Carbonic anhydrase inhibitors: Water-soluble 4-sulfamoylphenylthioureas as topical intraocular pressure-lowering agents with long-lasting effects

Article abstract of DOI:10.1021/jm001051+

A series of sulfonamides has been obtained by reaction of 4-isothiocyanatobenzenesulfonamide with amines, amino acids, and oligopeptides. The new thiourea derivatives showed strong affinities toward isozymes I, II, and IV of carbonic anhydrase (CA, EC 4.2.1.1). In vitro inhibitory power was good (in the low-nanomolar range) for the derivatives of β-phenylserine and α-proportionalto-phenylglycine, for those incorporating hydroxy and mercapto amino acids (Ser, Thr, Cys, Met), hydrophobic amino acids (Val, Leu, Ile), aromatic amino acids (Phe, His, Trp, Tyr, DOPA), and dicarboxylic amino acids as well as di/tri/tetrapeptides among others. Such CA inhibitors displayed very good water solubility (in the range of 2-3%) mainly as sodium (carboxylate) salts, with pH values of the obtained solutions being 6.5-7.0. Some of these preparations (such as the derivatives of Ser, β-Ph-Ser, Leu, Asn, etc.) strongly lowered intraocular pressure (IOP) when applied topically, directly into the normotensive/glaucomatous rabbit eye, as 2% water solutions. It is interesting to note that not all the powerful CA inhibitors designed in the present study showed topical IOP-lowering effects (such as, for instance, the Cys and Lys derivatives, devoid of such properties) whereas the Pro, Arg, and oligopeptidyl thiourea derivatives showed reduced efficacy when administered topically. This may be due to the very hydrophilic nature of some of these compounds, whereas inhibitors with balanced hydro- and liposolubility also showed optimal in vivo effects. The interesting pharmacological properties of this new type of CA inhibitors, correlated with the neutral pH of their solutions used in ophthalmologic applications, make them attractive candidates for developing novel antiglaucoma drugs devoid of major ocular side effects.

Full text of DOI:10.1021/jm001051+

4884
J . Med. Chem. 2000, 43, 4884-4892
Ca r bon ic An h yd r a se In h ibitor s: Wa ter -Solu ble 4-Su lfa m oylp h en ylth iou r ea s a s
Top ica l In tr a ocu la r P r essu r e-Low er in g Agen ts w ith Lon g-La stin g Effects
Angela Casini,† Andrea Scozzafava,† Francesco Mincione,‡ Luca Menabuoni,§ Marc A. Ilies,| and
Claudiu T. Supuran*,†
Laboratorio di Chimica Inorganica e Bioinorganica, Universita` degli Studi di Firenze, Via Gino Capponi 7, I-50121 Florence,
Italy, Istituto Oculistico, Universita` degli Studi di Firenze, Viale Morgagni 85, I-50134 Florence, Italy, Ospedale San Giovanni
di Dio, S.A. Oculistica, Via Torregalli 3, I-50123 Florence, Italy, and Department of Chemistry, Faculty of Biotechnologies,
University of Agricultural Sciences and Veterinary Medicine, B-dul Marasti nr. 59, 71331 Bucharest, Romania
Received August 4, 2000
A series of sulfonamides has been obtained by reaction of 4-isothiocyanatobenzenesulfonamide
with amines, amino acids, and oligopeptides. The new thiourea derivatives showed strong
affinities toward isozymes I, II, and IV of carbonic anhydrase (CA, EC 4.2.1.1). In vitro inhibitory
power was good (in the low-nanomolar range) for the derivatives of â-phenylserine and
R-phenylglycine, for those incorporating hydroxy and mercapto amino acids (Ser, Thr, Cys,
Met), hydrophobic amino acids (Val, Leu, Ile), aromatic amino acids (Phe, His, Trp, Tyr, DOPA),
and dicarboxylic amino acids as well as di/tri/tetrapeptides among others. Such CA inhibitors
displayed very good water solubility (in the range of 2-3%) mainly as sodium (carboxylate)
salts, with pH values of the obtained solutions being 6.5-7.0. Some of these preparations (such
as the derivatives of Ser, â-Ph-Ser, Leu, Asn, etc.) strongly lowered intraocular pressure (IOP)
when applied topically, directly into the normotensive/glaucomatous rabbit eye, as 2% water
solutions. It is interesting to note that not all the powerful CA inhibitors designed in the present
study showed topical IOP-lowering effects (such as, for instance, the Cys and Lys derivatives,
devoid of such properties) whereas the Pro, Arg, and oligopeptidyl thiourea derivatives showed
reduced efficacy when administered topically. This may be due to the very hydrophilic nature
of some of these compounds, whereas inhibitors with balanced hydro- and liposolubility also
showed optimal in vivo effects. The interesting pharmacological properties of this new type of
CA inhibitors, correlated with the neutral pH of their solutions used in ophthalmologic
applications, make them attractive candidates for developing novel antiglaucoma drugs devoid
of major ocular side effects.
In tr od u ction
Sulfonamide inhibitors of the zinc enzyme carbonic
anhydrase (CA, EC 4.2.1.1) are extensively used in
clinical medicine and as diagnostic tools, their main
applications being in the treatment of glaucoma and
macular edema.1-3 Several such drugs are presently
available, such as the recently introduced topical sul-
fonamides dorzolamide (1) and brinzolamide (2), in
addition to the classical, systemically acting inhibitors
acetazolamide (3), methazolamide (4), ethoxzolamide
(5), and dichlorophenamide (6), which have been em-
ployed clinically for more than 45 years.1-3
2 show a much more diminished number of side effects,
together with an efficient reduction of intraocular
pressure (IOP), when used alone or in combination with
other drugs, such as prostaglandin analogues, â-block-
ers, etc., due to inhibition of the enzymes (mainly
isozymes CA II and CA IV)1-3 present in the ciliary
processes, without appreciable inhibition of CAs from
other tissues/organs.1-4 These two sulfonamides 1 and
2 are both administered as hydrochloride salts, and this
leads to frequent stinging sensations, burning or red-
dening of the eye, blurred vision, pruritus, and other
local side effects. To reduce such inconveniences, many
tentatives were ultimately reported to design topical
antiglaucoma sulfonamides devoid of the above-men-
tioned side effects.5-8 Among them, one approach con-
sisted in attaching water-solubilizing “tails” to the
Systemic inhibitors possess many undesired side
effects due to inhibition of several CA isozymes (14 are
presently known in higher vertebrates)1 in tissues other
than the target one, i.e., the eye (more precisely, the
ciliary processes of the eye), and many agents such as
3-6 are presently used more in physiological studies
or as diagnostic tools than as antiglaucoma drugs.1 On
the other hand, the two topically acting inhibitors 1 and
* To whom correspondence should be addressed. Fax: +39-055-
2757555. Tel: +39-055-2757551. E-mail: cts@bio.chim.unifi.it.
† Laboratorio di Chimica Inorganica e Bioinorganica, Universita`
degli Studi di Firenze.
‡ Istituto Oculistico, Universita` degli Studi di Firenze.
§ S.A. Oculistica.
| University of Agricultural Sciences and Veterinary Medicine.
10.1021/jm001051+ CCC: $19.00 © 2000 American Chemical Society
Published on Web 11/10/2000

Water-Soluble 4-Sulfamoylphenylthioureas
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25 4885
molecules of aromatic/heterocyclic sulfonamides pos-
sessing amino, imino, or hydroxy moieties in their
molecules.6,7 Such tails included pyridinecarboximido,
carboxypyridinecarboxamido, quinolinesulfonamido, pi-
colinoyl, and isonicotinoyl, as well as amino acyl groups
among others, whereas ring systems which have been
derivatized by this procedure included 2-, 3-, or 4-ami-
nobenzenesulfonamides, 4-(ω-aminoalkyl)benzenesulfona-
mides, 3-halogenosubstituted-sulfanilamides, 1,3-ben-
zenedisulfonamides, 1,3,4-thiadiazole-2-sulfonamides,
and benzothiazole-2-sulfonamides, as well as thienothi-
opyran-2-sulfonamides among others, and had as the
main objective the possibility to formulate the eye drop
solutions at pH values close to neutrality.6-8
Continuing our previous research in this field,6-8 we
report here an alternative approach for obtaining water-
soluble, strong CA inhibitors with putative applications
as agents used in the treatment of ocular hypertension,
with potentially no side effects due to the acidic pH of
the eye drop solution. Thus, 4-isothiocyanatobenzene-
sulfonamide was reacted with amines, amino acids, or
oligopeptides, and the thioureas obtained in this way
showed excellent water solubilities either as sodium
salts (for the amino acid/oligopeptide derivatives) or as
hydrochlorides/triflates (in the case of the amine deriva-
tives). The nucleophiles used in the above-mentioned
syntheses were chosen in such a way as to possess pKa
values in the “physiological” range. More precisely, salts
of these new inhibitors applied into the eye of the
experimental animals generally possessed pH values in
the range 6.5-7.0. Such salts were applied topically into
the eye of normotensive or glaucomatous rabbits and
produced a powerful, long-lasting reduction of IOP.
CA In h ibitor y Activity. The new sulfonamides A7-
A47 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
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 relevant for their pharmacological
activity, such as buffer solubility or chloroform-buffer
partition coefficient, are shown in Table 2. The in vitro
transcorneal accession rates (kin) of classical sulfona-
mides and topically active derivatives, such as dorzola-
mide 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 dorzolamide
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
compounds in ocular tissues and fluids after topical
administration in normotensive rabbits are shown in
Table 5.
Resu lts
Syn th esis. A large number of derivatives have been
prepared by reaction of 4-isothiocyanatobenzenesulfona-
mide A with amines 7-13, amino acids 14-38, or
oligopeptides 39-47.
Discu ssion
Ch em istr y. The synthesis of 4-isothiocyanatobenze-
nesulfonamide A has already been reported in 1946 by
McKee and Bost,9 in the search of more effective
antibacterial sulfonamides. Still, this highly versatile
compound has never been used for the preparation of
sulfonamides possessing CA inhibitory properties. Com-
pound A has been obtained by the reported literature
procedure,9 from sulfanilamide B and thiophosgene, and
was subsequently reacted with a large number of
amines, amino acids, and oligopeptides, of type 7-47,
leading to the new thioureas A7-A47 (Scheme 1).9,10
The reaction generally proceeded in very good yields
and without the formation of side products mainly for
amines and simple amino acids. Still, in the case of
oligopeptides (such as 39-47) used as nucleophiles in
these syntheses, a lot of tar was formed during the
reaction, and pure compounds A39-A47 could only be
obtained after preparative HPLC.
The newly obtained derivatives will be numbered as
A7-A47, denoting that the 4-H2NO2S-C6H4-NHCSNH-
group is attached to the corresponding moiety of the
starting nucleophile 7-47. For instance, A9 is the
thiourea obtained by the reaction of phenethylamine 9
with 4-isothiocyanatobenzenesulfonamide A; A14 is the
thiourea obtained by reaction of glycine 14 with 4-isothio-
cyanatobenzenesulfonamide A; A40 is the thiourea
obtained by reaction of carnosine 40 with 4-isothiocy-
anatobenzenesulfonamide A; etc.
The nucleophiles used for the preparation of the new
compounds reported here (7-47) were chosen in such

4886 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25
Casini et al.
Ta ble 1. CA Inhibition Data with Standard Inhibitors 1-6, 4-Isothiocyanatobenzenesulfonamide A, and New Sulfonamides A7-A47
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
dorzolamide, 1
acetazolamide, 3
methazolamide, 4
ethoxzolamide, 5
dichlorophenamide, 6
A
A7
A8
50000
900
780
25
1200
5000
135
124
125
92
9
12
14
8
43
220
240
13
380
300
76
77
75
56
29
A17
A18
A19
A20
A21
A22
A23
A24
A25
A26
A27
A28
A29
A30
A31
A32
45
40
28
24
30
25
27
23
21
22
35
27
38
40
110
32
11
6
3
2
4
5
4
5
5
4
8
6
10
9
33
15
15
9
A33
A34
A35
A36
A37
A38
A39
A40
A41
A42
A43
A44
A45
A46
A47
30
26
35
47
97
115
54
23
13
21
23
20
50
20
59
6
5
7
11
12
15
15
3
1
3
4
5
13
13
18
24
55
61
32
10
6
38
185
45
40
42
33
13
18
16
20
12
11
16
10
12
13
12
10
17
11
19
23
68
15
A9
A10
A11
A12
A13
A14
A15
A16
9
55
50
59
62
54
47
13
13
32
11
27
36
40
39
36
15
3
10
25
5
35
a
b
Human (cloned) isozymes. From bovine lung microsomes, by the esterase method.
Ta ble 2. Solubility, Chloroform-Buffer Partition Coefficients,
and in Vitro Corneal Permeability of Some Classical and New
Sulfonamide CA Inhibitors
Ta ble 3. Fall of IOP of Normotensive Rabbits (19.5 ( 2.1
mmHg)a
∆IOP ( SEb (mmHg)
c
kin × 103 (h-1
)
inhibitor
pH
30 min
60 min
90 min
solubilitya
(mM)
cornea
no
dorzolamide
A7
5.5
6.5
6.5
7.0
7.0
7.0
7.0
7.5
7.0
7.0
7.0
7.0
7.0
7.0
7.0
7.0
7.0
7.0
7.0
7.0
7.0
7.0
2.2 ( 0.15
0.8 ( 0.10
3.0 (0.15
4.3 (0.15
3.0 ( 0.20
4.0 ( 0.10
5.0 ( 0.25
7.0 (0.15
8.0 (0.15
9.0 ( 0.20
0.0
5.2 (0.25
2.0 ( 0.10
2.0 ( 0.15
7.0 ( 0.35
0.0
4.1 ( 0.15
4.0 ( 0.15
0.3 ( 0.25
8.0 ( 0.25
5.9 ( 0.20
5.0 ( 0.30
3.1 ( 0.15
2.3 ( 0.30
9.5 ( 0.20
10.0 ( 0.30
0.0
6.3 ( 0.30
4.0 ( 0.20
2.6 ( 0.25
10.1 ( 0.25
1.0 ( 0.25
4.6 ( 0.30
4.1 ( 0.20
0.0
2.7 ( 0.10
0.6 ( 0.20
0.0
compd
log Pb
intact
epithelium
dorzolamided
acetazolamide
methazolamide
ethoxzolamide
benzolamide
A18e
60
3.2
12
0.04
1.2
54
73
48
65
60
2.0e
0.001
0.06
3.0
0.37
1.90
27
0.1
5.9
3.4
4.9
0.3
0.1
3.1
2.2
5.2
7.0
13
A10
A11
A14
A15
A17
A18
A19
A20
A22
A25
A26
A27c
A28
A31
A32
A33
A37
A38
A40
A43
0.9 ( 0.10
0.5 ( 0.15
5.2 ( 0.40
2.9 ( 0.20
2.1 ( 0.10
9.1 ( 0.25
9.6 ( 0.20
0.0
6.0 ( 0.20
0.3 ( 0.15
2.0 ( 0.10
6.0 ( 0.30
1.0 ( 0.10
4.0 ( 0.20
3.0 ( 0.15
0.0
25
40
0.00001
3.652
1.459
2.314
0.09
0.05
1.759
0.725
0.4
14.3
8.0
12.3
0.7
0.8
7.8
5.0
A19e
A20e
A22e
A27e
A28e
51
52
A40e
a
b
Solubility in pH 7.40 buffer, at 25 °C. Chloroform-buffer
partition coefficient. c Determined as described in refs 28, 29. As
d
4.0 ( 0.15
4.0 ( 0.10
0.0
1.0 ( 0.15
1.0 ( 0.15
0.2 ( 0.15
hydrochloride, at pH 5.8. e As sodium salts, at pH 7.0.
a manner as to contain water-solubilizing moieties in
the presence of acids/bases, such as the pyridine or
imidazole moieties in the case of 7, 8, and 10 (hydro-
chlorides6,7 or triflates6 of A7, A8, and A10 would
presumably lead to water-soluble CA inhibitors, but the
compound unable to form such a salt - A9 - has also
been prepared for comparison and confirmed the hy-
pothesis mentioned above). Thioureas obtained from
other nucleophiles (A11-A47), on the other hand, easily
form water-soluble sodium salts due to the presence of
at least one carboxyl group in their molecules. It was
previously noted in this laboratory6a,11 that sulfona-
mides incorporating carboxylate moieties in their mol-
ecules might interact with a histidine cluster at the
entrance of the hCA II active site (this is the isozyme
that plays the most important function in aqueous
humor secretion).1-3 This cluster comprises the residues
His 64 (at the center of the active site cavity) and His 4
and His 3 (at the rim of it) as well as several residues
on the external surface of the enzyme, near the entrance
to the active site cavity (His 10, His 15, His 17; hCA I
numbering).11 All imidazolic side chains of these resi-
dues may interact (better when in charged state, as
imidazolium ions) with negatively charged inhibitors,
containing, for instance, carboxylate anions, leading in
this way to a supplementary stabilization of the enzyme-
1.2 ( 0.30
1.2 ( 0.30
0.2 ( 0.10
4.0 ( 0.20
4.0 ( 0.20
3.0 ( 0.20
a
After treatment with 1 drop (50 µL) of a 2% CA inhibitor
solution (as hydrochloride salt in the case of dorzolamide, A7, and
A10 and as sodium salts for the other derivatives, with the pH
value shown) directly into the eye at 30, 60, and 90 min after
b
administration. ∆ IOP ) IOPcontrol eye - IOPtreated eye (n ) 3). c Eye
irritation observed.
Ta ble 4. Fall of IOP of Glaucomatous Rabbits (33.5 ( 3.0
mmHg)a
∆IOP ( SEb (mmHg)
inhibitor
pH
30 min
60 min
90 min
1c
5.5
7.5
6.5
7.0
3.6 ( 0.20
10.4 ( 0.35
10.5 ( 0.25
10.0 ( 0.40
6.7 ( 0.30
15.0 ( 0.30
14.1 ( 0.35
13.0 ( 0.25
4.2 ( 0.15
17.5 ( 0.35
16.4 ( 0.20
15.5 ( 0.40
A19d
A20d
A28d
aAfter treatment with 1 drop (50 µL) of a 2% CA inhibitor
solution (with the pH value shown) directly into the eye at 30, 60,
b
and 90 min after administration. ∆IOP ) IOPcontrol
-
eye
d
IOPtreated
(n ) 3). c As hydrochloride salt. As sodium salt.
eye
inhibitor adduct.6 This is the reason so many compounds
of this type have been designed and prepared in this
study. The greatest majority of these new compounds
contain one COO- moiety (obviously, when in solution,

Water-Soluble 4-Sulfamoylphenylthioureas
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25 4887
literature.1,2,12,13 Thus, the four derivatives of aromatic/
heterocyclic amines A7-A10 (which are the most inef-
fective CA inhibitors among the new compounds re-
ported here) showed affinities of 33-45 nM against hCA
II, 56-76 nM against bCA IV, and 92-135 nM against
the slow isozyme hCA I. Increased inhibitory power was
observed for the derivatives obtained from A and
aminobenzoic acids 11-13 or amino acids 14-38. Thus,
the most active such compounds were those incorporat-
ing â-phenylserine (A20), R-phenylglycine (A18), hy-
droxy (Ser, Thr; A19-A21) and mercapto (Cys, Met;
A22, A23) amino acids, hydrophobic amino acids (Val,
Leu, Ile; A24-A26), aromatic amino acids (His, Phe,
Tyr, DOPA; A32-A35), and dicarboxylic (Asp, Glu, and
their corresponding amides Asn, Gln; A27-A30) amino
acid moieties. Such compounds possessed inhibition
constants in the range of 2-10 nM against hCA II, 9-23
nM against bCA IV, and 21-40 nM against hCA I (being
more active than the classical inhibitors of type 1-6
mentioned above, which have been used as standards
in these measurements, Table 1). Slightly less active
were, on the other hand, the derivatives of aminobenzoic
acids (A11-A13) and those of Gly, Ala, â-Ala, and
GABA (A14-A17), as well as those of Trp, Lys, and Arg
(A36-A38), with inhibition constants in the range of
11-20 nM against hCA II, 24-61 nM against bCA IV,
and 45-115 nM against hCA I (but one must mention
that these compounds have the same potency as aceta-
zolamide or methazolamide, clinically used CA inhibi-
tors). Among all the thioureas reported here, the most
ineffective CA inhibitor was the proline derivative A31,
probably due to its relatively bulky nature (this com-
pound is anyhow a strong inhibitor, comparable with
the clinically used dichlorophenamide 6). Strong inhibi-
tory properties were, on the other hand, observed for
the oligopeptidyl derivatives A39-A47. Thus, except for
the Gly-Gly (A39) and Asp-Asp (A45) derivatives (which
possess the same activity as methazolamide 4 against
hCA II), all the other oligopeptidyl thioureas prepared
in the present study were very potent inhibitors, with
inhibition constants in the range of 1-10 nM against
hCA II, 6-27 nM against bCA IV, and 13-59 nM
against hCA I. Very good inhibitory properties were
correlated with the presence of His moieties in such
oligopeptidyl thioureas (such as A40-A42). It is also
interesting to note the influence of the number of
aspartyl residues, in some of the thioureas prepared in
this study, on the CA inhibitory properties of the
obtained compounds. Thus, among the Asp, Asp-Asp,
and (Asp)4 derivatives, best activity was observed for
the simple mono derivative A27, whereas the most
ineffective inhibitor was the dipeptidyl derivative A45
(with the Ki against hCA II doubled as compared to that
for A27). On the other hand, the tetrapeptide derivative
A47 regained much of the hCA II inhibitory activity but
showed a much lower affinity for bCA IV and hCA I. It
is difficult to explain at the present moment this
intricate behavior. From all these data, the main SAR
conclusion is that best inhibitors are obtained when the
molecule of the thiourea is prolonged in the direction of
the axis passing through the Zn(II) ion of the enzyme,
the sulfonamide sulfur atom, and the thiourea nitrogen
atom of the inhibitor, as was in fact explained theoreti-
cally by QSAR studies form our group for other series
F igu r e 1. Effect of topically administered sulfonamide inhibi-
tors (2% water solutions) on the IOP of normotensive albino
rabbits: curve 1, dorzolamide (1) (hydrochloride salt, pH 5.5);
curve 2, compound A20 (sodium salt, pH 7.0); curve 3,
compound A25 (sodium salt, pH 7.0); curve 4, compound A28
(sodium salt, pH 7.0).
Ta ble 5. Ocular Tissue Concentrations after Corneal
Application of 1 drop (50 µL) of a 2% Solution of Inhibitors A20
and A28 in Albino Rabbitsa
drug concentration (µM)b
time (h)
cornea
aqueous humor
ciliary process
1 (HCl)
32 ( 3
21 ( 2
1
2
105 ( 5
39 ( 4
15 ( 3
6 ( 1
A20
1
2
163 ( 18
74 ( 5
306 ( 21
65 ( 5
53 ( 7
19 ( 2
A28
1
2
141 ( 15
54 ( 9
285 ( 14
43 ( 3
47 ( 5
12 ( 2
b
aWith dorzolamide hydrochloride 1 as standard. Mean
standard deviation (n ) 3).
(
Sch em e 1. Synthesis of 4-Isothiocyanatobenzene-
sulfonamide A and the Thioureas A7-A47
as sodium salts, at the pH at which the experiments
have been performed, i.e., 7.4 for the in vitro inhibition
measurements and 6.5-7.0 for the in vivo experiments),
but derivatives with two (A45) or four (A47) carboxylate
moieties were also obtained. As will be discussed
shortly, indeed, many such derivatives showed very good
CA inhibitory properties.
In Vitr o CA In h ibition . CA inhibition data against
three isozymes: hCA I, hCA II, and bCA IV (h ) human;
b ) bovine isozyme) for the prepared compounds and
standard inhibitors are shown in Table 1. All these
compounds showed better CA inhibitory properties as
compared to the parent sulfonamide A from which they
were obtained, which is a moderately weak inhibitor,
similarly to many benzene sulfonamides reported in the

4888 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25
Casini et al.
of sulfonamide CA inhibitors12,13 (the exceptions from
this rule are just the aspartyl derivatives mentioned
above: A27, A45, and A47). 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, since the sensitive
isozymes are only those involved in aqueous humor
secretion within the eye, i.e., CA II and CA IV.
the IOP lowering was 3.6 mmHg); at 60 min the IOP
lowering with the first derivatives mentioned above was
in the range of 13.0-15.0 mmHg (6.7 with 1), whereas
at 90 min it was of 15.5-17.5 mmHg (versus 4.2 mmHg
with 1). 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 6.5-7.0 pH units and
that no eye irritation was observed after topical admin-
istration.
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
many of the new compounds reported here, due to their
strong CA II and CA IV inhibitory properties: A7, A10,
A11, A14, A15, A17-A22, A25-A28, A31-A33, A37,
A38, A40, and A43. The following may be noted regard-
ing the data of Table 3: (i) Some of the new derivatives,
such as A19, A20, and A28, provoked a very strong and
rapid decrease of IOP when applied topically, with IOP
lowering of 7.0-9.0 mmHg at 30 min after administra-
tion (compared with 2.2 mmHg IOP lowering after
dorzolamide), which at 1 h amounted to 9.5-10.0 mmHg
(versus 4.1 mmHg of dorzolamide), and this potent effect
was maintained at 1.5 h (6.0-9.6 mmHg); furthermore,
IOP returned to baseline values 6 h after administra-
tion. Thus, such new inhibitors are much more potent
hypotensor agents, and their effect is much longer than
that of the standard drug dorzolamide (Figure 1). It is
interesting to note that two of these very effective IOP-
lowering agents mentioned above are the Ser and â-Ph-
Ser derivatives, whereas the third one is the Asn
derivative. More surprisingly, the very similar Asp
derivative (A27) is a modest IOP hypotensor agent, also
producing eye irritation when administered topically
(this was the only compound in this series that showed
this unpleasant side effect). This fact also demonstrates
that very small structural modifications (replacement
of the CONH2 moiety by the COOH one) lead to a
completely different pharmacological profile for the two
derivatives containing them, although their CA inhibi-
tory properties are rather similar. (ii) Compounds such
as A11, A14, A15, A17, A18, A32, and A33 showed an
IOP lowering of 3-7 mmHg at 30 min after administra-
tion (being more effective than dorzolamide), whereas
at 1 h their effect was in the range of 2.3-8 mmHg and
at 90 min of 0.5-5.2 mmHg. Thus, although more
effective than the standard drug at 30 min after
administration, many of these agents show a diminished
effect at later times, probably due to their washing away
from the ciliary processes. This may be due to an
improper balance between lipo- and hydrophilicity for
some of these compounds, as will be discussed shortly.
(iii) Derivatives such as A7, A10, A26, A38, A40, and
A43 showed a modest effect in the decrease of IOP after
administration, with IOP lowering of 0.2-3.0 mmHg
after 30 min, 0.2-4.0 mmHg after 60 min, and 0.0-4.0
mmHg after 90 min. (iv) Compounds such as A22, A31,
and A37 did not reduce IOP after topical administration
(or had a very modest such effect).
To this point it is important to return to the correla-
tion between the physicochemical properties of the new
CA inhibitors reported here (Table 2) and their in vivo
activity as topical hypotensor agents. One of the most
interesting observations of this work is that some of the
newly obtained compounds, although acting as very
potent hCA II (and bCA IV) inhibitors, showed an
unexpectedly small IOP-lowering effect via the topical
route, whereas structurally very similar derivatives
acted as strong hypotensors (compare A19 and A22,
which differ minimally by the presence of an OH moiety
in the first and an SH one in the second compound, or
the example mentioned above, the Asp and Asn deriva-
tives A27 and A28). Already Maren2 noted in his
classical reviews that the most restrictive conditions
needed for a sulfonamide to act as an effective IOP-
lowering agent are to possess modest (but not insignifi-
cant) lipid solubility (attributable to its un-ionized form)
accompanied by good water solubility (eventually con-
ferred by the presence of ionizable groups of appropriate
pKa). As seen from data of Table 2, the very active
compounds reported here that act as effective topical
hypotensors, such as A19, A20, and A28, possessed just
this type of balanced physicochemical properties, which
led to good accession rates across the cornea followed
by sustained inhibition of the ciliary processes enzyme.
In contrast, the Cys (A22) or Asp (A27) derivatives
mentioned above, although effective CA inhibitors and
possessing very good water solubility, possess too low
hydrophobicity (due to the presence of the second
ionizable moiety, SH in the first case and COOH in the
second one) and as a consequence an impaired penetra-
tion through the cornea (observe the low kin values of
Table 2, similar to those of benzolamide 48). The same
seems to be true for other derivatives investigated here,
such as the Lys, Arg, or oligopeptidyl derivatives (A37,
A38, A40, and A43), which again showed relatively
modest IOP lowering, although they were extremely
efficient in vitro CA inhibitors. One must also mention
that a very hydrophobic inhibitor (such as ethoxzola-
mide 6) is also ineffective topically, although it possesses
very high accession rates through the cornea. Still, this
property also allows its rapid diffusibility into red blood
cells, and in consequence its washing away from the
ciliary processes due to the blood circulation. This might
explain why some of the compounds reported here (such
as the R-Ph-Gly derivative A18 or the GABA derivative
A17) initially showed a potent IOP lowering (at 30 min
after administration), whereas this effect rapidly di-
minished at later periods after administration. One may
The same powerful IOP lowering after administration
of some derivatives prepared in the present study, such
as A19, A20, and A28, has been observed in glaucoma-
tous rabbits (Table 4). In this case, at 30 min after
topical administration pressure was lowered with 10.0-
10.4 mmHg by the new sulfonamides (with dorzolamide

Water-Soluble 4-Sulfamoylphenylthioureas
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25 4889
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. Sulfanilamide, thiophosgene,
and nucleophiles (amines, amino acids, and oligopeptides) used
in the synthesis were of highest purity, commercially available
compounds (from Sigma-Aldrich, Fluka, E. Merck or Acros).
Acetonitrile, acetone (E. Merck) and other solvents used in the
synthesis were doubly distilled and kept on molecular sieves
in order to maintain them in anhydrous conditions.
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
A7-A47. An amount of 0.53 g (2.5 mmol) of 4-isothiocyana-
tobenzenesulfonamide A9 and the stoichiometric amount of
nucleophile 7-47 were suspended in 50-100 mL of dry
acetone or acetonitrile and heated at reflux for 2-8 h (TLC
control). The solvent was evaporated, and the crude product
either recrystallized from ethanol or ethanol-water (for the
largest majority of thioureas described here) or purified by
preparative HPLC in the case when the reaction mixture
contained appreciable amounts of impurities (as evidenced by
TLC). This was particularly the case for the oligopeptidyl
thioureas A39-A47. Conditions used for the purification
were: C18 reversed-phase µ-Bondapack or Dynamax-60A (25
× 250 mm) preparative columns; 90% acetonitrile/8% methanol/
2% water, 30 mL/min.
observe that A18 was the most hydrophobic among the
inhibitors investigated in some detail in this study
(Table 2), and this probably leads not only to very good
accession rates across the cornea but also to its rapid
clearing from the ciliary processes, similarly to ethox-
zolamide (6) mentioned above. Thus, this series of CA
inhibitors proves in a clear-cut manner that in order to
obtain hypotensors with prolonged duration of action
via the topical route, the physicochemical and enzyme
inhibitory properties must be fine-tuned in a very
precise manner, and such compounds should possess a
proper balance between lipo- and hydrophobicity, in
addition to acting as potent hCA II inhibitors and
possessing acceptable water solubility.
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 A20 and A28. It is seen that 1 and 2 h after
topical administration of the drug, high levels of inhibi-
tors 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/fluids is of 99.5-99.9%,2 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 285-305 µM
were detected 1 h after administration), whereas dor-
zolamide reaches much lower such concentrations (32
µM after 1 h). High concentrations of the inhibitor were
maintained 2 h after administration too. Thus, the
strong and long-lasting IOP-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.
1-(4-Su lfa m oylp h en yl)-3-[(1H-im id a zol-4-yl)eth yl]th io-
u r ea (h ista m in e d er iva tive, A10): white crystals, mp 148-9
°C (ethanol-water 1:1, v/v). IR (KBr), cm-1: 1150 (SO2sym),
1245 (thioamide III), 1375 (SO2as), 1550 (thioamide II), 1680
1
(thioamide I), 3060 (NH). H NMR (DMSO-d6), δ, ppm: 2.49
(t, 2H, J ) 7.0, Hst CH2); 2.99 (t, 2H, J ) 7.0, Hst CSNHCH2);
7.34 (m, 1H, imidazole CH); 7.52 (s, 2H, SO2NH2), 7.65 (d, 3J HH
) 8.1, 2H, Hortho of H2NO2SC6H4), 7.94 (d, 3J HH ) 8.1, 2H, Hmeta
of H2NO2SC6H4); 8.21 (br s, 2H, NHCSNH); 8.35 (s, 1H,
imidazole CH); 8.80 (s, 1H, imidazole NH). 13C NMR (DMSO-
d6), δ, ppm: 33.3 (s, CH2 of Hst); 37.6 (s, CH2 of Hst); 123.7 (s,
C-4 of Hst); 130.9 (s, Cmeta of H2NO2SC6H4), 134.2 (s, C-5 of
Hst); 135.0 (s, Cortho of H2NO2SC6H4), 137.3 (s, C-2 of Hst);
139.4 (s, NHCSNH), 144.6 (s, Cipso of H2NO2SC6H4), 148.0 (s,
Cpara of H2NO2SC6H4). Anal. Found: C, 44.13; H, 4.30; N, 21.39.
C15H15N5O2S2 requires C, 44.29; H, 4.65; N, 21.52.
1-(4-Su lfam oylph en yl)-3-(car boxym eth yl)th iou r ea (gly-
cin e d er iva tive, A14): white crystals, mp 214-6 °C (ethanol-
water 1:2, v/v). IR (KBr), cm-1: 1153 (SO2sym), 1240 (thioa-
mide III), 1375 (SO2as), 1550 (thioamide II), 1682 (thioamide
I), 1754 (COOH), 3065 (NH). 1H NMR (DMSO-d6), δ, ppm: 3.67
Con clu sion s
We report here a novel class of sulfonamides, obtained
by reaction of 4-isothiocyanatobenzenesulfonamide with
amines, amino acids, or oligopeptides. 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 acting
as efficient topical IOP-lowering agents. Some of these
new CA inhibitors possessed affinities in the 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 as compared to dorzola-
mide. The new compounds reported here might lead to
the development of more efficient antiglaucoma drugs
from the class of the sulfonamide CA inhibitors.
3
(s, 2H, CH2 of Gly); 7.54 (s, 2H, SO2NH2), 7.63 (d, J HH ) 8.1,
3
2H, Hortho of H2NO2SC6H4), 7.94 (d, J HH ) 8.1, 2H, Hmeta of
H2NO2SC6H4); 8.21 (br s, 2H, NHCSNH); 10.63 (br s, 1H,
COOH). 13C NMR (DMSO-d6), δ, ppm: 40.8 (s, CH2 of Gly);
131.5 (s, Cmeta of H2NO2SC6H4), 135.4 (s, Cortho of H2NO2SC6H4),
139.7 (s, NHCSNH), 144.1 (s, Cipso of H2NO2SC6H4), 147.3 (s,
Cpara of H2NO2SC6H4); 179.6 (COOH). Anal. Found: C, 37.48;
H, 3.75; N, 14.26. C9H11N3O4S2 requires C, 37.36; H, 3.83; N,
14.52.
1-(4-Su lfa m oylp h en yl)-3-(1-ca r b oxy-2-m et h ylb u t yl)-
th iou r ea (isoleu cin e d er iva tive, A26): white crystals, mp
179-81 °C (ethanol-water 1:1, v/v). IR (KBr), cm-1: 1151
(SO2sym), 1244 (thioamide III), 1376 (SO2as), 1557 (thioamide
II), 1676 (thioamide I), 1750 (COOH), 3065 (NH). 1H NMR
3
(DMSO-d6), δ, ppm: 1.15 (d, J HH ) 6.5, 3H, CH3 of Ile), 1.21
3
(t, 3H, J HH ) 6.7, CH3 of Et moiety of Ile); 1.54 (m, 2H, CH2
of Ile); 3.22 (m, 1H, EtCH(Me)- of Ile); 3.75 (m, 1H, NHCHCO
3
of Ile), 7.51 (s, 2H, SO2NH2), 7.66 (d, J HH ) 8.0, 2H, Hortho of
Exp er im en ta l Section
3
H2NO2SC6H4), 7.92 (d, J HH ) 8.0, 2H, Hmeta of H2NO2SC6H4);
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%
8.20 (br s, 2H, NHCSNH); 10.42 (br s, 1H, COOH); 13C NMR
(DMSO-d6), δ, ppm: 21.7 (s, CHCH3 of Ile), 22.6 (s, CH3CH2
of Ile). 31.4 (s, CH2 of Ile); 34.5 (s, CH(CH3)2 of Leu), 46.4 (s,
EtCH(Me)- of Ile), 55.0 (s, NHCHCH2 of Ile), 131.3 (s, Cmeta of
H2NO2SC6H4), 135.5 (s, Cortho of H2NO2SC6H4), 139.9 (s,
NHCSNH), 144.3 (s, Cipso of H2NO2SC6H4), 147.8 (s, Cpara of
H2NO2SC6H4); 179.1 (COOH). Anal. Found: C, 45.33; H, 5.30;
N, 11.97. C13H19N3O4S2 requires C, 45.20; H, 5.54; N, 12.16.

4890 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25
Casini et al.
1-(4-Su lfa m oylp h en yl)-3-(1-ca r b oxy-2-p h en ylet h yl)-
th iou r ea (p h en yla la n in e d er iva tive, A33): white crystals,
mp 223-4 °C (ethanol-water 1:1, v/v). IR (KBr), cm-1: 1148
(SO2sym), 1243 (thioamide III), 1376 (SO2as), 1550 (thioamide
II), 1684 (thioamide I), 1751 (COOH), 3065 (NH). 1H NMR
(DMSO-d6), δ, ppm: 3.10-3.55 (m, 2H, CH2CH of Phe), 4.08
isozyme has a decreased esterase activity21 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 (3 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 of inhibitors (2%, by
weight, as hydrochlorides or sodium carboxylates) were ob-
tained 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.22,23 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 are 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 eye and control eye, in this way
minimizing the diurnal, seasonal, and interindividual varia-
tions commonly observed in the rabbit.22,23 All data are
expressed as mean ( SE, using a one-tailed t-test.
3
3
(dd, J HH ) 5.0, J HH ) 7.8, 1H, CH2CH of Phe), 7.29-7.51
3
(m, 5H, Harom of Phe);7.57 (s, 2H, SO2NH2), 7.63 (d, J HH
)
3
8.1, 2H, Hortho of H2NO2SC6H4), 7.96 (d, J HH ) 8.1, 2H, Hmeta
of H2NO2SC6H4); 8.20 (br s, 2H, NHCSNH); 10.71 (br s, 1H,
COOH). 13C NMR (DMSO-d6), δ, ppm: 41.5 (s, CH2CH of Phe),
59.3 (s, CH2CH of Phe), 131.5 (s, Cmeta of H2NO2SC6H4), 133.8
(s, Cmeta of Phe), 134.4 (s, Cortho of Phe), 135.4 (s, Cortho of H2-
NO2SC6H4), 139.7 (s, NHCSNH), 141.5 (s, Cipso of Phe), 144.1
(s, Cipso of H2NO2SC6H4), 145.6 (s, Cpara of Phe), 147.3 (s, Cpara
of H2NO2SC6H4); 179.3 (COOH). Anal. Found: C, 50.49; H,
4.60; N, 10.86. C16H17N3O4S2 requires C, 50.65; H, 4.52; N,
11.07.
4-Su lfa m oylp h en ylth iou r eid o-â-a la n ylh istid in e (ca r -
n osin e d er iva tive, A40): white crystals, mp 127-8 °C. IR
(KBr), cm-1: 1156 (SO2sym), 1240 (thioamide III),1287 (amide
I), 1375 (SO2as), 1540 (amide I), 1550 (thioamide II), 1683
(thioamide I), 1720 (amide I), 1750 (COOH), 3065 (NH). 1H
NMR (DMSO-d6), δ, ppm: 2.79-2.88 (m, 2H, CH2 of â-Ala),
3.11-3.26 (m, 2H, CH2 of â-Ala), 3.34-3.45 (m, 2H, CHCH2
of His), 4.57-4.63 (m, 1H, CHCH2 of His), 7.33 (s, 1H, CH-5
3
of His), 7.54 (s, 2H, SO2NH2), 7.63 (d, J HH ) 8.1, 2H, Hortho of
3
H2NO2SC6H4), 7.94 (d, J HH ) 8.1, 2H, Hmeta of H2NO2SC6H4);
8.21 (br s, 2H, NHCSNH); 8.29 (br s, 1H, CONH); 8.35 (s, 1H,
CH-2 of His); 8.80 (s, 1H, imidazole NH from His)10.63 (br s,
1H, COOH). 13C NMR (DMSO-d6), δ, ppm: 33.3 (s, CH2 of His),
37.4 (s, NHCH2CH2 of â-Ala), 40.8 (s, CH2CH2CO of â-Ala),
59.6 (s, CHCH2 of His), 122.2 (s, C-4 of His), 131.5 (s, Cmeta of
H2NO2SC6H4), 134.2 (s, C-5 of His), 135.3 (s, Cortho of H2NO2-
SC6H4), 137.5 (s, C-2 of His), 139.4 (s, NHCSNH), 144.1 (s,
Cipso of H2NO2SC6H4), 147.1 (s, Cpara of H2NO2SC6H4); 175.6
(s, CH2CO of â-Ala), 180.1 (COOH). Anal. Found: C, 43.54;
H, 4.33; N, 19.00. C16H20N6O5S2 requires C, 43.63; H, 4.58; N,
19.08.
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.24 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 injection of R-chymotrypsin.
CA In h ibition . hCA I and hCA II cDNAs were expressed
in Escherichia coli strain BL21 (DE3) from the plasmids pACA/
hCA I and pACA/hCA II described by Lindskog’s group.14 Cell
growth conditions were those described in ref 15 and enzymes
were purified by affinity chromatography according to the
method of Khalifah et al.16 Enzyme concentrations 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, respectively, based on Mr ) 28.85 kDa for CA
I and 29.3 kDa for CA II, respectively.17,18 bCA IV was isolated
from bovine lung microsomes as described by Maren et al.,
and its concentration has been determined by titration with
ethoxzolamide.19
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.20 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.20 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.20 Enzyme concentrations were
3.6 nM for CA II, 9.1 nM for CA I, and 30 nM for CA IV (this
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.22,23 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 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 tissues from 4 eyes (average weight of
8 mg/eye) were 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 in the presence
of the 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 22, 23.
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/

Water-Soluble 4-Sulfamoylphenylthioureas
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25 4891
(8) (a) Supuran, C. T.; Briganti, F.; Menabuoni, L.; Mincione, G.;
Mincione, F.; Scozzafava, A. Carbonic anhydrase inhibitors. Part
78. Synthesis of water-soluble sulfonamides incorporating â-ala-
nyl moieties, possessing long lasting-intraocular pressure lower-
ing properties via the topical route. Eur. J . Med. Chem. 2000,
35, 309-321. (b) Mincione, G.; Menabuoni, L.; Briganti, F.;
Mincione, F.; Scozzafava, A.; Supuran, C. T. Carbonic anhydrase
inhibitors. Part 79. Synthesis and ocular pharmacology of
topically acting sulfonamides incorporating GABA moieties in
their molecule, with long-lasting intraocular pressure lowering
properties. Eur. J . Pharm. Sci. 1999, 9, 185-199.
(9) McKee, R. L.; Bost, R. W. p-Substituted phenyl isothiocyanates
and some related thioureas. J . Am. Chem. Soc. 1946, 68, 2506-
2507.
(10) (a) Browne, D. W.; Dyson, G. M. The inhibitory effect of
substituents in chemical reactions. Part II. The reactivity of the
isothiocyano group in substituted arylthiocarbimides. J . Chem.
Soc. 1931, 3285-33909. (b) Browne, D. W.; Dyson, G. M. The
inhibitory effect of substituents in chemical reactions. Part III.
The reactivity of the isothiocyanato group in substituted arylth-
iocarbimides. J . Chem. Soc. 1934, 178-179.
(11) 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.
(12) (a) Supuran, C. T.; Clare, B. W. Carbonic anhydrase inhibitors
Part 24. A quantitative structure-activity relationship study of
positively charged sulfonamide inhibitors. Eur. J . Med. Chem.
1995, 30, 687-696. (b) Maren, T. H.; Clare, B. W.; Supuran, C.
T. Structure-activity studies of sulfonamide carbonic anhydrase
inhibitors. Roum. Chem. Quart. Rev. 1994, 2, 259-282. (c) Clare,
B. W.; Supuran, C. T. Carbonic anhydrase inhibitors Part 41.
Quantitative structure-activity correlations involving kinetic
rate constants of 20 sulfonamides from a noncongeneric series.
Eur. J . Med. Chem. 1997, 32, 311-319. (d) Supuran, C. T.; Clare,
B. W. Carbonic anhydrase inhibitors Part 47. Quantum chemical
mL), A ) absorbance of standard solution, A′ ) absorbance of
saturated solution, C′ ) concentration of saturated solution
(mg/mL).25
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.26
Tr a n scor n ea l P en etr a tion of Dr u gs. The method of
Maren et al.27 with the modifications of Pierce’s group28,29 (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
of 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 both by the HPLC method of Pierce et al.28,29 or
enzymatically.27 The results of the drug analyses were used
to calculate the rate constant of transfer across the cornea (kin).
As described by Pierce,28,29 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 by
EU Grant ERB CIPDCT 940051 and by a grant from
the Italian CNR - Target Project Biotechnology.
Refer en ces
(1) (a) Supuran, C. T.; Scozzafava, A. Carbonic anhydrase inhibitors
and their therapeutic potential. Exp. Opin. Ther. Patents 2000,
10, 575-600. (b) Supuran, C. T. Carbonic anhydrase inhibitors.
In Carbonic Anhydrase and Modulation of Physiologic and
Pathologic Processes in the Organism; Puscas, I., Ed.; Helicon:
Timisoara, Romania, 1994; pp 29-111.
(2) (a) Maren, T. H. The links among biochemistry, physiology and
pharmacology in carbonic anhydrase mediated systems. In
Carbonic Anhydrase - From Biochemistry and Genetics to
Physiology and Clinical Medicine; Botre´, F., Gros, G., Storey,
B. T., Eds.; VCH: Weinheim, Germany, 1991; pp 186-207. (b)
Maren, T. H. The development of topical carbonic anhydrase
inhibitors. J . Glaucoma 1995, 4, 49-62.
(3) Sugrue, M. F. Pharmacological and ocular hypotensive properties
of topical carbonic anhydrase inhibitors. Prog. Ret. Eye Res.
2000, 19, 87-112.
quantitative structure-activity relationships for
a group of
sulfanilamide Schiff base inhibitors of carbonic anhydrase. Eur.
J . Med. Chem. 1998, 33, 489-500.
(13) (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. (c) Clare, B. W.;
Supuran, C. T. Carbonic anhydrase inhibitors. Part 86. A QSAR
Study on some Sulfonamide Drugs which Lower Intra-Ocular
Pressure. Eur. J . Med. Chem. 2000, 35, 859-865.
(4) 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.
(5) 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.
(14) 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 anhydrase - From biochemistry and
genetics to physiology and clinical medicine; Botre`, F., Gros, G.,
Storey, B. T. Eds.; VCH: Weinheim, Germany, 1991; pp 1-13.
(15) 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.
(16) 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.
(17) Lindskog, S.; Coleman, J . E. The catalytic mechanism of carbonic
anhydrase. Proc. Natl. Acad Sci. U.S.A. 1964, 70, 2505-2508.
(18) 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.
(19) 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.
(20) Pocker, Y.; Stone, J . T. The catalytic versatility of erythrocyte
carbonic anhydrase. III. Kinetic studies of the enzyme-catalyzed
hydrolysis of p-nitrophenyl acetate. Biochemistry 1967, 6, 668-
678.
(21) 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.
(22) 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.
(6) (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 in-
traocular pressure-lowering properties via the topical route. J .
Med. Chem. 1999, 42, 3690-3700.
(7) (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) 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 furan-, thiophene- and pyrrole-carboxamido groups
possess strong topical intraocular pressure lowering properties
as aqueous suspensions. Bioorg. Med. Chem. 2000, 8, 2145-
2155.

4892 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25
Casini et al.
(23) Brechue, W. F.; Maren, T. H. pH and drug ionization affects
ocular pressure lowering of topical carbonic anhydrase inhibitors.
Invest. Ophthalmol. Vis. Sci. 1993, 34, 2581-2587.
(24) Melena, J .; Santafe, J .; Segarra-Domenech, J .; Puras, G. Aqueous
humor dynamics in alpha-chymotrypsin-induced ocular hyper-
tensive rabbits. J . Ocul. Pharmacol. Ther. 1999, 15, 19-27.
(25) 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.
(26) Scozzafava, A.; Briganti, F.; Ilies, M. A.; Supuran, C. T. Carbonic
anhydrase inhibitors: Synthesis of membrane-impermeant low
molecular weight sulfonamides possessing in vivo selectivity for
the membrane-bound versus the cytosolic isozymes. J . Med.
Chem. 2000, 43, 292-300.
(27) 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.
(28) 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.
(29) Sharir, M.; Pierce, W. M. J r.; Chen, D.; Zimmerman, T. J .
Pharmacokinetics, acid-base balance and intraocular pressure
effects of ethyloxaloylazolamide - A novel topically active
carbonic anhydrase inhibitor. Exp. Eye Res. 1994, 58, 107-116.
J M001051+