(M=Re or 99mTc) arylsulfonamide, arylsulfamide, and arylsulfamate conjugates for selective targeting of human carbonic anhydrase IX

Article abstract of DOI:10.1002/anie.201107333

Enhanced receptor selectivity: Carbonic anhydrase inhibitors are relevant for both cancer diagnosis and therapy. Combining non-radioactive Re compounds with their radioactive ^99mTc homologs enables the use of identical molecules for therapy and imaging (theragnostic). The syntheses and in vitro evaluation of [(Cp-R)M(CO)₃] (Cp=cyclopentadienyl, M=Re, ^99mTc) with R being a highly potent carbonic-anhydrase-targeting vector is reported (see picture). Copyright

Full text of DOI:10.1002/anie.201107333

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DOI: 10.1002/anie.201107333
Bio-organometallic Chemistry
[(Cp-R)M(CO)3] (M = Re or 99mTc) Arylsulfonamide, Arylsulfamide,
and Arylsulfamate Conjugates for Selective Targeting of Human
Carbonic Anhydrase IX**
Daniel Can, Bernhard Spingler, Paul Schmutz, Filipa Mendes, Paula Raposinho,
Cꢀlia Fernandes, Fabrizio Carta, Alessio Innocenti, Isabel Santos, Claudiu T. Supuran, and
Roger Alberto*
Diagnosis and treatment of diseases requires molecules
designed for targeting specific receptors. Since nature has
developed a realm of functionally and structurally similar
receptors, selectivity for a specific target is a key criterion for
inhibitors.[1] Inhibitors are typically organic but Meggers et al.
showed that high selectivity does not only depend on
intermolecular interactions but also on a directed 3D arrange-
ment of different functionalities as exemplified with organo-
metallic protein kinase inhibitors.[2–4] Inert complexes offer
the opportunity to populate biologically relevant space. Thus,
bio-organometallic complexes are versatile chemical
probes[2,5–8] as pioneered by for example, Jaouen and co-
workers who developed ferrocene-based, selective estrogen
receptor inhibitors.[9–11] Replacing a phenyl ring in for
example, tamoxifen by [(Cp-R)Re(CO)3] (CP = cyclopenta-
dienyl) resulted in retention of high affinity for the estrogen
receptor.[12] These organometallic compounds have therapeu-
tic potential but their use for in vivo diagnosis is limited since
radionuclides such as 18F for PET (positron emission tomog-
raphy) cannot be introduced without alteration of chemical
authenticity.
We recently introduced a strategy for the aqueous syntheses
of [(Cp-R)99mTc(CO)3] (“R” = target-specific moiety). In this
report, we present Re bio-organometallic carbonic anhydrase
inhibitors (CAI) with nanomolar affinities for specific CA
subtypes. The 99mTc complexes have been prepared and
complement the Re congeners as identical, diagnostic agents.
Carbonic anhydrases (CAs) are Zn enzymes catalyzing
the formation of carbonic acid from CO2 and water.[18] In
mammals, 16 different membrane-bound, cytosolic or mito-
chondrial isozymes are known.[18,19] CAs are attractive from
a pharmaceutical point of view because of hypoxia-induced
overexpression of hCA IX and hCA XII (hCA = human
carbonic anhydrase) in many malignancies, including
cancer.[18,20–23] Therefore, CAs are targets for cancer diagnosis
and therapy. However, the large number of isozymes and
diffuse localization impede selective accumulation of inhib-
itors.[19] Several fluorescent sulfonamide- or sulfamate-based
CAIs, radio-iodinated monoclonal antibodies[24–27] but only
one 99mTc-labeled inhibitor have been reported.[28]
Following the concept of extended 3D space population,
we synthesized four new arylsulfonamide, -sulfamide, and
-sulfamate based CAIs with the [(Cp-R)M(CO)3]-motif (M =
Re or 99mTc) and evaluated their affinity to CA isoforms
(Scheme 1). Compounds 2 and 3 were structurally character-
ized (see the Supporting Information).
Binding studies of complexes 1–4 with 12 CA isozymes
showed inhibition constants in the low nanomolar range for
some of the isoforms (Table 1). Such low Ki values are
uncommonly rare for bio-organometallic compounds.[30] The
hCA II, VI, VII, IX, XII, and XIII are considered as
sulfonamide- and sulfamate-avid isoforms[18] which is consis-
Given that Cp-based complexes can replace phenyl rings
without affecting the bioactivity, identical compounds for
combined therapy and noninvasive diagnosis are desirable for
theragnostics.[13,14] Rhenium and technetium belong to the
same triad. Whereas Re-based compounds can be used for
therapy, their 99mTc homologous serve as imaging agents in
single photon emission computed tomography (SPECT).[15–17]
[*] D. Can, Priv.-Doz. Dr. B. Spingler, Dr. P. Schmutz,
Prof. Dr. R. Alberto
Institute of Inorganic Chemistry, University of Zurich
Winterthurerstrasse 190, 8057 Zurich (Switzerland)
E-mail: ariel@aci.uzh.ch
Dr. F. Mendes, Dr. P. Raposinho, Dr. C. Fernandes,
Prof. Dr. I. Santos
Instituto Tecnolꢀgico e Nuclear
Estrada Nacional 10, 2686-953 Sacavꢁm (Portugal)
F. Carta, A. Innocenti, Prof. Dr. C. T. Supuran
Universita degli Studi di Firenze, Polo Scientifico
Laboratorio di ChimiCABioinorganica
50019 Sesto Fiorentino, Florence (Italy)
[**] We thank the University of Zurich and the SLS for providing
beamtime. Work by the Supuran lab was financed by an EU FP7
grant (Metoxia).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201107333.
Scheme 1. Structures of cyclopentadienyl-based CAIs.
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Table 1: Inhibition data of complexes 1–4 against selected CA isozymes
in comparison with acetazolamide (AZA).
pressed CAs leads to a remarkable extracellular pH drop in
tumor tissue (to around 6 as compared to 7.4 in normal
tissues).[18,28,31,32] Consequently, strong CA inhibition will
reduce acidification. To confirm this hypothesis, hCA IX-
induced extracellular acidification was quantified before and
after exposure to the inhibitors 3 and 4. The three different
human carcinoma cell models cervical adenocarcinoma
HeLa, breast adenocarcinoma MDA MB 231 and colon
adenocarcinoma HT29 were studied. This panel of cell lines
was assessed by western blot for the levels of hCA IX
expression under normoxic and hypoxic conditions
(Figure 2). The HeLa and HT29 carcinoma cells showed
Compound
Ki [nm][a]
1
2
3
4
AZA
CAI
CAII
39
15.1
2570
4590
35.5
41.1
124
104
22.1
21.3
5.2
2775
250
12
74
63
54
11
2.5
25
5.7
27.4
32.9
113
105
10.6
7.6
3.7
4.5
25.3
33.8
109
102
14.5
10.1
7
4.4
CAIV
CAVA
CAVB
CAVI
CAVII
CAIX
CAXII
CAXIII
CAXIV
CAXV
467
481
305
497
360
43
6.7
482
20.1
313
6.9
78.5
7.9
62.1
4.1
28.4
56.8
5.4
12.5
17
41
72
36.8
[a] The data was determined by the CA-catalyzed hydration of CO2.
tent with the observed values for inhibitors 1–4. Our Ki values
for the various isoforms compare well to those of the
acetazolamide (AZA) standard and are for some isoforms
even better. In addition, distinct selectivity for hCA IX, XII,
and XIVover the other isoforms was observed (Figure 1). The
Ki ratios (hCA II/hCA IX) of 4.0 (for 4) or 7.0, respectively
Figure 2. Western blot analysis of hCA IX expression in normoxic N
(21% O2, 24 h) and hypoxic H (1% O2, 24 h) MDA MB 231, HeLa,
and HT29 carcinoma cells using a polyclonal hCA IX antibody. Actin
was used as loading control.
elevated levels of hCA IX under hypoxic conditions while
MDA MB 231 did not. Only HT29 cells expressed hCA IX
under normoxic conditions, albeit at a lower amount than
under reduced oxygen levels. This basal expression in HT29
cells is not surprising, and might be caused by the cluster-
shaped growth of these cells.
To minimize potential cytotoxicity, acidification experi-
ments were performed at a 0.5 mm of the Re complexes 3 and
4 (Figure 3). Hypoxic incubation of HeLa and HT29 cells led
Figure 1. Graphical representation of Ki values of compounds 1 and 2
for selected isozymes of hCAs.
(for 2 and 3), indicate a clear preference for hCA IX,
overexpressed in certain tumors, over the physiologically
dominant hCA II. Compound 1 showed the most pronounced
selectivity pattern (solid line in Figure 1) and strongly favors
hCA II, IX, and XIV. Compounds 3 and 4 behave similarly to
2. Data are shown in the Supporting Information. In contrast,
Ki values of the AZA standard do not show such a distinct
preference pattern for any of the isoforms. It has only
a slightly higher affinity for hCA II over hCA IX (hCA II/
hCA IX = 0.5).
Ki values for 1–4 are, thus, in the same range as found for
organic inhibitors[18] but much better than those reported for
the aforementioned organometallic CA inhibitors,[28,29] indi-
cating smaller and more compact [(Cp-R)M(CO)3] (M = Re
or 99mTc) to be favored over bulky complexes.
CAs are amongst the most effective catalysts in biological
systems (kcat/KM > 108 mÀ1).[19] Due to increased carbonic acid
formation, the high activity of hypoxia-induced, overex-
Figure 3. Effect of the reference sulfonamides and compounds 3 and 4
on the reduction of extracellular acidification in HeLa and HT29 cell
lines. Cells were incubated for 24 h. The data show the mean Æ
standard deviation of 4 replicates and are plotted as difference
between pHe values [DpH=pH(after incubation)ÀpH(before incuba-
tion)] measured in the absence (control) or in the presence of the
sulfonamides.
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to the expected extracellular acidification by around 0.5 to 0.7
pH units, caused by overexpression of hCA IX. Under
normoxic conditions, a pH drop of only about 0.1 for HeLa
(no overexpression) and 0.2 for HT29 (slight expression) was
found.
The presence of the reference inhibitors mafenide and
sulfanilamide under hypoxia caused a significant reduction of
acidification; more pronounced for HeLa than for HT29 cells
(Figure 3A compare with 3C). Complexes 3 and 4 showed
a distinct reduction of extracellular acidification, in hypoxia
comparable or even better than the standards, confirming
CA IX inhibition. The normoxic extracellular pH was not
significantly affected by incubation with either compound,
indicating specific interaction with hCA IX. In agreement
with the lack of hCA IX expression, MDA MB 231 cells did
not show a pH difference for cells grown under normoxia or
hypoxia.
major conformation of the [(Cp)Re(CO)3] unit, including the
amide linker, does not interact with either the protein or
water molecules. However, there is a hydrophobic interaction
between the [(Cp)Re(CO)3] moiety and the hydrophobic part
of Phe131, Leu198, and Pro202. Further details are given in
the Supporting Information.
Compounds 1–4 are strong organometallic CAIs. They
follow the concept of increased and matched space occupa-
tion in the binding pocket.[3] To extend the concept from
therapy to combined therapy and imaging, the preparation of
the 99mTc homologues from water is mandatory.[6] [(Cp-
R)99mTc(CO)3] type complexes are accessible from (HCp-
R)2 dimers and [99mTcO4]À.[36–38] For extending this approach,
we prepared the 99mTc analogs of 3 and 4, namely. Their
authenticity was assessed by comparing the HPLC retention
times of 3/6 and 4/8 (Figure 5). The “one pot” reactions of
[
99mTcO4]À with ligands 5 and 7 (millimolar concentrations)
To assess the binding mode of the Re-based CAIs with
proteins, crystals of hCA II were grown with the hanging drop
vapor diffusion method. The crystals were soaked with the
respective inhibitor and gave diffraction data until 1.2 ꢀ
resolution. The electron density of the complete complex 4
could be traced in the binding pocket of hCA II. An electron
density map is shown in Figure 4A. The binding mode is
reminiscent to the arrangement as seen in other hCA II
structures.[33,34] Avvaru et al. defined two binding pockets I
and II for elongate hCA II inhibitors.[35] Complex 4 binds in
the hydrophobic pocket II (see the Supporting Information).
The deprotonated nitrogen of the arylsulfonamide terminus
coordinates to the Zn atom in the active site (Figure 4B). The
Figure 5. i) Isolink Kit, TcO4À. Superposition of HPLC traces. Top: UV
trace showing ligands 5 and 7 and Re complexes 3 and 4, respectively.
Bottom: g trace of the 99mTc complexes 6 and 8.
gave nearly quantitative yields at 908C after 60–90 min. Based
on these 99mTc syntheses, macroscopic amounts of the Re
complexes 1–4 for therapy can be combined with homologous
99mTc compounds for accompanying diagnosis. It should be
emphasized that the phenyl–[(Cp-R)M(CO)3] analogy is not
limited to CAIs but can be extended to any pharmacophore
with a corresponding structural subunit.
In conclusion, four new piano-stool-type Re complexes
with pendent arylsulfonamides, -sulfamides, and -sulfamates
were synthesized. They inhibit CAs with nanomolar affinities.
Particularly strong inhibition was found for pharmaceutically
relevant isozymes hCA IX and hCA XII. Compound 1
showed superior CA isoform selectivity than the AZA
standard. Enhanced receptor selectivity because of better
space occupation is a preponderance of bio-organometallic
compounds. These CAIs are potential therapeutics for CA
overexpression related diseases. For imaging purposes, the
99mTc homologues of 3 and 4 were synthesized. Inert bio-
organometallic 99mTc and Re compounds behave biologically
identical, thus, these potent CAIs can be applied in combi-
nation with each other, macroscopic amounts of Re for
Figure 4. A) Crystal structure of 4 bound to hCA II (only the major
conformation of 4 is shown). A) Electron density map of 4, super-
imposed with the stick model of the inhibitor bound to the active site
of hCA II. Hydrophobic interactions can be observed between the
[(Cp)Re(CO)3] moiety and Phe131, Leu198, and Pro202. B) Detail of
the binding cavity of hCA II with 4. The Zn atom is shown as a green
sphere.
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Received: October 18, 2011
Revised: January 24, 2012
Published online: February 17, 2012
Keywords: bio-organometallic chemistry ·
.
carbonic anhydrase inhibitors · radiopharmaceuticals ·
sulfonamides · technetium
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