Synthesis of 1-(4-aminosulfonylphenyl)-3,5-diarylpyrazoline derivatives as potent antiinflammatory and antimicrobial agents

Article abstract of DOI:10.1007/s00044-011-9823-x

A new series of 1-(4-aminosulfonylphenyl)-3, 5-diaryl pyrazolines (5) was synthesized by the reaction of appropriate chalcones 3 with 4- hydrazinobenzenesulfonamide hydrochloride (4) in ethanol in the presence of catalytic amount of acetic acid. All newly

Full text of DOI:10.1007/s00044-011-9823-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:2945–2954
DOI 10.1007/s00044-011-9823-x
ORIGINAL RESEARCH
Synthesis of 1-(4-aminosulfonylphenyl)-3,5-diarylpyrazoline
derivatives as potent antiinflammatory and antimicrobial agents
•
Pawan K. Sharma Satish Kumar
•
•
Pawan Kumar Pawan Kaushik Chetan Sharma
•
•
•
Dhirender Kaushik Kamal R. Aneja
Received: 11 May 2011 / Accepted: 19 October 2011 / Published online: 3 November 2011
Ó Springer Science+Business Media, LLC 2011
Abstract A new series of 1-(4-aminosulfonylphenyl)-3,
5-diaryl pyrazolines (5) was synthesized by the reaction of
appropriate chalcones 3 with 4-hydrazinobenzenesulfona-
mide hydrochloride (4) in ethanol in the presence of catalytic
amount of acetic acid. All newly synthesized compounds
were in vivo evaluated for their antiinflammatory activity
using carrageenan-induced rat paw edema assay. Five of the
newly synthesized compounds, 5d, 5g, 5h, 5i, and 5m dis-
played significant antiinflammatory activity, greater than
55% inhibition 3 h after the carrageenan injection. All the
newly synthesized compounds were evaluated for their in
vitro antimicrobial activity against two Gram-positive bac-
teria (Staphylococcus aureus and Bacillus subtilis); two
Gram-negative bacteria (Escherichia coli and Pseudomonas
aeruginosa), and two yeast (Candida albicans and Saccha-
romyces cerevisiae). Some of the newly synthesized com-
pounds 5a, 5f, 5g, 5h, 5j, and 5k showed excellent antifungal
activity, greater than the reference drug amphotericin B,
against Saccharomyces cerevisiae.
Keywords Chalcones ꢀ
4-Hydrazinobenzenesulfonamide hydrochloride ꢀ
1-(4-aminosulfonylphenyl)-3 ꢀ 5-Diaryl-2-pyrazoline ꢀ
Antiinflammatory ꢀ Antimicrobial
Introduction
Five-membered heterocycles with a conserved vicinal diaryl
substitution pattern are ideal representatives of a recurring
core structure that is found in numerous biologically active
compounds, including cyclooxygenase inhibitors, kinase
inhibitors, GPCR antagonists and even agonists, phospha-
tase inhibitors, and dopamine transporter inhibitors (Muller,
2003). Pyrazolines are prominent nitrogen containing five-
membered heterocyclic compounds possessing broad spec-
trum of biological activities like antimicrobial (Manna and
Agrawal, 2009; Karthikeyan et al., 2007; Zitouni et al.,
2005), antiinflammatory (Rathish et al., 2009; Barsoum
et al., 2006; Rani et al., 2004; Fioravanti et al., 2010), LDL-
oxidation inhibitors (Jeong et al., 2004), antidepressants
(Zitouni et al., 2000), etc. Amongst a large array of medic-
inally important pyrazoline derivatives, 1,3,5-trisubstituted
pyrazolines occupy a unique position and their evaluation as
antiinflammatory agents has attracted much attention in the
recent past (Rathish et al., 2009; Reddy et al., 2008; Amir
et al., 2008).
P. K. Sharma (&) ꢀ S. Kumar ꢀ P. Kumar
Department of Chemistry, Kurukshetra University,
Kurukshetra 136119, Haryana, India
e-mail: pksharma@kuk.ac.in
Motivated by these findings coupled with our ongoing
interest (Sharma et al., 1998, 2010, 2011; Sharma and
Sawhney, 1993, 1997) in the field of nitrogen-containing
heterocycles as antiinflammatory agents, it was designed
to synthesize a new series of pyrazoline derivatives,
1-(4-aminosulfonylphenyl)-3,5-diarylpyrazolines as poten-
tial dual antimicrobial–antiinflammatory agents. The sub-
stitution pattern of the pyrazoline ring was rationalized so
P. Kaushik ꢀ D. Kaushik
Institute of Pharmaceutical Sciences, Kurukshetra Univeristy,
Kurukshetra 136119, India
C. Sharma ꢀ K. R. Aneja
Department of Microbiology, Kurukshetra Univeristy,
Kurukshetra 136119, India
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Med Chem Res (2012) 21:2945–2954
as to be correlated to the diaryl heterocycles template of
compounds that are known to act selectively as COX-2
inhibitors such as celecoxib (Penning et al., 1997).
of sulfanilamide followed by reduction of the corresponding
diazonium salt with stannous chloride (Soliman, 1979).
Spectral (1H NMR, 13C NMR, and IR) and elemental anal-
ysis data of the newly synthesized compounds were in full
1
agreement with the proposed structures. From H NMR
Result and discussion
spectra, all chalcones were found to be geometrically pure
and with trans-configuration (JHa–Hb = 15.30–15.90
Hz). Two vinyl protons (CH=CH) of the chalcones appeared
in the aromatic region as doublets around d 8.32–7.81 and d
7.92–7.40. In general, the characteristic signals in 1H NMR
of target pyrazolines are two protons attached to C4 and one
proton attached to C5 of pyrazoline ring exhibiting a typical
ABX splitting pattern in the form of three doublets of dou-
blets. Methine proton of pyrazolines resonates at around d
5.55 as a doublet of doublet with coupling constants of nearly
12.0 and 6.0 Hz. The two methylene protons displayed two
signals; a doublet of doublet at around d 4.00 with coupling
constants of nearly 17.0 and 12.0 Hz and a doublet of doublet
at around d 3.30 merging with the water signal from DMSO-
d6 in most of the cases. Presence of methoxy group can be
ascertained by the presence of signal around d 3.80–3.90.
Chemistry
The target compounds 1-(4-aminosulfonyl)phenyl-3,
5-diarylpyrazolines (5a–m) were synthesized by following
the synthetic route outlined in Scheme 1. The intermediate
chalcones 3 were synthesized by base catalyzed Claisen-
Schmidt condensation reaction of the appropriate acetoph-
enones 1 with substituted benzaldehydes 2 (Boumendjel
et al., 2008). The target 1-(4-aminosulfonyl)phenyl-3,
5-diarylpyrazolines (5a–m) were obtained by condensation
of appropriate chalcones 3 and 4-hydrazinobenzenesulfo-
namide hydrochloride (4) in ethanol containing a catalytic
amount of glacial acetic acid. 4-Hydrazinobenzenesulfona-
midehydrochloride (4)in turn was prepared via diazotization
O
R3
O
O
R3
R4
R5
R4
R5
H
CH3
KOH,CH3OH
R2
R1
R2
R1
R6
R6
1
3
2
3
H2NO2S
NHNH2.HCl
R1
C2H5OH/H+
N
N
R2
R6
R5
SO2NH2
R3
R4
4
5
Compound
3,5
a
b
c
d
e
f
g
h
i
j
k
l
m
R1
R2
R3
R4
H
F
H
F
H
F
H
F
H
F
H
H
H
H
H
OH
OH
OH
Cl
Cl
Cl
Cl
Cl
H
OCH3 OCH3 OCH3
OCH3 OCH3 OCH3
H
H
OCH3 OCH3 OCH3
H
OCH3 OCH3
H
OCH3
H
H
OCH3 OCH3 OCH3
H
H
OCH3 OCH3 OCH3
H
OCH3
R5
R6
H
H
OCH3
H
H
OCH3
H
H
H
H
OCH3
H
H
OCH3
H
H
H
H
OCH3
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
Scheme 1 Synthesis of 5a–5m from acetophenones (1)
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Med Chem Res (2012) 21:2945–2954
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13C NMR spectral chemical shift values of carbon atoms C-4
(42–45 ppm) and C-5 (56–65 ppm) of pyrazoline ring, and IR
spectral values of the compounds (N–H stretch around 3,325
and 3,240 cm-1, C=N stretch around 1,590 cm-1 and SO2
stretch around 1,330 and 1,150 cm-1) further supported the
structures of newly synthesized compounds.
other compounds 5a, 5c, 5e, 5i, and 5k showed distinctive
pharmacokinetic profiles as revealed from their potent and
rapid onset of action with percentage activity of
47.8–59.3%. After 2 h, three compounds, 5d, 5i, 5m
showed significant antiinflammatory activity ranging from
53.5 to 64.6% inhibition as compared with Indomethacin
(81.9%). Considering the fact that carrageenan-induced
paw edema assay is a biphasic event (Vinegar et al., 1969)
involving the release of histamine and serotonin as the
mediators of inflammation in the first phase which nor-
mally lasts for about 2 h after the carrageenan injection
(Vinegar et al., 1969), AI activity up to 2 h after carra-
geenan injection can be attributed to the antihistamine and
antiserotonin activity of the synthesized compounds. The
second phase which generally operates between 2 and 4 h
after the carrageenan injection involves prostaglandins as
the mediators for the inflammation (Vinegar et al., 1969).
Thus, any AI activity in this period can be attributed to the
inhibition of prostaglandin synthesis. A careful study of the
results shown in Table 1 revealed that compound 5i
showed potent antiinflammatory activity (68.3%) compa-
rable with Indomethacin (77.2%), whereas compounds 5a,
5d, 5g, 5h, and 5m displayed good antiinflammatory
activity (53.8–62.8%) 3 h after the carrageenan injection.
However, none of them was found to be superior over the
reference drug. Good AI activity in the second phase of
Biological evaluation
In vivo antiinflammatory activity
All the newly synthesized compounds 5a–m were screened
for their in vivo antiinflammatory activity by Carrageenan-
induced rat paw edema assay in male Wistar albino rats.
The protocol of animal experiments has been approved by
the Institutional Animal Ethics Committee (IAEC). The
antiinflammatory activity was then calculated 1–4 h after
induction and presented in Table 1 as the mean paw vol-
ume (ml) in addition to the percentage antiinflammatory
activity (AI%).
In the present study, the antiinflammatory activity of test
compounds relative to the reference drug at different time
intervals indicated the following: after 1 h, compound 5b
and 5d were nearly as effective in inhibiting the paw edema
with percentage activity of 63.7 and 62.3%, respectively,
when compared with that of Indomethacin (69.6%). Five
Table 1 In vivo antiinflammatory (AI) activity of compounds 5a–5m in carrageenan-induced rat paw edema assay (acute inflammatory model)
Compounda
Volume of edema (ml)b and % AIc
1 h
2 h
3 h
4 h
Control
0.69 0.12
1.27 0.09
1.80 0.03
1.94 0.02
Indomethacin
0.21 0.06** (69.6)
0.28 0.05** (59.4)
0.25 0.07** (63.7)
0.34 0.01** (50.7)
0.26 0.04** (62.3)
0.35 0.10** (49.3)
0.56 0.04 (18.8)
0.39 0.05 (43.5)
0.59 0.11 (14.5)
0.29 0.09** (57.9)
0.61 0.05 (11.6)
0.36 0.11** (47.8)
0.46 0.04 (33.3)
0.47 0.43 (31.9)
0.23 0.02** (81.9)
0.76 0.07* (40.1)
0.82 0.08* (35.4)
0.88 0.06 (30.7)
0.59 0.12** (53.5)
0.85 0.10 (33.1)
0.82 0.12* (35.4)
0.65 0.09** (48.8)
0.65 0.07** (48.8)
0.45 0.09** (64.6)
0.93 0.01 (26.8)
0.95 0.09 (25.2)
1.06 0.12 (16.5)
0.54 0.22** (57.5)
0.41 0.01** (77.2)
0.83 0.03** (53.8)
0.94 0.01** (47.7)
0.93 0.10** (48.3)
0.67 0.09** (62.8)
0.88 0.04** (51.1)
0.97 0.03** (46.1)
0.77 0.09** (57.2)
0.74 0.05** (58.9)
0.57 0.25** (68.3)
0.97 0.03** (46.1)
0.99 0.17** (45.0)
1.07 0.06** (40.6)
0.67 0.15** (62.8)
0.49 0.01** (74.7)
0.94 0.22** (51.5)
1.01 0.08** (47.9)
1.13 0.06** (41.7)
0.91 0.50**(53.1)
0.97 0.22** (50.0)
1.40 0.12 (27.8)
1.30 0.22** (32.9)
1.06 0.14** (45.4)
0.98 0.14** (49.5)
1.04 0.12** (46.4)
1.09 0.08** (43.8)
1.22 0.08 (37.1)
0.97 0.12** (50.0)
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
* Significantly different compared to respective control values, P \ 0.05
** Significantly different compared to respective control values, P \ 0.01
a
Dose level: test compounds (50 mg/kg body wt), indomethacin (4 mg/kg body wt)
b
Values are expressed as mean SEM and analyzed by ANOVA
c
Values in parentheses (percentage antiinflammatory activity, Al%)
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Table 2 In vitro antimicrobial
activity of compounds 5a–5m
using agar well diffusion
method
Compound
Diameter of zone of inhibition in mma
Staphylococcus
aureus
Bacillus
subtilis
Saccharomyces
cerevisiae
Candida
albicans
5a
17.6 (128)
15.3 (128)
13.6 (256)
14.0 (256)
12.6 ([256)
13.6 (256)
18.3 (64)
15.3 (128)
13.6 (256)
12.0 ([256)
18.6 (64)
15.6 (128)
13.3 (256)
26.6 (5)
18.3 (64)
16.3 (128)
15.0 (128)
15.3 (128)
13.6 (256)
15.0 (128)
19.3 (64)
18.6 (64)
17.3 (128)
13.6 ([256)
20.6 (32)
18.6 (64)
14.3 (256)
24.0 (5)
15.6 (64)
13.6 (128)
5b
–
–
5c
–
–
5d
–
–
5e
–
–
5f
17.3 (32)
16.0 (64)
17.6 (32)
–
15.3 (64)
13.6 (128)
14.3 (128)
–
5g
5h
5i
5j
16.3 (64)
15.6 (64)
–
12.6 ([256)
14.3 (128)
–
Nt not tested; – no activity
5k
Values in parentheses; MIC
(lg/ml) = minimum inhibitory
concentration
5l
5m
–
–
a
Values, including diameter of
the well (8 mm), are means of
three replicates
Ciprofloxacin
Amphotericin B
Nt
Nt
Nt
Nt
13.6 (100)
14.3 (100)
biphasic carrageenan edema assay indicates the capability
of the tested compounds to inhibit prostaglandin synthesis.
This can be attributed to their ability to bind cyclooxy-
genase (COX), an enzyme responsible for the synthesis of
prostaglandins. From the available data, no correlation can
be drawn between the positional preference of substituents
and AI activity.
activity against the tested bacterium, the results against two
fungal strains were encouraging. Six (5a, 5f, 5g, 5h, 5j, 5k)
out of 13 tested compounds (5a–5m) showed antifungal
activity with MIC in the range of 32–64 lg/ml that is better
than the reference drug amphotericin B which showed the
MIC of 100 against S. cerevisiae. However, only one of the
tested compounds 5f was found to be better than the ref-
erence drug amphotericin B against Candida albicans.
Four compounds (5a, 5g, 5h, 5k) showed antifungal
activity comparable to standard drug used against
C. albicans. Though no clear correlation between the
position of the substituents and antifungal activity could be
drawn, compounds having an ortho methoxy group in the
C5-phenyl were found to be exhibiting better antifungal
activity.
In vitro antimicrobial activity (bacteria and yeasts)
All the target compounds 5a–m were screened for their
antibacterial and antifungal activity against Staphylococcus
aureus (MTCC 96) and Bacillus subtilis (MTCC 121)
representing Gram-positive bacteria, Escherichia coli
(MTCC 1652) and Pseudomonas aeruginosa (MTCC 741)
representing Gram-negative bacteria, and two yeast, Can-
dida albicans (MTCC 227) and Saccharomyces cerevisiae
(MTCC 170). All the microbial cultures were procured
from Microbial Type Culture Collection (MTCC), IM-
TECH, Chandigarh. The bacteria were subcultured on
Nutrient agar whereas yeast on Malt yeast agar.
Conclusion
The objective of the synthesis and biological evaluation of
a new series of 1-(4-aminosulfonylphenyl)-3,5-diarylpy-
razolines (5) was to find dual antiinflammatory–antimi-
crobial agents. Amongst the tested compound, 5i showed
pronounced antiinflammatory activity (68.3% inhibition
after 3h) and some of compounds, 5d, 5g, 5h, and 5m
exhibited good antiinflammatory activity C55%. Though
the tested compounds failed to give encouraging results in
terms of their antibacterial properties, the antifungal
activity results are promising and demand further investi-
gations in this area. For instance, six (5a, 5f, 5g, 5h, 5j, 5k)
out of 13 tested compounds (5a–m) showed better
Most of the tested chemical compounds possessed
moderate antibacterial activity against both the Gram-
positive (Staphylococcus aureus and Bacillus subtilis)
bacteria (Table 2). However, all of the tested compounds
were found to be inactive (data not shown here) against
Gram-negative bacteria (E. coli and P. aeruginosa).
Amongst the tested compounds, 5k exhibited the lowest
MIC of 32 lg/ml against B. subtilis, whereas 5g showed
MIC of 64 lg/ml against S. aureus and B. subtilis. Though
the tested compounds failed to show any appreciable
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2949
(E)-1-(4-fluorophenyl)-3-(2,4-dimethoxyphenyl)prop-
2-en-1-one (3b)
antifungal activity than the reference drug amphotericin B
against yeast S. cerevisiae. It is interesting to note that the
best compound in terms of AI activity (5i) failed to show
antifungal activity and the best compound in terms of
antifungal activity (5f) failed to show appreciable AI
activity.
m.p. 134–135°C (Lit. 136°C) (Bandgar et al., 2010), yield
83%; H NMR (300 MHz, CDCl3): 8.08 (d, J = 15.6 Hz,
1
1H, COCH=CH), 8.07–8.03 (m, 2H, Ar–H), 7.56 (d,
J = 15.6 Hz, 1H, COCH=CH), 7.21–7.16 (m, 3H, Ar–H),
6.95 (dd, J = 2.4, 9.0 Hz, 1H, Ar–H), 6.90 (d, J = 9.0 Hz,
1H, Ar–H), 3.89 (s, 3H, OCH3), 3.84 (s, 3H, OCH3).
Experimental protocols
Melting points were determined in open glass capillaries in
an electrical melting point apparatus and are uncorrected.
The infrared (IR) spectra were recorded on Shimadzu-21
FT-IR or Perkin-Elmer IR Spectrophotometer using the
KBr pellet technique. 1H NMR and 13C NMR spectra were
recorded either in pure DMSO-d6 or in CDCl3 on Bruker
NMR spectrometers at 300 and 75.5 MHz, respectively,
using tetramethylsilane (TMS) as internal standard.
Chemical shifts are expressed in d, ppm. 1H NMR data are
reported in order: multiplicity (br, broad; s, singlet; d,
doublet; t, triplet; m, multiplet; *exchangeable by D2O),
approximate coupling constant in Hertz and number of
(E)-1-(4-fluorophenyl)-3-(2,5-dimethoxyphenyl)prop-
2-en-1-one (3c)
m.p. 110–112°C, yield 86%; 1H NMR (300 MHz, CDCl3):
8.09 (d, J = 16.2 Hz, 1H, COCH=CH), 8.08–8.04 (m, 2H,
Ar–H), 7.57 (d, J = 15.9 Hz, 1H, COCH=CH), 7.22–7.16
(m, 3H, Ar–H), 6.97 (dd, J = 2.4, 9.0 Hz, 1H, Ar–H), 6.90
(d, J = 9.0 Hz, 1H, Ar–H), 3.89 (s, 3H, OCH3), 3.84 (s, 3H,
OCH3).
1
(E)-1-(4-fluorophenyl)-3-(3,4-dimethoxyphenyl)prop-
2-en-1-one (3d)
protons. The purity of the compounds was checked by H
NMR and thin layer chromatography (TLC) on silica gel
plates using a mixture of petroleum ether and ethyl acetate
as eluent. Iodine or UV lamp was used as a visualizing
agent.
m.p. 82–83°C (Lit. 82°C) (Dhar and Singh, 1971), yield
79%; H NMR (300 MHz, CDCl3): 8.06 (t, J = 7.2 Hz,
1
2H, Ar–H), 7.77 (d, J = 15.6 Hz, 1H, COCH=CH), 7.37
(d, J = 15.6 Hz, 1H, COCH=CH), 7.25–7.17 (m, 4H, Ar–
H), 6.91 (d, J = 8.1 Hz, 1H, Ar–H), 3.96 (s, 3H, OCH3),
3.94 (s, 3H, OCH3).
General procedure for the synthesis of chalcones
3a–3m
To a stirred methanolic solution of NaOH (10 mmol) and
appropriate benzaldehyde (1 mmol) at ice-cold condition,
appropriate acetophenone (1.05 mmol) was added over
15 min. The reaction mixture was stirred for 18 h at
ambient temperature. The course of the reaction was
monitored with TLC. The contents of the flask were poured
into crushed ice and neutralized with dil. HCl that resulted
in precipitation of the yellowish solid. The solid obtained
was filtered, washed with water, and dried. The crude was
crystallized from ethanol–chloroform (8:2) to afford the
target chalcones 3 as solid material in excellent yield.
(E)-1-(4-fluorophenyl)-3-(3,5-dimethoxyphenyl)prop-
2-en-1-one (3e)
1
m.p. 87–88°C, yield 80%; H NMR (300 MHz, CDCl3):
8.06 (dd, J = 5.7, 8.4 Hz, 2H, Ar–H), 7.74 (d,
J = 15.6 Hz, 1H, –COCH=CH), 7.46 (d, J = 15.6 Hz, 1H,
–COCH=CH), 7.19 (t, J = 8.4Hz, 2H, Ar–H), 6.79 (d,
J = 1.5 Hz, 2H, Ar–H), 6.57–6.55 (m, 1H, Ar–H), 3.85 (s,
6H, 29 OCH3).
(E)-1-(4-fluorophenyl)-3-(2,3-dimethoxyphenyl)prop-
2-en-1-one (3a)
(E)-1-(4-chlorophenyl)-3-(2,3-dimethoxyphenyl)prop-
2-en-1-one (3f)
m.p. 68–69°C (Lit. 69°C) (Dhar and Singh, 1971), yield
88%; H NMR (300 MHz, CDCl3): 8.12 (d, J = 15.9 Hz,
1
m.p. 107–108°C (Lit. 109.5°C) (Tsatsas et al., 1960), yield
89%; H NMR (300 MHz, CDCl3): 8.09 (d, J = 15.6 Hz,
1
1H, COCH=CH), 8.09–8.05 (m, 2H, Ar–H), 7.59 (d,
J = 15.9 Hz, 1H, COCH=CH), 7.19 (t, J = 8.7 Hz, 2H,
Ar–H), 7.12 (t, J = 8.1 Hz, 2H, Ar–H), 6.99 (d,
J = 8.1 Hz, 1H, Ar–H), 3.91 (s, 3H, OCH3), 3.90 (s, 3H,
OCH3).
1H, COCH=CH), 7.98–7.92 (m, 3H, Ar–H), 7.58–7.42 (m,
2H, Ar–H), 7.51 (d, J = 15.3 Hz, 1H, COCH=CH), 7.46
(s, 1H, Ar–H), 6.56 (d, J = 8.4 Hz, 1H, Ar–H), 6.50 (s. 1H,
Ar–H), 3.92 (s, 3H, OCH3), 3.88 (s, 3H, OCH3).
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(E)-1-(4-chlorophenyl)-3-(2,4-dimethoxyphenyl)prop-
2-en-1-one (3g)
(E)-1-(2-hydroxy-4-methoxyphenyl)-3-
(2,4-dimethoxyphenyl)prop-2-en-1-one (3l)
m.p. 119–120°C (Lit. 120°C) (Bandgar et al., 2010), yield
92%; H NMR (300 MHz, CDCl3): 8.07 (d, J = 15.6 Hz,
m.p. 155–157°C (Lit. 158°C) (Boumendjel et al., 2008),
yield 87%; H NMR (300 MHz, CDCl3): 13.68 (s, 1H,
1
1
1H, COCH=CH), 7.96 (d, J = 8.1 Hz, 2H, Ar–H), 7.58 (d,
J = 8.4 Hz, 2H, Ar–H), 7.51 (d, J = 15.3 Hz, 1H,
COCH=CH), 7.46 (s, 1H, Ar–H), 6.56 (d, J = 8.4 Hz, 1H,
Ar–H), 6.50 (s. 1H, Ar–H), 3.92 (s, 3H, OCH3), 3.88 (s,
3H, OCH3).
OH), 8.11 (d, J = 15.3 Hz, 1H, COCH=CH–), 7.82–7.79
(m, 1H, Ar–H), 7.59 (d, J = 15.9 Hz, 1H, COCH=CH),
7.55 (d, J = 9.0 Hz, 1H, Ar–H), 6.52 (dd, J = 2.4, 8.7 Hz,
1H, Ar–H), 6.47–6.44 (m, 3H, Ar–H), 3.90 (s, 3H, OCH3),
3.84 (s, 3H, OCH3), 3.83 (s, 3H, OCH3).
(E)-1-(4-chlorophenyl)-3-(2,5-dimethoxyphenyl)prop-
2-en-1-one (3h)
(E)-1-(2-hydroxy-4-methoxyphenyl)-3-
(3,5-dimethoxyphenyl)prop-2-en-1-one (3m)
m.p. 56–58°C (Lit. 57°C, with decomposition) (Chaaban
et al., 2007), yield 86%; 1H NMR (300 MHz, CDCl3): 8.09
(d, J = 15.9 Hz, 1H, COCH=CH), 7.97 (d, J = 8.4 Hz,
2H, Ar–H), 7.55 (d, J = 15.9 Hz, 1H, COCH=CH), 7.48
(d, J = 8.4 Hz, 2H, Ar–H), 7.17 (s, 1H, Ar–H), 6.97 (dd,
J = 2.1, 8.7 Hz, 1H, Ar–H), 6.90 (d. J = 9.0 Hz, 1H, Ar–
H), 3.89 (s, 3H, OCH3), 3.84 (s, 3H, OCH3).
m.p. 148–150°C (Lit. 150°C) (Boumendjel et al., 2008),
yield 85%; H NMR (300 MHz, CDCl3): 13.39 (s, 1H,
1
–OH), 7.80 (d, 1H, J = 9.0 Hz, Ar–H), 7.77 (d,
J = 15.6 Hz, 1H, COCH=CH), 7.49 (d, J = 15.6 Hz, 1H,
COCH=CH), 6.75 (d, J = 2.1 Hz, 1H, Ar–H), 6.49 (dd,
J = 2.1, 9.0 Hz, 1H, Ar–H), 6.45 (s, 2H, Ar–H), 3.84 (s,
3H, OCH3), 3.82 (s, 6H, 29 OCH3).
(E)-1-(4-chlorophenyl)-3-(3,4-dimethoxyphenyl)prop-
2-en-1-one (3i)
General procedure for the synthesis of
1-(4-aminosulfonylphenyl)-3,5-diarylpyrazolines (5)
m.p. 108–109°C (Lit. 107–109°C) (Singh and Malik,
1994), yield 82%; H NMR (300 MHz, CDCl3): 7.97 (d,
1
Ethanolic solution of chalcone 3 (1.00 mmol) and 4-hydra-
zinobenzenesulfonamide hydrochloride 4 (1.10 mmol) in
presence of catalytic amount of glacial acetic acid was
refluxed for 12 h. The course of the reaction was monitored
by TLC. The contents of reaction flask were concentrated
and left overnight resulting in the formation of crystals. The
solid so obtained was filtered, dried over vacuum pump, and
crystallized from ethanol to afford the target pyrazolines 5 as
fluffy colorless crystals in excellent yield.
J = 7.8 Hz, 2H, Ar–H), 7.78 (d, J = 15.6 Hz, 1H,
COCH=CH), 7.49 (d, J = 7.8 Hz, 2H, Ar–H), 7.35 (d,
J = 15.6 Hz, 1H, COCH=CH), 6.25 (d, J = 8.4 Hz, 1H,
Ar–H), 7.17 (s, 1H, Ar–H), 6.92 (d, J = 8.4 Hz, 1H, Ar–
H), 3.97 (s, 3H, OCH3), 3.96 (s, 3H, OCH3).
(E)-1-(4-chlorophenyl)-3-(3,5-dimethoxyphenyl)prop-
2-en-1-one (3j)
1
1-(4-aminosulfonylphenyl)-3-(4-fluorophenyl)-5-
(2,3-dimethoxyphenyl) pyrazoline (5a)
m.p. 92–94°C, yield 87%; H NMR (300 MHz, CDCl3):
7.98 (d, J = 8.4 Hz, 2H, Ar–H), 7.75 (d, J = 15.6 Hz, 1H,
COCH=CH), 7.45 (d, J = 15.6 Hz, 1H, COCH=CH), 7.50
(d, J = 8.4 Hz, 2H, Ar–H), 6.79 (d, J = 2.4 Hz, 2H, Ar–
H), 6.56 (s, 1H, Ar–H), 3.86 (s, 6H, 29 OCH3).
m.p. 220–222°C, yield 82%; IR (cm-1): 3315 and 3235 (b,
N–H), 1595 (m, C=N), 1322 and 1151 (s, SO2); 1H NMR (300
MHz, DMSO-d6): d7.84 (t, J = 6.3 Hz, 2H, Ar–H), 7.60 (d,
J = 8.1 Hz, 2H, Ar–H), 7.29 (t, J = 8.1 Hz, 2H, Ar–H),
7.04–6.96 (m, 5H, Ar–H, SO2NH2), 6.58 (s, 1H, Ar–H), 5.72
(dd, J = 4.8, 12.3 Hz, 1H, pyrazoline-H), 3.97 (dd, J = 12.6,
17.4 Hz, 1H, pyrazoline-H), 3.84 (s, 3H, OCH3), 3.81 (s, 3H,
(E)-1-(2-hydroxy-4-methoxyphenyl)-3-
(2,3-dimethoxyphenyl)prop-2-en-1-one (3k)
m.p. 129–130°C (Lit. 130°C) (Boumendjel et al., 2008),
yield 91%; H NMR (300 MHz, CDCl3): 13.43 (s, 1H,
OCH3), 3.16 (dd, J = 4.8, 17.1 Hz, 1H, pyrazoline-H); 13
C
1
NMR (75.5 MHz, DMSO-d6): 163.1 (d, 1JC–F = 247.6 Hz),
153.1, 149.6, 146.3, 146.1, 134.9, 133.4, 128.9 (d,
OH), 8.12 (d, J = 15.6 Hz, 1H, COCH=CH), 7.78 (d,
J = 9.9 Hz, 1H, Ar–H), 7.62 (d, J = 15.9 Hz, 1H,
COCH=CH), 7.21 (s, 1H, Ar–H), 7.06 (t, J = 8.1 Hz, 1H,
Ar–H), 6.93 (d, J = 8.4 Hz, 1H, Ar–H), 6.45–6.42 (m, 2H,
Ar–H), 3.86 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 3.81 (s,
3H, OCH3).
3
4JC–F = 3.0 Hz), 128.6 (d, JC–F = 8.3 Hz), 127.6, 124.9,
118.3, 116.1 (d, 2JC–F = 21.7 Hz), 112.8, 112.1, 60.71, 58.4
(C5-pyrazoline), 56.1 (OCH3), 42.3 (C4-pyrazoline). Anal.
Calcd. for C23H22FN3O4S: N, 9.23: found: N, 9.31.
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Med Chem Res (2012) 21:2945–2954
2951
128.7 (d, 3JC–F = 8.3 Hz), 127.5, 117.9, 116.2 (d, 2JC–F
=
21.9 Hz), 112.7, 112.5, 110.3, 62.9 (C5-pyrazoline), 55.9
(OCH3), 55.9 (OCH3), 43.6 (C4-pyrazoline). Anal. Calcd.
for C23H22FN3O4S: N, 9.23: found: N, 9.40.
1-(4-aminosulfonylphenyl)-3-(4-fluorophenyl)-5-
(2,4-dimethoxyphenyl) pyrazoline (5b)
m.p. 178–180°C, yield 79%; IR (cm-1): 3320 and 3236 (b,
N–H), 1596 (m, C=N), 1324 and 1155 (s, SO2); H NMR
1
(300 MHz, DMSO-d6): d 7.82 (t, J = 6.6 Hz, 2H, Ar–H),
7.60 (d, J = 8.4 Hz, 2H, Ar–H), 7.27 (d, J = 8.4 Hz, 2H,
Ar–H), 7.04 (s, 2H, –SO2NH2), 7.00 (d, J = 8.4 Hz, 2H,
Ar–H), 6.77 (d, J = 8.1 Hz, 1H, Ar–H), 6.64 (s, 1H,
Ar–H), 6.41 (d, J = 8.4 Hz, 1H, Ar–H), 5.65 (dd, J = 4.5,
12.0 Hz, 1H, pyrazoline-H), 3.88 (s, 3H, OCH3), 3.71 (s,
1-(4-aminosulfonylphenyl)-3-(4-fluorophenyl)-5-
(3,5-dimethoxyphenyl)pyrazoline (5e)
m.p. 210–212°C, yield 80%; IR (cm-1): 3328 and 3245 (b,
N–H), 1596 (m, C=N), 1320 and 1151(s, SO2); H NMR
1
(300 MHz, DMSO-d6): d 7.84 (dd, J = 5.7, 8.4 Hz, 2H,
Ar–H), 7.61 (d, J = 9.0 Hz, 2H, Ar–H), 7.29 (t,
J = 9.0 Hz, 2H, Ar–H), 7.08 (d, J = 9.0 Hz, 2H, Ar–H),
7.03 (s, 2H, SO2NH2), 6.39 (s, 3H, Ar–H), 5.54 (dd,
J = 5.4, 12.0 Hz, 1H, pyrazoline-H), 3.94 (dd, J = 12.0,
17.7 Hz, 1H, pyrazoline-H), 3.69 (s, 6H, 29 OCH3), 3.20
(dd, J = 5.4, 17.7 Hz, 1H, pyrazoline-H); 13C NMR (75.5
3H, OCH3), 3.07 (dd, J = 4.5, 17.7 Hz, 1H, pyrazoline-H);
13C NMR (75.5 MHz, DMSO-d6): 163.5 (d, JC–F
1
=
246 Hz), 160.5, 157.6, 149.7, 146.2, 133.2, 129.0, 128.6 (d,
3JC–F = 8.3 Hz), 127.1, 120.9, 116.1 (d, 2JC–F = 21.7 Hz),
112.1, 105.5, 99.4, 57.6 (C5-pyrazoline), 56.2 (OCH3),
55.6 (OCH3), 42.4 (C4-pyrazoline). Anal. Calcd. for
C23H22FN3O4S: N, 9.23: found: N, 9.34.
1
MHz, DMSO-d6): 161.5, 159.1 (d, JC–F = 246.9 Hz),
3
149.5, 146.5, 144.5, 133.5, 128.8 (d, JC–F = 9.1 Hz),
127.6, 116.2 (d, 2JC–F = 21.9 Hz), 112.5, 104.2, 99.1, 63.0
(C5-pyrazoline), 55.6 (OCH3), 43.4 (C4-pyrazoline). Anal.
Calcd. for C23H22FN3O4S: N, 9.23: found: N, 9.38.
1-(4-aminosulfonylphenyl)-3-(4-fluorophenyl)-5-
(2,5-dimethoxyphenyl) pyrazoline (5c)
m.p. 84–86°C, yield 87%; IR (cm-1): 3322 and 3240 (b, N–
H), 1595 (m, C=N), 1315 and 1145 (s, SO2); 1H NMR (300
MHz, DMSO-d6): d 7.86–7.81 (m, 2H, Ar–H), 7.61 (d,
J = 8.7 Hz, 2H, Ar–H), 7.27 (t, J = 8.7 Hz, 2H, Ar–H),
7.04–7.00 (m, 5H, Ar–H, –SO2NH2), 6.82 (dd, J = 2.7,
9.0 Hz, 1H, Ar–H), 6.41 (s, 1H, Ar–H), 5.69 (dd, J = 4.8,
12.0 Hz, 1H, pyrazoline-H), 3.93 (dd, J = 12.0, 17.4 Hz,
1H, pyrazoline-H), 3.84 (s, 3H, OCH3), 3.56 (s, 3H, OCH3),
3.11 (dd, J = 5.1, 17.7 Hz, 1H, pyrazoline-H); 13C NMR
1-(4-aminosulfonylphenyl)-3-(4-chlorophenyl)-5-
(2,3-dimethoxyphenyl)pyrazoline (5f)
m.p. 230–232°C, yield 82%; IR (cm-1): 3325 and 3240 (b,
N–H), 1597 (m, C=N), 1319 and 1149 (s, SO2); H NMR
1
(300 MHz, DMSO-d6): d 7.80 (d, J = 8.4 Hz, 2H, Ar–H),
7.60 (d, J = 8.7 Hz, 2H, Ar–H), 7.50 (d, J = 8.7 Hz, 2H,
Ar–H), 7.06–7.03 (m, 4H, Ar–H, –SO2NH2), 6.97 (d,
J = 4.8 Hz, 2H, Ar–H), 6.60–6.57 (m, 1H, Ar–H), 5.74
(dd, J = 5.1, 12.0 Hz, 1H, pyrazoline-H), 3.97 (dd,
J = 12.0, 18.0 Hz, 1H, pyrazoline-H), 3.83 (s, 3H, OCH3),
3.81 (s, 3H, OCH3), 3.16 (dd, J = 5.4, 18.0 Hz, 1H, pyr-
azoline-H); 13C NMR (75 MHz, DMSO-d6): 153.2, 149.4,
146.1, 134.8, 134.1, 133.6, 131.2, 129.2, 128.2, 127.7,
124.9, 118.4, 112.9, 112.3, 60.7 (C5-pyrazoline), 58.6
(OCH3), 56.1 (OCH3), 49.1, 42.5 (C4-pyrazoline). Anal.
Calcd. for C23H22ClN3O4S: N, 8.90: found: N, 8.79.
1
(75 MHz, DMSO-d6): 163.1 (d, JC–F = 245.3 Hz), 153.7,
4
150.6, 149.8, 146.3, 133.2, 129.1, 128.9 (d, JC–F
3
2
= 3.0 Hz), 128.7 (d, JC–F = 8.3 Hz), 116.2 (d, JC–F
= 21.7 Hz), 113.2, 112.8, 112.2, 58.0 (C5-pyrazoline), 56.6
(OCH3), 55.7 (OCH3), 42.3 (C4-pyrazoline). Anal. Calcd.
for C23H22FN3O4S: N, 9.23: found: N, 9.28.
1-(4-aminosulfonylphenyl)-3-(4-fluorophenyl)-5-
(3,4-dimethoxyphenyl)pyrazoline (5d)
m.p. 198–200°C, yield 81%; IR (cm-1): 3320 and 3235 (b,
N–H), 1596 (m, C=N), 1325 and 1155 (s, SO2); H NMR
1-(4-aminosulfonylphenyl)-3-(4-chlorophenyl)-5-
(2,4-dimethoxyphenyl)pyrazoline (5g)
1
(300 MHz, DMSO-d6): d 7.84–7.82 (m, 2H, Ar–H), 7.60 (d,
J = 9.0 Hz, 2H, Ar–H), 7.29 (t, J = 9.0 Hz, 2H, Ar–H),
7.09 (d, J = 9.0 Hz, 2H, Ar–H), 7.04 (s, 2H, SO2NH2), 6.94
(d, J = 1.8 Hz, 1H, Ar–H), 6.88 (d, J = 8.4 Hz, 1H, Ar–H),
6.68 (dd, J = 1.8, 8.4 Hz, 1H, Ar–H), 5.54 (dd, J = 5.4,
12.0 Hz, 1H, pyrazoline-H), 3.93 (dd, J = 12.0, 17.7 Hz,
1H, pyrazoline-H), 3.72 (s, 3H, OCH3), 3.69 (s, 3H, OCH3),
3.19 (dd, J = 5.4, 17.4 Hz, 1H, pyrazoline-H); 13C NMR
(75.5 MHz, DMSO-d6): 163.1 (d, 1JC–F = 246.9 Hz), 149.6,
149.3, 148.6, 146.6, 134.4, 133.5, 128.9 (d, 4JC–F = 3.0 Hz),
m.p. 208–210°C, yield 86%; IR (cm-1): 3320 and 3235 (b,
N–H), 1598 (m, C=N), 1326 and 1153 (s, SO2); H NMR
1
(300 MHz, DMSO-d6): d 7.75 (d, J = 7.8 Hz, 2H, Ar–H),
7.69 (d, J = 8.4 Hz, 2H, Ar–H), 7.43 (d, J = 8.1 Hz, 2H,
Ar–H), 7.01–6.98 (m, 4H, Ar–H, –SO2NH2), 6.78 (d,
J = 8.4 Hz, 1H, Ar–H), 6.60 (s, 1H, Ar–H), 6.38 (d,
8.4 Hz, 1H, Ar–H), 5.64 (dd, J = 4.5, 11.7 Hz, 1H, pyr-
azoline-H), 3.88 (s, 3H, OCH3), 3.71 (s, 3H, OCH3), 3.05
(dd, J = 4.5, 17.7 Hz, 1H, pyrazoline-H); 13C NMR (75
123

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Med Chem Res (2012) 21:2945–2954
MHz, DMSO-d6): 160.6, 157.5, 149.2, 146.2, 134.2, 133.5,
131.3, 129.0, 127.9, 127.6, 127.1, 120.8, 112.2, 105.4,
99.3, 57.8 (C5-pyrazoline), 56.1 (OCH3), 55.6 (OCH3),
42.2 (C4-pyrazoline). Anal. Calcd. for C23H22ClN3O4S: N,
8.90: found: N, 8.96.
161.5, 148.9, 146.4, 144.3, 134.3, 133.8, 131.1, 129.1,
127.9, 127.6, 112.5, 104.1, 99.2, 63.3 (C5-pyrazoline), 55.5
(OCH3), 43.2 (C4-pyrazoline). Anal. Calcd. for C23H22
ClN3O4S: N, 8.90: found: N, 8.96.
1-(4-aminosulfonylphenyl)-3-(2-hydroxy-
4-methoxyphenyl)-5-(2,3-dimethoxyphenyl)pyrazoline (5k)
1-(4-aminosulfonylphenyl)-3-(4-chlorophenyl)-
5-(2,5-dimethoxyphenyl)pyrazoline (5h)
m. p. 218–220°C, yield 88%; IR (cm-1): 3326 and 3238 (b,
N–H), 1602 (m, C=N), 1325 and 1156 (s, SO2); H NMR
1
m.p. 228–230°C, yield 78%; IR (cm-1): 3327 and 3236 (b,
N–H), 1591 (m, C=N), 1325 and 1154 (s, SO2); H NMR
1
(300 MHz, DMSO-d6): d 10.53 (s, 1H, OH), 7.62 (d,
J = 8.6 Hz, 2H, Ar–H), 7.41 (d, J = 8.6 Hz, 1H, Ar–H),
7.05 (s, 2H, –SO2NH2), 6.98 (d, J = 4.8 Hz, 2H, Ar–H),
6.93 (d, J = 8.7 Hz, 2H, Ar–H), 6.65–6.52 (m, 3H, Ar–H),
5.65 (dd, J = 5.2, 12.1 Hz, 1H, pyrazoline-H), 4.06 (dd,
J = 12.2, 17.7 Hz, 1H, pyrazoline-H), 3.82 (s, 6H, 29
OCH3), 3.78 (s, 3H, OCH3), 3.30 (dd, J = 5.2, 17.9 Hz, 1H,
pyrazoline-H); 13C NMR (75 MHz, DMSO-d6): 161.8,
158.2, 152.9, 152.6, 145.9, 145.5, 134.5, 133.2, 129.8, 127.6,
124.7, 118.2, 112.7, 111.6, 109.8, 106.7, 101.3, 60.4
(C5-pyrazoline), 56.8 (OCH3), 55.9 (OCH3), 55.6 (OCH3),
43.5 (C4-pyrazoline). Anal. Calcd. for C24H25N3O6S: N,
8.69: found: N, 8.78.
(300 MHz, DMSO-d6): d 7.79 (d, J = 7.8 Hz, 2H, Ar–H),
7.62 (d, J = 8.1 Hz, 2H, Ar–H), 7.49 (d, J = 7.8 Hz, 2H,
Ar–H), 7.04–6.02 (m, 5H, Ar–H, –SO2NH2), 6.82 (d,
J = 9.0 Hz, 1H, Ar–H), 6.42 (s, 1H, Ar–H), 5.71 (dd,
J = 4.5, 12.6 Hz, 1H, pyrazoline-H), 3.92 (dd, J = 12.6,
17.4 Hz, 1H, pyrazoline-H), 3.84 (s, 3H, OCH3), 3.56 (s,
3H, OCH3), 3.10 (dd, J = 4.5, 17.4 Hz, 1H, pyrazoline-H);
13C NMR (75 MHz, DMSO-d6): 153.7, 150.6, 149.6,
146.2, 134.2, 133.7, 131.2, 129.9, 129.2, 128.2, 127.7,
113.2, 112.8, 112.3, 58.2 (C5-pyrazoline), 56.6 (OCH3),
55.7 (OCH3), 42.1 (C4-pyrazoline). Anal. Calcd. for
C23H22ClN3O4S: N, 8.90: found: N, 8.81.
1-(4-aminosulfonylphenyl)-3-(4-chlorophenyl)-
5-(3,4-dimethoxyphenyl)pyrazoline (5i)
1-(4-aminosulfonylphenyl)-3-(2-hydroxy-4-
methoxyphenyl)-5-(2,4-dimethoxyphenyl)pyrazoline (5l)
m.p. 184–186°C, yield 85%; IR (cm-1): 3328 and 3235 (b,
N–H), 1595 (m, C=N), 1321 and 1151 (s, SO2); H NMR
m.p. 228–230°C, yield 84%; IR (cm-1): 3327 and 3243 (b,
N–H), 1599 (m, C=N), 1321 and 1152 (s, SO2); H NMR
1
1
(300 MHz, DMSO-d6): d 7.62 (d, J = 7.8 Hz, 2H, Ar–H),
7.59 (d, J = 6.6 Hz, 2H, Ar–H), 7.29 (d, J = 8.7 Hz, 2H,
Ar–H), 7.00 (d, J = 8.4 Hz, 2H, Ar–H), 6.74 (s, 2H, –
SO2NH2), 6.66 (s, 1H, Ar–H), 5.23 (dd, J = 6.0, 12.0 Hz,
1H, pyrazoline-H), 3.83 (s, 3H, OCH3), 3.77 (s, 3H,
OCH3), 3.05 (dd, J = 4.5 Hz, 17.7 Hz, 1H, Ar–H); 13C
NMR (75 MHz, DMSO-d6): 149.7, 148.6, 148.1, 146.8,
134.9, 133.6, 132.2, 130.6, 128.8, 127.2, 126.9, 117.8,
112.5, 111.7, 108.5, 63.6 (C5-pyrazoline), 55.9 (OCH3),
55.8 (OCH3), 43.5 (C4-pyrazoline). Anal. Calcd. for
C23H22ClN3O4S: N, 8.90: found: N, 9.02.
(300 MHz, DMSO-d6): d 10.52 (s, 1H, OH), 7.61 (d,
J = 8.1 Hz, 2H, Ar–H), 7.40 (d, J = 8.4 Hz, 1H, Ar–H),
7.06 (s, 2H, –SO2NH2), 7.01–6.96 (m, 3H, Ar–H), 6.88 (d,
J = 8.1 Hz, 1H, Ar–H), 6.70 (d, J = 8.1 Hz, 1H, Ar–H),
6.56–6.52 (m, 2H, Ar–H), 5.46 (dd, J = 5.4, 11.7 Hz, 1H,
pyrazoline-H), 4.02 (dd, J = 12.3, 17.7 Hz, 1H, pyrazoline-
H), 3.78 (s, 3H, OCH3), 3.73 (s, 3H, OCH3), 3.69 (s, 3H,
OCH3), 3.29 (dd, J = 5.1, 18 Hz, 1H, pyrazoline-H); 13C
NMR (75 MHz, DMSO-d6): 162.2, 158.5, 152.6, 149.6,
148.6, 146.2, 134.2, 133.6, 130.1, 127.7, 118.0, 112.6, 112.3,
110.3, 107.0, 101.6, 61.6 (C5-pyrazoline), 56.0 (OCH3), 55.9
(OCH3), 55.8 (OCH3), 44.7 (C4-pyrazoline). Anal. Calcd.
for C24H25N3O6S: N, 8.69: found: N, 8.64.
1-(4-aminosulfonylphenyl)-3-(4-chlorophenyl)-
5-(3,5-dimethoxphenyl) pyrazoline (5j)
1-(4-aminosulfonylphenyl)-3-(2-hydroxy-
4-methoxyphenyl)-5-(3,5-dimethoxyphenyl)pyrazoline (5m)
m.p. 182–184°C, yield 76%; IR (cm-1): 3326 and 3238 (b,
N–H), 1589 (m, C=N), 1324 and 1143 (s, SO2); H NMR
1
(300 MHz, DMSO-d6): d 7.76 (d, J = 7.8 Hz, 2H, Ar–H),
7.61 (d, J = 8.1 Hz, 2H, Ar–H), 7.44 (d, J = 7.5 Hz, 2H,
Ar–H), 7.07 (d, J = 8.1 Hz, 2H, Ar–H), 6.98 (s, 2H,
–SO2NH2), 6.37 (s, 3H, Ar–H), 5.76 (dd, J = 5.1, 12.0 Hz,
1H, pyrazoline-H), 4.02 (dd, J = 12.0, 17.4 Hz, 1H, pyr-
azoline-H), 3.68 (s, 6H, 29 OCH3), 3.16 (dd, J = 5.1,
17.7 Hz, 1H, Ar–H); 13C NMR (75 MHz, DMSO-d6):
m.p. 232–234°C, yield 83%; IR (cm-1): 3329 & 3247 (b,
N–H), 1605 (m, C=N), 1323 and 1157 (s, SO2); H NMR
1
(300 MHz, DMSO-d6): d 10.47 (s, 1H, OH), 7.63 (d,
J = 8.4 Hz, 2H, Ar–H), 7.41 (d, J = 8.4 Hz, 1H, Ar–H),
7.06 (s, 2H, -SO2NH2), 6.99 (d, J = 8.7 Hz, 2H, Ar–H),
6.56 (s, 1H, Ar–H), 6.53 (d, J = 8.7 Hz, 1H, Ar–H), 6.41
(s, 3H, Ar–H), 5.46 (dd, J = 6.6, 11.7 Hz, 1H, pyrazoline-
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Med Chem Res (2012) 21:2945–2954
2953
H), 4.02 (dd, J = 12.0, 17.7 Hz, 1H, pyrazoline-H), 3.78
(s, 3H, OCH3), 3.73 (s, 6H, 29 OCH3), 3.31 (merged with
HOD peak of DMSO-d6, 1H, pyrazoline-H); 13C NMR
(75.5 MHz, DMSO-d6): 162.2, 161.5, 158.5, 152.6, 146.1,
144.3, 133.7, 130.1, 127.7, 112.3, 110.1, 107.0, 104.3,
101.6, 99.2, 61.7 (C5-pyrazoline), 55.8 (OCH3), 55.6
(OCH3), 44.6 (C4-pyrazoline). Anal. Calcd. for C24H25
N3O6S: N, 8.69: found: N, 8.83.
suspension of approximately 1.5 9 108 cfu/ml. 20 ml of
agar medium was poured into each Petri plate and plates
were swabbed with 100 ll inocula of the test microor-
ganisms and kept for 15 min for adsorption. Using sterile
cork borer of 8 mm diameter, wells were bored into the
seeded agar plates and these were loaded with a 100 ll
volume with concentration of 4.0 mg/ml of each com-
pound reconstituted in the dimethylsulphoxide (DMSO).
All the plates were incubated at 37°C for 24 h. Antimi-
crobial activity of each compound was evaluated by
measuring the zone of growth inhibition against the test
organisms with zone reader (Hi Antibiotic zone scale).
DMSO was used as a negative control, whereas Cipro-
floxacin was used as positive control for bacteria and
amphotericin B for yeast. This procedure was performed
in three replicate plates for each organism (Ahmad and
Beg, 2001; Andrews, 2001).
Pharmacological assay
Carrageenan-induced rat paw edema assay
Male Wistar albino rats weighing 200–500 g were used
throughout the study. They were kept in the animal house
under standard conditions of light and temperature with free
accesstofoodandwater.Foodwaswithdrawn12 hbeforeand
during experimental hours. The animals were randomly
divided into groups each consisting of six rats. One group of
six rats was kept as control and received tween 80 (95:5).
Another group received the standard drug indomethacin at a
dose of 4 mg/kg body weight, i.p. Other groups of rats were
administered the test compounds at a dose of 50 mg/kg body
weight orally. A mark was made on the left hind paw just
beyond the tibiotarsal articulation, so that every time the paw
was dipped up to fixed mark and constant paw volume was
ensured. Paw volumes were measured using a plethysmom-
eter (model 7140, Ugo Basile, Italy). Thirty minutes after
administrationoftest and standarddrugs, 0.1 ml, of1%w/vof
carrageenan suspension in normal saline was injected into
subplanter region of the left hind paw of all the animals. The
initial paw volume was measured within 30 s of the injection
and remeasured again 1, 2, 3, and 4 h after administration of
carrageenan. The edema was expressed as an increase in the
volume of paw, and the percentage of edema inhibition for
each rat and each group was obtained as follows:
MIC of the various compounds against bacterial and
yeast strains was tested through a modified agar well dif-
fusion method (Okeke et al., 2001). In this method, a
twofold serial dilution of each chemically synthesized
compound was prepared by first reconstituting the com-
pound in DMSO followed by dilution in sterile distilled
water to achieve a decreasing concentration range of
256–0.5 lg/ml. A 100 ll volume of each dilution was
introduced into wells (in triplicate) in the agar plates
already seeded with 100 ll of standardized inoculum
(106 cfu/ml) of the test microbial strain. All test plates were
incubated aerobically at 37°C for 24 h and observed for the
inhibition zones. MIC, taken as the lowest concentration of
the chemical compound that completely inhibited the
growth of the microbe, showed by a clear zone of inhibi-
tion, was recorded for each test organism. Ciprofloxacin
and amphotericin B was used as positive control, while
DMSO as negative control.
% inhibition ¼ ½ðVt ꢁ V0Þ control ꢁ ðVt ꢁ V0Þ tested compound =ðVt ꢁ V0Þ controlꢂ ꢃ 100
Acknowledgments One of the authors (S.K.) is grateful to the
where Vt is the volume of edema at specific time interval and
V0 is volume of edema at zero time interval.
University Grants Commission (UGC), New Delhi for the award of
senior research fellowship. Defence Research and Development
Organization (DRDO), New Delhi is acknowledged for financial
support in the form of a research project.
In vitro antimicrobial assay
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