Synthesis and in vitro photodynamic activity of new hexadeca-carboxy phthalocyanines

Article abstract of DOI:10.1039/b405868b

Two new hexadeca-carboxy phthalocyanines have been synthesised and evaluated for their photodynamic activities; the zinc(II) analogue exhibits a high class-A scavenger-receptor mediated photocytotoxicity towards the J774 marine macrophage cell line.

Full text of DOI:10.1039/b405868b

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Synthesis and in vitro photodynamic activity of new hexadeca-carboxy
phthalocyanines{
Chi-Fung Choi,a Po-Ting Tsang,b Jian-Dong Huang,a Elaine Y. M. Chan,b Wing-Hung Ko,c
Wing-Ping Fong*b and Dennis K. P. Ng*a
a Department of Chemistry, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China.
E-mail: dkpn@cuhk.edu.hk
b Department of Biochemistry, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China.
E-mail: wpfong@cuhk.edu.hk
c Department of Physiology, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China
Received (in Cambridge, UK) 21st April 2004, Accepted 6th July 2004
First published as an Advance Article on the web 25th August 2004
Two new hexadeca-carboxy phthalocyanines have been
synthesised and evaluated for their photodynamic activities;
the zinc(II) analogue exhibits a high class-A scavenger-
receptor mediated photocytotoxicity towards the J774 murine
macrophage cell line.
Photodynamic therapy (PDT) has been an experimental clinical
modality for the treatment of a range of cancer and wet age-related
macular degeneration for the past two decades.1 It involves three
individually non-toxic components, namely photosensitisers, light
and oxygen, that are combined to cause cellular and tissue damage.
The efficacy of PDT depends on several interdependent factors,
among which photosensitisers certainly play a decisive role.
Although Photofrin1 remains the most widely used photosensi-
tiser, it suffers from a high degree of chemical heterogeneity, poor
absorption of tissue-penetrating red light and long-lasting
cutaneous photosensitivity.2 Significant efforts have therefore
been put in the development of new photosensitisers which have
better photophysical properties, greater tumour specificity and less
skin photosensitivity.3 Owing to their many desirable character-
istics, phthalocyanines have emerged to be a promising class of
second-generation photosensitisers.3,4 As part of our continuing
interest in the development of phthalocyanine-based photosensi-
tisers,5 we describe herein the preparation and in vitro photo-
dynamic activities of two new phthalocyanines with 16 -COOH
substituents.6 In contrast to many previously described photo-
sensitisers such as the sulfonated and hydroxy zinc(II) and
aluminium(III) phthalocyanines, and the well-known silicon(IV)
phthalocyanine Pc4,3,4 these compounds do not have structural
isomers and contain a large number of negatively charged
carboxylate moieties upon deprotonation. These highly charged
species can target tumour-associated macrophages via the
scavenger receptor without the need of conjugation of any bio-
logical ligands.7
Scheme 1 shows the synthetic route to prepare these hexadeca-
carboxy phthalocyanines. Treatment of the readily available 4,5-
dichlorophthalonitrile (1)6b with dimethyl 5-hydroxyisophthalate
(2) in the presence of K2CO3 gave the disubstituted product 3 in
good yield. Cyclisation of 3 using lithium in 1-pentanol, followed
by alkaline hydrolysis with LiOH in a mixture of THF–MeOH–
H2O and acidification with HCl resulted in the isolation of
the metal-free hexadeca-carboxy phthalocyanine 4. Attempts to
prepare the zinc analogue by treating 4 with Zn(OAc)2?2H2O in
DMF were not successful initially. The reaction led to precipitation
which might be due to complexation between the peripheral
COOH groups and the zinc(II) ions. This problem could be
circumvented by the addition of NH4Cl, which suppresses this
complexation, allowing the zinc(II) ions to be incorporated into the
Scheme 1
phthalocyanine core. Phthalocyanine 5 was successfully prepared in
this manner in 89% yield (see the characterization data for 3–5 in
ESI{).8
The UV–Vis spectra of 4 and 5 in THF were typical for non-
aggregated phthalocyanines. In the presence of NaOH, these
compounds were also soluble in water giving similar spectra
properties (see their UV–Vis spectra in ESI{). The absorption
spectrum of 5 showed the Q band at 673 nm. Under slightly acidic
conditions (pH ~ 6.5–6.8), another broad band at ca. 630 nm was
also seen, which can be attributed to the aggregated species.8 At pH
w 7, a sharp Q band was observed. This together with the strong
fluorescence emission at 684 nm [quantum yield (Wf) ~ 0.26]9
suggests that this compound is relatively free from aggregation
under alkaline conditions. The metal-free phthalocyanine 4
behaved similarly with a Wf value of 0.12. It is worth noting that
phthalocyanines are usually highly aggregated in aqueous media
due to the strong hydrophobic interactions.8 The non-aggregated
nature of 4 and 5 (in deprotonated form) can be ascribed to the
inherent repulsion of the highly negatively charged molecules. This
property is particularly important for photosensitising applications.
To evaluate the photosensitising efficiency of these two novel
phthalocyanines, their singlet oxygen quantum yields (WD) were
determined by a steady-state method using 1,3-diphenylisobenzo-
furan as the scavenger.10 It was found that both compounds are
{ Electronic supplementary information (ESI) available: characterization
data for 3–5, UV–Vis spectra of 4 and 5 in the presence of NaOH in water,
and photodynamic effect of 4 on HepG2 and J774. See http://www.rsc.org/
suppdata/cc/b4/b405868b/
2 2 3 6
C h e m . C o m m u n . , 2 0 0 4 , 2 2 3 6 – 2 2 3 7
T h i s j o u r n a l i s ß T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4

View Online
Fig. 1 Effect of 5 on HepG2 (triangles) and J774 (circles) in the absence
(open symbols) and presence (closed symbols) of light. For the latter,
Fig. 2 Effect of poly I on the photodynamic activities of 5 on J774 cells.
J774 macrophages were incubated for 2 hours with various concentrations
of 5 in the absence ($) and presence of 25 mg cm23 (+) or 50 mg cm23 poly
I (&). Cells were illuminated and cytotoxicity was measured. Data are
expressed as mean ¡ SD (n ~ 5).
the cells were illuminated with a red light (l w 610 nm, 40 mW cm22
,
48 J cm22). Data are expressed as mean ¡ SD (n ~ 5).
singlet-oxygen generators. The WD value of the zinc(II) phthalo-
cyanine 5 (0.40 in DMF) is significantly higher than that for the
metal-free counterpart 4 (0.27).9
second-generation photosensitisers. The zinc(II) analogue exhibits a
high and selective photocytotoxicity against J774 cells of macro-
phage origin via receptor-mediated endocytosis without the need of
any bioconjugation.
We thank the Croucher Foundation for a Chinese Visitorship to
Dr J.-D. Huang.
The photodynamic activities of phthalocyanines 4 and 5 (in
deprotonated form) were investigated against two different cell
lines, namely murine macrophage J774 and human hepatocellular
carcinoma HepG2.11 The cells were incubated with different
concentrations of 4 or 5 for 2 hours. Cell viability was determined
after 20 hours by the colorimetric MTT assay.5 In the absence of
light, both compounds were not cytotoxic. The two cell lines
differed, however, in their response after illumination. While
HepG2 was resistant to the phototoxic effect of 4 and 5 at
concentrations below 4 mM, J774 was very sensitive. As shown in
Fig. 1, the percent viability of cells decreases gradually with increase
in the concentration of 5. Fifty percent cell death can be observed
around 1 mM of 5. The photodynamic activity of 4, however, was
somewhat lower. About 4 mM of 4 was needed to induce 50% of
cell death (ESI{).
Notes and references
1 D. E. J. G. J. Dolmans, D. Fukumura and R. K. Jain, Nature Rev.
Cancer, 2003, 3, 380.
2 G. Jori, J. Photochem. Photobiol. A: Chem., 1992, 62, 371.
3 H. Ali and J. E. van Lier, Chem. Rev., 1999, 99, 2379; E. S. Nyman and
P. H. Hynninen, J. Photochem. Photobiol. B: Biol., 2004, 73, 1.
4 E. A. Lukyanets,J. Porphyrins Phthalocyanines, 1999, 3, 424;C. M.Allen,
W. M. Sharman and J. E. van Lier, J. Porphyrins Phthalocyanines, 2001,
5, 161.
The J774 murine macrophage cell line has been used as a model
system in the study of PDT, particularly in scavenger-receptor
targeted PDT.12 Covalent conjugation of photodynamic drugs
to scavenger receptor ligands, for example, maleylated serum
albumin, has been used successfully to increase their uptake into
macrophage. J774 and HepG2 cells differ in the types of scavenger
receptor they possess. For example, it is known that class-A
scavenger receptor is expressed in J774 cells but not in HepG2
cells.12,13 This, together with the observations that 4 and 5 act
specifically on J774 but not HepG2, led to the speculation that
class-A scavenger receptor was involved in mediating the
cytotoxicity of the photosensitisers on J774. To show this, a com-
petitive assay was performed in the presence of polyinosinic acid
(poly I), a ligand of class-A scavenger receptor which is known for
its high capacity to bind a broad range of polyanionic molecules,
including modified lipoproteins, polyribonucleotides and poly-
saccharides.14 As shown in Fig. 2, poly I inhibits the cytotoxicity of
5 in a dose-dependent manner. Co-incubation with 50 mg cm23
poly I can increase the cell viability from ca. 20% to ca. 80% in the
presence of 4 mM of 5. These results show that the poly I-sensitive
class-A scavenger receptor is responsible for the uptake of 5, and
subsequently, the cell death in J774 cells. Although 5 did not kill the
tumour cell directly, the killing effect of macrophage is just as
important as the tumour-associated macrophages can help the
tumour to grow and spread,12 for example, they produce mediators
that increase the degree of angiogenesis.
5 See for example: J.-D. Huang, S. Wang, P.-C. Lo, W.-P. Fong, W.-H. Ko
and D. K. P. Ng, New J. Chem., 2004, 28, 348; P.-C. Lo, J.-D. Huang,
D. Y. Y. Cheng, E. Y. M. Chan, W.-P. Fong, W.-H. Ko and D. K. P. Ng,
Chem. Eur. J., in press.
6 Phthalocyanines with 4- or 8-COOH substituents have been described
previously. See for example: (a) N. Kobayashi, H. Shirai and N. Hojo,
J. Chem. Soc., Dalton Trans., 1984, 2107; (b) D. Wo¨hrle, M. Eskes,
K. Shigehara and A. Yamada, Synthesis, 1993, 194; (c) A. Suchan,
J. Hurek, W. Waclawek, J. Nackiewicz and K. Ejsmont, Polish J. Chem.,
1999, 73, 2013.
7 W. M. Sharman, J. E. van Lier and C. M. Allen, Adv. Drug Delivery
Rev., 2004, 56, 53.
8 X.-y. Li, X. He, A. C. H. Ng, C. Wu and D. K. P. Ng, Macromolecules,
2000, 33, 2119 and references cited therein.
9 The unsubstituted zinc(II) phthalocyanine was used as reference (Wf ~
0.30 in 1-chloronaphthalene, WD ~ 0.55 in DMF). See A. C. H. Ng,
X.-y. Li and D. K. P. Ng, Macromolecules, 1999, 32, 5292.
10 W. Spiller, H. Kliesch, D. Wo¨hrle, S. Hackbarth, B. Ro¨der and
G. Schnurpfeil, J. Porphyrins Phthalocyanines, 1998, 2, 145.
11 Compound 4 (or 5) was dissolved in 0.01 M aqueous NaOH and DMF
(9 : 1 v/v) with the aid of ultrasound to form a 0.9 mM stock solution. It
was then diluted with phosphate buffered saline to 80 mM and filtered
through a 0.45 mm filter. The solution was further diluted with RPMI
medium 1640 supplemented with 10% fetal calf serum to different
concentrations (pH ~ 7.4).
12 M. R. Hamblin, J. L. Miller and B. Ortel, Photochem. Photobiol., 2000,
72, 533.
13 D. Rhainds, L. Falstrault, C. Tremblay and L. Brissette, Eur. J. Biochem.,
1999, 261, 227.
In summary, we have prepared two novel hexadeca-carboxy
phthalocyanines, which have many desirable characteristics as
14 H. E. de Vries, A. C. E. Moor, T. M. A. R. Dubbelman, T. C. van Berkel
and J. Kuiper, J. Pharmacol. Exp. Ther., 1999, 289, 528.
C h e m . C o m m u n . , 2 0 0 4 , 2 2 3 6 – 2 2 3 7
2 2 3 7