Isatin-pyrazole benzenesulfonamide hybrids potently inhibit tumor-associated carbonic anhydrase isoforms IX and XII

Article abstract of DOI:10.1016/j.ejmech.2015.09.021

New series of benzenesulfonamide derivatives incorporating pyrazole and isatin moieties were prepared using celecoxib as lead molecule. Biological evaluation of the target compounds was performed against the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) and more precisely against the human isoforms hCA I, II (cytosolic), IX and XII (transmembrane, tumor-associated enzymes). Most of the tested compounds efficiently inhibited hCA I, II and IX, with K_Is of 2.5-102 nM, being more effective than the reference drug acetazolamide. Compounds 11e, 11f, 16e and 16f were found to inhibit hCA XII with Ki of 3.7, 6.5, 5.4 and 7.2 nM, respectively. Compounds 11e and 16e, with 5-NO₂substitution on the isatin ring, were found to be selective inhibitors of hCA IX and hCA XII. Docking studies revealed that the NO₂group of both compounds participate in interactions with Asp132 within the hCA IX active site, and with residues Lys67 and Asp130 in hCA XII, respectively.

Full text of DOI:10.1016/j.ejmech.2015.09.021

Accepted Manuscript
Isatin-pyrazole benzenesulfonamide hybrids potently inhibit tumor-associated
carbonic anhydrase isoforms IX and XII
Hany S. Ibrahim, Sahar M. Abou-Seri, Muhammet Tanc, Mahmoud M. Elaasser,
Hatem A. Abdel-Aziz, Claudiu T. Supuran
PII:
S0223-5234(15)30265-8
10.1016/j.ejmech.2015.09.021
EJMECH 8118
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 8 June 2015
Revised Date: 12 September 2015
Accepted Date: 14 September 2015
Please cite this article as: H.S. Ibrahim, S.M. Abou-Seri, M. Tanc, M.M. Elaasser, H.A. Abdel-Aziz,
C.T. Supuran, Isatin-pyrazole benzenesulfonamide hybrids potently inhibit tumor-associated carbonic
anhydrase isoforms IX and XII, European Journal of Medicinal Chemistry (2015), doi: 10.1016/
j.ejmech.2015.09.021.
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ACCEPTED MANUSCRIPT
Graphical abstract
Isatin-pyrazolebenzenesulfonamide hybrids potently inhibit tumor-associated
carbonic anhydrase isoforms IX and XII
a
b
c
d
Hany S. Ibrahim , Sahar M. Abou-Seri , Muhammet Tanc , Mahmoud M. Elaasser ,
e,f,*
c,*
Hatem A. Abdel-Aziz and Claudiu T. Supuran
R
R
R
O
N
Ar
N
NH
N
O
O
N
NH
NH
NH
H
N
NH
N
Ar
H N
2
O
O
O
N
N
N
O
S
N
O
O S
O
S
H N
O
O
(
5a-d)
Ar = Ph
R = H, Cl, Br, CH3
(11a-d)
Ar = Ph
NH2
H N
2
2
(16a-d)
R = H, Cl, Br, OCH3
R = H, Cl, Br, OCH3

ACCEPTED MANUSCRIPT
Isatin-pyrazole benzenesulfonamide hybrids potently inhibit tumor-
associated carbonic anhydrase isoforms IX and XII
a
b
c
Hany S. Ibrahim , Sahar M. Abou-Seri , Muhammet Tanc , Mahmoud M.
d
e,f,*
c,*
Elaasser , Hatem A. Abdel-Aziz and Claudiu T. Supuran
a
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Egyptian Russian University, Badr City, Cairo
1
1829, Egypt
b
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University,
Kasr El-Aini Street, Cairo, P.O. Box, 11562, Egypt
c
Neurofarba Department, Sezione di Scienze Farmaceutiche, Università degli di Studi di Firenze, Via U. Schiff 6,
5
0019 Sesto Fiorentino, Florence, Italy
d
The Regional Center for Mycology and Biotechnology, Al-Azhar University, Cairo, Egypt
e
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh
1
1451, Saudi Arabia
f
Department of Applied Organic Chemistry Department, National Research Center, Dokki, Cairo 12622, Egypt
ABSTRACT
New series of benzenesulfonamide derivatives incorporating pyrazole and isatin moieties were
prepared using celecoxib as lead molecule. Biological evaluation of the target compounds was
performed against the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) and more precisely
against the human isoforms hCA I, II (cytosolic), IX and XII (transmembrane, tumor-associated
enzymes). Most of the tested compounds efficiently inhibited hCA I, II and IX, with K s of 2.5-
I
1
02 nM, being more effective than the reference drug acetazolamide. Compounds 11e, 11f, 16e
and 16f were found to inhibit hCA XII with Ki of 3.7, 6.5, 5.4 and 7.2 nM, respectively.
Compounds 11e and 16e, with 5-NO substitution on the isatin ring, were found to be selective
2
inhibitors of hCA IX and hCA XII. Docking studies revealed that the NO group of both
2
compounds participate in interactions with Asp132 within the hCA IX active site, and with
residues Lys67 and Asp130 in hCA XII, respectively.
_
___________
Keywords: isatin; pyrazole; sulfonamide; carbonic anhydrase inhibitor; molecular docking.
1

ACCEPTED MANUSCRIPT
*
Corresponding authors:Tel.: +966-146-73764; fax: +966-146-76220; e-
mail:hatem_741@yahoo.com (HAA); Phone: +39-055-4573005; fax: +39-055-4573385; E-
mail: claudiu.supuran@unifi.it (CTS).
2

ACCEPTED MANUSCRIPT
1-Introduction
The sulfonamides and their isosters such as the sulfamates and sulfamides, are well
known carbonic anhydrase (CA, EC 4.2.1.1) inhibitors (CAIs) and are in clinical use for almost
0 years for the treatment of glaucoma, obesity, epilepsy and as diuretics [1]. The large use of
7
CAIs for pharmaceutical applications relies on the wide distribution of the 15 human (h) CA
isoforms within different tissues as well as on their involvement in many
physiological/pathological conditions. Antiglaucoma CAI-drugs mainly target CA II, IV and XII;
the diuretics CA II, IV, XII and XIV; the antiepileptics CA VII and XIV [2-5]. The selective
inhibition of CA IX and XII produces significaant antitumor and antimetastatic effects. However
the main drawback associated to the use of sulfonamide CAIs is represented by the lack of
selectivity in inhibiting the various isoforms, thus leading to a plethora of side effects [6, 7]. In
this contest many efforts have been made for the development of isoform-selective CAIs, and
some remarkable results have been achieved in the last 15 years since the introduction of the tail
approach [6-8]. Currently a sulfonamide CA IX inhibitor (SLC-0111) entered in Phase I clinical
studies for the treatment of hypoxic, advanced stage solid tumors [8-10]. Furthermore, novel CAI
classes such as the polyamines,[11] phenols,[12] dithiocarbamates,[13] xanthates[14] coumarins,
thiocoumarins, 2-thioxo-coumarins and coumarinoximes [1, 4, 15-17] were discovered and the
inhibition mechanisms of many of these compounds were explained by using kinetic,
spectroscopic and X-ray crystallographic techniques [2, 6, 12, 13].
Recently, benzyl aniline sulfonamides such as I were reported as hCA IX inhibitors (Ki
=
1.8-27 nM) [18]. The same group developed similar compounds such as the cyclic form II,
which selectively inhibited hCA IX (with Ki of 13-27 nM) versus hCA I/II [19]. The lead
molecule of these derivatives was celecoxib III which was demonstrated to be a strong hCA IX
(Ki = 16 nM) and hCA II (Ki = 21 nM) inhibitor by one of our groups [20]. In addition, other
studies were reported on five membered heterocyclic N-benzene sulfonamide IV possessing an
amino group instead of the aryl one found in compounds II and III. Compound IV showed
excellent inhibitory action against hCA IX (Ki = 6.3 nM) and hCA XII (Ki = 0.74 nM) [21].
(Figure 1)
Figure 1
3

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A series of 4-(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl)-1-(5-substituted-oxoindolin-3-
ylidene)semicarbazides V was recently evaluated as carbonic anhydrase inhibitors and displayed
2
interesting activity against hCA I and IX (X = NO , Ki = 5.95 and 1.25 µM, respectively) [22].
The Schiff base VI showed a potent inhibitory activity against hCA IX (Ki = 1.1 nM) and had a
high selectivity for isoform hCA IX compared to the cytosolic isozymes hCA I and hCA II [23].
(
Figure 1).
Based on these literature data, we report here the synthesis of three novel series of isatin-
pyrazole-benzenesulfonamide hybrids 5a-d, 11a-f and 16a-f as CAIs. The design of the new
hybrids relies on grafting the oxindole hydrazine carbonyl moiety from the isatin semicarbazide
derivatives V to the 5-phenyl-1H-pyrazol-1-yl)benzenesulfonamide scaffold found compounds II
and III, at either position 3 (5a-d ) or 4 (11a-f) of the pyrazole ring. The additive effect of
combining these pharmacophore moieties might produce compounds with high CA inhibitory
activity. Furthermore, the structure modification in the third series 16a-f involved replacement of
the 5-phenyl ring in 11a-f by an amino group in analogy to the potent CAI IV mentioned above
(
Figure 1).
2
-Results and Discussion
2
.1. Chemistry
The synthesis of the first series of sulfonamides, 5a-d, is presented in Scheme 1. The
classical Claisen condensation of acetophenone (1) with diethyl oxalate in the presence of
sodium ethoxide gave ethyl 2,4-dioxo-4-phenylbutanoate (2). The regioselective cyclization of
butanoate 2 with 4-aminosulfonylphenylhydrazine [24] was achieved in acetic acid, yielding
ethyl 5-phenyl-1-(4-sulfamoylphenyl)-1H-pyrazole-3-carboxylate (3) which was then reacted
with hydrazine hydrate to afford hydrazide 4. Hydrazones 5a-d were obtained by refluxing
hydrazide 4 with the appropriate isatin derivative in ethanol, in the presence of catalytic amounts
of acetic acid.
Scheme 1
4

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The IR spectrum of the unreported hitherto 4-(3-(hydrazinecarbonyl)-5-phenyl-1H-pyrazol-
1
-yl)benzenesulfonamide (4) showed absorption bands due to NH and NH groups at 3334-3196
2
-1
-1
cm , beside the absorption peak of C=O group at 1674 cm and two absorption bands of the SO
2
-1
1
group at 1319 and 1153 cm . Its H-NMR spectrum revealed three D O exchangeable singlet
2
signals corresponding to SO NH , NH-NH and NH-NH at
δ
7.47, 9.61 and 4.62, respectively.
δ 7.04.
2
2
2
2
The characteristic singlet signal of H-4 of pyrazole appeared at
1
The structure hydrazones 5a-d was confirmed by their H-NMR spectra which revealed the
disappearance of the hydrazide NH signal in compound 4 in addition to the appearance of the
2
1
signal of NH group of isatin moiety in the range
δ
10.74-11.33. The H-NMR spectra of 5a-d
showed downfield shifts of the hydrazide NH signal, which appeared in the range
δ
11.59-14.16
group)
157.50 and
20.48 ppm.
Preparation of the second series of sulfonamides (11a-f) was achieved as illustrated in
1
ppm. Moreover, the H-NMR spectrum of 5d showed a signal of aliphatic protons (CH
3
1
3
at
δ 2.30 ppm. The C-NMR spectrum of 5d showed the signals of C=O groups at δ
1
62.61 ppm, in addition to the signal of the aliphatic carbon (CH group) at
δ
3
Scheme 2. Ethyl benzoyl acetate (7) was synthesized by condensation of ethyl acetoacetate (6)
with benzoyl chloride in sodium ethoxide followed by hydrolysis in the presence of acqueous
NH
3
and NH
4
Cl. Refluxing
7
with DMF-DMA afforded ethyl 2-benzoyl-3-
(dimethylamino)acrylate (8) which was employed in the next step without further purification.
The reaction of the latter enaminone with 4-aminosulfonylphenylhydrazine hydrochloride in
refluxing ethanol produced the pyrazole ester 9. The ester 9 was subjected to hydrazinolysis by
fusion with hydrazine hydrate to give the corresponding hydrazide 10. Hydrazones 11a-f were
synthesized by the reaction of hydrazide 10 with the appropriate isatin derivative in ethanol and
in the presence of catalytic amount of acetic acid.
Scheme 2
1
The H NMR spectra of ester 9, hydrazide 10 and hydrazones 11a-f showed the characteristic
signal of H-3 of the pyrazole ring in the region
δ = 8.19-8.83 ppm. However, single crystal X-
ray analysis of hydrazide 10 gave an absolute confirmation for the structure of the latter
compound and excluded the other possible positional isomer (Figure 2) and (see also
supplementary Figure 1).
5

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Figure 2
1
The H-NMR spectrum of 11d revealed the appearance of a signal due to the aliphatic proton of
the OCH group at δ 3.77 ppm, whereas the carbon of the same group appeared at δ 55.58 ppm in
3
1
3
1
the C-NMR spectrum. The H-NMR spectrum of 11f presented one singlet signal characteristic
of benzylic protons ( at δ 4.93 ppm), whilst the carbon of the same group appeared at δ 42.96
1
3
ppm in the C NMR spectrum.
The synthetic pathway of the third series of sulfonamides, 16a-f, is depicted in Scheme 3.
The reaction of ethyl cyanoacetate (12) with triethyl orthoformate in the presence of acetic
anhydride generated ethyl (ethoxyethylene)cyanoacetate (13) which was then converted to
pyrazole 14 by treatment with 4-aminosulfonylphenylhydrazine in a mixture of acetic acid and
water (5:1). The structure of compound 14 was confirmed by X-ray crystallography (Figure 3)
and (supplementary Figure 2). Consequently, hydrazinolysis of the latter ester led to the
formation of hydrazide 15 which reacted with different isatins in refluxing ethanol to yield
hydrazones 16a-f.
Scheme 3
Figure 3
The IR spectra of compounds 16a-f contained bands of the NH
2
and NH groups in the range
-1
1
of 3120-3421 cm . Their H-NMR spectra had D O exchangeable signals related to 5-amino
2
protons at
of the sulfonamide group at around
1.50 ppm and the hydrazide NH proton in the range
spectroscopy revealed the characteristic signal of H-3 of the pyrazole in the range
δ 6.52-7.07 ppm, in addition to D O exchangeable singlet signals attributed to protons
2
δ
7.49 ppm, isatin NH proton (in 16a-e) in the range δ 10.55-
1
1
δ
11.24-13.10 ppm. In this series, H-NMR
8.20-9.18
ppm, while the C-NMR spectra showed a characteristic signal due to the C-5 of pyrazole ring
between 94.24-95.72 ppm.
Compound 16d had a signal of aliphatic protons (OCH group) at
δ
1
3
δ
1
δ
3.79 ppm in its H-NMR
3
13
spectrum and at
δ
56.09 ppm in its C-NMR spectrum. The benzylic protons of 16f appeared at
6

ACCEPTED MANUSCRIPT
1
13
δ
5.02 ppm in H NMR and the benzylic carbon was detected at
δ 43.00 ppm in the C-NMR
spectrum.
2
.2. Carbonic anhydrase inhibition
Inhibition data against four physiologically relevant hCA isoforms, hCA I, II (cytosolic) as
well as hCA IX and XII (transmembrane, tumor-associated isoforms), are shown in Table 1 and
were determined by a stopped-flow CO hydrase assays.[25]
The following SAR is evident from the data of Table 1:
i) The slow cytosolic isoform hCA I was effectively inhibited by sulfonamides 5, 11 and16
2
(
reported here, with K s ranging between 5.2 and 102 nM. Otherwise, 5b which was slightly less
I
effective (K of 102 nM) all the other compounds were low nanomolar inhibitors of this isoform
I
whose physiologic function is still not well understood. Acetazolamide, a clinically used
sulfonamide, was a much weaker CAI compared to the new compounds reported here (K of 250
I
nM).
(ii) hCA II, the physiologically dominant isoform was highly inhibited by all the compounds
reported here, with low nanomolar efficacy (K s ranging between 2.9 and 31.3 nM), making the
I
SAR discussion almost impossible since all scaffolds led to extremely effective hCA II inhibitors
(Except for compounds11e, 11f, 16e and 16f which were fairly less active than AAZ and 5a that
had the same efficacy as AAZ, the other compounds were much better hCA II inhibitors
compared to the standard drug, Table 1).
(iii) The tumor-associated hCA IX was also a highly inhibited by sulfonamides reported
here, with K s ranging between 2.5 and 52.9 nM. A part for 11a which was slightly less effective
I
as hCA IX inhibitors, all other synthesized derivatives showed inhibition constants ≤20 nM,
being thus highly effective for inhibiting this tumor-associated enzyme, a validated antitumor
target.
(
iv) hCA XII was also inhibited by sulfonamides reported here with K s ranging between 3.7 and
I
2
44 nM (Table 1). Except for compounds 11e, 11f, 16e and 16f, hCA XII was less efficiently
inhibited by the new derivatives than the other three isoforms. In fact compound 11e showed
higher activity than AAZ (K = 3.7 and 5.7 nM, respectively), whereas 11f, 16e and 16f had
I
comparable potency to the reference drug (K = 6.5, 5.4 and 7.2 nM, respectively).
I
7

ACCEPTED MANUSCRIPT
Table 1
It can be observed that moving the oxindole hydrazine carbonyl moiety on the pyrazole ring
from position 3 in 5a-d to position 4 in 11a-d and 16a-d enhanced the inhibitory activity against
hCA I, II and XII isoforms. On the other hand, no clear relationship was observed between the
enzyme inhibitory activity of different isoforms and replacement of the phenyl ring at position 5
of the pyrazole moiety in 11a-f with an amino group in 16a-f.
Regarding the effect of the substitution pattern on the isatin moiety, it was observed that the
2
introduction of a NO group to position 5 of the isatin led to compounds which preferentially
inhibited hCA IX and hCA XII over hCA I and hCA II, as evident for derivatives 11e and 16e.
Meanwhile, N-benzyl substitution on the isatin moiety, as in 11f and 16f, led to an increased
affinity of these derivatives for hCA XII.
2
.3. Molecular Docking Studies
Docking studies were employed to analyze the binding pattern of compounds 11e and 16e to
the tumor associated hCA IX and hCA XII isoforms. These studies revealed significant
information about the binding mode of these compounds, and showed the crucial role of the
sulfonamide as a zinc binding group [19] . Docking of compound 11e within the active site of
hCA IX revealed the same important role of the deprotonated sulfonamide moiety, which
interacts with zinc ion and the neighbor residue Leu198 (Figure 4A), whereas for 16e, the
sulfonamide group form H-bonds with Thr199 beside the coordination bond to the Zn(II) ion
(
Figure 4B). Moreover, the 5-NO substituent of isatin moiety presented an important role in the
2
interaction of both compounds with hCA IX by forming an electrostatic bond with Asp132
Figure 4A and 4B).
(
Figure 4
As for hCA IX; the binding of 11e and 16e to hCA XII is meainly influenced by the
deprotonated sulfonamide group acting as zinc binding moiety and H-bonds with the conserved
residue in all α-CAs, Thr199. The NO group showed its significant role by participating in
electrostatic interactions with Lys67 (compound 11e) or Asp130 (compound 16e) (Figure 5A and
B). In contrast to the binding to hCA IX, the isatin moiety of these compounds displayed extra
2
5
8

ACCEPTED MANUSCRIPT
H-bond interactions with the hCA XII active site. For example, the isatin moiety in 11e was able
to H-bond with Ser132 (Figure 5A). In compound 16e, the isatin moiety formed two hydrogen
bonds with Asn62 and Gln92 (Figure 5B). The additional interaction of the isatin moiety with
hCA XII active site may explain the higher inhibition of these compounds against hCA XII
compared to hCA XI.
Figure 5
Both compounds displayed CDOCKER interaction energy (11e; -50.23 Kcal/mol and -44.67
Kcal/mol) (16e; -49.78 Kcal/mol and -45.64 Kcal/mol) higher than AAZ (-24.63 Kcal/mol and -
2
4.67 Kcal/mol) upon interaction with hCA IX and hCA XII, respectively.
3
. Conclusion
Stimulated by the reported activity of several N-benzene sulfonamide pyrazoles and isatins as
CAIs, new series of isatin-pyrazole-benzenesulfonamide hybrids 5a-d, 11a-f and 16a-f were
designed and synthesized as inhibitors of several CA isoforms. The structure of the new
compounds was confirmed by spectral methods and intermediates 10 and 14 were confirmed by
using X-ray crystallography. Biological evaluation of the new compounds was performed against
hCA I, II, IX and XII. Most of the tested compounds efficiently inhibited hCA I, II and IX (K =
i
2
.5-102 nM) being more effective than the reference drug acetazolamide (AAZ). On the other
hand, they inhibited hCA XII to a lesser extent except for compounds 11e, 11f, 16e and 16f (K
.7, 6.5, 5.4 and 7.2 nM, respectively). Sulfonamides 11e and 16e with a 5-NO moiety on the
i
=
3
2
isatin ring were found to preferentially inhibit hCA IX and hCA XII. Docking studies were
performed to investigate the role of the NO group and revealed that it can form electrostatic
2
interaction with Asp 132 in hCA IX, and with Lys67/Asp130 during inhibition of hCA XII.
These results indicate that, the new hybrids provide an efficient pharmacophore to design CAIs,
yet further investigations are required to improve their selectivity toward the tumor-associated
isoforms hCA IX and XII.
4
. Experimental
4
.1. Chemistry
Unless otherwise noted, all materials were obtained from commercial suppliers and
used without further purification. Melting points were determined on Stuart SMP3
9

ACCEPTED MANUSCRIPT
version 5 digital melting point apparatus and were uncorrected. Elemental microanalyses
were performed at the Regional Center for Mycology and Biotechnology, Al-Azhar
University. The NMR spectra were recorded for some compounds on a Varian Mercury
1
13
VX-300 NMR spectrometer. H spectra were run at 300 MHz and C spectra were run at
7
4
5 MHz. For other compounds, the NMR spectra were recorded on Bruker Avance III
1
13
00 MHz high performance digital FT-NMR spectrophotometer ( H: 400, C: 100 MHz).
and were related to that of the solvents. Mass spectra were
Chemical shifts are quoted in
δ
recorded using Hewlett Packard Varian (Varian, Polo, USA), Shimadzu Gas
Chromatograph Mass spectrometer-QP 1000 EX (Shimadzu, Kyoto, Japan) and Finnegan
MAT, SSQ 7000 mass spectrophotometer at 70 eV. IR spectra were recorded on Bruker
FT-IR spectrophotometer as potassium bromide discs. Compounds
[19], 13 [29], 15 [30] were prepared and confirmed as reported.
2[26], 7[27], 8[28],
9
4
.1.1. Synthesis of ethyl 5-phenyl-1-(4-sulfamoylphenyl)-1H-pyrazole-3-carboxylate (3).
The diketoester, ethyl 2,4-dioxo-4-phenylbutanoate (2) (4 mmol, 0.88 g) was dissolved in acetic
acid (15 mL) and then a solution of 4-aminosulfonylphenylhydrazine (4 mmol, 0.75 g) in ethanol
(20 ml) was added. The reaction mixture was refluxed for 1 h. The formed precipitate was
filtered, dried and recrystallized from ethanol to yield compound 3. All spectral data coincide
with those reported [19].
4
.1.2. Synthesis of 4-(3-(hydrazinecarbonyl)-5-phenyl-1H-pyrazol-1-yl)benzenesulfonamide (4).
The ester 3 (10 mmol, 3.71 g) was refluxed in hydrazine hydrate (10 mL) and the reaction was
followed by TLC. After complete reaction (3 h), the mixture was poured onto ice and stirred for
1
h with addition of few drops of acetic acid. The formed precipitate was filtered off, washed
o
with diethyl ether, dried and recrystallized from ethanol. Beige crystals, 61% yield; mp 265 C.
-1
1
IR (KBr) νmax/cm 3334-3196 (NH
NMR (DMSO-d , 400 MHz) 4.61 (s, 2H, NH
of pyrazole), 7.29-7.31 (m, 2H, Ar-H), 7.38-7.46 (m, 3H, Ar-H), 7.51 (s, 2H, SO NH , D O
2
, NH), 1674 (C=O), 1593 (C=N), 1319, 1153 (SO
2
). H
6
δ
2
hydrazide, D O exchangeable), 7.06 (s, 1H, H-4
2
2
2
2
exchangeable), 7.53 (d, 2H, J = 8.7 Hz, Ar-H), 7.88 (d, 2H, J = 8.7 Hz, Ar-H), 9.69 (s, 1H, NH
1
3
hydrazide, D
2
O exchangeable). C NMR (DMSO-d
6
, 100 MHz)
δ
108.69, 125.99, 127.16,
+
1
29.18, 129.32, 129.49, 129.56, 142.07, 143.81, 144.64, 147.32, 161.07. MS m/z [%] 357 [M ,
1
0

ACCEPTED MANUSCRIPT
9
.84], 326 [100]. Anal. Calcd for C16
15 5 3
H N O S (357.39): C, 53.77; H, 4.23; N, 19.60; S, 8.97.
Found: C, 53.93; H, 4.29; N, 19.78; S, 9.04.
4
.1.3. General procedure for synthesis of compounds (5a-d).
To a solution of hydrazide 4 (10 mmol, 0.36 g) in ethanol (20 mL), the appropriate isatin (10
mmol) was added followed by a catalytic amount of acetic acid (0.5 mL) then the mixture was
refluxed for 1 h. The formed precipitate was filtered off, washed with hot ethanol and
recrystallized from DMF/ EtOH to give the targeted compounds 5a-d.
4
.1.3.1.4-(3-(2-(2-Oxoindolin-3-ylidene)hydrazine-1-carbonyl)-5-phenyl-1H-pyrazol-1-
o
-1
yl)benzenesulfonamide (5a). Yellow powder, 89 % yield; mp > 300 C. IR (KBr) νmax/cm 3361-
1
3
184 (NH , NH), 1732, 1701 (C=O), 1516 (C=N), 1325, 1155 (SO ). H NMR (DMSO-d , 400
2
2
6
MHz)
δ 6.95 (d, 1H, J = 7.8 Hz, H-7 isatin), 7.10 (t, 1H, J = 7.8 Hz, H-5 isatin), 7.28 (s, 1H, H-4
of pyrazole), 7.34-7.38 (m, 2H, Ar-H), 7.40-7.45 (m, 4H, Ar-H), 7.53 (s, 2H, SO NH , D O
2
2
2
exchangeable), 7.57 (d, 2H, J = 8.7 Hz, Ar-H), 7.65 (d, 1H, J = 7.8 Hz, H-4 isatin), 7.93 (d, 2H, J
=
8.7 Hz, Ar-H), 10.88, 11.22 (2s, 1H, NH isatin, D O exchangeable), 11.50, 14.16 (2s, 1H, NH
2
1
3
2 6
hydrazone, D O exchangeable). C NMR (DMSO-d , 100 MHz) δ 109.61, 111.54, 116.10,
1
1
3
20.37, 121.44, 122.52, 123.10, 126.09, 126.44, 127.35, 129.03, 129.32, 129.72, 132.22, 133.42,
+
38.45, 141.84, 143.05, 144.40, 145.95, 146.09, 158.02, 163.05. MS m/z [%] 486 [M , 8.62],
26 [100]. Anal. Calcd for C24 S (486.51): C, 59.25; H, 3.73; N, 17.27; S, 6.59. Found:
18 6 4
H N O
C, 59.37; H, 3.76; N, 17.41; S, 6.67.
4
.1.3.2.4-(3-(2-(5-Chloro-2-oxoindolin-3-ylidene)hydrazine-1-carbonyl)-5-phenyl-1H-pyrazol-1-
o
-1
yl)benzenesulfonamide (5b). Yellow powder, 56 % yield; mp > 300 C. IR (KBr) νmax/cm 3396-
1
3
221 (NH
MHz)
H, Ar-H), 7.54 (s, 2H, SO NH , D O exchangeable), 7.58 (d, 1H, J = 8.9Hz, H-4 isatin), 7.64
2
, NH), 1720, 1697 (C=O), 1517 (C=N), 1340, 1157 (SO
2
). H NMR (DMSO-d
6
, 400
δ
6.97 (dd, 1H, J = 8.3, 3.6 Hz, H-7 of isatin), 7.31 (s, 1H, H-4 of pyrazole), 7.34-7.49 (m,
6
2
2
2
2
(d, 1H, J = 8.6 Hz, Ar-H), 7.90 (d, 2H, J = 8.6 Hz, Ar-H), 11.00, 11.34 (2s, 1H, NH isatin, D O
1
3
exchangeable), 11.69, 14.13 (2s, 1H, NH hydrazone, D O exchangeable). C NMR (DMSO-d ,
2
6
1
1
00 MHz)
δ 109.98, 112.84, 117.26, 120.97, 122.12, 125.82, 126.16, 126.31, 126.46,127.20,
27.35, 129.14, 129.34, 129.42, 129.48, 131.26, 132.72, 137.61, 141.72, 143.12, 144.25,
1
1

ACCEPTED MANUSCRIPT
+
+
1
45.63,162.89, 164.77. MS m/z [%] 522 [M +2, 6.34], 520 [M , 18.60], 222 [100]. Anal. Calcd
for C H ClN O S (520.95): C, 55.33; H, 3.29; N, 16.13; S, 6.15. Found: C, 55.51; H, 3.27; N,
2
4
17
6
4
1
6.29; S, 6.22.
4
.1.3.3.4-(3-(2-(5-Bromo-2-oxoindolin-3-ylidene)hydrazine-1-carbonyl)-5-phenyl-1H-pyrazol-1-
o
-1
yl)benzenesulfonamide (5c). Yellow powder, 87 % yield; mp > 300 C. IR (KBr) νmax/cm 3367-
1
3
219 (NH , NH), 1725, 1699 (C=O), 1516 (C=N), 1325, 1157 (SO ). H NMR (DMSO-d , 300
2
2
6
MHz)
δ
6.94 (dd, 1H, J = 8.4, 4.4 Hz, H-7 of isatin), 7.29 (s, 1H, H-4 of pyrazole), 7.35-7.49 (m,
NH , D O exchangeable), 7.54-7.89 (m, 5H, Ar-H), 10.94, 11.30 (2s,
H, NH isatin, D O exchangeable), 11.61, 14.08 (2s, 1H, NH hydrazone, D O exchangeable).
6
1
H, Ar-H), 7.48 (s, 2H, SO
2
2
2
2
2
+
+
MS m/z [%] 566 [M +2, 5.41], 564 [M , 6.16], 326 [100]. Anal. Calcd for C H BrN O S
2
4
17
6
4
(565.40): C, 50.98; H, 3.03; N, 14.86; S, 5.67. Found: C, 51.16; H, 3.04; N, 14.95; S, 5.72.
4
1
3
3
.1.3.4. 4-(3-(2-(5-Methyl-2-oxoindolin-3-ylidene)hydrazine-1-carbonyl)-5-phenyl-1H-pyrazol-
o
-1
-yl)benzenesulfonamide (5d). Yellow powder, 77 % yield; mp > 300 C. IR (KBr) νmax/cm
1
344-3215 (NH , NH), 1714, 1685 (C=O), 1517 (C=N), 1340, 1166 (SO ). H NMR (DMSO-d ,
2
2
6
00 MHz)
δ
2.30 (s, 3H, CH
3
), 6.83 (d, 1H, J = 7.9 Hz, H-7 of isatin), 7.17 (d, 1H, J = 7.9 Hz,
NH
D O exchangeable), 7.57 (d, 2H, J = 8.1 Hz, Ar-H), 7.65 (d, 1H, J = 7.8 Hz, H-4 isatin), 7.91 (d,
H-6 of isatin), 7.26 (s, 1H, H-4 of pyrazole), 7.27-7.35 (m, 5H, Ar-H), 7.49 (s, 2H, SO
2
2
,
2
2
1
1
1
H, J = 8.7 Hz, Ar-H), 10.81, 11.22 (2s, 1H, NH isatin, D
2
O exchangeable), 11.54, 14.13 (2s,
1
3
H, NH hydrazone, D O exchangeable). C NMR (DMSO-d , 75 MHz) δ 20.48 (CH ), 109.07,
2
6
3
10.87, 119.88, 121.29, 125.37, 125.90, 126.69, 128.54, 129.21, 131.68, 132.13, 138.08, 140.28,
+
41.34, 143.91, 145.43, 157.50, 162.61. MS m/z [%] 500 [M , 5.60], 222 [100]. Anal. Calcd for
C H N O S (500.53): C, 59.99; H, 4.03; N, 16.79; S, 6.41. Found: C, 60.23; H, 4.11; N, 16.93;
25
20
6
4
S, 6.46.
4
.1.5. Synthesis of 4-(4-(hydrazinecarbonyl)-5-phenyl-1H-pyrazol-1-yl)benzenesulfonamide (10).
Ethyl 5-phenyl-1-(4-sulfamoylphenyl)-1H-pyrazole-4-carboxylate (9) (10 mmol, 3.71 g) was
refluxed with 15 mL of hydrazine hydrate for 3 h. After checking the end of the reaction using
TLC, the mixture was poured on ice, stirred for 1 h with addition of few drops of acetic acid. The
formed precipitate was filtered off, washed with diethyl ether, dried and recrystallized from
1
2

ACCEPTED MANUSCRIPT
o
-1
ethanol. Violet crystals, 77 % yield; mp 263-265 C. IR (KBr) νmax/cm 3398-3219 (2NH
2
, NH),
1
1
647 (C=O), 1560 (C=N), 1328, 1157 (SO
2
). H NMR (DMSO-d
6
, 400 MHz)
δ
4.42 (s, 2H, NH
2
hydrazone, D O exchangeable), 7.31-7.33 (m, 2H, Ar-H), 7.35-7.40 (m, 5H, Ar-H), 7.46 (s, 2H,
2
SO NH , D O exchangeable), 7.79 (d, 2H, J = 8.7 Hz, Ar-H), 8.21 (s, 1H, H-3 of pyrazole), 9.40
2
2
2
1
3
(
s, 1H, NH hydrazone, D
2
O exchangeable). C NMR (DMSO-d
6
, 100 MHz) δ 117.13, 125.82,
1
3
26.59, 128.54, 129.23, 129.45, 130.98, 140.35, 141.96, 143.49, 143.70, 162.23. MS m/z [%]
+
57 [M , 10.98], 326 [100]. Anal. Calcd for C H N O S (357.39): C, 53.77; H, 4.23; N, 19.60;
1
6
15
5
3
S, 8.97. Found: C, 53.91; H, 4.30; N, 19.76; S, 9.06.
4
.1.6. General procedure for synthesis of compounds (11a-f).
To a solution of 4-(4-(hydrazinecarbonyl)-5-phenyl-1H-pyrazol-1-yl)benzenesulfonamide (10)
10 mmol, 0.36 g) in 20 mL ethanol, 10 mmol of 5-(un)substituted isatin or N-benzyl isatin was
added followed by catalytic amount of acetic acid (0.5 ml). The reaction mixture was refluxed for
h. The formed precipitate, in case of 11a-e, was filtered, washed with hot ethanol and
recrystallized from DMF/ EtOH to give the targeted compounds 11a-e. Concerning compound
(
1
1
1f, the precipitate formed after cooling was filtered and recrystallized from DMF/ EtOH.
4
.1.6.1.4-(4-(2-(2-Oxoindolin-3-ylidene)hydrazine-1-carbonyl)-5-phenyl-1H-pyrazol-1-
o
-1
yl)benzenesulfonamide (11a). Yellow powder, 83 % yield; mp > 300 C. IR (KBr) νmax/cm
1
3
278- 3136 (NH
, 300 MHz)
2
, 2NH), 1710-1681 (C=O), 1552 (C=N), 1320, 1153 (SO
2
). H NMR (DMSO-
d
6
δ
6.85 (d, 1H, J = 7.8 Hz, H-7 isatin), 7.05 (t, 1H, J = 7.8, H-5 isatin), 7.30-7.53
(
m, 9H, Ar-H), 7.50 (s, 2H, SO NH , D O exchangeable), 7.81 (d, 2H, J = 8.4 Hz, Ar-H), 8.34,
2 2 2
8
.47 (2s, 1H, H-3 of pyrazole), 10.74,11.20 (2s, 1H, NH isatin, D O exchangeable), 10.95, 13.10
2
1
3
(
2s, 1H, NH hydrazone, D O exchangeable). C NMR (DMSO-d , 75 MHz) δ 110.43, 111.06,
2
6
1
1
15.21, 119.71, 120.77, 121.58, 122.58, 125.77, 125.88, 126.47, 128.01, 129.54, 130.34, 131.52,
+
32.41, 141.13, 142.11, 143.37, 162.46, 164.60. MS m/z [%] 486 [M , 7.98], 222 [100]. Anal.
Calcd for C H N O S (486.51): C, 59.25; H, 3.73; N, 17.27; S, 6.59. Found: C, 59.37; H, 3.76;
2
4
18
6
4
N, 17.39; S, 6.64.
4
.1.6.2.4-(4-(2-(5-Chloro-2-oxoindolin-3-ylidene)hydrazine-1-carbonyl)-5-phenyl-1H-pyrazol-1-
o
-1
yl)benzenesulfonamide (11b). Yellow powder, 61 % yield; mp > 300 C. IR (KBr) νmax/cm
1
3

ACCEPTED MANUSCRIPT
1
3
4
7
8
1
1
1
410-3186 (NH
00 MHz)
.42 (m, 8H, Ar-H), 7.47 (s, 2H, SO NH , D O exchangeable), 7.77 (d, 2H, J = 8.7 Hz, Ar-H),
2
, NH), 1712-1698 (C=O), 1506 (C=N), 1350, 1165 (SO
2 6
). H NMR (DMSO-d ,
δ
6.87 (d, 1H, J = 8.4 Hz, H-7 of isatin), 6.93 (d, 1H, J = 8.4 Hz, H-6 of isatin), 7.33-
2
2
2
.38, 8.48 (2s, 1H, H-3 of pyrazole), 10.88, 11.33 (2s, 1H, NH isatin, D O exchangeable), 11.35,
2
1
3
3.02 (2s, 1H, NH hydrazone, D O exchangeable). C NMR (DMSO-d , 100 MHz) δ 112.26,
2
6
13.09, 116.73, 120.81, 121.92, 126.08, 126.28, 126.41, 127.04, 127.22, 128.80, 128.96, 129.79,
30.71, 130.84, 131.40, 132.26, 141.38, 141.71, 142.81, 143.84, 143.96, 162.76, 164.92. MS m/z
+
+
[
%] 522 [M +2, 3.22], 520 [M , 9.01], 326 [100]. Anal. Calcd for C H ClN O S (520.95): C,
24 17 6 4
5
5.33; H, 3.29; N, 16.13; S, 6.15. Found: C, 55.71; H, 3.34; N, 16.29; S, 6.21.
4
.1.6.3.4-(4-(2-(5-Bromo-2-oxoindolin-3-ylidene)hydrazine-1-carbonyl)-5-phenyl-1H-pyrazol-1-
o
-1
yl)benzenesulfonamide (11c). Yellow powder, 87 % yield; mp > 300 C. IR (KBr) νmax/cm
1
3
4
7
8
1
1
1
412-3178 (NH , NH), 1712-1683 (C=O), 1506 (C=N), 1350, 1166 (SO ). H NMR (DMSO-d ,
2
2
6
00 MHz)
.45 (m, 8H, Ar-H), 7.51 (s, 2H, SO
.38, 8.47 (2s, 1H, H-3 of pyrazole), 10.89, 11.23 (2s, 1H, NH isatin, D O exchangeable), 11.35,
δ
6.82 (d, 1H, J = 8.4 Hz, H-7 of isatin), 6.88 (d, 1H, J = 8.4 Hz, H-6 of isatin), 7.38-
2
NH , D O exchangeable), 7.82 (d, 2H, J = 8.7 Hz, Ar-H),
2
2
2
1
3
3.01 (2s, 1H, NH hydrazone, D O exchangeable). C NMR (DMSO-d , 100 MHz) δ 112.74,
2
6
13.53, 113.74, 114.79, 117.20, 122.33, 123.55, 126.28, 126.41, 127.04, 128.87, 128.96, 129.76,
30.69, 130.83, 134.18, 135.06, 141.60, 141.75, 142.74, 143.84, 143.95, 162.61, 164.79. MS m/z
+
+
[
6 4
%] 566 [M +2, 3.32], 564 [M , 3.56], 326 [100]. Anal. Calcd for C24H17BrN O S (565.40): C,
5
0.98; H, 3.03; N, 14.86; S, 5.67. Found: C, 51.08; H, 3.09; N, 14.97; S, 5.73.
4
.1.6.4. 4-(4-(2-(5-Methoxy-2-oxoindolin-3-ylidene)hydrazine-1-carbonyl)-5-phenyl-1H-pyrazol-
o
-
1
-yl)benzenesulfonamide (11d). Orange powder, 81 % yield; mp > 300 C. IR (KBr) νmax/cm
1
1
3
414-3190 (NH
00 MHz) 3.77 (s, 3H, OCH
H, Ar-H), 7.30-7.43 (m, 6H, Ar-H), 7.45 (s, 2H, SO NH , D O exchangeable), 7.80 (d, 2H, J =
2
, NH), 1722-1690 (C=O), 1512 (C=N), 1322, 1156 (SO
2
). H NMR (DMSO-d
6
,
3
1
8
δ
3
), 6.81 (dd, 1H, J = 11.4, 8.6 Hz, H-7 of isatin), 6.91-7.08 (m,
2
2
2
2
.7 Hz, Ar-H), 8.34, 8.49 (2s, 1H, H-3 of pyrazole), 10.55, 11.02 (2s, 1H, NH isatin, D O
1
3
exchangeable), 11.31, 13.12 (2s, 1H, NH hydrazone, D O exchangeable). C NMR (DMSO-d ,
2
6
7
1
5 MHz) δ 55.58, 105.73, 110.91, 111.91, 112.39, 115.65, 118.23, 120.46, 125.74, 126.50,
28.29, 129.23, 130.23, 135.90, 137.35, 141.14, 142.19, 143.31, 154.45, 155.32, 162.60, 164.74.
1
4

ACCEPTED MANUSCRIPT
+
20 6 5
MS m/z [%] 516 [M , 7.62], 326 [100]. Anal. Calcd for C25H N O S (516.53): C, 58.13; H,
3
.90; N, 16.27; S, 6.21. Found: C, 58.30; H, 3.96; N, 16.38; S, 6.32.
4
.1.6.5. 4-(4-(2-(5-Nitro-2-oxoindolin-3-ylidene)hydrazine-1-carbonyl)-5-phenyl-1H-pyrazol-1-
o
-1
yl)benzenesulfonamide (11e). Yellow powder, 92 % yield; mp > 300 C. IR (KBr) νmax/cm
1
3
4
367-3105 (NH
2
, NH), 1716-1685 (C=O), 1523 (C=N), 1338, 1165 (SO
2
). H NMR (DMSO-d
6
,
00 MHz)
δ
7.08 (dd, 1H, J = 19.2, 8.7 Hz, H-7 of isatin), 7.33-7.46 (m, 5H, Ar-H), 7.47 (s, 2H,
SO NH , D O exchangeable), 7.82 (d, 2H, J = 8.7 Hz, Ar-H), 8.16-8.42 (m, 4H, Ar- H), 8.49,
2
2
2
8
.83 (2s, 1H, H-3 of pyrazole), 10.47, 11.86 (2s, 1H, NH isatin, D
2
O exchangeable), 11.90, 12.86
1
3
(
2s, 1H, NH hydrazone, D O exchangeable). C NMR (DMSO-d , 100 MHz) δ 111.02, 111.84,
2
6
1
1
5
15.44, 116.15, 120.94, 122.07, 126.30, 126.43, 127.03, 128.74, 129.02, 129.65, 130.70, 130.82,
41.57, 141.68, 142.35, 143.22, 143.86, 144.00, 147.78, 149.60, 163.20, 165.40. MS m/z [%]
+
31 [M , 9.67], 324 [30], 125 [100]. Anal. Calcd for C H N O S (531.50): C, 54.24; H, 3.22;
2
4
17
7
6
N, 18.45; S, 6.03. Found: C, 54.51; H, 3.28; N, 18.63; S, 6.11.
4
.1.6.6. 4-(4-(2-(1-benzyl-2-oxoindolin-3-ylidene)hydrazine-1-carbonyl)-5-phenyl-1H-pyrazol-1-
o
-1
yl)benzenesulfonamide (11f). Orange powder, 89 % yield; mp = 210-212 C. IR (KBr) νmax/cm
1
3
4
7
313-3194 (NH , NH), 1674-1612 (C=O), 1554 (C=N), 1342, 1168 (SO ). H NMR (DMSO-d ,
2
2
6
00 MHz)
δ 4.93 (s, 2H, benzylic CH ), 7.04 (d, 1H, J = 8.7 Hz, H-7 of isatin), 7.11 (t, 1H, J =
2
.5 Hz, H-5 of isatin), 7.25-7.40 (m, 11H, Ar-H), 7.47 (d, 2H, J = 8.4 Hz, Ar-H), 7.52 (s, 2H,
SO NH , D O exchangeable), 7.56 (d, 1H, J = 7.4 Hz, Ar-H), 7.85 (d, 2H, J = 8.4 Hz, Ar-H),
2
2
2
8
.39, 8.57 (2s, 1H, H-3 of pyrazole), 12.00, 13.02 (2s, 1H, NH hydrazone, D O exchangeable).
2
1
3
6
C NMR (DMSO-d , 100 MHz) δ 42.96, 110.82, 115.85, 119.69, 121.13, 123.74, 126.37,
1
1
27.02, 127.96, 128.15, 128.30, 128.99, 129.18, 130.05, 130.89, 131.84, 136.10, 141.62, 142.89,
+
43.97, 145.40, 161.08, 172.52. MS m/z [%] 576 [M , 2.03], 144 [100]. Anal. Calcd for
C H N O S (576.63): C, 64.57; H, 4.20; N, 14.57; S, 5.56. Found: C, 64.74; H, 4.27; N, 14.74;
31
24
6
4
S, 5.61.
4
.1.8. Synthesis of ethyl 5-amino-1-(4-sulfamoylphenyl)-1H-pyrazole-4-carboxylate (14).
Ethyl 2-cyano-3-ethoxyacrylate (13) (10 mmol, 1.69 g) and 4-aminobenzenesulfonamide
hydrochloride (10 mmol, 2.23 g) were refluxed in a mixture of acetic acid and water (5:1) for 4
1
5

ACCEPTED MANUSCRIPT
h. The reaction mixture was poured on ice and stirred for 1 h. The given precipitate was filtered,
washed with water, dried and recrystallized from ethanol. The experimental data were given as
reported [31].
4
.1.9. General procedure for synthesis of compounds (16a-f).
In 50 mL round flask, 4-(4-(hydrazinecarbonyl)-5-amino-1H-pyrazol-1-yl)benzenesulfonamide 15
10 mmol, 0.3 g) was dissolved in ethanol (20 mL) followed by the addition of the appropriate
(
isatin derivative (10 mmol). Reflux was performed after the addition of a catalytic amount of
acetic acid (0.5 mL) for 1 h. The formed precipitate, in case of 16a-e, was filtered washed with
hot ethanol and recrystallized from DMF / EtOH to give the targeted compounds 16a-e.
Concerning compound 16f, the precipitate formed after cooling was filtered and recrystallized
from DMF / EtOH.
4
.1.9.1.4-(5-Amino-4-(2-(2-oxoindolin-3-ylidene)hydrazine-1-carbonyl)-1H-pyrazol-1-
o
-1
yl)benzenesulfonamide (16a). Yellow powder, 76 % yield; mp > 300 C. IR (KBr) νmax/cm
1
3
3
1
7
385-3182 (NH
00 MHz) 6.52 (s, 2H, NH
H, Ar-H), 7.45, 7.50 (2s, 2H, SO NH , D O exchangeable), 7.57 (d, 2H, J = 7.5 Hz, Ar-H),
2
, NH), 1718-1690 (C=O), 1531 (C=N), 1321, 1153 (SO
2
). H NMR (DMSO-d
6
,
δ
2
, D
2
O exchangeable), 6.87-7.17 (m, 3H, Ar-H), 7.37 (t, J = 7.8 Hz,
2
2
2
.76-8.12 (m , 2H, Ar-H), 8.53, 9.18 (2s, 1H, H-3 of pyrazole), 10.79, 11.14 (2s, 1H, NH isatin,
1
3
D O exchangeable), 11.24, 12.96 (2s, 1H, NH hydrazone, D O exchangeable). C NMR
2
2
(
DMSO-d , 75 MHz) δ 95.48, 110.51, 111.04, 115.43, 120.01, 120.55, 121.71, 122.55, 123.52,
6
1
25.97, 126.93, 127.03, 131.12, 132.11, 138.77, 140.20, 142.02, 143.57, 151.60, 162.79, 165.02.
+
MS m/z [%] 425 [M , 30.01], 265 [100]. Anal. Calcd for C H N O S (425.42): C, 50.82; H,
1
8
15
7
4
3
.55; N, 23.05; S, 7.54. Found: C, 51.04; H, 3.53; N, 23.28; S, 7.63.
4
.1.9.2.4-(5-Amino-4-(2-(5-chloro-2-oxoindolin-3-ylidene)hydrazine-1-carbonyl)-1H-pyrazol-1-
o
-1
yl)benzenesulfonamide (16b). Yellow powder, 70 % yield; mp > 300 C. IR (KBr) νmax/cm
1
3
3
1
390-3182 (NH , NH), 1725-1664 (C=O), 1521 (C=N), 1305, 1165 (SO ). H NMR (DMSO-d ,
2
2
6
00 MHz)
δ 6.52 (s, 2H, NH , D O exchangeable), 6.83-7.05 (m, 2H, Ar-H), 7.34 (t, J = 7.8 Hz,
2 2
H, Ar-H), 7.50, 7.63 (2s, 2H, SO
2
2 2
NH , D O exchangeable), 7.82 (t, J = 6.6 Hz, 2H, Ar-H),7.99
(d, J = 8.4 Hz, 2H, Ar-H), 8.20, 8.46 (2s, 1H, H-3 of pyrazole), 10.80, 11.34 (2s, 1H, NH isatin,
1
6

ACCEPTED MANUSCRIPT
1
3
D
2
O exchangeable), 12.85 (s, 1H, NH hydrazone, D
2 6
O exchangeable). C NMR (DMSO-d , 75
MHz) δ 94.86, 112.43, 120.12, 121.66, 122.64, 123.25, 126.74, 127.01, 130.39, 133.30, 140.18,
+
+
1
40.58, 142.60, 151.68, 162.53. MS m/z [%] 461 [M +2, 3.22], 459 [M , 9.10], 265 [100]. Anal.
Calcd for C18 S (459.87): C, 47.01; H, 3.07; N, 21.32; S, 6.97. Found: C, 47.14; H,
7 4
H14ClN O
3
.09; N, 21.57; S, 7.04.
4
.1.9.3.4-(5-Amino-4-(2-(5-Bromo-2-oxoindolin-3-ylidene)hydrazine-1-carbonyl)-1H-pyrazol-1-
o
-1
yl)benzenesulfonamide (16c). Yellow powder, 79 % yield; mp > 300 C. IR (KBr) νmax/cm
1
3
3
412-3178 (NH
00 MHz)
2
, NH), 1712-1683 (C=O), 1506 (C=N), 1350, 1166 (SO
2
). H NMR (DMSO-d
6
,
δ
6.52 (s, 2H, NH , D O exchangeable), 6.86-7.02 (m, 3H, Ar-H), 7.49 (s, 2H,
2 2
SO NH , D O exchangeable), 7.70-7.85 (m, 2H, Ar-H), 7.99 (d, 2H, J = 8.4 Hz, Ar-H), 8.20,
2
2
2
8
1
1
.57 (2s, 1H, H-3 of pyrazole), 10.89, 11.33 (2s, 1H, NH isatin, D
2
O exchangeable), 12.84 (s,
1
3
H, NH hydrazone, D O exchangeable). C NMR (DMSO-d , 75 MHz) δ 94.24, 112.87, 114.35,
2
6
22.05, 122.84 123.45, 127.01, 133.17, 140.18, 140.95, 142.58, 151.68, 162.38. MS m/z [%] 505
+
+
[
M +2, 3.67], 503 [M , 3.69], 222 [100]. Anal. Calcd for C H BrN O S (504.32): C, 42.87; H,
18 14 7 4
2
.80; N, 19.44; S, 6.36. Found: C, 42.99; H, 2.79; N, 19.62; S, 6.39.
4
1
3
4
.1.9.4. 4-(5-Amino-4-(2-(5-methoxy-2-oxoindolin-3-ylidene)hydrazine-1-carbonyl)-1H-pyrazol-
o
-1
-yl)benzenesulfonamide (16d). Orange powder, 85 % yield; mp > 300 C. IR (KBr) νmax/cm
1
414-3190 (NH , NH), 1722-1690 (C=O), 1512 (C=N), 1322, 1156 (SO ). H NMR (DMSO-d ,
2
2
6
00 MHz)
δ 3.80 (s, 3H, OCH ), 6.82 (d, 1H, J = 8.4 Hz, H-7 isatin), 6.96 (d, 2H, J = 10.2 Hz,
3
Ar- H), 7.07 (s, 2H, NH , D O exchangeable), 7.49 (s, 2H, SO NH , D O exchangeable), 7.83 (d,
2
2
2
2
2
2
1
H, J = 8.4 Hz, Ar-H), 8.00 (d, 2H, J = 8.4 Hz, Ar-H), 8.61 (s, 1H, H-3 of pyrazole), 10.61,
1.07 (s, 1H, NH isatin, D O exchangeable), 11.38, 13.02 (2s, 1H, NH hydrazone, D O
2
2
1
3
exchangeable). C NMR (DMSO-d
6
, 100 MHz) δ 56.41, 95.92, 111.50, 112.13, 116.20, 118.52,
1
1
21.22, 123.18, 123.97, 127.51, 137.77, 140.83, 142.96, 143.31, 152.46, 155.11, 155.83, 165.69,
+
66.47. MS m/z [%] 455 [M , 9.06], 223 [100]. Anal. Calcd for C H N O S (455.45): C, 50.11;
19
17
7
5
H, 3.76; N, 21.53; S, 7.04. Found: C, 50.27; H, 3.83; N, 21.75; S, 7.13.
4
.1.9.5.4-(5-Amino-4-(2-(5-Nitro-2-oxoindolin-3-ylidene)hydrazine-1-carbonyl)-1H-pyrazol-1-
o
-1
yl)benzenesulfonamide (16e). Yellow powder, 92 % yield; mp > 300 C. IR (KBr) νmax/cm
1
7

ACCEPTED MANUSCRIPT
1
3
4
421-3120 (NH
2
2 6
, NH), 1732-1666 (C=O), 1523 (C=N), 1327, 1165 (SO ). H NMR (DMSO-d ,
00 MHz)
δ
2 2
6.79-7.14 (m, 2H, Ar-H), 7.07 (s, 2H, NH , D O exchangeable), 7.51 (s, 2H,
SO NH , D O exchangeable), 7.82 (d, 2H, J = 8.0 Hz, Ar-H), 7.99 (d, 2H, J = 8.0 Hz, Ar-H),
2
2
2
8
.10-8.39 (m, 1H, Ar- H), 8.57, 9.16 (2s, 1H, H-3 of pyrazole), 10.98, 11.50 (2s, 1H, NH isatin,
1
3
D O exchangeable), 11.95, 12.78 (2s, 1H, NH hydrazone, D O exchangeable). C NMR
2
2
(
DMSO-d , 100 MHz) δ 95.72, 110.97, 115.64, 121.89, 123.44, 124.02, 124.07, 127.51, 128.48,
6
+
1
2
33.07, 140.75, 142.52, 143.03, 143.19, 149.49, 152.61, 165.94. MS m/z [%] 470 [M , 8.97],
22 [100]. Anal. Calcd for C H N O S (470.42): C, 45.96; H, 3.00; N, 23.82; S, 6.82. Found:
1
8
14
8
6
C, 46.31; H, 2.98; N, 23.94; S, 6.93.
4
.1.9.6.4-(5-Amino-4-(2-(1-benzyl-2-oxoindolin-3-ylidene)hydrazine-1-carbonyl)-1H-pyrazol-1-
o
-1
yl)benzenesulfonamide (16f). Yellow powder, 88 % yield; mp = 270-272 C. IR (KBr) νmax/cm
1
3
4
8
2
313-3194 (NH , NH), 1732-1674 (C=O), 1554 (C=N), 1342, 1168 (SO ). H NMR (DMSO-d ,
2
2
6
00 MHz)
δ 5.02 (s, 2H, benzylic CH ), 7.03 (s, 2H, NH , D O exchangeable), 7.04 (d, 1H, J =
2 2 2
.0 Hz, H-7 of isatin), 7.14 (t, 1H, J = 7.6 Hz, H-5 of isatin), 7.27-7.42 (m, 6H, Ar-H), 7.53 (s,
H, SO NH , D O exchangeable), 7.70 (d, 1H, J = 7.4 Hz, Ar-H), 7.83 (d, 2H, J = 8.7 Hz, Ar-
2
2
2
H), 8.00 (d, 2H, J = 8.7 Hz, Ar-H), 8.21 (s, 1H, H-3 of pyrazole), 12.88 (s, 1H, NH hydrazone,
1
3
2 6
D O exchangeable). C NMR (DMSO-d , 100 MHz) δ 43.00, 95.51, 110.81, 119.98, 120.90,
1
1
23.70, 124.01, 127.52, 127.86, 128.09, 129.19, 131.42, 133.89, 136.18, 140.66, 142.72, 143.13,
+
52.13, 161.41. MS m/z [%] 515 [M , 10.12], 326 [100]. Anal. Calcd for C25
H
21
N
7
O
4
S (515.55):
C, 58.24; H, 4.11; N, 19.02; S, 6.22. Found: C, 58.43; H, 4.16; N, 19.26; S, 6.25.
4
.2. Carbonic anhydrase inhibition
4
.2.1. CA inhibitory assay
An SX.18MV-R Applied Photophysics stopped-flow instrument was used for assaying
the CA-catalyzed CO hydration activity by using the method of Khalifah [33]. Inhibitor
2
and enzyme were preincubated for 6 h. IC values were obtained from dose response
50
curves working at seven different concentrations of test compound (from 0.1 nM to 50
M), by fitting the curves using PRISM (www.graphpad.com) and non-linear least
squares methods, values representing the mean of at least three different determinations,
as described earlier by us.[32, 33] The inhibition constants (K ) were then derived by
µ
I
1
8

ACCEPTED MANUSCRIPT
using the Cheng-Prusoff equation, as follows:
i m
K = IC50/(1 + [S]/K ) where [S] represents
the CO concentration at which the measurement was carried out, and K the
2
m
concentration of substrate at which the enzyme activity is at half maximal. All enzymes
used were recombinant, produced in E.coli as reported earlier.[34, 35] The concentrations
of enzymes used in the assay were: hCA I, 1031 nM; hCA II, 8.4 nM; hCA IX, 7.8 nM
and hCA XII, 10.4 nM.
4
.3. X-Ray Crystallography
4
.3.1. General Data for compound 10. Single crystals for compounds 10 were obtained by slow
evaporation from ethanol. A good crystal with a suitable size was selected for analysis.
Crystallographic data for the structure 10 has been deposited with the Cambridge
Crystallographic Data Center (CCDC) under the numbers CCDC 1053077. Data were collected
on a Bruker APEX-II CCD diffractometer equipped with graphite monochromatic Cu K
ꢀ
radiation (ꢁ = 1.54178 Å) at 296 (2) K. Cell refinement and data reduction were done by Bruker
SAINT; program used to solve structure and refine structure is SHELXS-97 [36]. The final
refinement was performed by full-matrix least-squares techniques with anisotropic thermal data
for non-hydrogen atoms on ꢂ2. All the hydrogen atoms were placed in calculated positions and
constrained to ride on their parent atoms. Multiscan absorption correction was applied by the use
of SADABS software.
4
.3.2. General Data for compound 14. Single crystals for compound 14 were obtained by slow
evaporation from ethanol. A good crystal with a suitable size was selected for analysis.
Crystallographic data for the structure 14 has been deposited with the Cambridge
Crystallographic Data Center (CCDC) under the numbers CCDC 1063099. All diagrams and
calculations were performed using maXus [37]. Data were collected on a KappaCCD
diffractometer equipped with graphite monochromatic Mo Ka radiation, λ = 0.71073 Å at 298 (2)
K. Cell refinement and data reduction were done by HKL SCALEPACK (Otwinowski & Minor
1
997); program used to solve structure and refine structure is SHELXS-97 [36]. The final
refinement was performed by full-matrix least-squares techniques with anisotropic thermal data
for non-hydrogen atoms on ꢂ2. All the hydrogen atoms were placed in calculated positions and
1
9

ACCEPTED MANUSCRIPT
constrained to ride on their parent atoms. Multiscan absorption correction was applied by the use
of SADABS software.
4
.4. Molecular Docking Studies
The molecular docking of the tested compounds was performed using Discovery Studio 4
/
CDOCKER protocol (Accelrys Software Inc.). The protein crystallographic structure, hCA IX
(
PDB id: 3IAI) and hCA XII (PDB id: 1JD0) was downloaded from the Protein Data Bank
PDB). The protein was prepared for docking process according to the standard protein
(
preparation procedure integrated in Accelry’s discovery studio 4 and prepared by prepare protein
protocol. Docked compounds were drawn and prepared by prepare ligand protocol to generate
3
D structure and refined using CHARMM force field with full potential. Docking simulations
were run using CDOCKER protocol where maximum bad orientations was 800 and orientation
vdW energy threshold was 300. Simulated annealing simulation would be then carried out
consisting of a heating phase 700 K with 2,000 steps and a cooling phase back to 5,000 steps.
The binding energy was calculated as a score to rank the docking poses. The top 10 docking
poses would be finally saved. Docking poses were ranked according to their –CDOCKER
interaction energy, and the top pose was chosen for analysis of interactions for each compound.
Acknowledgements
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Cairo, Egypt
and department of Pharmaceutical Chemistry, Faculty of Pharmacy, Egyptian Russian
University, Cairo, Egypt, are highly appreciated for supporting this work. The authors would like
to extend their sincere appreciation to the Deanship of Scientific Research at King Saud
University for its funding of this research through the Research Group Project no. PRG-1436-05.
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2
0

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[
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[
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2

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bacterial and cyclin-dependent kinases (CDKs) inhibitory activities, Med. Chem. Res., 20 (2011)
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08-420.
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30] H. Ashour, A.E.A. Wahab, Synthesis and Biological Evaluation of Novel Pyrazoles and
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3
[
[
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2
3

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6
[
for the solution and refinement of crystal structures, Bruker Nonius, The Netherlands,
MacScience, Japan and The University of Glasgow, (1999).
2
4

ACCEPTED MANUSCRIPT
OH
HO
F3C
Ar
*
*
Ar
COOEt
N
N
CN
NH2
N
N
HN
N
Ar
Ar
O
O
O
O
O
O
S
S
S
S
O
O
NH2
NH2
NH2
NH2
II)
IV
)
(
I)
(
Celecoxib (III)
(
Ar = Ph
Ar = sub. phenyl
Ar = 4-Me-C H - Ar = 4-Br-C H -
6
4
6
4
H
N
N
S
O
S
O
O
S
H N
2
R
CH3
N
H
N
N
N
H
O
O
N
N
H
H
(
V)
R = H, Br, NO2
(VI)
R
R1
R1
O
N
N
Ar
N
NH
O
O
N
NH
NH
N
H
N
R2
N
N
Ar
H N
2
O
O
O
N
(
5a-d)
Ar = Ph
R = H, Cl, Br, CH3
N
N
O
N
O
O S
O
S
O
O
(16a-f)
S
(
11a-f)
NH2
H N
2
H N
2
1
1a-e; R = H, Cl, Br, OCH , NO ; R = H
1 3 2 2
Ar = Ph
1
6a-e; R = H, Cl, Br, OCH , NO ; R
1 3 2 2
1
1f; R = H; R = -CH C H
1
2
2
6
5.
1
6f; R = H; R = -CH C H
1 2 2 6 5
Ar = Ph
Figure 1. Chemical structure of compounds I-VI and the targeted hybrids 5a-d, 11a-f and 16a-f.
2
5

ACCEPTED MANUSCRIPT
Figure 2. ORTEP diagram of compound 10.
Figure 3. ORTEP diagram of compound 14.
2
6

ACCEPTED MANUSCRIPT
(A)
(B)
Figure 4. (A) 3D diagram for interaction of compound 11e with hCA IX (PDB id: 3IAI)
showing sulfonamide as zinc binding group and nitro group interacting with Asp132 with
electrostatic bond. (B) 3D diagram for interaction of compound 16e with hCA IX (PDB id: 3IAI)
showing sulfonamide as zinc binding group and nitro group interacting with Asp132 with
electrostatic bond. In these diagrams, the whole protein was displayed as a tube except the
interacting amino acids were displayed as stick. Hydrogen bond was represented by green dots,
electrostatic bond was represented by orange dots, Pi-hydrophobic interaction was represented
by pink dots and metallic bond was represented by grey dots.
2
7

ACCEPTED MANUSCRIPT
(A)
(B)
Figure 5
. (A) 3D diagram for interaction of compound 11e with hCA XII (PDB id: 1JD0)
showing sulfonamide as zinc binding group and nitro group interacting with Lys67 with
electrostatic bond. (B) 3D diagram for interaction of compound 16e with hCA XII (PDB id:
1
JD0) showing sulfonamide as zinc binding group and nitro group interacting with Asp130 with
electrostatic bond. In these diagrams, the whole protein was displayed as a tube except the
interacting amino acids were displayed as stick. Hydrogen bond was represented by green dots,
electrostatic bond was represented by orange dots, Pi-hydrophobic interaction was represented
by pink dots and metallic bond was represented by grey dots.
2
8