Synthesis and evaluation of technetium-99m- and rhenium-labeled inhibitors of the prostate-specific membrane antigen (PSMA)

Article abstract of DOI:10.1021/jm800111u

The prostate-specific membrane antigen (PSMA) is increasingly recognized as a viable target for imaging and therapy of cancer. We prepared seven ^99mTc/Re-labeled compounds by attaching known Tc/Re chelating agents to an amino-functionalized PSMA inhibitor (lys-NHCONH-glu) with or without a variable length linker moiety. K_i values ranged from 0.17 to 199 nM. Ex vivo biodistribution and in vivo imaging demonstrated the degree of specific binding to engineered PSMA+ PC3 PIP tumors. PC3-PIP cells are derived from PC3 that have been transduced with the gene for PSMA. Despite demonstrating nearly the lowest PSMA inhibitory potency of this series, [^99mTc(CO) ₃(L1)]⁺ (L1 = (2-pyridylmethyl)₂N(CH ₂)₄CH(CO₂H)-NHCO-(CH₂) ₆CO-NH-lys-NHCONH-glu) showed the highest, most selective PIP tumor uptake, at 7.9 ± 4.0% injected dose per gram of tissue at 30 min postinjection. Radioactivity cleared from nontarget tissues to produce a PIP to flu (PSMA-PC3) ratio of 44:1 at 120 min postinjection. PSMA can accommodate the steric requirements of ^99mTc/Re complexes within PSMA inhibitors, the best results achieved with a linker moiety between the ε amine of the urea lysine and the chelator.

Full text of DOI:10.1021/jm800111u

4504
J. Med. Chem. 2008, 51, 4504–4517
Synthesis and Evaluation of Technetium-99m- and Rhenium-Labeled Inhibitors of the
Prostate-Specific Membrane Antigen (PSMA)
Sangeeta R. Banerjee,† Catherine A. Foss,† Mark Castanares,‡ Ronnie C. Mease,† Youngjoo Byun,† James J. Fox,† John Hilton,†
Shawn E. Lupold,§ Alan P. Kozikowski,| and Martin G. Pomper*,†,‡
Russell H. Morgan Department of Radiology and Radiological Sciences, Department of Pharmacology & Molecular Sciences, Department of
Urology, Johns Hopkins Medical Institutions, Baltimore, Maryland 21231, and Department of Medicinal Chemistry and Pharmacognosy,
UniVersity of Illinois at Chicago, Illinois 60612
ReceiVed February 2, 2008
The prostate-specific membrane antigen (PSMA) is increasingly recognized as a viable target for imaging
and therapy of cancer. We prepared seven 99mTc/Re-labeled compounds by attaching known Tc/Re chelating
agents to an amino-functionalized PSMA inhibitor (lys-NHCONH-glu) with or without a variable length
linker moiety. Ki values ranged from 0.17 to 199 nM. Ex vivo biodistribution and in vivo imaging
demonstrated the degree of specific binding to engineered PSMA+ PC3 PIP tumors. PC3-PIP cells are
derived from PC3 that have been transduced with the gene for PSMA. Despite demonstrating nearly the
lowest PSMA inhibitory potency of this series, [99mTc(CO)3(L1)]+ (L1 ) (2-pyridylmethyl)2N(CH2)4CH(CO2H)-
NHCO-(CH2)6CO-NH-lys-NHCONH-glu) showed the highest, most selective PIP tumor uptake, at 7.9 (
4.0% injected dose per gram of tissue at 30 min postinjection. Radioactivity cleared from nontarget tissues
to produce a PIP to flu (PSMA-PC3) ratio of 44:1 at 120 min postinjection. PSMA can accommodate the
steric requirements of 99mTc/Re complexes within PSMA inhibitors, the best results achieved with a linker
moiety between the ε amine of the urea lysine and the chelator.
Introduction
series of low-molecular-weight imaging agents targets the
prostate-specific membrane antigen (PSMA).13–16
Prostate cancer (PCa) is the leading cancer in the U.S.
population and the second leading cause of cancer-related death
in men.1 By the time of diagnosis, only one-half of PCa tumors
are clinically localized and one-half of those represent extra-
capsular spread. Currently anatomic methods, such as computed
tomography (CT), magnetic resonance (MR) imaging, and
ultrasound, predominate for clinical imaging of prostate cancer.
The radiolabeled monoclonal antibody [111In]ProstaScint has
also been used, however, this agent tends to produce images
that are challenging to interpret.2–4 Low-molecular-weight,
radiopharmaceutical-based imaging agents may provide superior
pharmacokinetics for imaging than radiolabeled antibodies,
which tend to have long circulation times and delayed clearance
from nontarget tissues. A variety of experimental low-molecular-
weight PCa imaging agents are currently being pursued clini-
cally, including radiolabeled choline analogues,5–9 [18F]fluo-
rodihydrotestosterone ([18F]FDHT),10 anti-1-amino-3-[18F]-
fluorocyclobutyl-1-carboxylic acid (anti[18F]F-FACBC),11 [11C]ac-
etate, and 1-(2-deoxy-2-[18F]fluoro-L-arabinofuranosyl)-5-me-
thyluracil ([18F]FMAU).12 Each operates by a different mech-
anism and has certain advantages, e.g., low urinary excretion
for [11C]choline, and disadvantages, such as the short physical
half-life of positron-emitting radionuclides. A promising new
PSMA is a type II integral membrane protein that has
abundant and restricted expression on the surface of PCa,
particularly in androgen-independent, advanced, and metastatic
disease.17 The latter is important because almost all PCa
becomes androgen independent. It is also expressed within the
endothelium of most solid tumors other than prostate.18 PSMA
possesses the criteria of a promising target for therapy, i.e.,
abundant and restricted (to prostate) expression at all stages of
the disease, presentation at the cell surface but not shed into
the circulation, and association with enzymatic or signaling
activity.17 The PSMA gene is located on the short arm of
chromosome 11 and functions both as a folate hydrolase and
neuropeptidase. It is the neuropeptidase function that is equiva-
lent to glutamate carboxypeptidase II (GCPII), which is referred
to as the “brain PSMA”,17 and may modulate glutamatergic
transmission by cleaving N-acetylaspartylglutamate (NAAG) to
N-acetylaspartate (NAA) and glutamate.19 There are up to 106
PSMA molecules per cancer cell, further suggesting it as an
ideal target for imaging and therapy with radionuclide-based
techniques.20
Recently, we have demonstrated selective imaging of xe-
nografts that express PSMA using small animal positron
emission tomography (PET) and single photon emission com-
puted tomography (SPECT) and the urea-based PSMA inhibitors
N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-(S)-[11C]methyl-L-
cysteine, [11C]DCMC, N-[N-[(S)-1,3-dicarboxypropyl]carbam-
* To whom correspondence should be addressed. Phone: 410-955-2789.
Fax: 443-817-0990. E-mail: mpomper@jhmi.edu. Address: Martin G.
Pomper, M.D., Ph.D., Johns Hopkins Medical Institutions, 1550 Orleans
Street, 492 CRB II, Baltimore, MD 21231.
a Abbreviations: PSMA, prostate-specific membrane antigen; GCPII,
glutamate carboxypeptidase II; NAALADase, N-acetylated-R-linked acidic
dipeptidase; DCMC, N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-(S)-methyl-
L-cysteine; DCIT, N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-(S)-3-iodo-L-
tyrosine; DCFBC, and N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-(S)-4-
fluorobenzyl-L-cysteine; SAAC, single amino acid chelate; DCL, N-[N-[(S)-
1,3-dicarboxypropyl]carbamoyl]-(S)-L-lysine; PMPA, 2-(phosphonomethyl)pen-
tanedioic acid.
†
Russell H. Morgan Department of Radiology and Radiological Sciences,
Johns Hopkins Medical Institutions.
‡
Department of Pharmacology & Molecular Sciences, Johns Hopkins
Medical Institutions.
§
Department of Urology, Johns Hopkins Medical Institutions.
Department of Medicinal Chemistry and Pharmacognosy, University
|
of Illinois at Chicago.
10.1021/jm800111u CCC: $40.75
2008 American Chemical Society
Published on Web 07/19/2008

Tc-99m- and Re-Labeled Inhibitors of PSMA
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4505
Chart 2. Urea-Based PSMA Ligands L1-L7
Chart 1. Urea-Based PSMA Inhibitorsa
a
[
125I]DCIT ) N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-(S)-3-[125I]iodo-
L-tyrosine, [11C]DCMC ) N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-(S)-
[
11C]methyl-L-cysteine, [18F]DCFBC ) N-[N-[(S)-1,3-cicarboxypropyl]car-
bamoyl]-(S)-4-[18F]fluorobenzyl-L-cysteine, DCL ) N-[N-[(S)-1,3-dicar-
boxypropyl]carbamoyl]-(S)-lysine.
oyl]-(S)-3-[125I]iodo-L-tyrosine, [125I]DCIT, and N-[N-[(S)-1,3-
dicarboxypropyl]carbamoyl]-(S)-4-[18F]fluorobenzyl-L-
cysteine, [18F]DCFBC (Chart 1).13–15 These results encouraged
us to investigate the possibility of developing 99mTc-labeled,
urea-based inhibitors as PCa imaging agents. Although positron-
emitting radionuclides are increasingly used in clinical medicine,
99mTc remains the radionuclide of choice for clinical scinti-
graphic imaging because of its favorable physical properties (t1/2
) 6 h, Eγ ) 140 keV), low cost, and widespread availability.
The development of technetium complexes as radiopharmaceu-
ticals is facilitated by the use of rhenium, the group VIIB
congener of technetium. Rhenium generally produces complexes
with similar physical properties to those of technetium and is
often used as a nonradioactive alternative to technetium for
large-scale synthesis and structural characterization.
Here we describe the development of low-molecular-weight,
urea-based inhibitors incorporating tridentate chelators for
binding of the {M(CO)3}+ core, (M ) 99mTc, 186,188Re), while
retaining high affinity to PSMA. Because of high stability and
favorable labeling characteristics, the organometallic Re(I)(CO)3/
99mTc(I)(CO)3 approach represents an attractive radiolabeling
strategy. A number of tridentate chelates with different sets of
nitrogen, sulfur, oxygen donor atoms are known to form highly
stable complexes with the {M(CO)3}+ cores.21,22 Among them,
the single amino acid chelate concept,23,24 (SAAC), has proved
useful for designing our new urea-based inhibitors. We describe
the synthesis and in vitro binding of seven 99mTc/Re-based
radiopharmaceuticals formed from ligands L1-L7 (Chart 2)
as well as ex vivo biodistribution and in vivo imaging studies
of selected agents.
moiety of the inhibitors has a predisposition to orient within
the narrow S1′ pocket, whereas the remainder of the molecule
resides within the large S1 pocket. These observations are
similar for PSMA X-ray crystal structures upon cocrystallization
with compounds in the urea series, namely, DCMC, DCIT, and
DCFBC (Chart 1) (data not shown). In the current study, we
sought to synthesize a conjugate between a glutamate-contain-
ing, urea-based inhibitor and known chelators of [Re(I)(CO)3
]+/[99mTc(I)(CO)3]+. In the design of these new conjugates, it
was important to optimize the interaction between PSMA and
the bulky chelator. Considering the ∼9 Å diameter of the
rhenium tricarbonyl and technetium tricarbonyl coordination
spheres with pyridyl-based chelates, determined from reported
X-ray crystal structures,29,30 the calculated average volume of
the metal tricarbonyl core with the bispyridyl chelate was found
to be ∼378 Å3. To enable high-affinity binding of our putative
imaging agents to PSMA, we envisioned the need to attach a
functionalized methylene linker (>20 Å) to the remainder of
the molecule from the R-carbon of the urea function. Accord-
ingly, we have designed three sets of compounds, each with a
different linker length: L1-L3, with a linker of ∼31.5 Å, L4
and L7, with linker length of ∼33 Å, and L5 and L6, with
linker lengths of ∼22 Å and 7.7 Å, respectively (Chart 2). We
hypothesized that the metal chelate portion of the compound
should remain outside of the 20 Å tunnel as long as the
Chemistry
Structure-Based Design of PSMA Binding Inhibitors.
Recently, several high-resolution X-ray crystal structures of
GCPII (PSMA) with known, low-molecular-weight, glutamate-
containing inhibitors of PSMA have been reported.25–28 Those
structural studies established that the binding site of PSMA
contains a binuclear zinc ion and two substrate binding pockets,
i.e., an S1 (nonpharmacophore) pocket and an S1′ (pharma-
cophore) pocket. The active site also contains a chloride ion in
the S1 pocket. In the vicinity of the S1 pocket resides a funnel-
shaped tunnel with a depth of approximately 20 Å and a width
of 8-9 Å.25 Similarly, a narrow cavity is present near the S1′
pocket.27 Moreover, it has been determined that the glutamate

4506 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Banerjee et al.
Scheme 1
boxaldehyde and sodium triacetoxyborohydride followed by
NHS ester formation with N-hydroxysuccinimide in the presence
of O-benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-
phosphate (HBTU). Compound L5 was obtained by reacting
12 with 5 in CH2Cl2 and triethylamine, followed by deprotection
of the PMB groups using TFA/CH2Cl2. Compound L6 was
prepared by reductive amination of 1 using pyridine-2-carbox-
aldehyde and sodium triacetoxyborohydride followed by depro-
tection of the PMB groups using TFA/CH2Cl2 (Scheme 5).
Synthesis of Rhenium Analogs (ReL1-ReL7). Synthesis
of compounds [Re(CO)3L]+/0 (L ) L1-L7) was performed by
ligand exchange reaction using the rhenium tricarbonyl precursor
[Re(CO)3(H2O)3]Br37 as shown in Scheme 6 for L1. Equimolar
quantities of the ligand (L1-L7) and the precursor were refluxed
under argon for 3 h to afford the corresponding rhenium
complex in quantitative yield in each case. The complexation
was monitored by high-performance liquid chromatography
(HPLC). All complexes were purified via HPLC and character-
ized by standard spectroscopic methods.
glutamate moiety is maintained within the S1′ pocket, permitting
interaction between the inhibitor and PSMA.
Modeling of L1 in the Active Site of PSMA. For molecular
modeling studies of L1 with PSMA, a recently published crystal
structure of PSMA in complex with (S)-2-(4-iodobenzylphospho-
nomethyl)-pentanedioic acid (GPI-18431) (PDB ID: 2C6C) was
used.25 Initially, we carried out docking studies of L1 with the
active site of 2C6C using LigandFit and CHARMm-based
CDOCKER protocols implemented in Discovery Studio 1.7 (DS
1.7, Accelrys Inc.). However, none of them produced docked
poses in the active site because of the bulkiness of L1. Therefore,
we employed an alternative way to elucidate a potential binding
mode of L1. PSMA crystal structures with several ligands
including GPI-18431, 2-(phosphonomethyl)pentanedioic acid (2-
PMPA) and glutamate showed that the glutamate portion of
these compounds within the S1′-pocket virtually overlap,
suggesting that the orientation of the glutamate moiety is
unchanged despite a variety of structural motifs concurrently
within the S1-pocket. That was no surprise, as we expected the
glutamate portion of L1 to orient in the S1′-pocket in a fashion
similar to that of the known PSMA inhibitors (e.g., GPI-18431
and 2-PMPA). In particular, the R-carboxylate of glutamate,
which interacts with Arg 210, is known to be essential for PSMA
binding.38 We superimposed L1 with GPI-18431 using four
tether attachment points in glutamate (N-CH(COO-)-CH2, see
Supporting Information, Figure 1). Coordinates of the super-
imposed L1 were transferred and merged in the apo-form of
2C6C in which the ligand GPI-18431 was removed.
Synthesis of Urea-linked Chelators. We elected to develop
a new series of PSMA inhibitors containing lysine (Chart 1,
DCL) in order to utilize the free ε amine of lysine for
conjugation or derivatization with a suitable metal chelating
group. Compound 1 (Scheme 1) is a key intermediate, integral
to synthesis of all of the putative imaging agents described. The
protected lysine analogue 2 was prepared in two steps. Com-
mercially available Nε-Boc-NR-Fmoc-L-lysine was reacted with
4-methoxybenzyl chloride and cesium carbonate in N, N-
dimethylformamide (DMF), followed by removal of the Fmoc
group using 20% piperidine in DMF. Flash chromatography
provided the desired compound 2 in 80% yield. Urea 3 was
obtained by treating bis-4-methoxybenzyl-L-glutamate·HCl,
31,32
4
with triphosgene and triethylamine at -78 °C followed
by in situ trapping of the isocyanate intermediate by addition
of 2. Selective cleavage of the N-Boc group of 3 with
p-toluenesulfonic acid in ethanol/ethyl acetate33,34 produced 5.
Basic extraction of a solution of 5 in CH2Cl2 gave the free base
1. The p-methoxybenzyl (PMB) group was conveniently
removed at room temperature by using trifluoroacetic acid
(TFA)/anisole or TFA/CH2Cl2 solution in the final step after
performing the required conjugation.
The synthesis of the chelators and their conjugation with
intermediate 1 are presented in Schemes 2–6. Compound 1 was
used to attach different linkers as well as metal chelators to
generate a new series of PSMA inhibitors, L1-L7, for
coordination of {99mTc(CO)3}+/{Re(CO)3}+. Key N-hydrox-
ysuccinimide (NHS) ester intermediate 6, shown in Scheme 2,
was prepared by conjugation of 1 with excess disuccinimidyl
suberate (DSS) in DMF. Compound 6 was then reacted with
three different bispyridyl chelators, 7, 9, and 13, a bisquinoline
chelator, 8, and a monopyridyl monoacid chelator, 11, to prepare
L1-L4, and L7. Chelators 7, 8, 9, and 13 were prepared
according to published procedures.35
Synthesis of the monopyridyl monoacid chelator was per-
formed by modification of a previously described procedure
(Scheme 3). Compound 10 was prepared according to a
previously reported method.36 Reductive amination of 10, using
glyoxylic acid in presence of sodium triacetoxyborohydride in
dichloroethane, followed by removal of the protecting group
using a solution of TFA/CH2Cl2 at ambient temperature,
produced 11. The synthesis of L5 is outlined in Scheme 4.
Compound 12 was prepared by reductive amination of com-
mercially available 8-aminocaproic acid with pyridine-2-car-
Molecular dynamics simulation of the merged PSMA/L1
complex was performed with Generalized Born with a simple
Switching (GBSW) as an implicit solvent model. Amino acid
residues within 7 Å of L1 remained flexible while all other
amino acids were constrained. As shown in Supporting Informa-
tion Figure 2 (see Supporting Information), the location of the
carboxylic acid in the lysine portion of L1 dramatically changed
and strongly interacted with two arginines (Arg 534 and Arg
536, Figure 1A) after molecular dynamics simulation, while the
two carboxylic acids of glutamate changed only slightly. The
linear-type linker of initial L1 was grooved for maximizing
interaction with the tunnel region of PSMA, i.e., the flexible
linear-type linker of initial L1 adopted a compact conformation,
thus enhancing the interaction of L1 with the tunnel region of
PSMA after molecular dynamics simulations (Figure 1B and
Supporting Information Figure 2). From this PSMA/L1 model,
the R-carboxylate of glutamate demonstrated hydrogen bonding
interactions with Arg 210, Tyr 552, and Tyr 700 and the
γ-carboxylate did similarly with Asn 257 and Lys 699. In

Tc-99m- and Re-Labeled Inhibitors of PSMA
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4507
Scheme 2
Scheme 3
Scheme 5
Scheme 4
Scheme 6
to those of the free ligands and [99mTc(CO)3(OH2)3]+, enabling
the clear separation of the radiotracers.
Electronic Properties of ReL2. Bisquinoline ligand L2
allows for the preparation of isostructural fluorescent {Re-
(CO)3}+ core complexes and radioactive {99mTc(CO)3}+ core
complexes. Consequently, the fluorescent properties of ReL2
were investigated to determine whether the rhenium-based
complexes possess suitable characteristics for use as biological
probes.39,40 The electronic spectrum of ReL2 exhibited absor-
addition, the two NH groups of the urea contribute to interaction
with Gly 518.
Radiochemistry. Radiolabeling with [99mTc(CO)3(OH2)3]+
was performed using the commercially available Isolink kit at
ligand concentrations of 10-5-10-6 M with incubation times
of 30 min at 95 °C. Adducts were produced in high radiochemi-
cal yield (>70%) and purity (>98%). Formation of the
bance at 321 nm with an extinction coefficient of 17200 M-1
.
Excitation of ReL2 at 321 nm provides an intense fluorescence
emission at 550 nm. The large Stokes shift is characteristic of
this class of fluorophore.41 The emission peak is assigned to a
[
99mTc(CO)3L]+/0 (TcL1-TcL7) complexes resulted in a sig-
nificant shift in the HPLC retention times (to longer) compared

4508 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Banerjee et al.
Figure 1. Binding mode of L1 to the active site of PSMA (A). The corresponding contour map is shown in (B).
Table 1. PSMA Inhibitory Activity and Calculated ClogD
longer than that provided by lysine itself, as the Re-chelated
version of L6 displays the lowest PSMA inhibitory activity of
all compounds measured and was not capable of imaging
PMSA+ tumors (data not shown). Compound L5 demonstrates
that linkers containing seven methylene units between the
chelator nitrogen and the amide carbonyl provide compounds
of low nanomolar Ki. Longer linkers can also be accommodated
easily (L4 and L7). Introduction of rhenium does not cause a
consistent change in inhibitory activity with Re-labeled versions
only of L1, L4, and L5, demonstrating higher inhibitory
activities than the corresponding unchelated compounds. Intro-
duction of the Re(CO)3 core/moiety to chelators of this class
forces the chelator into a facial configuration, with unpredictable
effects on binding to the active site. Placing the carboxylate
adjacent to the chelator (L3), rather than adjacent to the amide
nitrogen on the linker (L1) caused an increase in inhibitory
activity of over an order of magnitude for the unchelated
versions, although the Re-labeled versions were comparable.
The bisquinoline chelator, which is much less polar than the
bispyridyl, provides correspondingly stronger PSMA inhibitory
activity. Replacing one of the pyridines with a carboxylic acid
moiety (L7 to L4) causes a 6-fold increase in inhibitory activity
for the unchelated molecules but a 12-fold decrease in activity
for the more biologically relevant Re-labeled compounds.
Because of the many structural perturbations provided in this
small structure-activity series, it is difficult to determine the
effect of linker chain length other than to say that a linker
beyond the lysine moiety itself is necessary for productive
interaction with PSMA.
Ki [nM]
95% CIa
ClogD
L1
ReL1
L2
ReL2
L3
ReL3
L4
ReL4
L5
ReL5
L6
ReL6
L7
ReL7
PMPA
15.25
10.75
0.17
0.50
1.08
10.34
2.54
0.17
1.86
0.91
7.53
199.56
0.45
2.06
0.20
7.93
3.81
0.05
0.07
0.14
3.76
0.60
0.08
0.21
0.44
5.65
135.26
0.25
0.25
0.06
-7.69
-3.91
-7.19
-6.13
-5.05
-6.25
-5.61
-8.65
a Confidence interval.
3MLCT [dπ(Re) f π*(ligand)] excited state on the basis of
previous spectroscopic studies of Re(I) tricarbonyl complexes.41–44
The fluorescence lifetime for ReL2 is 11.8 µs (λem ) 550 nm)
in ethylene glycol under an argon atmosphere, which is
sufficiently long to overcome the effects of endogenous
fluorescence. Cellular autofluorescence can complicate in vitro
imaging studies, however, because it occurs on the nanosecond
time scale, it can be eliminated using time-gating techniques
so long as the probe under investigation has a sufficiently long
lifetime. The fluorescence quantum yield of ReL2 of 0.018 in
ethylene glycol under argon is low but comparable to those
reported for other transition-metal band fluorescence probes.44,45
Ex Vivo Biodistribution. Compounds [99mTc]L1-L3 were
assessed for their pharmacokinetics ex vivo in severe-combined
immunodeficient (SCID) mice bearing both PSMA+ PC3 PIP
and PSMA-flu xenografts.14,18 Tables 2–4 show the percent
injected dose per gram (%ID/g) uptake values in selected organs
Biological Results
In Vitro Binding Studies. The relative binding affinities of
L1-L7 and ReL1-ReL7 were determined using the N-acety-
lated-R-linked acidic dipeptidase (NAALADase) assay as previ-
ously described.46,47 The data are presented in Table 1, with
corresponding structures shown in Chart 2. As all compounds
possess the lys-NHCONH-glu motif, structural variation derives
from (a) the length of the linker between the chelator and the
amide carbonyl carbon attached to the lysine moiety, (b) the
chelator, which may be either the bispyridyl, bisquinoline, or
mixed (monopyridyl monocarboxyl) functional groups, (c) the
presence or absence of a second amide function between the
chelator and the first amidesattached to the lysine moiety, and
(d) the presence or absence of a carboxyl group either adjacent
to the chelator or adjacent to the second (linker) amide group.
Immediately evident is the need for a methylene chain length
for compounds
[
99mTc]L1-L3, respectively. Compound
[
99mTc]L1 showed clear PSMA-dependent binding in PC3 PIP
tumor xenografts, reaching a maximum uptake, among times
investigated, of 7.87 ( 3.95%ID/g at 30 min postinjection (pi).
PSMA+ tumor to PSMA- tumor (PIP:flu) uptake ratios ranged
from 23 at 30 min pi to a high of 68 at 300 min pi. The
distribution within normal organs and tissues was also favorable,
with low nonspecific tissue uptake and rapid clearance. The
highest nonspecific uptake observed was in the spleen at 30
min pi and was 10.59 ( 6.05%ID/g, which decreased to 1.81
( 1.10 by 60 min pi. Kidney uptake, chiefly due to high
expression of PSMA within proximal tubules,48 was expectedly

Tc-99m- and Re-Labeled Inhibitors of PSMA
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4509
Table 2. Biodistribution of [99mTc]L1 in Tumor Bearing Micea
0.25%ID/g. That is significantly lower than the uptake observed
for [99mTc]L1 in the PC3 PIP tumor. The PIP:muscle ratios were
also significantly lower, achieving a maximum value of only
7.7 at 60 min pi as opposed to a maximum of 41.4 for [99mTc]L1
at 120 min pi. We speculate that the added lipophilicity of the
bisquinoline moiety contributes to additional nonspecific binding
(note relatively high liver uptake at 60 min pi (1.15 (
0.33%ID/g for [99mTc]L2 vs 0.25 ( 0.15%ID/g for [99mTc]L1)
as well as the very high spleen uptake at that same time point
(15.32 ( 6.64%ID/g)). Spleen had not yet reached equilibrium
during the 60 min time course of this study. Kidney uptake at
60 min pi was 86.0 ( 13.9%ID/g, similar in value to that seen
for [99mTc]L1.
30 min
60 min
120 min
300 min
blood
heart
lung
0.54 ( 0.39
0.19 ( 0.13
0.64 ( 0.23
1.49 ( 1.12
0.35 ( 0.15
0.18 ( 0.10
10.59 ( 6.05
0.36 ( 0.14
0.11 ( 0.04
0.04 ( 0.02
0.18 ( 0.06
0.25 ( 0.15
0.17 ( 0.00
0.05 ( 0.02
1.81 ( 1.10
0.11 ( 0.03
0.02 ( 0.01 0.01 ( 0.00
0.02 ( 0.01 0.01 ( 0.00
0.05 ( 0.00 0.04 ( 0.06
0.08 ( 0.04 0.04 ( 0.01
0.41 ( 0.61 0.03 ( 0.01
0.01 ( 0.01 0.00 ( 0.00
0.59 ( 0.29 0.07 ( 0.04
0.05 ( 0.07 0.01 ( 0.00
liver
stomach
pancreas
spleen
fat
kidney
muscle
95.66 ( 22.06 68.54 ( 8.32 10.08 ( 5.71 1.26 ( 0.67
0.39 ( 0.12 0.25 ( 0.15 0.056 ( 0.04 0.04 ( 0.05
1.29 ( 0.76 0.38 ( 0.13 0.03 ( 0.01
2.28 ( 2.03 16.02 ( 12.39 1.30 ( 2.00 0.10 ( 0.09
small intestine 5.87 ( 2.35
large intestine
bladder
PC-3 PIP
PC-3 flu
PIP:muscle
flu:muscle
PIP:flu
2.31 ( 0.88
7.87 ( 3.95
0.34 ( 0.15
20
2.19 ( 1.78
3.86 ( 0.57
0.16 ( 0.08
15
5.01 ( 8.18 0.80 ( 1.33
2.31 ( 0.84 0.84 ( 0.51
0.05 ( 0.02 0.01 ( 0.01
The last compound to undergo detailed, ex vivo biodistribu-
tion assay, [99mTc]L3, also demonstrated PSMA-dependent
tumor uptake, displaying highest PSMA+ PIP uptake at 30 min
pi (11.56 ( 2.86% ID/g) (Table 4). PIP:flu ratios were highest
at 30 min pi at 21.99 and then held steady at around 5:1 through
300 min pi. In this regard, both [99mTc]L2 and [99mTc]L3 are
inferior in providing high PIP:flu ratiossthe key criterion for
PSMA-mediated imagingsas compared with [99mTc]L1. Com-
pound [99mTc]L3 exhibited a similar trend in liver, lung, and
spleen as [99mTc]L1 and [99mTc]L2. Radiotracer uptake within
spleen and liver (nonspecific binding) were also very high for
41
0.9
44
23
0.3
68
0.9
0.6
23
25
a Values expressed are in % ID/g ( standard deviation. N ) 4 for all
tissues.
Table 3. Biodistribution of [99mTc]L2 in Tumor Bearing Micea
30 min
60 min
blood
heart
lung
liver
stomach
pancreas
spleen
kidney
muscle
small intestine
large intestine
bladder
PC-3 PIP
PC-3 flu
PIP:muscle
flu:muscle
PIP:flu
0.28 ( 0.05
0.23 ( 0.01
0.82 ( 0.17
1.75 ( 0.40
0.45 ( 0.12
0.35 ( 0.20
10.36 ( 9.64
47.86 ( 8.88
0.54 ( 0.27
5.22 ( 1.92
1.25 ( 1.21
0.46 ( 0.31
1.09 ( 0.61
0.34 ( 0.18
2
0.36 ( 0.11
0.22 ( 0.06
0.69 ( 0.14
1.15 ( 0.33
0.36 ( 0.30
0.34 ( 0.16
15.32 ( 6.64
86.02 ( 13.93
0.26 ( 0.11
2.35 ( 1.90
0.53 ( 0.42
0.39 ( 0.18
2.04 ( 0.25
0.46 ( 0.17
8
[
99mTc]L3. PSMA-mediated kidney uptake was also similar to
the other compounds of this class and peaked at 178.56 ( 35.45
at 60 min pi.
The remaining compounds of this series ([99mTc]L4-L7)
underwent imaging (see below), however, the images suggested
that these compounds did not warrant further ex vivo analysis.
Metabolism. Except for mouse kidney extracted 60 min after
injection of [99mTc]L1, which contained 2% of its extracted
radioactivity as a polar metabolite, all of the other tissue extracts,
plasma, and urine at 30 and 60 min postinjection contained
100% of the chromatographed radioactivity as the parent
compound.
0.6
3
2
4
a Values in percent injected dose per gram ( standard deviation. N ) 4
for all tissues.
Microscopy. Coordination of the bisquinoline moiety of L2
with Re(CO)3 renders this complex fluorescent. Accordingly,
we performed microscopy using ReL2 in live cells (Figure 2).
Because the Stokes shift for ReL2 is relatively large, it was
necessary to excite at 494 nm and collect emission fluorescence
at 628 nm. Efforts to excite at 321 nm, where the quantum
efficiency for this ligand was expected to be highest, resulted
in extreme autofluorescence and no useable data. Excitation in
the green region of the spectrum, however, led to a weak but
observable fluorescent signal from within the PSMA+ PC3 PIP
cells. This result provides visual confirmation of internalization
of low-molecular-weight ligands for PSMA. The mechanism
of internalization of PSMA has been studied previously,
however, only antibodies and antibody conjugates have been
used rather than small molecules.49,50
Table 4. Biodistribution of [99mTc]L3 in Tumor Bearing Micea
30 min
60 min
120 min
300 min
blood
heart
lung
0.68 ( 0.19
0.51 ( 0.13
2.48 ( 0.95
1.47 ( 0.14
0.74 ( 0.15
0.61 ( 0.14
1.81 ( 1.61
1.56 ( 1.05
3.14 ( 1.82
2.85 ( 1.85
3.87 ( 3.02
5.71 ( 4.68
0.08 ( 0.05 0.02 ( 0.00
0.04 ( 0.01 0.04 ( 0.01
0.13 ( 0.01 0.07 ( 0.00
0.22 ( 0.05 0.17 ( 0.01
0.36 ( 0.19 0.12 ( 0.06
0.12 ( 0.08 0.05 ( 0.00
liver
stomach
pancreas
spleen
fat
kidney
muscle
small intestine 10.62 ( 5.30
large intestine
bladder
PC-3 PIP
PC-3 flu
PIP:muscle
flu:muscle
PIP:flu
32.07 ( 16.36 25.90 ( 10.08 0.98 ( 0.25 0.42 ( 0.07
0.59 ( 0.17 4.67 ( 5.89 0.04 ( 0.01 0.02 ( 0.00
163.57 ( 29.62 178.56 ( 35.45 29.87 ( 27.09 1.91 ( 0.45
0.92 ( 0.25
1.42 ( 1.32
21.03 ( 4.46
6.49 ( 4.91
10.38 ( 6.28 21.63 ( 35.22 0.43 ( 0.19
6.59 ( 5.22
1.53 ( 1.69
0.73 ( 0.25 0.04 ( 0.01
0.58 ( 0.23 0.28 ( 0.20
0.80 ( 0.40 0.53 ( 0.24
1.64 ( 0.71
3.30 ( 1.06
11.56 ( 2.86
0.53 ( 0.15
13
1.89 ( 0.21 0.75 ( 0.55
0.32 ( 0.27 0.18 ( 0.17
Imaging. SPECT-CT imaging was carried out in SCID mice.
Each mouse had a PSMA+ PC3 PIP and PSMA- PC3 flu
xenograft in opposite, upper flanks. All radioligands were
screened this way, and the results obtained were used to
determine whether ex vivo biodistribution assay would add
further information. Figure 3 shows early, rendered images of
mice with radioligands that demonstrated positive PIP tumor
uptake. Mice were injected intravenously with 0.5-1 mCi
(19-37 MBq) of the corresponding 99mTc-labeled compound
and were imaged at 45 min pi. Successful radioligands enabled
visualization of both the PIP tumor and the kidneys, each of
which expresses PSMA. Compounds [99mTc]L1-L4 yielded
positive scans with distinguishing features. Compound
5
1
4
3
0.4
6
18
4
0.6
23
4
a Values in percent injected dose per gram ( standard deviation. N ) 4
for all tissues.
high and peaked at 95.66 ( 22.06%ID/g at 30 min and cleared
to 1.26 ( 0.67%ID/g by 300 min pi.
Compound [99mTc]L2 was also assayed for its pharmacoki-
netic characteristics in tumor-bearing mice, although only at
30 and 60 min pi. Table 3 shows the %ID/g of uptake for this
radioligand. As for [99mTc]L1, [99mTc]L2 showed PSMA-
dependent tumor uptake, which peaked at 60 min pi at 2.04 (

4510 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Banerjee et al.
Figure 2. Fluorescence microscopy of PSMA+ PC-3 PIP cells and PSMA- PC-3 flu cells using ReL2.
Figure 3. SPECT-CT imaging of tumor bearing mice with [99mTc]L1-L4 (A-D, respectively). Dual pinhole SPECT-CT of PC-3 PIP and PC-3
flu tumor bearing mice. Mice were injected with 0.5-1 mCi (19-37 MBq) of radiopharmaceutical iv followed by a 45 min uptake period. Note
essentially no uptake in the PSMA- flu tumors in each case. Abdominal radioactivity is primarily due to uptake within liver, spleen and kidneys.
The horizontal lines in B are due to a reconstruction artifact at the boundaries of the field-of-view. PIP ) PC-3 PIP; flu ) PC-3 flu; GB )
gallbladder in C; red circles highlight the location of the kidneys in D; L ) left, R ) right.
Compound [99mTc]L5 produced images qualitatively similar
to [99mTc]L4. Compounds [99mTc]L6 and [99mTc]L7 failed to
show any PIP tumor uptake and instead showed only liver and
kidney uptake (data not shown).
As a further test of in vivo binding specificity, we performed
a blocking study using [99mTc]L1 in an LNCaP (PSMA+)
prostate tumor model but first pretreating the animal with 50
mg/kg of the potent, selective PSMA inhibitor, 2-(phospho-
nomethyl)pentanedioic acid (PMPA).15,51 Figure 5 shows that
PMPA is capable of eliminating binding of [99mTc]L1 not only
to tumor but also to the renal cortex, another site of specific
binding for radiopharmaceuticals of this class. These results
provide one more check on in vivo binding selectivity, using a
blocking agent from a different chemical class than the urea-
based inhibitors, and in a different, well-established, PSMA+
prostate tumor.
Figure 4. SPECT-CT imaging of tumor bearing mice with [99mTc]L1
and [99mTc]L3 (A and B, respectively). Dual pinhole SPECT-CT of
PC-3 PIP and PC-3 flu tumor bearing mice. Mice were injected with
0.5-1 mCi (19-37 MBq) of radiopharmaceutical iv followed by a
3.5-4 h uptake period. Note lack of radiopharmaceutical outside of
tumor in (A); however, the kidneys are outside of the field of view.
Discussion
[
99mTc]L1 showed a strongly positive PIP tumor, gallbladder
uptake, and clear visualization of the kidneys. Compound
Despite advances using a variety of imaging modalities, most
notably MR spectroscopy, clinically viable molecular imaging
of PCa has remained elusive. FDG-PET, which has worked so
well not only for identification of primary and metastatic tumors
but also for therapeutic monitoring, has largely failed in the
case of PCa, perhaps due to the relatively low rate of metabolism
of these tumors compared to other epithelial cancers. Although
iterative reconstruction with anatomic coregistration can improve
ProstaScint imaging52 and using a radiolabeled version of the
J591 human monoclonal antibody against an extracellular
epitope of PSMA show some promise,53,54 these agents will be
fraught with the same disadvantages of all intact antibodies for
[
99mTc]L2 showed weak PIP tumor uptake, strong gallbladder
uptake and kidney uptake. Compound [99mTc]L3 showed strong
PIP tumor uptake, despite the small size of the tumor, gallblad-
der uptake, and clear visualization of the kidneys. Compound
[
99mTc]L4 showed elevated PIP tumor uptake as well as high
liver and kidney uptake. Images obtained several hours after
injection of [99mTc]L1 or [99mTc]L3 demonstrated higher
contrast of tumor with respect to background (Figure 4), with
very little radioactivity evident outside of the tumor for
[
99mTc]L1.

Tc-99m- and Re-Labeled Inhibitors of PSMA
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4511
degree of lipophilicity had a significant effect on both the in
vitro binding as well as the in vivo imaging, with ReL2
demonstrating 20-fold higher PSMA inhibitory activity than
ReL1, but 6-fold lower PIP:flu at 1 h postinjection and
significantly more liver and spleen uptake for the 99mTc analogue
(Chart 2, Table 1, Figure 3). PIP and flu tumors are derived
from PC3 human prostate cancer cells that differ only in their
expression of PSMA (PIP ) PSMA+, flu ) PSMA-).14,18
Another way to alter the lipophilicity of these complexes is to
introduce a carboxylic acid moiety to various positions on the
linker. Moving the linker carboxylic acid to the carbon adjacent
to the chelator nitrogen caused the binding affinity to drop
(ReL3) and provide lower PIP:flu and higher liver and spleen
uptake than compound [99mTc]L1 (Figure 3). In this series of
three compounds, [99mTc]L1 has the best properties for imaging
in vivo despite its relatively low PSMA inhibitory potency.
Compounds ReL4 and ReL7 enable comparison of the
bispyridyl and monopyridyl-monoacid chelators, respectively
(Chart 2). Compound ReL4 had a PSMA inhibitory potency of
about 12-fold higher than that of ReL7. Although ex vivo
biodistribution assays were not performed for these two
compounds, [99mTc]L4 demonstrated strong uptake in PIP as
opposed to flu tumors, but there was also significant uptake
within liver, an undesirable imaging characteristic perhaps due
to the increased lipophilicity of this compound relative to
Figure 5. SPECT-CT imaging of LNCaP (PSMA+) tumor bearing
mice with [99mTc]L1 with (left) and without (right) blockade of PSMA
using the potent, selective PSMA inhibitor, PMPA, as the blocking
agent. Lack of radiopharmaceutical in both the tumor and kidneys
(another PSMA+ site) upon cotreatment with PMPA provides a further
check on PSMA-specific binding. Images were acquired from 30-60
min postinjection. T ) tumor; K ) kidney.
imaging, namely slow clearance from blood and nonspecific
sites. Nevertheless, these antibodies bind to what we consider
an ideal target for prostate cancer imaging and therapys PSMA.
The radiopharmaceuticals we describe here are part of a series
of new low-molecular-weight PSMA-based imaging agents.
They are all based on ureas first described by Kozikowski et
al., which are synthetically accessible, high-affinity inhibitors
of PSMA.55 We have previously demonstrated the specific
binding of suitably functionalized ureas to PSMA for imaging
with SPECT and PET. However, those agents were radiolabeled
with either 125I, 11C, or 18F.13–15 Iodine-125-labeled agents can
be used in conjunction with high-resolution small animal
imaging devices to study experimental models and the isotope
can be switched to 123I or 124I for human SPECT or PET,
respectively. However, those isotopes are expensive ($1000/
mCi for [124I]NaI) and can be difficult to obtain on short notice.
Carbon-11 is largely an experimental radionuclide for use only
at centers that have a cyclotron in-house. Fluorine-18-labeled
radiopharmaceuticals can be shipped limited distances, but those
compounds will be of relatively low specific radioactivity upon
arrival at the site of usage. Fluorine-18 also requires a cyclotron
for production. For these reasons, the ready availability (via
generators delivered to nuclear medicine departments daily) and
ideal imaging characteristics of 99mTc, we have embarked on a
program to synthesize 99mTc-labeled PSMA-based imaging
agents. We have found that using SAAC technology developed
by one of us (SRB)56 in collaboration with Molecular Insight
Pharmaceuticals, Inc. (Cambridge, MA), 99mTc can be readily
incorporated in a sterically unobtrusive manner to these PSMA-
binding ureas. Because Tc has no stable isotope, we used the
group VIIB congener Re for the PSMA inhibitory studies.
We synthesized seven compounds, designated L1-L7, in
both their Re- and 99mTc-labeled forms. All seven compounds
derive from DCL (Chart 1), with different linkers between the
ε amine of lysine and the chelator. Using SAAC technology,
three different chelators were generated, namely the bispyridyl,
the bisquinoline, and monopyridyl-monoacid. The primary
rationale for the use of these different chelators was to exploit
their differing degrees of steric bulk and lipophilicity. Both L1
and L2 provide cationic complexes upon complexation with the
organometallic 99mTc(CO)3/Re(CO)3 core. On the other hand,
L4 offers a neutral complex for the metal tricarbonyl core.
Compound L2 provides the most lipophilic agent (Table 1). That
[
99mTc]L1 and [99mTc]L3, which have a slightly shorter linker
length and incorporate a linker acid moiety (Figure 3). Com-
pound [99mTc]L7 demonstrated no evidence of specific PIP
tumor uptake and showed only radioactivity within the liver.
Compound L5 has no amide or carboxylic acid within the
linker and L6 has the bispyridyl incorporated into the ε amine
of lysine (Chart 2). The linker chain of L5 is six carbons shorter
than that of the L1-L3 series. Compound ReL6 demonstrated
very low PSMA inhibitory activity, the lowest in the entire
series, and [99mTc]L6 showed no PIP tumor-specific uptake.
In this series, [99mTc]L1 and ReL2 have emerged as providing
utility for imaging prostate cancer in vivo and in vitro,
respectively. Compound [99mTc]L1 is a promising clinical
candidate because of its synthetic accessibility, very high target
to nontarget ratio (PIP:flu ) 44:1 at 2 h postinjection), rapid
washout kinetics, metabolic stability, and the many salutary
characteristics of 99mTc discussed above. The initial indication
for its use would be to study patients who have undergone
prostatectomy in whom a rising prostate-specific antigen (PSA)
is detectedsthe same indication as for ProstaScint. Compound
ReL2 has documented the internalization of PSMA after binding
of a low-molecular-weight agent to the active site (Figure 2).
This compound could be used to study the kinetics of PSMA
internalization. The internalization of compounds of this class
suggests the development of the corresponding radiotherapeutic
analogs. Future studies will involve further alterations in linker
length and lipophilicity in an effort to binding to liver and other
nonspecific sites.
Experimental Section
General Procedures. All reactions were performed under a
nitrogen atmosphere unless otherwise noted. Solvents and chemicals
obtained from commercial sources were of analytical grade or better
and used without further purification. All experiments were
performed in duplicate or triplicate to ensure reproducibility.
Analytical thin-layer chromatography (TLC) was performed using
Aldrich aluminum-backed 0.2 mm silica gel Z19, 329-1 plates and
visualized by ultraviolet light (254 nm), I2, and 1% ninhydrin in
EtOH. Flash chromatography was performed using silica gel
purchased from Bodman (Aston, PA), MP SiliTech 32-63 D 60

4512 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Banerjee et al.
Å. In most cases, product isolation consisted of removing of the
solvent from the reaction mixture, extracting with an organic
solvent, washing with water and brine, drying with anhydrous
sodium sulfate, filtering, and concentrating the filtrate. The use of
such workup conditions will be indicated by the phrase “product
isolation” (which is followed, in parentheses, by the extracting
solvent). Purification in most cases was achieved by flash chro-
matography and is signified by the term “flash chromatography”
(which is followed, in parentheses, by the elution solvent used).
Melting points were measured using a Mel-Temp apparatus and
are uncorrected. 1H NMR spectra were recorded on either a Varian
Mercury 400 MHz or on a Bruker Ultrashield 400 MHz spectrom-
eter. Chemical shifts (δ) are reported in ppm downfield by reference
to proton resonances resulting from incomplete deuteration of the
NMR solvent. Low resolution ESI mass spectra were obtained on
a Bruker Daltonics Esquire 3000 Plus spectrometer. Higher-
resolution FAB mass spectra were obtained on a JEOL JMS-
AX505HA mass spectrometer in the mass spectrometer facility at
the University of Notre Dame. Optical rotation was measured on a
Jasco P-1010 polarimeter. Infrared spectra were obtained on a
Bruker Tensor 27 spectrometer. High-performance liquid chroma-
tography (HPLC) purification of L1-L7 and ReL1-ReL7 using
a Phenomenex C18 Luna 10 × 250 mm2 column was performed
on a Waters 600E Delta LC system with a Waters 486 tunable
absorbance UV/vis detector, both controlled by Empower software.
Purification of ReL1-L7 and [99mTc]L1-L7 by HPLC was
performed using the following isocratic conditions: method 1, the
mobile phase was 65% solvent A (0.1% TFA in water) and 35%
solvent B (0.1% TFA in CH3CN), flow rate 2 mL/min; method 2,
mobile phase was 65% solvent A and 35% solvent B, flow rate 4
mL/min; method 3, mobile phase was 70% solvent A and 30%
solvent B, flow rate 2 mL/min. Eluant was monitored at 254 and
220 nm. For radiosynthetic purification, HPLC was performed with
a Waters Chromatography Division HPLC system equipped with
two model 510EF pumps, a model 680 automated gradient
controller, a model 490 UV absorbance detector, and a Bioscan
NaI scintillation detector connected to a Bioscan Flow-count system.
The output from the UV detector and the Flow-count radiodetector
system were fed into a Gateway 2000 P5-133 computer fitted with
an IN/US System, Inc. computer card and analyzed using Winflow
software (IN/US). Absorption spectra were collected using a
Hewlett-Packard 8453 spectrophotometer. The Isolink kit was a
generous gift from Mallinckrodt-Tyco Health Care (St. Louis, MO,
USA).
2-{3-[1-p-Methoxybenzylcarboxylate-(5-t-butylcarbamylpentyl)]-
ureido}-di-p-methoxybenzyl pentanedioate (3). Bis-4-methoxyben-
zyl-L-glutamate ·HCl 4 (3.6 g, 8.5mmol)31,32 was placed in a flame-
dried three-neck round-bottom flask under nitrogen and dissolved
in 15 mL CH2Cl2. Triphosgene (0.833 g, 2.8 mmol) was placed in
a vial, dissolved in 3 mL CH2Cl2 and added to the three neck flask.
The flask was cooled to -77 °C (dry ice ethanol slurry) under
nitrogen. To this was slowly added triethylamine (12 mL, 85 mmol
in 10 mL CH2Cl2). The reaction mixture was stirred at -77 °C for
1 h, allowed to warm to room temperature and was stirred for 30
min at rt. To this was added compound 2 (3.1 g, 8.5 mmol in 7
mL CH2Cl2). The resulting mixture was stirred overnight. Product
isolation (CH2Cl2, water, NaCl, Na2SO4) followed by flash chro-
matography (20/80 EtOAc/CH2Cl2) afforded an oil that solidified
upon standing. Yield: 4.135 g, 5.3 mmol, 62.3%. TLC Rf ) 0.47
1
(20/80 EtOAc/CH2Cl2). H NMR (CDCl3) δ: 7.26-7.2 (m, 6H),
6.86-6.80 (m, 6H), 5.89 (m, 2H), 5.12-5.0 (m, 6H), 4.51 (m, 1H),
4.45 (m, 1H), 3.77 (s, 9H), 2.98 (m, 2H), 2.36 (m, 2H), 2.12 (m,
1H), 1.92 (m, 1H), 1.70 (m, 1H), 1.58 (m, 1H), 1.4 (m, 11H), 1.24
(m, 3H). ESIMS m/z: 780 [M + 1]+. HRFAB+-MS: calcd for
C41H54N3O12, 780.3679, [M + 1]+, observed 780.3685 [M + 1]+.
25[R]D ) -3.440 (0.12, DMF).
p-Toluenesulfonate salt of 2-{3-[1-p-methoxybenzylcarboxylate-
(5-aminopentyl)]-ureido}-di-p-methoxybenzyl pentanedioate (5). A
solution of 3 (2 g, 2.6 mmol) dissolved in 20 mL EtOAc was cooled
to 0-2 °C in an ice bath and p-toluenesulfonic acid monohydrate
(0.49 g, 2.6 mmol) in 5 mL of absolute ethanol was added. The
cooling bath was removed, and the reaction mixture was allowed
to warm to room temperature for 2 h. The reaction mixture was
then concentrated to a thick oil under reduced pressure, and the
mixture was purified with flash chromatography using 10/90 MeOH/
CH2Cl2 to afford product as colorless solid in 45% (0.98 g,
1.15mmol) yield. TLC Rf ) 0.47 (10/90 MeOH/CH2Cl2). 1H NMR
(CDCl3): δ 7.68 (d, J ) 8.0 Hz, 2H), 7.66-7.57 (s, broad, 3H),
7.22-7.13 (m, 6H), 7.0 (d, J ) 7.2 Hz, 2H), 6.84-6.76 (m, 6H),
6.34 (s broad, 2H), 5.06-4.88 (m, 6H), 4.44 (m, 1H), 4.32 (m,
1H), 3.76 (s, 3H), 3.73 (s, 6H), 2.86 (s, broad, 2H), 2.3-2.24
(singlet on top of multiplet, 5H), 2.08-1.99 (m, 1H), 1.82-1.72
(m, 1H), 1.64-1.3 (m, 6H). ESIMS m/z: 680 [M+ + 1]. HRFAB+-
MS: calcd for C36H46N3O10, 680.3178 [M+]; found, 680.3177.
2-{3-[1-p-Methoxybenzylcarboxylate-(5-aminopentyl)]-ureido}-
di-p-methoxybenzyl pentanedioate (1). A solution of 5 (0.15 g, 0.17
mmol in 50 mL CH2Cl2) was placed in a separatory funnel, washed
with 100 mL of 0.5 M NaHCO3. The organic layer was collected,
dried over anhydrous Na2SO4, filtered, and concentrated to a yellow
film (0.107 g, 0.09 mmol, 52.5%). TLC Rf ) 0.40 (10/90 MeOH/
2-Amino-6-tert-butoxycarbonylamino-hexanoic acid 4-methoxy-
benzyl ester (2). Compound 2 was prepared in two steps. Into a
250 mL, flame-dried three-necked round-bottom flask under
nitrogen was placed Nε-Boc-NR-Fmoc-L-lysine (7.0 g, 15 mmol)
and 60 mL of dry DMF. To this was added cesium carbonate (7 g,
21 mmol) and 4-methoxybenzyl chloride (2.5 g, 16 mmol). The
suspension was stirred at room temperature under nitrogen for 4 h,
then filtered and washed with ethyl acetate. Product isolation
(EtOAc, 5% Na2CO3, water, Na2SO4) followed by recrystallization
from 60/40 (v/v) hexane/EtOAc gave 2 crops of a colorless solid.
mp 118-120 °C. TLC Rf ) 0.33 (70/30 hexane/EtOAc). Yield:
1
CH2Cl2). H NMR (CDCl3): δ 7.2-7.12 (m, 6H), 6.8-6.72 (m,
6H), 5.84 (s broad, 2H), 5.04-4.90 (m, 6H), 4.44-4.34 (m, 2H),
3.7 (m, 9H), 2.6 (s broad, 2H), 2.3 (m, 2H), 2.06 (m, 1H), 1.85 (m,
1H), 1.66 (m, 1H), 1.55 (m, 1H), 1.44-1.12 (m, 4H). ESIMS: 680
[M + 1]+ Compound 1 was used immediately in the next step.
.
2-{3-[5-[7-(2,5-Dioxo-pyrrolidin-1-yloxycarbonyl)-heptanoylamino]-
1-(4-methoxy-benzyloxycarbonyl)-pentyl]-ureido}-pentanedioic Acid
Bis-(4-methoxy-benzyl) Ester (6). A 100 mL round-bottom flask
was flame-dried under N2, after which 1 (0.08 g, 0.12 mmol) was
added and then dissolved in 10 mL of dry DMF. This solution was
added dropwise to a solution of suberic acid bis-(N-hydroxysuc-
cinimide ester), DSS (0.13 g, 0.35 mmol in 10 mL DMF), at room
temperature with mild stirring. After 2 h, the volume of the solution
was reduced under vacuum, and the colorless solid residue was
kept under high vacuum for 2 h further to remove traces of DMF.
The residue was dissolved in 1 mL of CH2Cl2 and was loaded onto
silica gel column (1 in. × 12 in.). Initially, the column was eluted
with 40/60 CH3CN/CH2Cl2 to remove excess DSS followed by 50/
50 CH3CN/CH2Cl2 to afford the product as a colorless solid. Yield:
0.062 mg, 0.07 mmol, 56.6%. TLC Rf ) 0.47 (5/95 MeOH/CH2Cl2).
1H NMR(CDCl3): δ 7.26 (m, 6H), 6.86 (m, 6H), 5.91 (m, 1H),
5.37 (m, 4H), 5.03 (m, 6H), 4.43 (m, 3H), 3.79 (s, 9H), 3.31 (m,
4H), 2.82 (s, 4H), 2.58 (t, J ) 8.4 Hz, 2H), 2.37 (m, 2H), 2.14 (m,
1
8.22 g, 14 mmol, 93.43%. H NMR(CDCl3) δ: 7.75 (d, J ) 7.2
Hz, 2H), 7.55 (d, J ) 7.2 Hz, 2H), 7.38 (t, J ) 7.5 Hz, 2H),
7.32-7.20 (m, 4H), 6.85-6.80 (m, 2H), 5.4 (d, J ) 7.6 Hz, 1H),
5.18-5.00 (m, 2H), 4.44-4.38 (m, 3H), 4.17 (t, J ) 6.0 Hz 1H),
3.80-3.70 (m, 4H), 3.00 (m, 2H), 1.90-1.11 (m, 15H). ESIMS
m/z: 588.40 [M + 1]+. Into a flame-dried round-bottom flask was
placed 5.0 g (8.54 mmol) of the fully protected analogue of 2. This
was dissolved in 60 mL of a 20% solution of piperidine in DMF.
The reaction was stirred at room temperature for 2 h. Product
isolation (CH2Cl2, water, Na2SO4) followed by flash chromatog-
raphy (4/96 MeOH/CHCl3) afforded a pure 2 as an oil (2.59 g,
7.07 mmol) in 83% yield. (TLC Rf ) 0.42 in 5/95 MeOH/CH2Cl2).
1H NMR (CDCl3) δ: 7.29 (d, J ) 7.2 Hz, 2H), 6.90 (d, J ) 7.2
Hz, 2H), 5.09 (m, 2H), 4.44-4.24 (m, 1H), 3.83 (s, 3H), 2.76-58
(m, 3H), 2.11-1.34 (m, 16H). ESIMS m/z: 367[M + 1]+ for
C19H31N2O5.

Tc-99m- and Re-Labeled Inhibitors of PSMA
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4513
4H), 1.71-1.21 (m, 9H). ESIMS m/z: 933 [M + 1]. (HRFAB+-
MS): calcd for C48H61N4O15 [M + 1]+, 933.4133; found, 933.4142
[M + 1]+.
Compounds L1-L3 were prepared by following the same
general synthetic procedure as shown in Scheme 2 for L1 as a
representative case.
g, 3.3 mmol) was added to the solution in small portions and stirred
overnight at ambient temperature. Product isolation (CH2Cl2, water,
NaCl, Na2SO4) afforded crude compound that was used in the next
step without further purification. Yield: 0.483 g, 1.32 mmol, 78.6%.
1
TLC Rf )0.37 (10/90 MeOH/CH2Cl2). H NMR (CDCl3): δ 8.62
(m, 1H), 7.75 (m, 1H), 7.32 (m, 2H), 4.56 (bs, 1H), 4.04 (s, 2H),
3.46 (s, 2H), 3.14 (m, 2H), 2.78 (m, 2H), 2.09 (m,1H), 1.74-1.16
(m, 16H). ESIMS m/z: 366.7 [M + 1], 388.5 [M + Na].The removal
of t-Boc was performed by dissolving the crude compound (0.483
g, 1.32 mmol) in an ice-cold solution of 10 mL 1/1 TFA/CH2Cl2.
The reaction mixture was allowed to stir at room temperature for
4 h. The solution was evaporated under reduced pressure and dried
under vacuum to provide a colorless solid of 11 and was used
2-[3-(5-{7-[5-(Bis-pyridin-2-ylmethyl-amino)-1-carboxy-pentyl-
carbamoyl]-heptanoylamino}-1-carboxy-pentyl)-ureido]-pentanedio-
ic Acid (L1). A solution of 735 (0.035 g, 0.107 mmol in 0.5 mL
MeOH) was added to a stirred solution of 6 (0.100 g, 0.107 mmol
in 6 mL dry DMF) at room temperature followed by the addition
of 0.2 mL NEt3. The reaction mixture was stirred for 10 h at room
temperature. The reaction mixture was then concentrated under
reduced pressure. Product isolation (CH2Cl2, water, Na2SO4)
followed by flash chromatography (50/50 MeOH/CH2Cl2) afforded
the intermediate compound as a colorless solid in 74% yield (0.090
g, 0.08 mmol). TLC Rf ) 0.45 (40/60 MeOH/CH2Cl2). ESIMS m/z:
1146.7 [M + 1]+, 1168.7 [M + Na]+. The above intermediate
compound (20 mg, 0.017 mmol) was dissolved in an ice-cold
solution of TFA (7 mL) and anisole (0.3 mL) and was stirred for
10 min. The ice bath was removed and the solution was allowed
to warm to room temperature with continued stirring for another
10 min. The solution was evaporated under reduced pressure, and
the light-brown residue was dried under high vacuum for 2 h. The
residue was washed with diethyl ether (3 × 5 mL) and water (10
× 2 mL) to produce crude L1. Yield: 9 mg, 0.011 mmol, 65%.
The colorless product was dried under vacuum and purified further
by HPLC using 75/25 water (0.1% TFA)/acetonitrile (0.1%TFA)
as mobile phase, flow rate 2 mL/min; Rt ) 14 min. 1H NMR (D2O):
δ 8.71 (d, J ) 5.6 Hz, 2H), 8.52 (t, J ) 8 Hz, 2H), 8.05 (d, J )
8 Hz, 2H), 7.96 (t, J ) 6.8 Hz, 2H), 4.32-4.18 (m, 7H), 3.80-3.70
(m, 1H), 3.18 (t, J ) 6 Hz, 2H), 2.69 (m, 2H), 2.51 (t, J ) 7.6 Hz,
2H), 2.40-2.18 (m, 25H). 13C NMR (D2O): δ 177.2, 177.13,
163.10, 162.8, 159.2, 152.4, 146.1, 142.3, 126.8, 126.1, 55.8, 54.4,
46.6, 38.8, 35.6, 35.2, 30.6, 30.0, 29.9, 27.7, 27.6, 26.3, 25.2, 25.0,
24.3, 22.4, 22.3. ESIMS m/z: 786 [M + 1]. HRFAB+-MS: calcd
for C38H56N7O11 [M + 1], 786.4038; found, 786.4033.
2-[3-(5-{7-[5-(Bis-quinolin-2-ylmethyl-amino)-1-carboxy-pentyl-
carbamoyl]-heptanoylamino}-1-carboxy-pentyl)-ureido]-pentanedio-
ic acid (L2). Compound L2 was obtained by reacting compound
839 with compound 6 similarly as described above for L1. HPLC
purification was done by using 70/30 water (0.1% TFA)/CH3CN
(0.1% TFA) as mobile phase, flow rate 4 mL/min; Rt ) 9 min. 1H
NMR(D2O/CD3CN 2/1): δ 8.81 (d, J ) 8.0 Hz, 2H), 8.42 (d, J )
8.4 Hz, 2H), 8.33 (d, J ) 8.4 Hz, 2H), 8.22 (t, J ) 7.6 Hz, 2H),
8.05 (t, J ) 7.8 Hz, 2H), 7.97 (d, J ) 8.4 Hz, 2H), 5.11 (s, 4H),
4.66-4.64 (m, 1H), 4.57-4.54 (m, 1H), 4.47-4.46 (m, 1H), 3.71
(m, 2H), 3.51-3.43 (m, 3H), 2.77 (t, J ) 7.6 Hz, 2H), 2.51 (m,
3H) 2.20-1.5 (m, 26H). ESIMS m/z: 902 [M + H2O]. HRFAB+-
MS: C46H60N7O12 [M + H2O], 902.4300; found, 902.4290.
2-[3-(5-{7-[5-(Bis-pyridin-2-ylmethyl-amino)-5-carboxy-pentyl-
carbamoyl]-heptanoylamino}-1-carboxy-pentyl)-ureido]-pentanedio-
ic acid (L3). Compound L3 was prepared by reacting compound
935 with compound 6 similarly as described above for L1. HPLC
purification was done by using 75/25 water (0.1% TFA)/CH3CN
(0.1% TFA) as mobile phase, flow rate 2 mL/min, Rt ) 8 min. 1H
NMR (D2O): δ 8.68 (d, J ) 6.0 Hz, 2H), 8.50 (t, J ) 7.6 Hz, 2H),
8.06 (d, J ) 5.6 Hz, J ) 7.9 Hz, 2H), 7.94 (t, J ) 6.4 Hz, 2H),
4.32-4.37 (m, 4H), 4.25 (m, 1H), 4.18 (m, 1H), 3.48 (t, J ) 7.2
Hz, 1H), 3.16 (m, 2H), 2.69 (m, 2H), 2.48 (t, J ) 7.2 Hz, 2H),
2.18-2.15 (m, 5H), 1.97-1.20 (m, 21H). ESIMS m/z: 786 [M +
1]+. HRFAB+-MS: calcd for C38H55N7O11, 786.4038 [M + 1];
found, 786.4032.
1
without further purification. Yield: 0.315 g, 1.19 mmol, 90%. H
NMR (MeOH-d4): δ 8.52 (d, J ) 5.6 Hz 1H), 7.78 (t, J ) 7.6 Hz,
1H), 7.54 (d, J ) 7.4 Hz, 1H), 7.29 (t, J ) 7.2 Hz, 1H), 3.78 (s,
2H), 3.22 (s, 2H), 2.85 (t, J ) 8.0 Hz, 2H), 2.5 (t, 2H), 1.72-1.20
(m, 8H). ESIMS m/z: 266.3 [M + 1]+, 288.3 [M + Na]+.
Compound L4 was prepared by coupling compound 6 with
compound 11. Compound L4 was purified by HPLC using 76/24
water (0.1% TFA)/CH3CN (0.1% TFA) as the mobile phase, flow
rate: 2 mL/min, Rt ) 10.2 min. 1H NMR (D2O): δ 8.62 (d, J ) 5.6
Hz, 1H), 8.15 (t, J ) 6.4 Hz,1H), 7.95 (d, J ) 7.6 Hz, 1H), 7.88
(t, J ) 6.4 Hz, 1H), 4.25 (m, 1H), 4.23-4.1 (m, 2H), 3.35 (m,
2H), 3.25-3.31 (m, 5H), 2.84 (m, 1H), 2.52 (t, J ) 6.8 Hz, 2H,)
2.27 (m, 6H), 1.64-1.21 (m, 23H). ESIMS m/z: 723 [M + 1]+and
745.7 for [M + Na]+. HRFAB+-MS: calcd for C35H55N6O11,
723.3929 [M + 1]; found, 723.3912.
2-(3-{5-[8-(Bis-pyridin-2-ylmethyl-amino)-octanoylamino]-1-car-
boxy-pentyl}-ureido)-pentanedioic Acid (L5). To a solution of
8-(bis-pyridin-2-ylmethyl-amino)-octanoic acid35 (0.9 g, 2.6 mmol,
15 mL DMF) was added O-benzotriazol-1-yl-N, N, N′, N′-
tetramethyluronium hexafluorophosphate (1.49 g, 3.9 mmol) and
N-hydroxysuccinimide (0.36 g, 3.1 mmol). The reaction mixture
was stirred at room temperature for 16 h. After removing solvent
under reduced pressure, the crude product was purified by flash
chromatography (10/90 MeOH /CH2Cl2) to give 12 as a thick,
colorless liquid. Yield: 0.75 g, 0.17 mmol, 65%. 1H NMR (CDCl3):
δ 8.58 (d, J ) 4.8 Hz, 2H), 7.78 (t, J ) 8.0 Hz, 2H), 7.50 (d, J )
7.6 Hz, 2H), 7.32 (t, J ) 8.0 Hz, 2H), 4.62 (s, 4H), 3.27 (t, J ) 7.6
Hz, 2H), 2.82 (s, 4H), 2.58 (d, J ) 7.2 Hz, 2H), 1.82-1.66 (m,
4H), 1.33-1.28(m, 6H). ESIMS: 439 [M + 1]+. To a solution of
12 (0.052 g, 0.11 mmol in 7 mL CH2Cl2) was added 5 (0.1 g, 0.11
mmol), followed by NEt3 (0.2 mL, 1.4 mmol). The reaction mixture
was stirred at room temperature for 5 h and then concentrated under
reduced pressure. Product isolation (EtOAc, water, NaCl, Na2SO4)
followed by flash chromatography (50/50 MeOH/CH2Cl2) afforded
pure compound the 4-methoxybenzyl ester of L5 in 51% (0.060 g,
0.056 mmol) yield. Cleavage of the PMB groups by stirring for
2 h in 1/1 TFA/CH2Cl2 followed by removal of solvent gave a
solid residue. The residue was dissolved in 7 mL water, washed
with 3 × 10 mL CH2Cl2 and the water layer concentrated under
vacuum to provide crude L5. The compound was further purified
by HPLC with 80/20 water (0.1% TFA)/CH3CN (0.1% TFA)
solution as the mobile phase. The flow rate was 3 mL/min, Rt ) 8
min. 1H NMR (D2O): δ 8.74 (d, J ) 6.0 Hz, 2H); 8.52 (t, J ) 8.0
Hz, 2H), 8.04 (d, J ) 8.0 Hz, 2H), 7.95 (t, J ) 6.4 Hz, 2H), 4.32
(s, 4H), 4.23 (s, 1H), 4.14 (s, 1H), 3.24 (t, J ) 6.4 Hz, 2H), 2.67
(t, J ) 7.6 Hz, 2H), 2.49 (t, J ) 7.2 Hz, 2H), 2.16 (m, 3H), 1.95
(m, 1H), 1.79 (m, 1H), 1.68 (m, 1H), 1.6-1.0 (m, 14H). ESIMS
m/z: 643 [M + 1]+. HRFAB+-MS: calcd for C32H47N6O8, 643.3455
[M + 1]; found 643.3463.
2-{3-[5-(Bis-pyridin-2-ylmethyl-amino)-1-carboxy-pentyl]-ure-
ido}-pentanedioic acid (L6). To a solution of pyridine-2-aldehyde
(50 mg, 0.44 mmol in 4 mL CH2Cl2) was added a solution of 1
(100 mg, 0.147 mmol in 4 mL CH2Cl2). This was stirred at ambient
temperature for 2 h. The reaction mixture was cooled to 0 °C, and
sodium triacetoxyborohydride (93 mg, 0.44 mmol) was then added
with stirring for an additional 3 h while warming to ambient
temperature. Product isolation (CH2Cl2, water, NaCl, Na2SO4)
followed by flash chromatography (10/90 MeOH/CH2Cl2) afforded
2-[3-(1-Carboxy-5-{7-[6-(carboxymethyl-pyridin-2-ylmethyl-
amino)-hexylcarbamoyl]-heptanoylamino}-pentyl)-ureido]-pen-
tanedioic acid (L4). Compound 10 was prepared following a
published procedure.36 Compound 11 was prepared as follows: to
a solution of compound 10 (0.517 g, 1.7 mmol) in 10 mL of CH2Cl2
was added a solution of glyoxylic acid monohydrate (1.55 g, 1.68
mmol in 1 mL of MeOH containing activated molecular sieves)
and was stirred for 30 min. Sodium triacetoxyborohydride (0.712

4514 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Banerjee et al.
a colorless solid as the tri-PMB ester of L6. Removal of the PMB
groups was effected by dissolving in 5 mL of 50/50 TFA/CH2Cl2
and was stirred at room temperature for 2 h. The resulting solution
was concentrated to provide a colorless solid. The solid was
dissolved in 3 mL water and washed with 5 × 5 mL CH2Cl2. The
water layer was concentrated to provide a solid. Yield: 132 mg,
0.26 mmol, 61%. The product was purified by HPLC using 85/15
water (0.1% TFA)/CH3CN (0.1% TFA) solution as mobile phase.
Flow rate was 3 mL/min, Rt )13 min. 1H NMR (D2O): δ 8.78 (d,
J ) 5.2 Hz, 2H), 7.89 (t, J ) 7.7 Hz, 2H), 7.49 (d, J ) 7.6 Hz,
2H), 7.34 (t, J ) 6.4 Hz, 2H), 4.75-4.62 (m, 4H), 4.45-4.22 (m,
2H), 2.75 (m, 2H), 2.55 (t, J ) 6.6 Hz, 2H), 2.2-1.01 (m, 7H).
ESIMS: 502 [M + 1]+. HRFAB+: for C24H31N5O7 calcd, 501.2224;
found, 502.2296.
2H), 3.53 (m, 2H), 3.35 (t, J ) 7.8 Hz, 2H), 2.72 (m, 2H),
2.46-1.30 (bm, 30H). ESIMS m/z: 993 [M + 1]+. HRFAB+-MS:
calcd for C37H54N6O14Re 993.3255 [M + 1]+; found, 993.3237.
Tricarbonyl(2-(3-{5-[8-(bis-pyridin-2-ylmethyl-amino)-octanoy-
lamino]-1-carboxy-pentyl}-ureido)-pentanedioic acid)rhenium bro-
mide (ReL5). For HPLC purification, method 1 was used. Rt ) 17
min. 1H NMR (5/1 D2O/CH3CN): δ 9.23 (d, J ) 5.6 Hz, 2H), 8.34
(t, J ) 8.0 Hz, 2H), 7.72 (d, J ) 8 Hz, 2H), 7.77 (t, J ) 6.4 Hz,
2H), 5.13 (m, 4H), 4.66 (m, 1H), 4.58 (m, 1H), 4.16 (m, 2H), 3.56
(t, J ) 6.8 Hz, 2H), 2.86 (t, J ) 7.6 Hz, 2H), 2.59-1.6 (m, 20H).
ESIMS m/z: 913 [M]+. HRFAB+-MS: calcd for C35H46N6O11Re,
[M]+ 914.2860; found, 914.2833.
Tricarbonyl(2-{3-[5-(bis-pyridin-2-ylmethyl-amino)-1-carboxy-
pentyl]-ureido}-pentanedioic acid)rhenium bromide (ReL6). For
1
2-[3-(5-{7-[6-(Bis-pyridin-2-ylmethyl-amino)-hexylcarbamoyl]-
heptanoylamino}-1-carboxy-pentyl)-ureido]-pentanedioic acid (L7).
Compound L7 was prepared by reacting 1335 with 6 similarly as
described above for L1. HPLC purification was performed using
75/25 water (0.1% TFA)/CH3CN (0.1% TFA) as mobile phase, flow
HPLC purification, method 1 was used. Rt ) 10.1 min. H NMR
(CD3CN): δ 9.12 (d, J ) 5.6 Hz, 2H), 8.22 (t, J ) 7.7 Hz, 2H),
7.80 (d, J ) 8.0 Hz, 2H), 7.65 (t, J ) 6.5 Hz, 2H), 5.03 (m, 4H),
4.59-4.58 (m, 2H), 4.08 (m, 2H), 2.79 (t, J ) 7.6 Hz, 2H),
2.34-2.24 (m, 6H), 1.82-1.80 (m, 2H). ESIMS: 502 [M]+.
HRFAB+: calcd for C72H31N5O10Re, 772.1628; found, 772.1632.
Tricarbonyl(2-[3-(5-{7-[6-(Bis-pyridin-2-ylmethyl-amino)-hexy-
lcarbamoyl]-heptanoylamino}-1-carboxy-pentyl)-ureido]-pentanedio-
ic acid)rhenium bromide (ReL7). For HPLC purification, method
1
rate 3 mL/min, Rt) 5.5 min. H NMR (D2O): δ 8.65 (d, J ) 5.2
Hz, 2H), 7.99 (t, J ) 8.6 Hz, 2H), 7.59-7.54 (m, 4H), 4.59 (s,
4H), 4.21-4.14 (m, 2H), 3.29-3.17 (m, 7H), 2.48 (t, J ) 7.6 Hz,
2H), 2.27-2.22 (m, 6H), 1.82-1.32 (m,19H). ESIMS m/z: 756
[M+1]+. HRFAB+-MS: calcd for C38H55N7O11, 756.4296 [M +
1]; found, 756.4032.
1
1 was used. Rt ) 18.0 min. H NMR (5/1 D2O/CD3CN): δ 9.43
(d, J ) 5.2 Hz, 2H), 8.56 (t, J ) 8.6 Hz, 2H), 8.10 (d, J ) 7.6 Hz,
2H), 7.97 (t, J ) 6.4 Hz, 2H), 5.38-5.29 (m, 4H), 4.80-4.35 (m,
2H), 3.80-3.72 (m, 5H), 3.05 (t, J ) 7.6 Hz, 2H), 2.8-1.82 (m,
27H).
All rhenium compounds were synthesized as for ReL1, for which
a detailed example is provided below.
Tricarbonyl (2-[3-(5-{7-[5-(bis-pyridin-2-ylmethyl-amino)-1-car-
boxy-pentylcarbamoyl]-heptanoylamino}-1-carboxy-pentyl)-ureido]-
pentanedioic acid) rhenium bromide (ReL1). Compound L1 (0.058
g, 0.074 mmol) was dissolved in 10 mL of water. A solution of
[Re(CO)3(H2O)3]Br37 (0.029 mg in 0.5 mL methanol) was added
and the reaction mixture was refluxed for 4 h. The solution was
concentrated to provide a colorless solid that was washed with 3
× 10 mL diethyl ether, 3 × 10 mL CH2Cl2, and finally with water.
Products were dried under vacuum and purified by HPLC method
Radiochemistry. Compounds L1-L7 were synthesized in ra-
dioactive (99mTc-labeled) form using the same general method as
described below for [99mTc]L1. All Tc-99m-labeled compounds
were synthesized in radiochemical yields of >70% and radiochemi-
cal purities of >98%. [99mTc(CO)3(H2O)3]+ preparation, typical
-
example: 11.2 mCi (in 1 mL saline) 99mTcO4 was added to the
Isolink kit and the reaction mixture was heated in a water bath at
95 °C for 30 min then allowed to cool to room temperature. 99mTcL
preparation, typical example: 500 µL of the [99mTc(CO)3(H2O)3]+
solution (2.3 mCi) was neutralized with 50 µL 1(N) HCl. To this
was added a 200 µL of phosphate-buffered saline (PBS) solution
and 300 µL of a solution of L1 (4 mg, 5.09 µmol in 2.5 mL water).
This was kept at 95 °C for 30 min. The vial was cooled for 5 min
at room temperature. This was diluted with 750 µL of the HPLC
mobile phase and purified by radio-HPLC (method 1). The major
radioactive peak constituting desired product (1.6 mCi) eluted at
14 min. The acidic eluate was neutralized with 100 µL 0.1 M
NaHCO3 solution and the volume was reduced to 400 µL, pH 8
under reduced pressure. This was diluted with PBS to the desired
radioactivity concentration for ex vivo biodistribution and imaging
studies. Radiochemical yield [99mTc]L1: 82.05%. Radiochemical
purity ) 98.99%.
Fluorescence Spectra. Fluorescence spectra were recorded using
a Varian Cary Eclipse fluorescence spectrophotometer using with
321 nm excitation from a Xenon arc lamp. Compound ReL2 was
dissolved in ethylene glycol. Measurements were performed under
air or after argon purging of the solution. Lifetime measurements
were performed using a model D2, ISS, Inc. frequency domain
spectrofluorimeter. The excitation wavelength was 370 nm from
the UV LED. The fluorescence intensity data were collected through
a bandpass filter in the spectral region 540-600 nm. Luminescence
quantum yields were measured by the optical dilute method57 using
an aerated aqueous solution of [Ru(bpy)3]Cl2 (φ ) 0.028, excitation
wavelength at 455 nm)58 as the standard solution.
1
1. Rt ) 12 min. H NMR (5/1 D2O/CH3CN): δ 9.31 (d, J ) 5.4
Hz, 2H,), 8.64 (t, J ) 8 Hz, 2H), 7.88 (d, J ) 8 Hz, 2H), 7.85 (t,
J ) 8 Hz, 2H,), 5.25-5.18 (m, 4H), 4.26 (m, 2H), 3.61 (t, J ) 5.2
Hz, 2H), 2.75 (t, J ) 6.4 Hz, 2H), 2.66 (t, J ) 7.2 Hz, 2H),
2.55-2.45 (m, 27H). ESIMS m/z: 1056 [M+]. HRFAB+-MS: calcd
for C41H55N7O14Re [M+], 1056.3364; found, 1056.3350 [M+]. IR
ν (cm-1) [Re(CO)3]+: 2030, 1912.
Tricarbonyl(2-[3-(5-{7-[5-(bis-quinolin-2-ylmethyl-amino)-1-car-
boxy-pentylcarbamoyl]-heptanoylamino}-1-carboxy-pentyl)-ureido]-
pentanedioic acid) rhenium bromide (ReL2). For HPLC purifica-
tion, method 2 was used. Rt ) 16 min. 1H NMR (1/1 D2O/CD3CN):
δ 8.81-8.74 (m, 4H), 8.30 (d, J ) 8.0 Hz, 2H), 8.16 (t, J ) 8.0
Hz, 2H), 7.99 (t, J ) 7.2 Hz, 2H), 7.90 (d, J ) 8.0 Hz, 2H),
5.42-5.37 (m, 2H), 5.38-5.22 (m, 2H), 4.68-4.64 (m, 1H),
4.53-4.51 (m, 1H), 4.45-4.42 (m, 1H), 4.06-4.04 (m, 2H), 3.37
(t, J ) 6.8 Hz, 2H), 2.72 (t, J ) 7.2 Hz, 2H), 2.51 (t, J ) 7.2 Hz,
2H), 2.40-1.30 (bm, 24H). ESIMS m/z: 1156 [M]+. HRFAB+-
MS: calcd for C49H59N7O14Re [M]+, 1156.3677; found, 1156.3662
[M+]. IR ν(cm-1) [Re(CO)3]+: 2028, 1900.
Tricarbonyl(2-[3-(5-{7-[5-(bis-pyridin-2-ylmethyl-amino)-5-car-
boxy-pentylcarbamoyl]-heptanoylamino}-1-carboxy-pentyl)-ureido]-
pentanedioic acid)rehenium bromide (ReL3). For HPLC purifica-
tion method 3 was used. Flow rate was 2 mL/min, Rt ) 11.5 min.
1H NMR (5/1 D2O/CH3CN): δ 9.26 (d, J ) 5.6 Hz, 1H), 9.20 (d,
J ) 5.6 Hz, 1H), 8.36 (t, J ) 8.0 Hz, 2H), 7.90 (m, 2H), 7.83 (m,
2H), 5.31-5.03 (m, 4H), 3.67 (t, J ) 6.8 Hz, 2H), 3.53 (t, J ) 6.6
Hz, 2H), 3.15 (t, 2H), 2.40-1.30 (bm, 29H). ESIMS m/z: 1056
[M]+. HRFAB+-MS: calcd for C41H55N7O14Re [M]+, 1056.3364;
found, 1056.3350 [M+].
NAALADase Assay. NAAG hydrolysis was performed es-
sentially as described previously.46,47 In short, LNCaP cell extracts
were prepared by sonication in NAALADase buffer [50 mM Tris
(pH 7.4) and 0.5% Triton X-100]. Cell lysates were incubated with
or without inhibitor at 37 °C for 10 min. Following the incubation,
the radiolabeled substrate N-acetyl-L-aspartyl-L-(3,4-3H)glutamate
(NEN Life Science Products, Boston, MA) was added to a final
concentration of 30 nM at 37 °C for 10-15 min. The reaction was
stopped by the addition of an equal volume of ice-cold 100 mM
Tricarbonyl(2-[3-(1-carboxy-5-{7-[6-(carboxymethyl-pyridin-2-
ylmethyl-amino)-hexylcarbamoyl]-heptanoylamino}-pentyl)-ureido]-
pentanedioic acid) rhenium (ReL4). For HPLC purification, method
1 was used. Rt ) 18 min. (D2O:CH3CN(5:1)): δ 9.29 (d, J ) 5.6
Hz, 1H), 9.22 (d, J ) 8.0 Hz, 1H), 8.88 (d, J ) 8.0 Hz, 1H), 7.84
(t, J ) 8.0 Hz, 1H), 5.31-5.03 (m, 2H), 4.67 (m, 2H), 4.25 (m,

Tc-99m- and Re-Labeled Inhibitors of PSMA
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4515
sodium phosphate and 2 mM EDTA. Products were partitioned by
AG 1-X8 formate resin (Bio-Rad Laboratories) anion exchange
chromatography, eluted with 1 M sodium formate, and quantified
by liquid scintillation counting. Inhibition curves were determined
using semilog plots and IC50 values determined at the concentration
at which enzyme activity was inhibited by 50%. Assays were
performed in triplicate with the entire inhibition study being repeated
at least once to confirm affinity and mode of inhibition. Data were
collected during linear phase of hydrolysis (i.e., <20% cleavage
of total substrate). Enzyme inhibitory constants (Ki values) were
generated using the Cheng-Prusoff conversion.59
then performed as described above, with both animals on the
scanner gantry.
Metabolite Studies. Male CD-1 mice (Charles River Laborato-
ries) were injected with 15 µCi (555 kBq) of [99mTc]L1 in saline
via the tail vein. Mice were sacrificed at either 30 min or 1 h
postinjection by cervical dislocation and their blood and selected
organs were removed. Blood samples were withdrawn using
heparinized syringes, and tissues were placed on ice prior to manual
homogenization in PBS, pH 7.4. Plasma and tissue homogenates
in PBS were centrifuged for 2 min at 13000g at ambient temper-
ature. A portion of the supernatant was diluted to 4 mL in 8 M
urea containing 50 mg citric acid. Urine samples were added directly
to 4 mL of the acidified urea solution. Samples then underwent
separation by HPLC as previously described.60 Briefly, the 4 mL
sample in 8 M acidified urea was passed through a capture column
(Strata-X, 19 mm × 4.4 mm, Phenomenex, Torrance, CA) at 2
mL/min followed by 1% acetonitrile in water to wash plasma
proteins from the column. The effluent from the capture column,
containing only highly polar components, flowed through a dual
BGO detector (Bioscan, Washington, DC) operating in the diode
mode. The solvent was then switched to 30% acetonitrile:50 mM
phosphate buffer at pH 2.4 (2 mL/min) for elution of the
radiolabeled components previously bound to the capture column
onto the analytical column (Synergi Polar-RP 250 mm × 4.6 mm
10 µm particle size Phenomenex).
In Vitro Fluorescence Microscopy of ReL2 in PC3 PIP and
PC3 flu Cells. Compound L2, when bound to the [Re(I)(CO)3]+
core, was hypothesized to be fluorescent as the corresponding
bisquinoline chelator is known to have fluorescent properties.30,39,40,61
Following fresh preparation of ReL2, 10000 PC3 PIP and PC3 flu
cells were seeded separately into each of four wells of a Laboratory-
Tek II 8-well chamber slide (Fisher Scientific). The cells were
cultured as described above and were allowed to attach to the
bottom of the wells overnight at 37 °C in 5% CO2 in air. Serially
diluted aliquots of ReL2 were added to the media in six of the
wells such that wells contained 500 nM, 250 nM or 125 nM ReL2
with two remaining free of fluorophore. The cells were then returned
to the incubator for one hour to enable binding. Each well was
then carefully washed by removing the supernatant followed by
addition of warm culture media for 30 s. The wash media was then
removed and added to the contents of the well chambers. Dako
Cytomation mounting medium was then applied and a glass
coverslip was added. The mounting medium was allowed to dry at
ambient temperature for 20 min prior to storage of the slide at 4
°C overnight. The cells were then viewed using an Olympus BX61
fluorescence microscope equipped with a Semrock DAPI/FITC/
Texas Red triple filter cube. Excitation was at 494 nm with
collection of emitted fluorescence at 628 nm.
Cell Culture and Ex Vivo Biodistribution. PSMA+ PC3 PIP
cells (human metastatic [bone] prostate carcinoma) engineered to
express PSMA stably and PSMA- PC3 flu cells were generously
provided by Warren Heston (Cleveland Clinic). Cells were cultured
in T175 flasks using RPMI 1640 medium (Sigma) supplemented
with 10% FBS and Penicillin/Streptomycin (100 U/mL/100 µg/
mL) at 37 °C in 5% CO2 in air. When a sufficient number of cells
were present in culture, the cells were trypsinized and formulated
in sterile Hanks buffered saline solution (Sigma, HBSS) and counted
using a hemocytometer and trypan blue dye to confirm cell viability.
Typically, 2-5 × 106 cells were injected subcutaneously such that
PC3 PIP cells were injected behind the left shoulder and PC3 flu
cells were injected behind the right shoulder of male severe-
combined immunodeficient mice (SCID). All in vivo experimental
procedures were undertaken in compliance with United States laws
governing animal experimentation and were approved by the Johns
Hopkins University Institutional Animal Care and Use Committee.
Mice were used when the tumors reached 3-7 mm in diameter for
either ex vivo biodistribution studies or in vivo SPECT-CT.
The xenograft-bearing mice (17-20 g) were injected via the tail
vein with 3.70 MBq (100 µCi) of [99mTc]L1-L4 in 200 µL of
saline. Blood was collected immediately after sacrifice (cervical
dislocation) by cardiac puncture and heart, lung, liver, stomach,
pancreas, spleen, white fat, kidney, muscle, small intestine, large
intestine, urinary bladder, and tumor xenografts were harvested,
weighed, and counted in an automated gamma counter (LKB
Wallace 1282 Compugamma CS Universal Gamma Counter).
Animals were sacrificed at 30, 60, 120, and 300 min postinjection
(n ) 4 per time point). Tissue radiopharmaceutical uptake values
were calculated as percent injected dose per gram (% ID/g) as
compared with a 1:10 diluted standard dose. The urinary bladder
was emptied and water washed and then dried prior to weighing
and counting.
SPECT-CT Imaging of PC3 PIP and PC3 flu Xenografts.
Compounds L1-L4 were studied with imaging. Xenograft models
were generated as described above. Mice were anesthetized using
1% isoflurane gas in oxygen flowing at 0.6 L/min prior to and
during radiopharmaceutical injection. Mice were injected via the
tail vein with approximately 480 µCi (17.76 MBq) of either L1,
L2, L3, or L4 formulated in 200 µL of PBS, pH 7. Allowing for
15 min of radiopharmaceutical uptake, anesthetized mice were
placed on the scanner gantry and secured with medical tape while
the anesthetic flow was increased to 0.8 L/min. Body temperature
of the mice was maintained by covering them with several layers
of Chux disposable pads in addition to keeping them illuminated
with a dissection lamp during scanning. A Gamma Medica
(Northridge, CA) X-SPECT scanner equipped with two opposing
low-energy 0.5 mm aperature pinholes and tunable CT was used
for all scans. Mice were scanned over 180° in 5.5°, 30 s increments.
A CT scan was performed prior to scintigraphy for both anatomical
coregistration and attenuation correction. Data were reconstructed
and fused using commercial software from the vendor (Gamma
Medica), which includes a 2D-OSEM algorithm.
Acknowledgment. This work was supported by NIH
CA92871, CA1114111, EB005324, and the AdMeTech Foun-
dation. We also thank Dr. Warren Heston for providing the PC3
PIP and flu cells.
Supporting Information Available: The procedure describing
the molecular modeling studies, an overlay figure of L1 with GPI-
18413 (Supporting Information Figure 1) and a superimposed image
of L1 before and after molecular dynamics simulation (Supporting
Information Figure 2). This material is available free of charge via
the Internet at http://pubs.acs.org.
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