Inhibition of β-carbonic anhydrases with ureido-substituted benzenesulfonamides

Article abstract of DOI:10.1016/j.bmcl.2010.11.064

A series of sulfonamides was prepared by reaction of sulfanilamide with aryl/alkyl isocyanates. The ureido-substituted benzenesulfonamides showed a very interesting profile for the inhibition of several carbonic anhydrases (CAs, EC 4.2.1.1) such as the human hCA II and three β-CAs from pathogenic fungal or bacterial species. The Candida albicans enzyme was inhibited with potencies in the range of 3.4-3970 nM, whereas the Mycobacterium tuberculosis enzymes Rv1284 and Rv3273 were inhibited with K_is in the range of 4.8-6500 nM and of 6.4-6850 nM, respectively. The structure-activity relationship for this class of inhibitors is rather complex, but the main features associated with effective inhibition of both α- and β-CAs investigated here have been delineated. The nature of the moiety substituting the second ureido nitrogen is the determining factor in controlling the inhibitory power, probably due to the flexibility of the ureido linker and the possibility of this moiety to orientate in different subpockets of the active site cavities of these enzymes.

Full text of DOI:10.1016/j.bmcl.2010.11.064

Bioorganic & Medicinal Chemistry Letters 21 (2011) 102–105
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Inhibition of b-carbonic anhydrases with ureido-substituted
benzenesulfonamides
⇑
Fabio Pacchiano, Fabrizio Carta, Daniela Vullo, Andrea Scozzafava, Claudiu T. Supuran
Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 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 sulfonamides was prepared by reaction of sulfanilamide with aryl/alkyl isocyanates. The ure-
ido-substituted benzenesulfonamides showed a very interesting profile for the inhibition of several car-
bonic anhydrases (CAs, EC 4.2.1.1) such as the human hCA II and three b-CAs from pathogenic fungal or
bacterial species. The Candida albicans enzyme was inhibited with potencies in the range of 3.4–3970 nM,
whereas the Mycobacterium tuberculosis enzymes Rv1284 and Rv3273 were inhibited with Kis in the
range of 4.8–6500 nM and of 6.4–6850 nM, respectively. The structure–activity relationship for this class
Received 20 October 2010
Revised 15 November 2010
Accepted 15 November 2010
Available online 20 November 2010
Keywords:
Beta-carbonic anhydrase
Candida albicans
Mycobacterium tuberculosis
Sulfonamide
Isoform selective inhibitor
of inhibitors is rather complex, but the main features associated with effective inhibition of both a- and
b-CAs investigated here have been delineated. The nature of the moiety substituting the second ureido
nitrogen is the determining factor in controlling the inhibitory power, probably due to the flexibility of
the ureido linker and the possibility of this moiety to orientate in different subpockets of the active site
cavities of these enzymes.
Ó 2010 Elsevier Ltd. All rights reserved.
The carbonic anhydrases (CAs, EC 4.2.1.1) enzymes are widely
distributed throughout the phylogenetic tree, with five genetically
cerevisiae)10,11 were also characterized, being shown to be suscep-
tible to inhibition with the main classes of CA inhibitors (CAIs), that
is, the inorganic anions and the sulfonamides and their bioisoster-
es.6–11 Two other b-CAs from Mycobacterium tuberculosis were suc-
cessfully cloned and crystallized by Suarez Covarrubias et al.12,13
being denominated Rv1284 and Rv3588c. Our group identified,
cloned and characterized the third such enzyme, encoded by the
gene Rv3273.14 The catalytic activity and inhibition with sulfona-
mides/sulfamates of all these b-class enzymes from bacterial and
fungal parasites have been reported, but very few low nanomolar
inhibitors were detected so far for most of them.11,14,15 Sulfon-
unrelated classes (a-, b-, c
-, d- and f-) known to date.1 These pro-
teins which catalyze the interconversion between carbon dioxide
and bicarbonate, with release of a proton, are not only involved
in pH homeostasis and regulation, but also in biosynthetic reac-
tions, such as gluconeogenesis and ureagenesis among others (in
animals), CO2 fixation (in plants and algae), electrolyte secretion
in a variety of tissues/organs, with many of the 16 mammalian
CA isozymes being established drug targets for the design of
diuretics, antiglaucoma, antiepileptic, antiobesity and/or antican-
cer agents.1–3 While the
a
-CA family is mainly present (but not
amide/sulfamate CAIs targeting various mammalian a-CAs, such
exclusive to) in mammals and it has been thoroughly investigated
as acetazolamide 1, dichlorophenamide 2 or sulfanilamide 3, have
been in clinical use for more than 50 years,1 but such compounds
generally show much less effective inhibition of the b-class en-
zymes.11 Furthermore, they generally do not possess the appropri-
ate pharmacological properties (e.g., good penetration through
bacterial walls/membranes) to effectively impair the growth of
many such pathogens (or they show weak such properties, as
shown by work on H. pylori and B. suis enzymes mentioned
above).5,6 Thus, in order to understand whether b-CAs may consti-
tute drug targets for developing antiinfective agents, it is essential
to design compounds with different affinities and pharmacological
properties compared to the classical CAIs of types 1–3.
Here we report the synthesis and inhibition studies of several
b-CAs from fungal or bacterial pathogens with a large series of
ureido-substituted sulfonamides obtained from sulfanilamide 3
as lead molecule. A large series of 4-ureido-substituted benzene-
sulfonamides 4–30 was prepared by reaction of sulfanilamide 3
from the drug design viewpoint,1–3 only recently CAs belonging to
the b- and
c
-CA families, which are widespread in bacteria and
-CAs), respectively,
fungi (the b-CAs) and Archaea (the b- and
c
started to be considered for such a purpose. Thus, a b-CA present
in the gastric pathogen Helicobacter pylori was recently shown to
be a possible target for gastric drugs,4,5 with several low nanomo-
lar inhibitors detected, which effectively inhibited the in vitro and
in vivo growth of the pathogen,4,5 whereas another enzyme from
Brucella suis was also shown to be inhibited strongly by some sul-
fonamide derivatives, leading to impairment of bacterial growth.6
The fungal b-CAs from Candida albicans7 and Cryptococcus neofor-
mans8,9 (as well as the related enzyme present in Saccharomyces
⇑
Corresponding author. Tel.: +39 055 457 3005; fax: +39 055 4573385.
E-mail address: claudiu.supuran@unifi.it (C.T. Supuran).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.11.064

F. Pacchiano et al. / Bioorg. Med. Chem. Lett. 21 (2011) 102–105
103
Table 1
SO2NH2
SO2NH2
Inhibition of the b-CAs from C. albicans, the mycobacterial enzymes Rv1284 and
Rv3273, and human hCA II with ureido-sulfonamides 4–30, sulfanilamide 3 and
acetazolamide AAZ, by a stopped-flow CO2 hydrase assay21
O
N
N
N
SO2NH2
Cl
SO2NH2
S
H
O
Cl
NH2
R
N
N
H
SO2NH2
1
2
H
3
4-30
with aryl/alkyl-isocyanates A1-A27 (Scheme 1).16 The chemical
diversity of these derivatives was generated by varying the nature
of the starting isocyanate A1-A27, since, as shown by a recent
X-ray crystallographic work on five of these derivatives for which
the structure in complex with the human isoforms hCA II has been
reported,17 was determined that it is the nature of the R group that
greatly influences the binding to the enzyme. For compounds7, 13,
16, 25 and 2817 in adduct with hCA II, it has been observed that the
benzenesulfonamide fragment binds in the usual manner, coordi-
nating to the zinc ion as SO2NHꢀ moiety. The phenylsulfamoyl part
of the inhibitor was superimposable between all these adducts and
bound in the usual way,18 filling the middle of the active site
cavity.17,18
However the different R moieties present in these compounds
were observed to bind towards the external part of the enzyme ac-
tive site, occupying various subpockets, and none of them was
superimposable with each other. This phenomenon is probably
due to the rather flexible nature of the ureido linker connecting
the benzenesulfonamide with the R group in this series of com-
pounds, and may also explain a fact reported earlier by our
group,19 that such ureido-substituted sulfonamides (different of
the compounds investigated here) may show selective inhibition
of isoform hCA I over the dominant one hCA II. Normally, it is
hCA II having higher affinity for sulfonamides compared to hCA I,
at least for most of the clinically used derivatives.1–3
A large variety of aromatic and aliphatic R moieties have been
incorporated in the new CAIs 4–30 reported here, in order to
expand the structure–activity relationship (SAR) insight regarding
their interactions with various CA isoforms belonging to the
b-class. The synthesis of such compounds is a one-step process,
generally occurring in high yield, being possible to apply it for
the preparation of a large number of derivatives.16 It should be
noted that most of the CAIs of sulfonamide type investigated ear-
lier1–3 contained CONH or SO2NH linkers between the sulfonamide
head moiety and the R group, instead of the ureido one present in
4–30. These different linkers allow less flexibility for the inhibitor
scaffold, and this is probably the reason why most such sulfona-
mides bind in the cannonical hydrophobic pocket of hCA II and also
generally do not show isoform selectivity.18,20We have investi-
gated the inhibition of the dominant, human isoform hCA II
(offtarget) as well as three b-CAs from pathogenic organisms (fungi
and bacteria) with derivatives 4–30 reported here (Table 1).21,22
The following SAR could be observed from data of Table 1:
No.
R
Kia (nM)
hCA II
C. albicans
Rv1284
Rv3273
3
4
5
6
7
–(Sulfanilamide)
Ph
PhCH2
Ph2CH
4-FC6H4
Cl-FC6H4
4-BrC6H4
4-IC6H4
4-CF3C6H4
3,5-(CF3)2C6H3
C6F5
2-MeOC6H4
4-AcC6H4
2-i-PrC6H4
4-i-PrC6H4
4-n-BuC6H4
4-n-BuOC6H4
4-n-octyl-C6H4
4-NCC6H4
2-NCC6H4
4-PhOC6H4
2-PhC6H4
3-O2NC6H4
4-MeO-2-MeC6H3
9H-Fluoren-2-yl
Cyclopentyl
3,5-Me2C6H3
Indan-5-yl
240
3730
2200
3725
96
781
1290
2634
1150
75
1086
395
547
9230
356
440
4330
6.4
4.8
7.5
6.8
6240
758
717
6380
53.0
63.1
87.1
56.4
533
481
6.4
603
74.6
6850
57.5
63.7
63.0
81.2
64.4
463
818
748
6.5
3970
42.1
62.0
45.0
42.5
61.0
58.5
38.0
702
29.1
3450
355
49.9
50.6
45.9
53.5
326
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
—
69.3
65.7
5.0
50
4070
1060
3.3
5005
2485
2.1
9600
64.7
2.4
85
9.7
15
3310
908
226
1765
8.9
473
470
5610
5690
4760
35.2
6500
50.2
534
560
5590
67.1
49.1
728
509
315
486
481
3.4
297
40.1
53.2
573
70.3
370
733
768
632
104
565
65.9
71.8
132
AAZ
12
a
Errors were in the range of 10% of the reported data, from three different
assays.
4-substituted phenyl moieties or the indane ring. It is inter-
esting to note that 16 and 19 contain in their molecule the
rather bulky, lipophilic i-Pr, n-BuO or biphenyl moieties,
which might be considered too bulky to fit well within the
hCA II active site. However as shown here and in the crystal-
lographic, preliminary communication,17 the i-Pr moiety of
16 makes favorable hydrophobic contacts in a patch within
the enzyme active site never seen earlier to accommodate
inhibitors, also participating to a
P stacking with Phe131.
This particular binding mode explains the excellent hCA II
inhibitory properties of 16 (Ki of 3.3 nM). However, four
other derivatives of the series investigated here, that is, 7,
13, 25 and 28, exploited different binding pockets within
the hCA II active site,17 and also possessed diverse inhibitory
power compared to 16, with inhibition constants in the
range of 15–226 nM (Table 1). Another series of investigated
sulfonamides showed medium potency hCA II inhibition,
with Kis in the range of 15–96 nM. These compounds ( 7,
12, 13, 21, 23 and 25) also incorporate aromatic R groups,
such as 4-fluorophenyl, 3,5-di(trifluoromethyl)phenyl,
pentafluorophenyl, 4-cyanophenyl, 4-phenoxyphenyl or
3-nitrophenyl. The remaining derivatives in this series
showed lower hCA II inhibitory properties, with Kis in the
range of 226–9600 nM. The least effective hCA II inhibitor
was the derivative incorporating the long 4-n-octylphenyl
moiety (20). It should be also noted that sulfanilamide 3
was a medium potency hCA II inhibitor (Ki of 240 nM)
whereas acetazolamide 1 a potent one (Ki of 12 nM).
(i) the offtarget isoform, the cytosolic hCA II, was inhibited with
potencies ranging from the low nanomolar to the micromo-
lar by ureidosulfonamides 4–30. Compounds 16, 19, 22, 24
and 30 were very potent hCA II inhibitors, with Kis in the
range of 2.1–9.7 nM. These derivatives incorporate 2- or
O
MeCN
H2N
SO2NH2
+ RNCO
R
N
N
SO2NH2
H
H
A1-A27
3
4-30
Scheme 1. Preparation of ureidosulfonamides 4–30 from sulfanilamide 3.

104
F. Pacchiano et al. / Bioorg. Med. Chem. Lett. 21 (2011) 102–105
(ii) the b-CA from C. albicans was inhibited by the ureido-
enhanced b-CA inhibitory properties compared to the lead
3, which was a very ineffective inhibitor of this enzyme
(Table 1). Several of the new derivatives were medium
potency ( 4, 5, 11, 12, 14, 22–24 and 27–30) or ineffective
(6 and 16) inhibitors of the Rv3273 enzyme. As for the other
sulfonamides 4–30 with a variable potency, with inhibition
constants in the range of 3.4–3970 nM, compared to sulfanil-
amide 3 which was a weak inhibitor (Ki of 1086 nM) and
acetazoalmide 1 which was a medium potency inhibitor (Ki
of 132 nM). Thus, strong inhibition has been observed for
derivatives 7–13, 15, 18–21, 23, 25, 26, 29 and 30, which
possessed inhibition constants in the range of 3.4–71.8 nM.
These compounds incorporate 3- or 4-substituted phenyl
moieties as R groups, such as 4-halogenophenyl, 4-trifluoro-
methylphenyl, 4-acetylphenyl-; 4-n-butyl/butoxyphenyl,
4-cyanophenyl-; 4-phenoxyphenyl-, 3-nitrophenyl-, as well
as 3,5- or 2,4-disubstituted-phenyl moieties (except 30 which
possesses an indane moiety and 13 which possesses the
pentafluorophenyl moiety). It is obvious that a rather large
number of substitution patterns lead to effective C. albicans
b-CA inhibitors. The best such inhibitor was 23, incorporating
the elongated 4-phenoxyphenylureido moiety which, with an
inhibition constant of 3.4 nM, is the best inhibitor of this
enzyme ever reported in the literature.8 Other substitution
patterns present in these compounds led to less effective
inhibitors. For example, derivatives 4–6, 14, 16, 17, 27 and
28, having aryl, aralkyl or cycloalkyl R moieties, are weak C.
albicans CAIs, with inhibition constants in the range of 297–
3970 nM (Table 1). As for hCA II; small variations in the nature
of the R moietyleads to very differentinhibitory properties for
this series of compounds, with highly effective, medium
potency and ineffectiveCAIs detected in this congenericseries
of sulfonamides.
a
- or b-CAs investigated here, it may be observed that all
types of activities were detected against Rv3273 for the
sulfonamides synthesized in this work.
(v) the selectivity ratios of the new sulfonamides investigated
here, for the inhibition of the target versus offtarget CAs is
a rather complex issue, due to the particular SAR discussed
above for each particular enzyme. However, there are inter-
esting aspects that may be evidenced. For example, several
compounds such as 16, 22 and 24 showed low nanomolar
hCA II inhibitory activity but were much less effective as
b-CA inhibitors and can be considered as selective for the
inhibition of the
a- versus b-CAs. Sulfonamide 23 on the
other hand was a C. albicans CA selective inhibitor, with
selectivity ratios for inhibiting the fungal enzyme over the
mammalian one hCA II of 25, and for inhibiting the fungal
over mycobacterial enzymes of 164.7 and of 240.5, respec-
tively. An inhibitor which was selective for the Rv1284
enzyme has also been discovered, 8, which had selectivity
ratios for inhibiting Rv1284 over hCA II of 162.7, for inhibit-
ing Rv1284 over the fungal enzyme of 12.9 and for inhibiting
Rv1284 over Rv3273 of 13.1. For Rv3273 the most selective
inhibitor was 25, with selectivity ratios of 2.3 over hCA II, of
6.1 over the C. albicans enzyme, and of 10.3 over Rv1284.
These selectivity ratios are less high than for the other two
investigate b-CAs, but these are the first ever reported CAIs
showing this interesting profile and may be useful for under-
standing in greater detail the physiological role of some of
these enzymes.
(iii) the mycobacterial enzyme Rv1284 was very weakly inhib-
ited by sulfanilamide 3 and weakly by acetazolamide 1, with
inhibition constants in the range of 481–9230 nM (Table 1).
However the ureido-benzenesulfonamides 4–30 reported
here generally showed a better inhibitory action against this
enzyme, with Kis in the range of 4.8–6500 nM. Thus, a num-
ber of derivatives, such as 7–10, and 13, were the best
Rv1284 inhibitors in this series, with Kis in the range of
4.8–7.5 nM. All of them incorporate 4-halogenosubstituted
phenyl or pentafluorophenyl R moieties. Another subseries
of these sulfonamides, such as 11, 12, 19, 21, 25 and 26,
showed slightly less effective inhibitory activity, with Kis
in the range of 35.2–69.3 nM. They incorporate mono- or
disubstituted phenyl R moieties containing trifluoromethyl,
nitro, cyano, and alkoxy groups. Medium potency inhibition
has been observed with compounds such as 4, 5, 14, 15, 22,
23, 27–30, which incorporate aryl, aralkyl or cycloalkyl R
moieties. The least effective inhibitors were 6, 16–18, 20
and 23, most of them incorporating rather bulky R groups
(diphenylmethyl, 2-i-Pr-phenyl, 4-n-Bu-phenyl, 4-n-octyl-
phenyl, etc.), which showed micromolar affinity for this
In conclusion, we report here a series of sulfonamides prepared
by reaction of sulfanilamide with aryl/alkyl isocyanates. The
ureido-substituted benzenesulfonamides showed a very interest-
ing profile for the inhibition of hCAII and three b-CAs from
pathogenic fungal or bacterial species. The C. albicans enzyme
was inhibited with potencies in the range of 3.4–3970 nM, whereas
the mycobacterial ones Rv1284 and Rv3273 with Kis in the range of
4.8–6500 nM and of 6.4–6850 nM, respectively. The structure–
activity relationship for this class of inhibitors is rather complex
but the main features associated with effective inhibition of both
a
- and each b-CAs investigated here have been delineated. The
nature of the R moiety substituting the second ureido nitrogen is
the determining factor in controlling the inhibitory power, proba-
bly due to the flexibility of the ureido linker and the possibility of
the R group to orientate in different subpockets of the active site
cavity of these enzymes.
enzyme (Kis in the range of 4.33–6.50 lM).
(iv) the mycobacterial enzyme Rv3273 was inhibited by the new
compounds 4–30 reported here with Kis in the range of 6.4–
6850 nM, whereas the lead 3 and AAZ were ineffective and
Acknowledgements
This research was financed in part by a grant of the 7th FP of EU
(Metoxia project). We are grateful to Professor Fritz A. Muhlschlegel
for the gift of the C. albicans enzyme used in this work.
medium potency inhibitors, respectively (Ki of 6.24 lM for
3 and of 104 nM for 1). The most effective CAIs against this
enzyme were 13 and 25, with Kis in the range of
6.4–6.5 nM. The incorporate the pentafluorophenyl and
3-nitrophenyl R moieties, respectively. A rather large groups
of compounds showed medium potency inhibitory activity
against Rv3273, with Kis in the range of 53.0–87.1 nM, and
they include the halogenophenyl-substituted derivatives
7–10, the 4-acetylphenyl group of 15, and the alkyl-/alkoxy-
phenyl or cyanophenyl moieties present in 17–21. The
disubstituted compound 26 also belong to this category.
Again a rather large number of R moieties lead to highly
References and notes
1. (a) Supuran, C. T. Nat. Rev. Drug Disc. 2008, 7, 168; (b) Supuran, C. T. Bioorg. Med.
Chem. Lett. 2010, 20, 3467; (c) Supuran, C. T.; Scozzafava, A.; Casini, A. Med. Res.
Rev. 2003, 23, 146.
2. (a) Elleuche, S.; Pöggeler, S. Microbiology 2010, 156, 23; (b) Krungkrai, J.;
Krungkrai, S. R.; Supuran, C. T. Curr. Top. Med. Chem. 2007, 7, 909; (c) Supuran,
C. T. Curr. Pharm. Des. 2008, 14, 641.
3. (a) Smith, K. S.; Jakubzick, C.; Whittam, T. S.; Ferry, J. G. Proc. Natl. Acad. Sci.
U.S.A. 1999, 96, 15184; (b) Rowlett, R. S. Biochim. Biophys. Acta 2010, 1804, 362.

F. Pacchiano et al. / Bioorg. Med. Chem. Lett. 21 (2011) 102–105
105
4-{[(20-Methoxyphenyl)amino]carbonyl)}aminobenzenesulfonamide (8): mp 234–
236 °C; silica gel TLC Rf 0.47 (ethyl acetate/petroleum ether 33%); mmax (KBr)
cmꢀ1, 3362 (N–H urea), 2838 (C–H aliphatic), 1684 (C@O urea), 1592 (aro-
matic); dH (400 MHz, DMSO-d6) 3.92 (3H, s, CH3), 6.94 (1H, ddd, J 8.2 7.4 1.4,
40-H), 7.01 (1H, ddd, J 8.0 7.4 1.6, 50-H), 7.07 (1H, dd, J 8.2 1.2, 30-H), 7.23
(2H, s, SO2NH2), 7.64 (2H, d, J 8.8, 2 ꢁ 2-H), 7.77 (2H, d, J 8.8, 2 ꢁ 3-H), 8.16
(1H, dd, J 8.0 1.6, 60-H), 8.38 (1H, s, NH), 9.73 (1H, s, NH); dC (100 MHz,
DMSO-d6) 153.0 (C@O, urea), 148.7, 143.8, 137.7, 129.2, 127.8, 123.2, 121.5,
119.4, 118.1, 111.7, 56.7 (CH3).
4. Nishimori, I.; Minakuchi, T.; Kohsaki, T.; Onishi, S.; Takeuchi, H.; Vullo, D.;
Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2007, 17, 3585.
5. Nishimori, I.; Onishi, S.; Takeuchi, H.; Supuran, C. T. Curr. Pharm. Des. 2008, 14,
622.
6. (a) Joseph, P.; Turtaut, F.; Ouahrani-Bettache, S.; Montero, J.-L.; Nishimori, I.;
Minakuchi, T.; Vullo, D.; Scozzafava, A.; Köhler, S.; Winum, J.-Y.; Supuran, C. T. J.
Med. Chem. 2010, 53, 2277; (b) Vullo, D.; Nishimori, I.; Scozzafava, A.; Köhler, S.;
Winum, J.-Y.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2010, 20, 2178.
7. Klengel, T.; Liang, W. J.; Chaloupka, J.; Ruoff, C.; Schropel, K.; Naglik, J. R.;
Eckert, S. E.; Mogensen, E. G.; Haynes, K.; Tuite, M. F.; Levin, L. R.; Buck, J.;
Mühlschlegel, F. A. Curr. Biol. 2005, 15, 2021.
8. (a) Innocenti, A.; Hall, R. A.; Schlicker, C.; Scozzafava, A.; Steegborn, C.;
Mühlschlegel, F. A.; Supuran, C. T. Bioorg. Med. Chem. 2009, 17, 4503; (b)
Schlicker, C.; Hall, R. A.; Vullo, D.; Middelhaufe, S.; Gertz, M.; Supuran, C. T.;
Mühlschlegel, F. A.; Steegborn, C. J. Mol. Biol. 2009, 385, 1207.
9. (a) Innocenti, A.; Mühlschlegel, F. A.; Hall, R. A.; Steegborn, C.; Scozzafava, A.;
Supuran, C. T. Bioorg. Med. Chem. Lett. 2008, 18, 5066; (b) Innocenti, A.; Hall, R.
A.; Schlicker, C.; Mühlschlegel, F. A.; Supuran, C. T. Bioorg. Med. Chem. 2009, 17,
2654.
10. Isik, S.; Kockar, F.; Aydin, M.; Arslan, O.; Ozensoy Guler, O.; Innocenti, A.;
Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. 2009, 17, 1158.
11. (a) Nishimori, I.; Minakuchi, T.; Maresca, A.; Carta, F.; Scozzafava, A.; Supuran,
C. T. Curr. Pharm. Des., in press.; (b) Winum, J. Y.; Köhler, S.; Supuran, C. T. Curr.
Pharm. Des., in press.; (c) Isik, S.; Guler, O. O.; Kockar, F.; Aydin, M.; Arslan, O.;
Supuran, C. T. Curr. Pharm. Des., in press.
12. Suarez Covarrubias, A.; Larsson, A. M.; Hogbom, M.; Lindberg, J.; Bergfors, T.;
Bjorkelid, C.; Mowbray, S. L.; Unge, T.; Jones, T. A. J. Biol. Chem. 2005, 280,
18782.
13. Suarez Covarrubias, A.; Bergfors, T.; Jones, T. A.; Hogbom, M. J. Biol. Chem. 2006,
281, 4993.
14. Nishimori, I.; Minakuchi, T.; Vullo, D.; Scozzafava, A.; Innocenti, A.; Supuran, C.
T. J. Med. Chem. 2009, 52, 3116.
17. Pacchiano, F.; Aggarwal, M.; Avvaru, B. S.; Robbins, A. H.; Scozzafava, A.;
McKenna, R.; Supuran, C. T. Chem. Commun. (Camb). 2010, 46, 8371.
18. 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.
19. (a) Supuran, C. T.; Scozzafava, A.; Jurca, B. C.; Ilies, M. A. Eur. J. Med. Chem. 1998,
33, 83; (b) Scozzafava, A.; Supuran, C. T. J. Enzyme Inhib. 1999, 14, 343.
20. (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)DiFiore, A.;
Monti, S. M.;Innocenti, A.; Winum,J.-Y.;De Simone, G.;Supuran, C. T. Bioorg. Med.
Chem. Lett. 2010, 20, 3601; (c) Wagner, J.; Avvaru, B. S.; Robbins, A. H.; Scozzafava,
A.; Supuran, C. T.; McKenna, R. Bioorg. Med. Chem. 2010, 18, 4873.
21. 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 10–20 mM Hepes (pH
7.5, for
a-CAs) or TRIS (pH 8.3 for b-CAs) as buffers, and 20 mM Na2SO4 (for
a
-CAs) or 10–20 mM NaCl—for b-CAs (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 (10 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
complex. The inhibition constants were obtained by non-linear least-squares
methods using PRISM 3, whereas the kinetic parameters for the uninhibited
enzymes from Lineweaver–Burk plots, as reported earlier,14,15 and represent
the mean from at least three different determinations.
15. (a) Minakuchi, T.; Nishimori, I.; Vullo, D.; Scozzafava, A.; Supuran, C. T. J. Med.
Chem. 2009, 52, 2226; (b) Güzel, Ö.; Maresca, A.; Scozzafava, A.; Salman, A.;
Balaban, A. T.; Supuran, C. T. J. Med. Chem. 2009, 52, 4063–4067; (c) Carta, F.;
Maresca, A.; Suarez Covarrubias, A.; Mowbray, S. L.; Jones, T. A.; Supuran, C. T.
Bioorg. Med. Chem. Lett. 2009, 19, 6649–6654.
16. General procedure for the preparation of compounds 4–30. Sulfanilamide 3
(2.90 mmols) was dissolved in acetonitrile (20–30 mL) and then treated with a
stoichiometric amount of commercially available isocyanate A1–A27. The
mixture was stirred at rt or heated at 50 °C for 2 h, until completion (TLC
monitoring). The heavy precipitate formed was filtered-off, washed with
diethyl ether (100 ml), dried under vacuo, and recrystallized. For example 8
22. The
a- and b-CAs used in this work were recombinant enzymes obtained as
was obtained by reaction of sulfanilamide
3 (0.50 g; 2.90 mmols) with
reported earlier.7,8,14,15
2-methoxyphenyl isocyanate (0.43 g; 2.90 mmols). The reaction was stirred
at rt overnight, treated as described above, to give 8 as a white solid in 40.4%
yield.
2
1
3
H2NO2S
O
4
N
NH
1'
H
O
2'
6'
5'
3'
4'
8