Synthesis of boron cluster lipids: Closo-dodecaborate as an alternative hydrophilic function of boronated liposomes for neutron capture therapy

Article abstract of DOI:10.1021/ol062840+

(Chemical Equation Presented) We succeeded in the synthesis of the double-tailed boron cluster lipids 4a-c and 5a-c, which have a B ₁₂H₁₁S moiety as a hydrophilic function, by S-alkylation of B₁₂H₁₁SH (BSH) with bromoacetyl and chloroacetocarbamate derivatives of diacylglycerols for a liposomal boron delivery system on neutron capture therapy. Calcein encapsulation experiments revealed that the liposomes, prepared from the boron cluster lipid 4b, DMPC, PEG-DSPE, and cholesterol, are stable at 37°C in FBS solution for 24 h.

Full text of DOI:10.1021/ol062840+

ORGANIC
LETTERS
2007
Vol. 9, No. 2
323-326
Synthesis of Boron Cluster Lipids:
closo-Dodecaborate as an Alternative
Hydrophilic Function of Boronated
Liposomes for Neutron Capture Therapy
Jong-Dae Lee, Manabu Ueno, Yusuke Miyajima, and Hiroyuki Nakamura*
Department of Chemistry, Faculty of Science, Gakushuin UniVersity, Mejiro,
Toshima-ku, Tokyo 171-8588, Japan
hiroyuki.nakamura@gakushuin.ac.jp
Received November 22, 2006
ABSTRACT
We succeeded in the synthesis of the double-tailed boron cluster lipids 4a−c and 5a−c, which have a B12H11S moiety as a hydrophilic function,
by S-alkylation of B12H11SH (BSH) with bromoacetyl and chloroacetocarbamate derivatives of diacylglycerols for a liposomal boron delivery
system on neutron capture therapy. Calcein encapsulation experiments revealed that the liposomes, prepared from the boron cluster lipid 4b,
DMPC, PEG-DSPE, and cholesterol, are stable at 37 °C in FBS solution for 24 h.
Boron neutron capture therapy (BNCT) is a binary cancer
treatment based on the nuclear reaction of two essentially
nontoxic species, 10B and thermal neutrons.1 The neutron
capture reaction by 10B produces an R-particle and a lithium-7
ion bearing approximately 2.4 MeV, and these high linear
energy transfer particles afford precise cell killing.2-4
Therefore, high accumulation and selective delivery of boron
into the tumor tissue are the most important requirements to
achieve efficient neutron capture therapy of cancers. There
are three most important parameters for development of
boron compounds: (1) achieving tumor concentrations in
the range of 20-35 µg 10B/g; (2) a tumor/normal tissue
differential greater than 3-5; (3) sufficiently low toxicity.2,5
Recent promising approaches that meet these requirements
entail the use of small boron molecules2,6 and boron-
conjugated biological complexes, such as monoclonal anti-
bodies,7 the epidermal growth factor,8 and carborane oligo-
(6) Our recent papers the for development of small boron molecules:
(a) Nakamura, H.; Fukuda, H.; Girald, F.; Kobayashi, T.; Hiratsuka, J.;
Akaizawa, T.; Nemoto, H.; Cai, J.; Yoshida, K.; Yamamoto, Y. Chem.
Pharm. Bull. 2000, 48, 1034. (b) Takahashi, K.; Nakamura, H.; Furumoto,
S.; Yamamoto, K.; Fukuda, H.; Matsumura, A.; Yamamoto, Y. Bioorg.
Med. Chem. 2005, 13, 735. (c) Tietze, L. F.; Bothe, U.; Griesbach, U.;
Nakaichi, M.; Hasegawa, T.; Nakamura, H.; Yamamoto, Y. Chem. Bio.
Chem. 2001, 2, 326. (d) Lee, C.-H.; Oh, J. M.; Lee, J.-D.; Nakamura, H.;
Ko, J.; Kang, S.-O. Synlett 2006, 275. (e) Nakamura, H.; Kuroda, H.; Saito,
H.; Suzuki, R.; Yamoti, T.; Maruyama, K.; Haga, T. Chem. Med. Chem.
2006, 1, 729.
(7) (a) Goldenberg, D. M.; Sharkey, R. M.; Primus, F. J.; Mizusawa, E.
A.; Hawthorne, M. F. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 560. (b) Alam,
F.; Soloway, A. H.; McGuire, J. E.; Barth, R. F.; Carey, W. E.; Adams, D.
J. Med. Chem. 1985, 28, 522. (c) Pak, R. H.; Primus, F. J.; Rickard-Dickson,
K. J.; Ng, L. L.; Kane, R. R.; Hawthorne, M. F. Proc. Natl. Acad. Sci.
U.S.A. 1995, 92, 6986.
(1) Locher, G. L. Am. J. Roentgenol. 1936, 36, 1.
(2) Soloway, A. H.; Tjarks, W.; Barnum, B. A.; Rong, F-G.; Barth, R.
F.; Codogni, I. M.; Wilson, J. G. Chem. ReV. 1998, 98, 1515.
(3) Barth, R. F.; Soloway, A. H.; Fairchild, R. G. Cancer Res. 1990, 50,
1061.
(4) Hawthorne, M. F. Angew. Chem., Int. Ed. Engl. 1993, 32, 950.
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1992, 70, 2995.
(8) (a) Gedda, L.; Olsson, P.; Carlsson, J. Bioconjugate Chem. 1996, 7,
584. (b) Yang, W.; Barth, R. F.; Adams, D. M.; Soloway, A. H. Cancer
Res. 1997, 57, 4333.
10.1021/ol062840+ CCC: $37.00
© 2007 American Chemical Society
Published on Web 12/20/2006

mers.9 We have focused on boronated liposomes for the
boron delivery system. 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, functionalization of liposomes is
possible by combination of lipid contents. Therefore, boron
and drugs may be simultaneously delivered to tumor tissues
for BNCT and chemotherapy of cancers. Hawthorne and co-
workers first introduced nido-carborane as a hydrophilic
moiety into the amphiphile 1 (Figure 1) and examined
toxicity and thus has been utilized for clinical treatment of
BNCT. However, few synthetic examples of BSH derivatives
as boron carriers have been reported so far due to the
difficulty of their functionalizations.14,15 Gabel and co-
workers reported a novel “BSH activation” method, which
enabled us to conduct S-alkylation of BSH under mild
conditions.16 In this paper, we report synthesis of closo-
dodecaborate containing boron lipids 4a-c and 5a-c and
their liposomal property (Scheme 1). Our design of the boron
Scheme 1
Figure 1. Structures of nido-carborane lipids.
lipids is based on biomimetic composition of phosphatidyl-
cholines to meet a sufficiently low toxic requirement.
Synthesis of the hydrophobic tail functions of 4 is shown
in Scheme 2. Reaction of the chiral alcohol 6 with 1.2 equiv
liposomal boron delivery in mice using 1 and distearoylphos-
phatidylcholine (DSPC).10 We recently developed the nido-
carborane lipid 2, which has a double-tailed moiety conju-
gated with nido-carborane as a hydrophilic function.11 We
investigated active targeting of the boronated liposomes to
solid tumors by functionalization of transferrin on the surface
of their liposomes and achieved a boron concentration of
22 µg of 10B/g of tumor by the injection of the liposomes at
7.2 mg of 10B/kg of body weight with longer survival rates
of tumor-bearing mice after BNCT.12 However, injection at
higher boron concentrations resulted in mortality. Hawthorne
and co-workers also recently reported synthesis of the nido-
carborane lipid 3 and its unilamellar liposomes. They pointed
out the high toxicity of the lipid 3 liposomes.13
Scheme 2
To overcome this problem, we focused on mercapto-
undecahydrododecaborate (B12H11SH2-, BSH) as an alterna-
tive hydrophilic function of boron lipids. BSH is a water-
soluble divalent anion cluster with significantly lowered
(9) (a) Cai, J.; Soloway, A. H.; Barth, R. F.; Adams, D. M.; Hariharan,
J. R.; Wyzlic, I. M.; Radcliffe, K. J. Med. Chem. 1997, 40, 3887. (b)
Fulcrand-El Kattan, G.; Lesnikowski, Z. J.; Yao, S.; Tanious, F.; Wilson,
W. D.; Schinazi, R. F. J. Am. Chem. Soc. 1994, 116, 7494. (c) Nakanishi,
A.; Guan, L.; Kane, R. R.; Kasamatsu, H.; Hawthorne, M. F. Proc. Natl.
Acad. Sci. U.S.A. 1999, 96, 238.
(10) (a) Feakes, D. A.; Shelly, K.; Hawthorne, M. F. Proc. Natl. Acad.
Sci. U.S.A. 1995, 92, 1367. (b) Watson-Clark, R. A.; Banquerigo, M. L.;
Shelly, K.; Hawthorn, M. F.; Brahn, E. Proc. Natl. Acad. Sci. U.S.A. 1998,
95, 2531.
(11) Nakamura, H.; Miyajima, Y.; Takei, T.; Kasaoka, T.; Maruyama,
K. Chem. Commun. 2004, 1910.
(12) Miyajima, Y.; Nakamura, H.; Kuwata, Y.; Lee, J.-D.; Masunaga,
S.; Ono, K.; Maruyama, K. Bioconjugate Chem. 2006, 17, 1314.
(13) Li, T.; Hamdi, J.; Hawthorne, M. F. Bioconjugate Chem. 2006, 17,
15.
of bromoacetyl bromide gave the ester 7, quantitatively, and
the deprotection of 7 was carried out using catalytic amounts
of p-TsOH in MeOH to give the corresponding diol 8 in
(14) (a) Holmberg, A.; Meurling, L. Bioconjugate Chem. 1993, 4, 570.
(b) Swenson, D. H.; Laster, B. H.; Metzger, R. L. J. Med. Chem. 1996, 39,
1540. (c) Sano, T. Bioconjugate Chem. 1999, 10, 905. (d) Hoffmann, S.;
Justus, E.; Ratajski, M.; Lork, E.; Gabel, D. J. Organomet. Chem. 2005,
690, 2757.
(15) Kultyshev, R. G.; Liu, J.; Liu, S.; Tjarks, W.; Soloway, A. H.; Shore,
S. G. J. Am. Chem. Soc. 2002, 124, 2614.
324
Org. Lett., Vol. 9, No. 2, 2007

38% yield. The ester formation of the diol 8 with various
carboxylic acids was promoted by dicyclohexylcarbodiimide
in the presence of catalytic amounts of N,N-dimethylamino-
pyridine in CH2Cl2 to afford the precursors 9a-c in 61-
75% yields.
Scheme 4
Synthesis of the hydrophobic tail functions of 5 is shown
in Scheme 3. We first examined the reaction of 6 with
Scheme 3
4b (1:1 - X:0.1:X ) 0-1, molar ratio), by the reverse-phase
evaporation (REV) method.17 The liposomes obtained were
subjected 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 PBS buffer. Liposome
size was measured with an electrophoretic light scattering
spectrophotometer. The size distribution of the liposomes
composed of 4b is shown in Figure 2. The sizes of maximum
chloroacetyl isocyanate followed by deprotection of the acetal
group; however, the chloroacetylcarbamate moiety decom-
posed under the acidic condition of p-TsOH in MeOH.
Therefore, 6 was protected with benzylbromide using NaH
and the resulting dioxolane 10 was converted into the diol
11 using aqueous AcOH in 83% yield. The ester formation
of 11 with various carboxylic acids was carried out in a
similar manner to give 12a-c, quantitatively. Deprotection
of the benzyl group of 12a-c by hydrogenation gave the
corresponding alcohols 13a-c (89->99% yields), which
reacted with chloroacetyl isocyanate in CH2Cl2 to give 14a-c
in 74-98% yields.
Introduction of BSH into the hydrophobic tail functions 9
and 12 was examined using the “activated BSH (15)”, which
was prepared according to Gabel's protocol, as shown in
Scheme 4. S-Alkylation of 15 with 9a-c proceeded in
acetonitrile at 70 °C for 12-24 h, giving the corresponding
S-dialkylated products 16a-c, which were immediately
treated with tetramethylammonium hydroxide (1 equiv) in
acetone to give 4a-c in 76-91% yields, as tetramethyl-
ammonium salts. In a similar manner, 5a-c were obtained
from 12a-c in 54-83% yields.
Figure 2. Size distributions of the boronated liposomes after
extrusion. 4b ratio: X ) 0.25 (red), 0.5 (green), 0.75 (blue), and 1
(black).
distribution of the liposomes against 4b contents were 103
(X ) 0.25), 105 (X ) 0.5), 102 (X ) 0.75), and 108 nm (X
) 1) with 0.121, 0.092, 0.106, and 0.089 as polydispersity
index (PDI) values, respectively. The liposomes composed
of 4a, 4c, and 5a-c also gave similar size distributions (the
data are not shown).
We next investigated the time-dependent stability of the
boronated lipsomes of 4b in fetal bovine serum (FBS).
Fluorescent probes, such as calcein, are self-quenching at
high concentrations, and the leakage of these fluorophores
into the external medium results in the relief of self-
quenching and an increase in the fluorescence. Therefore,
We examined formation and membrane stability of the
liposomes using the boron cluster lipid 4b. Liposomes were
prepared from cholesterol, dimyristoylphosphatidylcholine
(DMPC), polyethyleneglycol-conjugated distearoylphospha-
tidylethanolamine (PEG-DSPE), and the boron cluster lipid
(16) (a) Gabel, D.; Moller, D.; Harfst, S.; Rosler, J.; Ketz, H. Inorg.
Chem. 1993, 32, 2276. (b) Lechtenberg, B.; Gabel, D. J. Organomet. Chem.
2005, 690, 2780. (c) Azev, Y.; Lork, E.; Duelcks, T.; Gabel, D. Tetrahedron
Lett. 2004, 45, 3249.
(17) Szoka, F.; Papahadjopoulos, D. Proc. Natl. Acad. Sci. U.S.A. 1978,
75, 4194.
Org. Lett., Vol. 9, No. 2, 2007
325

stirring. The fluorescence of the FBS solutions was measured
at 0-24 h. The results of the liposomes of 4b with various
ratios (X ) 0.25, 0.5, and 0.75) are shown in Figure 3. No
increase in the fluorescence intensity of the FBS solutions
was observed within 24 h; therefore, the boronated liposomes
were stable in the FBS solution at 37 °C at least for 24 h.
However, the increase of the fluorescence intensity was
observed in the case of the liposome prepared from choles-
terol, PEG-DSPE, and 4b (X ) 1).
Furthermore, to investigate the accumulation ratios of the
boron cluster lipid 4b and phospholipids including DMPC
and PEG-DSPE on liposome formation, we determined boron
and phosphorus concentrations of the liposome solutions by
inductively coupled plasma atomic emission spectroscopy
(ICP-AES). The concentration ratios of 4b to the phospho-
lipids on the liposomes prepared from cholesterol, DMPC,
PEG-DSPE, and 4b (1:1 - X:0.1:X, molar ratio) were 0.84
(X ) 0.25) and 2.6 (X ) 0.50). Therefore, it was observed
that the boron lipid 4b was incorporated into the liposome
membranes at a higher concentration than phospholipids.
In conclusion, we succeeded in the synthesis of the double-
tailed boron cluster lipids 4 and 5, which have a B12H11S
moiety as a hydrophilic function, by S-alkylation of BSH
with bromoacetyl and chloroacetocarbamate derivatives of
diacylglycerols. The liposomes prepared from the boron
cluster lipid 4b, DMPC, PEG-DSPE, and cholesterol were
stable in FBS. We also investigated toxicity of the boron
liposomes and found that no mouse died after injection with
the boron liposomes at a dose of 15 mg of boron per kilogram
of weight for up to three weeks, although 50% of the mice
injected with the boron liposomes prepared from the lipid 2
died at a dose of 14 mg of boron per kilogram of weight
within 48 h (n ) 4). In vivo biodistribution and BNCT
studies of the boronated liposomes of 4 and 5 are in progress
in our laboratory.
Figure 3. Time-dependent fluorescence intensities of calcein-
encapsulated liposomes composed of 4b with various ratios (X: (a)
) 0.25, (b) ) 0.5, (c) ) 0.75) in FBS. Fluorescence intensity is
plotted on the vertical axis, and incubation time is plotted on the
horizontal axis. The black plots show the fluorescence intensity of
the FBS solution containing liposomes, and the white plots show
that of the solution after destruction of liposomes by the addition
of Triton X-100.
Acknowledgment. This work was supported by The New
Energy and Industrial Technology Development Organization
(NEDO) research project for developing a hospital-based
accelerator for boron neutron capture therapy with an
advanced drug delivery system. We thank Prof. D. Gabel
(University of Bremen) for helpful suggestions on the BSH
activation.
the release of aqueous contents of liposomes can be
monitored by an increase of fluorescent intensity.18 We
prepared the boronated liposomes at various concentrations
of the boron cluster lipid 4b using calcein (100 mM), and a
liposome solution (the volume ratio of FBS/liposome solution
) 9:1) was added to FBS and incubated at 37 °C with
Supporting Information Available: Detailed experi-
mental procedures and characterization data for compounds
4, 5, and 7-14. This material is available free of charge via
the Internet at http://pubs.acs.org.
(18) Du¨zgu¨nes¸, N.; Bagatolli, L. A.; Meers, P.; Oh, Y.-K.; Straubinger,
R. M. In Liposomes; Torchilin, V. P., Weissig, V., Eds.; Oxford: Oxford,
2002; pp 105-119.
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