Università degli Studi di Torino
Scuola di Dottorato in Scienza ed Alta Tecnologia
Indirizzo Scienze Chimiche
Ciclo XII
New Antitumoral Agents for the use in the Boron
Neutron Capture therapy (BNCT).
Relazione sull’attività svolta nel primo anno di Dottorato dal
dottorando:
Antonio Toppino
Attività svolta presso il Dipartimento: Chimica generale ed
organica applicata, Chimica I.F.M.
Relatore: dott. ssa Annamaria Deagostino
dott. Carlo Nervi
Direttore della Scuola di Dottorato SAT: prof. Elio Giamello
Coordinatore dell’Indirizzo: prof. Paolo Venturello
Anno Accademico 2006-2007
Scuola di Dottorato SAT- Indirizzo Scienze Chimiche
Introduction
Boron neutron capture therapy (BNCT) is a binary approach to cancer
treatment originally proposed by Locher in 1936. It is based on the 10B (n,
α)7Li reaction (figure 1), which occurs when boron-10, which has a large
capture cross section relative to the more abundant endogenous nuclei (1H,
12
C, 31P, 14N), is exposed to thermal neutrons. BNCT is referred to as a
binary therapy because the individual components (i.e. the boron atoms
and the neutrons) are innocent in themselves. In combination, however,
they have the potential to create a highly selective therapy because the
daughters of the boron neutron capture reaction, alpha particles and
lithium ions, traverse a distance which is only slightly less than the
diameter of a typical cell, thereby confining the citoxicity to the single
boron-containing cell.1
Figure 1 10B neutron capture reaction
In order to achieve successful therapy, BNCT agents must be able to
selectively deliver considerable quantities of boron to the tumor cells. It is
generally accepted that a successful therapy requires between 10 and 30
mg of 10B per g of tumor cells (109 10B atoms/cell). However, this amount is
reduced substantially if the boron is concentrated in or near the cell
nucleus. The appreciable amount of boron is required to minimize the
contribution of radiation dose derived from the capture of neutrons by
endogenous nuclei, 14N and 1H.2
BNCT agents must also clear the blood rapidly to avoid inducing necrosis in
the vasculature. The optimal tumor:blood ratio is around 5:1. Another
obvious, but not necessarily easily addressable requirement, is the
knowledge of the exact boron concentration in cells, so the neutron
irradiation can be precisely guided.
Dicarba-closo-dodecaboranes (carboranes) have been among the most
attractive boron clusters for the linkage to targeting moieties due to their
ready incorporation into organic molecules, high boron content, chemical
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Scuola di Dottorato SAT- Indirizzo Scienze Chimiche
and hydrolytic stability, hydrophobic character, and in most cases, their
negative charge.3
Carboranes have the general formula C2B10H12. They exist as ortho (1),
meta (2) and para (3) isomers, which differ in the relative positions of the
carbon atoms in the cluster. The structures of the three isomers and the
IUPAC numbering scheme for ortho-carborane are shown in Fig. 2
Figura 2 Ortho (1), meta (2) and para-Carborane (3).
Several functionalised carboranes have been synthesised bearing different
biological vectors e.g. nucleic acid precursors,4 amino acids, peptides,5
phospholipids,6 carbohydrates,7 lipoproteins,8 porphyrins,9 DNA alkylators,
DNA intercalators, DNA groove binders,10 polyamines,11 oligonucleotides,
monoclonal–bispecific antibodies12 and growth factors.13
10
B and 11B are both NMR active nuclei. Unfortunately their short relaxation
times cause signals to broaden and decay rapidly. This makes it nearly
impossible to use standard clinical magnetic resonance (MR) scanners and
pulse sequences to image the distribution of boron compounds in vivo.14
MRI techniques normally employ nuclei with better imaging characteristics,
such as 19F, or agents containing paramagnetic metals. Tatham et al.15
investigated the potential of MRI to measure the concentration of boron in
a complex containing both Gd and a carborane cluster. The Gd amount in a
sample, which can be calculated-based on changes in T1, is directly related
to the amount of boron, so long as the complex remains intact. The
archetype Gd–BNCT complex was [Gd(III)–DTPA–Carborane] (figure 3).16
Figure 3 [Gd(III)–DTPA–Carborane]
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Scuola di Dottorato SAT- Indirizzo Scienze Chimiche
In order to overcome the problems of incorporation and biological
evaluation of carboranes in tumor cells, we propose the synthesis of a
bifunctionalised carborane, which bears on one carbon atom a biological
vector and on the other carbon atom a MRI probe to evaluate the
biodistribution.17 With this goal in mind we have synthesised a carborane
linked to an amino and a carboxylic group protected with two orthogonal
protecting groups, which can be bonded to several biological vectors and
MRI contrast agents, creating a certain number of derivatives for MRI
guided BNCT.
Initially, we chose a palmitil chain as the lipophilic precursor delivery agent
that could interact with LDL (low-density lipoprotein), which is the real
biological vector. Kahl18 first proposed and studied targeting methods using
LDL as a boron carrier. Because the amount of new membrane synthesised
in rapidly growing cancer cells is considerable, they consume large
amounts of cholesterol. LDL is the major component of the cholesteroltransport pathway, LDL receptors are much more active on many types of
cancer cells than on the corresponding normal cells. Thus, LDL and other
molecules carried by LDL would be expected to accumulate preferentially in
tumor cells.19
As the MRI contrast medium we picked Gd(III)-DOTAMA-C6-NH2 shown in
figure 4, a derivative of DOTA (4,7,10 tetraazacyclododecane-1,4,7,10tetrayltetraacetic acid), one of the most efficient MRI contrast agent.
Figure 4 [Gd-DOTA]-.
Results and discussion
In order to obtain a bifunctionalised o-carborane with an amino and a
carboxylic group orthogonally protected, it was necessary to prepare a
suitable internal alkyne from the commercially available 3-butyn-1-ol,
which was protected with benzyl bromide.
Unfortunately all attemps to alkylate protected butynol 2 with various
electrophiles (alkyl halides and epoxides) and under different
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Scuola di Dottorato SAT- Indirizzo Scienze Chimiche
experimental conditions were unsuccessful. As shown in Scheme 1 only
paraformaldehyde, as reported from Quintana et al.,20 led to the desired
5-benziloxy-pentyn-1-ol 3. The product was confirmed by the
disappearance of the triplet centered at 2.08 ppm, relevant to the
acetilenic proton, in the 1H NMR spectrum, and the appearance of the
singlet at 4.23 ppm which corresponds to the HOCH2– group.
In the first synthetic way, we thought of synthesising the o-carborane
exploiting the alkyne 3, which bears one protected and one unprotected
alcoholic group.
a
OH
HO
b
OBz
c
OBz
2
1
3
HO
OBz
O
f,g
d
4
N
O
N3
OBz
OBz
5
6
e
H2N
h
OBz
= B−H
=C
7
Scheme 1 Synthesis of C-aminomethyl-C’-2-ethylbenzyl ether-o-carborane (7). Reaction
conditions and yields: a) BnBr, NaH, THF, rt (90%); b) BuLi, (CH2O)n, THF, –20 °C → rt
(65%); c) B10H14, (bmim)+Cl-, toluene, 120 °C (61%); d) oxalyl chloride, DMF, CH2Cl2, Kphtalimide, CH3CN, 65 °C (60%); e) NaBH4, iPrOH; HCl, CF3COOH, (0%); f) TEA, MsCl, ethyl
ether (70%);g) NaN3, DMF (50%); h) LiAlH4, THF, 0 °C (0%); PhP3, THF, H2O, RT (0%).
In order to insert the carboranic cage, alkyne 3 was allowed to react
with decaborane in a biphasic system, ionic liquid (bmim)+Cl- and
toluene, without the need of any catalyst, following the procedure
proposed by Sneddon et al.21. This gives, after chromatographic
purification, compound 4 in a 61% yield. For the sake of obtaining an
amino group, we planned to utilize the Vilsmeier reaction.22 This
procedure consists of the treatment of 4 with (chloromethylene)dimethylammonium chloride, generated from oxalyl chloride
and DMF and subsequently with potassium phthalimide at 65 °C,
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Scuola di Dottorato SAT- Indirizzo Scienze Chimiche
providing compound 5. The isolation of 5, after chromatographic
purification, was confirmed by the appearance of a 4 H atom multiplet
at 7.83 ppm in 1H NMR spectrum corresponding to the phtalimide
group.
Unfortunately it wasn’t possibile to obtain the amine 7 by the use of
NaBH4 followed by an hydrolysis in acidic conditions.23 Then we decided
to transform alcohol 4 into the corisponding azide 6, by its mesilate and
successively to reduce it to amine 7. Two different reduction methods
were tried: treatment with LiAlH424 and PPh3 in neutral and basic
conditions (Staudinger reaction)25 but both were unsuccessfull.
Furthermore azide derivative 6 was instable and impossibile to recover.
HO
a
MsO
OBz
OBz
3
b
8
N3
c
OBz
9
H2N
OBz
10
Scheme 2 Synthesis of 5-aminopent-3-in-1-ol (10). Reaction conditions and yields: a)
MsCl, TEA, Et2O, 0 °C (95%); b) NaN3, DMF, rt (92%);c) SnCl2, MeOH, rt (99%).
Afterwards we applied the same strategy to alkyne 3 instead of
carborane 4. We converted alcohol 3 into the corrisponding mesilate 826
by treatment with triethylamine and methanesulfonyl chloride at 0°C
(95%) and successively 8 was transformed into stable azide 9 using
NaN3 in DMF at room temperature (92%). Finally, treatment with SnCl2
at room temperature27 gave 5-benziloxy-pent-2-ynilamine 10 (99%).
The overall yield of the process was 86% (Scheme 2). Reaction
outcomes were confirmed by 1H NMR analysis: the singlet at 4.23 ppm,
relevant to the HOCH2– group, shifted to 4.53 ppm for mesilate 8, and
to 3.91 and 3.39 ppm for azide 9 and amine 10, respectively.
Amine 10 was protected as BOC derivative 11, using (BOC)2O and neat
sulfamic acid28 (99%) and then allowed to react with decaborane
(Scheme 3). The reaction was carried out using 1.5 equivalents of
alkyne, differently to the synthesis of carborane 4. Carborane 12 was
isolated in a 41% yield, after chromatographic purification. The
structure of compound 12 was confirmed on the basis of the 13C NMR
spectrum. In particular, the signals relevant to the sp carbons of alkyne
11 centered at 77.16 and 77.25 ppm were shifted to 78.45 and 79.68
ppm respectively in the cage of carborane derivative 12. All quaternary
C chemical shifts were confirmed by DEPT experiments.
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Scuola di Dottorato SAT- Indirizzo Scienze Chimiche
Boc
H2N
OBz
a
Boc
N
H
OBz
10
H
N
OBz
b
11
H2N
OBz
c
d
12
H
N
C15H31
O
13
OBz
14
H
N
C15H31
O
OH
e
= B−H
=C
15
Scheme 3 Synthesis of C-palmitilamidomethyl-C’-2-hydroxyethyl –o-carborane (15). Reaction
conditions and yields: a) (BOC)2O, NH2SO3H, rt (99%); b) B10H14, (bmim)+Cl-, toluene, 120 °C
(41%); c) CH2Cl2, CF3COOH, rt (85%); d) CDMT, N-methylmorfoline, palmitic acid, CH2Cl2, –5
°C → rt (40%); e) H2, Pd/C, CH3Cl/MeOH, rt (88%).
After the BOC deprotection, in a 1:1 mixture of CF3COOH–CH2Cl2 at
room temperature (85%), the resulting amino carborane 13 was
coupled with palmitic acid, as a lipophilic chain, according to the
procedure proposed by Kamiński,29 affording palmitil amide 14 (40%
yield, after chromatographic purification). In this coupling reaction the
fatty acid was activated in CH2Cl2 at 0°C, in nearly 4 h, with CDMT (2chloro-4,6-dimethoxy-1,3,5-triazine)
in
the
presence
of
Nmethylmorfoline and then allowed to react with amine 13. 13C NMR
analysis confirmed the proposed structure, in particular the signals
relevant to the quaternary carbon atoms of the carboranic cage shifted
to 78.77 and 78.98 respectively.
At this point it was possible to go onto the functionalisation of the other
arm of the o-carborane: the benzylic protecting group was removed by
Pd/C catalysed hydrogenation (88%), and alcohol 15 was readly
oxidised to the corrisponding carboxilic acid 16 by CrO3 in a acetonesulfuric acid solution30 at room temperature, in a 71% yield, as shown
in Scheme 4.
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H
N
C15H31
H
N
C15H31
O
O
a
OH
OH
O
16
15
H
N
C15H31
O
H
N
O
5N
H
O
b
17
H
N
O
O
c, d
18
N
N
N
t-Bu-OOC
H
N
C15H31
COO-t-Bu
N
COO-t-Bu
O
5N
H
-
OOC
COON
N
N
Gd3+
N
COO-
Scheme 4 Synthesis of C-palmitilamidomethyl–C’-Gd-DOTAMA-C6-o-carborane (18). Reaction
conditions and yields: a) CrO3, acetone, H2SO4 3M, rt (71%); b) CDMT, N-methylmorfoline,
DOTAMA-C6-NH2, CH2Cl2, 5 °C→rt (35%); c) CH2Cl2, CF3COOH, Et3SiH, rt (), d) Gd(III)Cl3, H2O
().
The structure of derivative 16 was confirmed by the 1H and 13C NMR
spectra: in particular, the 1H NMR analysis indicates the disappearance
of the signal at 3.84 ppm assigned to the HOCH2- group, while 13C
spectrum shows, on one side, a new signal centered at 170.00 ppm
relevant to the carboxilic group and, on the other side, the shift of the
signals of the quaternary carboranic carbons from 78.47 and 78.92 ppm
to 74.07 and 78.98 ppm. Carboxilic derivative 16 was subsequentely
coupled without purification, to the suitable DOTAMA(ter-Bu)3-C6-NH2
(DOTA with a six carbon atom spacer to distance the carborane from
the complex in order to facilitate cell internalisation) , to produce the
desired bifunctionalised o-carborane 17, using the previous coupling
method.29 The structure of the target compound was confirmed by an
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ESI mass spectrum that clearly shows the MH+ peak (1124.43).
Conclusions
In the first year of my Ph.D course C-(2-benzyloxy)-ethyl-C’-terbutoxyamidomethyl-o-carborane 12 was synthesised, a versatile
intermediate, which can be readily functionalised with different
biological vectors and MRI contrast agents to build a series of
substituted o-carboranes. A very lipophilic palmitil chain was chosen as
the first delivery agent, because of his affinity with LDL. On the other
side carborane was bonded to DOTAMA(ter-Bu)3-C6-NH2, which allows
the quantitative determination of boron in cells. In the next steps DOTA
proteting groups have to be removed and Gd–DOTA complex has to be
generated in order to use the final compound 18 in biological tests.
In the second year we intend to exploit compound 12 to functionalise
carborane with other biological vectors such as glutamine and
cholesterol and other MRI contrast agents.
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References
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Activities
Schools
9 XXXII Corso Estivo “A. CORBELLA”. Seminari di Sintesi Organica.
Gargnano (BS), Palazzo Feltrinelli, 18-22 Giugno 2007 (8 CFU).
Congresses
9 XXXI Convegno Nazionale della Divisione di Chimica Organica della
Società Chimica Italiana. Rende, 10-14 Settembre 2007 (2 CFU).
9 7° Sigma Aldrich Young Chemists Symposium: 7° S.A.Y.C.S.
Riccione, Palazzo del Turismo, 22-24 Ottobre 2007
Seminaries
9 “Organometallic Reagents in Organic Synthesis”. Torino, 17 Aprile
2007. (2 CFU).
Courses
9 “Electron Magnetic Resonance Techniques: Methology and
Applications in Chemistry and Material Science” (4 CFU), by: Elio
Giamello, Maria Cristina Paganini, Mario Chiesa (Dipartimento di
Chimica IFM).
Poster presentations
9 “Sintesi di carborani funzionalizzati per applicazioni BNCT”. Rende,
10-14 Settembre e Riccione, 22-24 Ottobre.
Oral communications
9 “Sintesi di un carborano bifunzionalizzato con acido Palmitico e
DOTA per applicazione BNCT-MRI.” Riccione, 22-24 Ottobre.
Publications
9
“Synthesis
of
Gd(III)-C-palmitilamidomethyl-C’-DOTAMA-C6-ocarboranes: new agent for MRI guided BNCT.” Silvio Aime,
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Scuola di Dottorato SAT- Indirizzo Scienze Chimiche
Alessandro Barge, Antonella Crivello, Annamaria Deagostino, Roberto
Gobetto, Carlo Nervi, Cristina Prandi, Antonio Toppino, and Paolo
Venturello. In preparation..
9
"Carborane Derivatives for BNCT Applications: Hunting the
Selectivity Toward Tumor Cells". Silvio Aime, Alessandro Barge,
Antonella Crivello, Annamaria Deagostino, Roberto Gobetto, Carlo Nervi,
Antonio Toppino, and Paolo Venturello. In preparation..
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