Carborane-based carbonic anhydrase inhibitors

Article abstract of DOI:10.1002/anie.201307583

CA inhibitors: Human carbonic anhydrases (CAs) are diagnostic and therapeutic targets. Various carborane cages are shown to act as active-site-directed inhibitors, and substitution with a sulfamide group and other substituents leads to compounds with high selectivity towards the cancer-specific isozyme IX. Crystal structures of the carboranes in the active site provide information that can be applied to the structure-based design of specific inhibitors. Copyright

Full text of DOI:10.1002/anie.201307583

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DOI: 10.1002/anie.201307583
Enzyme inhibition
Carborane-Based Carbonic Anhydrase Inhibitors**
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´
Jirꢀ Brynda, Pavel Mader, Vꢁclav Sꢀcha, Milan Fꢁbry, Kristyna Poncovꢁ, Mario Bakardiev,
ˇ
ˇ
Bohumꢀr Grꢂner, Petr Cꢀgler, and Pavlꢀna Rezꢁcovꢁ*
Human carbonic anhydrases (CAs) are zinc metalloenzymes
that play an important role in many physiological processes.
To date, 15 human CA isozymes with different subcellular
localization and tissue expression profiles have been identi-
fied. Vast experimental evidence also suggests the involve-
ment of CAs in various pathological processes (e.g., tumor-
igenicity, obesity, and epilepsy). Many CA isozymes are thus
recognized as diagnostic and therapeutic targets.[1]
About 30 CA inhibitors are used clinically, for example, as
anti-glaucoma drugs (targeting CAII, CAIV, and CAXII),
anti-convulsants (targeting CAII, CAVII, and CAXIV) and
anti-obesity agents (targeting CAVA and CAVB).[2] Recently,
other isozymes, namely the neuronal CAVII and CAXIV, and
the cancer-associated forms CAIX and CAXII, have been
validated as targets for inhibitor development.[3]
The traditional CA inhibitors contain a sulfonamide or
sulfamide moiety that coordinates the zinc cation located in
the CA catalytic site.[4] Most of the currently used CA
inhibitors lack selectivity, and their use causes numerous
unwanted side effects. A current challenge is the design of
compounds that can inhibit specific isozymes. Although the
conical active-site clefts of different human CA isoezymes are
conserved, variations exist in the amino acid residues at the
entrance to the active site. As a result of their differing in
shape and hydrophobicity, these surface pockets can be
exploited to design specific inhibitors.[5,6]
effectively based on three-dimensional scaffolds rather than
flat structures.
Carboranes, icosahedral clusters containing boron,
carbon, and hydrogen are bulky pharmacophores used to
replace various hydrophobic structures in biologically active
molecules.[8,9] The 12-vertex carboranes increase the in vivo
stability and bioavailability of biologically active molecules
and enhance the hydrophobic interactions between them and
their receptors.[10] They are an abiotic species that are very
stable towards catabolism and degradation by enzymes and
thus the use of boron clusters as components of new
pharmacological agents has been increasing.[11–15]
With the help of manual molecular docking into the active
site of CAII, we designed 1a, which contains a sulfamide
group connected to a carborane cluster intended to optimally
fill the enzyme active site. The length of the linker between
the sulfamide group and carborane cluster was chosen based
on comparison with the structures of isoquinoline sulfon-
amide inhibitors.[7] Attachment of the sulfamide moiety was
accomplished by using a transamination reaction between
aminomethylcarborane 1b and sulfamide (Scheme 1).
Structural analysis of CAII in complex with numerous
inhibitors revealed two general binding modes, each involving
a distinct site within the enzyme active site cavity.[7] This led us
to hypothesize that CA inhibitors could be designed more
Scheme 1. Preparation of 1a by heating 1b with sulfamide in dioxane.
Vertices represent BH, black spheres CH or C if substituted.
ˇ
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[*] Dr. J. Brynda, Dr. P. Mader, Dr. M. Fꢀbry, Dr. P. Rezꢀcovꢀ
Compound 1a showed inhibitory activity toward CAII
(Ki value of 0.7 mm) and showed almost 2-fold higher activity
toward the tumor-associated isoform CAIX (Ki of 0.38 mm).
The crystal structure of 1a in complex with CAII
determined at 1.35 ꢀ resolution (PDB code 4 MDG) con-
firmed that the inhibitor binds in the enzyme active site as
predicted (Figure 1) and revealed key interactions responsi-
ble for inhibitor binding and enzyme inhibition.
The sulfamide moiety of 1a proved to be the anchoring
group that completes the coordination sphere of Zn2+ in the
active site and makes the polar interactions with Thr199 that
are typical of other CA inhibitors.[4] An additional polar
interaction is a hydrogen bond between a linker NH group
and the side chain Og of Thr200 (Figure 1a). Further
interactions of the inhibitor with the active site cavity are
mediated through van der Waals interactions between the
carborane cluster and amino acid residues Gln92, His94,
Phe131, Leu198, and Thr200. For a complete list of inter-
actions, see Table S1 in the Supporting Information. Com-
pound 1a fills the proximal active site cavity of CAII, but
Institute of Molecular Genetics
Academy of Sciences of the Czech Republic, v.v.i.
Vꢁdenskꢀ 1083, 142 20 Prague 4 (Czech Republic)
ˇ
E-mail: rezacova@uochb.cas.cz
ˇ
ˇ
Dr. J. Brynda, K. Poncovꢀ, Dr. P. Cꢁgler, Dr. P. Rezꢀcovꢀ
Institute of Organic Chemistry and Biochemistry
Academy of Sciences of the Czech Republic, v.v.i.
Flemingovo nꢀm. 2, 16610 Prague 6 (Czech Republic)
E-mail: cigler@uochb.cas.cz
ˇ
Dr. V. Sꢁcha, Dr. M. Bakardiev, Dr. B. Grꢂner
Institute of Inorganic Chemistry
Academy of Sciences of the Czech Republic, v.v.i.
ˇ
ˇ
250 68 Rez u Prahy (Czech Republic)
E-mail: gruner@iic.cas.cz
[**] This work was supported by Grant Agency of the Academy of
Sciences of the Czech Republic (project IAAX00320901), Technology
Agency of the Czech Republic (project TE01020028) and in part by
research projects RVO 68378050, 61388963, and 61388980 awarded
by the Academy of Sciences of the Czech Republic.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201307583.
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Figure 1. Crystal structure of CAII in complex with 1a. a) The protein is
shown as a ribbon diagram; residues involved in interactions with the
Zn2+ ion (gray sphere) and 1a are shown as stick representations.
C green, B pink, S yellow, O red, and N blue atoms are shown. Polar
interactions are represented by blue dashed lines; Zn2+ ion coordina-
tion is shown as black dashed lines. b) Top view into the active site,
shown as a surface representation. 1a is shown as a space filling
model.
some side pockets at the entrance of the active site could
potentially be targeted by cluster substituents (Figure 1b).
Next, we considered several modifications and prepared
a series of novel boron cage compounds combining various
carborane clusters with a polar sulfamide moiety (Figure 2).
Compounds 2a–10a were prepared in high yields using the
transamination reaction depicted in Scheme 1.
Our pilot series of ten CA inhibitors includes the parent
compounds 1a and 4a, which contain one alkyl sulfamide
group and icosahedral ortho- and meta-carborane units,
respectively. A structurally similar, charged derivative based
on the [CB11H12]À cage was also prepared (6a). Closo-
carboranes containing either hydrophobic (2a and 3a) or
hydrophilic (second sulfamide moiety; 5a) substituents and
their charged 11-vertex nido counterparts (7a–10a) complete
this structurally diverse series.
Figure 2. Structures of compounds used in this study. Amino groups
attached to the anionic clusters (6b–10b) are protonated, forming
zwitterions. 3 was prepared as an inseparable mixture of two isomers
(termed 3m and 3n). The substituent in 10b is located at boron B(10)
sitting in the open face and not at a carbon as in 7.
Table 1: In vitro inhibition of selected carbonic anhydrase isozymes.
The compounds were tested for inhibition of the human
CA isozymes CAII and CAIX using a colorimetric enzymatic
assay. Most of the compounds tested displayed good CA
inhibition profiles, with Ki values in the low micromolar or
submicromolar range (Table 1). Almost all of the compounds
showed selectivity for the cancer-specific CAIX isozyme over
the more abundant variant CAII.
Analysis of crystal structures of CAII in complex with 4a
and 7a (PDB codes 4MDL and 4MDM) confirmed that these
compounds are positioned similarly in the enzyme active site
to 1a. In fact, 4a binds to CAII identically to 1a in terms of
conformation and interactions (Figure 3a).
Compound
Ki (CAII)
[mm]
Ki (CAIX)
[mM]
Selectivity
Index[a]
1a
2a
0.70Æ0.14
348Æ113
0.51Æ0.08
1.16Æ0.24
0.38Æ0.14
8.57Æ2.24
6.79Æ2.01
9.00Æ2.23
2.71Æ0.47
132Æ19
0.38Æ0.11
161Æ37
1.8
2.2
1.2
1.0
1.7
3.9
1.3
3.9
18.1
5.1
3ma + 3na
0.43Æ0.17
1.12Æ0.20
0.23Æ0.04
2.20Æ0.48
5.09Æ2.38
2.32Æ1.02
0.15Æ0.06
26.04Æ2.93
4a
5a
6a
7a
8a
9a
10a
Compound 7a binds CAII differently and, unlike 1a and
4a, does not form a hydrogen bond between its linker NH
group and the side chain Og of Thr200 (Figure 3b). The
distance between the position of the linker NH group in 1a
and 7a is 0.18 ꢀ. The nido-carborane cluster interacts with
His64, Gln92, His94, Leu198, Thr200, and Pro201 (for
a complete list of interactions see Table S1 in Supporting
Information).
[a] Selectivity index is the ratio between Ki (CAII) and Ki (CAIX).
the Ki values obtained for 1a, 4a, and 7a. The o-carborane
derivative 1a has slightly better inhibitory potency toward
CA isozymes than the m-carborane derivative 4a. This could
result from the presence of a slightly acidic CH group in the
position vicinal to the substituted carbon in 1a. Compound 7a
(containing 7,8-nido-carborane) is a less efficient inhibitor of
CA isozymes than the closo-carborane derivatives; however,
it shows a slightly different binding mode. As revealed by the
In our pilot series of carborane-based CA inhibitors, we
exploited four different carborane cluster types. The effect of
cluster type on inhibition can be deduced from comparison of
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binding. In CAIX, this pocket contains Leu91 and Val131
and is more spacious.
In 5a, an additional sulfamide group was attached by
a methylene linker to the meta position of the meta-carborane
cluster. This increased the inhibitory potency compared to
that of the parent m-closo-carborane by a factor of approx-
imately 2. This increase can be attributed to the presence of
two sulfamide groups that can anchor the compound into the
catalytic site of the enzyme.
The effect of the linker connecting the cluster to the
sulfamide group can be deduced from comparison of the
inhibitory properties of 8a and 9a, and 7a and 10a; two pairs
of compounds with the same carborane cage but with linkers
differing in length and nature.
Compound 8a contains the methylene linker designed in
the initial docking. Increasing the length of the aliphatic
linker to a propylene moiety improved the inhibitory potency
of 9a and made it almost 5-fold more selective toward CAIX.
Increasing the length of the cluster-sulfamide linker by two
methylene groups probably allows accommodation of the
nido cluster with its phenyl substituent in side pockets at the
entrance of the enzyme active site (Figure S3 in the Support-
ing Information). With a CAIX/CAII selectivity index of 18.1,
Compound 9a showed the highest selectivity observed within
the entire series.
For the parental nido cluster, we therefore tested an even
longer, 7-atom linker (10a). This elongation, however, sub-
stantially decreased the inhibitory efficiency of the compound
compared to that of the cluster bearing a short methylene
linker (7a). A possible explanation for this decrease, based on
this compoundꢁs similarity to ionic metallacarboranes bearing
a similar linker, is that complexation with sodium cations may
lead to a rigid, crown-ether-like arrangement.[16] This would
decrease the compoundꢁs solubility and its ability to interact
with the catalytic cleft. Our observations suggest that both the
length and the nature of the cluster-to-sulfamide linker are
crucial for inhibitory efficacy and should be carefully tuned in
future design.
In conclusion, our results suggest that carborane-based
compounds are promising lead structures for the develop-
ment of inhibitors of CA isozymes. Our experiments demon-
strated that various types of hydrophobic, space-filling
carborane clusters can be accommodated in the CA active
site and that substitution with an appropriately attached
sulfamide group and other substituents leads to compounds
with high selectivity for the cancer-specific CAIX isozyme
over the widespread CAII isozyme. Crystal structures con-
firmed our hypothesis that three-dimensional scaffolds could
be efficiently used in CA inhibitors and provided structural
information that can be applied to the structure-based design
of specific CAIX inhibitors.
Figure 3. Crystal structure of CAII in complex with 4a (a) or 7a (b).
The protein is shown as ribbon diagram; residues involved in
interactions are shown as stick representations. Polar interactions are
represented by dashed blue lines; Zn2+ ion coordination is represented
by dashed black lines. The binding mode of 1a (black line) is
superimposed on the crystal structures.
co-crystal structure, the nido cluster 7a has the possibility to
adjust its position within the active site, which confers
a potential advantage in that it can accommodate additional
substituents that might affect selectivity toward a particular
CA isozyme.
To explore a greater diversity of carborane clusters, we
also prepared 6a, which contains a 1-carbadodecaborate ion
[CB11H12]À. The removal of one methylene group from the
linker and the presence of a negatively charged cluster led to
a decrease in efficiency but a substantial increase in selectivity
index compared to 1a, 4a, and 7a. This demonstrates that
a relatively subtle change in structure can strongly influence
inhibitor interactions with a particular CA isozyme.
In our initial molecular design, we hypothesized that
tuning compound selectivity to a certain CA isozyme could be
achieved by cluster substitutions. In our series, we exploited
a phenyl ring as a substituent in synthetically accessible
positions on the cluster. Analysis of the crystal structure of 1a
in complex with CAII suggested that a phenyl ring in the
ortho position on the closo-carborane cluster would have
steric clashes with amino acids lining the entrance to the
enzyme active site. Indeed, this detrimental effect on
inhibitory efficiency was illustrated by the 400-fold greater
Ki value obtained for inhibition of CAII with 2a. On the other
hand, the phenyl substituent placed in the meta-position, as in
3ma and 3na, seems not to experience any steric clash with
the bottom of the active site, and the inhibitory potencies
toward CAII and CAIX remain virtually unchanged, with
Ki values close to those of parent compound 1a.
The nido-carborane cluster was substituted with a phenyl
ring in the ortho position, leading to 8a. We did not observe
a loss of inhibitory potency as a result of the addition of
a phenyl ring in this position. Derivative 8a shows only
a slightly increased Ki value for CAIX, while the Ki value for
CAII is lower than that of parent compound 7a. The smaller
nido cluster is apparently able to adjust its position in the
active site cavity; the phenyl ring can thus be accommodated
in a side pocket. Inspection of the crystal structure of CAII in
complex with 7a suggests that a pocket formed by Ile91 and
Phe131 (Figure 1b) would be accessible for substituent
Experimental Section
Experimental procedures including detailed synthetic procedures for
the sulfamide derivatives and their precursors are described in the
Supporting Information. Briefly, the sulfamide compounds were
prepared from their respective amine or ammonium carborane
derivatives (1b–10b). Compounds 1b[17] and 6b[18] were prepared
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according to known procedures. 4b and 5b were prepared by
lithiation of 1,7-carborane, reaction with bromomethyl phthalimide,
and removal of the phtalimido group by hydrazine hydrate.[19–22]
Derivatives 2b and 3b were synthesized similarly, but the final
steps involved reduction with NaBH4 and subsequent hydrolysis.
Although this pathway has been described as failing for 1-amino-
methyl-o-carboranes,[20] we have demonstrated the feasibility of this
approach for compounds 2b and 3b bearing methylene connectors.
During synthesis of 3b, an equilibrium mixture of two isomers formed
after lithiation of 9-C6H5-1,2-C2B10H11 and subsequent reaction with
bromomethyl phthalimide as a result of the presence of two available
CH sites with similar reactivity. The isolated mixture of the 9- and 12-
phenyl isomers (in a nearly 1:1 ratio) could not be separated by liquid
chromatography either as phthalimido-protected amines or after
deprotection. This isomeric mixture was therefore used for the
synthesis of sulfamide derivatives 3ma and 3na. Derivatives 8b and
9b were obtained using hydrazine hydrate to convert 1-alkylphthal-
imido-2-phenyl-closo-1,2-carboranes into their respective 11-vertex
nido-[C2B9H12]À ion species, as described in a previous report.[17] For
the preparation of 7b, we used a new straightforward one-step
deboronation[8]/amination of 1-BrCH2-1,2-C2B10H11. The reaction
proceeds even with 24 % aqueous NH4OH, producing a mixture of
the target nido-derivative 7b along with two other boron-substituted
isomeric species (7-CH3-9-NH3-1,7-C2B9H10, 7-CH3-11-NH3-1,7-
C2B9H10) in lower yield (ca. 35%). Compound 7b could be isolated
in good yield (62%) by column chromatography. Compound 10b was
prepared by cleavage of the dioxane derivative 10-O(CH2CH2)2O-
C2B9H11 with ammonia, as described in a previous report.[23]
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Addition of the sulfamide end group was accomplished with high
yields by heating the respective amines with sulfamide in dioxane (see
Scheme 1). Potassium carbonate was used for the deprotonation of
the ammonium groups or to release the respective amines from their
hydrochlorides in situ. Nevertheless, the attempted synthesis of 1-
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Received: August 28, 2013
Published online: November 4, 2013
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Keywords: carbonic anhydrases · carboranes · drug discovery ·
inhibitors · structure elucidation
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