Novel triazole-sulfonamide bearing pyrimidine moieties with carbonic anhydrase inhibitory action: Design, synthesis, computational and enzyme inhibition studies

Article abstract of DOI:10.1016/j.bmcl.2021.128249

A series of new triazole-sulfonamide bearing pyrimidine derivatives were designed and synthesized via click chemistry. All new compounds (SH-1 to SH-28) were validated by ¹HNMR, ¹³CNMR, HRMS, and SH-3 was further structurally validated by X-Ray single diffraction study. These compounds (SH-1 to SH-28) were tested as inhibitors of human carbonic anhydrase (hCA) isoforms, such as hCA I, II, IX and XII, using a stopped flow CO₂ hydrase assay. Most of the compounds exhibited significant inhibitory activity against hCA II and weak inhibitory activity against hCA I. The target compounds also displayed moderate to excellent inhibitory activity against tumor-related hCAs IX and XII. Some compounds, e.g., SH-20 (K_i = 9.4 nM), SH-26 (K_i = 1.8 nM) and SH-28 (K_i = 0.82 nM) exhibited excellent inhibitory activity and selectivity profile against hCAs XII over IX. SH-23 displayed promising inhibitory activity and selectivity profile against both tumor-related hCAs IX (K_i = 2.9 nM) as well as XII (K_i = 0.82 nM) over hCA I and II. To understand the molecular interactions, molecular docking study of compounds SH-20, SH-23, SH-26 and SH-28 with hCA XII and SH-23 also with hCA IX were performed. The computational study evidenced favorable interaction between the inhibitors and active residues of both proteins. Some of these derivatives are promising leads for the development of selective, anticancer agents based on CA inhibitors.

Full text of DOI:10.1016/j.bmcl.2021.128249

Bioorg. Med. Chem. Lett. 48 (2021) 128249
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel triazole-sulfonamide bearing pyrimidine moieties with carbonic
anhydrase inhibitory action: Design, synthesis, computational and enzyme
inhibition studies
,
,*
Shoaib Manzoora, Andrea Petrenib, Md Kausar Razac, Claudiu T. Supuranb *, Nasimul Hodaa
a Drug Design and Synthesis Laboratory, Department of Chemistry, Jamia Millia Islamia, New Delhi 110025, India
b University of Florence, Department of Neuroscience Psychology, Drug Research and Child’s Health, Section of Pharmaceutical and Nutraceutical Sciences, Via Ugo
Schiff 6, 50019 Sesto Fiorentino, Italy
c Department of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore 560012, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of new triazole-sulfonamide bearing pyrimidine derivatives were designed and synthesized via click
chemistry. All new compounds (SH-1 to SH-28) were validated by 1HNMR, 13CNMR, HRMS, and SH-3 was
further structurally validated by X-Ray single diffraction study. These compounds (SH-1 to SH-28) were tested as
inhibitors of human carbonic anhydrase (hCA) isoforms, such as hCA I, II, IX and XII, using a stopped flow CO2
hydrase assay. Most of the compounds exhibited significant inhibitory activity against hCA II and weak inhibitory
activity against hCA I. The target compounds also displayed moderate to excellent inhibitory activity against
tumor-related hCAs IX and XII. Some compounds, e.g., SH-20 (Ki = 9.4 nM), SH-26 (Ki = 1.8 nM) and SH-28 (Ki
= 0.82 nM) exhibited excellent inhibitory activity and selectivity profile against hCAs XII over IX. SH-23 dis-
played promising inhibitory activity and selectivity profile against both tumor-related hCAs IX (Ki = 2.9 nM) as
well as XII (Ki = 0.82 nM) over hCA I and II. To understand the molecular interactions, molecular docking study
of compounds SH-20, SH-23, SH-26 and SH-28 with hCA XII and SH-23 also with hCA IX were performed. The
computational study evidenced favorable interaction between the inhibitors and active residues of both proteins.
Some of these derivatives are promising leads for the development of selective, anticancer agents based on CA
inhibitors.
Triazole
Pyrimidine
Sulfonamide
Carbonic anhydrase inhibitors
Docking
Introduction
ciliary processes of eye. Other isoforms have a comparatively restricted
tissue distribution e.g., CA IX, in normal physiologic conditions, is
Carbonic anhydrases (CAs, EC 4.2.1.1) found in all organisms are a
superfamily of zinc metalloenzymes which catalyzes the reversible hy-
mainly found in the epithelium lining the stomach and small in-
testines1,9. CAs plays a key role in various pathological conditions and
currently the investigation of CA inhibition is a very active research area
taking account of the presence of multiple isoforms of this enzyme. In
various pathological conditions CAs were identified to play crucial roles,
for example in some central nervous system (CNS) diseases, glaucoma,
cancer, and obesity1,10–12. In recent years, hCA IX and XII became
fascinating targets for the discovery of new anti-proliferative com-
pounds, due to their significant role in the survival, proliferation and
spread of cancer cells. In hypoxic conditions, hCA IX and hCA XII
participate in the regulation of extracellular and intracellular pH as well
as the metabolism of the tumor cell, and therefore, their selective inhi-
dration of CO2 and H2O to HCO–3 and H+ ions (CO2 + H2O ⇌ HCOꢀ3
+
H+). Currently, 15 different isoforms of CA in humans have been
discovered and these isoforms have different subcellular localizations,
among which hCA I-III, VII and XIII are cytosolic, hCA IV, IX, XII, and
XIV are membrane bound, hCA VA and VB are mitochondrial, isoform VI
is secreted whereas the CA isoforms VIII, X and XI are acatalytic.
Through any of these CA isoforms, cells can easily regulate the extra-
cellular and intracellular pH and CO2/HCOꢀ3 pools1–8. The distribution
of human CA isoforms varies from one isoform to another, with some
isoforms being particularly abundant in most cells and tissues (e.g., CA I,
CA II), whereas CA IV is particularly abundant in kidneys, lungs, and
bition triggers interesting antitumor clinical effects12–13
.
* Corresponding authors.
E-mail addresses: shoaibchem19@gmail.com (S. Manzoor), claudiu.supuran@unifi.it (C.T. Supuran), nhoda@jmi.ac.in (N. Hoda).
https://doi.org/10.1016/j.bmcl.2021.128249
Received 5 May 2021; Received in revised form 26 June 2021; Accepted 1 July 2021
Available online 6 July 2021
0960-894X/© 2021 Elsevier Ltd. All rights reserved.

S. Manzoor et al.
Bioorganic & Medicinal Chemistry Letters 48 (2021) 128249
The primary sulfonamide scaffold plays a significant role in CA in-
(CH2O–) linker will give better flexibility for inhibition of CAs.
hibition, being among the versatile scaffolds used to build up selective
and potent CA inhibitors1–2,14–17. Currently, >30 sulfonamide-based
compounds are in clinical use with few examples such as acetazol-
amide (AAZ) A, methazolamide B, ethoxzolamide C, celecoxib D4,
dichlorophenamide E, dorzolamide F (Fig. 1). In deprotonated form, the
sulfonamide derivatives bind to CAs at the active site zinc (II) ion
through the primary nitrogen atom of the sulfonamide moiety.
Depending upon the modifications of the sulfonamide scaffolds, addi-
tional interactions with hydrophilic and hydrophobic amino acids resi-
dues occur within the CA active site. Therefore, novel CA inhibitors can
be designed and synthesized using the benzenesulfonamide as head
groups by incorporating a variety of substitution patterns on these
The synthesis of triazole-sulfonamide based pyrimidine derivatives
(SH-1 to SH-28) is shown in scheme 1 and 2. The new derivatives (SH-1
to SH-28) were synthesized from commercially available benzene-
sulfonamide. As shown in Scheme 1, two distinct synthetic methods
were used to obtain 2,4-dichloropyrimidine derivatives 5 (a and b). 2,4-
dichloro pyrimidine 5a was obtained from commercially available uracil
(4a) refluxed in POCl3. 2,4-dichloro-6-methylpyrimidine (5b) was ob-
tained in multiple steps as shown in scheme 1. The ethyl acetoacetate (1)
and thiourea (2) were reacted in water in presence of K2CO3 and conc.
HCl to give 6-methyl-2-thioxo-2,3-dihydropyrimidin-4(1H)-one (3). 6-
methyluracil (4) was obtained from 6-methyl-2-thioxo-2,3-dihydropyri-
midin-4(1H)-one (3) refluxed in chloroacetic acid and water in presence
of HCl. 6-methyluracil (4b) was refluxed in POCl3 to obtain 2,4-
dichloro-6-methylpyrimidine (5b). Both 2,4-dichloropyrimidines 5(a
and b) were reacted with propargyl alcohol in DMF to obtain in-
termediates 6 (a and b) in presence of K2CO3 as a base.
scaffolds and on their tails10,18–21
.
Click chemistry has been widely used to achieve CA inhibitors
belonging to the sulfonamide class. Copper-catalyzed azideꢀ alkyne
cycloadditions (CuAAC) have achieved a significant role due to their
short reaction time, good yields, and their modularity21–25. Triazole-
sulfonamide scaffolds incorporating substituted aromatic aryl or het-
erocyclic groups by means of uncleavable flexible linkers of the ether
(CH2O–), or amine (CH2NH) types led obtained to effective CA
As shown in scheme 2, benzenesulfonamide azide (8) was obtained
from commercially available benzenesulfonamide (7). The intermediate
triazole-benzenesulfonamide pyrimidine 9 (a and b) were synthesized
by click chemistry from intermediate 6(a and b) and freshly prepared
benzenesulfonamide azides (8) in the presence of CuSO4⋅5H2O and so-
dium ascorbate. The intermediate triazole-benzenesulfonamide pyrim-
idine 9(a and b) was reacted in dry DMF with different substituted
secondary amines using K2CO3 as base, affording the target triazole-
sulfonamide based pyrimidine derivatives (SH-1 to SH-28). The syn-
thesized compounds were purified by using flash column chromatog-
raphy. The target compounds (SH-1 to SH-28) were well validated by
using mass spectroscopy, 1H and 13C nuclear magnetic resonance (NMR)
and SH-3 was further structurally validated by X-Ray single diffraction
study. The purity of the all the final compounds was analyzed by UPLC
(Ultra Performance Liquid Chromatography) and was > 94%.
inhibitors26
.
For the design of new CA inhibitors, in the present work the triazole-
sulfonamide scaffold was fused with substituted pyrimidine rings
through linkers of the CH2O– type, for enhancing the flexibility, which
may produce significant interaction within the CA active sites, as well as
selectivity profiles for the various isoforms (Fig. 2). The increasing
flexibility of inhibitors may increase degrees of freedom and decrease
molecular tension, whereas aromatic tails can improve the interaction
with favorable sub pocket of the enzymes27. The incorporation of
substituted pyrimidine ring as tails to classical benezenesulfonamide
scaffolds was investigated, in order to assure effective binding with
various CA isoenzymes. The pyrimidine hybrids are the most common
nitrogen based heterocycle in nature and are associated in various
cellular and metabolic processes28–30. Pyrimidine like hybrids have been
developed that exhibit potential anti-tumor and anti-neurodegenerative
activity31–33. Pyrimidine derivatives were shown to inhibit cytosolic
hCA I, hCA II, hCA IV and hCA IX, which may be of interest for the
Compound SH-3 was structurally characterized by single crystal X-
ray crystallography (Fig. 3 and Table 1 and Figs. S85 and S86, sup-
porting information). The SH-3 compound crystallized in P121/c1
space group of the monoclinic system. In unit-cell packing diagram of
SH-3 compound exhibited the hydrogen bonding interaction between
the hydrogen atom of the sulfamoyl (N-7) and the ethereal oxygen (O-1)
at a distance of 2.436 Å. The hydrogen of pyrimidine at C-9 also dis-
played the hydrogen bonding interaction with oxygen atoms of sulfa-
moyl group at a distance of 2.367 Å. The unit cell packing diagrams of
SH-3 showed short contact bonding with their adjacent atoms. The unit
cell packing diagram of SH-3 compound are given in supporting
development of tighter-binding inhibitors34–35
.
Hence, we reported a new series of triazole-sulfonamide based py-
rimidine derivatives, that were designed, synthesized, and tested for
their inhibitory activity against hCA I, II, IX and XII. It was expected that
the introduction of substituted pyrimidine as a tail along with ether
Fig. 1. Clinically used sulfonamide-based CA inhibitors A-F.
2

S. Manzoor et al.
Bioorganic & Medicinal Chemistry Letters 48 (2021) 128249
Fig. 2. Design strategy of novel CA inhibitors.
Scheme 1. Reagents and conditions: (i) H2O, K2CO3, Conc. HCl, 100 ◦C; (ii) Chloroacetic acid, water, conc. HCl, reflux; (iii) POCl3, Reflux, 3–4 h; (iv) propargyl
alcohol, K2CO3, DMF, 60–70 ◦C, N2.
information (Figs. S85 and S86, Supporting information).
contain N-methylpiperazine (SH-2) linked with sulfamoylphenyl-
pyrimidine and N-ethylpiperazine (SH-19) with sulfamoylphenyl-
6-methylpyrimidine displayed significant inhibitory activity to-
wards hCA I with Ki value 41.5 and 44.3 nM respectively. Moreover,
the rest of the compounds showed weak inhibitions against hCA I.
2. hCA II: Most of the synthesized compounds showed effective inhib-
itory activity against hCA II isoform (Ki values ranging from 0.31 to
34.1 nM). However, derivatives which contain p-methox-
yphenylpiperazine (SH-6) and 2-piperazinylpyridine (SH-10) linked
with sulfamoylphenyl-pyrimidine and 2-piperazin-1-ylnicotinoni-
trile (SH-18) and p-chlorobenzylpiperazine (SH-23) with
sulfamoylphenyl-6-methylpyrimidine showed satisfactory inhibitory
activity between nanomolar range (Ki = 13.5 to 34.1 nM), which are
slightly weaker inhibitors compared to the AAZ standard drug (Ki =
12.0 nM). While the rest of the compounds showed strong inhibitions
against hCA II. It is noteworthy that few compounds were found to
act as subnanomolar hCA II inhibitors, e.g., SH-7 (Ki = 0.59 nM), SH-
11 (Ki = 0.80 nM), SH-13 (Ki = 0.65 nM), SH-22 (Ki = 0.92 nM) and
The synthesized new triazole-sulfonamide linked with pyrimidine
derivatives (SH-1 to SH-28) were assessed for their inhibition potential
against four human CA isoforms, namely, the cytosolic isoforms, hCA I
and hCA II and the trans-membrane tumor-related isoforms, hCA IX and
hCA XII, by a stopped flow CO2 hydrase assay method using acetazol-
amide as standard drug. The following structureꢀ activity relationship
(SAR) were highlighted from CA inhibition data of Table 2.
1. hCA I: All the synthesized compounds (SH-1 to SH-28) against
ubiquitous cytosolic hCA I displayed inhibitory activity between
nanomolar range (Ki = 41.5 to 94.8 nM) to micromolar range (Ki =
181.8 to 6745 nM). The compounds morpholine (SH-1), piperidine
(SH-3) and ethyl piperazine-1-carboxylate (SH-12) bearing
sulfamoylphenyl-pyrimidine and N-methylpiperazine (SH-16) and
ethyl piperazine-1-carboxylate (SH-26) bearing sulfamoylphenyl-6-
methylpyrimidine displayed satisfactory inhibition toward hCA I
with Ki value ranging from 61.9 to 94.8 nM. However, the derivatives
3

S. Manzoor et al.
Bioorganic & Medicinal Chemistry Letters 48 (2021) 128249
Scheme 2. Reagents and conditions: (i) NaNO2, HCl 6 N; NaN3, 2 h, rt, 100 ◦C; (ii) CuSO4⋅5H2O, sodium ascorbate, H2O:THF, 50–60 ◦C, 3 h; (iii) different
secondary amines, K2CO3, DMF, 3–4 h, 70–80 ◦C.
Fig. 3. ORTEP diagram of compounds SH-3 (50% probability level of thermal ellipsoids). Color codes: Carbon, light blue; Nitrogen: lavender; Oxygen: red and S,
light yellow. Hydrogen atoms are hidden for clarity.
SH-27 (Ki = 0.83 nM). The compounds benzylpiperazine and o-
methoxyphenylpiperazine bearing sulfamoylphenyl-pyrimidine
were found to acts as promising hCA II inhibitors at low nanomolar
range, e.g., SH-8 (Ki = 1.4 nM) and SH-14 (Ki = 1.2 nM).
and 465.7 nM respectively displayed weaker inhibitory activity
against hCA IX.
4. hCA XII: Most of the compounds displayed significant inhibitory
activity against tumor-related hCA XII isoform with Ki values ranging
from 0.36 to 90.5 nM. However, Compounds SH-3, SH-4, SH-5, SH-
12, SH-15, SH-16, SH-19, SH-23, SH-26, and SH-28 showed effec-
tive inhibitory potency in the subnanomolar range (0.36 to 0.90 nM)
against hCA XII isoform as compared to AAZ standard drug (5.7 nM).
Derivatives which contain p-methoxyphenylpiperazine (SH-20), o-
methoxyphenylpiperazine (SH-28), and ethyl piperazine-1-
carboxylate (SH-26) with sulfamoylphenyl-6-methylpyrimidine dis-
played as significant hCA XII inhibitors. The compounds SH-20 was
16-fold, SH-26 was 37-fold and SH-28 was 44-fold more selective
against tumor-related hCA XII isoform than tumor-related hCA IX
isoform. Moreover, the p-chlorobenzylpiperazine substituted deriv-
ative SH-23 displayed promising hCA XII inhibitor, which possessed
subnanomolar activity (0.82 nM) along with significant selectivity
towards hCA I and hCA II. Derivative SH-23 showed 1057-fold
selectivity over hCA I and > 41-fold selectivity over hCA II. In
addition, compounds SH-6, SH-10 and SH-25 showed satisfactory
3. hCA IX: The tumor-related isoform hCA IX was inhibited by all the
synthesized compounds (SH-1 to SH-28) in the 1.6–465.7 nM range.
Derivatives which contain ethyl piperazine-1-carboxylate (SH-12)
with sulfamoylphenyl-pyrimidine and p-chlorobenzylpiperazine
(SH-23) and 2-piperazinylpyridine (SH-24) with sulfamoylphenyl-6-
methylpyrimidine displayed strong inhibitory activity against hCA
IX. Interestingly, p-chlorobenzylpiperazine substituted derivative
SH-23 appeared as an excellent hCA IX inhibitor, along with high
selectivity towards hCA I and hCA II. Derivative SH-23 displayed >
299-fold selectivity over hCA I and > 11-fold selectivity over hCA II.
Compounds SH-2, SH-9, SH-11, SH-13, SH-15, SH-16, SH-18, SH-
20, SH-25, and SH-26 displayed nearly the same inhibition
(24.1–33.4 nM) of hCA IX to that of the standard drug AZZ (25.0 nM).
Only three derivatives, 2-piperazin-1-ylnicotinonitrile (SH-4),
bulkier diphenyl-piperazine (SH-7) in series
I and p-fluo-
rophenylpiperazine (SH-27) in series II, with Ki values 155.2, 192.0
4

S. Manzoor et al.
Bioorganic & Medicinal Chemistry Letters 48 (2021) 128249
Table 1
Table 2
Selected crystal data and structure refinement for 4-(4-(((2-(piperidin-1-yl)
Inhibition data of human hCA isoforms I, II, IX and XII for compounds SH-1 to
SH-28 using as AAZ standard drug.
pyrimidin-4-yl)oxy)methyl)-1H-1,2,3-triazol-1-yl)benzenesulfonamide (SH-
3).
Compd
R
R1
Ki (nM)a
hCA I
81.7
SH-3
hCA II
hCA IX
hCA XII
Empirical formula
Fw/g Mꢀ 1
Crystal system
Space group
a/Å
C36H42N14O6S2
415.16
SH-1
SH-2
SH-3
SH-4
H
H
H
H
0.43
42.5
78.9
monoclinic
P121/c1
41.5
89.6
628.9
0.63
0.31
0.66
30.4
41.3
90.5
0.81
0.87
8.748(9)
b/Å
10.2760(10)
16.4122(15)
90
c/Å
155.2
◦
α
/
β/◦
106.155(3)
90
SH-5
SH-6
H
H
6173
0.86
13.5
22.7
51.4
0.71
9.4
γ/◦
V/Å3
Z
2237.3(4)
2
830.9
T, K
298(2)
ρ
calcd/Mg mꢀ 3
1.233
SH-7
H
563.4
0.59
192.0
78.8
λ/Å (Mo-K
α
)
0.71073 Å
3937/9/270
872
Data/restraints/param
F(000)
GOF
SH-8
SH-9
H
H
315.0
6745
1.4
9.1
39.1
32.2
5.3
1.107
R(Fo),a I > 2
σ
(I) [wR(Fo)b]
0.0595 [0.1665]
0.0825 [0.1772]
0.431, ꢀ 0.420
A ¼ 0.1020B = 0.3210
66.6
R (all data) [wR (all data)]
Largest diff peak, hole (e Åꢀ 3
SH-10
SH-11
SH-12
H
H
H
845.8
441.3
85.4
16.8
0.80
3.4
16.8
26.3
1.6
9.5
70.7
0.76
w ¼ 1/[ (
σ
Fo)2 þ (AP)2 þ (BP)]
∑
∑
a
R = iFoi - iFcii/ iFoi
∑
∑
wR = { [w(Fo2 - Fc22]/ [w(Fo)2]}1/2, where P = (Fo + 2Fc2/3.
b
2
inhibitory activity in the nanomolar range (Ki = 7.4–9.5 nM), which
are slightly weaker inhibitors compared to the AAZ standard drug.
Compounds SH-1, SH-2, SH-7, SH-9, SH-11, SH-13, SH-14, SH-18,
SH-21, and SH-27 displayed weaker inhibitory activity against hCA
XII with Ki values ranging from 38.4 to 78.9 nM.
SH-13
SH-14
H
H
1610
0.65
1.2
25.8
44.7
48.8
64.4
243.6
SH-15
SH-16
SH-17
SH-18
CH3
CH3
CH3
CH3
181.8
61.9
0.87
0.61
6.5
30.2
24.1
35.8
32.0
0.69
0.36
23.6
68.9
It is clearly evoked by SAR study that the ether linker containing
triazole sulfamoylphenyl based pyrimidine derivatives showed signifi-
cant activity towards tumor-related hCAs IX and XII. The most active
compounds SH-20, SH-26 and SH-28 bestowed significant inhibition for
hCA XII isoform. In addition, SH-23 displayed excellent inhibition for
both hCA IX as well as hCA XII.
737.3
2324
28.5
SH-19
SH-20
CH3
CH3
44.3
0.32
4.7
16.1
29.7
0.55
1.8
In this study, a molecular docking study was performed to analyze
the interaction of some of the new sulfonamides within the active site of
hCA IX and hCA XII, for rationalizing the drug-receptor interaction.
However, binding free energy and interaction of the best posed com-
pounds were investigated by AutoDock vina program. In our study,
compounds SH-20, SH-26 and SH-28 showed significant selectivity to-
wards hCA XII, and SH-23 exhibited significant selectivity towards both
hCA IX and hCA XII, was taken account for this study. The compounds
SH-20, SH-26 and SH-28 were thus docked into hCA XII and SH-23 were
docked into hCA IX and hCA XII using AutoDock vina software 1.5.7
(Figs. 4 and 5). The molecular docking study revealed that these
triazole-sulfonamide based pyrimidine compounds displayed higher
binding affinity towards hCA IX and hCA XII and benzenesulfonamide
moiety accommodates exactly into the active catalytic pocket towards
zinc ion.
866.7
SH-21
CH3
4646
8.5
21.4
38.4
SH-22
SH-23
CH3
CH3
391.4
867.4
0.92
34.1
41.1
2.9
22.0
0.82
SH-24
SH-25
SH-26
CH3
CH3
CH3
652.7
5583
94.8
7.1
3.7
3.9
9.1
32.8
33.4
5.6
7.4
0.90
SH-27
SH-28
CH3
CH3
3254
0.83
4.0
465.7
36.3
47.4
0.82
It has been found that compounds SH-20, SH-26 and SH-28 has best
scores with ꢀ 8.4, ꢀ 7.6 and ꢀ 8.7 kcal/mol towards hCA XII, respec-
tively. The compounds are stabilized by hydrogen bond interaction be-
tween the –NH of sulfonamide with Thr199 amino acid residue in Fig. 4
(A, B and C). These compounds are further stabilized by hydrogen bond
interaction between sulfonamide oxygen with the peptide –NH of
Thr199 and Thr200 residues. The nitrogen rich triazole moiety of the
SH-20 (Fig. 4A), SH-26 (Fig. 4B) and SH-28 (Fig. 4C) compounds
formed a short contacting hydrogen bond with Asn62, Lys67 and Gln92
residues in hCA XII. The tail portion (piperazine based derivatives)
exhibited van der walls interaction with the residues Thr91, Ala131 and
Ser132, which might involve in selectivity of compounds towards hCA
428.3
AAZ
250.0
12.0
25.0
5.7
a
Mean from 3 different assays, by a stopped flow technique (errors were in the
range of ± 5–10% of the reported values).
5

S. Manzoor et al.
Bioorganic & Medicinal Chemistry Letters 48 (2021) 128249
Fig. 4. Compound SH-20 (A), SH-26 (B), SH-28 (C) best docking pose in hCA XII (pdb: 1JDO).
Fig. 5. (A) Compound SH-23 best docking pose in hCA IX (pdb: 3IAI); (B) Compound SH-23 best docking pose in hCA XII (pdb:1JDO).
XII. In addition, oxygen of methoxy group of SH-20 and carbonyl oxygen
group of SH-28 displayed hydrogen bond with Tyr123 and Thr91 resi-
dues in hCA XII respectively and nitrogen of pyrimidine moiety of SH-26
also exhibited hydrogen bond with Ser132.
Similarly, SH-23 has best scores with ꢀ 7.1 and ꢀ 8.2 kcal/mol to-
wards hCA IX and hCA XII, respectively. The compound SH-23 is sta-
bilized by hydrogen bond interaction between the –NH of sulfonamide
with Thr199 residue in hCA IX and hCA XII in Fig. 5 (A and B). The SH-
6

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Bioorganic & Medicinal Chemistry Letters 48 (2021) 128249
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23 compound is further stabilized by hydrogen bond interaction be-
tween sulfonamide oxygen with the peptide –NH of Thr199 in hCA IX
and with Thr199 and Thr200 in hCA XII. The electron rich pyrimidine
moiety of the SH-23 in hCA IX displayed hydrogen bond interaction with
Leu70 side chain (Fig. 5A) and triazole moiety of SH-23 in hCA XII also
formed hydrogen bonds with Asn62, Lys67 and Gln92 residues (Fig.
5B). Therefore, docking study confirms the importance of triazole and
pyrimidine derivatives in generating selective and potency between CA
enzymes.
[7] Smith KS, Jakubzick C, Whittam TS, Ferry JG. Carbonic anhydrase is an ancient
enzyme widespread in prokaryotes. Proc Natl Acad Sci. 1999;96(26):15184–15189.
https://doi.org/10.1073/pnas.96.26.15184.
[8] Supuran CT. Structure-based drug discovery of carbonic anhydrase inhibitors.
J Enzyme Inhib Med Chem. 2012;27(6):759–772. https://doi.org/10.3109/
14756366.2012.672983.
In summary, a series of new designed triazole-sulfonamide bearing
pyrimidine derivatives were synthesized via click chemistry. All the
synthesized compounds (SH-1 to SH-28) were validated by 1HNMR,
13CNMR, HRMS, and SH-3 was structurally validated by X-Ray single
diffraction analysis. The target compounds (SH-1 to SH-28) were tested
on hCA isoforms like hCA I, II, IX and XII, using a stopped flow CO2
hydrase assay. Most of the compounds displayed poor inhibitory activity
against hCA I (Ki 41.5 to 5583 nM) and significant inhibitory activity
towards hCA II (Ki 0.31 to 34.1 nM). The compounds also show mod-
erate to excellent inhibitory activity against tumor-related hCAs IX (Ki
1.6 to 465.7 nM) and CA XII (Ki 0.36 to 90.5 nM). Among all compounds,
SH-20, SH-26 and SH-28 compounds exhibited significant inhibitory
activity and selectivity profile against the tumor-related hCA XII. How-
ever, SH-20 was 16-fold, SH-26 was 37-fold and SH-28 was 44-fold
more selective against hCA XII than hCA IX. In addition, SH-23 dis-
played excellent inhibitory activity and selectivity profile against both
tumor-related hCAs IX (Ki 2.9 nM) as well as XII (Ki 0.82 nM) over hCA I
and hCA II. Molecular docking study of SH-20, SH-23, SH-26 and SH-28
with hCA XII and SH-23 with hCA IX were performed by AutoDock Vina,
and their binding modes signifies their significant interaction with
active residues of protein. The docking study implies that benzene-
sulfonamide moiety accommodates exactly into the active catalytic
pocket towards zinc ion of CA. These results revealed that these triazole-
sulfonamide based pyrimidine compounds displayed higher binding
affinity towards hCA IX and hCA XII. Therefore, these derivatives SH-20,
SH-23, SH-26 and SH-28 could be promising leads for the development
of selective and potential inhibitors as anticancer agents.
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inhibitors. Biphenylsulfonamides with inhibitory action towards the
transmembrane, tumor-associated isozymes IX possess cytotoxic activity against
human colon, lung and breast cancer cell lines. J Enzyme Inhib Med Chem. 2009;24
(2):499–505. https://doi.org/10.1080/14756360802218441.
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for Biophysical and Physical-Organic Studies of Proteins and Proteinꢀ Ligand
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[16] McKenna, R. Carbonic Anhydrase: Its Inhibitors and Activators. CRC Enzyme
Inhibitors Series, Volume 1 Edited by Claudiu T. Supuran, Andrea Scozzafava
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Declaration of Competing Interest
[19] De Simone G, Monti SM, Alterio V, et al. Crystal structure of the most catalytically
effective carbonic anhydrase enzyme known, SazCA from the thermophilic
bacterium Sulfurihydrogenibium azorense. Bioorg Med Chem Lett. 2015;25(9):
2002–2006. https://doi.org/10.1016/j.bmcl.2015.02.068.
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.
[20] Fisher SZ, Govindasamy L, Boyle N, et al. X-ray crystallographic studies reveal that
the incorporation of spacer groups in carbonic anhydrase inhibitors causes
alternate binding modes. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2006;62
(7):618–622. https://doi.org/10.1107/S1744309106020446.
Acknowledgments
[21] Di Fiore A, Capasso C, De Luca V, et al. X-ray structure of the first ‘extremo-
α
-carbonic anhydrase’, a dimeric enzyme from the thermophilic bacterium
Sulfurihydrogenibium yellowstonense YO3AOP1. Acta Crystallogr D Biol Crystallogr.
2013;69(6):1150–1159. https://doi.org/10.1107/S0907444913007208.
[22] Mocharla VP, Colasson B, Lee LV, et al. In Situ Click Chemistry: Enzyme-Generated
Inhibitors of Carbonic Anhydrase II. Angew Chemie Int Ed. 2005;44:116–120.
https://doi.org/10.1002/anie.200461580.
Shoaib Manzoor (S. M.) is extremely thankful to University Grants
Commission, Government of India for financial assistance through
Central University Ph.D. Students Fellowship.
[23] Morris JC, Chiche J, Grellier C, et al. Targeting Hypoxic Tumor Cell Viability with
Carbohydrate-Based Carbonic Anhydrase IX and XII Inhibitors. J Med Chem. 2011;
54(19):6905–6918. https://doi.org/10.1021/jm200892s.
Appendix A. Supplementary data
[24] Wilkinson BL, Bornaghi LF, Houston TA, et al. Carbonic Anhydrase Inhibitors:
Inhibition of Isozymes I, II, and IX with Triazole-Linked O -Glycosides of Benzene
Sulfonamides. J Med Chem. 2007;50:1651–1657. https://doi.org/10.1021/
jm061320h.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmcl.2021.128249.
[25] Wilkinson BL, Innocenti A, Vullo D, Supuran CT, Poulsen S-A. Inhibition of
Carbonic Anhydrases with Glycosyltriazole Benzene Sulfonamides. J Med Chem.
2008;51(6):1945–1953. https://doi.org/10.1021/jm701426t.
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