CA2135158A1 - Macromolecular structures for boron neutron-capture therapy - Google Patents

Abstract

A general synthetic method has been developed for the rapid and efficient production of a variety of boron-rich macromolecules suitable for conjugation with of inclusion in receptor-mediated delivery systems as well as other delivery systems. Preparation techniques have been developed to yield precisely ordered oligophosphates which are soluble, hydrophilic, may be homogenous, and may be prepared with a variety of functional groups.

Description

21~51$8 EXPRESS MAIL: TB565605546US
1, PATENT
7618.FD2600 MACROMOLECULAR SL~U~LU~ES FOR BORON NEUTRON-CAPTURE
THERAPY
This invention was made with support under Grant Numbers CA31753-09, CAS3870-01 and CA09306-01 from the National Institutes of Health, U.S. Department of Health and Human Resources. Accordingly, the U.S
government has certain rights in the invention.
8ACR~ROUND OF THE lN v~. llON
Boron neutron capture therapy (BNCT) is a binary approach to cancer therapy based on the capture of low-energy neutrons by lB, which results in the emission of the cytotoxic 7Li+ nuclei and ~-particles (l0B(n,~)7Li~).
Tumor-directed antibodies or their immunoreactive fragments are attractive candidates for the selective delivery of lB for BNCT, provided that about 1000 10B
atoms can be attached to each immunoreactive protein without significantly altering its biological properties. A number of attempts have been made to lin~ quantities of boron with tumor-directed antibodies, but these have not been succesful in delivering therapeutic quantities of lB 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 have 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 21351~8 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 "~ovel 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.
Patent 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.

2135158 ' 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 lB 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.

213~1~8 DET~T~-~n D~8CRIP~ION 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-l, 2-C2BI~2 cage, while nido-carborane refers to derivatives of the [nido-7,8-C2B~I]~ cage fragment.
801ution 8Ynthesis 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, (197~), which is herein incorporated by reference. In accordance with another aspect of the present invention, lipophilic ~,oH,o ~ ~ - H~c_c H

~C ~ HH ~ .~
s~l 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., - 21351~8 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-butyldimethylsilyl-trifluoromethanesulfonate) 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 diol. ' 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 HO~-~C-C~-~OH TBDMSOTf, HO''~'~Co~'~'~OT8DMS
\/ 2~6~ din~. \ /
B1CH10 CH2CI2 B1OHla Sch~D

of the dichlorophosphite coupling reaction (entry #4 of Table I) indicate that this method is a preferred embodiment of the present invention.

coupii~ rea5~ntS! _ ~ O-P-O~~~C\o C'~ ~OTBDMS
isobutanol OR B10H10 5A R = ~Cl-Ph 5b R = ~ yano~thyl SCHE~E ~

21~5158 Tabl- I: Yields for the 8ynthesis of the 8tructure 5 Compound Under Various Conditions.
Entry Coupling Reagent R Yield 1 C12P(O)O~ 2-ClC~ 25%

2 (BTO)2P(O)OR~ " 47%

3 Cl2PORb " 68~

4 Cl2PORC " 88%
5ClP(OR)N(i-Pr)2tC NC(CH2)2- 54%C
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/H2O/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) Yield from two steps.

5~ ~C3~3CO2H: ~HF: H7Q y~O_p_O---- B10~0 OH ., ~04~ 0 C\O/ n~OR \OB/~ OTBDMS

7 n=1, R~2-CIC"H~
8 n_2, R~2-clc~H~
sc~ lV

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 may also be performed by employing a hydroxyl protecting group other than the tert-butyldimethylsilyl group. For example, in a preferred embodiment of the present invention, a relatively labile protecting group such as lo dimethoxytrityl may be used.
In accordance with another aspect of the present invention, the phosphate-protecting groups may be 5 syn-pyridine-2-aWaxime, ~f O-P-O----C\o~C--`OTBDMS
t~t,~". :~.tl guanidin~ ~ Na' g BloH10 pyrrolidine. ~f O- Na~ C\O~C--OTBDMS

~~C\o/C----OTBDMS
-t B~H~0- Na~

SCH~ V

removed from these structures, and the closo-carboranes can be converted to the anionic nido-derivatives.
Removal of the phosphate-protecting groups is done under the standard conditions that are well known to one of ordinary skill in the art. By examining the reactions of the structure Sa monophosphate in a model 21~5158 study, we found that the 2-chlorophenyl phosphate-protecting group could be efficiently removed under standard conditions. Accordingly, treatment of the structure 5a compound with syn-pyridine-2-aldoxime and tetramethylguanidine in THF at room temperature followed by a cation exchange (Na+ form cation exchange resin) afforded the structure 9 sodium salt at an 87%
yield (Scheme V). When this anion was suspended in neat pyrrolidine at room temperature (1 hr) the cl oso-carborane was converted to the nido-carboranyl phosphate at a 71% crude yield (structure 10, Scheme V). A small amount of the structure 11 alkene appears to have also been isolated in this process. Milder conditions would afford substantially higher yields.
The isolation of the structure 10 pho--phate, which is extremely hydrophilic, demonstrates the utility of the present invention's approach to the synthesis of boron-rich macromolecules.

~xperimental Discussion of 8O1ution 8ynthesis lH NMR spectra were recorded on a Bruker AF-200 spectrometer, operating at 200.132 MHz or a Bruker AM-360 spectrometer, operating at 360.134 MHz. 13C NMR
spectra were also recorded on the AF-200 and AM-360, operating at 50.323 and 90.556, respectively. llB NMR
spectra were recorded on a Bruker AM-S00 spectrometer operating at 160.463 MHz. 31p NMR spectra were recorded on the AM-360 operating at 145.785 MHz. Infrared spectra were recorded on a Beckman FTIR spectrometer as a liquid film (neat) or a Nujol mull. Melting points were obtained on a Thomas Hoover "uni-melt" capillary melting point apparatus. HI-RES FAB mass spectra were performed by the University of California at Riverside Mass Spectrometry Facility and obtained on a VG
Analytical ZAB mass spectrometer using a _-nitrobenzyl alcohol matrix.
IH NMR, l3C NMR, IlB and 31p are reported in parts per million (~). The following abbreviations are used: s = singlet; d = doublet; t = triplet; q = quartet; and m = multiplet. IR data are reported in wave numbers (cm~
1). The following abbreviations are used to indicate qualitative intensities: vs = very strong; s = strong;
m = medium; w = weak; and br = broad.
Thin layer chromatography (TLC) was performed using plates from EM Science (silica gel 60 F254; layer thickness 0.2 mm). Visualization was accomplished using ultraviolet light and/or by staining with an 2 1 ~ 8 aqueous potassium permanganate solution (5.0 g KMnO4, 20 g K2CO3, 5.0 mL 5% NaOH, 300 mL H2O). Separation via flash column chromatography was possible using a 6 inch column (3 inch diameter) of silica gel (grade 60, 230-400 mesh, 60 A). Solvent systems were reported asvolume percent mixtures. All reagents were obtained from commercial sources and were used without further purification unless otherwise noted.

0 Ex~mple 1: Di-O-tert-butyldimethylsilyl-bis-hydroxypropyl-ortho-carborane and O-tert-butyldimethylsilyl-bis-hydroxypropyl-ortho-carborane 4:

Under nitrogen, 0.100 g (0.380 mmol) of bis-hydroxypropyl-o~o-carborane 3 was dissolved in a 1:1 solvent mixture of dry methylene chloride and dry diethyl ether at room temperature. Next, 0.0400 mL
(0.380 mmol) of 2,6-lutidine and 0.0900 mL (0.380 mmol) of t~t-butyldimethylsilyl trifluoromethanesulfonate 98%
were added. The reaction mixture was stirred at room temperature for two hours before being quenched with saturated NaHCO3. The resulting aqueous mixture was then extracted twice with ether. The ether extracts were collected, dried over MgSO4, filtered and concentrated on the rotary evaporator. The resulting residue was next purified by flash chromatography using a solvent system which consisted of EtOAc:Hexanes 1:1.
In this manner, 0.039 g (0.0799 mmol, 21.0%) of the diprotected product (R~0.8), m.p. 108-110C, and 0.068 ~1~5158 _ g (0.182 mmol, 47.9%) of the monoprotected product ~
(R=O.S), m.p. 49-51C, were isolated. Final elution of the flash column with ethanol allowed for the recovery of starting material 3.

~$~ \of~si l e~10H10 360 MHz IH NMR (CDCl3) ~ (ppm):

0.0338 (s, 12H, H-3) 0.874 (s, 18H, H-1) 1.69-1.77 (m,4H, H-5) 2.24-2.29 (m, 4H, H-6) 3.59 (t, 4H, J=5.7 Hz, H-4) 90MHz l3C NMR (CDCl3) ~ (ppm):

79.71, 61.62, 32.76, 31.72, 25.83, 18.18, 13.72 ~\0, 7 B10H1o - 21~5158 360 MHz IH NMR (CDC13) ~ (ppm):

0.0316 (s, 6H, H-2) 0.868 (s, 9H, H-l) 1.69-1.75 (m, 2H, H-7) 1.76-1.82 (m, 2H, H-4) 2.26-2.32 (m, 4H, H-S and H-6) 3.60 (t, 2H, J=5.7 Hz, H-3) 3.63 (dd, 2H, J=5.6 Hz, J=9.9 Hz, H-8) 90 Mhz l3C NMR ICDCl3) ~ (ppm):
lS 79.73, 79.49, 61.59, 61.45, 32.71, 32.42, 31.67, 31.59, 25.80, 18.15, 14.14 IR ~nujol): 3356 (br) cm~l, 2589 (s) cm~l, 1256 (s) cm -1, 1387 (br) cm~l NFGATIVF HI-RE8 FAB-M8 for cl~Bloo2si: m/e 376.3571[M-~. Found: m/e 376.3584. ~=1.2 mmu (3.3 ppm) Protected monophosphate Sa:
The compound Sa was synthesized following the method proposed by R. L. Letsinger, et al. Under nitrogen, 0.092 mL (0.590 mmol) of 2-chlorophenyl dichlorophosphite was added to a dry 50 mL schlenk flask cooled to -78C. In a separate flask, 4 was dissolved in dry THF (10 mL) before 0.224 mL (1.90 mmol) of 2,6-lutidine was added. The resulting THF
mixture was then added dropwise to the phosphite and stirred at -78C for 10 minutes. Then O.OS9 mL (0.640 mmol) of isobutanol was added and stirred for 20 minutes at -78C after which it was allowed to stir at room temperature for S minutes. Next an excess of O.lM
I2 (3.05g in THF:pyridine:H20; 80:40:2) was added. This mixture was then extracted twice with 100 mL aliquots ether. The ether layers were then washed with 10%
Na2S2O3 followed by saturated NaC1. Next the organic layers were collected, dried over MgSO4 and filtered.
The ether solvent was stripped off on the rotary evaporator to give a yellow residue which was purified 2~35158 on flash silica gel using a solvent system which consisted of EtOAc:Hexanes 1:1. In this manner, 0.290 g (0.468 mmol, 88.3%) of the desired product 5a (R~0.5) was recovered as a yellow oil.
5 6 7 g 10 lZ ~ 13 3\ H/~

5a 360 Mhz IH NMR (CDCl3) ~ (ppm):

0.0264 (s, 6H, H-6 0.863 (s, 9H, H-5) 0.942 (d, 6H, J=6.7 Hz, H-15) 1.66-1.76 (m, lH, H-14) 1.90-2.01 (m, 4H, H-8 and H-ll) 2.22-2.30 (m, 4H, H-9 and H-10) 3.58 (t, 2H, J=5.7 Hz, H-7) 3.96 (td, 2H, J=2.7 Hz, J=6.5 Hz, H-12) 4.18 (dd, 2H, J=6.0 Hz, J=13.0 Hz, H-13) 7.13 (t, lH, J=7.6 Hz, H-3) 7.25 (t, lH, J=7.6-8.0 Hz, H-2) 7.42 (d, lH, J=8.1 Hz, H-4) 7.43 (d, lH, J=8.1 Hz, H-l) 90 MHZ ~3C NMR (CDCl3) ~ (ppm):
146.6, 130.7, 127.9, 126.0, 125.3, 121.3, 79.71, 78.61, 74.99, 74.92, 67.23, 67.16, 61.48, 32.77, 31.65, 31.21, 30.22, 30.14, 29.05, 28.97, 25.80, 18.51, 18.15, 13.74 IR (ne~t): 2594 (s) cm-~, 1259 (s) cm~l FAB-~8 for C~ DC10sP8i: m/e 621 ~M+l]

-- 213~158 Hydroxy monophosphate 6:
The deprotection of Sa afforded compound 6. 0.211 g (0.340 mmol) of 5a was suspended in acetic acid-water-tetrahydrofuran (3:1:1). The reaction mixture was S allowed to stir at room temperature until Sa went into solution. The reaction was quenched thoroughly with saturated NaHCO3 and then extracted twice with 200 mL
aliquots of ether. The ether extracts were then collected, dried over MgSO4, filtered and concentrated on the rotary evaporator. The resulting residue was purified using flash chromatography. The column was first eluted with EtOAc:Hexanes 1:1. Next the same column was eluted with 100% EtOAc. Concentration of the fractions from the second elution gave 0.117 g lS (0.231 mmol, 68.0%) of 6 as a yellow oil.
r 9 7 6 4 1~) 3 0~ ~ 2\- 1 B10H10 ~) d b 21~5158 360 MHz IH NMR (CDCl3) ~ (ppm):
0.941 (d, 6H, J=6.7 Hz, H-l) 1.66-1.77 (m, lH, H-2) 1.92-2.07 (m, 4H, H-5 and H-8) 2.31-2.37 (m, 4H, H-6 and H-7) 3.59 (t, 2H, J=5.4 Hz, H-9) 3.95 (td, 2H, J=2.9 Hz, J=6.5 Hz, H-4) 4.22 (dd, 2H, J=2.8 Hz, J=7.0 Hz, H-3) 7.15 (t, lH, J=7.8 Hz, H-b) 7.26 (t, lH, J=8.0 Hz, H-c) 7.41 (d, lH, J=8.3 Hz, H-a) 7.43 (d, lH, J=8.2 Hz, H-d) 90 ~Ihz l3C NHR (CDCl3) 6 (ppm):
146.7, 130.7, 128.0, 126.2, 125.5, 121.3, 79.97, 78.42, 75.21, 75.13, 67.58, 61.25, 32.69, 31.91, 31.04, 30.20, 29.05, 18.49 145 M~z 3Ip N~R (CDCl3) ~ ~ppm):
External Reference H3P04/D20 -9.220 External Reference H3P04/CDCl3 -6.584 IR (neat): 2584 (s) cm~~, 1263 (s) cm~~
HI-RES FAB-MS for Cl8H36BIoCl05P: m/e 508.2919 [M-].
Found: m/e 508.289. ~=3.2 mmu (6.4 ppm).

Protected diphosphate 7:
The compound 7 was synthesized in a manner similar to that of 5a. 0.051 mL (0.330 mmol) of 2-chlorophenyl dichlorophosphite was placed in a schlenk flask, under nitrogen, and cooled to -78C. In a separate flask, 0.151 g (0.290 mmol) of 6, dissolved in 10 mL dry THF, and 0.125 mL (1.10 mmol) of 2,6-lutidine were combined and added dropwise to the phosphite. The resulting mixture was stirred at -78C for 10 minutes before 0.134g (0.360 mmol) of ~ in dry THF- was added and stirred 20 minutes longer before the cold bath was removed. After 5 minutes, an excess of O.lM I2 (3-05 g in pyridine:THF:H20; 40:80:2) was introduced.
Extraction with ether followed. The ether extracts were washed once with 10% Na2S203, once with saturated NaCl, dried over MgS04 and filtered. Solvent ether was then stripped off under reduced pressure. The crude product was chromatrographed on flash silica gel using a solvent system of EtOAc:Hexanes 1:1. In this manner, 0.155 g (0.147 mmol, 50.8%) of the desired product 7 was isolated.

360 MHz lH NMR (CDCl3) ~ (ppm):

0.0252 (s, 6H, H-16) 0.862 (s, 9H, H-17) 0.936 (d, 6H, J=6.6 Hz, H-l) 1.63-1.75 (m, lH, H-2) 1.92-2.05 (m, 8H, H-5, H-8, H-11 and H-14) 2.17-2.31 (m, 8H, H-6, H-7, H-12 and H-13) 3.57 (t, 2H, J=5.7 Hz, H-15) 3.95 (td, 2H, J=2.7 Hz, J=6.6 Hz, H-4) 4.17 (dd, 6H, J=6.0 Hz, J=10.1 Hz, H-3, H-9 and H-10) 7.15 (dd, 2H, J=7.8 Hz, J=18 Hz, H-c and H-g) 7.26 (dd, 2H, J=6.3-6.7 Hz, J=14 Hz, H-b and H-f) 7.42 (d, 4H, J=8.0 Hz, H-a, H-d, H-e and H-h) 16 15 13 12 ~0 ~ g 7 6 ~ G 3 C g - 90 MHz ~3C NMR (CDCl3) ~ (ppm):
146.3, 146.2, 130.8, 130.7, 128.1, 128.0, 126.4, 126.1, 125.3, 125.2, 121.4, 121.3, 79.72, 78.62, 78.49, 78.04, 75.05, 74.97, 67.57, 67.50, 67.44, 67.37, 67.19, 67.12, 61.46, 32.77, 31.64, 31.19, 31.06, 30.23, 30.16, 29.68, 29.05, 28.97, 25.82, 18.52, 18.16, 14.10 145 MHz 31p NMR (CDCl3) ~ (ppm):
External Reference H3PO4/CDCl3 -5.63, -5.66 NEGATIVE FAB-MS for C38H76B2ocl2o9p2si m/e 1053 Hydroxy diphosphate:
This compound was prepared in the same manner as 6.
0.176 g (0.167 mmol) of 7 was suspended in 100 mL of CH3COOH:THF:H2O 3:1:1 and stirred at room temperature until all was in solution. The reaction was quenched with saturated NaHCO3 and extracted with ether. The ether extracts were collected, dried over MgSO4 and filtered. The solvent ether was then removed. The crude product was purified on flash silica gel. The column was first eluted with EtOAc:Hexanes 1:1 and then with 100~ EtOAc. Concentration of the EtOAc fractions afforded 0.099 g (0.105 mmol, 63.1~) of the desired compound as a yellow oil.

HO~
B1oH10 ~C B~OH10 360 MHz 1H NNR (CDCl3) ~ (ppm):

0.939 (d, 6H, J=6.5 Hz, H-1) 1.66-1.74 (m, lH, H-2) 1.86-2.07 (m, 8H, H-5, H-8, H-11 and H-14) 2.17-2.33 (m, 8H, H-6, H-7, H-12 and H-13) 3.55 (t, 2H, J=5.7 Hz, H-15) 3.95 (td, 2H, J=2.2 Hz, J=6.5 Hz, H-4) 4.19 ~dd, 6H, J=6.6 Hz, J=12.7 Hz, H-3, H-9 and H-10) 7.16 (dd, 2H, J=7.8 Hz, J=16.0 Hz, H-c and H-g) - 21~5158 7.27 (dd, 2H, J=6.8 Hz, J=15.0 Hz, H-b and H-f) 7.36-7.4s (m, 4H, H-a, H-d, H-e and H-h) go MHz l3C NMR ~CDC13) ~ ~ppm):
146.5, 146.2, 130.8, 130.7, 128.2, 128.0, 126.5, 126.1, 125.3, 125.2, 121.5, 121.3, 79.88, 78.60, 78.54, 78.42, 75.10, 67.80, 67.73, 67.59, 67.27, 60.98, 32.56, 31.79, 31.12, 30.97, 30.15, 30.09, 28.99, 28.91, 28.51, 18.44 1~5 Mhz 31p NNR ~CDCl3) t ~ppm):
External Reference H3PO4/D2O -8.874, -8.454, -8.662, -8.694, -8.763 IR (neat): 3462 (br) cm~l, 2593 (s) cm~l, 1234 (s) cm~
NEGATIVE ~I-RES FAB-M8 for ~aB20Cl29P2 m/e 942-5107 Found: m/e 942.513. ~ = 2.4 mmu (2.5 ppm).
Triphosphate 8:
The compound 8 was synthesized in a manner similar to that of 7. In a schlenk flask, 0.017 mL
(O.110 mmol) of 2-chlorophenyl dichlorophosphite was cooled to -78C under nitrogen. 0.095 g (0.100 mmol) of the hydroxy diphosphate was dissolved in 5 mL dry THF before 0.042 mL (0.360 mmol) of 2, 6-lutidine was added. This THF solution was then added dropwise to the phosphite and stirred at -78C for 5 minutes. Next 0.045 g (0.120 mmol) of 4 dissolved in 5 mL dry THF was added and stirred at -78C for 20 minutes. The reaction mixture was then allowed to stir at room temperature for 5 minutes before an excess of 0.lM I2 (3.05 g in THF:pyridine:H2O; 80:40:2) was added. The resulting solution was extracted with ether. The ether extracts were then washed with 10% Na2S2O3 and saturated NaCl, dried over MgSO4 and filtered. After the solvent was removed, the crude product was columned on flash silica gel. The column was first eluted with EtOAc:Hexanes 1:1 and then 100% EtOAc. Concentration of the EtOAc fractions gave 0.042 g (0.028 mmol, 28.3%) of 8 as a yellow oil.

æ 21 19 1- ~6 0 ~5 ~ 12 1 O ~ 7 6 ~ 0 3 23 ,S ~ 1 360 MHz IH NMR ~CDCl3) 6 (ppm): r 0.0252 (s, 6H, H-22) 0.861 (s, 9H, H-23) - 10 0.933 (d, 6H, J=6.7 Hz, H-l) 1.67-1.75 (m, lH, H-2) 1.83-2.03 (m, 12H, H-5, H-8, H-ll, H-14, H-17 and H-20) 2.17-2.31 (m, 12H, H-6, H-7, H-12, H-13, H-18 and H-l9) 3.57 (t, 2H, J=5.7 Hz, H-21) 3.95 (d, 2H, J=2.6 Hz, J=6.6 Hz, H-4) 4.15-4.20 (m, 10H, H-3, H-9, H-10, H-15 and H-16) 7.13-7.18 (m, 3H, H-c, H-g and H-k) 7.23-7.29 (M, 3H, H-b, H-f and H-j) 7.39-744 (m, 6H, H-a, H-d, H-e, H-h, H-i and H-l) 90 ~E8 ~3C NMR (CDCl3) 6 ~ppm):
146.3, 146.7, 130.8, 130.7, 128.4, 128.2, 128.0, 126.5, 126.1, 125.3, 121.5, 121.4, 78.64, 78.53, 75.06, 74.99, 67.53, 67.14, 61.47, 32.76, 31.65, 31.16, 31.03, 30.20, 30.13, 29.05, 28.97, 25.81, 18.51, 14.11 35 145 M~8 3Ip NMR (CDCl3) 6 (ppm):
External Reference H3PO4/D2O -8.212, -8.240, -8.258 -N~GATIVE FAB-N8 for C5,~-R~Cl3OI3P38i: m/e 1488 [M-].

S Anionic monophosphate 9:
Deprotection of 5a provided compound 9. 0.0116 g (0.950 mmol) of 2-pyridinealdoxime and 0.120 mL
(0.820 mmol) of 1,1,3,3-tetramethyl guanidine were dissolved in 2.87 mL of dry dioxane:acetonitrile (1:1).
This solution was then added to Sa (0.115 g, 0.190 mmol). The reaction mixture was allowed to stir at room temperature for 28 hours. Next Bio. RAD. AGSOW-x8 ion-exchange resin (50-100 mesh; 22 g) ammonium form was added and stirred for 30 minutes. The resin was then filtered off and washed with tetrahydrofuran. The THF was removed under vacuum. The resulting residue was then purified on flash silica gel. The column was first eluted with CHC13:MeOH 8:2 followed by 100% MeOH. -In this manner, 0.070g (0.133 mmol, 71.4%) of 9 was formed.

6 7 g ~0 12 ~ 13 B\

I~H4 360 MHs IH NNR (CDCl3) ~ (ppm): -0.0460 (s, 6H, H-6) 0.878 (s, 9H, H-5) 0.918, (d, 6H, J=6.2 Hz, H-lS) 1.70-1.90 (br m, 5H, H-8, H-14 and H-11) 2.16-2.43 (br m, 4H, H-9 and H-10) 3.52-3.69 (br m, 4H, H-7 and H-12) 3.77-3.91 (br m, 2H, H-13) 90 MHz 13C NMR (CDCl3) ~ (ppm):
80.10, 79.10, 72.80, 64.80, 61.60, 33.19, 31.91, 30.14, 29.35, 26.72, 25.87, 19.13, 18.19, 14.11 145 MHz 31p NMR (CDC13) ~ (ppm):
External Reference H3PO4/D20 -4.238 160 MHz llB NMR ~ (ppm):
-4.336, -9.860 NEGATIVE HI-RES FAB-MS for Cl8H~6BloOsPSi: m/e 511.3782 [M-]. Found: m/e 511.3808. ~ = 2.5 mmu (4.9 ppm).

nido-Anionic monophosphate 10:
Degradation of closo-9 with pyrrolidine provided nido-10 0.070 g (0.133 mmol) of 9 was treated with 0.570 mL (6.83 mmol) of pyrrolidine and stirred at room temperature for one hour. Afterwards, the pyrrolidine was removed in vacuo. The resulting residue proved to be the pyrrolidinium salt of nido-10 (0.055 g, O. 093 mmol, 70. 5% crude).

5 6 7 9 1O 12 V ~3 T ~ ~ B\~H)/ 11 ( ~~

' 3C0 ~I}Iz IH MR (CDCl3) ~ (ppm):
0.0122 (s, 6H, H-6) 0.857 (s, 9H, H-S) 9.907 (d, 6H, J=6.7 Hz, H-15) 1.59-1.71 (m, lH, H-14) 1.75-1.90 (m, 4H, H-8 and H-ll) 1.96-2.03 (m, 4H, H-9 and H-10) 3.21 (t, 2H, J=6.8 Hz, H-7) 3.50-3.54 (m, 2H, H-12) 3.67-3.79 (m, 2H, H-13) 90 ~HZ ~3C NMR (CDCl3) ~ (ppm):
72.20, 65.30, 53.68, 31.89, 30.84, 30.33, 29.32, 26.70, 25.97, 19.04, 18.30, 14.08 1~.5 MH8 31p N~lR (CDCl3) ~ (ppm):
External Reference H3PO4/D20 -1. 246 160 ~I8 IIB N~IR (CDCl3) ~ (ppm):
-11.23, -17.77, -34.76, -37.55 NBGATIVIS HI-RE8 FAB-M8 for Cl~. R9o5P8i: m/e 500.3689 tM-]-Found: m/e 500.3714. ~ =2.4 mmu (4.9 ppm) .

- 21~5158 General DNA Synthesis The chemical synthesis of DNA customarily involves the repetitive coupling of suitably functionalized nucleosides, with the growing polymer remaining attached to a solid support throughout the synthesis.
Each step of this synthesis has been extensively studied, resulting in the development of an overall procedure that is fast (just minutes per monomer), efficient (coupling efficiency routinely >99%), and amenable to automation. Techniques for automated DNA
synthesis are well known in the art and are described in, for example, Gait, M.J. (ed.) Oligonucleotide Synthesis: A Practical Approach, (1984), which is herein incorporated by reference. A general scheme for the most common method of DNA synthesis, using B-cyanoethyl protected diisopropylaminophosphoramidites, is shown below (Scheme VI). In general, this method involves:
a) removal of an -OH protecting group from a polymer supported monomer;
b) coupling of the resulting free -OH with a protected diisopropylaminophosphoramidite utilizing tetrazole as an acid catalyst;
c) oxidation of the initially formed phosphite triester to form the phosphate triester;
d) acetate ester formation on unreacted -OH groups (blocking);
e) acid deprotection of the newly introduced dimethoxytrityl ether protecting groups;
f) repetition of steps (b) - (f) until complete - oligomer has been assembled; and g) removal of oligomer from the solid support using NH40H, with concomittant removal of the phosphate protecting groups.

This method can be readily adapted for use with other phosphoramidites (dimethylamino etc.), different phosphate protecting groups (methoxy vs. B-cyanoethyl), and alternate activation/oxidation strategies (H-2~435158 NCCHzCHzO
(i PrkN~P-b) ~ ~
, ~ ~ ODMTr o~1 OCH7CH7CN
(Sur~7~ O (~_uv~ e~tra)zob ~ ~ ~ i-O
f~o - l Fo~ d) acetic anhydride ~_ ~3 ODlUTr~3 OH ~ ~3 C --OMTr \~epeatcycie t~ H

¦ l < n ~ -150 ¦ ~O ~ j- o ~ OH ~ ,OCH2CH7CN
~atcomdebon o~ ' ~O~Ho~gomersynthesl~ ~~ ¦ O ~ \
n (~ O)~H

Scheme VI

phosphonate chemistry). In general, however, Scheme VI
describes the most common method of DNA synthesis.

In accordance with an aspect of the present invention, the DNA synthesis chemistry described in Scheme VI has the general requirements that a candidate monomer for this method of oligophosphate synthesis should contain both a dimethoxytrityl protected alcohol and a ~-cyanoethyl-diisopropylamino-phosphoramidite. A
schematic of oligophosphate synthesis in accordance with an aspect of the present invention involving a NccH2cH2ol ~I~Pr)2N P`o b) (~
~ f~\ O ODMTr ~O
1~ SU¢~I . O Suwcn )-- Telrazole ,~
(~ ~ ) d) ~ébc anhydride (~, o o - P o ~,~ a --DMTr ODMTr OH \ OCH2CH2CN
\repeat c~e HO (~ ~I H' (~) ~ (NH40H~ o~q o~O~H
j l<n<-~50 ¦ O~P-O~O~H oligome a~ynlhe~ OCH2 ~ O- NH~' ~n Scheme VII
generalized monomer is shown in Scheme VII.

SOLID-PHASE BORON-RICH OLIGOMER ~YNln~SIS
Boron-rich oligophosphates were synthesized on a one micromole scale using a Applied Biosystems DNA
Synthesizer, Model 391. Standard concentrations of reagents were used: The monomer was dissolved to a concentration of 0.1 M in CH3CN; the deprotecting solution was 3% trichloroacetic acid in dichloromethane; the capping solutions were an 8:1:1 - 213~158 mixture of THF:lutidine:acetic anhydride and 1.2%
dimethylaminopyridine in THF; the oxidant was 0.5 M I2 in methanol, pyridine and water (7:2:1). All steps were of standard duration; for example, the coupling time was 15 seconds.
Oligomers containing up to 40 carboranyl monomers were synthesized with coupling efficiencies of greater than 99%. The interspersion of these oligomers with a number of non-carboranyl monomers was performed. These monomers included linking groups (amines and thiols), fluorescent labels, a biotin derivative, and thymidine.
These oligomers were isolated from the synthesis support by treatment of the supported polymer with NH40H
for five minutes at room temperature. The water soluble oligomers were shown to contain closo-carboranes by IlB NMR. Extended treatment of the oligomers with NH40H (30 minutes at 80) resulted in complete conversion of the boron cages to the nido-carborane analogues, as determined by IlB NMR. These compounds have been shown to be of the correct composition by negative-ion electrosp-ay mass-spectral measurements. The homogeneity of these oligomers was demonstrated by polyacrylamide gel electrophoresis (20%
gel, 7 M urea).
PHOSPHORAMIDITE MONOMER SYNTHESIS
The phosphoramidite monomers required for oligophosphate synthesis in accordance with an aspect of the present invention are simple derivatives of diols. In a broad aspect of the present invention, almost any compound with two hydroxyl groups can be converted into an appropriate monomer. Dihydroxy compounds are converted into appropriate monomers by:

-a) monoprotection with the dimethoxytrityl protecting group; and b) conversion of remaining hydroxyl group to the phosphoramidite by treatment with chloro ~-cyanoethyl-N,N-diisopropylaminophosphite.

NCcH2cH20 OH OH (I Pt)2N-P`o $ pyridine l$~ NCCH2cH20 $
OH ODMTr (~PrkN `Cl OD~Tr Scheme VIII

.

Scheme VIII shows a general example of a monomer synthesis in accordance with the present invention.
BORON-RICH DIOL SYNTHESIS
In accordance with a preferred embodiment of the HO '` ` '--C C'--f OH1) ~MTr apyri~ule DMTrO~~C~,C~~`O' `N(i-Pr)z B1OH,o 2) NCCH2CH20loHlo A (IP~)2N~P`CI MA ~-4~h o~r~l) E~(j.Pr2), CH2CI2 Scheme IX
present invention, the monomers used in oligophosphate synthesis contain substantial quantities of boron. As such, certain aspects of this invention are related to methods for the production of boron-rich diols and 213~1~8 compounds that can be converted into appropriate monomers (~-cyanoethyldiisopropylamino-phosphoramidites) using the procedures of one aspect of the present invention.
The synthesis of boron-rich oligophosphates in accordance with the present invention uses boron-rich diols as the ultimate monomeric materials. In a preferred embodiment of the preseent invention, these diols are further derivatized to afford the starting materials for oligomer synthesis. The process for making a number of these diols is illustrated in the schematic formalism well-known to those of ordinary skill in the art.

- ~135158 PROCEDURES FOR DIOL SYNTHESIS

A) c o~c a~ ~ ~ HiO(CH~ ~ ~ (CH~nOH
b~ ~ C) H B1oHlo B) RX HO ~ HO~NHR U HO--~NR2 9~ HO--f NR

a~ E~C~CO2EI
C) RX b~ IIAIH~ ~ HO RX,~H ~ HO~OH

a) O~~~o U~
HO'~f O-R

E) RX j~o H~ f NHR Ho~o~-Np~ HO~yNRJ
~ HO H10 HO
HO

F) ~co2~ ~ ~HO~f N R

G) RC02H HO~NH2 ~,~ o HO

1~ ~ H. ak~l~ UYI R~ CH2~n~
X baYin~ ~cup R~ C~n,C
~1, ~, o~ a~S, OT~ H,o DELIVERY SYSTEMS
The oligomers described in accordance with certain aspects of the present invention may be used as agents for the concentration of boron in tumor cells for the boron neutron capture therapy of cancer. These oligomers may be suitable for use with a variety of targeting methods or delivery systems.

1. Unbound Delivery Strategies The term "unbound" is used in accordance with an aspect of the present invention to indicate that no covalent bonds are formed between a boron-rich macromolecule formed in accordance with an aspect of the present invention and a delivery vehicle as specified in accordance with the teachings of the present invention.

A) Non-Targeted Tumor Preferential Accumulation of Macromolecules.
Macromolecules are known to accumulate preferentially in a tumor. As such, one aspect of the present invention uses such tendencies to direct macromolecules formed in accordance with the present invention to tumors independent of tumor-targeting delivery vehicles.

~1'35158 B) Liposomal Delivery.
Further to an aspect of the present invention, liposomes have been developed that may be used to deliver boron-rich molecules to tumor cells.
Encapsulation of the boron-rich oligophosphates in liposomes is facilitated by the high water solubility of certain of the oligomers of the present invention.
A further advantage of the present invention is that, for a number of these molecules, the diffusion of these macromolecules away from the tumor cells subsequent to delivery is substantially slowed in certain embodiments by virtue of their size.

C) Avidin/Biotin Based Delivery ~d ~ }
~3~
~' ~rq ~N

Scheme X

In accordance with the teachings of the present invention, Biotin-substituted oligomers are prepared either by attaching biotin residues during solid-phase synthesis (a num~er of reagents are commercially available for this very purpose) or by post-synthetic modification of functionalized oligomers with suitable 21351~8 biotin derivatives (e.g. via the reaction of amine-substituted oligomers with active esters of biotin). A
preferred embodiment of the present invention uses the biotin/avidin interaction as a mode of delivery. The high affinity of this attraction as well as the tetrameric nature of avidin (or strepavidin), allows multiple oligomers to be localized by one avidin. In accordance with this embodiment, avidin is prelocalized in tumors. Several methods for accomplishing this goal are known. The overall concept is shown in Scheme X.

D) Sense/Antisense Oligonucleotide-Based Delivery In accordance with a further aspect of the present invention, sequences of DNA oligomers are appended to the boron-rich oligophosphates. This linking strategy is used in a preferred embodiment of the present r~
nu~
~ I I I I
a b u a ~ ~ b <~ ~ b b ~ ~ -I ll L

Tum TV~
~bd Scheme Xl 21~5~58 invention, particularly with respect to the solid-phase synthesis, which is performed on instruments that are already optimized for the synthesis of DNA oligomers.
A complementary strand of DNA is attached to the tumor targeting antibody. Scheme XI gives a general description of this method.

2. Covalently Bound Delivery Systems A) Synthesis of Reactive Oligomers In accordance with the present invention, covalent bonds are formed between an oligomer and a delivery vehicle through the synthesis of oligomers containing Ol~on~r tr~n / O
solutb- ~nlhk .~ R- -O~O-P--0~011 o~. OPg NC o p`o--~N ~

bl 1~ ~J~r, U~Tt=~Cid bbb f ~ ptOl C~O grDup c~ Ck~CC02H

R~O~O-P~;O~O-~-O--~'NH2 P~ O--CN

Scheme Xll 21351~8 reactive functional groups. Amine groups are extremeIy versatile functional handles for conjugation reactions.
Terminal or internal amine groups can readily be incorporated during the solid-phase synthesis of the boron-rich oligophosphates in accordance with the present invention. Preferably, commercially available O~n~r f~ / O ~
~o p ~ t ~- ~~ H

HO~ ~N ~
t j~ 2 a~ o ~~~ ~Tr ~ EU(~-~2 b) tZ ~b~
c) a~HCCO,H

R~O~O-P~O ~O-P-O--~, YH2 OP~ OPII

Scheme Xlll reagents are used for this purpose. The same reagents are used in accordance with a different aspect of the present invention to append amine groups to oligophosphates synthesized in solution (Scheme XII).
In an alternative embodiment, protected amino-alcohols are used in the normal one-pot coupling reaction currently being used for the solution oligophosphate synthesis (Scheme XIII).

~13515~

Oligomer from solution synthesi~
(R=capping group, ~ , O
Pg-protecting group) RtO~ 0 ~c O
`o~s~s~ - o~r + ~ t ) 12 commercially available thiol c) C~ linker, DMT = acid fabile dimethoxytrityl protecting group thiol is masked as a disulfide R~O~O- P~O~--o-P-O--/--P~ ~, ~C~

Scheme XIV

J o ~
Oligomer from R~O~O-P~O ~OH
solution synthesis ~P9 n ~R=capping group, /~
Pg-protecting group) HO~ ~S,~h 00 PgO
~ Et~Pr)2 PgO
C1 ' O ~S - S~ r + ~ 2 ~\
la c~ C~ chlorophosphite formed 'In situ', thiol masked as disulfide R~O~0-P~ o ~-'P9 n ~b Scheme XV

A second class of readily available functional handles used in accordance with the teachings of the present invention for attachment of delivery vehicles are the thiols. Although thiols are extremely reactive, they are remarkably selective. Again, it is preferable to use any of a number of commercial reagents to attach thiols to oligophosphates during solid-phase synthesis. These reagents can also be used to functionalize oligophosphates synthesized in solution (see Schemes XIV and XV). In accordance with r O, R--O~O-P -O~O-P-O~
P~ ~ n P

Traut's R~ag~nt Nlt ~S

O ' O H
R--O~O r o~o-P-o~~~ ~~s~
~ ~ n P~ ~N

Scheme XVI

an alternate embodiment of the present invention, thiols are introduced through the reaction of various amine-reactive thiolating reagents (such as Traut's -Reagent, Scheme XVI), which would convert amine-substituted oligomers to thiol substituted analogues. A
third method for introducing reactive thiol handles on the oligophosphates would involve the synthesis of oligomers containing one or more phosphorothioate group (Scheme XVII). In accordance with a preferred embodiment of the present invention, this class of compounds may routinely be made on solid-phase synthesis instruments or in solution.

- B) Conjugation Chemistry With the oligophosphates fitted with reactive functional groups a number of different methods are used in accordance with the present invention to attach the oligophosphate to a desired delivery vehicle. The two main classes of conjugation reactions practiced in accordance with the present invention are those involving bifunctional linking molecules and those that involve direct conjugation.
i) Bifunctional crosslinking reagents. A large variety of bifunctional crosslinking reagents are either commercially available or easily synthesized and their chemistry is well known. Heterobifunctional crosslinking reagents are effective at coupling two components with similar reactive groups - a thiol containing boron-rich oligophosphate and a thiol containing targeting molecule, or an amine containing boron-rich oligophosphate and an amine containing 'O
R-O--P-O ~0---H
, OPg ~ n intermediate a'.30phosphale- a) P-O~ODMTr R=solid support or capping group PgO
+ tetrazole b) suH~ dlion reagent c) Cl2HCCO2H

d) rernoval o~ ~hosphate protecting groups ~O ' O - ~
R-O---P-O~O---P-O~OH
,~M' ~ n S M~

monopho-~pho~oll ~ te Scheme XVII

targeting molecule. Homobifunctional crosslinking - reagents are effective at coupling components with orthogonally reactive functional groups - amine containing boron-rich oligophosphates with thiol-containing targeting molecules, for example.
ii~ Direct Conjugation. A multitude of methods are available for the direct conjugation of functionalized oligomers to tumor-targeting molecules. Amine containing oligomers can be readily coupled with active esters derived from carboxylic acids to form amides, - 21~5158 with aldehydes to form Schiff Bases (and amines upon subsequent reduction), with epoxides to form ~-hydroxy amines, and with carbons containing good leaving groups to form secondary, tertiary, or quaternary amines.
In accordance with a different aspect of the present invention, thiol containing oligomers are coupled directly to tumor-targeting molecules containing appropriate functional groups. Maleamides react very selectively with thiols at pH's around 6, and thiols readily alkylate electrophilic carbon centers (especially ~-haloacetate esters and amides).
In a different preferred embodiment, alkylation reactions are preformed with phosphorothioates. One preferred reaction mode using the thiol group takes advantage of its ability to be oxidatively coupled with another thiol, forming disulfide compounds. In another embodiment of the present invention, a disulfide bridged species is formed with a thiol exchange of a free thiol with an activated disulfide.

Conjugation Strategies for Tumor-Targeting Agents In accordance with the present invention, immunoprotein based delivery systems are used to deliver boron-rich macromolecules, including whole monoclonal IgG molecules, IgG derived fragments (F(ab') 2 and Fab'), and smaller engineered fragments (Fab-SH
'single chain antibody'). The conjugation of boron-rich oligomers with these compounds could occur through - 21351~8 free amine groups found on these proteins or through free thiols, either those revealed via antibody reduction, those created by amine modification, or those included in the design of engineered fragments.
Another embodiment of the present invention uses an aldehyde at the conjugation site, which is revealed upon oxidation of the carbohydrates found on intact IgG
antibodies, which is then reacted with amine substituted oligomers.
Regulatory peptides may be also used as delivery vehicles in accordance with the present invention.
These compounds are conjugated through their free amine groups or through endogeneous or created thiols. Other small molecules known to accumulate in tumors are conjugated with boron-rich oligophosphates by taking account of endogeneous binding sites. For example, amino-substituted oligomers are attached to free carboxyl groups found on porphyrins in accordance with -a preferred embodiment. In general, the flexibility of the synthesis of the boron-rich oligomers allows for conjugation to a wide variety of molecules employing a spectrum of coupling strategies.
Boron-rich compounds are those that have more than ten percent by weight boron. In a preferred embodiment of the present invention, compounds with in excess of 20~ boron by weight are preferred.
To achieve their full potential as 10B delivery vehicles, these building-block molecules must be 213~158 enriched in the lB-isotope. Enrichment to 95-96~ 10B is commonly employed since the ultimate source of boron, boric acid, is commercially available at this level of isotope purity. All of the carborane molecules discussed herein can be derived starting from boric acid, or more directly from lB2H6. Accordingly, the production of enriched carboranes is readily achievable. The added cost of the enrichment process may be determinative in establishing which methodology is most cost-effective for production.
While the present invention has been described with reference to specific preferred embodiments thereof, it will be understood by those skilled in this art that various changes may be made without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt the invention to a given situation without departing from its essential teachings.

Claims (75)

What is claimed is:

1. A method of preparing a boron-rich oligophosphate comprising the steps of:
(i) preparing a dihydroxy compound which comprises boron;
(ii) converting said dihydroxy compound to a mono-hydroxy-protected dihydroxy compound;
(iii) reacting said mono-hydroxy-protected dihydroxy compound with a coupling agent comprising phosphorus to form a monomer unit; and (iv) reacting at least a plurality of said monomer units to form an oligophosphate.

2. The method of claim 1 wherein a plurality of homogeneous oligophosphates are formed.

3. A method of coupling 10B with a tumor targeting delivery vehicle for BNCT of cancer, comprising the steps of:
(i) preparing a boron-rich oligophosphate according to the method of claim 1; and (ii) coupling said boron-rich oligophosphate with a tumor targeting vehicle.

4. The method of claim 3 wherein said tumor targeting vehicle comprises a protein based delivery system.

5. The method of claim 4 wherein said protein based delivery system comprises an antibody.

6. The method of claim 5 wherein said antibody is specific to a tumor antigen.

7. A method of preparing a boron-rich oligophosphate comprising the steps of:
(i) forming a carboranyl diol;
(ii) converting said diol to a mono-hydroxy-protected diol;
(iii) converting said mono-hydroxy-protected diol to a phosphoramidite or a H-phosphonate monomer; and (iv) reacting at least a plurality of said monomers to form an oligophosphate.

8. A method for forming a boron-rich oligophosphate comprising the steps of:
(i) forming an o-carborane diol;
(ii) converting said o-carborane diol to a mono-hydroxy-protected carboranyl diol;
(iii) treating said mono-hydroxy-protected carboranyl diol with a phosphate coupling reagent thereby producing a mono-hydroxy-protected phophotriester;
(iv) converting a phosphotriester produced in step (iii) to an alcohol by removing the hydroxy-protecting group; and (v) condensing an alcohol produced in step (iv) with a mono-hydroxyl-protected diol produced in step (ii) using a phosphate coupling reagent to produce an oligophosphate.

9. A boron-rich oligophosphate which comprises a plurality of monomer units comprising boron, said monomer units being linked by phosphate units.

10. The boron-rich oligophosphate of claim 9 comprising at least two dihydroxy carborane derivatives as monomer.units.

11. The boron-rich oligophosphate of claim 10 wherein said dihydroxy carborane derivatives are derived from a material selected from the group consisting of 1,2-carborane, 1,7-carborane and 1,12-carborane.

12. The boron-rich oligophosphate of claim 10 wherein said monomer units are selected from the group consisting of closo-carborane derivatives, nido-carborane derivatives and mixtures thereof.

13. The boron-rich oligophosphate of claim 10 wherein said dihydroxy carborane derivative is enriched in the 10B isomer

14. The boron-rich oligophosphate of claim 13 wherein said boron includes up to about 96% by weight 10B.

15. The boron-rich oligophosphate of claim 10 comprising at least 10% by weight 10B.

16. The boron-rich oligophosphate of claim 10 comprising up to 150 said monomer units.

17. The boron-rich oligophosphate of claim 9, wherein said phosphate units are protected with 2-chlorophenyl group or 2-cyanoethyl group.

18. The boron-rich oligophosphate of claim 9 further comprising at least one monomer unit that does not contain boron.

19. The boron-rich oligophosphate of claim 18, wherein said monomer unit that does not contain boron comprises a material selected from amine groups, thiol groups, nucleotide segments, biotin derivatives and thymidine.

20. A boron-rich oligophosphate prepared by a method as claimed in claim 1.

21. A boron-rich oligophosphate having the structure A--L-(-B--L-) m-(-C--L-)n-D

wherein A is a hydroxyl containing unit selected from the group consisting of carboranyl residues, fluorescent groups, radiometal-based groups, amine groups, thiol groups, nucleotide segments, thymidine, biotin derivatives and hapten groups, B is a boron-containing diol residue formed from a monomer unit selected from the group consisting of , , , , , , , , , , and C and D are individually a monomer unit comprising a material selected from the group consisting of carboranyl residues, fluorescent groups, radiometal-based groups, amine groups, thiol groups, nucleotide segments, thymidine, biotin derivatives and hapten groups, L is an unprotected or protected a phosphate linking group, and m, n are integers, with m + n being an integer from 2 to 150.

22. The method of claim 1, wherein said dihydroxy compound is a dihydroxy carborane derivative.

23. The method of claim 22, wherein said dihydroxy carborane derivative is derived from 1,2-carborane, 1,7- carborane and 1,12-carborane.

24. The method of claim 23, wherein said dihydroxy compound is prepared by condensing dilithio-o-carborane with excess trimethylene oxide.

25. The method of claim 1, wherein prior to step (ii) the dihydroxy derivative is reacted with a protective group to produce a mono-hydroxy-protected dihydroxy derivative.

26. The method of claim 25, wherein said protective group is tert-butyl-dimethylsilyl or dimethoxytrityl.

27. The method of claim 1, wherein said coupling agent comprises a material selected from the group consisting of a chlorophosphite, a dichlorophosphite, a chlorophosphate, a dichlorophosphate, a chloro-H-phosphonate and a chlorophosphoramidite.

28. The method of claim 1, wherein said oligophosphate comprises up to about 150 monomer units.

29. The method of claim 1, wherein in step (iv) said monomer units are reacted with at least one non-carboranyl monomer unit.

30. The method of claim 1, wherein in step (iv) said monomer units are reacted with at least one monomer unit that does not contain boron.

31. The method of claim 29, wherein said monomer unit that does not contain boron comprises a material selected from amine groups, thiol groups, nucleotide segments, biotin derivatives and thymidine.

32. A method of producing a therapeutic agent for BNCT of cancer, comprising the step of combining a boron-rich oligophosphate as claimed in claim 9 with a tumor targeting vehicle.

33. The method of claim 32, wherein prior to said combining step a molecule capable of binding biotin is localized in said tumor targeting vehicle and said boron-rich oligophosphate is biotinylated, and wherein said combining step is carried out by interaction between said molecule and biotin.

34. The method of claim 33, wherein said molecule is avidin or streptavidin.

35. The method of claim 32, wherein prior to said combining step a DNA oligomer sequence is attached to said boron-rich oligophosphate and a complementary DNA
oligomer sequence is attached to said tumor targeting vehicle, and wherein said combining step is carried out by hybridization of said complementary DNA strands.

36. The method of claim 32 further comprising the step of preparing a boron-rich oligophosphate comprising a reactive functional group prior to said combining step.

37. The method of claim 36, wherein said reactive functional group comprises a molecule suitable for conjugation selected from the group consisting of an amine group and a thiol group.

38. The method of claim 36, wherein said combining step comprises the step of crosslinking said reactive functional group to a corresponding functional group of said tumor targeting vehicle using a bifunctional crosslinking reagent.

39. The method of claim 36, wherein said combining step comprises the step of coupling said reactive functional group to a corresponding functional group of said tumor targeting vehicle.

40. The method of claim 32 wherein said tumor targeting vehicle comprises a molecule capable of accumulating in tumors, and said molecule has an endogenous binding site to which said oligophosphate can be attached.

41. The method of claim 32 wherein said tumor targeting vehicle comprises a peptide-based delivery system.

42. The method of claim 41, wherein said peptide-based delivery system comprises a regulatory peptide.

43. The method of claim 32 wherein said tumor targeting vehicle comprises a protein-based delivery system.

44. The method of claim 43, wherein said protein-based delivery system comprises an antibody.

45. The method of claim 44, wherein said antibody is specific to a tumor antigen.

46. The method of claim 44, wherein said antibody comprises a molecule selected from the group consisting of whole monoclonal antibodies, antibody derived fragments, and engineered proteins comprising an antigen-binding site.

47. The method of claim 3, wherein said tumor targeting vehicle comprises a peptide-based delivery system.

48. The method of claim 47, wherein said peptide comprises a regulatory peptide.

49. The method of claim 3, wherein step (iv) of said method of claim 1, said monomer units are reacted with at least one non-carboranyl monomer unit.

50. The method of claim 3, wherein step (iv) of said method of claim 1, said monomer units are reacted with at least one monomer unit that does not contain boron.

51. The method of claim 5 wherein said antibody comprises a molecule selected from the group consisting of whole monoclonal antibodies, antibody derived fragments, and engineered proteins comprising an antigen-binding site.

52. The method of claim 7, wherein step (iv) is carried out using a DNA synthesizer.

53. The method of claim 52, wherein in step (iv) said monomers are coupled with at least one non-carboranyl monomer.

54. The method of claim 7, wherein step (iv) said monomer units are reacted with at least one non-carboranyl monomer unit.

55. The method of claim 7, wherein in step (iv) said monomer units are reacted with at least one monomer unit that does not contain boron.

56. The method of claim 7, wherein said carboranyl diol is derived from a matereial selected from the group consisting of 1,2-carborane, 1,7-carborane and 1,12-carborane.

57. The method of claim 7, wherein said oligophosphate comprises up to 150 carboranyl monomers.

58. The method of claim 7, wherein said monomers are selected from the group consisting of closo-carboranes, nido-carboranes and mixtures thereof.

59. The method of claim 8, wherein said dihydroxy carborane derivative is a derivative of 1,2-carborane, 1,7-carborane and 1,12-carborane.

60. The method of claim 8, wherein said o-carborane diol is a closo-carborane or a nido-carborane.

61. The method of claim 8 further comprising the step of making a mono-hydroxy-protected carboranyl diol before step (iii).

62. The method of claim 61 wherein a dimethoxytrityl group is used as a protecting group.

63. The method of claim 8 wherein said coupling reagent comprises a dichlorophosphite.

64. The method of claim 8 wherein said coupling reagent comprises an H-phosphonate.

65. The method of claim 8 wherein step (iv) said alcohol is coupled with at least one non-carboranyl monomer.

66. The method of claim 8 wherein step (iv) said alcohol is coupled with at least one monomer unit that does not contain boron.

67. A boron neutron capture therapy method which comprises the step of delivering to a tumor a therapeutically effective amount of a boron-rich oligophosphate as claimed in claim 9.

68. The method as claimed in claim 67, wherein said oligophosphate is delivered to said tumor without use of a tumor targeting delivery vehicle.

69. The method as claimed in claim 67, wherein said oligophosphate is encapsulated in a liposome.

70. The method as claimed in claim 67 wherein said oligophosphate is delivered to said tumor cell in combination with a tumor targeting delivery vehicle.

71. The method as claimed in claim 70 wherein said tumor targeting delivery vehicle comprises a protein-based vehicle.

72. The method as claimed in claim 71 wherein said protein-based vehicle is selected from the group consisting of whole monoclonal antibodies, antibody derived fragments, and engineered proteins comprising an antigen-binding site.

73. The method as claimed in claim 70 wherein said tumor targeting delivery vehicle comprises a regulatory peptide.

74. The method as claimed in claim 70 wherein said tumor targeting delivery vehicle comprises a molecule capable of accumulating in tumors.

75. A therapeutic agent for BNCT of cancer comprising a boron-rich oligophosphate as claimed in claim 9 and a tumor targeting vehicle.