Hypoxia-targeting carbonic anhydrase IX inhibitors by a new series of nitroimidazole-sulfonamides/sulfamides/sulfamates

Article abstract of DOI:10.1021/jm4009532

A series of nitroimidazoles incorporating sulfonamide/sulfamide/sulfamate moieties were designed and synthesized as radio/chemosensitizing agent targeting the tumor-associated carbonic anhydrase (CA) isoforms IX and XII. Most of the new compounds were nanomolar inhibitors of these isoforms. Crystallographic studies on the complex of hCA II with the lead sulfamide derivative of this series clarified the binding mode of this type of inhibitors in the enzyme active site cavity. Some of the best nitroimidazole CA IX inhibitors showed significant activity in vitro by reducing hypoxia-induced extracellular acidosis in HT-29 and HeLa cell lines. In vivo testing of the lead molecule in the sulfamide series, in cotreatment with doxorubicin, demonstrated a chemosensitization of CA IX containing tumors. Such CA inhibitors, specifically targeting the tumor-associated isoforms, are candidates for novel treatment strategies against hypoxic tumors overexpressing extracellular CA isozymes.

Full text of DOI:10.1021/jm4009532

Article
pubs.acs.org/jmc
Hypoxia-Targeting Carbonic Anhydrase IX Inhibitors by a New Series
of Nitroimidazole-Sulfonamides/Sulfamides/Sulfamates
Marouan Rami,† Ludwig Dubois,‡ Nanda-Kumar Parvathaneni,†,‡ Vincenzo Alterio,§
Simon J. A. van Kuijk,‡ Simona Maria Monti,§ Philippe Lambin,‡ Giuseppina De Simone,§
Claudiu T. Supuran,∥ and Jean-Yves Winum*,†
†Institut des Biomolec
Nationale Super
́
ules Max Mousseron (IBMM) UMR 5247 CNRS-UM1-UM2 Bat
ieure de Chimie de Montpellier, 8 rue de l’Ecole Normale, 34296 Montpellier Cedex, France
‡Department of Radiation Oncology (MAASTRO Lab), GROWSchool for Oncology and Developmental Biology, Maastricht
̂
iment de Recherche Max Mousseron, Ecole
́
University Medical Centre, Universiteitssingel 50/23, PO Box 616, 6200 MD, Maastricht, The Netherlands
§Istituto di Biostrutture e BioimmaginiCNR, via Mezzocannone 16, 80134 Naples, Italy
∥Neurofarba Department, Section of Pharmaceutical Sciences, Universita
Fiorentino (Florence), Italy
̀
degli Studi di Firenze, Via Ugo Schiff 6, 50019 Sesto
ABSTRACT: A series of nitroimidazoles incorporating
sulfonamide/sulfamide/sulfamate moieties were designed and
synthesized as radio/chemosensitizing agent targeting the
tumor-associated carbonic anhydrase (CA) isoforms IX and
XII. Most of the new compounds were nanomolar inhibitors of
these isoforms. Crystallographic studies on the complex of
hCA II with the lead sulfamide derivative of this series clarified
the binding mode of this type of inhibitors in the enzyme
active site cavity. Some of the best nitroimidazole CA IX
inhibitors showed significant activity in vitro by reducing
hypoxia-induced extracellular acidosis in HT-29 and HeLa cell lines. In vivo testing of the lead molecule in the sulfamide series, in
cotreatment with doxorubicin, demonstrated a chemosensitization of CA IX containing tumors. Such CA inhibitors, specifically
targeting the tumor-associated isoforms, are candidates for novel treatment strategies against hypoxic tumors overexpressing
extracellular CA isozymes.
To improve the potency of cancer treatment with CA
inhibitors, we have recently focused our attention on the
development of dual drugs able to inhibit selectively CA IX and
to enhance hypoxic tumor sensitization toward therapy. To this
aim, we decided to utilize nitroimidazole scaffolds as they are
well-known as radiosensitizers and have been subject of
investigation for several decades with a number of such
derivatives being clinically used.11 Thus, the present article
deals with the development of novel nitroimidazole compounds
whose properties have been optimized by conjugation with
pharmacophoric moieties targeting CA IX. In particular, we
report here the synthesis and carbonic anhydrase inhibitory
activity of a panel of sulfonamide/sulfamide/sulfamate
derivatives containing 2- or 5-nitroimidazoles moieties. The
crystallographic structures of the lead sulfamide derivative in
adduct with isoform II was studied. Validation in in vitro
studies identified compounds that selectively inhibit CA IX
activity, thereby reducing hypoxia-induced extracellular acidosis.
From this inhibitor library a lead compound was taken forward
into in vivo, demonstrating to be able to chemosensitize tumors
INTRODUCTION
■
Hypoxia is a strong oncogenic phenotype occurring in all solid
tumors which is responsible for enhanced malignancy and is
associated with resistance to ionizing radiation and chemo-
therapy. This inherent feature provides significant opportunities
for drug discovery especially for specific tumor-targeting
agents.1,2
Among the proteins whose expression is induced by hypoxia,
via the hypoxia inducible factor 1α (HIF-1α), membrane-
associated carbonic anhydrase IX (CA IX, (CA, EC 4.2.1.1))3 is
the most strongly induced one in human cancer cells.4 CA IX is
also the most active CA isoform for the CO2 hydration
reaction, playing a major role in regulating the tumor acid−base
balance.4,5 It is strongly overexpressed in a broad range of
tumor types, and its expression negatively correlates with the
prognosis of cancer patients. Moreover, this isoform exhibits a
very limited expression in normal tissues, thus its inhibition
may lead to significantly fewer side effects compared to classical
anticancer agents in clinical use.4
CA IX is now a validated antitumor target, and its inhibition
with antibodies, sulfonamide, and coumarin inhibitors has been
undoubtedly proven to reverse the effect of tumor acidification,
leading to the inhibition of the cancer cells growth in vivo.4,6−10
Received: June 25, 2013
Published: October 15, 2013
© 2013 American Chemical Society
8512
dx.doi.org/10.1021/jm4009532 | J. Med. Chem. 2013, 56, 8512−8520

Journal of Medicinal Chemistry
Article
a
Scheme 1
a
Reagents and conditions: (i) 1 equiv of 2-nitroimidazole, 1 equiv of tert-butyl bromoacetate, 4 equiv of potassium carbonate, MeCN, RT, 1 night;
(ii) cocktail of trifluoroacetic acid/water/thioanisole 95/2.5/2.5 v/v, rt, 1 night; (iii) 1 equiv of 4-dimethylaminopyridine (DMAP), 1 equiv of 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N,N-dimethylacetamide (DMA), RT, 2 days.
a
(HT-29 colorectal carcinoma) to clinically available treatment
schedules.
Scheme 2
RESULTS AND DISCUSSION
■
Several studies have emphasized that specific inhibitors of CA
IX activity are promising to pursue for their tumor-specific
therapeutic properties also in combination with conventional
therapies.3−5 A different approach to sensitize tumors is the use
of nitroimidazole, although high clinical toxicity has been
observed upon therapeutic efficacy.11,12 An important requisite
for newly designed compounds is therefore the more specific
targeting toward the hypoxic tumor regions using lower doses.
On the basis of these considerations, the rationale of this
work for designing new CAIs was to incorporate in the same
scaffold two different functionalities: (i) the sulfonamide/
sulfamate/sulfamide one which is responsible for binding to the
Zn(II) within the CA active site and hence inhibition of the
enzyme and (ii) a nitroimidazole moiety which is responsible
for radiosensitization in tumors (such moiety is found in agents
such as Pimonidazole which has proved to be an effective and
nontoxic hypoxia marker for several human squamous cell
carcinomas). A comparable approach has been in fact reported
earlier by one of our groups13 by combining benzenesulfona-
mide and aromatic nitro derivatives in the same molecule.
Indeed, the derivatives of this type reported by D’Ambrosio et
al.13 were highly effective CA IX/XII inhibitors.
a
Reagents and conditions: (i) 1 equiv of 1-(2-aminoethyl)-2-methyl-5-
nitroimidazole dihydrochloride monohydrate, 1 equiv SCN-Ph-
SO2NH2, 2 equiv of triethylamine, MeCN, RT, 1 h.
Sulfamide derivative 7 and methylsulfonamide analogue 13
(Scheme 3) were obtained from the same starting material 5 by
a procedure previously reported by this group.16 Sulfamate
analogue 9 was prepared starting from the metronidazole 8
a
Scheme 3
Chemistry. Different libraries of inhibitors were synthesized
by incorporating 2- or 5-nitroimidazole moieties. Starting from
the 2-nitroimidazole 1, we introduced the acetic acid moiety on
position 1 of the imidazole ring by reacting tert-butyl
bromoacetate on 1 in basic medium followed by deprotection
of the resulting ester 2 by trifluoroacetic acid to give compound
3. The latter was then reacted with different amino-
benzenesulfonamide derivatives using carboodiimide as a
coupling agent to give compounds 4 in good yield (Scheme
1).10,11
The synthesis of the 5-nitroimidazole series was realized by
following classical strategies used for the preparation of CA
inhibitors. Starting from the amine analogue of metronidazole
5, we reacted different benzene sulfonamide isothiocyanates to
lead to the series of thiourea compounds 6 (Scheme 2).14,15
a
Reagents and conditions: (i) 1 equiv of 1-(2-aminoethyl)-2-methyl-5-
nitroimidazole dihydrochloride monohydrate, 4 equiv of triethylamine,
1 equiv of chlorosulfonylisocyanate, 1 equiv of tert-butanol, CH2Cl2, rt,
1 h; (ii) trifluoroacetic acid−CH2Cl2 7:3 v/v, RT, 6 h; (iii) 2 equiv of
methane sulfonyl chloride, 2 equiv of triethylamine, CH2Cl2.
8513
dx.doi.org/10.1021/jm4009532 | J. Med. Chem. 2013, 56, 8512−8520

Journal of Medicinal Chemistry
Article
using a direct sulfamoylation procedure as described previously
(Scheme 4).17
as negative control molecules, acted as effective inhibitors
against isoforms hCA I, II, IX, and XII.
Against the abundant, cytosolic isoform hCA I, the new
compounds reported here behaved as medium/weak potency
inhibitors, with KIs in the range of 79 nM to 9.57 μM. For the
benzenesulfonamides 4 and 6, the main factor influencing hCA
I inhibition was the substitution pattern of the benzenesulfo-
namide and the length of the linker between this and the rest of
the molecule. Indeed, for compounds of subseries 4, the
sulfanilamide derivative 4a was a much weaker hCA I inhibitor
compared to the congeners with a larger linker (n = 1 and 2)
between the benzenesulfonamide functionality and the nitro-
azole-acetamido moiety. Indeed, the derivatives 4b and 4c
having these longer linkers, as well as the metanilamide
derivative 4d, were much more effective hCA I inhibitors (KIs
of 79−107 nM) compared to the sulfanilamide derivative 4a
(KI of 3203 nM). In the case of the thioureas 6, the SAR is
slightly different in the sense that the least effective derivative
was the homosulfanilamide derivative 6b (KI of 483 nM),
whereas the remaining ones (6a, 6c, and 6d) showed a similar
behavior of medium potency inhibitors (KI of 79−105 nM).
Finally, sulfamates, sulfamides, and methylsulfonamide 7, 9, and
11−15, were either very weak hCA I inhibitors or devoid of
such an activity (Table 1).
a
Scheme 4
a
Reagents and conditions: (i) 1 equiv of 2-methyl-5-nitro-1-
imidazolylethanol, N,N-dimethylacetamide, 3 equiv sulfamoyl chloride,
RT, 1 night.
Compounds 11 and 12 were obtained as the same manners
as compounds 7 and 13 starting from compound 10 obtained
as previously described by Hoigebazar et al.18 (Scheme 5).
a
Scheme 5
The physiologically dominant off-target isoform hCA II was
highly inhibited by most of the new sulfonamides reported here
(4b−4d, 6a−6d), which had KIs in the range of 2.9−7.4 nM
(better than that of AAZ of 12 nM). Compounds in the
sulfamide and sulfamate series were medium potency inhibitor
(KIs of 33.8−58 nM) except 7, which showed activity in the
same range as AAZ. Derivatives 12 and 13 were not inhibitory
also against this isoform (Table 1). Again, SAR is
straightforward, as for the discussion of hCA I inhibition
above. For amides 4, the length of the spacer between the
benzenesulfonamide and nitroazole functionalities was the main
factor influencing activity, whereas for thioureas 6, all
substitution patterns were equal in generating potent hCA II
inhibitors. The aliphatic derivatives 7−15 possessing a ZBG
were effective or medium potency hCA II inhibitors, whereas
those without such a moiety (the methylsulfonamides) were
ineffective as hCA II inhibitors.
a
Reagents and conditions: (i) 1 equiv of 2-nitroimidazole, dry DMF, 1
equiv K2CO3, 1.5 equiv tert-butyl 2-bromoethylcarbamate; (ii)
trifluoroacetic acid−CH2Cl2 8:2 v/v, RT, 2 h; (iii) 4 equiv of
triethylamine, 1.5 equiv of chlorosulfonylisocyanate, 1.5 equiv of tert-
butanol, CH2Cl2, rt, 12 h; (iv) trifluoroacetic acid−CH2Cl2 8:2 v/v,
RT, 2 h; (v) 2 equiv of methane sulfonyl chloride, 2 equiv of
triethylamine, CH2Cl2.
Sulfamide 15 was prepared from the commercially available
compound 14 following the sulfamoylation process used for
compound 7 (Scheme 6).
a
Scheme 6
The tumor-associated transmembrane isoforms hCA IX and
hCA XII were both potently inhibited by all sulfonamides series
4 and 6 (except 4a) reported here, which showed inhibition
constants <10 nM, more precisely, in the range of 7.2−8.3 nM
against hCA IX, and of 6.7−8.5 against hCA XII, respectively.
Sulfamides 7, 11, and 15 and sulfamate 9 displayed also high
potency at nanomolar levels for these isoforms. SAR for hCA
IX inhibition is similar to what is discussed above for hCA I and
II inhibition, whereas in the case of hCA XII, all compounds
(except those without a ZBG, i.e., 12 and 13) were highly
effective inhibitors, proving that all substitution patterns
explored here led to highly effective CAIs.
Selectivity ratios for inhibiting hCA IX over hCA II and hCA
XII over hCA II were in the range of 0.4−5. The inhibition
profiles of CA II and IX (or CA II and XII) were generally
rather similar for all the series of compounds; indeed, only
inhibitors 4a, 4b, and 9 were shown to have a moderate CA IX/
XII selectivity. Nevertheless, considering the difficulty to obtain
small compounds with a better affinity for the tumor-associated
isozymes over CA II, the selectivity obtained for these series is
comparable or better with those of all the clinically used CA
a
Reagents and conditions: (i) 4 equiv of triethylamine, 1.5 equiv of
chlorosulfonylisocyanate, 1.5 equiv of tert-butanol, CH2Cl2, rt, 12 h;
(ii) trifluoroacetic acid−CH2Cl2 8:2 v/v, RT, 2 h.
The new compounds were characterized extensively by
spectral and physicochemical methods which confirmed their
structures
CA Inhibition Assays. All compounds reported here were
assayed as inhibitors of four physiologically relevant CA
isoforms, the cytosolic hCA I and II (h = human isoform),
and the transmembrane, tumor-associated hCA IX and XII
using the CO2 hydrase assay (Table 1).19 The clinically
employed sulfonamide acetazolamide (AAZ, 5-acetamido-1,3,4-
thiadiazole-2-sulfonamide) has been used as standard in these
measurements for comparison reasons.
Analysis of these inhibition data shows a very interesting
inhibition profile for this newly designed series of compounds.
Indeed, all compounds, except 12 and 13 which were prepared
8514
dx.doi.org/10.1021/jm4009532 | J. Med. Chem. 2013, 56, 8512−8520

Journal of Medicinal Chemistry
Article
Table 1. hCA I, II, IX, and XII Inhibition Data with Compounds 4−15 and Standard Inhibitor Acetazolamide AAZ by a
a
,
Stopped-Flow, CO2 Hydration Assay Method19
b
KI (nM)
selectivity ratios
c
c
d
d
compd
hCA I
hCA II
hCA IX
hCA XII
5.6
64
8.1
KI hCA II/KI hCA IX
KI hCA II/KI hCA XII
AAZ
4a
4b
4c
4d
6a
6b
6c
6d
7
250
3203
107
12
330
37
25
70
0.48
4.71
4.68
0.6
2.14
5.15
4.56
0.71
0.47
0.68
0.96
0.34
0.86
1.24
3.79
1.52
7.9
8.0
7.3
7.3
7.2
8.3
7.8
20.4
8.3
41
79
4.8
3.8
6.7
8.0
8.0
7.7
8.5
7.6
8.1
8.9
101
0.52
0.75
1.02
0.35
0.84
0.49
4.07
1.41
105
5.5
483
7.4
79
2.9
84
6.6
9576
4435
9120
>20000
>20000
8718
10.1
33.8
58
9
11
12
13
15
38
>20000
>20000
>20000
>20000
37
>20000
52
>20000
21
2.47
1.40
a
b
Selectivity ratios for the inhibition of the tumor-associated (CA IX and XII) over the cytosolic (CA II) isozyme are also reported. Errors in the
c
d
range of 5−10% of the reported value from three different determinations. Full length, cytosolic isoform. Catalytic domain, recombinant enzyme.
inhibitors which have selectivity ratios for the inhibition of CA
II over CA IX <1.
In Vitro Extracellular Acidification Tests. To investigate
the efficacy of different dual targeting compounds, their effects
on hypoxia-induced extracellular acidification was evaluated by
measuring changes in pH before and after exposure to the dual
compounds.20 For this purpose, HeLa cervical and HT29
colorectal carcinoma cells were grown under ambient air or
lowered oxygen concentrations and exposed to compounds in
0.1 and 1 mM concentrations. HeLa demonstrated increased
CA IX expression upon hypoxia, while HT29 also had a high
expression under ambient air.20 Previously, we have demon-
strated that inhibitor binding to CA IX requires both its
expression and its activation and this was only observed
exclusively during hypoxia, irrespective of the CA IX expression
levels.9,20 The 5-nitroimidazole series was selected for further
investigation based on the more favorable toxicity profile
compared with 2-nitroimidazoles.12 For all compounds, a
concentration of 1 mM resulted in a significant reduction (P <
0.05) in hypoxia-induced extracellular acidosis, while the effect
on cells exposed to ambient air was negligible (Figure 1). Only
for 7 a significant (P < 0.05) dose-dependent effect was
observed, while a lower concentration was not effective for the
other compounds. For 6c, a strong alkalization was observed
only for the HeLa cells. Previously, we have shown that for the
(20%) or hypoxia (0.2%) upon treatment with dual targeting
Figure 1. Extracellular acidification of cells exposed to normoxia
single targeting compound 13 and 15, only 15 was able to
reduce the extracellular acidification.21 These data indicated
that the efficacy of the dual targeting compound is attributed to
the CA IX inhibiting moiety. On the basis of these and the
current results, 7 was selected as the lead compound for further
in vivo investigations.
compounds (0.1 or 1 mM). Data are expressed as the difference
between medium pH values (ΔpHe = pH after incubation − pH
before incubation) and show the mean
SD of at least three
independent experiments. Asterisks indicate significant difference (*P
< 0.05).
In Vivo Tests. To investigate whether 7 exerted a
chemosensitizing effect, HT-29 tumor bearing mice were
treated with 7 according to previously described strategies9,10,21
and subsequently exposed to chemotherapy. 7-treated (P <
0.01) or doxorubicin-treated (P < 0.001) mice demonstrated a
significantly reduction in tumor growth, with an average time to
reach 4× starting volume (T4×SV) of 25.43 and 23.39 days for
7 and doxorubicin, respectively, compared to vehicle treatment
only (14.07 days). T4×SV was further enhanced (40.66 days, P
< 0.05) upon combination of 7 with doxorubicin (Figure 2).
None of the treatment schedules caused observable toxicity
assessed by body weight loss. Furthermore, recently we have
demonstrated that 7 also exerted a radiosensitizing effect in a
CA IX dependent manner. A significantly higher sensitization
enhancement ratio (SER) was observed upon pretreatment
with 7 before radiotherapy compared to vehicle pretreatment
only for CA IX expressing tumors.21 Derivative 7 sensitizes
tumors toward radiotherapy and doxorubicin-based chemo-
8515
dx.doi.org/10.1021/jm4009532 | J. Med. Chem. 2013, 56, 8512−8520

Journal of Medicinal Chemistry
Article
Figure 2. 7 (10 mg/kg 3 days) and doxorubicin (5 mg/kg at day 3) were injected intravenously when tumors reached a volume of 250 mm3. The
treatment schedule was repeated for three consecutive weeks. Tumor growth was monitored until reaching 4× the volume at treatment time
(T4×SV). Data represent the mean SD of 8−11 independent animals. Asterisks indicate statistical significance (*P < 0.05; **P < 0.01).
therapy, indicating the potential utility of nitroimidazole-based
CA IX targeting compounds as novel anticancer agents. The
sensitization effects of the “dual drug” were markedly higher
compared with single CA IX targeting agents9 or nitro-
imidazole-based drugs.12
Table 2. Crystal Parameters, Data Collection, and
Refinement Statistics
a
Crystal Parameters
space group
P21
a (Å)
42.17
41.22
71.95
104.22
1
X-ray Crystallography. To determine the molecular
features responsible of the CA inhibitory properties of the
dual drug 7, the crystal structure of such molecule in complex
with the cytosolic dominant isoform hCA II has been solved.
Crystals of the hCA II−7 adduct were isomorphous with those
of the native protein,22 allowing for the analysis of the three-
dimensional structure by difference Fourier techniques. Data
collection and refinement statistics are shown in Table 2.
Inhibitor binding did not generate significant changes in hCA
II structure, indeed, the rmsd for the superposition of the
corresponding Cα atoms between the native enzyme and the
enzyme−inhibitor adduct was 0.3 Å. The analysis of the
electron density maps showed the presence of one inhibitor
molecule bound within the active site. The main protein−
inhibitor interactions are schematically depicted in Figure 3.
Analysis of this figure reveals that the tetrahedral coordination
geometry of the Zn2+ ion and the key hydrogen bonds between
the sulfamide moiety of the inhibitor and the enzyme active site
are all retained with respect to other hCA II−sulfamide
complexes solved so far.23,24 In particular, the ionized sulfamide
NH− group coordinates to Zn2+ ion and donates a hydrogen
bond to Thr199OG1, while one of the two sulfamide oxygens
accepts a hydrogen bond from the backbone NH group of
Thr199. An additional H-bond interaction is observed between
the Thr200OG1 atom and the second nitrogen atom of the
sulfamide moiety. The imidazole ring of the inhibitor is located
in the middle of the active site cavity, with the nitro group
oriented toward the hydrophilic part,25 establishing strong
direct hydrogen bond interactions with residues Thr200 and
His64, stabilized in its in conformation (Figure 3). The
inhibitor binding is further stabilized by additional water
mediated hydrogen bonds and van der Waals interactions with
residues of enzyme active site.
b (Å)
c (Å)
γ (deg)
no. of independent molecules
Data Collection Statistics
resolution (Å)
wavelength (Å)
temperature (K)
20.00−1.85
1.54178
100
b
Rmerge (%)
4.7 (13.2)
25.7 (6.6)
94974
mean I/σ(I)
total reflections
unique reflections
redundancy (%)
completeness (%)
Refinement
20161
4.7 (2.4)
97.2 (82.2)
resolution (Å)
20.00−1.85
16.3
c
Rfactor (%)
c
Rfree (%)
19.6
RMSD from ideal geometry
bond lengths (Å)
bond angles (deg)
no. of protein atoms
no. of water molecules
no. of inhibitor atoms
average B factor (Å2)
all atoms
0.011
1.7
2177
199
16
14.1
13.6
20.1
22.9
protein atoms
inhibitor atoms
water molecules
a
Values in parentheses refer to the highest resolution shell (1.92−1.85
b
c
Å). Rmerge = ∑hkl ∑i|Ii(hkl) − ⟨I(hkl)⟩|/∑hkl∑iIi(hkl). Rfactor = ∑hkl||
Fo(hkl)| − |Fc(hkl)||/∑hkl|Fo(hkl)|; Rfree calculated with 5% of data
withheld from refinement.
CONCLUSION
■
We designed and synthesized a new class of CA IX inhibitors
containing nitroimidazole moieties. These molecules showed
efficacy in vitro for the inhibition of extracellular tumor
acidification in two cell lines overexpressing CA IX, namely the
colorectal HT-29 and the cervical HeLa carcinoma cell lines. In
both tumor types, a significant reduction of tumor acidosis was
detected. Moreover, one of the lead molecules, the sulfamide
derivative 7, was demonstrated to enhance sensitization toward
radiotherapy and chemotherapy with doxorubicin in vivo. The
X-ray crystal structure of the hCA II−7 adduct was also solved,
showing that the binding of 7 in the middle of the enzyme
active site cavity was mainly driven by the canonical
interactions of the sulfamide group, two H-bond interactions
between the nitro group of 7 and residues His64 and Thr200,
8516
dx.doi.org/10.1021/jm4009532 | J. Med. Chem. 2013, 56, 8512−8520

Journal of Medicinal Chemistry
Article
(2-Nitro-imidazol-1-yl) Acetic Acid (3). Compound 2 (2.5g) was
dissolved in 20 mL of a cocktail of TFA−water−thioanisole 95:2.5:2.5
and stirred at room temperature for one night. The mixture was then
concentrated under vacuum and coevaporated several times with
diethyl ether until formation of a powder. After filtration, the
precipitate was washed with dichloromethane and acetonitrile to give
1
quantitatively the expected product; mp 143 °C (decomposition). H
NMR (DMSO-d6, 400 MHz) δ 5.21 (s, 2H), 7.21 (d, 1H, J = 1.01
Hz), 7.64 (d, 1H, J = 1.01 Hz). 13C (DMSO-d6, 101 MHz) δ 50.65,
127.69, 128.44, 168.56, 168.57. MS (ESI+/ESI−) m/z 170.12 [M −
H]−, 341.05 [2M − H]−, 194.14 [M + Na]+.
General Procedure for the Preparation of Compounds (4a−d).
The aminoalkylbenzene sulfonamide (1.17 mmol, 1 equiv), 4-
dimethylaminopyridine (1.17 mmol, 1 equiv), and 1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide (1.17 mmol, 1 equiv) were
added to a solution of compound 3 (1.17 mmol, 1 equiv) in 8 mL
of N,N-dimethylacetamide. The mixture was stirred two days at room
temperature, then diluted with ethyl acetate and washed three times
with water. The organic layer was dried over anhydrous magnesium
sulfate, filtered, and concentrated under vacuum. The residue was then
purified by chromatography on silica gel using methylene chloride−
methanol 98:2 v/v as eluent.
2-(2-Nitro-imidazol-1-yl)-N-(4-sulfamoylphenyl)acetamide (4a).
Yield 68%; mp 163−165 °C. 1H NMR (DMSO-d6, 400 MHz) δ
5.36 (s, 2H), 7.24 (d, 1H, J = 1.01 Hz), 7.28 (s, 2H), 7.67 (d, 1H, J =
1.01 Hz), 7.70 (d, 2H, J = 8.9 Hz), 7.77 (d, 2H, J = 8.9 Hz), 10.7 (s,
1H). 13C (DMSO-d6, 101 MHz) δ 52.20, 118.70, 126.81, 127.57,
128.84, 138.76, 141.18, 144.79, 165.02. MS (ESI+/ESI−) m/z 324.09
[M − H]−, 359.92 [M + Cl]−, 649.15 [2M − H]−, 685.01 [2M + Cl]−,
348.14 [M + Na]+. HRMS (ESI) [M + H]+ calculated for
[C11H12N5O5S]+, 326.0559; found, 326.0563.
Figure 3. Active site region in the hCAII−7 complex. Active site Zn2+
coordination and direct hydrogen bonds between inhibitor and
enzyme residues are reported as dashed lines. The σ-A weighted |2Fo −
Fc| simulated annealing omit map (at 1.0σ) relative to the inhibitor
molecule is also shown.
2-(2-Nitro-imidazol-1-yl)-N-(4-sulfamoylbenzyl)acetamide (4b).
Yield 83%; mp 181−183 °C. 1H NMR (DMSO-d6, 400 MHz) δ
4.37 (d, 2H, J = 5.7 Hz), 5.22 (s, 2H), 7.18 (d, 1H, J = 1.01 Hz), 7.35
(s, 2H), 7.43 (d, 2H, J = 8.4 Hz), 7.67 (d, 1H, J = 1.01 Hz), 7.76 (d,
2H, J = 8.4 Hz), 9.15 (t, 1H, J = 6.06 Hz). 13C (DMSO-d6, 101 MHz)
δ 41.81, 51.55, 106.87, 125.58, 127.39, 138.91, 142.66, 142.99, 156.82,
165.88. MS (ESI+/ESI−) m/z 338.15 [M − H]−, 374.22 [M + Cl]−,
713.16 [2M + Cl]−, 340.15 [M + H]+, 362.17 [M + Na]+. HRMS
(ESI) [M + H]+ calculated for [C12H14N5O5S]+, 340.0716; found,
340.0723.
as well as several water mediated hydrogen bonds and
hydrophobic interactions. Altogether, these data indicate the
potential utility of nitroimidazole compounds and open the way
to their use as novel anticancer agents targeting the tumor-
associated CA isoforms IX and XII.
EXPERIMENTAL SECTION
■
2-(2-Nitro-imidazol-1-yl)-N-[2-(4-sulfamoylphenyl)ethyl]-
Chemistry. General. All reagents and solvents were of commercial
quality and used without further purification unless otherwise
specified. All reactions were carried out under an inert atmosphere
of nitrogen. TLC analyses were performed on silica gel 60 F254 plates
(Merck Art. no. 1.05554). Spots were visualized under 254 nm UV
illumination or by ninhydrin solution spraying. Melting points were
1
acetamide (4c). Yield 89%; mp 139−141 °C. H NMR (DMSO-d6,
400 MHz) δ 2.80 (t, 2H, J = 6.9 Hz), 3.16 (m 2H), 5.07 (s, 2H), 7.18
(d, 1H, J = 1.01 Hz), 7.30 (s, 2H), 7.40 (d, 2H, J = 8.2 Hz), 7.61 (d,
1H, J = 1.01 Hz), 7.74 (d, 2H, J = 8.2 Hz), 8.46 (t, 1H, J = 5.6 Hz).
13C (DMSO-d6, 101 MHz) δ 34.59, 51.49, 125.64, 127.38, 128.74,
129.09, 142.05, 143.39, 144.87, 165.57. MS (ESI+/ESI−) m/z 352.19
[M − H]−, 388.07 [M + Cl]−, 354.12 [M + H]+, 376.09 [M + Na]+,
729.21 [2M + Na]+. HRMS (ESI) [M + H]+ calculated for
[C13H16N5O5S]+, 354.0872; found, 354.0873.
determined on a Buchi Melting Point 510 and are uncorrected. 1H and
̈
13C NMR spectra were recorded on Bruker DRX-400 spectrometer
using DMSO-d6 as solvent and tetramethylsilane as internal standard.
For 1H NMR spectra, chemical shifts are expressed in δ (ppm)
downfield from tetramethylsilane, and coupling constants (J) are
expressed in hertz. Electron Ionization mass spectra were recorded in
positive or negative mode on a Water MicroMass ZQ. All compounds
that were tested in the biological assays were analyzed by high-
resolution ESI mass spectra (HRMS) using on a Q-ToF I mass
spectrometer fitted with an electrospray ion source in order to confirm
the purity of >95%.
2-(2-Nitro-imidazol-1-yl)-N-(3-sulfamoylphenyl)acetamide (4d).
Yield 79%; mp 195−197 °C. 1H NMR (DMSO-d6, 400 MHz) δ
5.35 (s, 2H), 7.24 (d, 1H, J = 1.01 Hz), 7.38 (s, 2H), 7.52 (m, 2H),
7.65 (m, 1H), 7.67 (d, 1H, J = 1.01 Hz), 8.14 (s, 1H), 10.81 (s, 1H).
13C (DMSO-d6, 101 MHz) δ 52.16, 116.10, 120.72, 121.92, 127.59,
128.86, 129.67, 138.67, 144.72, 164.87, 167.75. MS (ESI+/ESI−) m/z
324.24 [M − H]−, 360.18 [M + Cl]−, 685.13 [2M + Cl]−, 326.24 [M
+ H]+, 348.07 [M + Na]+, 364.17 [M + K]+, 673.18 [2M + Na]+.
HRMS (ESI) [M + H]+ calculated for [C11H12N5O5S]+, 326.0559;
found, 326.0553.
tert-Butyl-(2-nitro-imidazol-1-yl) Acetate (2). A solution of tert-
butylbromoacetate (17.7 mmol, 1 equiv) in 10 mL of acetonitrile was
added dropwise to a mixture of 2-nitroimidazole 1 (17,7 mmol, 1
equiv) and anhydrous potassium carbonate (70.74 mmol, 4 equiv) in
20 mL of acetonitrile. The mixture was stirred one night at room
temperature and concentrated under vacuum. The residue was purified
by chromatography on silica gel using a mixture CH2Cl2/MeOH 95/5
as eluent to give the expected compound as white powder in 71%
yield; mp 95−97 °C. 1H NMR (DMSO-d6, 400 MHz) δ 1.46 (s, 9H),
4.99 (s, 2H), 7.06 (d, 1H, J = 1.01 Hz), 7.17 (d, 1H, J = 1.01 Hz). MS
(ESI+/ESI−) m/z 226.15 [M − H]−, 262.13 [M + Cl]−, 250.20 [M +
H]+.
General Procedure for the Preparation of Compounds (6a−d).
Isothiocyanate (0.76 mmol, 1 equiv) was added to a solution of the
commercially available compound 5 (0.76 mmol, 1 equiv) in 10 mL of
acetonitrile. The reaction was stirred for one hour at room
temperature and then filtered. The filtrate was concentrated under
vacuum and the residue obtained purified by chromatography on silica
gel using methylene chloride−methanol 95:5 as eluent.
N-(4-Sulfamoylphenyl)-N-((2-aminoethyl)-2-methyl-5-nitroimi-
dazolyl) Thiourea (6a). Yield 72%; mp 186−188 °C. 1H NMR
8517
dx.doi.org/10.1021/jm4009532 | J. Med. Chem. 2013, 56, 8512−8520

Journal of Medicinal Chemistry
Article
(DMSO-d6, 400 MHz) δ 2.42 (s, 3H), 3.91 (m, 2H), 4.5 (t, 2H, J =
5.68 Hz), 7.3 (s, 2H), 7.46 (d, 2H, J = 8.7 Hz), 7.71 (d, 2H, J = 8.7
Hz), 8.05 (s, 2H), 10.0 (s, 1H). 13C (DMSO-d6, 101 MHz) δ 13.81,
42.87, 44.86, 122.46, 126.30, 133.19, 138.66, 139.14, 141.88, 151.35,
180.88. MS (ESI+/ESI−) m/z 385.17 [M + H]+, 407.07 [M + Na]+,
769.22 [2M + H]+, 383.21 [M − H]−, 419.18 [M + Cl]−, 767.16. [2M
− H]−. HRMS (ESI) [M + H]+ calculated for [C13H17N6O4S2]+,
385.0753; found, 385.0756.
Hz), 7.57 (s, 2H), 8.06 (s, 1H). 13C (DMSO-d6, 101 MHz) δ 14.04,
44.98, 57.21, 133.10, 138.32, 151.82. MS (ESI+/ESI−) m/z 250.3 [M +
H]+, 272.32 [M + Na]+, 521.30 [2M + Na]+,770.16 [3M + Na]+.
HRMS (ESI) [M + H]+ calculated for [C6H11N4O5S]+, 251.0450;
found, 251.0456.
2-(2-Nitro-imidazol-1-yl)ethylamine (10). tert-Butyl 2- bromoe-
thylcarbamate (3.3 mmol, 1.5 equiv) was added at room temperature
to a solution of 2-nitro imidazole (2.2 mmol, 1 equiv) and K2CO3 (2.2
mmol, 1 equiv) in 3 mL of DMF. The reaction mixture was stirred at
room temperature overnight then filtered, and the filtrate was
concentrated under vacuum. The solid obtained was dissolved with
ethyl acetate and washed with water. The organic layer was dried over
anhydrous sodium sulfate and concentrated. The residue was purified
on silica column chromatography using methylene chloride−methanol
9:1 as eluent. The pure Boc protected compound was then dissolved
in 20% trifluoroacetic acid−methylene chloride (16 equiv TFA)
solution and stirred at room temperature for 2 h. The reaction mixture
was then concentrated in vacuo and coevaporated with methanol. The
pure expected compound (under trifluoroacetate salt) was then
obtained after precipitation in diethyl ether and filtration. Yield 40%;
N-(4-Sulfamoylbenzyl)-N-((2-aminoethyl)-2-methyl-5-nitroimida-
zolyl) Thiourea (6b). Yield 82%; mp 67−69 °C. 1H NMR (DMSO-d6,
400 MHz) δ 2.37 (s, 3H), 2.87 (m, 2H), 4.43 (t, 2H, J = 5.18 Hz),
4.70 (br s, 2H), 7.32 (s, 2H), 7.34 (d, 2H, J = 8.4 Hz), 7.68 (s, 1H),
7.75 (d, 2H, J = 8.4 Hz), 8.03 (s, 1H), 8.15 (s, 1H). 13C (DMSO-d6,
101 MHz) δ 13.84, 30.64, 42.70, 45.44, 125.52, 127.23, 133.17, 138.61,
142.48, 151.41, 181.44. MS (ESI+/ESI−) m/z 399.23 [M + H]+,
421.16 [M + Na]+, 797.08 [2M + H]+, 819.26 [2M + Na]+, 397.10 [M
− H]−, 433.09 [M + Cl]−, 795.33 [2M − H]−. HRMS (ESI) [M +
H]+ calculated for [C14H19N6O4S2]+, 399.0909; found, 399.0912.
N-(4-Sulfamoylphenylethyl)-N-((2-aminoethyl)-2-methyl-5-nitroi-
midazolyl) thiourea (6c). Yield 86%; mp 75−77 °C. 1H NMR
(DMSO-d6, 400 MHz) δ 2.35 (s, 3H), 2.83 (m, 2H), 3.62 (m, 2H),
3.82 (m, 2H), 4.41 (m, 2H), 7.31 (s, 2H), 7.37 (d, 2H, J = 8.2 Hz),
7.52 (s, 1H), 7.63 (s, 1H), 7.74 (d, 2H, J = 8.2 Hz), 8.03 (s, 1H). 13C
(DMSO-d6, 101 MHz) δ 13.78, 30.64, 45.4, 125.65, 129.05, 133.18,
138.54, 142.03, 143.45, 151.42, 180.83. MS (ESI+/ESI−) m/z 413.06
[M + H]+, 435.02 [M + Na]+, 825.09 [2M + H]+, 847.21 [2M + Na]+,
411.06 [M − H]−, 447.20 [M + Cl]−, 822.99 [2M − H]−, 859.26 [2M
+ Cl]−. HRMS (ESI) [M + H]+ calculated for [C15H21N6O4S2]+,
413.1066; found, 413.1069.
N-(3-Sulfamoylphenyl)-N-((2-aminoethyl)-2-methyl-5-nitroimi-
dazolyl) Thiourea (6d). Yield 75%; mp 66−68 °C. 1H NMR (DMSO-
d6, 400 MHz) δ 2.43 (s, 3H), 3.91 (m, 2H), 4.49 (t, 2H, J = 5.68 Hz),
7.39 (s, 2H), 7.51 (m, 1H), 7.55 (s, 1H), 7.57 (m, 1H), 7.81 (s, 1H,
1H), 7.93 (m, 1H), 8.04 (s, 1H), 9.94 (s, 1H). 13C (DMSO-d6, 101
MHz) δ 13.81, 42.82, 44.99, 120.29, 121.39, 126.62, 129.09, 133.18,
138.65, 139.51, 144.35, 151.37, 181.28. MS (ESI+/ESI−) m/z 385.23
[M + H]+, 406.94 [M + Na]+, 791.19 [2M + Na]+, 383.12 [M − H]−,
419.09 [M + Cl]−, 767.26 [2M − H]−. HRMS (ESI) [M + H]+
calculated for [C13H17N6O4S2]+, 385.0753; found, 385.0746.
1
mp 166−168 °C. H NMR (DMSO-d6, 400 MHz) δ 3.33 (t, 2H, J =
6.0 Hz), 4.64 (t, 2H, J = 6.0 Hz), 7.23 (d, 1H, J = 0.6 Hz), 7.65 (d, 1, J
= 0.5 Hz), 8.17 (s, 2H). MS (ESI+) m/z 157.09 [M + H]+, 313.35 [2M
+ H]+.
N-[2-(2-Nitro-imidazol-1-yl)ethyl]sulfamide (11). Same protocol as
for the synthesis of compound 7 starting from 10. Overall yield 70%;
mp 78−80 °C. 1H NMR (DMSO-d6, 400 MHz) δ 3.39 (q, 2H, J = 6.2
Hz), 4.46 (t, 2H, J = 5.9 Hz), 6.64 (s, 2H), 7.18 (d, 1H, J = 1 Hz), 7.23
(t, 1H, J = 6.2 Hz), 7.59 (d, 1H, J = 1 Hz). 13C (DMSO-d6, 101 MHz)
δ 41.85, 49.45, 127.63, 128.52. MS (ESI+/ESI−) m/z 236.15 [M + H]+.
HRMS (ESI) [M + H]+ calculated for [C5H10N5O4S]+, 236.0453;
found, 236.0456.
N-Methanesulfonyl 2-(2-Nitro-imidazol-1-yl)ethylamine (12).
Compound 10 (1 equiv) was suspended in methylene chloride and
triethylamine (2 equiv 2.56 mmol) was added at 0 °C.
Methanesulfonyl chloride (2 equiv 2.56 mmol) was added dropwise
to the resulting solution. Reaction was monitored by TLC until
completion. Then the reaction mixture was diluted with methylene
chloride and washed with water. The organic phase was dried over
sodium sulfate and concentrated under vacuum. The residue was
purified on silica gel column chromatography (eluent methylene
chloride−methanol 9:1 v/v) to afford the pure expected compound as
N-[2-(2-Methyl-5-nitro-imidazol-1-yl)ethyl]sulfamide (7). A sol-
ution of chlorosulfonyl isocyanate (4.59 mmol, 1.2 equiv) and tert-
butanol (4.59 mmol, 1.2 equiv) in 2 mL of methylene chloride
(prepared ab initio) was added to a solution of 5 (3.83 mmol, 1 equiv)
and triethylamine (30.63 mmol, 4 equiv) in 10 mL of methylene
chloride. The mixture was stirred at room temperature for one hour,
then diluted with ethyl acetate and washed with water. The organic
layer was dried over anhydrous sodium sulfate, filtered, and
concentrated under vacuum. The residue was purified by chromatog-
raphy on silica gel using methylene chloride−methanol 98:2 as eluent.
This intermediate was then diluted in a solution of trifluoroacetic acid
in methylene chloride (30% volume) and stirred at room temperature
for 6 h. The mixture was then concentrated under vacuum and
coevaporated several times with diethyl ether to give the expected
sulfamide as a white powder. Overall yield 70%; mp 122 °C. 1H NMR
(DMSO-d6, 400 MHz) δ 2.52 (s, 3H), 3.26 (m, 2H), 4.37 (t, 2H, J =
5.81 Hz), 6.65 (s, 2H), 6.86 (s, 1H), 8.1 (s, 1H). 13C (DMSO-d6, 101
MHz) δ 14.03, 41.8, 46.0, 132.68, 138.26, 151.65. MS (ESI+/ESI−) m/
z 250.19 [M + H]+, 272.34 [M + Na]+, 499.32 [2M + H]+, 249.09 [M
− H]−, 284.12 [M + Cl]−, 533.14 [2M + Cl]−. HRMS (ESI) [M +
H]+ calculated for [C6H12N5O4S]+, 250.0610; found, 250.0616.
N-[2-(2-Methyl-5-nitro-imidazol-1-yl)ethyl]sulfamate (9). Sulfa-
moyl chloride (5.25 mmol, 3 equiv) was added to a solution of the
commercially available compound 8 (1.75 mmol, 1 equiv) in N,N-
dimethylacetamide. The mixture was stirred at room temperature for
one night, then diluted with ethyl acetate and washed three times with
water. The organic layer was dried over anhydrous magnesium sulfate,
filtered, and concentrated under vacuum. The residue was purified by
chromatography on silica gel using methylene chloride−methanol 9:1
1
a white powder. Yield 45%; mp 122−125 °C. H NMR (DMSO-d6,
400 MHz) δ 2.87 (s, 3H), 3.39 (q, 2H, J = 6.2 Hz), 4.47 (t, 2H, J = 5.9
Hz), 7.19 (d, 1H, J = 1.0 Hz), 7.24 (t, 1H, J = 6.2 Hz), 7.60 (d, 1H, J =
1.0 Hz). 13C (DMSO-d6, 101 MHz) δ 128.52, 127.63, 49.45, 41.85,
39.64. MS (ESI+/ESI−) m/z 235.13 [M + H]+. HRMS (ESI) [M +
H]+ calculated for [C6H11N4O4S]+, 235.0501; found, 235.0507.
N-Methanesulfonyl 1-(2-Aminoethyl)-2-methyl-5-nitroimidazole
(13). Same protocol as for the synthesis of compound 12 starting from
1
5. Yield 44%; mp 132−135 °C. H NMR (DMSO-d6, 400 MHz) δ
2.47 (s, 3H), 2.87 (s, 3H), 3.33 (q, 2H, J = 6.2 Hz), 4.34 (t, 2H, J = 5.9
Hz), 7.33 (t, 1H, J = 6.4 Hz), 8.05 (s, 1H). 13C (DMSO-d6, 101 MHz)
δ 151.72, 138.35, 133.17, 46.33, 41.66, 39.40, 14.16. MS (ESI+/ESI−)
m/z 249.20 [M + H]+. HRMS (ESI) [M + H]+ calculated for
[C7H13N4O4S]+, 249.0658; found, 249.0657.
N-[2-(2-Methyl-imidazol-1-yl)propyl]sulfamide (15). Same proto-
col as for the synthesis of compound 7 starting from 14. Overall yield
1
71%; mp 133−135 °C. H NMR (DMSO-d6, 400 MHz) δ 2.47 (s,
3H), 2.86 (m, 2H), 3.38 (q, 2H, J = 6.1 Hz), 4.46 (t, 2H, J = 5.9 Hz),
6.64 (s, 2H), 7.18 (d, 1H, J = 1.0 Hz), 7.23 (t, 1H, J = 6.2 Hz), 7.59 (d,
1H, J = 1.0 Hz). 13C (DMSO-d6, 101 MHz) δ 151.72, 138.35, 133.17,
46.33, 41.66, 38.81, 14.16. MS (ESI+/ESI−) m/z 219.28 [M + H]+.
HRMS (ESI) [M + H]+ calculated for [C7H15N4O2S]+, 219.0916;
found, 219.0917.
CA Inhibition Assays. An Applied Photophysics stopped-flow
instrument was used for assaying the CA-catalyzed CO2 hydration
activity.19 Phenol red (at a concentration of 0.2 mM) was used as an
indicator, working at the absorbance maximum of 557 nm, with 20
mM Hepes (pH 7.5) as buffer and 20 mM Na2SO4 (for maintaining
1
as eluent. Yield 81%; mp 166−168 °C. H NMR (DMSO-d6, 400
MHz) δ 2.45 (s, 3H), 4.35 (t, 2H, J = 5.05 Hz), 4.61 (t, 2H, J = 5.05
8518
dx.doi.org/10.1021/jm4009532 | J. Med. Chem. 2013, 56, 8512−8520

Journal of Medicinal Chemistry
Article
constant the ionic strength), following 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−deionized water, and dilutions up to 0.01 nM were done
thereafter with distilled−deionized water. Inhibitor and enzyme
solutions were preincubated together for 15 min at room temperature
prior to assay to allow for the formation of the E−I complex. The
inhibition constants were obtained by nonlinear least-squares methods
using PRISM 3 and represent the mean from at least three different
determinations. CA isoforms were recombinant ones obtained in
house as reported earlier.
Biological Assays. Cells. 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. Low oxygen conditions
were acquired in a hypoxic workstation (Ruskinn INVIVO2 1000).
The atmosphere in the chamber consisted of 0.2% O2 (hypoxia), 5%
CO2, and residual N2. In parallel, normoxic (20% O2) dishes were
incubated in air with 5% CO2. pH of the culture medium was
immediately measured at the end of each experiment as previously
described.9,20
Compounds Preparation. Compounds were dissolved in culture
medium containing 1% DMSO at the indicated final concentrations
just before addition to the cells. For the animal experiments, 7 was
dissolved in NaCl 0.9% containing 1% DMSO to a final concentration
of 10 mg/kg and injected intravenously via a lateral tail vein.
Animals. Cells were resuspended in Matrigel (BD Biosciences) and
injected subcutaneously into the lateral flank of adult NMRI-nu mice
(28−32 g). Intravenous 7 and doxorubicin treatment started at a
tumor volume of 250 mm3 for 3 days (10 mg/kg daily) and at day 3 (5
mg/kg), respectively. This schedule was repeated during 3 weeks.
Tumor growth was monitored until reaching 4× the volume at
treatment time (T4×SV), and treatment toxicity was scored by body
weight measurements. All experiments were in accordance with local
institutional guidelines for animal welfare and were approved by the
Animal Ethical Committee of the University (2008-025).
Statistics. All statistical analyses were performed with GraphPad
Prism version 5.03 for Windows (GraphPad Software, 2009,
California, USA). A nonparametric Mann−Whitney U test for small
groups was used to determine the statistical significance of differences
between two independent groups of variables. For all tests, a P < 0.05
was considered significant.
coordinates set for further refinement. Restraints on inhibitor bond
angles and distances were taken from the Cambridge Structural
Database,29 and standard restraints were used on protein bond angles
and distances throughout refinement. Water molecules were built into
peaks >3σ in |Fo| − |Fc| maps that demonstrated appropriate hydrogen-
bonding geometry. Final crystallographic Rfactor and Rfree values were
0.163 and 0.196, respectively. Statistics for refinement are summarized
in Table 2.
ASSOCIATED CONTENT
Accession Codes
Coordinates and structure factors have been deposited with the
Protein Data Bank (accession code 4MO8).
■
AUTHOR INFORMATION
Corresponding Author
*Phone: 33-467-147234. E-mail: jean-yves.winum@univ-
montp2.fr.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work has been funded with the support of the EU 6th
framework Program (Euroxy project ref 2003-502932), the EU
7th framework (Metoxia project ref 2008-222741), the KWF
UM2012-5394, the NGI Life Science Pre-Seed grant 93613002,
and also the CNRS/CNR (CoopIntEER program, grant no.
131999)
REFERENCES
■
(1) Wilson, W. R.; Hay, M. P. Targeting hypoxia in cancer therapy.
Nature Rev. Cancer 2011, 11, 393−410.
(2) Bennewith, K. L.; Dedhar, S. Targeting hypoxic tumour cells to
overcome metastasis. BMC Cancer 2011, 11, 504.
(3) Supuran, C. T. Carbonic anhydrases: novel therapeutic
applications for inhibitors and activators. Nature Rev. Drug Discovery
2008, 7, 168−181.
(4) McDonald, P. C.; Winum, J.-Y.; Supuran, C. T.; Dedhar, S.
Recent developments in targeting carbonic anhydrase IX for cancer
therapeutics. Oncotarget 2012, 3, 84−97.
(5) Neri, D.; Supuran, C. T. Interfering with pH regulation in
tumours as a therapeutic strategy. Nature Rev. Drug Discovery 2011, 10,
767−777.
(6) Pacchiano, F.; Carta, F.; McDonald, P. C.; Lou, Y.; Vullo, D.;
Scozzafava, A.; Dedhar, S.; Supuran, C. T. Ureido-substituted
benzenesulfonamides potently inhibit carbonic anhydrase IX and
show antimetastatic activity in a model of breast cancer metastasis. J.
Med. Chem. 2011, 54, 1896−1902.
(7) Touisni, N.; Maresca, A.; McDonald, P. C.; Lou, Y.; Scozzafava,
A.; Dedhar, S.; Winum, J-Y; Supuran, C. T. Glycosyl coumarin
carbonic anhydrase IX and XII inhibitors strongly attenuate the growth
of primary breast tumors. J. Med. Chem. 2011, 54, 8271−8277.
(8) Lou, Y.; McDonald, P. C.; Oloumi, A.; Chia, S.; Ostlund, C.;
Ahmadi, A.; Kyle, A.; Auf dem Keller, U.; Leung, S.; Huntsman, D.;
Clarke, B.; Sutherland, B. W.; Waterhouse, D.; Bally, M.; Roskelley, C.;
Overall, C. M.; Minchinton, A.; Pacchiano, F.; Carta, F.; Scozzafava,
A.; Touisni, N.; Winum, J.-Y.; Supuran, C. T.; Dedhar, S. Targeting
tumor hypoxia: suppression of breast tumor growth and metastasis by
novel carbonic anhydrase IX inhibitors. Cancer Res. 2011, 71, 3364−
3376.
X-ray Studies. The hCA II−7 complex was obtained by adding a 5
molar excess of inhibitor to a 10 mg/mL protein solution in 20 mM
Tris-HCl pH 8, 0.1% DMSO. Crystals of the complex were obtained
using the hanging drop vapor diffusion technique. In particular 2 μL of
complex solution and 2 μL of precipitant solution (1.4 M Na-citrate,
100 mM Tris-HCl pH 8.0) were mixed and suspended over a reservoir
containing 1 mL of precipitant solution at 20 °C. Crystals grew within
3 days. A complete data set was collected at 1.85 Å resolution from a
single crystal, at 100 K, with a copper rotating anode generator
developed by Rigaku and equipped with Rigaku Saturn CCD detector.
Prior to cryogenic freezing, crystals were transferred to the precipitant
solution with the addition of 20% (v/v) glycerol. Diffracted intensities
were processed using the HKL2000 crystallographic data reduction
package (Denzo/Scalepack).26 Crystal parameters and data processing
statistics are summarized in Table 2. The structure of the complex was
analyzed by difference Fourier techniques using hCA II crystallized in
the P21 space group (PDB code 1CA2)22 as the starting model after
deletion of nonprotein atoms. An initial round of rigid body
refinement followed by simulated annealing and individual B factor
refinement was performed using the program CNS.27 Model
visualization and rebuilding was performed using the graphics program
O.28 After an initial refinement, limited to the enzyme structure, a
model for the inhibitor was easily built and introduced into the atomic
(9) Dubois, L.; Peeters, S.; Lieuwes, N. G.; Geusens, N.; Thiry, A.;
Wigfield, S.; Carta, F.; McIntyre, A.; Scozzafava, A.; Dogne, J.-M.;
́
Supuran, C. T.; Harris, A. L.; Masereel, B.; Lambin, P. Specific
inhibition of carbonic anhydrase IX activity enhances the in vivo
therapeutic effect of tumor irradiation. Radiother. Oncol. 2011, 99,
424−431.
8519
dx.doi.org/10.1021/jm4009532 | J. Med. Chem. 2013, 56, 8512−8520

Journal of Medicinal Chemistry
Article
(10) Gieling, R. G.; Babur, M.; Mamnani, L.; Burrows, N.; Telfer, B.
A.; Carta, F.; Winum, J.-Y.; Scozzafava, A.; Supuran, C. T.; Williams, K.
J. Antimetastatic effect of sulfamate carbonic anhydrase IX inhibitors in
breast carcinoma xenografts. J. Med. Chem. 2012, 55, 5591−600.
(11) Liu, K.; Zhu, H. L. Nitroimidazoles as anti-tumor agents. Anti-
Cancer Agents Med. Chem. 2011, 11, 687−691.
(25) De Simone, G.; Alterio, V.; Supuran, C. T. Exploiting the
hydrophobic and hydrophilic binding sites for designing carbonic
anhydrase inhibitors. Expert Opin. Drug Discovery 2013, 8, 793−810.
(26) Otwinowski, Z.; Minor, W. Processing of X-ray diffraction data
collected in oscillation mode. Methods Enzymol. 1997, 276, 307−326.
(27) Brunger, A. T.; Adams, P. D.; Clore, G. M.; DeLano, W. L.;
̈
Gros, P.; Grosse-Kunstleve, R. W.; Jiang, J. S.; Kuszewski, J.; Nilges,
M.; Pannu, N. S.; Read, R. J.; Rice, L. M.; Simonson, T.; Warren, G. L.
Acta Crystallogr., Sect. D: Biol. Crystallogr. 1998, 54, 905−921.
(28) Jones, T. A.; Zou, J. Y.; Cowan, S. W.; Kjeldgaard, M. Acta
Crystallogr. Sect. A: Found. Crystallogr. 1991, 47, 110−119.
(29) Allen, F. H. The Cambridge Structural Database: a quarter of a
million crystal structures and rising. Acta Crystallogr., Sect. B: Struct. Sci.
2002, 58, 380−388.
(12) Overgaard, J.; Overgaard, M.; Nielsen, O. S.; Pedersen, A. K.;
Timothy, A. R. A comparative investigation of nimorazole and
misonidazole as hypoxic radiosensitizers in a C3H mammary
carcinoma in vivo. Br. J. Cancer 1982, 46, 904−911.
́
(13) D’Ambrosio, K.; Vitale, R. M.; Dogne, J. M.; Masereel, B.;
Innocenti, A.; Scozzafava, A.; De Simone, G.; Supuran, C. T. Carbonic
anhydrase inhibitors: bioreductive nitro-containing sulfonamides with
selectivity for targeting the tumor associated isoforms IX and XII. J.
Med. Chem. 2008, 51, 3230−3237.
(14) Rami, M.; Cecchi, A.; Montero, J.-L.; Innocenti, A.; Vullo, D.;
Scozzafava, A.; Winum, J.-Y.; Supuran, C. T. Carbonic anhydrase
inhibitors: design of membrane-impermeant copper(II) complexes of
DTPA-, DOTA-, and TETA-tailed sulfonamides targeting the tumor-
associated transmembrane isoform IX. ChemMedChem 2008, 3, 1780−
1788.
(15) Rami, M.; Montero, J.-L.; Dubois, L.; Lambin, P.; Scozzafava, A.;
Winum, J.-Y.; Supuran, C. T. Carbonic anhydrase inhibitors: Gd(III)
complexes of DOTA− and TETA−sulfonamide conjugates targeting
the tumor associated carbonic anhydrase isozymes IX and XII. New J.
Chem. 2010, 34, 2139−2144.
(16) D’Ambrosio, K.; Smaine, F. Z.; Carta, F.; De Simone, G.;
Winum, J.-Y.; Supuran, C. T. Development of potent carbonic
anhydrase inhibitors incorporating both sulfonamide and sulfamide
groups. J. Med. Chem. 2012, 55, 6776−6783.
(17) Winum, J.-Y.; Carta, F.; Ward, C.; Mullen, P.; Harrison, D.;
Langdon, S. P.; Cecchi, A.; Scozzafava, A.; Kunkler, I.; Supuran, C. T.
Ureido-substituted sulfamates show potent carbonic anhydrase IX
inhibitory and antiproliferative activities against breast cancer cell lines.
Bioorg. Med. Chem. Lett. 2012, 22, 4681−4685.
(18) Hoigebazar, L.; Jeong, J. M.; Choi, S. Y.; Choi, J. Y.; Shetty, D.;
Lee, Y. S.; Lee, D. S.; Chung, J. K.; Lee, M. C.; Chung, Y. K. Synthesis
and characterization of nitroimidazole derivatives for 68Ga-labeling and
testing in tumor xenografted mice. J. Med. Chem. 2010, 53, 6378−
6385.
(19) Khalifah, R. G. The carbon dioxide hydration activity of carbonic
anhydrase. I. Stop-flow kinetic studies on the native human
isoenzymes B and C. J. Biol. Chem. 1971, 246, 2561−2573.
(20) Dubois, L.; Douma, K.; Supuran, C. T.; Chiu, R. K.; van
́
Zandvoort, M. A.; Pastorekova, S.; Scozzafava, A.; Wouters, B. G.;
Lambin, P. Imaging the hypoxia surrogate marker CA IX requires
expression and catalytic activity for binding fluorescent sulfonamide
inhibitors. Radiother. Oncol. 2007, 83, 367−373.
(21) Dubois, L.; Peeters, S. G.; van Kuijk, S. J.; Yaromina, A.;
Lieuwes, N. G.; Saraya, R.; Biemans, R.; Rami, M.; Parvathaneni, N. K.;
Vullo, D.; Vooijs, M.; Supuran, C. T.; Winum, J.-Y.; Lambin, P.
Targeting carbonic anhydrase IX by nitroimidazole based sulfamides
enhances the therapeutic effect of tumor irradiation: a new concept of
dual targeting drugs. Radiother. Oncol. 2013, 108, 523−528.
(22) Eriksson, A. E.; Jones, T. A.; Liljas, A. Refined structure of
human carbonic anhydrase II at 2.0 Å resolution. Proteins 1988, 4,
274−282.
(23) Winum, J.-Y.; Temperini, C.; El Cheikh, K.; Innocenti, A.; Vullo,
D.; Ciattini, S.; Montero, J. L.; Scozzafava, A.; Supuran, C. T. Carbonic
anhydrase inhibitors: clash with Ala65 as a means for designing
inhibitors with low affinity for the ubiquitous isozyme II, exemplified
by the crystal structure of the topiramate sulfamide analogue. J. Med.
Chem. 2006, 49, 7024−7031.
(24) Di Fiore, A.; Monti, S. M.; Innocenti, A.; Winum, J. Y.; De
Simone, G.; Supuran, C. T. Carbonic anhydrase inhibitors: crystallo-
graphic and solution binding studies for the interaction of a boron-
containing aromatic sulfamide with mammalian isoforms I−XV. Bioorg.
Med. Chem. Lett. 2010, 20, 3601−3605.
8520
dx.doi.org/10.1021/jm4009532 | J. Med. Chem. 2013, 56, 8512−8520