Novel diamide-based benzenesulfonamides as selective carbonic anhydrase ix inhibitors endowed with antitumor activity: Synthesis, biological evaluation and in silico insights

Article abstract of DOI:10.3390/ijms20102484

In this work, we present the synthesis and biological evaluation of novel series of diamide-based benzenesulfonamides 5a-h as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) isoforms hCA I, II, IX and XII. The target tumor-associated i

Full text of DOI:10.3390/ijms20102484

International Journal of
Molecular Sciences
Article
Novel Diamide-Based Benzenesulfonamides as
Selective Carbonic Anhydrase IX Inhibitors Endowed
with Antitumor Activity: Synthesis, Biological
Evaluation and In Silico Insights
Mohamed A. Abdelrahman 1, Wagdy M. Eldehna 2,*, Alessio Nocentini 3,4, Silvia Bua 3
Sara T. Al-Rashood 5, Ghada S. Hassan 6 , Alessandro Bonardi 3,4
,
,
Abdulrahman A. Almehizia 5 , Hamad M. Alkahtani 5, Amal Alharbi 5, Paola Gratteri 4 and
Claudiu T. Supuran 3,
*
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Egyptian Russian University, Badr City,
Cairo 11829, Egypt; mohamed.ashraf.eru@gmail.com
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kafrelsheikh University, Kafrelsheikh 33516,
Egypt
Department of NEUROFARBA, Section of Pharmaceutical and Nutraceutical Sciences, University of
Florence, Polo Scientifico, Via U. Schiff 6, Sesto Fiorentino, 50019 Firenze, Italy;
alessio.nocentini@unifi.it (A.N.); silvia.bua@unifi.it (S.B.); alessandro.bonardi@unifi.it (A.B.)
Department of NEUROFARBA, Section of Pharmaceutical and Nutraceutical Sciences, Laboratory of
Molecular Modeling Cheminformatics & QSAR, University of Florence, Polo Scientifico, Via U. Schiff 6, Sesto
Fiorentino, 50019 Firenze, Italy; paola.gratteri@unifi.it
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh
11451, Saudi Arabia; salrashood@ksu.edu.sa (S.T.A.-R.); mehizia@ksu.edu.sa (A.A.A.);
haalkahtani@ksu.edu.sa (H.M.A.); amal.harbi@gmail.com (A.A.)
2
3
4
5
6
Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt;
ghadak25@yahoo.com
*
Correspondence: wagdy2000@gmail.com (W.M.E.); claudiu.supuran@unifi.it (C.T.S.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 29 April 2019; Accepted: 17 May 2019; Published: 20 May 2019
Abstract: In this work, we present the synthesis and biological evaluation of novel series of
diamide-based benzenesulfonamides 5a–h as inhibitors of the metalloenzyme carbonic anhydrase
(CA, EC 4.2.1.1) isoforms hCA I, II, IX and XII. The target tumor-associated isoforms hCA IX and
XII were undeniably the most affected ones (KIs: 8.3–123.3 and 9.8–134.5 nM, respectively). Notably,
diamides 5a and 5h stood out as a single-digit nanomolar hCA IX inhibitors (KIs = 8.8 and 8.3 nM).
The SAR outcomes highlighted that bioisosteric replacement of the benzylidene moiety, compounds
5a–g, with the hetero 2-furylidene moiety, compound 5h, achieved the best IX/I and IX/II selectivity
herein reported with SIs of 985 and 13.8, respectively. Molecular docking simulations of the prepared
diamides within CA IX active site revealed the ability of 5h to establish an additional H-bond between
the heterocyclic oxygen and HE/Gln67. Moreover, benzenesulfonamides 5a, 5b and 5h were evaluated
for their antitumor activity against renal cancer UO-31 cell line. Compound 5h was the most potent
derivative with about 1.5-fold more enhanced activity (IC50 = 4.89
±
0.22 µM) than the reference drug
Staurosporine (IC50 = 7.25 0.43 M). Moreover, 5a and 5h were able to induce apoptosis in UO-31
±
µ
cells as evidenced by the significant increase in the percent of annexinV-FITC positive apoptotic cells
by 22.5- and 26.5-folds, respectively.
Keywords: anticancer activity; diamide-based benzenesulfonamides; molecular docking; selective
hCAIX inhibitors; synthesis
Int. J. Mol. Sci. 2019, 20, 2484; doi:10.3390/ijms20102484
www.mdpi.com/journal/ijms

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1. Introduction
Carbonic anhydrases (CA) are zinc metalloenzymes that play a pivotal role in most living
organisms catalyzing the interconversion of carbon dioxide and water to bicarbonate and protons [ ].
Seven distinct CA families ( -, -, -, -, -, - and -CAs) are known to date [ ]. Human encode
15 different -CA isoforms among which some are cytosolic (CA I, CA II, CA III, CA VII and CA
XIII), others are membrane bound (CA IV, CA IX, CA XII, CA XIV and CA XV), two of them are
mitochondrial (CA VA and CA VB), and one isozyme is secreted in saliva (CA VI) [ ]. They are
involved in numerous physiological and pathological processes such as gluconeogenesis, lipogenesis,
ureagenesis and tumorigenicity [ ]. While inhibitors targeting hCA I are useful in retinal and cerebral
1
α
β
γ
δ
ζ
η
θ
1,2
α
2
2
edema, inhibitors targeting hCA II are used as diuretics, in the management of edema, as antiglaucoma
agents, antiepileptic drugs, and also for the treatment of altitude sickness [1,2].
The hCA IX expression is usually induced by hypoxia in certain types of solid tumors, such as
glioma, breast cancer and colon carcinoma [
with remarkable suppression of the growth of both primary tumor stages as well as metastases which
makes the enzyme a validated target for the treatment of diverse cancers [ ]. Accordingly, discovery of
3,4]. Besides, inhibition of hCA IX was strongly associated
5
isoform hCA IX selective inhibitors stands out as a crucial step to establish a promising cancer therapy
devoid of the classical adverse effects owing to isoforms hCA I and II inhibition.
While different CA inhibition mechanisms are known, the zinc binder sulfonamides undoubtedly
represent the most important class, with many derivatives in clinical use for decades or clinical
development in the last period, such as SLC-0111 (Figure 1) [6]. The CA inhibition mechanism with
sulfonamides is mainly mediated by coordination of its deprotonated form (SO2NH–) to the positively
charged Zn(II) ion from the enzyme active site. Moreover, the sulfonamide moiety engages two
H-bonds: the NH– group acts as donor, while the S=O as acceptor with T199 OG1 atom and backbone
NH respectively. These binding features are common among the active site architectures for all the
α-class fifteen human isozymes.
Figure 1. Structures of some reported benzenesulfonamides-based CAIs (
compounds (5a–g and 5h).
I–IV) and the target
Several approaches have been adopted for obtaining isoform-selective CAIs, with the “tail
approach” being the most successful and exploited one (Figure 1) [ ]. It consists in appending
7–9
“tails”, with a diverse chemical nature, to the aromatic/heterocyclic ring possessing the zinc binding
sulfonamide group, in order to interact with amino acid residues from the middle and the rim parts
of the active site cavity. In this context, several sulfonamide-based derivatives were successfully
developed as isoform hCA IX selective inhibitors through utilization of “tail approach” (compounds
Figure 1II–IV) [7–9].

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Pursuing on our effort towards development of selective hCA IX inhibitors [10
–
13], herein we
present the synthesis, and biological evaluation of novel series of diamide-based benzenesulfonamides
5a–h. The herein reported diamides possess a zinc anchoring moiety of the benzenesulfonamide type
that was linked to benzylidene tails incorporating halogen, methyl or methoxy substituents to ensure
suited SAR exploration regarding the hydrophobic region of the binding cleft. Also, the bioisosteric
replacement approach was adopted to replace the benzylidene tails (compounds 5a–g) with the hetero
2-furylidene one (compound 5h, Figure 1). The amide functionality of the appended benzamide
moiety was expected to engage contact with the amino acids residues of the “hydrophilic” region of
the active site.
All the newly-synthesized diamides 5a–h were characterized and biologically tested against a
panel of hCA I, II, IX and XII isoforms, using stopped-flow CO2 hydrase assay. Moreover, diamides
5a, 5b and 5h were evaluated for their anti-proliferative activity against renal cancer UO-31 cell
line. Compound 5h was further investigated for its apoptosis induction potential in UO-31 cells.
Furthermore, molecular docking investigations were carried out to provide insights for the binding
interactions of the herein reported benzenesulfonamides within hCA isozymes II and IX.
2. Results and Discussion
2.1. Chemistry
The synthetic pathway adopted for the preparation of the target diamide-based benzenesulfonamides
5a–h was depicted in Scheme 1. The azlactone intermediates 3a–h were prepared by the Erlenmeyer–
Plöchl synthesis [14,15] through reaction of hippuric acid 1 with different benzaldehyde derivatives
2a–g or 5-methylfurfural 2h in acetic anhydride in the presence of Hünig’s base [16]. Then, the target
diamide-based benzenesulfonamides 5a–h were obtained via reaction of azlactone intermediates 3a–h
with p-aminobenzenesulfonamide 4 in glacial acetic acid in the presence of sodium acetate to afford the
corresponding target benzenesulfonamides 5a–h with 80–93% yield (Scheme 1).
Scheme 1. Synthesis of diamide-based benzenesulfonamides 5a–h; Reagents and conditions: (i) Acetic
anhydride/Hünig’s base/reflux 3 hrs., (ii) Glacial acetic acid/CH3COONa/reflux 4 h.

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The elemental and spectral data supported the structures of the target benzenesulfonamides
5a–h. IR spectra of 5a–h revealed the presence of bands for (NH2) group at 3316–3249 cm−1, and
bands of (C=O) group around 1629–1636 cm−1. Also, their IR spectra displayed two bands of (SO2) at
(1350–1358) and (1157–1168) cm−1. Furthermore, the 1H NMR spectra of benzenesulfonamides 5a–h
displayed singlet signal of D2O exchangeable NH2 protons of salfamoyl group at
In addition to presence of olifenic signal at 7.04–7.32 ppm, and two D2O exchangeable NH protons
at 9.85–10.16 ppm and 10.23–10.65 ppm. On the other hand, compounds 5d–g were confirmed by
presence of aliphatic signals of; CH3 protons at 2.28–2.36 ppm for 5d, OCH3 protons at 3.74–3.83 ppm
for 5e–g. Moreover, 13C NMR spectra of compounds 5a–h displayed two signals for C=O groups
at
164.52–169.74 ppm, also, 13C NMR spectra for 5d–g displayed the aliphatic signals of CH3 at
δ 7.25–7.49 ppm.
δ
δ
δ
δ
δ
δ
δ
21.38–21.75 ppm for 5d, and OCH3 at δ 55.52–56.11 ppm for 5f and 5g.
2.2. Biological Evaluation
2.2.1. Carbonic Anhydrase Inhibition
The CA inhibitory activity of all the newly prepared diamide-based benzenesulfonamides 5a–h
was evaluated towards the cytosolic isoforms hCA I and II, as well as towards the transmembrane
tumor-associated isoforms hCA IX and XII using an applied photophysics stopped-flow instrument
for assaying the CA-catalyzed CO2 hydration activity [17]. The inhibitory activities were compared to
acetazolamide (AAZ), a clinically used standard CA inhibitor. The following SAR could be derived
from the results in Table 1:
Table 1. Inhibition data of human CA isoforms hCA I, II, IX and XII for diamide-based
benzenesulfonamides 5a–h, determined by stopped-flow CO2 hydrase assay, using acetazolamide
(AAZ) as a standard drug.
KI (nM)*
Comp.
R1
R2
R
hCA I
hCA II
hCA IX
hCA XII
5a
5b
5c
5d
5e
5f
5g
5h
AAZ
H
Cl
H
H
H
OCH3
H
-
-
H
H
H
H
H
H
OCH3
Cl
Cl
Br
CH3
OCH3
OCH3
OCH3
-
3955.7
5977.6
2397.8
796.1
1006.4
5132.7
2207.7
8175.4
250.0
68.3
223.9
251.9
94.4
127.7
294.2
448.0
114.8
12.0
8.8
18.3
33.5
62.1
78.0
73.7
123.3
8.3
16.1
10.5
55.4
60.2
42.8
134.5
9.8
-
-
57.9
5.7
-
25.0
* Mean from 3 different assays, by a stopped flow technique (errors were in the range of
reported values).
±
5–10 % of the
(i) The cytosolic isoform hCA I was weekly inhibited by most of the diamide-based
benzenesulfonamides 5a–h with inhibition constants (KIs) in the micromolar range, in detail, between
1.006 and 8.175
µM, except for the p-tolyl analogue 5d which arose as a nanomolar hCA I inhibitor with
a KI equals 796.1 nM, that represents 3-fold decreased activity to the reference drug AAZ (KI = 250 nM).

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(ii) The physiologically dominant isoform hCA II was moderately inhibited by the target diamides
5a–h with KIs spanning in the nanomolar range: 68.3–448 nM, Table 1. The SAR outcomes hinted
that mono substitution of the benzylidene moiety with 4-Cl or 4-CH3 substituents (compounds 5a
and 5d; KIs = 68.3 and 94.4 nM, respectively) is more beneficial for the activity against hCA II than
di-substitution with Cl or OCH3 substituents (compounds 5b, 5f and 5g; KIs = 223.9, 294.2 and 448.0 nM,
respectively).
(iii) The target tumor-associated isoform hCA IX was efficiently inhibited by the prepared
diamide-based benzenesulfonamides 5a–h (KI values ranging between 8.3 and 78 nM, Table 1), apart
from the 3,4-(OCH3)2 benzylidene-bearing sulfonamide 5g, which possessed a slightly reduced
inhibitory activity (KI = 123.3 nM). Notably, diamides 5a and 5h stood out as a single-digit nanomolar
hCA IX inhibitors with KI values = 8.8 and 8.3 nM, respectively, which are about three times more
potent than the standard drug AAZ (KI = 25 nM). Besides, diamide 5b (KI = 18.3 nM) was 1.4 times
more active than AAZ.
Regarding the impact of substitution of the benzylidene moiety, the hCA IX inhibitory activities
were decreased in the following order: 4-Cl > 2,4-(Cl)2 > 4-Br > 4-CH3 > 2,4-(OCH3)2 > 4-OCH3
> 3,4-(OCH3)2. This order of activity highlighted that incorporation of the electron-withdrawing
halogens (compounds 5a–c; KIs: 8.8–33.5 nM) is more advantageous for the inhibitory activity toward
hCA IX than the electron-donating methyl or methoxy groups (compounds 5d–g; KIs: 62.1–123.3 nM).
It is noteworthy that bioisosteric replacement of the substituted benzylidene moiety (compounds
5a–g; KIs: 8.8–123.3 nM) with a hetero 2-furylidene moiety (compound 5h; KI = 8.3 nM) resulted in a
1.1-fold to 14.9-fold efficacy enhancement, which could be attributed to an extra hydrogen bonding
within the active site through the heterocyclic oxygen atom.
(iv) The data displayed in Table 1 ascribed to the prepared diamide-based benzenesulfonamides
good efficacy in inhibiting the transmembrane tumor-associated isoform hCA XII. The inhibition
profiles were found to be rather flat, since the measured KIs ranged between 9.8 and 60.2 nM, aside
from diamide 5f whose efficacy raised to slightly higher concentration (KI = 134.5 nM). In particular,
diamide 5g was emerged as the only single-digit nanomolar hCA XII inhibitor in this study (KI =
9.8 nM).
Concerning the effect of substitution of the benzylidene moiety, the hCA XII inhibitory activities
were decreased in the order of: 3,4-(OCH3)2 > 2,4-(Cl)2 > 4-Cl > 4-OCH3 > 4-Br > 4-CH3 > 2,4-(OCH3)2.
It is worth stressing that, that bioisosteric replacement of the substituted benzylidene moiety with the
2-furylidene moiety elicited a worsening of effectiveness against hCA XII, unlike arose for hCA IX.
(v) Exploration of the the CA inhibitory trends in Table 1 revealed that all the examined
diamide-based benzenesulfonamides 5a–h displayed interesting selectivity towards hCA IX over hCA
I spanning in the range 12.8–985 (Table 2). Also, the examined diamides possessed good hCA IX/II
selectivity indexes spanning in the range 3.6–13.8, apart from diamides 5d and 5e which displayed
modest selectivity (SI = 1.4 and 1.5, respectively).
Table 2. Selectivity ratios for the inhibition of hCA IX and XII over hCA I and II for diamide-based
benzenesulfonamides 5a–h and acetazolamide.
Cmpd
I/IX
II/IX
I/XII
II/XII
5a
5b
5c
5d
5e
5f
5g
5h
AAZ
449.5
326.6
71.6
12.8
12.9
69.9
17.9
985
7.8
12.2
7.5
1.5
1.4
4
3.6
13.8
0.5
245.7
569.2
43.3
13.2
23.5
4.2
21.3
4.5
1.7
3
2.2
45.7
2
38.2
225.3
141.2
43.9
10.0
2.2

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Indeed, it is worth highlighting that bioisosteric replacement of the benzylidene moiety with the
hetero 2-furylidene moiety, compound 5h, achieved the best IX/I and IX/II selectivity herein reported
with SIs of 985 and 13.8, respectively.
2.2.2. Antitumor Activity
Antitumor Activity towards 60 Cancer Cell Lines (NCI, USA)
First, all diamide-based benzenesulfonamides herein reported 5a–h were screened for their
antitumor activity at one dose (10−5 M) assay against a panel of sixty cancer cell lines through the
National Cancer Institute (NCI) Developmental Therapeutic Program (www.dtp.nci.nih.gov), according
to US-NCI protocol. The cell growth and cell viability were evaluated using the sulforhodamine B
(SRB) colorimetric assay [18–20].
The obtained data were reported as mean-graph of the percentage growth of the different treated
tumour cells (Supplementary Materials). Exploration of results for this assay confirmed that the
examined diamide-based benzenesulfonamides 5a–h had non-significant activity towards most NCI
cancer cell lines. Of particular interest, all the prepared diamides displayed interesting selectivity
towards renal cancer UO-31 cell line with percentage growth inhibition (GI%) range of 13–35% (Figure 2).
Compound 5h was found to be the most potent member against UO-31 cells, in this assay, with GI%
equals 35.
40
35
30
25
20
15
10
5
0
5a
5b
5c
5d
5e
5f
5g
5h
Figure 2. Percentage growth inhibition (GI%) for the target diamide-based benzenesulfonamides 5a–h
towards the renal cancer UO-31 cell line.
Anti-Proliferative Activity towards Renal Cancer UO-31 Cell Line
As diamide-based benzenesulfonamides 5a, 5b and 5h emerged as low-nanomolar hCA IX
inhibitors (KIs: of 8.3–18.3 nM) with good hCA IX/I and IX/II selectivity (SI: 326.6–985 and 7.8–13.8,
respectively), they were selected to be evaluated for their antitumor activity against renal cancer UO-31
cell line, using the protocol of MTT colorimetric assay as reported by T. Mosmann [21]. Staurosporine
was adopted in the experiment as a reference anticancer drug. The results are presented as IC50 values
and displayed in Table 3.

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Table 3. In vitro anti-proliferative activity of diamide-based benzenesulfonamides 5a, 5b and 5h against
renal UO-31 cancer cell line.
IC50 (µM) a
Compound
UO-31
5a
5b
5h
6.53 ± 0.38
16.68 ± 0.92
4.89 ± 0.22
7.25 ± 0.43
Staurosporine
a
IC50 values are the mean ± S.D. of three separate experiments.
The results for the MTT assay presented in Table 3 revealed that that the examined diamides
possessed good anti-proliferative activity towards UO-31 cell line (IC50 range: 4.89 0.22–16.68
0.92 M). Notably, diamide-based sulfonamide 5h was the most potent derivative with about 1.5-fold
more enhanced activity (IC50 = 4.89 0.22 M) than the reference drug Staurosporine (IC50 = 7.25
0.43 M). Also, compound 5a showed comparable activity (IC50 = 6.53 0.38 M) to the reference
±
±
µ
±
µ
±
µ
±
µ
drug Staurosporine.
Cell Cycle Analysis
The effect of diamide-based benzenesulfonamides 5a and 5h on cell cycle progression was
examined in renal UO-31 cancer cells, through a DNA flow cytometric assay (Figure 3). The UO-31
cells were treated with diamides 5a and 5h at their IC50 concentrations (IC50 = 6.53
0.22 µM) for 24 h.
±
0.38 and 4.89
±
5h
5a
Control
Figure 3. Effect of diamide-based benzenesulfonamides 5a and 5h on the phases of cell cycle of
UO-31 cells.
The outcomes of this flow cytometric assay (Figure 3) suggested that exposure of renal UO-31
cells to diamides 5a and 5h led to a significant increase in the percentage of cells at Sub-G1 by 14-
and 15.5-folds, respectively, with concurrent significant arrest in the G2-M phase by 2.2- and 2.5-folds,
respectively, compared to control, which could be considered as significant remarks for sulfonamides
5a and 5h to provoke apoptosis in renal UO-31 cells.

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Annexin V-FITC Apoptosis Assay
To explore whether the anti-proliferative activity of diamide-based sulfonamides 5a and 5h is
consistent with the induction of apoptosis within the UO-31 cancer cells, Annexin V-FITC/propidium
iodide (AV/PI) dual staining assay was performed by flow cytometry (Figure 4).
5h
Control
5a
Figure 4. Effect of diamide-based benzenesulfonamides 5a and 5h on the percentage of annexin
V-FITC-positive staining in UO-31 cells. The experiments were done in triplicates. The four quadrants
identified as: LL, viable; LR, early apoptotic; UR, late apoptotic; UL, necrotic.
The results suggested that diamides 5a and 5h could induce the apoptosis of UO-31 cells as
evidenced by the significant increase in the percent of annexin V-FITC-positive apoptotic cells, including
both the early (from 0.85% to 2.64% for 5a, and from 0.95% to 8.41% for 5h) and late apoptotic (from
0.29% to 22.96% for 5a, and from 0.29% to 21.85% for 5h) phases (UR + LR), which represents about
22.5- and 26.5-folds total increase as compared with the control.
2.3. Molecular Modelling Study
To provide insights into the binding interactions of the reported diamide-based benzenesulfonamides
within hCA isozymes II and IX (PDB 5LJT [22] and 5FL4 [23]), docking and MM-GBSA-based
refinements were performed. As expected, in all docking solutions found for compounds 5a–h, the
benzenesulfonamide is placed deeply into the active site region of both isozymes, with the zinc-binding
group coordinating to the metal ion through its negatively charged nitrogen. Moreover, the sulfonamide
moiety engages two H-bonds: the NH– group acts as donor, while the S=O as acceptor with T199 OG1
atom and backbone NH respectively. The phenyl ring accommodates in the hydrophobic region lined
by V121, V143 and L198.
In hCA II, the carbonyl group of the amidic linker of derivatives 5a–h accepts a H-bond by the
HE-Q92, orienting the arylidene moiety in a cleft defined by N67, Q92, I91 and F131 residues, where

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compounds 5a–g can engage
π-
π
interactions. The benzoyl moiety accessed in a lipophilic pocket
situated just above the H64, whose imidazole NH acts as H-bond donor towards the oxygen atom of
the carbonyl group (Figure 5A).
Figure 5. Docking of 5b (purple) and 5h (violet) in hCA II (A) and in hCA IX (B) active sites.
As for hCA II, a similar set of interactions is also found for the hCA IX isoform. However, because
of the substitution V131/F131 (hCA XI/II), the arylidene moiety accommodates in a wider cavity, more
suited to the size of the group. As a result, the latter makes more effective interactions with the receptor
counterpart, thus probably contributing to the 1.5 to 14-fold hCA IX/II selectivity of compounds 5a–h.
The methylfuryl derivative 5h further shows an additional H-bond between the heterocyclic oxygen
and Q67 HE atom (Figure 5B).
3. Materials and Methods
3.1. Chemistry
3.1.1. General
Melting points were measured with a Stuart melting point apparatus and were uncorrected.
The NMR spectra were recorded by Varian Gemini-400BB 400 MHz FT-NMR spectrometers (Varian
Inc., Palo Alto, CA). 1H and 13C spectra were run at 400 and 100 MHz, respectively, in deuterated
dimethylsulphoxide (DMSO-d6). All coupling constant (J) values are given in hertz. Chemical shifts
(δC) are reported relative to DMSO-d6 as internal standards. The abbreviations used are as follows:
s, singlet; d, doublet; m, multiplet. IR spectra were recorded with a Bruker FT-IR spectrophotometer.
Reaction courses and product mixtures were routinely monitored by thin layer chromatography (TLC)
on silica gel precoated F254 Merck plates. Unless otherwise noted, all solvents and reagents were
commercially available and were used without further purification. Azlactones 3a–g were previously
reported [24,25].
3.1.2. General Procedure for Preparation of Target Diamide-Based Benzenesulfonamides 5a–h
A mixture of p-aminobenzenesulfonamide 4 (0.17 g, 1 mmol), sodium acetate (0.08 g, 1 mmol)
and the appropriate azlactone derivative 3a–h (1 mmol) in glacial acetic acid was heated under reflux
for 4 h. The solid product obtained upon cooling was filtered off and recrystallized from acetone to
produce the corresponding benzenesulfonamides 5a–h, with 80–93% yield.

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N-(1-(4-Chlorophenyl)-3-oxo-3-((4-sulfamoylphenyl)amino)prop-1-en-2-yl)benzamide (5a)
White crystal (yield 93%), m.p. 255–258 ◦C; IR (KBr,
ν
cm−1): 3311, 3255 (NH, NH2), 1704, 1639
(2C=O) and 1316, 1156 (SO2); 1H NMR (DMSO-d6)
δ
ppm: 7.10 (s, 1H, olefinic), 7.24 (s, 2H, NH2 D2O
exchangeable), 7.44 (d, 2H, J = 8.4 Hz, H-3, H-5 of C6H5), 7.49 (t, 1H, J = 8.0 Hz, H-4 of C6H5), 7.51 (d,
2H, J = 8.0 Hz, H-3, H-5 of 4-Cl-C6H4), 7.62 (d, 2H, J = 8.4 Hz, H-2, H-6 of C6H5), 7.74 (d, 2H, J = 8.8
Hz, H-2, H-6 of sulfonamide), 7.85 (d, 2H, J = 8.8 Hz, H-3, H-5 of sulfonamide), 7.98 (d, 2H, J = 8.0 Hz,
H-2, H-6 of 4-Cl-C6H4), 10.15 (s, 1H, NH D2O exchangeable), 10.53 (s, 1H, NH D2O exchangeable); 13
C
NMR (DMSO-d6)
δ ppm: 119.93, 126.92, 127.37, 128.39, 128.88, 129.08, 131.60, 131.93, 132.39, 133.57,
133.60, 133.66, 138.97, 142.70, 165.13, 166.48; HRMS (ESI) for C22H19ClN3O4S, calcd 456.07793, found
456.07800 [M+H]+; Anal. calcd. for C22H18ClN3O4S (455.91): C, 57.96; H, 3.98; N, 9.22. Found C, 58.17;
H, 3.89; N, 9.11.
N-(1-(2,4-Dichlorophenyl)-3-oxo-3-((4-sulfamoylphenyl)amino)prop-1-en-2-yl)benzamide (5b)
White crystal (yield 91%), m.p. 265–267 ◦C; IR (KBr,
ν
cm−1): 3303, 3249 (NH, NH2), 1711, 1629
(2C=O) and 1310, 1153 (SO2); 1H NMR (DMSO-d6)
δ
ppm: 7.04 (s, 1H, olefinic), 7.25 (s, 2H, NH2 D2O
exchangeable), 7.40 (dd, 1H, J = 2.0 Hz, J = 8.4 Hz, H-5 of 2,4(Cl)2-C6H3), 7.46 (t, 1H, J = 8.0 Hz, H-4 of
C6H5), 7.48 (d, 1H, J = 8.0 Hz, H-6 of 2,4(Cl)2-C6H3), 7.55 (d, 2H, J = 8.0 Hz, H-3, H-5 of C6H5), 7.71 (s,
1H, H-3 of 2,4(Cl)2-C6H3), 7.75 (d, 2H, J = 8.8 Hz, H-2, H-6 of sulfonamide), 7.86 (d, 2H, J = 8.8 Hz, H-3,
H-5 of sulfonamide), 7.91 (d, 2H, J = 7.6 Hz, H-2, H-6 of C6H5), 10.14 (s, 1H, NH D2O exchangeable),
10.57 (s, 1H, NH D2O exchangeable); 13C NMR (DMSO-d6)
δ ppm: 120.08, 122.58, 126.95, 127.98, 128.41,
128.83, 129.50, 131.53, 132.19, 132.45, 133.54, 133.95, 134.06, 134.49, 139.18, 142.44, 164.52, 166.46; Anal.
calcd. for C22H17Cl2N3O4S (490.36): C, 53.89; H, 3.49; N, 8.57. Found C, 53.65; H, 3.48; N, 8.68.
N-(1-(4-Bromophenyl)-3-oxo-3-((4-sulfamoylphenyl)amino)prop-1-en-2-yl)benzamide (5c)
Yellow powder (yield 80%), m.p. 210–212 ◦C; IR (KBr,
ν
cm−1): 3312, 3250 (NH, NH2), 1704, 1636
(2C=O) and 1316, 1156 (SO2); 1H NMR (DMSO-d6)
δ
ppm: 7.07 (s, 1H, olefinic), 7.29 (s, 2H, NH2 D2O
exchangeable), 7.42 (t, 1H, J = 8.0 Hz, H-4 of C6H5), 7.45- 7.52 (m, 2H, H-3, H-5 of C6H5), 7.52–7.58 (m,
2H, H-3, H-5 of 4-Br-C6H4), 7.70, 8.28 (d, 2H, H-2, H-6 of C6H5), 7.74 (d, 2H, H-2, H-6 of sulfonamide),
7.85 (d, 2H, H-3, H-5 of sulfonamide), 7.98 (d, 2H, J = 8.0 Hz, H-2, H-6 of 4-Br-C6H4), 10.61 (s, 2H, NH
D2O exchangeable); 13C NMR (DMSO-d6)
δ ppm: 119.89, 122.28, 124.82, 126.91, 126.99, 127.07, 127.14,
127.51, 128.23, 128.40, 128.65, 128.79, 128.84, 128.93, 129.02, 129.46, 130.49, 131.65, 131.83, 131.98, 132.20,
132.22, 132.32, 132.43, 133.69, 133.77, 133.98, 134.52, 137.59, 138.90, 139.18, 142.76, 144.17, 161.30, 165.24,
166.52, 169.69; Anal. calcd. for C22H18BrN3O4S (500.37): C, 52.81; H, 3.63; N, 8.40. Found C, 60.02; H,
3.60; N, 8.31.
N-(3-Oxo-3-((4-sulfamoylphenyl)amino)-1-(p-tolyl)prop-1-en-2-yl)benzamide (5d)
Yellow powder (yield 87%), m.p. 255–258 ◦C; IR (KBr,
ν
cm−1): 3313, 3251 (NH, NH2), 1701, 1639
(2C=O) and 1319, 1157 (SO2); 1H NMR (DMSO-d6)
δ
ppm: 2.28, 2.36 (2s, H, CH3), 7.13, 7.24 (2s, 1H,
olefinic), 7.27 (s, 2H, NH2 D2O exchangeable), 7.18, 8.00 (2d, 2H, J = 7.6, H-3, H-5 of C6H5), 7.31, 8.23
(2d, 2H, J = 8.4 Hz, H-2, H-6 of C6H5), 7.39–7.51 (m, 4H, Ar-H of 4-CH3-C6H4), 7.49, 7.53 (2t, 1H, J = 8.0
Hz, H-4 of C6H5), 7.74 (d, 2H, H-2, H-6 of sulfonamide), 7.85 (d, 2H, H-3, H-5 of sulfonamide), 10.09 (s,
1H, NH D2O exchangeable), 10.45 (s, 1H, NH D2O exchangeable); 13C NMR (DMSO-d6)
δ ppm: 21.38,
21.75 (CH3), 119.95, 126.88, 127.10, 128.34, 128.60, 128.85, 128.96, 129.01, 129.38, 129.63, 130.01, 130.07,
130.36, 131.72, 131.77, 131.97, 132.28, 132.92, 133.85, 137.74, 137.93, 138.86, 139.02, 141.51, 142.77, 144.04,
160.23, 165.31, 166.44, 169.74; HRMS (ESI) for C23H22N3O4S, calcd 436.13255, found 436.13251 [M+H]+;
Anal. calcd. for C23H21N3O4S (435.50): C, 63.43; H, 4.86; N, 9.65. Found C, 63.57; H, 4.83; N, 9.58.

Int. J. Mol. Sci. 2019, 20, 2484
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N-(1-(4-Methoxyphenyl)-3-oxo-3-((4-sulfamoylphenyl)amino)prop-1-en-2-yl)benzamide (5e)
Yellow powder (yield 88%), m.p. 265–268 ◦C; IR (KBr,
ν
cm−1): 3309, 3247 (NH, NH2), 1703, 1634
(2C=O) and 1314, 1154 (SO2); 1H NMR (DMSO-d6)
δ
ppm: 3.74, 3.83 (2s, 3H, OCH3), 6.93, 8.33 (2d,
2H, J = 8.8 Hz, H-3, H-5 of C6H5), 7.07, 8.04 (2d, 2H, J = 8.4 Hz, H-2, H-6 of C6H5), 7.18, 7.32 (2s, 1H,
olefinic), 7.39–7.45 (m, 4H, H-3, H-5 and H-2, H-6 of 4-OCH3-C6H4), 7.47 (t, 1H, J = 8.0 Hz, H-4 of
C6H5), 7.49 (s, 2H, NH2 D2O exchangeable), 7.54, 7.72 (2d, 2H, J = 8.8 Hz, H-2, H-6 of sulfonamide),
7.58, 7.85 (2d, 2H, J = 8.8 Hz, H-3, H-5 of sulfonamide), 10.95 (s, 2H, NH D2O exchangeable); Anal.
calcd. for C23H21N3O5S (451.50): C, 61.19; H, 4.69; N, 9.31. Found C, 60.88; H, 4.65; N, 9.30.
N-(1-(2,4-Dimethoxyphenyl)-3-oxo-3-((4-sulfamoylphenyl)amino)prop-1-en-2-yl)benzamide (5f)
Yellow powder (yield 85%), m.p. 245–250 ◦C; IR (KBr,
ν
cm−1): 3410, 3294 (NH, NH2), 1701, 1639
1
(2C=O) and 1369, 1161 (SO2); H NMR (DMSO-d6)
δ ppm: 3.75 (s, 3H, OCH3), 3.81 (s, 3H, OCH3),
6.65 (s, 1H, H-3 of (OCH3)2-C6H3), 7.42 (dd, 2H, J = 2.4 Hz, J = 9.2 Hz, H-5, H-6 of (OCH3)2-C6H3),
7.41–7.47 (m, 4H, H-3, H-5 of C6H4 and NH2 D2O exchangeable), 7.49 (t, 1H, J = 8.0 Hz, H-4 of C6H5),
7.55 (s, 1H, olefinic), 7.63 (d, 2H, H-2, H-6 of C6H5), 7.72 (d, 2H, J = 8.8 Hz, H-2, H-6 of sulfonamide),
7.85 (d, 2H, J = 8.8 Hz, H-3, H-5 of sulfonamide), 10.16 (s, 1H, NH D2O exchangeable), 10.65 (s, 1H,
NH D2O exchangeable); Anal. calcd. for C24H23N3O6S (481.52): C, 59.87; H, 4.81; N, 8.73. Found C,
60.09; H, 4.83; N, 8.67.
N-(1-(3,4-Dimethoxyphenyl)-3-oxo-3-((4-sulfamoylphenyl)amino)prop-1-en-2-yl)benzamide (5g)
Yellow powder (yield 90%), m.p. 250–253 ◦C; IR (KBr,
ν
cm−1): 3413, 3292 (NH, NH2), 1701, 1639
1
(2C=O) and 1369, 1161 (SO2); H NMR (DMSO-d6)
δ ppm: 3.75 (s, 3H, OCH3), 3.78 (s, 3H, OCH3),
6.97, 8.20 (d, 2H, H-3, H-5 of C6H5), 7.21–7.29 (m, 4H, H-5, H-6 of (OCH3)2-C6H3 and NH2 D2O
exchangeable), 7.43–7.46 (m, 2H, H-3 of (OCH3)2-C6H3 and olefinic), 7.48, 7.51 (t, 1H, J = 8.0 Hz, H-4 of
C6H5), 7.57, 8.04 (d, 2H, J = 8.4 Hz, H-2, H-6 of C6H5), 7.74 (d, 2H, J = 8.8 Hz, H-2, H-6 of sulfonamide),
7.86 (d, 2H, J = 8.8 Hz, H-3, H-5 of sulfonamide), 10.07 (s, 1H, NH D2O exchangeable), 10.36 (s, 1H, NH
D2O exchangeable); 13C NMR (DMSO-d6)
δ ppm: 55.52 (OCH3), 55.95 (OCH3), 112.00, 112.95, 120.07,
124.11, 126.86, 127.02, 127.11, 128.31, 128.57, 128.75, 128.82, 129.04, 129.22, 130.30, 132.31, 133.83, 138.84,
142.77, 148.73, 150.03, 165.28, 166.37; Anal. calcd. for C24H23N3O6S (481.52): C, 59.87; H, 4.81; N, 8.73.
Found C, 60.13; H, 4.77; N, 8.64.
N-(1-(5-Methylfuran-2-yl)-3-oxo-3-((4-sulfamoylphenyl)amino)prop-1-en-2-yl)benzamide (5h)
Orange powder (yield 87%), m.p. 240–243 ◦C; IR (KBr,
ν
cm−1): 3413, 3295 (NH, NH2), 1706, 1636
(2C=O) and 1366, 1160 (SO2); 1H NMR (DMSO-d6)
δ
ppm: 2.20, 2.40 (2s, 1H, CH3), 6.23 (d, 1H, H-4
furan), 6.46, 8.13 (2d, 2H, H-3, H-5 of C6H5), 6.69 (d, 1H, H-3 furan), 7.05, 7.12 (2s, 1H, olefinic), 7.23 (s,
2H, NH2 D2O exchangeable), 7.33–7.47 (m, 2H, H-2, H-6 of C6H5), 7.48 (t, 1H, J = 8.0 Hz, H-4 of C6H5),
7.74 (d, 2H, H-2, H-6 of sulfonamide), 7.83–7.86 (m, 2H, H-3, H-5 of sulfonamide), 9.85 (s, 1H, NH D2O
exchangeable), 10.23 (s, 1H, NH D2O exchangeable); 13C NMR (DMSO-d6)
δ ppm: 13.89 (CH3), 14.30
(CH3), 109.31, 111.59, 115.59, 116.81, 118.58, 120.17, 121.62, 122.08, 122.46, 126.32, 126.83, 127.06, 127.10,
128.34, 128.71, 128.93, 128.99, 129.02, 129.31, 131.42, 131.83, 132.13, 134.60, 135.05, 137.78, 138.91, 142.61,
144.01, 148.66, 149.53, 149.74, 154.60, 156.33, 157.81, 158.18, 158.88, 164.41, 166.35, 166.43, 169.07; Anal.
calcd. for C21H19N3O5S (425.46): C, 59.28; H, 4.50; N, 9.88. Found C, 59.15; H, 4.46; N, 9.97.
3.2. Biological Evaluation
3.2.1. CA Inhibitory Assay
An Applied Photophysics stopped-flow instrument was used for assaying the CA catalysed CO2
hydration activity, as reported earlier [17]. The inhibition constants were obtained by non-linear
least-squares methods using PRISM 3 and the Cheng-Prusoff equation as reported earlier, and represent

Int. J. Mol. Sci. 2019, 20, 2484
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the mean from at least three different determinations. The four tested CA isofoms were recombinant
ones obtained in-house as reported earlier [26–29].
3.2.2. Anticancer Activity towards 60 Cancer Cell Lines (NCI, Bethesda, MD, USA)
This assay was carried out according to the protocol of the Drug Evaluation Branch, NCI,
Bethesda [30–32]. The cell growth and cell viability were evaluated using the sulforhodamine B
(SRB) [26] colorimetric assay, as reported earlier [33,34].
3.2.3. Antiproliferative Activity towards Renal Cancer UO-31 Cell Line
Renal cancer UO-31 cell line was obtained from American Type Culture Collection (ATCC). UO-31
cells were seeded into 96-well plates at 1.8 x 104/well. Cells were grown in DMEM, and supplemented
with 10% heat-inactivated fetal bovine serum, 1% L-glutamine (2.5 mM), HEPES buffer (10 mM),
50 µ
g/mL gentamycin. All cells were maintained at 37 ◦C in a humidified atmosphere with 5% CO2.
Cytotoxic activity of diamide-based benzenesulfonamides 5a, 5b and 5h was evaluated following the
the protocol of MTT colorimetric assay, as reported previously [35,36].
3.2.4. Cell Cycle Analysis
Renal cancer UO-31 cells were treated with diamide-based benzenesulfonamide 5h at its IC50
concentration (IC50 = 4.89
buffered saline (PBS). Then, the treated cells were collected by centrifugation, fixed in ice-cold 70%
(v/v) ethanol, washed with PBS, re-suspended with RNase (100 g/mL), stained with PI (40 g/mL),
±
0.22 µM) for 24 h, and then cells were washed with ice-cold phosphate
µ
µ
and analyzed by flow cytometry using FACS Calibur (Becton Dickinson, BD, USA). The cell cycle
distributions were calculated utilizing CellQuest software 5.1 (Becton Dickinson) [37,38].
3.2.5. Annexin V-FITC Apoptosis Assay
Assay of the phosphatidylserine externalization was carried out using Annexin V-FITC/PI
apoptosis detection kit (BD Biosciences, San Jose, CA, USA) following the manufacturer’s instructions,
as reported previously [39,40].
3.2.6. Molecular Docking Simulations
Preparation of the crystal structure of both hCAII (PDB 5LJT [32]) and hCAIX (PDB 5FL4 [33])
was performed using the Protein Preparation Wizard tool implemented in Maestro-Schrödinger suite,
assigning bond orders, adding hydrogens, deleting water molecules, and optimizing H-bonding
networks [41]. Energy minimization protocol with a root mean square deviation (RMSD) value of
0.30 was applied using an Optimized Potentials for Liquid Simulation (OPLS3) force field. For the
simulations with sulfonate derivatives, 5JLT and 5FL4 were prepared adding the zinc-bound water
molecule as fourth ligand of the metal tetrahedral coordination sphere. 3D ligand structures were
prepared by Maestro [43a] and evaluated for their ionization states at pH 7.4
±
0.5 with Epik [41b].
OPLS3 force field in Macromodel [41e] was used for energy minimization for a maximum number
of 2500 conjugate gradient iteration and setting a convergence criterion of 0.05 kcal mol-1Å-1. The
docking grid was centered on the center of mass of the co-crystallized ligands, and was Glide used with
default settings. Ligands were docked with the standard precision mode (SP) of Glide [41f] and the
best 5 poses of each molecule retained as output. The best pose for each compound, evaluated in terms
of coordination, hydrogen bond interactions and hydrophobic contacts, was refined by Prime [41d]
with a VSGB solvatation model considering the target flexible within 3Å around the ligand [42–44].
4. Conclusions
In summary, this study reports the synthesis of novel series of diamide-based benzenesulfonamides
5a–h. The prepared diamides were examined as inhibitors of CA isoforms hCA I, II, IX and XII, using

Int. J. Mol. Sci. 2019, 20, 2484
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a stopped-flow CO2 hydrase assay. All the tested isoforms were inhibited by the diamide-based
benzenesulfonamides 5a–h herein reported in variable degrees; hCA I was inhibited with KIs in the
range of 796.1–8175nM, hCA II in the range of 68.3–448 nM; hCA IX in the range of 8.3–123.3 nM,
whereas hCA XII in the range of 9.8–134.5 nM. Notably, diamides 5a and 5h stood out as a single-digit
nanomolar hCA IX inhibitors with KI values = 8.8 and 8.3 nM, respectively, which are about three
times more potent than the standard drug AAZ (KI = 25 nM). All the examined diamides 5a–h
displayed interesting selectivity towards hCA IX over hCA I with SIs spanning in the range 12.8–985.
Also, they possessed good hCA IX/II SIs in the range 3.6–13.8, apart from diamides 5d and 5e which
exhibited modest selectivity (SI = 1.4 and 1.5, respectively). Interestingly, bioisosteric replacement of
the benzylidene moiety with the hetero 2-furylidene moiety, compound 5h, achieved the best IX/I and
IX/II selectivity herein reported with SIs of 985 and 13.8, respectively. Molecular docking simulations
of the prepared diamides in CA IX active site unveiled the ability of 5h to establish an additional
H-bond between the heterocyclic oxygen and HE/Gln67. Moreover, benzenesulfonamides 5a, 5b and
5h were selected to be in vitro evaluated for their antitumor activity against renal cancer UO-31 cell
line. Notably, 5h was the most potent derivative with about 1.5-fold more enhanced activity (IC50
=
4.89 0.22 M) than the reference drug Staurosporine (IC50 = 7.25 0.43 M). Furthermore, 5a and 5h
±
µ
±
µ
were able to induce apoptosis in UO-31 cells as evidenced by the significant increase in the percent
of annexinV-FITC positive apoptotic cells by 22.5- and 26.5-folds, respectively, as compared with
the control.
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/10/
2484/s1.
Author Contributions: Conceptualization, W.M.E.; Data curation, A.N. and S.B.; Formal analysis, G.S.H.; Funding
acquisition, S.T.A.-R. and A.A.A.; Methodology, Mohamed A.A., W.M.E., A.N., S.B., S.T.A.-R., G.S.H., A.B., A.A.
and P.G.; Project administration, W.M.E.; Resources, S.T.A.-R. and H.M.A.; Supervision, C.T.S.; Writing–original
draft, M.A.A., G.S.H., A.B., A.A.A., H.M.A., A.A. and P.G.; Writing–review & editing, W.M.E., A.N. and C.T.S.
Funding: The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at
King Saud University for its funding of this research through the Research Group Project no. RG-1439–65.
Conflicts of Interest: The authors declare no conflict of interest.
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