Synthesis, in vitro, and in vivo characterization of an integrin αvβ3-targeted molecular probe for optical imaging of tumor

Article abstract of DOI:10.1016/j.bmc.2005.03.024

Integrin α_vβ₃ is a widely-recognized target for the development of targeted molecular probes for imaging pathological conditions. α_vβ₃ is a cell-surface receptor protein that is upregulated in various pathological conditions including osteoporosis, rheumatoid arthritis, macular degeneration, and cancer. The synthesis of an α_vβ₃-targeted optical probe 7 from compound 1, and its in vitro and in vivo characterization is described. A series of aliphatic carbamate derivatives of the potent non-peptide integrin antagonist 1 was synthesized and the binding affinity to α _vβ₃ was determined in both enzyme linked immunosorbent assay (ELISA) and cell adhesion inhibition assays. The hydrophobic carbamate-linked appendages improved the binding affinity of the parent compound for α_vβ₃ by 2-20 times. A Boc-protected neopentyl derivative in the series is shown to have the best binding affinity to α_vβ₃ (IC₅₀ = 0.72 nM) when compared to compound 1 as well as to c-RGDfV. Optical probe 7 utilizes the neopentyl linker and demonstrates increased binding affinity and significant tumor cell uptake in vitro as well as specific tumor accumulation and retention in vivo. These results illustrate the potential of employing integrin-targeted molecular probes based on 1 to image a multitude of diseases associated with α_vβ₃ overexpression.

Full text of DOI:10.1016/j.bmc.2005.03.024

Bioorganic & Medicinal Chemistry 13 (2005) 3763–3771
Synthesis, in vitro, and in vivo characterization of an integrin
avb3-targeted molecular probe for optical imaging of tumor
Christopher A. Burnett,a, Jianwu Xie,a, Jade Quijano,a Zhimin Shen,b Finie Hunter,a
a
Monica Bur,a KingC. P. Li and S. Narasimhan Danthia,*
aMolecular Imaging Laboratory, Clinical Center, National Institutes of Allergies and Infectious Diseases,
National Institutes of Health, 10 Center Drive, Bethesda, MD 20892, USA
bVaccine Research Center, National Institutes of Health, 40 Convent Drive, Bethesda, MD 20892, USA
Received 23 December 2004; revised 8 March 2005; accepted 11 March 2005
Available online 6 April 2005
Abstract—Integrin avb3 is a widely-recognized target for the development of targeted molecular probes for imaging pathological
conditions. avb3 is a cell-surface receptor protein that is upregulated in various pathological conditions including osteoporosis, rheu-
matoid arthritis, macular degeneration, and cancer. The synthesis of an avb3-targeted optical probe 7 from compound 1, and its in
vitro and in vivo characterization is described. A series of aliphatic carbamate derivatives of the potent non-peptide integrin antag-
onist 1 was synthesized and the bindingaffinity to avb3 was determined in both enzyme linked immunosorbent assay (ELISA) and
cell adhesion inhibition assays. The hydrophobic carbamate-linked appendages improved the binding affinity of the parent com-
pound for avb3 by 2–20 times. A Boc-protected neopentyl derivative in the series is shown to have the best bindingaffinity to
avb3 (IC50 = 0.72 nM) when compared to compound 1 as well as to c-RGDfV. Optical probe 7 utilizes the neopentyl linker and
demonstrates increased bindingaffinity and significant tumor cell uptake in vitro as well as specific tumor accumulation and reten-
tion in vivo. These results illustrate the potential of employing integrin-targeted molecular probes based on 1 to image a multitude of
diseases associated with avb3 overexpression.
Published by Elsevier Ltd.
1. Introduction
duced by ligand binding to integrins invoke signaling
cascades inside the cell that regulate gene expression,
activate kinases, and direct cytoskeletal organization.4
Alternatively, internal cellular activation can produce
both conformational changes and multimeric clustering
of the integrins, which results in non-constitutive bind-
The integrins are a family of cell-surface glycoproteins
that mediate cell survival, proliferation, and cell migra-
tion through explicit non-covalent interactions with
endogenous extra cellular matrix (ECM) proteins. To
date, 19 alpha and 8 beta mammalian subunits have
been identified in this class and their distinct pair-wise
combinations consequently provide 25 unique heterodi-
meric transmembrane receptor proteins.1 Investigations
into the basic structure of the integrins utilizing electron
microscopy2 reveal that these proteins consist of a large
N-terminal extra cellular domain, a membrane-spanning
region, and a short cytoplasmic tail. The structure of the
integrins allows them to function as bidirectional cellu-
lar signal transducers.3 Conformational changes in-
5
ingto ligands, ECM components, as well as other cells.
The type and degree of the signaling event is determined
by the conformation and nature of the ligand and is regu-
lated by divalent cations bound to metal ion-dependent
adhesion sites on the integrin receptor.6
The vital role integrins play in fundamental cellular pro-
cesses renders them as attractive targets for the develop-
ment of treatments for a variety of diseases. Amongthe
more prominent targets is the integrin avb3, which has
been implicated in the formation of new blood vessels
(angiogenesis) and tissue remodeling in major diseases
such as osteoporosis,7 rheumatoid arthritis,8 macular
degeneration,9 and cancer.10 The avb3 integrin receptor
has also been shown to mediate the internalization of
rotavirus11 and adenovirus.12 Integrin avb3 binds to
Keywords: Integrin avb3; Tumor; Molecular probe; Imaging.
*
Correspondingauthor. Tel.: +1 301 451 5170; fax: +1 301 435
2714; e-mail: ndanthi@cc.nih.gov
Both authors contributed equally.
0968-0896/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmc.2005.03.024

3764
C. A. Burnett et al. / Bioorg. Med. Chem. 13 (2005) 3763–3771
the natural ligands vitronectin, fibrinogen, osteopontin,
and bone sialoprotein by recognizing the amino acid se-
quence Arg-Gly-Asp (RGD).13 Several research groups
have synthesized peptide and peptidomimetic small mole-
cule antagonists based on this motif that demonstrate
remarkable affinity and selectivity to avb3.14
imaging or therapy. The payloads include fluorescent
dyes for optical imaging, gadolinium complexes for
MRI, 111In complexes for SPECT, 18F-labeled probes
for PET, as well as cytotoxic agents or genes for ther-
apy. Several research groups have demonstrated the util-
ity of couplingpeptide or peptidomimetic liagnds of
avb3 to molecular beacons for use in non-invasive in
vivo imaging of tumor-related angiogenesis.20–23 18F-la-
beled RGD-containingcyclic peptides have been applied
as PET probes.20 DOTA-conjugated non-peptide inte-
grin antagonists chelating 111In or 90Y have been used
for avb3-targeted SPECT imaging as well as therapy,
respectively.21 MRI probes have been developed for in
vivo imaging of tumor models in animals by attaching
avb3 specific antibodies to paramagnetic nanoparticles.22
Most recently, several research groups have prepared
avb3-targeted near-infrared probes utilizing the cyclic
peptide c-RGDyK as the targeting agent for optical imag-
Targeting the integrin avb3 by directly interferingwith
its function is potentially a very effective strategy for
the treatment of several diseases.15 Administration of
small molecule antagonists of avb3 has been shown to re-
duce bone resorption in animal models of osteoporo-
sis.16 The monoclonal antibody Vitaxin as well as the
potent cyclic peptide c-[RGDf(N-Me)V] are currently
in clinical trials for avb3-targeted anti-angiogenic cancer
therapy.17 Angiogenesis is a result of multiple biochem-
ical pathways17a,18 and inhibitingsolely avb3 might be a
less effective strategy for anti-angiogenic therapy. Re-
cently, the integrin antagonist 1 (Scheme 1) was em-
ployed as the targeting agent for targeted gene delivery
to tumor neovasculature.19 Transfection of a mutant
Raf gene to the tumor associated endothelium led to
tumor cell apoptosis and regression of primary and met-
astatic tumors in M21 tumor-bearingmice. Research in
our laboratory is focused on developing avb3-targeted
low molecular weight compounds and nanoparticle
based therapeutic materials as well as imaging probes.
Apart from the prospective use of the avb3 antagonists
as anti-angiogenic agents, we are also developing novel
delivery systems that use avb3 antagonists as targeting
agents while simultaneously carrying a payload for
23
ingin vivo. In this paper we describe the synthesis of
novel carbamate linkers of a known integrin antagonist
1 and also the synthesis, in vitro, and in vivo characteri-
zation of an integrin avb3 probe 7 derived from com-
pound 1. Compound 7 represents the first example of
a non-peptide integrin avb3-targeted optical probe. The
convergent synthesis of 1, a nanomolar inhibitor of
avb3, has been previously described as a component of
a liposomal-based nanoparticle system.19 The cen-
trally-constrained benzoylamino-3-propionic acid scaf-
fold originally developed by Duggan et al.24 provides
optimum spacingbetween the acidic and basic portions
of this RGD mimic. Previous work demonstrated that
when the amine 1 was attached to a nanoparticle system
through an amide linkage, it retained significant binding
affinity to avb3 after conjugation, however extensive
structure–activity studies on the amine terminus of 1
were not examined.19 The current study focuses on
determiningthe structure–activity relationship potential
at the ethylamine terminus of the molecule and exploit-
ingthis potential by attachinga molecular beacon for
optical imaging. Herein, we describe the synthesis of a
series of aliphatic carbamate derivatives of 1 that have
enhanced bindingaffinity for avb3 and the development
of an integrin-targeted optical probe 7, synthesized from
6a, for in vivo tumor imaging. The in vitro binding affin-
ity of 7 for avb3 and the in vivo biodistribution of 7 in a
mouse melanoma model are also described.
O
H
N
R
R
H
N
R
R
b)
OH
N
O
Y
n
Y
n
N
2a-d Y= H
3a-d Y= Boc
4a-d Y= Boc
a)
Cmpd
n
R
a
b
c
d
1
0
1
2
CH3
H
H
H
H
N
H
N
N
N
O
O
HN
HN
2. Results and discussion
2.1. Synthesis of carbamate-linked derivatives
c)
4a-d
HN
O
HN
O
CO2H
CO2H
O
S
The synthesis of the carbamate-linked derivatives of 1
(Scheme 1) begins with commercially available amino
alcohols 2a–d. The amines were first protected as their
t-butoxycarbonyl derivatives 3a–d by the method de-
scribed by Ensch and Hesse,25 and their purity was veri-
fied by NMR.26 Protection was followed by activation
of the alcohols as monocarbonylimidazoles 4a–d. The
activated alcohols were then coupled to the free amine
1 in DMSO to provide carbamates 5a–d. In each case,
selective deprotection of the Boc carbamate in the pres-
ence of neopentyl, ethyl, propyl, or butyl carbamate was
H
N
H
N
R
R
O
S
HN
O
HN
NH2
Y
n
O
O
O
5a-d Y= Boc
6a-d Y= H
1
d)
Scheme 1. Synthesis of compounds. Reagents and conditions: (a)
(Boc)2O, Et3N, CH2Cl2; (b) 1,10-carbonyldiimidazole, DIEA, CH2Cl2;
(c) DIEA, DMSO, 70 ꢀC; (d) 4 M HCl, dioxane, 0 ꢀC. Yield 5a–d:
66%, 23%, 36%, and 33%, respectively. Compounds 6a–d were
obtained as hydrochloride salts in quantitative yield.

C. A. Burnett et al. / Bioorg. Med. Chem. 13 (2005) 3763–3771
3765
H
N
H
N
N
N
O
O
HO
HN
HN
O
O
OH
FITC, DIEA
HN
O
DMSO, 40°C
HN
O
CO2H
S
NH
NH
CO2H
H
N
O
H
N
O
S
O
S
HN
O
NH3Cl
HN
O
O
O
6a
O
O
7
Scheme 2. Synthesis of compound 7.
achieved with anhydrous HCl in dioxane at 0 ꢀC to pro-
vide amines 6a–d. Followinglyophilization, no appre-
ciable amount of unreacted 1 was detected after
deprotection reactions.27 Compound 7 was synthesized
(Scheme 2) by reactingthe amine 6a and the commer-
cially available fluorescein isothiocyanate. All intermedi-
ates and final compounds were characterized by 1H
NMR, 13C NMR, and high-resolution mass spectro-
metry.
sults of both the ELISA and cell adhesion assays show
that the aliphatic carbamate derivatives of the parent
compound 1 had equal or increased bindingaffinity
for avb3. In the ELISA, the Boc-protected neopentyl
derivative 5a had an IC50 of 0.72 nM compared to
14.1 nM for compound 1. The propyl derivative 5c,
which had the same chain length but less steric bulk than
5a had only slightly decreased activity.
Deprotection to reveal the terminal amine resulted in a
net decrease in activity for all four linker lengths studied,
however the neopentyl 6a retained superior activity in
this free amine series. The most conspicuous decrease
in activity is observed for the deprotection of the propyl
derivative indicating the geminal methyl groups of the
neopentyl derivatives favor interaction with the recep-
tor. The trends observed in the ELISA are mirrored in
the cell adhesion assay results. Most notable is the dra-
matic increase in activity seen for 5a when compared to
1. This is particularly encouraging as the cell adhesion
assay is designed to more closely resemble activity in
vivo when compared to the ELISA. Furthermore, both
neopentyl derivatives 5a and 6a show superior avb3
bindingaffinity when compared to the potent cyclic pep-
tide c-[RGDfV] in our assay system.
2.2. In vitro binding affinity assay
The integrin antagonist compounds were evaluated for
their ability to competitively inhibit the attachment of
the natural ligand vitronectin to purified human avb3
by ELISA and avb3-expressingM21 melanoma cells in
cell adhesion assays. ELISA measured competitive bind-
ingof the integrin antagonist and biotinylated human
vitronectin for the immobilized receptor avb3. For cell
adhesion assays, the M21 cells were first incubated with
the integrin antagonists then subsequently added to 96-
well plates previously coated with human vitronectin.
Inhibition of cell adhesion efficiency was measured by
the detection of the remainingcells stained with crystal
violet after plate washing. The results are summarized
in Table 1. Inhibition of cell adhesion is expressed as a
ratio (Q) relative to the parent compound 1 due to the
inherent variability of the cells used in the assay. The re-
2.3. In vitro fluorescence microscopy
Further studies with fluorescence microscopy were used
to determine the cellular distribution of 7 in the context
of avb3-expressingcells (M21) in vitro. The M21 cells
were incubated for 5 min followingthe addition of
500 lL of 7 (50 lg/mL). Cells were mounted and coun-
terstained with 40,6-diamidino-2-phenylindole (DAPI)
to delineate the vessels and observed with fluorescence
microscopy. Negative controls were given the same con-
centration of FITC only. Figure 1 summarizes the re-
sults, which suggest that the signal from 7 is found on
the cell membrane surface compared to controls, which
demonstrate non-specific accumulation of FITC outside
the cell.
Table 1. In vitro evaluation of carbamate-linked integrin antagonists
Compds
R
n
Y
ELISA
IC50, nMa
Cell
adhesion
Qb
1
5a
—
—
1
—
–CO2C(CH3)3
14.1 ( 1.4)
—
CH3
H
0.72 ( 0.08) 65.8
1.70 ( 0.20) 5.8
1.02 ( 0.12) 22.8
5b
5c
0
–CO2C(CH3)3
–CO2C(CH3)3
H
1
5d
6a
H
2
–CO2C(CH3)3
H
1.34 ( 0.14)
2.94 ( 0.19)
3.64 ( 0.30)
6.68 ( 0.54)
3.13 ( 0.33)
3.4 ( 0.33)
6.41 ( 0.49)
4.7
5.4
4.0
3.9
1.8
CH3
H
1
6b
6c
0
H
H
1
H
6d
7
H
2
H
CH3
—
1
—
Fluorescein
—
—
—
2.4. In vivo tumor accumulation and biodistribution
c-[RGDfV]c
a Values are averages of at least three determinations.
Optical imaging of excised organs was accomplished by
usinga multispectral imagingsystem usingtunable filter
technology optically coupled to a CCD to generate
b Ratio of the IC50 of 1 to test compound per individual determination.
c Purchased from Bachem Bioscience.

3766
C. A. Burnett et al. / Bioorg. Med. Chem. 13 (2005) 3763–3771
sectioned, mounted, and counterstained with DAPI
and observed under a fluorescence microscope. The re-
sults are summarized in Figure 4 and they suggest the
intracellular accumulation of compound 7 in avb3-
expressingcancer cells.
3. Conclusion
In conclusion, attachment of aliphatic carbamate linkers
to integrin antagonist 1 resulted in a series of com-
pounds with enhanced bindingaffinity to avb3. The neo-
pentyl derivative 5a, with the most lipophilic and bulky
linker, was the most efficacious inhibitor of the series in
both ELISA and cell adhesion assays. In addition, in
vitro and in vivo data suggest compound 7 binds to
and accumulates in avb3-expressingtumor cells. Biodis-
tribution data obtained from optical imaging of ex vivo
tumors and various tissues also suggest the preferential
bindingof compound 7 in avb3-expressingtumor cells
even after partial hepatobiliary clearance. The terminal
amine of compound 6a can be functionalized to conju-
gate molecular beacons. Using the amine functionality
in compound 6a, integrin avb3-targeted small molecule
imaging probes for other imaging modalities including
PET, SPECT, and MRI are currently beingdeveloped
by conjugating appropriate molecular beacons to 6a.
Figure 1. Bar represents 20 lM. Fluorescence microscopy of in vitro
cell assay of compound 7 and FITC. M21 human melanoma cells
expressing avb3 were cultured in glass chambers. Cells were given fresh
media 30 min prior to treatment. Compound 7 (500 lL, 50 lg/mL) was
added to each glass chamber and left to incubate for 5 min. Cells were
washed with warm PBS three times and subsequently mounted,
counterstained with DAPI and observed under a fluorescence micro-
scope. Control chambers were given the same concentration of FITC
without compound 7. Signal from compound 7 is present on the cell
membrane surface compared to controls, which demonstrate non-
specific accumulation of FITC signal outside the cell. (A) Cell uptake
of compound 7; (B) cell uptake of the negative control FITC.
multispectral data. Duringimage acquisition, FITC was
characteristically excited at 494 nm although emission
data were collected from 500 to 950 nm includingdata
from FITCÕs characteristic emission at 518 nm. Because
fluorescein derivates are best suited for imaging superfi-
cial tissues due to the shallow penetration of blue light in
tissue, tumors and various organs were excised to obtain
more accurate biodistribution data.28 For in vivo optical
imaging, the M21 cells were injected into the right flanks
of mice and allowed to grow for 14–21 days into a pal-
pable mass. Compound 7 was injected into the lateral
tail vein of M21 tumor-bearingmice. Controls were
M21 tumor-bearingmice that received no injection.
Preferential retention of the agent in the tumor was fast
and showed marked signal in the tumor at 30 min com-
pared to other organs. The signal remained pronounced
in the tumor and showed decreased signal in the liver at
4 h. The results are summarized in Figure 2. Qualitative
data from the images in Figure 2 are summarized in Fig-
ure 3. At 30 min, the signal intensity in the tumor is
nearly three times that found in the liver and muscle.
At 4 h, the signal intensity in the tumor is nearly four
times that in the liver. These results suggest that com-
pound 7 rapidly accumulates in tumor tissue and contin-
ues to stay bound to avb3-expressingcancer cells despite
partial clearingby the hepatobiliary system after 4 h.
This rapid accumulation of compound 7 in the cancer
cell is consistent with in vitro data aforementioned,
which demonstrates 7 bindingto the cancer cell mem-
brane surface after 5 min. To further elucidate the accu-
mulation of compound 7 in avb3-expressingtumors in
4. Experimental
4.1. General
All solvents and reagents were purchased from commer-
cial sources and used without further purification. Infra-
red spectra were prepared as KBr pellets and recorded
on a Perkin–Elmer Spectrum GX FT-IR spectropho-
1
tometer. H NMR and 13C NMR data were recorded
on a Bruker Ultrashield 300 instrument at 300 MHz
for proton and 75 MHz for carbon usingDMSO- d6 or
CDCl3. Mass spectral data for compounds 4a–d were re-
corded on a PE/SCIEX API 2000 instrument whereas
high and low resolution mass spectral data for all other
compounds were recorded on a JEOL JMS HX110
instrument usingpositive mode FAB with NBA ( meta-
nitro benzoic acid) matrix. Purified human integrin
avb3 and purified human vitronectin were purchased
from Chemicon International (Temecula, CA). c-
[RGDfV] was purchased from Bachem Bioscience, Inc.
(Kingof Prussia, PA) and was used as acquired. For
in vitro assay screening, stock solutions of integrin
antagonistic substances were prepared in anhydrous
DMSO and diluted with purified water. Chemilumines-
cence for ELISA assays and A590 for cell adhesion as-
says were recorded on a Wallac Victor2 1420 Multilabel
Counter.
4.2. Representative procedure for synthesis of N-Boc-
aminoalkyl-carbonyl-imidazoles
vivo, M21 tumor-bearingmice were injected with
7
and subsequently received a lateral tail vein injection
of rhodamine–lectin 2 h later, just prior to eutha-
nization. Rhodamine–lectin is used to delineate the ves-
sels duringthe microscopic imaign.g Tumors were
4.2.1.
3-(Boc-amino)-neopentyl-1-O-carbonylimidazole
(4a). N,N-Diisopropylethylamine (1.7 mL, 9.85 mmol)
was added to a stirred solution of 3a (2.00 g, 9.85 mmol)

C. A. Burnett et al. / Bioorg. Med. Chem. 13 (2005) 3763–3771
3767
Figure 2. Biodistribution of compound 7 in ex vivo tissues. Imaging of excised tumors and organs 30 min and 4 h post-injection of compound 7 in
M21 avb3-expressing tumor-bearing mice. The organs include tumor, heart, lung, liver, spleen, kidney, and skeletal muscle. Compound 7 is excreted
through the hepatobiliary and genitourinary system. Compound 7 retains signal in the tumor even after partial clearance through the hepatobiliary
system at 4 h. Left panels show the tissue layout, right panels show the results from optical imaging.
in anhydrous CH2Cl2 (8 mL) under argon. To this solu-
tion, 1,10-carbonyldiimidazole (2.80 g, 17.3 mmol) in
CH2Cl2 (22 mL) was added as a slurry and this mixture
was stirred at room temperature for 3 h. The mixture
was diluted to 50 mL with CH2Cl2, cooled to 0 ꢀC,
and washed with ice-cold water (2 · 50 mL). The solvent
was dried over Na2SO4, evaporated under reduced
pressure, and dried under high vacuum to give 4a
(2.72 g, 9.16 mmol, 93%) as a white solid. IR (KBr,
cmꢀ1) 3367w, 3142m, 1759s, 1709s, 1474s, 1303s,
1179s, 1006m. 1H NMR (300 MHz, DMSO-d6): 8.28
(s, 1H), 7.61 (s, 1H), 7.08 (s, 1H), 7.00 (t, J = 6.4 Hz,
1H), 4.04 (s, 2H), 2.95 (d, J = 6.4 Hz, 2H), 1.35 (s,
9H), 0.92 (s, 6H). 13C NMR (75 MHz, DMSO-d6):
156.5, 148.8, 137.7, 130.6, 117.9, 78.0, 73.6, 47.2, 36.0,
28.6, 22.6. MS (electrospray): m/z 298 (100, [M+H]+,
calcd 298).
4.2.2. 2-(Boc-amino)-ethyl-1-O-carbonylimidazole (4b).
White solid. Yield 90%. IR (KBr, cmꢀ1) 3403s, 2980m,
1748s, 1713s, 1526s, 1322s, 1166s, 1006m. 1H NMR
(300 MHz, DMSO-d6): 8.27 (s, 1H), 7.61 (s, 1H), 7.15

3768
C. A. Burnett et al. / Bioorg. Med. Chem. 13 (2005) 3763–3771
DMSO-d6): 156.1, 148.8, 137.6, 130.1, 117.9, 77.9,
68.3, 38.6, 28.7, 26.1, 25.7. MS (electrospray): m/z 284
Tissue Signal Intensity
180
(100, [M+H]+, calcd 284).
30 min
4 hours
160
140
120
100
80
4.3. Representative procedure for coupling IA 1 with N-
Boc-aminoalkyl-carbonyl-imidazoles
60
40
20
0
4.3.1. 4-[2-(3,4,5,6-Tetrahydropyrimidin-2-ylamino)ethyl-
oxy]benzoyl-2-(S)-[N-(3-butyloxycarbonylamino-neopenta-
1-carbamyl)]-aminoethylsulfonylamino-b-alanine (5a). To
a mixture of 4a (0.42 g, 1.42 mmol) in anhydrous
DMSO (12 mL) under argon, was added (0.50 g,
1.02 mmol) of 1, as the hydrochloride salt, followed by
addition of N,N-diisopropylethylamine (0.53 mL,
3.05 mmol). The mixture was stirred under argon at
70 ꢀC for 18 h. Excess solvent was removed by rotary
evaporation and the remainingresidue was washed with
water (2 · 5 mL) then with EtOAc (2 · 3 mL). The
remainingsolid was dissolved in hot EtOH, cooled
and the resultingprecipitate was collected by vacuum
filtration then dried under high vacuum to give 5a
(0.65 g, 0.95 mmol, 66%) as an off-white solid. IR (KBr,
cmꢀ1) 3323s, 3051m, 2973s, 2884m, 1704s, 1650s,
tumor heart
lung
liver spleen kidney muscle
Tissue Type
Figure 3. Qualitative data of the biodistribution of compound 7 in ex
vivo tissues. Signal intensity was measured from images obtained of
various ex vivo tissues, one animal for each time point, including
tumor, heart, lung, liver, spleen, kidney, and skeletal muscle. Signal
from compound 7 is most pronounced in avb3-expressingtumor tissue
at 30 min and 4 h post-injection even after clearance of signal from the
liver due to hepatobiliary clearance.
1
(t, J = 5.7 Hz, 1H), 7.07 (s, 1H), 4.33 (t, J = 5.1 Hz, 2H),
3.33 (m, 2H), 1.36 (s, 9H). 13C NMR (75 MHz, CDCl3):
155.8, 148.6, 137.2, 130.7, 117.2, 80.0, 67.4, 39.4, 28.3.
MS (electrospray): m/z 256 (100, [M+H]+, calcd 256).
1607s, 1533m, 1505s, 1319s, 1146s, 1057m. H NMR
(300 MHz, DMSO-d6): 8.86 (br, 1H), 8.58 (br, 2H),
8.29 (br, 1H), 7.73 (d, J = 8.6 Hz, 2H), 7.29 (t,
J = 5.3 Hz, 1H), 7.02 (br, 1H), 6.92 (d, J = 8.6 Hz,
2H), 6.79 (t, J = 5.8 Hz, 1H), 4.04 (t, J = 4.8 Hz, 2H),
3.73 (m, 1H), 3.67 (s, 2H), 3.55 (m, 2H), 3.47 (m, 2H),
3.37 (t, J = 7.0 Hz, 2H), 3.24 (br, 4H), 3.18 (m, 2H),
2.82 (d, J = 5.8 Hz, 2H), 1.80 (m, 2H), 1.37 (s, 9H),
0.79 (s, 6H). 13C NMR (75 MHz, DMSO-d6): 173.5,
165.8, 160.8, 156.8, 156.5, 153.6, 129.2, 127.7, 114.5,
77.9, 70.5, 66.5, 56.6, 51.8, 47.5, 43.3, 38.4, 36.2, 35.9,
28.7, 22.6, 20.2, 15.6. MS (FAB, NBA): m/z 686 (100,
[M+H]+). HR-MS (FAB, NBA): m/z 686.3181
([M+H]+, C29H48N7O10S, calcd 686.3184).
4.2.3. 3-(Boc-amino)-propyl-1-O-carbonylimidazole (4c).
White solid. Yield 92%. IR (KBr, cmꢀ1) 3337s,
3141m, 2977m, 1753s, 1721s, 1520s, 1415m, 1160s,
1
1016m. H NMR (300 MHz, DMSO-d6): 8.27 (s, 1H),
7.60 (s, 1H), 7.08 (s, 1H), 6.94 (t, J = 5.5 Hz, 1H), 4.37
(t, J = 6.2 Hz, 2H), 3.09 (m, 2H), 1.84 (m, 2H), 1.35 (s,
9H). 13C NMR (75 MHz, CDCl3): 155.9, 148.7, 137.1,
130.5, 117.1, 79.5, 66.0, 37.1, 29.2, 28.3. MS (electro-
spray): m/z 270 (100, [M+H]+, calcd 270).
4.2.4. 4-(Boc-amino)-butyl-1-O-carbonylimidazole (4d).
)
4.3.2. 4-[2-(3,4,5,6-Tetrahydropyrimidin-2-ylamino)ethyl-
oxy]benzoyl-2-(S)-[N-(2-butyloxycarbonylamino-ethyl-1-
carbamyl)]-aminoethylsulfonylamino-b-alanine (5b). White
powder. Yield 23%. IR (KBr, cmꢀ1) 3380s, 3048w,
2976m, 2886w, 1702s, 1652s, 1607s, 1529m, 1367m,
1254s, 1056m. 1H NMR (300 MHz, DMSO-d6): 8.73
(br, 1H), 8.48 (br, 2H), 8.35 (br, 1H), 7.72 (d,
Clear oil. Yield 90%. IR (KBr, cmꢀ1
3407s,
2976m, 2934m, 1764s, 1712s, 1526s, 1475m, 1283s,
1174s, 1004m. 1H NMR (300 MHz, DMSO-d6): 8.26
(s, 1H), 7.60 (s, 1H), 7.08 (s, 1H), 6.84 (t, J = 5.8 Hz,
1H), 4.37 (t, J = 6.3 Hz, 2H), 2.97 (m, 2H), 1.72 (m,
2H), 1.51 (m, 2H), 1.37 (s, 9H). 13C NMR (75 MHz,
Figure 4. Bar represents 50 lM. Fluorescence microscopy of M21 avb3-expressingtumors treated with compound 7. M21 (avb3 expressing) tumor-
bearingmice were injected with 7 and subsequently received a tail vein injection of rhodamine–lectin 2 h later, just prior to euthanization. Tumors
were sectioned, mounted, and counterstained with DAPI and observed under a fluorescence microscope. Results suggest the intracellular
accumulation of compound 7 in avb3-expressingcancer cells. (A) Compound 7; (B) DAPI; (C) rhodamine–lectin; (D) merge.

C. A. Burnett et al. / Bioorg. Med. Chem. 13 (2005) 3763–3771
3769
J = 8.6 Hz, 2H), 7.36 (t, J = 5.9 Hz, 1H), 6.96 (br, 1H),
6.92 (d, J = 8.6 Hz, 2H), 6.90 (t, J = 5.8 Hz, 1H), 4.04
(t, J = 5.3 Hz, 2H), 3.91 (t, J = 5.5 Hz, 2H), 3.63 (m,
1H), 3.55 (m, 2H), 3.47 (m, 2H), 3.34 (br, 2H), 3.24
(br, 4H), 3.16 (m, 2H), 3.11 (m, 2H), 1.81 (m, 2H), 1.37
(s, 9H). MS (FAB, NBA): m/z 644 (100, [M+H]+). HR-
2H), 3.26 (br, 4H), 3.17 (m, 2H), 2.70 (br, 2H), 1.82
(m, 2H), 0.94 (s, 6H). MS (FAB, NBA): m/z 586 (100,
[M+H]+). HR-MS (FAB, NBA): m/z 586.2657
([M+H]+, C24H40N7O8S, calcd 586.2659).
4.4.2. 4-[2-(3,4,5,6-Tetrahydropyrimidin-2-ylamino)ethyl-
oxy]benzoyl-2-(S)-[N-(2-amino-ethyl-1-carbamyl)]-amino-
ethylsulfonylamino-b-alanine hydrochloride (6b). 1H
NMR (300 MHz, DMSO-d6): 12.97 (br, 1H), 8.50 (br,
1H), 8.06 (br, 2H), 7.97 (br, 2H), 7.90 (br, 1H), 7.84
(d, J = 8.7 Hz, 2H), 7.59 (t, J = 5.5 Hz, 1H), 7.21 (t,
J = 5.0 Hz, 1H), 7.02 (d, J = 8.7 Hz, 2H), 4.14 (br,
2H), 4.12 (br, 2H), 3.70 (m, 1H), 3.53 (m, 2H), 3.51
(br, 2H), 3.49 (m, 2H), 3.26 (br, 4H), 3.17 (m, 2H),
3.03 (m, 2H), 1.82 (m, 2H). MS (FAB, NBA): m/z 544
(100, [M+H]+). HR-MS (FAB, NBA): m/z 544.2187
([M+H]+, C21H34N7O8S, calcd 544.2189).
MS
C26H42N7O10S, calcd 644.2714).
(FAB,
NBA):
m/z
644.2716
([M+H]+,
4.3.3. 4-[2-(3,4,5,6-Tetrahydropyrimidin-2-ylamino)ethyl-
oxy]benzoyl-2-(S)-[N-(3-butyloxycarbonylamino-propyl-
1-carbamyl)]-aminoethylsulfonylamino-b-alanine
(5c).
Off-white powder. Yield 36%. IR (KBr, cmꢀ1) 3380s,
2976m, 2928m, 2881w, 1687m, 1647s, 1607s, 1504s,
1367m, 1255s, 1178m, 1054w. 1H NMR (300 MHz,
DMSO-d6): 9.07 (br, 1H), 8.67 (br, 2H), 8.28 (br, 1H),
7.72 (d, J = 8.6 Hz, 2H), 7.34 (t, J = 5.3 Hz, 1H), 7.01
(br, 1H), 6.90 (d, J = 8.6 Hz, 2H), 6.85 (t, J = 5.5 Hz,
1H), 4.02 (t, J = 4.6 Hz, 2H), 3.92 (t, J = 6.2 Hz, 2H),
3.67 (m, 1H), 3.55 (m, 2H), 3.47 (m, 2H), 3.34 (br,
2H), 3.23 (br, 4H), 3.18 (m, 2H), 2.96 (m, 2H), 1.80
(m, 2H), 1.63 (m, 2H), 1.37 (s, 9H). 13C NMR
(75 MHz, DMSO-d6): 173.7, 165.7, 160.7, 156.6, 156.0,
153.7, 129.2, 127.7, 114.5, 78.0, 66.5, 62.3, 56.7, 51.6,
43.5, 38.4, 37.3, 35.9, 29.7, 28.7, 20.2 (21 of 22 reso-
nances observed). MS (FAB, NBA): m/z 658 (100,
[M+H]+). HR-MS (FAB, NBA): m/z 658.2872
([M+H]+, C27H44N7O10S, calcd 658.2870).
4.4.3. 4-[2-(3,4,5,6-Tetrahydropyrimidin-2-ylamino)ethyl-
oxy]benzoyl-2-(S)-[N-(3-amino-propyl-1-carbamyl)]-amino-
ethylsulfonylamino-b-alanine hydrochloride (6c). 1H
NMR (300 MHz, DMSO-d6): 12.96 (br, 1H), 8.50 (br,
1H), 7.95 (br, 2H), 7.91 (br, 2H), 7.87 (br, 1H), 7.83
(d, J = 8.6 Hz, 2H), 7.57 (t, J = 5.1 Hz, 1H), 7.29 (t,
J = 4.9 Hz, 1H), 7.02 (d, J = 8.6 Hz, 2H), 4.12 (t,
J = 4.7 Hz, 2H), 4.01 (t, J = 5.5 Hz, 2H), 3.70 (m, 1H),
3.55 (m, 2H), 3.50 (br, 2H), 3.48 (m, 2H), 3.26 (br,
4H), 3.15 (m, 2H), 2.84 (m, 2H), 1.84 (m, 2H), 1.82
(m, 2H). MS (FAB, NBA): m/z 558 (100, [M+H]+).
HR-MS (FAB, NBA): m/z 558.2344 ([M+H]+,
C22H36N7O8S, calcd 558.2346).
4.3.4. 4-[2-(3,4,5,6-Tetrahydropyrimidin-2-ylamino)ethyl-
oxy]benzoyl-2-(S)-[N-(4-butyloxycarbonylamino-butyl-1-
carbamyl)]-aminoethylsulfonylamino-b-alanine (5d). Off-
white powder. Yield 33%. IR (KBr, cmꢀ1) 3380s,
3051w, 2975m, 2935m, 2874m, 1687m, 1647s, 1607s,
1504s, 1366m, 1255s, 1148m, 1055m. 1H NMR
(300 MHz, DMSO-d6): 9.06 (br, 1H), 8.66 (br, 2H),
8.28 (br, 1H), 7.72 (d, J = 8.2 Hz, 2H), 7.33 (t,
J = 5.1 Hz, 1H), 6.98 (br, 1H), 6.90 (d, J = 8.2 Hz,
2H), 6.83 (t, J = 5.5 Hz, 1H), 4.02 (t, J = 4.7 Hz, 2H),
3.91 (t, J = 5.9 Hz, 2H), 3.66 (m, 1H), 3.55 (m, 2H),
3.46 (m, 2H), 3.41 (br, 2H), 3.23 (br, 4H), 3.18 (m,
2H), 2.90 (m, 2H), 1.80 (m, 2H), 1.49 (m, 2H), 1.47
(m, 2H), 1.36 (s, 9H). MS (FAB, NBA): m/z 672 (100,
[M+H]+). HR-MS (FAB, NBA): m/z 672.3029
([M+H]+, C28H46N7O10S, calcd 672.3027).
4.4.4. 4-[2-(3,4,5,6-Tetrahydropyrimidin-2-ylamino)ethyl-
oxy]benzoyl-2-(S)-[N-(4-amino-butyl-1-carbamyl)]-amino-
ethylsulfonylamino-b-alanine hydrochloride (6d). 1H
NMR (300 MHz, DMSO-d6): 12.96 (br, 1H), 8.50 (br,
1H), 8.02 (br, 2H), 7.95 (br, 2H), 7.88 (br, 1H), 7.84
(d, J = 8.6 Hz, 2H), 7.63 (t, J = 5.7 Hz, 1H), 7.22 (t,
J = 5.3 Hz, 1H), 7.01 (d, J = 8.6 Hz, 2H), 4.12 (br,
2H), 3.95 (br, 2H), 3.68 (m, 1H), 3.54 (m, 2H), 3.51
(br, 2H), 3.48 (m, 2H), 3.26 (br, 4H), 3.15 (m, 2H),
2.79 (m, 2H), 1.81 (m, 2H), 1.66 (m, 2H), 1.61 (m,
2H). MS (FAB, NBA): m/z 572 (100, [M+H]+). HR-
MS (FAB,
C23H38N7O8S, calcd 572.2502).
NBA): m/z 572.2507 ([M+H]+,
4.4. Representative procedure for deprotection of butyl-
oxycarbonyl integrin antagonists
4.5. 4-[2-(3,4,5,6-Tetrahydropyrimidin-2-ylamino)ethyl-
oxy]benzoyl-2-(S)-[N-(3-amino-neopenta-1-carbamyl)]-
aminoethylsulfonylamino-b-alanine fluorescein thiourea
(7)
4.4.1. 4-[2-(3,4,5,6-Tetrahydropyrimidin-2-ylamino)ethyl-
oxy]benzoyl-2-(S)-[N-(3-amino-neopenta-1-carbamyl)]-amino-
ethylsulfonylamino-b-alanine hydrochloride (6a). A mix-
ture of 5a (0.10 g, 0.14 mmol) and 4 M HCl in dioxane
(50 mL) was stirred under argon at 0 ꢀC for 3 h. The
heterogeneous mixture was frozen at ꢀ80 ꢀC then
lyophilized to dryness to give 6a in quantitative yield
To a mixture of 6a (0.11 g, 0.17 mmol) in anhydrous
DMSO (10 mL) under argon, was added N,N-diisoprop-
ylethylamine (0.06 mL, 0.34 mmol) followed by addi-
tion of fluorescein isothiocyanate (0.07 g, 0.19 mmol).
This mixture was stirred under argon at 40 ꢀC for
18 h. Excess solvent was removed by rotary evaporation
and the remainingresidue was purified by reverse phase
HPLC (4:1 MeOH–H2O, 1 mL/min, tR 10.5 min, C18
Microsorb, Varian, Inc.). The collected fractions were
combined and excess solvent was removed by rotary
evaporation followed by high vacuum to give 7
1
as a hydrochloride salt. H NMR (300 MHz, DMSO-
d6): 12.97 (br, 1H), 8.49 (br, 1H), 7.99 (br, 2H), 7.95
(br, 2H), 7.88 (br, 1H), 7.84 (d, J = 8.3 Hz, 2H), 7.60
(t, J = 5.1 Hz, 1H), 7.40 (t, J = 5.0 Hz, 1H), 7.02 (d,
J = 8.3 Hz, 2H), 4.12 (t, J = 6.4 Hz, 2H), 3.79 (s, 2H),
3.70 (m, 1H), 3.53 (m, 2H), 3.51 (br, 2H), 3.48 (m,

3770
C. A. Burnett et al. / Bioorg. Med. Chem. 13 (2005) 3763–3771
1
(0.04 g, 0.04 mmol, 24%) as a dark orange powder. H
NMR (300 MHz, MeOD-d4): 8.18 (s, 1H), 7.83 (d,
J = 8.0 Hz, 1H), 7.68 (d, J = 8.5 Hz, 2H), 7.11 (d,
J = 8.0 Hz, 1H), 7.10 (m, 2H), 6.82 (d, J = 8.5 Hz,
2H), 6.50 (s, 2H), 6.49 (m, 2H), 4.02 (t, J = 6.0 Hz,
1H), 3.92 (t, J = 4.5 Hz, 2H), 3.89 (s, 2H), 3.83 (m,
2H), 3.74 (s, 2H), 3.68 (d, J = 6.0 Hz, 2H), 3.62 (br,
2H), 3.43 (t, J = 5.9 Hz, 2H), 3.18 (t, J = 5.6 Hz, 4H),
1.85 (m, 2H), 1.05 (s, 6H). MS (electrospray): m/z 1020
(100, [M+2NaꢀH]+), m/z 973 (100, [MꢀH]ꢀ). HR-MS
(FAB, NBA): m/z 975.3021 ([M+H]+, C45H50N13O8S2,
calcd 975.2939).
mounted, counterstained with DAPI and observed
under a fluorescence microscope. Negative control
chambers were given the same concentration of FITC
only.
4.7. In vivo procedures
4.7.1. Animal protocols. All studies were conducted in
compliance with the National Institutes of Health Clini-
cal Center Animal Use and Care CommitteeÕs require-
ments for the care and use of laboratory animals in
research. The human melanoma (M21, received from
Dr. David Cheresh of Scripps Research Institutes, La
Jolla, CA) expressing avb3 was induced by subcutaneous
injection in the right flank. Palpable masses were de-
tected at 14–21 days post implant. The animals were
manually restrained duringtail vein injection. All tail
vein injections were carried out with a 30 gauge needle
placed in a lateral tail vein. Each animal weighing about
18–20 greceived 100 lL of a 6 mg/mL solution of com-
pound 7. In experiments where animals were given a
rhodamine–lectin injection to outline tumor vasculature,
mice were given 250 lL of rhodamine–lectin (5 mg/mL)
through the lateral tail vein.
4.6. In vitro assay procedures
4.6.1. ELISA. Purified integrin avb3 protein (Chemicon
International, Temecula, CA) was applied to 96-well
polystyrene microtiter plates at 0.1 lg/well. After over-
night incubation at 4 ꢀC the plates were washed, and
then blocked with milk solution (KPL, Inc., Gaithers-
burg, MD) at room temperature for 2 h. The blocking
buffer was removed, and the plates were inoculated in
quadruplicate with integrin antagonistic substances with
a typical startingconcentration of 1.25 lM. Serial dilu-
tions were prepared in the 96-well plates usingmulti-
channel pipettes. To each well was added biotinylated
vitronectin solution (0.1 lg/well) as standard competi-
tor. The plates were incubated at room temperature
for 3 h, washed, and the bound vitronectin was detected
4.7.2. Imaging procedure. A simple noninvasive in vivo
multispectral fluorescence imaging apparatus employed
to assess the localization and distribution of various
fluorophores has been previously described.29 Laser
light of the appropriate incident wavelength to excite
the fluorophore was launched into a fiber optic bundle.
A defocusinglens in position after the bundle expanded
the beam such that the entire mouse was illuminated. An
argon laser excited the fluorescein compound at the
appropriate wavelength of 494 nm and a 515 nm long
pass filter was used to detect all emissions past 515 nm
up to 950 nm. The detector was a CRI, Inc. Maestro
CCD camera. An interference filter in front of the
CCD (515 nm for fluorescein) allowed for detection of
the emitted fluorescent light only. Images were acquired
and processed usingMaestro acquisition and analysis
software from CRI, Inc. Typically, control and treated
ex vivo tumor and organ tissue was imaged concurrently
under fixed settings. Data analysis consisted of subtract-
ing(pixel by pixel) backrgound auto fluorescence in
control tissues from treated tissue in the same image
and displayingthe results in gray scale or false color.
Semi-quantitative data were obtained by usingImageJ
analysis software to semi-quantitate the signal intensity
values recorded in Maestro acquisition and analysis
software.
usingNeutrAvidin-HRP conjuagte at 0.01
lg/well
(Pierce, Rockford, IL) and LumiGlo chemiluminescent
substrate system (KPL, Inc., Gaithersburg, MD). The
luminescence was read usinga Wallac Counter. The
concentration of inhibitor producing50% inhibition
(IC50) of vitronectin bindingto avb3 was calculated
based on a curve fittingmodel usingKaleidaGraph 3.5
(Synergy Software, Reading, PA).
4.6.2. Cell adhesion assay. Integrin antagonistic sub-
stances were incubated with human melanoma M21 cells
expressing avb3 integrin proteins on their surface. At a
startingconcentration of 0.5 mM, serial dilutions of
the integrin antagonist/cell culture mixture were added
to 96-well plates previously coated with purified human
vitronectin (Chemicon International, Temecula, CA)
and blocked with BSA. After incubation at 30 ꢀC for
1 h, the plates were washed with distilled water to re-
move the unbound cells. The bound cells were then
stained with 0.05% Crystal Violet, washed again with
distilled water, and lysed with 1% SDS. Absorption @
590 nm was measured on a Wallac Counter and the con-
centration of inhibitor producing50% inhibition (IC
)
50
of vitronectin bindingto avb3 is calculated based on a
curve fittingmodel usingKaleidaGraph 3.5 (Syneryg
Software, Reading, PA).
Acknowledgements
We would like to thank Dr. David Cheresh of Scripps
Research Institutes, La Jolla, CA for kindly providing
the M21 cells and the Michigan State University Mass
Spectrometry Facility for low and high resolution mass
spectral analysis of integrin antagonist compounds. J.Q.
would like to thank the Howard Hughes Medical Insti-
tute—National Institutes of Health Research Scholars
Program for their support.
4.6.3. Cell uptake assay. M21 human melanoma cells
expressing avb3 were cultured in glass chambers. Cells
were given fresh media 30 min prior to treatment. Com-
pound 7 (500 lL, 50 lg/mL) was added to each glass
chamber and left to incubate for 5 min. Cells were
washed with warm PBS three times and subsequently

C. A. Burnett et al. / Bioorg. Med. Chem. 13 (2005) 3763–3771
3771
Ruminski, P. G.; Teitelbaum, S. L. J. Clin. Invest. 1997,
99, 2284–2292.
References and notes
17. (a) Eskens, F. A. L. M. Br. J. Cancer 2004, 90, 1–7; (b)
Buerkle, M. A.; Pahernik, S. A.; Sutter, A.; Jonczyk, A.;
Messmer, K.; Dellian, M. Br. J. Cancer 2002, 86, 788–
795.
1. Takagi, J.; Springer, T. A. Immunol. Rev. 2002, 186, 141–
163.
2. Humphries, M. J. Biochem. Soc. Trans. 2000, 28, 311–339.
3. Arnaout, M. A. Immunol. Rev. 2002, 186, 125–140.
4. Yamada, K. M.; Geiger, B. Curr. Opin. Cell Biol. 1997, 9,
76–85.
18. Eliceiri, B. Circ. Res. 2001, 89, 1104–1110.
19. Hood, J. D.; Bednarski, M.; Frausto, R.; Guccione, S.;
Reisfeld, R. A.; Xiang, R.; Cheresh, D. A. Science 2002,
296, 2404–2407.
5. Calderwood, D. A.; Shattil, S. J.; Ginsberg, M. H. J. Biol.
Chem. 2000, 275, 22607–22610.
20. (a) Haubner, R.; Wester, H.-J.; Weber, W. A.; Mang, C.;
Ziegler, S. I.; Goodman, S. L.; Senekowitsch-Schmidtke,
R.; Kessler, H.; Schwaiger, M. Cancer Res. 2001, 61,
1781–1785; (b) Ogawa, M.; Hatano, K.; Oishi, S.;
Kawasumi, Y.; Fujii, N.; Kawaguchi, M.; Doi, R.;
Imamura, M.; Yamamoto, M.; Ajito, K.; Mukai, T.; Saji,
H.; Ito, K. Nucl. Med. Biol. 2003, 30, 1–9.
6. (a) Plow, E. F.; Haas, T. A.; Zhang, L.; Loftus, J.; Smith,
J. W. J. Biol. Chem. 2000, 275, 21785–21788; (b) Xiong,
J.-P.; Stehle, T.; Zhang, R.; Dunker, R.; Joachimiak, A.;
Frech, M.; Goodman, S. L.; Arnaout, M. A. Science 2002,
296, 151–155.
7. Rodan, S. B.; Rodan, G. A. J. Endocrinol. 1997, 154, S47–
S56.
21. (a) Harris, T. D.; Kalogeropoulos, S.; Nguyen, T.; Liu, S.;
Bartis, J.; Ellars, C.; Edwards, S.; Onthank, D.; Silvia, P.;
Yalamanchili, P.; Robinson, S.; Lazewatsky, J.; Barrett,
J.; Bozarth, J. Cancer Biother. Radiopharm. 2003, 18, 627–
641; (b) Liu, S.; Harris, T. D.; Ellars, C. E.; Edwards, D.
S. Bioconjugate Chem. 2003, 14, 1030–1037; (c) Onthank,
D. C.; Liu, S.; Silva, P. J.; Barrett, J. A.; Harris, T. D.;
Robinson, S. P.; Edwards, D. S. Bioconjugate Chem. 2004,
15, 235–241.
8. Storgard, C. M.; Stupack, D. G.; Jonczyk, A.; Goodman,
S. L.; Fox, R. I.; Cheresh, D. A. J. Clin. Invest. 1999, 103,
47–54.
9. Friedlander, M.; Theesfeld, C. L.; Sugita, M.; Fruttinger,
M.; Thomas, M. A.; Chang, S.; Cheresh, D. A. Proc. Natl.
Acad. Sci. U.S.A. 1996, 93, 9764–9769.
10. (a) Eliceiri, B. P.; Cheresh, D. A. J. Clin. Invest. 1999, 103,
1227–1230; (b) Brooks, P. C.; Clark, R. A.; Cheresh, D. A.
Science 1994, 264, 569–571.
22. (a) Sipkins, D. A.; Cheresh, D. A.; Kazemi, M. R.; Nevin,
L. M.; Bednarski, M. D.; Li, K. C. P. Nat. Med. 1998, 4,
623–626; (b) Winter, P. M.; Caruthers, S. D.; Kassner, A.;
Harris, T. D.; Chinen, L. K.; Allen, J. S.; Lacy, E. K.;
Zhang, H.; Robertson, J. D.; Wickline, S. A.; Lanza, G.
M. Cancer Res. 2003, 63, 5838–5843.
23. (a) Bloch, S.; Liang, K.; Dorshow, R. B.; Ye, Y.; Achilefu,
S. I. Proc. SPIE 2004, 5329, 222–228; (b) Wang, W.; Ke,
S.; Wu, Q.; Charnsangavej, C.; Gurfinkel, M.; Gelovani, J.
G.; Abbruzzese, J. L.; Sevick-Muraca, E. M.; Li, C. Mol.
Imaging 2004, 3, 343–351; (c) Chen, X.; Conti, P. S.;
Moats, R. A. Cancer Res. 2004, 64, 8009–8014.
´
´
´
11. Guerrero, C. A.; Mendez, E.; Zarate, S.; Isa, P.; Lopez, S.;
Arlas, C. F. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 14644–
14649.
12. Wickham, T. J.; Mathias, P.; Cheresh, D. A.; Nemerow,
G. R. Cell 1993, 73, 309–319.
13. (a) Pierschbacher, M. D.; Ruoslahti, E. Nature 1984, 309,
30–33; (b) Ruoslahti, E.; Pierschbacher, M. D. Cell 1986,
44, 517–518.
14. (a) Haubner, R.; Gratias, R.; Diefenbach, B.; Goodman,
S. L.; Jonczyk, A.; Kessler, H. J. Am. Chem. Soc. 1996,
118, 7461–7472; (b) Duggan, M. E.; Hutchinson, J. H.
Exp. Opin. Ther. Pat. 2000, 10, 1367–1383.
24. Duggan, M. E.; Duong, L. T.; Fisher, J. E.; Hamill, T. G.;
Hoffman, W. F.; Huff, J. R.; Ihle, N. C.; Leu, C.-T.; Nagy,
R. M.; Perkins, J. J.; Rodan, S. B.; Wesolowski, G.;
Whitman, D. B.; Zartman, A. E.; Rodan, G. A.; Hartman,
G. D. J. Med. Chem. 2000, 43, 3736–3745.
25. Ensch, C.; Hesse, M. Helv. Chim. Acta 2003, 86, 233–246.
26. (a) Blaszykowski, C.; Dhimane, A.-L.; Fensterbank, L.;
Malacria, M. Org. Lett. 2003, 5, 1341–1344; (b) Mattingly,
P. G. Synthesis 1990, 4, 366–368; (c) Gray, C. J.; Parker,
R. C. Tetrahedron 1975, 31, 2940–2943; (d) Lee, B. H.;
Miller, M. J. J. Org. Chem. 1983, 48, 24–31.
15. (a) Folkman, J. Nat. Med. 1995, 1, 27–31; (b) Schnitzer, J.
E. N. Engl. J. Med. 1998, 339, 472–474; (c) Arap, W.;
Pasqualini, R.; Ruoslahti, E. Science 1998, 279, 377–380.
16. (a) Coleman, P. J.; Brashear, K. M.; Askew, B. C.;
Hutchinson, J. H.; McVean, C. A.; Duong, L. T.; Feuston,
B. P.; Fernandez-Metzler, C.; Gentile, M. A.; Hartman,
G. D.; Kimmel, D. B.; Leu, C.-T.; Lipfert, L.; Merkle, K.;
Pennypacker, B.; Prueksaritanont, T.; Rodan, G. A.;
Wesolowski, G. A.; Rodan, S. B.; Duggan, M. E. J. Med.
Chem. 2004, 47, 4829–4837; (b) Hutchinson, J. H.;
Halczenko, W.; Brashear, K. M.; Breslin, M. J.; Coleman,
P. J.; Duong, L. T.; Fernandez-Metzler, C.; Gentile, M.
A.; Fisher, J. E.; Hartman, G. D.; Huff, J. R.; Kimmel, D.
B.; Leu, C.-T.; Meissner, R. S.; Merkle, K.; Nagy, R.;
Pennypacker, B.; Perkins, J. J.; Prueksaritanont, T.;
Rodan, G. A.; Varga, S. L.; Wesolowski, G. A.; Zartman,
A. E.; Rodan, S. B.; Duggan, M. E. J. Med. Chem. 2003,
46, 4790–4798; (c) Engleman, V. W.; Nickols, G. A.; Ross,
F. P.; Horton, M. A.; Griggs, D. W.; Settle, S. L.;
27. The chiral purity of the deprotected compounds 6a–d is
currently under investigation.
28. Achilefu, S.; Jimenez, H. N.; Dorshow, R. B.; Bugaj, J. E.;
Webb, E. G.; Wilhelm, R. R.; Rajagopalan, R.; Johler, J.;
Erion, J. L. J. Med. Chem. 2002, 45, 2003–2015.
29. Levenson, R.; Mansfield, J. R. Abstract ID 184, The Thrid
Annual Meeting of the Society of Molecular Imaging, Sep
9–12, 2004.