Bile Acid Derivatives of 5-Amino-1,3,4-thiadiazole-2-sulfonamide as New Carbonic Anhydrase Inhibitors: Synthesis and Investigation of Inhibition Effects

Article abstract of DOI:10.1016/S0968-0896(02)00104-9

Bile acid amides (cholan-24-amides) of 5-substituted 1,3,4-thiadiazole-2-sulfonamide have been prepared from lithocholic, deoxycholic, cholic and dehydrocholic acids. Besides, the alcohol functional groups on the cholane ring systems were protected with acetyl group. Amides of the protected cholanes of lithocholic and cholic acids were also synthesized. Later, inhibition effects of these compounds on human carbonic anhydrase isozymes (HCA-I and II) have been investigated in vitro. For the most active compounds, inhibition constants ranged from 66 to 190 nM for HCA-II with I₅₀ (molarity of inhibitor producing a 50 percent inhibition of CA activity). In addition, in vivo studies were performed for the synthesized compounds in Sprague-Dawley rats. The compounds (11 and 18) showed especially significant inhibition efficacy (p < 0.001).

Full text of DOI:10.1016/S0968-0896(02)00104-9

Bioorganic & Medicinal Chemistry 10 (2002) 2561–2567
Bile Acid Derivatives of 5-Amino-1,3,4-thiadiazole-2-sulfonamide
as New Carbonic Anhydrase Inhibitors: Synthesis and
Investigation of Inhibition Effects
.
lu,* O. Irfan Ku
¨
Metin Bu
¨
lbu
¨
l, Nurullah Sarac¸
og
˘
¨
˘
frevioglu and Mehmet C¸
iftc¸
i
Faculty of Science and Arts, Department of Chemistry, Atatu¨rk University, 25240 Erzurum, Turkey
Received 4 February 2002; accepted 22 March 2002
Abstract—Bile acid amides (cholan-24-amides) of 5-substituted 1,3,4-thiadiazole-2-sulfonamide have been prepared from litho-
cholic, deoxycholic, cholic and dehydrocholic acids. Besides, the alcohol functional groups on the cholane ring systems were pro-
tected with acetyl group. Amides of the protected cholanes of lithocholic and cholic acids were also synthesized. Later, inhibition
effects of these compounds on human carbonic anhydrase isozymes (HCA-I and II) have been investigated in vitro. For the most
active compounds, inhibition constants ranged from 66 to 190 nM for HCA-II with I50 (molarity of inhibitor producing a 50%
inhibition of CA activity). In addition, in vivo studies were performed for the synthesized compounds in Sprague–Dawley rats. The
compounds (11 and 18) showed especially significant inhibition efficacy (p<0.001). # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
brinzolamide 2 are two topical CA inhibitors.2a Acet-
azolamide 3, a CA inhibitor, possessing several biome-
dical applications, is also known to increase the cerebral
Carbonic anhydrases (EC. 4.2.1.1) are a family of zinc
metalloenzymes that catalyze the reversible hydration of
carbon dioxide in a two-step reaction to yield bicarbo-
nate and proton. A number of 14 different carbonic
anhydrase (CA) isozymes were described up to now in
higher vertebrates, including humans. The isoenzymes
relevant to the human eye are CA-I, CA-II and CA-IV.
The isoenzymes CA-I and CA-II are cytosolic within the
7
blood flow when administered orally or intravenously.
The acetazolamide 3 as a therapeutically effective inhi-
bitor of the enzyme has encouraged synthesis of further
structural derivatives with the aim of studying the
effects of these changes on its inhibitory activity.
1
In this paper, we report the synthesis of new CA inhi-
bitors with 5-substituted-1,3,4-thiadiazole and both in
vitro and in vivo inhibition study of them on CA-I and
CA-II, because several important physiological and
physio-pathological functions are played by CA iso-
2
cell whereas CA-IV is membrane bound.
Carbonic anhydrase inhibitors, which reduce aqueous
production with a corresponding decrease in intraocular
pressure (IOP), have been used as ocular hypotensive
8
enzymes. Along with the use of CA inhibitors as glau-
coma therapy reagents, a recent discovery is also
connected with the involvement of inhibitors of CA
3
agents for the treatment of glaucoma. Glaucoma is a
4
term encompassing several diseases. The risk factors
9
for glaucoma disease include age, race, ocular hyper-
tension, severe myopia and a family history of glau-
coma. Of these, the strongest risk factors are age, race
and ocular hypertension. An elevated IOP was formerly
isoenzymes in cancer. Furthermore, some sulfonamides
have also applications as diagnostic tools in positron
emission tomography (PET) and magnetic resonance
1
0
imaging (MRI). Sulfonamides with the 1,3,4-thiadia-
zole ring are well known as inhibitors of the carbonic
2
a,5
synonymous with glaucoma.
1
1
anhydrase enzyme. We investigated the role of the
substituted lipid moiety attached to a 1,3,4-thiadiazole
ring on CA inhbition. Lithocholic (5), cholic (6),
deoxycholic (7), and dehydrocholic (8) acids were cho-
sen as lipid moieties. Bile acids have proved especially
useful because of their availability and useful levels of
The 1,3,4-thiadiazole ring is also known to possess sev-
eral biological activities and the antibacterial properties
have been largely described. Dorzolamide 1 and
6
1
2
functionalization. On the other hand, the codirected
hydroxyl groups present in most bile acids may be
*
0
Corresponding author. Tel.: +90-442-231-4425; fax: +90-442-236-
948; e-mail: nsarac@atauni.edu.tr
0
968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00104-9

2562
M. Bu¨lbu¨l et al. / Bioorg. Med. Chem. 10 (2002) 2561–2567
1
3
exploited in podand-type receptors, linear dimeric
the synthesis of the target inhibitors, the conversion of
the bile acids to the concerning amides is required for
activation of the side-chain carboxyls. For activation,
the bile acids were reacted by ethyl chloro formiate
(ECF) in the presence of N-methyl morpholine (Scheme
1
4
15
hosts, or ‘facial amphiphiles’. In addition, bile acids
are natural ligands specifically recognized by hepatitic
cells and are amphiphilic molecules that undergo a bio-
1
8
1
6
logical recycling during enterohepatic circulation.
1). Later, the activated carboxyl groups of the bile acids
were treated with 2-amino-1,3,4-thiadiazole-5-sulfona-
mide to obtain the desired amides (9–11). In order to
compare the inhibition effect on CA, the hydroxyl
groups of the cholane ring were protected by using ace-
tyl as a protecting group. For this reason, the bile acids
5, 6 and 7 were acetylated using acetyl chloride at room
temperature to afford the protected compounds 12, 13 and
Synthesis
The key compound 2-amino-1,3,4-thiadiazole-5-sulfo-
namide (4) required for the synthesis of inhibitors was
prepared by acidic hydrolysis of 2-acetilamino-1,3,4-
thiadiazole-5-sulfonamide (3).17 In order to approach
2
Scheme 1. (a) Ethyl chloro formate, N-methyl morpholine, THF; (b) 4, reflux; (c) AcCl, rt; (d) SOCl , 4, THF.

M. Bu¨lbu¨l et al. / Bioorg. Med. Chem. 10 (2002) 2561–2567
2563
14 which were converted into the amides 15 and 17
except for 16 as described in Scheme 1.
in a myriad of side effects, which include general malaise,
fatigue, depression, loss of appetite, gastrointestinal
disturbances, weight loss, parestheasias and renal
2
a,23
We next turned our attention to the preparation of the
amide 18 of the amino thiadiazole 4 with the dehydro-
cholic acid (8) containing triketone. For the preparation
of the amide, 8 was converted into the corresponding
calculi.
Unfortunately, because systemic therapy
with parenteral sulfonamides and their derivatives leads to
significant side effects, these undesirable side effects
obligate synthesis of new sulfonamide derivatives. There
are two main approaches used for the drug design of
acyl chloride with SOCl , and was then reacted with 4 in
2
8
dry THF. The structures of these compounds have been
1
effective antiglaucoma sulfonamides. The first is ‘ring’
13
elucidated on the basis of IR, H and C NMRdata,
and satisfactory mass spectral analysis.
approach, used for the synthesis of dorzolamide and
brinzolamide, consisted in exploring a great variety of
ring systems on which the sulfonamide group have been
8
,24
attached.
The second approach is ‘tail’, consisted in
attaching water solubilizing tails to different scaffolds of
well-known aromatic/heterocylic sulfonamides posses-
sing affinity for the CA active side.
Biochemistry
8
,25
The isoenzymes CA-I and CA-II were purified from
human erythrocytes by Sepharose-4B-l-tyrosine-p-ami-
nobenzensulfonamide affinity column. In vitro inhibi-
1
9
We have synthesized amides of 5-amino-1,3,4-thiadia-
zole-2-sulfonamide with some bile acids as candidate
inhibitors for glaucoma therapy as shown in Scheme 1.
Since the difference in interaction between the enzyme
and inhibitors can be rationalized from the varied shape
of inhibitors, three kind compounds having different
functional group and different number were synthe-
sized. The first framework contains mono, di and tri
alcohol groups on a bile acid. As a second skeleton, in
order to investigate the effect on inhibition activity, the
alcohol functional group/groups were protected with
acetyl. The last compound was also obtained from
dehydrocholic acid containing tri ketone.
tion study of newly synthesized compounds (9, 10, 11,
1
5, 17 and 18) and acetazolamide (3) are summarized in
Table 1. Results are expressed as I50 value. In addition,
a total of 42 adult rats (200–250 g) for in vivo studies
were used. They were divided into six groups and each
group had seven rats. The synthesized compounds were
injected intraperitionally into each rat. Blood samples
were taken from each rat at 2, 4 and 6 h after injection.
As a result, hemolysate was prepared as in vivo studies.
Carbonic anhydrase activity was assayed by the hydra-
tion of CO according to the method of Wilbur–Ander-
2
son at in vitro and in vivo studies. Analyses of the data
obtained from in vivo studies were made by test of t,
and were given as XÆSD.
For the synthesized compounds, the inhibitor con-
centrations causing up to 50% inhibition (I50 values)
were determined from regression analysis graphs. I50
values obtained for HCA-I and HCA-II purified by
affinity chromatography are shown in Table 1. Accord-
ing to in vitro studies, while the compound 9 has higher
I50 values than acetazolamide (3) on CA-I, the other
compounds (10, 11, 15, 17 and 18) have lower I50 values
than 3 on CA-I. The compound 9 containing one
hydroxyl group on cholane ring has no effect on CA-II.
For this reason, we think that 10, 11 and 17 in these
compounds on CA-II have the best inhibitor effect.
Although the compound 18 possessing tri ketone has
lower I50 values than 3 on both CA-I and CA-II, 18 is a
weaker inhibitor than the other inhibitors.
Results and Discussion
Current therapeutic intervention for glaucoma is pri-
marily directed toward physiological mechanism that
leads to the reduction of IOP in an effort to prevent
2
0
nerve fiber damage. Carbonic anhydrase is intimately
involved in the production of aqueous humor, and the
inhibitors of this enzyme are effective in lowering
intraocular pressure by reducing the production of
2
1
aqueous humor. Oral CA inhibitors have been in
clinical use for over 40 years following the observation
that orally administrated acetazolamide lowered the IOP
2
a,22
of glaucoma patients.
Systemically administered CA
inhibitors are very effective ocular hypotensive agents.
However, the extraocular inhibition of the enzyme results
For the in vivo studies, six groups of 42 adult rats for
each inhibitor were used. For compound 9, it was
observed that the activity of control, which did not
contain any inhibitor, were determined as 70663.67Æ
À1
Table 1.
synthesized compounds on carbonic anhydrase isozymes
I50 values which were obtained from in vitro studies for the
3324 EU (gHb) . Then the inhibitor injection was per-
formed on the control groups intraperitoneally. The
activities of the groups after the inhibitor injection were
measured at 2, 4 and 6 h, and % inhibition of the corres-
ponding activities were observed as 29% (p<0.001),
I50 (nM)
Compound
HCA-I
HCA-II
1
2% (p<0.05) and 14% (p<0.05) (Table 2). These
9
No effect
150
300
3500
93
66
1500
66
1400
2600
results indicated that compound 9 has inhibition effect.
Thus, in vivo studies related to this inhibitor does not
support the results of in vitro studies.
10
11
15
17
18
3
190
1100
1900
2600
In an analogous manner for in vivo studies we have
investigated the inhibition of the following compounds

2564
M. Bu¨lbu¨l et al. / Bioorg. Med. Chem. 10 (2002) 2561–2567
Table 2. Statistical values obtained from in vivo studies for the syn-
thesized compounds
melting apparatus. Infrared spectra were obtained from
1
KBr pellets for solids on a regular instrument. H NMR
1
3
and C NMRspectra were recorded on 200 and
0 MHz Varian spectrometer and are reported in d units
Compound Time (h) XÆSD EU (gHb)À1 % Inhibition
p
5
with SiMe as internal standard. Mass Spectrometry
9
Control
70663.67Æ3324
50173.67Æ4335
62046.00Æ7386
61051.83Æ7903
—
29
12
14
—
4
2
4
6
<0.001
<0.05
<0.05
was performed on a Micromass’LCT using lockspray
ionization.
10
Control
75054.50Æ6573
41259.67Æ6084
30 282.00Æ4773
4 4733.83Æ8712
—
45
60
41
—
2
4
6
<0.001
<0.001
<0.05
5
2
1
- (3ꢀ - Hydroxy - 5 - ꢁ - cholanamido) - 1,3,4- thiadiazole-
-sulfonamide (9). Lithocholic acid (5) (500 mg,
.33 mmol) was dissolved in 50 mL of dry and freshly
1
1
1
1
1
5
7
8
Control
6 8376.83Æ4256
35 771.66Æ5996
39222.00Æ5356
39033.83Æ5484
73981.33Æ8567
48970.83Æ5141
41650.17Æ5387
57222.67Æ4076
74158.83Æ2835
43837.00Æ3020
44678.00Æ4643
73890.17Æ10382
78117.17Æ3137
47479.17Æ6376
36341.67Æ7831
40998.50Æ5315
—
48
43
43
—
2
4
6
<0.001
<0.001
<0.001
distilled THF. To this solution, N-methyl morpholin
(
(
148 mg, 1.46 mmol) and ethyl chloro formiate (ECF)
159 mg, 1.46 mmol) were added. After the solution was
Control
—
34
44
23
—
stirred for 24h at room temperature, 263 mg (1.46 mmol)
of 5-amino-1,3,4-thiadiazole-2-sulfonamide was added
to the reaction mixture. The resulting mixture was
refluxed for 48 h, cooled and the precipitated solid was
filtrated. After removal of the solvent, the residue was
2
4
6
<0.001
<0.001
<0.05
Control
—
41
40
1
—
2
4
6
<0.001
<0.001
>0.05
dissolved in 50 mL of CHCl . The organic layer was
3
washed with NaOH solution (1 M, 2Â50 mL) and water
Control
—
39
54
48
—
(2Â50 mL) and dried over CaCl . The chloroform was
2
2
4
6
<0.001
<0.001
<0.001
removed and the residue (750 mg) was crystallized from
ethanol: colorless solid 9 (640 mg, 85%); mp 165 C. H
ꢀ
1
NMR(200 MHz, DMSO- d ): d 8.35 (m, 2H), 4.50 (m,
6
1
3
1H), 1.93–0.82 (m, 30H), 0.89 (s, 3H), 0.63 (s, 3H);
C
1
0–18. In all cases, we have observed the different
NMR(50 MHz, DMSO- d ): d 174.48, 166.08, 162.90,
6
results for the corresponding compounds. These results
indicated that the compounds 11 and 18 have similar
inhibition effect and the found values for 11 and 18 at
71.68, 57.90, 57.30, 44.12, 43.35, 38.11, 37.21, 36.97,
36.70, 36.02 (2C), 33.76, 32.50, 32.20, 29.48, 28.70,
27.96, 25.64, 25.06 (2C), 22.23, 20.01, 13.67; IR(KBr,
cm ): 3643, 3565, 3181, 2953, 2876, 1676, 1651, 1548,
1446, 1395, 1344, 1293, 1268, 1191, 1089, 1038, 961.
À1
2
, 4 and 6 h (p<0.001) are significant. While 11 shows
inhibition effect at both in vivo and in vitro, 18 only
shows inhibition effect in vivo. In the current study, our
data showed that the inhibitors (10, 15 and 17) decrease
the activation of CA enzyme in vivo studies during 2, 4
and 6 h. But the compound 17 has no inhibition effect at
5-(3ꢀ,12ꢀ-Dihydroxy-5-ꢁ-cholanamido)-1,3,4-thiadia-
zole-2-sulfonamide (10). The cholanamide 10 was pre-
pared from deoxycholic acid (500 mg, 1.2 mmol) as
described for the preparation of 9. The residue (600 mg)
was crystallized from ethanol: colorless solid 10
6
h (p>0.05). We compared the results of in vivo and in
vitro studies for 10, 15 and 17. In vivo studies related to
0, 15 and 17 provided evidence similar to data
ꢀ
1
1
(500 mg, 90%); mp 155 C. H NMR(200 MHz,
observed in vitro studies.
DMSO-d ): d 8.34 (m, 2H), 4.9 (m, 1H), 4.24 (m, 1H),
6
3
.81 (m, 1H), 0.81–1.80 (m, 29H), 0.86 (s, 3H), 0.61 (s,
1
3
In conclusion, in the present study, we report here the
preparation of 5-(5-b-cholanamido)-1,3,4-thiadiazole-2-
sulfonamide derivatives substituted 3, 7 and 12a-. They
were obtained by attaching bile acid moieties to the 5-
amino-1,3,4-thiadiazole-2-sulfonamide incorporating free
amino group. Also this work is aimed at the investigation
of the structure–action relationship on in vitro and in
vivo CA inhibition of the synthesized compounds. For
the most active compounds, the in vitro data showed
that inhibition constants ranged from 66 to 190 nM for
HCA-II with I . Especially, the in vivo data showed
3H), C NMR(50 MHz, DMSO- d ): d 174.54, 166.09,
6
162.90, 72.83, 71.78, 49.29, 47.96, 47.85, 43.46, 38.14,
37.49, 36.97, 36.78, 35.64, 34.78, 33.85, 32.61, 32.07,
30.43, 28.90, 28.81, 27.91, 25.27, 24.89, 18.83, 14.25, IR
(KBr, cm ): 3565, 3514, 3412, 3259, 2953, 2876, 1753,
1676, 1651, 1574, 1446, 1395, 1370, 1319, 1268, 1191,
1114, 1063, 961, MS (CI) m/z 555.2617 (M+1).
À1
5-(3ꢀ,7ꢀ,12ꢀ-Trihydroxy-5-ꢁ-cholanamido)-1,3,4-thia-
diazole-2-sulfonamide (11). The cholanamide 11 was
prepared from cholic acid (500 mg, 1.2 mmol) as descri-
bed for the preparation of 9. The residue (800 mg) was
crystallized from ethanol: colorless solid 11 (600 mg,
5
0
that the presence of trihydroxy-, and triketone-groups
on cholane ring resulted in better inhibition effect than
the other substituents.
ꢀ
1
87%); mp 182 C. H NMR(200 MHz, DMSO- d ): d
6
8
3
3
1
.36 (m, 2H), 4.40 (m, 1H), 4.19 (m, 1H), 4.07 (m, 1H),
.80 (m, 1H), 3.62 (m, 1H), 2.19–0.89 (m, 30H), 0.82 (s,
1
3
H), 0.60 (s, 3H), C NMR(50 MHz, DMSO- d ): d
6
Experimental
74.59, 166.06, 162.90, 72.81, 72.25, 68.06, 47.84, 47.59,
Solvents were concentrated at reduced pressure. Melting
points were determined on a Thomas-Hoover capillary
43.32, 43.18, 37.11, 36.97, 36.87, 36.67, 36.18, 33.82,
32.63, 32.18, 30.36, 30.24, 29.01, 28.03, 24.59, 24.41,

M. Bu¨lbu¨l et al. / Bioorg. Med. Chem. 10 (2002) 2561–2567
2565
À1
À1
1
1
1
8.84, 14.13, IR(KBr, cm ): 3412, 2953, 2876, 1753,
676, 1651, 1548, 1472, 1446, 1370, 1319, 1268, 1191,
089, 1038, 936, MS (CI) m/z 571.2617 (M+1).
22.77, 18.96, 13.66, IR(KBr, cm ): 3464, 3412, 2953,
2876, 1753, 1651, 1446,1395,1268, 1038, 961.
5
-(3ꢀ,7ꢀ,12ꢀ-triacetoxy-5-ꢁ-cholanamido)-1,3,4-Thia-
3
ꢀ-Acetoxy-5-ꢁ-cholan-24-oic acid (12). Lithocholic
diazole-2-sulfonamide (17). The cholanamide 17 was
prepared from 3a,7a,12a-triacetoxy-5-b-cholan-24-oic
acid (14) (500 mg, 0.94 mmol) as described for the pre-
paration of 9. The residue (700 mg) was crystallized
from ethanol: yellow solid 17 (470 mg, 72%); mp
acid (500 mg, 1.33 mmol) was dissolved in 5 mL of acetyl
chloride and the resulting solution was stirred for 12 h
at room temperature. The excessive acetyl chloride was
removed under reduced pressure and the residue
ꢀ
1
(
1
680 mg) was recrystallized from ethanol as a white solid
ꢀ
185 C. H NMR(200 MHz, CDCl ), d 5.04 (m, 1H),
3
1
2 (510 mg, 92%); mp 80 C. H NMR(200 MHz,
4.85 (m, 1H), 4.50 (m, 1H), 2.11 (s, 3H), 2.07 (s, 3H),
2.01 (s, 3H), 2.29–0.80 (m, 27H), 0.90 (s, 3H), 0.71 (s,
DMSO-d ): d 4.71 (m, 1H), 2.92–2.65 (m, 1H), 2.02 (s,
6
1
3
13
3
H), 2.22–0.61 (m, 30H), 0.92 (s, 3H), 0.64 (s, 3H),
C
3H), C NMR(50 MHz, DMSO- d ), d 181.01, 174.91,
6
NMR(50 MHz, CDCl ): d 176.10, 172.54, 76.36, 58.50,
171.80 (2C), 171.69, 171.62, 77.15, 75.83, 72.48, 49.36,
47.07, 45.40, 42.99, 39.82, 36.67, 36.58, 36.33, 33.54,
33.27, 32.74, 32.57, 30.84, 29.17, 28.88, 27.50, 24.83,
24.57, 23.41, 23.25, 23.18, 19.53, 14.25, IR(KBr, cm ):
3489, 3412, 2978, 2876, 1753, 1651, 1625, 1548, 1472,
3
5
3
2
3
1
7.89, 46.40, 44.82, 43.91, 42.45, 42.15, 37.82, 37.05,
6.97, 36.60, 34.29, 33.11, 30.15, 29.01, 28.66, 28.32,
6.14, 25.30, 23.41, 22.84, 20.24, 14.04, IR(KBr, cm ):
565, 3463, 3438, 2953, 2876, 1753, 1651, 1625, 1446,
395, 1370, 1319, 1268,1217, 1191, 1089, 1038, 987.
À1
À1
1446, 1395, 1268, 1191, 1089, 1038, MS (CI) m/z
697.2941 (M+1).
5
-(3ꢀ-Acetoxy-5-ꢁ-cholanamido)-1,3,4-thiadiazole-2-sul-
fonamide (15). The acetoxy cholanamide 15 was pre-
pared from 3a-acetoxy-5-b-cholan-24-oic acid (12)
5-(3,7,12-Trioxo-5-ꢁ-cholanamido)-1,3,4-thiadiazole-2-
sulfonamide (18). Method A: The cholanamide 18 was
prepared from dehydrocholic acid (500 mg, 1.24 mmol)
as described for the preparation of 9. The residue
(450 mg) was crystallized from ethanol: yellow solid 18
(
500 mg, 1.20 mmol) as described for the preparation of
9
H O: white solid 15 (510 mg, 92%); mp 165 C.
. The residue (800 mg) was crystallized from ethanol/
ꢀ
1
H
2
ꢀ
NMR(200 MHz, DMSO- d ): d 8.32 (m, 2H), 4.61 (m,
(340 mg, 60%); mp 180 C. Method B: A solution of
SOCl (31 mg, 0.26 mmol) in THF (5 mL) was added
6
1
H), 1.98 (s, 3H), 1.92–1.01 (m, 31H), 0.91 (s, 3H), 0.63
3
2
1
(
s, 3H), C NMR(50 MHz, DMSO- d ): d 174.45,
dropwise into a stirred solution of dehydrocholic acid
(100 mg, 0.25 mmol) and 5-amino-1,3,4-thiadiazole-2-
sulfonamide (4) (45 mg, 0.25 mmol) in dry THF (15 mL)
during 10 min. After the addition was complete, stirring
was continued for 12 h. The THF was removed under
reduced pressure and the residue was crystallized from
6
1
4
3
2
2
1
71.49, 166.10, 162.90, 75.31, 57.71, 57.33, 44.12, 43.03,
2.10, 41.68, 37.15, 36.68, 36.35, 35.98, 33.80, 33.73,
2.52, 29.47, 28.41, 28.09, 27.75, 25.61, 24.81, 22.85,
2.24, 20.02, 13.67, IR(KBr, cm ): 3285, 3208, 3080,
953, 2876, 1753, 1702, 1548, 1472, 1370, 1268, 1191,
114, 1038, 987, 936.
À1
1
ethanol: 18 (115 mg, 82%). H NMR(200 MHz,
DMSO-d ): d 2.68–0.73 (m, 27H), 1.32 (s, 3H), 0.99 (s,
6
1
3
3
ꢀ,12ꢀ-Diacetoxy-5-ꢁ-cholan-24-oic acid (13). The dia-
3H), C NMR(50 MHz, DMSO- d ): d 213.64, 211.28,
6
cetoxy cholanoic acid 13 was prepared from deoxy-
cholic acid (500 mg, 1.20 mmol) as described for the
preparation of 12. The residue (720 mg) was crystallized
211.21, 176.50, 174.93, 173.43, 58.05, 53.00, 49.78,
47.78, 47.19, 46.34, 45.87, 44.33, 40.17, 37.94, 37.43,
36.77, 36.38, 32.82, 32.14, 29.02, 26.39, 22.95, 20.42,
13.22, IR(KBr, cm ): 3565, 3489, 3438, 3259, 2978,
2876, 1727, 1651,1625, 1548, 1472, 1395, 1293, 1191,
1114, 1063, 961.
À1
from ethanol/H O: white solid 13 (450 mg, 75%); mp
2
ꢀ
.59 (m, 1H), 2.05 (s, 3H), 1.98 (s, 3H), 2.56–0.69 (m,
1
2
4
2
35 C. H NMR(200 MHz, DMSO- d ): d 4.97 (s, 1H),
6
9H), 0.89 (s, 3H), 0.70 (s, 3H), 13C NMR(50 MHz,
DMSO-d ): d 174.94, 171.55, 171.40, 76.72, 75.11, 50.85,
6
In vitro studies
4
3
2
3
9
8.84, 46.35, 42.89, 36.89, 36.02, 35.87, 35.68, 35.39,
3.69, 32.35, 32.24, 28.60, 28.23, 27.95, 27.36, 26.92,
4.81, 24.51, 22.88, 22.72, 18.97, 13.86, IR(KBr, cm ):
464, 2953, 2876, 1753, 1472, 1395,1268, 1140, 1114,
61, 783.
Purification of carbonic anhydrase I and II from human
erythrocytes. Erythrocytes were purified from human
blood. The blood samples were centrifuged at 1500 rpm
for 20 min and plasma and buffy coat were removed.
After the packed red cells were washed with NaCl
(0.9%), the erythrocytes were hemolyzed with 1.5
volumes of ice-cold water. Cell membranes were
À1
3ꢀ,7ꢀ,12ꢀ-Triacetoxy-5-ꢁ-cholan-24-oic acid (14). The
triacetoxy cholanoic acid 14 was prepared from cholic
ꢀ
acid (500 mg, 1.22 mmol) as described for the prepara-
tion of 12. The residue (680 mg) was crystallized from
removed by centrifugation at 4 C, 20,000 rpm for
30 min. The pH of hemolysate was brought to 8.7 with
solid Tris. The hemolysate was applied to affinity col-
umn having a structure of Sepharose-4B-l-tyrosine-p-
aminobenzensulfonamide and equilibrated with 25 mM
Tris–HCl/0.1 M Na SO (pH 8.7). The affinity gel was
ꢀ
ethanol/H O: white solid 14 (520 mg, 79%); mp 115 C.
2
1
H NMR(200 MHz, DMSO- d ), d 4.98 (m, 1H), 4.80
6
(
3
m, 1H), 4.45 (m, 1H), 2.09 (s, 3H), 2.02 (s, 3H), 1.99 (s,
3
1
H), 2.31–0.74 (m, 27H), 0.91 (s, 3H), 0.70 (s, 3H),
C
2
4
NMR(50 MHz, DMSO- d ), d 174.93, 171.57, 171.50,
washed with solution of 25 mM Tris–HCl/22 mM
Na SO (pH 8.7). CA-I and CA-II isozymes were eluted
with the solution of 1 M NaCl/25 mM Na HPO (pH
6
1
4
3
71.41, 76.36, 75.12, 71.97, 48.72, 46.41, 44.82, 42.01,
1.60, 38.66, 36.15, 35.84, 35.71 32.56, 32.32, 32.16,
0.12, 28.45, 28.25, 26.88, 24.03, 23.94, 23.02, 22.93,
2
4
2
4
6.3) and 0.1 M NaCH COO/0.5 M NaClO (pH 5.6),
3
4

2566
M. Bu¨lbu¨l et al. / Bioorg. Med. Chem. 10 (2002) 2561–2567
respectively. The absorbance at 280 nm was used to
monitor protein in the column effluents. For protein
content estimation, the method of Coomassie Brillant
Blue was used with bovine serum albumin as a standard.
Acknowledgements
The authors are indebted to the Department of Chem-
¨
istry (Ataturk University) for financial support of this
work, to Prof. Claudiu T. Supuran (Florence, Italy) for
providing his articles, to Dr. Gerald Draeger (Hann-
over, Germany) for mass spectral analysis and to Dr.
Cavit Kazaz for H/ C NMRspectral analysis
(Erzurum, Turkey).
Measurement of CA activity. CO -hydratase activity of
2
the enzyme was determined at 0 C in a veronal buffer
(
saturated carbon dioxide solution as substrate in a final
volume of 4.2 mL. The time (in seconds) taken for the
solution to change from blue to green was measured.
The enzyme unit (EU) is the enzyme amount resulting in
ꢀ
pH=8.15) with Brom Timol Blue as indicator and
1
13
References and Notes
5
Activity as an enzyme unit was calculated by using the
0% decreased of the nonenzymatic reaction time.
1
1
2
. Supuran, C. T.; Scozzafava, A. Exp. Opin. Ther. Pat. 2002,
2, 217.
. (a) Sugrue, M. F. Prog. Retinal Eye Res. 2000, 19 (1), 87.
equation (t Àt /t ) where t and t are times for pH
0
c
c
o
c
change of the nonenzymatic and enzymatic reactions,
respectively.
(
¨
b) Wistrand, P. J.; Schenholm, M.; Lonnerholm, G. Invest.
Ophthalmol. Vis. Sci. 1986, 27, 410. (c) Hageman, G. S.; Zhu,
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50
values were assayed by following the hydration of CO2.
I50 values for the synthesized compounds and acetazol-
amide (3) were determined on CA-I and CA-II. In order
to determine I50 values, a saturated solution of CO was
2
used as substrate. To a total volume of 1.7 mL of var-
ious aliquots (10–250 mL) of a suitable concentration of
inhibitors and water were added to 1 mL of Tris–HCl,
6
. Ashour, F. A.; Habib, N. S.; Taibbi, M.; Dine, S.; Dine, A.
Farmaco 1990, 45, 134.
. (a) Friberg, L.; Kastrup, J.; Rizzi, D.; Jensen, J. B.; Lassen,
7
0
.1 mL of Brom Timol Blue and 0.1 mL enzyme solu-
N. A. Am. J. Physiol. 1990, 258, 954. (b) Vostrup, S.; Henrik-
sen, L.; Paulson, O. B. J. Clin. Invest. 1984, 74, 1634.
tion. The reaction was started by adding 2.5 mL of CO2
solution. The activity was determined by using the
equation (t Àt /t ) where t and t are times for pH
8
. Supuran, C. T.; Scozzafava, A. Curr. Med. Chem. Imm.,
Endoc. Metab. Agents 2001, 1, 61.
. Supuran, C. T.; Briganti, F.; Tilli, S.; Chegwidden, W. R.;
0
c
c
0
c
change of enzymatic reactions for each inhibitor con-
centration, respectively. Regression analysis graphs
were drawn by using inhibitor concentration values by a
statistical packing program on a computer. I , the
9
Scozzafava, A. Bioorg. Med. Chem. 2001, 9, 703.
10. Supuran, C. T.; Scozzafava, A. Exp. Opin. Ther. Pat.
2000, 10, 575.
50
1
1. (a) Supuran, C. T. Roum. Chem. Q. Rev. 1993, 1, 77. (b)
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activity against inhibitor concentration.
(
1
Chemistry; Pergamon: Oxford, 1996. (b) Davis, A. P. Chem.
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In vivo inhibitor studies
Forty-two adult male Spargue–Dawley rats with a
weight from 200 to 250 g were used for the experiment.
All of the animals were fed with standard laboratory
chow and water before the experiment. The animal
1
4. (a) McKenna, J.; McKenna, J. M.; Thornthwaite, D. W.
laboratory was windowless with automatic temperature
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J. Chem. Soc., Chem. Commun. 1977, 809. (b) Janout, V.;
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(
22Æ1 C) and lighting controls (14 h light/10 h dark).
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fasted and allowed access to water ad lib. All of the ani-
mals, seven in each group, were housed in different cages.
1
5. (a) Chengy, Y.; Ho, D. M.; Gottlieb, C. R.; Kahne, D.;
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1
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For the control, 100 mL of blood samples were taken
from tail vein before inhibitor administration, which were
placed into test tubes containing EDTA. Then, 250 mg/kg
inhibitors (9–11, 15–18) were injected intraperitoneally
into each rat group. Blood samples were taken from each
rat at 2, 4 and 6 h after injection. Hemolysate was pre-
pared as described for in vitro studies. CA activity was
measured with the Wilbur and Anderson method.
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1
˘
.
¨
¨
Kufreviog
˘
2
Statistical analysis. Results were given as meanÆSD.
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values less than 0.001 were considered as indicative of
significance.
2
Chen, H.-H.; Gross, S.; Liao, J.; McLaughlin, M.; Dean, T.;
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