686
M.B. Ballatore et al. / European Journal of Medicinal Chemistry 83 (2014) 685e694
Generally, effective photosensitizers for PDI exhibit a high ab-
from Analtech (Newark, DE, USA) were used. Solvents (GR grade)
from Merck (Darmstadt, Germany) were distilled. Ultrapure water
was obtained from a Labconco (Kansas, MO, USA) equipment model
90901-01.
sorption coefficient in the visible region of the spectrum and a long
lifetime of triplet excited state to produce efficiently ROS. Thus,
several porphyrins with intrinsic positive charges meet these re-
quirements and they have been successfully tested as photo-
inactivating agents against microorganisms [3,4]. Also, an
interesting behavior was observed in fullerene C60 linked to an
electron donor molecular structure, such as porphyrins [7]. These
dyads present higher capacity to form photoinduced charge-
separated state [8e10]. Therefore, the electron transfer process
can compete with the O2(1Dg) production [11]. Previous studies
revealed that fullerene C60 covalently attached to a porphyrin
substituted by methoxy groups and its metal complex with Zn(II)
were effective photosensitizers to kill Hep-2 human larynx carci-
noma cell line [12,13]. Thus, depending on the microenvironment
where the photosensitizer is localized, this compound could pro-
duce a biological photodamage through either a O2(1Dg)-mediated
photoreaction process or a free radical mechanism under low ox-
ygen concentration. Hence, these dyads have offered a promising
molecular architecture for photosensitizer agents that can produce
cellular inactivation under anoxic conditions [12,13].
2.2. Synthesis
5,10,15,20-Tetrakis(4-methoxyphenyl)porphyrin (TMP) was
purchased from Aldrich. 4-(5,5-Dimethyl-1,3-dioxan-2-yl)benzal-
dehyde 2, N-methyl-2-phenylfulleropyrrolidine (MPC60), N,N-
dimethyl-2-(40-acetamidophenyl)fulleropyrrolidinium (DAC6þ0) and
5,10,15,20-tetrakis[3-(N-ethyl-N-methylcarbazoyl)]porphyrin
(TCP4þ) were synthesized as previously described [18e21]. Syn-
thesis procedures of porphyrin derivatives and dyads are shown in
Schemes 1 and 2.
2.2.1. meso-(N-Ethyl-3-carbazoyl)dipyrromethane 1
A
solution of N-ethyl-3-carbazolecarboxaldehyde (1.01 g,
4.52 mmol), pyrrole (50.0 mL, 722.75 mmol) and TFA (65 L,
m
0.85 mmol) was treated following the methodology reported
before [15]. After work up, this approach yielded 1.36 g (89%) of
dipyrromethane 1.
In the present study, a novel porphyrinefullerene C60 dyad 5
was synthetized, which contain three carbazoyl groups attached to
the tetrapyrrolic macrocycle at the meso positions. Many
condensed heterocyclic compounds containing a carbazole nucleus
have been reported to develop a broad range of potent biological
activities [14]. Also, porphyrins connected to peripheral carbazole
units represent efficient light-harvesting structures, where the
carbazole groups can act as antenna at lower wavelengths [15,16].
The nitrogen atoms in the carbazole units were used to obtain dyad
6 bearing cationic groups on the periphery of the macrocycle. The
formation of cationic amphiphilic photosensitizers has several
interesting features that make these compounds attractive photo-
sensitizers for a variety of biological systems [4,17]. The spectro-
scopic and photodynamic properties of these dyads were studied in
homogeneous media of different polarities and in a simple bio-
mimetic system formed by reverse micelles. The results obtained
for these dyads were compared with those of porphyrin and
fullerene reference compounds. Photodynamic action was then
evaluated in vitro for inactivation of S. aureus cells.
2.2.2. 5-(4-(5,5-Dimethyl-1,3-dioxan-2-yl)phenyl)-10,15,20-tris[3-
(N-ethylcarbazoyl)]porphyrin 3
A solution of N-ethyl-3-carbazolecarboxaldehyde (289 mg,
1.29 mmol), benzaldehyde 2 (355 mg, 1.61 mmol) and dipyrro-
methane 1 (1.10 g, 3.24 mmol) in 365 mL of chloroform was purged
with argon for 30 min. Then, trifluoroacetic acid (TFA, 500
mL,
6.50 mmol) was added. The solution was stirred for 30 min at room
temperature and after that triethylamine (TEA, 1 mL, 7.20 mmol)
was added. Then, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone
(DDQ, 2.50 g, 11.00 mmol) was added and the mixture was stirred
for an additional 3 h at room temperature. The solvent was evap-
orated under reduced pressure. Flash column chromatography
(silica gel, cyclohexane/chloroform (1:9) to chloroform gradient)
yielded 251 mg (18%) of pure porphyrin 3 as second moving purple
band. TLC (chloroform) analysis Rf ¼ 0.42. 1H NMR (CDCl3, TMS)
d
[ppm]: ꢀ2.48 (2H, pyrrole NH); 0.92 (s, 3H, eCH3); 1.29 (s, 3H,
eCH3); 1.69 (t, 9H, J ¼ 7.1 Hz, carbazoleeCH3); 3.85 (d, 2H, eCH2e,
J ¼ 10.8 Hz); 3.97 (d, 2H, eCH2e, J ¼ 10.8 Hz); 4.63 (q, 6H, J ¼ 7.1 Hz
carbazole-CH2); 5.75 (s, 1H, eCHe); 7.31 (m, 3H, Ar carbazole); 7.59
(m, 6H, Ar carbazole); 7.76 (d, 3H, Ar carbazole, J ¼ 8.2 Hz); 7.93 (d,
2H, 5-Ar 3, 5-H, J ¼ 7.9 Hz); 8.22 (d, 3H, Ar carbazole, J ¼ 8.0 Hz);
8.31 (d, 2H, 5-Ar 2, 6-H, J ¼ 7.9 Hz); 8.38 (d, 3H, Ar carbazole,
J ¼ 8.2 Hz); 8.88 (d, 2H, pyrrole, J ¼ 5.0 Hz); 8.92 (m, 6H, pyrrole);
8.99 (s, 3H, Ar carbazole). ESI-MS [m/z] 1080.4965 [MþH]þ
(1079.4887 calculated for C74H61N7O2). Also, the condensation
under these conditions produced 5,10,15,20-tetrakis[3-(N-ethyl-
carbazoyl)]porphyrin (TCP), which was obtained as the first moving
purple band (84 mg, 12%). Spectroscopic data of TCP agree with
those previously reported [15].
2. Materials and methods
2.1. General
Proton nuclear magnetic resonance spectra were performed on
a FT-NMR Bruker Avance DPX400 spectrometer at 400 MHz. Mass
Spectra were recorded on a Bruker micrOTOF-QII (Bruker Daltonics,
MA, USA) equipped with an atmospheric pressure photoionization
(APPI) source. FT-IR spectra were recorded on a Nicolet Impact 400
(Madison, WI, USA). Absorption and fluorescence spectra were
carried out in a Shimadzu UV-2401PC spectrometer (Shimadzu
Corporation, Tokyo, Japan) and on a Spex FluoroMax spectrofluo-
rometer (Horiba Jobin Yvon Inc, Edison, NJ, USA), respectively. The
visible light source used to irradiate cell suspensions was a Nova-
mat 130 AF (Braun Photo Technik, Nürnberg, Germany) slide pro-
jector containing a 150 W lamp. A 2.5 cm glass cuvette filled with
water was used to remove the heat from the lamp. A wavelength
range between 350 and 800 nm was selected by optical filters. The
fluence rate was determined as 90 mW/cm2 (Radiometer Laser
Mate-Q, Coherent, Santa Clara, CA, USA).
2.2.3. 5-(4-Formylphenyl)-10,15,20-tris[3-(N-ethylcarbazoyl)]
porphyrin 4
Porphyrin 3 (261 mg, 0.24 mmol) was hydrolyzed with 5 mL of
TFA in 12 mL of a heterogeneous mixture of chloroform/water (1:1).
The reaction was stirred at room temperature for 30 h. Then, the
mixture was washed with water (10 mL each) for two times, the
organic phase neutralized with TEA and washed with water (8 mL).
Removal of the solvent under vacuum yielded 228 mg (95%) of the
desired porphyrin 4. TLC (chloroform/TEA 5%) analysis Rf ¼ 0.86. 1H
All the chemicals from Aldrich (Milwaukee, WI, USA) were used
without further purification. Sodium bis(2-ethylhexyl)sulfosucci-
nate (AOT) from Sigma (St. Louis, MO, USA) was dried under vac-
uum. Silica gel thin-layer chromatography (TLC) plates 250 microns
NMR (CDCl3, TMS)
d
[ppm]: ꢀ2.50 (2H, pyrrole NH); 1.68 (t, 9H,
J ¼ 7.1 Hz, carbazole-CH3); 4.63 (q, 6H, J ¼ 7.1 Hz carbazole-CH2);
7.29 (m, 3H, Ar carbazole); 7.58 (m, 6H, Ar carbazole); 7.75 (d, 3H, Ar