Synthesis of dodecaborate-conjugated cholesterols for efficient boron delivery in neutron capture therapy

Article abstract of DOI:10.1016/j.tetlet.2007.03.043

Dodecaborate-conjugated cholesterols 3a-c were synthesized for liposomal boron delivery systems in neutron capture therapy. The current synthesis is based on the S-alkylation protocol of the cyanoethyl-protected BSH with alkyl halides. The dodecaborate-conjugated cholesterol 3a liposome, which was prepared from dimyristoylphosphatidylcholine (DMPC), cholesterol, dodecaborate-conjugated cholesterol 3a, and polyethyleneglycol-conjugated distearoylphosphatidylethanolamine (PEG-DSPE) (1:0.5:0.5:0.1), exhibited higher cytotoxicity than BSH at the same boron concentration and IC₅₀ values of the 3a liposome and BSH toward colon 26 cells were estimated as 25 and 78 ppm of boron concentration, respectively.

Full text of DOI:10.1016/j.tetlet.2007.03.043

Tetrahedron Letters 48 (2007) 3151–3154
Synthesis of dodecaborate-conjugated cholesterols for
efficient boron delivery in neutron capture therapy
Hiroyuki Nakamura,a,* Manabu Ueno,a Jong-Dae Lee,a Hyun Seung Ban,a Eugen Justus,b
Ping Fanb and Detlef Gabelb
aDepartment of Chemistry, Faculty of Science, Gakushuin University, Mejiro, Tokyo 171-8588, Japan
bDepartment of Chemistry, University of Bremen, D-28334 Bremen, Germany
Received 15 February 2007; revised 3 March 2007; accepted 8 March 2007
Available online 12 March 2007
Abstract—Dodecaborate-conjugated cholesterols 3a–c were synthesized for liposomal boron delivery systems in neutron capture
therapy. The current synthesis is based on the S-alkylation protocol of the cyanoethyl-protected BSH with alkyl halides. The dode-
caborate-conjugated cholesterol 3a liposome, which was prepared from dimyristoylphosphatidylcholine (DMPC), cholesterol, dode-
caborate-conjugated cholesterol 3a, and polyethyleneglycol-conjugated distearoylphosphatidylethanolamine (PEG-DSPE)
(1:0.5:0.5:0.1), exhibited higher cytotoxicity than BSH at the same boron concentration and IC50 values of the 3a liposome and
BSH toward colon 26 cells were estimated as 25 and 78 ppm of boron concentration, respectively.
Ó 2007 Elsevier Ltd. All rights reserved.
Boron neutron capture therapy (BNCT) is a binary can-
cer treatment based on the nuclear reaction of two
NBu4
10
essentially nontoxic species, B and thermal neutrons.1
The high neutron capture cross section of 10B allows us
to generate an a-particle and a lithium-7 ion bearing
approximately 2.4 MeV by neutron capture reaction,
and these high linear energy transfer particles afford pre-
cise cell killing.2–5 We have focused on boronated lipo-
somes for boron delivery system.6,7 A system involving
the accumulation of boron in the liposomal bilayer is
highly potent, because drugs can be encapsulated into
the vacant inner cell of a liposome. Furthermore, it is
possible to functionalize liposomes by combination of li-
pid contents. Therefore, boron and drugs may be simul-
taneously delivered to tumor tissues for BNCT and
chemotherapy of cancers. We recently reported boron
ion cluster lipids which have a double-tailed moiety
conjugated with boron ion clusters such as nido-
carborane8,9 and closo-dodecaborate10 as a hydrophilic
function, and investigated the stability, boron biodistri-
bution, and BNCT effect of their liposomes.9,11 Carbo-
rane-conjugated cholesterols (1)12 and carborane-
containing cholesterol mimic (2)13,14 have been devel-
oped as an alternative content of liposomal membranes
as shown in Scheme 1.
H
O
H
O
1
= C
= BH
2
HO
2
Y
O
S
O
lipophilic part
hydrophilic part
Y = CH
3a:
b:
c:
2
= BH
= B
Y = CH2CH2
Y = NHCOCH2
Scheme 1. Structures of boron cluster-conjugated cholesterols and the
mimic.
*
We focused on the structure of mercaptoundecahydro-
dodecaborate (BSH), which has been utilized for clinical
Corresponding author. E-mail: hiroyuki.nakamura@gakushuin.ac.
jp
0040-4039/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2007.03.043

3152
H. Nakamura et al. / Tetrahedron Letters 48 (2007) 3151–3154
treatment on BNCT, as a water-soluble boron cluster of
low toxicity. In this Letter, we report the synthesis of
dodecaborate-conjugated cholesterols 3a–c (Scheme 1)
for liposomal boron delivery systems. The current syn-
thetic strategy is based on the S-alkylation protocol of
the cyanoethyl-protected BSH with alkyl halides.15
4a-c
"protected BSH"
MeCN
We first introduced the various linkers at the hydroxy
group of cholesterol as shown in Scheme 2. Cholesterol
was treated with bromoacetyl bromide in the presence of
pyridine as a base to give the corresponding ester 4a and
with bromopropionyl chloride in the presence of trieth-
ylamine to 4b in 83% and 81% yields, respectively. In a
similar manner, chloroacetylcarbamate 4c was also syn-
thesized by treating with chloroacetyl isocyanate in the
presence of trimethylamine.
Y
O
S
NC
O
5a: Y = CH2
5b: Y = CH2CH2
5c: Y = NHCOCH2
Me4NOH
acetone
2
We next examined the introduction of BSH into the cho-
lesterol derivatives 4 as shown in Scheme 3. The S-alkyl-
ation of the protected BSH with 4a–c proceeded in
acetonitrile and the resulting sulfoniums 5a–c were trea-
ted with an equivalent of tetramethylammonium
hydroxide in acetone to give the corresponding thio-
esters 3a–c in 73%, 54%, and 47% yields, respectively,
in two steps.
Y
O
S
O
3a-c
Scheme 3. Synthesis of dodecaborate-conjugated cholesterols 3a–c.
A typical procedure for the synthesis of 3a from 4a is as
follows: To a mixture of the protected BSH (200 mg,
0.51 mmol) in acetonitrile (10 mL) was added 4a
(275 mg, 0.55 mmol) in THF (10 mL) at room tempera-
ture, and the reaction mixture was heated at 60 °C and
stirred for 14 h. The solvent was removed and the resi-
due was purified by column chromatography on silica
gel with ethyl acetate/acetone (5:1) to give 5a, as a white
solid, quantitatively. Sulfonium salt 5a obtained was
dissolved in acetone (5 mL) and a methanol solution
of tetramethylammonium hydroxide (25% w/w, 186
mg) was then added with stirring. The white precipitate
was filtered, washed with acetone, and dried in vacuo to
give 3a in 73% yield as a white solid.16
The 2D 13C/1H NMR spectrum of 3b and the assign-
ment are shown in Figure 1 and Table 1, respectively.
1
Whereas monodimensional H spectra did not resolve
the resonances of the extra protons of the linker, this
could be achieved with the two-dimensional spectrum.
We next examined the cytotoxicity of liposomes contain-
ing 3a. The liposome was prepared from dimyristoyl-
phosphatidylcholine (DMPC), cholesterol, dodecaborate-
conjugated cholesterol 3a, and polyethyleneglycol-
conjugated distearoylphosphatidylethanolamine (PEG-
DSPE) (1:0.5:0.5:0.1), by the reverse-phase evaporation
(REV) method.17 The liposomes obtained were sub-
jected to extrusion 10 times through a polycarbonate
membrane of 100 nm pore size, using an extruder device
thermostated at 60 °C. Purification was accomplished by
ultracentrifuging at 200,000g for 60 min at 4 °C, and the
pellets obtained were resuspended in cell culture med-
ium. Boron concentration was determined by induc-
tively coupled plasma atomic emission spectroscopy
(ICP-AES). Figure 2 shows dose-dependent cell survival
curves using colon 26 mouse colonectal cancer cells. The
dodecaborate-conjugated cholesterol 3a liposome exhib-
ited higher cytotoxicity than BSH at the same boron
concentration and IC50 values of the 3a liposome
and BSH were estimated as 25 and 78 ppm of boron
concentration, respectively. The higher cytotoxicity of
cholesterol 3a liposome may be due to the higher lipo-
philicity and higher molecular weight of 3a (and its lipo-
some) in comparison with BSH.18 It should be noted
that total dose volume of cholesterol 3a liposome for
administration was much larger than that of BSH
although boron concentrations were similar.
O
Br
4a: Y = CH2
b: Y = CH2CH2
O
Br
Y
Br
, py , CH2Cl2
O
cholesterol
N C
O
Cl
59%
, TEA , CH2Cl2
O
H
N
O
Cl
O
O
4c
Scheme 2. Synthesis of cholesterol-linker conjugates 4a–c.

H. Nakamura et al. / Tetrahedron Letters 48 (2007) 3151–3154
3153
(ppm)
16
24
32
40
48
56
(ppm)
2.4
2.0
1.6
1.2
0.8
Figure 1. HSQC 13C/1H spectrum of 3b.
Table 1. HSQC 13C/1H spectrum of 3b as tetramethylammonium salt
120
100
80
60
40
20
0
50% 3a liposome
BSH
Assignment
13C d (ppm)
1H d (ppm)
1 CH2
2 CH2
3 CH–C–CO
4 C2
5 @Cq
6 @CH
7 CH2
8 CH
37.8
28.5
74.1
38.85
141.1
123.0
32.5
1.87/1.11
1.80/1.59
4.47
2.29
—
5.38
1.97/1.53
1.48
32.6
9 CH
51.0
0.96
10 Cq
37.4
—
100 CH3
11 CH2
12 CH2
13 Cq
19.7
21.7
40.5
43.0
1.03
1.50
2.02/1.17
—
10
100
Boron conc. (ppm)
1000
130 CH3
14 CH
15 CH2
16 CH2
17 CH
18 CH
180 CH3
19 CH2
20 CH2
21 CH2
22 CH
23 CH3
S–CH2
CH2–CO
N(CH3)4
12.1
57.6
24.4
28.9
56.9
36.5
19.0
36.9
24.9
40.1
28.6
23.0/22.7
28.3
0.70
1.04
1.36/1.16
1.84/1.28
1.11
1.39
0.93
1.37/1.01
1.58/1.06
1.13
1.52
0.864/0.860
2.52
2.51
3.11
Figure 2. Cell survivals of colon 26 mouse colorectal cancer cells with
the 3a liposome and BSH after incubation for 3 days.
Acknowledgment
Part of this project was supported by JSPS under the
Japan–Germany Research Cooperative Program.
38.8
56.2/56.1/56.0
References and notes
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orate-conjugated cholesterols 3a–c. The BSH protection
protocol was effective for conjugation of BSH into
cholesterol. Liposomal formulations of 3a–c and
in vivo biodistribution of their liposomes are now under
investigation.

3154
H. Nakamura et al. / Tetrahedron Letters 48 (2007) 3151–3154
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