Synthesis of Tc-99m labeled glucosamino-Asp-cyclic(Arg-Gly-Asp-d-Phe-Lys) as a potential angiogenesis imaging agent

Article abstract of DOI:10.1016/j.bmc.2007.08.054

Angiogenesis imaging agents for single photon emission computed tomography (SPECT) play a role in diagnosing tumor-induced angiogenesis as well as tumor metastasis. We synthesized and evaluated radiolabeled RGD glycopeptides by incorporation of the [

Full text of DOI:10.1016/j.bmc.2007.08.054

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 15 (2007) 7755–7764
Synthesis of Tc-99m labeled glucosamino-Asp-cyclic(Arg-
Gly-Asp-D-Phe-Lys) as a potential angiogenesis imaging agent
Byung Chul Lee,a Hyun Ju Sung,a Ji Sun Kim,a Kyung-Ho Jung,b
Yearn Seong Choe,b Kyung-Han Leeb and Dae Yoon Chia,*
aDepartment of Chemistry, Inha University, 253 Yonghyundong, Namgu, Inchon 402-751, Republic of Korea
bDepartment of Nuclear Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine,
50 Ilwon-dong Kangnam-ku, Seoul 135-710, Republic of Korea
Received 21 April 2007; revised 22 August 2007; accepted 28 August 2007
Available online 1 September 2007
Abstract—Angiogenesis imaging agents for single photon emission computed tomography (SPECT) play a role in diagnosing
tumor-induced angiogenesis as well as tumor metastasis. We synthesized and evaluated radiolabeled RGD glycopeptides by incor-
poration of the [99mTc(CO)3(H2O)3]+. 99mTc labeled glucosamino-D-c(RGDfK) ([99mTc]2) was prepared in 90–93% radiochemical
yields (decay corrected). In vitro cell binding assays demonstrated selective binding [99mTc]2 to human umbilical vein endothelial
(HUVE) cells, with inhibition of binding to 37.3% of control levels by 10 lM of cold authentic compounds. In addition,
[
labeled RGD glycopeptides may have value for non-invasive assessment of angiogenesis.
99mTc]2 was shown to have high binding affinity to purified avb3 integrin (IC50 = 1.5 nM). These results suggest that these radio-
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
this RGD sequence (cRGD) as shown in Figure 1. These
include cRGD glycopeptides such as cRGD[125I]yV,
cRGD[125I]yK(SAA), and [18F]galacto-cRGDfK, as
avb3 integrin antagonists labeled with iodine-125 or
fluorine-18.15–18 More recently, an RGD radiotracer
composed of 1,4,7,10-tetraazadodecane-N,N0,N00,N000-
tetraacetic acid (DOTA) or hydrazinonicotinamide
(HYNIC) conjugate chelated with 111In, 90Y, or 99mTc
has been introduced.19,20
Integrins are a superfamily of heterodimeric transmem-
brane glycoproteins that consist of 19 a-subunits and
8 b-subunits known to date. These receptors play a ma-
jor role in cellular adhesion, migration, and signal trans-
duction.1–3 Among them, avb3 integrin is involved in
cell–cell and cell–matrix interaction, and plays a
particularly important role in tumor metastasis and
angiogenesis.4,5 In addition to being implicated in can-
cer progression, avb3 integrin expression is also involved
in improvement and healing of ischemic lesions.6,7
Blocking of avb3 integrins leads to apoptosis of endothe-
lial cells and inhibition of tumor blood vessel forma-
tion.8 The binding region in the avb3 integrin contains
the tripeptide amino acid sequence: arginine (R), glycine
(G), and aspartic acid (D) which is commonly called
RGD.9–14 As such, most angiogenesis imaging agents
that have been developed contain a cyclic form of
The design of new radiolabeled derivatives containing a
RGD moiety should include the following criteria: low
hepatic uptake, high selectivity toward avb3 integrin,
and preparation by simple radiolabeling method. With
these criteria, we synthesized new radiolabeled RGD
analog. To increase hepatic clearance, a glucosamino
group was inserted in the target molecule, which could
be used to improve the pharmacokinetics.16–18,21,22
Linkage of the prosthetic group and a glucosamino
group via an Asp residue to Lys of the RGD moiety
should have little influence on the selectivity of RGD
binding to avb3 integrin. Radiolabeled cRGD deriva-
tives were prepared using incorporation of tricarbonyl
technetium-99m to NO2 ligand attached to the amino
terminal of an Asp residue.
Keywords: Angiogenesis; Radiolabeled RGD; Cyclic RGD; AlphV
beta3 integrin; Technetium-99m.
*
Corresponding author. Tel.: +82 32 860 7686; fax: +82 32 867
5604; e-mail: dychi@inha.ac.kr
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.08.054

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B. C. Lee et al. / Bioorg. Med. Chem. 15 (2007) 7755–7764
HO
OH
HO
OH
18F
AcHN
O
HO
O
O
OH
O
HN
O
OH
OH
NH
NH
*I
*I
CO NH
CO NH
CO NH
O
O
O
HN
O
HN
O
N
H
HN
O
H
O
N
N
H
N
O
N
N
O
H
N
H
H
N
O
O
H
H
NH2
NH
O
H
N
NH2
NH
NH2
NH
HO
HO
HO
NH
NH
NH
O
O
O
cRGD[123I]yK(SAA)
[18F] galacto-cRGDfK
cRGD[123I]yV
O
NH
NH2
NH
O
O
O
N
N
H2N
O
N
N
H
H
H
H
HO
NH
NH
HN
O
O
NH
HN
HN
OH
O
O
O
O
HN
O
N
H
N
H
NH
O
O
NH
O
O
O
N
N
M
N
O
N
O
O
M = 111In
O
111In-DOTA-E-[cRGDfV]2
Figure 1. Radiolabeled cRGD derivatives.
Radiolabeled RGD analog was synthesized to allow
labeling with technetium-99m as SPECT tracers. Single
photon emitting tracers have advantages because of
the wide availability of SPECT cameras. Moreover,
technetium-99m is desirable due to its ideal physical
properties (141 keV, t1/2 = 6 h) and ready availability.23,24
pound as a glucose moiety.16,17 Treatment of N-acetyl-
D-glucosamine with acetyl chloride in methanol,
followed by direct benzylation at 3-OH, 4-OH, and 6-
OH, resulted in formation of methylation compound 5
at 1-OH, which avoids the reduction to a pentose form
Tricarbonyl technetium-99m is an attractive core for the
introduction of 99mTc into biomolecules because of its
high chemical stability and small size. [99mTc(H2O)3-
OH
M =99m Tc or Re
HO
O
Glucosamino
(CO)3]+ can be readily generated from 99mTcO4 and
ꢀ
O
HO
O
O
C
CO gas in the presence of NaBH4.25–28 Therapeutic
radionuclides such as the b-emitting rhenium-186 and
rhenium-188 [186/188Re(H2O)3(CO)3]+ could be applied
by the coordination with the same class of ligands used
in [99mTc(H2O)3(CO)3]+ reagent.29
NH
OC
OC
O
H
N
M
N
O
Asp (D)
O
Prosthetic groups
and linkage
O
O
O
NH
Lys (K)
In this work, we designed a technetium-99m labeled
RGD glycopeptide ([99mTc]Tc labeled glucosamino-D-
c(RGDfK), [99mTc]2) as a SPECT agent for angiogene-
sis imaging (Fig. 2).
D-Phe (f)
CO NH
O
HN
O
H
O
N
cRGDfK
N
H
O
H
N
NH
NH
HO
NH
Asp (D)
O
2. Results and discussion
2.1. Chemical synthesis
Arg (R)
Gly (G)
Figure 2. Structures of target compounds. Target compounds are
divided into three parts: glucosamino moiety, prosthetic group with
linkage, and cyclic RGD moiety.
To improve pharmacokinetics of the target compound,
we introduced applicative N-D-glucosamine to the com-

B. C. Lee et al. / Bioorg. Med. Chem. 15 (2007) 7755–7764
7757
under the condition of the following steps as shown in
Scheme 1.
the avb3 integrin.15 The glucosamino aspartic derivative
11 was further linked with cyclic-R(Pbf)-G-D(OtBu)-f-
K-NH2 using an amide bond condensation as shown
in Scheme 3. The protecting groups of compounds
12—2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfo-
nyl (Pbf) and tert-butyl groups at the side chain of Arg
and Asp, respectively—were removed by the treatment
of a mixture of trifluoroacetic acid/HSCH2CH2SH/
H2O (95:2.5:2.5) to obtain the desired compounds 13.
Mono acetyl protected amino group of compound 5
was further protected with tert-butyloxycarbonyl
(Boc) group in dry THF in the presence of 4-dimethyl-
aminopyridine (DMAP), so that N-acetyl group can be
readily removed under mild condition. Free amine 7
could not be obtained by direct hydrolysis of N-acet-
amide 5. This methodology has already been applied
for the synthesis of amide-linked amino acid.30–34 The
N-deacetylation in NaOMe followed by acidic hydroly-
sis in trifluoroacetic acid (TFA) of 6 afforded the free
amine 7 in 95% yield. The amide condensation between
compound 7 and Fmoc-Asp(OtBu)-OH gave product 8
in 71% yield.
The precursor 14 was prepared from the compound
13 in the presence of 10% palladium on charcoal with-
in a hydrogen atmosphere (Scheme 4). The debenzly-
ation was confirmed by HPLC. After the mixture
was filtered through a short plug filled with C-18 sil-
ica gel (3 cm), the solvent was evaporated in vacuo,
the resulting compound 14 was purified by reverse
phase HPLC.
After removal of the 9-fluorenylmethoxycarbonyl
(Fmoc) group, the amino group of compound 9 was
linked with a technetium pocket, N-a-bis(benzyloxycar-
bonylmethyl)-glycine-OH (4) from glycine tert-butyl es-
ter and benzyl bromoacetate, as shown in Scheme 2.
Re coordination reaction with the precursor 14 was per-
formed in a water/methanol mixture (1:1) at 65 ꢁC using
(NEt4)2[ReCl3(CO)3].27,35,36 The reaction was also mon-
itored by HPLC until the compound 14 peak
disappeared.
The small size of N-bis(hydroxycarbonylmethyl)-
[
99mTc]tricarbonyl led us to design and synthesize a tech-
netium-99m labeled cRGD derivative as an avb3 integrin
antagonist. tert-Butyl esters of compound 10 were sub-
jected to acid hydrolysis to afford acid compound 11.
It has been shown that the addition of Lys residue to
the RGD derivative has no influence on its affinity to
Incorporation of tricarbonyl technetium-99m into the
compound 14 using [99mTc(H2O)3(CO)3]+ was carried
out in aqueous media. The precursor fac-[99mTc(H2O)3-
(CO)3]+ was readily prepared using a known method.25–27
The radiochemical yield of [99mTc]2 was 90–92%.
BnO
O
N
OtBu
i
ii
OtBu
OH
O
OBn
H2N
HCl
O
O
O
HO
HO
BnO
BnO
O
O
i
ii
OBn
OH
NHAc
3
NHAc
5
OBn
BnO
O
O
OBn
OBn
BnO
O
N
BnO
BnO
BnO
BnO
BnO
O
O
N
O
iii
O
OH
iv
iii
NH
H
N
BnO
O
O
O
O
O
O
NAc
NH2
O
O
OBn
O
OBn
O
4
6
7
10
OBn
OBn
OBn
BnO
BnO
BnO
BnO
O
O
O
O
O
v
BnO
O
O
BnO
O
iv
BnO
O
O
N
NH
NH
NH
H
N
COOtBu
O
O
COOtBu
FmocHN
H2N
O
8
9
OBn
OH
Scheme 1. Reagents and conditions: (i) 1—MeOH, AcCl, 0–80 ꢁC, 1 h,
2—BnBr, NaOH/DMF, rt, 12 h; (ii) Boc2O, DMAP, THF, 80 ꢁC, N2,
18 h; (iii) 1—NaOMe/MeOH, rt, N2, 2.5 h, 2—TFA/CH2Cl2, rt, 2 h;
(iv) Fmoc-Asp(OtBu)-OH, HOBT, TBTU, DIEA, DMF, rt, N2, 16 h;
(v) 20% piperidine/DMF, rt, 20 min.
11
Scheme 2. Reagents and conditions: (i) benzyl bromoacetate, DIEA,
CH2Cl2, 0 ꢁC, 6 h; (ii) TFA/CH2Cl2, rt, 2 h; (iii) 9, HOBT, TBTU,
DIEA, DMF, rt, N2, 12 h; (iv) TFA/CH2Cl2, rt, 2 h.

7758
B. C. Lee et al. / Bioorg. Med. Chem. 15 (2007) 7755–7764
OBn
OBn
BnO
O
BnO
BnO
O
O
BnO
O
OBn
BnO
O
O
N
BnO
O
O
N
NH
NH
H
N
H
N
BnO
BnO
O
O
O
O
O
O
O
O
ii
BnO
O
N
i
NH
OBn
OBn
NH
NH
H
N
O
O
O
O
OBn
OH
CO NH
CO NH
O
O
HN
O
HN
O
11
H
O
H
O
N
N
H
H
N
O
O
H
N
NHPbf
NH
H
NH
NH
O
N
HO
N
NH
NH
O
O
12
13
Scheme 3. Reagents and conditions: (i) cyclic-Arg(Pbf)-Gly-Asp(OtBu)-D-Phe-Lys-NH2, HOBT, TBTU, DIEA, DMF, rt, N2, 12 h;
(ii) trifluoroacetic acid/HSCH2CH2SH/water (95:2.5:2.5), rt, 24 h.
2.2. In vitro endothelial cell binding assay of [99mTc]2, and
cRGD[125I]yV
shown in Figure 4. Inhibition studies demonstrated high
specificity of [99mTc]2 binding to avb3 integrin of HUVE
cells. Cell bound levels decreased by 30.5% and 34.8% of
control levels in the presence of 10 lM of cRGDyV and
cold authentic compound 1, respectively. If [99mTc]2 re-
mains to cell surface receptors, the results of this study
suggest that [99mTc]2 has high non-specific binding to
the cells. In spite of this possibility, cell bound levels de-
creased around 30% of control levels in the presence of
10 lM of cRGDyV and cold authentic compound 1,
Accumulation of radioactivity in human umbilical vein
endothelial (HUVE) cells and ECV304 cells was evalu-
ated in a paired format to compare [99mTc]2 uptake levels
with that of cRGD[125I]yV. The binding characteristics
of our radiotracers were therefore compared to that of
cRGD[125I]yV, a radiotracer known to exhibit high affin-
ity and selective binding to avb3 integrin.15 HUVE cells
were incubated with [99mTc]2, or cRGD[125I]yV for
60 min, then counted for bound radioactivity. Results
were expressed as % cell bound fractions of added radio-
activity calculated from standards.
[
99mTc]2 re are quite high binding selectivity toward
avb3 integrin.
2.5. Determination of IC50 value of glucosamino [99mTc]-
D(RGDfK) binding to purified avb3 integrin
As shown in Figure 3, [99mTc]2 was shown to have
significantly higher uptake levels at 60 min than
Competitive cell binding assays revealed a dose-depen-
dent inhibition of [99mTc]2 binding to immobilized
avb3 integrin by non-radiolabeled cRGDyV. Binding
in the presence of 1 lM cold RGD was markedly re-
duced to 8.4 0.4% of control levels (p < 0.00001), indi-
cating very low levels of non-specific binding. A sigmoid
dose–response curve was obtained from the binding
experiment from which high binding affinity with an
apparent IC50 value of 1.5 nM was measured (Fig. 6).
that of cRGD[125I]yV ([99mTc]2/cRGD[125I]yV = 2.9%ID
(2.3:0.8)). This tendency was also seen in HUVEC-de-
rived, ECV304 cells ([99mTc]2/cRGD[125I]yV = 3.3%ID
(1.8:0.55)). Thus, endothelial binding of [99mTc]2 was
higher than cRGD[125I]yV, despite a more complicated
structure.
2.3. In vitro stability studies
Percentage of the remaining [99mTc]2 was 98% after
240 min and over 95% even after 12 h when incubated
in human serum at 37 ꢁC and analyzed by radio-TLC,
indicating a high in vitro stability of radiotracer as
shown in Figure 5. This result means that the techne-
tium core of complex as well as RGD fraction is very
stable as similar result of technetium core itself which
was previously reported.37
3. Conclusion
Glucosamino-(Re)-D-c(RGDfK) (1) were synthesized
using prosthetic groups linked to Asp-Lys in the RGD
moiety with a glucosamino group followed by Re coor-
dination, which should have little influence on selective
binding to avb3 integrin. [99mTc]2 was efficiently incor-
porated using tricarbonyl technetium-99m to NO2 li-
gand attached to an amino terminal of an Asp residue
in 90–92% radiochemical yield. [99mTc]2 was shown to
bind to human endothelial cells in avb3 integrin specific
manners and with excellent binding affinities. Recently,
in vivo work of [99mTc]2 and its applications were pub-
2.4. Binding competition of labeled RGD ([99mTc]2) with
cRGDyV or authentic compound (1)
Competition binding assays were carried out on HUVE
cells with cold authentic compounds or cRGDyV as

B. C. Lee et al. / Bioorg. Med. Chem. 15 (2007) 7755–7764
7759
OH
O
3.0
2.5
2.0
1.5
HO
HO
O
HO
O
O
N
NH
H
N
O
O
O
i
OH
NH
13
1.0
0.5
0.0
CO NH
O
HN
O
H
O
N
N
H
N
O
H
NH
NH
Figure 4. Binding competition of labeled RGD ([99mTc]2) and
cRGDyV or 1. HUVE cell binding levels are expressed as percent of
added dose (%ID). [99mTc]2 was incubated with the cells for 60 min in
the presence or absence of 10 lM of either cold cRGDyV or authentic
compound 1; [99mTc]2 uptake levels in the absence of competitors
(closed bar); in the presence of cold cRGDyV (open bar); and in the
presence of 1 (left-handed striped bar).
HO
NH
O
14
OH
O
HO
HO
O
O
O
C
M
O
NH
OC
OC
O
H
N
N
O
O
O
100
90
O
NH
ii
80
CO NH
O
HN
O
70
H
O
N
N
30
60
120
Time (minute)
240
720
H
O
H
N
NH
NH
HO
NH
Figure 5. In vitro serum stability of [99mTc]2. The radiotracer (0.074–
0.74 MBq) was incubated in human serum at 37 ꢁC, and the percentage
of intact radioactivity was analyzed by radio-TLC at 30, 60, 120, 240,
and 720 min.
O
1, M = 185/187Re
2, M = 99mTc
Scheme 4. Reagents and conditions: (i) 10% Pd/C, H2, AcOH/water, rt,
12 h; (ii) (NEt4)2[ReCl3(CO)3], H2O, 60 ꢁC, 4 h, or 99mTc(CO)3ðH2OÞ
þ
,
3
H2O, 75 ꢁC, 30 min.
3
2.5
2
1.5
1
0.5
0
Figure 6. Specific endothelial binding characteristics of [
99mTc]2.
Competitive binding experiment results show concentration depen-
dent inhibition of [99mTc]2 binding to purified avb3 integrin by
excess non-radiolabeled cRGDyV, and a sigmoidal dose–response
curve with IC50 measurements. Data are expressed as means SE of
triplicate samples.
Figure 3. Endothelial cell binding of radiotracers. ECV304 cell binding
levels are expressed as percent of added dose (%ID); closed bar:
cRGD[125I]yV was incubated for 60 min; right-handed striped bar:
99mTc]2 was incubated for 60 min. HUVE cell binding levels are
expressed as percent of added dose (%ID); open bar: cRGD[125I]yV
was incubated for 60 min; left-handed striped bar: [99mTc]2 was
incubated for 60 min.
lished elsewhere.38 In addition, the synthesis and prepa-
ration of [99mTc]2 are emphasized in this work. Thus,
this radioprobe deserves further studies to evaluate their
utilities for SPECT imaging of angiogenesis.
[

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B. C. Lee et al. / Bioorg. Med. Chem. 15 (2007) 7755–7764
4. Experimental
55.7, 54.9, 28.0; MS (CI) m/z 428 (M+H+), 400, 372
(100), 326, 280, 236, 91; HRMS (CI) calcd for
4.1. Materials and general methods
C24H30NO6 (M+H+) 428.2076; found: 428.2073.
Solvents and reagents were purchased from the follow-
ing commercial sources: Sigma–Aldrich Company (Mil-
waukee, WI, USA) and Tokyo Chemical Industry
(Tokyo, Japan). Fmoc amino acids were purchased
from Advanced Chemtech (Louisville, KY, USA),
NovaBiochem (Bad Soden, Germany), and Bead Tech
(Seoul, Korea). The purified avb3 integrin was pur-
chased from Chemicon (CC1019, Temecula, CA,
4.3. N-a-Bis(benzyloxycarbonylmethyl)glycine (4)
To a stirred solution of Boc-protected 3 (500 mg,
1.17 mmol) in CH2Cl2 (20 mL) at room temperature
was added 10 mL of trifluoroacetic acid (TFA). The
mixture was stirred at room temperature for 2 h and
then the solvent was removed by azeotropy with toluene
(10 mL) and dried in vacuo. The crude product was
purified by flash column chromatography (MeOH/
CH2Cl2 = 5:95) to give compound 4 (399 mg, 92%) as
1
USA). H and 13C NMR spectra were recorded on a
Varian Gemini–400 (Palo Alto, CA, USA), and chemi-
cal shifts were reported in parts per million (ppm, d
units). Electron impact (EI) and chemical ionization
1
a yellow oil: H NMR (400 MHz, CDCl3) d 9.45 (br,
1H, Gly-OH), 7.38–7.33 (m, 10H, 2Ph), 5.17 (s, 4H,
2PhCH2), 3.66 (s, 4H, CH2), 3.57 (s, 3H, Gly-Ha); 13C
NMR (100 MHz, CDCl3) d 172.4, 171.1, 134.9, 128.63,
128.59, 128.4, 67.2, 57.3, 56.1; MS (FAB) m/z 372
(M+H+), 326, 280, 236, 91 (100), 57. HRMS (FAB)
calcd for C20H22NO6 (M+H+) 372.1447; found:
372.1437.
(CI) mass spectra were obtained on
a GC/MS
QP5050A spectrometer (Shimadzu, Kyoto, Japan). Fast
atom bombardment (FAB) mass spectra were obtained
on a JMS 700 (Jeol Ltd, Tokyo, Japan). MALDI-TOF
mass spectra were performed on Voyager-DE STR
MALDI-TOF mass spectrometer (San Francisco, CA,
USA). HPLC was carried out on a Thermo Separation
Products System (Fremont, CA, USA) with a semipre-
parative column (Alltech Econosil silica gel, 10 l,
10 · 250 mm) or an analytical column (YMC C18, 5 l,
4.6 · 250 mm). The eluant was simultaneously moni-
tored by a UV detector (254 nm) and a NaI(Tl) radioac-
tivity detector. TLC was performed on Merck F254 silica
plates and analyzed on a Bioscan radio-TLC scanner
(Washington DC, USA). In vitro incubation was carried
out at 37 ꢁC using a block heater (Digi-Block Labora-
tory Device Inc., Holliston, MA, USA). Na99mTcO4
was eluted on a daily basis from 99Mo/99mTc generators
(DuPont Pharmaceuticals Co., Delaware, USA, and
Daiichi Radioisotope Labs., Chiba, Japan). Radioactiv-
ity was measured in a dose calibrator (Perkin-Elmer,
Wellesley, MA, USA).
4.4. Methyl 2-deoxy-3,4,6-tri-O-benzyl-2-(N-acetly,N-
tert-butyloxycarbonylamino)-b-D-glucopyranoside (6)
A solution of 5 (700 mg, 1.39 mmol), DMAP (169 mg,
1.39 mmol), and Boc2O (742 mg, 3.47 mmol) in dry
THF (20 mL) was heated at 75 ꢁC under N2 gas. An
additional amount of 385 mg (1.80 mmol) of Boc2O
and 55 mg (0.45 mmol) of DMAP was added every
6 h. After the mixture was left for 12 h at 75 ꢁC, the
reaction mixture was cooled at room temperature and
50 mL of CH2Cl2 was added. The organic layer was
washed with 0.5 M H2SO4 (15 mL), water (15 mL),
and saturated NaHCO3 (20 mL), respectively. The or-
ganic layer was dried over anhydrous sodium sulfate
and concentrated. The crude product was purified by
flash column chromatography (EtOAc/hexane = 20:80)
to give 6 (714 mg, 85%) as a yellow oil; 1H NMR
(400 MHz, CDCl3) d 7.36–7.15 (m, 15H, 3Ph), 4.85 (s,
2H, PhCH2), 4.79 (d, J = 10.9 Hz, PhCH2), 4.72 (d,
J = 3.6 Hz, 1H), 4.47–4.62 (m, 2H), 4.53 (d,
J = 11.2 Hz, 1H, PhCH2), 4.52 (d, J = 12.4 Hz, 1H,
PhCH2), 3.85–3.82 (m, 1H, H-2), 3.78–3.66 (m, 3H, H-
3, H-4, H-5), 3.32 (s, 3H, OCH3), 2.22 (s, 3H, NAc),
1.50 (s, 9H, 3CH3); 13C NMR (100 MHz, CDCl3)
d172.28, 153.88, 139.24, 138.12, 137.83, 128.24, 128.22,
128.09, 127.68, 127.62, 127.51, 127.10, 126.90, 99.30,
83.48, 79.67, 79.47, 74.63, 73.98, 73.32, 70.51, 68.42,
54.82, 53.99, 28.26; MS (FAB) m/z 606 (M+H+); HRMS
calcd for C35H44O8N 606.3067; found: 606.3079.
The glucosamino derivatives 5 and 7 were prepared
according to the literatures30–34 and the cyclic pentapep-
tides; cyclic-Arg(Pbf)-Gly-Asp(OtBu)-D-Phe-Lys-NH2
and cRGD[125I]yV, were prepared as reported by Haub-
ner et al.15,16
4.2. N-Bis(benzyloxycarbonylmethyl)glycine(OtBu) (3)
To a solution of glycine tert-butyl ester hydrochloride
(1.05 g, 6.08 mmol) in CH3CN (30 mL) were added ben-
zyl bromoacetate (2.31 mL, 13.9 mmol) and N,N-diiso-
propylethylamine (DIEA, 5.30 mL, 30.4 mmol) at
0 ꢁC. The reaction mixture was stirred for 5 h and al-
lowed to attain room temperature. After saturated aque-
ous NaHCO3 (10 mL) was poured, the crude product
was extracted with CH2Cl2 (2· 25 mL). The organic
layer was dried with anhydrous sodium sulfate and con-
centrated. The crude product was purified by flash col-
umn chromatography (EtOAc/hexane = 20:80) to give
compound 3 (2.28 g, 88%) as a pale yellow oil: 1H
NMR (400 MHz, CDCl3) d 7.36–7.33 (m, 10H, 2Ph),
5.14 (s, 4H, PhCH2), 3.72 (s, 4H, CH2), 3.58 (s, 3H,
Gly-Ha), 1.44 (s, 9H, OtBu); 13C NMR (100 MHz,
CDCl3) d 170.6, 169.9, 135.5, 128.5, 128.2, 81.2, 66.3,
4.5. Methyl 2-deoxy-3,4,6-tri-O-benzyl-2-N-[N-a-Fmoc-
L-Asp(OtBu)]amino-a-D-glucopyranoside (8)
To
0.304 mmol), 1-hydroxybenzotriazole (HOBT, 49.4 mg,
0.365 mmol), and
O-(benzotriazol-1-yl)-N,N,N0,N0-
a
solution of Fmoc-Asp(OtBu)-OH (123 mg,
tetramethyluronium tetrafluoroborate (TBTU, 117.3 mg,
0.365 mmol) in DMF (10 mL), the known compound 7
(141 mg, 0.304 mmol) and DIEA (69 lL, 0.396 mmol) in
dry CH2Cl2 (3 mL) was added at room temperature under

B. C. Lee et al. / Bioorg. Med. Chem. 15 (2007) 7755–7764
7761
N2 gas. After the reaction mixture was stirred for 12 h,
CH2Cl2 was evaporated. The crude product was extracted
with EtOAc (2· 25 mL) and washed the saturated ammo-
nium chloride (15 mL). The organic layer was dried over
anhydrous sodium sulfate and concentrated. The crude
product was purified by flash column chromatography
(EtOAc/hexane = 40:60) to give compound 8 (185 mg,
71%) as a white solid (mp 148.4–149 ꢁC): 1H NMR
(400 MHz, CDCl3) d 7.76 (d, J = 7.6 Hz, 2H, Fmoc), 7.56
(t, J = 7.0 Hz, 2H, Fmoc), 7.40 (t, J = 7.4 Hz, 2H, Fmoc),
7.36–7.11 (m, 17H, Fmoc, 3Ph), 6.69 (d, J = 9.6 Hz, 1H,
NHCO), 6.02 (d, J = 8.0 Hz, 1H, NHCO), 4.76 (d,
J = 10.8 Hz, 2H), 4.66–4.11 (m, 10H, PhCH2, Asp-Ha,
Fmoc), 3.76–3.67 (m, 5H, H-3, 4, 5, H-6, H-60), 3.33 (s,
NMR (400 MHz, CDCl3) d 7.41–7.12 (m, 15H, 3Ph),
4.84 (d, J = 11.6 Hz, 1H, PhCH2), 4.80 (d, J = 10.8 Hz,
1H, PhCH2), 4.66–4.50 (m, 5H, PhCH2, H-1), 4.27–
4.22 (m, 1H, Asp-Ha), 3.77–3.69 (m, 5H, H-3, H-4, H-
5, H-6, H-60), 3.47 (dd, J = 8.0, 3.4 Hz, 1H, H-2), 3.35
(s, 3H, OCH3), 2.80 (dd, J = 16.0, 3.6 Hz, 1H, Asp-
0
Hb), 2.43 (dd, J = 16.0, 9.2 Hz, 1H, Asp-Hb ), 1.54 (br,
2H, NH2), 1.43 (s, 9H, OtBu); 13C NMR (100 MHz,
CDCl3) d 173.2, 171.1, 138.5, 137.93, 137.90, 128.29,
128.25, 127.9, 127.8, 127.6, 127.51, 127.49, 98.7, 80.94,
80.85, 78.3, 74.9, 73.3, 70.6, 68.4, 55.0, 52.3, 52.0, 40.3,
28.0; MS (MALDI) m/z 673 (M+K+), 657 (M+Na+,
100). HRMS (FAB) calcd for C36H47N2O8 (M+H+)
635.3332; found: 635.3350.
3H, OCH3), 2.84 (dd, J = 16.0, 4.4 Hz, 1H, Asp-Hb), 2.48
0
(dd, J = 16.0, 4.4 Hz, 1 H, Asp-Hb ), 1.42 (s, 9H, OtBu);
13C NMR (100 MHz, CDCl3) d 171.3, 170.2, 155.9,
143.6, 141.2, 138.3, 138.0, 137.9, 128.3, 127.8, 127.74,
127.70, 127.6, 127.50, 127.46, 127.0, 125.0, 124.9, 120.0,
98.6, 81.7, 80.8, 77.9, 74.8, 73.3, 70.6, 68.4, 67.1, 55.1,
52.8, 51.0, 46.9, 37.3, 31.5, 27.9; MS (MALDI) m/z 879
(M+Na+, 100), 656. HRMS (FAB) calcd for
C51H57N2O10 (M+H+) 857.4013; found: 857.4009.
4.8. Methyl 2-deoxy-3,4,6-tri-O-benzyl-2-N-{N-a-[N-a-
bis(benzyloxycarbonylmethyl)-gly]-L-Asp-OH}amino-a-
D-glucopyranoside (11)
To a solution of compound 10 (370 mg, 0.375 mmol) in
CH2Cl2 (12 mL) was added trifluoroacetic acid (5 mL).
The reaction mixture was stirred at room temperature
for 2.5 h. The mixture was quenched with 15 mL of
water and extracted with CH2Cl2 (2· 25 mL). The ob-
tained organic layer was dried over anhydrous sodium
sulfate and evaporated under reduced pressure. The res-
idue was purified with flash silica gel column chromatog-
raphy (MeOH/CH2Cl2 = 7:93) to give compound 11
4.6. Methyl 2-deoxy-3,4,6-tri-O-benzyl-2-N-{N-a-[N-a-
bis(benzyloxycarbonylmethyl)-Gly]-L-Asp(OtBu)}amino-
a-D-glucopyranoside (10)
1
Compound 10 was purified by flash column chromatog-
raphy (EtOAc/hexane = 40:60) to give colorless oil: H
(345 mg, 99%) as a colorless oil: H NMR (400 MHz,
1
CDCl3) d 8.55 (d, J = 8.8 Hz, 1H, CONH), 7.39–7.21
(m, 23H, Ph), 7.16–7.14 (m, 2H, Ph), 7.02 (d,
J = 9.2 Hz, 1H, CONH), 5.15 (s, 2H, CH2, PhCH2),
5.14 (s, 2H, CH2, PhCH2), 4.80 (d, J = 11.2 Hz, 1H,
PhCH2), 4.76–4.71 (m, 2H, PhCH2, H-1), 4.67 (d,
J = 12.0 Hz, 1H, PhCH2), 4.62 (d, J = 12.0 Hz, 1H,
PhCH2), 4.52 (d, J = 12.0 Hz, 1H, PhCH2), 4.50 (d,
J = 11.2 Hz, 1H, PhCH2), 4.31–4.25 (m, 1H, Asp-Ha),
3.83 (m, 1H, H-2), 3.84–3.64 (m, 5H, H-3, 4, 5, H-6,
H-60), 3.59 (s, 2H), 3.57 (s, 2H), 3.42 (d, J = 8.0 Hz,
2H, Gly-Ha), 3.28 (s, 3H, OCH3), 2.84 (dd, J = 16.8,
5.6 Hz, 0 1H, Asp-Hb), 2.76 (dd, J = 16.8, 6.0 Hz, 1H,
Asp-Hb ); 13C NMR (100 MHz, CDCl3) d 174.2, 171.5,
170.8, 170.6, 138.3, 138.1, 138.0, 135.1, 128.6 (·2),
128.5, 128.28 (·3), 128.26, 127.79, 127.73, 127.70,
127.6, 127.5, 98.6, 80.5, 78.1, 75.0, 74.8, 73.3, 70.7,
68.6, 66.9, 58.4, 56.0, 55.1, 53.2, 49.1, 35.6; MS (MAL-
DI) m/z 954 (M+Na+, 100). HRMS (FAB) calcd for
C52H58N3O13 (M+H+) 932.3970; found: 932.3961.
NMR (400 MHz, CDCl3) d 8.39 (d, J = 9.2 Hz, 1H,
CONH), 7.38–7.20 (m, 23H, Ph), 7.14–7.11 (m, 2H,
Ph), 6.91 (d, J = 9.6 Hz, 1H, CONH), 5.15 (s, 2H,
CH2, PhCH2), 5.14 (s, 2H, CH2, PhCH2), 4.79 (d,
J = 10.8 Hz, 1H, PhCH2), 4.77–4.70 (m, 3H, PhCH2,
H-1), 4.62 (d, J = 10.8 Hz, 1H, PhCH2), 4.52 (d,
J = 12.0 Hz, 2H, PhCH2), 4.33–4.27 (m, 1H, Asp-Ha),
3.84–3.64 (m, 6H, H-2, H-3, 4, 5, H-6, H-60), 3.61 (s,
2H), 3.59 (s, 2H), 3.41 (d, J = 2.0 Hz, 2H, Gly-Ha),
3.30 (s, 3H, OCH3), 2.79 (dd, J = 16.8, 5.8 H0z, 1H,
Asp-Hb), 2.63 (dd, J = 16.8, 6.0 Hz, 1H, Asp-Hb ), 1.40
(s, 9H, OtBu); 13C NMR (100 MHz, CDCl3) d 171.2,
170.7, 170.6, 170.5, 170.4, 138.4, 138.2, 138.0, 135.1,
128.7, 128.5, 128.31, 128.28, 127.79, 127.76, 127.70,
127.59, 127.57, 127.50, 98.7, 81.2, 80.7, 78.0, 74.9,
74.8, 73.4, 70.7, 68.9, 66.8, 58.3, 55.9, 55.1, 53.1, 49.2,
37.0, 28.0; MS (MALDI) m/z 1010 (M+Na+, 100),
954. HRMS (FAB) calcd for C56H66N3O13 (M+H+)
988.4596; found: 988.4618.
4.9. Tri-O-benzylglucosamino-[N-a-bis(benzyloxycarbonyl-
4.7. Methyl 2-deoxy-3,4,6-tri-O-benzyl-2-N-[NH2-
Asp(OtBu)]amino-a-D-glucopyranoside (9)
methyl)-Gly]-Asp-Arg(Pbf)-Gly-Asp(OtBu)-D-Phe-Lys (12)
To a solution of compound 11 (279 mg, 0.30 mmol),
HOBT (48 mg, 0.36 mmol), and TBTU (115 mg, 0.36
mmol) in DMF (15 mL), was added a mixture of cyc-
lic-R(Pbf)-G-D(OtBu)-f-K-NH2 (273 mg, 0.30 mmol)
and DIEA (67 lL, 0.39 mmol) in DMF (3 mL) at room
temperature under N2 gas. After the reaction mixture
was stirred for 12 h, the solvent was removed under re-
duced pressure. Water (5 mL) was added to a small por-
tion of mixture in DMF, the white solid appeared. This
crude peptide was isolated by centrifugation and then
the desired compound 12 was washed with 5% ethanol
A solution of compound 8 (184 mg, 0.215 mmol) in 20%
piperidine in DMF (5 mL) was stirred at room temper-
ature. After 20 min, the crude product was extracted
with EtOAc (2· 25 mL) and the organic layer was
washed with the saturated ammonium chloride
(20 mL) to remove DMF. After the organic layer was
dried over anhydrous sodium sulfate and concentrated,
the crude product was purified by flash column chroma-
tography (EtOAc/hexane = 80:20) to give compound 9
1
(134 mg, 98%) as a white solid (mp 70.2–71.9 ꢁC): H

7762
B. C. Lee et al. / Bioorg. Med. Chem. 15 (2007) 7755–7764
in diethyl ether (3· 10 mL) and dried in vacuo. Analyt-
ical data were as follows: MS (MALDI) m/z = 1849
(M+Na+, 100).
The residue was triturated with H2O (200 lL) and loaded
on a C-18 Sep-Pak cartridge, washed with H2O (1 mL)
and then methanol (1.5 mL). The product was dried
under a stream of N2 and diluted with PBS (pH 7.4).
The overall radiochemical yield was 90–93%.
4.10. Tri-O-benzylglucosamino-[N-a-bis(benzyloxycar-
bonylmethyl)-Gly]-Asp-Arg-Gly-Asp-D-Phe-Lys (13)
4.14. Cell culture
To a solution of compound 12 (200 mg, 0.109 mmol) in
a solution of trifluoroacetic acid/HSCH2CH2SH/H2O
(5 mL, 95:2.5:2.5) was stirred at room temperature for
24 h. The reaction mixture was filtered with reaction sol-
vent (2· 5 mL) and then the solvent was evaporated un-
der reduced pressure. To get the solid, diethyl ether was
added to the reaction mixture. The resulting solid was
filtered and washed with diethyl ether. After the solid
was dried under reduced pressure it gave compound 13
(88 mg, 53%) as a white solid; Analytical data were as
follows: MS (MALDI) m/z = 1517 (M+H+, 100), 1539
(M+Na+).
Human umbilical vein endothelial (HUVE) cells, known
to express avb3 integrin,39–42 were cultured in EGM
(10 mL FBS + 0.2 mL hydrocortisone + 2 mL human
recombinant
epidermal
growth
factor + 0.5 mL
VEGF + 0.5 mL R3-IGF-1 + 0.5 mL ascorbic acid +
0.5 mL GA-1000 + 0.5 mL heparin) supplemented with
10% FBS, in a 37 ꢁC incubator with 5% CO2. Endothe-
lium-derived ECV304 cells were cultured in M199 media
supplemented with 10% FBS and antibiotic–antimycotics
in a 37 ꢁC incubator with 5% CO2.
4.15. In vitro cell binding assays of labeled RGDs
4.11. Glucosamino-[N-a-bis(hydroxycarbonylmethyl)-
Gly]-Asp-Arg-Gly-Asp-D-Phe-Lys (14)
For binding experiments, cells were harvested with
0.25% trypsin–1 mM EDTA, washed, and resuspended
in D-PBS at a concentration of 2–3 · 106 cells per mL
based on trypan blue dye exclusion assays. One hundred
microliter of cell suspension in Eppendorf tubes was
To a solution of 13 (172 mg, 0.114 mmol) in a solution
of AcOH (5 mL) and H2O (5 mL) was added 269 mg
(0.228 mmol) of the 10% palladium on charcoal under
N2 gas. After gas atmosphere was exchanged from N2
to H2 gas, the reaction mixture was stirred for 12 h at
room temperature. The reaction mixture was filtered
through Celite layer and then the solvent was removed
by azeotropy with toluene (5 mL) under reduced pres-
sure. The crude mixture was purified by reverse phase
HPLC. The desired compound 14 (84.5 mg, 83%) was
obtained as a white solid. Analytical data were as fol-
lows: MS (MALDI) m/z = 1068 (M+H+, 100), 1090
(M+Na+).
incubated with
[
99mTc]2, or cRGD[125I]yV (0.037
MBq/tube) at 37 ꢁC for 60 min. At the end of the incu-
bation, cells were washed twice with 1 mL of D-PBS and
underwent measurement of radioactivity on high-energy
gamma counter along with standards.
4.16. Competitive cell binding experiments of labeled
RGD, cRGDyV, and cold authentic compounds
Specificity of endothelial cell binding was evaluated by
incubation of cells with [99mTc]2 (0.037 MBq/tube) at
37 ꢁC for 60 min as above, but in the presence of
10 lM of cRGDyV or cold authentic compound 1. At
the end of the incubation, cells were washed twice with
D-PBS and measured for radioactive counts on a high-
energy gamma counter along with standards.
4.12. Glucosamino (Re)-D-c(RGDfK) (1)
To a solution of compound 14 (10 mg, 9.37 lmol) in a
solution of H2O/MeOH (5 mL, 1:1) mixture was added
6.3 mg (10.3 lmol) of (NEt4)2[ReCl3(CO)3]. The reac-
tion mixture was stirred at 65 ꢁC for 1.5 h. The solution
was removed under reduced pressure and then the prod-
uct 1 was purified with a reverse pre-column (0–50%
CH3CN/0.1% TFA in H2O, 214 nm, 40 min). Analytical
data were as follows: MS (MALDI) m/z = 1337
(M+H+). HRMS (FAB) calcd for C47H65N12O22 187Re
(M+H+) 1337.3972; found: 1337.3956.
4.17. In vitro stability studies
An aliquot (0.074–0.74 MBq) of [99mTc]2 in 10% ethanol
saline was mixed with 1 mL of human serum and incu-
bated at 37 ꢁC. The solution was analyzed at intervals
of 30, 60, 120, 240, and 720 min by radio-TLC using
n-butanol/water/acetic acid (3:1:1) as the developing
solvents.43
4.13. Radiochemical synthesis of glucosamino [99mTc]-D-
c(RGDfK) ([99mTc]2)
4.18. Purified avb3 integrin binding assays
[
99mTc(H2O)3(CO)3]+ was prepared according to the lit-
erature.25–27 A solution of 200 lL of a mixture
99mTc(H2O)3(CO)3]+ (0.18–0.37 GBq) in saline was
To assess affinities of the radioprobes, binding assays
were performed on microtiter plates with avb3 integrin
protein immobilized to the floor. Briefly, purified avb3
integrin diluted to 10 lg/mL in phosphate buffer (pH
5.8) was aliquoted to a 96-well microtiter plate in vol-
umes of 50 lL/well (Medisorp, Nunc, Rochester, NY,
USA). After overnight adhesion at 4 ꢁC, the wells were
blocked with blocking buffer containing 2.5 mg/mL
casein for 30 min at 4 ꢁC, then washed with PBS con-
[
added to the precursor 14 (0.5 mg, 0.47 lmol) in H2O
(300 lL). The reaction mixture was stirred at 75 ꢁC for
30 min and then cooled in an ice bath. The radiotracer
was purified by HPLC at a flowrate of 1.5 mL/min using
a solvent gradient (0–50% CH3CN/0.1% TFA in H2O for
30.1 min). The desired fraction of [99mTc]2 was collected
from HPLC and concentrated under reduced pressure.

B. C. Lee et al. / Bioorg. Med. Chem. 15 (2007) 7755–7764
7763
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experiments, 40 lL of binding solution (PBS containing
0.1% bovine serum albumin) containing 370 kBq of
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Schmidtke, R.; Diefenbach, B.; Kessler, H.; Stocklin, G.;
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[
99mTc]2 was applied to each well. Various concentra-
tions of non-radiolabeled cRGDyV (0, 1, 10, 100,
1000, and 10,000 nM) were added to obtain competitive
binding data. After incubation at room temperature for
3 h, each well was washed twice with PBS, separated
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Acknowledgments
20. Liu, S.; Hsieh, W.-Y.; Kim, Y.-S.; Mohammed, S. I.
Bioconjug. Chem. 2005, 16, 1580–1588.
This work was supported by the National Mid- and
Long-term
M20243010001-04A0701-00110 and M10420000010-
04N20001010 of the Korean Ministry of Science and
Technology. High resolution mass spectra were carried
out at the Korea Basic Science Institute (Daegu, Korea).
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