Design, synthesis, biological evaluation, and computational studies of novel tri-aryl imidazole-benzene sulfonamide hybrids as promising selective carbonic anhydrase IX and XII inhibitors

Article abstract of DOI:10.3390/molecules26164718

A novel series of tri-aryl imidazole derivatives 5a–n carrying benzene sulfonamide moiety has been designed for their selective inhibitory against hCA IX and XII activity. Six compounds were found to be potent and selective CA IX inhibitors with the order of 5g > 5b > 5d > 5e > 5g > 5n (Ki = 0.3–1.3 μM, and selectivity ratio for hCA IX over hCA XII = 5–12) relative to acetazolamide (Ki = 0.03 μM, and selectivity ratio for hCA IX over hCA XII = 0.20). The previous sixth inhibitors have been further investigated for their anti-proliferative activity against four different cancer cell lines using MTT assay. Compounds 5g and 5b demonstrated higher antiproliferative activity than other tested compounds (with GI₅₀ = 2.3 and 2.8 M, respectively) in comparison to doxorubicin (GI₅₀ = 1.1 M). Docking studies of these two compounds adopted orientation and binding interactions with a higher liability to enter the active side pocket CA-IX selectively similar to that of ligand 9FK. Molecular modelling simulation showed good agreement with the acquired biological evaluation.

Full text of DOI:10.3390/molecules26164718

molecules
Article
Design, Synthesis, Biological Evaluation, and Computational
Studies of Novel Tri-Aryl Imidazole-Benzene Sulfonamide
Hybrids as Promising Selective Carbonic Anhydrase IX and
XII Inhibitors
Lamya H. Al-Wahaibi 1,*, Bahaa G. M. Youssif 2,
* ,
, Ehab S. Taher 3, Ahmed H. Abdelazeem 4,5
Antar A. Abdelhamid 6,7 and Adel A. Marzouk 8
1
Department of Chemistry, College of Sciences, Princess Nourah Bint Abdulrahman University, P.O. Box 84428,
Riyadh 11564, Saudi Arabia
Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt
Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Al-Azhar University,
Assiut 71524, Egypt; ehaborganic@yahoo.com
Department of Medicinal Chemistry, Faculty of Pharmacy, Beni-Suef University, Beni-Suef 62514, Egypt;
ahmed.abdelazeem@pharm.bsu.edu.eg
Department of Pharmaceutical Sciences, College of Pharmacy, Riyadh Elm University,
Riyadh 11681, Saudi Arabia
Department of Chemistry, Faculty of Science, Sohag University, Sohag 82524, Egypt; drantar25@yahoo.com
Chemistry Department, Faculty of Science, Albaha University, P.O. Box 1988, Albaha 65731, Saudi Arabia
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Al-Azhar University, Assiut 71524, Egypt;
adelmarzouk@azhar.edu.eg
2
3
4
5
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
6
7
8
Citation: Al-Wahaibi, L.H.; Youssif,
B.G.M.; Taher, E.S.; Abdelazeem,
A.H.; Abdelhamid, A.A.; Marzouk,
A.A. Design, Synthesis, Biological
Evaluation, and Computational
Studies of Novel Tri-Aryl
*
Correspondence: lhalwahaibi@pnu.edu.sa (L.H.A.-W.); bgyoussif@ju.edu.sa (B.G.M.Y.);
Tel.: +966-554101115 (L.H.A.-W.); +20-1098294419 (B.G.M.Y.)
Abstract: A novel series of tri-aryl imidazole derivatives 5a–n carrying benzene sulfonamide moiety
Imidazole-Benzene Sulfonamide
Hybrids as Promising Selective
Carbonic Anhydrase IX and XII
Inhibitors. Molecules 2021, 26, 4718.
https://doi.org/10.3390/
has been designed for their selective inhibitory against hCA IX and XII activity. Six compounds
were found to be potent and selective CA IX inhibitors with the order of 5g > 5b > 5d > 5e > 5g > 5n
(Ki = 0.3–1.3 µM, and selectivity ratio for hCA IX over hCA XII = 5–12) relative to acetazolamide
(Ki = 0.03 µM, and selectivity ratio for hCA IX over hCA XII = 0.20). The previous sixth inhibitors
have been further investigated for their anti-proliferative activity against four different cancer cell
lines using MTT assay. Compounds 5g and 5b demonstrated higher antiproliferative activity than
other tested compounds (with GI50 = 2.3 and 2.8 M, respectively) in comparison to doxorubicin
(GI50 = 1.1 M). Docking studies of these two compounds adopted orientation and binding inter-
actions with a higher liability to enter the active side pocket CA-IX selectively similar to that of
ligand 9FK. Molecular modelling simulation showed good agreement with the acquired biological
evaluation.
molecules26164718
Academic Editor: George
Mihai Nitulescu
Received: 28 June 2021
Accepted: 2 August 2021
Published: 4 August 2021
Keywords: imidazole; benzene sulfonamide; carbonic anhydrase IX; XII
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1. Introduction
Carbonic anhydrases (CAs) are metalloenzymes that catalyze the reversible hydration
of CO2 into protons and bicarbonate [1]. CAs are involved in many physiological and
pathological processes, including respiration and the transport of CO2 and bicarbonate
between metabolizing tissues and the lungs, electrolyte secretion in various tissues and
organs, biosynthetic reactions bone resorption, calcification, and tumor growth. [1]. As a
result, several CA isoforms involved in these processes are important therapeutic targets,
and their inhibitors have a high potential for pharmacological intervention in a variety
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
of diseases such as glaucoma, obesity, osteoporosis, and cancer [
2
,3]. There are now 16
different mammalian—CA isoforms recognized, including human isoforms [
4]. These CAs
Molecules 2021, 26, 4718. https://doi.org/10.3390/molecules26164718
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 4718
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are classified as cytosolic (CA I, CA II, CA III, CA VII, and CA XIII), membrane bound
(CA IV, CA IX, CA XII, CA XIV, and CA XV), and mitochondrial (CA VA and CA VB).
Cytosolic hCA I and II are the most common isoforms, and inhibitors of these isoforms are
primarily utilized as antiglaucoma, diuretics, antiepileptic, antiedema medications, and
for the treatment of altitude sickness [
mostly focused on the hCA IX and XII isoforms [
found in the GI mucosa and malignancies [ ]. CA IX is involved in a number of functions,
1
,4,5
]. However, in recent years, cancer research has
6,7
]. CA IX is a transmembrane isoform
8
including carcinogenesis, pH control, tumor cell proliferation and migration, and cell
adhesion. CA IX expression is regulated by tumor hypoxia, which has been identified
as a risk factor for the progression of a variety of malignancies [9,10]. CA IX inhibition
could be a promising anticancer therapeutic target. Similarly, the CA XII isoform has been
linked to the progression of many cancers [11]. CA XII is a transmembrane isoform found
in a variety of organs including the eye, malignancies, prostate, ovaries, intestines, and
kidney. Because of its expression in numerous organs, it has been linked to a variety of
malignancies [12,13]. Because hCA IX/XII have been shown to play a significant role
in tumor growth and metastasis, they have been identified as promising targets for the
development of novel antineoplastic drugs. The biggest impediment to developing new
cancer therapies by targeting hCA IX/XII occurred as a result of nonselective inhibition of
hCA I/ II isoforms, which was thought to be the main cause of the undesired side effects
reported with these nonselective inhibitors [1,5]. The majority of the investigated inhibitors
against one or more hCAs are sulfonamide-containing aromatic/heterocyclic compounds,
where the sulfonamide moiety acts as a zinc binding group (ZBG) and the heterocyclic
scaffold is thought to selectively promote interactions with isoform unique residues at
the active site cavity [5,14]. SLC-0111 (Figure 1), a sulfonamide derivative with strong
selectivity against the CA IX isoform, has entered clinical trials as a single agent for solid
tumors [15] or as part of a combination therapy for pancreatic ductal carcinoma [16].
Figure 1. Structure of SLC-0111, compound I and IIIa–f.
In recent years, the aryl imidazole scaffold has gained recognition as a helpful and
promising scaffold for the design and development of effective CA inhibitors. The syn-
thesis and biological evaluation of novel 4-phenyl-imidazol-2(3H)-one derivatives as CA

Molecules 2021, 26, 4718
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inhibitors were described by Congiu et al. [17], (Compound
I
, Figure 1). These imidazole
compounds were discovered to be selective nanomolar inhibitors of CAs IX and XII. An-
other class of 1,2,4-trisubstituted imidazoles with a 4-benzylidene moiety and primary and
secondary sulfonamide groups that act as dual carbonic anhydrase (isoforms I, II, IV, and
IX) and p38-MAPK inhibitors were designed and synthesized (IIa–f in Figure 1) [18]. The
results of this study’s SAR revealed that the presence of a primary sulfamoyl group is vital
for the CA inhibitory activity.
Encouraged by the facts mentioned above and mindful of the significance of continu-
ous development of selective hCA IX inhibitors to feed into anticancer discovery pipeline,
we report the synthesis of a new series of 2,4,5-triphenylimidazole-benzensulfonamides
5a–n (Scaffold A, Figure 2) using a one-pot single-step multicomponent reaction including
primary aromatic amine (sulfanilamide), aldehydes, and benzil. The newly synthesized
compounds contain primary sulfonamide group as Zinc-binding group (ZBG). The substi-
tution pattern on the pendant 2-aryl moiety was chosen to provide a variety of lipophilic
and electronic environments that would affect the CA inhibitory action of 5a
–n. All
the synthesized imidazoles 5a were characterized and biologically tested against the
–
n
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. A
molecular docking study for the prepared imidazoles within CA IX active sites was carried
out to rationalize the obtained results. Moreover, the most potent and selective hCA IX
inhibitors 5b, 5d, 5e, 5g, 5j and 5n were assessed for their antiproliferative activity against
four different cancer cell lines.
Figure 2. Rational design and structural modification of Scaffold A.
2. Results and Discussion
2.1. Chemistry
Tri-aryl imidazole-benzene sulphonamide hybrids 5a
a one-pot single-step multicomponent reaction involving primary aromatic amine (sul-
fanilamide), aldehydes 4a , benzil, and ammonium acetate in the presence of diethyl
–n were synthesized by adopting
–n
ammonium hydrogen sulfate (ionic liquid) as shown in Scheme 1.
With simple work-up processes and good yields of the target compounds, the reaction
time ranged from 15 to 30 min. All the newly synthesized compounds were analyzed by
IR, 1H NMR, 13C NMR, and elemental analyses. In the 1H NMR spectra of chemical 5i
(Ar = 3,4-dimethoxyphenyl), two singlet signals were common for methoxy groups: one at
3.52 ppm (OCH3) and the second at 3.76 ppm (OCH3) protons, as well as a doublet signal
at 7.77–7.79 of 2H associated to NH2 of the sulfonamide moiety. Element analyses were
used to confirm the purity of the produced compounds, and the results agreed with the
products’ molecular formula.

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Scheme 1. Synthesis of compounds 5a–n.
2.2. Biology
Newly synthesized hybrids 5a
–n
were tested for their inhibitory activity against four
physiologically and pharmacologically CA isoforms, namely cytosolic hCA I and II, and
tumor-associated isoforms hCA IX and XII, using a CO2 hydrase stop-flow assay [19].
Acetazolamide (AAZ), clinically used sulphonamide, was used as the standard drug. The
inhibition data of 5a
shown to inhibit the cytosolic isoforms hCA I and hCA II (Ki > 100
showed a number of inhibitory profiles against the transmembrane tumor-associated iso-
form hCA IX. Compounds 5g 5b 5d 5e 5j and 5n had sub micromolar inhibitory potencies
against hCA IX (Kis) of 0.32, 0.36, 0.72, 0.84, 1.10, and 1.3 M, respectively, with a pro-
–
n
were cited in Table 1. Generally, 5a
–
n
compounds have not been
µM). Compounds 5a
–n
,
,
,
,
µ
nounced selectivity for hCA IX over hCA XII (12, 11.50, 7.00, 6.50, 5.60, and 5.0, respectively)
in comparison to AAZ (selectivity ratio for hCA IX over hCA XII = 0.20) [20,21]. In addition,
compounds 5a
brane tumor-associated isoform, hCA XII, with Kis below 7
Among these compounds, compounds 5g (Ar = 2-MeO-phenyl) and 5b (Ar = 2-Br-phenyl)
showed promising inhibitory activity with a Ki value of 3.7 and 4.6 M, respectively. The
–
n
displayed a variable degree of inhibition against the other transmem-
µM for all these compounds.
µ
ortho substitution in the aryl moiety appears to correlate with higher inhibitory effects
against both hCA IX and XII. Notably, the activity increased with (Ar) in the order of
2-MeO-phenyl > 2-Br-phenyl > 2-HO-phenyl > 2-NO2-phenyl > 2-thienyl. In comparison
to 5e (Ar = 2-HO-phenyl, Ki = 0.8 µM), compound 5f (Ar = 4-HO-phenyl) had a 10-fold
lower inhibitory activity on hCA IX (Ki = 8.4 M). The similar pattern holds true for 5g
(Ar = 2-MeO-phenyl) and 5h (Ar = 4-MeO-phenyl), with 5h being 22-fold less reactive than
the ortho-derivative. From these results, 5a
–n derivatives exhibited selective inhibition of
hCA IX and XII compared to hCA I and II in the micromolar range.

Molecules 2021, 26, 4718
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Finally, the inhibitory activities of the newly synthesized 5a
–
n
against hCAs could
serve as a preliminary study for future biological studies on these new scaffolds.
Table 1. Inhibition of hCA isoforms
I
,
II
,
IX and XII with compounds 5a
–
n
and acetazolamide (AAZ).
Selectivity Ratio
hCA XII/ hCA IX
Compound
hCA I
hCA II
hCA IX
hCA XII
5a
5b
5c
5d
5e
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.25
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.012
2.9
0.4
8.2
0.7
0.8
8.4
0.3
7.4
9.2
1.1
9.3
8.5
8.8
1.3
0.026
5.5
4.6
3.7
4.9
5.2
7.1
3.7
3.9
6.6
6.2
6.8
6.8
6.2
6.4
0.006
1.90
11.50
0.45
7.00
6.50
0.90
12.00
0.50
0.70
5.60
0.70
0.80
0.70
5.00
0.20
5f
5g
5h
5i
5j
5k
5l
5m
5n
AAZ
2.2.1. Structure Activity Relationship Study
To the best of our knowledge, the existence of ortho-position in the aryl moiety for our
rationalized compounds has been correlated crucially to this higher selectivity. Results
depicted that the structure–activity relationship of our synthesized targeted compounds
are as following (Figure 2):
Occupying the ortho-position with lipophilic, as well as more of the electron-donating
group, i.e., 5g (Ar = 2-MeO-phenyl, Ki values 0.3 and 3.7 µM respectively, against hCA IX
and XII) has showed excellent selectivity.
The replacement of this group with a bromine atom, such as 5b (Ar = 2-Br-phenyl,
Ki values = 0.4 and 4.6 M, respectively) has also shown an experienced selective inhi-
µ
bition. This might be attributed to the advantages of electron donating (+M) rather than
withdrawing (-I) for such an atom.
Similarly, the ortho-2,6-dichloro 5d (Ar = 2,6-diCl-phenyl, Ki values = 0.7 and 4.9 µM,
respectively) has also presented desirable selectivity with the consideration of higher (+M)
for the bromine than the chlorine atom, albeit with the existence of two chlorine atoms.
Changing the methoxy group into a hydrophilic one, i.e., 5e (Ar =2-HO-phenyl,
Ki values = 0.8 and 5.2 µM, respectively) has likely displayed acceptable selectivity.
Installment of strong electron withdrawing atom at the same position or alteration of
the aryl group to hetero-aryl one, i.e., 5j (Ar =2-NO2-phenyl, Ki value = 1.1 and 6.2
µM,
respectively) and 5n (Ar = thienyl, Ki value = 1.3 and 6.4
µM, respectively) has offered a
reasonable selectivity.
Attempting of exploring the para position for such hybrids seemed unlikely to be
useful for this purpose, such as compound 5f (Ar = 4-HO-phenyl) which had a 10-fold
lower inhibitory activity on hCA IX (Ki = 8.4
µM) in comparison with 5e (Ar = 2-HO-
phenyl, Ki = 0.8 µM). Similarly, 5h (Ar = 4-MeO-phenyl, Ki = 7.4
µM) displayed 22-fold
less reactivity in comparison to the relevant ortho-derivative 5g (Ar = 2-MeO-phenyl,
Ki = 0.3 µM).
2.2.2. In Vitro Anticancer Activity
The antiproliferative activity of 5b, 5d, 5e, 5g, 5j and 5n against four human cancer
cell lines including pancreatic cancer cell line (Panc-1), breast cancer cell line (MCF-7),
colon cancer cell line (HT-29) and epithelial cancer cell line (A-549) using MTT assay and
doxorubicin was applied as the reference compound [22,23]. Graph Pad Prism software

Molecules 2021, 26, 4718
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(Graph Pad Software, San Diego, CA, USA) was used to calculate the median inhibition
concentration (IC50) and results are recorded in Table 2.
Table 2. Antiproliferative activity of compounds 5b, 5d, 5e, 5g, 5j, 5n and Doxorubicin.
Antiproliferative Activity IC50 ± SEM (µM)
Comp.
Cell Viability %
A-549
MCF-7
Panc-1
HT-29
Average
5b
5d
5e
5g
5j
5n
91
87
86
90
88
90
-
2.8 ± 0.5
3.1 ± 0.6
4.4 ± 0.7
2.4 ± 0.5
4.6 ± 0.6
4.7 ± 0.6
1.2 ± 0.8
2.7 ± 0.4
3.9 ± 0.4
3.6 ± 0.8
2.1 ± 0.2
3.6 ± 0.2
4.6 ± 0.6
0.9± 0.6
2.5 ± 0.2
3.8 ± 0.3
3.7 ± 0.5
2.2 ± 0.4
4.6 ± 0.9
4.8 ± 0.3
1.4 ±0.6
3.1 ± 0.1
3.1 ± 0.4
3.9 ± 0.6
2.4 ± 0.4
4.8 ± 0.4
4.8 ± 0.2
1.0 ± 0.8
2.8
3.5
3.9
2.3
4.4
4.7
1.1
Doxorubicin
The o-methoxy derivative 5g (Ar = 2-Meo-phenyl) was the most potent of all tested
derivatives, with mean GI50 value of 2.3
µM against the four cell lines, in comparison
with the reference doxorubicin (GI50 = 1.1
µM). Compound 5b (Ar = 2-Br-phenyl) showed
higher antiproliferative activity compared to 5d (Ar = 2,6-dichlorophenyl) with GI50 of
2.8 and 3.5 M, respectively, against the tested cell lines, suggesting the importance of the
µ
ortho-methoxy group for the antiproliferative activity, Table 2.
Despite having a 10-fold lower efficacy than acetazolamide in enzymatic assays and a
2-fold lower efficacy than doxorubicin in cellular assays, the newly synthesised compounds
5b
potent derivatives.
The o-methoxy derivative 5g (Ar = 2-Meo-phenyl) was the most potent of all tested
derivatives, with mean GI50 value of 2.3 M against the four cell lines, in comparison
to the reference doxorubicin (GI50 = 1.1 M). Compound 5b (Ar = 2-Br-phenyl) showed
higher antiproliferative activity compared to 5d (Ar = 2,6-dichlorophenyl) with GI50 of
2.8 and 3.5 M, respectively, against the tested cell lines, suggesting the importance of the
, 5d, 5e, 5g, 5j and 5n could serve as a starting point for future development of more
µ
µ
µ
ortho-methoxy group for the antiproliferative activity, Table 2.
2.3. Docking Study
Carbonic anhydrases are a ubiquitous metalloenzyme family containing zinc in their
active sites. CA-IX is a well-known tumor-associated isoenzyme which is highly overex-
pressed in different types of cancers, with a limited number in normal tissues [24]. Thus, it
is considered a promising drug target for treating cancer. Inspired by the highest inhibitory
activity exerted by some of our compounds 5a–n against CA-IX over the other CA iso-
forms, a docking simulation was performed in order to better understand the molecular
mechanisms of inhibition and to determine the binding pattern and the key interactions of
the newly synthesized inhibitors within the catalytic active site of CA-IX. It is important to
know that the catalytic site of CA IX is comprised of two distinct regions with a Zn2+ atom
located at the bottom of the active site. The first region is a hydrophobic defined by Leu-91,
Val-121, Val-131, Leu-135, Leu-141, Val-143, Leu-198, and Pro-202 amino acid residues while
the second region is a hydrophilic identified by Asn-62, His-64, Ser-65, Gla-67, Thr-69, and
Gln-92 residues [25].
The docking process was conducted using LibDock tool embedded in the Discovery
Studio software 2.5 (San Diego, CA, USA). The 3D crystal structure of CA-IX enzyme
(PDB ID: 5FL4, resolution = 1.82 Å) in complex with 2-thiophene-sulfonamide ligand
(
9FK), which acts as a highly efficient inhibitor, has been retrieved from Protein Data Bank
(https://www.rcsb.org/structure/5FL4. Accessed on 10 June 2021) [26]. The CA-IX protein
structure is a dimer where only one chain was used for the docking study. The amino acid
residues within a distance of 10 Å around the co-crystallized ligand in the active pocket
were isolated and the key interactions and ligands orientation were analyzed. Ligands
and protein files were prepared according to the previous reports [27,28]. The 2D and

Molecules 2021, 26, 4718
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3D binding modes of 9FK and compounds 5a
–n
were visualized using Discovery Studio
Visualizer [29].
The docking results of some representative compounds (5g, 5b, 5d, 5k, and 5i) selected
according to the activity range (high, moderate, and low) were analyzed and depicted in
Figures 3–6 in comparison with the co-crystalized sulfonamide ligand 9FK. Interestingly,
the docking results were in agreement with the results of the in vitro CA-IX inhibitory
assay. It is well-reported that sulfonamide or sulfamate moiety is a crucial and essential
structural feature in potent and specific inhibitors of CA-IX inhibitors where it coordinates
the zinc ion in active site and inhibits the enzymatic activity [30–34]. Therefore, the new
compounds contain a sulfonamide as a core component owing to their significant inhibitory
activity against CA-IX isoform. Inspection of the binding patterns of compounds 5g and
5b with the highest activity (IC50 = 0.32 and 0.36 µM, respectively) revealed that they bind
very similarly within the CA-IX active pocket with a very similar binding mechanism and
orientation to that of co-crystalized ligand 9FK. The deprotonated sulfonamide moiety
was positioned toward the hydrophobic region in the active site, where its nitrogen atom
formed a coordination bond with the Zn2+ cation such as 9FK and became involved in
a H-bond with His94 amino acid. Moreover, the two oxygen atoms of the SO2 group in
compound 5g formed a network of critical H-bonding interactions with key residues in the
active site, including Leu199 Thr200 and Thr201 in addition to one more optimal H-bond
between the methoxy group and Gln92 amino acid, exceeding the number of H-bond
interactions formed by ligand 9FK. However, the other two unsubstituted phenyl groups
of 5g, equivalent to the naphthyl moiety of 9FK, were positioned at the entrance of the
catalytic active site cleft forming some van der Waals and hydrophobic interactions with
Pro202 and Pro203 residues. The 2D and 3D binding interactions of 5g and 9FK within the
active site of CA-IX enzyme were shown in Figure 3A–D.
In Figure 4A, compound 5b showed almost an identical binding mode engaging
similar interactions to that exerted by 5g. The sulfonamide moiety was positioned towards
the bottom of the active site where a coordination bond was formed in addition to the
same network H-bonding interactions with the well-known key amino acid residues, such
as His94, Leu199, Thr200 and Thr201. The absence of the crucial H-bond with Gln92
emerged as a result of replacing the methoxy group with a bromo moiety in 5b, owing
to the very slight difference in activities between the two superior compounds 5g and
5b. The superimposition of 9FK, 5g and 5b, as shown in Figure 4B, suggested a similar
orientation and binding pattern of the three ligands, explaining the superior activity with
the same inhibition mechanism inside CA-IX catalytic pocket. Meanwhile, the docking of
5d revealed an exhibition of disposition similar to 5g forming a coordination bond with
Zn2+ ion through the one oxygen of sulfonamide instead of the nitrogen atom. Additionally,
the absence of some key H-bonds with Gln92, His94, Leu199 and Thr201 residues was
observed. In addition, the change in the methoxy group with dichloro atoms, i.e., 5d
,
resulted in some clashes in the hydrophobic region of the CA-IX active site. These reasons
can obviously clarify the moderate inhibitory activity of 5d (IC50 = 0.72
the remarkable activity of 5g (IC50 = 0.32 µM).
µM) compared to
Investigation of docking results of compounds 5k and 5i, which had minimal potency
(IC50 = 9.3 and 9.2 M), revealed a completely inverse orientation and binding mode
µ
compared to the co-crystallized ligand 9FK or the active compounds such as 5g and 5b. It
was found that the sulfonamide moiety was pushed outside the active site in the opposite
side of the Zn2+ cation location and the two unsubstituted phenyl groups were occupied
the hydrophobic region near the bottom of the catalytic active site. This inverse disposition
resulted in a real loss of the crucial H-bonding interactions with the key-amino acids (His94,
Leu199, Thr200 and Thr201) and the important coordination bond with Zn2+ cation which
in turn caused the remarkable reduction in the inhibitory activity of the corresponding
ligands 5k and 5i, Figure 5A–D. It was worth noting that these two respected derivatives,
5k and 5i, bearing the bulkiest substituents on one of the phenyl groups (dimethylamine
or dimethoxy, respectively) in the series. Based on that, it was conceptualized that these

Molecules 2021, 26, 4718
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bulky groups could not be accommodated within the active site and did not coordinate
with the Zn2+ ion. Accordingly, these groups preferred to protrude outside the catalytic
active site owing to the sharp decrease in their activity in comparison with 9FK, 5g and
5b. Figure 5D showed this disparity clearly through the superimposition of 5k and the
co-crystallized ligand 9FK.
Figure 3. Comparison of docking and 2/3D binding modes of compound 5g (stick with carbons colored in orange) and
co-crystallized inhibitor 9FK (stick with carbons colored in green) within the catalytic active site of CA-IX isoenzyme (PDB
code: 5FL4); (
mode of 9FK showing different types of interactions inside the active site of CA-IX isoenzyme; (
compound 5g into active site of CA-IX isoenzyme; ( ) 2D docking mode of 5g showing different types of interactions inside
A
) 3D binding mode of the co-crystallized ligand 9FK into active site of CA-IX isoenzyme; (
B) 2D docking
C
) 3D binding mode of
D
the active site of CA-IX isoenzyme. Zn2+ cation is presented as a blue sphere in coordination with His94, His96, His119 and
-NHSO2 shown in dashed dark blue lines. H-bonds are represented as dashed green lines. All hydrogens were removed for
the purpose of clarity.

Molecules 2021, 26, 4718
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Figure 4. Comparison of docking and 2/3D binding modes of compound 5b (stick with carbons colored in yellow) and
compound 5d (stick with carbons colored in grey) within the catalytic active site of CA-IX isoenzyme (PDB code: 5FL4); (
3D binding mode of compound 5b into active site of CA-IX isoenzyme; ( ) the superimposition of 5g 5b and 9FK docked
poses within the catalytic active site of CA-IX isoenzyme; ( ) 3D binding mode of compound 5d into active site of CA-IX
isoenzyme; ( ) 2D docking mode of 5d showing different types of interactions inside the active site of CA-IX isoenzyme.
A)
B
,
C
D
Zn2+ cation is presented as a blue sphere in coordination with His94, His96, His119 and -NHSO2 shown in dashed dark
blue lines. H-bonds were represented as dashed green lines. All hydrogens were removed for the purpose of clarity.
To conclude our study, and pointing out the variation in the biological results in a
collective way, the top docking poses 5g
active pocket and displayed in Figure 6A–C. Obviously, the poses 5g
,
5b and 9FK were overlaid into CA-IX catalytic
5b and 9FK were
,
found to fit nicely and demonstrated good shapes complementarity with the active site
of the enzyme, while compounds 5k exhibited a completely converse orientation without
forming the essential interactions required for the CA-IX inhibition mechanism. Taken
together, the docking study provided a clear explanation for the in vitro CA-IX assay results
and pointed out the right positioning, crucial structural features and key-interactions of
potent CA-IX inhibitors that greatly contribute to designing and developing potential leads
for CA-IX as a promising anticancer drug target.

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Figure 5. The docking and 2/3D binding modes of compound 5k (stick with carbons colored in blue) and compound 5i
(stick with carbons colored in cyan) within the catalytic active site of CA-IX isoenzyme (PDB code: 5FL4); ( ) 3D binding
mode of compound 5k into active site of CA-IX isoenzyme; ( ) 2D docking mode of 5i showing different types of interactions
inside the active site of CA-IX isoenzyme; ( ) 3D binding mode of compound 5i into active site of CA-IX isoenzyme; (D)
A
B
C
2D docking mode of 5i showing different types of interactions inside the active site of CA-IX isoenzyme. Zn2+ cation
is presented as a blue sphere in coordination with His94, His96, His119 and -NHSO2 shown in dashed dark blue lines.
H-bonds are represented as dashed green lines. All hydrogens were removed for the purpose of clarity.
Figure 6. Overlay of the docked poses 5d
isoenzyme (PDB code: 5FL4). The protein is represented as: (
secondary structure displayed in a solid ribbon (cartoon) style; (
,
5k
,
5i and the co-crystallized ligand 9FK into the catalytic active site of CA-IX
) solid surface colored according to atom charges; (
) the window shows 3D magnification of superimposition
A
C
B)
of the four docked poses within the catalytic active site of CA-IX isoenzyme. The poses were rendered as stick model colored
in green (9FK), blue (5d), orange (5k) and yellow (5i). Zn2+ cation is presented as a blue sphere. H-bonds are represented as
dashed green lines. All hydrogens were removed for the purpose of clarity.

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3. Materials and Methods
3.1. Chemistry
General Details: See Appendix A.
General Procedure for Synthesis of 4-(4,5-Diphenyl-2-(substituted
benzene)-1H-imidazol-1-yl)benzene Sulfonamides 5a–n
In a rounded 50 mL flask, benzil
1
(2.10 g, 5mmol) was added to an equivalent amount
(0.77 g, 10 mmol), the appropriate
of sulfanilamide (1.72 g, 10 mmol), ammonium acetate
2
3
aldehyde 4a–n (10 mmol), and diethyl ammonium hydrogen sulfate (0.51 g, 3 mmol). The
mixture was refluxed in an oil bath with stirring at 110 ◦C for about 15 min. The reaction
was monitored with TLC using chloroform: ethanol (9:1). The reaction mixture was poured
into iced water and the resulting precipitate was filtered off, washed with water, and then
dried. The crude product was crystallized from ethanol.
4-(2,4,5-Triphenyl-1H-imidazol-1-yl)benzene sulfonamide 5a
Yellowish white solid, Yield: 70%; m.p.: 210–212 ◦C; 1H NMR (400 MHz, DMSO)
7.20–7.30 (m, 5H, ArH), 7.34–7.46 (m, 12H, ArH), 7.54 (d, J = 7.1 Hz, 2H, ArH), 7.76 (d,
J = 7.1 Hz, 2H, NH2) ppm; 13C NMR (100 MHz, DMSO)
: 146.77, 144.60, 139.81, 137.71,
δ:
δ
134.68, 131.67, 131.55, 130.67, 130.59, 129.80, 129.10, 129.02, 128.74, 128.64, 127.06, 126.93,
126.89 ppm. Anal. Calcd for C27H21N3O2S (451.14): C, 71.82; H, 4.69; N, 9.31. Found: C,
71.86; H, 4.67; N, 9.33%.
4-(2-(2-Bromophenyl)-4,5-diphenyl-1H-imidazol-1-yl)benzene sulfonamide 5b
White solid, Yield: 78%; m.p.: 220–222 ◦C; 1H NMR (400 MHz, DMSO)
(m, 3H, ArH), 7.34–7.38 (m, 3H, ArH), 7.43–7.54 (m, 4H, ArH), 7.61–7.67 (m, 6H, ArH),
7.68–7.96 (4H, 2ArH + NH2) ppm; 13C NMR (100 MHz, DMSO)
: 146.13, 144.20, 138.82,
δ: 7.20–7.30
δ
137.36, 136.38, 135.93, 134.45, 133.69, 132.88, 132.79, 131.92, 131.45, 130.03, 129.93, 129.26,
129.18, 128.86, 128.67, 127.94, 127.88, 127.13, 126.92, 126.53, 124.32, 121.72, 113.03 ppm. Anal.
Calcd for C27H20BrN3O2S (529.05): C, 61.14; H, 3.80; N, 7.92. Found: C, 61.11; H, 3.78; N,
7.89%.
4-(2-(4-Chlorophenyl)-4,5-diphenyl-1H-imidazol-1-yl)benzene sulfonamide 5c
◦
Yellowish solid, Yield: 75%; m.p.: 230–232 C; 1H NMR (400 MHz, DMSO)
(m, 5H, ArH), 7.35–7.40 (m, 7H, ArH), 7.45–7.53 (m, 6H, ArH), 7.77 (d, J = 8.1 Hz, 2H, NH2)
ppm; 13C NMR (100 MHz, DMSO)
: 145.64, 144.73, 139.56, 137.88, 134.51, 133.92, 131.81,
δ: 7.20–7.29
δ
131.64, 130.64, 130.42, 129.76, 129.48, 129.23, 129.11, 128.86, 128.65, 127.14, 127.05, 126.88
ppm. Anal. Calcd for C27H20ClN3O2S (485.10): C, 66.73; H, 4.15; N, 8.65. Found: C, 66.70;
H, 4.12; N, 8.62%.
4-(2-(2,6-Dichlorophenyl)-4,5-diphenyl-1H-imidazol-1-yl)benzene sulfonamide 5d
Yellow solid, Yield: 80%; m.p.: 214–216 ◦C; 1H NMR (400 MHz, DMSO)
(m, 7H, ArH), 7.61–7.65 (m, 4H, 2ArH+ NH2), 7.78–7.80 (m, 8H, 8ArH) ppm.13C NMR (100
MHz, DMSO) : 195.24, 135.86, 132.83, 131.98, 131.17, 129.97, 129.88, 129.00, 128.52, 127.93,
δ: 7.45–7.49
δ
127.43, 126.91, 113.08, 111.91, 111.54 ppm. Anal. Calcd for C27H19Cl2N3O2S (519.06): C,
62.31; H, 3.68; N, 8.07. Found: C, 62.33; H, 3.70; N, 8.10%.
4-(2-(2-Hydroxyphenyl)-4,5-diphenyl-1H-imidazol-1-yl)benzene sulfonamide 5e
◦
Whitish solid, Yield: 74%; m.p.: 250–252 C; 1H NMR (400 MHz, DMSO)
NH2), 6.72–6.76 (m, 4H, ArH), 7.00–7.02 (m, 2H, ArH), 7.40–7.64 (m, 12H, ArH), 8.42 (broad
s, 1H, OH) ppm; 13C NMR (100 MHz, DMSO)
: 165.59, 155.50, 139.09, 135.07, 134.64,
δ: 5.90 (s, 2H,
δ
131.55, 129.39, 128.76, 128.44, 128.01, 127.31, 126.68, 122.85, 119.25, 115.76 ppm. Anal. Calcd
for C27H21N3O2S (467.13): C, 69.36; H, 4.53; N, 8.99. Found: C, 69.39; H, 4.57; N, 9.02%.

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4-(2-(4-Hydroxyphenyl)-4,5-diphenyl-1H-imidazol-1-yl)benzene sulfonamide 5f
Pale yellow solid, Yield: 68%; m.p.: 240–242 ◦C; 1H NMR (400 MHz, DMSO)
δ: 6.73
(d, J = 8.6 Hz, 2H, ArH), 7.18–7.27 (m, 7H, ArH), 7.40 (d, J = 8.4, 5H, ArH), 7.48–7.54 (m,
4H, ArH), 7.78 (d J = 8.4, 2H, NH2), 9.76 (s, 1H, OH) ppm; 13C NMR (100 MHz, DMSO)
δ
:
158.29, 147.25, 144.42, 140.08, 137.39, 134.84, 131.67, 130.84, 130.81, 130.56, 129.78, 129.04,
128.57, 126.92, 121.49, 115.62 ppm. Anal. Calcd for C27H21N3O2S (467.13): C, 69.36; H, 4.53;
N, 8.99. Found: C, 69.38; H, 4.56; N, 9.01%.
4-(2-(2-Methoxyphenyl)-4,5-diphenyl-1H-imidazol-1-yl)benzene sulfonamide 5g
White solid, Yield: 82%; m.p.: 268–270 ◦C; 1H NMR (400 MHz, DMSO)
(m, 2H, ArH), 7.17–7.27 (m, 11H, ArH), 7.34–7.41 (m, 4H, ArH), 7.48–7.57 (m, 3H, ArH),
7.61–7.64 (m, 2H, NH2); 13C NMR (100 MHz, DMSO)
: 156.95, 145.40, 143.64, 139.78,
δ: 6.68–7.06
δ
137.54, 134.80, 132.53, 131.62, 131.48, 130.70, 129.95, 129.19, 129.01, 128.61, 128.47, 126.90,
126.13, 120.89, 120.07, 111.67, 55.09 ppm. Anal. Calcd for C28H23N3O3S (481.15): C, 69.84;
H, 4.81; N, 8.73. Found: C, 69.87; H, 4.82; N, 8.72%.
4-(2-(4-Methoxyphenyl)-4,5-diphenyl-1H-imidazol-1-yl)benzene sulfonamide 5h
Yellow solid, Yield: 85%; m.p.: 235–237 ◦C; 1H NMR (400 MHz, DMSO)
OCH3), 6.88 (d, J = 8.2 Hz, 2H, ArH), 7.18–7.33 (m, 12H, ArH), 7.47 (d, J = 20.8 Hz and 7.9
Hz, 4H, ArH), 7.74 (d, J = 8.1 Hz, 2H, NH2) ppm; 13C NMR (100 MHz, DMSO)
: 159.89,
δ: 3.76 (s, 3H,
δ
146.72, 144.47, 139.94, 137.40, 134.76, 131.66, 131.11, 130.70, 130.40, 129.84, 129.08, 128.61,
126.92, 126.83, 123.04, 114.24, 55.66 ppm. Anal. Calcd for C28H23N3O3S (481.15): C, 69.84;
H, 4.81; N, 8.73. Found: C, 69.87; H, 4.87; N, 8.77%.
4-(2-(3,4-Dimethoxyphenyl)-4,5-diphenyl-1H-imidazol-1-yl)benzene sulfonamide 5i
◦
Pale yellow solid, Yield: 73%; m.p.: 260–262 C; 1H NMR (400 MHz, DMSO)
3H, CH3), 3.76 (s, 3H, CH3), 6.84–7.05 (3H, ArH), 7.26–7.35 (9H, ArH), 7.46–7.53 (m, 5H,
ArH), 7.77–7.79 (d, J = 8.5 Hz, 2H, NH2) ppm; 13C NMR (100 MHz, DMSO)
: 149.57, 148.53,
δ: 3.52 (s,
δ
146.68, 144.59, 140.10, 137.41, 134.75, 131.66, 131.14, 130.66, 129.97, 129.10, 128.62, 126.94,
126.90, 122.98, 121.81, 112.53, 112.00, 55.97, 55.66 ppm. Anal. Calcd for C29H25N3O4S
(511.16): C, 68.08; H, 4.93; N, 8.21. Found: C, 68.12; H, 4.97; N, 8.24%.
4-(2-(2-Nitrophenyl)-4,5-diphenyl-1H-imidazol-1-yl)benzene sulfonamide 5j
Yellowish solid, Yield: 86%; m.p.: 280–282 ◦C; 1H NMR (400 MHz, DMSO)
(m, 10H, ArH), 7.45–7.47 (m, 2H, ArH), 7.64–7.76 (m, 5H, ArH), 7.90–8.16 (m, 3H, 1ArH + NH2
δ: 7.18–7.38
)
ppm. Anal. Calcd for C27H20N4O4S (496.12): C, 65.31; H, 4.06; N, 11.28. Found: C, 65.35; H,
4.10; N, 11.24%.
4-(2-(4-(Dimethylamino)phenyl)-4,5-diphenyl-1H-imidazol-1-yl)benzene sulfonamide 5k
White solid, Yield: 76%; m.p.: 224–226 ◦C; 1H NMR (400 MHz, DMSO)
CH3), 3.03 (s, 3H, CH3), 6.58–6.79 (m, 3H, ArH), 7.19–7.49 (m, 15H, ArH), 7.68–7.75 (m, 2H,
NH2) ppm.13C NMR (100 MHz, DMSO)
: 152.32, 150.63, 147.59, 144.25, 140.31, 137.29,
δ: 2.88 (s, 3H,
δ
134.83, 132.05, 131.66, 130.81, 130.71, 130.61, 129.96, 129.83, 129.02, 128.56, 127.87, 126.89,
117.73, 113.09, 111.87, 111.56, 39.24 ppm. Anal. Calcd for C29H26N4O2S (494.18): C, 70.42;
H, 5.30; N, 11.33. Found: C, 70.46; H, 5.33; N, 11.34%.
4-(4,5-Diphenyl-1-(4-sulfamoylphenyl)-1H-imidazol-2-yl)benzoic acid 5l
◦
1
Pale yellow solid, Yield: 90%; m.p.: 200–202 C; H NMR (400 MHz, DMSO)
7.20–7.37 (m, 5H, ArH), 7.40–7.51 (m, 4H, ArH), 7.57–7.69 (m, 4H, ArH), 7.77–6.88 (m, 3H,
ArH), 7.93–8.23 (m, 5H, 3ArH+ NH2 + OH) ppm; 13C NMR (100 MHz, DMSO)
167.25,
145.71, 144.76, 139.56, 138.16, 135.96, 134.44, 132.21, 131.64, 130.94, 130.29, 130.03, 129.96,
δ:
δ

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129.77, 129.67, 129.31, 129.14, 128.88, 128.69, 127.23, 127.10, 126.90 ppm. Anal. Calcd for
C28H21N3O4S (495.13): C, 67.87; H, 4.27; N, 8.48. Found: C, 67.90; H, 4.30; N, 8.50%.
Ethyl 4-(4,5-diphenyl-1-(4-sulfamoylphenyl)-1H-imidazol-2-yl)benzoate 5m
◦
1
White solid, Yield: 88%; m.p.: 216–218 C; H NMR (DMSO, 400 MHz)
J = 7.0 Hz, 3H, CH3), 3.36–3.48 (m, 2H, CH2), 7.21–7.88 (m, 20H, 18 ArH+ NH2) ppm.13C
NMR (100 MHz, DMSO) : 167.26, 145.75, 144.79, 139.58, 138.18, 134.44, 132.22, 131.65,
δ: 1.07 (t,
δ
131.08, 130.32, 129.76, 129.63, 129.28, 129.12, 128.86, 128.66, 127.21, 127.07, 126.91, 56.53,
18.95 ppm. Anal. Calcd for C30H25N3O4S (523.16): C, 68.82; H, 4.81; N, 8.03. Found: C,
68.86; H, 4.83; N, 7.99%.
4-(4,5-Diphenyl-2-(thiophen-2-yl)-1H-imidazol-1-yl)benzene sulfonamide 5n
◦
1
Yellowish white solid, Yield: 92%; m.p.: 290–292 C; H NMR (400 MHz, DMSO)
δ
: 6.58 (d, J = 3.2, 1H, thienyl-H), 6.95–7.32 (m, 10H, 3H thienyl and 7ArH) 7.52 (dd,
J = 17.0 Hz and 7.52 Hz, 4H, ArH), 7.64 (d, J = 8.3, 2H, ArH), 7.86 (d, J = 8.3, 2H, NH2) ppm;
13C NMR (100 MHz, DMSO)
δ
: 145.45, 141.80, 139.31, 137.72, 135.93, 134.34, 132.95, 131.59,
130.30, 130.20, 129.99, 129.95, 129.64, 129.22, 129.08, 128.65, 127.92, 127.23, 126.85, 126.39
ppm. Anal. Calcd for C25H19N3O2S2 (457.09): C, 65.62; H, 4.19; N, 9.18. Found: C, 65.66; H,
4.23; N, 9.22%.
3.2. Biology
3.2.1. Carbonic Anhydrase Assay
The ability of imidazoles 5a–n toward inhibition of hCA I, II (cytosolic) as well as hCA
IX and XII (transmembrane, tumor-associated isoforms) using stopped-flow CO2 hydrase
assay was evaluated [19]. See Appendix A.
3.2.2. Cell Viability Assay
Cell viability assay was carried out using human mammary gland epithelial cell line
(MCF-10A) [20]. MCF-10A cells were incubated with compounds 5b, 5d, 5e, 5g, 5j and 5n
for 4 days and MTT assay [21] was used to determine the viability of cells. See Appendix A.
3.2.3. Antiproliferative Activity
The antiproliferative activity of 5b, 5d, 5e, 5g, 5j and 5n against four human cancer
cell lines including pancreatic cancer cell line (Panc-1), breast cancer cell line (MCF-7),
colon cancer cell line (HT-29) and epithelial cancer cell line (A-549) using MTT assay and
doxorubicin was applied as the reference compound [22,23]. See Appendix A.
4. Conclusions
A novel tranche of triaryl imidazole derivatives bearing benzene sulfonamide moiety
has been rationalized and synthesized. The ability of imidazoles 5a–n toward inhibition
of hCA I, II (cytosolic) as well as hCA IX and XII (transmembrane, tumor-associated
isoforms) using stopped-flow CO2 hydrase assay was evaluated. Results displayed that
our targets 5a
II in the micromolar range. The most potent and selective hCA IX inhibitors 5g
5e 5j and 5n with Ki values of 0.3, 0.4, 0.7, 0.8, 1.1 and 1.3 M, respectively, were further
–
n
had selective inhibition of hCA IX and XII in comparable with hCA I and
,
5b 5d,
,
,
µ
assessed for their anti-proliferative activity against four different cancer cell lines using
MTT assay in comparison to doxorubicin as reference standard. Anti-proliferative assay
displayed that compound 5g showed higher inhibitory activity other tested ones in the
order of 5g
> 5b > 5d > 5e > 5j > 5n. Molecular docking study demonstrated high docking
scores and good binding interactions of the most active compounds 5g and 5b within
the hCA-IX active pocket with adoption of similar orientation to that of co-crystalized
ligand 9FK. This concludes our assumption of the impact of electronic rich environment of
ortho-position rather than other ones (meta- and para-) and its impact as a pivotal feature

Molecules 2021, 26, 4718
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for developing highly selective hCA IX and XII inhibitors. Overall, our novel hybrids have
opened the door to a new significant approach for the imidazole scaffold being engaged
with benzene sulfonamide via a strategy that appeared to be little studied up until now.
Indeed, compounds 5g and 5b are likely to be prospective lead candidates for further
consideration and optimization to explore more about the structure–activity relationship
and release novel, potent hCA IX and XII selective inhibitors agent.
Author Contributions: Conceptualization, B.G.M.Y., E.S.T. and A.A.M.; software, A.A.A. and A.H.A.;
validation, B.G.M.Y., E.S.T. and A.A.M.; formal analysis, L.H.A.-W.; investigation, B.G.M.Y., E.S.T. and
A.A.M.; resources, L.H.A.-W.; data curation, A.A.A. and A.H.A.; writing—original draft preparation,
B.G.M.Y., E.S.T., A.H.A. and A.A.M.; writing—review and editing, B.G.M.Y.; visualization, A.A.A.
and A.H.A.; supervision, E.S.T.; project administration, B.G.M.Y. and A.A.M.; funding acquisition,
L.H.A.-W. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the Deanship of Scientific Research at Princess Nourah bint
Abdulrahman University through the Fast-track Research Funding Program.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are not available from the authors.
Appendix A. Experimental
Appendix A.1. Chemistry
General Details: Melting points were determined with a Gallenkamp (London, UK)
melting point apparatus and are uncorrected. Infrared spectra (νmax) were recorded
on Bruker Vector, 22FT-IR [Fourier Transform Infrared (FTIR), Germany] spectrometer.
1H NMR and 13C NMR spectra were recorded at room temperature on a Varian Gemini-
400 (400 MHz, Foster City, CA, USA) spectrometer operating at 400 MHz for proton and
100 MHz for carbon nuclei using dimethylsulphoxide and/or (DMSO/D2O) as solvent
and tetramethylsilane (TMS) as an internal standard (chemical shift in
signal due to residual [(CH3)2SO] appearing at H 2.50 and the central resonance of the
C 39.0 were used to reference 1H and 13C NMR
) [multiplicity,
δ, ppm). The
δ
[(CD3)2SO] “multiplet” appearing at
spectra, respectively. H NMR data are recorded as follows: chemical shift (
δ
1
δ
coupling constant(s) J (Hz), relative integral] where multiplicity is defined as s = singlet;
d = doublet; t = triplet; q = quartet; and m = multiplet or combinations of the above. High-
resolution measurements were conducted on a time-of-flight instrument. High-resolution
EI mass spectra were recorded on a magnetic-sector machine. All the results of elemental
analyses corresponded to the calculated values within experimental error. Progress of the
reaction was monitored by thin-layer chromatography (TLC) using precoated TLC sheets
with Ultraviolet (UV) fluorescent silica gel (Merck 60F254) and spots were visualized by
iodine vapours or irradiation with UV light (254 nm). All the starting materials and reagents
were generally commercially available and purchased from Sigma-Aldrich (Kenilworth,
NJ, USA) or Lancaster Synthesis Corporation (Lancashire, UK).
Appendix A.2. In Vitro Carbonic Anhydrase Inhibitory Assay
The carbonic anhydrase catalyzed CO2 hydration actions for all new compounds
herein reported have were assayed utilizing an instrument of Applied Photophysics
stopped-flow. The enzymes are recombinant proteins prepared in our lab. Phenol red (at a
concentration of 0.2 mM) has been used as indicator, working at the absorbance maximum
of 557 nm, with 20 mM Hepes (pH 7.5) as buffer, and 20 mM Na2SO4 (for maintaining
constant the ionic strength), following the initial rates of the CA-catalyzed CO2 hydration
reaction for a period of 10–100 s. The CO2 concentrations ranged from 1.7 to 17 mM for

Molecules 2021, 26, 4718
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the determination of the kinetic parameters and inhibition constants. For each inhibitor
at least six traces of the initial 5–10% of the reaction have been used for determining the
initial velocity. The uncatalyzed rates were determined in the same manner and subtracted
from the total observed rates. Stock solutions of inhibitor (0.1 mM) were prepared in
distilled-deionized water and dilutions up to 0.01 nM were done thereafter with the assay
buffer. Inhibitor and enzyme solutions were preincubated together for 15 min at room
temperature prior to assay, in order to allow for the formation of the E-I complex. The
inhibition constants were obtained by non-linear least-squares methods using PRISM 3
and the Cheng-Prusoff equation, and represent the mean from at least three different
determinations.
Appendix A.3. Cytotoxic Activity Using MTT Assay and Evaluation of IC50
Appendix A.3.1. MTT Assay
MTT assay was carried out to study the effect of compounds on mammary epithelial
cells (MCF-10A) [20
modified Eagle’s medium (DMEM)/Ham’s F-12 medium (1:1) supplemented with epider-
mal growth factor (20 ng/mL), hydrocortisone (500 ng/mL), insulin (10 g/mL), 2 mM
,21]. The medium in which cells were propagated contained Dulbecco’s
µ
glutamine and 10% fetal calf serum. After every 2–3 days, the cells were passaged using
trypsin ethylenediamine tetra acetic acid (EDTA). The cells were seeded at a density of
104 cells mL−1 in flat-bottomed culture plates containing 96 wells each. After 24 h, medium
was removed from the plates and the compounds in (in 0.1% DMSO) were added (in
200 µL medium to yield a final concentration of 0.1% v/v) to the wells of plates. A single
compound was designated with four wells followed by incubation of plates for 96 h at
37 ◦C. After incubation, medium was removed completely from the plates followed by
addition of MTT (0.4 mg/mL in medium) to each well and subsequent incubation of plates
for 3 h. MTT (along with the medium) was removed and DMSO (150 µL) was added
to each well of the culture plates, followed by vortexing and subsequent measurement
of absorbance (at 540 nm) using microplate reader. The data are shown as percentage
inhibition of proliferation in comparison with controls containing 0.1% DMSO.
Appendix A.3.2. Assay for Antiproliferative Effect
To explore the antiproliferative potential of compounds MTT assay was performed
according to previously reported procedure [22,23] using different cell lines To explore
the antiproliferative potential of compounds propidium iodide fluorescence assay was
performed using different cell lines. To calculate the total nuclear DNA, a fluorescent dye
(propidium iodide, PI) is used which can attach to the DNA, thus offering a quick and
precise technique. PI cannot pass through the cell membrane and its signal intensity can
be considered as directly proportional to quantity of cellular DNA. Cells whose cell mem-
branes are damaged or have changed permeability are counted as dead ones. The assay was
performed by seeding the cells of different cell lines at a density of 3000–7500 cells/well
◦
(in 200
µ
L medium) in culture plates followed by incubation for 24 h at 37 C in humidified
5% CO2/95% air atmospheric conditions. The medium was removed; the compounds were
added to the plates at 10 M concentrations (in 0.1% DMSO) in triplicates, followed by
incubation for 48 h. DMSO (0.1%) was used as control. After incubation, medium was
removed followed by the addition of PI (25 L, 50 g/mL in water/medium) to each well
of the plates. At
µ
µ
µ
−
80 ◦C, the plates were allowed to freeze for 24 h, followed by thawing
at 25 ◦C. A fluorometer (Polar-Star BMG Tech, Offenburg, Germany) was used to record
the readings at excitation and emission wavelengths of 530 and 620 nm for each well. The
percentage cytotoxicity of compounds was calculated using the following formula:
%Cytotoxicity = (Ac − Atc)/Ac × 100
where Atc = Absorbance of treated cells and Ac = Absorbance of control. Erlotinib was
used as positive control in the assay.

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Appendix A.4. Statistical Analysis
Computerized Prism 5 program was used to statistically analyzed data using one-way
ANOVA test followed by Tukey’s as post ANOVA for multiple comparison at p
Data were presented as mean ± SEM.
≤
0.05.
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