Effects of new 5-amino-1,3,4-thiadiazole-2-sulfonamide derivatives on human carbonic anhydrase isozymes

Article abstract of DOI:10.1016/j.bmc.2009.03.048

Pyrazole carboxylic acid amides of 5-amino-1,3,4-thiadiazole-2-sulfonamide 1 (inhibitor 1) were synthesized from 4-benzoyl-1-(4-nitrophenyl)-5-phenyl-1H-pyrazole-3-carbonyl chloride and 4-benzoyl-1-(3-nitrophenyl)-5-phenyl-1H-pyrazole-3-carbonyl chloride

Full text of DOI:10.1016/j.bmc.2009.03.048

Bioorganic & Medicinal Chemistry 17 (2009) 3295–3301
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Effects of new 5-amino-1,3,4-thiadiazole-2-sulfonamide derivatives
on human carbonic anhydrase isozymes
*
Rahmi Kasımog˘ulları , Metin Bülbül, Hatice Günhan, Hülya Güleryüz
Dumlupinar University, Art and Science Faculty, Department of Chemistry, 43100 Kutahya, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
Pyrazole carboxylic acid amides of 5-amino-1,3,4-thiadiazole-2-sulfonamide 1 (inhibitor 1) were synthe-
sized from 4-benzoyl-1-(4-nitrophenyl)-5-phenyl-1H-pyrazole-3-carbonyl chloride and 4-benzoyl-1-(3-
nitrophenyl)-5-phenyl-1H-pyrazole-3-carbonyl chloride compounds. Human carbonic anhydrase
isoenzymes (hCA-I and hCA-II) were purified from erythrocyte cells by the affinity chromatography.
The inhibitory effects of inhibitor 1, acetazolamide (AAZ), and of 16 newly synthesized amides (8–11,
12a–f, 13a–c, 14a–b, and 15) on hydratase and esterase activities of these isoenzymes have been studied
in vitro. The average IC50 values of the new compounds (8–11,12a–f, 13a–c, 14a–b, and 15) for hydratase
Received 17 December 2008
Revised 16 March 2009
Accepted 23 March 2009
Available online 28 March 2009
Keywords:
Carbonic anhydrase
Pyrazole carboxylic acids
Sulfonamide
activity ranged from 3.25 to 4.75
of the same inhibitors for esterase activity were in the range of 2.7–6.6
of inhibitor 10, which did not inhibit the esterase activity of hCA-I) and of 0.013–4.2
lM for hCA-I and from 0.055 to 2.6
l
M for hCA-II. The mean IC50 values
M for hCA-I (with the exception
M for hCA-II. The Ki
l
l
IC50 value
values for new compounds (8–11, 12a–f, 13a–c, 14a–b, and 15) were observed well below that of the
parent compound inhibitor 1 and were also comparable to that of AAZ under the same experimental con-
ditions. The comparison of newly synthesized amides to inhibitor 1 and to AAZ indicated that the new
derivatives preferentially inhibit hCA-II and are more potent inhibitors of hCA-II than the parent inhibitor
1 and AAZ.
Hydratase and esterase activities
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
5-Amino-1,3,4-thiadiazole-2-sulfonamide 1 has several biologi-
cal activities and antibacterial properties.10 Several derivatives of
Carbonic anhydrase (CA; EC 4.2.1.1) catalyzes the reversible
hydration of carbon dioxide to bicarbonate and protons, a very
simple but critically important physiological reaction for organ-
isms.1–6 In animals, the primary function of the enzyme is to trans-
port CO2 out of the cells and also to maintain acid–base balance in
blood and other tissues by converting CO2 to bicarbonate. CA is a
zinc-containing enzyme and zinc ion prosthetic group is coordi-
nated by histidine side chains in the active site. This enzyme has
16 different isoenzymes presently known in human. Several of
these isoenzymes (CA-II and CA-IV) are expressed in human
eyes.7–10
Glaucoma is a group of diseases characterized by gradual loss of
visual field due to an elevation in intraocular pressure (IOP); the
disease is the second leading cause of blindness worldwide.10,11
Since carbonic anhydrase inhibitors have been shown to reduce
intraocular pressure exclusively by lowering the aqueous humor
flow, these compounds have been used for the treatment of glau-
coma for years.12,13 Sulfonamides are the best-known inhibitors
of carbonic anhydrase enzyme, currently used for the treatment
of glaucoma in clinical medicine.1–14
this drug have been synthesized and used in treatment of glau-
coma; one of them is acetazolamide (AAZ). AAZ is used systemat-
ically (i.e., an oral medication) and reduce intraocular pressure
by lowering the fluid formation in the eye. A number of side effects
of this drug are however experienced such as numbness and tin-
gling in the fingers and toes, taste alterations, blurred vision, kid-
ney stones, and an increase in urination. Dorzolamide (DZA) and
brinzolamide (BRZ) are two other carbonic anydrase inhibitors, for-
mulated for topical ophthalmic use (see Fig. 1 for the struc-
tures).1,15,16 Nevertheless, people taking these two drugs also
experience side effects as stinging, burning, blurred vision, upset
stomach, dry eye, headache or dizziness.1,17–19 The purpose of
the present study was thus to synthesize and investigate new
inhibitors of carbonic anydrase isoenzymes with potential use in
the treatment of glaucoma.
2. Results and discussion
Treatment of glaucoma is primarily aimed at decreasing the
intraocular pressure, which may lead to optic nerve damage and
subsequently to vision loss. Several carbonic anydrase inhibitors
in use lower the intraocular pressure by decreasing the aqueous
* Corresponding author. Tel.: +90 274 265 2031 3204; fax: +90 274 265 2056.
E-mail address: rahmikasimoglu@hotmail.com (R. Kasımog˘ulları).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.03.048

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R. Kasımog˘ulları et al. / Bioorg. Med. Chem. 17 (2009) 3295–3301
N
N
Some hydrazo derivatives (12a–f) were produced in high yields
N
N
by reaction of compound 8 with aliphatic 1,3-dicarbonile com-
pounds which include active C–H protons.23 In these compounds,
because of unshared electron couples of nitrogen, proton which
was transferred to base results in the resonance form; therefore
the resulting products may have tautomeric structures as azo (–
N@N–) or hydrazo (–NH–N@C).24 In the literature, it has been re-
ported that such compounds in solid phases and in chloroform
have only keto-hydrazo tautomeric structures because of intermo-
lecular hydrogen bonds.25 In our research, when the results of 1H
NMR spectrums are examined, it is obvious that signals of 12a–f
compounds at d = 11.88–11.27 ppm intervals are caused by hydro-
gen atoms in covalent linkage with nitrogens. Thus, these com-
pounds including aliphatic active C–H groups prefer keto-
hydrazo (–NH–N@C) tautomeric structures after their coupling.
Compound 13a was synthesized in 34% yield by interference of
diazonium compound with KCN, and compound 13b was produced
in 65% yield by interference of diazonium compound with KI. As
source of CN, CuCN was also used along with KCN. Therein, Cu+
ion acts as a catalyzer. For transfer of ion of Iꢀ which is a strong
nucleophile, KI was sufficient without any catalyzer. After heating
of diazonium compound with H2O to 100 °C, compound 13c was
produced in 53% yield. Structures of compounds were evaluated
with spectral data. Compound 8 gave azo compounds by diazo cou-
pling reactions with aromatic compounds like phenol or b-naph-
thol which are very active. Finally compound 14a was produced
in 34% yield, and derivatives of compound 14b were produced in
43% yield. Structures of compounds were confirmed by spectral
data (see Experimental), and compound 15 was produced in 57%
yield by reaction of compound 8 with acetonylchloride in DMF at
60 °C for 72 h.26 Syntheses of compounds 12a–f, 13a–c, 14a–b,
and 15 from compound 8 are concluded in Scheme 2.
SO2NH2
SO2NH2
H2N
H3CCOHN
S
S
1
AAZ
NHCH2CH3
NHCH2CH3
SO2NH2
SO2NH2
N
S
S
S
S
CH3O(CH2)3
H3C
O
O
O
O
DZA
BRZ
Figure 1. Structures of several CA inhibitors used in glaucoma treatment.
humor production.1 However, as mentioned in Introduction, all
medications whether topical or oral have some benefits as well
as some side effects. Because of the ocular side effects after admin-
istration of the sulfonamide derivatives, the development of new
carbonic anhydrase inhibitors as candidate drugs with fewer side
effects will likely become valuable for the treatment of glaucoma.
In this study, 16 derivatives of sulfanomides have been synthesized
and their effects on purified human carbonic anydrase I and II iso-
enzymes have been investigated.
In order to synthesize new compounds, first activated pyrazole
carboxylic acids were reacted with inhibitor 1, and 4 new amide
derivatives (named compound 4–7) were obtained (Scheme 1).
After that, using these newly synthesized amide derivatives, our
target inhibitors were prepared by reducing NO2 groups to NH2
groups (the target compounds are named 8–11).20,21
In our work, diazotization method was generally used to synthe-
size new derivatives of compound 8 which is an aromatic primer
amine. At first, as described previously, diazonium salt of compound
wasprepared.22 Then, couplingand replacement reactionswere per-
formed. During reactions, temperature was controlled (0–5 °C).
Some pH intervals were evaluated during the experiments and
it is observed that the best pH value was around 3.5–4. Products
are generally colorful, reaction conditions are mild, and processes
are simple. The pureness of each product was checked by TLC
workings which were performed for reactions.
Next, effects of these new inhibitors on human carbonic anhyd-
rases I (hCA-I) and II (hCA-II) purified from red blood cells were
investigated. The CA isozymes have been purified to homogeneity
by affinity chromatography.
The concentration required to inhibit hCA-I and hCA-II activities
of the purified proteins by 50% (IC50) inhibition constant Ki was
determined for each compound. The IC50 and Ki values were used
to compare the inhibitory potential of parent inhibitor 1, AAZ,
and the newly synthesized derivatives (8–11, 12a–f, 13a–c, 14a–
O
O
O
O
Cl
R
OH
R
N
N
H2N
SO2NH2
SOCl2
N
N
S
Ph
Ph
N
Ar
3
N
Ar
2
N
S
N
O
O
N
N
O
O
R
R
SO2NH2
SO2NH2
S
N
N
H
H
Ph
Ph
N
N
Na2Sx9H2O
N
N
Ar
R
R
Ar
NO2
NH2
8
9
Ph
Ph
Ph-NH2(3)
Ph-NH2(4)
Ph-NO2(3)
Ph-NO2(4)
Ph-NO2(3)
Ph-NO2(4)
4
5
6
7
Ph
Ph
8-11
4-7
10 OEt Ph-NH2(3)
11 OEt Ph-NH2(4)
OEt
OEt
Scheme 1. Synthesis of compounds 8–11.

R. Kasımog˘ulları et al. / Bioorg. Med. Chem. 17 (2009) 3295–3301
3297
N
N
SO2NH2
Ph
SO2NH2
N
N
R1
R2
Ph
CH3
OEt
Ph
O
N
12
a
b
c
d
e
f
O
O
O
S
S
O
O
CH3
CH3
OEt
OEt
Ph
OC(CH3)3
NH
NH
R1
R2
Ph
Ph
N
Ph
N
N
Ph
CH3
O
O
N
O
N
R1
R1
N
N
H
R2
R2
Ph
O
N
12
SO2NH2
N
O
O
S
NH
X
N
N
O
O
13
X
N
SO2NH2
Ph
Ph
N
H
Ph
S
N
a
b
c
CN
I
OH
N
N
SO2NH2
N
N
8
O
O
S
X
Ph
NH
13
Ar
14
NH2
N
Ph
N
a
OH
OH
Ar
b
N
Ph
O
Ph
N
Ar
N
O
14
H
N
N
N
CH3COCH2Cl
N
N
S
O
CH3
SO2NH2
15
CH3
Scheme 2. Synthesis of compounds 12a–f, 13a–c, 14a–b, and 15.
b, and 15) on hCA-I and hCA-II. As seen in the Table 1, the IC50 val-
ues of the new compounds (8–11,12a–f, 13a–c, 14a–b, and 15) are
lower than the IC50 values of inhibitor 1 and AAZ for hydratase
activity of erythrocyte hCA-II. IC50 determination indicates that
about 82% of esterase activity of hCA-II was lost in the presence
(Table 1); our synthesized compounds are more efficient than ini-
tial compounds on hCA-II enzyme. When IC50 values of inhibitors
on hydratase activity of hCA-I were examined, compounds 12a–
12f, 13a–13c, 14a, 14b, and 15 of our synthesized compounds
are more effective as inhibitors than initial compounds (1 and
AAZ).
of 0.065 lM of inhibitor 11.
As we examine IC50 value of our synthesized compounds on
hydratase activity of enzyme obtained after inhibition experiments
As we examine IC50 values of synthesized compounds on ester-
ase activity of carbonic anhydrase (Table 1); Compounds 8, 10, 11,
Table 1
IC50 and Ki values of hCA-I and hCA-II isoenzyme hydratase and esterase activities obtained after inhibition experiments
_
Inhibitor
Hydratase IC50
(l
M)
Esterase IC50
(l
M)
Ki values (lM)
hCA-I
hCA-II
hCA-I
hCA-II
hCA-I
hCA-II
1
AAZ
8
9
10
3.25 1.77
3.75 3.18
4.75 0.35
4.2 0.14
3.25 0.21
4.1 0.07
0.42–0.13
0.44–0.36
0.14–0.055
0.56–0.43
0.21–0.39
0.02–0.065
0.25–0.158
0,8–0.52
2.9 0.57
3.9 2.26
2.6 0.57
1.59 0.30
0.055 0.01
0.08 0.014
0.77–0.59
0.09–0.085
0.80–0.71
0.80–0.6
0.22–0.87
0.53–0.41
0.08–0,04
0,48–0.13
0.05–0.01
0,04–0.06
0.02–0.08
0.32–0.96
2.6 1.98
2.9 0.85
2.7 0.71
4.3 0.42
0.44–0.21
6.6 2.69
0.41–0.9
3.6 0.57
8.3 0.99
2.8 0.42
4.2 0.28
0.013 0.001
0.015 0.004
0.55–0.78
0,18–0.33
0,1–0,094
0,04–0.07
0.02–0.05
0,06–0.055
0,32–0.25
0,36–0.92
0.26–0.31
0,081–0.022
0.01–0.03
0.1–0.23
5.7–4.2
6.1–3.5
3.09–2.7
2.42–3.5
no
8.2–6.3
2–3.2
0.561–0.33
1.56–4.6
4.09–6.4
1.24–2.5
0.2–079
0.41–0.122
0.20–0.166
0.61–0.58
0.04–0.06
0.57–0.02
0,41–0.32
0,38–0.136
0.22–0.83
0.034–0.05
0.02–0.06
0.4–0.21
11
2.9–4.7
0,3–0.8
12a
12b
12c
12d
12e
12f
13a
13b
13c
14a
14b
15
0.8–0.45
0.24–0.4
1.31–0.75
0.09–0.081
0.43–0.2
0.17–0.15
0.55–0.45
2,534–1.863
0.53–0.4
0.714–0.523
0.12–0.15
0.39–0.22
0,085–0.08
0,097–0.14
0.25–0.08
0,14–0.09
0,419–0.33
3,798–2.65
0.39–0.8
0,575–0.374
0.21–0.55
0.2–0.35
0.6–1.3
0.17–0.65
0.31–0.08
0.4–0.12

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12a–12f, 13a–13c, 14a, 14b, and 15 are more effective than initial
compounds (1 and AAZ). Especially compounds 10, 11, 12d, 12e,
12f, 14a, and 14b are the most effective compounds. When we
examine hCA-I esterase IC50 values, compounds 10, 12a–f, 13a,
13c, 14a, 14b, and 15 are more effective than initial compounds
(1 and AAZ). Especially 12c and 12d compounds are the most effec-
tive compounds on this enzyme.
When the effects of synthesized compounds on Ki values (Table
1) were investigated, hCA-II Ki values of these compounds 8, 11,
12a–f, 13a–c, 14a, 14b, and 15 are lower than initial compounds
of 1 and AAZ, indicating that they are more effective. Especially
compounds 12e, 14a, and 14b are the most effective compounds.
Similarly, hCA-I Ki values of synthesized compounds are less than
Ki values of initial compounds, therefore their potential inhibitory
effects are quite higher. As seen in Table 1, 12c and 12d are the
most effective inhibitors.
(m, 14H, ArH); 13C NMR (100 MHz, DMSO-d6) d (ppm): 190.58
(benzoyl C@O), 165.36 (amide C@O), 161.44 and 160.44 (thiadiazol
C-2 and C-5), 152.20, 148.80, 144.57, 143.36, 137.58, 134.10,
130.48, 130.13, 129.64, 129.06, 127.65, 127.40, 127.30, 122.91,
114.40; IR(KBr) (m
, cmꢀ1): 3312–3216, 3026, 1740, 1665, 1511,
1426, 1365, 1294, 1238, 1172, 1062; MS(CI) m/z 546.1(M+1); 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, 11.60.
3.2.3. Ethyl-1-(3-aminophenyl)-5-phenyl-3-(5-sulfamoyl-1,3,4-
thiadiazole-2-ylcarbamoyl)-1H-pyrazole-4-carboxylate (10)
(410 mg, 76%); Mp 253 °C; 1H NMR (400 MHz, DMSO-d6) d
(ppm): 13.60 (m, 1H, CONH), 1.01 (t, 3H, –CH3), 4.09 (q, 2H,
–CH2), 6.52 (m, 2H, Ar-NH2), 8.42 (m, 2H, –SO2NH2), 7.44–
6.78 (m, 9H, ArH); 13C NMR (100 MHz, DMSO-d6) d (ppm):
164.57 (ester C@O), 164.10 (amide C@O), 163.65 and 161.36
(thiadiazol C-2 and C-5), 152.21, 144.49, 144.09, 139.70,
130.57, 130.37, 129.94, 129.71, 128.67, 128.63, 128.57, 116.19,
If we discuss the results in general, it can be said that our syn-
thesized compounds show more effective inhibition property than
initial compounds, so they can be seen as a candidate medicine for
treatment of glaucoma.
114.66, 62.50, 13.95; IR(KBr)
(m,
cmꢀ1): 3451, 2969, 2877,
1740, 1676, 1566, 1490, 1436, 1375, 1327, 1222, 1108, 1057;
MS(CI) m/z 514.2 (M+1); Anal. Calcd for C21H19N7O5S2: C,
49.11; H, 3.73; N, 19.09; S, 12.49. Found: C, 49.10; H, 3.83;
N, 19.12; S, 12.35.
3. Experimental protocols
3.1. Syntheses
3.2.4. Ethyl-1-(4-aminophenyl)-5-phenyl-3-(5-sulfamoyl-1,3,4-
thiadiazole-2-ylcarbamoyl)-1H-pyrazole-4-carboxylate (11)
(364 mg, 71%); Mp 275 °C; 1H NMR (400 MHz, DMSO-d6) d
(ppm): 13.94 (m, 1H, CONH), 0.99 (t, 3H, CH3), 4.11 (q, 2H, CH2),
6.50 (m, 2H, Ar-NH2), 8.43 (m, 2H, SO2NH2), 7.40–6.68 (m, 9H,
ArH); 13C NMR (100 MHz, DMSO-d6) d (ppm): 165.55 (ester
C@O), 161.77 (amide C@O), 161.41 and 160.99 (thiadiazol C-2
and C-5), 147.26, 146.57, 146.11, 143.30, 130.80, 130.51, 128.95,
Melting points were measured with Barnstead Electrothermal
9200 apparatus, and were uncorrected. IR spectrum data of com-
pounds were determined by Mattson 1000 FT-IR using KBr pellets.
1H NMR and 13C NMR spectra were evaluated by BRUKER DPX-400,
(400 MHz), and High Performance Digital FT NMR (100 MHz) spec-
trometers. Mass spectra data were determined by Varian Mat III
80 eV. At the end of the each experiments TLC was performed using
DC Alufolien Kiesegel 60F/254 Merck and Camag TLC devices. Ele-
mental analyses were carried out on a Leco CHNS-932 instrument.
127.50, 126.85, 125.10, 114.80, 61.24, 13.94; IR(KBr) (m
, cmꢀ1):
3310–3214, 2967, 2740, 1738, 1659, 1524, 1495, 1440, 1365,
1327, 1251, 1174, 1105, 1065; MS(CI) m/z 514 (M+1); Anal. Calcd
for C21H19N7O5S2: C, 49.11; H, 3.73; N, 19.09; S, 12.49. Found: C,
49.23; H, 3.71; N, 18.97; S, 12.45.
3.2. General procedure for synthesis of compounds 8–11
Na2Sꢁ9H2O (1 mmol) and sulfur (S, 2 mmol) were stirred and
dissolved by boiling in 20 ml of water. This solution (sodium poly-
sulfur) was then added dropwise to a stirred solution of warm
compound 4, 5, 6, or 7 (1 mmol) in methanol–water. This mixture
was refluxed for 45 min and then HCl solution was added to this
mixture, refluxed again for 15 min. Precipitated solid was filtrated
and ammonia was added to the solution. The mixture was incu-
bated overnight and finally the solid products were filtrated and
crystallized from ethanol–water mixture. Finally, target inhibitors
(8–11) were obtained by reducing NO2 to NH2 groups.
3.3. General procedure for synthesis of compounds 12a–f
Sodium acetate (3 g) was dissolved in 10 ml H2O and 2 ml HCl.
Aromatic amine (1 mmol) and alcohol were added to mixture until
it dissolved. Temperature was adjusted to 0–5 °C. Solution of
NaNO2 (83 mg, 1.2 mmol) in 10 aqua was added to mixture slowly,
and process of diazotization was performed. In another place, aro-
matic and b-diketone (1 mmol) compounds were dissolved in eth-
anol and added to diazonium salt dropwise which was prepared
previously. Final colored precipitate was filtered by trompe, and
purified from ethyl alcohol.
3.2.1. 1-(3-Aminophenyl)-4-benzoyl-5-phenyl-N-(5-sulfamoyl-
1,3,4-thiadiazole-2-yl)-1H-pyrazole-3-carboxamide (8)
3.3.1. 4-Benzoyl-1-(3-(2-(1,3-dioxo-1-phenylbutan-2-ylidene)
hydrazinyl)phenyl)-5-phenyl-N-(5-sulfamoyl-1,3,4-thiadiazol-
2-yl)-1H-pyrazole-3-carboxamide (12a)
(480 mg, 88%); Mp 216 °C; 1H NMR (400 MHz, DMSO-d6) d
(ppm): 13.20 (m, 1H, CONH), 5.42 (m, 2H, Ar-NH2), 8.35 (m, 2H, –
SO2NH2), 7.79–6.92 (m, 14H, ArH); 13C NMR (100 MHz, DMSO-d6)
d (ppm): 190.80 (Benzoyl C@O), 165.28 (Amide C@O), 161.43 and
160.54 (thiadiazol C-2 and C-5), 148.82, 144.34, 142.41, 137.77,
137.69, 133.96, 130.23, 130.14, 129.58, 129.06, 128.84, 128.20,
(488 mg, 68%); Mp 174 °C; 1H NMR (400 MHz, DMSO-d6) d
(ppm): 13.82 (s, 1H, CONH), 11.27 (s, 1H, Ar-NH–N@C), 8.38 (s,
2H, SO2NH2), 2.50 (s, 3H, CH3), 7.95–7.08 (m, 19H, ArH); 13C
NMR (100 MHz, DMSO-d6)
d (ppm): 196.52 (acetyl C@O),
127.92, 127.43, 127.34, 122.40, 114.36, IR(KBr) (m
, cmꢀ1): 3351,
1740, 1668, 1494, 1426, 1354, 1295, 1248, 1167, 1060; MS(CI) m/z
546.1(M+1); Anal. Calcd for C25H19N7O4S2: C, 55.04; H, 3.51; N,
17.97; S, 11.75. Found: C, 55.35; H, 3.45; N, 17.82; S, 11.55.
195.39 and 190.56 (benzoyl C@O), 165.35 (amide C@O), 161.39
and 160.38 (thiadiazol C-2 and C-5), 25.47 (CH3), 144.40,
144.27, 143.28, 140.14, 139.71, 137.63, 135.88, 134.94, 134.08,
130.64, 130.48, 130.15, 130.04, 129.64, 129.55, 129.20, 129.09,
128.96, 127.89, 123.03, 120.17, 112.14; IR(KBr) (m
, cmꢀ1): 3381,
3.2.2. 1-(4-Aminophenyl)-4-benzoyl-5–phenyl-N-(5-sulfamoyl-
1,3,4-thiadiazole-2-yl)-1H-pyrazole-3-carboxamide (9)
3250, 3063, 2899, 2833, 1667, 1601, 1519, 1450, 1425, 1364,
1322, 1299, 1217, 1174; Anal. Calcd for C35H26N8O6S2: C,
58.49; H, 3.65; N, 15.59; S, 8.92. Found: C, 58.55; H, 3.68; N,
15.65; S, 8.89.
(465 mg, 85%); Mp 183 °C; 1H NMR (400 MHz, DMSO-d6) d
(ppm): 6.51 (m, 2H, Ar-NH2), 8.37 (m, 2H, SO2NH2), 7.80–6.82

R. Kasımog˘ulları et al. / Bioorg. Med. Chem. 17 (2009) 3295–3301
3299
3.3.2. 4-Benzoyl-1-(3-((2,4-dioxopentan-3-yl)diazenyl)phenyl)-
5-phenyl-N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)-1H-pyrazole-3-
carboxamide (12b)
3.3.6. tert-Butyl2-((3-(4-benzoyl-5-phenyl-3-(5-sulfamoyl-
1,3,4-thiadiazol-2-ylcarbamoyl)-1H-pyrazol-1-yl)
phenyl)diazenyl)-3-oxobutanoate (12f)
(328 mg, 50%); Mp 175 °C; 1H NMR (400 MHz, DMSO-d6) d
(ppm): 13.77 (s, 1H, CONH), 8.37 (s, 2H, –SO2NH2), 2.45, 2.29 (s,
6H, CH3), 7.81–7.01 (m, 14H, ArH); 13C NMR NMR (100 MHz,
DMSO-d6) d (ppm): 197.56 and 196.82 (acetyl C@O), 190.52 (ben-
zoyl C@O), 165.33 (amide C@O), 161.39 and 160.35 (thiadiazol C-2
and C-5), 31.70 and 26.76 (CH3), 144.55, 144.48, 143.40, 142.97,
139.86, 139.58, 137.63, 137.59, 134.61, 134.06, 130.51, 130.14,
(285 mg, 40%), Mp 217 °C; 1H NMR (400 MHz, DMSO-d6) d
(ppm): 13.79 (m, 1H, CONH), 11.59 (s, 1H, Ar-NH–N@C), 8.36 (s,
2H, –SO2NH2), 2.27 (s, 3H, CH3), 1.52 (m, 9H, C(CH3)3), 7.79–7.13
(m, 14H, ArH); 13C NMR (100 MHz, DMSO-d6) d (ppm): 194.31
(acetyl C@O), 190.55 (benzoyl C@O), 165.34 (ester C@O), 161.95
(amide C@O), 161.39 and 160.38 (thiadiazol C-2 and C-5), 28,36
and 26,48 (CH3), 83,59 (C(CH3)3), 144.46, 143.58, 143.26, 139.74,
137.63, 134.08, 132.38, 130.56, 130.50, 130.45, 130.16, 129.62,
129.63, 129.07, 127.79, 127.67, 123.10, 122.93; IR(KBr) (m
, cmꢀ1):
3380, 3232, 3061, 2889, 2838, 1677, 1598, 1519, 1498, 1448,
1427, 1363, 1302, 1251, 1175; MS(CI) m/z 657.1 (M+1); Anal. Calcd
for C30H24N8O6S2: C, 54.87; H, 3.68; N, 17.06; S, 9.77.Found: C,
54.72; H, 3.85; N, 17.11; S, 9.75.
129.08, 127.83, 123.01, 120.97, 116.25, 112.77; IR(KBr) (m
, cmꢀ1):
3414, 3064, 2900, 2833, 1686, 1609, 1525, 1498, 1450, 1427,
1368, 1321, 1299, 1217, 1174; Anal. Calcd for C33H30N8O7S2: C,
55.45; H, 4.23; N, 15.68; S, 8.97. Found: C, 55.39; H, 4.28; N,
15.56; S, 9.05.
3.3.3. Diethyl-2-((3-(4-benzoyl-5-phenyl-3-(5-sulfamoyl-1,3,4-
thiadiazol-2-ylcarbamoyl)-1H-pyrazol-1-yl)phenyl)
diazenyl)malonate (12c)
3.3.7. 4-Benzoyl-1-(3-cyanophenyl)–5-phenyl-N-(5-sulfamoyl-
1,3,4-thiadiazol-2-yl)-1H-pyrazole-3-carboxamide (13a)
(343 mg, 48%); Mp 181 °C; 1H NMR (400 MHz, DMSO-d6) d
(ppm): 13.80 (m, 1H, CONH), 8.38 (s, 2H, SO2NH2), 4.32 (q, 2H,
CH2), 3.50 (s, 1H, N-CH), 1.25 (t, 3H, CH3), 7.80–7.15 (m, 14H,
ArH); 13C NMR (100 MHz, DMSO-d6) d (ppm): 190.52, (benzoyl
C@O), 170.42 (ester C@O), 165.30 (amide C@O), 161.38 and
160.40 (thiadiazol C-2 and C-5), 15.3 (CH3), 46.03 (CH2), 61.82
(CH), 144.40, 143.70, 139.71, 137.61, 137.30, 134.04, 133.05,
130.56, 130.30, 130.05, 129.60, 129.09, 128.90, 128.88, 128.74,
Diazonium salt solution of aromatic amine compound was pre-
pared according to method in General procedure-II. A solution of
CuCN and KCN which was prepared in another vessel was heated
to 60 °C, and added slowly to diazonium salt solution prepared be-
fore. After complete mixing at same temperature, mixture was
cooled to room temperature. Precipitated yellow product was fil-
tered and purified from alcohol. (189 mg, 34%); Mp 173 °C; 1H
NMR (400 MHz, DMSO-d6) d (ppm): 13.80 (m, 1H, CONH), 8.39
(s, 2H, –SO2NH2), 7.81–7.28 (m, 15H, ArH); 13C NMR (100 MHz,
DMSO-d6) d (ppm): 190.52 (benzoyl C@O), 165.35 (amide C@O),
161.42 and 160.42 (thiadiazol C-2 and C-5), 152.07, 144.48,
143.25, 141.12, 139.68, 138.82, 137.63, 134.06, 130.53, 130.27,
129.62, 129.08, 128.97, 127.88, 126.26, 123.07, 122,92, 116.13;
127.65, 122.94; IR(KBr) (m
, cmꢀ1): 3468, 3380, 3162, 3060, 2899,
1683, 1612, 1596, 1525, 1496, 1448, 1427, 1367, 1323, 1301,
1216, 1174; MS(CI) m/z 717.2 (M+1); Anal. Calcd for C32H28N8O8S2:
C, 53.62; H, 3.94; N, 15.63; S, 8.95. Found: C, 53.48; H, 3.99; N,
15.61; S, 8.87.
IR(KBr) (m
, cmꢀ1): 3415, 3062, 2108 (CN), 1688, 1598, 1522,
3.3.4. Ethyl-2-((3-(4-benzoyl-5-phenyl-3-(5-sulfamoyl-1,3,4-
thiadiazol-2-ylcarbamoyl)-1H-pyrazol-1-yl)phenyl) diazenyl)-3-
oxo-3-phenylpropanoate (12d)
1496, 1449, 1426, 1366, 1299, 1244, 1174; Anal. Calcd for
C26H17N7O4S2: C, 56.21; H, 3.08; N, 17.65; S, 11.54. Found: C,
56.20; H, 3.15; N, 17.59; S, 11.58.
(381 mg, 51%); Mp 145 °C; 1H NMR (400 MHz, DMSO-d6) d
(ppm): 13.79 (m, 1H, CONH), 11.99 (s, 1H, Ar-NH–N@C), 8.37 (s,
2H, –SO2NH2), 4.20 (q, 2H, CH2), 3.82 (s, 1H, N-CH), 1.26 (t, 3H,
CH3), 7.54–7.06 (m, 19H, ArH); 13C NMR (100 MHz, DMSO-d6) d
(ppm): 190.46 and 189.26 (benzoyl C@O), 165.34 (ester C@O),
162.70 (amide C@O), 161.38 and 160.40 (thiadiazol C-2 and C-5),
14.33 (CH3), 46.03 (CH2), 61.80 (CH), 144.43, 143.71, 139.71,
137.61, 137.32, 134.09, 133.07, 130.52, 130.38, 130.32, 130.04,
129.60, 129.30, 129.08, 129.01, 128.94, 128.88, 128.73, 127.64,
3.3.8. 4-Benzoyl-1-(3-iodophenyl)-5-phenyl-N-(5-sulfamoyl-
1,3,4-thiadiazol-2-yl)-1H-pyrazole-3-carboxamide (13b)
Diazonium salt solution of aromatic amine compound was pre-
pared according to method in General procedure-II. In another ves-
sel, KI (656 mg, 1 mmol) was dissolved in ethanol and cooled to 0–
5 °C and added dropwise to diazonium salt solution which was
prepared before. Precipitated yellow precipitate was filtered with
tromp and purified from alcohol. (426 mg, 65%); Mp 191 °C; 1H
NMR NMR (400 MHz, DMSO-d6) d (ppm): 13.20 (m, 1H, CONH),
8.37 (s, 2H, SO2NH2), 7.81–6.52 (m, 14H, ArH); 13C NMR
(100 MHz, DMSO-d6) d (ppm): 190.52 (benzoyl C@O), 165.34
(amide C@O), 161.43 and 160.38 (thiadiazol C-2 and C-5),
144.48, 143.25, 143.06, 139.70, 139.61, 137.63, 134.07, 130.57,
130.26, 129.98, 129.83, 129.63, 129.07, 128.92, 127.91, 123.09,
122.94, 120.79, 115.96, 112.91 IR(KBr) (m
, cmꢀ1): 3379, 3242,
3062, 2903, 2829, 1670, 1599, 1526, 1497, 1448, 1425, 1368,
1323, 1299, 1218, 1175; MS(CI) m/z 749.2 (M+1); Anal. Calcd for
C36H28N8O7S2: C, 57.74; H, 3.77; N, 14.96; S, 8.56. Found: C,
57.78; H, 3.97; N, 14.90; S, 8.51.
3.3.5. 4-Benzoyl-1-(3-((1,3-dioxo-1,3-diphenylpropan-2-yl)
diazenyl)phenyl)-5-phenyl-N-(5-sulfamoyl-1,3,4-thiadiazol-2-
yl)-1H-pyrazole-3-carboxamide (12e)
122.59; IR(KBr) (m
, cmꢀ1): 3377, 3061, 2904, 1687, 1609, 1523,
1497, 1449, 1425, 1367, 1298, 1235, 1174; MS(CI) m/z 657.0
(M+1); Anal. Calcd for C25H17IN6O4S2: C, 45.74; H, 2.61; N, 12.80;
S, 9.77. Found: C, 45.80; H, 2.55; N, 12.81; S, 9.91.
(686 mg, 88%); Mp 128 °C; 1H NMR (400 MHz, DMSO-d6) d
(ppm): 13.80 (m, 1H, CONH), 11.76 (s, 1H, Ar-NH–N@C) 8.40 (s,
2H, SO2NH2), 4.89 (s, 1H, N–CH), 8.19–7.02 (m, 24H, ArH); 13C
NMR (100 MHz, DMSO-d6) d (ppm): 190.53 and 185.80 (benzoyl
C@O), 165.35 (amide C@O), 161.39 and 160.30 (thiadiazol C-2
and C-5), 93.73 (CH), 144.53, 143.39, 139.59, 137.60, 135.04,
134.05, 133.50, 130.60, 130.47, 130.13, 130.04, 129.63, 129.54,
129.32, 129.07, 129.04, 128.97, 128.64, 127.89, 127.69, 122.93,
3.3.9. 4-Benzoyl-1-(3-hydroxyphenyl)–5–phenyl-N-(5-
sulfamoyl-1,3,4-thiadiazol-2-yl)-1H-pyrazole-3-carboxamide
(13C)
Diazonium salt solution of aromatic amine compound was pre-
pared according to method in General procedure-II. Prepared dia-
zonium solution was heated in water bath at 100 °C until nitride
gas output had stopped. After cooling of mixture to room temper-
ature, pH adjustment (pH 3–4) was performed. After sometime,
precipitated product was filtered with tromp. Produced yellow so-
lid was purified from ethanol. (289 mg, 53%); Mp 200 °C; 1H NMR
IR(KBr) (m
, cmꢀ1): 3468, 3380, 3231, 3061, 2885, 2831, 1680,
1598, 1523, 1495, 1427, 1367, 1301, 1224, 1174; MS(CI) m/z
781.2 (M+1); Anal. Calcd for C40H28N8O6S2: C, 61.53; H, 3.61; N,
14.35; S, 8.21. Found: C, 61.42; H, 3.68; N, 14.27; S, 8.35.

3300
R. Kasımog˘ulları et al. / Bioorg. Med. Chem. 17 (2009) 3295–3301
(400 MHz, DMSO-d6) d (ppm): 13.80 (m, 1H, CONH), 8.37 (s, 2H, -
SO2NH2), 7.81–6.92 (m, 14H, ArH); 13C NMR (100 MHz, DMSO-d6) d
(ppm): 190.52 (benzoyl C@O), 165.30 (amide C@O), 161.43 and
160.37 (thiadiazol C-2 and C-5), 152.13 (@C–OH), 144.48, 143.23,
139.70, 137.62, 134.07, 130.56, 130.26, 129.89, 129.77, 129.63,
3.4. Purification of carbonic anhydrases I and II from human
erythrocytes
Carbonic anhydrases I and II were purified as described previ-
ously.27,28 Briefly, human blood was centrifuged at 1500 rpm for
20 min, and after removal of the plasma, the erythrocytes were
washed with an isotonic solution (0.9% NaCl). After that, the eryth-
rocytes were lysed with 1.5 volume of ice-cold water. The lysate
was centrifuged at 20000 rpm for 30 min to remove cell mem-
branes and non-lysed cells. The pH of the supernatant was ad-
justed to 8.7 and was then loaded onto an affinity column
129.08, 128.91, 127.90, 127.41, 123.08, 114.97; IR(KBr) (m
, cmꢀ1):
3370, 3241, 3061, 1688, 1600, 1521, 1495, 1448, 1426, 1366,
1300, 1237, 1173; MS(CI) m/z 546.1; Anal. Calcd for C25H18N6O5S2:
C, 54.94; H, 3.32; N, 15.38; S, 11.73. Found: C, 54.80; H, 3.45; N,
15.36; S, 11.79.
3.3.10. 4-Benzoyl-1-(3-((4-hydroxyphenyl)diazenyl)phenyl)-5-
phenyl-N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)-1H-pyrazole-3-
carboxamide (14a)
containing Sepharose-4B-L-tyrosine-p-aminobenzene sulfonamide
as the binding group. After extensive washing with 25 mM Tris–
HCl/22 mM Na2SO4 (pH 8.7), the hCA-I and II isozymes were eluted
with 1.0 M NaCl/25 mM Na2HPO4 (pH 6.3) and 0.1 M CH3COONa/
0.5 M NaClO4 (pH 5.6). Amount of purified protein was estimated
by the Bradford method29 and SDS–PAGE was carried out to deter-
mine whether the eluate contained the enzyme.30
Produced according to method in general procedure-II, (221 mg,
34%); Mp 207 °C; 1H NMR (400 MHz, DMSO-d6) d (ppm): 12.63 (m,
1H, CONH), 8.37 (s, 2H, SO2NH2), 10.05 (m, 1H, Ar-OH), 8.02–7.29
(m, 18H, ArH); 13C NMR (100 MHz, DMSO-d6) d (ppm): 190.51
(benzoyl C@O), 165.35 (amide C@O), 161.42 and 160.39 (thiadiazol
C-2 and C-5), 160.30 (@C–OH), 145.80, 144.51, 143.23, 139.70,
137.64, 134.07, 132.05, 130.93, 130.27, 130.18, 130.06, 129.64,
129.08, 128.03, 127.90, 126.26, 125.67, 123.30, 123.09, 116.58;
3.5. Determination of hydratase and esterase activities
CO2-hydratase activity of the enzyme was determined at 0 °C in
a veronal buffer (pH 8.15) with pH-state method. Enzyme activity
was calculated by using the equation (t0 ꢀ tc/tc) where to and tc are
the time taken for the pH change of the non-enzymatic and enzy-
matic reactions, respectively.30,31 Esterase activities of carbonic
anhydrase enzymes were assayed by the hydrolysis of p-nitrophe-
nylacetate. The absorbance was determined at 348 nm after
3 min.32 The % carbonic anhydrase activity values were assayed
by following the hydration of CO2. IC50 values for inhibitor 1,
AAZ, and the synthesized compounds (8–11) were determined
on hCA-I and hCA-II by measuring hydratase activity in the pres-
ence of various inhibitor concentrations. Regression analysis
graphs were drawn by plotting inhibitor concentrations versus en-
zyme activity by using Microsoft Excel Program. The data were
then fitted with non-linear regression using the second degree
polynomial equation. The IC50 value was obtained by solving the
equation derived from the second degree polynomial.31–36
IR(KBr) (m
, cmꢀ1): 3384, 3064, 1689, 1597, 1522, 1500, 1449,
1426, 1365, 1301, 1245, 1214, 1174; MS(CI) m/z 651.2 (M+1); Anal.
Calcd for C31H22N8O5S2: C, 57.22; H, 3.41; N, 17.22; S, 9.86. Found:
C, 57.15; H, 3.53; N, 17.15; S, 9.93.
3.3.11. 4-Benzoyl-1-(3-((2–hydroxynaphthalen–1-yl)diazenyl)
phenyl)-5-phenyl-N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)-1H-
pyrazole-3-carboxamide (14b)
Produced according to method in general procedure-II;
(300 mg, 43%); Mp 195 °C; 1H NMR (400 MHz, DMSO-d6)
d
(ppm): 13.80 (m, 1H, CONH), 8.39 (s, 2H, SO2NH2), 6.86 (d, 1H,
Ar-OH), 8.26–7.14 (m, 20H, ArH); 13C NMR (100 MHz, DMSO-d6)
d (ppm): 190.54 (Benzoyl C@O), 165.34 (Amide C@O), 162.72
(@C–OH), 161.45 and 160.36 (Thiadiazol C-2 and C-5), 145.53,
144.46, 143.25, 141.53, 140.08, 137.69, 134.12, 132.95, 130.93,
130.32, 130.18, 130.06, 129.77, 129.68, 129.46, 129.11, 128.39,
127.89, 126.82, 124.74, 124.50, 123.29, 122.11, 120.12, 114.76,
106.27; IR(KBr) (
m
, cmꢀ1): 3416, 3062, 1687, 1601, 1518, 1451,
3.6. Ki Determination
1426, 1364, 1298, 1254, 1206, 1174; MS(CI) m/z 701.0 (M+1); Anal.
Calcd for C35H24N8O5S2: C, 59.99; H, 3.45; N, 15.99; S, 9.15. Found:
C, 60.18; H, 3.43; N, 15.90; S, 9.19.
To determine Ki value as well as the inhibition type, three differ-
ent inhibitor concentrations giving 30%, 50%, and 70% inhibition
(2.4 lM, 4.8 lM, and 7.3 lM, respectively) were selected. At each
3.3.12. 4-Benzoyl-1-(2-methyl-1-(2-oxopropyl)-1H-indol-4-yl)-
5-phenyl-N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)-1H-pyrazole-3-
carboxamide (15)
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 mM0) and the data were linear-
ized with Lineweaver-Burke plot for Vmax (apparent Vmax) and the
Ki determination.31 Enzyme activity was also measured in the pres-
ence of the same substrate concentrations but in the absence of
Aromatic compound of amine (1 mmol, 545 mg) was dissolved
in 15 ml DMF. Chloroacetone (0,182 ml) was added into mixture.
Reaction was maintained at 60 °C for three days in back cooler
which has CaCl2 headpiece. After evaporation of solvent of dark
colored mixture in rotary evaporator, remainder dense liquid was
added to water and mixed. Granulated product was filtered with
trompt and purified from ethanol. (310 mg 57%); Mp 183 °C; 1H
NMR (400 MHz, DMSO-d6) d (ppm): 13.80 (m, 1H, CONH), 8.36
(s, 2H, SO2NH2), 4.35 (s, 2H, CH2), 2.51 and 2.24 (s, 6H, CH3),
7.79–7.22 (m, 13H, ArH); 13C NMR (100 MHz, DMSO-d6) d (ppm):
202.20 (acetyl C@O), 190.56 (benzoyl C@O), 165.33 (amide C@O),
161.41 and 160.46 (thiadiazol C-2 and C-5), 44.25 (CH2), 31.24
and 29.08 (CH3), 143.44, 139.34, 137.61, 134.03, 131.89, 130.98,
130.41, 130.15, 129.96, 129.65, 129.07, 129.01, 127.76, 127.43,
any inhibitor to determine the Vmax
.
Acknowledgment
This study was funded by The Scientific and Research Council of
Turkey (TÜBITAK) with Grant No. TBAG-2376 and 106T180.
References and notes
1. Supuran, C. T. Nat. Rev. Drug Disc. 2008, 7, 168.
_
2. Bülbül, M.; Saraçog˘lu, N.; Küfreviog˘lu, Ö. I.; Çiftçi, M. Bioorg. Med. Chem. 2002,
10, 2561.
, cmꢀ1): 3378,
3. Bülbül, M.; Kasımog˘ulları, R.; Küfreviog˘lu, Ö. I. J. Enzyme Inhib. Med. Chem.
_
123.45, 122.88, 121.50, 121.04, 117.03; IR(KBr) (
m
2008, 23, 895.
3064, 2903, 1685, 1606, 1524, 1496, 1449, 1424, 1363, 1301,
1241, 1173; MS(CI) m/z 640.15 (M+1); Anal. Calcd for
C31H25N7O5S2: C, 58.20; H, 3.94; N, 15.33; S, 10.02. Found: C,
58.35; H, 3.87; N, 15.28; S, 10.12.
4. Nuti, E.; Orlandini, E.; Nencetti, S.; Rossello, A.; Innocenti, A.; Scozzafava, A.;
Supuran, C. T. Bioorg. Med. Chem. 2007, 15, 2298.
5. Özensoy, Ö.; Kockar, F.; Arslan, O.; Isık, S.; Supuran, C. T.; Lyon, M. Clin. Biochem.
2007, 40, 835.

R. Kasımog˘ulları et al. / Bioorg. Med. Chem. 17 (2009) 3295–3301
3301
6. Supuran, C. T.; Scozzafava, A. Bioorg. Med. Chem. 2007, 15, 4336.
7. Di Fiore, A.; Scozzafava, A.; Winum, J. Y.; Montero, J. L.; Pedone, C.; Supuran, C.
T.; Simone, G. D. Bioorg. Med. Chem. Lett. 2007, 17, 1726.
8. Innocenti, A.; Vullo, D.; Pastorek, J.; Scozzafava, A.; Pastorekova, S.; Nishimori,
I.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2007, 17, 1532.
9. Winum, J. Y.; Thiry, A.; Cheikh, K. E.; Dogné, J. M.; Montero, J. L.; Vullo, D.;
Scozzafava, A.; Masereel, B.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2007, 17,
2685.
21. Sßener, A.; Kasımog˘ulları, R.; Sener, M. K.; Genç, H. Chem. Heterocycl. Compd.
2004, 8, 1201.
22. Koçyig˘it Kaymakçıog˘lu, B.; Rollas, S. Farmaco 2002, 57, 595.
23. Stevens, H. R.; Ward, F. W. J. Chem. Soc. 1924, 125, 1324.
24. Baraldi, P. G.; Manfredini, S.; Romagnoli, R.; Stevanato, L.; Zaid, A. N.;
Manservigi, R. Nucleosides Nucleotides 1998, 17, 2165.
25. Hatheway, G. J.; Hansch, C.; Kim, K. H.; Milstein, S. R.; Schimidt, C. L.; Smith, R.
N.; Quin, F. R. J. Med. Chem. 1978, 21, 563.
10. Scozzafava, A.; Banciu, M. D.; Popescu, A.; Supuran, C. T. J. Enzyme Inhib. Med.
Chem. 2000, 15, 443.
26. Gangjee, A. et al Bioorg. Med. Chem. Lett. 2005, 15, 2225.
27. Arslan, O.; Nalbantog˘lu, B.; Demir, N.; Özdemir, H.; Küfreviog˘lu, Ö. I. Trop. J.
_
11. Schuman, J. S. Clin. Ther. 2000, 22, 2.
Med. Sci. 1996, 26, 163.
12. Wilkinson, B. L.; Bornaghi, L. F.; Houston, T. A.; Innocenti, A.; Vullo, D.; Supuran,
C. T.; Poulsen, S. A. Bioorg. Med. Chem. Lett. 2007, 17, 987.
13. Santos, M. A.; Marques, S.; Vullo, D.; Innocenti, A.; Scozzafava, A.; Supuran, C. T.
Bioorg. Med. Chem. Lett. 2007, 17, 1538.
14. Ashour, F. A.; Habib, N. S.; Taibbi, M.; Dine, S.; Dine, A. Farmaco 1990, 45, 134.
15. Sabri, K.; Levin, A. V. J. Am. Assoc. Pediatr. Ophthalmol. Strab. 2006, 10, 464.
16. Aggarwal, D.; Pal, D.; Mitra, A. K.; Kaur, I. P. Int. J. Pharm. 2007, 338, 21.
17. Sugrue, M. F. Prog. Retin. Eye Res. 2000, 19, 87.
28. Rickli, E. E.; Ghazanfar, S. A.; Gibbson, B. H.; Edsall, J. T. J. Biol. Chem. 1964, 239,
1065.
29. Bradford, M. M. Anal. Biochem. 1976, 72, 248.
30. Laemmli, D. K. Nature 1970, 227, 680.
31. Wilbur, K. M.; Anderson, N. G. J. Biol. Chem. 1976, 176, 147.
32. Landolfi, C.; Marchetti, M.; Ciocci, G.; Milanese, C. J. Pharmacol. Toxicol. Methods
1998, 38, 169.
_
33. Bülbül, M.; Hisar, O.; Beydemir, Sß.; Çiftçi, M.; Küfreviog˘lu, Ö. I. J. Enzyme Inhib.
18. Ingram, C. J.; Brubaker, R. F. Am. J. Ophthalmol. 1999, 128, 3.
19. Konowal, A.; Morrison, J. C.; Brown, S. V. L.; Cooke, D. L.; Maguire, L. J.; Verdier,
D. V.; Fraunfelder, F. T.; Dennis, R. F.; Epstein, R. J. Am. J. Ophthalmol. 1998, 127,
4.
Med. Chem. 2003, 18, 371.
34. Çiftçi, M.; Bülbül, M.; Gül, M.; Gümüsßtekin, K.; Dane, Sß.; Süleyman, H. J. Enzyme
Inhib. Med. Chem. 2005, 20, 103.
35. Hisar, O.; Beydemir, Sß.; Bülbül, M.; Yanık, T. J. Appl. Anim. Res. 2006, 30, 185.
36. Winum, J. Y.; Cecchi, A.; Montero, J. L.; Innocenti, A.; Scozzafava, A.; Supuran, C.
T. Bioorg. Med. Chem. Lett. 2005, 15, 3302.
_
20. Sßener, A.; Kasımog˘ulları, R.; Sener, M. K.; Bildirici, I.; Akçamur, Y. J. Heterocycl.
Chem. 2002, 39, 869.