Synthesis and biological evaluation of methoxyphenyl porphyrin derivatives as potential photodynamic agents

Article abstract of DOI:10.1016/S0968-0896(01)00138-9

A new meso-2,4,6-trimethoxyphenyl porphyrin covalently linked to a 2′,6′-dinitro-4′-trifluoromethylphenyl group by an amine bond 5 and its metal complex with Cd(II) 6 was prepared. The photodynamic activities of 5 and 6 were evaluated in vitro on Hep-2 ce

Full text of DOI:10.1016/S0968-0896(01)00138-9

Bioorganic& Medicinal Chemistry 9 (2001) 1943–1949
Synthesis and Biological Evaluation of Methoxyphenyl Porphyrin
Derivatives as Potential Photodynamic Agents
M. Elisa Milanesio,a Flavia S. Moran,b E. Ines Yslas,b M. Gabriela Alvarez,b
Viviana Rivarolab and Edgardo N. Durantinia,*
a
´
Departamento de Quımica y Fısica, Universidad Nacional de Rıo Cuarto, Agencia Postal Nro 3, 5800 Rıo Cuarto, Argentina
´
´
´
b
´
´
´
Departamento de Biologıa Molecular, Universidad Nacional de Rıo Cuarto, Agencia Postal Nro 3, 5800 Rıo Cuarto, Argentina
Received 16 September 2000; accepted 4 January 2001
Abstract—A new meso-2,4,6-trimethoxyphenyl porphyrin covalently linked to a 20,60-dinitro-40-trifluoromethylphenyl group by an
amine bond 5 and its metal complex with Cd(II) 6 was prepared. The photodynamicactivities of 5 and 6 were evaluated in vitro on
Hep-2 cells. A considerable increase in the photocytotoxic effect was found for 6, which has higher singlet molecular oxygen,
O2(1Ág), production. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
pounds to investigate the theoretical and instrumental
aspects of PDT.12À14
The tetrapyrrolic macrocycles play highly diverse roles
in biological systems.1 One of more recent and promis-
ing applications of porphyrins in medicine is in the
detection and cure of tumors.2,3 Photodynamictherapy
(PDT) is based on the principle that a photosensitizer
become concentrated in tumor cells, upon subsequent
irradiation with visible light in the presence of oxygen,
specifically inactive neoplastic cells.4,5
This paper reports a convenient procedure for the
synthesis of a new porphyrin dyad 5, which contain
three meso-2,4,6-trimethoxyphenyl groups on the por-
phyrin ring. The macrocycle is covalently attached to a
20,60-dinitro-40-trifluorophenyl group by an amine bond.
The synthesis of this type of compound requires access
to asymmetrically substituted porphyrins.11,15 Thus,
porphyrins bearing two different types of meso-sub-
stituents can be prepared by a binary mixed aldehyde
condensation.16 This approach is statistical in nature
and usually a multiple porphyrin products are obtained.
Usually, six porphyrins are formed; the workup is not
simple because of the tar present and the yields are very
poor.15 More direct approaches to porphyrins bearing
three identical meso-phenyl substituents and one differ-
ent are provided by condensation of dipyrromethane
with aldehyde.15,17 By application of this method, 5-(4-
acetaminophenyl)-10,15,20-tris(2,4,6-trimethoxyphenyl)
porphyrin (3) was obtained, which was hydrolyzed to
give amino porphyrin 4. Dyad 5 was obtained by
means of a chloro-substitution reaction under two-
phase catalysis conditions, reacting amino porphyrin
4 and 1-chloro-2,6-dinitro-4-trifluoromethylbencene. In
this heterogeneous system, the presence of potassium
hydroxide activates the amino group on the por-
phyrin 4 to aromatic nucleophilic substitution reac-
tions.18 This method provides a general procedure for
the synthesis of porphyrin dyads bearing an amine
linkage.
Basically, two types of reactions can occur after photo-
activation of the photosensitizer. One involves the gen-
eration of free radicals (type I photochemical reaction)
and the other, the production of singlet molecular oxy-
gen, O2(1Ág) (type II), is the main species responsible
for cell inactivation.5 Evidence favors the role of the
type II process in cells, although the photodynamic
process of the sensitizers on neoplastic tissues is still not
well understood.6
Recently, several porphyrin derivatives covalently
linked to actives molecules have been synthesized with a
potential use in the treatment of tumors.7À11 Studies of
triplet state and in biological media carried out with a
series of 5,10,15,20-tetrakis (methoxyphenyl) porphyrins
have shown that these syntheticporphyrins are effective
photosensitizers which can be used as model com-
*Corresponding author. Tel.:+54-358-467-6157; fax: +54-358-467-
6233; e-mail: edurantini@exa.unrc.edu.ar
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00138-9

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M. E. Milanesio et al. / Bioorg. Med. Chem. 9 (2001) 1943–1949
On the other hand, the spectroscopic properties and
quantum yield of singlet molecular oxygen, O2(1Ág),
production (ÈÁ) of porphyrins can be largely changed
by forming complexes with metals.19À22 In particular,
the metal complex with Cd(II) produces a considerable
increase in ÈÁ and a high absorption coefficient in the
visible region of the spectrum, mainly in the photo-
therapeuticwindow (600–1000 nm). Taking into
account these properties, the ÈÁ value of dyad 5 was
considerably increased by forming its metal complex
with Cd(II) to form dyad 6. Absorption and emission
spectroscopic properties of the dyads were determined
in pure solvent. The mediation of O2(1Ág) in the photo-
oxidation mechanism was analyzed in tetrahydrofuran-
bearing photo-oxidizable substrate. In the biological
system, the response of Hep-2 human larynx-carcinoma
cell line to cytotoxicity photo-induced by the porphyrins
was evaluated. The survival of the irradiated cells, pre-
viously treated with the porphyrin, was dependent on
both light dose and sensitizer photochemical properties.
The effect shows a correlation between the O2(1Ág)
production and the photodynamic activity upon Hep-2
cells. Therefore, the intermediacy of O2(1Ág) appears to
be mainly responsible for the photocytotoxic activities
of these porphyrins. The results contribute to under-
standing the photodynamicproecss yielded by these
agents and the sensitivity of Hep-2 cells to this photo-
damage.
condition, pyrrole serves as the reactant in excess and as
the solvent for the reaction, giving direct formation of
dipyrromethane (Scheme 1). The brown crude solution
was washed with dilute aqueous NaOH. The dipyrro-
methane 1 was isolated yielded 80% by flash chroma-
tography on silica gel in a mildly basic medium, using n-
hexane/ethyl acetate/triethylamine (80/20/1) as eluent.
Triethylamine (ffi1%) was added to prevent decomposi-
tion of the dipyrromethane on silica column, which is
slightly acidic.15,17 Dipyrromethane 1 is stable in the
purified form upon storage at 0 ꢀC in nitrogen atmo-
sphere and absence of light. Performing high purity
dipyrromethane 1 is essential for its application in the
synthesis of substituted porphyrin.
Porphyrin formation. Scheme 2 shows the condensation
of dipyrromethane 1 with 2,4,6-trimethoxybenzaldehyde
and 4-acetamidobenzaldehyde to form porphyrins. The
reaction was performed using catalytic among of
BF.3O(C2H5)2 and chloroform as solvent at room tem-
perature. The reaction mixture was subject to oxidation
with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ).
Two porphyrins were easily separated by flash chroma-
tography, the first less polar moving band correspond-
ing to meso-tetrakis(2,4,6-trimethoxyphenyl) porphyrin
(2) and the second 5-(4-acetamidophenyl)-10,15,20-
tris(2,4,6-trimethoxyphenyl) porphyrin (3). The desired
amide porphyrin 3 was obtained in 16% yield. Finally,
basichydrolysis of amide porphyrin 3 with aqueous
KOH in mixture THF/methanol (2:1) affords 78% of 5-
(4-aminophenyl)-10,15,20-tris-(2,4,6-trimethoxyphenyl)
porphyrin (4).
Results and Discussion
Synthesis
Porphyrin dyad. Coupling of amine porphyrin 4 and 1-
chloro-2,6-dinitro-4-trifluorometilbenzene by amine
The 5-(4-aminophenyl)-10,15,20-tris(2,4,6-trimethoxy-
phenyl)porphyrin (4) was synthesized from dipyrro-
methane derivate and the appropriate aldehydes
(Scheme 2).
Dipyrromethane formation. Aldehyde and pyrrole
undergo acid-catalyzed condensation at room tempera-
ture.15,17,23 The condensation of 2,4,6-trimethoxy-
benzaldehyde with a large excess of pyrrole (1:45
aldehyde/pyrrole mol ratio) catalyzed by trifluoroacetic
acid
affords
meso-2,4,6-trimethoxyphenyldipyrro-
methane (1) (Scheme 1). The reaction mixture was stir-
red for 20 min at room temperature resulting in
complete disappearance of aldehyde. In this reaction
Scheme 1.
Scheme 2.

M. E. Milanesio et al. / Bioorg. Med. Chem. 9 (2001) 1943–1949
1945
bond allows formation of new dyads 5, as shown in
Scheme 3. To synthesize the dyad 5, the nucleophylic
aromaticsubstitution reatcion was performed under
two phase catalytic conditions, using tetra-
butylammonium bromide (TBAB) as catalyst. When the
reactants were mixed in homogeneous media (chloro-
form) without potassium hydroxide, no reaction was
observed after 2 h of stirring at room temperature.
However, when the solid potassium hydroxide was
added, the reaction takes place, and after 1.5 h 90% of
the dyad 5 is formed. The basicmedium activates the
–NH2 group to nucleophilic reaction with chloro aro-
porphyrins show the typical Soret and Q-bands char-
acteristic of free-base and Cd(II) metalloporphyrins (Table
1).21 The relative intensity of the Q-bands for 2 and 5, show
an etio-type spectrum (EVI>EIII>EII>EI).11,24 Also, as can
be observed in Table 1, dyad 6 presents a value of E
higher than 2 and 5 at l>600 nm.
The steady-state fluorescence emission spectra of these
porphyrins present two maxima, as shown in Table 1.
The same values of lem were obtained exciting the sam-
ple at the wavelength of maximum absorption of the
Soret and the Q-bands. The two bands are characteristic
for similar porphyrins and they have been assigned to
Q(0-0) and Q(0-1) transitions.19 A small Stokes shift is
expected for tetraphenylporphyrin derivatives indicating
that the spectroscopic energy is nearly identical to the
relaxed energy of the singlet state.19 The fluorescence
quantum yields (fF) of the porphyrins were calculated
by steady state comparative method using TPP as a
reference.25,26 The values of fF=0.053ꢁ0.002,
0.032ꢁ0.002 and 0.011ꢁ0.001 were obtained in tetra-
hydrofuran for porphyrin 2, dyads 5 and 6, respectively.
These results are consistent with the obtained for similar
metalloporphyrins.19
18
maticsubstrates.
Finally, dyad 5 was treated with cadmium acetate in
basicmedium to afford metal complex Cd(II), dyad 6, in
62% of yield (Scheme 3).
Spectroscopic studies and singlet molecular oxygen
production
The absorption spectrum of porphyrin 2, dyads 5 and 6,
in dichloromethane are summarized in Table 1. These
The aerobicirradiation with monohcromaticlight
(l=515 nm) of photosensitizer in tetrahydrofuran was
performed in the presence of 9,10-dimethylanthracene
(DMA). This substrate quenches O2(1Ág) by exclusively
chemical reaction.27 A time-dependent decrease in the
DMA concentration was observed by following a
decrease in its absorbance. The photo-process follows
first-order kinetics with respect to DMA concentration.
Figure 1 shows the semi-logarithmicplots describing the
progress of the reaction for DMA, using different pho-
tosensitizers. From these plots the values of the observed
rate constant (kobs) were calculate for DMA. The results
of kobs were (9.34ꢁ0.05)ꢂ10À5, (7.35ꢁ0.04)ꢂ10À5
,
(3.62ꢁ0.04)ꢂ10À5 and (7.68ꢁ0.05)ꢂ10À5 for TPP,
porphyrin 2, dyad 5 and 6, respectively. The quantum
yield of O2(1Ág) production (ÈÁ) was calculated from
the slopes of the plots for the porphyrin compared with
the corresponding slope obtained for the reference
(TPP). The values of ÈÁ in tetrahydrofuran were 0.49,
0.24 and 0.51 for 2, 5 and 6, respectively. As can be
expected, a considerable increase in ÈÁ was obtained by
forming a metal complex of dyad 5 with Cd(II).19,20
Studies in vitro on Hep-2 cells
Since these porphyrins are water-insoluble, they were
added to the cell cultures from a liposomal solution of
l,d-a-dipalmitoyl phosphatidylethanolamina bearing
20% mols of cholesterol. The use of phospholipid lipo-
Scheme 3.
Table 1. Absorption and fluorescence data for porphyrin 2 and dyads 5 and 6
Absorption lmax (nm) (e [MÀ1cmÀ1])a
Fluorescence lmax
b
Porphyrin
2
418 (179,490)
420 (216,950)
435 (226,320)
512 (10,190)
515 (14,270)
570 (12,920)
545 (3350)
550 (5770)
610 (6620)
592 (3150)
592 (5110)
646 (940)
650 (2470)
650
652
624
719
719
668
Dyad 5
CdDyad 6
aDichloromethane.
bTetrahydrofuran.

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M. E. Milanesio et al. / Bioorg. Med. Chem. 9 (2001) 1943–1949
somes as vehicles to transport hydrophobic photo-
sensitizers could constitute an advantage for its uses in
PDT, since liposomes optimizes the release of lipophilic
sensitizer to LDL.28,29
line treated with dyad 5 and its Cd(II) complex 6,
respectively.
On the other hand, when the cell cultures do not treated
with the sensitizer were irradiated with visible light, no
significant lethality was found (Fig. 3, control). There-
fore, the cell mortality obtained after irradiation of the
cultures treated with the porphyrin is due to the photo-
sensitization effect of the agent produced by visible
light. Also, the dyad uptake for the Hep-2 cell was ana-
lyzed by fluorescence. A similar value of ꢃ0.003 mmol/
106 cells was found for these agents.
Cell toxicity induced by the photosensitizers was first
analyzed in dark condition. The Hep-2 cells were trea-
ted with different concentration of the sensitizer for 24 h
at 37 ꢀC. The result are shown in Figure 2. No toxicity
in terms of cell survival (measured by microscopy in the
presence of TB) was detected at 0.1 and 1 mM of dyad 5
and 6, while similar cytotoxicity was found incubating
the cells with 10 mM of these dyads. However, porphyrin
2 was significantly toxic at every evaluated concentra-
tion (Fig. 2). Therefore, dyad 5 and 6 where selected for
the photodynamicstudies in biological medium. After
the combined treatment of cells with 1 mM of the dyad
for 24 h, the culture plates were irradiated with visible
light. The irradiation system used in these studies is
described in experimental section. The corresponding
survival curve for each photosensitizer is shown in Fig-
ure 3. As expected, Hep-2 cell inactivation depends on
the light dose employed. After an irradiation of ꢃ50 J/
cm2, the survival fractions were 0.52 and 0.16 for cell
The results of cytotoxicity (Fig. 3) show higher cell
mortality with an increase of the O2(1Ág) production by
the porphyrins. From the cell inactivation curves, the
irradiation time required to inactivate 50% of cell
population (t50) were obtained (48 and 12 min for 5 and
6, respectively). Taking into account the light intensity
at the treatment site (see experimental section), the cor-
responding light dose (D50) were calculated given values
of 52 and 13 J/cm2. Under these conditions, the cyto-
toxic effect considerably is increases with dyad 6, corre-
lating with the sensitizer production of O2(1Ág).
In conclusion, the following two basic steps were used
sequentially in the synthesis: (1) meso-(2,4,6-trimethoxy-
phenyl) dipyrromethane was formed from correspon-
dent benzaldehyde derivative and pyrrole catalyzed by
acid; (2) condensation of dipyrromethane with appro-
priate benzaldehydes yields the desired asymmetricpor-
phyrins 3, with an appreciable yields of 16%. The
resultant amido porphyrins 3 can be hydrolyzed to
amine porphyrin 4 by heat in tetrahydrofuran/metha-
nol/KOH medium. This macrocycle was covalently
attached to a phenyl group bearing electron-with-
drawing –NO2 and –CF3 groups by amine bond. Two-
phase catalysis conditions allow performing the amine
linkage with high yield due to the nucleophilic activa-
tion of the –NH2 group. The presence of methoxy
groups appears to be beneficial from the standpoint of
tumor localization.30 For this reason, extra methoxy
Figure 1. First-order plots for the photooxidation of DMA (45 mM)
photosensitized by TPP (&), dyad 5 (*), Cddyad 6 (!) and por-
phyrin 2 (~) in tetrahydrofuran.
Figure 3. Inactivation of the Hep-2 cells incubated with 1 mM of
dyad for 24 h and exposed to different light doses with visible light;
(*) dyad 5; (!) Cddyad 6; (*) corresponding controls in dark
conditions. Values represent meanstandard deviation of three separate
experiments.
Figure 2. Cytotoxic effect of the photosensitizers on Hep-2 cells incu-
bated with different concentration of porphyrin for 24 h at 37 ꢀC under
dark. Values represent meanꢁstandard deviation of three separate
experiments.

M. E. Milanesio et al. / Bioorg. Med. Chem. 9 (2001) 1943–1949
1947
groups were included in the porphyrin moiety of this
dyad. On the other hand, a lipophilictrifluoromethyl
group gives an amphiphilic character of this agent. The
influence of the trifluoromethyl group in biologically
active molecules is often associated with the increased
lipophilicity that this substituent imparts.31 This present
strategy may be easily used for preparation of other
similar dyad derivatives. The applications of these ways
of reactions to the synthesis of potential photo-
therapeutic agents could be of considerable interest for
PDT. In biological medium, the behavior of these pho-
tosensitizers indicated that the photodynamic effect
correlates with the O2(1Ág) production, and therefore,
these dyads appear to perform its photocytotoxic activ-
ities on Hep-2 cells through a mechanism which
involves mainly intermediacy of O2(1Ág).
(ppm) 3.73 (s, 6H, Ar–OCH3), 3.81 (s, 3H, Ar–OCH3),
5.91 (m, 2H, pyrrole-H), 6.07 (s, 1H, meso-H), 6.10 (q,
2H, pyrrole-H), 6.20 (s, 2H, Ar 3,5-H), 6.63 (m, 2H,
pyrrole-H); 8.47 (s, brs, 2H, pyrrole NH). MS (m/z) 312
(M+) (312.1475 calcd for C18H20N2O3). Anal. calcd C
69.21, H 6.45, N 8.97; found C 69.52, H 6.28, N 8.80.
5-(4-Aminophenyl)-10,15,20-tris(2,4,6-trimethoxylphenyl)
porphyrin (4).
benzaldehyde
A solution of 2,4,6-trimethoxyl-
(0.57 g, 2.91 mmol), 4-acetamido-
benzaldehyde (0.66 g, 4.07 mmol) and meso-(2,4,6-
trimethoxylphenyl)dipyrrometane 1 (2.00 g, 4.76 mmol)
in 500 mL of chloroform was purged with nitrogen for
15 min. Then BF.3O(C2H5)2 (2.0 mmol, 0.80 mL of 2.5 M
stock solution in chloroform) was added. The solution
was stirred for 60 min at room temperature. Then,
DDQ (1.02 g, 4.50 mmol) was added and the mixture
was stirred for an additional 1 h at room temperature.
The solvent was removed under reduced pressure and
flash column chromatography (silica gel, dichloro-
methane/methanol gradient) yielded 438 mg (16%) of
the pure selected 5-(4-acetamidophenyl)-10,15,20-
tris(2,4,6-trimethoxylphenyl) porphyrin 3 as second
porphyrin moving band. TLC analysis (dichloro-
methane/methanol 5%) Rf 0.45. MS (m/z) 941 (M+)
(941.3638 calculated for C55H51N5O10). To a solution of
amido-porphyrin 3 (200 mg, 0.21 mmol) in 40 mL of
THF was added 40 mL of saturated solution of KOH in
methanol. The reaction mixture was stirred under a
nitrogen atmosphere for 18 h at 60 ꢀC. Then, the mix-
ture was diluted with dichloromethane and washed with
aqueous sodium carbonate. The organic solvent was
evaporated under reduced pressure. Flash column
chromatography (silica gel, dichloromethane/methanol
0.5%) afforded 147 mg 78% of the desired 5-(4-amino-
phenyl)-10,15,20-tris-(2,4,6-methoxylphenyl) porphyrin
4. TLC (silica gel) Rf (dichloromethane/methanol
5%)=0.80. 1H NMR (CDCl3, TMS) d (ppm) À2.70
(brs, 2H, pyrrole N–H), 3.88 (s, brs, 2H, Ar–NH2), 4.09
(s, 24H, Ar–OCH3), 4.17 (s, 9H, Ar–OCH3), 6.55 (s, 6H,
10,15,20-Ar 3,5-H) 7.05 (d, 2H, J=8.3 Hz, 5-Ar 3,5-H),
7.97 (d, 2H, J=8.3 Hz, 5-Ar 2,6-H); 8.75–8.95 (m, 8H,
pyrrole). MS m/z 899 (M+) (899.3533 calcd for
C53H49N5O9). Anal. calcd C 70.73, H 5.49, N 7.78;
found C 70.38, H 5.70, N 7.64.
Experimental
General
UV–visible and fluorescence spectra were recorded on a
Shimadzu UV-2401PC spectrometer and on a Spex
1
FluoroMax fluorometer, respectively. HNMR spectra
was recorded on a Varian Gemini spectrometer at
300 MHz. Mass spectra were taken with a Varian Matt
312 operating in EI mode at 70 eV and a VestecLaser
TecResearhc Instrument by laser desorption time-of-
flight mass spectroscopy. Uniplate Silica gel GHLF 250
microns thin layer chromatography plates from Ana-
ltech and silica gel 200–400 Mesh for column chroma-
tography from Aldrich were used.
All the chemicals from Aldrich were used without fur-
ther purification. d,l-a-Dipalmitoyl phosphatidyletha-
nolamine from Sigma was used in liposome preparation.
Solvents from Merck (GR grade) were distilled and
storage over 4 A molecule sieves and sodium bicarbo-
nate. Tetrahydrofuran was distilled from lithium alumi-
num hydride under an argon atmosphere.
Synthesis
meso-(2,4,6-Trimethoxylphenyl)dipyrrometane (1).
A
solution of 2,4,6-trimethoxybenzaldehyde (1.76 g,
9 mmol) and pyrrole (25.0 mL, 360 mmol) was degassed
by bubbling with nitrogen for 15 min. Then tri-
fluoroacetic acid (139 mL, 1.8 mmol) was added. The
solution was stirred for 20 min at room temperature, at
which point no starting aldehyde was shown by TLC
5,10,15,20-tetrakis(2,4,6-trimethoxylphenyl) porphyrin
(2). Porphyrin 2 was obtained as the first porphyrin-
moving band in the flash column chromatography
described above for porphyrin 3. TLC (silica gel) Rf
(dichloromethane/methanol 5%)=0.82. 1H NMR
(CDCl3, TMS) d (ppm) À2.70 (brs, 2H, pyrrole N–H),
4.08 (s, 32H, Ar–OCH3), 4.16 (s, 12H, Ar–OCH3), 6.55
(s, 6H, 10,15,20-Ar-3,5-H); 8.75–8.95 (m, 8H, pyrrole).
MS m/z 974 (M+) (974.3740 calcd for C56H54N4O12).
Anal. calcd C 68.98, H 5.58, N 5.75; found C 68.87, H
5.52, N 5.81.
analysis
(cyclohexane/ethyl
acetate/triethylamine
80:20:1). The mixture was diluted with dichloromethane
(100 mL), washed with aqueous 0.1 M NaOH (50 mL)
and then washed with water. The organicphase was
dried with MgSO4, filtered and the solvent was removed
under reduced pressure. The unreacted pyrrole was
removed by vacuum distillation at room temperature.
The product was purified by flash chromatography
(silica gel, cyclohexane/ethyl acetate/triethylamine
80:20:1) yielded 2.25 g (80%) of the pure dipyrrometane
1. TLC (silica gel) Rf (cyclohexane/ethyl acetate/trie-
thylamine 80:20:1)=0.25. 1H NMR (CDCl3, TMS) d
5-[4-N-(N-20,60-dinitro-40-trifluoromethylphenyl) amino-
phenyl]-10,15,20-tris(2,4,6-trimethoxyphenyl) porphyrin
(dyad 5). Amino porphyrin 4 (50 mg, 0.056 mmol),
1-chloro-2,6-dinitro-4-trifluorometilbenzene (38 mg,
0.140 mmol) and tetrabutylammonium bromide (TBAB)

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M. E. Milanesio et al. / Bioorg. Med. Chem. 9 (2001) 1943–1949
(45 mg, 0.140 mmol) were dissolved in 40 mL of chloro-
form. Then, 500 mg of potassium hydroxide was added
and the mixture was stirred for 1.5 h at room tempera-
ture. The crude mixture was neutralized with hydro-
chloric acid (0.1 N) and then extracted with three
portions of chloroform (30 mL each). The organic phase
was dried with MgSO4, filtered and the solvents
removed under reduced pressure. Flash column chro-
matography (silica gel, chloroform/methanol gradient)
afforded 57 mg (90%) of pure dyad 5. TLC (silica gel) Rf
(dichloromethane/methanol 3%)=0.84 and Rf (di-
chloromethane)=0.64. 1H NMR (CDCl3, TMS) d
(ppm) À2.76 (brs, 2H, pyrrole N–H); 4.10 (s, 24H, Ar–
OCH3); 4.19 (s, 9H, Ar–OCH3); 6.57 (s, 6H, 10,15,20-
Ar 3,5-H); 6.75 (d, 2H, J=8.0 Hz, 5-Ar 3,5-H); 7.70 (d,
2H, J=8.0 Hz, 5-Ar 2,6-H); 8.50 (s, 2H, Ar 30,40-H);
8.75–8.90 (m, 8H, pyrrole); 10.30 (s, brs, 1H,-NH); MS
m/z 1133 (M+) (1133.3421 calcd for C60H50F3N7O13).
Anal. calcd. C 63.55, H 4.44, N 8.65; found C 63.43, H
4.51, N 8.70.
bance 0.1, l=515 nm) in tetrahydrofuran were irra-
diated with monochromatic light at l=515 nm. The
DMA photooxidation was measured by the decrease of
the absorbance at lmax=378 nm. The observed rate
constants (kobs) were obtained by a linear least-squares
fit of the semilogarithmicplot of Ln A /A versus time.
0
TPP in tetrahydrofuran was used as the standard
(ÈÁ=0.62).33 Measurements of sample and reference
under similar conditions afforded ÈÁ for porphyrins 2,
dyads 5 and 6 by direct comparison of the slopes in the
linear region of the plots. All the experiment were per-
formed at 25.0ꢁ0.5 ꢀC. The pooled standard deviation
of the kineticdata, using different prepared samples,
was less than 5%.
Liposome preparation
The incorporation of the porphyrins into the phospho-
lipid bilayer of the d,l-a-dipalmitoyl phosphatidyletha-
nolamine was achieved by a modification of the ethanol
injection procedure of Kremer et al.34 Typically, 2 mL
Cadmium (II) 5-[4-N-(N-20,60-dinitro-40-trifluoromethyl-
phenyl) aminophenyl]-10,15,20-tris(2,4,6-trimethoxylphe-
nyl) porphyrin (dyad 6). Dyad 5 (20 mg, 0.017 mmol) in
pyridine (8 mL) and saturated potassium hydroxide in
methanol (4 mL) was treated with cadmium acetate
dihydrate (18 mg, 0.067 mmol). After 2 h reflux, the
solution was cooled and treated with of water (30 mL).
The organicphase was extracted with three portions of
dichloromethane (15 mL each) and the solvents
removed under vacuum to give 13 mg (62%) of pure Cd
(II) porphyrin (5). MS m/z 1244 (M+) (1243.7374 calcd
for C60H48F3N7O13Cd). Anal. calcd C 57.91, H 3.89, N
7.88; found C 57.98, H 3.75, N 7.80.
of a solution bearing 9.60 mM of phospholipid,
1.91 mM of cholesterol and 0.27 mM of porphyrin in
ethanol-tetrahydrofuran binary mixture (1:1, vol/vol)
was injected into 10 mL of phosphate-buffered saline
(PBS) at 80 ꢀC. The injection was performed at a speed
28
of 50 mL/min with magneticstirring.
Cell culture
The Hep-2 human larynx-carcinoma cell line (Asocia-
cion Banco Argentino de Celulas, ABAC, Instituto
Nacinal de Enfermedades Virales Humanas, Pergamino,
Argentina) was maintained frozen in liquid nitrogen.
The cells were grown as a monolayer employing Dul-
becco’s modified Eagle’s medium (DMEM) containing
10% fetal calf serum (FCS) and 50 mg gentamycin as
antibiotic. The cells were incubated at 37 ꢀC in a humi-
dified 5% CO2 atmosphere and the medium was chan-
ged daily. The cell line was routinely checked for the
absence of mycoplasma contamination.
Irradiation
The light source used was a Kodak slide projector
equipped with a 150 W lamp. The light was filtered
through a 3 cm water layer to absorb heat and a glass
filter. A wavelength range between 350 and 800 nm was
selected by optical filters.32 The light intensity at the
treatment site was 18.0 mW/cm2 (Radiometer Laser
Mate-Q, Coherent).
Cell photosensitization studies and quantification
An appropriate number of cells (ꢃ1ꢂ106 cells) were
inoculated in 25 cm2 culture flasks and incubated to
obtain nearly confluent cell layers. Then, an appropriate
amount of the porphyrin, incorporated into liposomes,
was added to the culture flask bearing 5 mL of the
medium. The cells were treated with sensitizer for 24 h
in dark condition. Afterwards, the medium containing
the photosensitizer was discarded. Cells were washed
three times with medium and kept in 5 mL of it. The
dishes were exposed for different time intervals to visible
light. After each irradiation time, the viability of the
cells was estimated by microscopy with trypan blue (TB)
exclusion test using a Neubauer chamber counter.32 The
same procedure without irradiation was carried out for
determining dark toxicity. The uptake was determined
adding 1.0 mL of 4% sodium dodecyl sulphate (SDS,
Merck) to 1 mL of cellular suspension. The mixture was
incubated further for 15 min (in the dark and room
temperature) and centrifuged at 9000 rpm for 30 min.
Spectroscopic studies
Absorption spectra were recorded at 25.0ꢁ0.5 ꢀC using
1 cm path length cells. Wavelength maxima (lmax) were
measured by taking the mid-point between the two
positions of spectrum where the absorbance of the band
is equal to 0.9 Amax. The fluorescence quantum yield
(fF) of porphyrins were calculated by comparison of the
area below the corrected emission spectrum in tetra-
hydrofuran with that of tetraphenylporphyrin (TPP) as
a fluorescence standard, exciting at lex=550 nm.25
A
value of fF=0.10 for TPP in tetrahydrofuran was cal-
culated by the comparison with the fluorescence spec-
trum in toluene using fF=0.1126 and taking into
account the refractive index of the solvents.25 Photo-
oxidation 9,10-dimethylanthracene (DMA) was used to
determine O2(1Ág) production by the photosensitizers.
Solutions of DMA (45 mM) and photosensitizer (absor-

M. E. Milanesio et al. / Bioorg. Med. Chem. 9 (2001) 1943–1949
1949
The concentration of sensitizer in the supernatant was
measured by spectrofluorimetry in solution of 2% SDS
in PBS. The fluorescence values obtained from each
sample were referred to the total number of cells con-
tained in the suspension. The concentration of the por-
phyrin in this sample was estimated by comparison with
a calibration curve obtained with standard solutions of
the sensitizer in 2% SDS ([sensitizer]ꢃ0.1–5 mM). Four
culture flasks were used for each incubation time. Any
experiments were compared with a culture control
without photosensitizer.
11. Durantini, E. N. J. Porphyrins Phthalocyanines. 2000, 4,
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Acknowledgements
The authors are grateful to Consejo Nacional de Inves-
tigaciones Cientıficas
y Tecnicas (CONICET) of
Argentina, Agencia Nacional de Promocion Cientıfica y
Tecnologica FONCYT and SECYT of the Universidad
Nacional de Rıo Cuarto for financial support. V.R. and
E.N.D. hold a position of Researchers at CONICET.
M.G.A. thanks CONICET for a research fellowship.
22. Garbo, G. M.; Fingar, V. H.; Wieman, T. J.; Noakes,
E. B., III; Haydon, P. S.; Cerrito, P. B.; Kessel, D. H.; Mor-
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