Synthesis, biological evaluation and docking study of maleimide derivatives bearing benzenesulfonamide as selective COX-2 inhibitors and anti-inflammatory agents

Article abstract of DOI:10.1016/j.bmc.2015.07.070

A series of maleimide analogs bearing benzenesulfonamide were synthesized (4a-r). The anti-inflammatory activity of synthesized derivatives was evaluated using carrageenan induced rat paw edema model. COX-1 and COX-2 potency was evaluated through in vitro

Full text of DOI:10.1016/j.bmc.2015.07.070

Accepted Manuscript
Synthesis, biological evaluation and docking study of maleimide derivatives
bearing benzenesulfonamide as selective COX-2 inhibitors and anti-inflamma-
tory agents
Sandip D. Firke, Sanjay B. Bari
PII:
S0968-0896(15)00654-9
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.07.070
BMC 12499
Reference:
Bioorganic & Medicinal Chemistry
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Received Date:
Revised Date:
Accepted Date:
24 June 2015
30 July 2015
30 July 2015
Please cite this article as: Firke, S.D., Bari, S.B., Synthesis, biological evaluation and docking study of maleimide
derivatives bearing benzenesulfonamide as selective COX-2 inhibitors and anti-inflammatory agents, Bioorganic
& Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.07.070
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Synthesis, biological evaluation and docking study of maleimide derivatives bearing
benzenesulfonamide as selective COX-2 inhibitors and anti-inflammatory agents
Sandip D. Firke1*, Sanjay B. Bari2
1R. C. Patel Institute of Pharmaceutical Education and Research, Shirpur
2H. R. Patel Institute of Pharmaceutical Education and Research, Shirpur
*Corresponding Author.
Sandip D. Firke,
Assistant Professor
Department of Pharmaceutical
Chemistry,R. C. Patel Institute of Pharmaceutical
Education and Research Shirpur, Dist Dhule 425 405
Tel: +91-8055917640 Fax: +912563251808
E-mail address: sandipfirke@rediffmail.com (Mr. Sandip D Firke)
1

Abstract
A series of maleimide analogs bearing benzenesulfonamide were synthesized (4a-r). The anti-
inflammatory activity of synthesized derivatives was evaluated using carageenan induced rat
paw edema model. COX-1 and COX-2 potency was evaluated through invitrocyclooxygenase
assays. The results revealed that, compounds 4a, 4h, 4j, 4k and 4r had potent COX-2 percentage
inhibition as well as invivo anti- inflammatory activity. The potent compound 4j was docked into
the COX-2 active site to determine the probable binding model. The results of invivo and invitro
studies demonstrate that phenyl ring with electron withdrawing groups on maleimide ring would
generate more potent anti- inflammatory agents. Thus, these compounds can serve as potential
leads for further anti-inflammatory studies.
Keywords: Maleimide, COX-2, Ulcerogenic effects, Docking, Carageenan paw edema.
2

1. Introduction:
Inflammation is a localized physical condition with heat, swelling, redness and usually pain. It is
mediated by the release of proinflammatory mediators (bradykinin, and cytokines), which in turn
increases the rate of prostaglandin synthesis.[1, 2] Nonsteroidal anti-inflammatory drugs
(NSAIDs) inhibit the biosynthesis of prostaglandins (PGs) by inhibiting cyclooxygenases
(COX). It exists in two isoforms; a constitutive form (COX-1) and an inducible form (COX-
2).[3, 4]COX-1 enzyme is responsible for maintenance of gastric integrity and kidney function
whereas COX-2 is involved in the inflammation and pain.[5, 6]
Literature revealed that many NSAIDs including aspirin and indomethacin were found to interact
with these enzymesnonselectively and inhibit both COX-1 and COX-2.[7]Non-selective inhibitor
aspirin inhibits COX-1 more strongly than COX-2[7] and reduces the production of
prostaglandin E2 (PG-E2) and prostacyclin (PG-I2) which has an adverse ulcerogenic
effects.[8]COX-2 selective inhibitors used for treating inflammation and they have been shown a
much lower gastrointestinal side effects.[9, 10] However, COX-2 selective inhibitors found to
possesses cardiovascular side effects,[11]it gives an idea about the need to discover novel
scaffolds with COX-2 inhibitory activity and evaluate their anti-inflammatory effects.
Consequently, selective COX-2 inhibitorshave been developed to diminish the risk of unwanted
side effects.
So far, various classes of cyclic imides like maleimide, succimimde, phthalimide have been
received great attention due to their anti-inflammatory, antihyperlipidemic and antitumor
activities.[12-15] Recently, it was reported that N-substituted cyclic imide derivatives afforded a
new scaffold for small molecule COX-2 inhibitors which should offer opportunities for various
kinds of structural development.[16]In addition to this, N-substituted cyclic imide derivatives
3

possess remarkable anti-inflammatory activity through inhibition of tumor necrosis factor alpha
(TNF-α) and interleukin-6 (IL-6).
On the other hand, sulfonamide has been consistently rewarded as promising molecule because
of its broad spectrum of pharmacological activities like antibacterial[17],carbonic anhydrase
inhibitors[18, 19], hypoglycemic[20, 21], antithyroid[22] and antiproton activities.[23-
25]Moreover, among the highly marketed COX-2 inhibitors that comprise the sulfonamide
moiety, celecoxib and SC-558 (Fig.1) are the major determinant for COX-2 selectivity and in-
vivo efficacy.[13, 15, 26]Promising anti-inflammatory activity of some maleimide as well as
sulfonamide derivatives (Fig.2) prompted us to investigate them further. However, a maleimide
ring has not been appearing to be linked with benzenesulfonamide so far. Based on the literature
information, we decided to combine maleimide ring and benzenesulfonamide moiety to get new
compounds, which is prerequisite for anti-inflammatory activity. Based on the above mentioned
fact, in the proposed study, we aim to investigatewhether maleimide analogs bearing
benzenesulfonamide shows anti-inflammatory activity and cyclooxygenase inhibitory activity.
4

2
2
2
2
3
3
3
2
2
2
2
3
Figure 1. Structures of COX-2 inhibitors containing sulfonamide moiety
5

3
3
2
2
2
2
3
2
2
Figure 2 Common features present in COX-2 inhibitors and targeted compounds
2. Results and discussion
2.1 Chemistry
Synthesis of the titled compounds (4a-r) has been carried out as being depicted in scheme 1. The
intermediate 3, 4-dichloromaleimido benzenesulfonamide (3) was obtained by reacting 3, 4-
dichloromaleic anhydride(1) with benzenesulfonamide in glacial acetic acid at 90°C. The IR
spectra of dichloromaleimido benzenesulfonamide (3) showed the presence of the NH2
absorption band in 3458 cm-1 and SO2 absorption bands in 1344cm-1, 1158 cm-1 region, which
6

1
confirms the presence of free SO2NH2whereas its HNMR revealed the singlet of -NH2 protons
at δ7.36. Furthermore, the substitution of only one chlorine atom of compound 3 by appropriate
aryl/alkylamine in dimethyl formamide (DMF) at room temperature probably occurs by a
mechanism of addition-elimination, which loses a chlorine atom to form the desired compounds
4a-r.
The IR spectra of desired derivatives 4a-r exhibited –NH stretching vibrations in the region of
1
3364-3200cm-1. The HNMR spectrum exhibited singlet signal due to-NH proton of substituted
anilineat δ 12.92-8.15 and -NH2 of -SO2NH2 at δ7.40. Most of the synthesized compounds are
reported for the first time and gave satisfactory spectroscopic data.All the synthesized
compounds were characterized by their physical and spectral data.
2.2 Biological evaluation
2.2.1 In vitrocyclooxygenase (COX) inhibition
The ability of thesynthesized compounds(4a-r) to inhibit ovine COX-1 and human recombinant
COX-2 isozymes was determined using an ovine COX-1/COX-2 assay kit (catalog no. 560101,
Cayman Chemicals Inc., Ann Arbor, MI, USA).The % inhibition values calculated represents
means of two determinations (Table 1). Moreover, the selectivity indices (SI values) defined as
COX-2/COX-1 were calculated and compared with that of the standard selective COX-2
inhibitor; celecoxib. We estimated COX inhibition at a concentration of 1µM and eliminated
those derivatives that did not have an inhibitory action on either COX-1 or COX-2. Results
demonstrate that most of the synthesized compounds exhibited remarkable and selective
inhibition of COX-2 and weak inhibition of COX-1. Among these synthesized derivatives,
compounds 4a, 4h, 4j, 4k, 4q, and 4r showed potent % inhibitory activity. An analysis of
cyclooxygenase inhibitory activity of compounds 4a-r showed the subsequent structure activity
7

relationship.The activities of compounds may be due to the sulfonamide moieties, which may
interact with the active site of the COX-2 enzyme.[27]The unsubstituted 3, 4-dichloromaleimido
benzenesulfonamide (3) showed poor COX-2 inhibition (7.37%). The substitution on the phenyl
ring attached at the fourth position of maleimide ring was a determinant of COX-2 inhibitory
potency and selectivity of 3-chloro-4-substitutedmaleimido benzenesulfonamide derivatives(4a-
r). The results confirmed that the introduction of electronegative groups to meta position
improves the COX-2 inhibitory activities than para position. The results also indicated that
replacement of substituted anilines by cyclic amines also affect the COX-2 inhibitory activities.
Table 1
In vitro COX-1/COX-2 enzyme inhibition assay of the compounds
SIb
% inhibition (1µM)
Compound No.
(COX-2/COX-1)
0.611
44.52
2.60
COX-1
12.05
0.89
9.37
6.25
0.89
0.44
0.89
1.33
0.89
7.14
0.89
0.89
4.01
0.44
4.01
5.35
COX-2
7.373
39.63
24.42
10.14
17.05
6.45
3
4a
4b
4c
4d
4e
4f
1.62
19.15
14.65
11.9
10.6
4g
4h
4i
27.65
42.40
15.67
49.31
44.20
27.19
6.912
27.19
13.82
20.78
47.64
2.19
4j
55.40
49.66
6.78
4k
4l
4m
4n
4o
15.70
6.78
2.57
8

4p
4q
1.78
8.03
0.89
0.45
23.04
30.41
39.63
75.12
12.94
3.78
4r
44.52
166.93
Celecoxib
a% inhibition values represents means of two determinations using an ovine COX-1/COX-2 assay kit (catalog no.
560101, Cayman Chemicals Inc., Ann Arbor, MI, USA). bSelectivity index (COX-2/COX-1).
2.2.2 In vivoanti-inflammatory activity
Compounds with good COX-2 inhibitory activity were selected for in-vivoanti-inflammatory
activity and examined by carrageenan-induced rat paw edema method (Table 2).These
derivatives exhibited varying degree of anti-inflammatory activity (35% - 80%, 56% - 81% at 3h
and 62% - 82% at 5h). Compound 4jhas showed maximum activity at 5h (comparable to that of
celecoxib at 5h). Compounds4h, 4k and 4r showed promising activity.
Table 2In vivo anti-inflammatory activity against carrageenan-induced rat paw edema of the
compounds
Dose /kg
b.w.
% inhibition
3h
Compound
1h
5h
Controla
10ml/kg
20mg
20mg
20mg
20mg
20mg
20mg
0
0
0
Celecoxib
92.17
35.37
69.12
79.78
72.96
69.18
100
100
4a
4h
4j
56.79
70.41
81.01
71.55
71.03
62.13
72.14
82.56
77.32
74.91
4k
4r
avehicle (1% Carboxymethyl cellulose (CMC) in water in a volume of 10 mL/kg p.o.)
2.2.3 Ulcerogenic Potential
The ulcerogenic potential of most potent compound4jwas evaluated and compared to celecoxib
(20mg/kg). From the data obtained, Table 3, it has been observed that compound 4jshoweda
9

lower ulcer index than celecoxib. Compound 4j showed a good ulcer index (8.3) when
compared with that of celecoxib (11.9).
Table 3Ulcerogenic effect of 4j in rats (20 mg/kg)
Average
Compound
No.
No. of
% of incidence
divided by 10
Average
severity
Ulcer Index
animals
no. of ulcer
(Ulcer score)
Controla
Celecoxib
4j
05
05
05
0
0
4
0
0
10
06
1.5
1.1
11.9
8.3
1.2
a control group received 7% gum acacia (suspending vehicle) orally
2.3 Molecular docking study
A molecular docking study helps to understand the ligand-protein interactions and selectivity in
detail. Towards this goal, docking studies were carried out on the active site of COX-2 protein,
(PDB code: 6COX[28])co-crystal structure of SC-558 as a template, using Glide (V5.5) module
in Schrodinger’s molecular modeling suite.It was observed that compound 4j(Figure 3), SC-558
(Figure4) and celecoxib (Figure 5) showed hydrogen bonding interactions, hydrophilic
interactions and hydrophobic interactions at the active site of the COX. SC558 and celecoxib
showed hydrophobic interactions with Ala527, Val103, Leu525, Leu503, Phe523, Gly526, Phe357,
Tyr355, Lys358, Asn382, Gln350, Val349, Tyr385, His356, Gly354, Leu352 and less hydrophilic
interactions.
It was observed that, one of the O-atom and -NH2 of the SO2NH2 moiety of compound
4jexhibitedhydrogen bonding interaction with Gln192, Ala516 and the phenyl ring bearing
sulfonamide showed hydrophobic interactions with Leu359, Leu352, Gln350, His356, Tyr355, Met522,
Tyr504, Ala527, and Phe523.Since the structure is polar the magnitude of hydrophilic interactions is
more as compare to hydrophobic interactions. This may be the reason behind the low glide score
10

of compound 4j (glide score -8.80) as compared toSC558 (glide score -10.73) and celecoxib
(glide score -10.85).
Figure 3. The 3D image of 6COX-4j complex constructed using Glide (V5.5) module in Schrodinger’s molecular
modeling suite. The hydrogen bonds between one of the O-atoms of SO2NH2 with Ala516 and -NH2 of the SO2NH2
with Gln192 are shown in yellow dotted lines.
11

Figure 4. The 3D image of 6COX-SC558 complex constructed using Glide (V5.5) module in Schrodinger’s
molecular modeling suite. Hydrogen bond between -NH2 of the SO2NH2 with Gln192 is shown in yellow dotted lines.
12

Figure 5. The 3D image of 6COX-Celecoxib complex constructed using Glide (V5.5) module in Schrodinger’s
molecular modeling suite.
3 Conclusion
In summary, we have reporteda simple and efficient method for the synthesis of 3-chloro-4-
substitutedmaleimido benzenesulfonamide derivatives with excellent yield. All the synthesized
compounds have been screened for their invitro and selected compounds forinvivo anti-
inflammatory activity. In the newly synthesized compounds, 4h, 4j, 4k and 4r were found to
have moderate to goodanti-inflammatory activity.Moderate in vivo and in vitro activity of these
compounds may be due to poor hydrophobic interaction with the COX-2 active site. The overall
studies inferred that compound 4j rendered it as a hit molecule for the further development of
more potent anti-inflammatory agent. The in vivo and in vitro studies of these
compoundsevidenced that, the electron withdrawing groups enhances the anti- inflammatory
13

activity, which might be served as new template in the synthesis and development of potent
therapeutics.
4. Experimental
4.1 Chemistry
All the chemicals are purchased from Sigma Aldrich, Avera chemicals and solvents are reagent
grade and used without purification. The melting points were determined on Analab Melting
point apparatus in open capillary tubes and were uncorrected. The IR spectra in KBr were
recorded with the Schimadzu IR 48 spectrophotometer and were reported in cm-1. The 1H NMR,
13C spectra (DMSO-d6) were recorded on BrukerAvance II spectrometer. The chemical shifts are
expressed in δ values (ppm). The abbreviation s = singlet, d= doublet, and m= multiplet were
used throughout. Mass spectra were recorded on Waters Q time of flight MS-ES-1.0 1e4 mass
spectrometer. All reactions were followed by TLC (silica gel 60 F254 Merck) and visualization
was accomplished with UV light (254 or 366 nm).
4.1.1. General Procedure for the synthesis of 3,4-dichloromaleimido sulfonamide[29] (4)
The sulfonamide intermediate was prepared by the condensation of the sulfanilamide (2) and the
dichloromaleic anhydride (1) in glacial acetic acid solution. The procedure used to prepare the
3,4-dichloromaleimido sulfonamide (3) consisted in the addition of 0.1 mole of the sulfanilamide
(2) compound in 25-30 mL of glacial acetic acid to a solution of 0.1 mole of the dichloromaleic
anhydride (1) in 50-75 mL of glacial acetic acid at room temperature. The temperature was
gradually increased to reflux and the refluxing was continued for 15-30 min. Dilute the cooled
reaction mixture with water to obtain the 3,4-dichloromaleimido sulfonamide. Yield (%): 90%;
mp (°C): > 300°C; IR (KBr, cm-1):3458(NH2), 3126(Ar, C-H), 1734(C=O), 1594(Ar C=C),
1344,1158(SO2); 1H NMR (400MHz, DMSO-d6) ppm: 7.92(d, J = 8.52Hz, 2H, Ar), 7.53(d, J =
14

8.52, 2H, Ar), 7.36(br s, 2H, NH2); 13C NMR (100MHz, DMSO-d6) ppm: 161.6 (2; C=O imide),
143.6 (2; =C-Cl), 133.63(SO2-C), 132.93(=C-N), 126.78, 126.57(4C; Ar); m/z 319.2 [M+H]+.
4.1.2. General procedure for the synthesis of compounds (4a-r)
Compounds 4a-r were obtained by reaction of 3,4-dichloromaleimido sulfonamide (3)
(0.009mole), with appropriate amines (0.011mole) in 5mL DMF, which were stirred for
overnight at room temperature and then poured onto ice-cold water. The precipitate obtained was
filtered off, washed with water and dried. The crude product was purified by column
chromatography with 10% methanol in chloroform as an eluent.
4.1.2.1. 4-(3-chloro-2,5-dioxo-4-(phenylamino)-2,5-dihydro-1H-pyrrol-1-yl)benzenesulfonamide
(4a).Yield (%): 90%; mp (°C): 255-257°C; IR (KBr, cm-1):3457(NH), 1757(C=O), 1589(Ar
1
C=C), 1348,1156(SO2); H NMR (400MHz, DMSO-d6) ppm: 10.01(s, 1H, NH), 7.96(d, J =
13
8.0Hz, 2H, Ar), 7.62(d, J = 8.56Hz, 2H, Ar), 7.4(s, 2H, NH2), 7.39(d, J = 8.24Hz, 2H, Ar); C
NMR (100MHz, DMSO-d6) ppm: 165.64(C=O imide), 164.04(C=O imide), 142.65(C-N),
138.33(C-SO2), 136.48(=C-NH), 134.35(C-NH, Ar), 123.95–128.10(9C; Ar), 92.51(=C-Cl); m/z
378.2[M+H]+.
4.1.2.2.
4-(3-chloro-4-(4-chlorophenylamino)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-
yl)benzenesulfonamide (4b).Yield (%): 86%; mp (°C): 263-265°C; IR (KBr, cm-1):3452(NH),
1761(C=O), 1591(Ar C=C), 1340, 1152(SO2); 1H NMR (400MHz, DMSO-d6) ppm: 8.15(s, 1H,
NH), 7.99(d, 2H, J = 7.76Hz, 2H, Ar), 7.61(d, J = 8.56Hz, 2H, Ar), 7.4(s, 2H, NH2),7.37(d, J =
8.56Hz, 2H, Ar), 7.23(d, J = 8.76Hz, 2H, Ar); 13C NMR (100MHz, DMSO-d6) ppm:
165.43(C=O imide), 163.92(C=O imide), 143.63(C-N), 138.01(C-SO2), 135.42(=C-NH),
134.24(C-NH, Ar), 125.21–126.39(8C, Ar), 120.15(C-Cl, Ar), 93.53(=C-Cl); m/z 411.96
[M+H]+.
15

4.1.2.3. 4-(3-chloro-2,5-dioxo-4-(o-tolylamino)-2,5-dihydro-1H-pyrrol-1-yl)benzenesulfonamide
(4c). Yield (%): 69%; mp (°C): 273-275°C; IR (KBr, cm-1):3412, 3307(NH), 1720(C=O),
1593(Ar C=C), 1389,1170(SO2); 1H NMR (400MHz, DMSO-d6) ppm: 9.7(s, 1H, NH), 7.96(d, J
= 8.64, 2H, Ar), 7.63 (d, J = 9.08, 2H, Ar), 7.38(s, 2H, NH2), 7.27(m,4H,Ar), 2.29 (s,3H, CH3);
13C NMR (100MHz, DMSO-d6) ppm: 165.69 (C=O imide), 163.71(C=O imide), 142.56(C-N),
139.96(C-SO2), 135.16(=C-NH), 134.73 (C-NH, Ar), 130.02(C-CH3, Ar), 125.64 – 127.32(8C,
Ar), 90.52(=C-Cl), 17.30(CH3); m/z 392.3 [M+H]+.
4.1.2.4.
4-(3-chloro-2,5-dioxo-4-(4-nitrophenylamino)-2,5-dihydro-1H-pyrrol-1-
yl)benzenesulfonamide (4d). Yield (%): 80.7%; mp (°C): 295-297°C; IR (KBr, cm-1):3374,
1
3267(NH), 1741(C=O), 1509(Ar C=C), 1311,1162(SO2); H NMR (400MHz, DMSO-d6) ppm:
8.22(s, 1H, NH),7.98(d, J= 8.6Hz, 2H, Ar), 7.9(d, J = 8.92Hz, 2H, Ar), 7.59(d, J = 8.36Hz, 2H,
Ar), 7.42(s, 2H, NH2),6.60(d, J = 9.16Hz, 2H, Ar); 13C NMR (100MHz, DMSO-d6) ppm:
165.42(C=O imide), 162.96(C=O imide), 158.69(C-NO2), 143.65(C-N), 138.32(C-SO2),
133.63(=C-NH), 132.92(C-NH, Ar), 126.13 – 126.79(8C, Ar), 112.27(=C-Cl); m/z
421.2[M+H]+.
4.1.2.5.
4-(3-chloro-2,5-dioxo-4-(piperidin-1-yl)-2,5-dihydro-1H-pyrrol-1-
yl)benzenesulfonamide (4e).Yield (%): 86.3%; mp (°C): 193-195°C; IR (KBr, cm-1):3334(NH),
2936, 2854(C-H),1778(C=O), 1511(Ar, C=C), 1337,1162(SO2); 1H NMR (400MHz, DMSO-d6)
ppm: 7.92(d, J = 8.64Hz, 2H, Ar), 7.53(d, J = 8.6Hz, 2H, Ar), 7.38(s, 2H, NH2), 3.9(s, 4H, CH2),
1.69 (s, 6H, CH2); 13C NMR (100MHz, DMSO-d6) ppm: 164.48(C=O imide), 163.55(C=O
imide), 143.66(C-N), 142.62(C-SO2), 134.34(-C-N, piperidine), 126.22–126.50(4C, Ar),
92.26(=C-Cl), 23.44, 26.33, 49.19 (CH2, piperidine); m/z 370.3[M+H]+.
16

4.1.2.6.
4-(3-chloro-2,5-dioxo-4-(4-methoxyphenylamino)-2,5-dihydro-1H-pyrrol-1-
yl)benzenesulfonamide (4f).Yield (%): 92%; mp (°C): 252-255°C; IR (KBr, cm-1):3368(NH),
1
1709(C=O), 1403(Ar C=C), 1329,1162(SO2); H NMR (400MHz, DMSO-d6) ppm: 9.86(s, 1H,
NH), 7.96(d, J = 8.6Hz, 2H, Ar), 7.61(d, J = 8.6Hz,2H, Ar), 7.37(s, 2H, NH2), 7.18(d, J =
8.84Hz, 2H, Ar), 6.93(d, J = 8.88Hz, 2H, Ar), 3.78(s, 3H, OCH3); 13C NMR (100MHz, DMSO-
d6) ppm: 165.70(C=O imide), 163.94(C=O imide), 157.20(C-OCH3), 142.57(C-N), 138.86(C-
SO2), 134.40(=C-NH), 132.93(C-NH, Ar), 125.83-129.18(8C, Ar), 90.72(C-Cl), 55.15(OCH3);
m/z 406.2[M+H]+.
4.1.2.7.
4-(3-chloro-2,5-dioxo-4-(4-fluorophenylamino)-2,5-dihydro-1H-pyrrol-1-
yl)benzenesulfonamide (4g). Yield (%): 69%; mp (°C): 243-245°C; IR (KBr, cm-1):3454 (NH),
1762(C=O), 1592(Ar C=C), 1341, 1154(SO2); 1H NMR (400MHz, DMSO-d6) ppm: 9.90(s, 1H,
NH), 7.90(d, J = 8.88Hz, 2H, Ar), 7.54(d, J = 8.6Hz, 2H, Ar), 7.28(s, 2H, NH2),7.06(d, J =
8.6Hz, 2H, Ar); 13C NMR (100MHz, DMSO-d6) ppm: 165.50(C=O imide), 163.88(C=O imide),
158.58(C-F, Ar), 142.58(C-N), 138.40(C-SO2), 134.24(=C-NH), 132.60(C-HN, Ar), 125.99-
126.08(8C, Ar), 125.71, 125.90(CH-Ar-F), 114.66 – 114.96(CH-Ar-F), 92.22(=C-Cl); m/z
395.20 [M+H]+.
4.1.2.8.
4-(3-chloro-4-(3-chlorophenylamino)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-
yl)benzenesulfonamide (4h).Yield (%): 74.51%; mp (°C): 252-255°C; IR (KBr, cm-1):3302
1
(NH), 1713(C=O), 1410 (Ar C=C), 1332,1162 (SO2); H NMR (400MHz, DMSO-d6) ppm:
8.58(s, 1H, NH), 7.97(d, J = 8.64Hz, 2H, Ar), 7.62(d, J = 8.6Hz, 2H, Ar), 7.42-7.32(m, 4H, Ar),
.25(s, 2H, NH2);13CNMR(100MHz, DMSO-d6) ppm: 165.50(C=O imide), 163.88(C=O imide),
158.58(C-F, Ar), 142.58(C-N), 138.40(C-SO2), 134.24(=C-NH), 132.60(C-HN, Ar), 125.99-
17

126.08(8C, Ar), 125.71, 125.90(CH-Ar-F), 114.66-114.96(CH-Ar-F), 92.22(=C-Cl); m/z 411.96
[M+H]+
4.1.2.9.
4-(3-chloro-2,5-dioxo-4-(2-methoxyphenylamino)-2,5-dihydro-1H-pyrrol-1-
yl)benzenesulfonamide (4i). Yield (%): 76%; mp (°C): 280-282°C; IR (KBr, cm-1):3360(NH),
1770,(C=O), 1594(Ar C=C), 1328,1152 (SO2); 1H NMR (400MHz, DMSO-d6) ppm: 9.52(s, 1H,
NH), 7.98(d, J = 8.6Hz, 2H, Ar), 7.62(d, J = 8.68Hz, 2H, Ar), 7.39(s, 2H, NH2), 7.30(t, J =
7.44Hz, J = 7.56Hz, 1H, Ar), 7.22(d, J = 7.72Hz, 1H, Ar), 7.07(d, J = 8.0Hz, 2H, Ar), 3.82(s,
3H, OCH3); 13C NMR(100MHz, DMSO-d6) ppm: 165.58(C=O imide), 163.72(C=O imide),
154.11(C-OCH3), 142.57(C-N), 139.50(C-SO2), 134.35(=C-NH), 132.91(C-NH, Ar) 126.09–
127.95(8C, Ar), 91.61(=C-Cl), 55.39(OCH3); m/z 406.2[M+H]+.
4.1.2.10.
4-(3-chloro-2,5-dioxo-4-(3-fluorophenylamino)-2,5-dihydro-1H-pyrrol-1-
yl)benzenesulfonamide (4j). Yield (%): 53%, mp (°C): 268-270°C, IR (KBr, cm-1):3260(NH),
1765(C=O), 1589(Ar C=C), 1348, 1154(SO2); 1H NMR (400MHz, DMSO-d6) ppm: 9.89(s, 1H,
NH), 7.99(d, J = 7.6Hz, 2H, Ar) 7.62(d, J = 7.7Hz, 2H, Ar), 7.43(m, 4H, Ar), 7.29(s, 2H, NH2);
13C NMR (100MHz, DMSO-d6) ppm: 165.42 (C=O imide), 163.65(C=O imide), 159.14(C-F,
Ar), 142.40(C-N), 138.37(C-SO2), 134.34 (=C-NH, Ar), 132.72(C-NH, Ar), 125.88-126.36(8C,
Ar), 125.58, 125.76(CH, Ar-F), 113.96, 114.26(CH, Ar-F), 92.18(=C-Cl); m/z 395.20 [M+H]+.
4.1.2.11.
4-(3-chloro-2,5-dioxo-4-(3-methoxyphenylamino)-2,5-dihydro-1H-pyrrol-1-
yl)benzenesulfonamide (4k). Yield (%): 73.68%; mp (°C): 280-282°C; IR (KBr, cm-1):3252(NH),
1
1761(C=O), 1589(Ar C=C), 1349,1151(SO2); H NMR (400MHz, DMSO-d6) ppm: 9.53(s, 1H,
NH), 7.98(d, J = 8.6Hz, 2H, Ar), 7.63(d, J = 8.64Hz, 2H, Ar), 7.38(s, 2H, NH2), 7.31(t, J =
7.48Hz, J = 7.44Hz, 1H, Ar), 7.23(d, J = 7.72Hz, 1H, Ar), 7.07(d, J = 8.0Hz, 2H, Ar), 3.81(s,
13
3H, OCH3); C NMR (100MHz, DMSO-d6) ppm: 165.68(C=O imide), 163.80(C=O imide),
18

154.28(C-OCH3), 142.61(C-N), 139.56(C-SO2), 134.30(=C-NH), 132.86(C-NH, Ar), 126.06–
127.86(8C, Ar), 91.72(=C-Cl), 55.28(OCH3); m/z 406.2[M+H]+.
4.1.2.12.
4-(3-chloro-4-(2,3-dichlorophenylamino)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-
yl)benzenesulfonamide (4l). Yield (%): 90%; mp (°C): 286-288°C; IR (KBr, cm-1):3258(NH),
1762(C=O), 1590(Ar C=C), 1343,1149 (SO2); 1H NMR (400MHz, DMSO-d6) ppm: 9.98(s, 1H,
NH), 7.96(d, J = 8.6Hz, 2H, Ar), 7.64(d, J = 8.64Hz, 2H, Ar), 7.42(m, 3H, Ar), 7.3(s, 2H, NH2);
13C NMR (100MHz, DMSO-d6) ppm: 165.61(C=O imide), 163.69(C=O imide), 142.53(C-N),
139.52(C-SO2), 134.29(=C-NH), 123.8 and 132.64(2C-Cl, Ar), 132.71(C-NH, Ar), 124.93–
126.86(7C, Ar), 89.34(=C-Cl); m/z 466.70 [M+H]+.
4.1.2.13.
4-(3-chloro-4-(3,5-dimethylphenylamino)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-
yl)benzenesulfonamide (4m).Yield (%): 68%, mp (°C): 276-278°C, IR (KBr, cm-1):3278(NH),
1750(C=O), 1589 (Ar C=C), 1351,1142(SO2), 1H NMR (400MHz, DMSO-d6) ppm: 9.69(s, 1H,
NH), 7.96(d, J = 8.64Hz, 2H, Ar), 7.63(d, J = 9.08Hz, 2H, Ar), 7,59(d, J = 8.6Hz, 2H, Ar),
7.53(d, J = 8.6Hz, 1H, Ar), 7.37(s, 2H, NH2), 2.32(s,6H, CH3); 13C NMR (100MHz, DMSO-d6)
ppm: 165.11(C=O imide), 162.86(C=O imide), 142.55(C-N), 139.60(C-SO2),134.36 (=C-NH),
132.72(C-NH, Ar), 130.18 and 130.34 (2C-CH3,Ar), 120.86 – 126.36(7C, Ar), 94.32(=C-Cl),
21.3(2CH3); m/z 405.10[M+H]+.
4.1.2.14. 4-(3-chloro-2,5-dioxo--4-(4-(trifluoromethyl)phenylamino)- 2,5-dihydro-1H-pyrrol-1-
yl)benzenesulfonamide (4n). Yield (%): 70.7%; mp (°C): 270-272°C; IR (KBr, cm-1):3460 (NH),
1761(C=O), 1589 (Ar C=C), 1339,1140 (SO2); 1H NMR (400MHz, DMSO-d6) ppm: 9.96(s, 1H,
NH), 7.89(d, J = 8.6Hz, 2H, Ar), 7.61(d, J = 8.6Hz, 2H, Ar), 7.36(s 2H, NH2), 7.18(d, J =
13
8.84Hz, 2H, Ar), 6.93(d, J = 8.88Hz, 2H, Ar); C NMR (100MHz, DMSO-d6) ppm: 164.98
(C=O imide), 163.02 (C=O imide), 142.62(C-N), 139.71(C-SO2), 134.21(=C-NH), 132.61(C-
19

NH. Ar), 130.21(C-CF3), 125.09-126.32 (8C, Ar), 121.68(CF3), 90.91(=C-Cl); m/z
445.21[M+H]+.
4.1.2.15.
4-(3-chloro-4-(2,4-dimethylphenylamino)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-
yl)benzenesulfonamide (4o). Yield (%): 57.8%; mp (°C): 280-282°C; IR (KBr, cm-1):3248(NH),
1
1756(C=O), 1591(Ar C=C), 1348,1137(SO2); H NMR (400MHz, DMSO-d6) ppm: 9.78(s, 1H,
NH), 7.97(d, J = 8.4Hz, 2H, Ar), 7.69(d, J = 8.4Hz, 2H, Ar), 7.59(d, J = 7.8Hz, 2H, Ar), 7.54(d,
13
J = 7.8Hz, 2H, Ar), 7.31(s, 2H, NH2), 2.31(s,6H, CH3); C NMR (100MHz, DMSO-d6) ppm:
165.18(C=O imide), 163.84(C=O imide), 142.59(C-N), 139.51(C-SO2), 134.28 (=C-NH),
132.61(C-NH, Ar), 130.21 and 130.41(2C-CH3, Ar), 120.82–126.39(7C, Ar), 93.18(=C-Cl),
21.36(2CH3); m/z 405.10[M+H]+.
4.1.2.16.
4-(3-(2-aminophenylamino)-4-chloro-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-
yl)benzenesulfonamide (4p). Yield (%): 72%, mp (°C): 289-291°C, IR (KBr, cm-1):3223(NH),
1779(C=O), 1589(Ar C=C), 1343,1135(SO2), 1H NMR (400MHz, DMSO-d6) ppm: 12.92(s, 1H,
NH), 8.00(d, J = 8.68Hz, 2H, Ar), 7.81(d, J = 8.68Hz, 2H, Ar), 7.66(d, J = 9.2Hz, 1H, Ar),
7.61(d, J = 9.32Hz ,1H, Ar), 7.45(d, J = 8.48Hz, 1H, Ar), 7.41(s, 2H, NH2), 7.29(t, J = 8.28Hz, J
13
= 8.36Hz, 1H, Ar); C NMR (100MHz, DMSO-d6) ppm: 165.21(C=O imide), 161.28(C=O
imide), 154.80(C-NO2, Ar), 141.62(C-N), 138.33 (C-SO2),134.26(=C-NH),132.56(C-NH, Ar),
132.12 (C-Cl, Ar), 125.21–129.63(7C, Ar), 91.26(=C-Cl); m/z 455.65[M+H]+.
4.1.2.17.
4-(3-chloro-4-(3,4-dichlorophenylamino)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-
yl)benzenesulfonamide (4q). Yield (%): 78%; mp (°C): 278-280°C; IR (KBr, cm-1):3197(NH),
1781(C=O), 1591(Ar C=C), 1352,1139(SO2); 1H NMR (400MHz, DMSO-d6) ppm: 10.01(s, 1H,
NH), 7.96(d, J = 8.6Hz, 2H, Ar), 7.64(d, J = 8.6Hz , 2H, Ar), 7.41(m, 3H, Ar), 7.29(s, 2H, NH2);
13C NMR (100MHz, DMSO-d6) ppm: 161.31(C=O imide), 159.26(C=O imide), 143.48(C-N),
20

138.29(C-SO2), 134.22 (=C-NH), 132.28(C-CH, Ar), 123.19 and 132.18(2C-Cl, Ar),125.89 –
126.68(7C, Ar), 90.03(=C-Cl); m/z 466.70 [M+H]+.
4.1.2.18.
4-(3-chloro-2,5-dioxo-4-(2-fluorophenylamino)-2,5-dihydro-1H-pyrrol-1-
yl)benzenesulfonamide (4r). Yield (%): 53%; mp (°C): 268-270°C; IR (KBr, cm-1):3242(NH),
1762(C=O), 1591(Ar C=C), 1338, 1141(SO2); 1H NMR (400MHz, DMSO-d6) ppm: 9.83(s, 1H,
NH), 7.95(d, J = 7.6Hz, 2H, Ar),7.61(d, J = 7.7Hz, 2H, Ar), 7.48(m, 4H, Ar), 7.31(s, 2H, NH2);
13C NMR (100MHz, DMSO-d6) ppm: 165.37(C=O imide), 163.62(C=O imide), 158.34(C-F,
Ar), 142.42(C-N), 138.39(C-SO2), 134.41(=C-NH), 132.61(C-NH, Ar), 125.71–126.18(8C, Ar),
125.71, 125.48(CH, Ar-F), 116.14-115.23(CH, Ar-F), 92.01(=C-Cl); m/z 395.20 [M+H]+.
4.2 Pharmacological Evaluation
4.2.1 Invitrocyclooxygenase (COX)inhibition assay
The ability of the test compounds4a-r to inhibit ovine COX-1 and human recombinant COX-2
was determined by using a colorimetric enzyme assay kit. (Catalog No. 760131, Cayman
Chemicals Inc., Ann Arbor, MI, USA) according to the manufacturer’s assay protocol.
Colorimetric Inhibitor Screening Assay measures the peroxidase component of COXs. The
peroxidase activity is assayed colorimetrically by monitoring the appearance of oxidized
N,N,N’,N’-tetramethyl-p-phenylenediamine (TMPD) at 590 nm.[30] Inhibition of COX activity,
by a variety of selective and nonselective inhibitors, showed potencies similar to those observed
with other in vitro methods. Stock solutions of test compounds were dissolved in a minimum
volume of DMSO. Briefly, to a series of supplied reagents, buffer solution (0.1 M Tris-HCl, pH
8), heme and either COX-1 or COX-2 enzyme were mixed with test compounds. These solutions
were incubated for a period of 5 min at 25°C, after which colorimetric substrate TMPD and
quickly arachidonic acid were added. Shake the solutions for a few seconds and then incubate for
21

25°C for 2 min. The product of this reaction produces a blue colour that absorbs at 590nm. The
intensity of the blue colour was determined by a microplate reader (power wave XS2). The
calculations were performed as per kit guidelines.
4.2.2In vivo anti-inflammatory activity
4.2.2.1 Animals:
Adult male Wister albino rats (150-200 g) were used to study the anti-inflammatory activity and
ulcerogenic liabilities. The animalswere maintained under standard laboratory conditions (light
period of 12 h/day and temperature 27 2°C), with access to food and water. The experimental
procedures were carried out in strict compliance with the Institutional Animal Ethics Committee
regulations. All experiments were performed according to the guidelines for the care of
laboratory animals. [31]
4.2.2.2 Carageenan-induced hind paw edema method:
Carageenan-induced hind paw edema method[32] was used for evaluating anti-inflammatory
activity. Wistar albino rats (150-200g) were fasted for 24h and were divided into the following
groups: carageenan control, test compounds, celecoxib as standards, each comprising six
animals. The volume of the right hind paw of rat was measured by plethysmometer (UGO
Basile, Italy). One group of rats, which served as control was given vehicle (1% Carboxymethyl
cellulose (CMC) in water in a volume of 10 mL/kg p.o.) only. Test compounds (20 mg/kg b.w.)
and celecoxib (20 mg/kg b.w.) suspended in vehicle (10 mL/ kg) were administered orally to the
respective groups. After 30 min of administration of the drug and compounds, carageenan
solution in normal salinewas injectedinto the planter surface right hind paw of each animal. The
volume of swelling of the right hind paw of each rat was measured after 1, 3, and5h. The mean
22

increase in the volume of the right hind paw was compared with control and standard. The
percentage inhibition of paw edema was calculated as follows,
Reduction of inflammation = [1 - (Vt/Vc) × 100]
Where, Vt is the change in paw volume in the test compound treated group, and Vc, is the
changein paw volume in the control group.
4.2.2.3 Ulcerogenic activity
Male albino rats weighing 150-200 g were fasted for 12h prior to drug administration. Water was
supplied adlibitum. The animals were divided into three equal groups (each of four). The first
group received 7% gum acacia (suspending vehicle) orally once a day and was left as a control,
whereas the other groups received the reference drugs and test compound4j with a dose of
20mg/kg/day orally. The test compounds were administered once a day for three successive
days. Animals were sacrificed by diethyl ether 6h after the last dose and the animals were killed
by an overdose of ether 6h after the last dose. The stomachs were removed, opened along the
greater curvature, and examined for ulceration. The ulcer score was calculated according to the
method of Vijaya and Mishra [33]: 0.0- normal (no injury); 0.5- latent injury; 1.0- slight injury
(two to three dotted lines); 2.0- severe injury (continuous lined injury or five to six dotted
injuries); 3.0- very severe injury (several continuous lined injuries); 4.0- widespread lined injury.
The ulcer index was calculated according to the method of Robert et al[34]. The degree of
ulcerogenic effect was expressed in terms of:
I.
The percentage incidence of ulcers in each group ofanimals divided by 10.
The average number of ulcers per stomach.
II.
III.
The average severity of ulcers by visual observation.
23

Finally, the ulcer index was expressed as the summationvalue of the above three items.Results
are tabulated in Table 3.
4.3 Molecular docking studies
4.3.1 Preparation of Target Protein X-ray Structure
The crystal structure ofCOX-2 in complex with SC-558 inhibitor (PDB code: 6COX,
http://www.pdb.org/) was selected as the protein target model in this study. Using Schrodinger
software[35] hydrogens were added and enzyme structure was subjected to a refinement protocol
in which the constraints on the enzyme were gradually removed.
4.3.2 Ligands Preparation
LigPrep (v2.2) module in the Schrodinger molecular modeling suit was used for ligand
preparation, which produced low energy conformations of ligand using OPLS 2005 force field.
The bond orders of these ligands were fixed and the ligands ‘cleaned’ through LigPrep
specifying a pH value of 7.0.
4.3.3 Protein-Ligand docking using Glide
Compound 4j was docked into the active site of COX-2. The best docking conditions which
succeeded to retrieve the pose of the co-crystallized ligands were used.Glide provides three
different levels of docking precisions viz. High Throughput Virtual Screening, HTVS; Standard
precision, SP and Extra precision, XP. We carried out our calculations using XP docking mode
as the tool for better refinement.
Acknowledgements
The authors are grateful to, Dr. S.J. Surana, Principal, R.C. Patel Institute of Pharmaceutical
Education and Research, Shirpur, (MS), India for providing Molecular docking programme
Glide. Dr. C. R. Patil Professor, Department of Pharmacology, R.C. Patel Institute of
24

Pharmaceutical Education and Research, Shirpur, (MS), India for their help in the evaluation of
the anti-inflammatory activity. The authors are also thankful to, SAIF, Chandigarh for providing
NMR and Mass spectra.
25

2
2
2
2
2
2
6
5
6 4
6
4
3 6 4
3
6
4
6 3
2
6
4
3 6 3
5
10
3
3
6
6
4
4
6
4
3 6 3
2
2
6
4
2
6
4
6
4
6 3
3
6 4
26

References
[1] J.G. Black, Microbiology Principles and Explorations 8th edition, Wiley2012.
[2] S.M. Jachak, Cyclooxygenase inhibitory natural products: current status, Current medicinal
chemistry, 13 (2006) 659-678.
[3] L.J. Marnett, A.S. Kalgutkar, Cyclooxygenase 2 inhibitors: discovery, selectivity and the
future, Trends in pharmacological sciences, 20 (1999) 465-469.
[4] G. Dannhardt, W. Kiefer, Cyclooxygenase inhibitors–current status and future prospects,
European journal of medicinal chemistry, 36 (2001) 109-126.
[5] W.L. Smith, D.L. Dewitt, Prostaglandin endoperoxide H synthases-1 and -2, Adv Immunol,
62 (1996) 167-215.
[6] H.R. Herschman, Prostaglandin synthase 2, Biochim Biophys Acta, 1299 (1996) 125-140.
[7] E.A. Meade, W.L. Smith, D.L. Dewitt, Differential inhibition of prostaglandin endoperoxide
synthase (cyclooxygenase) isozymes by aspirin and other non-steroidal anti-inflammatory drugs,
Journal of Biological Chemistry, 268 (1993) 6610-6614.
[8] M.C. Allison, A.G. Howatson, C.J. Torrance, F.D. Lee, R.I. Russell, Gastrointestinal damage
associated with the use of nonsteroidal antiinflammatory drugs, New England Journal of
Medicine, 327 (1992) 749-754.
[9] A. Zarghi, R. Ghodsi, E. Azizi, B. Daraie, M. Hedayati, O.G. Dadrass, Synthesis and
biological evaluation of new 4-carboxyl quinoline derivatives as cyclooxygenase-2 inhibitors,
Bioorganic & medicinal chemistry, 17 (2009) 5312-5317.
[10] T.D. Penning, J.J. Talley, S.R. Bertenshaw, J.S. Carter, P.W. Collins, S. Docter, M.J.
Graneto, L.F. Lee, J.W. Malecha, J.M. Miyashiro, Synthesis and biological evaluation of the 1,
5-diarylpyrazole class of cyclooxygenase-2 inhibitors: identification of 4-[5-(4-methylphenyl)-3-
27

(trifluoromethyl)-1 H-pyrazol-1-yl] benzenesulfonamide (SC-58635, celecoxib), Journal of
medicinal chemistry, 40 (1997) 1347-1365.
[11] M.J. Stillman, M.T. Stillman, Choosing nonselective NSAIDs and selective COX-2
inhibitors in the elderly, (2007).
[12] I.A. Al-Suwaidan, A.M. Alanazi, A.S. El-Azab, A.M. Al-Obaid, K.E. ElTahir, A.R.
Maarouf, M.A.A. El-Enin, A.-M. Alaa, Molecular design, synthesis and biological evaluation of
cyclic imides bearing benzenesulfonamide fragment as potential COX-2 inhibitors. Part 2,
Bioorganic & medicinal chemistry letters, 23 (2013) 2601-2605.
[13] S. Bano, K. Javed, S. Ahmad, I. Rathish, S. Singh, M. Chaitanya, K. Arunasree, M. Alam,
Synthesis of some novel chalcones, flavanones and flavones and evaluation of their anti-
inflammatory activity, European journal of medicinal chemistry, 65 (2013) 51-59.
[14] M. Abdel-Aziz, E.A. Beshr, I.M. Abdel-Rahman, K. Ozadali, O.U. Tan, O.M. Aly, 1-(4-
Methoxyphenyl)-5-(3, 4, 5-trimethoxyphenyl)-1H-1, 2, 4-triazole-3-carboxamides: Synthesis,
molecular modeling, evaluation of their anti-inflammatory activity and ulcerogenicity, European
journal of medicinal chemistry, 77 (2014) 155-165.
[15] Z. Liu, L. Tang, P. Zou, Y. Zhang, Z. Wang, Q. Fang, L. Jiang, G. Chen, Z. Xu, H. Zhang,
Synthesis and biological evaluation of allylated and prenylated mono-carbonyl analogs of
curcumin as anti-inflammatory agents, European journal of medicinal chemistry, 74 (2014) 671-
682.
[16] M. Unnikrishnan, M. Rao, Antiinflammatory activity of methionine, methionine sulfoxide
and methionine sulfone, Agents and actions, 31 (1990) 110-112.
[17] J. Drews, Drug discovery: a historical perspective, Science, 287 (2000) 1960-1964.
28

[18] C.T. Supuran, A. Scozzafava, Carbonic anhydrase inhibitors and their therapeutic potential,
Expert Opinion on Therapeutic Patents, 10 (2000) 575-600.
[19] A. Weber, A. Casini, A. Heine, D. Kuhn, C.T. Supuran, A. Scozzafava, G. Klebe,
Unexpected nanomolar inhibition of carbonic anhydrase by COX-2-selective celecoxib: new
pharmacological opportunities due to related binding site recognition, Journal of medicinal
chemistry, 47 (2004) 550-557.
[20] A.E. Boyd, Sulfonylurea receptors, ion channels, and fruit flies, Diabetes, 37 (1988) 847-
850.
[21] T.H. Maren, Relations between structure and biological activity of sulfonamides, Annual
review of pharmacology and toxicology, 16 (1976) 309-327.
[22] C. Thornber, Isosterism and molecular modification in drug design, Chem. Soc. Rev., 8
(1979) 563-580.
[23] R.C. Ogden, C.W. Flexner, Protease inhibitors in AIDS therapy, Marcel Dekker New York,
USA2001.
[24] C.T. Supuran, A. Scozzafava, A. Mastrolorenzo, Bacterial proteases: current therapeutic use
and future prospects for the development of new antibiotics, Expert Opinion on Therapeutic
Patents, 11 (2001) 221-259.
[25] A. Scozzafava, C.T. Supuran, Carbonic Anhydrase and Matrix Metalloproteinase Inhibitors:
Sulfonylated Amino Acid Hydroxamates with Mmp Inhibitory Properties Act as Efficient
inhibitors of CA Isozymes I, II, and IV, and N-Hydroxysulfonamides Inhibit Both These Zinc
Enzymes 1, Journal of medicinal Chemistry, 43 (2000) 3677-3687.
[26] R. Aggarwal, A. Bansal, I. Rozas, B. Kelly, P. Kaushik, D. Kaushik, Synthesis, biological
evaluation and molecular modeling study of 5-trifluoromethyl-Δ2-pyrazoline and isomeric 5/3-
29