Design and synthesis of a siderophore conjugate as a potent PSMA inhibitor and potential diagnostic agent for prostate cancer

Article abstract of DOI:10.1016/j.bmc.2007.11.030

A siderophore conjugate was designed as a potential PSMA inhibitor and diagnostic agent for prostate cancer. A semi-rigid spacer was incorporated to avoid competitive participation of iron binding by the enzyme inhibitor relative to the siderophore component. Biological test results showed that, even with the extended scaffold, this compound is a potent PSMA inhibitor with an IC₅₀ of 4 nM. This siderophore conjugate may be useful for detection of prostate-derived cancer cells by magnetic resonance imaging (MRI).

Full text of DOI:10.1016/j.bmc.2007.11.030

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 1648–1657
Design and synthesis of a siderophore conjugate as a potent
PSMA inhibitor and potential diagnostic agent for prostate cancer
Pingyu Ding,a Paul Helquista and Marvin J. Millera,b,
*
a251 Nieuwland Science Hall, Department of Chemistry and Biochemistry and Notre Dame Cancer Research Center,
University of Notre Dame, Notre Dame, IN 46556, USA
bLeibniz-Institute for Natural Product Research and Infection Biology, Hans Kno¨ll Institute,
Beutenbergstrasse 11a, 07745 Jena, Germany
Received 8 October 2007; revised 8 November 2007; accepted 9 November 2007
Available online 17 November 2007
Abstract—A siderophore conjugate was designed as a potential PSMA inhibitor and diagnostic agent for prostate cancer. A semi-
rigid spacer was incorporated to avoid competitive participation of iron binding by the enzyme inhibitor relative to the siderophore
component. Biological test results showed that, even with the extended scaffold, this compound is a potent PSMA inhibitor with an
IC50 of 4 nM. This siderophore conjugate may be useful for detection of prostate-derived cancer cells by magnetic resonance imag-
ing (MRI).
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
extensive structure–activity relationship (SAR) studies
have been carried out and some potent inhibitors of
NAALADase, such as urea-based compounds5 and
GPI5232,6 were identified. Recent studies on the two
enantiomers of 2-PMPA and GPI5232 have clearly dem-
onstrated that potent inhibition of NAALADase is spe-
cific to the (S)-enantiomer, which has an absolute
configuration corresponding to L-glutamate.7 The high
potency of these phosphinate-based inhibitors can be
attributed to the strong chelation of the phosphonate
group to an active site zinc ion as well as to the interac-
tion of the glutamate portion of the inhibitor with the
glutamate recognition site of NAALADase.
In the brain, NAALADase (N-acetylated a-linked acidic
dipeptidase) releases N-acetyl aspartate and glutamate
from both the neuronal peptide N-acetylaspartylgluta-
mate (NAAG) and folate polyglutamate.1 NAAG is
the most abundant and widely distributed peptide trans-
mitter in the mammalian nervous system, and NAALA-
Dase is believed to play a key role in modulating the
release of glutamate. As a therapeutic target, NAALA-
Dase inhibition has been suggested to have potential
benefits over the existing receptor-based strategies be-
cause it represents an upstream mechanism for regula-
tion of synaptic glutamate that could reduce
transmission at a number of glutamatergic receptors
rather than inhibiting a single receptor subtype.2 There-
fore, NAALADase represents an intriguing target for
drug discovery aimed at unmet medical needs.3
A protein having NAALADase activity is also expressed
in the human prostate parenchyma, from where it was
first cloned and named prostate-specific membrane anti-
gen (PSMA).8 PSMA is a 110 kDa type II transmem-
brane protein that is expressed by prostate tumor cells
and as such is a clear cellular target for the development
of methods for reliable diagnosis of prostate cancer and
for selective drug delivery.8c–g The X-ray crystal struc-
ture of PSMA has been elucidated and its catalytic
mechanism has been proposed.9 PSMA is highly homol-
ogous to NAALADase, and inhibitors of NAALADase
also strongly bind to PSMA. For example, phosphinate-
based inhibitor VA-033 (2, IC50 = 12.5 nM) was found
to serve as a potent inhibitor of NAALADase activity
2-PMPA (1) is a potent and selective NAALADase
inhibitor and has been shown to provide significant pro-
tection against injury in rats after transient middle cere-
bral artery occlusion.4 Using 2-PMPA as a template,
Keywords: PSMA; NAALADase; Siderophore conjugate; Inhibitor.
*
Corresponding author. Tel.: +1 574 631 7058; fax: +1 574 631
6652; e-mail: mmiller1@nd.edu
0968-0896/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.11.030

P. Ding et al. / Bioorg. Med. Chem. 16 (2008) 1648–1657
1649
associated with PSMA that is expressed on LNCaP hu-
man prostate cancer cells and by tumor cells in vivo.10
The structurally similar phenylalkylphosphonamidates
had been reported by Berkman’s group as potent PSMA
inhibitors.11 Very recently, we reported some enantio-
merically pure phosphinate-based analogues 3 as potent
NAALADase inhibitors.12 In the next stage of our
investigations, we wished to design a suitably labeled
PSMA inhibitor that could be employed as a possible
diagnostic agent for prostate cancer cells.
ylate groups of the inhibitor may displace one of the
hydroxamate groups of the siderophore to chelate phys-
iological Fe(III) or added Gd(III). Presumably, this
interaction may impair the ability of the inhibitor to
bind to the active site of PSMA. Related competitive
binding interactions have been documented in sidero-
phore analogues containing catechol-salicylate moie-
ties.14 We therefore considered using different linker
groups to further restrict the conformations of the ana-
logues, and incorporation of linker 5 was anticipated to
allow metal chelation of the siderophore portion while
allowing the glutamate-derived hydroxyphosphinyl por-
tion to remain free to bind to the PSMA active site. The
proposed compound (6) may therefore be useful for
detection of prostate-derived cells by MRI if inclusion
of the extended linker would not have a detrimental ef-
fect on normal PSMA binding. Herein, we describe the
synthesis of 6 and assay results to indicate that com-
pounds of this type can be potent PSMA inhibitors
(Figs. 1 and 2).
As a labeling strategy, we chose to employ a sidero-
phore. Such compounds are well known to form highly
stable complexes with Fe(III) and Gd(III), and the
resulting metal complexes can be detected by magnetic
resonance imaging (MRI).13 We and others have re-
ported examples of Fe and Gd binders directed toward
prostate cancer detection.12 Our initial compound was
an analogue of 3 with R being a form of siderophore
4.12a However, closer consideration of its structural fea-
tures indicated that this type of analogue is subject to
conformational effects on activity. In particular, we
hypothesized that a longer, rigidified linker was needed
between the PSMA binding region and the siderophore
region to avoid interfering interactions between these
two groups. The risk is that otherwise one of the carbox-
2. Results and discussion
The retrosynthetic analysis of conjugate 6 is shown in
Scheme 1. Compound 6 could be assembled from the
CO2H
CO2H
CO2H
CO2H
CO2H
O
P
OH
O
P
OH
O
P
OH
n
( )
O
O
R
HO
CO2H
3
2 (VA-033)
1 (2-PMPA)
Figure 1. Inhibitors of NAALADase and PSMA.
O
N
O
OH
O
H
N
Ac
OH
N
N
H
H
O
O
3
5
4
O
N
OH
CO2H
O
P
OH
O
H
N
Ac
O
CO2H
N
H
O
H
N
O
3
N
H
O
6
Figure 2. Proposed siderophore-including inhibitor 6, its precursor siderophore component 4, and linker 5.

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P. Ding et al. / Bioorg. Med. Chem. 16 (2008) 1648–1657
O
N
OH
CO2H
CO2H
O
O
H
N
P
Ac
O
N
H
O
OH
H
N
O
3
N
H
O
6
O
N
O
OH
CO2H
CO2H
O
H2N
H2N
H2N
O
OH
OH
Ac
OH
3
P
N
H
O
OH
O
O
A
B
C
D
Scheme 1. Retrosynthetic analysis of proposed conjugate 6.
following four fragments: tripeptide component A, ami-
no acid component B, glycine C, and phosphonate D.
tripeptide methyl ester 10 in 85% yield. Saponification
of the methyl ester gave tripeptide acid 11 in quantita-
tive yield. Again, 13C NMR revealed three sharp singlets
at 53.56 ppm, 52.51 ppm, and 52.04 ppm assigned to the
three stereogenic methine carbons, indicating that, with-
in the limits of detection, only one diastereomer was
produced (Scheme 2).
The synthesis began with the removal of the Cbz group
of fully protected amino acid 715 by treatment with 33%
HBr/acetic acid. The free amine was released from the
resulting HBr salt and coupled to dipeptide acid 816 to
produce tripeptide methyl ester 9 in 90% yield. 13C
NMR analysis of tripeptide 9 indicated three sharp sig-
nals at 53.36 ppm, 52.38 ppm, and 51.43 ppm for the
stereogenic methine carbons, supporting the formation
of a single diastereomer. Removal of the Cbz group with
33% HBr/acetic acid and subsequent acetylation gave
Several approaches were tried to prepare the linker.
Since the linker includes carbonyl, carboxylic acid, and
free amino groups, caution had to be taken to choose
suitable protecting groups for these functional groups.
The successful route is summarized in Scheme 3. Ethyl
O
N
OBn
OBn
OBn
N
O
O
N
O
8
Cbz
N
OH
2
N
H
OBn
HBr-HOAc
O
.
HOAt, EDCI, DIPEA
90%
CbzHN
CO2Me
H2N
O
CO2Me
Cbz
HBr
OMe
3
N
H
7
O
9
O
N
N
OBn
OBn
LiOH, THF-H2O
100%
1. HBr-HOAc, CH2Cl2
2. Ac2O, Pyr., CH2Cl2
85%
Ac
Ac
OMe
3
OH
N
H
N
H
O
O
3
10
11
Scheme 2. Synthesis of tripeptide 11.

P. Ding et al. / Bioorg. Med. Chem. 16 (2008) 1648–1657
1651
O
O
EtOH (H+)
98%
(CH2OH)2, PTSA (cat.)
HO2C
EtO2C
HO
benzene
Br
Br
Br
12
13
O
TBSCl, Imid.
O
O
LiAlH4, 0
ºC/THF
O
96%
EtO2C
TBSO
TBSO
65%
(two steps)
Br
14
15
O
O
O
O
O
NaClO-H2O2-NaH2PO4
1.
n
-BuLi, -78 ºC
TBSO
95%
2. DMF
90%
CHO
Br
17
16
O
O
O
CH2N2, Et2O
93%
TBSO
TBAF, THF
82%
CO2H
CO2Me
O
18
19
O
O
O
1. MsCl, Pyr., CH2Cl2
HO
N3
2. NaN3, DMF
82%
CO2Me
CO2Me
20
21
O
O
HS(CH2)3SH, Et3N
H2N
MeOH
92%
CO2Me
22
Scheme 3. Synthesis of linker 22.
ester 13 was obtained in 97% yield by heating a solu-
tion of 12 at reflux in ethanol in the presence of a cat-
alytic amount of H2SO4.17 Treatment of 13 with
ethylene glycol in the presence of a catalytic amount
of p-toluenesulfonic acid (PTSA) gave ketal 14 as an
inseparable mixture with some unconsumed starting
material 13. The mixture was then subjected to reduc-
tion by 2 equiv of LiAlH4 at 0 °C in THF. The result-
ing alcohol 15 was isolated in 65% overall yield for the
two steps. Alcohol 15 was then protected with a tert-
butyldimethylsilyl (TBS) group to afford 16 in 96%
yield. Upon treatment of 16 with 1.1 equiv of n-BuLi
in THF at ꢀ78 °C for 30 min, the resulting aryllithium
was quenched with DMF to give aldehyde 17 in 90%
isolated yield. Oxidation with sodium chlorite—hydro-
gen peroxide afforded acid 18 in 90% yield.18 Next,
methylation with an ethereal solution of diazomethane
provided methyl ester 19 in 93% yield.19 After removal
of the TBDMS protecting group by TBAF in THF,
alcohol 20 was obtained in 82% yield. Azide 21 was
prepared from alcohol 20 via its mesylate in 82%
yield. Reduction of azide 21 proved to be difficult.
We first tried to use Ph3P/H2O or Pd–C/H2,20 but
both methods led to the formation of complicated
product mixtures. In the end, HS(CH2)3SH was used
as a reducing reagent in the presence of Et3N to pro-
duce free amine 22 in 92% yield.21
Free amine 22 was coupled to tripeptide acid 11 to give
23 in 89% yield (Scheme 4). In the next deprotection
step, we first used p-toluenesulfonic acid (PTSA) in
wet acetone,22 but the reaction was incomplete after
heating at reflux overnight, and isolation of the product
from the unconsumed starting material proved to be dif-
ficult. Alternatively, when HOAc–THF–H2O (v/v/v,
3:1:1) was used,23 the reaction was incomplete even
when the reaction mixture was heated at 70 °C for
24 h, and the increased reaction time led to some decom-
position. We finally succeeded with the deprotection by
using neat formic acid to obtain ketone 24 in 91%
yield.24 Subsequent hydrolysis readily provided acid 25
(Scheme 5).
Removal of the N-Boc group of 2612 provided the free
amine, which was used immediately in a coupling reac-
tion with acid 25 to provide the final precursor 27.

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P. Ding et al. / Bioorg. Med. Chem. 16 (2008) 1648–1657
O
N
OBn
HOAt, EDCI, DIPEA
89%
HCO2H
91%
22
11
+
O
H
N
O
Ac
N
H
O
3
CO2Me
23
O
O
N
N
OBn
OBn
O
O
LiOH, THF-H2O
90%
H
N
H
N
Ac
Ac
N
N
H
H
O
O
3
3
CO2Me
CO2H
25
24
Scheme 4.
CO2Bn
CO2Bn
1. TFA
2. Na2CO3
H
N
BocHN
O
P
OBn
O
25, HOAt, EDCI, DIPEA,
O
77%
26
O
N
OBn
CO2Bn
O
O
H
N
P
Ac
N
Pd-C, H2
98%
O
CO2Bn
6
O
OBn
H
H
N
O
3
N
H
O
27
Scheme 5.
The last global deprotection provided the target mole-
cule 6 in 98% yield.
phore component, this compound revealed outstanding
activity in a biological assay. This observation has dem-
onstrated that a large, added component such as tripep-
tide 4 is well tolerated upon conjugation with the active
phosphonic acid derivative 3. MRI experimental studies
of iron and gadolinium complexes of 6 and related ana-
logues will test the concept of selective detection of pros-
tate cancer cells through use of magnetic resonance
imaging of suitably functionalized PSMA binding agents.
The results will facilitate future design of additional ana-
logues as diagnostic agents for prostate cancer and, by
logical extension, as therapeutic agents for selective drug
delivery.
The in vitro NAALADase inhibitory potency of the syn-
thesized inhibitor was measured at Guilford Pharmaceu-
ticals Inc. using N-acetyl-L-aspartyl-[3H]-L-glutamate as a
substrate and human recombinant NAALADase25 as
previously reported.26 2-PMPA (1) with an IC50 of
0.3 nM was used as an assay reference. Results of the bio-
logical assays showed that designed conjugate 6 retains
potent activity with an IC50 of 4 nM. The iron and gado-
linium complexes of this compound are the subject of sep-
arate studies that will be reported in due course.
3. Conclusion
4. Experimental
4.1. General
In summary, a siderophore-containing, enantiomerically
pure phosphonic acid 6 conjugate has been designed and
synthesized as a potential diagnostic agent for prostate
cancer. Despite incorporation of an extended linker to
promote iron binding only by the trihydroxamate sidero-
All reactions were carried out under argon by using
standard techniques. Solvents were dried under nitrogen
by standard procedures, distilled before use, and stored

P. Ding et al. / Bioorg. Med. Chem. 16 (2008) 1648–1657
1653
under argon. NMR spectra were recorded on a Varian
Unityplus 300 MHz spectrometer or Varian Inova
500 MHz spectrometer. Optical rotations were recorded
on a Perkin-Elmer model 343 polarimeter. Mass spectra
were recorded on a JEOL JMS-AX505 HA Double Sec-
tor mass spectrometer. The chemical shifts of 31P NMR
spectra are reported in relation to 85% H3PO4.
7.24 (d, J = 8 Hz, 1H), 6.76 (br s, 1H), 4.84–4.78 (m,
4H), 4.70–4.58 (m, 2H), 4.50–4.44 (m, 1H), 4.02 (br s,
2H), 3.72–3.50 (m, 4H), 3.63 (s, 3H), 2.10 (s, 3H), 2.09
(s, 3H), 2.06 (s, 3H), 1.97 (s, 3H), 1.82–1.50 (m, 12H);
13C NMR (125 MHz, CDCl3) d: 173.1, 172.4, 172.3,
171.8, 170.4, 134.3, 129.33, 129.31, 129.1, 129.0, 128.8,
76.3, 52.2, 52.0, 51.8, 51.5, 44.7, 43.9, 43.5, 30.1, 29.5,
28.9, 23.3, 23.2, 23.19, 23.11, 20.5; HR-FABMS Calcd
for C45H61N6O11 (M+H)+ 861.4398. Found: 861.4379.
4.1.1. Methyl N5-acetyl-N5-(benzyloxy)-N2-(benzyloxy-
carbonyl)-L-ornithyl-N5-acetyl-N5-(benzyloxy)-L-ornithyl-
N5-acetyl-N5-(benzyloxy)-L-ornithinate (9). To a solution
of 714 (835 mg, 1.95 mmol) in CH2Cl2 (20 mL) was
added 33% HBr glacial acetic acid solution (8 mL).
The reaction mixture was stirred at 25 °C for 30 min un-
der argon, then diluted with CHCl3 (40 mL), and con-
centrated in vacuo. The resulting oil was redissolved in
and concentrated twice each using CHCl3 (30 mL) and
toluene (30 mL). The resulting residue was dissolved in
CH3CN (60 mL), and 8 (1.1 g, 1.62 mmol) and diisopro-
pylethylamine (0.65 mL, 3.73 mmol) were added, fol-
lowed by the addition of HOAt (242 mg, 1.78 mmol)
and EDCI (342 mg, 1.78 mmol). The reaction mixture
was stirred at 25 °C for 48 h, diluted with ethyl acetate
(300 mL), and then washed with 1 N HCl (15 mL), sat-
urated aq NaHCO3 (15 mL), and brine. After being
dried, filtered, and concentrated, the residue was puri-
fied by flash column chromatography, eluting with
4.1.3. N5-Acetyl-N5-(benzyloxy)-N2-acetyl-L-ornithyl-N5-
acetyl-N5-(benzyloxy)-L-ornithyl-N5-acetyl-N5-(benzyl-
oxy)-L-ornithine (11). To a solution of 10 (150 mg,
0.174 mmol) in THF–H2O (v/v = 1:1, 6 mL) was added
LiOHÆH2O (22 mg, 0.522 mmol). The reaction mixture
was stirred at 25 °C for 1 h and then diluted with EtOAc
(50 mL) and 1 N HCl (5 mL). The aq layer was ex-
tracted with EtOAc twice. The combined organic layers
were washed with brine. After the resulting solution was
dried, filtered, and concentrated, the product was ob-
tained in 100% yield (148 mg) as a colorless syrup:
20
D
½aꢁ +7.4° (c = 0.50, CH2Cl2); 1H NMR (500 MHz,
CDCl3) d: 7.68 (br s, 1H), 7.50 (br s, 1H), 7.36 (br s,
15H), 7.12 (br s, 1H), 4.82–4.76 (m, 6H), 4.70–4.60 (m,
2H), 4.40–4.36 (m, 1H), 4.00 (br s, 1H), 3.84 (br s,
1H), 3.64 (br s, 2H), 3.52 (br s, 2H), 2.06 (s, 3H),
2.055 (s, 3H), 2.050 (s, 3H), 1.95 (s, 3H), 1.88–1.56 (m,
12H); 13C NMR (125 MHz, CDCl3) d: 173.5, 173.1,
172.9, 172.6, 172.3, 170.9, 134.2, 129.37, 129.35, 129.0,
128.8, 76.37, 76.31, 52.56, 52.51, 51.8, 44.8, 44.4, 43.7,
30.3, 30.0, 29.8, 28.5, 23.4, 23.16, 23.11, 20.5; HR-FAB-
MS Calcd for C44H59N6O11 (M+H)+ 847.4242. Found:
847.4268.
EtOAc–CH3OH (100:3 ! 100:5) to provide 1.4 g
20
(90%) of the product as a colorless oil. ½aꢁ ꢀ2.8°
D
(c = 1.6, CH2Cl2); 1H NMR (500 MHz, CDCl3) d:
7.42–7.30 (m, 20H), 7.22 (d, J = 8 Hz, 1H), 7.12 (d,
J = 8 Hz, 1H), 5.63 (d, J = 8 Hz, 1H), 5.09 (s, 2H),
4.90–4.74 (m, 6H), 4.62–4.58 (m, 1H), 4.50–4.38 (m,
2H), 4.22–4.02 (m, 2H), 3.78–3.60 (m, 7H), 3.60–3.42
(m, 2H), 2.13 (s, 3H), 2.11 (s, 3H), 2.08 (s, 3H), 1.88–
1.48 (m, 12); 13C NMR (125 MHz, CDCl3) d: 173.4,
172.6, 172.4, 171.9, 156.5, 136.5, 134.4, 134.3, 129.45,
129.41, 129.25, 129.20, 128.96, 128.94, 128.92, 128.6,
128.2, 128.1, 76.5, 67.0, 53.3, 52.6, 52.4, 52.1, 51.4,
44.7, 43.8, 43.4, 30.8, 29.7, 29.3, 29.0, 23.4, 23.3, 23.2,
20.6; HR-FABMS Calcd for C51H65N6O12 (M+H)+
953.4660. Found: 953.4698.
4.1.4. Ethyl 4-(4-bromophenyl)-4-oxobutanoate (13). To a
solution of 3-(4-bromobenzoyl)propionic acid (25 g,
93.9 mmol) in ethanol (180 mL) was added concd
H2SO4 (2.5 mL), and the reaction mixture was heated
at reflux for 3 h. After the mixture was cooled to
25 °C, the excess ethanol was evaporated, the residue
was dissolved in ethyl acetate (200 mL), and the organic
solution was washed with 10% aqueous NaHCO3 and
brine. After the resulting solution was dried, filtered,
and evaporated, the product was obtained as a white so-
lid in 97% yield (28.01 g): mp 57–58 °C (lit. 58–59 °C)13;
1H NMR (300 MHz, CDCl3) d: 7.82 (2H, d, J = 8.7 Hz),
7.58 (2H, d, J = 8.7 Hz), 4.14 (2H, q, J = 7.2 Hz), 3.24
(2H, t, J = 6.6 Hz), 2.72 (2H, t, J = 6.6 Hz), 1.24 (2H,
t, J = 7.2 Hz).
4.1.2. Methyl N5-acetyl-N5-(benzyloxy)-N2-acetyl-L-orni-
thyl-N5-acetyl-N5-(benzyloxy)-L-ornithyl-N5-acetyl-N5-
(benzyloxy)-L-ornithinate (10). To a solution of 9
(175 mg, 0.183 mmol) in CH2Cl2 (10 mL) was added a
33% HBr glacial acetic acid solution (3 mL). The reac-
tion mixture was stirred at 25 °C for 30 min under ar-
gon, diluted with CHCl3 (20 mL), concentrated in
vacuo, and redissolved in and concentrated twice each
using CHCl3 (15 mL) and toluene (15 mL). The result-
ing residue was dissolved in CH2Cl2 (15 mL). Pyridine
(0.2 mL) and acetic anhydride (0.2 mL) were added at
0 °C, and the resulting mixture was stirred at 25 °C over-
night, then diluted with CH2Cl2, and washed with 1 N
HCl, 10% aq Na2CO3, and brine. After the resulting
solution was dried, filtered, and concentrated, the crude
product was purified by flash chromatography, eluting
with EtOAc–CH3OH (10:1 ! 8:1) to afford 135 mg
4.1.5. 1,3-Dioxolane-2-(4-bromophenyl)-2-propanol (15).
To a solution of 13 (8 g, 28.05 mmol) in benzene
(100 mL) were added ethylene glycol (3.85 mL,
70.1 mmol) and p-toluenesulfonic acid (80 mg,
0.42 mmol), and the reaction mixture was heated at re-
flux for 16 h. After cooling to 25 °C, the reaction mix-
ture was washed with 10% aq NaHCO3 and brine.
After the resulting solution was dried, filtered, and evap-
orated, the crude product 14 was obtained as a mixture
with some unconsumed 13.
(85%) of the product as a white solid: mp 91–92 °C,
20
½aꢁ ꢀ7.9° (c = 0.9, CH2Cl2); 1H NMR (500 MHz,
The above crude product was dissolved in dry THF
(100 mL). LiAlH4 (1.84 g, 48.6 mmol) was then added
D
CDCl3) d: 7.37 (br s, 10H), 7.31 (d, J = 8 Hz, 1H),

1654
P. Ding et al. / Bioorg. Med. Chem. 16 (2008) 1648–1657
portionwise at 0 °C. The resulting mixture was stirred at
0 °C for 2 h. Then H2O (2.4 mL) was added slowly, fol-
lowed by 15% NaOH solution (2.4 mL) and H2O
(8 mL). After stirring for another 30 min at 0 °C, the
mixture was filtered through a Celite pad to remove
the resulting white solid. The solid was washed with
THF. The combined organic layers were concentrated
to provide the crude product which was purified by flash
column chromatography on silica gel (1:1 hexanes/
EtOAc) to afford 4.47 g (65%) of the product as a color-
4.1.8. 2-(4-Carboxylic-phenyl)-2-(3-tert-butyl-dimethylsi-
lyloxy)propyl-1,3-dioxolane (18). To a solution of 17
(3.1 g, 8.84 mmol) in CH3CN (60 mL) was added a solu-
tion of NaH2PO4 (610 mg, 4.42 mmol) in water (12 mL)
at 0 °C, followed by the addition of 30% H2O2 (1.64 mL,
15.92 mmol) and
a solution of NaClO2 (1.60 g,
17.68 mmol) in water (12 mL). The reaction mixture
was stirred at 25 °C for 1 h (tlc showed the conversion
to be complete). Na2SO3 (2.23 g, 17.68 mmol) was
added to destroy the excess HOCl and H2O2. Five min-
utes later, 10% citric acid solution was added to adjust
the pH to 2, followed by the addition of EtOAc
(50 mL). The aq layer was extracted with EtOAc twice.
The combined organic extracts were washed with cold
water and brine. After being dried, filtered, and concen-
trated, the acid was obtained in 95% yield (3.08 g) as a
white solid: mp 103–104 °C; 1H NMR (500 MHz,
CDCl3) d: 8.08 (2H, d, J = 8.7 Hz), 7.57 (2H, d,
J = 8.7 Hz), 4.07–4.02 (2H, m), 3.79–3.75 (2H, m), 3.58
(2H, t, J = 6.6 Hz), 1.98–1.92 (2H, m), 1.61–1.51 (2H,
m), 0.86 (9H, s), 0.01 (6H, s); 13C NMR (125 MHz,
CDCl3) d: 171.96, 148.95, 130.34, 129.01, 110.37,
64.86, 63.19, 36.81, 27.13, 26.14, 18.53, ꢀ5.10; HR-
FABMS Calcd for C19H31O5Si (M+H)+ 367.1941.
Found: 367.1938.
1
less oil. H NMR (300 MHz, CDCl3) d: 7.46 (2H, d,
J = 8.7 Hz), 7.31 (2H, d, J = 8.7 Hz), 4.04–3.99 (2H,
m), 3.76–3.72 (2H, m), 3.60 (2H, t, J = 6.6 Hz), 2.07
(1H, m, OH), 2.00–1.92 (2H, m), 1.63–1.54 (2H, m);
13C NMR (75 MHz, CDCl3) d: 131.51, 128.31, 127.83,
125.93, 122.24, 110.20, 64.80, 64.73, 62.96, 37.17,
27.00; HR-FABMS Calcd for C12H14BrO3 (MꢀH)+
285.0126. Found: 285.0112.
4.1.6. 2-(4-Bromo-phenyl)-2-(3-tert-butyl-dimethylsilyl-
oxy)propyl-1,3-dioxolane (16). To a solution of 15
(3.3 g, 11.49 mmol) in DMF (45 mL) were added
TBDMSCl (2.77 g, 18.38 mmol) and imidazole (1.96 g,
28.73 mmol), and the reaction mixture was stirred at
25 °C overnight, then diluted with CH2Cl2, and washed
with cold H2O and brine. After the resulting solution
was dried, filtered, and evaporated, the residual DMF
was evaporated under high vacuum. The remaining res-
idue was purified by flash column chromatography on
silica gel (hexanes/EtOAc 12:1 ! 8:1) to afford 4.42 g
(96%) of the product as a colorless oil. 1H NMR
(500 MHz, CDCl3) d: 7.44 (2H, d, J = 8.7 Hz), 7.31
(2H, d, J = 8.7 Hz), 4.02–3.97 (2H, m), 3.76–3.72 (2H,
m), 3.56 (2H, t, J = 6.6 Hz), 1.96–1.87 (2H, m), 1.59–
1.49 (2H, m), 0.86 (9H, s), 0.01 (6H, s); 13C NMR
(125 MHz, CDCl3) d: 131.35, 128.15, 127.80, 125.90,
122.00, 110.32, 64.72, 63.30, 36.93, 27.22, 26.10, 18.43,
ꢀ5.15; HR-FABMS Calcd for C18H28BrO3Si (MꢀH)+
399.0991. Found: 399.0974.
4.1.9. 2-(4-Methoxycarbonyl-phenyl)-2-(3-tert-butyl-dim-
ethylsilyloxy)propyl-1,3-dioxolane (19). To a solution of
18 (2.6 g, 7.1 mmol) in diethyl ether (30 mL) at ꢀ5 °C
was added a freshly prepared ethereal solution of diazo-
methane in small portions until the gas evolution ceased,
and the solution became yellow. After the mixture was
stirred for another 10 min, excess diazomethane was de-
stroyed by addition of acetic acid dropwise until the yel-
low color disappeared. The volatiles were evaporated,
and the residue was purified by flash column chromatog-
raphy on silica gel (hexanes/EtOAc 5:1) to afford 2.5 g
(93%) of the product as a colorless oil. 1H NMR
(500 MHz, CDCl3) d: 8.01 (2H, d, J = 8.7 Hz), 7.52
(2H, d, J = 8.7 Hz), 4.07–3.99 (2H, m), 3.85 (3H, s),
3.79–3.73 (2H, m), 3.57 (2H, t, J = 6.6 Hz), 1.95–1.92
(2H, m), 1.58–1.52 (2H, m), 0.80 (9H, s), ꢀ0.05 (6H,
s); 13C NMR (125 MHz, CDCl3) d: 167.03, 147.93,
129.80, 129.62, 126.01, 110.31, 64.76, 63.11, 52.23,
36.79, 27.10, 26.08, 18.45, ꢀ5.16; HR-FABMS Calcd
for C20H33O5Si (M+H)+ 381.2097. Found: 381.2093.
4.1.7. 2-(4-Carbaldehyde-phenyl)-2-(3-tert-butyl-dimeth-
ylsilyloxy)propyl-1,3-dioxolane (17). To a solution of
16 (4.1 g, 10.21 mmol) in THF (50 mL) at ꢀ78 °C was
added dropwise a 1.6 M n-BuLi solution in hexanes
(7 mL, 11.22 mmol) by syringe. After the addition, the
mixture was stirred at ꢀ78 °C for another 40 min before
DMF (2.36 mL, 30.64 mmol) was added. The reaction
mixture was then warmed to 25 °C slowly. The volatiles
were evaporated, and the residue was dissolved in ethyl
acetate and washed with cold H2O and brine. After the
resulting solution was dried, filtered, and concentrated,
the residue was purified by flash column chromatogra-
phy on silica gel (hexanes/EtOAc 10:1 ! 7:1) to afford
3.32 g (90%) of the product as a white solid: mp 49–
50 °C; 1H NMR (300 MHz, CDCl3) d: 9.98 (1H, s,
CHO), 7.84 (2H, d, J = 8.7 Hz), 7.61 (2H, d,
J = 8.7 Hz), 4.04–3.99 (2H, m), 3.77–3.72 (2H, m), 3.56
(2H, t, J = 6.6 Hz), 1.95–1.90 (2H, m), 1.59–1.49 (2H,
m), 0.84 (9H, s), 0.01 (6H, s); 13C NMR (75 MHz,
CDCl3) d: 191.92, 149.87, 136.37, 129.79, 126.73,
110.40, 64.95, 63.14, 36.90, 27.21, 26.14, 18.49, ꢀ5.11;
HR-FABMS Calcd for C19H31O4Si (M+H)+ 351.1992.
Found: 351.2014.
4.1.10. 1,3-Dioxolane-2-(4-methoxycarbonyl-phenyl)-2-
propanol (20). To a solution of 19 (700 mg, 1.84 mmol)
in THF (10 mL) was added a 1.0 M TBAF solution in
THF (5.5 mL, 5.5 mmol), the reaction mixture was stir-
red at 25 °C overnight. The volatiles were evaporated.
The residue was dissolved in CH2Cl2 and washed with
cold water and brine. After the resulting solution was
dried, filtered, and evaporated, the residue was purified
by flash chromatography on silica gel (hexanes/EtOAc
2:1 ! 1:3) to provide 400 mg (82%) of the product as
a colorless oil. 1H NMR (300 MHz, CDCl3) d: 8.01
(2H, d, J = 8.4 Hz), 7.52 (2H, d, J = 8.4 Hz), 4.06–4.02
(2H, m), 3.91 (3H, s), 3.78–3.73 (2H, m), 3.62 (2H, t,
J = 6.3 Hz), 2.02–1.97 (3H, m), 1.67–1.58 (2H, m); 13C
NMR (75 MHz, CDCl3) d: 167.05, 147.82, 130.17,
129.75, 126.07, 110.32, 64.89, 62.97, 52.18, 37.07,

P. Ding et al. / Bioorg. Med. Chem. 16 (2008) 1648–1657
1655
26.98; HR-FABMS Calcd for C14H17O5 (MꢀH)+
m), 7.21–7.18 (1H, m), 6.84–6.82 (1H, m), 6.74–6.72
(1H, m), 4.83–4.75 (6H, m), 4.50–4.39 (3H, br s), 4.02–
3.90 (3H, m), 3.88 (3H, s), 3.87–3.42 (6H, m), 3.21–
3.17 (2H, m), 2.09 (3H, s), 2.07 (3H, s), 2.02 (3H, s),
1.97 (3H, s), 1.90–1.48 (16H, m); 13C NMR (75 MHz,
CDCl3) d: 173.12, 172.93, 172.62, 172.34, 171.74,
171.46, 171.21, 166.96, 147.87, 134.35, 129.77, 129.67,
129.63, 129.57, 129.37, 129.35, 129.17, 129.15, 129.10,
128.88, 128.86, 125.93, 109.96, 76.43, 76.42, 76.37,
64.73, 53.33, 53.23, 52.41, 52.29, 52.19, 44.47, 44.21,
39.36, 37.52, 29.54, 29.44, 28.48, 23.74, 23.57, 23.23,
23.16, 20.59, 20.53; HR-FABMS Calcd for
C58H76N7O14 (M+H)+ 1094.5450. Found: 1094.5477.
265.1076. Found: 265.1072.
4.1.11. 1,3-Dioxolane-2-(4-methoxycarbonyl-phenyl)-2-
propyl azide (21). To a solution of 20 (350 mg,
1.31 mmol) in CH2Cl2 (10 mL) were added pyridine
(0.637 mL, 7.88 mmol) and methanesulfonyl chloride
(0.4 mL, 5.25 mmol) at 0 °C. After stirring at 0 °C for
30 min, the reaction mixture was stirred at 25 °C for
an additional 15 h. It was then diluted with CH2Cl2
(70 mL) and washed with cold water (10 mL) and brine
(10 mL). After the resulting solution was dried, filtered,
and concentrated, the residue was dissolved in DMF
(10 mL), and NaN3 (513 mg, 7.88 mmol) was added.
The reaction mixture was stirred at 25 °C for 36 h, di-
luted with CH2Cl2, and washed with cold water twice.
After the resulting solution was dried, filtered, and con-
centrated, the residue was purified by flash chromatog-
raphy, eluting with hexanes/EtOAc (12:1 ! 6:1 ! 4:1)
to afford 315 mg (82%) of the product as a colorless
oil. 1H NMR (500 MHz, CDCl3) d: 7.98 (2H, d,
J = 8.0 Hz), 7.49 (2H, d, J = 8.0 Hz), 4.05–3.98 (2H,
m), 3.88 (3H, s), 3.80–3.75 (2H, m), 3.25 (2H, t,
J = 6.5 Hz), 1.94–1.91 (2H, m), 1.70–1.59 (2H, m); 13C
NMR (125 MHz, CDCl3) d: 166.83, 147.46, 129.93,
129.64, 125.83, 109.74, 64.74, 52.17, 51.36, 37.22,
23.25; HR-FABMS Calcd for C14H18N3O4 (M+H)+
292.1297. Found: 292.1292.
4.1.14. N5-Acetyl-N5-(benzyloxy)-N2-acetyl-L-ornithyl-
N5-acetyl-N5-(benzyloxy)-L-ornithyl-N5-acetyl-N5-(ben-
zyloxy)-L-ornithyl-N-[4-(4-methoxycarbonyl-phenyl)-4-
oxobutyl]-amide (24). To a 10-mL flask containing 23
(80 mg, 0.073 mmol) was added HCO2H (6.6 mL), and
the reaction mixture was stirred at 25 °C for 3 h until
the conversion was complete as indicated by tlc. The vol-
atiles were evaporated, and the residue was purified by
flash column chromatography, eluting with CH2Cl2/
CH3OH (100:6) to provide 70 mg (91%) of the product
20
as a white solid: mp 59–60 °C, ½aꢁ ꢀ9.1° (c = 1.10,
D
CH2Cl2); 1H NMR (500 MHz, CDCl3) d: 8.09 (2H,
J = 9.0 Hz), 8.99 (2H, J = 9.0 Hz), 7.78 (1H, br s), 7.40–
7.25 (15H, m), 6.97–6.90 (2H, m), 4.84–4.76 (6H, m),
4.48–4.39 (1H, m), 4.37–4.25 (2H, m), 3.94 (3H, s),
3.90–3.51 (6H, m), 3.40–3.26 (2H, m), 3.12–2.99 (2H,
m), 2.60 (1H, br s), 2.11 (3H, s), 2.09 (3H, s), 2.04 (3H,
s), 1.98 (3H, s), 1.97–1.53 (14H, m); 13C NMR
(125 MHz, CDCl3) d: 199.59, 173.48, 173.22, 173.07,
172.62, 171.87, 166.42, 140.28, 134.36, 134.21, 133.76,
129.87, 129.41, 129.38, 129.25, 129.15, 128.92, 128.87,
128.18, 76.45, 76.42, 54.25, 53.03, 52.55, 44.81, 44.13,
38.77, 36.20, 29.05, 28.83, 27.65, 24.08, 23.88, 23.81,
23.73, 23.13, 20.70, 20.60, 20.55; HR-FABMS Calcd for
C56H72N7O13 (M+H)+ 1050.5188. Found: 1050.5238.
4.1.12. 1,3-Dioxolane-2-(4-methoxycarbonyl-phenyl)-2-
propyl amine (22). To a solution of 21 (80 mg,
0.274 mmol) in methanol (5 mL) were added 1,3-pro-
panedithiol (138 lL, 1.373 mmol) and Et3N (191 lL,
1.373 mmol) under an atmosphere of N2. The reaction
mixture was stirred at 25 °C for 19 h, and the volatiles
were then evaporated. The residue was purified by flash
chromatography, eluting with CH2Cl2/MeOH/Et3N
(100:8.3:4) to afford 67 mg (92%) of the product as a yel-
1
low oil. H NMR (500 MHz, CD3OD) d: 7.99 (2H, d,
J = 8.0 Hz), 7.56 (2H, d, J = 8.0 Hz), 4.05–3.99 (2H,
m), 3.91 (3H, s), 3.81–3.75 (2H, m), 2.64 (2H, t,
J = 7.5 Hz), 1.93 (2H, t, J = 8.0 Hz), 1.59–1.50 (2H,
m); 13C NMR (125 MHz, CD3OD) d: 168.31, 149.51,
131.03, 130.56, 127.23, 111.20, 65.91, 52.80, 42.51,
38.74, 27.66; HR-FABMS Calcd for C14H20NO4
(M+H)+ 266.1392. Found: 266.1375.
4.1.15. N5-Acetyl-N5-(benzyloxy)-N2-acetyl-L-ornithyl-
N5-acetyl-N5-(benzyloxy)-L-ornithyl-N5-acetyl-N5-(ben-
zyloxy)-L-ornithyl-N-[4-(4-carboxy-phenyl)-4-oxobutyl]-
amide (25). To a solution of 24 (66 mg, 0.063 mmol) in
THF–H2O solution (1:1, 5 mL) was added LiOHÆH2O
(6.6 mg, 0.157 mmol). The reaction mixture was stirred
at 25 °C for 1 h and diluted with EtOAc (40 mL), fol-
lowed by the addition of 1 N HCl (1 mL). The layers
were separated, and the aq layer was extracted with
EtOAc twice. The combined organic layers were washed
with brine, dried, filtered, and concentrated to afford
4.1.13. N5-Acetyl-N5-(benzyloxy)-N2-acetyl-L-ornithyl-
N5-acetyl-N5-(benzyloxy)-L-ornithyl-N5-acetyl-N5-(ben-
zyloxy)-L-ornithyl-N-[1,3-dioxolane-2-(4-methoxycar-
bonyl-phenyl)-2-propyl]-amide (23). To a suspension of
11 (108 mg, 0.127 mmol) and 22 (37 mg, 0.141 mmol)
in CH3CN (5 mL) were added diisopropylethylamine
(29 lL, 0.166 mmol), HOAt (19.2 mg, 0.141 mmol),
and EDCI (27 mg, 0.141 mmol). The reaction mixture
was stirred at 25 °C for 22 h. The volatiles were evapo-
rated, and the residue was purified by flash column chro-
matography, eluting with EtOAc–CH3OH (10:1) and
then CH2Cl2/CH3OH (100:6.5) to provide 124 mg
58 mg (90%) of the product as a white solid: mp 77–
20
D
78 °C, ½aꢁ
ꢀ12.5° (c = 1.0, CH2Cl2); 1H NMR
(500 MHz, CDCl3) d: 8.02 (2H, J = 8.0 Hz), 7.93 (2H,
J = 8.0 Hz), 7.88 (1H, br s), 7.40–7.26 (15H, m), 7.23–
7.05 (2H, m), 4.85–4.72 (6H, m), 4.57–4.38 (3H, m),
3.96–3.50 (6H, m), 3.40–3.31 (2H, m), 3.12–2.96 (2H,
m), 2.10 (3H, s), 2.08 (3H, s), 2.06 (3H, s), 2.00 (3H,
s), 1.97–1.58 (14H, m); 13C NMR (125 MHz, CDCl3)
d: 199.50, 173.54, 173.30, 172.16, 172.86, 172.32,
172.26, 171.96, 168.00, 140.17, 134.20, 134.05, 130.19,
129.42, 129.25, 129.20, 128.91, 128.11, 76.45, 53.93,
53.04, 44.68, 44.30, 44.21, 38.87, 36.09, 30.44, 29.01,
(89%) of the product as a white amorphous solid: mp
20
83–84 °C, ½aꢁ ꢀ40.6° (c = 0.70, CH2Cl2); 1H NMR
D
(300 MHz, CDCl3) d: 7.96 (2H, d, J = 8.0 Hz), 7.54
(1H, br s), 7.47 (2H, d, J = 8.0 Hz), 7.40–7.32 (15H,

1656
P. Ding et al. / Bioorg. Med. Chem. 16 (2008) 1648–1657
28.10, 23.90, 23.73, 23.11, 20.56; HR-FABMS Calcd for
C55H70N7O13 (M+H)+ 1036.5032. Found: 1036.4990.
the biological assays. We appreciate Prof. Olaf Wiest’s
group for their advice on structural modifications. We
gratefully acknowledge financial support by the Depart-
ment of Defense and NIH (AI054193 (MJM, PD)) and
collaboration with the Walther Cancer Institute. We
also appreciate the use of the NMR facilities provided
by the Lizzadro Magnetic Center and the mass spec-
trometry services (Dr. W. Boggess and Ms. N. Sevova)
provided by the University of Notre Dame. M.J.M.
gratefully acknowledges the Hans-Ko¨ll Institute and
the University of Notre Dame for support of a sabbati-
cal leave.
4.1.16. Compound 27. To a solution of 2612 (60 mg,
0.0776 mmol) in CH2Cl2 (5 mL) was added TFA
(0.4 mL). The resulting reaction mixture was stirred at
25 °C for 1 h, then diluted with CH2Cl2 (100 mL), and
washed with saturated aq Na2CO3 (15 mL). The aq
layer was extracted with CH2Cl2 twice. The combined
extracts were washed with brine (10 mL). After the solu-
tion was dried (MgSO4), filtered, and concentrated, the
resulting amine was obtained as a colorless oil (30 mg).
To a suspension of the amine (30 mg, 0.0445 mmol) and
acid 25 (40 mg, 0.0386 mmol) in CH3CN (4 mL) were
added diisopropylethylamine (10 lL, 0.0535 mmol),
HOAt (7.5 mg, 0.0535 mmol), and EDCI (11 mg,
0.0535 mmol). The reaction mixture was stirred at
25 °C for 24 h. The volatiles were evaporated, and the
residue was purified by flash column chromatography,
eluting with CH2Cl2–MeOH (100:8) to provide 50 mg
(77%) of the product as a white solid: mp 83–85 °C;
1H NMR (500 MHz, CDCl3) d: 9.20 (1H, br s), 8.07
(1H, br s), 8.02 (2H, d, J = 8.5 Hz), 7.96 (2H, d,
J = 8.5 Hz), 7.70 (1H, br s), 7.49 (2H, d, J = 8.0 Hz),
7.43–7.21 (30H, m), 7.08 (2H, dd, J = 8.0, 3.5 Hz),
6.91 (1H, br s), 5.07–4.78 (12H, m), 4.46–4.22 (5H, m),
4.16–4.00 (3H, m), 3.97–3.76 (3H, m), 3.64–3.40 (4H,
m), 3.26–2.96 (3H, m), 2.88–2.74 (3H, m), 2.52–2.20
(5H, m), 2.11 (3H, s), 2.079 (3H, s), 2.075 (3H, s), 1.96
(3H, s), 1.95–1.22 (17H, m); 31P NMR (121 MHz,
CDCl3) d: 29.04; ESI-MS Calcd for C92H109N9O20P
(M+H)+ 1690.75. Found: 1690.
References and notes
1. Carter, R. E.; Coyle, J. T. In Handbook of Proteolytic
Enzymes; Barrett, A. J., Rawlings, N. D., Woessner, J. F.,
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2. (a) Bruno, V.; Battaglia, G.; Copani, A.; Giffard, R. G.;
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Pharmacol. 1995, 7, 1906; (b) Choi, D. W.; Rothman, S.
M. Annu. Rev. Neurosci. 1990, 13, 171.
3. Zhou, J.; Neale, J. H.; Pomper, M. G.; Kozikowski, A. P.
Nat. Rev. Drug Discov. 2005, 4, 1015.
4. Jackson, P. F.; Cole, D. C.; Slusher, B. S.; Stetz, S. L.;
Ross, L. E.; Donzanti, B. A.; Trainor, D. A. J. Med.
Chem. 1996, 39, 619.
5. (a) Kozikowski, A. P.; Zhang, J.; Nan, F.; Petukhov, P.
A.; Grajkowska, E.; Wroblewski, J. T.; Yamamoto, T.;
Bzdega, T.; Wroblewska, B.; Neale, J. H. J. Med. Chem.
2004, 47, 1729–1738; (b) Kozikowski, A. P.; Nan, F.;
Conti, P.; Zhang, J.; Ramadan, E.; Bzdega, T.; Wroble-
wska, B.; Neale, J. H.; Pshenichkin, S.; Wroblewski, J. T.
J. Med. Chem. 2001, 44, 298.
6. Jackson, P. F.; Tays, K. L.; Maclin, K. M.; Ko, Y.-S.;
Li, W.; Vitharana, D.; Tsukamoto, T.; Stoermer, D.; Lu,
X.-C. M.; Wozniak, K.; Slusher, B. S. J. Med. Chem.
2001, 44, 4170.
7. (a) Vitharana, D.; France, J. E.; Scarpetti, D.; Bonneville,
G. W.; Majer, P.; Tsukamoto, T. Tetrahedron: Asymmetry
2002, 13, 1609; (b) Tsukamoto, T.; Majer, P.; Vitharana,
D.; Ni, C.; Hin, B.; Lu, X.-C. M.; Thomas, A. G.;
Wozniak, K. M.; Calvin, D. C.; Wu, Y.; Slusher, B. S.;
Scarpetti, D.; Bonneville, G. W. J. Med. Chem. 2005, 48,
2319.
8. (a) Carter, R. E.; Feldman, A. R.; Coyle, J. T. Proc. Natl.
Acad. Sci. 1996, 93, 749; (b) Tiffany, C. W.; Lapidus, R.
G.; Merion, A.; Calvin, D. C.; Slusher, B. S. Prostate
1999, 39, 28; (c) Asatiani, E.; Gelmann, E. P. Expert Opin.
Ther. Targets 2005, 9, 283; (d) Elsasser-Beile, U.; Wolf, P.;
Gierschner, D.; Buhler, P.; Schultze-Seemann, W.; Wet-
terauer, U. Prostate 2006, 66, 1359; (e) Farokhzad, O. C.;
Cheng, J. J.; Teply, B. A.; Sherifi, I.; Jon, S.; Kantoff, P.
W.; Richie, J. P.; Langer, R. Proc. Natl. Acad. Sci. U.S.A.
2006, 103, 6315; (f) Chu, T. C.; Shieh, F.; Lavery, L. A.;
Levy, M.; Richards-Kortum, R.; Korgel, B. A.; Ellington,
A. D. Biosens. Bioelectron. 2006, 21, 1859; (g) Misra, P.;
Humblet, V.; Pannier, N.; Maison, W.; Frangioni, J. V.
J. Nucl. Med. 2007, 48, 1379.
9. (a) Davis, M. I.; Bennett, M. J.; Thomas, L. M.;
Bjorkman, P. J. Proc. Natl. Acad. Sci. U.S.A. 2005, 102,
5981; (b) Oliver, A. J.; Wiest, O.; Helquist, P.; Miller, M.
J.; Tenniswood, M. Bioorg. Med. Chem. 2003, 11, 4455.
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Y.; Zhai, H.; Chen, X.; Shen, T. Y.; Tenniswood, M.
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4.1.17. Compound 6. To a solution of 27 (30 mg,
0.018 mmol) in methanol (5 mL) was added 10% Pd–C
(10 mg), the flask was purged with argon, then the argon
was replaced with H2, and the reaction mixture was stir-
red under H2 atmosphere for 2 h. After the mixture was
filtered through filter paper, the solvent was evaporated
from the filtrate to provide 20 mg (98%) of the product
20
D
as a yellow solid: mp 57–59 °C, ½aꢁ ꢀ8.3° (c = ꢀ0.5,
1
MeOH); H NMR (500 MHz, CD3OD) d: 7.82 (2H, d,
J = 7.0 Hz), 7.50 (2H, d, J = 6.0 Hz), 7.30 (2H, d,
J = 7.0 Hz), 7.22 (2H, d, J = 6.0 Hz), 4.39–4.04 (6H,
m), 4.73–3.50 (7H, m), 3.33–3.30 (2H, m), 3.25–3.17
(2H, s), 2.96–2.88 (2H, m), 2.72–2.62 (3H, m), 2.40–
2.23 (2H, m), 2.092 (3H, s), 2.090 (3H, s), 2.08 (3H, s),
1.98 (3H, s), 1.96–1.25 (16H, m); 13C NMR (125 MHz,
CD3OD) d: 178.01, 176.64, 174.98, 174.20, 174.04,
173.99, 173.92, 173.83, 170.63, 169.96, 148.29, 138.19,
135.41, 132.66, 130.70, 129.86, 128.78, 121.53, 67.00,
55.22, 55.01, 54.83, 54.71, 52.27, 44.65, 40.85, 40.21,
37.63, 36.41, 35.08, 32.41, 31.03, 30.89, 30.65, 30.61,
30.37, 30.02, 29.92, 29.77, 29.64, 29.44, 28.73; 31P
NMR (121 MHz, CDCl3) d: 26.98; FAB-MS Calcd for
C50H73NaN9O20P (M+H+Na)+ 1173.46. Found: 1174.
Acknowledgments
We thank Dr. Camilo Rojas and Dr. Vincent Kalish at
Guilford Pharmaceutical Inc. for generously providing

P. Ding et al. / Bioorg. Med. Chem. 16 (2008) 1648–1657
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