Structure–activity relationships of succinimidyl-Cys-C(O)-Glu derivatives with different near-infrared fluorophores as optical imaging probes for prostate-specific membrane antigen

Article abstract of DOI:10.1016/j.bmc.2018.03.015

Prostate-specific membrane antigen (PSMA), which is overexpressed in malignant prostate cancer (PCa), is an ideal target for imaging and therapy of PCa. We previously reported a PSMA imaging probe, 800CW-SCE, based on succinimidyl-Cys-C(O)-Glu (SCE) for o

Full text of DOI:10.1016/j.bmc.2018.03.015

Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure–activity relationships of succinimidyl-Cys-C(O)-Glu
derivatives with different near-infrared fluorophores as optical imaging
probes for prostate-specific membrane antigen
a
a
a
b
b
Daiko Matsuoka , Hiroyuki Watanabe , Yoichi Shimizu , Hiroyuki Kimura , Yusuke Yagi ,
a
a
a,
⇑
Ryoko Kawai , Masahiro Ono , Hideo Saji
a
Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, 46-29, Yoshida Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan
Department of Analytical and Bioinorganic Chemistry, Kyoto Pharmaceutical University, 5 Nakauchi-cho, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan
b
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), which is overexpressed in malignant prostate cancer (PCa),
is an ideal target for imaging and therapy of PCa. We previously reported a PSMA imaging probe, 800CW-
SCE, based on succinimidyl-Cys-C(O)-Glu (SCE) for optical imaging of PCa. In this study, we investigated
the structure–activity relationships of novel SCE derivatives with five different near-infrared (NIR) fluo-
rophores (IRDye 680LT, IRDye 750, Indocyanine Green, Cyanine 5.5, and Cyanine 7) as optical imaging
probes targeting PSMA. An in vitro binding assay revealed that 800CW-SCE, 680LT-SCE, and 750-SCE
exhibited higher binding affinity than 2-PMPA, which is known as a PSMA inhibitor. These three SCE
derivatives were internalized into PSMA-positive cells (LNCaP cells) but not into PSMA-negative cells
Received 15 December 2017
Revised 9 March 2018
Accepted 9 March 2018
Available online xxxx
Keywords:
Succinimidyl-cysteine-glutamate
Prostate-specific membrane antigen
Optical imaging
(PC-3 cells). In the in vivo imaging study, 800CW-SCE and 750-SCE were highly accumulated in LNCaP
tumors but not in PC-3 tumors, and the ratio of LNCaP/PC-3 accumulation of 800CW-SCE was higher than
that of 750-SCE. The present study may provide valuable molecular design information for the future
development of new PSMA imaging probes based on the SCE scaffold.
IRDye 800CW
IRDye 680
IRDye 750
Ó 2018 Elsevier Ltd. All rights reserved.
1
. Introduction
to change to membrane-bound protein in PCa.16 The level of PSMA
expression rises with tumor aggressiveness, metastatic disease,
1
4,17,18
Prostate cancer (PCa) is one of the most prevalent cancers
and disease recurrence.
Thus, PSMA has gained increasing
1
worldwide. Although most patients will have indolent cancers, a
few men with aggressive forms of PCa experience metastasis
reaching the stage of castrate-resistant metastatic PCa within a
few years, which is the main cause of disease-related morbidity
attention as a target molecule for imaging and therapy.
For the detection of PSMA, optical imaging is an extremely
attractive tool in both preclinical and clinical settings due to its
easy usage and acceptable sensitivity. Especially, intraoperative
near-infrared (NIR) fluorescence imaging provides information
about cancer localization and excision margins by detecting fluo-
rescent signals from imaging probes that can label target
2
–4
and mortality.
For surgeons, evaluation of the localization and
invasion of PCa during surgery is challenging because cancers are
often invisible.
tumor recurrence and subsequent metastasis.
5
,6
Failure to completely resect a tumor leads to
7–11
19,20
The develop-
molecules.
ment of intraoperative diagnostic tools that will improve the qual-
ity of prostatectomies by visualization of aggressive PCa for
prevention of biochemical recurrence is therefore of utmost
importance.
A number of low-molecular-weight PSMA imaging probes for
2
1
nuclear medical imaging have been developed, and promising
6
8
22 68
23
18
probes, e.g., Ga-PSMA-11,
Ga-PSMA-617, and F-PSMA-
2
4
1007, have been used in clinical trials. However, there has been
no report on the development of promising NIR fluorescent probes
targeting PSMA for optical imaging that can be utilized clinically. In
basic evaluations, some groups reported the development of NIR
fluorescent probes based on phosphoramidates derivatives.2
Regarding urea-based glutamate heterodimers, some groups
including Ying Chen et al. and Kai Bao et al. reported on Lys-C
The overexpression of prostate-specific membrane antigen
2,13
(
PSMA), a membrane-type zinc protease,1
is one of the most
PSMA is located in
14,15
common phenomena in patients with PCa.
5,26
the cytosol in normal prostate cells and its localization is known
⇑
Corresponding author.
E-mail address: hsaji@pharm.kyoto-u.ac.jp (H. Saji).
2
7,28
(O)-Glu derivatives.
Another group, Xinning Wang et al.,
https://doi.org/10.1016/j.bmc.2018.03.015
968-0896/Ó 2018 Elsevier Ltd. All rights reserved.
0
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2
D. Matsuoka et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
0
reported
(
the
Glu-C(O)-Glu -Amc-Ahx-Glu-Glu-Glu-Lys-NH
2
logical polar surface area (TPSA) of SCE derivatives were assessed.
Cys-C(O)-Glu is a negatively charged small molecule with TPSA of
191.83 and three anionic sulfonic acid groups. Thus, the overall
hydrophilicity and polarity of the mixed products were increased
compared to the unconjugated NIR fluorophores. 800CW-SCE,
680LT-SCE, and 750-SCE had the highest hydrophilicity and
strong polarity among the six compounds. Optical properties,
including maximum excitation wavelength (kEx), maximum
emission wavelength (kEm), and quantum yield, of unconjugated
NIR fluorophores and SCE derivatives were also assessed
(Tables 1 and S1). The optical properties of all mixed products
were consistent with those of the respective unconjugated NIR
fluorophores, which suggests that Cys-C(O)-Glu does not affect
the optical properties of the dye.
2
9
PSMA-1) derivatives.
Against this background, we aimed to develop several imaging
probes targeting PSMA based on Cys-C(O)-Glu for optical imaging.
We formerly found that the succinimidyl group was important for
high affinity to PSMA; the binding affinity of succinimidyl-Cys-C
(
O)-Glu (SCE) was approximately six times higher than that of
3
0–32
Cys-C(O)-Glu alone.
Based on this knowledge, we previously
reported that a new SCE-based compound, 800CW-SCE, which
includes IRDye 800CW as a NIR fluorophore moiety, had potential
as an PSMA imaging probe for optical imaging under both in vitro
and in vivo conditions.33 In an in vitro cellular uptake study,
8
00CW-SCE was internalized into PSMA-positive cells (LNCaP
cells) but not into PSMA-negative cells (PC-3 cells). In an in vivo
imaging study using tumor-bearing mice, 800CW-SCE exhibited
PSMA-specific accumulation to LNCaP tumors, and the probe
showed the highest accumulation ratio of LNCaP/PC-3 tumors at
2.3. Inhibition assay
2
4 h after intravenous injection with a low background signal from
To determine the binding affinity of the newly synthesized
compounds to PSMA, we conducted competition binding studies
the whole body.
1
25
43
Interestingly, 800CW-SCE showed different pharmacokinetics
with
LNCaP cells. 2-PMPA, known as a PSMA inhibitor, was chosen
as a reference compound. The inhibition constant (K ) values
I-DCIT, known as a PSMA ligand, for PSMA-positive
1
23
99m
18
from
I-IGLCE,
Tc-TMCE, and F-10a, which are the SCE-
based nuclear medical imaging probes for PSMA that we developed
previously.
i
3
0–32
These findings suggested that the differences in
summarized in Table 2 were calculated from the IC50 values of
competitive binding curves of these compounds. The affinity of
SCE derivatives for PSMA varied, with K values of 13.2 to 579
i
nM. Among these compounds, the affinities of 800CW-SCE,
680LT-SCE, and 750-SCE were approximately 7.8, 7.2, and 3.5
times higher than that of 2-PMPA, respectively. In contrast, the
affinities of ICG-SCE, Cy 5.5-SCE, and Cy 7-SCE were approxi-
mately 2.4, 3.9, and 5.6 times lower than 2-PMPA, respectively.
Recent papers reported some PSMA imaging probes containing
signal-emitting moiety affected the pharmacokinetics of the imag-
ing probes, and SCE derivatives with different NIR fluorophores
may show better pharmacokinetics than 800CW-SCE. This possibil-
ity led us to develop other SCE-based compounds with different
fluorophores from IRDye 800CW.
IRDye 800CW is a cyanine-type NIR fluorophore and is already
employed in clinical studies.3
4,35
This time, we chose five cyanine-
type NIR fluorophores: IRDye 680LT, IRDye 750, Indocyanine Green
ICG), Cyanine 5.5 (Cy 5.5), and Cyanine 7 (Cy 7). ICG was the first
(
Cy 5.5 and Cy
7
as NIR fluorophore moieties for optical
36
27,29
FDA-approved NIR fluorophore for clinical use, having been used
in intraoperative NIR fluorescence imaging for image-guided sur-
gery for half a century. Regarding Cy 5.5 and Cy 7, these Cy X ser-
ies are fluorophores non-sulfonated cyanine dyes and are
imaging,
obtaining excellent results including high affinity
to PSMA. However, these probes are different from Cy 5.5-SCE
and Cy 7-SCE in their structures of the NIR-dye and PSMA-
targeting core. First, Cy 5.5 and Cy 7 used by Ying Chen et al.
3
7
2
7
conjugatable with targeting ligands for structural and functional
are highly sulfonated dyes. Second, PSMA-1-Cy5.5 reported by
Xinning Wang et al. is based on PSMA-1 which is a much larger
imaging of tumors and other tissues.3
8,39
They have been thor-
2
9
oughly investigated in various fields including not only PSMA
PSMA-targeting molecule than SCE. These differences should be
considered against the result that Cy 5.5-SCE and Cy 7-SCE
showed lower affinity than 2-PMPA.
imaging but also MT1-MMP and CD13 imaging.1
9,40,41
IRDye
4
2
6
80LT and IRDye 750 are mainly used in in vitro setting, and
there are no reports about conjugation with PSMA targeting ligand.
In this study, we designed and synthesized five SCE derivatives
with the fluorophores described above, and investigated their
structure–activity relationships as optical imaging probes for
PSMA (Fig. 1).
Interestingly, the affinities of 800CW-SCE, 680LT-SCE, and
750-SCE were approximately 10–30 times higher than that of
unconjugated asymmetric urea (Cys-C(O)-Glu) in spite of the very
low affinity of the unconjugated dye itself (Table S2). Some
reports stated that a bulky dye conjugation to the small ligand
did not interfere with but instead improved or maintained the
binding affinity to PSMA compared to the unconjugated ligand
2
. Results and discussion
44
itself. Other reports described that high negative charges (net
charge ꢀ 3) and high polarity (TPSA > 365) contribute to the
2.1. Synthesis
28,29
binding affinity to the PSMA.
i
Similarly, K values seemed to
correlate with TPSA and numbers of anionic sulfonic acid groups
in our study (Fig. S1). This importance of a bulky fluorophore,
especially anionic sulfonic acid groups of NIR fluorophores, was
also suggested in an in silico docking simulation (Fig. 2A-a and
B-a). The NIR fluorophore of 800CW-SCE, which had the best
affinity to PSMA, was directed to the arene-binding site in
As shown in Scheme 1, five SCE derivatives were designed and
synthesized by a coupling reaction between the thiol group of the
asymmetric urea and the maleimide group of each near-infrared
(
NIR) fluorophore, as in the design strategy of 800CW-SCE reported
3
3
previously. This labeling reaction proceeded under mild condi-
tions (pH 6–8, room temperature) to afford high synthetic yields
0
addition to interactions between the SCE moiety and S1
(
total yields were 66–96%). The SCE derivatives were purified by
reverse-phase HPLC with higher purity than 92–99% after
purification.
glutamate recognition pocket (Fig. 2A-b and A-c). In contrast,
the NIR fluorophore of ICG-SCE, which had a much lower affinity
to PSMA than 2-PMPA, did not interact with the arene-binding
site (Fig. 2B-b and B-c). These findings indicate that fluorophore
moieties, particularly sulfonic acid groups of the dye, may play
an important role in the binding to PSMA. These results also
suggest that 800CW-SCE, 680LT-SCE, and 750-SCE may possess
sufficient affinity to image PSMA in vivo.
2
.2. Physicochemical and optical properties of SCE derivatives
As shown in Table 1, the physicochemical properties, including
retention times (Rt) in the reverse-phase HPLC analysis and topo-
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D. Matsuoka et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
3
Fig. 1. Design strategy for novel PSMA-targeting SCE derivatives for optical imaging. §Data were reported previously.33
Scheme 1. Conjugation of Cys-C(O)-Glu with NIR fluorophores.
Table 1
Physicochemical and optical properties of SCE derivatives.
Rt* (min)
TPSA (Å2)
y
kEx (nm)
k
Em (nm)
Quantum yield (U)
Compound
00CW-SCE§
80LT-SCE
50-SCE
26.16
24.18
27.96
40.42
43.33
43.93
à
449.31
548.82
440.08
276.97
225.76
225.76
775
680
764
788
680
748
800
700
785
818
703
774
0.025
0.025
0.03
0.018
0.019
0.029
8
6
7
à
à
à
ICG-SCE
Cy 5.5-SCE
Cy 7-SCE
–
–
*
à
–
Retention times (Rt) were analyzed by reverse-phase HPLC under the following conditions: Cosmosil 5C18-AR-II 250 ꢁ 4.6 mm column, 10 mM or 50 mM triethy-
y
lammonium acetate buffer (pH 7.0) and MeOH; gradient of MeOH, 0 min = 5%, 5 min = 5%, 45 min = 100%; flow rate, 1 mL/min. Topological polar surface area (TPSA) were
calculated with Molecular Operating Environment (MOE). Data were reported previously.
§
33
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4
D. Matsuoka et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
Table 2
a high background signal from the whole body. As for 680LT-SCE,
the probe visualized neither the LNCaP tumors nor the PC-3 tumors
because the compound exhibited a fairly high background signal
from the whole body over time (LNCaP/PC-3 ratio = 0.91 at 24 h)
(Figs. 4B, 5B, and F). A recent paper regarding two PSMA-1 based
probes, PSMA-1-IR800 and PSMA-1-Cy5.5, described the polarity
of the fluorophores significantly influencing the pharmacokinetic
profile; high polarity and low hydrophobicity of the dye led to fast
Comparison of binding affinities of SCE derivatives to
PSMA.
Compound
K
i
(nM)*
8
6
7
00CW-SCE
80LT-SCE
50-SCE
13.2 ± 2.35
14.4 ± 6.49
29.1 ± 14.8
245 ± 64.5
400 ± 78.2
579 ± 68.3
103 ± 55.2
ICG-SCE
Cy 5.5-SCE
Cy 7-SCE
2
9,44
accumulation and fast washout.
These findings are reasonable
2-PMPA
because the NIR fluorophore moiety is much larger than the PSMA-
targeting core in molecular size. However, the physicochemical
properties of IRDye 680LT are very different from those of IRDye
*
Values are expressed as the means ± standard
errors of 3–6 independent determinations.
8
00CW and IRDye 750, the former having much larger molecular
size and much stronger polarity. Thus, IRDye 680LT is not suited
to demonstrate PSMA-targeting ability by introducing the SCE.
ICG-SCE, which had lower affinity than 2-PMPA, did not accumu-
late to any tumors (Figs. 4D, 5D, and H). ICG-SCE was cleared from
the body soon after intravenous administration, and tumor accu-
mulation was not seen even at the early time points (Fig. S3). These
results suggested that the introduction of SCE to ICG had almost no
effect on the pharmacokinetics of unconjugated ICG (Fig. S5).
2
.4. Cellular uptake study
To image PSMA-positive tumors in vivo, a high binding affinity
for PSMA greater than or close to 2-PMPA and a sufficient binding
selectivity of the compounds to PSMA are required. We performed
in vitro cellular uptake studies using LNCaP cells (PSMA-positive
cells) and PC-3 cells (PSMA-negative cells) to assess whether
6
80LT-SCE, 750-SCE, and ICG-SCE could bind specifically to PSMA
on the cellular surface (Fig. 3). We previously reported that
00CW-SCE bound to LNCaP cells but not to PC-3 cells in a
2.6. Biodistribution study
8
3
3
PSMA-specific manner. Here, 680LT-SCE and 750-SCE similarly
bound to and were internalized into LNCaP cells but not into PC-
An ex vivo biodistribution study was carried out to evaluate the
distribution patterns of 800CW-SCE, 680LT-SCE, 750-SCE, and ICG-
SCE to other organs. An acceptable NIR fluorescent probe for imag-
ing PSMA in in vivo and intraoperatively requires PSMA-selective
tumor accumulation and low accumulation to other organs. To
directly compare the distributions of the four compounds, the
results normalized to muscle are shown in Figs. 6 and 7, and quan-
titative data including all organs are listed in Table S3. Among the
four SCE derivatives, 800CW-SCE and 750-SCE displayed PSMA-
specific accumulation to the LNCaP tumors. 750-SCE distributed
to other organs such as the heart, lung, stomach, intestine, and
liver in addition to accumulating in the LNCaP tumors at 12 h after
intravenous administration. The heart and lung initially showed
high uptake but washout with time while the stomach, intestine,
and liver showed high accumulations over time until 24 h. In con-
trast, 800CW-SCE was almost cleared from these organs at 24 h
after intravenous injection, suggesting that 800CW-SCE exhibited
favorable in vivo pharmacokinetics compared to 750-SCE. 680LT-
SCE distributed to various organs including spleen, heart, lung,
stomach, intestine, and liver at not only 12 h but also 24 h after
administration, and these distributions were not affected by coin-
jection of 2-PMPA, indicating non-specific distribution. ICG-SCE
showed similar distribution to unconjugated ICG, i.e., ICG-SCE
accumulated to the liver soon after injection, and then cleared to
the intestine. Thus, IRDye 680LT and ICG were not suitable as
NIR fluorophore moieties for introduction to Cys-C(O)-Glu.
3
cells (Fig. 3A and B). These uptakes into LNCaP cells were signif-
icantly inhibited by 2-PMPA, suggesting that the bindings of these
two compounds to LNCaP cells were highly specific to PSMA. How-
ever, ICG-SCE bound slightly not only to LNCaP cells but also to PC-
3
cells, and these bindings were absolutely not affected by coincu-
bation with 2-PMPA, which suggested that the binding of ICG-SCE
to these cells was independent of the expression of PSMA (Fig. 3C).
In the evaluation of unconjugated IRDye 800CW-Carboxylate and
ICG under the same conditions, IRDye 800CW-Carboxylate was
internalized into neither LNCaP cells nor PC-3 cells regardless of
coincubation with 2-PMPA, while ICG bound slightly to both LNCaP
cells and PC-3 cells in a PSMA-independent manner like ICG-SCE
(Fig. S2). Consequently, the three compounds whose binding affini-
ties were higher than that of 2-PMPA could bind to PSMA and be
internalized PSMA-specifically into LNCaP cells. These findings
suggest that SCE moiety may play an important role in the selec-
tive binding to PSMA, and also suggest that 800CW-SCE, 680LT-
SCE, and 750-SCE may possess sufficient selectivity to image PSMA
in vivo.
2.5. In vivo imaging study
To evaluate the tumor accumulation and in vivo behavior of
00CW-SCE, 680LT-SCE, 750-SCE, and ICG-SCE, we conducted an
8
in vivo fluorescence imaging study using mice bearing both LNCaP
and PC-3 tumors. 800CW-SCE accumulated highly to LNCaP
tumors but not to PC-3 tumors (LNCaP/PC-3 ratio = 2.77 at 24 h),
and the accumulation was significantly suppressed by coinjection
of 2-PMPA, as described previously (Figs. 4A, 5A, and E).33 Tumor
accumulation of unconjugated IRDye 800CW-Carboxylate was also
assessed in this study, but the unconjugated fluorophore did not
accumulate to any tumors (Fig. S4), suggesting that SCE moiety
played an important role in the acquisition of PSMA-targeting abil-
ity. 750-SCE clearly visualized the LNCaP tumors at 12 and 24 h
after intravenous administration (LNCaP/PC-3 ratio = 2.23 at 24
h), but the compound also accumulated slightly in the PC-3 tumors
due to various factors including inadequate affinity to PSMA and
enhanced permeability and retention effects (EPR effect)
Together with the results of the in vitro binding study, the find-
ings in the biodistribution study suggest that a high binding affin-
ity to PSMA is prerequisite for a PSMA imaging probe. However, the
overall in vivo pharmacokinetics were mediated by the physico-
chemical properties of NIR fluorophores introduced to the SCE scaf-
fold because the NIR fluorophores had much larger molecular sizes
than the SCE moiety. Recently, some papers reported that various
linkers between NIR fluorophores and PSMA-targeting cores affect
the binding affinity, selectivity, and in vivo pharmacokinetics of
compounds. Therefore, to further advance molecular design for
the development of more useful imaging probes targeting PSMA,
optimization of the linker length between the NIR fluorophores
and SCE moiety in 800CW-SCE may be valuable, improving both
the binding affinity for PSMA and the accumulation to LNCaP
tumors.
4
4
(Figs. 4C, 5C, and G). 750-SCE also showed another drawback, i.e.,
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D. Matsuoka et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
5
Fig. 2. In silico docking simulation. Binding structures of 800CW-SCE (A) and ICG-SCE (B) with PSMA (PDB code 2XEG).47 (a) Overview of PSMA showing internal inhibitor-
0
binding cavity comprising S1 pocket, zinc (II) active site, hydrophobic pocket accessory to the S1 site, and arene-binding site. Close-up views of the arene-binding site (b), S1
0
site, and S1 pocket (c) of PSMA complex. 800CW-SCE and ICG-SCE are shown by thick sticks, and colored green. The side chains of residues contacted closely to ligands are
represented by sticks. The side chains of PSMA related to the arginine patch (R463, R534, and R536), and arene-binding site (R511 and W541) are colored blue, and yellow,
respectively. The backbone of PSMA is colored gray. Hydrogen bonds are indicated by dashed lines. The zinc ions in the active site are represented by pink spheres. Ligand
efficiency (LE) is defined as the affinity of a ligand to the PSMA divided by the number of heavy atoms in the molecule (LE values; 800CW-SCE: ꢂ0.224, ICG-SCE: ꢂ0.132).
3
. Conclusion
tant role in conferring high binding affinity to PSMA, which is pre-
requisite for high binding selectivity to PSMA. The in vivo imaging
study demonstrated that the pharmacokinetics of the imaging
probes differ greatly with respect to the differences of NIR fluo-
rophore moiety because of the latter’s much larger molecular size
compared to PSMA-targeting core. In the series of experiments,
800CW-SCE showed highly selective binding affinity to PSMA
In the present study, we designed and synthesized five new SCE
derivatives with various NIR fluorophores in addition to 800CW-
SCE, and evaluated their in vivo pharmacokinetics in addition to
their in vitro binding affinities and selectivities to PSMA. In vitro
studies revealed that the NIR fluorophore moiety plays an impor-
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D. Matsuoka et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
Fig. 3. In vitro cellular uptake study. The uptake of 680LT-SCE (A), 750-SCE (B), and ICG-SCE (C) into each cell after incubation with or without 2-PMPA was evaluated.
Representative images of Hoechst 33342 (left) and indicated compounds (right) are shown. Bar = 50 mm.
Fig. 4. In vivo imaging study. In vivo images of mice at 4, 8, 12, and 24 h postinjection of 800CW-SCE (A), 680LT-SCE (B), 750-SCE (C), and ICG-SCE (D) with or without 2-PMPA.
Representative images are shown of n = 4 or 5. §Data were reported previously.33
in vitro and its highly selective accumulation to LNCaP tumors
in vivo. These results suggested that further structural optimization
based on 800CW-SCE may lead to more useful NIR fluorescent
imaging probes for PSMA.
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D. Matsuoka et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
7
Fig. 5. Quantitative analysis of in vivo imaging study. (A–D) Dynamic changes of signal intensity in LNCaP and PC-3 tumors in mice injected with indicated compounds with
8
2
2
or without 2-PMPA. Signal intensity values are ꢁ 10 (p/s/cm /sr)/(mW/cm ). (E–H) Signal intensity ratios of LNCaP tumors to PC-3 tumors. Values represent mean ± SD of 4
animals. *P < 0.05 vs PC-3 without 2-PMPA; P < 0.05 vs LNCaP with 2-PMPA; P < 0.05 vs LNCaP/PC-3 ratio with 2-PMPA. §Data were reported previously.
y
à
33
4
. Materials and methods
hydroxide (NaOH) solution were purchased from Nacalai Tesque
Inc. (Kyoto, Japan). Deionized-destilled water (ddH O) was pre-
2
4
.1. General
pared with an MQ Integra15 (Nihon Millipore, Tokyo, Japan). Mass
spectra were recorded on an LCMS-2020 (Shimadzu Corporation).
The DCIT and its precursor were synthesized by a newly-developed
method (Scheme S1).
IRDye 800CW-Maleimide, IRDye 680LT-Maleimide, IRDye 750-
Maleimide, and IRDye 800CW-Carboxylate were purchased from
LI-COR Biosciences (NE, USA). ICG-Maleimide was purchased from
Goryo Chemical, Inc (Sapporo, Japan). Cy 5.5-Maleimide and Cy 7-
Maleimide were purchased from Lumiprobe Corporation (MD,
USA). Cys-C(O)-Glu was purchased from KNC Laboratories Co.,
Ltd. (Kobe, Japan). High-performance liquid chromatography
4.2. Synthesis
4.2.1. 680LT-SCE (2b)
A solution of Cys-C(O)-Glu (0.35 mg, 1.20
(0.030 mL) was adjusted to pH 6.0–8.0 with 1 N NaOH, and IRDye
680LT-Maleimide (0.53 mg, 0.40 mol) in DMSO (0.040 mL) was
2
lmol) in ddH O
(
HPLC) purifications were performed on a LC-20AD (Shimadzu Cor-
poration, Kyoto, Japan) equipped with an SPD-20A UV detector (
20 and 254 nm; Shimadzu Corporation). Indocyanine Green (ICG),
dimethyl sulfoxide (DMSO), methanol (MeOH), and sodium
q;
l
2
added to the solution. After 15 min at room temperature, the
reaction mixture was purified by HPLC to give 0.57 mg (88.4%)
Please cite this article in press as: Matsuoka D., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.03.015

8
D. Matsuoka et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
Fig. 6. Ex vivo imaging study. Representative fluorescence images of tumors and various organs obtained at 12 and 24 h after injection of 800CW-SCE (A), 680LT-SCE (B), 750-
SCE (C), and ICG-SCE (D) with or without 2-PMPA. (E) The arrangements of tumors and various organs. Representative images of n = 4 or 5. §Data were reported previously.
33
of 680LT-SCE under the following conditions: Cosmosil 5C18-AR-II
50 ꢁ 4.6 mm column, A = 10 mM triethylammonium acetate
buffer (pH 7.0), B = MeOH; gradient, 0 min = 5% B, 5 min = 5% B,
4.2.4. Cy 5.5-SCE (2e)
2
The same reaction as described above to prepare 680LT-SCE was
used. The reaction mixture was purified by HPLC to give 0.33 mg
(66.0%) of Cy 5.5-SCE under the following conditions: Cosmosil
5C18-AR-II 250 ꢁ 4.6 mm column, A = 50 mM triethylammonium
acetate buffer (pH 7.0), B = MeOH; gradient, 0 min = 5% B, 5 min
4
2
1
5 min = 100% B; flow rate, 1 mL/min (retention time =
ꢂ
4.18 min). MS (ESI) calcd for C65
74
H N
6
O
28
S
7
[M] 1610.8, found
ꢂ
611.0 [M] .
=
4
9
5% B, 45 min = 100% B; flow rate, 1 mL/min (retention time =
ꢂ
3.93 min). MS (ESI) calcd for C55
H
63
N
6
O
10
S
1
[M] 999.2, found
4
.2.2. 750-SCE (2c)
ꢂ
99.4 [M] .
The same reaction and purification method as described
above to prepare 680LT-SCE was used, and 0.28 mg of
7
2
1
50-SCE was obtained in 96.8% yield (retention time =
4.2.5. Cy 7-SCE (2f)
ꢂ
7.96 min). MS (ESI) calcd for C64
H
80
N
6
O
S
22 5
[M] 1444.7, found
The same reaction and purification method as described above
to prepare Cy 5.5-SCE was used, and 0.36 mg of Cy 7-SCE was
obtained in 74.5% yield (retention time = 43.33 min). MS (ESI) calcd
ꢂ
444.2 [M] .
ꢂ
ꢂ
65 6 10 1
for C52H N O S [M] 965.2, found 965.4 [M] .
4.2.3. ICG-SCE (2d)
The same reaction and purification method as described
above to prepare 680LT-SCE was used, and 0.45 mg of ICG-
4.3. Optical property analysis
SCE was obtained in 83.6% yield (retention time = 40.42 min).
+
MS (ESI) calcd for
C
60
70
H N
6
O
13
S
2
1147.4 [M+H] , found
All optical measurements were performed at 37 °C in 100%
MeOH. Fluorescence spectra for all compounds were measured
+
1
147.7 [M+H] .
Please cite this article in press as: Matsuoka D., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.03.015

D. Matsuoka et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
9
Fig. 7. Quantitative analysis of ex vivo imaging study. Biodistribution data at 12 h (A) and 24 h (B) postinjection of 800CW-SCE, 680LT-SCE, 750-SCE, and ICG-SCE with or
without 2-PMPA from regions of interest (ROI) drawn over organs displayed in ex vivo images corrected by weight of organ (g) and normalized to muscle. Values are
expressed as the means ± SD. *P < 0.05 vs LNCaP with 2-PMPA; P < 0.05 vs LNCaP without 2-PMPA; P < 0.05 vs LNCaP without 2-PMPA. §Data were reported previously.
y
à
33
using an RF-6000 (Shimadzu Corporation). The fluorescence quan-
tum yields were determined using an ICG (
= 0.016)45 or Cy 5 (
0.027) as the standard. The fluorescence intensity data were
measured in the spectral region of 650–900 nm, excited at 600 or
l
L of 125I-DCIT (15.5 kBq/mL) and the test compound were added
to each well, and the plates were incubated at 37 °C for 1 h. After
incubation, each well was washed twice with 500 L of fresh assay
medium, and then the cells were lysed with 0.2 N NaOH. Radioac-
tivity bound to the cells was measured with a -counter. IC50 val-
ues were calculated using the GraphPad Prism program
GraphPad Software, Inc., CA, USA). K values were calculated using
U
U
4
6
=
l
7
30 nm, over which the quantum yield was integrated.
c
5
(
i
4.4. Cell lines
the Cheng–Prusoff equation.
Two human prostate carcinoma cell lines were purchased from
DS Pharma Biomedical (Osaka, Japan): LNCaP (PSMA-positive) and
PC-3 (PSMA-negative). The cells were cultured in Roswell Park
Memorial Institute 1640 (RPMI 1640; Nacalai Tesque Inc.) medium
supplemented with 10% fetal bovine serum, 1.0 mM glutamine,
and antibiotics (100 U/mL penicillin/10 mg/mL streptomycin) in a
4.6. In silico docking study
Docking simulations were performed with Molecular Operating
Environment (MOE, Chemical Computing Group., Quebec, Canada)
version 2014.09. As the initial structure, we selected PSMA in com-
plex with one of the known PSMA inhibitors, antibody-recruiting
molecules targeting prostate cancer (ARM-P4), (PDB code:
humidified CO
.5. In vitro cellular binding assay
The affinities of six SCE derivatives, 2-PMPA (Tocris Bioscience,
2 2
incubator (37 °C/5% CO ).
4
7
2
XEG). First, hydrogen atoms were added to the crystal structure
4
of PSMA by Protonate 3D module in MOE. Next, conformational
searches of 800CW-SCE and ICG-SCE were performed using the
systematic search module in MOE. The binding structures were
evaluated with interaction energies.
BRS, UK), Cys-C(O)-Glu, and unconjugated fluorophores were
determined by an in vitro binding inhibition assay, as previously
3
0
reported. The radiosynthesis of N-[N-[(S)-1,3-dicarboxypropyl]-
1
25
125
carbamoyl]-S-3-[ I]iodo-
L
-tyrosine
(
I-DCIT) was performed
4.7. In vitro cellular uptake study
3
0
according to the same method, as described previously, and the
compound was used as the radioligand for the inhibition assay.
5
LNCaP and PC-3 cells (5.0 ꢁ 10 ) were plated on a covered
6
LNCaP cells in 12-well plates (1.0 ꢁ 10 cells/well) were incu-
glass-bottomed culture well and incubated for 24 h at 37 °C and
bated for 48 h (37 °C/5% CO
2
). The medium was removed, and then
2
5% CO . The medium was removed, and each well was washed with
each well was washed twice with 1 mL of assay medium (RPMI
1 mL of assay medium (RPMI 1640 supplemented with 0.5% bovine
serum albumin). 680LT-SCE, 750-SCE, ICG-SCE, and unconjugated
1
640 supplemented with 0.5% bovine serum albumin). Then 500
Please cite this article in press as: Matsuoka D., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.03.015

1
0
D. Matsuoka et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
fluorophores (5.0 mM) were then added to each well, and the plates
were incubated at 37 °C for 180 min followed by washing three
times with assay medium. Nonspecific binding was evaluated by
Acknowledgements
This work was supported by a Grant-in-Aid for Scientific
Research (B) (Grant No. 15H04637) and for Young Scientists (B)
(Grant No. 23791413) from the Japan Society for the Promotion
of Science (JSPS).
0
adding 2-PMPA (250 mM). Cell nuclei were then stained with 2 -
0
(
4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5 -bi-1H-benzimi-
dazole (Hoechst 33342; Dojindo, Kumamoto, Japan) followed by
cell fixing using 4% paraformaldehyde, and fluorescence micro-
scopy was performed using an Olympus X81 microscope (Olympus
Ò
America, Inc., NY, USA) under a 40 ꢁ objective lens. MetaMorph
A. Supplementary data
software (Molecular Devices, LLC, CA, USA) was used for imaging
analysis.
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.bmc.2018.03.015.
4.8. Mouse model
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