Synthesis and crystallographic analysis of new sulfonamides incorporating NO-donating moieties with potent antiglaucoma action

Article abstract of DOI:10.1016/j.bmcl.2011.04.046

Several aromatic/heterocyclic sulfonamide scaffolds have been used to synthesize compounds incorporating NO-donating moieties of the nitrate ester type, which have been investigated for the inhibition of five physiologically relevant human carbonic anhydr

Full text of DOI:10.1016/j.bmcl.2011.04.046

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3216–3221
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and crystallographic analysis of new sulfonamides incorporating
NO-donating moieties with potent antiglaucoma action q
Francesco Mincione a, Francesca Benedini b, Stefano Biondi b, Alessandro Cecchi c, Claudia Temperini c,
c,
Giuseppe Formicola d, Ilaria Pacileo d, Andrea Scozzafava c, Emanuela Masini d, , Claudiu T. Supuran
⇑
⇑
a U.O. Oculistica Az. USL 3, Val di Nievole, Ospedale di Pescia, Pescia, Italy
b NicOx Research Institute, Via Ariosto 21, 20091 Bresso, Milan, Italy
c Università degli Studi di Firenze, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, I-50019 Sesto Fiorentino (Firenze), Italy
d Università degli Studi di Firenze, Department of Preclinical and Clinical Pharmacology, Viale Pieraccini 6, 50123 Florence, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Several aromatic/heterocyclic sulfonamide scaffolds have been used to synthesize compounds incorpo-
rating NO-donating moieties of the nitrate ester type, which have been investigated for the inhibition
of five physiologically relevant human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms: hCA I (offtarget),
II, IV and XII (antiglaucoma targets) and IX (antitumor target). Some of the new compounds showed effec-
tive in vitro inhibition of the target isoforms involved in glaucoma, and the X-ray crystal structure of one
of them revealed factors associated with the marked inhibitory activity. In an animal model of ocular
hypertension, one of the new compounds was twice more effective than dorzolamide in reducing ele-
vated intraocular pressure characteristic of this disease, anticipating their potential for the treatment
of glaucoma.
Received 11 March 2011
Revised 7 April 2011
Accepted 12 April 2011
Available online 20 April 2011
Keywords:
Carbonic anhydrase
Sulfonamide
Nitrate ester
NO
Ó 2011 Elsevier Ltd. All rights reserved.
Antiglaucoma drug
X-ray crystallography
Inhibitors of zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1)
have various clinical applications as diuretic, antiglaucoma, anti-
obesity or antitumor drugs.1 Various CA isoforms are responsible
for specific physiological functions, and drugs with such a diversity
of actions target different isozymes of the 15 presently known in
humans.2–6 Sulfonamides and sulfamates constitute the principal
type of classical CA inhibitors (CAIs),1 whereas other chemotypes
(coumarins, polyamines and phenols), were only recently investi-
gated in detail for such an action.7–10 The inhibitors of the sulfon-
amide/sulfamate type bind as anions to the catalytically critical
Zn(II) ion from the enzyme active site, also participating in many
other interaction (hydrogen bond networks; van der Waals con-
mides.11 Recently, the various classes of non-classical inhibitors
have also been investigated from the crystallographic viewpoint,
allowing for a better understanding of the various inhibition mech-
anisms of these enzymes.11
Several sulfonamide CAIs such as acetazolamide AAZ, methazo-
lamide MZA, ethoxzolamide EZA or dichlorophenamide DCP, clin-
ically have been used as systemically acting antiglaucoma agents
for more than 50 years.12 Although glaucoma treatment with such
CAIs is effective in reducing elevated intraocular pressure (IOP),
systemic administration of such drugs leads to a wide range of
side-effects due to inhibition of the enzyme present in other tis-
sues (kidneys, red cells, stomach, lungs, etc.) than the eye.12 In-
deed, these sulfonamides are potent inhibitors of most of the 13
catalytically active isoforms described so far in mammals.1 The
possibility of the topical administration of classical drugs from this
class (AAZ, MZA, EZA or DCP) was investigated extensively in the
50s and 60s, but negative results have been constantly obtained,
and for more than 40 years it was considered that CAIs could only
be given systemically.1,12 In the mid 90s the first clinically used,
topically effective antiglaucoma sulfonamide, dorzolamide DZA,
has been discovered.12 The approach for achieving this compound
(and also brinzolamide BRZ, the second such clinically used phar-
macological agent)12 is known as the ‘ring approach’, as it involved
the exploration of a wide range of ring systems to which sulfamoyl
moieties were incorporated.1–6 We have explored thereafter an
tacts,
p-stacking, etc.) with amino acid residues from the hydro-
phobic and hydrophilic halves of the enzyme active site, as
shown by X-ray crystallographic studies of enzyme–inhibitor com-
plexes.1–6 X-ray crystal structures are available for many adducts
of several isozymes (i.e., CA I, II, IV, VA, VII, IX, XII, XIII and XIV)
mostly with sulfonamides, and with several sulfamates/sulfa-
q
Coordinates and structure factors have been deposited in the Protein Data Bank
as entry 3NI5.
⇑
Corresponding authors. Tel.: +39 055 427 1233; fax: +39 055 427 1280 (E.M.);
tel.: +39 055 457 3005; fax: +39 055 457 3385 (C.T.S.).
E-mail addresses: emnuela.masini@unifi.it (E. Masini), claudiu.supuran@unifi.it
(C.T. Supuran).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.04.046

F. Mincione et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3216–3221
3217
Me
N
N
N
N
N
EtO
S
SO2NH2
MeCONH
SO2NH2
MeCON
SO2NH2
S
S
EZA
AAZ
MZA
NHEt
NHEt
SO2NH2
SO2NH2
SO2NH2
N
O
S
MeO(CH2)3
S
S
Me
S
Cl
SO2NH2
O
O
O
Cl
DZA
BRZ
DCP
O
O
S
O
S
A
B
C
D
E
R = CO(CH2)5ONO2 (NCX 274)
S
O
NH2
R = CO-p-(C6H4)-CH2ONO2 (NCX 265)
R = COO(CH2)3ONO2 (NCX 278)
R = COO(CH2)4ONO2 (NCX 245)
RN
A-E
R = COOCH2CH(ONO2)CH2ONO2 (NCX 201)
alternative approach (the ‘tail approach’) for the design of topically
acting antiglaucoma sulfonamides, which consisted in attaching
tails that will induce the desired physico-chemical properties (such
as for example water solubility, enhanced penetrability through
the cornea, etc.) to scaffolds of simple aromatic/heterocyclic sul-
fonamides incorporating derivatizable moieties of the amino/hy-
droxy/carboxy type.6
We recently applied this approach to investigate some dorzola-
mide derivatives incorporating NO-donating moieties of types A–E
(NCX201–NCX278).13 NO-donors are in fact a relatively underex-
plored class in the field of ocular therapeutics, despite the ubiqui-
tous presence of NO targets in all eye compartments devoted to
aqueous humor production and drainage.14 It has been observed
that hypertensive glaucoma patients have a decreased NO/cGMP
content in the aqueous humor15 and that some NO-donors have
been shown to decrease IOP in normal and pathological condi-
tions.12,15,16 Compounds of types A–E investigated earlier13
showed interesting CA inhibitory and IOP lowering effects in nor-
motensive rabbits. A similar activity has been reported for prosta-
glandins derivatized with NO-donating moieties by NicOx in
collaboration with Pfizer.14b
4-carboxy-benzenesulfonamide 1, 3- or 4-carboxy-benzolamides
2 and 3, 4-(2carboxyethyl)-benzenesulfonamide 4 and 4-hydroxy-
benzenesulfonamide 5.17 The carboxylic or phenol moieties of 1–5
have been derivatized to introduce NO-containing donor groups of
types a–e, which incorporate either aliphatic C3–C5 moieties or
the aromatic ones of type e and f (ferrulic acid derivative).18
The preparation of some of the new compounds reported here is
depicted in Scheme 1. For simplicity, the sulfonamide incorporat-
ing the NO-donating moieties will be denominated by a combina-
tion of figures (indicating the original sulfonamide 1–5 from which
it has been prepared) and letters, indicating the NO-donating moi-
ety which has been used to derivatize the sulfonamide. For exam-
ple, 1e is the ester of the sulfonamide 1 with the mononitrate of
1,4-di(hydroxymethyl)-benzene e.19
The aliphatic nitrate esters, such as 1b, were prepared by reac-
tion of the carboxylate potassium salt of sulfonamide 1 and the
bromo nitrate ester 6 (in the presence of sodium iodide),18 whereas
esterification of carboxylic acids (of types 1–4) with the ferrulic
acid nitrate ester 7 (NCX 2057)18 in the presence of carbodiimides,
afforded derivatives such as 2f, shown in Scheme 1. Alternatively,
some of these esters have been prepared by reaction of the carbox-
ylate caesium salt of 1 with 1 equiv of 1,4-di(chloromethyl)ben-
In the present study our aim was to prepare new sulfonamide
CAIs incorporating NO-releasing moieties. Sulfonamides have been
chosen for this purpose as they are the most investigated class of
CAIs. The hydroxyl or carboxyl moieties present in some aro-
matic/heteroaromatic sulfonamides were chosen to be derivatized
by means of ester moieties which can release NO and sulfonamides
in vivo, both of which have beneficial effects in controlling glau-
coma.12–15
zene, leading to
a monochlorinated intermediate which by
reaction with silver nitrate in acetonitrile was transformed to the
nitrate ester 1e (Scheme 1). The phenol 5 has been derivatized in
a rather similar manner: reaction with 4-bromobutanoic acid in
the presence of carbodiimides led to the bromo derivative 10
which has been converted to the nitrate ester 5a by reaction with
silver nitrate in acetonitrile (Scheme 1).19 All compounds reported
here were characterized by physico-chemical methods which con-
firmed their structures.19
Five different sulfonamide scaffolds have been used to prepare
the new CAIs incorporating NO-donating moieties reported here:
Inhibition data against five physiologically relevant CA isoforms
(i.e., hCA I and II—cytosolic isoforms; hCA IV—membrane-associ-
ated one; hCA IX and XII; transmembrane enzymes)1 with
compounds 1b–5c reported here and standard sulfonamide CAIs
(AAZ, DCP and DZA) are shown in Table 1.20
SO2NH2
SO2NH2
SO2NH2
N
N
O
O
S
N
SO2NH2
S
HOOC
H
It may be observed that against the slow cytosolic isoform hCA I
(an offtarget enzyme for antiglaucoma agents)1 compound 1f was a
strong inhibitor (KI of 15 nM), 2b, 3b, 3f and 5b showed effective
inhibition (KIs in the range of 32–43 nM) whereas the remaining
compounds were less effective inhibitors (KIs in the range of
137–950 nM) similar to the clinically used sulfonamides.
The main target isoform for antiglaucoma sulfonamides is hCA
II.1 Data of Table 1 show that several of the new sulfonamides re-
ported here, such as1b, 1f, 2b, 2f, 3b, 3f and 5b acted as very good
inhibitors of this isozyme, with KIs in the range of 10–33 nM, in the
COOH
(CH2)2COOH
OH
2: 3-COOH
3: 4-COOH
4
1
5
ONO2
ONO2
O
O
(CH2)n
(CH2)4ONO2
a: n = 0
b: n = 1
c: n = 2
OMe
e
f

3218
F. Mincione et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3216–3221
SO2NH2
SO2NH2
ONO2
NaI, K2CO3
DMA
+
Br
6
COOH
ONO2
O
O
1
1b
O
N
N
O
O
(CH2)4ONO2
+
HOOC
S
O
N
SO2NH2
EDCI, DMAP
S
H
2f
HO
DMF
OMe
7
2
Cl
SO2NH2
+
1. Cs2CO3, DMF
2. AgNO3, MeCN
1e
COOH
1
Cl
8
SO2NH2
SO2NH2
EDCI, DMAP
DMA
AgNO3, MeCN
5a
+ Br(CH2)3COOH
9
OH
O
O
Br
5
10
Scheme 1. Synthesis of some of the sulfonamides incorporating NO-donating moieties of type 1–5(a–f). DMA, dimethylacetmide; EDCI, ethyl-diisoprylaminecarbodiimide,
DMAP, 4-dimethylaminopyridine; DMF, dimethylformamide.
Table 1
hCA I, II, IX and XII inhibition data with sulfonamides 1b–5c and AAZ, DCP and DZA as
clinically used drugs from Table 1. The remaining new compounds
were on the other hand less effective as inhibitors of this isoform,
with KIs in the range of 76–345 nM.
standard inhibitors, by a stopped-flow CO2 hydration assay method20
Compd
Ki* (nM)
The tumor associated isoforms hCA IX and XII were also inhib-
ited by all these compounds. Thus, against hCA IX the best inhibi-
tors were 1f, 3b, 3f, and 5a, which showed KIs in the range of 10–
25 nM. Another group of derivatives (4f, 5a and 5c) were medium
efficiency inhibitors (KIs in the range of 83–93 nM) whereas the
remaining ones were medium efficacy compounds (KIs in the range
of 165–253 nM). hCA XII has been shown to be overexpressed in
the eyes of glaucomatous patients,21 and inhibition of this enzyme
may be relevant for developing novel antiglaucoma agents. Indeed,
several of the new sulfonamides reported here (1b, 1f, 2b, 2f, 3b, 3f
and 5b) showed KIs in the range of 19–43 nM for inhibiting this
isoform. The remaining derivatives were less effective hCA XII
inhibitors (KIs in the range of 83–147 nM).
hCA Ia
hCA IIa
hCA IVa
hCA IXb
hCA XIIb
1b
1e
1f
2b
2f
3b
3f
189
242
15
40
137
32
18
198
10
28
33
10
14
101
88
12
71
12
38
9
44
345
47
154
225
46
165
253
11
235
177
10
11
93
83
25
30
147
19
31
42
31
43
87
83
43
38
4f
5a
395
587
41
950
250
1200
50000
139
148
76
89
74
5b
5c
AAZ
DCP
DZA
29
85
25
50
52
116
5.7
50
15000
8500
3.5
a
Full length, cytosolic isoform.
Catalytic domain, recombinant enzyme.
b
N N
O
O
S
HO
*
H
Errors in the range of 5–10% of the reported value, from three different
N
SO2NH2
S
H
O
determinations.
O
O
O
H
ONO2
same range as those of the systemically used antiglaucoma sulfon-
amides AAZ and DCP or the topically acting one DZA.1 It may be
observed that all sulfonamide scaffolds 1–5 and most NO-contain-
ing tails may lead to effective CAIs targeting hCA II. However, the
esters 1e, 4f, 5a and 5c were less active as hCA II inhibitors
compared to the above mentioned derivatives, showing KIs in the
range of 71–198 nM (Table 1).hCA IV, which is also found in the cil-
iary processes within the eyes,1,12 was also effectively inhibited by
some of these new compounds reported here, such as 1b, 1f, 3b
and 3f (KIs in the range of 38–47 nM). All these compounds were
thus much more effective hCA IV inhibitors compared to the
11
NEt2
12
In order to better understand the inhibition of CAs with these
compounds, we resolved the X-ray crystal structure of the target
isoform hCA II complexed with sulfonamide 2b (Figs. 1 and 2).
Crystallographic parameters and refinement statistics for the hCA
II—2b adduct are shown in Table 2. The electron density of almost
all atoms present in the sulfonamide 2b bound to hCA II is shown
in Figure 1, whereas the interactions that the compound makes
with the active site of the enzyme are detailed in Figure 2.22–26

F. Mincione et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3216–3221
3219
Table 2
Crystallographic parameters and refinement statistics for the hCA II—2b
adduct
Parameter
Value
Crystal parameter
Space group
P21
Cell parameters
a = 41.8 Å
b = 42.1 Å
c = 72.0 Å
b = 104.4°
Data collection statistics (20.0–2.1 Å)
No. of total reflections
No. of unique reflections
Completenessa (%)
F2/sig(F2)
37945
13657
97.3 (98.5)
18.9 (4.0)
4.2 (31.5)
R-sym (%)
Refinement statistics (20.0–2.1 Å)
R-factor (%)
22.9
27.0
0.013
1.7
R-freeb (%)
Rmsd of bonds from ideality (Å)
Rmsd of angles from ideality (°)
a
Values in parenthesis relate to the highest resolution shell (2.2–
2.1 Å).
b
Calculated using 5% of data.
Figure 1. Stick representation of compound 2b (yellow) bound in the active site of
hCA II. The electron density is represented by a 2
r
-weighted 2Fo À Fc Fourier map
(pink mesh). The Zn(II) ion (violet sphere), its three His ligands, and residues
Thr199, Thr200 and Gln92 are also shown.
Figure 3. Superposition of the hCA II adducts of sulfonamides 2b (in yellow) and 11
(sky blue).5d The Zn(II) ion is the violet sphere and the enzyme backbone is shown
as green ribbon. Only the 1,3,4-thiadiazolyl-sulfamoyl fragments of the two
inhibitors are superposable.
meta-substituent of the phenyl moiety present in 2b (compared
to the para-one in 11) the conformation of 2b and 11 are quite dif-
ferent when bound to the hCA II active site (Fig. 3). Indeed, in the
superposition shown in Fig. 3 of the hCA II—2b and hCA II—11 ad-
ducts, it may be observed that the amino-1,3,4-thiadiazolyl-2-sul-
famoyl moieties of the two inhibitors 2b and 11 are almost
completely superposable. However the terminal fragments of the
two inhibitors (5-SO2NH moiety and 3-substituted phenyl in 2b
vs 4-substituted phenyl in 11) bind in completely different regions
of the enzyme active site. This allows the 1,4-phenylene moiety of
inhibitor 11 to participate in an edge-to-face stacking with residue
Phe131 from the hCA II active site, whereas this interaction is ab-
sent for the inhibitor 2b, which due to the meta-substituent is ori-
entated in a diverse region of the active site, being too far from the
phenyl moiety of Phe131 for participating in the edge-to-face
stacking interaction. This probably explains why 11 with a KI of
1.4 nM against hCA II5d is 12.8 times a more potent inhibitor com-
pared to 2b (KI of 18 nM).
Figure 2. View of compound 2b (yellow) bound within the hCA II active site. The
Zn(II) ion (violet sphere), its three His ligands (His94, 96 and 119), and residues
Thr199, Thr200 and Gln92 with which the inhibitor interacts, are also shown (CPK
colors). The protein scaffold is shown as the green ribbon.
As for other hCA II-sulfonamide adducts investigated ear-
lier,3a,11,27,28 the deprotonated sulfonamide moiety of the inhibitor
is coordinated to the Zn(II) ion at a distance of 1.96 Å. The same NH
moiety makes a hydrogen bond with the OH of Thr199 (of 2.86 Å).
Furthermore, the two endocyclic nitrogen of the 1,3,4-thidiazole
ring participate in two hydrogen bonds (of 2.50–2.80 Å) with the
OH of Thr200, as reported earlier for a structurally related com-
pound, derivative 11, investigated by Abbate et al.5d One oxygen
of the secondary SO2 moiety of inhibitor 2b makes a hydrogen
bond (of 2.97 Å) with the NH2 of Gln92. However, due to the

3220
F. Mincione et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3216–3221
Compound 1b incorporating a simple 4-carboxybenzenesulf-
and XII (antiglaucoma targets) and IX (antitumor target). Some of
the new compounds showed effective in vitro inhibition of the tar-
get isoforms involved in glaucoma, and the X-ray crystal structure
of one of them also revealed factors associated with the strong
inhibitory activity. In an animal model of ocular hypertension,
one of the new compounds was twice more effective than dorzola-
mide in reducing elevated intraocular pressure typical of this dis-
ease, anticipating the potential of such compounds for the
treatment of glaucoma or elevated IOP.
onamide scaffold and the C4 nitrate ester moiety was chosen for
in vivo studies, due to the good inhibition observed against the
antiglaucoma target isoforms hCA II, IV and XII (KIs in the range
of 18–44 nM, Table 1) and high water solubility (in the range of
2%), compared to other sulfonamides prepared in this study (e.g.,
1f was a better in vitro CAI compared to 1b but had a very low
water solubility (>0.3%), which made it difficult to formulate the
drug for topical administration). Furthermore, 1b is not a strong
inhibitor of hCA I and IX, enzymes not involved in glaucoma. In
the previous work13 we have showed that in vivo the sulfonamides
incorporating nitrate ester moieties release NO and sulfonamides
which both have beneficial effects on the control of intraocular
pressure (IOP).
Table 3 shows the IOP data of hypertensive New Zealand rabbits
treated topically with vehicle, the standard drug dorzolamide (2%),
the NO-donating nitrate ester without CA inhibitory properties iso-
sorbide mononitrate 12 (2%), as well as the NO-donating sulfon-
amide 1b reported here (2%). It may be observed that after
induction of ocular hypertension with carbomer, the IOP of the
experimental animals raised from basal values of 14.7–18.2 mm
Hg to values of 32.4–35.4 mm Hg. Treatment with vehicle had no
effect on the IOP, whereas treatment with DZA led to a maximal
IOP decrease of 7.4 mm Hg, at 60 min post-administration of the
drug directly into the eye (Table 3). Treatment with the nitrate es-
ter 12 had a similar effect, with an IOP reduction of 6.8 mm Hg, but
the maximal effect was observed at 30 min post-administration of
the eye drops. The NO-donating sulfonamide 1b at 2% showed a
very strong IOP lowering, of 15.7 mm Hg, the maximal effect being
observed at 1 h post-administration, as for the standard drug DZA.
These data clearly show that the NO-donating sulfonamide 1b is
very effective in reducing elevated IOP in this animal model of
glaucoma, with an IOP reduction more than twice that shown by
the clinically used drug DZA. Its effect is a summation of the sul-
fonamide CA inhibition effect (as exemplified by the dorzolamide
IOP reduction) and the NO-mediated effects (as exemplified by
the non-CA inhibiting nitrate 12).
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C. T. J. Enzyme Inhib. 1999, 14, 457; (c) Scozzafava, A.; Menabuoni, L.; Mincione,
F.; Briganti, F.; Mincione, G.; Supuran, C. T. J. Med. Chem. 1999, 42, 2641.
7. Carta, F.; Temperini, C.; Innocenti, A.; Scozzafava, A.; Kaila, K.; Supuran, C. T. J.
Med. Chem. 2010, 53, 5511.
In conclusion, we report here a new series of sulfonamides
incorporating NO-donating moieties of the ester type in their mol-
ecule. Several sulfonamide scaffolds have been used to synthesize
these derivatives, which have been investigated for the inhibition
of five physiologically relevant isoforms, hCA I (offtarget), II, IV
8. (a) Maresca, A.; Temperini, C.; Vu, H.; Pham, N. B.; Poulsen, S. A.; Scozzafava, A.;
Quinn, R. J.; Supuran, C. T. J. Am. Chem. Soc. 2009, 131, 3057; (b) Maresca, A.;
Temperini, C.; Pochet, L.; Masereel, B.; Scozzafava, A.; Supuran, C. T. J. Med.
Chem. 2010, 53, 335.
9. (a) Maresca, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2010, 20, 4511; (b)
Maresca, A.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2010, 20,
7255.
Table 3
Intraocular pressure (IOP) lowering effects of 2% dorzolamide DZA, isosorbide
mononitrate 12, and sulfonamide 1b in carbomer-induced ocular hypertensive New
Zealand white rabbits
Compound* Basal IOP After
mm Hg carbomer
mm Hg
Post-
DD IOP** Tmax
***
10. Innocenti, A.; Vullo, D.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett.
2008, 18, 1583.
treatment
(À)mm
min
IOP#
Hg
11. (a) Pastorekova, S.; Parkkila, S.; Pastorek, J.; Supuran, C. T. J. Enzyme Inhib. Med.
Chem. 2004, 19, 199; (b) Alterio, V.; Di Fiore, A.; D’Ambrosio, K.; Supuran, C. T.;
De Simone, G. X-ray Crystallography of CA Inhibitors and its Importance in
Drug Design. In Drug Design of Zinc-Enzyme Inhibitors: Functional, Structural, and
Disease Applications; Supuran, C. T., Winum, J. Y., Eds.; Wiley: Hoboken, 2009;
pp 73–138.
mm Hg
Vehicle
DZA
12
17.9 0.3 32.4 1.3
14.7 0.9 35.4 0.8
16.8 0.3 35.3 1.2
18.2 0.7 34.2 1.4
31.8 1.9
27.1 1.2
28.4 1.6
20.6 1.8
0
0.9
—
7.4 1.9 60
6.8 0.5 30
15.7 1.8 60
1b
12. (a) Wistrand, P. J.; Lindqvist, A. In Carbonic Anhydrase—From Biochemistry and
Genetics to Physiology and Clinical Medicine; Botrè, F., Gros, G., Storey, B. T., Eds.;
VCH: Weinhein, 1991; pp 352–378; (b) Mincione, F.; Scozzafava, A.; Supuran,
C. T. Antiglaucoma Carbonic Anhydrase Inhibitors as Ophthalomologic Drugs.
In Drug Design of Zinc-Enzyme Inhibitors: Functional, Structural, and Disease
Applications; Supuran, C. T., Winum, J. Y., Eds.; Wiley: Hoboken (NJ), 2009; pp
139–154.
13. Steele, R. M.; Batugo, M. R.; Benedini, F.; Biondi, S.; Borghi, V.; Carzaniga, L.;
Impagnatiello, F.; Miglietta, D.; Chong, W. K. M.; Rajapakse, R.; Cecchi, A.;
Temperini, C.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2009, 19, 6565.
14. (a) Impagnatiello, F.; Borghi, V.; Gale, D. C.; Batugo, M.; Gazzetta, M.; Brambilla,
S.; Carreiro, S. T.; Chong, W. K.; Prasanna, G.; Chiroli, V.; Ongini, E.; Krauss, A. H.
Exp. Eye Res., in press.; (b) Krauss, A. H.; Impagnatiello, F.; Toris, C. B.; Gale, D.
C.; Prasanna, G.; Borghi, V.; Chiroli, V.; Chong, W. K.; Carreiro, S. T.; Ongini, E.
Exp. Eye Res., in press.
*
All drugs were administered at the final concentration of 2% in a volume of 50 ll.
IOP was recorded before treatment (basal IOP) and at 30, 60, 90, 180 and 240 min
thereafter, using an applanation tonometer (Tono-Pen XL-Medtronic, Solan). Values
are reported as mean SEM of 8 rabbits per group. Carbomer 0.25% (THEA Phar-
maceutical S.R.) 0.1 ml was introduced bilateraly into anterior chamber of pre-
anesthetized rabbits (Zoletil 100, 0.10 mg/Kg b.w.) for inducing the ocular hyper-
tension, 2–3 weeks before the treatment.
#
Post-treatment IOP is that reflecting the lowest measurement recorded over the
observation period.
**
DD IOP reflects the maximal difference recorded in drug-treated versus vehicle
(Cremophor EL 5% and DMSO 0.4% in phosphate buffer pH 6.00 at room
temperature).
***
The time after administration when the maximal IOP lowering was achieved.

F. Mincione et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3216–3221
3221
15. Galassi, F.; Renieri, G.; Sodi, A.; Ucci, F.; Vannozzi, L.; Masini, E. Br. J.
Ophthalmol. 2004, 88, 757.
16. Kotikoski, H.; Vapaatalo, H.; Oksala, O. Curr. Eye Res. 2003, 26, 119.
17. Sulfonamides 2, 3 were prepared as reported earlier5d whereas 1, 4 and 5 are
commercially available from Sigma–Aldrich (Milan, Italy).
18. The nitrate esters 6 and 7 were prepared as described earlier (Lazzarato, L.;
Donnola, M.; Rolando, B.; Marini, E.; Cena, C.; Corazzi, G.; Guaita, E.; Morini, G.;
Fruttero, R.; Gasco, A.; Biondi, S.; Ongini, E. J. Med. Chem. 2008, 51, 1894),
complex. The inhibition constants were obtained by non-linear least-squares
methods using PRISM 3, as reported earlier,13 and represent the mean from at
least three different determinations. CA isoforms were recombinant ones
obtained in house as reported earlier.7–10
.
21. Liao, S. Y.; Ivanov, S.; Ivanova, A.; Ghosh, S.; Cote, M. A.; Keefe, K.; Coca-Prados,
M.; Stanbridge, E. J.; Lerman, M. I. J. Med. Genet. 2003, 40, 257.
22. The hCA II-2b complex was crystallized as previously described.13 Diffraction
data were collected under cryogenic conditions (100 K) on a CCD Detector KM4
whereas the remaining ones were obtained in
commercially available halogeno derivatives and silver nitrate (all reagents
were from Sigma–Aldrich, Milan, Italy).
a
similar manner from
CCD/Sapphire using Cu K
determined to be: a = 41.8 Å, b = 42.1 Å, c = 72.0 Å and
a
radiation (1.5418 Å). The unit cell dimensions were
= 90°, b = 104.4° in
a = c
the space group P21 Data were processed with CrysAlis RED (Oxford Diffraction
2006).23 The structure was analyzed by difference Fourier technique, using the
PDB file 1CA2 as starting model. The refinement was carried out with the
19. 4-(Nitrooxy)butyl
4-(aminosulfonyl)benzoate
1b:
4-Carboxy-
benzenesulfonamide (200 mg, 0.99 mmol) was dissolved in 10 ml of DMA.
The mixture was stirred and sodium iodide (149 mg, 1.0 mmol) and potassium
carbonate (276 mg, 2.0 mmol) were added, followed by
program REFMAC5;
24 model building and map inspections were performing using
a
solution of 4-
the COOT program.25 The final model of the complex had an R-factor of 22.9%
and R-free 27.0% in the resolution range 20.0–2.1 Å. The correctness of
stereochemistry was finally checked using PROCHECK.26 Coordinates and
structure factors have been deposited within the Protein Data Bank, accession
code 3NI5.
bromobutyl nitrate (20% w/w in CH2Cl2). This solution was added dropwise for
2 h, then the suspension was stirred at room temperature for 24 h. 100 ml of
AcOEt was added and the organic solution was extracted with H2O, HCl 5% and
again water. The organic layer was dried over anhydrous sodium sulfate and
evaporated. The solid obtained was purified by flash-chromatography (CH2Cl2/
MeOH 10:1). 1H NMR (300 MHz, DMSO-d6), d: 1.82–2.14 (m, 4H,
23. Oxford Diffraction. CrysAlis RED. Version 1.171.32.2.; Oxford Diffraction Ltd:
Oxford, UK, 2006.
24. Jones, T. A.; Zhou, J. Y.; Cowan, S. W.; Kjeldoard, M. Acta Crystallogr., Sect. A
1991, 47, 110.
25. Emsley, P.; Cowtan, K. Acta Crystallogr., Sect. D 2004, 60, 2126.
26. Laskowski, R. A.; MacArthur, M. W.; Moss, D. S.; Thornton, J. M. J. Appl.
Crystallogr. 1993, 26, 283.
27. (a) Di Fiore, A.; De Simone, G.; Menchise, V.; Pedone, C.; Casini, A.; Scozzafava,
A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2005, 15, 1937; (b) Menchise, V.; De
Simone, G.; Alterio, V.; Di Fiore, A.; Pedone, C.; Scozzafava, A.; Supuran, C. T. J.
Med. Chem. 2005, 48, 5721.
28. (a) Avvaru, B. S.; Wagner, J. M.; Maresca, A.; Scozzafava, A.; Robbins, A. H.;
Supuran, C. T.; McKenna, R. Bioorg. Med. Chem. Lett. 2010, 20, 4376; (b)
Baranauskiene, L.; Hilvo, M.; Matuliene, J.; Golovenko, D.; Manakova, E.;
Dudutiene, V.; Michailoviene, V.; Torresan, J.; Jachno, J.; Parkkila, S.; Maresca,
A.; Supuran, C. T.; Grazulis, S.; Matulis, D. J. Enzyme Inhib. Med. Chem. 2010, 25,
863; (c) Di Fiore, A.; Monti, S. M.; Innocenti, A.; Winum, J.-Y.; De Simone, G.;
Supuran, C. T. Bioorg. Med. Chem. Lett. 2010, 20, 3601; Nishimori, I.; Vullo, D.;
Innocenti, A.; Scozzafava, A.; Mastrolorenzo, A.; Supuran, C. T. Bioorg. Med.
Chem. Lett. 2005, 15, 3828.
3
CH2CH2ONO2); 4.32 (t, 2H, OCO–CH2, JHH = 7.1 Hz); 4.60 (t, 2H, –CH2ONO2;
3JHH = 6.2 Hz); 7.56 (2H, d, J 8.8, 2 Â 3-H), 7.98 (2H, d, J 8.8, 2 Â 2-H), 8.13 (2H,
s, SO2NH2); 13C NMR (300 MHz, DMSO-d6), d 22.1, 30.2, 71,8, 123,4, 127.8,
129.2, 137.7, 143.8, 182.9 (C@O); ESI+ m/z = 319 (M+H+).
20. Khalifah, R. G. J. Biol. Chem. 1971, 246, 2561. An Applied Photophysics stopped-
flow instrument has been used for assaying the CA catalysed CO2 hydration
activity. Phenol red (at a concentration of 0.2 mM) has been used as indicator,
working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5)
as buffer, and 20 mM Na2SO4 (for maintaining constant the ionic strength),
following the initial rates of the 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 inhibition constants. For each
inhibitor at least six traces of the initial 5–10% of the reaction have been used
for determining the initial velocity. The uncatalyzed rates were determined in
the same manner and subtracted from the total observed rates. Stock solutions
of inhibitor (0.1 mM) were prepared in distilled–deionized water and dilutions
up to 0.01 nM were done thereafter with distilled–deionized water. Inhibitor
and enzyme solutions were preincubated together for 15 min at room
temperature (prior to assay, in order to allow for the formation of the E–I