Synthesis of 5-amino-1,3,4-thiadiazole-2-sulphonamide derivatives and their inhibition effects on human carbonic anhydrase isozymes

Article abstract of DOI:10.3109/14756366.2010.491795

In this study, some novel inhibitors were synthesised from the further stage reactions of 4-benzoyl-1-(4-nitrophenyl)-5-phenyl-1H-pyrazole-3-carbonyl chloride with 5-amino-1,3,4-thiadiazole-2-sulphonamide 1 (inhibitor 1). They were characterised by elemen

Full text of DOI:10.3109/14756366.2010.491795

Journal of Enzyme Inhibition and Medicinal Chemistry, 2011; 26(2): 231–237
© 2011 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2010.491795
rESEarCH artICLE
Synthesis of 5-amino-1,3,4-thiadiazole-2-sulphonamide
derivatives and their inhibition effects on human carbonic
anhydrase isozymes
Rahmi Kasımoğulları1, Metin Bülbül1, Samet Mert1, and Hülya Güleryüz
Dumlupinar University, Faculty of Arts and Sciences, Department of Chemistry, Kutahya, Turkey
abstract
In this study, some novel inhibitors were synthesised from the further stage reactions of 4-benzoyl-1-(4-nitrophenyl)-
5-phenyl-1H-pyrazole-3-carbonyl chloride with 5-amino-1,3,4-thiadiazole-2-sulphonamide 1 (inhibitor 1). They were
characterised by elemental and spectral (1H NMR, 13C NMR, IR) analyses. Human carbonic anhydrase isoenzymes
(hCA-I and hCA-II) were purified from erythrocyte cells by affinity chromatography. The inhibitory effects of inhibitor
1, acetazolamide (2) and the 11 newly synthesised amides (8–18) on the hydratase and esterase activities of these
isoenzymes (hCA-I and hCA-II) were studied in vitro. In relation to these activities, the inhibition equilibrium constants
(Ki) were determined. The Ki values for the new compounds (8–18) were observed to be well below that of the parent
compound inhibitor 1 and were also compared to 2 under the same experimental conditions. The comparison of the
newly synthesised amides to inhibitor 1 and to 2 indicated that the new derivatives preferentially inhibited hCA-II
and were more potent inhibitors of hCA-II than the parent inhibitor 1 and 2.
Keywords: Pyrazole, pyrazole-3-carboxylic acid, inhibition effect, 1,3,4-thiadiazole-2-sulphonamide, antiglaucoma
Introduction
and used in the treatment of glaucoma and one of them
is acetazolamide (2). Compound 2 is used systematically
(i.e. an oral medication) and reduces intraocular pres-
sure by lowering fluid formation in the eye. A number of
side effects of this drug are however experienced such
e sulphonamides constitute an important class of drugs
which show many pharmacologic activities such as anti-
bacterial,antihypertensive,antiviral,anti-thyroid,diuretic,
hypoglycaemic and anti-tumour activity. Furthermore the
sulphonamides are the best-known inhibitors of the car-
bonic anhydrase enzyme, currently used for the treatment
of glaucoma in clinical medicine [1–20].
Glaucoma is a group of diseases characterised by
a gradual loss of the visual field due to an elevation in
intraocular pressure (IOP) and is the second leading cause
of blindness worldwide [14,15]. Since carbonic anhydrase
inhibitors have been shown to reduce intraocular pres-
sure exclusively by lowering the aqueous humour flow,
these compounds have been used for the treatment of
glaucoma for years [16,17].
O
N
N
N
N
H3C
H2N
SO2NH2
N
H
SO2NH2
S
S
1
2
SO2NH2
N
SO2NH2
Cl
SO2NH2
S
EtO
Cl
3
4
5-amino-1,3,4-thiadiazole-2-sulphonamide (1) has
several biological activities and antibacterial properties
[21].Severalderivativesofthisdrughavebeensynthesised
Figure 1. Structures of several CA inhibitors used in glaucoma
treatment.
Address for Correspondence: Rahmi Kasımoğulları, Dumlupinar University, Faculty of Arts and Sciences, Department of Chemistry,
Kutahya, Turkey. Tel: +90 274 265 2031 3204; Fax: +90 274 265 2056; E-mail: rahmikasimoglu@hotmail.com
(Received 08 March 2010; revised 27 April 2010; accepted 04 May 2010)
231

232 Rahmi Kasımoğulları et al.
as numbness and tingling in the fingers and toes, taste
alterations, blurred vision, kidney stones and an increase
in urination. Ethoxzolamide (3) and dichlorophenamide
(4) are two other carbonic anydrase inhibitors, formu-
lated for topical ophthalmic use (see Figure 1 for the
structures) [22,23]. Nevertheless, people taking these
two drugs also experience side effects as stinging, burn-
ing, blurred vision, upset stomach, dry eye, headache or
dizziness [22,24–26]. e purpose of the present study
was to synthesise and investigate some new inhibitors of
carbonic anydrase isoenzymes with a potential use in the
treatment of glaucoma.
had been prepared previously. Finally, the coloured pre-
cipitate was filtered and purified from the ethanol.
4-Benzoyl-1-(4-((2-hydroxynaphthalen-1-yl)diazenyl)
phenyl)-5-phenyl-N-(5-sulphamoyl-1,3,4-thiadiazol-2-yl)-1H-
pyrazole-3-carboxamide (8)
is was produced according to the method in the gen-
eral procedure; 567 mg, 81%; mp 284–285°C; IR (ν, cm−1):
3388 (OH), 3200 (NH), 3027 (Ar CH), 1686 (C=O), 1597–
1
1448 (Ar C=C and C=N); H NMR (400 MHz, DMSO–d6)
δ (ppm): 15.7 (s, 1H, Ar–OH), 14.19 (br, s, 1H, CONH),
8.39 (s, 2H, SO2NH2), 8.56–6.69 (m, 20H, ArH); 13C NMR
(100 MHz, DMSO–d6) δ (ppm): 190.55 (benzoyl C=O),
165.35 (amide C=O), 161.44 and 160.35 (thiadiazole
C–2 and C–5), 144.74 (=C–OH), 144.47, 143.22, 141.61,
137.69, 137.13, 134.1, 133.09, 130.37, 130.34, 130.32,
130.21, 130.12, 129.75, 129.66, 129.48, 129.09, 128.42,
127.88, 127.47, 126.81, 125, 123.12, 122.13, 119.22; Anal.
Calcd. for C35H24N8O5S2: C, 59.99; H, 3.45; N, 15.99; S, 9.15.
Found: C, 59.82; H, 3.49; N, 16.03; S, 9.11.
materials and methods
General
e chemical compounds used in this research were of
analytical purity and the solvents were purified by using
the appropriate purifying agents and distillation. All melt-
ing points were recorded on a Barnstead Electrothermal
9200 apparatus and were uncorrected. e IR spectrum
data of the compounds were determined by a Mattson
1000 FT-IR using KBr pellets. 1H NMR and 13C NMR spec-
tra were evaluated by Bruker DPX-400, (400MHz) and
High Performance Digital FT-NMR (100 MHz) spectrom-
eters. At the end of the each experiment TLC was per-
formed using DC Alufolien Kiesegel 60F/254 Merck and
Camag TLC devices. Elemental analyses were carried out
on a Leco CHNS-932 instrument.
4-Benzoyl-1-(4-((4-hydroxyphenyl)diazenyl)phenyl)-5-phenyl-
N-(5-sulphamoyl-1,3,4-thiadiazol-2-yl)-1H-pyrazole-3-
carboxamide (9)
is was produced according to the method in the gen-
eral procedure; 429 mg, 66%; mp 175–177°C; IR (ν, cm−1):
3375 (OH), 3195 (NH), 3028 (Ar CH), 1679 (C=O), 1595–
1447 (Ar C=C and C=N); 1H NMR (400 MHz, DMSO–d6) δ
(ppm): 13.2 (s, 1H, CONH), 8.55–7.83 (br, s, 1H, Ar–OH),
7.79 (s, 2H, SO2NH2), 7.5–6.89 (m, 18H, ArH); 13C NMR
(100 MHz, DMSO–d6) δ (ppm): 195.63 (benzoyl C=O),
169.18 (amide C=O), 165.56 and 165.35 (thiadiazole C–2
and C–5), 148.96 (=C–OH), 148.61, 143.49, 142.48, 138.15,
138.11, 134.57, 134.29, 134.15, 133.9, 133.54, 133.43,
133.28, 132.5, 130.25, 130.21, 127.58, 121.53, 120.89; Anal.
Calcd. for C31H22N8O5S2: C, 57.22; H, 3.41; N, 17.22; S, 9.86.
Found: C, 57.11; H, 3.45; N, 17.21; S, 9.92.
Synthesis of organic compounds
1-(4-Aminophenyl)-4-benzoyl-5-phenyl-N-(5-
sulphamoyl-1,3,4-thiadiazole-2-yl)-1H-pyrazole-3-
carboxamide (7)
e starting compound (7) containing aromatic primary
amine function was prepared as described previously
[2] and crystallised from methanol. 465 mg, 85%; mp
183°C; IR (ν, cm−1): 3312–3216 (NH), 3026 (Ar CH), 1665
(C=O), 1596–1426 (Ar C=C and C=N); 1H NMR (400 MHz,
DMSO–d6) δ (ppm): 6.51 (m, 2H, Ar NH2), 8.37 (m, 2H,
SO2NH2), 7.8–6.82 (m, 14H, ArH); 13C NMR (100 MHz,
DMSO–d6) δ (ppm): 190.58 (benzoyl C=O), 165.36 (amide
C=O), 161.44 and 160.44 (thiadiazole C–2 and C–5), 152.2,
148.8, 144.57, 143.36, 137.58, 134.1, 130.48, 130.13, 129.64,
129.06, 127.65, 127.4, 127.3, 122.91, 114.4; Anal. Calcd. for
C25H19N7O4S2: C, 55.04; H, 3.51; N, 17.97; S, 11.75. Found:
C, 55.32; H, 3.59; N, 17.75; S, 11.6.
4-Benzoyl-1-(4-iodophenyl)-5-phenyl-N-(5-sulphamoyl-1,3,4-
thiadiazol-2-yl)-1H-pyrazole-3-carboxamide (10)
e diazonium salt solution of the aromatic amine com-
pound was prepared according to the method in the gen-
eral procedure. e KI (166 mg, 1 mmol) was dissolved in
ethanol and cooled to 0–5°C and added dropwise to the
diazonium salt solution which was prepared previously.
e precipitated yellow product was filtered and purified
from ethanol. 400 mg, 61%; mp 160–162°C; IR (ν, cm−1):
3320 (NH), 3005 (Ar CH), 1672 (C=O), 1595–1447 (Ar C=C
and C=N); 1H NMR (400 MHz, CDCl3) δ (ppm): 12.15 (br,
s, 1H, CONH), 7.77 (s, 2H, SO2NH2), 7.61–6.75 (m, 14H,
ArH); 13C NMR (100 MHz, CDCl3) δ (ppm): 190.5 (benzoyl
C=O), 165.45 (amide C=O), 161.43 and 160.38 (thiadiazol
C–2 and C–5), 97.09 (ArC–I), 144.48, 143.25, 143.06, 139.7,
139.61, 137.63, 134.07, 130.26, 129.98, 129.83, 129.63,
129.07, 128.92, 122.5; Anal. Calcd. for C25H17IN6O4S2: C,
45.74; H, 2.61; N, 12.80; S, 9.77. Found: C, 45.63; H, 2.64;
N, 12.82; S, 9.81.
General procedure for the synthesis of compounds 8–17
Sodium acetate (3 g) was dissolved in 10 mL H2O and
2 mL HCl. Aromatic amine (1 mmol) and alcohol were
added to the mixture until it dissolved. e temperature
was adjusted to 0–5°C. A solution of NaNO2 (83 mg, 1.2
mmol) in 10 mL H2O was added to the mixture slowly and
the process of diazotisation was performed. e aromatic
and β-diketone (1 mmol) compounds were dissolved in
ethanol and added dropwise to the diazonium salt which
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis of 5-amino-1,3,4-thiadiazole-2-sulphonamide derivatives 233
(q, 2H, OCH2), 1.32(t, 3H, CH3), 7.78–7.08 (m, 19H, ArH);
13C NMR (100 MHz, CDCl3) δ (ppm): 191.39 and 189.46
(benzoyl C=O), 164.63 (ester C=O), 163.59 (amide C=O),
161.67 and 158.64 (thiadiazole C–2 and C–5), 61.72
4-benzoyl-1-(4-(2-(2,4-dioxopentan-3-ylidene)
hydrazinyl)phenyl)-5-phenyl-N-(5-sulphamoyl-1,3,4-
thiadiazol-2-yl)-1H-pyrazole-3-carboxamide (11)
433mg, 66%; mp 271–273°C; IR (ν, cm−1): 3170 (NH), 3028
(CH2), 13.99 (CH3), 144.65, 142.09, 141.85, 137.17, 137.08,
134.73, 134.04, 133.71, 132.87, 130.33, 129.8, 129.68,
128.81, 128.69, 128.53, 128.45, 128.17, 127.91, 127.27,
126.47, 122.59, 115,51; Anal. Calcd. for C36H28N8O7S2: C,
57.74; H, 3.77; N, 14.96; S, 8.56. Found: C, 57.61; H, 3.79;
N, 14.99; S, 8.54.
(Ar CH), 2971 (aliphatic CH), 1686 (C=O), 1596–1445 (Ar
C=C and C=N); H NMR (400MHz, DMSO–d6) δ (ppm):
1
13.81 (s, 1H, CONH), 8.36 (s, 2H, SO2NH2), 2.48 and 2.41
(s, 6H, 2CH3), 7.82–7.24 (m, 14H, ArH); 13C NMR (100MHz,
DMSO–d6) δ (ppm): 197.49 and 196.87 (acetyl C=O),
190.56 (benzoyl C=O), 165.34 (amide C=O), 161.42 and
160.36 (thiadiazole C–2 and C–5), 31.71 and 26.87 (CH3),
144.49, 143.06, 142.59, 137.68, 135.31, 134.79, 134.06,
130.29, 130.02, 129.63, 129.08, 129.03, 127.85, 127.54,
122.91, 116.91; Anal. Calcd. for C30H24N8O6S2: C, 54.87; H,
3.68; N, 17.06; S, 9.77. Found: C, 54.80; H, 3.69; N, 17.08;
S, 9.75.
Ethyl 2-(2-(4-(4-benzoyl-5-phenyl-3-(5-sulphamoyl-
1,3,4-thiadiazol-2-ylcarbamoyl)-1H-pyrazol-1-yl)phenyl)
hydrazono)-3-oxobutanoate (15)
439mg, 64%; mp 193–195°C; IR (ν, cm−1): 3212 (NH), 3018
(Ar CH), 2971 (aliphatic CH), 1664 (C=O), 1600–1427 (Ar
1
C=C and C=N); H NMR (400MHz, DMSO–d6) δ (ppm):
14.02 (br, s, 1H, CONH), 11.73 (s, 1H, Ar–NH–N=C), 8.33
(s, 2H, SO2NH2), 4.32 (q, 2H, OCH2), 2.51 (s, 3H, COCH3)
1.28(t, 3H, CH3), 7.79–7.08 (m, 14H, ArH); 13C NMR
(100MHz, DMSO–d6) δ (ppm): 194.29 (acetyl C=O),
190.72 (benzoyl C=O), 164.86 (ester C=O), 162.86 (amide
C=O), 162.15 and 160.23 (thiadiazole C–2 and C–5), 61.79
(CH2), 25.79 (COCH3), 14.32 (CH3), 154.77, 144.29, 143.31,
137.81, 137.62, 133.96, 133.19, 130.25, 129.88, 129.59,
129.25, 129.04, 128, 127.51, 122.72, 115,67; Anal. Calcd. for
C31H26N8O7S2: C, 54.22; H, 3.82; N, 16.32; S, 9.34. Found: C,
54.15; H, 3.82; N, 16.30; S, 9.37.
4-Benzoyl-1-(4-(2-(1,3-dioxo-1-phenylbutan-2-ylidene)
hydrazinyl)phenyl)-5-phenyl-N-(5-sulphamoyl-1,3,4-
thiadiazol-2-yl)-1H-pyrazole-3-carboxamide (12)
445mg, 62%; mp 187–190 °C; IR (ν, cm−1): 3190 (NH), 3005
(Ar CH), 2971 (aliphatic CH), 1674 (C=O), 1597–1447 (Ar
1
C=C and C=N); H NMR (400MHz, DMSO–d6) δ (ppm):
13.86 (br, s, 1H, CONH), 11.31 (s, 1H, Ar–NH−N=C), 8.35
(s, 2H, SO2NH2), 1.92 (s, 3H, CH3), 7.81–7.11 (m, 19H,
ArH); 13C NMR (100MHz, DMSO–d6) δ (ppm): 196.6
(acetyl C=O), 195.43 and 190.66 (benzoyl C=O), 165.11
(amide C=O), 161.74 and 160.7 (thiadiazole C–2 and C–5),
25.42 (CH3), 144.38, 143.85, 140.27, 137.74, 136.01, 134.91,
134.01, 133.45, 130.58, 130.26, 129.9, 129.6, 129.52, 129.25,
129.06, 128.99, 128.4, 127.96, 127.48, 122.86, 122,72,
115.15; Anal. Calcd. for C35H26N8O6S2: C, 58.49; H, 3.65; N,
15.59; S, 8.92. Found: C, 58.38; H, 3.68; N, 15.63; S, 8.96.
Diethyl 2-(2-(4-(4-benzoyl-5-phenyl-3-(5-sulphamoyl-
1,3,4-thiadiazol-2-ylcarbamoyl)-1H-pyrazol-1-yl)phenyl)
hydrazono)malonate (16)
358mg, 50%; mp 170–172°C; IR (ν, cm−1): 3184 (NH), 3019
(Ar CH), 2970 (aliphatic CH), 1740 (C=O), 1610–1429 (Ar
1
C=C and C=N); H NMR (400MHz, DMSO–d6) δ (ppm):
4-Benzoyl-1-(4-(2-(1,3-dioxo-1,3-diphenylpropan-2-ylidene)
hydrazinyl)phenyl)-5-phenyl-N-(5-sulphamoyl-1,3,4-
thiadiazol-2-yl)-1H-pyrazole-3-carboxamide (13)
12 (br, s, 1H, CONH), 8.3 (s, 2H, SO2NH2), 4.34–4.12 (q,
4H, 2OCH2), 1.28(t, 6H, 2CH3), 7.79–6.55 (m, 14H, ArH);
13C NMR (100MHz, DMSO–d6) δ (ppm): 190.79 (benzoyl
C=O), 172.50 (ester C=O), 164.59 (amide C=O), 162.7 and
161.25 (thiadiazole C–2 and C–5), 61.89 and 61.31 (CH2),
14.56 and 14.32 (CH3), 149.85, 144.17, 138.93, 137.86,
133.92, 130.24, 129.89, 129.59, 129.37, 129.03, 128.96,
128.81, 128.04, 127.26, 126.22, 122.86, 122.27, 113,67; Anal.
Calcd. for C32H28N8O8S2: C, 53.62; H, 3.94; N, 15.63; S, 8.95.
Found: C, 53.49; H, 3.97; N, 15.65; S, 8.92.
656 mg, 84%; mp 181–182°C; IR (ν, cm−1): 3180 (NH),
3027 (Ar CH), 1656 (C=O), 1596–1448 (Ar C=C and
1
C=N); H NMR (400 MHz, DMSO–d6) δ (ppm): 13.7 (br,
s, 1H, CONH), 11.7 (s, 1H, Ar–NH–N=C), 8.35 (s, 2H,
SO2NH2), 8.2–7.2 (m, 24H, ArH); 13C NMR (100 MHz,
DMSO–d6) δ (ppm): 194.17, 191.01 and 190.59 (benzoyl
C=O), 165.31 (amide C=O), 161.44 and 160.38 (thiadiaz-
ole C–2 and C–5), 144.39, 143.69, 142.89, 138.5, 137.68,
137.31, 136.44, 135.04, 134.62, 134.06, 133.78, 133.52,
133.02, 130.57, 130.27, 129.95, 129.61, 129.53, 129.33,
129.12, 128.99, 128.67, 127.9, 127.46, 122.79, 115,51;
Anal. Calcd. for C40H28N8O6S2: C, 61.53; H, 3.61; N, 14.35;
S, 8.21. Found: C, 61.45; H, 3.64; N, 14.35; S, 8.22.
tert-Butyl 2-(2-(4-(4-benzoyl-5-phenyl-3-(5-sulphamoyl-
1,3,4-thiadiazol-2-ylcarbamoyl)-1H-pyrazol-1-yl)phenyl)
hydrazono)-3-oxobutanoate (17)
366mg, 51%; mp 206–207°C; IR (ν, cm−1): 3190 (NH), 3025
(Ar CH), 2974 (aliphatic CH), 1673 (C=O), 1600–1426 (Ar
1
C=C and C=N); H NMR (400MHz, DMSO–d6) δ (ppm):
Ethyl 2-(2-(4-(4-benzoyl-5-phenyl-3-(5-sulphamoyl-
1,3,4-thiadiazol-2-ylcarbamoyl)-1H-pyrazol-1-yl)phenyl)
hydrazono)-3-oxo-3-phenylpropanoate (14)
14.18 (s, 1H, CONH), 11.92 (s, 1H, Ar–NH–N=C), 8.2 (s, 2H,
SO2NH2), 2.37 (s, 3H, CH3), 1.53 (s, 9H, C(CH3)3), 7.76–6.55
(m, 14H, ArH); 13C NMR (100MHz, DMSO–d6) δ (ppm):
193.88 (acetyl C=O), 190.46 (benzoyl C=O), 165.26 (ester
C=O), 162.03 (amide C=O), 161.38 and 160.06 (thiadiaz-
ole C–2 and C–5), 83.29 (OC(CH3)3), 28.32 (OC(CH3)3),
434 mg, 58%; mp 133–135°C; IR (ν, cm−1): 3197 (NH),
3027 (Ar CH), 2971 (aliphatic CH), 1664 (C=O), 1597–
1
1448 (Ar C=C and C=N); H NMR (400 MHz, CDCl3) δ
(ppm): 12.62 (s, 1H, CONH), 8.14 (s, 2H, SO2NH2), 4.36
© 2011 Informa UK, Ltd.

234 Rahmi Kasımoğulları et al.
26.66 (CH3), 144.11, 142.95, 142.81, 137.64, 133.74, 130.02,
129.47, 128.81, 128.78, 128.73, 127.74, 126.93, 126.87,
122.83, 115.79, 113.8; Anal. Calcd. for C33H30N8O7S2: C,
55.45; H, 4.23; N, 15.68; S, 8.97. Found: C, 55.32; H, 4.23;
N, 15.71; S, 9.02.
respectively. e IC50 values (the concentration of inhibitor
producing a 50% inhibition of CA activity) were obtained
in vitro for the synthesised compounds (8–18), with 1 and
2 as the control compounds.
Esterase activity assay
4-Benzoyl-1-(2-methyl-1-(2-oxopropyl)-1H-indol-5-yl)-5-
phenyl-N-(5-sulphamoyl-1,3,4-thiadiazol-2-yl)-1H-pyrazole-
3-carboxamide (18)
Carbonic anhydrase activity was assayed by following the
change in absorbance at 348nm of 4-nitrophenylacetate
(NPA) to 4-nitrophenylate ion over a period of 3min at 25°C
using a spectrophotometer (CHEBIOS UV-vis) according
to the method described in the literature [32,33]. e enzy-
matic reaction, in a total volume of 3mL, contained 1.4mL
of 0.05M tris–SO4 buffer (pH 7.4), 1mL of 3mM 4-nitrophe-
nylacetate, 0.5mL H2O and 0.1mL enzyme solution. A ref-
erence measurement was obtained by preparing the same
cuvette without any enzyme solution. e IC50 values were
obtained in vitro for the synthesised compounds (8–18),
with 1 and 2 as the control compounds.
e aromatic amine compound (1 mmol, 545mg) was dis-
solved in 15mL DMF and chloroacetone (0.182mL) was
added to the mixture. e reaction was maintained at 60°C
for three days in a condenser with a CaCl2 headpiece. After
the evaporation of the solvent of the dark coloured mixture
in a rotary evaporator, the remainder composed of a dense
liquid was added to water and mixed. e granulated
product was filtered and purified from ethanol. 537mg,
84%; mp 158–160°C; IR (ν, cm−1): 3240 (NH), 3059 (Ar CH),
2922 (aliphatic CH), 1689 (C=O), 1598–1448 (Ar C=C and
C=N); 1H NMR (400MHz, DMSO–d6) δ (ppm): 13.78 (br, s,
1H, CONH), 8.37 (s, 2H, SO2NH2), 6.64 (s, 1H, indole CH),
2.89 (s, 2H, CH2), 2.51 and 2.24 (s, 6H, 2CH3), 8.08–7.28
(m, 13H, ArH); 13C NMR (100MHz, DMSO–d6) δ (ppm):
203.14 (acetyl C=O), 190.52 (benzoyl C=O), 165.33 (amide
C=O), 162.79 and 161.41 (thiadiazole C–2 and C–5), 48.81
(CH2), 36.27 and 31.25 (CH3), 146.62, 144.48, 143.15, 137.75,
137.64, 134.1, 130.29, 130.21, 130.05, 129.92, 129.71, 129.63,
129.07, 128.93, 128.04, 127.33, 126.58, 123.76, 123.07; Anal.
Calcd. for C31H25N7O5S2: C, 58.20; H, 3.94; N, 15.33; S, 10.02.
Found: C, 58.07; H, 3.98; N, 15.35; S, 10.03.
Determination of Ki values
e Ki values were determined as described in the lit-
erature [34–38]. According to this study, in its first part,
the IC50 values were obtained as in vitro. IC50 values of
the inhibitors (for the synthesised compounds 8–18,
with 1 and 2 as the control compounds) were assayed
by the hydrolysis of 4-nitrophenylacetate on the esterase
activities of the CA isoenzymes in the presence of vari-
ous inhibitor concentrations. e absorbance was deter-
mined at 348 nm after 3 min [34]. Regression analysis
graphs were drawn by the plotting inhibitor concentra-
tions versus the enzyme activity by using the Microsoft
Excel Program. In the second part of the study, the con-
centrations of the inhibitors which gave results of 30%,
50% and 70% inhibition on the isoenzymes were deter-
mined at five different substrate concentrations. At each
of these inhibitor concentrations (30%, 50% and 70%),
the enzyme activity was then measured in the presence
of the various substrate concentrations given above and
the data were linearised with a Lineweaver–Burk plot in
order to obtain the Ki value.
Purification of isoenzymes hCA-I and II from human
erythrocytes
In order to purify the hCA-I and II isoenzymes, first
human blood was centrifuged at 1500 rpm for 20 min and
after the removal of the plasma, the erythrocytes were
washed with an isotonic solution (0.9% NaCl). After that,
the erythrocytes were lysed with 1.5 volume of ice-cold
water. e lysate was centrifuged at 20 000rpm for 30 min
to remove the cell membranes and non-lysed cells. e
pH of the supernatant was adjusted to 8.7 with tris and
loaded onto an affinity column containing Sepharose-
4B-L-tyrosine-p-aminobenzene sulphonamide as the
binding group. After extensive washing with 25 mM tris-
HCl/22 mM Na2SO4 (pH 8.7), the hCA-I and II isoenzymes
were eluted with 1 M NaCl/ 25 mM Na2HPO4 (pH 6.3) and
0.1 M CH3COONa/0.5 M NaClO4 (pH 5.6) [27,28]. e
amount of purified protein was estimated by the Bradford
method [29] and SDS-PAGE was carried out to determine
whether the elute contained the enzyme [30].
results and discussion
e treatment of glaucoma primarily aims at decreas-
ing the intraocular pressure, which may lead to optic
nerve damage and subsequently to vision loss. Several
of the carbonic anhydrase inhibitors currently in use act
by lowering the intraocular pressure by decreasing the
aqueous humor production [22]. However, all medica-
tions, whether topical or oral, have some benefits as well
as some side effects. Because of the ocular side effects
after administration of the sulphonamide derivatives, the
development of new carbonic anhydrase inhibitors as
candidate drugs with fewer side effects will increasingly
become valuable for the treatment of glaucoma. In this
study, 11 derivatives of sulphonamides were synthesised
and their effects on purified human carbonic anhydrase
isoenzymes (hCA-I and hCA-II) were also investigated.
Hydratase activity assay
Carbonic anhydrase (CA) activity was assayed by following
the hydration of CO2 according to the method described
by Wilbur and Anderson [31]. e CO2-hydratase activ-
ity as an enzyme unit (EU) was calculated by using the
_
equation ((t0 tc)/tc) where t0 and tc were the times for pH
change of the nonenzymatic and the enzymatic reactions,
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis of 5-amino-1,3,4-thiadiazole-2-sulphonamide derivatives 235
ranges were tested and the best yield was observed in the
Chemistry
range of pH=3.5−4. e products were mostly coloured.
e purity of each product was checked by TLC which
was performed for each of the reactions. e structures of
the compounds were characterised by spectral data (see
experimental). Compound 7 gave azo compounds by the
diazo coupling reaction with aromatic compounds such
as phenol or β–naphthol. Compound 8 was produced in
81% yield and the derivatives of compound 9 were pro-
duced in a 66% yield. Compound 10 was produced in 61%
yield by interference of the diazonium compound with KI.
For the transfer of ion of I¯, known as a strong nucleophile,
KI was sufficient without any need for a catalyser.
In order to synthesise the new compounds, firstly the acti-
vated pyrazole carboxylic acid was reacted with inhibitor
1 and a new amide derivative (6) was obtained (Scheme
1). After that, using this newly synthesised amide deriva-
tive, our target inhibitor was prepared by reducing the
NO2 group to the NH2 group (7) [39,40].
In our work, the diazotisation method was generally
used to synthesise some new derivatives of compound 7
which was an aromatic primer amine. As described previ-
ously, the diazonium salt of the compound was prepared
[2] and then the coupling and replacement reactions
were performed. During the reactions, the temperature
was controlled (0–5°C). In our experiments, different pH
SO2NH2
SO2NH2
S
S
O
O
N
O
O
N
O
N
O
N
Ph
N
Ph
Ph
OH
Ph
N
H
Na2Sx9H2O
HCl
N
H
SOCl2
N
N
N
N
Ph
N
N
Ph
SO2NH2
N
H2N
S
NO2
5
NO2
6
NH2
7
Scheme 1. Synthesis of title compound.
SO2NH2
SO2NH2
SO2NH2
S
N
N
S
N
N
O
O
N
O
O
N
S
N
N
O
O
N
Ph
Ph
Ph
Ph
NH
N
H
Ph
Ph
NH
O
Ar
Cl
OH
OH
N
N
H3C
DMF, 60 °C
8
N
N
9
NH2
7
N
CH3
Ar
N
Ar
O
O
0–5 °C
CH3
HCl
0 °C
8, 9
NaNO2
18
CH3COONa
SO2NH2
SO2NH2
S
N
N
SO2NH2
O
N
N
N
S
Ph
NH
S
N
R2
R1
O
O
N
N
O
O
O
O
11 CH3
CH3
Ph
Ph
N
Ph
NH
Ph
Ph
H
CH3
N
N
Ph
Ph
Ph
OEt
OEt
12
R2
R1
KI
Ph
13
N
0–5 °C
N
OEt
CH3
OEt
14
15
16
17
0–5 °C
N
I
N
O
OC(CH3)3 CH3
N2+Cl−
H
O
R1
R2
10
11–17
Scheme 2. Synthesis of compounds 8-18.
© 2011 Informa UK, Ltd.

236 Rahmi Kasımoğulları et al.
lower than 1 and 2 (2.8 and 3.5 μM for hCA–I and 7.4
and 7.9 μM for hCA–II respectively). Consequently the
novel compounds have significant Kİ values. The Kİ
values of some of the compounds (9–17) ranged from
0.095 to 1.532 for hCA-I. The Kİ values of some of the
compounds (8–12, 14–17) were also ranged from 0.018
to 0.848 for hCA-II (Table 1).
Generally our synthesised compounds showed
remarkable inhibition effects on hCA-I and hCA-II and
showing higher inhibition than 1 and 2. Hence these
compounds may be candidates for the treatment of
glaucoma.
Table 1. IC50 and Ki values of hCA-I and hCA-II isoenzyme
hydratase and esterase activities obtained after the inhibition
experiments.
Hydratase IC50
(μM)
Esterase IC50 (μM)
Ki values (μM)
İnhibitor
hCA-I hCA-II hCA-I hCA-II
hCA-I hCA-II
1
2
8
9
10
11
12
13
14
15
16
17
18
3.35
4.2
2.75
4.02
2.4
0.8
0.18
0.08
0.16
1
2.8
7.4
2.7
3.5
3.5
7.9
3.3
7.75
2.86
0.2
4.75
0.23
0.35
0.57
1
2.1
5.83
0.095
0.36
0.572
1.532
1.018
1.532
1.19
0.3
0.848
0.018
0.056
0.103
0.244
3.184
0.498
0.125
0.065
0.108
3.916
0.03
0.05
0.13
0.37
1.42
0.85
0.14
0.07
0.08
1.74
0.27
0.33
0.164
0.7
4.8
declaration of interest
1.32
1.21
0.85
0.78
0.75
1.3
0.8
1
0.8
0.91
0.38
0.33
2.4
is research was funded by TUBITAK (e Foundation of
Scientific and Technological Research of Turkey, 106T180)
(Ankara, Turkey).
0.2
0.6
0.31
6.671
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