Note: Descriptions are shown in the official language in which they were submitted.
CA 02515495 2005-08-09
r
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B&P File No. 3244-118
TITLE: METHODS FOR PREPARING METAL-CARBORANE
COMPLEXES FOR RADIOIMAGING AND RADIOTHERAPY
FIELD OF THE INVENTION
The present invention relates to improved methods for the preparation of
radiopharmaceuticals, in particular carborane complexes of technetium and
rhenium.
BACKGROUND OF THE INVENTION
Cyclopentadienide (Cpy) complexes of 99"'Tc (Ey = 141 keV, t~~2 = 6.02 h),
the most widely used radionuclide in diagnostic medicine (Jurisson, S. S.;
Lydon,
J. D. Chem. Rev. 1999, 99, 2205) are attractive synthons for the development
of
organometallic radiopharmaceuticals because of the metal complexes' small size
and stability. A number of synthetic approaches to CpTc(CO)3 and related
derivatives have been developed [(a) Wenzel, M. J. Labelled Compd.
Radiopharm. 1992, 31, 641. (b) Spradau, T. W.; Katzenellenbogen, J. A.
Organometallics 1998, 17, 2009. (c) Cesati, R. R., III; Katzenellenbogen, J.
A. J.
Am. Chem. Soc. 2001, 123, 4093. (d) Minutolo, F.; Katzenellenbogen, J. A. J.
Am. Chem. Soc. 1998, 120, 4514. (e) Minutolo, F.; Katzenellenbogen, J. A.
Angew. Chem., Int. Ed. 1999, 38, 1617. (f) Spradau, T. W.; Edwards, W. B.;
Anderson, C. J.; Welch, M. J.; Katzenellenbogen, J. A. Nucl. Med. Biol. 1999,
26,
1. (g) Cesati, R. R., III; Tamagnan, G.; Baldwin, R. M.; Zoghbi, S. S.; Innis,
R. B.;
Kula, N. S.; Baldessarini, R. J.; Katzenellenbogen, J. A. Bioconjugate Chem.
2002, 13, 29. (h) Thei, M.; Kothari, K.; Pillai, M. R. A.; Hassan, A.; Sarma,
H. D.;
Chaudhari, P. R.; Unnikrishnan, T. P.; Korde, A.; Azzouz, Z. J. Labelled
Compd.
Radiopharm. 2001, 44, 603.]; however, because cyclopentadiene (Cp) does not
react efficiently with metals in aqueous media and because its conjugate base
oligomerizes in water, these methods typically require the use of organic
solvents, harsh reagents and reaction conditions, and/or multiple synthetic
steps,
CA 02515495 2005-08-09
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which limits their applicability for routine clinical use. More recently,
Alberto and
colleagues showed that introduction of an electron withdrawing carbonyl
substituent on the Cp ring stabilizes the conjugate base to the extent that it
facilitates the direct synthesis of RC(O)CpM(CO)3 (M - Re, 99Tc, 9smTc)
complexes in water in reasonable yields (Wald, J.; Alberto, R.; Ortner, K.;
Candreia, L. Angew. Chem., Int. Ed. 2001, 40, 3062). Half-sandwich complexes
of technetium linked to a serotonergic ligand were prepared using this
approach
(Bernard, J.; Ortner, K.; Spingler, B.; Pietzsch, H.-J.; Alberto, R. Inorg.
Chem.
2003, 42, 1014).
As an initial step toward establishing a new class of Tc organometallic
radiopharmaceuticals, the present inventors developed a method for preparing
9sTc(I) (E~,aX = 294 keV, t~~2 = 2.13 x 105 yr) and Re(I)-carborane complexes
in
water [(a) Valliant, J. F.; Morel, P.; Schaffer, P.; Kaldis, J. H. Inorg.
Chem. 2002,
41, 628; (b) Valliant, J. F.; Morel, P.; Schaffer, P.; Sogbein, O.O. U.S.
Patent
Application Publication No. 2003-0668271, published April 10, 2002)]. The
carborane ligands, which are prepared by deboronation of dicarba-
closododecaboranes followed by deprotonation of the resulting nido-carboranes,
are isolobal to Cp , but, unlike the quintessential organometallic ligand,
they are
highly effective at forming metal complexes in water (Grimes, R. N. Coord. ,
Chem. Rev. 2000, 200-202, 773). One further advantage to using carboranes
over more traditional ligands is that they can be readily functionalized with
a wide
range of different groups at select vertices regioselectively (Valliant, J.
F.;
Guenther, K. J.; King, A. S.; Morel, P.; Schaffer, P.; Sogbein, O. O.;
Stephenson,
A. Coord. Chem. Rev. 2002, 232, 173). This affords a tremendous amount of
flexibility when designing novel radiopharmaceuticals [(a) Hawthorne, M. F.;
Maderna, A. Chem. Rev. 1999, 99, 3421. (b) Wilbur, D. S.; Chyan, M.-K.;
Hamlin,
D. K.; Kegley, B. B.; Risler, R.; Pathare, P. M.; Quinn, J.; Vessella, R. L.;
Foulon,
C.; Zalutsky, M.; Wedge, T. J.; Hawthorne, M. F. Bioconjugate Chem. 2004, 15,
203. (c) Eriksson, L.; Tolmachev, V.; Sjo~~berg, S. J. Labelled Compd.
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Radiopharm. 2003, 46, 623.]
Metallocarboranes are typically prepared in the presence of strong bases
in order to remove the bridging proton on the nido-carborane ligand [(a)
Hawthorne, M. F.; Young, D. C.; Andrews, T. D.; Howe, D. V.; Pilling, R. L.;
Pitts,
A. D.; Reintjes, M.; Warren, L. F., Jr.; Wegner, P. A. J. Am. Chem. Soc. 1968,
90,
879. (b) Hawthorne, M. F.; Varadarajan, A.; Knobler, C. B.; Chakrabarti, S.;
Paxton, R. J.; Beatty, B. G.; Curtis, F. L. J. Am. Chem. Soc. 1990, 112, 5365.
(c)
Valliant, J. F.; Morel, P.; Schaffer, P.; Sogbein, O.O. U.S. Patent
Application
Publication No. 2003-0668271, published April 10, 2002)]. The 99Tc-carborane
complexes reported previously were prepared in water in the presence of KOH or
Na2C03 and [9sTc(CO)3Br3]2-. When the analogous reactions were carried out at
the tracer level with [99"'Tc(CO)s(OH2)3]+ [(a) Alberto, R.; Schibli, R.;
Egli, A.;
Schubiger, P. A. J. Am. Chem. Soc. 1998, 120, 7987. (b) Alberto, R.; Ortner,
K.;
Wheatly, N.; Schibli, R.; Schubiger, A. P. J. Am. Chem. Soc. 2001, 123, 3135.]
the yields of the desired products were low (less than 10%) with the major
reaction product being 99"'TCO4. Changing the amount of ligand, reaction time,
pH, or temperature did not produce yields comparable to those observed for
reactions performed at the macroscopic scale with 99Tc or cold Re.
Reports of the direct formation of a metallocarborane from the
corresponding closo-isomer are rare (Hawthorne, M. F. J. Organomef. Chem.
1975, 100, 97.) and there currently exists no method to carry out such a
reaction
in water.
There remains a need for an efficient synthesis of metallocarboranes, in
particular of Tc and Re, in aqueous solutions at the tracer levels that
provides
good yields of the desired products.
SUMMARY OF THE INVENTION
A new method for the preparation of metalfocarboranes in water under
mild reaction conditions has been developed. Three nido-carborane ligands were
reacted with [Re(CO)3Br3]2- in the presence of aqueous potassium fluoride, a
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hard base, to give the corresponding ~5-Re(CO)3-carborane complexes. The use
of KF as a base afforded the desired Re-metallocarboranes in good yields while
avoiding the formation of Re clusters, which are byproducts commonly observed
when reactions are carried out in the presence of strong, soft aqueous bases.
The reaction was also performed at the tracer level producing the first 99mTc-
carborane complex, which was isolated in 80% radiochemical yield following a
simple Sep-PakT"" purification process. The resulting organometallic complex
was stable to cysteine and histidine challenges for more than 24 hours. It was
also found that it is possible to prepare the metallocarboranes directly from
the
closo-carboranes, in a single step, using substantially the same conditions as
for
the nido-carboranes.
Accordingly, the present invention relates to a method of preparing metal-
carborane complexes comprising reacting a salt of the formula:
[M(CO)3(Xm)3]~~ + 3m),
wherein M is selected from a radioisotope of rhenium (Re), technetium (Tc) and
any other radioisotope binding in the same fashion, X is the same or different
and
is, independently, any suitable ligand and m is the formal charge for ligand
X,
with a nido-carborane or a closo-carborane in the presence of a hard base. In
an
embodiment of the invention, the base is a source of fluoride (F-), for
example
potassium fluoride. In a further embodiment of the invention, the metal, M, is
selected from radioisotopes of Tc and Re.
The present invention also includes a kit for use in the preparation of the
salts of formula [M(CO)3(Xm)3]~' + sm), wherein M is a radioisotope of Tc or
Re and
X is H20, comprising potassium boranocarbonate (K2H3BC02), Na2Ba0~.10H20,
Na2C03 and a hard base, for example a source of F-.
The present invention also relates to the use of a fluoride anion for
stabilizing a salt of formula:
~M(C~)3(Xm)3~~~ + 3m)~
wherein M is selected from a radioisotope of rhenium (Re), technetium (Tc) and
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any other radioisotope binding in the same fashion, X is the same or different
and
is, independently, any suitable ligand and m is the formal charge for ligand
X.
The present invention also relates to a method for stabilizing a salt of the
formula:
[M(CO)3(Xm)3]~~ + 3m),
wherein M is selected from a radioisotope of rhenium (Re), technetium (Tc) and
any other radioisotope binding in the same fashion, X is the same or different
and
is, independently, any suitable ligand and m is the formal charge for ligand
X,
comprising combining the salt with a fluoride anion.
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:
Fig. 1 shows 'y-HPLC radiochromatograms of (A) the crude reaction mixture
after
3 h at 85 °C; (B) Sep-Pak purified 2d; and (C) UV-HPLC chromatogram of
the Re
standard 2b.
Fig. 2 shows ~y-HPLC radiochromatograms of 2d after 24 h of L-cysteine (A) and
L-histidine (B) challenges.
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Fig. 3 showns a X-Ray structure of 2c showing 30% thermal probability
ellipsoids.
Fig. 4 shows y-HPLC radiochromatograms showing the conversion of [99"'Tc04]-
(tR = 11 min) to [99"'Tc(CO)3(OH2)3]+ (tR = 4.9 min) with increasing fluoride
ion
concentrations (method C elution conditions): 260 mM (front), 404 mM (second
from front), 500 nM (second from back), 1215 nM (back).
Fig. 5 shows y-HPLC radiochromatograms showing the conversion of [9smTc04]-
(tR = 11 min) to [99mTc(CO)3(OH2)3]+ (4.9 min) at different pH values (method
C
elution conditions):10-10.5 (front), 9-9.5 (second from front), 8-8.5 (second
from
back), 7-7.5 (back).
Fig. 6 shows [Front] y-HPLC radiochromatogram of the crude reaction mixture
after 3 hours at 85°C; [Middle] y-HPLC radiochromatogram of Sep-Pak~
purified
23b; [Back] UV-HPLC chromatogram of the Re-standard (method A elution
conditions).
Fig. 7 shows formation of 23b as a function of pH and ligand concentration for
the reaction of 1 b with [99"'Tc(CO)3(OH2)3]+ after 3 hours at ligand
concentrations
of: (~) 10-4 M, (~) 10-3 M, and (~) 10-2 M.
Fig. 8 shows y-HPLC radiochromatograms showing the formation of 23b at: [A]
mM NaF, [B] 50 mM NaF, [C) 100 mM NaF, [D] 500 mM NaF, and [E] 1000
mM NaF (pH z 12; [L] = 10-3 M) (method A elution conditions).
Fig. 9 shows [A] y-HPLC radiochromatogram of the crude reaction mixture after
90 minutes; [B] y-HPLC radiochromatogram of Sep-Pak~ purified 5; [C] UV-
chromatogram of the Re-standard (method A elution conditions).
DETAILED DESCRIPTION OF THE INVENTION
The present inventors believed that the unacceptably low yields of 99Tc-
carborane complexes prepared in water in the presence of KOH or Na2C03 and
[9 1 C(CO)3Br3]2- obtained at the tracer level might have been due to the
highly
CA 02515495 2005-08-09
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basic reaction conditions which can cause decomposition of
[99"'TC(CO)3(OH2)3]+
prior to complex formation. In light of these results, a search for a base
that
would remove the bridging hydrogen on the carborane, which is needed for
efficient complexation, but that would not cause premature decomposition of
the
Tc starting material was initiated.
Fluoride ion (pKb ~ 10.8) is one of the few bases that does not react with
the M(CO)3+ core (M = Re, Tc), making it a plausible candidate for preparing
metallocarboranes in aqueous solutions (Salignac, B.; Grundler, P. V.;
Cayemittes, S.; Frey, U.; Scopelliti, R.; Merbach, A. E.; Hedinger, R.;
Hegetschweiler, K.; Alberto, R.; Prinz, U.; Raabe, G.; Ko"lle, U.; Hall, S.
Inorg.
Chem. 2003, 42, 3516.). Bases that are commonly used to prepare
metallocarboranes (n-BuLi, NaH, TIOEt, and NaOH) [(a) Hawthorne, M. F.;
Andrews, T. D. J. Am. Chem. Soc. 1965, 87, 2496. (b) Ellis, D. D.; Jelliss, P.
A.;
Stone, F. G. A. Organometallics 1999, 18, 4982.] are much stronger and softer
than fluoride; consequently, the question remained as to whether or not an
aqueous fluoride solution would be able to generate sufficient amounts of the
deprotonated nido-carborane, otherwise referred to as the nido-dicarbollide
dianion, to afford good yields of the desired products. To probe the
feasibility of
using fluoride as a base, the nido-carborane ligands 1a and 1b were reacted
with
[NEt4]2[Re(CO)3Br3] in the presence of 100 mM KF at 85 °C (Scheme 1).
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Scheme 1
CO
1- ~'3C: v CO
H "~,~ ~ tit
Ki', ~-i.,t~. ~' ..
f:%f -~. y,~G..
/Mf,CCJ ~~t~OH),,~.
IaR~R 2aR=H,P.i--R~
1lJ R = ,~rtv.,.~r~'~.~~~~'1 i.L1 R = Ci~'-~,r~.r~'i~u:~k.~~. iYl = ~?~
1C R - CH~:H .C;HFt~tt~l~ 20 R = CH ~~H,~t'i~tetHtwl~l. M ~: R
~d R ~ ~i-i~(;tlaCO.,h-~, hfl = <~~~:~C
It has been previously demonstrated that these particular carborane
ligands, under strongly basic conditions, react with the Re(CO)3+ core to give
the
corresponding rl5-metal complexes in good yield. After heating, the desired
products, 2a and 2b, were obtained in 34% and 50% isolated yields,
respectively.
In the case of the reaction involving 1 b, analysis of the crude reaction
mixture by
HPLC indicated a yield of greater than 80%; however, difficulties were
encountered in separating the different salts of 2b, which in turn reduced the
overall isolated yield. The yield of the unsubstituted carborane was also
compromised by the desire to isolate a single salt and by the low solubility
of the
Iigand in aqueous KF. Nonetheless, the yields of the Re-metallocarboranes were
still an improvement over those values reported for the direct synthesis of
CpRe(CO)3 type complexes in water. One major advantage to using fluoride as
a base is that it does not promote the formation of polynuclear hydrolysis
products. [Re(CO)3(OH2)3]+, which is formed when [Re(CO)3Br3]2- is dissolved
in
dilute aqueous solutions, is stable below pH 6. In the presence of hydroxide
ion,
deprotonation of the metal bound water molecules occurs, which in turn leads
to
the formation of Re clusters like [Re3(CO)g(,u2-OH)3(,u3-OH)]- [(a) Alberto,
R.; Egli,
A.; Abram, U.; Hegetschweiler, K.; Gramlich, V.; Schubiger, P. A. J. Chem.
Soc.,
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Dalton Trans. 1994, 2815. (b) Egli, A.; Hegetschweiler, K.; Alberto, R.;
Abram,
U.; Schibli, R.; Hedinger, R.; Gramlich, V.; Kissner, R.; Schubiger, P. A.
Organometallics 1997, 16, 1833.] Until now, the formation of these metal
clusters complicated the use of ligands, like carboranes, which require the
removal of weakly acidic protons prior to complexation. For example, when
compound 1c, which is a carborane analogue of the monoamine oxidase-B
(MAO-B) inhibitor N,N-dimethyl-3-phenylpropylamine (Ding, C. Z.; Lu, Z.;
Nishimura, K.; Silverman, R. B. J. Med. Chem. 1993, 36, 1711.), was reacted
with [NEt4]2[Re(CO)sBr3] in the presence of aqueous NaOH (pH 12), the major
products were a series of rhenium clusters. When 1c and [NEt4]2[Re(CO)3Br3]
were combined in the presence of 100 mM KF and the mixture was heated for 13
h, analysis of the crude reaction mixture by electrospray mass spectrometry
showed only the desired product and no evidence of any cluster formation. The
pH of the reaction mixture was subsequently adjusted to 5 by the dropwise
addition of 1 M HCI, and the product 2c, as the internal salt, was isolated in
good
yield (70%). Compound 2c, which is a novel metallocarborane derivative, is
stable in the solid state and in solution. The IR shows the characteristic B-H
stretch at 2526 cm-', which is not significantly shifted from that of the
ligand. The
CO peaks appear at 2006 and 1899 cm-' with relative intensities that are
consistent with the local symmetry of the metal complex. The electrospray mass
spectrum showed the predicted molecular ion having the appropriate isotope
distribution while'H, "B, and'3C NMR spectra were consistent with the
structure
of the target compound.
Accordingly, the present invention relates to a method of preparing metal-
carborane complexes comprising reacting a salt of the formula:
[M(CO)3(Xm)3]~~ + sm)
wherein M is selected from a radioisotope of rhenium (Re), technetium (Tc) and
any other radioisotope binding in the same fashion, X is the same or different
and
is, independently, any suitable ligand and m is the formal charge for ligand
X,
CA 02515495 2005-08-09
with a nido-carborane in the presence of a hard base.
In embodiments of the present invention, the nido-carborane used can be
an ortho, para or meta isomer. An example using a meta isomer is given in
Scheme 2.
Scheme 2
~ _ o co
H ~ ~II OC .. C
H . C OMe KOH, EtOH, H20 H C. C- v _OH [M(CO)3(OHZ)~]r, NaF C M C OH
H
reflux, 12 hrs ~85°C, 1.5 h
M = ssmTc
5
It was also found that it is possible to obtain the desired metal-carboranes
as previously defined by using the closo-carboranes directly as an alternative
to
the use of the isolated nido-carboranes. In fact, it has been found that it is
possible to prepare, one pot, the desired metal-carboranes directly form the
closo-boranes.
Accordingly, the present invention also relates to a method of preparing
metal-carborane complexes comprising reacting a salt of the formula:
~M(C~)3(Xm)3~~~ +3m)~
wherein M is selected from a radioisotope of rhenium (Re), technetium (Tc) and
any other radioisotope binding in the same fashion, X is the same or different
and
is, independently, any suitable ligand and m is the formal charge for ligand
X,
with a c/oso-carborane in the presence of a hard base.
This approach offers a number of advantages. In fact, it represents a
reduction in the number of steps necessary to prepare the desired compounds
and it eliminates one of the counterions present in solution.
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11
As shown in Scheme 4, compound 2b was obtained directly from the
closo-carborane 6. As comparison, this compound was also prepared according
to the process defined in Scheme 3.
Scheme 3
NEt4' O~ NEt4.
C\ ~ H ~ OC M C
OH TEAF, THF C.C OH TEAF(aq), EtOH, 100_°C ~ C OH
80°~ H [NEtd)z(M(CO)38ra), 19 h H
g ~b 2b
Scheme 4
NEt4'
CO
O OC = C
C I I MM
~C~OH TEAF, EtOH/H20 C.C OH
H
(NEt4]z[M(CO)3Br3], 100°C
M = Re 2b
6
In embodiments of the present invention, the salts of the formula
[M(CO)3(X"')3~(~ + sm) include those where M is selected from a radioisotope
of
rhenium, technetium and any other radioisotope binding in the same fashion. A
person skilled in the art would be able to determine which radioisotopes bind
in
the same fashion as Tc and Re. Examples include radioisotopes of Rh, Cr, Mo,
Mn, Os, Ir and Ru. The ligand "X"'" may be the same or different (i.e. there
may
be three different "X" ligands, or one different "X" ligand and two the same
or all
three "X" ligands may be the same) and is, independently, any 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/or H20 (m = 0). A person skilled in the art would
know
CA 02515495 2005-08-09
12
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,
RCN and RNC, wherein R is an alkyl (including cycloalkyl) or aryl group, for
example methyl, ethyl, n-butyl, t-butyl and phenyl or R is any biomolecule.
The
present invention extends to cover the use of salts of the formula
jM(CO)3(X"')s]~'
+ 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]~~ +
3m)
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. In an embodiment of the invention, M is selected from
radioisotopes of Re and Tc and all three X ligands are H20 or Br .
The salts of the formula [M(CO)s(X"')s]~' + 3"'> may be prepared using
methods known in the art. For example, salts of the formula [M(CO)3Br3]2~ (M =
Re, 9sTc) may be prepared as described in Alberto et al. (Alberto, R.;
Schibli, R.;
Egli, A.; Schubiger, P.A.; Herrmann, W.A.; Artus, G.; Abram, U.; Kaden, T.A.
J.
Organomet. Chem. 1995, 493, 119-127.) 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-, para- and meta-carborane. 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.
CA 02515495 2005-08-09
13
In order to be useful as a radiopharmaceutical ligand, it is desirable that a
means to conjugate the carborane-M(CO)3 unit to targeting biomolecules be
available. To this end one or more linker moieties may be incorporated into
the
nido-carborane. Accordingly, the terms "nido-carborane" and "closo-carborane"
include those derived from unsubstituted and substituted carboranes, in
particular carboranes in which a linker group has been attached to one or more
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
terms
"nido-carborane" and "closo-carborane" further include those derwed from
carboranes with one or more linker groups with a biological targeting molecule
attached thereto.
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,
an
aldehyde or other electrophilic group on a biological targeting ligand.
Examples of biological targeting ligands include, but are not limited to,
small molecules having specificity for a specific receptor, immunoproteins,
oligopeptides, sugars, cocaine analogues, oligopeptides and polypeptides such
as epidermal growth factor. The applications of radiolabelled boron clusters
to
the diagnosis and treatment of cancer has been recently reviewed (Hawthorne,
M.F.; Varadarajan, A.; Knobler, C.B.; Chakrabarti, S. J. Am Chem. Soc. 1990,
112,
CA 02515495 2005-08-09
14
5365.).
The terms "nido-carborane" and "closo-carborane" further include 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 publications: Valliant et al. J. Org. Chem. 2002, 67, 383-387, and
Valliant, et al., Coord. Chem. Rev. 2002, 232, 173-230. and references cited
therein, the contents of which are incorporated herein by reference.
In an embodiment of the present invention, the nido-carborane is selected
from compounds 1 a, 1 b and 1 c. The present invention also includes the novel
nido-carborane 1c and its corresponding metal carborane complex 2c and 2d. It
also includes the novel nido-carborane 9 and 20 and their corresponding metal
carborane complex 10 and 21.
In another embodiment of the present invention, the closo-carborane is
selected from compounds 3, 6, 8, 11, 12, 15, 17 and 19.
The nido-carboranes can be prepared using procedures known in the art.
For example, from the corresponding ortho-, para- and meta-carboranes by a two
step process involving deboronation, to provide the dicarba-nido-undecaborate,
(for example, [nido-(C2B9H~2)]-), followed by deprotonation using a hard base.
The deboronation reaction may be effected using a base under a variety of
conditions [(a) Barth, R.F.; Adams, D.M.;Soloway, A.H.; Mechetner, E.B.;Alam,
F.; Anisuzzaman, A.K.M. Anal. Chem. 1991, 63, 890. (b) Imahori, Y.; Ueda, S.;
Ohmori, Y.; Sakae, K.; Kusuki, T.; Kobayashi, T.; Takagaki, M.; Ono, K.; Ido,
T.;Fujii, T. Jpn Clin. Cancer Res. 1998, 4(8), 1833.]. For example, ortho-
carborane, para-carborane or meta-carborane may be heated to reflux with
CA 02515495 2005-08-09
potassium hydroxide in an alcoholic solvent, such as ethanol. Other bases that
can be used include secondary amines, such as pyrolidine, and fluoride. The
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.
The base used for the deprotonation of the dicarba-nido-undecaborate, is
suitably a hard base. "Hard" and "soft" are well known terms to describe acids
and bases, known as the Hard Soft Acid Base (HSAB) Theory. A person skilled
in the art would readily recognize bases that are classified as hard and soft.
Hard bases such as fluoride do not react with the soft acid technetium (or
rhenium) centre. Hard bases such as fluoride will form hydrogen bonds with the
metal bound water but will not deprotonate them because they are not strong
bases and therefore will not promote premature degradation of the M(CO)3+
core.
Suitable hard bases include, but are not limited to, 02-, CI-, F-, CH3C00-,
N03 ,
C104 , S042-, NH3 and RNH2, wherein R is any suitable alkyl (including
cycloalkyl)
or aryl group, for example methyl, ethyl, butyl, t-butyl, phenyl and the like.
In a
further embodiment of the present invention, the base is a source of fluoride
(F-),
for example, Z+F-, wherein Z+ is any suitable cation, for example K+.
The term "about" as used herein means within experimental error.
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,
Hawthorne, M.F.; Maderna, A. Chem. Rev. 1999, 99, 3421-3434 or Hawthorne,
M.F.; Varadarajan, A.; Knobler, C.B.; Chakrabarti, S. J. Am Chem. Soc. 1990,
112,
5365.). For example, ortho-carboranes are readily synthesized from the
reaction
of an appropriately substituted acetylene with various nitrite and sulfide
adducts
of decaborane (B~oH~4) (Grimes, R.N. Carboranes, Academic Press, N.Y. , 1970;
Bregadze, V.I. Chem. Rev. 1992, 92, 209.). 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
CA 02515495 2005-08-09
16
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. The preparation of compounds 1a and 1b has
been previously described (Valliant, J. F.; Morel, P.; Schaffer, P.; Sogbein,
O.O.
U.S. Patent Application Publication No. 2003-0668271, published April 10,
2002).
The preparation of the metal-carborane complexes may be effected by
reacting a nido-carborane with a salt of [M(CO)3(X"')s]~' + 3"'> in aqueous
solutions.
It is an embodimenfi of the present invention that the reaction is carried out
in an
aqueous solution of the hard base, for example aqueous KF, suitably 100 mM
aqueous KF. The reaction mixture may be warmed and allowed to proceed for a
time period of about 30 minutes to about 48 hours, suitably about 1 hour to
about
24 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 method
involves generating the nido-dicarbollide dianion from the corresponding
dicarba-
nido-undecaborate by treatment with the hard base, for example potassium
fluoruide (KF), in aqueous solution, for example aqueous KF solution, and this
solution is warmed to a temperature of about 60-100 °C, suitably about
80-90 °C,
more suitably about 85 °C, and an aqueous solution of a salt of the
formula
[M(CO)3(X"')3]~' + sm>, for example an aqueous KF solution, and warmed to a
temperature of about 60-100 °C, suitably about 80-90 °C, more
suitably about 85
°C, is added to the solution of the nido-dicarbollide dianion and the
temperature
maintained for the entire reaction time. The reaction may also be carried out
in a
microwave as is well known in the art of radiochemical preparation. In further
embodiments of the present invention, the reactions are performed in an inert
atmosphere, for example under argon (Ar) or nitrogen (N2) gas. In still
further
embodiments of the invention, the reactions are performed at tracer levels. By
CA 02515495 2005-08-09
17
"tracer levels" it is meant that the amount of radiolabeled substances is such
that
it does not have an effect on the system under study. Typical tracer levels
are,
for example, in the range of
10-6 to 10'2 M. The desired product may be purified by any known means,
suitably using high performance liquid chromatography (HPLC). As previously
indicated, the preparation of the metal-carborane complexes can alternatively
be
carried out by using the corresponding closo-carboranes instead of the nido-
carboranes.
With the success of the reactions involving rhenium, attempts were made
to label the bifunctional ligand 1 b with ~99"'TC(CO)3(OH2)3~+, which was
prepared
using commercially available carbonyl labeling kits. 99"'TCO4 (370-740 MBq; 10-
20 mCi) was added to the kit and [99mTC(CO)3(OH2)3]+ prepared according to the
commercial protocol. After reacting a ligand/KF mixture with
[99"'TC(CO)3(OH2)3]+,
surprisingly, only a small quantity of the desired product was obtained (5%
radiochemical yield). After 6 h of heating, the main reaction constituents
were
unreacted starting materials and 99""Tc04 .
Commercially available kits contain sodium tartrate to prevent premature
decomposition of [99mTc(CO)3(OH2)3]+. Thinking that the chelate was
interfering
with the fluoride mediated complexation reaction, a kit in which KF was
substituted for tartrate was prepared. [99"'Tc(CO)3(OH2)3)+ was successfully
prepared using the "KF kit" formulation (see Example 4) in comparable purity
and
yield to the product from the commercial kit. It should also be noted that
~99mTc(CO)3(OH2)3)+ prepared in the presence of KF was stable for greater than
6
h, which is comparable to the stability of the product from the tartrate
formulation.
Accordingly, the present invention also includes kits for use in the
preparation of the salts of formula [M(CO)3(X"')3]~' + sm>, wherein M is a
radioisotope of Tc or Reand X is H20, comprising potassium boranocarbonate
(K2H3BC02), Na2B40~.10H20, a hard base and a suitable buffering reagent, for
example Na2C03. In an embodiment of the invention, the amounts of K2H3BC02,
CA 02515495 2005-08-09
18
Na2B40~.10Hz0, base and buffering reagent present are in the ratio of about
8:1:6:5. In a further embodiment of the invention, the hard base is a source
of F-.
In a typical preparation, an aqueous solution of M04 , wherein M is a
radioisotope of Tc, for example 99mTc or Re, is added to the kit via syringe,
suitably at elevated temperatures, for example about 60-100 °C, more
suitably
about 65-75 °C, under an inert atmosphere over a period of about 10
minutes to
about 2 hours, suitably about 30 minutes to 1.5 hours, more suitably about 1
hour, to generate the desired salt of the formula[M(CO)s(X"')3]~' + 3m),
wherein M is
a radioisotope of Tc or Re and X is H20. The reaction may also be performed in
a microwave.
In further embodiments of the invention, the kit ingredients are packaged
in a suitable container, for example, a glass vial with a rubber stopper as a
top,
and the container is sold, optionally with directions for use.
Compound 1 b, in the presence of KF, was added to [99"'Tc(CO)3(OH2)3]+
and the mixture heated for 3 h. The crude y~HPLC of the reaction mixture
showed
an appreciable amount of the desired product and only small amounts of
residual
starting material and 99"'TCOq . The metallocarborane 2d was obtained in 80%
isolated yield following purification using a C~$ Sep-Pak. The Sep-Pak
procedure
was able to separate 99"'Tc04 and the excess carborane ligand used during the
labeling reaction from the desired product. The ~HPLC traces of the crude
reaction mixture at 3h, the purified product 2d, and the UV trace of the Re
standard 2b are shown in Fig. 1. The small difference in retention times
between
the reference standard and the product is associated with the distance between
the UV and y detectors which are connected in series.
To demonstrate the stability of 2d, the purified product was incubated
separately with a 1000-fold excess of cysteine and histidine. Ligand challenge
experiments are routinely used in radiopharmaceutical chemistry to determine
the likelihood of a compound remaining intact in vivo where there is an
abundance of competing thiol and amine ligands. After incubation at 37
°C in
CA 02515495 2005-08-09
19
phosphate buffered saline (pH = 7.2) for 24 h, the radiochromatograms from
both
experiments indicated that greater than 94% of the product remained unchanged
(Fig. 2). These results strongly suggest that the 99mTc metallocarborane
complexes are sufficiently robust to be used as synthons for preparing
radiopharmaceuticals.
The mechanism of the fluoride mediated reaction may not necessarily be a
simple acid-base reaction given the weak basicity and acidity of KF and the
bridging hydrogen on the carborane, respectively. While not wishing to be
limited
by theory, two plausible alternatives could be (1) initial ~3-coordination of
the
M(CO)3+ core to the nido-carborane thereby causing a concomitant increase in
the acidity of the bridging hydrogen or (2) the presence of KF in solution
could
generate small quantities of HF, which react with a boron hydride leading to
the
formation of hydrogen gas and the dicarbollide dianion. The exact details of
the
mechanism notwithstanding, the reported experiments clearly demonstrate that a
hard base such as fluoride, unlike hydroxide ion, does not cause premature
decomposition of [M(CO)3(OH2)3]+ (M = 99"'Tc, Re) thereby allowing for the
preparation of metallocarboranes at both the macroscopic and tracer levels in
water.
As shown in Scheme 3, compound 6 was treated with TEAF in wet THF
following the methodology developed by Fox et al. Polyhedron 1997, 16, 2499.
Compound 1b was isolated in 64% yield following chromatographic purification
which was needed to remove unreacted starting material. Compound 1b was
subsequently reacted with [NEt4]2[Re(CO)3Br3] in a 500 mM solution of TEAF in
water/EtOH and the mixture heated to reflux. After approximately 19 hours the
product, compound 2b, was isolated in 61 % yield. As shown in Scheme 4,
compound 6 was combined with a slight excess of [NEt4]2[Re(CO)3Br3] in a
solution of 500 mM TEAF containing a small quantity of absolute ethanol. The
heterogeneous suspension was heated at 100°C and after 30 hours TLC
CA 02515495 2005-08-09
indicated complete consumption of 6. Extraction followed by chromatography
lead to isolation of the desired product in 70% yield.
To determine if substituents bearing good donor atoms would impact
complexation yields, a pyridine substituted carborane was prepared following a
literature procedure ((a) Wang, X.; Jin, G.-X. Organometallics 2004, 23, 6319
(b)
Alekseyeva, E.S.; Batsanov, A.S.; Boyd, L.A.; Fox, M.A.; Hibbert, T.G.;
Howard,
J.A.K.; MacBride, J.A.H.; Mackinnon, A.; Wade, K. Dalton Trans. 2003, 3, 475).
Pyridines, which are excellent ligands for Re(I), have been used to construct
a
number of Re(I) and Tc(I) bifunctional chelators. Pyridine substituted
compounds
have the added attraction that they can also be used to prepare piperidine
derivatives as a means of targeting specific neuroreceptors. The nido-
carborane
9 was reacted with [NEt4]2[Re(CO)3Br3] in the presence of 100 mM KF and the
solution heated to reflux for 24 hours. After the addition of HCI to form the
internal salt, the product was isolated in excellent yield (85%) by extraction
into
dichloromethane (Scheme 5).
Scheme 5
co
H 1 OC '- CO H
H ~ M
C \C N~ KOH, EtOH . N~ 5 1. M CO Br . N~ s
H I I ( )s ~_1z- C.C H
2 /4 p /
heat s KF, 24 hrs, 100°C
2. HCI
g g 10
CA 02515495 2005-08-09
21
The MS of 10 is consistent with the formation of the n5-rhenacarborane
complex as opposed to a compound in which Re is coordinated to the pyridine
nitrogen. The 'H NMR showed some minor shifts in the aromatic region of the
NMR spectrum compared to that for the starting material, which is expected
given the proximity of the pyridine ring to the carborane cage. The 'H NMR did
not show any evidence of the bridging H-atom on the cluster which further
supports our hypothesis that metal complex resides on the carborane. The
~'B{'H}NMR of 10 showed six peaks with some overlapping signals that are for
the most part shifted to higher frequency compared to that of the starting
material.
The higher yield of 10 with respect to the other Re-carborane complexes
(vide infra) may be associated with the formation of a kinetic product
involving
coordination of the pyridyl group to rhenium, which helps prevent the
formation of
metal clusters. This is analogous to the addition of co-ligands to
formulations for
preparing Tc(~ complexes as a way of preventing the formation of Tc02. A
further advantage is gained if coordination to pyridine takes place initially
in that
the intermediate complex would situate the fac-[Re(CO)3]+ core in close
proximity
to the open C2B3 face of the cluster. Alternatively, the pyridine nitrogen may
simply facilitate deprotonation of the bridging H-atom during the complexation
process. One clear practical advantage is the ability to form the internal
salt of 10
which avoided problems associated with the presence of different counter ions.
A carborane bearing a pendent tertiary amine was also prepared and the
corresponding [Re(CO)3]+ complex generated. The target Re metallocarborane
was prepared from the dimethylamino nido-carborane ligand 1c (Scheme 6),
which was synthesized in 58% yield by reacting the iodoalkyl-carborane 11 with
an ethanolic solution of dimethylamine overnight at room temperature.
Compound 1c and [NEt4]2[Re(CO)3Br3] were then combined in 500 mM aqueous
KF and the resultant heterogeneous mixture heated to 100°C. After 13
hours, the
product was isolated as an internal salt by adjusting the pH to 3 by the
dropwise
CA 02515495 2005-08-09
22
addition of HCI. The product was subsequently purified by column
chromatography through silica gel to give 2c in 72% yield.
Scheme 6
co
OC = CO
H H ~ Re H
~I -H I 1) [Re(CO) Br ]z' ~H I
HNMe2 NMez ~' C.C NMez
a U
KF, 100°C
EtOH
2) HCI
11 1~ 2c
The FTIR of 2c showed the characteristic C---O stretches at 2005 and
1899 cm-1 while the B-H stretch (2526 cm-1) was not significantly shifted from
that
of the free ligand. The 1H NMR revealed that the methylene protons directly
adjacent to the cage are diastereotopic and appear as two distinct multiplets
at
1.53 and 1.68 ppm. The protons in the methylene group adjacent to the amine in
contrast are homotopic. The presence of the protonated amine is evident in
that
the N-methyl groups, which appear at 3.18 ppm, are split into a doublet from
the
adjacent N-H group. The amino N-H proton appears as a broad singlet at 5.70
ppm. Its identity was confirmed by adding a small quantity of CD30D to the NMR
sample which caused the resonance to disappear due to exchange.
X-ray quality crystals of 2c (see Fig. 3) were obtained using a 1:1 (v/v)
solution of dichloromethane and methanol. The structure exhibits the tripodal
fac-
[Re(CO)s]+ core with one CO ligand nearly eclipsing the carborane C-H bond in
the solid state (Fig. 3). The aliphatic chain almost completely bisects the OC-
Re-CO bond angle and extends away from the carborane cage. The average
Re-B bond distance is 2.322(14) A which is identical to the Re-Ccage distance
(2.32(14) A). Crystallographic data for 2c is summarized in Tables 1 and 2.
CA 02515495 2005-08-09
23
Table 1. Crystal and structure refinement data for 2c
Empirical formula C~oH23BsN03Re
Formula weight 488.78
Temperature 173(2) K
Wavelength 0.71073 A
Crystal system Orthorhombic
Space group P2~2~2,
Unit cell dimensions a = 8.982(2) A a = 90.
b = 11.563(3) A [i = 90.
c=16.811(4)A y=90.
Volume 1746.1 (7) A3
Z 4
Density (calculated) 1.859 Mg/m3
Absorption coefficient 6.966 mm-'
F(000) 936
Crystal size 0.07 x 0.06 x 0.01 mm3
B range for data collection2.14 to 24.00.
Index ranges -10<_h<_9,-13<_k<_13,-19<_I<_19
Reflections collected 11192
Independent reflections 2709 [R(int) = 0.1363]
Completeness to A = 24.00 99.2
Absorption correction semi-empirical based on equivalents
Refinement method Full-matrix least-squares on
F2
Data / restraints / parameters2709 / 39 / 138
Goodness-of-fit on F2 1.102
Final R indices [I>2E(I)] R1 = 0.0684, wR2 = 0.0896
R indices (all data) R1 = 0.0982, wR2 = 0.0959
Absolute structure parameter0.01 (3)
Extinction coefficient 0.00076(15)
Largest diff. peak and 1.929 and -2.593 e.A-3
hole
CA 02515495 2005-08-09
24
Table 2 Selected bond lengths and angles for compound 2c
Re(1 )-C(3)1.845(17)
Re( 1 )-C(1.907(
1 ) 16)
Re(1)-C(2)1.962(17)
Re(1)-B(4)2.275(17)
Re(1)-B(7)2.33(2)
Re(1)-C(2')2.34(2)
Re(1)-C(1')2.348(19)
Re( 1 )-B(2.361
11 ) ( 19)
C(1)-O(1) 1.168(17)
C(3)-O(3) 1.183(18)
C(2)-O(2) 1.115(17)
C(2')-C(1')1.73(2)
C(1')-C(1A)1.54(2)
C(3A)-N(1)1.522(18)
C(5A)-N(1)1.497(19)
N(1 )-C(4A)1.489(18)
C(3)-Re(1)-C(1)86.6(7)
C(3)-Re(1)-C(2)92.7(7)
C(1)-Re(1)-C(2)88.6(7)
C(3)-Re(1 )-C(2')82.2(7)
C(1)-Re(1)-C(2')140.2(6)
C(2)-Re(1)-C(2')129.8(7)
C(3)-Re(1)-C(1')111.1(7)
C( 1 )-Re( 161.6(7)
1 )-C( 1')
C(2)-Re(1)-C(1')95.4(7)
C(2')-Re(1)-C(1')43.4(6)
O(1)-C(1)-Re(1)174.4(19)
O(3)-C(3)-Re(1)176.4(15)
O(2)-C(2)-Re(1)174.2(14)
C(1A)-C(1')-C(2')124.1
(15)
C(1A)-C(1')-Re(1)109.9(11)
C(2')-C(1')-Re(168.1 (10)
)
C(1')-C(1A)-C(2A)115.3(12)
C(2A)-C(3A)-N(1)111.4(11)
C(4A)-N(1)-C(5A)109.4(13)
C(4A)-N(1)-C(3A)111.3(10)
C(5A)-N(1)-C(3A)111.3(12)
CA 02515495 2005-08-09
Compound 12, which was prepared following literature methods
(Giovenzana, G. B.; Lay, L.; Monti, D.; Palmisano, G.; Panza, L.; Tetrahedron
1999, 55, 14123), was converted to the corresponding nido-caborane as both
the potassium and TEA salts. Formation of the potassium salt involved treating
compound 12 with KOH in ethanol and heating the mixture to reflux for 12
hours.
This method also resulted in the simultaneous deprotection of the acetate
esters
on C-2, 3, 4 and 6. The nido-carboranyl glucose derivative 13a could then be
extracted into methanol, ethanol, acetone or tetrahydrofuran thereby
separating
the product from residual salts. Further purification was accomplished using
silica
gel column chromatography and the desired product, 13a, was obtained as a
glassy solid in 83% yield (see Scheme 7).
Scheme 7
H OAc H OH
HO a s HO _
Ac0 O 1) KOH, EtOH, reflux HO s p s H
Ac0 ~ ~~. , 2) COx(9) HO ~ 2 ~ i -CSC
OAc H 3 H OH H
3) 1M HCI (pH=4) H
H 12 H 4) [NEt4]Br (13b only) H
13a (K+ salt)
13b (NEt4 salt)
CO _
H OH OC CO I
HO M
[NEt4]2[M(CO)3Br3], TEAF HO ~ O~C.
HO C. ,
100°C, 7 d H O~ H
H 14 H
M=Re
The 1H NMR of 13a showed the existence of two diastereomers, which
arise as a consequence of the fact that during degradation of mono-substituted
ortho-carboranes two enantiomers (diastereomers in the case of 13a) are
formed. Two distinct signals for the anomeric proton were detected at 4.51 and
4.41 ppm while two pairs of doublets arising from the protons of the C-1
CA 02515495 2005-08-09
26
substituent group were also observed. The formation of the nido-carborane was
evident in that there was a broad signal at -2.5 ppm which is associated with
the
hydrogen atom that is bound to the open C2B3 face of the nido-carborane cage.
The '3C NMR spectrum of 13a also indicated a mixture of diastereomers. For
instance, there were two signals associated with the anomeric carbon atom at
103.41 and 103.01 ppm and pairs of signals corresponding to the C-3 and C-5
carbon atoms.
Compound 13a and [NEt4]2[Re(CO)3Br3] were combined in 1.0 M aqueous
KF and the reaction mixture heated to reflux for 24 hrs. The mass spectrum
showed the presence of the ligand mass and the target mass, plus some
rhenium cluster species which appeared at m/z = 590 and 633, and at 878.
Reduction of the concentration of KF to 0.1 M in subsequent reactions appeared
to eliminate these undesired products however LC-MS analysis as a function of
time showed only very small quantities of the product after 48 hours. After a
period of seven days, the peak corresponding to the starting material had
diminished almost completely, while the peak corresponding to 14 increased
accordingly. The reaction lead to the formation of K~ and NEt4+ salts of the
desired complex, which were unfortunately inseparable. To simplify
purification,
the NEt4+ salt (13b) was prepared and the reaction repeated using TEAF as the
base. Semi-preparative HPLC was employed to isolate pure 14 in 16% yield. The
low yield of the target was somewhat surprising given that the analytical HPLC
of
the crude reaction mixture indicated a much higher yield than what was
actually
isolated.
The IR spectrum 14 featured the characteristic O-H stretch at 3425 cm'',
B-H stretches at 2537 cm-' and C°O stretches at 1999 and 1898 cm-
'. The
electrospray mass spectrum of the purified product showed the target mass with
an isotopic distribution characteristic of a ReB9 cluster. The 'H and '3C NMR
spectra, interestingly, appeared to indicate the formation of unequal amounts
of
the two diastereomers of 14. The anomeric doublets at 4.28 and 4.19 ppm for
CA 02515495 2005-08-09
27
example appeared with integration ratios of approximately 10:1 in favour of
the
lower frequency signal. The '1B{1H} NMR spectrum of 14 showed 7 signals,
which appeared at -5.82, -7.65, -8.78, -11.62, -18.35, -19.55, and -20.13 ppm,
with the peaks at -8.78 and -11.62 ppm consisting of two overlapping signals.
The signals in the ~~Bf'H} spectrum for 14 were shifted to higher frequency
versus those in 13b.
The long reaction time needed to achieve reasonable yields of 14 could be
the result of the formation of an intermediate complex between the rhenium
tricarbonyl core and multiple glucose hydroxyl groups. It is reasonable to
expect
that the products) of the rhenium core and the glucose hydroxyl groups would
form at a rate that is faster than the formation of the metallocarborane.
Separate
attempts to isolate a Re-glucose complex however were unsuccessful. With
respect to the observed isomer ratio, stereoselectivity in the complexation
reaction is improbable. It is more likely that one isomer was enriched during
HPLC purification.
One of the attractive features of carboranes is that they can be derivatized
at both of the cage carbon atoms selectively as a means of preparing unique
targeting agents. Endo et al. have utilized this feature to prepare a series
of
diphenyl substituted carboranes as estrogen agonists and antagonists ((a)
Endo,
Y.; lijima, T.; Yamakoshi, Y.; Fukusawa, H.; Miyaura, C.; Inada, M.; Kubo, A.;
Itai,
A.; Chem. Biol. 2001, 8, 341 (b) Endo, Y.; lijima, T.; Yamakoshi, Y.; Kubo,
A.;
Itai, A. Bioorg. Med. Chem. Lett. 1999, 9, 3313). The metallocarborane
complexes of related analogues could serve as a novel class of inorganic
antiestrogens ((a) Le Bideau, F.; Salmain, M.; Top, S.; Jaouen, G. Chem. Eur.
J.
2001, 7, 2289 (b) Jaouen, G.; Top, S.; Vessieres, A.; Alberto, R. J.
Organomet.
Chem. 2000, 600, 23 (c) Jaouen, G.; Top, S.; Vessieres, A.; Leclercq, G.;
McGlinchey, M.J. Current Med. Chem. 2004, 11, 2505) or as radiotracers for
imaging estrogen receptor positive tumours (Mull, E.S.; Sattigeri, V.J.;
Rodriguez,
A.L.; Katzenellenbogen, J.A. Bioorg. Med. Chem. 2002, 10, 1381.) One important
CA 02515495 2005-08-09
28
consideration for disubstituted derivatives is that the steric hindrance could
reduce the yields of the Re complexes. As a consequence, a model compound
was prepared and its reactivity towards complexation with the [Re(CO)s]+ core
investigated.
Compound 15 was prepared following literature procedures and the synthesis
of the target metal complex 16 carried out directly from the closo-carborane
(Scheme
8). The reaction was performed at reflux using an excess of Re and 500 mM
sodium
fluoride. After two days, HPLC showed complete consumption of 15 with the
target
compound being the major product. Compound 16 was obtained via silica gel
chromatography as a yellow oil in 51 % yield.
Scheme 8
\ OH CO~ Na' OH
OC M COI \
C MCO1 HO1.~
~3~ 2 ~,1~
NaF, H,,O, heat I \
M=Re
15 16
~= BH
The IR and mass spectra of the Re complex are consistent with the
proposed structure of 16. The 1H NMR spectrum is relatively uncomplicated and
shows that the each of the aromatic protons exist in a unique environment. In
contrast, the 13C NMR spectrum showed multiple environments for most carbon
atoms. This is not unexpected as showed that from an orbital overlap
perspective, the most favorable conformation of the aryl rings is parallel to
the
binding face of the carborane ((a) Lewis, Z.G.; Welch, A.J.; J. Organomet.
Chem.
1992, 430, C45 (b) Robertson, S.; Ellis, D.; McGrath, T.D; Rosair, G.M.;
Welch,
A.J. Polyherdron 2003, 22, 1293.). In diaryl carboranes however, interaction
between the rings prevents a parallel arrangement. As such, the rings can
adopt
multiple 8 values between 5 and 40° where A is defined as the angle
between the
CA 02515495 2005-08-09
29
plane made by the ring and the plane defined by the two carbon vertices and
the
bond to the substituent.
There are three isomeric forms of dicarbaclosododecaborane, which differ
in the relative positions of the carbon atoms in the cluster. Sandwich
complexes
of nido-carboranes derived from the ortho-isomer are widespread while
analogous complexes derived from meta-carboranes are comparatively less
common. Investigating complexation reactions with the meta isomer, [nido-7,9-
C2BgH~2J- is important because the bonding face of the carborane has a smaller
dipole than in the case of [nido-7,8-C2B9H~2]-. This difference could result
in more
stable metal complexes and higher yields of the desired product. Furthermore,
the different relative positions of the carbon atoms in the cluster offers a
way to
vary the spatial orientations of targeting entities attached to disubstituted
carboranes in order to achieve favourable receptor binding interactions.
To determine if the fluoride mediated reaction would work with the meta
isomer, the ester 18 (Scheme 9) was prepared and reacted with the [Re(CO)3]+
core. Substituted meta-carboranes are prepared by deprotonating one of the CH
vertices of the cluster followed by treatment with an electrophile. In the
example
presented here, meta-carborane 17 was treated with n-BuLi followed by methyl
3-bromopropionate (Cai, J.; Nemoto, H.; Singaram, B.; Yamamoto, Y.
Tetrahedron Lett. 1996, 37, 3383). Compound 18 was purified by silica gel
chromatography and recrystallization giving the final product in 46% yield.
The
complexation reaction was carried out using the closo-isomer to allow for
direct
comparison to the synthesis of compound 2b (Scheme 4). Because
saponification of 18 routinely lead to a mixture of the closo and nido acids,
the
methyl ester itself was used for the complexation reaction. The Re complex was
prepared successfully by heating the closo-carborane 18 with
[NEt4][Re(CO)sBr3]
in a solution of 500 mM TEAF(aq)/absolute EtOH (9:1 v/v) at 100°C for
22 hours.
The meta-rhenacarborane 7 was obtained as a brownish coloured solid in 57%
yield after acid hydrolysis of the methyl ester using HCI(aq), which was done
to
CA 02515495 2005-08-09
facilitate direct comparison of the spectral data to that of compound 2b.
Scheme 9
CO ~ ~NEt4
,H COzMe OC M CO
~C 1. nBuLi, Ef20, -10°C ~C~ 1. [NEt4]z[Re(CO)3Br3], TEAF ~COzH
H C. 100°C, 22 h C _C
2. BrCHzCHzC(O)OCH3
reflux, 2h 2. HCI
17 18 (46%) M = Re
The IR spectrum of compound 7 was nearly identical to that of the ortho-
isomer with the primary difference being the position of the two C---O
stretches
which appeared at 2032 and 1915 cm-' in 7 versus 1998 and 1893 cm-' for 2b.
The 'H NMR spectrum of 7 was also similar to compound 2b where the
methylene protons directly adjacent to the carborane cage appeared as two sets
of triplets at 2.22 ppm and 2.09 ppm. The carborane C-H was a broad singlet at
1.74 ppm and the protons from the NEt4+ cation were visible as a
characteristic
quartet (3.48 ppm) and triplet (1.39 ppm). The '3C{'H~ NMR spectrum of 7
showed a single resonance for the C---O carbon atoms at 200.4 ppm which was
not significantly shifted from that of the ortho-analogue (199.9 ppm). As
expected, the "B{'H} NMR spectrum of 7 was significantly different than that
for
2b. The difference in the chemical shift particularly for the highest field
resonances for 2b in comparison to 7 reflect the differences in the frontier
molecular orbitals of the C2B3 bonding faces of the two carborane isomers ((a)
Batsanov, A.S.; Eva, P.A.; Fox, M.A.; Howard, J.A.K.; Hughes, A.K.; Johnson,
A.L.; Martin, A.M.; Wade, K. Dalton Trans. 2000, 3519 (b) Hermanek, S. Inorg.
Chim. Acta. 1999, 289, 20). The stability of the resulting complex, at least
qualitatively, is comparable to that of the ortho-analogue.
CA 02515495 2005-08-09
31
A Cbz-protected hydrazine derivative of meta-carborane 19 (Scheme 10)
was prepared using a modification of our published methodology for the Boc
analogue. The Cbz protecting group was used in place of Boc groups as we
discovered that fluoride in the presence of Re(I) can facilitate deprotection
of the
t-butylcarbamate (Routier, S.; Sauge, L.; Ayerbe, N.; Coudert, G.; Merour, J.-
Y.
Tetrahedron Lett. 2002, 43, 589) even in water, which resulted in the
formation of
mixtures that included hydrazine-Re complexes. The Cbz-protected hydrazine
carborane was subsequently degraded to the corresponding nido-carborane
using TEAF in THF in excellent yield. Compound 20 was reacted with
[NEt4]2[Re(CO)3Br3] in varying amounts of TEAF. IR and MS experiments before
and after purification indicated the presence of the desired product 21.
Scheme 10
NEt,' CO ~ NEta.
Cbz Cbz OCR ,, CO
C~N~NH T~'F H ~, . C~N~NH TEAF, reflux (18 h) H M .,,CAN Nbz Cbz
H '° H
Cbz THF, 80°C Cbz [NEtb]2[M(CO)3Br3]
M=Re
19 2p 21
In another experiment, in order to determine if [Tc(CO)s(OH2)s]+ could be
prepared in the presence of fluoride, and to see if the product would react
with a
carborane ligand, a series of preliminary reactions were performed using 99Tc
(Epmax = 0.294 MeV, t,,Z= 2.11x105 yr). This particular isotope of technetium
allows
reactions to be carried out on a macroscopic scale (i.e. mmol) so that the
products can be fully characterized by conventional methods (NMR, mass
spectrometry etc.). To this end, [99Tc(CO)3(OH2)3]+ was synthesized by
reacting
9sTc04 with a mixture of NaBH4, Na2C03 and KF and heating the reaction to
100°C for 30 minutes (Scheme 11 ). The final concentration of KF in the
reaction
vial was 0.1 M. In a separate vial, compound 1 b was incubated with 0.1 M KF
at
CA 02515495 2005-08-09
32
85°C for one hour and subsequently added via syringe to the vial
containing
[99Tc(CO)s(OH2)3]+ and the reaction progress monitored by HPLC. An important
observation was that pre-incubating the ligand with fluoride prior to
complexation
afforded better yields of the desired product for reasons that at present are
not
obvious.
Scheme 11
0
H ~ ~ -
.C_ v _OH CO
O
NaBHe, CO H 1 b OC
M
C
NaZC03, KF or C
OH
Na[MOa - [M(CO)3(H20)3]' ,
C H
Naz[BH3 COZ], NaF, KF or NaF, 85C
NaZC03, NazB40~ M = saTc
(22a)
M = eamTC (22b)
M
=
aaTc
(23a)
M
=
a9"'Tc
(23b)
The radiochromatogram ((3- detection) of the crude reaction mixture after
14 hours showed a dominant peak at 19.1 minutes, which corresponded to that
for the rhenium analogue. Despite the residual [9sTc(CO)3(OH2)s]+ present in
the
mixture, the product was readily isolated by HPLC in 25% yield. The negative
ion
electrospray mass spectrum was consistent with the target mass while the IR
spectrum showed the expected features. This includes the carboxylic acid O-H
stretch at 3451 cm-', the carborane B-H stretch at 2550 cm-', and the C---O
stretches at 2017 and 1928 cm-'
Based on the success in preparing [99TC(CO)3(OH2)3]+ from NaBH4 in the
presence of fluoride, the reaction was repeated at the tracer level using
99mTcO4 .
HPLC (y-detection) showed that after 30 minutes the desired product was the
main reaction component with unreacted 99mTcOa and [99mTc(CO)3(OH2)3]+ being
the only impurities. Interestingly, extended incubation of the reaction
mixture at
elevated temperatures for over three hours resulted in only a small amount of
decomposition of the technetium starting material. This observation is in
contrast
to [99"'Tc(CO)3(OH2)3]+ prepared in the absence of tartrate and fluoride which
CA 02515495 2005-08-09
33
decomposed to a much greater extent under the same conditions suggesting that
fluoride ion has a unique stabilizing effect on the [Tc(CO)3]+ core.
The commercial kit that is normally used to prepare [99"'Tc(CO)3(OH2)3]+
consists of Na2[BH3~C02], Na/K-tartrate, Na2B40~~10H20 and Na2C03 and the
ratio of each component had been established for optimal formation of the
99"'Tc-
trisaquo species. The key ingredient is the boranocarbonate anion (BH3~C02)2_,
which acts as both the reducing agent (replacing NaBH4), and the in situ
source
of CO (Alberto, R.; Ortner, K.; Wheatley, N.; Schibli, R.; Schubiger, A.P. J.
Am.
Chem. Soc. 2001, 123, 3135). As mentioned previously, when [99TC(CO)3(OH2)3]+
prepared from the commercial kit was combined with a nido-carborane, the
desired metallocarborane was not detected. Varying the reaction conditions,
including the temperature and amount of ligand, did not facilitate the
formation of
the desired products. Because the boranocarbonate kit is so convenient for the
routine preparation of [99"'Tc(CO)3(OH2)3]+ and avoids the need to use carbon
monoxide, attempts were made to develop the equivalent fluoride-based kits.
Tables 3, 4 and 5 contain the results from the various experiments that
were performed to optimize the radiochemical yield of [99mTc(CO)s(OH2)3]+
using
fluoride in place of tartrate. Simply replacing tartrate in the commercial kit
formulation with an equivalent amount of fluoride did not afford good yields
of the
desired product. Increasing the quantity of boranocarbonate however, had a
dramatic impact on the yield of [99mTC(CO)3(OH2)3]+ (entries 4 - 6), which
improved to 55%. After establishing the need to increase the amount of
boranocarbonate, the next step was to adjust the quantity of borate. Borate
acts
to degrade excess boranocarbonate and prevent unwanted side-reactions with
the Tc(I) cation once it has formed. The correct quantity of borate is crucial
since
an excess would act to degrade boranocarbonate too rapidly, whereas an
insufficient quantity might lead to unwanted side-products. The commercial kit
utilizes the decahydrate, Na2B40~.10H20, and its molar ratio to Na2[BH3~COZ]
is
approximately 1:6. Using this ratio as a starting point, only poor yields of
the
CA 02515495 2005-08-09
34
~99mTC(CO)3(OH2)3~+ cation were obtained. However, subsequent experiments
established that higher yields of [99"'Tc(CO)3(OH2)3]+ could be achieved when
using reduced amounts of the anhydrous salt Na2B40~ instead of the
decahydrated form (entries 7 - 9). In the end, the optimal ratio of anhydrous
sodium borate to boranocarbonate was determined to be approximately 1:5,
which prompted an investigation of the effect of fluoride ion concentration on
the
yield of [99'"Tc(CO)3(OH2)3]+.
Table 3 Radiochemical yield of 22b using different formulations (pH a 12).
Expt # 1 2 3 4 5 6 7 8 9 10 11 12
Reage
Kz[BH3~C02]4.0 4.0 4.05.5 7.5 8.5 8.5 8.5 8.5 8.5 8.5 8.5
m)
Na2B40~ 3.5 4.5 3.03.0 3.0 3.0 2.5 2.2 1.9 1.9 1.9 1.9
(m )
NaF mg) 2.5 2.5 2.52.5 2.5 2.5 2.5 2.5 2.5 3.5 5.5 7.5
NaZC03 4.0 4.0 4.04.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0
m
Yield (%) 20 15 25 35 55 55 75 80 80 80 85 85
Table 4 Radiochemical yield of 22b as a function of fluoride concentration (pH
z
12).
Expt. 13 14 15 16 17
Reagent
K2[BH3.C02]8.5 8.5 8.5 8.5 8.5
mg)
NaZB40~ 1.9 1.9 1.9 1.9 1.9
m)
NaF (m 8.5 9.5 10.5 11.5 25.5
)
NazC03 4.0 4.0 4.0 4.0 4.0
(m )
Yield (%) 90 95 95 95 95
~
CA 02515495 2005-08-09
Table 5 Radiochemical yield of 22b as a function of different pH values.
Expt. 18 19 20 21
(10-10.5)(9-9.5)(8-8.5)(7-7.5)
(pH)
Reagent
Kz(BH3.C02]8.5 8.5 8.5 8.5
(mg)
NaZB40~ 1.9 1.9 1.9 1.9
(mg)
NaF (m 10.5 10.5 10.5 10.5
)
NaHzP04'Hz0 0 7.8 8.0
0 (mg)
NaZHP04 3.0 0 6.9 7.1
m)
NaHC03 5.0 5.2 0 0
(mg)
Na2COs 0 4.0 0 0
(m )
Yield (%) 95 95 95 95
It has been demonstrated that for certain ligands, increasing the
concentration of fluoride from 0.1 to 1.0 M improved the yields of
rhenacarboranes so long as the ligands remain soluble. Since the method for
ssmTc-metallocarborane formation involved essentially the same procedure, it
seemed logical that higher quantities of fluoride ion would also improve the
average yield of [99"'Tc(CO)3(OH2)s]+. To test this hypothesis, a series of
experiments were performed where the quantity of fluoride ion was increased
incrementally (Table 3: entries 10 - 12; Table 4: entries 13 - 16), which as
predicted, gave corresponding improvements to the yield of
(99"'Tc(CO)3(OH2)s]+.
This is illustrated in Fig. 4, which shows the y-HPLC radiochromatograms from
experiments involving different amounts of fluoride.
CA 02515495 2005-08-09
36
Entry 11 (260 mM NaF), showed high conversion of [99"'TCO4]- to
[99"'Tc(CO)3(OH2)3]+, however some unidentifiable peaks appeared in the HPLC
radiochromatogram (3.3 min, 5.5 min, and small peaks around 17 and 18 min).
These peaks were not present for the reactions which employed 500 mM
fluoride. Fluoride ion concentrations greater than this value produced the
same
results hence further increasing the amount of fluoride was unnecessary.
With the existing commercial kit, to investigate the influence of pH on the
efficiency of labelling it is necessary to adjust the pH of the solution after
the
synthesis of [99mTc(CO)3(OH2)3]+. A more convenient approach would be to
design kits that would afford the 99"'Tc-trisaquaion at specific pH values
directly.
Besides convenience, this approach would also create the opportunity for "one
pot" labelling procedures for biomolecules that are sensitive to high pH. To
date
however, ~99"'TC(CO)3(OH2)3]+ has only been prepared under highly basic
reaction conditions. With the stabilizing influence of fluoride being
apparent,
attempts were made to prepare [99"'Tc(CO)3(OH2)3]+ directly at various pH
values.
A series of reactions were performed in different buffers were the pH of
the mixtures were tested prior to and after formation of 22b. This approach
led to
the successful development of several unique formulations for the preparation
of
~ssm-I-C(CO)3(OH2)3]+ at pH values ranging from 7 to 10.5. As illustrated in
Table 5
(entries 18 - 21 ), the radiochemical yields of ~99mTC(CO)3(OH2)3)+ were
unaffected by changes in pH. The y-HPLC radiochromatograms from these
entries are shown in Fig. 5. This set of experiments represents the first
report of
the direct formation of [99"'TC(CO)3(OH2)3]+ from conditions that are not
highly
alkaline (pH a 12) which makes developing one pot kits for pH sensitive
biomolecules a real possibility.
CA 02515495 2005-08-09
37
One aspect that was not discussed was the influence of temperature. The
formation of the [99"'Tc(CO)3(OH2)3]+ cation using the commercial kit is
normally
achieved at 100°C. In the experiments described above, heating to
reflux
resulted in poor yields of the 99"'Tc-trisaquaion. The optimal temperature for
the
fluoride mediated formation of [99"'Tc(CO)s(OH2)3]+ was between 65 and
70°C.
Presumably, the stabilizing influence of fluoride ion is diminished at
temperatures
above 70°C leading to more rapid decomposition of ~99mTC(CO)3(OH2)3~+.
After establishing the necessary parameters needed to produce
~99m-~-C(CO)3(OH2)3]+ in high yield, the synthesis of 99"'Tc-metallocarboranes
was
investigated. The initial experiments were performed with compound 1 b (Scheme
11 ) because the carborane is soluble in aqueous solutions and because the Re
and 99Tc-analogues which were prepared previously could be used as well
characterized reference standards. [99"'Tc(CO)3(OH2)3]+ was prepared from 185 -
740 MBq (5 - 20 mCi) of [g9"'TcO4]-, using the optimal conditions described
above. Compound 1 b, which had been pre-incubated with fluoride ion, was
subsequently added directly into the reaction vial. The temperature was raised
to
85°C from the initial 70°C used to prepare [99"'TC(CO)3(OH2)3)+,
and the reaction
progress monitored by HPLC (Fig. 6). After 3 hours the reaction was complete
with the major product being the Tc-carborane with the minor impurities being
~99mTc(CO)3(OH2)3~+ and ~99mTCO4~ .
The next step involved separating 23b from residual [99mTc(CO)3(OH2)3]+,
Na[99"'TcO4], and unreacted ligand. This could have been performed by semi-
preparative HPLC however this approach is time consuming and impractical for
routine clinical use. A convenient alternative involved solid-phase extraction
using a commercially available C~$ Sep-Pak~ cartridge. In the procedure
described here, it was necessary to first condition the Sep-Pak~ with
acetonitrile,
ethanol, and 10 mM HCI. The entire crude reaction mixture was then loaded onto
the column and eluted with 10 mM HCI. Residual [99"'TCO4]- and
[99"'TC(CO)3(OH2)3~+ are both removed under these conditions, while the target
CA 02515495 2005-08-09
38
complex remains on the Sep-Pak~. After elution with acetonitrile, the product
was
obtained in greater than 99% purity (Fig. 6).
One important feature of this purification protocol is that it results in the
removal of excess ligand giving the product in high effective specific
activity. The
importance of this result lies in the fact that removal of residual ligand
from the
purified product assists in the prevention of unwanted biological effects once
the
tracer is administered. Furthermore, if a ligand and its ssmTc-complex have
similar affinities for a particular receptor, then the excess unlabelled
ligand can
prevent binding of the ss"'Tc-labelled compound to the target.
Another important criterion that needed to be evaluated was stability. In
vivo, there are many endogenous ligands that can degrade ss"'Tc complexes
through transmetalation reactions (K. Schwochau Angew. Chem. Int. Ed. 1994,
33, 2258). To test the stability of the ssmTc-metallocarborane towards ligand
exchange, compound 23b was incubated with a 1000-fold excess of cysteine and
histidine in separate experiments. After incubation at 37°C in
phosphate buffered
saline (PBS, pH=7.2) for 24 hours, the y-radiochromatograms from both
experiments indicated greater than 99% of the product remained intact. The
stability of the complexes supports the potential use of ss"'Tc-
metallocarboranes
as synthons for preparing radiopharmaceuticals.
To study the factors that impact the yield of 23b, pH, ligand concentration,
and fluoride ion concentration, were systematically varied. With the exception
of
the ligand concentration, these adjustments were made to the kit used to
prepare
[ssmTC(CO)3(OH2)3~+, taking advantage of the initial work on developing
different
fluoride kits, which allow for direct control of pH and fluoride ion
concentration.
The mild reaction conditions afforded by the fluoride-based method for the
preparation of the rhenacarboranes cannot necessarily be directly correlated
to
chemistry at the tracer level. The discrepancy lies in the fact that at the
tracer
level, ~ssmTC(CO)3(OH2)3]+ is normally prepared under highly basic conditions
whereas, the corresponding rhenacarboranes are prepared effectively at neutral
CA 02515495 2005-08-09
39
pH. Since the initial method used to prepare 99"'Tc-metallocarboranes involved
direct incubation of the nido-carborane ligand with [99"'Tc(CO)3(OH2)3]+
without
any adjustment of pH, the question remained as to whether fluoride mediates
the
metallation reaction or whether product formation was a consequence of the
high
pH.
To investigate the impact of basicity, labelling reactions were performed at
various pH values using a fixed fluoride ion concentration of 0.1 M and
reaction
time of 3 hours. As Fig. 7 illustrates, the yield of 23b increases with
increasing
pH and that a high pH is needed to afford good yields of the 99"'Tc-
metallocarborane, which is not the case for rhenium at the macroscopic level.
The results do demonstrate that it is possible, albeit in reduced yields, to
prepare
metallocarboranes at low pH, which will be important in cases where base
sensitive targeting vectors are attached to the cluster. These results also
indicate
that fluoride ion is clearly involved in mediating the formation of 23b since
a yield
of 40% was observed at neutral pH at a ligand concentration of 10-2 M. In the
absence of fluoride under the same conditions, no product was detected.
Fig. 7 also summarizes the results of a series of experiments that were
performed to evaluate the effect of changing the concentration of the ligand
on
the yield of the Tc-metallocarborane. The results clearly demonstrate that the
concentration of the ligand does play a significant role since the percent
conversion roughly doubles when the ligand concentration is increased to 10-2
M
from 10'4 M. The importance of a low ligand concentration for preparations
that
do not involve further purification lies predominantly in minimizing side
effects
and/or receptor saturation once the radiotracer is administered in vivo. In
this
respect, both the Cp- and the [nido-7,8-C2B9H~2]~ anion are less efficient
than the
bi- and tridentate chelates for Tc(I) where Schibli and coworkers have
demonstrated that mild radiolabelling conditions (30 min, 75 °C) can be
used to
prepare complexes of [99"'TC(CO)3(OH2)3]+ with tridentate chelates in yields
of ~
95% at ligand concentrations ranging from 10-4 to 10-6 M (Schibli, R.; Bella,
R.L.;
CA 02515495 2005-08-09
Alberto, R.; Garcia-Garayoa, E.; Ortner, K.; Abram, U.; Schubiger, P.A.
Bioconjugate Chem. 2000, 11, 345). In the case of the AcCp system, the
concentration of the ligand used was 10-3 to 10-4 M. Although carborane
concentrations of 10-3 to 10-4 M do not produce as high a yield as the AcCp
system, this limitation is overcome since excess ligand can readily be removed
using the simple purification method described above. The general utility of
this
approach for purifying other carboranes requires further investigation.
Another important factor that was investigated was the impact of changing
the fluoride ion concentration on the yield of 99"'Tc-metallocarborane
formation. A
series of experiments were performed where [99mTc(CO)3(OH2)3]+ was prepared
in the presence of varying amounts of fluoride ion (10, 100, 500, and 1000 mM
NaF) followed by the addition of compound 1b. The progress of the reaction was
monitored by HPLC (Fig. 8), which showed that increasing the fluoride ion
concentration increases the yield of the product. This is consistent with the
results observed for rhenium.
As shown in Scheme 2, compound 4 (At a ligand concentration of 10-3 M)
was combined with [99"'TC(CO)3(OH2)3~+ (85°C, pH 12) in 0.5 M fluoride.
Under
these conditions, nearly quantitative conversion of 4 to the desired product 5
was
observed in the HPLC radiochromatogram in approximately half of the reaction
time (1.5 hours) required to achieve the maximum yield of compound 23b (Fig.
9). Afterwards, the Sep-Pak~ purification protocol described above was
employed to isolate compound 5 in 70% yield free from residual ligand.
Cysteine
and histidine challenge experiments were performed under the same conditions
used for 23b where there was no sign of decomposition.
In summary, a novel strategy for the preparation of Re, 99"'Tc and other
metallocarboranes in water under mild reaction conditions has been developed.
The reported Tc complexes are attractive synthons for the preparation of
organometallic radiopharmaceuticals because of their inertness, relatively
small
size, and ease of derivatization. Furthermore, with the hard base reaction in
CA 02515495 2005-08-09
41
hand, it is now possible to use the numerous carborane derivatives that have
been designed to target tumors for boron neutron capture therapy as the basis
for designing novel 99mTc radiopharmaceuticals (Soloway, A. H.; Tjarks, W.;
Barnum, A.; Rong, F.-G.; Barth, R. F.; Codogni, I. M.; Wilson, J. G. Chem.
ReV.
1998, 98, 1515).
A synthetic strategy was used to prepare metal complexes of a wide
variety of both ortho and meta carborane derivatives. It was also shown that
metallocarboranes can be prepared from both nido-carboranes and closo-
carboranes. The fluoride-based approach is mild compared to traditional
synthetic methods and should be adaptable for the preparation of the
corresponding 99"'Tc-carborane complexes.
It was also shown that aqueous fluoride can be used to facilitate the
preparation of [99"'Tc(CO)3(OH2)s]+ and Tc-labeled carboranes. Simple
formulations were developed that allow for the synthesis of
[99"'Tc(CO)3(OH2)3]+
at specific pH values including those that are much less basic than what is
produced using the commercially available kit. In terms of the radiosynthesis
of
99"'Tc-metallocarboranes, a small collection of ligands were successfully
labeled
and the products isolated free from any unreacted ligand. The products
displayed
resistance to ligand exchange by both cysteine and histidine. It was
determined
that a number of factors, most notably ligand and fluoride concentrations and
pH
influence the overall yield of the reaction. Having the ability to label
carboranes
with Tc creates the means to explore the possibility of using the substantial
number of targeted carborane derivatives that have been developed for boron
neutron capture therapy ((a) Soloway, A.H.; Tjarks, W.; Barnum, A.; Rong, F.-
G.;
Barth, R.F.; Codogni, I.M.; Wilson, J.G. Chem. Rev. 1998, 98, 1515 (b)
Valliant,
J.F.; Guenther, K.J.; King, A.S.; Morel, P.; Schaffer, P.; Sogbein, O.O.;
Stephenson, A. Coord. Chem. Rev. 2002, 232, 173) as the basis for designing
novel radiopharmaceuticals for imaging tumours. Furthermore, it may also be
possible to use fluoride to generate "one pot" kits for preparing Tc(I)
CA 02515495 2005-08-09
42
radiopharmaceuticals and for improving the labelling yields of other ligand
systems and bioconjugates, which did not show satisfactory conversion to the
desired products when reactions were carried out using the existing commercial
kit.
The following non-limiting examples are illustrative of the present
invention:
EXAMPLES
Materials and Instrumentations: Potassium boranocarbonate (K2H3BC02) was
prepared following literature procedures (Alberto, R.; Ortner, K.; Wheatly,
N.;
Schibli, R.; Schubiger, A.P.; J. Am. Chem. Soc. 2001, 123, 3135). Analytical
TLC's were performed on silica gel 60-F254 (Merck) and boron compounds were
visualized with 0.1 % PdCl2 in hydrochloric acid (3.0 M), which upon heating
gave
dark brown spots. NMR spectroscopy was performed on a Bruker Avarice AV600
spectrometer at ambient temperature. The chemical shifts (b) for'H and'3C were
recorded relative to solvent peaks as internal standards. BF3-Et20 was used as
the reference standard for ~'B spectra. Electrospray ionization (ESI) mass
spectrometry experiments were performed on a Micromass Quattro Ultima
instrument where samples were dissolved in 1:1 CHsOH/ H20 or 1:1 CH3CN/H20
mixtures. High resolution MS was obtained using FAB MS and a Waters-
Micromass Q-TOF Ultima Global instrument. IR spectra were run on a Bio-Rad
FTS-40 FTIR spectrometer. HPLC experiments were pertormea on a vanan
Prostar Model 230 instrument, fitted with a Varian Pro Star model 330 PDA
detector and an IN/US y-RAM gamma detector. The wavelength for detection
was set at ~, = 254 rim and the dwell time in the gamma detector was 1 s in a
10
~.L loop. All runs were performed using a Varian DYNAMAX (250 mm x 4.6 mm),
MicroSorb-MV analytical column (300 A - 5~,m, RP-C18). The elution conditions
consisted of: Method A: Solvent A = 0.1 % TFA in H20, Solvent B = 0.1 % TFA in
CH3CN: Gradient Elution: 0 - 3 min, 100% A to 95% A; 3 - 6 min, 75% A; 6 - 9
CA 02515495 2005-08-09
43
min, 66% A; 9 - 20 min, 0% A; 20 - 22 min, 0% A; 22 - 24 min, 95% A; 24 - 25
min, 100 % A. Method B (which was used for the histidine and cysteine
challenge
experiments): Solvent A = H20, Solvent B = CH3CN: Gradient Elution: same as
above. Method C: Solvent A = tetraethylammonuium phosphate (TEAP; pH = 2 -
2.5), Solvent B = CH30H: Gradient Elution: 0 - 3 min, 100% A; 3 - 6 min, 100%
A
to 75% A; 6 - 9 min, 75% A to 67% A; 9 - 20 min, 67% A to 0% A; 20 - 22 min,
0% A; 22 - 25 min, 0% A to 100% A; 25 - 30 min, 100 % A. The flow rate for all
methods was set at 1 mL/min.
Caution: 99"'Tc is a y-emitter (141 KeV) with a half life of 6 hours, which
should
only be handled in appropriately shielded and licensed laboratories.
Example 1: Synthesis of 2a
In two separate vials compound 1a (0.11 g, 0.64 mmol) and [NEt4]2[Re(CO)3Br3]
(0.49 g, 0.64 mmol) were suspended in 100 mM solution of aqueous KF (5.0 mL)
and heated to 85 °C under Ar. After one hour, the solution containing
[NEt4]2[Re(CO)3Br3] was added dropwise to the solution containing the nido-
carborane while maintaining the temperature at 85 °C. After 3.5 hours,
the
heterogeneous suspension became clear and the reaction was allowed to
proceed at 85°C for 18 hours. After cooling the clear yellow solution
to room
temperature a precipitate formed which was collected by filtration. The
desired
product was isolated by preparative TLC (92:8 CH2C12/CH30H + 0.1 % AcOH).
TLC analysis indicated additional product remained in the aqueous layer.
Consequently, the filtrate was concentrated under reduced pressure, and the
resulting colourless solid washed with THF. An additional aliquot of sample
from
the THF solution was isolated by preparative TLC. Yield: 0.12 g, 34 %; TLC Rf
(17:3 CH2C12/CH30H + 0.1 % AcOH) = 0.45; 'H NMR (acetonitrile-d3, 600.13
MHz): 8 0.5 - 1.7 (br, BH), 1.29 (br, CH3), 1.94 (br, CH2), 2.34 (br, CH);
'3C~'H}
NMR (acetonitrile-d3, 150.92 MHz): b 33.53, 40.72, 55.0, 199.47; ~1B~1H~ NMR
(CD2C12/acetonitrile-d3 + 1 drop of DMSO-dfi, 160.46 MHz): b -6.86, -10.50, -
14.71, -21.73, -23.12; FTIR (KBr, cm-'): 3048 (s), 2924 (s), 2558 (s), 2510
(s),
CA 02515495 2005-08-09
44
1893 (s); MS (ESI): m/z 403.1 [M-]; HRMS m/z calculated for C5B9H»03Re:
403.1157 gmol-'; Observed: 403.1159.
Example 2: Synthesis of 2b
In two separate vials compound 1 b (0.065 g, 0.23 mmol) and
[NEt4]2[Re(CO)sBr3]
(0.18 g, 0.23 mmol) were suspended in a 100 mM solution of aqueous KF (2.5
mL) and heated to 85°C under Ar. After one hour the solution containing
the
[NEt4]2[Re(CO)3Br3] was added dropwise to the solution containing the nido-
carborane while maintaining the temperature at 85°C. After 30 minutes,
the
heterogeneous mixture became clear and the reaction was allowed to proceed at
85°C overnight. After cooling to room temperature, the pH was adjusted
to 3
using 12 M HCI. The aqueous solution was then passed through a fritted funnel
containing Celite yielding a brown solid upon concentration of the filtrate
under
reduced pressure. The colourless product was purified by flash column
chromatography (gradient elution: 19:1 CH2C12/CH30H to 4:1 CH2C12/CHsOH) on
silica gel (0.068 g, 50%). TLC Rf (4:1 CH2C12/CHsOH) = 0.44; 'H NMR (GD30D,
600.13 MHz): 8 1.27 (t, J = 7.2 Hz, CH3), 2.07 (m, CH2CH2C02H), 2.29 (m,
CH2CH2C02H), 3.27 (t, NCH2); ~3C(~H) NMR (CD30D, 150.92 MHz): 8 7.59,
29.04, 35.27, 36.50, 42.44, 46.21, 53.25, 176.89, 199.81, 200.66; ~~B(~H} NMR
(CD30D, 160.46 MHz): 8 -10.89, -13.79, -16.87, -18.24, -21.82, -33.151, -
37.04;
FTIR (KBr, crri ~): 3620 (w), 2928 (m), 2531 (s), 2007 (s), 1907 (s); MS
(ESI): m/z
474.71 [M-]; HRMS m/z calculated for CgH~5 B905Re: 475.1299 gmol-'; Observed
475.1302; HPLC (method A): tR = 18.3 min.
Example 3: Synthesis of 2c
Compound 1c (0.15 g, 0.70 mmol) was dissolved in a 1:1 mixture of aqueous
100 mM KF and methanol (10 mL) and the temperature elevated to 85 °C
for 1
hour. [NEt4]2[Re(CO)sBr3] (0.54 g, 0.70 mmol) was dissolved in distilled de-
ionized water and the temperature elevated to 85°C under Ar(g). After 1
hour, the
solution containing 1c was added to the solution containing
[NEt4]2[Re(CO)3Br3]
and the reaction heated to reflux. After 13 hours, the reaction was allowed to
cool
CA 02515495 2005-08-09
to room temperature. The resulting heterogeneous solution was acidified with 1
M HCI (3 mL) and subsequently extracted with 50 mL of ethyl acetate. The ethyl
acetate was extracted with 1 M HCI (2 x 50 mL), dried over anhydrous MgS04
and concentrated under reduced pressure giving an off-white solid. The solid
was
re-dissolved in CH3CN/H20 (2:3 v/v mixture, 10 mL) and purified using gel
permeation chromatography (Sephadex G-10; column volume of 12.5 cm x 1.5
cm; flow rate of 1.5 mL/min) yielding a white solid (0.24 g, 70 %). TLC Rf
(17:3
CH2C12/CH30H+ 0.1 % AcOH) - 0.33; m.p.: 240°C (decomp.); 'H NMR
(acetoneds, CD30D, 600.13 MHz): 8 1.52 (m, CH2CH2CH2), 1.54 (s, CH), 1.66
(m, CH2CH2CH2), 1.94 (m, CH2(CH2)2N), 3.15 (m, N(CH3)2), 3.23 (m,
(CHZ)2CH2NH), 5.75 (s, NH); '3Cf'H} NMR (acetone-ds, 150.92 MHz): 8 26.92,
36.67, 44.14, 59.41, 199.68; "B{'H} NMR (acetone-ds, 192.54 MHz): b
-10.22, -10.75, -13.37, -15.26, -18.69, -21.04, -32.53, -36.43.; IR (KBr, cm-
'):
3446 (w), 3148 (w), 2526 (s), 2006 (s), 1899 (s); HRMS (FAB): m/z calculated
for
C~oH2sgsNOsRe: 487.2121 gmol-1; Observed 487.2114.
Example 4: Synthesis of 2d
A penicillin vial (10 mL) containing freshly prepared potassium
boranocarbonate
(K2H3BC02), (8.5 mg, 62.5 pmol), Na2B40~.10H20 (2.9 mg, 7.6 pmol), KF (2.8
mg, 48.2 pmol) and Na2C03 (4.0 mg, 37.7 pmol) was capped with a rubber
stopper and flushed with N2(g) for 45 minutes. 99'"Tc04 (10 -20 mCi, 370 - 740
MBq) in 500 NL of saline was added via a syringe, and the mixture slowly
heated
to 70°C over a period of 1 hour. After cooling in an ice bath, an
aliquot was taken
to verify formation of [Tc(CO)3(OH2)3]+. HPLC (method A): tR = 12.5 min; Yield
>_
95 %. In a separate penicillin vial, K[C2B9H~2(CHZ)2COZK] (5.0 mg, 17.6 Nmol)
was suspended in 250 ~,L of a 100 mM de-gassed aqueous solution of KF and
the vial capped and flushed with N2. The mixture was heated at 85°C for
one
hour prior to adding the solution containing the ligand to
[99"'Tc(CO)3(OH2)3]+ via
syringe. After combining the solutions the temperature was raised to
85°C for 3
hours. The reaction was subsequently cooled on an ice-bath, loaded onto a pre-
CA 02515495 2005-08-09
46
conditioned Sep-Pak, which was eluted slowly with 10 mM HCI (7 mL), 1:1
acetonitrile/10 mM HCI (2 mL), and finally acetonitrile (10 mL). The product,
which was in the acetonitrile washing, was isolated in 80% radiochemical yield
in
>99% purity as determined by analytical HPLC. HPLC (method A): tR = 18.4 min.
Example 5: Synthesis of 4
Compound 3 (0.10 g, 0.434 mmol) and powdered potassium hydroxide (0.20 g,
3.4 mmol) were combined in a 95:5 (v/v) mixture of absolute ethanol/ddH20 (8
mL) at room temperature under N2(g). The suspension was heated to 90°C
for 12
hours, cooled to room temperature and COz(g) bubbled through the .
homogeneous solution to precipitate the excess KOH as K2COs. The
heterogeneous mixture was passed through a fritted funnel packed with Celite,
which was subsequently washed with absolute ethanol (3 x 10 mL). After
concentration of the filtrate under reduced pressure, the mixture was purified
by
flash chromatography through silica gel (isochratic elution: 1:9 CH30H/CH2C12
+
0.1 % AcOH). The oily residue was suspended in ddH20 (10 mL) and lyophilized
at -80 °C to yield a white solid (0.049 g, 50 %). TLC Rf (85:15
CH2C12/CH30H +
0.1 % AcOH) = 0.28; 'H NMR (500.13 MHz, 5:1 D20/CD30D): b -0.5 - 2.8 (bm,
BH), 0.94 (m, CH2), 1.08 (m, CH2); '3C{'H} NMR (50.3 MHz, CD30D): S 18.27,
23.47, 32.35, 58.28, 180.44; "B{'H} NMR (160.5 MHz, CD30D): b -5.14, -6.41, -
22.20, -23.19, -35.11, -36.14; FTIR (KBr, cm-'): v 3220, 2533, 1429; ESMS
(negative ion): 205.48 [M-H]-.
Example 6: Synthesis of 2b from the closo-carborane
Compound 6 (0.050 g, 0.231 mmol) and [NEt4]2[Re(CO)sBrs] (0.196 g, 0.254
mmol) were combined in a 10 mL penicillin vial, sealed with rubber septum and
aluminum cap, and then flushed with N2(g) for 10 minutes. A solution
containing
500 mM TEAF(aq)/absolute EtOH (9:1 v/v) was added (1.0 mL) and the resultant
suspension heated to 100°C. After 30 hours, the heat was removed and
the
mixture acidified by the addition of 12 M HCI. CH3CN was subsequently added
(1.0 mL) and the vial vigorously shaken for 5 minutes. The mixture was frozen
at
CA 02515495 2005-08-09
47
-5 °C overnight in a freezer resulting in a biphasic mixture with the
organic layer
portioned on top of the frozen aqueous layer. The organic layer was decanted
and concentrated yielding a brown viscous oil. The product was isolated by
flash
column chromatography through silica gel (gradient elution: 95:5 CH2C12/CH30H
to 90:10 CH2C12/CH30H) as a cream coloured solid upon concentration under
reduced pressure (0.098 g, 70%). TLC Rf (4:1 CH2C12/CH30H) = 0.44; mp > 140
°C (decomp.); 'H NMR (300.13 MHz, acetone-ds): b 1.15 (m, CH3), 1.81
(m,
CH2), 2.33 (m, CH2), 3.23 (t, CH3); '3C{'H} NMR (151 MHz, acetone-ds): b 7.59,
29.04, 35.27, 36.50, 37.03, 42.44, 46.21, 53.25, 176.89, 199.81, 200.66; "B{'
H}
NMR (160.5 MHz, acetone-ds): S 18.82, -5.79, -8.13, -10.46, -11.87, -18.56, -
20.02, -22.17; FTIR (KBr, cm-'): v 2542, 1998, 1893; HRMS (ESI-Q-TOF): Calcd
for B9C8H,505Re: 474.7085. Found: 475.1302.
Example 7: Synthesis of 9
Compound 8 (0.100 g, 0.426 mmol) was combined with potassium hydroxide
(100 mg, 1.42 mmol) and dissolved in absolute ethanol (2.5 mL). The reaction
mixture was heated at reflux for 24 hrs, the temperature lowered to room
temperature and C02(g) passed through the solution, resulting in the formation
of
a thick white precipitate. The heterogeneous mixture was filtered, and the
clear,
colorless eluent concentrated in vacuo giving a viscous, opaque oil. The crude
oil
was dissolved in distilled, deionized water (3 mL) and lyophilized giving the
product as a white solid (>99 %). TLC Rf (22% MeOH in CH2C12) = 0.56; mp >
225°C (decomp.);'H NMR (600 MHz, CD30D): b 8.37 (d, J= 4.59, 1H, H-5),
7.71
(m, 1 H, H-3), 7.25 (d, J = 7.51, 1 H, H-2), 7.14 (m, 1 H, H-4), 3.68 (bs,
carborane
CH), 3.036 (AB, J = -15.3, 1 H, CH2), 2.78 (AB, 1 H, CH2), 0 - 2.7 (br m, BH);
'3C~'H}NMR (151 MHz, CD30D): b 162.0, 146.53, 136.49, 123.22, 120.63, 57.9,
46.53, 44.90; "B{'H} NMR (160.5 MHz, CD30D): b -8.89, -9.96, -11.96, -14.40, -
17.29, -18.25, -19.90, -31.86, -35.44; FTIR (KBr, cm~'): v 2990, 2940, 2517,
1749; HRMS (ESMS-QTOF) calcd for CBH~~B9N: 225.2241. Found: 225.2246.
CA 02515495 2005-08-09
48
Example 8: X-Ray Crystallography of 2c
X-ray diffraction data for compound 2c was collected on a single crystal grown
from a CH30H/CH2C12 mixture (1:1 vJv) (0.07 x 0.06 x 0.01 mm3). Data was
collected on a p4 Bruker diffractometer fitted with a rotating anode, a Bruker
SMART-1 K CCD (charge coupled device) area detector and an Oxford
Cryostream cooling system. Diffraction data was collected using the program
SMART with graphite-monochromated Mo-Ka X-radiation (~, = 0.71073 A) and a
single crystal of 2c mounted on the tip of a glass fibre. The crystal to
detector
distance was 4.987 cm. Initially, accurate unit cell parameters were
determined
at 173 K with better than 0.9 A resolution from a least squares fit of the
strong
reflections, collected by a 12° scan in 40 frames using the SMART
software.
Data were obtained from a chosen number of centered reflections of the setting
angles (x, ~, and 28) in reciprocal space with truncation of the data (28 =
48°),
using least squares, due to disorder in the high angle reflections. Data
reduction
was carried out using the SAINT program applying polarization and Lorentz
corrections to the integrated diffraction spots. The raw frame data and the
structure was solved from direct methods and refined by full-matrix least
squares
on F2 using the Bruker SHELXTL PLUS package. Corrections were made for
decay and an empirical absorption correction was made with SADABS program
based on redundant reflections. Additionally, after completion of data
collection,
the first 50 frames were re-acquired for the improvement of the decay
correction.
All non-hydrogen atoms were refined with anisotropic atomic displacement
parameters giving rise to the prescribed R, values except for the carbonyl
atoms
(C,, C2, C3, O~, 02, and 03), which were refined isotropically due to
positional
disorder. All hydrogen atoms were assigned based on the difference map and
added as fixed contributors at calculated points with isotropic thermal
parameters
based on their respective carbon atoms. Crystallographic data is presented in
Table 1. The structure is shown in Fig. 3.
CA 02515495 2005-08-09
49
Example 9: Synthesis of 10
Compound 9 (11.4 mg, 0.044 mmol) and [NEt4]2[Re(CO)3Br3] (32 mg, 0.042
mmol) were combined and dissolved in 100 mM aqueous potassium fluoride (2
mL) and the mixture heated to reflux for 24 hrs. Upon cooling to room
temperature, the pH was adjusted using 0.1 M HCI (final pH ~1 ) and the
mixture
extracted with CH2C12 (3 x 10 mL). All organic portions were combined, dried
over sodium sulphate, and the solvent removed by rotary evaporation giving
pure
(19 mg, 85%), as an off-white semi-solid. No further purification was
required.
TLC Rf (18% MeOH in CH2C12) = 0.05;'H NMR (600 MHz, CD30D): b 8.57 (d, H-
5), 7.91 (m, 1 H, H-3), 7.41 (m, 2H, H-2, H-4), 3.69 (bs, carborane CH), 3.31
(m,
CH2), 2.1-1.01 (bm, BH); '3C{'H} NMR (50.3 MHz, CD30D): b 199.90, 155.32,
146.38, 142.49, 130.32, 126.43, 50.37, 45.17, 29.25; "B~'H}NMR (192.54 MHz,
CD30D) b -7.88, -11.44, -14.11, -16.77, -18.20, -19.96; IR (KBr, cm''): v
2546,
1993, 1885, 1626; HRMS (ESMS-QTOF) Cafcd for C"H,sOsB9NRe: 494.1581.
Found: 494.1584.
Example 10: Synthesis of 11
1-(3'-Chloropropyl)-1,2-dicarba-closo-dodecaborane (1.23 g, 5.57 mmol) and
sodium iodide (1.67 g, 11.14 mmol) were combined in dry acetone (150 mL)
under nitrogen and heated to reflux for 22 hours. A precipitate appeared,
which
upon completion of the reaction, was collected by filtration through a medium
porosity fritted funnel. The residue was washed with diethyl ether (3 x 25 mL)
and
all organic fractions pooled and concentrated under reduced pressure giving an
off-white solid. The solid was subsequently dissolved in diethyl ether (25
mL),
which was extracted with 0.1 M sodium thiosulfate (2 x 25 mL). The aqueous
layer was further extracted with ether (2 x 20 mL) and the organic fractions
combined, dried over Na2S04, filtered and the filtrate concentrated to dryness
under reduced pressure yielding a white solid. The product was purified by
flash
column chromatography (isocratic elution: 100% CHC13) through silica gel to
give
a white solid (1.42 g, 81%). TLC Rf (CHC13) = 0.56; 'H NMR (200.13 MHz,
CA 02515495 2005-08-09
CDC13): b 0.8 - 3.6 (bm, BH), 1.91 (m, CH2), 2.33 (m, CH2), 3.08 (t, CH2),
3.51
(bs, carborane CH); '3C{'H} NMR (50.3 MHz, CDC13): 5 3.61, 32.33, 38.84,
61.50, 73.77; "B{'H) NMR (160.5 MHz, CDC13): 5 -2.65, -6.05, -9.64, -12.12, -
12.52, -13.46; FTIR (KBr, cm-'): v 2959, 2591; MS (EI): m/z = 312 [M]~+, 183
[M-
I]+.
Example 11: Synthesis of 13a
Compound 12 (0.506 g, 1.00 mmol) and KOH (1.2 g, 22 mmol,) were dissolved in
absolute ethanol (20 mL) and the mixture heated to overnight. The reaction was
cooled to room temperature and the excess KOH was precipitated as K2COs by
passing a stream of C02 gas through the solution. The solid was removed by
filtration and the residue washed with cold ethanol (20 mL). The combined
filtrates were concentrated under reduced pressure yielding a white solid,
which
was dissolved in distilled water, and the pH adjusted to approximately 4 by
the
dropwise addition of 1 M HCI. The solution was again concentrated to a white
solid by rotary evaporation. The product was purified by silica gel column
chromatography using a gradient of 50% to 70% acetone in CH2C12. A white solid
was obtained by evaporating ether solutions of the product fraction. Yield:
83%
(0.3 g). TLC Rf (25% CH30H / CH2C12) = 0.38; 'H NMR (500 MHz, CD30D): b
4.51 (d, H-1, 3J~,2 = 7.8 Hz ), 4.41 (d, H-1 ',3J~ ~,2~ - 7.8 Hz), 4.01 (d, 1
H,
OCH2C°a9eC°a9eH, 2J~a,~b = - 10.9 Hz, H-7a), 4.01 (2dd, H-6a,6a'
), 3.88 (d, 1 H,
2J~a~,~t,~ _ -10.7 Hz, H-7a'), 3.86 (2dd, H-6b,6b'), 3.77 (d, H-7b'), 3.64 (d,
H-7b),
3.54 - 3.48 (m, H-3,3', H-4,4'), 3.40 (m, H-5,5'), 3.34 (m, H-2,2'), 2.07 (br
s,
OCH2C~ageCcageH~ H-9,9'), 2.14 -0.30 (br, m, B-H), -2.5 (br, B-H-B); '3C NMR
(126 MHz, CD30D): S 103.41, 103.01 (C-1,1'), 78.46 (C-7,7'), 77.12, 77.01 (C-
3,3'), 77.84, 77.74 (C-5,5'), 75.16 (C-2,2'), 71.55 (C-4,4'), 62.51 (C-6,6');
"B
NMR (160 MHz, CDsOD): b -10.83, -16.72, -21.87, -33.02, -37.50; IR (KBr, cm-
'):
v 3429, 2526 ; HRMS (EI): Calcd for C9H24B906: 326.2455. Found: 326.2452.
CA 02515495 2005-08-09
51
Example 12: Synthesis of 13b
Compound 13a (0.103 g, 0.282 mmol) was dissolved in distilled deionized water
(1 mL) and placed in a water / ice bath. Tetraethylammonium bromide in water
(2.0 M; 141 ~,L, 0.282 mmol) was whereupon a white precipitate formed. After
allow the precipitate to congeal, the solid was collected by vacuum filtration
and
dried using a lyophilizer. The product (0.075 g, 58%) was a white solid. TLC
Rf
(25% CH30H / CH2Ci2) = 0.54; 'H NMR (500 MHz, acetone-ds): b 4.35 (d, 1 H, H-
1, 3.J~.2 - 7.8 Hz), 4.25 (d, 1 H, H-1 ', 3J~ ~,2~ - 7.8 Hz), 3.84 (d, 1 H,
OCHHCca9eC~a9eH, H-7a, ZJ~a,~t, _ -10.9 HZ), 3.81 (2dd, 2H, H-6a, 6a'), 3.70
(d,
1 H, OCHHC~a9eCcageH, H-7a', 2J7a~,7b' _ -10.7 Hz), 3.71 (2dd, 2H, H-6b, 6b'),
3.59
(d, 1 H, OCHHCcageCcageH, H-7b'), 3.49 - 3.40 (m, 3H, H-3,3', OCHHCca9eCca9eH,
H-7b), 3.46 (q, 8H, (CH3CH2)4N+, s J = Hz), 3.40 (m, 2H, H-4,4'), 3.26 (m, 2H,
H-
5,5'), 3.20 (m, 2H, H-2,2'), 1.90 (br s, 2H, OCH2Cca9eCca9eH, H-8,8'), 1.39
(tt,
12H, (CH3CH2)4N+); '3C NMR (126 MHz, acetone-ds): ~ 103.07 (C-1), 102.58 (C-
1 '), 77.96, 77.85 (OCH2CcageCca9eH, C-7,7'), 77.43, 77.37 (C-3,3'), 77.28,
77.20
(C-5,5'), 74.85 (C-2,2'), 71.61 (C-4,4'), 62.70 (C-6,6'), 53.12 ((CH3CH2)4N+),
7.76 ((CH3CH2)4N+); '~B NMR (160 MHz, acetone-ds): -8.86, -15.99, -21.01,
-31.52, -35.85; FTIR (KBr, cm-'): v 3417 (s, br, O-H), 2526 (s, B-H); HRMS
(EI):
Calcd for C9H24B9O6: 326.2455. Found: 326.2462.
Example 13: Synthesis of 14
Compound 13b (0.21 g, 0.46 mmol), TEAF (0.35 g, 2.32 mmol), and
[NEt4]2[Re(CO)3Br3] (0.432 g, 5.61 mmol) were dissolved in distilled water (10
mL) and the mixture heated to reflux for seven days. Analytical HPLC indicated
complete consumption of the starting material and LC-MS indicated that the
major peak in the chromatogram corresponded to the target mass. Semi-
preparative HPLC (80:20 to 54:46 H20 : AcN, t= 20 min) was used to isolate the
product. Yield: 45 mg (16%); ~H NMR (600 MHz, CD3CN): b 4.19 (d, 1 H, 3J~,2 =
7.6 Hz, H-1), 3.90 (2d, 2H, 2J7a,7b = -10.8 Hz, H-7a), 3.69 (dd, 1H, 2J6a.sb =
-11.5
Hz, H-6a), 3.56 (m, 2H, H-6b, 7b), 3.28 (pt, 1 H, H-3), 3.22 (pt, 1 H, H-4),
3.16 (q,
CA 02515495 2005-08-09
52
m, NCH2CH3, H-5), 3.11 (pt, 1 H, H-2), 1.81 (br S, 1 H, OCH2CcaseCcaseH, H-9),
1.21 (t, NCH2CH3); '3C NMR (151 MHz, CD3CN): b 200.39 (C---O), 103.68 (C-1),
77.43 (C-3), 77.19 (C-5), 75.82 (C-7), 74.74 (C-4), 62.72 (C-6), 53.06
(NCH2CH3), 7.67 (NCH2CH3); 11Bj1Hl NMR (192 MHz, CD3CN): b -5.82, -7.65, -
8.78, -11.62, -18.35, -19.55, -20.113; FJTIR (KBr, cm-'): v 3425, 2537, 1999,
1898;
HRMS (ES-QTOF): Calcd for C~zH23BsOsRe: 595.1794. Found: 595.1785.
Example 14: Synthesis of 16
Aqueous sodium fluoride (500 mM, 5 mL) was added to compound 15 (50 mg, 0.21
mmol) along with 3 equivalents of [Re(CO)3(OH2)3]Br (258 mg, 0.62 mmol) and
the
mixture heated to reflux for two days. After allowing the reaction to cool to
room
temperature, the mixture was acidified with 10 M HCI (5 mL). The solution was
subsequently diluted with acetonitrife (10 mL) and cooled at -10 °C
until the organic
layer separated. The acetonitrile layer was removed by pipet and concentrated
under
reduced pressure. The product was isolated by silica gel chromatography (5%
methanol/chloroform) as a dark brown oil (70 mg, 51 °!o). TLC Rf (10%
methanol/chloroform) = 0.15; 'H NMR (600 MHz, CD30D): b 7.58 (d, 1 H, H-aryl),
7.38
(d, 1 H, H-aryl), 7.15 (m, 1 H, H-aryl), 7.066 (d, J = 7.8 Hz, 1 H, H-aryl),
6.98 (t, J = 7.8
Hz, 1 H, H-aryl), 6.91 (d, J = 15 Hz, 1 H, H-aryl), 6.84 (t, J = 7.8 Hz, 1 H,
H-aryl), 6.59
(d, J = 12.2 Hz, 1 H, H-aryl), 6.43 (d, J = 8.4 Hz, 1 H, H-aryl); '3C{'H} NMR
(151 MHz,
CD30D): b 206.01, 156.85, 154.73, 149.24, 144.42, 140.88, 135.56, 132.36,
130.66,
129.71, 128.20, 127.81, 127.22, 126.58, 125.71, 124.88, 114.57, 114.07, 58.54,
58.38, 57.11, 56.85;'18{'H} NMR (160 MHz, CD30D): b -9.04, -11.61, -15.80, -
19.57,
-20.46, -21.93; FTIR (KBr, cm~'): v 3440, 2557, 2001, 1900, 1615, 1513; ESMS
(negative ion): 571.2 [M]-.
CA 02515495 2005-08-09
53
Example 15: Synthesis of 18
n-BuLi (2.77 mL, 6.93 mmol; 2.5 M in hexanes) was added dropwise to a rapidly
stirring solution of 1,7-dicarba-closo-dodecaborane (compound 17) (1.0 g, 6.93
mmol) in dry diethyl ether (125 mL) at -10°C under a nitrogen
atmosphere. The
reaction mixture, which was maintained at -10°C for 45 minutes, was
subsequently added dropwise over 15 minutes to a stirring solution of methyl 3-
bromopropionate (823 ~,L, 1.27 g, 7.63 mmol) in dry diethyl ether (125 mL) at -
10
°C under nitrogen. The temperature was maintained for an additional 30
minutes
at -10 °C then brought to reflux. After 2.5 hours, the crude reaction
was
concentrated under reduced pressure yielding a viscous oil, which was re-
suspended in diethyl ether (75 mL) and extracted with acidified brine (pH =
0.1; 3
x 75 mL). The organic layer was dried over MgS04 and the solvent removed
under reduced pressure giving a pale yellow oil, and the target was isolated
by
silica gel chromatography (gradient elution: 5% ethyl acetate to 10% ethyl
acetate in hexanes). The desired product was recrystalized from a solution of
CH3CN/acetone (6:1) yielding a powdery white solid (0.76 g, 46%). TLC Rf (5:95
EtOAc/hexanes) = 0.13; 'H NMR (300.13 MHz, CDC13): b 1.0 - 3.0 (bm, BH),
1.73 (m, CH2), 2.44 (m, CH2) 2.89 (s, CH3), 3.52 (bs, carborane CH); ~3C{~H}
NMR (50.3 MHz, CDC13): b 28.04, 34.05, 41.56, 41.96, 80.53, 173.93; ~~B{~H~
NMR (96.3 MHz, CDC13,): i5 -7.08, -10.93, -13.72, -17.41; FTIR (KBr, crri'): v
3067, 2603, 1728; MS (ESMS): 273.3 [M+K]+.
Example 16: Synthesis of 7
Compound 18 (0.050 g, 0.217 mmol) and (NEt4]2[Re(CO)3Br3] (0.184 g, 0.234
mmol) were combined in a 10 mL penicillin vial, sealed with rubber septum and
aluminum cap, and then flushed with N2(g) for 10 minutes. A solution
containing
500 mM TEAF(aq)/absolute EtOH (9:1 v/v) Was added (1.0 mL) and the resultant
suspension heated to 100 °C. After 22 hours, the heat was removed and
the
mixture acidified by the addition of 12 M HCI. CH3CN was subsequently added
(1.0 mL) and the vial vigorously shaken for 5 minutes. The mixture was frozen
at
CA 02515495 2005-08-09
54
-5 °C overnight in a freezer resulting in a biphasic mixture with the
organic layer
portioned on top of the frozen aqueous layer. The organic layer was decanted
and concentrated yielding a brown viscous oil. The product was isolated by
flash
column chromatography through silica gel (gradient elution: CH2C12 to 90:10
CH2C12/CHsOH) as a cream coloured solid (0.061 g, 57%). TLC Rf (85:15
CH2C12/CH30H + 0.1% AcOH) = 0.44; 'H NMR (500.13 MHz, 5:1 CD30D-
acetone-d6): S 1.0 - 3.0 (b, BH), 1.39 (t, 3J = 5.8 Hz, CH3), 1.74 (bs,
carborane
CH), 2.09, 2.22 (t, CH2), 2.33, 2.39 (m, CH2) 3.48 (q, 3J = 7.2 Hz, NCH2);
~3C{'H}
NMR (125.77 MHz, acetone-ds): b 7.47, 33.37, 37.59, 52.77, 53.69, 85.31,
172.73, 200.38; "B{'H} NMR (160.46 MHz, CD30D): S -6.68, -10.81, -13.06, -
16.51, -18.44, -22.05; FTIR (KBr, cm-'): v 2032, 1915; HRMS (ESI-Q-TOF):
Calcd for B9C9H~405Re: 475.1370. Found: 475.1404.
Example 17: Synthesis of 19
n-BuLi (2.77 mL, 6.93 mmol, 2.5 M in hexanes) was added dropwise to a rapidly
stirring solution of meta-carborane (1.0 g, 6.93 mmol) in dry diethyl ether
(125
mL) at -10 °C under a nitrogen atmosphere. The reaction mixture, which
was
maintained at -10 °C for 45 minutes, was subsequently added dropwise
over 15
minutes to a solution of di-benzyl-azodicarboxylate (DBzAD) (2.30 g, 7.7 mmol)
in dry diethyl ether (125 mL) at -10 °C under nitrogen. The reaction
was stirred
for 1 hour, whereupon it was heated to reflux 2.5 hours. After cooling to room
temperature, the reaction was concentrated under reduced pressure yielding a
viscous oil, which was taken up in ethyl acetate (75 mL) and washed with 1.0 M
HCI (3 x 75 mL). The organic layer was dried over MgSOa and the solvent
removed under reduced pressure to yield a viscous oil, which was purified by
chromatography on silica gel (isocratic elution: 1:4 Et20/p.Et20) giving the
final
product as a white solid (1.81 g, 59%). TLC Rf (3:7 Et20/p.Et20) = 0.40; mp. _
101-104 °C; 'H NMR (300.13 MHz, CDC13): b 0.9 - 3.3 (bm, BH), 2.88 (s,
CH),
5.07 - 5.09 (m, CH2), 6.65 (bs, NH), 7.18 - 7.30 (m, C6H5); '3C~'H} NMR (50.3
MHz, acetone-ds): b 35.49, 52.88, 68.20, 69.25, 128.25, 128.57, 134.69,
152.85,
CA 02515495 2005-08-09
155.01; "B{'H} NMR (96.3 MHz, CDC13): b -5.60, -11.45, -12.90, -15.65; FTIR
(KBr): v 3310, 3053, 2632, 1745; HRMS (ESI-Q-TOF): Calcd for C~$B~oH2sNzOa:
442.1265. Found: 441.1297.
Example 18: Synthesis of 20
Compound 19 (0.10 g, 0.23 mmol) and TEAF (0.15 g, 0.90 mmol) were
suspended in wet THF (2.3 mL) and the mixture heated to 80°C for 18
hours.
The mixture was subsequently cooled to room temperature, concentrated to
dryness and purified by flash chromatography with silica gel (isocratic
elution: 1:9
CH30H/CH2C12), to give the desired product as a flaky cream coloured solid
(0.124 g, 98%). TLC Rf (85:15 CH2C12/CH30H -~ 0.1% ACOH) = 0.48; 'H NMR
(300.13 MHz, acetone-ds): b 0.5 - 2.5 (bm BH), 1.30 (m, CH3), 3.34 (m, CH2),
3.65 (s, NH), 5.11 (m, CH2), 7.26(m, C6H5); 13C(~H} NMR (50.3 MHz, acetone-
ds,): b 7.55, 15.46, 49.73, 52.80, 65.94, 66.69, 66.98, 67.54, 127.72, 128.09,
128.32, 128.45, 128.86, 129.01, 137.76, 137.96, 156.18, 157.20; "B{'H} NMR
(96.3 MHz, acetone-ds): S 19.65, 18.93, -2.85, -7.07, -20.43, -23.28, -34.13, -
37.72; FTIR (KBr, cm''): v 3364, 3002, 2535, 1755, 1706; HRMS (ESI-Q-TOF):
Calcd for C~sB~oH26N20s: 432.2780. Found: 432.27
82.
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. Where a term in the present
application
is found to be defined differently in a document incorporated herein by
reference,
the definition provided herein is to serve as the definition for the term.
