Development of 99mTc-labeled asymmetric urea derivatives that target prostate-specific membrane antigen for single-photon emission computed tomography imaging

Article abstract of DOI:10.1016/j.bmc.2016.03.051

Prostate-specific membrane antigen (PSMA) is expressed strongly in prostate cancers and is, therefore, an attractive diagnostic and radioimmunotherapeutic target. In contrast to previous reports of PMSA-targeting ^99mTc-tricarbonyl complexes that are cationic or lack a charge, no anionic ^99mTc-tricarbonyl complexes have been reported. Notably, the hydrophilicity conferred by both cationic and anionic charges leads to rapid hepatobiliary clearance, whereas an anionic charge might better enhance renal clearance relative to a cationic charge. Therefore, an improvement in rapid clearance would be expected with either cationic or anionic charges, particularly anionic charges. In this study, we designed and synthesized a novel anionic ^99mTc-tricarbonyl complex ([^99mTc]TMCE) and evaluated its use as a single-photon emission computed tomography (SPECT) imaging probe for PSMA detection. Direct synthesis of [^99mTc]TMCE from dimethyl iminodiacetate, which contains both the asymmetric urea and succinimidyl moiety important for PSMA binding, was performed using our microwave-assisted one-pot procedure. The chelate formation was successfully achieved even though the precursor included a complicated bioactive moiety. The radiochemical yield of [^99mTc]TMCE was 12-17%, with a radiochemical purity greater than 98% after HPLC purification. [^99mTc]TMCE showed high affinity in vitro, with high accumulation in LNCaP tumors and low hepatic retention in biodistribution and SPECT/CT studies. These findings warrant further evaluation of [^99mTc]TMCE as an imaging agent and support the benefit of this strategy for the design of other PSMA imaging probes.

Full text of DOI:10.1016/j.bmc.2016.03.051

Bioorganic & Medicinal Chemistry 24 (2016) 2251–2256
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Development of 99mTc-labeled asymmetric urea derivatives that
target prostate-specific membrane antigen for single-photon
emission computed tomography imaging
Hiroyuki Kimura a,b, Sotaro Sampei a,y, Daiko Matsuoka a, Naoya Harada a, Hiroyuki Watanabe a,
Kenji Arimitsu c, Masahiro Ono a, Hideo Saji a,
⇑
a Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, 46-29, Yoshida Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan
b Department of Analytical and Bioinorganic Chemistry, Kyoto Pharmaceutical University, 5 Nakauchi-cho, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan
c School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women’s University, 11-68 Koshien Kyuban-cho, Nishinomiya, Hyogo 663-8179, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Prostate-specific membrane antigen (PSMA) is expressed strongly in prostate cancers and is, therefore, an
attractive diagnostic and radioimmunotherapeutic target. In contrast to previous reports of PMSA-target-
ing 99mTc-tricarbonyl complexes that are cationic or lack a charge, no anionic 99mTc-tricarbonyl com-
plexes have been reported. Notably, the hydrophilicity conferred by both cationic and anionic charges
leads to rapid hepatobiliary clearance, whereas an anionic charge might better enhance renal clearance
relative to a cationic charge. Therefore, an improvement in rapid clearance would be expected with either
cationic or anionic charges, particularly anionic charges. In this study, we designed and synthesized a
novel anionic 99mTc-tricarbonyl complex ([99mTc]TMCE) and evaluated its use as a single-photon emis-
sion computed tomography (SPECT) imaging probe for PSMA detection. Direct synthesis of [99mTc]
TMCE from dimethyl iminodiacetate, which contains both the asymmetric urea and succinimidyl moiety
important for PSMA binding, was performed using our microwave-assisted one-pot procedure. The che-
late formation was successfully achieved even though the precursor included a complicated bioactive
moiety. The radiochemical yield of [99mTc]TMCE was 12–17%, with a radiochemical purity greater than
98% after HPLC purification. [99mTc]TMCE showed high affinity in vitro, with high accumulation in
LNCaP tumors and low hepatic retention in biodistribution and SPECT/CT studies. These findings warrant
further evaluation of [99mTc]TMCE as an imaging agent and support the benefit of this strategy for the
design of other PSMA imaging probes.
Received 19 February 2016
Accepted 29 March 2016
Available online 30 March 2016
Keywords:
Prostate-specific membrane antigen
Single-photon emission computed
tomography
Anionic 99mTc-tricarbonyl complex
Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction
prostate cancer detection, staging, prognosis, and post-therapy
monitoring.
Prostate cancer is the most common malignancy in men in Eur-
ope and the United States.1 Although prostate-specific antigen
(PSA), a serum marker of prostate cancer, is useful for the early
diagnosis of prostate cancer,2 PSA levels provide no information
about the location, progression, and spread of prostate cancer. At
present, diagnostic imaging technologies, such as magnetic reso-
nance imaging (MRI), computed tomography (CT), positron emis-
sion tomography (PET), and single-photon emission computed
tomography (SPECT), are used to detect primary and metastatic
prostate cancer. In particular, imaging is clinically important for
Prostate-specific membrane antigen (PSMA), which is abun-
dantly expressed in prostate cancer, is an attractive diagnostic
and radioimmunotherapeutic target.3,4 Although PSMA is
expressed in the normal prostate tissue, as well as in primary pros-
tate cancer and lymph node metastases of prostate cancer,3 PSMA
expression 10- to 100-fold higher in prostate cancer than in normal
prostate tissue.5 Because PSMA is a membrane-anchored protein,
the locations of metastases of prostate cancer can be diagnosed
by PSMA imaging. Moreover, PSMA expression is upregulated in
patients with hormone-refractory prostate cancer.6
PSMA is homologous to glutamate carboxypeptidase-II,7–9 and
asymmetric urea compounds (X-CO-Glu; X represents the amino
acid) have been reported as glutamate carboxypeptidase-II (GCP-
II) inhibitors.10 We previously reported a drug design strategy for
⇑
Corresponding author. Tel.: +81 75 753 4566; fax: +81 75 753 4568.
E-mail address: hsaji@pharm.kyoto-u.ac.jp (H. Saji).
y
Contributed equally to this work.
http://dx.doi.org/10.1016/j.bmc.2016.03.051
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.

2252
H. Kimura et al. / Bioorg. Med. Chem. 24 (2016) 2251–2256
CO
OC
O
O
N
99mTc
OC
O
CO2H
O
O
S
O
S
123I
N
H
N
O
N
H
N
O
CO2H
CO2H
CO2H
CO2H
O
O
O
O
O
HO2C
N
H
N
H
HO2C
N
H
N
H
H
H
H
H
[
123I]IGLCE
[
99mTc]TMCE
Figure 1. Chemical structures of [123I]IGLCE and [99mTc]TMCE.
single-photon emission computed tomography (SPECT) using a
nucleophilic conjugate addition between maleimide-based
reagents and the thiol group of (S)-2-[3-[(R)-1-carboxy-2-mercap-
toethyl]ureido-pentanedioic acid (Cys-CO-Glu)].11 In that report,
we additionally identified a succinimidyl moiety as an important
PSMA-binding moiety, and prepared a novel PSMA-targeting agent
([123I]IGLCE; Fig. 1).11 However, [123I]IGLCE exhibited high hepatic
uptake and slow gastrointestinal clearance, characteristics that
would contraindicate the use of this agent for prostate cancer
imaging as prostate cancer most often metastasizes to the lower
spine, pelvis, and lymph nodes within the abdominal cavity. There-
fore, we concluded that decreased lipophilicity would be required
to develop more useful PSMA imaging probes.
2. Results and discussion
2.1. Drug design
We previously reported our work with the novel PSMA imaging
probes, [123I]IGLCE.11 Because [123I]IGLCE exhibited a high affinity
for PSMA, we hypothesized the existence of interactions between
[
123I]IGLCE and PSMA. We then identified a succinimidyl moiety
as an important factor in this high-affinity interaction through
structure–activity relationship and docking simulation studies.
However, [123I]IGLCE exhibited reduced tumor accumulation when
compared to previously reported PSMA probes, such as [123I]MIP-
1072,17,18 even though [123I]IGLCE had higher affinity for PSMA
than did [123I]MIP-1072.11
We considered that this lower tumor accumulation might be
attributable to the lower hydrophilicity of [123I]IGLCE, as [123I]
IGLCE exhibited high hepatic uptake in a biodistribution study.
High hepatic uptake likely led to rapid blood clearance and thus
prevented PSMA imaging probes from migrating to tumors
formed by LNCaP prostate adenocarcinoma cells. Therefore, we
concluded that both high affinity and increased hydrophilicity
should be conserved in order to improve tumor accumulation
and reduce the hepatic uptake of [123I]IGLCE. Therefore, we
designed an anionic 99mTc-tricarbonyl complex with high
hydrophilicity due to strong polarity and electric charges with
the intent to further decrease non-target tissue uptake and
enhance renal clearance.
Technetium-99m (99mTc) is the most frequently used nuclide
for SPECT imaging because it possesses radiochemical characteris-
tics such as a suitable gamma emission energy level (141 keV) and
adequate half-life (6.01 h). In addition, 99mTc is easily prepared
using a 99Mo–99mTc generator, which yields the 99mTc-containing
molecule Na99mTcO4. Monovalent 99mTc-tricarbonyl complexes
are expected to be available in the field of radiopharmaceuticals
because of their compact structure, which allows linkages with
low-molecular-weight bioactive ligands that have little influence
on physiological activities and stability in a living body.12,13 These
factors led to our decision to use 99mTc-tricarbonyl complexes in
this study.
Previous reports have described several 99mTc-tricarbonyl
PSMA-targeting agents,14–16 and suggested that higher
hydrophilicity, in addition to high affinity, could lead to
a
lower uptake in non-target tissues, such as the liver, and high
uptake in target tissues.14,15 Although several PSMA-targeting
99mTc-tricarbonyl complexes were developed,14–16 none of these
agents have been anionic 99mTc-labeled complexes. Accordingly,
we designed a novel anionic 99mTc-tricarbonyl complex ([99mTc]
TMCE; Fig. 1) and evaluated its binding affinity for PSMA
and biodistribution to confirm the importance of affinity,
hydrophilicity, and electric charge in the pharmacokinetics of
PSMA imaging probes.
2.2. Chemistry and radiochemistry
As shown in Scheme 1, the target asymmetric urea derivative 7,
a
99mTc-chelating precursor, was synthesized from a 4-aminobu-
tanoic acid (1). After esterification of 1, benzyl ester 2 was reacted
with methyl bromoacetate to yield iminodiacetate 3. Deprotection
of the benzyl group in 3 and subsequent condensation with N-
hydroxysuccinimide yielded active ester 5. Subsequently, 5 was
reacted with N-(5-aminopentyl)maleimide to result in amide 6,
OBn
(a)
MeO2C
MeO2C
N
OH
OBn
(b)
H2N
+H3N
Cl
O
O
O
1
2
3
O
OH
O
(d)
(c)
MeO2C
MeO2C
N
(e)
MeO2C
N
N
O
O
MeO2C
O
4
5
MeO2C
MeO2C
O
O
O
N
N
H
N
H
N
(f)
N
CO2H
CO2H
MeO2C
N
O
S
O
O
O
MeO2C
6
7
HO2C
N
H
N
H
H
H
Scheme 1. Synthesis of precursor 7. Reagents and conditions: (a) SOCl2, benzyl alcohol, 87%; (b) BrCH2COOCH3, DMF, 57%; (c) Pd/C, under H2, EtOAc, 85%; (d) NHS, WSCI,
DMF, 57%; (e) N-(5-aminopentyl)maleimide hydrochloride, DIEA, CH3CN, 90%; (f) Cys-CO-Glu, NaOH, H2O, CH3CN, 19%.

H. Kimura et al. / Bioorg. Med. Chem. 24 (2016) 2251–2256
2253
MeO2C
MeO2C
O
O
N
N
N
H
CO2H
O
S
O
7
HO2C
N
H
N
H
CO2H
H
H
Isolink kit
100 oC
Na99mTcO4
[
99mTc(CO)3(H2O)3]+
H2O
or
Microwave (110 oC)
[Re(CO)3(H2O)3]+
CO
OC
OC
O
O
N
M
O
O
O
N
N
H
Figure 2. In vitro selectivity assay. The uptake of [99mTc]TMCE into each cell after
incubation with or without 2-PMPA was evaluated; values are presented in units of
% ID/mg protein. Uptake was significantly higher into LNCaP (PSMA-positive) cells
than into PC-3 cells (PSMA-negative) and inhibited LNCaP cells (n = 3, *P <0.05).
CO2H
CO2H
O
S
O
O
HO2C
N
H
N
H
H
H
[
99mTc]TMCE
: M = 99mTc
Re-TMCE
: M = Re
Scheme 2. Microwave-assisted synthesis of [99mTc]TMCE and Re-TMCE.
of which the maleimide moiety was found to be important for con-
jugation with asymmetric urea and high affinity for PSMA.11 The
conjugate addition of the thiol group from Cys-CO-Glu to the mal-
eimide moiety in 6 formed the desired asymmetric urea derivative,
7, as a chelating precursor.
[
99mTc]TMCE was synthesized from precursor 7 and [99mTc
(CO)3(H2O)3]+ using a previously described microwave-assisted
one-pot procedure (Scheme 2).19 Generally, 99mTc-labeled reac-
tions cannot be performed using a conventional heating method
such as oil bath heating because of precursor decomposition.
Because our method does not incorporate conventional hydrolysis
procedures or a long reaction process, we expected that it would
eliminate decomposition of the bioactive ligand and subsequent
decreases in chemical yield and reproducibility. Indeed, the reac-
tion yielded [99mTc]TMCE with a radiochemical yield of 12–17%
(calculated from Na99mTcO4). The radiochemical purity after HPLC
purification was greater than 98%, and HPLC analysis of the puri-
fied [99mTc]TMCE showed a single peak (tR: 26.5 min) that closely
corresponded to Re-TMCE (tR: 26.7 min), which was synthesized
from [Re(CO)3(H2O)3]+ using a similar method. In summary, we
succeeded in synthesizing a novel anionic 99mTc-tricarbonyl com-
plex from a precursor with the complicated structure required
for PSMA binding through a microwave-assisted one-pot synthesis
procedure.
Figure 3. In vitro inhibition assay. Displacement curves of three radiolabeled
probes, [99mTc]TMCE, [125I]IGLCE, and [125I]DCIT, in an inhibition assay based on the
binding of 2-PMPA to PSMA. Values are shown as the means standard errors of
three independent experiments.
results suggest that all three probes have a similarly high affinity
for PSMA.
2.4. In vivo biodistribution and blocking studies
An in vivo biodistribution study of [99mTc]TMCE was performed
using LNCaP tumor-bearing mouse models to confirm the viability
of our design strategy. The biodistribution data of [99mTc]TMCE and
the tumor-to-blood, -muscle, and -liver ratios are shown in Table 1.
Table 1
In vivo biodistribution of [99mTc]TMCE: Each mouse was sacrificed at 5 min, 30 min,
or 2 h after injection. Accumulation in each tissue (except for stomach) was
evaluated; all values are presented in units of % ID/g tissue. Accumulation in the
stomach is presented in units of % ID/tissue. Values are expressed as means standard
deviations. Ratios are based on the % ID/g tissue values
2.3. In vitro cell binding assay (selectivity and inhibition assays)
The uptake of [99mTc]TMCE into LNCaP cells was evaluated to
clarify whether [99mTc]TMCE binds to membranous PSMA (see
Fig. 2). This uptake was significantly inhibited by the GCP-II inhibi-
tor, 2-PMPA. In addition, uptake into LNCaP cells was significantly
higher than that into PC-3 prostate cancer cells, and uptake into
the latter cells was non-significantly inhibited by 2-PMPA. These
results indicate that uptake of [99mTc]TMCE into LNCaP cells only
resulted from binding to PSMA.
Tissues (% ID/g)
Time after injection
30
5
120
0.6 0.1
Blood
Heart
Lung
Liver
11.4 2.3
4.1 1.1
9.6 1.2
5.7 1.0
3.2 0.5
1.7 0.4
3.0 0.3
2.3 0.3
136.0 6.4
0.4 0.2
2.3 0.2
18.8 4.6
1.5 0.2
1.0 0.6
12.8 2.2
4.1 1.3
16.8 10.5
5.6 1.5
0.2 0.0
0.4 0.0
1.1 0.1
56.8 20.6
0.1 0.0
1.7 0.4
4.9 3.8
0.3 0.1
0.3 0.1
5.0 2.7
9.3 5.8
14.4 2.7
4.4 2.4
To further evaluate the quantitative binding affinity of [99mTc]
TMCE for PSMA, we performed an inhibition binding assay in
LNCaP cells, using 2-PMPA as a competitive ligand. We evaluated
the binding affinities of [99mTc]TMCE, [125I]IGLCE, and [125I]DCIT21
for PSMA. The data (analyzed using Prism 5 software; GraphPad,
Inc., San Diego, CA, USA) are shown in Figure 3. 2-PMPA competed
with the uptake of all three PSMA-targeting probes into LNCaP
cells, with 2-IC50 values of 3.4, 0.20, and 0.080 nM in the presence
of [99mTc]TMCE, [125I]IGLCE, and [125I]DCIT, respectively. These
Kidney
124.9 26.2
1.1 0.2
3.3 0.8
15.9 4.6
3.8 1.0
1.5 0.5
4.0 1.2
0.4 0.2
3.0 1.5
0.7 0.3
Stomach
Intestine
Spleen
Pancreas
Muscle
LNCaP
LNCaP/blood
LNCaP/muscle
LNCaP/liver

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H. Kimura et al. / Bioorg. Med. Chem. 24 (2016) 2251–2256
the other hand, [99mTc]L8, a 99mTc-tricarbonyl complex without a
net charge, exhibited high tumor uptake, but slow hepatobiliary
and renal clearance.16 These results indicate that the hydrophilicity
associated with both cationic and anionic charges leads to rapid
hepatobiliary clearance and that anionic charges might enhance
renal clearance to a greater extent than cationic charges. Therefore,
more rapid clearance would be necessary for cationic or anionic
charges, particularly anionic charges.
2.5. SPECT study
A SPECT/CT study was performed on the basis of the biodistri-
bution study results (see Fig. 5). SPECT scanning was initiated
30 min and 2 h after [99mTc]TMCE injection because the tumor
uptake and tumor/blood ratio, respectively, were highest at these
time points. [99mTc]TMCE clearly distinguished LNCaP tumors from
PC-3 tumors on SPECT/CT images at both time points after injec-
tion. SPECT images additionally revealed that by 2 h postinjection,
Figure 4. In vivo blocking study. All mice were sacrificed 2 h after injection.
Accumulation in each tissue (except for stomach) was evaluated and is presented in
units of % ID/g tissue. Accumulation in the stomach is presented as % ID/tissue.
[
99mTc]TMCE exhibited good tumor uptake by LNCaP tumors
(12.8% ID/g at 30 min), and this accumulation was maintained for
at least 2 h. Because high uptake in LNCaP tumors was observed,
a blocking study was performed to confirm whether this tumor
uptake was due to PSMA-specific binding. Tumor uptake was sig-
nificantly inhibited (0.10% ID/g at 2 h) following co-injection of
[
99mTc]TMCE had been sufficiently excreted via the bladder to
allow clear SPECT images. These results correlated well with those
of the biodistribution study and suggested that [99mTc]TMCE
would be an efficient PSMA imaging probe.
*
the GCP-II inhibitor 2-PMPA (Fig. 4; n = 3, P <0.05).
As expected, [99mTc]TMCE exhibited lower hepatic accumu-
3. Conclusion
lation (1.1% ID/g at 2 h), when compared
[
123I]IGLCE, and
hepatic accumulation was not blocked in the blocking study,
suggesting that the hepatic accumulation of [99mTc]TMCE was
not due to PSMA-specific binding and that high hydrophilicity
led to low hepatic accumulation. [99mTc]TMCE also displayed
the expected enhanced hepatobiliary clearance. However,
remarkably enhanced renal clearance was not observed, and
the renal uptake of [99mTc]TMCE began to decrease at 2 h after
injection.
In this study, we designed and synthesized a novel 99mTc-
labeled PSMA-targeting agent via microwave-assisted synthesis
of [99mTc(CO)3(H2O)3]+ and dimethyl iminodiacetate derivative 7,
which was designed using [123I]IGLCE as a model structure. Our
microwave-assisted procedure was found to be applicable to
99mTc-complex formation using a complicated bioactive precursor
such as the asymmetric urea 7. [99mTc]TMCE exhibited high affinity
for PSMA in vitro, as well as high accumulation in LNCaP tumors
and low hepatic retention in the biodistribution and SPECT/CT
studies. In this study, the hydrophilicity of both cationic and anio-
nic charges led to rapid hepatobiliary clearance, although anionic
charges might enhance renal clearance to a greater extent. These
findings warrant the further evaluation of [99mTc]TMCE as an imag-
ing agent and support the benefit of this strategy for the design of
other PSMA imaging probes.
In accordance with a previous study, higher uptake of 99mTc-
MIP-1404, a cationic 99mTc-tricarbonyl complex, than of [99mTc]
TMCE was observed in the LNCaP tumor xenograft, and the rate
of uptake increased until 2 h after injection.14 Moreover, the
uptake of 99mTc-MIP-1404 in kidney only began to decrease at
4 h after injection, after maintenance for 2 h after injection.14 On
4. Experimental
4.1. General
W-Prep 2XY (Yamazen Corporation, Osaka, Japan) was used for
silica gel column chromatography on a Hi Flash silica gel column
(40 mm, 60 Å; Yamazen). Silica gel 60 F254 plates (0.5 mm; Merck
KGaA, Darmstadt, Germany) were used for preparative thin layer
chromatography (PTLC). An LC-20AD (Shimadzu Corporation,
Kyoto, Japan) equipped with an SPD-20A UV detector (q; 220
and 254 nm; Shimadzu) and an NDW-351 radioisotope detector
(Hitachi Aloka Medical, Ltd, Tokyo, Japan) was used for high-per-
formance liquid chromatography (HPLC). The eluent comprised a
binary mixture of 0.1% trifluoroacetic acid (TFA) in H2O (solvent
A) and 0.1% TFA in methanol (solvent B). Nanopure water was pre-
pared with an MQ Integra15 (Nihon Millipore, Tokyo, Japan). Mass
spectra were recorded on an LCMS-2010 EV (Shimadzu). 1H NMR
spectra were recorded on an LNM-AL500 (JEOL), using CDCl3 or
D2O (Euriso-top, Saint-Aubin, France) as the solvent and tetram-
ethylsilane or residual solvent as the internal standard. Microwave
reactions were performed using a CEM Discover. All chemicals
used in the syntheses were reagent grade. N-(5-Aminopentyl)-
maleimide hydrochloride was synthesized according to previously
reported methods.11
Figure 5. SPECT/CT images of [99mTc]TMCE. (A and B) coronal SPECT/CT images of
[
and D) transverse SPECT/CT images were obtained at 30 min (C) and 2 h (D) after
injection. In all images, [99mTc]TMCE clearly illuminated LNCaP tumors (red arrow)
but did not illuminate PC-3 tumors (green arrow).
99mTc]TMCE distribution were obtained at 30 min (A) and 2 h (B) after injection. (C

H. Kimura et al. / Bioorg. Med. Chem. 24 (2016) 2251–2256
2255
4.2. Animal experiments
yield 6 (695 mg, 89.8% yield). 1H NMR (400 MHz, CDCl3): d 6.70
(s, 2H), 6.17 (s, 1H) 3.71 (s, 6H), 3.50–3.53 (m, 4H), 3.20 (dd,
J = 6.96, 13.33 Hz, 2H), 2.73 (t, J = 6.38 Hz, 2H), 2.27 (t, J = 6.96 Hz,
2H), 1.75–1.81 (m, 2H), 1.57–1.64 (m, 4H), 1.48–1.56 (m, 2H),
1.24–1.34 (m, 2H); ESI-MS m/z: 412 [M+H]+.
Six-week-old male ddY mice were obtained from Japan SLC
(Kyoto, Japan). C.B.-17/Icr +/+ Jcl mice and C.B.-17/Icr scid/scid Jcl
mice were purchased from CLEA Japan (Tokyo, Japan). Animal
studies were conducted in accordance with institutional guideli-
nes, and the experimental procedures were approved by the Kyoto
University Animal Care Committee.
4.3.6. N-[(1R)-N-({2-[N-(5-{4-[Bis(2-methoxy-2-oxoethyl)
amino]-1-oxobut-1-yl}amino)pentyl]succinimid-3-yl}thio)-1-
carboxyethyl]carbamoyl-L-glutamic acid (7)
4.3. Synthesis
Product 6 (695 mg, 1.7 mmol) was added to a solution of Cys-
CO-Glu (547.4 mg, 1.9 mmol) in aqueous MeCN (3.0 mL). The mix-
ture was neutralized with 1 N NaOH and stirred overnight. Product
7 was purified by HPLC under the following conditions: YMC-Pack
ODS-AQ 20-mm ꢁ 250-mm column, rate of 5 mL/min, solvent A/B
gradient of 40/60 (0 min) to 90/10 (60 min), and 40–90% gradient
of 0.1% TFA in methanol over 60 min (220.1 mg, 18.5% yield). The
following characteristics were determined: 1H NMR (400 MHz,
D2O): d 4.45 (d, J = 4.58 Hz, 1H), 4.17–4.23 (m, 5H), 3.94–4.00 (m,
1H), 3.75 (s, 6H), 3.41 (t, J = 6.87 Hz, 2H), 3.31 (t, J = 7.16 Hz, 2H),
3.16–3.25 (m, 1H), 3.13 (t, J = 5.15 Hz, 1H), 3.07 (t, J = 6.87 Hz,
2H), 2.96–3.01 (m, 1H), 2.60 (dq, J = 18.90, 4.01 Hz, 1H), 2.42 (t,
J = 7.16 Hz, 2H), 2.35 (t, J = 6.59 Hz, 2H), 2.09 (td, J = 13.60,
6.87 Hz, 1H), 1.84–1.96 (m, 3H), 1.85–1.88 (m, 3H), 1.38–1.50 (m,
4H), 1.14–1.20 (m, 2H); ESI-MS m/z: 706 [M+H]+.
4.3.1. 4-(Benzyloxy)-4-oxobutan-1-aminium chloride (2)
4-Aminobutanoic acid (1.03 g, 10 mmol) was added to benzyl
alcohol (10 mL) on the ice. Thionyl chloride (1.1 mL, 1.5 mmol)
was then added to the solution at 0 °C and stirred for 2 h at room
temperature. After the solution changed to yellow, the solvent was
evaporated and recrystallized to yield 2 (2.00 g, 87.0% yield), with
the following characteristics: 1H NMR (400 MHz, CDCl3) d: 8.23 (s,
3H), 7.25–7.37 (m, 5H), 5.07 (s, 2H), 3.09 (br, 2H), 2.50 (br, 2H),
2.10 (br, 2H); ESI-MS m/z: 194 [MꢀCl]+.
4.3.2. Benzyl 4-[bis(2-methyoxy-2-oxoethyl)amino]butanoate
(3)
Methyl bromoacetate (0.42 mL, 4.5 mmol) was added to a solu-
tion of 2 (345 mg, 1.5 mmol) in DMF (3.5 mL). The reaction mixture
was then stirred overnight at 80 °C, quenched with water, and
extracted with EtOAc. The resultant extract was washed with brine
and dried over Na2SO4. The solvent was removed using a rotary
vacuum evaporator, and the residue was purified by silica gel chro-
matography (Hexane/EtOAc = 2:1) to yield 3 (289 mg, 57.0% yield)
with the following characteristics: 1H NMR (400 MHz, CDCl3) d:
7.35–7.36 (m, 5H), 5.11 (s, 2H), 3.69 (s, 6H), 3.53 (s, 4H), 2.75 (t,
J = 6.96 Hz, 2H), 2.44 (t, J = 7.54, 2H), 1.77–1.84 (m, 2H); ESI-MS
m/z: 338 [M+H]+.
4.3.7. Re-TMCE
First, 50
l
L of [Re(CO)3(H2O)3]+ were added to a solution of 7
mol) in water (450 L); the resulting mixture was
(3.5 mg, 5.0
l
l
neutralized with 1 N NaOH and heated in a microwave reactor at
110 °C, 17 bars (max), and 300 W (max) for 5 min. Product 8 was
purified by HPLC under the following conditions: 5C18-AR-II 4.6-
mm ꢁ 150-mm column, rate of 1.0 ml/min, and solvent A/B gradi-
ent of 85/15 (0 min) to 20/80 (60 min). ESI-MS m/z: 945 [MꢀH]ꢀ.
4.4. Radiosynthesis
4.3.3. 4-[Bis(2-methoxy-2-oxoethyl)amino]butanoic acid (4)
Palladium on carbon (100 mg) was added to a solution of 3
(441 mg, 1.3 mmol) in EtOAc (5.0 mL). The reaction was stirred
for 1 h under hydrogen atmosphere and filtered through celite,
and the solvent was removed using a rotary vacuum evaporator
to obtain 4 (274 mg, 85.3% yield), with the following characteris-
tics: 1H NMR (400 MHz, CDCl3) d: 3.72 (s, 6H), 3.58 (s, 4H), 2.82
(t, J = 6.38 Hz, 2H), 2.52 (t, J = 6.67 Hz, 2H), 1.77–1.84 (m, 2H);
ESI-MS m/z: 248 [M+H]+.
In the present study, [99mTc(CO)3(H2O)3]+ was prepared as pre-
viously reported.15 Briefly, Na99mTcO4 (37 MBq) solution was
added to an IsoLink kitTM (Mallinckrodt Medical, Netherlands) and
heated at 100 °C for 20 min.19 First, 450 L of [99mTc(CO)3(H2O)3]+
l
was added to 50 lL of a solution of 7 (1.0 mM) in water. Next, the
mixture was neutralized with 3 N HCl and heated in a microwave
reactor at 110 °C, 17 bar (max), and 300 W (max) for 5 min. Purifi-
cation was conducted using preparative HPLC (5C18-AR-II 4.6-
mm ꢁ 150-mm column, rate of 1.0 ml/min, solvent A/B gradient
of 85/15 (0 min) to 20/80 (60 min)). [99mTc]TMCE was obtained
with a radiochemical yield of 14% (5.2 MBq, overall decay cor-
rected) and the radiochemical purity of >98%.
4.3.4. Succinimidyl 4-[bis(2-methoxy-2-oxoethyl)amino]
butanoate (5)
N-Hydroxysuccinimide (380 mg, 3.3 mmol) and WSCI (630 mg,
3.3 mmol) were added to a solution of 4 (816 mg, 3.3 mmol) in
DMF (10 mL). The reaction mixture was stirred overnight under
an argon atmosphere at room temperature, quenched with satu-
rated aqueous NaHCO3, and extracted with EtOAc. The extract
was washed with brine and dried over Na2SO4. The solvent was
removed using a rotary vacuum evaporator, and the residue were
purified via silica gel chromatography (Hexane/EtOAc = 1:1) to
obtain 5 (646 mg, 56.9% yield). ESI-MS m/z: 345 [M+H]+ and 343
[MꢀH]ꢀ.
4.5. Cell lines and mouse models
Two human prostate carcinoma cell lines were purchased from
DS Pharma Biomedical (Osaka, Japan): LNCaP (PSMA-positive) and
PC-3 (PSMA-negative).6 The cells were cultured in Roswell Park
Memorial Institute 1640 (RPMI 1640) medium supplemented with
10% fetal bovine serum, glutamine, and antibiotics (penicillin/
streptomycin) in a humidified CO2 incubator (37 °C/5% CO2), as
previously reported.20
4.3.5. N-[5-(Maleimidyl)pentyl]-4-[bis(2-methoxy-2-oxoethyl)
amino]butanamide (6)
To a solution of 5 (646 mg, 1.9 mmol) in MeCN (30 mL), N-(5-
aminopentyl)maleimide hydrochloride (411 mg, 1.9 mmol) and
Cultured cells were treated with 2.5 g/L trypsin/1 mM EDTA and
re-suspended in phosphate-buffered saline. Subsequently, C.B.-17/
Icr +/+ Jcl mice and C.B.-17/Icr scid/scid Jcl mice were each injected
with 100 l
L of a 1:1 mixture of cell suspension and BD MatrigelTM
DIEA (647
lL, 3.8 mmol) were added. After the reaction was stirred
Basement Membrane Matrix (1–5 ꢁ 106 cells/mouse) in the right
(PC-3) or left (LNCaP) flank. Tumor-bearing mice were used for
studies when tumors reached a diameter of approximately 10–
15 mm.
for 4 h, the reaction mixture was quenched with water and
extracted with EtOAc. The extract was washed with brine and
dried over Na2SO4. The solvent was removed under vacuum to

2256
H. Kimura et al. / Bioorg. Med. Chem. 24 (2016) 2251–2256
4.6. In vitro cell binding assay (selectivity assay)
vein. The mice were placed on a heating pad to maintain body
temperature throughout the procedure.
SPECT scans were acquired over periods of 30–64 and
120–154 min after injection; CT scans were performed for
To confirm that [99mTc]TMCE binds selectively to PSMA, we per-
formed cell uptake assays. LNCaP and PC-3 cells were incubated in
12-well plates (4 ꢁ 105 cells/well) for 48 h at 37 °C and 5% CO2. The
medium was removed, and each well was washed twice with
anatomic reference (50
In the SPECT studies, all projection data were acquired using a
20% energy window centered at 140 keV for 99mTc, with
a
lm spatial resolution, 60 kV, and 310 lA).
500
l
L of assay medium (RPMI 1640 supplemented with 0.5%
bovine serum albumin). Next, 500
l
L of [99mTc]TMCE (final con-
35-mm radius of rotation, 360° circular orbit, 60-s projection time,
and 32 projection angles. Single-pinhole collimators (1.0-mm
diameter, 9.0-mm focal length) were used. SPECT images were
reconstructed using three-dimensional ordered-subset expectation
maximization, and CT images were reconstructed using a modified
three-dimensional cone-beam Feldkamp algorithm resulting in a
centration, 74 kBq/mL) were added to each well, and the plates
were incubated at 37 °C for 1 h. Nonspecific binding was evaluated
by adding 1.0 mM 2-(phosphonomethyl)pentanedioic acid (2-
PMPA), a GCP-II inhibitor. After incubation, each well was washed
twice with 500
with 0.2 N NaOH. Radioactivity bound to cells was measured with
-counter.
lL of fresh assay medium, and the cells were lysed
0.177 ꢁ 0.177 ꢁ 0.177 mm3 voxel size for
a
512 ꢁ 512 ꢁ 512
a
c
image volume. Acquired SPECT and CT data sets were processed
using AMIRA software (version 5.1; FEI Company, Hillsboro,
Oregon, USA).
4.7. In vitro cell binding assay (inhibition assay)
The affinities of the novel compounds [125I]IGLCE and [125I]DCIT
were determined through an in vitro inhibition binding assay, as
previously reported.11 Specifically, we compared the half-maximal
inhibitory concentration (IC50) values of [99mTc]TMCE with the
above-mentioned compounds to confirm the high affinity of
4.11. Statistical analysis
GraphPad Prism 5 software (GraphPad Software Inc., San Diego,
CA, USA) was used for the statistical analyses. The Bonferroni
multiple-comparison test was used to assess statistical differences
in the biodistribution study. A P value of <0.05 was considered
statistically significant.
[
99mTc]TMCE for PSMA. LNCaP cells were incubated in 12-well
plates (4 ꢁ 105 cells/well) for 48 h at 37 °C and 5% CO2. The med-
ium was removed, and each well was washed twice with 500 lL
of assay medium. Next, 500
([99mTc]TMCE, [125I]IGLCE, and [125I]DCIT;21 final concentration,
74 kBq/mL) and 2-PMPA (final concentration, 10 p–10 M), a
GCP-II inhibitor, were added to each well, and the plates were
incubated at 37 °C for 1 h. After incubation, each well was washed
l
L of the radiolabeled compound
Acknowledgments
l
This work was partly supported by the Practical Research for
Innovative Cancer Control from the Japan Agency for Medical
Research and Development (AMED), a Grant-in-Aid for Young
Scientists (A) Grant Number 25713046 from the Japan Society for
the Promotion of Science.
twice with 500
with 0.2 N NaOH. Radioactivity bound to cells was measured with
-counter. Finally, we calculated the IC50 values using GraphPad
lL of fresh assay medium, and the cells were lysed
a
c
Prism 5.0.
References and notes
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4.8. In vivo biodistribution study
The distribution of [99mTc]TMCE in vivo was directly evaluated
in model mice after determining a sufficiently high-affinity level.
[
99mTc]TMCE (100
lL) was injected via the tail vein into tumor-
bearing mice weighing 20–24 g (n = 3 for each time point). Mice
were sacrificed by decapitation at the following fixed time points
post-injection: 5 min, 30 min, and 2 h. Tissue weights and radioac-
tivity levels were measured, and [99mTc]TMCE uptake was evalu-
ated as a percentage of the injected dose per gram of tissue (%
ID/g).
4.9. In vivo blocking study
A blocking study was performed to confirm in vivo PSMA-speci-
fic binding. Briefly, 100 l
L of [99mTc]TMCE were coinjected with 2-
PMPA (10 mg/kg) into tumor bearing mice (weight 22–24 g) via
the tail vein. Mice were decapitated 2 h after injection. Tissue
weights and radioactivity levels were measured, and [99mTc]TMCE
uptake was evaluated as% ID/g.
4.10. Small animal SPECT imaging
SPECT/CT studies were performed using a small animal scanner
(FX3300 Imager; SII NanoTechnology Inc., Northridge, CA, USA).
LNCaP and PC-3 tumor-bearing mice (20–25 g) were anesthetized
with isoflurane (2.5% in an air mixture). Each mouse was injected
with [99mTc]TMCE (9.45 MBq in 0.3 mL isotonic saline) via the tail