Inhibition of α-class cytosolic human carbonic anhydrases I, II, IX and XII, and β-class fungal enzymes by carboxylic acids and their derivatives: New isoform-I selective nanomolar inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.07.094

The members of a focused series of carboxylic acids and of their derivatives (esters, amides and metal complexes) have been investigated as inhibitors of the main cytosolic/transmembrane carbonic anhydrase isoforms, CA I, II, IX and XII, belonging to the mammalian α-class of CAs. These enzymes are present in red blood cells in submillimolar concentration, and typical sulfonamide CA inhibitors do not selectively inhibit any of them. Among such isozymes, the isoform-I is an 'orphan' target that mediates hemorrhagic retinal and cerebral vascular permeability, involved in retinal and cerebral disease. In the present study, we identified the first selective CA I nanomolar inhibitors, that displayed activity against other isozymes in micromolar/millimolar concentration range. Selective CA II over CA I inhibition has also been observed with some diketo acids/metal complexes. Few diketo acid derivatives showed inhibition activities against the fungal β-class enzymes from Candida albicans and Cryptococcus neoformans in low micromolar concentration range. Prediction of drug-like properties for the most interesting compounds suggests a favorable bioavailability.

Full text of DOI:10.1016/j.bmcl.2012.07.094

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5801–5806
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Inhibition of
a-class cytosolic human carbonic anhydrases I, II, IX and XII,
and b-class fungal enzymes by carboxylic acids and their derivatives: New
isoform-I selective nanomolar inhibitors
Mario Sechi a, , Alessio Innocenti b, Nicolino Pala a, Dominga Rogolino c, Mauro Carcelli c,
⇑
Andrea Scozzafava b, Claudiu T. Supuran b,
⇑
a Dipartimento di Chimica e Farmacia, Università di Sassari, Via Muroni 23/A, 07100 Sassari, Italy
b Dipartimento di Chimica, Laboratorio di Chimica Bioinorganica, Università degli Studi di Firenze, Rm 188, Via della Lastruccia 3, Polo Scientifico, I 50019 Sesto Fiorentino
(Firenze), Italy
c Dipartimento di Chimica, Università di Parma, Parco Area delle Scienze 17/A, 43124 Parma, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The members of a focused series of carboxylic acids and of their derivatives (esters, amides and metal
complexes) have been investigated as inhibitors of the main cytosolic/transmembrane carbonic anhy-
drase isoforms, CA I, II, IX and XII, belonging to the mammalian a-class of CAs. These enzymes are present
Received 25 June 2012
Revised 23 July 2012
Accepted 25 July 2012
Available online 2 August 2012
in red blood cells in submillimolar concentration, and typical sulfonamide CA inhibitors do not selectively
inhibit any of them. Among such isozymes, the isoform-I is an ‘orphan’ target that mediates hemorrhagic
retinal and cerebral vascular permeability, involved in retinal and cerebral disease. In the present study,
we identified the first selective CA I nanomolar inhibitors, that displayed activity against other isozymes
in micromolar/millimolar concentration range. Selective CA II over CA I inhibition has also been observed
with some diketo acids/metal complexes. Few diketo acid derivatives showed inhibition activities against
the fungal b-class enzymes from Candida albicans and Cryptococcus neoformans in low micromolar con-
centration range. Prediction of drug-like properties for the most interesting compounds suggests a favor-
able bioavailability.
Keywords:
CA inhibitors
CA I isoform
Carboxylic acids/carboxylates
Metal complexes
5-(1-Alyl-1H-indol-3-yl)-1H-pyrazole-3-
carboxylic acids
(Z)-Methyl 2-hydroxy-2-(2-oxo-1-
substituted-indolin-3-ylidene)acetates
Ó 2012 Elsevier Ltd. All rights reserved.
Carbonic anhydrases (CAs, EC 4.2.1.1), as isoforms I and II, are
highly abundant in red blood cells (and in gastrointestinal tract)
of vertebrates, arriving at concentrations as high as 0.2 mM, and
having an important role in CO2 transport to the lungs, and in blood
pH homeostasis.1,2 These two cytosolic isoforms are the dominant
ones among the 16 CAs found to date in various cell/tissues in
vertebrates.3–7 This family of metalloproteins uses Zn2+ as metal
cofactor and catalyzes the simple reaction between carbon dioxide
phosphoinositol glycan anchors to the plasma membranes (CA IV
and XV) or they are transmembrane proteins with extracellular ac-
tive sites (CA IX, XII and XIV).3–7 Many of the CA isozymes are
important therapeutic targets, and are involved in several physio-
logical/pathological processes, with the potential to be inhibited/
activated for treating a large range of disorders.3–10 For example,
CA II plays a role in bicarbonate production in the eye and it is there-
fore a validated target for the therapy of glaucoma.3,4 CA IX and CA
XII are extracellularly localized mainly on hypoxic tumor cells,8–10
where they are involved in tumorigenesis by regulating pH inside
and outside the cancer cell,5 thus interfering with phosphorylation
processes or by playing a role in the cell–cell adhesion.5,10 There-
fore, they provide a target for cancer therapy because of their spec-
ificity towards the hypoxic tumor cells. Concerning the isozyme CA
I, that is considered an ‘orphan’ target, it has been shown that the
presence of extracellular CA I inside either the blood–retinal or
blood–brain barrier can induce vasogenic edema.11 Increased reti-
nal vascular permeability contributes to the pathogenesis of prolif-
erative diabetic retinopathy and diabetic macular edema, leading
causes of vision loss in working-age adults. Elevated levels of extra-
cellular CA I in vitreous from individuals with diabetic retinopathy
suggest that retinal hemorrhage and erythrocyte lysis contribute to
and water with generation of bicarbonate and protons:
À 3–6
CO2+H2O = H++HCO3
.
In humans, in addition to blood, CAs are
present in a large variety of tissues including gastrointestinal and
reproductive tracts, central nervous system, kidneys, lungs, skin
and eyes.3–6 The different isozymes are localized in various cellular
compartments with CA I, CA II, CA III, CA VII and CA XIII being found
in the cytosol, two isoforms (CA VA and VB) are present in
mitochondria and one isoform (CA VI) is secreted in saliva and milk.
Several other CA isoforms are either associated through
⇑
Corresponding authors. Tel.: +39 079 228 753; fax: +39 079 228 745 (M.S.); tel.:
+39 055 4573005; fax: +39 055 4573385 (C.T.S.).
E-mail addresses: mario.sechi@uniss.it (M. Sechi), claudiu.supuran@unifi.it (C.T.
Supuran).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.07.094

5802
M. Sechi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5801–5806
the diabetic vitreous proteome. Alkalinization of vitreous induced
by CA I affects some biological activities, such as kallikrein activity
and its generation of factor XIIa, revealing a new pathway for con-
tact system activation. Thus, the release of erythrocyte CA I may ac-
count for the increased vascular permeability and edema in diabetic
retinopathy or following subdural hematoma. Therefore, inhibition
of extracellular CA I is a major challenge, and could provide new
therapeutic opportunities for the treatment of hemorrhage-induced
retinal and cerebral edema.11
As far as the known CA inhibitors (CAIs) are concerned, the sul-
fonamides and their isosteres (sulfamates, sulfamides, etc.) have
been clinically used for more than 60 years and constitute the most
investigated chemical classes of inhibitors.3–5,12 However, these
compounds indiscriminately inhibit many of the 16 CA isoforms
known in mammals. Thus, efforts have been made to find new
CAIs, in order: (a) to explore molecular diversities and to discover
original pharmacophores, (b) to identify novel inhibitors that
selectively inhibit each isoform, and (c) to develop hit/lead com-
pounds able to interfere with CA ‘orphan’ targets, such as the CA
isoform-I. Recently, the inhibitors belonging to the coumarin fam-
ily were discovered as mechanism-based inhibitors, which act as
prodrugs and bind in a very different mode compared to sulfona-
mides and their isosteres (I, Fig. 1).13,14
for the CAs. Previously, a series of aromatic and aliphatic carboxyl-
ates (V, Fig. 1) were evaluated for the inhibition of both mammalian
a
-class CA isoforms (CA I, II, IX and XII)19 and b-CAs from patho-
genic fungi and bacteria.20 Among various CA isoforms, only hCA
IV resulted sensitive to several carboxylate derivatives, whereas
other ones (i.e. hCA II, V, and IX) were not inhibited by such
compounds.
Considering that carboxylic acids and several of their deriva-
tives are chemically accessible, thus offering a wide and unex-
plored chemical space for designing enzyme inhibitors, we
selected a series of carboxylic/carboxylates based compounds to
be submitted to an extensive biological screening.
First, an in-house library of ꢀ150 compounds was built by
choosing structural backbones carrying such carboxylic/carboxyl-
ate functionalities (Fig. 2, stages 1 and 2), and having potential as
CAIs on the base of their previously evaluated chelating ability to-
wards metal cofactors of other metalloenzymes.21–25 Then, after a
preliminary screening against CA I/CA II isozymes (data not pre-
sented), the most interesting compounds 1–18 were selected (
Fig. 2, stage 3, and Fig. 3) and submitted to further investigation.
Most of the studied compounds were previously reported (1–3,
5, 6, 9–18);21–25 4 was commercially available,26 whereas 7 and 8
have been newly obtained. Briefly, the (alkyl)indol-yl-pyrazole-3-
carboxylic acids 7 and 8a were obtained by treating under alkaline
conditions the respective esters 21 and 8b, which were synthesized
by cyclocondensation of b-diketoesters 20a,b with hydrazine hy-
drate (Scheme 1).27
The trifluoro-dihydroxy-propanone moiety (II, Fig. 1), discov-
ered using combinated ligand- and pharmacophore-based virtual
screening approaches, represents another interesting bioisosteric
alternative to the sulfonamido-based functionalities.15 Some poly-
amines (such as spermine and its derivatives, III),16 as well as a
range of natural product phenols/carboxylic acids (IV),17,18 were
also investigated (Fig. 1), and showed interesting properties and
novel putative mechanisms of inhibition.
These intermediates were easily prepared by oxalylation of the
corresponding ketones 19a,b in the presence of sodium methoxide
in methanol under reflux conditions. 19a and 19b were synthe-
sized following a reported procedure.21,24
In this context, carboxylate-based compounds represent inter-
esting chemotypes for CA inhibition, as the COOÀ is an effective
zinc-binding group in many zinc enzymes, but it is less investigated
Compounds 1–18 were tested for the ability to inhibit CA I, II, IX
and XII
a-isoforms and b-CAs Nce103 and Can2 catalytic activities,
obtained by a stopped-flow technique as previously described.28,29
R1
R4
R1
R4
I
CH3 OH
H3C
R2
R3
R2
R3
CF3
OH
OH
H3C
COOH
O
O
OH
O
CH3
O
O
O
II
R3
N
H
N
R1
(n) NH
NH2
N
H
(n)
O
N
(n)
R4
H2N
N
R2
H
III
OH
HO
HO
O
OH
O
OH
O
O
OCH3
HO
HO
O
HO
O
OH
OH
CH3
OH
HO
OH
O
OH
O
OH
IV
O
O
OH
O
HO
O
O
O
OH
O
O
HO
N
OH
H3C
OH
H3C
OH
Cl
N
H
R
OH
H3C
O
O
HO
CH3
OH
HO
CH3
OH
H3C
OH
N
H
N
O
N
N
COOH
O
OH
CH3
N
NH
OH
O
O
OH
F
V
Figure 1. Representative structures of nonsulfonamide-containing CAIs.

M. Sechi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5801–5806
5803
Figure 2. Schematization of the screening workflow. Stage 1: throughput screening on in-house chemical library. Stage 2: selection of aromatic/heteroaromatic backbone
with carboxylic acid/carboxylate-based functionalities. Stage 3: intensive inhibition study and identification of hit compounds.
In general, almost all tested compounds showed inhibitory
activity in nanomolar/micromolar range, as reported in Table 1.
In particular, the b-diketo acids 1, 2a, 3, and the b-diketo ester
2b shared a similar CA inhibitory profile across all isoforms, with
of magnitude. It is interesting to note that coordination of metal
ions by b-diketo acid 1 or 2a to form a bimetallic complex led to
an increase in potency against CA II and to a loss in activity versus
b-Can2 enzyme.
quite selectivity of 1 and 2a for the b-form Can2 (KI = 0.89
The fumaric acid derivative 4 displayed an interesting inhibition
l
M).
Among the tested compounds, the most potent CA I inhibitors
(i.e. the heteroaryl-pyrazole carboxylic acid 7 and the hydroxy-
oxoindolin-ylidene carboxylate 10b) could interact with the Zn(II)
ion involving their carboxylate functionalities, similarly to sulfon-
amides and related inhibitors, as revealed by X-ray in co-crystals of
hCA II with other carboxylates.18 However, such molecules bear
other atoms/groups that may participate to the inhibition mecha-
nism. In particular, 7 presents two nitrogen atoms in the pyrazole
ring, which can modulate the interaction with the zinc ions and/or
with the amino acid pattern within the enzyme active site. In this
direction, also compound 10b, with additional hydroxy and oxo
functionalities, can share a favorable binding motif for selective
anti-CA I activity. Introduction of additional groups in a particular
orientation with respect to the carboxylate function appears a suit-
able strategy to enhance the carboxylates binding ability.
The difference in activities showed by 7 and by the other mem-
bers of the pyrazole carboxylic acid class (8a and 8b) can be ex-
plained by considering that at the end of their indolic backbone
is placed an aromatic bulky group, that can give unfavorable inter-
actions and/or steric hindrance between the aryl/heterocyclic
backbone and the amino acid residues at the entrance of the cavity
of the active site. Since this part of the protein CA I is the most var-
iable one among the different CA isoforms, specific structural
determinants and favorable combination of physicochemical fea-
tures, in term of electronic and steric properties, are required.
However, more studies are needed to carefully investigate the
mechanism of action. The possibility that such inhibitors would
behave differently, for example by establishing different putative
binding modes, should be also considered.
potency against CA II in nanomolar concentration (KI = 0.045 lM),
as already evidenced for some related compounds.19 The triketo-
based compound 6a exhibited a certain selectivity for hCAs IX
and XII (KIs = 0.72 and 0.77 lM, respectively), in line with those
of other related derivatives.20 Importantly, the 5-(1-ethyl-1H-in-
dol-3-yl)-1H-pyrazole-3-carboxylic acid (7) proved to be the most
potent and selective compound tested toward CA
(KIs = 0.042 M), with a very high CA I versus CA II selectivity
(KI = 1829 M for CA II, SI, selectivity index = >40,000), without it
I isoform
l
l
significantly affects the catalytic activities of the other isozymes.
The N-benzyl ester and acid homologues 8a and 8b did not show
significant inhibition profiles versus CA I: when a substitution of
the ethyl with a benzyl occurs, the difference in size and shape
can play, together with the hydrophobic properties, an important
role in the CA inhibitory activity for this set of compounds. Again,
interesting CA I nanomolar inhibition and selectivity (SI for CA I vs
CA II = ꢀ85 and ꢀ27, for 10b and 10a, respectively) have been
shown by compounds bearing a hydroxy-oxoindolin-ylidene car-
boxylate pharmacophore. The N-phenyl ester 10b resulted about
20-fold more active than the unsubstituted one 10a (CA
I
KIs = 0.049 vs 1.05 M, for 10b and 10a, respectively). The addition
l
of a phenyl group on the oxoindole ring significantly enhances the
potency, while the replacement of the ester with a carboxylic acid
functionality dramatically leads to a decrease in CA I inhibitory
activity (CA II KI = 38.0
pyrrole-2,5-dione derivative 11 demonstrated a selective inhibi-
tion for CA II in low micromolar concentration (CA II KI = 0.63 M),
lM, for 9). Moreover, the 3-hydroxy-1H-
l
whereas the inhibition potency against the other enzymes ranged
from 6.65 to 8.75 micromolar concentration. Finally, a selective
nanomolar inhibition for CA II isozyme has been detected for the
diketo acid-metal complexes 15 and 17 with Mn(II) (CA II
KI = 0.042 lM) and Zn(II) (CA II KI = 0.043 lM), respectively,
whereas the complexes 16 and 18 as well as the mononuclear
Since the valuation of pharmacokinetic, pharmacodynamic and
toxicity is also essential to address to a successful drug discovery
process and to predict the therapeutic potential of a drug candi-
date,30–32 calculation of selective physicochemical properties
important for absorption, distribution, metabolism, excretion,
and toxicity (ADME-Tox) for the most interesting CA I inhibitors
7 and 10b was performed. In this context, Lipinski’s rule-of-five
Mg(II)-complex 14 resulted less potent of about one/two orders

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M. Sechi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5801–5806
HO
O
OH
COOH
COOH
O
COOR
OH
N
O
O
OH
3
OH
a: R = H
HO
2
N
1
b: R = Me
F
OH
O
4
O
CH3
O
O
CH3O
OH
O
OH
COOH
O
a: R = H
H3COOC
N
CH3
6
b: R = Me
OH
OR
5
O
COOR
N
N
R
COOH
HOOC
N
HN
OH
HN
a: R = H
10
O
b: R = Ph
N
H
a: R = H
b: R = Me
N
7
8
9
N
H3C
O
H
N
OH
OH
O
HO
HO
HN
O
HO
HO
HN
O
N
H
OH
13
N
H
12
N
11
H3C
O
O
O
O
H3COOC
O
O
O
O
Mn
Ni
Mn
Ni
O
Mg
O
O
O
O
O
O
15
16
O
O
14
O
O
O
COOCH3
F
O
O
O
Z
n
O
O
O
N
O
O
O
O
N
Zn
Zn
Zn
O
O
O
17
O
O
18
F
O
Figure 3. Compounds selected in this study.
12% NaOH, reflux
7
21
from
H
O
O
O
N
N
.
Dimethyloxalate
NH2NH2 H2O
COOCH3
COOCH3
CH3
EtOH, reflux
MeONa / MeOH,
reflux
21
: R = Et
N
R
N
R
N
R
8b: R = Bn
19a,b
20a,b
a: R = Et
b: R = Bn
12% NaOH, reflux
8a
from 8b
Scheme 1. Synthetic route for the preparation of compounds 7 and 8.
(i.e. compounds are considered likely to be well absorbed when
they possess cLogP <5, molecular weight <500, number H-bond
acceptors <10, and number of H-bond donors <5)33 is a simplified
method to predict the solubility and the membrane permeability of
a small molecule. Further studies suggest that compounds with
polar surface area (PSA) <60 Å will likely have good absorption
(>90%), whereas other ones with PSA >140 Å are predicted to be
poorly absorbed (<10%).34–36 As shown in Table 2, the calculated
atom-based values (i.e. molecular weight, H-bond acceptor and do-
nor counts, the cLogP, miLogP and TPSA)37 for both compounds fall

M. Sechi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5801–5806
5805
Table 1
we observed a selective CA II over CA I inhibition by some tested
metal complexes, as well as a certain inhibition activity of the fun-
gal b-class enzymes from Candida albicans and Cryptococcus neofor-
mans by few diketo acid derivatives. Interestingly, computational
simulations suggested that the most interesting compounds 7
and 10b can meet desirable ADME criteria and favorable pharma-
cokinetic properties for further development. In addition, due to its
selectivity index between CA I/CA II (SI = >40,000), compound 7
shows a potential therapeutic window.
We highlighted that the discovered heteroaryl-pyrazole carbox-
ylic acids and the hydroxy-oxoindolin-ylidene carboxylates can
represent novel promising lead compounds for the development
of potent and selective CA I inhibitors, and as such further preclin-
ical studies, also devoted to elucidate their inhibition mechanism,
are warranted.
Human CA I, II, IX and XII, and b-CAs (Nce103 and Can2) from pathogenic fungi and
bacteria inhibition data for compounds 1–18 by a stopped flow CO2 hydrase assay at
pH 7.5 and 20 °C. The sulfonamide inhibitors AAZ and DCP were also included as
standards
Compound
KI (
l
M)a
hCA I
hCA II
hCA IX
hCA XII
Nce 103
Can2
1
3.35
5.07
5.53
2.78
4.41
7.50
4.02
1.20
0.042
6.20
1.65
38.0
1.05
0.049
7.53
3.60
1.85
4.15
4.59
4.34
2.73
3.40
0.25
1.20
4.30
2.65
3.27
1.93
0.045
5.20
4.35
4.55
1820
3.62
1.19
4.36
28.5
4.14
0.63
1.45
3.02
2.60
0.042
0.70
0.043
0.85
0.012
0.038
6.79
6.93
6.86
8.28
6.70
7.75
7.94
8.51
7.64
7.15
13.9
0.77
7.05
7.78
3.24
12.8
0.48
6.96
5.03
8.66
14.3
9.24
6.38
7.26
6.19
8.29
8.04
0.006
0.050
8.76
9.27
9.39
8.81
8.28
12.3
8.45
8.36
8.94
8.47
15.6
7.62
7.75
8.44
8.40
>20
0.89
0.94
9.73
9.63
10.2
16.3
10.4
9.85
9.69
10.4
15.8
9.33
10.4
10.1
8.75
>20
>20
>20
>20
>20
>20
>20
0.010
0.087
2a
2b
3
4
5
16.7
6a
6b
7
8a
8b
9
10a
10b
11
12
13
14
15
16
17
18
AAZ
DCP
0.72
7.57
7.79
6.12
9.24
7.68
8.02
Acknowledgements
8.05
6.65
16.23
15.10
9.23
9.14
8.73
8.70
10.24
0.025
0.050
M.S. is grateful to Fondazione Banco di Sardegna for its partial
financial support. The work in CS’s group was supported by Metox-
ia (European 7th framework programme).
>20
18.5
12.2
13.6
11.7
15.9
0.132
1.07
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within the desirable range for good absorption and membrane per-
meability, therefore suggesting a favorable bioavailability. The se-
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properties, thus making them suitable for further optimization
and experimental evaluations.
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with various molecular diversities, have been investigated as
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inhibitors of the a-class of CA I, II, IX and XII isoforms, and of the
fungal b-class enzymes from Candida albicans and Cryptococcus
neoformans. Since CAIs with peculiar selectivity for each isozyme
are of paramount importance, we made a major effort in identify-
ing novel CAIs differing from the well-known sulfonamide, sulfa-
mate and sulfamides, endowed with selectivity against relatively
unexplored targets, such as CA I. This isoform is involved in retinal
and cerebral disease, and we pursued the hypothesis that proteins
downstream of CA I could represent therapeutic targets for inhib-
iting CA I–induced vascular permeability. The present study re-
ports on the first prototype of CAIs targeting the CA-I isoform,
carrying an original pharmacophore able to selectively interfere
with such isozyme in nanomolar concentration range. Moreover,
11. Gao, B. B.; Clermont, A.; Rook, S.; Fonda, S. J.; Srinivasan, V. J.; Wojtkowski, M.;
Fujimoto, J. G.; Avery, R. L.; Arrigg, P. G.; Bursell, S. E.; Aiello, L. P.; Feener, E. P.
Nature Med. 2007, 13, 181.
12. (a) Supuran, C. T. Curr. Pharm. Des. 2008, 14, 641; (b) De Simone, G.; Di Fiore, A.;
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13. 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.
14. Carta, F.; Temperini, C.; Innocenti, A.; Scozzafava, A.; Kaila, K.; Supuran, C. T. J.
Med. Chem. 2010, 53, 5511.
15. Pala, N.; Dallocchio, R.; Dessì, A.; Brancale, A.; Carta, F.; Ihm, S.; Maresca, A.;
Sechi, M.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2011, 21, 2515.
16. Innocenti, A.; Vullo, D.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett.
2008, 18, 1583.
17. Davis, R. A.; Hofmann, A.; Osman, A.; Hall, R. A.; Mühlschlegel, F. A.; Vullo, D.;
Innocenti, A.; Supuran, C. T.; Poulsen, S. A. J. Med. Chem. 2011, 54, 1682.
18. De Simone, G.; Supuran, C. T. J. Inorg. Biochem. 2012, 111, 117.
19. (a) Innocenti, A.; Vullo, D.; Scozzafava, A.; Casey, J. R.; Supuran, C. T. Bioorg.
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20. (a) Innocenti, A.; Hall, R. A.; Schlicker, C.; Mühlschlegel, F. A.; Supuran, C. T.
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Table 2
Physicochemical properties of selected compounds 7 and 10b
Compound
MW
HBA
HBD
Rbond
cLogPa
miLogPb
TPSAc
7
10b
255.3
295.3
4
3
2
1
3
3
3.65
2.12
2.397
2.167
70.916
68.538
Abbreviations: MW, molecular weight; HBA, number of hydrogen bond acceptors;
HBD, number of hydrogen bond donors; Rbond, number of rotatable bonds; cLogP,
log octanol–water partition coefficient; miLogP, logP prediction based on group
contributions; TPSA, topological polar surface area.
a
Parameters calculated by ChemDraw Ultra 2005.
Parameters calculated by miLogP 2.2 method implemented in Molinspiration
b
Cheminformatics 2012 software.
23. Sechi, M.; Sannia, L.; Carta, F.; Palomba, M.; Dallocchio, R.; Dessì, A.; Derudas,
M.; Zawahir, Z.; Neamati, N. Antiviral Chem. Chemother. 2005, 16, 41.
c
Parameters calculated by Molinspiration Cheminformatics 2012.

5806
M. Sechi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5801–5806
24. Sechi, M.; Sannia, L.; Orecchioni, M.; Carta, F.; Paglietti, G.; Neamati, N. J.
Heterocycl. Chem. 2003, 40, 1097.
and 1H, NH), 7.15–7.40 (m, 7H, ArH), 7.03 (s, 1H, ArH), 5.41 (s, 2H, CH2), 3.51
(br s, 1H, OH); GC/MS: m/z 317 (M+).
25. Sechi, M.; Bacchi, A.; Carcelli, M.; Compari, C.; Duce, E.; Fisicaro, E.; Rogolino,
D.; Gates, P.; Derudas, M.; Al-Mawsawi, L. Q.; Neamati, N. J. Med. Chem. 2006,
49, 4248.
26. Provided from Sigma–Aldrich. CAS Number: 199926–38-0; PubChem
Substance ID 24893359.
28. Khalifah, R. G. J. Biol. Chem. 1971, 246, 2561.
29. Enzymatic evaluation. An Applied Photophysics stopped-flow instrument has
been used for assaying the CA-catalyzed CO2 hydration activity.29 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) as buffer,
and 20 mM Na2SO4 (for maintaining a constant 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 achieved thereafter with distilled-deionized water. Inhibitor and
enzyme solutions were preincubated together for 15 min (data not showed)
and for 6 h at room temperature prior to the assay, to allow formation of the E-I
complex. The inhibition constants were obtained by nonlinear least- squares
methods using PRISM 3, whereas the kinetic parameters for the uninhibited
enzymes from Lineweaver-Burk plots, as reported previously,13 represent the
means from at least three different determinations.
27. General procedure for the preparation of the esters 21 and 8b. For general
experimental information see in Ref. 24 A solution of the methyl 2,4-dioxo-4-
phenylbutanoates (20a, b)21 (4.85 mmol) and hydrazine hydrochloride
(3 mole equiv) in ethanol (20 mL) was refluxed for 5 (for 21) or 7 (for 8b)
hours. After evaporation in vacuo a pale yellow solid was obtained that was
purified by silica flash chromathography (hexane/ethyl acetate = 8:2). The
product was recrystallized from water–ethanol to give 21 and 8b as yellow
crystals. Methyl 5-(1-ethyl-1H-indol-3-yl)-1H-pyrazole-3-carboxylate (21). Yield:
57%, mp 174–175 °C; IR (Nujol): 1730 (C@O, ester) cmÀ1 1H NMR (CDCl3): d
;
7.9–8.0 (m, 1H, ArH), 7.48 (s, 1H, NH), 7.20–7.40 (m, 4H, ArH), 7.08 (s, 1H, ArH),
4.22 (q, 2H, CH2), 3.95 (s, 3H, CH3), 1.55 (t, 3H, CH3); GC/MS:m/z 269 (M+).
Methyl 5-(1-benzyl-1H-indol-3-yl)-1H-pyrazole-3-carboxylate (8b). Yield: 65%,
mp 221–222 °C; IR (Nujol): 1720 (C@O, ester) cmÀ1 1H NMR (CDCl3): d 7.90–
;
8.00 (m, 1H, ArH), 7.74 (s, 1H, NH), 7.54 (s, 2H, ArH), 7.15–7.40 (m, 7H, ArH),
7.07 (s, 1H, ArH), 5.38 (s, 2H, CH2), 3.93 (s, 3H, CH3); GC/MS:m/z 331 (M+).
General procedure for the preparation of the acids 7 and 8a. A solution of the
appropriate ester (21 and 8b) (1.3 mmol) and 12% NaOH pellets (4 equiv) in
ethanol (40 mL) was stirred under reflux for 2 h. Then, water was added and
the solution was acidified with 6 N HCl. A white precipitate was filtered,
recrystallized from water/ethanol and collected. 5-(1-ethyl-1H-indol-3-yl)-1H-
pyrazole-3-carboxylic acid (7). Yield: 79%, mp 280–281 °C; IR (Nujol): 1690
30. Egan, W. J.; Merz, K. M., Jr; Baldwin, J. J. Med. Chem. 2000, 43, 3867.
31. Egan, W. J.; Lauri, G. Adv. Drug Deliv. Rev. 2002, 54, 273.
32. Clark, D. E.; Grootenhuis, P. D. Curr. Top. Med. Chem. 2003, 3, 1193.
33. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Deliv. Rev.
1997, 23, 3.
34. Palm, K.; Luthman, K.; Ungell, A. L.; Strandlund, G.; Artursson, P. J. Pharm. Sci.
1996, 85, 32.
(C@O, acid) cmÀ1 1H NMR (CDCl3): d 8.00–8.05 (m, 1H, ArH), 7.60 (s, 1H, NH),
;
7.15–7.45 (m, 4H, ArH), 7.08 (s, 1H, ArH), 4.22 (q, 2H, CH2), 2.71 (s, 1H, OH),
1.54 (t, 3H, CH3); GC/MS: m/z 255 (M+). Acido 5 5-(1-benzyl-1H-indol-3-yl)-1H-
pyrazole-3-carboxylic acid (8a). Yield: 67%, mp 234–235 °C; IR (Nujol): 1690
35. Palm, K.; Stenberg, P.; Luthman, K.; Artursson, P. Pharm. Res. 1997, 14, 568.
36. Palm, K.; Luthman, K.; Ungell, A. L.; Strandlund, G.; Beigi, F.; Lundahl, P.;
Artursson, P. J. Med. Chem. 1998, 41, 5382.
(C@O, acid) cmÀ1
;
1H NMR (CDCl3): d 7.90–8.05 (m, 1H, ArH), 7.77 (m, 2H, ArH
37. Ertl, P.; Rohde, B.; Selzer, P. J. Med. Chem. 2000, 43, 3714.