Synthesis and characterization of a boronated metallophthalocyanine for boron neutron capture therapy

Article abstract of DOI:10.1021/ic9600240

Synthesis of the first fully characterized, water-soluble boronated phthalocyanine is reported. Reaction of 4-nitrophthalonitrile with dimethyl malonate in the presence of base yielded dimethyl (3,4-dicyanophenyl)malonate which was converted into dimethyl (3,4-dicyanophenyl)propargylmalonate by sequential treatment with potassium hydroxide and propargyl bromide. Formation of the o-carborane cage was accomplished by reaction of the alkyne with decaborane in acetonitrile at reflux. High-temperature solid state condensation of the resulting o-carboranylphthalonitrile with cobalt(II) chloride followed by ester deprotection and cation exchange provided the water-soluble closo-carbonylphthalocyanine product. The product contains 40 boron atoms (27% boron by weight) and may be useful as a tumor-seeking boron delivery agent for boron neutron capture therapy of cancer.

Full text of DOI:10.1021/ic9600240

3878
Inorg. Chem. 1996, 35, 3878-3880
Synthesis and Characterization of a Boronated Metallophthalocyanine for Boron Neutron
Capture Therapy
Stephen B. Kahl* and Jing Li
Department of Pharmaceutical Chemistry, University of California,
San Francisco, California 94143-0446
ReceiVed January 10, 1996X
Synthesis of the first fully characterized, water-soluble boronated phthalocyanine is reported. Reaction of
4-nitrophthalonitrile with dimethyl malonate in the presence of base yielded dimethyl (3,4-dicyanophenyl)malonate
which was converted into dimethyl (3,4-dicyanophenyl)propargylmalonate by sequential treatment with potassium
hydroxide and propargyl bromide. Formation of the o-carborane cage was accomplished by reaction of the alkyne
with decaborane in acetonitrile at reflux. High-temperature solid state condensation of the resulting o-
carboranylphthalonitrile with cobalt(II) chloride followed by ester deprotection and cation exchange provided the
water-soluble closo-carbonylphthalocyanine product. The product contains 40 boron atoms (27% boron by weight)
and may be useful as a tumor-seeking boron delivery agent for boron neutron capture therapy of cancer.
was purchased from Dexsil Corp. and was sublimed prior to use. All
other reagents and chemicals were purchased from Aldrich, and all
reactions were carried out under high-purity argon unless otherwise
indicated. High-resolution mass spectra (HRMS) were recorded on a
VG-70SE mass spectrometer in EI mode for molecules less than 900
amu. Liquid secondary ion mass spectra (LSIMS) were recorded in a
nitrobenzyl alcohol matrix on a VG-70SE mass spectrometer for
molecules of more than 1000 amu. Infrared (IR) spectra were recorded
on a Nicolet Impact 400 infrared spectrophotometer using KBr disks.
Nuclear magnetic resonance (NMR) spectra for proton and carbon were
recorded on a GE 300 NMR spectrometer with TMS as an internal
standard. Ultraviolet-visible spectra (UV-vis) were recorded on a
HP8452A diode array spectrophotometer. Melting points (mp) were
measured by a Thomas Hoover capillary melting point apparatus and
are uncorrected. Chromatography was performed with silica gel (Merck
grade 60) from Aldrich. Microanalyses were performed by the
Microanalysis Laboratory of College of Chemistry, University of
California, Berkeley, CA.
Dimethyl (3,4-Dicyanophenyl)propargylmalonate, 4. To a sus-
pension of compound 3 (3 g, 0.01 mol) in acetone (70 mL) was added
propargyl bromide (1.8 g, 80% in toluene, 0.012 mol). The suspension
was stirred and heated to reflux under argon for 20 h. The potassium
bromide precipitate was filtered off and acetone was removed in Vacuo
to give an oily residue. Chromatography of the oily residue using
hexane/THF (6:1) as the eluant generated 2.4 g (83%) of dimethyl (3,4-
dicyanophenyl)propargylmalonate 4 (mp: 90-91 °C). HRMS for
C16H12N2O4 (M+): calcd, 296.0797; found, 296.0789 (∆ ) 0.8 ppm).
1H NMR (CDCl3-d1): δ 8.02 (d, J ) 1.5 Hz, H), 7.89, 7.87 (2dd, J )
1.5 Hz, J ) 8.4 Hz, H), 7.81 (d, J ) 8.4 Hz, H), 3.84 (s, 6H), 3.23 (d,
J ) 2.4 Hz, 2H), 2.09 (t, J ) 2.4 Hz, H). 13C NMR (CDCl3-d1): δ
168.1, 141.2, 138.1, 133.9, 133.2, 133.1, 115.7, 115.2, 115.0, 114.9,
73.5, 61.6, 53.8, 25.7. IR (KBr disk): 2240 cm-1 (CN), 1733 cm-1
(CdO).
Introduction
Boron neutron capture therapy (BNCT) is a binary method
for cancer therapy which allows selective tumor irradiation. The
principle of BNCT is based on the interaction of a nonradioac-
tive 10B nucleus with low-energy (thermal) neutrons to generate
4
7
cytotoxic particles, He and Li, of ∼2.3 MeV kinetic energy.
These fission fragments have mean free paths approximately
equivalent to the average cell diameter, thus confining their
considerable radiation damage to the immediate vicinity of cells
in which they are located.1 It has been calculated that a
minimum cellular 10B concentration of between 10 and 30 µg
per gram of tumor is necessary for clinically effective BNCT.1
The challenge for chemists, therefore, is to synthesize 10B-
containing compounds capable of specific localization and
retention in tumor cells at or above these levels while simul-
taneously clearing normal, nontarget tissues and blood. Certain
phthalocyanines (Pc’s) have been proven to localize and persist
in various solid tumors, and metallophthalocyanines have been
used, in Vitro and in ViVo, as photosensitizers for photodynamic
therapy (PDT).2,3 Thus, the use of a metallophthalocyanine to
deliver 10B atoms to a tumor might be a viable strategy for
meeting this challenge. Phthalocyanines usually exhibit notori-
ous insolubility in both water and organic solvents that precludes
adequate purification and characterization, so the key issue is
to synthesize a boronated phthalocyanine having both lipophilic
and hydrophilic substituents capable of enhancing the solubility
of boronated phthalocyanines in therapeutic solutions. The first
water-soluble boronated phthalocyanine, bearing only one closo-
carborane cage, was synthesized by Soloway et al.4 However,
no detailed information on its purification and characterization
was reported.
Dimethyl (3,4-dicyanophenyl)(o-carboranylmethyl)malonate, 6.
To a two-necked round-bottomed flask charged with B10H14, 5 (0.82
g, 0.0067 mol), was added 50 mL of anhydrous acetonitrile by syringe.
The solution was heated to reflux under argon for 1 h and then allowed
to cool to room temperature. Compound 4 (2.0 g, 0.0067 mol) was
added, and the resulting solution was futher refluxed under argon for
40 h or until TLC analysis indicated that no compoud 4 could be
observed in the solution. Acetonitrile was evaporated in Vacuo to give
a waxy white solid. The solid was dissolved in a small volume of
methanol and then adsorbed on a small portion silica gel and loaded
onto a silica gel column which was eluted with hexane and THF (4:1).
The first fraction was collected to give 1.4 g of the desired product 6
Experimental Section
Dimethyl (3,4-dicyanophenyl)malonate, 2, and its potassium salt,
3, were synthesized according to literature procedures.5 4-Nitrophtha-
lonitrile was purchased from Tokyo Chemical Inc. (TCI). Decaborane
X Abstract published in AdVance ACS Abstracts, June 1, 1996.
(1) Hawthorne, M. F. Angew. Chem., Int. Ed. Engl. 1993, 32, 950.
(2) van Lier, J. E. In Photodynamic Therapy of Neoplastic Disease; Kessel,
D., Ed.; CRC Press: Boca Raton, FL, 1990; Vol. 1, p 279.
(3) Rosenthal, I. Photochem. Photobiol. 1991, 31, 102.
(4) Alam, F.; Soloway, A. H.; Bapat, B. V.; Barth, R. E.; Adams, D. M.
Basic Life Sci. 1989, 50, 107.
(5) Roze, M. P.; Berzin’sh, E. L.; Neiland, O. Ya. Zh. Org. Khim. 1992,
28, 827.
S0020-1669(96)00024-9 CCC: $12.00 © 1996 American Chemical Society

Boronated Phthalocyanine for Cancer Therapy
Inorganic Chemistry, Vol. 35, No. 13, 1996 3879
Scheme 1
Scheme 2
(50% yield) as a light yellow solid (mp: 184-185 °C). HRMS for
C16H22B10N2O4 (M+): calcd, 414.2582; found, 414.2564 (∆ ) 1.8 ppm).
1H NMR (CDCl3-d1): δ 8.16 (dd, J ) 1.5 Hz, H), 7.99, 7.96 (2dd, J
) 1.5 Hz, J ) 8.4 Hz, H), 7.83 (d, J ) 8.4 Hz, H), 3.82 (s, 6H), 3.41
(s, 2H), 2.0 (br, 10H), 1.3 (s, H). 13C NMR (CDCl3-d1): δ 167.4,
140.5, 133.5, 133.4, 132.9, 116.3, 115.9, 114.8, 114.6, 70.5, 60.9, 54.1,
43.5, 26.4. IR (KBr disk): 2629 and 2582 cm-1 (BH), 2232 cm-1
(CN), 1748 cm-1 (CdO).
Sodium Salt of (2,9,17,23-Tetrakis(1-carboxyl-2-(o-carbonyl)-
ethyl)phthalocyanato)cobalt(II), 9. To a 25 mL single-necked flask
charged with anhydrous ethanol (10 mL) was added cut sodium pieces
(53 mg, 2.3 mmol) with stirring under argon. After the sodium was
completely dissolved, compound 7 (80 mg, 0.05 mmol) was added
under argon. The resulting blue solution was stirred at ambient
temperature under argon for 4 days. The solution was concentrated in
Vacuo and the residue dissolved in water. A trace amount of
undissolved particles was filtered off. The water layer was acidified
with 0.1 M aqueous HCl solution. The precipitated deep blue product
was filtered off and dissolved in a solution of acetone/water (8:2), passed
through an ion exchange column packed with Dowex 50WX2-400 ion
exchange resin in Na+ form, and eluted with water. Acetone was
evaporated by rotary evaporator and water was lyophilized to pro-
duce the sodium salt 9 in 60% yield as a blue solid. UV-vis (H2O):
λ (nm) (log ꢀ) 636 (5.4), 290 (5.6), 192 (5.8). Anal. Calcd for
C52H68B40N8O8CoNa4‚6H2O: C, 38.45; H, 4.96; N, 6.90. Found: C,
38.49; H, 4.89; N, 6.75.
(2,9,17,23-Tetrakis(1,1-(dimethoxycarbonyl)-2-(o-carboranyl)eth-
yl)phthalocyanato)cobalt(II), 7. A mixture of compound 6 (300 mg,
0.17 mmol) and anhydrous CoCl2 (98 mg, 0.77 mmol) was pulverized
and transferred to a 20 mL single-necked flask. The mixture was heated
to 200 °C under argon on an oil bath in about 40 min and kept at 200
°C for 2 h. The resulting dark blue solid was chromatographed and
eluted with a solution of toluene and ethyl acetate (100:3) to give 84
mg of the desired product 7 (27% yield) as a dark blue solid. MS:
m/z 1717 (MH+, 94%). IR (KBr disk): 2587 cm-1 (BH), 1739 cm-1
(CO). UV-vis (CH2Cl2): λ (nm) (log ꢀ) 266 (3.9), 328 (3.9), 602
(3.6), 630 (3.6), 670 (4.3). Anal. Calcd for C64H88B40N8O16Co: C,
44.78; H, 5.16; N, 6.53. Found: C, 44.80; H, 5.32; N, 6.42.
(2,9,17-Tris(1,1-(dimethoxycarbonyl)-2-(o-carboranyl)ethyl)-23-
(1-methoxycarbonyl)-2-((o-carboranyl)ethyl)phthalocyanato)cobalt-
(II), 8. A mixture of compound 6 (300 mg, 0.17 mmol) and anhydrous
CoCl2 (93 mg, 0.73 mmol) was ground to a fine powder and then
transferred to a 20 mL single-necked flask. The mixture was quickly
heated to 210 °C and kept at 210-220 °C for 8 h under argon. A dark
blue solid was formed. The solid was chromatographed using a solution
of toluene and dichloromethane (100:3) as the eluant to give 80 mg of
the desired product 8 (20% yield) as a blue solid. MS: m/z 1659 (MH+,
60%). IR (KBr disk): 2589 cm-1 (BH), 1739 cm-1 (CO). UV-vis
(CH2Cl2): λ (nm) (log ꢀ) 206 (4.0), 328 (3.9), 604 (3.6), 668 (4.3).
Anal. Calcd for C62H86B40N8O14Co: C, 44.89; H, 5.22; N, 6.75.
Found: C, 44.91; H, 5.12; N, 6.81.
Results and Discussion
In this paper we report the synthesis and characterization of
the first boronated metallophthalocyanine in which four closo-
carborane cages are covalently linked to the periphery of the
phthalocyanine ring. Water-solubility is obtained by hydrolyz-
ing eight methyl ester groups followed by ion exchange to the
sodium salt. Schemes 1 and 2 present the synthetic procedure
used to prepare this water-soluble boronated metallophthalo-
cyanine.
Substituted phthalocyanines are generally prepared either by
solid state condensation of a substituted phthalonitrile in the
presence of a metal template or by derivatization of a preformed

3880 Inorganic Chemistry, Vol. 35, No. 13, 1996
Kahl and Li
phthalocyanine. Two strategies are then obvious for the
preparation of a boronated phthalocyanine: (1) attach the boron
at an early stage by placing the boron moiety on the phthaloni-
trile or (2) add the boron group to a reactive functional group
on a suitable preformed phthalocyanine derivative. A further
consideration in choosing from these two strategies is the need
to achieve a modicum of aqueous solubility in the final product.
The second strategy is attractive in that, in principle, the essential
boron group(s) can be added in a high-yield step at the
Attempts to hydrolyze the ester groups of compound 7 under
either mild or extreme acidic conditions failed. Hydrolysis
under basic conditions (sodium ethoxide in absolute ethanol),
however, was successful and was accompanied by decarboxyl-
ation to give the tetraacid upon acidification. This compound
was not isolated for characterization, but was immediately
converted to the highly water-soluble sodium salt 9 by cationic
exchange. Compound 8 was hydrolyzed under the same
conditions to give compound 9 after ion exchange. Elemental
analysis of compound 9 illustrated that partial decarboxylation
occurs under these conditions. Decarboxylation of carborane
esters under similar conditions has been reported by Zakharkin
et al. who reported that overnight room temperature treatment
of ethyl 3-(2-phenylcarboranyl)propionate with potassium hy-
droxide in absolute ethanol produced 1-ethyl-2-phenylcarbo-
rane.6 We have also previously observed decarboxylation of
diethyl 2,2-bis(carboranylmethyl)malonate to 2,2-bis(carbora-
nylmethyl)acetic acid with sodium ethoxide in absolute ethanol.7
No degradation of the carborane cages of 7 or 8 to the nido
open cage form was observed under these conditions as
evidenced by the infrared spectrum of compound 9 which
exhibited a B-H absorption band at 2587 cm-1 identical to the
starting material 7. Opening of the closo-carborane cage
culmination of the route, an important consideration when 10
B
reagents are to be used. However, the solubility requirement
led us to adopt the former approach since it appeared possible
to incorporate a protected solubilizing group, the methyl esters,
into the phthalonitrile and then deprotect in a later step.
Synthesis of dimethyl malonate, 2, from commercially
available nitrophthalonitrile, 1, was readily accomplished by
literature techniques.5 The yield of 2 was comparable to the
reported yield of 40%. Conversion of 2 to the desired
propargylphthalonitrile, 4, was readily carried out in 83% yield
by conversion to the potassium malonate salt, 3, followed by
reaction with propargyl bromide. Addition of the boron was
made at this stage by condensation of decaborane (B10H14), 5,
with the ethynyl group of 4. It was found that in situ conversion
of 5 to its bis(acetonitrile) adduct of decaborane followed by
reaction with the alkyne resulted in higher product yields and
shorter reaction times than reacting the preformed bis(acetoni-
trile) adduct with the alkyne in either THF or toluene. Carbo-
rane phthalonitrile 6 was isolated in 50% yield after chroma-
tography, a typical yield for this type of condensation.
generally shifts this absorption to the vicinity of 2450 cm-1
.
11B NMR spectra of 7 and 9 were substantially broadened by
the presence of the paramagnetic Co(II) and were not useful in
establishing the nature of the cage in 9.
Conclusions
Compound 9 is the first fully characterized boronated
phthalocyanine to be reported and contains four closo carborane
cages covalently linked to the phthalocyanine periphery. Sub-
stantial aqueous solubility is obtained through the presence of
four carboxylate residues as their Na+ salts. This boronated
phthalocyanine may be useful as a tumor-selective sensitizer
for boron neutron capture therapy using thermal neutrons to
activate 10B through the 10B(n,R)7Li reaction, or in photody-
namic therapy by activation of the nitrogen macrocycle with
light. Studies evaluating the in Vitro and in ViVo localizing
ability of 9 are currently being carried out and will be reported
in an appropriate venue in the near future. The synthesis of
other boronated phthalocyanines in which the boron cages are
added at a later stage to preformed derivatized phthalocyanines
is currently being explored and will also be reported in the near
future.
Solid state condensation of boronated phthalonitrile 6 to
boronated phthalocyanine 7 was carried out by heating a
pulverized mixture of 6 and cobalt(II) chloride at 200 °C under
an argon atmosphere for 2 h. The relatively low yield of 7
(27%), while typical of this type of reaction, illustrates again
the potential advantages of adding the boron at a later stage.
When 6 and cobalt(II) chloride were quickly heated to 210 °C
and kept at this temperature for 8 h, a different phthalocyanine
was produced whose elemental composition and mass spectrum
corresponded to partially decarboxylated phthalocyanine 8.
Boronated metallophthalocyanines 7 and 8 were fully char-
acterized by their spectroscopic data and elemental analyses.
The liquid secondary ion mass spectra (LSIMS) of 7 and 8 in
a nitrobenzyl alcohol (NBA) matrix show molecular ion clusters
(MH+) at nominal m/z 1717 and 1659 corresponding to the
formulas C64H88B40N8O16Co and C62H86B40N8O14Co, respec-
tively. The clusters of the theoretical molecular ions for these
formulae are almost identical to ion clusters observed at m/z
1717 and 1659, respectively. The infrared spectra of 7 and 8
Acknowledgment. We gratefully acknowledge the support
of this work by the National Cancer Institute in the form of
Grant CA 53901. Mass spectra were provided by the University
of California, San Francisco, Mass Spectrometry Facility (A.
L. Burlingame, Director), funded by the Biomedical Technology
Program of the National Center for Research Resources, Grants
NIH NCRR BRTP RR04112 and NIH NCRR BRTP RR01614.
exhibit strong B-H absorption bands at 2587 and 2590 cm-1
,
respectively, and a strong carbonyl absorption band at 1739
cm-1. The UV-vis spectra of 7 and 8 in dichloromethane show
absorption maxima at 670 and 668 nm, respectively. Both
compounds presumably exist as a complex set of positional
isomers since the dysymmetric phthalonitrile starting material
6 can condense in either of two orientations. However, no
isomeric separation was observed on thin layer, column, or
HPLC chromatography.
IC9600240
(6) Zakharkin, L. I.; Chapovaski, Yu. A.; Brattsev, V. A.; Stanko, V. I.
Zh. Obshch. Khim. 1960, 36, 878.
(7) Kahl, S. B. Unpublished observation.