Photochemistry und Photobiology Vol. 51, No. 3. pp, 351-356. 1990 Printed in Great Britain. All rights reserved

0031-8655/90 $03.00+0.00 Copyright 0 1990 Pergamon Press plc

SYNTHESIS OF POSITIVELY CHARGED PHTHALOCYANINES AND THEIR ACTIVITY I N THE PHOTODYNAMIC THERAPY OF CANCER CELLS D. W~HRLE*', N. ISKANDER',G. GRASCHEW~, H. S I N N ~E., A. FRIEDRICH*, W. MAIERBORST~, J. STERN'and P. SCHLAG.' 'Institut fur Organische und Makromolekulare Chemie, Universitat Bremen, 2800 Bremen 33, ZDeutsches Krebsforschungszentrum, Institut fur Biochemie, Institut fur Radiologie und Pathophysiologie, 6900 Heidelberg 1 and Thirurgische Universitatsklinik, Universitat Heidelberg. 6900 Heidelberg I , W . Germany (Received 26 May 1989; accepted 5 September 1989')

Abstract-Positively charged zinc containing or metal free phthalocyanines 6a-c and 7a-c were prepared via a three step procedure starting from 4-nitrophthalonitrile. The phthalocyanines contain alkyl chains of different length in order to influence the hydrophilic 1's lipophilic character of the compounds. The partition between a hydrophilic (water) and lipophilic (octanol-I) phase was determined, and the photoredox activities were investigated. Initial results on the photodynamic activity of these compounds were compared with those of Dougherty's Photofrin I1 on different malignant and non-malignant cell lines (XP 2YMAma1, CX1, HeLa, S180 and N 0 1 7 ) . Positively charged phthalocyanines in vitro showed a higher photodynamic activity than Photofrin 11.

stituted phthalocyanine in oleum. Differently sulfonated products can be separated by high performance liquid chromatography (HPLC)t. Less hydrophilic phthalocyanines possess only one or two sulfonic acid groups. The less photodynamically active but more hydrophilic tetrasulfonated phthalocyanine is prepared using only 4-sulfophthalic acid. Negatively charged phthalocyanines exhibit the advantage of easy preparation but the disadvantage of an extended purification procedure for chelates containing less than four sulfonic acid groups. Sulfonated phthalocyanines strongly aggregate in water (Bernauer and Fallab, 1961). It was shown that the formation of monomers in cells results in production of singlet oxygen upon irradiation (Spikes and Bommer, 1986; Wu et al., 1985; Langlois et a l . , 1986; Rosenthal et al., 1986). Electron transfer from the excited state to various molecules also has been reported but usually in the absence of oxygen (Darwent et a l . , 1982; Wohrle, 1986; Wohrle et al., 1988a). Progress in the use of phthalocyanines depends on the preparation of new, well-defined compounds. Recently Leznoff er al. (1989) described the synthesis and photocytotoxicity of positively charged phthalocyanines. This paper describes the synthesis and properties of positively charged phthalocyanines for phototherapy. A new concept was introduced in this work by changing the balance of the hydrophilic/lipophilic character of the compounds. Alkyl chains of different length were covalently incorporated in the positively-charged phthalocyanines. The complexes were investigated for their photosensitizing activity in photoelectron transfer reactions and their partition between a water and a octanol-1 phase. The photodynamic activity in vitra

INTRODUCTION

Interest in using phthalocyanines as active photosensitizers in the photodynamic therapy of cancer is increasing (Spikes, 1986). Phthalocyanines offer more favourable spectral properties with Q-band absorptions around 680 nm compared with HpD with lowest energy absorptions at around 630 nm. This property allows better skin penetration by the light source. The photochemistry of phthalocyanines is well investigated (Darwent et al., 1982; Simon and Andre, 1985; Wohrle, 1986; Wohrle et al., 1988a). Phthalocyanines which contain metal ions in a closed-shell arrangement such as Zn(I1) and AI(II1) exhibit long triplet lifetimes, high triplet quantum yields and therefore a high probability of energy or electron transfer. Previously, sulfonated phthalocyanine complexes of zinc and aluminium were investigated as photosensitizers for cancer therapy (Spikes, 1986; Lier et a l . , 1988). Sulfonated phthalocyanine metal complexes containing different numbers of sulfonic acid groups are obtained by a condensation procedure using mixtures of 4-sulfophthalic and phthalic acid, or by direct sulfonation of the unsub-~

~~~~~~~

*To whom correspondence should be addressed. ?Abbreviations: C X l , human colon carcinoma; DMF. N,N-dimethylformamide; DMSO, dimethylsulfoxide; E D T A , disodium ethylendinitrilotetraacetate; EJ 28, human bladder carcinoma; FTIR, Fourier transform infrared; HAM'S F 12, tissue culture medium; HeLa, human cervix adenocarcinoma; HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonicacid; HPLC, high performance liquid chromatography; M V + , methylviologen; N017, normal human fibroblast; P A , photodynamic activity in vitro; S180, mouse mamma carcinoma; TLC, thin layer chromatography; XP 29MAma1, Xeroderma pigmentosum human malignant cell line. PAP 51:3-G

35 1

D. W ~ H R LetEa / .

352

of these compounds was determined in comparison t o that of Dougherty's Photofrin I1 on different malignant and non-malignant cell lines. MATERIALS AND METHODS

The solvents and reagents were of GR grade and used without further purification. 4-nitrophthalonitrile was a gift of Toyo Ink. MFG (Tokyo, Japan). This compound was additionally prepared as described (Wohrle and Schulte, 1988). Fourier transform infrared spectra were recorded on a Nicolet 5-DX instrument and electronic spectra on a Perkin-Elmer Lamda 9. M solutions of 6a-c resp. 7a-c Partition coeficient. were prepared in 10 mP of 0.01 M Tris-buffer (pH 7; Merck AG, Darmstadt, W. Germany). This solution was intensively shaken with 10 mP of octanol-1. From the difference in extinction values at A,; of the Tris-buffer solutions before and after shaking, the distribution coefof the phthalocyanines were ficients (K=E,,,,,,,,,IE,;,,,,) calculated. Photoredox measurements. Measurements were carried out in a quartz cell (1 x 1 cm) at 297 K under argon. Typically, a solution (3 mP) containing sensitizer mol4- I), EDTA (disodium ethylenedinitrilotetraacetate; lo-' mo1.P-l) and MVZ+(l,l'-dimethyl-4,4'-bipyridinium dichloride, methylviologen; mol-P- ') with N,N-dimethylformamide (DMF)/water (vol. ratio 9: 1) was used. The quartz cell was placed into the light beam path of a UVlVIS spectrophotometer with DMFiwater as reference. Perpendicularly to the analytical light beam path, the quartz cell was irradiated with visible light (4W700 nm) by opening a shutter. A 12 V, 100 W halogen lamp (power supply, Oriel 6329; lamp housing, Oriel 6385, condensing lamp assembly, fl0.75) with IR- and UV-cut-off filters (Oriel 5148 and 5740) was used as the light source. The light intensity was calibrated with a bolometer (Kipp and Zonen, CAl-754399) and adjusted to 150 mW cm-'. Irradiation and registration of the spectra were performed automatically (by a connected computer). The concentration of M V was calculated from the increased absorbance at 610 nm, E being 13 700 C.mol-l.cm-l. Photodynamic activity in vitro. Photodynamic efficiency of phthalocyanines was investigated in vitro (Graschew et al., 1988) in five malignant and one non-malignant human cell lines: XP 29MAmal-Xeroderma pigmentosum human malignant cell line; CX1-human colon carcinoma; HeLa-human cervix adenocarcinoma; EJ 2 R h u m a n bladder carcinoma; S18C-mouse mamma carcinoma; N017-normal human fibroblast. The cells were cultured in HAM'S F 12 (Fa. Biochrom) with 10% fetal calf serum (Fa. Biochrom), buffered with 20 mM HEPES (Fa. Boehringer) to pH 7.4 in culture flasks (Fa. Nunc, flask volume-50 cm3). Flasks were inoculated with 2 x lo5 cells and the medium was changed every third day. Confluency was reached after 6-7 days. The photosensitizers were added to the exponentially growing cultures (2-3 day after inoculation) to a final concentration of 10 kg/mt (total volume 15 me). All subsequent handling of the cultures was performed in the dark. The medium was discarded 24 h later. The cells were washed twice with Ringer-solution and 20 mM HEPES-buffer, pH 7.4, 37°C (Fresenius, W. Germany) and supplied with fresh photosensitizer-free medium. Four hours later the medium was changed again. The cells containing only now tightly bound photosensitizers were irradiated at 37°C with light (610-700 nm), dose rate 15 mWicm', total dose 30 J/cm'. (Light source: filtered xenon arc lamp 100 W, Fa. Karl Zeiss, W. Germany.) The lamp was equipped with combined orange and chemical thermal filters (complex salt of CuSO,). Light intensity at different wavelengths (61C700 nm) and across the beam was homogenous (the differences were not higher than l0Y0). For each cell type

and compound tested six culture flasks were needed. For each culture flask 12 areas (1 mm diam.) were irradiated simultaneously. The cell numbers (after conventional staining with trypan blue solution 12-15 h after irradiation, and the unstained cell numbers at the irradiated areas were determined microscopically) in the control culture flasks (treated with photosensitizers, but not irradiated) were determined at the same time, so that the influence of dark toxicity of the photosensitizers are taken into account. Under the experimental conditions and concentration of the tested photosensitizers the dark toxicity was negligible. The photodynamic activity in vitro (PA) was calculated by: Nci - NPi PA (Yo) = 100. -__ N,, where N,,

mean unstained cell number per investigated area of the control culture flasks (treated with different photosensitizers, but not irradiated) and NPi = mean unstained cell number per irradiated area in photosensitizers treated and irradiated culture flasks. =

The relative error of all measurements did not exceed 1&15%. 4-(3-Pyridyfoxy)phthafonitrile (2). 4-Nitrophthalonitrile (1) (8.7 g, 50 mmol) and an excess of 3-hydroxypyridine (7.13 g, 75 mmol) were dissolved in 100 mC of dry dirnethylsulfoxide (DMSO) under inert gas. Dry potassium carbonate (13.8 g, 100 mmol) was added. The same amount of potassium carbonate was added after 4 and 24 h stirring at room temperature (RT). After 48 h the mixture was added to 500 me 1 M hydrochloric acid. The isolated and dried reaction product was dissolved under addition of active charcoal in hot ethanol and precipitated after filtration by addition of water. Yield 8.8 g (80%); m.p. 124°C. Thin layer chromatography (TLC) on silica gel in methanol: Rf= 0.757. IH NMR spectrum (DMSO) d6): 6 8.53 (d,lH), 8.51 (q,lH), 8.12 (d,lH), 7.90 (d,lH), 7.70 (4d,lH), 7.50 (d,lH), 7.48 (d,lH). IR (KBr): 2230 (C-N), 1280 and 1256 (Ar-O-Ar) cm-I. MS (20 eV): miz 221 (loo%, M+). 4- (2-Dimethylaminoethoxy)phthalodinitri/e (3). 4-Nitrophthalonitrile (1) (8.7 g, 50 mrnol) and dry potassium carbonate (13.8 g, 100 mmol) were dissolved in 60 mC DMF. Under inert gas 2-dimethylaminoethanol (10 me, 100 mmol) was added. After stirring for 20 h at RT the green colored solution was added to 400 mY water. Some green precipitate was filtered and the water solution extracted five times with 25 me toluene. The solution was passed through a A1,03 column (2 cm diam. 25 cm long), the toluene was evaporated and the residue crystallized by addition of n-heptane. The compound was dissolved in methanol and precipitated by addition of water. Yield 1.7 g (16%); m.p. 50°C. TLC on silicagel in methanol: R, = 0.286. 'H NMR spectrum (CDCI,): 6 2.42 (s,6H), 2.86

(t,2H),4.26(t,2H),7.29(d,lH),7.33(s,lH),7.74(d,lH). IR (KBr): 2238 (C-N), 1230 (Ar-O-CH,) cm-I. MS (20 eV): mlz 215 (100Y0, M+). 2, 9, 16, 23-Tetrakis(3-pyridyloxy)phthalocyaninezinc(lI) complex (4Zn). 2 (0.5 g, 2.26 mmol) were mixed with zinc(I1) acetate dihydrate (0.25 g, 1.14 mmol). The mixture was transferred to a glass ampoule. After evacuating and flushing with argon, the tube was sealed i. vac. (lo-' Torr = 1.33 Pa). The ampoule was heated for 4 h at 200°C. The reaction product was intensively treated with ethanol in a Soxhlet apparatus. The dark green residue was pure as judged by TLC. Yield 0.43 g (85%). IR (KBr): 1263 and 1236 (Ar-0-Ar), 1046 cm-' (Zn-N). UV/VIS (DMF): Mnm = 670, 601, 346. Elemental analysis: found: C 64.95, H 3.0, N 17.61; calculated: C 65.45, H 3.38, N 17.61.

Positive phthalocyanines in PDT

2,9,16,23Tetrakis(3-pyridy1oxy)phthalocyanine (42H). 2 (0.5 g, 2.26 mmol) was heated in the presence of 1.5diazabicyclo[4,3,0] nonen-5 (DBN) (65 Fe, 0.565 mmol) in a closed ampoule as described above to yield a dark green product. Yield 0.46 g (91'/0). IR (KBr): 1227 (Ar-0-Ar), 1012 cm-' (N-H). UVIVIS (DMF): AInm = 700, 670, 609, 348. Elemental analysis: found: C 69.5, H 4.20, N 18.05; calculated: C 70.1, H 3.85, N 18.87. 2,9,16,23Tetrakis[2-(2-dirnethylamino)ethoxy]-phthalocyaninezinc(l1) complex ( S Z n ) . 3 (0.8 g, 3.7 mmol) and zinc(I1) acetate dihydrate (0.4 g, 1.85 mmol) was dissolved in 60 m t 2-dimethylamino-ethanol and heated under ammonia bubbling for 24 h at 130°C. Subsequently the solution was added to 400 mY water. The isolated solid was treated intensively with diethylether in a Soxhlet apparatus to yield a dark green product. Yield 0.4 g (33%). IR (KBr): 1231 (Ar-0-CH,), 1051 cm-I (Zn-N). UVIVIS (DMF): AInm = 673, 604, 345. Elemental analysis: found: C 60.3, H 5.1, N 17.8; calculated: C 61.9, H 6.0, N 18.0. General procedure for the ulkylution of 4 and 5 to 6a-c and 7a-c with methyliodine, 1-hexyliodine and l-dodecyliodine. 4 or S(O.l g) were dissolved in 15 mC DMF. A 40fold molar excess of alkyl iodines were added and the solution was heated for 24 h at different temperatures (6aZn, 7aZn-20°C; 6a2H-50"C; QbZn, 6cZn--100°C; 7bZn, 7cZn-110°C). The liquids were evaporated i.vac. and the reaction products were washed with diethylether and acetone. After dissolving in water and filtration the products were isolated by freeze drying. Purity was controlled by TLC. Yields: 6aZn 84%,6a2H 20%. 6bZn 29%, 6cZn 38%, 7aZn 78%, 7bZn 5%, 7cZn 87%. Elemental analysis 6aZn: C 44.82, H 3.10, N 10.59; calculated: C 44.16, H 2.90, N 11.04; 6a2H: C 44.30, H 3.60, N 10.73; calculated: C 44.11, H 3.18, N 11.52; 7aZn: C 41.21, H 4.25, N 10.98; calculated: C 41.69, H 4.58, N 11.22. UVI VIS: AInm (dl mol-' cm-l) = 6aZn (DMF) 668 (94 200). 602, 334; 6aZn (isotonic NaCI) 664 (sh), 629 (30 141), 338; 6a2H (DMF) 690 (90 700), 660, 341; 6a2H (isotonic NaCI), 628 (15 800). 325; 6bZn (DMF) 669 (53 000). 605 (9200), 345; 6bZn (isotonic NaCI) 664 (sh), 629 (60 500), 338; 6cZn (DMF) 673 (107 loo), 604 (19 200), 347; 7aZn (DMF) 671 (43 600). 601, 351; 7aZn (isotonic NaC1) 670 (sh), 633 (12 200). 337; 7bZn (DMF) 675 (107 500), 602 (21 NO), 352; 7bZn (isotonic NaCI) 675 (sh), 633 (39 400), 338; 7cZn (DMF) 676 (54 200). 610, 340. ' H NMR spectrum 6bZn (DMSO d6): 6 9.5-8.1 (broad signals, 28H), 4.75 (8H), 2.05 (8H). 1.35 (24H), 0.85 (12H).

353

phthalocyanines 4Zn and 42H. The dinitrile 3 was converted, in the presence of zinc(I1) acetate dihydrate in 2-dimethylaminoethanol under ammonia bubbling, to the compound 5Zn with a yield of around 30%. The positively charged phthalocyanines 6a-c and 7a-c were obtained by alkylation reactions of 4 resp. 5 with an excess of methyliodine, hexyliodine or dodecyliodine in DMF. The solubility in water decreases with increasing chain length of the alkyl substituent: 6a, 7a excellent solubility; 6b, 7b good solubility; 6c, 7c nearly insoluble. In chloroform only 6c, 7c are soluble. D M F is a good solvent for all compounds. The purity of the compound was controlled by TLC, elemental analysis and partly by 'H NMR. Constitutional isomers are possible for &7, as for compounds of similar structure (Wohrle et al., 1985). TLC and 'H NMR gave no hints whether isomers exist. Charged phthalocyanines easily aggregate in aqueous solution. Aggregation mainly to dimers was investigated in detail employing negatively charged phthalocyanines such as sulfonated derivatives (Abel et al., 1976; Bernauer and Fallab, 1961; Yang et al., 1985). The concentration dependent aggregation is reduced in

1

1

Q

RESULTS

Synthesis and characterization

The positively charged phthalocyanines 6 and 7 were prepared by a three step procedure. Commercially available 4nitrophthalonitrile (1) is a suitable starting material for various substituted phthalonitriles. By a nucleophilic displacement reaction of the nitro group various nucleophiles can be introduced. The reaction of 1 with 3-hydroxypyridine or 3,-dimethylaminoethanol in polar solvents in the presence of K2C03 lead to 4-(3-pyridyloxy)phthalonitrile (2) resp. 4-(2-dimethylaminoethoxy)phthalonitrile (3). The dinitriles 2, 3 are suitable starting materials for the syntheses of phthalocyanines 4, 5 . The cyclotetramerization of 2 with or without zinc(I1) acetate in the presence of an organic base in a closed bomb vessel results in nearly quantitative yields in the formation of the

Q-' R-J

-0-Q \

i

I

-CL -C..

-GI,. -C*.L*

Scheme 1

D. WBHRI.E

354

et

al.

0 54

,

;

P

I

n

a 01

00 300

-----_LOO

/

500

-600

700

800

h (nml

Figure 1. UViVIS spectra of 6bZn (concentration mol [ - I ) in aqueous isotonic NaCI/DMF (vol. ratio 1:2) (-) and aqueous isotonic NaCl (----). Table 1. Distribution coefficients of positively charged phthalocyanines between octanol-1 and 0.01 M Tris-buffer pH 7.0

Scheme 2

organic solvents such as pyridine, DMF and ethanol Also adsorption respectively covalent binding at macromolecular compounds and addition of detergents lead to a decrease of dimerization (Darwent et al., 1982; Wohrle et al., 1988b). The existence of monomers in biological surroundings is important because intermolecular quenching of the excited triplet state occurs in dimers resulting in shorter triplet life time of the sensitizer and reduced sensitizer activity (Wohrle et al., 1988a). All synthesized water soluble phthalocyanines 6a, b and 7a, b exhibit in water at concentrations of 10-4-10-h mol t - ' a pronounced monomer/dimer equilibrium whereas in organic solvents (DMF, chloroform, ethanol) only the monomer exists. Figure 1 shows a typical of the monomer comexample for 6bZn with ,A,, pound at 669 nm and the A, of the dimer at 629 nm. Table 1 lists the distribution coefficients of positively charged phthalocyanines between octanol-1 and 0.01 M Tris-buffer pH 7.0. As expected there is an increase of equilibrium concentration in octanol-1 with increasing chain length of the alkyl rests. Therefore a directed hydrophilic/lipophilic

Phthalocyanine

Distribution coefficient

6aZn 6a2H 6bZn 6cZn 7aZn 7bZn 7cZn

0.21 0.05 0.64 0.78 0.13 0.22 9.02

balance which should be important for the cell uptake can be achieved. The photoredox behavior of all synthesized phthalocyanines upon irradiation with visible light was investigated in DMF/water (vol. ratio 9:l) in the presence of an excess of EDTA as donor and methylviologen (MV2+) as acceptor. These photoreactions are different from those assumed to occur in cells (singlet oxygen as photoproduct). In the presence of an acceptor and a donor, photoelectron transfer under formation of reduced acceptor ( M V ) and oxidized donor (EDTA,,) is observed (Darwent et al., 1982; Wohrle, 1986; Wohrle et al., 1988a). But the investigation of photoelectron transfer reactions easily leads to clear hints of sensitizer ability of the synthesized compounds. It is important to note that the water soluble 6a, b and 7a, b exhibit no sensitizer activity in water or isotonic NaCl in the presence of MV2+ and EDTA. As mentioned before these phthalocyanines strongly aggregate in aqueous solution. This results in an intermolecular quenching of the triplet states (dissipation of the energy of the excited state) (Darwent etal., 1982; Wohrle et al., 1988a). In the DMF/water mixture used all phthalocyanines are monomers at concentrations of mol [ - I . Figure 2 shows the formation of M V as a function of irradiation time. All phthalocyanines exhibit photoredox activities. In the case of 7cZn about 50% of employed MV2+ was reduced after 5 min irradiation with 150 mW cm-*. It can be seen that the alkylated compounds 6a-c, 7a-c show a higher rate of MV' formation than the non-alkylated compounds 4 and 5 . Zinc

355

Positive phthalocyanines in PDT

Photofrin I1 the latter shows the lowest PA (28-41%). The complexes 6cZn and 7cZn containing long chain dodecyl rests could not be measured as a consequence of low water solubility.

30

/

--.20 -

DISCUSSION

0

E

VI

c-

+

>

E

10

0

1

2

3

4

5

t lmin) Figure 2. Photoreduction of MV'+ to MV' in the presence of EDTA in DMFiwater (vol. ratio (9:l) (for conditions see "Materials and Methods").

complexes are superior to metal free ligands (Wohrle et al., 1985). The fact that phthalocyanines containing the dimethylaminoethoxy substituent exhibit a better photoredox activity than those containing a pyridyloxy substituent cannot be explained this time. Figure 3 gives a survey of the results obtained in in vitro experiments. 6bZn and 7bZn exhibit the highest photodynamic activity in vitro in all cell lines (61-78% and 60-75%). The PA in vitro of 6a2H, 6aZn and 7aZn was lower. However, comparing phthalocyanines to the well established

Positively charged phthalocyanines are obtained by simple three step procedures. As expected, water insoluble phthalocyanines exhibit strong aggregation in aqueous solutions. The lipophilic character increases with increasing chain length of substituting alkyl groups. Being dissolved as monomers, phthalocyanines are characterized as active sensitizers. One reason for higher PA in vitro of the new photosensitizers in comparison to that of Photofrin I1 could be that they are bound more tightly to cellular components (e.g. higher capability for intercalation) so that the concentration of these compounds in cells was higher after the washing procedures than that of Photofrin I1 (work in progress). No significant difference was observed in the uptake of photosensitizers by malignant cells compared to normal cell line. A higher uptake of the new photosensitizers into cells is supported by the fact that their P A in vitro increases with their lipophility (compare 7bZn and 6bZn, Fig. 3 and Table 1). A good correlation was observed between the photodynamic activity in vitro and the distribution coefficients of the tested photosensitizers (cf. Table 1 and Fig. 3). Compounds with a long aliphatic side chain at the quarternary nitrogen of the pyridine or the amino-groups yielded higher PA in vitro than those which had only a methyl group as substituent (cf. 7aZn, 6aZn and 6a2H, Fig. 3). The absorption maxima of the new compounds are around 680 nm. The light intensity of 680 nm was not higher than 630 nm. Further investigations on the distribution of the new photosensitizers in normal and tumor tissues in vivo with heterotransplanted human tumors in nude mice and determination of the singlet oxygen yield will give valuable information on the structure-reactivity relationship of these compounds in photodynamic therapy. REFERENCES

XP 29P1Amwl

CXl

NO17

Hsla

EJ28

980

Coll lines

Figure 3 Photodynamic activity in vrfro of various photosensitizers' Photofrin 11, 7aZn. 7bZn, 6aZn. 6bZn and 6a2H The relative error of all measurements was not higher than I&150/,

Abel, E. W., J . M. Pratt and R. Whelan (1976) The association of cobalt(I1)tetrasulphophthalocyanine. J . Chem. SOC. Dalton Trans. 509-514. Bernauer, K. and S. Fallab (1961) Phthalocyanine in wapriger Losung. Helv. Chim. Acta 44, 1287-1292. Darwent, J . R., P. Douglas, A. Harriman, G . Porter and M.-C. Richoux (1982) Metal phthalocyanines and porphyrins as photosensitizers for the reduction of water to hydrogen. Coord. Chem. Rev. 44,83-126. Graschew, G., M. Shopova, G. Anastassova, A. Chakarova and Ch. Getov (1988) Sensitivity of individual tumors to photodynamic therapy. Lasers Med. Sci. 3, 233-238. Langlois, R., H. Ali, N . Brasseur, J . R. Wagner and J . E . Lier (1986) Biological activities of phthalocyanines-

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D. WiinRLE ei at.

IV. Photochem. Photobiol. 44, 117-125. Leznoff, C. C., S. Vigh, P. I. Svirskaya, S. Greenberg, D. M. Drew, E. Ben Hur and I. Rosenthal (1989) Synthesis and photocytotoxicity of some new substituted phthalocyanines. Photochem. Photobiol. 49,279-284. van Lier, J. E., N. Brasseur, B. Paquette, J . R. Wagner, J . Ali, R. Langlois and J . Rousseau (1988) Phthalocyanines as sensitizer for photodynamic therapy of cancer. In Photosensitization, Molecular, Cellular and Medical Aspects (Edited by G. Moreno, R. H. Pottier and T G. Truscott). NATO AS1 Series, Vol. H 15, pp. 435-444. Springer, Berlin. Rosenthal, I., C. M. Krishna, P. Riesz and E. Ben-Hur (1986) The role of molecular oxygen in the photodynamic effect of phthalocyanines. Radial. Res. 107, 136- 142. Simon, J. and J. J. Andre (1985) Molecular Semiconductors, Photoelectrical Properties and Solar Ceffs.Springer, Berlin. Spikes, J. D. (1986) Phthalocyanines as photosensitizers in biological system and for the photodynamic therapy of tumors. Photochem. Photobiol. 43, 691-699. Spikes, J. D. and J. C.Bommer (1986) Zinc phthalocyanine as sensitizer for biomolecules. Inf. J . Radiat. Biol. 50, 41-45.

Wohrle, D. (1986) Phthalocyanine-ein System ungewohnlicher Struktur und Eigenschaften. Kontakte (Merck) 1, 24-34. Wohrle, D.,J . Gitzel, I. Okura and S. Aono (1985) Photoredoxproperties of tetra-2,3-pyridinoporphyrazines. J . Chem. SOC.,Perkin Trans. 2 , 1171-1178. Wohrle, D., J. Gitzel, G. Krawczyk, E. Tsuchida, H. Ohno, I. Okura and T. Nishisaka (1988a) Synthesis, redox behaviour, sensitizer activity and oxygen transfer of covalently bound polymeric porphyrines. J. Macromol. Sci. Chem. A25, 1227-1254. Wohrle, D.,G . Krawczyk and M. Paliuras (1988b) Polymeric bound porphyrins and their precursors, 7. Makromol. Chem. 189, 1013-1018. Wohrle, D.and B. Schulte (1988) Polymeric phthalocyanines and their precursors 14. Makromol. Chem. 189, 1167-1 187. Wu, S . , H. Zhang, G. Cui, D . Xu and H. Xu (1985) A study on the ability of some phthalocyanine compounds for photogenerating singlet oxygen. Acfa Chim. Sinica 43, 21-25. Yang, Y.-C., J . R. Ward and R. P. Seiders (1985) Dimerization of cobalt(I1) tetrasulfonated phthalocyanines in water and aqueous alcoholic solution. Inorg. Chem. 24, 1765-1 769.