Discovery of a novel series of indolylchalcone-benzenesulfonamide hybrids acting as selective carbonic anhydrase II inhibitors

Article abstract of DOI:10.1016/j.bioorg.2021.104647

The primary sulfonamide group is one of the most efficient zinc binding group (ZBG) for designing carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. In the present study primary sulfonamide linked with indolylchalcone were designed. The newly synthesized molecules (5a-r) were examined against four human (h) CA isoforms (hCA I, hCA II, hCA IX and hCA XIII). These sulfonamides showed good inhibition activity against isoforms hCA I, hCA II and hCA XIII. Compound 5i (2.3 nM), 5m (2.4 nM), 5o (3.6 nM) and 5q (7.0 nM) were more potent than standard drug AAZ (12.1 nM) against isoform hCA II, respectively. Most of the other compounds in the present series inhibited hCA XIII and hCA IX in the range of 50 nM ? 100 nM.

Full text of DOI:10.1016/j.bioorg.2021.104647

Bioorganic Chemistry 108 (2021) 104647
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Discovery of a novel series of indolylchalcone-benzenesulfonamide hybrids
acting as selective carbonic anhydrase II inhibitors
Priti Singha, Parvatha Purnachander Yadava, Baijayantimala Swaina, Pavitra S. Thackera,
,
,c,*
Andrea Angelib, Claudiu T. Supuranb *, Mohammed Arifuddina
a Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), Balanagar, Hyderabad 500037, India
b
`
UniversitadegliStudi di Firenze, Neurofarba Dept, Sezione di ScienzeFarmaceutiche e, Nutraceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino, Florence, Italy
c Department of Chemistry, Anwarul Uloom College, 11-3-918, New Malleypally, Hyderabad-500001, T. S, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
The primary sulfonamide group is one of the most efficient zinc binding group (ZBG) for designing carbonic
anhydrase (CA, EC 4.2.1.1) inhibitors. In the present study primary sulfonamide linked with indolylchalcone
were designed. The newly synthesized molecules (5a-r) were examined against four human (h) CA isoforms (hCA
I, hCA II, hCA IX and hCA XIII). These sulfonamides showed good inhibition activity against isoforms hCA I, hCA
II and hCA XIII. Compound 5i (2.3 nM), 5m (2.4 nM), 5o (3.6 nM) and 5q (7.0 nM) were more potent than
standard drug AAZ (12.1 nM) against isoform hCA II, respectively. Most of the other compounds in the present
series inhibited hCA XIII and hCA IX in the range of 50 nM ꢀ 100 nM.
Carbonic anhydrase
Knoevenagel condensation
Acetazolamide
Indolylchalcone- benzenesulfonamide hybrids
Selective hCA II inhibition
1. Introduction
strong affinities for isoforms II, VA, VB, VI, VII, IX, XII, XIII and XIV
[6–7]. Various other reports also claim that indole containing moieties
Carbonic anhydrase (CA, EC 4.2.1.1) is a well-explored enzyme in
the pharmacological research field. Inhibition of the different isoforms
of carbonic anhydrase (CA) has been used in the treatment of a variety of
disorders such as edema, osteoporosis, glaucoma, epilepsy, and obesity
for decades, whereas applications in the field of cancer started to be
considered in the last 15 years. These is due to the fact that CAs are
involved in various important physiological functions such as meta-
bolism, respiration, pH regulation, ion transport, bone resorption and
secretion of gastric juice and other fluids (e.g., aqueous humor in the
eye, cerebrospinal fluid, etc.) [1–3]. A total of 16 isozymes have been
have significant inhibitory properties against CA (Fig. 1) [8–10]. Simi-
larly, the chalcone is a well-known moiety which posses various bio-
logical properties. [11]
Sulfonamides represent a significant class of biologically active
compounds and most of the human carbonic anhydrase isoforms (hCA)
are inhibited by aromatic sulfonamides [12]. Aromatic sulfonamides are
known as specific and strong inhibitors of CA isoforms [13–14] and
considered as prototype drugs for additional modification and devel-
opment of new and more potent inhibitors (Fig. 1) [14] But lack of
selectivity in inhibiting various isoforms is the major drawback with the
use of CAIs, that results from structural similarity and identical subcel-
lular localization of these isoforms producing undesirable side effects
[15,16].
previously reported as members of the mammalian α-CA family. CAs I,
II, III, VII, and XIII are cytosolic isoenzymes, CAs VA and VB are local-
ized in the mitochondria, CA VI is a unique secreted isozyme, CAs IX, XII,
and XIV are transmembrane proteins, and CAs IV and XV are GPI-
anchored to the cell membrane [4]. Exploration of CA inhibitors has
been done by many researchers but still many aspects have remained to
be investigated.
In the last two decades, CAIs research has been focused on designing
isoform-selective sulfonamide inhibitors by employing two principal
methods: the ring and the tail approaches. The first resides in modu-
lating the ring (chiefly its chemical nature) directly connected to the
sulfonamide ZBG, whereas the other consists of attaching different tails
to the aromatic/heterocyclic ring carrying various ZBGs like sulfon-
amide, sulfamide, sulfamate, carboxylate, hydroxamate, or
At least 25 clinically used drugs have been reported to possess sig-
nificant CA inhibitory properties [5]. Indisulam is an indole-containing
anti-cancer drug in clinical trials which strongly inhibits CA, having
* Corresponding authors at: Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), Balanagar, Hyderabad
500037, India.
E-mail addresses: claudiu.supuran@unifi.it (C.T. Supuran), arifabib@gmail.com (M. Arifuddin).
https://doi.org/10.1016/j.bioorg.2021.104647
Received 2 December 2020; Received in revised form 11 December 2020; Accepted 6 January 2021
Available online 19 January 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

P. Singh et al.
Bioorganic Chemistry 108 (2021) 104647
dithiocarbamate. This enabled modulation of the interactions that the
ligand establishes with the middle and outer parts of the active site
cavity, which are the most variable regions among the 16 hCA isoforms
mentioned above, and led to a variety of isoform-selective CAIs.
In this article we explore indolylchalcone moiety using tail approach
as CA inhibitors. In our previous research work [17], we have explored
indolychalcone linked to benzsulfonamide moiety through a triazole
linker and they were extremely good inhibitor of selectively hCA I in
subnanomolar range. In addition, various reports were found which
stated that presence of an amide bond showed potent inhibition of CA
isoforms (Fig. 1). In the present study the target molecules were
designed by replacing the triazole linker with an amide linker in such a
way that it contains benzsulfonamide as head, indolylcalcone as tail and
an amide bond as linker and to test these synthesized compounds against
CA isoforms, hCAs I, II, IX and XIII for their inhibitory activity (Fig. 2).
the final target compounds (5a-r) by using reported literature method
[20]. The structures of all the compounds (5a-r) were elucidated on the
basis of 1H NMR, 13C NMR and HRMS.
2.2. Carbonic anhydrase inhibition
The newly synthesized compounds (5a-r) have been assayed for their
inhibition against the four isoforms, the cytosolic isoforms, hCA I, hCA II
and hCA XIII as well as the trans-membrane tumor associated isoform,
hCA IX by means of stopped flow carbon dioxide assay [21]. Here
acetazolamide (AAZ) was taken as the standard drug. The result of the
synthesized compounds against different hCA isoforms are summarized
in Table 1:-
From the data given in the Table 1 the following inferences were
made:-
2. Results and discussion
1) The ubiquitous cytosolic isoform hCA I was weakly inhibited by all
the synthesized compounds with Ki values ranging from 166.9 nM ꢀ
2.1. Chemistry
6.6 μM. The 2-bromo substituted derivative (5 m) and 3,4,5-trime-
thoxy substituted derivative (5o) exhibited the best inhibition
amongst all the compounds with Ki values of 166.9 nM and 673 nM
respectively.
The synthetic strategy employed for the synthesis of the target
compounds is depicted in Scheme 1. The chalcones (2a-r) were syn-
thesized by Knoevenagel condensation by reacting indole-3-
carboxaldehyde with different acetophenones using piperidine as the
base and ethanol as solvent [18]. On the other hand the sulfanilamide
(3) was N-acetylated with chloroacetyl chloride to get (4) [19] which
was subsequently subjected to nucleophilic substitution with indo-
lylchalcones (2a-r) in the presence of K2CO3 in DMF as solvent to afford
2) All the synthesized compounds were strongly inhibited the physio-
logically dominant isoform hCA II with Ki values ranging between
2.3 nM and 136.8 nM. Among all the synthesized compounds, the
compounds 5i (2.3 nM), 5 m (2.4 nM), 5o (3.6 nM) and 5q (7.0 nM)
were found to be more potent hCA II inhibitors compared to the
standard AAZ. The compound 5i (Ki = 2.3 nM) and 5 m (2.4 nM)
Fig. 1. Structure of clinically used drugs containing amide bond.
2

P. Singh et al.
Bioorganic Chemistry 108 (2021) 104647
Fig. 2. Rational to design target molecule.
Scheme 1. Synthetic Route for (E)-2-(3-(3-oxo-3-phenylprop-1-en-1-yl)-1H-indol-1-yl)-N-(4-sulfamoylphenyl)acetamides. Reagents and conditions: a) Piperidine
(0.5 equiv), EtOH, reflux, 24 h, b) 2-chloro-N-(4-sulfamoylphenyl)acetamide 4 (1 equiv), K2CO3 (3 equiv), DMF, 60 ◦C, C) K2CO3, acetone, 0 ◦C 1 h.
were 5 times more potent than AAZ (Ki = 12.1 nM) against hCA II.
Similarly, the compound 5q (7.0 nM) was 2 times more active than
AAZ. Apart from them other compounds also inhibited hCA II with Ki
values < 100 nM (except 5f {136.6 nM}).
4) The cytosolic isoform hCA XIII was also inhibited by the compounds
5o, 5p and 5r in a low to moderate nanomolar range with Ki values
of 18.4 nM, 18.5 nM and 19.4 nM respectively compared to standard
AAZ (17 nM). The most potent inhibitor against this isoform was
found to be the compound 5o, 5p and 5r with 3,4,5-trimethoxy, 4-
trifluoromethyl and 3-chloro substitution respectively.
3) The tumor-associated isoform hCA IX was also inhibited by all the
synthesized compounds (5a-r) in the 29 nM to 160 nM range. Among
all the compounds, the 9 compounds i.e., 5a (32.4 nM), 5e (30.3
nM), 5f (32.1 nM), 5 g (29.9 nM), 5i (31.0 nM), 5 m (32.4 nM), 5p
(31.5 nM), 5q (29.8 nM) and 5r (31.0 nM) inhibition constant values
with compare to standard AAZ (25.8 nM).
3. Conclusion
Novel indolychalcone-benzsulfonamide hybrids linked with an
3

P. Singh et al.
Bioorganic Chemistry 108 (2021) 104647
Table 1
Table 1 (continued)
KI (nM)*
Inhibition of hCA isoforms I, II, IX and XIII with compounds 5a-r and acet-
Cmp
5p
R
hCA I
3625
hCAII
60.2
hCA IX
31.5
hCA XIII
18.4
5q
5r
775.3
6630
250.0
7.0
29.8
31.0
25.8
42.9
19.4
17.0
azolamide (AAZ) as standard inhibitor
66.1
12.1
KI (nM)*
AAZ
*
Cmp
5a
R
hCA I
5702
hCAII
97.6
hCA IX
32.4
hCA XIII
50.0
Mean from 3 different assays, by a stopped flow technique (errors were
5b
5c
5d
5e
2498
1520
3505
3765
27.5
26.6
30.7
53.5
72.1
61.2
159.9
30.3
114.3
47.0
i31.0n the range of ± 5–10% of the reported values)
amide bond (5a-r) were synthesized. All the newly synthesized com-
pounds were evaluated for their CA inhibitory profile against cytosolic
isoforms (hCA I, II and XIII) and membrane-bound isoform (hCA IX).
Most of them were found to be more selective inhibitors of hCA II with
compare to other hCA isoforms hCA I, hCA IX and hCA XIII. The results
displayed 4 compounds 5i (2.3 nM), 5 m (2.4 nM), 5o (3.6 nM) and 5q
(7.0 nM)) displayed more potencies than AAZ against hCA II and
remaining were active under 100 nM range except 5f (Ki 136.8 nM). In
the case of hCA XIII, the compounds 5o, 5p and 5r showed the best
inhibition with Ki values of 18.4 nM, 18.5 nM and 19.4 nM respectively.
In the case of hCA IX, nine compounds exhibited the best inhibition with
Ki values < 50 nM. Whereas, all these compounds showed weak po-
tencies against hCA I with submicromolar range. Thus, 5i (2.3 nM), 5 m
(2.4 nM), 5o (3.6 nM) and 5q (7.0 nM)) will be taken as lead for further
development of potent hCA II inhibitors that target ocular disorders and
cancer via their structural modifications.
146.6
117.6
5f
5764
136.8
37.5
32.1
29.9
110.8
127.9
5 g
964.1
5 h
5i
3463
3744
2019
4086
21.4
2.3
139.5
31.0
51.4
4. Experimental section
120.2
163.8
63.8
4.1. General
All solvents were purified and dried using standard methods prior to
use. Commercially available reagents were used without further purifi-
cation. All reactions involving air- or moisture sensitive compounds
were performed under a nitrogen atmosphere using dried glassware and
syringe techniques to transfer solutions. Analytical thin-layer chroma-
tography (TLC) was carried out on Merck silica gel 60F-254 aluminium
plates. Melting points were determined on Stuart digital melting-point
apparatus/SMP 30 in open capillary tubes and uncorrected. Nuclear
magnetic resonance (1H NMR, 13C NMR) spectra were recorded using
an Avance Brucker 500 MHz, 125 MHz spectrometer in DMSO‑d6.
Chemical shifts reported in parts per million (ppm) with TMS as an in-
ternal reference, and the coupling constants (J) expressed in hertz (Hz).
Splitting patterns are denoted as follows: s, singlet; d, doublet; t, triplet;
m, multiplet; dd, doublet of doublet. HRMS were determined with
Agilent QTOF mass spectrometer 6540 series instrument and were
performed in the ESI techniques at 70 eV.
5j
30.7
62.1
106.1
88.3
5 k
5 l
840.0
166.9
18.9
2.4
106.8
32.4
74.3
5 m
184.4
5n
5o
4640
94.4
3.6
90.0
40.4
18.5
4.2. General procedures for the synthesis of compounds
672.3
107.8
4.2.1. General procedure for synthesis of indolylchalcone (2a-r)
A mixture of indole-3-carboxaldehyde 1 (1 equiv) was treated with
substituted acetophenones (1 equiv), piperidine (0.5 equiv) in ethanol
and refluxed for 24 h at 60 ◦C. The progress of the reaction was moni-
tored by TLC. After the completion of the reaction the crushed-ice was
added into the reaction mixture and neutralized with glacial acetic acid
4

P. Singh et al.
Bioorganic Chemistry 108 (2021) 104647
to obtian solid product 2. The solid formed was filtered under high
vacuum and washed with hexane and dried over to get the product in
80–90% yield.
111.3, 49.8, 21.6; HRMS (ESI) m/z calcd for C26H23N3O4SNa [M + Na+]
496.1320, found 496.1307.
4.2.3.5. (E)-2-(3-(3-(3-bromophenyl)-3-oxoprop-1-en-1-yl)-1H-indol-1-
4.2.2. General procedure for synthesis of 2-chloro-(4-sulfamoylphenyl)
acetamide (4)
yl)-N-(4-sulfamoylphenyl). acetamide (5e): Yellow solid, (yield 82%); m.
◦
p 270–272 C; FT-IR (
ν
cmꢀ 1): 3269, 1679, 1650, 1591, 1526, 1394,
1289, 1156; 1H NMR (500 MHz, DMSO‑d6, TMS) δ (ppm): 10.80 (s, 1H),
8.22 (d, J = 11.3 Hz, 2H), 8.15 (dd, J = 8.8, 4.9 Hz, 2H), 8.08 (d, J =
15.4 Hz, 1H), 7.85 (d, J = 8.4 Hz, 1H), 7.80–7.75 (m, 4H), 7.66 (d, J =
15.4 Hz, 1H), 7.59–7.53 (m, 2H), 7.33–7.29 (m, 2H), 7.26 (s, 2H), 5.23
(s, 2H); 13C NMR (125 MHz, DMSO‑d6, TMS) δ (ppm): 188.26, 166.61,
149.94, 143.99, 141.93, 140.40, 139.30, 138.68, 138.36, 129.98,
127.29, 126.04, 124.26, 23.65, 122.23, 121.15, 122.10, 119.36, 116.18,
112.92, 112.32, 49.9; HRMS (ESI) m/z calcd for C25H20BrN3O4S [M +
H+]; 538.0436, found 538.0432.
To a solution of Sulfanilamide 3 (1 equiv) and K2CO3 (2 equiv) in 20
ml acetone, 2-chloroacetylchloride (2 equiv) was added drop-wise. The
reaction mixture was stirred at 0 0C for 1 h and then left to warm at room
temperature. After completion of reaction filter and dry it washed with
ethanol yields 88%.
4.2.3. General procedure for synthesis of (E)-2-(3-oxo-phenylprop-1-en-
yl)-1H-indolyl)-N-(4-sulfamoylphenyl)acetamide (5a-r)
To a mixture of indolylchalcone (2a-r) in DMF solvent add K2CO3 (3
equiv) and heated for 30 min at 50 ◦C. After dissolving add 2-chloro-(4-
sulfamoylphenyl)acetamide 4 (1 equiv). Then stir it at rt for 6 h, after the
completion of the reaction the crushed-ice was added into the reaction
mixture. The solid formed was filter out and dry it to get desired product
in 70–86% yields.
4.2.3.6. (E)-2-(3-(3-(4-methyl-2-nitrophenyl)-3-oxoprop-1-en-1-yl)-1H-
indol-1-yl)-N-(4-sulfamoylphenyl)acetamide (5f). Red solid (yield 82%);
m.p: 275–277 ◦C; FT-IR (
ν
cmꢀ 1): 3266, 1685, 1596, 1526, 1524, 1396,
1334, 1159; 1H NMR (500 MHz, DMSO‑d6, TMS) δ (ppm): 10.82 (s, 1H),
8.62 (d, J = 1.5 Hz, 1H), 8.40 (dd, J = 8.0, 1.5 Hz, 1H), 8.23 (s, 1H), 8.18
(dd, J = 6.1, 2.5 Hz, 1H), 8.13 (d, J = 15.4 Hz, 1H), 7.78 (q, J = 9.0 Hz,
4H), 7.72 (s, 1H), 7.70 (d, J = 5.6 Hz, 1H), 7.58 (dd, J = 6.4, 2.3 Hz, 1H),
7.35 – 7.29 (m, 2H), 7.28 (s, 2H), 5.24 (s, 2H), 2.62 (s, 3H); 13C NMR
(125 MHz, DMSO‑d6, TMS) δ (ppm): 188.3, 166.6, 141.9, 139.3, 138.6,
137.9, 137.6, 132.2, 130.7, 127.3, 126.9, 126.1, 123.5, 122.0, 121.0,
119.3, 116.1, 112.9, 111.37, 49.9; HRMS (ESI) m/z calcd for
4.2.3.1. (E)-2-(3-(3-(4-chlorophenyl)-3-oxoprop-1-en-1-yl)-1H-indol-1-
yl)-N-(4-sulfamoylphenyl). acetamide (5a): Yellow solid (yield 82%); m.
p: 289–290 C; FT-IR (
ν
cmꢀ 1): 3267, 1681, 1651, 1588, 1564, 1524,
◦
1400, 1205, 1150; 1H NMR (500 MHz, DMSO‑d6, TMS) δ (ppm):
10.84–10.77 (m, 1H), 8.20–8.03 (m, 5H), 7.82–7.73 (m, 4H), 7.70–7.61
(m, 3H), 7.60–7.54 (m, 1H), 7.35–7.23 (m, 4H), 5.28–5.18 (m, 2H); 13
C
C
26H22N4O6S [M + H+]; 519.1338, found 519.1347.
NMR (125 MHz, DMSO‑d6) δ (ppm): 188.1, 166.6, 141.9, 139.2, 138.6,
137.8, 137.5, 130.5, 129.2, 127.3, 126.0, 123.5, 122.0, 121.0, 119.3,
116.1, 112.9, 111.3, 49.8; HRMS (ESI) m/z calculated for
4.2.3.7. (E)-2-(3-(3-(2,4-dichlorophenyl)-3-oxoprop-1-en-1-yl)-1H-indol-
C
25H20ClN3O4S 494.0941 [M + H+]; found 494.0955.
1-yl)-N-(4-sulfamoylphenyl). acetamide (5 g): Yellow solid (yield 82%);
m.p: 269–271 ◦C; FT-IR (
ν
cmꢀ 1):3268, 1683, 1591, 1522, 1395, 1254,
1156; 1H NMR (500 MHz, DMSO‑d6, TMS) δ (ppm): 10.80 (s, 1H), 8.10
(s, 1H), 7.98 (d, J = 7.6 Hz, 1H), 7.77 (dd, J = 20.0, 8.1 Hz, 5H), 7.66 (d,
J = 15.9 Hz, 1H), 7.58 (dd, J = 12.6, 6.2 Hz, 3H), 7.33–7.25 (m, 4H),
7.06 (d, J = 16.0 Hz, 1H), 5.20 (s, 2H); 13C NMR (125 MHz, DMSO‑d6,
TMS) δ (ppm): 188.1, 166.6, 141.9, 139.2, 138.6, 137.8, 137.5, 130.5,
129.2, 127.3, 126.0, 123.5, 122.0, 121.0, 119.3, 116.1, 112.9, 111.3,
49.8; HRMS (ESI) m/z: calcd for C25H19Cl2N3O4S [M + H+] 528.0552,
found 528.0549.
4.2.3.2. (E)-2-(3-(3-(4-nitrophenyl)-3-oxoprop-1-en-1-yl)-1H-indol-1-yl)-
N-(4-sulfamoylphenyl). acetamide (5b): Orange solid (yield 84%); m.p:
260–261 ◦C; FT-IR (
ν
cmꢀ 1): 3272, 1681, 1651, 1583, 1520, 1343,1155;
1H NMR (500 MHz, DMSO‑d6, TMS) δ (ppm): 10.86 (s, 1H), 8.37 (q, J =
8.8 Hz, 4H), 8.22 (s, 1H), 8.19–8.15 (m, 1H), 8.11 (d, J = 15.4 Hz, 1H),
7.78 (q, J = 9.0 Hz, 4H), 7.68 (d, J = 15.4 Hz, 1H), 7.60–7.57 (m, 1H),
7.35–7.30 (m, 2H), 7.28 (d, J = 11.7 Hz, 2H), 5.24 (s, 2H); 13C NMR
(125 MHz, DMSO‑d6, TMS) δ (ppm): 188.2, 166.6, 149.9, 143.9, 141.9,
140.4, 139.3, 138.6, 138.3, 129.9, 127.2, 126.0, 124.2, 123.6, 122.23,
121.1, 119.3, 116.1, 112.9, 49.9; HRMS (ESI) m/z calcd for
4.2.3.8. (E)-2-(3-(3-(3,4-dimethoxyphenyl)-3-oxoprop-1-en-1-yl)-1H-
C
25H20N4O6S [M + H+] 505.1182; found 505.1177.
indol-1-yl)-N-(4-sulfamoylphenyl). acetamide (5 h): Yellow solid (yield
82%); m.p: 268–270 ◦C; FT-IR (
ν
cmꢀ 1): 3321, 1689, 1646, 1596, 1529,
1388, 1273, 1126; 1H NMR (500 MHz, DMSO‑d6, TMS) δ (ppm): 10.82
(s, 1H), 8.15 (s, 1H), 8.11 (dd, J = 6.2, 2.3 Hz, 1H), 8.01 (d, J = 15.4 Hz,
1H), 7.88 (dd, J = 8.4, 1.8 Hz, 1H), 7.78 (q, J = 9.1 Hz, 4H), 7.71 (d, J =
15.5 Hz, 1H), 7.61 (d, J = 1.7 Hz, 1H), 7.58 – 7.55 (m, 1H), 7.31 – 7.26
(m, 4H), 7.12 (d, J = 8.5 Hz, 1H), 5.22 (s, 2H), 3.88 (d, J = 3.2 Hz, 6H);
13C NMR (125 MHz, DMSO‑d6, TMS) δ (ppm): 188.2, 166.6, 149.9,
143.9, 141.9, 140.4, 139.3, 138.6, 138.3, 129.9, 127.2, 126.0, 124.2,
123.6, 122.2, 121.1, 119.3, 116.1, 112.9, 49.9; HRMS (ESI) m/z calcd
for C27H25N3O6S [M + H+] 520.1542; found 520.1557.
4.2.3.3. (E)-2-(3-(3-(4-bromophenyl)-3-oxoprop-1-en-1-yl)-1H-indol-1-
yl)-N-(4-sulfamoylphenyl). acetamide (5c): Yellow solid (yield 82%); m.
◦
p: 270–272 C; FT-IR (
ν
cmꢀ 1): 3266, 1681, 1650, 1586, 1525, 1397,
1290, 1156; 1H NMR (500 MHz, DMSO‑d6, TMS) δ (ppm): 10.81 (s, 1H),
8.18 (s, 1H), 8.15 (d, J = 6.9 Hz, 1H), 8.07 (t, J = 11.7 Hz, 3H), 7.78 (q,
J = 8.9 Hz, 6H), 7.66 (d, J = 15.4 Hz, 1H), 7.57 (d, J = 7.3 Hz, 1H), 7.30
(dd, J = 15.5, 9.9 Hz, 4H), 5.23 (s, 2H); 13C NMR (125 MHz, DMSO‑d6,
TMS) δ (ppm): 188.3, 166.6, 141.9, 139.3, 138.6, 137.9, 137.6, 132.2,
130.7, 127.3, 126.9, 126.1, 123.5, 122.0, 121.0, 119.3, 116.1, 112.9,
111.3, 49.9; HRMS (ESI) m/z calcd for C25H20BrN3O4S [M + H+]
540.0416; found 540.0446.
4.2.3.9. (E)-2-(3-(3-oxo-3-(3-(trifluoromethyl)phenyl)prop-1-en-1-yl)-
1H-indol-1-yl)-N-(4-sulfamoyl-phenyl)acetamide (5i). Yellow solid (yield
82%); m.p: 284–286 ◦C; FT-IR (
ν
cmꢀ 1): 3268, 1680, 1650, 1525, 1390,
4.2.3.4. (E)-2-(3-(3-oxo-3-(p-tolyl)prop-1-en-1-yl)-1H-indol-1-yl)-N-(4-
1156; 1H NMR (500 MHz, DMSO‑d6, TMS) δ (ppm): 10.83 (s, 1H), 8.49
(d, J = 7.6 Hz, 1H), 8.35 (s, 1H), 8.24 (s, 1H), 8.14 (dd, J = 23.0, 11.0
Hz, 2H), 8.02 (d, J = 7.6 Hz, 1H), 7.86–7.71 (m, 6H), 7.58 (d, J = 8.1 Hz,
1H), 7.34–7.26 (m, 4H), 5.24 (s, 2H); 13C NMR (125 MHz, DMSO‑d6,
TMS) δ (ppm): 188.3, 166.6, 141.9, 139.3, 138.6, 137.9, 137.6, 132.2,
130.7, 127.3, 126.9, 126.1, 123.5, 122.0, 121.0, 119.3, 116.1, 112.9,
111.3, 49.9; HRMS (ESI) m/z calcd for C26H20F3N3O4S [M + H+]
528.1205; found 528.1215.
sulfamoylphenyl)acetamide (5d). Yellow solid (yield 82%); m.p:
268–270 ◦C; FT-IR (
ν
cmꢀ 1): 3272, 1657, 1651, 1591, 1524, 1393, 1290,
1
1156; H NMR (500 MHz, DMSO‑d6, TMS) δ (ppm): 11.00–10.71 (m,
1H), 8.19–8.11 (m, 2H), 8.10–8.00 (m, 3H), 7.85–7.75 (m, 4H),
7.72–7.65 (m, 1H), 7.61–7.55 (m, 1H), 7.43–7.25 (m, 6H), 5.32–5.16
(m, 2H), 2.45–2.37 (m, 3H); 13C NMR (125 MHz, DMSO‑d6, TMS) δ
(ppm): 188.8, 166.7, 143.2, 141.9, 139.2, 138.5, 138.2, 137.1, 136.3,
129.7, 128.7, 127.3, 126.1, 123.4, 121.9, 120.9, 119.3, 116.5, 112.8,
5

P. Singh et al.
Bioorganic Chemistry 108 (2021) 104647
4.2.3.10. (E)-2-(3-(3-(3-fluorophenyl)-3-oxoprop-1-en-1-yl)-1H-indol-1-
’’, 2H), 3.93 (s, 6H), 3.78 (s, 3H); 13C NMR (125 MHz, DMSO‑d6, TMS) δ
(ppm): 188.2, 166.6, 149.9, 143.9, 141.9, 140.4, 139.3, 138.6, 138.3,
129.9, 127.2, 126.0, 124.2, 123.6, 122.2, 121.1, 119.3, 116.1, 112.9,
49.9; HRMS (ESI) m/z calcd for C28H27N3O7S [M + H+] 550.1648; found
550.1675.
yl)-N-(4-sulfamoylphenyl). acetamide (5j): Yellow solid (yield 84%); m.
p: 268–270 ◦C; FT-IR (
ν
cmꢀ 1): 3317, 1693, 1524, 1313, 1288, 1152; 1H
NMR (500 MHz, DMSO‑d6, TMS) δ (ppm): 10.82 (s, 1H), 8.20 (s, 1H),
8.19–8.16 (m, 1H), 8.06 (s, 1H), 8.03–8.00 (m, 1H), 7.93–7.90 (m, 1H),
7.78 (dd, J = 15.2, 9.0 Hz, 5H), 7.67 (d, J = 15.5 Hz, 1H), 7.60–7.55 (m,
1H), 7.54–7.48 (m, 1H), 7.31 (d, J = 5.4 Hz, 2H), 7.27 (s, 2H), 5.23 (s,
2H); 13C NMR (125 MHz, DMSO‑d6, TMS) δ (ppm): 188.1, 166.6, 141.9,
139.2, 138.6, 137.8, 137.5, 130.5, 129.2, 127.3, 126.0, 123.5, 122.0,
121.0, 119.3, 116.1, 112.9, 111.3, 49.8; HRMS (ESI) m/z calcd for
4.2.3.16. (E)-2-(3-(3-oxo-3-(4-(trifluoromethyl)phenyl)prop-1-en-1-yl)-
1H-indol-1-yl)-N-(4-sulfamoyl phenyl)acetamide (5p). Yellow solid,
(yield 83%); m.p: 253–255 ◦C; FT-IR (
ν
cmꢀ 1): 3264, 1681, 1590, 1527,
1321, 1158, 1123; 1H NMR (500 MHz, DMSO‑d6, TMS) δ (ppm): 10.90
(s, 1H), 8.32 (s, J = 8.0 Hz, 1H), 8.22 (s, 1H), 8.16 (s, J = 6.9 Hz, 1H),
8.10 (d, J = 15.5 Hz, 1H), 7.94 (d, J = 8.2 Hz, 2H), 7.79 (q, J = 9.2 Hz,
4H), 7.74 (s, 1H), 7.69 (d, J = 15.4 Hz, 2H), 7.59 (d, J = 7.1 Hz, 1H),
7.33–7.27 (t, 3H), 5.25 (s, 2H); 13C NMR (125 MHz, DMSO‑d6, TMS) δ
(ppm): 188.3, 166.6, 141.9, 139.3, 138.6, 137.9, 137.6, 132.2, 130.7,
127.3, 126.9, 126.1, 123.5, 122.0, 121.0, 119.3, 116.1, 112.9, 111.3,
49.9; HRMS (ESI) m/z calcd for C26H20F3N3O4S [M + H+] 528.1205;
found 528.1226.
C
25H20FN3O4S [M + H+] 478.1237, found 478.1246.
4.2.3.11. (E)-2-(3-(3-(3,5-bis(trifluoromethyl)phenyl)-3-oxoprop-1-en-1-
yl)-1H-indol-1-yl)-N-(4-sulfamoylphenyl)acetamide (5 k). Yellow solid
(yield 87%); m.p 290–292 ◦C; FT-IR (
ν
cmꢀ 1): 3264, 1676, 1568, 1523,
1279, 1156, 1125; 1H NMR (500 MHz, DMSO‑d6, TMS) δ (ppm): 10.83
(s, 1H), 8.69 (s, 2H), 8.40 (s, 1H), 8.29 (s, 1H), 8.18 (dd, J = 9.6, 5.5 Hz,
2H), 7.78 (dd, J = 15.1, 8.0 Hz, 5H), 7.61–7.57 (m, 1H), 7.34–7.30 (m,
2H), 7.27 (s, 2H), 5.25 (s, 2H); 13C NMR (125 MHz, DMSO‑d6, TMS) δ
(ppm): 188.1, 166.6, 141.9, 139.2, 138.6, 137.8, 137.5, 130.5, 129.2,
127.3, 126.0, 123.5, 122.0, 121.0, 119.3, 116.1, 112.9, 111.3, 49.8;
HRMS (ESI) m/z calcd for C27H19F6N3O4S [M + Na+] 618.0898; found
618.0923.
4.2.3.17. (E)-2-(3-(3-(2-chlorophenyl)-3-oxoprop-1-en-1-yl)-1H-indol-1-
yl)-N-(4-sulfamoylphenyl). acetamide (5q): Yellow solid (yield 82%); m.
◦
p: 272–274 C; FT-IR (
ν
cmꢀ 1):3272, 1657, 1651, 1591, 1524, 1393,
1290, 1156; 1H NMR (500 MHz, DMSO‑d6, TMS) δ (ppm): 10.79 (s, 1H),
8.09 (s, 1H), 7.96 (d, J = 7.4 Hz, 1H), 7.77 (dd, J = 19.9, 8.1 Hz, 4H),
7.66–7.49 (m, 6H), 7.29 (d, J = 18.1 Hz, 4H), 7.07 (d, J = 16.0 Hz, 1H),
5.19 (s, 2H); 13C NMR (125 MHz, DMSO‑d6, TMS) δ (ppm): 188.1, 166.6,
141.9, 139.2, 138.6, 137.8, 137.5, 130.5, 129.2, 127.3, 126.0, 123.5,
122.0, 121.0, 119.3, 116.1, 112.9, 111.3, 49.8; HRMS (ESI) m/z calcd
for C25H20ClN3O4S [M + H+] 494.0941; found 494.0967.
4.2.3.12. (E)-2-(3-(3-(benzo[d][1,3]dioxol-5-yl)-3-oxoprop-1-en-1-yl)-
1H-indol-1-yl)-N-(4-sulfamoyl phenyl)acetamide (5 l). Yellow solid (yield
82%); m.p: 250–252 ◦C; FT-IR (
ν
cmꢀ 1): 3268, 1686, 1525, 1288, 1244,
1155; 1H NMR (500 MHz, DMSO‑d6, TMS) δ (ppm): 10.82 (s, 1H),
8.17–8.13 (m, 2H), 8.01 (d, J = 15.4 Hz, 1H), 7.84 (dd, J = 8.2, 1.5 Hz,
2H), 7.81–7.74 (m, 3H), 7.68 (s, 1H), 7.66–7.64 (m, 1H), 7.56 (d, J =
7.2 Hz, 1H), 7.32–7.27 (m, 4H), 7.09 (d, J = 8.1 Hz, 1H), 6.17 (s, 2H),
5.22 (s, 2H); 13C NMR (125 MHz, DMSO‑d6, TMS) δ (ppm): 188.1, 166.6,
141.9, 139.2, 138.6, 137.8, 137.5, 130.5, 129.2, 127.3, 126.0, 123.5,
122.0, 121.0, 119.3, 116.1, 112.9, 111.3, 49.8; HRMS (ESI) m/z calcd
for C26H21N3O6S [M + H+] 504.1229, found 504.1240.
4.2.3.18. (E)-2-(3-(3-(3-chlorophenyl)-3-oxoprop-1-en-1-yl)-1H-indol-1-
yl)-N-(4-sulfamoylphenyl). acetamide (5r): Yellow solid (yield 81%); m.
p: 264–266 ◦C; FT-IR (
ν
cmꢀ 1): 3260, 1679, 1522, 1391, 1287, 1152; 1H
NMR (500 MHz, DMSO‑d6, TMS) δ (ppm):10.87–10.78 (s, 1H),
8.24–8.20 (s, 1H), 8.19–8.15 (s, 1H), 8.15–8.09 (d, 2H), 8.09–8.05 (s,
1H), 7.82–7.75 (m, 4H), 7.74–7.71 (s, 1H), 7.70–7.65 (s, 1H), 7.64–7.60
(s, 1H), 7.60–7.56 (s, 1H), 7.35–7.27 (m, 4H), 5.27–5.21 (d, 2H); 13C
NMR (125 MHz, DMSO‑d6, TMS) δ (ppm): 187.9, 166.6, 141.9, 140.7,
139.5, 139.2, 138.5, 137.7, 128.1, 127.3, 126.1, 123.5, 122.1, 121.1,
119.3, 115.9, 112.9, 111.3, 49.8; HRMS (ESI) m/z calcd for
4.2.3.13. (E)-2-(3-(3-(2-bromophenyl)-3-oxoprop-1-en-1-yl)-1H-indol-1-
yl)-N-(4-sulfamoyl phenyl)acetamide (5 m). Yellow solid (yield 82%); m.
p 251–252 ◦C; FT-IR (
ν
cmꢀ 1): 3282, 1682, 1593, 1521, 1315, 1156; 1H
NMR (500 MHz, DMSO‑d6, TMS) δ (ppm): 10.79 (s, 1H), 8.09 (s, 1H),
7.96 (d, J = 7.5 Hz, 1H), 7.81–7.72 (m, 5H), 7.62–7.51 (m, 4H),
7.48–7.44 (m, 1H), 7.29 (dt, J = 19.5, 9.7 Hz, 4H), 7.04 (d, J = 16.0 Hz,
1H), 5.19 (s, 2H); 13C NMR (125 MHz, DMSO‑d6, TMS) δ (ppm): 188.1,
166.6, 141.9, 139.2, 138.6, 137.8, 137.5, 130.5, 129.2, 127.3, 126.0,
123.5, 122.0, 121.0, 119.3, 116.1, 112.9, 111.3, 49.8; HRMS (ESI) m/z
calcd for C25H20BrN3O4S [M + Na+] 562.0235; found 562.0265.
C
25H20ClN3O4S [M + H+] 494.0941, found 494.0952.
4.3. Carbonic anhydrase inhibition assay
An SX.18MV-R Applied Photophysics (Oxford, UK) stopped-flow
instrument has been used to assay the catalytic/inhibition of various
CA isozymes.[22] Phenol Red (at a concentration of 0.2 mM) has been
used as indicator, working at the absorbance maximum of 557 nm, with
10 mM Hepes (pH 7.4) as buffer, 0.1 M Na2SO4 or NaClO4 (for main-
taining constant the ionic strength; these anions are not inhibitory in the
used concentration), following the CA-catalyzed CO2 hydration reaction
for a period of 5–10 s. Saturated CO2 solutions in water at 25 ◦C were
used as substrate. Stock solutions of inhibitors were prepared at a con-
4.2.3.14. (E)-2-(3-(3-(3-nitrophenyl)-3-oxoprop-1-en-1-yl)-1H-indol-1-
yl)-N-(4-sulfamoylphenyl). acetamide (5n): Orange solid (yield 86%); m.
◦
p 260–261 C; FT-IR (
ν
cmꢀ 1): 3272, 1676, 1586, 1525, 1393, 1290,
1157; 1H NMR (500 MHz, DMSO‑d6, TMS) δ (ppm): 10.83 (s, 1H), 8.78
(s, 1H), 8.62 (d, J = 7.6 Hz, 1H), 8.48 (d, J = 7.2 Hz, 1H), 8.25 (s, 1H),
8.17 (dd, J = 15.9, 11.9 Hz, 2H), 7.89 (t, J = 7.9 Hz, 1H), 7.76 (dt, J =
18.9, 12.2 Hz, 5H), 7.59 (d, J = 6.9 Hz, 1H), 7.31 (dd, J = 14.3, 10.7 Hz,
4H), 5.25 (s, 2H); 13C NMR (125 MHz, DMSO‑d6, TMS) δ (ppm): 188.2,
166.6, 149.9, 143.9, 141.9, 140.4 139.3, 138.6, 138.3, 129.9, 127.2,
126.0, 124.2, 123.6, 122.2, 121.1, 119.3, 116.1, 112.9, 49.9; HRMS
(ESI) m/z calcd for C25H20N4O6S [M + H+] 505.1182; found 505.1190.
centration of 10 μM (in DMSO-water 1:1, v/v) and dilutions up to 0.01
nM done with the assay buffer mentioned above. At least 7 different
inhibitor concentrations have been used for measuring the inhibition
constant. Inhibitor and enzyme solutions were pre-incubated together
for 10 min at room temperature prior to assay, in order to allow for the
formation of the E-I complex. Triplicate experiments were done for each
inhibitor concentration, and the values reported throughout the paper
are the mean of such results. The inhibition constants were obtained by
non-linear least-squares methods using the Cheng-Prusoff equation, as
reported earlier [23] and represent the mean from at least three different
determinations. All CA isozymes used here were recombinant proteins
obtained as reported earlier by our group [24–26].
4.2.3.15. (E)-2-(3-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-en-1-yl)-1H-
indol-1-yl)-N-(4-sulfamoyl phenyl)acetamide (5o). Brown solid, yield
83%; m.p 262–264 ◦C; FT-IR (
ν
cmꢀ 1):3272, 1657, 1651, 1591, 1524,
1393, 1290, 1156; 1H NMR (500 MHz, DMSO‑d6, TMS) δ (ppm):
10.91–10.77 (m, 1H), 8.20 (s, 1H), 8.06 (s, 2H), 7.78 (d, J = 5.6 Hz, 4H),
7.69 (s, 1H), 7.58 (d, J = 5.6 Hz, 1H), 7.40 (s, 2H), 7.29 (s, 4H), 5.24 (s/
6

P. Singh et al.
Bioorganic Chemistry 108 (2021) 104647
b) M. Bozdag, F. Varta, M. Ceruso, M. Ferrroni, P.C. McDonald, S. Dedhar, C.
T. Supuran, Discovery of 4-Hydroxy-3-(3-(phenylureido)benzenesulfonamides as
SLC-0111 Analogues for the Treatment of Hypoxic Tumors Over-expressing
Carbonic Anhydrase IX, J. Med. Chem. 61 (2018) 6328–6338.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
[14] F. Carta, C.T. Supuran, A. Scozzafava, Sulfonamides and their isosters as carbonic
anhydrase inhibitors, Future Med. Chem. 6 (2014) 1149–1165.
[15] C.T. Supuran, Advances in structure-based drug discovery of carbonic anhydrase
inhibitors, Expert. Opin. Drug. Discov. 12 (2017) 61–88.
Acknowledgements
[16] V. Alterio, A.D. Fiore, K.D. Ambrosio, C.T. Supuran, G.D. Simone, Multiple Binding
Modes of Inhibitors to Carbonic Anhydrases: How to Design Specific Drugs
Targeting 15 Different Isoforms? Expert. Opin. Drug. Discov. 112 (2012)
4421–4468.
PS, PPY, BS and PST are thankful to Department of Pharmaceuticals,
Ministry of Chemicals and Fertilizers, Govt. of India, New Delhi for the
award of NIPER fellowship.
[17] P. Singh, B. Swain, P.S. Thacker, D.K. Sigalapalli, P. Yadav, A. Angeli, C.
T. Supuran, M., Arifuddin) Synthesis and carbonic anhydrase inhibition studies of
sulfonamide based indole- 1,2,3-triazole chalcone hybrids, Bioorg. Chem. 99
(2020), 103839.
Appendix A. Supplementary data
[18] F. Manna, F. Chimenti, A. Bolasco, B. Bizzarri, W. Filippelli, A. Filippelli,
L. Gagliardi, Anti-inflammatory, analgesic and antipyretic 4,6-disubstituted 3-
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[19] Abdul-Malek S. Altamimi, Ahmed M. Alafeefy, A. Balode, I. Vozny, A. Pustenko,
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.104647.
ˇ
Mohey Eldin El Shikh, F. A. S. Alasmary, Sherif A. Abdel-Gawad & R. Zalubovskis.
Symmetric molecules with 1,4-triazole moieties as potent inhibitors of tumour-
associated lactate dehydrogenase-A, J. Enzyme Inhib. Med. Chem. 33 (2018) 147-
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