Phototoxic activity and DNA interactions of water-soluble porphyrins and their rhenium(I) conjugates

Article abstract of DOI:10.1002/cmdc.201500288

In the search for alternative photosensitizers for use in photodynamic therapy (PDT), herein we describe two new water-soluble porphyrins, a neutral fourfold-symmetric compound and a +3-charged tris-methylpyridinium derivative, in which either four or one

Full text of DOI:10.1002/cmdc.201500288

DOI: 10.1002/cmdc.201500288
Full Papers
Phototoxic Activity and DNA Interactions of Water-Soluble
Porphyrins and Their Rhenium(I) Conjugates
[
a]
[a]
[b]
[c]
[c]
Giuliana Mion, Teresa Gianferrara,* Alberta Bergamo, Gilles Gasser,* Vanessa Pierroz,
[c]
[d]
[d]
[a]
Riccardo Rubbiani, Ramon Vilar,* Anna Leczkowska, and Enzo Alessio
In the search for alternative photosensitizers for use in photo-
dynamic therapy (PDT), herein we describe two new water-
soluble porphyrins, a neutral fourfold-symmetric compound
and a +3-charged tris-methylpyridinium derivative, in which
either four or one [1,4,7]-triazacyclononane (TACN) units are
connected to the porphyrin macrocycle through a hydrophilic
epithelial cells) are reported. Three of the compounds are not
cytotoxic in the dark up to 100 mm, and the fourfold-symmetric
couple revealed very good phototoxic indexes (PIs). The intra-
cellular localization of all derivatives was studied in HeLa cells
by confocal fluorescence microscopy. Although low nuclear lo-
calization was observed for some of them, it still prompted us
to investigate their capacity to bind both quadruplex and
duplex DNA; we observed significant selectivity in the tris-
methylpyridinium derivatives for G-quadruplex interactions.
I
linker; we also report their corresponding tetracationic Re con-
jugates. The in vitro (photo)toxic effects of the compounds
toward the human cell lines HeLa (cervical cancer), H460M2
(
non-small-cell lung carcinoma), and HBL-100 (non-tumorigenic
Introduction
[
6–10]
1
Photodynamic therapy (PDT) is emerging as a particularly at-
tractive clinical method to treat cancers, infectious diseases,
ry responses.
As a consequence of its high reactivity, O2
has a short lifetime in the physiological environment, and its
[1–4]
[11–13]
and a series of local afflictions.
This therapeutic strategy is
action occurs close to the site of formation within the cell.
an alternative or adjuvant to other therapy modalities such as
Therefore, besides being typically noninvasive (e.g., relative to
[5]
[14–17]
surgery, chemotherapy, or radiotherapy. The basic principle of
PDT is the light-mediated activation of a nontoxic tumor-local-
ized photosensitizer (PS). Thus, upon irradiation at a specific
desired wavelength, the PS reaches an excited triplet state
surgery),
PDT treatment offers several advantages over
current cancer treatments, namely: 1) the possibility to induce
cell death with precise spatial and temporal control, 2) low sys-
temic toxicity, as the cytotoxic effects are triggered only in se-
[
18]
(
PS*). PS* can transfer electrons or protons to substrates in
lectively irradiated tissues, 3) the possibility to be repeated
several times at the same site, and 4) the possibility to exploit
the inherent fluorescence properties of PSs in photodynamic
close proximity, giving rise to radical anions or cations, respec-
tively, that are subsequently scavenged by oxygen to form re-
active oxygen species (ROS, type I mechanism). As an alterna-
tive, PS* can transfer its energy directly to the ground state of
[
19,20]
diagnosis (PDD).
The most frequently used PSs in PDT are porphyrins and por-
phyrinoids, because they preferentially accumulate in tumor
3
1
the molecular triplet oxygen ( O ) to form singlet oxygen ( O ,
2
2
[
2,21,22]
type II mechanism), a very reactive and toxic form of oxygen.
tissues and are efficient singlet oxygen generators.
There
1
O is believed to be the major cytotoxic agent in PDT. It is
2
has been a recent growth in interest in the preparation of
metal–porphyrin conjugates as potential cytotoxic and photo-
cytotoxic agents. Such conjugates might combine the photo-
toxicity of the porphyrin chromophore with the cytotoxicity of
the metal fragment for additive antitumor effects. In addition,
these conjugates might have increased tumor selectivity. The
first metal–porphyrin conjugates developed for biomedical ap-
able to induce cell death by apoptosis and necrosis, immuno-
logical effects, vascular shutdown, and can trigger inflammato-
[
a] G. Mion, Dr. T. Gianferrara, Prof. E. Alessio
Department of Chemical & Pharmaceutical Sciences
Università degli Studi di Trieste, P.le Europa 1, 34127 Trieste (Italy)
E-mail: gianfer@units.it
II
plications were porphyrins functionalized with Pt fragments
[
b] Dr. A. Bergamo
[
23–26]
structurally related to cisplatin and carboplatin.
More
Callerio Foundation Onlus, Via A. Fleming 22–31, 34127 Trieste (Italy)
II
recent examples concern several Ru –porphyrin conjugates de-
[
c] Prof. G. Gasser, Dr. V. Pierroz, Dr. R. Rubbiani
Department of Chemistry, University of Zurich
Winterthurerstrasse 190, 8057 Zurich (Switzerland)
E-mail: gilles.gasser@chem.uzh.ch
[
27]
veloped in particular by some of us, and by Therrien and co-
[
28,29]
workers,
that possess promising phototoxicity induced by
visible light. Furthermore, the water solubility of the porphyr-
ins can be considerably improved by an appropriate choice of
the peripheral metal fragments. This is an essential feature for
clinical application; indeed, the low water solubility of synthet-
[
d] Prof. R. Vilar, Dr. A. Leczkowska
Department of Chemistry, Imperial College London, London SW7 2AZ (UK)
E-mail: r.vilar@imperial.ac.uk
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201500288.
ChemMedChem 2015, 10, 1901 – 1914
1901
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
1
ic porphyrins is a substantial limitation, as it may impair both
their administration and efficacy.
and singlet oxygen ( O ). These species can oxidize purine and
2
pyrimidine bases, damage the DNA at apurinic/apyrimidinic
(abasic) sites, and can also give rise to single-strand (SSBs) and
More recently, we described the preparation and characteri-
zation of two novel water-soluble porphyrins bearing three
meso-pyridyl rings and one peripheral chelator, which was
either a diethylenetriamine unit or a bipyridyl fragment. Both
[
45]
double-strand breaks (DSBs).
For all the reasons detailed above, we decided to investigate
tetrapyrrolic chromophores suitable for conjugation with Re
fragments to be used in both therapy and diagnosis of cancer.
Herein we describe the synthesis, characterization, cellular lo-
calization, evaluation of the affinity for both quadruplex and
duplex DNA, and (photo)toxic behavior of two novel porphyr-
ins functionalized with either four or one [1,4,7]-triazacyclono-
nane (TACN) peripheral units—the neutral 1 and the +3-
9
9m
chelators were suitable for complexation of the
{
Tc/
Technetium is the most widely used
radionuclide in diagnosis, and rhenium is its heavier homo-
+
[30,31]
Re(CO)3} moieties.
99m
logue. The properties of the “cold” Re and of the “hot”
Tc
complexes are assumed to be very similar. By applying the
I
99m
I
matched pair paradigm, the Re / Tc –porphyrin conjugates
may act as multifunctional agents endowed either with diag-
I
charged 3—as well as of their corresponding tetracationic Re
conjugates 2 and 4 (Figure 1). Porphyrins 1 and 3 were specifi-
cally designed for binding a facial metal fragment such as Re/
9
9m
[32]
nostic ( Tc) or with therapeutic properties (Re). In principle,
9
9m
I
radioimaging performed on a labeled
Tc –porphyrin might
allow noninvasive, in vivo localization of the corresponding
PDT active “cold” Re conjugate.
Tc(CO) . In fact, the neutral TACN chelating unit matches the
3
binding preferences of those metal fragments, providing a rela-
tively rigid set of three thermodynamically and kinetically
stable bonds. Hence, 1 and 3 are an evolution of two mono-
functionalized porphyrins previously synthesized by us, which
Whereas metal complexes based on platinum and rutheni-
um have been studied extensively as anticancer agents, rela-
tively few rhenium complexes have been explored as potential
anticancer agents, although they possess excellent properties
bear a bidentate (bypyridyl) or tridentate (diethylenetriamine)
[33,34]
[30,31]
for developing a novel class of antineoplastic drug.
Nota-
ligand, respectively (Figure 2a,c).
Of note, the open-chain
bly, certain organometallic rhenium complexes have been
found to have interesting luminescence properties which allow
their intracellular distribution as well as their mechanism of
diethylenetriamine ligand has a poorer geometrical match
than TACN and, being flexible, is a less efficient binder than
TACN. The bidentate bipyridyl ligand is hydrophobic and
leaves a hydrolysable position upon coordination with the Re/
[35]
action to be followed by emission microscopy. But the most
attractive property of Re compounds is the possibility to
obtain the corresponding “hot” analogues, suitable for therapy,
Tc(CO) fragment (Figure 2d). The coordination chemistry of
3
I
I
Re/Tc with TACN as a ligand is well documented, and a variety
of complexes containing rhenium, as well as technetium, in
the formal oxidation state I+ with TACN have been report-
186
188
by using Re and Re radionuclides.
The ability of cationic porphyrins and their metal complexes
[26,36]
[46–48]
to interact with nucleic acids is well documented,
and one
ed.
of the most extensively investigated compounds is meso-tetra-
kis-(4-N-methylpyridiniumyl)porphyrin (TMPyP). The original
work of Fiel and co-workers on the interactions of TMPyP with Results and Discussion
DNA was aimed at developing DNA-targeting photosensitizers,
Synthesis and characterization
as selective photocleavage of DNA in tumor cells might result
[36a,37]
in lethal damage to the cells.
Furthermore, cationic por-
Porphyrins 1 and 3 bear four or one TACN ligands, respectively,
which are attached at phenyl rings at the meso positions
(Figure 1). To prepare them, a suitably functionalized TACN de-
rivative, 2-(diBoc)TACN acetic acid (5), was synthesized in good
yield by selective 1,4-diprotection of the amino groups of
phyrins were recently exploited for selective recognition of
[
38–42]
noncanonical DNA structures called G-quadruplexes.
The
formation of G-quadruplex DNA structures in vivo has been as-
sociated with a number of key biological processes such as
regulation of gene expression and telomere maintenance. In
general, cationic porphyrins with relatively long and flexible
side arms are better binders of G-quartets than TMPyP4, pre-
sumably because they might permit stacking of the aromatic
porphyrin core on the G-tetrad, minimizing steric clashes with
[
49]
TACN, followed by reaction with benzyl bromoacetate and
reductive deprotection with H on Pd/C (Supporting Informa-
2
tion Scheme S1). The synthetic procedures to obtain 1 and 3
start from two precursors that bear either four (6, Scheme 1)
or one (8, Scheme 2) hydrophilic 2,2’-(ethylenedioxy)diethyla-
mine chains on meso-phenyl rings (para position) on the mac-
rocycles. The multistep syntheses of 6 and 8 were described
[
40]
the G-tetrad edges. Furthermore, there is mounting interest
in synthetizing cationic asymmetric porphyrins as G-quadru-
plex ligands, in particular to obtain molecules endowed with
an increased overall affinity for the telomeric structures by
[
27,30]
previously.
The hydroxybenzotriazole (HOBt) ester of 5 was
then coupled with the terminal amino group of either 6 or 8
in anhydrous DMF to give 7 (66% yield) and 9 (66% yield), re-
spectively. These intermediates were quantitatively deprotect-
ed using trifluoroacetic acid (TFA) in CH Cl , and treated with
[43]
virtue of additional interactions. Due to the important bio-
logical roles associated to G-quadruplexes, they are potential
[44]
targets for anticancer drug candidates.
Furthermore, it is
2
2
well known that DNA can be damaged by exogenous and en-
dogenous oxidative stress induced by reactive oxygen (ROS)
and nitrogen species (RNS). ROS include superoxide anion radi-
triethylamine (TEA) to give the neutral tetrafunctionalized por-
phyrin 1 or the +3-charged monofunctionalized porphyrin 3,
respectively. Both porphyrins were characterized by UV/Vis,
À
À
1
cal (O C), hydrogen peroxide (H O ), hydroxyl radicals (OH C)
H NMR, H–H COSY, HSQC spectroscopy, electrospray mass
2
2
2
ChemMedChem 2015, 10, 1901 – 1914
www.chemmedchem.org
1902
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
Figure 1. Structures of porphyrins and Re–porphyrin conjugates 1–4 used in this study with NMR labeling scheme.
I
spectrometry (Figures S3, S4, S8, and S9 in the Supporting In-
formation), and by ESI HRMS, and were found to be well solu-
ble in water.
with the Re precursor fac-[Re(CO) (dmso-O) ](CF SO ). Whereas
3
3
3
3
2 was obtained by holding the starting materials at reflux in
anhydrous methanol (60% yield; Scheme 1), a microwave reac-
tion at 110 8C in methanol was necessary to afford conjugate 4
in good yield (73%; Scheme 2). Both reactions were monitored
The TACN moieties provide three N atoms in facial geometry,
and are thus particularly well suited for binding the fac-
+
1
{
Re(CO)3} fragment. To obtain the corresponding rhenium
by TLC and by recording H NMR spectra of the reaction mix-
conjugates, 2 and 4 (Figure 1), each porphyrin was reacted
tures at time intervals (Figures S5 and S14, Supporting Informa-
ChemMedChem 2015, 10, 1901 – 1914
www.chemmedchem.org
1903
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
99m
[30,31]
Figure 2. Structures of previously described Re/ Tc–porphyrin conjugates.
tion). The resonances of Hd and NH of TACN (see Figure 1 for
labeling scheme) move significantly downfield upon Re coordi-
nation, and are thus diagnostic of product formation.
both H460M2 and HBL-100 cell lines, with IC50 values of 7.4
and 33.7 mm, respectively, but not toward HeLa cells at the
maximum concentration used. This result is consistent with ob-
+
[27,50,51]
Notably, upon coordination of 1 and 3 to the fac-{Re(CO)3}
servations reported previously
that tetrasubstituted por-
II
fragment, both conjugates become tetracationic. The rhenium
conjugates 2 and 4 were characterized by one- and two-di-
mensional NMR (Figures S6 and S7, Figures S10–13, Supporting
Information) and IR spectroscopy. All compounds are well (1, 3,
and 4), or at least appreciably (2), soluble in water, besides
being soluble in DMSO.
phyrin–Ru conjugates typically show remarkable cytotoxicity
in the absence of light. The negligible dark cytotoxicity of the
I
Re conjugates 4 is consistent with what was found for mono-
substituted porphyrin–Ru conjugates by several research
II
[
51–53]
groups.
After exposure to red light, the monofunctional-
ized compounds 3 and 4 were consistently less active than the
tetrafunctionalized compounds 1 and 2 under the same exper-
imental conditions used for determining phototoxicity. In par-
ticular, compounds 3 and 4 are not effective at the lowest
(Photo)toxicity studies
À2
The (photo)toxic activity of compounds 1–4 was assessed on
three different cell lines, namely the cervical cancer cell line
HeLa, the lung cancer cell line H460M2, and the non-tumori-
genic cell line HBL-100. Effects on cell growth were evaluated
both in the dark and upon irradiation with red light (l=
light dose (1 Jcm ) on all the cell lines (IC50 values >100 mm).
Instead, an activity from mild to moderate was always ob-
À2
served at 10 Jcm , with a greater effectiveness of both por-
phyrins against the H460M2 tumor cell line (IC : 13 and 12 mm
50
for 3 and 4, respectively).
6
2
50 nm) for each compound. Cell cultures were exposed for
A comparison between compounds 1 and 2 reveals a greater
4 h at concentrations ranging from 0.1 to 100 mm, and were
phototoxic effect of the latter against the HeLa cell line at all
À2
À2
then irradiated with a fluence rate of 14 mWcm and light
light doses (e.g., PI at 10 Jcm >7.8 and >71.4, respectively,
À2
doses from 1 to 10 Jcm . As a control, we could show that
Table 1). Conjugate 2 showed a PI value of >38.5 upon irradia-
À2
the light doses used in this study do not influence the prolifer-
ation of untreated cells. Cells treated with the same concentra-
tions of test compounds and kept in the dark were used as
controls for phototoxicity. The (photo)toxicity of compounds
tion at 5 Jcm , which is consistent with the PIs of known PSs,
[
21]
photofrin and hypericin (>10 and >43, respectively, at the
[
54]
same light dose).
However, under the same experimental
conditions, a much higher PI (>200) was determined for the
porphyrin bearing a diethylenetriamine ligand (Figure 2a). On
the other hand, porphyrin 1 is >77-fold more active after irra-
1
–4 against the selected cell lines at increasing total light
doses is reported in Table 1.
À2
The phototoxicity of compounds 1 and 2 was found to in-
crease upon to the total light dose used, as shown by the
dose–effect curves on HeLa and H460M2 cells (Figure 3). By ir-
diation at 10 Jcm on H460M2 cells, whereas 2 has a much
À2
lower PI (>14.8, both at 5 and 10 Jcm , Table 1). The cytotox-
icity of conjugate 2 on H460M2 cells in the dark, with an IC50
value very close to that found after photo-irradiation, indicates
a different sensitivity between the two cell lines. These results
suggest that the Re–porphyrin conjugate 2 is not suitable for
non-small-cell lung cancers. However, it could be used in PDT
for cervical cancer and other tumor types that similarly show
radiating tumor cells at increasing total light doses from 1 to
À2
1
0 Jcm , the dose–response curve shifts to the left, and the
IC50 value correspondingly decreases.
All compounds were found to be non-cytotoxic in the dark,
with the exception of 2, which proved to be cytotoxic toward
ChemMedChem 2015, 10, 1901 – 1914
www.chemmedchem.org
1904
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
Scheme 1. Synthetic route to 1 and 2. Reagents and conditions: a) DMAP, EDCI, HOBt, DMF, 24 h, RT, 66%; b) CH
d) fac-[Re(CO) (dmso-O) ](CF SO ), anhydrous CH OH, 48 h, 708C, reflux, 65%.
2 2 3 2
Cl , TFA, 3 h, RT; c) CH OH, TEA, Et O, 97%;
3
3
3
3
3
ChemMedChem 2015, 10, 1901 – 1914
www.chemmedchem.org
1905
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
2 2 3 2
Scheme 2. Synthetic route to 3 and 4. Reagents and conditions: a) DMAP, EDCI, HOBt, DMF, MW 6 min, 608C, 66%; b) TFA, CH Cl , 3 h, RT; c) TEA, CH OH/Et O,
9
8%; d) fac-[Re(CO) (dmso-O) ](CF SO ), CH OH, MW 10 min, 110 8C, 73%.
3
3
3
3
3
Table 1. IC50 [mm] values and phototoxic indexes (PI) in HeLa, H460M2, and HBL-100 cells treated for 24 h with compounds 1–4 (0.1, 1, 3, 10, 30, and
[a]
1
00 mm) and then exposed to increasing doses of red light (l=650 nm).
À2
[b]
À2
[b]
À2
[b]
Cell line
HeLa
Compd
Dark
>100
>100
>100
>100
1 Jcm
>100
PI
5 Jcm
>100
PI
10 Jcm
PI
[c]
[c]
1
2
3
4
ND
>3
ND
ND
ND
12.9Æ3.0
1.4Æ1.3
100Æ41
73Æ19
>7.8
>71.4
>1
32.6Æ4.4
>100
>100
2.6Æ1.6
>100
ꢀ100
>38.5
[
c]
c]
[c]
ND
[
[c]
ND
>1.4
H460M2
HBL-100
1
2
3
4
>100
35.5Æ7.7
2.8Æ1.4
>100
>100
>2.8
>2.6
4.3Æ1.3
0.5Æ0.2
>23.3
>14.8
ND
1.3Æ0.6
0.5Æ0.2
13Æ1
>77
7.4Æ2.0
>14.8
>7.7
>8.3
[c]
[c]
>100
>100
ND
ND
ꢀ100
[c]
58Æ9
>1.7
12Æ5
1
2
3
4
>100
82.0Æ7.4
9.4Æ3.4
>100
>100
>1.2
>3.6
4.8Æ2.9
1.0Æ0.3
>20.8
>33.7
>1.3
1.4Æ0.9
0.5Æ0.1
>71.4
>67.4
>4.3
33.7Æ14.5
[
c]
>100
>100
ND
ND
76.6Æ1.6
75.4Æ0.1
23.4Æ12.0
42.8Æ5.3
[
c]
>1.3
>2.3
[
a] Cells were exposed for 24 h to each compound at concentrations ranging from 0.1 to 100 mm. Cells were then irradiated at l=650 nm with a fluence
À2
À2
rate of 14 mWcm for increasing time intervals (71 s; 5 min, 57 s; 11 min, 54 s) corresponding to total light doses of 1, 5, or 10 Jcm . These light doses
do not affect proliferation of untreated cells in control experiments. Cell cytotoxicity was determined by MTT assay 24 h post-irradiation. Cells treated with
the same concentrations of the test compounds, but kept in the dark, were used as controls. IC50 values were calculated from dose–effect curves and are
the mean ÆSD of at least three separate experiments. Experiments were conducted in quadruplicate and repeated thrice. Statistical analyses (not reported
in the table): ANOVA and Tukey–Kramer post-test. [b] PI=IC50 in the dark/IC50 upon irradiation. [c] Not determinable.
ChemMedChem 2015, 10, 1901 – 1914
www.chemmedchem.org
1906
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
Figure 3. Light dose–effect curves for 1 and 2 as representative porphyrins. HeLa and H460M2 cells were exposed to doses from 0.1 to 100 mm for 24 h, then
À2
À2
cells were irradiated at l=650 nm with a fluence rate of 14 mWcm and total light doses ranging from 1 to 10 Jcm . Cell cytotoxicity was determined 24 h
after the end of irradiation by MTT test.
1
a different sensitivity between dark and irradiated conditions.
Of note, in compound 4, the rhenium fragment does not en-
hance phototoxicity relative to 3, suggesting that other fea-
tures (hydrophilicity, permanent positive charges, one flexible
arm, etc.) play a major role in determining their phototoxic
properties.
of their F values. Beside being the best O generator, uncon-
D
2
jugated porphyrin 1 presents other advantageous features. It is
not cytotoxic in the dark toward all three cell lines investigat-
ed, and after irradiation, its IC50 values on H460M2 cells de-
crease by two orders of magnitude.
As previously outlined, the prevalent mechanism of action in
Cellular localization studies
1
PDT is type II and involves the generation of O after photoex-
2
1
citation of the PS. For this reason, the O quantum yield (F )
The cellular localization of porphyrins 1 and 3 as well as their
2
D
I
was evaluated for compounds 1–4 in DMSO. Usually, a clinically
Re conjugates 2 and 4 was investigated by confocal fluores-
[55]
useful PS has F value of ~0.5. Among the investigated por-
cence microscopy in HeLa cells. The results are shown in
Figure 4. After incubation for 2 h at a moderate concentration
of 20 mm, only compound 2 displayed pronounced cellular in-
ternalization. These data are in agreement with the antiproli-
ferative experiments. However, upon light irradiation com-
pounds 1 and 4 showed toxicity, indicative of probable uptake.
Consequently, to investigate their localization we increased the
treatment concentration to 100 mm. Porphyrin 1 displayed the
lowest accumulation signal inside the cell, with red lumines-
cent speckles localized in the cytoplasm (Figure 4a). Com-
pounds 2–4 showed a similar pattern with a diffused and
more intense luminescence localized in the cytoplasm and to
a very minor extent in the nucleoli (Figure 4, white arrows), as
indicated by comparison with the 4’-6-diamino-2-phenyindole
(DAPI) staining (Figure 4b,c,d). The intense red luminescence
D
phyrins, the best singlet oxygen generator is 1 (F =0.63),
D
while compounds 2–4 have much lower singlet oxygen quan-
tum yields (F =0.31, 0.20, and 0.19 for 2, 3, and 4, respective-
D
1
ly). However, despite being the best O generator, 1 shows
2
lower potency than 2 in the phototoxicity assay, probably due
to its low cellular uptake (see below). Instead, the low FD
value of 3 and 4 might be related to their low activity on all
cell lines after light irradiation. It is important to note that FD
values were determined in DMSO solutions and that a totally
different environment surrounds the compound under in vitro
conditions, so that other parameters—and in particular their
cellular uptake—might be more important for the activity of
the compounds. In other words, the phototoxic potencies of
porphyrins 1–4 are not precisely predictable only on the basis
ChemMedChem 2015, 10, 1901 – 1914
www.chemmedchem.org
1907
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
[
59]
complexes in tumor cells, showing that a variety of com-
plexes are internalized and accumulate in cytoplasmic organ-
elles, but only a small number reaches the nucleus. Even if lo-
calization might be explained in terms of simple chemical pro-
cesses such as ionic, hydrophobic, and electrostatic hydrogen
bonding interactions, in some cases, it is difficult to rationalize
[
59c]
the experimental results on these bases.
DNA binding studies
The ability of compounds 1–4 to recognize and bind to DNA
structures (quadruplex-forming sequences: Myc22 and H-telo;
and a duplex: ds-26) was investigated using emission spectros-
copy (Figures S15 and S16, Supporting Information). In all
cases, interaction with DNA causes red shifting of the excita-
tion/absorption bands of each of the four porphyrins (Dlꢁ
2
0 nm), while different changes were observed in the emission
spectra: no shift for 1, a blue shift for 2, and red shifts for 3
and 4. The latter may be indicative of p–p stacking interac-
tions. Besides these spectroscopic shifts, the compounds also
display changes in emission intensity upon addition of differ-
ent DNA sequences (see Figure 5).
In the case of 2, 3, and 4, enhancement in the emission is
more pronounced for quadruplex DNA than ds26. In contrast,
compound 1 does not display selectivity for the quadruplex se-
quences under study versus ds26. We also carried out studies
using calf thymus (ct)-DNA. As can be seen from Figure 5, the
four compounds show greater enhancement of emission in
the presence of quadruplexes than with ct-DNA.
Figure 4. Fluorescence confocal microscopy images showing the pattern of
HeLa cells incubated for 2 h with a) 100 mm 1, b) 20 mm 2, c) 100 mm 3, and
d) 100 mm 4, fixed in formaldehyde and stained with mounting solution con-
The effect of the compounds on thermal stability of Myc22
DNA was investigated further by using variable-temperature
circular dichroism (CD). The experiments were carried out for
Myc22 (5 mm) alone and in the presence of two equivalents of
each compound (Figure 6). Of the four studied compounds, 3
and 4 were found to have the greatest stabilization effect on
À1
taining DAPI (Vectashield, 1.5 mgmL ). White arrows indicate the nucleoli.
of 2 (incubated at a concentration of 20 mm against 100 mm for
1
, 3, and 4) suggests that this conjugate is more efficiently
Myc22 (DT =19 and 208C for 3 and 4, respectively). Lower
m
taken up inside the cell than the other three compounds used
in this study.
DTm was observed for 1 (68C) and, surprisingly, 2 showed no
It is worth outlining that the nuclear localization of the Re
conjugates, although very low, is extremely interesting in view
of the Re/Tc matched pair paradigm. In fact, among radioactive
99m
sources, Tc emits four low-energy auger electrons per decay.
Auger electron emission is a high linear energy transfer (LET)
process that creates high ionization density and can cause ex-
[56]
tensive DNA fragmentation that is difficult to repair, leading
[
57]
to cell death. Auger electrons have the advantage of an ex-
tremely short effective range, which helps minimize tissue
damage if delivered directly to the nuclei of cancerous cells.
99m
Thus, a
Tc compound that localizes in the nucleus, besides
being used as an imaging agent, might be itself a therapeutic
agent in targeted radiotherapy for treating multiple common
oncologic situations. Santos et al. showed that a Re tricarbonyl
complex containing a pyrazolyldiamine chelator-bearing acri-
9
9m
dine derivative and its “hot” analogue Tc are internalized sig-
nificantly and are retained in the nuclei of B16F1 murine mela-
Figure 5. Bar chart showing changes in the emission intensity (determined
by integration of the emission between l=600–850 nm) of the compounds
[
58]
noma cells. So far, only a few reports have been published
that deal with the cellular localization of rhenium tricarbonyl
(Tris/KCl buffer) upon addition of 20 equiv Myc22, H-telo, ds-26, and ct-DNA;
l
ex: 1, 430 nm; 2, 430 nm; 3, 445 nm; 4, 445 nm.
ChemMedChem 2015, 10, 1901 – 1914
www.chemmedchem.org
1908
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
Figure 6. CD thermal denaturation profiles of Myc22 (5 mm) alone and in the presence of 2 equiv 1, 2, 3, and 4. All measurements were carried out in lithium
cacodylate buffer (10 mm Li cacodylate, 99 mm LiCl, 1 mm KCl, pH 7.4); each experiment was performed in triplicate.
stabilization of Myc22 under the experimental conditions used
in this experiment. The apparent lack of DNA stabilization by
this compound could be due to its lower solubility in aqueous
media and possibly partial precipitation of this compound
during the experiment (although this was not observed by
visual inspection).
same light dose used in the phototoxicity experiments
À2
(10 Jcm ). A negative control of the plasmid treated with 1–4
(10 mm) in the dark was used for comparative purposes. As
shown in Figure 7, the complexes are all able to photocleave
plasmid DNA in a concentration-dependent manner. Notably,
1 and 2 demonstrated a mild effect just at 10 mm with attenua-
tion of the supercoiled band and an increase in intensity of
the nicked band. Of utmost interest, 3 and 4 showed a marked
photocleavage effect already at 0.5 mm. This is most likely due
to the intercalative potential of the porphyrin core and the
presence of three positive charges, which are thought to maxi-
mize the DNA–complex interaction. DNA treated in the dark
with the compounds (dark sample) did not show any signifi-
cant alterations of the supercoiled form. Notably, complex 2
showed a shift of the supercoiled and nicked bands. This effect
could be related to the intercalation of the porphyrin with the
plasmid DNA.
DNA photocleavage
Taking into account their favorable uptake into the nucleus,
their interactions with different topologies of DNA, and their
phototoxic activity, we decided to investigate the effect of 1–4
on plasmid DNA upon light irradiation. In these experiments,
circular plasmid DNA was treated with the target porphyrins
and then irradiated. If the compounds induce DNA damage,
a decrease in intensity of the supercoiled (intact form) band
and the formation of single-strand breaks (nicked form) or
double-strand breaks (linear form) bands, which migrate
slower in the gel, would be observed. Supercoiled pUC18 plas-
mid was treated with increasing concentrations of the porphyr-
ins (0.5–10 mm) and incubated at room temperature for 20 min.
Then, taking advantage of the pronounced Soret band of 1–4,
we irradiated the samples at 420 nm for 22 min to achieve the
Conclusions
Herein we report the preparation, in reasonable overall yields,
of two novel versatile porphyrins, namely the tetrafunctional-
ized compound 1 and the monofunctionalized 3 using multi-
ChemMedChem 2015, 10, 1901 – 1914
www.chemmedchem.org
1909
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
À2
Figure 7. DNA photocleavage experiments of pUC18 plasmid treated with 1–4, in the dark and upon light irradiation at l=420 nm for 22 min (10 Jcm );
untr.=DNA untreated; concentrations are expressed in mm.
step syntheses. These porphyrins bear either four (1) or one (3)
chelating TACN fragments at the meso positions connected
through flexible and hydrophilic linkers. We demonstrated that
whereas the presence of the metal center did not substantially
affect the photoactivity of the monofunctionalized porphyrin 3
on all the cell lines studied in this work. These overall results
do not clearly indicate the role of the metal center in influenc-
ing the phototoxic properties of porphyrins, but at the same
time suggest that it does not represent a negative factor.
+
both porphyrins 1 and 3 can bind the fac-{Re(CO)3} fragment
to give the tetracationic conjugates 2 and 4, respectively, in
good yields. The presence of one or four hydrophilic spacers
containing the ethylenedioxy groups, as well as the positive
charges on the complexes contribute to the water solubility of
the unconjugated porphyrins as well as of their corresponding
99m
Therefore, the possibility of obtaining the corresponding
Tc
conjugates is a very interesting perspective to develop new
therapeutic agents. We are currently assessing this opportunity,
and our results will be published in due course.
Re derivatives. Because the Re(CO) fragment is rather hydro-
3
phobic, conjugate 2 is only slightly soluble in water, even if it
bears four positive charges.
All compounds were investigated for in vitro cell growth in- Experimental Section
hibition toward cervical cancer (HeLa), lung cancer (H460M2),
Synthesis and characterization
and non-tumorigenic (HBL-100) cell lines. Importantly, all com-
One- and two-dimensional (H–H COSY and HSQC) NMR spectra
pounds are nontoxic up to a concentration of 100 mm in the
dark on the cell lines studied in this work, except for the Re
conjugate 2, which has IC50 values of ~7 and 34 mm on
H460M2 and HBL-100 cells, respectively. Upon light irradiation
at l=650 nm, compounds 1 and 2 showed much better pho-
totoxic properties than the monofunctionalized 3 and 4. In
particular, Re–porphyrin conjugate 2 showed remarkable pho-
totoxicity on HeLa cells, similar to hypericin and photofrin. The
phototoxic activity of 2 and its lack of dark toxicity on HeLa
cells suggest a selective use of this compound in all tumor cell
lines that show a selective sensitivity between the dark and ir-
radiation experiments. The unconjugated parent porphyrin
1
were recorded on a Varian 500 spectrometer (500 MHz for H,
1
9
4
70 MHz for F). All spectra were run at room temperature.
1
H NMR chemical shifts were referenced to the peak of residual
non-deuterated solvent (d=7.26 for CDCl , 3.31 for CD OD, 4.34
3
3
19
for CD CN, 2.50 for [D ]DMSO). F NMR shifts are referenced to
3
6
CFCl3 as internal standard. UV/Vis spectra were obtained at T=
258C on a Jasco V-500 UV/Vis spectrophotometer equipped with
a Peltier temperature controller, using 1.0 cm path-length quartz
cuvettes (3.0 mL). IR spectra were recorded on a PerkinElmer Spec-
trum RXI FTIR spectrophotometer. Mass spectra were obtained by
positive and negative ESI-MS using a Bruker Esquire ESI-MS instru-
ment. ESI HRMS were obtained using a Bruker maXis instrument.
Column chromatography was performed on silica gel 60 Š (Merck,
230–400 mesh ASTM), eluting with chloroform/ethanol mixtures as
specified below. The reactions were monitored by TLC (silica gel/
UV 254, 0.25 mm, glass or aluminum support).
1
>
had excellent phototoxic activity on H460M2 cells, with a PI
À2
77 at a light dose of 10 Jcm . Notably, the light dose used
during the course of our experiments is similar to, or even
lower than, that used for related compounds. This clearly guar-
antees the absence of damage to untreated cells.
I
Porphyrin intermediates 6 and 8 and Re precursor fac-[Re-
[
60]
(
CO) (dmso-O) ](CF SO ) were prepared according to published
The intracellular localization studies in HeLa cells outlined
a minimal cellular penetration for 1 that might be related to its
low photoactivity on this cell line. Instead, the Re conjugate 2
showed very good cellular uptake. This might explain its better
3
3
3
3
[
27,30,61]
procedures.
All chemicals were purchased from Sigma–Al-
drich and were used without further purification unless otherwise
specified.
The porphyrins and their Re conjugates precipitate with variable
amounts of crystallization solvent, depending on the batch. For
this reason elemental analysis of such conjugates did not afford re-
liable and reproducible results, and the values are not reported
here (typically, some of the elemental analysis values, especially for
C, differ from calculated values by >0.5%). Nevertheless, the purity
calculated from elemental analysis data was always >95%, and
the proposed formulae are all consistent with NMR, ESI MS and ESI
photoactivity on the same cell line, although 2 is less efficient
1
at producing O than 1. Compounds 3 and 4 were found to
2
be more efficient than 1 and 2 at binding DNA (with some se-
lectivity for quadruplex versus duplex) as well as in DNA pho-
tocleavage.
Upon Re coordination, we observed an enhancement in the
phototoxic activity of the tetrafunctionalized porphyrin 1,
ChemMedChem 2015, 10, 1901 – 1914
www.chemmedchem.org
1910
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
1
3
HRMS spectra. Of note, the C NMR spectra are not reported, as
(CDCl ): d=8.83 (s, 8H, bH), 8.27 (dd, 16 H mPh+oPh), 7.74 (m,
3
the solubility of compounds 1–4 in CD OD at the maximum extent
2H, NHCO), 7.66 (m, 2H, NHCO), 7.38 (m, 4H, NHCO), 3.83 (m, 8H,
3
was insufficient to observe all the carbon resonances, especially of
the quaternary carbons, even after an accumulation time of 72 h.
CH NH+8H, CH O), 3.72 (m, 16H, CH O), 3.62 (m, 8H, CH O), 3.50
2
2
2
2
(m, 16H, CH c+CH NH), 3.35 (m, 16H, CH b), 3.21 (m, 8H, CH d),
2
2
2
2
2
.67 (m, 16H, CH a), 1.48, 1,46, 1.42 (s, 72H, CH Boc), À2,78 ppm
2
3
Di-tert-butyl-[1,4,7]-triazacyclononane-1,4-dicarboxylate (diBoc-
TACN) (10): To a solution of TACN (500.5 mg, 3.87 mmol) in CHCl3
(
(
brs, 2H, NH); UV/Vis (CH Cl ): l (relative intensity %)=419.5
max
100), 515.2 (4.3), 550.2 (2.1), 590.2 (1.5), 645.9 nm (1.1).
2
2
(
10 mL), TEA (744 mL, 5.34 mmol) was added. To this transparent
solution a solution of (Boc) O (1.53 g, 7.00 mmol) in CHCl (20 mL)
Porphyrin 1: Porphyrin 7 (14.54 mg, 0.0052 mmol) was dissolved
2
3
was added dropwise under an argon atmosphere in 4 h at room
temperature. The reaction mixture was stirred overnight under an
argon atmosphere at room temperature. The reaction was moni-
in CH Cl (1.5 mL) to obtain a purple solution. To this solution TFA
2
2
(1.5 mL) was added and the solution turned deep green. The solu-
tion was stirred for 4 h shielded from light, and then the solvent
was removed under reduced pressure until the elimination of TFA
was complete, to give a green–blue product. The porphyrin as TFA
salt (0.0052 mmol) was dissolved in the minimum amount of
tored by TLC (EtOAc/EtOH 10:1, R =0.3). The solvent was removed
f
under reduced pressure to give the product as a white oil. The
product was dissolved in EtOAc (100 mL) and then washed with
4
% NaHCO3 (100 mL) and brine (100 mL”2). The organic phase
CH OH to obtain a deep green solution. To this solution 30 mL
3
was washed with 10% aqueous citric acid (100 mL”3) and the
product moved in the water phase. NaOH 10% was added under
ice-cooling until the water phase was adjusted to pH 10 and the
product was extracted with CH Cl (100 mL”3). The organic solu-
(0.20 mmol) of TEA were added and the solution turned purple,
that indicated the pH changed to basic. The product was precipi-
tated by adding Et O dropwise to the solution. The solid was ex-
2
tensively washed with Et O and dried under vacuum. (10 mg, yield
2
2
2
1
tion was dried over Na SO and the solvent was removed under re-
97%); H NMR (CD OD): d=8.88 (brs, 8H, bH), 8.34 (d, 8H, H Ph,
2
4
3
duced pressure to give the product as a white oil. Yield: 718 mg
J=8.2 Hz), 8.30 (d, 8H, H Ph, J=8.2 Hz), 3.83 (m, 8H, CH O), 3.78
2
1
(
56%); H NMR (CDCl ): d=3.49 (m, 2H, CH c), 3.43 (m, 2H, CH c),
(m, 16H, CH O+CH NH), 3.73 (m, 8H, CH O), 3.64 (m, 8H, CH O),
3
2
2
2
2
2
2
3
1
.31 (m, 2H, CH2 b), 3.25 (m, 2H, CH2 b), 2.94 (m, 4H, CH2 a),
3.47 (m, 16H, CH d+CH NH), 3.22 (m, 16H, CH c), 3.04 (m, 16H,
2 2 2
+
À3
.48 ppm (s, 18H, CH Boc); ESI-MS m/z 330.2 [M+H] , 325.2 [M+
CH b), 2.86 ppm (m, 16H, CH a); UV/Vis (CH OH): l (e”10 )=
3
2
2
3
max
3
+
À1
À1
Na] .
416 (250), 513 (11), 547 (4.9), 590 (2.8) 645 nm (2.4 dm mol cm );
ESI-MS m/z 1989.9 [M+H] ; ESI HRMS calcd for [C104H146N O ]/z
M+3H]
+
24
16
1
,4-Bis(tert-butyloxycarbonyl)-1,4,7-triazacyclononane-7-benzyla-
cetate (diBocTACNCbz) (11): To solution of 10 (718 mg,
.18 mmol) and TEA (608 mL, 4.36 mmol) in CHCl (12 mL) a solution
3+
4+
[
4
663.37829, found 663.71765, calcd for [M+4H]
a
5+
97.78372 found 498.04085, calcd for [M+5H] 398.42697 found
2
3
3
98.63419.
of benzyl bromoacetate (1.26 g, 5.50 mmol) in CHCl (23 mL) was
3
I
added dropwise under ice-cooling in 1 h. The solution was stirred
for 48 h at room temperature and the reaction was monitored by
TLC (CH Cl /EtOH 95:5, R =0.6). The solvent was removed under
Porphyrin–Re conjugate 2: Porphyrin 1 (25.08 mg, 0.0126 mmol)
was dissolved in anhydrous CH OH (20 mL). To this solution fac-
3
[Re(CO) (dmso-O) ](CF SO ) (49.91 mg, 0.0764 mmol, 6 equiv) was
2
2
f
3
3
3
3
reduced pressure to give a yellow oil that was purified by column
chromatography (CH Cl /EtOH, 97:3, then 95:5) to afford the pure
added. The stirred mixture was held at reflux under argon and was
shielded from light for 48 h. The reaction was monitored by
2
2
1
1
product as a yellow oil. Yield: 926 mg (89%); H NMR (CDCl ): d=
H NMR spectroscopy after 6, 13, and 30 h and by TLC (CH CN/
3
3
7
4
.35 (m, 5H, H Ph), 5.13 (t, 2H, CH e), 3.49 (s, 2H, CH d), 3.45 (m,
H O/KNO R =0.46). After the solvent was removed under reduced
2
2
2
3
f
H, CH c), 3.26 (m, 2H, CH b), 3.20 (m, 2H, CH b), 2.84 (m, 4H,
pressure, the solid was dissolved in the minimum amount of
CH OH and precipitated by adding Et O dropwise. The product
2
2
2
CH a), 1.46 (s, 9H, CH Boc), 1.45 ppm (s, 9H, CH Boc); ESI-MS m/z
4
2
3
3
3
2
+
+
+
78.3 [M+H] , 500.3 [M+Na] , 516.3 [M+K] .
was then extensively washed with Et O and CH Cl to give a brown
2 2 2
1
product (30.1 mg, yield 65%); H NMR (CD OD): d=8.32 (dd, 16H,
3
1
,4-Bis(tert-butyloxycarbonyl)-1,4,7-triazacyclononane-7-acetic
oPh+mPh), 8.89 (brs, 8H, bH), 6.83 (s, 8H, NH TACN), 4.16 (s, 8H,
CH d), 3.83 (m, 8H, CH O), 3.78 (m, 16H, CH O+CH NHCO), 3.72
acid (2-(diBoc)TACN acetic acid) (5): To a deoxygenated solution
of 11 (926 mg, 1.94 mmol) in CH OH (25 mL) Pd/C was slowly
added, then the reaction flask was purged with H several times.
After 24 h the catalyst was removed by filtration over a Celite pad
and the solvent was evaporated under reduced pressure to give
a white crystalline solid. TLC: CH Cl /EtOH 9:1, R =0.3; Yield:
2
2
2
2
3
(
m, 8H, CH O), 3.63 (t, 8H, CH O, J=5.5 Hz), 3.44 (t, 8H, CH NHCO,
2 2 2
2
J=5.5 Hz), 3.23 (m, 16H, CH a+CH TACN), 3.13 (m, 8H, CH a),
2
2
2
2
.90 (m, 8H, CH TACN), 2.70 (m, 8H, CH TACN), 2.59 (m, 8H, CH
2 2 2
À3
TACN); UV/Vis (CH OH): l
(e”10 )=416 (217), 516 (10), 554
3
max
2
2
f
3
À1
À1
(
[
5.0), 596 (3.1), 649 nm (2.3 dm mol cm ); ESI HRMS calcd for
C120H170N O Re ]/z [M]
1
6
86 mg (91%); H NMR (CDCl ): d=3.56–3.18 (m, 8H, CH b+CH
4+
3
2
2
785.77116, found 785.77425; IR (KBr):
24
28
4
c), 3.39 (s, 2H, CH d) 2.77–2.65 (m, 4H, CH a) 1.48 (s, 9H, CH
Boc), 1.49 ppm (s, 9H, CH Boc); ESI-MS m/z (negative mode) 386.1
À1
2
2
3
n˜ =2028 (CO), 1910 cm (CO).
3
À
+
+
[MÀH] , (positive mode): 388.2 [M+H] , 410.2 [M+Na] .
Boc-protected porphyrin 9: To a solution of 5 (14.34 mg,
0
(
0
.0370 mmol, 1.44 equiv) in anhydrous DMF (1.5 mL) HOBt
7.52 mg, 0.0556 mmol, 2.17 equiv) and EDCI (10.64 mg,
.0555 mmol, 2.17 equiv) were added and the resulting solution
was stirred for 30 min. To this solution a purple solution of the por-
phyrin (30.07 mg, 0.0256 mmol) and DMAP (7.65 mg,
.0626 mmol, 2.44 equiv) in anhydrous DMF (1.5 mL) was added.
Boc-protected porphyrin 7: To solution of (54.44 mg,
a
5
0
.14 mmol) in anhydrous DMF (3 mL), HOBt (28.51 mg, 0.21 mmol)
and N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride
EDCI) (40.6 mg, 0.21 mmol) were added. The solution was stirred
for 30 min, then purple solution of the porphyrin
0.0234 mmol) and DMAP (28.80 mg, 0.23 mmol) in anhydrous
DMF (4 mL) was added. The reaction was monitored by TLC
CH Cl /EtOH 9:1, R =0.3). The reaction solution was stirred over-
(
8
a
6
0
(
The coupling was performed in a microwave oven reactor (ramp
time: 10 s, hold time: 6 min, T=608C, P=1720 kPa, power: 30 W).
The reaction was monitored by TLC (CH CN/KNO /H O 4:0.3:1, R =
(
2
2
f
3
3
2
f
night at room temperature shielded from light. The product was
purified by column chromatography (CH Cl /EtOH 90:10, then
8
0
.38). The charged porphyrin 9 was purified by repeated precipita-
2
2
tion (CH OH/Et O and CH CN/Et O) and extensive washing with
1
3
2
3
2
5:15) to obtain 43.2 mg of a purple solid (66% yield); H NMR
Et O and CH Cl to remove the excess either of reagents and of
2
2
2
ChemMedChem 2015, 10, 1901 – 1914
www.chemmedchem.org
1911
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
1
À1
À1
the ligand. Yield: 65% (26.27 mg); H NMR (CD ,CN): d=9.12 (m,
FBS (Gibco), HEPES (Euroclone), 100 UImL penicillin, 100 mgmL
3
6
8
3
H, 2,6py), 9.07 (m, 6H, bH), 8.95 (m, 2H, bH), 8.82 (m, 6H, 3,5py),
.30 (dd, 4H, oPh+mPh), 4.70 (s, 3H, CH py), 4.69 (s, 6H, CH py),
streptomycin, 2 mm l-glutamine. HBL-100 non-tumorigenic epithe-
3
3
lial cells were grown in McCoy’s (Sigma) supplemented with 10%
À1
À1
.75 (m, 2H, CH O), 3.70 (m, 4H, CH O+CH NH), 3.65 (m, 2H,
FBS (Gibco), 100 UImL
penicillin, 100 mgmL
streptomycin,
2
2
2
CH O), 3.55 (t, 2H, CH O, J=5.6 Hz), 3.37 (m, 6H, CH NH+CH c),
2 mm l-glutamine. The cells were cultured at 378C and in 5% CO2
2
2
2
2
3
1
.23 (m, 4H, CH b), 3.09 (m, 2H, CH d), 2.59 (m, 4H, CH a), 1.43,
humidified atmosphere.
2
2
2
À
.42, 1.36 (s, 18H, CH Boc), 2.96 ppm (s, 2H, NH pyrrole); UV/Vis
3
Cell phototoxicity: Cells were sown at 10000 per well on 96-well
plates and allowed to grow 24 h. Then they were incubated for
(
CH OH): l (relative intensity %)=422 (100), 516 (8.3), 553 (3.9),
3 max
+
2+
5
89 (3.3), 648 nm (1.3); ESI-MS m/z 1205.7 [M] , 603.1 [M] .
2
4 h with 0.1–100 mm solutions of each compound, obtained by
Porphyrin 3: To a solution of porphyrin 9 (26.27 mg, 0.0166 mmol)
serial dilutions of stock solutions (freshly prepared in DMSO at
a concentration of 10 m) with complete medium. Maximum
À2
in CH OH (2 mL) TFA (800 mL) was added. The solution was stirred
3
for 3 h shielded from light, and then the solvent was removed
under reduced pressure until the complete elimination of TFA, to
give the product as TFA salt. The deep green–brown solid was dis-
DMSO concentration in the cell incubation medium was ꢂ0.3% v/
v. Thereafter, the media containing compounds were replaced with
drug-free medium and cells were irradiated at l=650 nm at a flu-
À2
solved in 1 mL CH OH. To this solution 10 mL TEA were added. The
ence rate of 14 mWcm for a time such that the total light dose
3
À2
product was precipitated by adding Et O dropwise to the solution
was either 1, 5, or 10 Jcm (71 s; 5 min, 57 s; or 11 min, 54 s). The
2
and was then extensively washed with Et O. The product was ob-
illumination was performed with a LED board equipped with dedi-
cated software. The LED board and software were assembled and
set up by F. Armani and G. Verona (A.P.L. Laboratory of the Depart-
ment of Engineering and Architecture at the University of Trieste).
The LED board is equipped with 96 red LEDs (L-53SRC-E, King-
bright) arranged in order to fit with the 96 wells of the cell culture
plates. The emitted power (mW) at the end of the optical fiber was
measured with an Ophir NOVA Laser Measurement power meter.
Control experiments performed in the absence of any photosensi-
2
1
tained as a purple solid (21.83 mg, yield 98%); H NMR (CD CN):
3
d=9.13 (d, 6H, 2,6py, J=6.4 Hz), 9.06 (m, 6H, bH), 8.98 (m, 2H,
bH), 8.83 (d, 6H, 3,5py, J=6.5 Hz), 8.30 (dd, 4H, oPh+mPh), 7.83 (t,
NHCO, 1H, J=5.4 Hz), 4.69 (s, 3H, CH py), 4.70 (s, 6H, CH py), 3.79
3
3
(
t, 2H, CH O, J=5.4 Hz), 3.72 (m, 4H, CH O+CH NH), 3.66 (m, 2H,
2 2 2
CH O), 3.58 (t, 2H, CH 2, J=5.5 Hz), 3.37 (s, 2H, CH d), 3.38 (m,
2
2
2
2
4
H, CH NH), 3.29 (m, 4H, CH b), 3.02 (m, 4H, CH c), 2.82 ppm (t,
2 2 2
H, CH2 a, J=5.4 Hz); TLC: R =0.06 CH CN/KNO /H O 4:0.3:1;
f
3
3
sat
2
À3
À2
UV/Vis (CH CN): l
(e”10 )=423 (157), 517 (15), 553 (8.3), 590
tizer indicated that light doses up to 10 Jcm cause no evident
3
max
3
À1
À1
19
(
6.4), 646 nm (3.2 dm mol cm );
F NMR (CD CN): d=
cell damage. A plate similarly treated, but not exposed to light was
used as reference for dark cytotoxicity under the same experimen-
tal conditions. Analysis of cell phototoxicity by MTT assay was per-
formed after further 24 h of incubation and compared with the
values of control cells without light irradiation. Briefly, MTT dis-
3
À
+
2+
À75.62 ppm (CF COO ); ESI-MS m/z 1005.6 [M] , 502.8 [M] ; ESI
3
3
+
HRMS calcd for [C H N O ]/z [M] 335.17451, found 335.17451.
5
9
65 12
4
I
Porphyrin–Re conjugate 4: Porphyrin 3 (14.32 mg, 0.0106 mmol)
was dissolved in anhydrous CH OH (5 mL). To this solution fac-[Re(-
3
À1
solved in phosphate-buffered saline (PBS, 5 mgmL ) was added
CO) (dmso-O) ](CF SO ) (10.27 mg, 0.0157 mmol, 1.5 equiv) was
3
3
3
3
(
10 mL per 100 mL medium) to all wells, and the plates were then
added. The reaction was performed in a microwave oven reactor
first step: T=658C, ramp time: 10 min, hold time: 30 s, P=
720 kPa, power: 25 W; second step: T=110 8C, ramp time: 10 min,
incubated at 378C with 5% CO and 100% relative humidity for
2
(
1
4
h. After this time, the medium was discarded, and 200 mL DMSO
[
62]
were added to each well according to the method of Alley et al.
hold time: 10 min, P=1720 kPa, power: 30 W). The reaction was
monitored by TLC (CH CN/KNO /H O, 4:0.3:1, R =0.32). The solvent
Absorbance units were measured at l=570 nm on a SpectraCount
Packard instrument (Meriden, CT, USA). IC50 values were calculated
from dose–effect curves and are the mean ÆSD of at least three
separate experiments. The fitting procedure applied is a nonlinear
regression performed with GraphPad Prism version 6 for Mac OS X
version 6.0b (GraphPad Software, San Diego, CA, USA). Experiments
were conducted in quadruplicate and repeated thrice.
3
3
2
f
was then removed under reduced pressure. The product was dis-
solved in the minimum amount of CH OH and precipitated with
3
Et O, then it was washed with Et O and CH Cl to remove excess
2
2
2
2
complex to obtain 13.76 mg of the pure product (yield 73%);
1
H NMR (CD CN): d=9.12 (d, 6H, 2,6py, J=6.2 Hz), 9.06 (m, 6H,
3
bH), 8.98 (m, 2H, bH), 8.83 (d, 6H, 3,5py, J=6.5 Hz), 8.31 (dd, 4H,
oPh+mPh), 7.66 (t, NHCO, 1H, J=6.2 Hz), 7.12 (t, NHCO, 1H, J=
Statistical analysis: Data obtained in the experiments were subject-
ed to statistical analysis of variance (ANOVA) and Tukey–Kramer
post-test, or to unpaired t test performed using GraphPad InStat
version 3.06 for Windows (GraphPad Software, San Diego, CA,
USA).
7
.5 Hz), 5.90 (m, 2H, NH TACN), 4.96 (m, 9H, CH py), 4.12 (s, 2H,
3
CH d), 3.78 (t, 2H, CH O, J=5.5 Hz), 3.72 (m, 4H, CH O+CH 8),
2
2
2
2
3
1
2
.66 (m, 2H, CH O), 3.57 (t, 2H, CH 2J=5.5 Hz), 3.38 (m, 2H, CH
2 2 2
), 3.31 (m, 2H, CH c), 3.21 (m, 4H, CH a), 3.00 (m, 2H, CH b),
2
2
2
.72 (m, 2H, CH c), 2.63 ppm (m, 2H,CH b); UV/Vis (CH CN): l
max
2
2
3
À3
Singlet oxygen production: The quantum yield (F ) of singlet
D
(e”10 )=424 (222), 517 (15), 553 (6.0), 590 (4.5), 646 nm
3
À1
À1
19
À
oxygen generated by compounds 1–4 upon photoexcitation was
measured using 9,10-dimethylanthracene (DMA) as substrate. Typi-
(
0.7 dm mol cm );
F NMR (CD CN): d=À75.30 (CF COO ),
3
3
À
4+
À79.28 ppm (CF SO ); ESI HRMS calcd for [C H N O Re]/z [M]
3
3
63 71 12
7
À1
cally, 3 mL of 0.133 mm CHCl solution of DMA and 0.4 mL solution
3
3
23.62774, found 323.62772; IR (KBr): n˜ =1977 (CO), 2104 cm
of the porphyrin (0.4 A at Soret band maximum, ꢁ10 mm) in
DMSO were placed in a luminescence quartz cuvette of 1 cm opti-
cal path and irradiated in a RPR100 Rayonet Chamber Reactor
(
CO).
(
Southern New England Ultraviolet Company) complete with two
Biological methods
lamps of l=420 nm light for different periods of time. The fluence
rate was 2.57 mWcm . The first-order rate constant of the photo-
À2
Cell culture: Human cervical carcinoma cells (HeLa) were cultured
in DMEM (Euroclone) supplemented with 5% fetal bovine serum
1
oxidation of DMA by O was obtained by plotting A ÀA as a func-
2
0
À1
À1
tion of the irradiation time t, in which A and A represent the ab-
0
(
FBS, Gibco), 100 UImL
penicillin, 100 mgmL
streptomycin,
sorbance intensity at time 0 and at time t, respectively. The rate
constant was then converted into O quantum yield by compari-
2
mm l-glutamine. The non-small-cell lung carcinoma cell line
1
2
(
H460M2) was grown in RPMI (Euroclone) supplemented with 5%
ChemMedChem 2015, 10, 1901 – 1914
www.chemmedchem.org
1912
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
son with the rate constant for DMA photo-oxidation sensitized by
Acknowledgements
TPP, (for which F was shown to be 0.55) and with the absorbance
D
correction factor I=I *(1À10ÀAl), where I is the light intensity of
0
0
Financial Support from the Swiss National Science Foundation
Professorships PP00P2_133568 and PP00P2_157545 to G.G.), the
the irradiation source in the irradiation interval, and A is the ab-
sorbance of the sample at wavelength l.
(
Stiftung für Wissenschaftliche Forschung of the University of
Zurich (G.G.), the UBS Promedica Stiftung (R.R. and G.G), the No-
vartis Jubilee Foundation (R.R and G.G.), the Casali Foundation
Fluorescence microscopy: Cells were grown on 18 mm Menzel glass
5
À1
coverslips (Menzel, Germany) at a density of 2.5”10 cellsmL and
incubated with the indicated compound. Upon 2 h treatment, cells
were fixed for 15 min at room temperature in 4% formaldehyde
solution (4% formaldehyde (w/v) in 1”PBS) and mounted on mi-
croscopy slides. Fixed cells were examined with a CLSM Leica SP5
(
(
T.G.), the Beneficentia Stiftung (E.A.), the University of Zurich
G.G.), the UK’s Engineering and Physical Sciences Research Coun-
cil grant number EP/H005285/1 (A.L. and R.V.), and the COST
Action CM1105 (G.G., R.V., A.B., and E.A.) is gratefully acknowl-
edged. The authors thank the Center for Microscopy and Image
Analysis of the University of Zurich for access to state-of-the-art
equipment.
confocal microscope (DAPI l =405 nm, lem =430–500 nm; por-
ex
phyrin l =514 nm, lem =600–700 nm) using 63”1.20 oil-immer-
ex
sion lenses.
DNA preparation: All oligonucleotides, namely Myc22 (5’-TGA GGG
TGG GTA GGG TGG GTA A-3’), H-telo (5’-AGG GTT AGG GTT AGG
GTT AGG G-3’), and ds-26 (5’-CAA TCG GAT CGA ATT CGA TCC GAT
TG-3’) were purchased from Eurogentec and used without further
purification. ct-DNA was purchased from Sigma–Aldrich and used
as received. The oligonucleotides (for both emission and CD spec-
troscopic studies) were dissolved in Milli-Q water to yield 1 mm
stock solution which was stored at À208C and defrosted on the
day when a given experiment was carried out. The 1 mm oligonu-
cleotide stock solution was further diluted using appropriate buffer
to the desired concentration on the day of the experiment. The
concentration of oligonucleotides was determined by UV/Vis spec-
troscopy using appropriate extinction coefficients at l=260 nm.
For the emission titrations, concentration of the compound being
studied was kept constant (at 5 mm); the concentration of DNA
added during the titrations (after appropriate dilutions from the
stock solution) ranged between 0 and 40 mm. For the variable-tem-
perature CD spectroscopic studies the final concentrations of DNA
and compound were 5 and 10 mm, respectively.
Keywords: antitumor agents · photodynamic therapy (PDT) ·
phototoxicity · porphyrins · rhenium
[
1] P. Agostinis, K. Berg, K. A. Cengel, T. H. Foster, A. W. Girotti, S. O. Goll-
nick, S. M. Hahn, M. R. Hamblin, A. Juzeniene, D. Kessel, M. Korbelik, J.
Moan, P. Mroz, D. Nowis, J. Piette, B. C. Wilson, J. Golab, Ca-Cancer J.
Clin. 2011, 61, 250–281.
[2] D. E. J. G. J. Dolmans, D. Fukumura, R. K. Jain, Nat. Rev. Cancer 2003, 3,
380–387.
[3] M. R. Hamblin, T. Hasan, Photochem. Photobiol. Sci. 2004, 3, 436–450.
[4] T. Maisch, Mini-Rev. Med. Chem. 2009, 9, 974–983.
[5] T. J. Dougherty, C. J. Gomer, B. W. Henderson, G. Jori, D. Kessel, M. Kor-
belik, J. Moan, Q. Peng, J. Natl. Cancer Inst. 1998, 90, 889–905.
[6] R. Bhuvaneswari, Y. Y. Gan, K. C. Soo, M. Olivo, Cell. Mol. Life Sci. 2009,
6
6, 2275–2283.
[
[
[
7] A. P. Castano, P. Mroz, M. R. Hamblin, Nat. Rev. Cancer 2006, 6, 535–545.
8] N. L. Oleinick, H. H. Evans, Radiat. Res. 1998, 150, S146–S156.
9] R. Bonnett, Chem. Soc. Rev. 1995, 24, 19–33.
Compound preparation for DNA binding studies: Compounds were
dissolved in Milli-Q water to yield 1 mm solutions (except for 2,
which was dissolved in DMSO). These solutions were diluted with
the buffer to give a final concentration of 5 mm for the emission ti-
trations and 10 mm variable-temperature CD studies. Unless stated
otherwise, all measurements were performed in Tris·HCl (50 mm)
buffer containing 100 mm KCl (pH 7.4). Emission and excitation
spectra were recorded on Varian Cary Eclipse Spectrometer. Circu-
lar dichroism measurements were carried out using a Jasco J-715
Spectropolarimeter.
[
10] A. Leonidova, V. Pierroz, R. Rubbiani, Y. Lan, A. G. Schmitz, A. Kaech,
R. K. O. Sigel, S. Ferrari, G. Gasser, Chem. Sci. 2014, 5, 4044–4056.
11] J. Moan, K. Berg, Photochem. Photobiol. 1991, 53, 549–553.
[
[
12] E. Skovsen, J. W. Snyder, J. D. Lambert, P. R. Ogilby, J. Phys. Chem. B
2005, 109, 8570–8573.
[13] J. Moan, P. Juzenas, J. Environ. Pathol. Toxicol. Oncol. 2006, 25, 29–50.
[14] A. M. R. Fisher, A. L. Murphree, C. J. Gomer, Lasers Surg. Med. 1995, 17,
2
–31.
[
[
15] G. Palumbo, Expert Opin. Drug Delivery 2007, 4, 131–148.
16] A. Juarranz, P. Jaen, F. Sanz-Rodriguez, J. Cuevas, S. Gonzalez, Clin.
Transl. Oncol. 2008, 10, 148–154.
DNA photocleavage: DNA photocleavage experiments were per-
formed according to a method reported recently by our group.
More specifically, supercoiled pUC18 plasmid (0.20 mg) was incu-
bated with 1–4 at increasing concentrations in buffer (50 mm
[17] R. Bonnett, Chemical Aspects of Photodynamic Therapy, Gordon and
Breach Science Publishers, London, 2000.
[63]
[
[
18] R. R. Allison, C. H. Sibata, Photodiagn. Photodyn. Ther. 2010, 7, 61–75.
19] F. H. J. Figge, G. S. Weiland, L. O. J. Manganiello, Proc. Soc. Exp. Biol. Med.
1
948, 68, 640–641.
20] R. Ackroyd, C. Kelty, N. Brown, M. Reed, Photochem. Photobiol. 2001, 74,
56–669.
21] F. S. De Rosa, M. V. L. B. Bentley, Pharm. Res. 2000, 17, 1447–1455.
Tris·HCl, 18 mm NaCl, pH 7.2) and at l=420 nm for 22 min
[
[
À2
(
10 Jcm ) in a RPR100 Rayonet Chamber Reactor (Southern New
6
England Ultraviolet Company). A series of negative controls of the
plasmid treated with the highest concentrations of 1–4 in the dark
was used for comparative purposes. After irradiation the samples
were added with loading buffer (250 mg xylene cyanol in 33 mL
[22] M. G. H. Vicente, Curr. Med. Chem. Anti-Cancer Agents 2001, 1, 175–194.
[23] a) C. Lottner, R. Knuechel, G. Bernhardt, H. Brunner, Cancer Lett. 2004,
203, 171–180; b) C. Lottner, K.-C. Bart, G. Bernhardt, H. Brunner, J. Med.
Chem. 2002, 45, 2064–2078; c) C. Lottner, K.-C. Bart, G. Bernhardt, H.
Brunner, J. Med. Chem. 2002, 45, 2079–2089; d) H. Brunner, K.-M. Schell-
erer, B. Treittinger, Inorg. Chim. Acta 1997, 264, 67–79; e) H. Brunner, H.
Obermeier, Angew. Chem. Int. Ed. Engl. 1994, 33, 2214–2215; Angew.
Chem. 1994, 106, 2305–2306.
1
50 mm Tris·HCl buffer, pH 7.6) and analyzed by electrophoresis in
agarose 0.8% in 1”TBE (diluted from a 10” solution of 108 g
Tris·HCl, and 55 g of H BO in 900 mL H O) at 70 V (BioRad Power
3
3
2
Pack 1000) for 1.5 h. The gel was pre-stained with GelRed (1:10000,
Biotium), photographed and quantified with an AlphaDigiDoc 1000
CCD camera (Buchner Biotec AG) and AlphaImager software (ver-
sion 1.3.0.7.).
[
24] a) R. Song, Y.-S. Kim, C. O. Lee, Y. S. Sohn, Tetrahedron Lett. 2003, 44,
537–1540; b) R. Song, Y.-S. Kim, Y. S. Sohn, J. Inorg. Biochem. 2002, 89,
1
83–88.
[25] L. Monsffl-Scolaro, C. Donato, M. Castriciano, A. Romeo, R. Romeo, Inorg.
Chim. Acta 2000, 300–302, 978–986.
ChemMedChem 2015, 10, 1901 – 1914
www.chemmedchem.org
1913
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
[
[
[
[
[
[
[
26] A. Naik, R. Rubbiani, G. Gasser, B. Spingler, Angew. Chem. Int. Ed. 2014,
3, 6938–6941; Angew. Chem. 2014, 126, 7058–7061.
27] T. Gianferrara, A. Bergamo, I. Bratsos, B. Milani, C. Spagnul, G. Sava, E.
Alessio, J. Med. Chem. 2010, 53, 4678–4690.
28] F. Schmitt, N. P. E. Barry, L. Juillerat-Jeanneret, B. Therrien, Bioorg. Med.
Chem. Lett. 2012, 22, 178–180.
29] F. Schmitt, P. Govindaswamy, G. Süss-Fink, W. H. Ang, P. J. Dyson, L. Juil-
lerat-Jeanneret, B. Therrien, J. Med. Chem. 2008, 51, 1811–1816.
30] C. Spagnul, R. Alberto, G. Gasser, S. Ferrari, V. Pierroz, A. Bergamo, T.
Gianferrara, E. Alessio, J. Inorg. Biochem. 2013, 122, 57–65.
31] T. Gianferrara, C. Spagnul, R. Alberto, G. Gasser, S. Ferrari, V. Pierroz, A.
Bergamo, E. Alessio, ChemMedChem 2014, 9, 1231–1237.
32] R. Alberto in Bioinorganic Medicinal Chemistry (Ed.: E. Alessio), Wiley-
VCH, Weinheim, 2011, pp. 253–282.
33] A. Leonidova, G. Gasser, ACS Chem. Biol. 2014, 9, 2180–2193.
34] P. Collery, G. Bastian, F. Santoni, A. Mohsen, M. M. Wei, T. Collery, A.
Tomas, D. Desmaele, J. D’Angelo, Anticancer Res. 2014, 34, 1679–1690.
35] I. Kitanovic, S. Can, H. Alborzinia, A. Kitanovic, V. Pierroz, A. Leonidova,
A. Pinto, R. Molteni, B. Spingler, S. Ferrari, A. Steffen, N. Metzler-Nolte, S.
Wçlfl, G. Gasser, Chem. Eur. J. 2014, 20, 2496–2507.
[46] R. Alberto, W. A. Herrmann, P. Kiprof, F. Baumgartner, Inorg. Chem. 1992,
31, 895–899.
[47] H. Braband, S. Imstepf, M. Benz, B. Spingler, R. Alberto, Inorg. Chem.
2012, 51, 4051–4057.
[48] C. Pomp, S. Drüeke, H. J. Küppers, K. Wieghardt, C. Kruger, B. Nuber, J.
Weiss, Z. Naturforsch. B 1988, 43, 299–305.
[49] a) A. C. Benniston, P. Gunning, R. D. Peacock, J. Org. Chem. 2005, 70,
115–130; b) S. Kimura, E. Bill, E. Bothe, T. Weyhermuller, K. Wieghardt, J.
Am. Chem. Soc. 2001, 123, 6025–6039.
[50] T. Gianferrara, I. Bratsos, E. Iengo, B. Milani, A. O sˇ tri c´ , C. Spagnul, E. Zan-
grando, E. Alessio, Dalton Trans. 2009, 10742–10756.
[51] F. Schmitt, P. Govindaswamy, O. Zava, G. Süss-Fink, L. Juillerat-Jeanneret,
B. Therrien, J. Biol. Inorg. Chem. 2009, 14, 101–109.
[52] K. Davia, D. King, Y. Hong, S. Swavey, Inorg. Chem. Commun. 2008, 11,
584–586.
5
[
[
[53] a) C.-T. Poon, P.-S. Chan, C. Man, F.-L. Jiang, R. N. S. Wong, N.-K. Mak,
D. W. J. Kwong, S.-W. Tsao, W.-K. Wong, J. Inorg. Biochem. 2010, 104, 62–
70; b) J.-X. Zhang, J.-W. Zhou, C.-F. Chan, T. C.-K. Lau, D. W. J. Kwong, H.-
L. Tam, N.-K. Mak, K.-L. Wong, W.-K. Wong, Bioconjugate Chem. 2012, 23,
1623–1638.
[
[
36] a) R. J. J. Fiel, J. Biomol. Struct. Dyn. 1989, 6, 1259–1274; b) R. F. Paster-
nack, E. J. Gibbs, Met. Ions Biol. Syst. 1996, 33, 367–397; c) G. Pratviel, J.
Bernadou, B. Meunier, Met. Ions Biol. Syst. 1996, 33, 399–426; d) L. G.
Marzilli, New J. Chem. 1990, 14, 409–420.
37] a) R. Fiel, B. Jenkins, J. Alderfer in The 23rd Jerusalem Symposium in
Quantum Chemistry and Biochemistry, Vol. 23 (Eds.: B. Pullman, J. Jort-
ner), Kluwer Academic Publishers, Netherlands, 1990, pp. 385–399;
b) R. J. Fiel, N. Datta-Gupta, E. H. Mark, J. C. Howard, Cancer Res. 1981,
[54] E. Delaey, F. van Laar, D. De Vos, A. Kamuhabwa, P. Jacobs, P. A. de Witte,
J. Photochem. Photobiol. B 2000, 55, 27–36.
[55] M. Ochsner, J. Photochem. Photobiol. B 1997, 39, 1–18.
[56] a) B. M. Sutherland, P. V. Bennett, M. Saparbaev, J. C. Sutherland, J. Laval,
Radiat. Prot. Dosim. 2001, 97, 33–38; b) S. Britz-Cunningham, S. Adel-
stein, J. Nucl. Med. 2003, 44, 1945–1961; c) M. Hada, A. G. Georgakilas,
J. Radiat. Res. 2008, 49, 203–210; d) N. Shikazono, M. Noguchi, K. Fujii,
A. Urushibara, A. Yokoya, J. Radiat. Res. 2009, 50, 27–36.
[
[
57] a) A. I. Kassis, J. Nucl. Med. 2003, 44, 1479–1481; b) M. L. Schipper,
C. G. U. Riese, S. Seitz, A. Weber, M. Behe, T. Schurrat, N. Schramm, B.
Keil, H. Alfke, T. M. Behr, Eur. J. Nucl. Med. Mol. Imaging 2007, 34, 638–
4
1, 3543–3545.
38] a) H. Han, D. R. Langley, A. Rangan, L. H. Hurley, J. Am. Chem. Soc. 2001,
23, 8902–8913; b) J. Seenisamy, S. Bashyam, V. Gokhale, H. Vankayala-
[
1
6
2
50; c) A. A. S. Tavares, J. M. R. S. Tavares, Int. J. Radiat. Biol. 2010, 86,
61–270.
pati, D. Sun, A. Siddiqui-Jain, N. Streiner, K. Shin-Ya, E. White, W. D.
Wilson, L. H. Hurley, J. Am. Chem. Soc. 2005, 127, 2944–2959.
[
[
58] T. Esteves, C. Xavier, S. Gama, F. Mendes, P. D. Raposinho, F. Marques, A.
[
[
39] a) L. Martino, B. Pagano, I. Fotticchia, S. Neidle, C. J. Giancola, J. Phys.
Chem. B 2009, 113, 14779–14786; b) A. Cummaro, I. Fotticchia, M. Fran-
ceschin, C. Giancola, L. Petraccone, Biochimie 2011, 93, 1392–1400.
40] a) I. M. Dixon, F. Lopez, A. M. Tejera, J. P. Est›ve, M. A. Blasco, G. Pratviel,
J. Am. Chem. Soc. 2007, 129, 1502–1503; b) C. Romera, O. Bombarde, R.
Bonnet, D. Gomez, P. Dumy, P. Calsou, J. F. Gwan, J. H. Lin, E. Defrancq,
G. Pratviel, Biochimie 2011, 93, 1310–1317.
Paulo, J. Costa Pessoa, J. Rino, G. Viola, I. Santos, Org. Biomol. Chem.
2
010, 8, 4104–4116.
59] a) R. G. Balasingham, M. P. Coogan, F. L. Thorp-Greenwood, Dalton Trans.
011, 40, 11663–11674; b) F. L. Thorp-Greenwood, M. P. Coogan, L.
2
Mishra, N. Kumari, G. Raic, S. Saripella, New J. Chem. 2012, 36, 64–72;
c) V. Fernµndez-Moreira, F. L. Thorp-Greenwood, A. J. Amoroso, J. Cable,
J. B. Court, V. Gray, A. J. Hayes, R. L. Jenkins, B. M. Kariuki, D. Lloyd, C. O.
Millet, C. Ff. Williams, M. P. Coogan, Org. Biomol. Chem. 2010, 8, 3888–
[
[
41] a) D. F. Shi, R. T. Wheelhouse, D. Sun, L. H. Hurley, J. Med. Chem. 2001,
4
4, 4509–4523; b) E. P. Cohn, K. L. Wu, T. R. Pettus, N. O. Reich, J. Med.
3
901.
60] M. Casanova, E. Zangrando, F. Munini, E. Iengo, E. Alessio, Dalton Trans.
006, 5033–5045.
Chem. 2012, 55, 3678–3686.
[
[
[
42] a) A. Siddiqui-Jain, C. L. Grand, D. J. Bearss, L. H. Hurley, Proc. Natl. Acad.
Sci. USA 2002, 99, 11593–11598; b) C. L. Grand, H. Han, R. M. MuÇoz, S.
Weitman, D. D. Von Hoff, L. H. Hurley, D. J. Bearss, Mol. Cancer Ther.
2
61] P. D. Beer, J. Cadman, J. M. Lloris, R. Martínez-MµÇez, J. Soto, T. Pardo,
M. D. Marcos, J. Chem. Soc. Dalton Trans. 2000, 1805–1812.
62] M. C. Alley, D. A. Scudiero, A. Monks, M. L. Hursey, M. J. Czerwinski, D. L.
Fine, B. J. Abbott, J. G. Mayo, R. H. Schoemaker, M. R. Boyd, Cancer Res.
2002, 1, 565–573.
[
[
43] I. M. Dixon, F. Lopez, J.-P. Esteve, A. M. Tejera, M. A. Blasco, G. Pratviel, B.
Meunier, ChemBioChem 2005, 6, 123–132.
1988, 48, 589–601.
44] a) S. Balasubramanian, S. Neidle, Curr. Opin. Chem. Biol. 2009, 13, 345–
[
63] C. Mari, V. Pierroz, R. Rubbiani, M. Patra, J. Hess, B. Spingler, L. Oehning-
3
53; b) S. Balasubramanian, L. H. Hurley, S. Neidle, Nat. Rev. Drug Discov-
ery 2011, 10, 261–275; c) G. W. Collie, G. N. Parkinson, Chem. Soc. Rev.
011, 40, 5867–5892; d) M. Düchler, J. Drug Targeting 2012, 20, 389–
00.
er, J. Schur, I. Ott, L. Salassa, S. Ferrari, G. Gasser, Chem. Eur. J. 2014, 20,
14421–14436.
2
4
[
45] a) T. B. Krystona, A. B. Georgieva, P. Pissis, A. G. Georgakilas, Mutation
Res. 2011, 711, 193–201; b) R. De Bont, N. van Larebeke, Mutagenesis
Received: July 3, 2015
Revised: August 13, 2015
2004, 19, 169–185.
Published online on September 2, 2015
ChemMedChem 2015, 10, 1901 – 1914
www.chemmedchem.org
1914
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim