Selenols: a new class of carbonic anhydrase inhibitors

Article abstract of DOI:10.1039/c8cc08562e

Stable aryl selenols were obtained through a convenient procedure. Selenols represent a new chemotype acting as carbonic anhydrase inhibitors (CAIs), inhibiting four human isoforms, CAs I, II, VII and the tumor-associated CA IX. X-ray crystallographic, physical and computational studies provided insights into the binding mode of this conceptually new class of CAIs.

Full text of DOI:10.1039/c8cc08562e

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Selenols: a new class of carbonic anhydrase
inhibitors†
Cite this: Chem. Commun., 2019,
55, 648
Andrea Angeli,‡a Damiano Tanini, ‡b Alessio Nocentini,ac Antonella Capperucci,b
Marta Ferraroni,b Paola Gratteri
and Claudiu T. Supuran
*
ac
a
Received 31st October 2018,
Accepted 3rd December 2018
DOI: 10.1039/c8cc08562e
rsc.li/chemcomm
Stable aryl selenols were obtained through a convenient procedure. SeC (pKa B 5.8) more nucleophilic than the thiol group of Cys
Selenols represent a new chemotype acting as carbonic anhydrase (pKa B 8.3). Moreover, selenols with a low pKa value,9 under
inhibitors (CAIs), inhibiting four human isoforms, CAs I, II, VII and physiological conditions (pH B 7.4), were proved to be almost fully
the tumor-associated CA IX. X-ray crystallographic, physical and present as selenolates (R-SeÀ), whereas the majority of thiols are
computational studies provided insights into the binding mode of present in the non-ionized form (R-SH). Taking into account the
this conceptually new class of CAIs.
soft character of the selenium atom, the polarity of the Se–H bond,
and the affinity of selenium compounds for zinc finger proteins,10
Carbonic anhydrase (CA, EC 4.2.2.1) is a metalloenzyme that selenols could represent a new chemotype as human (h) CA
catalyzes a very simple reaction: the hydration of carbon inhibitors (CAIs). Indeed, both phenols (ArOH) and aryl thiols
dioxide to bicarbonate and a proton.1 This reaction plays an (ArSH) exert CA inhibitory activity.11,12 Despite the interest in the
important role in many physiological and pathological processes study of the behaviour of selenols, their use is often limited by a
associated with pH control, ion transport, and fluid secretion.2 not straightforward preparation, mainly due to their instability. In
Their inhibition is an important target correlated with the fact selenols are known to be rather unstable, showing a high
treatment of several diseases such as glaucoma,3 obesity,4 and tendency to be oxidized to diselenides. Nonetheless, the synthesis
epilepsy.5 More recently CA inhibition was validated as a new of a limited number of substituted aryl selenols was described
approach to fight tumors and metastases.6 In this context, during in the literature. Aryl selenols can be obtained, although often
the past decades, organoselenium derivatives attracted increasing in rather low yields, using Grignard reagents13 or organolithium
interest in identifying free radical scavengers or antioxidant agents derivatives.14 On the other hand, aryl selenolates can be easily
involved in the protection against oxidative damage.7 Oxidative generated by reducing the corresponding diselenides, and sodium
stress, induced by the generation of reactive oxygen species borohydride is usually the reagent of choice to generate selenolates
(ROS), is considered a major causative factor of many diseases from diselenides. Nonetheless, to the best of our knowledge, a
including diabetes, cardiovascular diseases, and several neuro- NaBH4 based protocol has not been applied to the synthesis and
degenerative conditions and is reported to affect all phases of the isolation of aryl selenols. Thus, we sought to evaluate the
the oncogenic process including initiation, promotion, and efficiency of different proton sources in order to protonate aryl
progression.8 The biological significance of selenols was predo- selenolates to access stable aryl selenols. With this purpose,
minantly dependent on their incorporation in proteins, as seleno- diphenyl diselenide was reacted with NaBH4 in THF or EtOH
cysteine (SeC). The different atomic structure and composition of and on the basis of previous results related to the synthesis of
selenium compared with sulfur makes the selenol (SeH) group of stable alkyl selenols,15 the corresponding selenolate was then
treated with different organic or inorganic proton sources. The
results of this investigation are reported in Table S1 (ESI†). As
can be noted, the nature of the acid is crucial in order to obtain
a University of Florence, NEUROFARBA Department, Sezione di Scienze
Farmaceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino, Florence, Italy.
stable aryl selenols. Higher isolated yields were found when the
reaction was carried out at 0 1C in dry EtOH, using citric acid as
the H+ source. Having demonstrated the possibility of obtaining
a stable aryl selenol through this easy and convenient procedure,
we sought to synthesize a series of substituted selenols in order to
investigate their activity as hCA inhibitors. Thus, different
diaryl diselenides were reacted under the conditions described
E-mail: claudiu.supuran@unifi.it
b University of Florence, Department of Chemistry ‘‘Ugo Schiff’’, Via della Lastruccia
13, I-50019 Sesto Fiorentino, Italy
c University of Florence, NEUROFARBA – Pharmaceutical and Nutraceutical Section,
Laboratory of Molecular Modeling Cheminformatics & QSAR, Via Ugo Schiff 6,
50019 Sesto Fiorentino, Florence, Italy
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c8cc08562e
‡ These authors contributed equally to this work.
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Table 1 Synthesis of substituted aryl selenols 2a–2j
Table 2 Inhibition data of human CA isoforms I, II, VII and IX with
compounds 2a–j and compared to the standard CAI acetazolamide
(AAZ) by a stopped flow CO2 hydrase assay16
Kia (mM)
Cmp.
hCA I
hCA II
hCA VII
hCA IX
Entry
1
Diselenide
Selenol
Yield (%)
76
2a
2b
2c
2d
2e
2f
2g
2h
2i
6.7
0.85
2.8
2.2
0.49
3.6
93.8
0.76
6.4
0.44
0.20
0.28
3.0
9.6
0.20
3.3
0.18
0.46
22.4
0.26
7.5
3.2
6.1
51.0
0.87
0.52
0.28
7.1
3.7
0.25
51.1
0.72
3.6
0.95
26.0
3.9
460
0.84
5.4
4.5
6.8
18.2
0.002
2
3
56
82
2j
AAZ
0.012
a
Mean from 3 different assays by a stopped flow technique (errors were
in the range of Æ5–10% of the reported values).
4
5
85a
95
types of CAIs, such as sulfonamides, inorganic complexing
anions, phenols, or thiophenols, an incubation time of 15 min
is allowed for the formation of the enzyme–inhibitor adduct.17
Working under the same conditions, the data from Table 2
indicate phenyl selenol (2a) as a promising scaffold to develop
nonclassical inhibitors of the tumor associated CA (i.e. isoform
IX). In fact, most of the tested derivatives inhibited hCA IX at
nanomolar concentrations. In contrast, the cytosolic hCA isoforms
(I, II and VII) proved to be less sensitive to selenols (in the
micromolar range). Additional moieties on the benzene scaffold
modulate the selectivity towards the different CA isoforms.
Introduction of substituents at position 4, as in compounds
2d, 2g, and 2j, generally led to a loss of selectivity against the
membrane isoform hCA IX. 4-Methylbenzeneselenol 2g showed
remarkable inhibition for hCA I. On the other hand, replace-
ment of the methyl moiety with the methoxy one (2d) decreased
the efficacy of inhibition for this isoform.
6
7
66a
60a
8
9
78
84
10
64a
The introduction of a fluorine atom in the para position (2j)
led to a six fold increase of the inhibition selectivity against the
two dominant cytosolic isoforms hCAs I and II, over the tumor
associated one (hCA IX). The inhibition potency instead
increased noticeably for all isoforms considered here when
a
Less than 5% of the corresponding diselenide was detected.
in Table 1, finding a clean and high yielding entry to aryl the substituents were present at position 2 of the phenyl ring
selenols 2a–j bearing different groups at selected positions of (2b and 2f). Furthermore, these compounds were found to be
the aromatic ring (Table 1).
two-fold more selective against the tumor-associated membrane
Having in hand a series of differently substituted aryl isoform over the cytosolic ones. An interesting inhibition profile
selenols, we then moved to study their activity as CAIs. In order was observed for compound 2c, in which the methoxy moiety
to demonstrate the new chemotype as CAIs, we tested the was placed at position 3 of the phenyl scaffold. This placement
corresponding diselenides 1a–j which resulted to be ineffective led to the best result for obtaining selectivity against hCA IX,
against all isoforms tested here (data are reported in Table S2, which was over 10-fold when compared with the other isoforms
ESI†). Moreover, 77Se NMR spectra acquired under the inhibition considered here. The presence of two additional methoxy
conditions showed that the half-life of aryl selenols ranges from groups in 4 and 5 positions (compound 2e) resulted in a
30 to 40 minutes. An appreciable decomposition did not occur remarkable decrease in inhibition potency for hCAs I, II, and
during the short time of the biological assays. Inhibition data for IX and a complete loss of activity against hCA VII. On the other
compounds 2a–j against the human CA isoforms I, II, VII and IX hand, compound 2h with two methyl moieties in positions 2
are presented in Table 2. The data have been obtained by a and 6 showed the best substitution pattern for increasing the
stopped-flow technique monitoring the production of H+ from inhibition potency against hCA IX (Ki 0.18 mM). A further
the CA catalysed hydration of CO2.16 When assaying all other interesting inhibition profile was observed for the naphthyl
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scaffold 2i. This compound showed a high nanomolar activity deprotonated form at a pH of 7.4.12 This choice was indeed
(Ki 0.46 mM) for the membrane associated IX isoform also with confirmed by X-ray evidence. Derivatives reported in Table 2
remarkable selectivity, since it was 50 fold more active over hCA were subjected to QM geometry optimization (B3LYP/cc/pvtz-f)
II (Table 2). In order to carry out a more thorough and targeted and ESP charge computation prior to docking the molecules
investigation on the activity of selenols, the X-ray structure of 2a into the active sites of hCAs II and IX. Due to the shortened
in complex with both hCAs I and II was determined at 1.64 and C–Se bond length of poses resulting from docking QM investi-
1.44 Å resolution, respectively, and compared with the structure gations were undertaken and the targets broken down into
of the same enzymes in complex with phenol and thiophenol as smaller model systems each comprising, in addition to the
ligands.11,12 Our adducts revealed the binding mode of selenols selenol derivatives, some residues of the hCA binding cleft. All
to be different with respect to the typical binding mode of residues found to form non-bonding interactions with selenols
phenols. According to the reported literature, phenols were were considered in the model definition and capped with
found anchored to the non-protein zinc ligand by a hydrogen typical end groups (N67, Q92, V121, V143, W209, F131, L141,
bond involving the H atom of the inhibitor and the donor OH L198, H64, T199, and T200 in hCA II and Q71, Q92, V121, V142,
from the zinc hydroxide.11 Contrarily, we observed that the W210, V130, L140, L199, H68, T200, and T201 in hCA IX). These
selenol was bound directly to the Zn(II) ion from the CA active so defined model systems were checked with DFT (B3LYP/
site with its usual tetrahedral geometry, similar to the binding LACVP*+) calculation investigating the ligand conformations
observed for thiolates12 (Fig. 1A and B). In particular the selenol that corresponded to the best docking poses (Fig. 2).
2a scaffold makes hydrophobic interactions with Ala122, Val144
In all derivatives, the negatively charged selenium atom
and Leu199 within the active site of hCA I. Furthermore, the indistinctly completes the Zn coordination sphere occupying
imidazole ring of His95 was also involved in a p-stacking the fourth coordination position. The theoretical prediction of
interaction with the aromatic ring of the inhibitor (Fig. 1A).
the positioning of 2a in hCA II matches the experimental one
The crystal structures of hCA II (Fig. 1B) also revealed a subsequently obtained by crystallography (RMSD 0.54 Å, Fig. S1,
similar binding mode for selenol 2a, which was observed to be ESI†), thus supporting the reliability of the results found
directly bound to the Zn(II) ion. However, the electron density in silico. Based on the substituents appended at the benzene
relative to the inhibitor was less clear, indicative of a less ring, the Se–Zn coordination properties (Se–Zn bond length and
ordered position of the inhibitor when compared to the hCA I Zn–Se–C angle) slightly differ among compounds 2a–j, ranging
complex (Fig. 1B). This might be justified by the fact that 2a is in the intervals reported in Table S4, ESI.† The coordination
bound less effectively to this isoform, since a limited number of patterns of the binding conformations obtained from docking
interactions among the ligand and the amino acid residues were compared with data from experimentally determined
occurred (i.e. 2a showed hydrophobic interactions only with structures from CSD (Cambridge Structural Database)18 and
Val122 and Leu198; Fig. 1B). In an earlier stage of this study, with the coordination parameters in the PDB ID 2OSF (thiophenol
when the X-ray structure of 2a in complex with CA II had not yet derived from tioxolone) and 3WC5 (the unique selenate R-SeÀ/
been solved, in silico investigations were undertaken to predict carboxypeptidase B complex reported in the PDB database. In that
the binding mode of aromatic selenols 2a–j to hCA II and hCA structure, the aliphatic selenate coordinates two zinc ions).19 The
IX. The PhSeÀ form of the selenol derivatives was used in the results of this comparison point out the conformational strains of
computations due to the benzeneselenol 4 thiophenol 4 the docked poses of derivatives 2a–j, which may have an influence
phenol acidic trend (calculated pKa values of 5.9, 6.6 and 9.9, on the binding entropy contributions that is not explicitly accounted
respectively) and to the experimental evidence that thiophenols for in the used G-score function. The deviation of the angle
directly coordinate the Zn ion in the hCA II binding cleft in the from the CSD search reference value (103.531) is greater in the
case of the binding of the derivatives in CA II than in CA IX
(Fig. S2, ESI†).
Fig. 1 (A) Active site region of the hCA I/2a adduct (PDB: 6HWZ). Inhibitor
showed as a sA-weighted |Fo À Fc| density map at 2.0s. (B) Active site
region of the hCA II/2a adduct (PDB: 6HX5). Inhibitor showed as a
sA-weighted |Fo À Fc| density map at 2.0s. van der Waals interactions,
p-stacking interactions and the active site Zn2+-ion coordination are also
Fig. 2 2D representation of the binding mode of benzeneselenols 2a–j
shown and labelled in blue, magenta and red respectively.
within hCA II/IX. Residues in round brackets refer to hCA IX.
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Cassa di Risparmio di Firenze, Italy, is gratefully acknowledged for a
grant to A. N. (ECR 2016.0774).
Conflicts of interest
There are no conflicts to declare.
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