Application of hydrazino and hydrazido linkers to connect benzenesulfonamides with hydrophilic/phobic tails for targeting the middle region of human carbonic anhydrases active site: Selective inhibitors of hCA IX

Article abstract of DOI:10.1016/j.ejmech.2019.06.081

Herein we report the design and synthesis of three different sets of novel benzenesulfonamides (5a-e, 7a-e and 10a-d) incorporating hydrophilic/hydrophobic tails by hydrazido or hydrazino linkers. The newly synthesized benzenesulfonamides were examined in vitro for their inhibitory activity towards four human (h) carbonic anhydrase (hCA, EC 4.2.1.1) isoforms, hCA I, II, IX and XII using a stopped-flow CO₂ hydrase assay. All these isoforms were inhibited by the sulfonamides (5a-e, 7a-e and 10a-d) with variable degrees in the following K_I ranges: 76.8–357.4 nM for hCA I, 8.2–94.6 nM for hCA II, 2.0–46.3 nM for hCA XI, and 8.3–88.3 nM for hCA XII. The sulfonamide 7d exhibited potent anti-proliferative activity against breast MCF-7 cancer cell line under both normoxic and hypoxic conditions with IC₅₀ values equal 3.32 ± 0.06 and 8.53 ± 0.32 μM, respectively, which are comparable to the reference drug doxorubicin (IC₅₀ = 2.36 ± 0.04 and 8.39 ± 0.25 μM, respectively). Furthermore, 7d was screened for cell cycle disturbance and apoptosis induction in MCF-7 cells. It was found to persuade cell cycle arrest at G2-M stage as well as to alter the Sub-G1 phase, also, 7d resulted in a significant increase in the percent of annexinV-FITC positive apoptotic cells from 1.03 to 18.54%. Molecular docking study was carried out for 7d within the hCA IX and hCA XII active sites to rationalize the obtained inhibition results.

Full text of DOI:10.1016/j.ejmech.2019.06.081

Accepted Manuscript
Application of hydrazino and hydrazido linkers to connect benzenesulfonamides with
hydrophilic/phobic tails for targeting the middle region of human carbonic anhydrases
active site: Selective inhibitors of hCA IX
Heba Abdelrasheed Allam, Samar H. Fahim, Mahmoud F.Abo-Ashour, Alessio
Nocentini, Mohamed E. Elbakry, Mohamed A. Abdelrahman, Wagdy M. Eldehna,
Hany S. Ibrahim, Claudiu T. Supuran
PII:
S0223-5234(19)30608-7
DOI:
https://doi.org/10.1016/j.ejmech.2019.06.081
EJMECH 11484
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 18 May 2019
Revised Date: 27 June 2019
Accepted Date: 27 June 2019
Please cite this article as: H.A. Allam, S.H. Fahim, M. F.Abo-Ashour, A. Nocentini, M.E. Elbakry, M.A.
Abdelrahman, W.M. Eldehna, H.S. Ibrahim, C.T. Supuran, Application of hydrazino and hydrazido
linkers to connect benzenesulfonamides with hydrophilic/phobic tails for targeting the middle region of
human carbonic anhydrases active site: Selective inhibitors of hCA IX, European Journal of Medicinal
Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.06.081.
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ACCEPTED MANUSCRIPT
Graphical Abstract

ACCEPTED MANUSCRIPT
Application of hydrazino and hydrazido linkers to connect
benzenesulfonamides with hydrophilic/phobic tails for targeting the middle
region of human carbonic anhydrases active site: selective inhibitors of hCA
IX.
1
1
2
3
Heba Abdelrasheed Allam, Samar H. Fahim, Mahmoud F. Abo-Ashour, Alessio Nocentini,
2
2
4
2,*
Mohamed E. Elbakry, Mohamed A. Abdelrahman, Wagdy M. Eldehna, Hany S. Ibrahim,
3
,*
and Claudiu T. Supuran
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Egyptian Russian University, Badr
2
City, Cairo 11829, Egypt
3
Department of NEUROFARBA, Section of Pharmaceutical and Nutraceutical Sciences, Laboratory of
Molecular Modeling Cheminformatics & QSAR, University of Florence, Polo Scientifico, Via U. Schiff
6
, 50019, Sesto Fiorentino, Firenze, Italy
4
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kafrelsheikh University,
Kafrelsheikh, Egypt
ABSTRACT
Herein we report the design and synthesis of three different sets of novel benzenesulfonamides
(5a-e, 7a-e and 10a-d) incorporating hydrophilic/hydrophobic tails by hydrazido or hydrazino
linkers. The newly synthesized benzenesulfonamides were examined in vitro for their
inhibitory activity towards four human (h) carbonic anhydrase (hCA, EC 4.2.1.1) isoforms,
hCA I, II, IX and XII using a stopped-flow CO hydrase assay. All these isoforms were
2
inhibited by the sulfonamides (5a-e, 7a-e and 10a-d) with variable degrees in the following KI
ranges: 76.8–357.4 nM for hCA I, 8.2–94.6 nM for hCA II, 2.0–46.3 nM for hCA XI, and 8.3–
8
8.3 nM for hCA XII. The sulfonamide 7d exhibited potent anti-proliferative activity against
breast MCF-7 cancer cell line under both normoxic and hypoxic conditions with IC values
5
0
equal 3.32 ± 0.06 and 8.53 ± 0.32µM, respectively, which are comparable to the reference drug
doxorubicin (IC50 = 2.36 ± 0.04 and 8.39 ± 0.25 µM, respectively). Furthermore, 7d was
screened for cell cycle disturbance and apoptosis induction in MCF-7 cells. It was found to
persuade cell cycle arrest at G2-M stage as well as to alter the Sub-G1 phase, also, 7d resulted
in a significant increase in the percent of annexinV-FITC positive apoptotic cells from 1.03 to
1
8.54%. Molecular docking study was carried out for 7d within the hCA IX and hCA XII
active sites to rationalize the obtained inhibition results.
_______
_
1

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Keywords: Benzenesulfonamides; Selective hCA IX inhibitors; Molecular modeling; Synthesis;
Anticancer activity.
*
Corresponding authors. E-mail addresses: hany.s.ibrahim@gmail.com (HSI).
claudiu.supuran@unifi.it (CTS).
2

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1
.
Introduction
Carbonic anhydrases (CAs, EC 4.2.1.1) catalyze the hydration of CO to bicarbonate and
2
proton as well as other hydrolytic reactions [1]. Up to now, seven genetic families have been
discovered, the α-, β, γ-, δ-, ζ-, η-, and θ-CAs [1]. Fifteen different α-CA isoforms have been
identified and characterized in human (h) so far, of which twelve are catalytically active and
three are inactive (CA-related proteins) [2]. hCAs are involved in a plethora of physiological
-
and pathological processes, such as gas exchange, transport of CO and HCO across
2
3
membranes,
biosynthetic
reactions,
acid-base
tuning,
calcification
and
tumorigenesis/metastatization [2,3]. The latter processes see isoforms hCA IX and XII
implicated in the tumor cells pH regulatory machinery, with their inhibition having been
validated as a winning anti-tumor strategy [3]. Notably, hCA IX expression is strictly related to
cancer and poor prognosis in tumors, while hCA XII is present in many normal tissues and
organs, such as endometrium, colon, kidney and eye. hCA I and II are cytosolic ubiquitous
isoforms to whom most CA activity in human body is due. Their inhibition, unless desired,
should be avoided to overcome the unpleasant side effects due to promiscuous inhibition [3].
Literature survey regarding several hCA inhibitors (hCAIs) revealed that the main skeleton of
these compounds consists of a certain linker between a benzene sulfonamide group on one side
and another aryl or heteroaryl moiety on the other side. The previous finding was concluded
from studying the structure of different potent hCAs inhibitors as shown in Fig. 1. For example,
a ureido linker was incorporated between benzene sulfonamide group and 4-flourophenyl group
in SLC-0111 (I); a synthetic compound in phase I/II clinical trials in combination therapy for
the treatment of metastatic solid tumors, with Ki (hCA IX) = 45 nM and Ki (hCA XII) = 4.5
nM [4-8]. The ureido linker was again involved in other hCA inhibitors with a heteroaryl
oxindole moiety attached in the other side as in compounds IIa (hCA IX; Ki = 5.3 nM, hCA
XII; Ki = 6.3 nM) and IIb (hCA IX; Ki = 14.8 nM, hCA XII; Ki = 0.64 nM (Fig. 1) [9,10]. In
other hCA inhibitors, the ureido linker was replaced by a hydrazido linker as represented in
compound III (Ki (hCA IX) = 8.9 nM; Ki (hCA XII) = 9.2 nM), compound IV (Ki (hCA IX) =
1
7.3 nM) and compound V (Ki (hCA IX) = 30.4 nM) [11-13]. The approach of using a linker
between a benzene sulfonamide moiety and aryl or heteroaryl moiety was applied again but
through using a hydrazine linker as shown in compound VI (hCA IX; Ki = 5.3 nM) (Fig. 1)
[
14].
In this study, our target is to synthesize three series of hCA inhibitors using the
aforementioned approach. A hydrazido group was used as a linker between a benzene
3

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sulfonamide moiety from one side and a para substituted phenyl group at the other side as in
the target compounds 7a-e. The choice of the other side moiety was based upon the same
rational of compound VII (hCA; II Ki = 0.5 nM, hCA VII; Ki = 0.45 nM) (Fig. 1) [15] which
assumes that mixing hydrophilic/hydrophobic rings in this region will allow the synthesized
compounds to target both the hydrophilic and the hydrophobic regions in the middle of hCA
enzyme. Consequently, p-substituted phenyl ring with aliphatic secondary amines were
employed in this region to achieve the previous rational. Two other series 5a-e and 10a-d
containing a hydrazine linker, again coupled with mixed hydrophilic/hydrophobic moieties
were synthesized (Fig. 1). The target compounds were biologically evaluated against different
isoforms of hCA and were tested for their cytotoxicity against cancer cells under hypoxic
conditions. Moreover, molecular docking studies were performed to rationalize their biological
activities.
4

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Fig. 1. Structures of some reported CA inhibitors I-VII, and the targeted sulfonamides 5a-e,
7
a-e and 10a-d.
2
.
Results and Discussion
2
.1. Chemistry
5

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As a starting point for preparation of the targeted compounds 5a-e, 7a-e and 10a-d, p-
substituted aldehydes (3a-e) (Scheme 1) and p-substituted acetophenone derivatives (9a-d)
(Scheme 2) were synthesized from p-fluorobenzaldehyde and p-fluoroacetophenone through
nucleophilic substitution reactions via different secondary amines where the amines acted as
nucleophiles in exchange of the fluorine atom as reported [16, 17]. Consequently, the
aforementioned p-substituted aldehydes (3a-e) were reacted with both 4-hydrazinobenzene
sulfonamide (4) and 4-sulfamidobenzoic acid hydrazide (6) to give the corresponding
hydrazones 5a-e and hydrazides 7a-e, respectively (Scheme 1).
Scheme 1. Synthetic scheme for compounds 5a-e and 7a-e. Reagents and conditions: (i)
K CO / DMSO/ reflux for 3-5 h; (ii) EtOH/ AcOH / reflux for 3 h; (iii) AcOH / reflux for 2
2
3
h.
On the other hand, the previously synthesized p-substituted acetophenone derivatives
(9a-d) were subjected to the reaction with both 4-hydrazinobenzene sulfonamide (4) and 4-
sulfamidobenzoic acid hydrazide (6) using the same conditions adopted in the reaction with
aldehydes (3a-e) as in scheme 1. However, we managed to obtain the corresponding
6

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hydrazones 10a-d only and not the hydrazides (scheme 2). Further trials were performed to
get the corresponding hydrazides using conditions that are more drastic. But unfortunately
we failed to obtain the desired hydrazides even in the presence of strong dehydrating agents
as conc. H SO or ZnCl .
2
4
2
Scheme 2. Synthetic scheme for compounds 10a-d. Reagents and conditions: (i) K CO /
2
3
DMSO / reflux for 6 h; (ii) EtOH/AcOH / reflux for 3 h.
2
.2. Biological Evaluation.
2
.2.1. Carbonic anhydrase inhibition
All the newly prepared sulfonamides (5a–e, 7a–e and 10a–d) were evaluated for their
ability to inhibit hCA isoforms, hCA I and II (cytosolic) as well as hCA IX and XII
transmembrane, tumor associated isoforms) via a stopped flow CO hydrase assay [18] using
(
2
acetazolamide (AAZ) as a standard inhibitor. The following SAR can be gathered from the
data shown in Table 1:
7

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Table 1: Inhibition data of human CA isoforms hCA I, II, IX and XII with compounds
reported here and the standard sulfonamide inhibitor acetazolamide (AAZ) by a stopped flow
CO hydrase assay.
2
K (nM)*
I
Compound
hCA I
hCA II
hCA IX
hCA XII
50.2 ± 4.2
29.6 ± 1.5
13.1 ± 0.8
31.6 ± 2.4
88.3 ± 6.2
10.1 ± 0.7
10.1 ± 1.2
9.2 ± 0.7
8.3 ± 0.6
51.4 ± 4.5
36.4 ± 2.2
8.7 ± 0.6
9.7 ± 0.4
9.2 ± 0.5
5.7 ± 0.4
5
a
96.2 ± 5.8
100.8 ± 6.9
87.4 ± 5.1
29.9 ± 2.0
24.0 ± 1.8
34.5 ± 2.6
17.9 ± 1.2
94.6 ± 5.3
12.5 ± 1.1
9.3 ± 0.7
19.4 ± 1.2
20.9 ± 1.3
16.5 ± 1.1
14.6 ± 0.9
46.3 ± 2.7
3.1 ± 0.2
3.3 ± 0.2
8.6 ± 0.5
10.1 ± 0.8
23.7 ± 0.1
18.3 ± 1.2
7.2 ± 0.5
8.9 ± 0.6
2.0 ± 0.1
25 ± 1.4
5
b
5
c
5
d
76.8 ± 4.3
5
e
240.4 ± 16.7
347.3 ± 27.0
152.9 ± 14.2
175.4 ± 11.3
129.8 ± 10.5
357.4 ± 24.8
95.4 ± 7.1
7
a
7
b
7
c
15.6 ± 1.2
8.2 ± 0.6
7
d
7
e
62.3 ± 4.3
57.7 ± 5.0
69.2 ± 4.7
65.6 ± 3.9
89.5 ± 5.8
12 ± 0.8
1
0a
0b
0c
0d
AAZ
1
110.6 ± 9.5
149.5 ± 13.2
266.1 ± 19.4
250 ± 15
1
1
*
Inhibition data are expressed as means ± SEM of 3 different assays.
(i) The ubiquitous hCA I is quite effectively inhibited by all reported sulfonamides. The
inhibition constants (K s) span in the range 76.8-357.3 nM. Notably, unsubstituted
I
hydrazones 5a-e are slightly more effective (K s 76.8-100.8 nM, except 5e) than hydrazides
I
7
a-e and methylhydrazones 10a-d. Within subsets 5a-e and 7a-e, compounds e bearing a
benzotriazole tail feel the effect of the steric hindrance of the pendant and show K s of 240.4
I
and 357.4 nM. All other heterocycles incorporating compounds show similar inhibition to
hCA I.
(ii) The other ubiquitous and physiologically relevant isoform, hCA II, is potently inhibited
by most of the reported compounds (K s 8.2-94.6 nM). In particular, hydrazides 7a-e act as
I
the most potent hCA II inhibitors in this study, showing K s in the low nanomolar range (8.2-
I
1
5.6 nM) with the exception of 7e whose K rises to 62.3 nM likely because of steric
I
hindrance. As a general trend, hydrazones 5a-e and 10a-d show a partially lowered inhibitory
efficacy compared to hydrazide compounds. One could speculate about a H-bond in the
ligand-target adduct where the carbonyl group of hydrazides acts as acceptor with an active
site residue of hCA II enhancing the binding with respect to hydrazone compounds. The
addition of a methyl group to the hydrazone linker (10a-d) further decreases the binding with
respect to unsubstituted derivatives (5a-d) probably arousing unfavorable clashes to the
8

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binding of 5a-d. The benzotriazole group of compound 5e elicits a worsening of the
inhibitory activity to 94.6 nM.
(iii) The tumor-associated isoforms hCA IX and XII are potently inhibited in a low
nanomolar range by the screened sulfonamides. Against hCA IX, K s reach one-digit
I
nanomolar values with hydrazides 7a-c (3.1-8.6 nM) and methylhydrazones 10b-d (2.0-8.9
nM). The unsubstituted hydrazones 5a-e show the worst hCA IX inhibition though remaining
very potent inhibitors (K s 14.6-46.3 nM). The rather flat inhibition reported with different
I
substituents disables to devise more accurate SAR.
(
iv) hCA XII is also inhibited with single-digit K s by hydrazides 7c and 7d (8.3-9.2 nM) and
I
methylhydrazones 10b-d (8.7-9.7 nM). Increasing the size of the tail such as in the
benzotriazole derivatives 5e and 7e decreases the inhibition efficacy to 88.3 and 51.4 nM,
respectively.
(v) the selectivity index (SI) data reported in Table 2 show that while actually all compounds
are selective CAIs for the tumor-associated isoforms over hCA I (SI in the range 4.8-133.1
for hCA IX and 1.9-34.4 for hCA XII), significant selectivity for the target CAs over hCA II
exist only within the subset of methylhydrazones 10a-d. In detail, the four compounds
exhibit a II/IX selectivity ranging between 3.2 and 44.8, whereas 10b-d are 7- to 10-fold
II/XII selective CAIs. The latter compounds could represent interesting candidates for further
anti-tumor assessments.
Table 2: Selectivity index (SI) of sulfonamides reported here for hCA IX and XII over hCA I
and II calculated as ratio KI off-target CA / KI target CA
.
SI (KI off-target CA / KI target CA
)
Compound
I / IX
I / XII
II / IX
II/ XII
5
a
5.0
4.8
1.9
3.4
1.5
1.1
2.1
1.2
2.0
4.0
2.8
1.8
0.8
2.6
3.2
9.6
0.6
0.8
2.6
0.6
1.1
1.2
0.9
1.7
1.0
1.2
1.6
8.0
5b
5
c
5.3
6.7
5d
5.3
2.4
5
e
5.2
2.7
7a
112.0
46.3
20.4
12.9
15.1
5.2
34.4
15.1
19.1
15.6
7.0
7b
7
c
7d
7
e
10a
2.6
10b
15.4
12.7
9

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1
0c
0d
AAZ
16.8
133.1
10.0
15.4
28.9
43.9
7.4
44.8
0.5
6.8
9.7
2.1
1
2
.2.2. In vitro anti-proliferative activity towards breast cancer MCF-7 cell line
On account of hCA IX high expression in different breast malignancies, hCA IX has been
correlated with the prognosis and therapy outcomes in breast cancer patients [19].
Herein, the sulfonamides 7d and 10d were evaluated for their anti-proliferative activities
towards breast cancer MCF-7 cell line using doxorubicin as positive control, under normoxic
and hypoxixc conditions adopting the MTT colorimetric assay as reported by T. Mosmann
[
20]. Induction of hypoxia was chemically established via the usage of cobalt (II) chloride
hexahydrate as a chemical inducer of HIF-1α. The results were expressed as median growth
inhibitory concentration (IC ) values that represented the concentration of compound
5
0
required to produce a 50% inhibition of cell growth after 48 h of incubation, compared to the
untreated controls (Table 3).
Table 3. In vitro anti-proliferative activity of sulfonamides 7d and 10d against breast MCF-7
cancer cell line.
a
Comp.
IC50 (µM)
Normoxia
3.32 ± 0.06
7.72 ± 0.22
2.36 ± 0.04
Hypoxia
7
d
8.53 ± 0.32
17.34 ± 0.66
8.39 ± 0.25
10d
Dox.
a
IC values are the mean ± S.D. of three separate experiments.
5
0
As displayed in Table 3, sulfonamide 7d displayed potent anti-proliferative activities
under both normoxic and hypoxic conditions with IC values equal 3.32 ± 0.06 and 8.53 ±
5
0
0
.32µM, respectively, which are comparable to the reference drug doxorubicin (IC = 2.36 ±
50
0
.04 and 8.39 ± 0.25µM, respectively). Whereas, the sulfonamide 10d exhibited moderate
activity against MCF-7 cell line with IC values equal 7.72 ± 0.22 and 17.34 ± 0.66 µM
5
0
under normoxic and hypoxic conditions, respectively.
2
.2.3. Cell Cycle Analysis
In the current study, the sulfonamide 7d was examined for its effect on the cell cycle
progression in MCF-7 cells (Fig. 2). This impact was illustrated by DNA flow cytometric
1
0

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analysis; where MCF-7 cells were treated with compound 7d at its IC concentration (IC =
5
0
50
3
.32 µM).
Fig. 2. Effect of sulfonamide 7d on the phases of cell cycle of MCF-7 cells.
As displayed in Fig. 2, the assay outcomes highlighted that exposure of MCF-7 cells to
sulfonamide 7d resulted in a significant increase in the percentage of cells at Sub-G by more
1
than 10-folds, with concurrent significant arrest in the G -M phase by 14-folds in comparison
2
to the control.
2
.2.4. AnnexinV-FITC/Propidium Iodide Analysis of Apoptosis.
AnnexinV-FITC/PI dual staining assay was utilized to evaluate the impact of
sulfonamide 7d on both early and late apoptosis percentages in MCF-7 cells (Fig. 3, Table
).
4
1
1

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Control
7d
Fig. 3. Effect of sulfonamide 7d on the percentage of annexin V-FITC-positive staining in MCF-7
cells. The experiments were done in triplicates. The four quadrants identified as: LL, viable; LR,
early apoptotic; UR, late apoptotic; UL, necrotic.
As shown in Fig. 3, the AnnexinV assay results revealed that treatment of MCF-7 cells
with sulfonamide 7d led to a significant increase in the percentage of annexinV-FITC-
positive apoptotic cells for both the early (LR; from 1.05% to 4.29%) and late apoptotic (LR;
from 1.05% to 4.29%) phases, which represents more than tenfold total increase compared to
the control (Table 4).
Table 4. Distribution of apoptotic cells in the AnnexinV-FITC/PI dual staining assay in
MCF-7 cells.
Early Apoptosis
Late Apoptosis
(Upper Right %)
11.93
Total
(L.R % + U.R %)
16.22
Comp.
(
Lower Right %)
4.29
7
d
Control
1
.05
0.49
1.54
3
.
Molecular docking studies
Regarding the inhibitory activity of the sulfonamide 7d against both hCA IX and hCA
XII, docking simulations were performed in order to obtain a detailed explanation about the
method of its interaction with the active site of both isoforms. Molecular docking simulations
of the sulfonamide 7d inside the active site of hCA IX (PDB code 5FL4) confirmed the role
of the different parts of this compound in its binding with the receptor as discussed in the
introduction section. At first, the sulfonamide group played an important role as ZBG by
interacting with the catalytic zinc atom through a metallic bond, beside formation of H-bond
with Thr200. In addition, the carbonyl group within the hydrazide moiety accepted a H-bond
1
2

ACCEPTED MANUSCRIPT
from GLN92. The terminal imidazole moiety in its protonated state was able to form ionic
bond with carboxylate ion of Glu 137 (Fig. 4).
Fig. 4. Predicted binding orientations of compound 7d within the active site of CA IX (PDB code:
5
FL4). Hydrogen bonds and salt bridged are represented by red and green dashed lines, respectively.
Concerning docking simulations of compound 7d within hCA XII active site (PDB code
1
JD0), the sulfonamide group played the same role as in hCA IX. Here, we can find the
hydrazide linker did not show any interaction inside the active site, but the terminal
imidazole moiety stabilized the binding orientation by forming an anchoring π- π interaction
with Tyr20 as illustrated in Fig. 5.
Fig. 5. Predicted binding orientations of compound 7d within the active site of CA XII (PDB code:
1
JD0). Hydrogen bonds and π-π interactions are represented by red and black dashed lines,
respectively.
4
. Conclusions
In conclusion, we report here the design and synthesis of three series of novel
sulfonamides (5a-e, 7a-e and 10a-d) incorporating mixed hydrophilic/hydrophobic tails
linked to benzenesulfonamide (as zinc anchoring moieties) through hydrazide and
hydrazine linkers. The structures of the novel derivatives were confirmed by different
1
3

ACCEPTED MANUSCRIPT
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-e, 7a-e and 10a-d, in variable degrees with
the following K s ranges: 76.8–357.4 nM for hCA I, 8.2–94.6 nM for hCA II, 2.0–46.3 nM
I
for hCA XI, and 8.3–88.3 nM for hCA XII. Best activity was observed towards the tumor
associated isoform hCA IX, where compounds 7a-c and 10b-d emerged as single-digit
nanomolar hCA IX inhibitors (K s in the range of 2.0–8.9 nM). Besides, all the remaining
I
compounds, except 5e, were more potent than AAZ with K s in a range of 10.1–23.7 nM.
I
On the other hand, the sulfonamide 7d displayed potent anti-proliferative activity against
breast MCF-7 cancer cell line under both normoxic and hypoxic conditions (IC = 3.32 ±
5
0
0
.06 and 8.53 ± 0.32 µM, respectively), which is comparable to the reference drug
doxorubicin (IC = 2.36 ± 0.04 and 8.39 ± 0.25µM, respectively). Furthermore, 7d was
5
0
screened for cell cycle disturbance and apoptosis induction in MCF-7 cells. It was found to
persuade cell cycle arrest at G2-M stage as well as to alter the Sub-G1 phase, also, 7d
resulted in a significant increase in the percentage of annexinV-FITC positive apoptotic
cells from 1.03 to 18.54%. Finally, a molecular docking study for 7d was carried out within
the hCA IX and hCA XII active sites to rationalize the obtained inhibition results.
5
5
5
. Experimental
.1. Chemistry
.1.1. General
Melting points were determined using Stuart SMP10 digital melting point apparatus and
were uncorrected. Infrared (IR) Spectra were recorded as KBr disks using a Shimadzu FT-IR
400S infrared spectrophotometer. Mass spectra were measured as m/z (Intensity %) using
Thermo Scientific ISOLT mass spectroscopy at the Regional Center for Mycology and
8
1
13
Biotechnology.NMR spectra were recorded on a Bruker Ascend 400/ R ( H: 400, C: 100
1
13
MHz) spectrometer. H NMR spectra were run at 400 MHz and C spectra were run at 100
MHz in deuterated dimethylsulfoxide (DMSO-d ). Chemical shifts are expressed in δ values
6
(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 performed at the Regional Center for Mycology and Biotechnology, Al-Azhar
University, Egypt. Analytical thin layer chromatography (TLC) on silica gel pre-coated F254
1
4

ACCEPTED MANUSCRIPT
Merck plates containing UV indicator was employed routinely to follow the course of
reactions. Unless otherwise noted, all solvents and reagents were commercially available and
used without further purification. Synthesis and analytical data of compounds 3a-e [16], 4 [21],
6
[11] and 9a-d [16, 17] were found as reported.
5
.1.2. General procedure for the preparation of compounds 5a-e.
To a solution of the appropriate substituted aldehydes 3a-e (1 mmol) in absolute ethanol in
the presence of two drops of glacial acetic acid, 4-hydrazinobenzenesulfonamide hydrochloride
(0.223 g, 1 mmol) was added. The reaction mixture was heated under reflux for 3-6 hours.
4
The obtained solid was collected by filtration, dried and recrystallized from ethanol/DMF to
afford compounds 5a-e.
5
.1.2.1. 4-(2-(4-(pyrrolidin-1-yl)benzylidene)hydrazinyl)benzenesulfonamide (5a).
Purple
-1
crystals, (yield 61%), m.p. 236-239°C; IR (KBr, νmax / cm ): 3448, 3325, 3248 (NH & NH),
2
1
1
3
330 and 1153 (SO ); H NMR (DMSO-d )
δ
ppm: 1.96-1.97 (m, 4H, pyrrolidine Hs), 3.28-
2
6
.34 (m, 4H, pyrrolidine Hs), 6.57 (d, J = 8.6 Hz, 2H, Ar-H), 7.02 (s, 2H, D O exchangable, -
2
SO NH ), 7.07 (d, J = 8.7 Hz, 2H, Ar-H), 7.50 (d, J = 8.6 Hz, 2H, Ar-H), 7.63 (d, J = 8.8 Hz,
2
2
1
3
2
H, Ar-H), 7.84 (s, 1H, = C-H), 10.44 (s, 1H, D O exchangable, -NH); C NMR (DMSO-d ) δ
2 6
ppm: 25.40, 47.73 (pyrrolidine carbons), 111.07, 111.74, 112.09, 122.47, 127.90, 132.63,
+
1
5
41.11, 148.54, 148.71; ESI MS m/z: 344 [M] ; Anal. Calcd. for C H N O S (344.43): C,
1
7
20
4
2
9.28; H, 5.85; N, 16.27; Found C, 59.14; H, 6.01; N, 16.43.
5
.1.2.2. 4-(2-(4-(piperidin-1-yl)benzylidene)hydrazinyl)benzenesulfonamide (5b). Brick red
-1
powder, (yield 55%), m.p. 246-248°C; IR (KBr, νmax / cm ): 3402, 3325, 3236 (NH & NH),
2
1
1
276 and 1141 (SO ); H NMR (DMSO-d )
δ
ppm: 1.63-1.66 (m, 2H, piperidine Hs), 1.85-1.88
2
6
(
m, 4H, piperidine Hs), 3.44-3.46 (m, 4H, piperidine Hs), 7.08 (s, 2H, D O exchangable, -
2
SO NH ), 7.16 (d, J = 8.4 Hz, 2H, Ar-H), 7.59-7.75 (m, 6H, Ar-H), 7.95 (s, 1H, = C-H), 10.90
2
2
1
3
(
s, 1H, D O exchangable, -NH); C NMR (DMSO-d ) δ ppm: 22.00, 24.05, 54.50 (piperidine
2
6
carbons) 111.74, 120.53, 120.80, 127.70, 127.93, 133.97, 138.28, 144.50, 148.12; ESI MS m/z:
+
3
58 [M] ; Anal. Calcd. for C H N O S (358.46): C, 60.31; H, 6.19; N, 15.63; Found C,
18
22
4
2
6
0.48; H, 6.34; N, 15.46.
1
5

ACCEPTED MANUSCRIPT
.1.2.3. 4-(2-(4-morpholinobenzylidene)hydrazinyl)benzenesulfonamide (5c). Grey powder,
5
-1
(
yield 62%), m.p. > 300°C; IR (KBr, νmax / cm ): 3348, 3263, 3205 (NH & NH), 1327 and
2
1
1
4
161 (SO ); H NMR (DMSO-d )
δ
ppm: 3.18 (t, J = 4.7 Hz, 4H, morpholine Hs), 3.75 (t, J =
2
6
.7 Hz, 4H, morpholine Hs), 6.98 (d, J = 8.8 Hz, 2H, Ar-H), 7.04 (s, 2H, D O exchangable, -
2
SO NH ), 7.10 (d, J = 8.7 Hz, 2H, Ar-H), 7.56 (d, J = 8.7 Hz, 2H, Ar-H), 7.64 (d, J = 8.8 Hz,
2
2
1
3
2
H, Ar-H), 7.96 (s, 1H, = C-H), 10.57 (s, 1H, D O exchangable, -NH); C NMR (DMSO-d ) δ
2 6
ppm: 48.28, 66.45 (morpholine carbons), 111.28, 115.14, 126.31, 127.61, 127.92, 133.13,
+
1
5
40.10, 148.54, 151.74; ESI MS m/z: 360 [M] ; Anal. Calcd. for C H N O S (360.43): C,
1
7
20
4
3
6.65; H, 5.59; N, 15.54; Found C, 56.79; H, 5.67; N, 15.70.
5
.1.2.4. 4-(2-(4-(1H-imidazol-1-yl)benzylidene)hydrazinyl)benzenesulfonamide (5d). Yellow
-1
powder, (yield 61%), m.p. 270-272°C; IR (KBr, νmax / cm ): 3394, 3305, 3217 (NH & NH),
2
1
1
7
7
1
1
323 and 1141 (SO ); H NMR (DMSO-d )
δ
ppm: 7.13 (s, 2H, D O exchangable, -SO NH ),
2
6
2
2
2
.24 (d, J = 8.6 Hz, 2H, Ar-H), 7.69 (d, J = 8.6 Hz, 2H, Ar-H), 7.87 (d, J = 8.6 Hz, 2H, Ar-H),
.91-7.93 (m, 3H, Ar-H), 8.10 (s, 1H, = C-H), 8.34 (s, 1H, Ar-H), 9.79 (s, 1H, Ar-H), 11.37 (s,
1
3
H, D O exchangable, -NH); C NMR (DMSO-d ) δ ppm: 111.92, 121.06, 121.62, 122.66,
2
6
+
27.66, 127.91, 134.29, 134.84, 136.91, 137.52, 148.02; ESI MS m/z: 341 [M] ; Anal. Calcd.
for C H N O S (341.39): C, 56.29; H, 4.43; N, 20.51; Found C, 56.45; H, 4.61; N, 20.79.
1
6
15
5
2
5
.1.2.5. 4-(2-(4-(1H-benzo[d][1,2,3]triazol-1-yl)benzylidene)hydrazinyl)benzenesulfonamide
-1
(
5e). Canary yellow powder, (yield 45%), m.p. 230-232°C; IR (KBr, νmax / cm ): 3309, 3282,
1
3
232 (NH & NH), 1327 and 1153 (SO ); H NMR (DMSO-d )
δ
ppm: 7.11 (s, 2H, D O
2
2
6
2
exchangable, -SO NH ), 7.22 (d, J = 8.6 Hz, 2H, Ar-H), 7.53-7.57 (m, 1H, Ar-H), 7.71 (d, J =
2
2
8
1
1
1
1
2
.5 Hz, 2H, Ar-H), 7.95 (d, J = 8.5 Hz, 2H, Ar-H), 8.01 (d, J = 8.4 Hz, 2H, Ar-H), 8.05 (m,
H, Ar-H), 8.08 (s, 1H, =C-H), 8.22 (d, J = 8.4 Hz, 1H, Ar-H), 8.37 (d, J = 8.5 Hz, 1H, Ar-H),
1
3
0.99 (s, 1H, D O exchangable, -NH); C NMR (DMSO-d ) δ ppm: 111.54, 111.93, 118.63,
2
6
20.23, 121.02, 123.38, 125.33, 127.99, 129.31, 132.14, 134.38, 138.04, 144.99, 146.27,
+
48.03; ESI MS m/z: 392 [M] ; Anal. Calcd. for C H N O S (392.44): C, 58.15; H, 4.11; N,
19
16
6
2
1.42; Found C, 58.32; H, 4.27; N, 21.68.
5
.1.3. General procedure for the preparation of compounds 7a-e.
To a solution of the appropriate substituted aldehydes 3a-e (1 mmol) in glacial acetic acid,
-(hydrazinecarbonyl)benzenesulfonamide 6 (0.215 g, 1 mmol) was added. The reaction
4
1
6

ACCEPTED MANUSCRIPT
mixture was heated under reflux for 2-4 hours.. The solid formed was collected by filtration,
dried and recrystallized from ethanol/DMF to afford compounds 7a-e.
5
.1.3.1. 4-(2-(4-(Pyrrolidin-1-yl)benzylidene)hydrazine-1-carbonyl)benzenesulfonamide (7a).
-1
Yellowish brown powder, (yield 70%), m.p. > 300°C; IR (KBr, νmax / cm ): 3305, 3194, 3113
1
(
NH & NH), 1342 and 1161 (SO ); H NMR (DMSO-d )
δ
ppm: 1.96-1.98 (m, 4H, pyrrolidine
2
2
6
Hs), 3.28-3.31 (m, 4H, pyrrolidine Hs), 6.61 (d, J = 8.6 Hz, 2H, Ar-H), 7.51 (s, 2H, D O
2
exchangable, -SO NH ), 7.55 (d, J = 8.6 Hz, 2H, Ar-H), 7.95 (d, J = 8.3 Hz, 2H, Ar-H), 8.05
2
2
1
3
(
d, J = 8.3 Hz, 2H, Ar-H), 8.31 (s, 1H, = C-H), 11.67 (s, 1H, D O exchangable, -NH); C NMR
2
(
DMSO-d ) δ ppm: 25.43, 47.71 (pyrrolidine carbons), 112.06, 121.09, 126.17, 128.64, 129.24,
6
+
1
37.19, 146.81, 149.54, 150.11, 162.08 (C=O); ESI MS m/z: 372 [M] ; Anal. Calcd. for
C H N O S (372.44): C, 58.05; H, 5.41; N, 15.04; Found C, 58.23; H, 5.66; N, 15.28.
18
20
4
3
5
.1.3.2. 4-(2-(4-(Piperidin-1-yl)benzylidene)hydrazine-1-carbonyl)benzenesulfonamide (7b).
-1
Green powder, (yield 61%), m.p. 269-271°C; IR (KBr, νmax / cm ): 3379, 3305, 3267 (NH &
2
1
NH), 1338 and 1161 (SO ); H NMR (DMSO-d )
δ ppm: 1.54-1.62 (m, 6H, piperidine Hs),
2
6
3
.17-3.32 (m, 4H, piperidine Hs), 6.99 (d, J = 8.7 Hz, 2H, Ar-H), 7.52 (s, 2H, D O
2
exchangable, -SO NH ), 7.57 (d, J = 8.6 Hz, 2H, Ar-H), 7.95 (d, J = 8.4 Hz, 2H, Ar-H), 8.06
2
2
1
3
(
d, J = 8.3 Hz, 2H, Ar-H), 8.33 (s, 1H, = C-H), 11.75 (s, 1H, D O exchangable, -NH); C NMR
2
(
DMSO-d ) δ ppm: 24.39, 25.42, 48.81 (piperidine carbons), 115.01, 123.50, 126.19, 128.68,
6
+
1
29.03, 137.07, 146.89, 149.50, 152.97, 162.22 (C=O); ESI MS m/z: 386 [M] ; Anal. Calcd.
for C H N O S (386.47): C, 59.05; H, 5.74; N, 14.50; Found C, 58.89; H, 5.87; N, 14.71.
1
9
22
4
3
5
.1.3.3. 4-(2-(4-Morpholinobenzylidene)hydrazine-1-carbonyl)benzenesulfonamide (7c). Buff
-1
powder, (yield 85%), m.p. 290-292°C; IR (KBr, νmax / cm ): 3417, 3317, 3205 (NH & NH),
2
1
1
3
342 and 1165 (SO ); H NMR (DMSO-d )
δ
ppm: 3.22-3.25 (m, 4H, morpholine Hs), 3.73-
2
6
.76 (m, 4H, morpholine Hs), 7.03 (d, J = 8.6 Hz, 2H, Ar-H), 7.52 (s, 2H, D O exchangable, -
2
SO NH ), 7.61 (d, J = 8.5 Hz, 2H, Ar-H), 7.95 (d, J = 8.2 Hz, 2H, Ar-H), 8.06 (d, J = 8.3 Hz,
2
2
1
3
2
H, Ar-H), 8.35 (s, 1H, = C-H), 11.79 (s, 1H, D O exchangable, -NH); C NMR (DMSO-d ) δ
2 6
ppm: 47.87, 66.41 (morpholine carbons), 114.81, 124.70, 126.20, 128.70, 128.94, 137.02,
+
1
46.92, 149.33, 152.81, 162.31 (C=O); ESI MS m/z: 388 [M] ; Anal. Calcd. for C H N O S
1
8
20
4
4
(388.44): C, 55.66; H, 5.19; N, 14.42; Found C, 55.87; H, 5.31; N, 14.70.
5
.1.3.4. 4-(2-(4-(1H-Imidazol-1-yl)benzylidene)hydrazine-1-carbonyl)benzenesulfonamide (7d).
-1
White powder, (yield 56%), m.p. > 300°C; IR (KBr, νmax / cm ): 3280, 3221, 3136 (NH &
2
1
7

ACCEPTED MANUSCRIPT
1
NH), 1307 and 1165 (SO ); H NMR (DMSO-d )
δ ppm: 7.15 (s, 1H, Ar-H), 7.54 (s, 2H, D O
2
6
2
exchangable, -SO NH ), 7.80 (d, J = 8.3 Hz, 2H, Ar-H), 7.85 (s, 1H, Ar-H), 7.90 (d, J = 8.4 Hz,
2
2
2
8
1
1
5
H, Ar-H), 7.98 (d, J = 8.2 Hz, 2H, Ar-H), 8.10 (d, J = 8.2 Hz, 2H, Ar-H), 8.38 (s, 1H, Ar-H),
1
3
.51 (s, 1H, = C-H), 12.08 (s, 1H, D O exchangable, -NH); C NMR (DMSO-d ) δ ppm:
2
6
18.30, 120.89, 126.26, 128.84, 129.15, 130.60, 133.02, 136.02, 136.72, 138.48, 147.17,
+
47.92, 162.70 (C=O); ESI MS m/z: 369 [M] ; Anal. Calcd. for C H N O S (369.40): C,
1
7
15
5
3
5.28; H, 4.09; N, 18.96; Found C, 55.46; H, 4.32; N, 19.21.
5
.1.3.5. 4-(2-(4-(1H-Benzo[d][1,2,3]triazol-1-yl)benzylidene)hydrazine-1-carbonyl)benzene
-1
sulfonamide (7e). Yellowish white powder, (yield 87%), m.p. > 300°C; IR (KBr, νmax / cm ):
1
3
417, 3344, 3244 (NH & NH), 1334 and 1161 (SO ); H NMR (DMSO-d )
δ
ppm: 7.56-7.58
2
2
6
(
m, 4H, Ar-H and D O exchangable, -SO NH ), 7.72 (t, J = 7.6 Hz, 1H, Ar-H), 7.99 (d, J = 8.0
2 2 2
Hz, 2H, Ar-H), 8.03-8.13 (m, 3H, Ar-H), 8.12 (d, J = 8.1 Hz, 2H, Ar-H), 8.22 (d, J = 8.3 Hz,
1
H, Ar-H), 8.44 (d, J = 8.4 Hz, 1H, Ar-H), 8.60 (s, 1H, = CH), 12.18 (s, 1H, D O exchangable,
2
1
3
-
NH); C NMR (DMSO-d ) δ ppm: 111.60, 118.71, 120.29, 121.10, 123.28, 125.42, 126.29,
6
1
28.47, 128.89, 129.27, 132.03, 134.80, 136.66, 137.91, 145.07, 146.33, 147.68, 162.78
+
(
C=O); ESI MS m/z: 420 [M] ; Anal. Calcd. for C H N O S (420.45): C, 57.13; H, 3.84; N,
20
16
6
3
1
9.99; Found C, 57.40; H, 4.02; N, 20.16.
5
.1.4. General procedure for the preparation of compounds 10a-d.
To a solution of the appropriate substituted acetophenones 9a-d (1 mmol) in absolute
ethanol in the presence of catalytic amount of glacial acetic acid, 4-
hydrazinobenzenesulfonamide hydrochloride (0.223 g, 1 mmol) was added. The reaction
mixture was heated under reflux for 6-8 hours.. The solid formed was collected by filtration,
dried and recrystallized from ethanol/DMF to afford compounds 10a-d.
5
.1.4.1. 4-(2-(1-(4-(Pyrrolidin-1-yl)phenyl)ethylidene)hydrazinyl)benzenesulfonamide (10a).
-1
Yellowish brown powder, (yield 45%), m.p. 291-292°C; IR (KBr, νmax / cm ): 3479, 3336,
1
3
259 (NH & NH), 1315 and 1145 (SO ); H NMR (DMSO-d )
δ
ppm: 1.97-2.00 (m, 4H,
2
2
6
pyrrolidine Hs), 2.24 (s, 3H, CH ), 3.29 (m, 4H, pyrrolidine Hs), 6.63 (d, J = 8.3 Hz, 2H, Ar-
3
H), 7.05 (s, 2H, D O exchangable, -SO NH ), 7.26 (d, J = 8.7 Hz, 2H, Ar-H), 7.65 (d, J = 8.7
2
2
2
1
3
Hz, 2H, Ar-H), 7.69 (d, J = 8.6 Hz, 2H, Ar-H), 9.53 (s, 1H, D O exchangable, -NH); C NMR
2
(
DMSO-d ) δ ppm: 13.48 (-CH ), 25.35, 26.32, 47.71, 47.99 (pyrrolidinecarbons),111.14,
6 3
1
12.01, 113.72, 124.57, 126.30, 127.49, 130.84, 133.09, 136.76, 145.25, 147.97, 149.43,
1
8

ACCEPTED MANUSCRIPT
+
1
1
51.18; ESI MS m/z: 358 [M] ; Anal. Calcd. for C H N O S (358.46): C, 60.31; H, 6.19; N,
18
22
4
2
5.63; Found C, 60.58; H, 6.22; N, 15.80.
5
.1.4.2. 4-(2-(1-(4-(Piperidin-1-yl)phenyl)ethylidene)hydrazinyl) benzenesulfonamide (10b).
-1
Black crystals, (yield 57%), m.p. 240-242°C; IR (KBr, νmax / cm ): 3421, 3197, 3132 (NH &
2
1
NH), 1330 and 1157 (SO ); H NMR (DMSO-d )
δ ppm: 1.59-1.71 (m, 4H, piperidine Hs),
2
6
2
7
7
.43 (s, 3H, CH ), 3.29-3.44 (m, 6H, piperidine Hs), 7.18 (s, 2H, D O exchangable, -SO NH ),
3 2 2 2
.47 (d, J = 8.8 Hz, 2H, Ar-H), 7.58 (d, J = 8.7 Hz, 2H, Ar-H), 7.79 (d, J = 8.3 Hz, 2H, Ar-H),
1
3
.85 (d, J = 8.3 Hz, 2H, Ar-H) , 8.86 (s, 1H, D O exchangable, -NH); C NMR (DMSO-d ) δ
2
6
ppm: 24.32 (-CH ), 25.20, 26.52, 48.54 (piperidinecarbons), 113.72, 125.50, 126.04, 127.15,
3
+
1
29.39, 130.03, 130.67, 132.25, 141.49, 142.67, 143.10, 144.60; ESI MS m/z: 372 [M] ; Anal.
Calcd. for C H N O S (372.49): C, 61.27; H, 6.49; N, 15.04; Found C, 61.51; H, 6.37; N,
19
24
4
2
1
5.29.
5
.1.4.3. 4-(2-(1-(4-Morpholinophenyl)ethylidene)hydrazinyl)benzenesulfonamide (10c). Brown
-1
powder, (yield 50%), m.p. 255-257°C; IR (KBr, νmax / cm ): 3394, 3336, 3275 (NH & NH),
2
1
1
334 and 1149 (SO ); H NMR (DMSO-d )
δ
ppm: 2.25 (s, 3H, CH ), 3.18 (t, J = 4.3 Hz, 4H,
2
6
3
morpholine Hs), 3.77 (t, J = 4.4 Hz, 4H, morpholine Hs), 7.00 (d, J = 8.7 Hz, 2H, Ar-H), 7.05
s, 2H, D O exchangable, -SO NH ), 7.29 (d, J = 8.7 Hz, 2H, Ar-H), 7.66 (d, J = 8.7 Hz, 2H,
(
2
2
2
1
3
Ar-H), 7.72 (d, J = 8.7 Hz, 2H, Ar-H), 9.60 (s, 1H, D O exchangable, -NH); C NMR (DMSO-
2
d ) δ ppm: 13.58 (-CH ), 49.87, 65.89 (morpholine carbons), 112.30, 113.51, 116.53, 126.99,
6
3
+
1
27.68, 130.50, 132.35, 133.68, 143.74, 149.18; ESI MS m/z: 374 [M] ; Anal. Calcd. for
C H N O S (374.46): C, 57.74; H, 5.92; N, 14.96; Found C, 58.01; H, 5.87; N, 15.12.
18
22
4
3
5
.1.4.4. 4-(2-(1-(4-(4-Methylpiperazin-1-yl)phenyl)ethylidene)hydrazinyl)benzenesulfonamide
-
1
(
10d). Buff powder, (yield 46%), m.p. 281-283°C; IR (KBr, νmax / cm ): 3421, 3282, 3205
1
(
NH & NH), 1330 and 1138 (SO ); H NMR (DMSO-d )
δ
ppm: 2.26 (s, 3H, CH ), 2.77 (s,
2
2
6
3
3
H, CH ), 3.34-3.46 (m, 8H, piperazine Hs), 7.02 (d, J = 8.6 Hz, 2H, Ar-H), 7.06 (s, 2H, D O
3 2
exchangable, -SO NH ), 7.30 (d, J = 8.6 Hz, 2H, Ar-H), 7.66 (d, J = 8.6 Hz, 2H, Ar-H), 7.73
2
2
1
3
(
d, J = 8.5 Hz, 2H, Ar-H), 9.64 (s, 1H, D O exchangable, -NH); C NMR (DMSO-d ) δ ppm:
2
6
1
1
3.60, 19.02 (2 -CH ), 42.54, 45.63, 52.49, 56.49 (piperazine carbons), 112.21, 115.78,
3
+
26.96, 127.68, 130.77, 133.58, 143.85, 149.25, 149.82; ESI MS m/z: 387 [M] ; Anal. Calcd.
for C H N O S (387.50): C, 58.89; H, 6.50; N, 18.07; Found C, 59.12; H, 6.37; N, 18.40.
1
9
25
5
2
5
.2. Biological Evaluation
1
9

ACCEPTED MANUSCRIPT
5
.2.1. CA inhibitory assay
An Applied Photophysics stopped-flow instrument has been used for assaying the CA
catalysed CO hydration activity, as reported earlier [18]. The inhibition constants were
2
obtained by non-linear least-squares methods using PRISM 3 and the Cheng-Prusoff equation
as reported earlier [22,23], and represent the mean from at least three different
determinations. The four tested CA isofoms were recombinant ones obtained in-house as
reported earlier [24-26].
5
.2.2. Anti-proliferative activity towards MCF-7 breast cancer cell line
Breast cancer MCF-7 cell line was obtained from American Type Culture Collection
(ATCC). The cells were propagated in DMEM supplemented with 10% heat-inactivated fetal
bovine serum, 1% L-glutamine (2.5 mM), HEPES buffer (10 mM), 50 µg/mL gentamycin.
The hypoxia inducer CoCl (100 µM) was in case of the hypoxic condition. All cells were
2
maintained at 37 C in a humidified atmosphere with 5% CO . Cytotoxicity was determined
2
following the MTT assay, as reported earlier [20, 27].
5
.2.3. Cell cycle analysis
MCF-7 cells were treated with sulfonamide 7d for 24 h at its IC concentration then cells
5
0
were washed with ice-cold phosphate buffered saline (PBS). The treated cells were collected
by centrifugation, fixed in ice-cold 70% (v/v) ethanol, washed with PBS, re-suspended with
1
00 µg/mL RNase, stained with 40 µg/mL PI, and analyzed by flow cytometry using FACS
Calibur (Becton Dickinson, BD, USA). The cell cycle distributions were calculated using
CellQuest software 5.1 (Becton Dickinson) [28, 29].
5
.2.4. Annexin V-FITC apoptosis assay
Phosphatidylserine externalization was assayed using Annexin V-FITC/PI apoptosis
detection kit (BD Biosciences, USA) according to the manufacturer’s instructions, as reported
earlier [28, 29].
5
.3. Computational studies
The crystal structures of hCA IX (PDB 5FL4) [30] and hCA XII (PDB 1JD0) [31] were
obtained from protein databank and were prepared according to the Protein Preparation wizard
in Maestro - Schrödinger suite 2017-1 [32] in order to adding missing atoms by Prime, assining
the protonation states, optimization of the hydrogen bonds and refinement process by running a
restrained minimization (OPLS3 force field). 3D ligand structures were drawn and prepared
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minimized using LigPrep [33] in order to predict ionization states for the ligands using a pH of
7
.0 ± 0.5. Before docking process, the grid box was prepared at size 20 Å. Zn atom was chosen
as a metal constrained. In docking simulations were done using Glide, the protein was treated
as rigid, compounds were flexibly docked, and scoring was assigned according to the xtra
precision (XP) mode.
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[
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Highlights
-
-
-
-
-
Three sets of novel benzenesulfonamides (5a-e, 7a-e and 10a-d) were designed and synthesized.
Inhibitory activity of the prepared sulfonamides was evaluated toward hCA I, II, IX and XII.
hCA IX was efficiently inhibited by all sulfonamides with K s in the range of 2.0–46.3 nM.
I
Antiproliferative and proapoptotic activities towards breast cancer MCF-7 cells were examined.
Docking was done to provide insights in the binding interactions of the reported sulfonamides.