Glucose-functionalized amino-OPEs as biocompatible photosensitizers in PDT

Article abstract of DOI:10.1016/j.ejmech.2016.01.041

Photodynamic therapy (PDT) is a minimally invasive procedure that can provide a selective eradication of neoplastic diseases by the combined effect of a photosensitizer, light and oxygen. New amino oligo(phenylene-ethynylene)s (OPEs), bearing hydrophilic

Full text of DOI:10.1016/j.ejmech.2016.01.041

European Journal of Medicinal Chemistry 111 (2016) 58e71
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Glucose-functionalized amino-OPEs as biocompatible
photosensitizers in PDT
,
1
b
,
1
a
c
Elisa Deni a , Alicia Zamarron , Paola Bonaccorsi , M. Carmen Carreno ,
ꢀ
~
b
a
a
ꢀ
Angeles Juarranz , Fausto Puntoriero , Maria Teresa Sciortino ,
María Ribagorda c *, Anna Barattucci a
,
, **
a
ꢁ
Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali- ChiBioFarAm, Universita di Messina, Messina, Italy
Departamento de Biología, Universidad Autonoma de Madrid, Madrid, Spain
Departamento de Química Organica, Universidad Autonoma de Madrid, Madrid, Spain
b
c
ꢀ
ꢀ
ꢀ
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 a minimally invasive procedure that can provide a selective eradication of
Received 10 December 2015
Received in revised form
21 December 2015
Accepted 21 January 2016
Available online 28 January 2016
neoplastic diseases by the combined effect of
a photosensitizer, light and oxygen. New amino
oligo(phenylene-ethynylene)s (OPEs), bearing hydrophilic glucoside terminations, have been prepared,
characterized and tested as photosensitizers in PDT. The effectiveness of these compounds in combi-
nation with UVA light has been checked on two tumor cell lines (HEp-2 and HeLa cells, derived from a
larynx carcinoma and a cervical carcinoma, respectively). The compounds triggered a mitotic blockage
that led to the cell death, being the effect active up to 3 mm concentration. The photophysical properties
Keywords:
Phothodynamic therapy
Phothosensitizer
Oligo(phenylene ethynylene)
Singlet oxygen production
Glucose base photosensitizers
of OPEs, such as high quantum yield, stability, singlet oxygen production, biocompatibility, easy cell-
internalization and very good response even at low concentration, make them promising photosensi-
tizers in the application of PDT.
© 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction
respond to other topical treatments [3,4]. PDT is based on the
specific accumulation of a photosensitizer (PS) in the target tissue,
Phototherapy is a minimally invasive therapeutic procedure
already approved for the treatment of various oncological and
non-oncological pathologies of skin [1,2]. Variants of phototherapy
include targeted ultraviolet B (UVB) phototherapy, topical psoralen
plus ultraviolet A (PUVA), and photodynamic therapy (PDT). All of
them are localized forms of phototherapy, either as first-line
treatment or as complementary treatment for those that do not
followed by irradiation with light at a wavelength matching the
absorption spectrum of the PS. Upon light absorption, the PS
transits from its ground state to an unstable excited singlet state,
from which it can decay, either to the ground-state or to the
excited triplet state. In this long-lived excited triplet state, the PS
is able to produce singlet oxygen (1O2) which, in turn, gives rise to
other reactive oxygen species (ROS), such as peroxides, superoxide
anions or hydroxyl radicals. Taking into account that 1O2 has a
lifetime of approximately 40 ns, it acts near to the site where it is
generated. ROS are able to react directly with a biological sub-
strate, oxidizing vital cellular components inducing an acute cell
stress response culminating in cellular death, mainly by apoptosis
and/or necrosis. The cell death pathway depends, among other
factors, on the nature and intracellular localization of the PS and
on tumor properties [1,5].
Abbreviations: OPE, oligo(phenylene ethynylene); PDT, phothodynamic therapy;
PS, phothosensitizer; UV, ultraviolet light; UVA, ultraviolet light A; UVB, ultraviolet
light B; PUVA, psolaren plus ultraviolet A therapy; ROS, reactive oxygen species;
LD50, lethal dose 50%; LD90, lethal dose 90%; MTT, 3-[4,5-dimethylthiazol-2-yl]-
2,5-diphenyltetrazolium bromide; AO-EtBr, acridine orange-ethidium bromide;
MTs, microtubules.
* Corresponding author.
** Corresponding author. Dipartimento di Scienze Chimiche, Biologiche, Farm-
Due to their selectivity towards tumor tissue in PDT, porphyrins
and porphyrin-related macrocycles are at the forefront of PDT [6].
Several compounds belonging to a first generation of PSs, such as
the hematoporphyrin derivative HpD (Photofrin) [1,5], are already
ꢁ
aceutiche ed Ambientali- ChiBioFarAm, Universita di Messina, Messina, Italy.
E-mail addresses: maria.ribagorda@uam.es (M. Ribagorda), abarattucci@unime.
it (A. Barattucci).
1
The first and second authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ejmech.2016.01.041
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

E. Deni et al. / European Journal of Medicinal Chemistry 111 (2016) 58e71
59
in clinical use or in clinical trials to treat cancer patients and are
carbohydrate metabolism (Warburg effect) [23].
2. Results and discussion
approved by the Food and Drug Administration for the palliative
treatment of obstructive lung and esophageal cancers. A second-
generation of PSs, with improved pharmacokinetics and reduced
skin photosensitivity, includes aminolevulinic acid (ALA; a pro-PS),
widely used for the treatment of non melanoma skin cancer
(NMSC), benzoporphyrin derivative (BPD), silicon phthalocyanine
(Pc4) or m-tetrahydroxyphenylchlorin (mTHPC) [1,2]. In the case of
PUVA, psoralens are excited by UVA light at suberythemogenic
doses and have been used to treat determined skin diseases,
including psoriasis, vitiligo and mycosis fungoides [7e9].
2.1. Chemistry
The target dimethylamino-derived OPE glucosides 1 and 2,
having one or two N,N-dimethylamino substituted aryl moieties
respectively, are depicted in Fig. 1.
The synthesis of compound 1 was completed following a strat-
egy previously reported by us [22], based on copper-free Sonoga-
shira type coupling to assemble the triple bonds to the adequately
substituted aromatic rings, in a convergent manner (Scheme 1).
Thus, compound 1 was prepared from the key intermediate N,N-
dimethyl-2,5-bis[(trimethylsilyl)ethynyl] aniline 4, which allowed
a double Pd (0) catalyzed cross-coupling reaction with iodo aryl
derivative 7, incorporating the 2,5-dimethoxy aryl substituents as
well as the acetylated glucoside methylene ethynyl moiety, leading
to the final carbon skeleton in one step. Precursor 4 was prepared in
55% isolated yield, using a Pd(0) cross-coupling reaction between
N,N-dimethyl-2,5-dibromoaniline 3 and an excess of ethynyl-
trimethylsilane, whereas compound 7 was efficiently formed by Pd
mediated cross-coupling of 1,4-diiodo-2,5-dimethoxybenzene 5
[24] and tetraacetylpropynyl glucoside 6 [25]. The use of 2 equiva-
lents of the diiododerivative 5 was essential to achieve a high yield
for the preparation of 7. Inspired by the work of Mori [26], and Naso
[27] we successfully achieved the cross-coupling reaction between
4 and 7 (2 equiv) in 61% yield using [Pd(PPh3)4] as catalyst in the
presence of Ag2O, which was known to act as activator facilitating
coupling between aromatic iodides and silane derivatives. Final
deprotection of acetylated glucoside moieties gave the desired
gluco-OPE 1.
Preparation of gluco-OPE 2 is based on a convergent strategy
that allows a double assembly of a gluco-2-propynyl aryl ethyny-
lene framework with the aryl central core 1,4-diiodo-2,5-
dimethoxy benzene 5 (Scheme 2). It is noteworthy to mention
that the Pd(0) mediated coupling reaction of 6 and 2,5-dibromo
aryl derivative 3, [28] in an appropriate molecular ratio (1:1),
exclusively led to the mono substituted regioisomer 8, [29]
resulting from reaction on the less sterically hindered 5-bromo
substituent situated meta to the bulky dimethylamino group of 3,
in a 60% yield. The palladium catalyzed cross-coupling reaction
between 8 and an excess of commercially available trimethylsily-
lacetylene gave rise to the key intermediate 9 in high yield under
very mild conditions. The final Ag2O-modified Pd(0) mediated
coupling [26,27] between 9 (2 equiv) and 1,4-diiodo-2,5-dimethoxy
benzene 5 allowed a double assembly that directly lead to the
desired carbon skeleton of OPE 10. All the newly synthesized in-
termediates 8, 9 and 10 could be purified by conventional flash
column chromatography and are stable and easy to handle. Finally,
quantitative deacetylation of 10 in the presence of an excess of
aqueous ammonia gave the gluco-OPE 2 as a brilliant yellow solid,
easily separated from residual acetamide by subsequent washings
with MeOH, in almost quantitative yield.
Photosensitizers based on amphiphilic skeletons, bearing lipo-
philic and hydrophilic moieties, are in continuous development
due to their biological applications. For instance, symmetric and
asymmetrically substituted phthalocyanines bearing hydrophilic D-
galactose units have shown to be efficient PSs towards HeLa cells
[10], while glucose-appended Iron(III) complexes caused HeLa cells
apoptosis by the generation of ROS on irradiation [11].
The ideal photosensitizers [12] should possess high photo-
activity and selective photocytotoxicity for sick skin cells but low
dark toxicity, activation at discrete wavelengths, efficient and fast
distribution and elimination from tissues and chemical and phys-
ical stability. Although a significant number of PS are synthesized
each year, the development of new targets that fulfill all the re-
quirements continues to be an appealing area of research in pho-
totherapy [13]. Oligo(phenylene-ethynylene)s (OPEs) are
luminescent linear oligomers with extended conjugated aromatic
and ethynylenic moieties. Due to their intriguing optical and elec-
tronic properties [14], OPEs have lately gained a prominent role in
the design of organic materials such as sensing or electronic organic
devices. On the other hand, there are only few examples of their use
in biological fields. For instance, OPEs incorporating cationic ends
have been shown to behave as light-activated biocides, killing some
specific kind of bacteria, as a result of the production of singlet
oxygen after irradiation [15]. Galactose-functionalized oligomers
have been shown to act as inhibitors of Pseudomonas aeruginosa
lectin LecA [16]. N-Hydroxy succinimidoyl esters incorporated to
OPEs were reported to behave as luminescent labeling probes for
proteins [17]. Aryleneethynylene compound bearing two polar
sulfonate groups has been reported as effective fluorescent markers
for liposomes and mammalian cell membranes [18]. In spite of the
encouraging electronic properties displayed by the OPEs, to our
knowledge, there are no precedents of their use as photosensitizers
in photodynamic therapy.
Following our biological studies of carbohydrate based conju-
gated systems [19e21], we recently prepared several end-glucose
functionalized OPEs and reported their ability to permeate the
cellular cancer membrane and to localize in cytoplasmic organelles
[22]. Few structural features were found to be crucial to reach an
efficient cytoplasmatic OPE dye: three aryl ethenyl conjugated
units, two hydrophilic glucose molecules at both ends of the OPE
skeleton and, a dimethylamino substituent at the central aryl ring
of the conjugated system (see compound 1 in Fig. 1). Preliminary
biological evaluation tests demonstrated that these OPE derivatives
were non-cytotoxic. Taking into account these precedents, we
decided to study the behavior of two OPE derivatives, as bio-
compatible photosensitizers targets in PDT (Fig. 1). Herein we
report full details of the preparation of end-glucose OPE derivative
1 and a new derivative 2, the photophysical studies and in vitro
toxicity, either in the absence of light or under light irradiation,
against HeLa and HEp-2 tumor cell lines. The presence of hydro-
phobic (aryl conjugated fragments) and hydrophilic (glucose)
moieties in these structures was expected to facilitate cellular
membrane permeation. Moreover, the terminal glucose molecules
were chosen considering the enhanced glycolytic process of cancer
2.2. Photophysical properties
The absorption and photophysical data of the final OPE gluco-
sides 1 and 2 in aqueous solution are summarized in Table 1. For
comparison purposes, the data of all 2,5-dimethoxy aryl ring gluco-
OPE 11 [22] (Fig. 2), previously studied by us are also included.
The absorption spectra of the OPEs 1 and 2, in aqueous solution
(pH ¼ 7.2, buffer phosphate, see Fig. 3), are characterized by intense
broad low-energy absorption bands centered at 388 nm and at
392 nm, respectively with ε values in the range 104e105 Mꢀ1 cmꢀ1
.

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E. Deni et al. / European Journal of Medicinal Chemistry 111 (2016) 58e71
Fig. 1. Structure of gluco-OPEs 1 and 2.
Scheme 1. Synthesis of gluco-OPE 1.
According with literature [30], these absorptions in the UV region
are due to spin-allowed * transitions and are attributed to
evaluation of singlet oxygen production by excitation of the oligo-
mers 1, 2 and 11. The singlet-oxygen generation was evaluated by a
chemical method using uric acid (UA) as detector and the methy-
lene blue species as a reference photosensitizer [31,32]. Under
irradiation, at neutral pH, uric acid is stable and shows one ab-
sorption band centered at 292 nm. When singlet oxygen (or ROS) is
photo-produced, uric acid is irreversibly oxidized as highlighted by
the reduced intensity of its absorption band. The decay curves of
the UA absorption band (lmax 292 nm) as a function of the irradi-
ation time in the presence of 1, 2 and the model 11 are reported in
Fig. 4. A strong decay was observed upon irradiation of the uric acid
solution in the presence of OPE-1 at 380 nm, and a moderate decay
was observed in the presence of OPE-2 upon irradiation at 390 nm.
On the contrary, the UA absorption band remained unchanged
upon irradiation at 380 nm in the presence of OPE-11. These pho-
tophysical results are in agreement with the singlet oxygen quan-
tum yields previously obtained for 1 (F1O2 0.15) and 2 (F1O2 0.09)
pep
Internal Change Transfer (ICT) transitions. The excitation of com-
pounds 1 and 2 in the range of 280e420 nm allowed the appear-
ance of emission bands in the blue region of spectrum.
Interestingly, the results presented in Table 1 revealed clear dif-
ferences between the photophysical properties of 1 and 2. In
particular, moving from 1 to 2, the emission quantum yield
increased, from
state lifetime, from
both quantum yields were reduced with respect to the analogous
model compound 11 (
F
¼ 0.57 (1) to
F
¼ 0.74 (2), as well as the excited
t
/ns ¼ 2.47 (1) to
t
/ns 3.54 (2). Remarkably,
F
¼ 0.85) in which no dimethylamino group
is present. This behavior could suggest that the excited singlet
states decay faster in 1 because, in this case, the rate of inter-system
crossing to the triplet excited state (responsible for sensing singlet
oxygen) is enhanced with respect to 2 and 11.
On the basis of this assumption, we performed an indirect

E. Deni et al. / European Journal of Medicinal Chemistry 111 (2016) 58e71
61
Scheme 2. Synthesis of gluco-OPE 2.
indicated that signal emitted by treated HaCaT, HeLa and HEp-
2 cells was due to the intracellular accumulation of the PSs, con-
firming the cellular uptakes of both compounds. In order to
determine the places where PSs were located, cells were incubated
with known markers for endoplasmic reticulum (ER-Tracker®) and
Golgi apparatus (NBD®) (Fig. 5B). Comparison of the images ob-
tained after incubation with 1 and 2 with those obtained with the
known probes for specific organelles, confirmed that compounds 1
and 2 are localized mainly in Golgi apparatus and endoplasmic
reticulum. It is important to indicate that no differences were found
in the intracellular localization of OPE-1 or 2 in the three cell types.
Table 1
Spectroscopic and photophysical data of 1, 2 and 11 in buffered aqueous solution.a
OPEs
Absorption
Luminescence at 298 K
b
lmax nm (ε/Mꢀ1 cmꢀ1
)
lmax nm
F
t
/ns
F
1O2
1
388 (38900)
392 (40000)
390 (49500)
472
474
433c
0.57
0.74
0.85c
2.47
3.54
0.98c
0.15
0.09
e
2
11c
a
Buffer phosphate aqueous solution, pH ¼ 7.2.
b
For the absorption, the maxima or shoulders of the lower energy bands are
given.
c
Data from Ref. [22].
2.4. Effects induced by 1 and 2 e PDT on cell survival
(see Table 1), confirming that the reduced luminescence quantum
yield for 1 could be ascribed to a more efficient sensitization of the
triplet excited state of these species.
The action of a PS on cells is related to the production of 1O2 and/
or other ROS generated after light irradiation [1,15,35]. Therefore,
we next evaluated the toxicity of compounds 1 and 2 first in the
absence of light and then after UV irradiation. The toxicity experi-
ments were performed by incubation of HaCaT, HeLa or HEp-2 cells
with 1 or 2 (ranging from 3$10ꢀ6 to 2.5$10ꢀ5 M) in the dark. In the
absence of light the toxicity for both OPEs 1 and 2, was dependent
on the PS concentration (Table 2). Thus, concentrations of 3$10ꢀ6
and 5$10ꢀ6 M of both PSs did not induce any relevant toxic effect
(survival rates above 90% in all cell lines), and higher PS concen-
trations (1$10ꢀ5 and 2.5$10ꢀ5 M) caused a variable cell damage
depending on the cell type (survival percentages between 60 and
85%). The dark toxicity levels obtained for both OPEs 1 and 2 are
similar to other reported PSs tested in vitro, including porphyrin
derivatives and phtalocyanines [36,37].
2.3. Biological study. Subcellular localization of PSs
The efficiency of a photosensitizer for PDT is related to its sub-
cellular localization [33,34]. Therefore, we first evaluated the cell
internalization and localization of OPE compounds 1 and 2. The
subcellular localization of 1 and 2 in HaCaT, HeLa and HEp-2 cells
was analyzed by fluorescence microscopy under UV excitation light
(360e370 nm) (Fig. 5A). Similar results were observed under blue
light excitation (460e490 nm) (Fig. S15, ESI). The intracellular
luminescent signals of 1 and 2 were observed after 6 h of incuba-
tion with 1$10ꢀ6 M of both compounds. Corresponding controls
(without PSs incubation) of each cell type were also performed. In
the three cell lines, after the incubation with PSs, an intense signal
was visualized around the nucleus in a reticular pattern, as well as
in a yuxtanuclear position. By contrast, control cells showed a very
low signal, corresponding to mitochondrial autofluorescence. This
We next evaluated the effect of UVA irradiation on the cell
cultures, first without the presence of the OPEs. As can be seen in
Table 3, none of the tested doses (ranging from 0.25 to 2 J/cm2)
induced substantial cytotoxic effects, the survival rates of HaCaT,
HeLa and HEp-2 after UVA light irradiation (2 J/cm2) were 97.2%,

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E. Deni et al. / European Journal of Medicinal Chemistry 111 (2016) 58e71
Fig. 2. Structure of gluco-OPEs 1, 2 and 11.
3$10ꢀ6 and 5$10ꢀ6 M of both compounds and 0.25, 0.5 and 1 J/cm2
of UVA light to evaluate the synergic effect of the PSs compounds 1
and 2 plus UVA irradiation on the three cell lines. The photody-
namic treatments, carried out by incubation with OPE-1 or OPE-2
for 6 h followed by UVA exposure, induced cytotoxic effects in
HaCaT, HeLa and HEp-2, depending on the PS concentration and the
UVA dose. Cell survival rates after PDT (Fig. 6A), measured by MTT
assay, were significantly decreased (P < 0.01) in all the cell lines
with respect to untreated controls. Viability after PDT were similar
for both PSs, since no significant differences between 1 and 2 were
found. However, HeLa cells tended to be slightly more sensitive to
PDT than HaCaT and HEp-2. The LD50 dose (treatment conditions
that induced approximately a 50% of cell death) was reached with
concentrations of 3$10ꢀ6 of 1 or 2 followed by 0.25 J/cm2 of UVA
radiation. The other tested conditions caused a lethality rate greater
than 50%. In the case of 1, values lower than 10% of cell survival
(LD90) were obtained after cell treatment with 2.5$10ꢀ5 M and 2 J/
cm2 of UVA light (Fig. S16, ESI); similar results were found for 2. All
these findings indicated that compounds 1 and 2 were not toxic for
the cells in the absence of light at concentrations lower than
5$10ꢀ6 M, confirming that the cytotoxic effect when UVA light was
applied was due to the production of reactive oxygen species. These
cytotoxic values are comparable with other PSs used in PDT under
UVA light irradiation, such as curcumin [38], hypericin or psoralen
[39]. Both PSs reached a good photodynamic effect when combined
with UVA light, but we did not observe selectively for tumor HeLa
and HEp-2 cells compared to non-tumor HaCaT cell line.
Fig. 3. Absorption (solid line) and emission (dotted line) spectra of 1 (green), 2 (red)
and 11 (blue) in aqueous phosphate buffered solution, pH ¼ 7.2. (For interpretation of
the references to colour in this figure legend, the reader is referred to the web version
of this article.)
The cell morphology was also analyzed 24 h after phototreat-
ments, using phase contrast microscopy (Fig. 6B). This analysis
revealed that 1 and 2-based PDT induced notable changes in the
morphology of HaCaT, HeLa and HEp-2 cells. These alterations were
dependent on the treatment conditions (PS concentration and UVA
light dose) and did not differ when 1 or 2 were used. Under a LD50
conditions, HaCaT cells showed cytoplasmic retraction and cell
elongation, as well as the appearance of apoptotic (with membrane
blebbing) and necrotic (with loss of membrane integrity) figures.
The tumoral cell lines (HeLa and HEp-2) also suffered retraction of
their cytoplasm, cellular stretching and a strongly cytoplasmic
vacuolization, which is indicative of cell degeneration processes. In
addition, we noticed the apparition of a large amount of rounded
Fig. 4. Degradation of uric acid (lmax ¼ 292 nm) versus Time using as sensitizer OPE-1
(green squares, 5$10ꢀ5 M) irradiated at 380 nm, OPE-2 (red squares, 5$10ꢀ5 M) irra-
diated at 390 nm and the model OPE-11 (blue squares, 5$10ꢀ5 M) irradiated at 380 nm.
(For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
94.0% and 95.1%, respectively.
According to the above results we selected concentrations of

E. Deni et al. / European Journal of Medicinal Chemistry 111 (2016) 58e71
63
Fig. 5. (A) Subcellular localization of OPE-1 and OPE-2 in HaCaT, HeLa and Hep-2 cells. After 6 h of incubation with 1$10ꢀ6 M of PSs, a blue fluorescence was observed under UV
excitation light. This fluorescence had a reticular distribution and also appeared in a yuxtanuclear position. (B) Intracellular localization of ER and Golgi apparatus in HaCaT, HeLa
and Hep-2 cells. Cells were incubated with known markers for endoplasmic reticulum (ER-Tracker) and Golgi apparatus (NBD-Golgi). ER showed a blue fluorescent signal under UV
excitation light (
l
¼ 360e370 nm) and Golgi apparatus emitted a green fluorescence under blue excitation light (
l
¼ 460e490 nm). Scale bar: 20
mm. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of this article.)
Table 2
Dark toxicity: Cell survival percentage after incubation with compounds 1 and 2 in the absence of light.a
[PS]
HaCaT
1
HeLa
1
HEp-2
1
2
2
2
3$10ꢀ6
5$10ꢀ6
1$10ꢀ5
M
M
M
92.36 1.6
91.41 2.4
71.55 3.2
61.18 1.8
91.45 1.3
90.93 2.0
70.23 2.7
59.72 2.8
96.38 1.6
95.43 1.7
82.21 2.5
77.76 2.7
96.61 2.9
93.52 2.2
76.89 3.0
66.65 3.1
97.22 1.4
97.73 1.0
95.72 2.4
80.07 2.8
70.19 1.2
96.28 2.0
85.63 2.9
81.49 1.9
2.5$10ꢀ5
M
a
Cells were incubated with 3$10ꢀ6, 5$10ꢀ6, 1$10ꢀ5 and 2.5$10ꢀ5 M of 1 or 2 for 6 h in the dark. Cell survival was evaluated after 24 h by MTT assay.
cells, indicating a possible mitotic blockage, as well as a large
number of cells detached from the substrate, floating on culture
medium. All the changes observed confirm the results obtained
from cell viability assays.
When the treatment conditions induced a 90% of cell death
(LD90), deep deformations of cell membrane were seen with both
PSs and in the three types of cells analyzed (Fig. S16, ESI).
Nuclear morphology was also evaluated 24 h after PDT by
€
Hoechst-33258 staining and fluorescence microscopy (Fig. 7AeB).
A nuclear retraction was observed in the three cell lines when
subjected to LD50 dose of both phototreatments, in comparison to
untreated cells, as well as the appearance of apoptotic and necrotic
morphologies. Furthermore, a great amount of mitotic nuclei was
observed in HeLa and HEp-2 cell lines, most of them abnormal

64
E. Deni et al. / European Journal of Medicinal Chemistry 111 (2016) 58e71
Table 3
mitotic cells with abnormal metaphase plates was also calculated
Cell survival percentage after UVA light irradiation.a
(Fig. 9B). In the same way, PDT of HeLa and HEp-2 cells subjected to
LD50 of 1 or 2 presented a significantly higher percentage of altered
metaphases in comparison with untreated cells, while photo-
treatments did not induce aberrant metaphases in HaCaT cells.
These findings suggest that MTs could be a target of photo-
treatments with compounds 1 and 2 plus UVA light, which cause
the disorganization and collapse of MTs network and, in the case of
tumoral cell lines, also the incorrect functioning of mitotic appa-
ratus and thus the appearance of aberrant metaphases.
UVA dose
HaCaT
HeLa
HEp-2
0.25 J/cm2
0.5 J/cm2
1 J/cm2
98.76 1.0
98.33 1.1
97.92 2.1
97.2 1.2
99.01 0.9
97.50 2.0
96.79 1.7
94.0 2.3
98.11 1.8
98.42 1.3
97.33 1.5
95.2 1.6
2 J/cm2
a
Cells were exposed to UVA light at the indicated doses (0.25, 0.5, 1 and 2 J/cm
[2]). Cell survival was evaluated 24 h after irradiation by MTT assay.
metaphases.
3. Conclusions
2.5. Acridine orange/ethidium bromide staining
In summary, we have prepared two dimethylamino (NMe2) aryl
substituted OPE glucosides 1 and 2, by an efficient Pd(0) catalyzed
cross-coupling reaction in the presence of Ag2O, as a key step. OPE
glucosides 1 and 2 have been studied from the photophysical and
biological point of view, and have been tested as photosensitizers in
photodynamic therapy. These compounds have shown to be highly
The cytotoxicity of LD50 of 1 and 2 in PDT was also evaluated
in vivo using acridine orange/ethidium bromide staining (AO/EtBr)
(Fig. 8). This method allowed us to distinguish between viable,
rounded, apoptotic and necrotic cells (Fig. S17, ESI). As shown in
Fig. 8A, the PDT induced evident alterations in the morphology of
HaCaT, HeLa and HEp-2 cells, in comparison with their control
counterparts. These morphological changes were similar for both
PSs. After PDT, HaCaT cultures showed an increase of apoptotic and
necrotic cells. In HeLa and HEp-2 cultures, besides apoptotic and
necrotic figures, a large number of rounded cells densely stained as
green, were observed. In order to determine the exact percentage of
each cellular condition, a cell counting was performed. The per-
centage of cells in each established condition was similar for 1
(Fig. 8B) and 2 (Fig. 8C) phototreatments. HaCaT cells showed a
significant higher proportion of apoptotic and necrotic figures in
comparison with tumoral cell lines. By contrast, in the case of HeLa
and HEp-2, cell counting revealed a significant increase (P < 0.01) of
rounded cells as compared to HaCaT and to its control counterparts.
All of these results suggest that photodynamic treatment could be
triggering a mitotic blockage in tumoral cell lines, while in HaCaT
mainly induce apoptotic processes.
luminescent and bio-compatible.
A good dark toxicity were
measured for OPE-1 and 2 in the range of known PSs such as
porphyrin and phatalocyanine derivatives. Compounds 1 and 2 are
susceptible to be excited by UVA light and produced a high
photodynamic effect causing tumoral cell death even with low
singlet oxygen production. This photodynamic effect seems to
induce damage in the MTs network of tumoral cells, triggering a
blockage in the metaphase of cell cycle, which can lead to cell
death. The glucose-ends OPE tested have been able to produce
singlet oxygen with moderated efficiencies. The study presented
here provides the first use of OPEs as photosensitizers and could be
a starting point to extend the application of these luminescent
oligomers in photodynamic therapy.
4. Experimental section
4.1. General synthetic methods
2.6. Cytoskeleton of microtubules as a target for 1 or 2-PDT
Solvents were purified according to standard procedures. All of
the reactions were monitored by TLC on commercially available
precoated plates (silica gel 60 F254), and the products were visu-
alized with vanillin [1 g dissolved in MeOH (60 mL) and conc. H2SO4
(0.6 mL)] and UV lamp. Silica gel 60 was used for column chro-
matography. Proton (1H) and carbon (13C) NMR spectra were
recorded on a Varian 500 spectrometer (at 500 MHz for 1H; and
125 MHz for 13C) or a AV-300 Bruker (at 300 MHz for 1H; and
75 MHz for 13C) using CDCl3 as solvent, unless differently stated.
To get insight into the mechanisms by which PDT using 1 or 2 as
PSs reach their effects on HaCaT, HeLa and HEp-2 cells, we also
evaluated the changes induced by PDT in the cytoskeleton of mi-
crotubules (MTs) (Fig. 9). MTs are cytoskeletal polymers essential
for cell structure, cell division and intracellular transport and their
role in tumor progression makes them a key target for anti-cancer
therapies, including PDT [12,40,41]. As shown in Fig. 9A, the
interphasic HaCaT, HeLa and HEp-2 cells showed a well-developed
MT network and no alterations were observed in the mitotic
spindle of dividing cells. However, cells showed a clear increase in
microtubular changes 24 h after phototreatments with LD50 doses.
Most HaCaT cells retracted their cytoplasm and thus showed an
altered and disorganized MT network. Moreover, apoptotic mor-
phologies were also observed. With respect to HeLa and HEp-
2 cells, in addition to the described alterations, there was also an
increase in the number of dividing cells and many of them showed
modifications of the spindle apparatus, such as the presence of
extrapoles (three and four-poles spindles) and the consequent ab-
normalities in the distribution of chromosomes in the metaphase
plate.
Chemical shifts are given in parts per million (ppm) (d relative to
residual solvent peak for 1H and 13C), coupling constants (J) are
given in hertz, and the attributions are supported by heteronuclear
single-quantum coherence (HSQC), Heteronuclear Multiple Bond
Correlation (HMBC) and correlation spectroscopy (COSY) experi-
ments. Chemical shifts are reported in ppm relative to CDCl3 (d
7.26 ppm). For simplification purpose, assigned 1H and 13C NMR
signals follow the numeration depicted in Scheme 2 (see also ESI).
Mass spectra (m/z) and HRMS were recorded under the conditions
of electron impact (EI) and electrospray (ES) in a Waters VG Auto-
spect spectrometer. Melting points were obtained in open capillary
tubes and are uncorrected. Tested compounds 1 and 2 were ꢁ95%
pure as determined by qNMR analysis (see ESI), on Varian 500
spectrometer, according to the quantification protocol described by
Henderson [42].
In order to determine the percentage of cells in possible mitotic
blockage, mitotic index was quantified 24 h after 1 and 2 photo-
treatments (Fig. 9B). As shown in Fig. 9, after 1 and 2-PDT in LD50
conditions, mitotic index of HeLa and HEp-2 was significantly
higher as compared to untreated (control) cells. By contrast, mitotic
index of HaCaT cells did not change after PDT. The percentage of
4.1.1. 3-[4-Bromo-3-(dimethylamino)phenyl]-2-propyn-1-yl-b-D-
glucopyranoside-2,3,4,6-tetraacetate (8)
To a flask were added Pd(PPh3)4 (0.45 g, 0.39 mmol, 0.15 equiv),

E. Deni et al. / European Journal of Medicinal Chemistry 111 (2016) 58e71
65
A
HaCaT
HeLa
HEp-2
I
120
100
80
60
40
20
0
120
100
80
60
40
20
0
120
100
80
60
40
20
0
***
***
***
***
***
***
***
***
***
***
***
***
***
***
***
***
***
***
C
0.25
0.5
1
C
0.25
0.5
1
C
0.25
0.5
1
II
120
100
80
60
40
20
0
120
100
80
60
40
20
0
120
100
80
60
40
20
0
***
***
***
***
***
***
***
***
***
***
***
***
***
***
***
***
***
***
C
0.25
0.5
1
C
0.25
0.5
1
C
0.25
0.5
1
+ UVA (J/cm2)
+ UVA (J/cm2)
+ UVA (J/cm2)
C (control, untreated cells)
1
2
B
HaCaT
HeLa
HEp-2
1
2
Fig. 6. Cell survival rates (A) and morphological changes (B) after OPE-1 and OPE-2 based PDT. I: 3 ꢂ 10ꢀ6 M of PSs. II: 5 ꢂ 10ꢀ6 M of PSs. (A) Both 1 and 2-PDT, in all of the treatment
conditions applied, caused a significant decrease in HaCaT, HeLa and HEp-2 survival with respect to untreated controls (P < 0.01). No significant differences were found between 1

66
E. Deni et al. / European Journal of Medicinal Chemistry 111 (2016) 58e71
€
Fig. 7. Effect of phototreatments in the nuclear morphology of HaCat, HeLa and Hep-2 cells. Nuclei morphology was observed 24 h after PDT using Hoechst-33258 staining and
fluorescence microscopy (A). The controls (untreated cells) of the three cell lines displayed well defined interphasic and mitotic (*) nuclei. Nevertheless, when treated with OPE-1 or
OPE-2 PDT (LD50 dose), cultures showed an increment of apoptotic and necrotic nuclei. Also, in HeLa and HEp-2 cultures an increase of mitotic cells was observed. (B) Details of
normal (a) and altered (b) mitotic figures, as well as apoptotic (c) and necrotic (d) cells. Morphologies corresponding to abnormal mitosis, apoptosis and necrosis were found in
treated cell cultures, both in cells attached to the substrate and cells detached from it.
6 (1.00 g, 2.59 mmol, 1 equiv) and 3 (1.45 g, 5.18 mmol, 2 equiv),
capped with a rubber septum and evacuated. After backfilling with
N2, this process was repeated three times. To the flask were added
dry DMF (20 mL) and Et3N (20 mL) at room temperature. The re-
action mixture was heated at 65 ꢃC, and maintained under
continuous stirring for 4 h, until the disappearance of compound 6
by TLC (Hexane/EtOAc 70:30). Solvents were removed under
reduced pressure and the solid residue was dissolved in CH2Cl2 ad
filtered on celite. The volatiles were removed in vacuo and the re-
action crude was purified by flash chromatography on silica gel,
using Hexane/EtOAc (90:10) as eluant, giving compound 8 as a pale
12H, 4 ꢂ CH3CO). 13C NMR:
d
170.6, 170.2 and 169.4 (4 ꢂ CO), 151.9
(C-3), 134.0 (C-5), 126.8 and 123.6 (C-2 and C-6), 121.9 and 119.9 (C-
1 and C-4), 98.4 (C-10), 86.3 and 83.9 (C^C), 72.7 (C-30), 71.8 (C-50),
71.1 (C-20), 68.2 (C-40), 61.8 (C-60), 56.8 (CH2C≡), 44.0 ([N(CH3)2]),
20.6 and 20.5 (4 ꢂ CH3CO). Calc. for C25H30BrNO10 583.11; found
positive ESI-MS: [M-Hþ ] ¼ 584.1114.
4.1.2. 3-[3-(Dimethylamino)-4-[2-(trimethylsilyl)ethynyl]phenyl]-
2-propyn-1-yl-b-D-glucopyranoside-2,3,4,6-tetraacetate (9)
To a flask were added Pd(PPh3)4 (0.27 g, 0.23 mmol, 0.15 equiv),
8 (0.91 g, 1.55 mmol, 1 equiv) and ethynyltrimethylsilane (1.31 mL,
9.3 mmol, 6 equiv), capped with a rubber septum and evacuated.
After backfilling with N2, this process was repeated three times. To
the flask were added dry DMF (11 mL) and Et3N (11 mL) at room
temperature. The mixture was heated at 65 ꢃC, and maintained
under continuous stirring for 24 h, until the disappearance of
compound 8 by TLC (Hexane/EtOAc 70:30). Solvents were removed
in vacuo and the solid residue was dissolved in CH2Cl2 ad filtered on
yellow oil (0.91 g, 1.55 mmol, 60%). TLC: Rf 0.60. 1H NMR:
d 7.48 (d,
J5,6 ¼ 8.1, 1H, H-5), 7.11 (d, J2,6 ¼ 1.0, 1H, H-2), 6.93 (dd, J5,6 ¼ 8.1,
J2,6 ¼ 1.0, 1H, H-6), 5.25 (t, J2 ,3 ¼ J3 ,4 ¼ 9.3, 1H, H-30), 5.10 (t,
0
0
0
0
J3 ,4 ¼ J4 ,5’ ¼ 9.3, 1H, H-40), 5.02 (bdd, J1 ,2 ¼ 8.3 J2 ,3 ¼ 9.3, 1H, H-2 ),
0
0
0
0
0
0
0
0
0
0
0
4.80 (d, J1 ,2 ¼ 8.3, 1H, H-1 ), 4.56 (s, 2H, CH2C≡), 4.27 and 4.14 (split
0
0
0
0
0
0
0
AB system, J5 ,6A ¼ 4.2, J5 ,6B ¼ 1.8, J6A ,6B ¼ 12.6, 2H, H2-6 ), 3.79 (m,
1H, H-50), 2.78 (s, 6H, [N(CH3)2]), 2.06, 2.03, 2.01 and 1.99 (four s,
and 2-based PDT. (B) Images correspond to cells treated with 3$10ꢀ6 M of 1 or 2 followed by 0.25 J/cm2 of UVA light (LD50 dose). PDT induced visible alterations in cell morphology.
In the case of HaCaT cells, PDT using OPE-1 and OPE-2 triggered the appearance of apoptotic/necrotic cells. In HeLa and HEp-2 cells, in addition to apoptotic/necrotic figures, both
treatments induced a large number of cells with rounded morphology (potential mitotic blockage) and cells detached from substrate. Scale bar: 30 mm.

E. Deni et al. / European Journal of Medicinal Chemistry 111 (2016) 58e71
67
A
HaCaT
HeLa
HEp-2
*
B
C
Rounded
Apoptotic
Necrotic
100
90
80
70
60
50
40
30
20
10
0
100
90
80
70
60
50
40
30
20
10
0
10
9
8
7
6
5
4
3
2
1
0
***
***
***
***
***
***
**
***
***
***
***
HaCaT
HeLa
Hep-2
HaCaT
HeLa
Hep-2
HaCaT
HeLa
Hep-2
Rounded
Apoptotic
Necrotic
100
90
80
70
60
50
40
30
20
10
0
10
9
8
7
6
5
4
3
2
1
0
100
90
80
70
60
50
40
30
20
10
0
***
***
***
***
*
***
***
**
***
***
***
***
HaCaT
HeLa
Hep-2
HaCaT
HeLa
Hep-2
HaCaT
HeLa
Hep-2
0,5 J/cm2
0,25 J/cm2
Fig. 8. AO/EtBr staining. A: HaCaT, HeLa and HEp-2 cells were in vivo stained with AO/EtBr 24 h after PDT with 1 and 2 in LD50 conditions. While untreated cells (control) showed a
normal morphology, cells subjected to PDT presented morphological alterations, similar for both PSs. In the case of HaCaT cells, the treatments induced apoptotic (*) and necrotic
figures (arrow). In the tumoral cells (HeLa and HEp-2), in addition to apoptotic a necrotic cells, PDT triggered the appearance of a great number of cells with rounded morphology
(arrowhead) (potential mitotic blockage). Scale bar: 20 mm. BeC: Graphs shows the percentages of rounded, apoptotic and necrotic cells 24 h after LD50 conditions of 1 (B) and 2 (C)
mediated PDT. The number of cells with rounded morphology (possible mitotic arrest) were significantly increased in tumoral cells (HeLa and HEp-2) with respect to HaCaT cells. By
contrast, the amount of apoptotic and necrotic morphologies was significantly higher in HaCat than in HeLa and HEp-2 cells. *: significant P < 0.1; **: significant P < 0.05; ***:
significant P < 0.01.

68
E. Deni et al. / European Journal of Medicinal Chemistry 111 (2016) 58e71
Fig. 9. Effects of OPE-1 or OPE-2 based PDT in microtubules from cytoskeleton (LD50 conditions). A: Microtubules from cytoskeleton of HaCaT, HeLa and HEp-2 cells were analyzed
by immunofluorescence of -tubulin 24 h after 1 and 2-PDT. Untreated (control) cells showed a well-developed MT network, while MTs of cells subjected to PDT appeared
disorganized. In addition, the increased number of dividing cells in tumoral lines presented alterations in the mitotic apparatus and abnormalities in the distribution of chro-
mosomes in the metaphase plate (*). Scale bar: 10 m. B: Mitotic index and altered metaphases after 1 and 2-based PDT.
a
m
0
0
0
0
0
0
celite. The volatiles were removed under vacuum and the crude
purified by flash chromatography on silica gel using Hexane/EtOAc
(90:10) as eluant to give compound 9 as a yellow oil (0.77 g,
5.24 (t, J2 ,3 ¼ J3 ,4 ¼ 9.3, 1H, H-30), 5.09 (t, J3 ,4’ ¼ J4 ,5’ ¼ 9.3, 1H, H-
40), 5.01 (bdd, J1 ,2 ¼ 8.1 J2 ,3 ¼ 9.3, 1H, H-20), 4.82 (d, J1 ,2 ¼ 8.1, 1H,
0
0
0
0
0
0
H-10), 4.57 (s, 2H, CH2C≡), 4.27 and 4.15 (split AB system,
1.27 mmol, 82%). TLC: Rf 0.55. 1H NMR:
d
7.31 (d, J5,6 ¼ 7.8, 1H, H-5),
J5 ,6A ¼ 4.5, J5 ,6B ¼ 2.1, J6A ,6B ¼ 12.3, 2H, H2-60), 3.74 (m, 1H, H-50),
2.94 (s, 6H, [N(CH3)2]), 2.06, 2.03, 2.01 and 1.99 (four s, 12H, 4 ꢂ
0
0
0
0
0
0
6.86 (d, J2,6 ¼ 1.2, 1H, H-2), 6.87 (dd, J5,6 ¼ 7.8, J2,6 ¼ 1.2, 1H, H-6),

E. Deni et al. / European Journal of Medicinal Chemistry 111 (2016) 58e71
69
CH3CO), 0.24 (s, 9H, [Si(CH3)3]). 13C NMR:
d
170.6, 170.2, 169.4 and
4.2. Singlet oxygen evaluation
169.3 (4 ꢂ CO), 154.8 (C-3), 134.8 (C-5), 123.2 and 119.8 (C-2 and C-
6), 122.8 and 116.2 (C-1 and C-4), 104.0, 101.7, 87.2 and 84.4 (C^C),
98.4 (C-10), 72.8 (C-30), 71.9 (C-50), 71.1 (C-20),68.3 (C-40), 61.8 (C-60),
56.9 (CH2C≡), 43.1 [N(CH3)2], 21.0, 20.7 and 20.5 (4 ꢂ CH3CO), 0.2
([Si(CH3)3]). Calc. for C30H39NO10Si 601.23; found positive ESI-MS:
[M-Hþ] ¼ 602.2422.
A direct comparison between the oligomers and Methylene blue
species in solution by monitoring the UA photooxidation is not
possible, because different excitation wavelengths were used for
the photoproduction of 1O2. Therefore, the efficiency of singlet-
oxygen deliver was calculated by normalizing for the photon flux
of the lamp at the lambda used for excitation (400 nm for OPEs and
600 nm for the methylene blue).
4.1.3. (2,5-Dimethoxy-1,4-phenylene)bis[2,1-ethynediyl[2-
(dimethylamino)-4,1-phenylene]-2-propyne-3,1-diyl]-bis-
b
-
D
-
The absorption spectra were recorded in ultrapure spectro-
scopic solvents. UV/Vis absorption spectra were taken on a Jasco V-
560 spectrophotometer. For steady-state luminescence measure-
ments, a Jobin Yvon-Spex Fluoromax 2 spectrofluorimeter was
used, equipped with a Hamamatsu R3896 photomultiplier. The
spectra were corrected for photomultiplier response using software
purchased with the fluorimeter. For the luminescence lifetimes, an
Edinburgh OB 900 time-correlated single-photon-counting spec-
trometer was used. As excitation sources, a Hamamatsu PLP 2 laser
diode (59 ps pulse width at 408 nm) and/or the nitrogen discharge
(pulse width 2 ns at 337 nm) were employed. Emission quantum
yields for acetonitrile deaerated solutions were determined using
the optically diluted method [43]. As luminescence quantum yield
standards we used an air equilibrated ethanol solution of anthra-
cene (0.2) [44]. Experimental uncertainties on the absorption and
photophysical data are as follows: absorption maxima, 2 nm; molar
absorption, 15%; luminescence maxima, 4 nm; luminescence life-
times, 10%; luminescence quantum yields, 20%.
glucopyranoside-2,20,3,30,4,40,6,60-octaacetate (10)
To a flask were added Pd(PPh3)4 (0.11 g, 0.09 mmol, 0.15 equiv),
Ag2O (0.30 g, 1.28 mmol, 2 equiv), 9 (0.77 g, 1.28 mmol, 2 equiv) and
1,4-diiodo-2,5-dimethoxybenzene 5 (0.25 g, 0.64 mmol, 1 equiv),
capped with a rubber septum and evacuated. After backfilling with
N2, this process was repeated three times. To the flask were added
dry DMF (10 mL) and dry THF (5 mL). The reaction mixture was
heated at 70 ꢃC for 7 h, until the disappearance of compound 9 by
TLC (Hexane/EtOAc 50:50). Solvents were removed in vacuo and the
solid residue was dissolved in CH2Cl2 ad filtered on celite. The
volatiles were removed under reduced pression and the crude
purified by column chromatography on silica gel using Hexane/
EtOAc (60:40) as eluant to give compound 10 as a brilliant yellow
oil (0.43 g, 0.36 mmol, 57%). TLC: Rf 0.35. 1H NMR:
d 7.45 (d,
0
0
0
0
0
J5 ,6 ¼ 7.8, 2H, 2 ꢂ H-5 ), 6.99 (s, 2H, H-3 and H-6), 6.97 (d, J2 ,6 ¼ 1.2,
2H, 2xH-20), 6.96 (dd, J5 ,6 ¼ 7.8, J2 ,6 ¼ 1.2, 2H, 2xH-60), 5.28 (t,
0
0
0
0
J2 ,3 ¼ J3 ,4 ¼ 9.3, 2H, 2xH-300), 5.12 (t, J3 ,4’’ ¼ J4 ,5’’ ¼ 9.3, 2H, 2xH-
00 00
00 00
00
00
400), 5.04 (bdd, J1 ,2 ¼ 8.2 J2 ,3 ¼ 9.3, 2H, 2 ꢂ H-200), 4.85 (d,
00 00
00 00
J1 ,2’’ ¼ 8.2, 2H, 2xH-100), 4.60 (s, 4H, 2xCH2C≡), 4.29 and 4.17 (split
5. Biological study. Materials and methods
00
AB system, J5 ,6A ¼ 4.4, J5 ,6B ¼ 2.5, J6A ,6B ¼ 12.2, 4H, 2xH2-600),
00
00
00
00
00
00
3.89 (s, 6H, 2xOCH3), 3.77 (m, 2H, 2xH-500), 3.03 (s, 12H, 2ꢂ
5.1. Cell cultures
[N(CH3)2]), 2.08, 2.05, 2.03 and 2.02 (four s, 24H, 8 ꢂ CH3CO). 13
C
NMR:
d
170.6, 170.2, 169.4 and 169.3 (8 ꢂ CO), 154.3 and 153.9 (C-2,
The human carcinoma cells used in this study were HEp-2
(derived from a larynx carcinoma) and HeLa (derived from a cer-
vical carcinoma) cells. The spontaneously transformed HaCaT cell
line, obtained from human keratinocytes, has been used as control
of nontumorogenic cells. HeLa and HaCaT cells were cultured using
Dulbecco's modified Eagle's medium (DMEM), supplemented with
10% (v/v) fetal bovine serum (FBS), 50 units/mL penicillin and
C-5, C-30), 134.3 (C-50), 123.5 (C-60), 122.6 (C-40), 119.9 (C-20), 115.5,
114.9 and 114.8 (C-1, C-3, C-4 and C-6), 113.6 (C-10), 98.4 (C-100), 98.1,
93.0, 87.2 and 84.4 (C^C), 72.7 (C-300), 71.8 (C-500), 71.1 (C-200),68.3
(C-400), 61.7 (C-600), 56.9 (CH2C≡), 56.3 (OCH3), 43.3 [N(CH3)2], 20.7
and 20.5 (8 ꢂ CH3CO). Calc. for C62H68N2O22 1192.43: found positive
MALDI [Mþ] ¼ 1192.4; Purity by qNMR: 96.3%.
50 mg/mL streptomycin. HEp-2 cells were cultured in RPMI medium
4.1.4. (2,5-Dimethoxy-1,4-phenylene)bis[2,1-ethynediyl[2-
(dimethylamino)-4,1-phenylene]-2-propyne-3,1-diyl]-bis-b-D-
glucopyranoside (2)
also supplemented with FBS, penicillin and streptomycin (all from
GE Healthcare Life Sciences, HyClone Laboratories, Logan, Utah,
USA). Cell cultures were performed under standard conditions at
37 ꢃC, 95% of humidity and 5% of CO2 and the medium was changed
each two days.
To a flask was added 10 (0.43 g, 0.36 mmol) and dissolved in a
mixture of MeOH (15 mL) and THF (15 mL). A large excess (9 mL) of
aqueous ammonia was added to the obtained solution and the re-
action was maintained under continuous stirring overnight at RT.
Solvents were removed under reduced pressure and the undesired
acetamide was eliminated by a series of MeOH washings, with the
final obtaining of compound 2 as a brilliant yellow solid (0.30 g,
0.35 mmol, 97%). TLC: Rf 0.05 (CHCl3/MeOH 80:20)0. Mp 123e125 ꢃC.
5.2. Photosensitizers administration
Stock solutions of both 1 and 2 were prepared in DMSO (Pan-
reac) at a concentration of 10ꢀ3 M. The work solutions were ob-
tained by dissolving the compounds in DMEM/RPMI with 1% FBS.
The final concentration of DMSO was always lower than 0.5% (v/v),
and the lack of toxicity of this solvent for the cells was also tested
and confirmed. All the treatments were performed when cultures
reached around 60e70% of confluence.
1H NMR (dmso-d6):
d
7.40 (d, J5 ,6 ¼ 8.7, 2H, 2xH-5 ), 7.12 (s, 2H, H-3
0
0
and H-6), 6.95 (m, 4H, 2xH-20 and 2xH-60), 5.14 (d, J2 ,OH ¼ 4.4, 2H,
00
2xOH-200), 4.97 and 4.92 (two d, J3 , OH ¼ J4 ,OH ¼ 4.4, 4H, 2xOH-300
00
00
and 2xOH-400), 4.64 and 4.51 (AB system and m, Jgem ¼00 15.7, 6H, 2 ꢂ
CH2C≡ and 2 ꢂ OH-600), 4.31 (d, 2H, J1 ,2 ¼ 7.8, 2xH-1 ), 3.83 (s, 6H,
00 00
2xOCH3), 3.70 and 3.46 (split AB m, 4H, 2 ꢂ H2-600), 3.16e2.98 (m,
8H, 2xH-200, 2xH-300, 2xH- 400, 2xH-500), 2.96 (s, 12H, 2ꢂ[N(CH3)2]).
5.3. Intracellular localization of 1 and 2
13C NMR (dmso-d6):
d
153.9 and 153.5 (C-2, C-5 and C-30), 134.4 (C-
For the analysis of 1 and 2 subcellular localization, the cells were
plated on glass coverslips placed into wells and incubated with
1 ꢂ 10ꢀ6 M of 1 or 2 for 6 h at 37 ꢃC. After incubation, cells were
washed twice with PBS, mounted on slides with a drop of PBS and
immediately observed under the fluorescence microscope coupled
to a digital capture camera (Olympus BX61) using the appropriate
filters of excitation light (UV, 365 nm, exciting filter UG-1). In
50), 131.5 (C-10), 122.8, 122.6, 119.5 and 119.4 (C-20and C-60), 114.9
and 114.8 (C-3 and C-6),113.7 and 112.9 (C-1, C-4 and C-40),101.2 (C-
100), 94.2, 92.8, 87.1 and 85.6 (C^C), 77.0 and 76.5 (C-300 and C-500),
73.3 (C-200), 70.0 (C-400), 61.3 (C-600), 56.2 (CH2C≡), 55.7 (OCH3), 42.6
[N(CH3)2]. Calc. for C46H52N2O14 856.34; found positive ESI-MS: [M-
Hþ] ¼ 857.3515; Purity by qNMR: 96.3%.

70
E. Deni et al. / European Journal of Medicinal Chemistry 111 (2016) 58e71
addition, to better determine the intracellular localization of 1 or 2,
the distribution pattern of both compounds was compared with
that of specific organelles. For this purpose, cells were incubated
with known fluorescent probes for endoplasmic reticulum (ER-
Tracker, Molecular Probes; Eugene, OR) and Golgi apparatus (NBD,
Molecular Probes; Eugene, OR). The fluorescent probes were used
at the concentrations indicated by the suppliers and were incu-
bated for 30 min. After incubation, cells were washed twice in PBS
and observed under fluorescence microscope. The fluorescence
excitation and emission wavelengths of ER-Tracker were 374 and
430 nm respectively. In the case of NBD were 466 and 536 nm
respectively.
(possible mitotic arrest and densely stained as green); (3) apoptotic
cells (early apoptosis: densely stained as green but showing orange
fragments; late apoptosis: densely stained as orange and
condensed chromatin) and (4) necrotic cells (uniformly stained as
orange-red stained and no chromatin condensation) (Fig. S17, ESI).
5.7. Immunofluorescence
For immunodetection of a-tubulin, cells were raised on cover-
slips and subjected to PDT and, 24 h after treatment, were fixed and
permeabilized in methanol (ꢀ20 ꢃC) for 7 min. Then, cells were
washed twice in PBS and incubated with
a -tubulin primary anti-
body (Sigma-Aldrich. St. Louis, MO) for 1 h at 37 ꢃC. After washing,
cells were incubated with Alexa Fluor 488-conjugated goat anti-
mouse IgG secondary antibody (1:250) (Invitrogen, California,
USA) for 45 min at 37 ꢃC, washed in PBS and mounted in Prolong
Gold Antifade Reagent (Invitrogen, California, USA).
5.4. Photodynamic treatments
Cells seeded on 24 well plates were incubated for 6 h with
variable concentrations of 1 or 2 (from 3$10ꢀ6 M to 2.5$10ꢀ5 M).
Afterwards, the media were removing, replaced by fresh media and
cells were irradiated with different UVA light doses, ranging from
5.8. Microscopical observations and statistical analysis
0.25 to 2 J/cm2 (UVA source: CAMAG,
l
¼ 300e400 nm, power:
15.9 W/m2 at 10 cm from the source). After irradiation, cells were
washed three times with PBS, complete DMEM or RPMI was added,
and were maintained in the incubator for 24 h until evaluation. To
assess the possible dark cytotoxic effect of both PSs, cells were
incubated in the dark for 18 h with different PS concentrations. In
the same way, to test the effect of UVA light alone, cells were
subjected to different light doses (0.25e2 J/cm2).
Microscopic observation was carried out using a fluorescence
microscope (Olympus BX61) equipped with the following filter sets
for fluorescence microscopy: ultraviolet (UV, 365 nm, exciting filter
UG-1), blue (450e490 nm, exciting filter BP 490), and green
(545 nm, exciting filter BP 545). Images were obtained with the
digital camera Olympus CCD DP70 and processed using the Adobe
PhotoShop CS5 extended version 12.0 software (Adobe Systems
Inc., USA).
5.5. Morphological studies
Data are expressed as the mean value
standard deviations
(SD). Statistical analysis was carried out with SPSS Statistics 20.0
(IBM®). The statistical significance was determined using the T test
for independent samples (*: P < 0.1; **: P < 0.05; ***: P < 0.01).
Changes in general cell morphology and nuclear morphology
after PDT were analyzed by phase contrast microscopy and
€
Hoescht-33258 staining, respectively. In the second case, cells were
fixed in formaldehyde (3.7% in PBS) for 15 min, washed and stained
with 2.5 mg/mL Hoescht- 33258 dye (H-33258) (Sigma) for 5 min.
Acknowledgment
After washing with distilled water, preparations were mounted
with ProLong® Gold Antifade Reagent (Invitrogen). Death type was
determinate analyzing nuclei morphology according to morpho-
logical criteria previously published [45].
We thank Prof. Anna Notti for the very helpful discussion on
qNMR analyses. We thank MIUR, Italy (PRIN 20109Z2XRJ_010),
Spanish MINECO grant FIS PI12/01253, CTQ2011-24783, CTQ2014-
ꢀ
53894-R and Comunidad Autonoma de Madrid grant SkinModel
CAM S10/BMD-2359.
5.6. Viability assays
Appendix A. Supplementary data
Cell viability was assessed 24 h after treatments using the MTT
assay and the acridine orange (AO)/ethidium bromide (EtBr)
method. Regarding the MTT assay, following treatments, 3-[4,5-
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.01.041
dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide
Sigma, St Louis, USA) solution was added to each well at a final
concentration of 50
g/mL, and plates were incubated at 37 ꢃC for
(MTT
References
m
4 h. The formazan crystals were dissolved in DMSO and the
absorbance at 542 nm was measured using a spectrophotometer
(Espectra Fluor 4, Tecan). The results were expressed as cell survival
percentage of control (cell survival (%) ¼ (mean OD value of PDT-
treated cells/mean OD value of control cells) x 100%).
The AO/EB method was applied according as previously
described by Liu and cols [46]. This method allowed us to confirm
the tendency of the cell death rate detected by MTT assay and,
moreover, to determine approximately the type of cell death
induced by PDT. AO is a vital dye able to stain nuclear DNA across an
intact cell membrane, while EB can only stains cells that have lost
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