Synthesis and carbonic anhydrase inhibition studies of sulfonamide based indole-1,2,3-triazole chalcone hybrids

Article abstract of DOI:10.1016/j.bioorg.2020.103839

Sulfonamide is one of the most promising classes of classical carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. A novel series of indolylchalcones incorporating benzenesulfonamide-1,2,3-triazole (6a-q) has been synthesized by click chemistry reaction and in

Full text of DOI:10.1016/j.bioorg.2020.103839

Journal Pre-proofs
Synthesis and carbonic anhydrase inhibition studies of sulfonamide based in-
dole-1,2,3-triazole chalcone hybrids
Priti Singh, Baijayantimala Swain, Pavitra S. Thacker, Dilep Kumar
Sigalapalli, P. Purnachander Yadav, Andrea Angeli, Claudiu T. Supuran,
Mohammed Arifuddin
PII:
S0045-2068(20)30336-9
DOI:
https://doi.org/10.1016/j.bioorg.2020.103839
YBIOO 103839
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
10 February 2020
31 March 2020
6 April 2020
Please cite this article as: P. Singh, B. Swain, P.S. Thacker, D. Kumar Sigalapalli, P. Purnachander Yadav, A.
Angeli, C.T. Supuran, M. Arifuddin, Synthesis and carbonic anhydrase inhibition studies of sulfonamide based
indole-1,2,3-triazole chalcone hybrids, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.
2020.103839
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover
page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version
will undergo additional copyediting, typesetting and review before it is published in its final form, but we are
providing this version to give early visibility of the article. Please note that, during the production process, errors
may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier Inc.

Synthesis and carbonic anhydrase inhibition studies of sulfonamide based
indole-1,2,3-triazole chalcone hybrids
Priti Singha, Baijayantimala Swaina, Pavitra S. Thackera, Dilep Kumar Sigalapallia, P.
Purnachander Yadava, Andrea Angelic, Claudiu T. Supuranc* and Mohammed Arifuddina,b*
.
aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Balanagar, Hyderabad 500037, India
bDepartment of Chemistry, Anwarul Uloom College, 11-3-918, New Malleypally, Hyderabad-
500001, T. S., India (Present Address)
cUniversitàdegliStudi di Firenze, Neurofarba Dept., Sezione di ScienzeFarmaceutiche e
Nutraceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino, Florence, Italy
ABSTRACT. Sulfonamide is one of the most promising classes of classical carbonic anhydrase
(CA, EC 4.2.1.1) inhibitors.
A
novel series of indolylchalcones incorporating
benzenesulfonamide-1,2,3-triazole (6a-q) has been synthesized by click chemistry reaction and
investigated for hCA inhibitory activity against a panel of human carbonic anhydrases (hCAs).
Most of these newly synthesized compounds exhibited interesting inhibition constants, in the
nanomolar range, with some derivatives being more potent than the standard drug acetazolamide
(AAZ) on hCA I isoform. Among the tested compounds, the compounds 6d (18.8 nM), 6q (38.3
nM) and 6e (50.4 nM) were 13, 6 and 5 times more potent than AAZ against hCA I isoform,
respectively. Compounds 6o, 6m and 6f efficiently inhibited isoform hCA XII, with KIs in the
range of 10 – 41.9 nM. Several compounds were also active against isoforms hCA II and hCA IX,
with KIs under 100 nM. These indolylchalcone-benzenesulfonamide-1,2,3-triazole hybrids may
be considered as potential leads for hCA I-selective inhibitors.
Keywords: Carbonic anhydrase, Knoevenagel condensation, Acetazolamide, indolylchalcone-
benzenesulfonamide-1,2,3-triazole hybrids, selective hCA I inhibition.
*Corresponding author: Email ID: arifabib@gmail.com (Mohammed Arifuddin); claudiu.supuran@unifi.it (C.T.Supuran)

1. Introduction
A massive research activity has been reported in the field of carbonic anhydrase inhibition.
Carbonic anhydrases (CAs; also known as carbonate dehydratases EC 4.2.1.1), are
metalloenzymes, as their active site contains a metal ion. The main function of this enzyme is to
catalyze the reversible inter-conversion of carbon dioxide and water to bicarbonate and protons as
shown by the following equation 1 [1].
−
CO2 + H2O ↔ HCO3 + H+
(Eq. 1)
Most of the CA isozymes are involved in important physiological functions such as
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]. Therefore, their inhibitors
constitute an important class of therapeutic agents to treat a range of disorders including edema,
glaucoma, obesity, cancer, epilepsy and osteoporosis [2].
There are several classes of CA inhibitors (CAIs): such as i) metal ion binders which
include the sulfonamides and their isosteres sulfamates/sulfamides, dithiocarbamates, mercaptans
and hydroxamates; ii) compounds anchoring to the zinc-coordinated water molecule/hydroxide ion
like phenols, carboxylates, polyamines, esters and iii) coumarins and related compounds such as
sulfocoumarins, which bind even further away from the metal ion [3].
The primary sulfonamide group plays an important role in CA inhibition mechanism, with
more than 20 compounds in clinical use, with: “acetazolamide (AAZ)” A, methazolamide B,
ethoxzolamide C, celecoxib D [4], dichlorophenamide E, dorzolamide F, brinzolamide (Fig 1) to
cite a few. Furthermore, compounds such as sulpiride, zonisamide, topiramate etc., are in clinical
use as anti-epileptics or antidepressant agents [1a]. They function by binding to the zinc metal ion,
in their active deprotonated form, as sulfonamidate [5].

N
S
N
S
N
S
N
O
N
O
SO2NH2
SO2NH2
SO2NH2
O
N
H
N
C
B
A
SO2NH2
NH
S
SO2NH2
N
N
F3C
SO2NH2
S
Cl
SO2NH2
O
O
Cl
D
F
E
Fig-1: Structure of clinically used sulfonamide containing drugs
There are eight CA families identified up until now among various living species like
bacteria, archaea and eukarya. The α-CAs are expressed primarily in vertebrates, plants, and algae;
β in bacteria, algae, and fungi; γ in archaea and bacteria; two minor forms, δ and ζ, are expressed
in marine diatoms; and η in protozoa and the θ- and -classes are found in marine diatoms such as
Phaeodactylum tricornutum, and related species [6]. In humans, 15 CA isoforms are found which
differ not only in tissue localization but also in enzymatic efficiency: CA I, CA II, CA III, CA VII,
CA XIII are cytosolic; CA IV, CA IX, CA XII, CA XIV, membrane-bound; CA Va and CA Vb
mitochondrial and CA VI secreted primarily in saliva.
Indisulam (E7070), an anticancer agent incorporating a chloro-indolyl sulfonamide (Fig 2)
was in phase II clinical studies till recently, and it inhibits several CA isoform, including the tumor-
associated one, CA IX (CAIX) [7,8]. The ureido benzenesulfonamide compound SLC-0111 (Fig
2), is currently in Phase I/II clinical trials for the treatment of advanced metastatic solid tumors
[9,10]. The 1,2,3-triazole scaffold has received considerable attention due to its diverse biological
activities such as antimicrobial, anti-allergic, analgesic, anti-HIV, anti-inflammatory, anticancer,
anti-malarial and anti-tuberculosis [11]. In addition, many research groups have also explored
carbonic anhydrase inhibitory activity of 1,2,3-triazole [12,13,14]. Chalcone has also found to be
active against hCAs [15,16]. Hence, in the present study we decided to incorporate these 4
scaffolds i.e., indole, chalcone, triazole and sulfonamide, to synthesize our target molecules.

R2
O
R1
Chal
cone
SO2NH2
R3
R4
Indole
O
O
N
N
S
O
HN
R5
Cl
S
O
O
N
N
H
MeO
N
Indisulam
N
N
F3C
Triazole
H
N
H
N
Sulfonamide
N
H2NO2S
Target molecule
N
O
N
F
H2NO2S
O S
O
SLC-0111
O
Fig-2: Rationale for designing the target molecules
2. Result and discussion
2.1. Chemistry
The synthetic strategy employed for the synthesis of the target compounds is depicted in
Scheme 1. The chalcones (2a-q) were synthesized by Knoevenagel condensation by reacting
indole-3-carboxaldehyde with different acetophenones using piperidine as the base and ethanol as
solvent [17]. In the next step the obtained chalcones (2a-q) were propargylated using propargyl
bromide and anhydrous K2CO3 in dry DMF to get intermediates (3a-q) [18]. On the other side the
benzenesulfonamide azide (5) was prepared from sulfanilamide through diazotization reaction
using conc. HCl, NaNO2 and NaN3, which was finally subjected to click reaction with substituted
intermediates (3a-q) in the presence of CuSO4 and sodium ascorbate in tBuOH and H2O (1:1) as
solvent to afford the final target products (6a-q) by using reported literature method [19]. The
1
13
structures of all the compounds (6a-q) were elucidated on the basis of H NMR, C NMR, and
HRMS.

O
a
O
O
O
R
R
R
b
N
N
N
H
H
3
1
2
c
H2N
N3
d
SO2NH2
SO2NH2
4
5
N
N
N
O
R
N
H2NO2S
6 a-q
Scheme 1: General synthetic scheme for the synthesis of benzenesulfonamide triazole linked to
indolylchalcone. Reagents and conditions: a) Piperidine (0.5 equiv), EtOH, reflux, 24 h; b) (i)
K2CO3 (3 equiv), DMF, 700C, 3-4 h; (ii) Propargyl bromide (1 mmol); (c) 5, CuSO4 (5 mol%),
t
Sodium ascorbate (10 mol%), BuOH:H2O (1:1); d) (i) HCl, NaNO2 (1 equiv), 00C, 30 min; (ii)
Sodium azide (1.6 equiv), 00C, 4 h.
2.2. Carbonic anhydrase inhibition
The 17 newly synthesized compounds (6a-q) have been tested for the inhibition against
the four physiologically and pharmacologically significant isoforms, the cytosolic isoforms, hCA
I and hCA II and the trans-membrane tumor associated isoforms, hCA IX and hCA XII, by means
of stopped flow carbon dioxide assay [20]. Here acetazolamide (AAZ) was taken as the standard
drug. The screening result of the synthesized compounds against different hCA isoforms can be
drawn, from the results shown in Table 1:-

Table 1. Ki value of compounds (6a- q) obtained from stopped flow carbon dioxide assay against
hCA isoforms I, II, IX and XII.
H2NO2S
N
N
N
N
O
R
KI (nM)*
hCA I
Compoun
hCA II
hCAIX
hCAXII
R
d
6a
6b
6c
6d
6e
6f
6g
6h
6i
Phenyl
89.0±7
468.4±24
69.3±4
73.3±5
94.5±3
522.4±27
554.0±38
73.8±4
4-Chlorophenyl
596.3±32 861.2±49
501.5±24 341.2±17
4-Fluorophenyl
4-Bromophenyl
18.8±1.1 1721±124 375.1±21 283.9±21
4-Methylphenyl
50.4±4
36.2±3
88.1±7
615.1±34
240.9±16
54.5±3
41.9±1
3-Bromophenyl
5500±214
4-Methoxyphenyl
4-Methyl-3-nitrophenyl
2,4-Dichlorophenyl
3,4-Dimethoxyphenyl
3-(Trifluoromethyl)phenyl
3-Fluorophenyl
771.7±25 416.5±22 1397±121
548.5±30 88.5±6 187.0±9
241.4±15 881.9±62
54.7±4 84.7±5
2800±126 7738±340
60.9±4 192.3±15
81.1±4
450.7±21
763.6±31 257.8±17
6j
490.3±41
1439±78
888.0±54
50.3±3
82.0±4
70.6±2
6k
6i
3,5-
6m
6n
2247±157 7589±420 500.7±29
36.9±1.5
Bis(trifluoromethyl)phenyl
3,4-
78.7±3.6
722.5±31
85.0±5
92.4±5
(Methylenedioxy)phenyl
2-Bromophenyl
6o
6p
6q
829.8±45 9688±310 757.8±20
10.0±0.4
3,4,5-Trimethoxyphenyl
3-Methoxyphenyl
405.2±21 943.0±30
767.5±41 1534±120
940.1±40 192.8±9.5
38.3±2
716.4±27
O
S
N
N
O
NH2
AAZ
250±11
12.1±0.5
25.8±1.1
5.7±0.3
S
N
O
H
* Mean  standard error (from 3 different assays, by a stopped flow technique).

From the data given in the Table 1 the following inferences were made:-
1) The cytosolic isoform hCA I was strongly inhibited by all the synthesized compounds with
Kis ranging between 18.8 nM to 5.5 μM. Among all the synthesized compounds, 6d, 6q,
6e and 6j were found to be more potent hCA I inhibitors compared to the standard AAZ.
The compound 6d (Ki = 18.8 nM) was 13 times more active than AAZ (Ki = 250 nM)
whereas compound 6q (Ki = 38.3 nM) was found to be 6 times more active than AAZ
against hCA I. Similarly, the compound 6e (Ki= 50.4 nM) was 5 times more active than
AAZ against hCA I isoforms. It was also noticed that the 4-bromo (6d), 4-methyl (6e), and
3-methoxy (6q) substituted derivatives were showing better inhibition as compared to
electron withdrawing derivatives (Table-1).
2) The other cytosolic isoform hCA II was also inhibited by few compounds in moderate
nanomolar range with Ki values ranging between 36.2-88 nM. Compound 6e having a p-
methyl substitution was found to be more potent amongst all with Ki= 36.2 nM. Three other
compounds i.e., 6j, 6f and 6h were also inhibited hCA II isoform with Ki values of 84.7
nM, 88.1 nM and 88.5 nM respectively.
3) The tumor associated isoform hCA IX was weakly inhibited by all the synthesized
compounds with Ki values ranging from 69.3 nM-1.4 μM. The 4-chloro substituted
derivative (6b) and 3,4-(Methylenedioxy) substituted derivative (6n) exhibited the best
inhibition amongst all the compounds with Ki values of 73.3 and 85 nM respectively
(Table-1).
4) The other tumor-associated isoform hCA XII, was also inhibited by the compounds 6o, 6m
and 6f in a low to moderate nanomolar range with Ki values of 10nM, 36.9 nM and 41.9
nM respectively. The most potent inhibitor against this isoform was found to be the
compound 6o with 2-bromo substitution.
2.3. Molecular docking study
The molecular docking results along with the major interactions for 6d, 6q and co-crystallized
ligand with human hCA I enzyme is depicted in Table 2. From the molecular docking studies, it
was observed that 6d and 6q were well accommodated in the active pocket of hCA I. In case of
the most active compound 6d has shown two hydrogen bond interactions with the active residues

Thr199 and His200. Fig 3 illustrates the predicted binding mode and the detailed protein-inhibitor
interactions of compound 6d within the active site of hCA I. One of the oxygen atom of
sulfonamide moiety acts as hydrogen bond acceptor and formed two H-bond interactions with
Thr199 and His200 with a distance of 1.93 and 3.98 Å, respectively. The other oxygen atom and
nitrogen atom of sulfonamide moiety established a metal ion interaction with “Zn”. The triazole
core of 6d has made one arene-arene (π-π) interaction with Phe91. Additionally, several
hydrophobic interactions were observed between the compound 6d and the active site residues,
e.g., Ile60, Val62, Phe91, Ala121, Leu131, Ala132, Ala135, Leu141, Val143, Leu198, Pro202 and
Trp209 that stabilized the binding of the compound 6d in the active site of hCA I.
Similarly, Compound 6q also showed good docking score and more protein-ligand interactions
with the active site residues of hCA I. Fig 4 demonstrates the possible binding mode and protein-
ligand interactions of 6q with the active site of hCA I. On the basis of our molecular docking study,
it could be deduced that the active compounds elicited potency via occupying the active pocket of
hCA I and making favourable interactions with its key residues.
Fig 3. Binding pose of the most potent compound 6d (orange colour stick) at the active site of
hCA I (PDB ID: 1AZM). The red and purple dashed lines represent hydrogen bond and metal ion
interactions, respectively.

Fig 4. Binding pose of 6q (orange colour stick) at the active site of hCA I (PDB ID: 1AZM). The
red and purple dashed lines represent hydrogen bond and metal ion interactions, respectively.
Table 2. GLIDE docking results for compound 6d, 6q and co-crystallized ligand at the active
site of human carbonic anhydrase I (PDB ID: 1AZM).
S.
no
Ligand
Name
Dockin
g score
Interactions
Hydrophobic
H-
π-π
Metal ion
interaction
with “Zn”
bonds
1
2
3
6d
6q
-7.915 Thr199, Phe91 Ile60, Val62, Phe91, Ala121,
Positive
Positive
Positive
His200
Leu131, Ala132, Ala135,
Leu141, Val143, Leu198,
Pro202, Trp209
-7.269 Thr199, Phe91 Val62, Phe91, Ala121, Leu131,
His200 Ala132, Ala135, Leu141,
Val143,
Trp209
Leu198,
Pro202,
Co-crystal
(acetazolami
de)
-8.520 Thr199, His20 Phe91, Leu131, Ala 135,
His200
0
Leu141, Val143, Leu198,
Pro202, Trp209

3. Conclusion
In the present work, novel indolylchalcone linked benzenesulfonamide-1,2,3-triazole
hybrids (6a-q) were designed, synthesized and evaluated for their ability to inhibit
pharmacologically significant human carbonic anhydrases hCA I, hCA II, hCA IX and hCAXII.
Most of the synthesized compounds showed good inhibition profile against all the four isoforms,
with KI values less than 100 nM. Against hCA I the compounds 6d, 6e, 6j and 6q were found to
be more potent hCA I inhibitors compared to the standard AAZ with KI values of 18.8 nM, 38.3
nM, 50.4 nM and 54.7 nM respectively. Against hCA II, compound 6e showed the best inhibition
with a KI value of 36.3 nM. Against hCA IX the compounds 6b and 6n exhibited the best inhibition
amongst all with Ki values of 73.3 and 85 nM respectively. Against hCA XII, compound 6o
showed the best Ki values of 10 nM. Hence based on the results, it can be concluded that the
compounds containing electron donating groups (-CH3 (6d) and -OCH3 (6e)) as well as compounds
containing bromo substitution on the phenyl ring (6q) of chalcone showed good inhibitory activity
as compared to compounds containing electron withdrawing groups. Thus it is anticipated that the
compounds 6d, 6q, 6e and 6j which are more potent than standard AAZ could be considered as
potential lead molecules for the design and development of selective hCA I inhibitors.
4. Experimental:
4.1. General
All the commercially available reagents were used without further purification. Solvents
were dried and distilled wherever necessary prior to use, using standard methods. All the air and
moisture sensitive reactions were performed under inert conditions using clean and dried glassware
and syringe technique to transfer solutions. Reactions were monitored by TLC using Merck silica
gel 60F-254 plates. Melting points were acquired on Stuart digital melting point apparatus / SMP
30 in open capillary tubes and uncorrected. Nuclear Magnetic Resonance (1H NMR and 13C NMR)
spectra were recorded by using an Avancebruker 500MHz and 125MHz spectrometer in DMSO-
d6 as solvent and trimethylsilane (TMS) as internal standard. Chemical shifts are reported as δ
values in parts per million (ppm) and coupling constants (J) are expressed in Hz. Multiplicities are
described as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), dd (doublet of doublets).
HRMS were determined with Agilent QTOF mass spectrometer 6540 series instrument and were
performed using ESI techniques at 70 eV.

4.1.1 General procedure for synthesis of indolylchalcones (2a-q):
A mixture of indole-3-carboxaldehyde 1 (1 equiv) was refluxed with substituted
acetophenones (1 equiv) in presence of piperidine (0.5 equiv) in ethanol for 24 h at 600C under
stirring. The reaction was monitored by TLC and after completion of the reaction crushed-ice was
added into the reaction mixture and neutralized with glacial acetic acid by monitoring with pH
paper, which resulted in the formation of a solid product 2. The solid formed was filtered using
vacuum filtration and washed with hexane and dried to get the product in 80-90% yield.
4.1.2 General procedure for synthesis of (E)-3-(1-(prop-2-yn-1-yl)-1H-indol-3-ylchalcones (3a-
q):
Anhydrous K2CO3 (3 equiv) was suspended in dry DMF (5 mL) followed by the addition
of compound 2a-q (1 equiv). The resulting mixture was stirred for 1 h at 600C. Then propargyl
bromide (1 equiv) was added drop wise. Reaction mixture was stirred for 2-4 h at rt. After the
completion of the reaction as monitored by TLC, ice-cool water (10 mL) was added to the reaction
mixture and the formed precipitate was filtered off to get the product in 85-90% yield.
4.1.3 General procedure for synthesis of (6a- q):
(E)-3-(1-(prop-2-yn-1-yl)-1H-indol-3-ylchalcones
3a-q
(1
equiv)
and
4-
azidobenzenesulfonamide (5) (1.08 equiv) were dissolved in tBuOH/H2O (1:1, 5mL), followed by
the addition of CuSO4.5H2O (5 mol%) and sodium ascorbate (10 mol%). The resultant solution
was stirred at 500C till completion of the reaction (TLC monitoring). Solvents were removed under
vacuum and the residue was purified by column chromatography using silica gel (60-120 mesh)
as the stationary phase and 1:3 Hex:Ethylacetate as mobile phase. Pure products (6a-q) were
obtained as yellow solids with 63- 95% yield.
(E)-4-(4-((3-(3-oxo-3-phenylprop-1-en-1-yl)-1H-indol-1-yl)methyl)-1H-1,2,3-triazol-1-
1
yl)benzenesulfonamide (5a): Yellow solid; yield 80%, m.p 225-2270C; H NMR (500 MHz,
DMSO) δ 8.99 (s, 1H), 8.29 (s, 1H), 8.15 – 7.98 (m, 8H), 7.77 (d, J = 8.0 Hz, 1H), 7.66 (dd, J =
13
20.3, 11.5 Hz, 2H), 7.57 (t, J = 7.5 Hz, 2H), 7.51 (s, 2H), 7.31 (m, 2H), 5.68 (s, 2H). C NMR
(125 MHz, DMSO) δ 189.34, 144.56, 144.44, 138.96, 138.89, 138.59, 137.65, 135.86, 132.91,

129.16, 128.61, 127.95, 126.39, 123.46, 122.76, 122.10, 121.06, 120.88, 116.65, 113.00, 111.68,
41.69. HRMS (ESI) m/z: [M+H]+ calculated for C26H21N5O3S 484.1443, found 484.1441.
(E)-4-(4-((3-(3-(4-chlorophenyl)-3-oxoprop-1-en-1-yl)-1H-indol-1-yl)methyl)-1H-1,2,3-triazol-
1
1-yl)benzenesulfonamide (5b):Yellow solid; yield 95%, m.p 280-2820C; H NMR (500 MHz,
DMSO) δ 9.00 (s, 1H), 8.31 (s, 1H), 8.21 – 7.96 (m, 8H), 7.77 (d, J = 7.8 Hz, 1H), 7.70 – 7.60 (m,
3H), 7.53 (s, 2H), 7.32 (dd, J = 14.1, 7.4 Hz, 2H), 5.68 (s, 2H). 13C NMR (125 MHz, DMSO) δ
188.14, 144.51, 144.45, 139.12, 138.95, 137.81, 137.67, 137.53, 136.17, 130.56, 129.24, 127.95,
126.36, 123.52, 122.77, 122.15, 121.12, 120.88, 116.17, 113.01, 111.71, 41.70. HRMS (ESI) m/z:
[M+H]+ calculated for C26H20ClN5O3S 518.1054, found 518.1060.
(E)-4-(4-((3-(3-(4-fluorophenyl)-3-oxoprop-1-en-1-yl)-1H-indol-1-yl)methyl)-1H-1,2,3-triazol-1-
1
yl)benzenesulfonamide (5c): Yellow solid; yield 89%, m.p 261-2630C; H NMR (500 MHz,
DMSO) δ 9.00 (s, 1H), 8.30 (s, 1H), 8.26 – 8.20 (m, 2H), 8.13 (dd, J = 16.6, 8.2 Hz, 3H), 8.03 (dd,
J = 20.7, 12.0 Hz, 3H), 7.77 (d, J = 8.1 Hz, 1H), 7.68 (d, J = 15.5 Hz, 1H), 7.53 (s, 2H), 7.42 –
7.27 (m, 4H), 5.68 (s, 2H). 13C NMR (125 MHz, DMSO) δ 187.83, 166.15, 164.16, 144.54, 144.44,
138.95, 138.77, 137.65, 135.98, 135.47, 131.57, 131.49, 127.95, 126.36, 123.48, 122.76, 122.10,
121.12, 120.88, 116.27, 116.18, 116.00, 113.00, 111.67, 41.68. HRMS (ESI) m/z: [M+H]+
calculated for C26H20FN5O3S 502.1349, found 502.1365.
(E)-4-(4-((3-(3-(4-bromophenyl)-3-oxoprop-1-en-1-yl)-1H-indol-1-yl)methyl)-1H-1,2,3-triazol-
1
1-yl)benzenesulfonamide (5d): Yellow solid; yield 70%, m.p 276-2780C; H NMR (500 MHz,
DMSO) δ 9.00 (s, 1H), 8.31 (s, 1H), 8.16 – 7.99 (m, 8H), 7.78 (d, J = 8.3 Hz, 3H), 7.65 (d, J =
13
15.4 Hz, 1H), 7.52 (s, 2H), 7.32 (dd, J = 14.1, 7.7 Hz, 2H), 5.68 (s, 2H). C NMR (125 MHz,
DMSO) δ 188.32, 144.51, 144.44, 139.15, 138.94, 137.86, 137.67, 136.20, 132.19, 130.70, 127.95,
126.91, 126.35, 123.52, 122.77, 122.15, 121.12, 120.88, 116.13, 113.01, 111.71, 41.70. HRMS
(ESI) m/z: [M+Na]+ calculated for C26H20BrN5NaO3S 586.0347, found 586.0350
(E)-4-(4-((3-(3-oxo-3-(p-tolyl)prop-1-en-1-yl)-1H-indol-1-yl)methyl)-1H-1,2,3-triazol-1-
1
yl)benzenesulfonamide(5e): Yellow solid; yield 65%,m.p 265-2670C; H NMR (500 MHz,
DMSO) δ 8.99 (s, 1H), 8.28 (s, 1H), 8.15 – 7.98 (m, 8H), 7.76 (d, J = 7.9 Hz, 1H), 7.68 (d, J =
15.5 Hz, 1H), 7.52 (s, 2H), 7.32 (ddd, J = 19.9, 17.5, 7.4 Hz, 4H), 5.67 (s, 2H), 2.42 (s, 3H). 13C
NMR (125 MHz, DMSO) δ 188.80, 144.59, 144.45, 143.19, 138.95, 138.13, 137.62, 136.31,

135.58, 129.72, 128.75, 127.94, 126.42, 123.41, 122.74, 122.04, 121.00, 120.88, 116.69, 112.99,
111.64, 41.68, 21.62. HRMS (ESI) m/z: [M+Na]+ calculated for C27H23N5NaO3S 520.1419, found
520.1419.
(E)-4-(4-((3-(3-(3-bromophenyl)-3-oxoprop-1-en-1-yl)-1H-indol-1-yl)methyl)-1H-1,2,3-triazol-
1
1-yl)benzenesulfonamide (5f): Yellow solid; yield 75%, m.p 222-2240C; H NMR (500 MHz,
DMSO) δ 8.99 (s, 1H), 8.34 (s, 1H), 8.23 (s, 1H), 8.17 – 7.98 (m, 7H), 7.84 (d, J = 7.4 Hz, 1H),
7.77 (d, J = 7.8 Hz, 1H), 7.65 (d, J = 15.4 Hz, 1H), 7.57 – 7.48 (m, 3H), 7.31 (dd, J = 12.3, 7.8 Hz,
2H), 5.68 (s, 2H).13C NMR (125 MHz, DMSO) δ 187.96, 144.45, 141.00, 139.38, 138.95, 137.64,
136.14, 135.51, 131.40, 131.03, 127.95, 127.70, 126.45, 123.53, 122.77, 122.69, 122.17, 121.08,
120.88, 116.11, 113.03, 111.70, 41.74. HRMS (ESI) m/z: [M+Na]+ calculated for C26H20BrN5O3S
586.0347, found 586.0350
(E)-4-(4-((3-(3-(3-methoxyphenyl)-3-oxoprop-1-en-1-yl)-1H-indol-1-yl)methyl)-1H-1,2,3-triazol-
1
1-yl)benzenesulfonamide (5g): Yellow solid; yield 70%, m.p 244-2460C; H NMR (500 MHz,
DMSO) δ 8.98 (s, 10H), 8.26 (s, 10H), 8.12 (dd, J = 16.5, 8.7 Hz, 52H), 8.05 – 7.96 (m, 32H),
7.76 (d, J = 8.0 Hz, 11H), 7.69 (d, J = 15.5 Hz, 11H), 7.51 (s, 22H), 7.30 (dt, J = 14.6, 7.1 Hz,
22H), 7.09 (d, J = 8.6 Hz, 21H), 5.67 (s, 20H), 3.87 (s, 31H).13C NMR (125 MHz, DMSO) δ
187.67, 163.23, 144.61, 144.45, 138.96, 137.60, 135.34, 131.65, 130.90, 127.95, 126.43, 123.37,
122.73, 121.97, 120.99, 120.88, 116.66, 114.40, 113.02, 111.61, 55.97, 41.66. HRMS (ESI) m/z:
[M+H]+ calculated for C27H23N5O3S 514.1549, found 514.1547.
(E)-4-(4-((3-(3-(4-chloro-3-nitrophenyl)-3-oxoprop-1-en-1-yl)-1H-indol-1-yl)methyl)-1H-1,2,3-
1
triazol-1-yl)benzenesulfonamide (5h): Yellow solid; yield 76%, m.p 205-2070C; H NMR (500
MHz, DMSO) δ 9.01 (s, 1H), 8.61 (s, 1H), 8.38 (d, J = 14.0 Hz, 2H), 8.10 (t, J = 36.8 Hz, 6H),
7.75 (d, J = 27.2 Hz, 3H), 7.53 (s, 2H), 7.33 (s, 2H), 5.70 (s, 2H), 2.62 (s, 3H).13C NMR (125
MHz, DMSO) δ 187.00, 149.81, 144.48, 144.41, 139.78, 138.93, 137.75, 137.65, 137.28, 136.52,
133.75, 132.81, 127.95, 126.38, 124.21, 123.59, 122.80, 122.23, 121.18, 120.88, 115.59, 113.04,
111.75, 41.73, 19.95. HRMS (ESI) m/z: [M+H]+ calculated for C27H23N6O5S 543.1451, found
543.1446.
(E)-4-(4-((3-(3-(2,4-dichlorophenyl)-3-oxoprop-1-en-1-yl)-1H-indol-1-yl)methyl)-1H-1,2,3-
1
triazol-1-yl)benzenesulfonamide (5i): Yellow solid; yield 83%, m.p. 236-2380C; H NMR (500

MHz, DMSO) δ 8.98 (s, 1H), 8.21 (s, 1H), 8.10 (d, J = 8.4 Hz, 2H), 7.99 (dd, J = 16.9, 8.0 Hz,
3H), 7.77 (d, J = 8.4 Hz, 2H), 7.59 (dd, J = 40.6, 20.6 Hz, 5H), 7.30 (dt, J = 31.4, 7.3 Hz, 2H),
7.04 (d, J = 16.0 Hz, 1H), 5.65 (s, 2H).13C NMR (125 MHz, DMSO) δ 192.43, 144.44, 144.33,
141.85, 138.93, 138.79, 137.79, 136.86, 135.39, 131.55, 130.95, 129.97, 128.05, 127.95, 126.03,
123.66, 122.84, 122.41, 121.04, 120.88, 112.46, 111.84, 41.68. HRMS (ESI) m/z: [M+H]+
calculated for C26H20Cl2N5O3S 552.0664, found 552.0654.
(E)-4-(4-((3-(3-(3,4-dimethoxyphenyl)-3-oxoprop-1-en-1-yl)-1H-indol-1-yl)methyl)-1H-1,2,3-
1
triazol-1-yl)benzenesulfonamide (5j):Yellow solid; yield 71%,m.p. 194-1960C; H NMR (500
MHz, DMSO) δ 8.99 (s, 1H), 8.27 (s, 1H), 8.05 (d, J = 41.8 Hz, 6H), 7.86 (s, 1H), 7.78 – 7.66 (m,
2H), 7.59 (s, 1H), 7.51 (s, 2H), 7.30 (s, 2H), 7.11 (d, J = 7.1 Hz, 1H), 5.67 (s, 2H), 3.87 (s, 6H).13C
NMR (125 MHz, DMSO) δ 187.70, 153.23, 149.29, 144.45, 138.96, 137.58, 137.46, 135.20,
131.77, 127.94, 126.48, 123.35, 123.16, 122.73, 121.97, 120.88, 116.69, 113.01, 111.61, 111.44,
111.27, 56.24, 56.09, 41.67. HRMS (ESI) m/z: [M+H]+ calculated for C28H26N5O5S 544.1655,
found 544.1650.
(E)-4-(4-((3-(3-oxo-3-(3-(trifluoromethyl)phenyl)prop-1-en-1-yl)-1H-indol-1-yl)methyl)-1H-
1,2,3-triazol-1-yl)benzenesulfonamide (5k): Yellow solid; yield 74%, m.p. 219-2210C; 1H NMR
(500 MHz, DMSO) δ 8.99 (s, 1H), 8.47 (d, J = 7.0 Hz, 1H), 8.34 (d, J = 9.8 Hz, 2H), 8.12 (dd, J
= 17.8, 9.9 Hz, 4H), 8.01 (s, 3H), 7.83 (d, J = 7.3 Hz, 1H), 7.74 (dd, J = 30.0, 11.4 Hz, 2H), 7.50
(s, 2H), 7.37 – 7.26 (m, 2H), 5.69 (s, 2H).13C NMR (125 MHz, DMSO) δ 188.04, 144.49, 144.42,
139.71, 138.93, 137.64, 136.35, 132.71, 130.51, 129.25, 127.95, 126.43, 124.85, 123.56, 122.79,
122.22, 121.10, 120.88, 115.94, 113.01, 111.74, 41.74. HRMS (ESI) m/z: [M+H]+ calculated for
C27H21FN5O3S 552.1317, found 552.1315.
(E)-4-(4-((3-(3-(3-fluorophenyl)-3-oxoprop-1-en-1-yl)-1H-indol-1-yl)methyl)-1H-1,2,3-triazol-1-
1
yl)benzenesulfonamide (5l): Yellow solid; yield 82%, m.p. 223-2250C; H NMR (500 MHz,
DMSO) δ 8.99 (s, 1H), 8.32 (s, 1H), 8.15 (d, J = 7.7 Hz, 1H), 8.09 (dd, J = 15.1, 12.2 Hz, 3H),
8.04 – 7.96 (m, 3H), 7.90 (d, J = 9.6 Hz, 1H), 7.77 (d, J = 8.0 Hz, 1H), 7.69 – 7.60 (m, 2H), 7.54
13
– 7.46 (m, 3H), 7.32 (dt, J = 21.1, 7.1 Hz, 2H), 5.68 (s, 2H). C NMR (125 MHz, DMSO) δ
188.04, 163.81, 161.86, 144.51, 144.45, 141.26, 141.21, 139.32, 138.95, 137.67, 136.21, 131.34,
131.27, 127.95, 126.39, 124.81, 123.53, 122.77, 122.17, 121.17, 120.89, 119.85, 119.68, 116.17,

115.15, 114.98, 113.03, 111.68, 41.72. HRMS (ESI) m/z: [M+H]+ calculated for C26H21FN5O3S
502.1349, found 502.1351.
(E)-4-(4-((3-(3-(3,5-bis(trifluoromethyl)phenyl)-3-oxoprop-1-en-1-yl)-1H-indol-1-yl)methyl)-1H-
1,2,3-triazol-1-yl)benzenesulfonamide (5m): Yellow solid; yield 77%, m.p. 277-2790C; 1H NMR
(500 MHz, DMSO) δ 9.00 (s, 1H), 8.68 (s, 2H), 8.40 (d, J = 11.5 Hz, 2H), 8.23 – 8.07 (m, 4H),
8.01 (d, J = 8.2 Hz, 2H), 7.85 – 7.72 (m, 2H), 7.50 (s, 2H), 7.41 – 7.28 (m, 2H), 5.70 (s, 2H). 13C
NMR (125 MHz, DMSO) δ 186.74, 144.42, 140.94, 140.55, 138.92, 137.59, 136.50, 131.62,
131.36, 131.09, 130.83, 129.10, 127.95, 127.55, 126.89, 126.56, 126.02, 124.72, 123.63, 122.81,
122.55, 122.27, 121.06, 120.38, 115.51, 113.09, 111.76, 41.79. HRMS (ESI) m/z: [M+Na]+
calculated for C28H19F6N5O3S 642.1001, found 642.1007
(E)-4-(4-((3-(3-(1,3-dihydroisobenzofuran-5-yl)-3-oxoprop-1-en-1-yl)-1H-indol-1-yl)methyl)-
1
1H-1,2,3-triazol-1-yl)benzenesulfonamide (5n): Yellow solid; yield 80%, m.p. 241-2430C; H
NMR (500 MHz, DMSO) δ 8.98 (s, 1H), 8.27 (s, 1H), 8.06 (d, J = 50.4 Hz, 6H), 7.68 (dd, J =
109.0, 45.9 Hz, 6H), 7.31 (s, 2H), 7.08 (s, 1H), 6.16 (s, 2H), 5.67 (s, 2H).13C NMR (125 MHz,
DMSO) δ 187.25, 151.47, 148.38, 144.44, 138.96, 137.88, 137.59, 135.42, 133.51, 127.94, 126.44,
124.80, 123.38, 122.73, 121.98, 121.05, 120.88, 116.49, 113.03, 111.59, 108.53, 108.22, 102.38,
41.67. HRMS (ESI) m/z: [M+H]+ calculated for C27H22ClN5O5S 528.1342, found 528.1343.
(E)-4-(4-((3-(3-(2-bromophenyl)-3-oxoprop-1-en-1-yl)-1H-indol-1-yl)methyl)-1H-1,2,3-triazol-
1
1-yl)benzenesulfonamide (5o): Yellow solid; yield 72%, m.p. 182-1840C; H NMR (500 MHz,
DMSO) δ 8.99 (s, 1H), 8.34 (s, 1H), 8.23 (s, 1H), 8.17 – 7.98 (m, 7H), 7.84 (d, J = 7.4 Hz, 1H),
7.77 (d, J = 7.8 Hz, 1H), 7.65 (d, J = 15.4 Hz, 1H), 7.57 – 7.48 (m, 3H), 7.31 (dd, J = 12.3, 7.8 Hz,
2H), 5.68 (s, 2H).13C NMR (125 MHz, DMSO) δ 187.96, 144.45, 141.00, 139.38, 138.95, 137.64,
136.14, 135.51, 131.40, 131.03, 127.95, 127.70, 126.45, 123.53, 122.77, 122.69, 122.17, 121.08,
120.88, 116.11, 113.03, 111.70, 41.74. HRMS (ESI) m/z: [M+H]+ calculated for C26H21BrN5O3S
564.0528, found 564.0534.
(E)-4-(4-((3-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-en-1-yl)-1H-indol-1-yl)methyl)-1H-1,2,3-
1
triazol-1-yl)benzenesulfonamide (5p): Yellow solid; yield 73%, m.p. 244-2460C; H NMR (500
MHz, DMSO) δ 9.00 (s, 1H), 8.32 (s, 1H), 8.04 (dd, J = 33.2, 15.7 Hz, 6H), 7.76 (d, J = 7.4 Hz,
1H), 7.68 (s, 1H), 7.52 (s, 2H), 7.42 – 7.24 (m, 4H), 5.68 (s, 2H), 3.91 (s, 6H), 3.77 (s, 3H).13C

NMR (125 MHz, DMSO) δ 188.34, 153.34, 144.57, 144.45, 142.03, 138.95, 138.15, 137.55,
135.27, 134.39, 127.94, 126.58, 123.39, 122.74, 122.05, 120.88, 116.79, 112.98, 111.64, 106.40,
60.66, 56.69, 56.55, 41.71. HRMS (ESI) m/z: [M+H]+ calculated for C29H28N5O6 574.1760, found
574.1762
(E)-4-(4-((3-(3-(3-methoxyphenyl)-3-oxoprop-1-en-1-yl)-1H-indol-1-yl)methyl)-1H-1,2,3-triazol-
1
1-yl)benzenesulfonamide (5q): Yellow solid; yield 80%, m.p. 203-2050C; H NMR (500 MHz,
DMSO) δ 8.98 (s, 1H), 8.26 (s, 1H), 8.12 (dd, J = 16.5, 8.7 Hz, 52H), 8.05 – 7.96 (m, 32H), 7.76
(d, J = 8.0 Hz, 11H), 7.69 (d, J = 15.5 Hz, 11H), 7.51 (s, 22H), 7.30 (dt, J = 14.6, 7.1 Hz, 22H),
7.09 (d, J = 8.6 Hz, 21H), 5.67 (s, 20H), 3.87 (s, 31H).13C NMR (125 MHz, DMSO) δ 187.67,
163.23, 144.61, 144.45, 138.96, 137.60, 135.34, 131.65, 130.90, 127.95, 126.43, 123.37, 122.73,
121.97, 120.99, 120.88, 116.66, 114.40, 113.02, 111.61, 55.97, 41.66. HRMS (ESI) m/z: [M+H]+
calculated for C27H23N5O3S 514.1549, found 514.1540.
4.1.4. 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 [21]. 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 maintaining 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 250C were used
as substrate. Stock solutions of inhibitors were prepared at a concentration 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 preincubated 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 [22], 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 [23-24]

4.2 Computational studies
To rationalize the experimental results obtained, molecular docking studies were performed on
representative compounds 6d, 6q and co-crystallized ligand (acetazolamide) on the active site of
human carbonic anhydrase I (hCA I) enzyme by employing GLIDE docking module of
Schrödinger suite 2019-1 [25]. The protein crystal structure of hCA I in complex with
acetazolamide was retrieved from the RCSB Protein Data Bank (PDB code: 1AZM, resolution
2ꢀÅ) [26]. Next, protein was prepared by using Protein Preparation Wizard of Maeströ. Missing
amino acid residues and side chains were added using Prime module of Maeströ. The bound co-
crystallized ligand was used to define the active site in hCA I. All the ligand molecules were built
and optimized using Maestro Molecule Builder and OPLS_2005 force field in LigPrep modules
of Schrödinger software, respectively. Further, the prepared ligands were docked at the active site
of hCA I.
Acknowledgements
PS, 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. The Italian Ministry for
University and Research (MIUR) is gratefully acknowledged for a grant to CTS (PRIN: rot.
2017XYBP2R).
Conflict of Interest
The authors declare no conflict of interest.
References
1. C. T. Supuran, Carbonic anhydrases: novel therapeutic applications for inhibitors and
activators, Nat. Rev. Drug Discov. 7 (2008) 161-181.
2. B. Cornelio, M. L. Cochard, M. Ceruso, M. Ferraroni, G. A. Rance, F. Carta, A. N.
Khlobystov, A. Fontana, C. T. Supuran and J. Sapi, 4-Arylbenzenesulfonamides as human
carbonic anhydrase inhibitors (hCAIs): synthesis by Pd nanocatalyst-mediated Suzuki-

Miyaura reaction, enzyme inhibition and X-ray crystallographic studies J. Med. Chem. 59
(2016) 721-732.
3. G. D. Simone, V. Alterio & C. T. Supuran, Exploiting the hydrophobic and hydrophilic
binding sites for designing carbonic anhydrase inhibitors, Expert Opin. Drug Discov. 8
(2013) 793-810.
4. A. Weber, A. Casini, A. Heine, D. Kuhn, C. T. Supuran, A. Scozzafava and G. Klebe,
Unexpected Nanomolar Inhibition of Carbonic Anhydrase by COX-2-Selective Celecoxib:
New Pharmacological Opportunities Due to Related Binding Site Recognition, J. Med.
Chem. 47 (2004) 550-557.
5. C. T. Supuran, Carbon- versus sulphur-based zinc binding groupsfor carbonic anhydrase
inhibitors, J. Enzyme Inhib. Med. Chem. 33 (2018) 485–495.
6. A. Rogato, S. D. Prete, A. Nocentini, V. Carginale, C. T. Supuran and C. Capasso,
Phaeodactylum tricornutum as a model organism for testing the membrane penetrability of
sulphonamide carbonic anhydrase inhibitors, Journal of Enzyme Inhibition and Medicinal
Chemistry, 34: (2019) 510-518.
7. C. T. Supuran, Indisulam: an anticancer sulfonamide in clinical trial, Expert Opin. Investig.
Drugs. 12 (2003) 283-287.
8. C. Dittrich, A. S. Zandvliet, M. Gneist, A. D. R. Huitema, A. A. J King and J. Wanders, A
phase I and pharmacokinetic study of indisulam in combination with carboplatin, Br. J.
Cancer. 96 (2007) 559–566.
9. W. M. Eldehna, M. F. Abo-Ashour, A. Nocentini, R. S. El-Haggar, S. Bua, A. Bonardi, S.
T. Al-Rashood, G. S. Hassan, P. Gratteri, H. A. Abdel-Aziz and C. T. Supuran,
Enhancement of tail hydrophobic interactions within the carbonic anhydrase lX active site
via extension: Design and synthesis of novel N-substituted isatins-SLC-0111 hybrids as
carbonic anhydrase inhibitors and antitumour agents. Eur. J. Med. Chem. 162 (2019) 147-
160.
10. M. Bozdag, F. Varta, M. Ceruso, M. Ferrroni, P. C. McDonald, S. Dedhar and 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.

11. R. Kumar, L. Vats, S. Bua, C. T. Supuran and P. K. Sharma, Design and synthesis of novel
benzenesulfonamide containing 1,2,3,-triazoles as potent human carbonic anhydrase
isoforms I, II, lV and lX inhibitors. Bioorg. Chem.77 (2018) 542-547.
12. R. Kumar, L. Vats, S. Bua, C. T. Supuran, P. K. Sharma, Design and synthesis of novel
benzenesulfonamide containing 1,2,3-triazoles as potent human carbonic anhydrase
isoforms I, II, IV and IX inhibitors, Eur. J. Med. Chem. 155 (2018) 545-551.
13. A. Srinivas, P. V. Sri Ramya. A. Andrea, C. T. Supuran and M. Arifuddin, Novel
sulfocoumarin/coumarin/4-sulfamoylphenyl bearing indazole-3-carboxamide hybrids:
Synthesis and Selective inhibition of tumor associated carbonic anhydrase isozymes IX
and XII. ChemMedChem. 12 (2017) 1578-1584.
14. L. Vats, V. Sharma, A. Angeli, R. Kumar, C. T. Supuran and P. K. Sharma, Synthesis of
novel 4-functionalized 1,5-diaryl-1,2,3-triazoles containing benzesulfonamide moiety as
carbonic anhydrase l, ll, lV, and lX inhibitors. Eur. J. Med. Chem. 150 (2018) 678-686.
15. P. V. Sri Ramya, A. Angapelly, C. S. Digwal, M. Ariffudin, N. B. Bathini, S. T. Suura,
A. Kamal, A. A. Angeli and C. T. Supuran. Discovery of curcumin inspired sulfonamide
derivatives as a new class of carbonic anhydrase isoforms l, ll, lX, and Xll inhibitors. J.
Enzyme Inhib. Med. Chem. 32 (2017) 1274-1283.
16. T. Arslana, E. A. Turkoglu, M. Şenturk and C. T. Supuran, Synthesis and carbonic
anhydrase inhibitory properties of novel chalcone substituted benzenesulfonamides,
Bioorg. Med. Chem. Lett, 26 (2016) 5867-5870.
17. F. Manna, F. Chimenti, A. Bolasco, B. Bizzarri, W. Filippelli, A. Filippelli and L.
Gagliardi, Anti-inflammatory, analgesic and antipyretic 4,6-disubstituted 3-cyano-2-
aminopyridines, Eur. J. Med. Chem. 34 (1999) 245−254
18. A. Nagarsenkar, S. K. Prajapti, S. D. Guggilapu, S. Birineni, S. S. Kotapalli, R. Ummanni,
and B. N. Babu, Investigation of triazole-linked indole and oxindole glycoconjugates as
potential anticancer agents: novel Akt/PKB signaling pathway inhibitors, Med,chem.,commun.
7 (2016) 646-653.
19. B. L. Wilkinson, L. F. Bornaghi, T. A. Houston, A. Nocentini, C. T. Supuran and S. A.
Poulsen, A novel class of carbonic anhydrase inhibitors: glycoconjugate benzene
sulfonamides prepared by “clicktailing”, J. Med. Chem. 49 (2006) 6539-6548.

20. R. G. Khalifah, The carbon dioxide hydration activity of carbonic anhydrase. I. Stop-flow
kinetic studies on the native human isoenzymes B and C, J. Biol. Chem. 246 (1971) 2561-
2573.
21. (a) M. Bozdag, M. Ferraroni, F. Carta, D. Vullo, L. Lucarini, E. Orlandini, A. Rossello, E.
Nuti, A. Scozzafava, E. Masini and C. T. Supuran, Structural Insights on Carbonic
Anhydrase Inhibitory Action, Isoform Selectivity and Potency of Sulfonamides and
Coumarins Incorporating Arylsulfonylureido Groups, J. Med.Chem. 57 (2014) 9152 -
9167.(b) A. Grandane, M. Tanc, L. D. C. Mannelli, F. Carta, C. Ghelardini, R. Zalubovskis
and C. T. Supuran, Substituted Sulfocoumarins Are Selective Carbonic Anhdydrase IX and
XII Inhibitors with Significant Cytotoxicity against Colorectal Cancer Cells, J. Med.
Chem. 58 (2015) 3975 - 3983.
22. (a) N. Korkmaz, O. A. Obaidi, M. Senturk, D. Astley, D. Ekinci and C. T. Supuran,
Synthesis and biological activity of novel thiourea derivatives as carbonic anhydrase
inhibitors, J. Enzyme Inhib. Med. Chem. 30 (2015) 75-80. (b) A. Akdemir, C. D. Monte,
S. Carradori and C. T. Supuran, Computational investigation of the selectivity of salen and
tetrahydro salen compounds towards the tumor-associated hCA XII isozyme, J. Enzyme
Inhib. Med. Chem. 30 (2015) 114 -118. (c) I. Nishimori, D. Vullo, A. Innocenti, A.
Scozzafava, A. Mastrolorenzo and C. T. Supuran, Carbonic Anhydrase Inhibitors. The
Mitochondrial Isozyme VB as a New Target for Sulfonamide and Sulfamate Inhibitors, J.
Med. Chem. 48 (2005) 7860-7866.
23. (a) H. Gocer, A. S. Akincioglu, G. I. Gulcin and C. T. Supuran, Carbonic anhydrase and
acetylcholinesterase inhibitory effects of carbamates and sulfamoyl carbamates, J. Enzyme
Inhib. Med.Chem. 30 (2015) 316-320.(b) M. Ceruso, M. Bragagni, Z. AlOthman, S. M.
Osman and C. T. Supuran, New series of sulfonamides containing amino acid moiety act
as effective and selective inhibitors of tumor-associated carbonic anhydrase XII, J. Enzyme
Inhib. Med. Chem. 30 (2015) 430-434. (c) R. Z. Emameh, L. Syrjanen, H. Barker, C. T.
Supuran and S. Parkkila, Drosophila melanogaster: a model organism for controlling
Dipteran vectors and pests, J. Enzyme Inhib. Med. Chem. 30 (2015) 505 -513.(d) A. M.
Alafeefy, M. Ceruso, A.M.S. Al-Tamimi, S. D. Prete, C. T. Supuran and C. Capasso,
Inhibition studies of quinazoline-sulfonamide derivatives against the g-CA (PgiCA) from

the pathogenic bacterium, Porphyromonas gingivalis, J. Enzyme Inhib. Med. Chem. 30
(2015) 592-596.
24. (a) C. T. Supuran, M. Barboiu, C. Luca, E. Pop, M. E. Brewster and A. Dinculescu,
Carbonic anhydrase activators. Part 14. Syntheses of mono and bis pyridinium salt
derivatives of 2-amino-5 (2-aminoethyl)- and 2-amino-5(3-aminopropyl)-1,3,4-thiadiazole
and their interaction with isozyme II, Eur. J. Med. Chem. 31 (1996) 597-606.
(b) L. Puccetti, G. Fasolis, D. Vullo, Z. H. Chohan, A. Scozzafava and C. T. Supuran,
Carbonic anhydrase inhibitors. Inhibition of cytosolic/tumor-associated carbonic
anhydrase isozymes I, II, IX, and XII with Schiff’s bases incorporating chromone and
aromatic sulfonamide moieties, and their zinc complexes, Bioorg. Med. Chem. Lett. 15
(2005) 3096-3101. (c) C. T. Supuran, A. Nicolae and A. Popescu, Carbonic anhydrase
inhibitors. Part 35. Synthesis of Schiff bases derived from sulfanilamide and aromatic
aldehydes: the first inhibitors with equally high affinity towards cytosolic and membrane-
bound isozymes, Eur. J. Med. Chem. 31 (1996) 431-438.
25. Schrödinger Release 2019-1: Maestro, Schrödinger, LLC, New York, NY, 2019.
26. S. Chakravarty, K. K. Kannan, Drug-protein interactions: Refined structures of three
sulfonamide drug complexes of human carbonic anhydrase I enzyme, J. Mol. Biol. 1994,
243, 298-309.

Highlights
 17 novel compound of indole-1,2,3-triazole chalcones (6a-q) hybrid were synthesized
 The synthesized compounds were screened for their CA inhibition potency against four
human CA isoforms hCA I, II, IX, XII
 Compound 6d (18.8 nm), 6q (38.3 nm) and 6e (50.4 nm) are 13, 6 and 5 times more potent
than standard AAZ against hCA I.
 Compound 6o, 6m and 6f also inhibit isoform hCA XII in the range of 10 nm, 36.9 nm and
41.9 nm respectively
A novel series of indolylchalcone linked with benzenesulfonamide-1,2,3-triazoles (6a-q) has been
synthesized by click chemistry reaction and investigated for their hCA inhibitory activity against
a panel of human carbonic anhydrases (CA; EC 4.2.1.1). The hCA inhibition assay results shows
that these compounds were inhibiting all the four isoforms, hCA I, II, IX and XII. Among all the
compounds tested the compound 6d (18.8 nM), 6q (38.3 nM) and 6e (50.4 nM) are 13, 6 and 5
times more potent than standard AAZ respectively against hCA I isoform. The compound 6o, 6m
and 6f also inhibited isoform hCA XII in the range of 10 nM, 36.9 nM and 41.9 nM respectively.
Few of the compounds are also active in against isoform hCA II and isoform hCA IX under 100
nM ranges.
OMe
Br
Br
O
O
O
SO2NH2
N
SO2NH2
SO2NH2
N
N
N
N
N
N
N
N
N
N
N
hCA I Ki = 18.8 nm
hCA I Ki = 38.3 nm
hCA XII Ki = 10 nm
All other are agreed for this work to be published and have no conflict of interest