A series of halogenated heterodimeric inhibitors of prostate specific membrane antigen (PSMA) as radiolabeled probes for targeting prostate cancer

Article abstract of DOI:10.1021/jm800994j

Prostate specific membrane antigen (PSMA) is a validated molecular marker for prostate cancer. A series of glutamate-urea (Glu-urea-X) heterodimeric inhibitors of PSMA were designed and synthesized where X = ε-N-(o-I, m-I, p-I, p-Br, o-Cl, m-Cl, p-Cl, p-F

Full text of DOI:10.1021/jm800994j

J. Med. Chem. 2009, 52, 347–357
347
A Series of Halogenated Heterodimeric Inhibitors of Prostate Specific Membrane Antigen
(PSMA) as Radiolabeled Probes for Targeting Prostate Cancer
K. P. Maresca,† S. M. Hillier,† F. J. Femia,† D. Keith,† C. Barone,† J. L. Joyal,† C. N. Zimmerman,† A. P. Kozikowski,‡
J. A. Barrett,† W. C. Eckelman,† and J. W. Babich*,†
Molecular Insight Pharmaceuticals, Inc., 160 Second Street, Cambridge, Massachusetts 02142, and Department of Medicinal Chemistry and
Pharmacognocy, UniVersity of Illinois at Chicago, Chicago, Illinois 60612
ReceiVed August 6, 2008
Prostate specific membrane antigen (PSMA) is a validated molecular marker for prostate cancer. A series
of glutamate-urea (Glu-urea-X) heterodimeric inhibitors of PSMA were designed and synthesized where
X ) ε-N-(o-I, m-I, p-I, p-Br, o-Cl, m-Cl, p-Cl, p-F, H)-benzyl-Lys and ε-(p-I, p-Br, p-Cl, p-F, H)-phenylureido-
Lys. The affinities for PSMA were determined by screening in a competitive binding assay. PSMA binding
of the benzyllysine series was significantly affected by the nature of the halogen substituent (IC50 values, Cl
< I ) Br , F ) H) and the ring position of the halogen atom (IC50 values, p-I < o-I , m-I). The halogen
atom had little affect on the binding affinity in the para substituted phenylureido-Lys series. Two lead iodine
compounds were radiolabeled with 123I and 131I and demonstrated specific PSMA binding on human prostate
cancer cells, warranting evaluation as radioligands for the detection, staging, and monitoring of prostate
cancer.
Introduction
capable of sequential removal of the poly γ-glutamyl termini.4,5
It has been reported that overexpression of PSMA in primary
prostate cancer correlates with other adverse traditional prog-
nostic factors and independently predicts disease outcome.6
Since PSMA is expressed by virtually all prostate cancers and
its expression is further increased in poorly differentiated,
metastatic, and hormone-refractory carcinomas,7-9 it is a very
attractive target for developing radiopharmaceuticals for the
diagnosis, staging, and treatment of the disease.
Currently, an indium-111 (111In) radiolabeled anti-PSMA
monoclonal antibody (mAb) 7E11-C5.3 (Capromab Pendetide,
Cytogen Corporation) is used to detect soft tissue metastasis
and recurrence of prostate cancer. This antibody targets the
intracellular domain of PSMA and is thought to bind mostly to
necrotic cells of prostate tumors.10 More recently, Bander et
al. have developed and radiolabeled monoclonal antibodies that
bind to the extracellular domain of PSMA which have entered
into various clinical trials including targeted radiotherapy of
metastatic prostate cancer.11
At least 250 000 men in the U.S. are diagnosed with prostate
cancer annually, and it is estimated that the disease will affect
one in six men between the ages of 60 and 80, leading to
approximately 30 000 deaths per year. The cancer-related
mortality from the disease is second only to lung cancer.1,2 The
global costs associated with prostate cancer are estimated at
$15-20 billion with about half of the spending in the U.S. alone.
The management of the prostate cancer patient is challenging,
as there are numerous clinical factors and treatment options to
consider in deciding on the optimal therapy for a given patient.
Since men are living much longer with the disease because of
early detection, clinical decision making may have long-term
consequences. Accurately defining the extent of disease burden
and accurately defining the aggressiveness of the disease at
diagnosis are important factors in treatment selection. Hence,
the ability to visualize disease is increasingly important for
informing therapeutic selection and treatment planning. New
imaging agents that will more accurately detect and stage the
disease, as well as monitor response to therapy, will enable
improved disease management allowing better patient out-
come and quality of life. Current imaging techniques offer some
opportunity to visualize disease in various parts of the body,
but none of them provide both highly specific and sensitive
detection of metastatic prostate cancer. A sensitive and specific
means of imaging tumor burden throughout the body, in both
soft tissue and bone, is the goal of our current effort.
While monoclonal antibodies offer potential for tumor
targeting, long circulating half-life and poor tumor penetrability
limit their effectiveness as diagnostic and therapeutic radiop-
harmaceuticals.12 For this reason, there have been only limited
clinical successes to date, mostly in the treatment of blood-
borne cancers such as non-Hodgkin’s lymphoma.13 Small
molecules offer significant advantages over antibodies for
targeting solid tumors.14 They can be designed with affinities
similar to those of monoclonal antibodies. Small molecules
exhibit enhanced diffusibility to the extravascular space and
faster blood clearance than antibodies, thus resulting in lower
background signal. In addition, the opportunity to synthesize
analogues exhibiting diverse chemical properties allows alter-
ation of binding affinity as well as pharmacokinetics. Thus,
radiolabeled small molecule radiotracers that bind PSMA may
offer the preferred approach.15-19
PSMA is an attractive target for the detection of primary and
metastatic prostate cancer. PSMA, also known as folate hydro-
lase I or glutamate carboxypeptidase II, is a transmembrane,
750 amino acid type II glycoprotein that is primarily expressed
in normal human prostate epithelium and is up-regulated in
prostate cancer, including metastatic disease.3 PSMA is a unique
exopeptidase with reactivity toward poly γ-glutamated folates
* To whom correspondence should be addressed. Phone: 617-492-5554.
Fax: 617-492-5664. E-mail: jbabich@molecularinsight.com.
†
PSMA is highly homologous to N-acetylated R-linked acidic
dipeptidase (NAALADase), a neuropeptidase that produces the
Molecular Insight Pharmaceuticals, Inc.
University of Illinois at Chicago.
‡
10.1021/jm800994j CCC: $40.75
2009 American Chemical Society
Published on Web 12/29/2008

348 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2
Maresca et al.
Figure 1. Hydrolytic cleavage of NAAG by NAALADase.
four iodine-containing molecules that may be applicable for
radioimaging or radiotherapy of prostate cancer. Two of the
lead iodine containing compounds have been prepared as the
radioiodinated (123I and 131I) analogues in high radiochemical
yields, high radiochemical purity, and high specific activity and
have demonstrated prolonged radiochemical stability at elevated
temperature.
Figure 2. Substrate (a, b) and transition state (c) analogues of
NAALADase based on the structure of NAAG.
Results and Discussion
neurotransmitter glutamate and N-acetylaspartate (NAA) through
the hydrolysis of N-acetylaspartylglutamate (NAAG) (Figure
1).20
Chemistry. Synthesis of Glu-Urea-Lys (4). The key inter-
mediate (4), utilized to prepare the PSMA inhibitors described,
was prepared by either route A or route B as depicted in Scheme
1. In route A, 1, the di-tert-butyl ester of glutamic acid, was
reacted with carbonyldiimidazole (CDIa) under anhydrous
conditions in the presence of triethylamine (TEA) to form the
intermediate acylimidazole derivative 2. This intermediate was
activated with methyl triflate (MeOTf) to afford the activated
methylimidazolium salt, 2B, which reacted readily with (S)-
tert-butyl 2-amino-6-(benzyloxycarbonylamino)hexanoate (Cbz-
Lys-Ot-Bu) to accomplish formation of the heterodimer. The
removal of the carboxybenzyloxy (Cbz) protecting group via
hydrogenolysis afforded 4 in moderate yield. Alternatively, 4
was prepared via the isocyanate (1B), which was generated in
situ from 1 upon treatment with triphosgene. Subsequent reaction
with Cbz-Lys-Ot-Bu followed by removal of the Cbz protecting
group by hydrogenolysis afforded 4 in good yield.
Synthesis of Glu-Urea-Lys(N-benzyl-X) Analogues (6).
Compounds 11-19, of the general structure 6, were prepared
utilizing the route depicted in Scheme 2. The key synthetic
intermediate (4) was reacted with the appropriate aldehyde to
form the intermediate Schiff base, which was not isolated but
directly reduced in situ with sodium triacetoxyborohydride to
afford the desired tri-tert-butyl ester protected benzylamines (5).
The tert-butyl ester protecting groups were removed using TFA.
Following deprotection, the samples were purified by high
pressure liquid chromatography (HPLC) or recrystallized to
afford the desired products (6) in overall yields of 20-90%.
Synthesis of Glu-Urea-Ureido(Phenyl-X) Analogues. Com-
pounds of the general structure 8 were prepared by the route
depicted in Scheme 3. The key synthetic intermediate (4) was
reacted with the appropriate phenyl isocyanate at room tem-
perature to afford the desired protected intermediates (7) in good
yields. The tert-butyl ester protecting groups were removed with
TFA. Following deprotection, the sample was purified by
column chromatography or recrystallized to afford the desired
products (8) in overall yields of 40-90%.
Analysis of the recently reported crystal structure of PSMA
has aided in the understanding of the critical interactions of
potent inhibitors within the active site of the enzyme and has
led to the design and synthesis of several classes of NAALA-
Dase inhibitors that are substrate or transition state analogues,21
including the amide (Figure 2a), urea (Figure 2b) substrate
analogues, phosphinate (Figure 2c, where X ) CH2), and
phosphonate (Figure 2c, where X) O) transition state analogues.
Our attention focused on analogues of the urea-linked
dipeptide NAALADase inhibitors described by Kozikowski and
co-workers (Figure 2b).22,23 The original structures were
developed in which two amino acids were joined through the
R-amino groups by a urea linkage, with the rationale that the
urea group would serve as a suitable replacement for the central
phosphinate/phosphonate backbone present in the early lead
structures described by Kozikowski and Barinka.23-26 On the
basis of the inhibitory potency of the different isomers of the
glutamate-C(O)-glutamatecoreandsubsequentstructure-activity
relationship (SAR) studies with other amino acids, information
was obtained regarding structural modifications to the pharma-
cophore and the effect on binding to the enzyme active site.27
Recently, two radiolabeled urea-based analogues have been
reported, N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-(S)-[11C]m-
ethyl-L-cysteine ([11C]DCMC) and N-[N-[(S)-1,3-dicarboxypro-
pyl]carbamoyl]-(S)-3-iodo-L-tyrosine ([125I]DCIT).28,29 These
compounds exhibit specific uptake in human prostate cancer
xenografts.
Here, we describe the synthesis and in vitro evaluation of
novel Glu-urea-X heterodimers as PSMA inhibitors, where X
is a derivatized lysine (Lys). These molecules are prepared
through the modification of the ε-amino group of the Lys residue
of a protected Glu-urea-Lys heterodimer by conversion to
halogen substituted benzylamines, benzamides and phenylureas.
These in vitro studies explored the electronic, steric and
regiochemistry properties of the halogen atoms on the phenyl
ring. In addition, these experiments probed the functionality of
the linker bridge from the lysine nitrogen to the aromatic ring.
Consequently, a preliminary understanding of the structural
requirements for PSMA binding of these novel halogenated Glu-
urea-X heterodimers was developed. The study resulted in the
identification of several potent inhibitors of PSMA, including
a Abbreviations: DCM, dichloromethane; EA, ethyl acetate; Hex, hex-
anes; DCE, dichloroethane; DMF, dimethylformamide; TFA, trifluoroacetic
acid; THF, tetrahydrofuran; CDI, carbonyldiimidazole; DMAP, dimethy-
laminopyridine; TEA, triethylamine; DIPEA, diisopropylethylamine; MeOTf,
methyl trifluoromethanesulfonate; Glu, glutamic acid; Boc, tert-butoxycar-
bonyl; Cbz, benzyloxycarbonyl; SWFI, sterile water for injection; RCY,
radiochemical yield; RCP, radiochemical purity; Lys, Lysine.

Inhibitors of PSMA for Targeting Prostate Cancer
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2 349
Scheme 1. General Pathway for the Synthesis of the Key Intermediate (4) Utilized in the Synthesis of Glu-Urea-Lys Analogues, via the
Acyl Imidazole Intermediate (Route A) and the Isocyanate Intermediate (Route B)
Scheme 2. General Pathway for the Synthesis of Halogenated Glu-Urea-Lys (N-benzyl-X) Analogues (6)
Scheme 3. General Pathway for the Synthesis of Halogenated Glu-Urea-Ureido (Phenyl-X) Analogues
Radiochemistry. 131I-DCIT was used as a radioligand in the
competitive binding studies and was prepared following
the published literature procedure on the radioiodination of the
tyrosine precursor (S)-2-(3-((S)-1-carboxy-2-(4-hydroxyphenyl)-
ethyl)ureido)pentanedioic acid (DCT),28 utilizing Na131I as the
radioiodine source and iodogen (Pierce) as the oxidant.30 The
8 day half-life of 131I permits the screening of multiple
compounds over several days utilizing the same preparation of
the radioligand.
The radioiodine labeled compounds (S)-2-(3-((S)-1-carboxy-
5-(4-iodobenzylamino)pentyl)ureido)pentanedioic acid (11) and
(S)-2-(3-((S)-1-carboxy-5-(3-(4-iodophenyl)ureido)pentyl)ure-
ido)pentanedioic acid (20) were prepared by iododestannylation
of the trimethylstannyl precursors (S)-di-tert-butyl 2-(3-((S)-1-
tert-butoxy-1-oxo-6-(4-(trimethylstannyl)benzylamino)hexan-2-
yl)ureido)pentanedioate (29) and (S)-di-tert-butyl 2-(3-((S)-1-
tert-butoxy-1-oxo-6-(3-(4-(trimethylstannyl)phenyl)ureido)hexan-
2-yl)ureido)pentanedioate (30), respectively, to form both
[
123I]11 and [123I]20 as depicted in Scheme 4. The synthesis of
29 followed the chemistry previously described to prepare
compounds 11-19 in Scheme 2. Compound 30 was prepared
from tert-butyl protected 20 by palladium catalyzed deiodostan-
nylation utilizing hexamethylditin as the stannane source.
Radioiododestannylation afforded the 123I labeled tri-tert-butyl
ester which was deprotected with TFA to afford the desired
radioiodinated inhibitors.
Binding of Glu-Urea-X Analogues. Two series of Glu-
urea-X heterodimers were prepared. The first was benzylamines

350 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2
Scheme 4. Iododestannylation To Form [123I]11 and [123I]20
Maresca et al.
where X ) ε-N-(o-I, m-I, p-I, p-Br, o-Cl, m-Cl, p-Cl, p-F, H),
series structure A. The second was phenylureido analogues
where X ) ε-(p-I, p-Br, p-Cl, p-F, H), series structure B. All
of the compounds, as shown in Figure 3, comprised a Glu linked
through a urea to the R-amine of a modified Lys derivative.
The Glu residue contributes two of the three carboxylic acids
essential for binding to the PSMA active site.22 The modified
Lys residue contributes the third free carboxylic acid functional-
ity and the ε-amine which was used as the synthetic handle
that led to the rapid synthesis of the two series of compounds
to establish the initial SAR.
The compounds were evaluated in a competitive binding assay
using 131I-DCIT as the radioligand for binding to PSMA on
LNCaP cells. This method of screening compounds was selected
rather than the conventional NAALADase enzymatic assay used
previously21 for several reasons: the throughput is greater, the
Figure 3. Structures of the Glu-urea-X heterodimers.

Inhibitors of PSMA for Targeting Prostate Cancer
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2 351
Table 1. Summary of the in Vitro Cell Binding Data of the Glu-Urea-X
Derivatives
away from the ε-lysine amine and appears to be well tolerated,
as evidenced by the m-iodo analogue (9S,13S)-1-(3-iodophenyl)-
3,11-dioxo-2,4,10,12-tetraazapentadecane-9,13,15-tricarboxyl-
ic acid (25) (IC50 ) 18 nM).
The replacement of the urea moiety with a sulfonamide as
shown by (S)-2-(3-((S)-1-carboxy-5-(4-iodophenylsulfonami-
do)pentyl)ureido)pentanedioic acid (26) was also tested. The
para iodo analogue 26 was prepared and represents a slightly
less lipophilic analogue of 20 (calculated log P ) 0.63 and 0.81
for 26 and 20, respectively). This demonstrated that the
replacement of the urea functionality with a sulfonamide moiety
is an allowed substitution that did not alter the potency of the
compound (IC50 value of 10 nM).
To test the tolerance of bulk off of the ε-lysine amine in the
benzylamine Glu-urea-X series, the 2-naphthyl analogue (S)-
2-(3-((S)-1-carboxy-5-(naphthalen-1-ylmethylamino)pentyl)ure-
ido)pentanedioic acid (27) was prepared. The addition of the
fused unsubstituted napthalene ring afforded an increase in
affinity when compared to the simple phenyl substituted
analogue (S)-2-(3-((S)-5-(benzylamino)-1-carboxypentyl)ure-
ido)pentanedioic acid (19), resulting in a 20-fold increase in
binding affinity (IC50 ) 154 nM). The 2-naphthyl substituent
apparently contributes detrimental bulk as well as potential
interference with the presumed hydrogen bond formed with the
ε-nitrogen of the lysine.
Four compounds that contain an aryl iodide moiety, 11, 20,
25, and 26, hold significant potential as radiopharmaceuticals
for imaging (123I-labeled) and therapy (131I-labeled) of PSMA
positive prostate cancer. Iodine-123, with its relatively short half-
life (13.2 h) and 159 keV γ photons, is an ideal radionuclide
for imaging by single-photon emission computed tomography
(SPECT).31,32 In contrast, iodine-131, with an 8 day half-life
and both γ and ꢀ emissions, offers the potential for therapy.33,34
Two of the lead iodine-containing compounds, 11 and 20, were
selected based on favorable in vitro activity for further study
and were radiolabeled with 123I and 131I using the trimethyl-
stannane precursors 29 and 30, respectively.
The synthesis of the radioiodine labeled compounds used
standard literature procedures starting with the trimethylstannyl
precursors.35,36 The lead compounds were radioiodinated in
g60% radiochemical yield (RCY) with >95% radiochemical
purity (RCP) following purification by reverse phase high
pressure liquid chromatography (RP-HPLC) with a specific
activity of >4000 mCi/µmol. The identities of the radioiodinated
products were confirmed by RP-HPLC through correlation of
the retention time of the radioiodinated products with that of
the corresponding nonradioiodinated material following co-
injection, as shown for 20 in Figure 4.
The specificity for binding of the most potent radioiodinated
compound 20 to PSMA on LNCaP cells was tested in a direct
binding assay. The compound was incubated with LNCaP cells
or the PSMA deficient prostate cancer cell line, PC3, in the
presence or absence of homologous nonradiolabeled 20 or
2-(phosphonomethyl)pentanedioic acid (PMPA), a structurally
unrelated NAALADase inhibitor. Approximately 400 fmol per
million cells of 20 bound to LNCaP cells, but little binding was
observed with PC3 cells. Binding was blocked by either
unlabeled 20 or PMPA (Figure 5), demonstrating the specificity
of the interaction.
compound
structure series
R
IC50 (nM)
9
NA
NA
p-I
>3000
498
22
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
A
A
A
A
A
A
A
A
A
B
B
B
B
B
C
D
A
o-I
37
443
43
m-I
p-Br
p-Cl
o-Cl
m-Cl
p-F
H
p-I
p-Br
p-Cl
p-F
2
245
277
1200
2960
10
2
4
3
12
H
m-I
p-I
2-naphthyl
18
10
154
assay assesses binding to native protein on intact prostate cancer
cells as opposed to the activity of solubilized protein in a cell
lysate, and lead compounds can be subsequently radiolabeled
and retested in the same assay prior to proceeding to tumor
uptake studies in rodents. In general, this method of in vitro
screening may better reflect the in vivo setting.
One of the first Glu-urea-X heterodimers prepared and
analyzed was (S)-2-(3-(4-iodobenzyl)ureido)pentanedioic acid
(9). The poor affinity (IC50 > 3000 nM) shown in Table 1
confirms earlier reports of the necessity of the third carboxyl
group for high affinity binding to the PSMA active site.
In the case of the benzylamine Glu-urea-X heterodimers
series, compounds 11-19, binding was dependent upon the
halogen substituent and their position on the aryl ring (Table
1). For the para-substituted analogues, the IC50 values followed
the trend Cl < I ) Br , F ) H. This relationship appears to
favor the larger less electron negative halogen atoms, with the
exception of (S)-2-(3-((S)-1-carboxy-5-(4-chlorobenzylamino)-
pentyl)ureido)pentanedioic acid (15) where an intermediate size
coupled with the electron poor nature of the chlorine atom is
the preferred substituent in the para-position of this series. The
binding affinity was also strongly influenced by the position of
the halogen atom on the aryl ring. For the iodine substituted
benzylamine series, the para- and ortho-substituted analogues
11 and 12 exhibited IC50 values of 22 and 37 nM, respectively,
whereas the meta-substituted analogue 13 was much less active
(IC50 value of 443 nM). For the chlorine substituted compounds,
the para analogue (15) was the most potent in the series (IC50
value of 2 nM), while the activities of both the ortho (16) and
the meta (17) analogues were significantly decreased (IC50 of
245 and 277 nM, respectively).
In the case of the para-substituted phenylureido series B,
compounds 20-24, the results demonstrated that the type of
halogen atom had little effect on binding. Whether the lack of
a clear relationship between the halogen atom and the affinity
for PSMA is due to the hydrogen bonding ability of the urea
functionality that has been introduced into the analogues or
simply the increase in tether length due to the additional atom
that exists between the ε-lysine amine and the aromatic ring is
still to be determined.
The relationship between the halogen substituent and affinity
in the substituted phenylureido series B was further investigated
with the addition of a methylene spacer (series C). In this case,
the additional methylene group further extends the phenyl ring
The 123I radiolabeled compounds (11 and 20) demonstrated
prolonged stability at elevated temperature, an important
characteristic for potential radiopharmaceuticals. Following
radiolabeling and purification, the product was stored at 40 °C
for 48 h, after which HPLC analysis was performed to assess

352 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2
Maresca et al.
Figure 4. UV-visible chromatogram (upper trace) and radiochromatogram (lower trace) of 20 after RP-HPLC purification demonstrating >95%
RCP of the eluted product, upon co-injection.
Figure 5. Direct binding analysis of radioiodinated 20.
the retention of the radiolabel to the molecules, as well as any
product degradation. The results of the stability study demon-
strated no significant degradation of the products at room or
elevated temperature over time, as shown in Table 2.
pounds, (S)-2-(3-((S)-1-carboxy-5-(4-iodobenzylamino)penty-
l)ureido)pentanedioic acid (11) and (S)-2-(3-((S)-1-carboxy-5-
(3-(4-iodophenyl)ureido)pentyl)ureido)pentanedioic acid (20),
were radiolabeled with both 123I and 131I using the trimethyl-
stannane precursors in high radiochemical yield. The radioio-
dinated molecules demonstrated high affinity and selectivity for
PSMA on LNCaP cells. These data have led to the advancement
of these compounds as clinical development candidates that may
eventually permit the more accurate detection of metastatic
disease, improve staging, and enable the monitoring of response
to systemic therapy.
Conclusions
In summary, a series of novel Glu-urea-X heterodimers for
targeting PSMA were successfully synthesized using the Glu-
urea-Lys building block (S,S)-2-[3-(5-amino-1-carboxypenty-
l)ureido]pentanedioic acid (10). The molecules were evaluated
in vitro, and a preliminary understanding of the structural
requirements for high affinity PSMA binding was established
by systematically substituting halogen atoms on the aryl ring,
thereby demonstrating the impact of both the nature of the
halogen atom and its position on the aryl ring. Altering the
halogen substituent produced changes in the molecule’s overall
hydrophobicity, steric bulk, and electronic properties that
potentially influenced the interactions with the binding pocket
of PSMA. The study resulted in the identification of several
potent inhibitors of PSMA. Two of the lead iodinated com-
Experimental Methods
General Methods. All reactions were carried out in dry
glassware under an atmosphere of argon unless otherwise noted.
Reactions were purified by column chromatography under medium
pressure using a Biotage SP4 or by preparative high pressure liquid
chromatography using a Varian Prostar 210 preparative HPLC
system equipped with a semipreparative Vydac C18 reverse-phase
column (250 mm × 10 mm × 5 µm) connected to a Varian Prostar
model 320 UV-visible detector and monitored at a wavelength of

Inhibitors of PSMA for Targeting Prostate Cancer
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2 353
Table 2. Stability of 123I Radiolabeled 11 and 20
pentanedioic acid di-tert-butyl ester (3) (630 mg, 1.0 mmol) in
ethanol (20 mL) was added ammonium formate (630 mg, 10 eqv)
followed by 10% Pd-C, and the suspension was allowed to stand
with occasional agitation overnight until complete. The mixture was
filtered through Celite and concentrated to afford the desired product
11
20
time
(h)
retention
(%)
retention
at 40 °C (%)
retention
(%)
retention
at 40 °C (%)
1
0
24
48
94.5
94.0
94.1
94.2
93.6
93.2
94.4
93.9
93.4
94.4
93.6
92.7
(479 mg, 0.98 mmol, 98%) as a waxy solid. H NMR (400 MHz,
CDCl3) δ 7.15-6.0 (bm, 4H, NHs), 4.29 (m, 2H), 3.02 (m, 2H),
2.33 (m, 2H), 2.06-1.47 (m, 8H), 1.45-1.40 (m, 27H, t-Bu’s).
ESMS m/z: 488 (M + H)+. Anal. (C24H45N3O7 ·0.35TFA) C, H,
N.
254 nm. The final product purifications were achieved using a binary
solvent gradient of 5-50% B over 21 min (A ) water + 0.1%
TFA, B ) acetonitrile + 0.1% TFA). Analytical HPLC of the
radioiodinated compounds was performed using the same method
with an analytical Vydac C18 reverse-phase column (250 mm ×
4.6 mm × 5 µm). 1H NMR spectra were obtained on a Bruker 400
MHz instrument. Spectra are reported as ppm δ and are referenced
to the solvent resonances in CDCl3, DMSO-d6, or methanol-d4.
Elemental analysis was performed by Prevalere Life Sciences, Inc.
High-resolution mass spectra were determined by M-Scan Inc. using
positive ion electrospray with a Q-Tof API US hybrid quadrupole/
time-of-flight mass spectrometer. All solvents were purchased from
Sigma-Aldrich. Reagents were purchased from Sigma Aldrich,
Bachem, Akaal, or Anaspec.
(S)-2-[(Imidazole-1-carbonyl)amino]pentanedioic Acid Di-tert-
butyl Ester (2). To a suspension of L-di-tert-butyl glutamate
hydrochloride (15.0 g, 51 mmol) in DCM (150 mL) cooled to 0
°C was added TEA (18 mL) and DMAP (250 mg). After the mixture
was stirred for 5 min, CDI (9.0 g, 56 mmol) was added and the
mixture was stirred overnight with warming to room temperature.
The mixture was diluted with DCM (150 mL) and washed with
saturated sodium bicarbonate (60 mL), water (2 × 100 mL), and
brine (100 mL). The organic layer was dried over sodium sulfate
and concentrated to afford the crude product as a semisolid, which
slowly solidified upon standing. The crude material was triturated
with hexane/ethyl acetate to afford a white solid which was filtered,
washed with hexane (100 mL), and dried to afford the desired
(S)-2-(3-(4-Iodobenzyl)ureido)pentanedioic Acid (9). To a sus-
pension of (S)-2-[(imidazole-1-carbonyl)amino]pentanedioic acid
di-tert-butyl ester (2) (0.13 g, 0.37 mmol) in DCE (10 mL) cooled
to 0 °C was added methyl triflate (MeOTf) (0.061 g, 0.37 mmol)
and TEA (0.11 g, 1.1 mmol). After the mixture was stirred for 30
min, 4-iodobenzylamine (110 mg, 0.40 mmol) was added and the
mixture was stirred for 3 h at 50 °C. The mixture was evaporated
to dryness and purified via the Biotage column using a gradient
method with 5-50% B where solvent A was methylene chloride
and solvent B was methanol. The purified compound (56 mg, 0.11
mmol) was deprotected using 1:1 TFA/water mixture with the
mixture stirring overnight. Following aqueous extraction, the
compound was purified by HPLC using a Vydac C18 column
employing a gradient method of 5-50% acetonitrile containing
0.1% TFA and water containing 0.1% TFA resulting in the desired
product (67 mg, 0.002 mmol, 15%) as a white solid. 1H NMR (400
MHz, DMSO-d6) δ 7.6 (s, 2H), 7.1 (br, 2H), 6.5 (m, 1H), 6.3 (d,
1H), 4.15 (m, 2H), 4.06 (m, 1H), 2.22 (m, 2H), 1.9 (m, 1H), 1.68
(m, 1H). Anal. (C13H15IN2O5 ·0.2TFA) C, H, N.
(S)-2-(3-((S)-5-Amino-1-carboxypentyl)ureido)pentanedioic Acid
(10). The compound (S)-2-[3-(5-amino-1-tert-butoxycarbonylpen-
tyl)ureido]pentanedioic acid di-tert-butyl ester (4) (68 mg, 0.140
mmol) was deprotected using the previously described methods to
yield the desired product (31 mg, 0.098 mmol, 70%) as an off-
white solid. 1H NMR (400 MHz, DMSO-d6) δ 12.4 (bs, 3Η), 6.35
(s, 2H), 4.1 (m, 2H), 3.6 (m, 2H), 2.7 (d, 2H), 2.1 (m, 2H), 1.9 (m,
2H), 1.65 (m, 2H), 1.45 (m, 2H), 1.35 (m, 2H). Anal. (C24H45N3O7 ·
0.35TFA·3 tert-butyl) C, H, N.
1
product (15.9 g, 45 mmol, 88%) as a white solid. H NMR (400
MHz, DMSO-d6) δ 7.63 (s, 1H), 7.00 (br, 2H), 6.31 (d, J ) 8.00
Hz, 1H), 4.02 (m, 1H), 2.19 (m, 2H), 1.86 (m, 1H), 1.67 (m, 1H),
1.39 (s, 9H), 1.38 (s, 9H). ESMS m/z: 354 (M + H)+.
(S)-2-(3-((S)-1-Carboxy-5-(4-iodobenzylamino)pentyl)ureido)-
pentanedioic Acid (11). To a solution of (S)-2-[3-(5-amino-1-
carboxypentyl)ureido]pentanedioic acid di-tert-butyl ester (4) (90
mg, 0.185 mmol) dissolved in DCE (2 mL) was added 4-iodoben-
zaldehyde (36 mg, 0.155 mmol). The mixture was heated at 50 °C
for 1 h whereupon sodium triacetoxyborohydride (36 mg, 0.185
mmol) was added. The mixture was heated for 12 h and was
subsequently evaporated to dryness and purified utilizing a Biotage
SP4 with a gradient method of 5-50% methanol in DCM. The
purified compound (24 mg, 0.034 mmol) was deprotected by
treatment with TFA/DCM 1:1 (1 mL), and the mixture was stirred
at room temperature for 18 h. Following aqueous extraction the
compound was purified by HPLC using a Vydac C18 column
employing a gradient method of 5-50% acetonitrile containing
0.1% TFA and water containing 0.1% TFA resulting in the desired
(S)-2-[3((S)-(5-Benzyloxycarbonylamino)-1-tert-butoxycarbon-
ylpentylureido]pentanedioic Acid Di-tert-butyl Ester (3). Route
A. To a solution of 2 (1 g, 2.82 mmol) in DCE (10 mL) at 0 °C
was added MeOTf (0.47 g, 2.85 mmol) and TEA (0.57 g, 5.65
mmol). After the solution was stirred for 30 min, Cbz-Lys-Ot-Bu
(1.06 g, 2.82 mmol) was added in one portion and allowed to stir
for 1 h at 40 °C. The mixture was concentrated to dryness and
purified by column chromatography (SiO2) to afford the desired
product as a white solid (1.37 g, 79%). 1H NMR (400 MHz, CDCl3)
δ 7.34 (m, 5H), 5.33-5.28 (m, 3H), 5.08 (d, J ) 7.4 Hz, 2H),
4.38-4.29 (m, 2H), 3.15 (m, 2H), 2.32-2.01 (m, 2H), 1.90-1.50
(m, 8H), 1.43-1.40 (m, 27H, t-Bu’s). ESMS m/z: 622 (M + H)+.
Route B. In a round-bottom flask triphosgene (2.9 g, 10 mmol)
was suspended in DCM (50 mL) and stirred at 0 °C. A solution of
Cbz-Lys-Ot-Bu (9.1 g, 27 mmol) and DIPEA (10.4 mL, 60 mmol)
in DCM (50 mL) was added dropwise to the triphosgene solution
over 2.5 h. A solution of L-glutamic acid di-tert-butyl ester
hydrochloride (8.0 g, 27 mmol) containing DIPEA (10.4 mL, 60
mmol) and DCM (50 mL) was then added in one portion and
allowed to stir for 45 min. The mixture was concentrated to dryness,
diluted with 150 mL of ethyl acetate, washed with 2 N NaHSO4 (2
× 200 mL), brine (150 mL), and dried over sodium sulfate to yield
a yellow oil. Purification by column chromatography (SiO2) afforded
the desired product as clear oil which upon standing solidifies to a
white solid (12.0 g, 19.4 mmol, 72%). 1H NMR (400 MHz, CDCl3)
δ 7.34 (m, 5H), 5.33-5.28 (m, 3H), 5.08 (d, J ) 7.4 Hz, 2H),
4.38-4.29 (m, 2H), 3.15 (m, 2H), 2.32-2.01 (m, 2H), 1.90-1.50
(m, 8H), 1.43-1.40 (m, 27H, t-Bu’s). ESMS m/z: 622 (M + H)+.
2-[3-(5-Amino-1-tert-butoxycarbonylpentyl)ureido]pen-
tanedioic Acid Di-tert-butyl Ester (4). To a solution of (S)-2-[3-
(5-benzyloxycarbonylamino-1-tert-butoxycarbonylpentyl)ureido]-
1
product (12 mg, 66%) as an off-white solid. H NMR (400 MHz,
DMSO-d6) δ 12.4 (bs, 3Η), 8.8 (s, 2H), 7.8 (d, 2H), 7.27 (d, 2H),
6.35 (s, 2H), 4.1 (m, 4H), 2.89 (s, 2H), 2.2 (d, 2H), 1.90 (m, 1H),
1.65 (m, 4H), 1.35 (m, 2H). 13C NMR (75 MHz, DMSO-d6) δ
173.2, 173.0, 172.5, 156.2, 136.6, 131.3, 131.0, 95.1, 51.9, 51.6,
49.3, 46.4, 31.7, 30.0, 27.7, 25.2, 22.4. ESMS m/z: 536 (M + H)+.
Anal. (C19H26IN3O7 ·1.0TFA) C, H, N.
(S)-2-(3-((S)-1-Carboxy-5-(2-iodobenzylamino)pentyl)ureido)-
pentanedioic Acid (12). Following the same procedure as utilized
in the preparation of 11, the target compound was prepared from
4 reacted with 2-iodobenzaldehyde to yield the desired product (108
mg, 35%) as an off-white solid. 1H NMR (400 MHz, DMSO-d6) δ
12.4 (s, 3H), 8.8 (s, H), 7.94 (m, 1H), 7.5 (m, 1H), 7.16 (t, H),
6.38 (m, 2H), 4.15 (m, 5H), 3.06 (s, 2H), 2.85 (s, 1H), 2.2 (m,
2H), 1.90 (m, 1H), 1.70 (m, 2H), 1.50 (s, 2H), 1.35 (m, 2H). ESMS
m/z: 536 (M + H)+. Anal. (C19H26IN3O7 ·0.8TFA) C, H, N.

354 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2
Maresca et al.
(S)-2-(3-((S)-1-Carboxy-5-(3-iodobenzylamino)pentyl)ureido)-
pentanedioic Acid (13). Following the same procedure in the
preparation of 11, the target compound was prepared from 4 reacted
with 3-iodobenzaldehyde to yield the desired product (4 mg, 53%)
mg, 0.41 mmol) was added and stirred for 3 h. The reaction mixture
was evaporated to dryness and the crude mixture dissolved in
methanol (5 mL) and added dropwise to rapidly stirring water (20
mL) to afford a white precipitate which was collected and washed
with water (20 mL) and dried to afford the desired tri-tert-butyl
ester as a white solid. The purified compound was treated with
TFA/DCM 1:1 (1 mL) and stirred at room temperature for 18 h.
Following aqueous extraction the compound was purified by HPLC
using a Vydac C18 column employing a gradient method of 5-50%
acetonitrile containing 0.1% TFA and water containing 0.1% TFA,
resulting in the desired product (158 mg, 53%) as an off-white solid.
1H NMR (400 MHz, DMSO-d6) δ 8.51 (s, 1H), 7.5 (d, 2H), 7.22
(d, 2H), 6.3 (t, 2H), 6.16 (t, 1H), 4.05 (m, 2H), 3.05 (m, 2H), 2.24
(m, 2H), 1.9 (m, 1H), 1.68 (m, 2H), 1.52 (m, 1H), 1.38 (m, 2H),
1.28 (m, 2H), (3 CO2H not seen). 13C NMR (75 MHz, DMSO-d6)
δ 173.3, 173.0, 172.5, 156.2, 153.9, 139.6, 136.2, 119.2, 83.0, 52.2,
51.5, 39.8, 31.9, 30.0, 29.6, 27.6, 22.8. ESMS m/z: 565 (M + H)+.
Anal. (C19H25IN4O8 ·0.7TFA) C, H, N.
1
as an off-white solid. H NMR (400 MHz, DMSO-d6) δ 12.4 (s,
3H), 8.7 (s, 2H), 7.9 (s, 1H), 7.8 (d, 1H), 7.44 (d, 1H), 7.22 (t,
1H), 6.25 (s, 2H), 4.09 (m, 5H), 2.89 (s, 1H), 2.75 (s, 1H), 2.2 (d,
2H), 1.90 (m, 2H), 1.65 (m, 2H), 1.40 (m, 2H). Anal.
(C19H26IN3O7 ·1.0TFA) C, H, N.
(S)-2-(3-((S)-1-Carboxy-5-(4-bromobenzylamino)pentyl)ure-
ido)pentanedioic Acid (14). Following the same procedure as
utilized in the preparation of 11, the target compound was prepared
from 4 reacted with 4-bromobenzaldehyde to yield the desired
product (100 mg, 45%) as an off-white solid. Di-tert-butyl ester:
1H NMR (400 MHz, CDCl3) δ 7.52 (d, 2H), 7.32 (d,2H), 6.28 (m,
2H), 3.98 (m, 2H), 2.55 (t, 2H), 2.48 (t, 2H), 2.22 (m, 2H), 1.85
(m, 1H), 1.62 (m, 2H), 1.45 (m, 2H), 1.37 (s, 27H), 1.28 (m, 2H).
ESMS m/z: 642(M + H)+. The compound was deprotected using
the previously described methods. ESMS m/z: 474 (M + H)+. Anal.
(C19H26BrN3O7 ·1.3TFA) C, H, N.
(S)-2-(3-((S)-1-Carboxy-5-(3-(4-bromophenyl)ureido)pentyl)ure-
ido)pentanedioic Acid (21). Following the same procedure as
utilized in the preparation of 20, the target compound was prepared
from 4 reacted with 4-bromophenyl isocyanate to afford the desired
(S)-2-(3-((S)-1-Carboxy-5-(4-chlorobenzylamino)pentyl)ure-
ido)pentanedioic Acid (15). Following the same procedure as
utilized in the preparation of 11, the target compound was prepared
from 4 reacted with 4-chlorobenzaldehyde to yield the desired
1
product. H NMR (400 MHz, DMSO-d6) δ 12.5 (s, 3H), 8.55 (s,
1H), 7.35 (d, 4H), 6.30 (t, 2H), 6.18 (t, 1H), 4.08 (m, 2H), 3.05
(m, 2H), 2.22 (m, 2H), 1.90 (m, 1H), 1.68 (m, 2H), 1.52 (m, 1H),
1.40 (m, 2H), 1.30 (m, 2H). ESMS m/z: 518 (M + H)+. Anal.
(C19H25BrN4O8 ·0.25H2O) C, H, N.
1
product (10 mg, 66%) as an off-white solid. H NMR (400 MHz,
DMSO-d6) δ 12.3 (bs, 3Η), 7.5 (m, 4H), 6.30 (s, 2H), 4.1 (m, 4H),
2.89 (s, 2H), 2.23 (d, 2H), 1.90 (m, 1H), 1.65 (m, 4H), 1.32 (m,
2H). ESMS m/z: 444 (M + H)+. Anal. (C19H26ClN3O7 ·0.8TFA)
C, H, N.
(S)-2-(3-((S)-1-Carboxy-5-(2-chlorobenzylamino)pentyl)ure-
ido)pentanedioic Acid (16). Following the same procedure as
utilized in the preparation of 11, the target compound was prepared
from 4 reacted with 2-chlorobenzaldehyde to yield the desired
product (100 mg, 45%) as an off-white solid. 1H NMR (400 MHz,
DMSO-d6) δ 9.0 (br, 3H), 7.63 (d, 1H), 7.2 (m, 2H), 7.15 (d, 1H),
6.30 (d, 2H), 4.1 (m, 4H), 2.9 (br, 2H), 2.2 (m, 2H), 1.90 (m, 2H),
1.60 (m, 4H), 1.35 (m, 2H). ESMS m/z: 444 (M + H)+. Anal.
(C19H26ClN3O7 ·0.8TFA) C, H, N.
(S)-2-(3-((S)-1-Carboxy-5-(3-(4-chlorophenyl)ureido)pentyl)ure-
ido)pentanedioic Acid (22). Following the same procedure as
utilized in the preparation of 20, the target compound was prepared
from 4 reacted with 4-chlorophenyl isocyanate to afford the desired
1
product (147 mg, 51%). H NMR (400 MHz, DMSO-d6) δ 12.5
(s, 3H), 8.35 (s, 1H), 7.40 (dd, 2H), 7.19 (dd, 2H), 6.30 (t, 2H),
6.10 (t, 1H), 4.08 (m, 2H), 3.05 (m, 2H), 2.32 (m, 2H), 1.90 (m,
1H), 1.68 (m, 2H), 1.52 (m, 1H), 1.40 (m, 2H), 1.30 (m, 2H). ESMS
m/z: 474 (M + H)+. Anal. (C19H25ClN4O8 ·0.25TFA) C, H, N.
(S)-2-(3-((S)-1-Carboxy-5-(3-(4-fluorophenyl)ureido)pentyl)ure-
ido)pentanedioic Acid (23). Following the same procedure as
utilized in the preparation of 20, the target compound was prepared
from 4 reacted with 4-fluorophenyl isocyanate to afford the desired
(S)-2-(3-((S)-1-Carboxy-5-(3-chlorobenzylamino)pentyl)ure-
ido)pentanedioic Acid (17). Following the same procedure as
utilized in the preparation of 11, the target compound was prepared
from 4 reacted with 3-chlorobenzaldehyde to yield the desired
product (200 mg, 90%) as an off-white solid. 1H NMR (400 MHz,
DMSO-d6) 8.9 (br, 3H), 7.6 (s, H), 7.43 (m, 3H), 6.39 (br, 2H),
4.1 (m, 4H), 2.9 (br, 2H), 2.2 (m, 2H), 1.90 (m, 2H), 1.60 (m, 4H),
1
product. H NMR (400 MHz, DMSO-d6) δ 12.5 (s, 3H), 8.40 (s,
1H), 7.40 (dd, 2H), 7.05 (dd, 2H), 6.30 (t, 2H), 6.10 (t, 1H), 4.1
(m, 2H), 3.05 (m, 2H), 2.20 (m, 2H), 1.90 (m, 1H), 1.70 (m, 2H),
1.52 (m, 1H), 1.40 (m, 2H), 1.30 (m, 2H). HRMS (ES+) calcd for
C19H25FN4O8 (M + H)+ 457.1656, found 457.1735. Anal.
(C19H25FN4O8) C, H, N.
1.35 (m, 2H). ESMS m/z: 444 (M
(C19H26ClN3O7 ·0.8TFA) C, H, N.
+
H)+. Anal.
(S)-2-(3-((S)-1-Carboxy-5-(3-phenylureido)pentyl)ureido)pen-
tanedioic Acid (24). Following the same procedure as utilized in
the preparation of 20, the target compound was prepared from 4
reacted with phenyl isocyanate to afford the desired product (124
mg, 52%). 1H NMR (400 MHz, DMSO-d6) δ 12.5 (s, 3H), 8.54 (s,
1H), 7.40 (dd, 2H), 7.26 (dd, 2H), 6.30 (t, 2H), 6.17 (t, 1H), 4.05
(m, 2H), 3.05 (m, 2H), 2.44 (m, 2H), 1.90 (m, 1H), 1.68 (m, 2H)
1.52 (m, 1H), 1.40 (m, 2H), 1.29 (m, 2H). ESMS m/z: 439 (M +
H)+. Anal. (C19H26N4O8 ·0.2TFA) C, H, N.
(9S,13S)-1-(3-iodophenyl)-3,11-dioxo-2,4,10,12-tetraazapentade-
cane-9,13,15-tricarboxylic Acid (25). In a round-bottom flask,
triphosgene (55 mg, 0.19 mmol) was dissolved in DCM (10 mL)
and cooled to 0 °C. To the solution was added 4 (300 mg, 0.62
mmol) in DCM (50 mL) followed by TEA (0.14 mL, 1.4 mmol).
The mixture was stirred for 3 h whereupon the mixture was
evaporated to dryness and the crude mixture dissolved in methanol
(5 mL) and purified via column chromatography (SiO2) using an
increasing amount of methanol in methylene chloride. The purified
compound was treated with TFA/DCM 1:1 (1 mL) and stirred at
room temperature for 18 h. Following aqueous extraction the
compound was purified by HPLC using a Vydac C18 column
employing a gradient method of 5-50% acetonitrile containing
0.1% TFA and water containing 0.1% TFA, resulting in the desired
product (14 mg, 32%) as a yellow oil. 1H NMR (400 MHz, DMSO-
d6) δ 12.4 (bs, 3H), 7.6 (m, 2H), 7.22 (m, H), 7.12 (m, 1H), 6.31
(S)-2-(3-((S)-1-Carboxy-5-(4-fluorobenzylamino)pentyl)ure-
ido)pentanedioic Acid (18). Following the same procedure as
utilized in the preparation of 11, the target compound was prepared
from 4 reacted with 4-fluorobenzaldehyde to yield the desired
product (100 mg, 45%) as an off-white solid. The compound was
deprotected using the previously described methods. 1H NMR (400
MHz, DMSO-d6) δ 12.4 (s, 2H), 8.7 (s, 1H), 7.5 (m, 2H), 7.3 (m,
2H), 6.35 (m, 2H), 5.2 (bs, H), 4.1 (m, 3H), 3.4 (s, 4H), 2.9 (s,
1H), 2.2 (d, 2H), 1.90 (m, 1H), 1.60 (m, 4H), 1.35 (m, 2H). ESMS
m/z: 428 (M + H)+, 450 (M + Na). Anal. (C19H26FN3O7 ·3TFA)
C. Calcd H, 3.80; found 4.51. Calcd N, 5.46; found 6.12.
(S)-2-(3-((S)-5-(Benzylamino)-1-carboxypentyl)ureido)pen-
tanedioic Acid (19). Following the same procedure as utilized in
the preparation of 11, the target compound was prepared from 4
reacted with benzaldehyde to yield the desired product (5 mg, 47%)
1
as an off-white solid. H NMR (400 MHz, DMSO-d6) δ 12.4 (bs,
2H), 7.5 (m, 5H), 6.3 (m, 2H), 4.1 (m, 2H), 2.9 (s, 1H), 2.2 (d, H),
1.90 (m, 1H), 1.60 (m, 4H), 1.35 (m, 2H). ESMS m/z: 410 (M +
H)+. Anal. (C19H27N3O7 ·0.8TFA) C, H. Calcd N, 10.58; found 7.89.
(S)-2-(3-((S)-1-Carboxy-5-(3-(4-iodophenyl)ureido)pentyl)ure-
ido)pentanedioic Acid (20). In a round-bottom flask 4-iodophenyl
isocyanate (100 mg, 0.41 mmol) is dissolved in DCM (10 mL)
containing TEA (0.057 mL, 0.4 mmol). (S)-2-[3-(5-Amino-1-
carboxypentyl)ureido]pentanedioic acid di-tert-butyl ester (4)(200

Inhibitors of PSMA for Targeting Prostate Cancer
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2 355
(m, 3H), 6.01 (s, 1H), 4.15 (s, 2H), 4.06 (m, 2H), 2.95 (s, 2H),
2.22 (m, 2H), 1.93 (m, 1H), 1.75 (m, 2H), 1.52 (m, 1H), 1.42 (m,
2H), 1.31 (m, 2H). ESMS m/z: 579 (M
(C20H27IN4O8 ·0.8TFA) C, H, N.
2H), 1.92-1.75 (m, 4H), 1.71-1.15 (m, 7H), 1.35 (m, 27H, t-Bu’s),
0.23 (s, 9H). ESMS m/z: 771 (Sn cluster). Anal. (C34H58N4O8Sn)
C, H, N.
General Radiochemistry. All reactions were carried out in a
laboratory designed and licensed for use of radioactivity. All iodine
isotopes were purchased from MDS Nordion as NaI in mild sodium
hydroxide solutions or in dry form concentrated from aqueous
sodium hydroxide. All solvents (JT Baker) were purchased from
Fisher Scientific.
The radioiodinated compounds were purified on a Varian Prostar
210 HPLC system using an analytical Vydac C18 column (250
mm × 4.6 mm × 5 µm) connected to a Varian Prostar model 320
UV-visible detector monitoring at 254 nm. The final products were
purified using a binary solvent gradient of 5-50% buffer B in buffer
A over 21 min (A ) water containing 0.1% TFA, B ) acetonitrile
containing 0.1% TFA). Analytical HPLC of the compounds was
performed using the same method.
+
H)+. Anal.
(S)-2-(3-((S)-1-Carboxy-5-(4-iodophenylsulfonamido)pentyl)-
ureido)pentanedioic Acid (26). In a round-bottom flask 2-[3-(5-
amino-1-carboxypentyl)ureido]pentanedioic acid di-tert-butyl ester
(4) (300 mg, 0.62 mmol) was suspended in water (10 mL) and 1,4
dioxane (10 mL), and TEA (1.75 mL, 1.25 mmol) was added
followed by 4-iodobenzenesulfonyl chloride. The mixture stirred
overnight at 50 °C. The reaction mixture was evaporated to dryness,
taken up in DCM, and purified via column chromatography (SiO2)
using an increasing amount of methanol in methylene chloride to
afford the desired product (375 mg, 80%) as a clear oil. The purified
compound was deprotected using 1:1 TFA/methylene chloride
mixture stirring for 18 h. Following aqueous extraction, the
compound was purified by HPLC using a Vydac C18 column
employing a gradient method of 5-50% acetonitrile containing
0.1% TFA and water containing 0.1% TFA to afford the desired
product (270 mg, 90%) as an off-white solid. 1H NMR (400 MHz,
DMSO-d6) δ 7.97 (d, 2H), 7.68 (t, 1H), 7.53(d, 2H), 6.35 (dd, 2H),
4.10 (m, 1H), 4.00 (m, 1H), 2.65 (m, 2H), 2.22 (m, 2H), 1.91 (m,
1H), 1.71 (m, 1H), 1.55 (m, 1H), 1.45 (m, 1H), 1.35 (m, 2H), 1.25
(m, 2H), (3 CO2H not seen). HRMS (ES+) calcd for C18H24N3O9S
(M + H)+ 586.03, found 586.0356.
(S)-2-(3-((S)-1-Carboxy-5-(naphthalen-1-ylmethylamino)pentyl)-
ureido)pentanedioic Acid (27). Following the same procedure as
utilized in preparation of 11, the target compound was prepared
from 4 reacted with 2-naphthaldehyde to yield the desired product
(51 mg, 70%) as a light tan solid. 1H NMR (400 MHz, DMSO-d6)
8.9 (br, 3H), 7.95 (m, 5H), 7.62 (m, 2H), 6.35 (br, 2H), 4.11 (m,
4H), 2.91 (br, 2H), 2.55 (m, 2H), 2.25 (m, 2H), 1.70 (m, 4H), 1.35
(m, 2H). ESMS m/z: 460 (M + H)+. Anal. (C23H29N3O7 ·1.0TFA)
C, H, N.
Radiolabeling Procedure for the Synthesis 123I-DCIT. The
DCIT, utilized as a reference standard, was radiolabeled following
standard techniques30 employing the tyrosine precursor (S)-2-(3-
((S)-1-carboxy-2-(4-hydroxyphenyl)ethyl)ureido)pentanedioic acid
(DCT) and either Na131I or Na123I. The reactions utilized Iodogen
tubes as the source of the oxidant for the radioiodination.
Preparation of the iodine-131 labeled compound (131I-DCIT) was
achieved by adding 100 µL of Na131I in 0.1 N NaOH to a phosphate
buffered solution (pH 7.2) containing DCT (1 mg/mL) in an Iodogen
tube (Pierce). The mixture was vortexed for 3 min and allowed to
stand at room temperature for 20 min. Upon completion of the
reaction, the solution was purified by HPLC using a gradient method
of water, 0.1% TFA, and methanol, RCY > 80%, RCP > 95%).
Radiolabeling Procedure for the Synthesis 123I-(S)-2-(3-((S)-1-
carboxy-5-(4-iodobenzylamino)pentyl)ureido)pentanedioic Acid (11).
Into a 5 mL vial containing [123I]NaI (300 mCi) was added 100 µL
of sterile water for injection (SWFI), followed by 305 µL of an
acid solution [acetic acid (300 µL) and sulfuric acid (5 µL)],
followed by 300 µL of oxidant [acetic acid (0.2 mL) and 30%
hydrogen peroxide (0.335 mL) brought to a final volume of 5 mL
with SWFI], to which was added 150 µL of (S)-di-tert-butyl 2-(3-
((S)-1-tert-butoxy-1-oxo-6-(4-(trimethylstannyl)benzylamino)hexan-
2-yl)ureido)pentanedioate (29) (1 mg/mL solution in ethanol). The
mixture was vortexed for 2 min and allowed to incubate at room
temperature for an additional 30 min. The reaction was quenched
with 200 µL of 0.1 M sodium thiosulfate. The product was then
diluted in 18 mL of SWFI and loaded onto a C18 Sep Pak Plus
column. The column was washed with 60 mL of SWFI to remove
unreacted radioiodine and inorganic and organic salts. The 123I-
(S)-2-(3-((S)-1-carboxy-5-(4-iodobenzylamino)pentyl)ureido)penta-
nedioic acid (11) ester was eluted from the column using 6 mL of
ethanol. The resulting solution, containing the ester of 123I-11, was
evaporated to dryness under a stream of nitrogen and the residue
dissolved in DCM (0.5 mL). TFA (2 mL) was added to remove
the ester protecting groups. The solution was then incubated at room
temperature for 40 min. After the deprotection was complete, the
solution was evaporated to dryness under a stream of nitrogen and
the residue was dissolved in the formulation matrix of 2% gentisate
and 5% ascorbate, pH 5. The radiochemical yields ranged from
50% to 70%, RCP > 90%, specific activity of g4000 mCi/µmol.
Radiolabeling Procedure for the Synthesis 123I-(S)-2-(3-((S)-1-
Carboxy-5-(3-(4-iodophenyl)ureido)pentyl)ureido)pentanedioic Acid
(20). In a 5 mL vial containing [123I]NaI (300 mCi) was added sterile
water for injection (SWFI) (50 µL), 50% sulfuric acid in SWFI
(50 µL), oxidant (100 µL) [which was prepared fresh via the
incubation of acetic acid (0.2 mL) and 30% hydrogen peroxide
(0.335 mL) followed by dilution to a final volume of 5 mL with
SWFI], acetonitrile (0.5 mL), and (S)-di-tert-butyl 2-(3-((S)-1-tert-
butoxy-1-oxo-6-(3-(4-(trimethylstannyl)phenyl)ureido)hexan-2-yl)ure-
ido)pentanedioate (30) (100 µL of a 1 mg/mL solution in
acetonitrile). The mixture was vortexed for 1 min and allowed to
incubate at room temperature for an additional 10 min. The reaction
was quenched with 200 µL of 0.1 M sodium thiosulfate. The
product was then diluted in 18 mL of SWFI and loaded onto a
4-Trimethylstannanylbenzaldehyde (28). To a solution of 4-io-
dobenzaldehyde (1.92 g, 8.27 mmol) in dry dioxane (60 mL) was
added hexamethylditin (4.1 mL, 19.8 mmol) followed by
Pd(Ph3P)Cl2 (150 mg), and the reaction mixture was heated for
3 h under reflux until judged complete. The mixture was filtered
through Celite and purified by column chromatography (SiO2) using
hexanes/ethyl acetate (9/1) as eluent to afford the product (2.24 g,
1
98%) as a clear oil. H NMR (400 MHz, CDCl3) δ 9.97 (s, 1H),
7.81 (d, J ) 7.8 Hz, 2H), 7.72 (d, J ) 7.8 Hz, 2H), 0.29 (s, 9H).
ESMS m/z: 268 (Sn cluster).
(S)-Di-tert-butyl 2-(3-((S)-1-tert-Butoxy-1-oxo-6-(4-(trimethyl-
stannyl)benzylamino)hexan-2-yl)ureido)pentanedioate (29). Fol-
lowing the same procedure as utilized in preparation of 11, the
target compound was prepared from 4 reacted with 28 to afford
the desired product (88 mg, 38%) as a thick syrup which solidifies
1
on standing. H NMR (400 MHz, DMSO-d6) δ 7.48 (d, J ) 7.4
Hz, 2H), 7.30 (d, J ) 7.4 Hz, 2H), 6.27 (m, 2H, NH’s), 3.96 (m,
4H), 2.74 (bm, 2H), 2.21 (m, 2H), 1.87 (m, 2H), 1.65-1.19 (m,
7H), 1.35 (m, 27H, t-Bu’s), 0.23 (s, 9H). ESMS m/z: 742 (Sn
cluster). Anal. (C34H59N3O7Sn) C, H, N.
(S)-Di-tert-butyl 2-(3-((S)-1-tert-Butoxy-1-oxo-6-(3-(4-(trimeth-
ylstannyl)phenyl)ureido)hexan-2-yl)ureido)pentanedioate (30). To
a solution of tert-butyl protected 20 (227 mg, 0.31mmol) dissolved
in anhydrous dioxane (20 mL) was added hexamethylditin (256
mg, 0.78 mmol) at room temperature. Dichlorobis(triphenylphos-
pnine)palladium(II) (23 mg, 0.031mmol) was added in one portion
to the mixture, and the mixture was heated to 80 °C and stirred for
1.5 h. The reaction mixture was cooled to room temperature and
the dioxane removed in vacuo. The residue was dissolved in DCM
(25 mL) and filtered through a pad of Celite. The volume of the
filtrate was reduced and the crude product purified by column
chromatography (SiO2) utilizing ethyl acetate/hexane (9:1) as the
1
eluent to afford the desired product (94 mg, 41%). H NMR (400
MHz, DMSO-d6) δ 7.48 (d, J ) 7.4 Hz, 2H), 7.30 (d, J ) 7.4 Hz,
2H), 6.27 (m, 2H, NHs), 3.96 (m, 2H), 3.69 (bs, 2H), 2.25 (m,

356 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2
Maresca et al.
C18 Sep Pak Plus column. The column was washed with 60 mL
of SWFI to remove unreacted radioiodine and inorganic and organic
salts. The 123I-(S)-2-(3-((S)-1-carboxy-5-(3-(4-iodophenyl)ureido)-
pentyl)ureido)pentanedioic acid (20) ester was eluted from the
column using 4 mL of ethanol. The resulting solution, containing
the tri-tert-butyl ester of 123I-20, was evaporated to dryness under
a stream of nitrogen. The residue was dissolved in DCM (0.5 mL),
and TFA (2 mL) was added to cleave the tert-butyl ester protecting
groups. The deprotection solution was incubated at room temper-
ature for 45 min. After the deprotection was complete, the solution
was evaporated to dryness under a stream of nitrogen. The product
was dissolved in 50% acetonitrile/water and purified on a C18 Sep
Pak Plus column using a gradient of SWFI containing 0.1% acetic
acid and ethanol, and the product was obtained upon elution with
4 mL of 100% ethanol. The solution was evaporated to dryness
under a stream of nitrogen, and the residue was dissolved in a
formulation matrix of 2% gentisate and 5% ascorbate/ascorbic acid,
pH 5. The radiochemical yields ranged from 50% to 70%, RCP >
90%, specific activity of g4000 mCi/µmol.
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Preparation of the Ascorbate/Ascorbic Acid/Gentisate Excipi-
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