Synthesis, antioxidant and carbonic anhydrase I and II inhibitory activities of novel sulphonamide-substituted coumarylthiazole derivatives

Article abstract of DOI:10.3109/14756366.2015.1077823

New secondary benzenesulphonamide-substituted coumarylthiazole derivatives were synthesized and their inhibitory effects on purified carbonic anhydrase I and II were evaluated using CO₂ as a substrate. The result showed that all the synthesized

Full text of DOI:10.3109/14756366.2015.1077823

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–8
2015 Taylor & Francis. DOI: 10.3109/14756366.2015.1077823
!
RESEARCH ARTICLE
Synthesis, antioxidant and carbonic anhydrase I and II inhibitory
activities of novel sulphonamide-substituted coumarylthiazole
derivatives
1
2
3
3
3
3
Belma Zengin Kurt , Fatih S o¨ nmez , C¸ i g˘ dem Bilen , Adem Ergun , Nahit Gen c¸ er , Oktay Arslan , and
Mustafa Kucukislamoglu
4
1
2
Department of Medicinal Chemistry, Faculty of Pharmacy, Bezmialem Vakif University, Istanbul, Turkey, Pamukova Vocational High School,
3
4
Sakarya University, Sakarya, Turkey, Department of Chemistry, Faculty of Art and Sciences, Balikesir University, Balikesir, Turkey, and Department
of Chemistry, Faculty of Arts and Science, Sakarya University, Sakarya, Turkey
Abstract
New secondary benzenesulphonamide-substituted coumarylthiazole derivatives were synthe- ABTS, antioxidant, carbonic anhydrase,
sized and their inhibitory effects on purified carbonic anhydrase I and II were evaluated using
CO as a substrate. The result showed that all the synthesized compounds exhibited inhibitory
Keywords
coumarin, CUPRAC, sulphonamide
2
activity on both hCA I and hCA II with N-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-yl)naphthalene-2-
sulphonamide (5f, IC50 value of 5.63 and 8.48 mM, against hCA I and hCA II, respectively) as the
strongest inhibitor revealed from this study. Structure–activity relationship revealed that the
inhibitory activity of the synthesized compounds is related to the type of the halogen and bulky
substituent on the phenyl ring. In addition, the cupric reducing antioxidant capacities (CUPRAC)
and ABTS cation radical scavenging abilities of the synthesized compounds were assayed.
History
Received 8 June 2015
Revised 25 July 2015
Accepted 26 July 2015
Published online 24 August 2015
4
-methoxy-N-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-yl)benzenesulphonamide (5e) exhibited the
strongest ABTS and CUPRAC activity with IC50 value of 48.83 mM and A0.50 value of 23.29 mM,
respectively.
Introduction
enzyme, the CA enzyme family continues to capture the attention
of drug discovery scientists and clinicians as the knowledge
regarding the therapeutic implications associated with this
The carbonic anhydrases (CAs; EC 4.2.1.1) are a superfamily
of metalloenzymes, which catalyse the interconversion between
CO2 and HCO ꢀ by using a metal hydroxide nucleophilic
4,13,14
enzyme class continues to grow
.
3
1
–4
The CA inhibitor (CAI) targeting enzymes from mammals,
which belong to the a-CA has been classified as (i) metal ion
binders [inorganic anions; sulphonamides and their isosteres (such
as the sulfamates, sulfamides, N-hydroxy-sulphonamides)]; (ii)
compounds which anchor to the zinc-coordinated water molecule/
hydroxide ion (phenols, polyamines, sulfocoumarins, etc.); (iii)
compounds occluding the entrance of the active site (coumarins
and their isosteres); and (iv) compounds which bind in an
mechanism . These metalloenzymes are known in nature as
five different, genetically distinct families, the a-, b-, g-, d- and
5
–7
f-CAs . Additionally, a new genetic family of CAs, which was
called the Z-CA class, was discovered by Supuran’s group in the
8
last year . The a-, b-, d-CAs use Zn(II) ions at the active site, the
g-CAs are Fe(II) enzymes [but they are active also with bound
Zn(II) or Co(II) ions], while the f-class uses Cd(II) or Zn(II) to
perform the physiologic reaction catalysis . Sixteen different
a-CA isoforms were isolated in mammals, where they play crucial
physiological roles. Some of them are cytosolic (CA I, CA II, CA
III, CA VII, CA XIII), others are membrane-bound (CA IV, CA
IX, CA XII, CA XIV and CA XV), CA VA and CA VB are
mitochondrial, and CA VI is secreted in saliva and milk . The
isozyme CA I is found in many tissues, and a presented study
from Gao et al. demonstrated that this enzyme is involved in
retinal and cerebral edema, and its inhibition may be a valuable
tool for fighting these conditions. The CA II is involved in several
diseases, such as glaucoma, edema, epilepsy, and probably
5
–7
unknown manner (secondary/tertiary sulphonamides, imatinib,
15–22
nilotinib, etc.)
.
Sulphonamides are the best known CAIs and are used for the
treatment of glaucoma in medicinal chemistry. Members of this
class include aromatic, heterocyclic or aliphatic primary sul-
9
,10
23,24
phonamides, but most drugs belong to the heterocyclic class
The coumarin is a common moiety found in many biologically
.
1
1
active natural and therapeutic products and thus represents a very
25–27
important pharmacophore
. Besides the numerous activities of
coumarin compounds, they were recently shown to constitute a
28–30
1
2
novel class of inhibitors of the metalloenzyme CA
.
altitude sickness . Although there are many studies on this
31
In addition, Supuran et al. reported that the hydrolysis of
the sulfocoumarin to the vinyl sulfonic acid is mediated by the
zinc hydroxide nucleophile from the CA active site, as for
the coumarins which are transformed to 2-hydroxycinnamic acids.
Interestingly, this sulfonic acid moiety is not coordinated to the
Address for correspondence: Fatih S o¨ nmez, Sakarya University,
Pamukova Vocational High School, 54900, Sakarya, Turkey. Tel: + 90-
2
64-2953378. Fax: + 90-264-2953679. E-mail: fsonmez@sakarya.edu.tr

2
B. Z. Kurt et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
Figure 1. Design strategy of the reported
compounds.
OH
N
N
O
O
S
SO NH
2
N
2
S
H
O
O
OMe
R
Asetazoleamide (AAZ) derivatives as CAI
modification
Coumarin derivatives as CAI
O
O
S
O
S
O
S
N
H
S
N
N
H
N
R
O
O
R
A
Designed Compounds
Zn(II) ion but it is anchored to the zinc-bound water molecule/ most of the hydrogen bromide, then cooled and filtered. The solid
3
1
hydroxide ion .
was washed with ether and recrystallizated with acetic acid.
In this work, novel sulphonamides substituted coumarylthiazole About 2.60 g product was obtained in 98% yield. Spectral data of
3
3
were synthesized and their antioxidant activities and inhibitory this compound matched with the literature .
effects on the activity of purified human carbonic anhydrase (hCA)
I and II were evaluated. In the present study, our main aim is to General procedure for the synthesis of 3-(2-amino-1,3-
check whether the new secondary sulphonamide derivatives thiazol-4-yl)coumarin (4)
substituted coumarin modified by the addition of thiazole ring
Thiourea (5 mmol) was added to the solution of 3 (5 mmol) in
(A) (Figure 1) may show higher inhibitory effect on CA enzymes
with respect to the compounds previously investigated.
boiling ethanol (20 mL). The mixture was refluxed for 1 h, then
cooled and neutralized with aqueous ammonia. The precipitate
was filtered off, washed with ethanol and used directly without
crystallization or other purification. About 1.098 g product was
obtained in 90% yield. Spectral data of this compound matched
Methods
Chemistry
3
4
with the literature .
Melting points were taken on a Barnstead Electrothermal 9200
(Staffordshire, UK). IR spectra were registered on a Shimadzu
Prestige-21 (200 VCE) spectrometer (Columbia, MD). H and
1
General procedure for the synthesis of sulphonamides
substituted coumarylthiazole (5a–j)
1
3
C-NMR spectra were registered on a Varian Infinity Plus
13
1
spectrometer at 300 and at 75 Hz, respectively. H and
C
To a solution of 4 (1 mmol) in dry pyridine was added
chemical shifts are referenced to the internal deuterated solvent. sulfonylchloride derivatives (1 mmol). The mixture was refluxed
The elemental analyses were carried out with a Leco CHNS-932 for 12 h with stirring, than cooled and added to ice water and 10%
(St. Joseph, MI) instrument. Spectrophotometric analyses were HCl. The product was filtered off, washed with water and dried
performed by a BioTek Power Wave XS (BioTek, Winooski, VT). under vacuum. The products were recrystallizated from ethanol
Sepharose 4B, L-tyrosine, sulphonamide, synthetic starting mater- over 98% purity. Compounds 5a–j were obtained with 68–82%
ial, reagents and solvents were purchased from Merck, Alfa Easer, yields.
Sigma-Aldrich and Fluka.
4-Methyl-N-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-yl)benzenesul-
phonamide (5a)
General procedure for the synthesis of
-acetylcoumarin (2)
3
ꢁ
Yellow powder, 78% yield (310 mg), m.p.: 235–237 C; IR: 3366
A mixture of benzaldehyde (3 mmol), reactive methylene com- 3287, 3042, 1712, 1634, 1599, 1522, 1365, 1271, 1134, 1081,
ꢀ
1 1
pound (3 mmol) and L-proline (10 mol%) was heated under neat 765 cm ; H-NMR (DMSO-d , 300 MHz) d/ppm: 2.51 (3H, s),
6
conditions for 0.5 h. The reaction was monitored by thin-layer 7.37–7.48 (4H, m), 7.57 (1H, s), 7.67 (2H, t, J ¼ 8.8 Hz), 7.77
1
3
chromatography (TLC). After completion of reaction, the reaction (2H, t, J ¼ 7.9 Hz), 8.63 (1H, s); C NMR (DMSO-d , 75 MHz)
6
mixture was cooled and recrystallized from ethanol to get pure d/ppm: 21.6, 109.5, 116.7, 119.1, 119.4, 125.7, 126.7, 129.5,
crystalline 3-acetylcoumarin (2) in 92% yield (0.52 g). Spectral 130.2, 133.0, 133.3, 139.7, 139.8, 143.3, 153.0, 158.9, 169.5.
3
data of this compound matched with the literature .
2
Anal. Calcd. for C H N O S : C, 57.27; H, 3.54; N, 7.03;
19 14 2 4 2
found: C, 57.24; H, 3.50; N, 7.10.
General procedure for the synthesis of 3-(bromoacetyl)-
coumarin (3)
2,5-Dimethyl-N-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-yl)benzene-
sulphonamide (5b)
To a solution of 2 (0.01 mol) in 20 mL chloroform was added
0.01 mol of bromine in 5 mL chloroform, with intermittent Orange powder, 82% yield (338 mg), m.p.: 160–162 C; IR: 3328,
shaking and warming to decompose an addition product. The 3124, 2965, 1710, 1607, 1528, 1361, 1292, 1131, 1060, 794,
1 1
mixture was heated for fifteen minutes on a water-bath to expel 593 cm ; H-NMR (DMSO-d , 300 MHz) d/ppm: 2.35 (3H, s),
ꢁ
ꢀ
6

DOI: 10.3109/14756366.2015.1077823
Novel sulphonamide-substituted coumarylthiazole derivatives
3
2
.58 (3H, s), 7.24–7.38 (2H, m), 7.43 (2H, t, J ¼ 7.9 Hz), 7.64 (DMSO-d , 75 MHz) d/ppm: 111.3, 113.4, 113.7, 116.2, 116.7,
6
1
3
(1H, s), 7.69 (2H, t, J ¼ 7.9 Hz), 7.77 (1H, s), 8.48 (1H, s); C- 119.0, 120.0, 120.3, 122.8, 125.7, 129.5, 132.2, 132.3, 133.3,
NMR (DMSO-d , 75 MHz) d/ppm: 20.2, 21.1, 111.0, 116.7, 139.7, 144.7, 144.8, 153.0, 158.3, 160.7, 163.9, 168.9. Anal.
6
1
1
19.0, 125.7, 126.2, 128.8, 129.5, 132.9, 133.3, 133.4, 134.0, Calcd. for C H FN O S : C, 53.72; H, 2.76; N, 6.96; found: C,
35.8, 139.7, 140.1, 142.9, 153.0, 158.3, 168.1. Anal. Calcd. for 53.74; H, 2.75; N, 6.94.
1
8
11
2
4
2
C H N O S : C, 58.24; H, 3.91; N, 6.79; found: C, 58.28; H,
2
0 16 2 4 2
3
.95; N, 6.75.
4-Chloro-N-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-yl)benzenesul-
phonamide (5h)
4
-Isopropyl-N-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-yl)benzene-
sulphonamide (5c)
ꢁ
Yellow powder, 82% yield (342 mg), m.p.: 264–266 C; IR: 3145,
3
9
043, 1720, 1567, 1526, 1440, 1362, 1306, 1267, 1139, 1086,
ꢀ1
ꢁ
1
Orange powder, 80% yield (340 mg), m.p.: 182–184 C; IR: 3276,
28, 752 cm ; H-NMR (DMSO-d , 300 MHz) d/ppm: 7.39–7.51
6
3
9
142, 2960, 1712, 1606, 1524, 1454, 1360, 1306, 1148, 1090,
ꢀ
(
3H, m), 7.64–7.70 (4H, m), 7.85 (2H, d, J ¼ 8.2 Hz), 8.48 (1H, s);
1 1
38, 755, 569 cm ; H-NMR (DMSO-d , 300 MHz) d/ppm: 1.21
1
3
6
C-NMR (DMSO-d , 75 MHz) d/ppm: 111.3, 116.8, 119.0,
6
(
(
(
6H, d, J ¼ 5.8 Hz), 2.93–2.99 (1H, m), 7.37–7.51 (4H, m), 7.63
1
1
25.8, 128.5, 129.9, 133.4, 137.8, 139.8, 140.2, 141.7, 144.7,
53.1, 158.4, 162.7, 168.4. Anal. Calcd. for C H ClN O S : C,
51.61; H, 2.65; N, 6.69; found: C, 51.63; H, 2.62; N, 6.67.
1H, s), 7.69 (2H, d, J ¼ 7.6 Hz), 7.79 (2H, d, J ¼ 8.5 Hz), 8.46
1
8
11
2 4 2
1
3
1H, s); C-NMR (DMSO-d , 75 MHz) d/ppm: 24.1, 34.0, 111.2,
6
1
1
16.7, 119.9, 125.7, 126.8, 127.6, 129.5, 132.1, 133.3, 136.8,
39.6, 140.0, 143.8, 153.0, 158.4, 168.1. Anal. Calcd. for
4-Bromo-N-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-yl)benzenesul-
phonamide (5i)
C H N O S : C, 59.14; H, 4.25; N, 6.57; found: C, 59.10; H,
2
1 18 2 4 2
4
.28; N, 6.55.
ꢁ
Reddish powder, 76% yield (351 mg), m.p.: 266–268 C; IR:
3
1083, 936, 759, 553 cm
275, 3043, 1726, 1605, 1529, 1441, 1366, 1307, 1269, 1139,
1
4-(Tert-butyl)-N-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-yl)benze-
nesulphonamide (5d)
ꢀ
1
;
H-NMR (DMSO-d , 300 MHz)
6
d/ppm: 7.35–7.45 (4H, m), 7.50–7.76 (5H, m), 8.45 (1H, s);
ꢁ
Orange powder, 72% yield (316 mg), m.p.: 250–252 C; IR: 3368, 13
C-NMR (DMSO-d , 75 MHz) d/ppm: 111.2, 116.2, 116.8,
6
2
5
7
8
1
1
961, 1716, 1518, 1442, 1361, 1287, 1145, 1106, 1084, 755,
78 cm ; H-NMR (DMSO-d , 300 MHz) d/ppm: 1.28 (9H, s),
1
1
4
19.0, 122.7, 125.7, 128.6, 129.6, 132.8, 133.4, 139.8, 141.0,
53.1, 158.3, 162.4, 168.6. Anal. Calcd. for C H BrN O S : C,
6.66; H, 2.39; N, 6.05; found: C, 46.64; H, 2.35; N, 6.08.
ꢀ1 1
6
1
8
11
2 4 2
.34–7.51 (3H, m), 7.58–7.69 (4H, m), 7.79 (2H, d, J ¼ 8.7 Hz ),
1
3
.46 (1H, s); C-NMR (DMSO-d , 75 MHz) d/ppm: 31.4, 35.4,
6
09.4, 111.2, 116.4, 116.7, 119.0, 125.3, 125.7, 126.5, 126.8,
27.7, 129.5, 133.3, 139.6, 153.0, 158.4, 168.1. Anal. Calcd. for
C H N O S : C, 59.98; H, 4.58; N, 6.36; found: C, 59.95; H,
2
4-Iodo-N-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-yl)benzenesulpho-
namide (5j)
2 20 2 4 2
ꢁ
4
.56; N, 6.38.
Dark yellow powder, 68% yield (347 mg), m.p.: 244–246 C; IR:
3
1086, 941, 759, 554 cm
382, 3154, 3042, 1701, 1605, 1532, 1438, 1361, 1265, 1140,
1
ꢀ
1
4-Methoxy-N-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-yl)benzene-
sulphonamide (5e)
;
H-NMR (DMSO-d , 300 MHz)
6
d/ppm: 7.36–7.45 (2H, m), 7.58–7.67 (5H, m), 7.94 (2H, d,
13
J ¼ 7.6 Hz), 8.46 (1H, s); C-NMR (DMSO-d , 75 MHz) d/ppm:
ꢁ
6
Yellow powder, 75% yield (310 mg), m.p.: 259–261 C; IR: 3333,
1
11.2, 116.8, 119.0, 125.7, 128.3, 129.6, 133.4, 137.5, 138.6,
3
9
117, 2943, 1712, 1521, 1438, 1365, 1304, 1261, 1140, 1088,
ꢀ
1
1
139.8, 142.2, 147.4, 153.1, 158.4, 161.1, 168.6. Anal. Calcd. for
C H IN O S : C, 42.36; H, 2.17; N, 5.49; found: C, 42.39; H,
2.15; N, 5.47.
27, 749, 556 cm ; H-NMR (DMSO-d , 300 MHz) d/ppm: 3.82
6
1
8
11
2 4 2
(
(
(
3H, s), 7.10 (2H, d, J ¼ 7.9 Hz), 7.37–7.46 (3H, m), 7.64–7.69
1
3
2H, m), 7.81 (2H, d, J ¼ 8.2 Hz), 8.46 (1H, s); C-NMR
DMSO-d , 75 MHz) d/ppm: 56.3, 109.4, 111.3, 114.9, 116.7,
6
Preparation and purification of haemolysate from blood
red cells
1
1
3
19.1, 125.4, 125.7, 128.8, 129.5, 133.2, 134.1, 139.6, 153.0,
58.4, 162.8, 168.1. Anal. Calcd. for C H N O S : C, 55.06; H,
.40; N, 6.76; found: C, 55.09; H, 3.45; N, 6.75.
1
9 14 2 5 2
Preparation and purification of haemolysate from blood red cells
3
5
made by the literature . Blood samples (25 mL) were taken from
healthy human volunteers. They were anticoagulated with acid–
N-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-yl)naphthalene-2-sulpho-
namide (5f)
ꢁ
citrate–dextrose, centrifuged at 2000 g for 20 min at 4 C and the
ꢁ
supernatant was removed. The packed erythrocytes were washed
three times with 0.9% NaCl and then haemolysed in cold water.
The ghosts and any intact cells were removed by centrifugation at
Cream powder, 70% yield (304 mg), m.p.: 251–253 C; IR: 3292,
3
6
122, 3049, 1709, 1599, 1441, 1367, 1301, 1115, 1078, 922, 752,
69 cm ; H-NMR (DMSO-d , 300 MHz) d/ppm: 7.39 (2H, t,
ꢀ1 1
6
ꢁ
2
000 g for 25 min at 4 C, and the pH of the haemolysate was
J ¼ 8.4 Hz), 7.62–7.64 (5H, m), 7.84 (1H, d, J ¼ 8.7 Hz), 8.00
1H, d, J ¼ 7.9 Hz), 8.09 (1H, d, J ¼ 8.7 Hz), 8.17 (1H, d,
adjusted to pH 8.5 with solid Tris-base. The 25 mL haemolysate
was applied to an affinity column containing L-tyrosine-sul-
phonamide-Sepharose-4B equilibrated with 25 mM Tris–HCl/
(
1
3
J ¼ 6.7 Hz), 8.42 (1H, s), 8.52 (1H, s); C-NMR (DMSO-d ,
6
7
1
1
6
5 MHz) d/ppm: 111.3, 116.7, 119.0, 122.8, 125.7, 127.1, 128.2,
28.4, 129.3, 129.5, 129.9, 130.0, 132.3, 133.3, 134.8, 139.3,
39.7, 153.0, 158.4, 164.8. Anal. Calcd. for C H N O S : C,
0
of 25 mM Tris–HCl/22 mM Na SO (pH 8.5). The human CA
.1 M Na SO (pH 8.5). The affinity gel was washed with 50 mL
2 4
2
4
2
2 14 2 4 2
(
hCA) isozymes were eluted with 0.1 M NaCl/25 mM Na HPO4
0.82; H, 3.25; N, 6.45; found: C, 60.84; H, 3.28; N, 6.40.
2
(
pH 6.3) and 0.1 M CH COONa/0.5 M NaClO (pH 5.6), which
3
4
recovered hCA-I and hCA-II, respectively. Fractions of 3 mL were
collected and their absorbance measured at 280 nm.
4-fluoro-N-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-yl)benzenesul-
phonamide (5g)
ꢁ
Reddish powder, 74% yield (297 mg), m.p.: 248–250 C; IR:
CA enzyme assay
3
9
7
299, 3123, 3073, 1702, 1608, 1524, 1432, 1366, 1292, 1069,
ꢀ
1
;
1
21, 753, 582 cm
.38–7.55 (3H, m), 7.60–7.74 (6H, m), 8.49 (1H, s); C-NMR on determination of the time required for the pH to decrease from
H-NMR (DMSO-d , 300 MHz) d/ppm: CA activity was measured by the Maren method which is based
6
1
3

4
1
B. Z. Kurt et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
3
0.0 to 7.4 due to CO hydration . The assay solution was 0.5 M Different volumes (1000 mg/L and 54.5 mL) of the sample were
6
2
Na CO /0.1 M NaHCO (pH 10.0) and Phenol Red was added as added to a solution prepared by adding 61.0 mL of 10 mM CuCl2,
2
3
3
the pH indicator. CO -hydratase activity [enzyme units (EU)] was 61.0 mL 7.5 mM neocuproine and 61.0 mL of 1.0 mM
2
calculated by using the equation t ꢀt /t where t and t are the NH CH COO buffer (pH 7), respectively. The absorbance was
0
c
c
0
c
4
3
times for pH change of the non-enzymatic and the enzymatic measured at room temperature at 450 nm, after an hour. The
reactions, respectively.
results were calculated as A0.50. Propyl alcohol was used as a
solvent to controls.
In vitro inhibition studies
Results and discussion
For the inhibition studies of sulphonamides, five different
concentrations of these compounds were added to the medium
in 4.2 mL of total reaction volume including the enzyme solution.
Duration (in seconds) of the colour change from red to yellow in
solution was measured in a 10-ml glass tube with 1 cm diameter.
CA enzyme activity without a compound solution was accepted as
2
3
Sulphonamide derivatives are known as the strongest CAIs . The
asetazolamide (AAZ) derivatives have also been reported as
4
potent CAI . Our design strategy is that the 1,3,4-thidiazole ring
of AAZ was modified to thiazole ring and was added to
sulphonamides in order to increase inhibitory activity by forming
hydrogen bond. On the other hand, we think that the presence of
coumarin moiety contributes to inhibitor activity by hydrolysing
to 2-hydroxycinnamic acids or interacting with the enzyme active
sites (Figure 1).
The synthetic procedures employed to obtain the target
compounds 5a–j are depicted in Scheme 1. Compound 4 was
synthesized from salicylaldehydes in three steps according to the
literature , and then it was reacted with various benzenesulfonyl
choloride derivatives in pyridine to get product sulphonamides
substituted coumarylthiazole (5a–j).
All the new compounds were characterized by H-NMR, C-
NMR, IR and elemental analysis. In the infrared spectra of the
synthesized compounds, it was possible to observe the absorp-
1
00% activity. All compounds were tested in triplicate at each
concentration used. Activity % values of CA for different
concentrations of each compound were determined by regression
analysis using Microsoft Office 2000 Excel. For the compounds
having an inhibition affect, the inhibitor concentration causing up
to 50% inhibition (IC50 values) was determined from the graphs.
3
9
Antioxidant activity assays
In CUPRAC assay, the absorbance values were used to calculate
for A0.50, but in ABTS assay, inhibition (%) values were used to
calculate for IC .
1
13
5
0
ꢀ
1
ABTS cation radical decolourization assay
tions ꢃ3300 cm relating to NH stretch of sulphonamide groups,
ꢀ
1
ꢂ+
ꢃ1520 cm relating to C¼N stretch for thiazole, absorptions in
ABTS scavenging activities of the synthesized compounds were
ꢀ
1
3
determined as previously reported . The solution of ABTS
7
ꢂ+ ꢃ1710 cm from coumarin carbonyl moiety stretch and absorp-
ꢀ
2
1
0
tions ꢃ1360 cm relating to SO
antisym stretch in sulphona-
radical was generated by dissolving 19.2 mg of 2,2 -azino-bis(3-
1
mides. From the H-NMR spectra, the resonance due to the
hydrogen attached to the sulphonamide nitrogen was not detected.
The signals for aromatic hydrogens were observed between 7.10
and 8.17 ppm, the signal of proton at thiazole ring was detected at
ꢃ8.48 ppm. From the C-NMR spectra, the signals can be seen
ꢃ158 and 168 ppm relating to coumarin carbonyl and thiazole
ring, respectively.
For evaluation, the physiologically relevant human CA
isozyme hCA I and II inhibitory activity, all the synthesized
compounds were subjected to CA inhibition assay with CO as a
ethylbenzothiazoline-6-sulphonic acid) (7 mM ABTS) and 3.3 mg
K S O in distilled water (5 mL). This solution was kept in dark
2
2 3
for 24 h at room temperature, and the absorbance of the solution
was fixed to ꢃ0.70 at 734 nm by dilution. The solutions of the
samples were prepared in n-propanol at a concentration of
1
3
1
000 mg/mL. The absorbance was measured at room temperature
ꢂ+
at 734 nm, after 6 min from ABTS addition. The decrease in the
absorption was used to calculate the activities. The results were
expressed as IC . Propyl alcohol was used as a control solvent.
5
0
2
substrate.
The result showed that all synthesized compounds (5a–j)
inhibited the hCA I and II enzyme activity. The IC50 values for
Cupric reducing antioxidant capacity assay (CUPRAC)
Cupric reducing antioxidant capacities of the synthesized com-
pounds were determined in accordance with the literature hCA I and II inhibitions are summarized in Table 1. The IC50
3
8
method . The solutions of compounds and standards were values were between 5.63 and 22.63 mM for hCA I and between
prepared in n-propanol at a concentration of 1000 mg/mL.
8
.48 and 23.87 mM for hCA II inhibitory activity. Among
O
CHO
COMe
COMe
Br
L-Proline
Br2
OH
COOEt
Solvent free
CHCI3
reflux, 15min.
O
O
80-90°C
O
O
1
2
3
O
O
S
O
S
N
S
S
S
R
R
Cl
NH2
N
H
O
H N
NH2
N
2
pyridine, 60°C
EtOH, reflux, 1h
O
O
O
O
4
5a-j
Scheme 1. Synthesis of new sulphonamides substituted coumarylthiazole (5a–j) derivatives.

DOI: 10.3109/14756366.2015.1077823
Novel sulphonamide-substituted coumarylthiazole derivatives
5
Table 1. In vitro inhibition IC50 and A0.50 values (mM) of sulphonamides substituted coumarylthiazole (5a–j) for
hCA I and hCA II and antioxidant activities.
O
O
S
N
S
R
N
H
O
O
5a-j
ꢂ+
hCA I
IC50 (lM)
hCA II
IC50 (lM)
ABTS
IC50 (mM)*
CUPRAC
Compound
R
A
0.50 (mM)y
61.36 ± 0.31
5
a
22.63
23.87
70.95 ± 2.51
5
b
8.64
6.46
11.68
54.77 ± 2.87
116.62 ± 1.56
40.26 ± 0.40
43.70 ± 0.32
5
5
5
c
d
e
11.12
10.91
18.00
6.21
104.77 ± 2.08
48.83 ± 1.38
49.71 ± 0.30
23.29 ± 0.02
10.90
H CO
3
5
f
5.63
8.48
64.42 ± 3.06
50.45 ± 2.98
80.62 ± 0.33
81.19 ± 0.40
F
5
g
16.21
18.30
Cl
Br
I
5
5
5
h
9.55
7.67
5.88
15.72
11.35
8.69
60.40 ± 1.11
81.29 ± 1.38
87.65 ± 1.12
71.06 ± 0.34
49.58 ± 0.30
41.96 ± 0.39
i
j
Quercetinz
15.49 ± 2.33
18.47 ± 0.04
*
IC50 values represent the mean ± standard error of mean of three parallel measurements (p50.05).
yA0.50 values represent the mean ± standard error of mean of three parallel measurements (p50.05).
zStandard.

6
B. Z. Kurt et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
OH
O
OH
synthesized compounds, 5f (IC50 ¼ 5.63 and 8.48 mM, for hCA I
and hCA II, respectively) showed the highest inhibitory activity
against hCA I and II. Most of the primary sulphonamide
compounds are mentioned very potent inhibitor of the cytosolic
CA
COO−
O
O
O
OH
rt, pH 7.4
4-6h
6
isoform hCAs . All the synthesized sulphonamides in this study
A
A1
B1
are moderate inhibitory activity for the hCAs, because they are
secondary sulphonamides and contain big bulky groups. Up to
now, many sulphonamide and coumarin derivatives have been
CA
2
8–30,40–45
COO−
synthesized as strong CAIs by Supuran’s group
. The
O
O
rt, pH 7.4
-6h
OH
4
inhibition values of them ranged from low micromolar to
nanomolar. These compounds have stronger inhibitory effect
than the sulphonamides substituted coumarylthiazole in this study.
But, 5f (IC50 ¼ 5.63 and 8.48 mM, for hCA I and hCA II,
respectively) showed higher inhibitory properties against hCA I
and II compared to some sulphonamide and coumarin derivatives,
B
Figure 2. Formation of 2-hydroxy-cinnamic acids A1 and B1 by the CA-
28,41,42
mediated hydrolysis of coumarin A and B
.
4
6–51
reported in our previously study
between 6.79 and 620 mM, for hCA I; between 6.54 and 51.45 mM, So we suppose that the bulky substituents (such as tert-butyl,
for hCA II.
naphthalene and iodine), increasing inhibitory activity, could
, with an IC50 value of
The following structure–activity relationship (SAR) observa- locate at the entrance of the enzyme active site. We also think that
tions can be drawn from data of Table 1: (i) the best inhibitor the nitrogen of the thiazole ring of the synthesized compounds
among the newly synthesized and investigated compounds was contribute to inhibition by forming hydrogen bonds.
naphthalene substituted derivative (5f) for hCA I and II; (ii) the
alkyl series at the phenyl ring showed a qualitative relationship
Antioxidant activity assay
between increasing inhibitory activity and bulky group for hCA I
and II [5d (R ¼ 4-tert-butyl-benzene, IC ¼ 6.21 and 10.91 mM It is known that many natural and synthetic coumarin, sulphona-
5
0
for hCA I and II, respectively)45c (R ¼ 4-isopropyl-benzene, mide and thiazole derivatives have various pharmacological
IC ¼ 6.46 and 11.12 mM for hCA I and II, respectively)45b properties. Among these properties, their antioxidant effects
5
0
5
2–54
53
. Also, Gocer et al. have recently reported
(
R ¼ 2,5-dimethyl-benzene, IC ¼ 8.64 and 11.68 mM for hCA I were examined
5
0
and II, respectively)45a (R ¼ 4-methyl-benzene, IC ¼ 22.63 that some sulphonamides demonstrated effective antioxidant
5
0
2
+
ꢂ+
reducing capabilities and ABTS ). In
and 23.87 mM for hCA I and II, respectively)]; and (iii) the properties (for Cu
inhibitory activity on both hCA I and II seems to be strongly addition, Supuran’s group investigated the the interaction of CA
dependent on the size and polarizability of the halogen substituent isozymes with some antioxidant compounds, such as resveratrol,
at the para-position of the phenyl ring [for size and polarizability, dobutamine, curcumin, catechin, silymarin, salicyclates and
I4Br4Cl4F, for inhibitory activity, 5j (R ¼ 4-iodobenzene, quercetin. It was reported that all these antioxidant compounds
5
5–58
IC ¼ 5.88 and 8.69 mM for hCA I and II, respectively)45i showed effective hCA I and II inhibitory activity
. In view of
5
0
(
R ¼ 4-bromobenzene, IC ¼ 7.67 and 11.35 mM for hCA I and these findings, it was considered that the synthesized compounds
50
5
0
II, respectively)45h (R ¼ 4-cholorobenzene, IC ¼ 9.55 and might possess certain antioxidant activity due to include sul-
1
5.72 mM for hCA I and II, respectively)45g (R ¼ 4-fluoroben- phonamide, coumarin and thiazole moiety containing sulfonyl,
zene, IC ¼ 16.21 and 18.30 mM for hCA I and II, respectively)]. carbonyl, imine and methoxy groups.
5
0
According to SAR study, it is clear that the bulky substituents The ABTS method is based on the ability of hydrogen or
such as tert-butyl, naphthalene and iodine) increase inhibitory electron-donating antioxidants to decolourize the performed
activity of the compound due to steric effect. The sulphonamide radical monocation of 2,2 -azino-bis(3-ethylbenzthiazoline-6-sul-
drug binds in deprotonated form to the catalytically critical Zn(II) fonic acid) generated due to oxidation of ABTS with potassium
(
0
3
7
ion, also participating in extensive hydrogen bond and van der persulfate . The results in Table 1 indicated that all synthesized
Waals interactions with amino acid residues both in the hydro- compounds exhibited moderate radical scavenging ability. 5e and
phobic and hydrophilic halves of the enzyme active site, as shown 5g exhibited the strongest ABTS activity with IC50 values of
by X-ray crystallographic studies of enzyme–inhibitor com- 48.83 and 50.45 mM, respectively. These potencies of 5e and 5g
6
plexes . The inhibition mechanism of coumarins is different was 3.3-fold less than that of quercetin (IC50 ¼ 15.49 mM) used as
compared to that of the classical CAIs of the sulphonamide the reference compound.
4
type . Several kinetic and X-ray crystallographic studies have
0
The halogen substituent at the para-position of the phenyl ring
revealed that coumarins are mechanism-based inhibitors, which showed an inverse relationship for increasing ABTS activity with
undergo hydrolysis under the influence of the zinc hydroxide, growing size and polarizability [for size and polarizability,
nucleophilically active species of the enzyme, with generation of I4Br4Cl4F; for ABTS activity, 5j (R ¼ 4-iodobenzene,
2
8,41,42
substituted-2-hydroxycinnamic acids (Figure 2)
. Supuran’s IC50 ¼ 87.65 mM)55i
(R ¼ 4-bromobenzene,
IC
¼
50
group reported that inhibitor (as for coumarin/sulfocoumarin) and 81.29 mM)55h (R ¼ 4-cholorobenzene, IC ¼ 60.40 mM)55g
5
0
enzyme solutions were pre-incubated together for ꢃ6 h prior to (R ¼ 4-fluorobenzene, IC ¼ 50.45 mM)].
5
0
assay in order to allow for the formation of the EꢀI complex or
CUPRAC assays have a distinct advantage over other electron-
3
1
for the eventual active site mediated hydrolysis of the inhibitor . transfer based assays (e.g. Folin, FRAP, ABTS, DPPH). This
On the other hand, the two endocyclic nitrogen of the 1,3,4- advantage is its realistic pH close to that physiological, favourable
thidiazole ring of AAZ derivatives participates in two hydrogen redox potential, accessibility and stability of reagents and
4
bonds with the OH of Thr200 .
Based on the above consideration, we consider that the ones . The cupric reducing antioxidant capacities of the
applicability to lipophilic antioxidants as well as hydrophilic
3
8
coumarin ring should not undergo ring opening without pre- synthesized compounds (5a–j) were determined according to
3
8
incubation on enzyme and inhibitor. Also it is difficult that the the literature method using quercetin as the reference com-
sulphonamide moiety of the synthesized compounds could bind to pound. Among the synthesized compounds, only 5e
zinc cation in traditional manner due to contain big bulky groups. (A0.50 ¼ 23.29 mM) showed close cupric reducing antioxidant

DOI: 10.3109/14756366.2015.1077823
Novel sulphonamide-substituted coumarylthiazole derivatives
7
activity to quercetin (A0.50 ¼ 18.47 mM). The others have less the 14. Krall N, Pretto F, Decurtins W, et al. A small-molecule drug
tumors. Angew Chem Int Ed 2014;53;4231–5.
conjugate for the treatment of carbonic anhydrase IX expressing
cupric reducing antioxidant capacity than quercetin.
Interestingly, in contrast ABTS activity, the CUPRAC activity
1
5. Congiu C, Onnis V, Balboni G, Supuran CT. Synthesis and carbonic
anhydrase I, II, IX and XII inhibition studies of 4-N,N-disubstituted
sulfanilamides incorporating 4,4,4-trifluoro-3-oxo-but-1-enyl, phe-
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Chem Lett 2014;24:1776–9.
seems to be strongly dependent on the increasing size and
polarizability of the halogen substituent at the para-position
of the phenyl ring [for size and polarizability, I4Br4Cl4F;
for CUPRAC activity, 5j (R ¼ 4-iodobenzene, A0.50
¼
4
1.96 mM)45i (R ¼ 4-bromobenzene,
R ¼ 4-cholorobenzene,
benzene, A ¼ 81.19 mM)].
A
¼ 49.58 mM)45h 16. Durdagi S, Senturk M, Ekinci D, et al. Kinetic and docking studies
0
.50
of phenol-based inhibitors of carbonic anhydrase isoforms I, II, IX
and XII evidence a new binding mode within the enzyme active site.
Bioorg Med Chem 2011;19:1381–9.
(
A
¼ 71.06 mM)45g (R ¼ 4-fluoro-
0
.50
0
.50
1
1
7. De Simone G, Alterio V, Supuran CT. Exploiting the hydrophobic
and hydrophilic binding sites for designing carbonic anhydrase
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Conclusions
A series of new secondary benzenesulphonamide substituted
coumarylthiazole derivatives (5a–j) was synthesized and their
activities as hCA I and hCA II inhibitors and structure–activity 19. Aggarwal M, Kondeti B, McKenna R. Anticonvulsant/antiepileptic
relationship were examined. All synthesized compounds inhibited
hCA I and II. 5f exhibited the strongest inhibition against hCA I
and II with IC50 value of 5.63 and 848 mM, respectively. The
SARs revealed that the inhibitory activity of the synthesized
compounds could also be affected by the type of the halogen
substituent on the phenyl ring. Also the bulky substituents (such
as tert-butyl, naphthalene and iodine) increase inhibitory activity
of the compounds due to steric effect. Additionaly, the
synthesized compounds showed moderate antioxidant activity.
carbonic anhydrase inhibitors: a patent review. Expert Opin Ther Pat
013;23:717–24.
0. Liu F, Martin-Mingot A, Lecornu e´ F, et al. Carbonic Anhydrases
inhibitory effects of new benzenesulfonamides synthesized by
using superacid chemistry. J Enzyme Inhib Med Chem 2012;27:
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2
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86–91.
1. Metayer B, Mingot A, Vullo D, et al. New superacid synthesized
(fluorinated) tertiary benzenesulfonamides acting as selective hCA
IX inhibitors: toward a new mode of carbonic anhydrase inhibition
by sulphonamides. Chem Commun 2013;49:6015–17.
2. Metayer B, Martin-Mingot A, Vullo D, et al. Superacid synthesized
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Declaration of interest
The authors report no conflicts of interest. The authors alone are
responsible for the content and writing of this article.
This work was supported by the Sakarya Research Fund of the
Sakarya University (2014-28-00-001).
7
540–9.
2
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inhibitors. Drug design of zinc-enzyme inhibitors: functional,
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