Metallation of pentaphyrin with Lu(III) dramatically increases reactive-oxygen species production and cell phototoxicity

Article abstract of DOI:10.1016/j.ejmech.2010.12.007

Photodynamic therapy (PDT) is an emerging cancer treatment modality based on the excitation of a nontoxic photosensitizer with harmless visible light to produce reactive-oxygen species (ROS) that induce apoptosis and/or necrosis in cancer cells. As the ef

Full text of DOI:10.1016/j.ejmech.2010.12.007

European Journal of Medicinal Chemistry 46 (2011) 712e720
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Metallation of pentaphyrin with Lu(III) dramatically increases reactive-oxygen
species production and cell phototoxicity
,
a,
Maurizio Ballico a, Valentina Rapozzi b, Luigi E. Xodo b , Clara Comuzzi
**
*
a Department of Chemical Science, University of Udine, Via del Cotonificio, 108, 33100 Udine, Italy
b Department of Biomedical Science and Technology, University of Udine, P.le Kolbe, 4, 33100 Udine, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Photodynamic therapy (PDT) is an emerging cancer treatment modality based on the excitation of
a nontoxic photosensitizer with harmless visible light to produce reactive-oxygen species (ROS) that
induce apoptosis and/or necrosis in cancer cells. As the efficacy of this therapy strongly depends of the
nature of the photosensitizer, there is a great interest to develop new photoactive molecules. Here we
report for the first time the synthesis, characterization and bioactivity of metal complexes between the
non-aromatic expanded porphyrin, namely 20-[[40-(Trimethylsilyl)ethoxycarbonyl]phenyl-2,13-
dimethyl-3,12-diethyl-[24] iso-pentaphyrin (PCRed) and Zn(II) [Zn(II)ePCRed] or Lu(III) [Lu(III)ePCRed].
The complexation of these two diamagnetic heavy metal ions to PCRed improved the properties of the
free photosensitizer as a PDT drug. We discovered that the 1:1 complex between PCRed and Lu(III)
significantly increases the cellular uptake, ROS production and antiproliferative capacity in four cancer
cell lines. Our study shows that metal complexation is a useful strategy to potentiate iso-pentaphyrin as
a PDT drug.
Received 16 July 2010
Received in revised form
24 November 2010
Accepted 7 December 2010
Available online 14 December 2010
Keywords:
Pentaphyrin
Lu(III)
PDT
ROS
Cell proliferation
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
transparent and efficiently generates ROS and singlet oxygen (1O2)
is a potential PDT agent. The photosensitizer most widely used in
Large polypyrrole macrocycles or expanded porphyrins,
composed by five or more pyrrole rings, are versatile ligands
capable to coordinate almost all metals and semimetals of the
periodic table [1] and act as receptors of anions [2] or neutral
molecules [3]. Expanded porphyrins may be used for a wide range
of applications including lanthanide and actinide complexation [4],
anion binding and optics [1,5], biomedical applications as magnetic
resonance imaging [4] (MRI, paramagnetic contrast agents), anti-
sense-targeted RNA [6], X-ray radiation sensitization [4g] and
visible-light-based photodynamic therapy (PDT) [7]. PDT is a local
treatment designed to eliminate abnormal tissue lesions including
cancer [8]. It requires the administration to the tumor tissue of
a photosensitizer agent, which becomes active when it is irradiated
with light. The activated photosensitizer transfers energy to
molecular oxygen to generate reactive-oxygen species (ROS), in
particular singlet oxygen, that causes cell death [9]. A macrocycle
that strongly adsorbs in the region where the body is most
clinic is Photofrin, a defined mixture of haematoporphirin [8].
However, in the last decade many new compounds have been
produced and tested and nowadays PDT has the potential to
become a major treatment in cancer therapy. Among these new
drugs, expanded porphyrins have been successfully tested as PDT
photosensitizers [10]. Expanded porphyrins of the type [1.1.1.1.1],
called pentaphyrins, have been not thoroughly investigated. These
compounds contain five pyrrolic rings linked by means of meso-
like bridges, which make the macrocycle flexible and capable of
assuming different conformations. We have previously reported
the synthesis and photosensitizing properties of two [1.1.1.1.1]-
macrocycles characterized by a different oxidation state: non-
aromatic iso-pentaphyrin with 24
p-electrons and aromatic
pentaphyrin with 22 -electrons [11]. To improve their phototox-
p
icity we decided to coordinate the pentaphyrin macrocycle to
diamagnetic metal ions. We reasoned that the insertion of a metal
into the central core of the expanded porphyrin should produce
two main effects: the shift of the absorption peaks to longer
wavelengths, i.e. into the ideal spectral region (650e800 nm) and
an increased production of ROS and 1O2 due to heavy metal effects.
In fact, the presence of a central metal ion can strongly influence
the photokilling efficiency of a photosensitizer [10b, 12]. To
* Corresponding author. Tel.: þ39 0432 558823; fax: þ39 0432 558803.
** Corresponding author. Fax: þ39 0432 494301.
E-mail addresses: luigi.xodo@uniud.it (L.E. Xodo), clara.comuzzi@uniud.it
(C. Comuzzi).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.12.007

M. Ballico et al. / European Journal of Medicinal Chemistry 46 (2011) 712e720
713
understand the heavy metal effect, one should consider the
mechanism of singlet oxygen formation. Upon activation, the
photosensitizer is promoted from its ground state (S0) to the first
excited singlet state (S1) from which it can change its spin multi-
plicity to a triplet exited state (T1) through intersystem crossing
(ISC). Energy transfer to triplet oxygen to yield highly reactive
singlet oxygen occurs when the triplet state of the photosensitizer
is significantly populated. The efficiency of singlet oxygen
production [13] is directly related to the quantum yield of triplet
state of the photosensitizer and its lifetime. In this mechanism ISC
is a key parameter. Some diamagnetic metals are known to enhance
ISC and to elongate triplet lifetime of the photosensitizer. For the
series of diamagnetic metals including Y, In, Lu and Cd,
the production of singlet oxygen was found proportional to the
increase in atomic number: a result that is interpreted as a heavy
atom effect [14]. Instead, paramagnetic species deactivate the
excited state, reduce the excited-state lifetime and prevent
photochemical reactions from taking place [13]. In fact, it has been
demonstrated that photophysical parameters that are directly
associated with the intrinsic decay rates of the singlet excited state
or fluorescence lifetimes, are enhanced by coordination of para-
magnetic metal ions. So, the coordination of paramagnetic metals
to photosensitizers decreases the singlet oxygen production.
However, there are exceptions to this general behavior especially
revealed for porphyrins and phthalocyanines [10b,12,13].
N
H
N
H
NH
O
HN
O
NH
NH
HN
N
1
OH
O
HO
O
1) TFA, CH Cl ,
2h, r.t.
H
H
2) DDQ, CH Cl ,
30 min., r.t.
HN
NH
Si
O
O
4
Si
O
O
2
DDQ, CH2Cl2
30 min., r.t.
N
NH
NH
HN
N
TFA, CH2Cl2,
2h, r.t.
Here we describe the synthesis of two new macrocycles belonging
to the class of [1.1.1.1.1]-pentaphyrins, namely the partially oxidized
pentaporphynogen 3 and the non-aromatic PCRed 4 (with 24
Si
O
O
3
structure of one of the possible
tautomers
p-electrons) (Scheme 1).
We have modified the original structure of the pentaphyrins by
Scheme 1. Synthesis of the PCRed 4, and the partially oxidized pentaporphynogen 3.
replacing the diethyl-pyrrole with an unsubstituted pyrrole and by
introducing a carboxylic group on the phenyl substituent. These
two changes were made to eliminate the time-consuming
synthesis of 3,4-diethyl-pyrrole, improve the solubility in polar
solvents of these molecules and allow the conjugation of PCRed
with polyethylene glycol (PEG) to obtained a photosensitizer with
a superior bioavailability. To study the heavy metal effect on PCRed
we coordinate it to Zn(II) and Lu(III) (5, 6) (Schemes 2 and 3). The
new compounds have been characterized by spectroscopic anal-
yses, while the stoichiometry of the complexes was determined by
mass spectrometry (ESI-MS and EI). The biological activity of these
new compounds was tested in four different cell lines in terms of
ROS production and phototoxicity.
water and methanol adducts could be seen (m/z 712 ([MH þ H2O]þ)
and 726 ([MH þ CH3OH]þ). Finally the UVevis spectrum recorded
in CH2Cl2 exhibits a strong Soret-like band at 485 nm (log
3
¼ 4.39)
and a Q-band at 814 nm (log
3
¼ 3.71) (Supporting information S1).
Partially oxidized pentaporphynogen 3 was obtained following
a modified McDonald-type acid-catalyzed [3 þ 2] condensation
seen for 4, but the oxidation step with DDQ was omitted and the
workup of the molecule was performed at the end of the cycliza-
tion. Compound 3 was isolated to investigate the mechanism of
[3
þ
2] cyclization and successive macrocycle oxidation, to
synthesize a highly flexible molecule able to coordinate metal ions
of different dimension more easily than oxidized pentaphyrins.
The (þ)-ESI-MS spectrum (in CH3OH) displays the protonated
molecular ion peak at m/z 696 ([MH]þ), suggesting the presence of
a reduced form of the pentaphyrin and adducts with m/z 714
([MH þ H2O]þ) and 728 ([MH þ CH3OH]þ).
2. Results and discussion
2.1. Synthesis and characterization of the PCReds
The complexity of the 1H NMR spectrum reveals that the
partially oxidized pentaporphynogen 3 is a very flexible macrocycle
The synthesis of PCRed 4 was performed following a modified
McDonald-type acid-catalyzed [3 þ 2] condensation strategy as
shown in Scheme 1 [11]. PCRed was obtained as a green metallic
film in 50% yield and characterized by a combination of ESI mass
spectrometry, NMR and UVevis spectroscopy. 1H NMR of 4 in
CD3OD shows the non-aromatic character of this molecule: meso-
N
NH
Zn
NH
NH
HN
N
N
N
CH are detected at
substituents appears at
nance at 1.20 ppm. In the CDCl3 spectrum, NH protons were
detected at 11.41, 10.21 and as a large signal in the region from
d
6.52 and 5.60 ppm, CH2 quartet of the ethyl
Zn(OAc)2
NH
NH
d
3.13 ppm, while the singlet methyl reso-
CHCl3, CH3OH
72h, r.t.
d
d
8.80 to 8.10 ppm (overlapped to the aromatic signals); the absence
of upfield shift of the nitrogen protons indicate the lack of a large
diamagnetic ring current characteristic of aromatic porphyrins.
The (þ)-ESI mass spectrometry studies confirmed the proposed
Si
Si
O
O
O
O
5
3
structure of one of the possible
tautomers
structure for
4 and showed that this macrocycle can form
complexes with small neutral molecules: depending on sample
preparation, peaks in the mass spectrograms corresponding to both
Scheme 2. Simultaneous oxidation/metallation of the partially oxidized pentapor-
phynogen 3 with zinc acetate: synthesis of the Zn(II)ePCRed 5.

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M. Ballico et al. / European Journal of Medicinal Chemistry 46 (2011) 712e720
The (þ)-ESI-MS spectrum in CH3OH of 5 shows the molecular ion
peak at m/z 756 ([M]þ) (Supporting information S3), which was
identified to be a 1:1 complex between Zn(II) and PCRed 4 (Scheme
2). The ESI-MS spectrum of 5 shows signals at m/z 774 and 788
corresponding to adducts with H2O and CH3OH, respectively. The
addition of formic acid (0.2%) to a solution of 5 in MeOH resulted in
a rapid demetallation. Indeed, (þ)-ESI-MS mass spectrum of this
solution reveals the complete disappearance of the peaks relative to
the complex and the appearance (as main peaks) of the signals
attributable to the PCRed 4 (in particular peaks at m/z 694 ([MH]þ)
and 726 ([MH þ CH3OH]þ). This is a further evidence that, during the
synthesisof 5, the coordination of the metal occurs together with the
oxidation of the macrocycle. The 1H NMR spectrum of 5 in CD3OD
shows that the coordination of Zn(II) does not cause drastic changes
in the pentaphyrin structure: the only signal that undergoes
N
N
NH
NH
HN
N
N
N
Lu
Lu(OAc)3.4H2O
CHCl3, CH3OH
72h, r.t.
or
48h, 60°C
HN
N
Si
Si
O
O
O
O
3
6
structure of one of the possible
tautomers
Scheme 3. Simultaneous oxidation/metallation of the partially oxidized pentapor-
phynogen 3 with Lutetium acetate hydrate: synthesis of the Lu(III)ePCRed 6.
a downfield shift is the meso-CH from
d 6.52 (4) to 7.97 (5) ppm,
that exists in solution in several rapidly exchanging tautomers and
conformers with comparable stability. None of these structures is
fully conjugated. We identified multiple peaks attributable to the
same protons in different isomers where the macrocycle conjuga-
tion is interrupted. For example, the 1H NMR spectrum in CDCl3
while -pyrrolic, phenylic and other meso-CH protons do not
b
change. This simple NMR pattern suggests that the symmetry of the
macrocycle is retained by the complexation of the zinc ion
(Supporting information S4, S5). The signals of the alkyl substituents
in
the ethyl-CH2 protons was downfield shifted from
and singlet CH3 from 1.26 to 2.05 ppm. This indicates that the
b
-position are those most affected bycomplexation: the quartet of
showed several signals in the region between
due to exchanging NH protons and various doublets attributable to
the -pyrrolic protons of the isomers in the region between 6.0
and 8.0 ppm.
The UVevis spectrum in CH2Cl2 displays two large bands at
300 nm (log ¼ 4.21), and a single broad Q-
¼ 4.16) and 482 nm (log
type band at 802 nm (log
¼ 3.93). Interestingly, the intensity of the
d 12.0 and 10.0 ppm
d
3.16 to 4.10 ppm
d
b
d
pyrrols are directly involved in the coordination to Zn(II). The
absence in 2D NMR NOESY spectra of anycorrelation between meso-
CH and b-pyrrolic protons is a further indication of the high distor-
tion from planarity of this complex. The 1H NMR spectra of complex
5 in CDCl3 suggest the existence of a mixture of isomeric structures
rapidly exchanging on the NMR time scale. The spectrum shows the
3
3
3
Q-type band is greater in compound 3 then in 4. The partly oxidized
pentaporphynogen 3 was converted into 4 by using DDQ as an
oxidizing agent. It is interesting to note that the [3 þ 2] condensation
did not give the pentaporphynogen, but only a more stable oxidized
macrocycle (partly oxidized pentaporphynogen 3). The fluorescence
spectrum of 4 in methanol shows a broad emission band between
500 and 550 nm, with a maximum around 540 nm (Ex 480 nm)
(Supporting information S2). Its intensity is indirectly proportional
to pentaphyrin concentration (from 1.78 ꢀ 10ꢁ5 M to 5.18 ꢀ 10ꢁ7 M).
In CH2Cl2 the fluorescence spectrum does not change, showing
a broad band with a maximum at 525 nm (lex ¼ 480 nm) but, in this
case, the intensity is directly proportional with concentration (from
5.74 ꢀ 10ꢁ7 M to 1.78 ꢀ 10ꢁ5 M).
NH of 5 centered at d 10.03 (1H) and 8.0 (2H) as broad signals. NMR
and mass spectra indicate that 4 loses only one proton upon
complexation with Zn(II), as previously observed [14]. Finally, the
UVevis Soret band of Zn(II)ePCRed 5 in CH2Cl2 is bathocromatically
shifted at 491 nm (log
3
¼ 4.33), while the Q-band is at 817 nm
(log
3
¼ 3.37) (Supporting information S1).
We also inserted Lu(III) into 3 using conditions similar to those
used for the complexation of Zn(II). We used Lu(OAc)3$4H2O and Lu
(NO3)3$5H2O and performed the reactions in THF or CHCl3/CH3OH
mixtures, either at room temperature or at 60 ꢂC (Scheme 3). EI-MS
spectra show that PCRed 4 forms with Lu(III) a 1:1 complex (6). The
1H NMR spectra of 6 are similar to those obtained for 5. CD3OD
spectra show that meso-CH protons appear at
-pyrrolic signals at 9.29, 6.88 and 5.90 ppm; CH2 of ethyl groups
and singlet CH3 are detected at 3.23 and 1.20 ppm respectively.
d 7.81 and 5.60 ppm,
2.2. Metallation chemistry of PCReds
b
d
d
The size of the central cavity of PCRed is expected to be similar
to that of sapphyrin (w5.5 Å). Compared to porphyrins, the pres-
ence of an additional nitrogen donor allows this molecule to act as
The 1H NMR spectrum of 6 in CDCl3 shows only one NH signal at
9.83 ppm (1H). The others NH signals at 11.41 ppm (1H), 10.21 ppm
(1H) and 8.80e8.10 ppm (overlapped to the aromatic signals) dis-
appeared from the spectrum upon Lu(III) complexation. This result
suggests that the coordination of Lu(III) causes the ligand to lose
three protons leading to a neutral complex. This is the reason why
we could not follow the metal complexation by ESI-MS. The
treatment with formic acid produces a partial demetallation of the
complex which releases free PCRed 4. This reaction was easily fol-
lowed by ESI-MS as in the spectrum appeared the characteristic
peaks of 4 at m/z 694 ([MH]þ) and 726 ([MH þ CH3OH]þ). The
UVevis spectrum of complex 6 in CH2Cl2 (Supporting information
S1) shows a pattern completely different from that of complex 5,
Zn(II)ePCRed, with broad bands between 300 and 600 nm: at
a
pentadentate ligand, potentially capable of coordinating
a plethora of metal cations. To investigate the effects of heavy
metals we synthesized complexes between PCRed and Zn(II) and
Lu(III). We used these metal ions because diamagnetic Zn(II)
coordinated to phthalocyanines and porphyrins strongly increased
their photosensitizing property [15]; while Lu(III) having a higher
atomic weight provides a good system to investigate the heavy
metal effect. It should be remembered that LuTex is one of the most
efficient photosensitizers known up to date [16].
PCRedemetal complexes were synthesized by direct reaction
between Zn(II) or Lu(III) and 4. However, a simplified synthetic route
allows to insert, by simultaneous oxidation and metallation reac-
tions, the metals on 3 and to obtain complexes 5 and 6. The treat-
ment in air of 3 dissolved in CHCl3 with an excess of anhydrous Zn(II)
acetate in CH3OH, at room temperature for 48 h, resulted in the
formation of complex 5, which was characterized by UVevis spec-
troscopy, 1H NMR, 2D COSY and NOESY measurements. The stoi-
chiometry of the complex was established by mass spectrometry.
339 nm (log
(log
3
¼ 4.25), at 406 nm (log
3
¼ 4.15) and at 498 nm
3
¼ 3.99). These bands are blue shifted compared to 5 and this
can be ascribed to the formation of aggregates in this solvent [17].
However, the addition of small amount of TFA to complex 6 in
dichloromethane causes the appearance of the Soret band at
480 nm (Supporting information S1 insert). Complexes 5 and 6
show broad fluorescence emission spectra in the region between

M. Ballico et al. / European Journal of Medicinal Chemistry 46 (2011) 712e720
715
500 and 550 nm, when excited at 480 nm (Supporting information
metal complexes 5 and 6 (15
mM). It can be seen that the fluores-
S6 and S7). Fluorescence spectra of the Lu(III)ePCRed 6 shows two
peaks at 518 and 558 nm in methanol (Ex 480 nm). Instead, in PBS
buffer the fluorescence emission of 5 and 6, in the region between
500 and 550 nm, is quenched and comparable to that of 4 in the
same medium.
cence associated with the cells treated with 4, 5 and 6 was 8, 10 and
40 FU, respectively. These results show that all three PCReds have
the capacity to penetrate the cell membrane, but the complexation
to Zn(II) and Lu(III) significantly increases the uptake. The higher
uptake of 6 compared to 5 can be rationalized by considering that
Lu(III)ePCRed is a neutral compound, while Zn(II)ePCRed is
a cationic species. Neutral molecules being more lipophylic are
expected to penetrate the cell membrane easier than charged
molecules. In order to investigate the intracellular distribution of
the PCReds, we performed confocal laser microscopy experiments
with B78-H1 cells. Fig. 1b shows the green fluorescence emitted by
the cells treated with 4, 5 and 6. The fluorescence distribution
suggests that the two metal complexes locate in both the nucleus
and cytoplasm, probably with a slight preference for the latter.
Instead, PCRed 4 appears clearly more localized in the cytoplasm, in
keeping with previous data obtained with HeLa cells [11a]. The
decrease of fluorescence intensity from 6 to 4 correlates with the
flow cytometry data showing that the decrease of uptake follows
the order 6 > 5 > 4.
2.3. Phototoxicity of metallated PCRed
To evaluate the photosensitizing activity of the PCRedemetal
complexes we first investigated their uptake in B78-H1 cells (Fig.1).
We found a clear dependence of the cellular uptake from the charge
of the metal complexes. As PCRed 4, upon excitation at 488 nm,
emits green fluorescence, we measured by flow cytometry (Fig. 1a)
the fluorescence of B78-H1 cells treated for 24 h with PCRed 4 or its
The photodynamic effect promoted by the complexes between
PCRed and Zn(II) or Lu(III) was tested in four cancer cell lines:
B78-H1 murine amelanotic clone derived from melanoma B16;
HeLa human cervix adenocarcinoma; HepG2 human hepatic
carcinoma and MCF-7 human breast cancer. Fig. 2 shows that in
HeLa and B78-H1 cells, compounds 4, 5 and 6 are basically not
a
120
100
HeLa
80
60
40
20
0
b
120
B78-H1
100
80
60
40
20
0
Fig. 2. Cytotoxicity in the dark of compounds 4, 5 and 6 in HeLa (a) and B78-H1 (b)
cells. Numbers below bars indicate pentaphyrin concentrations M); NT means
(m
pentaphyrin untreated cells. Numbers at top bars indicate the pentaphyrin used in the
treatment (4, 5 or 6). Viable cells were measured with resazurin. Histograms report in
ordinate the percent of viable cells, i.e. the ratio RFUT/RFUC ꢀ 100, where RFUT is the
fluorescence of treated cells, while RFUC is the fluorescence of untreated cells. The data
are the means ꢃ sd of three experiments.
Fig. 1. (a) Cell cytometry of B78-H1 cells treated with 15 mM compounds 4, 5 and 6.
Fluorescence of cells treated with 4, 5 and 6 is 8, 10 and 40 FU, respectively; (b)
Confocal microscopy images of B78-H1 cells treated with 4, 5 and 6. Ex. 488 nm (Arg
laser); Em. 500e530 nm.

716
M. Ballico et al. / European Journal of Medicinal Chemistry 46 (2011) 712e720
cytotoxic in the dark, up to 25 mM (4 and 5) or 20 mM (6). A similar
result was obtained with the MCF-7 and HepG2 cells (data not
shown).
a
When PCRed 4 is irradiated with a halogen light at a fluence of
8 mW/cm2 (14 J/cm2), a slight cytotoxic effect is promoted at
photosensitizer concentrations higher than 25 mM (Figs. 3 and 4).
In contrast, the two metal complexes 5 and 6 displayed, after
irradiation, a stronger cytotoxic effect proportional to the atomic
weight of the metal ion (Lu(III)-PCRed 6 > Zn(II)ePCRed 5) [10,13].
These experiments were carried by treating the cells for 24 h with
increasing amounts of photosensitizer, followed by irradiation and
24 h-incubation in the dark before a resazurin proliferation assay
was performed. As a control the cells were treated with the same
amount of DMSO present in the PCReds treatment (PCReds were
dissolved in DMSO and diluted with cell medium). From these
plots we estimated the IC50 values reported in Table 1. These
values show that free PCRed 4 has little if no photoactivity. But
when it is coordinated to Zn(II), its light-induced cytotoxicity
slightly improved. In contrast, the complexation of PCRed with
Lu(III) dramatically increases the phototoxicity of complex 6, for
b
which we found a IC50 between 5 and 12 mM (values comparable
to those found for the previously tested aromatic pentaphyrin
analogues [11]).
In order to evaluate the photokilling mechanism triggered by
the photoactivated PCReds we measured in B78-H1 cells the levels
of ROS production 24 h after light activation. To this purpose we
carried out a standard CMeH2DCFDA assay based on the observa-
tion that the dye in its reduced form is not fluorescent, but it
becomes so when it is oxidized by ROS and the dye acetate groups
are removed by cellular esterases [18].
Fig. 4. Phototoxicity of PCReds (4, 5 and 6) in MCF-7 and HepG2 cells. % Viable cells in
samples treated with increasing amounts of pentaphyrins after light treatment (flu-
ence 14 J/cm2). Viable cells were measured with resazurin. Histograms report in
ordinate the percent of viable cells, i.e. the ratio RFUT/RFUC ꢀ 100, where RFUT is the
fluorescence of treated cells, while RFUC is the fluorescence of untreated cells. The data
are the means ꢃ sd of three experiments. Numbers below the bars indicate penta-
phyrin concentrations (mM); NT means pentaphyrin untreated cells. Numbers at top of
the bars indicate the pentaphyrin used in the treatment (4, 5 or 6).
a
The amount of fluorescence generated in the cells by the dye,
which is directly proportional to the ROS production, was measured
by FACS. The results obtained are in nice accord with the trend
observed on measuring the phototoxicity. The Lu(III)ePCRed 6
produces higher ROS levels then Zn(II)ePCRed 5 and PCRed 4
(Fig. 5). The amount of fluorescence generated in the cells by the
dye, which is directly proportional to ROS production, was
measured. It is worth to note that immediately after irradiation ROS
levels of 5 and 6 are similar while after 24 h the production of ROS
increased 8-fold with complex 6 and only 3-fold with 5.
The free ligand 4 produces very low levels of ROS, either
immediately or 24 h after irradiation. The data show that metal-
lated PCReds generate ROS with a slow rate, with an efficiency
proportional to the atomic weight of the metal cation.
b
It is worth noting that the insertion of Lu(III) into the macrocycle
transforms a compound that does not have any phototoxic activity
into an efficient photosensitizer. No structural changes of the
complexes can be invoked to explain this effect, as NMR data show
no relevant structural diversity between 6 and 5 (in methanol)
Table 1
Fig. 3. Phototoxicity of PCReds (4, 5 and 6) in HeLa and B78-H1 cells. % Viable cells in
samples treated with increasing amounts of pentaphyrin after light treatment (fluence
14 J/cm2). Viable cells were measured with resazurin. Histograms report in ordinate
the percent of viable cells, i.e. the ratio RFUT/RFUC ꢀ 100, where RFUT is the fluores-
cence of treated cells, while RFUC is the fluorescence of untreated cells. The data are
the means ꢃ sd of three experiments. Numbers below the bars indicate pentaphyrin
IC50 values of compounds 4, 5 and 6.
Cells
PCRed 4 (
m
M)
Zn(II)ePCRed 5 (
m
M)
Lu(III)ePCRed 6 (mM)
HeLa
40
>30
>30
>25
25
40
>25
25
5
6
11
12
B78-H1
HEPG2
MCF-7
concentrations (
mM); NT means pentaphyrin untreated cells. Numbers at top of the
bars indicate the pentaphyrin used in the treatment (4, 5 or 6).

M. Ballico et al. / European Journal of Medicinal Chemistry 46 (2011) 712e720
717
B78-H1 cells. The phototoxicity nicely correlates with the higher
capacity of the Lu(III)-complex 6 to generate ROS species compared
to free PCRed 4 and Zn(II)ePCRed 5. Metallation is a suitable
strategy to potentiate reduced pentaphyrins as PDT drugs.
after irradiation
4000
3000
2000
1000
0
24 h after irradiation
4. Experimental section
4.1. General remarks
Allsolventsandchemicals were of reagentgradequalityandwere
used as received from the suppliers. Mass spectra were recorded on
an ion trap Finnigan Mat GCQ (Finnigan MAT, Austin, Texas, USA),
operated in electron ionization mode, and on a Finnigan LXQ linear
ion trap Mass Spectrometer (Thermo Scientific, Waltham, MA, USA)
equipped with an ESI source. Experiments were carried out in the
positive ion mode. EI mass spectrums were performed on a Micro-
mass VG 7070 H Mass Spectrometer operating at 70 eV (Micromass
Ltd., Manchester, UK). Purifications were conducted on a VersaFlash
station (Supelco, 595 North Harrison Road, Bellefonte, PA (USA)),
supplied with semipreparative VersaPak Cartridges (23 ꢀ 53 mm,
running at 5 mL/min, Supelco, 595 North Harrison Road, Bellefonte,
NT
4
5
6
Fig. 5. Fluorescence measured by cell cytometry of B78-H1 cells treated for 24 h with
pentaphyrin concentrations near IC50 (30 M for 4; 25 M for 5; 3 M for 6), activated
with light and treated with 10 M CMeH2DCFDA immediately after activation or after
m
m
m
m
PA (USA)) containing multiuse end-capped C18 resin (20e45 mm
24 h. The increase of fluorescence is proportional to ROS production.
particle size), and eluted with water/acetonitrile 1:1 and 1% TFA. 1H
NMR spectra were recorded on a Bruker AM-200 (200 MHz).
Chemical shifts are given in ppm (d) relative to tetramethylsilane
except for the degree of deprotonation of the ligand due to coor-
dination of the metal ion. As the stability of these compounds in
physiological conditions was similar over a period of 48 h, the lower
activity of 5 compared to 6 cannot be ascribed to demetallation of
the complex. So, could this difference in activity be attributed to the
fact that complex 6 is taken up by the cells more efficiently than 4
or 5? This is unlikely because the experiment of Fig. 5 was per-
formed with concentrations of 5 and 6 close to their IC50 values. In
fact, the much higher production of ROS by 6 was observed at
a concentration 8-fold lower than that of 5.
(1H NMR) as an internal standard. UVevis spectra were measured on
a Varian Cary 50 (Palo Alto, CA, USA) spectrophotometer using 1.0 cm
quartz cuvettes. Fluorescence emission spectra were recorded on
a spectrofluorometer LS 50B Perkin Elmer in CH3OH, CH2Cl2 and PBS
in standard quartz cells using a lexc ¼ 480 nm.
4.2. Synthesis of photosensitizers
4.2.1. Synthesis of the partially oxidized pentaporphynogen 3
100 mg (0.25 mmol) of the tripyrrane diacid 1 was dissolved in
However, another phenomenon can account for the different
bioactivity of the PCReds. We found that ROS in B78-H1 cells are
produced with a slow rate, suggesting that the photodynamic
process, triggered by 5 and 6, is neither of Type I nor of Type II, as
these are fast [10a,19]. These mechanisms have been proposed for
photosensitizers in their final oxidation state (porphyrins, phtha-
locyanines, etc). In our systems we were able to rule out a Type II
mechanism by means of a 9,10-dimethylanthracene test [20]
(Supporting information S8) which demonstrated that compound
4 and 6 did not produce singlet oxygen by irradiation. Therefore,
the ROS production has to be ascribed to an electron transfer
process trigged by light, consistent with the fact that PCReds
molecules are in an intermediate oxidation state [11,21,22]. So, the
increase of the PDT effect caused by the coordination of a heavy
metal to the macrocycle cannot be due to an enhanced ISC, as
currently reported [10,12e14]. Instead, it is likely related to a vari-
ation of the oxidation potential within the complexes. These new
features induced by metallation open a new approach in the design
of efficient photosensitizers based on reduced-state expanded
porphyrins.
200
mL (2.60 mmol) of TFA and stirred for 10 min at room
temperature under N2. The mixture was diluted with dry CH2Cl2
(20 mL) and the protected dipyrrometane dialdehyde 2 (97 mg,
0.23 mmol), dissolved in dry CH2Cl2 (5 mL), was added. The mixture
was stirred at room temperature for 2 h under N2 with exclusion of
light. The resulting solution was then cooled in an ice bath and
neutralized with triethylamine (362 mL, 2.60 mmol). The mixture
was diluted with dry CH2Cl2 (30 mL) and extracted with brine
(3 ꢀ 50 mL). The evaporation of the solvent affords a dark residue,
that was washed with pentane (10 mL). Yield 121 mg (76%). 1H NMR
(major tautomer, 200 MHz, CDCl3, 20 ꢂC):
d
¼ 11.23, 10.70, 10.06 (br
s; NH), 9.22 (s; H pyrrole), 8.40e7.50 (br s; NH), 8.10 (d; H phenyl),
7.44 (d; H phenyl), 6.85 (d; H pyrrole), 6.58 (s; meso-CH), 6.05 (d; H
pyrrole), 5.93 (s; meso-CH), 5.63 (s; meso-CH), 4.41 (t; CH2CH2O),
4.1 (q; CH2CH3), 2.70e1.50 (m; CH2CH3, CH3), 1.12 (t; CH2CH2Si),
1.35 (m; CH2CH3), 0.082 ppm (s; Si(CH3)3); 1H NMR (major
tautomer, 200 MHz, CD3OD, 20 ꢂC):
d
¼ 9.29 (s; H pyrrole), 9.23 (s;
H pyrrole), 7.89 (d; H phenyl), 7.22 (d; H phenyl), 6.89 (d; H
pyrrole), 6.63 (s; meso-CH), 5.90 (d; H pyrrole), 5.60, 5.44, 5.40 (s;
meso-CH), 4.33 (t; CH2CH2O), 3.16 (q; CH2CH3), 1.26 (s; CH3), 1.04 (t;
CH2CH2Si), 0.78 (t; CH2CH3), ꢁ0.006 ppm (s; Si(CH3)3); UVevis
3. Conclusion
(CH2Cl2) lmax (nm): 300 (log
3
¼ 4.16), 482 (log
3
¼ 4.21), 802
(log
3
¼ 3.93); MS (ESI, positive mode, CH3OH): m/z 696 ([MH]þ),
In this study we describe a simple synthetic route to obtain
metal complexes of pentaphyrin with Zn(II) and Lu(III). These
complexes have been characterized by a number of techniques. We
found that metallation strongly affects the photosensitizing prop-
erty of the PCRed. While the free ligand (PCRed) did not show any
PDT activity in four cancer cell lines, the Lu(III)-complex promoted
714 ([MH þ H2O]þ), 728 ([MH þ CH3OH]þ).
4.2.2. Synthesis of PCRed 4
100 mg (0.25 mmol) of the tripyrrane diacid 1 was dissolved in
200 L (2.60 mmol) of TFA and kept under stirring for 10 min at
m
room temperature under N2. The mixture was diluted with dry
a strong PDT effect with an IC50 as low as 5e6
mM in HeLa and
CH2Cl2 (20 mL) and the protected dipyrrometane dialdehyde 2

718
M. Ballico et al. / European Journal of Medicinal Chemistry 46 (2011) 712e720
(97 mg, 0.23 mmol), dissolved in dry CH2Cl2 (5 mL), was added. The
mixture was stirred at room temperature for 2 h under N2 with
exclusion of light. The resulting solution was then cooled in an ice
in vacuo. The residue was dissolved in acetone (2 mL), addition of n-
pentane (10 mL) afforded a red-dark precipitate which was filtered
and dried under reduced pressure. Yield 44 mg (81%). 1H NMR
bath and neutralized with triethylamine (362
mL, 2.60 mmol). DDQ
(200 MHz, CDCl3, 20 ꢂC):
d
¼ 9.83 (br s, 1H; NH), 9.31 (s, 2H; H
(2,3-Dichloro-5,6-Dicyanobenzoquinone, 105 mg, 0.46 mmol) was
added and the ice bath removed. The mixture was stirred for
30 min at room temperature, diluted with dry CH2Cl2 (30 mL) and
extracted with brine (3 ꢀ 50 mL). Evaporation of the solvent affords
a dark metallic green film. The residue was purified by flash chro-
matography (VersaFlash). Yield 80 mg (50%). 1H NMR (200 MHz,
pyrrole), 8.00 (d, 2H; H phenyl), 7.99 (s, 2H; meso-CH), 7.27 (d, 2H;
H phenyl), 6.88 (d, 2H; H pyrrole), 6.06 (d, 2H; H pyrrole), 5.57 (s,
2H; meso-CH), 4.51 (q, 4H; CH2CH3), 4.42 (t, 2H; CH2CH2O), 1.26 (s,
6H; CH3), 1.12 (t, 2H; CH2CH2Si), 0.84 (t, 6H; CH2CH3), 0.075 ppm (s,
9H; Si(CH3)3); 1H NMR (200 MHz, CD3OD, 20 ꢂC):
d
¼ 9.29 (s, 2H; H
pyrrole), 7.89 (d, 2H; H phenyl), 7.81 (s, 2H; meso-CH), 7.21 (d, 2H;
H phenyl), 6.88 (d, 2H; H pyrrole), 5.90 (d, 2H; H pyrrole), 5.60 (s,
2H; meso-CH), 4.32 (t, 2H; CH2CH2O), 3.23 (q, 4H; CH2CH3), 1.20 (s,
6H; CH3), 1.04 (t, 2H; CH2CH2Si), 0.81 (t, 6H; CH2CH3), ꢁ0.007 ppm
CDCl3, 20 ꢂC):
d
¼ 11.40 (br s, 1H; NH), 10.21 (br s, 1H; NH), 9.27 (s,
2H; H pyrrole), 8.80e8.10 (br s, 2H; NH), 8.01 (d, 2H; H phenyl), 7.33
(d, 2H; H phenyl), 7.26 (s, 1H; meso-CH), 6.86 (d, 2H; H pyrrole),
6.05 (d, 2H; H pyrrole), 5.62 (s, 1H; meso-CH), 4.41 (t, 2H;
CH2CH2O), 3.12 (q, 4H; CH2CH3), 1.25 (s, 6H; CH3), 1.11 (t, 2H;
CH2CH2Si), 0.88 (t, 6H; CH2CH3), 0.08 ppm (s, 9H; Si(CH3)3); 1H
(s, 9H; Si(CH3)3); UVevis (CH2Cl2) lmax (nm): 339 nm (log
e
¼ 4.25),
406 nm (log
865 (3), 394 (100).
3
¼ 4.15), 498 nm (log
3
¼ 3.99); MS (EI, 70 eV): m/z (%):
NMR (200 MHz, CD3OD, 20 ꢂC):
d
¼ 9.28 (s, 2H; H pyrrole), 7.88 (d,
2H; H phenyl), 7.21 (d, 2H; H phenyl), 6.88 (d, 2H; H pyrrole), 6.52
(s, 2H; meso-CH), 5.90 (d, 2H; H pyrrole), 5.60 (s, 2H; meso-CH),
4.32 (t, 2H; CH2CH2O), 3.16 (q, 4H; CH2CH3), 1.23 (t, 6H; CH2CH3),
1.20 (s, 6H; CH3), 1.04 (t, 2H; CH2CH2Si), ꢁ0.009 ppm (s, 9H; Si
4.3. Cell lines
The HeLa human cervical cancer and the HepG2 hepatic carci-
noma cells were cultured in DMEM (high glucose); the B78-H1
amelanotic clone derived from the murine melanoma was cultured
in DMEM (low glucose) and the MCF-7 human breast cancer cell
line was cultured in RPMI. All media (CELBIO, Milan, Italy) con-
tained 10% fetal calf serum and were supplemented with antibiotics
(CH3)3); UVevis (CH2Cl2) lmax (nm): 485 (log
3
¼ 4.39), 814
(log
3
¼ 3.71); MS (ESI, positive mode, CH3OH): m/z 694 ([MH]þ),
726 ([MH þ CH3OH]þ).
4.2.3. Synthesis of Zn(II)ePCRed 5
Penicillin 100 U/ml, Streptomycin 100 mg/ml and Glutamine 2 mM
a) Partially oxidized pentaporphynogen 3 (50 mg, 0.072 mmol)
was dissolved in dry CHCl3 (70 mL) and stirred at room temperature
for 30 min. A solution of anhydrous zinc acetate (60 mg, 0.33 mmol)
and anhydrous sodium acetate (86 mg, 1.05 mmol) in methanol
(5 mL) was added, and the mixture was stirred at room
temperature for 48 h with exclusion of light. CHCl3 (70 mL) was
added, and the solution was washed with deionized water
(3 ꢀ 20 mL), dried over anhydrous sodium sulphate and evaporated
at room temperature in vacuo. The residue was dissolved in acetone
(2 mL), addition of n-pentane (10 mL) afforded a red-dark precip-
itate which was filtered and dried under reduced pressure. Yield
44 mg (81%). b) The preparation of 5 was carried out in a way
similar to that described in the method a, using compound 4
(20 mg, 0.029 mmol) instead of 3, and stirring the reaction mixture
at room temperature only for 24 h. Yield 14 mg (64%). 1H NMR
(CELBIO, Milan, Italy). All experiments were done using exponential
growth phase cells.
4.4. PDT treatment
Treatment with different PCReds was performed at dark for 24 h
and followed from irradiation with metal halogen white lamp at an
irradiance of 8 mW/cm2 for 60 min.
4.5. Determination of cell proliferation
The % of viable cells was determined by the resazurin assay
following the manufacturer’s instructions (Sigma). To reach expo-
nential growth phase, defined cell densities were seeded in a 96
multiwell plate: HeLa 5000 cells/well; HepG2 7000 cells/well;
B78-H1 5000 cells/well and MCF-7 7000 cell/well. Values were
obtained by spectrofluorometer Spectra Max Gemini XS (Molecular
Devices, Sunnyvale).
(200 MHz, CDCl3, 20 ꢂC):
d
¼ 10.03 (br s, 1H; NH), 9.26 (s, 2H; H
pyrrole), 9.0e7.0 (br s, 2H; NH), 8.02 (d, 2H; H phenyl), 7.97 (s, 1H;
meso-CH), 7.28 (d, 2H; H phenyl), 6.86 (d, 2H; H pyrrole), 6.04 (d,
2H; H pyrrole), 5.58 (s, 1H; meso-CH), 4.45 (t, 2H; CH2CH2O), 4.10
(q, 4H; CH2CH3), 2.05 (s, 6H; CH3), 1.43 (t, 2H; CH2CH2Si), 1.21 (t, 6H;
CH2CH3), 0.07 ppm (s, 9H; Si(CH3)3); 1H NMR (200 MHz, CD3OD,
4.6. ROS measurement
20 ꢂC):
d
¼ 9.29 (s, 2H; H pyrrole), 7.89 (d, 2H; H phenyl), 7.85 (s, 2H;
ROS levels were determined after PDT by incubating the cells in
white medium DMEM (Biowhittaker LONZA) without serum for
meso-CH), 7.22 (d, 2H; H phenyl), 6.89 (d, 2H; H pyrrole), 5.90 (d,
2H; H pyrrole), 5.60 (s, 2H; meso-CH), 4.33 (t, 2H; CH2CH2O), 4.01
(q, 4H; CH2CH3), 1.92 (s, 6H; CH3), 1.15 (t, 6H; CH2CH3), 1.04 (t, 2H;
CH2CH2Si), ꢁ0.006 ppm (s, 9H; Si(CH3)3); UVevis (CH2Cl2) lmax
30 min at 37 ꢂC with 5
m
M of 5-(and 6)-chloromethyl-20,70-
dichlorodihydrofluorescein diacetate, acetyl ester CMeH2DCFDA
(mixed isomers, C6827, Molecular Probes, Invitrogen).
CMeH2DCFDA was metabolized by non-specific esterases to the
non-fluorescence product, which was oxidized to the fluorescent
product, DCF, by ROS [18]. Then, the cells were washed twice in PBS,
trypsinized, resuspended in PBS and measured for the ROS content
by FACS (FACScan, Becton Dickinson).
(nm): 407 (log
3
¼ 4.13), 491 (log
3
¼ 4.33), 817 (log
3
¼ 3.37); MS
(ESI, positive mode, CH3OH): m/z 756 ([M]þ), 774 ([M þ H2O]þ), 788
([M þ CH3OH]þ).
4.2.4. Synthesis of Lu(III)-PCRed 6
Partially oxidized pentaporphynogen 3 (40 mg, 0.057 mmol)
was dissolved in dry CHCl3 (60 mL) and stirred at room temperature
for 30 min. A solution of Lu(OAc)3$4H2O (110 mg, 0.257 mmol) and
anhydrous sodium acetate (68 mg, 0.829 mmol) in methanol (8 mL)
was added, and the mixture was stirred at room temperature for
72 h with exclusion of light. CHCl3 (60 mL) was added, and the
solution was washed with deionized water (3 ꢀ 20 mL), dried over
anhydrous sodium sulphate and evaporated at room temperature
4.7. Confocal microscopy
To study the uptake, B78-H1 cells were plated (density of
1 ꢀ105) on coverslips (diameter 24 mm) and after 24 h treated with
4, 5 and 6 (15 mM) for other 24 h. The glasses were prepared. The
cells were washed twice with PBS, fixed with 3% paraformaldehyde
(PFA) in PBS for 20 min. After washing with 0.1 M glycine,

M. Ballico et al. / European Journal of Medicinal Chemistry 46 (2011) 712e720
719
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100 (0.1% in PBS), the cells were incubated with Hoechst to stain the
nuclei. The cells were analysed using a Leica TCSNT confocal laser
scanning system on an inverted microscope DMIRBE (Leica
Microsystems, Heidelberg, German). Green fluorescence was
excited with 488 nm line Argon-Ion laser and detected with
emission bandpass filters 500/530 nm.
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Acknowledgements
This study was supported by the Italian Ministry of University
and Scientific Research (PRIN 2007), FVG and PRISMA. We thank
Dr. M. Zacchigna (University of Trieste) for fluorescence
measurements.
Appendix. Supplementary data
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(PDT):
a short review on cellular mechanisms and cancer research