Design of multivalent galactosyl carborane as a targeting specific agent for potential application to boron neutron capture therapy

Article abstract of DOI:10.1039/c1cc14447b

A multivalent galactosyl carborane derivative 10 (dendritic glyco-borane, DGB) was synthesized and demonstrated as a potential cell-targeting agent in BNCT with HepG2 cells. DGB 10 improved the delivery of boron to HepG2 cells and neutron irradiation data

Full text of DOI:10.1039/c1cc14447b

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COMMUNICATION
Design of multivalent galactosyl carborane as a targeting specific agent
for potential application to boron neutron capture therapyw
Chian-Hui Lai,a Yu-Chuan Lin,b Fong-In Chou,b Chien-Fu Liang,a En-Wei Lin,a
Yung-Jen Chuangc and Chun-Cheng Lin*a
Received 21st July 2011, Accepted 7th November 2011
DOI: 10.1039/c1cc14447b
A multivalent galactosyl carborane derivative 10 (dendritic
glyco-borane, DGB) was synthesized and demonstrated as a
potential cell-targeting agent in BNCT with HepG2 cells. DGB
10 improved the delivery of boron to HepG2 cells and neutron
irradiation data show DGB 10 with ten-fold improvement at
killing the HepG2 cells over BSH.
or target ligands.1a Recently, many carborane-based derivatives
categorized as small molecules,2 polymers,3 dendrimers,4 and
liposomes5 have been designed and synthesized.
Cell surface carbohydrate–protein interactions are involved
in many biological events.6 For example, asialoglycoprotein
receptors (ASGP-Rs) found on the surface of hepatocyte cells
(1–5 Â 105 ASGP-R/cell7 for HepG2 cells) interact with the
nonreducing end of galactose (Gal) or N-acetyl-galactosamine
(GalNAc) moieties on glycoproteins and trigger the receptor-
mediated endocytotic uptake of glycoproteins into the cell.8
ASGP-R contains two H1 and H2 subunits and exhibits high
affinity for triantennary Gal/GalNAc ligands9 due to the
presence of multivalent interactions. The Kd of ASGP-R with
mono-, di-, tri-, and tetra-antennary galactosyl oligosaccharides
is B10À3, B10À6, B5 Â 10À9, and B10À9 M, respectively.9
Previously, we showed that a pre-assembled tri-antennary Gal
moiety on fluorescent magnetic nanoparticles (MNP) was a
better multivalent ligand for HepG2 cells uptake.10 Thus, the
similar tri-antennary galactosyl structure 4 (Scheme 1) was
used as targeting moiety to improve the specificity and water
solubility of the boron complex to reduce the cytotoxicity.2c
Previously, we applied neutron irradiation to investigate BPA,
BSH, and boric acid (BA) as potential boron carriers in the
treatment of hepatoma.11 To further increase the specificity and
intracellular uptake of the BNCT agent, we designed a novel,
potent BNCT agent, compound 10 (a dendritic glyco-borane,
DGB) as shown in Scheme 1. Nearly quantitative yields of DGB
10 were obtained by using Cu(I)-catalyzed azide–alkyne cyclo-
addition (CuAAC)12 in which the DGB contains two moieties,
a trivalent galactose residue for targeting and a trivalent
carborane as boron source. Subsequently, the effect of 10 on
BNCT was evaluated by irradiating cells with neutrons using
the Tsing-Hua Open Pool Reactor (THOR) at the National
Tsing-Hua University in Taiwan.
Boron neutron capture therapy (BNCT) is a binary radiation
therapy for cancer that involves the irradiation of non-radioactive
10B-enriched tumors with low energy thermal neutrons to yield
high linear energy transfer (LET) particles.1 Neutron capture
results in the formation of excited 11B nuclei that split into
4
7
highly energetic He2+ (a-particle) and Li3+ ions. Due to the
limited penetrable path length (5–9 mm, approximately the radius
of a typical cell) of high-LET particles into tissues, the destruc-
tive effects of high-LET particles are localized within cells that
contain boron atoms,1 which reduces damage to healthy tissues.
The success of BNCT treatment depends on the amount of
10B inside the tumor cell (15–35 mg of 10B per gram of tumor
tissue or 109 atoms of 10B per tumor cell) and the differential
uptake of boron in tumor and normal cells (greater than 3 : 1).1
Currently, only two boronated compounds are clinically used
for BNCT, sodium borocaptate (Na2B12H11SH or BSH) and
boronophenylalanine (BPA). However, neither agent can
selectively target tumor cells, which results in cytotoxicity and
limits their application. Thus, a well-designed BNCT agent with
a cell-targeting moiety could increase the amount of boron in
cancer cells and result in the selective destruction of malignant
cells. To enrich cancer cells with 10B atoms for BNCT, carborane
cages that contain ten boron atoms are ideal substrates
because they possess a large number of boron atoms per
molecule and can be readily conjugated with organic molecules
a Department of Chemistry, National Tsing Hua University,
101, Sec. 2, Kuang Fu Rd., Hsinchu 30013, Taiwan.
E-mail: cclin66@mx.nthu.edu.tw
The synthesis of DGB 10 is illustrated in Scheme 1. Trivalent
galactosyl alkyne 4 was obtained by reacting N,N-bis[carboxy-
methyl]-L-lysine (1)13 with 2,5-dioxopyrrolidin-1-yl pent-4-ynoate
(2)14 under basic conditions, followed by coupling with 6-amino-
hexyl-D-galactopyranoside (3)15 using HATU16 (46% yield over
two steps). Through the modification of literature procedures,2e,17
decaborane was reacted with N-(5-hexynyl) phthalimide (5)
under reflux to yield 6 (43%). Subsequently, the phthalyl
group of 6 was removed by stepwise cleavage,18 and sodium
b Nuclear Science and Technology Development Center,
and National Tsing Hua University, 101, Sec. 2, Kuang Fu Rd.,
Hsinchu 30013, Taiwan
c Department of Medical Science and Institute of Bioinformatics and
Structural Biology, National Tsing Hua University, 101, Sec. 2,
Kuang Fu Rd., Hsinchu 30013, Taiwan. Fax: +886 3-571-1082
w Electronic supplementary information (ESI) available: Experimental
details, characterization data and NMR spectra for new compounds.
See DOI: 10.1039/c1cc14447b
c
612 Chem. Commun., 2012, 48, 612–614
This journal is The Royal Society of Chemistry 2012

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Scheme 1 Synthesis of DGB 10. Reaction conditions: (a) (1) 2, DMF/H2O, NEt3, 4 1C, 16 h, 90%; (2) 3, HATU, DMF, NEt3, 45 1C, 16 h, 51%;
(b) B10H14, CH3CN, toluene, reflux, 4 h, 43%; (c) NaBH4, i-PrOH, rt, 22 h, 76%; (d) HCl/HOAc, 95 1C, 3 h, 95%; (e) (1) 8, DMF/H2O, NEt3, rt,
16 h, 88%; (2) 7, HBTU, DMF, 45 1C, 16 h, 53%; (f) 4, CuI, CuSO4, sodium ascorbate, NEt3, EtOH/H2O/tBuOH, rt, 65 h, 98%.
borohydride reduction and acidic hydrolysis were conducted
to obtain 7 (72% yield for two steps). Trivalent carborane
derivative 9 was synthesized using a procedure that was similar
to the method for the synthesis of compound 4. Compound 1
was treated with activated 8 (Scheme S2 in ESIw) and was
coupled with compound 7 under basic conditions. Finally,
trivalent galactosyl alkyne 4 was conjugated with trivalent
carborane azide 9 by CuAAC12 to give an almost quantitative
yield of DGB 10. The formation of compound 6 was evidenced
by the absence of an alkynyl proton peak and the presence of a
indicating that uptake occurs via ASGP-R mediated endocy-
tosis, not passive diffusion. The incubation time of compound
10 with HepG2 cells (72 h, Fig. S6 (ESIw)) was increased, and
enhanced cytotoxicity was observed, further confirming the
uptake of 10 by HepG2 cells. Notably, when cells were
incubated with low concentrations of boron-containing
compounds (r50 ppm) for 6 h, no significant cytotoxicity
was observed. For comparison, HepG2 cells were incubated
with BSH at boron concentrations of 25 and 50 ppm for 6h,
and cytotoxicity was not detected (Fig. S7, ESIw). In addition,
soft-agar colony formation assays were employed to show that
compound 10 (at boron concentrations of 12.5 and 50 ppm)
did not affect cell proliferation (Fig. S8, ESIw).
1
carborane CH resonance peak at 3.59 ppm in the H NMR
spectrum (CDCl3). Due to the low solubility of compounds 7
1
and 10 in CDCl3, the H NMR spectra of these compounds
were taken in CD3OD and the carborane CH resonance peaks
appeared at 4.55 ppm. Moreover, multiple broad peaks
appeared between 1.5 and 2.9 ppm, indicating the presence of
BH protons (see ESIw, the broad BH peaks were not integrated
The amount of compound 10 taken up by HepG2 cells was
determined by inductively coupled plasma–mass spectrometry
(ICP-MS). HepG2 cells were seeded and then cultured in
DMEM overnight. The cells were incubated in a solution of
DMEM containing compound 10 (boron concentration = 12.5,
25, and 50 ppm) and BSH (boron concentration = 50 ppm),
respectively. After 6 h of incubation, the cells were washed three
times with PBS buffer and were treated with a strong oxidant and
strong acid to determine the boron concentration by ICP-MS.
As illustrated in Fig. 1, HepG2 cells showed higher uptake of
compound 10. Under all of the studied conditions, more than
1010 boron atoms were delivered per HepG2 tumor cell.
Although only 19.6% of the boron in the carborane cage was
10B, this amount is sufficient for the success of BNCT. Using
equal boron concentration, the uptake efficiency of DGB 10 and
BSH into HepG2 cells was compared, and DGB 10 showed
20-fold greater boron uptake than that of BSH. Overall, DGB 10
showed selective targeting capability and highly efficient boron
delivery, which are requirements for potential BNCT agents.
Subsequently, the use of DGB 10 as a potential BNCT agent
for hepatoma was evaluated. HepG2 cells were treated with
DGB 10 at boron concentrations of 50 ppm for 6 h and were
irradiated with thermal neutrons at a neutron flux of 1.1 Â
1011 n cmÀ2 sÀ1 in the THOR. To compare the cell killing
1
in the H-NMR spectra). In addition, the 13C NMR spectrum
displayed a CH carborane resonance peak around 61 ppm and
a CB carborane resonance peak around 75 ppm. Notably, the
11B NMR spectra of carborane derivatives showed similar peak
patterns (see Fig. S1, ESIw). The naturally occurring isotopes of
boron (10B, 19.6% and 11B, 80.4%) yield characteristic isotope
patterns19 in the mass spectra of boronyl compounds; thus,
mass analysis was also applied to confirm the production of
carborane derivative 10 (Fig. S2, ESIw). The reverse HPLC
spectrum of compound 10 is shown in Fig. S3 (ESIw). The
IR spectra of the boronyl compounds displayed a strong
B–H stretch at approximately 2580 cmÀ1 (Fig. S4, ESIw). In
addition, the loss of an azido signal at 2100 cmÀ1 in the IR
spectrum of 10 indicated the success of CuACC.
The cytotoxicity of compound 10 was evaluated by con-
ducting MTT assays on HepG2 cells with ASGP-R and Hela
cells without ASGP-R. The results (Fig. S5, ESIw) revealed
that compound 10 had a concentration dependent cytotoxic
effect on HepG2 cells after 6 h of incubation. Alternatively,
compound 10 did not have cytotoxic effects on Hela cells,
c
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Chem. Commun., 2012, 48, 612–614 613

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di-dendrimer structure of 10 provides multivalent ligand receptor
interactions for selective cell targeting and enhances the delivery
of boron into cells. In addition, the use of galactose as a targeting
moiety increases the water solubility of the carborane cage.
This work was financially supported by Academia Sinica,
the National Tsing Hua University, and the National Science
Council, Taiwan.
Notes and references
Fig. 1 Average boron concentration obtained by ICP-MS per
HepG2 cell after incubation with compound 10 or BSH for 6 hours.
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1011 n cmÀ2 À1, respectively. Notably, when the dose of
s
irradiated thermal neutrons is increased, the cell lethal effect
is correspondingly increased, which is no dose contribution
from the BNCT.11 Compared to previous reports,11 DBG 10
provided superior cytotoxic effect at low irradiation flux
(1.66 Â 1011 n cmÀ2
s
À1). Furthermore, upon irradiation at
an intensity of 4.90 Â 1011 n cmÀ2, the surviving fraction of
cells treated with DBG 10 was only 0.45%. Thus, the results
clearly demonstrated that DBG 10 provides superior in vitro
cytotoxic effects and indicated that targeting is of great
importance in the development of BNCT agents.
The success of BNCT treatment ultimately depends on its
in vivo tumor-localizing property and its ability to enrich boron
atoms in tumors. In the present study, we demonstrated the
usefulness of DGB 10 as a potential boron delivery agent. The
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614 Chem. Commun., 2012, 48, 612–614
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