Synthesis, biological evaluation and in silico studies with 4-benzylidene-2-phenyl-5(4H)-imidazolone-based benzenesulfonamides as novel selective carbonic anhydrase IX inhibitors endowed with anticancer activity

Article abstract of DOI:10.1016/j.bioorg.2019.103102

In the presented work, we report the synthesis of a series of 4-benzylidene-2-phenyl-5(4H)-imidazolone-based benzenesulfonamides 7a-f via the Erlenmeyer–Pl?chl reaction. All the prepared imidazolones 7a-f were evaluated as inhibitors of human (h) carbonic anhydrases (CA, EC 4.2.1.1) cytosolic isoforms hCA I and II, as well as transmembrane tumor-associated isoforms hCA IX and XII. All the tested hCA isoforms were inhibited by the prepared imidazolones 7a-f in variable degrees with the following K_Is ranges: 673.2–8169 nM for hCA I, 61.2–592.1 nM for hCA II, 23–155.4 nM for hCA XI, and 21.8–179.6 nM for hCA XII. In particular, imidazolones 7a, 7e, and 7f exhibited good selectivity towards the tumor-associated isoforms (CAs IX and XII) over the off-target cytosolic (CAs I and II) with selectivity index (SI) in the range of 6.2–19.4 and 3.3–8, respectively. Moreover, imidazolones 7a-f were screened for their anticancer activity in one dose (10^?5 M) assay against a panel of 60 cancer cell lines according to US-NCI protocol. Furthermore, 7a, 7e and 7f were evaluated for their anti-proliferative activity against colorectal cancer HCT-116 and breast cancer MCF-7 cell lines. Furthermore, 7e and 7f were screened for cell cycle disturbance and apoptosis induction in HCT-116 cells. Finally, a molecular docking study was carried out to rationalize the obtained results.

Full text of DOI:10.1016/j.bioorg.2019.103102

Accepted Manuscript
Synthesis, biological evaluation and in silico studies with 4-benzylidene-2-phe-
nyl-5(4H)-imidazolone-based benzenesulfonamides as novel selective carbonic
anhydrase IX inhibitors endowed with anticancer activity
Wagdy M. Eldehna, Mohamed A. Abdelrahman, Alessio Nocentini, Silvia Bua,
Sara T. Al-Rashood, Ghada S. Hassan, Alessandro Bonardi, Abdulrahman A.
Almehizia, Hamad M. Alkahtani, Amal Alharbi, Paola Gratteri, Claudiu T.
Supuran
PII:
S0045-2068(19)30708-4
https://doi.org/10.1016/j.bioorg.2019.103102
103102
DOI:
Article Number:
Reference:
YBIOO 103102
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
2 May 2019
13 June 2019
1 July 2019
Please cite this article as: W.M. Eldehna, M.A. Abdelrahman, A. Nocentini, S. Bua, S.T. Al-Rashood, G.S. Hassan,
A. Bonardi, A.A. Almehizia, H.M. Alkahtani, A. Alharbi, P. Gratteri, C.T. Supuran, Synthesis, biological evaluation
and in silico studies with 4-benzylidene-2-phenyl-5(4H)-imidazolone-based benzenesulfonamides as novel
selective carbonic anhydrase IX inhibitors endowed with anticancer activity, Bioorganic Chemistry (2019), doi:
https://doi.org/10.1016/j.bioorg.2019.103102
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Synthesis, biological evaluation and in silico studies with 4-benzylidene-2-
phenyl-5(4H)-imidazolone-based benzenesulfonamides as novel selective
carbonic anhydrase IX inhibitors endowed with anticancer activity
Wagdy M. Eldehnaa,*, Mohamed A. Abdelrahmanb, Alessio Nocentinic,d, Silvia Buac, Sara T.
Al-Rashoode, Ghada S. Hassanf, Alessandro Bonardic,d, Abdulrahman A. Almehiziae, Hamad
M. Alkahtanie, Amal Alharbie, Paola Gratterid, Claudiu T. Supuranc,*
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Kafrelsheikh University,
Kafrelsheikh, Egypt
bDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Egyptian Russian University, Badr
City, Cairo 11829, Egypt
cDepartment of NEUROFARBA, Section of Pharmaceutical and Nutraceutical Sciences, University of
Florence, Polo Scientifico, Via U. Schiff 6, 50019, Sesto Fiorentino, Firenze, Italy
dDepartment 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
eDepartment of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box
2457, Riyadh 11451, Saudi Arabia.
fDepartment of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516,
Egypt
ABSTRACT
In the presented work, we report the synthesis of a series of 4-benzylidene-2-phenyl-5(4H)-
imidazolone-based benzenesulfonamides 7a-f via the Erlenmeyer–Plöchl reaction. All the
prepared imidazolones 7a-f were evaluated as inhibitors of human (h) carbonic anhydrases
(CA, EC 4.2.1.1) cytosolic isoforms hCA I and II, as well as transmembrane tumor-
associated isoforms hCA IX and XII. All the tested hCA isoforms were inhibited by the
prepared imidazolones 7a-f in variable degrees with the following KIs ranges: 673.2–8169
nM for hCA I, 61.2–592.1 nM for hCA II, 23–155.4 nM for hCA XI, and 21.8–179.6 nM for
hCA XII. In particular, imidazolones 7a, 7e, and 7f exhibited good selectivity towards the
tumor-associated isoforms (CAs IX and XII) over the off-target cytosolic (CAs I and II) with
selectivity index (SI) in the range of 6.2–19.4 and 3.3–8, respectively. Moreover,
imidazolones 7a-f were screened for their anticancer activity in one dose (10−5 M) assay
against a panel of 60 cancer cell lines according to US-NCI protocol. Furthermore, 7a, 7e and
7f were evaluated for their anti-proliferative activity against colorectal cancer HCT-116 and
breast cancer MCF-7 cell lines. Furthermore, 7e and 7f were screened for cell cycle
1

disturbance and apoptosis induction in HCT-116 cells. Finally, a molecular docking study
was carried out to rationalize the obtained results.
__________
Keywords: Anticancer activity; Benzenesulfonamides; Imidazolone; Molecular modeling;
Selective hCA IX inhibitors.
* Corresponding authors. E-mail addresses: wagdy2000@gmail.com (W.M. Eldehna),
claudiu.supuran@unifi.it (C.T. Supuran).
2

1. Introduction
Carbonic anhydrases (CA, EC 4.2.1.1) are a group of zinc containing metalloenzymes and
are vastly distributed in living organisms. CAs are distributed into seven classes, α, β, γ, δ, ζ,
η and θ. The hydration of carbon dioxide to produce bicarbonate and protons catalysed by the
CAs have critical role in various human physiological processes such as respiration, pH and
CO2 homeostasis, lipogenesis, gluconeogenesis and tumorigenicity [1]. The human carbonic
anhydrases (hCAs) belong to the α- class of carbonic anhydrases. This family of CA is
divided into fifteen isoforms, which exhibit distinct molecular attributes, protein structure,
kinetics, localization and catalytic behaviour [2, 3]. The isoforms can be listed as: CA I, CA
II, CA III, CA VII and CA XIII are cytosolic, CA IV, CA IX, CA XII, and CA XIV are
transmembrane bound, CA VA and VB mitochondrial and CA VI secreted in body fluids like
saliva and colostrum [4-8].
CAs IX and XII are well-known transmembrane CA isoforms which have shown
increased expression in hypoxia-induced tumor cells [9]. Human CA IX plays a great role in
tumor cell proliferation, pH regulation migration and adhesion, thus the inhibition of hCA IX
activity lead to a decrease of these processes and metastatic cascade [10]. Selective targeting
of membrane-bound isoform hCA IX has been advised as a promising strategy to halt the
growth of different solid tumours through suppressing distinctive tumour survival
mechanisms in hypoxic environment [9,10]. A leading pre-clinical proof-of-concept data and
one front-runner small molecule, SLC-0111, in Phase I/II clinical trials for the treatment of
advanced hypoxic tumours [11, 12] provides confidence that selective targeting of hCA
isoforms will guarantee clinical validation in the near future.
In the last few years, aryl imidazolone scaffold have attracted attention as an effective and
promising scaffold for the design and development of potent CA inhibitors. Congiu et al. [13]
reported the synthesis and biological evaluation of novel 4-phenyl-imidazol-2(3H)-one
derivatives as CA inhibitors (e.g. compound I in Fig. 1). These imidazol-2-ones emerged as
selective nanomolar CAs IX and XII inhibitors.
Awadallah and co-workers reported two studies about the development of novel
imidazolone-based benzenesulfonamides as CA inhibitors [14, 15]. In the first study, the 2-
phenyl-imidazol-5(1H)-one scaffold was condensed with a chromone moiety, (e.g. compound
II in Fig. 1), whereas in the second study the 2-phenyl-imidazol-5(1H)-one scaffold was
3

condensed with an indole moiety (e.g. compound III in Fig. 1). Uniquely the imidazolone-
chromone hybrids (compound II, Fig. 1) exhibited good activity and selectivity towards the
tumor-associated isoforms hCA IX and XII. Accordingly, they were evaluated for their anti-
proliferative and pro-apoptotic activities towards cancer MCF-7 and A-549 cell lines. In
2016, another study [16] explored the synthesis and biological evaluation of novel CA
inhibitors based on the 2-phenyl-imidazol-5(1H)-one scaffold (e.g. compound IV in Fig. 1),
hinting that grafting a benzylidene moiety at imidazolone C-4 is well tolerated rather than the
heteroaryl chromone moiety, and could result in more effective CA IX inhibitors.
.
O
O
O
O
O
S
O
S
O
S
O
NH
O
O
N
N
N
N
O
N
N
H2N
H2N
H2N
O
I
CAI=572
CAII=375
CAIX=34
CAXII=12
III
II
CAI=8003
CAII=75
CAIX=23.04
CAI=96.6
CAII=56.4
CAIX=318.4
CAXII=5.23
H
N
S
R
O
O
O
O
R1
R2
O
N
O
S
O
S
O
O
N
N
S
N
HN
H2N
N
N
OH
H2N
H2N
N
Cl
R3
R
O
7a-f
IV
CAI=702
CAII=288
CAIX=29.2
Va; R
=H
Vb; R
CAI=781.3
CAII=9.2
CAIX=354.3
=OCH3
CAI=85.0
CAII=0.83
CAIX= 12.4
Fig. 1. Structures of some reported imidazolone-based benzenesulfonamides I-V and the
target imidazolones 7a-f.
Latterly, the Supuran's research group reported a new series of 1,2,4-trisubstituted
imidazolin-5-ones bearing a 4-benzylidene moiety, and incorporating primary and secondary
sulfonamide groups as dual carbonic anhydrase (isoforms I, II, IV and IX) and p38-MAPK
inhibitors (e.g. compounds Va and Vb in Fig. 1) [17]. The SAR outcomes in this study
revealed that presence of the free primary sulfamoyl group is crucial for the CA inhibitory
activity. Conversely, it is noteworthy that incorporation of di-substituted phenyl moiety at C-
2 (3,4-dimethoxy) elicited a worsening of effectiveness towards isoform hCA IX, suggesting
that decreasing bulkiness of the substituent at C-2 of the imidazolone moiety is more
advantageous for CA IX inhibitory activity (e.g. compounds Va and Vb in Fig. 1).
4

Unfortunately, the four prepared primary sulfonamides reported in this study exhibited a high
selectivity towards the off-target cytosolic isoform CA II over the tumor-associated isoform
CA IX with IX/II selectivity indexes (SIs) spanning in the range of 10 – 175, though they
displayed good to moderate CA IX inhibitory activity (KIs values ranging between 12.4 and
902.4 nM).
Encouraged by the facts mentioned above and mindful of the significance of continuous
development of selective hCA IX inhibitors to feed into anticancer discovery pipeline, we
pursued on our efforts [18-20] to develop effective antitumor candidates that selectively
targeting the tumor-associated isoform hCA IX. In the presented work, we report the
synthesis of 4-benzylidene-2-phenyl-5(4H)-imidazolone-based benzenesulfonamides 7a-f via
the Erlenmeyer–Plöchl reaction (Fig. 1). Firstly, unsubstituted phenyl group (as in
compounds II-IV) was selected to substitute the C-2 of the imidazolone core. Also, different
un/substituted benzylidene derivatives were grafted at imidazolone C-4. The substitution
pattern on the pendant 4-benzylidene moieties was selected to ensure diverse lipophilic and
electronic environments that would manipulate the CA inhibitory activity of the target
imidazolones. All the synthesized imidazolones 7a-f were characterized and biologically
tested against the physiologically relevant hCA isoforms, hCA I, II (cytosolic) as well as hCA
IX and XII (transmembrane, tumor-associated isoforms) using stopped-flow CO2 hydrase
assay. As no attention was paid to investigate the potential binding mode of the reported
imidazolone-based sulfonamides (compounds I-V, Fig 1 [13-17]) within CAs active sites to
understand how they can inhibit CAs, a molecular docking study for the prepared
imidazolones within CA II and IX active sites was carried out to rationalize the obtained
results.
Moreover, imidazolones 7a-f were screened for their anticancer activity with one dose
(concentration 10−5 M) assay towards a panel of 60 cancer cell lines according to US-NCI
protocol. Furthermore, the most potent and selective hCA IX inhibitors were assessed for
their anti-proliferative activity towards breast cancer MCF-7 and colorectal cancer HCT-116
cell lines. Then, imidazolones 7e and 7f were examined for cell cycle disturbance and
apoptosis induction in colorectal cancer HCT-116 cells to acquire more mechanistic insights
into their antitumor activity.
2. Results and Discussion
5

2.1. Chemistry
The synthetic strategy for the synthesis of the target imidazolone-based
benzenesulfonamides 7a-f was illustrated in Scheme 1. The synthesis was started by
preparation of N-benzoylglycine (hippuric acid, 2) via N-acylation of glycine with benzoyl
chloride 1 as reported [21]. The key intermediates 4a-f were prepared through the
Erlenmeyer–Plöchl azlactone synthesis [22, 23]; where hippuric acid 2 was reacted with
different benzaldehydes 3a-f in acetic anhydride in the presence of Hünig's base [24]. The
key intermediates 4a-f were refluxed with sulfanilamide 5 in glacial acetic acid and fused
sodium acetate to furnish imidazolone-based benzenesulfonamides 7a-f with 68-77% yield
(Scheme 1).
..
R1
R
R
O
R2
R3
O
R1
R2
O
O
O
O
H2N
N
OH
3a-f
ii
OH
Cl
N
H
R3
i
O
2
4a-f
1
O
S
H2N
O
NH2
iii
5
O
O
S
R
H2N
O
R
R1
R2
O
R1
R2
O
S
N
O
H
-H2O
N
NH
N
O
H2N
R3
R3
7a-f
R1
H
H
Cl
CH3
6a-f
3, 7
a
b
c
d
R
H
H
H
H
R2
R3
H
F
H
H
H
H
H
H
H
OCH3
OCH3
e
f
OCH3 H
OCH3
H
OCH3
Scheme 1. Synthesis of imidazolone-based benzenesulfonamides 7a-f; Reagents and conditions: (i)
Aq. NaOH, r.t. 2 hrs., (ii) Acetic anhydride / Hünig's base / reflux 3 hrs., (iii) Glacial acetic acid /
CH3COONa / reflux 4 hrs.
6

Postulated structures of the prepared imidazolone-based benzenesulfonamides 7a-f were
in full agreement with their spectral and elemental analyses data.
IR spectra of sulfonamides 7a-f revealed the presence of characteristic bands of NH2
group (at 3316 – 3249 cm-1), C=O group (at 1636 – 1629 cm-1), and SO2 at (1358 – 1350 and
1
1186-1157 cm-1). Furthermore, H NMR spectra of imidazolones 7a-f displayed one D2O
exchangeable singlet signal assigned to (NH2) of sulfonamido group at δ 7.40-7.50 ppm. In
addition, compounds 7a-f were confirmed by presence of olifenic signal at δ 7.25-7.39 ppm,
whereas, 1H NMR spectra of 7d,e and f confirmed the presence of aliphatic signals of CH3 at
δ 1.84 ppm, two OCH3 at δ 3.77- 3.86 ppm and three OCH3 at δ 3.74- 3.84 ppm, respectively.
13
On the other hand, C NMR spectra of imidazolones 7a-f revealed presence of signal of
(C=O) group at δ 169.65-169.81 ppm, in addition to, aliphatic CH3 signals of 7d at δ 21.48
ppm and OCH3 signals of 7e,f at δ 55.84- 60.69 ppm.
2.2. Biological Evaluation
2.2.1. Carbonic anhydrase inhibition
The CA inhibitory effects of all the synthesized imidazolone-based benzenesulfonamides
7a-f were evaluated towards the physiologically relevant hCA isoforms, hCA I, II (cytosolic)
as well as hCA IX and XII (transmembrane, tumor-associated isoforms) using an applied
photophysics stopped-flow instrument for assaying the CA-catalyzed CO2 hydration activity
[25]. The inhibitory activities were compared to acetazolamide (AAZ), a clinically used
standard CA inhibitor. The following SAR is evident from the data of Table 1:
(i) The ubiquitous cytosolic isoform hCA I was inhibited by imidazolone-based
benzenesulfonamides 7a-f prepared in this study, with inhibition constants (KIs) ranging from
high nanomolar to low micromolar concentration, between 673.2 nM and 8.17 μM.
Incorporation of unsubstituted benzylidene moiety led to sulfonamide 7a with moderate
inhibitory activity against hCA I (KI = 692 nM). Since fluorine atom has a size and electronic
properties similar to those of hydrogen, it was introduced as an isostere to the hydrogen atom.
Sulfonamide 7b bears fluorine substituent at the 4-position and showed mild improvement in
the activity (KI = 673.2 nM). On the other hand, grafting 3-Cl, 3-CH3, 2,5-(OCH3)2 or 3,4,5-
(OCH3)3 substituents at the benzylidene moiety (compounds 7c-f; KIs = 823.1, 4411.8,
8169.6 and 5021.1 nM, respectively) resulted in 1.2-, 6.4-, 11.8- and 7.3-fold decreased
activity in comparison to their unsubstituted analogue 7a (KI = 692 nM).
7

(ii) The second physiologically dominant isoform examined here was hCA II. It was
evident from the obtained results that the prepared imidazolones 7a–f exhibited inhibition
constants ranging in the low-high nanomolar range, in detail, between 61.2 and 592.1 nM.
Imidazolones 7b and 7c bearing 4-F and 3-Cl substituents, respectively, were the most potent
hCA II inhibitors that displayed better activity (KIs = 83.7 and 61.2 nM, respectively) than
their unsubstituted counterpart 7a (KI = 362.7 nM), whereas, incorporation of 3-CH3
substituent led to sulfonamide 7d that displayed slight improvement in the inhibitory activity
(KI = 223.3 nM) than 7a, suggesting that substitution with halogens is more advantageous
than methyl group for hCA II inhibitory activity.
Similarly to the SAR for hCA I inhibition, incorporation of 2,5-(OCH3)2 or 3,4,5-(OCH3)3
substituents (compounds 7e, f; KIs = 445.5 and 592.1 nM, respectively) led to a decreased
inhibitory activity in comparison to unsubstituted member 7a (KI = 362.7 nM).
Table 1. Inhibition data of human CA isoforms hCA I, II, IX and XII for imidazolone-based
benzenesulfonamides 7a-f, using (AAZ) as a standard drug.
R
O
R1
O
O
S
N
N
R2
H2N
R3
7a-f
KI (nM)*
Comp.
R
R1
R2
R3
hCA I
692.0
hCA II
362.7
83.7
hCA IX
58.7
hCA XII
76.5
7a
H
H
H
H
H
H
H
F
H
H
H
H
7b
7c
673.2
51.1
62.1
Cl
H
H
H
823.1
61.2
155.4
79.1
21.8
7d
7e
CH3
H
4411.8
8169.6
5021.1
250.0
223.3
445.5
592.1
12.0
133.0
54.8
OCH3
OCH3
23.0
7f
H
-
OCH3 OCH3 OCH3
68.2
179.6
5.7
AAZ
-
-
-
25.0
* Mean from 3 different assays, by a stopped flow technique (errors were in the range of
 5-10 % of the reported values).
(iii) The in vitro kinetic data in Table 1 revealed that the tumor-associated isoform hCA
IX was efficiently inhibited by the prepared imidazolone-based benzenesulfonamides 7a-f
8

with KIs spanning in the nanomolar range: 23-79.1 nM, apart from imidazolone 7c which
possessed a slightly reduced inhibitory efficacy (KI = 155.4 nM). Nevertheless, among the
examined imidazolones, compound 7e proved to be the most active one in inhibiting hCA IX
in this study with KI value of 23 nM, which is more potent than the standard drug AAZ (KI =
25 nM). Concerning the effect of substitution of the benzylidene moiety, the hCA IX
inhibitory activities were decreased in the order of 2,5-(OCH3)2 > 4-F > 3,4,5-(OCH3)3 > 3-
CH3 > 3-Cl.
(iv) The second tumor-associated transmembrane isoform investigated here, hCA XII,
was effectively inhibited by all the synthesized imidazolones 7a-f, with KIs in the range of
21.8–179.6 nM. In particular, imidazolone 7c was the most potent hCA XII inhibitor (KI =
21.8 nM). The decreased KI values of the halogenated derivatives (compounds 7b and 7c; KIs
= 62.1 and 21.8 nM, respectively) than their unsubstituted counterpart 7a (KI = 76.5 nM),
highlighted that incorporation of halogens within the benzylidene moiety is beneficial for the
hCA XII inhibitory activity, as it was noticed for hCA II activity. On the contrary,
substitution of the benzylidene moiety with 3-CH3 or 3,4,5-(OCH3)3 groups decreased the
activity (compounds 7d and 7f; KIs = 133 and 179.6 nM, respectively). The order of activities
for the substituted imidazolone-based benzenesulfonamides towards hCA XII was decreased
in the order of 3-Cl > 2,5-(OCH3)2 > 4-F > 3-CH3 > 3,4,5-(OCH3)3.
Table 2. Selectivity ratios for the inhibition of hCA IX and XII over hCA I and II for
imidazolone-based benzenesulfonamides 7a-f and acetazolamide.
Cmpd
7a
I/IX
11.8
13.2
5.3
II/IX
6.2
I/XII
9
II/XII
4.7
1.3
2.8
1.7
8
7b
1.6
10.8
37.8
33.2
149.1
28
7c
0.4
7d
55.8
355
2.8
7e
19.4
8.7
7f
73.6
10.0
3.3
2.2
AAZ
0.5
43.9
9

(v) As a result of the profiles for inhibitory activities of the prepared imidazolone-based
benzenesulfonamides 7a-f (Table 1), the SI for each imidazolone-based benzenesulfonamide
was calculated and listed in Table 2. Concerning selectivity towards hCA IX and XII over
hCA I, all the evaluated imidazolones displayed excellent SIs spanning in the range of 5.3 –
73.6 and 9 – 149.1, respectively. Otherwise, only imidazolones 7a, 7e, and 7f exhibited good
selectivity towards hCA IX and XII over hCA II with SIs in the range of 6.2 – 19.4 and 3.3 –
8, respectively.
Of particular interest, substitution of the benzylidene moiety with 2,5-(OCH3)2 group,
imidazolone 7e, not only resulted in an enhancement of the inhibitory activity against hCA
IX and XII, but also led to an worsening of effectiveness towards hCA I and II, in
comparison to unsubstituted analogue 7a. Such trend for 7e resulted in the best selectivity
profile for inhibition of the tumor-associated isoforms hCAs IX and XII over the off-target
cytosolic hCAs I and II in this study (I/IX = 355, II/IX = 19.4, I/XII = 149.1 and II/XII = 8).
2.2.2. In Vitro Antitumor Activity towards 60 cancer cell lines (NCI, USA)
The structures of all the prepared sulfonamides 7a-f were submitted to the National
Cancer Institute (NCI) Developmental Therapeutic Program (www.dtp.nci.nih.gov), where
they were chosen to be in vitro evaluated for their antitumor activity. The selected
sulfonamides were examined at single dose (10−5 M) primary anticancer assay towards a
panel includes eighty five cancer lines. A 48 h drug exposure protocol was adopted, and
sulforhodamine B (SRB) assay [26-28] was utilized to evaluate the cell growth and cell
viability. The obtained data were reported as mean-graph of the percentage growth of the
different treated cancer cells and showed as percentage growth inhibition (GI%) caused by
the tested sulfonamides (Table 3). Exploration of results in Table 3 confirmed that the
examined imidazolone-based benzenesulfonamides exhibited distinctive patterns of
sensitivity and selectivity towards the different NCI cancer cell panels.
Close examination of the GI% values in Table 3, highlighted that imidazolone-based
benzenesulfonamides 7a, 7e and 7f were the most active anti-proliferative analogues in this
study with broad spectrum activity against numerous cancer cell lines that belong to different
tumor subpanels. The most susceptible cancer cell lines towards the impact of
benzenesulfonamides 7a, 7e and 7f were displayed in Figure 2.
Imidazolone 7a displayed broad spectrum activity towards forty one cancer cell lines
represent all subpanels except leukemia. In particular, imidazolone 7a exhibited a potent
10

growth inhibitory activity against non-small cell lung cancer (A549 and HOP-62), colon
cancer (HCT-116), CNS cancer (SNB-75), ovarian cancer (OVCAR-4 and OVCAR-8), renal
cancer (ACHN) and breast cancer (MCF7) cell lines with inhibition % 41, 44, 50, 73, 47, 42,
44 and 45, respectively. Moreover, 7a showed GI more than 25% over CNS cancer (SF-539
and U251), renal cancer (CAKI-1) and breast cancer (HS 578T and MDA-MB-468) cell
lines. Furthermore, imidazolone 7f possessed anti-proliferative activity towards thirty four
cancer cell lines (Table 3) representing all subpanels, with potent growth inhibitory impact
against CNS cancer (SNB-75), ovarian cancer (OVCAR-4 and OVCAR-8) and breast cancer
(HS 578T) cell lines with inhibition % equal 61, 43, 54 and 65, respectively. Also,
imidazolone 7f exerted anti-proliferative effect with GI more than 25% against leukemia
(SR), non-small cell lung cancer (HOP-62 and HOP-92), CNS cancer (SNB-19), melanoma
(UACC-257), renal cancer (CAKI-1) and breast cancer (MCF7) cell lines.
It is noteworthy that non-small cell lung cancer (A549 and NCI-H522), colon cancer
(HCT-116), CNS cancer (SNB-75), ovarian cancer (OVCAR-8), renal cancer (CAKI-1) and
breast cancer (MCF7 and HS 578T) cell lines were sensitive to all the tested midazolone-
based benzenesulfonamides (7a-f) with GI% range of 10–41%,13–27%, 10–50%, 10–73%,
11–54%, 14–33%, 12–45% and 10–65%, respectively.
Table 3. Percentage growth inhibition (GI %) of in vitro subpanel tumor cell lines at 10 μM
concentration for sulfonamides 7a-f.
Subpanel / Cell line
Compound a
7c
7a
7b
7d
7e
7f
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
-
-
-
-
-
-
41
-
-
10
13
-
16
18
29
35
-
68
22
-
-
-
10
10
-
31
25
-
10
-
-
-
-
15
29
20
20
16
-
-
-
10
38
10
-
30
22
-
A549/ATCC
EKVX
HOP-62
HOP-92
44
12
13
18
15
23
13
-
-
-
-
-
-
-
17
-
-
12
-
-
-
-
-
27
-
-
13
17
-
-
11
-
-
18
-
-
-
23
-
10
-
-
22
-
-
36
28
16
-
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
COLO 205
HCC-2998
HCT-116
-
16
23
-
-
18
-
50
10
17
21
11

HCT-15
HT29
KM12
15
14
11
-
22
23
34
19
73
31
-
20
-
-
-
-
-
13
-
10
-
-
25
22
12
-
-
-
-
-
17
-
-
-
14
-
-
-
14
28
20
51
-
-
18
-
14
-
-
10
-
-
SW-620
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
LOX IMVI
MALME-3M
M14
-
-
10
11
10
-
26
15
11
-
19
24
22
29
61
-
-
20
-
13
-
-
-
13
-
-
12
-
14
-
-
-
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
786-0
-
-
-
-
-
-
-
-
-
-
-
12
-
-
23
12
-
25
15
-
-
-
-
-
14
-
-
30
-
21
15
-
-
-
11
17
-
-
-
12
-
-
-
-
23
-
27
12
-
-
-
14
14
-
-
-
17
-
27
46
-
18
-
11
16
-
-
-
-
-
-
10
24
13
11
-
29
16
13
11
43
-
54
-
18
-
47
-
42
21
10
14
-
44
33
-
A498
ACHN
CAKI-1
RXF 393
SN12C
-
15
24
-
11
-
24
33
20
21
-
20
10
-
-
-
-
TK-10
-
UO-31
PC-3
23
16
12
-
20
19
-
-
41
25
24
16
DU-145
10
-
-
-
-
-
MCF7
MDA-MB-231
HS 578T
BT-549
T-47D
45
15
32
-
13
27
83
41
12
-
14
39
-
-
94
18
28
-
10
NTb
27
-
18
-
24
NTb
15
-
29
-
20
NTb
25
-
29
15
65
NTb
11
MDA-MB-468
15
85
34
Mean growth, %
Sensitive cell lines no.
a Only GI % higher than 10% are shown.
90
26
91
23
89
31
12

80
70
60
50
40
30
20
10
0
Fig. 2. The most susceptible NCI cancer cell lines towards the effect of target imidazolones
7a, 7e and 7f according to the GI%.
2.2.3 Anti-proliferative activity towards breast MCF-7 and colon HCT-116 cancer cell
lines
Association of CA IX overexpression with poor prognosis in breast and colon cancers is
well reported in the literature [29-31]. As imidazolones 7a, 7e and 7f displayed good
inhibitory activity towards tumor-associated hCA IX isoform, and good hCA II/IX
selectivity, they were selected to be evaluated for their anti-proliferative activity against
breast cancer MCF-7 and colon cancer HCT-116 cell lines using the MTT colorimetric
assay as described by T. Mosmann [32]. Staurosporine was used in this assay as a reference
antitumor drug. The results were presented as IC50 values which are the compounds
concentrations needed to produce a 50% inhibition of cell growth after 48 hours of
incubation, compared to untreated control (Table 4).
From the displayed results in Table 4, it was obvious that HCT-116 cells is more
sensitive to the influence of the examined imidazolones (7a, 7e and 7f) with IC50 values equal
13.51±0.77, 4.37±0.12 and 3.21±0.16 μM, respectively. In particular, sulfonamide 7f was the
most potent one with better activity (IC50 = 3.21 ± 0.16 μM) than that of the reference drug
Staurosporine (IC50 = 4.54 ± 0.17 μM).
13

Concerning activity towards breast cancer MCF-7 cells, imidazolones 7a emerged as the
most active one that exhibited potent anti-proliferative activity with IC50 value equals 7.63 ±
0.31 μM, which is better than that of the reference drug Staurosporine (IC50 = 10.41 ± 0.49
μM). Moreover, imidazolones 7e and 7f possessed moderate activity against MCF-7 cells
with IC50 values equal 14.57 ± 0.66 and 16.28 ± 0.84 μM, respectively.
Table 4. In vitro anti-proliferative activity of imidazolone-based benzenesulfonamides 7a, 7e
and 7f towards breast MCF-7 and colorectal HCT-116 cancer cell lines.
Comp.
IC50 (µM)a
HCT-116
MCF-7
7a
7.63 ± 0.31
14.57 ± 0.66
16.28 ± 0.84
10.41 ± 0.49
13.51 ± 0.77
4.37 ± 0.12
3.21 ± 0.16
4.54 ± 0.17
7e
7f
Staurosporine
a IC50 values are the mean ± S.D. of three separate experiments.
2.2.4. Cell Cycle Analysis
The effect of imidazolone-based benzenesulfonamides 7e and 7f on cell cycle
progression was evaluated in colorectal HCT-116 cells after 24 hours of treatment (Fig. 3).
This impact was determined through a DNA flow cytometric assay; where colorectal HCT-
116 cells were treated with imidazolones 7e and 7f at their IC50 concentrations (IC50 = 4.37
± 0.12 and 3.21 ± 0.16 µM, respectively).
As displayed in Fig. 3, this flow cytometric assay outcomes revealed that exposure of
colorectal HCT-116 cells to imidazolones 7e and 7f resulted in a significant increase in the
percentage of cells at Sub-G1 by 6- and 7.5-folds, respectively, with concurrent significant
arrest in the G2-M phase by 2.9- and 6.3-folds, respectively, compared to control. Both
alteration of the Sub-G1 phase and arrest of G2-M phase are considered to be significant
remarks for imidazolone-based benzenesulfonamides 7e and 7f to persuade apoptosis in
colorectal HCT-116 cells.
14

Fig. 3. Effect of imidazolone-based benzenesulfonamides 7e and 7f on the phases of cell cycle of
HCT-116 cells.
2.2.5. Annexin V-FITC Apoptosis Assay
Annexin V-FITC/propidium iodide (AV/PI) dual staining assay was carried out to
evaluate the impact of both imidazolones 7e and 7f on early and late apoptosis percentages
in colorectal HCT-116 cells (Fig. 4).
This analysis suggested that treatment of HCT-116 cells with imidazolones 7e and 7f
resulted in a significant increase in the percent of annexin V-FITC-positive apoptotic cells,
including both the early (from 0.95% to 5.09% for 7e, and from 0.95% to 7.52% for 7f) and
late apoptotic (from 0.38% to 4.4% for compound 7e, and from 0.38% to 6.21% for
compound 7f) phases (UR + LR), which represents about 7- and 10-folds total increase,
respectively, as compared with the control.
7f
Control
7e
15

Fig. 4. Effect of imidazolone-based benzenesulfonamides 7e and 7f on the percentage of annexin V-
FITC-positive staining in HCT-116 cells. The experiments were done in triplicates. The four
quadrants identified as: LL, viable; LR, early apoptotic; UR, late apoptotic; UL, necrotic.
2.3. Molecular modelling study
To point out the binding modes and to unveil the relationships between structural features
and inhibition profile of the herein reported compounds, docking and MM-GBSA-based
refinements within hCA isozymes II and IX (PDB 5LJT [33] and 5FL4 [34]) were performed.
As expected, in all compounds docking solutions the benzenesulfonamide accommodates
deeply into the active site region of both isozymes, with the negatively charged nitrogen of
zinc-binding group coordinating the metal atom. Furthermore, H-bonds are formed by the
NH- group and T199 (N-H…OG1) and by the S=O and T199 (S=O…H-N), while the phenyl
ring accommodates into an area defined by V121, V143 and L198. In hCA II, the
heterocyclic ring is stabilized by contacts with F131, V135 and L198, whereas the
unsubstituted phenyl ring of derivatives 7a-f, placed in a small cleft defined by the
hydrophilic residues N62, N67 and Q92, forms a π-π interaction with H64. The benzylidene
moiety is oriented toward the exit of the active site, assuming different orientations according
to the substituents decorating the ring (Fig. 5A).
Fig. 5. Docking of 7a (green) and 7f (blue) in hCA II (A) and in hCA IX (B) active sites.
In hCA IX, the carbonyl group of the heterocyclic ring of 7a-f is in H-bond distance with
T200 hydroxyl group, orienting the unsubstituted phenyl moiety towards a wide cleft lined by
16

L91, Q92, V121, V131, L140. It is likely that the enhanced hCA IX vs II inhibition profile of
the studied compounds derives from the better complementarity between the phenyl ring
features and the hydrophobic residues present in the aforesaid pocket of the hCA IX active
site. Again, the pendants of the benzylidene moiety determine the different orientations
assumed by this ring in the outer area of the active site (Fig. 5B).
3. Conclusion
All the tested hCA isoforms were inhibited by the prepared imidazolones 7a-f in variable
degrees with the following KIs ranges: 673.2–8169 nM for hCA I, 61.2–592.1 nM for hCA II,
23–155.4 nM for hCA XI, and 21.8–179.6 nM for hCA XII. Superiorly, imidazolone 7e
emerged as the most potent hCA IX inhibitor in this study with KI value equals 23 nM, which
is more potent than the standard drug AAZ (KI = 25 nM). Regarding selectivity, imidazolones
7a, 7e, and 7f exhibited good selectivity towards the tumor-associated isoforms (CAs IX &
XII) over the off-target cytosolic (CAs I & II) with SIs in the range of 6.2–19.4 and 3.3–8,
respectively. In particular, imidazolone 7e displayed the best selectivity profile for isoforms
hCAs IX and XII over hCAs I and II in this study (I/IX = 355, II/IX = 19.4, I/XII = 149.1 and
II/XII = 8). On the other hand, imidazolones 7a-f were screened for their anticancer activity
at one dose (10−5 M) assay towards a panel of 60 cancer cell lines according to US-NCI
protocol. Also, 7a, 7e and 7f were evaluated for their anti-proliferative activity against
colorectal cancer HCT-116 and breast cancer MCF-7 cell lines. Imidazolones 7e and 7f
possessed excellent activity against HCT-116 cells with IC50 values equal 4.37 ± 0.12 and
3.21 ± 0.16 μM, respectively. Accordingly, imidazolones 7e and 7f were screened for cell
cycle disturbance and apoptosis induction in HCT-116 cells. They were found to persuade
cell cycle arrest at G2-M stage as well as alter the Sub-G1 phase, in addition, they increased
the percent of annexinV-FITC positive apoptotic cells from 1.33% to 9.13% for compound
7e, and from 1.33% to 13.73% for compound 7f. Finally, a molecular docking study for the
prepared imidazolones within CA II and IX active sites was carried out (PDB; 5LJT and
5FL4, respectively) to rationalize the obtained results.
4. Experimental
4.1. Chemistry
4.1.1. General
17

Melting
points
were
measured
with
a
Stuart
melting
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). Chemical shifts (δH) are reported
relative to TMS as internal standard. 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 used without further purification. Compounds 4a-f are previously reported [35, 36].
4.1.2. General procedure for preparation of target imidazolone-based benzenesulfonamides
7a-f.
A mixture of equimolar quantities of azlactone 4a-f (2 mmol), sulfanilamide 5 (0.34 g, 2
mmol) and fused sodium acetate (0.16 g, 2 mmol) was refluxed in glacial acetic acid (5 mL)
for 4 hrs. The precipitated solid was collected by filtration while hot, washed with cold
methanol, dried and recrystallized from dioxan to furnish the corresponding imidazolone-
based benzenesulfonamides 7a-f with 68-77% yield.
4.1.2.1. 4-(4-Benzylidene-5-oxo-2-phenyl-4,5-dihydro-1H-imidazol-1-yl)benzenesulfonamide
(7a).
Yellow crystals (yield 77%), m.p. 211-213 °C; IR (KBr, ν cm-1): 3316 (NH2), 1705 (C=O)
and 1350, 1168 (SO2); 1H NMR (DMSO-d6)  ppm: 7.30 (s, 1H, olefinic), 7.40-7.44 (m, 4H,
Ar-H), 7.45-7.50 (m, 4H, Ar-H and D2O exchangeable NH2), 7.51-7.53 (m, 4H, Ar-H), 7.85
(d, 2H, J = 7.2 Hz, Ar-H), 8.34 (d, 2H, J = 7.2 Hz, Ar-H); 13C NMR (DMSO-d6)  ppm:
127.15, 128.65, 128.91, 129.04, 129.38, 129.46, 131.18, 132.11, 132.88, 134.48, 137.71,
138.71, 144.14, 160.86, 169.81; Anal. calcd. for C22H17N3O3S (403.46): C, 65.17; H, 4.25; N,
10.42. Found C, 65.01; H, 4.20; N, 10.38.
4.1.2.2.
4-(4-(4-Fluorobenzylidene)-5-oxo-2-phenyl-4,5-dihydro-1H-imidazol-1-
yl)benzenesulfonamide (7b).
18

Yellow crystals (yield 75%), m.p. 223-224 °C; IR (KBr, ν cm-1): 3310 (NH2), 1708 (C=O)
and 1358, 1158 (SO2); 1H NMR (DMSO-d6)  ppm: 7.33-7.52 (m, 12H; 1H olefinic, 9H Ar-
H and D2O exchangeable NH2), 7.85 (d, 2H, J = 8.4 Hz, Ar-H), 8.42-8.45 (m, 2H, Ar-H); 13C
NMR (DMSO-d6)  ppm: 116.42, 116.65, 127.13, 127.35, 128.62, 128.84, 129.01, 129.42,
131.19, 131.22, 132.10, 135.24, 135.33, 137.62, 138.29, 138.31, 144.15, 160.89, 160.91,
162.46, 164.95, 169.74; Anal. calcd. for C22H16FN3O3S (421.45): C, 62.70; H, 3.83; N, 9.97.
Found C, 62.42; H, 3.80; N, 10.03.
4.1.2.3.
4-(4-(3-Chlorobenzylidene)-5-oxo-2-phenyl-4,5-dihydro-1H-imidazol-1-
yl)benzenesulfonamide (7c).
White crystals (yield 70%), m.p. 250-252 °C; IR (KBr, ν cm-1): 3313 (NH2), 1701 (C=O) and
1350, 1168 (SO2); 1H NMR (DMSO-d6)  ppm: 7.31 (s, 1H, olefinic), 7.41-7.54 (m, 11H; 9H
Ar-H and D2O exchangeable NH2), 7.85 (d, 2H, J = 8.4 Hz, Ar-H), 8.29 (d, 1H, J = 6.4 Hz,
13
Ar-H), 8.44 (s, 1H, Ar-H); C NMR (DMSO-d6)  ppm: 126.51, 127.14, 128.66, 128.72,
129.08, 129.47, 130.59, 131.17, 131.30, 131.81, 132.29, 133.93, 136.53, 137.52, 139.70,
144.19, 161.77, 169.65; Anal. calcd. for C22H16ClN3O3S (437.90): C, 60.34; H, 3.68; N, 9.60.
Found C, 60.55; H, 3.64; N, 9.53.
4.1.2.4.
4-(4-(3-Methylbenzylidene)-5-oxo-2-phenyl-4,5-dihydro-1H-imidazol-1-
yl)benzenesulfonamide (7d).
Yellow crystals (yield 68%), m.p. 259-261 °C; IR (KBr, ν cm-1): 3312 (NH2), 1703 (C=O)
and 1353, 1165 (SO2); 1H NMR (DMSO-d6)  ppm: 2.38 (s, 3H, CH3), 7.25 (s, 1H, olefinic),
7.27 (d, 2H, J = 8.0 Hz, Ar-H), 7.38 (d, 2H, J = 8.0 Hz, Ar-H), 7.42 (s, 2H, D2O
exchangeable NH2), 744-7.53 (m, 5H, Ar-H), 7.85 (d, 2H, J = 7.2 Hz, Ar-H), 8.08 (s, 1H, Ar-
13
H), 8.21 (d, 1H, J = 7.6 Hz, Ar-H); C NMR (DMSO-d6)  ppm: 21.48 (CH3), 127.12,
128.61, 128.80, 128.90, 129.02, 129.28, 129.41, 130.00, 131.94, 132.05, 133.44, 134.38,
137.69, 138.46, 138.55, 144.08, 160.67, 169.78; Anal. calcd. for C23H19N3O3S (417.48): C,
66.17; H, 4.59; N, 10.07. Found C, 65.87; H, 4.63; N, 10.14.
4.1.2.5.
4-(4-(2,5-Dimethoxybenzylidene)-5-oxo-2-phenyl-4,5-dihydro-1H-imidazol-1-
yl)benzenesulfonamide (7e).
Orange crystals (yield 75%), m.p. 246-247 °C; IR (KBr, ν cm-1): 3310 (NH2), 1710 (C=O)
and 1358, 1163 (SO2); 1H NMR (DMSO-d6)  ppm: 3.77 (s, 3H, OCH3), 3.86 (s, 3H, OCH3),
19

7.07-7.08 (m, 2H, Ar-H), 7.39-7.52 (m, 9H, Ar-H), 7.56 (s, 1H, Ar-H), 7.85 (d, 2H, J = 8.4
13
Hz, Ar-H), 8.55 (d, 1H, J = 1.6 Hz, Ar-H); C NMR (DMSO-d6)  ppm: 55.84 (OCH3),
56.74 (OCH3), 113.05, 117.10, 118.91, 121.30, 123.15, 127.14, 128.61, 128.88, 129.07,
129.32, 132.10, 137.72, 138.18, 144.11, 153.46, 154.00, 160.46, 169.75; Anal. calcd. for
C24H21N3O5S (463.51): C, 62.19; H, 4.57; N, 9.07. Found C, 61.93; H, 4.60; N, 9.13.
4.1.2.6.
4-(5-Oxo-2-phenyl-4-(3,4,5-trimethoxybenzylidene)-4,5-dihydro-1H-imidazol-1-
yl)benzenesulfonamide (7f).
Orange crystals (yield 71%), m.p. 271-272 °C; IR (KBr, ν cm-1): 3303 (NH2), 1708 (C=O)
1
and 1357, 1157 (SO2); H NMR (DMSO-d6)  ppm: 3.74 (s, 3H, OCH3), 3.84 (s, 6H, 2
OCH3), 7.26 (s, 1H, olefinic), 7.39-7.53 (m, 9H; 7H Ar-H and D2O exchangeable NH2), 7.82
13
(s, 2H, Ar-H), 7.86 (d, 2H, J = 8.8 Hz, Ar-H); C NMR (DMSO-d6)  ppm: 56.37 (OCH3),
60.69 (OCH3), 110.58, 127.13, 128.59, 128.85, 128.92, 129.08, 29.27, 129.90, 132.06,
137.77, 137.78, 140.52, 144.10, 153.28, 159.99, 169.71; Anal. calcd. for C25H23N3O6S
(493.53): C, 60.84; H, 4.70; N, 8.51. Found C, 61.05; H, 4.68; N, 8.42.
4.2. Biological Evaluation
4.2.1. CA inhibitory assay
An Applied Photophysics stopped-flow instrument has been used for assaying the CA
catalysed CO2 hydration activity, as reported earlier [25]. The inhibition constants were
obtained by non-linear least-squares methods using PRISM 3 and the Cheng-Prusoff equation
as reported earlier, and represent the mean from at least three different determinations. The
four tested CA isofoms were recombinant ones obtained in-house as reported earlier [37-41].
4.2.2. In Vitro Antitumor Activity towards 60 cancer cell lines
The antitumor assay was performed according to the protocol of the Drug Evaluation
Branch, NCI, Bethesda [42-44]. A 48 h drug exposure protocol was adopted, and
sulforhodamine B (SRB) assay [26] was utilized to assess the cell growth and viability, as
reported earlier [45, 46].
4.2.3. Antiproliferative activity against MCF-7 and HCT-116 cell lines
Breast cancer MCF-7 and colorectal cancer HCT-116 cell lines were obtained from
American Type Culture Collection (ATCC). MCF-7 cells were grown in RPMI-1640 while
20

HCT-116 cells were grown in DMEM. The cells were supplemented with 10% heat-
inactivated fetal bovine serum, 1% L-glutamine (2.5 mM), HEPES buffer (10 mM), 50
μg/mL gentamycin. All cells were maintained at 37 C in a humidified atmosphere with 5%
CO2. Cytotoxicity was determined following the MTT assay, as reported earlier [47, 48].
4.2.4. Cell Cycle Analysis
Colorectal HCT-116 cells were treated with imidazolones 7e and 7f for 24 hour at their
IC50 concentration, and then cells were washed twice with ice-cold phosphate buffered saline
(PBS). Subsequently, the treated cells were collected by centrifugation, fixed in ice-cold 70%
(v/v) ethanol, washed with PBS, re-suspended with 100 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) [49,
50].
4.2.5. 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 [51, 52].
4.2.6. Molecular docking simulation
The crystal structure of hCA II (PDB 5LJT [33]) and hCA IX (PDB 5FL4 [34]) were
prepared using the Protein Preparation Wizard tool implemented in Maestro - Schrödinger
suite, assigning bond orders, adding hydrogens, deleting water molecules, and optimizing H-
bonding networks [53]. 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 [53a] and evaluated for
their ionization states at pH 7.4 ± 0.5 with Epik [53b]. OPLS3 force field in Macromodel
[53e] 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 Glide used with default
settings. Ligands were docked with the standard precision mode (SP) of Glide [53f] and the
best 5 poses of each molecule retained as output. The best pose for each compound, evaluated
21

in terms of coordination, hydrogen bond interactions and hydrophobic contacts, was refined
with Prime [53d] with a VSGB solvatation model considering the target flexible within 3Å
around the ligand [54-56].
Acknowledgements
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.
References
[1] C.T. Supuran, Carbonic anhydrases: from biomedical applications of the inhibitors and
activators to biotechnological use for CO2 capture, in, Taylor & Francis, 2013.
[2] C.T. Supuran, Carbonic anhydrases: novel therapeutic applications for inhibitors and
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28

Graphical abstract
Synthesis, biological evaluation and in silico studies with 4-benzylidene-2-
phenyl-5(4H)-imidazolone-based benzenesulfonamides as novel selective
carbonic anhydrase IX inhibitors endowed with anticancer activity
A series of 4-benzylidene-2-phenyl-5(4H)-imidazolone-based benzenesulfonamides 7a-f was
synthesized via the Erlenmeyer–Plöchl reaction. All the prepared imidazolones 7a-f were
evaluated as inhibitors of cytosolic isoforms hCA I and II, as well as transmembrane tumor-
associated isoforms hCA IX and XII.
O
O
O
O
S
N
N
H2N
O
7e
29