Synthesis and evaluation of sulfonamide-bearing thiazole as carbonic anhydrase isoforms hCA I and hCA II

Article abstract of DOI:10.3109/14756366.2015.1128426

Sulfonamide-bearing thiazole compounds were synthesized and their inhibitory effects on the activity of purified human carbonic anhydrase I and II were evaluated. Human carbonic anhydrase isoenzymes (hCA-I and hCA-II) were purified from erythrocyte cells

Full text of DOI:10.3109/14756366.2015.1128426

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Synthesis and evaluation of sulfonamide-bearing
thiazole as carbonic anhydrase isoforms hCA I and
hCA II
Soner Kılıcaslan, Mustafa Arslan, Zeynep Ruya, Çigdem Bilen, Adem Ergün,
Nahit Gençer & Oktay Arslan
To cite this article: Soner Kılıcaslan, Mustafa Arslan, Zeynep Ruya, Çigdem Bilen, Adem Ergün,
Nahit Gençer & Oktay Arslan (2016): Synthesis and evaluation of sulfonamide-bearing thiazole
as carbonic anhydrase isoforms hCA I and hCA II, Journal of Enzyme Inhibition and Medicinal
Chemistry, DOI: 10.3109/14756366.2015.1128426
To link to this article: http://dx.doi.org/10.3109/14756366.2015.1128426
Published online: 08 Jan 2016.
Submit your article to this journal
Article views: 1
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=ienz20
Download by: [Gazi University]
Date: 12 January 2016, At: 03:48

http://informahealthcare.com/enz
ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–6
2016 Taylor & Francis. DOI: 10.3109/14756366.2015.1128426
!
RESEARCH ARTICLE
Synthesis and evaluation of sulfonamide-bearing thiazole as carbonic
anhydrase isoforms hCA I and hCA II
Soner Kılıcaslan1, Mustafa Arslan1, Zeynep Ruya1, C¸igdem Bilen2, Adem Ergu¨n2, Nahit Genc¸er2, and Oktay Arslan2
1Chemistry Department, Faculty of Arts and Sciences, Sakarya University, Sakarya, Turkey and 2Chemistry Department, Faculty of Arts and Sciences,
Balikesir University, Balikesir, Turkey
Abstract
Keywords
Sulfonamide-bearing thiazole compounds were synthesized and their inhibitory effects on the
activity of purified human carbonic anhydrase I and II were evaluated. Human carbonic
anhydrase isoenzymes (hCA-I and hCA-II) were purified from erythrocyte cells by affinity
chromatography. The inhibitory effects of the 12 synthesized sulfonamide (5a–l) on the
hydratase and esterase activities of these isoenzymes (hCA-I and hCA-II) were studied in vitro. In
relation to these activities, the inhibition equilibrium constants (Ki) were determined. The
results showed that all the synthesized compounds inhibited the CA isoenzyme activity. Among
them 5b was found to be the most active (IC50 ¼0.35 mM; Ki: 0.33 mM) for hCA I and hCA II.
Carbonic anhydrase, enzyme inhibitor,
sulfonamide, thiazole
History
Received 16 June 2015
Revised 23 November 2015
Accepted 24 November 2015
Published online 7 January 2016
Introduction
Until now, 15 human CA (hCA) isoforms have been identified
that exhibit significant differences in catalytic activity, subcelluler
localization and tissue expression. Some isoforms are cytosolic
(CA I, CA II, CA III, CA VII and CA XIII), two are mitochondrial
(CA VA and CAVB), others are membrane bound (CA IV, CA IX,
CA XII and CA XIV), and one is secreted in saliva (CA VI). It has
been reported that the CA XV isoform is not expressed in humans
or in other primates, but it is abundant in rodents and other
Heterocyclic moieties, such as indoles, thiazolidine-4-ones,
pyrazoles, piperazines, pyridines, etc., always have considerable
attention due to their pharmacological activities1,2. Heterocycles
bearing one or more nitrogen and sulfur atoms have received more
attention recently. Among the most frequently encountered
heterocyclic compounds, thiazole and its derivatives play an
important role in nature. The heterocyclic compounds have broad
applications including the treatment of hypertension, bacterial and
HIV infections, allergies and as antibiotics and ligands for
estrogen receptors3,4. Beside, these compounds have used as
inhibitors against fructose 1,6-bisphosphatase5, tumor-associated
carbonic anhydrase isoforms hCA IX and hCA XII6, sphingosine
kinase7 and evaluation of in vitro anticancer activity8.
vertebrates14,15
.
These enzymes catalyze a very simple but critically important
physiological reaction, rapid interconversion of carbon dioxide
and water into protons and bicarbonate ions, in many physiolo-
gical/pathological processes open up widespread opportunities for
the development of diverse, specific inhibitors for clinical
applications16,17. In addition to this physiological reaction, the
reversible hydration of CO2 to bicarbonate, CAs catalyze several
other reactions, such as the hydration of cyanate or cyanamide to
carbamic acid, or urea, the aldehyde hydration to gem-diols, the
The carbonic anhydrases (CAs, EC 4.2.1.1) are superfamily of
metalloenzymes present in Archaea, prokaryotes and eukaryotes,
and in all life kingdoms with five genetically distinct families
described in various organisms. Evolutionarily unrelated gene
families are a-, b-, d-, g-, z-CAs9. a-, b-, d CAs contain zinc metal
at the active site. a-CAs are found in vertebrates, eubacteria, algae
and cytoplasm of green plants and the b-CAs are predominantly
found in eubacteria, algae and chloroplasts of both mono- as well
as dicotyledons10–12. g-CAs may have Fe(II) at the active site and
mainly are found in Archaea, some eubacteria and plants. Cd(II)
and Zn(II) ions are equally effective for catalysis in z-CAs
(diatoms encode). Three His residues coordinate the metal ion in
the a-, d-, g-class enzymes or one His and two Cys residues
coordinate it with the fourth ligand being a water molecule/
hydrolysis of carboxylic, or sulfonic and some others18,19
.
The importance of sulfonamide compounds in medicinal
chemistry cannot be ignored and the compounds are well-
known pharmaceutical agents and have gained much attention due
to their diverse biological activities in pharmaceutical as well as
in agricultural areas20. The sulfonamide compounds have a
number of biological activities, such as antibacterial21, anti-
inflammatory22, antitumor23, anticancer24 and carbonic anhydrase
inhibitory functions25.
Carbonic anhydrase inhibitors or activators have many medical
applications, such as in the treatment of glaucoma, in the
management of neurological disorders, in the treatment of
Alzheimer’s disease. Acetazolamide (AAZ), dorzalamide (DZA)
and brinzolamide (BRZ) are sulfonamide derivatives and used in
the treatment of glaucoma26–28. In this study, 12 sulfonamide-
bearing thiazole compounds were synthesized and their inhibitory
hydroxide ion in the b- and z-CAs9,13
.
Address for correspondence: Dr Nahit Genc¸er, PhD, Faculty of Arts and
Sciences, Chemistry Department, Balikesir University, Balikesir, 10145,
Turkey. E-mail: ngencer@balikesir.edu.tr

2
S. Kılıcaslan et al.
J Enzyme Inhib Med Chem, Early Online: 1–6
O
Scheme 1. Synthesis of sulfonamide-bearing
thiazole compounds.
Br
R3
R2
S
S
R1
R3
NH2
NH2
HN
N
R1
NH4SCN
2
R2
4
NH
SO2
NH2
SO2
NH2
SO2
NH2
5
3
1
5a
H
H
H
5b
H
5c
H
5d
H
H
F
5e
5f
H
H
5g
5h
5i
5j
5k
5l
R1
H
H
H
H
H
H
NO
H
H
NO
H
Cl
H
H
2
R2
R3
H
H
OH
OH
2
NO
C
Cl
OCH
CH
3
OH
H
Cl
N
2
3
effects on the activity of purified human carbonic anhydrase (2H, d), 7.80 (2H, d), 7.52 (2H, t), 7.38 (1H, t), 7.18 (1H, s,
(hCA) I and II were evaluated.
thiazole ring), 7.10 (2H, s, NH2); 13C NMR (75 MHz, DMSO-d6,
ppm): 162.9, 151.0, 144.5, 136.3, 134.9, 129.0, 128.1, 127.6,
126.2, 116.5, 103.7; Anal. Calcd.: C15H13N3O2S2: C, 54.36; H,
3.95; N, 12.68; O, 9.66; S, 19.35. Found: C, 54.87; H, 3.85; N,
12.98; O, 9.46; S, 19.05.
Materials and methods
The compounds were prepared from a mixture of sulfanilamide-
substituted thiourea and a-haloacetophenone derivatives in DMF-
Ethanol (1:1 volume) by heating for 5 h at 80 ꢀC, as shown in
Scheme 1. The prepared compounds were characterized by
1H NMR, 13C NMR, IR and elemental analysis.
4–(4–(4-Nitrophenyl)thiazol-2-ylamino)
benzenesulfonamide (5b)
Yield 87% (0.327 g), m.p. 284–86 ꢀC; IR (KBr, ꢀ, cm ꢁ 1): 3326
(NH), 3225 (NH2), 1738 (C ¼ N), 1333 (NO2), 1310 and 1156
General procedure
1
(S¼O); H NMR (300 MHz, DMSO-d6, ppm): 10.70 (1H, –NH),
Melting points were taken on
a Yanagimoto Barnstead
8.30 (2H, d), 8.28 (2H, d), 7.90 (2H, d), 7.82 (2H,d), 7.62 (1H, s,
thiazole ring), 7.18 (2H, s, NH2), 13C NMR (75 MHz, DMSO-d6,
ppm): 161.1, 150.2, 145.5, 139.3, 138.2, 130.8, 127.6, 127.1,
124.5, 116.8, 109.9; Anal. Calcd.: C15H12N4O4S2: C, 47.86; H,
3.21; N, 14.88; O, 17.00; S, 17.04. Found: C, 47.56; H, 3.41; N,
14.55; O, 17.42; S, 17.64.
Electrothermal (Surrey, UK) micromelting point apparatus and
are uncorrected. IR spectra were measured on a SHIMADZU
Prestige-21 (200VCE) (Kyoto, Japan) spectrometer. H- and 13C-
NMR spectra were measured on spectrometer at Varian Infinity
Plus 300 and at 75 Hz (Palo Alto, CA). 1H and 13C chemical shifts
are referenced to the internal deuteranated solvent. The elemental
analysis was carried out with a Leco CHNS-932 (St. Joseph, MI)
instrument. All chemicals were purchased from Merck
(Darmstadt, Germany), Alfa Aesar (Ward Hill, MA) and Sigma-
Aldrich (Taufkirchen, Germany).
1
4–(4–(4-Cyanophenyl)thiazol-2-ylamino)
benzenesulfonamide (5c)
Yield 82% (0.291 g), m.p. 297–98 ꢀC; IR (KBr, ꢀ, cm ꢁ 1): 3300
(NH), 3212 (NH2), 2226 (C\equivN), 1738 (C¼N), 1314 and 1155
(S¼O); 1HNMR (300 MHz, DMSO-d6, ppm): 10.58 (1H, s), 8.10
(2H, d), 7.98 (2H, d), 7.90 (2H, d), 7.79 (2H, ¼d), 7.40 (1H, s,
thiazole ring), 7.10 (2H, s, NH2), 13C NMR (75 MHz, DMSO-d6,
ppm): 162.6, 149.3, 144.3, 138.9, 136.3, 132.8, 127.5, 126.8,
119.3, 118.6, 116.7, 110.6; Anal. Calcd.: C16H12N4O2S2: C,
53.92; H, 3.39; N, 15.72; O, 8.98; S, 17.99. Found: C, 53.58; H,
3.62; N, 15.22; O, 8.65; S, 17.75.
General procedure for the synthesis of sulfanilamide
substituted thiourea
Sulfanilamide (20 mmol) was dissolved in a mixture of conc. HCl
(5 ml) and water (15 ml) by heating. The solution was cooled and
ammonium thiocyanate (26.3 mmol) in 15 ml water was added
slowly, and then the mixture was refluxed for 12 h at 100 ꢀC. Then
the reaction was completed, cooled and filtered off. The product
was crystallized with or through aqueous ethanol.
4–(4–(4-Fluorophenyl)thiazol-2-ylamino)
benzenesulfonamide (5d)
General procedure for the synthesis of sulfonamide-
bearing thiazole derivatives (5a–l)
Yield 92% (0.321 g), m.p. 233–35 ꢀC; IR (KBr, ꢀ, cm ꢁ 1) 3330
(NH), 3298 (NH2), 1738 (C¼N), 1316 and 1148 (S¼O); 1H NMR
(300 MHz, DMSO-d6, ppm): 10.70 (1H, s, NH), 8.0 (2H, m), 7.92
(2H, d) 7.88 (2H, d), 7.4 (1H, s, thiazole ring), 7.22 (2H,t), 7.20
(2H, d, NH2), 13C NMR (75 MHz, DMSO-d6, ppm): 163.0, 160.5,
149.9, 145.5, 132.6, 129.8, 127.6, 116.9, 116.5, 110.2, 102.3;
Anal. Calcd.: C15H12FN3O2S2: C, 51.56; H, 3.46; F, 5.44; N,
12.03; O, 9.16; S, 18.35. Found: C, 51.17; H, 3.25; F, 5.64; N,
12.53; O, 9.55; S, 18.62.
a-Haloacetophenone derivatives (1 mmol) and sulfanilamide-
substituted thiourea (1 mmol) were dissolved in DMF (6 ml) and
6 ml of ethanol was added to the mixture. Then, it was heated for 5
h at 80 ꢀC, after the reaction was completed. The mixture was
cooled to room temperature and poured into ice cold water. It was
then filtered and crystallized through acetone. H NMR and 13C
NMR spectra of 5a, 5h and 5l have been found to be consistent
with the literature5.
1
4–(4–(4-Chlorophenyl)thiazol-2-ylamino)
benzenesulfonamide (5e)
4–(4-Phenylthiazol-2-ylamino)benzene sulfonamide (5a)
Yield 85% (0.281 g), m.p. 243–44 ꢀC; IR (KBr, ꢀ, cm ꢁ 1): 3328
Yield 78% (0.277 g), m.p. 225–26 ꢀC; IR (KBr, ꢀ, cm ꢁ 1): 3320
(NH), 3310 (NH2) 1738 (C¼N), 1316 and 1147 (S¼O);
1
(NH), 3307 (NH2), 1602 (C¼ ) 1310 and 1150 (S¼O); H NMR
(300 MHz, DMSO-d6, ppm): 10.5 (1H, s, –NH), 7.90 (2H, d), 7.80

DOI: 10.3109/14756366.2015.1128426
Sulfonamide-bearing thiazole compounds
3
1H NMR (300 MHz, DMSO-d6, ppm): 10.55 (1H, s, –NH), 7.98 136.5, 132.5, 130.6, 127.7, 122.7, 120.7, 116.8, 107.5; Anal.
(2H, d), 7.95 (2H, d), 7.8 (2H, d), 7.40 (2H,d), 7.18 (1H, s, Calcd.: C15H12N4O4S2: C, 47.86; H, 3.21; N, 14.88; O, 17.00; S,
thiazole ring), 7.05 (2H, s, NH2) 13C NMR (75 MHz, DMSO-d6, 17.04. Found: C, 47.55; H, 3.41; N, 14.63; O, 17.48; S, 17.38.
ppm): 163.1, 149.8, 144.4, 136.4, 133.6, 133.0, 129.0, 127.7,
127.6, 116.6, 105.2; Anal. Calcd.: C15H12ClN3O2S2: C, 49.24; H, 4–(4-(2,4-Dichlorophenyl)thiazol-2-ylamino)
3.31; Cl, 9.69; N, 11.49; O, 8.75; S, 17.53. Found: C, 49.65; H, benzenesulfonamide (5k)
3.71; Cl, 9.38; N, 11.88; O, 8.34; S, 17.13.
Yield 93% (0.375 g), m.p. 200 ꢀC; IR (KBr, ꢀ, cm ꢁ 1): 3388 (NH),
3343 (NH2),1738 (C¼N), 1324 and 1142 (S¼O); 1H NMR
4–(4–(4-Methoxyphenyl)thiazol-2-ylamino)
(300 MHz, DMSO-d6, ppm): 10.68 (1H, s, –NH), 8.0 (1H, d), 7.82
benzenesulfonamide (5f)
(2H, d), 7.80 (2H, d), 7.55 (1H, s), 7.40 (1H, d), 7.38 (1H, s,
Yield 83% (0.299 g), m.p. 213–15 ꢀC; IR (KBr, ꢀ, cm ꢁ 1): 3337 thiazole ring), 7.10 (2H, d, NH2), 13C NMR (75 MHz, DMSO-d6,
(NH), 3300 (NH2), 1738 (C¼N), 1319 and 1146 (S¼O); 1H NMR ppm): 162.6, 162.0, 146.4, 144.4, 136.3, 133.3, 132.7, 132.2,
(300 MHz, DMSO-d6, ppm): 10.65 (1H, s, –NH), 7.95 (2H, d), 130.3, 127.7, 127.5, 116.6, 109.3; Anal. Calcd.:
7.93 (2H, d), 7.8 (2H, -d), 7.27 (2H, s, thiazole ring), 7.20 (2H, s, C15H11Cl2N3O2S2: C, 45.01; H, 2.77; Cl, 17.71; N, 10.50; O,
NH2), 7.0 (2H,d), 3.8(3H, s, OCH3), 13C NMR (75 MHz, DMSO- 7.99; S, 16.02. Found: C, 45.33; H, 2.57; Cl, 17.62; N, 10.33; O,
d6, ppm): 162.8, 159.5, 150.8, 144.5, 136.2, 129.8, 127.8, 127.6, 7.78; S, 16.42.
116.5, 114.3, 101.8, 55.6; Anal. Calcd.: C16H15N3O3S2: C, 53.17;
H, 4.18; N, 11.63; O, 13.28; S, 17.74. Found: C, 53.65; H, 4.52; 4–(4-(3,4-Dihydroxyphenyl)thiazol-2-ylamino)
N, 11.23; O, 13.62; S, 17.34.
benzenesulfonamide (5 l)
Yield 81% (0.294 g), m.p. 214–15 ꢀC; IR (KBr, ꢀ, cm ꢁ 1): 3400
(OH), 3390 (NH), 3340 (NH2), 1739 (C¼N), 1311 and 1152
4–(4-p-Tolylthiazol-2-ylamino)benzenesulfonamide (5 g)
Yield 89% (0.307 g), m.p. 241–42 ꢀC; IR (KBr, ꢀ, cm ꢁ 1): 3318 (S¼O); 1H NMR (300 MHz, DMSO-d6, ppm): 10.65 (1H, s,
(NH), 3300 (NH2), 1738 (C¼N), 1320 and 1150 (S¼O); 1H NMR –NH), 7.90 (2H, d), 7.78 (2H, d), 7.38 (1H, s, thiazole ring), 7.20
(300 MHz, DMSO-d6, ppm):10.50 (1H, s, NH), 7.90 (2H, d), 7.86 (1H, d), 7.10 (2H, s, NH2), 6.80 (1H, d), 13C NMR (75 MHz,
(2H, d), 7.80 (2H, d), 7.22 (2H, d), 7.12 (1H, s, thiazole ring), 7.10 DMSO-d6, ppm): 163.0, 150.3, 146.0, 145.7, 144.3, 136.6, 127.6,
(2H, s, NH2), 2.1 (3H, s, –CH3), 13C NMR (75 MHz, DMSO-d6, 126.1, 117.8, 117.0, 116.2, 114.1, 101.0; Anal. Calcd.:
ppm): 160.5, 149.6, 144.2, 137.5, 129.6, 127.5, 126.2, 118.6, C15H13N3O4S2: C, 49.57; H, 3.61; N, 11.56; O, 17.61; S, 17.65.
116.5, 110.8, 103.83, 21.8; Anal. Calcd.: C16H15N3O2S2: C, Found: C, 49.89; H, 3.42; N, 11.76; O, 17.95; S, 17.98.
55.63; H, 4.38; N, 12.16; O, 9.26; S, 18.56. Found: C, 55.94; H,
4.52; N, 12.28; O, 9.53; S, 18.15.
Preparation of hemolysate and purification from blood
red cells
4–(4–(4-Hydroxyphenyl)thiazol-2-ylamino)
benzenesulfonamide (5 h)
Blood samples (25 ml) were taken from healthy human volun-
teers. They were anticoagulated with acid-citrate-dextrose,
Yield 84% (0.291 g), m.p. 204–05 ꢀC; IR (KBr, ꢀ, cm ꢁ 1): 3394 centrifuged at 1000g for 20 min at 4 ꢀC and the supernatant was
(OH), 3312 (NH), 3225 (NH2), 1738 (C¼N), 1311 and 1150 removed. The packed erythrocytes were washed three times with
(S¼O); 1H NMR (300 MHz, DMSO-d6, ppm): 10.50 (1H, s, NH), 0.9% NaCl and then hemolysed in cold water. The ghosts and any
7.95 (2H, d), 7.90 (2H, d), 7.82 (2H, d), 7.22 (1H, s, thiazole ring), intact cells were removed by centrifugation at 3100g for 25 min at
7.12 (2H, s, NH2), 7.02 (2H, d), 13C NMR (75 MHz, DMSO-d6, 4 ꢀC, and the pH of the hemolysate was adjusted to pH 8.5 with
ppm): 160.4, 158.5, 148.2, 146.3, 129.7, 129.2, 128.5, 125.5, solid Tris-base. The 25 ml hemolysate was applied to an affinity
116.8, 116.2, 101.5; Anal. Calcd.: C15H13N3O3S2: C, 51.86; H, column containing L-tyrosine-sulfonamide-Sepharose-4B29 equi-
3.77; N, 12.10; O, 13.82; S, 18.46. Found: C, 51.45; H, 3.56; N, librated with 25 mMTris–HCl/0.1 M Na2SO4 (pH 8.5). The
12.40; O, 13.41; S, 18.66.
affinity gel was washed with 50 ml of 25 mM Tris–HCl/22 mM
Na2SO4 (pH 8.5). The human CA (hCA) isozymes were then
eluted with 0.1 M NaCl/25 mM Na2HPO4 (pH 6.3) and 0.1 M
CH3COONa/0.5 M NaClO4 (pH 5.6), which recovered hCA-I and
II, respectively. Fractions (3 ml) were collected and their
absorbance was measured at 280 nm.
4–(4-(2-Nitrophenyl)thiazol-2-ylamino)
benzenesulfonamide (5i)
Yield 84% (0.315 g), m.p. 263–64 ꢀC; IR (KBr, ꢀ, cm ꢁ 1): 3360
(NH), 3333 (NH2), 1738 (C¼N), 1333 (NO2), 1316 and 1145
(S¼O); 1H NMR (300 MHz, DMSO-d6, ppm): 10.68 (1H, s,
–NH), 8.79 (1H, d), 8.40 (1H, d), 8.20(1H, d), 7.9 (2H, d), 7.8
(2H, d), 7.72 (1H, –t), 7.70 (1H, s, thiazole ring), 13C NMR
(75 MHz, DMSO-d6, ppm): 163.3, 148.9, 148.5, 144.3, 136.7,
136.5, 132.4, 130.6, 127.6, 122.6, 1207, 116.7, 107.0; Anal.
Calcd.: C15H12N4O4S2: C, 47.86; H, 3.21; N, 14.88; O, 17.00; S,
17.04 Found: C, 47.55; H, 3.41; N, 15.23; O, 17.20; S, 17.34.
Hydratase activity assay
Carbonic anhydrase activity was measured by the Wilbur and
Anderson method, which is based on determination of the time
required for the pH to decrease from 10.0 to 7.4 due to CO2
hydration30. The assay solution was 0.5 M Na2CO3/0.1 M
NaHCO3 (pH 10.0) and Phenol Red was added as the pH
indicator. CO2-hydratase activity [enzyme units (EU)] was
calculated using the equation t0–tc/tc, where t0 and tc are the
times for pH change of the non-enzymatic and the enzymatic
reactions, respectively.
4–(4-(3-Nitrophenyl)thiazol-2-ylamino)
benzenesulfonamide (5j)
Yield 80% (0.300 g), m.p. 259 ꢀC; IR (KBr, ꢀ, cm ꢁ 1): 3360 (NH),
3334 (NH2), 1738 (C¼N), 1333 (NO2), 1316 and 1145(S¼O); 1H
NMR (300 MHz, DMSO-d6, ppm): 10.78 (1H, s, –NH), 8.78 (1H,
Esterase activity assay
s), 8.40 (1H, d), 8.20 (1H, d), 7.98 (2H, d), 7.90 (2H, d), 7.72 (1H, Carbonic anhydrase activity was assayed by following the change
t), 7.70 (1H, s, thiazole ring),7.20 (2H, s, NH2), 13C NMR in absorbance at 348nm of 4-nitrophenyl-acetate (NPA) to
(75 MHz, DMSO-d6, ppm): 163.4, 148.9, 148.5, 144.3, 136.8, 4-nitrophenylate ion over a period of 3 min at 25ꢀC using a

4
S. Kılıcaslan et al.
J Enzyme Inhib Med Chem, Early Online: 1–6
Table 1. IC50 (mM) values of the sulfonamide-bearing thiazole compounds.
Hydratase
hCA I IC50 (mM)
Hydratase
hCA II IC50 (mM)
Esterase
hCA I Ki (mM)
Esterase
hCA II Ki (mM)
5a–l Inhibitor
Inhibition type
5a
5b
5c
5d
5e
5f
5g
5h
5i
0.55
0.35
0.40
0.37
1.34
0.45
0.92
1.22
1.05
0.49
0.59
0.72
0.77
0.35
0.45
0.40
1.46
0.40
0.96
1.56
1.07
0.57
0.75
0.53
0.40
0.33
0.51
0.33
1.23
0.59
1.01
1.21
1.04
0.42
0.48
0.78
0.11
0.33
0.58
0.41
1.44
0.59
0.81
1.99
0.88
0.48
0.72
0.50
Noncompetitive
Noncompetitive
Noncompetitive
Noncompetitive
Noncompetitive
Noncompetitive
Noncompetitive
Noncompetitive
Noncompetitive
Noncompetitive
Noncompetitive
Noncompetitive
5j
5k
5l
spectrophotometer (HACH LANGE DV 6000 UV–VIS) according compounds (5a–l) inhibited the hCA I and II enzyme activity. The
to the method described in the literature31. The enzymatic reaction, inhibition values of the compounds against CAs are summarized
in a total volume of 3.0 ml, contained 1.4ml of 0.05 M Tris–SO4 in Table 1. We have determined the IC50 values of 0.35–11.34 mM
buffer (pH 7.4), 1 ml of 3 mM 4-nitrophenylacetate, 0.5 ml H2O and (Ki ¼ 0.33–1.21 mM) for hCA I and 0.3–1.56 mM (Ki ¼ 0.11–
0.1ml enzyme solution. A reference measurement was obtained by 1.99 mM) for hCA II. Among the compounds, 5b was found to be
preparing the same cuvette without enzyme solution. The inhibitory the most active for CAs (IC50: 0.35 mM for hCA I and hCA II).
effects of the sulfonamide derivatives were examined. All Sulfonamides are coordinated to the zinc (II) ion within the
compounds were tested in triplicate at each concentration level. hCAs active site, whereas their organic scaffolds fill the entire
Different concentrations of the compounds were used.
enzyme cavity, making an extensive series of van der Waals and
polar interactions with amino acid residues both at the bottom,
middle and entrance of the active site cavity33.
In vitro inhibition studies
There are many classes of compounds for CA inhibitors in the
literature: (i) sulfonamides and their isosteres (such as sulfamates,
sulfamides and similar derivatives) and metal complexing anions.
The primary sulfonamides34 (RSO2NH2) in clinical use for more
than 50 years as anti-glaucoma drugs are classical CA inhibitors
(CAI). In addition to the established role of these CAIs as anti-
glaucoma agents, it has been recently reported that they have
potential as anti-convulsant. Sulthiame, topiramate and zonisa-
mide are clinically used antiepileptics, showing potent inhibition
of many CA isozymes present in the brain. (ii) Polyamines35, such
as spermine, spermidine and congeners. (iii) Phenols36, and (iv)
the coumarins and thiocoumarins37, which have an inhibition
For the inhibition studies of sulfonamide, different concentrations
of these compounds were added to the enzyme. Activity percentage
values of CA for different concentrations of each sulfonamide were
determined by regression analysis using Microsoft Office 2000
Excel (Microsoft, Redmond, WA). CA enzyme activity without a
sulfonamide solution was accepted to be 100% activity. Inhibitory
effects of compounds 5a–l on enzyme activities were tested under
in vitro conditions; Ki values were calculated from the Lineweaver–
Burk graphs and are given in Table 128.
Results and discussion
For the evaluation of inhibitory effects of sulfonamides on mechanism not dependent of Zn(II), and bind (in hydrolyzed
physiologically relevant human CA isozymes hCAI and II, several form) in the same active site region as the activators, occluding
sulfonamide-bearing thiazole compounds were subjected to CA the entrance to the active site while the other three CAI groups
inhibition assay with CO2 as substrate.
bind to Zinc(II) ion or Zinc coordinated water molecule/hydroxide
Benzenesulfonamide derivatives 5a–l were prepared as shown ion and the recently reported class of effective CAIs, (v) urea/
in Scheme 1. Sulfanilamide thiourea was prepared from thiourea compounds38,39, (vi) b-lactames40. Sulfonamides have
sulfanilamide and ammoniumthiocyanate in HCl. The proposed important inhibitory effect on the CA enzyme due to their ability
compounds were prepared from the thiourea compound and to gain ionic structure easily and these compounds are very
a-haloacetophenone derivatives in DMF-Ethanol by heating. The important in medicinal chemistry and they constitute an important
1
prepared compounds were characterized by H NMR, 13C NMR, class of drugs used extensively as pharmaceutical and agricultural
IR and elemental analysis. From the 1H NMR spectra, the agents41. The following conclusions should be noted regarding the
hydrogen attached to the nitrogen resonances between 10.50 and CA inhibition data shown in Table 1.
11.00 ppm, sulfanilamide NH2 are seen at around 7.10 ppm and
The derivatives 5a–l investigated here showed inhibitory
the ¼CH proton peak on thiazol ring comes around 7.50 ppm. activity against both two investigated CA isozymes and the
From the 13C NMR spectra, thiazole ring C¼N and ¼C carbon affinity in the micromolar range for both cytosolic isozyme hCA I
atoms are seen at around 160 and 103 ppm, respectively. In the and CA II. The compounds showed IC50 in the range of 0.35–
infrared spectra of compounds 5a–l, it was possible to observe the 1.34 mM and 0.35–1.56 mM against hCA I and CA II, respectively.
absorptions between 3200 and 3400 cm ꢁ 1 relating to NH and Nitro group at the para-position of the phenyl ring exhibited a
NH2 peaks, As can be seen in the literature32, there are two peaks greater inhibitory effect than at the ortho and meta-position.
assigned to S¼O as symmetric and asymmetric stretching. The Flouro-substituted compound showed more inhibitory effect than
peaks of asymmetric and symmetric stretch are appeared around chloro, cyano, methoxy, methyl and unsubstituted compounds.
1300 and 1100 cm ꢁ 1, respectively. All spectra and elemental Flourophenylsulphamate adducts were reported that the sulpho-
analyses support the structure of the synthesized compounds.
mates possess a rather variable binding pattern within the hCA
For evaluating the inhibitory activity of compounds on CA, all active site42,43. It should also be mentioned that among the nitro
synthesized compounds were subjected to CA inhibition assay substituted compounds, o-nitro substituted compound (5i) seems
with CO2 as substrate. The results showed that all the synthesized to be much less effective inhibitors as compared to the meta (5j)

DOI: 10.3109/14756366.2015.1128426
Sulfonamide-bearing thiazole compounds
5
13. Del Prete S, Vullo D, Fisher GM, et al. Discovery of a new family of
carbonic anhysrases in tha malaria pathogen plasmodium falci-
parum-the Z-carbonic anhydrases. Bioorg Med Chem Lett 2014;24:
4389–96.
and para (5b) substituted corresponding compounds. According to
the literature44, this may be due to the sterical impairment of the
ortho-substituent for the binding of the compounds to the Zn(II)
ion within the enzyme active site. Ki values of the compounds for
˘
14. Demirdag R, C¸ omakli V, S¸ entu¨rk M, et al. Purification and
hCA
I
(Ki ¼ 0.33–1.21) are close to the clinically used
characterization of carbonic anhydrase from sheep kidney and
effects of sulfonamides on enzyme activity. Bioorg Med Chem 2013;
21:1522–5.
sulfonamide AZA (acetazolamide, Ki ¼ 0.250 mM) whereas the
values of the compounds for hCA II (Ki ¼ 0.11–1.99 mM) are
higher than AZA (Ki ¼ 0.012 mM)26.
15. Innocenti A, Durdagi S, Doostdar N, et al. Nanoscale enzyme
inhibitors: fullerenes inhibit carbonic anhydrase by occluding the
active site entrance. Bioorg Med Chem 2010;18:2822–8.
16. SitaRam Ceruso M, Khloya P, et al. Functionalized 1,3-diarylpyr-
azoles bearing 6-aminosulfonylbenzothiazole moiety as potent
inhibitors of carbonic anhydrase isoforms hCA I, II, IX and XII.
Bioorg Med Chem 2014;22:6945–52.
17. Ozensoy O, Arslan O, Kockar F. Differential in vitro inhibition
effects of some antibiotics on tumor associated carbonic anhydrase
isozymes of hCA-IX and hCA-XII. J Enzyme Inh Med Chem 2008;
23:579–85.
18. Briganti F, Mangani S, Scozzafava A, et al. Carbonic anhydrase
catalyzes cyanamide hydration to urea: is it mimicking the
physiological reaction? J Biol Inorg Chem 1999;4:528–36.
19. Guerri A, Briganti S, Scozzafava A, et al. Mechanism of cyanamide
hydration catalyzed by carbonic anhydrase II suggested by cryogenic
X-ray diffraction. Biochemistry 2000;39:12391–7.
20. Mustafa G, Khan IU, Ashraf M, et al. Synthesis of new sulfonamides
as lipoxygenase inhibitors. Bioorg Med Chem 2012;20:2535–9.
21. Gadad AK, Mahajanshetti CS, Nimbalkar S, Raichurkar A.
Synthesis and antibacterial activity of some 5-guanylhydrazone/
thiocyanato-6-arylimidazo[2,1-b]-1,3,4-thiadiazole-2-sulfonamide
derivatives. Eur J Med Chem 2000;35:853–67.
In conclusion, we have reported the synthesis, characterization
and evaluation of biological activity of a series of sulfonamide-
bearing thiazole compounds for the inhibition of the physiologi-
cally relevant CA isozymes. They inhibit the CAs with the
inhibition constants of 0.35–1.22 mM for hCA I and 0.35–1.56 mM
for hCA II. Compound 5b behaved as a good inhibitor on hCA I
and hCA II with an activation constant of 0.35 mM. Enzyme
inhibition is a very important issue for drug design and
biochemical applications45–48. The results put forward that new
sulfonamide derivatives inhibited the hCA I and II enzyme
activity. Therefore, our results suggested that the sulfonamide
derivatives may be used for some medical applications and they
may be taken for further evaluation in vivo studies. The inhibition
of the various enzymes in tissues may cause several disorders, but
may also be the opposite. Sometimes, enzyme inhibition may
need for the treatment of some diseases. In this regard, enzyme
inhibition studies are very critical in the new drug development.
Declaration of interest
22. Li JJ, Anderson D, Burton EG, et al. 12-Diarylcyclopentenes as
selective cyclooxygenase-2 inhibitors and orally active antiinflam-
matory agents. J Med Chem 1995;38:4570–8.
23. Yoshino H, Ueda N, Niijma J, et al. Novel sulfonamides as potential,
systemically active antitumor agents. J Med Chem 1992;35:2496–7.
24. Pacchiano F, Carta F, MCDonald PC, et al. Ureido-substituted
benzenesulfonamides potently inhibit carbonic anhydrase IX and
show antimetastatic activity in a model of breast cancer metastasis.
J Med Chem 2011;54:1896–902.
25. Renzi G, Scozzafava A, Supuran CT. Carbonic anhydrase inhibitors:
topical sulfonamide antiglaucoma agents incorporating secondary
amine moieties. Bioorg Med Chem Lett 2000;10:673–6.
26. Alp C, Maresca A, Alp NA, et al. Secondary/tertiary benzenesulfo-
namides with inhibitory action against the cytosolic human carbonic
anhydrase isoforms I and II. J Enzyme Inhib Med Chem 2013;28:
294–8.
27. Supuran CT. Carbonic anhydrases: novel therapeutic applications for
inhibitors and activators. Nat Rev Drug Discov 2008;7:168–81.
28. Fabrizi F, Mincione F, Somma T, et al. A new approach to
antiglaucoma drugs: carbonic anhydrase inhibitors with
or without NO donating moieties. Mechanism of action and
preliminary pharmacology. J Enzyme Inhib Med Chem 2012;27:
138–47.
29. Arslan O, Nalbantoglu B, Demir N, et al. A new method for the
purification of carbonic anhydrase isozymes by affinity chromato-
graphy. Turk J Med Sci 1996;26:163–6.
30. Wilbur KM, Anderson NG. Electrometric and colorimetric determi-
nation of carbonic anhydrase. J Biol Chem 1948;176:147–54.
31. Verpoorte JA, Mehta S, Edsall JT. Esterase activities of human
carbonic anhydrases B and C. J Biol Chem 1967;242:4221–9.
32. Sen E, Alim Z, Duran H, et al. Inhibitory effect of novel pyrazole
carboxamide derivatives on human carbonic anhydrase enzyme.
J Enzyme Inhib Med Chem 2013;28:328–36.
33. Maresca A, Temperini C, Pochet L, et al. Deciphering the
mechanism of carbonic anhydrase inhibition with coumarins and
thiocoumarins. J Med Chem 2010;53:335–44.
34. Supuran CT. Carbonic anhydrase inhibitors. Bioorg Med Chem Lett
2010;20:3467–74.
This work was supported by the Research Fund of the Sakarya
University (project no. 2014–02-04–004) and Balikesir University
Research Project (2015/234).
References
1. Ranga R, Sharma V, Kumar V. New thiazolidinly analogs containing
pyridine ring: synthesis, biological evaluation and QSAR studies.
Med Chem Res 2013;22:1538–48.
2. Marson CM. New and unusual scaffolds in medicinal chemistry.
Chem Soc Rev 2011;40:5514–33.
3. Patt WC, Hamilton HW, Taylor MD, et al. Structure activity
relationships of a series of 2-amino-4-thiazole containing renin
inhibitors. J Med Chem 1992;35:2562–72.
4. Jung KY, Kim SK, Gao ZG, et al. Structure-activity relationships of
thiazole and thiadiazole derivatives as potent and selective human
adenosine A3 receptor antagonists. Bioorg Med Chem 2004;12:
613–23.
5. Heng S, Gryncel KR, Kantrowitz ER. A library of novel allosteric
inhibitors against fructose 1,6-bisphosphatase. Bioorg Med Chem
2009;17:3916–22.
6. Ram S, Celik G, Khloya P, et al. Benenesulfonamide bearing 1,2,4-
triazole scaffolds as potent inhibitors of tumor associated carbonic
anhydrase isoform hCA IX and hCA XII. Bioorg Med Chem 2014;
22:1873–82.
7. Vogt D, Veber J, Ihlefeld K, et al. Desing, synthesis and evaluation
of 2-aminothiazole derivatives as sphingosine kinase inhibitors.
Bioorg Med Chem 2014;22:5354–67.
8. Wei MX, Reng L, Li XQ, et al. Synthesis of new chiral 2,5-
disubstituted 1,3,4-thiadiazoles possessing g-butenolide moiety and
preliminary evaluation of in vitro anticancer activity. Eur J Med
Chem 2009;44:3340–4.
9. Alterio V, Di Fiore A, D’Ambrosio K, et al. Multiple binding modes
of inhibitors to carbonic anhydrases: how to desing specific drugs
targeting 15 different isoforms? Chem Rev 2012;112:4421–68.
10. Supuran CT, Scozzafava A, Casini A. Carbonic anhydrase inhibitors.
Med Res Rev 2003;23:146–89.
11. Supuran CT, Scozzafava A. Applications of carbonic anhydrase
inhibitors and activators in therapy. Exp Opin Ther Patents 2002;12:
217–42.
12. Hewett-Emmett D. Evolution and distribution of the carbonic
anhydrase gene families. In: Chegwidden WR, Edwards Y, Carter N,
eds. The carbonic anhydrase – New horizons. Basel: Birkhauser
Verlag; 2000:29–78.
35. Carta F, Temperini C, Innocenti A, et al. Polyamines inhibit
carbonic anhydrases by anchoring to the zinc-coordinated water
molecule. J Med Chem 2010;53:5511–17.
36. Sarikaya SBO, Topal F, Sentu¨rk M, et al. In vitro inhibition of
a-carbonic anhydrase isozymes by some phenolic compounds.
Bioorg Med Chem Lett 2011;21:4259–68.

6
S. Kılıcaslan et al.
J Enzyme Inhib Med Chem, Early Online: 1–6
37. Maresca A, Temperini C, Vu H, et al. Non-zinc mediated inhibition
of carbonic anhydrases: coumarins are a new class of suicide
inhibitors. J Am Chem Soc 2009;131:3057–62.
activity and selectivity for the inhibition of the tumor-associated
isozymes IX and XII over the cytosolic ones I and II. Bioorg Med
Chem Lett 2009;19:5082–5.
38. Celik F, Arslan M, Yavuz E, et al. Synthesis and carbonic anhydrase 44. Winum JY, Dogne JM, Casini A, et al. Carbonic anhydrase
inhibitory properties of novel 1,4-dihydropyrimidinone substituted
diarylureas. J Enzyme Inhib Med Chem 2014;29:18–22.
39. Demirhan H, Arslan M, Kaya MO, et al. In vitro inhibition of
purified human carbonic anhydrase I and II by novel fluorene 45. Alim Z, Kilinc¸ N, Is¸gor MM, et al. Some anti-inflammatory agents
derivatives. Maced J Chem Chem Eng 2014;33:199–207.
40. Berber N, Arslan M, Nurcan Bilen C¸ , et al. Synthesis and evaluation
inhibitors: synthesis and inhibition of cytosolic membrane-asso-
ciated carbonic anhydrase isozymes I, II, and IX with sulfonamides
incorporating hydrazino moieties. J Med Chem 2005;48:2121–5.
_
¨
inhibit esterase activities of human carbonic anhydrase isoforms I
and II: an in vitro study. Chem Biol Drug Design 2015;86:857–63.
of new phthalazine substituted b-lactam derivatives as carbonic 46. Karatas MO, Alici B, Cakir U, et al. Synthesis and carbonic
anhydrase inhibitors. Russ J Bioorg Chem 2015;41:414–20.
41. Jain P, Saravanan C, Singh SK. Sulphonamides: deserving class as
MMP inhibitors? Eur J Med Chem 2013;60:89–100.
42. Kim CY, Chang JS, Doyon JB, et al. Contribution of fluorine to
protein-ligand affinity in the binding of fluoroaromatic inhibitors to
carbonic anhydrase II. J Am Chem Soc 2000;122:12125–34.
43. Winum JY, Innocenti A, Vullo D, et al. Carbonic anhydrase
inhibitors; fluorinated phenyl sulfamates show strong inhibitory
anhydrase inhibitory properties of novel coumarin derivatives.
J Enzyme Inhib Med Chem 2013;28:299–304.
47. Arslan M, Gencer N, Arslan O, et al. In vitro efficacy of some cattle
drugs on bovine serum paraoxonase 1 (PON1) activity. J Enzyme
Inhib Med Chem 2012;27:722–9.
48. Sayin D, Cakir DT, Gencer N, et al. Effects of some metals on
paraoxonase activity from shark Scyliorhinus canicu. J Enzyme
Inhib Med Chem 2012;27:595–8.