Biochem. J.

(1989) 261, 277-280 (Printed in Great Britain) 277

Hydroporphyrins of the meso-tetra(hydroxyphenyl)porphyrin series

as tumour photosensitizers
Raymond BONNETT,* Rosemary D. WHITE,* Una-Jane WINFIELD* and Morris C. BERENBAUMt
*Department of Chemistry, Queen Mary College, Mile End Road, London El 4NS, and tDepartment of Experimental
Pathology, St. Mary's Hospital Medical School, London W2 1PG, U.K.

Four new hydroporphyrins [the o, m and p isomers of 5,10,15,20-tetra(hydroxyphenyl)chlorin and

5,10,15,20-tetra(m-hydroxyphenyl)bacteriochlorin] related to the tetra(hydroxyphenyl)porphyrins have been
prepared. They show the expected strong absorption bands in the red region of the visible spectrum and are
found to be very effective tumour photosensitizers.

INTRODUCTION through tissue is very low at 400 nm, but reaches a

maximum in the 700-800 nm region (Wan et al., 1981).
There is currently worldwide activity in the develop- Absorption in the red has been increased by (i) modifying
ment of a phototherapy for cancer (Dougherty, 1987a). the substitution pattern (Bonnett et al., 1987) or (ii) by
An essential aspect of this effort is the design, synthesis reducing the porphyrin to the chlorin or bacteriochlorin
and biological assay in vivo of new photosensitizers. chromophore (Selman et al., 1987; Kessel & Smith,
New photosensitizers are needed because of the 1989) or (iii) by more deep-seated changes in the structure,
inadequacies of haematoporphyrin derivative (HpD), for example, to give phthalocyanine or naphthalocyanine
which has been most commonly used in both biochemical systems (Ben-Hur & Rosenthal, 1985; Chan et al., 1987).
and clinical studies in this area. HpD was first described Recently a series of porphyrins, namely the o, m and
by Lipson & Baldes (1960). It is prepared by the action p isomers of 5,10,15,20-tetra(hydroxyphenyl)porphyrin
of H2SO4 in acetic acid on haematoporphyrin at room (displayed structures 1, 2 and 3 respectively) has been
temperature. This gives a purple solid (HpD Stage I), reported which fall into the first modification category
which is a complex mixture, but consists largely of (Berenbaum et al., 1986). These substances showed
haematoporphyrin diacetate (Bonnett et al., 1981). For promising activity and tissue selectivity in photonecrosis.
injection, this material is treated with alkali to give HpD Thus the m-isomer (2) was found to be 25-30 times as
Stage II. The treatment causes hydrolysis and elimination potent as HpD in sensitizing tumours in the 'in vivo'
to give monomeric porphyrins such as haemato- bioassay.
porphyrin, hydroxyethyl(vinyl)deuteroporphyrin and On the basis of this substitution pattern, we now
protoporphyrin; at the same time, condensation- report the effect of a modification of the second category
polymerization reactions occur to give a high-molecular- to give chlorin (structures 4, 5 and 6) and bacteriochlorin
mass fraction (fraction D) in which the bulk of the (structure 7) analogues, a structural change which is
activity resides. We postulated that the composition of accompanied by a dramatic increase in photonecrotic
the material after treatment with base includes a mixture activity.
of dimers and oligomers involving ester, ether or carbon-
carbon linkages (Berenbaum et al., 1982). Although
there has been some support for various of these pro- MATERIALS AND METHODS
posals (Dougherty et al., 1984; Kessel, 1986; Scourides
et al., 1987; Keir et al., 1987), the limited information on Chlorins
the components of HpD Stage II indicates that it is a The porphyrin (1, 2 or 3; Bonnett et al., 1987) was
complex and variable mixture from which it has not yet reduced with di-imide using the method of Whitlock et
proved possible to isolate a single highly active compound al. (1969), without protection of the phenolic functions.
(Dougherty, 1987b). Thus the p isomer (3; 163 mg), p-toluenesulphonyl-
The need therefore arises for 'second-generation' hydrazide (90 mg), anhydrous K2CO3 (300 mg) and an-
photosensitizers which embody the following hydrous pyridine (11.25 ml) were stirred with N2 flushing
characteristics: (i) lack of toxicity in the dark; (ii) uniform for 20 min. The mixture was heated (100-105 °C) for
stable composition, and preferably a single substance; 6.5 h under N2; further quantities of the hydrazide (97 mg
(iii) selective photosensitization of tumour tissue, a in 0.3 ml of anhydrous pyridine on each occasion) were
feature which seems to be related to amphiphilic proper- added after 2 and 4 h. The reaction mixture was treated
ties; (iv) high triplet yield, with a triplet energy greater with ethyl acetate (75 ml) and distilled water (37.5 ml)
than 94 kJ mol ', the excitation energy for Ag singlet and digested on the steam bath for 1 h. The organic layer
oxygen; (v) absorption in the red. was separated and washed in turn with HCI (2 M, 75 ml),
The last criterion arises because, due to scattering and distilled water (75 ml) and saturated NaHCO3 solution
absorption effects, the transmission of visible light (75 ml). o-Chloranil (total 166 mg) was added in portions

Abbreviations used: abbreviations for the porphyrins are provided below the structural formulae and full chemical names are displayed in the text;
other abbreviation: HpD, haematoporphyrin derivative.

Vol. 261
 278 R. Bonnett and others

(1) 5,10,1 5,20-Tetra(o-hydroxyphenyl)porphyrin (2) 5,10,15,20-Tetra(m-hydroxyphenyl)porphyrin (3) 5,10,15,20-Tetra(p-hydroxyphenyl)porphyrin

(o-THPP) (m-TH PP) (p-THPP)

(4) 5,10,1 5,20-Tetra(o-hydroxyphenyl)chlorin (5) 5,10,1 5,20-Tetra(m-hydroxyphenyl)chlorin (6) 5,10,1 5,20-Tetra(p-hydroxyphenyl)chlorin

(o-TH PC) (m-TH PC) (p-TH PC)

Hl

(7) 5,10,1 5,20-Tetra(m-hydroxyphenyl)bacteriochlorin

(m-TH PBC)

to the stirred organic solution at room temperature until of S,10,15,20-tetra(p-hydroxyphenyl)chlorin (6) as a

the absorption peak at 735 nm had just disappeared.
-
purple solid. (Found, by fast-atom-bombardment mass
The solution was washed with aqueous NaHSO3 (5 %; spectrometry: M+ H+, 681.253; C44H32N404 + H
2 x 75 ml), distilled water (75 ml), NaOH (0.01 M, requires 681.250). Light-absorption maxima in methanol
100 ml), and saturated NaHCO3 (80 ml), and dried (over [c (molar absorption coefficient)]: 285 (17000), 295
anhydrous Na2SO4). The filtered solution was (16900), 418 (143000), 520 (10000), 550 (9000), 597
evaporated to dryness, and the residue was crystallized (5100) and 651 nm (18600 litre-mol-l-cm-1). N.m.r.: a
from methanol/water to give 77 mg (middle cut, 47 %) (p.p.m.) ([2H]chloroform/[2H6jdimethyl sulphoxide) 9.22
1989
Hydroporphyrins as tumour photosensitizers 279

Table 1. Tumour photonecrosis with chlorins and a bacteno- 26 %). (Found: M+ 682.260; C44H34N404 requires
chlorin of the meso-tetra(hydroxyphenyl) series 682.258). Light-absorption maxima in methanol (c): 352
(92000), 361 (114000), 372 (129000), 516 (50000) and
Dose Depth of tumour 735nm (91000 litre-mol- 'cm-1). N.m.r.: a (p.p.m.)
(umol/kg Wavelength* necrosis (mm) ([2H6]dimethyl sulphoxide) 9.69 (s, 4 x OH), 7.95 (d,
Photosensitizer body wt.) (nm) (mean + S.E.M.) J 2 Hz, pyrrole fl-H), 7.47, 7.21, 7.18, 7.04 m, benzenoid
H), 3.95 (s, 8 x CH2), and -1.54 (bs, 2 x NH). The
n.m.r. spectrum revealed minor contamination by the
o-THPC (4) 6.25 652 4.16 + 0.27 (14t) corresponding chlorin.
3.125 652 3.69 + 0.74(9)
1.56 652 0.31 +0.31 (8) Tumour necrosis
0.78 652 0 (8) The PC6 plasma cell tumour, obtained initially from
m-THPC (5) 0.75 652 5.41 +0.39 (19) the Chester Beatty Research Institute, was grown by
0.375 652 3.79 + 0.28 (6) inoculating (0.3-0.6) x 106 cells subcutaneously in female
0.2 652 0.13+0.05 (12) BALB/c mice. It was used about 2 weeks later, when it
0.1 652 0.042 + 0.0247(12) was 12-13 mm in its longest diameter and 6-7 mm deep.
p-THPC (6) 6.25 653 3.50 +0.54(10) Sensitizers were injected in dimethyl sulphoxide
3.125 653 2.13+0.50(10) intraperitoneally (2.5 ,ul g-1) and, 24 h later, skin over
m-THPBC (7) 6.25 741 Died within 2 h of the tumours was depilated, the mice anaesthetized and
3.125 741 irradiation, tumour tumours exposed to light (10 J. cm-2). Wavelengths for
1.562 741 visibly blackening illumination were the longest-wavelength absorption
0.39 741 5.22+ 1.21 (8) peaks in the red in solutions of the sensitizer in fetal-calf
0.19 741 0.11 ±0.05 (7) serum, and were 652-653 nm for the compounds 4-6 and
* The total energy administered was 10 J cm-2
throughout. 741 nm for compound 7. The light source was a copper
t Number of tumours in parentheses. vapour laser with an output of 10-12 W (Cu 10 laser:
Oxford Lasers, Oxford, U.K.) pumping a D2 10K dye
laser (Oxford Lasers). The dye was Rhodamine 640
(Applied Photophysics, The Royal Institution, London,
(s, 4 x OH), 8.62, 8.42, 8.23 (d, s, d, J 4.5 Hz, pyrrole ,- U.K.) for illumination at 652-653 nm and LDS-722
H), 7.90, 7.65 (d, d, J 8 Hz, 4-benzenoid H), 7.16 (two (Exciton, Dayton, OH, U.S.A.) for illumination at
overlapping d, J 8 Hz, benzenoid H), 4.17 (s, 2 x CHOI 741 nm. Light intensity at the tumour surface was kept
and -1.53 (bs, 2 x NH). below 0.3W cm-2, where thermal effects were un-
Similarly, the m isomer (5) was obtained from detectable.
methanol/water as a purple solid (37 %). (Found: At 24 h after illumination, 0.2 ml of 1 %0 Evans Blue
M+H+, 681.251). Light-absorption maxima in (Sigma) in saline was given intravenously and tumours
methanol (e): 284 (16900), 306 (15 600), 415 (146000), 516 were removed 1 h later and fixed in formol/saline. The
(11 000), 543 (7300), 591 (4400) and 650 nm (22400 fixed tumours were sliced at right angles to the surface,
litre mol-' cm-'). N.m.r.: a (p.p.m.) ([2H]chloroform/' and the depth of necrosis measured with a dissecting
[2H6]dimethyl sulphoxide) 9.16 (s, 4xOH), 8.66, 8.46, microscope fitted with an eyepiece graticule, as described
8.28 (d, s, d, J 4.5 Hz, pyrrole f-H), 7.1-7.7 (m, benzenoid elsewhere (Berenbaum et al., 1982).
H), 4.21 (s, 2 x CH2) and 1.64 (bs, 2 x NH).
The o-chlorin (4) was obtained as a purple crystalline RESULTS AND DISCUSSION
solid (64 %o) from methanol/water. Like the starting
porphyrin (1), it was a mixture of atropisomers (Gottwald Di-imide reduction of the porphyrins (1, 2 and 3) gives
& Ullmann, 1969), which were not individually isolated. a mixture of the corresponding dihydro- and tetrahydro-
RF (1 % methanol in chloroform; Merck silica gel) 0.23, porphyrins. Dehydrogenation with o-chloranil cleanly
0.29, 0.38, and 0.43. (Found: M+H+, 681). Light- removes the tetrahydro compound (Amax 735 nm) to
absorption maxima in methanol (c): 415 (90700), 515 give a useful route to the chlorins (4, 5 and 6) without the
(8400), 542 (5600), 597 (3800) and 651 nm (16000 necessity for protection of the phenolic functions. Under
litre mol-1 cm-l). N.m.r.: a (p.p.m.) ([2H6]dimethyl more forcing conditions, di-imide reduction of the m
sulphoxide) 9.49 (s, 4 x OH), 8.50, 8.25, 8.15 (d, s, d, J isomer (2) gives mainly the corresponding bacteriochlorin
5 Hz, pyrrole ,-H), 7.02-7.97 (m, benzenoid H), 4.12 (s, (7).
2xCH2), and -1.57 (s, 2 x NH). As expected, these compounds are soluble in polar
To obtain bacteriochlorin (7), m-THPP (109 mg) was solvents, and they all have strong absorption in the red
reduced with p-toluenesulphonylhydrazide (483 mg) in region of the visible spectrum. The chlorins have maxima
anhydrous pyridine (7.5 ml) as described above, except at - 650 nm with molar absorption coefficients of about
that further additions of the hydrazide (60 mg in 2 ml of 20000 litre- mol-1 cm-1; in the bacteriochlorin (7) the
anhydrous pyridine) were made every 1.5 h, and refluxing long-wavelen-gth band in methanol has shifted to 735 nm
was continued for a total of 12 h. Digestion was effected (741 in fetal-calf serum), with a much increased absorp-
with ethyl acetate (100 ml) and distilled water (50 ml), tion coefficient (91000 litre mol-' cm-').
and the separated organic layer was washed in turn with The tumour photonecrosis results are summarized in
HCI (2 M, 50 ml), H,PO4 (56 00, 4 x 50 ml), distilled water Table 1. Although the advantages of absorption in the
(50 ml) and saturated NaHCO3 (50 ml) and dried (over red region of the visible spectrum are well recognized,
anhydrous Na2SO4). The resulting solid was crystallized this is the first report of results for a series of reduced
from methanol/water to give 5,10,15,20-tetra-(m- porphyrins based on a porphyrin substitution pattern of
hydroxyphenyl)bacteriochlorin as a green solid (29 mg, established photobiological activity.
Vol. 261
280 R. Bonnett and others

All four sensitizers show considerable activity and, Bonnett, R., Ioannou, S., White, R. D., Winfield, U. J. &
even with these limited results, appear to be considerably Berenbaum, M. C. (1987) Photobiochem. Photobiophys.
more potent as tumour photosensitizers than are the Suppl. 45-56
corresponding tetra(hydroxyphenyl)porphyrins. This is Bonnett, T., Ridge, R. J., Scourides, P. A. & Berenbaum, M.
especially evident with the p and m isomers, where C. (1981) J. Chem. Soc. Perkin Trans. 1, 3135-3140
substantial tumour necrosis is produced with doses at Chan, W. S., Marshall, J. F. & Hart, I. R. (1987) Photochem.
which p- and m-tetra(hydroxy)phenylporphyrin are quite Photobiol. 46, 867-872
ineffective (Berenbaum et al., 1986). Whether these Dougherty, T. J. (1987a) Photochem. Photobiol. 45, 879-889
compounds will prove to be useful in clinical tumour Dougherty, T. J. (1987b) Photochem. Photobiol. 46, 569-573
phototherapy will depend largely on whether their effects Dougherty, T. J., Potter, W. R. & Weishaupt, K. R. (1984)
are relatively selective for tumours as compared with Adv. Exp. Med. Biol. 170, 301-314
normal tissues. Gottwald, L. K. & Ullman, E. F. (1969) Tetrahedron Lett.
3071-3074
Keir, W. F., Land, E. J., Mackennan, A. H., McGarvey, D. J.
We are grateful to Scotia Pharmaceuticals Ltd. and to the & Truscott, T. G. (1987) Photochem. Photobiol. 46, 587-589
Cancer Research Campaign for support. The new compounds Kessel, D. (1986) Photochem. Photobiol. 44, 193-196
described here are the subject of British Patent Application no. Kessel, D. & Smith, K. (1989) Photochem. Photobiol. 49,
88/05849. 157-160
Lipson, R. L. & Baldes, E. J. (1960) Arch. Dermatol. 82,
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Received 13 March 1989/12 April 1989; accepted 24 April 1989

1989