Synthesis of tetrasubstituted Zn(II)-phthalocyanines carrying four carboranyl-units as potential BNCT and PDT agents

Article abstract of DOI:10.1016/j.tetlet.2005.03.030

The synthesis of two tetrasubstituted zinc(II)phthalocyanines carrying four carbon-carbon linked o-carboranyl-units (40 boron atoms, 27.5% boron by weight) is presented. In an in vitro model test the new compounds showed a good photosensitizing efficiency

Full text of DOI:10.1016/j.tetlet.2005.03.030

Tetrahedron
Letters
Tetrahedron Letters 46 (2005) 2979–2982
Synthesis of tetrasubstituted Zn(II)-phthalocyanines carrying four
carboranyl-units as potential BNCT and PDT agents
Francesca Giuntini,a Yann Raoul,b Donata Dei,b,* Moira Municchi,b Giacomo Chiti,b
Clara Fabris,a Paolo Colautti,c Giulio Joria and Gabrio Roncuccib
a
`
Dipartimento di Biologia, Universita degli Studi di Padova, Via Trieste 75, 35121 Padova, Italy
bMolteni Farmaceutici, S.S, 67, Loc. Granatieri, 50018 Scandicci (FI), Italy
`
INFN-Laboratori Nazionali di Legnaro,Via dell’Universita 2, 35020 Legnaro (PD), Italy
c
Received 10 January 2005; revised 2 March 2005; accepted 7 March 2005
Available online 22 March 2005
Abstract—The synthesis of two tetrasubstituted zinc(II)phthalocyanines carrying four carbon–carbon linked o-carboranyl-units (40
boron atoms, 27.5% boron by weight) is presented. In an in vitro model test the new compounds showed a good photosensitizing
efficiency, that encourages further studies for their evaluation as possible BNCT–PDT sensitizers.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Photodynamic therapy (PDT) and boron neutron cap-
ture therapy (BNCT) are two binary therapeutic modal-
ities, which are currently under investigation for the
treatment of several kinds of malignancies. Both of them
rely on the interaction of two relatively harmless factors:
a photo- or radiosensitizing compound and an external
radiation. PDT treatment1,2 consists in loading the tar-
get cells with a photosensitizer that is able to generate
highly reactive species (mainly singlet oxygen) upon irra-
diation with light of the appropriate frequency. The
consequent oxidative modification of several subcellular
targets, including proteins and unsaturated lipids, in the
microenvironment of the photosensitizer causes cell
death, via either random necrosis or apoptosis. Simi-
larly, BNCT3–5 is based on the interaction of the non
radioactive 10B nucleus and a thermal neutron. Thus,
ticles through ionization processes is thus confined to
the cell they are generated in. The success of BNCT
treatment depends both on the selective delivery of the
sensitizers to the tumor tissue and on the possibility of
achieving a sufficiently high endocellular concentration
of 10B. It has been calculated that the minimum effective
concentration of 10B in the tumor is 20–30 lg per gram
of tissue.6 Due to their selectivity of accumulation in
tumor over many normal tissues, porphyrins, and phtha-
locyanines have recently been proposed as boron carri-
ers to target tumoral tissue in BNCT treatment, and
indeed a number of papers reported the synthesis of nat-
ural abundance carboranyl-containing porphyrins and
phthalocyanines as models to develop novel radiosensi-
tizers.7 As a part of our current interest in the synthesis
of phthalocyanines for biomedical applications,8–12 we
undertook the synthesis of two tetrasubstituted Zn(II)-
phthalocyanines containing four carborane cages, with
the purpose of developing a radio/photosensitizing agent
for the treatment of tumors by means of a combination
of BNCT and PDT. The photosensitizing efficiency of
the compounds obtained was evaluated in vitro.
a
10B-containing sensitizer, that can be selectively deli-
vered to the target cell, is administered; the subsequent
irradiation of the area with thermal neutrons causes
the 10B nucleus to split into high linear energy transfer
(LET) particles, namely an a particle and a lithium
ion; such particles deliver a relatively large amount of
energy (ꢀ2.3 MeV) in a mean free path of about
10 lm, which is equivalent to the average diameter of
a normal cell. The cytotoxic effect exerted by LET par-
As it is shown in Scheme 1, the benzylic alcohols 3 and 4
were prepared according the to literature procedures,13
starting from commercially available 3- and 4-nitroph-
thalonitriles 1 and 2, by base-catalyzed aromatic nucleo-
philic substitution in anhydrous DMSO.14 The synthesis
of benzylic bromides 5 and 6 was accomplished by
converting 3 and 4 into the corresponding mesylates
Keywords: Zinc(II)phthalocyanines; Carboranes; PDT; BNCT.
*
Corresponding author. Tel.: +39 0557361329; fax: +39
0557361288; e-mail: d.dei@moltenifarma.it
0040-4039/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2005.03.030

2980
F. Giuntini et al. / Tetrahedron Letters 46 (2005) 2979–2982
NC
b, c
a
NC
O
3, 4
NC
NC
NC
NC
NC
NC
f
OH
O
NO2
O
5, 6
1, 2
9, 10
Br
NC
NC
d
e
= C
O
= CH
= BH
7, 8
Scheme 1. Reagents and conditions: (a) 4-hydroxybenzylic alcohol, K2CO3, DMSO, 40 ꢁC; (b) MsCl, TEA, CH2Cl2, 0 ꢁC; (c) LiBr, THF, reflux; (d)
4-methylphenol, K2CO3, DMSO, 40 ꢁC; (e) NBS, BPO, ClCH2CH2Cl, reflux; (f) Li-o-carborane, THF, À78 ꢁC.
and by subsequent bromine substitution of the mesylate
group upon treatment with lithium bromide in refluxing
THF.15 An alternative route to obtain the benzylic
bromides 5 and 6 was also developed. According to this
approach, base-catalyzed aromatic nucleophilic substi-
tution of p-cresol on 3- and 4-nitrophthalonitriles 1
and 2 yielded 3- and 4-(4-methylphenoxy)-phthaloni-
triles 7 (93%) and 8 (92%), respectively. Conversion to
the corresponding bromides 5 and 6 was achieved in
excellent yields (95% and 94%, respectively), by bromin-
ation with N-bromosuccinimide in presence of benzoyl
peroxide.16 Reaction of the lithium salt of o-carbo-
rane17–19 with benzyl bromides 5 and 6 in anhydrous
THF led to 3- and 4-substituted phthalonitriles 9 and
10 in satisfactory yields (55% and 48%, respectively).
The condensations of the phthalonitriles 9 and 10 to
the corresponding Zn(II)-phthalocyanines 11 and 12
(1,8(11),15(18),22(25)- and 2,9(10),16(17),23(24)-tetra-
substituted, respectively), were carried out by heating a
finely ground mixture of 9 or 10 and zinc acetate at
220 ꢁC for several hours20 (Scheme 2). Compounds 11
and 12 were obtained as a statistical mixture of regio-
isomers, in 43% and 40% yield, respectively, such results
are in agreement with the typical yields reported for this
kind of reactions,21 confirming that the presence of the
carborane cage does not impair the formation of the
phthalocyanine. Any attempt to obtain phthalocyanines
11 and 12 by different methods,21–23 such as DBU-
mediated cyclization in DMF or lithium/alcoholate
method, led to the extensive decomposition of the starting
material supposedly because of the low stability of the
carborane cage towards strong basic conditions.24 Pht-
halocyanines 11 and 12 were characterized by means
of 1H and 13C NMR spectroscopy, UV–vis spectros-
copy, and mass spectrometry. 1H NMR spectra of com-
pounds 11 and 12 displayed the typical pattern of signals
of 1,8(11), 15(18),22(25)- and 2,9(10),16(17),23(24)-
tetrasubstituted phthalocyanines, respectively.25 The
absorption spectra of compounds 11 and 12 in DMF
are presented in Figure 1, showing the expected absorp-
tion bands of phthalocyanines, with the Soret band cen-
tered at 329 nm and 357 nm, respectively, and Q-bands,
respectively, at 690 nm and 677 nm; both of the values
concerning the Q-bands are in agreement with the liter-
ature data relative to the absorption properties of
1,8(11),15(18), 22(25)- and 2,9(10),16(17),23(24)-tetra-
substituted phthalocyanines.26 Compounds containing
a large number of boron atoms usually display complex
MS spectra.27 Indeed, in the ESI-MS spectra of com-
pounds 11 and 12 the molecular ion gave very large iso-
topic patterns, because of the presence of a zinc atom
and 40 boron atoms, both existing as several stable
isotopes. However, the experimental spectra were in
agreement with the calculated ones both for position
CN
O
ZnOAc2,
CN
O
N
220°C
O
N
N
N
N
N
O
Zn
N
N
9, 10
O
11, 12
= C
= CH
= BH
Scheme 2.

F. Giuntini et al. / Tetrahedron Letters 46 (2005) 2979–2982
2981
0.5
4
3
2
1
0
11
12
0.4
0.3
0.2
0.1
compound 11 : kν = 4.06x10-4s-1
compound 12 : kν = 3.33x10-4s-1
300
400
500
600
700
800
Wavelength (nm)
0.0
Figure 1. UV–vis absorption spectra of compounds 11 and 12 in
DMF.
0
200
400
600
800
1000
1200
irradiation time (s)
Figure 2. Plot of the decay of NATA fluorescence after predetermined
irradiation times.
of the peaks (m/z values) and relative intensities (isotopic
pattern). (See Supplementary data.) Phthalocyanines
11 and 12 proved to be soluble in several organic sol-
vents, such as dichloromethane, chloroform, DMSO,
THF, methanol, and DMF. The lack of solubility in
water exhibited both by 11 and 12, which might repre-
sent a severe drawback for future in vivo application,
can be overcome by incorporating the molecules in
liposomes (unpublished results).
weight and may be regarded both as models for sensitiz-
ers to be used in boron neutron capture therapy and as
photosensitizers for PDT, as well as for the BNCT–PDT
combined treatment of tumors. The photodynamic effi-
ciency of the compounds obtained was evaluated by
measuring the rate of the photosensitized oxidation of
a model substrate (NATA). The present results show
that the presence of four carborane cages does not
impair the ability of the phthalocyanine molecule to
act as a photosensitizer. Studies concerning the localiza-
tion in vitro and in vivo of these compounds, as well as
their photosensitizing activity on human fibroblasts are
currently ongoing in our laboratories.
The photodynamic efficiency of compounds 11 and 12
was determined by measuring the degradation of NATA
(N-acetyl-L-tryptophanamide). The indolic moiety of
tryptophan is known to react with a wide range of electro-
philes,28 and NATA is a derivative of tryptophan that can
be regarded as a suitable model substrate for in vitro stud-
ies of photosensitized oxidations,29 because it readily
reacts with the electrophiles generated during type I
(radical involving) and type II (singlet oxygen involving)
photodynamic processes. Typically, such photoprocesses
follow first-order kinetics with respect to the substrate
concentration.30 The rate of photodegradation of the
indolic ring of NATA can be followed by measuring
the decrease of its fluorescence emission intensity
(kem = 360 nm) as a function of the irradiation time in
the presence of the photosensitizer. In a typical experi-
ment, a solution of compound 11 or 12 (2 lM) and
NATA (10 lM) in DMF is irradiated with red light
(650–750 nm, fluence rate: 100 mW/cm2) and the decrease
of the concentration of NATA is followed by measuring
the decrease of the intensity of its fluorescence emission
in 300–400 nm range (kexc = 290 nm). The rate constant
of the photoprocess is obtained from the slope of the
semilogarithmic plot (Fig. 2). The values thus found for
compounds 11 and 12 are 4.06 · 10À4 sÀ1 and
3.33 · 10À4 sÀ1, respectively. In the same conditions, the
rate of the photoprocess we found using unsubstituted
Zn(II)-phthalocyanine as the photosensitizer is
2.70 · 10À4 sÀ1 (data not shown). Such values are compa-
rable with those found for the same substrate using other
photodynamically active sensitizers, namely hematopor-
phyrin31 and tetrasulfonated-Zn(II)-phthalocyanine.32
Acknowledgments
This work received financial support from Istituto
Nazionale di Fisica Nucleare (Italy), Project SPES.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.tetlet.
2005.03.030.
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