Carbonic anhydrase inhibitors. A general approach for the preparation of water-soluble sulfonamides incorporating polyamino-polycarboxylate tails and of their metal complexes possessing long-lasting, topical intraocular pressure-lowering properties

Article abstract of DOI:10.1021/jm0108202

Reaction of polyamino-polycarboxylic acids or their dianhydrides with aromatic/heterocyclic sulfonamides possessing a free amino/imino/hydrazino/hydroxy group afforded mono- and bissulfonamides containing polyamino-polycarboxylic acid moieties in their molecule. The acids/ anhydrides used in synthesis included IDA, NTA, EDDA, EDTA and EDTA dianhydride, DTPA and DTPA dianhydride, EGTA and EGTA dianhydride, and EDDHA, among others. All the newly prepared derivatives showed strong affinity toward isozymes I, II, and IV of carbonic anhydrase (CA). Metal complexes of the new compounds have also been prepared. Metal ions used in such preparations included di- and trivalent main-group and transition cations, such as Zn(II), Cu(II), Al(III), etc. Some of the new sulfonamides/disulfonamides obtained in this way, as well as their metal complexes, behaved as nanomolar CA inhibitors against isozymes II and IV, being slightly less effective in inhibiting isozyme I. Some of these sulfonamides as well as their metal complexes strongly lowered intraocular pressure (IOP) when applied topically, directly into the normotensive/glaucomatous rabbit eye, as 1-2% water solutions/ suspensions. The good water solubility of these sulfonamide CA inhibitors, correlated with the neutral pH of their water solutions used in the ophthalmologic applications and the long duration of action of the IOP-lowering effect, makes them interesting candidates for developing novel types of antiglaucoma drugs devoid of serious topical side effects.

Full text of DOI:10.1021/jm0108202

1466
J . Med. Chem. 2002, 45, 1466-1476
Ca r bon ic An h yd r a se In h ibitor s. A Gen er a l Ap p r oa ch for th e P r ep a r a tion of
Wa ter -Solu ble Su lfon a m id es In cor p or a tin g P olya m in o-P olyca r boxyla te Ta ils
a n d of Th eir Meta l Com p lexes P ossessin g Lon g-La stin g, Top ica l In tr a ocu la r
P r essu r e-Low er in g P r op er ties
Andrea Scozzafava,† Luca Menabuoni,‡ Francesco Mincione,§ and Claudiu T. Supuran*,†,|
Laboratorio di Chimica Inorganica e Bioinorganica, Universita` degli Studi di Firenze, Via Gino Capponi 7, I-50121,
Florence, Italy, Ospedale San Giovanni di Dio, U.O. Oculistica, Via Torregalli 3, I-50123, Florence, Italy,
Institute of Ophthalmology, Universita` degli Studi, Viale Morgagni 85, I-50134 Florence, Italy, U.O. Oculistica Az. USL 3,
Val di Nievole, Ospedale di Pescia, Pescia, Italy, and CSGI, c/ o Universita` degli Studi di Firenze, Via Gino Capponi 7,
I-50121 Florence, Italy
Received J anuary 15, 2001
Reaction of polyamino-polycarboxylic acids or their dianhydrides with aromatic/heterocyclic
sulfonamides possessing a free amino/imino/hydrazino/hydroxy group afforded mono- and bis-
sulfonamides containing polyamino-polycarboxylic acid moieties in their molecule. The acids/
anhydrides used in synthesis included IDA, NTA, EDDA, EDTA and EDTA dianhydride, DTPA
and DTPA dianhydride, EGTA and EGTA dianhydride, and EDDHA, among others. All the
newly prepared derivatives showed strong affinity toward isozymes I, II, and IV of carbonic
anhydrase (CA). Metal complexes of the new compounds have also been prepared. Metal ions
used in such preparations included di- and trivalent main-group and transition cations, such
as Zn(II), Cu(II), Al(III), etc. Some of the new sulfonamides/disulfonamides obtained in this
way, as well as their metal complexes, behaved as nanomolar CA inhibitors against isozymes
II and IV, being slightly less effective in inhibiting isozyme I. Some of these sulfonamides as
well as their metal complexes strongly lowered intraocular pressure (IOP) when applied
topically, directly into the normotensive/glaucomatous rabbit eye, as 1-2% water solutions/
suspensions. The good water solubility of these sulfonamide CA inhibitors, correlated with the
neutral pH of their water solutions used in the ophthalmologic applications and the long
duration of action of the IOP-lowering effect, makes them interesting candidates for developing
novel types of antiglaucoma drugs devoid of serious topical side effects.
In tr od u ction
applied directly into the eye. This route, discovered in
1983 by Maren’s group,5 was shortly followed by the
development of the first agents of this type, dorzolamide
DZA (clinically launched in 1995),6 followed soon there-
after (in 1999) by the structurally related brinzolamide
BRZ7 (Chart 1). These topically acting antiglaucoma
sulfonamides incorporate secondary amine moieties
because the required water solubility needed for effec-
tive topical action is achieved by using their hydrochlo-
ride salts.6,7 Still, this represents an undesired problem
because the pH of such solutions is rather acidic (pH
5.5) and consequently produces eye irritation after the
topical administration of the drug, as already reported
for many patients treated with dorzolamide (the use of
brinzolamide is rather recent, and few studies of the
side effects of this drug are available for the mo-
ment).1,6,7 In fact, the most common adverse effects after
topical dorzolamide treatment are local burning and
stinging of the eyes, reddening of the eyes, and blurred
vision and bitter taste,1,6,7 but more serious side effects,
such as contact allergy, nephrolithiasis, anorexia, de-
pression, and dementia, as well as irreversible corneal
decompensation in patients already presenting corneal
problems, were also reported with dorzolamide.8-11
Sulfonamides possessing carbonic anhydrase (CA, EC
4.2.1.1) inhibitory properties such as acetazolamide
(AAZ), methazolamide (MZA), ethoxzolamide (EZA), and
dichlorophenamide (DCP) (Chart 1) have been used for
more than 40 years as systemic eye-pressure-lowering
drugs in the treatment of open-angle glaucoma as well
as other diseases associated with acid/base secretory
disequilibria.1-3 Their ocular effects are due to inhibition
of at least two CA isozymes present within ciliary
processes of the eye, i.e., CA II and CA IV, followed by
a diminished secretion of bicarbonate and a 25-30%
reduction of aqueous humor secretion.1-3 The main
drawback of such agents is constituted by side effects
such as augmented diuresis, fatigue, paresthesias, an-
orexia, etc. due to CA inhibition in other tissues/organs
than the target one (CA, in the form of 14 isozymes, is
ubiquitous in vertebrates).4
The above-mentioned side effects are absent in the
case in which the inhibitor has topical activity, and is
* To whom correspondence should be addressed. Phone: +39-055-
2757551. Fax: +39-055-2757555. E-mail: claudiu.supuran@unifi.it.
† Laboratorio di Chimica Inorganica e Bioinorganica, Universita`
degli Studi di Firenze.
‡ Ospedale San Giovanni di Dio, U.O. Oculistica.
§ Institute of Ophthalmology, Universita` degli Studi and U.O.
Oculistica Az. USL 3.
The clinical importance of the two existing topical
antiglaucoma sulfonamides, and also their imperfections
due to many undesired topical side effects, fostered
| CSGI.
10.1021/jm0108202 CCC: $22.00 © 2002 American Chemical Society
Published on Web 02/22/2002

Preparation of Sulfonamides
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7 1467
Ch a r t 1
Ch a r t 2
much research in the synthesis and evaluation of novel
generation of such derivatives.12-18 The major side
effects of dorzolamide mentioned above might be avoided
for compounds that should not be administered as
hydrochloride salts, but this generally leads to a dras-
tical diminution of water solubility of such a sulfona-
mide.1,2,12 Here we propose a general approach for
obtaining water-soluble, high-affinity sulfonamide CA
inhibitors, which do not owe their water solubility to
formation of hydrochloride salts. Thus, in a preliminary
communication19 we recently showed that by attaching
diethylenetriaminopentaacetic acid moieties to the mol-
ecules of aromatic/heterocyclic sulfonamides, strong,
water-soluble (at pH 7-7.5) CA inhibitors may be
obtained, which showed promising properties as topi-
cally acting antiglaucoma agents in normotensive and
glaucomatous rabbits. Here, we extend the above-
mentioned approach, showing that by attachment of
different polyamino-polycarboxylic acid moieties to the
amino/imino/hydrazino/hydroxy moieties of aromatic/
heterocyclic sulfonamides (by their reaction with acids/
anhydrides such as IDA (iminodiacetic acid), NTA
(nitrilotriacetic acid), EDDA (ethylenediaminodiacetic
acid), EDTA (ethylenediaminotetraacetic acid) and EDTA
dianhydride, DTPA (diethylenetriaminopentaacetic acid)
and DTPA dianhydride, EGTA (ethylenebis(oxyethyl-
enenitrilo)tetraacetic acid) and EGTA dianhydride, and
EDDHA (ethylenediaminodihydroxyphenyldiacetic acid)),
both mono- as well as bis-sulfonamides with excellent
water solubility (at neutral pH values) may easily be
prepared. Furthermore, metal complexes of such deriva-
tives were also obtained because it was recently shown
that some Zn(II) or Cu(II) complexes of sulfonamides
devoid of topical activity as intraocular pressure-lower-
ing (IOP) drugs show themselves to be very strong in
such properties in experimental animals.20
Resu lts
Ch em istr y. The chemical structures of the carboxylic
acids 1-7 as well as those of sulfonamides A-T used
in the synthesis of the new compounds reported here
are shown in Charts 2 and 3, respectively. Because a
large number of such derivatives (monoamides/esters,
as well as diamides/esters) have been prepared, in the
following they will be abbreviated by using both a figure
designating the carboxylic acid from which they were
derived and a letter designating the sulfonamides at
which the polyamino-polycarboxylate moiety has been
attached. For example, 1A is the monoamide of imino-
diacetic acid 1 with orthanilamide A; 6M is the monoa-
mide of EGTA 6 with 5-amino-1,3,4-thiadiazole-2-
sulfonamide M; 6M2 is the bisamide of EGTA with the
sulfonamide M; 4S is the monoester of EDTA 4 with
4-(2-hydroxymethyl)benzenesulfonamide S; 4S2 is the
diester of EDTA 4 with 4-(2-hydroxymethyl)benzene-
sulfonamide S (see these in Chart 4). The whole series
of the monosulfonamides containing the seven carboxy-
lic acids and the 20 sulfonamides have been prepared
(140 derivatives). Bis-amides/esters derived from the
above-mentioned 20 sulfonamides and the following
acids EDTA, DTPA, and EGTA (60 compounds) have
also been obtained.
Two synthetic approaches have been used for the
preparation of the new sulfonamides reported here: (i)
reaction of polyamino-polycarboxylic acids 1-7 with
sulfonamides A-T in the presence of carbodiimides, as
reported previously by Whitesides17 or by this group;15,18
(ii) reaction of dianhydrides (such as EDTA dianhydride,

1468 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7
Scozzafava et al.
Ch a r t 3
DTPA dianhydride, and EGTA dianhydride) with sul-
fonamides A-T, in molar ratios of 1:2, when bis-
sulfonamides were obtained.19 The metal complexes
were then prepared by using sodium salts of the above-
mentioned sulfonamides as ligands and transition metal
salts (such as zinc(II) chloride, copper(II) sulfate, alu-
minum(III) sulfate, etc.), as reported previously.20-22
Ca r bon ic An h yd r a se In h ibitor y Activity. The
new sulfonamides reported here were assayed for
inhibition of three CA isozymes, two of them known to
play a critical role in aqueous humor formation (CA II
and CA IV), whereas the other one, CA I, is known to
be important for the possible systemic side effects of
such drugs (Table 1).1
Ch a r t 4
Tr a n scor n ea l P en etr a tion of Dr u gs. Some phys-
icochemical properties of several of the new sulfona-
mides reported here that are relevant for their phar-
macological activity, such as buffer solubility and
chloroform-buffer partition coefficient, are shown in
Table 2. The in vitro transcorneal accession rates (kin)
of classical sulfonamides and topically acting deriva-
tives, such as dorzolamide and some of the new com-
pounds reported in the present study, are also shown
in Table 2.
IOP Mea su r em en ts. In vivo IOP lowering data with
some of the most active CA inhibitors reported here, in
normotensive and glaucomatous rabbits after topical

Preparation of Sulfonamides
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7 1469
Ta ble 1. Inhibition Data for Some Derivatives Reported in the Present Paper (Data in Parentheses Represent Inhibition by the
Parent Sulfonamide A-T)
KI (nm)
KI (nm)
inhibitor
hCA Ia
hCA IIa
bCA IVb
220
240
13
380
43
45
1240 (1310)
1500 (2200)
450 (3000)
980 (3200)
279 (2800)
257 (2500)
145 (180)
165 (320)
57 (66)
114 (125)
107 (175)
145 (160)
265 (540)
120 (355)
48 (125)
14 (19)
inhibitor
hCA Ia
hCA IIa
bCA IVb
acetazolamide
methazolamide
ethoxzolamide
dichlorophenamide
dorzolamide
brinzolamide
1A
1B
1C
1D
1E
900
780
25
12
14
8
38
9
3
4C
4D
4E
4F
4G
4H
4I
4J
4K
4L
4M
4N
4O
4P
4Q
4R
4S
6300 (28000)
16000 (78500)
900 (25000)
650 (21000)
360 (8300)
700 (9800)
130 (6500)
123 (6000)
130 (6100)
210 (8400)
225 (8600)
215 (9300)
200 (455)
48 (70)
124 (300)
310 (320)
52 (170)
47 (160)
31 (60)
52 (110)
25 (40)
36 (70)
19 (28)
73 (75)
12 (60)
9 (19)
330 (3000)
750 (3200)
83 (2800)
75 (2500)
40 (180)
69 (320)
50 (66)
78 (125)
96 (175)
105 (160)
37 (540)
26 (355)
23 (125)
11 (19)
1200
>50000
27000 (45400)
23000 (25000)
21300 (28000)
32700 (78500)
3600 (25000)
3450 (21000)
1300 (8300)
975 (9800)
610 (6500)
515 (6000)
405 (6100)
400 (8400)
415 (8600)
710 (9300)
280 (455)
280 (295)
233 (240)
214 (300)
313 (320)
125 (170)
90 (160)
54 (60)
80 (110)
38 (40)
55 (70)
24 (28)
69 (75)
50 (60)
13 (19)
1.5 (3)
6 (9)
1F
1G
1H
1I
1J
1K
1L
1M
1N
1O
1P
1Q
1R
1S
2 (3)
3 (9)
2 (8)
43 (55)
39 (50)
10 (17)
8 (15)
1.5 (7)
104 (125)
89 (110)
230 (295)
190 (240)
87 (300)
300 (320)
36 (170)
33 (160)
20 (60)
31 (110)
15 (40)
23 (70)
13 (28)
24 (75)
6 (60)
7 (19)
1 (3)
2 (9)
1.5 (8)
1 (7)
78 (125)
71 (110)
20
6
8
850 (24000)
640 (18000)
8100 (45400)
7650 (25000)
5000 (28000)
12500 (78500)
510 (25000)
400 (21000)
240 (8300)
225 (9800)
105 (6500)
89 (6000)
65 (6100)
72 (8400)
80 (8600)
105 (9300)
125 (455)
33 (70)
335 (560)
310 (450)
650 (1310)
620 (2200)
230 (3000)
510 (3200)
62 (2800)
54 (2500)
35 (180)
50 (320)
34 (66)
45 (125)
51 (175)
69 (160)
12 (540)
17 (355)
10 (125)
9 (19)
4T
4A2
4B2
4C2
4D2
4E2
4F 2
4G2
4H2
4I2
4J 2
4K2
4L2
4M2
4N2
4O2
4P 2
4Q2
4R2
4S2
4T2
Zn-4C2
Zn-4E2
Zn-4F 2
Zn-4M2
Zn-4N2
Cu-4M2
Al-4M2
5A
5B
5C
5D
5E
5F
5G
5H
5I
5J
5K
5L
5M
5N
5O
5P
5Q
5R
65 (70)
50 (55)
46 (50)
5 (8)
6 (7)
14 (17)
12 (15)
1260 (24000)
1200 (18000)
25800 (45400)
23000 (25000)
20600 (28000)
30500 (78500)
3500 (25000)
3300 (21000)
1000 (8300)
960 (9800)
620 (6500)
510 (6000)
345 (6100)
300 (8400)
410 (8600)
520 (9300)
268 (455)
110 (125)
97 (110)
280 (295)
234 (240)
210 (300)
318 (320)
101 (170)
88 (160)
53 (60)
82 (110)
36 (40)
57 (70)
26 (28)
66 (75)
52 (60)
14 (19)
2 (3)
350 (560)
340 (450)
1200 (1310)
1520 (2200)
450 (3000)
930 (3200)
256 (2800)
240 (2500)
136 (180)
150 (320)
53 (66)
110 (125)
100 (175)
148 (160)
240 (540)
106 (355)
45 (125)
13 (19)
1T
2A
2B
2C
2D
2E
2F
2G
2H
2I
2J
2K
2L
2M
2N
2O
2P
2Q
2R
2S
37 (55)
25 (50)
460 (24000)
450 (18000)
450
8 (17)
6 (15)
200 (560)
165 (450)
210
45
39
40
40
39
51
10
13
62 (70)
48 (55)
44 (50)
7 (9)
6 (8)
5 (7)
0.8
0.7
0.6
0.9
5
8
6
8
13 (17)
10 (15)
1200 (24000)
1100 (18000)
20500 (45400)
13000 (25000)
12000 (28000)
21800 (78500)
1760 (25000)
1540 (21000)
780 (8300)
900 (9800)
380 (6500)
430 (6000)
290 (6100)
250 (8400)
250 (8600)
275 (9300)
240 (455)
56 (70)
45 (55)
43 (50)
1020 (24000)
1000 (18000)
9200 (45400)
10000 (25000)
103 (125)
94 (110)
286 (295)
235 (240)
200 (300)
315 (320)
81 (170)
80 (160)
49 (60)
78 (110)
35 (40)
50 (70)
24 (28)
68 (75)
53 (60)
12 (19)
2 (3)
7 (9)
5 (8)
4 (7)
106 (125)
95 (110)
280 (295)
230 (240)
350 (560)
320 (450)
1020 (1310)
1005 (2200)
365 (3000)
950 (3200)
245 (2800)
250 (2500)
103 (180)
155 (320)
54 (66)
115 (125)
110 (175)
135 (160)
130 (540)
76 (355)
39 (125)
12 (19)
2T
8600 (45400)
7200 (25000)
4100 (28000)
12000 (78500)
550 (25000)
290 (21000)
180 (8300)
200 (9800)
96 (6500)
79 (6000)
58 (6100)
87 (8400)
55 (8600)
62 (9300)
110 (455)
36 (70)
210 (295)
180 (240)
75 (300)
250 (320)
23 (170)
15 (160)
13 (60)
20 (110)
12 (40)
16 (70)
10 (28)
12 (75)
0.9 (60)
2 (19)
550 (1310)
500 (2200)
180 (3000)
630 (3200)
40 (2800)
36 (2500)
29 (180)
32 (320)
27 (66)
29 (125)
36 (175)
52 (160)
28 (540)
19 (355)
16 (125)
9 (19)
3A
3B
3C
3D
3E
3F
3G
3H
3I
3J
3K
3L
3M
3N
3O
3P
3Q
3R
3S
3T
4A
4B
1 (3)
0.8 (9)
0.8 (8)
0.6 (7)
80 (125)
61 (110)
175 (295)
155 (240)
64 (300)
32 (55)
30 (50)
6 (17)
5 (15)
11 (17)
9 (15)
350 (560)
330 (450)
800 (1310)
750 (2200)
5S
5T
5A2
5B2
5C2
510 (24000)
360 (18000)
5400 (45400)
6400 (25000)
3750 (28000)
265 (560)
190 (450)
330 (1310)
425 (2200)
156 (3000)

1470 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7
Scozzafava et al.
Ta ble 1. (Continued)
KI (nm)
KI (nm)
inhibitor
hCA Ia
hCA IIa
bCA IVb
inhibitor
hCA Ia
hCA IIa
bCA IVb
5D2
5E2
5F 2
5G2
5H2
5I2
9800 (78500)
380 (25000)
170 (21000)
135 (8300)
155 (9800)
79 (6500)
70 (6000)
43 (6100)
63 (8400)
50 (8600)
54 (9300)
102 (455)
25 (70)
21 (55)
16 (50)
325 (24000)
290 (18000)
350
39
36
40
43
215 (320)
19 (170)
8 (160)
7 (60)
15 (110)
10 (40)
9 (70)
9 (28)
11 (75)
1 (60)
1.5 (19)
0.6 (3)
0.5 (9)
0.6 (8)
0.5 (7)
43 (125)
34 (110)
16
430 (3200)
37 (2800)
25 (2500)
18 (180)
27 (320)
21 (66)
24 (125)
25 (175)
41 (160)
7 (540)
9 (355)
8 (125)
6 (19)
5 (17)
4 (15)
170 (560)
115 (450)
100
21
16
4
5
6C2
6D2
6E2
6F 2
6G2
6H2
6I2
3800 (28000)
9500 (78500)
350 (25000)
160 (21000)
155 (8300)
150 (9800)
85 (6500)
76 (6000)
49 (6100)
50 (8400)
55 (8600)
36 (9300)
76 (455)
23 (70)
21 (55)
15 (50)
335 (24000)
300 (18000)
300
35
30
60 (300)
210 (320)
15 (170)
7 (160)
7 (60)
9 (110)
12 (40)
8 (70)
150 (3000)
400 (3200)
39 (2800)
20 (2500)
16 (180)
22 (320)
25 (66)
20 (125)
21 (175)
36 (160)
4 (540)
5 (355)
6 (125)
4 (19)
5 (17)
5 (15)
150 (560)
96 (450)
76
16
12
8
7
5J 2
5K2
5L2
5M2
5N2
5O2
5P 2
5Q2
5R2
5S2
5T2
Zn-5C2
Zn-5E2
Zn-5F 2
Zn-5M2
Zn-5N2
Cu-5M2
Al-5M2
6A
6B
6C
6D
6E
6F
6G
6H
6I
6J
6K
6L
6M
6N
6O
6P
6Q
6R
6S
6T
6A2
6B2
6J 2
6K2
6L2
6M2
6N2
6O2
6P 2
6Q2
6R2
6S2
6T2
Zn-6C2
Zn-6E2
Zn-6F 2
Zn-6M2
Zn-6N2
Cu-6M2
Al-6M2
7A
7B
7C
7D
7E
7F
7G
7H
7I
10 (28)
13 (75)
2 (60)
1.1 (19)
0.5 (3)
0.5 (9)
0.6 (8)
0.6 (7)
38 (125)
33 (110)
11
9
6
0.7
0.8
9
7
0.5
0.4
0.3
0.8
27
32
21
19
31
44
3
6
0.2
0.7
2
3
8900 (45400)
7450 (25000)
4000 (28000)
12500 (78500)
610 (25000)
290 (21000)
210 (8300)
230 (9800)
78 (6500)
76 (6000)
50 (6100)
93 (8400)
54 (8600)
66 (9300)
120 (455)
39 (70)
200 (295)
195 (240)
79 (300)
265 (320)
30 (170)
18 (160)
17 (60)
16 (110)
10 (40)
13 (70)
12 (28)
16 (75)
2 (60)
2 (19)
1.5 (3)
1 (9)
0.9 (8)
0.8 (7)
72 (125)
56 (110)
160 (295)
145 (240)
545 (1310)
500 (2200)
170 (3000)
620 (3200)
48 (2800)
32 (2500)
31 (180)
30 (320)
32 (66)
24 (125)
35 (175)
65 (160)
21 (540)
16 (355)
13 (125)
7 (19)
12500 (45400)
10000 (25000)
11500 (28000)
21000 (78500)
1200 (25000)
1050 (21000)
450 (8300)
870 (9800)
175 (6500)
220 (6000)
250 (6100)
240 (8400)
245 (8600)
250 (9300)
210 (455)
52 (70)
280 (295)
235 (240)
175 (300)
310 (320)
69 (170)
61 (160)
43 (60)
67 (110)
34 (40)
47 (70)
23 (28)
65 (75)
54 (60)
13 (19)
3 (3)
800 (1310)
860 (2200)
320 (3000)
840 (3200)
135 (2800)
215 (2500)
79 (180)
108 (320)
56 (66)
90 (125)
105 (175)
123 (160)
105 (540)
57 (355)
35 (125)
13 (19)
12 (17)
7 (15)
7J
7K
7L
7M
7N
7O
7P
7Q
33 (55)
30 (50)
500 (24000)
430 (18000)
5600 (45400)
6150 (25000)
5 (17)
6 (15)
250 (560)
155 (450)
320 (1310)
400 (2200)
6 (9)
4 (8)
3 (7)
110 (125)
94 (110)
47 (55)
41 (50)
945 (24000)
760 (18000)
7R
7S
7T
320 (560)
300 (450)
a
b
Human (cloned) isozymes. From bovine lung microsomes, by the esterase method.4,5
administration of the drug, are shown in Tables 3 and
4, respectively.
to better understand the structure-activity relationship
in this class of CA inhibitors. Thus, several well-known
polyamino-polycarboxylic acids possessing metal com-
plexing moieties were chosen to be incorporated in the
molecules of the new sulfonamides reported here for at
least three reasons. (i) It was previously observed in this
laboratory23 that CA activators possessing “sticky” tails
such as EDTA and DTPA attached to the aliphatic
amino moiety of histamine (a much investigated CA
activator)24,25 possessed a very high affinity for isozymes
CA I, II, and IV. This has been explained as due to the
fact that the many heteroatoms present in these moi-
eties confer to the obtained compounds “sticky” proper-
ties; i.e., they are able to participate in a host of
interactions with amino acid residues from the rim of
the active site, ensuring thus the formation of very
stable E-A (enzyme-activator) adducts. Thus, it ap-
peared of interest to apply the same approach to the
design of CA inhibitors, and the strategy proved to be
of interest because the compounds incorporating DTPA
moieties reported in our preliminary communication19
The full time dependence of the IOP after dorzolamide
and some of the new compounds reported here in
normotensive albino rabbits is shown in Figure 1.
Distr ibu tion of Dr u gs in Ocu la r F lu id s a n d
Tissu es. Ex vivo distribution data of some active
compound in ocular tissues and fluids after the topical
administration in normotensive rabbits are shown in
Table 5.
Discu ssion
Ch em istr y. The preparation of sulfonamide CA
inhibitors incorporating amino acid moieties has thor-
oughly been studied by this,1,15 Whitesides,17 and Black-
burn’s groups.12 Still, compounds incorporating poly-
amino-polycarboxylic acid moieties in their molecule
(and more specifically DTPA moieties) have only re-
cently been reported in our preliminary communica-
tion.19 Here we extend that work, including a large
series of such derivatives in our study (of types 1-7),

Preparation of Sulfonamides
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7 1471
Ta ble 2. Solubility, Chloroform-Buffer Partition Coefficients,
and in Vitro Corneal Permeability of Some Sulfonamide CA
Inhibitors Reported in the Paper and Dorzolamide as Standard
Ta ble 4: Fall of IOP of Glaucomatous Rabbits (34.5 ( 2.0
mmHg) after Treatment with 1 Drop (50 µL) of Solution/
Suspension of 2% of CA Inhibitor (as Hydrochloride or Sodium
Salt, with the pH Value Shown below) Directly into the Eye at
30, 60, and 90 min after Administration
c
kin × 103 (h-1
)
solubility,a
mM
cornea
no
a
∆IOP (mmHg)
b
compound
pH
log P
intact epithelium
inhibitor
pH t ) 0 min t ) 30 min t ) 60 min t ) 90 min
dorzolamide DZA 5.5
60d
62e
37e
44e
65e
41e
32e
46e
48e
35e
59e
54e
50e
52e
47e
44e
2.0e
3.0
2.5
5.1
4.7
4.8
3.1
3.6
3.0
3.8
3.6
4.3
3.9
3.3
4.6
4.5
5.1
5.2
4.6
10.3
8.4
9.5
5.5
7.0
5.9
6.9
6.5
8.1
6.2
6.5
8.4
8.3
10.7
1O
7.4
7.4
7.4
7.4
7.4
7.4
7.4
7.4
7.4
7.4
7.4
7.4
7.4
7.4
7.4
1.230
1.925
1.166
1.750
1.333
1.764
1.150
0.983
1.456
1.375
1.961
1.747
1.446
1.534
2.354
dorzolamide 5.5
4N
0
0
0
0
0
0
0
3.6 ( 0.2
6.7 ( 0.3
4.2 ( 0.2
2R
3Q
4N
7.4
7.0
7.5
7.5
7.0
7.0
7.3 ( 0.1 11.3 ( 0.4 15.2 ( 0.4
6.0 ( 0.4 12.5( 0.2 11.0 ( 0.2
7.4 ( 0.3 13.0 ( 0.5 14.9 ( 0.5
8.8 ( 0.6 16.0 ( 0.4 17.5 ( 0.3
19.8 ( 0.7 19.2 ( 0.8 18.9 ( 0.5
7.6 ( 0.4 12.9 ( 0.5 15.1 ( 0.5
5C2
5M2
5N
Zn-5M2
6M
4N2
4K2
4O2
5C
b
a
∆IOP ) IOPcontrol
- IOPtreated eye; mean ( SEM (n ) 3).
eye
5C2
5M
5M2
6I
b
Suspension.
6M
6G2
7O
a
b
Solubility in pH 7.40 buffer, at 25 °C. Chloroform-buffer
partition coefficient.13 c Determined as described in ref 13. As
d
hydrochloride, at pH 5.8, from ref 6. e As sodium salts.
Ta ble 3. IOP Lowering in Normotensive Rabbits (23.0 ( 1.5
mmHg) after Treatment with 1 Drop (50 µL) of 2% Solution/
Suspension of CA Inhibitor (pH of the Solution Shown), at 30,
60, and 90 min after Administration Directly into the Eye
a
∆IOP (mmHg)
inhibitor
pH t ) 0 t ) 30 min t ) 60 min t ) 90 min
dorzolamide
5.5
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1.9 ( 0.2
2.9 ( 0.1
2.2 ( 0.2
2.6 ( 0.2
1.8 ( 0.2
6.0 (0.1
4.9 (0.1
3.1 ( 0.2
4.4 ( 0.2
3.1 ( 0.5
4.0 ( 0.3
3.2 ( 0.3
4.5 ( 0.3
4.9 ( 0.3
2.5 ( 0.3
9.3 ( 0.2
9.5 ( 0.2
4.5 ( 0.2
7.5 ( 0.2
6.8 ( 0.2
2.1 ( 0.2
6.3 ( 0.4
5.7 ( 0.3
6.2 ( 0.3
3.4 ( 0.3
12.2 ( 0.4
13.1 ( 0.5
7.2 ( 0.3
9.5 ( 0.3
4.5 ( 0.3
8.1 ( 0.6
9.5 ( 0.3
13.0 ( 0.4
7.2 ( 0.2
8.6 ( 0.2
12.1 ( 0.4
13.6 (0.5
13.5 (0.3
8.0 ( 0.4
brinzolamideb 5.5
1O
7.0
7.0
7.0
7.4
6.5
7.0
7.0
7.4
7.5
7.0
7.5
7.0
6.2
7.0
6.0
6.0
7.0
2Rc
F igu r e 1. Effect of topically administered sulfonamide inhibi-
tors (2% water solutions/suspensions) on the IOP of normo-
tensive albino rabbits: (1) dorzolamide (hydrochloride salt, pH
5.5); (2) brinzolamide (suspension, hydrochloride salt, pH 5.5);
(3) compound 5M2 (trisodium salt, pH 7.0); (4) compound Zn-
5M2 (suspension, pH 7.0); (5) compound 4N (trisodium salt,
pH 7.0); (6) compound Zn-4N (suspension, pH 7.0).
4K
4N
Zn-4Nb
5C
5C2
5M
5M2
Zn-5M2
5N
6.5 ( 0.1 13.0 ( 0.2
7.4 ( 0.2 14.5 ( 0.2
b
Ta ble 5. Ocular Tissue Concentrations (µM) after 1 and 2 h,
Following Corneal Application of 1 Drop (50 µL) of 2% Solution
of Compounds 5M2 and 6M in Normotensive Albino Rabbits
6.7 ( 0.1
1.2 ( 0.1
2.9 ( 0.1
4.6 ( 0.2
5.2 ( 0.1 10.3 ( 0.3
4.7 ( 0.2
2.5 ( 0.3
9.2 ( 0.3
3.9 ( 0.15
5.6 ( 0.1
8.9 ( 0.4
6I
Zn-6I
6M
drug concentrationa (µM)
Zn-6Mb
Cu-6Mb
9.6 ( 0.2
4.9 ( 0.3
compound time (h) cornea
aqueous humor ciliary process
7O
5M2
6M
1
2
1
2
150 ( 10
45 ( 5
173 ( 12
66 ( 5
241 ( 13
39 ( 3
268 ( 16
53 ( 3
50 ( 3
19 ( 1
59 ( 4
37 ( 3
a
∆IOP ) IOPcontrol eye - IOPtreated eye; mean ( SEM (n ) 3).
Suspension. c Eye reddening.
b
a
Mean ( SEM (n ) 3).
showed indeed very good CA inhibitory properties. (ii)
Sulfonamides incorporating polyamino-polycarboxylic
acid moieties would possess good water solubility in the
neutral pH range because of the buffering properties of
such moieties and the easy formation of water-soluble
sodium salts. This hypothesis was in fact verified during
the course of the preparation of the DTPA derivatives
mentioned above,19 which showed indeed excellent
water solubility at pH 7. (iii) The presence of polyamino-
polycarboxylic acid moieties in the molecules of such
sulfonamides leads to good metal-complexing properties
of these derivatives, and this may be important for the
design of coordination compounds as CA inhibitors.20,21
Indeed, it was recently reported that some Zn(II) and
Cu(II) complexes of heterocyclic sulfonamides possess
excellent IOP-lowering properties via the topical route,
although the parent sulfonamides from which the
complexes have been prepared are devoid of such
properties.20,21 It must be mentioned that metal com-
plexes of sulfonamides are generally 10-100 times
stronger CA inhibitors than the sulfonamides from
which they are prepared, a fact explained mechanisti-
cally at the molecular level by this group.1,2
Two series of derivatives incorporating polyamino-
polycarboxylic acid moieties are reported here: bis- and
monosulfonamides (Scheme 1). The following strategy
has been employed for preparing these compounds. It
was originally investigated whether the reaction of
anhydrides 8 (such as EDTA dianhydride; EGTA dian-
hydride and DTPA dianhydride) with aromatic/hetero-
cyclic sulfonamides A-T in molar ratios of 1:1 leads to
monosulfonamides. When these reactions were per-

1472 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7
Scozzafava et al.
Sch em e 1
moieties of the ligand are involved in the interaction
with the metal ion in such compounds, although it is
well established that the sulfonamide moiety, as well
as heteroatoms present in the heterocyclic moieties of
some of these derivatives, may indeed participate in
coordinating metal ions.20-22,27
Ca r bon ic An h yd r a se In h ibitor y Activity. The
data of Table 1 show that the new inhibitors prepared
by attaching polyamino-polycarboxylic acid moieties to
aromatic orheterocyclic sulfonamides A-T are more
effective compared to the parent sulfonamides from
which they were prepared toward 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) polyamino-polycarboxylic
acid moiety incorporated in their molecules with hydro-
philic patches at the entrance of the enzyme active site
as observed for inhibitors previously reported by Whi-
formed in a variety of conditions, it was observed that
only bis-sulfonamides (and unreacted anhydride/acid)
could be isolated. Thus, this strategy has been employed
only for the preparation of bis-sulfonamides, working
at molar ratios of anhydride 8 to sulfonamide A-T of
1:2 in DMF at room temperature (Scheme 1). To obtain
the monosulfonamides, a different strategy has been
employed. The conjugation of the sulfonamides A-T
with the polyamino-polycarboxylic acid 1-7 has been
performed in the presence of diisopropylcarbodiimide,
as previously reported for the synthesis of CA inhibitors
incorporating amino acyl or oligopeptidyl moieties.15
This reaction led to the preparation of monosulfona-
mides of types 1-7 (A-T) in high yields, without other
undesired side reactions (Scheme 1). The only complica-
tion consisted of the need to protect the secondary amino
moieties present in IDA 1 and EDDA 3, as well as the
phenolic OH moieties of EDDHA 7. This has been done
by using the tert-butoxycarbonyl (Boc) moiety as the
protecting group (the Boc derivatives were prepared
from Boc-On and compounds 1, 3, and 7),26 whereas the
deprotection (after the condensation step) has been
effected in the usual way, with trifluoroacetic acid (TFA)
in methylene chloride at room temperature.15 All 140
derivatives incorporating the seven distinct polyamino-
polycarboxylic acids 1-7 and the 20 sulfonamides A-T
were prepared according to this strategy (Table 1).
Metal complexes of some of the new sulfonamides
reported here and Zn(II), Cu(II), and Al(III) ions have
also been obtained. The synthesis has been done by
using the di- or trisodium carboxylate salts of the
sulfonamides and Zn(II), Cu(II), or Al(III) inorganic salts
(chlorides or sulfates) in molar ratios of sulfonamide
ligand to metal ion of 1:1, 1:2, or 1:3. The metal ions
included in these studies included Zn(II) and Cu(II)
because it was originally shown that complexes contain-
ing such cations act as efficient IOP-lowering agents.20
Al(III) complexes have also been prepared because we
recently showed that some Al(III) derivatives of hetero-
cyclic sulfonamides exhibit interesting pharmacological
properties as antacids in gastric acid disequilibria in
experimental animals.21 In all cases investigated here,
only complexes containing one metal ion per sulfona-
mide ligand moiety have been obtained, as determined
by elemental analysis and physicochemical investiga-
tions with these new complexes (see Experimental
Protocols for details). Presumably, only the carboxylate
17
tesides’ and our groups,1,2,13,15 and explained by de-
tailed QSAR models.28
The nature of the sulfonamide attached to the polyami-
no-polycarboxylic acid moiety in the new derivatives
reported here greatly influenced the CA inhibitory
power of these compounds. Among the synthesized
compounds, the heterocyclic sulfonamide derivatives
were the most active, followed by the aromatic sulfona-
mide derivatives. The efficiency of the obtained inhibitor
generally varied in the following way based on the
parent sulfonamide from which it was prepared: the
derivatives of p-hydrazino-benzenesulfonamide < the
orthanilamides < the metanilamides < the sulfanil-
amides < the homosulfanilamides < the p-aminoethyl-
benzenesulfonamides = the halogeno-substituted sul-
fanilamides = the 1,3-benzene-disulfonamides < the
1,3,4-thiadiazole-2-sulfonamides = 4-methyl-δ2-1,3,4-
thiadiazoline-2-sulfonamide = the benzothiazole-2-sul-
fonamides. The monoamides or esters were generally
less active than the corresponding bis-amides or esters.
The metal complexes of some bis-sulfonamides that
were also prepared were more active than the corre-
sponding ligands, as already reported in the litera-
ture.20,27 The Cu(II) complexes were more effective than
the Zn(II) derivatives, which in turn were more inhibi-
tory than the corresponding Al(III) complexes. The
nature of the acylating moiety also influenced the
inhibitory properties of these new sulfonamides, with
the following range of inhibitory effects for the corre-
sponding sulfonamides incorporating polyamino-poly-
carboxylic acid moieties: IDA derivatives < NTA de-
rivatives < EDDA derivatives < EDDHA derivatives <
EDTA derivatives < EGTA derivatives = DTPA deriva-
tives. All three CA isozymes investigated here were
susceptible to inhibition with this type of sulfonamide,
with hCA II and bCA IV the most sensitive, whereas
hCA I was generally less susceptible to inhibition
compared to the first two isozymes. Mention should be
made that the two susceptible isozymes (CA II and CA
IV) are just those involved in aqueous humor secretion,1
whereas CA I inhibition may explain the presence or
the lack of side effects due to inhibitor washed out from
the eye.19 Thus, our compounds, in contrast to dorzola-
mide, also inhibit the slow isozyme CA I, which may be
a positive property because once in the systemic circula-
tion (after being washed out from the eye after a

Preparation of Sulfonamides
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7 1473
prolonged use of eye drops containing such CA inhibi-
tors), they should bind to the predominant isozyme
present in blood, i.e., hCA I, and will thus inhibit to a
slighter degree the rapid isozyme hCA II, which is
involved in bicarbonate transport, electrolyte secretion,
and other vital physiological processes.1,2 Indeed, the
blood contains up to 150 µM of hCA I, whereas the hCA
II concentration is much lower, of about 20 µM.1,4 The
fact that dorzolamide shows so many systemic side
effects8-11 after topical administration may be due in
part to its low affinity for just hCA I.
normotensive rabbits after the topical administration
of two of the most potent topical inhibitors in the
prepared series. It can be observed that at 1 and 2 h
after topical administration of drug high levels of
inhibitors were found in the cornea, aqueous humor, and
ciliary processes. On the basis of the inhibition constant
of these compounds, the fractional inhibition estimated
in these tissues or fluids is 99.5-99.9%, indicating the
fact that the powerful IOP decrease observed is due to
CA inhibition.
Con clu sion s
Tr a n scor n ea l P en etr a tion of Dr u gs. The data of
Table 2 show that many of the new derivatives reported
here possess excellent water solubility in the neutral
pH range (pH around 7.4) as mono-, di-, tri-, or tetra-
sodium salts. This is correlated with a balanced hydro-
philicity and lipophilicity and, as a consequence, optimal
accession rates across the cornea, which is typical for
the effective topically acting sulfonamides.1,3,5 The
prepared metal complexes of such derivatives were, on
the other hand, less water-soluble (data not shown), but
because brinzolamide BRZ is administered as a suspen-
sion (because of its poor water solubility), we also used
water suspensions of some of these compounds for in
vivo experiments.
We report here a novel class of water-soluble, topically
acting antiglaucoma sulfonamides obtained by the “tail”
approach,1 which incorporate polyamino-polycarboxylic
moieties in their molecules, such as those of iminodi-
acetic acid, NTA, EDDA, e EDTA, DTPA, EGTA, and
EDDHA. Metal complexes (containing Zn(II), Cu(II),
and Al(III) ions) of such sulfonamides have also been
synthesized. Many of the newly reported derivatives
showed very good water solubility at nearly neutral pH
values, whereas their lipid solubility, hydrophobicity
(log P), and accession rates across the cornea were those
appropriate for efficient topical IOP-lowering agents.
Some of these new CA inhibitors, as well as their metal
complexes, possessed affinities in the low nanomolar
range for isozymes hCA II and bCA IV, acting as
effective enzyme inhibitors in vitro. Several of the most
active inhibitors strongly lowered IOP pressure in
normotensive and glaucomatous rabbits, showing a
prolonged duration of action compared to dorzolamide
or brinzolamide. The new compounds reported here
might lead to the development of more efficient anti-
glaucoma drugs from the class of the sulfonamide CA
inhibitors, with fewer side effects compared to the
presently available drugs.
IOP Mea su r em en ts. In vivo, in normotensive rab-
bits, some of the new mono- and bis-sulfonamides
reported here, such as 4N and its Zn(II) complex, 5C,
5C2, 5M, 5M2 and its Zn(II) complex, 5N, 6M and its
Zn(II) and Cu(II) complexes, etc., showed very effective
IOP lowering after topical administration, with pressure
reductions of 3.1-7.4 mmHg at half an hour (compared
to 1.9 mmHg for dorzolamide and 2.9 mmHg for brin-
zolamide), 4.5-14.5 mmHg at 1 h (4.0 for the first
standard drug and 3.2 mmHg for the second one), and
7.2-13.5 mmHg at 90 min after administration (com-
pared to 2.1 for dorzolamide and 6.3 for brinzolamide)
(Table 3 and Figure 1). Other derivatives, such as 1O,
2R, 4K, 6I, and 7O among others, showed more modest
IOP lowering, which was comparable to or better with
those produced by dorzolamide or brinzolamide. An
important feature of the new class of CA inhibitors
reported here is that IOP remained at much lower
values for longer periods (3-6 h) after their topical
administration, as compared to the standard drugs
(Figure 1). This was particularly true for some Zn(II)
complexes of these new sulfonamide CA inhibitors. IOP
generally returned to the baseline values after 5-6 h
after administration of these new topically acting sul-
fonamides/metal complexes reported here. The above
findings also apply to the glaucomatous rabbits experi-
ments (Table 4), in which the IOP reductions were much
more important compared to those seen in normotensive
rabbits. Thus, in glaucomatous animals, IOP reductions
of 6.0-19.8 mmHg were generally observed after 30 min
postadministration, whereas at 1 h, these amounted to
11.3-19.2 mmHg, and in the case of the Zn(II) complex
Zn-5M2, they remained at these low values for rather
prolonged periods (Table 4). Thus, all these derivatives
are longer lasting and much more effective IOP-lowering
agents compared to the clinically available drugs dor-
zolamide and brinzolamide.
Exp er im en ta l P r otocols
Ch em istr y. Melting points were recorded with a heating
plate microscope and are not corrected. IR spectra were
recorded in KBr pellets with a Carl Zeiss IR-80 instrument.
1H NMR spectra were recorded in DMSO-d6 or TFA as
solvents, with a Bruker CPX200 or Varian 300 instrument.
Chemical shifts are reported as δ values relative to Me4Si as
an internal standard. Elemental analyses were done by
combustion for C, H, and N with an automated Carlo Erba
analyzer, and gravimetrically for the metal ions, and were
(0.4% of the theoretical values. High-resolution mass spec-
troscopy (HRMS) was recorded with an AEI-MS 902 spectrom-
eter, using fast-atom bombardment (FAB) or electrospray
techniques. All reactions were monitored by thin-layer chro-
matography (TLC) using 0.25 mm precoated silica gel plates
(E. Merck). Analytical and preparative HPLC was performed
on a reversed-phase C18 Bondapack column, with a Beckman
EM-1760 instrument. Sulfonamides A-T used in the synthe-
ses were either commercially available compounds (from
Sigma-Aldrich, Milan, Italy, or Acros, Milan, Italy) or were
prepared as described previously.13,15,16 Dorzolamide was from
Merck & Co. (Trusopt eye drops), whereas brinzolamide was
from Alcon Laboratories (Azopt eye drops). Polyamino-poly-
carboxylic acids and anhydrides, carbodiimides, metal salts,
and other inorganic reagents were from Sigma-Aldrich, Fluka,
or E. Merck and were used without additional purification.
Acetone, acetonitrile, DMF, and other solvents (E. Merck) used
in the synthesis/chromatography were doubly distilled and
kept on molecular sieves in order to maintain them under
anhydrous conditions.
Distr ibu tion of Dr u gs in Ocu la r F lu id s a n d
Tissu es. Table 5 shows ex vivo data obtained in
Gen er a l P r oced u r e for th e P r ep a r a tion of Bis-su l-
fon a m id es. An amount of 15 mmol of EDTA, EGTA, or DTPA

1474 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7
Scozzafava et al.
dianhydride was added to a solution of 30 mmol of sulfonamide
A-T dissolved in 100 mL of anhydrous DMF. The mixture
was magnetically stirred at room temperature for 4 h, then
the reaction mixture was poured in 300 mL of methylene
chloride and the obtained solid was filtered and thoroughly
washed with methylene chloride and then with acetone. HPLC
purification was necessary in some cases and was done by
elution with potassium phosphate buffer-MeCN 2:1, v/v (10
mL/min), on a reversed-phase C18 Bondapack column.
zole); 170.9 (C-5 of thiadiazole); 176.41 (CONH); 180.82
(COOH). Anal. (C7H11N5O5S2) C, H, N.
4M. Mp 155-6 °C (dec). IR (KBr), cm-1: 1164 (SO2sym),
1360 (SO2as), 1561 (amide II), 1600 (amide I), 1765 (COOH),
3330 (NH, NH2). 1H NMR (D2O-KOD), 300 MHz, δ, ppm: 3.35
(6H, s, 3CH2COOH); 3.54 (4H, t, CH2CH2); 3.56 (2H, s, CH2-
CONH); the SO2NH2, CONH, and COOH protons are not seen
in this solvent mixture, being in fast exchange. 13C NMR
(D2O-KOD), δ, ppm: 53.24 (CH2CH2); 53.70 (CH2CH2); 60.18
(CH2CONH); 61.34 (CH2COOH); 159.5 (C-2 of thiadiazole);
170.3 (C-5 of thiadiazole); 176.41 (CONH); 180.82 (COOH).
Anal. (C12H18N6O9S2) C, H, N.
Gen er a l P r oced u r e for th e P r ep a r a tion of Com p ou n d s
1-7 (A-T). An amount of 1 mM sulfonamide A-T was
dissolved/suspended in 25 mL of anhydrous acetone and then
treated with the stoichiometric amount (1 mM) of (Boc-
protected) polyamino-polycarboxylic acid 1-7. An amount of
190 mg (1 mM) of EDCI‚HCl (or the equivalent amount of
diisopropylcarbodiimide) was then added, and the reaction
mixture was magnetically stirred at room temperature for 15
min. Then a total of 30 µL (2 mM) of triethylamine was added,
and stirring was continued for 8-16 h at 4 °C (TLC control).
The solvent was evaporated in vacuo, and the residue was
taken up in ethyl acetate (5 mL), poured into a 5% solution of
sodium bicarbonate (5 mL), brought to pH 7 with 1 N HCl,
and extracted with ethyl acetate. The combined organic layers
were dried over sodium sulfate and filtered, and the solvent
was removed in vacuo. The obtained oils were directly used
in the deprotection step or the intermediates were purified by
preparative HPLC as described below. The removal of the
protecting groups has been performed as described in the
literature.15-17 The crude Boc-protected intermediate was
dissolved in 20 mL of CH2Cl2, treated with 4 mL of trifluoro-
acetic acid (TFA), and stirred for 10 min at 0 °C. The solvent
was removed in vacuo, and the residue was concentrated from
water twice to remove excess TFA, giving thus the unprotected
derivative as a colorless syrup. The pure compounds 1-7 (A-
T) were obtained in pure form by means of preparative HPLC
(C18 reversed-phase µ-Bondapack or Dynamax-60A (25 mm ×
250 mm) columns, 90% acetonitrile/8% methanol/2% phosphate
buffer, pH 7.4, 10 mL/min).
4M2. Mp 176-8 °C (dec). IR (KBr), cm-1: 1163 (SO2sym),
1356 (SO2as), 1563 (amide II), 1600 (amide I), 1764 (COOH),
3330 (NH, NH2). 1H NMR (D2O-KOD), 300 MHz, δ, ppm: 3.33
(4H, s, 2CH2COOH); 3.54 (4H, t, CH2CH2); 3.60 (4H, s, 2CH2-
CONH); the SO2NH2, CONH and COOH protons are not seen
in this solvent mixture, being in fast exchange. 13C NMR
(D2O-KOD), δ, ppm: 53.19 (CH2CH2); 53.76 (CH2CH2); 60.23
(CH2CONH); 61.13 (CH2COOH); 159.3 (C-2 of thiadiazole);
170.5 (C-5 of thiadiazole); 176.28 (CONH); 180.70 (COOH).
Anal. (C14H20N10O10S4) C, H, N.
5F 2. Mp 121-2 °C (dec). IR (KBr), cm-1: 1173 (SO2sym),
1340 (SO2as), 1560 (amide II), 1600 (amide I), 1760 (COOH),
3335 (NH, NH2). 1H NMR (D2O-KOD), 300 MHz, δ, ppm: 2.90
(t, 2H, CH2 of aminoethyl-benzenesulfonamide, 7.2), 3.47 (q,
2H, CH2 of aminoethylbenzenesulfonamide 6.5), 3.20 (4H, t,
ethylenic CH2 near lateral nitrogens); 3.35 (4H, s, CH2 of the
lateral acetates); 3.48 (4H, t, ethylenic CH2 near central
nitrogen); 3.56 (4H, s, CH2 of the acetamido groups); 3.91 (2H,
s, CH2 of the central acetate); 7.42 (d, 2H, AA′BB′, 8.2), 7.75
(d, 2H, AA′BB′, 8.2). 13C NMR (D2O-KOD), δ, ppm: 36.15 (C-
10); 42.07 (C-9); 53.12 (C-4); 53.68 (C-3); 60.03 (C-2); 60.15 (C-
7); 61.05 (C-5); 128.12 (C-13); 132.47 (C-12); 146.79 (C-14);
147.20 (C-11), 177.04 (C-8); 180.53 (C-1); 181.20 (C-6). Anal.
(C30H43N7O12S2) C, H, N.
Th e Zn -5F 2 Com p lex. Mp >300 °C. IR(KBr), cm-1: 1170
(SO2sym), 1336 (SO2as), 1560 (amide II), 1610 (amide I), 1745
(COO-), 3335 (NH2). Anal. (C30H41N7O12S2Zn) Zn, C, H, N.
Gen er a l P r oced u r e for th e P r ep a r a tion of th e Coor -
d in a tion Com p ou n d s. The metal complexes of some of the
above-mentioned sulfonamides were prepared as exemplified
below for 5F 2 as ligand. A suspension of 14-[4-(aminosulfonyl)-
phenyl]-3-[2-[[2-[4-(aminosulfonyl)phenyl]ethyl]amino]-2-oxo-
ethyl]-6,9-bis(carboxymethyl)-11-oxo-3,6,9,12-tetraazatetrade-
canoic acid (5F 2, 1.51 g, 2 mmol) in 50 mL of water was treated
with a stoichiometric amount of 1 N NaOH solution in order
to obtain the disodium carboxylate salt. The obtained solution
was treated with a solution of ZnCl2 (1 mmol) in 5 mL of water,
maintaining the pH at 6.5. The reaction was monitored by
HPLC on a stationary phase of Lichrospher 100 RP-18.5 µm,
with a 250 mm × 4 mm column packed by E. Merck, at 40 °C.
Isocratic elution with a premixed mobile phase (1 g of octy-
lamine was added to 100 mL of acetonitrile mixed with 900
mL of water) has been performed. The eluent was buffered
with phosphoric acid, maintaining the pH at 6, and the flow
rate was 1.5 mL/min. After 4 h the solution was loaded onto
an Amberlite XAD 1600 resin column (250 mL) and eluted with
MeCN/water (1:10, v/v). The fractions containing the complex
were evaporated to give a white solid of the complex with an
overall yield of 95%. Similarly prepared were the Cu(II) and
Al(III) complexes of this and other sulfonamides, by using Cu-
(II) sulfate or Al(III) sulfate instead of Zn(II) chloride and
working at different ratios of metal salt to sulfonamide ligand
(between 1:1 and 1:3). In all cases only complexes with the
stoichiometry metal/sulfonamide of 1:1 were obtained.
En zym e P r ep a r a tion s. Human CA I and CA II cDNAs
were expressed in Escherichia coli strain BL21 (DE3) from the
plasmids pACA/hCA I and pACA/hCA II described by Lindskog
et al.29 (the two plasmids were a gift from Prof. Sven Lindskog,
Umea University, Sweden). Cell growth conditions were those
described by this group,30 and enzymes were purified by
affinity chromatography according to the method of Khalifah
et al.31 Enzyme concentrations were determined spectropho-
tometrically at 280 nm, utilizing a molar absorptivity of 49
mM-1 cm-1 for CA I and 54 mM-1 cm-1 for CA II, respectively,
based on Mr ) 28.85 kDa for CA I and 29.30 kDa for CA II,
respectively.32,33 CA IV was isolated from bovine lung mi-
crosomes as described by Maren et al., and its concentration
has been determined by titration with ethoxzolamide.34
Initial rates of 4-nitrophenyl acetate hydrolysis catalyzed
by different CA isozymes were monitored spectrophotometri-
cally, at 400 nm, with a Cary 3 instrument interfaced with an
IBM-compatible PC.35 Solutions of substrate were prepared
in anhydrous acetonitrile; the substrate concentrations varied
between 2 × 10-2 and 1 × 10-6 M, working at 25 °C. A molar
absorption coefficient ꢀ of 18 400 M-1 cm-1 was used for the
4-nitrophenolate formed by hydrolysis, under the conditions
of the experiments (pH 7.40), as reported in the literature.35
Nonenzymatic hydrolysis rates were always subtracted from
the observed rates. Duplicate experiments were done for each
inhibitor concentration, and the values reported throughout
the paper are the mean of such results. Stock solutions of
inhibitor (1 mM) were prepared in distilled-deionized water
with 10-20% (v/v) DMSO (which is not inhibitory at these
concentrations), and dilutions up to 0.01 nM were done
thereafter with distilled-deionized water. Inhibitor and en-
zyme solutions were preincubated together for 10 min at room
temperature prior to assay to allow for the formation of the
E-I complex. The inhibition constant KI was determined as
described by Pocker and Stone.35 Enzyme concentrations were
1N. Mp 184-5 °C (dec). IR (KBr), cm-1: 1175 (SO2sym),
1381 (SO2as), 1560 (amide II), 1603 (amide I), 1760 (COOH),
3334 (NH, NH2). 1H NMR (TFA), 300 MHz, δ, ppm: 3.30 (2H,
s, CH2COOH); 3.42 (2H, s, CH2CONH); 3.51 (s, 3H, N-Me);
7.25 (br s, 3H, CONH + SO2NH2); the NH and COOH protons
are not seen in this solvent, being in fast exchange. 13C NMR
(TFA), δ, ppm: 26.8 (Me); 53.24 (CH2CH2); 53.70 (CH2CH2);
60.18 (CH2CONH); 61.34 (CH2COOH); 158.7 (C-2 of thiadia-

Preparation of Sulfonamides
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7 1475
3.5 nM for hCA II, 12 nM for hCA I, and 36 nM for bCA IV
(this isozyme has a decreased esterase activity,36 and higher
concentrations had to be used for the measurements).
ously determined. Total solubility was determined by the
relationship C′ ) A′C/A, where C is the concentration of
standard solution (mg/mL), A is the absorbance of standard
solution, A′ is the absorbance of the saturated solution, and
C′ is the concentration of the saturated solution (mg/mL).40
P a r t it ion Coefficien t Det er m in a t ion s. Chloroform-
buffer partition coefficients were obtained by equilibrating the
test compound between chloroform and 0.1 ionic strength pH
7.4 phosphate buffer. The concentration in each phase was
determined by UV spectrophotometry or HPLC.15,16
Tr a n scor n ea l P en etr a tion of Dr u gs. The method of
Maren et al.5 with the modifications of Pierce’s group41,42 (for
the HPLC assay of sulfonamides) has been used. Excised
rabbit corneas with either intact or denuded epithelium were
used in these experiments. The pH was 7.4, and the exposed
area was 1.2 cm2. Drugs of concentrations 40-2000 µM were
placed in the epithelial chamber, and samples of fluid were
collected from the endothelial chamber at different intervals,
up to 4 h. Both chambers contained 6 mL. Drugs present in
these fluids were assayed by the HPLC method of Pierce et
al.41,42 or enzymatically.5 The results of the drug analyses were
used to calculate the rate constant of transfer across the cornea
(kin). As described by Pierce,41,42 this value was determined by
using the formula
Mea su r em en t of Ton om etr ic IOP . Adult male New
Zealand albino rabbits weighing 3-3.5 kg were used in the
experiments (three animals were used for each inhibitor
studied). The experimental procedures conform to the Associa-
tion for Research in Vision and Ophthalmology Resolution on
the use of animals. The rabbits were kept in individual cages
with food and water provided ad libitum. The animals were
maintained on a 12 h:12 h light/dark cycle in a temperature-
controlled room, at 22-26 °C. Solutions or suspensions of
inhibitors (2% by weight as hydrochlorides or sodium carboxy-
lates) were obtained in distilled-deionized water. The pH
values of these solutions were in the range 5.5-7.4.
IOP was measured using a Digilab 30R pneumatonometer
(BioRad, Cambridge, MA) as described by Maren’s group.37,38
The pressure readings were matched with two-point standard
pressure measurements at least twice each day using a Digilab
Calibration verifier. All IOP measurements were done by the
same investigator with the same tonometer. One drop of 0.2%
oxybuprocaine hydrochloride (novesine, Sandoz) diluted 1:1
with saline was instilled in each eye immediately before each
set of pressure measurements. IOP was measured three times
at each time interval, and the means were reported. IOP was
measured first immediately before drug administration, then
at 30 min after the instillation of the pharmacological agent,
and then each 30 min for a period of 4-6 h. For all IOP
experiments the drug was administered to only one eye,
leaving the contralateral eye as an untreated control. The
ocular hypotensive activity is expressed as the average differ-
ence in IOP between the treated and control eye, in this way
minimizing the diurnal, seasonal, and interindividual varia-
tions commonly observed in the rabbit.37,38 All data are
expressed as the mean ( SE, using a one-tailed t test. Ocular
hypertension was elicited in the right eye of albino rabbits by
the injection of R-chymotrypsin (from Sigma) as described by
Sugrue et al.39 The IOP of operated animals was checked after
approximately 4 weeks, and animals with an elevated pressure
of 30-36 mmHg were used at least 1 month after the injection
of R-chymotrypsin.
Dr u g Distr ibu tion in Ocu la r F lu id s a n d Tissu es. The
general procedure of Maren’s group has been followed.37,38 The
animals were killed with an intracardiac air injection. Aqueous
humor (both posterior and anterior chamber fluids) were
withdrawn. Then, the cornea and anterior uvea (iris plus
attached ciliary body) were dissected, rinsed well with water,
blotted, weighed, and put into 1-2 mL of water. For isolation
of the ciliary processes, intact anterior uvea rings were placed
on a Parafilm covered piece of polystyrene foam in a Petri dish.
The tissue has been wetted with normal saline and dissected
under a microscope when ciliary processes were liberated from
their attachment to the iris, cut, weighed, and put in 0.5 mL
of distilled water. The tissue from four eyes (average weight
of 8 mg/eye) was pooled for drug analysis. Samples were boiled
for 5 min (in order to denature CA and free the drug from the
E-I complex), diluted, and then incubated with a known
amount of enzyme. The activity of the free enzyme and in the
presence of the inhibitor was determined as described above.
A calibration curve has been used in order to determine the
fractional inhibition in the different tissues, as described in
refs 37 and 38.
Deter m in a tion of Wa ter (Bu ffer ) Solu bility. A standard
solution was prepared by dissolving a precisely weighed
amount (generally 1 mg) of inhibitor in 10 mL of methanol.
The UV absorption maximum of each compound has been
determined (with a Cary 3 spectrophotometer), eventually
diluting the solution (with MeOH) as necessary. A saturated
solution of each compound was then prepared by stirring
magnetically a small volume of 0.039 M phosphate buffer (pH
7.4) in the presence of excess inhibitor for 3 h. The obtained
saturated solution was filtered in order to remove solid
compound through a Millipore 0.45 µm filter and was scanned
by UV at the wavelength of the absorption maximum previ-
[drug]endo 60
kin(×103 h-1) )
× 1000
t
[drug]epi
where [drug]endo is the concentration of drug on the endothelial
side, [drug]epi is the concentration of drug on the epithelial side,
and t is time (in min).
Ack n ow led gm en t. This research was financed by
a grant from the Italian CNRsTarget Project Biotech-
nology and by CSGI (Florence, Italy). Thanks are
extended to Drs. M.A. Ilies, A. Casini, and M. Barboiu
for expert technical assistance.
Refer en ces
(1) (a) Supuran, C. T.; Scozzafava, A. Carbonic anhydrase inhibitors
and their therapeutic potential. Expert Opin. Ther. Pat. 2000,
10, 575-600. (b) Supuran, C. T.; Scozzafava, A. Carbonic
anhydrase inhibitors. Curr. Med. Chem.-Imm., Endoc., Metab.
Agents 2001, 1, 61-97.
(2) Supuran, C. T. Carbonic anhydrase inhibitors. In Carbonic
Anhydrase and Modulation of Physiologic and Pathologic Pro-
cesses in the Organism; Puscas, I., Ed.; Helicon: Timisoara,
Romania, 1994; pp 29-111.
(3) Maren, T. H. Carbonic anhydrase inhibition in ophthalmology:
Aqueous humor secretion and development of sulphonamide
inhibitors. In The Carbonic AnhydrasessNew Horizons; Birkha¨us-
er Verlag: Basel, 2000; pp 425-436.
(4) Chegwidden, W. R., Edwards, Y., Carter, N., Eds. The Carbonic
AnhydrasessNew Horizons; Birkha¨user Verlag: Basel, 2000; pp
1-610 and references therein..
(5) Maren, T. H.; J ankowska, L.; Sanyal, G.; Edelhauser, G. F. The
transcorneal permeability of sulfonamide carbonic anhydrase
inhibitors and their effect on aqueous humor secretion. Exp. Eye
Res. 1983, 36, 457-480.
(6) Sugrue M. F. Pharmacological and ocular hypotensive properties
of topical carbonic anhydrase inhibitors. Prog. Retinal Eye Res.
2000, 19, 87-112.
(7) Silver L. H. Dose-response evaluation of the ocular hypotensive
effect of brinzolamide ophthalmic suspension (Azopt). Brinzola-
mide dose-response study group. Surv. Ophthalmol. 2000, 44
(Suppl. 2), 147-153.
(8) Konowal, A.; Morrison, J . C.; Brown, S. V.; Cooke, D. L.; Maguire,
L. J .; Verdier DV, Fraunfelder, F. T.; Dennis, R. F.; Epstein, R.
J . Irreversible corneal decompensation in patients treated with
topical dorzolamide. Am. J . Ophthalmol. 1999, 127, 403-406.
(9) Aalto-Korte, K. Contact allergy to dorzolamide eyedrops. Contact
Dermatitis 1998, 39, 206.
(10) Carlsen, J .; Durcan, J .; Swartz, M.; Crandall, A. Nephrolithiasis
with dorzolamide. Arch. Ophthalmol. 1999, 117, 1087-1088.
(11) Thoe Schwartzenberg, G. W.; Trope, G. E. Anorexia, depression
and dementia induced by dorzolamide eyedrops (Trusopt). Can.
J . Ophthalmol. 1999, 34, 93-94.
(12) Mansoor, U. F.; Zhang, Y. R.; Blackburn, G. M. The design of
new carbonic anhydrase inhibitors. In The Carbonic Anhydrasess
New Horizons; Chegwidden W. R., Edwards, Y., Carter, N., Eds.;
Birkha¨user Verlag: Basel, 2000; pp 437-460.

1476 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7
Scozzafava et al.
(13) (a) Scozzafava, A.; Menabuoni, L.; Mincione, F.; Briganti, F.;
Mincione, G.; Supuran, C. T. Carbonic anhydrase inhibitors.
Perfluoroalkyl/aryl-substituted derivatives of aromatic/hetero-
cyclic sulfonamides as topical intraocular pressure lowering
agents with prolonged duration of action. J . Med. Chem. 2000,
43, 4542-4551. (b) Casini, A.; Scozzafava, A.; Mincione, F.;
Menabuoni, L.; Ilies, M. A.; Supuran, C. T. Carbonic anhydrase
inhibitors. Water soluble 4-sulfamoylphenyl-thioureas as topical
intraocular pressure lowering agents with long lasting effects.
J . Med. Chem. 2000, 43, 4884-4892.
(14) Chen, H. H.; Gross, S.; Liao, J .; McLaughlin, M.; Dean, T.; Sly,
W. S.; May, J . A. 2H-Thieno [3,2-e]- and [2,3-e]-1,2-thiazine-6-
sulfonamide 1,1-dioxides as ocular hypotensive agents: synthe-
sis, carbonic anhydrase inhibition and evaluation in the rabbit.
Bioorg. Med. Chem. 2000, 8, 957-975.
(15) (a) Scozzafava, A.; Menabuoni, L.; Mincione, F.; Briganti, F.;
Mincione, G.; Supuran, C. T. Carbonic anhydrase inhibitors:
Synthesis of water-soluble, topically effective intraocular pres-
sure lowering aromatic/heterocyclic sulfonamides containing
cationic or anionic moieties: Is the tail more important than
the ring? J . Med. Chem. 1999, 42, 2641-2650. (b) Scozzafava,
A.; Briganti, F.; Mincione, G.; Menabuoni, L.; Mincione, F.;
Supuran, C. T. Carbonic anhydrase inhibitors: Synthesis of
water-soluble, amino acyl/dipeptidyl sulfonamides possessing
long-lasting intraocular pressure-lowering properties via the
topical route. J . Med. Chem. 1999, 42, 3690-3700.
(16) (a) Borras, J .; Scozzafava, A.; Menabuoni, L.; Mincione, F.;
Briganti, F.; Mincione, G.; Supuran, C. T. Carbonic anhydrase
inhibitors: Synthesis of water-soluble, topically effective in-
traocular pressure lowering aromatic/heterocyclic sulfonamides
containing 8-quinoline-sulfonyl moieties: Is the tail more im-
portant than the ring? Bioorg. Med. Chem. 1999, 7, 2397-2406.
(b) Renzi, G.; Scozzafava, A.; Supuran, C. T. Carbonic anhydrase
inhibitors: Topical sulfonamide antiglaucoma agents incorporat-
ing secondary amine moieties. Bioorg. Med. Chem. Lett. 2000,
10, 673-676. (c) Supuran, C. T.; Briganti, F.; Tilli, S.; Chegwid-
den, W. R.; Scozzafava, A. Carbonic anhydrase inhibitors:
Sulfonamides as antitumor agents? Bioorg. Med. Chem. 2001,
9, 703-714.
(26) Itoh, M.; Hagiwara, D.; Kamiya, T. A new reagent for tert-
butoxycarbonylation: 2- tert-Butoxycarbonyloxyimino-2-phenyl-
acetonitrile. Org. Synth. 1980, 59, 95-101.
(27) (a) Alzuet, G.; Ferrer, S.; Borras, J .; Supuran, C. T. Complexes
of heterocyclic sulfonamidessA class of dual carbonic anhydrase
inhibitors. Roum. Chem. Q. Rev. 1994, 2, 283-300. (b) Borja,
P.; Alzuet, G.; Server-Carrio`, J .; Borras, J .; Supuran, C. T. Zinc
complexes of carbonic anhydrase inhibitors. Crystal structure
of [Zn(5-amino-1,3,4-thiadiazole-2-sulfonamidate)2(NH3)]‚H2O.
Carbonic anhydrase inhibitory activity. Main Group Met. Chem.
1998, 21, 279-292. (c) Alzuet, G.; Casanova, J .; Borras, J .;
Garcia-Granda, S.; Gutie´rrez-Rodriguez, A.; Supuran, C. T.
Copper complexes modelling the interaction between benzola-
mide and Cu-substituted carbonic anhydrase. Crystal structure
of Cu(bz)(NH3)4 complex. Inorg. Chim. Acta 1998, 273, 334-338.
(28) (a) Supuran, C. T.; Clare, B. W. Carbonic anhydrase inhibitors.
Part 57. Quantum chemical QSAR of a group of 1,3,4-thiadiazole
and 1,3,4-thiadiazoline disulfonamides with carbonic anhydrase
inhibitory properties. Eur. J . Med. Chem. 1999, 34, 41-50. (b)
Clare, B. W.; Supuran, C. T. Carbonic anhydrase inhibitors. Part
61. Quantum Chemical QSAR of a group of benzenedisulfona-
mides. Eur. J . Med. Chem. 1999, 34, 463-474.
(29) Lindskog, S.; Behravan, G.; Engstrand, C.; Forsman, C.; J onsson,
B. H.; Liang, Z.; Ren, X.; Xue, Y. Structure-function relations
in human carbonic anhydrase II as studied by site-directed
mutagenesis. In Carbonic anhydrasesFrom biochemistry and
genetics to physiology and clinical medicine; Botre`, F., Gros, G.,
Storey, B. T., Eds.; VCH: Weinheim, 1991; pp 1-13.
(30) Behravan, G.; J onsson, B. H.; Lindskog, S. Fine tuning of the
catalytic properties of carbonic anhydrase. Studies of a Thr200-
His variant of human isoenzyme II. Eur. J . Biochem. 1990, 190,
351-357.
(31) Khalifah, R. G.; Strader, D. J .; Bryant, S. H.; Gibson, S. M.
Carbon-13 nuclear magnetic resonance probe of active site
ionization of human carbonic anhydrase B. Biochemistry 1977,
16, 2241-2247.
(32) Lindskog, S. Coleman, J . E. The catalytic mechanism of carbonic
anhydrase. Proc. Natl. Acad. Sci. U.S.A. 1964, 70, 2505-2508.
(33) Steiner, H.; J onsson, B. H.; Lindskog, S. The catalytic mecha-
nism of carbonic anhydrase. Hydrogen-isotope effects on the
kinetic parameters of the human C isoenzyme. Eur. J . Biochem.
1975, 59, 253-259.
(34) Maren, T. H.; Wynns, G. C.; Wistrand, P. J . Chemical properties
of carbonic anhydrase IV, the membrane-bound enzyme. Mol.
Pharmacol. 1993, 44, 901-906.
(35) Pocker, Y.; Stone, J . T. The catalytic versatility of erythrocyte
carbonic anhydrase. 3. Kinetic studies of the enzyme-catalyzed
hydrolysis of p-nitrophenyl acetate. Biochemistry 1967, 6, 668-
678.
(36) Baird, T. T.; Waheed, A.; Okuyama, T.; Sly, W. S.; Fierke, C. A.
Catalysis and inhibition of human carbonic anhydrase IV.
Biochemistry 1997, 36, 2669-2678.
(37) Maren, T. H.; Brechue, W. F.; Bar-Ilan, A. Relations among IOP
reduction, ocular disposition and pharmacology of the carbonic
anhydrase inhibitor ethoxzolamide. Exp. Eye Res. 1992, 55, 73-
79.
(38) Brechue, W. F.; Maren, T. H. pH and drug ionization affects
ocular pressure lowering of topical carbonic anhydrase inhibitors.
Invest. Ophthalmol. Visual Sci. 1993, 34, 2581-2587.
(39) Sugrue, M. F.; Gautheron, P.; Mallorga, P.; Nolan, T. E.;
Graham, S. L.; Schwam, H.; Shepard, K. L.; Smith, R. L. L-662,-
583 is a topically effective ocular hypotensive carbonic anhydrase
inhibitor in experimental animals. Br. J . Pharmacol. 1990, 99,
59-64.
(40) Supuran, C. T.; Ilies, M. A.; Scozzafava, A. Carbonic anhydrase
inhibitors. Part 29. Interaction of isozymes I, II and IV with
benzolamide-like derivatives. Eur. J . Med. Chem. 1998, 33, 739-
751.
(41) Pierce, W. M., J r.; Sharir, M.; Waite, K. J .; Chen, D.; Kaysinger,
K. K. Topically active ocular carbonic anhydrase inhibitors:
Novel biscarbonylamidothiadiazole sulfonamides as ocular hy-
potensive agents. Proc. Soc. Exp. Biol. Med. 1993, 203, 360-
365.
(17) J ain, A.; Whitesides, G. M.; Alexander, R. S.; Christianson, D.
W. Identification of two hydrophobic patches in the active-site
cavity of human carbonic anhydrase II by solution-phase and
solid-state studies and their use in the development of tight-
binding inhibitors. J . Med. Chem. 1994, 37, 2100-2105.
(18) Ilies, M.; Supuran, C. T.; Scozzafava, A.; Casini, A.; Mincione,
F.; Menabuoni, L.; Caproiu, M. T.; Maganu, M.; Banciu. M. D.
Carbonic anhydrase inhibitors. Sulfonamides incorporating fu-
ran-, thiophene-, and pyrrole-carboxamido groups possess strong
topical intraocular pressure lowering properties as aqueous
suspensions, Bioorg. Med. Chem. 2000, 8, 2145-2155.
(19) Scozzafava, A.; Menabuoni, L.; Mincione, F.; Mincione, G.;
Supuran, C. T. Carbonic anhydrase inhibitors. Synthesis of
sulfonamides incorporating dtpa tails and of their zinc complexes
with powerful topical antiglaucoma properties. Bioorg. Med.
Chem. Lett. 2001, 11, 575-582.
(20) Supuran, C. T.; Mincione, F.; Scozzafava, A.; Briganti, F.;
Mincione, G.; Ilies, M. A. Carbonic anhydrase inhibitors. Part
52. Metal complexes of heterocyclic sulfonamides: A new class
of strong topical intraocular pressure-lowering agents with
potential use as antiglaucoma drugs. Eur. J . Med. Chem. 1998,
33, 247-254.
(21) Ilies, M. A.; Scozzafava, A.; Supuran C. T. Carbonic anhydrase
inhibitors. Part 91. Metal complexes of heterocyclic sulfonamides
as potential pharmacological agents in the treatment of gastric
acid secretion imbalances. Met.-Based Drugs 2000, 7, 57-62.
(22) (a) Mastrolorenzo, A.; Scozzafava, A.; Supuran, C. T. Antifungal
activity of silver and zinc complexes of sulfadrug derivatives
incorporating arylsulfonylureido moieties. Eur. J . Pharm. Sci.
2000, 11, 99-107. (b) Mastrolorenzo, A.; Scozzafava, A.; Supu-
ran, C. T. Antifungal activity of Ag(I) and Zn(II) complexes of
aminobenzolamide (5-sulfanilylamido-1,3,4-thiadiazole-2-sul-
fonamide) derivatives. J . Enzyme Inhib. 2000, 15, 517-531.
(23) Scozzafava, A.; Supuran, C. T. Carbonic anhydrase activators.
Part 21. Novel activators of isozymes I, II and IV incorporating
carboxamido- and ureido histamine moieties. Eur. J . Med. Chem.
2000, 35, 31-39.
(24) Briganti, F.; Mangani, S.; Orioli, P.; Scozzafava, A.; Vernaglione,
G.; Supuran, C. T. Carbonic anhydrase activators: X-ray
crystallographic and spectroscopic investigations for the interac-
tion of isozymes I and II with histamine. Biochemistry 1997, 36,
10384-10392.
(25) Supuran, C. T.; Scozzafava, A. Activation of carbonic anhydrase
isozymes. In The Carbonic AnhydrasessNew Horizons; Cheg-
widden, W. R., Carter, N., Edwards, Y., Eds.; Birkhauser
Verlag: Basel, Switzerland, 2000; pp 197-219.
(42) Sharir, M.; Pierce, W. M., J r.; Chen, D.; Zimmerman, T. J .
Pharmacokinetics, acid-base balance and intraocular pressure
effects of ethyloxaloylazolamidesA novel topically active car-
bonic anhydrase inhibitor. Exp. Eye Res. 1994, 58, 107-116.
J M0108202