Radiohalogenated prostate-specific membrane antigen (PSMA)-based ureas as imaging agents for prostate cancer

Article abstract of DOI:10.1021/jm801055h

To extend our development of new imaging agents targeting the prostate-specific membrane antigen (PSMA), we have used the versatile intermediate 2-[3-(5-amino-1-carboxy-pentyl)-ureido]-pentanedioic acid (Lys-C(O)-Glu), which allows ready incorporation of

Full text of DOI:10.1021/jm801055h

J. Med. Chem. 2008, 51, 7933–7943
7933
Radiohalogenated Prostate-Specific Membrane Antigen (PSMA)-Based Ureas as Imaging Agents
for Prostate Cancer
Ying Chen,† Catherine A. Foss,† Youngjoo Byun,† Sridhar Nimmagadda,† Mrudula Pullambhatla,† James J. Fox,†
Mark Castanares,‡ Shawn E. Lupold,§ John W. Babich,| Ronnie C. Mease,† 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, Molecular Insight Pharmaceuticals, Inc., Cambridge, Massachusetts 02142
ReceiVed August 24, 2008
To extend our development of new imaging agents targeting the prostate-specific membrane antigen (PSMA),
we have used the versatile intermediate 2-[3-(5-amino-1-carboxy-pentyl)-ureido]-pentanedioic acid (Lys-
C(O)-Glu), which allows ready incorporation of radiohalogens for single photon emission computed
tomography (SPECT) and positron emission tomography (PET). We prepared 2-[3-[1-carboxy-5-(4-[125I]iodo-
benzoylamino)-pentyl]-ureido]-pentanedioic acid ([125I]3), 2-[3-[1-carboxy-5-(4-[18F]fluoro-benzoylamino)-
pentyl]-ureido]-pentanedioic acid ([18F]6), and 2-(3-[1-carboxy-5-[(5-[125I]iodo-pyridine-3-carbonyl)-amino]-
pentyl]-ureido)-pentanedioic acid ([125I]8) in 65-80% (nondecay-corrected), 30-35% (decay corrected),
and 59-75% (nondecay-corrected) radiochemical yields. Compound [125I]3 demonstrated 8.8 ( 4.7% injected
dose per gram (%ID/g) within PSMA+ PC-3 PIP tumor at 30 min postinjection, which persisted, with clear
delineation of the tumor by SPECT. Similar tumor uptake values at early time points were demonstrated for
[18F]6 (using PET) and [125I]8. Because of the many radiohalogenated moieties that can be attached via the
ε amino group, the intermediate Lys-C(O)-Glu is an attractive template upon which to develop new imaging
agents for prostate cancer.
Introduction
standing tumor physiology, enabling more accurate prognosis
and therapeutic monitoring.
Prostate cancer (PCaa) is the second leading cause of cancer-
related death in men.1 Localization of tumor as well as
determination of the total body burden of PCa have important
implications for therapy, particularly as new combination and
focal therapies become available. Also critically needed are
targeted agents that can provide a readout on the biology of the
tumor, with the ability to predict which will lie dormant and
which will develop into aggressive, metastatic disease. The
current clinical standard for localizing cancer, including PCa,
is shifting from the anatomic techniques of computed tomog-
raphy (CT) and magnetic resonance (MR) imaging to more
physiologically relevant methods that employ molecular imag-
ing, such as MR spectroscopy,2 single photon emission com-
puted tomography (SPECT), and positron emission tomography
(PET).3,4 Those newer methods, which utilize molecular imag-
ing, may provide the biological readout necessary for under-
Because of the relatively low metabolism of PCa, PET with
[18F]fluorodeoxyglucose (FDG-PET) has proved ineffectual in
this disease. Other promising, experimental radiopharmaceuticals
for imaging PCa are emerging, including those of the choline
series,5-9 radiolabeled acetates,10 anti-1-amino-3-[18F]fluoro-
cyclobutyl-1-carboxylic acid (anti[18F]F-FACBC),11,12 1-(2-
deoxy-2-[18F]fluoro-L-arabinofuranosyl)-5-methyluracil ([18F]F-
MAU),13 and [18F]fluorodihydrotestosterone ([18F]FDHT).14
Each has its benefits and detriments, with no single agent ideal,
i.e., easy to synthesize, little metabolism, and demonstrating
tumor-specific uptake, in all PCa phenotypes.
Overexpressed on most solid tumor neovasculature15 as well
as in prostate cancer, the prostate-specific membrane antigen
(PSMA) is becoming an attractive target for cancer imaging
and therapy.16,17 ProstaScint is an 111In-labeled monoclonal
antibody against PSMA that is clinically available for imaging
PCa.18 ProstaScint and radiolabeled variations of this antibody
are fraught with long circulation times and poor target to
nontarget tissue contrast, limiting the utility of these agents.19-21
Previously, we reported the synthesis of several low molecular
weight, urea-based PSMA inhibitors for imaging PCa. Those
compounds include N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-
(S)-[11C]methyl-L-cysteine ([11C]DCMC),22 N-[N-[(S)-1,3-di-
carboxypropyl]carbamoyl]-(S)-3-[125I]iodo-L-tyrosine ([125I]D-
CIT),23 N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-(S)-4-[18F]-
fluorobenzyl-L-cysteine ([18F]DCFBC),24 and [99mTc](2-pyri-
dylmethyl)2N(CH2)4CH-(CO2H)NHCO-(CH2)6CO-NH-lys-NH-
CONH-glu) ([99mTc]L1)25 (Figure 1). Here we report the
molecular modeling, radiosynthesis, in vitro PSMA inhibitory
potency, tissue uptake selectivity, and preclinical imaging of
the latest compounds in our series of low molecular weight
agents for imaging PCa. Those agents include: 2-[3-[1-carboxy-
5-(4-[125I]iodo-benzoylamino)-pentyl]-ureido]-pentanedioic acid
* To whom correspondence should be addressed. Phone: 410-955-2789.
Fax: 443-956-5055. E-mail: mpomper@jhmi.edu. Address: Johns Hopkins
Medical Institutions, 1550 Orleans Street, 492 CRB II, Baltimore, MD
21231.
†
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.
Molecular Insight Pharmaceuticals, Inc.
|
a Abbreviations: PSMA, prostate-specific membrane antigen; GCPII,
glutamate carboxypeptidase II; NAAG, N-acetylaspartylglutamate; NAALA-
Dase, N-acetylated-R-linked acidic dipeptidase; DCMC, N-[N-[(S)-1,3-di-
carboxypropyl]carbamoyl]-(S)-methyl-L-cysteine; DCIT, N-[N-[(S)-1,3-
dicarboxypropyl]carbamoyl]-(S)-3-iodo-L-tyrosine; DCFBC, N-[N-[(S)-1,3-
dicarboxypropyl]carbamoyl]-(S)-4-fluorobenzyl-L-cysteine; DCL, N-[N-[(S)-
1,3-dicarboxypropyl]carbamoyl]-(S)-L-lysine; L1, (2-pyridylmethyl)2N(CH2)4-
CH(CO2H)NHCO-(CH2)6CO-NH-lys-NHCONH-glu); 2-PMPA, 2-(phospho-
nomethyl)pentanedioic acid; SPECT, single photon emission computed
tomography; PET, positron emission tomography; PCa, prostate cancer; SD,
standard deviation.
10.1021/jm801055h CCC: $40.75
2008 American Chemical Society
Published on Web 12/03/2008

7934 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Chen et al.
Scheme 1a
Figure 1. Radiolabeled PSMA inhibitors.
([125I]3), 2-[3-[1-carboxy-5-(4-[18F]fluoro-benzoylamino)-pen-
tyl]-ureido]-pentanedioic acid ([18F]6), and 2-(3-[1-carboxy-5-
[(5-[125I]iodo-pyridine-3-carbonyl)-amino]-pentyl]-ureido)-pen-
tanedioic acid ([125I]8).
Results and Discussion
Design of PSMA Inhibitors. As mentioned above, we
previously reported SPECT and PET imaging probes of urea-
based PSMA inhibitors including [125I]DCIT, [11C]DCMC,
[18F]DCFBC, and [99mTc]L1.
125I]DCIT is a derivative of a tyrosine-glutamate urea,
a (a) N-hydroxysuccinimidyl-4-iodobenzoate, triethylamine, CH2Cl2; (b)
TFA, anisole; (c) N-hydroxysuccinimidyl-4-tributylstannylbenzoate, tri-
ethylamine, CH2Cl2; (d) [125I]NaI, N-chlorosuccinimide, HOAc, MeOH; (e)
TFA, anisole.
[
whereas [11C]DCMC and [18F]DCFBC are derivatives of a
cysteine-glutamate urea. Compound [99mTc]L1 consists of Lys-
C(O)-Glu to which a chelate at the terminus of a linker moiety
has been attached. In this work, we chose to investigate further
derivatives of Lys-C(O)-Glu in order to (1) take advantage of
the many radiohalogenation methods and radiohalogenated
prosthetic groups developed previously for reaction with the
ε-amino group of lysine residues26 and (2) increase the structural
diversity of this class of PSMA inhibitors. We accordingly
designed the syntheses of 3, 6, and 8 to utilize the tri-p-
methoxybenzyl (tri-PMB) ester of 2-[3-(5-amino-1-carboxy-
pentyl-ureido)]-pentanedioic acid as a common starting material.
Scheme 2a
Chemistry
Synthesis. The synthesis of 3, 6, and 8 are shown in Schemes
1, 2, and 3, respectively. N-hydroxysuccinimidyl-4-iodoben-
zoate27 and N-hydroxysuccinimidyl-4-fluorobenzoate28 were
reacted with the tri-PMB ester of Lys-C(O)-Glu 125 to provide
2 (Scheme 1) and 5 (Scheme 2), respectively. The PMB groups
were then removed using trifluoroacetic acid (TFA)/anisole to
yield compounds 3 and 6, respectively. N-Hydroxysuccinimidyl-
5-(tri-n-butylstannyl)-3-pyridinecarboxylate29 was reacted with
1 to give 7 (Scheme 3), followed by iododestannylation and
ester cleavage to give 8.
Modeling of Inhibitors in the Active Site of PSMA.
Recently determined GCPII (PSMA) crystal structures made it
possible to perform structure-based molecular modeling studies
with 3, 6, and 8.30-32 An X-ray structure of PSMA complexed
with 2-PMPA (PDB ID: 2PVW) was used for modeling due to
the high resolution of this structure (1.71 Å) and the small
number of missing amino acid residues.32 Interestingly, PSMA
exists in two conformations including a “binding” conformer
and a “stacking” conformer in the arginine-rich region (S1-
binding pocket) of the active site.33 We carried out docking
studies of 3, 6, and 8 with both conformers using a CHARMm-
based CDOCKER module implemented in Discovery Studio
2.0 (Accelrys Inc., San Diego, CA).34 Details of that procedure
a (a) N-hydroxysuccinimidyl-4-fluorobenzoate, triethylamine, CH2Cl2; (b)
TFA, anisole; (c) N-hydroxysuccinimidyl- 4-[18F]fluorobenzoate, triethy-
lamine, CH2Cl2; (d) TFA, anisole.
are available in the Supporting Information. Analysis of the
docked poses demonstrated that potential binding modes of the
three compounds were very similar to that of 2-PMPA, showing
that the glutamate moiety of the three compounds completely
overlapped with the pentanedioic acid moiety of 2-PMPA,
irrespective of which conformer was under study (Figures 1
and 2 in the Supporting Information). The only differences
among the three compounds with respect to their binding to
either conformer were observed in the arginine patch. The

Radiohalogenated PSMA-Based Ureas as Imaging Agents
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7935
Scheme 3a
region (Figure 2b), while the 4-iodophenyl group of 3 was
projected into the subpocket. On the basis of in vitro PSMA
inhibitory activities and molecular modeling studies, it appears
that ligand interaction with the subpocket of the S1 binding site
contributes more to binding affinity than does interaction with
the tunnel region.
Radiochemistry. As shown in Scheme 1, [125I]3 was prepared
via iododestannylation of the corresponding tri-n-butylstannyl
precursor 4 followed by deprotection. The final product was
obtained after purification by radio-high performance liquid
chromatography (radio-HPLC). The radiochemical yield of this
reaction ranged from 65-80% (n ) 3). Specific radioactivity
was >700 Ci/mmol (25.9 GBq/µmol).
The radiosynthesis of [18F]6 is shown in Scheme 2. N-Hy-
droxysuccinimidyl-4-[18F]iodobenzoate ([18F]SFB) was prepared
by the procedure of Chen et al.35 [18F]SFB was reacted with 1,
followed by deprotection and purification by radio-HPLC to give
[18F]6. The decay-corrected radiochemical yield of [18F]6 ranged
from 30-35% based on starting [18F]fluoride (n ) 3). The mean
synthesis time was 180 min from the time of addition of
[18F]fluoride. Starting from 40 mCi of [18F]fluoride, the specific
radioactivity of [18F]6 was 250-300 Ci/mmol (9.1-11.1 GBq/
µmol).
a (a) N-hydroxysuccinimidyl-5-(tri-n-butylstannyl)-3-pyridinecarboxylate,
triethylamine; CH2Cl2; (b) NaI, N-chlorosuccinimide, HOAc, MeOH; (c)
TFA, anisole; (d) [125I]NaI, N-chlorosuccinimide, HOAc, MeOH; (e) TFA,
CH2Cl2.
As shown in Scheme 3, [125I]8 was prepared using radio-
iododestannylation of the corresponding tri-n-butylstannyl pre-
cursor 7 followed by deprotection and purification by radio-
HPLC. The radiochemical yields of this reaction ranged from
59-75% (n ) 3). Specific radioactivity was >2000 Ci/mmol
(74.0 GBq/µmol).
subpocket generated by three arginines (Arg 463, 534, and 536)
in the binding conformer can accommodate the 4-fluorophenyl
substituent completely and the 4-iodophenyl and 5-iodo-3-
pyridyl groups partially, while all three projected into the tunnel
region when docked with the stacking conformer. CDOCKER
scores of the best poses with the binding conformer of PSMA
(83.177 for 3, 83.172 for 6, 87.610 for 8) were higher than those
with the stacking conformer (82.193 for 3, 75.32 for 6, 78.134
for 8), indicating that the binding conformer of PSMA likely
generates a more stable complex with 3, 6, and 8 than does the
stacking conformer.
We also resolved the PSMA crystal structure complexed with
3 (PDB ID: 3D7H). Details of PSMA cocrystallized with 3, as
well as with other urea-based PSMA inhibitors such as DCIT,
DCMC, and DCFBC, are described elsewhere (in press). As
predicted from modeling studies, only the binding conformation
of the arginine patch region was found in PSMA complexed
with 3. To elucidate potential binding modes of the other two
compounds (6 and 8), we carried out docking studies using the
3D coordinates of 3D7H in the presence or in the absence of
water molecules in the active site. The best poses of 3, 6, and
8 from the docking studies using the CDOCKER module are
shown in Figure 2a, overlaid with crystal ligand, i.e., the
compound as it is cocrystallized with PSMA, of 3. As shown
in Figure 2a, all of the radionuclide-bearing moieties (4-
iodophenyl, 4-fluorophenyl, and 5-iodo-3-pyridyl) were located
within the arginine patch of the S1 binding site. 4-Iodophenyl
and 4-fluorophenyl groups protruded deeply within the sub-
pocket compared to the 5-iodo-3-pyridyl moiety. CDOCKER
scores of the three poses are ordered 3 (80.63) > 6 (72.39) >
8 (69.78). Detailed polar interaction of crystal ligand 3 and the
best poses of 6 and 8 with PSMA are available in the Supporting
Information. The cationic sidechains of Arg 463 and Arg 534
provide cation-π interactions with the aromatic rings of 3, 6,
and 8, leading to stabilization of the ligand within the subpocket.
Docking results of PSMA without water molecules in the active
site showed that the radionuclide-bearing moieties of 6 and 8
were outside of the subpocket and projected into the tunnel
Biological Results
In Vitro Binding. The NAALADase inhibition assay was
undertaken to determine the Ki value for 3, 6, and 8.36 The
concentration of each compound was varied from 0.01 to 1000
nM against a fixed amount of [3H]NAAG (30 nM). The
NAALADase (PSMA) was prepared from LNCaP cell lysates.
The percentage of the enzymatic cleavage product,
[3H]glutamate, produced was measured by scintillation counting
and was plotted against the logarithmic concentration of the
compound under study. Linear regression of the resulting data
were solved for 50% [3H]glutamate production (50% inhibition)
and resulted in Ki values of 0.010 nM for 3, 0.256 nM for 6,
and 0.351 nM for 8 (Table 1). These results are similar to other
compounds of this class.37 Compounds 3, 6, and 8 were also
evaluated using a fluorescence-based inhibition assay37 as a
second check on affinity and gave Ki values of 0.010, 0.194,
and 0.557 nM, respectively.
Ex Vivo Biodistribution and Imaging. Compound [125I]3
was injected intravenously into severe-combined immunodefi-
cient (SCID) mice bearing both PC-3 PIP (PSMA+) and PC-3
flu (PSMA-) xenografts. Table 2 outlines the ex vivo rodent
tissue distribution results of [125I]3. The blood, kidney, urinary
bladder, spleen, and PSMA+ PC-3 PIP tumor display high
uptake at the initial, 30 min postinjection (pi) time point. By
60 min pi, the uptake in PSMA+ PC-3 PIP tumor achieved its
highest absolute value. The kidney achieved its maximal uptake
at 24 h pi. The values noted in the kidney are largely due to
specific binding rather than renal clearance due to the expression
of high amounts of PSMA in the proximal renal tubule.38,39
The PSMA+ PC-3 PIP:kidney ratio remained constant (1:10)
over 48 h. The spleen also exhibited high uptake (141 ( 14%
ID/g) at 1 h, perhaps due to the presence of GCPIII, a close
homologue of GCPII/PSMA.40 We speculate that [125I]3 may
bind to GCPIII as well as to GCPII, as demonstrated by several

7936 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Chen et al.
Figure 2. (a) Overlay of the best poses for 3, 6, and 8 with the crystal ligand, i.e., 3, as it is cocrystallized with PSMA (3, green), in the presence
of water molecules in the active site of PSMA (PDB ID: 3D7H). (b) Overlay of the best poses (3, 6, and 8) with the crystal ligand (3, green) in
the absence of water molecules in the active site of PSMA. Orange sphere (zinc ions), yellow spheres (chloride ion).
compounds of this class has been demonstrated previously.22,25,24
We did not perform site-specific blockade studies, as we have
previously proved the PSMA status of these tumor lines
(PSMA+ PC-3 PIP and flu) by Western blotting (data not shown)
and have other publications demonstrating PSMA-selective
targeting and blockade in this model.23,25
Table 1. PSMA in Vitro Inhibitory Activities and Calculated ClogD
Values
compd
Ki [nM]a
SDb
Ki [nM]c
SDb
ClogD
3
6
8
0.010
0.256
0.351
0.003
0.038
0.257
0.010
0.194
0.557
0.004
0.134
0.265
-5.16
-5.64
-5.88
a obtained from the NAALADase (radiometric) assay. b 95% confidence
interval. c obtained from a fluorescence-based assay.
Compound [125I]3 was injected intravenously into a SCID
mouse bearing both PSMA+ PC-3 PIP and PC-3 flu xenografts
followed by SPECT-CT imaging. Figure 3 shows a SPECT-
CT image of radiopharmaceutical uptake at 4 h pi. Note the
intense uptake in the PSMA+ PC-3 PIP and absence of uptake
in the PC-3 flu tumor. The small amount of radiopharmaceutical
uptake seen in the liver, with no concurrent gastrointestinal
uptake, is likely due to the hydrophilic nature of [125I]3 (ClogD
) -5.16 at pH 7.4), and is consistent with the ex vivo
biodistribution data. The very intense uptake in the kidneys is
also consistent with the ex vivo biodistribution data.
other PSMA ligands.41 However, unlike the kidneys, there was
slow washout from the spleen over time. Urinary bladder uptake
represents excretion at all time points, i.e., there was no specific
binding to bladder wall. Tumor uptake indicated a high degree
of specificity represented by the PSMA+ PIP:flu ratio of 11:1
at 60 min, rising to 140:1 at 48 h. Radiopharmaceutical uptake
within tumor relative to other organs also increased with time,
providing high PSMA+ PIP:organ ratios. We did not remove
bone as part of our ex vivo assay, however, no bone uptake
was noted on imaging (see below). Little metabolism for

Radiohalogenated PSMA-Based Ureas as Imaging Agents
Table 2. Ex Vivo Biodistribution of [125I]3 in Tumor-Bearing Micea b
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7937
,
30 min
60 min
2 h
5 h
12 h
24 h
48 h
blood
heart
lung
liver
0.9 ( 0.8 (10)
2.7 ( 0.9 (3)
5.8 ( 2.4 (1.5)
7.7 ( 3.1 (1)
1.5 ( 0.9 (6)
2.0 ( 0.3 (4)
83 ( 8 (0.1)
4.5 ( 1.1 (2)
119 ( 15 (0.1)
2.7 ( 2.4 (3)
4.9 ( 1.9 (2)
1.4 ( 0.6 (6)
5.2 ( 1.7 (2)
8.8 ( 4.7
0.6 ( 0.2 (22)
1.9 ( 0.3 (7)
4.5 ( 0.5 (3)
7.5 ( 0.8 (2)
1.5 ( 0.3 (9)
2.3 ( 0.3 (6)
141 ( 14 (0.1)
5.6 ( 1.0 (2)
121 ( 17 (0.1)
0.8 ( 0.2 (17)
3.8 ( 0.4 (3.5)
1.0 ( 0.2 (13.5)
6.1 ( 0.8 (2)
13.5 ( 2.1
0.3 ( 0.1 (36)
1.4 ( 0.7 (8)
3.6 ( 2.8 (3)
5.9 ( 3.5 (2)
1.6 ( 1.5 (8)
2.0 ( 0.7 (6)
104 ( 43 (0.1)
6.2 ( 0.8 (2)
111 ( 34 (0.1)
0.6 ( 0.2 (21)
1.5 ( 0.3 (8)
0.6 ( 0.1 (21)
4.0 ( 2.6 (3)
11.8 ( 5.6
0.4 ( 0.2 (31)
0.9 ( 0.2 (14)
3.5 ( 0.6 (3.5)
4.5 ( 1.0 (3)
0.8 ( 0.03 (15)
1.6 ( 0.5 (8)
119 ( 9 (0.1)
6.8 ( 1.3 (2)
132 ( 12 (0.1)
0.4 ( 0.1 (31)
1.5 ( 0.2 (8)
0.7 ( 0.6 (17)
3.0 ( 1.7 (4)
12.4 ( 6.4
0.1 ( 0.03 (125)
0.5 ( 0.2 (25)
2.6 ( 0.8 (5)
1.4 ( 0.2 (9)
0.3 ( 0.06 (39)
0.6 ( 0.2 (21)
69 ( 39 (0.2)
3.8 ( 0.8 (3)
169 ( 29 (0.1
1.0 ( 0.1 (12.5)
1.0 ( 0.4 (12.5)
0.6 ( 0.2 (21)
0.8 ( 0.3 (16)
12.5 ( 4.8
ND
ND
0.5 ( 0.2 (28)
1.1 ( 0.8 (12)
1.4 ( 0.6 (9)
0.7 ( 0.6 (18)
1.2 ( 0.7 (11)
39 ( 6 (0.3)
1.6 ( 1.9 (8)
234 ( 140 (0.1)
0.4 ( 0.1 (33)
0.25 ( 0.1 (54)
1.6 ( 1.9 (9)
0.2 ( 0.2 (64)
13.4 ( 5.1
0.1 ( 0.1 (100)
0.5 ( 0.04 (22)
0.7 ( 0.1 (16)
0.4 ( 0.2 (25)
1.1 ( 0.6 (10)
22 ( 8.6 (0.5)
2.8 ( 0.7 (4)
101 ( 30 (0.1)
0.25 ( 0.03 (44)
0.1 ( 0.1 (110)
0.15 ( 0.1 (73)
0.3 ( 0.04 (37)
11.0 ( 0.2
stomach
pancreas
spleen
fat
kidney
muscle
small int.
large int.
bladder
PC-3 PIP
PC-3 flu
PIP:flu
1.8 ( 1.0
1.2 ( 0.3
0.7 ( 0.3
0.6 ( 0.05
0.3 ( 0.1
0.1 ( 0.06
134
0.08 ( 0.06
138
5
11
17
21
42
b
a Values are in % ID/g ( SD; ND ) not determined; n ) 4 except at 48 h where n ) 3. int. ) intestine. PSMA+ PC-3 PIP tumor:organ ratios are in
parentheses.
Table 3. Ex Vivo Biodistribution of [18F]6 in Tumor-Bearing Micea b
,
30 min
60 min
2 h
5 h
blood
heart
lung
2.5 ( 1.7 (3)
0.7 ( 0.5 (9)
0.4 ( 0.2 (9) 0.03 ( 0.00 (117)
0.8 ( 0.1 (11) 0.2 ( 0.02 (35) 0.15 ( 0.05 (25) 0.03 ( 0.01 (117)
1.7 ( 0.3 (5)
8.7 ( 1.8 (1)
0.5 ( 0.1 (13) 1.0 ( 0.9 (4)
5.8 ( 0.6 (1) 11.7 ( 7.0 (0.3) 1.0 ( 0.6 (3.5)
0.1 ( 0.1 (35)
liver
stomach 0.8 ( 0.1 (11) 0.25 ( 0.1 (26) 0.3 ( 0.1 (13) 0.04 ( 0.02 (88)
pancreas 0.8 ( 0.1 (11) 0.3 ( 0.1 (21) 0.15 ( 0.03 (25) 0.05 ( 0.03 (70)
spleen
kidney
muscle
small int. 1.7 ( 0.7 (5)
large int. 1.2 ( 0.8 (7)
bladder
PC-3 PIP 8.6 ( 3.1
PC-3 flu 0.8 ( 0.3
PIP:flu
12.7 ( 0.4 (0.7) 7.2 ( 1.6 (1)
4.4 ( 1.2 (1)
29 ( 12 (0.1)
1.0 ( 0.6 (3.5)
14 ( 9 (0.3)
72 ( 3 (0.1)
2.4 ( 3.1 (4)
48 ( 5 (0.1)
0.5 ( 0.7 (13) 0.2 ( 0.1 (19) 0.1 ( 0.1 (35)
0.6 ( 0.2 (11) 0.3 ( 0.2 (12) 0.1 ( 0.04 (35)
0.5 ( 0.1 (13) 0.2 ( 0.1 (19) 0.6 ( 0.6 (6)
17 ( 21 (0.4)
6.4 ( 0.9
0.3 ( 0.05
21
6.8 ( 3.5 (1)
11 ( 8 (0.3)
3.7 ( 1.2
0.25 ( 0.1
15
5.2 ( 8.9 (0.7)
3.5 ( 2.3
0.1 ( 0.1
35
11
b
a Values are in % ID/g ( SD, n ) 4, int. ) intestine. PSMA+ PC-3
PIP tumor:organ ratios are in parentheses.
10) over the length of the biodistribution. Uptake in and washout
from nonspecific tissues were low and fast, respectively. Only
the liver and spleen displayed uptake values that rivaled those
seen in PSMA+ PC-3 PIP tumor, although this radioactivity
cleared with time. Compared to [18F]DCFBC,24 [18F]6 had
similar PSMA+ PC-3 PIP tumor uptake but slower clearance
from nontarget tissues, including liver, spleen, and kidney.
Compound [18F]6 was injected intravenously into a tumor-
bearing mouse for PET imaging. Figure 4 shows the averaged
results of the dynamic scan from 94-120 min pi. The uptake
pattern for [18F]6 is very similar to that seen for [125I]3: easily
observed within PSMA+ PIP tumor, none within flu tumor, high
renal uptake, and a modest degree of liver uptake. That result
was expected due to similar ClogD values for [18F]6 (-5.64 at
pH 7.4) and [125I]3. The urinary bladder was visualized at 2 h
pi due to the continually accumulating presence of radioactive
urine, however, as with [125I]3, specific binding to the bladder
wall was not demonstrated.
Table 4 outlines the ex vivo rodent tissue distribution results
of [125I]8. The liver, spleen, kidney, and PSMA+ PC-3 PIP tumor
displayed high uptake at the initial, 30 min pi, time point. By
60 min pi, the kidney displayed the highest uptake while that
in PSMA+ PC-3 PIP tumor remained steady, showing a similar
value to that at 30 min. By 24 h, there was complete clearance
of radioactivity from the individual, nontarget organs. The values
noted in the kidney are largely due to specific binding rather
than renal clearance, as for the other radiopharmaceuticals
discussed above. Urinary bladder uptake represented excretion
at all time points, i.e., there was no specific binding to bladder
wall, while tumor uptake demonstrated a high degree of
Figure 3. [125I]3 SPECT-CT in PCa tumor models (4 h postinjection).
Note uptake within the PSMA+ PIP tumor only. Uptake within kidneys
is due in large measure to the specific binding of [125I]3 to renal cortex.
Table 3 illustrates tissue uptake of [18F]6. This radiophar-
maceutical also displayed rapid, specific uptake within PSMA+
PIP tumors (8.6 ( 3.1% ID/g at 30 min pi). At 1 h pi, the
PSMA+ PC-3 PIP:PC-3 flu ratio was 21:1. As with [125I]3, the
PSMA+ PC-3 PIP:kidney ratio remained roughly constant (1:

7938 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Chen et al.
For example, [18F]FDHT can be used to study the androgen
receptor status of tumors.14 Likewise, the PSMA-based agents
described here can report on the presence of this marker, which
is increasingly recognized as an important prognostic determi-
nant in PCa.42 It is also the target for a variety of new PCa
therapies.43
Among the three compounds discussed here, namely [125I]3,
[18F]6, and [125I]8, [125I]3, and [125I]8 are designed for SPECT
imaging, if 123I is substituted for the 125I used in this experimental
study, while [18F]6 is designed for PET. In principle, all three
compounds can be used for PET if the two radioiodinated
compounds are labeled with 124I. The two radioiodinated
compounds were prepared in high yield and specific radioactiv-
ity, while [18F]6 was prepared in high radiochemical yield, but
the specific activity was relatively low, due to the low amount
of starting [18F]F- (40 mCi) used in the manual synthesis here
described. The specific radioactivity of the starting [18F]F- can
be improved by using higher levels of [18F]F- and a remote
synthesis. In each case, the radiosynthesis was straightforward,
as [125I]3 and [18F]6 could be synthesized by coupling the
intermediate Lys-C(O)-Glu with the corresponding N-hydrox-
ysuccinimidyl benzoate, while a similar approach was used for
[
125I]8, merely replacing the benzoate with a pyridinecarboxylate
moiety. The synthesis of both the halobenzoate and pyridin-
ecarboxylate radionuclide-bearing precursors have been pub-
lished,44,45,28,30 while the Lys-C(O)-Glu urea precursor has been
used by us previously to develop SPECT agents.25 The relative
ease of synthesis of compounds of this class suggests that they
will be amenable for use at a wide variety of imaging centers.
Not surprisingly, 3, 6, and 8 assume similar conformations
within the PSMA active site. The radionuclide-bearing moiety
resides within the arginine patch region of the S1 binding site
in each case, however, that of 8 is not as deep within the pocket
(Figure 2a). Compound 3 has been cocrystallized with PSMA
(in press), and the aromatic phenyl ring for that compound shows
productive cation-π stacking interactions with guanidine func-
tions of Arg 463 and Arg 534 of the enzyme, whereas the
pyridine moiety of 8 forms a less productive cation-π interac-
tion. However, unlike 3 or 6, 8 was found to be able to interact
with Asp 465 (via the pyridine nitrogen) and a water molecule
(via the carbonyl group of 5-iodonicotinamido moiety) in the
S1′ subpocket (see Supporting Information, Figure 5). Those
additional interactions likely offset the less productive cation-π
interaction geometry of 8, providing a high-affinity interaction
and an imaging agent that gives clear delineation of tumor
(Figure 5). While 6 adopts a very similar conformation within
the active site to 3, it binds with lower affinity, likely due to
additional interactions of the iodine within the positively charged
arginine patch for 3. In all, the affinities of 3, 6, and 8 (Table
1) track with predictions based on molecular modeling.
Figure 4. [18F]6 PET coregistered to CT in PCa tumor models (∼100
min postinjection). Note uptake within the PSMA+ PIP tumor only.
Uptake within kidneys is due in large measure to the specific binding
of [125I]3 to renal cortex. There is more intense tumor uptake and less
liver seen with this agent than with [125I]3.
specificity represented by the PSMA+ PC-3 PIP:PC-3 flu uptake
ratio of 18:1 at 30 min, rising to 47:1 at 8 h. In addition, the
radiopharmaceutical uptake within tumor relative to other organs
increased with time due to the rapid clearance from normal
organs. That was most noticeable in the clearance from the
spleen, where the PSMA+ PC-3 PIP:spleen ratio increased from
0.5:1 at 0.5 h to 3:1 at 4 h compared to [125I]3, where the ratios
only increased from 1:10 at 0.5 h to 1:5 at 48 h.
Compound [125I]8 was injected intravenously into a SCID
mouse bearing an LNCaP (PSMA+) tumor followed by SPECT-
CT imaging. Figure 5 shows a SPECT-CT image of radiop-
harmaceutical uptake at 4 h pi. Tumor uptake was high, with
little uptake in normal tissues. At 4 h pi, the radioactivity in
the tumor appeared even more intense than that in the kidneys.
Regarding ex vivo biodistribution, [125I]3 demonstrated only
about twice the tumor uptake at 1 h pi as [18F]6, while its affinity
is about 25 times higher. The target to nontarget (PSMA+ PC-
3PIP:PC-3flu) ratios for [18F]6, however were higher than that
for [125I]3 at 0.5-1 h pi, reflecting lower nonspecific binding,
however the two compounds are equivalent by 2 h. Target to
nontarget ratios rise to approximately 140 at 48 h pi for [125I]3
and to 35 at 5 h pi for [18F]6. Compound [125I]8 differs from
Discussion
Unlike many other cancers, PCa is particularly difficult to
detect using existing molecular imaging radiotracers. There are
several reasons for that, including the relatively slow growth
and metabolic rate of PCa compared to other malignancies as
well as the small size of the organ and proximity to the urinary
bladder, into which most radiopharmaceuticals are eventually
excreted. Molecular imaging may provide a way not only to
detect tumor in vivo but also to provide information regarding
the biology of the lesion if a mechanism-specific agent is used.
[
125I]3 in that the aromatic ring is a pyridine and the iodine is
substituted at the 3-position. At 1 h pi [125I]8 demonstrated a
similarly high tumor uptake (12.1 ( 4.9%ID/g) compared to
[
125I]3 but had a much higher target to nontarget tissue ratio at
that time (20), which rose to 47 at 8 h pi. Compound [125I]3
took 12 h to reach that ratio. Interestingly, the affinity of [125I]8

Radiohalogenated PSMA-Based Ureas as Imaging Agents
Table 4. Ex Vivo Biodistribution of [125I]8 in Tumor-Bearing Micea b
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7939
,
organ
blood
heart
lung
liver
stomach
pancreas
spleen
fat
kidney
muscle
small int.
large int.
bladder
PC-3 PIP
PC-3 flu
PIP:flu
30 min
60 min
4 h
8 h
24 h
2.5 ( 1.4 (6)
0.9 ( 0.2 (16)
2.7 ( 3.2 (5)
8.2 ( 1.2 (2)
0.8 ( 0.3 (18)
0.9 ( 0.2 (15)
26 ( 12 (0.5)
3.5 ( 0.2 (4)
160 ( 27 (0.1)
1.9 ( 2.4 (7)
0.8 ( 0.2 (18)
0.9 ( 0.4 (16)
1.9 ( 0.4 (8)
14.2 ( 9.5
1.1 ( 0.5 (11)
0.4 ( 0.3 (30)
2.3 ( 1.0 (5)
9.7 ( 1.2 (1)
0.5 ( 0.3 (23)
0.8 ( 0.3 (16)
13.0 ( 6.8 (1)
1.4 ( 0.5 (8)
205 ( 46 (0.06)
0.9 ( 0.7 (13)
1.1 ( 1.3 (11)
0.6 ( 0.3 (21)
4.8 ( 4.9 (2.5)
12.1 ( 4.9
0.25 ( 0.2 (17)
0.06 ( 0.02 (20)
0.3 ( 0.1 (14)
4.8 ( 0.9 (1)
0.1 ( 0.05 (42)
0.3 ( 0.3 (14)
1.25 ( 0.4 (3)
0.04 ( 0.1 (105)
71 ( 27 (0.06)
0.2 ( 0.3 (21)
0.2 ( 0.05 (21)
0.2 ( 0.1 (21)
7.0 ( 3.6 (0.6)
4.2 ( 1.5
0.06 ( 0.01 (23)
0.02 ( 0.01 (70)
0.1 ( 0.05 (12)
1.6 ( 0.4 (1)
0.06 ( 0.02 (23)
0.03 ( 0.00 (47)
0.5 ( 0.2 (3)
0.2 ( 0.2 (7)
24 ( 10 (0.06)
0.1 ( 0.1 (14)
0.1 ( 0.02 (14)
0.1 ( 0.1 (14)
2.8 ( 2.1 (0.5)
1.4 ( 0.4
0.00 ( 0.00
0.00 ( 0.00
0.01 ( 0.00 (9)
0.04 ( 0.03 (2)
0.01 ( 0.01 (9)
0.00 ( 0.00
0.03 ( 0.02 (3)
0.00 ( 0.01
0.97 ( 0.94 (0.1)
0.00 ( 0.00
0.01 ( 0.01 (9)
0.00 ( 0.00
0.05 ( 0.01 (2)
0.09 ( 0.04
0.00 ( 0.00
0.8 ( 0.1
18
0.6 ( 0.25
0.1 ( 0.02
0.03 ( 0.01
20
42
47
b
a Values are in % ID/g ( SD; n ) 4; int. ) intestine. PSMA+ PC-3 PIP tumor:organ ratios are in parentheses.
compounds of this series, we again show that there is not a
clear relationship between affinity and in vivo tumor uptake
selectivity. Notably, the affinities of these compounds are quite
high (Table 1) and the tumors are clearly delineated (Figures
3, 5).
On the basis of the in vivo imaging studies, [125I]3 and [125I]8
have appropriate pharmacokinetics and imaging characteristics
for imaging of PSMA+ prostate cancer with SPECT when
radiolabeled with 123I, while [18F]6 and [124I]3 would be useful
for PET. All compounds demonstrate high target to nontarget
tissue ratios relatively soon after injection, making any of them
convenient for clinical use. The syntheses are also adaptable to
an automated format. Although our previous 18F-labeled inhibi-
tor, [18F]DCFBC,24 produced higher PSMA+ PC-3 PIP:organ
ratios than [18F]6, [18F]6 can be prepared in higher radiochemical
yield. Additionally, automated syntheses of [18F]SFB, the
precursor for [18F]6, have been reported, suggesting that the
radiosynthesis of [18F]6 may be easier to automate.46,47 Radiation
dosimetry and toxicity studies of this latest series of compounds
are currently under way.
Experimental Section
General Procedures. All reagents and solvents were purchased
from either Sigma-Aldrich (Milwaukee, WI) or Fisher Scientific
(Pittsburgh, PA). The tosylate salt of 1 was prepared according to
a reported procedure.25 1H NMR spectra were obtained on a Varian
Mercury 400 mHz or a Bruker Avance 400 mHz spectrometer. ESI
mass spectra were obtained on an API 150EX or a Bruker Esquire
3000 plus system. High-resolution mass spectra (HRMS) were
performed on a JEOL JMS-AX505HA mass spectrometer in the
Mass Spectrometry Facility at the University of Notre Dame. HPLC
purification of nonradioactive compounds was performed on a
Waters 625 LC system with a Waters 490E multiwavelength UV/
vis detector (Milford, MA).
[
125I]NaI was purchased from MP Biomedicals (Costa Mesa, CA).
[18F]Fluoride was produced by 18 MeV proton bombardment of a
high pressure [18O]H2O target using a General Electric PETtrace
biomedical cyclotron (Milwaukee, WI). Solid-phase extraction
cartridges (C18 plus, Sep-Pak) were purchased from Waters As-
sociates. Reverse phase radio-HPLC purification of [125I]3 and
Figure 5. [125I]8 SPECT-CT in PSMA+ LNCaP tumors (4 h postin-
jection). Note intense uptake within tumor. A similar result was obtained
for PSMA+ PIP but not PSMA- flu tumors (data not shown). There is
less renal and liver uptake with this agent than the halobenzoylated
analogues, [125I]3 and [18F]6, respectively.
[
125I]8 were performed using a Waters 510 pump, Waters 490E
variable wavelength UV/vis detector at 254 nm, and a Bioscan Flow
Count PMT radioactivity detector (Washington, DC). Reverse phase
radio-HPLC purification of [18F]6 was performed using a Varian
Prostar System with a Bioscan Flow Count PMT radioactivity
detector. Radioactivity was measured in a Capintec CRC-10R dose
calibrator (Ramsey, NJ). The specific radioactivity was calculated
as the radioactivity eluting at the retention time of product during
is the lowest among the three compounds tested (Table 1),
however, compared with [125I]3, it provided a higher target to
nontarget ratio at 1 h pi, equivalent to that of [125I]3 at 2-5 h
pi. As demonstrated in our previous work with 99mTc-labeled

7940 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Chen et al.
the preparative HPLC purification divided by the mass correspond-
ing to the area under the curve of the UV absorption.
2.07-2.09 (m, 1H), 1.79-1.90 (m, 2H), 1.55-1.69 (m, 3H),
1.39-1.40 (m, 2H). ESI-Mass calcd for C19H24FN3O8Na [M + Na]+
464.1, found 464.4. FAB-HRMS calcd for C19H25FN3O8 [M + H]+
442.1626, found 442.1646.
2-[3-[5-(4-Iodo-benzoylamino)-1-(4-methoxy-benzyloxycarbo-
nyl)-pentyl]-ureido]-pentanedioic Acid Bis-(4-methoxy-benzyl)
ester (2). To a solution of 1 (0.126 g, 0.148 mmol) in CH2Cl2 (4
mL) was added triethylamine (0.1 mL, 0.712 mmol), followed by
N-hydroxysuccinimidyl-4-iodobenzoate48 (0.073 g, 0.212 mmol).
After stirring for 2 h at room temperature, the solvent was evaporated
on a rotary evaporator. The crude material was purified on a silica
column using methanol/methylene chloride (5:95) to afford 0.127 g
(94%) of 2. 1H NMR (400 MHz, CDCl3) δ 7.69 (d, J ) 8.8 Hz, 2H),
7.49 (d, J ) 8.8 Hz, 2H), 7.17-7.26 (m, 6H), 6.77-6.86 (m, 7H),
5.37-5.46 (m, 2H), 4.93-5.09 (m, 6H), 4.32-4.40 (m, 2H), 3.76-3.77
(m, 9H), 3.30-3.33 (m, 2H), 2.30-2.36 (m, 2H), 2.07-2.12 (m, 1H),
1.84-1.92 (m, 1H), 1.70-1.79 (m, 1H), 1.49-1.57 (m, 3H), 1.25-1.33
(m, 2H). ESI-Mass calcd for C43H48IN3O11Na [M + Na]+ 932.2, found
932.7.
2-(3-[1-(4-Methoxy-benzyloxycarbonyl)-5-[(5-tributylstanna-
nyl-pyridine-3-carbonyl)-amino]-pentyl]-ureido)-pentanedioic Acid
Bis-(4-methoxy-benzyl) Ester (7). To a solution of 1 (0.120 g,
0.148 mmol) in CH2Cl2 (2 mL) was added triethylamine (0.1 mL,
0.712 mmol), followed by N-hydroxysuccinimidyl-5-(tri-n-butyl-
stannyl)-3-pyridinecarboxylate50,51 (0.075 g, 0.147 mmol). After
stirring for 30 min at room temperature, the crude material was
purified on a silica column using methanol/methylene chloride (5:
1
95) to afford 0.115 g (76%) of 7. H NMR (400 MHz, CDCl3) δ
8.85 (s, 1H), 8.65 (s, 1H), 8.19 (s, 1H), 7.19-7.24 (m, 6H),
6.81-6.85 (m, 6H), 6.65 (m, 1H), 5.32-5.35 (m, 1H), 5.22-5.25
(m, 1H), 4.96-5.10 (m, 6H), 4.40-4.47 (m, 2H), 3.70-3.77 (m,
9H), 3.34 (m, 2H), 2.35-2.39 (m, 2H), 2.10-2.15 (m, 1H),
1.90-1.94 (m, 1H), 1.72-1.79 (m, 1H), 1.46-1.59 (m, 9H),
1.27-1.36 (m, 8H), 1.02-1.25 (m, 6H), 0.84-0.87 (m, 9H). ESI-
Mass calcd for C54H75N4O11Sn [M + H]+ 1075.4, found 1075.5.
2-(3-[1-Carboxy-5-[(5-iodo-pyridine-3-carbonyl)-amino]-pen-
tyl]-ureido)-pentanedioic Acid (8). To a solution of 7 (0.025 g,
0.023 mmol) in 2 mL of methanol was added 0.020 mL of acetic
acid and sodium iodide (0.017 g, 0.113 mmol), followed by
N-chlorosuccinimide (0.025 g, 0.187 mmol). After 20 min at room
temperature, the solvent was removed under a stream of nitrogen.
A solution of TFA in CH2Cl2 (1:1, 2 mL) was then added to the
residue. After 1 h at room temperature, 8 (0.008 g, 62%) was
isolated by HPLC (Econosphere C18 10 µ, 250 mm × 10 mm,
H2O/CH3CN/TFA (85/15/0.1), 4 mL/min, product peak eluting at
10 min). 1H NMR (400 MHz, D2O) δ 9.00-9.15 (m, 3H),
4.18-4.24 (m, 2H), 3.40-3.41 (m, 2H), 2.45-2.49 (m, 2H),
2.12-2.13 (m, 1H), 1.85-1.97 (m, 2H), 1.64-1.73 (m, 3H), 1.44
(m, 2H). ESI-Mass calcd for C18H24IN4O8 [M + H]+ 551.1, found
551.0. FAB-HRMS calcd for C18H24IN4O8 [M + H]+ 551.0639,
found 551.0607.
2-[3-[1-Carboxy-5-(4-iodo-benzoylamino)-pentyl]-ureido]-
pentanedioic Acid (3). A solution of 3% anisole in TFA (15 mL)
was added to 2 (0.117 g, 0.129 mmol) at 0 °C. The mixture was
stirred at room temperature for 30 min and then concentrated on a
rotary evaporator. The crude material was purified by HPLC
(Econosil C18 10 µ, 250 mm × 10 mm, H2O/CH3CN/TFA (70/
30/0.1), 4 mL/min, 3 eluting at 11 min) to afford 0.040 g (57%) of
1
3. H NMR (400 MHz, D2O:CD3CN ) 1:1 (v/v)) δ 7.79 (d, J )
8.0 Hz, 2H), 7.46 (d, J ) 8.0 Hz, 2H), 4.08-4.16 (m, 2H), 3.26
(m, 2H), 2.35 (m, 2H), 2.00-2.03 (m, 1H), 1.72-1.84 (m, 2H),
1.52-1.62 (m, 3H), 1.34-1.36 (m, 2H). ESI-Mass calcd for
C19H24IN3O8Na [M + Na]+ 572.1, found 572.0. FAB-HRMS calcd
for C19H25IN3O8 [M + H]+ 550.0686, found 550.0648.
2-[3-[1-(4-Methoxy-benzyloxycarbonyl)-5-(4-tributylstanna-
nyl-benzoylamino)-pentyl]-ureido]-pentanedioic Acid Bis-(4-
methoxy-benzyl) Ester (4). To a solution of 1 (0.120 g, 0.148
mmol) in CH2Cl2 (6 mL) was added triethylamine (0.1 mL, 0.712
mmol), followed by N-hydroxysuccinimidyl-4-tributylstannylben-
zoate48 (0.075 g, 0.147 mmol). After stirring for 2 h at room
temperature, the reaction mixture was condensed on a rotary
evaporator. The crude material was purified on a silica column using
methanol/methylene chloride (5:95) to afford 0.130 g (86%) of 4.
1H NMR (400 MHz, CDCl3) δ 7.68 (d, J ) 8.4 Hz, 2H), 7.49 (d,
J ) 7.6 Hz, 2H), 7.18-7.24 (m, 6H), 6.80-6.85 (m, 6H), 6.47
(m, 1H), 5.44-5.47 (m, 2H), 4.95-5.09 (m, 6H), 4.41-4.45 (m,
2H), 3.76-3.77 (m, 9H), 3.32-3.38 (m, 2H), 2.35-2.37 (m, 2H),
2.08-2.16 (m, 1H), 1.90-1.94 (m, 1H), 1.70-1.79 (m, 1H),
1.45-1.64 (m, 9H), 1.24-1.30 (m, 8H), 1.01-1.06 (m, 6H),
0.85-0.87 (m, 9H). ESI-Mass calcd for C55H75N3O11SnNa [M +
Na]+ 1096.4, found 1096.7.
2-[3-[5-(4-Fluoro-benzoylamino)-1-(4-methoxy-benzyloxycar-
bonyl)-pentyl]-ureido]-pentanedioic Acid Bis-(4-methoxy-ben-
zyl) Ester (5). To a solution of 1 (0.140 g, 0.164 mmol) in CH2Cl2
(4 mL) was added triethylamine (0.1 mL, 0.712 mmol), followed
by N-hydroxysuccinimidyl-4-fluorobenzoate49 (0.043 g, 0.181
mmol). After stirring for 2 h at room temperature, the solvent was
evaporated on a rotary evaporator. The crude material was purified
by on a silica column using methanol/methylene chloride (5:95) to
afford 0.120 g (91%) of 5. 1H NMR (400 MHz, CDCl3) δ 7.78 (m,
2H), 7.16-7.24 (m, 6H), 7.01 (m, 2H), 6.80-6.85 (m, 7H), 5.51-
5.64 (m, 2H), 4.93-5.09 (m, 6H), 4.34-4.40 (m, 2H), 3.75-3.77
(m, 9H), 3.28-3.34 (m, 2H), 2.26-2.38 (m, 2H), 2.04-2.15 (m,
1H), 1.82-1.91 (m, 1H), 1.68-1.74 (m, 1H), 1.44-1.57 (m, 3H),
1.25-1.33 (m, 2H). ESI-Mass calcd for C43H48FN3O11Na [M +
Na]+ 824.3, found 824.7.
Radiochemistry. 2-[3-[1-Carboxy-5-(4-[125I]iodo-benzoylamino)-
pentyl]-ureido]-pentanedioic Acid ([125I]3). To a solution of 4 (1
mg, 0.932 µmol) in 0.1 mL of methanol was added 0.001 mL of
acetic acid and 1-4 µL (1-4 mCi) (37-148 MBq) [125I]NaI
solution, followed by N-chlorosuccinimide (0.25 mg, 0.187 µmol)
in 0.025 mL of methanol. After stirring at room temperature for
20 min, the solvent was removed under a stream of N2. A solution
of 3% anisole in TFA (0.1 mL) was then added to the residue.
After 5 min at room temperature, solvent was removed under a
stream of N2 and then [125I]3 was isolated by radio-HPLC (Econosil
C18 10 µ, 250 mm × 4.6 mm, H2O/CH3CN/TFA (72/28/0.1), 1
mL/min). The HPLC eluate containing [125I]3 was neutralized with
1 M NaHCO3, concentrated under vacuum to dryness, reconstituted
in PBS (pH 7.4), and passed through a 0.22 µm syringe filter into
an evaculated sterile vial. The radiochemical yield ranged from
65-80% and the specific activity was >700 Ci/mmol (25.9 GBq/
µmol).
N-Hydroxysuccinimidyl-4-[18F]fluorobenzoate([18F]SFB).[18F]-
SFB was synthesized according to a literature procedure30 and
purified by radio-HPLC (Econosphere C18 10 µ, 250 mm × 10
mm, H2O/CH3CN/TFA (75/25/0.1), 5 mL/min, product peak eluting
at 19 min).
2-[3-[1-Carboxy-5-(4-[18F]fluoro-benzoylamino)-pentyl]-ure-
ido]-pentanedioic Acid ([18F]6). In a vial containing 2 mg of 1
and 0.002 mL of triethylamine was added 9 mCi (333 MBq)
[18F]SFB in 2 mL of CH2Cl2. The reaction was heated at 45 °C for
20 min, followed by removal of solvent under a stream of nitrogen,
addition of 0.1 mL of 3% anisole/TFA, and heating at 45 °C for 5
min. Solvent was then removed under a stream of N2. The final
product was obtained after radio-HPLC purification (Econosphere
C18 10 µ, 250 mm × 10 mm, H2O/CH3CN/TFA [80/20/0.1], 4
mL/min), and was neutralized, concentrated, and filtered as above.
The radiochemical yield ranged from 30-35% (decayed corrected)
and the specific radioactivity ranged from 250 to 300 Ci/mmol
(9.1-11.1 GBq/µmol).
2-[3-[1-Carboxy-5-(4-fluoro-benzoylamino)-pentyl]-ureido]-
pentanedioic Acid (6). A solution of 3% anisole in TFA (15 mL)
was added to 5 (0.081 g, 0.1 mmol) at 0 °C. The mixture was stirred
at room temperature for 20 min then concentrated on a rotary
evaporator. The crude material was purified by HPLC (Econosil
C18 10 µ, 250 mm × 10 mm, H2O/CH3CN/TFA (75/25/0.1), 4
mL/min, 6 eluting at 9 min) to afford 0.035 g (79%) of 6. 1H NMR
(400 MHz, D2O) δ 7.66-7.69 (m, 2H), 7.11-7.16 (m, 2H),
4.12-4.19 (m, 2H), 3.28-3.31 (m, 2H), 2.39-2.43 (m, 2H),

Radiohalogenated PSMA-Based Ureas as Imaging Agents
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7941
2-(3-[1-Carboxy-5-[(5-[125I]iodo-pyridine-3-carbonyl)-amino]-
pentyl]-ureido)-pentanedioic Acid ([125I]8). To a solution of 7
(0.1 mg, 0.093 µmol) in 0.1 mL of methanol was added 0.001 mL
of acetic acid, 3 µL (2.7 mCi) (100 MBq) of [125I]NaI, followed
by N-chlorosuccinimide (0.03 mg, 0.225 µmol) in 0.010 mL of
methanol. After 20 min at room temperature, the solvent was
removed under a stream of nitrogen. A solution of 3% anisole in
TFA (0.1 mL) was then added to the residue. After 5 min at room
temperature, solvent was removed under a stream of N2 and then
pugamma CS, Pharmacia/LKB Nuclear, Inc., Gaithersburg, MD).
The percent-injected dose per gram of tissue (% ID/g) was
calculated by comparison with samples of a standard dilution of
the initial dose. All measurements were corrected for decay.
A single SCID mouse implanted with both a PSMA+ PC-3 PIP
and a PC-3 flu xenograft was injected intravenously with 1 mCi
(37 MBq) of [125I]3 in PBS. At 4 h pi, the mouse was anesthetized
with isoflurane and maintained under 1% isoflurane in oxygen. The
mouse was positioned on the X-SPECT (Gamma Medica, Northridge,
CA) gantry and was scanned using two low energy, high-resolution
pinhole collimators (Gamma Medica) rotating through 360° in 6°
increments for 45 s per increment. All gamma images were
reconstructedusingLumaGEMsoftware(GammaMedica,Northridge,
CA). Immediately following SPECT acquisition, the mice were then
scanned by CT (X-SPECT) over a 4.6 cm field-of-view using a
600 µA, 50 kV beam. The SPECT and CT data were then
coregistered using the supplier’s software (Gamma Medica) and
displayed using AMIDE (http://amide.sourceforge.net/). Data were
reconstructed using the ordered subsets-expectation maximization
(OS-EM) algorithm.
[
125I]8 was isolated by HPLC (Econosil C18 10 µ, 250 mm × 4.6
mm, H2O/CH3CN/TFA [85/15/0.1], 1 mL/min), neutralized, con-
centrated, and filtered as above. The radiochemical yield ranged
from 59-75%, and the specific activity was greater than 2000 Ci/
mmol (74.0 GBq/µmol).
NAALADase Assay. NAAG hydrolysis was performed es-
sentially as described previously.31,52 Briefly, 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
sodium phosphate and 2 mM EDTA. Products were partitioned by
AG 1-X8 formate resin (Bio-Rad Laboratories, Hercules, CA) 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 were 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 the linear phase of hydrolysis
(i.e., < 20% cleavage of total substrate). Enzyme inhibitory
constants (Ki values) were generated using the Cheng-Prusoff
conversion.53 Data analysis was performed using GraphPad Prism
version 4.00 for Windows (GraphPad Software, San Diego, CA).
PSMA activity was also determined using a fluorescence-based
assay according to a previously reported procedure.32 Briefly, lysates
of LNCaP cell extracts were incubated with inhibitor in the presence
of 4 µM NAAG. The amount of reduced glutamate was measured
by incubating with a working solution of the Amplex Red glutamic
acid kit (Molecular Probes Inc., Eugene, OR). The fluorescence
was measured with a VICTOR3V multilabel plate reader (Perkin-
Elmer Inc., Waltham, MA) with excitation at 490 nm and emission
at 642 nm.
Compound [18F]6. Ex vivo biodistribution proceeded as for
[
125I]3 with the following exceptions: Mice were injected with 100
µCi (3.7 MBq) of [18F]6 and uptake times were 30, 60, 120, and
300 min pi. In vivo PET-CT: A SCID mouse bearing subcutaneous
PSMA+ PC-3 PIP and PC-3 flu xenografts was anesthetized using
3% isoflurane in oxygen for induction and 1.5% isoflurane in
oxygen at 0.8 L/min flow for maintenance. The animal was placed
in the prone position on the gantry of a GE eXplore Vista small
animal PET scanner (GE Healthcare, Milwaukee, WI). The mouse
was injected intravenously with 200 µCi (7.4 MBq) of [18F]6,
followed by image acquisition using the following protocol: The
images were acquired as a pseudodynamic scan, i.e., a sequence
of successive whole-body images were acquired in three bed
positions for a total of 120 min. The dwell time at each position
was 5 min, such that a given bed position (or mouse organ) was
revisited every 15 min. An energy window of 250-700 keV was
used. Images were reconstructed using the FORE/2D-OSEM
method (2 iterations, 16 subsets) and included corrections for
radioactive decay, scanner dead time, and scattered radiation.
Compound [125I]8. PSMA+ PC-3 PIP and PC-3 flu xenograft-
bearing SCID mice were injected via the tail vein with 2 µCi (74
KBq) of [125I]8. Four mice each were sacrificed by cervical
dislocation at 30, 60, 240 min, 8 and 24 h pi. The heart, lungs,
liver, stomach, pancreas, spleen, fat, kidney, muscle, small and large
intestines, urinary bladder, and PSMA+ PC-3 PIP and flu tumors
were quickly removed. A 0.1 mL sample of blood was also
collected. All organs were weighed, and the tissue radioactivity
was measured with an automated gamma counter (1282 Com-
pugamma CS, Pharmacia/LKB Nuclear, Inc., Gaithersburg, MD).
The %ID/g was calculated by comparison with samples of a
standard dilution of the initial dose. All measurements were
corrected for decay.
Cell Lines and Mouse Models. PC-3 PIP (PSMA+) and PC-3
flu (PSMA-) cell lines were obtained from Dr. Warren Heston
(Cleveland Clinic) and were maintained as previously described.25
LNCaP cells were obtained from Dr. Ron Rodriguez (JHU) and
were maintained as described.25 All cells were grown to 80-90%
confluence before trypsinization and formulation in Hank’s Bal-
anced Salt Solution (HBSS, Sigma, St. Louis, MO) for implantation
into mice.
All animal studies were undertaken in full compliance with
institutional guidelines related to the conduct of animal experiments.
Male SCID mice (Charles River Laboratories, Wilmington, MA)
were implanted subcutaneously with 1-5 × 106 for PC-3s and 5
× 106 for LNCaPs forward of each shoulder. PSMA+ PC-3 PIP
cells were implanted behind the left shoulder, and PC-3 flu cells
were implanted behind the right shoulder. Mice were imaged or
used in biodistribution assays when the tumor xenografts reached
3-5 mm in diameter.
A single SCID mouse implanted with a LNCaP xenograft was
injected intravenously with 1 mCi (37 MBq) of [125I]8 in PBS. At
4 h pi, the mouse was anesthetized with isoflurane and maintained
under 1% isoflurane in oxygen. The mouse was positioned on the
X-SPECT (Gamma Medica, Northridge, CA) gantry and was
scanned using two low energy, high-resolution pinhole collimators
(Gamma Medica) rotating through 360° in 6° increments for 45 s
per increment. All gamma images were reconstructed using
LumaGEM software (Gamma Medica, Northridge, CA). Im-
mediately following SPECT acquisition, the mice were then scanned
by CT (X-SPECT) over a 4.6 cm field-of-view using a 600 µA, 50
kV beam. The SPECT and CT data were then coregistered using
the supplier’s software (Gamma Medica) and displayed using
AMIDE (http://amide.sourceforge.net/). Data were reconstructed
using the OS-EM algorithm.
Ex Vivo Biodistribution and Imaging. Compound [125I]3.
Xenograft-bearing SCID mice were injected via the tail vein with
2 µCi (74 Bq) of [125I]3. Four mice each were sacrificed by cervical
dislocation at 30, 60, 120, 300 min, 12, 24, and 48 h pi. The heart,
lungs, liver, stomach, pancreas, spleen, fat, kidney, muscle, small
and large intestines, urinary bladder, and PSMA+ PC-3 PIP and
flu tumors were quickly removed. A 0.1 mL sample of blood was
also collected. The organs were weighed, and the tissue radioactivity
was measured with an automated gamma counter (1282 Com-
Acknowledgment. This work was supported by NIH
CA92871, CA1114111, EB005324, and the AdMeTech Foun-
dation. We thank Gilbert Green for technical assistance. We

7942 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
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