Visualizing Tumors in Real Time: A Highly Sensitive PSMA Probe for NIR-II Imaging and Intraoperative Tumor Resection

Article abstract of DOI:10.1021/acs.jmedchem.1c00444

Owing to the complex anatomical structure, precise resection of a tumor while maintaining adjacent tissue is a challenge in radical prostatectomy for prostate cancer (PCa). Optical imaging in near-infrared window II (NIR-II) is a promising technology for intraoperative guidance, whereas there is no available probe for PCa yet. In this article, a novel probe (PSMA-1092) bearing two prostate-specific membrane antigen (PSMA) binding motifs was developed, displaying excellent optical properties (λmax = 1092 nm) and ultrahigh affinity (Ki = 80 pM) toward PSMA. The tumor was visualized with high resolution (tissue-to-normal tissue ratio = 7.62 ± 1.05) and clear margin by NIR-II imaging using PSMA-1092 in a mouse model. During the tumor resection, residual tumors missed by visible inspection were detected by the real-time imaging. Overall, PSMA-1092 displayed excellent performance in delineating the tumor margin and detecting residual tumors, demonstrating promising potential for precise PCa tumor resection in clinical practice.

Full text of DOI:10.1021/acs.jmedchem.1c00444

pubs.acs.org/jmc
Article
Visualizing Tumors in Real Time: A Highly Sensitive PSMA Probe for
NIR-II Imaging and Intraoperative Tumor Resection
Longfei Zhang, Xiaojing Shi, Yuying Li, Xiaojiang Duan, Zeyu Zhang, Hualong Fu, Xing Yang, Jie Tian,
Zhenhua Hu,* and Mengchao Cui*
Cite This: J. Med. Chem. 2021, 64, 7735−7745
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: Owing to the complex anatomical structure, precise
resection of a tumor while maintaining adjacent tissue is a challenge in
radical prostatectomy for prostate cancer (PCa). Optical imaging in
near-infrared window II (NIR-II) is a promising technology for
intraoperative guidance, whereas there is no available probe for PCa
yet. In this article, a novel probe (PSMA-1092) bearing two prostate-
specific membrane antigen (PSMA) binding motifs was developed,
displaying excellent optical properties (λmax = 1092 nm) and ultrahigh
affinity (Ki = 80 pM) toward PSMA. The tumor was visualized with
high resolution (tissue-to-normal tissue ratio = 7.62 1.05) and clear
margin by NIR-II imaging using PSMA-1092 in a mouse model.
During the tumor resection, residual tumors missed by visible
inspection were detected by the real-time imaging. Overall, PSMA-1092 displayed excellent performance in delineating the
tumor margin and detecting residual tumors, demonstrating promising potential for precise PCa tumor resection in clinical practice.
penetration capacity, high spatial and temporal resolution, and
powerful sensitivity.20−24 Based on the above advantages, optical
imaging in the NIR-II window is highly suitable for intra-
operative fluorescent imaging guidance of precise tumor
resection.25−28
INTRODUCTION
■
In the past decades, the incidence rates of prostate cancer (PCa)
have increased rapidly worldwide, and it has become one of the
leading causes of tumor-related deaths in males.1,2 Even though
several drugs for treating PCa have been developed,3−5 radical
prostatectomy is still the most effective treatment and first
choice for patients in early stages.6,7 Owing to the irregular and
indistinctive tumor boundary, a positive surgical margin in
radical prostatectomy specimens is a common issue,8 which may
increase the risk of recurrence and metastasis9−12 and thus
deteriorate the prognoses of patients.13 Moreover, excessive
resection could easily injure the abundant nerves and vessels in
this region, which could lead to various complications, such as
urinary incontinence and urethrostenosis.14,15 Therefore,
developing a novel imaging technology for aiding surgeons to
precisely delineate tumor margins and detect missed lesions
during surgery in real time with high resolution is highly
desirable and demanded.
Compared to the traditional imaging modalities, such as
computed tomography and magnetic resonance imaging, optical
imaging in the near-infrared window I (NIR-I, 650−900 nm)
has been demonstrated to have many advantages, for example,
being easy to implement, providing fast signal feedback, and
showing no exposure to radiation.16−19 According to recent
studies, the imaging quality can be dramatically improved by
detecting in the near-infrared window II (NIR-II, 1000−1700
nm) because of a lower level of tissue autofluorescence, minimal
photon scatting and photoabsorption, which leads to a deeper
After the report of the first small organic NIR-II fluorescent
probe in 2016,29 great efforts have been devoted to develop
probes and technologies for NIR-II imaging.30−34 In 2019,
Tian’s group developed a novel multispectral imaging instru-
ment, which could detect an optical signal in both NIR-I and
NIR-II regions, and successfully implemented it into the precise
tumor resection of patients with liver cancer in clinical
practice.35 Even though the novel imaging instrument
demonstrated good performance and successfully detected an
extrahepatic metastasis missed by the surgeon in one case, it still
suffered from some limitations, such as false positives, owing to
the lack of appropriate NIR-II agent. Hence, the development of
new NIR-II agents with excellent optical and biological
properties could effectively facilitate the clinical translation of
NIR-II imaging.
Received: March 11, 2021
Published: May 28, 2021
https://doi.org/10.1021/acs.jmedchem.1c00444
© 2021 American Chemical Society
J. Med. Chem. 2021, 64, 7735−7745
7735

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Figure 1. (a) Structures of previously reported fluorescent PSMA probes. Blue indicates the binding motif, and purple indicates the fluorescent
fluorophores in the NIR-I region. (b) Structure of the newly designed NIR-II PSMA probe.
In recent years, diverse NIR-II probes based on single-walled
carbon nanotubes, quantum dots, and rare-earth-doped nano-
particles have been developed and have demonstrated excellent
optical properties.36−38 However, owing to the long-term
retention in the reticuloendothelial system and the lack of
long-term safety study, it is almost impossible for these probes to
be applied in clinical practice.38,39 Oppositely, because of the
excellent biocompatibility and quick clearance capacity through
the metabolic system, the molecular probe is considered as a
promising candidate for biological imaging and attracts intense
focus. Even though some NIR-II organic molecular probes were
developed in recent years, the majority of them encounter
bottlenecks because of the lack of a definite target and poor
water solubility, which are an inevitable obstruction to clinical
transformation. Therefore, developing a novel NIR-II organic
molecular probe with high water solubility and certain targeting
site is urgent to address this dilemma.
Prostate-specific membrane antigen (PSMA) is a type II
transmembrane glycoprotein which is overexpressed on the cell
membrane of primary and metastatic prostate tumors, and the
PSMA expression level positively correlates to the stages of PCa
and Gleason score.9,40,41 As a result, PSMA attracts intense
attention as an effective biomarker for assessing PCa, and several
probes targeting it for optical imaging have been developed in
recent years (Figure 1a).12,42−46 In 2012, Pomper’s group
designed a series of fluorescent PSMA probes with different
linkers based on commercial fluorophores to explore the
relationship between linker length and affinities.47 Among
these probes, compounds CY7-2 and CY7-3, with long linkers,
displayed high affinities (Ki = 7 pM for CY7-2 and 5 pM for CY7-
3) to PSMA and excellent performance with in vivo imaging.
More recently, an IRDye800CW-based probe, L16, was
reported by Yang’s group, which demonstrated impressive
biological properties (Ki = 0.65 nM) and successfully visualized
the tumor in a mouse model at 4 h postinjection.48 A further ex
vivo biodistribution study indicated that the tumor-to-muscle
ratio could reach 19.0 at 24 h postinjection. However, these
probes suffer from the inherent drawbacks of NIR-I probes, such
as poor tissue-penetrating capability and limited resolution
because of their short fluorescence emission wavelengths
(emission maxima of CY7-3, CY7-2, and L16 in phosphate-
buffered saline (PBS) are 767, 767, and 800 nm, respectively).
In this study, a novel small organic NIR-II fluorescent probe
was designed with favorable optical properties, super water
solubility, and clinical transformation potential to provide
efficient fluorescent guidance in prostate tumor resection.
Considering that the donor−acceptor−donor (D−A−D) core
structure and appropriate electron acceptor and electron donor
moieties are the key factors to obtain NIR-II fluorescent
probes,49,50 the benzobis(1,2,5-thiadiazole) moiety was selected
as an excellent electron acceptor36,37 and conjugated two N-
methylaniline as electron donors. More importantly, two (((S)-
5-amino-1-carboxypentyl)carbamoyl)-L-glutamic acid (Glu-
urea-Lys) moieties as specific binding moieties toward PSMA
were incorporated into the N-methylamino group via a short
amide linker to enhance the binding affinity (Figure 1b). In
addition to the prominent binding ability, the three carboxy
groups on Glu-urea-Lys moieties could endow the probe with
7736
https://doi.org/10.1021/acs.jmedchem.1c00444
J. Med. Chem. 2021, 64, 7735−7745

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
a
Scheme 1. Synthetic Route of PSMA-1092
a
Reagents and conditions: (a) acrylic acid, acetic acid (20% in H2O), 100 °C, overnight, 94.5%; (b) tert-butanol, DCC, DMAP, CH2Cl2, room
temperature, overnight, 43.2%; (c) hexabutyldistannane, Pd(PPh3)4, toluene, N2, 120 °C, 2 h; (d) 4,7-dibromo-5,6-dinitrobenzo[c][1,2,5]-
thiadiazole, PdCl2(PPh3)2, toluene, N2, 120 °C, 2 h, 7.7%; (e) iron powder, acetic acid, 100 °C, 30 min, 36.2%; (f) PhNSO, TMSCl, dry pyridine,
80 °C, overnight, 27.2%; (g) TFA, CH2Cl2, room temperature, 4 h, 100%; (h) 2,3,5,6-tetrafluorophenol, DCC, CH2Cl2, room temperature, 5 h,
67.4%; (i) di-tert-butyl (((S)-6-amino-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)-L-glutamate, Et3N, CH2Cl2, room temperature, 6 h, 61.3%.
a
b
Figure 2. (a) Optical properties table of PSMA-1092. Absorption (λex1) and emission (λem1) maximum recorded in DMSO. Quantum yield
measured in PBS. cAbsorption (λex2) and emission (λem2) spectra determined in acid aqueous solution (pH 2.66). dKi value measured by the inhibition
assay using the lysate of LNCaP cell. eThe pKa value was calculated based on the Boltzmann curve. (b) UV−vis−NIR absorption and fluorescence
emission spectra of PSMA-1092 (10 μM) in DMSO. (c) Photostability of PSMA-1092 (10 μM) in DMSO under 727 nm continuous laser exposure.
(d) NIR-II imaging of PSMA-1092 (0.43 mM) in PBS with various LP filters. White dotted line contours the invisible solution.
7737
https://doi.org/10.1021/acs.jmedchem.1c00444
J. Med. Chem. 2021, 64, 7735−7745

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
high hydrophilicity, which could significantly improve its
solubility in aqueous solution.
general, the probes possess a favorable fluorescence emission
wavelength, a large Stokes shift, and excellent photostability
property, showing great potential for in vivo NIR-II imaging.
Interestingly, the fluorescence property of PSMA-1092
demonstrated a dramatic change in solutions with different
pH values (Figure 3a,b), which could be observed by the nake
eyes (Figure 3c). After being exposed to an acid solution, the
color of the PSMA-1092 solution changed to bright pink from
cyan under natural light, and a gradually increasing fluorescence
could be observed under 365 nm ultraviolet light (Figure 3d) or
in NIR-I imaging (Figure 3e). Fluorescence spectra indicated
that the fluorescence maxima shifted to 620 from 1092 nm in
RESULTS AND DISCUSSION
■
Synthesis. The synthetic route of PSMA-1092 is shown in
Scheme 1. Acrylic acid as a functional linker was reacted with 4-
bromo-N-methylaniline in aqueous acetic acid to provide
compound 2 with a high yield (94.5%). The carboxy group
was then protected with tert-butanol, which was stable in
subsequent reactions. After the bromine atom was replaced with
a tributyltin group, a Stille cross-coupling reaction was
performed between the tributyltin derivative and the commer-
cially available 4,7-dibromo-5,6-dinitrobenzo[c][1,2,5]-
thiadiazole in methylbenzene to provide compound 5 with
moderate yield (17.7%). Then nitro reduction and N-
thionylaniline-induced cyclization were used in sequence to
build the core structure of the NIR-II fluorophore (36.2 and
37.5% yield, respectively). After the tert-butanol groups in
compound 7 were removed by a TFA/DCM system, the carboxy
derivative was reacted with 2,3,5,6-tetrafluorophenol to produce
the active ester (compound 9) with a high yield (67.4%). Then
two tert-butanol-protected Glu-urea-Lys binding moieties were
conjugated with the NIR-II fluorophore via an amide bond to
give compound 10 (61.3% yield). The final product, PSMA-
1092, was obtained as a dark green crystal in high yield (98.4%)
after the deprotection of all tert-butyl groups. All intermediates
were characterized by 1H NMR and MS, and PSMA-1092 was
characterized by 1H NMR, 13C NMR, and HRMS. The purity of
PSMA-1092 was determined as 97.3% using high-performance
liquid chromatography (HPLC, Figure S1).
Optical Properties. Owing to the intense intramolecular
charge transfer (ICT) effect in the D−A−D structure, the
fluorescence emission peak of PSMA-1092 was measured at
1092 nm (DMSO, Figure 2a,b and Figures S2a−d and S4),
which was in the NIR-II region and similar to other probes based
on the benzobis(1,2,5-thiadiazole) fluorophore.20,36,39 More-
over, the probe demonstrated a large Stokes shift (365 nm,
DMSO, Figure 2a,b) compared to that in previously reported
NIR-II probes,36 which could decrease the self-quenching of the
imaging agent and avoid the influence of an excitation laser,
consequently leading to improved image quality in NIR-II
imaging. To verify the photostability of PSMA-1092, its DMSO
solution was exposed to 30 min continuous radiation under an
excitation laser (727 nm, 10 W). During the above process, no
obvious photodegradation was observed (Figure 2c), indicating
its outstanding light stability. The quantum yield of PSMA-1092
was measured as 6.66% in PBS, which is high enough for NIR-II
imaging and is expected to have a good performance in further
investigation. The NIR-II fluorescence signal of PSMA-1092 in
PBS was further compared under various long-pass (LP) filters
using an NIR-II imaging system. The PSMA-1092 solution was
visualized under 1000−1200 nm LP filters, whereas no signal
was detected with a 1300 or 1400 nm filter (Figure 2d).
Considering that the fluorescence signal under a 1000 nm LP
filter was stronger than others, it was selected for further in vivo
NIR-II imaging. To investigate the penetration ability of PSMA-
1092, the NIR-II images of its aqueous solution (0.5 mg/mL,
with 5% DMSO, filled in a capillary glass tube in advance) under
different thicknesses of chicken breast tissues were acquired. An
intense fluorescence signal of PSMA-1092 could be observed
under 2.98 mm of chicken breast tissue, and the signal could still
be detected when 3.72 mm of cover tissue was used (Figure
S2e), guaranteeing the sensitivity of our probe during surgery. In
Figure 3. (a) UV−vis−NIR absorption spectra and fluorescence
emission spectra of PSMA-1092 in aqueous solution with different pH
values. (b) Plots of the fluorescence (0.1 mM) signal intensities at 620
nm versus the pH values for PSMA-1092 within the range of 0−7.5.
The pKa values were calculated to be 1.95 based on the Boltzmann
fitting method, and the R2 was measured as 0.9984. (c,d) Photograph of
PSMA-1092 aqueous solution with different pH under natural light (c)
and 365 ultraviolet light (d). (e) NIR-I imaging of the above solutions.
(f) Possible protonation mechanism of PSMA-1092 in an acid
environment. (g) 1H NMR spectra (600 MHz, DMSO-d6) of PSMA-
1092 upon different amounts of deuterated TFA in 1.5 mL of DMSO-
d6. Arrows indicate the peaks with shifts.
7738
https://doi.org/10.1021/acs.jmedchem.1c00444
J. Med. Chem. 2021, 64, 7735−7745

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
acid conditions, and the fluorescence intensity at 620 nm was
greatly improved with the increasing acidity of the solution
(Figure 3a,b). When the pH of the solution changed from 7 to
0.3, the fluorescence intensity of PSMA-1092 increased by 85
times. Further quantitative analysis reveals that the signal
intensity of the new fluorescence maximum is linearly correlated
with the concentration of H+ within a limited pH range (pH 1−
2, Figure S3). In addition, plots of fluorescence intensities of
PSMA-1092 at 620 nm and the pH values are well fitted by the
Boltzmann curve (R2 = 0.9984), and the pKa value is calculated
to be 1.95 (Figure 3b). The pH response of PSMA-1092
highlighted its potential in a broader field, such as for
noninvasively measuring the pH value of gastric juice.51 The
tremendous wavelength shift, which was over a span from the
NIR-II region to NIR-I, could effectively avoid the influence of
the original wavelength and thus prompted the detection
accuracy. Further studies of pH response applications are still
underway.
1
To further investigate the mechanism of pH response, H
NMR titration with deuterated trifluoroacetic acid (TFA) was
performed. The protons in the aromatic ring and the N-alkyl
donor groups displayed gradual downfield shifts (8.18 to 8.49
ppm for H1, 6.94 to 7.82 ppm for H2, 3.02 to 3.39 ppm for H3,
3.05 to 3.18 ppm for H4, and 2.40 to 2.63 ppm for H5, Figure 3g)
with an increasing amount of deuterated TFA, indicating that
the electron density of the donor moiety was decreased. Based
on the above data, it could be inferred that the nitrogen on the
dialkylamino group tended to be protonated in an acid
environment (Figure 3f), which resulted in a lower density in
the electron acceptor moiety and thus inhibited the ICT effect. It
should be noted that a similar phenomenon was also observed in
other NIR-II fluorescent probes, in which they displayed a 150
or 120 nm hypochromic shift after exposure to an acid
environment; however, such a tremendous blue shift (472
nm) and linear correlation were first reported in this study.51,52
In Vitro Biological Properties. To quantitatively inves-
tigate the affinity of PSMA-1092 toward PSMA, an in vitro
competitive inhibition assay was performed according to
reported methods.48 Owing to the synergistic effect of two
PSMA targeting moieties, PSMA-1092 demonstrated ultrahigh
affinity to PSMA with a Ki of 80 pM, which was higher than that
of other PSMA inhibitors, such as ZJ-43 (3.53 nM, under the
same assay conditions) or L16 (0.65 nM).48 The high affinity of
the probe could effectively improve the detection sensitivity and
reduce nontarget uptake, which could lead to an excellent
performance in NIR-II imaging. To further evaluate the
biocompatibility of PSMA-1092, an MTT assay was performed
using PC3 cells. No apparent decrease in cell viability was
observed with the increasing concentration (up to 50 μM) of
PSMA-1092 (Figure S5), indicating the low toxicity of our
probe. Moreover, our probe demonstrated high stability in
blood; after being incubated with mice plasma for 24 h, there are
no decomposed compounds detected by the HPLC method
(Figure S6).
Figure 4. (a−h) NIR-II images of LNCaP tumor-bearing mice (n = 3)
after tail vein injection of PSMA-1092 (2.5 mg/kg) at different time
points. (i) Semiquantitative analysis of PSMA-1092 biodistribution in
major organs. (j) TNR (left Y axis, black line) and tumor uptake (right
axis, blue line) versus time curve after intravenous injection of PSMA-
1092 in tumor-bearing mice. (k) NIR-II images of LNCaP tumor-
bearing mice (n = 3) after injection of PSMA-1092 with or without 2-
PMPA at different time points. (l) Quantitative analysis of PSMA-1092
biodistribution in two group mice. (m) TNR versus time curve of two
group mice after tail vein injection of agents. Fluorescent intensities and
T/N ratios were measured in triplicate with results given as the mean
standard deviation. Significance was calculated by a Student’s t test.
***P < 0.001.
In Vivo NIR-II Imaging of Tumor-Bearing Mice. To
further test the in vivo performance of PSMA-1092,
subcutaneous LNCaP tumor-bearing mice (n = 3) were used
in NIR-II imaging. After the tail vein administration of PSMA-
1092 at a dose of 2.5 mg/kg body weight, NIR-II imaging was
acquired at different time points (1, 2, 4, 6, 8, 12, and 24 h,
Figure 4 and Figure S7) under 792 nm laser excitation at a power
density of 100 mW/cm2 (1000 nm long-pass filter, 50 ms
exposure time). As shown in Figure 4, PSMA-1092 accumulates
in the tumor and surrounding tissue within 2 h postinjection
(Figure 4c). After 4 h postinjection, the tumor was distinguished
from surrounding tissue with good contrast (Figure 4d), and the
high image quality was maintained at further time points (Figure
4e−h), indicating excellent imaging performance and high
7739
https://doi.org/10.1021/acs.jmedchem.1c00444
J. Med. Chem. 2021, 64, 7735−7745

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Figure 5. (a−d,f,g,i−m) Images of the intraoperative NIR-II imaging-guided precise tumor resection. (e,h) Histological study of two resected tumors.
Scale bar: 50 μm. (n) TNR value along the surgery (n = 1).
stability of PSMA-1092 in the mice model. To obtain more
detailed pharmacokinetic information, a semiquantitative
analysis of tumor-to-normal tissue ratio (TNR) based on the
NIR-II images was performed. The maximum tumor uptake was
reached at 6 h postinjection, whereas the peak value of TNR was
obtained at 12 h postinjection (7.62 1.05, Figure 4j), owing to
the clearance rate of PSMA-1092 in normal tissue being faster
than that in the tumor. According to the Rose criterion, an TNR
of 5 is necessary to differentiate image features with 100%
certainty.29,53 The TNR values of PSMA-1092 in NIR-II
imaging reached 5.99 0.72 at 8 h postinjection and maintained
a high level (7.62 1.05 for 12 h and 6.66 1.48 for 24 h
postinjection, Figure 4j) at later time points, which were both
suppressing the Rose criterion and enabling excellent NIR-II
image quality. Ex vivo imaging data were collected at 24 h
postinjection to study the distribution of PSMA-1092 in major
organs. In addition to the tumor, a high accumulation of PSMA-
1092 in the kidney and liver (Figures S8a,c,d and S10) was
observed, indicating PSMA-1092 is mainly metabolized by the
liver and excreted through the kidney system. More importantly,
without the interference of skin, the fluorescence signal of
muscle could rarely be detected (Figures S8b and S9), and the
TNR ratio was further enhanced, achieving a value of 9.44
(Figure 4i). To study the pharmacokinetics of PSMA-1092, an
in vivo blood clearance experiment was performed on ICR mice
(8 weeks old, n = 3). After intravenous administration of PSMA-
1092 (10 mg/kg), the fluorescence intensities of blood samples
collected from the above mice demonstrated a fast decline in the
beginning (0−7 h), and the downtrend tended to be slow after
10 h postinjection (Figure S11). The ultrahigh affinity (Ki = 80
pM) toward PSMA, moderate clearance rate, excellent NIR-II
imaging performance, and high tumor-to-muscle ratio of PSMA-
1092 highlight its potential utilization for in vivo imaging and
intraoperative fluorescence image-guided precise tumor resec-
tion in clinical practice.
To verify the specificity of PSMA-1092 for PSMA, a blocking
experiment was further performed on tumor-bearing mice (n = 3
per group). NIR-II images (792 nm laser excitation, 1000 nm LP
filter, and 50 ms exposure time) were obtained at selected time
points after the co-injection of 2-PMPA (a potent and selective
inhibitor of glutamate carboxypeptidase II with an IC50 of 300
pM, 34.3 mg/kg body weight) and PSMA-1092 (2.5 mg/kg
body weight).54,55 Tumor-bearing mice injected only PSMA-
1092 (2.5 mg/kg body weight) were assigned as the control
group to analyze the blocking data. As shown in Figure 4k and
Figure S12, in the blocking group, most of the tumor signal in
living mice could be inhibited by 2-PMPA, and semiquantitative
analysis indicated that the TNR values of the control group were
much higher than those of the blocking group after 6 h
postinjection (Figure 4m). Further ex vivo imaging demon-
strated that fluorescence signals from tumors and kidneys were
successfully reduced after co-injection of 2-PMPA and PSMA-
1092 (Figure 4l and Figure S13). These results well proved that
PSMA-1092 is a specific ligand for PSMA and has ultrahigh
7740
https://doi.org/10.1021/acs.jmedchem.1c00444
J. Med. Chem. 2021, 64, 7735−7745

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
affinity, which meets most of the requirements for a NIR-II
imaging probe.
1092, the tumor was successfully delineated with a high spatial
and temporal resolution (TNR = 9.81). After the resection of the
tumor, a residual tumor in the margin missed by visible
inspection was successfully detected by the NIR-II imaging,
which highlighted the powerful sensitivity of our probe. In
summary, as a molecular probe, PSMA-1092 meets the
requirements for intraoperative NIR-II image-guided precise
tumor resection, and it is a highly promising candidate for
clinical practice.
Intraoperative Fluorescence Image-Guided Precise
Tumor Resection. To verify the feasibility of PSMA-1092 in
intraoperative fluorescence image-guided precise tumor resec-
tion, living LNCaP tumor-bearing mice were selected to perform
tumor resection study under anesthesia. According to the above
imaging data, the optimal time window for surgery was selected
at 24 h postinjection of PSMA-1092 (2.5 mg/kg body weight)
by tail vein to obtain the lowest background signal. During the
entire operation, the mouse was anesthetized by the
combination gas of isoflurane and oxygen at a rate of 0.4 L/
min. The tumor was resected under the NIR-II navigation, and
the entire process was recorded by the imaging instrument
(Figure 5 and supplementary video). First, the skin around the
tumor region was dissected to expose the tumor (Figure 5b,c).
Semiquantitative analysis indicated that the TNR value was
increased after the incision (from 7.95 to 9.81, Figure 5n) since
the removal of covering skin. Then the tumor was resected
(Figure 5d,f,g) and thought to be completely removed by visible
inspection. However, an intense fluorescence signal was still
observed from the surrounding area (Figure 5i) with a TNR
value of 5.48 (Figure 5n), indicating there was still residual
tumor missed by the operation in margin. Guided by the NIR-II
fluorescent imaging, the residual tumor was further resected
(Figure 5j), while the healthy tissue was maintained as much as
possible, with the TNR value decreased to 3.12 (Figure 5n) after
resection. However, after the extended resection, a fluorescent
line could still be detected from the remaining tissue (Figure 5j).
More detailed inspection found a missed tiny tumor (Figure 5k,l,
indicated by red arrowhead), which was further removed with
no residual fluorescence signal observed by the imaging
instrument (TNR = 2.97, Figure 5m,n). Histological study
using hematoxylin and eosin (H&E) staining demonstrated that
the resected tumor was poorly differentiated carcinoma. Overall,
the outstanding tumor delineation capability highlights the
promising NIR-II small-molecule probe with clinical transla-
tional potential for fluorescence image-guided precise tumor
resection.
EXPERIMENTAL PROCEDURES
■
General Information. All reagents used for synthesis were
purchased commercially and used without further purification unless
specifically noted. Reaction processes were monitored by thin-layer
chromatography (TLC) on Merck silica gel 60 F254 plates (Germany).
After the reactions, the coarse productions were loaded on a Bonna-
Agela Technologies Co., Ltd. flash column silica-CS (20−80 g, 12.6 bar,
China) and purified by Bonna-Agela Technologies Co., Ltd. FLEXA
modular preparative chromatography system (China). 1H and 13C
NMR spectra were performed on a Bruker Avance III (400 or 100 MHz,
German), JEOL JNM-ECZ400R/S1 (400 or 100 MHz, Japan), or
JNMECZ600R/S3 (600 or 150 MHz, Japan) NMR spectrometer in
CDCl3, DMSO-d6, or trifluoroacetic acid-d solution at room temper-
ature unless further noted. Chemical shifts were reported in parts per
million (ppm) compared with tetramethylsilane (δ 0.00, s). Coupling
constants were reported in hertz (Hz). Multiplicities were reported as s
(singlet), d (doublet), t (triplet), and m (multiplet). MS spectra were
collected on Thermo Scientific LCQ FLEET (ESI) mass spectrometer
(USA). High-resolution mass spectra were recorded on Thermo
scientific Q-Ecactive (ESI) mass spectrometer (USA). The purity of
PSMA-1092 was determined by HPLC method, and the purity was
greater than 95%. UV−vis−NIR spectra were performed on a Shimadzu
UV-3600 UV−vis−NIR spectrophotometer (Japan). Fluorescence
spectra and fluorescence quantum yields were recorded on an
Edinburgh Instruments FLS980 fluorescence spectrometer (UK).
LNCaP and PC3 cell lines were obtained from the Chinese Academy
of Sciences Typical Culture Collection (Shanghai, China). The
quantitative binding affinities were determined by an in vitro inhibition
assay using the lysate of LNCaP cells according to previously reported
works.48 In vivo NIR-II imaging was performed on a Princeton
Instruments NIRvana 640 InGaAs charge-coupled device camera
(USA) coupled to a lens of a Schneider Kreuznach SWIRON 2.8/50
high transparency instrument in the NIR-II spectrum (Germany). A
Thorlabs FEL1000 LP 1000 nm long-pass filter (USA) was fixed on the
end of the lens to filter the excitation light. LNCaP tumor-bearing mice
were purchased from the Institute of Laboratory Animals Science,
CAMS & PUMC (China). The tissue embedding, sectioning, H&E
staining, and microscope photography were entrusted to Wuhan
Servicebio Technology Co., Ltd. (China).
Moreover, the excellent optical properties and high affinities
of PSMA-1092 make it possible to obtain a clear NIR-II image
within a short exposure time (50 ms), which ensures 20 frames
could be recorded in 1 s. The high frame rate of imaging could
guarantee fluent signal feedback in real time, which facilitates the
precise tumor resection in clinical practice.
Chemistry. 3-((4-Bromophenyl)(methyl)amino)propanoic Acid
(2). To a mixture of 1 (7.80 g, 40.00 mmol, 1 equiv) in acetic acid (20%
in H2O, 50 mL) was added acrylic acid (3.75 g, 52.00 mmol, 1.3 equiv).
The reaction was refluxed overnight and monitored by TLC. The
mixture was cooled to room temperature and extracted by CH2Cl2 (15
mL × 3). The combined organic layer was dried over MgSO4 and
concentrated in vacuum. The crude product was purified by silica gel
column chromatography (petroleum/ethyl acetate = 2/1, v/v) to give 2
as a yellow solid (9.70 g, 94.5%). 1H NMR (600 MHz, CDCl3): δ 7.32
(d, J = 9.0 Hz, 2H), 6.64 (d, J = 9.0 Hz, 2H), 3.65 (t, J = 7.1 Hz, 2H),
2.92 (s, 3H), 2.61 (t, J = 7.1 Hz, 2H). MS: m/z calcd for [M + H]+
258.0, found 258.0
tert-Butyl 3-((4-Bromophenyl)(methyl)amino)propanoate (3). To
a suspension of 2 (7.96 g, 31.00 mmol, 1 equiv) in CH2Cl2 (50 mL)
were added in succession tert-butanol (3.45 g, 46.52 mmol, 1.5 equiv),
dicyclohexylcarbodiimide (9.59 g, 46.50 mmol, 1.5 equiv) and 4-
dimethylaminopyridine (0.19 g, 1.60 mmol, 0.05 equiv). The mixture
was maintained at room temperature overnight and monitored by TLC.
The reaction mixture was filtrated, and the solution was concentrated in
vacuum. The crude product was purified by silica gel column
CONCLUSION
■
A novel fluorescent probe for NIR-II imaging of PCa tumors and
image-guided precise tumor resection was successfully designed,
synthesized, and evaluated. The probe demonstrated excellent
optical properties (λmax = 1092 nm in DMSO, Φ = 6.66% in
PBS), ideal water solubility, prominent biocompatibility, and
ultrahigh affinity (Ki = 80 pM) toward PSMA, which meets most
of the requirements for NIR-II imaging. Interestingly, PSMA-
1092 displayed a sensitive fluorescent response to the pH value
of the solution, and the fluorescence maxima shifted to 620 nm
(NIR-I) from its original wavelength (NIR-II) when transferred
into an acid environment. Moreover, PSMA-1092 exhibited
excellent performance in the in vivo imaging of PCa mice with a
good contrast and high resolution. The tumor was contoured
accurately at 4 h postinjection, and the maximum of TNR
reaches 7.62 1.05 at 12 h postinjection. More importantly, in
the intraoperative NIR-II image-guided surgery using PSMA-
7741
https://doi.org/10.1021/acs.jmedchem.1c00444
J. Med. Chem. 2021, 64, 7735−7745

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
chromatography (petroleum ether/ethyl acetate = 6/1, v/v) to give 3 as
a yellow oil (4.20 g, 43.2%). 1H NMR (400 MHz, CDCl3): δ 7.30 (d, J =
9.0 Hz, 2H), 6.62 (s, 2H), 3.60 (t, J = 7.2 Hz, 2H), 2.91 (s, 3H), 2.46 (t,
J = 7.2 Hz, 2H), 1.43 (s, 9H). 13C NMR (151 MHz, CDCl3): δ 171.49,
139.88, 131.93, 114.16, 108.61, 80.85, 55.82, 38.34, 35.00, 28.14.
HRMS: m/z calcd for [M + H]+ 314.0677, found 314.0750.
tert-Butyl 3-(Methyl(4-(tributylstannyl)phenyl)amino)-
propanoate (4). To the solution of 3 (4.00 g, 12.70 mmol, 1 equiv)
in toluene (50 mL) was added Pd(PPh3)4 (1.47 g, 1.27 mmol, 0.1
equiv). Hexabutyldistannane (7.43 g, 12.74 mmol, 1 equiv) was then
added under N2. The reaction was stirred at 120 °C for 2 h and
monitored by TLC. After being cooled to room temperature, to the
mixture was added H2O and extracted by diethyl ether (15 mL × 3).
The combined organic layers were dried over MgSO4 and concentrated
under vacuum to give 4 as a brown oil, which was used for the next step
without further purification.
152.54, 149.00, 133.55, 123.92, 119.36, 112.22, 48.43, 38.53, 32.10.
HRMS: m/z calcd for [M + H]+ 549.1300, found 549.1161.
Compound 9. To the suspension of 8 (25.1 mg, 0.046 mmol, 1
equiv) in CH2Cl2 (50 mL) were added 2,3,5,6-tetrafluorophenol (18.3
mg, 0.11 mmol, 2.4 equiv) and N,N′-dicyclohexylcarbodiimide (22.7
mg, 0.11 mmol, 2.4 equiv). The reaction mixture was stirred at room
temperature for 5 h and monitored by TLC. The solvent was removed
by evaporation in vacuum, and the residue was purified by silica gel
column chromatography (petroleum ether/CH2Cl2 = 1/5, v/v) to give
9 as a blue solid (25.9 mg, 67.4%). 1H NMR (400 MHz, CDCl3): δ 8.47
(d, J = 8.8 Hz, 2H), 7.87 (d, J = 8.5 Hz, 2H), 7.07−6.96 (m, 1H), 3.96
(t, J = 7.4 Hz, 2H), 3.41 (t, J = 7.3 Hz, 2H), 3.32 (s, 3H). 13C NMR (101
MHz, CDCl3): δ 168.19, 152.85, 148.44, 139.89, 133.23, 124.34,
120.03, 112.13, 103.68, 103.45, 102.98, 55.83, 38.83, 35.00. HRMS: m/
z calcd for [M + H]+ 845.1173, found 845.1249.
Compound 10. To the solution of 9 (25.9 mg, 0.031 mmol, 1 equiv)
in CH2Cl2 (50 mL) were added in succession tert-butyl Glu-urea-Lys
(35.9 mg, 0.074 mmol, 2.4 equiv) and Et3N (12.5 mg, 0.074 mmol, 2.4
equiv). The reaction mixture was stirred at room temperature for 6 h
and monitored by TLC. The solvent was removed by evaporation in
vacuum, and the residue was purified by silica gel column
chromatography (petroleum ether/ethyl acetate = 1/5, v/v) to give
10 as a green solid (28.5 mg, 61.3%). 1H NMR (400 MHz, CDCl3): δ
8.42 (d, J = 8.6 Hz, 2H), 8.05 (d, J = 8.4 Hz, 2H), 6.95 (s, 1H), 4.36−
4.28 (m, 2H), 3.98 (ddt, J = 34.8, 13.0, 6.9 Hz, 2H), 3.31 (s, 3H), 3.06
(s, 1H), 2.85 (s, 1H), 2.40 (t, J = 7.8 Hz, 2H), 2.17−2.02 (m, 2H),
1.97−1.54 (m, 6H), 1.49 (s, 2H), 1.43 (s, 9H), 1.41 (s, 9H), 1.41 (s,
9H). 13C NMR (101 MHz, CDCl3): δ 172.83, 172.49, 172.20, 171.58,
157.28, 82.09, 81.62, 80.50, 77.30, 67.98, 60.38, 53.39, 53.24, 39.19,
32.51, 31.69, 30.44, 28.81, 28.18, 28.09, 25.65, 22.62, 21.01, 14.22.
HRMS: m/z calcd for [M]+ 1531.8308, found 1531.7543.
Compound PSMA-1092. To the solution of 10 (28.5 mg, 0.019
mmol, 1 equiv) in 5 mL of CH2Cl2 was added 5 mL of TFA. The
reaction mixture was stirred at room temperature for 4 h. The solvent
was removed by evaporation in vacuum, and the residue was added to
10 mL of diethyl ether and a dark green precipitate was formed. Diethyl
ether was removed carefully, and 10 mL of petroleum ether was added
to wash the precipitate. After petroleum ether was removed, the
precipitate was allowed to be dried off to give PSMA-1092 as a green
solid (21.5 mg, 98.4%). 1H NMR (600 MHz, DMSO-d6): δ 8.18 (d, J =
8.5 Hz, 2H), 7.98 (t, J = 5.5 Hz, 1H), 6.95 (d, J = 8.6 Hz, 2H), 6.31 (m,
2H), 4.10 (td, J = 8.1, 5.2 Hz, 1H), 4.03 (q, J = 7.1 Hz, 3H), 3.72 (d, J =
7.2 Hz, 2H), 3.03 (d, J = 16.1 Hz, 5H), 2.40 (t, J = 7.0 Hz, 2H), 2.24
(qdd, J = 16.4, 9.1, 6.2 Hz, 2H), 1.91 (s, 2H), 1.75−1.61 (m, 2H), 1.40
(tt, J = 7.9, 4.5 Hz, 2H), 1.30 (q, J = 7.7, 5.6 Hz, 2H). 13C NMR (151
MHz, DMSO-d6): δ 174.43, 174.06, 173.61, 170.30, 170.24, 157.20,
151.91, 148.38, 132.93, 118.73, 111.48, 59.65, 52.18, 51.57, 48.46,
38.35, 37.94, 32.86, 31.72, 29.81, 28.67, 27.45, 22.57, 20.66, 13.99.
HRMS: m/z calcd for [M − H]− 1149.3848, found 1149.3775.
Method. Purity Determination. The purity of PSMA-1092 was
determined by HPLC. PSMA-1092 aqueous solution (500 μL,
containing 5% DMSO) was analyzed by a Bonna-Agela Technologies
Venusil MP C18 reverse-phase column (5 μm, 4.6 mm × 250 mm) and
eluted at a flow rate of 1.0 mL/min. Mobile phase A was water (with
0.1% TFA), and mobile phase B was acetonitrile (A/B = 73/27, v/v).
HPLC was performed on a Hitachi Primaide system (Japan) equipped
with an SPD-20A UV detector (λ = 600 nm).
Di-tert-butyl 3,3′-(((5,6-Dinitrobenzo[c][1,2,5]thiadiazole-4,7-
diyl)bis(4,1-phenylene))bis(methylazanediyl))dipropionate (5). To
the mixture of 4,7-dibromo-5,6-dinitrobenzo[c][1,2,5]thiadiazole
(1.23 g, 3.21 mmol, 1 equiv) and PdCl2(PPh3)2 (0.45 g, 0.64 mmol,
0.2 equiv) in toluene (50 mL) was added 4 under N2. The reaction was
stirred at 120 °C for 2 h and monitored by TLC. The solvent was
removed by evaporation in vacuum, and the residue was purified by
silica gel column chromatography (petroleum ether/ethyl acetate = 5/
1, v/v) to give 5 as a purplish red solid (171.0 mg, 7.7%). 1H NMR (600
MHz, CDCl3): δ 7.51 (d, J = 8.7 Hz, 2H), 6.93 (s, 2H), 3.72 (t, J = 7.2
Hz, 2H), 3.07 (s, 3H), 2.59 (t, J = 6.9 Hz, 2H), 1.45 (s, 9H). 13C NMR
(101 MHz, CDCl3): δ 171.36, 153.58, 149.95, 142.10, 130.71, 127.71,
117.77, 112.01, 81.04, 48.27, 38.36, 33.41, 28.17. HRMS: m/z calcd for
[M + H]+ 693.2628, found 693.2703.
Di-tert-butyl 3,3′-(((5,6-Diaminobenzo[c][1,2,5]thiadiazole-4,7-
diyl)bis(4,1-phenylene))bis(methylazanediyl))dipropionate (6). To
the solution of 5 (327.2 mg, 0.47 mmol, 1 equiv) in acetic acid (40 mL)
was added iron powder (1.32 g, 23.50 mmol, 50 equiv). The mixture
was stirred at 100 °C and monitored by TLC. After being cooled to
room temperature, the reaction was filtered and the filtrate was added to
ice water and neutralized by NaHCO3. The mixture was then extracted
by CH2Cl2 (15 mL × 3). The combined organic layers were dried over
Na2SO4 and concentrated under vacuum. The crude product was
purified by silica gel column chromatography (petroleum ether/ethyl
acetate = 3/1, v/v) to give 6 as a brown solid (106.8 mg, 36.2%). 1H
NMR (400 MHz, CDCl3): δ 7.47 (d, J = 7.9 Hz, 2H), 6.93 (s, 2H), 4.10
(s, 1H), 3.72 (t, J = 7.0 Hz, 2H), 3.04 (s, 3H), 2.57 (s, 2H), 1.47 (s, 9H).
13C NMR (151 MHz, CDCl3): δ 171.68, 151.75, 148.33, 138.18,
131.26, 122.64, 113.75, 112.88, 80.82, 48.66, 38.46, 33.31, 28.20.
HRMS: m/z calcd for [M + H]+ 633.3145, found 633.3221.
Compound 7. To the solution of 6 (106.5 mg, 0.17 mmol, 1 equiv)
in dry pyridine (20 mL) were added in succession PhNSO (165.6 mg,
1.19 mmol, 7 equiv) and TMSCl (2.40 g, 22.07 mmol, 130 equiv). The
reaction was stirred at 80 °C overnight and monitored by TLC. After
being cooled to room temperature, the above mixture was poured into
ice water and extracted by CH2Cl2 (15 mL × 3). The combined organic
layers were dried over Na2SO4 and concentrated under vacuum. The
residue was purified by silica gel column chromatography (petroleum
ether/ethyl acetate = 6/1, v/v) to give 7 as a green solid (30.5 mg,
27.2%). 1H NMR (400 MHz, CDCl3): δ 8.31 (d, J = 8.4 Hz, 2H), 7.14
(s, 2H), 3.78 (t, J = 7.3 Hz, 2H), 3.15 (s, 3H), 2.69 (s, 2H), 1.46 (s, 9H).
13C NMR (101 MHz, CDCl3): δ 171.55, 152.85, 148.84, 133.10,
123.81, 119.99, 112.01, 80.89, 48.53, 38.60, 33.35, 28.21. HRMS: m/z
calcd for [M + H]+ 661.2552, found 661.2625.
Compound 8. To the solution of 7 (30.5 mg, 0.046 mmol, 1 equiv)
in 5 mL of CH2Cl2 was added 5 mL of TFA. The reaction mixture was
stirred at room temperature for 4 h and monitored by TLC. The
solution was removed by evaporation in vacuum. The residue was
added to 15 mL of CH2Cl2 and ultrasonically vibrated for 5 min. The
solid was collected by filtration and washed with 5 mL of CH2Cl2 and 5
mL of petroleum ether. Compound 8 was obtained as a dark green solid
(25.1 mg, 100%). 1H NMR (400 MHz, DMSO-d6): δ 8.18 (d, J = 8.4
Hz, 2H), 6.96 (d, J = 8.4 Hz, 2H), 3.74 (t, J = 7.0 Hz, 2H), 3.04 (s, 3H),
2.56 (t, J = 7.0 Hz, 2H). 13C NMR (101 MHz, DMSO-d6): δ 173.60,
Fluorescence penetration: PSMA-1092 aqueous solution (0.5 mg/
mL, containing 5% DMSO) was filled into a capillary glass tube (1.5
mm × 100 mm) and fixed on the imaging platform. Different chicken
breast tissue with known thickness was covered upon the fixed capillary
glass tube, and the NIR-II images were acquired subsequently.
NMR Titration. PSMA-1092 (8.33 mg) was dissolved in 1.5 mL of
DMSO-d6, and its 1H NMR spectrum was recorded on a
JNMECZ600R/S3 NMR spectrometer (600 MHz, Japan) at 30 °C.
Then various amounts of TFA-d were added successively with the final
volumes of 20, 45, 80, 140, 210, 310, and 360 μL for each time. After
1
each addition of TFA-d, the H NMR spectrum of the sample was
recorded.
7742
https://doi.org/10.1021/acs.jmedchem.1c00444
J. Med. Chem. 2021, 64, 7735−7745

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Cell Culture. LNCaP and PC3 cells were cultured in Sigma RPMI-
1640 media (USA) supplemented with 10% fetal bovine serum and 1%
penicillin−streptomycin solution. All the cells were maintained in a
humidified environment with 5% CO2 at 37 °C in an incubator.
In Vitro Inhibition Assay. To quantitatively analyze the binding
affinity of PSMA-1092 to PSMA, an in vitro inhibition assay was
performed according to a previous study with slight modification.1 First,
12.5 μL of N-acetylaspartylglutamate (8 μM) and 12.5 μL of PSMA-
1092 solution (variable concentration covering 0.01 nM to 1 μM) were
added into the lysates of LNCaP cell extracts (25 μL), and the above
mixture was incubated for 120 min. The glutamate concentration was
measured by incubation with a working solution (50 μL) of the Amplex
Red glutamic acid kit for 30 min, and the fluorescence intensities were
recorded by a plate reader (excitation = 530 nm and emission = 590
nm). IC50 values were determined from the inhibition curve at the
concentration at which enzyme activity was inhibited by 50%, and
enzyme inhibitory constants (Ki values) were calculated using the
Cheng−Prusoff conversion. Assays were performed in triplicate.
MTT Assay. PC-3 cells were seeded into a 96-well plate and
incubated at 37 °C with 5% CO2 for 24 h. Then the cells were treated
with different concentration of PSMA-1092 (10.0, 20.0, 25.3, 40.0, and
50.0 μM) and PBS as the control for 24 incubation. After incubation,
MTT solution (5 mg/mL) was added, and the cells were kept
incubating for another 4 h. After the culture media were removed by
centrifugation, 300 μL of DMSO was added. The absorbance of each
well was measured at 570 nm using a 1420 Multiabel counter for
assessing the viability of living cells.
ASSOCIATED CONTENT
* Supporting Information
■
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00444.
1
Additional figures, H NMR, 13C NMR, and HRMS
spectra (PDF)
Tumor resection under NIR-II navigation video of
PSMA-1092 (AVI, AVI)
Molecular formula strings (CSV)
AUTHOR INFORMATION
Corresponding Authors
■
Mengchao Cui − Key Laboratory of Radiopharmaceuticals,
Ministry of Education, College of Chemistry, Beijing Normal
University, Beijing 100875, China; Center for Advanced
Materials Research, Advanced Institute of Natural Sciences,
Beijing Normal University at Zhuhai, Zhuhai 519087, China;
orcid.org/0000-0002-3488-7864; Phone: +86-
13811995064; Email: cmc@bnu.edu.cn
Zhenhua Hu − CAS Key Laboratory of Molecular Imaging,
Beijing Key Laboratory of Molecular Imaging, The State Key
Laboratory of Management and Control for Complex Systems,
Institute of Automation, Chinese Academy of Sciences, Beijing
100190, China; Phone: +86-10-82544536;
In Vitro Stability in Plasma. One hundred microliters of PSMA-
1092 aqueous solution was added into 1 mL of mice plasma (100 μM),
and the above mixture was incubated at 37 °C. A 100 μL sample was
collected at selected time points (1, 2, 3, 12, 24 h) and treated with 300
μL of acetonitrile. After centrifugation and filtration, 90 μL of
supernatant was collected and added 160 μL of H2O. The above
mixture was subjected to HPLC analysis, and the purities of PSMA-
1092 were determined using acetonitrile/H2O = 27/73 with a flow rate
of 1 mL/min.
In Vivo Blood Clearance. After intravenous administration of
PSMA-1092 (10 mg/kg, ICR, 8 weeks old), the blood sample was
collected at selected time points and stored in a centrifuge tube (2.5
mL) with heparin sodium. After centrifugation, the NIR-II images of 50
μL blood sample supernatants were acquired under the same condition
(100 mW/cm2 power density, 1000 nm long-pass filter, 500 ms
exposure time), and the fluorescence intensities were analyzed by
ImageJ.
In Vivo NIR-II Imaging. To perform in vivo NIR-II fluorescence
imaging, LNCap tumor-bearing mice were administered PSMA-1092
(2.5 mg/kg) intravenously, and the NIR-II imaging was performed at 1,
2, 4, 6, 8, 12, and 24 h postinjection under 792 nm laser excitation at a
power density of 100 mW/cm2 (1000 nm long-pass filter, 50 ms
exposure time). Mice were anesthetized by the combination gas of
isoflurane and oxygen at a rate of 0.4 L/min and remained under
anesthesia for the whole process. The NIR-II images were analyzed with
the open-source software ImageJ.
Intraoperative Image-Guided Tumor Resection. LNCap tumor-
bearing mice were intravenously injected with PSMA-1092 (2.5 mg/
kg) and received surgery at 24 h postinjection. Mice were anesthetized
by the combination gas of isoflurane and oxygen at a rate of 0.4 L/min
before the operation and remained under anesthesia for the surgery.
The tumor was removed under the guidance of NIR-II images, and the
whole process was recorded by the imaging instrument. The NIR-II
images were analyzed on the open-source software ImageJ.
Email: zhenhua.hu@ia.ac.cn
Authors
Longfei Zhang − Key Laboratory of Radiopharmaceuticals,
Ministry of Education, College of Chemistry, Beijing Normal
University, Beijing 100875, China
Xiaojing Shi − CAS Key Laboratory of Molecular Imaging,
Beijing Key Laboratory of Molecular Imaging, The State Key
Laboratory of Management and Control for Complex Systems,
Institute of Automation, Chinese Academy of Sciences, Beijing
100190, China
Yuying Li − Key Laboratory of Radiopharmaceuticals, Ministry
of Education, College of Chemistry, Beijing Normal University,
Beijing 100875, China
Xiaojiang Duan − Department of Nuclear Medicine, Peking
University First Hospital, Beijing 100034, China
Zeyu Zhang − School of Medical Science and Engineering,
Beihang University, Beijing 100191, China
Hualong Fu − Key Laboratory of Radiopharmaceuticals,
Ministry of Education, College of Chemistry, Beijing Normal
University, Beijing 100875, China
Xing Yang − Department of Nuclear Medicine, Peking
University First Hospital, Beijing 100034, China;
orcid.org/0000-0001-5186-4990
Jie Tian − CAS Key Laboratory of Molecular Imaging, Beijing
Key Laboratory of Molecular Imaging, The State Key
Laboratory of Management and Control for Complex Systems,
Institute of Automation, Chinese Academy of Sciences, Beijing
100190, China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00444
Data Availability. The data supporting the results of this study are
available in this article and its Supporting Information. The raw data are
available in at 10.6084/m9.figshare.13520072.v1.
Laboratory Animals. All protocols requiring the use of animals
conformed to the China Animal Management Regulations (2017
edition) and were approved by the animal care committee of Beijing
Normal University.
Author Contributions
M.C. and L.Z. conceived and designed the experiment. L.Z.
synthesized and characterized PSMA-1092. Optical properties
were determined by L.Z. MTT assay was performed by Y.L. In
vitro competitive inhibition assay was performed by X.D. Animal
studies were performed by L.Z., X.S., M.C., H.F., and Z.Z. M.C.,
7743
https://doi.org/10.1021/acs.jmedchem.1c00444
J. Med. Chem. 2021, 64, 7735−7745

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
(7) Fowler, J. E.; Terrell, F. L.; Renfroe, L. D. Co-Morbidities and
Survival of Men with Localized Prostate Cancer Treated with Surgery
or Radiation Therapy. J. Urol. 1996, 156, 1714−1718.
(8) Preston, M. A.; Breau, R. H.; Lantz, A. G.; Morash, C.; Gerridzen,
R. G.; Doucette, S.; Mallick, R.; Eastham, J. A.; Cagiannos, I. The
association between nerve sparing and a positive surgical margin during
radical prostatectomy. Urol. Oncol. 2015, 33 (1), 18.e1.
Z.H., L.Z., and X.S. analyzed the data. M.C., Z.H., X.Y., and J.T.
provided reagents, materials, and experiment instruments. M.C.,
Z.H., L.Z., X.S., and H.F. wrote the paper. L.Z. and X.S.
contributed equally to this work.
Notes
The authors declare no competing financial interest.
(9) Nakajima, T.; Mitsunaga, M.; Bander, N. H.; Heston, W. D.;
Choyke, P. L.; Kobayashi, H. Targeted, activatable, in vivo fluorescence
imaging of prostate-specific membrane antigen (PSMA) positive
tumors using the quenched humanized J591 antibody-indocyanine
green (ICG) conjugate. Bioconjugate Chem. 2011, 22 (8), 1700−1705.
(10) Stephenson, A. J.; Eggener, S. E.; Hernandez, A. V.; Klein, E. A.;
Kattan, M. W.; Wood, D. P., Jr.; Rabah, D. M.; Eastham, J. A.; Scardino,
P. T. Do margins matter? The influence of positive surgical margins on
prostate cancer-specific mortality. Eur. Urol. 2014, 65 (4), 675−680.
(11) Eastham, J. A.; Kuroiwa, K.; Ohori, M.; Serio, A. M.; Gorbonos,
A.; Maru, N.; Vickers, A. J.; Slawin, K. M.; Wheeler, T. M.; Reuter, V. E.;
Scardino, P. T. Prognostic significance of location of positive margins in
radical prostatectomy specimens. Urology 2007, 70 (5), 965−969.
(12) Hensbergen, A. W.; van Willigen, D. M.; van Beurden, F.; van
Leeuwen, P. J.; Buckle, T.; Schottelius, M.; Maurer, T.; Wester, H.-J.;
van Leeuwen, F. W. B. Image-Guided Surgery: Are We Getting the
Most Out of Small-Molecule Prostate-Specific-Membrane-Antigen-
Targeted Tracers? Bioconjugate Chem. 2020, 31 (2), 375−395.
(13) Andriole, G. L. Do Margins Matter? The Prognostic Significance
of Positive Surgical Margins in Radical Prostatectomy Specimens.
Yearb. Urol. 2008, 2008, 157−158.
ACKNOWLEDGMENTS
■
This work was supported by the National Natural Science
Foundation of China (NSFC) (Nos. U1967221, 22022601, and
81671759), Beijing Natural Science Foundation (7182089 and
JQ19027), Beijing Nova Program (Z181100006218046), and
the innovative research team of high-level local universities in
Shanghai.
ABBREVIATIONS
■
D−A−D, donor−acceptor−donor; DCC, N,N′-dicyclohexyl-
carbodiimide; DCM, dichloromethane; DMAP, 4-dimethyla-
minopyridine; DMSO, dimethyl sulfoxide; FDA, food and drug
administrator; Glu-urea-Lys, (S)-3-(carboxyformamido)-2-(3-
(carboxymethyl) ureido) propanoic acid; Hz, hertz; HRMS,
high-resolution mass spectrometry; ICG, indocyanine green;
ICT, intramolecular charge transfer; LP, long-pass; MS, mass
spectrometry; NIR-I, near-infrared window I; NIR-II, near-
infrared window II; PCa, prostate cancer; ppm, parts per million;
PhNSO, N-sulfinylaniline; PSMA, prostate-specific membrane
antigen; TLC, thin-layer chromatography; TMSCl, trimethyl
chlorosilane; TFA, trifluoroacetic acid; TNR, tumor-to-normal
tissue ratio
(14) Costello, A. J. Considering the role of radical prostatectomy in
21st century prostate cancer care. Nat. Rev. Urol. 2020, 17 (3), 177−
188.
(15) Smith, J. A. Quality of life and satisfaction with outcome among
prostate-cancer survivors. Urol. Oncol-semin. Ori. 2008, 26, 685.
(16) Cui, M. Past and recent progress of molecular imaging probes for
beta-amyloid plaques in the brain. Curr. Med. Chem. 2013, 21 (1), 82−
112.
(17) James, M. L.; Gambhir, S. S. A molecular imaging primer:
modalities, imaging agents, and applications. Physiol. Rev. 2012, 92 (2),
897−965.
(18) Hu, Z.; Zhao, M.; Qu, Y.; Zhang, X.; Zhang, M.; Liu, M.; Guo, H.;
Zhang, Z.; Wang, J.; Yang, W.; Tian, J. In Vivo 3-Dimensional
Radiopharmaceutical-Excited Fluorescence Tomography. J. Nucl. Med.
2017, 58 (1), 169−174.
(19) Liu, M.; Zheng, S.; Zhang, X.; Guo, H.; Shi, X.; Kang, X.; Qu, Y.;
Hu, Z.; Tian, J. Cerenkov luminescence imaging on evaluation of early
response to chemotherapy of drug-resistant gastric cancer. Nano-
medicine 2018, 14 (1), 205−213.
(20) Sun, Y.; Qu, C.; Chen, H.; He, M.; Tang, C.; Shou, K.; Hong, S.;
Yang, M.; Jiang, Y.; Ding, B.; Xiao, Y.; Xing, L.; Hong, X.; Cheng, Z.
Novel benzo-bis(1,2,5-thiadiazole) fluorophores for in vivo NIR-II
imaging of cancer. Chem. Sci. 2016, 7 (9), 6203−6207.
(21) Zhu, S.; Yang, Q.; Antaris, A. L.; Yue, J.; Ma, Z.; Wang, H.;
Huang, W.; Wan, H.; Wang, J.; Diao, S.; Zhang, B.; Li, X.; Zhong, Y.; Yu,
K.; Hong, G.; Luo, J.; Liang, Y.; Dai, H. Molecular imaging of biological
systems with a clickable dye in the broad 800- to 1,700-nm near-infrared
window. Proc. Natl. Acad. Sci. U. S. A. 2017, 114 (5), 962−967.
(22) Wan, H.; Yue, J.; Zhu, S.; Uno, T.; Zhang, X.; Yang, Q.; Yu, K.;
Hong, G.; Wang, J.; Li, L.; Ma, Z.; Gao, H.; Zhong, Y.; Su, J.; Antaris, A.
L.; Xia, Y.; Luo, J.; Liang, Y.; Dai, H. A bright organic NIR-II
nanofluorophore for three-dimensional imaging into biological tissues.
Nat. Commun. 2018, 9 (1), 1171.
(23) Ding, B.; Xiao, Y.; Zhou, H.; Zhang, X.; Qu, C.; Xu, F.; Deng, Z.;
Cheng, Z.; Hong, X. Polymethine Thiopyrylium Fluorophores with
Absorption beyond 1000 nm for Biological Imaging in the Second
Near-Infrared Subwindow. J. Med. Chem. 2019, 62 (4), 2049−2059.
(24) Hu, Z.; Chen, W. H.; Tian, J.; Cheng, Z. NIRF Nanoprobes for
Cancer Molecular Imaging: Approaching Clinic. Trends Mol. Med.
2020, 26 (5), 469−482.
REFERENCES
■
(1) Bray, F.; Ferlay, J.; Soerjomataram, I.; Siegel, R. L.; Torre, L. A.;
Jemal, A. Global cancer statistics 2018: GLOBOCAN estimates of
incidence and mortality worldwide for 36 cancers in 185 countries. Ca-
Cancer J. Clin. 2018, 68 (6), 394−424.
(2) Skolarus, T. A.; Wolf, A. M.; Erb, N. L.; Brooks, D. D.; Rivers, B.
M.; Underwood, W., 3rd; Salner, A. L.; Zelefsky, M. J.; Aragon-Ching, J.
B.; Slovin, S. F.; Wittmann, D. A.; Hoyt, M. A.; Sinibaldi, V. J.; Chodak,
G.; Pratt-Chapman, M. L.; Cowens-Alvarado, R. L. American Cancer
Society prostate cancer survivorship care guidelines. Ca-Cancer J. Clin.
2014, 64 (4), 225−249.
(3) Fizazi, K.; Scher, H. I.; Molina, A.; Logothetis, C. J.; Chi, K. N.;
Jones, R. J.; Staffurth, J. N.; North, S.; Vogelzang, N. J.; Saad, F.;
Mainwaring, P.; Harland, S.; Goodman, O. B.; Sternberg, C. N.; Li, J.
H.; Kheoh, T.; Haqq, C. M.; de Bono, J. S. Abiraterone acetate for
treatment of metastatic castration-resistant prostate cancer: final overall
survival analysis of the COU-AA-301 randomised, double-blind,
placebo-controlled phase 3 study. Lancet Oncol. 2012, 13 (10), 983−
992.
(4) Olson, W. C.; Heston, W. D.; Rajasekaran, A. K. Clinical trials of
cancer therapies targeting prostate-specific membrane antigen. Rev.
Recent Clin. Trials 2007, 2 (3), 182−190.
(5) Fizazi, K.; Tran, N.; Fein, L.; Matsubara, N.; Rodriguez-Antolin,
A.; Alekseev, B. Y; Ozguroglu, M.; Ye, D.; Feyerabend, S.; Protheroe,
A.; Sulur, G.; Luna, Y.; Li, S.; Mundle, S.; Chi, K. N Abiraterone acetate
plus prednisone in patients with newly diagnosed high-risk metastatic
castration-sensitive prostate cancer (LATITUDE): final overall survival
analysis of a randomised, double-blind, phase 3 trial. Lancet Oncol.
2019, 20 (5), 686−700.
(6) Lu-Yao, G. L.; Albertsen, P. C.; Moore, D. F.; Shih, W.; Lin, Y.;
DiPaola, R. S.; Yao, S. L. Fifteen-year survival outcomes following
primary androgen-deprivation therapy for localized prostate cancer.
JAMA Int. Med. 2014, 174 (9), 1460−1467.
7744
https://doi.org/10.1021/acs.jmedchem.1c00444
J. Med. Chem. 2021, 64, 7735−7745

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
(25) Sun, Y.; Zeng, X.; Xiao, Y.; Liu, C.; Zhu, H.; Zhou, H.; Chen, Z.;
Xu, F.; Wang, J.; Zhu, M.; Wu, J.; Tian, M.; Zhang, H.; Deng, Z.; Cheng,
Z.; Hong, X. Novel dual-function near-infrared II fluorescence and PET
probe for tumor delineation and image-guided surgery. Chem. Sci. 2018,
9 (8), 2092−2097.
(26) Sun, Y.; Ding, M.; Zeng, X.; Xiao, Y.; Wu, H.; Zhou, H.; Ding, B.;
Qu, C.; Hou, W.; Er-Bu, A.; Zhang, Y.; Cheng, Z.; Hong, X. Novel
bright-emission small-molecule NIR-II fluorophores for in vivo tumor
imaging and image-guided surgery. Chem. Sci. 2017, 8 (5), 3489−3493.
(27) Zhang, Z.; Cai, M.; Bao, C.; Hu, Z.; Tian, J. Endoscopic
Cerenkov luminescence imaging and image-guided tumor resection on
hepatocellular carcinoma-bearing mouse models. Nanomedicine 2019,
17, 62−70.
(28) Hu, Z.; Chi, C.; Liu, M.; Guo, H.; Zhang, Z.; Zeng, C.; Ye, J.;
Wang, J.; Tian, J.; Yang, W.; Xu, W. Nanoparticle-mediated
radiopharmaceutical-excited fluorescence molecular imaging allows
precise image-guided tumor-removal surgery. Nanomedicine 2017, 13
(4), 1323−1331.
(29) Antaris, A. L.; Chen, H.; Cheng, K.; Sun, Y.; Hong, G.; Qu, C.;
Diao, S.; Deng, Z.; Hu, X.; Zhang, B.; Zhang, X.; Yaghi, O. K.;
Alamparambil, Z. R.; Hong, X.; Cheng, Z.; Dai, H. A small-molecule
dye for NIR-II imaging. Nat. Mater. 2016, 15 (2), 235−242.
(30) Zhang, X. D.; Wang, H.; Antaris, A. L.; Li, L.; Diao, S.; Ma, R.;
Nguyen, A.; Hong, G.; Ma, Z.; Wang, J.; Zhu, S.; Castellano, J. M.;
Wyss-Coray, T.; Liang, Y.; Luo, J.; Dai, H. Traumatic Brain Injury
Imaging in the Second Near-Infrared Window with a Molecular
Fluorophore. Adv. Mater. 2016, 28 (32), 6872−6879.
(31) Antaris, A. L.; Chen, H.; Diao, S.; Ma, Z.; Zhang, Z.; Zhu, S.;
Wang, J.; Lozano, A. X.; Fan, Q.; Chew, L.; Zhu, M.; Cheng, K.; Hong,
X.; Dai, H.; Cheng, Z. A high quantum yield molecule-protein complex
fluorophore for near-infrared II imaging. Nat. Commun. 2017, 8, 15269.
(32) Feng, Y.; Zhu, S.; Antaris, A. L.; Chen, H.; Xiao, Y.; Lu, X.; Jiang,
L.; Diao, S.; Yu, K.; Wang, Y.; Herraiz, S.; Yue, J.; Hong, X.; Hong, G.;
Cheng, Z.; Dai, H.; Hsueh, A. J. Live imaging of follicle stimulating
hormone receptors in gonads and bones using near infrared II
fluorophore. Chem. Sci. 2017, 8 (5), 3703−3711.
(33) Lin, J.; Zeng, X.; Xiao, Y.; Tang, L.; Nong, J.; Liu, Y.; Zhou, H.;
Ding, B.; Xu, F.; Tong, H.; Deng, Z.; Hong, X. Novel near-infrared II
aggregation-induced emission dots for in vivo bioimaging. Chem. Sci.
2019, 10 (4), 1219−1226.
(41) Kawatani, M.; Yamamoto, K.; Yamada, D.; Kamiya, M.;
Miyakawa, J.; Miyama, Y.; Kojima, R.; Morikawa, T.; Kume, H.;
Urano, Y. Fluorescence Detection of Prostate Cancer by an Activatable
Fluorescence Probe for PSMA Carboxypeptidase Activity. J. Am. Chem.
Soc. 2019, 141 (26), 10409−10416.
(42) Wang, X.; Huang, S. S.; Heston, W. D.; Guo, H.; Wang, B. C.;
Basilion, J. P. Development of targeted near-infrared imaging agents for
prostate cancer. Mol. Cancer Ther. 2014, 13 (11), 2595−2606.
(43) Chen, Y.; Chatterjee, S.; Lisok, A.; Minn, I.; Pullambhatla, M.;
Wharram, B.; Wang, Y.; Jin, J.; Bhujwalla, Z. M.; Nimmagadda, S.;
Mease, R. C.; Pomper, M. G. A PSMA-targeted theranostic agent for
photodynamic therapy. J. Photochem. Photobiol., B 2017, 167, 111−116.
(44) Kwon, Y. D.; Oh, J. M.; La, M. T.; Chung, H. J.; Lee, S. J.; Chun,
S.; Lee, S. H.; Jeong, B. H.; Kim, H. K. Synthesis and Evaluation of
Multifunctional Fluorescent Inhibitors with Synergistic Interaction of
Prostate-Specific Membrane Antigen and Hypoxia for Prostate Cancer.
Bioconjugate Chem. 2019, 30 (1), 90−100.
(45) Kwon, Y. D.; Chung, H. J.; Lee, S. J.; Lee, S. H.; Jeong, B. H.; Kim,
H. K. Synthesis of novel multivalent fluorescent inhibitors with high
affinity to prostate cancer and their biological evaluation. Bioorg. Med.
Chem. Lett. 2018, 28 (4), 572−576.
(46) Shallal, H. M.; Minn, I.; Banerjee, S. R.; Lisok, A.; Mease, R. C.;
Pomper, M. G. Heterobivalent agents targeting PSMA and integrin-
alphavbeta3. Bioconjugate Chem. 2014, 25 (2), 393−405.
(47) Chen, Y.; Pullambhatla, M.; Banerjee, S. R.; Byun, Y.; Stathis, M.;
Rojas, C.; Slusher, B. S.; Mease, R. C.; Pomper, M. G. Synthesis and
Biological Evaluation of Low Molecular Weight Fluorescent Imaging
Agents for the Prostate-Specific Membrane Antigen. Bioconjugate
Chem. 2012, 23 (12), 2377−2385.
(48) Duan, X.; Liu, F.; Kwon, H.; Byun, Y.; Minn, I.; Cai, X.; Zhang, J.;
Pomper, M. G.; Yang, Z.; Xi, Z.; Yang, X. (S)-3-(Carboxyformamido)-
2-(3-(carboxymethyl)ureido)propanoic Acid as a Novel PSMA
Targeting Scaffold for Prostate Cancer Imaging. J. Med. Chem. 2020,
63 (7), 3563−3576.
(49) Staderini, M.; Martin, M. A.; Bolognesi, M. L.; Menendez, J. C.
Imaging of beta-amyloid plaques by near infrared fluorescent tracers: a
new frontier for chemical neuroscience. Chem. Soc. Rev. 2015, 44 (7),
1807−1819.
(50) Zhu, S.; Tian, R.; Antaris, A. L.; Chen, X.; Dai, H. Near-Infrared-
II Molecular Dyes for Cancer Imaging and Surgery. Adv. Mater. 2019,
31 (24), No. 1900321.
(34) Wang, F.; Wan, H.; Ma, Z.; Zhong, Y.; Sun, Q.; Tian, Y.; Qu, L.;
Du, H.; Zhang, M.; Li, L.; Ma, H.; Luo, J.; Liang, Y.; Li, W. J.; Hong, G.;
Liu, L.; Dai, H. Light-sheet microscopy in the near-infrared II window.
Nat. Methods 2019, 16 (6), 545−552.
(35) Hu, Z.; Fang, C.; Li, B.; Zhang, Z.; Cao, C.; Cai, M.; Su, S.; Sun,
X.; Shi, X.; Li, C.; Zhou, T.; Zhang, Y.; Chi, C.; He, P.; Xia, X.; Chen, Y.;
Gambhir, S. S.; Cheng, Z.; Tian, J. First-in-human liver-tumour surgery
guided by multispectral fluorescence imaging in the visible and near-
infrared-I/II windows. Nat. Biomed. Eng. 2020, 4 (3), 259−271.
(36) He, S.; Song, J.; Qu, J.; Cheng, Z. Crucial breakthrough of second
near-infrared biological window fluorophores: design and synthesis
toward multimodal imaging and theranostics. Chem. Soc. Rev. 2018, 47
(12), 4258−4278.
(37) Hong, G.; Antaris, A. L.; Dai, H. Near-infrared fluorophores for
biomedical imaging. Nat. Biomed. Eng. 2017, 1, 0010.
(38) Ding, F.; Zhan, Y.; Lu, X.; Sun, Y. Recent advances in near-
infrared II fluorophores for multifunctional biomedical imaging. Chem.
Sci. 2018, 9 (19), 4370−4380.
(39) Li, D.; Qu, C.; Liu, Q.; Wu, Y.; Hu, X.; Qian, K.; Chang, B.; He,
S.; Yuan, Y.; Li, Y.; Ko, T.; Yu, A.; Cheng, Z. Monitoring the Real-Time
Circulatory System-Related Physiological and Pathological Processes
In Vivo Using a Multifunctional NIR-II Probe. Adv. Funct. Mater. 2020,
30 (6), 1906343.
(51) Wang, S.; Fan, Y.; Li, D.; Sun, C.; Lei, Z.; Lu, L.; Wang, T.; Zhang,
F. Anti-quenching NIR-II molecular fluorophores for in vivo high-
contrast imaging and pH sensing. Nat. Commun. 2019, 10 (1), 1058.
(52) Qu, C.; Xiao, Y.; Zhou, H.; Ding, B.; Li, A.; Lin, J.; Zeng, X.;
Chen, H.; Qian, K.; Zhang, X.; Fang, W.; Wu, J.; Deng, Z.; Cheng, Z.;
Hong, X. Quaternary Ammonium Salt Based NIR-II Probes for In Vivo
Imaging. Adv. Opt. Mater. 2019, 7 (15), 1900229.
(53) Hong, G.; Robinson, J. T.; Zhang, Y.; Diao, S.; Antaris, A. L.;
Wang, Q.; Dai, H. In vivo fluorescence imaging with Ag2S quantum
dots in the second near-infrared region. Angew. Chem., Int. Ed. 2012, 51
(39), 9818−9821.
(54) Banerjee, S. R.; Foss, C. A.; Castanares, M.; Mease, R. C.; Byun,
Y.; Fox, J. J.; Hilton, J.; Lupold, S. E.; Kozikowski, A. P.; Pomper, M. G.
Synthesis and evaluation of technetium-99m- and rhenium-labeled
inhibitors of the prostate-specific membrane antigen (PSMA). J. Med.
Chem. 2008, 51 (15), 4504−4517.
(55) Rais, R.; Rojas, C.; Wozniak, K.; Wu, Y.; Zhao, M.; Tsukamoto,
T.; Rudek, M. A.; Slusher, B. S. Bioanalytical method for evaluating the
pharmacokinetics of the GCP-II inhibitor 2-phosphonomethyl
pentanedioic acid (2-PMPA). J. Pharm. Biomed. Anal. 2014, 88, 162−
169.
(40) Benesova, M.; Bauder-Wust, U.; Schafer, M.; Klika, K. D.; Mier,
W.; Haberkorn, U.; Kopka, K.; Eder, M. Linker Modification Strategies
To Control the Prostate-Specific Membrane Antigen (PSMA)-
Targeting and Pharmacokinetic Properties of DOTA-Conjugated
PSMA Inhibitors. J. Med. Chem. 2016, 59 (5), 1761−1775.
7745
https://doi.org/10.1021/acs.jmedchem.1c00444
J. Med. Chem. 2021, 64, 7735−7745