Structure-activity relationships of 2′,5′-oligoadenylate analogue modifications of prostate-specific membrane antigen (PSMA) antagonists

Article abstract of DOI:10.1080/15257770.2012.671988

□ Prostate-specific membrane antigen (PSMA) is an ideal biomarker for prostate cancer. A previously reported 2-5A conjugate RBI1033 (3) showed binding affinity more than 10 times higher than the parent urea-based compound (S)-2-(3-((S)-5-amino-1-carboxypentyl)ureido) pentanedioic acid (1). The purpose of this work is to further optimize the structure of 3 to identify highly selective ligands of PSMA. It was found that conjugates having 2-5A in their structure showed extraordinary improved binding affinity to PSMA compared with compound 1. Removal of 2-5A significantly reduced its biological activity. The results will provide a path to agents for targeted imaging and treatment of prostate cancer. Copyright Taylor and Francis Group, LLC.

Full text of DOI:10.1080/15257770.2012.671988

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Structure-Activity Relationships of 2′,5′-
Oligoadenylate Analogue Modifications
of Prostate-Specific Membrane Antigen
(PSMA) Antagonists
Xinning Wang a , Haibin Tian b , Zhenghong Lee b & Warren D. W.
Heston a
a Department of Cancer Biology, Cleveland Clinic, Cleveland, Ohio,
USA
b Department of Radiology, Case Western Reserve University,
Cleveland, Ohio, USA
Published online: 12 Apr 2012.
To cite this article: Xinning Wang , Haibin Tian , Zhenghong Lee & Warren D. W. Heston (2012):
Structure-Activity Relationships of 2′,5′-Oligoadenylate Analogue Modifications of Prostate-Specific
Membrane Antigen (PSMA) Antagonists, Nucleosides, Nucleotides and Nucleic Acids, 31:5, 432-444
To link to this article: http://dx.doi.org/10.1080/15257770.2012.671988
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Nucleosides, Nucleotides and Nucleic Acids, 31:432–444, 2012
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2012.671988
STRUCTURE-ACTIVITY RELATIONSHIPS OF 2ꢀ,5ꢀ-OLIGOADENYLATE
ANALOGUE MODIFICATIONS OF PROSTATE-SPECIFIC MEMBRANE
ANTIGEN (PSMA) ANTAGONISTS
Xinning Wang,1 Haibin Tian,2 Zhenghong Lee,2 and Warren D. W. Heston1
1Department of Cancer Biology, Cleveland Clinic, Cleveland, Ohio, USA
2Department of Radiology, Case Western Reserve University, Cleveland, Ohio, USA
Prostate-specific membrane antigen (PSMA) is an ideal biomarker for prostate cancer. A pre-
2
viously reported 2-5A conjugate RBI1033 (3) showed binding affinity more than 10 times higher
than the parent urea-based compound (S)-2-(3-((S)-5-amino-1-carboxypentyl)ureido) pentanedioic
acid (1). The purpose of this work is to further optimize the structure of 3 to identify highly selective
ligands of PSMA. It was found that conjugates having 2-5A in their structure showed extraordinary
improved binding affinity to PSMA compared with compound 1. Removal of 2-5A significantly re-
duced its biological activity. The results will provide a path to agents for targeted imaging and
treatment of prostate cancer.
Keywords PSMA; 2ꢁ, 5ꢁ-oligoadenylate conjugate; structure-activity relationship
ABBREVIATIONS
PSMA prostate-specific membrane antigen
Rt
r.t.
retention time
room temperature
SMCC succinimidyl 4-(N -maleimidomethyl)cyclohexane-1-carboxylate
2-5A
2ꢁ,5ꢁ-oligoadenylate
INTRODUCTION
According to the American Cancer Society, prostate cancer is the most
prevalent cancer in American males and represents their second leading
Received 16 September 2011; accepted 27 February 2012.
This work was supported by grants from Department of Defense (DOD) (W81XWH-07-1-0656, PI:
W. D. W. Heston; W81XWH-05-1-0600, PI: X. Wang), and in part by grant NIH/NCI U24 CA110943
(PI: J. Duerk). The authors also thank Dr. Steve Shin-lin Huang at Cleveland Clinic for help with the
manuscript.
Address correspondence to Warren D. W. Heston, Department of Cancer Biology, Cleveland Clinic,
9500 Euclid Avenue, Cleveland, OH 44195, USA. E-mail: hestonw@ccf.org.
432

PSMA Antagonists
433
cause of cancer-related death. Prostate cancer kills more than 200,000 men
annually worldwide. About 241,740 new cases of prostate cancer will be di-
agnosed, and about 28,170 men will die of the disease in the United States
in 2012.[1] Prostate-specific membrane antigen (PSMA) is a type II trans-
membrane protein consisting of 750 amino acids and having a molecular
weight of ∼110,000.[2,3] Immunohistochemical analysis of pathological spec-
imens indicates that PSMA is expressed by virtually all prostate cancers,
and its expression is further increased in poorly differentiated, metastatic,
and hormone-refractory carcinomas.[2,4,5] PSMA expression has also been
found in a variety of tumor, but not normal, vascular endothelium, which
further broadens its interest and potential applications in anti-angiogenic
therapies.[6,7] In contrast to other well-known prostate-restricted molecules
such as prostate-specific antigen (PSA) that are secretory proteins, PSMA is
a type II integral cell-surface membrane protein that is not secreted.[6] With
its abundant expression in most prostate cancers, PSMA would therefore
appear to be an ideal target for diagnosis and therapy.
The development of small molecules for targeted imaging and treat-
ment of prostate cancer has been based on the identification of enzy-
matic activity of PSMA. PSMA cleaves alpha-linked glutamates from N -
acetylaspartylglutamate.[8] It also has a unique folate hydrolase activity which
can remove the gamma-linked terminal glutamates from folate in a sequen-
tial fashion.[9] Highly potent ligands targeting PSMA have been described
recently.[10–14] To date, some of the best low weight inhibitors are highly
charged water-soluble agents which include the di-acid of glutamic acid
as part of their structure. One of the leading compounds is (S)-2-(3-((S)-5-
amino-1-carboxypentyl)ureido)pentanedioic acid (1 in Figure 1) containing
a central urea group.[11] It can be easily modified without compromising ac-
tivity through the –SH group. Selective imaging of PSMA-expressing tumor
xenografts has been reported by radiolabeling of the urea-based PSMA in-
hibitors.[15–20] Recently, a 2ꢁ,5ꢁ-oligoadenylate (2-5A) containing urea-based
analogue RBI1033 (3 in Figure 1) has been synthesized,[21] which showed
binding activity 10 times better than compound 1 itself. The purpose of
this study is to perform structure-activity relationship studies of 3 to iden-
tify highly selective ligands of PSMA that can be subsequently coupled to
imaging agents for systemic delivery to localized, primary and disseminated,
metastatic prostate tumors.
RESULTS AND DISCUSSION
Chemistry
Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC,
5 in Scheme 1) is a heterobifunctional linker. It can covalently
conjugate to amine- and sulfhydryl-containing molecules through its

434
X. Wang et al.
O
OH
OH
O
OH
OH
3H3C
HS
S
O
O
HO
HO
N
N
H
N
N
H
H
H
O
O
O
O
2
1
NH2
N
N
N
OH
N
NH2
HO P
O
O
N
N
S
N
HO
HO
O
P
S
N
NH2
N
O
O
N
N
HO
O
P
S
O
N
HO
O
O
N
O
OH
OH
S
OH
HO
O
P
S
O
O
H
N
O
HO
N
N
H
O
H
O
O
3
FIGURE 1 Structures of urea-based PSMA inhibitors 1–3.
NH2
N
O
N
N
O
NH2
N
OH
O
N
N
N
N
N
O
HO
P
O
O
OH
O
N
O
N
O
O
O
HO
P
O
O
NH
O
HO
O
O
(5)
HO P
O
O
NH2
HO
O
O
3
(i)
HO P
O
3
6
4
O
OH
HS
(ii)
O
1
HO
OH
N
H
N
H
NH2
N
O
O
N
N
OH
O
N
O
HO
P
N
O
O
OH
O
O
S
O
NH
O
HO
HO
O
HO
OH
P
N
N
H
H
3
O
O
O
O
7
SCHEME 1 Synthetic pathway of compound 7. (i) SMCC (5), 100 mM phosphate buffer, pH 7.0, r.t.
overnight; (ii) (S)-2-(3-((S)-5-amino-1-carboxypentyl)ureido)pentanedioic acid (1), 100 mM phosphate
buffer, pH 7.0, 1 hour at r.t.

PSMA Antagonists
435
N -hydroxysuccinimide (NHS) ester and maleimide group. Conjugation re-
action can be carried out either by reaction with amine group first (see
Scheme 1) or by reaction with sulfhydryl group first (see Scheme 2). Conju-
gate 7 was synthesized through reaction of 3ꢁ-C6-amine modified 2-5A trimer
4 (see Scheme 1) with excess amount of 5 in phosphate buffer to form inter-
mediate 6 first. After purification, 6 was reacted with sulfhydryl containing
compound 1 to get 7 (see Scheme 1). This method is the same as the one
used for the preparation of previously reported ligand 3.[21]
O
O
OH
N
HS
O
O
O
O
HO
OH
+
N
N
O
N
H
H
O
O
O
O
1
O
N
OH
OH
O
5
F
CH2NH
O
S
(i)
HO
N
N
H
H
O
O
O
O
O
13
N
OH
OH
F
CH2NH2
O
(iii)
O
O
S
O
N
O
O
HO
O
N
N
(iv) BocNH(CH2)2NH2
O
H
H
N
OH
O
O
NH2
N
O
O
S
N
8
BocNH(CH2)6NH
OH
OH
N
N
N
H
N
HO
O
[
HS
P
H
O
O
(ii)
O
O
14
(v)
O
O
NH2
HO
HO
O
]
P
n=1, 9
n=3, 10
n
O
S
S
NH2
O
N
OH
OH
O
N
O
HO
N
O
NH2(CH2)6NH
OH
P
N
N
N
H
N
H
N
O
O
HS
OH
OH
O
O
O
O
O
S
15
H
O
O
O
N
HO
HO
O
n
HO
N
N
P
O
H
H
O
O
n=1, 11
n=3, 12
S
O
(vi)
Br
S
N
H
F
NH2
N
N
OH
O
O
N
N
O
N
O
P
OH
OH
O
N
H
O
O
H
N
S
F
O
n=1, 16
n=3, 17
HO
HO
O
O
n
HO
N
N
P
S
O
H
H
O
O
SCHEME 2 Synthetic pathway of compounds 11–17. (i) DMF, 1 hour at r.t.; (ii) phosphorothioate
2-5A monomer (9) or trimer (10), DMF, r.t. overnight; (iii) 4-fluoro-benzylamine; (iv) N -Boc-1,6-
diaminohexane, DMF, r.t. overnight; (v) 4 M hydrochloric acid/dioxane, 1 hour at r.t.; (vi) N -(4-
fluorobenzyl)-2-bromoacetamide, methanol/phosphate buffer, pH 8.0, 120◦C, 30 minutes.
Phosphorothioate-containing conjugates 11 and 12 were prepared by
the reaction of 5 with excess amount of sulfhydryl-containing compound

436
X. Wang et al.
1 at room temperature (r.t.) for 1 hour followed by direct addition of
C6-amine modified phosphorothioate 2-5A monomer (9) or trimer (10)
(see Scheme 2). To avoid hydrolysis of NHS-ester group in 5, anhydrous
N ,N -dimethylformamide (DMF) was used as a solvent instead of phos-
phate buffer. Using the method as shown in Scheme 2, side reaction of
5ꢁ-phosphorothioate group with maleimide group of 5 was successfully pre-
vented and compounds 11 and 12 were obtained at good yield. Another
advantage of the method is that it did not require purification of the inter-
mediate reaction and therefore was more time saving.
To help investigate the importance of the 2-5A for the binding affinity,
compounds 13 and 15 omitting 2-5A part were synthesized. Both compounds
can be made by using either method as shown in Scheme 1 or 2 and no
purification is needed after first step coupling. Cleavage of the N -Boc group
in 14 was achieved by the addition of 4 M HCl in dioxane to get desired
compound 15 (see Scheme 2).
To get further evidence to support that the new ligands designed here
can be further terminal modified without losing their binding affinity, cold
compounds 16 and 17 of future 18F-radiolabeled analogues were synthe-
sized. Terminal labeling of 11 and 12 was performed by reaction with
N -(4-fluorobenzyl)-2-bromoacetamide (see Scheme 2). This is a common
method used to label oligonucleotides. The reaction happened quickly at
120◦C and gave 16 or 17 as the highly predominant product. Compared
with 11 and 12, compounds 16 and 17 have longer retention time (Rt)
due to increased lipophilicity following the addition of the halogenobenzyl
substitution.
Competitive Binding Studies
All new compounds synthesized were evaluated in a competitive bind-
ing assay using tritium labeled S-methylated derivative of 1 (N -[N -[(S)-1,3-
dicarboxypropyl]carbamoyl]-S-[3H]-methyl-l-cysteine, 2, in Figure 1) as the
radioligand to examine the ability of the novel compounds to compete bind-
ing with 2 on PSMA-expressing LNCaP cells. Parent compound 1 was used
as positive control. The results are summarized in Table 1.
Previously, we have reported that inclusion of a C3-amine modified 2-5A
trimer into 1 (compound 3) improved its binding affinity 10 times.[21] Here
we confirmed the observation by preparation of a series of analogues of
compound 3. In all the newly synthesized conjugates, a C6 linker was used to
replace the original C3 linker in compound 3 because our initial study found
that extension of the linker from C3 (compound 3) to C6 (compound 12)
improved the binding activity more than 100-fold. Our study showed that
compounds with 2-5A in their structures (compounds 7, 11, 12, 16, and 17)
had significantly improved binding activity compared with those omitting
2-5A (compounds 13 and 15). These results indicated that the integration

PSMA Antagonists
437
TABLE 1 Summary of competitive binding results of new conjugates. IC50 is
the concentration required to inhibit the binding of 3H-ZJ24 (2) by 50%.
Values are mean SD of three replicates
Compounds
IC50 (nM)
1
3
13.9 0.12
1.5 0.12
7
1.58 0.26
0.11 0.06
0.012 0.003
96.2 5.4
163.1 6.2
1.93 0.11
3.36 0.15
11
12
13
15
16
17
of 2-5A plays a crucial role in PSMA binding, and that part cannot be elim-
inated from structure. It was also found that the binding affinity increased
when the number of 2-5A increased from one (compound 11) to three (com-
pound 12). Another interesting finding was that free 5ꢁ-phosphorothioate
contributed to the activity greatly; replacement of 5ꢁ-phosphorothioate
group (12) with phosphate group (7) or substitution of the free 5ꢁ-
thioate group in 11 and 12 decreased their binding activity (16 and 17)
dramatically.
The crystal structure of PSMA has been well elucidated.[22–29] The active
site contains two zinc atoms, a smaller pharmacophore binding site (S1ꢁ) and
a larger nonpharmacophore pocket (S1). According to this elucidated crys-
tal structure of PSMA, the glutamate portion (P1ꢁ) of our compounds should
be undoubtedly located in the S1ꢁ pocket of PSMA, while the remainder of
the molecule (P1) should be oriented in the S1 pocket. The S1 pocket con-
˚
tains a ∼20 A deep tunnel that narrows gradually. Extension of C3 (3) to C6
(12) might have optimized the steric hindrance, thus leading to better fit and
binding characteristic. Similar observations have been reported by Kularatne
et al. recently.[30] They included an 8-aminooctanoic acid spacer in their lig-
ands and found that the linker improved binding potency more than 10-fold.
The S1 pocket of PSMA has an adjacent hydrophobic pocket and it is arginine
rich. It has been reported that introduction of a phenyl group can improve
the binding activity.[30,31] Similar to a phenyl group, an adenine group from
2-5A can form hydrophobic binding with the pocket. On the other side,
the negatively charged phosphorothioate groups of 2-5A can form strong
ion pairs with the positively charged guanidine groups of arginine. These
two factors working together offer the 2-5A containing compounds excel-
lent affinity. At this time, it is still not clear how the 5ꢁ-terminal phosphoro-
thioate affects the binding. One hypothesis is that it might interact with the
sulfhydryl group of cysteines in PSMA and form S–S bond, thus increasing

438
X. Wang et al.
its binding affinity. Our future work will be to perform docking studies to
help understand the binding characteristics of our conjugates.
As mentioned before, prostate cancer is the leading cancer in men and
the second most common cause of cancer-related death. Furthermore, by
the time of diagnosis, only half of the tumors are clinically localized and
half of these represent extracapsular spread. Sensitive, functional imaging
techniques are required to identify the extent of disease prior to therapy.
PSMA provides an excellent target for prostate tumor imaging and therapy
because of its transmembrane location and the fact that it is significantly
upregulated in prostate cancer metastasis relative to the few normal tissues
in which it resides at least 100-fold lower levels. Compounds 11 and 12 con-
tain a 5ꢁ-terminal phosphorothioate ester group that is active in nucleophilic
reaction. N -(4-fluorobenzyl)-2-bromoacetamide is suitable as the carrier of a
positron emitter to label phosphorothioate.[32] This terminal labeling offers
minimal structural perturbation, and more importantly, protects 2-5A from
terminal enzymatic degradation due to various exonuclease in vivo. After la-
beling, the corresponding cold compounds (16 and 17) lost 10- to 100-fold
of activity compared with unlabeled 11 and 12, but still had binding activ-
ities much better than parent compound 1. Parent compound 1 has been
reported to have a Ki at 1 nM in inhibiting PSMA’s enzymatic activity. Other
groups have tried N -[N -(S)-1,3-dicarboxypropyl]-S-[11C]methyl-l-cysteine
([11C]-DCMC, Ki = 3.1 nM),[18] N -[N -(S)-1,3-dicarboxypropyl]carbamoyl]-
4-[18F]fluorobenzyl-l-cysteine ([18F]-DCFBCA, IC50 = 13.9 nM),[20] and
123I-labled glutamtate-urea-lysine analogues ([123I]MIP-702, Ki = 4.6 nM;
[
123I]MIP-1095, Ki = 0.24 nM).[19] They all showed selectively high uptake
on PSMA-expressing tumor xenografts. Our compounds resemble part of
their structures and have better or comparable binding affinity to PSMA. In
in vitro cytotoxicity assay, none of the compounds showed any toxic effect
to LNCaP cells, which indicated their low toxicity (data not shown). These
facts together suggested that the future of using these ligands for imaging
should be very promising.
CONCLUSIONS
In summary, a series of novel urea-based PSMA ligand containing 4-
(N -maleimidomethyl)cyclohexane-1-amidate linkage has been synthesized.
Integration of 2-5A into the structure reveals outstanding activity in com-
petitive binding assay. Compared to antibodies, these conjugates can be
easily synthesized and are more cost effective. More importantly, as small
molecules, they should have favorable pharmacokinetic behaviors, such as
fast uptake and quick clearance. The results reported in this article provide
supporting evidence for future 18-F radiolabeling as potential diagnostic
imaging agents by targeting PSMA.

PSMA Antagonists
439
EXPERIMENTAL
General Methods
(S)-2-(3-((S)-5-amino-1-carboxypentyl)ureido)pentanedioic acid (1) was
custom made by Bachem Bioscience Inc. (Torrance, CA, USA) 3ꢁ-C6-
amine modified 2ꢁ,5ꢁ-oligoadenylate trimer{[(2R,5R)-5-(6-amino-9H-purin-
9-yl)-4-[({[2R,5R-5-(6-amino-9H-purin-yl)-4-[({[2R,5R-5-(6-amino-9H-purin-
yl)-4-({[(6-aminohexyl)oxy](hydroxy) phosphoryl} oxy)-3-hydroxyoxolan-
2-yl]-methoxy}(hydroxy)phosphoryl)oxy]-3-hydroxyoxolan-2-yl]methoxy}
(hydroxy) phosphoryl)oxy]-3-hydroxyoxolan-2-yl]methoxy}phosphonic acid
(4), phosphorothioate 2ꢁ,5ꢁ-oligoadenylate monomer thiophosphoric acid
O-(6-amino-hexyl) ester Oꢁ-[2-(6-amino-purin-9-yl)-4-hydroxy-5-thiophos
phonooxymethyl-tetrahydro-furan-3-yl] ester (9), and {[(2R,5R)-5-(6-
amino-9H-purin-9-yl)-4-[({[(2R,5R)-5-
(6-amino-9H-purin-9-yl)-4-[({[(2R,
5R)-5-(6-amino-9H-purin-9-yl)-4-({[(6-amino-hexyl)-oxy](hydroxy)sulfany
lidene-phosphanyl}oxy)-3-hydroxyoxolan-2-yl]methoxy} (hydroxy)sulfany
lidene-phosphanyl)oxy]-3-hydroxyoxolan-2-yl]methoxy}(hydroxy) sulfanyli
dene-phosphanyl)oxy]-3-hydroxyoxolan-2-yl]methoxy}(sulfanyl)phosphinic
acid (10) were custom synthesized by GeneACT Inc., Fukuoka, Japan.
Compound 2 was custom made by GE Healthcare Life Sciences (Piscat-
away, NJ, USA). SMCC was purchased from Pierce (Rockford, IL, USA).
N -succimimidyl 4-fluorobenzoate was ordered from ABX Advanced Bio-
chemical Compounds, Radeberg, Germany. All the other chemicals were
purchased from Sigma-Aldrich Inc., St. Louise, MO, USA. Proton nuclear
magnetic resonance (1H NMR) spectroscopy was obtained from a Varian
Inova NMR Spectrometer (Darmstadt, Germany) at a proton frequency
of 600 MHz. Deuterated chloroform was used as a solvent for obtaining
NMR spectra. High-resolution matrix-assisted laser desorption/ionization
mass (MALDI-MS) spectra were obtained from an Applied Biosystems 4800
MALDI TOF/TOF Analyzer (Carlsbad, CA, USA) in positive ion mode.
High-Performance Liquid Chromatography (HPLC)
All reactions were monitored by HPLC, which was performed on a Shi-
madzu HPLC system (Columbia, MD, USA) equipped with a SPD-20V promi-
nence UV/visible detector and monitored at a wavelength at 260 nm. The
gradient used was 0%–55% B over 45 minutes (A was 10 mM triethylam-
monium acetate, TEAA, pH 7.0; B was acetonitrile). Preparative HPLC was
achieved using SymmetryPrep C18 column (100 mm × 19 mm × 5 µm, Wa-
ters Corporation, Milford, MA, USA) at a flow rate of 3.0 mL/minute. Ana-
lytical HPLC was performed using an analytical Symmetry C18 column (150
mm × 4.6 mm × 5 µm, Waters Corporation) at a flow rate of 1 mL/minute.
Synthesis of {[(2R,5R)-5-(6-amino-9H-purin-9-yl)-4-[({[(2R,5R)-5-(6-
amino-9H-purin-9-yl)-4-[({[(2R,5R)-5-(6-amino-9H-purin-9-yl)-4-[({[6-({4-[(2,

440
X. Wang et al.
5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl]cyclohexyl}formamido)hexyl]
oxy}(hydroxy)phosphoryl)-oxy]-3-hydroxyoxolan-2-yl]methoxy}(hydroxy)
phosphoryl)oxy]-3-hydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)oxy]-
3-hydroxyoxolan-2-yl]methoxy}phosphonic acid (6). To a solution of
3ꢁ-C6-amine modified 2-5A trimer 4 (900 µg, 0.8 µmol) in 100 uL of 100 mM
phosphate buffer (pH 7.0) was added 10.7 mg of SMCC (5, 32 µmol) in
1 mL of 100 mM phosphate buffer (pH 7.0). After stirring at r.t. overnight,
the mixture was purified by preparative HPLC to obtain the desired product
at 72.8% yield (815 µg). Rt = 23.5 minutes. C48H65N17O25P4, MALDI-MS
m/z: 1404 (M+1).
Synthesis of 2-[({2-[(1-{[4-({6-[({[(2R,5R)-2-(6-amino-9H-purin-9-yl)-5-
{[({[(2R, 5R)-2-(6-amino-9H-purin-9-yl)-5-{[({[(2R,5R)-2-(6-amino-9H-purin-
9-yl)-4-hydroxy-5-[(phosphonooxy)methyl]oxolan-3-yl]oxy}(hydroxy)
phosphoryl)oxy]methyl}-4-hy-droxyoxolan-3-yl]oxy}(hydroxy)phosphoryl)
oxy]methyl}-4-hydroxyoxolan-3-yl]oxy} (hydroxy)phosphoryl)oxy]hexyl}car-
bamoyl)cyclohexyl]-methyl}-2,5-dioxopyrrolidin-3-yl)sulfanyl]-1-carboxy-
ethyl}carbamoyl)amino] pentanedioic acid (7). To a solution of 6 (300 µg,
0.2 µmol) in 400 µL of 100 mM phosphate buffer (pH 7.0) was added 500
µg of 1 (1.0 µmol). After stirring at r.t. for 1 hour, the mixture was purified
by preparative HPLC to get the final product at 89.8% yield (300 µg). Rt =
17.5 minutes. C57H79N19O32P4S, MALDI-MS m/z: 1698 (M+1).
Synthesis of 2-[3-(2-{1-[4-(6-{[2-(6-Amino-purin-9-yl)-4-hydroxy-5-thio-
phos-phonooxymethyl-tetrahydro-furan-3-yloxy]-hydroxy-thiophosphoryl-
oxy}hexylcarbamoyl)-cyclohexylmethyl]-2,5-dioxo-pyrrolidin-3-ylsulfanyl}-1-
carboxyethyl)ureido]-pentanedioic acid (11). SMCC 10 mg (0.03 mmol)
was dissolved in 500 uL DMF, then 17 mg of 1 (0.06 mmol) in 500 uL DMF
was added. After stirring at r.t. for 1 hour, 0.2 mg of 3ꢁ-C6-amine modified
phosphorothioate 2ꢁ,5ꢁ-oligoadenylate monomer 9 (0.0035 mmol) was
added. The mixture was stirred at r.t. overnight, then applied to preparative
HPLC to give the purified product at 90% yield (0.35 mg). Rt = 13.4
minutes. C37H55N9O18P2S3, MALDI-MS m/z: 1072 (M+1).
Synthesis of 2-[({2-[(1-{[4-({6-[({[(2R,5R)-2-(6-amino-9H-purin-9-yl)-5-
{[({[(2R,5R)-2-(6-amino-9H-purin-9-yl)-5-{[({[(2R,5R)-2-(6-amino-9H-purin-
9-yl)-4-hydroxy-5-({[hydroxy(sulfanyl)phosphoryl]oxy}methyl)oxolan-3-yl]
oxy} (hydroxy)sulfanylidene-phosphanyl)oxy]methyl}-4-hydroxyoxolan-3-yl]
oxy} (hydroxy)-sulfanylidene-phosphanyl)oxy]methyl}-4-hydroxyoxolan-
3-yl]oxy} (hydroxy) sulfanylidene-phosphanyl)oxy]hexyl}carbamoyl)
cyclohexyl]methyl}-2,5-dioxopyrrolidin-3-yl)sulfanyl]-1-carboxyethyl}
carbamoyl)amino]pentanedioic acid (12). The compound was synthesized
using the same method as 11 at a yield of 83%. Rt = 13.9 minutes.
C57H79N19O28P4S5, MALDI-MS m/z: 1762 (M+1).

PSMA Antagonists
441
Synthesis of 2-(3-{1-Carboxy-2-[1-(4-{[(4-fluoro-benzylamino)-methyl]-
carbamoyl}-cyclohexyl)-2,5-dioxo-pyrrolidin-3-ylsulfanyl]-ethyl}-ureido)-
pentanedioic acid (13). The compound was synthesized using the same
method as 11. Rt = 22.3 minutes. MALDI MS m/z: 639 (M+1).
Synthesis of 2-[3-(2-{1-[4-(6-tert-Butoxycarbonylamino-hexylcarbamoyl)-
cyclohexyl]-2,5-dioxo-pyrrolidin-3-ylsulfanyl}-1-carboxy-ethyl)-ureido]
pentanedioic acid (14). SMCC (1 mg, 0.003 mmol) was dissolved in 0.5
mL DMF, and then 1.7 mg of 1 (0.006 mmol) in 0.5 mL DMF was added.
After stirring at r.t. for 1 hour, N -Boc-1,6-diaminohexane (1.3 µL, 0.006
mmol) was added. The mixture was stirred at r.t. overnight, then applied to
preparative HPLC to get purified product with a yield of 84% (1.8 mg). Rt
= 22.1 minutes. C32H51N5O12S, MALDI-MS m/z: 752 (M+Na); 768 (M+K).
Synthesis of 6-{[4-(3-{2-Carboxy-2-[3-(1,3-dicarboxy-propyl)-ureido]-
ethylsulfanyl}-2,5-dioxo-pyrrolidin-1-yl)-cyclohexanecarbonyl]-amino}-hexyl
ammonium chloride (15). One milligram of 14 (0.0014 mmol) was dissolved
in 1 mL of 4 M HCl/dioxane. After stirring at r.t. for 1 hour, the solvent
was removed to give the dry product with a yield of 97% (0.9 mg). Rt = 13.5
minutes. C27H44ClN5O10S, MALDI-MS m/z: 666 (M+1).
Synthesis of N-(4-fluorobenzyl)-2-bromoacetamide.[32] To a solution con-
taining 4-fluorobenzylamine (1.25 g, 10 mmol) and N -methylmorpholine
(1.0 g, 10 mmol) in 25 mL of dichloromethane was added bromoacetyl bro-
mide (2.0 g, 20 mmol). After addition, the reaction mixture was stirred at
r.t. overnight. The mixture was then filtered and concentrated to dryness,
and the residue was purified on a silica column. Elution with hexane/ethyl
1
acetate (9:1) gave the pure product as light yellow solid (2.1 g, 87.5%). H
NMR (CDCl3) δ 7.25 (d, 2H), 7.04 (d, 2H), 4.45 (d, 2H), 3.93 (s, 2H).
Synthesis of 2-[({2-[(1-{[4-({6-[({[(2R,5R)-2-(6-amino-9H-purin-9-yl)-
5-[({[({[(4-fluorophenyl)methyl]carbamoyl}methyl)sulfanyl](hydroxy)
phosphoryl}oxy) methyl]-4-hydroxyoxolan-3-yl]oxy}(hydroxy)sulfanylidene-
phosphanyl)oxy]hexyl} carbamoyl)cyclohexyl]methyl}-2,5-dioxopyrrolidin-
3-yl)sulfanyl]-1-carboxyethyl} carbamoyl)amino]pentanedioic acid (16).[32]
To a solution of 11 (200 µg, 0.19 µmol) in 1 mL of methanol-phosphate
buffer (100 mM, pH 8.0) was added excess amount of N -(4-fluorobenzyl)-
2-bromoacetamide (240 µg, 0.95 µmol). The mixture was stirred at
120◦C for 30 minutes, then the solvent was evaporated and the products
were purified by preparative HPLC (214 µg, 91.3%). Rt = 27.5 minutes.
C46H63FN10O19P2S3, MALDI-MS m/z: 1237 (M+1).
Synthesis of 2-{[(1-carboxy-2-{[1-(2-methylbutyl)-2,5-dioxopyrrolidin-3-
yl]sulfanyl}ethyl)carbamoyl]amino}pentanedioic acid; {[(2R,5R)-5-(6-amino-
9H-purin-9-yl)-4-[({[(2R,5R)-5-(6-amino-9H-purin-9-yl)-4-[({[(2R,5R)-5-(6-
amino-9H-purin-9-yl)-3-hydroxy-4-{[hydroxy(sulfanylidene)-phosphanyl]oxy}
oxolan-2-yl] methoxy}(hydroxy)sulfanylidene-phosphanyl)oxy]-3-
hydroxyoxolan-2-yl]methoxy} (hydroxy)-sulfanylidene-phosphanyl)oxy]-

442
X. Wang et al.
3-hydroxyoxolan-2- yl]methoxy}[({[(4- fluorophenyl)-methyl]-carbamoyl}
methyl)sulfanyl]phosphinic acid (17). The compound was synthesized
using the same method as 16 with a yield at 86.3%. Rt = 11.6 minutes.
C66H87FN20O29P4S5, MALDI-MS m/z: 1927 (M+1).
Cell Culture
Prostate cancer cell line LNCaP was obtained from American Type Cul-
ture Collection (ATCC, Manassas, VA, USA). LNCaP cells were grown at
37◦C and 5% CO2 under a humidified atmosphere. Cells were maintained
in RPMI1640 medium supplemented with 2 mM l-glutamine and 10% fetal
bovin serum.
Competitive Binding Assay
Briefly, LNCaP cells (5 × 105) were incubated with different concentra-
tions of ligands in the presence of 12 nM 2 in a total volume of 300 µL for
1 hour at 37◦C. The mixture was centrifuged at 3000 g for 5 minutes at 4◦C,
then washed three time with 500 µL of cold PBS. Finally, 4 mL of EcoLume
cocktail (MP Biomedicals, Solon, OH, USA) was added, and radioactivity
was counted by scintillation counter. The concentration required to inhibit
50% of binding is determined (IC50) by GraphPad Prism 3.0.
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