Carbonic anhydrase inhibitors: Synthesis of water-soluble, aminoacyl/dipeptidyl sulfonamides possessing long-lasting intraocular pressure-lowering properties via the topical route

Article abstract of DOI:10.1021/jm9901879

Reaction of 26 aromatic/heterocyclic sulfonamides containing amino, imino, hydrazino, or hydroxyl groups with Boc-Gly, Boc-Sar, TrS-Crt, or Boc- Gly-Gly (Sar = sarcosine, N-Me-Gly; Crt = creatine, N-amidinosarcosine; TrS = tritylsulfenyl; Boc = tert-butoxycarbonyl) in the presence of carbodiimide derivatives afforded after removal of the protecting groups a series of water-soluble compounds (as salts of strong acids, such as hydrochloric, trifluoroacetic, or trifluoromethanesulfonic). The new derivatives were assayed as inhibitors of the zinc enzyme carbonic anhydrase (CA) and more precisely of three of its isozymes, CA I, II (cytosolic forms), and IV (membrane-bound form), involved in important physiological processes. Efficient inhibition was observed against all three isozymes and especially against CA II and IV (in the nanomolar range), the two isozymes known to play a critical role in aqueous humor secretion within the ciliary processes of the eye. Some of the best inhibitors synthesized were applied as 2% water solutions into the eye of normotensive or glaucomatous albino rabbits, when strong and long-lasting intraocular pressure (IOP) lowering was observed with many of them. Thus, the aminoacyl/dipeptidyl tail conferring water solubility to these sulfonamide CA inhibitors coupled with strong-enzyme inhibitory properties and balanced lipid solubility seem to be the key factors for obtaining compounds with effective topical antiglaucoma activity.

Full text of DOI:10.1021/jm9901879

3690
J . Med. Chem. 1999, 42, 3690-3700
Ca r bon ic An h yd r a se In h ibitor s: Syn th esis of Wa ter -Solu ble, Am in oa cyl/
Dip ep tid yl Su lfon a m id es P ossessin g Lon g-La stin g In tr a ocu la r
P r essu r e-Low er in g P r op er ties via th e Top ica l Rou te1
Andrea Scozzafava,† Fabrizio Briganti,† Giovanna Mincione,† Luca Menabuoni,‡ Francesco Mincione,§ and
Claudiu T. Supuran*,†
Laboratorio di Chimica Inorganica e Bioinorganica, Universita` degli Studi, Via Gino Capponi 7, I-50121 Florence, Italy,
S. O. Oculistica, Ospedale San Giovanni di Dio, Via Torregalli 3, I-50123 Florence, Italy, and Istituto Oculistico,
Universita` degli Studi, Viale Morgagni 85, I-50134 Florence, Italy
Received April 19, 1999
Reaction of 26 aromatic/heterocyclic sulfonamides containing amino, imino, hydrazino, or
hydroxyl groups with Boc-Gly, Boc-Sar, TrS-Crt, or Boc-Gly-Gly (Sar ) sarcosine, N-Me-Gly;
Crt ) creatine, N-amidinosarcosine; TrS ) tritylsulfenyl; Boc ) tert-butoxycarbonyl) in the
presence of carbodiimide derivatives afforded after removal of the protecting groups a series
of water-soluble compounds (as salts of strong acids, such as hydrochloric, trifluoroacetic, or
trifluoromethanesulfonic). The new derivatives were assayed as inhibitors of the zinc enzyme
carbonic anhydrase (CA) and more precisely of three of its isozymes, CA I, II (cytosolic forms),
and IV (membrane-bound form), involved in important physiological processes. Efficient
inhibition was observed against all three isozymes and especially against CA II and IV (in the
nanomolar range), the two isozymes known to play a critical role in aqueous humor secretion
within the ciliary processes of the eye. Some of the best inhibitors synthesized were applied as
2% water solutions into the eye of normotensive or glaucomatous albino rabbits, when strong
and long-lasting intraocular pressure (IOP) lowering was observed with many of them. Thus,
the aminoacyl/dipeptidyl tail conferring water solubility to these sulfonamide CA inhibitors
coupled with strong enzyme inhibitory properties and balanced lipid solubility seem to be the
key factors for obtaining compounds with effective topical antiglaucoma activity.
In tr od u ction
as hydrochloride salts (Chart 2). The use of such
hydrochloride salts is imposed by the need of ensuring
a good water solubility to the drugs, but in some cases
this represents an undesired complication, since the pH
of such solutions becomes acidic enough and conse-
quently produces eye irritation after the topical admin-
istration, as already reported for many patients treated
with dorzolamide.22 Moreover, the duration of action of
these drugs is generally short (2-3 h), and they must
be administered several times a day. It is thus of critical
importance to design novel types of water-soluble,
topically acting sulfonamide CA inhibitors in order to
obtain third-generation drugs (if one considers the
systemically administered sulfonamides such as aceta-
zolamide (28) or methazolamide (29) as the first-
generation CA inhibitors and the recently developed
dorzolamide (26) and brinzolamide (27) as the second-
generation ones) totally devoid of undesired side effects.
In previous contributions from this laboratory1-3 it
was shown that by attaching water-solubilizing tails
(such as 8-quinolinesulfonyl,1 nicotinoyl,2 isonicotinoyl,2
6-carboxypyridine-2-carboxamido,2,3 etc.) to the mole-
cules of aromatic/heterocyclic sulfonamides of types
1-26 (Chart 1), possessing free amino, hydrazino,
imino, or hydroxy groups, it is possible to obtain water-
soluble, efficient inhibitors of the zinc enzyme carbonic
anhydrase (CA, EC 4.2.1.1),4-8 some of which showed
promising antiglaucoma9 activity via the topical route
in experimental animals.
Indeed, CA inhibition in ocular tissues (mainly the
ciliary processes) with systemically or topically admin-
istered sulfonamide CA inhibitors is followed by an
effective reduction of intraocular pressure (IOP) due to
the reduced rate of bicarbonate secretion within the
aqueous humor.8-11 Since the systemic inhibitors gener-
ally produce undesired side effects due to inhibition of
different CA isozymes in other tissues than the eye,8-11
many efforts have been made in the last 15 years for
the development of water-soluble sulfonamide CA in-
hibitors that should be administered via the topical
route.12-20 Two such drugs are presently available,
dorzolamide (26)13,17 and brinzolamide (27),21 both used
The approach used by us for the design of topically
active IOP-lowering agents reported in this paper is
based on the interesting findings of Whitesides’ group
23,24 regarding the increase of the binding affinity to the
enzyme for compounds possessing secondary binding
sites adjacent to the sulfonamido one (which is the
primary binding moiety, since the ionized sulfonamide
is directly coordinated to the Zn(II) ion within the CA
active site).25,26 The above-mentioned findings23,24 (which
generally exploited hydrophobic interactions between
hydrophobic patches at the entrance of the hCA II active
site and similar moieties present in the inhibitor
* Correspondence author. Fax: +39-055-2757555. E-mail: cts@bio.
chim.unifi.it.
† Universita` degli Studi, Via Gino Capponi 7.
‡ Ospedale San Giovanni di Dio.
§ Universita` degli Studi, Viale Morgagni 85.
10.1021/jm9901879 CCC: $18.00 © 1999 American Chemical Society
Published on Web 08/10/1999

Carbonic Anhydrase Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3691
Ch a r t 1
F igu r e 1. hCA II-aminobenzolamide (16) complex. The Zn-
(II) ion (central sphere) and its three histidine ligands (His
94, 96, and 119), as well as the histidine cluster comprising
residues 3 (disordered in this structure), 4, 10, 15, 17, and 64,
are shown. The inhibitor molecule (shown in a ball-and-stick
form) is coordinated to the Zn(II) ion and interacts with some
of the residues of the hydrophilic cluster mentioned above. This
figure has been generated from the X-ray coordinates of Liljas
group25b by using the program Rasmol for Windows. This
structure is not deposited in the Brookhaven Protein Database.
are of interest for ophthalmologic applications (mention
should be made that the above-mentioned research-
ers23,24 were not interestedsas far as we knowsin the
antiglaucoma use of their compounds, but their studies
greatly contributed to a better understanding of CA
inhibition by sulfonamides).23-26 In this context we27,28
have recently evidenced by means of X-ray crystal-
lography a cluster of six histidine residues (His 3, His
4, His 10, His 15, His 17, and His 64) at the entrance of
the hCA II active site, some of which possess different
conformations (as shown by the above-mentioned tech-
nique)27,28 which could easily participate in hydrogen
bond formation (as well as other types of interactions)
with inhibitors possessing hydrophilic moieties grafted
to the aromatic/heterocyclic sulfonamide used as the
primary binding group. In fact, in the X-ray structure
of the adduct of aminobenzolamide (16) with hCA II
reported by Liljas’ group,25b it is possible to evidence
that the terminal part of the sulfonamide molecule is
placed in such a way to interact favorably with some of
the hydrophilic residues belonging to the histidine
cluster mentioned above (Figure 1).
In this paper we report the reaction of 26 aromatic/
heterocyclic sulfonamides containing a free amino,
imino, hydrazino, or hydroxyl group with protected
amino acid/dipeptide derivatives, such as Boc-Gly, Boc-
Sar, TrS-Crt, or Boc-Gly-Gly (Sar ) sarcosine, N-Me-
Gly; Crt ) creatine, N-amidinosarcosine; TrS ) trityl-
sulfenyl; Boc ) tert-butoxycarbonyl), in the presence of
carbodiimides, which afforded (after removal of the
protecting groups) a series of water-soluble (as hydro-
Ch a r t 2
molecule), combined with our strategy of ensuring water
solubility (as salts of a strong acid/base for instance),1-3
might lead to compounds with affinity in the nanomolar
range for the relevant CA isozymes involved in aqueous
humor secretion (CA II and IV). Obviously, to achieve
this goal, we used the opposite of the approach reported
by Whitesides’ group,23,24 i.e., taking advantage of the
hydrophilic interactions between secondary binding
sites and the enzyme, since just hydrophilic inhibitors

3692 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18
Scozzafava et al.
Ch a r t 3
Ch a r t 4
chloride, trifluoroacetate, or triflate salts) sulfonamides
with good CA inhibitory properties. Moreover, the
clinically used inhibitor dorzolamide (26) has been
derivatized similarly, at its secondary amino group, and
the obtained compounds also possessed good water
solubility (as salts of strong acids) and CA inhibitory
properties. The new compounds reported here were
tested for the inhibition of three CA isozymes: hCA I,
hCA II, and bCA IV (h ) human, b ) bovine isozymes).
Affinities in the nanomolar range were detected for
many compounds against isozymes II and IV, which are
those involved in aqueous humor secretion. The most
active derivatives were assayed in vivo in normotensive
and glaucomatous rabbits for their IOP-lowering prop-
erties. Very strong and long-lasting IOP lowering was
observed for many of them. Transcorneal accession rates
for some of the new compounds were also determined,
indicating a facilitated penetration of our compounds
as compared to other heterocyclic sulfonamides devoid
of topical activity. Our conclusion is that the aminoacyl/
dipeptidyl water-solubilizing tail is critical for the
topical activity of these compounds and that antiglau-
coma drugs might be obtained by this approach from
many other classes of sulfonamides and not only from
the thienothiopyransulfonamides and their derivatives.
Furthermore, the pH of the solutions of compounds (as
salts of strong acids) reported here is around 6.5-7.0,
making them much less irritating to the eye as com-
pared to the relatively highly acidic dorzolamide prepa-
rations (pH 5.5).
phenols with protected amino acid/dipeptide derivatives
(of type 30-33), followed by the usual deprotection steps
(TFA for eliminating the Boc moiety and dioxane-HCl
for the tritylsulfenyl one), as reported previously by
Whitesides23,24 or this group15,16 for structurally related
sulfonamides.
CA In h ibitor y Activity. Inhibition data against
three CA isozymes, hCA I, hCA II, and bCA IV, with
compounds A(1-26)-D(1-26) as well as the original
raw materials and standard sulfonamides of type 1-29
are shown in Table 1. The esterase activity of CA
isozymes against 4-nitrophenyl acetate as substrate has
been used in this assay (see Experimental Section for
details). Mention should be made that the Boc- or
tritylsulfenyl-protected intermediates prepared here
also possessed very good CA inhibitory properties (data
not shown).
Tr a n scor n ea l P en etr a tion of Dr u gs. Some phys-
icochemical properties of the new compounds, relevant
for their pharmacological activity, such as buffer solu-
bility or chloroform-buffer partition coefficient, are
shown in Table 2. The in vitro transcorneal accession
rates (kin) of classical sulfonamides and topically active
derivatives, such as dorzolamide 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 with
dorzolamide and some of the new compounds reported
here in normotensive albino rabbits is shown in Figure
2.
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. As a large number of derivatives are
reported here, each compound will be designated by a
letter identifying the amino acid/dipeptide from which
it is derived and a figure identifying the sulfonamide of
type 1-26 at which the aminoacyl/dipeptidyl moiety has
been attached. For instance, A3 is glycylsulfanilamide;
B6 is 4-(sarcosinylamidoethyl)benzenesulfonamide; C13
is 5-creatinyl-1,3,4-thiadiazole-2-sulfonamide; D26 is
the glycylglycinamido derivative of dorzolamide, etc.
(Chart 3).
The new compounds A(1-26)-D(1-26) were pre-
pared by reaction of the aromatic/heterocyclic sulfona-
mides 1-26 with the protected amino acid/dipeptide
derivatives of type 30-33 (originating from the four
amino acid/dipeptide derivatives A-D), followed by
removal of the protecting groups in the standard man-
ner (Chart 4).
Discu ssion
Ch em istr y. Although several thousand different
aromatic/heterocyclic sulfonamide CA inhibitors have
been synthesized in the last 45 years in search of diverse
pharmacological agents,4,10 the number of aminoacyl/
dipeptidyl derivatives is unexpectedly small. Thus,
Antonarolli et al.29 reported one such compound, 34
Nonexceptional routine synthetic procedures were
employed for the reaction of amines/imines/alcohols/

Carbonic Anhydrase Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3693
Ta ble 1. CA Inhibition Data with Standard Inhibitors, Parent
Sulfonamides 1-26, and New Derivatives (A-D)1-26
Reported in the Present Study Against Isozymes I, II, and IV
Ta ble 2. Solubility, Chloroform-Buffer Partition Coefficients,
and in Vitro Corneal Permeability of Some Sulfonamide CA
Inhibitors
Kia (nM)
Kia (nM)
c
kin × 103 (h-1
)
inhib-
inhib-
itor hCA Ib hCA IIb bCA IVc itor hCA Ib hCA IIb bCA IVc
solubilitya
(mM)
cornea
no
compd
log Pb intact epithelium
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
45400
25000
28000
78500
25000
21000
8300
9800
6500
6000
6100
8400
8600
9300
455
6
1
42
44
690
70
55
50
295
240
300
320
170
160
60
110
40
70
28
75
60
19
3
2
0.6
6
9
1310
2200
3000
3215
2800
2450
180
320
66
125
175
160
540
355
125
5
B14
B15
B16
B17
B18
B19
B20
B21
B22
B23
B24
B25
B26
C1
C2
C3
C4
C5
C6
C7
C8
C9
C10
C11
C12
C13
C14
C15
C16
C17
C18
C19
C20
C21
C22
C23
C24
C25
C26
D1
D2
D3
D4
D5
D6
D7
D8
D9
290
21
12
11
50
51
60
10
11
6
3
1.5
1.8
8
9
19
5
4
3
43
9
6
28 (acetazolamide)d
3.2
12
0.001
0.06
0.37
1.90
3.0
3.1
4.6
2.1
3.9
4.1
4.7
4.8
2.0
3.1
7.0
13
29 (methazolamide)d
7
26 (dorzolamide)
60e
65f
54f
59f
62f
73f
75f
59f
51f
50f
2.0e
5.2
5.4
7.9
6.0
7.8
7.9
8.5
11.5
5.0
6.4
62
61
60
14
30
15
123
112
30
205
177
140
245
71
64
39
60
62
69
51
42
35
41
8
A16
B12
B16
B26
C13
C15
D11
D16
D23
0.043
1.569
0.983
1.785
1.620
1.944
2.133
0.754
1.850
10
1500
1300
78
57
50
3
20300
16200
14600
20800
950
775
510
510
525
545
525
310
170
190
21
187
160
111
190
30
30
10
27
29
33
13
7
5
6
2
1.1
1.3
8
8
15
4
3
a
b
Solubility in pH 7.40 buffer, at 25 °C. Chloroform-buffer
partition coefficient. c Determined as described.11,52,53 Data from
d
0.8
50
53
154
19
17
ref 11. e As hydrochloride, at pH 5.8, from ref 17. f As hydrochloride
salt.
12
9
8
Ta ble 3. Fall of IOP of Normotensive Rabbits (20.2 ( 2.3
mmHg) after Treatment with 1 drop (50 µL) of a 2% Solution of
CA Inhibitor (as hydrochloride, trifluoroacetate, or triflate salt,
with pH value shown) Directly into the Eye, at 30, 60, and 90
min after Administration
7
15
24000
18000
50000
125
110
9
560
450
45
25
26
∆IOP (mmHg)a
27d
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
A26
B1
B2
B3
B4
B5
B6
B7
B8
B9
B10
B11
B12
B13
3.2
45.3
25000
18000
15000
25500
1400
850
540
600
600
610
520
360
350
335
21
210
200
124
250
40
39
12
41
38
45
13
10
9
245
240
175
300
82
76
46
78
73
74
62
60
55
70
10
9
10
86
79
75
72
69
23
180
155
35
205
195
146
280
74
70
46
62
64
inhibitor pH t ) 0 min t ) 30 min t ) 60 min t ) 90 min
12
11
46
45
53
10
11
9
5
6
26b
5.5
5.5
5.5
6.0
5.5
6.5
6.5
6.5
5.6
6.5
6.5
7.0
7.5
6.5
6.4
6.4
7.5
6.4
6.4
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2.2 ( 0.20
2.9 ( 0.25
5.0( 0.20
5.9 ( 0.25
2.5 ( 0.25
3.3 ( 0.30
5.5 ( 0.25
2.5 ( 0.15
3.1 ( 0.20
3.0 ( 0.15
4.1 ( 0.30 2.7 ( 0.25
5.8( 0.45 4.6 ( 0.30
9.3 ( 0.40 8.4 ( 0.35
9.5 ( 0.30 9.0 ( 0.25
5.2 ( 0.20 4.5 ( 0.12
6.2 ( 0.15 5.5 ( 0.30
9.9 ( 0.20 9.4 ( 0.30
6.3 ( 0.15 5.5 ( 0.25
7.0 ( 0.30 5.5 ( 0.30
7.2 ( 0.35 5.0 ( 0.20
A13b
A15c
A16d
B12b
B14b
B16c
B18d
B26b
C7b
51
57
40
12
15
11
89
86
27
240
235
154
300
75
70
45
65
70
71
56
54
45
44
12
6
3
1200
1000
75
41
40
2
C13c
C14b
C15d
C26b
D11c
D13d
D16b
D23b
D26b
6.5 ( 0.20 10.1 ( 0.30 9.1 ( 0.20
5.5 ( 0.15 9.2 ( 0.20 9.0 ( 0.30
6.6 ( 0.20 10.7 ( 0.20 9.8 ( 0.30
23500
17000
15000
25200
1360
800
540
550
570
590
500
360
320
280
21
200
200
121
245
36
35
11
36
36
40
12
9
10
4
2
14
15
62
60
61
17
16
9
5.0 ( 0.40
3.2 ( 0.25
5.6 ( 0.30
5.9 ( 0.20
3.3 ( 0.25
4.9 ( 0.35
9.1 ( 0.15 7.6 ( 0.25
7.0 ( 0.30 4.8 ( 0.35
9.0 ( 0.40 8.9 ( 0.30
8.8 ( 0.25 9.1 ( 0.35
7.0 ( 0.20 5.4 ( 0.40
8.7 ( 0.30 6.9 ( 0.30
3
12
13
23
9
9
8
73
64
6
187
180
115
193
33
32
10
28
30
36
14
9
D10
D11
D12
D13
D14
D15
D16
D17
D18
D19
D20
D21
D22
D23
D24
D25
D26
a
∆IOP ) IOPcontrol eye - IOPtreated eye; mean ( average spread
(n ) 3). As HCl salt. c As TFA salt. As triflate salt.
b
d
1800
1700
98
6
7
3
2
Ta ble 4. Fall of IOP of Glaucomatous Rabbits (36.3 ( 2.2
mmHg) after Treatment with 1 drop (50 µL) of a 2% Solution of
CA Inhibitor (as hydrochloride, trifluoroacetate, or triflate, with
pH value shown) Directly into the Eye, at 30, 60, and 90 min
after Administration
20300
16600
15000
22000
965
820
500
510
540
560
520
350
305
13
13
54
55
60
16
12
9
2
9
10
10
21
9
8
5
68
61
6
66
60
69
64
62
18
150
150
31
∆IOP (mmHg)a
inhibitor pH t ) 0 min t ) 30 min t ) 60 min t ) 90 min
A16d
B16c
C13c
C15d
D13d
D16b
6.0
6.5
6.5
7.5
6.4
7.5
0
0
0
0
0
0
7.9 ( 0.35 12.5 ( 0.30 14.0 ( 0.20
6.5 ( 0.50 11.3 ( 0.25 13.7 ( 0.15
8.5 ( 0.45 13.4 ( 0.20 14.5 ( 0.40
8.6 ( 0.30 14.2 ( 0.20 14.3 ( 0.35
7.7 ( 0.20 12.0 ( 0.45 13.8 ( 0.35
7.0 ( 0.25 11.8 ( 0.30 13.1 ( 0.30
70
55
50
39
1500
1500
90
6
a
Standard error for the determination of Ki values was 10-
20% (from 2 different assays). b Human (cloned) isozyme. c Isolated
a-dAs in Table 3.
d
from bovine lung microsomes. Data from ref 50.
(Chart 5); Blackburn’s group30 also synthesized three
structurally related derivatives, 35-37, whereas we
have recently reported the â-alanylamido-1,3,4-thiadia-
zole-2-sulfonamide (15),1 possessing very good water
solubility (as HCl salt) and strong IOP-lowering proper-
ties in normotensive and glaucomatous rabbits. All
compounds just mentioned, 15 and 34-37, are 1,3,4-
thiadiazole-2-sulfonamide derivatives, as this was the

3694 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18
Scozzafava et al.
Ch a r t 5
F igu r e 2. 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 C13 (trifluoroacetate salt, pH 6.50); curve
3, compound C15 (triflate salt, pH 7.6).
Ta ble 5. Ocular Tissue Concentrations after 1 and 2 h
following Corneal Application of 1 drop (50 µL) of a 2% Solution
of C13 (as trifluoroacetate) and C15 (as triflate salt) in
Normotensive Albino Rabbits
drug concentration (µM)a
inhibitor time (h)
cornea
aqueous humor ciliary process
Obviously, compounds such as 39 or 41 would be highly
water-soluble as sodium carboxylate salts, but presum-
ably the pH of such solutions would be in the alkaline
range. On the other hand, of all compounds 34-41
mentioned above, ophthalmologic applications have
been claimed only for those reported by Blackburn’s
group,30 but without any quantitative data, and by our
own report on compound 15 and some of its derivatives
(with detailed ocular pharmacological data).1 It ap-
peared thus of interest to study in greater detail
aminoacyl/dipeptidyl sulfonamide CA inhibitors ob-
tained in such a way as to possess free amino moieties
(or their variants, such as N-methylamino, N-meth-
ylguanidino, etc.). Based on previous QSAR studies from
this group on several series of CA inhibitors,32 it
emerged that the enhancement of CA inhibitory activity
is correlated with increased positive charges on the
heterocyclic/aromatic ring incorporated in such mole-
cules, as well as with “long” inhibitor molecules per se
(i.e., molecules extending on the direction passing
through the Zn(II) ion of the enzyme, the sulfonamide
nitrogen atom, and the long axis of the inhibitor).32
Examining different potential groups that might be
attached to aromatic/heterocyclic sulfonamide CA in-
hibitors, it appeared of great interest to use glycyl
moieties as well as some of their closely related variants,
such as sarcosinyl (Sar, N-methylglycine) and creatinyl
(Crt, N-amidinosarcosinyl) as well as glycyl-glycyl (Gly-
Gly). Thus, we attached these amino acid/dipeptide
derivatives (obviously protected in the appropriate
manner) to the amino, hydrazino, imino, or hydroxy
groups of sulfonamides 1-26.
C13
C15
1
2
1
2
172 ( 9
65 ( 8
184 ( 16
73 ( 11
308 ( 10
50 ( 5
54 ( 7
18 ( 2
69 ( 9
21 ( 4
325 ( 24
45 ( 6
a
Mean ( standard deviation (n ) 3).
ring system best studied, due to the fact that the
first-generation CA inhibitors derived from it (such as
acetazolamide (28) or methazolamide (29)) were suc-
cessful drugs, clinically used for more than 40 years.4,10
Compounds of a different nature were also reported
by Whitesides’ (synthesis) and Christianson’s (X-ray
crystallography) groups.23,24,26 Generally these were
derivatives of 4-carboxybenzenesulfonamide to which
oligopeptidyl moieties were attached, of type 38 (eight
such derivatives were reported) and 39 (seven such
derivatives were reported).23,24 In another series of such
derivatives, oligoethylene glycol units were attached to
4-carboxybenzenesulfonamide and the terminal hydroxy
moiety of the tail was derivatized by means of aminoacyl
moieties (six derivatives of type 40 were thus ob-
tained).23,24 Finally, Baldwin’s group31 reported several
structurally related inhibitors, of type 41, obtained
again from 4-carboxybenzenesulfonamide, by attaching
dipeptidyl moieties incorporating nipecotic acid at its
carboxy group. One should notice that almost all these
aminoacyl/peptidyl sulfonamide derivatives do not pos-
sess free amino groups, as these were derivatized by the
COOH moiety of the 4-carboxybenzenesulfonamide used
in their preparation (exceptions are the six compounds
of type 40). These free NH2 groups are of considerable
interest for the putative ophthalmologic applications of
such inhibitors, since they afford the formation of water-
soluble salts with strong acids, which are highly desir-
able for the topical administration of sulfonamide CA
inhibitors (for reasons little explained for the moment,
ophthalmologic applications of compounds with weakly
acidic pH are preferred over those of alkaline pH).
Reaction of sulfonamides 1-26 with the N-protected
derivatives 30-33 in the presence of EDCI (1-(3-
(dimethylamino)propyl)-3-ethylcarbodiimide) or diiso-
propylcarbodiimide afforded the new derivatives (A-
D)1-26, by a procedure relatively similar to that
reported by Whitesides’ group23,24 for preparation of the

Carbonic Anhydrase Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3695
4-carboxybenzenesulfonamide derivatives 38-40. Men-
tion should be made that the classical tert-butoxycar-
bonyl (t-Boc) protecting group was used for the prepa-
ration of derivatives of Gly, Sar, and GlyGly, whereas
in the case of creatine the guanidino moiety has been
protected by using the tritylsulfenyl group, successfully
used by us previously for obtaining histamine deriva-
tives with CA activatory properties.33 Removal of the
two protecting groups was achieved then in the standard
fashion, i.e., with trifluoroacetic acid (TFA) for t-Boc and
with HCl-dioxane for TrS.33
ally led to an increase of the CA inhibitory properties
for the obtained compound, as compared to the corre-
sponding parent sulfonamide. Particularly strong in-
hibitors were those derived from heterocyclic derivatives
(1,3,4-thiadiazoles, 1,3,4-thiadiazolines, benzothiazoles,
and thienothiopyransulfonamides). Slightly less active
were the 1,3-benzenedisulfonamide and 3-fluorosulfa-
nilamide derivatives, together with the pyrimidine-
substituted sulfanilamides of type 20 and the sulfa-
nilylsulfanilamides 18 and sulfanilylmetanilamides 19.
The simple aromatic derivatives were (as expected) less
active than the previously mentioned heterocyclic sul-
fonamides. The efficiency of the obtained inhibitor
generally varied in the following way, based on the
parent sulfonamide from which it was prepared: the
derivatives of p-hydrazinobenzenesulfonamide 4 < the
orthanilamides 1 = the metanilamides 2 < the sulfa-
nilamides 3 < the homosulfanilamides 5 < the p-
(aminoethyl)benzenesulfonamides 6 = the halogeno-
substituted sulfanilamides 7-10 = the 1,3-benzene-
disulfonamides 11 and 12 = the sulfanilylsulfanilamides
18 and the sulfanilylmetanilamides 19 < the 1,3,4-
thiadiazole-2-sulfonamides 13, 15, and 16 = the 4-methyl-
δ2-1,3,4-thiadiazoline-2-sulfonamides 14 and 17 = the
benzothiazole-2-sulfonamides 21-23 = the dorzolamide
derivatives 26. On the basis of the amino acid from
which they were obtained, the Gly (A type) derivatives
were less active than the GlyGly (D type) derivatives,
which in turn were less active than the sarcosine-based
inhibitors (B type compounds). The best inhibitors in
the whole series were those derived from creatine (C
type derivatives).
Salts of the new derivatives were prepared by reacting
the free bases mentioned above with a methanolic HCl
solution. Similarly were obtained triflate or trifluoro-
acetate salts, by reaction of the previously mentioned
amines with triflic acid or TFA in water as solvent.
These salts possessed good water solubility, generally
in the range of 2-5% (by weight), i.e., in the range of
50-80 mM (Table 2). Triflates, trifluoroacetates, and
hydrochlorides possessed relatively similar water solu-
bilities. The pH of such solution was in the range of 5.5-
7.5, making them appropriate for topical application into
the eye. As seen from the data of Table 2, some of the
newly obtained compounds for which detailed pharma-
cological data were obtained possessed a relatively
moderate lipid solubility, similarly to or slightly less
than that of dorzolamide (26). In fact, Maren10 noted in
his classical review that one of the conditions needed
for a sulfonamide to act as an effective topical IOP-
lowering agent is to possess a modest lipid solubility
(attributable to its un-ionized form), accompanied by a
good water solubility (conferred by the presence of
ionizable groups of appropriate pKa).10 To illustrate the
importance of water/lipid solubility and corneal acces-
sion rates for the topical activity of a sulfonamide CA
inhibitor, data for two topically inactive, historically
important compounds, acetazolamide (28) and meth-
azolamide (29), are also shown in Table 2. These two
classical high-affinity CA inhibitors (Ki of 8 nM against
hCA II for 28 and 7 nM for 29) possess a too low lipid
solubility (acetazolamide) or an acceptable one (meth-
azolamide) correlated with a relatively high water
solubility, but only as sodium salts, RSO2NHNa, and
are thus impossible to be adminsitered via the topical
route. Both are inactive topically due to poor penetration
across the cornea, which is basically due to their very
low water solubility (in the neutral form). As seen from
the data of Table 2, the compounds reported here
possessed excellent water solubility (as HCl salts),
balanced by a modest but not insignificant lipid solubil-
ity. Their accession rates across the cornea were thus
of the same order of magnitude as or slightly better
those of dorzolamide.
All three CA isozymes investigated here were sus-
ceptible to inhibition with this type of sulfonamides,
with hCA II and bCA IV the most sensitive, whereas
hCA I was generally less susceptible to inhibition as
compared to the first two isozymes.
IOP Low er in g in Nor m oten sive a n d Gla u com a -
tou s Ra bbits. The promising in vitro CA inhibitory
activity as well as other physicochemical properties
mentioned above for some of the newly prepared com-
pounds prompted us to investigate their effect in vivo
on the IOP after topical application directly into the eye
in normotensive and glaucomatous rabbits (frequently
used as an animal model of glaucoma).3-5,11-13
The compounds selected for in vivo studies were
among the most active in vitro inhibitors against hCA
II and IV, in the prepared series, and possessed other
favorable properties such as a moderate lipid solubility,
good accession rates across the cornea, etc. Such com-
pounds included among others: A13, A15, A16, B12,
B14, B16, B18, B26, C7, C13-C15, C26, D11, D13,
D16, D23, and D26. The following facts should be noted
regarding the IOP-lowering data of Table 3. Some of the
new compounds investigated in vivo, such as A13, B12,
B14, B18, B26, C7, D11, and D23, showed IOP-
lowering effects generally of the same order of magni-
tude (or slightly better than) as those of dorzolamide
(26). Thus, after 0.5 or 1 h after adminsitration of the
new drugs, the IOP-lowering values were around 2.5-
3.3 and 5.5-7.0 mmHg, respectively (for dorzolamide
they are 2.2 and 4.1 mmHg, respectively). An important
difference between the two groups of drugs appears at
longer periods after administration, since unlike dor-
The new compounds reported in the present work
were characterized by standard chemical and physical
methods (elemental analysis, within (0.4% of the
1
theoretical values; IR; H and 13C NMR spectroscopy)
that confirmed their structure (see Experimental Sec-
tion for details) and were assayed for inhibition of
isozymes hCA I, hCA II, and bCA IV (Table 1).
In Vitr o CA In h ibition . Inhibition data against
three CA isozymes, hCA I, hCA II, and bCA IV, with
the new derivatives (Table 1) prove that the aminoacyl/
dipeptidyl moiety attached to these sulfonamides gener-

3696 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18
Scozzafava et al.
zolamide, which diminishes its power of action to an IOP
lowering of 2.7 mmHg after 90 min, the new compounds
mentioned above maintained a more effective IOP
lowering, in the range of 4.5-5.5 mmHg, comparable
to that observed at 1 h after their administration. A
second group of inhibitors, such as A16, B16, C13, C15,
D13, and D16, showed much more effective IOP-
lowering effects as compared to dorzolamide (26), both
after 30 min from administration of the inhibitor within
the rabbit eye, as well as at longer times (1, 1.5, and
2-6 h, respectively). Thus, after 30 min, the IOP
lowering was in the range of 5.0-6.6 mmHg with the
new compounds mentioned above (and only 2.2 mmHg
with dorzolamide). At 1 h after administration, the new
compounds generally fared at least doubly as well as
the clinically used drug 26 (9.0-10.5 mmHg for the new
derivatives versus 4.1 mmHg for dorzolamide), and this
strong effect was maintained after another 0.5 h (whereas
it is halved in the case of 26, for which the pressure
decrease amounts to 2.7 mmHg after 90 min). Both
hydrochlorides as well as trifluoroacetate or triflate salts
of the new derivatives possessed similar IOP-lowering
properties.
Con clu sion s
We report here a general approach for the preparation
of water-soluble, topically effective antiglaucoma sul-
fonamides, by attaching aminoacyl/dipeptidyl water-
solubilizing moieties to well-known aromatic/heterocy-
clic sulfonamides. Ring systems which have been
derivatized by the above-mentioned procedures included
2-, 3-, or 4-aminobenzenesulfonamides; 4-(ω-aminoalkyl)-
benzenesulfonamides; 3-halogeno-substituted sulfanil-
amides; 1,3-benzenedisulfonamides; 1,3,4-thiadiazole-
2-sulfonamides; and benzothiazole-2-sulfonamides as
well as thienothiopyran-2-sulfonamides among others,
and they were chosen in such a way as to prove that
the proposed approach is a general one. The new
compounds formed water-soluble salts by reaction with
strong acids, such as hydrochloric, trifluoroacetic, or
trifluoromethanesulfonic acids, with protonation of the
amino/guanidino nitrogen atom(s). These salts pos-
sessed good water solubility, in the range of 2-5%,
whereas their lipid solubility and hydrophobicity (log
P) as well as accession rates across the cornea were
those appropriate for acting as efficient topical IOP-
lowering agents. The pH of such solutions was in the
range of 6.5-7.5. Many of the reported 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
dorzolamide. The new compounds reported here might
lead to the development of more efficient and inexpen-
sive antiglaucoma drugs (the presently available topical
antiglaucoma sulfonamides dorzolamide and brinzola-
mide are quite expensive drugs, whereas the patients
mostly affected are generally the elderly of more than
60 years).
An important feature of the new class of CA inhibitors
reported here is that IOP remained low for longer
periods (3-6 h) after their topical administration, as
compared to the standard drug dorzolamide (Figure 2).
As seen from Figure 2, compounds such as C13 or C15
possessed maximal IOP-lowering effects at 1 h after
administration, similarly to dorzolamide. The main
difference between them was that the new compounds
acted as much more potent IOP-lowering agents and
that at 3 or 4 h after administration (when the effects
of dorzolamide completely vanished) they still dimin-
ished eye pressure appreciably (5.0-6.5 mmHg). IOP
generally returned at the baseline values after 5-6 h
after administration of the drug. Thus, all these deriva-
tives are longer-lasting IOP-lowering agents as com-
pared to the clinically available drug dorzolamide.
Exp er im en ta l Section
Gen er a l. Melting points, heating plate microscope (not
corrected); IR spectra, KBr pellets, 400-4000 cm-1 Perkin-
Elmer 16PC FTIR spectrometer; 1H NMR spectra, Varian
300CXP apparatus (chemical shifts are expressed as δ values
relative to Me4Si as standard); elemental analysis, Carlo Erba
Instrument CHNS elemental analyzer model 1106. All reac-
tions were monitored by thin-layer chromatography (TLC)
using 0.25-mm precoated silica gel plates (E. Merck). Analyti-
cal and preparative HPLC was performed on a reversed-phase
The above findings also apply for the glaucomatous
rabbits experiments (Table 4), but the IOP values are
much more important as compared to those of normo-
tensive rabbits. Thus, IOP reductions of 6.5-8.6 mmHg
were generally observed after 30 min, whereas at longer
periods, these amounted to 12.0-14.5 mmHg. No im-
portant differences between the different salts were
observed. The long-lasting effect mentioned above has
also been evidenced for the glaucomatous rabbit experi-
ments (data not shown).
C
18 Bondapack column, with a Beckman EM-1760 instrument.
Sulfonamides 1-26 used in syntheses were either com-
mercially available compounds (from Sigma, Acros, or Aldrich)
or were prepared as described previously: 4-hydrazinoben-
zenesulfonamide (4) by diazotization of sulfanilamide followed
by reduction of the diazonium salt with tin(II) chloride;34
halogenosulfanilamides 7-10 by halogenation of sulfanilamide
as reported in the literature;35 compound 15 from 5-amino-
1,3,4-thiadiazole-2-sulfonamide (obtained from acetazola-
mide)36 by acylation with the phthalimido derivative of â-ala-
nine, followed by hydrazinolysis;1 imine 14 by deprotection of
methazolamide with concentrated hydrochloric acid;36 ami-
nobenzolamide 16 and the corresponding thiadiazoline 17 as
reported in ref 51; sulfanilylsulfanilamide 18 and its meta
derivative 19 as reported in ref 16. The benzothiazole-2-
sulfonamide derivatives 21-23 were prepared as described in
ref 37, whereas the alcohols 24 and 25 were prepared from
the corresponding amines by diazotization followed by hy-
drolysis of the diazonium salts. Dorzolamide (26) was prepared
as described in the literature38 or was obtained from Merck,
Sharp and Dohme. Amino acid and dipeptide derivatives; Boc-
Dr u g Distr ibu tion in Ocu la r F lu id s a n d Tissu es.
Table 5 shows ex vivo data obtained in normotensive
rabbits after the topical administration of two of the
most potent topical inhibitors in the prepared series,
i.e., compounds C13 and C15. It can be observed that
at 1 and 2 h after topical administration of the drug,
high levels of inhibitors were found in the cornea,
aqueous humor, and ciliary processes. On the basis of
the inhibition constant of these compounds (5 nM for
CA II for C13 and 2 nM for CA II for C15, respectively),
the fractional inhibition estimated in these tissues/fluids
is 99.5-99.9%, indicating the fact that the IOP decrease
is indeed due to CA inhibition.

Carbonic Anhydrase Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3697
On, tritylsulfenyl chloride, EDCI, diisopropylcarbodiimide,
TFA, triflic acid, and triethylamine were from Sigma Chemical
Co. Acetonitrile, dioxane (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.
(amide III), 1363 (SO2as), 1564 (amide II), 1715 (amide I), 3060
1
(NH), 3365 (NH2); H NMR (TFA) δ 3.23 (s, 3H, Me); 3.69 (s,
2H, CH2 of Crt); 7.25 (br s, 2H, SO2NH2); 13C NMR (TFA) δ
33.7 (s, Me of Crt); 40.2 (s, CH2 of Crt); 159.5 (C-2 of
thiadiazole); 160.8 (s, NC(dNH)NH2 of Crt), 170.5 (C-5 of
thiadiazole); 171.3 (CONH). Anal. Found: C, 24.69; H, 4.01;
N, 33.35. C6H11N7O3S2 Requires: C, 24.57; H, 3.78; N, 33.43.
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
(A-D)1-26. An amount of 1 mM sulfonamide 1-26 was
dissolved/suspended in 25 mL of anhydrous acetonitrile and
then treated with the stoichiometric amount (1 mM) of
protected amino acid/dipeptide 30-33. An amount of 190 mg
(1 mM) of EDCI‚HCl was then added, and the reaction mixture
was magnetically stirred at room temperature for 15 min; then
30 µL (2 mM) of triethylamine was added, and stirring was
continued for 8-16 h at 4 °C (TLC control). The solvent was
evaporated in vacuo and the residue taken up in ethyl acetate
(5 mL), poured into a 5% solution of sodium bicarbonate (5
mL), and extracted with ethyl acetate. The combined organic
layers were dried over sodium sulfate and filtered, and the
solvent was removed in vacuo. The obtained oils were directly
used in the deprotection step, or the intermediates were
recrystallized from ethanol-water or methanol. The removal
of the protecting groups was performed as described below.
For Boc-protected compounds: the crude intermediate was
dissolved in 20 mL of CH2Cl2, treated with 4 mL of TFA, and
stirred for 10 min at 0 °C. The solvent was removed in vacuo
and the residue concentrated from water twice to remove
excess TFA, thus giving the amine salt as a colorless syrup.
The pure compounds (A-D)1-26 were obtained by means of
preparative HPLC (C18 reversed-phase µ-Bondapack or Dy-
namax-60A (25 × 250 mm) columns; 90% acetonitrile/8%
methanol/2% water, 30 mL/min). For the tritylsulfenylated
compounds: the crude product was dissolved in 20 mL of
dioxane and treated with 25 mL of a 4 M HCl solution in
dioxane, followed by heating at 40 °C for 2-3 h (TLC control).
The solvent was evaporated in vacuo, the residue taken in 5%
solution of sodium bicarbonate (50 mL), and the TrSCl formed
in the reaction extracted with 2 × 50 mL of diethyl ether. The
aqueous phase was then worked up as described above for the
Boc-protected derivatives.
Hydrochlorides of the new derivatives were obtained from
the free bases and a methanolic HCl solution, in methanol as
solvent. The hydrochlorides precipitated by leaving the above
mixtures at 4 °C overnight. The hydrochlorides were analyzed
for the presence of Cl- by potentiometric titrations. The
obtained data were (0.5% of the theoretical data calculated
for the proposed formulas (data not shown). Triflate and
trifluoroacetate salts were similarly obtained from the free
bases and the stoichiometric amount of triflic acid/TFA, in
water as solvent.
4-Glycin a m id oben zen esu lfon a m id e (A3): white crys-
tals, mp 294-6 °C dec; IR (KBr) cm-1 1152 (SO2sym), 1293
(amide III), 1345 (SO2as), 1560 (amide II), 1710 (amide I), 3360
(NH, NH2); 1H NMR (TFA) δ 3.67 (s, 2H, CH2 of Gly); δA 7.18,
δB 7.75 (AA′BB’system, 4H, J AB ) 7.9, ArH from 4-sulfa-
moylphenyl); 7.59 (br s, 2H, SO2NH2) (the H2N and CONH
protons are in fast exchange with the solvent and are not seen
in TFA); 13C NMR (TFA) δ 40.8 (s, CH2 of Gly); 129.9; 132.5;
135.9; 142.5; 168.0 (CONH). Anal. Found: C, 42.13; H, 5.02;
N, 18.05. C8H11N3O3S Requires: C, 41.91; H, 4.84; N, 18.33.
5,6-Dih yd r o-4-(N-glycylglycin a m id o-N-eth yl)-6-m eth -
yl-4H-th ien o[2,3-b]th iop yr a n -2-su lfon a m id e 7,7-d ioxid e
(D26): white crystals, mp 288-90 °C dec; IR (KBr) cm-1 1133
(SO2sym), 1292 (amide III), 1345 (SO2as), 1565 (amide II), 1720
1
(amide I), 3060 (NH), 3365 (NH2); H NMR (TFA) δ 1.29 (d,
3H, Me); 1.39 (t, 3H, Me from ethyl);: 2.49 (m, 4H, J ) 7.0,
2CH2 of GlyGly); 2.55 (m, 1H, CH); 2.80 (m, 1H, CH); 3.05-
3.20 (m, 2H, CH2 from ethyl); 4.37 (m, 2H, CH2); 8.03 (s, 1H,
CH, ArH from thienyl); 8.25 (br s, 2H, SO2NH2); 13C NMR
(TFA) δ 10.0; 11.1; 30.6; 35.8 (s, CH2 of GlyGly); 40.8; 49.3;
51.5; 130.7; 137.5; 141.9; 149.8; 167.1 (CONH); 172.5 (CONH).
Anal. Found: C, 38.17; H, 5.23; N, 12.59. C14H22N4O6S3
Requires: C, 38.34; H, 5.06; N, 12.78.
En zym e P r ep a r a tion s. Human CA I and CA II cDNAs
were expressed in Escherichia coli strain BL21 (DE3) from the
plasmids pACA/hCA I and pACA/hCA II described by Lindskog
et al.39 (the two plasmids were a gift from Prof. Sven Lindskog,
Umea University, Sweden). Cell growth conditions were those
described by this group,40 and enzymes were purified by
affinity chromatography according to the method of Khalifah
et al.41 Enzyme concentrations were determined spectropho-
tometrically at 280 nm, utilizing a molar absorptivity of 49
mM-1‚cm-1 for CA I and 54 mM-1‚cm-1 for CA II, respectively,
based on Mr ) 28.85 kDa for CA I and 29.30 kDa for CA II,
respectively.42,43 CA IV was isolated from bovine lung mi-
crosomes as described by Maren et al., and its concentration
has been determined by titration with ethoxzolamide.44
Initial rates of 4-nitrophenyl acetate hydrolysis catalyzed
by different CA isozymes were monitored spectrophotometri-
cally, at 400 nm, with a Cary 3 instrument interfaced with an
IBM-compatible PC.45 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.45 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.45 Enzyme concentrations were
3.5 nM for hCA II, 12 nM for hCA I, and 36 nM for bCA IV
(this isozyme has a decreased esterase activity46 and higher
concentrations had to be used for the measurements).
Mea su r em en t of Ton om etr ic IOP . Adult male New
Zealand albino rabbits weighing 3-3.5 kg were used in the
experiments (three animals were used for each inhibitor
studied). The experimental procedures conform to the Associa-
tion for Research in Vision and Ophthalmology Resolution on
the use of animals. The rabbits were kept in individual cages
with food and water provided ad libitum. The animals were
maintained on a 12 h:12 h light/dark cycle in a temperature-
controlled room, at 22-26 °C. Solutions 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.
4-(Sa r cosin yla m id oet h yl)b en zen esu lfon a m id e (B6):
white crystals, mp 240-2 °C; IR (KBr) cm-1 1158 (SO2sym),
1295 (amide III), 1355 (SO2as), 1562 (amide II), 1711 (amide
1
3
I), 3365 (NH, NH2); H NMR (TFA) δ 3.03 (t, 2H, J HH ) 6.7,
RCH2); 3.13 (s, 3H, N-Me); 3.70 (s, 2H, CH2 of Sar); 3.78 (t,
2H, J HH ) 6.7, âCH2); δA 7.21, δB 7.79 (AA′BB’system, 4H,
3
J AB ) 8.2, ArH from 4-sulfamoylphenyl); 7.51 (br s, 2H, SO2-
NH2) (the H2N and CONH protons are in fast exchange with
the solvent and are not seen in TFA); 13C NMR (TFA) δ 30.3
(s, CH2 of aminoethyl); 33.7 (s, Me of Crt); 37.5 (s, CH2 of
aminoethyl); 41.5 (s, CH2 of Sar); 130.7; 132.8; 134.3; 145.5;
170.2 (CONH). Anal. Found: C, 48.54; H, 6.45; N, 15.33.
IOP was measured using a Digilab 30R pneumatonometer
(BioRad, Cambridge, MA) as described by Maren’s group.47,48
The pressure readings were matched with two-point standard
pressure measurements at least twice each day using a Digilab
C
11H17N3O3S Requires: C, 48.69; H, 6.32; N, 15.49.
5-Cr eatin yl-1,3,4-th iadiazole-2-su lfon am ide (C13): white
crystals, mp > 300 °C; IR (KBr) cm-1 1180 (SO2sym), 1291

3698 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18
Scozzafava et al.
Calibration verifier. All IOP measurements were done by the
same investigator with the same tonometer. One drop of 0.2%
oxybuprocaine hydrochloride (novesine, Sandoz) diluted 1:1
with saline was instilled in each eye immediately before each
set of pressure measurements. IOP was measured three times
at each time interval, and the means were reported. IOP was
measured first immediately before drug administration, then
at 30 min after the instillation of the pharmacological agent,
and then each 30 min for a period of 4-6 h. For all IOP
experiments 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.47,48 All data are expressed
as mean ( SE, using a one-tailed t-test. Ocular hypertension
was elicited in the right eye of albino rabbits by injection of
R-chymotrypsin (Sigma) as described by Sugrue et al.49 The
IOP of operated animals was checked after approximately 4
weeks, and animals with an elevated pressure of 30-36 mmHg
were used at least 1 month after the injection of R-chymo-
trypsin.
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.47,48 The
animals were killed with an intracardiac air injection. Aqueous
humor (both posterior and anterior chamber fluids) were
withdrawn. Then, the cornea and anterior uvea (iris plus
attached ciliary body) were dissected, rinsed well with water,
blotted, weighed, and put into 1-2 mL of water. For isolation
of the ciliary processes, intact anterior uvea rings were placed
on a Parafilm-covered piece of polystyrene foam in a Petri dish.
The tissue 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
distilled water. The tissue from 4 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 activities 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.47,48
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).51
et al.52,53 or enzymatically.5,11 The results of the drug analyses
were used to calculate the rate constant of transfer across the
cornea (kin). As described by Pierce,52,53 this value was deter-
mined 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. Thanks are addressed
to Drs. M. A. Ilies, B. Iorga, and M. Barboiu for expert
technical assistance and to Dr. A. Liljas for providing
the X-ray coordinates of the aminobenzolamide-hCA
II adduct.
Refer en ces
(1) This paper is part 77 of the series. Preceding part: Barboiu, M.;
Supuran, C. T.; Scozzafava, A.; Menabuoni, L.; Mincione, F.;
Briganti, F.; Mincione, G. Carbonic anhydrase inhibitors. Part
76. Synthesis of topically effective intraocular pressure lowering
agents derived from 5-(ω-aminoalkylcarboxamido)-1,3,4-thia-
diazole-2-sulfonamide. J . Enzyme Inhib. 1999, 14, in press.
(2) 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.
(3) Borras, J .; Scozzafava, A.; Menabuoni, L.; Mincione, F.; Briganti,
F.; Mincione, G.; Supuran, C. T. Carbonic anhydrase inhibi-
tors: Synthesis of water-soluble, topically effective intraocular
pressure lowering aromatic/heterocyclic sulfonamides containing
8-quinoline-sulfonyl moieties: is the tail more important than
the ring? Bioorg. Med. Chem. 1999, in press.
(4) Supuran, C. T. Carbonic anhydrase inhibitors. In Carbonic
Anhydrase and Modulation of Physiologic and Pathologic Pro-
cesses in the Organism; Puscas, I., Ed.; Helicon: Timisoara,
Roumania, 1994; pp 29-111.
(5) Supuran, C. T.; Scozzafava, A.; Ilies, M. A.; Iorga, B.; Cristea,
T.; Briganti, F.; Chiraleu, F.; Banciu, M. D. Carbonic anhydrase
inhibitors. Part 53. Synthesis of substituted-pyridinium deriva-
tives of aromatic sulfonamides: The first nonpolymeric mem-
brane-impermeable inhibitors with selectivity for isozyme IV.
Eur. J . Med. Chem. 1998, 33, 577-595. Supuran, C. T.; Minci-
one, F.; Scozzafava, A.; Briganti, F.; Mincione, G.; Ilies, M. A.
Carbonic anhydrase inhibitors. Part 52. Metal complexes of
heterocyclic sulfonamides - A new class of strong topical
intraocular pressure-lowering agents in rabbits. Eur. J . Med.
Chem. 1998, 33, 247-254.
(6) Maren, T. H. Carbonic anhydrase: general perspectives and
advances in glaucoma research. Drug Dev. Res. 1987, 10, 255-
276.
(7) Supuran, C. T.; Conroy, C. W.; Maren, T. H. Carbonic anhydrase
inhibitors: synthesis and inhibitory properties of 1,3,4-thiadia-
zole-2,5-bissulfonamide. Eur. J . Med. Chem. 1996, 31, 843-846.
(8) Maren, T. H. Carbonic anhydrase: Chemistry, physiology and
inhibition. Physiol. Rev. 1967, 47, 595-782.
(9) Becker, B. The mechanism of the fall of intraocular pressure
induced by the carbonic anhydrase inhibitor Diamox. Am. J .
Ophthalmol. 1955, 39, 177-183.
(10) Maren, T. H. The development of topical carbonic anhydrase
inhibitors. J . Glaucoma 1995, 4, 49-62.
(11) 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.
(12) Katritzky, A. R.; Caster, K. C.; Maren, T. H.; Conroy, C. W.; Bar-
Ilan, A. Synthesis and physicochemical properties of thiadiazolo-
[3,2-a]pyrimidinesulfonamides and thiadiazolo[3,2-a]triazine-
sulfonamides as candidates for topically effective carbonic
anhydrase inhibitors. J . Med. Chem. 1987, 30, 2058-2062.
(13) Ponticello, G. S.; Freedman, M. B.; Habecker, C. N.; Lyle, P. A.;
Schwam, H.; Varga, S. L.; Christy, M. E.; Randall, W. C.;
Baldwin, J . J . Thienothiopyran-2-sulfonamides: A novel class
of water-soluble carbonic anhydrase inhibitors. J . Med. Chem.
1987, 30, 591-597.
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.5,51
Tr a n scor n ea l P en etr a tion of Dr u gs. The method of
Maren et al.11 with the modifications of Pierce’s group52,53 (for
the HPLC assay of sulfonamides) was been 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 by both the HPLC method of Pierce
(14) Supuran, C. T.; Popescu, A.; Ilisiu, M.; Costandache, A.; Banciu,
M. D. Carbonic anhydrase inhibitors. Part 36. Inhibition of
isozymes I and II with Schiff bases derived from chalkones and
aromatic/heterocyclic sulfonamides. Eur. J . Med. Chem. 1996,

Carbonic Anhydrase Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3699
31, 439-448. Supuran, C. T.; Scozzafava, A.; Popescu, A.; Bobes-
Tureac, R.; Banciu, A.; Creanga, A.; Bobes-Tureac, G.; Banciu,
M. D. Carbonic anhydrase inhibitors Part 43. Schiff bases
derived from aromatic sulfonamides: towards more specific
inhibitors for membrane-bound versus cytosolic isozymes. Eur.
J . Med. Chem. 1997, 32, 445-452.
carbonic anhydrase. J . Med. Chem. 1993, 36, 126-133. Gao, J .
M.; Qiao, S.; Whitesides, G. M. Increasing binding constants of
ligands to carbonic anhydrase by using “greasy tails”. J . Med.
Chem. 1995, 38, 2292-2301. Gao, J . M.; Cheng, X. H.; Chen, R.
D.; Sigal, G. B.; Bruce, J . E.; Schwartz, B. L.; Hofstadler, S. A.;
Anderson, G. A.; Smith, R. D.; Whitesides, G. M. Screening
derivatized peptide libraries for tight binding inhibitors to
carbonic anhydrase II by electrospray ionization - mass spec-
troscopy. J . Med. Chem. 1996, 39, 1949-1955.
(15) Supuran, C. T.; Briganti, F.; Scozzafava, A. Sulfenamido-
sulfonamides as inhibitors of carbonic anhydrase isozymes I, II
and IV. J . Enzyme Inhib. 1997, 12, 175-190. Briganti, F.;
Pierattelli, R.; Scozzafava, A.; Supuran, C. T. Carbonic anhy-
drase inhibitors Part 37. Novel classes of isozyme I and II
inhibitors and their mechanism of action. Kinetic and spectro-
scopic investigations on native and cobalt-substituted enzymes.
Eur. J . Med. Chem. 1996, 31, 1001-1010. Mincione, F.; Mena-
buoni, L.; Briganti, F.; Mincione, G.; Scozzafava, A.; Supuran,
C. T. Carbonic anhydrase inhibitors: Inhibition of isozymes I,
II and IV with N-hydroxy-sulfonamides - A novel class of
intraocular pressure lowering agents. IV. J . Enzyme Inhib. 1998,
13, 267-284.
(16) Supuran, C. T.; Scozzafava, A. Novel aromatic/heterocyclic
sulfonamides and their metal complexes as inhibitors of carbonic
anhydrase isozymes I, II and IV. J . Enzyme Inhib. 1997, 12, 37-
51. Scozzafava, A.; Supuran, C. T. Carbonic anhydrase inhibi-
tors: Novel compounds containing S-NH moieties: sulfenamido-
sulfonamides, sulfenimido-sulfonamides and their interaction
with isozymes I, II and IV. J . Enzyme Inhib. 1998, 13, 419-
442.
(25) (a) Liljas, A.; Hakansson, K.; J onsson, B. H.; Xue, Y. Inhibition
and catalysis of carbonic anhydrase. Recent crystallographic
analyses. Eur. J . Biochem. 1994, 219, 1-10. (b) Vidgren, J .;
Svensson, A.; Liljas, A. Refined structure of the aminobenzola-
mide complex of human carbonic anhydrase II at 1.9 Å and
sulphonamide modelling of bovine carbonic anhydrase III. Int.
J . Biol. Macromol. 1993, 15, 97-100.
(26) Cappalonga, A. M.; Alexander, R. S.; Christianson, D. W.
Mapping protein-peptide affinity: Binding of peptidylsulfona-
mide inhibitors to human carbonic anhydrase II. J . Am. Chem.
Soc. 1994, 116, 5063-5068.
(27) 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.
(28) Briganti, F.; Iaconi, V.; Mangani, S.; Orioli, P.; Scozzafava, A.;
Vernaglione, G.; Supuran, C. T. A ternary complex of carbonic
anhydrase: X-ray crystallographic structure of the adduct of
human carbonic anhydrase II.with the activator phenylalanine
and the inhibitor azide. Inorg. Chim. Acta 1998, 275-276, 295-
300.
(29) Antonaroli, S.; Bianco, A.; Brufani, M.; Cellai, L.; Lo Baido, G.;
Potier, E.; Bonomi, L.; Perfetti, S.; Fiaschi, A. I.; Segre, G.
Acetazolamide-like carbonic anhydrase inhibitors with topical
ocular hypotensive activity. J . Med. Chem. 1992, 35, 2697-2703.
(30) J ayaweera, G. D. S. A.; MacNeil, S. A.; Trager, S. F.; Blackburn,
G. M. Synthesis of 2-substituted-1,3,4-thiadiazole-5-sulphona-
mides as novel water-soluble inhibitors of carbonic anhydrase.
Bioorg. Med. Chem. Lett. 1991, 1, 407-410.
(17) Ponticello, G. S.; Sugrue, M. F.; Plazonnet, B.; Durand-Cavagna,
G. Dorzolamide, a 40-year wait. From an oral to a topical
carbonic anhydrase inhibitor for the treatment of glaucoma.
Pharmacol. Biotechnol. 1998, 11, 555-574.
(18) Woltersdorf, O. W.; Schwam, H.; Bicking, J . B.; Brown, S. L.;
deSolms, S. J .; Fishman, D. R.; Graham, S. L.; Gautheron, P.
D.; Hoffman, J . M.; Larson, R. D.; Lee, W. S.; Michelson, S. R.;
Robb, C. M.; Share, C. N.; Shepard, K. L.; Smith, A. M.; Smith,
R. L.; Sondey, J . M.; Strohmeyer, K. M.; Sugrue, M. F.; Viader,
M. P. Topically active carbonic anhydrase inhibitors. 1. O-Acyl
derivatives of hydroxybenzothiazole-2-sulfonamide. J . Med.
Chem. 1989, 32, 2486-2492. Graham, S. L.; Shepard, K. L.;
Anderson, P. S.; Baldwin, J . J .; Best, D. B.; Christy, M. E.;
Freedman, M. B.; Gautheron, P.; Habecker, C. N.; Hoffman, J .
M.; Lyle, P. A.; Michelson, S. R.; Ponticello, G. S.; Robb, C. M.;
Schwam, H.; Smith, A. M.; Smith, R. L.; Sondey, J . M.;
Strohmaier, K. M.; Sugrue, M. F.; Varga, S. L. Topically active
carbonic anhydrase inhibitors. 2. Benzo[b]thiophenesulfonamide
derivatives with ocular hypotensive activity. J . Med. Chem.
1989, 32, 2548-2554.
(31) Burbaum, N. J .; Ohlmeyer, M. H. J .; Reader, J . C.; Henderson,
I.; Cillard, L. W.; Li, G.; Randle, T. L.; Sigal, N. H.; Chelsky, D.;
Baldwin, J . J . A paradigm for drug discovery employing encoded
combinatorial libraries. Proc. Natl. Acad. Sci. U.S.A. 1995, 92,
6027-6031.
(19) Hartman, G. D.; Halczenko, W. The synthesis of 5-alkylami-
nomethylthieno[2,3-b]pyrrole-5-sulfonamides. Heterocycles 1989,
29, 1943-1949. Prugh, J . D.; Hartman, G. D.; Mallorga, P. J .;
McKeever, B. M.; Michelson, S. R.; Murcko, M. A.; Schwam, H.;
Smith, R. L.; Sondey, J . M.; Springer, J . B.; Sugrue, M. F. New
isomeric classes of topically active ocular hypotensive carbonic
anhydrase inhibitors: 5-substituted thieno[2,3-b]thiophene-2-
sulfonamides and 5-substituted thieno[3,2-b]thiophene-2-sul-
fonamides. J . Med. Chem. 1991, 34, 1805-1818.
(20) Hartman, G. D.; Halczenko, W.; Prugh, J . D.; Smith, R. L.;
Sugrue, M. F.; Mallorga, P. J .; Michelson, S. R.; Randall, W. C.;
Schwam, H.; Sondey, J . M. Thieno[2,3-b]furan-2-sulfonamides
as topical carbonic anhydrase inhibitors. J . Med. Chem. 1992,
35, 3027-3033. Baldwin, J . J .; Ponticello, G. S.; Anderson, G.
S.; Christy, M. E.; Murcko, M. A.; Randall, W. C.; Schwam, H.;
Sugrue, M. F.; Springer, J . B.; Gautheron, P.; Grove, J .;
Mallorga, P.; Viader, M. P.; McKeever, B. M.; Navia, M. A.
Thienothiopyran-2-sulfonamides: Novel topically active carbonic
anhydrase inhibitors for the treatment of glaucoma. J . Med.
Chem. 1989, 32, 2510-2513.
(21) Silver, L. H. Clinical efficacy and safety of brinzolamide (Azopt),
a new topical carbonic anhydrase inhibitor for primary open-
angle glaucoma and ocular hypertension. Am. J . Ophthalmol.
1998, 126, 400-408.
(22) Balfour, J . A.; Wilde, M. I. Dorzolamide. A review of its
pharmacology and therapeutic potential in the management of
glaucoma and ocular hypertension. Drugs Aging 1997, 10, 384-
403. Aalto-Korte, K. Contact allergy to dorzolamide eyedrops.
Contact Dermatitis 1998, 39, 206.
(23) J ain, A.; Whitesides, G. M.; Alexander, R. S.; Christianson, D.
W. Identification of two hydrophobic patches in the active-site
cavity of human carbonic anhydrase II by solution-phase and
solid-state studies and their use in the development of tight-
binding inhibitors. J . Med. Chem. 1994, 37, 2100-2105. Boriack,
P. A.; Christianson, D. W.; Kingery-Wood, J .; Whitesides, G. M.
Secondary interactions significantly removed from the sulfona-
mide binding pocket of carbonic anhydrase II influence inhibitor
binding constants. J . Med. Chem. 1995, 38, 2286-2291.
(24) Avila, L. Z.; Chu, Y. H.; Blossey, E. C.; Whitesides, G. M. Use of
affinity capillary electrophoresis to determine kinetic and equi-
librium constants for binding of arylsulfonamides to bovine
(32) 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. 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. 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. Supuran, C. T.; Clare,
B. W. Carbonic anhydrase inhibitors Part 47. Quantum chemical
quantitative structure-activity relationships for
a group of
sulfanilamide Schiff base inhibitors of carbonic anhydrase. Eur.
J . Med. Chem. 1998, 33, 489-500.
(33) Supuran, C. T.; Scozzafava, A. Novel carbonic anhydrase isozymes
I, II and IV activators incorporating sulfonyl-histamino moieties.
Bioorg. Med. Chem. Lett. 1999, 9, 2043-2048.
(34) Crippa, G. B.; Maffei, S. Derivati solfonamidici del pirazolo. 1-(p-
solfonamidofenil)-3-metil-5-pirazolone. Gazz. Chim. Ital. 1941,
71, 97-99.
(35) Cingolani, E. Sulla alogenazione della p-aminobenzenesolfonam-
mide (derivatialogenati nucleari). Gazz. Chim. Ital. 1948, 78,
275-282.
(36) J itianu, A.; Ilies, M. A.; Scozzafava, A.; Supuran C. T. Complexes
with biologically active ligands. Part 8. Synthesis and carbonic
anhydrase inhibitory activity of 5-benzoylamido- and 5-(3-
nitrobenzoylamido)-1,3,4-thiadiazole-2-sulfonamide and their
metal complexes. Main Group Met. Chem. 1997, 20, 147-153.
(37) Eller, M. G.; Schoenwald, R. D.; Dixson, J . A.; Segarra, T.;
Barfknecht, C. F. Topical carbonic anhydrase inhibitors. III.
Optimization model for corneal penetration of ethoxzolamide
analogues. J . Pharm. Sci. 1985, 74, 155-160.
(38) Blacklock, T. J .; Sohar, P.; Butcher, J . W.; Lamanec, T.;
Grabowski, E. J . J . An enantioselective synthesis of the topically
active carbonic anhydrase inhibitor MK-0507: 5,6-dihydro-(S)-
4-(ethylamino)-(S)-6-methyl-4H-thieno[2,3-b]thiopyran-2-sul-
fonamide 7,7-dioxide hydrochloride. J . Org. Chem. 1993, 58,
1672-1679.

3700 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18
Scozzafava et al.
(39) 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, 1991; pp 1-13.
(40) 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.
(41) 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.
(42) Lindskog, S.; Coleman, J . E. The catalytic mechanism of carbonic
anhydrase. Proc. Natl. Acad Sci. U.S.A. 1964, 70, 2505-2508.
(43) 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.
(44) 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.
anhydrase inhibitor ethoxzolamide. Exp. Eye Res. 1992, 55, 73-
79.
(48) 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.
(49) Sugrue, M. F.; Gautheron, P.; Mallorga, P.; Nolan, T. E.;
Graham, S. L.; Schwam, H.; Shepard, K. L.; Smith, R. L. L-662,-
583 is a topically effective ocular hypotensive carbonic anhydrase
inhibitor in experimental animals. Br. J . Pharmacol. 1990, 99,
59-64.
(50) Stams, T.; Chen, Y.; Boriack-Sjodin, P. A.; Hurt, J . D.; Liao, J .;
May, J . A.; Dean, T.; Laipis, P.; Christianson, D. W. Structures
of murine carbonic anhydrase IV and human carbonic anhydrase
II complexed with brinzolamide: Molecular basis of isozyme-
drug discrimination. Protein Sci. 1998, 7, 556-563.
(51) 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.
(52) 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.
(53) 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.
(45) 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.
(46) 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.
(47) Maren, T. H.; Brechue, W. F.; Bar-Ilan, A. Relations among IOP
reduction, ocular disposition and pharmacology of the carbonic
J M9901879