Carbonic anhydrase inhibitors: 4-Sulfamoyl-benzenecarboxamides and 4-chloro-3-sulfamoyl-benzenecarboxamides with strong topical antiglaucoma properties

Article abstract of DOI:10.1016/S0960-894X(01)00303-1

Reaction of 4-carboxy-benzenesulfonamide or 4-chloro-3-sulfamoyl benzoic acid with carboxy-protected amino acids/dipeptides, or aromatic/heterocyclic sulfonamides/mercaptans afforded the corresponding benzene-carboxamide derivatives. These were tested as inhibitors of three carbonic anhydrase (CA) isozymes, CA I, II and IV. Some of the new derivatives showed affinity in the low nanomolar range for isozymes CA II and IV, involved in aqueous humor secretion within the eye, and were tested as topically acting anti-glaucoma agents, in normotensive and glaucomatoous rabbits. Good in vivo activity and prolonged duration of action has been observed for some of these derivatives, as compared to the clinically used drugs dorzolamide and brinzolamide. Some of the 4-chloro-3-sulfamoyl benzenecarboxamides reported here showed higher affinity for CA I than for the sulfonamide avid isozyme CA II.

Full text of DOI:10.1016/S0960-894X(01)00303-1

Bioorganic & Medicinal Chemistry Letters 11 (2001) 1787–1791
Carbonic Anhydrase Inhibitors: 4-Sulfamoyl-benzenecarboxamides
and 4-Chloro-3-sulfamoyl-benzenecarboxamides with Strong
Topical Antiglaucoma Properties
Francesco Mincione,a,b Michele Starnotti,a,b Luca Menabuoni,c Andrea Scozzafava,d
Angela Casinid and Claudiu T. Supurand,e,
*
a
`
Universita degli Studi, Istituto Oculistico, Viale Morgagni 85, I-50134 Firenze, Italy
bU.O. Oculistica Az. USL 3, Val di Nievole, Ospedale di Pescia, Pescia, Italy
cOspedale San Giovanni di Dio, U.O. Oculistica, Via Torregalli 3, I-50123, Firenze, Italy
Universita degli Studi, Laboratorio di Chimica Inorganica e Bioinorganica, Via Gino Capponi 7, I-50121, Firenze, Italy
d
`
e
`
CSGI, c/o Universita degli Studi di Firenze, Via Gino Capponi 7, I-50121, Firenze, Italy
Received 5 March 2001; revised 29 March 2001; accepted 27 April 2001
Abstract—Reaction of 4-carboxy-benzenesulfonamide or 4-chloro-3-sulfamoyl benzoic acid with carboxy-protected amino acids/
dipeptides, or aromatic/heterocyclic sulfonamides/mercaptans afforded the corresponding benzene-carboxamide derivatives. These
were tested as inhibitors of three carbonic anhydrase (CA) isozymes, CA I, II and IV. Some of the new derivatives showed affinity
in the low nanomolar range for isozymes CA II and IV, involved in aqueous humor secretion within the eye, and were tested as
topically acting anti-glaucoma agents, in normotensive and glaucomatoous rabbits. Good in vivo activity and prolonged duration
of action has been observed for some of these derivatives, as compared to the clinically used drugs dorzolamide and brinzolamide.
Some of the 4-chloro-3-sulfamoyl benzenecarboxamides reported here showed higher affinity for CA I than for the sulfonamide
avid isozyme CA II. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
due either to the acidic pH of their solutions (in the case
of 1) or to their low solubility and need to be adminis-
tered as suspensions (in the case of 2).1À3 Thus, much
research has been done in the last years for developing
alternative agents, devoid of the major draw-backs of
the dorzolamide-type agents.4À8
Inhibition of carbonic anhydrase (CA, EC 4.2.1.1) with
sulfonamides constitutes one of the most physiological
approaches for the treatment of glaucoma, a condition
associated with elevated intraocular pressure (IOP) due
to excessive bicarbonate secretion within the aqueous
humor.1À3 This secretion is mediated by isozymes CA II
and CA IV present within ciliary processes of the eye,
and its inhibition by topically acting sulfonamides, such
as dorzolamide 1 or brinzolamide 2 (which are low
nanomolar CA II inhibitors) are widely used clinically,
alone or in combination with other agents, such as b-
blockers, prostaglandin derivatives or a-adrenergic
agonists among others.1À3
The presently available topically acting sulfonamides 1
and 2 are imperfect due to many topical side effects
observed after their administration. These are mainly
A successful approach recently designed by us4a in order
to avoid many of the side effects of dorzolamide due to
the high acidity of its ophthalmologic solutions,
*Corresponding author. Tel.: +39-055-275-7551; fax: +39-055-275-
7555; e-mail: claudiu.supuran@unifit.it
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00303-1

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F. Mincione et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1787–1791
consisted of preparing water soluble sulfonamides
incorporating a 4-thioureido-benzenesulfonamide scaf-
fold. Some of these derivatives, such as 3 and 4, showed
high affinity for the enzyme, very good water solubility
(as sodium carboxylate salts) at pH values of 7.0–7.5,
and excellent IOP lowering properties in animal models
of glaucoma.4a Using derivatives 3 and 4 as lead mole-
cules, we report here a new approach for obtaining
nanomolar CA II and CA IV inhibitors with good water
solubility in the neutral pH range, also characterized by
good and long lasting in vivo properties as IOP low-
ering agents in glaucoma animal models.
affinity was much lower, of around 270–560 nM [for
compounds of the first series; see discussion for the sec-
ond series of derivatives (of type 9) against hCA I later
in the text]; (iii) several derivatives of the first series,
such as the Gly, b-Ala, Ser, Thr and Cys derivatives, as
well as the largest majority of the carboxamides of the
second series (such as 9a–9i and 9k–9v) were only mod-
erate hCA II/bCA IV inhibitors, with affinities in the
85–735 nM against the first isozyme and 170–600 nM
against the second one. The diminished affinity of deri-
vatives of type 9 as compared to the corresponding
inhibitors 8, towards isozymes II and IV, may be due to
the chlorine atom ortho to the sulfamoyl moiety present
in the first compounds, which is known to lead to a
steric hindrance when binding to the Zn(II) within the
enzyme active site,12,13 but it seems that this phenom-
enon is beneficial for the selectivity of such inhibitors
towards the isozyme I; (iv) a fascinating discovery
regards the activity of the second series of sulfona-
mides—some compounds of type 9—(as an entire class
of CA inhibitors) against isozyme I: these derivatives
are among the very few reported CA inhibitors1,2 that
possess a higher affinity for isozyme CA I as compared
to isozyme CA II. Indeed, as seen from the data of
Table 1, many of these compounds possess inhibition
constants of 21–305 nM against hCA I, and of 26–
490 nM towards hCA II, respectively. Sometimes, a
quite spectacular ‘selectivity’ is achieved, with the
His, Tyr and DOPA derivatives possessing an hCA I
inhibition constant around 100 nM, whereas the same
parameter against hCA II is of around 250 nM. We
consider these data highly significant, especially con-
sidering the fact that hCA I is a very abundant blood
protein (85% of the blood CA is constituted by this
isozyme) whose physiological role is largely unknown
for the moment.1,2,11
Reaction of 4-carboxybenzenesulfonamide 5 with car-
boxy-protected (as tert-butyl esters) amino acids/dipep-
tides 6 in the presence of carbodiimides (EDCI) and
hydroxybenzotriazole, afforded the corresponding car-
boxamides 7, which were deprotected with TFA fol-
lowing the standard procedure,6,7 leading to the
derivatives 8 (Scheme 1 and Table 1). Several structu-
rally related derivatives were obtained by using aro-
matic/heterocyclic sulfonamides/mercaptans possessing
free amino groups instead of amino acids in the cou-
pling reactions mentioned above (Table 1). Finally, by
using 4-chloro-3-sulfamoyl benzoic acid instead of 5, a
related series of amides 9 has been prepared (Table 1).10
It must be mentioned that structurally related deriva-
tives to 8 and 9 have been previously reported by
Whitesides’7 and Baldwin’s group,9 but as far as we
know, such derivatives were not investigated for their
IOP lowering properties (although some of them
showed excellent in vitro CA II inhibitory properties).7,9
The following may be commented regarding the inhibi-
tion data with the new sulfonamides 8 and 9 reported
here: (i) the dipeptide derivatives of 4-carboxy-
benzenesulfonamide 8x–8z, 8aa and 8ab, as well as the
sulfonamide/mercapto derivatives in both series (8ac–
8ag and 9af, 9ag) behave as very potent CA II and CA
IV inhibitors, with affinities in the low nanomolar range
for these isozymes (3–16 nM for hCA II, and 13–25 nM
for bCA IV, respectively) whereas generally they are
slightly less effective CA I inhibitors (this isozyme is
known to possess a lower affinity for sulfonamides);1À3,11
(ii) less effective inhibitors are the 4-sulfamoylbenzene-
carboxamides derived from GABA, Ala, Val, Leu, Ile,
a-phenyl-glycine, Met and dicarboxylic amino acids as
well as aromatic/heterocyclic amino acids (compounds
8r–8u), together with several derivatives belonging to
the second series, such as 9x–9z, 9aa–9ae. All these
inhibitors possessed affinities of 26–79 nM against hCA
II and 50–160 nM against bCA IV, whereas their hCA I
Some physicochemical properties of several strong in
vitro inhibitors were investigated in detail (Table 2),
showing that many of these new derivatives possess
excellent water solubility, at pH values in the range of
7.0–7.5 pH units. This is correlated with a balanced
hydro- and lipophilicity, and in consequence optimal
accession rates across the cornea, which is typical for
the effective topically acting sulfonamides.1À3 Two of
the investigated compounds (8ae and 9af) showed a very
low water solubility, but all the other physicochemical
properties were appropriate for acting as topical IOP
lowering agents. These compounds have been applied in
suspensions (similarly to brinzolamide 1) and indeed
they showed very good antiglaucoma activity in an ani-
mal model of the disease (Tables 3 and 4).
In vivo, in normotensive rabbits, some of the new sul-
fonamides reported here showed very effective IOP
lowering after topical administration, with pressure
reductions of 2.5–7.0 mmHg at 30 min (compared to
1.9 mmHg for dorzolamide and 2.9 mmHg for brinzol-
amide), 5.0–7.1 mmHg at 60 min (4.0 for 1, and 3.2for
2, respectively), 3.1–9.1 mmHg at 90 min, and 2.1–
9.5 mmHg at 120 min after administration (compared to
the much lower values for the standard drugs 1 and 2)
(Table 3). An important feature of the new class of CA
Scheme 1.

F. Mincione et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1787–1791
1789
Table 1. Inhibition data for some derivatives 8 and 9 reported in the paper, and standard inhibitors
No.
Inhibitor AA
KI (nM)
hCA Ia
hCA IIa
bCA IVb
1
2
Dorzolamide
Brinzolamide
Gly
50,000
—
9
3
85
85
73
79
74
53
48
36
120
21
115
110
66
43
45
200
190
120
75
150
90
86
75
325
62
350
230
150
150
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
8p
8q
8r
500
435
415
425
420
405
350
250
440
54
475
450
400
425
410
430
425
270
560
535
520
350
270
380
240
320
400
38
b-Ala
GABA
Ala
Val
Leu
Ile
a-Ph-Gly
Ser
b-Ph-Ser
Thr
Cys
Met
Asp
Asn
Glu
Gln
His
Phe
Tyr
DOPA
Gly-Gly
b-AlaHis
HisGly
HisPhe
AlaPhe
73
72155
74
75
160
160
125
40
8s
8t
52130
45
43
4250
16
10
9
12
9
10
7
5
3
120
105
8u
8v
8x
8y
8z
8aa
8ab
8ac
8ad
8ae
8af
8ag
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
9o
9p
9q
9r
21
19
23
24
16
2
16
13
15
14
600
610
540
475
360
330
360
240
395
LeuGly
–C6H4–SO2NH2 (p)
CH2–C6H4–SO2NH2 (p)
(CH2)2C6H4–SO2NH2 (p)
-1,3,4-thiadiazole–SO2NH2
-1,3,4-thiadiazole–SH
40
40
35
38
5
Gly
b-Ala
GABA
Ala
Val
Leu
Ile
a-Ph-Gly
Ser
b-Ph-Ser
Thr
Cys
Met
Asp
Asn
Glu
Gln
His
Phe
Tyr
210
205
200
175
130
125
150
105
170
37
320
300
240
285
240
305
250
103
135
115
100
69
450
450
400
420
330
305
280
160
290
6298
520
490
410
300
275
340
305
210
250
250
240
85
735
720
650
355
330
400
410
350
340
325
330
170
50
9s
9t
9u
9v
9x
DOPA
Gly-Gly
b-AlaHis
30
36
(Continued on next page)

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F. Mincione et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1787–1791
Table 1 (continued)
No.
Inhibitor AA
KI (nM)
hCA Ia
hCA IIa
bCA IVb
9y
9z
HisGly
HisPhe
AlaPhe
41
27
36
39
2
2
2
13
15
50
38
44
51
4
4
1
10
11
73
45
72
80
30
2
9aa
9ab
9ac
9ad
9ae
9af
9ag
LeuGly
–C6H4–SO2NH2 (p)
CH2–C6H4–SO2NH2 (p)
(CH2)2C6H4–SO2NH2 (p)
-1,3,4-thiadiazole–SO2NH2
-1,3,4-thiadiazole–SH
2
22
21
aHuman (cloned) isozymes.
bFrom bovine lung microsomes, by the esterase method.14
Table 2. Solubility, chloroform–buffer partition coefficients and in
vitro corneal permeability of some sulfonamide CA inhibitors reported
in the paper and dorzolamide 1 as standard
inhibitors reported here is that IOP remained low for
longer periods (3–5 h) after their topical administration,
as compared to the standard drug (data not shown).
The above findings also apply to the glaucomatous
rabbits experiments (Table 4) but the IOP reductions
were much more important as compared to those seen
in normotensive rabbits. Thus, IOP reductions of 6.4–
9.5 mmHg were observed after 30 min, whereas, at
60 min these amounted to 12.0–13.0 mmHg, at 90 min of
11.0–14.2mmHg, and at 120 min of 10.0–13.3 mmHg,
respectively. Thus, these derivatives are longer lasting
and more effective IOP lowering agents as compared
to the clinically available drugs dorzolamide and
brinzolamide.
Compound
Solubilitya (nM) Log Pb
kinÂ103 (hÀ1 c
)
Cornea intact No epithelium
Dorzolamide 1
8z
8ab
8ae
9af
60d
2.0
3.0
2.9
3.3
2.5
3.1
5.2
5.8
6.4
6.3
7.0
37e
1.735
1.821
1.433
1.893
34e
<0.1
<0.1
aSolubility in pH 7.40 buffer, at 25 ꢀC, determined as described in ref
13.
bChloroform–buffer partition coefficient, determined as described in
ref 13.
cDetermined as described in ref 4.
dAs hydrochloride, at pH 5.8, from ref 3.
eAs sodium salts.
In conclusion, we report here a novel class of powerful,
topically acting sulfonamide CA inhibitors, incorporating
Table 3. IOP lowering in normotensive rabbits (21.8Æ2.1 mmHg) after treatment with one drop (50 mL) 2% solution/suspension of CA inhibitor
(the pH of the solution also shown), at 30, 60, 90 and 120 min after administration directly into the eye14
Inhibitor
pH
ÁIOP (mmHg)a
t=60 min
t=0
t=30 min
t=90 min
t=120 min
Dorzolamide 1
Brinzolamide 2b
8z
8ab
8aeb
8ag
9afb
5.5
5.5
7.0
7.0
7.0
7.0
7.5
0
0
0
0
0
0
0
1.9Æ0.24.0
2.9Æ0.1
Æ0.3
3.2Æ0.3
Æ0.29.1
2.1Æ0.21.3
6.3Æ0.4
Æ0.3
Æ0.3
7.0Æ0.2
9.5Æ0.4
8.7Æ0.2
5.3Æ0.2
Æ0.1
4.2Æ0.26.5
3.3Æ0.3
5.2Æ0.4
5.3Æ0.26.7
5.0Æ0.23.1
7.1Æ0.3
7.8Æ0.3
Æ0.3
3.1Æ0.1
7.0Æ0.1
.1
Æ0.22
2.5Æ0.3
8.3Æ0.4
7.5Æ0.3
aÁIOP=IOPcontrol eyeÀIOPtreated eye; meanÆSEM (n=3 rabbits for each compound). Drug was administered in the left eye, whereas the con-
tralateral eye was used as control.
bSuspension.
Table 4. Fall of IOP of glaucomatous rabbits (30.5Æ3.0 mmHg), after treatment with one drop (50 mL) solution/suspension of 2% of CA inhibitor
(the pH value shown below) directly into the eye, at 30, 60, 90 and 120 min after administration
Inhibitor
PH
ÁIOP (mmHg)a
t=60 min
t=0
t=30 min
t=90 min
t=120 min
Dorzolamide
5.5
7.0
7.0
7.5
0
0
0
0
3.6Æ0.26.7
7.1Æ0.3
Æ0.3
12.0Æ0.3
12.3Æ0.5
Æ0.5
4.2Æ0.23.6
11.0Æ0.210.0
11.9Æ0.5
Æ0.3
Æ0.3
8z
8aeb
9afb
9.5Æ0.3
10.5Æ0.2
13.3Æ0.4
6.4Æ0.213.0
14.2Æ0.3
aÁIOP=IOPcontrol eyeÀIOPtreated eye; meanÆSEM (n=3 rabbits for each compound). Drug was administered in the left eye, whereas the con-
tralateral eye was used as control.
bSuspension.

F. Mincione et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1787–1791
1791
sulfamoyl-benzenecarboxamide moieties in their mole-
cules. Some of these inhibitors were very efficient IOP
lowering agents in normotensive and glaucomatous
rabbits after topical administration as water solutions/
suspensions. Furthermore, an entire class of sulfona-
mides possessing higher affinity for the slow isozyme
CA I as compared to the sulfonamide ‘avid’ one CA II
is reported.
D. L.; Maguire, L. J.; Verdier, D. V.; Fraunfelder, F. T.;
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10. For example: 258 mg (1 mmol) Phe t-butyl ester hydro-
chloride, 200 mg (1 mmol) of 4-carboxybenzenesulfonamide,
140 mg HOBT (1.1 mM) and 200 mg EDCI.HCl were sus-
pended in 50 mL of acetonitrile, and the required amount of
triethylamine added to the reaction mixture, which was stirred
at room temperature for 16 h (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 oils obtained
after evaporation of the solvent were directly used in the
deprotection step. The removal of the t-Bu groups has been
performed by treating the crude intermediate dissolved in
Acknowledgements
This research was financed by the EU grant ERB
CIPDCT 940051 and by a grant from the Italian CNR-
Target Project Biotechnology.
References and Notes
2 0 mL of CH Cl2, with 4 mL of trifluoroacetic acid (TFA) and
2
stirring for 60 min at 0 ꢀC. The solvent was removed in vacuo
and the residue concentrated from water twice to remove
excess TFA, giving thus the carboxamide as a colorless syrup.
The pure compound was obtained by means of preparative
HPLC (C18 reversed-phase m-Bondapack or Dynamax-60A
(25Â250 mm) columns; 90% acetonitrile/8% methanol/2%
water, 30 mL/min). Mp 229–231 ꢀC (ethanol–water 1:1, v/v).
IR (KBr), cmÀ1: 1148 (SO2sym), 1256 (amide III), 1370 (SOa2s),
1550 (amide II), 1695 (amide I), 1770 (COOH), 3065 (NH); 1H
NMR (DMSO-d6), d, ppm: 3.10–3.52(m, H2 , C H2CH of
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3
3
Phe), 4.11 (dd, JHH=5.0 Hz, JHH=7.8 Hz, 1H, CH2CH of
Phe), 7.29–7.51 (m, 5H, Harom of Phe), 7.50 (s, 2H, SO2NH2),
3
7.61 (d, JHH=8.1, 2H, Hortho of H2NO2SC6H4), 7.94 (d,
3JHH=8.1 Hz, 2H, Hmeta of H2NO2SC6H4), 8.12(br s, 1H,
CONH), 10.71 (br s, 1H, COOH); 13C NMR (DMSO-d6), d,
ppm: 41.3 (s, CH2CH of Phe), 59.8 (s, CH2CH of Phe), 131.5
(s, Cmeta of H2NO2SC6H4), 133.9 (s, Cmeta of Phe), 134.6 (s,
Cortho of Phe), 135.2(s, Cortho of H2NO2SC6H4), 141.7 (s, Cipso
of Phe), 144.2(s, Cipso of H2NO2SC6H4), 145.6 (s, Cpara of
Phe), 147.3 (s, Cpara of H2NO2SC6H4), 158.9 (s, CONH), 179.3
(COOH). Anal. found: C, 55.33; H, 4.60; N, 9.07%;
C16H16N2O5S requires C, 55.16; H, 4.63; N, 9.20%.
11. Supuran, C. T.; Scozzafava, A. In Carbonic Anhydrase
Activation; Chegwidden, W. R., Edwards, Y., Carter, N., Eds.;
The Carbonic Anhydrases—New Horizons; Birkhauser: Basel,
¨
2000; pp 197–220.
12. (a) Supuran, C. T.; Clare, B. W. Eur. J. Med. Chem. 1999,
34, 41. (b) Clare, B. W.; Supuran, C. T. Eur. J. Med. Chem.
1999, 34, 463. (c) Clare, B. W.; Supuran, C. T. Eur. J. Med.
Chem. 2000, 35, 859.
13. Supuran, C. T.; Ilies, M. A.; Scozzafava, A. Eur. J. Med.
Chem. 1998, 33, 739.
14. Dorzolamide hydrochloride was from Merck & Co.,
whereas brinzolamide hydrochloride from Alcon Labora-
tories. KI values were determined by the method described by:
Pocker, Y.; Stone, J. T. Biochemistry 1967, 6, 668, with the
modifications reported by: Briganti, F.; Mangani, S.; Orioli,
P.; Scozzafava, A.; Vernaglione, G.; Supuran, C. T. Biochem-
istry 1997, 36, 10384.
8. (a) Konowal, A.; Morrison, J. C.; Brown, S. V.; Cooke,