Multimeric xanthates as carbonic anhydrase inhibitors

Article abstract of DOI:10.3109/14756366.2015.1072177

The field of multivalent inhibition of enzymes is growing exponentially from the first reported multivalent effect on a glycosidase enzyme. However, the investigations have generally remained restricted to carbohydrate-processing enzymes. Carbonic anhydrases are ubiquitous metallo-enzymes involved in many key biological processes, that catalyze the reversible hydration/dehydration of CO₂/HCO₃. This study reports the first synthesis of multimeric xanthates addressing the selectivity and potency of CA multivalent inhibition. Six multivalent compounds containing three, four, and six xanthate moieties were prepared and assayed against four relevant CA isoforms together with their monovalent analogues. Some of the multimers were stronger inhibitors than the monomeric species. For hCA I, the two best molecules 18 and 20 showed an improvement of the ligand affinity of 4.8 and 2.3 per xanthate units (valence-corrected values), respectively, which corresponds to a clear multivalent effect. Moreover, the biochemical assays demonstrated that the multimeric presentation of xanthates, also affected the selectivity of the relative inhibition among the four CAs assayed.

Full text of DOI:10.3109/14756366.2015.1072177

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–7
2015 Taylor & Francis. DOI: 10.3109/14756366.2015.1072177
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RESEARCH ARTICLE
Multimeric xanthates as carbonic anhydrase inhibitors
1
2
2
Marta Abellan-Flos , Muhammet Tanc¸ , Claudiu T. Supuran , and Stephane P. Vincent
1
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´
1
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Laboratoire de Chimie Bio-Organique, Departement de Chimie, Academie Louvain, University of Namur (UNamur), Namur, Belgium and
´
2
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Dipartimento Neurofarba, Sezione di Scienze Farmaceutiche e Nutraceutiche, Universita degli Studi di Firenze, Florence, Italy
Abstract
Keywords
The field of multivalent inhibition of enzymes is growing exponentially from the first reported
multivalent effect on a glycosidase enzyme. However, the investigations have generally
remained restricted to carbohydrate-processing enzymes. Carbonic anhydrases are ubiquitous
metallo-enzymes involved in many key biological processes, that catalyze the reversible
hydration/dehydration of CO2=HCOꢀ3 . This study reports the first synthesis of multimeric
xanthates addressing the selectivity and potency of CA multivalent inhibition. Six multivalent
compounds containing three, four, and six xanthate moieties were prepared and assayed
against four relevant CA isoforms together with their monovalent analogues. Some of the
multimers were stronger inhibitors than the monomeric species. For hCA I, the two best
molecules 18 and 20 showed an improvement of the ligand affinity of 4.8 and 2.3 per xanthate
units (valence-corrected values), respectively, which corresponds to a clear multivalent effect.
Moreover, the biochemical assays demonstrated that the multimeric presentation of xanthates,
also affected the selectivity of the relative inhibition among the four CAs assayed.
Carbonic anhydrases, enzyme inhibitors,
multivalent inhibitors, xanthates
History
Received 13 June 2015
Revised 9 July 2015
Accepted 9 July 2015
Published online 3 August 2015
Introduction
acid residue Thr199 and other S interacts also through hydrogen
bond with the NH group of the same residue7. This binding mode
explains the good inhibition rates of these compounds for various
CA tested to date8. Three representative families containing this
ZBG, i.e. dithiocarbamates, trithiocarbonates, and xanthates
(Figure 1) have been intensively investigated.
They display a wide range of inhibitory powers depending on
the organic moiety present in the molecule and its orientation
within the active site cavity, which promote distinct interactions
with the surrounding amino acids7. Therefore, as the active site
of all the CA isoforms is highly conserved11, these kind of
recently discovered CAIs offer the possibility of designing more
selective and potent agents8. Herein we targeted the synthesis
and study of multivalent alkali-metal xanthates as CAIs from
distinct polyols.
Multivalency has gained much importance in the last years,
particularly in the fields of biomedicine and bioorganic synthesis.
The critical role of multivalency in essential biological phenom-
ena is only starting to be unravelled now12. Many natural
processes, particularly at the membranes interface, benefit from
the multivalent display of biological entities to boost the efficacy
of binding events. Especially, carbohydrate–protein interactions
are notably weak at the monomer level, but can be tremendously
enhanced if the ligand is displayed in a multivalent way13.
Scientists have extensively studied the biophysical interactions
among natural systems and created a plethora of artificial
multivalent systems in order to understand the underlying
mechanisms of multivalency14–22. The most scrutinized systems
are likely lectin–carbohydrate dyads. Lectins are non-processing
but only carbohydrate-binding proteins often expressing multiple
binding sites. Since some biologically relevant lectins are
involved in pathological processes, their inhibition can be of
Carbonic anhydrases (CAs, EC 4.2.1.1), which are ubiquitous
Zn-based metallo-enzymes catalyzing the reversible hydration/
dehydration of carbon dioxide/bicarbonate, play an essential role
in numerous physiological processes. In animals, they are critical
in vital processes such as respiration, pH regulation, biosynthesis
and calcification. Fifteen different human CA (hCA) isoforms and
CA-related proteins (CARPs), belonging to the a-class and
differing mainly in their location within the cell and tissues, have
been reported to date1. CA inhibitors (CAIs) are already employed
as diuretics and anti-glaucoma drugs. Furthermore, they are
promising as anticonvulsant, anti-obesity, anticancer, and anti-
virulent agents, among others2. Some CAIs, i.e. coumarins3 and
lacosamide4, inhibit the enzyme activity by occluding the
entrance of the catalytic site and ortho-substituted benzoic acid
derivatives can inhibit CA without entering the active site at all5.
However, most CAIs act by binding either to the Zn(II) cation,
inside of the active site and crucial for the catalytic activity, or to
the water molecule coordinated to it6. Namely, the classical
sulfonamides and their isosteres, phenols, thiophenols, polya-
mines, and metal complexing anions. Belonging to this last group
of anionic compounds, several families of CAIs displaying the
CSꢀ2 zinc-binding group (ZBG) have been recently reported7–10
By crystallography, it has been proven that this ZBG coordinates
the Zn(II) from the CA active site through one of the sulphur
atoms; the same S forms a hydrogen bond with the OH of amino
.
´
Address for correspondence: Stephane P. Vincent, Departement de
Chimie, Laboratoire de Chimie Bio-Organique, Academie Louvain,
University of Namur (UNamur), rue de Bruxelles 61, B-5000 Namur,
Belgium. Fax: +32 81 72 45 17. E-mail: stephane.vincent@unamur.be
´
´

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M. Abellan-Flos et al.
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J Enzyme Inhib Med Chem, Early Online: 1–7
therapeutic interest20–24. For instance, the adhesion of pathogenic spacer to which each inhibitor is grafted to the scaffold are
bacteria to cells can be inhibited to prevent the bacterial parameters that must be fine-tuned to optimize a multivalent
infections25–32. Nevertheless, the field of multivalent enzyme inhibition. Herein, we decided to explore both different multiva-
inhibition (MEI) has been barely explored until 2009, when a lent scaffolds and CAI. A small collection of xanthate-derivatized
clear multivalent effect (and an increase in selectivity) was compounds were prepared from multivalent polyol-scaffolds
determined in a-mannosidase inhibition by iminosugar clusters33. (Figure 2). The multivalent xanthates contained either three,
The term ‘‘multivalent effect’’, generally only employed with four or six functionalities. In order to asses a possible multivalent
carbohydrate-binding proteins, could then be applied to the field effect, the monovalent analogues for each of the linkers used were
of carbohydrate-processing enzymes. After this groundbreaking also prepared. All the compounds were assayed in vitro for
result, considerable amount of research has been reported, inhibition against the cytosolic hCA I and II as well as the
although almost exclusively with carbohydrate-processing transmembrane, tumor-associated hCA IX and XII. Interestingly,
enzymes such as glycosidases34–42 and glycosyltranferases43.
hCA IX and XII could be targeted by multivalency since
In a recent work, we reported the inhibitory study of coumarin- crystallographic studies have demonstrated that they both present
derivatized fullerene hexakis-adducts toward a set of biologically a dimeric extracellular domain45,46. Therefore, inhibition of these
relevant hCA isoforms44. These multimeric compounds did not two isoforms could be enhanced by multivalent mechanisms such
show strong multivalent effects, however they were always more as chelation if an appropriate design of the inhibitor is achieved.
potent than the respective monovalent analogues. Furthermore, Other few previous works have explored the multivalent inhibition
the selectivity of the multimeric species varied with respect to the of CAs. For instance, Whitesides and collaborators studied the
monovalent ones. MEI seems to be highly depending on the dependence of avidity on spacer length in bivalent model
structure of the multivalent inhibitor: the valency, the topology, systems47. Also, very recently, Winum and co-workers analyzed
the structure of the central core and the length and rigidity of the the inhibition of multivalent CAIs-decorated silica nanoparti-
cles48. Nevertheless, as far as we know, this study reports the first
synthesis of multimeric xanthates as CAIs, designed to address
the selectivity and the potency of CA multivalent inhibition.
Materials and methods
Chemistry
All the synthetic procedures are described in the Experimental
Figure 1. Metal complexing anions containing the ZBG CSꢀ2 : dithio-
Supporting Information (ESI). NMR spectra of all the non-
previously reported compounds are also shown in the ESI.
carbamates, trithiocarbonates and xanthates.
Figure 2. Multimeric compounds synthesized for this study and their monovalent counterparts.

DOI: 10.3109/14756366.2015.1072177
Multimeric xanthates as CA inhibitors
3
CA inhibition
pathways. Both molecules were azidated by substitution of the
chloride leaving group to provide 1 and 5, respectively, which
were engaged in the synthesis of two distinct types of molecules.
On the one hand, 1 and 5 were reacted with KOH and CS2 in
Et2O, as reported in the literature for simple hydroxyl groups8,
giving the desired monovalent xanthates 2 and 6 as yellowish
precipitates. In addition, 1 and 5 were clicked to 3-methoxyprop-
1-yne to afford 3 and 7, which were transformed in the
monovalent xanthates analogues 4 and 8, respectively, through
reaction with CS2 and KOH in DMSO/H2O 10:1. This last
procedure was optimized since the use of the same conditions as
for the monovalent analogues 2 and 6, did not afford the desired
analogues in good yields. Compounds 1–8 were assayed against
the hCAs studied to determine the importance of the presence of a
triazole moiety and to assess the existence of a possible
multivalent effect when compared to multimers.
An SX.18MV-R Applied Photophysics stopped-flow instrument
was used for assaying the CA-catalyzed CO2 hydration activity by
using the method of Khalifah49. Inhibitor and enzyme were pre-
incubated for 15 min prior to assay. IC50 values were obtained
from dose response curves working at seven different concentra-
tions of test compound (from 0.1 nM to 50 mM), by fitting the
curves using PRISM (www.graphpad.com) and non-linear least
squares methods, values representing the mean of at least three
different determinations, as described earlier by us50,51. The
inhibition constants (KI) were then derived by using the Cheng–
Prusoff equation, as follows: Ki ¼ IC50/(1 + [S]/Km) where [S]
represents the CO2 concentration at which the measurement was
carried out, and Km the concentration of substrate at which the
enzyme activity is at half maximal. All enzymes used were
recombinant, produced in E. coli as reported earlier. The
concentrations of enzymes used in the assay were: hCA I,
10.1 nM; hCA II, 8.4 nM; hCA IX, 7.9 nM and hCA XII,
The synthesis of the multivalent xanthates started with the
preparation of the multivalent alkyne-derivatized scaffolds that
would be later functionalized to finally provide the desired
xanthates clusters. As shown in Scheme 2, the three scaffolds
were obtained in one step from commercially available starting
materials in good to very good yields in comparison to previously
12.5 nM52,53
.
Results
reported reactions in bibliography54–56
.
Chemistry
The synthesis of the multivalent xanthates from the formerly
produced scaffolds was achieved in two steps, as depicted in
Scheme 3, since the direct click of the azidated xanthates 2 and 6
was not feasible. Indeed, xanthates strongly coordinates Cu, thus a
The preparation of the monovalent analogues, as shown in
Scheme 1, started from commercially available 3-chloropropan-1-
ol and 2-(2-chloroethoxy)ethan-1-ol and followed parallel
Scheme 1. Synthesis of the monovalent
analogues. Reagents and conditions:
(a) NaN3, H2O, 18 h, 80 ꢁC (1: from
3-chloropropan-1-ol, 95%; 5: from 2-(2-
chloroethoxy)ethan-1-ol, 99%); (b) CS2,
KOH, Et2O, 3 h, r.t. (2: from 1, 82%; 6: from
5, 83%); (c) 3-methoxyprop-1-yne, CuSO4/
NaAsc, THF/H2O, o/n, r.t. (3: from 2, 99%;
7: from 6, 99%); (d) CS2, KOH, DMSO/H2O
10:1, 2 h, r.t. (4: from 3, 84%; 8: from 7,
77%).
Scheme 2. Synthesis of the alkyne-deriva-
tized multivalent scaffolds S2, S4 and S6.

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M. Abellan-Flos et al.
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J Enzyme Inhib Med Chem, Early Online: 1–7
Scheme 3. Synthesis of the multivalent
xanthates. Reagents and conditions: (a) 1 or
5, CuSO4/NaAsc, 1,4-dioxane/H2O, o/n, r.t.
or 2 h at 80 ꢁC (9: from 1, 70%; 10: from 5,
67%; 11: from 1, 96%; 12: from 5, 79%; 13:
from 1, 90%; 14: from 5, 90%); (b) CS2,
KOH, DMSO/H2O, 2 h, r.t. (15: from 9, 90%;
16: from 10, 89%; 17: from 11, 97%;
18: from 12, 71%; 19: from 14, 74%;
20: from 14, 83%).
high excess of Cu complex must be added to catalyze the click side-products were always produced and were found impossible to
reaction but it cannot be fully removed afterwards. Therefore, the be separated by selective precipitation. Based on these observa-
first step was straightforward and involved the grafting of the two tions, we eventually developed a satisfactory procedure which
different hydroxyl azidated-linkers 1 and 5 through CuAAC included an excess of CS2, since it can be removed by
reaction. The click reaction was either realized overnight at r.t. evaporation, and one equivalent of KOH per hydroxyl groups.
or under microwave irradiation at 80 ꢁC for 2 h with similar Importantly, the KOH was finely crushed before the reaction and
outcomes. After purification of the products by simple a few drops of water were added to the reactant mixture at the
co-precipitation and removal of the residual Cu salts by beginning of the reaction. After 2 h at r.t., the reactions were
scavenging (QuadrasilMPÕ, Sigma Aldrich, St. Louis, MO), the complete and the products able to be selectively precipitated with
xanthates were prepared with the optimized conditions in very THF. The precipitates were washed three times with THF. To
good to excellent yields, considering the number of reactions per remove the last impurities and the traces of DMSO, the
molecule.
precipitates were dissolved in MeOH and rapidly precipitated
In order to develop an efficient methodology for the prepar- again with Et2O. The products were extensively dried under high
ation, isolation and characterization of the desired multivalent vacuum to remove any traces of solvent which could lead to
xanthates, an intensive optimization of the procedure was degradation. The characterization of all the molecules was carried
1
required. It is worth to note that, as commented above, xanthates out unambiguously by H NMR, 13C NMR and HRMS.
are not very stable molecules; they are known to be sensitive to
acidic pH, high temperature, humidity and time57. The degrad-
CA Inhibition assays
ation process occurs through complex radical reactions giving the
The monovalent compounds 1–8 and the multivalent compounds
9–20 were assayed in vitro for inhibition against the four
physiologically relevant CA isoforms, the cytosolic hCA I
and II as well as the transmembrane, tumor-associated hCA IX
and XII. The results for the compounds which presented some
inhibition are shown in Table 1. Acetazolamide (5-acetamido-
1,3,4-thiadiazole-2-sulfonamide) was used as a standard in the
assay49.
Among the monovalent compounds, the alcohols 1, 3, 5 and 7
did not show any inhibitory effects against the four CA isoforms,
as expected. The xanthates 2, 4, 6 and 8 on the other hand showed
inhibitory effects (Table 1). The isoform more sensitive to these
compounds was hCA I, for which the monovalent xanthates
showed KIs ranging between 7.5 and 51.7 mM. The organic
scaffold did not show much influence in the case of xanthates 2, 6
starting free hydroxyl and CS2 as products, among others.
Contrarily to the synthesis of the monovalent analogues, herein
it was not intended that the xanthate products precipitated during
the course of the reaction. Precipitation would provide a mixture
of partially functionalized polyols. Therefore, after screening of
solvents, DMSO was eventually selected as the preferred solvent
because it can solubilize the final compounds, thus avoiding
premature precipitation of partially functionalized polyols.
The completion of the reaction could be followed by 1H NMR
in DMSO-d6 thanks to the chemical shifts of the peak of the CH2
next to the hydroxyl groups (d & 3.60 ppm) which was converted
into -OCS2K (d & 4.30 ppm). For the first tests, 1 eq/-OH of KOH
and CS2 were added, but the reactions were very slow and not
complete after several days. When an excess of both KOH
and CS2 was used, the reactions were complete in 2 h, but some

DOI: 10.3109/14756366.2015.1072177
Multimeric xanthates as CA inhibitors
5
Table 1. Inhibition constants (KIs) by a stopped flow CO2 hydrase assay, with acetazolamide AAZ as standard drug48
.
Compound
Valency
(xanthate)
KI (mM)a
hCA II
hCA IX
hCA XII
hCA I
rpb
rp/nc
2
4
6
8
1
1
1
1
–
4
4
6
6
–
7.5
51.7
7.9
–
–
–
–
–
0.6
–
29
14
0.25
–
–
–
–
–
–
–
4.8
2.3
4100
4100
4100
4100
4100
4100
4100
68.9
4100
4100
4100
9.9
4100
4100
8.1
10.0
39.0
0.025
9.4
4100
93.9
4100
15.9
4100
4100
4100
4100
0.006
7.5
11
14
16
18
20
AAZ
70.8
82.9
4100
1.80
0.53
66.1
0.012
Bold numbers are the values for the compounds with a Ki5100 mM.
aMean from three different assays. Errors were in the range of 10% of the reported values (data not shown).
bRelative potency calculated as the ratio of KI values of the monovalent controls (4 or 8) to the multivalent compounds.
cCorrected relative potency (rp/valency) calculated as the ratio of rp to the valency of the multivalent compounds.
and 8, which had very similar inhibition constants whereas 4, available commercial materials, all the molecules, even the
which incorporates the substituted triazole moiety (present also in complex multimeric xanthates, were produced in three or less
1
8) but a shorter linker between the ring and the ZBG (compared steps. The characterization was performed unambiguously by H
to 8), was an order a magnitude a weaker CAI compared to the NMR, 13C NMR and HRMS.
other monovalent xanthates prepared here. hCA II was not at all
Some of the multivalent compounds, containing three, four,
inhibited by these xanthates (and alcohols) up to 100 mM and six xanthate moieties showed interesting CA inhibitory
concentrations in the assay system, and the same situation was activity, mainly against hCA I, an isoform whose physiologic
observed for the inhibition of hCA IX, except for 8, which was function is still poorly understood. Together with their monova-
a low micromolar inhibitor (KI of 9.9 mM). Against hCA XII lent analogues, some of the multimers were significantly stronger
xanthates 2 and 6 showed some degree of inhibition (KIs of 9.4 – inhibitors than the monomeric species although no strong
93.9 mM).
‘‘multivalent effect’’ was found in general (as compared to
For the multivalent compounds 9–20 reported here, the carbohydrate-lectin interactions). All four CA isoforms on which
inhibition data were again more interesting against the slow we tested the xanthates have been crystallized alone or in adduct
cytosolic isoform hCA I. Molecules 9, 10, 12, 13, 15, 17 and 19 with various classes of inhibitors. The two cytosolic isoforms
did not show any inhibitory effect. Against isoform hCA I, the hCA I and II are monomers whereas the two transmembrane ones
alcohol 11 and the xanthate 14 were weak micromolar inhibitors are dimers with two accessible active sites. However, as shown
(KIs of 70.8 – 82.9 mM). On the other hand, the polyxanthates from the data in the Table 1, both monomeric as well as dimeric
derived from S6, 18 and 20, showed low-micromolar or isoforms were inhibited in a rather similar manner by the
submicromolar inhibitory action (KIs of 0.53 – 1.80 mM). Thus, multivalent derivatives reported in the paper. The best hCA I
in this case a very interesting multivalent effect is observed for the inhibitors in the reported series had inhibition constants of 0.53 –
inhibition of hCA I, since the corresponding monomeric com- 1.80 mM, and they were two orders of magnitude less efficient
pounds were much less effective enzyme inhibitors compared to against the physiologically dominant cytosolic isoform hCA II.
the multivalent xanthates. Indeed, the relative potency values of Thus, an interesting class of hCA I-selective inhibitors was
18 and 20 were found to be 29 and 14, respectively (Table 1). detected in this work. Nonetheless, the biological assays
Taking into account the valency of these hexameric species, the demonstrated that the multimeric presentation of xanthates,
enhancement of the affinity per xanthate unit corresponds to 4.8 indeed affected the selectivity of the relative inhibition among
and 2.3, which is already significant for multivalent enzyme the four CAs assayed, and may open new directions in the
inhibitors. The same two xanthates, 18 and 20, were also research of isoform selective CAIs binding to the metal ion from
inhibiting hCA II, but orders of magnitude less efficiently the enzyme active site.
compared to hCA I (KIs of 66.1–68.9 mM). None of the other
multivalent derivatives possessed inhibitory activity against
this isoform. For hCA IX, three multivalent xanthates (16, 18
and 20) showed inhibition, with KIs ranging between 8.1 and
Acknowledgements
We are grateful to Dr. Abdellatif Tikad for technical support.
39.0 mM. Interestingly, hCA XII was not inhibited by the
multivalent xanthates, and only the alcohol 11 showed a weak
inhibitory action.
Declaration of interest
The authors report no conflict of interest. We thank the People
Programme (Marie Curie Actions) of the European Union’s Seventh
Framework Programme ‘‘DyNano’’ FP7/2007–2013/under REA grant
agreement no. 289033 for the financial support.
Discussion and conclusions
We designed monovalent alcohols/xanthates 1–8 and three series
of multivalent derivatives 9–20 in order to investigate multivalent
inhibition of CA isoforms involved in crucial physiologic and
pathologic processes. An optimized procedure for the synthesis of
multivalent xanthates was developed providing all the desired
molecules in very good to excellent yields. Starting from readily
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Supplementary material available online
Supplementary Information