4-Functionalized 1,3-diarylpyrazoles bearing benzenesulfonamide moiety as selective potent inhibitors of the tumor associated carbonic anhydrase isoforms IX and XII

Article abstract of DOI:10.1016/j.ejmech.2014.02.023

A library of 4-functionalized 1,3-diarylpyrazoles (3a-3h, 5a-5g and 6a-6g) was designed, synthesized and evaluated against four human carbonic anhydrase (CA, EC 4.2.1.1) isozymes representing two cytosolic isozymes hCA I and hCA II, and two transmembrane

Full text of DOI:10.1016/j.ejmech.2014.02.023

European Journal of Medicinal Chemistry 76 (2014) 284e290
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
4-Functionalized 1,3-diarylpyrazoles bearing benzenesulfonamide
moiety as selective potent inhibitors of the tumor associated carbonic
anhydrase isoforms IX and XII
,d,
Poonam Khloya a, Gulsah Celik b, SitaRam a, Daniela Vullo c, Claudiu T. Supuran c
**
,
,
Pawan K. Sharma a
*
a Department of Chemistry, Kurukshetra University, Kurukshetra 136119, India
b Chemistry Department, Balikesir University, 10145 Balikesir, Turkey
c Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino,
Florence, Italy
d Università degli Studi di Firenze, Neurofarba Dept., Section of Pharmaceutical and Nutraceutical Sciences, Via U. Schiff 6, 50019 Sesto Fiorentino,
Florence, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A library of 4-functionalized 1,3-diarylpyrazoles (3ae3h, 5ae5g and 6ae6g) was designed, synthesized
and evaluated against four human carbonic anhydrase (CA, EC 4.2.1.1) isozymes representing two
cytosolic isozymes hCA I and hCA II, and two transmembrane tumor associated ones, hCA IX and hCA XII.
All the twenty two tested compounds exhibited excellent CA activity profile against the four CA isozymes
when compared to the reference drug acetazolamide. Six of the tested compounds (3ae3b, 3f, 3h, 6a and
6b) displayed low nanomolar affinity (Ki < 5 nM) for hCA IX whereas seven compounds (3ae3b, 3de3f,
3h and 6f) displayed Ki < 10 nM against hCA XII. In addition, they acted as selective CA inhibitors of
isoforms IX and XII over the physiological isoforms I and II.
Received 3 December 2013
Received in revised form
5 February 2014
Accepted 8 February 2014
Available online 11 February 2014
Keyword:
Pyrazole
Ó 2014 Elsevier Masson SAS. All rights reserved.
Benzenesulfonamide
Carbonic anhydrase isoforms I, II, IX, XII
Acetazolamide
1. Introduction
Human carbonic anhydrase I (hCA I) is an erythrocyte-specific
enzyme and is a member of a multigene family occurring on the
Carbonic anhydrases (CAs, EC 4.2.1.1) belong to the group of zinc
metalloenzymes and play an important physiologic role by cata-
lyzing the reversible rapid hydrationedehydration reaction of car-
bon dioxide to bicarbonate anion and protons. 16 different isoforms
long arm of chromosome 8 [2]. CA II is ubiquitous housekeeping
cytosolic isozyme and is the most widely distributed CA in the eye,
kidney, central nervous system and inner ear besides being
involved in important metabolic processes such as respiration and
regulation of the acid/base homeostasy. On the other hand, CA IX
and XII are predominantly found in tumor cells and are involved in
cell proliferation and transformation which make them obvious
targets for the development of novel antitumor therapies [3].
Primary sulfonamides [4,5] were identified as the first and one of
of CAs belonging to the
now, which differ widely in their cellular localization and catalytic
activity. Different CAs belonging to the -class play vital roles in
a-class have been found in mammals till
a
various physiological processes, including respiration, calcification,
acid-base balance, bone resorption, etc [1]. They are also involved in
a number of biosynthetic pathways such as gluconeogenesis,
ureagenesis, and lipogenesis as well as in pathological disorders
including edema, glaucoma, obesity and epilepsy.
the most important class of a-family of CA inhibitors (CAIs) that has
given many clinically used drugs such as acetazolamide (AZA),
methazolamide (MZA), dichlorophenamide (DCP), ethoxolamide
(EZA), dorzolamide (DZA) and brinzolamide (BRZ) [6,7]. There has
been a flurry of activity in the field of benzenesulfonamide bearing
scaffold after recent reports indicating the anticancer potential
[8e10] of such compounds by way of being highly potent and iso-
form selective CAIs targeting two of the most investigated
mammalian CAs, that is, the tumor associated isoforms CA IX and XII.
* Corresponding author. Tel.: þ91 94164 57355; fax: þ91 1744 238277.
** Corresponding author. Tel.: þ39 055 4573005; fax: þ39 055 4573385.
E-mail addresses: claudiu.supuran@unifi.it (C.T. Supuran), pksharma@kuk.ac.in
(P.K. Sharma).
http://dx.doi.org/10.1016/j.ejmech.2014.02.023
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

P. Khloya et al. / European Journal of Medicinal Chemistry 76 (2014) 284e290
285
Furthermore, perusal of literature reveals that pyrazole scaffold
has been an important pharmacophore and privileged structure
present in various CA inhibitors [11e13]. Motivated by the above
findings and coupled with our ongoing research in the field of
pyrazoles and other heterocyclic compounds of potential medicinal
interest [14e19], we turned our attention towards the synthesis
and evaluation of 4-functionalized pyrazoles bearing primary sul-
fonamide group as novel CAIs. We were interested to investigate
the effect of a free amide group at position-4 of pyrazole and
substituting it further with an ester and a hydrazinocarbonyl group
[9,20,21] with a view to study its impact on enzyme selectivity. It is
pertinent to mention here that, to the best of our knowledge, 4-
functionalized pyrazoles has been evaluated as CA inhibitors for
the first time.
range
d 3.80e3.77. The target pyrazole-4-hydrazinocarbonyl com-
pounds (6ae6g) displayed two exchangeable singlets in the range
d
9.61e9.46 due to NH proton and d 4.57e4.48 due to NH2 protons
in their 1H NMR spectra whereas IR spectra displayed absorption
peak for C]O stretch at 1643-1620 cmꢀ1. The presence of sulfon-
amide group in all the target 4-functionalized pyrazole compounds
(3, 5, 6) was evident from a broad singlet, exchangeable in D2O,
appearing in the range
other aromatic protons also appeared at the expected values in
accordance with the proposed structure.
d
7.53e7.44 in their 1H NMR spectra. The
3. Biological evaluation
3.1. Carbonic anhydrase inhibition
In the present report, we have designed and synthesized a small
library of novel 4-functionalized 1,3-diarylpyrazole compounds
(3ae3h, 5ae5g and 6ae6g) bearing a primary sulfonamide group
on the phenyl ring at N-1 position of pyrazole scaffold with
different functionalities at C-4 such as carboxamide, ester, and
hydrazinocarbonyl (Fig 1) and evaluated them against four human
carbonic anhydrases hCA I, II, IX and XII.
All the twenty two newly synthesized 4-functionalized 1,3-
diarylpyrazoles (3ae3h, 5ae5g and 6ae6g) were screened
against two cytosolic CA isozymes hCA I and II as well as trans-
membrane tumor associated isozymes hCA IX and hCA XII by a
stopped-flow, CO2 hydrase assay method [24] and the CA inhibition
data is summarized in Table 1.
Results revealed that in general, all the tested compounds (3ae
3h, 5ae5g and 6ae6g) exhibited excellent CA activity profile.
Interestingly, most of the compounds were found to be more
potent, showing low nanomolar CA inhibition constant (Ki), when
compared to the reference drug acetazolamide (AZA). Moreover,
tested compounds in general exhibited better selectivity ratio for
target tumor associated isozymes hCA IX and hCA XII over off target
cytosolic isozymes hCA I and II.
Target compounds (3ae3h, 5ae5g, and 6ae6g) inhibited hCA I
isozyme with inhibition constants (Ki) in the range of 3.9e
377.0 nM, hCA II with Ki in the range of 4.0e681.0 nM, hCA IX with
Ki in the range of 2.1e49.3 nM and hCA XII with Ki in the range of
1.9e43.7 nM. As evident from Table 1, the hCA I isoform was
inhibited very well by almost all the tested compounds (3ae3h,
5ae5g, and 6ae6g) as compared to the standard drug AZA whereas
hCA II is mainly inhibited only by the compounds of series 3.
Interesting results regarding tumor isozymes hCA IX and XII with
these compounds have been observed.
2. Results and discussion
2.1. Chemistry
The synthetic route adopted for the synthesis of 4-
functionalized pyrazole compounds is illustrated in Scheme 1. 4-
Cyanopyrazoles (2), required for the synthesis of corresponding
4-carboxamide analogs (3), were prepared following our earlier
reported method [22]. Target pyrzole-4-carboxamides (3) were
obtained by the oxidation of 4-cyanopyrazoles with hydrogen
peroxide [23] in THF under basic conditions. On the other hand,
oxidation of 4-formylpyrazoles (1) with KMnO4 afforded the cor-
responding pyrazole-4-carboxylic acids 4 [22] which were trans-
formed to their methyl esters 5 by refluxing a methanolic solution
of 4 in the presence of a few drops of concentrated sulfuric acid. The
target pyrazole-4-hydrazinocarbonyl compounds (6) were ob-
tained by the condensation of methyl esters 5 with hydrazinee
hydrate in MeOH/THF.
The following features of SAR of 4-functionalized 1,3-
diarylpyrazole compounds (3ae3h, 5ae5g, and 6ae6g) have been
drawn against the inhibition of these four CA isozymes from the
data presented in Table 1.
Analytical and spectral data of the newly synthesized compounds
were in full agreement with the proposed structures. In general,
pyrazole-4-carboxamides (3ae3h) were characterized by two
exchangeable singlets in the range of
d 7.73e7.60 and 7.38e7.29
corresponding to NH and OH protons in their 1H NMR spectra besides
a sharp band around 2350 cmꢀ1 due to C^N stretching and a sharp
bandat1659e1651 cmꢀ1 correspondingtoC]OstretchintheirFT-IR.
The IR spectra of methyl esters of pyrazole-4-carboxylic acids
(5ae5g) exhibited a strong band in the range 1728e1697 cm1
attributed to C]O stretch while their 1H NMR spectra displayed
two characteristic sharp singlets due to C5eH of the pyrazole ring in
1. The entire class of pyrazole-4-carboxamide derivatives (3ae
3h) bearing primary sulfonamide as well as carboxamide
moieties (except 3g), displayed excellent CA inhibition pro-
file (Ki ¼ 2.1e7.1 nM) against the tumor-associated isozyme
hCA IX as compared to the standard drug acetazolamide
(Ki ¼ 25 nM).
2. Out of the pyrazole-4-carboxamide analogs (3ae3h), two
compounds 3f and 3h showed approximately 12 fold better
CA inhibition activity profile against hCA IX isozyme having
low nanomolar potency, Ki z 2.0 nM as compared to the AZA
whereas five other compounds (3ae3e) also showed multi
fold better nanomolar affinity against hCA IX with inhibition
constant, Ki < 7.1 nM.
the range
d 9.36e9.24 and methyl group in methyl ester in the
3. Nature of the substituents present on 4-position of the aro-
matic ring attached to 3-position of pyrazole scaffold does
not seem to follow any definite SAR pattern in terms of
electron donating/withdrawing influence on CA inhibition
profile against hCA IX.
4. Compounds 3ae3h also possessed appreciable CA inhibition
activity profile against hCA XII isozyme. Compound 3f was
found to be much more effective against hCA XII showing CA
Fig. 1. Design of the 4-functionalized 1,3-diarylpyrazole benzenesulfonamides (3ae3h,
5ae5g and 6ae6g) as CAIs.

286
P. Khloya et al. / European Journal of Medicinal Chemistry 76 (2014) 284e290
Scheme 1. Synthesis of target 4-functionalized pyrazoles (3, 5, 6). (i) THF/NH3/I2, stir; (ii) THF, NaOH/H2O2 stir; (iii) pyridine, KMnO4, stir; (iv) CH3OH/THF, H2SO4(catalytic), reflux;
(iv) CH3OH/THF, NH2NH2.H2O, reflux.
potency Ki ¼ 1.9 nM as shown in Table 1. Other compounds
3aeb, 3d, 3e in this series also exhibited better activity
(Ki < 5 nM) as compared to the standard drug acetazolamide
(Ki ¼ 5.7 nM).
(3, 5, 6), the trend of CA activity was found to be F > Cl > Br
against three isoforms hCA II, hCA IX and hCA XII.
7. CA II was moderately inhibited by the compounds 3ae3h.
8. Other series of pyrazole-4-hydrazinocarbonyl analogs (6ae
6g) also appreciably inhibited hCA IX and hCA XII isozymes
with inhibitory effects in nanomolar range.
9. Compounds 6a and 6b showed approximately 12 fold
superior CA inhibition profile against hCA IX isozyme as
compared to the standard drug AZA (Ki ¼ 25 nM). Indeed,
compound 6b was more effective with nanomolar inhibition
constant Ki ¼ 2.0 nM. Other analogs in this series except
6g also displayed high inhibition for hCA IX with Ki < 8.5 nM.
All the pyrazole-4-hydrazinocarbonyl compounds (6ae6g)
in general were found to be moderate inhibitors of CA II
and XII.
5. Interestingly, in the series of pyrazole-4-carboxamide com-
pounds (3ae3h), replacement of the aryl group with heter-
oaromatic thienyl (3h) at the 3-position of pyrazole scaffold
resulted in 12 fold better CA activity with inhibition constant
Ki ¼ 2.1 nM against hCA IX isozyme and 1.5 fold against hCA
XII isozymes (Ki ¼ 3.9 nM). This observation prompts us to
design another library of such compounds having various
heteroaromatics at C-3 of pyrazole and the results of such a
study would be reported in future publications.
6. The data in Table 1 leads to the conclusion that in case of
halogen analogs of the 4-functionalized pyrazole compounds
Table 1
Carbonic anhydrase activity of 4-functionalized pyrazole compounds (3ae3h, 5ae5g, and 6ae6g) using a stopped-flow, CO2 hydrase assay.
Compounds
Ki (nM)a
hCA I
hCA II
hCA IX
hCA XII
I/IX
I/XII
II/IX
4.88
1.33
5.86
1.42
0.86
1.73
7.36
4.52
14.72
25.88
32.5
24.56
33.85
33.19
130.96
3.32
II/XII
7.77
1.80
3.17
2.02
0.95
2.10
6.22
2.43
10.43
16.98
30.6
12.81
26.93
30.58
151.33
0.72
0.88
7.61
14.26
13.69
7.19
3a
3b
3c
3d
3e
3f
3g
3h
5a
5b
5c
5d
5e
5f
5g
6a
6b
6c
6d
6e
6f
77
68
257
32
33
54
21
5.6
34
10.1
4.5
4.0
4.3
4.2
5.8
7.1
5.2
2.3
49
2.1
16.3
12.4
10.0
22.8
17.1
9.4
5.2
2.8
2.0
6.9
2.7
3.1
10.7
5.0
4.7
1.9
17.906
16.19
44.31
4.50
6.34
23.47
7.69
18.57
1.31
2.29
0.83
7.85
0.56
1.28
7.65
2.82
4.80
13.18
0.84
0.54
6.545
0.649
10
28.518
21.93
24.01
6.40
7.02
28.42
6.50
10.0
0.93
1.50
0.78
4.09
4.46
1.18
8.84
0.61
0.92
3.74
0.41
377
39
361
9.5
58
3.9
21.5
28.4
8.3
179
9.6
12.1
39.8
7.9
9.6
91
7.1
3.9
36
32
250
240
321
325
560
579
312
681
9.3
23.0
18.9
10.6
43.7
21.5
10.2
4.5
12.9
10.4
24.3
17.1
13.0
8.9
9.2
4.6
185
244
178
64
71
12.1
26.81
29.04
25.07
11.636
1.44
8.4
7.1
5.5
49.3
25
0.30
4.044
2.37
6g
AZA
13.5
5.7
5.25
2.22
43.859
0.484
AZA ¼ Acetazolamide (reference compound).
a
Mean from 3 different assays, errors in the range of ꢁ5e10% of the reported value.

P. Khloya et al. / European Journal of Medicinal Chemistry 76 (2014) 284e290
287
10. Pyrazole carboxylates (5ae5g) were in general found to be
moderate inhibitors of hCA II and XII, with Ki value in the
range of 240e681 nM and 10.2e23.0 nM respectively. The
other two isoforms hCA IX and hCA XII were appreciably but
not significantly inhibited.
(s, ex, 1H, OH/NH), 7.51 (s, ex, 2H, SO2NH2), 7.45e7.42 (m, 3H, Are
H), 7.33 (s, ex, 1H, OH/NH); 13C NMR (DMSO-d6, 75.5 MHz):
164.7
(C]O), 152.3, 142.5, 132.6, 131.2, 128.9, 128.5, 127.9, 119.0, 118.9;
DART MS: m/z 343.20 [M þ H]þ, C16H14N4O3SHþ Calcd. 343.08.
d
11. A comparative study of pyrazole carboxamides (3), esters (5)
and carbohydrazides (6) revealed that in general the car-
boxamide analogs exhibited better CA activity profile against
hCA IX, XII and II.
5.1.1.2. 1-[4-(Aminosulfonyl)phenyl]-3-(4-methylphenyl)-1H-pyr-
azole-4-carboxamide (3b). Mp 248e251 ꢂC, yield 85%; IR (KBr,
cmꢀ1): 3387, 3279, 3194, 3140 (m, NeH stretch), 1651 (s, C]O
stretch),1589 (s, C]N stretch),1543 (s, NeH bend),1342 and 1157 (s,
SO2 stretch); 1H NMR (DMSO-d6, 300 MHz):
d 9.00 (s, 1H, pyrazole
4. Conclusions
C5eH), 8.08 (d, 2H, J ¼ 8.7 Hz, AreH), 7.98 (d, 2H, J ¼ 8.7 Hz, AreH),
7.76 (d, 2H, J ¼ 8.1 Hz, AreH), 7.64 (s, ex, 1H, OH/NH), 7.46 (s, ex, 2H,
SO2NH2), 7.33 (s, ex, 1H, OH/NH), 7.25 (d, 2H, J ¼ 8.1 Hz, AreH), 2.35
In the present paper, we report a series of twenty two com-
pounds of 4-functionalized 1,3-diarylpyrazole derivatives (3ae3h,
5ae5g and 6ae6g), which were evaluated against four isozymes,
hCA I, II, IX and XII. Most of the tested compounds exhibited
excellent CA inhibitory activity profile showing low nanomolar
potency against the tumor associated CA IX and XII. Some of the
tested compounds 3ae3f, 3h and 6ae6f showed low nanomolar
affinity (Ki < 10 nM) for CA IX while seven compounds (3ae3b, 3de
f, 3h and 6f) displayed Ki < 10 nM for CA XII. 4-Functionalized
pyrazoles derivatives (3ae3h, 5ae5g and 6ae6g) possessed high
degree of selectivity ratio for the inhibition of tumor associated CA
IX and XII over off target cytosolic isozymes I and II. In short, re-
ported compounds exhibited remarkable antitumor potential and
warranted further investigation to broaden the structure activity
relationship for this interesting class of sulfonamide CAIs.
(s, 3H, CH3); 13C NMR (DMSO-d6, 75.5 MHz):
d 164.9 (C]O), 152.2,
142.9, 141.5, 138.4, 131.2, 129.7, 129.1, 128.8, 127.9, 119.0, 118.8, 21.3
(CH3); DART MS: m/z 357.18 [M þ H]þ, C17H16N4O3SHþ Calcd. 357.09.
5.1.1.3. 1-[4-(Aminosulfonyl)phenyl]-3-(4-methoxyphenyl)-1H-pyr-
azole-4-carboxamide (3c). Mp 270e272 ꢂC, yield 94%; IR (KBr,
cmꢀ1): 3394, 3317, 3263, 3178 (m, NeH stretch), 1659 (s, C]O
stretch),1597 (s, C]N stretch),1543 (s, NeH bend),1327 and 1157 (s,
SO2 stretch); 1H NMR (DMSO-d6, 300 MHz):
d 8.99 (s, 1H, pyrazole
C5eH), 8.06 (d, 2H, J ¼ 8.4 Hz, AreH), 7.97 (d, 2H, J ¼ 8.7 Hz, AreH),
7.84 (d, 2H, J ¼ 8.7 Hz, AreH), 7.60 (s, ex, 1H, OH/NH), 7.38 (s, ex, 2H,
SO2NH2), 7.29 (s, ex, 1H, OH/NH), 7.00 (d, 2H, J ¼ 8.7 Hz, AreH), 3.81
(s, 3H, OCH3); 13C NMR (DMSO-d6, 75.5 MHz):
d 164.9 (C]O), 160.0,
142.3,141.6,131.2,130.3,127.9,118.9,118.6,113.9, 55.7 (OCH3); DART
MS: m/z 373.18 [M þ H]þ, C17H16N4O4SHþ Calcd. 373.09.
5. Experimental protocols
5.1.1.4. 1-[4-(Aminosulfonyl)phenyl]-3-(4-bromophenyl)-1H-pyr-
azole-4-carboxamide (3d). Mp 232e236 ꢂC, yield 95%; IR (KBr,
cmꢀ1): 3387, 3325, 3279, 3186 (m, NeH stretch), 1659 (s, C]O
stretch),1597 (s, C]N stretch),1543 (s, NeH bend),1366 and 1157 (s,
5.1. Chemistry
Melting points were determined in open capillaries in an elec-
trical apparatus and are uncorrected. The IR spectra in KBr were
recorded with ABB MB3000 DTGS IR instrument. 1H NMR and 13C
NMR spectra were recorded on Bruker instrument at 300 MHz and
75.5 MHz respectively using DMSO-d6 as a solvent. Chemical shifts
SO2 stretch);1HNMR(DMSO-d6, 300MHz):
d9.05(s,1H, pyrazoleC5e
H), 8.07 (d, 2H, J ¼ 8.7 Hz, AreH), 7.99 (d, 2H, J ¼ 8.7 Hz, AreH), 7.85 (d,
2H, J ¼ 8.4 Hz, AreH), 7.72 (s, ex, 1H, OH/NH), 7.65 (d, 2H, J ¼ 8.7 Hz,
AreH), 7.46 (s, ex, 2H, SO2NH2), 7.37 (s, ex, 1H, OH/NH); 13C NMR
are expressed in d, ppm. Mass spectra (DART-MS) were recorded on
(DMSO-d6, 75.5 MHz): d 164.5 (C]O), 151.2, 142.7, 141.4, 131.8, 131.5,
a JEOL-AccuTOF JMS-T100LC Mass spectrometer having a DART
(Direct Analysis in Real Time) source in ESþ mode. The purity of the
compounds was checked by 1H NMR and thin layer chromatog-
raphy (TLC) on silica gel plates using a mixture of petroleum ether
and ethyl acetate as eluent. Thin layer chromatography (TLC) ana-
lyses were performed on aluminum silica gel sheets 60 F254 plates
(Merck, Darmstadt, Germany) and spots were detected using a UV
lamp at 254 nm. Abbreviations ‘s’ for singlet, ‘m’ for multiplet, ‘ex’
for exchangeable proton are used for NMR assignments; ‘s’ for
strong and ‘m’ for medium for IR assignments.
131.5, 131.0, 128.0, 122.4, 119.1, 118.8; DART MS: m/z 420.12/422.14
[M þ H]þ/[M þ H þ 2]þ, C16H13BrN4O3SHþ Calcd. 420.99/422.01.
5.1.1.5. 1-[4-(Aminosulfonyl)phenyl]-3-(4-chlorophenyl)-1H-pyr-
azole-4-carboxamide (3e). Mp 240e242 ꢂC, yield 89%; IR (KBr,
cmꢀ1): 3387, 3325, 3239, 3217, 3186 (m, NeH stretch),1659 (s, C]O
stretch), 1597 (s, C]N stretch), 1504 (s, NeH bend), 1319 and 1157
(s, SO2 stretch); 1H NMR (DMSO-d6, 300 MHz):
d 9.06 (s, 1H, pyr-
azole C5eH), 8.08 (d, 2H, J ¼ 8.7 Hz, AreH), 7.99 (d, 2H, J ¼ 8.7 Hz,
AreH), 7.92 (d, 2H, J ¼ 8.4 Hz, AreH), 7.73 (s, ex,1H, OH/NH), 7.53 (s,
ex, 2H, SO2NH2), 7.49 (d, 2H, J ¼ 7.8 Hz, Ar), 7.38 (s, ex, 1H, OH/NH);
5.1.1. General procedure for conversion of 4-cyanopyrazoles (2) into
respective pyrazole-4-carboxamides (3)
13C NMR (DMSO-d6, 75.5 MHz):
d 164.5 (C]O), 151.2, 142.7, 141.4,
133.7, 131.5, 131.4, 130.7, 128.5, 128.0, 119.1, 118.8; DART MS: m/z
377.14/379.14 [M þ H]þ/[M þ H þ 2]þ, C16H13ClN4O3SHþ Calcd.
377.04/379.01.
To a stirred solution of appropriate 4-cyanopyrazole 2 [22]
(1.4 mmol) in THF (15 mL) was added aqueous NaOH (5 mmol)
followed by 30% H2O2 solution (5 mmol). Reaction mixture was
stirred overnight at room temperature whereupon it was poured
into ice cold water. The precipitates so obtained were filtered,
washed with excess of cold water, dried and crystallized from
aqueous THF to afford carboxamide derivatives 3 in excellent yields.
5.1.1.6. 1-[4-(Aminosulfonyl)phenyl]-3-(4-fluorophenyl)-1H-pyr-
azole-4-carboxamide (3f). Mp 230e234 ꢂC, yield 92%; IR (KBr,
cmꢀ1): 3379, 3310, 3186, 3078 (m, NeH stretch), 1659 (s, C]O
stretch), 1597 (s, C]N stretch), 1512 (s, NeH bend), 1335 and 1157
(s, SO2 stretch); 1H NMR (DMSO-d6, 300 MHz):
d 9.04 (s, 1H, pyr-
5.1.1.1. 1-[4-(Aminosulfonyl)phenyl]-3-phenyl-1H-pyrazole-4-
carboxamide (3a). Mp 238e240 ꢂC, yield 86%; IR (KBr, cmꢀ1): 3325
(m, NeH stretch),1651 (s, C]O stretch),1589 (s, C]N stretch),1520
(s, NeH bend), 1319, 1142 (s, SO2 stretch); 1H NMR (DMSO-d6,
azole C5eH), 8.07 (d, 2H, J ¼ 8.7 Hz, AreH), 7.98 (d, 2H, J ¼ 8.7 Hz,
AreH), 7.96e7.91 (m, 2H, AreH), 7.70 (s, ex, 1H, OH/NH), 7.47 (s, ex,
2H, SO2NH2), 7.36 (s, ex, 1H, OH/NH), 7.31e7.25 (m, 2H, AreH); 13
C
NMR (DMSO-d6, 75.5 MHz): d 164.6 (C]O), 151.4, 142.5, 141.5, 131.4,
3
2
300 MHz):
d
9.02 (s, 1H, pyrazole C5eH), 8.21 (d, 2H, J ¼ 8.7 Hz, Are
131.1 (d, JCF ¼ 8.3 Hz), 127.9, 119.0, 118.6, 115.3 (d, JCF ¼ 21.1 Hz);
H), 8.08 (d, 2H, J ¼ 8.4 Hz, AreH), 7.86 (d, 2H, J ¼ 6.6 Hz, AreH), 7.65
DART MS: m/z 361.16 [M þ H]þ, C16H13FN4O3SHþ Calcd. 361.07.

288
P. Khloya et al. / European Journal of Medicinal Chemistry 76 (2014) 284e290
5.1.1.7. 1-[4-(Aminosulfonyl)phenyl]-3-(4-nitrophenyl)-1H-pyrazole-
4-carboxamide (3g). Mp 310e314 ꢂC, yield 80%; IR (KBr, cmꢀ1):
3371, 3302 (m, NeH stretch), 1659 (s, C]O stretch), 1597 (s, C]N
stretch),1543 (s, NeH bend),1342 and 1165 (s, SO2 stretch); 1H NMR
130.9,127.8,124.3,119.6,113.8,113.6, 55.7 (OCH3), 51.9 (OCH3); DART
MS: m/z 388.18 [M þ H]þ, C18H17N3O5SHþ Calcd. 388.09.
5.1.2.4. Methyl 1-[4-(aminosulfonyl)phenyl]-3-(4-bromophenyl)-1H-
pyrazole-4-carboxylate (5d). Mp 190e192 ꢂC, yield 84%; IR (KBr,
cmꢀ1): 3317, 3240, 3132 (m, NeH stretch), 1697 (s, C]O stretch),
1597 (s, C]N stretch), 1535 (s, NeH bend), 1335 and 1165 (s, SO2
(DMSO-d6, 300 MHz):
J ¼ 8.1 Hz, AreH), 8.18 (d, 2H, J ¼ 8.1 Hz, AreH), 7.89 (m, 7H, Ar), 7.38
(s, ex, 1H, OH/NH); 13C NMR (DMSO-d6, 75.5 MHz):
164.5 (C]O),
d 9.07 (s, 1H, pyrazole C5eH), 8.29 (d, 2H,
d
150.2, 147.6, 146.4, 140.2, 139.1, 131.1, 130.1, 127.6, 123.7, 119.1, 118.9;
stretch); 1H NMR (DMSO-d6, 300 MHz):
d 9.27 (s, 1H, pyrazole C5e
DART MS: m/z 388.18 [M þ H]þ, C16H13N5O5SHþ Calcd. 388.06.
H), 8.19 (d, 2H, J ¼ 8.4 Hz, AreH), 7.97 (d, 2H, J ¼ 8.7 Hz, AreH), 7.78
(d, 2H, J ¼ 8.4 Hz, AreH), 7.66 (d, 2H, J ¼ 8.4 Hz, AreH), 7.48 (s, ex,
2H, SO2NH2), 3.77 (s, 3H, OCH3); 13C NMR (DMSO-d6, 75.5 MHz):
5.1.1.8. 1-[4-(Aminosulfonyl)phenyl]-3-(2-thienyl)-1H-pyrazole-4-
carboxamide (3h). Mp 248e250 ꢂC, yield 82%; IR (KBr, cmꢀ1): 3448,
3340, 3286, 3163 (m, NeH stretch), 1659 (s, C]O stretch), 1597 (s,
C]N stretch), 1528 (s, NeH bend), 1319 and 1157 (s, SO2 stretch);
d
162.9 (C]O), 152.7, 143.1, 141.1, 134.7, 131.6, 131.4, 131.2, 127.8,
122.9, 119.7, 113.9, 52.0 (OCH3); DART MS: m/z 435.12/437.13
[M þ H]þ/[M þ H þ 2]þ, C17H14BrN3O4SHþ Calcd. 435.99/437.88.
1H NMR (DMSO-d6, 300 MHz):
d 9.06 (s, 1H, pyrazole C5eH), 8.09
(d, 1H, J ¼ 3.3 Hz, thienyl), 8.03 (d, 2H, J ¼ 9.3 Hz, AreH), 7.99 (d, 2H,
J ¼ 9.0 Hz, AreH), 7.73 (s, ex, 1H, OH/NH), 7.58 (d, 1H, J ¼ 4.8 Hz,
thienyl), 7.47 (s, ex, 2H, SO2NH2), 7.38 (s, ex, 1H, OH/NH), 7.14e7.12
5.1.2.5. Methyl 1-[4-(aminosulfonyl)phenyl]-3-(4-chlorophenyl)-1H-
pyrazole-4-carboxylate (5e). Mp 206e208 ꢂC, yield 75%; IR (KBr,
cmꢀ1): 3387, 3302, 3124 (m, NeH stretch), 1705 (s, C]O stretch),
1597 (s, C]N stretch), 1535 (s, NeH bend), 1342 and 1157 (s, SO2
(m, 1H, thienyl); 13C NMR (DMSO-d6, 75.5 MHz):
d 164.4 (C]O),
147.1, 142.6, 141.2, 134.4, 131.7, 129.4, 128.0, 127.5, 119.0, 117.8; DART
stretch); 1H NMR (DMSO-d6, 300 MHz):
d 9.30 (s, 1H, pyrazole C5e
MS: m/z 349.13 [M þ H]þ, C14H12N4O3S2Hþ Calcd. 349.04.
H), 8.20 (d, 2H, J ¼ 8.7 Hz, AreH), 7.97 (d, 2H, J ¼ 8.7 Hz, AreH), 7.86
(d, 2H, J ¼ 8.7 Hz, AreH), 7.54 (d, 2H, J ¼ 8.4 Hz, AreH), 7.48 (s, ex,
2H, SO2NH2), 3.78 (s, 3H, OCH3); 13C NMR (DMSO-d6, 75.5 MHz):
5.1.2. General procedure for the synthesis of pyrazole-4-
carboxylates (5ae5g)
d
162.9 (C]O), 152.6, 143.1, 141.1, 134.8, 134.2, 131.3, 130.8, 128.5,
To a solution of pyrazole-4-carboxylic acid 4 [22] (1.2 mmol) in
20 mL MeOH:THF (6:4) was added conc. H2SO4 (1.5 mL) as a cata-
lyst. The reaction mixture was refluxed for 48 h. Progress of the
reaction was monitored by TLC. On completion of the reaction, the
reaction mixture was concentrated and allowed to cool to room
temperature. Solid thus obtained was filtered, washed with cold
water and crystallized from MeOH:THF (1:1) to afford the desired
compounds 5 in good yields.
127.8, 119.7, 113.9, 52.0 (OCH3); DART MS: m/z 392.14/394.13
[M þ H]þ/[M þ H þ 2]þ, C17H14ClN3O4SHþ Calcd. 392.04/394.05.
5.1.2.6. Methyl 1-[4-(aminosulfonyl)phenyl]-3-(4-fluorophenyl)-1H-
pyrazole-4-carboxylate (5f). Mp 228e230 ꢂC, yield 87%; IR (KBr,
cmꢀ1): 3618, 3240 (m, NeH stretch), 1697 (s, C]O stretch), 1597 (s,
C]N stretch),1528 (s, NeH bend),1342 and 1157 (s, SO2 stretch); 1H
NMR (DMSO-d6, 300 MHz): d 9.27 (s,1H, pyrazole C5eH), 8.20 (d, 2H,
J ¼ 8.7 Hz, AreH), 7.97 (d, 2H, J ¼ 8.7 Hz, AreH), 7.90e7.85 (m, 2H,
5.1.2.1. Methyl 1-[4-(aminosulfonyl)phenyl]-3-phenyl-1H-pyrazole-
4-carboxylate (5a). Mp 210e215 ꢂC, yield 85%; IR (KBr, cmꢀ1): 3240,
3124 (m, NeH stretch), 1697 (s, C]O stretch), 1597 (s, C]N
stretch), 1528 (s, NeH bend), 1350 and 1165 (s, SO2 stretch); 1H
AreH), 7.48 (s, ex, 2H, SO2NH2), 7.33e7.27 (m, 2H, AreH), 3.77 (s, 3H,
OCH3); 13C NMR (DMSO-d6, 75.5 MHz):
d
163.8 (d, 1JCF ¼ 245.4 Hz)),
162.9 (C]O), 152.9, 143.1, 141.2, 134.6, 131.8 (d, 3JCF ¼ 9.1 Hz), 128.4,
127.8,119.7,115.5 (d, 2JCF ¼ 21.1 Hz),113.8, 51.9 (OCH3); DART MS: m/z
376.17 [M þ H]þ, C17H14FN3O4SHþ Calcd. 376.07.
NMR (DMSO-d6, 300 MHz):
d 9.28 (s, 1H, pyrazole C5eH), 8.21 (d,
2H, J ¼ 8.7 Hz, AreH), 7.97 (d, 2H, J ¼ 9.0 Hz, AreH), 7.83e7.78 (m,
2H, AreH), 7.47e7.44 (m, 5H, AreH, SO2NH2), 3.77 (s, 3H, OCH3);
5.1.2.7. Methyl 1-[4-(aminosulfonyl)phenyl]-3-(4-nitrophenyl)-1H-
pyrazole-4-carboxylate (5g). Mp 220e224 ꢂC, yield 81%; IR (KBr,
cmꢀ1): 3325, 3256, 3152 (m, NeH stretch), 1705 (s, C]O stretch),
1597 (s, C]N stretch), 1528 (s, NeH bend), 1342 and 1157 (s, SO2
13C NMR (DMSO-d6, 75.5 MHz):
d 163.0 (C]O), 153.8, 143.0, 141.2,
134.6, 132.0, 129.5, 129.3, 128.4, 127.8, 119.7, 113.9, 51.9 (OCH3);
DART MS: m/z 358.11 [M þ H]þ, C17H15N3O4SHþ Calcd. 358.08.
stretch); 1H NMR (DMSO-d6, 300 MHz):
d 9.36 (s, 1H, pyrazole C5e
5.1.2.2. Methyl 1-[4-(aminosulfonyl)phenyl]-3-(4-methylphenyl)-1H-
pyrazole-4-carboxylate (5b). Mp 172e175 ꢂC, yield 80%; IR (KBr,
cmꢀ1): 3348, 3178, 3132, 3040 (m, NeH stretch), 1728 (s, C]O
stretch),1597 (s, C]N stretch),1528 (s, NeH bend),1335 and 1149 (s,
H), 8.33 (d, 2H, J ¼ 8.7 Hz, AreH), 8.23 (d, 2H, J ¼ 8.4 Hz, AreH), 8.13
(d, 2H, J ¼ 8.4 Hz, AreH), 7.98 (d, 2H, J ¼ 8.4 Hz, AreH), 7.49 (s, ex,
2H, SO2NH2), 3.80 (s, 3H, OCH3); 13C NMR (DMSO-d6, 75.5 MHz):
d
162.8 (C]O), 151.7, 147.9, 143.3, 141.0, 138.4, 135.0, 130.8, 127.8,
SO2 stretch); 1H NMR (DMSO-d6, 300 MHz):
d
9.25 (s, 1H, pyrazole
123.6, 119.9, 114.4, 52.2 (OCH3); DART MS: m/z 403.18 [M þ H]þ,
C5eH), 8.19 (d, 2H, J ¼ 9.0 Hz, AreH), 7.96 (d, 2H, J ¼ 8.7 Hz, AreH),
7.71 (d, 2H, J ¼ 8.1 Hz, AreH), 7.47 (s, ex, 2H, SO2NH2), 7.27 (d, 2H,
J ¼ 8.1 Hz, AreH), 3.77 (s, 3H, OCH3), 2.37 (s, 3H, CH3); 13C NMR
C
17H14N4O6SHþ Calcd. 403.06.
5.1.3. General procedure for the synthesis of pyrazole-4-
hydrazinocarbonyl derivatives (6ae6g)
(DMSO-d6, 75.5 MHz):
d 163.0 (C]O), 153.8, 142.9, 141.2, 138.8,
134.5, 129.4, 129.1, 129.0, 127.8, 119.6, 119.5, 113.8, 51.9 (OCH3), 21.4
To a solution of pyrazole-4-carboxylates 5 (1.4 mmol) in 20 mL
MeOH:THF (6:4) was added hydrazine hydrate 98% (1.6 mmol) and
resulting mixture was refluxed for 25e30 h. The progress of the
reaction was monitored by TLC. On completion of the reaction,
excess solvent was evaporated to reduce the volume to half
whereupon reaction mixture was allowed to cool to room tem-
perature and the solid thus obtained was filtered, washed with
water, dried and crystallized from MeOH:THF (1:1) to afford the
target compounds 6 in excellent yields.
(CH3); DART MS: m/z 372.19 [M þ H]þ, C18H17N3O4SHþ Calcd. 372.09.
5.1.2.3. Methyl 1-[4-(aminosulfonyl)phenyl]-3-(4-methoxyphenyl)-
1H-pyrazole-4-carboxylate (5c). Mp 179e180 ꢂC, yield 87%; IR (KBr,
cmꢀ1): 3348, 3240, 3140, 3109 (m, NeH stretch), 1713 (s, C]O
stretch),1597 (s, C]N stretch),1528 (s, NeH bend),1342 and 1149 (s,
SO2 stretch); 1H NMR (DMSO-d6, 300 MHz):
d 9.24 (s, 1H, pyrazole
C5eH), 8.19 (d, 2H, J ¼ 8.7 Hz, AreH), 7.96 (d, 2H, J ¼ 8.7 Hz, AreH),
7.78 (d, 2H, J ¼ 8.7 Hz, AreH), 7.48 (s, ex, 2H, SO2NH2), 7.02 (d, 2H,
J ¼ 8.7 Hz, AreH), 3.82 (s, 3H, OCH3), 3.77 (s, 3H, OCH3); 13C NMR
5.1.3.1. 4-[4-(Hydrazinocarbonyl)-3-phenyl-1H-pyrazole-1-yl]benze-
(DMSO-d6, 75.5 MHz):
d
163.1 (C]O),160.3,153.6,142.9,141.2,134.5,
nesulfonamide (6a). Mp 270e274 ꢂC; yield 99%; IR (KBr, cmꢀ1):

P. Khloya et al. / European Journal of Medicinal Chemistry 76 (2014) 284e290
289
3356, 3232, 3124, 3032 (m, NeH stretch), 1643 (s, C]O stretch),
1597 (s, C]N stretch), 1535 (s, NeH bend), 1335 and 1149 (s, SO2
J ¼ 8.7 Hz, AreH), 7.98 (d, 2H, J ¼ 8.7 Hz, AreH), 7.47 (s, ex, 2H,
SO2NH2), 7.31e7.25 (m, 2H, AreH), 4.57 (s, ex, 2H, NH2); 13C NMR
stretch); 1H NMR (DMSO-d6, 300 MHz):
d
9.49 (s, ex, 1H, NH), 8.94
(DMSO-d6, 75.5 MHz): d 162.8 (C]O),161.2,151.0,142.6,141.5,130.9,
(s, 1H, pyrazole C5eH), 8.09 (d, 2H, J ¼ 8.7 Hz, AreH), 7.98 (d, 2H,
J ¼ 9.0 Hz, AreH), 7.89e7.86 (m, 2H, AreH), 7.46e7.39 (m, 5H, Are
H, SO2NH2), 4.49 (s, ex, 2H, NH2); 13C NMR (DMSO-d6, 75.5 MHz):
130.8 (d, 3JCF ¼ 7.5 Hz), 128.9, 127.9, 119.0, 115.5 (d, 2JCF ¼ 21.1 Hz),
115.4; DART MS: m/z 376.17 [M þ H]þ, C16H14FN5O3SHþ Calcd.
376.08.
d
163.0 (C]O), 151.9, 142.5, 141.5, 132.4, 130.7, 129.0, 128.6, 128.6,
127.9, 119.0, 117.5; DART MS: m/z 358.21 [M þ H]þ, C16H15N5O3SHþ
5.1.3.7. 4-[4-(Hydrazinocarbonyl)-3-(4-nitrophenyl)-1H-pyrazole-1-
yl]benzenesulfonamide (6g). Mp 271e274 ꢂC, yield 94%; IR (KBr,
cmꢀ1): 3433, 3325, 3132, 3016 (m, NeH stretch), 1643 (s, C]O
stretch), 1597 (s, C]N stretch), 1520 (s, NeH bend), 1342 and 1165
Calcd. 358.09.
5.1.3.2. 4-[4-(Hydrazinocarbonyl)-3-(4-methylphenyl)-1H-pyrazole-
1-yl]benzenesulfonamide (6b). Mp 250e252 ꢂC, yield 90%; IR (KBr,
cmꢀ1): 3317, 3109, 3032 (m, NeH stretch), 1620 (s, C]O stretch),
1589 (s, C]N stretch), 1551 (s, NeH bend), 1342 and 1157 (s, SO2
(s, SO2 stretch); 1H NMR (DMSO-d6, 300 MHz):
d 9.61 (s, ex,1H, NH),
9.03 (s, 1H, pyrazole C5eH), 8.31 (d, 2H, J ¼ 8.4 Hz, AreH), 8.19 (d,
2H, J ¼ 8.7 Hz, AreH), 8.11 (d, 2H, J ¼ 8.4 Hz, AreH), 8.01 (d, 2H,
stretch); 1H NMR (DMSO-d6, 300 MHz):
d 9.46 (s, ex, 1H, NH), 8.91
J ¼ 8.7 Hz, AreH); 13C NMR (DMSO-d6, 75.5 MHz):
d 162.4, 147.7,
(s, 1H, pyrazole C5eH), 8.08 (d, 2H, J ¼ 8.7 Hz, AreH), 7.97 (d, 2H,
J ¼ 8.7 Hz, AreH), 7.77 (d, 2H, J ¼ 8.1 Hz, AreH), 7.24 (d, 2H,
J ¼ 7.8 Hz, AreH), 2.35 (s, 3H, CH3); 13C NMR (DMSO-d6, 75.5 MHz):
143.0, 142.0, 138.8, 131.3, 129.8, 128.0, 123.8, 119.4, 118.0; DART MS:
m/z 403.20 [M þ H]þ, C16H14N6O5SHþ Calcd. 403.07.
d
163.0 (C]O), 151.8, 142.4, 141.5, 138.4, 130.6, 129.6, 129.2, 128.5,
5.2. CA inhibition assay
127.9, 118.9, 117.4, 21.4 (CH3); DART MS: m/z 372.20 [M þ H]þ,
C
17H17N5O3SHþ Calcd. 372.11.
An SX.18MV-R Applied Photophysics (Oxford, UK) stopped-flow
instrument has been used to assay the catalytic/inhibition of
various CA isozymes as reported by Khalifah [24]. Phenol red (at a
concentration of 0.2 mM) has been used as indicator, working at the
absorbance maximum of 557 nM, with 10 mM Hepes (pH 7.4) as
buffer, 0.1 M Na2SO4 or NaClO4 (for maintaining constant the ionic
strength; these anions are not inhibitory in the used concentra-
tion), following the CA-catalyzed CO2 hydration reaction for a
period of 5e10 s. Saturated CO2 solutions in water at 25 ꢂC were
used as substrate. Stock solutions of inhibitors were prepared at a
concentration of 10 mM (in DMSOewater 1:1, v/v) and dilutions up
to 0.01 nM done with the assay buffer mentioned above. At least 7
different inhibitor concentrations have been used for measuring
the inhibition constant. Inhibitor and enzyme solutions were pre
incubated together for 10 min at room temperature prior to assay,
in order to allow for the formation of the EeI complex. Triplicate
experiments were done for each inhibitor concentration, and the
values reported throughout the paper are the mean of such results.
The inhibition constants were obtained by non-linear least squares
methods using PRISM 3, from Lineweaver burk plots as reported
earlier [25,26] and represent means from at least three different
determinations.
5.1.3.3. 4-[4-(Hydrazinocarbonyl)-3-(4-methoxyphenyl)-1H-pyr-
azole-1-yl]benzenesulfonamide (6c). Mp 240e242 ꢂC, yield 96%; IR
(KBr, cmꢀ1): 3865, 3842, 3742, 3649, 3618 (m, NeH stretch), 1643
(s, C]O stretch), 1551 (s, C]N stretch), 1520 (s, NeH bend), 1335
and 1157 (s, SO2 stretch); 1H NMR (DMSO-d6, 300 MHz):
d 9.46 (s,
ex, 1H, NH), 8.90 (s, 1H, pyrazole C5eH), 8.08 (d, 2H, J ¼ 9.0 Hz, Are
H), 7.97 (d, 2H, J ¼ 8.7 Hz, AreH), 7.85 (d, 2H, J ¼ 8.7 Hz, AreH), 7.45
(s, ex, 2H, SO2NH2), 6.99 (d, 2H, J ¼ 8.7 Hz, AreH), 4.48 (s, ex, 2H,
NH2), 3.80 (s, 3H, OCH3); 13C NMR (DMSO-d6, 75.5 MHz):
d 163.1
(C]O), 160.0, 142.3, 141.5, 130.6, 129.9, 127.9, 120.6, 118.9, 117.0,
114.0, 55.7 (OCH3); DART MS: m/z 388.21 [M þ H]þ, C17H17N5O4SHþ
Calcd.388.10.
5.1.3.4. 4-[3-(4-Bromophenyl)-4-(hydrazinocarbonyl)-1H-pyrazole-
1-yl]benzenesulfonamide (6d). Mp 282e284 ꢂC, yield 93%; IR (KBr,
cmꢀ1): 3742, 3618 (m, NeH stretch), 1620 (s, C]O stretch), 1551 (s,
C]N stretch), 1512 (s, NeH bend), 1335, 1165 (s, SO2 stretch); 1H
NMR (DMSO-d6, 300 MHz):
d 9.52 (s, ex, 1H, NH), 8.95 (s, 1H, pyr-
azole C5eH), 8.08 (d, 2H, J ¼ 8.1 Hz, AreH), 7.99 (d, 2H, J ¼ 8.1 Hz,
AreH), 7.86 (d, 2H, J ¼ 8.1 Hz, AreH), 7.64 (d, 2H, J ¼ 8.1 Hz, AreH),
7.45 (s, ex, 2H, SO2NH2), 4.49 (s, ex, 2H, NH2); 13C NMR (DMSO-d6,
75.5 MHz):
d 162.8 (C]O), 150.8, 142.7, 141.4, 131.6, 130.9, 130.7,
Acknowledgments
128.0, 122.5, 119.2, 117.4; DART MS: m/z 436.12/438.11 [M þ H]þ/
[M þ H þ 2]þ, C16H14BrN5O3SHþ Calcd. 436.00/438.03.
One of the authors (Poonam Khloya) is grateful to the Haryana
State Council for Science and Technology (HSCST), Panchkula
(Haryana), India and the other (SitaRam) to the Council of Scientific
and Industrial Research, New Delhi, India for the award of Senior
Research Fellowships. The authors are thankful to Sophisticated
Analytical Instrument Facility, Central Drug Research Institute,
Lucknow for providing Mass spectra. Work from CTS lab was
financed by an EU Project of the FP7 programme (Metoxia).
5.1.3.5. 4-[3-(4-Chlorophenyl)-4-(hydrazinocarbonyl)-1H-pyrazole-
1-yl]benzenesulfonamide (6e). Mp 268e270 ꢂC, yield 85%; IR (KBr,
cmꢀ1): 3325, 3217 (m, NeH stretch), 1620 (s, C]O stretch), 1551 (s,
C]N stretch), 1504 (s, NeH bend), 1327, 1157 (s, SO2 stretch); 1H
NMR (DMSO-d6, 300 MHz):
d 9.54 (s, ex, 1H, NH), 8.96 (s, 1H, pyr-
azole C5eH), 8.09 (d, 2H, J ¼ 8.7 Hz, AreH), 7.99 (d, 2H, J ¼ 8.7 Hz,
AreH), 7.93 (d, 2H, J ¼ 8.7 Hz, AreH), 7.53 (s, ex, 2H, SO2NH2), 7.50e
7.48 (m, 2H, AreH), 4.52 (s, ex, 2H, NH2); 13C NMR (DMSO-d6,
Appendix A. Supplementary data
75.5 MHz):
d 162.8 (C]O), 150.7, 142.7, 141.4, 133.8, 131.3, 130.9,
130.4, 128.7, 127.9, 119.1, 117.4; DART MS: m/z 392.20/394.15
[M þ H]þ/[M þ H þ 2]þ, C16H14Cl N5O3SHþ Calcd. 392.05/394.03.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.02.023.
5.1.3.6. 4-[3-(4-Fluorophenyl)-4-(hydrazinocarbonyl)-1H-pyrazole-
1-yl]benzenesulfonamide (6f). Mp 283e285 ꢂC, yield 96%; IR (KBr,
cmꢀ1): 3649, 3618, 3317 (m, NeH stretch), 1636 (s, C]O stretch),
1597 (s, C]N stretch), 1512 (s, NeH bend), 1311 and 1165 (s, SO2
References
[1] A. Thiry, J.M. Dogne, B. Masereel, C.T. Supuran, Trends in Pharmacological
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[2] J.H. Barlow, N. Lowe, Y.H. Edwards, P.H.W. Butterworth, Nucleic Acids
Research 15 (5) (1987) 2386.
stretch); 1H NMR (DMSO-d6, 300 MHz):
d
9.52 (s, ex,1H, NH), 8.95 (s,
1H, pyrazole C5eH), 8.20 (d, 2H, J ¼ 8.7 Hz, AreH), 8.09 (d, 2H,

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P. Khloya et al. / European Journal of Medicinal Chemistry 76 (2014) 284e290
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