Synthesis, biological evaluation, and docking studies of 3-(substituted)-aryl-5-(9-methyl-3-carbazole)-1H-2-pyrazolines as potent anti-inflammatory and antioxidant agents

Article abstract of DOI:10.1016/j.bmcl.2012.07.080

A novel series of 3-(substituted)-aryl-5-(9-methyl-3-carbazole)-1H-2- pyrazolines (5a-o) has been synthesized and the structures of newly synthesized compounds were characterized by IR, ¹H NMR and mass spectral analysis. All the synthesized compounds were evaluated for their in vitro and in vivo anti-inflammatory activity, and also for their antioxidant activity. Compounds 5b, 5c, 5d and 5n were found to be selective COX-2 inhibitors. Compound 5c was found to potent inhibitor of the carrageenin induced paw edema in rats. Most of the compounds exhibited good DPPH and superoxide radical scavenging activity, while compounds 5c, 5d, 5i and 5k exhibited good hydroxyl radical scavenging activity. Molecular docking result, along with the biological assay data, suggested that compound 5c was a potential anti-inflammatory agent.

Full text of DOI:10.1016/j.bmcl.2012.07.080

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5839–5844
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, biological evaluation, and docking studies of
3-(substituted)-aryl-5-(9-methyl-3-carbazole)-1H-2-pyrazolines as potent
anti-inflammatory and antioxidant agents
Babasaheb P. Bandgar a, , Laxman K. Adsul a, Hemant V. Chavan a, Shivkumar S. Jalde a,
⇑
Sadanand N. Shringare a, Rafique Shaikh b, Rohan J. Meshram b, Rajesh N. Gacche b, Vijay Masand c
a Medicinal Chemistry Research Laboratory, School of Chemical Sciences, Solapur University, Solapur 413 255, Maharashtra, India
b Biochemistry Research Laboratory, School of Life Sciences, S.R.T.M. University, Nanded 431 606, Maharashtra, India
c Department of Chemistry, Vidyabharati Mahavidyalaya, Camp, Amravati 444602, Maharashtra, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of 3-(substituted)-aryl-5-(9-methyl-3-carbazole)-1H-2-pyrazolines (5a–o) has been syn-
thesized and the structures of newly synthesized compounds were characterized by IR, 1H NMR and mass
spectral analysis. All the synthesized compounds were evaluated for their in vitro and in vivo anti-inflam-
matory activity, and also for their antioxidant activity. Compounds 5b, 5c, 5d and 5n were found to be
selective COX-2 inhibitors. Compound 5c was found to potent inhibitor of the carrageenin induced
paw edema in rats. Most of the compounds exhibited good DPPH and superoxide radical scavenging
activity, while compounds 5c, 5d, 5i and 5k exhibited good hydroxyl radical scavenging activity. Molec-
ular docking result, along with the biological assay data, suggested that compound 5c was a potential
anti-inflammatory agent.
Received 24 May 2012
Revised 6 July 2012
Accepted 24 July 2012
Available online 31 July 2012
Keywords:
Carbazole
Pyrazoline
Anti-inflammatory activity
Antioxidant
Ó 2012 Elsevier Ltd. All rights reserved.
Molecular docking
Inflammation is a multi-factorial process. It reflects the re-
sponse of the organism to the various stimuli and is related to
many disorders such as arthritis, asthma, and psoriasis which re-
quire prolonged or repeated treatment. Cyclooxygenase (COX),
the rate limiting enzyme of the prostanoid biosynthetic pathway,
catalyzes the conversion of arachidonic acid to important anti-
inflammatory mediators such as prostaglandins (PGs), prostacyclin
(PGI) and thromboxane (TXA2).1 The COX enzyme possesses two
distinct catalytic activities: (i) cyclooxygenase activity that cata-
lyzes the oxidation of arachidonic acid to produce hydroperoxy
endoperoxide (PGG2), and (ii) peroxidase activity that reduces
the endoperoxide (PGG2) to the hydroxyl endoperoxide (PGH2).
The PGH2 is transformed to a wide range of enzymatic and non-
enzymatic mechanisms into the primary prostanoids.
It is well known that cyclooxygenase exists in two isoforms,
cyclooxygenase 1 (COX-1) and cyclooxygenase 2 (COX-2). The
COX-1 is a constitutive enzyme and is responsible for the produc-
tion of cytoprotective prostaglandins in the gastrointestinal tract
(GI) and proaggregatory thromboxanes in blood platelets. How-
ever, COX-2 is an inducible enzyme, which is induced in response
to the release of several pro-inflammatory mediators such as TNF-
a
, IL-6, IL-1, LPS, carragenan, TPA, and histamine.2 Since COX-2 is
involved in the inflammation and the resulting pain, inhibiting
its enzymatic activity would be of therapeutic value. Many non-
steroidal anti-inflammatory drugs (NSAIDs) were found to interact
with these enzymes and inhibit their enzymatic activity.3 These
molecules include aspirin and indomethacin which are non-selec-
tive anti-inflammatory agents and inhibit both enzymes COX-1
and COX-2. Aspirin inhibits COX-1 more strongly than COX-23
and inhibition of COX-1 by aspirin reduces the production of
PGE2 and PGI2, which has an adverse ulcerogenic effect.4 The
COX-2 expressions was associated with inflammation and other
pathologies, such as cancer proliferation and have led to the devel-
opment of COX-2 selective inhibitors to improve the therapeutic
potency and reduce the classical side effects associated with the
use of conventional NSAIDs.5 Therefore, selective COX-2 inhibitors
(coxibs) with better safety profile have been marketed as a new
generation NSAIDs.6 But careful prospective examination of coxibs
has revealed unexpected cardiovascular adverse effects such as
myocardial infarction (MI).7 Therefore, development of novel com-
pounds having anti-inflammatory activity with an improved safety
profile is still of paramount importance.
Reactive oxygen species (ROS) are being formed during normal
cellular metabolism and they are known to contribute to healthy
functions in human health and development when they are not
excessive. Mammalian cells possess elaborate defense mechanism
⇑
Corresponding author. Tel./fax: +91 217 2351300.
E-mail address: bandgar_bp@yahoo.com (B.P. Bandgar).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.07.080

5840
B. P. Bandgar et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5839–5844
to detoxify free radicals. A balance between formation of ROS and
their detoxification is essential for normal cellular function. When
such a balance is disrupted as a result of excessive generation of
damaging species or low level of antioxidants, a cell enters in a
state of oxidative stress and is damaged.8 At high concentrations,
ROS can cause damage to cell structures, nucleic acids, lipids and
proteins.9 The hydroxyl radical is known to react with all the com-
ponents of DNA molecule, damaging both purine and pyrimidine
bases and also deoxyribose backbone.10 There are now increasing
experimental and clinical evidences showing the involvement of
oxidative stress induced by reactive oxygen and nitrogen species
in a variety of disorders including cancer,11 atherosclerosis,12 neu-
rodegenerative disorders and aging.13 Many studies have sug-
gested that agents with the ability to protect against ROS may be
therapeutically useful in the treatment of these diseases.
Pyrazolines and their derivatives are important nitrogen-con-
taining heterocyclic compounds and have been found to possess
a broad spectrum of biological activities such as antitumor,14
anti-inflammatory,15 MAO-B inhibitors,16 agonist of cannabinoid
receptors,17 and antioxidant.18 Out of these biological activities of
pyrazolines and their derivatives, anti-inflammatory activity is of
particular interest. Many pyrazolines and their derivatives have
found their clinical application as NSAIDs. The chemical structures
of the NSAIDs like antipyrene, phenylbutazone, celecoxib, moraz-
one, famprofazone and ramifonazone are shown in Figure 1.19 In
addition to these many pyrazoline derivatives are also reported
in the literature as having potent inflammatory activity.20–23
Carbazole and its derivatives are the important class of hetero-
cyclic compounds endowed with various pharmacological activi-
ties such as anti-cancer, antimicrobial, antiviral, anti-
inflammatory and antioxidant activity.24 Carbazole scaffold is pres-
ent in many drugs such as carvedilol, carazostatin and carprofen
(Fig. 2). Carvedilol, an antihypertensive drug acts as a non-specific
b-adrengic antagonist.25 Carprofen, a non-steroidal anti-inflamma-
tory drug (NSAID), is a selective COX-2 inhibitor.26 Carbazole sul-
fonamides (Fig. 2) are reported as a novel class of anti-mitotic
agents against solid tumors.27 3-Benzamidocarbazole (Fig. 2) has
been reported as an inhibitor of PGE2 Production.28
ment with hydrazine hydrate in ethanol at reflux temperature gave
the target compounds 5(a–o).31 All the synthesized compounds
were characterized by their physical and spectral data (IR, 1H
NMR and Mass spectroscopy). The IR spectra of the compounds
5(a–o) showed absorption due to –NH stretching at ꢀ3350 cmꢁ1
whereas, the –CH stretching band was seen around 2925–
3025 cmꢁ1. In the 1H NMR spectra of these compounds, the chiral
CH proton appeared as triplet, while the pro-chiral methylene pro-
tons appeared as two distinct doublets of a doublet thereby indi-
cating the magnetic non-equivalency of the two protons.
All the synthesized target compounds were evaluated for their
in vitro anti-inflammatory activity by COX-1 and COX-2 inhibi-
tion.32,33 The results of in vitro anti-inflammatory activity are pre-
sented in Table 1. Out of the 15 compounds, compounds 5b, 5c, 5d,
and 5n have shown potent COX-2 inhibition. Compounds 5f, 5i,
and 5o have shown moderate inhibition of COX-2. These com-
pounds have selectively inhibited enzyme COX-2 than that of
COX-1. Most of the target compounds were found to be inactive
against COX-1 except 5e, 5g and 5l. Compound 5l was found to
be effective inhibitor of both COX-1 and COX-2. The COX-2 selec-
tivity of these compounds was anticipated since these compounds
possess carbazole moiety similar to that of carprofen, which is a
COX-2 selective drug. The structure activity relationship (SAR)
study of these compounds revealed that, in general electron donat-
ing groups on aromatic ring enhances the COX-2 inhibition as com-
pared to the corresponding compounds containing electron
withdrawing groups with exception of the compound 5l. The
replacement of aromatic ring (5a) by heterocyclic ring like thio-
phene and pyridine revealed an increase in the COX-2 inhibition.
Methoxylated compounds 5b, 5c and 5d were most active com-
pounds of the series with 83.69, 88.43 and 86.58% inhibition of
COX-2, respectively and showed 3–4 times more selectivity to-
wards inhibition of COX-2 than that of COX-1.
To gain better understanding on the potency and SAR studies of
the studied compounds, we proceeded to examine the interactions
of the active compound 5c with COX-II crystal structure (PDB ID-
3NTG).41 The molecular docking was performed by inserting the
compound 5c into the binding site of COX-II. The binding modes
of compound 5c and COX-II were depicted in Figure 3. All the ami-
no acid residues which had interactions with COX-II were exhib-
ited in Figure 4. In the binding mode, compound 5c is potently
bound to the binding site of COX-II via hydrophobic interactions
In an ongoing study on the design and synthesis of biologically
active small molecules,29,30 herein we report the synthesis 3-
(substituted)-aryl-5-(9-methyl-3-carbazole)-1H-2-pyrazolines. All
the synthesized compounds were evaluated for their in vitro
anti-inflammatory and antioxidant activity.
and binding is stabilized by four hydrogen bond, one
p-cation
The target compounds were synthesized as shown in Scheme 1.
Carbazole (1) on methylation with methyl iodide gave 9-methyl
carbazole (2), which on Vilsmeier–Haack formylation gave 3-for-
myl-9-methylcarbazole (3). The 3-formyl-9-methylcarbazole (3)
on Claisen–Schimdt condensation with various aromatic acetophe-
nones and heteroaromatic acetophenones in aqueous sodium
hydroxide afforded compounds 4(a–o)30 which on further treat-
interaction and one -Sigma interaction. The oxygen atoms of
p
the methoxy groups on compound 5c formed two hydrogen bonds
with the Asp B:225 (bond length: 2.49 Å) and with Nag C:671
(bond length: 2.68 Å). The nitrogen atoms of the pyrazoline ring
on compound 5c formed two hydrogen bonds with the Leu
A:131 (bond length: 2.15 Å and 1.93 Å). The enzyme surface model
was showed in Figure 3b, which revealed that the molecule was
well embedded in the active pocket. This molecular docking result,
along with the biological assay data, suggested that compound 5c
was a potential inhibitor of COX-II.
The COX-II active compounds 5b, 5c, 5d and 5n from in vitro
study were subjected to in vivo inhibition of the carrageenin in-
duced paw edema in rats.34 The compounds examined in vivo
did not present toxic effects in doses up to 0.5 mmol/kg body
weight of the mouse. The results of in vivo study are presented
as the percentage of inhibition of induction of edema at the right
hind paw (Table 2). Edema was measured by weight increase of
the right hind paw in comparison to the uninjected left paw. All
the tested compounds have shown induced protection against car-
rageenin induced paw edema. Protection ranged up to 65% by
tested compounds, while the reference drug indomethacin exhib-
ited 47% protection at an equivalent dose. Compound 5c is the po-
tent followed by compounds 5d, 5b, and 5n.
O
O
O
N
N
N
N
N
N
O
N
O
Antipyrine
H2NO2S
Phenylbutazone
Morazone
N
HN
N
N
N
O
CF3
N
N
N
O
Celecoxib
Ramifenazone
Famprofazone
Figure 1. Some reported anti-inflammatory agents.

B. P. Bandgar et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5839–5844
5841
Cl
O
O
NH
OH
O
OH
O
N
H
N
H
OH
N
H
C7H15
Carvedilol
N
Carazostatin
Carprofen
O O
S
H
N
N
R
H
Cl
O
N
3-Benzamidocarbazole
Carbazole sulphonamides
Figure 2. Biologically active carbazole derivatives.
O
O
iii
i
ii
Ar
Ar
H
N
H
N
N
N
2
N
3
4a-o
iv
1
N
HN
5a-o
O
S
O
Ar = (a)
(c)
(d)
O
(e)
F
(b)
O
O
O
(h)
(j)
Cl
(f)
(k)
(g)
(i)
Cl
F
F
F
Cl
(m)
O2N
Cl
F
Cl
N
Cl
(n)
(o)
(l)
Br
Cl
Scheme 1. Reagents and conditions: (i) CH3I, NaH, DMF, rt, 3 h; (ii) DMF, POCl3, 75–80 °C, 3 h; (iii)Substitued acetophenones, NaOH, Ethanol, rt, 24 h; (iv) NH2NH2H2O,
Ethanol, Reflux, 8 h.
Table 1
All the synthesized target compounds were evaluated for DPPH
In vitro COX-1 and COX-2 enzyme inhibition assay data
radical scavenging activity35,36 and the results are presented in Ta-
Compound
% Inhibition (100
l
M)
Selectivity ratio
ble 3. Results of Table 3 show that almost all compounds have
shown good DPPH radical scavenging activity. Compounds 5i, 5k,
5n, 5d, 5c and 5f have shown excellent DPPH radical scavenging
activity (92.08–83.82%) as compared to ascorbic acid (91.52%),
while compounds 5m, 5b, 5l, 5a, 5g, 5h and 5e have shown good
activity (81.12–79.26%). The SAR study revealed that, compounds
with electron withdrawing groups on aromatic ring B show higher
DPPH radical scavenging activity than the compounds with elec-
tron donating groups with the exception of compound 5d. Dihalo-
gen substituted compounds have shown higher DPPH radical
scavenging activity than the monohalogen substituted compounds.
The replacement of aromatic ring by heterocyclic ring, in particular
by thiophene ring (5n), shows higher DPPH radical scavenging
activity, while with pyridine ring (5o), the DPPH radical scavenging
activity decreases. The higher antioxidant activity of these com-
pounds may be attributed to the presence of carbazole ring. Many
antioxidant molecules such as carvedilol and carzotasin have car-
bazole nucleus.
COX-1
COX-2
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
10.20
20.59
24.15
23.49
46.22
10.37
90.76
10.15
24.15
12.15
8.85
87.40
14.15
18.50
20.12
63.87
22.13
83.69
88.43
86.85
22.80
26.85
20.45
12.64
40.53
16.32
10.15
88.95
18.45
48.43
41.58
28.43
2.16
4.06
3.66
3.69
0.49
2.58
0.22
1.24
1.68
1.34
1.14
1.02
1.30
2.61
2.06
—
Indomethacin
The results summarized are the mean values of n = 3.
Selectivity ratio = COX-2% inhibition/COX-1% inhibition.

5842
B. P. Bandgar et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5839–5844
Figure 3. Dock poses of 5c in the active site of COX-II showing a hydrogen bond interaction. All the aromatic rings are deeply embedded into the hydrophobic pockets.
Table 3
Antioxidant (radical scavenging) activity
Compound
% Radical scavenging activity (100 lM)
DPPH radical
SOR radical
.OH radical
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
79.94
80.78
83.98
85.98
79.26
83.82
79.77
79.26
92.08
87.36
91.74
80.61
81.12
85.33
72.01
91.52
63.24
56.24
69.42
70.42
45.65
63.18
69.44
62.41
81.67
61.35
86.26
56.24
65.14
81.14
62.24
51.95
63.45
58.21
69.34
74.34
54.65
60.24
63.25
54.96
69.34
65.14
78.64
57.51
64.85
63.14
54.84
88.34
Ascorbic acid
The results summarized are the mean values of n = 3.
Figure 4. Interactions of compound 5c with COX-II
(88.34%), while rest of the compounds has shown moderate hydro-
xyl radical scavenging activity. In general, the SAR study revealed
that disubstituted compounds shows higher hydroxyl radical scav-
enging activity than the monosubstituted compounds.
Table 2
In vivo inhibition of the carrageenin induced paw edema
in rats
Compound
% Inhibitiona
As the radicals are involved in the inflammation, the anti-
inflammatory agents should have both radical scavenging activity
and anti-inflammatory activity. Majority of NSAIDs such as indo-
methacin, Ketorolac, tolmelin, and oxaprozin have both anti-
inflammatory and antioxidant activities.23 In case of our synthe-
sized compounds, compounds 5c and 5d have shown potent
COX-2 inhibition and antioxidant activity, and these compounds
could be considered as potential anti-inflammatory agents.
In conclusion, we have synthesized a novel series of 3-(substi-
5b
5c
5d
5n
51.4 3.1
60.3 4.5
54.3 1.1
42.2 1.8
47.0 1.1
Indomethacin
We also subjected all the compounds for superoxide37,38 and
hydroxyl radical scavenging activity39,40 and the results are shown
in Table 3. Almost all the test compounds showed potent superox-
ide radical scavenging activity. Compounds 5k, 5i, 5n, 5d and 5g
have shown potent superoxide radical scavenging activity
(86.26–69.42%) as compared to the standard ascorbic acid
(51.95%). In general, the SAR study revealed that dihalogen substi-
tuted compounds show higher superoxide radical scavenging
activity than the monohalogen substituted compounds. The target
compound with heteroaromatic ring such as thiophene (5n) shows
higher superoxide radical scavenging activity, while the one with
pyridine (5o) shows lower activity than the compound with
unsubstitued aromatic ring (5a).
tuted)-aryl-5-(9-methyl-3-carbazole)-1H-2-pyrazolines.
The
investigation of their in vitro anti-inflammatory and antioxidant
screening data revealed that out of the 15 compounds screened,
compounds 5b, 5c, 5d and 5n showed potent COX-2 inhibition.
From in vivo anti-inflammatory activity study, compound 5c was
found to be potent inhibitor of the carrageenin induced paw edema
in rats. Compounds 5i, 5k, 5n, 5d, 5c and 5f have shown potent
DPPH scavenging activity. Compounds 5k, 5i, 5n, 5d and 5g have
shown potent superoxide inhibition. Compounds 5k, 5d, 5i and
5c exhibited good hydroxyl radical scavenging activity. Com-
pounds 5c and 5d have shown potent COX-2 inhibition and antiox-
idant activity and these compounds could be considered as
potential anti-inflammatory agents. Furthermore, Molecular dock-
Compounds 5k, 5d, 5i and 5c exhibited good hydroxyl radical
scavenging activity as compared to the standard ascorbic acid

B. P. Bandgar et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5839–5844
5843
ppm): 2.95 (dd, 1H, J = 9 and 12 Hz, HCH), 3.51 (dd, 1H, J = 9 and 12 Hz, HCH),
3.86 (s, 3H, NCH3), 5.01 (t, 1H, J = 9 Hz, CH), 7.19 (t, 1H, J = 6 Hz, ArH), 7.28–7.60
ing result, along with the biological assay data, suggested that
compound 5c was a potential anti-inflammatory agent.
(m, 9H, ArH), 8.12 (d, 1H, J = 6 Hz, ArH), 8.16 (s, 1H, ArH), 10.21 (s, 1H, NH); 13
C
NMR (100 MHz, DMSO-d6, d in ppm): 142.1, 141.2, 136.4, 135.0, 128.7, 128.6,
127.9, 126.1, 125.0, 122.3, 122.1, 120.3, 119.4, 109.5, 109.0, 101.2, 65.1, 40.0,
29.1; MS (m/z) = 326.09 (m+1).
Acknowledgments
3-(3-(4-Methoxyphenyl)-4,5-dihydro-1H-pyrazol-5-yl)-9-methyl-9H-carbazole
(5b): Yield: 85% (white solid); mp: 160–162 °C; IR (KBr, cmꢁ1): 3335, 2930,
1622, 1595, 1586, 1552, 1485, 1358, 1264, 757; 1H NMR (300 MHz, DMSO-d6, d
in ppm): 2.92 (dd, 1H, J = 12 and 18 Hz, HCH), 3.48 (dd, 1H, J = 12 and 18 Hz,
HCH), 3.78 (s, 3H, OCH3), 3.87 (s, 3H, NCH3), 4.99 (t, 1H, J = 12 Hz, CH), 6.95 (d,
2H, J = 9 Hz, ArH) 7.19 (t, 1H, J = 6 Hz, ArH), 7.42–7.60 (m, 6H, ArH), 8.12 (d, 1H,
J = 6 Hz, ArH), 8.16 (s, 1H, ArH), 10.45 (s, 1H, NH); MS (m/z) = 356.19 (m+1).
3-(3-(3,4-Dimethoxyphenyl)-4,5-dihydro-1H-pyrazol-5-yl)-9-methyl-9H-
carbazole (5c): Yield: 80% (white solid); mp: 180–181 °C; IR (KBr, cmꢁ1): 3335,
2930, 1622, 1595, 1586, 1552, 1485, 1358, 1264, 757, 750, 727; 1H NMR
(300 MHz, DMSO-d6, d in ppm): 2.92 (dd, 1H, J = 9 and 12 Hz, HCH), 3.48 (dd,
1H, J = 9 and 12 Hz, HCH), 3.77 (s, 3H, OCH3), 3.79 (s, 3H, OCH3), 3.86 (s, 3H,
NCH3), 5.01 (t, 1H, J = 9 Hz, CH), 6.95 (d, 1H, J = 6 Hz, ArH), 7.10 (d, 1H, J = 6 Hz,
ArH), 7.19 (t, 1H, J = 6 Hz, ArH), 7.31 (s, 1H, ArH), 7.42–7.59 (m, 4H, ArH), 8.12
(d, 1H, J = 6 Hz, ArH), 8.16 (s, 1H, ArH), 10.45 (s, 1H, NH); MS (m/z) = 386.15
(m+1).
3-(3-(4-Fluorophenyl)-4,5-dihydro-1H-pyrazol-5-yl)-9-methyl-9H-carbazole
(5e): Yield: 80% (white solid); mp: 176–178 °C; IR (KBr, cmꢁ1): 3335, 2932,
1626, 1598, 1580, 1552, 1485, 1473, 1358, 1264, 757, 727; 1H NMR (300 MHz,
DMSO-d6, d in ppm): 2.93 (dd, 1H, J = 9 and 15 Hz, HCH), 3.49 (dd, 1H, J = 9 and
15 Hz, HCH), 3.85 (s, 3H, NCH3), 5.01 (t, 1H, J = 9 Hz, CH), 7.13–7.25 (m, 2H,
ArH), 7.40–7.70 (m, 7H, ArH), 8.10 (d, 1H, J = 6 Hz, ArH), 8.14 (s, 1H, ArH), 10.45
(s, 1H, NH); MS (m/z) = 344.05 (m+1).
H.V.C. grateful to Council of Scientific and Industrial Research
(CSIR), New Delhi, India for financial assistance in the form of SRF.
Supplementary data
Supplementary data associated with this article can be found, in
the
online
version,
at
http://dx.doi.org/10.1016/
j.bmcl.2012.07.080.
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3-(3-(3,4-Difluorophenyl)-4,5-dihydro-1H-pyrazol-5-yl)-9-methyl-9H-carbazole
(5g): Yield: 77% (white solid); mp: 174–177 °C; IR (KBr, cmꢁ1): 3331, 2932,
1626, 1598, 1586, 1552, 1485, 1473, 1358, 1264, 757, 740, 727; 1H NMR
(300 MHz, DMSO-d6, d in ppm): 2.95 (dd, 1H, J = 9 and 12 Hz, HCH), 3.50 (dd,
1H, J = 9 and 12 Hz, HCH), 3.86 (s, 3H, NCH3), 5.05 (t, 1H, J = 9 Hz, CH), 7.19 (t,
1H, J = 6 Hz, ArH), 7.42–7.51 (m, 3H, ArH), 7.58–7.69 (m, 3H, ArH), 8.02 (s, 1H,
ArH), 8.10 (d, 1H, J = 6 Hz, ArH), 8.14 (s, 1H, ArH), 10.45 (s, 1H, NH); MS (m/
z) = 362.15 (m+1).
13. Berliner, J. A.; Heinecke, J. W. Free Radic. Biol. Med. 1996, 20, 707.
14. Congiu, C.; Onnis, V.; Vescil, L.; Castorina, M.; Pisano, C. Bioorg. Med. Chem.
2010, 18, 6238.
3-(3-(4-Chlorophenyl)-4,5-dihydro-1H-pyrazol-5-yl)-9-methyl-9H-carbazole
(5h): Yield: 80% (white solid); mp: 167–169 °C; IR (KBr, cmꢁ1): 3335, 2934,
1625, 1595, 1580, 1550, 1485, 1473, 1358, 1260, 760; 1H NMR (300 MHz,
DMSO-d6, d in ppm): 2.95 (dd, 1H, J = 9 and 15 Hz, HCH), 3.50 (dd, 1H, J = 9 and
15 Hz, HCH), 3.89 (s, 3H, NCH3), 5.04 (t, 1H, J = 9 Hz, CH), 7.13–7.25 (m, 2H,
ArH), 7.40–7.70 (m, 7H, ArH), 8.12 (d, 1H, J = 6 Hz, ArH), 8.16 (s, 1H, ArH), 10.50
(s, 1H, NH); MS (m/z) = 360.15 (m+).
3-(3-(4-Bromophenyl)-4,5-dihydro-1H-pyrazol-5-yl)-9-methyl-9H-carbazole (5l):
Yield: 68% (off white solid); mp: 170–173 °C; IR (KBr, cmꢁ1): 3335, 2934, 1625,
1595, 1580, 1550, 1485, 1473, 1358, 1260, 760; 1H NMR (300 MHz, DMSO-d6, d
in ppm): 2.95 (dd, 1H, J = 12 and 18 Hz, HCH), 3.50 (dd, 1H, J = 12 and 18 Hz,
HCH), 3.86 (s, 3H, NCH3), 5.04 (t, 1H, J = 12 Hz, CH), 7.10–7.24 (m, 2H, ArH),
7.41–7.70 (m, 7H, ArH), 8.12 (d, 1H, J = 6 Hz, ArH), 8.16 (s, 1H, ArH), 10.50 (s,
1H, NH); MS (m/z) = 404.06 (m+).
15. Bansal, E.; Srivastava, V. K.; Kumar, A. Eur. J. Med. Chem. 2001, 36, 81.
16. Gokhan-Kelekci, N.; Yabanoglu, S.; Kupeli, E.; Salgin, U.; Ozgen, O.; Ucar, G.;
Yesilada, E.; Kendi, E.; Yesilade, A.; Bilgin, A. Bioorg. Med. Chem. 2007, 15, 5775.
17. Lange, J. H. M.; Coolen, H. K. A.; Van Stuivenberg, H. H.; Dijksman, J. A. R.;
Herremans, A. H. J.; Ronken, E.; Keizer, H. G.; Tipker, K.; McCreary, A. C.;
Veerman, W.; Wals, H. C.; Stork, B.; Verveer, P. C.; Den Hartog, A. P.; De Jong, N.
M. J.; Adolfs, T. J. P.; Hoogendoorn, J.; Kruse, C. G. J. Med. Chem. 2004, 47, 627.
18. Jeong, T.-S.; Kim, K. S.; Kim, J.-R.; Cho, K.-H.; Lee, S.; Lee, W. Bioorg. Med. Chem.
Lett. 2004, 14, 2719.
19. Reynold, J. E. F. Martindale. The extra pharmacopeia, 30th ed.; Pharmaceutical
press: London, 1993. p. 594.
20. Joshi, R. S.; Mandhane, P. G.; Diwakar, S. D.; Dhabade, S. K.; Gill, C. H. Bioorg.
Med. Chem. Lett. 2010, 20, 3721.
21. Rathish, I. G.; Javed, K.; Ahmad, S.; Bano, S.; Alam, M. S.; Pillai, K. K.; Singh, S.;
Bagchi, V. Bioorg. Med. Chem. Lett. 2009, 19, 255.
22. Fioravanti, R.; Bolasco, A.; Manna, F.; Rossi, F.; Orallo, F.; Ortuso, F.; Alcaro, S.;
Cirilli, R. Eur. J. Med. Chem. 2010, 12, 6135.
23. Parij, N.; Nagy, A. M.; Neve, J. Free Radic. Res. 1995, 23, 571.
24. Knolker, H.-J.; Reddy, K. R Chem. Rev. 2002, 102, 4303.
25. Noguchi, N.; Nishino, K.; Niki, E. Biochem. Pharmacol. 2000, 59, 1069.
26. Zall, A.; Kieser, D.; Hottecke, N.; Naumann, E. C.; Thomaszewski, B.; Schneider,
K.; Steinbacher, D. T.; Schubenel, R.; Masur, S.; Baumann, K.; Schmidt, B. Bioorg.
Med. Chem. 2011, 19, 4903.
27. Hu, L.; Li, Z.-R.; Li, Y.; Qu, J.; Ling, Y.-H.; Jiang, J.-D.; Bojkin, D. Med. Chem. 2006,
49, 6273.
28. Polenzani, L.; Mangano, G.; Coletta, I.; Alisi, M. A.; Cazzolla, N.; Fuelotti, G.;
Maugeri, C. WO Patent 122680 A1, 2006.
29. (a) Bandgar, B. P.; Sarangdhar, R. J.; Viswakarma, S.; Ali Ahamed, F. J. Med.
Chem. 2011, 54, 1191; (b) Bandgar, B. P.; Jalde, S. S.; Korbad, B. L.; Patil, S. A.;
Chavan, H. V.; Kinkar, S. N.; Adsul, L. K.; Shringare, S. N.; Nile, S. H. J. Enzyme
Inhib. Med. Chem. 2012, 27, 267.
9-Methyl-3-(3-(4-nitrophenyl)-4,5-dihydro-1H-pyrazol-5-yl)-9H-carbazole (5m):
Yield: 74% (pale yellow solid); mp: 156–158 °C; IR (KBr, cmꢁ1): 3334, 2926,
1594, 1551, 1509, 1483, 1330, 1248, 846, 750; 1H NMR (300 MHz, DMSO-d6, d
in ppm): 3.01 (dd, 1H, J = 12 and 18 Hz, HCH), 3.56 (dd, 1H, J = 12 and 18 Hz,
HCH), 3.85 (s, 3H, NCH3), 5.14 (t, 1H, J = 12 Hz, CH), 7.17 (t, 1H, J = 6 Hz, ArH),
7.40–7.49 (m, 2H, ArH), 7.56 (d, 1H, J = 9 Hz, ArH), 7.83 (d, 2H, J = 9 Hz, ArH),
8.09–8.15 (m, 2H, ArH), 8.21 (d, 2H, J = 9 Hz, ArH), 8.32 (s, 1H, ArH), 10.51 (s,
1H, NH); 13C NMR (100 MHz, DMSO-d6, d in ppm): 146.0, 145.7, 140.9, 140.1,
140.0, 132.9, 125.7, 124.6, 123.8, 121.8, 120.1, 118.7, 118.2, 109.1, 64.7, 40.1,
29.0; MS (m/z) = 371.10 (m+1).
9-Methyl-3-(3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-5-yl)-9H-carbazole (5n):
Yield: 62% (white solid); mp: 135–137 °C; IR (KBr, cmꢁ1): 3331, 2932, 1626,
1595, 1580, 1552, 1485, 1473, 1360, 1264, 760; 1H NMR (300 MHz, DMSO-d6, d
in ppm): 2.95 (dd, 1H, J = 9 and 12 Hz, HCH), 3.50 (dd, 1H, J = 9 and 12 Hz,
HCH), 3.86 (s, 3H, NCH3), 5.04 (t, 1H, J = 9 Hz, CH), 7.05 (t, 1H, J = 3 Hz, ArH),
7.10–7.21 (m, 2H, ArH), 7.41–7.60 (m, 5H, ArH), 8.13 (d, 1H, J = 6 Hz, ArH), 8.16
(s, 1H, ArH), 10.51 (s, 1H, NH); MS (m/z) = 332.08 (m+1).
32. Maurias, M. Bioorg. Med. Chem. 2004, 12, 5571.
33. COX inhibition assay: The assay was performed by using Colorimetric COX
(human ovine) inhibitor screening assay kit. Briefly, the reaction mixture
30. Bandgar, B. P.; Adsul, L. K.; Lonikar, S. V.; Chavan, H. V.; Shringare, S. N.; Patil, S.
A.; Jalde, S. S.; Koti, B. A.; Dhole, N. A.; Gacche, R. N.; Shirfule, A. J. Enzyme Inhib.
Med. Chem. 2012. http://dx.doi.org/10.3109/14756366.2012.663365.
31. General procedure for the synthesis of 3-(substituted)-aryl-5-(9-methyl-3-
carbazole)-1H-2-pyrazolines (5a–o): 1 mmol of chalcone 4(a–o) was dissolved
in 15 ml of ethanol. To this reaction mixture, 0.5 ml of hydrazine hydrate was
added. The reaction mass was heated at reflux for 6 h. After completion of
reaction (monitored by TLC), reaction mixture was cooled to room
temperature. At the end 10 ml cold water was slowly added to the flask and
the separated product was filtered off and washed with cold water for several
times. The crude compounds were recrystallized from ethanol to afford the
target compounds 5(a–o).
contains, 150
or COX-2), and 50
l
l of assay buffer, 10
ll of heme, 10 ll of enzyme (either COX-1
ll of sample (0.1 mM). The assay utilizes the peroxidase
component of the COX catalytic domain. The peroxidase activity can be
assayed colorimetrically by monitoring the appearance of oxidized N,N,N0,N0-
Tetra methyl-p-phenylenediamine (TMPD) at 590 nm. Indomethacin (0.1 mM)
was used as a standard drug.
34. Edema was induced in the right hind paw of mice (AKR) by the intradermal
injection of 0.05 mL of 2% carrageenin in water. Both sexes are used, but
pregnant females were excluded. Each group was composed of 6–10 animals.
The animals, which have been bred in our laboratory, were housed under
standard conditions and received a diet of commercial food pellets and water
prior to experimentation, but they were fasted during the experimental period.
The tested compounds (0.01 mmol/kg of body weight) were suspended in
water with a few drops of Tween-80 and ground in a mortar before use and
were given simultaneously with the carrageenin injection. The mice were
9-Methyl-3-(3-phenyl-4,5-dihydro-1H-pyrazol-5-yl)-9H-carbazole (5a): Yield:
78% (white solid); mp: 171–173 °C; IR (KBr, cmꢁ1): 3331, 2932, 1626, 1598,
1586, 1552, 1485, 1358, 1264, 757, 740, 727; 1H NMR (300 MHz, DMSO-d6, d in

5844
B. P. Bandgar et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5839–5844
reaction was initiated by adding 0.75 ml pf PMS (120
euthanized 3.5 h after the carrageenin injection. The difference between the
l
M) to the mixture. After
injected and uninjected paws was calculated for each animal. It was compared
with that in control animals (treated with water) and expressed as a percent
inhibition of the edema CPE% values (Table 3). Each experiment was performed
in duplicate and results are mean of two experiments with standard deviation
less than 10%.
5 min of incubation at room temperature the absorbance was recorded at
560 nm using spectrophotometer. Ascorbic acid (0.1 mM) was used as
reference compound.
39. Rollet-Labelle, E.; Gragane, M. S.; Elbim, C.; Marquetty, C.; Gougerot-Pocidalo,
M. A.; Pasquier, C. Free Radical Biol. Med. 1998, 24, 563.
35. (a) Blois, M. S. Nature 1958, 181, 1199; (b) Roberta, R.; Luciana, G.; Luciana, M.;
Glaucia, P. J. Food Sci. 2006, 71, 102.
36. DPPH radical scavenging assay: DPPH (1,1-diphenyl-2-picrylhydrazyl) radical
scavenging assay was carried out as per reported method with slight
40. Hydroxyl (OH) radical scavenging assay: The OH radical scavenging activity was
demonstrated with Fenton reaction. The reaction mixture contained 60
FeCl2 (1 mM), 90 l of 1,10-phenanthroline (1 mM), 2.4 ml of phosphate buffer
(0.2 M, pH: 7.8), 150 l of H2O2 (0.17 M) and 1.5 ml of individual sample
ll of
l
l
modifications. Briefly,
1
l
l
of test solution (0.1 mM) was added to equal
(0.1 mM). The reaction was started by adding H2O2, after 5 min incubation at
room temperature, the absorbance was recorded at 560 nm. Ascorbic acid
(0.1 mM) was used as reference compound.
quantity of 0.1 mM solution of DPPH in ethanol. After 20 min of incubation at
room temperature, the DPPH reduction was measured by reading the
absorbance at 517 nm. Ascorbic acid (0.1 mM) was used as reference
compound.
41. Molecular docking of compound 5c into the three-dimensional structure of
COX-II (PDB ID-3NTG) was carried out using MOE 2008, which has the
improved flexible docking as well as integration with a graphical interface
along with other modules, such as analysis, molecular mechanics, and
molecular dynamics. To get fruitful results we docked the ligand into active
site of receptor COX-II. A good number of X-ray crystallographically resolved
3D structures are available for COX-II, PDB ID-3NTG was chosen on the basis of
following two reasons: (1) X-ray diffraction resolution, 2.14 in present case. (2)
The pdb 3NTG contains crystal structure of COX-II with selective compound
23d-(R).
37. Liu, F.; Ooi, V.; Chang, S. T. Life Sci. 1997, 60, 763.
38. Superoxide radical (SOR) scavenging assay: The superoxide radical (SOR)
scavenging assay was performed by the method reported by Liu, et al.
superoxide anion radicals were generated in
a non-enzymatic Phenazine
methosulphate-Nicotinamide Adenine Dinucleotide (PMS-NADH) system
through the reaction of PMS, NADH and oxygen. It was assayed by the
reduction of Nitroblue tetrazolium (NBT). In this experiment superoxide anion
was generated in 3 ml of Tris HCl buffer (100 mM, pH 7.4) containing 0.75 ml of
NBT (300 lM), 0.75 ml of NADH (936 lM) and 0.3 ml of sample (0.1 mM). The