Carbonic anhydrase inhibitors. Inhibition of the β-class enzymes from the fungal pathogens Candida albicans and Cryptococcus neoformans with branched aliphatic/aromatic carboxylates and their derivatives

Article abstract of DOI:10.1016/j.bmcl.2011.02.057

The inhibition of the β-carbonic anhydrases (CAs, EC 4.2.1.1) from the pathogenic fungi Cryptococcus neoformans (Can2) and Candida albicans (Nce103) with a series of 25 branched aliphatic and aromatic carboxylates has been investigated. Human isoforms hCA I and II were also included in the study for comparison. Aliphatic carboxylates were generally millimolar hCA I and II inhibitors and low micromolar/submicromolar β-CA inhibitors. Aromatic carboxylates were micromolar inhibitors of the four enzymes but some of them showed low nanomolar activity against the fungal pathogenic enzymes. 4-Hydroxy- and 4-methoxy-benzoate inhibited Can2 with K_Is of 9.5-9.9 nM. The methyl esters, hydroxamates, hydrazides and carboxamides of some of these derivatives were also effective inhibitors of the α- and β-CAs investigated here.

Full text of DOI:10.1016/j.bmcl.2011.02.057

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2521–2526
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Carbonic anhydrase inhibitors. Inhibition of the b-class enzymes from the
fungal pathogens Candida albicans and Cryptococcus neoformans with
branched aliphatic/aromatic carboxylates and their derivatives
Fabrizio Carta a, Alessio Innocenti a, Rebecca A. Hall b, Fritz A. Mühlschlegel b, Andrea Scozzafava a,
Claudiu T. Supuran a,
⇑
a Università degli Studi di Firenze, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, I-50019 Sesto Fiorentino (Firenze), Italy
b School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
The inhibition of the b-carbonic anhydrases (CAs, EC 4.2.1.1) from the pathogenic fungi Cryptococcus
neoformans (Can2) and Candida albicans (Nce103) with a series of 25 branched aliphatic and aromatic
carboxylates has been investigated. Human isoforms hCA I and II were also included in the study for com-
parison. Aliphatic carboxylates were generally millimolar hCA I and II inhibitors and low micromolar/
submicromolar b-CA inhibitors. Aromatic carboxylates were micromolar inhibitors of the four enzymes
but some of them showed low nanomolar activity against the fungal pathogenic enzymes. 4-Hydroxy-
and 4-methoxy-benzoate inhibited Can2 with KIs of 9.5–9.9 nM. The methyl esters, hydroxamates, hydra-
Received 28 December 2010
Revised 10 February 2011
Accepted 12 February 2011
Available online 17 February 2011
Keywords:
Carbonic anhydrase
Carboxylate
Inhibitor
b-Class enzyme
Antifungals
zides and carboxamides of some of these derivatives were also effective inhibitors of the
investigated here.
a- and b-CAs
Ó 2011 Elsevier Ltd. All rights reserved.
Candida albicans
Cryptococcus neoformans
In previous work, the inhibition of various carbonic anhydrases
(CAs, EC 4.2.1.1) with a small number of carboxylates (C1–C5 ali-
phatic derivatives and several intermediates of the Krebs cycle)
has been investigated.1,2 These compounds are interesting as they
are a chemotype quite different from the sulfonamides, which are
the main class of inhibitors of these metalloenzymes and have the
potential to have specificity for the b-class CAs which are predom-
inantly found in microorganisms and not vertebrates, over the
in a high number of human pathogens, such as the fungi/yeasts
Candida albicans, Candida glabrata, Cryptococcus neoformans,
Saccharomyces cerevisiae,8–11 and the bacteria Helicobacter pylori,
Mycobacterium tuberculosis, Haemophilus influenzae, Brucella suis,
Streptococcus pneumoniae.10–13 Their inhibition with various
agents, such as sulfonamides, anions, carboxylates, and boronic
acids, has been explored in some detail and for some of these en-
zymes.8–17 Anti-infectives based on inhibition of b-CAs from path-
ogenic organisms have been proposed as alternative novel
therapeutic agents to treat bacterial/fungal infections,3,6 consider-
ing that many of the clinically used agents targeting these patho-
gens led to serious resistance problems.3,6,16,17
a
-class enzymes that are predominant in vertebrates, including
humans.3,4
The b-CAs are the most common class of CA enzymes among the
five genetically distinct families described so far ( -, b-, -, d-, and
a
c
f-CAs), and have been identified in various organisms all over the
phylogenetic tree.3–5 b-CAs have been described in a great number
of species belonging to the Archaea and Bacteria domains,3,5
whereas in Eukaryotes only yeasts, microscopic fungi and very re-
cently arthropods were shown to encode such enzymes.6–10 A high
One of the main problems in the design of CA inhibitors (CAIs)
as therapeutic agents is the inability to selectively target patho-
gen’s b-CA isoform without inhibiting the mammalian
a-CAs. To
date, only the coumarins,18,19 which act as prodrugs, are true selec-
tive CAIs.18,19 The selectivity of this class of drugs is achieved by
number of
a
-CA isoforms (16 CAs in non-primates and 15 in prima-
exploiting the esterase activity of the
a-class enzymes (present
tes),3 have been described and are well characterized for their fun-
only for them and not for the b-,
c
- and f-classes),20 to generate
damental physiological roles.3 b-CAs were recently characterized
substituted 2-hydroxycinnamic acids, which occlude the entrance
to the active site cavity.18–20
In order to find compounds selective for other CA families,
we decided to explore a range of other chemotypes, such as car-
boxylic acids, boronic acids, small inorganic (metal-complexing)
⇑
Corresponding author. Tel.: +39 055 4573005; fax: +39 055 4573385.
E-mail address: claudiu.supuran@unifi.it (C.T. Supuran).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.02.057

2522
F. Carta et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2521–2526
anions, phenols, etc., in addition to the sulfonamides and their
bioisosteres.1,3,11,12,14–17 Only a limited number of carboxylates
have been investigated up to now for the inhibition of the 16 mam-
b-CAs over human (h) hCA II, the physiologically dominant human
isoform. For comparison reasons, hCA I and hCA II inhibition data
with the same anions are provided. Indeed, hCA II is the best stud-
malian
a
-CA isoforms,1,3,14–17 and of the b-CAs from the pathogenic
ied a-CA, being ubiquitous in the human body and playing many
fungi C. albicans (Nce103) and C. neoformans (Can2), by these
groups.1 The investigated carboxylates included the C1–C5 ali-
phatic derivatives, several intermediates of the Krebs cycle (pyru-
vic, lactic, malonic, maleic and citric acids) as well as two
aromatic ones, benzoic acid and 2,3,5,6-tetrafluorobenzoic acid.1
In this Letter we extend the previous work, and investigate a series
of 25 monocarboxylic acids, including branched C3–C12 aliphatic
derivatives (compounds 1–11) and substituted benzoic acids (com-
pounds 12–27) for their interactions with the two enzymes from
the fungal pathogens mentioned above, as well as with the host,
physiologically dominant enzymes hCA I and II (h = human iso-
form; the inhibition of these enzymes was also not reported up
to now with the carboxylates investigated here).21,22 We also
investigated whether derivatization of the COOH moiety (leading
to methyl esters, hydroxamates, hydrazides and carboxamides) of
some of these derivatives influences the CA inhibitory properties
significantly.
important physiologic (or pathologic in cases of disbalances) func-
tions.3 Some of the hCA II inhibition data with these anions (e.g.,
formate, acetate, propionate and butyrate) were reported earlier,1b
and are presented here for comparison reasons. The other data are
new and are presented for the first time. Acetazolamide (5-acetam-
ido-1,3,4-thiadiazole-2-sulfonamide) AAZ, a clinically used CAI has
also been investigated as standard inhibitor.
The following may be observed regarding the inhibition of hCA
I, hCA II, Can2 and Nce103 with carboxylates 1–27 and some of the
carboxylic acid derivatives investigated here:
(i) The slow, ubiquitous cytosolic3 isoform hCA I was weakly
inhibited by the aliphatic carboxylates 1–11, as well as by formate,
propionate and butyrate, with KIs generally in the millimolar range
(101–1040
isoform were acetate, valerate, 4-methylvalerate and 2-methylh-
eptanoate, which showed KIs in the range of 10.8–98 M. It is
lM). The best aliphatic carboxylate inhibitors of this
l
rather striking that there is no regularity in the inhibition pattern
COOH
COOH
COOH
COOH
COOH
Isovaleric Acid
Isobutyric Acid
2-Methylvaleric Acid
3-Methylvaleric Acid
4-Methylvaleric Acid
1
2
4
5
3
COOH
COOH
COOH
4-Methylexanoic Acid
2-Methylexanoic Acid
2-Ethylexanoic Acid
6
8
7
COOH
COOH
COOH
2-Methyleptanoic Acid
2-Butyloctanoic Acid
4-Ethyloctanoic Acid
9
10
11
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
F
F
F
F
I
Br
NO2
OH
F
Cl
15
12
17
13
14
16
18
19
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
NH2
SO2NCl2
NMe2
OMe
NH2
NHAc
CN
Cl
20
21
22
24
23
27
26
25
Tables 1 and 2 show the inhibition data of the b-CAs from path-
ogenic fungi, Can2 and Nce103, with the carboxylates 1–27 men-
tioned above as well as selectivity ratios for the inhibition of the
when passing from formate (very weak inhibitor, KI of 1.04 mM)
to acetate (the best aliphatic inhibitor) and then to propionate
(the same type of activity as formate, KI of 1.01 mM). However

F. Carta et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2521–2526
2523
Table 1
Inhibition constants of carboxylates against isozymes hCA I and II (a-CA class), and b-CAs Can2 (from Cryptococcus neoformans) and Nce103 (from Candida albicans), for the CO2
hydration reaction, at 20 °C22
No.
Inhibitor§
KI#
(l
M)
Selectivity ratios
hCA II/Can2 hCA II/Nce103
hCA Ia
hCA IIa
Can2b
Nce103b
—
—
—
—
—
1
2
3
4
5
6
7
8
9
10
11
Formate
Acetate
Propionate
Butyrate
Valerate
Isobutyrate
Isovalerate
2-Methylvalerate
3-Methylvalerate
4-Methylvalerate
2-Methylhexanoate
4-Methylhexanoate
2-Ethylhexanoate
2-Methylheptanoate
4-Ethyloctanoate
2-Buyloctanoate
1040
10.8
1010
511
55.3
120
300
310
104
98
380
420
450
92
380
101
24000c
130c
960c
1032c
257c
350
100
113
281
320
103
112
93
53.5c
7.3c
35.1c
36.0c
51.5c
26.9c
94.0c
0.98
0.95
10.4
11.1
2.14
3.73
1.25
43.1
11.7
37.9
10.7
448
683
17.8
2.37
4.58
6.00
91.8
109.9
11.0
8.05
60.0
31.8
140.0
97.9
2.79
2.33
16.0
3.61
18.6
34.8
2.73
357.0
105.2
10.8
25.3
149.5
27.6
89.6
2.15
9.40
2.53
7.75
404c
225c
42.8c
3.81
0.91
10.2
34.9
5.33
3.23
0.80
0.95
39.3
41.2
5.18
110
96
83
Selectivity ratios for inhibition of b-CAs over hCA II are also given.
#
Errors were in the range of 3–5% of the reported values, from three different assays.22
§
As sodium salt.
a
Human cloned isoforms, assay at pH 7.5 (Hepes buffer).
This work, at pH 8.3 (Tris buffer), as b-CAs show low catalytic activity at pH values <8.0.
b
c
From Ref. 1.
Table 2
Inhibition constants of aromatic carboxylates against isozymes hCA I and II (a-CA class), and b-CAs Can2 (from Cryptococcus neoformans) and Nce103 (from Candida albicans), for
the CO2 hydration reaction, at 20 °C22
COOH
N
N
AcNH
SO2NH2
R
S
12-27
AAZ
No.
R§
KI#
(lM)
Selectivity ratios
hCA II/Nce103
hCA Ia
hCA IIa
Can2b
Nce103b
hCA II/Can2
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
AAZ
H
730
74
30c
1267c
49.1c
0.13
3.40
4.24
5.46
0.12
0.0095
0.0099
0.12
3.06
0.14
0.32
3.65
0.13
0.12
0.010
24.4c
61.6c
1.09
2.65
4.18
5.17
4.85
0.11
0.15
2.95
3.40
4.47
0.34
4.71
5.20
4.97
0.13
0.02
0.12
25.7
1.12
1.10
1.07
71.3
766.3
811.1
67.7
2.82
48.5
22.7
1.19
25.6
16.3
1.20
1.23
0.09
3.06
1.43
1.12
1.13
1.76
66.2
53.5
2.75
2.53
1.52
21.4
0.92
0.64
0.39
0.09
2,3,5,6-F4–
4-F–
4-Cl–
4-Br–
4-I–
4-O2N–
4-HO–
4-MeO–
4-H2N–
3-H2N–
4-NC–
4-AcHN–
4-ClCH2–
4-Me2N–
4-Cl2NSO2–
—
6c
7.39
7.61
8.00
8.18
8.80
7.61
9.06
8.40
8.79
6.92
4.04
3.88
3.94
3.88
0.25
3.34
3.81
4.70
5.85
8.56
7.28
8.03
8.13
8.63
6.79
7.28
4.36
3.33
1.96
0.012
The standard sulfonamide inhibitor acetazolamide (AAZ) was also included as standard. Selectivity ratios for inhibition of b-CAs over hCA II are also given.
#
Errors were in the range of 3–5% of the reported values, from three different assays.22
§
As sodium salt.
a
Human cloned isoforms, assay at pH 7.5 (Hepes buffer).
This work, at pH 8.3 (Tris buffer), as b-CAs show low catalytic activity at pH values <8.0.
b
c
From Ref. 1.
the longer chain and branched carboxylate then showed a more
regular inhibition pattern, most of them being weak inhibitors (Ta-
ble 1). Considering the aromatic carboxylates (Table 2), it may be
observed that benzoic acid 12 and its tetrafluoro derivative 13
are also rather ineffective inhibitors of this isoform (KIs in the
acids 14–27 show effective, low micromolar inhibitory properties,
with KIs in the range of 3.88–9.06 M. The carboxylate moiety is
-CAs23 (and also
l
known to bind zinc in monodentate fashion in
a
b-CAs).2 Indeed, in the hCA XIII23a and Can2 structures, an acetate
ion was found bound monodentately, by means of one oxygen
atom, to the catalytically crucial Zn(II) ion from the enzyme active
range of 74–730 lM), but all the remaining substituted benzoic

2524
F. Carta et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2521–2526
site. The methyl group on the other hand made not any relevant
interactions with amino acid residues from the active site,2,23 due
to its small moiety. However, it is expected that carboxylates with
a more complicated scaffold compared to acetate, will bind in the
same way to the metal ion, by coordinating through the COOÀ zinc-
binding function, whereas the organic scaffold will interact with
amino acid residues from the active site of the enzyme. This seems
to be the case for the carboxylates investigated here. It may be ob-
served from data of Tables 1 and 2 that these interactions seem to
be rather unfavourable for the aliphatic derivatives, but much
more favorable for the aromatic carboxylates 14–27.
9.5–9.9 nM (Table 2). As the selectivity ratios for inhibiting Can2
over hCA I and II were >80, it may be also concluded that these
compounds are Can2-selective, highly potent inhibitors (no sulfon-
amides with this potency as Can2 inhibitors were detected so far).2
Thus, a rather large range of 4-substituted-benoic acids showed
excellent Can2 inhibition and also a rather high degree of selectiv-
ity for the inhibition of the fungal over the host enzymes. Again the
range of selectivity ratios for the inhibition of Can2 over hC II varies
very much, between 0.02 (for 12) and 811.1 (for 20).
(iv) The fungal enzyme from C. albicans, Nce103, was weakly
inhibited by the previously investigated1 aliphatic carboxylates,
(ii) The physiologically dominant cytosolic isoform hCA II was
also weakly inhibited by the aliphatic, C1–C5 carboxylates (inves-
tigated earlier)1 as well as by compounds 1–11 investigated here,
with KIs in the range of 26.9–94.0 lM, whereas the branched car-
boxylates 1–11 investigated here showed an enhanced activity (Ta-
ble 1). Thus, two compounds, 8 and 10, were rather ineffective
which showed KIs in the range of 83–350
aliphatic hCA II inhibitors were 2-ethylhexanoate 8, 4-ethyloctano-
ate 10 and 2-butyloctanoate 11 (KIs in the range of 83–96 M),
whereas the least effective ones were 1 and 5 (KIs in the range of
l
M (Table 1). The best
Nce103 inhibitors (KIs of 37.9–43.1
phatic derivatives showed inhibition constants in the low micro-
molar range (KIs of 0.95–11.1 M). The best inhibitors in this
subseries were isobutyrate 1 and isovalerate 2 (KIs of 0.95–
0.98 M) whereas the least effective ones the longer chain com-
pounds 8–11. Thus, the SAR for the inhibition of Nce103 and
Can2, two ortholog enzymes, is quite different with this class of
CAIs (see discussion above for Can2 inhibition). For example, 8
was 45 times a better Can2 than Nce103 inhibitor (Table 1). How-
ever, as for Can2, the two benzoic acids 12 and 13 showed weak
lM) but the remaining ali-
l
l
320–350
(KI of 30
l
l
M). Benzoic acid 12 was a medium potency inhibitor
M) but its substituted derivatives 13–27 showed en-
M (Table
l
hanced inhibition, with KIs in the range of 1.96–8.63
l
2). Structure activity relationship (SAR) is not straightforward even
for this subseries of derivatives: for example the highly electroneg-
ative substituents (4-F, 4-Cl) and electron withdrawing (4-chloro-
sulfonyl) moieties led to derivatives with comparable activity (KIs
Nce103 inhibitory properties, with KIs of 24.4–61.6 lM. Selectivity
of 1.96–3.81
l
M for 14, 15 and 27). Thus, the pKa of the carboxylic
ratios for inhibiting Nce103 over hCA II vary considerably from
acid is not the only factor influencing biological activity. Probably
the various interactions in which all fragments present in the
inhibitor scaffold participate with amino acid residues and water
molecules from the active site influence the overall inhibitory
capacity of these compounds.
(iii) The fungal enzyme from C. neoformans, Can2, showed an
interesting inhibition profile with these carboxylates (Tables 1
and 2). Among the aliphatic derivatives investigated earlier,1 only
2.15 (for 8) to 683 (for formate).
The substituted benzoates of type 14–27 investigated here
showed effective inhibitory properties, with KIs in the range of
0.11–5.20 lM. Again, many substitution patterns at the aromatic
ring lead to effective CAIs, the best compounds being the 4-hydro-
xy-, 4-methoxy- and 4-acetamido-benzoic acids 19, 20 and 24,
with KIs of 110–340 nM (Table 2). The selectivity ratios for inhibit-
ing Nce103 over hCA II were <1 for some of the investigated com-
pounds (e.g., 13, 25–27 and acetazolamide) and varied between
1.52 and 66.2 for the remaining compounds (Table 2). SAR is not
straightforward, as already discussed for the inhibition of other
CAs with these compounds, and only the resolution of some
X-ray structures of adducts of this enzyme with such carboxylates
may shed some light in the drug design of carboxylate-based inhib-
itors of b-CAs.
acetate showed low micromolar inhibitory effects (KI of 7.3
with the other anions being quite weak inhibitors (KIs in the range
of 42.8–404 M). Among the branched aliphatic carboxylates
investigated here, three derivatives, 4, 9 and 10, showed weak
inhibitory activity, with KIs in the range of 34.9–41.2 M. The
remaining carboxylates, 1–3, 5–8 and 11, were more effective
Can2 inhibitors, with KIs in the range of 0.80–10.2 M (Table 1).
lM),
l
l
l
This is indeed a very interesting result, considering the fact that
all these carboxylates are much weaker inhibitors of the human
isoforms hCA I and II. For example, 4-methylhexanoate 7, with a
(v) As the free carboxylic acids investigated here showed no
antifungal activity in vivo (data not shown) we decided to investi-
gate whether reducing their polar character by derivatization of
the COOH moiety significantly influences the CA inhibitory proper-
ties. Thus, the methyl ester, hydroxamate, hydrazide and carbox-
amide of the aliphatic derivative 8 and the aromatic one 20 have
been prepared24–26 and investigated for the inhibition of the four
CAs (Table 3). To our greatest surprise, we noticed that this deriv-
atizations lead to a significant increase of the hCA I and II inhibi-
tory effects of the carboxylic acid derivatives over the free
carboxylate, mainly for the aliphatic compounds 8a–8d. Indeed,
they were one–two orders of magnitude better inhibitors com-
pared to the free acid. For the aromatic acid derivatives 20a–20d,
the enhancement of the inhibitory properties was observed but
this is modest compared to the activity of the free acid (17–18
times for the hydroxamate 20b over the acid 20, for hCA I and
II). As far as we know, this is the first study in which such an effect
has been reported.
KI of 0.80 lM for the inhibition of Can2, also has a selectivity ratio
for inhibiting Can2 over hCA I of 525, and of 140 for inhibiting Can2
over hCA II. The selectivity ratios of 2-ethylhexanoate 8 are of 473
(Can2 over hCA I) and of 97.9 for inhibiting Can2 over hCA II (Table
1). The position of the branching from the carboxylate function and
the size of the substituents thus significantly influenced the inhib-
itory power. For example, the isomeric acids 6 and 7 showed four
times differences of activity for the inhibition of Can2. It may be
also observed (Table 1) that the selectivity ratios for the inhibition
of Can2 over hCA II for these aliphatic derivatives varies in a rather
large range, between 2.33 (for 10) to 448 (for formate).
Whereas benzoic acid 12 and its tetrafluoroderivative 13 were
shown1 to be weak Can2 inhibitors (KIs of 48.1–1267
lM), the
substituted benzoates 14–27 showed highly enhanced inhibitory
properties (Table 2). Thus, compounds 15–17 (containing heavy
halogens), 22 (3-aminobenzoic acid) and 25 (4-chloromethyl
derivative) were the weakest inhibitor in the series, with KIs in
In the case of the pathogenic b-class enzymes Can2 and Nce103,
for the aliphatic acid derivatives 8a–8b, an enhancement of the CA
inhibitory properties has been also evidenced, which is rather
modest for Can2 but high (in the range of 7–14 times) for
Nce103 (Table 3). Indeed, for example the hydroxamate 8b was
14.1 times a better Nce103 inhibitor compared to the correspond-
ing acid 8. However, for the aromatic derivatives 20a–20d, the
the range of 3.40–5.46
(14, 18, 21, 23, 24, 26 and 27) showed strong Can2 inhibitory prop-
erties, with KIs in the range of 0.12–0.14 M. Two derivatives, the
lM. Most of the remaining derivatives
l
4-hydroxy- and 4-methoxy-benzoic acid 19 and 20 were on the
other hand low nanomolar can2 inhibitors, with KIs of

F. Carta et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2521–2526
2525
Table 3
Inhibition constants of carboxylic acid derivatives 8–8d and 20–20d against isozymes hCA I and II (
a
-CA class), and b-CAs Can2 (from Cryptococcus neoformans) and Nce103 (from
Candida albicans), for the CO2 hydration reaction, at 20 °C22
COX
COX
OMe
8 - 8d
20 - 20d
No.
X
KI#
(l
M)
Selectivity ratios
hCA Ia
hCA IIa
Can2b
Nce103b
hCA II/Can2
hCA II/Nce103
8
8a
8b
8c
8d
OH
450
93
0.95
0.74
0.29
0.23
43.1
5.60
3.05
4.56
5.70
0.15
0.61
0.47
0.81
0.54
97.9
2.77
8.96
9.43
8.84
811.1
52.7
6.33
66.1
35.2
2.15
0.36
0.85
0.47
0.60
53.5
5.78
0.95
7.09
4.03
OMe
NHOH
NHNH2
NH2
4.38
4.81
8.51
6.48
9.06
3.84
0.48
7.84
4.34
2.05
2.60
2.17
3.45
8.03
3.53
0.45
5.75
2.18
0.39
20
OH
0.0099
0.067
0.071
0.087
0.062
20a
20b
20c
20d
OMe
NHOH
NHNH2
NH2
Selectivity ratios for inhibition of b-CAs over hCA II are also given.
#
Errors were in the range of 3–5% of the reported values, from three different assays.22
Human cloned isoforms, assay at pH 7.5 (Hepes buffer).
This work, at pH 8.3 (Tris buffer), as b-CAs show low catalytic activity at pH values <8.0.
a
b
opposite was true, as the carboxylic acid derivatives were less
Cys127 and a water molecule in the uninhibited enzyme. This
water molecule/hydroxide ion is presumably replaced by the car-
boxylate inhibitor, generating Zn(II) in a stable tetrahedral geome-
try.27 Acetate binds in this way to the enzyme.2 In addition, the
organic scaffold of the inhibitor with its various moieties substitut-
ing it, surely interact with the active site channel (shown schemat-
ically by the two arches from Fig. 1) in different ways, explaining
both the difference of affinity of these inhibitors for Can2 and
Nce103 (since the amino acids are different in the two active sites),
as well as the wide range variation (from hundreds of micromolar
to low nanomolar affinities) of the inhibitory power for diverse
derivatives (e.g., aliphatic versus aromatic carboxylates; variously
substituted benzoic acids, etc.).
effective Can2 and Nce103 inhibitors compared to the free acid
20. The loss of activity was however not very high (Table 3), and
work is in progress in our laboratories to investigate whether such
compounds may be effective as antifungals in vivo. The selectivity
ratios for inhibiting Can2 over hCA II remained good for most of the
new derivatives, but the Nce103 over hCA II selectivity ratios were
less favorable for the aliphatic derivatives 8a–8d, with all of them
<1 (Table 3).
The proposed binding of one of the most effective CAIs detected
here for the b-CAs, that is, 4-hydroxybenzoate 19 (KI of 9.5 nM) to
Can2, based on the X-ray crystal structure of the enzyme,2 is shown
in Figure 1. The Zn(II) is coordinated by residues Cys68, His124,
In conclusion, in this Letter we studied the inhibition of the b-
CAs from the pathogenic fungi C. neoformans (Can2) and C. albicans
(Nce103) with a series of 25 branched aliphatic and aromatic car-
boxylates. hCA I and II were also included in the study for compar-
ison. Aliphatic carboxylates were generally ineffective (millimolar)
hCA I and II inhibitors and low micromolar/submicromolar b-CA
inhibitors. The aromatic carboxylates were micromolar inhibitors
of the four enzymes but some of them showed low nanomolar
activity against the fungal pathogenic enzymes. 4-Hydroxy- and
4-methoxy-benzoate inhibit Can2 with KIs of 9.5–9.9 nM. Interest-
ing structure–activity relationship for the inhibition of the four en-
zymes with these carboxylates have also been evidenced although
they are not always easy to be interpreted due to the scarcity of
crystal structures of b-CAs in adducts with inhibitors. Furthermore,
derivatization of the COOH moiety as methyl ester, hydroxamate,
hydrazide and carboxamide for both aliphatic and aromatic car-
boxylic acids led to very interesting CA inhibitory effects against
O
OH
-
O
NH
Zn2+
-
S
-
O
S
O
Cys127
N
NH
HN
N
H
Cys68
both the
a- and b-CAs investigated here, opening thus new re-
His124
O
search directions for the design of non-sulfonamide inhibitors tar-
geting these enzymes.
Figure 1. Proposed binding of 4-hydroxybenzoate 19 (KI of 9.5 nM) to Can2 based
on the X-ray crystal structure of the enzyme in adduct with acetate.2 The Zn(II) is
coordinated by residues Cys68, His124, Cys127 and
a water molecule in the
Acknowledgments
uninhibited enzyme, which is presumably replaced by the carboxylate inhibitor.2
The half circles represent the active site channel of the enzyme, which is aligned
with various residues in Can2 and Nce103, explaining the different affinity of the
variously substituted carboxylates for the two enzymes.
We thank Professor Clemens Steegborn (Bayreuth University,
Germany) for the gift of Can2 cDNA. This research was financed

2526
F. Carta et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2521–2526
16. (a) Supuran, C. T. Curr. Pharm. Des. 2010, 16, 3233; (b) Nishimori, I.; Minakuchi,
T.; Maresca, A.; Carta, F.; Scozzafava, A.; Supuran, C. T. Curr. Pharm. Des. 2010,
16, 3300; (c) Winum, J. Y.; Kohler, S.; Supuran, C. T. Curr. Pharm. Des. 2010, 16,
3310.
in part by a grant of the 7th Framework Programme of the
European Union (the Metoxia project to A.S. and C.T.S.), and by
MRC and bbsrc grants (to F.A.M.).
17. Burghout, P.; Vullo, D.; Scozzafava, A.; Hermans, P. W. M.; Supuran, C. T. Bioorg.
Med. Chem. 2011, 19, 243.
18. (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.
19. (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.
20. Innocenti, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2010, 20, 6208.
21. Buffers and carboxylates 1–27 (as sodium salts) were of highest purity
available, and were used without further purification, being purchased from
Sigma–Aldrich (Milan, Italy). hCA I and II were prepared in recombinant form
as described earlier,18 whereas Can2 and Nce103 were recombinant, purified
enzymes obtained as described by Mühlschlegel’s and Steegborn’s groups.6–8
22. 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
References and notes
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557 nm, with 10–20 mM Hepes (pH 7.5, for the a-CAs) or TRIS (pH 8.3, for the
b-CAs) as buffers, and 20 mM Na2SO4 or 20 mM NaClO4 (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 (1 mM) were prepared in
distilled–deionized water and dilutions up to 0.1 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,1,10,12,20 and represent the mean from at least three
different determinations.
23. (a) Di Fiore, A.; Monti, S. M.; Hilvo, M.; Parkkila, S.; Romano, V.; Scaloni, A.;
Pedone, C.; Scozzafava, A.; Supuran, C. T.; De Simone, G. Proteins 2009, 74, 164;
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24. Derivatives 8a–8d and 20a–20d have been prepared from the corresponding
acyl chlorides by reaction with nucleophiles or by direct esterification (for the
methyl esters) of the acids.
2-Ethylhexanehydrazide 8c: yield 68% yield; mp 95 °C (Lit.25 91–93 °C); silica
gel TLC Rf 0.15 (ethyl acetate/n-hexane 70% v/v); dH (400 MHz, DMSO-d6) 0.97
(6H, 2 Â CH3), 1.30 (4H, m), 1.51 (4H, m), 2.34 (1H, m, CH), 4.20 (2H, br s,
exchange with D2O, NH2), 9.60 (1H, s, exchange with D2O, CONH); dC (100 MHz,
DMSO-d6) 172.2 (C@O), 47.9, 32.0, 30.2, 26.5, 23.0, 16.2, 12.0.
N-Hydroxy-4-methoxybenzamide 20b: 55% yield; mp 157 °C (Lit.26 153–
155 °C); silica gel TLC Rf 0.16 (ethyl acetate/n-hexane 70% v/v); dH (400 MHz,
DMSO-d6) 3.83 (3H, s, CH3), 7.00 (2H, d, J 8.8, Ar-H), 7.82 (2H, d, J 8.8, Ar-H),
8.92 (1H, br s, exchange with D2O, NH), 10.30 (1H, s, exchange with D2O, OH);
dC (100 MHz, DMSO-d6) 169.3 (C@O), 164.0, 128.1, 127.1, 114.2, 56.0.
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Ghosh, A. J. Org. Chem. 2002, 67, 4833.
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