À0.18
À0.16
À0.14
À0.14
À0.16
À0.16
À0.15
À0.15
À0.17
À0.15
À0.17
À0.13
À0.11
À0.11
À0.14
À0.11
À0.40
À0.35
À0.34
À0.31
À0.37
À0.19
À0.23
À0.79
À0.16
À0.26
À0.18
À0.17
À0.18
À0.18
À0.18
À0.24
À0.21
À0.18
-0.20
À0.15
À0.26
À0.20
À0.19
À0.21
À0.20
À0.17
À0.25
À0.23
À0.20
À0.22
À0.20
À0.31
À0.26
À0.23
À0.25
À0.24
À0.21
À0.29
À0.25
À0.24
À0.26
À0.20
À0.30
À0.26
À0.23
À0.25
À0.26
À0.21
À0.19
À0.20
À0.20
À0.03
À0.17
À0.11
À0.09
À0.09
À0.10
À0.04
À0.18
À0.12
À0.10
À0.11
À0.28
À0.03
1.17
À0.23
À0.33
À0.27
À0.23
À0.25
À0.24
À0.23
À0.30
À0.25
À0.23
À0.25
À0.21
À0.30
À0.24
À0.21
À0.23
À0.33
À0.27
À0.23
À0.26
À0.25
À0.15
À0.28
À0.20
À0.16
À0.18
À0.17
À0.30
À0.42
À0.34
À0.28
À0.32
À0.21
À0.09
À0.02
+
+
+
+
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
+
a
+
a
+
À
+
a
AZT
+
MUT = mutagenic, TUMO = tumorigenic, IRRI = irritant, REP = reproductive effect. A dash (À) indicates no effect and a plus (+) indicates the effect.
GPCR = GPCR ligand, ICM = Ion channel modulator, KI = Kinase inhibitor, NRL = Nuclear receptor ligand, PI = Protease inhibitor and EI = Enzyme inhibitor.
Cip. = Ciprofloxacin, Flu. = Fluconazole, AZT = Zidovudine.
a
Slight effect.

M. J. Ahsan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7029–7035
7033
HIV activities against IIIB and ROD strains with their IC50 between
>4.83 M and >11.84 M and CC50 between 4.83 M and 11.84
respectively, 13 compounds showed moderate activities with their
IC50 below >40.38 M and CC50 below 40.38 M while rest of the
showed less activity. Anti-HIV activity against the HIV-I (IIIB
strain) and HIV-II (ROD strain) were obtained from supernatants
of persistently infected H9/IIIB cells. Multiplication in acutely
infected cells was based on inhibition of virus-induced cytopathog-
enicity in MT-4 cells. Cytotoxicity of compounds was based on
the viability of mock infected cells as monitored by the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
method.22–24
l
l
l
lM
l
l
compounds showed less activities. The most active compound of
the series was D07 however it is less active than the standard drug
zidovudine. All the compounds showed selective index (SI) of h1.
The SI is the ratio of CC50/IC50, a higher SI means a more selective
compounds. The maximum protection percent was found with the
compounds D04, D15, D21, D30 and D36 (between 30 and 39). The
anti-HIV activity screening data of the pyrazoline analogues are
given in Table 4. The Osiris calculation for toxicity prediction of
the compounds showed the compounds were less toxic than
the standard drug zidovudine, with the exception of some
compounds (D23–D27) which showed tumorigenic effect. In the
title compounds (D01–D43), the aryl group attached to pyrazoline
nucleus influenced the anti-HIV activity. The 3-aryl group with
2-chlorophenyl substitution showed maximum activity followed
by 4-pyridinyl. The compounds having 3,4-dimethoxyphenyl,
4-methoxyphenyl and 4-fluorophenyl (except compound D29)
11 Pyrazoline derivatives were taken for antimicrobial screen-
ing. The most active compounds among them was D41 which
showed minimum inhibitory concentration (MIC) of
against all bacterial strains and minimum bactericidal concentra-
tion (MBC) of 8 g/ml while 5 compounds showed moderate activ-
ity with MIC between 8 and 32 g/ml and rest of the compounds
4 lg/ml
l
l
showed less activity. The activity of compound D41 was found to
be comparable to the standard drug ciprofloxacin. The compounds
D34, D35, D40 and D41 showed bactericidal action while rests of
the compounds were bacteriostatic. The compound D38 showed
maximum antifungal activity with MIC between 2 and 4
lg/ml,
however less active than standard drug fluconazole, 2 compounds
Table 4
Anti-HIV activities of 3a,4-dihydro-3H-indeno[1,2-c]pyrazole-2-carboxamide/carbothioamide analogues (D01–D43)
Compd.
IIIB strain
M)
ROD strain
IC50
(
lM)
CC50
(
l
Max. Protection %
Av. IC50
(lM)
Av. CC50
(lM)
Max. Protection %
D01
D02
D03
D04
D05
D06
D07
D08
D09
D10
D11
D12
D13
D14
D15
D16
D17
D18
D19
D20
D21
D22
D23
D24
D25
D26
D27
D28
D29
D30
D31
D32
D33
D34
D35
D36
D37
D38
D39
D40
D41
D42
D43
AZT
>31.94
>9.09
31.94
9.09
2
2
4
11
3
7
4
2
2
4
2
1
2
8
15
3
3
4
5
2
12
7
1
1
4
16
5
4
3
11
4
5
4
3
3
6
2
>31.94
>9.09
31.94
9.09
2
1
14
37
7
9
5
2
2
>119.27
>208.21
>68.56
>131.84
>4.83
>22.93
>25.76
>105.98
>28.41
>26.47
>11.5
>108.24
>150.36
>86.03
>135.88
>29.46
>11.84
>179.29
>222.86
>35.81
>25.25
>10.95
>197.6
>144.71
>33.4
119.27
208.21
68.56
131.84
4.83
22.93
25.76
105.98
28.41
26.47
11.5
108.24
150.36
86.03
135.88
29.46
11.84
179.29
222.86
35.81
25.25
10.95
197.6
144.71
33.4
>119.27
>208.21
>68.56
>131.84
>4.83
>22.93
>25.76
>105.98
>28.41
>26.47
>11.5
>108.24
>150.36
>86.03
>135.88
>29.46
>11.84
>179.29
>222.86
>35.81
>25.25
>10.95
>197.6
>144.71
>33.4
119.27
208.21
68.56
131.84
4.83
22.93
25.76
105.98
28.41
26.47
11.5
108.24
150.36
86.03
135.88
29.46
11.84
179.29
222.86
35.81
25.25
10.95
197.6
144.71
33.4
9
19
1
2
16
39
10
5
2
1
7
38
10
4
5
12
16
7
9
4
37
12
6
5
3
>159.37
>9.36
159.37
9.36
>159.37
>9.36
159.37
9.36
>182.44
>211.45
>168.54
>40.38
>6.47
>150.79
>150.58
>259.28
>150.35
>36.94
>6.87
182.44
211.45
168.54
40.38
6.47
150.79
150.58
259.28
150.35
36.94
6.87
>182.44
>211.45
>168.54
>40.38
>6.47
>150.79
>150.58
>259.28
>150.35
>36.94
>6.87
182.44
211.45
168.54
40.38
6.47
150.79
150.58
259.28
150.35
36.94
6.87
9
30
11
6
3
1
3
2
0
5
4
2
90
>39.89
>158.43
>28.41
0.007
39.89
158.43
28.41
>93.55
>39.89
>158.43
>28.41
>0.009
39.89
158.43
28.41
>93.55
9
11
12
66
AZT = Zidovudine.
IC50: 50% effective concentration of the compounds required to achieve 50% protection of MT-4 cells against the cytopathogenic effect of HIV.
CC50: concentration that reduces the MT-4 cell viability by 50%.
IC50 and CC50: all data represent mean value of at least two or more separate experiments.

7034
M. J. Ahsan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7029–7035
showed moderate (D36 and D42) with MIC between 16 and 32
l
g/
(NCIM 3471) fungal strains. All the fungal strains were procured
from National Chemical Laboratory, Pune, India. The lowest con-
centration (highest dilution) required to arrest the growth of fun-
gus was regarded as minimum inhibitory concentration (MIC).
The lowest concentration (highest dilution) required to completely
kill the fungus was regarded as minimum fungicidal concentration
(MFC).
ml, and rest of the compounds showed less activity against fungal
strains. The compound D38 showed fungicidal action on various
fungal strains. The 3-aryl substitution with electronegative group
such as 4-fluorophenyl had more inhibitory activity than 2-chloro-
phenyl and 4-pyridinyl group against bacterial strains, while the
electron releasing group 4-methoxyphenyl showed more activity
than 3,4-dimethoxyphenyl against fungal strains. The antimicro-
bial screening data are given in Table 5 with standard drugs cipro-
floxacin and fluconazole for comparison. The antibacterial and
antifungal activity were done by disc-diffusion and agar diffusion
method respectively reported elsewhere.25–28 The pyrazoline ana-
logues were screened for antibacterial activity against Staphylococ-
cus aureus (NCIM 2079), Bacillus subtilis (NCIM 2439), Escherichia
coli (NCIM 5051) and Pseudomonas aeruginosa (ATCC 10145) bacte-
rial strains. All the bacterial strains were procured from National
Chemical Laboratory, Pune and American Type Culture Collection
(ATCC) and Gene Bank, Institute of Microbial Technology,
Chandigarh, India. Minimum inhibitory concentrations (MICs)
were determined by broth dilution technique. The lowest concen-
tration (highest dilution) required to arrest the growth of bacteria
was regarded as minimum inhibitory concentration (MIC). The
lowest concentration (highest dilution) required to completely kill
the bacteria was regarded as minimum bactericidal concentration
(MBC). The pyrazoline analogues were also screened for antifungal
activity against Aspergillus niger (NCIM 1196) and Candida albicans
Earlier we have reported the anticonvulsant activity of 14
pyrazoline analogues. Here we reported the anticonvulsant activity
of 6 more compounds. The compound, D09 showed 100% protection
(4/4, 0.25–0.5 h) and 75% protection (3/4, 1.0 h) against minimal
clonic seizure, without any toxicity. The compound, D08 showed
75% (3/4, 0.5 h) and 50% (2/4, 1.0 h) protection while compounds
D19, D21 and D22 showed 75% (3/4, 0.25 h), 50% (2/4, 0.5–1.0 h)
and 50% (2/4, 0.25 h) protection respectively against minimal clonic
seizure without any toxicities. Osiris calculations reveal that the
compounds have low toxicity profile was further proved by toxicity
(neurotoxicity) profile of some of the compounds (D08, D09 and
D19–D22) evaluated for anticonvulsant screening. The earlier
reported compounds had also low toxicity profile.12 The SAR was
developed for anticonvulsant activity showed that the compound
having 3-aryl group with 4-fluorophenyl, 3,4-dimethoxyphenyl,
4-pyridinyl, 4-methoxyphenyl and phenyl group showed good to
moderate activity. The anticonvulsant activity of some of the
pyrazoline analogues is given in Table 6. The proposed pharmaco-
phore model contains three binding site for interaction with a
Table 5
Antimicrobial screening of the 3a,4-dihydro-3H-indeno[1,2-c]pyrazole-2-carboxamide/carbothioamide analogues (D33–D43)
Compd.
Concentration in lg/ml
Antibacterial
Antifungal
S. aureus
MBC
B. subtilis
MBC
>512
E. coli
P. aeruginosa
A. niger
C. albicans
MIC
MIC
MIC
MBC
MIC
MBC
MIC
MFC
MIC
MFC
D33
D34
D35
D36
D37
D38
D39
D40
D41
D42
D43
Cip.
Flu.
16
16
8
128
256
64
32
16
4
>512
256
16
>512
>512
>512
>512
128
8
16
16
8
128
256
128
32
16
4
8
16
8
64
256
32
32
8
>512
256
16
>512
>512
>512
>512
128
8
16
16
8
>512
256
16
256
256
128
32
256
4
128
128
64
>512
>512
>512
512
>512
8
>512
>512
>512
512
>512
–
256
128
128
32
128
2
>512
>512
>512
512
>512
2
256
16
>512
>512
>512
>512
128
8
>512
>512
8
128
256
64
32
16
4
>512
>512
>512
>512
256
8
128
64
>512
>512
>512
512
>512
–
4
32
16
128
256
4
>512
>512
8
128
256
4
64
128
4
>512
>512
4
128
256
4
>512
>512
8
16
128
–
128
–
–
–
–
–
–
–
–
–
2
2
1
2
MIC = minimum inhibitory concentration, that is, the lowest concentration to completely inhibit bacterial and fungal growth.
MBC = minimum bactericidal concentration, that is, the lowest concentration to completely kill bacteria.
MFC = minimum fungicidal concentration, that is, the lowest concentration to completely kill fungus.
Cip. = Ciprofloxacin, Flu. = Fluconazole.
Ciprofloxacin and fluconazole were used as standard drugs.
Table 6
6 Hz psychomotor seizure test and toxicity of 3a,4-dihydro-3H-indeno[1,2-c]pyrazole-2-carboxamide analogues
Compd.
ADD no.
Intraperitoneal injection in mice
Time (h) to peak effecta (N/F)
Toxicityb
1.0
0.25
0.5
1.0
2.0
4.0
0.25
0.5
2.0
4.0
D08
D09
D19
D20
D21
D22
439069
439070
439064
439065
439066
439067
1/4
4/4
3/4
0/4
1/4
2/4
3/4
4/4
1/4
1/4
2/4
1/4
2/4
3/4
0/4
0/4
2/4
0/4
0/4
0/4
0/4
0/4
1/4
0/4
0/4
1/4
0/4
0/4
0/4
0/4
0/4
1/4
1/4
0/4
0/4
0/4
0/4
1/4
0/4
0/4
0/4
0/4
0/4
1/4
2/4
0/4
1/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
ADD no. = antiepileptic drug development number (National Institute of Neurodegenerative Disorders and Stroke, National Institute of Health, USA).
a
N/F = number of animals active or toxic over the number tested at a dose of 100 mg/kg.
Time interval in hours.
b

M. J. Ahsan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7029–7035
7035
3. Dawane, B. S.; Konda, S. G.; Mandawad, G. G.; Shaikh, B. M. Eur. J. Med. Chem.
2010, 45, 387.
4. Sharma, P. K.; Kumar, S.; Kumar, P.; Kaushik, P.; Kaushik, D.; Dhingra, Y.; Aneja,
K. R. Eur. J. Med. Chem. 2010, 45, 2650.
5. Bilgin, A. A.; Palaska, E.; Sunal, R. Drug Res. 1993, 43, 1041.
6. Turan-Zitouni, G.; Chevallet, P.; Kiliç, F. S.; Erol, K. Eur. J. Med. Chem. 2000, 35,
635.
7. Sim, M. M.; Ng, S. B.; Buss, A. D.; Crasta, S. C.; Goh, K. L.; Lee, S. K. Bioorg. Med.
Chem. Lett. 2002, 12, 697.
8. Ali, M. A.; Shaharyar, M.; Siddiqui, A. A. Eur. J. Med. Chem. 2007, 42, 268.
9. Ahsan, M. J.; Samy, G. J.; Dutt, K. R.; Agrawal, U. K.; Shankar, B.; Vyas, S.; Kaur,
R.; Yadav, G. Bioorg. Med. Chem. Lett. 2011, 21, 4451.
10. Ahsan, M. J.; Samy, G. J.; Khalilullah, H.; Bakht, M. A.; Hassan, M. Z. Eur. J. Med.
Chem. 2011, 46, 5694.
11. Ahsan, M. J.; Samy, G. J.; Soni, S.; Jain, N.; Kumar, L.; Sharma, L. K.; Yadav, H.;
Saini, L.; Kalyansingh, R. G.; Devenda, N. S.; Prasad, R.; Jain, C. B. Bioorg. Med.
Chem. Lett. 2011, 21, 5269.
12. Ahsan, M. J.; Samy, G. J.; Khalilullah, H.; Stable, J. P. J. Enzime. Inhib. Med. Chem.
in press. doi: http://dx.doi.org/10.3109/14756366.2012.663364.
13. Hadda, T. B.; Ali, M. A.; Masand, V.; Gharby, S.; Fergoug, T.; Warad, I. Med. Chem.
Res. in press. doi: http://dx.doi.org/10.1007/s00044-012-0143-6.
14. http://www.vcclab.org/lab/alogps/start.html.
15. Zhao, Y.; Abraham, M. H.; Le, J.; Hersey, A.; Luscombe, C. N.; Beck, G.;
Sherborne, B.; Cooper, I. Pharm. Res. 2002, 19, 1446.
16. Veber, D. F.; Johnson, S. R.; Cheng, H. Y.; Smith, B. R.; Ward, K. W.; Kapple, K. D.
J. Med. Chem. 2002, 45, 2615.
17. Refsgaard, H. H. F.; Jensen, B. F.; Brockhoff, P. B.; Padkjaer, S. B.; Guldbrandt, M.;
Christensen, M. S. J. Med. Chem. 2005, 48, 805.
18. Ertl, P.; Rohde, B.; Selzer, P. J. Med. Chem. 2000, 43, 3714.
19. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Delivery Rev.
2001, 46, 3.
macromolecular complex in vivo is well supported for the tautom-
erism I where electrons (of atom N) for donation is easily available
for binding with the receptor than in the tautomerism II where
steric hindrance may be observed due to the presence of H atom.12
The keto-imine (tautomerism I) form is predominant in the solid
state was supported by the anticonvulsant activity of pyrazoline
analogues. The anticonvulsant activity was evaluated according to
the Antiepileptic Drug Development Programme (ADD) protocol
reported elsewhere.29,30 Minimal motor impairment was measured
by the rotarod (neurotoxicity or toxicity) test. When a mouse
placed on a rod that rotates at a speed of 6 rpm, can maintain its
equilibrium for a long period of time. The compound was consid-
ered toxic if the treated animal falls off this rotating rod 3 times
during 1 min period. The toxicity study was well supported by
Osiris calculation for anticonvulsant screening also as all the
reported compounds were devoid of any toxicity.
The POMA analyses presented here emphasise the key struc-
tural features impacting the biological activities of pyrazoline
analogues. Moreover, they indicate constructive hints for the de-
sign of new analogues with improved activity. The models could
be used to design new ligands with better activity, before their
synthesis.
Acknowledgments
20. Molinspiration Chemoinformatics: http://www.molinspiration.com/cgi-bin/
properties.
Authors are thankful to the management people of Alwar Phar-
macy College, Alwar, Rajasthan, India for providing research facili-
ties. The authors are also thankful to Rega Institute for Medical
Research, Katholieke Universiteit Leuven, Belgium for anti-HIV
activity and National Institute of Neurological Disorders and Stroke
(NINDS), National Institute of Health (NIH), Bethesda, MD, USA for
anticonvulsant screening.
21. http://www.organic-chemistry.org/prog/peo/.
22. Pannecouque, C.; Daelemans, D.; De Clercq, E. Nat. Protocols 2008, 3, 427.
23. Di Santo, R.; Costi, R.; Artico, M.; Massa, S.; Ragno, R.; Marshall, G. R.; La Colla,
P. Bioorg. Med. Chem. 2002, 10, 2511.
24. Pauwels, R.; Balzarini, J.; Baba, M.; Snoeck, R.; Schols, D.; Herdewijn, P.;
Desmyter, J.; De Clercq, E. J. Virol. Methods 1988, 20, 309.
25. Collins, A. H. Microbiological Methods, 2nd ed.; Butterworth: London, 1976.
26. Cruickshank, R.; Duguid, J. P.; Marmion, B. P.; Swain, R. H. A. In Medicinal
Microbiology; Churchill Livingstone: London, 1975; Vol. II, 12th ed., 196.
27. Varma, R. S. In Antifungal Agents: Past, Present and Future Prospects; Varma, R. S.,
Ed.; National Academy of Chemistry & Biology: Lucknow, India, 1998.
28. Khan, Z. K. In-vitro and in-vivo screening techniques for bioactivity screening
and evaluation, In: Proc. Int. Workshop UNIDO-CDRI. 1997, 210.
29. Barton, M. E.; Klein, B. D.; Wolf, H. H.; White, H. S. Epilepsy Res. 2000, 47, 217.
30. Toman, J. E.; Everett, G. M.; Richards, R. K. Tex. Rep. Biol. Med. 1952, 10, 96.
References and notes
1. Budakoti, A.; Bhat, A. R.; Athar, F.; Azam, A. Eur. J. Med. Chem. 2008, 43, 1749.
2. Parmar, S. S.; Pandey, B. R.; Dwivedi, C.; Harbison, R. D. J. Pharm. Sci. 1974, 63,
1152.