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A.M. Slomp et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 156–161
yield), which was obtained by using pyrrole, p-nitrobenzaldehyde
and benzaldehyde in a 4:3:1 ratio. In this case, flash chromatogra-
phy of the reaction concentrated extracts gave an additional frac-
tion that was recrystallized to give the trans-A2B2 di-nitrated
analog (8, 12% yield). Amino-tetraarylporphyrin 9 was obtained
from 7 with Sn/HCl reactant (61% yield).27 Anionic tetra-sulfonated
porphyrin 6 (tetrasodium salt, 68% yield) was synthesized through
sulfonation of 1 with H2SO4 at 100 °C28 followed by workup involv-
ing cation exchange resin (Na+ form). Cationic porphyrins 12b–16b
(iodide salts) were synthesized through methylation of the
corresponding meso-substituted (4-pyridyl)porphyrins (12a–16a)
with methyl iodide. In turn, 12a–16a were obtained by means of
Adler-Longo29 conditions, with modifications of Zimmerman
et al. (2003)30 and Vandresen et al. (2016).31 Briefly, pyrrole, 4-pyr-
idine-carboxialdehyde and benzaldehyde (4:3:1) were refluxed in
propionic acid to give a mixture of porphyrins. Silica gel chro-
matography of the porphyrin mixture using chloroform as eluent
gave separately compounds 1, 12a and 13a with 0.3%, 0.9% and
1.1% yield, respectively. The silica gel column was then washed
with methanol and the washes were concentrated to give a residue
that was loaded to a second column. Elution with chloroform:
methanol (98:2) allowed the isolation of compounds 14a (1.0%),
15a (3.1%) and 16a (2.5%). Cationic porphyrin 11 was synthesized
as described by Gomes et al. (2011),32 which was based on the
nucleophilic displacement of the four p-fluorine atoms of the
5,10,15,20-tetraquis(pentafluorophenyl)porphyrin with 4-mercap-
topyridine, followed by cationization with methyl iodide. All
synthesis procedures and spectral data are detailed in Supplemen-
tary information.
Porphyrins 1–11 and 12b–16b were first evaluated in terms of
their photophysical aspects, including photostability and 1O2
assays. Both photophysical assays were conducted either in aque-
ous media (photostability assay)32 or in DMF (1O2 assay).33 Under
these conditions, porphyrins 1, 3, 5 and 12b could not be properly
dissolved, giving rise to aggregates. Attempts to make them soluble
using the help of an ultrasound bath was unsuccessful for some
cases. For this reason, one or both of the photophysical parameters
were not assayed for the mentioned compounds. Photostability
(Fig. 1) was verified by measuring the porphyrin Soret band extinc-
tion, under white light irradiation (100 mW/cm2), during 30 min.31
In general, there were no substantial changes in the Soret band
absorbance level of the studied porphyrins. Porphyrins 4 and 11,
which respectively decayed to 83 and 89% from the original absor-
bance, were the least photostable compounds. In this way, all com-
pounds herein evaluated were considered sufficiently photostable
to be properly evaluated through the following experiments.
The ability of the porphyrins to generate 1O2 was comparatively
estimated by the DPiBF photooxidation method.32 DPiBF absor-
bance decreasing (at 415 nm) was reported in terms of percentage
from the initial absorbance values, with lower percentages reflect-
ing higher 1O2 production (Fig. 2). All compounds evaluated were
capable of producing 1O2 in considerable amounts under 9 mW/
cm2 light intensity for 30 min irradiation time. Porphyrin 9 pro-
moted a 1O2 generation that resulted in 59% of DPiBF remaining
absorbance, while the other porphyrins were better 1O2 producers,
giving substantially lower absorbances after total irradiation time.
Porphyrin 14b was among the photosensitizers that decreased
DPiBF absorbance to 20% or less, with porphyrins 12b and 15b
being able to produce 1O2 to an extent that resulted in only 13%
and 12% of the remaining absorbance, respectively. These results
clearly indicated that the charged porphyrins (11, 6 and 12b–
16b) presented superior 1O2 producing properties, especially those
ones having the methylpyridinium group directly attached to the
meso carbons of the porphyrin ring (12b–16b).
After photophysical evaluation, 1–11 and 12b–16b were first
screened in terms of their photodynamic properties against HaCaT
cells in a relatively high porphyrin concentration (1 lM) under 100
mW/cm2 light irradiation for 30 min (fluence of 180 J/cm2). Table 1
presents values related to porphyrin photoactivity, expressed as
cell viability (%) after PDT, based on MTT assay. Non polar tetra-
kis-tetra(aryl)porphyrins 1–5 and 10, and amino porphyrin 9 were
unable decrease cell viability under the conditions evaluated.
Nitroporphyrins 8 and 9 promoted a very weak photodynamic
effect (93% and 95% of cell viability), with anionic porphyrin 6 giv-
ing somewhat a lower cell viability value (76%). Cationic deriva-
tives 11, 12b–16b were the most effective photosensitizers under
the conditions evaluated, which gave less than 30% cell viability.
These preliminary results suggest that the photoactivity on HaCaT
cells was related the presence charged moieties within porphyrin
structure. The inactivity or low activity of the neutral and anionic
analogs could be attributed to their inappropriate lipo-/hydrophilic
balance, which affects porphyrin solubilization or a proper interac-
tion with cell structures.
Cationic porphyrins 11, 12b–16b, were then more thoroughly
evaluated by both measuring their partition coefficient (Table 2)
and performing PDT with lower porphyrin concentrations (0.01,
0.03, 0.1 and 0.3 lM). Partition coefficient was determined using
an adaptation34 of the shake-flask method, which is based on the
partition between water and n-butanol. The values obtained as
log PBW were then converted into log POW through the equation
shown in the footnotes of Table 2. The Log POW values found for
the cationic porphyrins indicated that among 12b–16b,
hydrophilicity increases with charge number, where the
triply- and quadruply-charged compounds (15b and 16b) were
predominantly soluble in the water phase (negative log POW val-
ues), with the doubly-charged ones (13b and 14b) giving positive
log POW values. Log PBW or log POW of porphyrin 12b could not be
calculated because it remained exclusively in the n-butanol phase.
Fig. 1. Photostability of porphyrins 2, 4, 6, 7, 8, 9, 11, 13b, 14b, 15b and 16b after irradiation with white light (100 mW/cm2) for different periods of time.32 Percentages of
Soret band remaining absorbance, after 30 min irradiation, are indicated in parentheses. Lower percentages reflect lower photostabilities.
Slomp, Amanda M.
