Synthesis and selective inhibitory effects of some 2-oxindole benzenesulfonamide conjugates on human carbonic anhydrase isoforms CA I, CA II, CA IX and CAXII

Article abstract of DOI:10.1016/j.bioorg.2019.103514

Three series of 2-oxindole benzenesulfonamide conjugates with different linkers were prepared by the condensation reaction of isatin derivatives 1a-e with different benzenesulfonamides. They were screened for their ability to inhibit human (h) carbonic anhydrase (CA, EC 4.2.1.1) isoforms hCA I, hCA II, hCA IX and hCA XII. Many compounds revealed promising activity and selectivity toward CAI, CAII and CAIX compared to acetazolamide (AAZ) especially compounds 2b (K_I = 97.6, 8.0 nM against hCA I, hCA II, respectively) and 3a (K_I = 90.2, 6.5 and 21.4 nM against hCA I, hCA II and hCA IX, respectively) relative to AAZ (K_I = 250, 12 and 25 nM). Additionally, compound 4a revealed the highest activity against hCA II and hCA IX with K_I of 3.0 and 13.9 nM, respectively. Docking of 2b, 3a and 4a into the active site of CA I, II, IX and XII revealed binding mode comparable to AAZ confirming the inhibition results.

Full text of DOI:10.1016/j.bioorg.2019.103514

BioorganicChemistry95(2020)103514
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis and selective inhibitory effects of some 2-oxindole
benzenesulfonamide conjugates on human carbonic anhydrase isoforms CA
I, CA II, CA IX and CAXII
⁎
⁎⁎
Riham F. Georgea, , Mona F. Saida, Silvia Buab, Claudiu T. Supuranb,
a Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt
b University of Florence, Department of NEUROFARBA, Section of Pharmaceutical and Nutraceutical Sciences, via Ugo Schiff 6, 50019 Sesto Fiorentino, Florence, Italy
A R T I C L E I N F O
A B S T R A C T
Keywords:
Three series of 2-oxindole benzenesulfonamide conjugates with different linkers were prepared by the con-
densation reaction of isatin derivatives 1a-e with different benzenesulfonamides. They were screened for their
ability to inhibit human (h) carbonic anhydrase (CA, EC 4.2.1.1) isoforms hCA I, hCA II, hCA IX and hCA XII.
Many compounds revealed promising activity and selectivity toward CAI, CAII and CAIX compared to acet-
azolamide (AAZ) especially compounds 2b (KI = 97.6, 8.0 nM against hCA I, hCA II, respectively) and 3a
(KI = 90.2, 6.5 and 21.4 nM against hCA I, hCA II and hCA IX, respectively) relative to AAZ (KI = 250, 12 and
25 nM). Additionally, compound 4a revealed the highest activity against hCA II and hCA IX with KI of 3.0 and
13.9 nM, respectively. Docking of 2b, 3a and 4a into the active site of CA I, II, IX and XII revealed binding mode
comparable to AAZ confirming the inhibition results.
2-Oxindoles
Benzenesulfanilamides
Carbonic anhydrase inhibitors
Isoform selectivity
Molecular docking
1. Introduction
benzenesulfonamide and 2-oxindole scaffold resulted in CA inhibitors
with different selectivity profile [22–24]. 4-{2-[(5-Bromo-1-methyl-2-
Carbonic anhydrases (CAs, EC 4.2.1.1) are essential metalloenzymes
that catalyze the hydration process of carbon dioxide, thus, playing a
crucial role in regulation of the cellular pH, electrolyte secretion, bio-
synthetic reactions (gluconeogenesis and lipogenesis) and carcino-
genicity [1–4]. Furthermore, this enzyme family comprises of sixteen
isoforms CA I-XV belonging to α-class and they differ in location and
tissue distribution [5–7]. They represent targets for many bioactive
agents used as antiglaucoma [8,9], diuretics [10], antiepileptics
[11,12] and anticancer drugs [13–16]. Primary sulfonamide moiety is
considered as a fundamental pharmacophore for carbonic anhydrase
inhibitors (CA I, as acetazolamide AAZ, compound I), being responsible
for coordination with the enzyme metal ion (Zn2+) in all isoforms [17]
resulting in non-selective inhibition and various consequent side ef-
fects. Therefore, many approaches were directed toward the discovery
of different scaffolds bearing sulfonamide in order to obtain new iso-
form selective CA inhibitors [18–20]. Schiff bases of aromatic sulfo-
namides incorporating isatin moieties were reported and they showed
an interesting selectivity against specific CA isoforms, for example
compound II was selective inhibitor of the tumor associated CA IX with
KI of 1 nM [21]. Additionally, the presence of a spacer between
oxoindolin-3-ylidene)amino]ethyl}benzenesulfonamide (III) possessed
a high selectivity toward CA II (KI = 5.9 nM) [22], while compound IV
showed a remarkable selectivity toward CAXII isoform [23]. Ad-
ditionally, compound V revealed promising activity against CA IX with
KI = 8.9 nM [24]. Moreover, the hydrazine carbonyl-1-pyrazolyl as a
linker between 5-nitro-2-oxindole and benzenesulfonamide (VI) re-
ported an interesting selectivity against tumor associated isoforms CA
IX and CAXII with KI of 7.4 and 5.4 nM, respectively [25] (Fig. 1).
Inspired by the previous findings, this work focuses on the synthesis
of some Schiff bases 2a-e, 3a-e and 4a-e composed of isatin 1a or its N-
alkyl derivatives 1b-e as a scaffold that has been attached to benze-
nesulfonamide moiety through different spacers (Fig. 2). All the syn-
thesized compounds were tested for inhibition against different CA
isoforms (Cytosolic CA I, CA II and tumor associated CA IX and CAXII)
in order to study the effect of these structural changes on the obtained
activity and selectivity profile toward the tested isoforms.
⁎ Corresponding author.
⁎⁎ Corresponding author.
E-mail addresses: riham.eskandar@pharma.cu.edu.eg (R.F. George), claudiu.supuran@unifi.it (C.T. Supuran).
https://doi.org/10.1016/j.bioorg.2019.103514
Received 3 September 2019; Received in revised form 20 October 2019; Accepted 16 December 2019
Availableonline23December2019
0045-2068/©2019ElsevierInc.Allrightsreserved.

R.F. George, et al.
BioorganicChemistry95(2020)103514
Fig. 1. Structure of acetazolamide (AAZ, I) and isatin based benzenesulfonamides (II-VI) reported as CA inhibitors.
Fig. 2. Structure of the target 2-oxindole based benzenesulfonamides 2a-e, 3a-e and 4a-e designed in the present study.
2. Results and discussion
E/Z geometrical isomers. However, it was reported that E/Z ratio is
dependent on each isomer stability and NMR solvent [23,31,32]. This
fact was observed in many compounds as 2a-e, 3a-e and 4d,e which
were found in a mixture with higher concentration of Z isomer as evi-
denced from their 1H NMR spectra that revealed additional signals due
to both isomers, whereas, compounds 4a-c were isolated mainly as Z
isomers. For example, 1H NMR spectrum of compound 2e had two
singlets attributed to the benzylic methylene protons at 4.85 (E-isomer),
5.00 ppm (Z-isomer) with integration ratio 2.94:7.36, respectively (see
the experimental section).
2.1. Chemistry
The target 2-oxindole benzenesulfonamide conjugates were pre-
pared according to pathways outlined in Scheme 1. The key starting
substituted isatins (1b-e) were prepared according to the reported
method [26] by alkylation of commercially available isatin 1a with the
corresponding alkyl halide in dry N, N-dimethyl formamide (DMF) in
the presence of potassium carbonate. The intermediates 1a-e were
condensed with sulfanilamide under reflux in glacial acetic acid to af-
ford the reported 4-(2-oxindolin-3-ylideneamino)benzenesulfonamides
2a,b [21] and their new analogs 2c-e. Furthermore, 1a-e were con-
densed with the prepared 4-hydrazinylbenzenesulfonamide hydro-
chloride [27] at room temperature to give the known 4-[2-(2-oxindolin-
3-ylidene)hydrazinyl]benzenesulfonamide (3a) [28,29] and its new
congeners 3b-e. Finally, condensation of 1a-e with the prepared 4-(2-
hydrazinyl-2-oxoethylamino) benzenesulfonamide [30] in methanol at
room temperature gave 4-(2-oxindolin-3-ylidene)hydrazinyl-2-ox-
oethylamino benzenesulfonamide derivatives (4a-e). All new deriva-
tives were confirmed by spectral and elemental analyses as mentioned
in details in the experimental part.
2.2. Carbonic anhydrase inhibition
The target 2-oxindole based benzenesulfonamides 2a-e, 3a-e and
4a-e were screened for their ability to inhibit the cytosolic human
carbonic anhydrase isoforms (hCA I, hCA II) and the tumor associated
isoforms (hCA IX, hCAXII) by a stopped flow CO2 hydrase assay
[33–37]. Acetazolamide (AAZ) was used as a reference standard and the
obtained results as KIs (nM) are summarized in Table 1. From these
results, the following structure activity relationships can be concluded:
(i) Regarding the inhibition of the cytosolic isoform hCA I, it was
observed that the tested compounds revealed inhibition with KI
range 89.3–839.9 nM except compounds 3d,e and 4c-4e displayed
It is noteworthy that all target compounds 2a-e, 3a-e and 4a-e
contain an exocyclic C]N imine bond which results in their presence as
2

R.F. George, et al.
BioorganicChemistry95(2020)103514
Scheme 1. Synthetic pathways for the target compounds 2a-e, 3a-e and 4a-e.
lower activity with KI in the range of 2417.0–8165.2 nM.
reference standard acetazolamide (KI = 250 nM). Concerning the
effect of the spacer, it was found that generally compounds con-
taining benzenesulfonamide directly attached to 2-oxindole core
Furthermore, compounds 2b,c, 3a-3c and 4b exerted higher ac-
tivity (KI = 97.6, 95.3, 90.2, 89.3, 164.2 and 229.1 nM) than the
Table 1
Inhibition data of human CA isoforms hCA I, II, IX and XII with 2-oxindole based benzenesulfonamides 2a-e, 3a-e, 4a-e and the standard inhibitor Acetazolamide
(AAZ) by a stopped flow CO2 hydrase assay.
Compd ID
R
KIa (nM)
hCA I
Selectivity ratio
I/IX
hCA II
hCA IX
hCAXII
II/IX
I/XII
II/XII
2a
2b
2c
2d
2e
3a
3b
3c
3d
3e
4a
4b
4c
4d
4e
AAZ
H
377.9
97.6
21.5
8.0
135.4
838.5
30.5
672.8
565.1
21.4
852.1
23.6
15.9
597.3
13.9
32.1
16.0
262.0
16.4
25
82.8
34.3
76.4
71.4
42.3
275.4
57.4
462.9
955.9
71.5
602.9
61.7
92.2
192.1
283.2
5.7
2.79
0.16
0.009
0.85
0.40
0.08
0.30
0.09
0.87
4.91
0.56
0.21
2.23
9.66
1.18
12.46
0.48
4.56
2.84
1.25
5.19
6.40
0.33
1.56
0.35
1.76
33.8
1.39
3.71
54.97
42.51
19.59
43.86
0.26
0.23
0.34
3.82
1.10
0.02
1.33
0.04
0.08
4.65
0.005
1.16
1.67
1.61
0.72
2.10
CH3
0.12
C2H5
95.3
26.1
272.8
46.6
6.5
3.12
CH2CH]CH2
CH2C6H5
H
370.9
270.9
90.2
0.55
0.48
4.21
CH3
89.3
76.5
20.6
78.1
332.5
3.0
0.10
C2H5
164.2
1684.2
2417.0
839.9
229.1
5068.2
8165.9
5547.2
250
6.96
CH2CH]CH2
CH2C6H5
H
105.92
4.05
60.42
7.14
CH3
71.5
154.5
308.9
204.3
12
C2H5
316.76
31.17
338.24
10
CH2CH]CH2
CH2C6H5
–
a
Mean from 3 different assays, by a stopped flow technique (errors were in the range of 5–10% of the reported values).
3

R.F. George, et al.
BioorganicChemistry95(2020)103514
(2a-e) had higher activity than that with NH spacer (3a-e) which
docking of the co-crystallized ligand (acetazolamide) in the vicinity of
the binding site of the enzyme and by the ability of the docking pose to
reproduce all the key interactions accomplished by the co-crystallized
ligand (Fig. 2, supplementary materials). Docking of compounds 2b, 3a
and 4a into the binding pocket of CA I confirmed the higher inhibitory
activity of 2b and 3a compared to 4a. Thus, compounds 2b and 3a were
able to form Zn2+ coordinate bonds with their sulfonamide anion
(SeO−, S]NH) in addition to hydrogen bond formation with Gln92,
His200, Thr199 (in case of 2b, Fig. 3A) and with His200 and Thr199 (in
case of 3a, Fig. 4A). However, in case of compound 4a with longer
spacer between the sulfonamide moiety and isatin core that resulting in
orientation of the molecule slightly outside the pocket to bind with
Zn2+ via only one bond with S = NH along with hydrogen bond of S]
O with Leu198 and formation of another hydrogen bond between C]O
of isatin and His67 (Fig. 5A).
in turn were more active than those with 2-aminoacetamide spacer
(4a-e). Additionally, the effect of N-alkylation of 2-oxindole scaf-
fold on the activity can be summarized that in compounds 2a-e, N-
methyl 2b and its N-ethyl analog 2c exhibited higher activity than
N-allyl 2d, N-benzyl 2e or even the unsubstituted congener 2a.
While in case of compounds 3a-e, the unsubstituted 3a, N-methyl
3b and N-ethyl 3c derivatives showed the highest activity.
However, compounds 4a-e, only N-methyl derivative 4b exerted
the highest activity within this series.
(ii) The tested compounds showed higher inhibitory activity toward
hCA II isoform than hCA I with KI ranging 3.0–332.5 nM.
Compounds 2b, 3a and 4a reported better inhibition against CA II
(KI = 8.0, 6.5 and 3.0 nM) than AAZ (KI = 12 nM). It was noticed
that the spacer had no obvious impact on the activity while the N-
substitution affected the obtained activity. Thus, it was found that
generally the unsubstituted derivatives 3a and 4a revealed higher
activity than their N-substituted analogs 3b-e and 4b-e. N-methyl
derivative 2b was an exception as it had the highest activity within
the series 2a-e. Furthermore, the N-methyl (2b, 3b, 4b) and ethyl
substituents (2c,3c, 4c) were more favoured for activity than N-
allyl (2d, 3d, 4d) or N-benzyl analogs (2e, 3e, 4e).
Regarding the binding site of CAII, it was observed that the co-
crystallized AAZ binds to Zn2+ through coordinate bonds with its sul-
fonamide anion in addition to hydrogen bonds of S]O with Thr199 and
Leu198 (Fig. 1B and 3 in supplementary materials). As expected,
compounds 2b and 3a (KI = 8.0, 6.5 nM, respectivly) revealed similar
binding interactions as AAZ with Thr199 and Zn2+ through hydrogen
bond with S]O and coordinate bond with sulfonamide anion, respec-
tively. Moreover, the presence of additional arene-H bond of isatin with
Gln92 and arene-arene bond of phenyl ring with His94 in compound 3a
may explain the higher affinity than compound 2b (Figs. 3B and 4B).
However, the most active compound 4a (KI = 3.0 nM) showed the
same key interactions of metal Zn2+ coordinate bond and hydrogen
bond with Thr199 and the shorter bond length of metal complex may
reflect the stronger binding and hence the highest activity of 4a com-
pared to 2b and 3a (Fig. 5B).
(iii) Concerning the activity of the tested compounds against the tumor
associated hCA IX, it was found that they exhibited inhibition with
KI in the range of 13.9–852.1 nM. Compounds 3a, 3c, 3d, 4a, 4c
and 4e exerted higher activity (KI = 21.4, 23.6, 15.9, 13.9, 16.0
and 16.4 nM, respectively) compared to AAZ (KI = 25 nM). It was
observed that the presence of 2-aminoacetamido linker in com-
pounds 4a-e resulted in higher activity profile (4d was an excep-
tion) than NH spacer in 3a-e which in turn were more active than
2a-e. The N-alkyl substitution had no considerable impact on the
observed activity.
Concerning the CA IX isoform, the co-crystallized AAZ revealed
binding with Zn2+ via metal complex formation in addition to hy-
drogen bond with Thr199 and Thr200 (Fig. 1C and 4 in supplementary
materials). Compounds 3a and 4a with higher activity than AAZ
(KI = 21.4 and 13.9 nM, respectively) bind to CA IX in the same
manner (i.e. through Zn2+ metal coordination with sulfonamide anion
(SeO−, S]NH) along with hydrogen bond of S]O with Thr199,
Figs. 4C and 5C). Moreover, compound 4a forms an additional binding
of side chain CH2 and Gln92 explaining its higher activity than 3a. On
the other hand, the lower affinity of compound 2b (KI = 838.5 nM)
may be explained by its binding with Zn2+ through metal coordinate
with S]O, S]NH which is weaker than that resulted from attraction of
Zn2+ to the sulfonamide anion (Fig. 3C).
(iv) The tested compounds showed moderate inhibition toward hCAXII
with KI spanning between 34.3 and 955.9 nM compared to the
reference standard AAZ (KI = 5.7 nM). The benzenesulfonamides
2a-e exerted higher activity than compounds either with NH
spacer (3a-e) or those with 2-aminoacetamido linker (4a-e).
Generally, the N-methyl derivatives 2b, 3b and 4b revealed the
higher inhibitory activity in each series; thus, N-methyl alkylation
was well tolerated for good activity in all compounds with dif-
ferent linkers. Additionally, N-benzyl derivatives 2e and 3e dis-
played good activity with KI of 42.3 and 71.5 nM.
2.3. Molecular docking study
Finally, the binding of the co-crystallized AAZ to the active site of
CA XII showed the key interactions with Zn2+ as well as hydrogen
bonds with Thr199 and Thr200 (Fig. 1D and 5 in supplementary ma-
terials). Docking of compounds 2b, 3a and 4a into CA XII binding site
revealed similar binding to Zn2+ and hydrogen bond formation with
Thr199. In case of compound 2b, two additional hydrogen bonds with
Leu198 and Thr200 were observed explaining the higher affinity of 2b
to CA XII (KI = 34.3 nM) (Fig. 3D). While, compound 4a forms addi-
tional hydrogen bond with Thr200 in comparison to 3a, it exhibited
lower affinity than 3a. This may be due to the more stable and stronger
coordinate bond that was formed in case of 3a than 4a, as in compound
3a it was formed between charged SeO−, S]NH and Zn2+ with
shorter bond length (2.8, 0.99 Å, respectively, Fig. 4D), whereas, in
compound 4a, the coordinate bond was formed between uncharged S]
O, S]NH and Zn2+ with longer bond length (3.09, 2.15 Å, respectively,
Fig. 5D) (i.e. the higher the attraction forces between opposite charges,
and the shorter the bond length, the stronger the bond and the higher
the affinity to the binding site).
Compounds 2b, 3a and 4a as representatives from each synthesized
series and at the same time covered wide range of activity against the
tested CA isoforms were selected for molecular docking in the binding
site of CA I (PDBID: 3W6H), CA II (PDBID: 3HS4), CA IX (PDB ID: 3IAI)
and CA XII (PDB ID: 1JD0) co-crystallized with acetazolamide [38]. The
molecular docking was performed using Molecular Operating En-
vironment (MOE, 2015.10) software in order to rationalize the obtained
in vitro enzyme inhibitory activity. It was reported that the sulfonamide
group binds as the anion with Zn2+ of CA and binding in this form
requires deprotonation of the sulfonamide [39]. Therefore, all struc-
tures (AAZ, 2b, 3a and 4a) were drawn in their deprotonated forms and
the negative charge was automatically considered by the program to be
on the oxygen atom of the sulfonamide moiety (SeO−, S]NH) that was
more favorable and stable rather than its equivalent form (S]O,
SeNH−). The docking results were presented in Figs. 3–5 and Figs. 1–5
in supplementary materials.
The co-crystallized ligand acetazolamide, AAZ, was found to in-
teract with the key amino acids in the binding site of CA I (PDBID:
3W6H) through mainly metal Zn2+ coordination with S]NH and
SeO− in addition to hydrogen bond with Gln92 (Fig. 1A, supplemen-
tary materials). Furthermore, docking setup was first validated by self-
2.4. Physicochemical, ADME and pharmacokinetics prediction
Computational study for the prediction of physicochemical para-
meters, lipophilicity, solubility, pharmacokinetics and drug likeness of
4

R.F. George, et al.
BioorganicChemistry95(2020)103514
Fig. 3. 2D diagram of compound 2b showing its interaction with the CA I (A), CA II (B), CA IX (C) & CA XII (D) binding site.
the target compounds was conducted using the Swiss ADME www.
swissadme.ch/. The computed molecular properties are presented in
Tables 1–3, supplementary materials. From the obtained data, it can be
concluded that the target compounds were expected to have promising
physicochemical and pharmacokinetic properties. They exhibited a
predicted consensus Log Po/w values ranging from 0.46 to 2.54 and no
BBB permeability, therefore, they are expected to be safe with no CNS
side effects. Additionally, all compounds except 4b,c were not P-gly-
coprotein substrates. Furthermore, all compounds except 4a-e revealed
high GIT absorption, thus, they can be taken orally. Regarding their
ability to inhibit cytochrome P isoforms, they were not inhibitors for
CYP1A2 and CYP2D6, whereas, few compounds were found to inhibit
CYP2C19 (2e, 4e), CYP2C9 (2c-e, 3d,e, 4e) and CYP3A4 (2c-e, 3e, 4e),
therefore, all compounds except 2c-e, 3d,e and 4e do not have any
interactions with drugs metabolized by these isoforms. In addition, all
compounds fullfilled the druglikness criteria as described by Lipinski
and Ghose, while, according to Veber and Egan, only 4a-e were ex-
ceptions and only 4a was an exception in case of Muegge.
lipophilic agents.
3. Conclusion
Three series of 2-oxindole benzenesulfonamide conjugates without
or with NH and 2-aminoacetamido linkers (2a-e, 3a-e and 4a-e, re-
spectively) were synthesized and screened for their inhibitory activity
against hCA I, hCA II, hCA IX and hCA XII isoforms. Some tested
compounds revealed higher activity and selectivity than AAZ against
hCA I, hCA II and hCA IX especially compounds 2b (KI = 97.6, 8.0 nM
against hCA I, hCA II, respectively) and 3a (KI = 90.2, 6.5 and 21.4 nM)
relative to acetazolamide, AAZ (KI = 250, 12 and 25 nM) against hCA I,
hCA II and hCA IX, respectively. Additionally, compound 4a revealed
the highest activity against hCA II and hCA IX with KI of 3.0 and
13.9 nM, respectively. Thus, the linker between benzenesulfonamide
and 2-oxindole core played an obvious role in controlling the selectivity
profile of the prepared compounds towards different hCA isoforms.
Moreover, molecular docking of 2b, 3a and 4a in the active site of CA I,
CAII, CA IX and CA XII revealed the key interactions similar to the
reference standard acetazolamide and confirmed the obtained activity.
In summary, the most active compounds 2b, 3a and 4a have pro-
mising physicochemical and pharmacokinetic properties; only 4a has
low GIT absorption which can be improved by derivatization to more
5

R.F. George, et al.
BioorganicChemistry95(2020)103514
Fig. 4. 2D diagram of compound 3a showing its interaction with the CA I (A), CA II (B), CA IX (C) & CA XII (D) binding site.
4. Experimental
4.1.1.1. 4-(1-Ethyl-2-oxoindolin-3-ylideneamino)benzenesulfonamide
(2c). Orange crystals, yield 70%, m.p. 237–239 °C; IR (KBr, ʋcm−1):
3329, 3229 (NH2), 3094, 3055 (CH aromatic), 2986, 2939 (CH
aliphatic), 1724 (C]O), 1655 (NH bending), 1605–1543 (C]C),
1331, 1157 (SO2); 1H NMR (DMSO‑d6, 400 MHz): δ 1.15, 1.21 (2t,
J = 7.10 Hz, 3H, CH3CH2), 3.64, 3.79 (2q, J = 7.00 Hz, 2H, CH3CH2),
6.39, 7.13 (2d, J = 7.48 Hz, 1H, aromatic H), 6.82, 7.15 (2t,
J = 7.17 Hz, 1H, aromatic H), 7.18 (d, J = 8.32 Hz, 2H, aromatic
H), 7.20, 7.67 (2d, J = 7.36 Hz, 1H, aromatic H), 7.33, 7.39 (2s, 2H,
NH2, D2O exchangeable), 7.47, 7.58 (2t, J = 7.66 Hz, 1H, aromatic H),
7.76, 7.92 (2d, J = 8.32 Hz, 2H, aromatic H); 13C NMR (DMSO‑d6,
100 MHz): δ 12.8 (CH3CH2), 34.8 (CH3CH2), 110.8, 115.6, 118.0,
122.7, 123.4, 125.8, 128.0, 135.3, 140.9, 147.6, 153.6, 155.0, 157.0
(aromatic carbons), 162.0 (C]O); MS, m/z [%]: 329 [M+, 100], 330
[(M++1), 20], 331 [(M++2), 8]; Anal. Calcd. for C16H15N3O3S
(329.37): C, 58.34; H, 4.59; N, 12.76. Found: C, 58.09; H, 4.73; N,
12.94.
4.1. Chemistry
All chemicals were purchased from VWR International Merck, or
Sigma-Aldrich, Germany. Melting Points are uncorrected and were
carried out by open capillary tube method using Stuart SMP3 melting
point apparatus. Elemental Microanalyses were carried out at the
Regional Center for Mycology and Biotechnology, Al-Azhar University.
Infrared spectra were recorded on Shimadzu Infrared spectrometer IR
Affinity-1 (FTIR- 8400S-Kyoto-Japan), and expressed in wave number
(cm−1). 1H NMR and 13C NMR Spectra were recorded on Bruker High
Performance Digital FT-NMR Spectrometer Avance III 400 MHz, 13C,
100 MHz NMR spectrometer. Chemical shifts were expressed in δ units
and were related to that of the solvents. Mass Spectra were recorded
using ISQLT Thermo Scientific Mass spectrometer at The Regional
Center for Mycology and Biotechnology, Al-Azhar University. All the
reactions were monitored by TLC using silica gel F254 plates (Sigma-
Aldrich), using chloroform: methanol 9.5:0.5 as eluting system and
were visualized by UV-lamp. Compounds 1b-e [26], 2a,b [21], 3a
[28,29] and the reagents 4-hydrazinylbenzenesulfonamide hydro-
chloride [27] and 4-(2-hydrazinyl-2-oxoethylamino)benzenesulfona-
mide [30] were prepared according to their reported methods.
4.1.1.2. 4-(1-Allyl-2-oxoindolin-3-ylideneamino)benzenesulfonamide
(2d). Orange crystals, yield 65%, m.p. 208–209 °C; IR (KBr, ʋ cm−1):
3325, 3224 (NH2), 3086 (CH aromatic), 2985 (CH aliphatic), 1717 (C]
O), 1659 (NH bending), 1608–1554 (C]C), 1339, 1161 (SO2); 1H NMR
(DMSO‑d6, 400 MHz):
δ 4.25, 4.40 (2d, J = 4.66 Hz, 2H,
NCH2CH]CH2), 5.18–5.24, 5.28–5.33 (2m, 2H, NCH2CH]CH2),
5.78–5.86, 5.87–5.94 (2m, 1H, NCH2CH]CH2), 6.41, 7.06 (2d,
J = 7.60 Hz, 1H, aromatic H), 6.83, 7.16 (2t, J = 7.60 Hz, 1H,
aromatic H), 7.08, 7.69 (2d, J = 8.16 Hz, 1H, aromatic H), 7.14, 7.20
(2d, J = 8.40 Hz, 2H, aromatic H), 7.33, 7.40 (2s, 2H, NH2, D2O
exchangeable), 7.46, 7.56 (2t, J = 7.76 Hz, 1H, aromatic H), 7.77, 7.93
4.1.1. General procedure for the synthesis of compounds 2a-e
A mixture of equimolar amounts of the appropriate isatin deriva-
tives (1a-e) and sulfanilamide (2 mmol) was heated in glacial acetic
acid (5 mL) at 100 °C for 3 h. The mixture was cooled and the formed
precipitate was collected by filtration and washed with ethanol.
6

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BioorganicChemistry95(2020)103514
Fig. 5. 2D diagram of compound 4a showing its interaction with the CA I (A), CA II (B), CA IX (C) & CA XII (D) binding site.
(2d, J = 8.36 Hz, 2H, aromatic H); 13C NMR (DMSO‑d6, 100 MHz): δ
42.2 (NCH2CH]CH2), 115.5, 117.8, 117.9, 122.8, 123.4, 123.5, 126.7,
127.9, 131.8, 135.1, 140.9, 147.7, 154.8. 157.1 (aromatic and
NCH2CH]CH2), 162.1 (C]O); MS, m/z [%]: 341 [M+, 38], 342
[(M++1), 5], 343 [(M++2), 3], 65 [100]; Anal. Calcd. for
solid was filtered, washed with ethanol and recrystallized from ethanol.
4.1.2.1. 4-[2-(1-Methyl-2-oxoindolin-3-ylidene)hydrazinyl]
benzenesulfonamide (3b). Yellow crystals, yield 78%, m.p. 264–266 °C;
IR (KBr, ʋ cm−1): 3317, 3174 (NH, NH2), 3082 (CH aromatic), 2974
(CH aliphatic), 1708 (C]O), 1612 (NH bending), 1574–1508 (C]C),
1323, 1157 (SO2); 1H NMR (DMSO‑d6, 400 MHz): δ 3.20, 3.24 (2s, 3H,
NCH3), 7.08–7.11, 7.13–7.17 (2m, 2H, aromatic H), 7.26, 7.28 (2s, 2H,
NH2, D2O exchangeable), 7.36, 7.43 (2t, J = 7.70 Hz, 1H, aromatic H),
7.56, 7.63 (2d, J = 8.72 Hz, 2H, aromatic H), 7.60, 8.25 (2d,
J = 7.44 Hz, 1H, aromatic H), 7.81, 7.83 (2d, J = 8.64 Hz, 2H,
aromatic H), 10.75, 12.72 (2s, 1H, NH, D2O exchangeable); 13C NMR
(DMSO‑d6, 100 MHz): δ 25.8 (CH3), 114.2, 115.0, 115.7, 119.2, 122.2,
124.6, 127.6, 129.3, 131.1, 137.8, 138.0, 142.1, 145.5 (aromatic
carbons), 164.3 (C]O); MS, m/z [%]: 330 [M+, 100], 331 [(M++1),
45], 332 [(M++2), 17]; Anal. Calcd. for C15H14N4O3S (330.36): C,
54.53; H, 4.27; N, 16.96. Found: C, 54.80; H, 4.45; N, 16.85.
C
17H15N3O3S(341.38): C, 59.81; H, 4.43; N, 12.31. Found: C, 60.05;
H, 4.68; N, 12.17.
4.1.1.3. 4-(1-Benzyl-2-oxoindolin-3-ylideneamino)benzenesulfonamide
(2e). Orange crystals, yield 60%, m.p. 224–226 °C; IR (KBr, ʋ cm−1):
3298, 3217 (NH2), 3101, 3066 (CH aromatic), 2920, 2850 (CH
aliphatic), 1716 (C]O), 1651 (NH bending), 1608, 1585 (C]C),
1338, 1165 (SO2); 1H NMR (DMSO‑d6, 400 MHz): δ 4.85, 5.00 (2s,
2H, CH2), 6.42, 7.01 (2d, J = 7.56 Hz, 1H, aromatic H), 6.82, 7.15 (2t,
J = 7.62 Hz, 1H, aromatic H), 7.04, 7.70 (2d, J = 7.76 Hz, 1H,
aromatic H), 7.20, 7.24 (2d, J = 8.48 Hz, 2H, aromatic H), 7.30, 7.50
(2t, J = 7.56 Hz, 1H, aromatic H), 7.35–7.45 (m, 7H, 5 aromatic H,
NH2), 7.80, 7.94 (2d, J = 8.48 Hz, 2H, aromatic H); 13C NMR
(DMSO‑d6, 100 MHz): δ 43.4 (CH2), 115.7, 118.1, 122.9, 123.6,
125.7, 126.7, 127.8, 127.9, 129.2, 135.2, 136.2, 141.0, 147.6, 154.8,
157.5 (aromaticcarbons), 162.5 (C]O); MS, m/z [%]: 391 [M+, 13],
392 [(M+ +1), 4], 65 [100]; Anal. Calcd. for C21H17N3O3S (391.44): C,
64.43; H, 4.38; N, 10.73. Found: C, 64.21; H, 4.60; N, 10.97.
4.1.2.2. 4-[2-(1-Ethyl-2-oxoindolin-3-ylidene)hydrazinyl]
benzenesulfonamide (3c). Yellow crystals, yield 75%, m.p. 242–243 °C;
IR (KBr, ʋ cm−1): 3321, 3179 (NH, NH2), 3082, 3067 (CH aromatic),
2982, 2939 (CH aliphatic), 1701 (C]O), 1610 (NH bending),
1574–1508 (C]C), 1323, 1157 (SO2); 1H NMR (DMSO‑d6, 400 MHz):
δ 1.18, 1.20 (2t, J = 7.10 Hz, 3H, CH3CH2), 3.81, 4.10(2q, J = 7.10 Hz,
2H, CH3CH2), 7.13–7.17 (m, 2H, aromatic H), 7.25, 7.27 (2s, 2H, NH2,
D2O exchangeable), 7.38, 7.41 (2t, J = 7.76 Hz, 1H, aromatic H), 7.61,
7.63 (2d, J = 8.72 Hz, 2H, aromatic H), 7.82, 7.83 (2d, J = 8.68 Hz,
2H, aromatic H), 8.27 (d, J = 7.60 Hz, 1H, aromatic H), 10.78, 12.77
4.1.2. General procedure for the synthesis of compounds 3b-e.
A mixture of the appropriate isatin derivative (1b-e) (1 mmol) and
4-hydrazinylbenzenesulfonamide hydrochloride (0.22 g, 1 mmol) in
methanol (5 mL) was stirred at room temperature for 4 h. The obtained
7

R.F. George, et al.
BioorganicChemistry95(2020)103514
(2 s, 1H, NH, D2O exchangeable); 13C NMR (DMSO‑d6, 100 MHz): δ
13.3 (CH3CH2), 34.2 (CH3CH2), 109.8, 114.3, 119.5, 120.6, 122.9,
127.6, 127.8, 129.4, 129.7, 138.1, 141.0, 145.6 (aromatic carbons),
161.1 (C]O); MS, m/z [%]: 344 [M+, 100], 345 [(M++1), 17]; Anal.
Calcd. for C16H16N4O3S (344.39): C, 55.80; H, 4.68; N, 16.37. Found: C,
56.06; H, 4.82; N, 16.56.
373 [M+, 5], 374 [(M+ + 1), 2], 375 [(M++ 2), 2], 65 [100]; Anal.
Calcd. for C16H15N5O4S (373.39): C, 51.47; H, 4.05; N, 18.76. Found: C,
51.62; H, 4.23; N, 18.94.
4.1.3.2. 4-{2-[2-(1-Methyl-2-oxoindolin-3-ylidene)hydrazinyl]-2-
oxoethylamino}benzenesulfonamide (4b). Yellow crystals, yield 82%,
m.p. 202–204 °C; IR (KBr, ʋ cm−1): 3417, 3332, 3259 (2NH, NH2),
3078 (CH aromatic), 2943, 2909 (CH aliphatic), 1720, 1697 (2C]O),
1601 (NH bending), 1516 (C]C), 1334, 1153 (SO2); 1H NMR
(DMSO‑d6, 400 MHz): δ 3.19 (s, 3H, CH3), 4.35 (s, 2H, CH2), 6.71 (d,
J = 8.40 Hz, 3H, aromatic H), 6.95 (s, 2H, NH2, D2O exchangeable),
7.10–7.12 (m, 2H, aromatic H), 7.49 (t, J = 7.70 Hz, 1H, aromatic H),
7.54 (d, J = 8.52 Hz, 2H, aromatic H), 8.07 (s, 1H, NH, D2O
exchangeable), 11.32 (s, 1H, NH, D2O exchangeable); 13C NMR
(DMSO‑d6, 100 MHz): δ 26.5 (CH3), 45.1 (CH2), 109.8, 111.8, 115.0,
122.7, 126.2, 127.8, 131.4, 133.1, 142.8, 145.3, 151.4 (aromatic
carbons), 163.7 (C]O), 169.3 (C]O); MS, m/z [%]: 387 [M+, 50],
187 [1 0 0]; Anal. Calcd. for C17H17N5O4S (387.41): C, 52.70; H, 4.42;
N, 18.08. Found: C, 52.96; H, 4.63; N, 17.89.
4.1.2.3. 4-[2-(1-Allyl-2-oxoindolin-3-ylidene)hydrazinyl]
benzenesulfonamide (3d). Yellow crystals, yield 70%, m.p. 235–237 °C;
IR (KBr, ʋ cm−1): 3318, 3156 (NH, NH2), 3078 (CH aromatic), 2982
(CH aliphatic), 1709 (C]O), 1605 (NH bending), 1574–1508 (C]C),
1323, 1161 (SO2); 1H NMR (DMSO‑d6, 400 MHz): δ 4.40, 4.42 (2d,
J = 4.50 Hz, 2H, NCH2CH]CH2), 5.14–5.20 (2d, J = 11.84 Hz, 2H,
NCH2CH]CH2), 5.84–5.95 (m, 1H, NCH2CH]CH2), 7.03, 7.07 (2d,
J = 7.90 Hz, 1H, aromatic H), 7.13, 7.15 (2t, J = 7.38 Hz, 1H,
aromaticH), 7.26, 7.29 (2s, 2H, NH2, D2O exchangeable), 7.34, 7.40 (2t,
J = 7.70 Hz, 1H, aromatic H), 7.59, 7.65 (2d, J = 8.60 Hz, 2H,
aromatic H), 7.64, 8.29 (2d, J = 7.36 Hz, 1H, aromatic H), 7.82, 7.84
(2d, J = 8.48 Hz, 2H, aromatic H), 10.82, 12.73 (2s, 1H, NH, D2O
exchangeable); 13C NMR (DMSO‑d6, 100 MHz):
δ
41.6
(NCH2CH]CH2), 110.3, 114.3, 117.6, 119.4, 120.5, 123.0, 127.8,
129.1, 129.6, 132.2, 138.1, 141.2, 145.5 (aromatic and
NCH2CH = CH2), 161.1 (C]O); MS, m/z [%]: 356 [M+, 100], 357
[(M++ 1), 5], 358 [(M++ 2), 10]; Anal. Calcd. for C17H16N4O3S
(356.40): C, 57.29; H, 4.52; N, 15.72. Found: C, 57.48; H, 4.66; N,
15.69.
4.1.3.3. 4-{2-[2-(1-Ethyl-2-oxoindolin-3-ylidene)hydrazinyl]-2-
oxoethylamino}benzenesulfonamide (4c). Yellow crystals, yield 80%,
m.p. 218–219 °C; IR (KBr, ʋ cm−1): 3375, 3283, 3233 (2NH, NH2),
3071 (CH aromatic), 2986, 2939, 2885 (CH aliphatic), 1720 (2C]O),
1601 (NH bending), 1570–1512 (C]C), 1307, 1149 (SO2); 1H NMR
(DMSO‑d6, 400 MHz): δ 1.17 (t, J = 7.00 Hz, 3H, CH3CH2), 3.77 (q,
J = 6.90 Hz, 2H, CH3CH2), 4.36 (s, 2H, CH2), 6.71 (d, J = 8.40 Hz, 3H,
aromatic H), 6.95 (s, 2H, NH2, D2O exchangeable), 7.10 (t, J = 7.62 Hz,
1H, aromatic H), 7.17 (d, J = 7.92 Hz, 1H, aromatic H), 7.48 (t,
J = 7.82 Hz, 1H, aromatic H), 7.55 (d, J = 8.52 Hz, 2H, aromatic H),
8.08 (s, 1H, NH, D2O exchangeable), 11.33 (s, 1H, NH, D2O
exchangeable); 13C NMR (DMSO‑d6, 100 MHz): δ 13.0 (CH3CH2),
34.6 (CH3CH2), 45.0 (CH2), 109.9, 111.8, 115.2, 122.6, 126.4, 127.8,
131.4, 133.2, 143.0, 144.2, 151.4 (aromatic carbons), 163.4 (C]O),
169.4 (C]O); MS, m/z [%]: 401 [M+, 28], 106 [100]; Anal. Calcd. for
4.1.2.4. 4-[2-(1-Benzyl-2-oxoindolin-3-ylidene)hydrazinyl]
benzenesulfonamide (3e). Yellow crystals, yield 70%, m.p. 255–256 °C;
IR (KBr, ʋ cm−1): 3309 (NH, NH2), 3055, 3028 (CH aromatic), 2904
(CH aliphatic), 1701 (C]O), 1605 (NH bending), 1573–1508 (C]C),
1323, 1161 (SO2); 1H NMR (DMSO‑d6, 400 MHz): δ 5.00 (s, 2H, CH2),
7.02 (d, J = 7.88 Hz, 1H, aromatic H), 7.14 (t, J = 7.56 Hz, 1H,
aromaticH), 7.26 (br s, 3H, NH2, D2O exchangeable + 1H aromaticH),
7.32–7.37 (m, 5H, aromatic H), 7.63, 7.66 (2d, J = 8.76 Hz, 2H,
aromatic H), 7.82, 7.84 (2d, J = 8.72 Hz, 1H, aromatic H), 8.30 (d,
J = 7.56 Hz, 2H, aromatic H), 10.86, 12.77 (2s, 1H, NH, D2O
exchangeable); 13C NMR (DMSO‑d6, 100 MHz): δ 42.8 (CH2), 110.3,
114.4, 115.1, 120.6, 123.1, 127.6, 127.8, 127.9, 129.1, 129.2, 136.6,
138.2, 141.1, 145.5, 147.1, 161.5 (aromatic carbons), 164.5 (C]O);
MS, m/z [%]: 406 [M+, 87], 407 [(M++ 1), 9], 91 (100); Anal. Calcd.
for C21H18N4O3S (406.46): C, 62.05; H, 4.46; N, 13.78. Found: C, 62.31;
H, 4.67; N, 13.89.
C
18H19N5O4S (401.44): C, 53.85; H, 4.77; N, 17.45. Found: C, 53.71; H,
4.89; N, 17.68.
4.1.3.4. 4-{2-[2-(1-Allyl-2-oxoindolin-3-ylidene)hydrazinyl]-2-
oxoethylamino}benzenesulfonamide (4d). Yellow crystals, yield 78%,
m.p. 213–214 °C; IR (KBr, ʋ cm−1): 3379, 3345, 3291, 3240 (2NH,
NH2), 3071, 3032 (CH aromatic), 2901 (CH aliphatic), 1720 (2C]O),
1659 (NH bending), 1601, 1512 (C]C), 1315, 1150 (SO2); 1H NMR
(DMSO‑d6, 400 MHz):
δ 4.38–4.39 (m, 4H, CH2-NH + NCH2-
4.1.3. General procedure for the synthesis of compounds 4a-e.
A mixture of the isatin derivatives (1a-e) (1 mmol) and 4-((2-hy-
drazinyl-2-oxoethyl)amino)benzenesulfonamide (0.24 g, 1 mmol) was
dissolved in methanol (5 mL) in the presence of glacial acetic acid
(0.5 mL) and stirred at room temperature for 3 h. The obtained pre-
cipitate was filtered, washed with ethanol and recrystallized from
ethanol.
CH]CH2), 5.15–5.20 (m, 2H, NCH2-CH]CH2), 5.84–5.91 (m, 1H,
NCH2-CH]CH2), 6.61 (d, J = 8.44 Hz, 1H, aromatic H), 6.71 (d,
J = 8.32 Hz, 2H, aromatic H), 6,94, 6.96 (2s, 2H, NH2, D2O
exchangeable), 7.06 (d, J = 7.92 Hz, 1H, aromatic H), 7.12 (t,
J = 7.62 Hz, 1H, aromatic H), 7.47 (t, J = 7.74 Hz, 1H, aromatic
H), 7.51, 7.55 (2d, J = 8.56 Hz, 2H, aromatic H), 8.11 (s, 1H, NH, D2O
exchangeable), 11.33 (s, 1H, NH, D2O exchangeable); 13C NMR
(DMSO‑d6, 100 MHz): δ 41.9 (NCH2CH]CH2), 45.0 (CH2), 111.6,
111.7, 115.1, 117.4, 122.8, 123.6, 126.3, 127.6, 127.7, 131.2, 132.1,
133.0, 144.3, 151.4 (aromatic and NCH2CH = CH2), 163.5 (C]O),
169.3 (C]O); MS, m/z [%]: 413 [M+, 91], 392 [1 0 0]; Anal. Calcd. for
4.1.3.1. 4-{2-Oxo-2-[2-(2-oxoindolin-3-ylidene)hydrazinyl]ethylamino}
benzenesulfonamide (4a). Yellow crystals, yield 85%, m.p. 258–260 °C;
IR (KBr, ʋ cm−1): 3383, 3344, 3310, 3233 (3NH, NH2), 3078 (CH
aromatic), 2893, 2824 (CH aliphatic), 1720, 1697 (2C]O), 1658 (NH
bending), 1597–1512 (C]C), 1315, 1149 (SO2); 1H NMR (DMSO‑d6,
400 MHz): δ 4.34 (s, 2H, CH2), 6.61 (d, J = 8.56 Hz, 1H, aromatic H),
6.70 (d, J = 8.36 Hz, 2H, aromatic H), 6.90 (d, J = 7.80 Hz, 1H,
aromatic H), 6.95 (s, 2H, NH2, D2O exchangeable), 7.04 (t, J = 7.68 Hz,
1H, aromatic H), 7.39 (t, J = 7.68 Hz, 1H, aromatic H), 7.55 (d,
J = 8.56 Hz, 2H, aromatic H), 8.05 (s, 1H, NH, D2O exchangeable),
10.83 (s, 1H, NH, D2O exchangeable), 11.23 (s, 1H, NH, D2O
exchangeable); 13C NMR (DMSO‑d6, 100 MHz): δ 45.1 (CH2), 111.2,
111.6, 115.7, 122.2, 127.7, 127.8, 131.2, 131.4, 133.2, 142.9, 144.2,
151.5 (aromatic carbons), 165.1 (C]O), 169.2 (C]O); MS, m/z [%]:
C
19H19N5O4S (413.45): C, 55.19; H, 4.63; N, 16.94. Found: C, 55.46; H,
4.80; N, 16.82.
4.1.3.5. 4-{2-[2-(1-Benzyl-2-oxoindolin-3-ylidene)hydrazinyl]-2-
oxoethylamino}benzenesulfonamide (4e). Yellow crystals, yield 75%,
m.p. 213–215 °C; IR (KBr, ʋ cm−1): 3375, 3345, 3294, 3244 (2NH,
NH2), 3067, 3032 (CH aromatic), 2947 (CH aliphatic), 1724 (2C]O),
1659 (NH bending), 1601–1512 (C]C), 1315, 1149 (SO2); 1H NMR
(DMSO‑d6, 400 MHz): δ 4.38 (s, 2H, CH2NH), 4.98 (s, 2H, CH2 benzyl),
6.61 (d, J = 8.60 Hz, 1H, aromatic H), 6.72 (d, J = 8.48 Hz, 2H,
aromatic H), 6.94, 6.96 (2s, 2H, NH2, D2O exchangeable), 7.04 (d,
8

R.F. George, et al.
BioorganicChemistry95(2020)103514
J = 7.88 Hz, 1H, aromatic H), 7.10 (t, J = 7.60 Hz, 1H, aromatic H),
7.27–7.36 (m, 5H, aromatic H), 7.41 (t, J = 7.76 Hz, 1H, aromatic H),
7.52, 7.55 (2d, J = 8.60 Hz, 2H, aromatic H), 8.12 (s, 1H, NH, D2O
exchangeable), 11.35 (s, 1H, NH, D2O exchangeable); 13C NMR
(DMSO‑d6, 100 MHz): δ 43.1 (CH2 benzyl), 45.0 (CH2), 110.4, 111.6,
111.8, 115.2, 122.9, 126.4, 127.6, 127.7, 128.0, 129.2, 131.2, 133.0,
136.4, 144.1, 151.4, 151.5 (aromatic carbons), 164.0 (C]O), 169.3
(C]O); MS, m/z [%]: 463 [M+, 4], 65 [100]; Anal. Calcd. for
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Appendix A. Supplementary material
C
23H21N5O4S (463.51): C, 59.60; H, 4.57; N, 15.11. Found: C, 59.83;
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2019.103514.
H, 4.71; N, 15.34.
References
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All the molecular modeling studies were carried out using Molecular
Operating Environment (MOE, 2015.10) software. All minimizations
were performed with MOE until an RMSD gradient of 0.05 kcal∙mol−1
Å−1 with MMFF94x force field and the partial charges were auto-
matically calculated. The X-ray crystallographic structure of human
carbonic anhydrase isoforms I, II, IX and XII co-crystalized with acet-
azolamide (PDB ID: 3W6H, 3HS4, 3IAI and 1JD0, respectively) were
downloaded from the protein data bank [38]. Water molecules which
are not involved in the binding were removed. The protein was pre-
pared for the docking study using Protonate 3D protocol in MOE with
default options. The 3D structures of the compounds were drawn by
using the MOE of (Chemical Computing Group software, Canada)
drawing tool bar. The Lowest energy conformer of the compounds
(global-minima) was docked into the active site. All structures were
drawn with deprotonation of their sulfonamide moiety and the negative
charge was localized automatically by the program on SeO−. The co-
crystalized ligand (acetazolamide) was used to define the binding site
for docking. Triangle Matcher placement method and London dG
scoring function were used for docking. Docking setup was first vali-
dated by self-docking of the co-crystallized ligand (acetazolamide) in
the vicinity of the binding site of the enzyme and by the ability of the
docking pose to reproduce all the key interactions accomplished by the
co-crystallized ligand with the hot spots in the active site. The validated
setup was then used in predicting the ligand-receptor interactions at the
binding site for the compounds of interest.
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