Carbonic anhydrase inhibitors. Preparation of potent sulfonamides inhibitors incorporating bile acid tails.

Article abstract of DOI:10.1016/S0960-894X(02)00252-4

Reaction of TBDMS-protected bile acids (cholic, chenodeoxycholic, deoxycholic, lithocholic, ursodeoxycholic acids) or dehydrocholic acid with aromatic/heterocyclic sulfonamides possessing free amino/hydroxy moieties, in the presence of carbodiimides, afforded after deprotection of the OTBDMS ethers, a series of sulfonamides incorporating bile acid moieties in their molecules. Many such derivatives showed strong inhibitory properties against three isozymes of carbonic anhydrase (CA, EC 4.2.1.1), that is CA I, II and IV, zinc enzymes playing critical roles in many pathologies, and which represent interesting targets for developing diverse pharmacological agents. Some of the most active derivatives, incorporating 1,3,4-thiadiazole-2-sulfonamide or benzothiazole-2-sulfonamide functionalities in their molecules, showed low nanomolar affinity for CA II and CAIV. Furthermore, the bioavailability of these derivatives in rabbits is comparable to that of acetazolamide, being in the range of 85-90%, showing them as promising candidates for systemically acting CA inhibitors.

Full text of DOI:10.1016/S0960-894X(02)00252-4

Bioorganic & Medicinal Chemistry Letters 12 (2002) 1551–1557
Carbonic Anhydrase Inhibitors. Preparation of Potent
Sulfonamides Inhibitors Incorporating Bile Acid Tails
Andrea Scozzafava and Claudiu T. Supuran*
`
Universita degli Studi di Firenze, Dipartimento di Chimica, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3,
I-50019 Sesto Fiorentino, Firenze, Italy
Received 20 February 2002; revised 16 April 2002; accepted 18April 2002
Abstract—Reaction of TBDMS-protected bile acids (cholic, chenodeoxycholic, deoxycholic, lithocholic, ursodeoxycholic acids) or
dehydrocholic acid with aromatic/heterocyclic sulfonamides possessing free amino/hydroxy moieties, in the presence of carbodii-
mides, afforded after deprotection of the OTBDMS ethers, a series of sulfonamides incorporating bile acid moieties in their mole-
cules. Many such derivatives showed strong inhibitory properties against three isozymes of carbonic anhydrase (CA, EC 4.2.1.1),
that is CA I, II and IV, zinc enzymes playing critical roles in many pathologies, and which represent interesting targets for devel-
oping diverse pharmacological agents. Some of the most active derivatives, incorporating 1,3,4-thiadiazole-2-sulfonamide or ben-
zothiazole-2-sulfonamide functionalities in their molecules, showed low nanomolar affinity for CA II and CAIV. Furthermore, the
bioavailability of these derivatives in rabbits is comparable to that of acetazolamide, being in the range of 85–90%, showing them
as promising candidates for systemically acting CA inhibitors. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
among which open-angle glaucoma, epilepsy and other
neuromuscular diseases, acid/base secretory dis-
equilibria and osteoporosis are the most common
ones.1ꢀ3 The main drawback of systemically used agents
(such as the first four sulfonamides mentioned above) is
constituted by side effects such as augmented diuresis,
fatigue, paresthesias, anorexia and so on due to CA
inhibition in other tissues/organs than the target one
Sulfonamide inhibitors of carbonic anhydrase (CA, EC
4.2.1.1), such as acetazolamide AAZ, methazolamide
MZA, ethoxzolamide EZA, dichlorophenamide DCP,
dorzolamide DZA or brinzolamide BRZ have been
clinically used for a long period in the treatment/pre-
vention of a variety of diseases/pathological states
*Corresponding author. Tel.: +39-055-457-3005; fax: +39-055-457-3363; e-mail: claudiu.supuran@unifi.it
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00252-4

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A. Scozzafava, C. T. Supuran / Bioorg. Med. Chem. Lett. 12 (2002) 1551–1557
(CA, in the form of 14 isozymes, is ubiquitous in verte-
brates).4 The recently developed topically acting agents
DZA and BRZ, on the other hand, show better selec-
tivity, generally inhibiting only eye tissues CAs respon-
sible for the enhanced aqueous humor secretion and
increased intraocular pressure characteristic of glau-
coma, which represents the main risk factor of blindness
in the western countries.1ꢀ5 The ubiquity of these
enzymes in many tissues of higher vertebrates con-
stantly prompts much research for discovering novel
biomedical applications which may exploit their differ-
ential inhibition.6ꢀ16
interaction may contribute to the regulation of the
enzymatic activity along the enterohepatic circulation as
well as to some metabolic derangements in patients with
cholestatic liver disease.17 Since several CA isozymes
(such as CA I, II, IV and V) are highly abundant along
the gastro-intestinal tract (where they play important
roles in the control of ion and fluid transport processes
as well as acid-base homeostasy),3,18,19 it appeared of
interest to investigate in more detail whether the pres-
ence of bile acid moieties in the molecules of sulfon-
amide CA inhibitors may lead to novel biomedical
applications of these agents. It should be mentioned
that bile acids per se (such as derivatives 1–6) play
important physiological functions such as induction of
bile flow, feedback inhibition of bile acid synthesis,
It has been reported that bile acids/salts are inhibitors
of several CA isozymes, with speculation that this

A. Scozzafava, C. T. Supuran / Bioorg. Med. Chem. Lett. 12 (2002) 1551–1557
1553
modulation of cholesterol synthesis, elimination of cho-
lesterol (by solubilizing it in the bile and promoting its
intestinal excretion) and facilitation of adsorption of
lipids and fat-soluble vitamins, and are also used thera-
peutically for gallstone dissolution, the treatment of
primary biliary cyrrhosis and hepatobiliary compli-
cations of cystic fibrosis.20,21 Here, we report the syn-
thesis and CA inhibitory properties of a large series of
sulfonamides incorporating cholic, chenodeoxycholic,
deoxycholic, lithocholic, ursodeoxycholic and dehydro-
cholic acid (1–6) moieties in their molecules, as well as
preliminary in vivo bioavailability data of several such
compounds.
by the standard procedure, that is in the presence of
tetraalkylammonium fluoride,23 leading to the desired
products (1–6)A–T with excellent yields.24
Carbonic Anhydrase Inhibitory Activity
The data of Table 1 show that the new inhibitors pre-
pared by attaching bile acid moieties to aromatic/het-
erocyclic sulfonamides A–T, are more effective as
compared to the parent sulfonamides from which they
were prepared, towards the three investigated isozymes,
hCA I, hCA II and bCA IV. The enhanced inhibitory
power of these compounds is presumably due to the
interaction of the (long) bile acid moiety incorporated in
their molecules with hydrophilic/hydrophobic patches at
the entrance of the enzyme active site as observed for
inhibitors previously reported by Whitesides’25 and our
groups,1,2,7ꢀ16 and explained by detailed QSAR models.26
Chemistry
The chemical structure of the carboxylic acids 1–6 as
well as those of sulfonamides A–T used in the synthesis
of the new compounds reported here are shown.
The nature of the sulfonamide attached to the bile acid
moiety in the new derivatives reported here greatly
influenced the CA inhibitory power of these com-
pounds. Among the synthesized compounds, the
heterocyclic sulfonamide derivatives were the most
active, followed by the aromatic sulfonamide deriva-
tives. The efficiency of the obtained inhibitor generally
varied in the following way, based on the parent sul-
fonamide from which it was prepared: the derivatives of
p-hydrazino-benzenesulfonamide < the orthanilamides
< the metanilamides < the sulfanilamides<the homo-
sulfanilamides < the p-aminoethyl-benzenesulfonamides
ffi the halogeno-substituted sulfanilamides ffi the 1,3-
benzene-disulfonamides < the 1,3,4-thiadiazole-2-sul-
fonamides ffi 4-methyl-d2-1,3,4-thiadiazoline-2-sulfon-
amide ffi the benzothiazole-2-sulfonamides. The nature
of the acylating moiety also influenced the inhibitory
properties of these new sulfonamides, with the following
range of inhibitory effects for the corresponding sul-
fonamides incorporating polyamino-polycarboxylic acid
moieties: cholic acid derivatives <chenodeoxycholic
acid derivatives < deoxycholic acid derivatives <
lithocholic acid derivatives < ursodeoxycholic acid
derivatives < dehydrocholic acid derivatives. All three
CA isozymes investigated here were susceptible to
As a large number of such derivatives (amides/esters)
have been prepared, in the following they will be
abbreviated by using both a figure designating the car-
boxylic acid from which they were derived, as well as a
letter designating the sulfonamides to which the bile
acid moiety has been attached. For example: 1A is the
amide of cholic acid 1 with orthanilamide A; 6M is the
amide of dehydrocholic acid 6 with 5-amino-1,3,4-thia-
diazole-2-sulfonamide M; 4S is the ester of lithocholic
acid 4 with 4-(2-hydroxymethyl)-benzenesulfonamide S
and so on. The whole series of sulfonamides containing
the six carboxylic acids 1–6 and the 20 sulfonamides A–
T have been prepared (Table 1).
The synthesis has been done by applying previously
reported procedures,2,7,10ꢀ12 involving the reaction of
appropriately protected carboxylic acids with amino/
hydroxy-sulfonamides A–T, in the presence of carbodii-
mides (Scheme 1). The tert-butyl-dimethylsilyl ethers
were chosen as protection of the reactive hydroxyl moi-
eties present in bile acids 1–5,22 whereas no protecting
groups were necessary for the reaction of 6, which was
simply coupled with A–T in the presence of carbodi-
imides. The deprotection step has thereafter been done

1554
A. Scozzafava, C. T. Supuran / Bioorg. Med. Chem. Lett. 12 (2002) 1551–1557
Table 1. Inhibition data for derivatives (1–6)A–T reported in the present paper (data in parentheses represent inhibition by the parent sulfonamide
A–T) and standard sulfonamide CA inhibitors
Inhibitor
KI (nM)
hCA IIa
hCA Ia
bCA IVb
Acetazolamide
Methazolamide
Ethoxzolamide
Dichlorophenamide
Dorzolamide
Brinzolamide
1A
1B
1C
1D
1E
1F
1G
1H
1I
1J
1K
900
780
25
12
14
813
3838
9
3
220
240
1200
>50,000
0
43
45
—
25,500 (45,400)
23,000 (25,000)
21,000 (28,000)
32,100 (78,500)
3780 (25,000)
3400 (21,000)
1280 (8300)
970 (9800)
275 (295)
218(240)
210 (300)
315 (320)
120 (170)
86 (160)
50 (60)
82 (110)
35 (40)
53 (70)
21 (28)
56 (75)
50 (60)
12 (19)
1.2 (3)
1200 (1310)
1350 (2200)
430 (3000)
1050 (3200)
254 (2800)
220 (2500)
133 (180)
168 (320)
60 (66)
104 (125)
97 (175)
130 (160)
205 (540)
115 (355)
31 (125)
600 (6500)
510 (6000)
400 (6100)
400 (8400)
420 (8600)
700 (9300)
250 (455)
35 (70)
1L
1M
1N
1O
1P
1Q
1R
5 (9)
4 (8)
3 (7)
10 (19)
9 (17)
10 (15)
32 (55)
39 (50)
1S
1T
2A
2B
2C
2D
2E
1200 (24,000)
1150 (18,000)
22,100 (45,400)
21,400 (25,000)
20,000 (28,000)
27,500 (78,500)
3450 (25,000)
3300 (21,000)
1050 (8300)
950 (9800)
97 (125)
80 (110)
245 (295)
210 (240)
186 (300)
295 (320)
96 (170)
86 (160)
50 (60)
80 (110)
34 (40)
55 (70)
21 (28)
62 (75)
52 (60)
13 (19)
2 (3)
280 (560)
300 (450)
900 (1310)
1250 (2200)
420 (3000)
850 (3200)
244 (2800)
235 (2500)
128 (180)
150 (320)
53 (66)
110 (125)
95 (175)
143 (160)
240 (540)
100 (355)
45 (125)
2F
2G
2H
2I
2J
2K
617 (6500)
521 (6000)
343 (6100)
300 (8400)
410 (8600)
514 (9300)
268(455)
62 (70)
2L
2M
2N
2O
2P
2Q
2R
5.6 (9)
6.1 (8)
5.2 (7)
13 (19)
12 (17)
9 (15)
47 (55)
44 (50)
2S
2T
3A
3B
3C
3D
3E
1230 (24,000)
1100 (18,000)
20,100 (45,400)
12,700 (25,000)
12,000 (28,000)
21,300 (78,500)
1710 (25,000)
1500 (21,000)
780 (8300)
98(125)
90 (110)
280 (295)
230 (240)
210 (300)
310 (320)
80 (170)
77 (160)
42 (60)
77 (110)
31 (40)
47 (70)
20 (28)
55 (75)
50 (60)
11 (19)
2.1 (3)
340 (560)
320 (450)
975 (1310)
980 (2200)
355 (3000)
920 (3200)
233 (2800)
245 (2500)
96 (180)
3F
3G
3H
3I
3J
3K
890 (9800)
365 (6500)
415 (6000)
285 (6100)
225 (8400)
200 (8600)
240 (9300)
200 (455)
151 (320)
50 (66)
110 (125)
100 (175)
121 (160)
130 (540)
63 (355)
30 (125)
12 (19)
9 (17)
8(15)
3L
3M
3N
3O
3P
3Q
3R
56 (70)
39 (55)
37 (50)
6.5 (9)
4.8(8)
4.1 (7)
3S
3T
4A
4B
4C
4D
1000 (24,000)
885 (18,000)
9200 (45,400)
9500 (25,000)
6300 (28,000)
15,000 (78,500)
106 (125)
78 (110)
270 (295)
210 (240)
106 (300)
280 (320)
350 (560)
300 (450)
750 (1310)
740 (2200)
280 (3000)
650 (3200)
(continued on next page)

A. Scozzafava, C. T. Supuran / Bioorg. Med. Chem. Lett. 12 (2002) 1551–1557
1555
Table 1 (continued)
Inhibitor
KI (nM)
hCA Ia
hCA IIa
bCA IVb
4E
4F
4G
4H
4I
910 (25,000)
650 (21,000)
360 (8300)
720 (9800)
120 (6500)
120 (6000)
130 (6100)
200 (8400)
210 (8600)
205 (9300)
200 (455)
48 (170)
41 (160)
29 (60)
47 (110)
21 (40)
32 (70)
17 (28)
54 (75)
10 (60)
8(19)
83 (2800)
70 (2500)
36 (180)
54 (320)
53 (66)
69 (125)
87 (175)
82 (160)
28 (540)
21 (355)
19 (125)
9 (19)
4J
4K
4L
4M
4N
4O
4P
4Q
4R
4S
4T
5A
5B
5C
5D
5E
5F
5G
5H
5I
2.1 (3)
2.8(9)
2.0 (8)
1.4 (7)
48(70)
40 (55)
36 (50)
9 (17)
8(15)
800 (24,000)
625 (18,000)
8400 (45,400)
7050 (25,000)
4000 (28,000)
12,000 (78,500)
550 (25,000)
295 (21,000)
180 (8300)
190 (9800)
91 (6500)
92 (125)
81 (110)
180 (295)
157 (240)
71 (300)
240 (320)
21 (170)
14 (160)
11 (60)
17 (110)
10 (40)
13 (70)
10 (28)
9 (75)
0.9 (60)
2.0 (19)
1.3 (3)
0.6 (9)
0.7 (8)
0.6 (7)
75 (125)
58 (110)
170 (295)
156 (240)
70 (300)
260 (320)
26 (170)
18 (160)
15 (60)
13 (110)
9 (40)
313 (560)
310 (450)
500 (1310)
480 (2200)
164 (3000)
615 (3200)
40 (2800)
34 (2500)
24 (180)
30 (320)
23 (66)
20 (125)
29 (175)
46 (160)
16 (540)
17 (355)
11 (125)
9 (19)
5J
67 (6000)
59 (6100)
71 (8400)
48(8600)
54 (9300)
98(455)
30 (70)
31 (55)
30 (50)
5K
5L
5M
5N
5O
5P
5Q
5R
5S
5T
6A
6B
6C
6D
6E
6F
6G
6H
6I
6 (17)
5 (15)
500 (24,000)
350 (18,000)
8100 (45,400)
7440 (25,000)
3700 (28,000)
12,750 (78,500)
600 (25,000)
285 (21,000)
210 (8300)
200 (9800)
66 (6500)
245 (560)
160 (450)
505 (1310)
440 (2200)
147 (3000)
550 (3200)
40 (2800)
29 (2500)
28 (180)
30 (320)
28(66)
6J
61 (6000)
45 (6100)
77 (8400)
41 (8600)
29 (9300)
22 (455)
39 (70)
33 (55)
10 (70)
9 (28)
11 (75)
0.5 (60)
1.0 (19)
1.1 (3)
0.8(9)
0.6 (8)
0.8(7)
71 (125)
50 (110)
19 (125)
24 (175)
50 (160)
9 (540)
8(355)
7 (125)
7 (19)
5 (17)
6 (15)
200 (560)
123 (450)
6K
6L
6M
6N
6O
6P
6Q
6R
6S
6T
30 (50)
450 (24,000)
415 (18,000)
aHuman (cloned) isozymes.
bFrom bovine lung microsomes, by the esterase method.27
inhibition with this type of sulfonamides, with hCA II
and bCA IV the most sensitive, whereas hCA I was
generally less susceptible to inhibition as compared to
the first two isozymes.
ability. Acetazolamide, a widely clinically used CA
inhibitor has been used as standard in these experiments
(Table 2). Both at 1 h, as well as 2 h post administration,
red blood cells (containing 150 mM CA I and 10 mM CA
II)29 were saturated with sulfonamide, as determined
both by an HPLC as well as an electronic spectroscopic
method.28 Due to the increased liposolubility of the new
inhibitors as compared to acetazolamide, already at 1 h
Some of the most active inhibitors reported here, such
as 6M and 6Q were administered (via gavage) to albino
rabbits in order to investigate their systemic bioavail-

1556
A. Scozzafava, C. T. Supuran / Bioorg. Med. Chem. Lett. 12 (2002) 1551–1557
Scheme 1. Preparation of sulfonamides (1–6)A–T. Reagents and conditions are: TBDMSCl, tert-butyl-dimethylsilylchloride; DMAP, 4-dimethyl-
aminopyridine; DMF, room temperature, 4 h; EDAC, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, sulfonamide A–T, in DMF, room tem-
perature, 12–15 h; n-Bu4NF, tetra-n-butylammonium fluoride, 2 equivalents, in THF, room temperature, 2 h (in the above formulas X=O or NH; A
as in sulfonamides A–T).
post administration the maximal sulfonamide levels
were attained in the blood of the experimental animals
in the case of the new inhibitors 6M and 6Q, whereas
for acetazolamide, the same levels are attained only
after 2 h. Since the bioavailability of acetazolamide is
estimated to be of 85–90%,29 we speculate the same
order of magnitude for this parameter in the case of the
new sulfonamides reported here, which makes them
interesting leads for the investigation of different types
of biomedical applications employing systemic CA
inhibitors.
nanomolar range for isozymes hCA I, hCA II and bCA
IV, acting as effective enzyme inhibitors in vitro. In
vivo, in rabbits, the bioavailability of some of these
derivatives was in the range of 85–90%, proving that
such new compounds reported here might lead to the
development of other type of drugs from the class of the
sulfonamide CA inhibitors.
References and Notes
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Conclusions
We report here a novel class of sulfonamides obtained
by the ‘tail’ approach,1 which incorporate bile acid
moieties. They were obtained by coupling TBDMS-
protected bile acids with amino/hydroxysulfonamides in
the presence of carbodiimides, followed by deprotection
in the presence of tetraalkylammonium fluoride. Some
of these new CA inhibitors possessed affinities in the low
Table 2. Levels of sulfonamides (mM) in red blood cells of albino
rabbits, at 60 and 120 min post-administration of 50 mg/kg sulfon-
amides AAZ, 6M and 6Q
Inhibitor
[sulfonamide], mM*
t=60 min t=120 min
HPLCa HPLCa ESb
ESb
Acetazolamide
6M
6Q
136Æ7
155Æ4
156Æ6
140Æ4
151Æ5
158Æ9
160Æ8167
162Æ4
Æ5
159Æ3
163Æ5
159Æ5
aMeanÆstandard deviation (from three determinations) by the HPLC
bMeanÆstandard deviation (from three determinations) by the elec-
tronic spectroscopic method.
method.

A. Scozzafava, C. T. Supuran / Bioorg. Med. Chem. Lett. 12 (2002) 1551–1557
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Acids in Health and Disease. In Gastrointestinal Disease, 5th
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Philadelphia, 1993; p 127.
metric amount of Et3N (79 mL) was then added and the reac-
tion mixture stirred at room temperature for 12 h (TLC
control). The solvent was evaporated in vacuo, the residue
taken up with 25 mL of water, filtered, dried and crystallized
from EtOH (502 mg, yield of 89%). Colorless crystals, mp 191–
193 ꢁC, IR(KBr), cmꢀ1: 1164 (SO2sym), 1347 (SOa2s), 1561 (amide
1
II), 1607 (amide I), 1723 (C¼O); 3330 (NH, NH2); H NMR
(CDCl3), 300 MHz, d, ppm: 0.98(s, 3H); 1.32 (s, 3H); 0.74–2.71
(m, 27H); 7.35 (2H, br s, SO2NH2); 8.10 (br s, CONH); 13C
NMR (DMSO-d6), d, ppm: 13.24; 20.31; 22.89; 26.34; 28.97;
32.13; 32.82; 36.30; 36.75; 37.45; 37.90; 40.21; 44.25; 45.83;
46.30; 47.22; 47.78; 49.80; 53.12; 57.89; 173.49; 175.11; 176.50;
211.20; 211.32; 213.55. Anal. found C, 55.61; H, 6.24; N,
9.89%; C26H36N4O6S2 requires: C, 55.30; H, 6.43; N, 9.92%.
25. Jain, A.; Whitesides, G. M.; Alexander, R. S.; Chris-
tianson, D. W. J. Med. Chem. 1994, 37, 2100.
26. (a) Supuran, C. T.; Clare, B. W. Eur. J. Med. Chem. 1999,
34, 41. (b) Clare, B. W.; Supuran, C. T. Eur. J. Med. Chem.
1999, 34, 463.
27. A stopped flow variant of the Pocker and Stone spectro-
photometric method (Pocker, Y.; Stone, J. T. Biochemistry
1967, 6, 668) has been employed, using an SX.18MV-R
Applied Photophysics stopped flow instrument.
28. Supuran, C. T.; Scozzafava, A.; Ilies, M. A.; Iorga, B.;
Cristea, T.; Chiraleu, F.; Banciu, M. D. Eur. J. Med. Chem.
1998, 33, 577.
22. Corey, E. J.; Venkateswarlu, A. J. Am. Chem. Soc. 1972,
94, 6190.
23. Huff, B. E. In Handbook of Reagents for Organic Syn-
thesis—Activating Agents and Protecting Groups; Pearson, A.
J., Roush, W. J. Eds.; Wiley: Chichester, 1999; p 84.
24. An example of synthesis is illustrated below: an amount of
180 mg (1 mmol) 5-amino-1,3,4-thiadiazole-2-sulfonamide M,
29. Maren, T. H. Physiol. Rev. 1967, 47, 595.