Benzoxaborole as a new chemotype for carbonic anhydrase inhibition

Article abstract of DOI:10.1039/c6cc06399c

In this paper we report the synthesis of a series of benzoxaborole derivatives, their inhibition properties against some carbonic anhydrases (CAs), recognized as important drug targets, and the characterization of the binding mode of these molecules to the CA active site. Our data provide the first experimental evidence that benzoxaboroles can be efficiently used as CA inhibitors.

Full text of DOI:10.1039/c6cc06399c

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Cadoni, D. Esposito, D. Vullo, A. Di Fiore, S. M. Monti, A. Caporale, M. Ruvo, M. Sechi, P. Dumy, C.
Supuran, G. De Simone and J. Winum, Chem. Commun., 2016, DOI: 10.1039/C6CC06399C.
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Benzoxaborole as a New Chemotype for Carbonic Anhydrase
Inhibition
Vincenzo Alterio,†a Roberta Cadoni,†bc Davide Esposito,a Daniela Vullo,d Anna Di Fiore,a Simona
Maria Monti,a Andrea Caporale,a Menotti Ruvo,a Mario Sechi,c Pascal Dumy,b Claudiu T. Supuran,e
Giuseppina De Simone,*a and Jean-Yves Winum*b
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
associated isoforms hCA IX and hCA XII over the cytosolic,
more widespread hCA I and hCA II (off-target enzymes for
many pharmacological applications). These compounds were
also shown to significantly inhibit the growth of primary
tumors and metastases in vivo.2 Thus, research in this field is
still very active, with an increasing number of novel
chemotypes examined for their interaction with this
superfamily of enzymes.
In this paper we report the synthesis of a series of benzoxaborole
derivatives, their inhibition properties against some Carbonic
Anhydrases (CAs), recognized as important drug targets, and the
characterization of the binding mode of these molecules to the CA
active site. Our data provide the first experimental evidence that
benzoxaboroles can be efficiently used as CA inhibitors.
Carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous metallo-
enzymes, present in most living organisms, which catalyze the
reversible hydration of carbon dioxide to bicarbonate and
To date, boron-containing compounds have been only scarcely
investigated as CAIs. An inhibition activity in the micromolar
range against some hCAs has been shown by simple boronic
acids, incorporating aromatic, arylalkyl and arylalkenyl
-
proton (CO2 + H2O
ꢀ
HCO3 + H+).1 In humans 15 different
isoforms have been so far described, which show variable
catalytic properties, cellular localization and tissue distribution.
These enzymes are involved in a large number of physiological
processes and their abnormal levels and/or activities have
been often associated with different human diseases.
Consequently, many human CAs (hCAs) are interesting targets
for the design of inhibitors or activators with biomedical
applications.1
CA inhibitors (CAIs), which have initially found applications as
diuretics, antiglaucoma agents and antiepileptics, are now
emerging as pharmacological agents against cancer and
obesity.1 However, their use as drugs is limited by the lack of
selectivity against the different CA isoforms. In order to
improve the selectivity profiles, recent years saw an increasing
interest for the development of new CAIs, alternative to the
first generation inhibitors such as sulfonamides and
sulfamates. Thus, novel chemotypes, among which the
coumarins, thiocoumarins, sulfocoumarins, polyamines,
dithiocarbamates, hydroxamates and carboxylates, were
reported as CAIs and some of them were shown to possess
very interesting selectivity properties.1 For example, some
coumarins were demonstrated to inhibit specifically the tumor
4
moieties.3, Although some of these compounds have been
proposed as proteasome inhibitors,5 with bortezomib A quite
effective for the treatment of multiple myelomas and other
malignancies,6 many concerns, mainly related to the high
reactivity and metabolic instability, have been raised on the
8
use of such derivatives as drugs.7, Thus, alternative boron-
containing moieties to be incorporated in enzyme inhibitors
started to be ultimately considered, with benzoxaboroles
constituting an interesting such class of molecules. Indeed,
these compounds were found to have a very stable oxaborole
ring and a higher hydrolytic resistance of the boron–carbon
bond in comparison with the boronic acids.9 Many such
molecules do also possess strong anti-inflammatory, antifungal
and antibacterial activity.10-13
Considering our interest for alternative chemotypes (to the
sulfonamides) for the development of novel classes of CAIs, we
have investigated the capability of benzoxaboroles to inhibit
some CAs present in humans and recognized as potential drug
targets, such as isoforms hCA I, II, IX and XII. In particular,
starting from benzoxaborole 1 (Scheme 1) as lead molecule,
we synthesized a small library of urea- and thiourea-
benzoxaborole derivatives, tested their inhibition properties
against the above mentioned isoforms and elucidated their
interaction mode with the CA active site by means of X-ray
crystallographic studies. Our data provide the first
experimental evidence that benzoxaboroles can be efficiently
used as CAIs, thus representing an interesting scaffold for
developing new molecules with a therapeutic potential for the
treatment of various diseases.
A straightforward strategy was used to synthesize
benzoxaborole derivatives starting from 6-
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aminobenzo[c][1,2]oxaborol-1(3H)-ol 3 (Scheme 1). The latter properties. Indeed, these compounds inhibit hCA IX and XII
was prepared as described in the literature14-16 starting with with KIs ranging between 92-94 nM and 640-813 nM
the nitration in position 6 of commercially available 1,3- respectively, whereas their efficacy against hCA
dihydro-1-hydroxy-2,1-benzoxaborole with fuming nitric acid substantially unaltered. hCA II inhibition by these compounds
at –45 °C, followed by hydrogenation over palladium on is also an order of magnitude more efficient compared to the
carbon at room temperature. Compound was then reacted in parent compound . Ureas and thioureas show increased
acetone with either isocyanates or isothiocyanates to obtain hCA I inhibition compared to the simple derivatives 1-3, with
the ureas or thioureas , respectively (Scheme 1). KIs in the range of 98-654 nM. The hCA II inhibitory properties
Compounds were then assayed for their inhibitory properties are even more interesting, with ureas behaving as medium
against hCA I, II, IX and XII (Table 1). potency inhibitors and thioureas showing a weak activity (KIs
> 1 M). hCA IX is inhibited efficiently by two benzoxaborole
derivatives, the nitro compound and thiourea 5c, which have
I is
1
3
1
4
5
4
5
4
5
µ
2
KIs of 94 and 93 nM, respectively. The remaining
ureas/thioureas are much less effective hCA IX inhibitors, with
KIs of 336-1230 nM. The strongest inhibition is observed for
isoform hCA XII, for which 2, 4c, 4f and all thioureas 5 show KIs
in the range of 42-93 nM. The remaining compounds are less
effective, with KIs of 184-663 nM. Data show that even subtle
structural changes induce great alterations of inhibitory
properties against all investigated CA isoforms, with some
compounds able to reach nanomolar inhibition constants
against the studied isoforms (see for example 4f against hCA I,
hCA II and hCA XII). It is thus highly probable that much more
effective CAIs belonging to this new chemotype may be
designed by tailoring the nature and substitution pattern at
the heterocyclic ring system. These data prove the capability of
the newly developed molecules to act as CAIs, but do not
provide any information regarding their mechanism of action.
To clarify this important aspect, detailed X-ray crystallographic
studies were undertaken. In particular, the high-resolution
Scheme 1. Synthesis of benzoxaborole derivatives
Table 1. Inhibitory activity of compounds 1-3 4a-4f and 5a-5e against the
hCA isoforms I, II, IX and XII, determined by a Stopped-Flow, CO2 hydration
assay. Data relative to the standard CAI acetazolamide (AAZ) are also
shown for comparison.
,
KI (nM)a
hCA Ib
5690
6352
9434
654
235
487
557
613
98
548
355
417
380
258
250
hCA IIb
8180
504
hCA IXc
>50000
94
hCA XIIc
>50000
92
640
240
175
93
184
663
69
76
89
crystal structure of hCA II in complex with benzoxaborole 1,
1
2
3
the lead compound of the series here investigated, was solved.
hCA II was chosen as model isoform for crystallization, since it
readily forms crystals and many studies have been reported on
its adducts with different classes of inhibitors.1 Crystals of the
adduct were obtained following a procedure well described in
literature.19 In particular, native hCA II was crystallized by the
hanging drop vapor diffusion method at pH 8.7, and the
obtained crystals were soaked in the precipitant solution
containing the inhibitor at a concentration of 100 mM (see ESI
for details). The structure was refined by using REFMAC20 of
the CCP4 suite21 and the native hCA II (PDB code 1CA2)22 as an
initial model. Inspection of the electron density maps at
various stages of the crystallographic refinement revealed the
binding of the inhibitor to the enzyme active site in its
590
730
480
456
439
841
89
1148
1500
1838
1305
1500
12
813
4a
4b
4c
4d
4e
4f
5a
5b
5c
5d
5e
AAZ
1060
843
845
925
1230
414
436
336
93
610
336
25
71
42
89
5.7
[a] Errors in the range of ±5−10% of the reported value from three
different determinations. [b] Full length. [c] Catalytic domain.
tetrahedral anionic form 6 (Scheme 2). This finding is in
agreement with the observation that at the pH used for
Among the twelve catalytically active hCAs, these isoforms
were chosen for our studies since two of them, the cytosolic
hCA I and II, are widespread house-keeping enzymes involved
crystallization experiments (i.e., 8.7), compound
present in solution in this form (see Scheme 2).10
1 is mainly
in
a
host of physiologic processes,17 whereas the
transmembrane hCA IX and XII are tumor-associated enzymes,
abundant in hypoxic tumors and recently validated as
antitumor targets.18 Indeed, one CA IX/XII inhibitor, belonging
to the sulfonamide series, is currently in Phase I clinical trials
for the management of hypoxic, metastatic tumors.2 The data
Scheme 2. Lewis/Brønsted acidic properties of benzoxaborole.
Interestingly, two different binding modes, both characterized
by the coordination of the tetrahedral derivative to the
catalytic zinc ion, were observed (Fig. 1A and S1). In the first
one, hereafter indicated as binding mode A, one of the
hydroxyl groups of the benzoxaborole was anchored to the
catalytic zinc ion, completing its tetrahedral coordination (Fig.
1B), whereas, in the second one, hereafter indicated as
binding mode B, the inhibitor was bound to the zinc ion with
of Table 1 show that benzoxaborole
hCA I and II inhibitor, and does not significantly inhibit the
transmembrane isoforms hCA IX and XII (KI > 50 M).
However, the introduction of simple substituents of the NO2 or
NH2 type on the 6 position of the benzoxaborole ring, as in
and , leads to a dramatic change of the CA inhibitory
1 is a weak, micromolar
µ
2
3
2 | J. Name., 2012, 00, 1-4
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two of its oxygen atoms, generating a trigonal bipyramidal obtained at pH 7.4, benzoxaborole
COMMUNICATION
1
bound other sites on the
coordination geometry of zinc (Fig. 1C). In both binding modes, protein surface, beyond the active site. In particular, two
additional polar and van der Waals interactions contributed to molecules of in the trigonal planar form were present in a
1
the stabilization of the adduct (Fig. 1B and 1C).
cleft on the protein surface, delimited by residues Ser2, His3,
His4, Trp5, His10, Asn11, His15, Trp16, Lys18 and Asp19 (Fig.
2), whereas three other molecules formed adducts with three
histidines, highly exposed to the solvent (His3, His4 and His10),
at the amino-terminal part of the enzyme (Fig. 2). The
formation of such kind of dative adducts between boron
containing derivatives and His residues has been already
described for some protease inhibitors belonging to the
boronic acid class.24
The additional binding sites, observed in the structure at pH
7.4, were not present in that obtained at pH 8.7, and were
never reported for other classes of CAIs investigated through
X-ray crystallography. In such derivatives, one nitrogen atom
from the imidazole ring coordinates the boron atom of the
inhibitor as the fourth, tetrahedral ligand (Fig. 2). These
additional interactions could represent a limit in the use of
benzoxaboroles as drugs, indicating their propensity to react in
a non-specific way not only with the target protein but also
with other protein systems possibly present in vivo and
possessing accessible nucleophile residues. We should
however remark that the very large excess of
1 used in the
crystallization experiments, is likely unpractical in vivo,
suggesting that the occurrence of such unwished chemical
bonds and non covalent interactions is only governed by the in
vitro experimental conditions and might never take place at
the concentrations expected for a therapeutic use. Moreover,
Fig. 1 (A) σA-weighted |2Fo-Fc| map (contoured at 1.0 σ) relative to the inhibitor
molecule in the hCA II/1 adduct crystallized at pH 8.7. The two possible
coordination modes of the inhibitor are depicted. The zinc ion coordination is
drawn in black for the histidines, in blue for binding mode A and in orange for the occurrence of such adducts only in the structure at pH 7.4
binding mode B. (B) Detail of the interactions of inhibitor
site in binding mode A. (C) Detail of the interactions of inhibitor
active site in binding mode B. In (B) and (C) residues involved in polar and van
der Waals (distance < 4 Å) interactions are shown. Continuous lines indicate zinc
ion coordination, whereas dashed lines indicate hydrogen bond distances.
1
with enzyme active
1
strongly suggests that their formation is strictly related to the
presence of the neutral trigonal form of the inhibitor, which is
nearly absent at pH 8.7. Thus, it can be hypothesized that,
lowering the pKa of benzoxaborole derivative with proper
substituents of the aromatic ring,25 and consequently reducing
with enzyme
The observation that in both cases the inhibitor binding to the
enzyme was mediated by the tetrahedral anionic form
prompted us to investigate which was the binding mode of
the amount of the trigonal form of 1 present at physiological
pH, the formation of unspecific adducts as those shown in Fig.
2 may be suppressed.
6,
1
at physiological pH, where, on the basis of the equilibrium
reported in Scheme 2, the trigonal planar form of the inhibitor
was also present. To clarify this point, we set up new
crystallization conditions of the hCA II/1 adduct at pH 7.4 (see
the ESI for details). Interestingly, the analysis of the obtained
structure revealed that also in this case the inhibitor bound the
enzyme in its anionic tetrahedral form 6, adopting two binding
modes identical to those observed in the structure crystallized
at pH 8.7 (Fig. S2 and S3). Based on this finding we can clearly
conclude that the binding mode of benzoxaborole
enzyme active site is independent on pH and is always
associated to the anionic tetrahedral form , probably because
in this way the electron avid boron atom has eight electrons in
its last shell. However, the question whether only the anionic
tetrahedral form 6 can bind the active site or the trigonal
1 to the
6
Fig. 2 Solvent accessible surface of hCA II showing the binding sites of inhibitor
(A). The tetrahedral form coordinating the catalytic zinc ion is colored in red,
the trigonal form is drawn in blu, whereas the inhibitor molecules involved into
1
6
1
dative adducts with exposed histidine residues are shown in green (B).
planar form can bind and then is transformed in the
1
These findings may also constitute the basis for mapping
exposed His residues from proteins with an unresolved
structure, by means of benzoxaborole derivatives, which are
inexpensive, stable and easily identifiable via mass
spectrometric experiments.
tetrahedral one 6, by reaction with the hydroxide anion bound
to the zinc ion, remains unanswered. In any case, the
observation that the tetrahedral form is involved in the
mechanism of action of the benzoxaboroles is in agreement
with previously reported data on benzoxaboroles as inhibitors
of Leucyl-tRNA synthetase.12, 23
To verify if the substitution pattern on the benzoxaborole ring could
influence the binding mode of the inhibitors to the enzyme active
site, we also crystallized the best hCA II inhibitor of the series,
Interestingly, a careful analysis of the electron density maps in
other regions of the protein showed that in the structure
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Roskelley, C. M. Overall, A. Minchinton, F. Pacchiano, F.
Carta, A. Scozzafava, N. Touisni, J. Y. Winum, C. T. Supuran
and S. Dedhar, Cancer Res., 2011, 71, 3364.
namely compound 4f, in complex with its target enzyme (see ESI for
experimental details). Interestingly, analysis of the structure of the
hCA II/4f adduct showed that the bulky substituent of the
benzoxaborole ring has a great effect in influencing the interaction
of the inhibitor with the enzyme active site. Indeed, in this case only
one binding mode is observed (Fig. 3A and S4), which is completely
different from binding mode A of compound 1 (Fig. 3B) and rather
similar to binding mode B (Fig. 3C), even if some differences in the
orientation of the benzoxaborole ring can be observed also in this
case.
3. J. Y. Winum, A. Innocenti, A. Scozzafava, J. L. Montero and
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Fig. 3 (A) σA-weighted |2Fo-Fc| map (contoured at 1.0 σ) relative to the inhibitor
molecule in the hCA II/4f adduct. (B) Structural superposition of the hCA II/4f
adduct with hCA II/1
the hCA II/4f adduct with hCA II/
adduct in binding mode A. (C) Structural superposition of
adduct in binding mode B
1
.
13. A. Adamczyk-Woźniak, M. K. Cyrański, A. Żubrowska and A.
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It is worth noting that the inhibitor 4f cannot assume the
binding mode A when it interacts with hCA II active site, since
in such case the bulky tail in position 6 would clash with the
14. W. J. Lennarz and H. R. Snyder, J. Am. Chem. Soc., 1960, 82
2172.
,
hydrophobic pocket defined by residues Phe131, Leu141, 15. T. Akama, C. Dong, C. Virtucio, D. Sullivan, Y. Zhou, Y. K.
Val121 and Val143. The stabilization of only one binding mode
and the formation of a higher number of polar and van der
Waals interactions with enzyme active site (see Fig. S4) can be
considered as the main factors responsible with the improved
inhibition properties against hCA II of compound 4f with
respect to 1.
In conclusion, altogether data here reported provide evidence
that benzoxaboroles can be efficiently used as a new class of
Zhang, F. Rock, Y. Freund, L. Liu, W. Bu, A. Wu, X. Q. Fan
and K. Jarnagin, J. Pharmacol. Exp. Ther., 2013, 347, 615.
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19. A. Di Fiore, A. Vergara, M. Caterino, V. Alterio, S. M. Monti,
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CAIs, presenting interesting inhibition properties and
a
completely new binding mode to the CA active site. Moreover,
the substitution pattern at the benzoxaborole ring can be used
to finely modulate the affinity and the binding mode toward
the different CA isoforms. Finally, our data also suggest that
the high reactivity of benzoxaborole 1 towards His residues
may be useful to map the exposition of such amino acids in
other poorly investigated proteins.
This work was supported by a grants from CNR-DSB Progetto
Bandiera "InterOmics”, and from the Ligue contre le Cancer
(comité des Pyrénées-Orientales) and by the LabEx CheMISyst
(ANR-10-LABX-05-01).
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Noe, J. M. Sligar, Y. K. Zhang, C. J. Bacchi, N. Yarlett and R.
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4 | J. Name., 2012, 00, 1-4
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