Carbonic anhydrase inhibitors: Topically acting antiglaucoma sulfonamides incorporating esters and amides of 3- and 4-carboxybenzolamide

Article abstract of DOI:10.1016/S0960-894X(03)00580-8

Reaction of 3- and 4-carboxybenzenesulfonyl chloride with 5-amino-1,3,4-thiadiazole-2-sulfonamide/5-imino-4-methyl-δ ²-1,3,4-thiadiazoline-2-sulfonamide afforded two series of benzolamide analogues to which the carboxyl moiety has been derivatized as esters or amides, in order to reduce their very polar character. The new derivatives showed low nanomolar affinity for three carbonic anhydrase (CA) isozymes, CA I, II and IV, and were effective as topical antiglaucoma agents in normotensive rabbits. Efficacy of several of the new sulfonamides reported was better than that of the standard drugs dorzolamide and brinzolamide, whereas their duration of action was prolonged as compared to that of the clinically used drugs.

Full text of DOI:10.1016/S0960-894X(03)00580-8

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2867–2873
Carbonic Anhydrase Inhibitors: Topically Acting Antiglaucoma
Sulfonamides Incorporating Esters and Amides of 3- and
4-Carboxybenzolamide
Angela Casini,a Andrea Scozzafava,a Francesco Mincione,b Luca Menabuoni,c
Michele Starnottid and Claudiu T. Supurana,*
a
`
Universita degli Studi, Laboratorio di Chimica Inorganica e Bioinorganica, Via della Lastruccia 3, Rm. 188;
50019 Sesto Fiorentino, Florence, Italy
bU.O. Oculistica Az. USL 3, Val di Nievole, Ospedale di Pescia, Pescia, Italy
cCasa di Cura Villa Donatello, Piazza Donatello, 14, 50100 Florence, Italy
dClinica Oculistica, Viale Morgagni 85, I-50134 Florence, Italy
Received 15April 2003; revised 2 June 2003; accepted 3 June 2003
Abstract—Reaction of 3- and 4-carboxybenzenesulfonyl chloride with 5-amino-1,3,4-thiadiazole-2-sulfonamide/5-imino-4-methyl-
d2-1,3,4-thiadiazoline-2-sulfonamide afforded two series of benzolamide analogues to which the carboxyl moiety has been deriva-
tized as esters or amides, in order to reduce their very polar character. The new derivatives showed low nanomolar affinity for three
carbonic anhydrase (CA) isozymes, CA I, II and IV, and were effective as topical antiglaucoma agents in normotensive rabbits.
Efficacy of several of the new sulfonamides reported was better than that of the standard drugs dorzolamide and brinzolamide,
whereas their duration of action was prolonged as compared to that of the clinically used drugs.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
conversion between carbon dioxide and the bicarbonate
ion, and are thus involved in crucial physiological pro-
cesses connected with respiration and transport of CO2/
bicarbonate between metabolizing tissues and the lungs,
pH and CO2 homeostasis, electrolyte secretion in a
variety of tissues/organs, biosynthetic reactions (such as
the gluconeogenesis, lipogenesis and ureagenesis), bone
The carbonic anhydrases (CAs, EC 4.2.1.1) are ubiqui-
tous zinc enzymes, present in Archaea, prokaryotes and
eukariotes, being encoded by three distinct, evolution-
arily unrelated gene families: the a-CAs (present in ver-
tebrates, bacteria, algae and cytoplasm of green plants),
the b-CAs (predominantly in bacteria, algae and chloro-
plasts of both mono- as well as dicotyledons) and the
g-CAs (mainly in Archaea and some eubacteria),
respectively.1,2 In higher vertebrates, including humans,
14 different CA isozymes or CA-related proteins
(CARP) were described, with very different subcellular
localization and tissue distribution.1,2 Basically, there
are several cytosolic forms (CA I-III, CA VII), four
membrane-bound isozymes (CA IV, CA IX, CA XII
resorption, calcification, tumorigenicity, and many
1,2
other physiologic or pathologic processes.
Many of
these isozymes were important targets for the design of
inhibitors with clinical applications, such as for obtain-
ing different diuretic types, or drugs useful in the treat-
ment and prevention of a variety of diseases such as
glaucoma, epilepsy, congestive heart failure, mountain
sickness, gastric and duodenal ulcers, epilepsy and other
neurological disorders, or osteoporosis among others.1-3
Several such derivatives (acetazolamide AZA, methazo-
lamide MZA, ethoxzolamide EZA and dichloro-
phenamide DCP) were successfully used in clinical
medicine for the last 45years. More recently, dorzol-
amide DZA and brinzolamide BRZ have also found
important therapeutic applications as ocular (mainly
antiglaucoma) drugs.1ꢀ3
and CA XIV), one mitochondrial form (CA V) as well
1,2
as a secreted CA isozyme, CA VI. These enzymes
catalyze a very simple physiological reaction, the inter-
*Corresponding author. Tel.: +39-055-457-3005; fax: +39-055-457-
3385; e-mail: claudiu.supuran@unifi.it
0960-894X/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00580-8

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A. Casini et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2867–2873
Two main classes of CA inhibitors (CAIs) are known:
the metal complexing anions, and the unsubstituted
sulfonamides, which bind to the Zn(II) ion of the
enzyme either by substituting the non-protein zinc
ligand, or add to the metal coordination sphere gen-
erating trigonal-bipyramidal species1ꢀ3. Sulfonamides,
which are the most important CAIs bind in a tetra-
hedral geometry of the Zn(II) ion, in deprotonated
state, with the nitrogen atom of the sulfonamide moi-
ety coordinated to Zn(II); anions may bind either in
tetrahedral geometry of the metal ion, or as trigonal-
bipyramidal adducts.1ꢀ3 X-ray crystallographic struc-
tures are available for many adducts of sulfonamide
inhibitors with isozymes CA I, II and IV.2,4,5 In all these
adducts, the deprotonated sulfonamide is coordinated
to the Zn(II) ion of the enzyme, and its NH moiety
donates a hydrogen bond to the Og of Thr 199, which in
turn donates a hydrogen bond to the carboxylate group
of Glu 106. One of the oxygen atoms of the SO2NH moi-
ety also participates in a hydrogen bond with the back-
bone NH moiety of Thr 199, and extensive hydrophobic
and van der Waals interactions between the heterocyclic/
aromatic part of the inhibitor molecule and active site
amino acid residues, assure a strong affinity of sulfon-
amides to the CA active site (which may arrive to the
nanomolar range in some cases).1ꢀ5
carboxyphenylsulfonamide tails derivatized as esters
and amides, and were designed in such a way as to
assure a good penetration through the cornea, for
optimizing their topical antiglaucoma properties.
Chemistry
Benzolamide BZA, is a very potent CAI, and although
quite similar structurally with acetazolamide, AZA, the
CAI par excellence, it possesses different physico-chem-
ical properties, due to the presence of the second sulfo-
namide moiety in its molecule.11,12 In consequence,
BZA is much more polar than AZA, does not possess
antiglaucoma properties when given systemically (in
contrast to acetazolamide or methazolamide), and to a
certain extent, crosses biological membranes with much
more difficulty as compared to other CAIs in clinical
use.1,11 This was the rationale that lead to the proposal
that it acts as a renal-specific CAI, but no drug house
developed this agent for wide clinical use, and presently
BZA is still an orphan drug.11 Due to its good CA
inhibitory properties, as well as interesting physico-che-
mical properties, BZA has been used as lead for the
design of CAIs by our group.12 Indeed, a large number
of benzolamide analogues possessing diversely sub-
stituted phenyl moieties, as well as the corresponding 4-
methyl-thiadiazoline-sulfonamides (incorporating the
methazolamide scaffold) have been prepared and
assayed as CAIs.12 Some of these derivatives, such as
for example 5- (2 - carboxyphenylsulfonamido) - 1,3,4 -
thiadiazole-2-sulfonamide (2-carboxy-BZA) as well as
the corresponding 4-methyl-thiadiazoline derivative
showed sub-nanomolar affinity for hCA II (the main
enzyme involved in aqueous humor secretion within the
eye)1,2 and very good inhibition profiles against other
isozymes (such as CA I and IV), but were devoid of
topical antiglaucoma properties (unpublished results
from this laboratory).12 It appeared thus of interest on
the one hand to prepare the 3-carboxy- and 4-carboxy-
isomers of 2-carboxy-BZA, which were not reported in
In our laboratory an original approach has recently
been developed for obtaining high affinity CAIs with a
multitude of pharmacological uses, such as topically
acting antiglaucoma drugs, antitumor agents or diag-
nostic tools for PET among others.6ꢀ10 This approach
(the so-called ‘tail approach’) consists in using well-
known aromatic/heterocyclic sulfonamide scaffolds to
which tails that will induce water solubility or other
desired physico-chemical properties are attached at the
amino, hydroxy, imino or hydrazino moieties contained
in the precursor sulfonamides.6ꢀ10
In this paper, we report novel types of CAIs prepared
by the tail approach. These compounds incorporate

A. Casini et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2867–2873
2869
the preceding study,12 and on the other one, to reduce
their very polar character by derivatizing the carboxyl
moiety, in order to investigate whether this will lead to
enhanced ocular penetration, and thus topical anti-
glaucoma activity. Thus, a series of esters and amides of
3-carboxy-BZA, and 4-carboxy-BZA were prepared,
together with the corresponding thiadiazoline-sulfon-
amides. These compounds formally incorporate tails 1–
12 which were attached to heads A and B of the parent
sulfonamides from which acetazolamide and methazo-
lamide were derived. As for other compounds prepared
by the tail aproach,1 sulfonamides reported here are
denominated by a figure specifying the tail, followed by
a letter specifying the head to which this tail has been
attached. For example, 1A is the ethylene glycol ester of
3-carboxybenzolamide, whereas 12B is the 2-N,N-di-
ethylaminoethylamide of 5-(4-carboxybenzenesulfonyl-
imido)-4-methyl-d2-1,3,4-thiadiazoline-2-sulfonamide.
Compounds reported here were prepared as shown in
Scheme 1. The key intermediates 13–16 were obtained
by arylsulfonylation of 5-amino-1,3,4-thiadiazole-2-sul-
fonamide (A)/5-imino-4-methyl-d2-1,3,4-thiadiazoline-
2-sulfonamide (B) with 3- or 4-carboxyphenylsulfonyl
chloride, as reported previously for structurally related
benzolamide derivatives.7,12ꢀ15 Coupling of these 3- and
4-carboxybenzolamide derivatives 13–16 with different
alcohols/amines in the presence of carbodiimides (such
as EDCI) led to the new sulfonamides of types (1–
12)A,B, by the same procedure reported previously for
preparing topically actings antiglaucoma sulfonamides
incorporating amino acyl tails.6ꢀ10,16 In a variant of
these syntheses, the key intermediates 13–16 were con-
verted to the corresponding acyl chlorides by treatment
with thionyl chloride, and the obtained acyl chlorides
were then reacted with alcohols/amines in standard
conditions17 (Scheme 1). The presence of hydroxyethyl,

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A. Casini et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2867–2873
methoxyethyl, dialkylaminoethyl or dialkylaminoethoxy-
ethyl moieties in the tails 1–12 investigated here is con-
sidered beneficial for improving pharmacological
properties of these new CAIs, such as for example water
solubility, balanced water/lipid solubility; penetrability
of the drug through the cornea, etc., and this was shown
indeed to be the case (see later in the text).
for these isozymes, with Kis in the 10–23 nM range
against hCA I, 1.3–5.2 nM against hCA II and 6–14 nM
against bCA IV (Table 1).20
The following SAR can be also evidenced for the new
esters/amides (1–12)A,B: (i) the ethylene glycol esters
1A, 7A, 1B and 7B were generally less effective than the
corresponding methoxy derivatives (2A, 8A, 2B and
8B), which in turn were less effective than the corre-
sponding N,N-dimethylamino-substituted compounds
3A, 9A, 3B and 9B (against hCA II), but differences of
activity were rather small (ii) the presence of the methyl
group in the ester tail, such as in 4A,B and 10A,B did
not significantly influence the CA inhibitory activity of
these compounds, whereas the elongation of the tail,
such as in compounds 5A,B and 11A,B leads to some of
the best inhibitors in this series; (iii) the amides 6A,B
and 12A,B also led to some of the best inhibitors in this
series; (iv) the para-substituted derivatives were slightly
better inhibitors than the corresponding meta-sub-
stituted derivatives (with the exception of bCA IV, for
which the para-type tails were slightly less effective
CA inhibition
Inhibition data of Table 1 show that the new com-
pounds reported here act as very strong inhibitors of the
physiologically relevant isozymes I, II and IV, similarly
with the clinically used derivatives acetazolamide,
methazolamide, ethoxzolamide or benzolamide. Thus,
the parent carboxy-derivatives are already potent inhi-
bitors of all these isozymes, with inhibition constants in
the range of 45–73 nM against hCA I, 4.6–5.9 nM
against hCA II and 12–15nM against bCA IV. Enlarg-
ing the carboxyphenylsulfonyl tail as in esters/amides
(1–12)A,B generally leads to an increase of the affinity
Scheme 1.

A. Casini et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2867–2873
2871
Table 1. CA inhibition data with standard inhibitors, the parent
sulfonamides 13–16 and some of the new derivatives reported in the
present study, against isozymes I, II and IV
Table 2. Fall of IOP of normotensive rabbits (20.5ꢁ2.1 mm Hg),
after treatment with one drop (50 mL) 2% water solution of CA
inhibitor directly into the eye, 60 and 90 min after administration
Inhibitor
Ki (nM)a
hCA IIb
Inhibitor
pH
ÁIOP (mm Hg)a
30 min 60 min 90 min 240 min
hCA Ia
bCA IVc
t=0
AZA
MZA
EZA
BZA
DZA
BRZ
13
14
15
16
1A
2A
3A
4A
5A
6A
7A
8A
9A
10A
11A
12A
1B
2B
3B
4B
5B
6B
7B
8B
9B
10B
11B
12A
200
100
258
159
7
9
120
145
Dorzolamide 5.5
Brinzolamide 5.5
0
0
0
0
0
0
0
0
0
0
0
0
1.9ꢁ0.2 4.0ꢁ0.3
2.9ꢁ0.1 3.2ꢁ0.3
2.1ꢁ0.2
0
6.3ꢁ0.4 1.3ꢁ0.2
13
12
1A
2A
3A
4A
5A
6A
9A
10A
11A
12A
7
7
7
7
7
7
7
7
7
7
10.5ꢁ0.55 4.8ꢁ0.152.8 ꢁ0.2 0.5ꢁ0.1
0
6.0ꢁ0.3 2.5ꢁ0.2
4.5ꢁ0.154.9 ꢁ0.1
2.0ꢁ0.3
7.1ꢁ0.4 5.7ꢁ0.4
50,000
—
68
9
3
5.1
43
45
13
2.5ꢁ0.2 6.7ꢁ0.158.5 ꢁ0.2 7.9ꢁ0.5
4.9ꢁ0.3 4.5ꢁ0.3
6.5ꢁ0.4 6.9ꢁ0.2
4.1ꢁ0.3 3.3ꢁ0.2
9.1ꢁ0.58.0 ꢁ0.3
454.6
73
50
16
152.9
152.1
17
13
14
14
12
11
13
12
10
23
17
16
18
152.0
16
20
16
13
151.8
13
12
12
5.9
5.4
3.1
15
14
8
4.5ꢁ0.25.5
ꢁ0.258.0 ꢁ0.4 7.6ꢁ0.4
4.8ꢁ0.4 5.9ꢁ0.3
8.2ꢁ0.3 8.0ꢁ0.2
ꢁ0.2 6.3ꢁ0.4
2.7ꢁ0.1 3.9ꢁ0.1.755
8
7
3.6ꢁ0.2 8.5ꢁ0.3 10.1ꢁ0.4 8.7ꢁ0.4
2.0
1.6
1.7
2.9
2.8
1.510
1.4
1.3
1.4
5.2
3.3
2.9
2.2
8
6
7
11
10
aÁIOP=IOPcontrol eyeꢀIOPtreated eye; meanꢁSE (n=3).
effect lasted generally no longer than 2–3 h (data not
shown). Some of the new derivatives reported here, such
as 1A and to a lesser extent 2A showed a very sharp
decrease of IOP after 30 min post-administration, but
this good effect vanished rather quickly (in the next 30
min), probably because the inhibitor has been washed
away from the eye tissue due to the blood circulation.
Clearly such inhibitors did not possess the desired
physico-chemical properties for a prolonged duration of
action. On the contrary, several other compounds, such
as 6A, 9A, 10A and 12A among others, showed a good
IOP lowering effect after 30 min (around 3.6–6.5mm
Hg), which continued to increase both at one h, as well
as at 90 min post-administration, when the maximal
peak of IOP lowering has been achieved (of 8.0–10.1
mm Hg). These inhibitors may indeed be considered as
valuable candidates for achieving long-lasting IOP low-
ering after topical administration of sulfonamide CAIs.
Furthermore, the pH of the eye drops of new CAIs
reported here were neutral, as compared to the acidic
solutions of dorzolamide and brinzolamide (pH 5.5)
which produce eye irritation in many patients.22
10
7
8
10
9
10
12
8
1.9
4.0
3.1
2.4
7
14
12
14
13
1.7
1.7
9
8
aFrom triplicate experiments (errors in the range of 5-10% of the
reported value).
bHuman cloned isozyme.18
cIsolated from bovine lung.19
inhibitors); (v) compounds incorporating the 1,3,4-thi-
adiazole-2-sulfonamide moiety (head A) were more
effective CAIs as compared to the corresponding deri-
vatives incoporating the thiadiazoline-sulfonamide
scaffold (head B).
Conclusions
Topical antiglaucoma activity
Two new series of sulfonamides have been synthesized
by using the tail approach and benzolamide as lead
molecule. These are derivatives of 3-carboxy- and 4-
carboxy-benzolamide (as well as the homologous 4-
methyl-1,3,4-thiadiazoline-2-sulfonamides) to which the
carboxyl moiety has been either esterified or amidated
in order to reduce the very polar character of the lead,
which is ineffective as antiglaucoma agent. The new
sulfonamides reported showed low nanomolar activity
against isozymes CA I, II and IV, and some of them
were effective in normotensive rabbits in reducing IOP
after topical administration as 2% solutions. Their effi-
cacy was better than that of the standard drugs dorzol-
amide and brinzolamide, whereas the duration of action
was also prolonged as compared to that of the clinically
used drugs.
As seen from data of Table 2, in vivo, in normotensive
rabbits, some of the new sulfonamides reported here
(which possess very good water solubility—data not
shown) exhibited effective intraocular pressure (IOP)
lowering after topical administration, with pressure
reductions of 2.5–10.5 mm Hg at half an hour (compared
to 1.9 mm Hg for dorzolamide and 2.9 mm Hg for
brinzolamide), 2.5–8.5 mm Hg at 1 h (4.0 for DZA, and
3.2 for BRZ, respectively), 2.0–10.1 mm Hg at 90 min
(compared to 2.1 mm Hg for DZA, and 6.3 for BRZ).21
An important feature of some representatives of the new
class of CA inhibitors reported here (such as 6A, 9A,
10A and 12A among others) was that IOP remained low
for longer periods (3–5h) after their topical adminis-
tration, as compared to the standard drug, for which the

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A. Casini et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2867–2873
sulfonamide was dissolved in 30 mL NaOH 2.5N, under stir-
Acknowledgements
ring at room temperature. The obtained clear solution was
cooled at 10 ꢂC, then a 1.3 molar excess of the corresponding
3- or 4-carboxybenzenesulfonyl chloride (from Sigma-Aldrich,
Milan, Italy), together with 50 mL NaOH 5 N were added
simultaneously, in five portions, maintaining the temperature
at 10 ꢂC. The dark brown solution obtained was stirred over-
night at room temperature, then the pH was adjusted to 1.5–2
with 6 N HCl. The crude precipitate was filtered and sus-
pended in 100 mL HCl 1:1 in order to dissolve the unreacted
amine. The obtained solid was suspended in 80 mL of water
and treated dropwise with concentrated (25%) NH3 solution
until pH 9 was reached. The solution of the sulfonamide/car-
boxylate ammonium salt was brought dropwise to pH 1.5–2
with 6 N HCl. Recrystallization from 300 mL of water with
active charcoal (0.5–0.7 g) afforded the pure products, 13 and
14, respectively (overall yields were in the range of 30–40%).
4-Carboxybenzolamide 14, colorless crystals, mp 235–237 ꢂC
(dec.); 1H NMR (DMSO-d6, ppm): 7.31 (s, NH2); 7.39 (d,
ArH, AA0BB0, 8.4); 7.72 (d, ArH, AA0BB0, 8.4); 8.83 (br s,
SO2NH); 13C NMR (DMSO-d6, ppm): 168.08 (COOH),
166.17, 158.32 (Cthiadiazole), 144.70 (C-1 of 4-HOOC-C6H4),
134.52 (C-4 of 4-HOOC-C6H4), 130.26 (C-2 of 4-HOOC-
C6H4), 126.24 (C-3 of 4-HOOC-C6H4); elem. anal. data:
found, C, 29.46; H, 2.13; N, 15.10%; C9H8N4O6S3 requires: C,
29.67; H, 2.21; N, 15.38%.
This research was financed by CSGI and by the CNR—
Target Project Biotechnologies. Thanks are addressed
to Dr. Daniela Vullo for registering some NMR spectra
and to Dr. Monica Ilies for technical assistance.
References and Notes
1. (a) Supuran, C. T.; Scozzafava, A.; Casini, A. Med. Res.
Rev. 2003, 23, 146. (b) Supuran, C. T.; Scozzafava, A. Curr.
Med. Chem. Imm., Endoc., Metab. Agents 2001, 1, 61. (c)
Supuran, C. T.; Scozzafava, A. Exp. Opin. Ther. Pat. 2000, 10,
575. (d) Scozzafava, A.; Owa, T.; Mastrolorenzo, A.; Supuran,
C. T. Curr. Med. Chem. 2003, 10, 925.
2. (a) Christianson, D. W.; Cox, J. D. Annu. Rev. Biochem.
1999, 68, 33. (b) Liljas, A.; Hakansson, K.; Jonsson, B. H.;
Xue, Y. Eur. J. Biochem. 1994, 219, 1. (c) Gruneberg, S.;
Wendt, B.; Klebe, G. Angew. Chem. Int. Ed. 2001, 40, 389.
3. (a) Smith, K. S.; Ferry, J. G. FEMS Microbiol. Rev. 2000,
24, 335. (b) Chirica, L. C.; Elleby, B.; Lindskog, S. Biochim.
Biophys. Acta 2001, 1544, 55. (c) Elleby, B.; Chirica, L. C.; Tu,
C.; Zeppezauer, M.; Lindskog, S. Eur. J. Biochem. 2001, 268,
1613. (d) Krungkrai, S. R.; Suraveratum, N.; Rochanakij, S.;
Krungkrai, J. Int. J. Parasitol. 2001, 31, 661.
16. An amount of 1 mM 13–16 was dissolved in 25mL of
anhydrous acetonitrile and then treated with 2 mM of alcohol/
amine (ethylene glycol; 2-methoxyethanol; 2-N,N-dimethyl-
aminoethanol; 1-methyl-2-N,N-dimethylamino-ethanol; 2-(2-
4. Abbate, F.; Supuran, C. T.; Scozzafava, A.; Orioli, P.;
Stubbs, M.; Klebe, G. J. Med. Chem. 2002, 45, 3583.
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N,N-dimethylaminoethoxy)-ethanol;
2-N,N-diethylamino-
ethylamine, all commercially available from Sigma-Aldrich,
Milan, Italy). An amount of 190 mg (1 mM) of EDCI HCl was
then added and the reaction mixture was magnetically stirred
at room temperature for 15min, then 30 mL (2mM) of tri-
ethylamine were added and stirring was continued for 8–16 h
at 4 ꢂC (TLC control). The solvent was evaporated in vacuo
and the residue taken up in ethyl acetate (5mL), 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 removed in vacuo.
The obtained oils were were recrystallized from ethanol–water
or methanol, or purified by means of preparative HPLC (C18
reversed-phase m-Bondapack or Dynamax-60A (25ꢃ250 mm)
columns; 90% acetonitrile/8% methanol/2% water, 30 mL/
min). Yields were in the range of 50–54%. 12A, white crystals,
mp 197–9 ꢂC; 1H NMR (DMSO-d6, ppm): 1.86 (t, 6H, 2Me
from Et, 6.9 Hz); 2.84 (q, 4H, CH2 from 2 Et, 7.3 Hz); 2.90 (t,
2H, CH2CH2N, 7.2 Hz); 3.47 (q, 2H, CH2NH, 6.5Hz); 7.37
(d, ArH, AA0BB0, 8.4 Hz); 7.73 (d, ArH, AA0BB0, 8.4 Hz); 8.85
(br s, SO2NH); 13C NMR (DMSO-d6, ppm): 171.12 (CONH),
166.237, 158.31 (Cthiadiazole), 144.82 (C-1 of 4-HOOC-C6H4),
134.65(C-4 of 4-HOOC-C 6H4), 130.43 (C-2 of 4-HOOC-
C6H4), 124.98 (C-3 of 4-HOOC-C6H4); 42.35(CH 2); 42.12
(CH2); 40.81 (CH2); 16.54 (Me); elem. anal. data: found, C,
39.13; H, 4.68; N, 18.05%; C15H22N6O5S3 requires: C, 38.95;
H, 4.79; N, 18.17%.
17. Carboxyl-sulfonamides 13–16 (1 mM) were suspended in
20 mL of anhydrous benzene and a 2-fold excess of SOCl2 was
added. The reaction mixture was refluxed for 3–5h till the for-
mation of the acyl chloride was complete (TLC control), the
solvent and excess thionyl chloride were removed in vacuo, and
the obtained acyl halides dissolved in 10 mL of anhydrous ace-
tonitrile. To this solution was added the 2-fold excess amount of
alcohols/amines mentioned above, and the acylation reaction
was followed by TLC. The reaction mixture has been worked
out as described above. Yields were in the range of 70–75%.
18. Human CA I and CA II cDNAs were expressed in
Escherichia coli strain BL21 (DE3) from the plasmids pACA/
6. Scozzafava, A.; Menabuoni, L.; Mincione, F.; Briganti, F.;
Mincione, G.; Supuran, C. T. J. Med. Chem. 1999, 42, 2641.
7. Borras, J.; Scozzafava, A.; Menabuoni, L.; Mincione, F.;
Briganti, F.; Mincione, G.; Supuran, C. T. Bioorg. Med.
Chem. 1999, 7, 2397.
8. (a) Menabuoni, L.; Scozzafava, A.; Mincione, F.; Briganti,
F.; Mincione, G.; Supuran, C. T. J. Enz. Inhib. 1999, 14, 457.
(b) Supuran, C. T.; Scozzafava, A.; Menabuoni, L.; Mincione,
F.; Briganti, F.; Mincione, G. Eur. J. Med. Chem. 1999, 34,
799.
9. (a) Scozzafava, A.; Briganti, F.; Mincione, G.; Menabuoni,
L.; Mincione, F.; Supuran, C. T. J. Med. Chem. 1999, 42,
3690. (b) Supuran, C. T.; Scozzafava, A.; Menabuoni, L.;
Mincione, F.; Briganti, F.; Mincione, G. Eur. J. Pharm. Sci.
1999, 8, 317. (c) Barboiu, M.; Supuran, C. T.; Menabuoni, L.;
Scozzafava, A.; Mincione, F.; Briganti, F.; Mincione, G. J.
Enz. Inhib. 1999, 15, 23. (d) Scozzafava, A.; Briganti, F.;
Mincione, G.; Menabuoni, L.; Mincione, F.; Supuran, C. T. J.
Med. Chem. 1999, 42, 3690.
10. (a) Scozzafava, A.; Menabuoni, L.; Mincione, F.; Bri-
ganti, F.; Mincione, G.; Supuran, C. T. J. Med. Chem. 2000,
43, 4542. (b) Scozzafava, A.; Menabuoni, L.; Mincione, F.;
Mincione, G.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2001,
11, 575. (c) Casini, A.; Scozzafava, A.; Mincione, F.; Mena-
buoni, L.; Ilies, M. A.; Supuran, C. T. J. Med. Chem. 2000, 43,
4884. (d) Scozzafava, A.; Briganti, F.; Ilies, M. A.; Supuran,
C. T. J. Med. Chem. 2000, 43, 292.
11. Maren, T. H. Benzolamide A Renal Carbonic Anhydrase
Inhibitor. In Orphan Drugs, Karch, F. E. Ed., Dekker: New
York, 1982; p 89.
12. Supuran, C. T.; Ilies, M. A.; Scozzafava, A. Eur. J. Med.
Chem. 1998, 33, 739.
13. Supuran, C. T.; Clare, B. W. Eur. J. Med. Chem. 1999, 34,
41.
14. Clare, B. W.; Supuran, C. T. Eur. J. Med. Chem. 1999, 34,
463.
15. An amount of 10 g (55 mM) 5-amino-1,3,4-thiadiazole-2-

A. Casini et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2867–2873
2873
hCA I and pACA/hCA II described by: Lindskog, S.; Behra-
van, G.; Engstrand, C.; Forsman, C.; Jonsson, B. H.; Liang,
Z.; Ren, X.; Xue, Y. In Carbonic Anhydrase—From Biochem-
istry and Genetics to Physiology and Clinical Medicine, Botre,
F., Gros, G., Storey, B. T. Eds.: VCH, Weinheim 1991; p 1.
Cell growth conditions were those described by this group,
and enzymes were purified by affinity chromatography.
the Association 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 ꢂC. Solutions of inhi-
bitors (2%, by weight, as hydrochlorides) were obtained in
distilled deionized water. The pH of these solutions was in the
range of 6.5–7.0. IOP was measured using a Tono-Pen XL
tonometer (Medtronic Solan, USA) as reported earlier.9,10 The
pressure readings were matched with two-point standard
pressure measurements at least twice each day using a Digilab
Calibration verifier. All IOP measurements were done by the
same investigator with the same tonometer. IOP was measured
three times at each time interval, and the means reported. IOP
was measured first immediately before drug administration,
then at 30 min after the instillation of the pharmacological
agent, and then each 30 min for a period of 4–6 h. For all IOP
experiments drug was administered to only one eye, leaving
the contralateral eye as an untreated control. The ocular
hypotensive activity is expressed as the average difference in
IOP between the treated and control eye, in this way mini-
mizing the diurnal, seasonal and interindividual variations
commonly observed in the rabbit.9
Enzyme
concentrations
were
determined
spectro-
photometrically at 280 nm, utilizing a molar absorptivity of 49
mMꢀ1 cmꢀ1 for CA I and 54 mMꢀ1.cmꢀ1 for CA II,
respectively, based on Mr=28.85kDa for CA I, and 29.30
kDa for CA II, respectively. CA IV was isolated from
bovine lung microsomes as described by Maren et al.19 and
its concentration has been determined by titration with
ethoxzolamide.
19. Maren, T. H.; Wynns, G. C.; Wistrand, P. J. Mol. Phar-
macol. 1993, 44, 901.
20. A stopped flow variant of the Pocker and Stone spectro-
photometric method (Pocker, Y.; Stone, J. T. Biochemistry
1967, 6, 668) has been employed, using an SX.18MV-R
Applied Photophysics stopped flow instrument, as described
previously.9,10
21. Adult male New Zealand albino rabbits weighing 3–3.5kg
were used in the experiments (three animals were used for each
inhibitor studied). The experimental procedures conform to
22. (a) Sugrue, M. F. Progr. Ret. Eye Res. 2000, 19, 87. (b)
Silver, L. H. Surv. Ophthalmol. 2000, 44, 147.