Furazan and furoxan sulfonamides are strong α-carbonic anhydrase inhibitors and potential antiglaucoma agents

Article abstract of DOI:10.1016/j.bmc.2014.06.016

A series of furazan and furoxan sulfonamides were prepared and studied for their ability to inhibit human carbonic anhydrase (CA, EC 4.2.1.1) isoforms hCA I, hCA II, hCA IX, and hCA XII. The simple methyl substituted products 3-5 were potent inhibitors. Differing structural modifications of these leads had differing effects on potency and selectivity. In particular, products in which the sulfonamide group is separated from the hetero ring by a phenylene bridge retained high potency only on the hCA XII isoform. The sulfonamides 3-5 exerted intraocular pressure (IOP) lowering effects in vivo in hypertensive rabbits more efficiently than dorzolamide. Some other products (39-42), although less effective in vitro hCA II/XII inhibitors, also effectively lowered IOP in two different animal models of glaucoma.

Full text of DOI:10.1016/j.bmc.2014.06.016

Bioorganic & Medicinal Chemistry 22 (2014) 3913–3921
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Furazan and furoxan sulfonamides are strong
inhibitors and potential antiglaucoma agents
a-carbonic anhydrase
Konstantin Chegaev a, Loretta Lazzarato a, Yasinalli Tamboli a, Donatella Boschi a, Marco Blangetti a,
Andrea Scozzafava b, Fabrizio Carta b, Emanuela Masini c, Roberta Fruttero a, , Claudiu T. Supuran b,c,
⇑
⇑
,
Alberto Gasco a
a Dipartimento di Scienza e Tecnologia del Farmaco, Università degli Studi di Torino, via Pietro Giuria 9, 10125 Torino, Italy
b Università degli Studi di Firenze, Dipartimento di Chimica, Lab. Chimica Bioinorganica, Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy
c Università degli Studi di Firenze, NEUROFARBA Dept., Sezione di Scienze Farmaceutiche e Nutraceutiche, Via Ugo Schiff 6, I-50019 Sesto Fiorentino (Florence), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of furazan and furoxan sulfonamides were prepared and studied for their ability to inhibit human
carbonic anhydrase (CA, EC 4.2.1.1) isoforms hCA I, hCA II, hCA IX, and hCA XII. The simple methyl substi-
tuted products 3–5 were potent inhibitors. Differing structural modifications of these leads had differing
effects on potency and selectivity. In particular, products in which the sulfonamide group is separated
from the hetero ring by a phenylene bridge retained high potency only on the hCA XII isoform. The sul-
fonamides 3–5 exerted intraocular pressure (IOP) lowering effects in vivo in hypertensive rabbits more
efficiently than dorzolamide. Some other products (39–42), although less effective in vitro hCA II/XII
inhibitors, also effectively lowered IOP in two different animal models of glaucoma.
Received 24 April 2014
Revised 5 June 2014
Accepted 6 June 2014
Available online 17 June 2014
Keywords:
Carbonic anhydrase
Isoforms I, II, IX, XII
Sulfonamides
Glaucoma
Ó 2014 Elsevier Ltd. All rights reserved.
IOP
1. Introduction
CO2/bicarbonate, pH and CO2 homeostasis, electrolyte secretion
in various tissues and organs, biosynthetic reactions, bone remod-
Carbonic anhydrases (CAs, EC 4.2.1.1) are enzymes found in
both eukaryotic and prokaryotic cells, which catalyze the conver-
sion of carbon dioxide to bicarbonate and a proton (Eq. 1).
eling, and calcification. Inhibition of the CAs involved in these pro-
cesses is an important target for the treatment of a number of
disorders, including edema, glaucoma, obesity, osteoporosis, epi-
lepsy, pain, and cancer.3,4 Recently, CAs belonging not only to the
CO2 þ H2O ! HCOꢀ þ Hþ
ð1Þ
3
a-family but also to the b-, c-, d-, and f-families have been charac-
CAs evolved along at least five different lines, giving rise to the
distinct , b, , d, and f enzyme families. In mammals, 13 CA isoen-
terized in several pathogens, including protozoa, bacteria, fungi,
and parasites such as nematodes.2 An increasing number of studies
show that they are involved in the virulence and growth of all
these agents.5,6 These findings open exciting new perspectives for
the use of CAIs in treating infections and infestations.
a
c
zymes and 3 acatalytic CA-related proteins (CARPs) with different
subcellular localizations have been identified to date: CA I, CA II, CA
III, CA VII, CA XIII are cytosolic, CA IV, CA IX, CA XII, CA XIV, CA XV
(not expressed in humans) are membrane bound, CA VA, CA VB are
mitochondrial, CA VI is a secreted enzyme, while CARP VIII, CARP X,
CARP XI appear to be cytosolic proteins. These isoenzymes belong
Most CAs are metalloenzymes containing a Zn2+ ion in the
active site; primary sulfonamides are their classical inhibitors.
Crystallographic studies of adducts between members of this class
and several CA isoenzymes show that the nitrogen deprotonated
sulfonamide moiety binds the catalytic Zn2+ ion and simulta-
neously interacts with the Thr199 residue through two hydrogen
bonds.3 Some sulfonamide inhibitors are heterocyclic compounds,
in which the sulfonamide group is linked to the hetero ring, either
directly or through appropriate spacers.2 To our knowledge, no
examples of 1,2,5-oxadiazole (furazan) sulfonamide derivatives
or of the related 2-oxides (furoxans) have been reported thus
far. We recently described a synthetic method to obtain these
to the evolutional a-CA gene family, and are present in various tis-
sues, including the eyes, kidneys, lungs, the gastrointestinal and
reproductive tracts, and the CNS.1,2 CAs are associated with many
physiological processes related to respiration and the transport of
⇑
Corresponding authors. Tel.: +39 011 6707850 (R.F.); tel.: +39 055 4573729
(C.T.S.).
E-mail addresses: roberta.fruttero@unito.it (R. Fruttero), claudiu.supuran@unifi.
it (C.T. Supuran).
http://dx.doi.org/10.1016/j.bmc.2014.06.016
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

3914
K. Chegaev et al. / Bioorg. Med. Chem. 22 (2014) 3913–3921
Table 1
Data on the inhibition of hCA I, II (cytosolic) and IX, XII (transmembrane, tumor-associated isoforms) by a stopped-flow CO2 hydrase assay of compounds 3–8, 13a–f, 14–16, 21–
28, 37–44
Compd
Formula
Ki (nM)
hCA I
9.0
hCA II
8.6
hCA IX
7.9
hCA XII
5.9
SO2NH2
3
4
5
6
7
N
N
O
H2NO2S
11.0
10.1
11.1
41.5
4.7
4.2
7.3
6.5
6.3
7.1
8.2
7.6
7.9
9.9
N+
N
O
O
SO2NH2
N+
O
N
O
SO2NH2
Ph
10.5
38.2
N
N
O
H2NO2S
Ph
O
N+
N
O
SO2NH2
Ph
N
8
56.5
15.9
12.4
8.5
49.3
6.4
5.9
5.5
5.3
5.1
7.2
7.4
5.8
6.6
4.8
3.9
5.8
6.3
7.5
26.4
6.6
N+
O
O
SO2NH2
MeO
13a
13b
13c
13d
13e
13f
N
N
O
SO2NH2
EtO
N
N
O
SO2NH2
BuO
23.7
7.1
N
N
O
SO2NH2
i-BuO
16.0
19.8
21.1
N
N
O
SO2NH2
PhH2CO
7.9
N
N
O
SO2NH2
PhO
6.3
N
N
O
H2NO2S
Ph
14
15
16
620
667
563
51.2
56.9
49.5
62.9
62.8
58.0
6.5
8.3
7.1
N
N
O
H2NO2S
Ph
N+
N
O
O
SO2NH2
Ph
N
N+
O
O

K. Chegaev et al. / Bioorg. Med. Chem. 22 (2014) 3913–3921
3915
Table 1 (continued)
Compd
Formula
Ki (nM)
hCA I
492
hCA II
358
hCA IX
54.7
hCA XII
O2N
N
O
21
22
23
24
25
26
27
28
77.8
82.3
6.1
H2NO2S
H2NO2S
N
O2N
N
O
591
214
498
244
262
303
282
479
75.9
204
103
113
135
64.1
54.5
71.1
N+
O
PhO
N
O
H2NO2S
H2NO2S
N
PhO
N
O
63.9
6.3
N+
O
PhS
N
O
57.6
3.9
H2NO2S
H2NO2S
N
PhS
N
O
5037
63.6
3.8
N+
O
PhSO2
N
O
6.2
H2NO2S
H2NO2S
N
PhSO2
N
O
34.5
6.0
N+
O
N
N
N
O
N
37
38
39
387
354
337
340
285
146
110
47.9
49.8
70.3
72.5
28.4
H2NO2S
O
Ph
Ph
O
N+
H2NO2S
H2NO2S
O
O
O
O
O
O
N
H2NOC
N
O
N+
40
41
42
229
101
106
113
50
32.3
10.8
11.3
30.1
5.6
H2NO2S
H2NO2S
O
H2NOC
N
N
O
N
O
NC
O
N+
53
5.9
H2NO2S
H2NO2S
O
NC
O
SO2Ph
43
44
9.5
8.9
9.5
7.3
6.4
3.1
2.9
N
N
O
O
H2NO2S
O
SO2Ph
103
N
N+
O

3916
K. Chegaev et al. / Bioorg. Med. Chem. 22 (2014) 3913–3921
O
O
in Scheme 2. The action of chlorosulfonic acid on 4-nitro-3-phe-
O
O
S
O
S
S
O
S
S
nylfurazan (17) and on the related furoxan 18 affords the corre-
sponding chlorosulfonyl derivatives 19, 20, which are easily
transformed by action of ammonia into the sulfonamides 21, 22.
Nucleophilic displacement, under basic conditions, of the nitro
group by phenol and thiophenol yields the final products 23, 24
and 25, 26, respectively. Potassium permanganate oxidation of
the latter two substances in acetic acid solution gives the expected
target compounds 27, 28.
S
O
N
O
O
NH2
NH2
NH
NH
2, Brinzolamide (Azopt)
1, Dorzolamide (Trusopt)
Chart 1.
Furazans 37, 39, 41, in which the sulfonamide group is sepa-
rated from the variously-substituted furazan ring by a phenoxy-
methyl bridge, were prepared by reaction under basic conditions
of the 4-hydroxybenzenesulfonamide (29) with the appropriate
bromomethyl derivatives 30, 32, 34 (Scheme 3). Similarly, the
furoxans 38, 40, 42 were obtained from 31, 33, 35. The two phen-
ylsulfonylfurazanyloxy and 3-phenylsulfonylfuroxanyloxy para
substituted benzenesulfonamides 43, 44 were obtained from the
corresponding furazan 9 and furoxan 36, respectively. Compounds
37, 39, 41, 43 were previously used as intermediates for the prep-
aration of a series of hypoglycemic products described in a paper9
that gave no details of their preparation and structural character-
ization, which, conversely, are given here (see Section 4).
products, starting from the easily-accessible related phenylmerca-
pto compounds.7 The methyl and phenyl substituted compounds
are water soluble products and their pKas fall in the 6.80–7.16 range.
This paper reports the characterization of some furazan and
furoxan sulfonamides (Table 1) as inhibitors of human CA I and
CA II, the two isoforms most widespread in organs and tissues,
and as inhibitors of CA IX and CA XII, the two isoforms predomi-
nantly associated with and overexpressed in many tumors. It also
looks at a series of their phenylogues and related derivatives. The
results of a study aimed at evaluating the capacity of selected sul-
fonamides to lower intraocular pressure (IOP) in hyper- and nor-
mo-tensive rabbits are also discussed. This in vivo study was
designed in the light of the fact that most of the investigated sul-
fonamides, such as dorzolamide 1, and brinzolamide 2 (see Chart 1,
effectively inhibit CA II and CA XII, two isoforms associated with
aqueous humor secretion in the eye, and well-established anti-
glaucoma targets.6
2.2. Carbonic anhydrase inhibition
The sulfonamide products described here were tested by a
stopped flow assay10 for their ability to inhibit human (h) catalyt-
ically-active hCA I, hCA II, hCA IX, h CA XII isoforms. All the com-
pounds were able to inhibit the four CA isoforms, but the
strength of their inhibitory potency, evaluated as inhibition con-
stant Ki (nM), depended on their structure (see Table 1).
The simple methyl-substituted compounds 3–5 are potent
inhibitors of all four CA isoforms, since their Kis fall in the low
nanomolar range (4.2–11). The substitution of alkoxy moieties
for the methyl group in 3, to give 13a–f, does not substantially
modify this pattern; there is only a slight fall in the activity of
13e,f on the hCA I isoform, and in the activity of 13a,c on hCA XII
isoform. The phenyl substituted sulfonamides 6–8 behave similarly
to the analogue methyl derivatives, the only difference being that
the two furoxans 7, 8 are less active on both the hCA I and hCA II
isoforms (Ki range 41.5–56.5).
This preference for the hCA IX and hCA XII isoforms is interest-
ing, since both isoforms are overexpressed in many tumor spe-
cies.11,12 The sulfonamides 14–16 that are phenylogues of 6–8
retain strong inhibitory activity on the hCA XII isoform (Ki = 6.5–
8.3), while they display moderate (58.4–62.9) or weak (563–667)
activity, respectively, on the hCA II/hCA IX and hCA I isoforms.
The substitution in 14 and 16 of phenoxy, phenylthio, phenylsulfo-
nyl groups for the phenyl ring does not substantially modify this
picture, since compounds 23–28 are quite selective and potent
2. Results and discussion
2.1. Chemistry
The synthesis of the simple methyl or phenyl substituted furazan
and furoxan sulfonamides studied here (der.s 3–8, Table 1) is
described elsewhere.7 The alkyloxy substituted furazansulfona-
mides 13a–f were prepared by the procedure outlined in Scheme 1.
The action of benzyl mercaptan on 3,4-diphenylsulfonylfurazan (9)
dissolved in methylene chloride in the presence of triethylamine
gave rise to the 3-benzylthio-4-phenylsulfonylfurazan (10) subse-
quently transformed into the related alkyloxy 11a–f derivatives by
reaction with potassium tert-butoxide and the appropriate alcohols.
These intermediates were treated with chlorine in acetic acid solu-
tion to afford the corresponding sulphonyl chlorides 12a–f, which
yielded the expected sulfonamides when reacted with ammonia.
The three sulfonamides 14–16 (Table 1), which are phenylogues
of 6–8, are described elsewhere.8 The substitution in 14 and 16 of
nitro, phenoxy, phenylthio, and phenylsulfonyl groups for the phe-
nyl ring gives rise to the related furazan and furoxan derivatives
21–28, respectively. The synthesis of these products is reported
O
O
O
S
S
S
S
O
S
R
b)
a)
O
O
O
N
N
N
N
N
N
O
O
O
11a-11f
10
9
c)
a R = CH3
b R = C2H5
c R = n-C4H9
d R = iso-C4H9
e R = CH2Ph
f R = Ph
O
SO2NH2
O
R
SO2Cl
R
d)
N
N
N
N
O
O
13a-13f
12a-12f
Scheme 1. Reagents and conditions: (a) PhCH2SH, Et3N, CH2Cl2; (b) ROH, t-BuOK, THF; (c) Cl2, CH3COOH; (d) NH3.

K. Chegaev et al. / Bioorg. Med. Chem. 22 (2014) 3913–3921
3917
O
O
S
Cl
O
O
S
N+
O
O
O
a)
O
N+
+
N+
O
O
Cl
N
N+
(
)
n
O
N+
N
N+
O
N
(
O
)
(
O
)
19 n = 0
20 n = 1
19a n = 0
20a n = 1
n
n
O
O
17 n = 0
18 n = 1
b)
O
O
O
O
S
S
NH2
NH2
O
N+
c)
O
O
N+
(
N
N+
d)
N
(
O
)
n
)
n
O
21 n = 0
22 n = 1
O
O
23 n = 0
24 n = 1
O
O
O
O
S
S
NH2
NH2
O
e)
S
S
O
N+
N
N+
(
O
)
n
N
(
O
)
O
n
O
25 n = 0
26 n = 1
27 n = 0
28 n = 1
Scheme 2. Reagents and conditions: (a) ClSO3H; (b) THF, 33% NH3; (c) PhOH, NaOH, THF; (d) PhSH, NaOH, THF; e) KMnO4, CH3COOH.
O
NH2
S
O
NH2
O
S
( )m
N
R
X
O
a)
+
N+
( )m
N
R
(
O
)
O
n
O
OH
N+
(
O
)
n
O
29
37 R = Ph, n = 0, m = 1
38 R = Ph, n = 1, m = 1
39 R = CONH2, n = 0, m = 1
30 X = Br, R = Ph, n = 0, m = 1
31 X = Br, R = Ph, n = 1, m = 1
32 X = Br, R = CONH2, n = 0, m = 1
40 R = CONH2, n = 1, m = 1
33 X = Br, R = CONH2, n = 1, m = 1
41 R = CN, n = 0, m = 1
42 R = CN, n = 1, m = 1
43 R = SO2Ph, n = 0, m = 0
34 X = Br, R = CN, n = 0, m = 1
35 X = Br, R = CN, n = 1, m = 1
9
X = SO2Ph, R = SO2Ph, n = 0, m = 0
44 R = SO2Ph, n = 1, m = 0
36 X = SO2Ph, R = SO2Ph, n = 1, m = 0
Scheme 3. Reagents and conditions: (a) NaOH, EtOH for R = Ph, CONH2; Na2CO3, DMF for R = CN, SO2Ph.
inhibitors of the hCA XII, and display moderate or weak potency on
the remaining isoforms. Interestingly, the presence of the nitro
group as substituent at the hetero ring (21 and 22) switches off
the selective action on the hCA XII isoform. The suppression of
the phenylogy (cyclic vinylogy) in 14 and 15, following separation
of the benzenesulfonamide moiety from the heterocycle system
through an oxymethyl bridge, gives rise to 37 and 38, respectively,
in which the strong inhibitory action on hCA XII shown by the
leads is reduced. This action is maintained in the cyano analogues
41, 42, but not in the related amides 39, 40. Finally, the separation
of the benzenesulfonamide moiety by an oxy bridge in 27 and 28
gives rise to 43 and 44, respectively, which show strong inhibitory
potency on all the hCA isoforms considered (Ki = 2.9–9.5), with the
sole exception of the furoxan 44 on hCA I (Ki = 103).
worldwide.13 Current medical therapy of open-angle glaucoma
seeks to decrease the elevated IOP resulting from increased pro-
duction of aqueous humor by the ciliary body, and/or decreased
aqueous outflow through trabecular meshwork and the canal of
Schlemm or uveo-scleral route. Humor dynamics are principally
controlled by b-adrenergic receptors present on the ciliary epithe-
lium involved in aqueous production, and by prostaglandin PGF2
a
receptors, which control uveo-scleral drainage.14 Also significantly
involved in humor dynamics are the carbonic anhydrases (CAs), in
particular the CA II isoform, localized to both the pigmented and
the non-pigmented ciliary epithelium, as well as in the anterior
uvea of eye; they control bicarbonate and aqueous humor secre-
tion rates.2 CAIs, such as dorzolamide 1, and brinzolamide 2 (see
Chart 1), are currently used to reduce IOP in open-angle glaucoma
therapy.14 The three methyl substituted furazan and furoxan sul-
fonamides 3–5 were chosen for the in vivo IOP lowering studies
since they are freely soluble in water and trigger potent hCA II/
XII inhibition; dorzolamide was taken as reference compound.
Furoxan 5 possesses NO-dependent vasodilating properties, as
2.3. IOP lowering studies
One of the principal therapeutic applications of CAIs is in
treating glaucoma, one of major causes of visual impairment

3918
K. Chegaev et al. / Bioorg. Med. Chem. 22 (2014) 3913–3921
does its isomer 4, although to a much lesser extent.7 NO release
might bring additional beneficial effects on the IOP lowering prop-
erties of these products.15–17
studied here, showed IOP lowering in the 5–8 mm Hg range, peak-
ing at 2 h post-administration. The stronger hCA II/XII inhibitors 41
and 42 showed a more significant effect, lowering IOP by some
8–12 mm Hg, with maximal effect at 2 h post-administration
(Fig. 2).
In hypertensive rabbits (eye pressure of around 35–42 mm Hg,
generated as reported in Ref. 15) compounds 3–5, and dorzolamide
1 as standard drug, were investigated in a chronic administration
schedule, and the IOP measured for 48 h (Fig. 1). Dorzolamide
was moderately effective for the first 2–3 h post-administration,
whereas the sulfonamides 3–5 investigated here showed much
stronger IOP lowering activities. At the peak, a 10–13.5 mm Hg
decrease of IOP was observed, which was usually achieved at 3–
4 h post administration. For 5, this effect was present also at 8
and 24 h post-administration, making it a very interesting eye-
drop drug candidate (Fig. 1).
Although the sulfonamides 39–42 were less effective hCA II/XII
inhibitors than the methyl derivatives 3–5 discussed above, their
good water solubility, combined with the possibility to formulate
them as 2% eye drops, prompted us to investigate their IOP lower-
ing effects in another animal model of glaucoma, that is, a normo-
tensive rabbit model.17 As seen from Figure 2, dorzolamide 1 is
rather ineffective as an IOP lowering agent in this model, with only
a moderate effect, about 4 mm Hg, at 1 h post-administration. By
contrast the sulfonamides 39–42 showed much stronger IOP low-
ering effects than the standard drug. Products 39 and 40, which
were the weakest hCA II/XII inhibitors of the four compounds
3. Conclusions
Methylfurazan and methylfuroxan sulfonamides were found to
be potent inhibitors of the human hCA I, hCA II, hCA IX, hCA XII iso-
forms; the analogue phenyl derivatives retain high inhibitory
activity, particularly on the hCA IX, and hCA XII isoforms. Struc-
tural modification of these leads gave rise to products with modu-
lated activity and selectivity. In particular, differently-substituted
phenylogues of 3 and 5 showed high potency, particularly on the
hCA XII isoform. The finding that some of the sulfonamides inves-
tigated here possess stronger IOP lowering activity than dorzola-
mide, when tested in rabbits, indicates that these products, and
more in general the class of furazan and furoxan sulfonamide
derivatives, deserve additional study.
4. Experimental protocols
4.1. Chemistry
1H and 13C NMR spectra were recorded on a Bruker Avance 300,
at 300 and 75 MHz, respectively, using SiMe4 as internal standard.
Low resolution mass spectra were recorded with a Finnigan-Mat
TSQ-700. Melting points were determined with a capillary appara-
tus (Büchi 540). Flash column chromatography was performed on
silica gel (Merck Kieselgel 60, 230 ꢁ 400 mesh ASTM); PE stands
for 40–60 petroleum ether. The progress of the reactions was fol-
lowed by thin layer chromatography (TLC) on 5 cm ꢁ 20 cm plates
with a layer thickness of 0.2 mm. Anhydrous magnesium sulfate
was used as drying agent for the organic phases. Organic solvents
were removed under vacuum at 30 °C. Elemental analyses (C, H, N)
were performed by Section de Pharmacie, Service de Microanalyse
(Geneva) and REDOX (Monza): the results are within 0.4% of theo-
retical values. Compounds 3–8,7 9,18 14–16,8 17,19 18,19 29,20 32,21
33,22 34,21 35,23 and 3624 were obtained as described elsewhere.
ꢀme/min
0
0.5
1
2
3
4
5
6
7
8
24 48
2.0
0.0
3
-2.0
4
-4.0
-6.0
5
-8.0
vehicle
1
-10.0
-12.0
-14.0
-16.0
Figure 1.
in rabbit eyes treated with 50
2% as standard drug and vehicle. Errors were within 10ꢀ15% of the reported IOP
values (from three different measurements for each of the four animals in the study
group) and were statistically significant (p = 0.04 5 by the Student’s t test).
D
IOP (mmHg) versus time (h), in carbomer-induced ocular hypertension
L of 2% solution of compounds 3–5, dorzolamide 1 at
4.1.1. 3-(Benzylsulfanyl)-4-(phenylsulfonyl)-furazan (10)
l
To
a solution of 3,4-bis-(phenylsulfonyl)-furazan (1.05 g,
3.00 mmol) and triethylamine (0.45 mL, 3.2 mmol) in CH2Cl2
(25 mL), benzylmercaptan (0.35 mL, 3.0 mmol) was added and
the reaction mixture was stirred at room temperature (rt) over-
night. The following day, the reaction mixture was diluted with
CH2Cl2 (25 mL) and the organic phase was washed with H2O, HCl
1 N solution and NaHCO3 saturated solution, after which it was
dried and the solvent evaporated. The resulting white solid was
crystallized from EtOH to give the title compound. Yield 91%; mp
88–89 °C (EtOH). 1H NMR (CDCl3) d: 4.39 (s, 2H, CH2); 7.27–7.41
(m, 5H; CH2C6H5); 7.61 (t, 2H), 7.74 (t, 1H), 8.08 (d, 2H) (SO2C6H5);
13C NMR (CDCl3) d: 37.0, 128.2, 128.8, 129.2, 129.7, 134.6, 135.5,
137.9, 151.5, 155.2, 163.1. MS EI: 332 (M+).
4.1.2. General procedure for the synthesis of 4-alkyl/aryloxy-
furazan-3-sulfonamides (13a–f)
To a solution of the corresponding alcohol/phenol (10.0 mmol)
in dry THF (15 mL), under positive N2 pressure, t-BuOK (0.67 g,
6.0 mmol) was added and the reaction mixture stirred at rt until
it became transparent (ca. 15 min). Then 10 (6.0 mmol) was added
in one portion and stirring was continued for 1 h. The reaction mix-
ture was then poured into H2O (50 mL) and extracted with Et2O
(2 ꢁ 50 mL). The organic phase was washed with brine, dried and
evaporated. The resulting oil was dissolved in AcOH (15 mL) and
Figure 2.
D
IOP (mmHg) versus time (h) in hypertonic saline-induced ocular
L of 2% solution of compounds 39–42,
hypertension in rabbits, treated with 50
l
dorzolamide 1 at 2% as standard drug and vehicle. Errors were within 10ꢀ15% of the
reported IOP values (from three different measurements for each of the four
animals in the study group) and were statistically significant (p = 0.045 by the
Student’s t test).

K. Chegaev et al. / Bioorg. Med. Chem. 22 (2014) 3913–3921
3919
gaseous chlorine was bubbled through the solution for 1 h. The
resulting mixture was poured into H2O (50 mL) and extracted with
PE (2 ꢁ 50 mL). The organic phase was washed with brine, dried
and evaporated. The residue was cooled in an ice-bath and treated
with NH3 concd sol (2 mL) at 0 °C for 30 min. The mixture was then
diluted with HCl 4 N sol (10 mL) and extracted with EtOAc
(2 ꢁ 25 mL). The organic phase was washed with brine, dried and
evaporated. The resulting product was purified as described.
PE/EtOAC without heating to give the title compound as a white
solid, with 86% yield. Mp 141.5–142.5 °C. 1H NMR (DMSO-d6) d:
7.61 (s, 2H, NH2), 8.04 (m, 4H, C6H4). 13C NMR (DMSO-d6) d:
125.8, 130.1, 146.6, 149.8, 159.8. MS EI: 270 (M+).
4.1.2.8.
4-(4-Nitrofuroxan-3-yl)benzenesulfonamide
(22).
Benzensulfonylchloride 20a was prepared from 18 by action of
ClSO3H, following the procedure described above, with 34% yield.
The mixture of meta and para regioisomers was resolved by flash
chromatography (PE/EtOAc 90/10 v/v) to obtain the first eluted
20, as a white solid, 34% yield, and the second eluted meta isomer
20a as a white solid, 16% yield. Compound 20 (3.05 g, 10.0 mmol)
was immediately dissolved in dry THF (100 mL), and 32% NH3
(16 mL) was added. After 30 min the reaction was complete and
the solvent was evaporated under reduced pressure. The solid thus
obtained was taken up with H2O and extracted with EtOAc; the
organic layers were washed with brine, dried, and evaporated
under reduced pressure. The crude product was purified by flash
chromatography (PE/EtOAc 70/30 v/v) to give the title compound
as a yellow solid, 67% yield. Mp 138–139 °C (PE/EtOAc). 1H NMR
(DMSO-d6) d: 7.57 (s, 2H, NH2), 7.94 (d, 2H, C6H4), 8.03 (d, 2H,
C6H4). 13C NMR (DMSO-d6) d: 111.3, 124.8, 126.8, 131.7, 147.3,
159.7. MS EI: 286 (M+).
4.1.2.1. 4-Methoxyfurazan-3-sulfonamide (13a).
The result-
ing oil was purified by flash chromatography (eluent 95/5 v/v
CH2Cl2/EtOAc). Yield 68%; mp 71–72 °C (CCl4/1,2-dichloroetane).
1H NMR (DMSO-d6) d: 4.14 (s, 3H, CH3); 8.67 (s, 2H, NH2); 13C
NMR (DMSO-d6) d: 60.4, 150.0, 161.8. MS EI: 179 (M+).
4.1.2.2. 4-Ethoxyfurazan-3-sulfonamide (13b).
The result-
ing oil was purified by flash chromatography (eluent 8/2 v/v
PE/EtOAc). Yield 52%; mp 66–67.5 °C (CCl4/1,2-dichloroethane).
1H NMR (DMSO-d6) d: 1.42 (t, 3H, CH3); 4.48 (q, 2H, CH2); 8.67
(s, 2H, NH2); 13C NMR (DMSO-d6) d: 14.0, 69.6, 150.1, 160.8. MS
EI: 193 (M+).
4.1.2.3. 4-Butoxyfurazan-3-sulfonamide (13c).
The result-
ing oil was purified by flash chromatography (eluent 9/1 v/v
PE/EtOAc). Yield 50%; mp 85–86 °C (CCl4). 1H NMR (DMSO-d6) d:
0.92 (t, 3H, CH3); 1.38–1.50 (m, 2H, CH2); 1.74–1.83 (m, 2H,
CH2); 4.43 (t, 2H, CH2); 8.66 (s, 2H, NH2); 13C NMR (DMSO-d6) d:
13.3, 18.2, 29.9, 73.3, 150.1, 161.0. MS CI: 222 (M+H+).
4.1.3. General procedure for the synthesis of compounds 23–26
To a solution of the appropriate nitro derivative (2.0 mmol) in
acetone (8 mL), stirred at rt, a solution of phenol or thiophenol
(1.3 equiv) and NaOH 6 M (1.3 equiv) in H2O (10 mL) was slowly
added. After 6 h the acetone was evaporated and the residue was
taken up with H2O; the white solid thus obtained was filtered to
give the desired compound.
4.1.2.4. 4-(2-Methylpropoxy)-furazan-3-sulfonamide (13d).
The resulting oil was purified by flash chromatography (eluent
95/5 v/v CH2Cl2/EtOAc). Yield 52%; mp 96.5–97.5 °C (CCl4). 1H
NMR (DMSO-d6) d: 0.98 (d, 6H, 2CH3); 2.07–2.20 (m, 1H, CH);
4.21 (d, 2H, CH2); 8.66 (s, 2H, NH2); 13C NMR (DMSO-d6) d: 18.3,
27.2, 79.0, 150.1, 161.1. MS CI: 222 (M+H+).
4.1.3.1. 4-(4-Phenoxyfurazan-3-yl)benzenesulfonamide (23).
Yield 86%. Mp 184–184.5 °C (EtOH). 1H NMR (DMSO-d6) d: 7.36–
7.42 (m, 1H, C6H5), 7.62–7.64 (m, 6H, C6H5 + NH2), 8.08 (d, 2H,
C6H4), 8.25(d, 2H, C6H4). 13C NMR (DMSO-d6) d: 119.9, 126.6,
126.7, 127.3, 128.5, 130.3, 145.6, 146.4, 154.2, 163.3. MS EI: 317
(M+).
4.1.2.5.
4-Benzyloxyfurazan-3-sulfonamide
(13e).
The
resulting solid was crystallized from H2O. Yield 32%; mp 126–
127.5 °C (H2O). 1H NMR (DMSO-d6) d: 5.50 (s, 2H, CH2); 7.38–
7.54 (m, 5H, C6H5) 8.70 (s, 2H, NH2); 13C NMR (DMSO-d6) d: 74.4,
128.4, 128.5, 128.9, 134.3, 150.1, 160.7. MS CI: 256 (M+H+).
4.1.3.2. 3-(4-Phenoxyfuroxan-3-yl)benzenesulfonamide (24).
Yield 84%. Mp 178–179.5 °C (EtOH). 1H NMR (DMSO-d6) d: 7.38–
7.40 (m, 1H, C6H5), 7.54–7.59 (m, 6H, C6H5 + NH2), 8.08 (d, 2H,
C6H4), 8.25(d, 2H, C6H4). 13C NMR (DMSO-d6) d: 108.1, 120.4,
125.2, 126.4, 126.7, 127.4, 130.3, 145.7, 152.7, 162.2. MS EI: 333
(M+).
4.1.2.6. 4-Phenoxyfurazan-3-sulfonamide (13f).
The result-
ing solid was crystallized from H2O. Yield 14%; mp 143.5–145 °C
(H2O). 1H NMR (DMSO-d6) d: 7.34–7.61 (m, 5H, C6H5) 8.84 (s, 2H,
NH2); 13C NMR (DMSO-d6) d: 119.4, 126.6, 130.2, 150.7, 153.7,
160.6. MS EI: 241 (M+).
4.1.3.3. 4-(4-Phenylthiofurazan-3-yl)benzenesulfonamide (25).
Yield 93%. Mp 158–158.5 °C (EtOH). 1H NMR (DMSO-d6) d: 7.45–
7.61 (m, 7H, C6H5 + NH2), 8.02 (m, 4H, C6H4). 13C NMR (DMSO-d6)
d: 126.6, 127.5, 127.9, 129.2, 129.8, 130.1, 133.0, 146.4, 151.5,
152.7. MS EI: 333 (M+).
4.1.2.7.
4-(4-Nitrofurazan-3-yl)benzenesulfonamide
(21).
To a stirred solution of ClSO3H (100 mL) at 0 °C, compound 17
(10.00 g; 52.0 mmol) was added portionwise. When the addition
was complete, the mixture was allowed to reach rt, and stirred
for 4 h. The mixture was then added slowly to ice and the resulting
solution was extracted with CH2Cl2. The collected organic layers
were washed with H2O and brine, dried, and evaporated under
reduced pressure. The mixture of the resulting meta- and para-
regioisomers was resolved by flash chromatography (PE/EtOAc
90/10 v/v) to obtain the first eluted compound 19 as a white solid
with 13% yield, and the second eluted meta-isomer 19a as a white
solid, with 53% yield. Compound 19 (2.89 g, 10.0 mmol) was imme-
diately dissolved in dry THF (50 ml), and 32% NH3 (11 mL) was
added. After 30 min the reaction was complete and the solvent
was evaporated under reduced pressure. The solid thus obtained
was taken up with H2O and extracted with EtOAc; the organic
layers were washed with brine, dried, and evaporated under
reduced pressure. The crude product was crystallized from
4.1.3.4. 3-(4-Phenylthiofuroxan-3-yl)benzenesulfonamide (26).
Yield 91%. Mp 184–185 °C (EtOH). 1H NMR (DMSO-d6) d: 7.44–7.66
(m, 5H, C6H5), 7.61 (s, 2H, NH2), 8.03 (m, 4H, C6H4). 13C NMR
(DMSO-d6) d: 114.4, 125.2, 126.3, 126.6, 128.8, 129.8, 130.0,
133.0, 145.9, 154.2. MS EI: 349 (M+).
4.1.4. General procedure for the synthesis of compounds 27–28
To a solution of the appropriate thio derivative (3 mmol) in
AcOH (10–40 mL) stirred at 0 °C, KMnO4 (3 equiv) was added por-
tionwise. The mixture was stirred at rt until completion of the
reaction, after which water and Na2S2O3 were added portionwise
to turn the mixture white. The white solid thus obtained was fil-
tered to give the desired compound.

3920
K. Chegaev et al. / Bioorg. Med. Chem. 22 (2014) 3913–3921
4.1.4.1. 4-(4-Phenylsulfonylfurazan-3-yl)benzenesulfonamide
4.1.6.4. 4-(3-Carbamoylfuroxan-4-yl-methoxy)benzenesulfon-
(27).
Yield 94%. Mp 191.5–192 °C (EtOH). 1H NMR (DMSO-
amide (40).
Eluent (CH2Cl2/EtOAc 80/20 v/v); yield: 61%;
d6) d: 7.61 (s, 2H, NH2), 7.72–8.05 (m, 9H, C6H5 + C6H4). 13C NMR
(DMSO-d6) d: 126.0, 126.3, 129.0, 130.2, 130.5, 136.3, 137.0,
146.8, 151.6, 156.5. MS EI: 365 (M+).
mp 226.5 °C (EtOH /H2O 1/1 v/v). 1H NMR (DMSO-d6) d: 5.51 (s,
2H, –OCH2–), 7.22 (d, 2H, C6H4), 7.26 (s, 2H, NH2), 7.76 (d, 2H,
C6H4), 7.82 (br s, 1H, –CONH2), 8.50 (br s, 1H, –CONH2). 13C NMR
(DMSO-d6) d: 61.3, 110.4, 114.8, 127.6, 134.4, 137.0, 154.9, 159.7.
MS CI: 315 (M+H+).
4.1.4.2. 3-(4-Phenylsulfonylfuroxan-3-yl)benzenesulfonamide
(28).
Yield 85%. Mp 209–213 dec °C (iPrOH). 1H NMR
(DMSO-d6) d: 7.47–8.01 (m, 11H, C6H5 + C6H4 + NH2). 13C NMR
(DMSO-d6) d: 112.6, 124.1, 126.1, 129.2, 130.1, 130.5, 135.8,
136.3, 146.4, 158.5. MS EI: 381 (M+).
4.1.7. General procedure for the preparation of 41–44
To a solution of 29 (0.9 g, 5.2 mmol) in DMF (20 mL), Na2CO3
(0.55 g, 1 equiv) plus a solution of bromo derivative or bis-
phenylsulfonyl derivative (5.2 mmol) in DMF (10 mL) were added;
the resulting mixture was then stirred at rt for 4 h. The reaction
mixture was poured into water (50 mL) and extracted with EtOAc
(3 ꢁ 50 mL). The organic layers were dried, filtered and evaporated
under reduced pressure. The crude product was purified by flash
chromatography Chromatographic eluents and yields of the prod-
ucts were as follows.
4.1.5. General procedure for the synthesis of compounds 30–31
To a solution of the appropriate alcohol (0.52 mmol) in DMF
(5 mL) stirred at 0 °C, SOBr2 (1.5 equiv) was added and the mixture
was stirred at rt for 1 h. The mixture was then poured into H2O
(30 mL) and extracted with EtOAc (3 ꢁ 10 mL). The organic layers
were washed with NaHCO3 (3 ꢁ 10 mL), brine (20 mL), dried, fil-
tered and evaporated under reduced pressure The crude product
was purified by flash chromatography. Chromatographic eluents
and yields of the products were as follows.
4.1.7.1. 4-(3-Cyanofurazan-4-yl-methoxy) benzenesulfonamide
(41).
Eluent (PE/EtOAc 65/35 v/v); yield: 43%; mp 116 °C
(EtOH /H2O 1/1 v/v). 1H NMR (DMSO-d6) d: 5.71 (s, 2H, –OCH2–),
7.25–7.28 (m, 4H, C6H4 + NH2), 7.81 (d, 2H, C6H4). 13C NMR
(DMSO-d6) d: 59.6, 107.7, 114.8, 127.7, 133.0, 137.6, 154.0. 159.2.
MS CI: 281 (M+H+).
4.1.5.1. 4-Bromomethyl-3-phenylfurazan (30).
Eluent (PE/
EtOAc 90/10 v/v); yield 85%. 1H NMR (CDCl3) d: 4.67 (s, 2H, CH2),
7.54–7.59 (m, 3H, C6H5), 7.84–7.86 (m, 2H, C6H5). 13C NMR (CDCl3)
d: 17.5, 125.4, 128.7, 129.8, 131.4, 151.1, 153.9.
4.1.7.2. 4-(3-Cyanofuroxan-4-yl-methoxy)benzenesulfonamide
4.1.5.2. 4-Bromomethyl-3-phenylfuroxan (31).
Eluent (PE/
(42).
Eluent (PE/EtOAc 65/35 v/v); yield: 52%; mp 149 °C
EtOAc 85/15 v/v); yield 97%. 1H NMR (CDCl3) d: 4.53 (s, 2H, CH2),
7.59–7.46 (m, 3H, C6H5), 7.83–7.74 (m, 2H, C6H5). 13C NMR (CDCl3)
d: 19.8, 114.0, 122.2, 127.8, 129.6, 131.2, 154.0.
(EtOH /H2O 1/1 v/v). 1H NMR (DMSO-d6) d: 5.52 (s, 2H, –OCH2–),
7.20–7.29 (m, 4H, C6H4 + NH2), 7.81 (d, 2H, C6H4). 13C NMR
(DMSO-d6) d: 60.8, 98.1, 106.1, 114.9, 127.7, 137.6, 154.6, 159.2.
MS CI: 297 (M+H+).
4.1.6. General procedure for the synthesis of compounds 37–40
To a solution of 29 (0.69 g, 4.0 mmol) in ethanol (25 mL), 1 N
NaOH (2.7 mL, 0.7 equiv) plus the appropriate bromo derivative
(4.0 mmol) were added; the resulting mixture was then stirred at
50 °C for 6 h. The reaction mixture was poured into water
(50 mL) and extracted with EtOAc (3 ꢁ 50 mL). The organic layers
were dried, filtered and evaporated under reduced pressure. The
crude product was purified by flash chromatography. Chromato-
graphic eluents and yields of the products were as follows.
4.1.7.3.
amide (43).
4-(3-Phenylsulfonylfurazan-4-yloxy)benzenesulfon-
Eluent (PE/EtOAc 80/20 v/v); yield: 62%; mp
155 °C (EtOH /H2O 1/1 v/v). 1H NMR (DMSO-d6) d: 7.50 (s, 2H,
NH2), 7.63 (d, 2H, C6H4), 7.80 (t, 2H, C6H5), 7.92–7.96 (m, 3H,
C6H5), 8.16 (d, 2H, C6H4). 13C NMR (DMSO-d6) d: 119.7, 128.0,
128.7, 130.2, 136.3, 136.8, 142.2, 149.6, 155.4, 160.0. MS CI: 382
(M+H+).
4.1.7.4.
amide (44).
4-(3-Phenylsulfonylfuroxan-4-yloxy)benzenesulfon-
Eluent (PE/EtOAc 65/35 v/v); yield: 29%; mp
4.1.6.1. 4-(3-Phenylfurazan-4-yl-methoxy)benzenesulfonamide
173 °C (EtOH /H2O 1/1 v/v). 1H NMR (DMSO-d6) d: 7.48 (s, 2H,
NH2), 7.64 (d, 2H, C6H4), 7.76 (d, 2H, C6H5), 7.90–7.95 (m, 3H,
C6H5), 8.02 (d, 2H, C6H4). 13C NMR (DMSO d6) d: 111.3, 119.9,
127.9, 128.5, 129.9, 136.2, 136.6, 142.0, 154.8, 157.7. MS CI: 398
(M+H+).
(37).
Eluent (PE/EtOAc 65/35 v/v); yield: 50%; mp 175 °C
(EtOH /H2O 1/1 v/v). 1H NMR (DMSO-d6) d: 5.68 (s, 2H, –OCH2–),
7.19 (d, 2H, C6H4), 7.27 (s, 2H, NH2), 7.54–7.62 (m, 3H, C6H5),
7.80 (d, 2H, C6H4), 7.84–7.86 (m, 2H, C6H5). 13C NMR (DMSO-d6)
d: 59.3, 114.9, 124.6, 127.6, 127.6, 128.2, 129.1, 131.0, 137.3,
150.1, 153.8, 159.4. MS CI: 332 (M+H+).
4.2. CA inhibition
4.1.6.2. 4-(3-Phenylfuroxan-4-yl-methoxy)benzenesulfonamide
An Applied Photophysics stopped-flow instrument was used for
assaying CA-catalysed CO2 hydration activity.10 Phenol red (at a
concentration of 0.2 mM) was used as indicator, working at the
absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.4)
and 20 mM NaBF4 (to maintain constant ionic strength), monitor-
ing the initial rates of CA-catalyzed CO2 hydration reaction for a
period of 10–100 s. The CO2 concentrations ranged from 1.7 to
17 mM for the determination of the kinetic parameters and inhibi-
tion constants. For each inhibitor, at least six traces of the initial 5–
10% of the reaction were used to determine the initial velocity. The
uncatalyzed rates were determined similarly, and subtracted from
the total observed rates. Stock solutions of inhibitor (10 mM) were
prepared in distilled-deionized water and dilutions up to 0.01 nM
were prepared thereafter with distilled-deionized water. Inhibitor
and enzyme solutions were preincubated together for 15 min at
(38).
Eluent (PE/EtOAc 80/20 v/v); yield: 45%; mp 204 °C
(EtOH /H2O 1/1 v/v). 1H NMR (DMSO-d6) d: 5.55 (s, 2H, –OCH2–),
7.20 (d, 2H, C6H4), 7.28 (s, 2H, –NH2), 7.57–7.62 (m, 3H, C6H5),
7.76 (d, 2H, C6H4), 7.83–7.87 (m, 2H, C6H5). 13C NMR (DMSO-d6)
d: 61.2, 114.3, 115.0, 121.9, 127.8, 129.0, 130.8, 137.3, 153.7,
159.3. MS CI: 348 (M+H+).
4.1.6.3. 4-(3-Carbamoylfurazan-4-yl-methoxy)benzenesulfon-
amide (39).
Eluent (CH2Cl2/EtOAc 80/20 v/v); yield: 59%;
mp 183 °C (EtOH/H2O 1/1 v/v). 1H NMR (DMSO-d6) d: 5.61 (s, 2H,
–OCH2–), 7.20 (d, 2H, C6H4), 7.24 (s, 2H, –SO2NH2), 7.76 (d, 2H,
C6H4), 8.23 (br s, 1H, –CONH2), 8.61 (br s, 1H, –CONH2). 13C NMR
(DMSO-d6) d: 59.8, 114.8, 127.6, 137.0, 149.2, 152.0, 158.1, 159.7.
MS CI: 299 (M+H+).

K. Chegaev et al. / Bioorg. Med. Chem. 22 (2014) 3913–3921
3921
room temperature prior to the assay, in order to allow for forma-
tion of the E–I complex. The inhibition constants were calculated
by non-linear least-squares methods using PRISM 3 and the Prus-
off-Cheng equation, as reported elsewhere;17 figures reported are
means from at least three different determinations. All CAs were
recombinant proteins obtained as reported earlier by these
groups.25–29
and 300 min after drug administration. The treatment was applied
in three animals for each drug, in one eye, and compared to the
contralateral eye, treated with vehicle alone. A group of four
non-glaucomatous rabbits was treated with the study drugs and
used as control. At the end of the experiments, the animals were
killed with a lethal dose of Pentothal (Abbott S.p.A., Campoverde
di Aprilia, LT).
4.3. IOP-lowering studies in hypertonic saline-induced IOP raise
in rabbit
Acknowledgments
This work was financed in part by two EU Projects, Metoxia and
Dynano and was partially supported by a Grant of University of
Torino to R.F.
Male white New Zealand rabbits weighing 2–2.5 kg were used
in these studies. Animal were anaesthetized with Zoletil (Tileta-
mine chloride plus Zolazepam chloride, 3 mg/kg b.w., i.m.) and
injected with 0.1 mL hypertonic saline solution (5% in distilled
water) into the vitreous of both eyes. IOP was determined using
a tonometer (Tono-pen Avia Tonometer, Reichhert Inc. Depew,
NY 14043, USA) prior to hypertonic saline injection (basal) and
0.5, 1.0, 1.5, 3, and 6 h thereafter. Vehicle (phosphate buffer 7.00
plus DMSO 2%) or drugs were instilled immediately after the injec-
tion of hypertonic saline. Eyes were randomly assigned to different
groups. Vehicle or drug (0.50 mL) was directly instilled into the
conjunctival pocket at the desired doses (usually 2%).17
References and notes
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4.4. IOP-lowering studies in carbomer-induced IOP raise in
rabbit
Adult male New Zealand Albino rabbits weighing 2–2.5 kg were
employed in this study. The animals were utilized in groups of
eight for each specific treatment. The experimental procedures
conformed to the tenets of the Declaration of Helsinki and the
Guidelines for the Care and Use of Laboratory Animals, as adopted
and promulgated by the U.S. National Institute of Health, and were
conducted after authorization under the Italian regulations on the
protection of animals used for experimental and other scientific
purposes (DM 116/1992) as well as with the European Union Reg-
ulations (OJ of ECL 358/1, 12/12/1986); the experimental protocol
was approved by the Animal Care Committee of the University of
Florence (Florence, Italy). The rabbits were kept in individual
cages; food and water were provided ad libitum. The animals were
identified with a tattoo on the ear, numbered consecutively, and
maintained on a 12–12 h light/dark cycle in a temperature con-
trolled room (22–23 °C). All selected animals were examined
before the beginning of the study and were determined to be nor-
mal at ophthalmic and general health examinations. Glaucoma was
induced by injection of 0.1 mL 0.25% carbomer (Siccafluid, FarMil-
a—THEA Pharmaceuticals) into the anterior eye-chamber bilater-
ally, in rabbits anesthetized with tiletamine and zolazepam
(Zoletil 100, 0.05 mg/Kg b.w.) plus xilazine (Xilor 2%, 0.05 ml/Kg
b.w.) i.m., by the procedure described elsewhere.17 IOP was mea-
sured before carbomer injection and after 1, 2 and 4 h on the first
day, then thrice daily until stabilization, and every 24 h thereafter.
All rabbits treated with carbomer presented a net increase in IOP.
One drop of 0.2% oxybuprocaine hydrochloride (Novesine, Sandoz)
diluted 1:1 with sterile saline was instilled in each eye immedi-
ately before each set of pressure measurements. IOP was measured
using
a Tono-Pen XL tonometer (Medtronic Solan, USA) as
described elsewhere.17 The pressure readings were matched with
two-point standard pressure measurements at 1, 2, 4 and 8 h after
instillation of the drug, and once daily thereafter, using a Digilab
calibration verifier. All IOP measurements were done by the same
investigators using the same tonometer. As soon as a stable IOP
increase was achieved, the animals were treated with the study
drugs. The drugs’ efficacy at lowering IOP was evaluated after drug
administration throughout a four hour period, as follows: before
and immediately after administration, then 30, 60, 90, 120, 240
24. Farrar, W. W. J. Chem. Soc. 1964, 904.
25. Wagner, J. M.; Avvaru, B. S.; Robbins, A. H.; Scozzafava, A.; Supuran, C. T.;
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26. Pacchiano, F.; Aggarwal, M.; Avvaru, B. S.; Robbins, A. H.; Scozzafava, A.;
McKenna, R.; Supuran, C. T. Chem. Commun. (Camb.) 2010, 46, 8371.
27. Köhler, K.; Hillebrecht, A.; Wischeler, J. S.; Innocenti, A.; Heine, A.; Supuran, C.
T.; Klebe, G. Angew. Chem., Int. Ed. 2007, 46, 7697.
28. De Simone, G.; Vitale, R. M.; Di Fiore, A.; Pedone, C.; Scozzafava, A.; Montero, J.
L.; Winum, J. Y.; Supuran, C. T. J. Med. Chem. 2006, 49, 5544.
29. Biswas, S.; Aggarwal, M.; Guzel, O.; Scozzafava, A.; McKenna, R.; Supuran, C. T.
Bioorg. Med. Chem. 2011, 19, 3732.