Discovery of curcumin inspired sulfonamide derivatives as a new class of carbonic anhydrase isoforms I, II, IX, and XII inhibitors

Article abstract of DOI:10.1080/14756366.2017.1380638

A series of curcumin inspired sulfonamide derivatives was prepared from various chalcones and 4-sulfamoyl benzaldehyde via Claisen–Schmidt condensation. All new compounds were assayed as inhibitors of four human isoforms of the metalloenzyme carbonic anhydrase (hCA, EC 4.2.1.1) isoforms hCA I, II, IX and XII. Interesting inhibitory activities were observed against all these isoforms. hCA I, an isoform involved in several eye diseases was inhibited moderately with K_Is in the range of 191.8–904.2 nM, hCA II, an antiglaucoma drug target was highly inhibited by the new sulfonamides, with K_Is in the range of 0.75–8.8 nM. hCA IX, a tumor-associated isoform involved in cancer progression and metastatic spread was potently inhibited by the new sulfonamides, with K_Is in the range of 2.3–87.3 nM, whereas hCA XII, and antiglaucoma and anticancer drug target, was inhibited with K_Is in the range of 6.1–71.8 nM. It is noteworthy that one of the new compounds, 5d, was found to be almost 9 times more selective against hCA II (K_I = 0.89 nM) over hCA IX and hCA XII, whereas 5e was 3 and 70 times more selective against hCA II (K_I = 0.75 nM) over hCA IX and hCA XII, respectively.

Full text of DOI:10.1080/14756366.2017.1380638

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Discovery of curcumin inspired sulfonamide
derivatives as a new class of carbonic anhydrase
isoforms I, II, IX, and XII inhibitors
P. V. Sri Ramya, Srinivas Angapelly, Andrea Angeli, Chander Singh Digwal,
Mohammed Arifuddin, Bathini Nagendra Babu, Claudiu T. Supuran & Ahmed
Kamal
To cite this article: P. V. Sri Ramya, Srinivas Angapelly, Andrea Angeli, Chander Singh Digwal,
Mohammed Arifuddin, Bathini Nagendra Babu, Claudiu T. Supuran & Ahmed Kamal (2017)
Discovery of curcumin inspired sulfonamide derivatives as a new class of carbonic anhydrase
isoforms I, II, IX, and XII inhibitors, Journal of Enzyme Inhibition and Medicinal Chemistry, 32:1,
1274-1281, DOI: 10.1080/14756366.2017.1380638
To link to this article: http://dx.doi.org/10.1080/14756366.2017.1380638
© 2017 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group.
Published online: 02 Oct 2017.
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Date: 02 October 2017, At: 01:05

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, 2017
VOL. 32, NO. 1, 1274–1281
https://doi.org/10.1080/14756366.2017.1380638
RESEARCH PAPER
Discovery of curcumin inspired sulfonamide derivatives as a new class of carbonic
anhydrase isoforms I, II, IX, and XII inhibitors
P. V. Sri Ramyaa, Srinivas Angapellya, Andrea Angelib, Chander Singh Digwala, Mohammed Arifuddina,
Bathini Nagendra Babua, Claudiu T. Supuranb and Ahmed Kamala
b
aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad, India; NEUROFARBA
ꢀ
Department, Universita degli Studi di Firenze, Sezione di Scienze Farmaceutiche, Florence, Italy
ABSTRACT
ARTICLE HISTORY
Received 26 August 2017
Accepted 13 September 2017
A series of curcumin inspired sulfonamide derivatives was prepared from various chalcones and 4-sulfa-
moyl benzaldehyde via Claisen–Schmidt condensation. All new compounds were assayed as inhibitors of
four human isoforms of the metalloenzyme carbonic anhydrase (hCA, EC 4.2.1.1) isoforms hCA I, II, IX and
XII. Interesting inhibitory activities were observed against all these isoforms. hCA I, an isoform involved in
several eye diseases was inhibited moderately with KIs in the range of 191.8–904.2 nM, hCA II, an antiglau-
coma drug target was highly inhibited by the new sulfonamides, with KIs in the range of 0.75–8.8 nM. hCA
IX, a tumor-associated isoform involved in cancer progression and metastatic spread was potently inhibited
by the new sulfonamides, with KIs in the range of 2.3–87.3 nM, whereas hCA XII, and antiglaucoma and
anticancer drug target, was inhibited with KIs in the range of 6.1–71.8 nM. It is noteworthy that one of the
new compounds, 5d, was found to be almost 9 times more selective against hCA II (KI ¼ 0.89 nM) over
hCA IX and hCA XII, whereas 5e was 3 and 70 times more selective against hCA II (KI ¼ 0.75 nM) over hCA
IX and hCA XII, respectively.
KEYWORDS
Carbonic anhydrase;
isoforms I, II, IX and XII;
sulfonamide; curcumin
Introduction
hypertension (hCAs I, II, and VII), edema (hCA II, IV, XII, and XIV),
obesity (hCAs VA and VB), and osteoporosis (hCA IV and
XIV)9,11,14,15. Currently, the known carbonic anhydrase inhibitors
(CAIs) can be divided into various classes: those that coordinate to
the active site metal ion9 and those that do not interact with it16.
Primary sulfonamides are the central and historically most promin-
ent class of CAIs, being discovered in 1940, with many representa-
tives in clinical use for decades17. The sulfonamide functionality,
denoted as a zinc-binding group (ZBG), coordinates in deproto-
nated form to the Zn(II) ion within the hCA active sites and estab-
lishes hydrogen bonds with a residues nearby (e.g. Thr199 in
a-CAs)3,4. Such binding features are common among the active
site architectures of all the 15 human isozymes, that all belong to
the a-class9,14. Furthermore, mercaptophenols, ureates/hydroxa-
mates, metal complexing anion inhibitors, and the bioisosteres of
sulfonamides (such as sulfamates and sulfamides) exhibit CA
inhibitory activity following a similar mechanism9,16. Some of the
structures of CAIs [acetazolamide, methazolamide, brinzolamide,
dorzolamide (antiglaucoma drugs), celecoxib (COX-2 selective non-
steroidal anti-inflammatory drug), topiramate (anticonvulsant
drug), and indisulam (in clinical development as an anticancer
agent)] in clinical use/development are depicted in Figure 1. The
main drawback associated with the use of CAIs is their lack of
selectivity in inhibiting various isoforms, considering the fact that
many of these isoforms are rather similar from the structural view
Carbonic anhydrase (CAs, EC 4.2.1.1) enzymes are ubiquitous met-
alloproteins present in prokaryotes and eukaryotes, and they cata-
lyze the fundamental biochemical process of carbon dioxide
hydration (a reversible reaction producing a bicarbonate anion
and a proton), being therefore one of the principal regulators of
cellular pH homeostasis1. These enzymes also take part in several
vital biological processes such as carbon dioxide and bicarbonate
ion transport, respiration, electrolyte secretion, bone resorption,
gluconeogenesis, lipogenesis and ureagenesis among others2–6
.
Indeed, seven (a-, b-, c-, d-, f-, g-, and h-) genetically different CA
families are known so far, which incorporate different metal ions
at their active site including, Zn(II) (in all classes), Fe(II) (in the
c-CAs), Co(II) (in d-CAs), and Cd(II) (in f-CAs)7,8. To date, in mam-
mals, 15 a-CA isozymes have been identified, that differ in subcel-
lular localization, tissue distribution, and catalytic activity1,2. Some
of these isozymes are cytosolic (CA I, CA II, CA III, CA VII, and CA
XIII), others are membrane bound (CA IV, CA IX, CA XII, and CA
XIV), two are mitochondrial (CA VA and CA VB), and one is
secreted in saliva and milk (CA VI)2,9–12
.
The potential of this enzyme family as an important class of
biological targets for pharmacologic intervention was recognized
several decades ago13. An atypical expression level or dysregulated
activities of these enzymes were proven to be linked with several
human diseases, such as glaucoma (hCAs I, II, IV, and XII), cancer
(hCAs IX and XII), some central-nervous system syndromes, includ-
point and even subcellular localization3,4,9
, resulting thus in
ing epilepsy, neuropathic pain, and idiopathic intracranial undesired side effects3,4. Thus, it is still challenging to design
CONTACT Ahmed Kamal ahmedkamal@iict.res.in Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER),
ꢀ
NEUROFARBA Department, Universita degli Studi di Firenze, Sezione di Scienze Farmaceutiche,
Hyderabad, India; Claudiu T. Supuran
Florence, Italy
claudiu.supuran@unifi.it
ß 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distri-
bution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1275
(a)
(b)
(c)
(d)
(e)
(f)
(g)
Figure 1. Structures of some drugs and drug candidates possessing sulfonamide/sulfamate moiety.
(a)
(b)
(c)
Figure 2. Design of curcumin inspired sulfonamide derivatives as hCA inhibitors.
selective/specific agents with distinct inhibition profiles (inhibitors compounds such as resveratrol, catechin, silymarin, dobutamin,
or activators) for any of these isoforms. In the last decades, many and curcumin (KI ¼ 7.44 mM against hCA II isozyme) exhibit CA
inhibitory activity23. Curcumin, the main curcuminoid of popular
efforts have been carried out to design isoform-selective sulfona-
mide-inhibitors by employing two principal methods: the ring and
the tail approaches9,14,18,19. The first resides in modulating the
ring (chiefly its chemical nature) directly connected to the sulfona-
mide ZBG, whereas the latter consists in attaching different tails to
the aromatic/heterocyclic ring carrying various ZBGs, of the sul-
fonamide, sulfamide, sulfamate, carboxylate, hydroxamate, or
dithiocarbamate type3,4. This enabled modulation of the interac-
tions that the ligand establishes with the middle and outer parts
of the active site cavity, which are the most variable regions
among the 15 hCA isoforms mentioned above, and led to a var-
Indian spice turmeric (Curcuma longa), was reported to possess
neuroprotective properties, which may be effective in the preven-
tion and treatment of glaucoma24. Curcumin as well as its ana-
logues and 40-(phenylurenyl)chalcones (Figure 2) were proven to
act as CAIs25,26. Intrigued by these findings and in continuation of
our research into the synthesis and biological evaluation of curcu-
min inspired analogues27, herein, we wish to report curcumin
inspired sulfonamide derivatives as a new class of CAIs against iso-
forms I, II, IX, and XII.
iety of isoform-selective CAIs3,4,9
.
Materials and methods
Among the CA isoforms, CA I and CA II are the two major iso-
zymes present at high concentrations in the cytosol in erythro-
cytes, and CA II (together with CA IX) is the most active among all
the a-CAs2,9–12. Sulfonamides which are known as strong inhibitors
of CA II, have been utilized as commercial drugs (Figure 1, struc-
tures A, B, C, and D) to treat glaucoma or diuretics for a long
period20. In view of lack of selectivity and side effects associated
with the use of the existing drugs, exhaustive search for novel
CAIs is ongoing either through synthesis of new derivatives of
known drugs or from new molecular bases.
Chemistry
All reagents and solvents were obtained from commercial sup-
pliers and used without further purification. The reactions were
monitored by thin layer chromatography (TLC), using MERCK
pre-coated silica gel 60-F254 aluminum plates. Column chroma-
tography with 60–120 mesh silica gel was used as separation
and purification method. Ethyl acetate and hexane were used
as eluents. Melting points were obtained on Stuart digital melt-
Systematic search for new chemotypes may be tackled by ing-point apparatus/SMP 30 and were uncorrected. All IR spec-
investigating compounds from natural products21,22
.
Natural tra were recorded on a Perkin Elmer, FT-IR spectrometer using

1276
P. V. S. RAMYA ET AL.
KBr discs. 1H NMR spectra were recorded on an Avance NMR 1161.3, 1094.7; 1H NMR (500 MHz, DMSO-d6): d 7.98 (d, J ¼ 8.4 Hz,
instrument operated at 500 MHz. 13C NMR spectra were 2H), 7.89 (d, J ¼ 8.5 Hz, 2H), 7.84–7.75 (m, 4H), 7.52–7.43 (m, 3H),
recorded on an Avance NMR instrument operated at 125 MHz. 7.21 (d, J ¼ 16.0 Hz, 1H), 7.05 (d, J ¼ 8.8 Hz, 2H), 3.83 (s, 3H); 13C
NMR (125 MHz, DMSO-d6): d 188.7, 161.8, 145.5, 143.8, 140.8,
138.5, 130.9, 129.3, 128.2, 127.6, 126.6, 123.9, 115.0, 55.8; HRMS
(ESI): m/z Calcd for C18H18NO4S 344.0951, found 344.0950
[M þ H]þ.
Chemical shift values were reported in ppm with TMS as an
internal reference and J values were given in Hertz. The follow-
ing abbreviations were used for 1H NMR spectra to indicate
the signal multiplicity: s (singlet), d (doublet), dd (doublet of
doublet), and
m (multiplet). HRMS were determined with
Agilent QTOF mass spectrometer 6540 series instrument using
ESI technique.
General procedure for the synthesis of 3a–j
To a stirred solution of appropriately substituted benzaldehydes
1a–j (1 mmol) in ethanol (3 ml) was added 0.5 ml of acetone (2)
and 15% aqueous NaOH (1 ml) solution at 0 ꢀC. The reaction was
allowed to stir at room temperature till it was completed. The
reaction mixture was evaporated to dryness, extracted twice with
ethyl acetate, and the combined organic layers were dried over
anhydrous Na2SO4 and concentrated under reduced pressure. The
crude product was purified by column chromatography (Silica gel,
60–120 mesh, 9:1 hexane/ethyl acetate) to obtain the desired chal-
cones 3a–j in good to very good yields.
4-((1E,4E)-5-(2,4-dimethoxyphenyl)-3-oxopenta-1,4-dien-1-
yl)benzenesulfonamide (5c)
Yellow solid, yield 40%; mp: 185–186 ꢀC; IR (KBr, cmꢁ1): ꢀmax
3321.3, 3258.6, 2939.6, 2838.9, 1644.6, 1600.6, 1328.7, 1159.9,
1
1098.9; H NMR (500 MHz, DMSO-d6): d 7.97 (d, J ¼ 8.6 Hz, 2H), 7.93
(s, 1H), 7.88 (d, J ¼ 8.5 Hz, 3H), 7.79–7.74 (m, 3H), 7.39 (d,
J ¼ 16.1 Hz, 2H), 7.28 (d, J ¼ 16.0 Hz, 2H), 3.91 (s, 3H), 3.85 (s, 3H);
13C NMR (125 MHz, DMSO-d6): d 188.7, 163.6, 160.5, 145.5, 140.5,
138.6, 138.5, 130.8, 129.2, 129.0, 126.6, 123.5, 116.3, 106.9, 98.9,
56.3, 56.0; HRMS (ESI): m/z Calcd for C19H20NO5S 374.1057, found
374.1061 [M þ H]þ.
General procedure for the synthesis of 5a–j
To a stirred solution of chalcone 3a–j (0.5 mmol) in ethanol (3 ml)
was added 15% aqueous NaOH (1 ml) solution and aldehyde 4
(0.5 mmol) at 0 ꢀC. The resulting solution was stirred at room tem-
perature till the complete consumption of starting materials was
observed. The reaction mixture was evaporated to dryness,
extracted twice with ethyl acetate, and the combined organic
layers were dried over anhydrous Na2SO4 and concentrated under
reduced pressure. The crude mass was purified by recrystallization
in ethanol or ethyl acetate to give the pure product 5a–j in yields
of 35–45%.
4-((1E,4E)-5-(2,5-dimethoxyphenyl)-3-oxopenta-1,4-dien-1-
yl)benzenesulfonamide (5d)
Yellow solid, yield 38%; mp: 202–203 ꢀC; IR (KBr, cmꢁ1): ꢀmax
3364.1, 3258.2, 2943.2, 2836.0, 1654.0, 1597.9, 1326.5, 1158.5,
4-((1E,4E)-5-(4-isopropoxyphenyl)-3-oxopenta-1,4-dien-1-
yl)benzenesulfonamide (5a)
1
1096.1; H NMR (500 MHz, DMSO-d6): d 7.98 (d, J ¼ 8.6 Hz, 2H), 7.87
(d, J ¼ 8.5 Hz, 2H), 7.83–7.77 (m, 3H), 7.40 (d, J ¼ 16.0 Hz, 2H), 7.21
(d, J ¼ 16.1 Hz, 1H), 7.08–7.04 (m, 3H), 3.85 (s, 3H), 3.79 (s, 3H); 13C
NMR (125 MHz, DMSO-d6): d 188.7, 153.7, 153.2, 144.3, 143.3,
141.3, 138.0, 129.3, 126.6, 123.5, 118.5, 113.6, 113.5, 113.2, 56.5,
56.1; HRMS (ESI): m/z Calcd for C19H20NO5S 374.1057, found
374.1060 [M þ H]þ.
Yellow solid, yield 42%; mp: 163–165 ꢀC; IR (KBr, cmꢁ1): ꢀmax
3309.1, 3249.8, 2975.3, 2937.2, 1666.6, 1622.4, 1600.6, 1337.7,
1158.0, 1095.2; 1H NMR (500 MHz, DMSO-d6): d 7.98 (d, J ¼ 8.5 Hz,
2H), 7.88 (d, J ¼ 8.4 Hz, 2H), 7.83–7.72 (m, 4H), 7.50–7.41 (m, 3H),
7.20 (d, J ¼ 16.0 Hz, 1H), 7.01 (d, J ¼ 8.8 Hz, 2H), 4.76–4.69 (m, 1H),
1.30 (d, J ¼ 6.0 Hz, 6H); 13C NMR (125 MHz, DMSO-d6): d 189.1,
156.3, 144.2, 143.8, 138.5, 131.0, 130.0, 129.2, 126.6, 125.9, 125.2,
123.7, 116.3, 69.9, 22.3, 22.2; HRMS (ESI): m/z Calcd for C20H22NO4S
372.1264, found 372.1262 [M þ H]þ.
4-((1E,4E)-5-(3,4-dimethoxyphenyl)-3-oxopenta-1,4-dien-1-
yl)benzenesulfonamide (5e)
Yellow solid, yield 40%; mp: 198–200 ꢀC; IR (KBr, cmꢁ1): ꢀmax
3321.7, 3252.6, 3091.3, 2940.1, 2839.5, 1619.5, 1596.1, 1330.1,
1158.8, 1095.2; 1H NMR (500 MHz, DMSO-d6): d 7.99 (d, J ¼ 8.4 Hz,
2H), 7.89 (d, J ¼ 8.5 Hz, 2H), 7.84–7.74 (m, 4H), 7.51 (d, J ¼ 16.0 Hz,
2H), 7.23 (d, J ¼ 16.0 Hz, 2H), 7.06 (d, J ¼ 8.4 Hz, 1H), 3.85 (s, 3H),
4-((1E,4E)-5-(4-methoxyphenyl)-3-oxopenta-1,4-dien-1-
yl)benzenesulfonamide (5b)
Yellow solid, yield 39%; mp: 192–193 ꢀC; IR (KBr, cmꢁ1): ꢀmax 3.83 (s, 3H); 13C NMR (125 MHz, DMSO-d6): d 188.7, 151.7, 149.5,
3356.5, 3289.2, 3071.1, 2934.3, 2838.6, 1645.3, 1597.0, 1331.3, 145.5, 144.3, 140.7, 138.5, 130.5, 129.2, 128.1, 127.8, 126.8, 126.6,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1277
124.2, 123.9, 112.1, 111.0, 56.5, 56.0; HRMS (ESI): m/z Calcd for 4-((1E,4E)-5-(4-chlorophenyl)-3-oxopenta-1,4-dien-1-
C19H20NO5S 374.1057, found 374.1055 [M þ H]þ.
yl)benzenesulfonamide (5i)
Yellow solid, yield 43%; mp: 205–206 ꢀC; IR (KBr, cmꢁ1): ꢀmax
3306.9, 3238.4, 2962.1, 2881.8, 1611.9, 1597.2, 1327.0, 1157.7,
659.6; 1H NMR (500 MHz, DMSO-d6): d 7.99 (d, J ¼ 8.2 Hz, 2H), 7.89
(d, J ¼ 8.2 Hz, 2H), 7.86–7.81 (m, 4H), 7.55 (dd, J ¼ 8.2, 3.3 Hz, 2H),
7.50–7.44 (m, 2H), 7.39 (dd, J ¼ 16.0, 12.2 Hz, 2H); 13C NMR
(125 MHz, DMSO-d6): d 188.9, 145.6, 142.3, 142.0, 141.6, 138.3,
135.6, 135.5, 134.1, 130.7, 129.5, 129.3, 128.1, 126.7, 126.6; HRMS
(ESI): m/z Calcd for C17H15ClNO3S 348.0456, found 348.0448
[M þ H]þ.
4-((1E,4E)-3-oxo-5-(2,3,4-trimethoxyphenyl)penta-1,4-dien-1-
yl)benzenesulfonamide (5f)
Yellow solid, yield 35%; mp: 159–161 ꢀC; IR (KBr, cmꢁ1): ꢀmax
3305.9, 3241.6, 2974.1, 2948.4, 1652.1, 1592.9, 1334.4, 1156.5,
1090.8; 1H NMR (500 MHz, DMSO-d6): d 7.98 (d, J ¼ 8.4 Hz, 2H),
7.89–7.84 (m, 3H), 7.80 (d, J ¼ 16.1 Hz, 1H), 7.63 (d, J ¼ 8.9 Hz, 1H),
7.47 (s, 2H), 7.42–7.36 (m, 1H), 7.33 (d, J ¼ 16.0 Hz, 1H), 6.95 (d,
J ¼ 8.9 Hz, 1H), 3.88 (s, 6H), 3.78 (s, 3H); 13C NMR (125 MHz, DMSO-
d6): d 188.7, 156.2, 153.5, 145.5, 142.3, 140.9, 138.4, 138.1, 129.3,
129.0, 126.6, 124.5, 123.9, 121.3, 109.0, 61.9, 60.9, 56.5; HRMS (ESI):
m/z Calcd for C20H22NO6S 404.1162, found 404.1169 [M þ H]þ.
4-((1E,4E)-5-(2-fluoro-4-methoxyphenyl)-3-oxopenta-1,4-dien-1-
yl)benzenesulfonamide (5j)
Yellow solid, yield 40%; mp: 167–169 ꢀC; IR (KBr, cmꢁ1): ꢀmax
3289.8, 3241.1, 2944.6, 2845.9, 1628.8, 1615.3, 1320.3, 1272.0,
1157.4, 1091.3; 1H NMR (500 MHz, DMSO-d6): d 7.99 (d, J ¼ 8.5 Hz,
2H), 7.92–7.86 (m, 3H), 7.81 (d, J ¼ 16.1 Hz, 2H), 7.50–7.43 (m, 3H),
7.31 (d, J ¼ 16.1 Hz, 1H), 6.99 (dd, J ¼ 13.0, 2.5 Hz, 1H), 6.94–6.90
(m, 1H), 3.85 (s, 3H); 13C NMR (125 MHz, DMSO-d6): d 188.7, 161.7,
158.6, 145.6, 143.3, 141.5, 138.5, 130.8, 128.3, 126.6, 123.7, 116.8,
111.2, 106.7, 55.7; HRMS (ESI): m/z Calcd for C18H17FNO4S
362.0857, found 362.0856 [M þ H]þ.
4-((1E,4E)-3-oxo-5-(2,4,6-trimethoxyphenyl)penta-1,4-dien-1-
yl)benzenesulfonamide (5g)
Yellow solid, yield 43%; mp: 249–251 ꢀC; IR (KBr, cmꢁ1): ꢀmax
3304.9, 3253.2, 2969.1, 2951.9, 1616.8, 1591.7, 1335.2, 1157.2,
1090.1; 1H NMR (500 MHz, DMSO-d6): d 8.07–8.01 (m, 1H), 7.97 (d,
J ¼ 8.4 Hz, 2H), 7.86 (d, J ¼ 8.4 Hz, 2H), 7.64 (d, J ¼ 16.0 Hz, 1H),
7.46–7.39 (m, 3H), 7.35 (d, J ¼ 16.0 Hz, 1H), 6.32 (s, 2H), 3.91 (s, 6H),
3.86 (s, 3H); 13C NMR (125 MHz, DMSO-d6): d 189.4, 161.8, 160.4,
145.1, 141.1, 137.3, 130.9, 129.2, 128.8, 126.6, 123.6, 115.9, 104.8,
90.1, 55.3, 54.5; HRMS (ESI): m/z Calcd for C20H22NO6S 404.1162,
found 404.1166 [M þ H]þ.
CA inhibition assay
An Applied Photophysics stopped-flow instrument has been used
for assaying the CA catalyzed CO2 hydration activity29. 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
7.5) as buffer, and 20 mM Na2SO4 (for maintaining constant the
ionic strength), following the initial rates of the CA-catalysed CO2
hydration reaction for a period of 10–100 s. The CO2 concentra-
tions ranged from 1.7 to 17 mM for the determination of the kin-
etic parameters and inhibition constants. For each inhibitor at
least six traces of the initial 5–10% of the reaction have been used
for determining the initial velocity. The uncatalysed 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 0.01 nM were
done thereafter with the assay buffer. Inhibitor and enzyme solu-
tions were preincubated together for 15 min at room temperature
prior to assay, in order to allow for the formation of the E–I com-
plex. The inhibition constants were obtained by non-linear least-
squares methods using PRISM 3 and the Cheng–Prusoff equation,
as reported earlier30–32, and represent the mean from at least
three different determinations. All CA isofoms were recombinant
4-((1E,4E)-3-oxo-5-(3,4,5-trimethoxyphenyl)penta-1,4-dien-1-
yl)benzenesulfonamide (5h)
Yellow solid, yield 45%; mp: 242–244 ꢀC; IR (KBr, cmꢁ1): ꢀmax 3301.8,
3240.4, 2940.2, 2834.7, 1639.5, 1590.5, 1319.8, 1157.0, 1093.9; 1H
NMR (500 MHz, DMSO-d6): d 8.11–7.99 (m, 2H), 7.97 (d, J ¼ 8.5 Hz,
2H), 7.85 (d, J ¼ 8.4 Hz, 2H), 7.63 (d, J ¼ 15.9 Hz, 1H), 7.48–7.42 (m,
2H), 7.33 (d, J ¼ 16.0Hz, 1H), 6.93 (s, 2H), 3.91 (s, 6H), 3.86 (s, 3H);
13C NMR (125 MHz, DMSO-d6): d 188.6, 153.2, 143.3, 142.7, 138.6,
130.5, 129.3, 127.8, 126.8, 123.7, 119.1, 104.1, 60.6, 56.5; HRMS (ESI):
m/z Calcd for C20H22NO6S 404.1162, found 404.1164 [M þ H]þ.
ones obtained in-house as reported earlier30–32
.

1278
P. V. S. RAMYA ET AL.
and their activities were compared to the standard CA inhibitor
(CAI) acetazolamide (AAZ) (Table 1).
Results and discussion
Chemistry
We have investigated the novel series of sulfonamide deriva-
tives for their interaction with four hCAs of pharmacologic interest
(i.e. isoforms hCA I, II, IX, and XII), using a period of 15 min of incu-
bation of the enzyme and inhibitor solutions7,8,15. The following
structure–activity-relationship (SAR) may be noted regarding the
inhibition data of Table 1:
The synthetic route for the preparation of the target compounds,
4-((1E,4E)-3-oxo-5-phenylpenta-1,4-dien-1-yl)benzenesulfonamides
(5a–j) is illustrated in Scheme 1. In the first step, chalcones (3a–j)
were prepared by NaOH-catalyzed Claisen–Schmidt condensation
of a variety of benzaldehydes (1) with acetone28. The correspond-
ing chalcones were reacted with 4-sulfamoyl benzaldehyde (4)
using 15% NaOH by Claisen–Schmidt condensation to afford the
target compounds 5a–j in good yields. All the synthesized com-
pounds were purified by recrystallization in hot ethanol and well
characterized by spectroscopic techniques such as 1H, 13C NMR,
FT-IR and HRMS etc., which were in full accordance with the
1. Against the slow cytosolic isoform hCA I, almost all tested sul-
fonamide derivatives exhibited moderate inhibitory activity
with KI in the range 191.8–904.2 nM. Methoxy substituents on
the benzene ring showed an important role for the modula-
tion of inhibition. Among them, compound 5f incorporating
three MeO moieties in the 2, 3, 4 positions on the ring, prove
1
depicted structures. The H NMR spectrum of 4-((1E,4E)-5-(4-isopro-
poxyphenyl)-3-oxopenta-1,4-dien-1-yl)benzenesulfonamide
(5a)
showed a multiplet of isopropoxy (C–H) proton at d 4.76–4.69, a
doublet of two methyl group’s protons (total 6) of isopropoxy
group at d 1.30 and rest all protons appeared in the aromatic
region in the range of d 7.98–7.01. In the 13C NMR spectrum of
5a, the carbonyl carbon appeared at d 189.1 and the remaining
aromatic carbons appeared in the range of d 156.3–116.3. Nearly
Table 1. In vitro CA I, II, IX, and XII inhibition with compounds 5a–j and aceta-
zolamide (AAZ) as standard, by a stopped-flow, CO2 hydrase assay.29
Selectivity
KI (nM)a
hCA IX
ratiob
Compound
hCA I
hCA II
hCA XII
II/IX
II/XII
1
similar pattern was observed in H and 13C NMR spectra of all the
5a
5b
5c
5d
5e
5f
5g
5h
5i
417.4
286.1
848.1
703.7
294.8
191.8
904.2
594.6
372.6
246.2
250
7.9
2.1
4.7
0.89
0.75
3.0
0.87
8.8
2.3
8.6
7.5
8.4
2.3
2.4
2.4
7.8
6.1
6.3
7.4
3.43
0.24
0.62
0.10
0.32
1.25
0.36
1.12
0.09
0.13
0.46
1.29
0.33
0.63
0.10
0.01
0.04
0.11
0.35
0.26
0.12
2.12
other compounds (5b–j) of this series. In the FT-IR spectrum,
bands at 3309.1, 3249.8, 1337.7 and 1158.0 cmꢁ1 confirmed the
presence of sulfonamide whereas a band at 1666.6 cmꢁ1 con-
firmed the presence of ketone functionality in compound 5a. The
HRMS (ESI) of all the compounds showed an [M þ H]þ peak
equivalent to their molecular formulae.
8.1
52.2
71.8
7.4
25.0
31.1
68.0
5.7
8.1
8.2
12.1
87.3
61.0
25.8
5j
AAZ
CA inhibition
aMean from 3 different assays, by a stopped flow technique (errors were in the
range of 5–10% of the reported values).
All compounds 5a–j were tested in vitro for their inhibitory activity
against the physiologically relevant hCA isoforms I, II, IX, and XII
by means of the stopped-flow carbon dioxide hydration assay29
bSelectivity as determined by the ratio of Kis for hCA isozyme relative to hCA IX
and hCA XII.
O
O
O
i
R
+
R
2
1
3a-j
3f:
3g:
R = 2,3,4-tri-OCH3
R = 2,4,6-tri-OCH3
3a: R = 4-OCH(CH3)2
3b: R = 4-OCH3
3h: R = 3,4,5-tri-OCH3
3i: R = 4-Cl
3j: R = 2-F-4-OCH3
3c: R = 2,4-di-OCH3
3d: R = 2,5-di-OCH3
3e: R = 3,4-di-OCH3
O
O
O
ii
R
R
+
H2NO2S
H2NO2S
3a-j
4
5a-j
5f:
5g:
R = 2,3,4-tri-OCH3
R = 2,4,6-tri-OCH3
5a: R = 4-OCH(CH3)2
5b: R = 4-OCH3
5h: R = 3,4,5-tri-OCH3
5i: R = 4-Cl
5j: R = 2-F-4-OCH3
5c: R = 2,4-di-OCH3
5d: R = 2,5-di-OCH3
5e: R = 3,4-di-OCH3
Scheme 1. Synthesis of curcumin inspired sulfonamide derivatives (5a–j); Reagents and conditions: (i) 15% NaOH, ethanol, 0 ꢀC – rt, 2–3 h, 72–85%; (ii) 15% NaOH,
ethanol, 0 ꢀC – rt, 1–2 h, 35–45%.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1279
to possess the highest inhibitory potency with a KI of
191.8 nM.
Disclosure statement
No potential conflict of interest was reported by the authors.
2. hCA II, the dominant physiologic isoform, was effectively
inhibited by all compounds here considered, in the low nano-
molar range with KIs of 0.75–8.8 nM. Also for this isoform, the
presence and position of methoxy groups in the chalcone
synthon seems to play a crucial role for the inhibition
potency. Among compounds with two OCH3 groups, deriva-
tive 5c showed the “worst” inhibition profile, with a 5 times
decreased activity compared to 5d (4.7 nM and 0.89 nM), the
two compounds only differing by the position of the two
methoxy moieties in the second aryl functionality, the one
coming from the chalcone. In addition, the position of OCH3
groups for compounds 5f–h, with three substituents, plays an
important role in modulating the activity. However, all these
compounds were highly effective hCA II inhibitors (all of
them better than AAZ), with a very small variation of the KIs,
and more detailed structure activity relationship is impossible
to be delineated at this pot for a relatively small group of
derivatives.
3. hCA IX, the tumor-associated isoform, was inhibited by almost
all compounds reported here, in the low nanomolar range (KI
2.3–8.6 nM) except for compounds 5i and 5j which were
active in the medium nanomolar range (KI 61–87.3 nM). For
this transmembrane isoform, the methoxy groups present on
the scaffold did not influence significantly the inhibition pro-
file. The interesting case for hCA IX inhibition was the pres-
ence of halogens in compounds 5i and 5j, that decreased the
inhibition potency near 10 times compared to the other sul-
phonamides investigated here, which were devoid of such
moieties.
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