Synthesis of a Tyr3-octreotate conjugated closo-carborane [HC2B10H10]: a potential compound for boron neutron capture therapy

Article abstract of DOI:10.1016/j.tetlet.2003.10.048

A novel Tyr³-octreotate conjugated closo-carborane as a potential compound for boron neutron capture therapy was obtained via Fmoc solid phase peptide synthesis. The boron cluster [C2B10H11] was introduced through the reaction of 6,9-bi(acetonitrile)decaborane and 5-hexynoic acid yielding a new closo-carborane conjugated carboxylic acid which was coupled subsequently with solid phase conjugated Tyr³-octreotate. The final boron-containing peptide was purified by preparative reverse phase HPLC and structural identity was proved applying MALDI-TOF mass spectrometry.

Full text of DOI:10.1016/j.tetlet.2003.10.048

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 9143–9145
Synthesis of a Tyr3-octreotate conjugated closo-carborane
[HC2B10H10]: a potential compound for boron neutron
capture therapy
Esther Schirrmacher,a Ralf Schirrmacher,a,* Carmen Beck,a Walter Mier,b Norbert Trautmana
and Frank Ro¨scha
aInstitute of Nuclear Chemistry, University of Mainz, D-55177 Mainz, Germany
bUniversita¨tsklinikum Heidelberg, Department of Nuclear Medicine, D-69120 Heidelberg, Germany
Received 23 September 2003; revised 2 October 2003; accepted 7 October 2003
Abstract—A novel Tyr3-octreotate conjugated closo-carborane as a potential compound for boron neutron capture therapy was
obtained via Fmoc solid phase peptide synthesis. The boron cluster [C2B10H11] was introduced through the reaction of
6,9-bis(acetonitrile)decaborane and 5-hexynoic acid yielding a new closo-carborane conjugated carboxylic acid which was coupled
subsequently with solid phase conjugated Tyr3-octreotate. The final boron-containing peptide was purified by preparative reverse
phase HPLC and structural identity was proved applying MALDI-TOF mass spectrometry.
© 2003 Elsevier Ltd. All rights reserved.
Boron neutron capture therapy (BNCT) originates
from the idea of delivering a boron compound with
enriched 10B to tumour cells and subsequent exposure
of those cells to a thermal neutron beam inducing the
application of boron clusters in BNCT offers a new
approach for enhancing the boron density in the target
tissue. Some novel boron clusters containing
polyamines for example have been reported which dis-
played no distinct affinity to tumour cells, but may
function as building blocks for further syntheses.6,7
10B(n,a)7Li nuclear reaction.1 The energetic He and Li
products are responsible for injuring cancer cells. The
application of boron compounds for cancer treatment,
e.g. for high grade gliomas and anaplastic astrocytomas
has received attention due to the fact that these tumour
entities cannot be easily cured via surgery, chemother-
apy or conventional radiation therapy. Clinical treat-
ments of patients with brain tumours with BNCT in the
early 1960s failed due to a lack of boron compounds
with high selectivity for the target tissue. In addition,
neutron beams which could deposit the required ther-
mal neutron flux at depths greater than a few centime-
tres were not available.2 Today an increasing number of
boron compounds have been synthesised3 but among
these, only the p-dihydroxy-[10B]boryl-phenylalanine-
fructose complex (BPA-Fr) and [10B]BSH have been
applied in clinical trials.4,5 BPA-Fr has the disadvan-
tage of bearing only one boron atom, which is far from
being ideal considering that the concentration of 10B
should be exceptionally high in the target tissue. The
4
7
There is also a demand for novel targeting concepts for
delivering 10B-enriched compounds to tumours.
The use of peptides as targeting vectors is a promising
approach to assess tumours expressing specific peptide-
Keywords: closo-carborane; Tyr3-octreotate; boron neutron capture
therapy (BNCT); somatostatin receptor.
* Corresponding author. Tel.: +49-6131-3925371; fax: +49-6131-
3925253; e-mail: schirrmacher@mail.kernchemie.uni-mainz.de
Figure 1. The Tyr3-octreotate conjugated closo-carborane
[HC2B10H10] synthesised.
0040-4039/$ - see front matter © 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2003.10.048

9144
E. Schirrmacher et al. / Tetrahedron Letters 44 (2003) 9143–9145
receptors on the cell surface.8–10 Tyr3-octreotate [H-
Phe-cyclo[Cys-Tyr- -Trp-Lys-Thr-Cys]-Thr-OH] is
D-
a
D
somatostatin analogue with high affinity for the
somatostatin receptor subtype-2. This subtype is over-
expressed on the cell surface of several tumours of the
neuroendocrine system, making somatostatin analogues
eligible compounds for tumour targeting.11–13
A variety of radiolabelled octreotide and octreotate
derivatives are involved in clinical routine and clinical
research, both for diagnosis and radiotherapy.14 These
derivatives demonstrate selective accumulation in the
corresponding tumours. Recently, the pattern of cellu-
lar uptake of the radiolabelled compounds has been
investigated in detail. In particular, octreotate deriva-
tives have been proven to internalise effectively into
tumour cells.15,16 As a prerequisite for efficient BNCT is
an internalisation of the boron containing compound
into the target cell, Tyr3-octreotate-boron clusters seem
to be promising compounds for achieving a high con-
centration of 10B in the tumour cell.
Furthermore, Tyr3-octreotate can be labelled with
radionuclides such as 123I enabling the evaluation of the
biodistribution in vivo. Taking these considerations
into account, we synthesised compound 5 (Fig. 1) as a
candidate for the application of BNCT.
The synthesis of the boron cluster 5,6-dicarba-closo-
dodecaboranyl hexynoic acid (4) was achieved by react-
ing 6,9-bis(acetonitrile) decaborane (6)17 (Fig. 2) and
5-hexynoic acid methyl ester (2), which was synthesised
via esterification of 5-hexynoic acid (1) (Fluka, Ger-
many) with methanol under acidic conditions. The
reaction conditions for coupling followed Valliant et al.
for the synthesis of a closo-carborane analogue of
tamoxifen.18 Two molecules of acetonitrile were substi-
tuted by the alkynyl carbon atoms resulting in the
formation of the closo-carborane. The resulting boron
labelled ester 3 was converted into the free acid 4 by
treatment with 6N HCl and finally purified via column
chromatography. The Tyr3-octreotate was synthesised
applying standard Fmoc solid phase peptide synthe-
sis via a batchwise procedure, i.e. 4 molar equiv. of
the amino acid, 4 equiv. of 2-(1H-benzotriazole-1-
yl)-1,1,3,3-tetramethyluronium (HBTU), 8 equiv. of
diisopropylethylamine (DIPEA) in DMF at 25°C.
Figure
2. Synthesis
of
Tyr3-octreotate
conjugated
[HC2B10H10] closo-carborane. Reagents and conditions: (a)
methanol, H2SO4, reflux, 1 day, 80%; (b) B10H12(CH3CN)2,
toluene, argon, reflux, 95%; (c) 6N HCl, 74%; (d) 4 equiv. 4,
4 equiv. HBTU, 8 equiv. DIPEA, 17%.
ysis. Compound 5 was identified by MALDI-TOF mass
spectrometry (Table 1).21
Cyclisation of the linear octapeptide
D-Phe-Cys(Acm)-
In conclusion, the synthesis of a novel Tyr3-octreotate
Tyr(tBu)- -Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-
D
conjugated [HC2B10H10] closo-carborane
5
was
Thr(p-benzyloxyoxyphenylalcohol)-resin by formation
of the disulphide bridge was achieved with 4 equiv.
of thallium(III) trifluoroacetate in DMF at 25°C.19
The coupling of 4 to the peptide was performed
analogously yielding the final fully protected com-
pound 5 on the solid support, which was deprotected
and removed from the resin in one step applying
TFA:water:triisopropylsilane (95:2.5:2.5). The crude
boron cluster conjugated peptide 5 was precipitated by
the addition of cold diethyl ether and purified via
preparative gradient HPLC.20 Structural identities of
achieved in an overall yield of 17%.22 This compound
might serve as a new substance for BNCT due to (a)
its possible high affinity for somatostatin receptor
expressing tumours and (b) possible internalisation
into tumour tissue as well as (c) the number of boron
atoms, ten per targeting molecule. To assess the
biodistribution data after in vivo application in
humans, the labelling of this compound with radionu-
clides such as 123I is planned. Additionally, binding
experiments to prove the affinity of 5 to the human
somatostatin receptor subtype 2 are under investiga-
tion.
compounds 2, 3 and 4 were proven with H, 13C, and
1
11B NMR, FD mass spectrometry and elemental anal-

E. Schirrmacher et al. / Tetrahedron Letters 44 (2003) 9143–9145
9145
Table 1. Analytical data for compounds 2, 3 and 4 (1H, 13C, 11B NMR; FD MS, elemental analysis, melting points)
Compound
1H NMR
(400 MHz)
13C NMR
(400 MHz)
FD MS
11B NMRd
(400 MHz)
Elemental analysis
(%)
Melting points
(°C)c
b
2
l (CDCl3)=3.7 (s,
3H), 2.4 (t, 2H,
J=7.4 Hz), 2.2 (m,
2H), 1.95 (t, 1H,
J=2.7 Hz), 1.8 (m,
2H)
l (CDCl3)=3.7 (s,
3H), 3.6 (s, 1H),
3.2–2.4 (br s, 5H),
2.3 (t, 2H, J=7 Hz),
2.25 (t, 2H, J=8.5
Hz), 2.2–1.85 (br s,
3H), 1.8 (m, 2H),
1.75–1.4 (br s, 2H)
l (DMSO-d6)=5.2
(s, 1H), 2.3 (t, 2H,
J=8.5 Hz), 2.2 (t,
2H, J=7 Hz), 1.6
(m, 2H)
l
126.6 [M]+
–
Calcd: C, 66.65; H,
7.99. Found: C,
66.38; H, 7.82
–
(DMSO-d6)=172.9,
83.7, 71.8, 51.4,
32.2, 23.5, 17.2
3
l (CDCl3)=169.3,
74.4, 60.9, 51.6,
36.9, 32.5, 24.1
244.5 [M]+
l (CDCl3)=−2.3,
−5.7, −9.3, −11.5,
−12.2, −13.1
Calcd: C, 34.41; H,
8.25. Found: C,
34.63; H, 8.41
62
4a
l
230.5 [M]+
l (DMSO-d6)=−3.3, Calcd: C, 31.29; H,
−6.3, −9.9, −11.8,
−13.1
142
(DMSO-d6)=179.0,
76.4, 63.3, 36.1,
32.7, 24.5
7.88. Found: 31.21;
H, 7.97
a The 10 protons from the closo-carborane cluster were observed as a broad peak from 1.5 to 3.0 ppm.
b Compound 2 was a colourless liquid.
c Melting points are uncorrected.
d 11B-peaks were broad singlets and only the centre of the peak is displayed.
14. De Herder, W. W.; Hofland, L. J.; van der Lely, A. J.;
Lamberst, S. W. J. Endocrine-Related Cancer 2003, avail-
able online at http://www.endocrinology.org
15. De Jong, M.; Breeman, W. A.; Bakker, W. H.; Kooij, P.
P.; Bernard, B. F.; Hofland, L. J.; Visser, T. J.; Srini-
vasan, A.; Schmidt, M. A.; Erion, J. L.; Bugaj, J. E.;
Macke, H. R.; Krenning, E. P. Cancer Res. 1998, 58,
437–441.
16. Hofland, L. J.; Lamberts, S. W. J. Endocr. Rev. 2003, 24,
28–47.
17. Schaefer, R. J. Am. Chem. Soc. 1957, 79, 1006–1007.
18. Valliant, J. F.; Schaffer, P.; Stephenson, K. A.; Britten,
F. J. Org. Chem. 2002, 67, 383–387.
19. Graham, K. A. N.; Wang, Q.; Eisenhut, M.; Haberkorn,
U.; Mier, W. Tetrahedron Lett. 2002, 43, 5021–5024.
20. Column: LiChrosorb® RPselectB 250×10 (Merck Darm-
stadt, Germany), gradient eluent: 100% H2O+0.1% TFA
after 30 min 100% acetonitrile+0.1% TFA; retention time:
Rt=24 min. The octreotate derivative 5 was a white solid
after lyophilisation and the overall yield of the purified
peptide was 17%.
21. Molecular weight determinations were carried out by
MALDI-TOF mass spectrometry using a Micromass Tof-
specE. The main observed masses were m/z=1260.9 for
[10B211B8], m/z=1259.9 for [10B311B7] and m/z=1261.9
for [10B111B9]; calculated masses (with relative intensities):
1255.71 (0.1%); 1256.71 (0.5%); 1257.71 (2.6%); 1258.71
(8.7%); 1259.71 (20%); 1260.71 (30.2%); 1261.71 (27%);
1262.71 (10.9%); with the abundance of 19.9% 10B and
80.1% 11B
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