A class of sulfonamides as carbonic anhydrase I and II inhibitors

Article abstract of DOI:10.1080/14756366.2016.1198900

Four groups of novel sulfonamide derivatives: (i) acetoxybenzamide, (ii) triacetoxybenzamide, (iii) hydroxybenzamide and (iv) trihydroxybenzamide, all having thiazole, pyrimidine, pyridine, isoxazole and thiadiazole moieties were prepared and their inhibitory effects were studied on two metalloenzymes, i.e. carbonic anhydrase isozymes (hCA I and II), purified from human erythrocyte cells by Sepharose-4B-l-tyrosine-sulfanilamide affinity chromatography. These enzymes are present in almost all living organisms to catalyse the synthesis of bicarbonate ion (HCO₃ ^?) from carbon dioxide and water. The sulfonamide derivatives were found to be active against hCA I and II in the range of 2.62–136.54 and 5.74–210.58 nM, respectively.

Full text of DOI:10.1080/14756366.2016.1198900

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
A class of sulfonamides as carbonic anhydrase I
and II inhibitors
Taner Gokcen, Ilhami Gulcin, Turan Ozturk & Ahmet C. Goren
To cite this article: Taner Gokcen, Ilhami Gulcin, Turan Ozturk & Ahmet C. Goren (2016): A class
of sulfonamides as carbonic anhydrase I and II inhibitors, Journal of Enzyme Inhibition and
Medicinal Chemistry, DOI: 10.1080/14756366.2016.1198900
To link to this article: http://dx.doi.org/10.1080/14756366.2016.1198900
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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–9
2016 Informa UK Limited, trading as Taylor & Francis Group. DOI: 10.1080/14756366.2016.1198900
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RESEARCH ARTICLE
A class of sulfonamides as carbonic anhydrase I and II inhibitors
Taner Gokcen1,2, Ilhami Gulcin3,4, Turan Ozturk1,2, and Ahmet C. Goren1
1Chemistry Group Laboratories, TUBITAK UME, Gebze, Turkey, 2Department of Organic Chemistry, Faculty of Science, Istanbul Technical University,
Istanbul, Turkey, 3Department of Chemistry, Faculty of Science, Atatu¨rk University, Erzurum, Turkey, and 4Fetal Programming of Diseases Research
Chair, Zoology Department, College of Science, King Saud University, Riyadh, Saudi Arabia
Abstract
Keywords
Four groups of novel sulfonamide derivatives: (i) acetoxybenzamide, (ii) triacetoxybenzamide,
(iii) hydroxybenzamide and (iv) trihydroxybenzamide, all having thiazole, pyrimidine, pyridine,
isoxazole and thiadiazole moieties were prepared and their inhibitory effects were studied on
two metalloenzymes, i.e. carbonic anhydrase isozymes (hCA I and II), purified from human
erythrocyte cells by Sepharose-4B-L-tyrosine-sulfanilamide affinity chromatography. These
enzymes are present in almost all living organisms to catalyse the synthesis of bicarbonate ion
(HCO3ꢀ) from carbon dioxide and water. The sulfonamide derivatives were found to be active
against hCA I and II in the range of 2.62–136.54 and 5.74–210.58 nM, respectively.
Enzyme inhibition, gallic acid,
p-hydroxybenzoic acid
History
Received 20 March 2016
Revised 31 May 2016
Accepted 1 June 2016
Published online 23 June 2016
Introduction
The CA isozymes have an increasing attention as important
targets for designing inhibitors or activators for biomedical
applications including treatments of epilepsy, glaucoma, idio-
Carbonic anhydrases (CAs, EC 4.2.1.1) are zinc-containing
metalloenzymes that catalyse the reversible reaction of carbon
dioxide and water to produce bicarbonate (HCO3ꢀ) and proton
(H+)1–5. CAs play crucial pathophysiological roles including pH
regulation, CO2 homeostasis and biosynthetic reactions such as
gluconeogenesis, respiration, transport of CO2 and HCO3ꢀ among
metabolizing tissues, lung electrolyte secretion, bone resorption,
ureagenesis, lipogenesis, production of biological fluids, tumori-
genicity, cell adhesion, proliferation, calcification, and in the
growth and virulence of various fungal and bacterial pathogens6–
pathic intracranial hypertension and altitude sickness29–31
.
Sulfonamides (R-SO2NH2) have found widespread applications
in design of CA inhibitors as they are among the most
important groups for zinc ion (Zn2+) binding and most of the
clinically used CA inhibitors contain sulfonamide moieties32.
Since the first evidence of their CA inhibition, their physio-
logical, pharmacological and kinetic properties were largely
investigated32–36. Crystallographic studies of their adducts with
several CA isozymes elucidate the key factors responsible for
the binding of the sulfonamide moiety to the CA active site
and provide a rationale for the unique tailored properties of this
anchoring group32,37,38. Although binding of the sulfonamide
derivatives was indicated to be driven by coordination of the
deprotonated sulfonamide nitrogen to the catalytic zinc ion
(Zn2+), additional interactions with the hydrophilic and/or
hydrophobic region of the active site may take place, depend-
ing on the nature of the substituent group32,39–41. These studies
demonstrated that sulfonamide is an ideal ligand of the CA
active site as it combines the negative charge of the
deprotonated nitrogen with the zinc ion, and the presence of
a proton on the coordinated nitrogen atom provides Thr199
atom with a strong H-bond32.
Sulfonamides, which are the basis of several groups of drugs,
were reported to have significant inhibitory activity against many
CA classes. These studies are widely related with inhibition of
mammalian isoforms42,43. It was reported that the discovery of
isoform-selective CA inhibitors or at least organ-specific targeting
inhibitors would represent one of the most important aims of the
CA research, which has been very intense recently44. However,
there are critical barriers need to be addressed on design of CAI as
therapeutic agent such as high number of CA isoforms in human
body, their rather diffuse localization in many tissues and organs,
9
.
CA appears to be almost ubiquitously expressed in living
organisms. Thus far, six genetically distinct CA families are
known, including the a-, b-, g-, d-, z- and Z-class enzymes10–13
.
Currently, there are 16 known human CA isoforms, which differ
in their cellular localization and rate of enzymatic activity14. The
mammalian enzymes belong to the a-CA family and have
different functions, kinetic parameters, inhibitory properties and
cell and tissue localizations14–18. Humans encode 12 catalytically
active a-CA isozymes, many of which have been studied both
functionally and structurally. These CAs comprise CA I, II, III,
IV, VA, VB, VI, VII, IX, XII, XIII and XIV19. CAs I, II, III, VII
and XIII are cytosolic isozymes20–23, CAs VA and VB are
localized in mitochondria24,25, CA VI is a unique secreted
isozyme25, CAs IX, XII and XIV are transmembrane proteins26,27
and CAs IV and XV are glycosylphosphatidylinositol-anchored to
the cell membrane28.
,
Address for correspondence: Dr Ahmet C. Goren, Chemistry Group
Laboratories, TUBITAK UME, P.O. Box 54, 41470 Gebze, Kocaeli,
Turkey. Tel: +90 2626795000. Fax: +90 2626795001. E-mail:
ahmetceyhan.goren@tubitak.gov.tr

2
T. Gokcen et al.
J Enzyme Inhib Med Chem, Early Online: 1–9
and lack of isozyme selectivity of the currently available general application in medicinal chemistry to esterify polar
inhibitors of sulfonamide/sulfamates45,46
hydroxyl groups to convert them into prodrugs to promote
.
The natural phenolic acids, gallic (3,4,5-trihydroxybenzoic) physicochemical, biopharmaceutical and pharmacokinetic proper-
acid and p-hydroxybenzoic acid, found in many plants and used as ties, membrane permeability and oral absorption56–59. In this
preservatives in food, possess antibacterial, anticarcinogenic, study, we focus on inhibition of two metalloenzymes, CA
antioxidant, antifungal and antimutagenic activities, and cytotox- isozymes (hCA I and II), purified from human erythrocyte
icity toward tumor cells47–49. Innocenti et al. showed that cells by Sepharose-4B-L-tyrosine-sulphanilamide affinity
p-hydroxybenzoic acid had better inhibition properties on hCA I chromatography.
and hCA II compared with the natural products, polyphenols and
phenolic acids42. Caffeic acid phenethyl ester which is also a Methods
polyphenolic compound, was derived from natural products. It
Syntheses of the sulfonamide derivatives were conducted starting
possesses a range of biological activities50 against such as hCA I,
from 4-hydroxy 1a and 3,4,5-trihydroxy 1b benzoic acids
hCA II, hCA IX, hCA XII and comprises potent activities toward
(Scheme 1), which are naturally occurring compounds in many
HIV-1. Wang et al. showed that designed inhibitors, mimicing
plants and fruits60–63. Their reaction with acetic anhydride
caffeic acid phenethyl ester as bearing polyphenols at one side,
selectively produced 4-acetoxy 2a and 3,4,5-triacetoxy 2b ben-
sulphonamide moieties in the middle and various phenyl groups at
zoic acids, reactions of which with thionyl chloride yielded the
the other side can be effective integrase inhibitors51. The study
benzoyl chlorides 3a,b. Then, sulfonamides 4, possessing thia-
presented herein targets zinc ion (Zn2+) instead of Mg2+ or Mn2+
zole, pyrimidine, pyridine, isoxazole and thiadiazole moieties,
and replaced catechol with gallic acid and p-hydroxyphenolic
were reacted with the benzoyl chlorides 3a,b to obtain first two
acid. In place of sulfonamide linker and phenyl group, sulfona-
classes of sulfonamide derivatives: (i) acetoxybenzamides 5a–h
mide drugs having thiazole, pyrimidine, pyridine, isoxazole and
and (ii) triacetoxybenzamides 6a–h. Acid treatment of the first
thiadiazole moieties were introduced. Although, it was believed
sulfonamide derivatives gave the second two classes of the
that amine group of sulfonamide should be a primary agent acting
derivatives 7a–h and 8a–h. Compounds 5g64, 6c65, 8a65, 8c66 and
as a CA inhibitor, recent studies indicated that secondary and even
8h51 are listed in literature but no CA inhibition activity was
tertiary sulfonamides can act as affective CA inhibitors and they
reported before.
can be selective52–55. In this study, four groups of novel
sulfonamide derivatives: (i) acetoxybenzamide, (ii) triacetoxy-
Chemistry
benzamide (iii) hydroxybenzamide and (iv) trihydroxybenzamide,
all having thiazole, pyrimidine, pyridine, isoxazole and thiadia- All starting materials and the reagents were purchased from
zole moieties are disclosed. In addition to their hydroxyl forms, commercial suppliers. Progress of the reaction was monitored by
we decided to investigate their acetylated derivatives since it is a TLC using silica gel-60 F254 plates with detection by short wave
Scheme 1. Synthesis of the compounds.

DOI: 10.1080/14756366.2016.1198900
A class of sulfonamides as carbonic anhydrase I and II inhibitors
3
UV fluorescence (l ¼ 254 nm) and staining with cerium sulphate. N-(sulfadiazine)-p-acetoxybenzamide (5b)
Silica gel flash chromatography was performed using silica gel
White powder, yield 55%. m.p. 269–270 ꢁC. 1H NMR (600 MHz,
DMSO-d6) ꢀ (ppm): 11.71 (s, 1H), 10.61 (s, 1H), 8.51 (d,
J ¼ 4.8 Hz, 2H), 7.98 (m, 6H), 7.31 (d, J ¼ 8.7 Hz, 2H), 7.05
(t, J ¼ 4.8 Hz, 1H), 2.31 (s, 3H). 13C NMR (150 MHz, DMSO-d6)
ꢀ (ppm): 169.38, 165.74, 158.79, 157.37, 153.62, 143.55, 134.94,
132.41, 129.82, 129.14, 122.37, 119.95, 116.23, 21.32. HRMS
(ESI+): m/z calculated for C19H16N4O5S1Na1 [M + Na+]:
435.0734, found 435.0738.
1
60 A (230–400 mesh). H and 13C NMR spectra were recorded on
˚
1
a Varian 600 MHz spectrometer at 25 ꢁC. Chemical shifts for H
and 13C NMR spectra obtained in DMSO-d6 are reported in ppm
relative to residual solvent proton (ꢀ ¼ 2.50 ppm) and carbon
(ꢀ ¼ 39.52 ppm) signals, respectively. Multiplicity is indicated as
follows: s (singlet); d (doublet); t (triplet); m (multiplet). Coupling
constants are reported in hertz (Hz). High-resolution mass spectra
(HRMS) were recorded on BrukerMicroTOF-Q at positive electro
spray ionization (ESI+) mode. 1H and 13C NMR, and HRMS
spectra of compounds are provided in Supporting Information.
Melting points were determined by Barnstead Electrothermal
IA9200. Purities of compounds determined by C, H, N analysis by
Thermo Finnigan Flash 1112 EA Series and also by HPLC-UV at
270 nm and 495%.
N-(sulfamethazine)-p-acetoxybenzamide (5c)
Off white powder, yield 62%. m.p. 245–246 ꢁC. 1H NMR
(600 MHz, DMSO-d6)
ꢀ (ppm): 10.58 (s, 1H), 8.00 (d,
J ¼ 8.7 Hz, 2H), 7.98 (d, J ¼ 8.9 Hz, 2H), 7.94 (d, J ¼ 8.9 Hz,
2H), 7.31 (d, J ¼ 8.6 Hz, 2H), 6.77 (s, 1H), 2.31 (s, 3H), 2.26 (s,
6H). 13C NMR (150 MHz, DMSO-d6) ꢀ (ppm): 169.37, 167.72,
165.66, 157.08, 153.29, 132.41, 130.72, 129.79, 129.54, 122.35,
119.60, 114.19, 112.26, 23.54, 21.31. HRMS (ESI+): m/z
calculated for C21H21N4O5S1 [M + H+]: 441.1227, found
441.1214.
General procedure for acetylation of hydroxyl substituted
benzoic acids 1a,b
To the stirred solution of benzoic acid 1 (20 mmol) in acetic
anhydride (120 mmol), few drops of concentrated H2SO4 was
added67. A fast temperature rise was observed and after all the
solid was dissolved, the stirring was continued for an hour. Then,
water (100 mL) was added and the solution was stirred for further
2.5 h to remove any excess acetic anhydride left. The solid
precipitate was filtered, washed with water (2 ꢂ 50 mL) and dried
on filter by air suction for 10 min and under vacuum for overnight.
p-Acetoxybenzoic acid (2a). White solid, yield 90%. 1H NMR
(DMSO-d6, 600 MHz) ꢀ (ppm): 7.99 (d, J ¼ 8.6 Hz, 2H), 7.26 (d,
J ¼ 8.6 Hz, 2H), 2.28 (s, 3H).
N-(sulfapyridine)-p-acetoxybenzamide (5d)
White powder, yield 65%. m.p. 259–260 ꢁC. 1H NMR (600 MHz,
DMSO-d6) ꢀ (ppm): 10.57 (s, 1H), 8.03 (d, J ¼ 3.6 Hz, 1H), 7.99
(d, J ¼ 8.6 Hz, 2H), 7.92 (d, J ¼ 8.8 Hz, 2H), 7.87 (d, J ¼ 8.7 Hz,
2H), 7.75–7.68 (m, 1H), 7.31 (d, J ¼ 8.6 Hz, 2H), 7.15 (d,
J ¼ 8.6 Hz, 1H), 6.88 (t, J ¼ 5.9 Hz, 1H), 2.31 (s, 3H). 13C NMR
(150 MHz, DMSO-d6) ꢀ (ppm): 168.94, 165.23, 153.15, 152.96,
142.55, 140.11, 136.07, 132.00, 129.38, 127.64, 126.63, 121.91,
119.75, 115.78, 113.53, 20.88. HRMS (ESI+): m/z calculated for
C20H18N3O5S1 [M + H+]: 412.0962, found 412.0963.
3,4,5-Triacetoxybenzoic acid (2b). White solid, yield 85%.
1H NMR (DMSO-d6, 600 MHz) ꢀ (ppm): 7.72 (s, 2H), 2.30 (s,
3H), 2.27 (s, 6H).
N-(sulfisoxazole)-p-acetoxybenzamide (5e)
General procedure for preparation of acyl chlorides 3
Off white powder, yield 47%. m.p. 221–222 ꢁC. 1H NMR
(600 MHz, DMSO-d6) ꢀ (ppm): 10.68 (s, 1H), 8.02 (d, J ¼ 8.7 Hz,
2H), 8.00 (d, J ¼ 8.9 Hz, 2H), 7.76 (d, J ¼ 8.9 Hz, 2H), 7.32 (d,
J ¼ 8.7 Hz, 2H), 2.31 (s, 3H), 2.09 (s, 3H), 1.66 (s, 3H). 13C NMR
(150 MHz, DMSO-d6) ꢀ (ppm): 169.39, 165.81, 161.86, 155.94,
153.65, 143.95, 134.53, 132.34, 129.86, 128.20, 122.39, 120.42,
105.58, 21.33, 10.77, 6.33. HRMS (ESI+): m/z calculated
for C20H20N3O6S1 [M + H+]: 430.1067, found 430.1066.
A mixture of acetyl protected benzoic acid 2 (20 mmol) and
thionyl chloride (300 mmol) was heated at 80 ꢁC for 5 h. Excess of
thionyl chloride was then evaporated under reduced pressure at
50 ꢁC. To the residue was added acetone (20 mL) and the solution
was used for the next step without further purification51.
General procedure for preparation of N-(sulfonamide)-
acetoxybenzamide (5 and 6)
N-(sulfamethizole)-p-acetoxybenzamide (5f)
White powder, yield 58%. m.p. 244.5–246 ꢁC. 1H NMR
To a mixture of sulfonamide 4 (20 mmol) and pyridine (20 mmol)
in acetone (50 mL) was added dropwise acyl chloride 3 (20 mmol)
in acetone (20 mL) with stirring at 0 ꢁC, after which stirring was
continued at room temperature for overnight51. Progress of the
reaction was monitored by TLC, staining with ceric sulphate. The
precipitated products were filtered. Otherwise, the solvent was
evaporated under reduced pressure. The crude product was purified
by re-crystallization from ethanol or flash column chromatography
using ethyl acetate/hexane (1:1) mixture as an eluent.
(600 MHz, DMSO-d6)
ꢀ (ppm): 10.60 (s, 1H), 8.01 (d,
J ¼ 8.7 Hz, 2H), 7.95 (d, J ¼ 8.8 Hz, 2H), 7.78 (d, J ¼ 8.8 Hz,
2H), 7.31 (d, J ¼ 8.7 Hz, 2H), 2.47 (s, 3H), 2.31 (s, 3H). 13C NMR
(150 MHz, DMSO-d6) ꢀ (ppm): 169.37, 168.24, 165.65, 154.88,
153.60, 143.11, 136.74, 132.40, 129.80, 127.20, 122.36, 120.30,
21.32, 16.50. HRMS (ESI+): m/z calculated for C18H17N4O5S2
[M + H+]: 433.0635, found 433.0636.
N-(sulfathiazole)-p-acetoxybenzamide (5a)
N-(sulfamerazine)-p-acetoxybenzamide (5g)
Off white powder, yield 58%. m.p. 267–268 ꢁC. 1H NMR Off white powder, yield 62%. m.p. 260–261 ꢁC. 1H NMR
(600 MHz, DMSO-d6) (ppm): 10.57 (s, 1H), 8.00 (d, (600 MHz, DMSO-d6) ꢀ (ppm): 11.61 (s, 1H), 10.60 (s, 1H),
ꢀ
J ¼ 8.7 Hz, 2H), 7.93 (d, J ¼ 8.8 Hz, 2H), 7.79 (d, J ¼ 8.8 Hz, 8.33 (d, J ¼ 5.1 Hz, 1H), 8.00 (d, J ¼ 8.6 Hz, 2H), 7.98 (d,
2H), 7.31 (d, J ¼ 8.6 Hz, 2H), 7.25 (d, J ¼ 4.6 Hz, 1H), 6.82 (d, J ¼ 9.0 Hz, 2H), 7.95 (d, J ¼ 9.0 Hz, 2H), 7.31 (d, J ¼ 8.6 Hz, 2H),
J ¼ 4.6 Hz, 1H), 2.31 (s, 3H). 13C NMR (150 MHz, DMSO-d6) ꢀ 6.91 (d, J ¼ 5.1 Hz, 1H), 2.33 (s, 3H), 2.31 (s, 3H). 13C NMR
(ppm): 169.27, 169.16, 165.61, 153.57, 142.79, 137.21, 132.46, (150 MHz, DMSO-d6) ꢀ (ppm): 169.39, 168.80, 165.71, 158.10,
129.79, 127.23, 124.83, 122.36, 120.18, 108.55, 21.33. HRMS 156.98, 153.61, 143.43, 135.12, 132.41, 129.81, 129.34, 122.36,
(ESI+): m/z calculated for C18H16N3O5S2 [M + H+]: 418.0526, 119.80, 115.28, 23.69, 21.30. HRMS (ESI+): m/z calculated for
found 418.0526.
C19H19N3O8S1 [M + H+]: 449.0887, found 449.0890.

4
T. Gokcen et al.
J Enzyme Inhib Med Chem, Early Online: 1–9
N-(sulfanilamide)-p-acetoxybenzamide (5h)
White powder, yield 55%. m.p. 251–252 ꢁC. 1H NMR (600 MHz, Yellowish powder, yield 40%. m.p. 225–226 ꢁC. 1H NMR
(ppm): 10.68 (s, 1H), 7.94
N-(sulfamethizole)-3,4,5-triacetoxybenzamide (6f)
DMSO-d6) ꢀ (ppm): 10.58 (s, 1H), 8.03 (d, J ¼ 8.6 Hz, 2H), 7.95 (600 MHz, DMSO-d6)
ꢀ
(d, J ¼ 8.8 Hz, 2H), 7.82 (d, J ¼ 8.8 Hz, 2H), 7.32 (d, J ¼ 8.6 Hz, (d, J ¼ 8.7 Hz, 2H), 7.84 (s, 2H), 7.80 (d, J ¼ 8.7 Hz, 2H), 2.47
2H), 7.28 (s, 2H), 2.32 (s, 3H). 13C NMR (150 MHz, DMSO-d6) (s, 3H), 2.35 (s, 3H), 2.33 (s, 6H). 13C NMR (150 MHz, DMSO-
ꢀ (ppm): 169.38, 165.61, 153.58, 142.51, 139.20, 132.44, d6) ꢀ (ppm): 167.83 (d, J ¼ 22.4 Hz), 167.75–167.27 (m), 166.84
129.79, 126.96, 122.36, 120.22, 21.32. HRMS (ESI+): m/z (s), 163.46 (s), 154.39 (s), 143.06 (s), 142.19 (s), 137.49 (s),
calculated for C15H15N2O5S1 [M + H+]: 335.0696, found 136.56 (s), 132.30 (s), 126.69 (s), 120.63 (s), 120.01 (s), 20.21 (s),
335.0694.
19.77 (s), 15.97 (s). HRMS (ESI+): m/z calculated for
C22H20N4O9S2Na [M + Na+]: 571.0564, found 571.0564.
N-(sulfathiazole)-3,4,5-triacetoxybenzamide (6a)
Yellowish powder, yield 50%. m.p. 177–178 ꢁC. 1H NMR
N-(sulfamerazine)-3,4,5-triacetoxybenzamide (6g)
Yellowish powder, yield 42%. m.p. 234–236 ꢁC. 1H NMR
(600 MHz, DMSO-d6)
ꢀ (ppm): 10.63 (s, 1H), 7.90 (d,
J ¼ 8.9 Hz, 2H), 7.82 (s, 2H), 7.80 (d, J ¼ 8.8 Hz, 2H), 7.24
(d, J ¼ 4.6 Hz, 1H), 6.82 (d, J ¼ 4.6 Hz, 1H), 2.34 (s, 3H), 2.33
(s, 6H). 13C NMR (150 MHz, DMSO-d6) ꢀ (ppm): 168.77,
167.99, 166.94, 163.50, 143.14, 141.91, 137.55, 137.20, 132.46,
126.81, 124.59, 120.69, 119.95, 108.10, 20.31, 19.87. HRMS
(ESI+): m/z calculated for C22H20N3O9S2 [M + H+]: 534.0635,
found 534.0641.
(DMSO-d6, 600 MHz)
ꢀ (ppm): 10.66 (s, 1H), 8.33 (d,
J ¼ 5.1 Hz, 1H), 7.99 (d, J ¼ 8.9 Hz, 2H), 7.93 (d, J ¼ 8.9 Hz,
2H), 7.82 (s, 2H), 6.91 (d, J ¼ 5.1 Hz, 1H), 2.34 (s, 3H), 2.33 (s,
6H), 2.32 (s, 3H). 13C NMR (150 MHz, DMSO-d6) ꢀ (ppm):
168.26, 168.00, 166.95, 163.63, 157.53, 156.56, 143.17, 142.61,
137.61, 135.08, 132.45, 128.93, 120.75, 119.58, 114.85, 23.25,
20.31, 19.87. HRMS (ESI+): m/z calculated for C24H23N4O9S1
[M + H+]: 543.1180, found 543.1180.
N-(sulfadiazine)-3,4,5-triacetoxybenzamide (6b)
N-(sulfanilamide)-3,4,5-triacetoxybenzamide (6h)
Off white powder, yield 40%. m.p. 207–208 ꢁC. 1H NMR
White powder, yield 54%. m.p. 222–223 ꢁC. 1H NMR (DMSO-d6,
600 MHz) ꢀ (ppm): 10.61 (s, 1H), 7.89 (d, J ¼ 8.8 Hz, 2H), 7.81
(s, 2H), 7.79 (d, J ¼ 8.8 Hz, 2H), 7.26 (s, 2H), 2.32 (s, 3H), 2.30
(s, 6H). 13C NMR (150 MHz, DMSO-d6) ꢀ (ppm): 168.02, 166.96,
163.53, 143.17, 141.69, 139.09, 137.57, 132.45, 126.57, 120.71,
120.02, 20.33, 19.88. HRMS (ESI+): m/z calculated for
C19H18N2O9S1Na [M + Na+]: 473.0625, found 473.0625.
(600 MHz, DMSO-d6)
ꢀ (ppm): 10.64 (s, 1H), 8.48 (d,
J ¼ 4.8 Hz, 2H), 7.95 (d, J ¼ 8.9 Hz, 2H), 7.90 (d, J ¼ 8.9 Hz,
2H), 7.79 (s, 2H), 7.02 (t, J ¼ 4.9 Hz, 1H), 2.31 (s, 3H), 2.29 (s,
6H). 13C NMR (150 MHz, DMSO-d6) ꢀ (ppm): 168.43, 167.37,
164.08, 158.79, 157.33, 143.58, 143.13, 138.03, 135.30, 132.84,
129.13, 121.15, 120.16, 116.23, 20.73, 20.29. HRMS (ESI+): m/z
calculated for C23H21N4O9S1 [M + H+]: 529.1024, found
529.1015.
General procedure for N-(sulfonamide)-
hydroxybenzamide (7 and 8)
N-(sulfamethazine)-3,4,5-triacetoxybenzamide (6c)
White powder, yield 48%. m.p. 236–238 ꢁC. 1H NMR (600 MHz,
DMSO-d6) ꢀ (ppm): 10.64 (s, 1H), 7.99 (d, J ¼ 8.7 Hz, 2H), 7.91
(d, J ¼ 8.8 Hz, 2H), 7.82 (s, 2H), 6.76 (s, 1H), 2.34 (s, 3H), 2.33
(s, 6H), 2.26 (s, 6H). 13C NMR (150 MHz, DMSO-d6) ꢀ (ppm)
169.16, 168.43, 167.38, 164.01, 156.60, 143.58, 142.81, 139.52,
138.01, 135.80, 132.86, 129.55, 121.14, 119.80, 23.27, 21.45,
20.28. HRMS (ESI+): m/z calculated for C25H25N4O9S1
[M + H+]: 557.1337, found 557.1336.
N-(sulfonamide)-acetoxybenzamide 5 or 6 (1 g) was added to a
solution of tetrahydrofuran (10 mL), methanol (10 mL) and
concentrated hydrochloric acid (5 mL)53. The mixture stirred at
60 ꢁC for one hour. The solvent was evaporated under reduced
pressure and the crude product was purified by re-crystallization
from ethanol or by flash column chromatography using ethyl
acetate/hexane (1:1).
N-(sulfathiazole)-p-hydroxybenzamide (7a)
N-(sulfapyridine)-3,4,5-triacetoxybenzamide (6d)
Off white powder, yield 48%. m.p. 293–294 ꢁC. 1H NMR
(600 MHz, DMSO-d6) ꢀ (ppm): 12.68 (s, 1H), 10.28 (s, 1H),
10.16 (s, 1H), 7.91 (d, J ¼ 8.8 Hz, 2H), 7.86 (d, J ¼ 8.7 Hz, 2H),
7.76 (d, J ¼ 8.8 Hz, 2H), 7.24 (d, J ¼ 4.6 Hz, 1H), 6.87 (d,
J ¼ 8.7 Hz, 2H), 6.81 (d, J ¼ 4.6 Hz, 1H). 13C NMR (150 MHz,
DMSO-d6) ꢀ (ppm): 169.16, 165.85, 161.30, 143.19, 136.75,
130.35, 127.15, 125.33, 124.77, 120.02, 115.42, 108.48. HRMS
(ESI+): m/z calculated for C16H14N3O4S2 [M + H+]: 376.0420,
found 376.0419.
White powder, yield 62%. m.p. 213–214 ꢁC. 1H NMR (600 MHz,
DMSO-d6) ꢀ (ppm): 10.61 (s, 1H), 8.00 (s, 1H), 7.90–7.82 (m,
4H), 7.79 (s, 2H), 7.70 (t, J ¼ 8.0 Hz, 1H), 7.13 (d, J ¼ 8.6 Hz,
1H), 6.85 (t, J ¼ 6.3 Hz, 1H), 2.32 (s, 3H), 2.30 (s, 6H). 13C NMR
(150 MHz, DMSO-d6) ꢀ (ppm): 168.44, 167.38, 164.01, 153.47,
143.89, 143.58, 142.53, 140.66, 138.00, 136.94, 132.89, 128.07,
121.13, 120.38, 116.14, 114.04, 20.73, 20.28. HRMS (ESI+): m/z
calculated for C24H22N3O9S1 [M + H+]: 528.1071, found
528.1068.
N-(sulfisoxazole)-3,4,5-triacetoxybenzamide (6e)
N-(sulfadiazine)-p-hydroxybenzamide (7b)
White powder, yield 54%. m.p. 147–148 ꢁC. 1H NMR (600 MHz, White powder, yield 44%. m.p. 298–299 ꢁC. 1H NMR
(ppm): 10.33 (s, 1H), 8.51 (d,
DMSO-d6) ꢀ (ppm): 10.72 (s, 1H), 7.95 (d, J ¼ 8.8 Hz, 2H), 7.82 (600 MHz, DMSO-d6)
ꢀ
(s, 2H), 7.75 (d, J ¼ 8.8 Hz, 2H), 2.33 (s, 3H), 2.31 (s, 6H), 2.07 J ¼ 4.9 Hz, 2H), 7.94 (s, 4H), 7.85 (d, J ¼ 8.7 Hz, 2H), 7.04
(s, 3H), 1.63 (s, 3H). 13C NMR (150 MHz, DMSO-d6) ꢀ (ppm): (t, J ¼ 4.9 Hz, 1H), 6.87 (d, J ¼ 8.7 Hz, 2H). 13C NMR
168.02, 166.96, 163.74, 161.43, 155.51, 143.17, 143.13, 137.65, (150 MHz, DMSO-d6) ꢀ (ppm): 165.96, 161.37, 158.77,
134.48, 132.38, 127.80, 120.79, 120.21, 105.14, 20.33, 19.89, 157.38, 143.96, 134.42, 130.39, 129.05, 125.24, 119.79,
10.35, 5.90. HRMS (ESI+): m/z calculated for C24H23N3O10S1Na 116.21, 115.43. HRMS (ESI+): m/z calculated for
[M + Na+]: 568.0996, found 568.0997.
C17H15N4O4S1 [M + H+]: 371.0809, found 371.0817.

DOI: 10.1080/14756366.2016.1198900
A class of sulfonamides as carbonic anhydrase I and II inhibitors
5
N-(sulfamethazine)-p-hydroxybenzamide (7c)
J ¼ 8.8 Hz, 2H), 7.72 (d, J ¼ 8.8 Hz, 2H), 7.22 (d, J ¼ 4.6 Hz,
1H), 6.94 (s, 2H), 6.79 (d, J ¼ 4.6 Hz, 1H). 13C NMR (150 MHz,
DMSO-d6) ꢀ (ppm): 169.07, 166.31, 145.96, 143.30, 137.59,
136.54, 127.09, 124.84, 124,87, 119.95, 108.50, 107.85. HRMS
(ESI+): m/z calculated for C16H14N3O6S2 [M + H+]: 408.0319,
found 408.0315.
Off white powder, yield 48%. m.p. 162–163 ꢁC. 1H NMR
(600 MHz, DMSO-d6) ꢀ (ppm): 10.31 (s, 1H), 10.17 (s, 1H),
7.95 (q, J ¼ 9.0 Hz, 4H), 7.87 (d, J ¼ 8.7 Hz, 2H), 6.88 (d,
J ¼ 8.7 Hz, 2H), 6.76 (s, 1H), 2.26 (s, 6H). 13C NMR (150 MHz,
DMSO-d6) ꢀ (ppm): 167.79, 165.90, 161.34, 156.67, 143.66,
134.84, 130.36, 129.46, 125.25, 119.45, 115.42, 114.01, 23.33.
HRMS (ESI+): m/z calculated for C19H19N4O4S1 [M + H+]:
399.1122, found 399.1123.
N-(sulfadiazine)-3,4,5-trihydroxybenzamide (8b)
Off white powder, yield 43%. m.p. 244–245 ꢁC. 1H NMR
(600 MHz, DMSO-d6)
ꢀ (ppm): 10.26 (s, 1H), 8.50 (d,
N-(sulfapyridine)-p-hydroxybenzamide (7d)
J ¼ 4.9 Hz, 2H), 7.96–7.89 (m, 4H), 7.04 (t, J ¼ 4.9 Hz, 1H),
6.96 (s, 2H). 13C NMR (150 MHz, DMSO-d6) ꢀ (ppm): 166.41,
158.77, 157.36, 145.97, 144.06, 137.67, 134.25, 128.98, 124.77,
119.72, 116.21, 107.89. HRMS (ESI+): m/z calculated for
C17H15N4O6S1 [M + H+]: 403.0707, found 403.0707.
White powder, yield 50%. m.p. 282–283 ꢁC. 1H NMR (600 MHz,
DMSO-d6) ꢀ (ppm): 10.29 (s, 1H), 8.03 (s, 1H), 7.91 (d,
J ¼ 8.7 Hz, 2H), 7.84 (two doublets, 4H), 7.71 (d, 1H), 7.14 (d,
J ¼ 8.5 Hz, 1H), 6.87 (3H). 13C NMR (150 MHz, DMSO-d6) ꢀ
(ppm): 165.45, 160.93, 152.85, 143.94, 142.98, 140.02, 135.45,
129.91, 127.54, 124.76, 119.57, 115.42, 114.96, 113.43. HRMS
(ESI+): m/z calculated for C18H16N3O4S1 [M + H+]: 370.0856,
found 370.0856.
N-(sulfamethazine)-3,4,5-trihydroxybenzamide (8c)
Off white powder, yield 35%. m.p. 270.5–271.5 ꢁC. 1H NMR
(600 MHz, DMSO-d6) ꢀ (ppm): 10.23 (s, 1H), 7.96–7.89 (m, 4H),
6.96 (s, 2H), 6.75 (s, 1H), 2.25 (s, 6H). 13C NMR (150 MHz,
DMSO-d6) ꢀ (ppm): 167.47, 166.02, 156.29, 145.63, 143.43,
137.30, 134.30, 129.07, 124.45, 119.03, 113.68, 107.52, 22.99.
HRMS (ESI+): m/z calculated for C19H19N4O6S1 [M + H+]:
431.1020, found 431.1026.
N-(sulfisoxazole)-p-hydroxybenzamide (7e)
Off white powder, yield 41%. m.p. 235–236 ꢁC. 1H NMR
(600 MHz, DMSO-d6) ꢀ (ppm): 10.92 (s, 1H), 10.39 (s, 1H),
10.19 (s, 1H), 7.99 (d, J ¼ 8.8 Hz, 2H), 7.87 (d, J ¼ 8.6 Hz, 2H),
7.73 (d, J ¼ 8.8 Hz, 2H), 6.88 (d, J ¼ 8.6 Hz, 2H), 2.09 (s, 3H),
1.65 (s, 3H). 13C NMR (150 MHz, DMSO-d6) ꢀ (ppm): 166.02,
161.84, 161.42, 156.01, 144.37, 134.05, 130.44, 128.12, 125.19,
120.25, 115.46, 105.52, 10.77, 6.32. HRMS (ESI+): m/z
calculated for C18H17N3O5S1Na [M + Na+]: 410.0781, found
410.0781.
N-(sulfapyridine)-3,4,5-trihydroxybenzamide (8d)
White powder, yield 48%. m.p. 268–269 ꢁC. 1H NMR (600 MHz,
DMSO-d6) ꢀ (ppm): 10.21 (s, 1H), 8.04 (d, J ¼ 4.5 Hz, 1H), 7.92–
7.88 (m, 2H), 7.85–7.80 (m, 2H), 7.71 (ddd, J ¼ 8.9, 7.3, 1.9 Hz,
1H), 7.15 (d, J ¼ 8.6 Hz, 1H), 6.96 (s, 2H), 6.91–6.86 (m, 1H). 13C
NMR (150 MHz, DMSO-d6) ꢀ (ppm): 166.35, 153.26, 145.97,
143.53, 140.47, 137.63, 135.73, 129.22, 127.98, 124.83, 119.92,
116.40, 113.86, 107.83. HRMS (ESI+): m/z calculated for
C18H16N3O6S1 [M + H+]: 402.0754, found 402.0754.
N-(sulfamethizole)-p-hydroxybenzamide (7f)
Off white powder, yield 39%. m.p. 272–273 ꢁC. 1H NMR
(600 MHz, DMSO-d6) ꢀ (ppm): 10.29 (s, 1H), 10.14 (s, 1H),
7.91 (d, J ¼ 8.9 Hz, 2H), 7.84 (d, J ¼ 8.7 Hz, 2H), 7.73 (d,
J ¼ 8.8 Hz, 2H), 6.85 (d, J ¼ 8.7 Hz, 2H), 2.45 (s, 3H). 13C NMR
(150 MHz, DMSO-d6) ꢀ (ppm): 168.20, 165.89, 161.34, 154.85,
143.54, 136.26, 130.38, 127.13, 125.27, 120.14, 115.43, 16.51.
HRMS (ESI+): m/z calculated for C16H15N4O4S2 [M + H+]:
391.0529, found 391.0530.
N-(sulfisoxazole)-3,4,5-triacetoxybenzamide (8e)
White powder, yield 57%. m.p. 252–253 ꢁC. 1H NMR (600 MHz,
DMSO-d6) ꢀ (ppm): 10.93 (s, 1H), 10.32 (s, 1H), 7.98 (d,
J ¼ 8.9 Hz, 2H), 7.71 (d, J ¼ 8.9 Hz, 2H), 6.99 (s, 2H), 2.09 (s,
3H), 1.66 (s, 3H). 13C NMR (150 MHz, DMSO-d6) ꢀ (ppm):
166.45, 161.84, 156.01, 145.98, 144.48, 137.75, 133.90, 128.07,
124.68, 120.15, 107.85, 105.49, 10.77, 6.32. HRMS (ESI+): m/z
calculated for C18H17N3O7S1Na [M + Na+]: 442.0679, found
442.0675.
N-(sulfamerazine)-p-hydroxybenzamide (7g)
Off white powder, yield 50%. m.p. 271–272 ꢁC. 1H NMR
(600 MHz, DMSO-d6)
ꢀ (ppm): 10.36 (s, 1H), 8.33 (d,
J ¼ 5.1 Hz, 1H), 7.95 (s, 4H), 7.89–7.86 (m, 2H), 6.91 (d,
J ¼ 5.2 Hz, 1H), 6.90–6.87 (m, 2H), 2.32 (s, 3H). 13C NMR
(150 MHz, DMSO-d6) ꢀ (ppm): 168.65, 165.93, 161.40, 157.96,
157.00, 143.86, 134.56, 130.38, 129.22, 125.17, 119.65, 115.44,
115.35, 23.70. HRMS (ESI+): m/z calculated for C18H17N4O4S1
[M + H+]: 385.0965, found 385.0965.
N-(sulfamethizole)-3,4,5-trihydroxybenzamide (8f)
Off white powder, yield 62%. m.p. 177–178 ꢁC. 1H NMR
(600 MHz, DMSO-d6)
J ¼ 8.9 Hz, 2H), 7.73 (d, J ¼ 8.9 Hz, 2H), 6.96 (s, 2H), 2.46 (s,
3H). 13C NMR (150 MHz, DMSO-d6) ꢀ (ppm): 167.82, 165.98,
154.49, 145.57, 143.26, 137.27, 135.69, 126.71, 124.40, 119.69,
107.41, 16.13. HRMS (ESI+): m/z calculated for
C16H14N4O6S2Na [M + Na+]: 445.0247, found 445.0249.
ꢀ (ppm): 10.23 (s, 1H), 7.92 (d,
N-(sulfanilamide)-p-hydroxybenzamide (7h)
White powder, yield 47%. m.p. 308–309 ꢁC. 1H NMR (600 MHz,
DMSO-d6) ꢀ (ppm): 10.30 (s, 1H), 10.18 (s, 1H), 7.95 (d,
J ¼ 8.9 Hz, 2H), 7.89 (d, J ¼ 8.7 Hz, 2H), 7.80 (d, J ¼ 8.8 Hz, 2H),
7.26 (s, 2H), 6.89 (d, J ¼ 8.7 Hz, 2H). 13C NMR (150 MHz,
DMSO-d6) ꢀ (ppm): 165.52, 160.93, 142.56, 138.34, 129.99,
126.54, 124.96, 119.73, 115.08. HRMS (ESI+): m/z calculated for
C13H13N2O4S1 [M + H+]: 293.0591, found 293.0594.
N-(sulfamerazine)-3,4,5-trihydroxybenzamide (8g)
White powder, yield 54%. m.p. 214–215 ꢁC. 1H NMR (600 MHz,
DMSO-d6) ꢀ 8 ppm): 10.24 (s, 1H), 8.31 (d, J ¼ 5.1 Hz, 1H), 7.92
(s, 4H), 6.97 (s, 2H), 6.89 (d, J ¼ 5.2 Hz, 1H), 2.31 (s, 3H). 13C
NMR (150 MHz, DMSO-d6) ꢀ (ppm): 168.39, 166.09, 157.63,
156.66, 145.67, 143.63, 137.36, 134.09, 128.89, 124.46, 119.28,
114.99, 107.58, 23.40. HRMS (ESI+): m/z calculated for
C18H17N4O6S1 [M + H+]: 417.0863, found 417.0862.
N-(sulfathiazole)-3,4,5-trihydroxybenzamide (8a)
Off white powder, yield 50%. m.p. 289–290 ꢁC. 1H NMR
(600 MHz, DMSO-d6)
ꢀ (ppm): 10.19 (s, 1H), 7.88 (d,

6
T. Gokcen et al.
J Enzyme Inhib Med Chem, Early Online: 1–9
Table 1. IC50, Ki and the selectivity values of the compounds.
IC50 (nM)
Ki (nM)
hCA I
OH/Ac
hCA II
OH/Ac
Compounds
hCA I
hCA II
hCA I
hCA II
hCA II/hCA I
Sulfathiazole (4a)
77.01
17.83
22.35
31.51
36.47
77.01
7.59
16.12
40.76
49.51
36.47
9.07
12.38
10.89
27.72
34.65
11.59
18.24
8.35
9.25
57.75
7.38
23.89
9.93
4.99
17.24
9.48
7.07
9.54
7.04
17.92
13.26
5.44
77.01
15.78
10.66
49.51
43.31
86.63
40.98
9.91
63.01
63.01
115.51
47.69
8.77
43.94
49.51
20.38
53.35
10.19
43.42
40.65
77.01
40.43
7.62
47.76
40.65
87.39
24.36
49.68
34.48
29.62
13.89
38.78
20.82
21.07
36.28
100.73
17.21
11.91
53.31
12.73
31.51
99.01
57.75
31.51
15.17
136.54 24.21
12.71 0.04
12.21 3.03
27.91 1.38
26.33 2.75
46.82 3.49
6.17 0.01
11.82 1.64
27.38 1.09
62.74 20.42
22.98 5.32
7.01 0.01
12.43 2.20
9.37 0.03
27.18 1.53
23.79 4.70
7.49 0.04
15.69 3.80
2.62 0.05
6.89 0.01
60.11 6.11
4.82 0.01
14.75 0.74
7.37 0.04
5.75 0.02
19.08 0.07
9.26 0.06
2.75 0.05
7.81 0.02
4.69 0.04
11.14 0.01
14.19 0.01
3.67 0.02
6.01 0.01
11.25 0.04
20.76 0.04
13.57 0.06
11.69 0.01
4.06 0.80
6.27 0.01
39.11 3.41
57.75 7.71
35.69 12.03
32.12 5.85
19.92 0.16
118.45 14.90
8.85 0.03
9.11 1.34
51.69 16.52
64.75 15.30
107.43 62.80
39.39 0.03
7.34 0.74
68.52 22.74
146.74 93.16
134.36 47.61
137.96 0.64
5.74 1.17
16.84 0.02
44.78 5.57
14.54 1.12
19.15 0.03
8.29 2.33
37.69 0.18
36.41 0.13
210.58 75.36
25.15 0.13
10.24 1.96
77.45 0.24
39.41 0.14
31.39 0.04
29.85 0.06
22.61 0.08
70.12 0.44
77.86 0.25
26.56 0.02
64.82 0.30
17.37 0.02
18.97 0.01
44.76 0.05
120.22 0.25
33.98 0.07
14.92 0.02
52.15 4.56
15.73 0.08
41.29 29.92
104.61 30.64
37.96 1.48
38.16 12.05
9.76 0.03
0.87
0.70
0.75
1.85
2.46
2.29
6.38
0.62
2.50
2.34
5.85
19.68
0.46
1.80
1.65
0.61
2.56
0.53
14.39
5.28
3.50
5.22
0.69
10.51
6.85
1.65
3.22
8.22
8.98
16.60
2.38
4.57
4.73
3.16
3.98
5.79
2.50
1.28
12.84
2.51
1.06
1.81
1.06
1.19
0.49
Sulfathiazole-p-acetoxybenzamide (5a)
Sulfathiazole-3,4,5-triacetoxybenzamide (6a)
Sulfathiazole-p-hydroxybenzamide (7a)
Sulfathiazole-3,4,5-trihydroxybenzamide (8a)
Sulfadiazine (4b)
Sulfadiazine-p-acetoxybenzamide (5b)
Sulfadiazine-3,4,5-triacetoxybenzamide (6b)
Sulfadiazine-p-hydroxybenzamide (7b)
Sulfadiazine-3,4,5-trihydroxybenzamide (8b)
Sulfamethazine (4c)
Sulfamethazine-p-acetoxybenzamide (5c)
Sulfamethazine-3,4,5-triacetoxybenzamide (6c)
Sulfamethazine-p-hydroxybenzamide (7c)
Sulfamethazin-3,4,5-trihydroxybenzamide (8c)
Sulfapyridine (4d)
Sulfapyridine-p-acetoxybenzamide (5d)
Sulfapyridine-3,4,5-triacetoxybenzamide (6d)
Sulfapyridine-p-hydroxybenzamide (7d)
Sulfapyridine-3,4,5-trihydroxybenzamide (8d)
Sulfisoxazole (4e)
Sulfisoxazole-p-acetoxybenzamide (5e)
Sulfisoxazole-3,4,5-triacetoxybenzamide (6e)
Sulfisoxazole-p-hydroxybenzamide (7e)
Sulfisoxazole-3,4,5-trihydroxybenzamide (8e)
Sulfamethizole (4f)
Sulfamethizole-p-acetoxybenzamide (5f)
Sulfamethizole-3,4,5-triacetoxybenzamide (6f)
Sulfamethizole-p-hydroxybenzamide (7f)
Sulfamethizole-3,4,5-trihydroxybenzamide (8f)
Sulfamerazine (4g)
Sulfamerazine-p-acetoxybenzamide (5g)
Sulfamerazine-3,4,5-triacetoxybenzamide (6g)
Sulfamerazine-p-hydroxybenzamide (7g)
Sulfamerazine-3,4,5-trihydroxybenzamide (8g)
Sulfanilamide (4h)
Sulfanilamide-p-acetoxybenzamide (5h)
Sulfanilamide-3,4,5-triacetoxybenzamide (6h)
Sulfanilamide-p-hydroxybenzamide (7h)
Sulfanilamide-3,4,5-trihydroxybenzamide (8h)
p-Acetoxybenzoic acid (2a)
2.20
2.16
5.84
7.11
4.44
5.31
1.74
19.99
1.34
2.19
0.12
7.80
0.35
0.44
1.97
4.39
1.53
0.39
3.08
3.85
0.84
1.71
2.35
3.44
0.42
3.07
0.29
2.58
14.07
8.38
24.53
13.06
12.42
7.01
12.25
53.31
46.21
46.21
57.75
21.95
0.30
0.54
1.53
1.05
3,4,5-Triacetoxybenzamide acid (2b)
p-Hydroxybenzoic acid (1a)
3,4,5-Trihydroxybenzoic acid (1b)
AZA*
*Acetazolamide (AZA) was used as a standard inhibitor for both CA isozymes.
N-(sulfanilamide)-3,4,5-trihydroxybenzamide (8h)
prepared
Sepharose-4B-L-tyrosine-sulfanilamide
affinity
column68. The proteins from the column were spectrophotomet-
rically determined at 280 nm. For determination of the purity of
the isozymes, sodium dodecyl sulphate-polyacrylamide gel elec-
trophoresis (SDS-PAGE), having 10% and 3% acrylamide as an
eluent and packing gel, respectively, with 0.1% SDS, was
performed, through which a single band was observed for each
isozyme.
White powder, yield 40%. m.p. 307–308 ꢁC. 1H NMR (600 MHz,
DMSO-d6) ꢀ (ppm): 10.22 (s, 1H), 7.93 (d, J ¼ 8.8 Hz, 2H), 7.77
(d, J ¼ 8.8 Hz, 2H), 7.25 (s, 2H), 6.99 (s, 2H). 13C NMR
(150 MHz, DMSO-d6) ꢀ (ppm): 166.00, 145.63, 142.69, 138.22,
137.27, 126.52, 124.53, 119.66, 107.48. HRMS (ESI+): m/z
calculated for C13H13N2O6S1 [M + H+]: 325.0489, found
325.0477.
CA isozyme activities were determined following to the
methods described by Verpoorte et al.69 and the methods reported
previously. Absorbance change at 348 nm from p-nitrophenyla-
Carbonic anhydrase inhibition
CA isozymes (hCA I and II) were purified by Sepharose-4B-L- cetate (NPA) to p-nitrophenolate (NP) was recorded by 3 min
tyrosine-sulfanilamide affinity chromatography in a single puri- intervals at the room temperature (25 ꢁC) using a spectrophotom-
fication step2. The column material, Sepharose-4B-L-tyrosine- eter (Shimadzu, UV-VIS Spectrophotometer, UVmini-1240,
sulfanilamide, was prepared according to a reported method22,25
.
Kyoto, Japan). Quantity of the protein was measured spectro-
Thus, pH of the solution was adjusted to 8.7, using solid Tris. photometrically at 595 nm during the purification steps according
Subsequently, the supernatant was transferred to the previously to the Bradford method. As reported previously, bovine serum

DOI: 10.1080/14756366.2016.1198900
A class of sulfonamides as carbonic anhydrase I and II inhibitors
7
albumin (BSA) was used as a standard protein14. An activity hCA I, and compound 6c (N-(sulfamethazine)-3,4,5-triacetox-
(%)–[Sulfonamide] graph was depicted to determine the inhibition ybenzamide) with Ki value of 5.74 1.17 nM for hCA II.
effect of each sulfonamide derivative. For Ki values, three Although almost all the synthesized sulfonamides showed good
different sulfonamide derivatives were tested. NPA was used as inhibition activities compared with the used standard, AZA
a substrate at five different concentrations and Lineweaver–Burk (acetazolamide), as
curves were drawn as described previousely11.
a structure–activity relationship (SAR)
there is no clear cut evidence on the individual effects of
the introduced groups on the activities of the sulfonamides.
Results and discussion
Acknowledgements
The isozymes CA I and CA II, examined in this study, have
different activities. In mammals, CA II, which generally exists in
red blood cells in lower concentrations, has approximately 10
times higher activity compared with CA I30. Cytosolic isozyme
hCA I is ubiquitously expressed in body and available in high
concentrations in blood and gastrointestinal tract. As it was
demonstrated that this isozyme is involved in retinal and cerebral
edema, its inhibition could be a valuable tool for fighting the
condition. It is generally accepted that if Ki value of a tested
compound is less than 50 mM (Ki 4 50 mM), that compound is
considered to be inactive against hCA I55. The results presented in
Table 1 indicate that the new sulfonamide derivatives had
effective inhibition profile against slow cytosolic isoform hCA
I, and cytosolic dominant rapid isozymes hCA II. The cytosolic
isozyme hCA I was inhibited by all the synthesized sulfonamide
derivatives in nanomolar level, the Ki of which varied between
2.62 0.05 and 136.54 24.21 nM. On the other hand, acetazo-
lamide (AZA), considered being a broad-specificity CA inhibitor
owing to its widespread inhibition of CAs, showed Ki value of
19.92 0.16 nM against hCA I. Among the inhibitors, sulfona-
mide derivative 7d (N-(sulfapyridine)-p-hydroxybenzamide) was
found to be the best hCA I inhibitor with Ki of 2.62 0.05 nM.
The inhibition effects of the sulfonamide derivatives (6a–8h) were
found to be the same or lower than that of acetazolamide.
Comparison of hydroxyl groups with their acetylated derivatives,
in terms of hCA I inhibition, no clear trend was observed.
Compound 5c showed the best hCA I inhibition selectivity over
hCA II inhibition.
We thank Prof. Ramazan Altundas of Ataturk University for his useful
discussions, F. Melike Al of University of Applied Sciences (BeuthHS)
for her support for the synthetic studies, Dr. Meryem Topal for carbonic
anhydrase inhibition activity studies, Dr. Ilker Un and Muhiddin Cergel
for NMR spectrums and Gokhan Bilsel for HRMS spectrums.
Declaration of interest
The authors have declared no conflict of interest.
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Supplementary material available online