Tail approach synthesis of novel benzenesulfonamides incorporating 1,3,4-oxadiazole hybrids as potent inhibitor of carbonic anhydrase I, II, IX, and XII isoenzymes

Article abstract of DOI:10.1016/j.ejmech.2020.112219

Two new series of 1,3,4-oxadiazole benzenesulfonamide hybrids 3 and 4, having twenty novel compounds, have been designed and synthesized in order to assess their inhibition potential as CAIs against hCA I, II, IX, and XII. ‘Tail approach’ strategy has been used to design the aromatic sulfonamide scaffolds with carbonyl and amide linker. Excellent inhibitory activity against hCA I has been exhibited by compounds 3g and 4j, 3.5 magnitude of order better than reference drug AAZ (K_I = 250 nM). Moreover, compound 4j (K_I = 7.9 nM) effectively inhibited glaucoma-associated hCA II isoform as well as tumor-associated hCA IX isoform with K_I = 16.3 nM. Further hCA XII was weakly inhibited by all the compounds with K_I values ranging from 0.23 μM to 3.62 μM. Interestingly structure-activity relationship (SAR) study indicates that N-(3-nitrophenyl)-2-((5-(4-sulfamoylphenyl)-1,3,4-oxadiazol-2-yl)thio)acetamide (4j) is a potent compound to be investigated further for antiglaucoma and antitumor activity. The chemistry of the nature of different substitutions on the 1,3,4-oxadiazole bearing benzenesulfonamide substituted aromatic ring for potency and selectivity over one hCA isoform versus others is deliberated in the present study. In this context, the 1,3,4-oxadiazole motif can be a valuable tool worth developing for the procurement of novel and potent selective CAIs potentially useful for the management of a variety of diseases as chemotherapeutic agents.

Full text of DOI:10.1016/j.ejmech.2020.112219

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Tail approach synthesis of novel benzenesulfonamides incorporating 1,3,4-oxadiazole
hybrids as potent inhibitor of carbonic anhydrase I, II, IX, and XII isoenzymes
Vikas Sharma, Rajiv Kumar, Andrea Angeli, Claudiu T. Supuran, Pawan K. Sharma
PII:
S0223-5234(20)30186-0
DOI:
https://doi.org/10.1016/j.ejmech.2020.112219
EJMECH 112219
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 February 2020
Revised Date: 8 March 2020
Accepted Date: 8 March 2020
Please cite this article as: V. Sharma, R. Kumar, A. Angeli, C.T. Supuran, P.K. Sharma, Tail approach
synthesis of novel benzenesulfonamides incorporating 1,3,4-oxadiazole hybrids as potent inhibitor of
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Tail Approach Synthesis of Novel Benzenesulfonamides Incorporating 1,3,4-Oxadiazole
Hybrids as Potent Inhibitor of Carbonic Anhydrase I, II, IX, and XII
Vikas Sharma, Rajiv Kumar, Andrea Angeli, Claudiu T. Supuran, Pawan K. Sharma
Graphical Abstract

Tail Approach Synthesis of Novel Benzenesulfonamides Incorporating 1,3,4-Oxadiazole
Hybrids as Potent Inhibitor of Carbonic Anhydrase I, II, IX, and XII Isoenzymes
Vikas Sharmaa,b, Rajiv Kumara,c, Andrea Angelid, Claudiu T. Supurand,*, Pawan K. Sharmaa,*
aDepartment of Chemistry, Kurukshetra University, Kurukshetra-136119, India.
bPt. Chiranji Lal Sharma Government College, Karnal, Haryana, 132001, India.
cCh. Mani Ram Godara Government College for Women, Bhodia Khera, Fatehabad, Haryana,
125050, India.
dUniversità degli Studi di Firenze, Laboratorio di Chimica Bioinorganica, Rm 188, and
Neurofarba Department, Sezione di Scienze Farmaceutiche, Via U. Schiff 6, I-50019 Sesto
Fiorentino (Firenze), Italy.
*Corresponding authors: Tel.: +91 9416457355; Fax: +91 1744 238277; e-mail:
pksharma@kuk.ac.in (PKS); Tel/Fax: +39-055-4573005, e-mail: claudiu.supuran@unifi.it (CTS)
Abstract
Two new series of 1,3,4-oxadiazole benzenesulfonamide hybrids 3 and 4, having twenty novel
compounds, have been designed and synthesized in order to assess their inhibition potential as
CAIs against hCA I, II, IX, and XII. ‘Tail approach’ strategy has been used to design the
aromatic sulfonamide scaffolds with carbonyl and amide linker. Excellent inhibitory activity
against hCA I has been exhibited by compounds 3g and 4j, 3.5 magnitude of order better than
reference drug AAZ (KI = 250 nM). Moreover, compound 4j (KI = 7.9 nM) effectively inhibited
glaucoma-associated hCA II isoform as well as tumor-associated hCA IX isoform with KI = 16.3
nM. Further hCA XII was weakly inhibited by all the compounds with KI values ranging from
0.23 µM–3.62 µM. Interestingly structure-activity relationship (SAR) study indicates that N-(3-
nitrophenyl)-2-((5-(4-sulfamoylphenyl)-1,3,4-oxadiazol-2-yl)thio)acetamide (4j) is a potent
compound to be investigated further for antiglaucoma and antitumor activity. The chemistry of
the nature of different substitutions on the 1,3,4-oxadiazole bearing benzenesulfonamide
substituted aromatic ring for potency and selectivity over one hCA isoform versus others is
deliberated in the present study. In this context, the 1,3,4-oxadiazole motif can be a valuable tool
worth developing for the procurement of novel and potent selective CAIs potentially useful for
the management of a variety of diseases as chemotherapeutic agents.
Keywords: 1,3,4-Oxadiazole, Benzenesulfonamide, Carbonic anhydrase, CA isoforms.
1

Abbreviations: CA: Carbonic anhydrase; hCA: Human carbonic anhydrase; CAIs: Carbonic
anhydrase inhibitors; AAZ: Acetazolamide; KI: Inhibition constant; nM: Nanomolar; µM:
Micromolar.
1. Introduction
World Health Organization cited cancer as a major public health problem worldwide with one in
six deaths globally attributed to cancer [1]. Cancer is a generic term for an enormous cluster of
diseases that matures by the genetic and epigenetic mutations transforming normal healthy cells
into malignant phenotypes and the process is referred as metastasizing [1]. Undoubtedly,
investigations in the arena of pioneering anti-cancer drug discovery focused on cancer treatment
with more effective and less toxic agents are highly desired.
The carbonic anhydrases (CAs, EC 4.2.1.1) are metalloenzymes having the core of Zn2+ ion in
the active center present all over the phyla of the animal kingdom [2-6]. The CAs catalyze the
–
reversible and fundamental biochemical reaction, hydration of CO2 into HCO3 and H+ ions as
well as other hydrolytic reactions by the metal hydroxide nucleophilic mechanism [7, 8]. This
simple reaction is crucial for many physiological mechanisms including electrolyte secretion,
respiration, acid-base tuning, bone resorption, tumorigenesis, calcification and biosynthesis of
–
important molecules such as urea, glucose, and lipids, which require HCO3 as a substrate [9-11].
CAs have been developed as eight genetically different enzyme families ɑ-, β-, γ-, δ-, ζ-, η-, θ-,
and ι-CAs, [12, 13]. Further α-CA isoforms existing in human have been divided into sixteen
sub-isoforms that differ by molecular features, oligomeric arrangement, cellular localization,
distribution in organs and tissues, expression levels, kinetic properties and response to different
classes of inhibitors [14-18]. Various dysfunctions, and/or over-expression of hCAs in different
human physiological as well as pathological processes are responsible for many ailments in body
such as mental disorders (hCA II, VII, VIII, XIV), obesity (hCA VA, VB), edema (hCA I, II)
and glaucoma (hCA II, IV, XII) [19-21]. The eminent transmembrane isoforms hCA IX and XII
are overexpressed in hypoxic tumors, with limited expression in most normal cells [14, 19, 22,
23]. These tumor-associated proteins help in pH regulation in tumors, proliferation,
angiogenesis, and metastasis of variety of cancer cells, their selective inhibition can lead to the
development of new generation anticancer agents [24, 25]. It is noteworthy that ubiquitous hCA I
and II are the main off-target isoforms because these are involved in many physiological
processes [2].
2

Traditional primary carbonic anhydrase sulfonamide inhibitors have been used over the last few
decades in clinics for the treatment of glaucoma, epilepsy, obesity, and as diuretics [26].
Acetazolamide (AAZ), Methazolamide (MZA) and Ethoxzolamide (EZA) (Fig. 1) are the
prototypical first and second-generation drugs [19, 27]. The sulfonamide based hCA IX and XII
inhibitor SLC-0111 is under clinical investigations [28]. Since most of the CA epitopes are
nonspecific, a major challenge in therapeutic antitumor applications of hCA IX and XII
sulfonamide-based inhibitors is to the risk of a plethora of undesired side effects [29].
Fig. 1. Some sulfonamide-based drugs in clinical use or in clinical trials as potent CAIs.
The first choice in the field of specific CAI chemotypes is the zinc-binding group (ZBG) primary
sulfonamide [23]. The ring and tail approach strategies have been used for the design of isoform-
selective sulfonamide based CAIs [23]. The CAIs consisting of modulating moieties with various
steric demands were directly attached to the sulfonamide group and appended with different tails
to the aromatic/heterocyclic ring in the scaffold of the ZBG in order to target selectively the rim
of the active site of CA, a region with significant amino acid difference between isoenzymes
[30]. The sulphonamide moiety and the aromatic ring are attached through carbonyl/amide linker
(Fig. 2.).
Fig. 2. Tail approach as carbonyl/amide linker bearing benzenesulfonamide.
Several studies have been extensively performed on 1,3,4-oxadiazole moiety, which shows
diverse biological activities including antiviral [31], analgesic [32], antitumor [33], and anti-
inflammatory [34] activity. The biological potential of these heterocycles against cancer cells has
3

been reported with different mechanisms of action, such as inhibition of tubulin, mitogenesis,
angiogenesis, metastasis in tumors, focal adhesion kinase inhibition, telomerase inhibition,
interacting with several receptors involved in proliferation, cell growth, and DNA biosynthesis
[32, 35-39]. Azole group present in the 1,3,4-oxadiazole make it more lipophilic and, therefore,
more liable to pass through the cell membrane [40].
In previous years, our research group has explored N containing heterocyclic compounds such as
1,2,3-triazoles, 1,2,4 triazoles, pyrazoles and pyrazolines 1 containing benzenesulfonamides as
CAIs (Fig. 3) that show moderate to excellent inhibition potential against hCA IX and XII [41-
44]. Recently, we reported the synthesis of benzenesulfonamides containing triazole moietiy 2
that showed excellent inhibition against hCA I, II, IV, and IX [45]. In order to explore the
heterocyclic scaffolds as CAIs, we report herein the design and synthesis of new sets of twenty
novel 1,3,4-oxadiazole containing benzenesulfonamides 3a–j and 4a–j to study the effect of the
incorporation of amide and carbonyl linker between the aromatic ring and main 1,3,4-oxadiazole
benzenesulfonamide moiety on their inhibition potential against physiologically relevant
isoforms hCA I and II as well as tumor-associated isoforms hCA IX and XII (Fig. 3).
Fig. 3. Chemical structure of the sulfonamide CA inhibitors and derivatives incorporating the
pyrazolines and 1,2,3-triazole ring 1-2, together with the newly designed sulfonamides 3a–j and
4a–j.
2. Results and discussion
2.1. Chemistry
4

The synthesis of the novel derivatives of 1,3,4-oxadiazole bearing benzenesulfonamide 3a–j and
4a–j is outlined in Scheme 1. Initially, the 4-sulfamoylbenzoic acid (6) was prepared by the
oxidation of 4-methylbenzenesulfonamide (5) in the presence of KMnO4 that was further
converted into methyl 4-sulfamoylbenzoate (7) using usual Fischer esterification reaction [46].
Refluxing methyl ester derivatives with hydrazine monohydrate in ethanol afforded
corresponding hydrazide 8 [46]. The acid hydrazide 8 was cyclized using carbon disulfide and
potassium hydroxide to yield key intermediate 1,3,4-oxadiazole 9. Which was stirred with
differently substituted α-bomoacetophenones 10a–j in acetonitrile solvent in presence of
triethylamine resulting in formation of targeted compounds 3a–j while refluxing 9 with
differently substituted derivatives of α-chloro-N-phenylacetamide 11a–j in acetone using K2CO3
base yielded 4a–j. Differently substituted α-chloro-N-phenylacetamide 11a–j in turn were
prepared by the reaction of corresponding anilines with chloroacetyl chloride in DMF by stirring
for 8 hrs at room temperature [47].
13
1H NMR, C NMR, IR, and HRMS techniques were used for the characterization of the newly
synthesized compounds. According to the IR spectra of the synthesized 3a–j and 4a–j
derivatives, the presence of SO2NH2 group was confirmed by the absorption at about 3300 cm-1
appearing due to NH2 stretching as well as appearance of characteristic sharp IR absorption
bands at ~1333 cm-1 and ~1155 cm-1 for SO2 stretching. The presence of SO2NH2 group was
further confirmed by the appearance of a characteristic singlet of two protons at ~7.58 ppm in 1H
NMR spectra. Peaks at ~190 ppm in 3a–j and at ~164 ppm in 4a–j in their 13C NMR were
ascribed to the presence of C=O groups. Further, the IR absorption band from 1643 cm-1–1690
cm-1 in 3 and 4 confirms the presence of the C=O group in carbonyl and amide functional group.
1
From the H NMR spectra of 4a–j the signal for amide N–H protons was identified as singlet
which appeared downfield to aromatic protons in the range of 9.74 ppm–11.05 ppm. In addition,
the signal for aliphatic protons of methylene was observed as a singlet appearing in the range
1
4.37 ppm to 5.29 ppm integrating for two protons in H NMR spectra of compounds 3a–j and
4a–j. It was speculated from 1H NMR data that compounds 4a–j show some minor peaks which
are due to the dynamic equilibrium between imine-enol and keto-amine form [48]. The
duplication of each and every proton in NMR revealed the presence of two isomers in the
variable ratio in all the compounds but in the compound 4c the isomer presence was observed in
1
approximately equal amount (Fig. 4, supplementary file)[48]. For supporting the fact, H NMR
5

spectrum of representative compound 4c was taken in DMSO-d6 in the presence of TFA where
only one isomer was observed as duplication of peaks disappeared completely as the compound
was supposed to be entirely predisposed in one isomeric form due to restricted rotation about
(C=N) linkage (Fig. 4, supplementary file). From the data available in TFA it was predicted that
the compound 4c mainly exists in imine-enol from as the methylenec protons in the keto form
were supposed to resonate upfield (δH = 4.185 ppm) whereas in imine-enol form the same were
found to resonate downfield (δH = 4.345 ppm). In the 1H NMR spectra in TFA, the upfield (δH =
4.185 ppm) value for methylenec protons disappeared whereas the peak at δH = 4.3 ppm remains
as such confirming the presence of only one isomer in an acidic medium that is in imine-enol
form (Fig. 4, supplementary file).
6

Scheme 1. Synthesis of target compounds. Reagents and conditions: (i) KMnO4, H2O, reflux; (ii)
CH3OH, H2SO4, reflux; (iii) NH2NH2.H2O, EtOH, reflux; (iv) CS2, KOH, EtOH/THF, reflux; (v)
CH3CN, TEA, r.t.; (vi) Acetone, K2CO3, reflux.
2.2. CA inhibition studies
7

All the newly prepared sulfonamides 3a–j and 4a–j were screened for their inhibition
efficacy as CAIs against four physiologically relevant isoforms i.e. hCA I, II (cytosolic
enzymes), IX, and XII (transmembrane, tumor-associated isoforms) by stopped-flow
CO2 hydrase assay, using standard sulfonamide inhibitor acetazolamide (AAZ) as a
reference drug. The following structure-activity relationship (SAR) can be revealed from
the data shown in Table 1:
i.
The ubiquitous isoform hCA I was weakly inhibited by all the newly synthesized
sulfonamides with inhibition constants (KI) specifically spanning between 70.7 nM–
3.896 µM. The compounds 3a–f, 3h–j, and 4a–i showed low inhibitory activity while
compounds 3g (KI = 70.7 nM) and 4j (KI = 73.2 nM) displayed inhibitory values
better than the reference drug AAZ (KI = 250 nM). The structure-activity relationship
of hCA I indicated that introduction of electron-withdrawing groups, such as fluoro
and nitro group, at the para/meta-position enhanced the inhibition potential as
compared to electron releasing groups, such as methyl and methoxy group at para
position that possess a remarkably diminished inhibitory efficacy towards the hCA I.
Table 1
Inhibition data of human CA isoforms hCA I, II, IX, and XII with compounds reported here and
the standard sulfonamide inhibitor acetazolamide (AAZ) by a stopped-flow CO2 hydrase assay
[49].
KI (nM)a
Compounds
R
hCA I
1359
3324
3815
744.1
594.6
616.9
hCA II
346.8
147.8
157.8
69.4
hCA IX
756.5
201.1
448.6
1977
hCA XII
265.0
230.6
3618
H
4-CH3
4-OCH3
4-F
3a
3b
3c
3d
3e
3f
2239
4-Cl
67.3
1318
839.9
635.7
4-Br
67.0
1745
8

4-NO2
3-OCH3
3-Cl
70.7
469.6
543.5
669.4
542.5
739.0
2924
710.7
2080
3896
761.9
673.1
500.9
73.2
29.5
79.6
59.2
58.5
61.1
78.7
53.6
85.7
96.0
847.7
73.1
69.8
81.9
7.9
633.3
1328
1249
566.2
111.2
74.1
294.3
491.5
382.7
576.7
366.0
263.0
266.3
587.9
837.6
751.1
737.1
689.9
400.5
256.7
5.7
3g
3h
3i
3-NO2
H
3j
4a
4b
4c
4-CH3
4-OCH3
4-F
29.0
163.2
757.0
671.2
256.4
53.1
4d
4e
4-Cl
4-Br
4f
4-NO2
3-OCH3
3-Cl
4g
4h
4i
50.8
3-NO2
-
16.3
4j
250
12.1
25.8
AAZ
AAZ = acetazolamide, reference compound, a standard sulfonamide CAI, is also provided for
comparison.
aMean from 3 different assays, by a stopped-flow technique (errors were in the range of ±5-10% of the
reported values).
ii.
The physiologically dominant cytosolic isoform hCA II (associated with glaucoma)
was moderately inhibited by all the newly synthesized sulfonamides 3a–j and 4a–j
with inhibition constant ranging in between 7.9 nM–0.85 µM. Except for one
compound 4j (KI = 7.9 nM), which showed better inhibition potency than AAZ, all
others were found to be less potent inhibitors as compared to standard drug AAZ (KI
9

= 12.5 nM). The introduction of electron-withdrawing groups in phenyl ring in both
the series 3a–j and 4a–j in general increases the potency of inhibition against hCA II
as compared to electron-donating groups. However, sixteen compounds out of total of
twenty compounds were found to inhibit hCA II isoform with KI < 100 nM. Both the
series 3a–j and 4a–j, showed almost similar inhibition potential against hCA II
isoform whereas compound 4f (KI = 847.7 nM) was found to be exceptionally weak
inhibitor.
iii.
iv.
v.
The transmembrane tumor-associated isoform hCA IX was moderately inhibited by
most of the novel compounds herein reported. Only 3-nitro derivative 4j (KI = 16.3
nM) showed effective inhibitory efficiency better than the reference drug AAZ,
whereas compound 4c shows inhibition (KI = 29.0 nM) comparable to the reference
drug AAZ. The displacement of the nitro group from meta to para-position in 4g led
to a 16-fold reduction in inhibition activity. The replacement of nitro group in 4-
position with electron-donating groups, such as methyl and methoxy in 4a–j
compounds leads to an increase in inhibition potential against hCA IX. The novel
compounds 3a–j with carbonyl linker showed weak inhibition efficacy against hCA
IX.
The compounds 3a–j and 4a–j have shown variable and diverse inhibition against the
transmembrane tumor-associated isoform hCA XII, with KI spanning between 0.23
µM–3.62 µM. Compound 3b (KI = 0.23 µM) with a methyl group was found to be an
efficient inhibitor and the 4-methoxy substituted compound 3c (KI = 3.62 µM) was
the weakest inhibitor against hCA XII. On the other hand, in series 4a–j the 4-
methoxy substituted compound 4c was 13-fold efficient hCA XII inhibitor which
shows that 1,3,4-oxadiazole with amide tail/linker having low nanomolar affinity
against tumor-associated isoform hCA XII is better than the 1,3,4-oxadiazole with
carbonyl tail/linker compounds. The remaining derivatives inhibit hCA XII in a rather
narrow range that does not allow to compile further SAR.
In terms of structure-activity relationship, the compound 4j with amide linkage was
found to be a better inhibitor against the screened isoforms hCA I, II, and IX as
compared to compound 3j having carbonyl linkage. In 4a–j compounds there is a
significant increase in inhibition that may be due to the NH group that can lead to an
10

increase in polarity and extent of hydrogen bonding. It may indicate that the
inhibition potential depends upon the length of the tail and positioning of the linker
connecting the two pharmacophores.
Table 2
Selectivity ratios for inhibiting the tumor-associated isoforms hCA IX and XII over cytosolic
isoforms hCA I and II, with AAZ and compounds 3a–j and 4a–j.
Selectivity ratio*
Compounds
R
I/IX
3.812
16.529
8.504
0.376
0.4511
0.353
0.111
0.353
0.435
1.182
4.878
9.973
100.82
4.354
II/IX
0.458
0.734
0.351
0.035
0.051
0.038
0.046
0.059
0.047
0.103
0.549
1.062
1.848
0.525
I/XII
5.128
14.41
1.054
0.332
0.707
0.970
0.240
0.955
1.420
1.160
1.482
2.809
10.981
1.208
II/XII
1.354
0.640
0.043
0.030
0.080
0.105
0.100
0.161
0.154
0.101
0.166
0.299
0.201
0.145
H
3a
3b
3c
3d
3e
3f
4-CH3
4-OCH3
4-F
4-Cl
4-Br
4-NO2
3-OCH3
3-Cl
3g
3h
3i
3-NO2
H
3j
4a
4b
4c
4d
4-CH3
4-OCH3
4-F
11

4-Cl
4-Br
2.747
5.804
2.971
0.126
9.860
4.490
9.689
0.126
1.262
0.285
1.314
1.612
0.484
0.468
2.483
5.187
1.033
0.975
1.250
0.285
43.8
0.114
1.128
0.099
0.101
0.204
0.030
2.122
4e
4f
4-NO2
3-OCH3
3-Cl
4g
4h
4i
3-NO2
-
4j
AAZ
AAZ = acetazolamide, reference compound, a standard sulfonamide CAI, is also provided for
comparison.
*The KI ratios are indicative of isozyme selectivity: a weak selective inhibitor is characterized by a low-
value ratio.
vi.
The selectivity index (SI) for inhibiting the tumor-associated isoforms hCA IX and
XII over the off-targeted cytosolic isoforms hCA I and II have been presented in
Table 2. It is evident that the compounds didn’t show consistent behavior in their
potential against all the four tested isoforms (hCA I, II, IX, and XII). It can be
observed that some of the investigated derivatives showed promising levels of
selective inhibition of the transmembrane associated isoforms over the cytosolic
isoforms. It was also observed that compound 4c and 3b were exhibiting the best
selectivity for hCA IX and XII over hCA I. Indicating that the electron-donating
group at para-position having amide tail/linker in 1,3,4-oxadiazole
benzenesulfonamide leads to a potent precursor for designing the tumor-selective
hCA XI CAIs.
3. Conclusions
As a part of our research aiming to design selective novel carbonic anhydrase inhibitors, we have
synthesized two series of twenty compounds 3a–j and 4a–j containing 1,3,4-oxadiazole bearing
benzenesulfonamide. These compounds were examined for their inhibition potential against the
two dominant cytosolic isoforms hCA I/II and the tumor-associated isoforms hCA IX/XII and
12

compared with that of reference drug acetazolamide AAZ, a standard inhibitor. The hCA I was
relatively weakly inhibited by compounds 3a–f, 3h–j, and 4a–i with KI ranging from 469.6 nM–
3.89 µM except 3g (KI = 70.7 nM) and 4j (KI = 73.2 nM) which were potent inhibitors of hCA I
as compared to that of AAZ (KI = 250 nM). The compound N-(3-nitrophenyl)-2-((5-(4-
sulfamoylphenyl)-1,3,4-oxadiazol-2-yl)thio) (4j) exercised strong inhibition potential against
hCA II and tumor-associated isoform hCA IX, whereas compounds 3a–j and 4a–j were
moderately effective inhibitors of hCA XII. CA inhibition data demonstrated that 1,3,4-
oxadiazole bearing benzenesulfonamide with amide tail/linker was an effective inhibitor of hCA
IX as per SAR. Compound 4c was selective inhibitor of tumor-associated isoform hCA IX over
off target hCA I (Table 2). These results speculated that these molecules can be the choice of
future drug candidates targeting hypoxic tumors and can lead to design and optimization of
selective hCA inhibitors.
4. Experimental protocols
4.1. Chemistry
4.1.1. General
All the reagents and solvents were purchased from commercial suppliers and were used as
received unless otherwise indicated. All the solvents were dried and/or purified according to
standard procedures prior to use. All the air or moisture-sensitive reactions were performed
under a nitrogen atmosphere using dried glassware and syringes techniques to transfer solutions.
Analytical thin-layer chromatography (TLC) was performed on MERCK precoated silica gel on
F254 aluminium plates using a mixture of chloroform and methanol as eluent while UV lamp was
used to visualize the spots. Melting points were determined in open glass capillary tubes by
Precision Digital Melting Point apparatus (Popular India) and may be uncorrected values.
Infrared (IR) were recorded as KBr disks using an ABB MB 3000 DTGS infrared
1
13
spectrophotometer. Nuclear magnetic resonance H NMR and C NMR spectra were recorded
using Bruker Avance III 400 MHz and 100 MHZ respectively, using deuterated dimethyl
sulfoxide (DMSO-d6) and Me4Si (TMS) as internal standard at room temperature. Chemical
shifts are reported as δ values in parts per million (ppm) and the coupling constants (J) are
expressed in hertz (Hz). High-resolution mass spectra were obtained from a XEVO G2-S QTof
UPLC/MS spectrometer using acetonitrile as solvent. Splitting pattern are designated as follows:
singlet (s), doublet (d), doublet of doublet (dd), doublet of triplet (dt), triplet (t), quartet (q),
13

multiplet (m), exchangeable proton (ex) for NMR assignments and strong (s), medium (m),
broad (br) for IR assignments.
4.1.2. Synthesis of 4-(5-mercapto-1,3,4-oxadiazol-2-yl)benzenesulfonamide (9)
General procedure: To a mixture of 4-(hydrazinecarbonyl)benzenesulfonamide (8) [46] (4.65
mmol) in ethanol/THF (1:1) at room temp. was added a solution of KOH (4.65 mmol) in ethanol
(20 ml) followed by the addition of CS2 (23.25 mmol). Then the reaction mix. was refluxed for 8
hrs. The solvent was evaporated under vacuum and the residue was acidified with dil. HCl and
resulting white solid was collected, washed with water, and recrystallized with ethanol to give
final product 4-(5-mercapto-1,3,4-oxadiazol-2-yl)benzenesulfonamide.
4.1.2.1.
4-(5-mercapto-1,3,4-oxadiazol-2-yl)benzenesulfonamide (9).
o
Yield 86%; Colour: White; mp: 180–182 C; IR (KBr) (ν, cm-1): 3335, 3240 (m, N-H stretch),
1
2608 (s, S-H stretch), 1342, 1165 (s, SO2 stretch); H NMR (400 MHz, DMSO-d6) δH (ppm):
8.08 (d, J = 8.4 Hz, 2H, Ar), 8.00 (d, J = 7.2 Hz, 2H, Ar), 7.59 (s, 2H, SO2NH2); 13C NMR (100
MHz, DMSO-d6) δC (ppm): 178.11, 159.98, 147.26, 127.26, 127.14, 125.79; HRMS (ESI-MS)
m/z 257.9999 (M+H)+, C8H7N3O3S2H+, calcd. 257.9928.
4.1.3. Synthesis of 4-(5-((2-aryl)-2-oxoethyl)thio)-1,3,4-oxadiazol-2-yl)benzenesulfonamide
(3a–j)
General procedure: To
a
stirred mixture of 4-(5-mercapto-1,3,4-oxadiazol-2-
yl)benzenesulfonamide (9) (1.0 mmol) and differently substituted phenacyl bromide 10a–j (1.0
mmol) in acetonitrile, triethylamine (1.5 mmol) was added at room temp. and the immediately
formed precipitates were filtered off, washed with water and recrystallized with ethanol to give
desired product.
4.1.3.1.
4-(5-((2-oxo-2-phenylethyl)thio)-1,3,4-oxadiazol-2-yl)benzenesulfonamide (3a).
o
Yield 76%; Colour: White; mp: 247–249 C; IR (KBr) (ν, cm-1): 3348, 3248 (m, N-H stretch),
1
1651 (s, C=O stretch), 1335, 1165 (s, SO2 stretch); H NMR (400 MHz, DMSO-d6) δH (ppm):
8.15 (d, J = 8.4 Hz, 2H, Ar), 8.09 (d, J = 7.2 Hz, 2H, Ar), 8.00 (d, J = 8.4 Hz, 2H, Ar), 7.73 (t, J
= 7.2 Hz, 1H, Ar), 7.63–7.56 (m, 4H, Ar, SO2NH2), 5.22 (s, 2H, CH2); 13C NMR (100 MHz,
DMSO-d6) δC (ppm):192.56, 164.69, 147.22, 145.13, 133.02, 129.94, 129.10, 127.53, 127.16,
126.19, 41.05, 21.73; HRMS (ESI-MS) m/z 376.0345 (M+H)+, C16H13N3O4S2H+, calcd
376.3548.
14

4.1.3.2.
4-(5-((2-oxo-2-(p-tolyl)ethyl)thio)-1,3,4-oxadiazol-2-yl)benzenesulfonamide (3b).
o
Yield 68%; Colour: White; mp: 251–253 C; IR (KBr) (ν, cm-1): 3356, 3263 (m, N-H stretch),
1
1651 (s, C=O stretch), 1335, 1165 (s, SO2 stretch); H NMR (400 MHz, DMSO-d6) δH (ppm):
8.15 (d, J = 8.0 Hz, 2H, Ar), 8.00 (t, J = 8.0 Hz, 4H, Ar), 7.57 (s, 2H, SO2NH2), 7.40 (d, J = 8.0
13
Hz, 2H, Ar), 5.18 (s, 2H, CH2), 2.42 (s, 3H, CH3); C NMR (100 MHz, DMSO-d6) δC (ppm):
192.56, 164.69, 147.22, 145.12, 133.02, 129.94, 129.10, 127.53, 127.16, 126.19, 41.05, 21.72;
HRMS (ESI-MS) m/z 390.0514 (M+H)+, C17H15N3O4S2H+, calcd 390.0582.
4.1.3.3. 4-(5-((2-(4-methoxyphenyl)-2-oxoethyl)thio)-1,3,4-oxadiazol-2-yl)benzenesulfonamide
(3c).
o
Yield 73%; Colour: White; mp: 218–220 C; IR (KBr) (ν, cm-1): 3348, 3271 (m, N-H stretch),
1
1651 (s, C=O stretch), 1342, 1173 (s, SO2 stretch); H NMR (400 MHz, DMSO-d6) δH (ppm):
8.27 (d, J = 2.4 Hz, 1H, Ar), 8.17–8.10 (m, 4H, Ar), 8.01 (d, J = 8.4 Hz, 2H, Ar), 7.58 (s, 2H,
13
SO2NH2), 7.31 (d, J = 8.4 Hz, 1H, Ar), 5.17 (s, 2H, CH2), 3.99 (s, 3H, OCH3); C NMR (100
MHz, DMSO-d6) δC (ppm): 190.79, 164.75, 160.23, 147.23, 133.78. 130.84, 129.45, 127.55,
127.16, 126.18, 113.00, 111.50, 57.39; HRMS (ESI-MS) m/z 406.0471 (M+H)+,
C17H15N3O5S2H+, calcd 406.0531.
4.1.3.4.
4-(5-((2-(4-fluorophenyl)-2-oxoethyl)thio)-1,3,4-oxadiazol-2-
yl)benzenesulfonamide (3d).
o
Yield 82%; Colour: White; mp: 232–234 C; IR (KBr) (ν, cm-1): 3371, 3279 (m, N-H stretch),
1
1666 (s, C=O stretch), 1335, 1157 (s, SO2 stretch); H NMR (400 MHz, DMSO-d6) δH (ppm):
8.20–8.13 (m, 4H, Ar), 8.00 (d, J = 8.4 Hz, 2H, Ar), 7.57 (s, 2H, SO2NH2), 7.44 (t, J = 8.8, 2H,
Ar), 5.21 (s, 2H, CH2); 13C NMR (100 MHz, DMSO-d6) δC (ppm): 191.75, 165.96 (d,
4
3
1JCF = 251.5 Hz), 164.75, 164.59, 147.23, 132.30 (d, JCF = 2.7 Hz), 132.11 (d, JCF = 9.6 Hz),
2
127.54, 127.16, 126.18, 116.49 (d, JCF = 22 Hz ), 49.97; HRMS (ESI-MS) m/z 394.0257
(M+H)+, C16H12FN3O4S2H+, calcd 394.0331.
4.1.3.5.
4-(5-((2-(4-chlorophenyl)-2-oxoethyl)thio)-1,3,4-oxadiazol-2-
yl)benzenesulfonamide (3e).
o
Yield 72%; Colour: White; mp: 228–230 C; IR (KBr) (ν, cm-1): 3356, 3263 (m, N-H stretch),
1
1666 (s, C=O stretch), 1342, 1165 (s, SO2 stretch); H NMR (400 MHz, DMSO-d6) δH (ppm):
8.14 (d, J = 8.0 Hz, 2H, Ar), 8.10 (d, J = 8.4 Hz, 2H, Ar), 8.00 (d, J = 8.4 Hz, 2H, Ar), 7.68 (d, J
= 8.4 Hz, 2H, Ar), 7.57 (s, 2H, SO2NH2), 5.20 (s, 2H, CH2); 13C NMR (100 MHz, DMSO-d6) δC
15

(ppm): 192.23, 164.77, 164.53, 147.24, 139.45, 134.23, 130.91, 129.53, 127.54, 127.16, 126.17,
41.00; HRMS (ESI-MS) m/z 409.9961 (M+H)+, (M+H+2)+ 411.9934, C16H12ClN3O4S2H+, calcd
410.0036.
4.1.3.6.
4-(5-((2-(4-bromophenyl)-2-oxoethyl)thio)-1,3,4-oxadiazol-2-
yl)benzenesulfonamide (3f).
o
Yield 72%; Colour: Light yellow ; mp: 233–235 C; IR (KBr) (ν, cm-1): 3356, 3256 (m, N-H
stretch), 1659 (s, C=O stretch), 1350, 1165 (s, SO2 stretch) ; 1H NMR (400 MHz, DMSO-d6) δH
(ppm): 8.14 (d, J = 8.4 Hz, 2H, Ar), 8.02–8.00 (m, 4H, Ar), 7.82 (d, J = 8.4 Hz, 2H, Ar), 7.56 (s,
2H, SO2NH2), 5.19 (s, 2H, CH2); 13C NMR (100 MHz, DMSO-d6) δC (ppm): 192.44, 164.76,
164.50, 147.24, 134.55, 132.47, 130.94, 128.67, 127.52, 127.15, 126.17, 40.96; HRMS (ESI-
MS) m/z 453.9452 (M+H)+, (M+H+2)+ 455.9433, C16H12BrN3O4S2H+, calcd 453.9531.
4.1.3.7.
4-(5-((2-(4-nitrophenyl)-2-oxoethyl)thio)-1,3,4-oxadiazol-2-
yl)benzenesulfonamide (3g).
o
Yield 74%; Colour: White; mp: 232–234 C; IR (KBr) (ν, cm-1): 3340, 3271 (m, N-H stretch),
1
1690 (s, C=O stretch), 1319, 1157 (s, SO2 stretch); H NMR (400 MHz, DMSO-d6) δH (ppm):
8.41 (d, J = 8.4 Hz, 2H, Ar), 8.31 (d, J = 8.4 Hz, 2H, Ar), 8.15 (d, J = 8.4 Hz, 2H, Ar), 8.01 (d, J
= 8.4 Hz, 2H, Ar), 7.57 (s, 2H, SO2NH2), 5.28 (s, 2H, CH2); 13C NMR (100 MHz, DMSO-d6) δC
(ppm): 192.54, 164.84, 164.38, 150.82, 147.26, 140.18, 130.43, 127.56, 127.17, 126.16, 124.25,
41.29; HRMS (ESI-MS) m/z 421.0194 (M+H)+, C16H12N4O6S2H+, calcd 421.0276.
4.1.3.8.
4-(5-((2-(3-methoxyphenyl)-2-oxoethyl)thio)-1,3,4-oxadiazol-2-
yl)benzenesulfonamide (3h).
o
Yield 70%; Colour: Pale yellow; mp: 212–214 C; IR (KBr) (ν, cm-1): 3612 (br,O-H stretch),
1
3317, 3240 (m, N-H stretch), 1690 (s, C=O stretch), 1335, 1157 (s, SO2 stretch); H NMR (400
MHz, DMSO-d6) δH (ppm): 8.18–8.12 (m, 2H, Ar), 8.03–7.98 (m, 2H, Ar), 7.70–7.60 (m, 1H,
Ar), 7.60–7.50 (m, 4H, Ar, SO2NH2), 7.30 (dd, J = 0.8 Hz, J = 8.4 Hz, 1H, Ar), 5.20 (s, 2H,
CH2), 3.85 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6) δC (ppm): 192.92, 164.74, 164.61,
159.99, 147.23, 136.87, 130.61, 127.57, 127.53, 127.16, 121.48, 120.51, 113.52, 55.95, 41.09;
HRMS (ESI-MS) m/z 406.0452 (M+H)+, C17H15N3O5SH+, calcd 406.4608.
4.1.3.9.
4-(5-((2-(3-chlorophenyl)-2-oxoethyl)thio)-1,3,4-oxadiazol-2-
yl)benzenesulfonamide (3i).
16

o
Yield 69%; Colour: White; mp: 234–236 C; IR (KBr) (ν, cm-1): 3356, 3271 (m, N-H stretch),
1
1674 (s, C=O stretch), 1335, 1157 (s, SO2 stretch); H NMR (400 MHz, DMSO-d6) δH (ppm):
8.15 (d, J = 8.4 Hz, 2H, Ar), 8.11 (s, 1H, Ar), 8.05–7.99 (m, 3H, Ar), 7.80 (d, J = 8.0 Hz, 1H,
Ar), 7.64 (t, J = 8.04 Hz, 1H, Ar), 7.57 (s, 2H, SO2NH2), 5.21 (s, 2H, CH2); 13C NMR (100 MHz,
DMSO-d6) δC (ppm): 192.26, 164.80, 164.55, 147.25, 137.36, 134.31, 134.16, 131.41, 128.67,
127.62, 127.55, 127.16, 126.17, 40.95; HRMS (ESI-MS) m/z 409.9951 (M+H)+, (M+H+2)+
411.9924, C16H12ClN3O4S2H+, calcd 410.0036.
4.1.3.10.
4-(5-((2-(3-nitrophenyl)-2-oxoethyl)thio)-1,3,4-oxadiazol-2-
yl)benzenesulfonamide (3j).
o
Yield 76%; Colour: Off White; mp: 243–245 C; IR (KBr) (ν, cm-1): 3333, 3248 (m, N-H
1
stretch), 1682 (s, C=O stretch), 1335, 1157 (s, SO2 stretch); H NMR (400 MHz, DMSO-d6) δH
(ppm): 8.78 (t, J = 2.0 Hz, 1H, Ar), 8.57–8.50 (m, 2H, Ar), 8.16 (dd, J = 6.8, J = 1.6 Hz, 2H, Ar),
8.01 (dd, J = 6.8, J = 2.0 Hz, 2H, Ar), 7.91 (t, J = 8.0 Hz, 1H, Ar), 7.58 (s, 2H, SO2NH2), 5.31 (s,
13
2H, CH2); C NMR (100 MHz, DMSO-d6) δC (ppm): 191.97, 164.83, 164.36, 148.55, 147.22,
136.72, 135.14, 131.25, 128.59, 127.67, 127.15, 126.15, 123.34, 41.04; HRMS (ESI-MS) m/z
421.0194 (M+H)+, C16H12N4O6S2H+, calcd 421.0276.
4.1.4. Synthesis of N-aryl-2-((5-(4-sulfamoylphenyl)-1,3,4-oxadiazol-2-yl)thio)acetamide (4a–
j)
A mixture of 4-(5-mercapto-1,3,4-oxadiazol-2-yl)benzenesulfonamide (9) (1 mmol), various
substituted 2-chloro-N-phenylacetamide 11a–j (1 mmol) and K2CO3 (1.5 mmol) was refluxed in
acetone (15 ml) for 3–4 h. The reaction was monitored with TLC and after completion of the
reaction, excess of solvent was evaporated and diluted with water (about 100 ml). The
precipitated was separated out, filtered washed with water and recrystallized with an appropriate
solvent.
4.1.4.1.
N-phenyl-2-((5-(4-sulfamoylphenyl)-1,3,4-oxadiazol-2-yl)thio)acetamide (4a).
o
Yield 71%; Colour: Off White; mp: 243–245 C; IR (KBr) (ν, cm-1): 3348, 3248 (m, N-H
1
stretch), 1659 (s, C=O stretch), 1335, 1165 (s, SO2 stretch); H NMR (400 MHz, DMSO-d6) δH
(ppm): 10.46 (s, 1H, NH), 8.16 (d, J = 8.8 Hz, 2H, Ar), 7.99 (d, J = 8.8 Hz, 2H, Ar), 7.61–7.56
(m, 2H, Ar, 2H, SO2NH2), 7.33 (t, J = 8.0 Hz, 2H, Ar), 7.09 (t, J = 7.2 Hz, 1H, Ar), 4.38 (s, 2H,
CH2); 13C NMR (100 MHz, DMSO-d6) δC (ppm): 165.31, 164.75, 164.72, 147.19, 139.08,
17

129.36, 127.52, 127.16, 126.20, 124.22, 119.66, 37.32; HRMS (ESI-MS) m/z 391.0454 (M+H)+,
C16H14N4O4S2H+, calcd 391.0529.
4.1.4.2.
2-((5-(4-sulfamoylphenyl)-1,3,4-oxadiazol-2-yl)thio)-N-(p-tolyl)acetamide (4b).
o
Yield 74%; Colour: White; mp: 259–261 C; IR (KBr) (ν, cm-1): 3340, 3248 (m, N-H stretch),
1
1659 (s, C=O stretch), 1335, 1165 (s, SO2 stretch); H NMR (400 MHz, DMSO-d6) δH (ppm):
10.36 (s, 1H, NH), 8.15 (d, J = 8.4 Hz, 2H, Ar), 8.00 (d, J = 8.4 Hz, 2H, Ar), 7.57 (s, 2H,
SO2NH2), 7.47 (d, J = 8.0 Hz, 2H, Ar), 7.13 (d, J = 8.0 Hz, 2H, Ar), 4.36 (s, 2H, CH2), 2.26 (s,
13
3H, CH3); C NMR (100 MHz, DMSO-d6) δC (ppm): 165.01, 164.74, 147.22, 136.60, 133.18,
129.71, 128.37, 127.51, 127.16, 126.21, 119.67, 37.34, 20.92; HRMS (ESI-MS) m/z 405.0614
(M+H)+, C17H16N4O4S2H+, calcd 405.0686.
4.1.4.3.
N-(4-methoxyphenyl)-2-((5-(4-sulfamoylphenyl)-1,3,4-oxadiazol-2-
yl)thio)acetamide (4c).
o
Yield 68%; Colour: Grey; mp: 259–261 C; IR (KBr) (ν, cm-1): 3742 (br, O-H stretch), 3333,
1
3225 (m, N-H stretch), 1327, 1165 (s, SO2 stretch); H NMR (400 MHz, DMSO-d6) δH (ppm):
11.00 & 10.30 (s, 1H, NH/OH), 8.16 (d, J = 8.0 Hz, 1H, Ar), 8.00 (d, J = 8.4 Hz, 1H, Ar), 7.95
(d, J = 8.0 Hz, 1H, Ar), 7.90 (d, J = 8.4 Hz, 1H, Ar), 7.58 (s, 1H, Ar), 7.50 (d, J = 9.2 Hz, 2H,
SO2NH2), 7.28 (d, J = 8.8 Hz, 1H, Ar), 7.07 (d, J = 8.8 Hz, 1H, Ar), 6.90 (d, J = 8.8 Hz, 1H, Ar),
4.35 & 4.18 (s, 2H, CH2), 3.82 & 3.73 (s, 3H, OCH3); 13C NMR (100 MHz, DMSO-d6) δC
(ppm): 165.12, 160.40, 157.67, 146.38, 140.42, 134.02, 131.32, 127.21, 126.96, 126.66, 122.24,
117.11, 116.29, 37.29.; HRMS (ESI-MS) m/z 421.0561 (M+H)+, C17H16N4O5S2H+, calcd
421.0635.
4.1.4.4.
N-(4-fluorophenyl)-2-((5-(4-sulfamoylphenyl)-1,3,4-oxadiazol-2-
yl)thio)acetamide (4d).
o
Yield 68%; Colour: Off White; mp: 279–281 C; IR (KBr) (ν, cm-1): 3394, 3294 (m, N-H
1
stretch), 1636 (s, C=O stretch), 1342, 1165 (s, SO2 stretch); H NMR (400 MHz, DMSO-d6) δH
(ppm): 10.69 & 10.52 (s, 1H, NH/OH), 8.15 (d, J = 8.8 Hz, 2H, Ar), 8.00 (d, J = 8.8 Hz, J = 1.2
Hz, 2H, Ar), 7.95–7.89 (m, 1H, Ar), 7.63–7.57 (m, 3H, 2H, SO2NH2), 7.41 (t, J = 8.8 Hz, 1H,
Ar), 7.18 (t, J = 8.8 Hz, 1H, Ar), 4.37 (s, 2H, CH2); 13C NMR (100 MHz, DMSO-d6) δC (ppm):
3
165.68, 165.24, 164.79, 164.61, 147.20, 127.523, 127.164, 126.194, 121.47 (d, JCF = 7.8 Hz),
2
121.10, 120.08, 115.96 (d, JCF = 22.1 Hz ), 37.15; HRMS (ESI-MS) m/z 409.0361 (M+H)+,
C16H13FN4O4S2H+, calcd 409.0435.
18

4.1.4.5.
N-(4-chlorophenyl)-2-((5-(4-sulfamoylphenyl)-1,3,4-oxadiazol-2-
yl)thio)acetamide (4e).
o
Yield 70%; Colour: White ; mp: 257–259 C; IR (KBr) (ν, cm-1): 17, 3232 (m, N-H stretch),
1
1659 (s, C=O stretch), 1335, 1165 (s, SO2 stretch); H NMR (400 MHz, DMSO-d6) δH (ppm):
10.60 (s, 1H, NH), 8.15 (d, J = 8.4 Hz, 2H, Ar), 8.00 (d, J = 8.4 Hz, 2H, Ar), 7.62 (d, J = 8.8 Hz,
13
2H, Ar), 7.59 (s, 2H, SO2NH2), 7.39 (d, J = 8.8 Hz, 2H, Ar), 4.39 (s, 2H, CH2); C NMR (100
MHz, DMSO-d6) δC (ppm):165.49, 164.76, 164.67, 147.21, 138.04, 129.29, 127.77, 127.52,
127.16, 126.20, 121.21, 37.28; HRMS (ESI-MS) m/z 425.0065 (M+H)+, (M+H+2)+ 427.0036,
C16H13ClN4O4S2H+, calcd 425.0140.
4.1.4.6.
N-(4-bromophenyl)-2-((5-(4-sulfamoylphenyl)-1,3,4-oxadiazol-2-
yl)thio)acetamide (4f).
Yield 69%; Colour: Silver; mp: 279–281oC; IR (KBr) (ν, cm-1): 3340, 3263 (m, N-H stretch),
1
1659 (s, C=O stretch), 1327, 1165 (s, SO2 stretch); H NMR (400 MHz, DMSO-d6) δH (ppm):
10.59 (s, 1H, NH), 8.15 (d, J = 8.4 Hz, 2H, Ar), 8.00 (d, J = 8.4 Hz, 2H, Ar), 7.59–7.55 (m, 2H,
Ar, 2H, SO2NH2), 7.52 (d, J = 8.8 Hz, 2H, Ar), 4.38 (s, 2H, CH2); 13C NMR (100 MHz, DMSO-
d6) δC (ppm):165.50, 164.77, 164.66, 147.23, 138.46, 132.19, 127.52, 127.17, 126.20, 121.60,
115.82, 37.32; HRMS (ESI-MS) m/z 468.9561 (M+H)+, (M+H+2)+ 470.9542,
C16H13BrN4O4S2H+, calcd 468.9634.
4.1.4.7.
N-(4-nitrophenyl)-2-((5-(4-sulfamoylphenyl)-1,3,4-oxadiazol-2-yl)thio)acetamide
(4g).
o
Yield 73%; Colour: Yellow; mp: 234–236 C; IR (KBr) (ν, cm-1): 3302, 3254 (m, N-H stretch),
1
1690 (s, C=O stretch), 1319, 1157 (s, SO2 stretch); H NMR (400 MHz, DMSO-d6) δH (ppm):
11.05 (s, 1H, NH), 8.25 (d, J = 9.2 Hz, 2H, Ar), 8.15 (d, J = 8.4 Hz, 2H, Ar), 8.00 (d, J = 8.8 Hz,
13
2H, Ar), 7.85 (d, J = 9.2 Hz, 2H, Ar), 7.57 (s, 2H, SO2NH2), 4.45 (s, 2H, CH2); C NMR (100
MHz, DMSO-d6) δC (ppm): 166.42, 164.83, 164.57, 147.23, 145.14, 143.05, 127.53, 127.17,
126.18, 125.57, 119.47, 37.38; HRMS (ESI-MS) m/z 436.0310 (M+H)+, C16H13N5O6S2H+, calcd
436.0380.
4.1.4.8.
N-(3-methoxyphenyl)-2-((5-(4-sulfamoylphenyl)-1,3,4-oxadiazol-2-
yl)thio)acetamide (4h).
o
Yield 70%; Colour: White; mp: 228–230 C; IR (KBr) (ν, cm-1): 3325, 3232 (m, N-H stretch),
1
1643 (s, C=O stretch), 1327, 1157 (s, SO2 stretch); H NMR (400 MHz, DMSO-d6) δH (ppm):
10.44 (s, 1H, NH), 8.15 (d, J = 8.4 Hz, 2H, Ar), 8.00 (d, J = 8.4 Hz, 2H, Ar), 7.57 (s, 2H,
19

SO2NH2), 7.30–7.20 (m, 2H, Ar), 7.12 (d, J = 8.0 Hz, 1H, Ar), 6.68 (d, J = 8.0 Hz, 1H, Ar), 4.37
13
(s, 2H, CH2), 3.73 (s, 3H, CH3); C NMR (100 MHz, DMSO-d6) δC (ppm): δ 165.34, 164.70,
160.06, 147.23, 140.26, 130.18, 127.52, 127.16, 126.21, 111.92, 109.65, 105.48, 55.49, 37.40;
HRMS (ESI-MS) m/z 421.0560 (M+H)+, C17H16N4O5S2H+, calcd 421.0635.
4.1.4.9.
N-(3-chlorophenyl)-2-((5-(4-sulfamoylphenyl)-1,3,4-oxadiazol-2-
yl)thio)acetamide (4i).
o
Yield 70%; Colour: White; mp: 291–293 C; IR (KBr) (ν, cm-1): 3325, 3248 (m, N-H stretch),
1
1659 (s, C=O stretch), 1335, 1165 (s, SO2 stretch); H NMR (400 MHz, DMSO-d6) δH (ppm):
10.65 (s, 1H, NH), 8.15 (d, J = 8.4 Hz, 2H, Ar), 7.80 (t, J = 2.0 Hz, 1H, Ar), 7.58 (s, 2H,
SO2NH2), 7.47–7.43 (m, 1H, Ar), 7.38 (t, J = 8.0 Hz, 1H, Ar), 7.18–7.14 (m, 1H, Ar), 4.39 (s,
13
2H, CH2); C NMR (100 MHz, DMSO-d6) δC (ppm): 165.12, 160.40, 157.67, 146.38, 140.42,
134.02, 131.32, 127.21, 126.96, 126.66, 122.24, 117.11, 116.29, 37.29; HRMS (ESI-MS) m/z
425.0061 (M+H)+, (M+H+2)+ 427.0032, C16H13ClN4O4S2H+, calcd 425.0140.
4.1.4.10.
N-(3-nitrophenyl)-2-((5-(4-sulfamoylphenyl)-1,3,4-oxadiazol-2-yl)thio)acetamide
(4j).
o
Yield 72%; Colour: Pale Yellow; mp: 210–212 C; IR (KBr) (ν, cm-1): 3310, 3248 (m, N-H
1
stretch), 1666 (s, C=O stretch), 1335, 1165 (s, SO2 stretch); H NMR (400 MHz, DMSO-d6) δH
(ppm): 10.96 (s, 1H, NH), 8.63 (s, 1H, Ar), 8.16 (d, J = 8.4 Hz, 2H, Ar), 8.01 (d, J = 8.4 Hz, 2H,
Ar), 7.96 (dd, J = 8.4 Hz, J = 1.2 Hz, 1H, Ar), 7.92 (d, J = 7.6 Hz, 1H, Ar), 7.65 (t, J = 8.0 Hz,
13
1H, Ar), 7.57 (s, 2H, SO2NH2), 4.43 & 4.21 (s, 2H, CH2); C NMR (100 MHz, DMSO-d6) δC
(ppm):166.19, 164.82, 164.58, 148.50, 147.25, 140.18, 130.89, 127.53, 127.17, 126.19, 125.65,
118.76, 113.78, 37.24; HRMS (ESI-MS) m/z 436.0314 (M+H)+, C16H13N5O6S2H+, calcd
436.0380.
5. CA inhibition Assay
An SX.18MV-R Applied Photophysics (Oxford, UK) stopped-flow instrument has been used to
assay the inhibition of various CA isozymes [50]. Phenol Red (at a concentration of 0.2 mM) has
been used as an indicator, working at the absorbance maximum of 557 nm, with 10 mM Hepes
(pH 7.4) as a 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 the water at 25 °C were used as
substrate. Stock solutions of inhibitors were prepared at a concentration of 10 mM (in DMSO-
20

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 nonlinear least-squares methods using
the Cheng-Prusoff equation, as reported earlier, and represent the mean from at least three
different determinations [50]. All CA isozymes used here were recombinant proteins obtained as
reported earlier by our group.
Disclosure statement
The authors report no conflicts of interest. The authors alone are responsible for the content and
writing of this article.
Acknowledgment
One of the authors (Vikas Sharma) is grateful to the Council of Scientific and Industrial
Research, New Delhi, India for the award of Junior Research Fellowship and the other (Rajiv
Kumar), is thankful to University Grants Commission, New Delhi, India for the award of Senior
Research Fellowship. The authors are also thankful to Materials Research Centre, MNIT Jaipur
for providing HRMS facility and Guru Jambheshwar University of Science
Technology, Hisar-Haryana for NMR facility.
&
21

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25

Tail Approach Synthesis of Novel Benzenesulfonamides Incorporating 1,3,4-Oxadiazole
Hybrids as Potent Inhibitor of Carbonic Anhydrase I, II, IX, and XII
Vikas Sharma, Rajiv Kumar, Andrea Angeli, Claudiu T. Supuran, Pawan K. Sharma
Research Highlights
•
•
•
•
Two series of 1,3,4-oxadiazole based benzenesulfonamides 3a–j and 4a–j having carbonyl
and amide tail/linker were synthesized.
Inhibitory efficacy of 3a–j and 4a–j was evaluated towards hCA I, II, IX, and XII
isoforms.
Compound 4j (KI = 70.7 nM, 7.9 nM, 16.3 nM) emerged as the most potent inhibitor of
hCA I, II and IX respectively as compared to AAZ.
4c and 3b were found to be the most selective hCA IX and XII inhibitors over hCA I.

Tail Approach Synthesis of Novel Benzenesulfonamides Incorporating 1,3,4-Oxadiazole
Hybrids as Potent Inhibitor of Carbonic Anhydrase I, II, IX, and XII
Vikas Sharma, Rajiv Kumar, Andrea Angeli, Claudiu T. Supuran, Pawan K. Sharma
Declaration of interests
The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests:
One of the authors (Vikas Sharma) is grateful to the Council of Scientific and Industrial
Research, New Delhi, India for the award of Junior Research Fellowship and the other (Rajiv
Kumar), is thankful to University Grants Commission, New Delhi, India for the award of Senior
Research Fellowship.