Development of the 99mTc-Hydroxamamide Complex as a Probe Targeting Carbonic Anhydrase IX

Article abstract of DOI:10.1021/acs.molpharmaceut.8b01120

Carbonic anhydrase IX (CA-IX) is regarded as a favorable target for in vivo imaging because of its specific expression in hypoxic regions of tumors. Hypoxia assists tumor propagation and growth and is resistant to chemotherapy and radiotherapy. Here, we designed and synthesized [^99mTc]hydroxamamide ([^99mTc]Ham) and [^99mTc]methyl-substituted-hydroxamamide ([^99mTc]MHam) complexes including a bivalent CA-IX ligand, sulfonamide (SA), and ureidosulfonamide (UR). In a cell binding assay, [^99mTc]Ham complexes with bivalent SA ([^99mTc]SAB2A and [^99mTc]SAB2B) and UR ([^99mTc]URB2A and [^99mTc]URB2B) showed significantly greater uptake into CA-IX high-expressing (HT-29) cells than that into CA-IX low-expressing cells. Since the binding affinity of [^99mTc]URB2A and [^99mTc]URB2B for CA-IX was significantly higher than that of [^99mTc]SAB2A and [^99mTc]SAB2B, we additionally synthesized [^99mTc]MURB2 (a [^99mTc]MHam complex with bivalent UR) and evaluated the CA-IX-specific binding affinity of [^99mTc]URB2A, [^99mTc]URB2B, and [^99mTc]MURB2. Their uptake into HT-29 cells was reduced by the addition of a CA inhibitor, acetazolamide, suggesting their CA-IX-specific binding affinity. A biodistribution study in HT-29 tumor-bearing mice was carried out using [^99mTc]URB2A and [^99mTc]MURB2 with the highest specificity for HT-29 cells. [^99mTc]URB2A showed moderate tumor uptake and reduction by coinjection with acetazolamide; however, the tumor/blood ratio was insufficient for in vivo imaging. These results provided key information for the design of novel Ham-based imaging probes targeting CA-IX.

Full text of DOI:10.1021/acs.molpharmaceut.8b01120

Article
pubs.acs.org/molecularpharmaceutics
Cite This: Mol. Pharmaceutics XXXX, XXX, XXX−XXX
Development of the 99mTc-Hydroxamamide Complex as a Probe
Targeting Carbonic Anhydrase IX
Shimpei Iikuni,† Keiichi Tanimura,† Hiroyuki Watanabe, Yoichi Shimizu, Hideo Saji,
and Masahiro Ono*
Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida
Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan
S
* Supporting Information
ABSTRACT: Carbonic anhydrase IX (CA-IX) is regarded as a favorable
target for in vivo imaging because of its specific expression in hypoxic
regions of tumors. Hypoxia assists tumor propagation and growth and is
resistant to chemotherapy and radiotherapy. Here, we designed and
synthesized [99mTc]hydroxamamide ([99mTc]Ham) and [99mTc]methyl-
substituted-hydroxamamide ([99mTc]MHam) complexes including a
bivalent CA-IX ligand, sulfonamide (SA), and ureidosulfonamide (UR).
In a cell binding assay, [99mTc]Ham complexes with bivalent SA
([99mTc]SAB2A and [99mTc]SAB2B) and UR ([99mTc]URB2A and
[
99mTc]URB2B) showed significantly greater uptake into CA-IX high-
expressing (HT-29) cells than that into CA-IX low-expressing cells. Since the binding affinity of [99mTc]URB2A and
[
[
[
99mTc]URB2B for CA-IX was significantly higher than that of [99mTc]SAB2A and [99mTc]SAB2B, we additionally synthesized
99mTc]MURB2 (a [99mTc]MHam complex with bivalent UR) and evaluated the CA-IX-specific binding affinity of
99mTc]URB2A, [99mTc]URB2B, and [99mTc]MURB2. Their uptake into HT-29 cells was reduced by the addition of a CA
inhibitor, acetazolamide, suggesting their CA-IX-specific binding affinity. A biodistribution study in HT-29 tumor-bearing mice
was carried out using [99mTc]URB2A and [99mTc]MURB2 with the highest specificity for HT-29 cells. [99mTc]URB2A showed
moderate tumor uptake and reduction by coinjection with acetazolamide; however, the tumor/blood ratio was insufficient for in
vivo imaging. These results provided key information for the design of novel Ham-based imaging probes targeting CA-IX.
KEYWORDS: carbonic anhydrase IX, technetium-99m, hydroxamamide, imaging
developed for the in vivo imaging of CA-IX. Fluorine-18 and
gallium-68-labeled probes could be used to successfully
visualize a CA-IX high-expressing tumor in a mouse
model.23−25 However, these probes are solely used for positron
emission tomography (PET), which is only available at limited
facilities. Therefore, it is necessary to develop nuclear medical
imaging probes for single photon emission computed
tomography (SPECT), which is more versatile than PET.
We previously reported an indium-111-labeled probe for
SPECT imaging of CA-IX, which successfully visualized CA-IX
high-expressing tumors in mice.26 Here, we developed a
SPECT imaging probe including a more useful radiometal,
technetium-99m (99mTc).
INTRODUCTION
■
Carbonic anhydrase IX (CA-IX) is specifically expressed in
hypoxic regions of tumors. Hypoxia assists tumor propagation
and growth and is resistant to chemotherapy and radio-
therapy.1−10 CA-IX catalyzes the reversible reaction of carbon
dioxide with water to bicarbonate ions and protons to maintain
cellular acid−base homeostasis.1,3,4,7−9 The CA-IX expression
is controlled by hypoxia-inducible factor-1α (HIF-1α), which
is reduced by von Hippel Lindau (VHL).1,3−5,7,11,12 In hypoxic
regions of tumors, the degradation of HIF-1α through VHL is
suppressed, resulting in the overexpression of CA-
IX,1,3−5,7,11,12 which is observed in various tumors.13−19 On
the other hand, CA-IX expression is primarily limited to the
gastrointestinal tract in normal tissue;20 therefore, CA-IX is
regarded as a favorable target for the in vivo imaging of tumors.
Several antibody-based nuclear medical imaging probes
targeting CA-IX have been reported. Among them, iodine-124-
labeled girentuximab showed a favorable property for the in
vivo imaging of CA-IX, and clinical studies were performed to
evaluate its efficacy and safety.21,22 However, they showed
pharmacokinetic limitations, such as slow blood clearance and
nontargeted tissue and nonspecific normal organ uptake,
raising issues of a low signal-to-background ratio and high
radiation toxicity. Small-molecule probes have also been
99mTc is the mainly used radioisotope in clinical practice. It
is available from a commercial molybdenum-99/technetium-
99m generator column, and its 141-keV γ ray is nearly optimal
for imaging with SPECT. The half-life of 99mTc is 6 h, which is
moderate for in vivo imaging. Therefore, 99mTc has played an
important part in nuclear medicine. In order to apply 99mTc to
Received: October 26, 2018
Revised: February 8, 2019
Accepted: March 7, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acs.molpharmaceut.8b01120
Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Article
molecular imaging, it is necessary to incorporate a chelating
moiety into an imaging probe. Many effective coordination
molecules for 99mTc have been developed, such as type N2S2
diamidedithiols, tricarbonyl, hydrazinonicotinic acid
(HYNIC), and diethylenetriaminepentaacetic acid (DTPA).27
Among them, we have previously reported several imaging
probes based on hydroxamamide (Ham).28
Ham is a chelating agent including no thiol, which can form
the 99mTc complex with high stability and a high radiochemical
yield.29,30 It can be synthesized easily and simply from nitrile in
a one-step reaction. Moreover, Ham provides a 99mTc complex
consisting of two Ham ligands, indicating that Ham would be
helpful in the preparation of a 99mTc complex with a bivalent
targeting vector.30 Because enhancement in the binding of a
multivalent ligand compared with its monovalent ligand was
reported,31,32 the [99mTc]Ham complex with a bivalent
compound may be a candidate for in vivo imaging.
temperature, water (50 mL) was added. The mixture was
extracted with ethyl acetate (30 mL × 2). The organic phases
were combined, dried over MgSO4, and filtered. After
evaporation of the filtrate, the residue was purified by silica
gel chromatography (ethyl acetate/hexane = 9/1) to give 72
1
mg of 1 (34% yield). H NMR (400 MHz, dimethyl sulfoxide
(DMSO)-d6, δ): 5.96 (s, 2H), 7.39 (s, 2H), 7.81 (m, 4H), 9.89
(s, 1H). 13C NMR (100 MHz, DMSO-d6, δ): 125.5 (2C),
125.8 (2C), 136.5 (1C), 144.1 (1C), 149.9 (1C). MS (ESI)
m/z: 216.1 [M + H]+.
Synthesis of 4-{[(4′-Cyanophenyl)carbamoyl]amino}-
benzene-1-sulfonamide (2). To a solution of 4-amino-
benzene-1-sulfonamide (172 mg, 1 mmol) in DMSO (5 mL)
was added 1,1-carbonyldiimidazole (195 mg, 1.2 mmol). The
reaction mixture was stirred at room temperature overnight. 4-
Aminobenzonitrile (118 mg, 1 mmol) was added, and the
reaction mixture was stirred at room temperature for 6 h. After
lyophilization of the reaction mixture, the residue was purified
by silica gel chromatography (chloroform/methanol = 9/1) to
give 41 mg of 2 (13% yield). 1H NMR (400 MHz, DMSO-d6,
δ): 7.24 (s, 2H), 7.64 (m, 4H), 7.75 (m, 4H), 9.29 (s, 1H),
9.36 (s, 1H). 13C NMR (100 MHz, DMSO-d6, δ): 112.4 (1C),
117.9 (2C), 118.3 (2C), 119.3(1C), 126.9 (2C), 133.4 (2C),
137.4 (1C), 142.3 (1C), 143.9 (1C), 151.9 (1C). MS (ESI)
m/z: 317.1 [M + H]+.
Synthesis of N′-Hydroxy-4-{[(4-sulfamoylphenyl)-
carbamoyl]amino}benzene-1-carboximidamide (3). To a
solution of 2 (20 mg, 0.06 mmol) in EtOH (5 mL) were
added hydroxylamine hydrochloride (13 mg, 0.2 mmol) and
triethylamine (26 μL, 0.2 mmol). The reaction mixture was
stirred and heated to reflux overnight. After being cooled to
room temperature, water (50 mL) was added. The mixture was
extracted with ethyl acetate (30 mL × 2). The organic phases
were combined, dried over MgSO4, and filtered. After
evaporation of the filtrate, the residue was purified by silica
gel chromatography (chloroform/methanol = 9/1) to give 18
mg of 3 (84% yield). 1H NMR (400 MHz, DMSO-d6, δ): 6.69
(s, 2H), 7.23 (s, 2H), 7.53 (d, J = 9.2 Hz, 2H), 7.64 (t, J = 8.4
Hz, 4H), 7.75 (d, J = 8.4 Hz, 2H), 10.19 (s, 1H), 10.32 (s,
1H), 10.66 (s, 1H). 13C NMR (100 MHz, DMSO-d6, δ): 117.1
(3C), 117.3 (3C), 126.9 (3C), 127.5 (2C), 136.8 (1C), 142.9
(1C), 152.4 (1C). MS (ESI) m/z: 350.1 [M + H]+.
In this study, we designed [99mTc]Ham complexes with the
aim of selective and specific in vivo imaging of CA-IX. We
selected two types of structures as CA-IX ligands, sulfonamide
(SA) and ureidosulfonamide (UR), and synthesized [99mTc]-
SAB2 and [99mTc]URB2. SA is a common structure
incorporated in many types of public CA inhibitors, such as
acetazolamide. UR has been reported as a highly effective
inhibitor of CA-IX.33 The bulky structures of these [99mTc]-
Ham complexes may contribute to cell membrane imperme-
ability according to Lipinski’s rule of five (molecular weight
≥500),34 preventing them from binding to cytosolic CA
isozymes. We also synthesized [99mTc]BHam, the [99mTc]Ham
complex without CA-IX ligand, as a negative control. We
evaluated their binding affinity using CA-IX high-expressing
cells, HT-29, and RCC4 plus vector alone (RCC4-VA). After
the most effective compound ([99mTc]URB2) was identified,
we designed a UR derivative-incorporated N-methyl-substi-
tuted hydroxamamide (MHam), which provides a [99mTc]-
MHam complex constituting two N-methyl-substituted Ham
ligands with high stability,35 and synthesized [99mTc]MURB2.
After confirming its cellular binding affinity, we evaluated the
biological properties of [99mTc]URB2 and [99mTc]MURB2 in
HT-29 tumor-bearing mice.
EXPERIMENTAL SECTION
■
Synthesis of 4-({[4-(5-oxo-4,5-Dihydro-1,2,4-oxadiazol-3-
yl)phenyl]carbamoyl}amino)benzene-1-sulfonamide (4). To
a solution of 3 (104 mg, 0.30 mmol) in DMSO (10 mL) was
added 1,1-carbonyldiimidazole (58 mg, 0.36 mmol). The
reaction mixture was stirred and heated gradually from room
temperature to 95 °C for 6 h. The reaction mixture was
lyophilized and purified by silica gel chromatography (chloro-
General. All reagents were purchased and used without
further purification unless otherwise indicated. Na99mTcO4 was
purchased from Nihon Medi-Physics Co., Ltd. (Tokyo, Japan).
Silica gel column chromatography was performed with W-Prep
2XY (Yamazen, Osaka, Japan) on a Hi Flash silica gel column
1
(40 μm, 60 Å; Yamazen). H NMR and 13C NMR spectra
were recorded using a JNM-ECS400 (JEOL, Tokyo, Japan)
with tetramethylsilane as an internal standard. Coupling
constants are reported in Hertz. Multiplicity was defined as
singlet (s), doublet (d), triplet (t), or multiplet (m). Mass
spectra were obtained using a Shimadzu LCMS-2020 (an LC-
20AT pump with an SPD-20A UV detector, λ = 254 nm;
Shimadzu) with a Cosmosil C18 column (5C18-AR-II, 4.6 mm
× 150 mm; Nacalai Tesque, Kyoto, Japan) was used for high-
performance liquid chromatography (HPLC).
Chemistry. Synthesis of N′-Hydroxy-4-sulfamoylben-
zene-1-carboximidamide (1). To a solution of 4-cyanoben-
zene-1-sulfonamide (182 mg, 1 mmol) in EtOH (10 mL) were
added hydroxylamine hydrochloride (208 mg, 3 mmol) and
triethylamine (416 μL, 3 mmol). The reaction mixture was
heated to reflux overnight. After being cooled to room
1
form/methanol = 9/1) to give 46 mg of 4 (42% yield). H
NMR (400 MHz, DMSO-d6, δ): 7.08 (s, 1H), 7.23 (s, 2H),
7.62 (m, 4H), 7.73 (m, 4H), 9.40 (s, 1H), 9.44 (s, 1H). 13C
NMR (100 MHz, DMSO-d6, δ): 117.8 (2C), 118.2 (2C),
118.3 (1C), 126.9 (2C), 127.2 (2C), 137.1 (1C), 142.5 (1C),
142.7 (1C), 152.2 (1C), 159.3 (1C), 163.1 (1C). MS (ESI)
m/z: 376.0 [M + H]+.
Synthesis of 4-({[4-(4-Methyl-5-oxo-4,5-dihydro-1,2,4-ox-
adiazol-3-yl)phenyl]carbamoyl}amino)benzene-1-sulfona-
mide (5). Iodomethane (55 μL, 0.88 mmol) and potassium
carbonate (55 mg, 0.44 mmol) were added to a solution of 4
(55 mg, 0.15 mmol) in N,N-dimethylformamide (DMF) (5
mL). The reaction mixture was stirred at room temperature for
3 h. After the addition of water (50 mL), the mixture was
B
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Article
extracted with ethyl acetate (30 mL × 2). The organic phases
were combined, dried over MgSO4, and filtered. After
evaporation of the filtrate, the residue was purified by silica
gel chromatography (chloroform/methanol = 9/1) to give 19
mg of 5 (34% yield). 1H NMR (400 MHz, DMSO-d6, δ): 3.24
(s, 3H), 7.24 (s, 2H), 7.64 (m, 2H), 7.69 (m, 4H), 7.75 (m,
2H), 9.32 (s, 1H), 9.34 (s, 1H). 13C NMR (100 MHz, DMSO-
d6, δ): 29.7 (1C), 116.3 (2C), 117.8 (2C), 118.3 (1C), 126.9
(2C), 129.3 (2C), 137.2 (1C), 142.5 (1C), 142.9 (1C), 158.9
(1C), 159.5 (1C). MS (ESI) m/z: 390.2 [M + H]+.
S ynt he sis o f N ′- Hy droxy -N-met h yl- 4-{ [( 4-
sulfamoylphenyl)carbamoyl]amino}benzene-1-carboximi-
damide (6). Compound 5 (19 mg, 0.05 mmol) was dissolved
in 1 M NaOH (aq)/DMF (1/1, 10 mL). The reaction mixture
was stirred at 95 °C for 14 h. After neutralization with 1 M
HCl (aq) in an ice bath, the mixture was extracted with ethyl
acetate (30 mL × 2). The organic phases were combined,
dried over MgSO4, and filtered. After evaporation of the
filtrate, the residue was purified by silica gel chromatography
(chloroform/methanol = 9/1) to give 7 mg of 6 (38% yield).
1H NMR (400 MHz, DMSO-d6, δ): 3.51 (s, 3H), 5.68 (d, J =
5.2 Hz, 1H), 7.33 (d, J = 8.0 Hz, 4H), 7.50 (d, J = 8.0 Hz, 2H),
7.62 (d, J = 8.0 Hz, 2H), 7.73 (d, J = 8.0 Hz, 2H), 9.51 (s,
1H), 9.79 (s, 1H), 10.00 (s, 1H). 13C NMR (100 MHz,
DMSO-d6, δ): 29.7 (1C), 117.4 (2C), 117.8 (2C), 126.9 (2C),
128.8 (2C), 136.9 (1C), 139.9 (1C), 142.8 (1C), 152.3 (1C),
155.2 (1C). MS (ESI) m/z: 364.1 [M + H]+.
Synthesis of N′-Hydroxybenzenecarboximidamide (7). To
a solution of benzonitrile (102 μL, 1 mmol) in EtOH (10 mL)
were added hydroxylamine hydrochloride (208 mg, 3 mmol)
and triethylamine (416 μL, 3 mmol). The reaction mixture was
heated to reflux for 2 h. After being cooled to room
temperature, water (50 mL) was added. The mixture was
extracted with ethyl acetate (30 mL × 2). The organic phases
were combined, dried over MgSO4, and filtered. After
evaporation of the filtrate, the residue was purified by silica
gel chromatography (ethyl acetate/hexane = 3/2) to give 22
mg of 7 (16% yield). 1H NMR (400 MHz, DMSO-d6, δ): 5.85
(s, 2H), 7.37 (m, 3H), 7.67 (m, 2H), 9.63 (s, 1H). 13C NMR
(100 MHz, DMSO-d6, δ): 125.4 (2C), 128.1 (2C), 128.9
(1C), 133.4 (1C), 150.9 (1C). MS (ESI) m/z: 137.1 [M +
H]+.
(400 MHz, DMSO-d6, δ): 3.37 (s, 3H), 7.62 (m, 2H), 7.68
(m, 1H), 7.75 (m, 2H). 13C NMR (100 MHz, DMSO-d6, δ):
29.5 (1C), 123.3 (1C), 128.4 (2C), 129.2 (2C), 132.0 (1C),
159.1 (1C), 159.4 (1C). MS (ESI) m/z: 177.0 [M + H]+.
Synthesis of N′-Hydroxy-N-methylbenzenecarboximida-
mide (10). Compound 9 (115 mg, 0.65 mmol) was dissolved
in 1 M NaOH (aq)/DMF (1/1, 10 mL). The reaction mixture
was stirred at 95 °C for 14 h. After neutralization with 1 M
HCl (aq) in an ice bath, the mixture was extracted with ethyl
acetate (30 mL × 2). The organic phases were combined,
dried over MgSO4, and filtered. After evaporation of the
filtrate, the residue was purified by silica gel chromatography
(chloroform/methanol = 9/1) to give 76 mg of 10 (78%
1
yield). H NMR (400 MHz, DMSO-d6, δ): 3.34 (s, 3H), 5.76
(d, J = 5.2 Hz, 2H), 7.40 (s, 5H), 9.59 (s, 1H). 13C NMR (100
MHz, DMSO-d6, δ): 30.4 (1C), 128.2 (2C), 128.8 (2C), 132.6
(1C), 155.4 (1C). MS (ESI) m/z: 151.1 [M + H]+.
Radiolabeling. Na99mTcO4 solution (100 μL) and tin(II)
tartrate hydrate solution (3.0 mM in water, 30 μL) were added
to a solution of 1, 3, 6, 7, or 10 (1 mg) in acetic acid/EtOH
(1/4, 100 μL). After a 10 min incubation at room temperature,
the mixture was purified by reversed-phase HPLC (RP-HPLC)
on a Cosmosil C18 column using a mobile phase (10 mM
phosphate buffer (pH 7.4)/acetonitrile = 9/1 (0 min) to 1/1
(30 min) ([99mTc]SAB2) or 4/1 (0 min) to 3/2 (30 min)
([99mTc]URB2, [99mTc]BHam, [99mTc]MURB2, and [99mTc]-
MBHam)) at a flow rate of 1.0 mL/min. After the HPLC
purification, the elusion buffer containing the 99mTc-labeled
compound was subject to a stream of argon gas to remove
acetonitrile. Subsequently, the 99mTc-labeled compound was
extracted with ethyl acetate, and the extract was concentrated
through a stream of argon gas. The residue was used after
being resolved with a solvent for each experiment.
Cell Culture. HT-29 human colorectal carcinoma cells and
RCC4-VA renal cell carcinoma cells, which are VHL-deficient,
were used as CA-IX high-expressing cells, and MDA-MB-231
human breast adenocarcinoma cells and RCC4 plus VHL
(RCC4-VHL) renal cell carcinoma (cell line RCC4 stably
transfected with pcDNA3-VHL) cells were used as CA-IX low-
expressing cells. HT-29 and MDA-MB-231 cells were
purchased from Sumitomo Dainippon Pharma (Osaka,
Japan). RCC4-VA and RCC4-VHL cells were purchased
from DS Pharma Biomedical (Osaka, Japan). All cells were
cultured in Dulbecco’s modified Eagle’s medium (DMEM)
(4000 mg/L of glucose; Nacalai Tesque), including 10% fetal
bovine serum and a 100 U/mL concentration of penicillin and
streptomycin at 37 °C in an atmosphere containing 5% CO2.
Cell Binding Assay. Cells (HT-29, MDA-MB-231, RCC4-
VHL, and RCC4-VA) were incubated in 12-well plates (4 ×
105 cells/well) at 37 °C in 95% air/5% CO2 for 24 h. After
removing the medium, the 99mTc-labeled compound (37 kBq)
in DMEM (1 mL) was added to each well, and the plates were
incubated at 37 °C in 95% air/5% CO2 for 2 h. Nonspecific
binding of [99mTc]MURB2, [99mTc]MBHam, and [99mTc]-
URB2 was evaluated in the presence of acetazolamide (2.0
μM) using HT-29 and MDA-MB-231 cells. The half-maximal
inhibitory concentration (IC50) was calculated by the addition
of acetazolamide with increasing concentrations (2000, 400,
80, 16, 3.2, 0.64, 0.128, 0.0256, and 0.00512 nM) using HT-29
cells. After incubation, the wells were washed with 1 mL of
phosphate-buffered saline (pH 7.4) (Thermo Fisher Scientific,
Massachusetts, U.S.A.), and the cells were lysed with 1 M
NaOH (aq) (0.2 mL × 2). Radioactivity in the cell solution
Synthesis of 3-Phenyl-1,2,4-oxadiazol-5(4H)-one (8). To a
solution of 7 (1013 mg, 7.4 mmol) in DMSO (20 mL) was
added 1,1-carbonyldiimidazole (1446 mg, 8.9 mmol). The
reaction mixture was stirred and heated gradually from room
temperature to 95 °C for 6 h. The reaction mixture was
lyophilized and purified by silica gel chromatography (chloro-
1
form/methanol = 9/1) to give 360 mg of 8 (30% yield). H
NMR (400 MHz, DMSO-d6, δ): 7.60 (m, 3H), 7.82 (d, J = 7.2
Hz, 2H), 12.98 (s, 1H). 13C NMR (100 MHz, DMSO-d6, δ):
123.7 (1C), 126.4 (2C), 129.7 (2C), 132.6 (1C), 157.7 (1C),
160.4 (1C). MS (ESI) m/z: 163.0 [M + H]+.
Synthesis of 4-Methyl-3-phenyl-1,2,4-oxadiazol-5(4H)-
one (9). Iodomethane (232 μL, 3.72 mmol) and potassium
carbonate (257 mg, 1.86 mmol) were added to a solution of 8
(100 mg, 0.62 mmol) in DMF (5 mL). The reaction mixture
was stirred at room temperature for 3 h. After the addition of
water (50 mL), the mixture was extracted with ethyl acetate
(30 mL × 2). The organic phases were combined, dried over
MgSO4, and filtered. After evaporation of the filtrate, the
residue was purified by silica gel chromatography (chloroform/
1
methanol = 9/1) to give 61 mg of 9 (55% yield). H NMR
C
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Scheme 1. Synthetic Route for Ham and MHam Compounds
was measured with a γ counter (Wallac 2470 Wizard;
PerkinElmer, Massachusetts, U.S.A.). The protein concen-
tration in the cell solution was determined using a
bicinchoninic acid protein assay kit (Thermo Fisher Scientific).
Stability in Murine Plasma Assay. The experiments with
animals were conducted according to our institutional
guidelines and approved by Kyoto University Animal Care
Committee. ddY mice (male, 5 weeks old) were purchased
from Japan SLC, Inc. (Shizuoka, Japan). After collection of
plasma (200 μL) from ddY mice, [99mTc]MURB2 or
RESULTS AND DISCUSSION
■
Chemistry. Synthesis of the Ham and MHam compounds
is shown in Scheme 1. The UR derivative 2 was synthesized by
reacting 4-aminobenzonitrile and 4-aminobenzene-1-sulfona-
mide with 1,1-carbonyldiimidazole. The reaction of 4-
cyanobenzene-1-sulfonamide, 2, or benzonitrile with hydroxyl-
amine gave Ham compounds according to a previously
reported method,30 which afforded 1, 3, and 7 in yields of
34, 84, and 16%, respectively. The precursors for [99mTc]-
MURB2 (6) and [99mTc]MBHam (10) were prepared in three
steps from the precursors for [99mTc]URB2 (3) and [99mTc]-
BHam (7), respectively. The reaction of 3 or 7 with 1,1-
carbonyldiimidazole provided compound 4 or 8 in DMSO.
Methylation of compounds 4 and 8 with iodomethane
provided compounds 5 and 9, which were decyclized by
heating in alkaline solution to compounds 6 and 10,
respectively (Scheme 1).
[
99mTc]URB2 (740 kBq) was added, and the solution was
incubated at 37 °C for 1, 4, and 7 h. After the addition of
acetonitrile (200 μL), they were centrifuged (4000g, 5 min).
Subsequently, the supernatant was analyzed by RP-HPLC. The
analytical method of RP-HPLC was the same as that written in
the Radiolabeling section.
Biodistribution Assay. Nude mice (BALB/c nu/nu; male,
5 weeks old) were purchased from Japan SLC, Inc. HT-29 or
MDA-MB-231 cells (1 × 107) were injected subcutaneously
into the left flank of mice. After 4 weeks, [99mTc]URB2A or
Radiolabeling. 99mTc labeling was performed using 99mTc
pertechnetate, the Ham or MHam precursor, and tin(II)
tartrate hydrate (Scheme 2). [99mTc]SAB2, [99mTc]URB2, and
[
99mTc]BHam were synthesized from the precursors 1, 3, and
[
99mTc]MURB2 (480 kBq, 150 μL in saline supplemented with
7, respectively. The RP-HPLC analysis of the reaction mixture
indicated that two kinds of 99mTc-labeled compounds were
generated as reported in previous reports on [99mTc]Ham
complexes, suggesting that two specific isomers of them were
given by the 99mTc labeling reaction using Ham (Figure
1A).30,35 Hereinafter, the isomers observed at shorter retention
times on RP-HPLC are referred to as the A-form ([99mTc]-
SAB2A, [99mTc]URB2A, and [99mTc]BHamA), and the others
as the B-form ([99mTc]SAB2B, [99mTc]URB2B, and [99mTc]-
BHamB). The 99mTc labeling reaction with 6 and 10 provided
a single radioactive peak at a retention time of 12.2
([99mTc]MURB2) and 13.5 min ([99mTc]MBHam) on RP-
HPLC, respectively (Figure 1B). In contrast, the reaction with
3 or 7 rendered two peaks at retention times of 9.7 and 12.7
min ([99mTc]URB2) or 7.5 and 15.5 min ([99mTc]BHam)
(Figure 1A). This suggests that introduction of the methyl
group into Ham would be useful to obtain a single
0.1% Tween 80) was intravenously injected, and the mice were
sacrificed at 1, 3, 6, or 24 h post-injection (p.i.) (n = 5 per
time-point). In the blocking experiment, acetazolamide (10
mg/kg mouse) was added to the injection. After collection of
the blood, organs of interest (spleen, pancreas, stomach,
intestines, kidneys, liver, heart, lungs, brain, muscle, and
tumor) were removed and weighed. The radioactivity in each
sample was measured with a γ counter (PerkinElmer).
Moreover, the thyroid of MDA-MB-231 tumor-bearing mice
was collected at 24 h p.i., and radioactivity was measured. The
results are expressed as a percentage of the injected dose per
gram (% ID/g) for selected organs, except the stomach and
thyroid, whose results are expressed as a percentage of the
injected dose.
Statistical Analysis. The significance of differences was
assessed by a Student’s t-test. Differences at the 95%
confidence level (P < 0.05) were regarded as significant.
[
99mTc]MHam complex.35 All 99mTc-labeled compounds
were obtained in a 14−61% radiochemical yield and over
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Scheme 2. 99mTc Labeling Route and Structure of
[
95% radiochemical purity without contamination by the
precursors, which were observed at 0−5 min of the retention
times on RP-HPLC (Table S1).
99mTc]Ham and [99mTc]MHam Complexes
Cell Binding Assay. We previously evaluated in vitro
expression of CA-IX in HT-29, MDA-MB-231, RCC4-VHL,
and RCC4-VA cells by Western blotting. A high CA-IX
expression was observed in HT-29 and RCC4-VA, and almost
no expression was present in the others.26 Between the A-form
and B-form of all [99mTc]Ham complexes, cellular uptakes
were not markedly different. The uptake of each [99mTc]Ham
complex with a bivalent CA-IX ligand ([99mTc]SAB2A,
[
99mTc]SAB2B, [99mTc]URB2A, and [99mTc]URB2B) into
HT-29 cells was significantly greater than that into MDA-
MB-231 cells. Moreover, the uptake into RCC4-VA cells was
also significantly greater than that into RCC4-VHL cells,
indicating marked selectivity for the CA-IX high-expressing
cells (Figure 2). However, [99mTc]BHamA and [99mTc]-
Figure 2. In vitro uptake of [99mTc]Ham complexes into cells. Values
are expressed as the mean
experiments. *P < 0.05, and P < 0.005 (Student’s t-test).
standard error of six independent
†
BHamB, [99mTc]Ham complexes with no CA-IX ligand, also
bound to HT-29 cells. In contrast, both complexes showed
almost no binding to RCC4-VA cells, suggesting that uptakes
of [99mTc]BHamA and [99mTc]BHamB into HT-29 cells would
not be due to CA-IX. The uptakes of [99mTc]URB2A and
[
[
99mTc]URB2B were greater than those of [99mTc]SAB2A and
99mTc]SAB2B. These results corresponded with a report that
UR showed a much greater affinity for CA isozymes as
compared with nonsubstituted SA.33
After the most effective compound ([99mTc]URB2) was
identified, we evaluated the CA-IX-specific binding affinity of
[
[
99mTc]MURB2 and [99mTc]MBHam (a negative control of
99mTc]MURB2) (Figure 3). The uptake of [99mTc]MURB2
into HT-29 cells was significantly reduced by the addition of
acetazolamide, indicating that [99mTc]MURB2 CA-specifically
bound to HT-29 cells. On the other hand, [99mTc]MBHam
also bound to HT-29 cells; however, this uptake was not
reduced by the addition of acetazolamide, suggesting that the
uptake of [99mTc]MBHam into HT-29 cells is not CA-IX-
specific, as with [99mTc]BHamA and [99mTc]BHamB. The
uptakes of [99mTc]URB2A and [99mTc]URB2B were also
reduced by the addition of acetazolamide, supporting the CA-
IX-specific binding.
In the acetazolamide inhibition assay, IC50 values of
acetazolamide in the presence of [99mTc]MURB2, [99mTc]-
URB2A, and [99mTc]URB2B were not significantly different
(22.7, 38.2, and 33.5 nM, respectively), suggesting that N-
methylation of Ham would not affect the CA-IX binding
affinity (Table 1 and Figure 4).
Figure 1. Radiochromatograms of [99mTc]Ham (A) and [99mTc]-
MHam (B) complexes.
For application to further studies, we selected two effective
compounds, [99mTc]URB2A and [99mTc]MURB2, which
E
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were 13.1 and 16.3%, respectively (n = 3). Both compounds
showed over 80% radiochemical purity until 7 h (Table 2).
Table 2. In Vitro Stability of [99mTc]MURB2 and
a
[
99mTc]URB2 in Murine Plasma
radiochemical purity (%) at given incubation times
compound
1 h
4 h
7 h
[
[
99mTc]MURB2
99mTc]URB2
93.9 1.4
90.7 2.3
90.2 2.3
83.5 2.3
86.9 4.1
80.0 3.1
a
Values are expressed as the mean
standard error of six
independent experiments.
Biodistribution. We previously confirmed that high and
low levels of CA-IX expression were present in HT-29 and
MDA-MB-231 tumor lysate from model mice, respectively.
Thus, HT-29 and MDA-MB-231 tumors were used as CA-IX
high-expressing and low-expressing tumors in vivo, respec-
tively.26 We examined the stability of [99mTc]URB2A and
Figure 3. In vitro uptake of [99mTc]MURB2, [99mTc]MBHam,
[
as the mean standard error of six independent experiments. *P <
0.005 (Student’s t-test).
99mTc]URB2A, and [99mTc]URB2B into cells. Values are expressed
[
99mTc]MURB2 in saline supplemented with 0.1% Tween 80.
The HPLC analysis after incubation for 1 h at room
temperature showed no marked decomposition of [99mTc]-
URB2A or [99mTc]MURB2, indicating that they were stable in
the injectate. The uptake of [99mTc]URB2A and [99mTc]-
MURB2 in each organ was expressed as % ID/g (Figure 5, and
Table 1. Half-Maximal Inhibitory Concentration (IC50, nM)
for the Binding of Acetazolamide to CA-IX Determined
Using [99mTc]Ham and [99mTc]MHam Complexes as
Ligands
a
compound
IC50 of acetazolamide (nM)
[
[
[
99mTc]MURB2
99mTc]URB2A
99mTc]URB2B
22.7 7.5
38.2 10.5
33.5 6.8
a
Values are expressed as the mean
independent experiments.
standard error of six
Figure 4. Inhibition curves of [99mTc]MURB2, [99mTc]URB2A, and
99mTc]URB2B from the inhibition assay for binding acetazolamide to
[
Figure 5. Radioactivity of extracted organs after intravenous injection
of [99mTc]URB2A (A) and [99mTc]MURB2 (B) in the HT-29 tumor-
bearing mice. Values are expressed as the mean standard deviation
of five mice. *Coinjection of acetazolamide (10 mg/kg mouse).
CA-IX. Values are expressed as the mean
independent experiments.
standard error of six
§
‡
†Values are expressed as % injected dose. P < 0.05, and P < 0.001
(Student’s t-test).
showed higher CA-IX-specificity, determined by the ratio of
uptake without acetazolamide to that with 2.0 μM
acetazolamide into HT-29 cells (4.4, 3.9, and 1.9 for
Tables S2 and S3). Tumor uptake of [99mTc]URB2A was 3.44,
3.13, 2.75, and 2.18% ID/g at 1, 3, 6, and 24 h p.i., respectively.
This uptake was markedly greater than that previously reported
using 99mTc-labeled CA-IX probes in HT-29 tumor-bearing
mice.36,37 The in vivo blocking studies were performed at the
latest time to minimize the possibility of nontargeted
accumulation of the 99mTc-labeled compound. Coinjection
with acetazolamide (10 mg/kg mouse) significantly reduced
the accumulation of [99mTc]URB2A in the HT-29 tumor at 24
h p.i. (1.62% ID/g), indicating CA-specificity of [99mTc]-
[
99mTc]MURB2, [99mTc]URB2A, and [99mTc]URB2B, respec-
tively).
Stability in Murine Plasma Assay. The stability of
99mTc]URB2 and [99mTc]MURB2 in murine plasma was
[
examined after incubation at 37 °C for 1, 4, and 7 h. The
recovery rates of radioactivity in the supernatant after
centrifugation were 86.9 and 83.7% for [99mTc]URB2 and
[
99mTc]MURB2, respectively (n = 3). In other words, the
protein-bound rates of [99mTc]URB2 and [99mTc]MURB2
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URB2A. Moreover, the accumulation of [99mTc]URB2A in the
HT-29 tumor (2.18% ID/g at 24 h p.i.) was significantly
greater than that in the MDA-MB-231 tumor (0.77% ID/g at
that time), suggesting marked in vivo selectivity for the CA-IX
high-expressing tumor (Figure 6). On the other hand, high
activity in the stomach would not be caused by the
−
accumulation of 99mTcO4 following collapse of the 99mTc
complex, because high in vitro stability in murine plasma was
confirmed. In addition, no marked accumulation of [99mTc]-
URB2A or [99mTc]MURB2 in the thyroid at 24 h p.i. was
observed (<0.1% injected dose for all evaluated mice),
suggesting their limited decomposition to pertechnetate.
Moreover, in our previous study, a [99mTc]MHam complex
showed marked accumulation in the stomach as compared
with the corresponding [99mTc]Ham complex (unpublished
data), suggesting that [99mTc]MHam complexes may generally
accumulate in the stomach by an active system, although the
details are unclear. On the other hand, radioactivity retention
in the blood was not significantly changed by N-methyl-
substitution of Ham, and the tumor/blood ratio was 0.3, 0.4,
0.5, and 1.0 at 1, 3, 6, and 24 h p.i., respectively. These results
suggest that [99mTc]URB2A is more effective for in vivo CA-IX
imaging than [99mTc]MURB2.
CONCLUSION
Figure 6. Radioactivity of extracted tumors at 24 h p.i. of
■
[
99mTc]URB2A or [99mTc]MURB2 in the HT-29 or MDA-MB-231
We designed and synthesized [99mTc]Ham and [99mTc]MHam
complexes with bivalent SA and UR and evaluated their
binding to CA-IX-expressing cells. An in vitro cellular binding
assay showed that bivalent [99mTc]Ham and [99mTc]MHam
complexes exhibited specific binding affinity for CA-IX. After
that, we evaluated the biodistribution of [99mTc]URB2A, which
showed the highest cellular binding affinity, and moderate
tumor uptake of [99mTc]URB2A was observed and reduced by
coinjection with the CA inhibitor, acetazolamide. These
studies provided key information for the design of novel
Ham-based imaging probes targeting CA-IX.
tumor-bearing mice. Values are expressed as the mean
standard
deviation of five mice. *P < 0.01 (Student’s t-test).
blood retention was observed at all times, and the tumor/
blood ratio was 0.4, 0.6, 0.7, and 1.0 at 1, 3, 6, and 24 h p.i.,
respectively. The low ratio should be improved because it
causes a high background signal on in vivo imaging. One
strategy to solve this problem is connecting two Ham by an
ethylene or propylene linker, which showed shorter blood
retention in a previous report.38 Meanwhile, [99mTc]URB2A
showed high kidney uptake, which was significantly blocked by
acetazolamide coinjection. It was reported, that other trans-
membrane CA isozymes (CA-IV and CA-XIV) are expressed
in the kidney;39 therefore, the kidney uptake of [99mTc]URB2A
might be caused by binding to these CA isozymes. A recent
report suggested that expression levels of CA-IV and CA-XIV
might be lower than that of CA-IX, and that blocking of
binding sites in the kidney and other normal organs with
nonradioactive CA ligands at suitable concentrations led to a
higher tumor/normal organ ratio.40 Moreover, the decrease in
kidney uptake of [99mTc]URB2A might be influenced by the
diuretic effect of acetazolamide.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.molpharma-
ceut.8b01120.
Tables of radiochemical yield and radiochemical purity
of [99mTc]Ham and [99mTc]MHam complexes, and data
on the biodistribution of [99mTc]URB2A and [99mTc]-
MURB2 in HT-29 tumor-bearing mice (PDF)
Tumor uptake of [99mTc]MURB2 was 2.10, 1.87, 1.45, and
0.73% ID/g at 1, 3, 6, and 24 h p.i., respectively. In contrast to
AUTHOR INFORMATION
■
[
99mTc]URB2A, coinjection of acetazolamide did not signifi-
Corresponding Author
cantly reduce the accumulation of [99mTc]MURB2 in the HT-
29 tumor at 24 h p.i. (0.57% ID/g). Because the in vitro CA-
IX-selective binding affinity of [99mTc]MURB2 was confirmed
by a cell binding assay, this nonsignificance is caused by a
change in pharmacokinetics through the N-methyl-substitution
of Ham. The accumulation of radioactivity was decreased in
not only the tumor but also the normal organs, suggesting that
radioactivity circulation was insufficient to specifically accu-
mulate in the tumor at 24 h p.i. On the other hand, the in vivo
selectivity of [99mTc]MURB2 for the HT-29 tumor as
compared with the MDA-MB-231 tumor was demonstrated
at 24 h p.i. (0.73 and 0.43% ID/g for HT-29 and MDA-MB-
231 tumors, respectively) (Figure 6). Interestingly, the slight
structural difference of Ham markedly reduced the kidney
uptake at 1 and 3 h p.i., and alternatively increased the stomach
*Phone: +81-75-753-4556; Fax: +81-75-753-4568; E-mail:
ono@pharm.kyoto-u.ac.jp.
ORCID
Hideo Saji: 0000-0002-3077-9321
Masahiro Ono: 0000-0002-2497-039X
Author Contributions
†S.I. and K.T. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The study was partially supported by a Grant-in-Aid for
Scientific Research (B) (Grant 26293274) and a Japan Society
for the Promotion of Science (JSPS) Research Fellowship for
Young Scientists (16J05493).
−
uptake at 1 h p.i. Although it is well-known that 99mTcO4
predominantly accumulates in the stomach, the high radio-
G
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anhydrase IX expression, a novel surrogate marker of tumor hypoxia,
is associated with a poor prognosis in non-small-cell lung cancer. J.
Clin. Oncol. 2003, 21 (3), 473−82.
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DOI: 10.1021/acs.molpharmaceut.8b01120
Mol. Pharmaceutics XXXX, XXX, XXX−XXX