Carbonic anhydrase inhibitors: Gd(iii) complexes of DOTA- and TETA-sulfonamide conjugates targeting the tumor associated carbonic anhydrase isozymes IX and XII

Article abstract of DOI:10.1039/c0nj00214c

Gd(iii)-sulfonamide complexes incorporating macrocyclic rings of the DOTA/TETA type have been prepared and assayed for the inhibition of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). The cytosolic isoform, CA I, was poorly inhibited, whereas cytosolic CA II and transmembrane, tumor-associated CA IX and XII were inhibited in the low nanomolar range by the Gd(iii) complexes. Magnetic susceptibility and relaxivity measurements proved that the Gd(iii) complexes have comparable parameters to those of clinically used MRI contrast agents like Dotarem, Prohance and Omniscan in aqueous solution. Some Gd(iii) complexes were investigated for the inhibition of extracellular tumor acidification in two cell lines overexpressing CA IX, the colorectal HT-29 cell line and the cervical HeLa carcinoma cell line. In both tumor types, a slight but significant reduction of tumor acidosis was detected. Gd(iii)-sulfonamide conjugates may thus be of interest for developing imaging techniques or novel treatment strategies for the management of hypoxic tumors overexpressing extracellular CA isozymes such as CA IX and XII.

Full text of DOI:10.1039/c0nj00214c

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www.rsc.org/njc | New Journal of Chemistry
Carbonic anhydrase inhibitors: Gd(III) complexes of DOTA- and
TETA-sulfonamide conjugates targeting the tumor associated carbonic
anhydrase isozymes IX and XII
Marouan Rami,a Jean-Louis Montero,a Ludwig Dubois,b Philippe Lambin,b
Andrea Scozzafava,c Jean-Yves Winum*a and Claudiu T. Supuran*c
Received (in Montpellier, France) 22nd March 2010, Accepted 28th April 2010
DOI: 10.1039/c0nj00214c
Gd(III)-sulfonamide complexes incorporating macrocyclic rings of the DOTA/TETA type have
been prepared and assayed for the inhibition of the metalloenzyme carbonic anhydrase
(CA, EC 4.2.1.1). The cytosolic isoform, CA I, was poorly inhibited, whereas cytosolic CA II and
transmembrane, tumor-associated CA IX and XII were inhibited in the low nanomolar range by
the Gd(III) complexes. Magnetic susceptibility and relaxivity measurements proved that the
Gd(III) complexes have comparable parameters to those of clinically used MRI contrast agents like
Dotarem, Prohance and Omniscan in aqueous solution. Some Gd(III) complexes were investigated
for the inhibition of extracellular tumor acidification in two cell lines overexpressing CA IX, the
colorectal HT-29 cell line and the cervical HeLa carcinoma cell line. In both tumor types, a slight
but significant reduction of tumor acidosis was detected. Gd(III)-sulfonamide conjugates may thus
be of interest for developing imaging techniques or novel treatment strategies for the management
of hypoxic tumors overexpressing extracellular CA isozymes such as CA IX and XII.
biosynthetic reactions, which require bicarbonate as a substrate
(lipogenesis, gluconeogenesis and ureagenesis).1 Two of them,
Introduction
a-Carbonic anhydrases (CAs, EC 4.2.1.1) are widespread
CA IX and CA XII, are predominantly associated with and
metalloenzymes in higher vertebrates, including humans.1,2
overexpressed in many tumors, being involved in critical
16 isozymes have been characterized to date that differ in their
processes connected with cancer progression and responses
to therapy.1–3,11–14 CA IX is confined to a few normal tissues
sub-cellular localization, catalytic activity and susceptibility to
different classes of inhibitors. There are cytosolic isozymes
(stomach and body cavity lining) but it is ectopically induced
(CA I, CA II, CA III, CA VII and CA XIII), membrane bound
and highly overexpressed in many solid tumor types through
examples (CA IV, CA IX, CA XII and CA XIV), and
strong transcriptional activation by hypoxia, accomplished via
mitochondrial (CA VA and CA VB) and secreted (CA VI)
the hypoxia inducible factor-1 (HIF-1) transcription factor, as
isoforms.3–9 Three acatalytic forms, called CA-related proteins
shown schematically in Fig. 1.1,3–5,12–14
(CARPs) (CARP VIII, CARP X and CARP XI) are also
The expression of CA XII was detected in all tumor
known.1 In humans, CAs are present in a large variety of
categories, although the mean staining intensity was weaker
than for CA IX in all groups, except renal clear cell carcinomas.11
tissues, such as the gastrointestinal tract (GI), the reproductive
tract, the nervous system, kidneys, lungs, skin and
These two enzymes are multidomain proteins with the CA
eyes.1,2,6,10–12 Most CAs are very efficient catalysts for the
domain situated outside the cell and their CO2 hydrase
reversible hydration of carbon dioxide to bicarbonate and
catalytic activity being medium-to-high for CA XII and very
À
protons (CO2 + H2O " HCO3 + H+), which is the only
high for CA IX.8,15 The X-ray crystal structure of CA IX has
only recently been reported our group,15 allowing for
interesting drug design campaigns. Sulfonamides and their
isosteres (sulfamates and sulfamides) among others, constitute a
well-known class of CA inhibitors (CAIs), with some derivatives,
such as acetazolamide (AAZ) or ethoxzolamide (EZA), having
been used clinically for a long time for the management of CA
mis-balances different from cancer-related examples.1–7
physiological reaction in which they are involved.1
Many CA isoforms are involved in critical physiological
processes such as respiration and acid–base regulation,
electrolyte secretion, bone resorption, calcification and
a
´
Institut des Biomolecules Max Mousseron (IBMM), UMR 5247
CNRS-UM1-UM2, Baˆtiment de Recherche Max Mousseron,
´
Ecole Nationale Superieure de Chimie de Montpellier,
Many new inhibitors of both CA IX and XII have been
reported in recent years when connections between these CA
isozymes and hypoxic tumors began to emerge,1–10 with many
of these new generation inhibitors being specifically designed
for targeting tumor-associated CAs. Indeed, classical
CAIs, such as AAZ and EZA, indiscriminately inhibit all
CA isoforms,1 and this may constitute a disadvantage when
only tumor-associated examples must be targeted.
8 rue de l’Ecole Normale, 34296 Montpellier Cedex, France.
E-mail: jean-yves.winum@univ-montp2.fr
b Maastricht Radiation Oncology (MaastRO) Lab, GROW-School
for Oncology and Developmental Biology, University of Maastricht,
Maastricht, The Netherlands
c
`
Universita degli Studi di Firenze, Polo Scientifico, Laboratorio di
Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3,
50019 Sesto Fiorentino (Florence), Italy.
E-mail: claudiu.supuran@unifi.it
ꢀc
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agents decrease water signal as a consequence of the transfer
of saturated magnetization from the exchangeable protons of
the agent.24 Here, we report that Gd(III)-incorporating
conjugates of sulfonamides show a strong inhibition of the
tumor-associated CA isoforms CA IX and XII, decrease
tumor acidifications in cell cultures of HT-29 colorectal and
HeLa cervical cancer cell lines, and can also be used for MRI
purposes, making this class of conjugates of particular interest
for further studies.
Experimental
Synthesis
Sulfonamide ligands 1a–c and 2a–c were synthesized as
previously described by our group.25 A solution of Gd(III)
chloride hexahydrate (1.1 mmol) in de-ionized water (2 ml)
was added dropwise to a solution of ligand 1a–c (1 mmol) or
2a–c (1 mmol) dissolved in water (50 ml). The pH of the
aqueous solution was adjusted and maintained at pH 6.8 by
the constant addition of 2N NaOH. The solution was
vigorously stirred at room temperature and the formation of
the gadolinium complex monitored by ESI-MS. After 2 h,
the solution was concentrated to approximately 10 ml and
acetonitrile (50 ml) was added. The precipitate was filtered,
washed with water and ether, and dried over P2O5 to give the
expected gadolinium complexes (3a–c) or (4a–c) quantitatively
as a white solid.
Fig. 1 The mechanism of hypoxia-induced gene expression mediated
by the HIF transcription factor, leading to CA IX overexpression in
hypoxic tumors (HRE = hypoxia responsive element; PHD = prolyl
hydroxylase; VHL = von Hippel–Lindau protein).1
3a: mp > 205 1C, MS (ESI+/ESIÀ) m/z: 728.3 (M + H)+,
750.3, (M + Na)+, 726.2 (M À H)À; HRMS (M + H)+ calc.
for C23H34GdN6O9S 728.1349, found 728.11346; 3b:
mp > 205 1C, MS (ESI+/ESIÀ) m/z: 742.15 (M + H)+,
764.16, (M + Na)+, 740.27 (M À H)À; HRMS (M + H)+
calc. for C24H36GdN6O9S 742.1505, found 742.1508; 3c:
mp > 205 1C, MS (ESI+/ESIÀ) m/z: 714.25 (M + H)+,
736.21 (M + Na)+, 712.19 (M À H)À; HRMS (M + H)+
calc. for C22H32GdN6O9S 714.1192, found 714.1196; 4a:
mp > 205 1C, MS (ESI+/ESIÀ) m/z: 778.30 (M + Na)+,
778.21 (M + Na)+, 754.35 (M À H)À; HRMS (M + H)+
calc. for C25H38GdN6O9S 756.1662, found 756.1665; 4b:
mp > 205 1C, MS (ESI+/ESIÀ) m/z: 770.07 (M + H)+,
792.19 (M + Na)+, 768.12 (M À H)À; HRMS (M + H)+
calc. for C26H40GdN6O9S 770.1818, found 770.1816; 4c:
mp > 205 1C, MS (ESI+/ESIÀ) m/z: 742.21 (M + H)+,
764.11, (M + Na)+, 740.33 (M À H)À; HRMS (M + H)+
calc. for C24H36GdN6O9S 742.1505, found, 742.1507.
Recently our group reported some interesting CA IX
inhibitors (possessing fluoresceinthioureido moieties in their
molecules) that were critical for assessing the in vivo role of CA
IX/XII in tumor acidification processes.16–18 Such inhibitors
were shown to bind to CA IX only under hypoxia in vivo in cell
cultures or in animals bearing human tumors, followed by a
reduction of extracellular pH acidification in the tumor
tissues,16–18 properties which may be exploited for designing
diagnostic tools for the treatment or imaging of hypoxic
tumors.16–20 Indeed, we16–18 and Neri’s group19,20 recently
published proof of concept studies showing the possibility of
using CA IX/XII inhibitors both for the imaging and
treatment of hypoxic tumors.
Magnetic susceptibility measurements
Magnetic susceptibility data were collected using a Quantum
Design MPMS-XL SQUID magnetometer working in the
temperature range 1.8–350 K up to 5 T. The magnetic data
were corrected for the sample holder and the diamagnetism
contributions calculated from Pascal’s constants.26
Continuing our studies in the design of CA IX/XII inhibitors,
we report here conjugates of sulfonamides incorporating
macrocyclic rings that can bind gadolinium(III), a metal ion
possessing interesting properties for the magnetic resonance
imaging (MRI) of tumors and other diseases.21–23 Indeed,
paramagnetic lanthanide(III) complexes are used in two major
classes of MRI applications as contrast agents: the well-
established class of Gd-based agents and the emerging class
Relaxivity MRI protocols
For each compound, 10 samples were prepared with gadolinium
concentrations ranging from 0 to 1 mM in steps of 0.1 mM.
Measurements were performed on a 7 T Bruker Biospec 70/30
USR MRI system (Bruker Biospin GmbH, Ettlingen, Germany)
of chemical exchange saturation transfer (CEST) agents.24
A
Gd-based complex increases the water signal by enhancing the
longitudinal relaxation rate of water protons, whereas CEST
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interfaced to an AVANCE II console. A BGA12-S mini-imaging
gradient system (maximum gradient strength 720 mT mÀ1, slew
rate 6000 T mÀ1 sÀ1) was used, and images were acquired using a
7.2 cm inner diameter quadrature volume resonator.
non-linear least-squares methods using PRISM 3, as
reported earlier,25 and represent the mean from at least three
different determinations. KI is the equilibrium constant for the
dissociation of the E-I complex.
Longitudinal relaxation rates (R1 = 1/T1) were determined
using a series of inversion recovery measurements with
increasing inversion times of 200, 500, 750, 1000, 1500, 2000,
2500 and 3500 ms. Repetition and echo times were 7500 ms and
8.2 ms, respectively. Transverse relaxation rates (R2 = 1/T2)
were measured using a multi-slice, multi-echo spin echo
sequence, with a repetition time of 7500 ms, echo times
ranging from 10 to 120 ms and an echo interval of 10 ms.
For all images, 5 slices were recorded with a slice thickness of
1 mm and an interslice distance of 1.5 mm. The field-of-view
was 7 Â 7 cm2 and the acquisition matrix was 256 Â 256,
resulting in a spatial resolution of 0.27 Â 0.27 Â 1.0 mm3.
Regions of interest were drawn manually in MRIcro.27
All further data processing was performed in Matlab
(The MathWorks, Natick, MA). R1 values were determined
by non-linear curve fitting of the inversion recovery signal
intensity function (eqn 1):28
Cell culture and pH experiments
Exponentially growing colorectal (HT-29, ATCC HTB-38)
and cervical (HeLa, ATCC CCL-2) carcinoma cells were
cultured in Dulbecco’s modified Eagle’s medium supplemented
with 10% fetal bovine serum. Hypoxic conditions were
maintained in a hypoxic culture chamber (MACS VA500
micro-aerophilic workstation, Don Whitley Scientific, Shipley,
UK) consisting of an atmosphere of 0.2% O2, 5% CO2 and
residual N2. In parallel, normoxic dishes were incubated in air
with 5% CO2. The pH of the culture medium was immediately
measured at the end of each experiment and the data expressed
as delta pH (pH after incubation À pH before incubation).
The Gd compounds were dissolved in PBS containing 10%
DMSO at a 100 mM concentration and diluted in culture
medium to a final concentration of 1 mM just before adding to
the cells. The cells were incubated with the compounds for 24 h
during normoxia or hypoxia. In parallel, 4-aminoethylbenzene-
sulfonamide (S), a compound investigated earlier and shown to
decrease tumor acidification,16 was used as a positive control in
these experiments.
S = S0(1 À 2eÀT R + eÀT R
(1)
I
1
R 1
)
using the Levenberg–Marquardt optimization algorithm. S0 is
a scaling factor including proton density, pre-amplifier gain
and echo time. TI and TR are the inversion and repetition
times, respectively. R2 values were determined analogously by
non-linear fitting of the spin echo signal intensity function
(eqn 2):
Results and discussion
Chemistry
S = SeÀT R
(2)
Many sulfonamide/sulfamate/sulfamide CAIs have been
reported as inhibitors of both CA IX and XII in recent years,
in the search for derivatives with selectivity for tumor-
associated enzymes over other CAs involved in crucial
physiological processes.1–7 The compounds specifically
designed for targeting tumor-associated isoforms may be
classified as follows: (i) sulfonamides tagged with fluorescent
or other moieties (e.g., complexed metal ions, stable free
radicals, etc.), which make them useful for imaging
purposes;16–18 (ii) positively- or negatively-charged compounds
that cannot cross plasma membranes due to their charged
character and thus inhibit selectively only extracellular CAs,
among which are CA IX and XII;16,30 (iii) hypoxia-activatable
compounds that exploit the reducing conditions of hypoxic
tumors to convert an inactive prodrug into an active CAI;31
(iv) sugar-containing sulfonamides/sulfamates/sulfamides
that, due to their highly hydrophilic character, do not easily
cross membranes and thus possess an enhanced affinity for
extracellular CAs, such as CA IX and XII;32–34 (v) diverse
chemotypes other than the sulfonamides and their bioisosters,
such as phenols, coumarins and other compounds, have
recently been investigated as alternative CAIs to classical types
of inhibitor.35 Among the different approaches mentioned
above, we have observed that sulfonamides incorporating
macrocyclic rings of the DOTA and TETA type, as well as
their Cu(II) complexes, show excellent inhibitory activity and
selectivity for tumor-associated CAs over cytosolic isoforms
CA I and II.25 We thus decided to prepare the Gd(III)
derivatives of previously reported sulfonamides 1 and 2 in
E
2
Here, S0 is a scaling factor depending on proton density,
pre-amplifier gain and repetition time, and TE is the echo
time. The longitudinal relaxivity, r1, and transverse relaxivity,
r2, were given by the slope of a linear fit of R1 and R2 vs.
gadolinium concentration, respectively.
CA inhibition
An Applied Photophysics stopped-flow instrument was used
for assaying the CA-catalysed CO2 hydration activity.29
Phenol red (at a concentration of 0.2 mM) was used as an
indicator, working at an absorbance maximum of 557 nm with
20 mM HEPES (pH 7.5) as the buffer and 20 mM Na2SO4 (for
maintaining constant the ionic strength), to follow the initial
rates of the CA-catalyzed CO2 hydration reaction for a period
of 10–100 s. The CO2 concentrations ranged from 1.7 to
17 mM for the determination of the kinetic parameters and
inhibition constants. For each inhibitor, at least six traces of
the initial 5–10% of the reaction were used for determining the
initial velocity. The uncatalyzed rates were determined in the
same manner and subtracted from the total observed rates.
Stock solutions of inhibitor (0.1 mM) were prepared in
distilled and de-ionized water, and dilutions down to
0.01 nM were undertaken thereafter with further distilled
and de-ionized water. Inhibitor and enzyme solutions were
pre-incubated together for 15 min at room temperature
prior to assay in order to allow for the formation of the
E-I complex. The inhibition constant (KI) was obtained by
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Fig. 2 Magnetic susceptibility measurements for compounds 3b and 4b.
Table 1 Longitudinal (r1) and transverse (r2) relaxivities at 7 T and
20 1C for Gd(III) complexes 3b and 4b
À1
À1
r1/mMGd sÀ1
r2/mMGd sÀ1
3b
4b
3.6 Æ 0.2
3.2 Æ 0.1
12.7 Æ 1.1
21.8 Æ 0.9
Scheme 1 The synthesis of Gd complexes 3a–c and 4a–c.
order to investigate their interaction with these CA isoforms
(Scheme 1). This is the first detailed study of lanthanide-
containing CAIs; these compounds being the first conjugate
CAIs to be investigated as potential MRI agents.
20 1C. The relaxivities of both compounds are in line with the
known values of clinically-used MRI contrast agents like
Dotarem, Prohance and Omniscan in aqueous solution.22–24
They are typical of compounds incorporating this paramagnetic
ion, making these derivatives suitable for MRI purposes.24
The 12-membered ring DOTA and 14-membered ring
TETA, known to easily complex lanthanide(III) ions22–24 were
chosen as starting materials. These macrocycles also contain
four pendant arms substituting the four endocyclic nitrogen
atoms, three of which are of the methylcarboxylate type (for
effective binding of the Gd(III) ions) and one of which is
derivatized by means of aromatic sulfonamides, such as
homosulfanilamide (3a and 4a), 4-aminoethylbenzenesulfona-
mide (3b and 4b) or metanilamide (3c and 4c), which will
assure an interaction with the enzyme.1,10 Indeed, sulfonamides
in their deprotonated form complex Zn(II) ions from the CA
active site and usually bind with low nanomolar affinity to
many CA isoforms.1–10
Carbonic anhydrase inhibition
Inhibition data of four physiologically relevant CA isozymes,
the cytosolic, ubiquitous CA I and II (off-targets), and the
tumor-associated CA IX and XII, with compounds 1–4 are
shown in Table 2. The inhibition of standard sulfonamides
AAZ, EZA and S are also included in Table 2 for comparison.
The data for ligands 1 and 2 were reported earlier.25 The
following should be noted regarding CA inhibition with the
gadolinium complexes investigated here.
(i) The slow cytosolic isoform hCA I was not effectively
inhibited by Gd(III) complexes 3 and 4, which showed inhibition
constants in the range 380–7700 nM. This is a positive feature
for a compound which must show selectivity for the trans-
membrane isoforms and not inhibit too effectively the
cytosolic examples, such as CA I. It is, however, interesting
to note that the bulkier TETA derivatives were generally
better hCA I inhibitors compared to the corresponding DOTA
complexes. Furthermore, Gd(III) complexes 3 were generally
weaker inhibitors compared to corresponding ligands 1
(except 3a) for the DOTA derivatives, whereas they were more
inhibitory in the case of TETA complexes 4 (compared with
2). It is rather difficult to rationalize these data as no X-ray
crystal structures of the adducts of such compounds with any
CA isoform are known at present.
Gd(III) complexes 3 and 4 were prepared from the corres-
ponding macrocyclic ligands 1 and 2 by a reaction with Gd(III)
salts under controlled pH conditions, as reported in the
literature.21–24 Gd(III)-sulfonamide conjugates 3 and 4 were
characterized by standard procedures that established their
purity (of >99%) and stability in the conditions of the
experiments performed in vitro and ex vivo (see later in the
text). The absence of free gadolinium in the final compounds
was confirmed by the detection of free Gd(III) using a xylenol
orange indicator.23
Magnetic properties and susceptibility measurements
It was determined by means of magnetic measurements that
complexes 3b and 4b, as with all the Gd(III) derivatives
investigated so far,21–24 present
a
strong paramagnetic
(ii) The physiologically dominant cytosolic isoform hCA II
was, on the other hand, effectively inhibited by all complexes 3
and 4 (KI values of 2.0–17.7 nM), except 3c, which was a
medium potency inhibitor (KI of 110 nM, Table 2). In this
case, metal complexes 3 and 4 were much more effective hCA
II inhibitors compared to the corresponding ligands from
which they were obtained of type 1 and 2 (which showed
inhibition constants in the range 267–720 nM).25 Again, it is
difficult to rationalize these results without detailed X-ray
behavior (Fig. 2 and Table 1). The temperature dependencies
of wT and 1/c measured for these two compounds are shown
Fig. 2. For both compounds, the wT values at room temperature
were equal to 7.869 and 7.870 emu
K , which
molÀ1
corresponds to the calculated value for the one Gd3+ ion
(7.875 emu K molÀ1) per molecule of complex.24
Table 1 summarizes the ionic longitudinal and transverse
relaxivities for compounds 3b and 4b, as determined at 7 T and
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Table 2 The inhibition of human (h)CA isozymes I, II, IX and XII
with sulfonamides 1 and 2, and their corresponding Gd(III) complexes
3 and 4, by a stopped-flow, CO2 hydrase assay.29 Data for the
standard sulfonamide CAIs AAZ, EZA and S are also included for
comparison
isoforms, among which are also the tumor-associated
examples CA IX and XII.
Reduction of tumor acidosis
KI/nMa
As shown in Fig. 3, two of the effective hCA IX/XII inhibitors
detected here, i.e., 3b and 4b, were investigated for the inhibition
of extracellular tumor acidification in two cell lines: the
colorectal HT-29 and cervical HeLa carcinoma cell lines, both
of which overexpress high amounts of CA IX under hypoxic
conditions through the HIF-1 activation cascade described in
Fig. 1. S was used as a control in these experiments, being
shown earlier16–18 to reduce extracellular acidosis due to
CA IX inhibition.
hCA I
hCA II
hCA IX
hCA XII
Compound
1a
1b
1c
2a
2b
2c
3a
3b
3c
4a
4b
4c
AAZ
EZA
S
970
425
88
298
720
290
360
267
280
5.5
9.5
110
13
2.0
17.7
12
8
160
110
41
27
96
36
20
34
27
98
32
37
40
25
34
33
14
38
39
17
40
17
89
38
38
20
67
55
5.7
22
3.2
2130
390
367
680
1600
7700
560
380
750
250
25
It may be observed that in both cell lines, the three
investigated sulfonamides reduced acidosis only under hypoxic
conditions when CA IX/XII were overexpressed, due to the
binding of the inhibitor within the enzyme active site. In the
blank experiments, the medium was acidified by about 0.48 pH
units for the HT-29 cell line and by about 0.30 pH units for
the HeLa cell line. Inhibition of the enzyme by sulfonamides
lead to the slight but significant (P o 0.01) effect of reducing
this acidosis by around 0.10–0.20 pH units. Complex 4b was
the most effective compound against HT-29, whereas S was
the most effective against HeLa cells. Although modest, this
effect might be enhanced by using the complementary targeting
of the tumors by means of a combination therapy, in which the
CAI is combined either with radiation or with other antitumor
21000
a
Errors in the range Æ5% of the reported data from three different
assays.
crystal structures of the complexes of hCA II with this type of
inhibitor. Both DOTA-Gd(III) and TETA-Gd(III) conjugates
lead thus to very effective hCA II inhibitors. This enzyme is, in
fact, a drug target itself for obtaining diuretics, antiglaucoma
and/or antiobesity agents.1
(iii) The transmembrane, tumor-associated enzyme CA IX
was also inhibited by all Gd(III) complexes reported here, with
KI values in the range 27–98 nM (Table 2). The metal
complexes are thus more effective hCA IX inhibitors than
their parent ligands, from which they have been prepared.
Both the DOTA and TETA derivatives showed similar
efficacy, the main factor influencing the inhibitory activity
being the sulfonamide head group present in these molecules.
Indeed, the 4-substituted benzenesulfonamide derivatives
(3a, 3b, 4a and 4b) were slightly better inhibitors compared
to the 3-substituted benzenesulfonamides (3c and 4c) for both
sub-series. It should be noted that these metal complexes show
the same CA IX inhibitory activity as the clinically-used
sulfonamides AAZ and EZA.
(iv) hCA XII is also inhibited by Gd(III) complexes 3 and 4
reported here, with KI values in the range 20–89 nm, but the
complexes are generally weaker inhibitors compared to the
corresponding sulfonamide from which they were prepared.
Thus, both tumor-associated isoforms hCA IX and XII are
effectively inhibited by the Gd(III)-sulfonamide conjugates
reported here, but these compounds do not act as selective
inhibitors for tumor-associated CAs, as hCA II is also highly
inhibited by most of them. However, as cytosolic CAs seem to
be also involved in tumorigenesis,36 we decided to investigate
the activity of some of these Gd(III) derivatives in vitro in cell
cultures of hypoxic tumors overexpressing CAs. On the other
hand, the presence of the Gd(III) ions in the new compounds
reported here may lead to membrane-impermeable derivatives,
as reported earlier by our group for positively-charged
sulfonamides.37 Thus, ex vivo, the reported Gd(III)-sulfonamide
conjugates may prevalently inhibit only the transmembrane
Fig. 3 The decrease of tumor cell extracellular acidosis in HT-29 and
HeLa cell cultures treated with Gd(III) sulfonamide conjugates 4b,
3b and S as a standard16 under normoxic and hypoxic conditions.
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agents possessing a different mechanism of action.19 Studies
are in progress in several laboratories to demonstrate the
efficacy of such a combination therapy of CAIs with other
antitumor agents or radiation.
P. H. Maxwell, C. W. Pugh, P. J. Ratcliffe and A. L. Harris, Cancer
Res., 2000, 60, 7075.
13 M. C. Brahimi-Horn and J. Pouysse
165.
14 M. Bartosova
´
gur, Essays Biochem., 2007, 43,
´
,
S. Parkkila, K. Pohlodek, T. J. Karttunen,
S. Galbavy, V. Mucha, A. L. Harris, J. Pastorek and
S. Pastorekova, J. Pathol., 2002, 197, 314.
Conclusion
15 V. Alterio, M. Hilvo, A. Di Fiore, C. T. Supuran, P. Pan,
S. Parkkila, A. Scaloni, J. Pastorek, S. Pastorekova, C. Pedone,
A. Scozzafava, S. M. Monti and G. De Simone, Proc. Natl. Acad.
Sci. U. S. A., 2009, 106, 16233.
We have reported here for the first time that Gd(III)-sulfonamide
complexes incorporating the macrocyclic ring systems DOTA
or TETA are remarkable inhibitors of both tumor-associated
CA isozymes CA IX and XII, being able to reduce tumor
acidosis in vitro. This new class compound thus promises
applications in the diagnostic and treatment of hypoxic
tumors, which are largely non-responsive to classical chemo-
and radiotherapy.
16 E. Svastova
´
,
A. Hulıkova, M. Rafajova, M. Zat’ovicova,
´ ´ ´ ´
A. Casini, A. Cecchi, A. Scozzafava,
A. Gibadulinova
´
C. T. Supuran and J. Pastorek, FEBS Lett., 2004, 577, 439.
,
17 L. Dubois, K. Douma, C. T. Supuran, R. K. Chiu, M. A. M.J. van
Zandvoort, S. Pastorekova, A. Scozzafava, B. G. Wouters and
´
P. Lambin, Radiother. Oncol., 2007, 83, 367.
18 L. Dubois, N. G. Lieuwes, A. Maresca, A. Thiry, C. T. Supuran,
A. Scozzafava, B. G. Wouters and P. Lambin, Radiother. Oncol.,
2009, 92, 423.
19 J. K. J. Ahlskog, C. E. Dumelin, S. Trussel, J. Marlind and
¨
Acknowledgements
D. Neri, Bioorg. Med. Chem. Lett., 2009, 19, 4851.
20 J. K. Ahlskog, C. Schliemann, J. Marlind, U. Qureshi, A. Ammar,
R. B. Pedleym and D. Neri, Br. J. Cancer, 2009, 101, 645.
21 S. Aime and P. J. Caravan, J. Magn. Reson. Imaging, 2009, 30,
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22 L. Lattuada and G. Lux, Tetrahedron Lett., 2003, 44, 3893.
23 A. Barge, G. Cravotto, E. Gianolio and F. Fedeli, Contrast Media
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24 S. Aime, D. D. Castelli, S. G. Crich, E. Gianolio and E. Terreno,
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25 M. Rami, A. Cecchi, J.-L. Montero, A. Innocenti, D. Vullo,
A. Scozzafava, J.-Y. Winum and C. T. Supuran, ChemMedChem,
2008, 3, 1780.
This research was financed in part by a grant from the 6th
Framework Programme of the European Union (DeZnIT
project, to C. T. S. and A. S.) and by a 7th Framework
Programme of the European Union grant (METOXIA). The
authors thank Prof. Joulia Larionova (Institut Charles
Gerhardt, Montpellier, France) for performing the magnetic
measurements and Dr Marlies Oostendorp (University of
Maastricht, The Netherlands) for determining the relaxivity
values.
26 E. A. L. Boudreaux and N. Mulay, Theory and Applications of
Molecular Paramagnetism, John Wiley & Sons, New York, 1976,
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2144 | New J. Chem., 2010, 34, 2139–2144