Synthesis and carbonic anhydrase inhibitory properties of 1,3-dicarbonyl derivatives of methylaminobenzene-sulfonamide

Article abstract of DOI:10.3109/14756366.2012.757603

1,3-Dicarbonyl derivatives of methylaminobenzene-sulfonamide were synthesized and their inhibitory effects on the activity of purified human carbonic anhydrase (hCA) I and hCA II were evaluated. hCA I and hCA II from human erythrocytes were purified by a

Full text of DOI:10.3109/14756366.2012.757603

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J Enzyme Inhib Med Chem, 2014; 29(1): 132–136
2014 Informa UK Ltd. DOI: 10.3109/14756366.2012.757603
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RESEARCH ARTICLE
Synthesis and carbonic anhydrase inhibitory properties of
1,3-dicarbonyl derivatives of methylaminobenzene-sulfonamide
1
1
2
2
Tuna Demirci , Mustafa Arslan , C¸igdem Bilen , Dudu Demir , Nahit Genc¸er , and Oktay Arslan
2
2
˘
2
1Chemistry Department, Faculty of Arts and Sciences, Sakarya University, Sakarya, Turkey, and Chemistry Department, Faculty of Arts and
Sciences, Balikesir University, Balikesir, Turkey
Abstract
Keywords
1,3-Dicarbonyl derivatives of methylaminobenzene-sulfonamide were synthesized and their
inhibitory effects on the activity of purified human carbonic anhydrase (hCA) I and hCA II were
evaluated. hCA I and hCA II from human erythrocytes were purified by a simple one-step
procedure by using Sepharose 4B-L-tyrosine-sulfanilamide affinity column. Our results show
that the synthesized compounds inhibited the activity of carbonic anhydrase (CA) I and CA II.
Among them, 2b and 2e were found to be the most active (IC50 ¼ 2.12 and 2.52 mM) for hCA I
and hCA II, respectively.
Carbonic anhydrase, 1,3-dicarbonyl, enzyme
inhibitor, sulfonamide
History
Received 25 July 2012
Revised 7 December 2012
Accepted 7 December 2012
Published online 28 January 2013
Introduction
derivatives are well known pharmaceutical agents and have
gained much attention due to their diverse biological activities in
pharmaceutical as well as in agricultural areas15. Some of
1,3-diketone compounds and their barbituric and thiobarbituric
derivatives were prepared and urease inhibition studies were
reported16,17. The sulfonamide compounds have a number of
biological activities such as antibacterial18, insulin releasing19,
anti-inflammatory20, antitumor21 and CA inhibitory22.
Carbonic anhydrases (CAs, EC 4.2.1.1) are zinc metalloenzymes
that assist a very simple physiological reaction, rapid interconver-
sion of carbon dioxide and water into protons and bicarbonate
ions1. The zinc ion that is essential for catalysis is found within
the active site of the most CAs. The reaction is involved in many
pathological and physiological processes that include respiration
and transport of CO2 and bicarbonate between metabolizing
tissues and lungs, biosynthetic reactions, electrolyte secretion in
Sulfonamides are the best known inhibitors of CA enzymes
and used for the treatment of glaucoma in medicinal chemistry23.
Acetazolamide (AAZ), dorzalamide (DZA) and brinzolamide
(BRZ) are sulfonamide derivatives and used in the treatment of
glaucoma. However, the drugs have side effects such as numbness
and tingling in the fingers and toes, blurred vision, kidney stones,
an increase in urination, upset stomach, dry eye and headache or
various tissues and organs, pH and CO2 homeostasis2–4
.
Up to now, 16 human CA (hCA) isoforms have been identified
exhibiting significant differences in catalytic activity, subcellular
localization and tissues expression. They play important roles in
many of physiological processes such as several biochemical
pathways, cell proliferation, intra- and extracellular pH home-
ostasis and differentiation, and modulation of neuronal transmis-
sion5,6. In human, CAs are found in a variety of tissues such as
kidneys, lungs, eyes, skins, nerves systems and gastrointestinal
tract7. The different isozymes are found in different part of cell,
and hCA I and hCA II are localized in the cytosol2. Biological
activities of this metalloenzyme family have several medicinal
dizzines24,25
.
In this study, 1,3-dicarbonyl derivatives of methylaminoben-
zene-sulfonamide were synthesized and their inhibitory effects on
the activity of purified human hCA I and hCA II were evaluated.
Materials and methods
applications such as treatment of glaucoma, diuretics and The compounds were prepared from a mixture of sulfanilamide,
management of several neurological disorders, whereas several 1,3-dicorbonyl compounds and triethyl [orthoformate] in ethanol
agents are in clinical evaluations as antiobesity or antidrugs8.
by refluxing for 3 h and shown in Scheme 1. The prepared
1
There are abundant number of molecules containing sulfona- compounds were characterized by H NMR, 13C NMR, IR and
mide moiety9. The compounds have several applications such as elemental analysis.
modified oligonucleotides10, amines synthons11, peptidomi-
metics12, chiral auxilliaries13 and therapeutics14. Sulfonamide General procedure
Melting points were taken on
a Yanagimoto Barnstead
Electrothermal (Surrey, UK) micro-melting point apparatus and
were uncorrected. IR spectra were measured on a Shimadzu
Address for correspondence: Nahit Genc¸er, Chemistry Department,
Faculty of Arts and Sciences, Biochemistry Division, Balikesir
University, Cagis kampus, Balikesir 10145, Turkey. Tel: þ90-266-612-
1278. Fax: þ90-266-612-1215. E-mail: ngencer@balikesir.edu.tr
1
Prestige-21 (200 VCE) spectrometer (Kyoto, Japan). H and 13C
NMR spectra were measured on spectrometer at Varian Infinity

DOI: 10.3109/14756366.2012.757603
Synthesis and CA inhibitions of some sulfonamides 133
Scheme 1. Synthesis of 1,3-dicarbonyl derivatives of methylaminobenzene-sulfonamide derivatives.
Plus 300 and at 75 Hz (California), respectively. 1H and 13C j ¼ 13.9), 7.88 (2H, d, ¼CH, j ¼ 8.7), 7.45 (2H, d, ¼CH, j ¼ 8.7),
chemical shifts were referenced to the internal deuterated solvent. 7.22 (NH2), 1.86 (6H, s, –CH3), 13C NMR (75 MHz, DMSO-d6,
The elemental analysis was carried out with a Leco CHNS-932 ppm): 164.4, 163.3, 153.9, 141.9, 127,8, 119.9, 105,0, 88.5, 39.3,
instrument (St. Joseph, MI). Flash column chromatography was 27.2. Anal. Calcd for C13H14N2O6S (%): C, 47.85; H, 4.32; N,
performed using Merck silica gel 60 (230–400 mesh ASTM) 8.58; O, 29.42; S, 9.83. Found (%): C, 47.99; H, 4.39; N, 8.64; O,
(Darmstadt, Germany). All chemicals were purchased from 29.47; S, 9.89.
Merck (Darmstadt, Germany), Alfa Easer (Ward Hill, MA) and
Sigma-Aldirch (Taufkirchen, Germany).
Synthesis of 1,3-dicarbonyl substituted methylaminobenzene-
sulfonamide derivatives were prepared according to Scheme 1.
4-((4-(Furan-2-yl)-2,6-dioxocyclohexylidene)methylamino)ben-
zene-sulfonamide (2d)
Yield 75.3%, m.p. 278.3 ꢀC; IR (KBr, ꢀ, cmꢁ1): 3321 (NH2), 3180
(NH), 3082 (¼C–H, aromatic), 1596 (C¼O); 1H NMR (300 MHz,
DMSO-d6, ppm): 12.63 (1H, –NH, j ¼ 13.6), 8.53 (1H, d, ¼CH,
j ¼ 13.6), 7.83 (2H, d, ¼CH, j ¼ 8.7), 7.70 (2H, d, ¼CH, j ¼ 8.7),
7.52 (NH2), 6.41 (1H, s, ¼CH), 6.38 (1H, d, ¼CH, j ¼ 3.1), 6.16
General procedure for the synthesis of 1,3-dicarbonyl
substituted methylaminobenzene-sulfonamide
derivatives
A mixture of sulfanilamide (18.25 g, 0.106 mol), 1,3-dicarbonyl (1H, d, ¼CH, j ¼ 3.1), 3.51–3.59 (1H, m, –CH), 2.68–2.95 (4H,
compound (0.127 mol) and triethyl [orthoformate] (16 mL) in m, –CH2), 13C NMR (75 MHz, DMSO-d6, ppm): 198.7, 194.6,
ethanol (100 mL) was refluxed for 3 h. After cooling in room 156.8, 150.6, 142.7, 141.9, 141.7, 128.0, 119.4, 115.1, 110.6,
temperature, the product was filtered, washed with cold ethanol 105.7, 42.3, 42.0, 30.7. Anal. Calcd for C17H16N2O5S (%): C,
and air dried.
56.66; H, 4.47; N, 7.77; O, 22.20; S, 8.90. Found (%): C, 56.73; H,
4.52; N, 7.79; O, 22.26; S, 8.92.
4-((2,6-Dioxocyclohexylidene)methylamino)benzene-
sulfonamide (2a)
4-((1,3-Dimethyl-2,4,6-trioxo-tetrahydropyrimidin-5(6H)-ylide-
ne)methylamino)benzene-sulfonamide (2e)
Yield 85.8%, m.p. 270.2 ꢀC; IR (KBr, ꢀ, cmꢁ1): 3296 (NH2), 3203
(NH), 3072 (¼C–H, aromatic), 1653 (C¼O); 1H NMR (300 MHz, Yield 87.6%, m.p. 294.5 ꢀC; IR (KBr, ꢀ, cmꢁ1): 3307 (NH2), 3190
DMSO-d6, ppm): 12.62 (1H, d, –NH, j ¼ 13.5), 8.56 (1H, d, ¼CH, (NH), 3062 (¼C–H, aromatic), 1633 (C¼O); 1H NMR (300 MHz,
j ¼ 13.5), 7.84 (2H, d, ¼CH, j ¼ 8.7), 7.71 (2H, d, ¼CH, j ¼ 8.7), DMSO-d6, ppm): 11.98 (1H, d, –NH, j ¼ 13.9), 8.61 (1H, d, ¼CH,
7.41 (–NH2), 2.42 (2H, t, –CH2), 2.39 (2H, t, –CH2), 1.96 (2H, m, j ¼ 8.7), 7.86 (2H, d, ¼CH, j ¼ 8.7), 7.65 (2H, d, ¼CH, j ¼ 13.9),
–CH2), 13C NMR (75 MHz, DMSO-d6, ppm): 200.7, 196.4, 150.6, 7.43 (NH2), 3.21 (6H, s, N–CH3), 13C NMR (75 MHz, DMSO-d6,
142.0, 141.6, 128.0, 119.3, 111.0, 38.3, 37.9, 19.7. Anal. Calcd for ppm): 164.5, 162.4, 152.2, 151.95, 141.7, 141.6, 128.0, 119.3,
C13H14N2O4S (%): C, 53.05; H, 4.79; N, 9.52; O, 21.74; S, 10.89. 94.2, 28.3, 27.6. Anal. Calcd for C13H14N4O5S (%): C, 46.15; H,
Found (%): C, 53.18; H, 4.82; N, 9.59; O, 21.80; S, 10.96.
4.17; N, 16.56; O, 23.64; S, 9.48. Found (%): C, 46.21; H, 4.24; N,
16.59; O, 23.68; S, 9.52.
4-((4,4-Dimethyl-2,6-dioxocyclohexylidene)methylamino)
benzene-sulfonamide (2b)
4-((2,4,6-Trioxo-tetrahydropyrimidin-5(6H)-ylidene)methylami-
no)benzene-sulfonamide (2f)
Yield 87.2%, m.p. 305.4 ꢀC; IR (KBr, ꢀ, cmꢁ1): 3277 (NH2), 3203
(NH), 3065 (¼C–H, aromatic), 1666 (C¼O); 1H NMR (300 MHz, Yield 89.3%, m.p.4350 ꢀC; IR (KBr, ꢀ, cmꢁ1): 3346 (NH), 3209
DMSO-d6, ppm): 12.65 (1H, d, –NH, j ¼ 13.5), 8.50 (1H, d, ¼CH, (NH2), 3105 (NH), 3042 (¼C–H, aromatic), 1643 (C¼O); 1H
j ¼ 13.5), 7.80 (2H, d, ¼CH, j ¼ 7.9), 7.58 (2H, d, ¼CH, j ¼ 7.9), NMR (300 MHz, DMSO-d6, ppm): 11.95 (1H, d, –NH, j ¼ 13.7),
7.28 (NH2), 2.38 (2H, s, –CH2), 2.36 (2H, s, –CH2), 1.02 (6H, s, 11.12 (1H, –NH), 10.97 (1H, –NH), 8.64 (1H, d, ¼CH, j ¼ 13.7),
–CH3), 13C NMR (75 MHz, DMSO-d6, ppm): 199.9, 195.9, 150.0, 7.84 (2H, d, ¼CH, j ¼ 8.8), 7.72 (2H, d, ¼CH, j ¼ 8.8), 7.41
141.8, 141.5, 128.1, 118.9, 109.7, 51.7, 51.4, 39.2, 31.3, 28.5. Anal. (NH2), 13C NMR (75 MHz, DMSO-d6, ppm): 166.7, 164.1, 151.7,
Calcd for C15H18N2O4S (%): C, 55.88; H, 5.63; N, 8.69; O, 19.85; 151.3, 141.85, 141.4, 128.0, 119.2, 94.3. Anal. Calcd for
S, 9.95. Found (%): C, 55.99; H, 5.7; N, 8.71; O, 19.93; S, 9.98.
C11H10N4O5S (%): C, 42.58; H, 3.25; N, 18.06; O, 25.78; S,
10.33. Found (%): C, 42.68; H, 3.29; N, 18.16; O, 25.98; S, 10.55.
4-((2,2-Dimethyl-4,6-dioxo-1,3-dioxan-5-ylidene)methylamino)-
benzene-sulfonamide (2c)
4-((1,3-Dioxo-1H-inden-2(3H)-ylidene)methylamino)benzene-sul-
fonamide (2g)
Yield 80.6%, m.p. 263.2 ꢀC; IR (KBr, ꢀ, cmꢁ1): 3365 (NH2), 3269
(NH), 3075 (¼C–H, aromatic), 1631 (C¼O); 1H NMR (300 MHz, Yield 90.5%, m.p. 307.8 ꢀC; IR (KBr, ꢀ, cmꢁ1): 3367 (NH2), 3278
DMSO-d6, ppm): 11.25 (1H, d, –NH, j ¼ 13.9), 8.64 (1H, d, ¼CH, (NH), 3052 (¼C–H, aromatic), 1651 (C¼O); 1H NMR (300 MHz,

134 T. Demirci et al.
J Enzyme Inhib Med Chem, 2014; 29(1): 132–136
Table 1. The IC50 values of the synthesized compounds.
DMSO-d6, ppm): 11.25 (1H, d, –NH, j ¼ 13.8), 8.38 (1H, d, ¼CH,
j ¼ 13.8), 7.84 (2H, d, ¼CH), 7,80 (2H, d, ¼CH), 7.78 (2H, t,
¼CH), 7.72 (2H, t, ¼CH), 7.39 (NH2), 13C NMR (75 MHz,
DMSO-d6, ppm): 191.9, 189.6, 144.5, 142.4, 141.1, 140.2,
140.2, 134.9, 127.8, 122.5, 122.2, 119.0, 107.3. Anal.
Calcd for C16H12N2O4S (%): C, 58.53; H, 3.68; N, 8.53; O,
19.49; S, 9.77. Found (%): C, 58.65; H, 3.75; N, 8.61; O, 19.57;
S, 9.96.
Compounds
hCA I IC50 (mM)
hCA II IC50 (mM)
2a
2b
2c
2d
2e
2f
4.25
2.12
6.56
8.61
5.20
10.89
3.19
4.8
4.93
6.41
18.64
7.21
2.52
9.92
3.36
3.36
2g
2h
4-((2,5-Dioxocyclopentylidene)methylamino)benzene-
sulfonamide (2h)
Yield 78.7%, m.p. 290.7 ꢀC; IR (KBr, ꢀ, cmꢁ1): 3296 (NH2), 3203
(NH), 3076 (¼C–H, aromatic), 1593 (C¼O); 1H NMR (300 MHz,
DMSO-d6, ppm): 11.95 (1H, d, –NH, j ¼ 13.2), 8.27 (1H, d, ¼CH,
j ¼ 13.2), 7.80 (2H, d, ¼CH, j ¼ 6.7), 7.77 (2H, d, ¼CH, j ¼ 6.7),
7.36 (NH2), 2.39 (2H, d, –CH2), 2.36 (2H, d, –CH2). 13C NMR
(75 MHz, DMSO-d6, ppm): 205.4, 201.9, 147.2, 141.8, 127.8,
119.6, 109.7, 34.5, 33.9. Anal. Calcd for C12H12N2O4S (%): C,
51.42; H, 4.32; N, 9.99; O, 22.83; S, 11.44. Found (%): C, 51.56;
H, 4.36; N, 9.91; O, 22.94; S, 11.50.
The prepared compounds were characterized by 1H NMR, 13
C
1
NMR, IR and elemental analysis. From the H NMR spectra, the
hydrogen attached to the nitrogen resonances between 11.00 and
13.00 ppm, the ¼CH proton peak comes around 8.50 ppm and NH2
protons are seen between 7.00 and 7.50. From the 13C NMR
spectra, 1,3-diketone carbonyl and barbiturate carbonyl carbons
are observed at around 200 and 160 ppm, respectively. In the
infrared spectra of compounds 2a–h, it was possible to observe the
absorptions between 3200 and 3400 cmꢁ1 relating to NH stretch,
absorptions in 1590–1700 cmꢁ1 from carbonyl moiety stretching.
Sulfonamides are coordinated to the zinc (II) ion within the
hCAs active site, whereas its organic scaffold fills the entire
enzyme cavity, making an extensive series of van der Waals and
polar interactions with amino acid residues both at the bottom,
middle and entrance of the active site cavity27.
Inhibitors of CA play an important role in ophthalmology
where they are used to reduce elevated intraocular pressure (IOC).
Acetazolamide is the most widely used inhibitor and has
advantages over the others. It is 20 times less active against
CAI than against CAII in erythrocytes28. For evaluating the
physiologically relevant hCA isozymes hCA I and hCA II
inhibitory activity, several new sulfonamide compounds were
subjected to CA inhibition assay with CO2 as substrate. The
results showed that all the compounds inhibited the enzyme
activity and the inhibition constants against CAs were given in
Table 1. It is determined that the inhibition values are between
2.12 and 10.89 mM for hCA I and between 2.52 and 18.64 mM for
hCA II. Among the compounds, 2b and 2e were found to be the
most active for CAs.
Preparation of haemolysate and purification from blood
red cells
Blood samples (25 mL) were taken from healthy human
volunteers. They were anticoagulated with acid-citrate-dextrose,
centrifuged at 2000g 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 2000g for 25 min at
4 ꢀC, and the pH of the haemolysate was adjusted to pH 8.5 with
solid Tris-base. The 25 mL haemolysate was applied to an affinity
column containing L-tyrosine-sulfonamide-Sepharose-4B26 equi-
librated with 25 mM Tris–HCl/0.1 M Na2SO4 (pH 8.5). The
affinity gel was washed with 50 mL of 25 mM Tris–HCl/22 mM
Na2SO4 (pH8.5). The hCA isozymes were then eluted with 0.1 M
NaCl/25 mM Na2HPO4 (pH 6.3) and 0.1 M CH3COONa/0.5 M
NaClO4 (pH 5.6), which recovered hCA I and hCA II,
respectively. Fractions of 3 mL were collected and their
absorbance measured at 280 nm.
CA enzyme assay
CA activity was measured by the Maren method based on the
determination of the time required for the pH to decrease from
10.0 to 7.4 due to CO2 hydration19. The assay solution was 0.5 M
Na2CO3/0.1 M NaHCO3 (pH 10.0) and phenol red was added as
the pH indicator. CO2-hydratase activity (enzyme units (EU)) was
calculated by using the equation t0 ꢁ tc/tc where t0 and tc are the
times for pH change of the nonenzymatic and the enzymatic
reactions, respectively.
Although sulfonamide compounds are mentioned very potent
inhibitor of the cytosolic isoform hCAs29, all the synthesized
substituted sulfonamides are moderate inhibitory activity for the
hCAs. Substituents on the sulfonamides affect inhibition. In this
way, methyl groups in the 2b (2.12 mM for CA I) and 2e
(2.52 mM for CA II), and aromatic group in 2g (3.19 and
3.36 mM for hCA I and hCA II, respectively) make them strong
inhibitory effects on hCAs compared to the other synthesized
sulfonamides.
In vitro inhibition studies
Although the structures of CA I and CA II are similar, there
are several differences in the active sites of amino acids.
Particularly, hydrophobic residues (Phe131, Val135 and Leu204)
on the surface of CAII play an important stabilizing role when
interacting with the hydrophobic groups of sulfonamide
inhibitors30. The sulfonamides inhibit CAs by the coordination
of the sulfonamide moiety to the metal ion (Znþ2) from the
enzyme active site, and participation in hydrophobic and
hydrophilic interactions of the organic scaffold of the inhibitor
with amino acids residues are known to be involved in the
binding process of CAs31. It is assumed that the effects are the
results of hydrophobic and van der Waals interactions between
aromatic/aliphatic part of the inhibitor molecule and active site
of amino acid residues. The compounds have a lower affinity to
For the inhibition studies of sulfonamide, different concentrations
of these compounds were added to the enzyme. Activity
percentage values of CA for different concentrations of each
sulfonamide were determined by regression analysis using
Microsoft Office 2000 Excel (New York, NY). CA enzyme
activity without a sulfonamide solution was accepted to be 100%.
Results and discussion
For evaluating the physiologically relevant hCA isozymes hCA I
and hCA II inhibitory activity, several 1,3-dicarbonyl derivatives
of methylaminobenzene-sulfonamide were subjected to CA
inhibition assay with CO2 as substrate.

DOI: 10.3109/14756366.2012.757603
Synthesis and CA inhibitions of some sulfonamides 135
CA I and CA II compared to acetazolamide32 which is used in
the treatment of glaucoma.
thiocyanato-6-arylimidazo[2,1-b]-1,3,4-thiadiazole-2-sulfonamide
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19. Maren TH. A simplified micromethod for the determination of
In conclusion, we report here a series of 1,3-dicarbonyl
derivatives of methylaminobenzene-sulfonamide which have been
assayed for the inhibition of the physiologically relevant CA
isozymes. They inhibit the CAs with the inhibition constants of
2.12–10.89 mM for hCA I and 2.52–18.64 mM for hCA II.
Compounds 2b and 2e behaved as a moderate inhibitory activity
on hCA I and hCA II with an activation constant of 2.12 and
2.52 mM, respectively. Last but not the least, enzyme inhibition is
more important issue for drug design and biochemical applica-
tions33–46. The results put forward that new sulfonamide
derivatives inhibited the hCA I and hCA II enzyme activity.
Therefore, our results suggested that sulfonamide derivatives are
likely to be adopted as candidates to treat glaucoma and they may
be taken for further evaluation in in vivo studies.
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Declaration of interest
This work was supported by Research Fund of the Sakarya University.
Project Number: 2012-02-04-033.
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