Novel hydrazido benzenesulfonamides-isatin conjugates: Synthesis, carbonic anhydrase inhibitory activity and molecular modeling studies

Article abstract of DOI:10.1016/j.ejmech.2018.07.054

As a part of our ongoing efforts towards developing novel carbonic anhydrase inhibitors based on the isatin moiety, herein we report the synthesis and biological evaluation of novel sulfonamides (5a-h, 10a-g and 11a-c) incorporating substituted 2-indolinone moiety (as tail) linked to benzenesulfonamide (as zinc anchoring moiety) through a hydrazide linker. The synthesized sulfonamides were evaluated in vitro for their inhibitory activity against the following human (h) carbonic anhydrase (hCA, EC 4.2.1.1) isoforms, hCA I, II, IX and XII. All these isoforms were inhibited by the sulfonamides reported here in variable degrees. hCA I was inhibited with K_Is in the range of 671.8: 3549.5 nM, hCA II in the range of 36.8: 892.4 nM; hCA IX in the range of 8.9: 264.5 nM, whereas hCA XII in the range of 9.0: 78.1 nM. In particular, compound 10b emerged as a single-digit nanomolar hCA IX and XII inhibitor (8.9 and 9.2 nM, respectively). Molecular docking studies carried out for compound 10b within the hCA II, IX and XII active sites allowed us to rationalize the obtained inhibition results.

Full text of DOI:10.1016/j.ejmech.2018.07.054

Accepted Manuscript
Novel hydrazido benzenesulfonamides-isatin conjugates: Synthesis, carbonic
anhydrase inhibitory activity and molecular modeling studies
Mahmoud F. Abo-Ashour, Wagdy M. Eldehna, Alessio Nocentini, Hany S. Ibrahim,
Silvia Bua, Sahar M. Abou-Seri, Claudiu T. Supuran
PII:
S0223-5234(18)30618-4
10.1016/j.ejmech.2018.07.054
EJMECH 10589
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 June 2018
Revised Date: 16 July 2018
Accepted Date: 22 July 2018
Please cite this article as: M.F. Abo-Ashour, W.M. Eldehna, A. Nocentini, H.S. Ibrahim, S. Bua, S.M.
Abou-Seri, C.T. Supuran, Novel hydrazido benzenesulfonamides-isatin conjugates: Synthesis, carbonic
anhydrase inhibitory activity and molecular modeling studies, European Journal of Medicinal Chemistry
(2018), doi: 10.1016/j.ejmech.2018.07.054.
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ACCEPTED MANUSCRIPT
Graphical abstract
Novel hydrazido benzenesulfonamides-isatin conjugates: Synthesis, carbonic
anhydrase inhibitory activity and molecular modeling studies
Mahmoud F. Abo-Ashour, Wagdy M. Eldehna, Alessio Nocentini, Hany S. Ibrahim,
Silvia Bua, Sahar M. Abou-Seri, Claudiu T. Supuran
Three series of novel sulfonamides incorporating substituted isatin moieties linked to
benzenesulfonamide through hydrazide linkers were synthesized and evaluated for their inhibitory
activity against a panel of carbonic anhydrase isoforms hCA I, II, IX and XII. 10b emerged as a
single-digit nanomolar hCA IX and XII inhibitor (8.9 and 9.2 nM, respectively). Molecular
docking studies were carried out for 10b within the hCA II, IX and XII active sites
O
O
S NH2
O
NH
N
O
KI hCA I = 720.7 nM
KI hCA II = 76.2 nM
KI hCA IX = 8.9 nM
KI hCA XII = 9.2 nM
N
10b

ACCEPTED MANUSCRIPT
Novel hydrazido benzenesulfonamides-isatin conjugates: Synthesis,
carbonic anhydrase inhibitory activity and molecular modeling studies
a
c
a
Mahmoud F. Abo-Ashour , Wagdy M. Eldehna b,*, Alessio Nocentini , Hany S. Ibrahim ,
Silvia Bua c, Sahar M. Abou-Seri d,*, Claudiu T. Supuran c,**
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Egyptian Russian University, Badr City,
Cairo 11829, Egypt
b
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kafrelsheikh University, Kafrelsheikh,
Egypt
c
Department of NEUROFARBA, Section of Pharmaceutical and Nutraceutical Sciences, University of
Florence, Polo Scientifico, Via U. Schiff 6, 50019, Sesto Fiorentino, Firenze, Italy
e
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, El-kasr Elaini Street,
Cairo, Egypt.
ABSTRACT. As a part of our ongoing efforts towards developing novel carbonic
anhydrase inhibitors based on the isatin moiety, herein we report the synthesis and
biological evaluation of novel sulfonamides (5a-h, 10a-g and 11a-c) incorporating
substituted 2-indolinone moiety (as tail) linked to benzenesulfonamide (as zinc anchoring
moiety) through a hydrazide linker. The synthesized sulfonamides were evaluated in vitro
for their inhibitory activity against the following human (h) carbonic anhydrase (hCA,
EC 4.2.1.1) isoforms, hCA I, II, IX and XII. All these isoforms were inhibited by the
sulfonamides reported here in variable degrees. hCA I was inhibited with KIs in the range
of 671.8 : 3549.5 nM, hCA II in the range of 36.8 : 892.4 nM; hCA IX in the range of 8.9
: 264.5 nM, whereas hCA XII in the range of 9.0 : 78.1 nM. In particular, compound 10b
emerged as a single-digit nanomolar hCA IX and XII inhibitor (8.9 and 9.2 nM,
respectively). Molecular docking studies were carried out for compound 10b within the
hCA II, IX and XII active sites, allowed us to rationalize the obtained inhibition results.
__________
Keywords: Benzenesulfonamides; Isatin; Carbonic anhydrase inhibitors; Synthesis; Molecular docking.
* Corresponding authors. E-mail addresses: wagdy2000@gmail.com (W.M. Eldehna),
saharshaarawy_69@yahoo.com (S.M. Abou-Seri), claudiu.supuran@unifi.it (C.T.
Supuran).
1

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1. Introduction
Carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous metalloenzymes, present
throughout most living organisms and encoded by seven evolutionarily unrelated gene
families: the α-, β-, γ-, δ-, ζ-, η and θ-CAs. Conversion of CO2 to the bicarbonate ion and
protons is a plain physiological reaction that is effectively sped up by CAs [1-4]. This
simple reaction is required in many physiological processes, including pH and CO2
homeostasis, biosynthetic reactions, respiration and transport of CO2 and bicarbonate
between lungs and metabolizing tissues [1,2].
Many of the CA isozymes involved in these processes are significant therapeutic
targets with the potential to be inhibited to treat a wide range of disorders. Indeed CA
isoforms are involved in diverse physio/pathological conditions such as glaucoma (hCA
II, IV and XII), edema (hCA II, IV, XIV), central nervous system (CNS)-related
pathologies (hCA VII and XIV) and tumors (hCA IX and hCA XII) [1,2]. In detail,
human CA isozymes IX and XII are two tumor-associated proteins, being overexpressed
in many tumors and involved in serious processes related to cancer progression and
response to therapy. These two CA isozymes had been the recent target for anticancer
drug during last years [5-10].
Literature surveying revealed that isatin is a promising tail scaffold to design
compounds with interesting carbonic anhydrase inhibitory activity profiles towards
different CA isoforms. Several studies have developed diverse isatin-based derivatives as
potent CA inhibitors, compounds I-VI (Figure 1) [11-18].
Based on the aforementioned findings and as a part of our ongoing effort to develop
potent isatin-based CAIs [11-14], herein we report the synthesis and biological evaluation
of three novel series of sulfonamides (5a-h, 10a-g and 11a-c, Figure 1) featuring
benzenesulfonamide, a zinc anchoring moiety, conjugated with an isatin tail through
hydrazide linker (-NNHC=O). Two strategies were adopted to design the target
sulfonamides. The first one focused on grafting various substituents (compounds 5a-h) at
5-position of the isatin tail to ensure different electronic and lipophilic environments
which could manipulate the activity of the target sulfonamides, and to evaluate their
2

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influence on activity. In the second strategy, different N-alkyl (compounds 10a-g) and N-
benzyl (compounds 11a-c) substituents were incorporated into the isatin tail. The N-
substitution pattern on the isatin moieties was varied in a systemic fashion to define the
optimal length (methyl, ethyl, n-propyl, n-butyl), bulkiness (isopropyl) and unsaturation
(allyl, benzyl) which confer the best CA inhibitory effect.
All the newly synthesized sulfonamides were in vitro evaluated for their inhibitory
activity against a panel of hCA I, II, IX and XII isoforms, using stopped-flow CO2
hydrase assay. Molecular docking studies were carried out for compound 10b to justify
its activity through investigating its key interactions within the active site of hCA II (PDB
5LJT), hCA IX (PDB 5FL4) and hCA XII (PDB 1JD0) isoforms.
Figure 1. Structures of some reported isatin-based carbonic anhydrase inhibitors I-VI
and the target sulfonamides 5a-h, 10a-g and 11a-c.
2. Results and Discussion
2.1. Chemistry
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The synthesis of the new sulfonamides in this study is outlined in Schemes 1 and 2.
In Scheme 1, esterification of 4-sulfamoylbenzoic acid using methanol with catalytic
amount of H2SO4 under reflux afforded methyl 4-sulfamoylbenzoate 2 which converted
to 4-(hydrazinecarbonyl)benzenesulfonamide 3 by refluxing with hydrazine hydrate in
methanol. Hydrazide 3 condensed with different isatin derivatives 4a-h to afford the
target compounds 5a-h.
Scheme 1. Synthesis of target sulfonamides 5a-h; Reagents and conditions: (i) Methanol
/ H2SO4 (Cat.) / reflux 8 h, (ii) Hydrazine hydrate / methanol / reflux 4 h, (iii) Glacial
acetic acid / reflux 6 h.
In Scheme 2, N-substituted isatins 8a-g and 9a-c were furnished through NH
alkylation of isatin 4a with different alkyl bromides 6a-g or benzyl bromides 7a-c in
DMF in the presence of potassium carbonate, which subsequently reacted with the key
intermediate 4-(hydrazinecarbonyl)benzene-sulfonamide 3 to give the target compounds
10a-g and 11a-c respectively.
4

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O
O
N
H
4a
Br
R1
R
Br
i
6a-g
7a-c
O
O
O
O
N
N
R
8a-g
9a-c
O
NH2
R1
N
H
O
ii
S
O
3
3
ii
NH2
O O
O O
S
S
O
O
NH2
NH2
NH
O
N
NH
O
N
N
R
N
10a-g
R
10
a
11a-c
-CH3
-CH2CH3
R1
11
b
c
R1
-CH2CH2CH3
-CH(CH3)2
a H
d
e
b F
-CH2CH2CH2CH3
-CH2CH=CH2
c CH3
f
-CH2COOC2H5
g
Scheme 2. Synthesis of target sulfonamides 10a-g and 11a-c; Reagents and conditions:
(i) DMF / K2CO3 / KI (Cat.) / reflux 4 h, (ii) Glacial acetic acid / reflux 6 h.
The elemental and spectral data supported the structures of the target compounds 5a-
h, 10a-g and 11a-c. IR spectra of 5a-h, 10a-g and 11a-c revealed the presence of bands
of (NH2, NH) at 3405-3017 cm-1, also two bands of (C=O) around 1695-1650 cm-1, in
1
addition to two bands of (SO2) at 1023-1050 and 1308-1390 cm-1. Furthermore, the H
5

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NMR spectra of compounds 5a-h, 10a-g and 11a-c displayed singlet signal of D2O
exchangeable NH2 of sulfamoyl group and D2O exchangeable NH of hydrazide at δ 7.60-
7.62 ppm and δ 13.67-14.02 ppm respectively. While, compounds 5a-h were confirmed
with the presence of an additional signal of D2O exchangeable NH of isatin at δ 11.23-
12.01 ppm. The protons of aliphatic substituents in compounds 10a-g were detected at
expected chemical shift and integration, meanwhile compounds 11a-c were confirmed
with appearance of benzylic protons at δ 5.02-5.13 ppm. On the other hand, 13C NMR
spectra of compounds 5a-h, 10a-g and 11a-c revealed the presence of two signals of
(C=O) of hydrazid and isatin between δ 160-.07-162.67 and 163.10-163.95 ppm
respectively. While, compounds 11a-c were confirmed with appearance of benzylic
carbon at δ 42.34-43.20 ppm.
2.2. Carbonic anhydrase inhibition
The newly synthesized sulfonamides 5a-h, 10a-g and 11a-c were evaluated for their
ability to inhibit the physiologically relevant hCA isoforms, hCA I, II (cytosolic) as well
as hCA IX and XII (trans membrane, tumor associated isoforms) using acetazolamide
(AAZ) as standard inhibitor by a stopped flow CO2 hydras assay [19]. The following
structure–activity relationship (SAR) can be compiled from the inhibition data reported in
Table 1:
(i) The off-target isoform hCA I was weakly inhibited by sulfonamides 5a-h, 10a-g
and 11a-c reported here, with inhibition constants (KIs) ranging from high nanomolar to
low micromolar concentration, between 671.8 and 3.549 µM. Indeed, compounds 5a, 5b,
5e, 5h, 10a, 10e, 10f and 11a arose as the least hCA I inhibitors in this study with KIs in
the range (1.485-3.549 µM).
(ii) Inhibition of isoform hCA II was ranged from weak to potent inhibitory activity,
with KI values ranging between 36.8 and 892.4 nM. The 5-NO2 oxoindole derivative 5g
was the most active compound, followed by N-ethyl acetate derivative 10g with KI values
of 36.8 and 38.1 nM, respectively. On the other hand, the N-benzyl derivatives 11b and
11c displayed the modest activity with KI values of 784.1 and 892.4 nM, respectively.
6

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Table 1: Inhibition data of human CA isoforms hCA I, hCA II, hCA IX and hCA XII
with sulfonamides 5a-h, 10a-g and 11a-c determined by stopped-flow CO2 hydrase
assay, using acetazolamide (AAZ) as a standard drug.
KI (nM)
Comp. X
R/R1
hCA I
2587.3
3289.5
843.6
872.1
1864.2
899.7
896.3
3549.5
1832.6
720.7
900.9
991.9
1485.6
1816.0
979.3
2335.2
671.8
822.8
250
hCA II
741.7
235.1
465.0
488.1
530.3
95.1
hCA IX
173.6
32.3
hCA XII
43.9
9.1
H
-
5a
5-F
-
5b
5-Cl
-
31.5
15.3
40.2
48.7
24.7
9.0
5c
5-Br
-
101.2
196.9
264.5
23.8
5d
5e
5-CH3
-
5-OCH3
-
5f
5-NO2
-
36.8
5g
5,7-(CH3)2
-
598.6
160.9
76.2
715.6
30.6
53.9
65.6
9.2
5h
-
-
-
-
-
-
-
-
-
-
-
CH3
10a
10b
10c
10d
10e
10f
10g
11a
11b
11c
AAZ
CH2CH3
8.9
CH2CH2CH3
244.7
347.9
565.1
94.0
30.1
24.7
16.7
44.1
43.3
9.6
CH(CH3)2
177.2
27.4
CH2CH2CH2CH3
CH2CH=CH2
77.9
CH2COOC2H5
38.1
17.7
H
595.9
784.1
892.4
12
25.2
70.4
53.1
78.1
5.7
F
148.3
81.2
CH3
-
25
* Mean from 3 different assays, by a stopped flow technique (errors were in the range of
± 5-10 % of the reported values).
7

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(iii) All the investigated sulfonamides acted as good inhibitors for the tumor-
associated isoform hCA IX (KI values ranging between 8.9 and 264.5 nM). Superiorly,
conjugate 10b emerged as a single-digit nanomolar hCA IX inhibitor with KI value of 8.9
nM, which is 3-fold more potent than the standard drug AAZ (KI = 25 nM against hCA
IX). Also, compound 10g (KIs = 17.7 nM) was 1.5 times more active than AAZ. In
addition, compounds 5b, 5c, 5g, 10a, 10c, 10e and 11a were equipotent to AAZ with KIs
in the range 23.8-32.3 nM.
It is worth stressing that the inhibitory activity was affected the type of substituent at
position 5 of the isatin moiety. Grafting of electron-withdrawing groups enhanced the
activity against hCA IX (compounds 5b-d and 5g; KIs range 23.8-101.2 nM) compared to
unsubstituted analogue 5a (KIs =173.6 nM). Whereas, incorporation of electron-donating
groups resulted in a decreased activity (5e and 5f; KIs = 196.9 and 264.5 nM,
respectively) compared to the unsubstituted 5a. Thus, the order of activities of the 5-
substituted isatins 5b-g, was decreased in the order of NO2 > F ≈ Cl (small halogens) >
Br > CH3 > OCH3. In the same context, 5,7-disubstitution with methyl group, compound
5h, led to the weakest hCA IX inhibitor in this study with remarkable decrease in activity
(KIs =715.6 nM).
Regarding the impact of N-alkylation of isatin moiety in compounds 10; the obtained
results highlighted that incorporating different alkyl groups, except 10d, enhanced the
activity (KIs range 8.9-77.9 nM), in comparison to unsubstituted derivative 5a (KIs
=173.6 nM). It is noteworthy that ethyl group represented the optimal length which
confer the best hCA IX inhibitory effect in this study. Otherwise, both branching (as
compound 10d; KIs =177.2 nM) and unsaturation (as compound 10f; KIs =77.9 nM)
decreased activity, compared to compound 10c (KIs =30.1 nM). Finally, grafting N-
benzyl group, compound 11a, was well-tolerated (KIs =25.2 nM). Whereas, p-substitution
on the benzyl group with electron-withdrawing or electron-donating group, decreased the
inhibitory activity against hCA IX (Compounds 11b and 11c; KIs = 148.3 and 81.2 nM,
respectively).
(iv) The tumor-associated isoform hCA XII was undeniably the highly affected
isoform amongst the hCA isoforms considered here, with KIs in the range of 9.0-78.1 nM.
The most effective compounds were 5b, 5c, 5f, 5e and 10b, 10c, 10d and 10g with KIs in
8

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the range of 9.0-24.7 nM. Similar to the SAR for hCA IX inhibition; 5-substitution with
electron-withdrawing NO2 group and small halogens (as F and Cl) displayed an improved
activity (compounds 5b, 5c and 5g; KIs range 9.0-15.3 nM). In addition, 5-substitution
with electron-donating methyl group displayed a slight decreased activity (compound 5e;
KIs =48.7 nM) compare to unsubstituted analogue 5a (KIs =43.9 nM). In contrary,
grafting electron-donating methoxy group (compound 5f; KIs =24.7 nM) enhanced the
inhibitory activity towards hCA XII.
Interestingly, N-ethyl isatin counterpart 10b emerged as the most potent hCA XII
inhibitor (KIs =9.2 nM) among the N-substituted isatin series 10, as highlighted for hCA
IX inhibition. Moreover, unsaturated derivative 10f possessed lower inhibitory activity
(KIs =43.3 nM) than its saturated analogue 10c (KIs =24.7 nM). Unlike hCA IX, the
utilization of branched alkyl group enhanced the activity (KIs =16.7 nM) against hCA
XII. Finally, the N-benzyl derivatives 11a-c failed to improve the inhibitory activity
against hCA XII.
3. Molecular docking studies
Compound 10b was selected for molecular docking simulations to justify its activity
by investigating its key interactions within the active site of hCA II (PDB 5LJT [20]),
hCA IX (PDB 5FL4 [21]) and hCA XII (PDB 1JD0 [22]) isoforms. The top poses for
10b docked within the active site of hCA II, hCA IX and hCAXII were illustrated in
Figures 2, 3 and 4, respectively. As a result, the sulfonamide group played the usual role
as zinc-binding group (ZBG) for compound 10b with the three different hCA isoforms by
forming a metallic bond with Zn metal and hydrogen bond with Thr200. Carbonyl group
of oxindole moiety was involved in the interaction within the active site of hCA II and IX
isoforms by accepting a hydrogen bond from Gln92. The same interaction took place in
hCA XII active site by carbonyl group of hydrazide moiety. All of the aforementioned
contacts explained the inhibitory activity for compound 10b for the three different
isoforms.
Regarding the low inhibitory activity of compound 10b towards hCAII isoform, this
could be explained through the behavior of N-ethyl moiety of oxindole ring inside the
active site of the three isoforms. Within hCA II active site, the N-ethyl moiety was
9

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engaged to unfavorable interactions with Ile91 and Gln 92. Free rotation of this moiety
would not compensate this bad interaction, as it would result in another unfavorable
interaction with Asn67 (Figure 2). In contrast, N-ethyl moiety was not surrounded by any
clashes with amino acid in the active site of hCA IX as Leu91 was 4.06 Å away from N-
ethyl moiety (Figure 3). Moreover, within the active site of hCA XII, polar Ser132 and
Ser135 were facing N-ethyl moiety from one side, but free rotation of this group would
assure the absence of any unfavorable interactions with these amino acids (Figure 4).
Figure 2. Docking pose of compound 10b with the active site of hCA II (PDB 5LJT)
showing the unfavorable interactions of N-ethyl moiety of oxindole group with amino
acids Ile91 and Gln92 which is assigned by orange dashed lines.
Figure 3. Simulated binding pose of compound 10b with the active site of hCA IX
(PDB 5FL4) illustrating the common features for activity with no clashes around N-ethyl
moiety of oxindole group.
10

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Figure 4. Interaction of compound 10b with the key amino acids and Zn metal within
the active site of hCA XII (PDB 1JD0) showing that N-ethyl moiety of oxindole group is
hindered from one side only. Hydrogen bonds were represented as yellow dashed lines.
4. Conclusion
In summary, we report here the synthesis of three novel series of sulfonamides (5a-h,
10a-g and 11a-c), incorporating different isatin moieties linked to benzenesulfonamide
(as zinc anchoring moieties) through hydrazide linker. The structure of the novel
sulfonamides was confirmed by the different spectral and elemental analyses methods.
Biological evaluation of the newly prepared sulfonamides was performed against hCA I,
II, IX and XII. All the tested isoforms were inhibited by the synthesized sulfonamides 5a-
h, 10a-g and 11a-c, in variable degrees. Best activity was observed against both isoforms
hCA IX and XII. Sulfonamide 10b emerged as single-digit nanomolar hCA IX and XII
inhibitor (KIs = 8.9 and 9.2 nM, respectively). Finally, the inhibitory potency of the
synthesized sulfonamides was further investigated carrying out molecular docking for
sulfonamide 10b to rationalize the obtained inhibition results.
5. Experimental
5.1. Chemistry
5.1.1. General
Melting points were measured with a Stuart melting point apparatus and were
uncorrected. Infrared (IR) Spectra were recorded as KBr disks using Schimadzu FT-IR
11

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8400S spectrophotometer. Mass spectral data are given by GCMS-QP1000 EX
spectrometer at 70 e.V. NMR Spectra were recorded on Bruker AVF-400 spectrometer.
13
1H spectrum was run at 400 MHz and C spectrum was run at 100 MHz in deuterated
dimethylsulfoxide (DMSO-d6). Chemical shifts are expressed in values (ppm) using the
solvent peak as internal standard. All coupling constant (J) values are given in hertz. The
abbreviations used are as follows: s, singlet; d, doublet; m, multiplet. Elemental analyses
were carried out at the Regional Center for Microbiology and Biotechnology, Al-Azhar
University, Cairo, Egypt. High-resolution mass spectra (EI and ESI) were recorded using
a Bruker MicroTOF spectrometer. Analytical thin layer chromatography (TLC) on silica
gel plates containing UV indicator was employed routinely to follow the course of
reactions and to check the purity of products.
5.1.2. Synthesis of compounds 2 and 3
Compounds 2 and 3 were prepared according to the literature procedures [23].
5.1.3. Synthesis of N-substituted isatin derivatives 8a-g and 9a-c.
Isatin 4a (0.5 gm, 3.4 mmol) was stirred in DMF (15 mL) with (3.7 mmol) the
appropriate alkyl halide 6a-g or benzyl bromide derivative 9a-c in the presence of
potassium carbonate (7.4 mmol) and catalytic amount of potassium iodide at reflux
temperature for 4 hrs. After the reaction is completed, the mixture was poured into ice
water, the formed solid was collected, washed with water and recrystallized from ethanol
to furnish compounds 8a-g or 9a-c, respectively [24-27].
5.1.4.
Synthesis
of
4-[2-(2-oxoindolin-3-ylidene)hydrazine-1-
carbonyl]benzenesulfonamide derivatives 5a-h, 10a-g and 11a-c
To a hot solution of 4-(hydrazinecarbonyl)benzenesulfonamide 3 (0.25g, 1.16 mmol)
in glacial acetic acid (15 mL), the appropriate isatin derivative 4a-h, 8a-g or 9a-c (1.2
mmol) was added. This mixture was heated under reflux for 6 hrs. The formed precipitate
was filtered off while hot and washed with ethanol and petroleum ether then
recrystallized from DMF/ethanol to obtain the target compounds 5a-h, 10a-g and 11a-c.
5.1.4.1. 4-(2-(2-Oxoindolin-3-ylidene)hydrazine-1-carbonyl)benzenesulfonamide 5a.
12

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Yellow powder (yield 75%), m.p. Over 300 °C; IR(KBr, ν cm-1): 3210, 3150, 3018 (NH2,
1
2NH), 1690, 1652 (2C=O) and 1337, 1023 (SO2); H NMR (DMSO-d6, 400 MHz) δ
ppm: 6.97 (d, 1H, H-7 of isatin, J = 8 Hz), 7.11 (t, 1H, H-5 of isatin, J = 7.6 Hz), 7.40 (t,
1H, H-5 of isatin, J = 7.6 Hz), 7.61 (br s, 3H, H-4 of isatin, 2H, NH2, D2O
exchangeable), 8.02-8.09 (m, 4H, H-2,3,5 and 6 of phenyl ring of benzenesulfonamide),
11.33 (s, 1H, isatin NH, D2O exchangeable), 13.97 (s, 1H, hydrazide NH, D2O
13
exchangeable); C NMR (DMSO-d6,100 MHz) δ ppm: 111.79, 116.22, 120.16, 121.60,
123.32, 126.84, 128.79, 132.55, 134.35, 135.47, 139.28, 143.10, 147.96 (Aromatic
carbons), 162.67 (hydrazide C=O), 163.47 (isatin C=O); HRMS (ESI) for C15H11O4N4S,
calcd 343.05065, found 343.05029 [M+H] +.
5.1.4.2.
4-[2-(5-Fluoro-2-oxoindolin-3-ylidene)hydrazine-1-
carbonyl]benzenesulfonamide 5b.
Yellow powder (yield 65%), m.p. Over 300 °C; IR (KBr, ν cm-1): 3395, 3210, 3018
1
(NH2, 2NH), 1695, 1667 (2 C=O) and 1331, 1023 (SO2); H NMR (DMSO-d6, 400
MHz) δ ppm: 6.95 (dd, 1H, H-6 of isatin, J = 8.4 Hz), 7.23 (dt, 1H, H-7 of isatin, J =
8.8), 7.43 (d, 1H, H-4 of isatin, J = 6.8 Hz), 7.61 (s, 2H, NH2, D2O exchangeable), 8.03-
8.09 (m, 4H, H-2,3,5 and 6 of phenyl ring of benzenesulfonamide), 11.43 (s, 1H, isatin
NH, D2O exchangeable), 13.94 (s, 1H, hydrazide NH, D2O exchangeable); 13C NMR
(DMSO-d6, 100 MHz) δ ppm: 108.57, 108.83, 112.87, 112.95, 118.76, 119.00, 121.35,
126.86, 128.92, 135.26, 139.35, 148.03, 157.70 (aromatic carbons), 160.07 (hydrazide
C=O), 163.58 (isatin C=O); HRMS (ESI) for C₁₅H₁₀O₄N₄FS, calcd 361.04123, found
361.04106 [M+H] +.
5.1.4.3.
4-(2-(5-Chloro-2-oxoindolin-3-ylidene)hydrazine-1-
carbonyl)benzenesulfonamide 5c.
Red powder (yield 60%), m.p. Over 300 °C; IR (KBr, ν cm-1): 3395, 3232, 3018
(NH2,2NH), 1690, 1652 (2 C=O) and 1337, 1023 (SO2); 1H NMR (DMSO-d6, 400 MHz)
δ ppm: 6.97 (d, 1H, H-6 of isatin, J = 8.4 Hz), 7.44 (d, 1H, H-7 of isatin, J = 8.8 Hz),
7.59-7.61 (m, 3H, 1H of H-4 of isatin and 2H, NH2, D2O exchangeable), 8.03-8.09 (m,
4H, H-2,3,5 and 6 of phenyl ring of benzenesulfonamide), 11.52 (s, 1H, isatin NH, D2O
13

ACCEPTED MANUSCRIPT
exchangeable), 13.88 (s, 1H, hydrazide NH, D2O exchangeable); 13C NMR (DMSO-
d6,100 MHz) δ ppm: 111.39, 113.32, 117.98, 121.08, 121.87, 126.85, 127.42, 128.86,
129.02, 131.90, 135.22, 141.76, 148.03 (aromatic carbons), 162.07 (hydrazide C=O),
163.26 (isatin C=O), HRMS (ESI) for C15H10O4N4ClS, calcd 377.01168, found
377.01167 [M+H]+.
5.1.4.4.
4-[2-(5-Bromo-2-oxoindolin-3-ylidene)hydrazine-1-
carbonyl]benzenesulfonamide 5d.
Yellow powder (yield 70%), m.p. Over 300 °C; IR (KBr, ν cm-1): 3408, 3210, 3017
(NH2,2NH), 1690, 1671 (2C=O) and 1337, 1023 (SO2); 1H NMR (DMSO-d6, 400 MHz)
δ ppm: 6.92 (d, 1H, H-6 of isatin, J = 8.4 Hz), 7.56-7.61 (m, 3H, 1H of H-7 of isatin and
2H, NH2, D2O exchangeable), 7.69 (s, 1H, H-4 of isatin, Ar-H), 8.03-8.09 (m, 4H, H-
2,3,5 and 6 of phenyl ring of benzenesulfonamide), 11.50 (s, 1H, isatin NH, D2O
13
exchangeable), 13.88 (s, 1H, hydrazide NH, D2O exchangeable); C NMR (DMSO-d6,
100 MHz) δ ppm: 111.76, 113.76, 114.99, 118.76, 122.26, 123.79, 126.85, 128.77,
134.69, 135.21, 138.16, 142.13, 148.02 (aromatic carbons), 162.07 (hydrazide C=O),
163.10 (isatin C=O); HRMS (ESI) for C₁₅H₁₀O₄N₄BrS, calcd 420.96116, found
420.96126 [M+H]+.
5.1.4.5.
4-[2-(5-Methyl-2-oxoindolin-3-ylidene)hydrazine-1-
carbonyl]benzenesulfonamide 5e.
Yellow powder (yield 66%), m.p. Over 300°C; IR (KBr, ν cm-1): 3386, 3210, 3018 (NH2,
1
2NH), 1695, 1652 (2C=O) and 1337, 1023 (SO2); H NMR (DMSO-d6, 400 MHz) δ
ppm: 2.31 (s, 3H, CH3), 6.84 (d, 1H, H-6 of isatin, J = 8 Hz), 7.19 (d, 1H, H-7 of isatin, J
= 7.6 Hz), 7.43 (s, 1H, H-4 of isatin), 7.60 (s, 2H, NH2, D2O exchangeable), 8.02-8.08
(m, 4H, H-2,3,5 and 6 of phenyl ring of benzenesulfonamide), 11.29 (s, 1H, isatin NH,
D2O exchangeable), 13.97 (s, 1H, hydrazide NH, D2O exchangeable); 13C NMR (DMSO-
d6, 100 MHz) δ ppm: 20.98 (CH3), 111.54, 115.07, 120.13, 121.91, 126.83, 128.70,
132.41, 132.95, 135.46, 139.44, 140.82, 147.91, 153.50 (aromatic carbons), 162.48
(hydrazide C=O), 163.53 (isatin C=O); HRMS (ESI) for C₁₆H₁₃O₄N₄S, calcd 357.06630,
found 357.06635 [M+H]+.
14

ACCEPTED MANUSCRIPT
5.1.4.6.
4-[2-(5-Methoxy-2-oxoindolin-3-ylidene)hydrazine-1-
carbonyl]benzenesulfonamide 5f.
Red powder (yield 60%), m.p. Over 300 °C; IR (KBr, ν cm-1): 3393, 3210, 3018 (NH2,
1
2NH), 1692, 1654 (2C=O) and 1308, 1023 (SO2); H NMR (DMSO-d6, 400 MHz) δ
ppm: 3.79 (s, 3H, OCH3), 6.88 (d, 1H, H-6 of isatin, J = 8.4 Hz), 6.99 (d, 1H, H-7 of
isatin, J = 8.4 Hz), 7.15 (s, 1H, H-4 of isatin), 7.60 (s, 2H, NH2, D2O exchangeable),
8.02-8.08 (m, 4H, H-2,3,5 and 6 of phenyl ring of benzenesulfonamide), 11.23 (s, 1H,
isatin NH, D2O exchangeable), 14.02 (s, 1H, hydrazide NH, D2O exchangeable); 13C
NMR (DMSO-d6,100 MHz) δ ppm: 56.13 (O-CH3), 106.38, 111.85, 112.67, 118.84,
120.86, 126.87, 128.73, 135.40, 136.75, 140.61, 145.51, 148.01, 155.96 (aromatic
carbons), 161.58 (hydrazide C=O), 163.59 (isatin C=O); HRMS (ESI) for C₁₆H₁₃O₅N₄S,
calcd 373.06121, found 373.06106 [M+H]+.
5.1.4.7. 4-[2-(5-Nitro-2-oxoindolin-3-ylidene)hydrazine-1-carbonyl]benzenesulfonamide
5g.
Yellow powder (yield 70%), m.p. Over 300 °C; IR (KBr, ν cm-1): 3393, 3210, 3018
(NH2, 2NH), 1694, 1655 (2C=O) and 1382, 1048 (SO2); 1H NMR (DMSO-d6, 400 MHz)
δ ppm: 7.13 (d, 1H, H-7 of isatin, J = 8.4 Hz), 7.62 (s, 2H, NH2, D2O exchangeable),
8.04-8.09 (m, 4H, H-2,3,5 and 6 of phenyl ring of benzenesulfonamide), 8.24 (s, 1H, H-4
of isatin), 8.28 (d, 1H, H-6 of isatin, J = 8.8 Hz), 12.01 (s, 1H, isatin NH, D2O
exchangeable), 13.67 (s, 1H, hydrazide NH, D2O exchangeable); 13C NMR (DMSO-
d6,100 MHz) δ ppm: 110.29, 112.06, 116.35, 120.83, 126.87, 128.23, 128.99, 134.99,
137.48, 140.52, 143.35, 148.10, 148.15 (aromatic carbons), 160.34 (hydrazide C=O),
163.71 (isatin C=O); HRMS (ESI) for C₁₅H₁₀O₆N₅S, calcd 388.03573, found 388.03564
[M+H]+.
5.1.4.8.
4-[2-(5,7-Dimethyl-2-oxoindolin-3-ylidene)hydrazine-1-
carbonyl]benzenesulfonamide 5h.
Yellow powder (yield 85%), m.p. Over 300 °C; IR (KBr, ν cm-1): 3387, 3210, 3018
(NH2, 2NH), 1692, 1653 (2C=O) and 1361, 1023 (SO2); 1H NMR (DMSO-d6, 400 MHz)
δ ppm: 2.20 (s, 3H, CH3 of position 7 of isatin), 2.27 (s, 3H, CH3 of position 5 of isatin),
15

ACCEPTED MANUSCRIPT
7.04 (s, 1H, H-6 of isatin), 7.26 (s, 1H, H-4 of isatin), 7.60 (s, 2H, NH2, D2O
exchangeable), 8.02-8.08 (m, 4H, H-2,3,5 and 6 of phenyl ring of benzenesulfonamide),
11.31 (s, 1H, isatin NH, D2O exchangeable), 13.97 (s, 1H, hydrazide NH, D2O
exchangeable); 13C NMR (DMSO-d6, 100 MHz) δ ppm: 16.31 (CH3 of position 7 of
isatin), 20.89 (CH3 of position 5 of isatin), 116.33, 119.31, 119.88, 120.94, 126.84,
128.71, 131.47, 132.32, 134.38, 135.51, 139.47, 146.22, 147.96 (aromatic carbons),
162.67 (hydrazide C=O), 163.95 (isatin C=O); HRMS (ESI) for C₁₇H₁₇O₄N₄S, calcd
373.09650, found 373.09634 [M+H]+.
5.1.4.9.
4-[2-(1-Methyl-2-oxoindolin-3-ylidene)hydrazine-1-
carbonyl]benzenesulfonamide 10a.
Orange powder (yield 71%), m.p. 300 °C; IR (KBr, ν cm-1): 3400, 3019 (NH, NH2),
1
16935, 1652 (C=O) and 1380, 1043 (SO2); H NMR (DMSO-d6, 400 MHz) δ ppm: 3.35
(s, 3H, N-CH3), 7.16-7.20 (m, 2H, H-5,7 of isatin), 7.46 (t, 1H, H-6 of isatin, J = 7.6 Hz),
7.61 (br s, 3H,1H of H-4 of isatin, 2H of NH2, D2O exchangeable), 8.04-8.9 (m, 4H, H-
2,3,5 and 6 of phenyl ring of benzenesulfonamide), 13.88 (s, 1H, NH, D2O
exchangeable); 13C NMR (DMSO-d6,100 MHz) δ ppm: 26.21 (CH3), 110.55, 116.60,
119.41, 121.20, 123.82, 126.85, 128.77, 132.41, 135.31, 138.42, 144.27, 149.02, 154.22
(aromatic carbons), 161.59 (hydrazide C=O), 162.49 (isatin C=O); Anal. Calcd. for
C16H14N4O4S (358.37): C, 53.62; H, 3.94; N, 15.63; found C, 53.80; H, 3.90; N, 15.57.
5.1.4.10.
4-(2-(1-Ethyl-2-oxoindolin-3-ylidene)hydrazine-1-
carbonyl)benzenesulfonamide 10b.
Yellow powder (yield 70%), m.p. 270-273 °C; IR (KBr, ν cm-1): 3395, 3017 (NH, NH2),
1693, 1652 (C=O) and 1345, 1043 (SO2); 1H NMR (DMSO-d6, 400 MHz) δ ppm: 1.20 (t,
2H, N-CH2, J = 7.2 Hz), 3.77 (q, 3H, CH2-CH3, J = 7.2), 7.14 (t, 1H, H-5 of isatin, J =
7.6 Hz), 7.20 (d, 1H, H-7 of isatin, J = 8 Hz), 7.44 (t, 1H, H-6 of isatin, J = 7.6 Hz), 7.62
(br s, 3H,1H of H-4 of isatin and 2H of NH2, D2O exchangeable), 8.04-8.09 (m, 4H, H-
2,3,5 and 6 of phenyl ring of benzenesulfonamide), 13.88 (s, 1H, NH, D2O
exchangeable); 13C NMR (DMSO-d6,100 MHz) δ ppm: 13.01 (CH3-CH2), 34.62 (N-
CH2), 110.56, 116.60, 119.56, 121.40, 123.71, 126.84, 128.78 132.42, 135.30, 138.42,
143.19, 148.02, 154.22 (aromatic carbons), 161.21 (hydrazide C=O), 162.49 (isatin
16

ACCEPTED MANUSCRIPT
C=O); Anal. Calcd. for C17H16N4O4S (372.40): C, 54.83; H, 4.33; N, 15.05; found C,
54.72; H, 4.29; N, 15.11.
5.1.4.11.
4-[2-(2-Oxo-1-propylindolin-3-ylidene)hydrazine-1-
carbonyl]benzenesulfonamide 10c.
Yellow powder (yield 80%), m.p. 285-286 °C; IR (KBr, ν cm-1): 3405, 3017 (NH, NH2),
1693, 1654 (C=O) and 1382, 1047 (SO2); 1H NMR (DMSO-d6, 400 MHz) δ ppm: 0.90 (t,
3H, CH3-CH2, J = 7.6 Hz), 1.63-1.73 (m, 2H, CH3-CH2), 3.73 (t, 2H, N-CH2, J = 6.8
Hz), 7.17 (t, 1H, H-5 of isatin, J = 7.6 Hz), 7.25 (d, 1H, H-7 of isatin, J = 8 Hz), 7.47 (t,
1H, H-6 of isatin, J = 8 Hz), 7.61 (s, 2H, NH2, D2O exchangeable), 7.65 (d, 1H, H-4 of
isatin, J = 6.8 Hz), 8.03-8.10 (m, 4H, H-2,3,5 and 6 of phenyl ring of
benzenesulfonamide), 13.91 (s, 1H, hydrazide NH, D2O exchangeable); 13C NMR
(DMSO-d6,100 MHz) δ ppm: 11.66 (CH2-CH3), 20.88 (CH2-CH3), 41.32 (N-CH2),
110.81, 116.60, 119.51, 121.41, 123.76, 126.86, 128.81, 132.47, 135.31, 140.38, 143.69,
148.03, 154.22 (aromatic carbons), 161.62 (hydrazide C=O), 162.57 (isatin C=O); HRMS
(ESI) for C₁₈H₁₉O₄N₄S, calcd 387.11215, found 387.11266 [M+H]+.
5.1.4.12.
4-[2-(1-Isopropyl-2-oxoindolin-3-ylidene)hydrazine-1-
carbonyl]benzenesulfonamide 10d.
Yellow powder (yield 77%), m.p. 295-296 °C; IR (KBr, ν cm-1): 3400, 3017 (NH, NH2),
1
1690, 1650 (C=O) and 1380, 1040 (SO2); H NMR (DMSO-d6, 400 MHz) δ ppm: 1.47
(d, 6H, CH3-CH-CH3, J = 7.2 Hz), 4.55-4.63 (m, 1H, N-CH), 7.15 (t, 1H, H-5 of isatin, J
= 7.6 Hz), 7.34 (d, 1H, H-7 of isatin, J = 8 Hz), 7.45 (t, 1H, H-6 of isatin, J = 7.6 Hz),
7.61 (s, 2H, NH2, D2O exchangeable), 7.64 (d, 1H, H-4 of isatin, J = 7.2 Hz), 8.04-8.10
(m, 4H, H-2,3,5 and 6 of phenyl ring of benzenesulfonamide), 13.95 (s, 1H, NH, D2O
exchangeable); 13C NMR (DMSO-d6,100 MHz) δ ppm: 19.56 (CH3-CH-CH3), 44.41 (N-
CH), 111.69, 119.86, 116.60, 121.45, 123.48, 126.86, 128.80, 132.34, 135.38, 138.36,
142.88, 148.01, 154.22 (aromatic carbons), 161.40 (hydrazide C=O), 162.40 (isatin
C=O); Anal. Calcd. for C18H18N4O4S (386.43): C, 55.95; H, 4.70; N, 14.50; found C,
55.73; H, 4.66; N, 14.57.
17

ACCEPTED MANUSCRIPT
5.1.4.13.
carbonyl]benzenesulfonamide 10e.
4-[2-(1-Butyl-2-oxoindolin-3-ylidene)hydrazine-1-
Yellow powder (yield 70%), m.p. 272 °C; IR (KBr, ν cm-1): 3989, 3017 (NH, NH2),
1693, 1655 (C=O) and 1378, 1042 (SO2); 1H NMR (DMSO-d6, 400 MHz) δ ppm: 0.89 (t,
3H, CH2-CH3, J = 7.6 Hz), 1.32-1.40 (m, 2H, CH2-CH3), 1.60-1.67 (m, 2H, N-CH2-CH2),
3.75 (t, 2H, N-CH2, J = 7.2 Hz), 7.16 (t, 1H, H-5 of isatin, J = 7.6 Hz), 7.22 (d, 1H, H-7
of isatin, J = 8 Hz), 7.46 (t, 1H, H-6 of isatin, J = 8 Hz), 7.61 (s, 2H, NH2, D2O
exchangeable), 7.64 (d, 1H, H-4 of isatin, J = 7.2 Hz), 8.04-8.10 (m, 4H, H-2,3,5 and 6 of
phenyl ring of benzenesulfonamide), 13.89 (s, 1H, NH, D2O exchangeable); 13C NMR
(DMSO-d6,100 MHz) δ ppm: 14.05 (CH3-CH2), 20.03 (CH3-CH2), 29.55 (N-CH2-CH2),
41.18 (N-CH2-), 110.73, 116.60, 119.54, 121.38, 123.73, 126.85, 128.78, 138.36, 132.44,
135.32, 143.60, 148.03, 154.22 (aromatic carbons), 161.52 (hydrazide C=O), 162.40
(isatin C=O); Anal. Calcd. for C19H20N4O4S (400.45): C, 56.99; H, 5.03; N, 13.99; found
C, 56.85; H, 4.99; N, 13.93.
5.1.4.14. 4-[2-(1-Allyl-2-oxoindolin-3-ylidene)hydrazine-1-carbonyl]benzenesulfonamide
10f.
Yellow powder (yield 67%), m.p. 253-255 °C; IR (KBr, ν cm-1): 3405, 3017 (NH, NH2),
1693, 1654 (C=O) and 1390, 1050 (SO2); 1H NMR (DMSO-d6,400MHz) δ ppm: 4.42 (br
s, 2H, N-CH), 5.21-5.30 (m, 2H, CH2=CH), 5.86-5.96 (m, 1H, N-CH-CH), 7.11 (d, 1H,
H-7 of isatin, J = 8 Hz), 7.17 (t, 1H, H-5 of isatin, J = 7.6 Hz), 7.45 (t, 1H, H-6 of isatin,
J = 7.6 Hz), 7.62 (s, 2H, NH2, D2O exchangeable), 7.64 (d, 1H, H-4 of isatin, J = 6.4
Hz), 8.04-8.10 (m, 4H, H-2,3,5 and 6 of phenyl ring of benzenesulfonamide), 13.85 (s,
1H, NH, D2O exchangeable); 13C NMR (DMSO-d6,100 MHz) δ ppm: 41.91 (N-CH),
111.05, 116.60, 118.09, 119.56, 121.37, 123.86, 126.86, 128.81, 131.72, 132.34, 135.29,
138.18, 143.38, 148.04, 154.15 (aromatic carbons), 161.32 (hydrazide C=O), 162.40
(isatin C=O); Anal. Calcd. for C18H16N4O4S (384.41): C, 56.24; H, 4.20; N, 14.58; found
C, 56.45; H, 4.17; N, 14.52.
5.1.4.15. Ethyl-2-[2-oxo-3-(2-(4-sulfamoylbenzoyl)hydrazono)indolin-1-yl]acetate 10g.
18

ACCEPTED MANUSCRIPT
Yellow powder (yield 72%), m.p. 253-255 °C; IR (KBr, ν cm-1): 3405, 3018 (NH, NH2),
1706, 1693, 1654 (C=O) and 1381, 1023 (SO2); 1H NMR (DMSO-d6, 400 MHz) δ ppm:
1.22 (t, 3H, CH2-CH3, J = 7.2 Hz), 4.16 (q, 2H, CH2-CH3, J = 7.6 Hz), 4.74 (s, 2H, N-
CH3), 7.20-7.24 (m, 2H, H-5,7 of isatin), 7.47 (t, 1H, H-6 of isatin, J = 7.6 Hz), 7.61 (s,
2H, NH2, D2O exchangeable), 7.64 (d, 1H, H-4 of isatin, J = 7.2 Hz), 8.04-8.11 (m, 4H,
H-2,3,5 and 6 of phenyl ring of benzenesulfonamide), 13.85 (s, 1H, NH, D2O
exchangeable); 13C NMR (DMSO-d6,100 MHz) δ ppm: 14.48 (CH3), 56.51 (N-CH2),
61.94 (CH2-CH3), 110.91, 116.60, 119.34, 121.42, 124.17, 126.82, 128.90, 132.46,
135.24, 137.49, 143.31, 148.06, 154.22 (aromatic carbons), 161.40 (hydrazide C=O),
162.40 (isatin C=O), 167.85 (ester C=O); Anal. Calcd. for C19H18N4O6S (430.44): C,
53.02; H, 4.22; N, 13.02; found C, 52.83; H, 4.24; N, 12.95.
5.1.4.16.
4-[2-(1-Benzyl-2-oxoindolin-3-ylidene)hydrazine-1-
carbonyl]benzenesulfonamide 11a.
Yellow powder (yield 56%), m.p. 243-245 °C; IR (KBr, ν cm-1): 3955, 3017 (NH, NH2),
1701, 1654 (C=O) and 1385, 1023 (SO2); 1H NMR (DMSO-d6,400MHz) δ ppm: 5.03 (s,
2H, CH2-Ar), 7.07 (d, 1H, Ar-H, J = 7.6 Hz), 7.15 (t, 1H, Ar-H, J = 7.6 Hz), 7.29 (d, 1H,
Ar-H, J = 7.2 Hz), 7.34-7.43 (m, 5H, Ar-H), 7.62 (s, 2H, NH2, D2O exchangeable), 7.66
(d, 1H, H Ar-H, J = 6.4 Hz), 8.05-8.13 (m, 4H, H-2,3,5 and 6 of phenyl ring of
benzenesulfonamide), 13.85 (s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-d6,100
MHz) δ ppm: 43.2 (CH2), 111.07, 116.60, 119.68, 121.44, 123.97, 126.87, 127.63, 127.87
(2C), 128.15, 128.84, 129.21(2C), 132.33, 135.32, 136.01, 138.18, 143.32, 148.06
(aromatic carbons), 161.40 (hydrazide C=O), 162.70 (isatin C=O); Anal. Calcd. for
C22H18N4O4S (434.47): C, 60.82; H, 4.18; N, 12.90; found C, 60.61; H, 4.13; N, 12.96.
5.1.4.17.
4-[2-(1-(4-Fluorobenzyl)-2-oxoindolin-3-ylidene)hydrazine-1-
carbonyl]benzenesulfonamide 11b.
Yellow powder (yield 75%), m.p. 242-243 °C; IR (KBr, ν cm-1): 3398, 3017 (NH, NH2),
1
1690, 1654 (C=O) and 1314, 1023 (SO2); H NMR (DMSO-d6, 400 MHz) δ ppm: 5.02
(s, 2H, CH2-Ar), 7.10 (d, 1H, Ar-H, J = 7.6 Hz), 7.16-7.21 (m, 3H, Ar-H), 7.41-7.49 (m,
3H, Ar-H), 7.61-7.69 (m, 3H, 1H of Ar-H and 2H, NH2, D2O exchangeable), 8.04-8.12
19

ACCEPTED MANUSCRIPT
(m, 4H, H-2,3,5 and 6 of phenyl ring of benzenesulfonamide), 13.81 (s, 1H, NH, D2O
exchangeable); 13C NMR (DMSO-d6,100 MHz) δ ppm: 42.34 (CH2), 110.88, 111.05,
115.89, 116.10, 119.73, 121.48, 124.01, 126.88, 130.04 (2C), 130.12, 132.28, 133.11,
135.32 (2C),141.78, 143.21, 148.02, 155.49 (aromatic carbons), 161.63 (hydrazide C=O),
162.81 (isatin C=O); HRMS (ESI) for C22H18O4N4FS, calcd 453.10273, found 453.10302
[M+H]+.
5.1.4.18.
4-[2-(1-(4-Methylbenzyl)-2-oxoindolin-3-ylidene)hydrazine-1-
carbonyl]benzenesulfonamide 11c.
Yellow powder (yield 70%), m.p. 260-261 °C; IR (KBr, ν cm-1): 3405, 3017 (NH, NH2),
1
1693, 1654 (C=O) and 1382, 1047 (SO2); H NMR (DMSO-d6, 400 MHz) δ ppm: 2.51
(s, 3H, Ar-CH3), 5.13 (s, 2H, CH2-Ar), 7.04 (d, 1H, Ar-H, J = 8 Hz), 7.15 (t, 1H, Ar-H, J
= 7.2 Hz), 7.39 (t, 1H, Ar-H, J = 7.6 Hz), 7.60-7.63 (m, 4H, 2H of Ar-H and 2H, NH2,
D2O exchangeable), 7.66 (d, 1H, H-4 of isatin, J = 7.2 Hz), 7.82 (d, 2H, Ar-H, J = 8 Hz),
8.05-8.11 (m, 4H, H-2,3,5 and 6 of phenyl ring of benzenesulfonamide), 13.81 (s, 1H,
NH, D2O exchangeable); 13C NMR (DMSO-d6,100 MHz) δ ppm: 21.30 (CH3), 42.82
(CH2), 110.88, 110.99, 119.09, 119.79, 121.51, 124.10, 126.88, 128.71 (2C), 130.12,
132.36, 133.11 (2C), 135.28, 138.14, 141.78, 143.08, 148.06, 155.49 (aromatic carbons),
161.40 (hydrazide C=O), 162.81 (isatin C=O); Anal. Calcd. for C23H20N4O4S (448.50):
C, 61.60; H, 4.50; N, 12.49; found C, 61.44; H, 4.53; N, 12.56.
4.2. Biological Evaluation
4.2.1. CA inhibitory assay
An Applied Photophysics stopped-flow instrument has been used for assaying the CA
catalysed CO2 hydration activity, as reported earlier [19]. The inhibition constants were
obtained by non-linear least-squares methods using PRISM 3 and the Cheng-Prusoff
equation as reported earlier [28], and represent the mean from at least three different
determinations. The four tested CA isofoms were recombinant ones obtained in-house as
reported earlier [29].
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4.3. Molecular docking simulation
The crystal structure of hCA II (PDB 5LJT), hCA IX (PDB 5FL4) and hCA XII
(PDB 1JD0) was downloaded from protein databank and were prepared according to the
Protein Preparation wizard in Maestro - Schrödinger suite 2017-1 [30], using the default
values in the preprocess. The missing atoms appeared after this process and were
completed using Prime. Consequently, optimization of the hydrogen bonds was done
followed by refinement process by running a restrained minimization (OPLS3 force field)
which is controlled by cutoff RMSD value of heavy atoms reached 0.30 Å. 3D ligand
structures were drawn and prepared minimized using Maestro [31] (Schrödinger suite).
LigPrep [32] was used to predict ionization states for the ligands using a pH of 7.0 ± 0.5.
In docking simulations using Glide, the protein was treated as rigid, compounds were
flexibly docked, and scoring was assigned according to the standard precision (SP) mode.
Before docking process, the grid box was prepared according to the default size. The
constrained were assigned using the metallic bond for Zn metal.
References
[1] C. T. Supuran, Carbonic anhydrases: novel therapeutic applications for inhibitors and
activators. Nat. Rev. Drug Discovery 7 (2008) 168-181.
[2] V. Alterio, A. Di Fiore, K. D’Ambrosio, C.T. Supuran, G. De Simone, Multiple
binding modes of inhibitors to carbonic anhydrases: how to design specific drugs
targeting 15 different isoforms? Chem. Rev. 112 (2012) 4421-4468.
[3] Y. Xu, L. Feng, P.D. Jeffrey, Y. Shi, F.M. Morel, Structure and metal exchange in the
cadmium carbonic anhydrase of marine diatoms. Nature 452 (2008) 56-61.
[4] S. Del Prete, D. Vullo, G.M. Fisher, K. T. Andrews, S.A. Poulsen, C. Capasso, C.T.
Supuran, Discovery of a new family of carbonic anhydrases in the malaria pathogen
Plasmodium falciparum-the η-carbonic anhydrases. Bioorg. Med. Chem. Lett. 24 (2014)
4389-4396.
[5] D. Neri, C.T. Supuran, Interfering with pH regulation in tumours as a therapeutic
strategy. Nat. Rev. Drug Discov. 10 (2011) 767-777.
21

ACCEPTED MANUSCRIPT
[6] G. De Simone, C.T. Supuran, Carbonic anhydrase IX: Biochemical and
crystallographic characterization of a novel antitumor target. Biochim. Biophys. Acta
1804 (2010) 404-409.
[7] C.T. Supuran. How many carbonic anhydrase inhibition mechanisms exist? J Enzyme
Inhib Med Chem. 31 (2016) 345-360.
[8] C.T. Supuran, V. Alterio, A. Di Fiore, K. D’Ambrosio, F. Carta, S.M. Monti, G. De
Simone, Inhibition of carbonic anhydrase IX targets primary tumors, metastases, and
cancer stem cells: Three for the price of one. Med. Res. Rev. 2018;in press; doi :
10.1002/med.21497.
[9] C.T. Supuran, Carbonic Anhydrase Inhibition and the Management of Hypoxic
Tumors. Metabolites 7 (2017) 48.
[10] P.C. McDonald, J.Y. Winum, C.T. Supuran, S. Dedhar, Recent Developments in
Targeting Carbonic Anhydrase IX for Cancer Therapeutics. Oncotarget 3 (2012) 84-97.
[11] W.M. Eldehna, G.H. Al-Ansary, S. Bua, A. Nocentini, P. Gratteri, A. Altoukhy, H.
Ghabbour, H.Y. Ahmed, C.T. Supuran, Novel indolin-2-one-based sulfonamides as
carbonic anhydrase inhibitors: synthesis, in vitro biological evaluation against carbonic
anhydrases isoforms I, II, IV and VII and molecular docking studies, Eur. J. Med. Chem.
127 (2017) 521-530.
[12] W.M. Eldehna, M.F. Abo-Ashour, A.Nocentini, P.Gratteri, H.I. Eissa, M. Fares,
O.E.Ismael, H.A. Ghabbour, M.M. Elaasser, H.A. Abdel-Aziz, C.T Supuran, Novel 4/3-
((4-oxo-5-(2-oxoindolin-3-ylidene) thiazolidin-2-ylidene) amino) benzenesulfonamides:
Synthesis, carbonic anhydrase inhibitory activity, anticancer activity and molecular
modelling studies, Eur. J. Med. Chem. 139 (2017) 250-262.
[13] W.M. Eldehna, M. Fares, M. Ceruso, H.A. Ghabbour, S.M. Abou-Seri, H.A. Abdel-
Aziz, D.A. El Ella, C.T. Supuran, Amido/ureidosubstituted benzenesulfonamides-isatin
conjugates as low nanomolar/subnanomolar inhibitors of the tumor-associated carbonic
anhydrase isoform XII, Eur. J. Med. Chem. 110 (2016) 259-266.
22

ACCEPTED MANUSCRIPT
[14] 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, Eur. J. Med. Chem. 103 (2015) 583-593.
[15] O. Güzel-Akdemir, A. Akdemir, N. Karalı, C.T. Supuran, Discovery of novel isatin-
based sulfonamides with potent and selective inhibition of the tumor associated carbonic
anhydrase isoforms IX and XII, Org. Biomol. Chem. 13 (2015) 6493-6499.
[16] M.K. Abdel-Hamid, A.A. Abdel-Hafez, N.A. El-Koussi, N.M. Mahfouz, A.
Innocenti, C.T. Supuran, Design, synthesis, and docking studies of new 1,3,4-thiadiazole-
2-thione derivatives with carbonic anhydrase inhibitory activity, Bioorg. Med. Chem. 15
(2007) 6975-6984.
[17] N. Karalı, A. Akdemir, F. Göktaş, P.E. Elma, A. Angeli, M. Kızılırmak, C.T.
Supuran, Novel sulfonamide-containing 2-indolinones that selectively inhibit tumor-
associated alpha carbonic anhydrases, Bioorg. Med. Chem. 25 (2017) 3714-3718.
[18] C. Melis, R. Meleddu, A. Angeli, S. Distinto, G. Bianco, C. Capasso, F. Cottiglia, R.
Angius, C.T. Supuran, E. Maccioni, Isatin: a privileged scaffold for the design of
carbonic anhydrase inhibitors, J Enzyme Inhib Med Chem. 32 (2017) 68-73.
[19] (a) M. Fares, R.A. Eladwy, A. Nocentini, S.R.A. El Hadi, H.A. Ghabbour, A.
Abdel-Megeed, W.M. Eldehna, H.A. Abdel-Aziz, C.T. Supuran, Synthesis of bulky-
tailed sulfonamides incorporating pyrido [2, 3-d][1, 2, 4] triazolo [4, 3-a] pyrimidin-1
(5H)-yl) moieties and evaluation of their carbonic anhydrases I, II, IV and IX inhibitory
effects, Bioorg. Med. Chem. 25 (2017) 2210-2217. (b) H. S. Ibrahim, H.A. Allam, W R.
Mahmoud, A. Bonardi, A. Nocentini, P. Gratteri, E. S. Ibrahim, H. A. Abdel-Aziz, and C.
T. Supuran, Dual-tail arylsulfone-based benzenesulfonamides differently match the
hydrophobic and hydrophilic halves of human carbonic anhydrases active sites: Selective
inhibitors for the tumor-associated hCA IX isoform, Eur. J. Med. Chem. 152 (2018) 1-9.
[20] A. Nocentini, M. Ferraroni, F. Carta, M. Ceruso, P. Gratteri, C. Lanzi, E. Masini,
C.T. Supuran, Benzenesulfonamides incorporating flexible triazole moieties are highly
effective carbonic anhydrase inhibitors: synthesis and kinetic, crystallographic,
23

ACCEPTED MANUSCRIPT
computational, and intraocular pressure lowering investigations, J. Med. Chem., 59
(2016) 10692-10704.
[21] J. Leitans, A. Kazaks, A. Balode, J. Ivanova, R. Zalubovskis, C.T. Supuran, K. Tars,
Efficient expression and crystallization system of cancer-associated carbonic anhydrase
isoform IX, J. Med. Chem., 58 (2015) 9004-9009.
[22] D.A. Whittington, A. Waheed, B. Ulmasov, G.N. Shah, J.H. Grubb, W.S. Sly, D.W.
Christianson, Crystal structure of the dimeric extracellular domain of human carbonic
anhydrase XII, a bitopic membrane protein overexpressed in certain cancer tumor cells,
Proc. Natl. Acad. Sci., 98 (2001) 9545-9550.
[23] Bianco, G., Meleddu, R., Distinto, S., Cottiglia, F., Gaspari, M., Melis, Alcaro, S. N-
Acylbenzenesulfonamide Dihydro-1, 3, 4-oxadiazole Hybrids: Seeking Selectivity
toward Carbonic Anhydrase Isoforms. ACS Med Chem. Lett. 8 (2017) 792-796.
[24] W.M. Eldehna, M. Fares, H.S. Ibrahim, M.A. Alsherbiny, Mohamed H. Aly, H.A.
Ghabbour, H.A. Abdel-Aziz, Synthesis and Cytotoxic activity of Biphenylurea
derivatives containing indolin-2-one moieties, Molecules 21 (2016) 762.
[25] W.M. Eldehna, M.F. Abo-Ashour, H.S. Ibrahim, G.H. Al-Ansary, H.A. Ghabbour,
M.M.
Elaasser,
H.Y.
A.
Ahmed,
N.A.
Safwat,
Novel
[(3-
indolylmethylene)hydrazono]indolin-2-ones as apoptotic anti-proliferative agents: design,
synthesis and in vitro biological evaluation J. Enzyme Inhib. Med. Chem. 33 (2018) 686-
700.
[26] M.I. Attia, W.M. Eldehna, S.A. Afifi, A.B. Keeton, G.A. Piazza, H.A. Abdel-Aziz,
New hydrazonoindolin-2-ones: Synthesis, exploration of the possible anti-proliferative
mechanism of action and encapsulation into PLGA microspheres, PloS one, 12 (2017)
e0181241.
[27] W.M. Eldehna, H. Almahli, G.H. Al-Ansary, H.A. Ghabbour, M.H. Aly, O.E.
Ismael, A. Al-Dhfyan, H.A. Abdel-Aziz, Synthesis and in vitro antiproliferative activity
of some novel isatins conjugated with quinazoline/phthalazine hydrazines against triple-
24

ACCEPTED MANUSCRIPT
negative breast cancer MDA-MB-231 cells as apoptosis-inducing agents, J. Enzyme
Inhib. Med. Chem. 32 (2017) 600-613.
[28] (a) A. Nocentini, D. Moi, G. Balboni, S. Salvadori, V. Onnis, C.T. Supuran
Synthesis and biological evaluation of novel pyrazoline-based aromatic sulfamates with
potent carbonic anhydrase isoforms II, IV and IX inhibitory efficacy. Bioorg. Chem. 77
(2018) 633-639; (b) S. U. Mahmood, A. Saeed, S. Bua, A. Nocentini, P. Gratteri, C.T.
Supuran,
Synthesis, biological evaluation and computational studies of novel
iminothiazolidinone benzenesulfonamides as potent carbonic anhydrase II and IX
inhibitors. Bioorg. Chem. 77 (2018) 381-386.
[29] M. Nakai, J. Pan, K.S. Lin, J.R. Thompson, A. Nocentini, C.T. Supuran, Y.
Nakabayashi, T. Storr, Evaluation of 99mTc-sulfonamide and sulfocoumarin derivatives
for imaging carbonic anhydrase IX expression. J. Inorg. Biochem. 185 (2018) 185:63-70.
[30] L. Schrödinger, Schrödinger, LLC; New York, NY: 2015, PyMOL Molecular
Graphics System, Version, 1 (2016).
[31] Schrödinger Release 2018-1: Maestro, Schrödinger, LLC, New York, NY, 2018.
[32] Schrödinger Release 2018-1: LigPrep, Schrödinger, LLC, New York, NY, 2018.
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Highlights
- Three novel series of isatin-based sulfonamides (5a-h, 10a-g and 11a-c) were synthesized.
- Inhibitory activity of these sulfonamides was evaluated toward hCA I, II, IX and XII isoforms.
- 10b emerged as a single-digit nanomolar hCA IX and XII inhibitor (8.9 and 9.2 nM).
- Molecular docking studies were carried out for 10b within the hCA II, IX and XII active sites.