Synthesis and evaluation of cyclic RGD-boron cluster conjugates to develop tumor-selective boron carriers for boron neutron capture therapy

Article abstract of DOI:10.1016/j.bmc.2011.01.020

Boron-containing agents play a key role in successful boron neutron capture therapy (BNCT). Icosahedral boron cluster-Arg-Gly-Asp (RGD) peptide conjugates were designed, synthesized, and evaluated for the biodistribution to develop tumor-selective boron c

Full text of DOI:10.1016/j.bmc.2011.01.020

Bioorganic & Medicinal Chemistry 19 (2011) 1721–1728
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of cyclic RGD-boron cluster conjugates to develop
tumor-selective boron carriers for boron neutron capture therapy
Sadaaki Kimura a, , Shin-ichiro Masunaga b, Tomohiro Harada a, Yasuo Kawamura a, Satoshi Ueda a,
Kensuke Okuda a, Hideko Nagasawa a,
⇑
a Laboratory of Medicinal & Pharmaceutical Chemistry, Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi, Gifu 501-1196, Japan
b Radiation Oncology Research Laboratory, Research Reactor Institute, Kyoto University, Asashiro-nishi 2-1010, Kumadori-cho, Sennan-gun, Osaka 590-0494, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Boron-containing agents play a key role in successful boron neutron capture therapy (BNCT). Icosahedral
boron cluster-Arg-Gly-Asp (RGD) peptide conjugates were designed, synthesized, and evaluated for the
Received 23 December 2010
Revised 11 January 2011
Accepted 12 January 2011
Available online 18 January 2011
biodistribution to develop tumor-selective boron carriers. Integrin
tumor drug delivery because of its specific expression in proliferating endothelial and tumor cells of var-
ious origins. We, therefore, selected a c(RGDfK) moiety recognizing vb3 as an active tumor-targeting
avb3 is an attractive target for anti-
a
device to conjugate with icosahedral boron-10 clusters, disodium mercaptododecaborate (BSH) or o-car-
borane as a thermal neutron-sensitizing unit. Preparation of o-carborane derivatives involved microwave
Keywords:
Integrin avb3
Boron neutron capture therapy (BNCT)
o-Carborane
irradiation, and resulted in high yields in a short time. An in vitro cell adhesion assay on
U87MG and SCCVII cells demonstrated the high binding affinity of conjugates to integrin
(IC50 = 0.19–2.66 M). Biodistribution experiments using SCCVII-bearing mice indicated that GPU-201
avb3-positive
a
vb3
l
Cyclic RGD
showed comparable tumor uptake and a significantly longer retention in tumors compared with BSH.
These results suggest that GPU-201 is a promising candidate for use in BNCT.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
agent sodium–10B-dodecaborate conjugates.11 In the development
of these compounds, we focused on the tumor microenvironment
Boron neutron capture therapy (BNCT) is, in principle, the most
promising modality for cancer treatment provided that a sufficient
amount of boron-10 (10B) can be accumulated in the target tumor
and a sufficient number of very-low-energy thermal neutrons can
characterized by regions of hypoxia, low nutrition, and acidosis
due to incomplete blood-vessel networks as a general target of solid
tumors regardless of cancer types.12 Meanwhile several boron car-
riers such as molecular-targeted agents toward somatostatin,13,14
folate,15,16 transferrin,17 VEGF,16,18 or EGF16,19,20 receptors as well
as nucleoside,21 aminoacid,22 or porphyrin derivatives23,24 have
been developed as tumor-targeting BNCT agents.25
be delivered there. BNCT is based on the neutron capture and fis-
7
sion reaction of non-radioactive 10B, [10B (1n,
a)
Li]. This results
in selective boron-localized cell killing because the particles gener-
ated in this reaction have a short range of approximately 14
l
m
In recent years, great progress has been made towards targeting
(the approximate diameter of 1–2 cells), and carry high linear en-
ergy transfer.1,2 Therefore, tumor-selective delivery of a sufficient
amount of 10B atoms is essential for the success of BNCT.
integrins in cancer treatment.26 Integrins are a superfamily of hete-
rodimeric transmembrane receptors comprising 19 a-subunits and
9 b-subunits which participate in cell–cell and cell–matrix interac-
tions.27 Integrin regulates a diverse array of cellular functions re-
lated to the progression, angiogenesis, and metastasis in the
tumor microenvironment.28,29 Among the integrin family, integrin
Disodium mercaptododecaborate (BSH)3,4 and p-boronophenyl-
alanine (BPA)5,6 are available 10B carriers for clinical investigation,
and currently undergoing clinical trials7 for BNCT. Both boron car-
riers are, however, not ideal for delivering a therapeutic amount
of 10B throughout the target tumor because they tend to be washed
out immediately after injection.8,9 We recently developed tumor-
targeting 10B carriers such as 2-nitroimidazole-sodium borocap-
tate-10B conjugate10 and hypoxia-specific cytotoxic bioreductive
a
vb3 is the most attractive target for anti-tumor drug delivery,30
and tumor imaging31 with PET32–34 SPECT,35–37 optical imag-
ing,38,39 and MRI40 due to its specific expression on proliferating
endothelial cells and tumor cells of various origins.27,29 Therefore,
we developed new tumor-selective boron carriers bearing inte-
grin-binding Arg-Gly-Asp (RGD) consensus motifs for enhancing
the tumor-specific uptake of 10B atoms. We carried out the design,
synthesis, and pharmacokinetic evaluation of novel boron carriers
⇑
Corresponding author. Tel./fax: +81 58 230 8112.
E-mail address: hnagasawa@gifu-pu.ac.jp (H. Nagasawa).
Present address: Functional Imaging Division, National Cancer Center Hospital
East, Kashiwanoha 6-5-1, Kashiwa, Chiba 277-8577, Japan.
conjugated with cyclic RGD peptides directed to integrin avb3.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.01.020

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S. Kimura et al. / Bioorg. Med. Chem. 19 (2011) 1721–1728
treatment with 95% TFA to afford the desired c(RGDfK) in quantita-
2. Results and discussion
2.1. Molecular design
tive yield. Preparation of E[c(RGDfK)]2 was achieved by the reac-
tion of Boc-Glu(OSu)-OSu with the amino group of lysine residue
on c(RGDfK) and subsequent removal of the Boc group.
RGD peptide-BSH conjugates were prepared in a two-step pro-
cess: conjugation of peptide with linker-bearing maleimide moiety
and Michael addition of the SH group on BSH to the maleimide
moiety (Scheme 1). c(RGDfK) or E[c(RGDfK)]2 were conjugated
with maleimide linker 1 derived from b-alanine to afford 2 and
4, respectively. Subsequent reactions of maleimide on these cyclic
peptide-linker conjugates with BSH were carried out in phosphate
buffer (pH 7) to give 3: GPU-51 and 5: GPU-180. The Michael addi-
tion was successfully performed in aqueous media with low dimer-
ization of BSH to BSSB, which was a measurable side reaction in
organic media such as DMF or MeCN using an organic base. Simi-
larly, a cyclic RGD-BSH conjugate-bearing tranexamic acid-based
linker was synthesized. Maleimido-tranexamic acid linker 645
was converted to the corresponding activated ester 7, which was
conjugated with c(RGDfK) to give 8. Conjugate addition of BSH
was undertaken in the same manner to give 9: GPU-176. Attempts
to synthesize E[c(RGDfK)]2-BSH conjugate via a tranexamic acid
linker were not successful because of the instability of the isolated
product.
RGD is the consensus sequence of matrix proteins for binding
integrins at the cell surface. Kessler and coworkers developed
c(RGDfV) as the first highly active and selective a
vb3 antagonist,41
which acts as a lead structure for stereoisomeric peptidomimetics.
c(RGDfK), and c(RGDfC) were developed to be functionalized with
various linker molecules through the amino or mercapto group on
the lysine or cysteine residue.32 Therefore, we selected the
c(RGDfK) moiety as an active tumor-targeting device and two ico-
sahedral boron-10 clusters, that is, disodium mercaptododecab-
orate (BSH) or 1,2-dicarba-closo-dodecaborane (o-carborane), in
terms of the difference in physicochemical properties as a thermal
neutron-sensitizing unit. These tumor-targeting units and sensitiz-
ing units were conjugated through suitable linkers, which can con-
trol distance, orientation, flexibility, and physicochemical
properties (Fig. 1). More recently, multimeric RGD compounds
such as E[c(RGDfK)]2 or {E[c(RGDfK)]2}2 have been exploited to im-
prove the targeting potential.42–44 Hence, we also used RGD-di-
mers such as E[c(RGDfK)]2 to increase tumor accumulation. BSH
is an extremely hydrophilic dianion comprising 12 boron atoms
with a mercapto group, whereas o-carborane (icosahedra compris-
ing 10 boron atoms and two adjacent carbon atoms) is hydropho-
bic comparable with benzene. Various combinations of boron
clusters having different physicochemical properties and cyclic
RGDs through suitable linkers are expected to improve the phar-
macokinetics and tumor uptake of boron carriers.
The syntheses of c(RGDfK)-linked o-carborane from 5-hexyn-1-
ol (10) are summarized in Scheme 2. Protection of alcohol 10 with
a benzyl group was employed to afford the corresponding benzyl
ether 11. Subsequent formation of a carborane cage under micro-
wave irradiation gave carboranyl ether 12 in high yield within a
very short period (30 min) compared with the conventional syn-
thetic method (40%, 2.5 days). Hydrogenolysis of 12 and oxidiza-
46
tion of 13 with CrO3/H5IO6
afforded the corresponding
2.2. Chemistry
carboxylic acid 14. After transformation of 14 to activated ester
15, conjugation of c(RGDfK) or E[c(RGDfK)]2 with 15 produced
16: GPU-49 and 17: GPU-189, respectively. o-Carborane has two
adjacent carbon atoms thereby enabling construction of a different
type of RGD dimer by the attachment of c(RGDfK) to each carbon
atom via a linker. Lithiation of 12 followed by alkylation with 18
gave symmetrically 1,2-bifunctionalized o-carboarne 19, which
was deprotected by hydrogenolysis using cyclohexene under reflux
conditions. Oxidation of diol 20 afforded the corresponding di-car-
boxylic acid 21. Conversion to di-activated ester 22 using HOBt and
EDCI was performed; subsequent reaction with c(RGDfK) gave the
symmetric RGD-dimer 23: GPU-201. Similarly, the randomized
peptide sequence c(KGfDR) was coupled with di-activated ester
22 to give 24 GPU-205 as the corresponding negative control of
GPU-201.
To compare the effects of RGD-monomer and -dimer on integrin
selectivity and tumor uptake, c(RGDfK) and E[c(RGDfK)]2 were pre-
pared. Additionally, the randomized sequence peptide c(KGfDR)
was prepared as a control. To prepare cyclic RGD peptide, the lin-
ear-protected peptide, H-Asp(t-Bu)-D-Phe-Lys(Boc)-Arg(Pbf)-Gly-
OH, was obtained by assembling Fmoc-Gly-(2-Cl)Trt resin by the
conventional Fmoc strategy, and cleaving with 20% 1,1,1,3,3,3-
hexafluoroisopropanol in DCM. Cyclization of the linear-protected
peptide was carried out using diphenylphosphoryl azide in the
presence of NaHCO3 under high dilution conditions followed by
O
Active targeting moiety
cRGD
H
N
O
OH
NH
HN
O
O
2.3. Biology
NH
O
HN
O
peptide
HN
HN
2.3.1. Cell adhesion assay
NH2
HN
Binding affinities and specificities of all RGD-boron conjugates
c(RGDfK)
to integrin
vb3-positive U87MG human glioblastoma cells (see the western
blotting data in Fig. S1 in Supplementary data). Vitronectin is a
a
vb3 were evaluated using a cell adhesion assay33 on
Control of distance, orientation, flexibility
and physicochemical properties
a
O
Linker
27
O
specific ligand for integrin
avb3. All conjugates except the non-
N
N
n
RGD conjugate GPU-205 inhibited the adhesion of U87MG cells
on vitronectin-coated plates in a concentration-dependent manner
(Fig. 2). As shown in Figure 3C, IC50 values of BSH conjugates
H
S
O
closo-
1,2-dicarba-closo-
dodecaborate
dodecaborane
GPU-51, -176, and -180 were 1.08 0.34
0.44 0.04 M, and those of o-carborane conjugates GPU-49,
-189, and -201 were 2.66 0.85 M, 0.39 0.03 M, and 0.61
0.08
lM, 0.31 0.03 lM, and
2-
l
Boron
cluster
l
l
l
M, respectively, which were comparable to the values
reported for other multimeric RGD conjugates previously.33 Bind-
ing affinities of RGD-monomers GPU-49 and -51 except for
GPU-176 were lower than those of the RGD-dimers GPU-180,
-189 and -201. Multimeric RGD peptides can significantly enhance
10B source for BNCT
= C
Figure 1. Molecular design of boron carrier.

S. Kimura et al. / Bioorg. Med. Chem. 19 (2011) 1721–1728
1723
R
G
R
G
D
K
D
K
O
a
b
f
f
HN
HN
N
OSu
O
O
O
O
O
O
2-
S
N
O
N
1
2
3: GPU-51
O
2 Na+
R
G
R
c
G
D
H
N
H
N
K
f
K
D
O
O
f
d
H
N
H
N
R
O
R
K
O
N
HN
HN
G
K
O
O
2-
S
G
N
O
O
f
D
f
D
O
O
4
5: GPU-180
R
G
O
K
D
R
G
f
N
O
O
H
K
D
R
f
N
H
N
O
O
f
g
N
O
e
O
N
S
O
O
2-
8
6: R = OH
7: R = OSu
Na+
9: GPU-176
Scheme 1. Reagents and conditions: (a) c(RGDfK), DIPEA, DMF, rt, 84%; (b) B-10 enriched BSH, pH 7 phosphate buffer, rt, 56%; (c) E[c(RGDfK)2], DIPEA, DMF, rt, 86%; (d) B-10
enriched BSH, pH 7 phospate buffer, rt, 71%; (e) HOSu, EDCIꢀHCl, DCM, rt; (f) c(RGDfK), DIPEA, DMF, rt, 40% (from 6); (g) B-10 enriched BSH, pH 7 phosphate buffer, rt, 45%.
R
G
D
H
N
K
f
O
f
16: GPU-49
b
R
R
O
R
f
G
D
N
K
10: R = CH2OH
g
12: R = CH2OBn
H
N
H
a
c
11:
R = CH2OBn
13:
14:
R= CH2OH
R = CO2H
R
f
d
G
O
O
N
K
e
15: R = CO2Su
H
D
17: GPU-189
ZAA
R
XAA
Br
D
YAA
R
R
18
K
l
NH
O
HN
K
YAA
D
12
XAA
ZAA
O
h
19: R = CH2 OBn
i
20:
21:
22:
R = CH2OH
R = CO2H
R = CO2Su
j
23: GPU-201 (XAA = R, YAA = G, ZAA = f)
24: GPU-205 (XAA = G, YAA = f, ZAA = R)
k
Scheme 2. Reagents and conditions: (a) (i) NaH, THF, 0 °C to rt; (ii) BnBr, THF, rt, 52%; (b) B10H14, N,N-dimethylaniline, chlorobenzene, 80 °C, microwave, 88%; (c) H2, 10% Pd/
C, MeOH, AcOH, rt, 86%; (d) H5IO6, CrO3, wet MeCN, rt, quant.; (e) HOSu, EDCIꢀHCl, DCM, rt, 97%; (f) c(RGDfK), DIPEA, DMF, rt, 86%; (g) E[c(RGDfK)2], DIPEA, DMF, rt, 68%; (h) (i)
n-BuLi, THF, ꢁ40 °C to rt; (ii) 18, LiI, rt, 65%; (i) cyclohexene, Pd/C, EtOH, reflux, 88%; (j) H5IO6, CrO3, wet MeCN, rt, quant.; (k) HOSu, EDCIꢀHCl, DCM, rt; (l) cyclic peptide,
DIPEA, DMF, rt, 23: 46% (from 21), 24: 25% (from 21).

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S. Kimura et al. / Bioorg. Med. Chem. 19 (2011) 1721–1728
GPU-51 has an ethylene linker derived from b-alanine whereas
GPU-176 has cyclohexane linker that is more rigid and longer than
the ethylene linker, and restricts the flexibility and orientation of
molecule. Although the reason for enhanced binding affinity of
GPU-176 is unclear, we suspect it is because its cyclohexane linker
kept the boron cluster away from the RGD peptide to enhance
recognition of integrin
avb3. BSHs showed slightly higher binding
affinity than the corresponding o-carborane derivatives. We also
performed the cell adhesion assay of GPU-176 and -201 on the
mouse cell line SCCVII (squamous cell carcinoma derived from
C3H/He mice) expressing integrin
data). The IC50 values of GPU-176 and 201 were 0.19 0.08 and
0.68 0.03 M, respectively. The non-RGD derivative GPU-205
avb3 (Fig. S2 in Supplementary
l
did not inhibit attachment of the cells in the same way as the assay
for U87MG.
2.3.2. Biodistribution experiment
Figure 2. Cell adhesion assay on U87MG cells.
We examined RGD-boron cluster conjugates for the time-course
of change in boron concentration in solid tumors, brain, blood,
liver, muscle, and skin of mice bearing SCCVII tumors. Boron
concentrations in tissue were measured by inductively coupled
plasma-atomic emission spectroscopy (ICP-AES). All compounds
tested here should be enough stable during blood circulation for
integrin-binding affinity due to the polyvalency effect.42–44 How-
ever, the RGD-monomer GP-176 was found to be the most potent
inhibitor among all the conjugates tested here. The difference be-
tween GPU-51 and -176 is evident in the structure of the linker.
Figure 3. Tumor uptake of RGD conjugates and BSH after intravenous administration at a dose of 0.075 mmol/kg (A) and intraperitoneal administration (B). IC50 value in cell
adhesion assay on U87MG and retention time on ODS for hydrophobicity analysis (C).

S. Kimura et al. / Bioorg. Med. Chem. 19 (2011) 1721–1728
1725
at least 6 h based on the serum stability assay (Fig. S4 in Supple-
mentary data). Figure 3A shows the change in boron concentration
in tumors at various time points after intravenous administration
of each compound (see also Fig. S3 in Supplementary data for the
results in other tissues). All compounds except for BSH were
dissolved in physiological (0.9%) saline containing 10% 2-hydroxy-
propyl-b-cyclodextrin (HP-b-CD) as a solubilizing and dispersing
agent, and 0.075 mmol/kg was injected into the tail vein of each
mouse. Relatively large intracellular accumulations of boron
concentration in tumor for BNCT could not be performed in the
present study. Further studies are required to improve the solubil-
ity and bioavailability of boron conjugates.
3. Conclusion
We synthesized the cyclic RGD-boron cluster conjugates GPU-
49, -51, -176, -180, -189, and -201 to develop tumor-selective bor-
on carriers for BNCT. All RGD conjugates had high in vitro integrin
(approximately 20–30 l
g of 10B per gram of tumor) are necessary
avb3-binding affinity, and showed dose-dependent inhibition of
to produce cell death.47,48 However the dose of 0.075 mmol/kg in
this experiment was one-tenth the dose of BSH (0.75 mmol/kg)
previously reported to achieve a boron concentration of >20 lg/g
cell adhesion with IC50 values of 0.19–2.66 M in a vitronectin-
l
mediated cell adhesion assay on U87MG cells and SCCVII cells.
By analyses of in vivo biodistribution, RGD dimer-boron clusters
had higher tumor uptake and slower clearance than that of mono-
meric RGD conjugates. In particular, GPU-201 should be a promis-
ing candidate due to its dose dependent-tumor uptake and
significantly longer tumor retention than BSH. We shall evaluate
its radio-sensitizating effect under thermal neutrons beam expo-
sure in future studies.
in tumor by intraperitoneally administration.11 Furthermore, the
dosage was also the maximum dose at which a pharmacokinetics
response was measured, providing that drug associated toxicity,
precipitates and others were not found in case of an intravenous
injection. This is because the solubility of RGD-boron clusters in
water is relatively low. To evaluate hydrophobicity, all compounds
were analyzed by reverse-phase (RP)-HPLC and retention time in
gradient mode is shown in Figure 3C. The order of hydrophobicity
are predicted by the retention time was GPU-49 > -201 > -189 >
-176 > -180 > -51 > BSH, that is, all carborane derivatives were
more hydrophobic than BSH derivatives.
4. Experimental
4.1. General
Tumor uptake of BSH 15 min following injection was much
higher than that in other RGD conjugates, but BSH was rapidly
washed out from tumors. In contrast, GPU-201 and -189 (RGD-di-
mer and o-carborane conjugate, respectively) showed good accu-
mulation and retention in tumors. The tumor/blood ratio of GPU-
201 was nearly twice greater than that of BSH from 15 min to
60 min following intravenous injection. Dimeric RGD conjugates
showed higher boron concentrations in tumors than those of
monomeric RGD conjugates. Among dimeric RGD derivatives,
GPU-180 including BSH showed the lowest uptake in the same le-
vel of monomeric RGD derivatives due to its hydrophilic proper-
ties. Although boron concentrations were low in the brain, the
concentrations in muscle and skin were similar to those in the tu-
mors whereas those in the blood and liver were much higher than
those in the tumors (Fig. S3). In particular, the concentrations of
the monomeric RGD derivatives GPU-51 and -49 in the liver were
significantly higher irrespective of their hydrophobicity, which
might cause to be lower concentration in blood and tumor. Conse-
quently, GPU-201 maintained high boron concentrations in tumors
more effectively than any other compound.
We, therefore, examined the dose-dependence of GPU-201 on
tumor distribution after intraperitoneal injection (Fig. 3B). The bor-
on concentrations in tumors from 30 min to 120 min following
intraperitoneal injection of GPU-201 decreased to one-third of that
of BSH intravenous injection because of low bioavailability. How-
ever, the tumor retention of boron following intraperitoneal injec-
tion was higher than that following intravenous injection. The
boron concentrations in tumors at each time point following intra-
peritoneal injection increased dose-dependently. Interestingly,
boron concentrations in tumors increased till 120 min, whereas
BSH was eliminated promptly.
Protected amino acids, Cl-Trt(2-Cl) resins were purchased from
Novabiochem (Merck). Decaborane (14) (B10H14) and 10B-enriched
disodium mercaptododecaborate (BSH) were purchased from Kat-
Chem. Other reagents were purchased from Sigma–Aldrich, Wako
or TCI. RP-HPLC analyses were performed on 10-AD series (Shima-
dzu) equipped with Waters Symmetry C18 analytical column
(Waters, 4.6 ꢂ 75 mm, flow rate 1 mL/min). For preparative HPLC,
Bensil 5-C18 preparative column (Bentech, 20 ꢂ 315 mm, flow rate
6 mL/min) was employed. A solvent system consisting 0.1% TFA
solution (v/v) and 0.1% TFA in acetonitrile (v/v) or 10 mM pH 7.0
sodium phosphate buffer and acetonitrile were used for HPLC elu-
tion and the eluting products were detected by UV at 220 nm.
Microwave reactions were performed on Biotage Initiator 2.0 with
single mode operating at 2.45 GHz. Electron impact (EI) or fast
atom bombardment (FAB) mass spectra were recorded on a JEOL
JMS-SX102A mass spectrometer. High- and low-resolution mass
spectra using electron spray ionization (ESI) were obtained on
LCMS-IT-TOF (Shimadzu) and HP1100 series (Hewlett-Packard),
respectively. 1H and 13C NMR spectra were recorded using a JEOL
JNM-AL400 or JNM-EX400 spectrometer at 400 MHz (1H NMR)
and 100 MHz (13C NMR) frequency in CDCl3, d6-DMSO or d6-Ace-
tone. Chemical shifts for 1H NMR were referenced to tetramethyl-
silane (0.00 ppm). Chemical shifts for 13C NMR were calibrated to
the solvent signals (CDCl3: 77.0 ppm, d6-DMSO: 39.5 ppm, d6-Ace-
tone: 29.8 ppm). Infrared (IR) spectra were measured on a JASCO
IRA-100. Inductively coupled plasma-atomic emission spectrome-
try (ICP-AES) analyses were performed on Ultima2 (Horiba-Jobin-
Yvon) and the spectra was measured at 249.773 nm.
4.2. Synthesis of c(RGDfK) and c(KGfDR) using solid phase
peptide synthesis by Fmoc strategy
Tumor accumulations of dimeric RGD derivatives were higher
than those in monomeric RGD derivatives. However, the results
did not clarify the direct correlation between in vivo tumor accu-
mulation and in vitro integrin-binding affinity. To maximize BNCT
efficiency, the amount of 10B atom accumulated in tumor tissue
Linear-protected peptides were manually constructed on Fmoc-
Gly-Trt-resin (0.4–0.6 mmol/g). Fmoc-amino acids were coupled
with 3 equiv of N,N0-diisopropylcarbodiimide and HOBtꢀH2O in
DMF for 1–2 h. The Fmoc group was deprotected by treatment with
20% (v/v) piperidine in DMF for 20 min. Protected peptide linked to
resin was cleaved with 20% (v/v) 1,1,1,3,3,3-hexafluoroisopropanol
in DCM for 2 h. Filtration and removal of the solvent gave the crude
linear protected peptide. The residue was dissolved in DMF (3 mM
solution) and the solution was cooled to ꢁ40 °C. Diphenylphospho-
must be P20 lg/g of tissue with a ratio of tumor/blood concentra-
tion of 4:1.23 Though the tumor retention of boron was signifi-
cantly improved, the maximum boron concentration was >9 ppm
in tumors 120 min after intraperitoneal injection at 0.75 mmol/kg
of GPU-201. Due to low water-solubility, administering an ade-
quate dose of the boron conjugate to achieve an efficient boron

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S. Kimura et al. / Bioorg. Med. Chem. 19 (2011) 1721–1728
ryl azide (3 equiv) and NaHCO3 (5 equiv) were added to the solu-
tion at the same temperature and the solution was stirred for
36 h at room temperature. After filtration and evaporation of the
solvent, the residue was charged over basic alumina and eluted
with CHCl3/MeOH (9:1) to afford crude protected cyclic peptide.
The protected cyclic peptide was deprotected with 95% (v/v) TFA
for 2 h at room temperature. After concentration, the residue was
purified by preparative RP-HPLC to afford pure cyclic peptide as a
bistrifluroacetate salt of colorless freeze-dried amorphous. Yield:
c(RGDfK)ꢀ2TFA (99%): LRMS (ESI) m/z: [M+H]+ 604.3,
added 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydro-
chloride (EDCIꢀHCl) (0.939 g, 4.90 mmol) at room temperature
and stirred for 4 h. The solvent was removed and the residue was
dissolved in ethyl acetate. The solution was washed with 10% citric
acid (three times), satd NaHCO3 (three times) and brine, dried over
MgSO4 and concentrated to afford crude product 7 which was used
without further purification. To
(50 mg, 0.06 mmol) and 7 (13.4 mg, 0.04 mmol) in 2 mL of DMF
was added DIPEA (34.8 L, 0.20 mmol) and the solution was stirred
a
solution of c(RGDfK)ꢀ2TFA
l
at room temperature. After 9 h, the solvent was removed in vacuo.
Purification of the crude product by preparative RP-HPLC afforded
8 (19.1 mg, 40% for two-steps) as trifluoroacetate salt of colorless
+
c(KGfDR)ꢀ2TFA (42%): HRMS (ESI) m/z: calcd for C27H41N9O7
[M+H]+ 604.3202, found 604.3210.
freeze-dried amorphous. HRMS (FAB) m/z: calcd for
C
39H55
e
+
4.2.1. cyclo[
L
-Arginyl-
L
-glycyl-
L
-asparagyl-
D
-phenylalanyl-
L
-( N-
N10O10 [M+H]+ 823.4097, found 823.4091.
[3-(1-maleimido)propionyl])lysine] trifluoroacetate (2)
e
To a solution of c(RGDfK)ꢀ2TFA (0.333 g, 0.40 mmol) and 1
(0.107 g, 0.40 mmol) in 5 mL of DMF was added N,N-diisopropyl-
ethylamine (DIPEA) (0.279 mL, 1.60 mmol) and the solution was
stirred at room temperature. After 24 h, the solvent was removed
in vacuo. Purification of the crude product by preparative RP-HPLC
afforded 2 (0.293 g, 84%) as trifluoroacetate salt of colorless freeze-
dried amorphous. HRMS (FAB) m/z: calcd for C34H47N10O10 [M+H]+
755.3471, found 755.3485.
4.2.6. cyclo(
L
-Arginyl-
L
-glycyl-
L
-asparagyl-
D
-phenylalanyl-
L
-[ N-
(trans-[4-(1-[3-(undecahydrododecaborato)sulfanyl]succimido)
methyl]cyclohexylcarbonyl)]lysine) sodium salt: GPU-176 (9)
The linker conjugated peptide 8 (19.0 mg, 0.02 mmol) and
10BSH (12.6 mg, 0.06 mmol) were dissolved in 6 mL of 0.2 M so-
dium phosphate buffer (pH 7.0)/MeCN (= 2:1) solution under N2
atmosphere and the solution was stirred at room temperature for
36 h. Purification of the crude product by preparative RP-HPLC
afforded 9 (10.6 mg, 45%) as sodium salt of colorless freeze-dried
e
10
4.2.2. cyclo[
L
-Arginyl-
L
-glycyl-
L
- asparagyl-
D
-phenylalanyl-
L
-( N-
amorphous. HRMS (ESI) m/z: calcd for C39H66 B
N10O10S2ꢁ
12
[(1-[3-(undecahydrododecaborato)sulfanyl]succinimido)
propionyl])lysine] disodium salt: GPU-51 (3)
[M]2ꢁ 493.3124, found 493.3110.
The linker conjugated peptide 2 (127 mg, 0.14 mmol) and 10BSH
(88.3 mg, 0.42 mmol) were dissolved in 5 mL of 0.2 M sodium
phosphate buffer (pH 7.0)/MeCN (= 1:1) solution under N2 atmo-
sphere and the solution was stirred at room temperature for one
day. The reaction solution was charged on a pre-charged resin
(Waters OASIS HLB cartridge) and eluted with H2O/MeCN
(100:0–85:15) to give 3 (74.3 mg, 56%) as disodium salt of colorless
4.2.7. 1-(4-Benzyloxybutyl)-1,2-dicarba-closo-dodecaborane
(12)
A 0.5 mL microwave vial equipped with stir bar was charged with
6-benzyloxyhex-1-yne (11) (0.20 mmol, 37.6 mg), decaborane (14)
(0.20 mmol, 24.4 mg), N,N-dimethylaniline (48.5 mg, 0.40 mmol)
and chlorobenzene (0.4 mL). The solution was stirred for 30 s before
microwave irradiation and the reaction vial was capped. The reac-
tion mixture was then heated by microwave irradiation for 30 min
at 80 °C. After irradiation, the reaction mixture was cooled and the
residual decaborane was decomposed with small amount of metha-
nol. The reaction mixture was purified by silica-gel chromatography
(hexane/ethyl acetate/benzene = 100:1:1) to give 12 (53.9 mg, 88%)
as a yellow oil. 1H NMR (400 MHz, CDCl3): d 1.26–3.04 (br m, 10H),
1.50–1.55 (m, 4H), 2.17 (t, J = 7.9 Hz, 2H), 3.42 (t, J = 5.2 Hz, 2H),
3.48 (br s, 1H), 4.45 (s, 2H), 7.25–7.35 (m, 5H); 13C NMR (100 MHz,
CDCl3): d 26.1, 28.9, 37.7, 60.9, 69.2, 72.9, 75.3, 127.6 (two-Ar carbon
was overlapped), 128.4, 138.2.
freeze-dried amorphous. HRMS (ESI) m/z: calcd for
10
C
34H58 B
N10O10S2ꢁ [M]2ꢁ 459.2811, found 459.2811.
12
e
4.2.3. Bis[ N0-cyclo(
L
-arginyl-
L
-glycyl-
L-asparagyl-D-
a
phenylalanyl-
L-lysyl)] N-[3-(1-maleimido)propionyl]glutamide
bistrifluoroacetate (4)
To a solution of E[c(RGDfK)2]ꢀ3TFA (143 mg, 0.086 mmol) and 1
(26.6 mg, 0.10 mmol) in 5 mL of DMF was added DIPEA (0.418 mL,
2.40 mmol) and the solution was stirred at room temperature.
After 6 h, the solvent was removed in vacuo. Purification of the
crude product by preparative RP-HPLC afforded 4 (109 mg, 86%)
as bistrifluoroacetate salt of colorless freeze-dried amorphous.
HRMS (ESI) m/z: calcd for C66H93N20O19+ [M+H]+ 1469.6920, found
1469.6906.
4.2.8. N-Succinimidyl 4-[1-(1,2-dicarba-closo-dodecaboranyl)]
butanoate (15)
A solution of 14 (130 mg, 0.56 mmol), N-hydroxysuccinimide
(96.7 mg, 0.84 mmol) and EDCIꢀHCl (161 mg, 0.84 mmol) in 5 mL
of DCM was stirred for 5.5 h at room temperature. The solvent was
evaporated, water was added and extracted withether (threetimes).
The combined organic phase was washed with 10% citric acid
(twice), brine, satd NaHCO3 (twice) and brine, dried over MgSO4
andevaporatedto affordcrudeproduct. Purificationby silica-gelcol-
umnchromatography (hexane/ethyl acetate = 1:1) gave 15 (178 mg,
97%) as a white solid. 1H NMR (400 MHz, CDCl3): d 1.42–3.14 (m,
10H), 1.91–1.99 (m, 2H), 2.34–2.38 (m, 2H), 2.63 (t, J = 6.8 Hz, 2H),
2.84 (s, 4H), 3.65 (br s, 1H); 13C NMR (100 MHz, CDCl3): d 24.3,
25.6, 30.2, 36.4, 61.7, 74.1, 167.5, 169.0; HRMS (FAB) m/z: calcd for
e
4.2.4. Bis[ N0-cyclo(
L
-arginyl-
L
-glycyl-
L-asparagyl-D-
a
phenylalanyl-
L-lysyl)] N-[(1-[3-(undecahydrododecaborato)
sulfanyl]succinimido)propionyl]glutamide: GPU-180 (5)
The linker conjugated peptide 4 (73.0 mg, 0.043 mmol) and
10BSH (40.0 mg, 0.19 mmol) were dissolved in 4.5 mL of 0.2 M so-
dium phosphate buffer (pH 7.0)/MeCN (= 8:1) solution under N2
atmosphere and the solution was stirred at room temperature for
two days. Purification of the crude product by preparative RP-HPLC
afforded 5 (50 mg, 71%) of colorless freeze-dried amorphous.
10
HRMS (ESI) m/z: calcd for C66H104
B
12
N20O19Sꢁ [M]2ꢁ 816.4535,
found 816.4545.
C
10H22 B10NO4+ [M+H]+ 330.2474,, found 330.2468.
e
4.2.9. cyclo( -Arginyl- -glycyl- -asparagyl- -phenylalanyl-L-[ N-
e
4.2.5. cyclo(
L
-Arginyl-
L
-glycyl-L-asparagyl-D-phenylalanyl-
L
-[ N-
L
L
L
D
(trans-[4-(1-maleimido)methyl]cyclohexylcarbonyl)]lysine)
(4-[1-(1,2-dicarba-closo-dodecaboranyl)]butanoyl)]lysine)
trifluoroacetate (8)
trifluoroacetate: GPU-49 (16)
To
a
solution of
6
(0.387 g, 1.60 mmol) and N-hydroxy-
To a solution of c(RGDfK)ꢀ2TFA (49.9 mg, 0.06 mmol) and 15
succinimide (HOSu) (0.562 g, 4.90 mmol) in 19.6 mL of DCM was
(29.5 mg, 0.09 mmol) in 0.6 mL of DMF was added DIPEA (42 L,
l

S. Kimura et al. / Bioorg. Med. Chem. 19 (2011) 1721–1728
1727
0.24 mmol) and the solution was stirred for one day. Purification of
the crude product by preparative RP-HPLC afforded 16 (48.1 mg,
86%) as trifluoroacetate salt of colorless freeze-dried amorphous.
HRMS (ESI) m/z: calcd for C33H58B10N9O8+ [M+H]+ 818.5333, found
818.5393.
solution was washed with 10% citric acid (three times), satd NaH-
CO3 (three times) and brine, dried over MgSO4 and concentrated to
afford crude di-activated ester 22. To a solution of 22 (crude
80 mg) and c(RGDfK)ꢀ2TFA (416 mg, 0.50 mmol) in 3 mL of DMF
was added DIPEA (0.372 mL, 2.14 mmol) and stirred for 12 h at
room temperature. The solvent was removed in vacuo and the res-
idue was purified by preparative RP-HPLC to afford 23 (121 mg,
e
4.2.10. Bis[ N0-cyclo(
L
-arginyl-
L
-glycyl-
L
-asparagyl-
D
-phenyl-
a
alanyl-
L
-lysyl)] N-(4-[1-(1,2-dicarba-closo-dodecaboranyl)]
46% from 21) as bistrifluoroacetate salt of colorless freeze-dried
2+
butanoyl)glutamide bistrifluoroacetate: GPU-189 (17)
amorphous. HRMS (ESI) m/z: calcd for C64H104B10N18O16
To a solution of E[c(RGDfK)2]ꢀ3TFA (99.6 mg, 0.06 mmol) and 15
[M+2H]2+ 745.4399, found 745. 4447.
(16.4 mg, 0.05 mmol) in 1 mL of DMF was added DIPEA (93 lL,
e
0.54 mmol) and the solution stirred for 8 h. Purification of the
crude product by preparative RP-HPLC afforded 17 (60.2 mg,
68%) as bistrifluoroacetate salt of colorless freeze-dried amor-
4.2.15. Bis[ N0-cyclo(
L-lysyl-
L
-glycyl-
D
-phenylalanyl-
L
-asparagyl-
L
-arginyl)] 1,2-dicarba-closo-dodecaboranedibutanamide
bistrifluoroacetate: GPU-205 (24)
To a solution of 22 (51.1 mg, 0.10 mmol) and c(KGfDR)ꢀ2TFA
(249 mg, 0.30 mmol) in 7 mL of DMF was added DIPEA (174 L,
phous. HRMS (ESI) m/z: calcd for C65H105B10N19O17 [M+2H]2+
2+
766.4468, found 766.4495.
l
1.0 mmol) and the reaction mixture was stirred overnight at room
temperature. The solvent was removed in vacuo and the residue
was purified by preparative RP-HPLC to afford 24 (43.1 mg, 25%
from 21) as bistrifluoroacetate salt of colorless freeze-dried amor-
4.2.11. 1,2-Bis(4-benzyloxybutyl)-1,2-dicarba-closo-dodecabo-
rane (19)
To a solution of 12 (61.3 mg, 0.20 mmol) in 2 mL of THF was
phous. HRMS (ESI) m/z: calcd for C64H104B10N18O16 [[M+2H]2+
2+
added dropwise n-butyllithium (2.76 M in hexane; 94.2
l
L,
0.26 mmol) under Ar atmosphere at ꢁ40 °C and the reaction was
warmed to room temperature. Lithium iodide (5.3 mg, 0.04 mmol)
in 0.4 mL of THF and 18 (63.2 mg, 0.26 mmol) in 1 mL of THF were
successively added and the mixture was stirred overnight at room
temperature. The reaction was quenched with water and the prod-
uct was extracted with ether. The organic phase was washed with
water, brine and then dried over MgSO4 and concentrated under
reduced pressure. The residue was purified by preparative-TLC
with hexane/ethyl acetate (10:1) to afford 19 (60.8 mg, 65%) as a
yellow oil. 1H NMR (400 MHz, CDCl3): d 7.35–7.31 (m, 10 H), 4.74
(s, 4 H), 3.43 (t, J = 6.0 Hz, 4 H), 2.13–2.12 (m, 4 H), 1.59–1.57 (m,
8 H), 2.79–1.34 (br m, 10 H); 13C NMR (100 MHz, CDCl3): d 165.8,
745.4399, found 745.4458.
4.3. Cell adhesion assay
Ninety-six-well culture plates were coated with 100 lL of 2 lg/
mL vitronectin (AGC Techno Glass) in PBS at 4 °C overnight, and
treated with 2% bovine serum albumin (BSA) (Wako) in PBS.
U87MG cells or SCCVII cells were suspended at 2 ꢂ 105 cells/mL
in Minimum Essential Medium Eagle (MEM) (Sigma–Aldrich) con-
taining 1 mM MgCl2, 0.25 mM MnSO4, and 0.1% BSA. The suspen-
sion was incubated with varying concentrations of RGD
conjugates for 20 min at 37 °C. Hundred microliter per well (2 ꢂ
104 cells) were added to the wells and incubated for 1 h at 37 °C.
The wells were washed three times with PBS. Adherent cells were
then fixed for 15 min with 10% formalin and stained with 0.04%
crystal violet. Plates were washed three times with distilled water
138.2, 128.4, 127.6, 79.7, 73.0, 64.5, 34.8, 29.2, 26.7; HRMS (EI)
10
m/z: calcd for C24H40
B
[M]+ 468.4026, found 468.4034.
10
4.2.12. 1,2-Bis(4-hydroxybutyl)-1,2-dicarba-closo-dodecaborane
(20)
and the remaining bound dye was solubilized in 100 lL of 1% SDS
A mixture of 19 (93.7 mg, 0.20 mmol), 10% Pd/C (42.6 mg,
0.04 mmol), cyclohexene (2 ml), and ethanol (4 mL) was refluxed
overnight. The mixture was then filtered with celite pad and con-
centrated under reduced pressure to give 20 (50.9 mg, 88%) as a
(Wako) for 1 h. The number of cells was then evaluated by measur-
ing absorbance at 570 nm with a microplate reader.
4.3.1. Mice and tumors
white solid. 1H NMR (400 MHz, d6-Acetone):
d
3.58–3.55 (t,
SCCVII squamous cell carcinoma derived from C3H/He mice was
maintained in vitro in MEM (Sigma–Aldrich) containing 12.5% FBS
(Japan Bioserum). Cells were collected from exponentially growing
cultures, and 1 ꢂ 105 cells of each tumor were inoculated subcuta-
neously into both hind legs of 8–11-week-old syngeneic female
C3H/He mice (Japan Animal). Fourteen days after inoculation, each
tumor was approximately 1 cm in diameter. Mice were handled
according to the Recommendations for Handling of Laboratory Ani-
mals for Biomedical Research, complied by the Committee on
Safety and Ethical Handling Regulations for Laboratory Animal
Experiments, Kyoto University. All experimental procedures men-
tioned here were in accordance with institutional guidelines for
the care and use of laboratory animals in research.
J = 6.1 Hz, 4H), 2.40–2.36 (m, 4H), 1.70–1.62 (m, 4H), 1.58–1.51
(m, 4H), 4.48–1.00 (m, 10H); 13C NMR (100 MHz, d6-Acetone):
+
81.8, 61.6, 35.3, 32.7, 27.2; HRMS (EI) m/z: calcd for C10H28B10O2
[M+] 290.3014, found 290.3020.
4.2.13. 1,2-Dicarba-closo-dodecaboranedibutanoic acid (21)
To a solution of 20 (419 mg, 1.45 mmol) in 16.5 mL of wet
MeCN was added dropwise 16.5 mL of the H5IO6/CrO3 solution in
an ice bath.46 The solution was stirred for 2 h at 0 °C and overnight
at room temperature. The reaction mixture was purified by RP-
HPLC to give 21 (458 mg, quant.) as colorless freeze-dried amor-
phous. 1H NMR (400 MHz, d6-Acetone): d 1.21–3.05 (m, 10H),
1.68–1.75 (m, 4H), 2.24–2.33 (m, 8H), 10.43 (br s, 1H); 13C NMR
(100 MHz, d6-Acetone): 25.6, 32.8, 34.5, 81.3, 174.0; HRMS (ESI)
4.3.2. Biodistribution experiments
10
m/z: calcd for C10H25
B
10
O4 [M+H]+ 309.3041, found 309.3049.
+
All compounds were dissolved in physiological saline contain-
ing 10% HP-b-cyclodextrin (Nisshoku). They were administered
to the tumor-bearing mice via the intravenous or intraperitoneal
route. BSH dissolved in physiological saline was administrated
intravenously or intraperitoneally. At various time points following
BSH administration, mice were killed, and tumors and certain or-
gans were collected and weighed. Samples (0.2–0.5 g) in polyethyl-
ene vials were treated with 70% perchloric acid and 30% hydrogen
peroxide. The vials were then placed in shaking water bath for 24 h
e
4.2.14. Bis[ N0-cyclo(
L
-arginyl-
L
-glycyl-
L
-asparagyl-
D
-phenyl-
alanyl-L-lysyl)] 1,2-dicarba-closo-dodecaboranedibutanamide
bistrifluoroacetate: GPU-201 (23)
To a solution of 21 (458 mg, 1.45 mmol) and HOSu (460 mg,
4.0 mmol) in 40 mL of DCM was added EDCIꢀHCl (767 mg,
4.0 mmol) at room temperature and stirred for 12 h. The solvent
was removed and the residue was dissolved in ethyl acetate. The

1728
S. Kimura et al. / Bioorg. Med. Chem. 19 (2011) 1721–1728
16. Wu, G.; Barth, R. F.; Yang, W.; Lee, R. J.; Tjarks, W.; Backer, M. V.; Backer, J. M.
Anticancer Agents Med. Chem. 2006, 6, 167.
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Maruyama, K. Bioconjugate Chem. 2006, 17, 1314.
at 75 °C. The volume of the samples was precisely adjusted to 5–
10 L with distilled water. The solutions were then filtered
through a 0.22- m filter. Boron concentrations in the solution
l
l
18. Thirumamagal, B. T. S.; Zhao, X. B.; Bandyopadhyaya, A. K.; Narayanasamy, S.;
Johnsamuel, J.; Tiwari, R.; Golightly, D. W.; Patel, V.; Jehning, B. T.; Backer, M.
V.; Barth, R. F.; Lee, R. J.; Backer, J. M.; Tjarks, W. Bioconjugate Chem. 2006, 17,
1141.
were measured by ICP-AES.
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This research was supported in part by a Grant from The Saijiro
Endo Memorial Foundation for Science & Technology, the Sasaga-
wa Scientific Research Grant from The Japan Science Society (19-
621), and a Grant-in-Aid for Scientific Research (C) (19590102)
from the Japan Society for the Promotion of Science. The authors
thank Prof. Osamu Sakurada at Gifu University for the use of ICP-
AES, Prof. Mitsunori Kirihata at Osaka Prefecture University for
helpful suggestions about ICP-AES, and Ms. Emi Inaba for their
technical assistance.
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