Synthesis of novel sulfonamides under mild conditions with effective inhibitory activity against the carbonic anhydrase isoforms I and II

Article abstract of DOI:10.3109/14756366.2015.1134524

Novel sulfonamide derivatives 6a–i, as new carbonic anhydrase inhibitors which candidate for glaucoma treatment, were synthesized from the reactions of 4-amino-N-(4-sulfamoylphenyl) benzamide 4 and sulfonyl chloride derivatives 5a–i with high yield (71–90

Full text of DOI:10.3109/14756366.2015.1134524

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Synthesis of novel sulfonamides under mild
conditions with effective inhibitory activity against
the carbonic anhydrase isoforms I and II
Erhan Başar, Ekrem Tunca, Metin Bülbül & Muharrem Kaya
To cite this article: Erhan Başar, Ekrem Tunca, Metin Bülbül & Muharrem Kaya (2016):
Synthesis of novel sulfonamides under mild conditions with effective inhibitory activity
against the carbonic anhydrase isoforms I and II, Journal of Enzyme Inhibition and Medicinal
Chemistry, DOI: 10.3109/14756366.2015.1134524
To link to this article: http://dx.doi.org/10.3109/14756366.2015.1134524
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Date: 04 February 2016, At: 15:08

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–6
2016 Taylor & Francis. DOI: 10.3109/14756366.2015.1134524
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RESEARCH ARTICLE
Synthesis of novel sulfonamides under mild conditions with effective
inhibitory activity against the carbonic anhydrase isoforms I and II
Erhan Bas¸ar1, Ekrem Tunca2, Metin Bu¨lbu¨l2, and Muharrem Kaya2
2
1Chemistry Department and Biochemistry Department, Faculty of Arts and Science, Dumlupınar University, Ku¨tahya, Turkey
Abstract
Keywords
Novel sulfonamide derivatives 6a–i, as new carbonic anhydrase inhibitors which candidate for
glaucoma treatment, were synthesized from the reactions of 4-amino-N-(4-sulfamoylphenyl)
benzamide 4 and sulfonyl chloride derivatives 5a–i with high yield (71–90%). The structures of
Amidation reaction, carbonic anhydrase,
enzyme inhibition, glaucoma, sulfonamide
1
these compounds were confirmed by using spectral analysis (FT-IR, H NMR, 13C NMR, LC/MS
History
and HRMS). The inhibition effects of 6a–i on the hydratase and esterase activities of human
carbonic anhydrase isoenzymes, hCA I and II, which were purified from human erythrocytes
with SepharoseÕ4B-L-tyrosine-p-aminobenzene sulfonamide affinity chromatography, were
studied as in vitro, and IC50 and Ki values were determined. The results show that newly
synthesized compounds have quite powerful inhibitory properties.
Received 6 November 2015
Revised 14 December 2015
Accepted 15 December 2015
Published online 22 January 2016
Introduction
humor secretion. Therefore, the inhibition of hCA II is therapeutic
for glaucoma, characterized by the elevation of intraocular
Sulfonamide compounds are an important class of drugs for the
medicine industry and they possess various types of biological
activities such as antibacterial1, anticancer2, anticarbonic anhy-
drase for glaucoma treatment3–6, acetylcholinesterase inhibitor
agents for Alzheimer’s disease7,9, antiobesity10 and high-ceiling
diuretic11. They are commonly used in human and veterinary
medicine for therapeutic and prophylactic purposes to fight many
dangerous illnesses12.
pressure as
a result of excessive secretion of aqueous
humor19,20. Some sulfonamide class CA inhibitors (acetazolamide
(AAZ), dorzolamide (DZA) and brinzolamide (BRZ)) have been
developed for the treatment of glaucoma so far3. But they have
some side effects including metallic taste, depression, weight loss,
blurred vision, burning of the eye, etc.20 This situation reveals that
there is a need for the development of new inhibitory agents.
This paper reports the synthesis, characterization and inves-
tigation of inhibitory properties of some new sulfonamide
derivatives on hCA I and hCA II (ciliary process isozyme).
Inhibition effects of the compounds have been investigated under
in vitro conditions and structure–activity relationships of them
have been explained.
Carbonic anhydrase (CA, EC 4.2.1.1) is a metalloenzyme that
contains Zn2+ ion in its active site, which catalyzes the
interconversion between carbon dioxide and bicarbonate and
proton13. Although this reaction is simple, it plays an important
role in many physiological and pathological processes such as
transport of carbon dioxide and bicarbonate between metabolizing
tissues and lungs, electrolyte secretion, pH and carbon dioxide
homeostasis, some biosynthetic reactions, calcification and
tumorigenicity14. These enzymes were encoded by six different
Methods
unrelated gene families (a, b, g, d, z, Z) in living organisms15,16
.
Chemistry
Nowadays, it is known that there are 16 different isozymes of
mammalian CAs that belongs to a gene family17. Among these
isozymes CA I, CA II, CA III, CA VII and CA XIII are cytosolic;
CA IV, CA IX, CA XII, CA XIV and CA XV are membrane
bound; CA VA and CA VB are mitochondrial and CA VI is
secreted into saliva and milk15–17. CA I is a major CA isozyme
in most of the vertebrates and CA II is also present in human
eye15–18. hCA II is involved in ciliary processes for aqueous
The chemicals used in the synthesis of sulfonamide derivatives
were provided by Merck and Aldrich Chemical Company and
CNBr activated SepharoseÕ4B for affinity column and electro-
phoresis reagents were obtained from Sigma Chem. Co. All
chemicals and solvents used for the synthesis were spectroscopic
reagent grade. Melting points were measured on a Bibby
Scientific Stuart Digital, Advanced, SMP30 apparatus. Fourier
Transform Infrared (FT-IR) spectra were recorded on a Bruker
Optics, ALPHA FT-IR spectrometer. The 1H NMR, and 13C NMR
spectra were obtained with a Bruker AV 400 Ultra Shield
instrument in DMSO-d6 as solvent with trimethylsilane as the
internal reference, at 400 and 100 MHz, respectively. HRMS
spectra were detected by an Agilent Technologies 6530
Address for correspondence: M. Kaya, Biochemistry Department,
Faculty of Arts and Science, Dumlup{nar University, Ku¨tahya, Turkey.
Tel: + 90 274 2652031. Fax: + 90 274 2652056. E-mail:
muharrem.kaya@dpu.edu.tr

2
E. Bas¸ar et al.
J Enzyme Inhib Med Chem, Early Online: 1–6
Accurate-Mass Q-TOF LC/MS at the Advanced Technology 4-(4-Methoxyphenylsulfonamido)-N-(4-sulfamoylphenyl)-
Research Center of Dumlupinar University (ILTEM).
benzamide (6c)
As white solid, 200.75 mg, 87%, mp 259 ꢁC (dec.). IR (cmꢂ1):
3370, 3283 and 3260 w (–NH and –NH2), 3105 w (Ar–H), 2976 w
(C–H), 1651 s (C¼O), 1503 s (C¼C); 1H NMR (DMSO-d6,
General procedure for preparation of 4-nitro-N-
(4-sulfamoylphenyl)benzamide compound (3)
4-Aminobenzenesulfonamide (1) (1.739 g, 10.1 mmol), 4-nitro 400 MHz) ꢀ (ppm): 10.60 (s, 1H, NH), 10.33 (s, 1H, NH), 7.83 (d,
benzoylchloride (2) (1.856 g, 10 mmol), 3 mL dry triethylamine 2H, J ¼ 8.78 Hz, Ar–H), 7.78 (d, 2H, J ¼ 8.8 Hz, Ar–H), 7.73–
(TEA) and dry 30 mL THF were stirred for 5 h at room 7.70 (m, 4H, Ar–H), 7.21 (s, 2H, SO2NH2), 7.16 (d, 2H,
temperature. Afterwards the solvent was removed in vacuo and J ¼ 8.5 Hz, Ar–H), 7.02 (d, 2H, J ¼ 8.8 Hz, Ar–H), 3.72 (s, 3H,
the precipitated crude product was washed with 1000 mL distilled –CH3); 13C NMR (DMSO-d6, 100 MHz) ꢀ (ppm): 165.18, 162.61,
H2O. The product was purified by recrystallization from 142.16, 142.32, 138.55, 130.81, 129.17, 128.98, 126.49, 119.68,
ethanol4,5
.
118.04, 114.52, 55.65; HRMS (QTOF-ESI): m/z [M] calcd. for
C20H19N3O6S2: 461.0715; found [M ꢂ H]ꢂ: 460.0638.
Procedure for preparation of 4-amino-N-(4-
sulfamoylphenyl)benzamide compound (4)
4-(4-Methylphenylsulfonamido)-N-(4-sulfamoylphenyl)-
benzamide (6d)
Na2Sꢀ9H2O (1 mmol) and sulfur (2 mmol) were dissolved by
boiling 20 mL of water. This solution (sodium poly-sulfur) was
then added dropwise to a stirred and warm solution of 4-nitro-N-
(4-sulfamoylphenyl)benzamide (3) (1 mmol) in ethanol–water.
The progress of the reaction was monitored by TLC. Once the
reaction was completed, the mixture was cooled to room
temperature and solid was filtered off and washed with H2O.
The sulfonamide product was purified and recrystallized from the
ethanol (90%). The melting point of compound (4) was found to
As white solid, 182.65 mg, 82%, mp 289 ꢁC (dec.). IR (cmꢂ1):
3375, 3299 and 3276 w (–NH and –NH2), 3088 w (Ar–H), 1648 s
(C¼O), 1506 s (C¼C); 1H NMR (DMSO-d6, 400 MHz) ꢀ (ppm):
10.66 (s, 1H, NH), 10.33 (s, 1H, NH), 7.84–7.65 (m, 8H, Ar–H),
7.30 (d, 2H, J ¼ 8.0 Hz, Ar–H), 7.20 (s, 2H, SO2NH2), 7.16 (d,
2H, J ¼ 8.5 Hz, Ar–H), 2.27 (s, 3H, –CH3); 13C NMR (DMSO-d6,
100 MHz) ꢀ (ppm): 165.17, 143.63, 142.14, 141.19, 138.56,
136.38, 129.38, 129.83, 129.18, 126.76, 126.48, 119.67, 118.10,
20.94; HRMS (QTOF-ESI): m/z [M] calcd. for C20H19N3O5S2:
445.0766; found [M ꢂ H]ꢂ: 444.0689.
be 313 ꢁC4,5
.
General procedure for preparation of sulfonamide
derivatives 6a–i
4-(Naphthalene-2-sulfonamido)-N-(4-sulfamoylphenyl)-
benzamide (6e)
A mixture of a 4-amino-N-(4-sulfamoylphenyl)benzamide (4)
0.5 mmol (0.1455 g) and methanesulfonyl chloride (5a)
0.55 mmol (0.06325 g) in dry 5 mL pyridine were stirred for
4 h at room temperature. The progress of the reaction was
monitored by TLC. Once the reaction is completed, the solvent
was removed in vacuo and residue was washed with 1000 mL
water. The sulfonamide derivatives were obtained as pure
products.
As white solid, 204.65 mg, 85%, mp 254 ꢁC (dec.). IR (cmꢂ1):
3356 and 3324, 3262 and 3230 w (–NH and –NH2), 1653 s
(C¼O), 1506 s (C¼C); 1H NMR (DMSO-d6, 400 MHz) ꢀ (ppm):
10.96 (s, 1H, –NH), 10.39 (s, 1H, –NH), 8.60 (s, 1H, Ar–H), 8.19
(d, 1H, J ¼ 7.8 Hz, Ar–H), 8.13 (d, 1H, J ¼ 8.8 Hz, Ar–H), 8.02
(d, 1H, J ¼ 7.8 Hz, Ar–H), 7.91–7.79 (m, 7H, Ar–H), 7.73–7.65
(m, 2H, Ar–H), 7.32 (d, 2H, J ¼ 8.8, Ar–H) 7.30 (s, 2H, SO2NH2);
13C NMR (DMSO-d6, 100 MHz) ꢀ (ppm): 165.14, 142.13,
141.04, 138.54, 136.17, 134.33, 131. 50, 129.69, 129.39, 129.29,
129.20, 129.16, 128.29, 127.83, 127.80, 126.48, 121.85, 119.66,
118.19; HRMS (QTOF-ESI): m/z [M] calcd. for C23H19N3O5S2:
481.0766; found [M ꢂ H]ꢂ: 480.0690.
4-(Methylsulfonamido)-N-(4-sulfamoylphenyl)benzamide
(6a)
As white solid, 132.98 mg, 72%, mp 291 ꢁC (dec.). IR (cmꢂ1):
3377, 3306 and 3280 w (–NH and –NH2), 3039 w (Ar–H), 2939 w
(C–H), 1650 s (C¼O), 1509 s (C¼C); 1H NMR (DMSO-d6,
400 MHz) ꢀ (ppm): 10.48 (s, 1H, –NH), 10.27 (s, 1H, –NH),
8.00–7.95 (m, 4H, Ar–H), 7.83 (d, 2H, J ¼ 8.8 Hz, Ar–H), 7.34 (d,
N-(4-sulfamoylphenyl)-4-(2,4,6-trimethylphenylsulfonami-
do)benzamide (6f)
2H, J ¼ 8.8 Hz, Ar–H), 7.30 (s, 2H, SO2NH2), 3.12 (s, 3H, As white solid, 179.95 mg, 76%, mp 262 ꢁC (dec.). IR (cmꢂ1):
–CH3); 13C NMR (DMSO-d6, 100 MHz) ꢀ (ppm): 165.22, 142.19, 3378 and 3359, 3314 and 3285 w (–NH and –NH2), 3188 w (Ar–
141.84, 138.56, 128.93, 129.33, 126.51, 119.78, 117.82, 39.75; H), 2979 w (C–H), 1658 s (C¼O), 1508 s (C¼C); 1H NMR
HRMS (QTOF-ESI): m/z [M] calcd. for C14H15N3O5S2: (DMSO-d6, 400 MHz) ꢀ (ppm): 10.78 (s, 1H, NH), 10.37 (s, 1H,
369.0453; found [M ꢂ H]ꢂ: 368.0377.
NH), 7.91 (d, 2H, J ¼ 8.8 Hz, Ar–H), 7.85 (d, 2H, J ¼ 8.8 Hz, Ar–
H), 7.79 (d, 2H, J ¼ 8.8 Hz, Ar–H), 7.29 (s, 2H, SO2NH2), 7.10
(d, 2H, J ¼ 8.8 Hz, Ar–H), 7.05 (s, 2H, Ar–H), 2.63 (s, 6H, 2x–
CH3), 2.23 (s, 3H, –CH3); 13C NMR (DMSO-d6, 100 MHz) ꢀ
(ppm): 165.14, 142.43, 142.19, 141.18, 138.73, 136.51, 133.42,
128.62, 131.92, 129.22, 126.48, 119.60, 116.82, 22.38, 20.94;
HRMS (QTOF-ESI): m/z [M] calcd. for C22H23N3O5S2:
473.1079; found [M ꢂ H]ꢂ: 472.1001.
4-(Ethylsulfonamido)-N-(4-sulfamoylphenyl)benzamide
(6b)
As white solid, 141.87 mg, 74%, mp 311 ꢁC (dec.). IR (cmꢂ1):
3399, 3306 and 3280 w (–NH and –NH2), 3095 w (Ar–H), 2944 w
(C–H), 1650 s (C¼O), 1507 s (C¼C); 1H NMR (DMSO-d6,
400 MHz) ꢀ (ppm): 10.38 (s, 1H, –NH), 10.20 (s, 1H, –NH),
7.90–7.86 (m, 4H, Ar–H), 7.73 (d, 2H, J ¼ 9.0 Hz, Ar–H), 7.27
(d, 2H, J ¼ 8.8 Hz, Ar–H), 7.21 (s, 2H, SO2NH2), 3.13 (q, 2H,
J ¼ 7.36 Hz, –CH2), 1.14 (t, 3H, J ¼ 7.3 Hz, –CH3); 13C NMR
(DMSO-d6, 100 MHz) ꢀ (ppm): 165.22, 142.19, 141.90, 138.56,
128.88, 129.34, 126.49, 119.75, 117.65, 45.56, 8.02; HRMS
(QTOF-ESI): m/z [M] calcd. for C15H17N3O5S2: 383.0610; found
[M ꢂ H]ꢂ: 382.0535.
4-(3,5-Dichloro-2-hydroxyphenylsulfonamido)-N-(4-sulfa-
moylphenyl)benzamide (6g)
As white solid, 209.13 mg, 81%, mp 266 ꢁC (dec.). IR (cmꢂ1):
3390 br (OH), 3347, 3282 and 3250 w (–NH and –NH2), 3090 w
(Ar–H), 1653 s (C¼O), 1505 s (C¼C); 1H NMR (DMSO–d6,
400 MHz) ꢀ (ppm): 11.27 (s, 1H, OH), 10.85 (s, 1H, NH), 10.41

DOI: 10.3109/14756366.2015.1134524
Synthesis of novel sulfonamides under mild conditions
3
(s, 1H, NH), 7.91 (d, 2H, J ¼ 9.0 Hz, Ar–H), 7.87–7.85 (m, 3H, are times for pH change of the non-enzymatic and enzymatic
Ar–H), 7.80–7.78 (m, 3H, Ar–H), 7.28 (s, 2H, SO2NH2), 7.25 (d, reactions, respectively24.
2H, J ¼ 8.8 Hz, Ar–H); 13C NMR (DMSO-d6, 100 MHz) ꢀ (ppm):
Esterase activity was assayed by following the change in the
165.15, 150.26, 142.16, 140.66, 138.53, 129.25, 128.83, 123.88, absorbance at 348 nm of 4-nitrophenylacetate to 4-nitrophenylate
122.86, 134.21, 129.15, 128.41, 126.48, 119.64, 117.83; HRMS ion over a period of 3 min at 25 ꢁC using a spectrophotometer
(QTOF-ESI): m/z [M] calcd. for C19H15Cl2N3O6S2: 514.9779; according to the method described in the literature25. The
found [M ꢂ H]ꢂ: 513.9723.
enzymatic reaction, in a total volume of 3.0 mL, contained
1.4 mL of 0.05 M TRIS–SO4 buffer (pH 7.4), 1.0 mL of 3.0 mM
4-nitrophenylacetate, 0.5 mL H2O and 0.1 mL enzyme solution. A
reference measurement was obtained by preparing the same
cuvette without enzyme solution.
4-(Phenylsulfonamido)-N-(4-sulfamoylphenyl)benzamide
(6h)
As white solid, 153.17 mg, 71%, mp 279 ꢁC (dec.). IR (cmꢂ1):
3386 and 3274 w (–NH and –NH2), 3090 and 3066 w (Ar–H),
1649 s (C¼O), 1505 s (C¼C); 1H NMR (DMSO-d6, 400 MHz) ꢀ
Determination of IC50 and Ki values of the compounds
(ppm): 10.84 (s, 1H, NH), 10.43 (s, 1H, NH), 7.93–7.80 (m, 8H, To determine the IC50 values of the inhibitors, hydratase and
Ar–H), 7.67–7.58 (m, 3H, Ar–H), 7.30 (s, 2H, SO2NH2), 7.27 (d, esterase activities of CA isoenzymes were assayed in the presence
2H, J ¼ 8.5 Hz, Ar–H). 13C NMR (DMSO-d6, 100 MHz) ꢀ (ppm): of various inhibitor concentrations as mentioned above.
165.18, 142.15, 141.06, 139.25, 138.56, 133.21, 129.42, 129.21, Regression analysis graphs were drawn by plotting the percent
126.70, 126.50, 119.70, 118.25; HRMS (QTOF-ESI): m/z [M] enzyme activity versus inhibitor concentration and IC50 values
calcd. for C19H17N3O5S2: 431.0610; found [M ꢂ H]ꢂ: 430.0535. were calculated3,6
.
To determine Ki values as well as the inhibition type, three
different inhibitor concentrations giving 30%, 50% and 70%
inhibition were selected. At each of these inhibitor concentrations,
enzyme activity was measured in the presence of various substrate
concentrations (0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM and 0.7 mM)
and the data were linearized with Lineweaver–Burk plot for Vmax
and the Ki determination. Enzyme activity was also measured in
4-(4-Bromophenylsulfonamido)-N-(4-sulfamoylphenyl)-
benzamide (6i)
As white solid, 229.67 mg, 90%, mp 287 ꢁC (dec.). IR (cmꢂ1):
3417 and 3387, 3291 and 3273 w (–NH and –NH2), 3089 w (Ar–
H), 1649 s (C¼O), 1507 s (C¼C); H1 NMR (DMSO-d6,
400 MHz) ꢀ (ppm): 10.91 (s, 1H, NH), 10.45 (s, 1H, NH),
7.95–7.89 (m, 4H, Ar–H), 7.84–7.77 (m, 6H, Ar–H), 7.31 (s, 2H,
SO2NH2), 7.27 (d, 2H, J ¼ 8.5 Hz, Ar–H); 13C NMR (DMSO-d6,
100 MHz) ꢀ (ppm): 165.15, 142.14, 140.72, 138.58, 138.47,
129.74, 127.16, 132.53, 129.29, 128.72, 126.51, 119.73, 118.58;
the presence of the same substrate concentrations but in the
3,6
absence of any inhibitor to determine the Vmax
.
Statistical analysis
HRMS (QTOF-ESI): m/z [M] calcd. for C19H16BrN3O5S2: All the presented data were confirmed in at least three independ-
508.9715; found [M ꢂ H]ꢂ: 507.9619.
ent experiments and are expressed as the mean standard
deviation (SD). Data were analyzed by using a one-way analysis
of variance for multiple comparisons (SPSS 13.0, SPSS Inc.,
Chicago, IL). p 5 0.00001 was considered to be statistically
significant.
Purification of carbonic anhydrase I and II isoenzymes
from human erythrocytes
Erythrocytes were purified from human blood. The blood samples
were centrifuged at 1500 rpm for 20 min and plasma was
removed. Later, red cells were washed with isotonic solution
Result and discussion
(0.9% NaCl), and the erythrocytes were hemolyzed with 1.5 Chemistry
volumes of ice-cold water. Cell membranes were removed by
The general synthetic procedure to obtain sulfonamide derivatives
centrifugation at 4 ꢁC, 20 000 rpm for 30 min. The pH of
hemolysate was adjusted to 8.7 with solid TRIS (tris(hydrox-
ymethyl)aminomethane). The hemolysate was applied to affinity
column (SepharoseÕ4B-L-tyrosine-p-aminobenzene sulfonamide)
pre-equilibrated with 25.0 mM TRIS–HCl/0.1 M Na2SO4 (pH
8.7). After the extensive washing with a solution of 25.0 mM
TRIS–HCl/22.0 mM Na2SO4 (pH 8.7), the hCA I and hCA II
isoenzymes were eluted with the solution of 1.0 M NaCl/25.0 mM
Na2HPO4 (pH 6.3) and 0.1 M NaCH3COO/0.5 M NaClO4 (pH
5.6), respectively21. For quantitative protein determination, the
Bradford method was used with bovine serum albumin as a
standard22. Also purity control of the isoenzymes was performed
with SDS-PAGE after the purification23.
in three step reaction is described in Scheme 1. The 4-nitro-N-
(4-sulfamoylphenyl)benzamide (3) was prepared in the presence
of TEA in THF at room temperature3–5. The 4-amino-N-(4-
sulfamoylphenyl)benzamide (4) was obtained in polysulfide
solution by reduction of the nitro compound (3). Novel sulfona-
mide derivatives 6a–i were synthesized from the reactions of
4-amino-N-(4-sulfamoylphenyl)benzamide 4 and sulfonyl chlor-
ide derivatives 5a–i in pyridine.
The novel sulfonamide derivatives were obtained under mild
conditions, in short times and in high yields.
Spectral data are consistent with the chemical structures of the
compounds. When the IR spectrums were examined; the C¼O
groups stretching vibrations, which belong to the structure of
sulfonamide derivatives 6a–i, were observed between 1658 and
1648 cmꢂ1 (Supplemental Figures).
Determination of hydratase and esterase activities of
hCA I and hCA II
The CO2 hydratase activity of the enzyme was determined at 0 ꢁC 3390 cmꢂ1. Besides, sulfonamide derivatives 6a–i aromatic C–H
in a veronal buffer (pH 8.15) with the pH-stat method as the stretching bands and aliphatic C–H stretching bands were
indicator and saturated carbon dioxide solution as the substrate in observed between 3188–3039 cmꢂ1 and 2979–2939 cmꢂ1
Compound 6g aromatic O–H stretching band was observed at
,
a final volume of 4.2 mL. The time (in seconds) taken for the respectively. –NH and –NH2 groups stretching vibration bands
solution to change from pH 8.15 to pH 6.50 was measured. The that belong to these compounds 6a–i were observed in the region
enzyme unit (EU) is the enzyme amount that reduces the non- between 3399–3317 cmꢂ1 and 3314–3230 cmꢂ1, respect-
enzymatic reaction time by 50%. The activity of an enzyme unit ively26,27. Stretching vibration of the C¼C bonds in the aromatic
was calculated by using the equation ((t0ꢂtc)/tc), where t0 and tc structures was observed at 1509–1503 cmꢂ1. Symmetric and

4
E. Bas¸ar et al.
J Enzyme Inhib Med Chem, Early Online: 1–6
O
S
O
O
THF, rt
TEA
O
O2N
O2N
+
H2N
NH2
HN
3
S
NH2
Cl
O
1
2
O
Na2S.9H2O, S8
EtOH-H2O
O
O
O
HN
RSO2Cl
5a-i
R
O
S
H2N
S
HN
S
NH2
pyridine, rt
O
HN
4
NH2
O
O
O
6a-i
6a
CH3-
6b
C2H5-
6c
4-OCH3C6H4-
6d
4-CH3C6H4-
6e
2-Naphtyl
6f
2,4,6(CH3)C6H2-
6g
6h
6i
4-BrC6H4-
R
3,5-diCl,
C6H5-
2-OHC6H2-
Scheme 1. Synthesis of novel sulfonamide derivatives.
asymmetric vibration bands of SO2, existing in the structure of the compounds showed remarkable inhibition effects on the esterase
novel sulfonamide compounds were determined to give severe activities of hCA I and hCA II when compared with AAZ, which
vibrations in the range of 1159–1135 cmꢂ1 (Supplemental is intraocular pressure reducing agent.
Figures).
The compounds have inhibitory effects, with micromolar
1
The H NMR spectra of sulfonamide compounds (6a, 6c, 6d, range, on the hydratase activities of the isozymes. The IC50 values
and 6f), which belong to protons of the methyl groups, showed were in the range of 0.518–1.654 mM for hCA I and 0.138–
singlet peaks in between 2.27 and 3.72 ppm. The compound 6b 1.060 mM for hCA II. Compound 6c has the most powerful
ethyl group protons were observed in triplet peak at 1.14 ppm inhibition effect on the hydratase activity of hCA I (IC50 value
(3H) and quartet peak at 3.12 ppm (2H) (Supplemental Figures). 0.518 mM), whereas 6d is the strongest inhibitor for the hydratase
Aromatic protons of all the sulfonamide derivatives 6a–i activity of hCA II (IC50 value 0.138 mM). Also 6i has the weakest
showed signals in the region between 7.02 and 8.60 ppm. The inhibition effect on the hydratase activities of the isoforms, hCA I
compound 6g hydroxyl group proton was observed as a broad and II (IC50 values 1.654 mM and 1.060 mM, respectively).
peak at 11.27 ppm. SO2NH2 group protons of sulfonamide Synthesized compounds are the more potent inhibitors for
compounds 6a–i were observed as singlet peaks between 7.20 and hydratase activity of hCA II than the other isoform’s hydratase
7.31 ppm. The carboxamide group protons (–CONH–) of all activity. According to hydratase IC50 values, the inhibition
compounds 6a–i showed broad peaks between 10.20 and potentials were in the order of 6c46d46e434446f46a46g
10.96 ppm (Supplemental Figures).
46b46h46i for hCA I, and 6d46a46c4346e4446f46b
46g46h46i for hCA II.
When the inhibition effects of the compounds on the esterase
activities of the isozymes, hCA I and II were examined, a similar
situation to the inhibition of the hydratase activity could be
observed clearly. But the compounds showed more effective
inhibition, nearly in nanomolar range, on the esterase activities of
the isozymes. The IC50 values were in the range of 0.049–
0.276 mM for hCA I and 0.027–0.237 mM for hCA II. Unlike the
inhibition of the hydratase activity, newly synthesized compounds
6a–i have more powerful inhibition effects on the esterase
activities of hCA I and hCA II than the starting compounds 3 and
4. 6f, containing 2,4,6–trimethylphenyl, has the strongest inhib-
ition effect on the esterase activities of hCA I and II (IC50 values
0.049 and 0.027 mM, respectively). Also 6h and 6d have nearly
the same inhibition potentials on the isozymes (see Table 1).
These compounds contain phenyl and tolyl moieties, respectively.
Esterase IC50 values of 6d, 6f and 6h are close to each other.
Furthermore, their substituents are similar to each other as
structurally. Therefore it can be thought that these compounds
make similar interactions with the active site. However, contrary
to our expectations, the increase of the polarity in the substituents,
reduced the inhibitory potentials of the compounds. This situation
is clearly observed on 6c, 6g and 6i. Also the compounds
containing non-aromatic substituents, 6a and 6b, have weaker
inhibition potentials on the esterase activities of hCA I and hCA
II. The reason why compounds 6d, 6f and 6h, which have apolar
Carbonic anhydrase inhibition
In this part of the study we aimed to:
ꢃ investigate the inhibition potentials of synthesized compounds
on hCA I and II,
ꢃ compare the contribution of different functional groups on the
inhibition potentials of the compounds.
For this purpose, firstly, the carbonic anhydrase isozymes
(hCA I and II) were purified separately from human erythrocyte
cells with a single-step method, SepharoseÕ4B-p-aminobenzene
sulfonamide affinity chromatography. Purity control of the
isozymes was performed by SDS-PAGE and single bands were
observed and then the inhibition potentials of newly synthesized
compounds on hydratase and esterase activities of hCA I and II
were investigated as in vitro24,25
.
Kinetic studies showed that the compounds have inhibitory
properties on hydratase and esterase activities of hCA I and II
isoforms. The inhibition effects of synthesized sulfonamide
derivatives on the hydratase activities of the isozymes demon-
strate that these compounds are attached to zinc ion in the active
site16.
As shown in Table 1, synthesized derivatives have more
potential inhibition effects on hCA II than hCA I. Because hCA II
is a target enzyme for glaucoma treatment, more powerful
inhibition of this isozyme is clinically important. Also the

DOI: 10.3109/14756366.2015.1134524
Synthesis of novel sulfonamides under mild conditions
5
Table 1. The effects of synthesized compounds on hCA I and II isozymes under in vitro conditions.
a,b
Hydratase IC50 (mM)
a,b
Esterase IC50 (mM)
Kia,b (mM)
Compound
hCA I
hCA II
hCA I
hCA II
hCA I
hCA II
AAZ
3
4
0.224 0.0042
0.741 0.0011
0.860 0.0005
1.125 0.0003
1.248 0.0008
0.518 0.0001
0.547 0.0007
0.738 0.0004
0.925 0.0009
1.143 0.0002
1.416 0.0006
1.654 0.0004
0.198 0.0071
0.312 0.0002
0.370 0.0003
0.203 0.0004
0.418 0.0009
0.250 0.0006
0.138 0.0004
0.362 0.0007
0.394 0.0005
0.596 0.0002
0.620 0.0005
1.060 0.0001
0.321 0.0051
0.276 0.0003
0.184 0.0005
0.136 0.0005
0.160 0.0005
0.112 0.0005
0.058 0.0005
0.105 0.0005
0.049 0.0005
0.195 0.0005
0.053 0.0005
0.106 0.0005
0.212 0.0048
0.237 0.0012
0.085 0.0009
0.070 0.0009
0.082 0.0007
0.059 0.0001
0.032 0.0005
0.051 0.0005
0.027 0.0008
0.113 0.0002
0.029 0.0004
0.065 0.0006
0.210 0.0035
0.149 0.0009
0.081 0.0001
0.052 0.0002
0.065 0.0005
0.050 0.0005
0.025 0.0008
0.043 0.0003
0.020 0.0003
0.081 0.0006
0.022 0.0002
0.044 0.0004
0.120 0.0049
0.141 0.0008
0.048 0.0005
0.029 0.0005
0.036 0.0003
0.028 0.0007
0.015 0.0006
0.023 0.0005
0.011 0.0001
0.045 0.0004
0.013 0.0001
0.025 0.0008
6a
6b
6c
6d
6e
6f
6g
6h
6i
AAZ was used as a reference compound.
aMean standard error, from three different assays.
bp 5 0.00001 for all analysis.
3. Kaya M, Basar E, Cakir E, et al. Synthesis and characterization of
novel dioxoacridine sulfonamide derivatives as new carbonic
anhydrase inhibitors. J Enzym Inhib Med Chem 2012;27:509–14.
substituents, are potentially better inhibitors is that these
compounds better interact with the hydrophobic pocket of the
active site. A similar compound that has been studied previously
supports this claim and PDB code of this compound (3N3J) shows
how this compound interacts with this enzyme28.
_
4. Esirden I, Ulus R, Aday B, et al. Synthesis of novel acridine bis-
sulfonamides with effective inhibitory activity against the carbonic
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The esterase Ki values are in agreement with the esterase IC50
values. Therefore, the aforementioned structure–activity relation-
ships for esterase activity can also be said for Ki values. These
values were in the range of 0.020–0.149 mM for hCA I, and
0.011–0.141 mM for hCA II. hCA I and hCA II isozymes have
more affinity against 6d, 6f and 6h derivatives that have apolar
aromatic substituents (Ki values 0.025 0.008 mM,
0.020 0.003 mM and 0.022 0.002 mM for hCA I, and
0.015 0.006 mM, 0.011 0.001 mM and 0.013 0.001 mM for
hCA II, respectively). But the affinity of the isozymes against
inhibitors decreased in the presence of non-aromatic substituents
(6a and 6b) and polar aromatic substituents (6c, 6g and 6i).
According to Ki values, inhibition potentials were in the order of
6f46h46d46e46i46c & 6a46b46g & 443 for hCA I and
II isoforms.
_
˘
5. Ulus R, Yes¸ildag I, Tanc¸ M, et al. Synthesis of novel acridine and bis
acridine sulfonamides with effective inhibitory activity against the
cytosolic carbonic anhydrase isoforms II and VII. Bioorg Med Chem
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6. Yes¸ildag I, Ulus R, Basar E, et al. Facile, highly efficient, and clean
one-pot synthesis of acridine sulfonamide derivatives at room
temperature and their inhibition of human carbonic anhydrase
isoenzymes. Monatsh Chem 2014;145:1027–34.
¨
7. Goksu S, Naderi A, Akbaba Y, et al. Carbonic anhydrase inhibitory
properties of novel benzylsulfamides using molecular modeling and
experimental studies. Bioorg Chem 2014;56:75–82.
8. Bag S, Tulsan R, Sood A, et al. Sulfonamides as multifunctional
agents for Alzheimer’s disease. Bioorg Med Chem Lett 2015;25:
626–30.
9. Akincioglu, A, Gulcin, H, Durdagi, IS, et al. Discovery of potent
carbonic anhydrase and acetylcholine esterase inhibitors: novel
sulfamoylcarbamates and sulfamides derived from acetophenones.
Bioorg Med Chem 2015;23:3592–602.
10. Arechederra RL, Waheed A, Sly WS, et al. Effect of sulfonamides as
carbonic anhydrase VA and VB inhibitors on mitochondrial
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action: a patent and literature review (2005–2013). Expert Opin Ther
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Conclusion
Today, some carbonic anhydrase inhibitors for the treatment of
glaucoma are present on the market, but such drugs have many
side effects. Strong carbonic anhydrase inhibitors can be
developed to reduce dosage, and as a result of this, reduction of
side effects can be achieved. In the present study, the synthesized
compounds have remarkable inhibition effects on hCA I and II.
Consequently, these potential inhibitors may be sufficient to
reduce the dosage. So we can say that the synthesized inhibitors
can be candidates for in vivo studies in the treatment of glaucoma.
´
12. Pator-Navarro N, Gallego-Iglesias E, Maquieira A, Puchades R.
Development of a group-specific immunoassay for sulfonamides.
Application to bee honey analysis. Talanta 2007;71:923–33.
13. Carta F, Di Cesare Manneli L, Pinard M, et al. A class of
sulfonamide carbonic anhydrase inhibitors with neuropathic pain
modulating effects. Bioorg Med Chem 2015;23:1828–40.
_
´
14. Zołnowska B, Sławinski J, Pogorzelska A, et al. Carbonic anhydrase
inhibitors. Synthesis, and molecular structure of novel series N-
substituted
N0-(2-arylmethylthio-4-chloro-5-methylbenzenesulfo-
Declaration of interest
nyl)guanidines and their inhibition of human cytosolic isozymes I
and II and the transmembrane tumor-associated isozymes IX and
XII. Eur J Med Chem 2014;71:135–47.
This research was financed by Dumlupınar University Research
Fund (Grant No. 2014-20).
15. Sarikaya B, Ceruso M, Carta F, Supuran CT. Inhibition of carbonic
anhydrase isoforms I, II, IX and XII with novel Schiff bases:
identification of selective inhibitors for the tumor-associated isoforms
over the cytosolic ones. Bioorg Med Chem 2014;22:5883–90.
16. Alterio V, Di Fiore A, D’Ambrosio K, et al. Multiple binding modes
of inhibitors to carbonic anhydrases: how to design specific drugs
targeting 15 different isoforms? Chem Rev 2012;112:4421–68.
17. Chegaev K, Lazzarato L, Tamboli Y, et al. Furazan and furoxan
sulfonamides are strong a-carbonic anhydrase inhibitors and poten-
tial antiglaucoma agents. Bioorg Med Chem 2014;22:3913–21.
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