Hydroporphyrins as tumour photosensitizers: Synthesis and photophysical studies of 2,3-dihydro-5,15-di(3,5-dihydroxyphenyl) porphyrin

Article abstract of DOI:10.1016/S0960-894X(03)00002-7

The synthesis and characterization of 2,3-dihydro-5,15-di(3,5-dihydroxyphenyl) porphyrin is reported. The phototoxicity on C6 cell lines and the pharmacokinetics are also reported as preliminary results showing a very high tumor to skin ratio and short retention time in tissues, and thus promising activity in photodynamic therapy.

Full text of DOI:10.1016/S0960-894X(03)00002-7

Bioorganic & Medicinal Chemistry Letters 13 (2003) 833–835
Hydroporphyrins as Tumour Photosensitizers: Synthesis and
Photophysical Studies of 2,3-Dihydro-5,15-
di(3,5-dihydroxyphenyl) Porphyrin
Yann Ferrand,a Ludovic Bourre,b Gerard Simonneaux,a,* Sonia Thibaut,b
Fabrice Odobel,c Y. Lajatb and Thierry Patriceb,*
a
´
´
Laboratoire de Chimie Organometallique et Biologique, UMR CNRS 6509, Universite de Rennes1, 35042 Rennescedex, France
b
´
Departement Laser, Neurochirurgie, CHU Nantes, 44480 Nantes, France
` ´
Laboratoire de Synthese Organique, UMR CNRS 6513, Universite de Nantes, 44322 Nantes cedex 3, France
c
Received 22 October 2002; revised 6 December 2002; accepted 20 December 2002
Abstract—The synthesis and characterization of 2,3-dihydro-5,15-di(3,5-dihydroxyphenyl) porphyrin is reported. The phototoxicity
on C6 cell lines and the pharmacokinetics are also reported as preliminary results showing a very high tumor to skin ratio and short
retention time in tissues, and thus promising activity in photodynamic therapy.
# 2003 Elsevier Science Ltd. All rights reserved.
Recently, promising photosensitizers have been developed
for photodynamic therapy.1 Among these substances,
certain hydroxysubstituted meso-tetraarylchlorins showed
good activity.2,3 However one problem with these mole-
cules is the long retention time of the drug in normal
tissues, more particularly in the skin because after injec-
tion and treatment, the patient must be protected against
sun during a period up to several days. A possible solu-
tion is to use diphenylporphyrins instead of tetra-
phenylporphyrins as precursors. This class of porphyrins
was originally studied by several groups as models of the
photosynthetic centre.4 These compounds are stable and
now relatively easy to prepare in high yields. We pos-
tulated that (i), the diphenylporphyrin can be reduced
in chlorin in order to show absorption in the red and
(ii), the presence of two free meso positions will make
easier the biotransformation in order to decrease pro-
longed cutaneous photosensitivity, a major adverse
effect associated with tetraphenyl porphyrin series and
photofrin.
the meso position, a structural change which is accom-
panied by a short retention time in tissues together with
a promising activity. Cell uptake and photodynamic
properties are compared to those of the structurally
related meso-tetrahydroxyphenylchlorin (m-THPC), a
drug which is one of the most potent photosensitizers
discovered to date.5
The condensation of dipyrrylmethane 1 with 3,5-di-
methoxybenzaldehyde furnishes, upon oxidation with
o-chloranil, 5,15-di(3,5-methoxyphenyl)porphyrin 2 in
high yield (78%) under reaction conditions that ther-
modynamically favors the formation of the intermediate
porphyrinogen, as previously reported in the Lindsey
method.6 The porphyrin is then demethylated with
boron tribromide in dichloromethane to give 3 (yield 75%).
In the last step, diimide reduction of the diphenyl-
porphyrin gives a mixture of the corresponding dihydro
and tetrahydroporphyrins using the method of
Whitlock et al.7 Selective oxidation with o-chloranil
removes the bacteriochlorin. Actually, reduction of the
porphyrin gave first a mixture of tosylated inter-
mediates 4 and after addition of HCl N/10, the tetra-
hydroxy product 5 with the desired photophysical
properties. Interestingly, the yield obtained for the
chlorin is high (75%) and the formation of the corres-
ponding porphyrin in the last step is not observed.
Several attempts were made in order to isolate the
On the basis of these criteria, we now report the synth-
esis and the biophysical studies of these second genera-
tion photosensitizers bearing only two phenyl groups on
*Corresponding author. Tel.: +33-022-323-6285; fax: +33-022-323-
5637; e-mail: gerard.simonneaux@univ-rennes1.fr
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00002-7

834
Y. Ferrand et al. / Bioorg. Med. Chem. Lett. 13 (2003) 833–835
hydroxy form of the bacteriochlorin but it seems quite
unstable under acidic conditions. The preparation of
the compound 5 is summarized in Figure 1. Whilst this
work was in progress, a different diphenylchlorin8,9 has
been reported. The porphyrin was first prepared using
phenyldipyrrylmethane and trimethyl orthoformate
(yield: 18%). Two hydroxy groups were then added to
one pyrrole to give the chlorin.10,11 However, the pre-
sent procedure using condensation of aryl aldehyde
with unsubstituted dipyrrylmethane leads a better yield
than the synthesis using phenyldipyrrylmethane and
formaldehyde as precursors.10,11
properties were characterized by the partition coefficient
of the compound between the two non-miscible solvents
octanol and water. The octanol/water partition ratios
were 26 for compound 5 and 40 for m-THPC indicating
that 5 was more hydrophilic than m-THPC. The fluor-
escence spectrum of compound 5, in saline isotonic
solution (100 mg mLÀ1), showed a peak at 649 nm with
a shoulder between 700 and 750 nm, after excitation at
488 nm. The singlet oxygen quantum yield of 5 and
m-THPC was determined using perinaphthenone as
reference. As compared to perinaphthenone, the quan-
tum yield was 0.69 for 5 and 0.70 for m-THPC for the
highest laser energy in deuterated methanol, indicating a
similar efficiency for the two photosensitizers (Fig. 2).
As expected from the presence of four hydroxy groups,
the compound is weakly soluble in polar solvent such as
methanol and water. The lipophilic and hydrophilic
The intracellular localization patterns of the chlorin 5
(10 mg mLÀ1) were determined by confocal laser micro-
spectrofluorimetry, showing staining in C6 glioma cells
as a function of time, with a maximum of fluorescence
after 3 h. We employed the MTT assay to evaluate the
effect of the concentration on the phototoxic potential
of compound 5.3 The survival rates ranged from 100%
to 97% when cells were incubated for 5 h without light,
showing absence of toxicity. The photosensitizer dose
inducing a 90% death rate (LD90) for C6 cells was 4.8
mg mLÀ1 for 5 and 6.8 mg mLÀ1 for m-THPC after 5-h
incubation and 20 J/cm2 irradiation (Fig. 1).12 Thus 5
showed an efficacy similar to that of m-THPC. Com-
pound 5 was also tested for in-vivo photosensitizing
activity on human colon adenocarcinoma cell (HT-29)
tumour and it was found to show optimal photo-
dynamic response (40% of inhibition growth) for a
delay between injection and irradiation of 12 h while for
m-THPC optimal values were only observed for a delay
of 24 h using in both cases a dye dose of 5 mg kgÀ1 and
300 J cmÀ2 irradiation with laser light (650 nm, 300
mW). This suggests that 5 is rapidly distributed in tis-
sues and rapidly eliminated (vide infra). More impor-
tantly, the fluorescence levels recorded in tumour, skin
and muscle of mice were detected 3–144 h after 2 mg
kgÀ1 of 5 or m-THPC injection. It was found that the
maximum concentration is reached quickly and reten-
tion time in tissues is shorter than that of m-THPC. For
example, the concentration of 5 decreased rapidly in all
tissues after 24 h, and no sensitizer was detectable in
these tissues after 144 h (Fig. 3). In contrast, m-THPC
concentration in skin is about 100 times above what is
observed for 5 after 48 h. The shorter retention time of 5
in tissues can be explained by the presence of two free
meso positions13 which may facilitate the bio-
transformation of the drug and thus decrease prolonged
cutaneous photosensitivity. Further developments and
improvements of this approach are in progress using
various diphenylchlorins,14 and more extensive biological
studies will be reported elsewhere.
Figure 1. The synthesis of 5.
Figure 2. Toxicity and phototoxicity of compound 5 and m=THPC
in C6 cells determined by MTT assay. C6 cells were incubated for 5 h
2
. . . . ..
with photosensitizers and irradiated at 650 nm and 20 J/cm 5 (
. . . . . .
^
);
m-THPC (
~
) or not irradiated 5 (—^—); m-THPC (—~—).
References and Notes
1. Pandey, R. K.; Zheng, G. The Porphyrin Handbook 2000, 6,
157.
2. Bonnett, R.; White, D. R.; Winfield, U. J.; Berenbaum,
M. C. Biochem. J. 1989, 261, 277.
Figure 3. The fluorescence levels recorded in tumour (~), skin (&)
and muscle (^) of mice detected 3–144 h after 2 mg kgÀ1 of 5.

Y. Ferrand et al. / Bioorg. Med. Chem. Lett. 13 (2003) 833–835
835
3. Morlet, L.; Vonarx-Coinsmann, V.; Lenz, P.; Foultier,
M. T.; Xavier de Brito, L.; Stewart, C.; Patrice, T. J. Photo-
chem. Photobiol. 1995, 28, 25.
4. Anderson, H. L. J. Chem. Soc., Chem. Commun. 1999,
2323.
5. Bonnett, R.; Martinez, G. Org. Lett. 2002, 4, 2013.
6. Manka, J. S.; Lawrence, D. S. Tetrahedron Lett. 1989, 30,
6989.
7. Whitlock, H. W.; Hanauer, R.; Oester, M. Y.; Bower, B. K.
J. Am. Chem. Soc. 1969, 91, 7485.
13. Clarke, O. J.; Boyle, R. W. Tetrahedron Lett. 1998, 39, 7167.
14. Selected spectroscopic data:
5,15-Bis(3,5-dimethoxyphenyl)porphyrin (2). 1H NMR
(CF3COOD) ppm: 11.13 (2H-meso), 9.7 and 9.3 (2H, d, J=6
Hz, pyrrole), 7.9 (o-H, phenyl), 7.4 (p-phenyl), 4.2 (methoxy);
UV–Vis (CH2Cl2), max (nm): 407 (e 400 dm3 mmolÀ1 cmÀ1),
502 (e 5.0), 536 (e 4.9), 574 (e 4.9), 629 (e 1.3). FAB-MS: calcd
for C36H30N4O4: 582.2401 found: 582.2432 [M+H+].
5,15-Bis(3,5-dihydroxyphenyl)porphyrin (3). 1H NMR(ace-
tone d6): 10.55 (H meso), 9.6 and 9.3 (2H, d, J=6 Hz, pyrrole),
7.3 (o-H, phenyl), 6.9(p-phenyl), À3.1 (NH); UV–Vis (ace-
tone), lmax (nm): 402 (e 400 dm3 mmolÀ1 cmÀ1), 532 (e 5.0),
572 (e 4.9), 628 (e 1.4). FAB-MS: calcd for C32H22N4O4:
526.1641 found: 526.1657 [M+H]+.
8. Wang, T. Y.; Liu, H. L.; Chen, J. R.; Liu, F. G.; Guu, Y.;
Ma, J. S. Bioorg. Med. Chem. Lett. 2001, 11, 2049.
9. Wang, T. Y.; Chen, J. R.; Ma, J. S. Dyesand Pigments
2002, 52, 199.
10. Boyle, R. W.; Dolphin, D. J. Chem. Soc., Chem. Commun.
1994, 2463.
11. Sylva, A. M.; Tome, A. C.; Neves, M. G. Cavaleiro. Tet-
rahedron Lett. 2000, 41, 3065.
12. Morlet, L.; Vonarx, V.; Foultier, M. T.; Gouyette, A.;
Stewart, C.; Lenz, P.; Patrice, T. J. Photochem. Photobiol. B
1997, 39, 249.
2,3-dihydro-5,15-bis(3,5-dihydroxyphenyl)porphyrin (5). 1H
NMR(acetone, d6) ppm: 10.1 and 9.2 (2H-meso), 9.35–8.6
(6H, m, pyrrole), 7.2 and 6.95 (4H, d, o-phenyl), 6.85 and 6.75
(2H, t, p-phenyl), 4.65 and 4.45 (4H, m, pyrrolidine), À1.4 and
À1.9 (2H, s, NH); UV–Vis (acetone), lmax (nm): 395 (e 31.6
dm3 mmolÀ1 cmÀ1), 405 (e 162), 500 (e 12.6), 645 (e 40.1). FAB-
MS: calcd for C32H24N4O4: 528.1798 found: 528.1805 [M+].