Design, synthesis and biological evaluation of N-(5-methyl-isoxazol-3-yl/1,3,4-thiadiazol-2-yl)-4-(3-substitutedphenylureido) benzenesulfonamides as human carbonic anhydrase isoenzymes I, II, VII and XII inhibitors

Article abstract of DOI:10.1080/14756366.2016.1197221

A series of N-(5-methyl-isoxazol-3-yl/1,3,4-thiadiazol-2-yl)-4-(3-substitutedphenylureido) benzenesulfonamide derivatives has been designed, synthesized and screened for their in vitro human carbonic anhydrase (hCA; EC 4.2.1.1) inhibition potential. These

Full text of DOI:10.1080/14756366.2016.1197221

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Design, synthesis and biological evaluation of N-
(5-methyl-isoxazol-3-yl/1,3,4-thiadiazol-2-yl)-4-(3-
substitutedphenylureido) benzenesulfonamides
as human carbonic anhydrase isoenzymes I, II, VII
and XII inhibitors
Chandra Bhushan Mishra, Shikha Kumari, Andrea Angeli, Simona Maria
Monti, Martina Buonanno, Amresh Prakash, Manisha Tiwari & Claudiu T.
Supuran
To cite this article: Chandra Bhushan Mishra, Shikha Kumari, Andrea Angeli, Simona Maria
Monti, Martina Buonanno, Amresh Prakash, Manisha Tiwari & Claudiu T. Supuran (2016):
Design, synthesis and biological evaluation of N-(5-methyl-isoxazol-3-yl/1,3,4-thiadiazol-2-
yl)-4-(3-substitutedphenylureido) benzenesulfonamides as human carbonic anhydrase
isoenzymes I, II, VII and XII inhibitors, Journal of Enzyme Inhibition and Medicinal Chemistry,
DOI: 10.1080/14756366.2016.1197221
To link to this article: http://dx.doi.org/10.1080/14756366.2016.1197221
Published online: 17 Jun 2016.
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2016 Informa UK Limited, trading as Taylor & Francis Group. DOI: 10.1080/14756366.2016.1197221
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RESEARCH ARTICLE
Design, synthesis and biological evaluation of N-(5-methyl-isoxazol-3-yl/
1,3,4-thiadiazol-2-yl)-4-(3-substitutedphenylureido) benzenesulfona-
mides as human carbonic anhydrase isoenzymes I, II, VII and XII
inhibitors
Chandra Bhushan Mishra1, Shikha Kumari1, Andrea Angeli2, Simona Maria Monti3, Martina Buonanno3,
Amresh Prakash4, Manisha Tiwari1, and Claudiu T. Supuran2
1Bio-Organic Chemistry Laboratory, Dr. B. R. Ambedkar Centre for Biomedical Research, University of Delhi, Delhi, India, 2Dipartimento Neurofarba,
3
Universita degli Studi di Firenze, Sezione di Scienze Farmaceutiche e Nutraceutiche, Florence, Italy, Istituto di Biostrutture e Bioimmagini (IBB) CNR,
`
4
via Mezzocannone, Naples, Italy, and School of Computational and Integrative Sciences, Jawaharlal Nehru University, New Delhi, India
Abstract
Keywords
A series of N-(5-methyl-isoxazol-3-yl/1,3,4-thiadiazol-2-yl)-4-(3-substitutedphenylureido) benze-
nesulfonamide derivatives has been designed, synthesized and screened for their in vitro
human carbonic anhydrase (hCA; EC 4.2.1.1) inhibition potential. These newly synthesized
sulfonamide compounds were assessed against isoforms hCA I, II, VII and XII, with
acetazolamide (AAZ) as a reference compound. The majority of these compounds were
found quite weak inhibitor against all tested isoforms. Compound 15 showed a modest
inhibition potency against hCA I (Ki ¼73.7 mM) and hCA VII (Ki ¼85.8 mM). Compounds 19 and
25 exhibited hCA II inhibition with Ki values of 96.0 mM and 87.8 mM, respectively. The results of
the present study suggest that, although the synthesized derivatives have weak inhibitory
potential towards all investigated isoforms, some of them may serve as lead molecules for the
further development of selective inhibitors incorporating secondary sulfonamide functional-
ities, a class of inhibitors for which the inhibition mechanism is poorly understood.
Carbonic anhydrase, inhibitor, sulfonamide,
synthesis
History
Received 16 May 2016
Accepted 31 May 2016
Published online 16 June 2016
Introduction
processes such as CO2/bicarbonate transportation, respiration,
electrolyte secretion, gluoconeogensis, lipogenesis, ureagenesis,
bone resorption, neuronal excitability and tumorigenicity8. CAs
have profound involvement in many pathological conditions like
obesity, glaucoma, kidney dysfunction, osteoporosis, gastric
ulcers, migraine, epilepsy and cancer9. For example CA II
deficiency is the major defect in disease conditions such as
osteopetrosis, cerebral calcification and renal tubular acidosis10.
Another isoform, CA VII is implicated in generating neuronal
excitation leading to the development of seizures as well as in the
neuropathic pain control11. hCA II, IV and XII are druggable
targets for anti-glaucoma agents while, hCA VII and hCA XIV are
known drug targets for antiepileptic drugs. hCA IX and XII are
anti-tumor drug targets.At present, CAs are attractive therapeutic
targets to control these aforementioned diseases. The CAs are also
essential for other living organism including pathogens.
Acetazolamide (AAZ) is considered as an excellent CA
inhibitor (CAI) and is used for the treatment of glaucoma, epilepsy
and altitude sickness12. Several other CAIs such as ethoxolamide
(EZA), brinzolamide (BRZ), dorzolamide (DZA), methazolamide
(MZA), and dichlorophenamide (DCP) are useful clinically as anti-
glaucoma agents but, they are also used to treat various neuro-
logical as well as neuromuscular disorders such as epilepsy, genetic
hemiplegic migraine and ataxia, tardive diskinesia, hypokalemic
periodic paralysis, essential tremor and Parkinsons disease13.
Unfortunately, the presently used CAIs are non-selective to a
The carbonic anhydrases (CAs; EC 4.2.1.1) are a large group of
ubiquitous zinc containing enzymes, present in all mammals,
including human1. CAs are encoded by six distinct gene families
known as alpha (a), beta (b), gamma (g), delta (d) zeta (z) and eta
(Z) and all human CAs belongs to class alpha2–4. These enzymes
are well described by their different molecular features,
oligomeric arrangement as well as kinetic properties5. Till date,
a total number of 15 different human carbonic anhydrases (hCA)
isoforms have been discovered, among which CA I-III, CA VII
and CA XIII are present in cytosol; CA IV, CA IX, CA XII and
CA XIV are membrane bound; CA VA and CA VB are restricted
to the mitochondrion and CA VI is secreted in milk and saliva6.
Functionally, CAs are primarily involved in catalyzing the rapid
interconversion of CO2 and H2O to bicarbonate (HCOꢀ3 ) and
protons (H+) using a metal hydroxide nucleophilic mechanism7.
The hCAs play a pivotal role in a variety of physiological
Address for correspondence: Manisha Tiwari, Bio-Organic Chemistry
Laboratory, Dr. B. R. Ambedkar Centre for Biomedical Research,
University of Delhi,North Campus, Delhi, 110007, India, Tel: +91-
9999348938. Fax: +011-27666248. E-mail: mtiwari07@gmail.com
`
Claudiu T. Supuran, Universita degli Studi di Firenze, Dipartimento
Neurofarba, Sezione di Scienze Farmaceutiche e Nutraceutiche, via U.
Schiff 6, 50019 Sesto Fiorentino, Florence, Italy. Tel: +39-055-4573005.
Fax: +39-055-4573385. E-mail: claudiu.supuran@unifi.it

2
C. B. Mishra et al.
J Enzyme Inhib Med Chem, Early Online: 1–6
Figure 1. Sulfonamides CA inhibitors (acetazolamide, methazolamide and SLC-0111), sulfadrugs (sulfadiazine and sulfapyridine) and the designed
novel compounds 15–26.
particular CA isoform, which leads to the occurrence of peripheral General procedure for the synthesis of novel N-(5-methyl-
side effects. Therefore, the development of novel CAIs having high isoxazol-3-yl/1,3,4-thiadiazol-2-yl)-4–(3-substitutedpheny-
potency and selectivity against specific isoform still represents a lureido) benzenesulfonamides (15–26)
needful and challenging task to obtain newer compounds which
Sulfamethoxazole/sulfamethiazole (10 mmol) was fully dissolved
could replace the ‘‘old ones’’, in consequence avoiding side effects
in dried DMF and then subsequent isocyanate (10 mmol) was
as well as improving therapeutic safety.
added dropwise to the reaction mixture and heated at 90–100 ꢁC
Sulfonamides containing molecules have presented a remark-
for 5–6 h. The appeared precipitate was filtered and washed with
able history in CA inhibition. The primary sulfonamide group
hot petroleum ether as well as water. The obtained crude products
(-SO2NH2) is present in these molecules which bind as anions to
were purified by column chromatography using chloroform/
the Zn2+ ion in the enzyme active site with high affinity, and
methanol (98:02) as an eluent to furnish target compounds (15–
block catalysis14. In the past, several research reports showed that
26).The purity of the synthesized compounds was analyzed by
the urea functional group along with sulfonamide also seemed to
HPLC using acetonitrile/methanol (98:02) as mobile phase
be beneficial to generate effective CA inhibitory activity15. In the
(Scheme 1).
present study, we have used sulfamethoxazole/sulfamethiazole
to develop potent CAIs and also phenyl urea moiety was also
N-(5-Methyl-isoxazol-3-yl)-4–(3-phenyl-ureido)-benzene-
sulfonamide (15)
installed to this pharmacophore to develop new CAIs. Figure 1
showed some known CAIs and the molecular framework of our
synthesized compound. The synthesized derivatives have been
well characterized by using 1H NMR, 13C NMR and mass
spectroscopy. The purity was analyzed by HPLC. The esterase
activity was performed to study the potential of the newly
synthesized compounds (15–26) towards hCA I, hCA II, hCA VII
and hCA XII inhibition.
White solid; yield 1.74 g; mp 240–242 ꢁC; 1H NMR (DMSO-
d6,400 MHz): d 2.28 (s, 3H, CH3), 6.13 (s, 1H, CH), 6.98 (t, 1H,
Ar-H, J ¼ 7.2 Hz), 7.27 (t, 2H, Ar-H, J ¼ 7.6 Hz), 7.44 (d, 2H, Ar-
H, J ¼ 7.6 Hz), 7.63 (d, 2H, Ar-H, J ¼ 9.1 Hz), 7.75 (d, 2H, Ar-H,
J ¼ 7.7 Hz), 8.82 (s, 1H, NH), 9.15 (s, 1H, NH), 11.29 (s, 1H,
NH). 13C NMR (DMSO-d6): 12.08, 95.3, 117.7, 118.4, 122.3,
128.1, 128.8, 131.4, 139.1, 144.3, 152.1, 157.6, 170.2. LC–MS:
m/e; 372 (M+). HPLC purity: 97.6%.
Materials and methods
Chemistry
4-[3–(4-Fluoro-phenyl)-ureido]-N-(5-methyl-isoxazol-3-yl)-
benzenesulfonamide (16)
All the chemicals and reagents were obtained from Sigma Aldrich
(St. Louis, MO), Alfa Aesar (Massachusetts), S.D Fine Chemicals
(India) and Merck (Darmstadt, Germany) and solvents for
reaction medium were dried by standard methods. Analytical
thin layer chromatography (TLC) was performed on commercially
available silica gel (Kieselgel 60, F254) coated aluminum plates
(Merck). Column chromatography purification was performed
using silica gel Merck 100–200 mesh. Melting points were taken
in open capillaries using model KSPII, KRUSS, (Germany).
1H NMR and 13C NMR were recorded on Jeol-400 MHz High
Resolution NMR Spectrophotometer (JEOL USA, Inc., Peabody,
MA). Chemical shifts in 1H NMR are reported in parts per million
and coupling constant (J) in Hertz (Hz) using DMSO-d6 as
solvent. The splitting patterns are designated as follows: s, singlet;
White solid; yield 1.92 g; mp 228–230 ꢁC; 1H NMR (DMSO-
d6,400 MHz): d 2.28 (s, 3H, CH3), 6.12 (s, 1H, CH), 7.12 (t, 2H,
Ar-H, J ¼ 8.7 Hz), 7.43–7.47 (m, 2H, Ar-H), 7.61 (d, 2H, Ar-H,
J ¼ 9.1 Hz), 7.76 (d, 2H, Ar-H, J ¼ 8.4 Hz), 8.85 (s, 1H, NH), 9.10
(s, 1H, NH), 11.28 (s, 1H, NH). 13C NMR (DMSO-d6): 12.07,
95.3, 115.2, 115.5, 117.7, 120.2, 120.3, 128.1, 131.5, 135.4,
144.3, 152.2, 156.4, 157.6, 158.7, 170.2. LC–MS: m/e; 391
(M + 1). HPLC purity: 97.1%.
4-[3–(4-Chloro-phenyl)-ureido]-N-(5-methyl-isoxazol-3-yl)-
benzenesulfonamide (17)
d, doublet; t, triplet; q, quadruplet. Mass spectra were recorded on White solid; yield 2.35 g; mp 238–240 ꢁC; 1H NMR (DMSO-
an Agilent 6310 Ion trap LC/MS and the purity of final d6,400 MHz): d 2.28 (s, 3H, CH3), 6.12 (s, 1H, CH), 7.33 (d, 2H,
compounds was analyzed by using reverse phase HPLC Ar-H, J ¼ 8.4 Hz), 7.47 (d, 2H, Ar-H, J¼ 8.4 Hz), 7.62 (d, 2H, Ar-
(Shimadzu, Kyoto, Japan) with C-18 column.
H, J ¼ 9.1 Hz), 7.75 (d, 2H, Ar-H, J ¼ 8.4 Hz), 8.97 (s, 1H, NH),

DOI: 10.1080/14756366.2016.1197221
In vitro studies of benzenesulfonamides
3
9.20 (s, 1H, NH), 11.28 (s, 1H, NH). 13C NMR (DMSO-d6):
12.09, 95.4, 117.8, 120.0, 125.9, 128.1, 131.6, 138.2, 144.1,
152.1, 157.6, 170.2. LC–MS: m/e; 406 (M+). HPLC purity:
97.9%.
R1
O
NH2
NCO
HN
S
N
H
A
+
O
HN
S
R1
N-(5-Methyl-isoxazol-3-yl)-4-(3-p-tolyl-ureido)-benzene-
sulfonamide (18)
O
O
O
R
HN
3-14
R
White solid; yield 1.49 g; mp 233–235 ꢁC; 1H NMR (DMSO-
d6,400 MHz): d 2.23 (s, 3H, CH3), 2.28 (s, 3H, CH3), 6.12 (s, 1H,
CH), 7.08 (d, 2H, Ar-H, J ¼ 7.6 Hz), 7.32 (d, 2H, Ar-H,
J ¼ 8.4 Hz), 7.61 (d, 2H, Ar-H, J ¼ 9.1 Hz), 7.74 (d, 2H, Ar-H,
J ¼ 8.4 Hz), 8.71 (s, 1H, NH), 9.11 (s, 1H, NH), 11.20 (s, 1H,
NH). 13C NMR (DMSO-d6): 12.0, 20.3, 95.3, 117.3, 118.5, 128.1,
129.2, 131.2, 131.3, 136.6, 144.4, 152.1, 157.6, 170.2. LC–MS:
m/e; 386 (M+). HPLC purity: 99.4%.
1-2
15-26
Scheme 1. Synthesis of N-(5-methyl-isoxazol-3-yl/1,3,4-thiadiazol-2-yl)-
4-(3-substituted phenylureido)benzenesulfonamide derivatives. Reagent
and conditions: A. Dried DMF reflux, 5–6 h.
Compound number
R-Group
R1-Group
H
4-[3–(4-Methoxy-phenyl)-ureido]-N-(5-methyl-isoxazol-3-
yl)-benzenesulfonamide (19)
15
N
O
White solid; yield 1.84 g; mp 212–214 ꢁC; 1H NMR (DMSO-
d6,400 MHz): d 2.28 (s, 3H, CH3), 3.70 (s, 3H, CH3), 6.12 (s, 1H,
CH), 6.86 (d, 2H, Ar-H, J ¼ 8.4 Hz), 7.34 (d, 2H, Ar-H,
J ¼ 8.4 Hz), 7.61 (d, 2H, Ar-H, J ¼ 8.4 Hz), 7.73 (d, 2H, Ar-H,
J ¼ 9.1 Hz), 8.63 (s, 1H, NH), 9.08 (s, 1H, NH), 11.21 (s, 1H,
NH). 13C NMR (DMSO-d6): 12.0, 55.1, 95.3, 114.0, 117.5, 120.3,
128.1, 131.2, 132.1, 144.5, 152.3, 154.8, 157.6, 170.2. LC–MS:
m/e; 402 (M+). HPLC purity: 99.7%.
16
17
18
19
20
4-Fluoro
4-Chloro
4-Methyl
4-Methoxy
N
O
N
O
4-(3-Benzyl-ureido)-N-(5-methyl-isoxazol-3-yl)-benzene-
sulfonamide (20)
White solid; yield 2.12 g; mp 218–220 ꢁC; 1H NMR (DMSO-
d6,400 MHz): d 2.27 (s, 3H, CH3), 4.29 (d, 2H, CH2, J ¼ 6.1 Hz),
6.10 (s, 1H, CH), 6.82 (t, 1H, NH, J ¼ 5.7 Hz), 7.21–7.33 (m, 5H,
Ar-H), 7.57 (d, 2H, Ar-H, J ¼ 9.1 Hz), 7.69 (d, 2H, Ar-H,
J ¼ 8.4 Hz), 9.08 (s, 1H, NH), 11.22 (s, 1H, NH). 13C NMR
(DMSO-d6): 12.0, 42.7, 95.3, 117.1, 126.8, 127.1, 128.1, 128.3,
130.7, 139.9, 145.0, 154.7, 157.7, 170.2. LC–MS: m/e; 387
(M + 1). HPLC purity: 99.4%.
N
O
N
O
N-(5-methyl-[1,3,4]thiadiazol-2-yl)-4-(3-phenyl-ureido)-
benzenesulfonamide (21)
Benzyl
H
N
O
White solid; yield 2.16 g; mp 226–228 ꢁC; 1H NMR (DMSO-
d6,400 MHz): d 2.45 (s, 3H, CH3), 6.98 (t, 1H, Ar-H, J ¼ 7.6 Hz),
7.28 (t, 2H, Ar-H, J ¼ 7.9 Hz), 7.44 (d, 2H, Ar-H, J ¼ 8.4 Hz),
7.58 (d, 2H, Ar-H, J ¼ 9.1 Hz), 7.68 (d, 2H, Ar-H, J ¼ 9.1 Hz),
8.77 (s, 1H, NH), 9.09 (s, 1H, NH), 13.81 (s, 1H, NH). 13C NMR
(DMSO-d6): 16.1, 117.6, 118.4, 122.2, 127.1, 128.8, 134.3, 139.2,
143.5, 152.2, 154.3, 167.7. LC–MS: m/e; 390 (M + 1). HPLC
purity: 96.8%.
N
N
21
22
23
24
25
26
S
N
N
4-Fluoro
4-Chloro
4-Methyl
4-Methoxy
Benzyl
S
4-[3-(4-Fluoro-phenyl)-ureido]-N-(5-methyl-[1,3,4]thiadiazol-
2-yl)-benzene sulfonamide (22)
N
N
S
White solid; yield 1.64 g; mp 252–254 ꢁC; 1H NMR (DMSO-
d6,400 MHz): d 2.45 (s, 3H, CH3), 7.10–7.14 (m, 2H, Ar-H),
7.43–7.47 (m, 2H, Ar-H), 7.58 (d, 2H, Ar-H, J ¼ 8.3 Hz), 7.68 (d,
2H, Ar-H, J ¼ 8.4 Hz), 8.81 (s, 1H, NH), 9.10 (s, 1H, NH), 13.82
(s, 1H, NH). 13C NMR (DMSO-d6): 16.1, 115.2, 115.5, 117.6,
120.2, 120.3, 127.0, 134.4, 135.6, 143.5, 152.3, 154.4, 156.4,
158.8, 167.7. LC–MS: m/e; 408 (M + 1). HPLC purity: 99.5%.
N
N
S
N
N
S
4-[3–(4-Chloro-phenyl)-ureido]-N-(5-methyl-[1,3,4]thiadia-
zol-2-yl)-benzene sulfonamide (23)
N
N
White solid; yield 2.0 g; mp 275–277 ꢁC; 1H NMR (DMSO-
d6,400 MHz): d 2.44 (s, 3H, CH3), 7.32 (d, 2H, Ar-H, J ¼ 9.1 Hz),
S

4
C. B. Mishra et al.
J Enzyme Inhib Med Chem, Early Online: 1–6
7.48 (d, 2H, Ar-H, J ¼ 9.1 Hz), 7.59 (d, 2H, Ar-H, J ¼ 8.4 Hz), XII were recombinant enzymes produced as described earlier in
7.69 (d, 2H, Ar-H, J ¼ 9.1 Hz), 8.93 (s, 1H, NH), 9.14 (s, 1H, our laboratory17–32
.
NH), 13.8 (s, 1H, NH).13C NMR (DMSO-d6): 16.1, 117.7, 120.0,
125.8, 127.1, 128.7, 134.5, 138.3, 143.3, 152.1, 154.4, 167.7. LC– Result and discussion
MS: m/e; 423 (M+). HPLC purity: 98.6%.
Chemistry
N-(5-methyl-[1,3,4]thiadiazol-2-yl)-4-(3-p-tolyl-ureido)-
benzenesulfonamide (24)
The synthetic route utilized for the preparation of N-(5-methyl-
isoxazol-3-yl/1,3,4-thiadiazol-2-yl)-4–(3-substitutedphenylureido)
benzenesulfonamide derivativesis depicted in Scheme 1.
An equimolar ratio of sulfamethoxazole/sulfamethiazole and
substituted phenyl isocyantes were reacted in dry DMF to afford
N-(5-methyl-isoxazol-3-yl/1,3,4-thiadiazol-2-yl)-4–(3-substituted
phenylureido)benzenesulfonamide derivatives. All compounds
were purified by using column chromatography using chloro-
form/methanol as eluent. The purity of the final compounds was
analyzed by HPLC analysis using acetonitrile/methanol (98:02) as
mobile phase. All compounds presented percent purity more than
95% and were fully characterized by NMR and mass
spectroscopy.
White solid; yield 1.75 g; mp 258–260 ꢁC; 1H NMR (DMSO-
d6,400 MHz): d 2.23 (s, 3H, CH3), 2.45 (s, 3H, CH3), 7.08 (d, 2H,
Ar-H, J ¼ 8.4 Hz), 7.32 (d, 2H, Ar-H, J ¼ 8.4 Hz), 7.57 (d, 2H, Ar-
H, J ¼ 9.9 Hz), 7.67 (d, 2H, Ar-H, J ¼ 9.9 Hz), 8.66 (s, 1H, NH),
9.05 (s, 1H, NH), 13.8 (s, 1H, NH).13C NMR (DMSO-d6): 16.09,
20.3, 117.5, 118.5, 127.0, 129.2, 131.1, 134.1, 136.6, 143.5,
152.2, 154.3, 167.7. LC–MS: m/e; 403 (M+). HPLC purity:
96.7%.
4-[3-(4-Methoxy-phenyl)-ureido]-N-(5-methyl-[1,3,4]thia-
diazol-2-yl)-benzenesulfonamide (25)
We have used SLC-0111 as lead molecule for designing these
White solid; yield 2.0 g; mp 238–240 ꢁC; 1H NMR (DMSO- compounds33,34, as this derivative recently completed successfully
d6,400 MHz): d 2.48 (s, 3H, CH3), 3.70 (s, 3H, CH3), 6.86 (d, 2H, the Phase I clinical trials as an anti-tumor agent. In addition, a
Ar-H, J ¼ 9.1 Hz), 7.34 (d, 2H, Ar-H, J ¼ 9.1 Hz), 7.57 (d, 2H, Ar- range of secondary and tertiary sulfonamides has been investi-
H, J ¼ 8.4 Hz), 7.65 (d, 2H, Ar-H, J ¼ 8.4 Hz), 8.58 (s, 1H, NH), gated as CAIs recently, some of them showing effective and
9.02 (s, 1H, NH), 13.8 (s, 1H, NH).13C NMR (DMSO-d6): 16.0, selective inhibition of several important isoforms35–49. However,
55.1, 114.0, 117.4, 120.2, 127.0, 132.2, 134.0, 143.6, 152.3, the inhibition mechanism with secondary and tertiary sulfona-
154.7, 167.7. LC–MS: m/e; 419 (M+). HPLC purity: 99.3%.
mides is unknown, as no X-ray crystal structures of such
derivatives bound to the CA are available to date. Thus, the
sulfonamides reported here incorporate in their molecule the urea
fragment found in SLC-0111 and the secondary sulfonamide
moiety present in sulfa drugs and several recently investigated
4-(3-Benzyl-ureido)-N-(5-methyl-[1,3,4]thiadiazol-2-yl)-
benzenesulfonamide (26)
White solid; yield 2.2 g; mp 270–272 ꢁC; 1H NMR (DMSO-
d6,400 MHz): d 2.44 (s, 3H, CH3), 4.28 (q, 2H, CH2, J ¼ 6.0 Hz),
6.76 (t, 1H, NH, J ¼ 6.1 Hz), 7.20–7.33 (m, 5H, Ar-H), 7.53 (d,
2H, Ar-H, J ¼ 9.1 Hz), 7.62 (d, 2H, Ar-H, J ¼ 8.4 Hz), 9.02 (s, 1H,
CAIs35–49
.
In vitro carbonic anhydrase activity
NH), 13.8 (s, 1H, NH).13C NMR (DMSO-d6): 16.0, 42.7, 117.0, All the synthesized compounds (15–26) were studied for the
126.8, 127.0, 127.1, 128.3, 133.5, 140.0, 144.2, 154.2, 154.8, inhibition of four CA isozymes of human origin, i.e. hCA I, hCA
167.6. LC–MS: m/e; 404 (M + 1). HPLC purity: 99.3%.
II, hCA VII and hCA XII (Table 1). The following structure
activity relationship (SAR) was obtained by analyzing CA
inhibition data of Table 1:
Carbonic anhydrase inhibition assay
(1) In the isoxazole subseries (15–20) compound substituted
phenyl ring (compound 15) at the terminal end has mild hCA
inhibitory activity for both hCA I (Ki ¼73.7 mM)and hCA VII
(Ki ¼85.8 mM) isoforms.
An applied photophysics stopped-flow instrument has been used
for assaying the CA catalyzed CO2 hydration activity16. Phenol
red (at a concentration of 0.2 mM) has been used as indicator,
working at the absorbance maximum of 557 nm, with 20 mM
Hepes (pH 7.4) as buffer, and 20 mM Na2SO4 for maintaining
constant the ionic strength (this anion is not inhibitory and has a
KI4200 mM against these enzymes), following the initial rates of
the CA-catalyzed CO2 hydration reaction for a period of 10–100 s.
The CO2 concentrations ranged from 1.7 to 17 mM for the
determination of the kinetic parameters and inhibition constants.
For each measurement at least six traces of the initial 5–10% of
the reaction have been used for determining the initial velocity,
working with 10-fold decreasing inhibitor concentrations ranging
between 0.1 nM and 10–100 mM (depending on the inhibitor
potency, but at least five points at different inhibitor concentra-
tions were employed for determining the inhibition constants).
The uncatalyzed rates were determined in the same manner and
subtracted from the total observed rates. Stock solutions of
inhibitor (0.1 mM) were prepared in distilled-deionized water and
dilutions up to 0.1 nM were done thereafter with the assay buffer.
Inhibitor and enzyme solutions were preincubated together for
15 min at room temperature prior to assay, in order to allow for
the formation of the E-I complex. The inhibition constants were
obtained by non-linear least-squares methods using the Cheng–
Prusoff equation, and represent the mean from at least three
different determinations. The human isoforms hCA I, II, VII and
(2) In this subseries, compound substituted with para-methoxy
phenyl (compound 19) also exhibited low inhibition against
hCA II (Ki ¼96.0 mM). Moreover, this compound displayed a
Table 1. hCA I, II, VII and XII inhibition with compounds 15–26, with
AAZ as standard.
KI (mM)
Compound
hCA I
hCA II
hCA VII
hCA XII
15
16
17
18
19
20
21
22
23
24
25
26
AAZ
73.7
4100
4100
4100
4100
4100
4100
4100
4100
4100
4100
4100
0.25
4100
4100
4100
4100
96.0
4100
4100
4100
4100
4100
87.8
85.8
4100
4100
4100
4100
4100
4100
4100
4100
4100
4100
4100
0.005
4100
4100
4100
4100
4100
4100
4100
4100
4100
4100
4100
4100
0.006
4100
0.012

DOI: 10.1080/14756366.2016.1197221
In vitro studies of benzenesulfonamides
5
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cranial hypertension. Expert Rev Neurother 2015;15:851–6.
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Ki value of4100 mM towards hCA I, VII and XII, thus seems
to inactive for these isoforms.
The remaining compounds in this subseries did not produce
remarkable inhibitory potential against hCA I, II, VII and XII
isoforms.
(3) In thiadiazole subseries (21–26), compound 25 comprising
para-methoxy phenyl ring at its terminal end has shown a
modest inhibitory potential against hCA II with Ki value of
87.8 mM. This compound was inactive against hCA I, VII and
XII (Ki¼4100 mM).
(4) Overall, the SAR study indicates that substitutions on the
phenyl ring did not produce remarkable inhibitory potential
against tested hCA isoforms except para-methoxy substitu-
tion. Therefore, further structural modifications are neces-
sarily required to achieve more potent CAIs.
16. Khalifah RG. The carbon dioxide hydration activity of carbonic
anhydrase. I. Stop-flow kinetic studies on the native human
isoenzymes B and C. J Biol Chem 1971;246:2561–73.
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conjugates and 3-cyano-7-hydroxy-coumarin act as isoform-select-
ive carbonic anhydrase inhibitors. J Enzyme Inhib Med Chem 2013;
28:397–401.
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Conclusions
A set of N-(5-methyl-isoxazol-3-yl/1,3,4-thiadiazol-2-yl)-4–(3-
substitutedphenylureido) benzenesulfonamide derivatives were
synthesized in a good yield and were characterized by using
NMR and mass spectroscopy techniques. All compounds were
subjected for their in-vitro CA inhibitory activity and only
compound 15 showed CA inhibition against hCA I and hCA VII
isoform. The selectivity ratio of compounds 19 and 25 for hCA II
isoform makes them interesting leads for the development of
potent and selective hCA II inhibitor.
19. Innocenti A, Casini A, Alcaro MC, et al. Carbonic anhydrase
inhibitors: the first on-resin screening of a 4-sulfamoylphe-
nylthiourea library. J Med Chem 2004;47:5224–9.
20. Casini A, Scozzafava A, Mincione F, et al. Carbonic anhydrase
inhibitors: water-soluble 4-sulfamoylphenylthioureas as topical
intraocular pressure-lowering agents with long-lasting effects.
J Med Chem 2000;43:4884–92.
21. Mincione F, Starnotti M, Masini E, et al. Carbonic anhydrase
inhibitors: design of thioureido sulfonamides with potent isozyme II
and XII inhibitory properties and intraocular pressure lowering
activity in a rabbit model of glaucoma. Bioorg Med ChemLett 2005;
15:3821–7.
Acknowledgements
The University Science Instrumentation Center (USIC), University of
Delhi is acknowledged for providing NMR and Mass spectral character-
ization of the synthesized compounds.
Declaration of interest
22. Supuran CT, Scozzafava A, Bogdan C, et al. Carbonic
anhydrase inhibitors – Part 49: Synthesis of substituted ureido
and thioureido derivatives of aromatic/heterocyclic sulfonamides
with increased affinities for isozyme I. Eur J Med Chem 1998;33:
83–93.
23. Avvaru BS, Wagner JM, Maresca A, et al. Carbonic anhydrase
inhibitors. The X-ray crystal structure of human isoform II in adduct
with an adamantyl analogue of acetazolamide resides in a less
utilized binding pocket than most hydrophobic inhibitors. Bioorg
Med ChemLett 2010;20:4376–81.
Chandra Bhushan Mishra (DSK-PDF) and Shikha Kumari are thankful to
the University Grant Commission for providing financial support. Author
Amresh Prakash is thankful to SERB-YSS for financial support. Manisha
Tiwari deeply acknowledges the financial support to carry out this
research work. Work from Supuran laboratory was financed by two EU
grants (Dynano and Metoxia).
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