α-Diketocarboxylic Acids and Their Esters Act as Carbonic Anhydrase IX and XII Selective Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.9b00023

Among human carbonic anhydrase (CA) inhibitors, the α,I-diketocarboxylic acids and esters are still poorly investigated. Here, we report the first compounds of this class (1-6) acting as potent inhibitors at low nanomolar level against the cancer-related

Full text of DOI:10.1021/acsmedchemlett.9b00023

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Letter
#,#-Diketocarboxylic acids and their esters act as
carbonic anhydrase IX and XII selective inhibitors
Alessio Nocentini, Alessia Lucidi, Francesca Perut, Annamaria Massa, Daniela Tomaselli,
Paola Gratteri, Nicola Baldini, Dante Rotili, Antonello Mai, and Claudiu T Supuran
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00023 • Publication Date (Web): 19 Feb 2019
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α,γ-Diketocarboxylic acids and their esters act as carbonic anhydrase
IX and XII selective inhibitors
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†,≠
§
^
^
§
Alessio Nocentini, Alessia Lucidi, Francesca Perut, Annamaria Massa, Daniela Tomaselli, Paola
≠
^,+
,§
,§
,†
Gratteri, Nicola Baldini, Dante Rotili,* Antonello Mai,* and Claudiu T. Supuran*
†Department of NEUROFARBA, Section of Pharmaceutical and Nutraceutical Sciences, University of Florence, Polo
Scientifico, Via U. Schiff 6, 50019, Sesto Fiorentino, Firenze, Italy.
≠Department of NEUROFARBA, Section of Pharmaceutical and Nutraceutical Sciences, Laboratory of Molecular Modeling
Cheminformatics & QSAR, University of Florence, Polo Scientifico, Via U. Schiff 6, 50019, Sesto Fiorentino, Firenze, Italy.
§Dipartimento di Chimica e Tecnologie del Farmaco "Sapienza" Università di Roma, 00185 Rome, Italy.
^Laboratorio di Fisiopatologia ortopedica e medicina rigenerativa, IRCCS Istituto Ortopedico Rizzoli, via di Barbiano 1/10,
4
0136, Bologna, Italy
+Department of Biomedical and Neuromotor Sciences, University of Bologna, 40123 Bologna, Italy.
KEYWORDS: Metalloenzyme, carbonic anhydrase, carboxylic acid, selective inhibition, antitumor.
ABSTRACT: Among human carbonic anhydrase (CA) inhibitors, the α,γ-diketocarboxylic acids and esters are still poorly
investigated. Here we report the first compounds of this class (1-6) acting as potent inhibitors at low nanomolar level against the
cancer-related human CA IX and XII, and 2-3 magnitude orders selective towards the cytosolic isoforms hCA I and II. At
enzymatic level, the α,γ-diketoacids 1-3 were more effective inhibitors compared to the corresponding ethyl esters 4-6. The phenyl-
and α–naphthyl-containing compounds (1, 3, 4, and 6) behaved as dual hCA IX/XII inhibitors, while the β–naphthyl analogues (2
and 5) exhibited hCA IX-selective inhibition. In MG63 and HOS osteosarcoma (OS) cell lines, the ethyl esters 5 and 6 displayed
dose-dependent reduction of viability and proliferation after 72 h treatment, with 6 being more potent than 5 likely for its dual hCA
IX/XII inhibition.
for the design of inhibitors with diverse profiles for the many
Carbonic anhydrase (CA) inhibitors (CAIs) possess a range of
CA isoforms present in vertebrates (15 α-class isoforms) or
pharmacologic applications for the treatment and preventions
other organisms (with 7 distinct CA genetic families present in
of diseases connected with enhanced secretion of electrolytes,
5,6
organisms all over the tree of life). Indeed, several drug
1
-4
pH regulation, metabolic modulation, etc. In fact, many
CAIs are used as diuretics, antiglaucoma agents, antiepileptics,
design studies of carboxylic acids as CAIs were reported in the
last years based on aromatic/heterocyclic scaffolds, with
1
-4
antiobesity and antitumor drugs. In addition to primary
sulfonamides and their isosteres (sulfamates and sulfamides),
several other chemo types with CA inhibitory properties and
diverse mechanisms of CA inhibition were reported in the last
several such derivatives also being characterized by X-ray
11-14
crystallographic techniques.
In some cases, highly
12-14
isoform-selective inhibitors were detected,
as well as
compounds with enhanced inhibitory action against
5
,6
years. Indeed, sulfonamides and their congeners, as well as
dithio-/monothiocarbamates, xanthates, hydroxamates and
some carboxylates coordinate the zinc ion from the enzyme
14
pathogenic versus human enzymes.
O
OH
COOR
5
,6
active site potently inhibiting the CA catalytic activity.
Compounds that inhibit CAs by anchoring to the zinc-
coordinated water molecule are the phenols, polyamines,
sulfocoumarins, some carboxylic acid esters and the
O
R1
N
H
7
,8
thioxocoumarins, whereas the coumarins show a distinct CA
inhibition mechanism, acting as prodrug inhibitors, and after
hydrolysis to the corresponding 2-hydroxycinnamic acids,
1-6
Figure 1. New α,γ-diketocarboxylic acids and ethyl esters 1-6
as CAIs (R = H, ethyl; R = phenyl, α- or β-naphthyl).
9,10
occlude the entrance to the active site. The fourth inhibition
1
mechanism, out of the active site binding, was also reported
1
1
for a carboxylic acid derivative. Thus, from the above data it
is possible to observe that the carboxylates and their
derivatives may interact with these enzymes by at least four
different inhibition mechanisms, making them of great interest
With the only exception of the carboxylic acid derivatives
reported by Sechi et al., which showed inhibitory activity in
the low micromolar/submicromolar range against a panel of
1
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human and fungal CAs,14 the α,γ-diketo acid scaffold has
Table 1. Inhibitory Potencies of Derivatives 1-6 versus
hCA I, II, IX and XII
1
2
3
4
5
6
7
8
9
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5
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5
5
6
poorly been investigated for the interaction with CAs so far.
Therefore, we designed a few 6-aromatically substituted-2,4-
dioxo-5-hexenoic acids and their ethyl esters 1-6 (Figure 1) as
potential CAIs, studied their inhibition mechanism by using
kinetic and computational techniques, and evaluated their
effects in two human osteosarcoma (OS) cell lines, MG63 and
HOS, highly expressing the cancer-related CA isoforms IX
and XII.
O
OH
O
COOR
R1
N
H
1
-6
a
I
K (nM)
cpd
R
R
1
The general synthetic pathway followed for the preparation of
derivatives 1-6 is depicted in the Scheme 1. The (E)-4-(4-
nitrophenyl)but-3-en-2-one 7, prepared as reported in
hCA I
>10000
8981
hCA II hCA IXhCA XII
0
1
2
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5
6
7
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9
0
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9
0
1
H
H
Ph
β-Naph
5034
2084
2580
>10000
9443
8654
12
12.5
7.5
1
5
literature, was dissolved in ethanol and treated with a
solution of stannous chloride dihydrate in concentrated
hydrochloric acid at 0 °C to obtain the reduced product (E)-4-
2
11.3 767.6
(4-aminophenyl)but-3-en-2-one 8, that was then subjected to
acylation reaction with the appropriate commercial aroyl
chloride in the presence of triethylamine in dry DCM at 0 °C
in order to afford the intermediates 9-11. The compounds 4-6,
obtained by treating 9-11 with diethyl oxalate in the presence
of sodium ethoxide in dry THF at room temperature under
nitrogen atmosphere, were finally hydrolyzed to the
corresponding α,γ-diketocarboxylic acids 1-3 with 0.7 M
lithium hydroxide in a mixture of THF and methanol.
3
4
H
α-Naph >10000
12.8
90.2
8.7
ethyl
Ph
7331
6178
34.6
5
ethyl β-Naph
12.6 249.1
6
ethyl α-Naph >10000
37.9
25
8.9
5.7
Scheme 1. Synthetic Route to Compounds 1-6a
AAZ
250
O
O
aMean from three different assays, by a stopped flow technique
(errors were in the range of ± 5-10% of the reported values).
O
CH3
b
O
CH3
a
CH3
O2N
R =
R
N
H2N
H
7
8
9-11
Carboxylic acids and mostly esters are known to be relatively less
effective CA inhibitors than sulfonamides, with most derivatives
of these classes of compounds acting in the micromolar range.11-14
The present results against the cytosolic isoforms confirms these
evidences. No significant difference stood out between acids 1-3
and esters 4-6 in the inhibition of hCA I. Conversely, hCA II was
more than two-fold better inhibited by compounds 1-3 than their
ethyl esters. Switching the phenyl group to α- or β-naphthyl
moiety does not affect the inhibitory properties against hCA I and
II within the two subsets of molecules. (ii) The tumor-associated
hCA IX and XII are potently inhibited by both subsets of
derivatives. Compounds 1-6 inhibited hCA IX in the low
c
1, 4, 9
, 5, 10
2
O
OH
O
OH
OH
OEt
O
d
O
3, 6, 11
O
O
R
N
1-3
R
N
4-6
H
H
aReagents and conditions: (a) 2.8 M SnCl
°C, 24 h; (b) appropriate aroyl chloride, Et
C, 2-24 h; (c) diethyl oxalate, EtONa, dry THF, N
atmosphere, rt, 3-5 h; (d) 0.7 M LiOH, THF/MeOH, rt, 6-8 h.
in 37% HCl, EtOH,
N, dry DCM, 0
2
0
°
3
2
Full experimental details regarding the synthesis and
physicochemical characterization of final (1-6) and
intermediate (8-11) compounds are outlined in Supporting
Information.
nanomolar range, exhibiting K
0.2 nM. The nature of the outer aromatic ring does not affect the
hCA IX inhibitory properties of acid derivatives 1-3, being their
values not significantly diverse (11.3-12.8 nM). Solely the β-
naphthyl ethyl ester 5 exhibits analog K (12.6 nM) to carboxylic
I
values spanning between 11.3 and
9
K
I
The CA inhibition profile of compounds 1-6 was evaluated by
1
6
applying a stopped flow CO
2
hydrase assay against four
I
physiologically significant CA isoforms (I, II, IX and XII), in
comparison with acetazolamide (AAZ) as reference drug.
acids 1-3, whereas the switching of the aromatic moiety to the
phenyl (4) and α-naphthyl (6) ones worsens the inhibitory activity
by three- (37.9 nM) or seven-fold (90.2 nM), respectively. (iii)
The following SAR can be gathered from the inhibition data
shown in Table 1: (i) The cytosolic hCA I and II are the least
inhibited isoforms by α,γ-diketocarboxylic acids 1-3 and their
ethyl esters 4-6. Most derivatives act as low micromolar
With the exception of the β-naphthyl derivatives 2 and 5 (K
I
values = 767.6 and 249.1 nM, respectively), the hCA XII is
potently inhibited by the other screened derivatives that show
comparable inhibitory properties in the low nanomolar range (7.5-
34.6 nM) independently from the nature of the zinc binding group
(ZBG). Anyway, it should be noted that the phenyl ester
derivative 4 is four-fold less effective than its carboxylic acid
cognate 1. (iv) A clear selectivity of action against hCA IX and
I
inhibitors, with inhibition constant (K ) values spanning
between 2.08 and 9.4 µM. Compounds 1, 3 and 6 do not
inhibit hCA I below 10 µM and 4 exhibit the same behavior
against hCA II.
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XII over the cytosolic hCA I and II stands out from the data in coordination bond stabilization which enhances the hCA IX
Table 1 for most of the reported derivatives, with β-naphthyl inhibition efficacy of the compounds more than two-orders of
derivatives 2 and 5 that can be considered hCA IX-selective magnitude if compared to that towards hCA II.
inhibitors.
Chemotypes endowed with a selectivity ratio spanning between 2
The in silico assessment of the binding mode of compounds 2 and and 3 orders of magnitude for hCA IX and XII over both I and II
1
2
3
4
5
6
7
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9
1
1
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1
1
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5
2
into the hCA II and IX highlights that 2-hydroxy-4-oxohexa- have a great potential as starting points for the design of novel
,5-dienoic acids and their ethyl esters are able to occupy the CAIs as anti-tumor agents devoid of undesired side effect related
catalytic region of the binding pockets by coordinating the zinc to promiscuous activity. Since in a previous paper we have
ion and interacting with residues nearby. Docking studies demonstrated that there is a strong rationale for the use of CA IX
followed by a refinement in a VSGB solvent model show that the inhibitors in human OS models,17 we tested the β-naphthyl
coordination occurs between the metal ion and the deprotonated derivatives 2 and 5 together with the α-naphthyl ethyl ester 6 (0-
carboxylic group of 2 or the oxo-group of the ethyl ester of 5 100 µM, 72 h) in two different OS cell lines (MG63 and HOS),
0
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(Figure 2). The molecular architecture of the two active sites that highly express CA IX and/or XII (see Figure S1 in
thoroughly affects the binding modes. The hCA II/IX Supporting Information). Likely for the reduced cell permeability
Phe131/Val130 mutation modulates the H-bonds network that the due to its acidic nature, 2 did not show any inhibitory effect on
2-hydroxy-4-oxohexa-2,5-dien portions can form within the OS cell growth, while the two ethyl esters 5 and 6 affected MG63
pockets. The presence of several H-bonds effectively reinforces and HOS cell viability in a dose-dependent way (Figure 3). In
the carboxylate coordination to the metal ion of 2 and 5 in hCA particular, 5 decreased by 50% the viability of both the tested cell
IX (Figure 2A-B). The carboxy or carbethoxy moieties of 2 and 5, lines at 50 µM, while 6 at 25 µM arrested the viability of MG63
respectively, accept two H-bonds from the backbone NH of and HOS cells at 16.4 and 19.7%, respectively, thus being the
Thr200 and Thr201. The side chain OH group of Thr201 is most potent of the two compounds.
involved in an interesting pattern of H-bonds with the 2-hydroxy-
4
-oxohexa-2,5-diene portions of the ligands under investigation,
MG63 cells
HOS cells
acting both as donor and acceptor group. In particular, the OH
group acts as both H-bond donor towards the 2-hydroxy and to
the 4-oxo moieties of 2, while it participates to a three center H-
bond involving the analogue deprotonated groups in 5. The ethyl
moiety of 5 accommodates into the pocket lined by Val121,
Val142 and Trp210. The naphthamidophenyl fragment of both
molecules orients towards the hydrophobic half of hCA IX active
site, with van der Waals interactions taking place with Val130,
Asp131 and Arg129.
150
100
6
5
2
150
100
6
5
2
50
50
0
0
0
12
.5 25
50100
0
12
.5 25
50100
0
12
.5 25
50100
0
12
.5 25 5010
0
0
12
.5 25 5010
0
0
12
.5 25 5010
0
concentration, M
concentration, M
Figure 3. Cell viability assay. The treatment of OS cells with
the two ethyl esters 5 and 6 for 72 h reduced cell viability in a
dose-dependent way, as revealed by the indirect acid
phosphatase viability assay. Treatment with 2 did not produce
any cytotoxicity on OS cells.
In order to confirm this viability indirect test, the effectiveness
of 5 and 6 was then evaluated by erythrosine B dye vital staining
in both MG63 and HOS OS cell lines. Both compounds
displayed strong antiproliferative effects in the low micromolar
range after 72 h treatment (Figure 4), with 6 [IC50 values = 14.7
µM (MG63) and 15.6 µM (HOS)] being more potent than 5
[IC50 values = 45.8 µM (MG63) and 19.2 µM (HOS)]. When
tested in the same cell lines to determine their putative effect on
apoptosis induction through detection of caspase 3 and 7
Figure 2. Docking of 2 (A) and 5 (B) into hCA IX. Docking of 2
into hCA II (C).
The above mentioned hCA II/IX Phe131/Val130 mutation makes
the hCA II binding site less roomy if compared to that one of
hCA IX preventing, de facto, the positioning of the ligand as activity, neither 6 (at 25 µM) or 5 (at 50 µM) induced any pro-
described for hCA IX. Nonetheless, the carboxylates maintain the apoptotic activity (see Figure S2 in Supporting Information).
Thus, the decreased number of cells after compound 5 and 6
treatment could be a consequence of a cell cycle arrest.
zinc-coordination and a H-bond with the backbone NH Thr200.
The 2-hydroxy-4-oxohexa-2,5-diene portions lack the proper H-
bond distances with Thr201 because of the rotation undergone by
the ligands to accommodate the naphthamidophenyl core towards
His64, Ans62 and Asn67 (Figure 1C). These evidences support
the observed CA IX/II selective inhibition profiles. In fact, the
binding mode of 2 within the hCA II active site prevents the
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compound 5
compound 6
Experimental procedures for the synthesis and physicochemical
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characterization of all new compounds; material and methods for
CA inhibition assays, in silico studies, cell based assays (PDF)
The Supporting Information is available free of charge on the
ACS Publications website.
HOS
HOS
MG63
MG63
60
40
20
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AUTHOR INFORMATION
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15
30
45
60
0
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60
Corresponding Author
*DR, Phone: +39 06 49913237. E-mail: dante.rotili@uniroma1.it.

M

M
*
AM, Phone: +39 06 49913391. E-mail:
antonello.mai@uniroma1.it. *CTS, Phone: +39 055 4573729. E-
mail: claudiu.supuran@unifi.it.
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Figure 4. Cell proliferation assay. The percentage of cell growth in
respect to untreated cells is significantly lower (p<0.05) after the
treatment with compound 5 or 6 for all the doses tested.
Author Contributions
A.N., D.R., A.Mai, F.P., N.B. and C.T.S. assisted manuscript
preparation, data interpretation, and project overview. A.L., D.T.
and D.R., compound synthesis. A.N., P.G., and C.T.S. in vitro
profiling for hCAs inhibition and molecular modelling. F.P., A.M.
and N.B., biological assays on OS cell lines. D.R., A.Mai and
C.T.S. conceived the study and followed the whole project. All
authors have approved the final version of the manuscript.
The evidence that the dual CA IX/XII inhibitor α-naphthyl ester 6
resulted more effective than the CA IX-selective β-naphthyl
regioisomer 5 in inhibiting the proliferation of both the OS cell
lines suggests the importance of the simultaneous inhibition of
both the hCA IX and XII isoforms, at least in this context.
ACKNOWLEDGMENT
This work was supported by funds from PRIN 2015
In summary, we have reported herein a new series of α,γ-
diketocarboxylic acids and ethyl esters (1-6) potent at nanomolar
level and selective towards the human CA isoforms IX and XII,
highly expressed in tumor cells, and scarcely potent against the
cytosolic CAs I and II. This unique inhibitory profile displayed by
1-6 is different from previously reported α,γ-diketoacids and
esters (see compounds 1-3, 6a,b in Ref. 14) which showed
unselective, low micromolar inhibition against hCA I, II. IX, and
XII, with only one compound (6a in Ref. 14) potent against hCA
IX and XII at submicromolar level.14
In general, among the new α,γ-diketoacids the carboxylic acids 1-
displayed similar or higher potency than the corresponding
ethyl esters 4-6 against the hCA IX and XII, and the phenyl- and
α-naphthyl-substituted compounds of the two series (1, 3, 4, 6)
behaved as dual hCA IX/XII inhibitors, while the β-naphthyl
analogues (2 and 5) exhibited hCA IX-selective inhibition.
Molecular modelling studies performed on 2 and 5 docked into
hCA II and hCA IX highlighted their mechanism of action, due to
coordination between the enzyme zinc ion and the carboxylate
group of 2 or the carbonyl group of 5. In comparison with the
binding mode within the hCA II active site, the better
accommodation (more interactions and H-bonds) of the ligands
into the hCA IX binding site accounted for their higher selectivity
towards hCA IX. When tested in MG63 and HOS OS cells to
determine their effects on cell viability, 2 was totally inactive
likely due to its acidic nature (low cell permeability), while 5 and
6 displayed dose-dependent reduction of viability after 72 h
treatment. As antiproliferative agents, the α-naphthyl ester 6 was
more potent than the β-naphthyl analogue 5, probably because 6
was a dual hCA IX/XII inhibitor, while 5 exerted selective hCA
IX inhibition.
(prot.20152TE5PK) (A.M.), AIRC IG 2016 (n. 19162) (A.M.),
and The Italian Ministry of the Health, Financial Support for
Scientific Research (‘5 per 1000 2015’ (N.B.).
ABBREVIATIONS
AAZ, acetazolamide; CA, carbonic anhydrase; CAI, carbonic
anhydrase inhibitors; OS, osteosarcoma.
REFERENCES
1. Supuran, C.T. Carbonic anhydrase inhibitors and their potential in a
range of therapeutic areas. Expert Opin. Ther. Pat. 2018, 28, 709-712.
2. Nocentini, A.; Supuran, C.T. Carbonic anhydrase inhibitors as
antitumor/antimetastatic agents: a patent review (2008-2018). Expert
Opin. Ther. Pat. 2018, 28, 729-740.
3. Supuran, C.T. Applications of carbonic anhydrases inhibitors in
renal and central nervous system diseases. Expert Opin. Ther. Pat.
2018, 28, 713-721.
3
4
. Supuran, C.T. Carbonic anhydrases: novel therapeutic applications
for inhibitors and activators. Nat. Rev. Drug Discov. 2008, 7, 168-
1
5
81.
. Supuran, C.T. Structure and function of carbonic anhydrases.
Biochem. J. 2016, 473, 2023-2032.
6. Supuran, C.T. How many carbonic anhydrase inhibition
mechanisms exist? J. Enzyme Inhib. Med. Chem. 2016, 31, 345-360.
7
. Supuran, C.T. Advances in structure-based drug discovery of
carbonic anhydrase inhibitors. Expert Opin. Drug. Discov. 2017, 12,
1-88.
6
8. Lopez, M.; Vu, H.; Wang, C.K.; Wolf, M.G.; Groenhof, G.;
Innocenti, A.; Supuran, C.T.; Poulsen, S.A. Promiscuity of carbonic
anhydrase II. Unexpected ester hydrolysis of carbohydrate-based
sulfamate inhibitors. J. Am. Chem. Soc. 2011, 133, 18452-18462.
9
. Maresca, A.; Temperini, C.; Vu, H.; Pham, N.B.; Poulsen, S.A.;
Scozzafava, A.; Quinn, R.J.; Supuran, C.T. Non-zinc mediated
inhibition of carbonic anhydrases: coumarins are a new class of
suicide inhibitors. J. Am. Chem. Soc. 2009, 131, 3057-3062.
1
0. Davis, R.A.; Vullo, D.; Maresca, A.; Supuran, C.T.; Poulsen, S.A.
Natural product coumarins that inhibit human carbonic anhydrases.
Bioorg. Med. Chem. 2013, 21, 1539-1543.
11. D'Ambrosio, K.; Carradori, S.; Monti, S.M.; Buonanno, M.; Secci,
D.; Vullo, D.; Supuran, C.T.; De Simone, G. Out of the active site
ASSOCIATED CONTENT
Supporting Information
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ACS Medicinal Chemistry Letters
binding pocket for carbonic anhydrase inhibitors. Chem. Commun.
(Camb.) 2015, 51, 302-305
2. Langella, E.; D'Ambrosio, K.; D'Ascenzio, M.; Carradori, S.;
Monti, S.M.; Supuran, C.T.; De Simone, G. combined
crystallographic and theoretical study explains the capability of
carboxylic acids to adopt multiple binding modes in the active site of
Carbonic Anhydrases. Chemistry. 2016, 22, 97-100.
carboxylic acids and their derivatives: new isoform-I selective
nanomolar inhibitors. Bioorg. Med. Chem. Lett. 2012, 22, 5801-5806.
15. Shokat, K.; Uno, T.; Schultz, P. G. mechanistic studies of an
antibody-catalyzed elimination-reaction. J. Am. Chem. Soc. 1994,
116, 2261-2270.
16. Khalifah, R.G. The carbon dioxide hydration activity of carbonic
anhydrase. I. Stop-flow kinetic studies on the native human
isoenzymes B and C. J. Biol. Chem. 1971, 246, 2561-2573.
17. Perut, F.; Carta, F.; Bonuccelli, G.; Grisendi, G.; Di Pompo, G.;
Avnet, S.; Sbrana, F.V.; Hosogi, S.; Dominici, M.; Kusuzaki, K.;
Supuran, C.T.; Baldini, N. Carbonic anhydrase IX inhibition is an
effective strategy for osteosarcoma treatment. Expert Opin. Ther.
Targets. 2015, 19, 1593-1605.
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3. Cadoni, R.; Pala, N.; Lomelino, C.; Mahon, B.P.; McKenna, R.;
Dallocchio, R.; Dessì, A.; Carcelli, M.; Rogolino, D.; Sanna, V.;
Rassu, M.; Iaccarino, C.; Vullo, D.; Supuran, C.T.; Sechi, M.
Exploring Heteroaryl-pyrazole carboxylic acids as human Carbonic
Anhydrase XII inhibitors. ACS Med. Chem. Lett. 2017, 8, 941-946.
1
4. Sechi, M.; Innocenti, A.; Pala, N.; Rogolino, D.; Carcelli, M.;
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Scozzafava, A.; Supuran, C.T. Inhibition of α-class cytosolic human
carbonic anhydrases I, II, IX and XII, and β-class fungal enzymes by
TOC Graphic
O
OH
COOH
O
OH
COOEt
O
O
N
N
H
H
2
5 = -Naph
= -Naph
6
2
5
6
hCA I
hCA II
hCA IX
hCA XII 767.6
8981
2084
11.3
6178 >10000
2
5
6
antiproliferative
effect in OS cells:
IC50 (M), 72h
9443
8654
37.9
8.9
MG63 no effect 45.8
HOS no effect 19.2
14.7
15.6
K , nM
I
12.6
249.1
5
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