Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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B&P File No. 3244-70
TITLE: METAL-CARBORANE COMPLEXES FOR RADIOIMAGING
AND RADIOTHERAPY AND METHODS FOR THEIR PREPARATION
FIELD OF THE INVENTION
The present invention relates to new radiopharmaceuticals, in
particular carborane complexes of technetium and rhenium, and new methods
for their preparation.
BACKGROUND OF THE INVENTION
The synthesis of low oxidation state technetium (Tc) and rhenium (Re)
radiopharmaceuticals has become feasible recently because of the ease with
which fac-[M(CO)3X3]" (M = Tc, Re; X = CI or Br, n= -2; X= H20, n = +1 ) can
be prepared at the tracer level,' using 99"'TC ('y, t»2 = 6.02 h), '$6Re (~-,
t1,2 =
91 h) and '88Re (~~, t~,2 = 17 h), and on a macroscopic scale2 using 99Tc (a~,
t~,Z = 2.12 x 105 yr) and ~85,187Re, Consequently, several publications
describing the preparation of technetium(I) and rhenium(I)
radiopharmaceuticals have appeared in the literature.3 These complexes are
typically composed of bidentate4 or tridentate chelates5 or organometallic
ligands.s
There has been particular interest lately in preparing substituted ~5-
cyclopentadienyltricarbonyl rhenium and technetium complexes because of
their unique physical and chemical properties compared with Tc(V) and Re(V)
coordination complexes, which, at present, are more commonly used to
develop new radiopharmaceuticals. The main obstacle to using
cyclopentadiene (Cp) as the core of bifunctional radiopharmaceutical ligands
has been the lack of a mild and direct method for the synthesis of the metal
complexes, which is readily adaptable for use at the tracer level. Top et al.'
reported an elegant method for preparing rhenium(I) complexes of substituted
cyclopentadienes through an exchange reaction with
cyciopentadienyltricarbonyl manganese derivatives while Katzenellenbogen's8
group has reported a one pot procedure for preparing
cyclopentadienyltricarbonylrhenium through the in situ generation of Cp-
trialkylstannane derivatives. The involvedness of the procedures and, in the
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latter case, the poor yields of synthesis in concert with the required use of
potentially toxic reagents, at present, limit the widespread use of Cp as a
bifunctional radionuclide ligand.
The carborane, 7,8-dicarba-nido-undecaborate, when deprotonated,
has an open pentagonal face that can form metallocene-type sandwich complexes
with analogy to the formally isolobal Cp ion.9 As a result, rl5-carborane
complexes with a variety of metals have been reported, two of the earliest
being [Re(CO)3(rl5-7,8-C2BgH~~)]~ (1, Figure 1) and [Mn(CO)3(~5-7,8-C2BgH~~)]-
,10
The original method for the preparation of [Re(CO)3(rl5-7,8-C2BgH~~)]~
involved
reacting [nido-7,8-(C2B9H»)]2-, the dicarbollide dianion, with Re(CO)5Br.
Ellis et
al. reported an improved synthetic procedure starting with [ReBr(CO)3(THF)2]
prepared in situ from ReBr(CO)5 under anhydrous conditions." The
corresponding reaction with technetium was never reported, which may have been
a consequence of radioactivity issues and/or the fact that the synthesis of
Tc(CO)5Br is not uncomplicated. Convenient methods for preparing M(CO)5Br (M
= 9~"'Tc and '~~'~Re) in aqueous solutions at the tracer level suitable for
routine
radiolabeling experiments do not, at present, exist.
There is a need for new methodologies for the synthesis of
radiopharmaceuticals, in particular radiopharmaceuticals of r~5-carborane
complexes of technetium and rhenium, that are suitable for routine
radiolabeling experiments (i.e. can be performed in aqueous solutions at
tracer levels).
SUMMARY OF THE INVENTION
~5-Technetium and rhenium carborane ~-complexes, including
bifunctional derivatives, were prepared in high yield from the reaction of fac-
[M(CO)3Br3]2- and fac-[M(CO)s(OHz)3]'+ (M- Re, Tc) with a dicarbollide
dianion ([nido-(CzBgH»)]2'), and functional derivatives thereof. The products,
[M(CO)3(rl5-C2BgH10)]-, and corresponding functional derivatives, which
include
the first examples of Tc-carborane complexes, where characterized by multi-
NMR spectroscopy, X-ray crystallography and mass spectrometry.
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The present invention therefore provides a method for preparing metal-
carborane complexes comprising reacting a salt of the formula:
[M(C~)3(Xm)3]~1 + 3m)~
wherein M is selected from a radioisotope of rhenium, technetium and any
other radioisotope that binds in a similar fashion, X is any suitable iigand
and
m is the formal charge for ligand X, with a nido-dicarbollide dianion of the
formula [nido-(C2B9H~~)]2-, and functional derivatives thereof. The functional
derivatives preferably include nido-dicarbollide dianions in which one or more
linker group has been incorporated within the structure. The linker serves to
connect the metal-carborane complex with a biological targeting ligand.
Alternatively, the biological targeting ligand may have the metal carborane
complex incorporated within its structure or directly attached thereto.
The present invention also relates to novel metal-carborane
complexes. Therefore there is provided a metal carborane complex
comprising the formula [M(CO)3(~5-7,8-C2BgH~p)]~, wherein M is a radioisotope
of technetium; a metal-carborane complex comprising the formula [M(CO)3(~5-
7,9-C2B9H~o)]-, wherein M is selected from a radioisotope of technetium and
rhenium, and functional derivatives thereof. The invention also includes a
metal-carborane complex having the formula [M(CO)3(rl5-7-R-7,8-CZB9H,o)]-,
(M(CO)3(rl5-8-R'-7,8-C2BsH~o)J or (M(CO)3(rl5-7-R,8-R'-7,8-C2BsH~o)] , wherein
M
is selected from a radioisotope of technetium and rhenium and R and R' are
independently selected from (CHZ)~C02H, NH2 and NH-NH2, wherein n is 0-50;
and a metal-carborane complex having the formula [M(CO)3(rl5-7-R-7,9-
CzBsH~o)1, [M(CO)s(~15-9-R~-~,9-CzBsH~o)] or [M(CO)s(~15-7-R,9-R'-7,9-
CZBsH~o)]
wherein M is selected from a radioisotope of technetium and rhenium and R
and R' are independently selected from (CH2)~C02H, NH2 or NH-NH2, wherein
n is 0-50.
The invention also includes the use of the metal-carborane complexes
prepared using the method of the invention to prepare radiopharmaceuticals.
The invention also includes radiopharmaceutical compositions comprising a
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metal carborane complex prepared using the method of the invention and a
pharmaceutically acceptable carrier.
Other features and advantages of the present invention will become
apparent from the following detailed description. It should be understood,
however, that the detailed description and the specific examples while
indicating preferred embodiments of the invention are given by way of
illustration only, since various changes and modifications within the spirit
and
scope of the invention will become apparent to those skilled in the art from
this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention will now be described in relation to the drawings in
which:
Figure 1 is a schematic showing the conversion of an ortho-carborane to the
corresponding nido-dicarbollide dianion, followed by reaction with a
[M(CO)3X3]2~ salt to provide Tc and Re carborane complexes;
Figure 2 is a schematic showing the preparation of Tc and Re carborane
complexes in which a linker group has been incorporated into the carborane
moiety; and
Figure 3 is a schematic showing the preparation of a bifunctional carborane.
DETAILED DESCRIPTION OF THE INVENTION
Carboranes, which can be readily derivatized with a range of functional
groups, have been used successfully as carriers of a variety of different
radionuclides including some radiometals.'2 This is typified by the report by
Hawthorne et al. describing the synthesis of a pyrazole-bridged dicarbollide
or
"Venus Flytrap" ligand,'3 which was used to prepare bifunctional complexes of
5'Co (y, t~,2 = 271d). The synthesis of technetium-carborane complexes have
thus far not been reported despite the fact that 99"'Tc is the most widely
used
radionuclide in diagnostic medicine.'4 The present inventors have now
prepared rl5-technetium and rhenium carborane ~-complexes, including
bifunctional derivatives, in high yield from the reaction of fac-[M(CO)3Br3]2-
and
fac-[Re(CO)3(OH2)3]2+ (M = 9gTc, Re) with a dicarbollide dianion ([nido-
(C2B9H~~)]z-, and functional derivatives thereof.
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Accordingly, the present invention provides a method for preparing
metal-carborane complexes comprising reacting a salt of the formula
[M(CO)3(X"')3]~' + smy wherein M is selected from a radioisotope of rhenium,
technetium and any other radioisotope that binds in a similar fashion, X is
any
suitable ligand and m is the formal charge for ligand X, with a nido-
dicarbollide dianion of the formula [nido-(C2B9H~~)]2-, and functional
derivatives thereof.
In embodiments of the present invention, the salts of the formula
[M(CO)3(X"')3]~' + 3m) include those where M is selected from a radioisotope
of
rhenium, technetium and any other radioisotope that binds in a similar
fashion. A person skilled in the art would be able to determine which
radioisotopes bind in a similar fashion to Tc and Re. Examples include
radioisotopes of Rh, Cr, Mo, Mn, Os, Ir and Ru. The ligand "Xm" may be any
such suitable ligand, including, for example, CI- (m = -1 ), Br (m = -1 ), PR3
(m
= 0), RCN (m = 0), NOxy (x = 1, 2; y = 1, -1 ) and Hz0 (m = 0). A person
skilled
in the art would know which ligands are suitable for use in the salts of the
formula [M(CO)3(X"')3]~~ + 3m), based on those known in the art. A suitable
ligand will be compatible with the reaction conditions used in the method of
the invention. It will be appreciated that one or more of the "CO" ligands
many be substituted with any ligand that is isoelectronic and isolobal
therewith. Examples of such ligands include NO+, PR3, and RCN. The
present invention extends to cover the use of salts of the formula
[M(CO)3(X"')3]~~ + 3m) in which one or more of the CO ligands has been
substituted with a ligand that is isoelectronic and isolobal therewith. An
example of such a complex wherein one CO has been replaced with NO+ is
found in Rattat et al. Cancer Biotherapy & Radiopharmaceuticals, 2001, 16(4),
339-343. A person skilled in the art would also understand that the salts of
the formula [M(CO)3(X"')3]~' + 3"'~ may require one or more counterions to
balance the charge on the complex. Any such counterion compatible with the
reaction conditions may be used in the method of the invention.
The salts of the formula [M(CO)3(X'")3]~~ + sm> may be prepared using
methods known in the art. For example, salts of the formula [M(CO)3Br3]2' (M
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= Re, 99Tc) may be prepared as described in Alberto et al. 1995.'5 In general
these compounds may be prepared, for example, using low temperature
reductions of [NBu4][M04] in the presence of CO. The reducing agent may be
any suitable reagent, such as a boron hydride, including BH3-THF and NaBH4.
The nido-dicarbolide dianion may be derived from the possible isomers
of carborane, for example, ortho- and meta-carborane, which provide the
corresponding 7,8- and 7,9-nido-dicarbollide dianions respectively. The nido
dicarbollide dianions may also be used in optically pure form or as a racemic
mixture. The invention includes the use of all isomers and mixtures thereof in
any proportion
The nido-dicarbollide dianions can be prepared using procedures
known in the art. For example, from the corresponding ortho- and meta-
carboranes by a two step process involving deboronation, to provide the
dicarba-nido-undecaborate, [nido-(C2B9H~2)]-, followed by deprotonation. The
deboronation reaction may be effected using a base under a variety of
conditions.'6~" For example, ortho-carborane or meta-carborane may be
heated to reflux with potassium hydroxide in an alcoholic solvent, such as
ethanol. Other bases that can be used include secondary amines, such as
pyrolidine, and fluoride. The 7,8-dicarba-nido-undecaborate or 7,9- dicarba-
nido-undecaborate product may be isolated as a salt, for example an
ammonium salt such as trimethylammonium, or a phosphonium salt such as
methyl triphenylphosphonium, using standard procedures. Deprotonation of
the dicarba-nido-undecaborates, to yield [nido-(CZB9H")]z-, may be
accomplished using a strong base, such as TIOEt. The conversion of
orthocarborane 2 to 7,8-dicarba-nido-undecaborate, trimethylammonium salt,
3, followed by the reaction of 3 with TIOEt to provide the nido-dicarbollide
dianion 4, is depicted in Figure 1 as a representative example.
In order to be useful as a radiopharmaceutical ligand, a means to
conjugate the carborane-M(CO)3 unit to targeting biomolecules must be
available. To this end one or more linker moieties may be incorporated into
the nido-dicarbollide dianions. Accordingly, the term "functional derivatives"
includes carboranes in which a linker group has been attached to one or more
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of the carbon and/or boron atoms. The one or more linkers may be the same
or different. In embodiments of the present invention, the one or more linker
groups are attached to the carbon atoms in the carborane. In further
embodiments, one linker group is attached to one of the carbon atoms in the
carborane. The term "functional derivative" further includes one or more
linker
groups with a biological targeting molecule attached thereto.
When the metal-carborane complex includes one or more linker groups
attached to a biological targeting ligand, the compounds prepared using the
method of the invention may be represented as:
M/Carborane-(R)p-Lig,
wherein M is selected from radioisotopes of Tc, Re and any other radioisotope
that binds in a similar fashion (for example'°5Rh), R is a linker
group, p is 0-1
and Lig is a ligand having specificity for a biological target. As used
herein,
the term "linker group" means any functional grouping that allows the metal-
carborane complex to be conjugated to a biological target ligand. Generally,
the linker group will have a reactive functional group at the end opposed to
the metal-carborane complex, to allow reaction with (and therefore
conjugation to) a reactive functional grouping on the biological target
ligand.
The one or more linker groups may be the same or different. The specific
linker groups used herein comprise a carboxylic acid which is capable of
reacting with, for example, free amino, hydroxy or thiol groups on a
biological
targeting ligand and a hydrazino group, which is capable of reacting with, for
example, a carboxylic acid or other electrophilic group on a biological
targeting ligand. Therefore, in embodiments of the present invention, the
linker group is selected from (CH2)~C02H, where n is 0-50, NH2 and NH-NH2.
In embodiments of the invention, n is 0-10. In further embodiments, n is 0-5.
Examples of biological targeting ligands include, but are not limited to,
small molecules having specificity for a specific receptor, immunoproteins,
oligopeptides, sugars, cocacin analogues and polypeptides such as epidermal
growth factor. The applications of radiolabelled boron clusters to the
diagnosis and treatment of cancer has been recently reviewed.'3
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The term "functional derivative" further includes metal carborane
complexes that have been incorporated within the structure of a biological
targeting ligand. When the metal-carborane complex is incorporated within
the structure of a biological targeting ligand, the ligand is preferably a
compound having a functional group that is structurally and electronically
similar to the carborane moiety. Examples of such functional groups include
phenyl and adamantyl groups. An example of such a ligand is the
antiestrogen, tamoxifen. The preparation of carborane analogs of tamoxifen
is described in inventor Valliant's publication Valliant et al. J. Org. Chem.
2002, 67, 383-387.
Functionalization of the carborane may be performed before or after
the formation of the metal complex using methods known in the art (see for
example, references 12 and 13). For example, ortho-carboranes are readily
synthesized from the reaction of an appropriately substituted acetylene with
various nitrite and sulfide adducts of decaborane (B~pH~4).'$ The linker group
may be incorporated into the carborane by judicious choice of the starting
acetylene compound. Hydrophilic groups on the acetylenic compounds
should be protected in order that the synthetic sequence will produce the
desired ortho-carborane. Also, the linker group may be modified using
standard procedures at any stage during the preparation of the metal-
carborane complex, including modification of the complex itself. As a
representative example, the nido-acid 10 (linker = (CHz)ZCOzH) was prepared
(Figure 2) and its ability to complex rhenium and technetium evaluated. The
acid 9, prepared by hydrogenation of the benzyl ester 8, was converted to
nido-anion 10, as a mixture of enantiomers, using the aforementioned
KOH/EtOH procedure, and the product isolated as the methyl
triphenylphosphonium salt (Figure 2). Alternatively, the carbon and boron
centers of the carboranes may be directly functionalized. For example, one or
both of the carbon centers may be deprotonated using a strong base such as
an alkyl lithium, for example butyllithium, and the resulting anion reacted
with
any electrophilic reagent. An example of this is shown in Figure 3 where the
ortho-carborane 2 was difunctionalized by first reacting with one equivalent
of
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butyllithium followed by treatment with di-tert-butyl azodicarboxylate.
Treatment with a second equivalent of butyl lithium followed by reaction with
C02, provided, after acidification, the hydrazino carborane carboxylic acid
13.
The same reaction sequence was successfully performed on the
corresponding meta-carborane 14. In general, the B-H vertexes of the
carboranes may also be functionalised by electrophilic substitution reactions
reminiscent of aromatic hydrocarbon reactions.
The preparation of the metal-carborane complexes may be effected by
reacting a nido-dicarboilide dianion with a salt of [M(CO)3(Xm)3]~' + 3'"~ in
either
aqueous or polar organic solvents, such as tetrahydrofuran (THF). The
reaction mixture may be warmed and allowed to proceed for a time period of
about 1 hour to about 48 hours. The extent of the reaction can be monitored
by thin layer chromatography (TLC), therefore a person skilled in the art
would
be able to determine when the reaction was complete and adjust the reaction
time and temperature accordingly. In an embodiment of the invention, the
nido-dicarbollide dianion is generated from the corresponding dicarba-nido-
undecaborate by treatment with a strong base such as TIOEt or potassium
hydroxide (KOH) and a solution of a salt of the formula [M(CO)3(Xm)3]~' + 3m)
IS
mixed with the nido-dicarbollide dianion directly (i.e. without isolation of
the
nido-dicarbollide dianion). In further embodiments, the nido-dicarbollide
dianion is present in excess amounts. It is also possible to generate the nido-
dicarbollide dianion directly from the corresponding ortho-carborane in the
presence of a metal complex using a known in situ deboronation reaction (see
Hawthorne, M.F. J. Organomet. Chem. 1975, 100, 97-110). This reaction
may be used to generate the nido-dicarbollide dianion directly from the ortho
carborane in the presence of the salt of the formula [M(CO)3(X"')3]~' + 3m),
As a representative example, the addition of the nido-dicarbollide
dianion 4 to a THF solution of [M(CO)3Br3]2- is shown in Figure 1. After
gentle
reflux overnight the product was isolated by preparative TLC. In the example
reported herein, the yield of the technetium-carborane complex 5 was
approximately 80% while the Re complex 1 was consistently isolated in nearly
quantitative yield. Negative ion electrospray mass spectrometry indicated the
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presence of the two target compounds each containing the expected isotopic
distributions. The v (CO) absorptions for the Re-carborane complex were
2009 cm-', 1896 cm-' and 1865 cm'', which was identical to that previously
reported for 1.'9 Compound 5 exhibited v (CO) absorbances at 2016 cm-',
1918 cm-', and 1908 cm-' respectively. The B-H stretching frequencies were
broad (va~erage (1) = 2556 cm-', vaverage (5) = 2523 cm-') and within the
expected range for related metallocarboranes including [Re(CO)3(rl5-7-
CB~°H»)]2- reported by Blandford et a1.2° The "B NMR of 1
showed peaks
ranging from -24.16 to -7.99 ppm and were identical to the shifts reported
previously. The "B NMR for 5 was, as expected, similar to that of 1.
The method of the invention was also shown to work with the nido-
dicarbollide dianion having a linker group incorporated within its structure.
As
shown in Figure 2, a slight excess of compound 10 was treated with two
equivalents of TIOEt followed by the addition of the limiting reagent,
[Re(CO)3Br3]2-. The product, 11, isolated by preparative TLC in 99% yield.
The Tc analogue 12 was prepared analogously and isolated in 93% yield.
Compounds 11 and 12 were characterized by MS, ' H, '3C and "B
NMR, IR, HPLC and in the case of compound 11 by elemental analysis. All
results were in agreement with the proposed structures. The IR of 11 and 12
exhibited B-H stretches at similar frequencies (2524 cm-' and 2529 cm-') and
the'H and'3C NMR spectra were, as expected, similar. The "B NMR for 11
and 12 showed substantial overlap and, due to the loss of symmetry,
increased complexity compared to that for compounds 1 and 5. The
preparation of compounds 11 and 12 was also effected in aqueous solution
(Example 10), confirming the applicability of the method of the invention to
the
preparation of radiopharmaceuticals at tracer levels. Further confirmation of
this was obtained by preparing complexes 5 and 12 at tracer levels (see
Example 16).
The stability of the Re and Tc carborane complexes were evaluated
using a cysteine challenge experiment.2' Compound 1, for example, was
incubated with a thousand fold excess of cysteine in a 1:1 (v/v) phosphate
buffer (pH = 7.4)-ethanol mixture at 37°C over 24 hours. HPLC, FTIR,
and
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electrospray mass spectrometry experiments indicated that the complexes
showed no signs of decomposition over the entire duration of the experiment.
Compounds of the Invention
The present invention also relates to novel metal-carborane
complexes. Therefore there is provided a metal carborane complex having
the formula [M(CO)3(rl5-7,8-C2B9H~o)]~ wherein M is a radioisotope of
technetium; and a metal-carborane complex having the formulae and
[M(CO)3(~5-7,9-C2B9H~o)]~, wherein M is selected from a radioisotope of
technetium and rhenium, and functional derivatives thereof.
In specific embodiments of the present invention, the functional
derivative has the formula [M(CO)s(rl5-7-R-7,8-CzB9H~o)]-, [M(CO)3(rl5-8-R'-
7,8-
C2B9Hio)]- or [M(CO)3(rl5-7-R,8-R'-7,8-C2B9H~o)]-, wherein M is selected from
a
radioisotope of technetium and rhenium and R and R' are independently
selected from (CHZ)nC02H, NH2 and NH-NH2, n is 0-50. In more specific
embodiments of the present invention, the functional derivative has the
formula [M(CO)3(~5-7-R-7,8-CZBgH~o)]~, wherein R is selected from (CH2)~C02H,
NH2 and NH-NH2, and n is 0-10. In even more specific embodiments, the
functional derivative has the formula [M(CO)s(rl5-7-R-7,8-C2BgH~p)]-, wherein
R
is (CHZ)nC02H and n is 0-10. In further embodiments, the present invention
includes metal-carborane complexes having the formula [M(CO)s(rl5-7-R-7,9-
CZBsH~o)] ~ [M(CO)s(~15-9-R~-~,9-C2BsH~o)l or [M(CO)3(~15-~-R,9-R'-7,9-
CZBsHIO)]
wherein M is selected from a radioisotope of technetium and rhenium and R
and R' are independently selected from (CH2)~C02H, NH2 and NH-NH2, and n
is 0-50.
In further embodiments of the present invention, the functional
derivatives comprise a carborane selected from the group consisting of
compound 13 (Figure 3) and 14 (Example 14), and the corresponding
deprotected compounds.
Some of the compounds of the invention may have at least one
asymmetric center. Where the compounds according to the invention have
one asymmetric center, they may exist as enantiomers. Where the
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compounds of the invention possess two or more asymmetric centers, they
may additionally exist as diastereomers. It is to be understood that all such
isomers and mixtures thereof in any proportion are encompassed within the
scope of the present invention.
Uses
The invention also includes the use of the metal-carborane complexes
prepared using the method of the invention for the preparation of
radiopharmaceuticals. In particular, the functional derivatives of the metal
carborane complexes may be conjugated to a biological targeting ligand and
the resulting complex used as a radiopharmaceutical or to prepare a
pharmaceutical composition by mixing with a pharmaceutically acceptable
carrier or diluent.
Accordingly, the present invention provides a method of preparing a
radiopharmaceutical comprising conjugating a metal carborane complex
prepared using the method of the invention to a biological targeting ligand.
The invention also includes a method of preparing a radiopharmaceutical
composition comprising conjugating a metal carborane complex prepared
using the method of the invention to a biological targeting ligand and
combining with a pharmaceutically acceptable carrier or diluent. In an
embodiment of the invention, the metal carborane complex is selected from
the novel metal carborane complexes of the invention.
In a further embodiment of the present invention, there is included a
use of a metal-carborane complex prepared using the method of the invention
as a radiopharmaceutical. Therefore the present invention also includes
radiopharmaceutical compositions comprising a metal carborane complex
prepared using the method of the invention and a pharmaceutically
acceptable carrier.
The compositions containing the metal carborane complexes prepared
using the method of the invention can be prepared by known methods for the
preparation of pharmaceutically acceptable compositions which can be
administered to subjects, such that an effective amount of the active
substance is combined in a mixture with a pharmaceutically acceptable
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vehicle. Suitable vehicles are described, for example, in Remington's
Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack
Publishing Company, Easton, Pa., USA 1985). On this basis, the
compositions include, albeit not exclusively, solutions of the substances in
association with one or more pharmaceutically acceptable vehicles or
diluents, and contained in buffered solutions with a suitable pH and iso-
osmotic with the physiological fluids.
The term an "effective amount" " of an agent as used herein is that
amount sufficient to effect beneficial or desired results, and, as such, an
"effective amount" depends upon the context in which it is being applied. For
example, in the context of administering an agent for use as a
radiopharmaceutical or for radioimaging, an effective amount of an agent is,
for example, an amount sufficient to achieve effective image of the tissue or
organ of interest.
In accordance with the methods of the invention, the described
radiopharmaceutical compositions may be administered to a patient in a
variety of forms depending on the radiopharmaceutical application, as will be
understood by those skilled in the art.
The dosage of the compositions of the invention can vary depending on
many factors such as the pharmacodynamic properties of the active
ingredient, the mode of administration, the age, health and weight of the
recipient, the nature and extent of the symptoms, the frequency of the
treatment and the type of concurrent treatment, if any, and the clearance rate
of the compound in the animal to be treated. One of skill in the art can
determine the appropriate dosage based on the above factors.
The following non-limiting examples are illustrative of the present
invention:
EXAMPLES
Materials and General Procedures for Examples 1-11
All reactions were carried out under an atmosphere of dry argon or
nitrogen using standard Schlenk line techniques. With the exception of
decaborane, which was purchased from Katchem Ltd. and used as received,
CA 02387436 2002-05-24
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reagents were obtained from Aldrich Chemical Co. The reagent, [nido-7,8-
C2BsH~2]- was prepared as previously described.z2 Petroleum ether refers to
that fraction of boiling point 40-60°C. THF was purified by
distillation on
benzophenone-sodium under nitrogen while acetonitrile was distilled from
CaH2 under N2. TLC was performed using Merck silica gel F-254 on
aluminum plates. Solvent systems are reported as v/v mixtures. Compounds
were visualized using a 0.1 % mixture of PdClz in hydrochloric acid (3 M)
followed by heating with a heat gun. Merck 60 GF2sa (7730) on silica gel
plates and Silicycle silica gel (70-230 mesh) were used for preparative thin
layer chromatography and flash chromatography respectively.
The NMR spectra were recorded at ambient temperature on Bruker
AV200 and AV300 spectrometers. Chemical shifts (S) are relative to
tetramethylsilane as internal standard for'H NMR and BF3.OEt2 as external
reference for "B NMR. CI refers to chemical ionization mass spectrometry,
Ei refers to electron impact mass spectrometry and ES refers to electrospray
mass spectrometry, which was performed on a Fisons Platform quadrupole
instrument using samples dissolved in 50/50 CH30H-H20 mixtures. 1R
spectra were run on a Bio-Rad FTS-40 FT FTIR spectrometer. HPLC
experiments were run on a Varian Prostar Model 230 HPLC using a
MicroSorb-MV column (25 cm, 8 ~c, G6 10002-4) with a mobile phase
consisting of 70% CH30H-30% H20. The flow rate was maintained at 0.5
mL/min.
Caution: 99Tc is a weak remitter (E= 292 keV, t,~2= 2.12 x 105 yrs).
Example 1: Synthesis of (NEta]2[M(CO)3Br3]
The title compound was prepared using the method reported in Alberto et al.
1995.'5 The pattern of the vC0 IR stretches for [NEt4][M(CO)3Br3] (M = Re,
99Tc) prepared were indicative of the C3" symmetry of the products and the
stretching frequencies were similar to the reported values for the
corresponding chloro complexes.'5
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Example 1A: Synthesis of [NEt4][Re(CO)3(rl5-7,8-C2BsH~~)], Compound 1
[(CH3)3NH][nido-7,8-CZB9H~2] (96.8 mg, 0.5 mmol) was dissolved in
THF (20 mL) under argon and cooled to -20 °C with an acetone-dry
ice bath.
TIOEt (267 mg, 76 p,L, 1.05 mmol) was added using an Eppendorff pipet
previously filled with argon. With vigorous stirring under argon and protected
from light, the reaction mixture was allowed to warm to room temperature over
1 hr. The ensuing precipitate was isolated by a combination of careful
decantation and extraction of the remaining solvent using a syringe. The
precipitate was rinsed twice with pentane (7 mL), then dried under vacuum.
To the yellow solid, [NEt4]2[ReBr3(CO)3] (193 mg, 0.25 mmol) and THF (10
mL) were added under argon and the mixture heated to reflux overnight. After
cooling to room temperature, the heterogenous solution was filtered through
celite, and the residue rinsed with THF. The filtrate was concentrated under
reduced pressure and the product isolated from the resulting yellow oil by
either flash chromatography or preparative thin layer chromatography
(CH2C12). The product, a colourless solid was further purified by
recrystalization from a dichloromethane-ether mixture (124 mg, 93 %). Mp:
220°C (decomp.); TLC Rf 0.41 (CH2C12); IR (KBr): v 3043, 3014, 2996,
2588,
2556, 2528, 2510, 2494, 2009, 1896, 1865; 'H NMR (200 MHz, CD2C12): 8
3.19 (q, J= 7.3 Hz, 8H, CH2), 2.97 (br s, 2H, CH), 1.33 (m, 12H, CH3); '3C
NMR (50.3 MHz, CDZC12): 8 198.8, 53.1, 32.7, 7.8; "B (96.3 MHz, CD2C12): 8-
7.99, -11.62, -15.83, -22.92, -24.16; MS (ES, negative): m/z = 403.1 [M]-;
Anal.
Calcd. for C,3H3,03B9ReN (532.89): C, 29.30; H, 5.86. Found: C, 29.43; H,
6.08.
Example Z: Synthesis of [NEtd](Tc(CO)3(rl5-7,H-C2B9H11)], Compound 5
[(CH3)3NH][nido-7,8-C2B9H~2] (48 mg, 0.25 mmol) was dissolved in
THF (10 mL) under nitrogen and cooled to -30 °C with an acetone-
dry ice
bath. TIOEt (134 mg, 38 ~L, 0.525 mmol) was added using an Eppendorff
pipette previously filled with nitrogen. With vigorous stirring under nitrogen
and protected from light, the reaction mixture was allowed to warm to room
temperature over an hour. The ensuing precipitate was isolated by a
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combination of careful decantation and extraction of the remaining solvent
using a syringe. The precipitate was rinsed once with THF (5 mL) then dried
under vacuum. To the yellow solid [NEt4]2[TcBr3(CO)3] (85 mg, 0.125 mmol)
and THF (5 mL) were added under nitrogen and the mixture refluxed
overnight. After cooling to room temperature, the solution was filtered and
the
residue rinsed with THF. The filtrate was concentrated under reduced
pressure and the product, a beige powder (45 mg, 80%), was isolated by
preparative thin layer chromatography (CH2C12). TLC Rf 0.25 (CH2C12/MeOH:
4/1); IR (KBr): v 2959, 2581, 2523, 2503, 2016, 1918, 1908; 'H NMR (200
MHz, CD2C12): 8 3.20 (q, J = 7.3 Hz, 8H, CHZ), 2.72 (br s, 2H, CH), 1.34 (m,
12H, CH3); '3C NMR (50.3 MHz, CDZCIZ): 8 53.2, 35.3, 7.80; "B (96.3 MHz,
CD2C12): 8 -7.52, -8.22, -10.18, -13.26, -20.75, -22.42; MS (ES, negative):
m/z
= 315.0[M]-.
Example 3: Synthesis of Benzyl Pent-4-ynoate,Z3 Compound 7
4-pentynoic acid (5.0 g, 48.42 mmol) was dissolved in a solution of dry
acetonitrile (100 mL) followed by the dropwise addition of 1,8-
diazabicyclo[5,4,0]undec-7-ene (DBU) (8.38 mL, 53.79 mmol). Freshly
distilled benzyl bromide (6.68 mL, 56.07 mmol) was added slowly to the
stirring mixture, and the reaction maintained at room temperature overnight.
The reaction mixture was transferred to a separatory funnel and diluted with
diethyl ether (60 mL). The organic layer was washed with 1 M HCI (3 x 40
mL), and all aqueous layers combined, and further washed with ether (3 x 60
mL). The organic fractions were combined, dried over magnesium sulfate,
filtered, and the solvent removed in vacuo giving a light yellow oil. Vacuum
distillation (2 mm Hg/ b.p. 111°C) yielded the desired product (8.57 g,
94%).
TLC Rr 0.67 (1:1 ether:hexanes); IR (neat): v 3297, 3090, 3068, 2956, 2928,
2897, 2122, 1739, 1164; 'H NMR (200 MHz, CDC13): 8 7.45-7.26 (m, 5H, H-
aryl), 5.14 (s, 2H, CHzPh), 2.56 (m, 4H, CH2CH2), 1.96 (br s, 1H, CH); '3C
NMR (50 MHz, CDC13): 8 171.49, 135.65, 128.47, 128.14, 82.32, 69.04,
66.41, 33.25, 14.23; MS (E1): m/z = 189[M+H]+
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Example 4: Synthesis of Benzyl 3-(1',2'-dicarboclosododecaboran-1'-
yl)propanoate, Compound 8
Decaborane (3.25 g, 26.6 mmol) was dissolved in dry acetonitrile (50
mL) and stirred under nitrogen for 13 hours prior to the dropwise addition of
4
(5.0 g, 26.6 mmol). The reaction mixture was brought to reflux for four days
after which the solution was allowed to cool to room temperature and the
solvent removed in vacuo giving a yellow oil. The oil was taken up in
diethylether (100 mL) and washed three times with 1 M NaOH (50 mL). The
organic layer was collected, dried over magnesium sulfate, filtered and the
solvent removed in vacuo. The resulting oil was taken up in a minimal
amount of hexanes and subjected to silica gel chromatography with hexanes-
ethyl acetate (9:1 ) as the eluent. The product (4.89 g, 60%) was isolated as
a
white amorphous solid. m.p. = 80-81°C; TLC Rf 0.58 (1:1 ether:hexanes);
IR
(KBr): v 3064, 3034, 2945, 2929, 2879, 2583, 1729; 'H NMR (300 MHz,
CDCi3): 8 7.41-7.34 (m, 5H, H-aryl), 5.14 (s, 2H, PhCH2), 3.69 (br s, 1 H,
CH),
2.59 (m, 4H, CH2CH2); '3C NMR (75.5 MHz, CDC13): b 171.3, 135.3, 128.8,
128.7, 128.6, 74.1, 67.2, 61.6, 33.6, 32.8; "B NMR (160 MHz, CDC13): 8 -
2.50, -5.97, -9.85, -12.01, -12.68, -13.37; MS (CI): m/z = 324[M + NH3].
Example 5: Synthesis of 3-(1',2'-dicarbaclosododecaboran-1'-
yl)propanoic acid, Compound 9
The reaction vessel was charged with 10% palladium on carbon (320
mg) and compound 8 (3.21 g, 10.47 mmol) in absolute ethanol (50 mL) and
glacial acetic acid (2 drops). In a Parr Hydrogenator, the mixture was
subjected to an atmosphere of hydrogen (2 atm.) with shaking for 2 hours.
The mixture was filtered through Celite, and the residue washed with absolute
ethanol (3 x 20 mL). The filtrates were combined, the solvent evaporated in
vacuo and the resulting solid dried under high vacuum (2.30 g, 99%). m.p. _
145-146 °C; TLC Rf 0.39 (1:1 ether:hexanes); IR (KBr): v 3448, 3054,
2999,
2929, 2859, 2586, 1702; 'H NMR (300 MHz, CDC13): 8 3.67 (br s, 1H, CH),
2.65-2.54 (m, 4H, CH2CH2);'3C NMR (75.5 MHz, CDC13): 8 177.1, 73.7, 61.7,
33.3, 32.5; "B NMR (160 MHz, CDC13): b -2.51, -5.96, -9.80, -11.93, -12.57, -
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13.31; MS (ES, negative): m/z = 215.1[M-H+]-.
Example 6: Synthesis of (PMePhs]rac-[nido-7-(CHZCH2COzH)-7,8-
C2B91"111~~ Compound 10
Compound 9 (216 mg, 1.0 mmol) was added to a solution of potassium
hydroxide (297 mg, 4.5 mmol) in absolute ethanol (6.7 mL) and the reaction
mixture heated to reflex for 24 hrs. After cooling to room temperature, carbon
dioxide was bubbled through the solution to precipitate the excess of KOH as
potassium carbonate. The white suspension was filtrated through celite and
the residue rinsed with absolute ethanol. After evaporation of the filtrate
under reduced pressure, the resulting oil was acidified to pH 2 with HCI (1.0
M). The water was removed under vacuum and the resulting white solid re-
dissolved in distilled water (4 mL) and a 2.0M aqueous solution of MePPh3Br
(547 mg, 1.5 mmol) was added dropwise. The solution was cooled to 0°C
and the resulting precipitate isolated by filtration. The residue, after
iyophilization overnight, was dissolved in CH2C12, filtered through a plug of
glass wool, and, after removal of the solvent, the resulting solid was
recrystallized from a mixture of CH2C12 and petroleum ether yielding the
product as a colourless solid (486 mg, >99%). m.p. 160°C decomp; TLC Rf
0.82 (4:1 CH2C12:CH30H); IR (KBr): v 3440, 3059, 2984, 2915, 2855, 2512,
1711, 1438, 1115, 900; 'H NMR (200 MHz, CD2C12): 8 7.88-7.57 (m, 15H, H-
aryl), 2.84 (d, J = 13 Hz, 3H, P-CH3), 2.43-2.33 (m, 2H, CHZ), 1.90-1.73 (m,
2H, CHZ), 1.66 (br s, 1 H, CH); '3C NMR (50.3 MHz, CD2C12): 8 179.0, 136.1,
133.7, 133.5, 131.3, 131.0, 35.9, 34.5, 11.2, 10.0; "B (CD2C12): 8 -11.57, -
14.40, -17.48, -19.09, -22.43, -34.05, -38.00; MS (ES, negative): m/z = 205.2
[M]-; Anal. Calcd. for C24H~02PB9, (482.80): C, 59.71; H, 7.10. Found: C,
56.55; H, 6.94.
Example 7: Synthesis of [PMePh3]rac-[Re(CO)3(rl5-7-(CHZCHZC02H)-7,8-
C2B91"110~~~ Compound 11
Compound 10 (97 mg, 0.2 mmol) was dissolved in THF (8 mL) under
argon and cooled to -20 °C. TIOEt (107 mg, 30.4 wL, 0.42 mmol) was
added
dropwise using an Eppendorff syringe previously filled with argon. With
vigorous stirring under argon and protected from light, the reaction mixture
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was warmed to room temperature over 2 hours. Pentane (10 mL) was added
to the heterogenous mixture and the solution cooled with an acetone-dry ice
bath. The ensuing precipitate was isolated by a combination of careful
decantation and extraction of the remaining solvent using a syringe. The
precipitate was rinsed twice with pentane (10 mL) and then dried under
vacuum. To the yellow solid, [NEt4)2[ReBr3(CO)s] (77 mg, 0.1 mmoi) and THF
(6.5 mL) were added and the reaction mixture heated to reflux overnight
under argon. After cooling to room temperature, the solution was acidified to
pH 2 with HCI (1 M), filtered through celite, and the residue washed with a
1:1
mixture of THF/1 M HCI (5 mL). The filtrate was concentrated under reduced
pressure and the resulting brown oil triturated with dichloromethane (5 mL),
and the ensuing precipitate removed by filtration. The filtrate was
concentrated under reduced pressure and the product, a yellowish powder
(74 mg, 99%), was isolated by thin layer chromatography using an eluent
composed of CH2C12-CH30H (97:3). The TLC silica was extracted with
CH2C12, THF and MeOH, m.p. 140°C (decomp.); TLC Rf 0.70 (9:1
CH2C12/MeOH); IR (KBr): v: 3629, 3433, 3060, 2962, 2919, 2871, 2524, 2002,
1893, 1707, 1589, 1439; 'H NMR (200 MHz, CD2C12): S 8.07-7.26 (m, 15H, H-
aryl), 3.24 (br s, 1 H, CH), 2.75 (d, J= 13.2 Hz, P-CH3), 2.50-2.45 (m, 2H,
CH2), 2.24-2.11 (m, 2H, CH2); '3C NMR (50.3 MHz, CD2C12): 8 199.0, 180.0,
135.9, 133.5, 133.3, 132.7, 131.1, 130.9, 129.5, 129.2, 125.8, 119.7, 117.9,
34.66, 31.17, 30.64; "B (96.3 MHz, CD2C12): 8 -7.37, -10.82, -12.11, -18.03, -
19.70, -22.93, -34.60, -38.41; MS (ES, negative): m/z = 475.1 [M]-; Anal.
Calcd. for CZ~H330sPBsFte, (752.03): C, 43.12; H, 4.42. Found: C, 42.68; H,
4.87.
Example 8: Synthesis of [PMePh3]rac-[Tc(CO)3(rl5-7-(CH2CH2C02H)-7,8-
C2B91"~10~~~ Compound 12
Compound 10 (57 mg, 0.117 mmol) was dissolved in THF (5 mL) under
nitrogen and cooled to -20 °C. TIOEt (94 mg, 27 ~,L, 0.369 mmol) was
added
using an Eppendorff syringe previously filled up with nitrogen. With vigorous
stirring under nitrogen and protected from light the reaction mixture was
allowed to warm to room temperature over two hours. The ensuing
CA 02387436 2002-05-24
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precipitate was isolated by a combination of careful decantation and
extraction of the remaining solvent using a syringe. The precipitate was
rinsed twice with THF and dried under vacuum. To the yellow solid
[NEt4j2[TcBr3(CO)3] (40 mg, 0.059 mmol) and THF (7 mL) were added under
nitrogen and the mixture heated to reflux overnight. After cooling to room
temperature, the solution was acidified to pH 2 with HCI (1 M), filtered
through
celite, and the residue rinsed with a 1:1 mixture of THF/1 M HCI. The filtrate
was concentrated under reduced pressure and the product, a beige powder,
was isolated by thin layer chromatography using an eluent composed of
CH2C12-CH30H (97:3). The TLC silica was extracted with CH2C12, THF and
CH30H (93%). TLC Rf 0.64 (9:1 CH2C12/CH30H); IR (KBr) v 3627; 3295,
3059, 2956, 2917, 2870, 2529, 2009, 1911, 1727; 'H NMR (200 MHz,
CDZC12): 8 7.76-7.44 (m, 15H, H-aryl), 2.25 (m, 4H, CHzCHz), 2.00 (d, J = 13.2
Hz, 3H, P-CH3); "B (96.3 MHz, CD2C12): 8 -6.81, -8.92, -10.81, -12.25, -14.13,
-18.62, -21.77; MS (ES, negative): m/z = 388.1 [M]-.
Example 9: Synthesis of (Na]rac-[Re(CO)3(rl5-7-(CHzCH2C02H)-7,8-
C2B91"~10~]24
A sample of [PMePh3]rac-[Re(CO)s(~5-7-(CH2CH2C02H)-7,8-CZB9HIO)]
was dissolved in a mixture of CH3CN-H20 (60:40) and subjected to ion
exchange on a strong cation-exchange resin column (MALLINCKRODT
Amberlite CG-120 100-200 mesh). The volatiles were removed by
evaporation under reduced pressure, and the residue dried on a lyophilizer to
yield the respective sodium salt. HPLC tR = 6.21 (95.2%); MS (ES, negative):
m/z = 475.1 [M]-.
Example 10: Synthesis of [K]rac-[Re(CO)3[~5-7-(CH2CH2C02Hj-7,8-
C2B91"'110~~ in water
The sodium salt of compound 10 (171 mg, 0.7 mmol) and potassium
hydroxide (370 mg, 5.6 mmol) were dissolved in deionized water (3 mL).
[NEta]2[ReBr3(CO)3] (1.1 mg, 1.4 mmol) was added and the reaction mixture
was heated under reflux overnight. After cooling to room temperature, the
solution was acidified to pH 1 using HCI (1 M), and the heterogenous solution
filtered through celite. The filtrate was evaporated, re-dissolved in water
and
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the pH adjusted to between 5 and 6 with HCI (6 M). The resulting precipitate
was filtered through Celite and the solution evaporated under reduced
pressure. The remaining residue was further lyophilized leaving a white
powder. HPLC tR = 6.72 {[K]rac-[Re(CO)3[~~-7-(CH2CH2C02H)-7,8-C2B9H~o]},
tR = 7.01 {[K]rac-[nido-7-(CH2CH2C02H)-7,8-C2B9H~~]}; MS (ES, negative):
m/z = 475.1 [M]-.
Materials and General Procedures for Examples 11-16
All commercial reagents were used as supplied. THF and Et20 were
distilled under nitrogen from sodium and benzophenone while CH2Ci2 was
distilled from CaHz. Ortho-carboranes were purchased from Katchem Ltd.
(Czech. Rep.), while mefa-carborane was purchased from Dexsil Corp.
(Hamden, CT). C02(g), which was generated by sublimation of C02(s), was
passed through a column of Drierite~ prior to its addition to a reaction.
Analytical TLC was performed on silica gel 60-F2sa (Merck). Boron
compounds were visualized with 0.1 % PdCl2 in hydrochloric acid (3.0 M),
which upon heating gave dark brown spots. Hydrazine derivatives were
visualized using a ninhydrin solution, which consisted of 0.3% of ninhydrin in
n-butanol containing 3% acetic acid.
NMR spectroscopy experiments were performed on Bruker Avance
AV300 and DRX500 spectrometers. TMS and BF3-Et20 were used as internal
standards for'H and "B spectra respectively. For NMR assignments, b refers
to broad signals, s refers to singlets and m refers to multiplets.
Electrospray
mass spectrometry experiments were performed on a Fisons Platform
quadrupole instrument. Samples were dissolved in 50.50 CH3CN/H20 and, for
compounds run in negative ion detection mode, one drop of 0.10 M NH40H
was added. Microanalyses were performed by Guelph Chemical Laboratories
(Guelph, Ontario, Canada). 1R spectra were run on a Bio-Rad FTS-40 FTIR
spectrometer.
Example 11: Synthesis of 1-[(N,N'((tert-butyloxy)carbonyl)hydrazino)]-
7,8-dicarba-closo-dodecaborane, 15.
MeLi (4.70 mL, 6.53 mmol, 1.39 M in diethyl ether) was added
dropwise over 5 minutes to a solution of 7,8-dicarba-closo-dodecaborane (2,
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1.00 g, 6.93 mmol) in ether (125 mL) at 0°C. The reaction was
maintained at
0°C for an additional 45 minutes at which time, the solution was added
dropwise over 15 minutes to a solution of DBAD (4.00 g, 17.37 mmol) in ether
(100 mL) under argon atmosphere. After the complete addition of the
carborane, the reaction heated to reflux for 2.5 hours at which point the
reaction was quenched with the addition of water (100 mL). The solvent was
removed by rotary evaporation, and the resulting solution acidified through
dropwise addition of HCI (1.0 M) affording a white precipitate. The solution
was extracted with ethyl acetate (3 x 100 mL) and the organic layers
combined, dried over MgS04, and the solvent removed by rotary evaporation.
Residual water was removed by the addition and subsequent evaporation of
9:1 CHC13/toluene mixtures (5 x 50 mL). Excess DBAD was removed by
triturating the yellow solid with cold pentane (5 x 50 mL), leaving a
colourless
solid, which was further purified by silica gel chromatography (gradient
elution; 100% hexanes to 1:3 diethyl ether/hexanes). The main product
isolated from the column was recrystallized from petroleum ether affording a
white solid (2.01 g, 84%). TLC Rf (1:5 diethyl ether/pet. ether) = 0.38; mp
171
- 174°C (decomp.); 'H NMR (acetone-ds, 300 MHz): 8 1.391, 1.422 (s,
CH3),
0.91-3.1 (b, BH), 5.084, 5.233 (bs, CH), 8.241, 8.808 (bs, NH); '3C NMR
(acetone-ds, 125.77 MHz): 8 27.08, 27.46 (CH3), 63.86 (CH), 81.12, 83.80
(C(CH3)s), 87.44 (CN), 151.69 (NCO), 155.01 (NHCO); "B{'H} NMR (CDC13,
96.3 MHz): S -3.51, -5.00, -7.43, -11.03, -12.66, -15.05; IR (KBr, cm-'):
3321,
3077, 2612, 1754, 1719; MS/ESI: 373.3(M-H]- with the expected isotopic
distribution; Anal. Calcd for C~ZB~oH3oN204: C, 38.49; H, 8.07; N, 7.48.
Found:
C, 38.10; H, 8.12; N, 7.40.
Example 12: Synthesis of 7-((N,N'((tert-butyloxy)carbonyl)hydrazino)]-
7,8-dicarba-closo-dodecaborane-8-carboxylic acid, 13
n-BuLi (1.75 mL, 2.80 mmol, 1.6 M in hexanes) was added dropwise
over a period of 5 minutes to a solution of compound 15 (0.50 g, 1.34 mmol)
in 5:1 THF/ether (60 mL) at 0°C. After 45 minutes the temperature was
lowered to -78°C and C02 bubbled into the solution for 3.5 hours. The
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temperature was allowed to warm to room temperature and the solvent
removed in vacuo. Water (50 mL) was added and the solution acidified by
dropwise addition of HCI (1.0 M) until a precipitate appeared (pH = 3). The
solution was extracted with ethyl acetate (3 x 50 mL), and the organic layers
combined, dried over MgS04, and evaporated leaving a colourless oil.
Suspension of the oil in a 4:1 mixture of pentane/ether afforded a white
precipitate, which was washed with the pentane/ether mixture (4 x 50 mL)
leaving the product as a white solid (371 mg, 65%); TLC Rf (1:9
CH30H/CH2C12) = 0.14; mp 131°C (decomp.); 'H NMR (acetone-ds, 300
MHz): 8 1.438, 1.472 (s, CH3), 1.220-2.810 (b, BH), 8.335, 8.904 (s, NH); '3C
NMR (acetone-ds + 2 drops of DMSO-d6, 50.3 MHz): 8 27.50, 27.80, 27.95
(CH3), 63.57, 64.76 (CC02H), 80.90, 81.16, 83.79 (C(CH3)3), 87.83 (CN),
151.74 (BocC(O)), 154.00 (COzH), 155.04 (BocC(O)); "B{'H~ NMR (acetone-
ds, 96.3 MHz): S -3.58, -6.20, -11.05, -13.49; IR (KBr): 3380, 2578, 1713,
1650. MS/ESI: 835.5 [2M-H]-, 417.3[M-H]-, 408.4 [M-B]~, 373 [M-C02H]- with
the expected isotopic distribution; Anal. Calcd for C~3H30B10N206. C, 37.31;
H,
7.23. Found: C, 34.33; H, 7.59.
Example 13: Synthesis of 1-[(N,N'((tert-butyloxy)carbonyl)hydrazino)~-
7,9-dicarba-closo-dodecaborane.
n-BuLi (5.00 mL, 6.93 mmol; 1.39 M in hexanes) was added to 7,9-
dicarba-closo-dodecaborane (meta-carborane, 1.00 g, 6.93 mmol) in dry
diethyl ether (125 mL) at 0°C under argon. After 45 minutes, the
solution
containing the anion was added slowly to DBAD (3.19 g, 13.86 mmol) in dry
diethyl ether (100 mL). The reaction was subsequently heated under reflux for
2.5 hours whereupon it was cooled to room temperature and quenched by the
addition of water (10 mL). Diethyl ether was removed under reduced pressure
and the mixture extracted with ethyl acetate (100 mL), which in turn was
washed with water (1 x 100 mL), 0.1 M HCI (2 x 100 mL), and brine (2 x 100
mL). The organic layers were combined, dried over MgS04 and the solvent
concentrated under reduced pressure. The product, an amorphous white solid
(1.94 g, 75%), was further purified by silica gel chromatography (gradient
elution; from petroleum ether to 1:9 ethyl acetate/petroleum ether): TLC Rf
CA 02387436 2002-05-24
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(1:9 ethyl acetate/pet. ether) = 0.36; mp 143-144°C; 'H NMR (acetone-
ds, 300
MHz): b 1.242, 1.280 (s, CH3), 1.198-2.87 (b, BH), 3.541, 4.445 (bs, CH),
8.056, 8.503 (bs, NH); '3C NMR (acetone-ds, 125.77 MHz): 8 27.22, 27.57
(CH3), 53.12 (CH), 80.22, 80.78, 82.30, 82.51 (C(CH3)3), 89.37 (CN), 151.70,
151.87 (BocC(O)), 154.25, 154.50 (BocC(O)); "B{'H} NMR (acetone-ds, 96.3
MHz): S -4.79, -11.27, -12.97, -15.68; IR (KBr): 3317, 3061, 2972, 2607, 1739,
1722.1; MS/ESI: 373.3[M-H]-'; Anal. Calcd for C~2B10H30N204. C, 38.49; H,
8.07; N, 7.48. Found: C, 38.91; H, 8.42; N, 7.47.
Example 14: Synthesis of 1-[(N,N'((tert-butyloxy)carbonyl)hydrazino)]-
7,9-dicarba-closo-dodecaborane-7-carboxylic acid, 14.
n-BuLi (1.75 mL, 2.80 mmol) was added dropwise over a period of 5
minutes to the product from Example 13 (0.50 g, 1.34 mmol) in dry THF (100
mL) under argon at 0°C for 45 minutes. The temperature of the reaction
was
subsequently lowered to -78°C and dry COz(g) was bubbled into the
solution
with rigorous stirring. After 3.5 hours, the reaction was allowed to warm to
room temperature and acidified with 1 M HCI (pH = 3). The solvent was
removed under reduced pressure and the yellowish residue re-dissolved in
ethyl acetate (50 mL) and extracted with brine (3 x 50 mL). The organic phase
was dried over MgS04 and evaporated to dryness. The resulting yellow oil
was further purified by flash chromatography through silica gel (gradient
elution; from CH2C12 to 5:95 CH30H/CH2C12) yielding an amorphous solid
(0.41 g, 73%): TLC Rf (1:4 CH30H/ CH2C12) = 0.57; mp 156-159°C;'H NMR
(acetone-ds, 300 MHz): S 1.353, 1.391, 1.430 (s, CH3), 0.95-4.25 (b, BH),
8.064, 8.507 (bs, NH); '3C NMR (acetone-ds, 125.77 MHz): 8 27.21, 27.55
(CH3), 70.12 (CC02H), 80.45, 81.09, 82.66, 82.84 (C(CH3)3), 89.12 (CN),
151.81, 154.52 (BocC(O)), 161.80 (C02H); "B{'H} NMR (acetone-ds, 96.3
MHz): 8 -4.36, -8.90, -10.52, -12.76; IR (KBr): 3317, 3185, 2986, 2939, 2617,
1753, 1721; MS/ESI: 373.3[M-H]', 835.5[2M-H]-; Anal. Calcd for
C13B10H30N2Og: C, 37.31; H, 7.23; N, 6.69. Found: C, 36.96; H, 7.61; N, 6.20.
CA 02387436 2002-05-24
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Example 15: General Method for the Preparation of the nido-Derivatives
The nido salts of compounds 13 and 14 may be prepared by refluxing
the corresponding closo compounds in KOH and EtOH for 24 hours. The
remaining KOH is quenched with water and the products may be isolated as
colourless solids by precipitation as their [MePPh3]+ salts.
Example 16: Syntheses Of Na[9smTc(CO)3(rl5-7,8-C2BgH~~)] and Na{rac-
~99mTC~CO~3~'n 5-7-CH2CHZC02H-7,8-CZB9H~o)]} from Na99"'Tc04
In a 10 mL sealed serum vial were introduced NaBH4 (5 mg, 130 pmol,
22.106 eq) and NaZC03 (4 mg, 38 p,mol, = 6.106 eq). The vial was flushed
with CO for 10-20 min. Na99"'Tc04 (3 mCi) dissolved in saline (3 mL of 0.9
w/w NaCI in water) was added. The reaction was heated 40 min at 85°C
(temperature of the water bath). For safety reason, a 20 mL guard syringe
was kept through the septum to buffer the H2 release. The colorless solution
was cooled down to room temperature using an ice bath. A sample was taken
up to observe the formation of [Tc(CO)3(OH2)]+ by TLC scanning (95:5
CH30H/ 12M HCI, Rf ~ 0.4). The solution was splinted into two aliquots (1.5
mCi, 1.5 mL) and added to two 10 mL serum vials containing
(Me3NH)(C2B9H~2) (193.53 g/mol, 105 eq, 0.289 pmol, 56 pg) or K(C2B9H»-
CH2-CH2-C02H) (244.57 g/mol, 105 eq, 0.289 ~mol, 71 p,g). The vials were
heated at 85°C for 2 h and cooled down to room temperature with an ice
bath.
The solutions were acidified with 1 M HCI and analyzed by TLC scanning and
HPLC according to the conditions below. The TLC and HPLC retention times
and profiles of the radioactive compounds were identical to authentic
standards prepared and characterized separately.
(Me3NH)(CZBgH12) K(C2BaH~~-CH2-CHZ-C02H)
Acidification to pH ~ 7 Acidification to pH ~ 2
TLC scanning (9:1 CH2CI2/CH30H)TLC scanning (9:1 CHzCIz/CH30H)
Rf
Rf = 0.40 = 0.40
HLPC (40:60 CH30H/H20) HLPC (70:30 CH30H/H20)
CA 02387436 2002-05-24
-26-
Note:
1. HPLCs ran on an analytical Varian HPLC connected to a beta-radiation
detector and equipped with a C~a semi-Preparative MicroSorb 250x10 300-
column. All runs were isocratic at 1.5 mL/min.
5 2. Control injection of Na99"'Tc04 in HPLC (40:60 CH30H/H20) and (70:30
CH30H/Hz0)
3. Control injection of Na[Re(CO)3(CZB9H~~)] in HPLC (40:60 CH30H/Hz0)
4. Control injection of (Me3NH)(CzB9H~z) in HPLC (40:60 CH30H/HZO)
5. Control injection of Na[Re(CO)3(C2BsH~~-CHz-CHz-COOH)] in HPLC
(70:30 CH30H/H20)
6. Control injection of K(C2B9H ~ ~-CHz-CHz-COZH) in HPLC (70:30
CH30H/H20)
While the present invention has been described with reference to what
are presently considered to be the preferred examples, it is to be understood
that the invention is not limited to the disclosed examples. To the contrary,
the
invention is intended to cover various modifications and equivalent
arrangements included within the spirit and scope of the appended claims.
All publications, patents and patent applications are herein
incorporated by reference in their entirety to the same extent as if each
individual publication, patent or patent application was specifically and
individually indicated to be incorporated by reference in its entirety.
CA 02387436 2002-05-24
27
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