Macromolecular structures for boron neutron-capture therapy

Details Macromolecular structures for boron neutron-capture therapy Macromolecular structures for boron neutron-capture therapy

1999-01-05

2001-06-19

Lambkin, Deborah C. (Department: 1626)

Organic compounds -- part of the class 532-570 series

Organic compounds

Phosphorus esters

Reexamination Certificate

active

06248916

ABSTRACT:

BACKGROUND OF THE INVENTION
Boron neutron capture therapy (BNCT) is a binary approach to cancer therapy based on the capture of low-energy neutrons by
10
B, which results in the emission of the cytotoxic
7
Li
+
nuclei and &agr;-particles (
10
B(n,&agr;)
7
Li
+
). Tumor-directed antibodies or their immunoreactive fragments are attractive candidates for the selective delivery of
10
B for BNCT, provided that about 1000
10
B atoms can be attached to each immunoreactive protein without significantly altering its biological properties. A number of attempts have been made to link quantities of boron with tumor-directed antibodies, but these have not been successful in delivering therapeutic quantities of
10
B to tumor cells. One such attempt proceeded by randomly conjugating whole monoclonal antibodies (Mabs) with large numbers of small boron-containing compounds. other attempts have been directed to attaching limited numbers of heterogeneous or homogeneous boron-rich polymers. Variability in these studies has limited the progress realized using these techniques.
These studies have also produced disappointing results. For example, an article by Barth, et al., entitled “Conjugation, Purification, and Characterization of Boronated Monoclonal Antibodies for use in Neutron Capture Therapy,” describes a delivery system based on attaching a large number of small boron-containing molecules to an antibody. This study indicated that the boronated antibody had a lower level of specificity for tumor tissue than that typical for a native antibody. Studies, using boronated carboranyl peptides, such as that described by Paxton, et al. in an article entitled “Carboranyl Peptide-Antibody Conjugates for Neutron-Capture Therapy: Preparation, Characterization, and in Vivo Evaluation,” have also shown a reduced specificity for boronated antibodies.
An article by Varadarajan, et al., entitled “Novel Carboranyl Amino Acids and Peptides: Reagents for Antibody Modification and Subsequent Neutron-Capture Studies,” investigated the use of caged boron molecules coupled to peptides. This technique proved unsatisfactory because of excessive hydrophobic bonding between the peptide and the antibody delivery system.
In addition to the poor results obtained using these techniques, these synthesis techniques are frequently slow, sometimes taking weeks to produce a single delivery system. Moreover, if there is to be an eventual commercialization of this technology, a more manufacturable and predictable process must be developed. Little work has been reported on the use of carboranyl derivatives in oligophosphates. One reported use of a carboranyl derivative is in U.S. Pat. No. 4,399,817 to Benedict entitled “Boron Containing Polyphosphonates for the Treatment of Cancer Tumors.” The Benedict reference describes the use of boronated polyphosphonates to delivery boron to calcified tumors. Some of the compounds described incorporate carboranyl derivatives, but these compounds only incorporate carboranyl as an end group and not as a monomer within a oligophosphate.
It is therefore an object of the present invention to produce an phosphate-based boron-rich oligomer that is substantially hydrophilic. It is a further object of this invention to develop a synthesis process which utilizes the substantial technical sophistication of standard DNA synthesis techniques.
SUMMARY OF THE INVENTION
One aspect of the present invention relates to a method of preparing a boron-rich oligophosphate including the steps of preparing a dihydroxy carborane derivative; and forming an oligomer structure having at least two dihydroxy carborane derivatives as monomer units.
Another aspect of the present invention relates to a boron-rich oligophosphate which includes at least two dihydroxy carborane derivatives as monomer units.
Another aspect of the present invention relates to a method of coupling
10
B with a tumor targeting delivery vehicle for BNCT of cancer, comprising the steps of preparing an oligomer having at least two dihydroxy carborane derivatives as monomer units and coupling the oligomer with a preselected tumor targeting vehicle.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to the use of boron-rich oligophosphates in boron neutron-capture therapy (BNCT) of cancer. Although a number of the embodiments of the present invention are described in terms of preparing an antibody-based delivery vehicle, the present invention is also directed to the use of boron-rich oligophosphates without a delivery vehicle, and to the use of boron-rich oligophosphates with a variety of other delivery vehicles.
By way of terminology, the terms closo-carborane, o-carborane, or carboranyl refer to derivatives of the closo-1,2-C
2
B
10
H
12
cage, while nido-carborane refers to derivatives of the [nido-7,8-C
2
B
9
H
11
]
−
cage fragment.
Solution Synthesis
The present invention is directed to the use of derivatives of o-carborane (structure 1) and one aspect of the present invention utilizes these relatively stable boron-rich compounds
because they can be readily functionalized. Synthesis of the carboranes is described in Grimes,
Carboranes
, (1970), which is herein incorporated by reference. In accordance with another aspect of the present invention, lipophilic closo-carborane derivatives are converted under mild conditions to stable anionic nido-carborane derivatives (structure 2, Scheme 1) which exhibit enhanced hydrophilicity. The papers by Hawthorne, et al.,
Inorg. Chem
., 4, 1675 (1965), and by Wiesbock, et al.,
J. Am. Chem. Soc
. 86, 1643-1644, describe this synthesis process and are herein incorporated by reference. With reference to Scheme II, oligophosphates formed in accordance with one aspect of the present invention are derived from the structure 3, or o-carborane diol, which can be prepared by the condensation of dilithio-o-carborane with an excess of trimethylene oxide (yield+90%).
Treatment of the structure 3 diol with one equivalent of TBDMSOTf (tert-butyldimethylsilyltrifluoromethanesulfonate) affords the structure 4 molecule (at a 48% yield by Scheme II) after chromotographic purification of the statistically protected mixture. Materials removed in the chromatographic purification process included a mixture of mono- and diprotected products and unreacted dial. The coupling of the structure 4 monoprotected o-carboranyl diol with isobutanol was then examined under a variety of conditions (Scheme III). The experimental results of these coupling reactions are summarized in Table I. The simplicity, speed, economy and efficiency of the dichiorophosphite coupling reaction (entry #4 of Table I) indicate that this method is a preferred embodiment of the present invention.
TABLE I
Yields for the Synthesis of the Structure 5 Compound
Under Various Conditions.
Entry
Coupling Reagent
R
Yield
1
Cl
2
P(O)OR
2-ClC
6
H
4
-
25%
2
(BTO)
2
P(O)OR
a
″
47%
3
Cl
2
POR
b
″
68%
4
Cl
2
POR
c
″
88%
5
ClP(OR)N(i-Pr)
2
d,c
NC(CH
2
)
2
-
54%
e
Notes:
a
BT = benzotriazole;
b
The initially formed phosphite triester was oxidized in situ with aqueous iodine (0.1 M);
c
The initially formed phosphite triester was oxidized in situ with 0.1 M iodine in
# THF/H
2
O/2,6-Lutidine (40/1/10);
d
The intermediate phosphoramidite was isolated in 90% yield, and was coupled with isobutanol in the presence of tetrazole;
e
Yieid from two steps.
In accordance with an aspect of the present invention, reaction of the monoprotected o-carboranyl diol 4 with isobutanol under a variety of conditions yields the structure 5 phosphotriester. The structure 5a phosphotriester may be converted under acidolytic conditions to the structure 6 alcohol. The structure 6 alcohol may be condensed with another portion of the structure 4 alcohol (monoprotected diol) to produce the structure 7 diphosphate at a moderate yield (35% from two steps, Scheme IV). A second iteration of deprotection and coupling provided the structure 8 triphosphate in a low but reproducible yield (18% from two steps). This process

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