Carbonic anhydrase inhibitors: Perfluoroalkyl/aryl-substituted derivatives of aromatic/heterocyclic sulfonamides as topical intraocular pressure-lowering agents with prolonged duration of action

Article abstract of DOI:10.1021/jm000296j

Reaction of perfluoroalkyl/arylsulfonyl chlorides or perfluoroalkyl/arylcarbonyl chlorides with aromatic/heterocyclic sulfonamides possessing a free amino/imino/hydrazino/hydroxy group afforded compounds with the general formula C_xF_yZ-A-SO₂NH₂, where Z = SO₂NH, SO₃, CONH, or CO₂ and A = aromatic/heterocyclic moiety. The sulfonyl chlorides used in synthesis included: CF₃SO₂Cl, n-C₄F₉SO₂Cl, n-C₈F₁₇SO₂Cl, and C₆F₅SO₂Cl, whereas the acyl chlorides were C₈F₁₇COCl and C₆F₅COCl. A total of 25 different sulfonamides have been derivatized by means of the above-mentioned perfluorosulfonyl/acyl halides. These new series of sulfonamides showed strong affinities toward isozymes I, II, and IV of carbonic anhydrase (CA). For a given sulfonamide derivatized by the above procedures, inhibitory power was greater for the alkyl/arylsulfonylated compounds, as compared to the corresponding perfluoroalkyl/arylcarbonylated ones. In vitro inhibitory activity generally increased with the number of carbon atoms in the molecule of the acylating/sulfonylating agent, with a maximum for the perfluorophenylsulfonylated and perfluorobenzoylated derivatives. Some of the prepared CA inhibitors displayed very good water solubility (in the range of 2%) and strongly lowered intraocular pressure (IOP) when applied topically, directly into the normotensive/glaucomatous rabbit eye, as 2% water solutions. The good water solubility of these new classes of CA inhibitors, correlated with the neutral pH of their solutions used in the ophthalmologic applications, makes them attractive candidates for developing novel types of antiglaucoma drugs devoid of unpleasant ocular side effects.

Full text of DOI:10.1021/jm000296j

4542
J . Med. Chem. 2000, 43, 4542-4551
Ca r bon ic An h yd r a se In h ibitor s: P er flu or oa lk yl/Ar yl-Su bstitu ted Der iva tives of
Ar om a tic/Heter ocyclic Su lfon a m id es a s Top ica l In tr a ocu la r P r essu r e-Low er in g
Agen ts w ith P r olon ged Du r a tion of Action
Andrea Scozzafava,† Luca Menabuoni,‡ Francesco Mincione,# Fabrizio Briganti,† Giovanna 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, S. A. Oculistica, Via Torregalli 3, I-50123 Florence, Italy, and
Istituto Oculistico, Universita` degli Studi di Firenze, Viale Morgagni 85, I-50134 Florence, Italy
Received J uly 13, 2000
Reaction of perfluoroalkyl/arylsulfonyl chlorides or perfluoroalkyl/arylcarbonyl chlorides with
aromatic/heterocyclic sulfonamides possessing a free amino/imino/hydrazino/hydroxy group
afforded compounds with the general formula CxFyZ-A-SO2NH2, where Z ) SO2NH, SO3, CONH,
or CO2 and A ) aromatic/heterocyclic moiety. The sulfonyl chlorides used in synthesis
included: CF3SO2Cl, n-C4F9SO2Cl, n-C8F17SO2Cl, and C6F5SO2Cl, whereas the acyl chlorides
were C8F17COCl and C6F5COCl. A total of 25 different sulfonamides have been derivatized by
means of the above-mentioned perfluorosulfonyl/acyl halides. These new series of sulfonamides
showed strong affinities toward isozymes I, II, and IV of carbonic anhydrase (CA). For a given
sulfonamide derivatized by the above procedures, inhibitory power was greater for the alkyl/
arylsulfonylated compounds, as compared to the corresponding perfluoroalkyl/arylcarbonylated
ones. In vitro inhibitory activity generally increased with the number of carbon atoms in the
molecule of the acylating/sulfonylating agent, with a maximum for the perfluorophenylsulfon-
ylated and perfluorobenzoylated derivatives. Some of the prepared CA inhibitors displayed
very good water solubility (in the range of 2%) and strongly lowered intraocular pressure (IOP)
when applied topically, directly into the normotensive/glaucomatous rabbit eye, as 2% water
solutions. The good water solubility of these new classes of CA inhibitors, correlated with the
neutral pH of their solutions used in the ophthalmologic applications, makes them attractive
candidates for developing novel types of antiglaucoma drugs devoid of unpleasant ocular side
effects.
In tr od u ction
Sulfonamides possessing carbonic anhydrase (CA; EC
4.2.1.1) inhibitory properties1,2 such as acetazolamide
(AAZ), methazolamide (MZA), ethoxzolamide (EZA), and
dichlorophenamide (DCP) have been used for more than
45 years as pressure-lowering systemic drugs in the
treatment of open-angle glaucoma as well as other
diseases associated with acid base/secretory disequilib-
ria.3,4 Recently, they started to show increasing interest
as potential agents for the treatment of macular edema,
a condition for which no effective therapy was known
up to now.1 The ocular effects of such sulfonamides are
due to inhibition of at least two CA isozymes present
within cilliary processes of the eye, i.e., CA II and CA
IV, which is followed by a diminished secretion of
bicarbonate and reduction of aqueous humor secretion.5-7
Their main drawback is constituted by systemic side
effects such as fatigue, augmented diuresis, or pares-
thesias, due to CA inhibition in other tissues/organs
applied directly into the eye. This route, discovered in
than the target one, i.e., the eye.8
1983 by Maren’s group,9 was shortly followed by the
The above-mentioned side effects are absent in the
development of the first clinical agents of this type,
case in which the inhibitor has topical activity and is
dorzolamide (DZA)10 as well as the structurally related
brinzolamide (BRZ), several years later.11 The clinical
* To whom correspondence should be addressed. Tel: +39-055-
success of topical antiglaucoma CA inhibitors fostered
much research in the synthesis and evaluation of other
types of such compounds.12-16 These two drugs men-
tioned above are effective in reducing intraocular pres-
2757551. Fax: +39-055-2757555. E-mail: cts@bio.chim.unifi.it.
† Laboratorio di Chimica Inorganica e Bioinorganica, Universita`
degli Studi di Firenze.
‡ Ospedale San Giovanni di Dio.
#
Istituto Oculistico, Universita` degli Studi di Firenze.
10.1021/jm000296j CCC: $19.00 © 2000 American Chemical Society
Published on Web 10/28/2000

Carbonic Anhydrase Inhibitors
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 23 4543
sure (IOP) and show fewer side effects as compared to
the systemically applied sulfonamides. The observed
side effects after topical sulfonamides include stinging,
burning or reddening of the eye, blurred vision, pruritus,
and bitter taste.6,7 All but the last are probably due to
the fact that DZA (the best studied topical CA inhibitor)
is the salt of a weak base with a very strong acid, so
that the pH of the drug solution is rather acidic
(generally around 5.5). The last side effect mentioned
above is probably due to drug-laden lachrymal fluid
draining into the oropharynx and inhibition of CA
present in the saliva (CA VI) and the taste buds (CA II
and CA VI), with the consequent accumulation of
bicarbonate, and was seen with both systemic as well
as topical CA inhibitors.6,7 Less is known for the
moment regarding the side effects of BRZ, but it
seems that this drug produces less stinging (being
administered at pH 7.5) but more blurred vision as
compared to DZA.7 Unfortunately, DZA also showed
some more serious side effects, such as contact allergy,
nephrolithiasis, anorexia, depression, and dementia as
well as irreversible corneal decompensation in patients
who already had corneal problems.17 Thus, new types
of topically effective CA inhibitors as possible antiglau-
coma agents are being investigated,12-16 and an alter-
native approach (to the thienothiopyran sulfonamides
and their analogues) was recently reported, consisted
in attaching water-solubilizing moieties to the molecules
of aromatic/heterocyclic sulfonamides.12,13 Such moieties
included pyridinecarboximido, carboxypyridinecarbox-
amido, quinolinesulfonamido, picolinoyl, and isonico-
tinoyl, as well as amino acyl groups among others,
whereas ring systems which have been derivatized by
using the above-mentioned moieties included: 2-, 3-, or
4-aminobenzenesulfonamides, 4-(ω-aminoalkyl)benzene-
sulfonamides, 3-halogeno-substituted-sulfanilamides,
1,3-benzenedisulfonamides, 1,3,4-thiadiazole-2-sulfon-
amides, benzothiazole-2-sulfonamides, and thienothio-
pyran-2-sulfonamides among others, and were chosen
in such a way as to demonstrate that the proposed “tail”
approach is a general one.12,13
perfluoroalkyl/arylcarbonyl chlorides (of types A-F )
with aromatic/heterocyclic sulfonamides containing a
free amino/imino/hydrazino/hydroxy group in their mol-
ecule of type 1-25 (the synthesis of perfluorobenzol-
amide C13 has not been reported previously in the
mentioned work) (Chart 1).18
Resu lts
Syn th esis. As a large number of derivatives (150
compounds) have been prepared, in the following, they
will be abbreviated by using both a letter, designating
the perfluorosulfonic/carboxylic acid moiety, as well as
a number, designating the sulfonamide from which the
new compounds were derived. For instance: A1 is the
trifluoromethylsulfonyl derivative of orthanilamide 1;
C13 is the perfluorophenylsulfonylamido derivative of
5-amino-1,3,4-thiadiazole-2-sulfonamide 13, (perfluoro-
benzolamide); D24 is the perfluorooctylsulfonylated
derivative of 4-(2-hydroxymethyl)benzenesulfonamide
(24); F 14 is the perfluorobenzoyl derivative of 5-imino-
4-methyl-2-sulfonamido-δ2-1,3,4-thiadiazoline (see the
structures of these derivatives below). The whole series
of compounds from 6 acylating/sulfonylating agents and
the 25 initial sulfonamides 1-25 has been prepared by
reported procedures from Whitesides’ laboratory14,15 or
from our laboratory,12,13 in order to select valuable
inhibitors for the in vivo studies (Table 1).
CA 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
acqueous 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).
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 compounds
reported here, relevant for their pharmacological activ-
ity, such as buffer solubility or chloroform-buffer parti-
tion coefficient, are shown in Table 2. The in vitro
transcorneal accession rates (kin) of classical sulfon-
amides and topically active derivatives, such as DZA
and some of the new compounds 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
administration of the drug, are shown in Tables 3 and
4, respectively. The full time dependence of the IOP
after DZA and some of the new compounds reported
here in normotensive albino rabbits is shown in Figure 1.
Dist r ib u t ion of Dr u gs in Ocu la r F lu id s a n d
Tissu es. Ex vivo distribution data of some active com-
As mentioned above, the two topically active anti-
glaucoma drugs used clinically, DZA and BRZ, are both
secondary amines, and the required water solubility
needed for effective topical action is achieved by using
their hydrochloride salts. Still, in some cases this
represents an undesired problem, since the pH of such
solutions is acidic enough and produces eye irritation
after topical administration, which is in fact the side
effect most frequently reported after the use of these
drugs. Such side effects might be avoided for compounds
that do not need to be administered as hydrochloride
salts, but this generally leads to a drastical diminution
of water solubility of such sulfonamides. Here we
propose a novel approach for obtaining water-soluble,
high-affinity sulfonamide CA inhibitors, which do not
owe their water solubility to formation of hydrochloride
salts. Thus, during our work18 for the synthesis of
benzolamide-like sulfonamide CA inhibitors with ap-
plications in positron emission tomography (PET), it was
observed that perfluorobenzolamide possesses an un-
expectedly high water solubility. Starting from this
compound, a large series of derivatives were obtained
by reaction of perfluoroalkyl/arylsulfonyl halides or

4544 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 23
Scozzafava et al.
Ch a r t 1
pounds in ocular tissues and fluids after topical admin-
istration in normotensive rabbits are shown in Table 5.
2, 4) act as potent CA inhibitors, but only the trifluoro-
methanesulfonamide mentioned previously has been
investigated for its IOP-lowering effects in normotensive
rabbits. One must also mention that CA inhibitors
Discu ssion
Ch em istr y. Although the lead molecule for obtaining
this series of CA inhibitors was perfluorobenzolamide
C13 (initially prepared in a study18 in which sulfon-
amides for applications in PET studies were being
designed), historically, the topical route for administra-
tion of sulfonamides has been discovered by Maren’s
group working with trifluoromethazolamide 26.9 Still,
trifluoromethazolamide, as well as the structurally
related trifluoroacetazolamide 27, is unstable, undergo-
ing spontaneous hydrolysis with a half-life of 45 min at
neutral pH and could not be developed for clinical use.9
Also having a similar fate is the other fluoro-containing
sulfonamide investigated up to now, trifluoromethane-
sulfonamide (CF3SO2NH2, 28) which has been shown
by Maren’s group19 and by Kvam20 to act as an effective
IOP-lowering agent but which also proved to be chemi-
cally unstable, being hydrolyzed to triflic acid (CF3-
SO3H) which is a very strong acid.19 In the same study,
Maren and Conroy19 showed that other perfluoroalkyl-
sulfonamides as well as perchloroalkylsulfonamides of
the general formula CnX2n+1SO2NH2 (X ) F, Cl; n ) 1,
F igu r e 1. Effect of topically administered sulfonamide inhibi-
tors (2% water solutions) on the IOP of normotensive albino
rabbits: curve 1, DZA 1 (hydrochloride salt, pH 5.5); curve 2,
compound C14 (pH 6.50); curve 3, compound C13 (pH 7.5).

Carbonic Anhydrase Inhibitors
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 23 4545
Ta ble 1. CA Inhibition Data with Standard Inhibitors and the New Sulfonamides Reported in the Present Study
Ki (nM)
Ki (nM)
Ki (nM)
inhibitor
hCA Ia
hCA IIa
bCA IVb
inhibitor
hCA Ia
hCA IIa
bCA IVb
inhibitor
hCA Ia
hCA IIa
bCA IVb
AZA
MZA
EZA
DCP
DZA
A1
A2
A3
A4
A5
A6
A7
A8
A9
A10
A11
A12
A13
A14
A15
A16
A17
A18
A19
A20
A21
A22
A23
A24
A25
B1
B2
B3
B4
B5
B6
B7
B8
B9
900
780
25
12
14
8
38
9
220
240
13
380
43
B23
B24
B25
C1
C2
C3
C4
C5
C6
C7
19
24500
20900
1390
1100
890
1000
790
760
0.7
460
385
24
10
10
16
15
15
9
110
125
150
15
5
0.3
0.3
0.4
1.0
1.5
8
6
10550
9200
680
500
410
500
240
230
140
540
550
675
142
26
D24
D25
E1
E2
E3
E4
E5
E6
E7
15000
22000
1800
1600
1400
1850
1000
950
1400
2300
2500
3000
7000
810
540
440
103
69
66
107
62
55
51
55
69
70
72
35
5
10900
12300
1500
935
850
840
750
520
550
600
620
680
830
130
13
1200
50000
175000
188400
165500
155500
137800
81300
100000
180000
200000
210000
5500
550
59800
2000
3000
1000
1000
4000
5000
8000
70
20450
18700
10900
14800
5000
600
500
730
1040
1300
430
90
100
24
13
3
5
21
23
25
0.9
0.9
1
5100
550
250
170
160
180
150
150
98
425
485
650
51
97000
95300
64600
60780
24500
50000
29000
42000
47000
49000
725
150
600
32
710
E8
E9
C8
C9
1450
1500
1755
1500
300
210
250
200
24
21
125
150
325
41
25
18
290
300
1500
1400
1140
1680
1000
965
1450
2480
2550
3400
8000
890
400
380
300
800
1100
3000
3600
4500
49
28
17
E10
E11
E12
E13
E14
E15
E16
E17
E18
E19
E20
E21
E22
E23
E24
E25
F 1
F 2
F 3
F 4
F 5
F 6
F 7
F 8
F 9
F 10
F 11
F 12
F 13
F 14
F 15
F 16
F 17
F 18
F 19
F 20
F 21
F 22
F 23
F 24
F 25
C10
C11
C12
C13
C14
C15
C16
C17
C18
C19
C20
C21
C22
C23
C24
C25
D1
D2
D3
D4
D5
D6
D7
D8
D9
D10
D11
D12
D13
D14
D15
D16
D17
D18
D19
D20
D21
D22
D23
300
330
300
600
6
9
9
8
9
15
16
24
6
2
18
8
13
12
25
30
36
9
1.5
2
11
16
22
1
900
40
25
33
40
45
39
8
10
2400
3000
4400
45
37
24
15000
20000
1560
1400
950
1500
980
900
760
1080
1870
2560
1900
750
250
270
300
1500
1600
6600
8000
7700
120
105
65
1200
1250
8
11
0.2
0.3
0.5
40
35
96
67
62
110
60
54
125
550
680
755
98
32
3
3
2
2
3
12
21
20
0.6
0.5
0.6
5
6
7
3
2
14
7
550
410
35
19
17
23
20
17
15
125
156
230
38
12
2
11000
12000
810
650
600
980
350
310
185
590
865
1040
420
54
10
8
10
32
29
39
55
62
8
9
11
410
400
55
50
100
103
1200
870
760
860
705
470
500
10300
12500
15000
765
155
12
7
135000
80000
22000
18400
15200
15000
13000
12500
1070
10200
20300
23400
5100
400
360
350
300
900
1000
3600
4000
6000
54
24100
45000
900
590
480
600
255
240
200
880
900
1040
160
43
B10
B11
B12
B13
B14
B15
B16
B17
B18
B19
B20
B21
B22
1.5
2
8
8
2
2
3
2
4
15
20
14
9
21
25
29
33
34
7
9
10
11
17
24
31
36
30
7
18
36
27
0.5
0.6
0.7
54
50
20
0.5
0.5
8
5
6
7
29
a
b
Human (cloned) isozymes. From bovine lung microsomes, by the esterase method.
containing fluorine atoms were recently investigated
by Whitesides’ 15b and J ain’s21 groups, who reported
derivatives of types 29 and 30, respectively, showing
good hCA II inhibitory properties, but which were not
tested for their efects on the IOP.
Considering these data, it appeared of interest to
design inhibitors that would contain perfluoroalkyl/
arylsulfonylamido and perfluoroacyl moieties attached
to an aromatic/heterocyclic ring also bearing an unsub-
stituted SO2NH2 group. Mention should be made that
based on empirical considerations, one should expect
increased hydrolytic stability for the CnF2n+1SO2NH
moiety when attached to an aromatic/heterocyclic ring,
as compared to a CF3CONH one (which precluded with
the clinical development of derivatives 26 and 27).
Fortunately these initial hypotheses proved to be correct
(see later in the text).
Reactions of sulfonyl chlorides/sulfonic acid anhydrides
or acyl chlorides with amino-containing sulfonamides
were previously reported, both in the heterocyclic series
(such as 5-amino-1,3,4-thiadiazole-2-sulfonamide 13 or
5-imino-4-methyl-2-sulfonamido-δ2-1,3,4-thiadiazoline 14)
by Vaughan et al.22 as well as in the aromatic series by

4546 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 23
Scozzafava et al.
Ta ble 5. Ocular Tissue Concentrations after 1 and 2 h
following Corneal Application of 1 drop (50 µL) of a 2% Solution
of Inhibitors C13,14 in Albino Rabbits
Ta ble 2. Solubility, Chloroform-Buffer Partition Coefficients,
and in Vitro Corneal Permeability of Some Sulfonamide CA
Inhibitors
c
kin × 103 (h-1
)
drug concentration (µM)a
solubilitya
(mM)
compd
log Pb
cornea intact
no epithelium
time (h)
cornea
aqueous humor
ciliary process
AAZd
MZAd
DZA
A14
B13
C13
C14
D16
E16
F 13
3.2
12
0.001
0.06
0.37
1.90
3.0
3.1
4.3
4.5
4.9
4.1
3.2
4.8
7.0
13
DZA‚HCl
32 ( 3
1
2
105 ( 5
39 ( 4
15 ( 3
6 ( 1
60e
69
2.0e
5.2
4.8
7.0
8.7
8.8
7.5
8.0
10.1
21 ( 2
1.012
1.525
2.113
2.347
1.689
1.436
2.245
C13
324 ( 18
42 ( 3
50
62
66
47
54
43
1
2
176 ( 15
69 ( 5
45 ( 6
12.5 ( 1
C14
296 ( 10
39 ( 3
1
2
150 ( 11
36 ( 5
38 ( 3
7.1 ( 0.8
a
b
a
Solubility in pH 7.40 buffer, at 25 °C. Chloroform-buffer
Mean ( standard deviation (n ) 3). With DZA‚HCl as
partition coefficient. c Determined as described in refs 12, 44, 45.
standard.
Data from ref 9. e As hydrochloride, at pH 5.8, from ref 12.
d
developed by us, consisting of a combination of the
two methods described above. Thus, we observed that
a large number of amino/imino/hydrazino/hydroxy-
containing aromatic/heterocyclic sulfonamides are easily
derivatized by sulfonyl/acyl halides in acqueous acetone,
working at temperatures between 4 and 25 °C. This
strategy has been perticularly useful for obtaining
the fluoro-containing sulfonamides reported here (see
Experimental Section for details). Derivatization of
amino, alcoholic, phenolic, or imino/hydrazino moieties
present in the raw material sulfonamides 1-25 has
been achieved the above-mentioned procedures too, but
generally, the reactions of amines could be controlled
better than those of the corresponding phenols/alcohols,
leading to fewer side products. The new compounds
reported here were characterized by standard chemical
(elemental analysis) and spectroscopic (IR, 1H NMR,
13C NMR, 19F NMR) procedures that confirmed their
structure (see Experimental Section for details).
Ta ble 3. Fall of IOP of Normotensive Rabbits (21.5 ( 2.0
mmHg) after Treatment with 1 drop (50 µL) of a 2% Solution of
CA Inhibitor Directly into the Eyea
∆IOP ( SEb (mmHg)
inhibitor
pH
30 min
60 min
90 min
DZA
A14
B13
B14
C13
C14
C15
C21c
D13
D14
F 13
F 14
5.5
6.5
7.0
7.0
7.5
6.5
7.0
7.0
7.0
7.0
7.0
7.0
2.2 ( 0.15
3.2 ( 0.15
2.8 ( 0.20
3.2 ( 0.20
4.9 ( 0.30
4.7 ( 0.25
4.0 ( 0.15
3.0 ( 0.20
4.6 ( 0.30
5.2 ( 0.30
2.2 ( 0.25
3.2 ( 0.15
4.1 ( 0.15
7.3 ( 0.25
8.5 ( 0.25
8.9 ( 0.30
12.5 ( 0.40
11.3 ( 0.35
7.5 ( 0.20
8.0 ( 0.30
8.9 ( 0.35
10.3 ( 0.10
7.3 ( 0.35
8.6 ( 0.25
2.7 ( 0.10
4.9 ( 0.25
5.6 ( 0.30
7.1 ( 0.30
10.2 ( 0.40
9.1 ( 0.50
6.1 ( 0.35
6.6 ( 0.25
7.2 ( 0.40
8.1 ( 0.35
6.5 ( 0.20
7.7 ( 0.40
a
Solution was the hydrochloride salt in the case of DZA and
neutral compounds for the other derivatives, with the pH value
shown. Decrease in IOP measured 30, 60, and 90 min after
b
administration. ∆IOP ) IOPcontrol eye - IOPtreated eye (n ) 3). c Eye
In Vitr o CA In h ibition . CA inhibition data against
three isozymes (hCA I, hCA II, and bCA IV) for the pre-
pared compounds and standard inhibitors are shown in
Table 1. All these compounds act as better CA inhibitors
than the parent sulfonamides from which they were
obtained (data not shown), the most efficient ones
including the heterocyclic sulfonamide derivatives,
such as the 1,3,4-thiadiazole-2-sulfonamides 13 and
15 and the corresponding thiadiazolines 14, as well
as the benzothiazole-2-sulfonamide derivatives 21-23.
In the aromatic sulfonamide series, compounds with
different types of activity were obtained, from moder-
ately active (derivatives of orthanilamide 1, metanil-
amide 2, sulfanilamide 3, etc.) to active (derivatives
of halogenosulfanilamides 7-10 or the benzene-1,3-
disulfonamides 12 and 13). The same pattern of inhibi-
tion has in fact been observed previously, with other
sulfonamides designed by the above-mentioned tail
strategy, and this fact has been discussed previously in
greater detail.12,13 The most important factor influencing
the biological activity of these compounds was the
nature of the acylating/sulfonylating moiety containing
the perfluoroalkyl/aryl groups. Thus, for a given sul-
fonamide, inhibitory activity generally varied in the
following manner: C6F5SO2 > C4F9SO2 = C6F5CO >
C8F17SO2 = C8F17CO > CF3SO2.
reddening was observed.
Ta ble 4. Fall of IOP of Glaucomatous Rabbits (31.9 ( 3.0
mmHg) after Treatment with 1 drop (50 µL) of a 2% Solution of
CA Inhibitor Directly into the Eyea
∆IOP ( SEb (mmHg)
inhibitor
pH
30 min
60 min
90 min
DZAc
C13
C14
D14
5.5
7.5
6.5
7.0
3.6 ( 0.20
8.3 ( 0.30
7.1 ( 0.25
6.8 ( 0.30
6.7 ( 0.30
15.7 ( 0.25
14.2 ( 0.30
13.0 ( 0.45
4.2 ( 0.15
17.4 ( 0.30
16.9 ( 0.40
15.5 ( 0.30
a
Solution had the pH value shown. Decrease in IOP measured
b
30, 60, and 90 min after administration. ∆IOP ) IOPcontrol eye
IOPtreated
-
(n ) 3). c As hydrochloride salt.
eye
this group,12,13,18,23 and were applied for the synthesis
of derivatives (A-F )1-25 described in the present
paper. Generally, in the aromatic series, best results
were obtained working in anhydrous pyridine as sol-
vent, for both acylation and alkyl/arylsulfonylation
reactions, whereas in the heterocyclic sulfonamides
series things were more complicated. Thus, Schotten-
Baumann conditions were efficient only when working
with aromatic sulfonyl/acyl halides (case in which the
reactions performed in pyridine led to formation of tar
and small yields in the desired acylated/arylsulfonylated
compounds). Conversely, aliphatic sulfonyl/acyl halides
reacted better in anhydrous pyridine than in Schotten-
Baumann conditions. In the present study both these
methods have been used, together with a new procedure
Among the three CA isozymes investigated here, the
most susceptible to inhibition was hCA II, followed by
bCA IV and then hCA I. This is of great importance,

Carbonic Anhydrase Inhibitors
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 23 4547
since the sensitive isozymes are just those involved
in aqueous humor secretion within the eye, i.e., CA II
and CA IV.
min, which was in the range of 7.3-12.5 mmHg. At later
times after the topical administration their effect was
much more pronounced than that of the clinically used
drug (for instance at 90 min the IOP lowering was in
the range of 4.9-10.2 mmHg with derivatives such as
A14, B13,14, C13-15, D13,14, etc.; see Table 3) and
in several cases the return at the basal IOP occurred
after 5-6 h post-administration, in contrast to DZA for
which the return occurred after 3 h (Figure 1). This fact
might be extremely important for the pharmacology of
a putative drug of the class described here, as it would
imply administration of the eye drops only two times
daily, in contrast to DZA which was initially adminis-
tered four times a day and then three times daily (which
remains the mostly used administration schedule pres-
ently).7
Quantitative detailed measurements of water solubil-
ity, log P, and corneal accession rates were performed
for several compounds reported here: A14, B13, C13,14,
D16, E16, F 13,14. An important factor influencing the
pharmacological properties of a topical CA inhibitor is
represented by its water solubility. The presence of
perfluoroalkyl/arylsulfonyl moieties in the compounds
reported here led to highly increased water solubility
as compared to that of the starting sulfonamide (data
not shown) from which it was prepared. Inhibitors
containing the perfluoroalkyl/arylcarbonyl moiety
were slightly less hydrosoluble than the corresponding
sulfonylated ones, but they possessed good enough
solubility for their ocular topical application. As seen
from the data of Table 2, the newly obtained compounds
also possess moderate lipid solubility, similar to or
slightly better than that of DRZ, and this is also of great
importance for the topical antiglaucoma effect. In fact,
Maren4 noted in his classical review that one of the
conditions needed for a sulfonamide to act as an effective
IOP-lowering agent is to possess (modest) lipid solubility
(attributable to its un-ionized form), accompanied by
good water solubility (eventually conferred by the pres-
ence of ionizable groups of appropriate pKa). As seen
from data of Table 2, the compounds reported here
possessed just this type of balanced physicochemical
properties. The very good hydrosolubility of these per-
fluoro derivatives is rather surprising (considering their
liposolubility), but a similar case was also encountered
by Maren’s group for analogues of MZA possessing
longer aliphatic chains instead of the acetyl moiety, with
the propionyl derivative 3 times more soluble than the
acetyl one. The accession rates across the cornea of the
new compounds investigated here were of the same
order of magnitude (or slightly better) as those of DZA,
as a consequence of their favorable physicochemical
properties mentioned above. Thus, 2% water solutions
of some of these new inhibitors could be obtained
without any problems, and these solutions were stable
for months at room temperature, with no hydrolysis of
the sulfonylated compound detected by means of TLC
or HPLC (data not shown). Thus, in contrast to trifluoro-
methazolamide 26, the compounds reported here are
chemically stable in water solutions.
The same powerful IOP lowering after administration
of perfluorosulfonamide derivatives has been observed
in glaucomatous rabbits (Table 4). In this case, at 30
min after topical administration IOP was lowered with
6.8-8.3 mmHg by the new sulfonamides (with DZA the
IOP lowering was of 3.6 mmHg); at 60 min the IOP
lowering with the first derivatives mentioned above was
in the range of 13.0-15.7 mmHg (6.7 with DZA),
whereas at 90 min it was 15.5-17.4 mmHg (versus 4.2
mmHg with DZA). We stress again that the pH of all
solutions of the new inhibitors described here, used in
the in vivo experiments, were in the range of 6.5-7.5
pH units and that no eye irritation has been observed
(except for a benzothiazole derivative, C21, in which
probably an allergic reaction occurred, as reported for
similar benzothiazole- or benzothiophenesulfonamides
by the Merck group).25 In fact it has been established
that such electrophilic sulfonamides undergo a nucleo-
philic substitution reaction of the sulfonamido group by
reduced glutathione in vivo, which leads to the above-
mentioned allergy, which in our case has been observed
only with the benzothiazole derivative C21 but not with
other perfluorosulfonamides. Thus, the prerequisites
that solutions of such perfluorosulfonamides will pro-
voke less (or no) eye irritation as compared to the very
acidic DZA solutions are met, as one may expect from
these in vivo data in experimental animals.
Dr u g Distr ibu tion in Ocu la r F lu id s a n d Tissu es.
In Table 5 the drug distribution in ocular fluids and
tissues is shown, after the topical administration of
compounds C13,14. It is seen that 1 and 2 h after topical
administration of the drug, high levels of inhibitors were
found in the cornea, aqueous humor, and ciliary pro-
cesses. Based on the inhibition constant of these com-
pounds, the fractional inhibition estimated in these
tissues/fluids is of 99.5-99.9%,4 proving the fact that
the IOP decrease is indeed due to CA inhibition.
Furthermore, as seen from the data of Table 5, the new
compounds reported here tend to concentrate in the
aqueous humor (concentrations of around 295-325 µM
were detected 1 h after administration), whereas DZA
reaches much lower concentrations (32 µM after 1 h).
High concentrations of the inhibitor were maintained
2 h after administration too. Concentrations of the new
compounds C13,14 in the cornea and ciliary processes
are also enhanced as compared to those of DZA, but the
differences are not so dramatic as those from the
aqueous humor. Thus, the strong and long-lasting IOP-
IOP Low er in g in Nor m oten sive a n d Gla u com a -
tou s Ra bbits. In vivo IOP-lowering experiments were
done in normotensive and glaucomatous rabbits, with
several of the new compounds (A14, B13,14, C13-
15,21, D13,14, F 13,14, etc.) which were among the
strongest hCA II and bCA IV inhibitors in the obtained
series. From data of Table 3 it is seen that similarly to
DZA, the new perfluorosulfonamides possess strong
topical activity in lowering IOP in normotensive rabbits.
IOP lowering observed with some of the most active CA
inhibitors reported here was much more accentuated
than that with the standard, clinically used inhibitor
DZA. Thus, DZA reaches a maximal IOP lowering at
60 min postadministration, which is of 4.1 mmHg, but
after 90 min, this effect is further reduced to an IOP
lowering of only 2.7 mmHg. The compounds reported
here also showed their maximal IOP lowering after 60

4548 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 23
Scozzafava et al.
Gen er a l P r oced u r e for P r ep a r a tion of P er flu or in a ted
Com p ou n d s. Meth od A (Sch otten -Ba u m a n n syn th esis):
5 mmol of sulfonamide 1-25 (such as 5-amino-1,3,4-thiadia-
zole-2-sulfonamide 13 or 5-imino-4-methyl-2-sulfonamido-δ2-
1,3,4-thiadiazoline 14) was dissolved in a 15-mL solution of
2.5 M NaOH and cooled to 2-5 °C in a salt-ice bath. 5 mmol
of sulfonyl/acyl chloride A-F was added in small portions,
concomitantly with 10 mL of a 2 M NaOH solution, maintain-
ing the temperature under 10 °C. The reaction mixture was
then stirred at room temperature for 5-10 h (TLC control),
then the pH was adjusted to 2 with 5 N HCl, and the
precipitated sulfonamides were filtered and recrystallized from
aqueous ethanol.
Meth od B: As above, but pyridine was used as solvent and
no other base was necessary. After the reaction was performed,
the excess pyridine was evaporated in vacuo, the reaction
mixture was poured in 50 mL of ice + water, and the
precipitated sulfonamides were recrystallized as described
above or purified by preparative HPLC.
Meth od C: The sulfonamide to be derivatized was sus-
pended/dissolved in a 1:1 mixture of acetone-water and the
stoichiometric amounts of sulfonyl chloride and base were
added concomitantly. The used base was NaOH, NaHCO3,
Et3N, pyridine, etc. Good results were generally obtained when
working with sodium bicarbonate as base. The reaction
mixture was magnetically stirred at room temperature for
several hours, then the solvent evaporated and the reaction
products recrystallized as described above.
4-Tr iflu or om eth a n esu lfon a m id oben zen esu lfon a m id e,
A3: white crystals, mp 290-1 °C dec; IR (KBr) cm-1 1152
and 1176 (SO2sym), 1345 and 1382 (SO2as), 3360 (NH, NH2);
1H NMR (DMSO-d6) δ 7.33 (s, 2H, SO2NH2), 7.80 (d, 2H,
AA′BB′, 8.9), 7.95 (d, 2H, AA′BB′, 8.9), 8.15 (br s, SO2NH);
13C NMR (DMSO-d6) δ 45.8 (CF3), 126.67 (C2/C3-Ph), 128.26
(C3/C2-Ph), 139.49 (C1/C4-Ph), 141.80 (C4/C1-Ph). Anal.
(C7H7F3N3O4S2) C, H, N.
lowering properties of the new compounds reported here
are probably due to this concentrating effect reached
mainly in the aqueous humor, but which is also present
in the cornea and ciliary processes. The mechanism by
which such high concentrations of active compounds
reach these tissues is unknown at the moment.
Con clu sion s
We report here a novel class of water-soluble, topically
very effective antiglaucoma sulfonamides, obtained by
attaching perfluoroalkyl/arylsulfonyl or perfluoroalkyl/
arylcarbonyl moieties to well-known aromatic/hetero-
cyclic sulfonamides incorporating free amino, imino,
hydrazino, or hydroxy groups. Ring systems which have
been derivatized by the above-mentioned procedures
included: 2-, 3-, or 4-aminobenzenesulfonamides, 4-(ω-
aminoalkyl)benzenesulfonamides, 3-halogeno-substituted-
sulfanilamides, 1,3-benzenedisulfonamides, 1,3,4-thia-
diazole-2-sulfonamides, benzothiazole-2-sulfonamides,
and thienothiopyran-2-sulfonamides, among others. Many
of the newly reported derivatives showed very good
water solubility, in the range of 2-5% at neutral pH
values, whereas their lipid solubility, hydrophobicity
(log P), and accession rates across the cornea were those
appropriate for acting as efficient topical IOP-lowering
agents. Many such inhibitors possessed affinities in the
nanomolar range for isozymes hCA II and bCA IV,
acting as effective enzyme inhibitors in vitro. Some of
the most active inhibitors strongly lowered IOP in
normotensive and glaucomatous rabbits, showing a
prolonged duration of action as compared to DZA. The
new compounds reported here might lead to the devel-
opment of more efficient antiglaucoma drugs from the
class of sulfonamide CA inhibitors.
4-(P er flu or obu tylsu lfon yla m id oeth yl)ben zen esu lfon -
a m id e, B6: white crystals, mp 234-6 °C; IR (KBr) cm-1 1158
and 1169 (SO2sym), 1355 and 1374 (SO2as), 3365 (NH, NH2);
1H NMR (DMSO-d6) δ 2.91 (t, 2H, 7.2, RCH2), 3.49 (q, 2H, 6.4,
âCH2), 7.30 (s, 2H, SO2NH2), 7.44 (d, 2H, AA′BB′, 8.3), 7.78
(d, 2H, AA′BB′, 8.1), 8.19 (br s, SO2NH); 13C NMR (DMSO-d6)
δ 34.80 (CH2-Ph), 40.28 (N-CH2), 127.99 (C2/C3-Ph), 129.13
(C3/C2-Ph), 139.90 (C1/C4-Ph), 143.73 (C4/C1-Ph). Anal.
(C12H11F9N2O4S2) C, H, N.
Exp er im en ta l Section
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 as solvent, with
a Bruker CPX200 or Varian 300 instrument. Chemical shifts
are reported as δ values, relative to Me4Si as internal
standard. Elemental analyses were done by combustion for C,
H, N with an automated Carlo Erba analyzer and were (0.4%
of the theoretical values. All reactions were monitored by thin-
layer chromatography (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 1-25 used
in synthesis were either commercially available compounds
(from Sigma-Aldrich or Acros) or were prepared as described
previously: 4-hydrazinobenzenesulfonamide 4 by diazotization
of sulfanilamide followed by reduction of the diazonium salt
with tin(II) chloride;26 halogenosulfanilamides 9-12 by halo-
genation of sulfanilamide as reported in the literature;27
compound 15 from 5-amino-1,3,4-thiadiazole-2-sulfonamide
(obtained from AAZ)28 by acylation with the phthalimido
derivative of â-alanine, followed by hydrazinolysis;29 whereas
imine 14 by deprotection of MZA with concentrated hydro-
chloric acid.30 The benzothiazole-2-sulfonamide derivatives
18-20 were prepared as described in ref 31, whereas the
alcohols 21 and 22 from the corresponding amines by diazo-
tization followed by hydrolysis of the diazonium salts. DZA
was prepared as described in the literature.32 Perfluorosulfonyl
halides and perfluoroacyl halides were from Sigma-Aldrich.
Acetonitrile (E. Merck) or other solvents used in the synthesis
were doubly distilled and kept on molecular sieves in order to
maintain them in anhydrous conditions.
5-P er flu or op h en ylsu lfon yla m id o-1,3,4-t h ia d ia zole-2-
su lfon a m id e, C13: white crystals, mp 260-2 °C; IR
(KBr) cm-1 1151 and 1180 (SO2sym), 1363 and 1376 (SO2as),
3060 (NH), 3365 (NH2); 1H NMR (DMSO-d6) δ 7.25 (br s, 2H,
SO2NH2), 9.15 (br s, 1H, SO2NH); 13C NMR (DMSO-d6) δ
124.8, 127.9, 138.6, 159.5 (C-2 of thiadiazole), 170.0 (C-5 of
thiadiazole); 19F NMR (DMSO-d6) δ -134.0 (2,6-F2), -143.7
(4-F), -158.9 (3,5-F2). Anal. (C8H3F5N4O4S3) C, H, N.
4-Meth yl-5-p er flu or op h en ylca r boxim id o-δ2-1,3,4-th ia -
d ia zolin e-2-su lfon a m id e, F 14: white crystals, mp 243-4 °C;
IR (KBr) cm-1 1146 (SO2sym), 1375 (SO2as), 1550 (amide II),
1
1610 (amide I); 3060 (NH), 3365 (NH2); H NMR (DMSO-d6)
δ 3.54 (s, 3H, Me), 7.13 (br s, 2H, SO2NH2), 9.15 (br s, 1H,
SO2NH); 13C NMR (DMSO-d6) δ 31.5 (Me), 120.7, 124.3, 135.9,
164.91 (C-thiadiazoline), 164.94 (C-thiadiazoline); 19F NMR
(DMSO-d6) δ -137.8 (2,6-F2), -149.1 (4-F), -158.2 (3,5-F2).
Anal. (C10H5F5N4O3S2) C, H, N.
CA In h ibition . Human CA I and CA II cDNAs were ex-
pressed in Escherichia coli strain BL21 (DE3) from the plas-
mids pACA/hCA I and pACA/hCA II described by Lindskog’s
group.33 Cell growth conditions were those described in ref
34 and enzymes were purified by affinity chromatography
according to the method of Khalifah et al.35 Enzyme concentra-
tions were determined spectrophotometrically 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, based on Mr ) 28.85 kDa for CA I
and 29.3 kDa for CA II.36,37 bCA IV was isolated from bovine
lung microsomes as described by Maren et al., and its
concentration has been determined by titration with EZA.38

Carbonic Anhydrase Inhibitors
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 23 4549
Initial rates of 4-nitrophenylacetate hydrolysis catalyzed by
different CA isozymes were monitored spectrophotometrically,
at 400 nm, with a Cary 3 instrument interfaced with an
IBM-compatible PC.39 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, in the conditions of the
experiments (pH 7.40), as reported in the literature.39 Non-
enzymatic 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.39 Enzyme concentrations were
3.3 nM for CA II, 10 nM for CA I, and 34 nM for CA IV (this
isozyme has a decreased esterase activity40 and higher con-
centrations had to be used for the measurements). 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 conformed to the As-
sociation 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 temper-
ature-controlled room, at 22-26 °C. Solutions of inhibitors (2%,
by weight, as hydrochlorides, triflates, or sodium carboxylates)
were obtained in distilled-deionized water. The pH of these
solutions was in the range of 5.5-8.4.
Mea su r em en t of Ton om etr ic IOP . IOP was measured
using a Digilab 30R pneumatonometer (BioRad, Cambridge,
MA) as described by Maren’s group.41,42 The pressure readings
were matched with two-point standard pressure measure-
ments 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 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 drug was administered to only one eye, leaving
the contralateral eye as an untreated control. The ocular
hypotensive activity is expressed as the average difference in
IOP between the treated and control eye, in this way minimiz-
ing the diurnal, seasonal, and interindividual variations
commonly observed in the rabbit.41,42 All data are expressed
as mean ( SE, using a one-tailed t-test.
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 drug from the E‚I
complex), diluted and then incubated with a known amount
of enzyme. The activity of the free enzyme and the activity in
the presence of inhibitor were determined as described above.
A calibration curve was used in order to determine the
fractional inhibition in the different tissues, as described in
refs 41, 42.
Deter m in a tion of Wa ter (Bu ffer ) Solu bility. A standard
solution was prepared by dissolving a precisely weighted
amount (generally 1 mg) of inhibitor in 10 mL of methanol.
The UV absorption maximum of each compound was deter-
mined (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 scanned by UV at the
wavelength of the absorption maximum previously deter-
mined. Total solubility was determined by the relationship:
C′ ) A′C/A
where C ) concentration of standard solution (mg/mL), A )
absorbance of standard solution, A′ ) absorbance of the
saturated solution, C′ ) concentration of the saturated solution
(mg/mL).18
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.18
Tr a n scor n ea l P en etr a tion of Dr u gs. The method of
Maren et al.9 with the modifications of Pierce’s group44,45 (for
the HPLC assay of sulfonamides) was used. Excised rabbit
corneas with either intact or denuded epithelium were used
in these experiments. The pH was 7.4 and exposed area was
1.2 cm2. Concentrations of drug of 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.44,45 or
enzymatically.9 The results of the drug analyses were used to
calculate the rate constant of transfer across the cornea (kin).
As described by Pierce,44,45 this value was determined by using
the formula:
kin (×103 h-1) ) [drug]endo/[drug]epi × 60/t × 1000
where [drug]endo ) concentration of drug on endothelial side,
[drug]epi ) concentration of drug on epithelial side, t ) time
(in min).
Ack n ow led gm en t. This research was financed in
part by EU Grant ERB CIPDCT 940051. The financial
support of the Italian CNR-Target Project Biotechnol-
ogy is also gratefully acknowledged. Thanks are ad-
dressed to Drs. M. A. Ilies and M. Barboiu for expert
technical assistance.
Ocular hypertension was elicited in the right eye of albino
rabbits by the injection of R-chymotrypsin (from Sigma) as
described by Melena et al.43 The IOP of operated animals was
checked after approximately 4 weeks, and animals with an
elevated pressure of 30-35 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 was followed.41,42 The
animals were killed with an intracardiac injection. Aqueous
humor (both posterior and anterior chamber fluids) was
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 was 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
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and their therapeutic potential. Exp. Opin. Ther. Patents 2000,
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(2) Supuran, C. T. Carbonic anhydrase inhibitors. In Carbonic
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(3) Maren, T. H. The links among biochemistry, physiology and
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4550 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 23
Scozzafava et al.
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