Tuning the Hydrophobicity of a Mitochondria-Targeted NO Photodonor

Article abstract of DOI:10.1002/cmdc.201800088

A few compounds in which the nitric oxide (NO) photodonor N-[4-nitro-3-(trifluoromethyl)phenyl]propane-1,3-diamine is joined to the mitochondria-targeting alkyltriphenylphosphonium moiety via flexible spacers of variable length were synthesized. The lipop

Full text of DOI:10.1002/cmdc.201800088

Accepted Article
Title: Tuning the Hydrophobicity of a Mitochondria-Targeted NO-
Photodonor
Authors: Loretta Lazzarato, Federica Sodano, Barbara Rolando,
Francesca Spyrakis, Mariacristina Failla, Elena Gazzano,
Chiara Riganti, Roberta Fruttero, Alberto Gasco, and
Salvatore Sortino
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Tuning the Hydrophobicity of a Mitochondria-Targeted NO-
Photodonor
Federica Sodano,[a] Barbara Rolando,[a] Francesca Spyrakis,[a] Mariacristina Failla,[b] Loretta
Lazzarato,*[a] Elena Gazzano,[c] Chiara Riganti,*[c] Roberta Fruttero,[a] Alberto Gasco,[a] and Salvatore
Sortino*[b]
[a]
F. Sodano ORCID: 0000000320134893, Prof. B. Rolando ORCID: 0000-0001-6138-1503, Prof. F. Spyrakis ORCID: 0000-0002-4016-227X, Prof. L.
Lazzarato ORCID: 0000-0001-7100-8593, Prof. R. Fruttero ORCID: 0000-0001-6726-1254, Prof. A. Gasco
Department of Science and Drug Technology
University of Torino
Via Pietro Giuria 9, 10125 Torino (Italy)
E-mail: loretta.lazzarato@unito.it
[b]
[c]
M. Failla, Prof. S. Sortino ORCID: 0000-0002-2086-1276
Laboratory of Photochemistry, Department of Drug Sciences
University of Catania
95125 Catania (Italy)
E-mail: ssortino@unict.it
Dr. E. Gazzano ORCID:, Prof. C. Riganti ORCID: 0000-0001-9787-4836
Department of Oncology
University of Torino
Via Santena 5/bis, 10126 Torino (Italy)
E-mail: chiara.riganti@unito.it
Supporting information for this article is given via a link at the end of the document.
Abstract: A few compounds in which the NO-photodonor N–[4-nitro-
3-(trifluoromethyl)phenyl]propane-1,3-diamine has been joined to the
targeting mitochondria ligand alkyltriphenylphosphonium moiety,
through flexible spacers of variable length have been synthesized.
Lipophilicity of the products was evaluated by measuring their partition
coefficients in n-octanol/water. The obtained values, markedly lower
than those calculated, are in keeping with the likely collapsed
conformation assumed by the compounds in solution as suggested by
molecular dynamic simulations. The capability of the compounds to
release NO under visible light irradiation was evaluated by measuring
the nitrite production by means of the Griess reaction. The
accumulation of the compounds inside the mitochondria of human
lung adenocarcinoma A549 cells was assessed using UPLC MS.
Interestingly, compound 13 displayed both the highest accumulation
value and high toxicity towards A549 cells upon irradiation-mediated
NO release in mitochondria.
In contrast to traditional anticancer photodynamic therapy (PDT)[6]
based on the production of singlet oxygen (1O2),[6] the so-called
NO-photodynamic therapy (NOPDT)[3] does not require O2
availability to function. In particular, NOPDT may be useful in the
treatment of hypoxic tumors which are not responsive to classical
PDT.[7]
Targeting mitochondria for cancer therapy is also currently
generating great interest,[8] and the role that NO plays in these
organelles has received particular attention.[9,10] NO may be
formed in mitochondria by NOS (nitric oxide synthase) isoforms
(eNOS, nNOS, iNOS), that are attached to the cytoplasmic face
of the outer mitochondrial membrane and also, possibly, by a
specific NOS isoform, referred to as mitochondrial NOS (mit NOS),
located within these organelles. Low levels of NO up-regulate
cellular respiration and stimulate mitochondrial biogenesis. On
the other hand, high levels of NO induce toxicity as this free
radical directly binds and inhibits crucial mitochondrial enzymes,
such as aconitase, a Fe-containing enzyme involved in the
tricarboxylic acid cycle, and respiratory chain complexes
containing iron and iron-sulphur centres, namely complexes I, III
and IV. Furthermore, high NO levels produce reactive nitrogen
(RNS), and oxygen (ROS), species and trigger mitochondrial
apoptosis.[9,10] The mitochondrial accumulation of NOPDs has
therefore been proposed as an effective strategy for the design of
new anticancer drugs. This approach has been created by linking
the NOPDs to vectors that display high tropism for these
organelles.[11-15]
Introduction
Nitric oxide (NO), is an endogenous messenger that is
ubiquitously produced by mammalian tissues and is involved in
many physiological and pathophysiological processes.[1] There is
a significant amount of experimental evidence showing that low
levels (pM-nM concentration) of NO encourage cancer growth,
while high levels (M concentration) reduce cancer progression.[2]
The photogeneration of NO achieved by using suitable NO
photodonors (NOPDs), has recently received a great deal of
attention as potential new anticancer therapy.[3] Indeed, unlike
classical NO-donors,[4] these light-activated precursors allow the
action of NO to be confined within the irradiated area with high
spatial precision and its dosage to be controlled with great
accuracy by tuning the duration and intensity of the irradiation.[5]
NOPD class nitro derivatives that bear appropriate substituents at
the o- and p-positions have received particular attention. Under
the action of violet light, these compounds undergo a nitro-to-
nitrite rearrangement, followed by O-NO bond cleavage with
consequent generation of NO and a phenoxy radical and,
eventually, the formation of non-toxic phenol compounds.[16-17]
The flexibility of the o-CF3 substituted p-nitroaniline derivatives
1
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means that they are suited to this approach as their structures
make their derivatization possible by simple synthetic methods.
Rhodamine derivatives and triphenylphosphonium compounds
have been used as vectors. The finished products are lipophilic
cations with a largely delocalised positive charge that can enter
mitochondria by binding the outer surface of the inner membrane.
The high potential across this phospholipid bilayer (Δψm= 150-180
mV), drives their passage to the inner surface where they are
desorbed and accumulated in the mitochondrial matrix.[18]
Although most solid tumors obtain their energy from aerobic
glycolysis (Warburg effect), mitochondria still play a key role in
tumor growth.[19] Active mitochondria favor tumorigenesis, cell
migration, metastasis[20,21] and drug resistance.[22,23] By contrast,
impairing oxidative phosphorylation in mitochondria reduces
cancer cell proliferation and invasion, and triggers apoptosis.[19]
Cancer cell mitochondria have higher transmembrane potentials
than non-transformed cells.[24] This feature indicates that
mitochondrial activity is higher in tumor cells and this fact can be
exploited to increase the selectivity of tumor cell killing by
delivering drugs into the mitochondria.
adenocarcinoma cells, both in the dark and when irradiated with
the violet light.
Results and Discussion
Synthesis and spectroscopic properties
The compounds studied in this work were obtained through the
synthetic pathways reported in Scheme 1. The new NOPD 2 was
prepared via the acetylation of 1, synthesized according to
literature,[28] with acetic anhydride in dry CH2Cl2 (DCM), in the
presence of triethylamine. The aliphatic carboxylic acids bearing
the triphenylphosphonium moiety in the ω-position 7-10 were
obtained from the reaction of related ω-bromo substituted acids
3-6 with triphenylphosfine (PPh3), in either refluxing toluene or
acetonitrile. Finally, target products 11-14 were synthesized via
the coupling of 1 with the appropriate ω-triphenylphosphonium
substituted acid in DMF and DCM, in the presence of N,N'-
dicyclohexylcarbodiimide (DCC), and N-hydroxysuccinimide
(NHS).
As a further development of our work on NOPDs that are
structurally related to o-(trifluoro)methyl-p- nitroaniline,[15,25-27] we
have designed a series of new compounds in which N–[4-nitro-3-
(trifluoromethyl)phenyl]propane-1,3-diamine (1, Scheme 1), is
linked through an amide bridge to alkyltriphenylphosphonium
chains of variable length. This paper reports the synthesis, NO-
photorelease and partition coefficient (log Poct/w), of these
compounds as well as those of the acetyl derivative of 1 (2,
Scheme 1), used as suitable reference model. Furthermore, we
analyse how lipophilicity affects the compounds' mitochondrial
uptake in human lung adenocarcinoma A549 cells. Compound 13,
which shows the greatest tropism for mitochondria, was also
investigated as regard its toxicity against human lung
The UV-vis spectra, recorded in phosphate buffer saline (PBS)
solutions, of all the compounds are characterized, in the visible
region, by the dominant absorption of the nitroaniline-
chromophore centred at 402 nm (see Figure S1a in Supporting
Information).The molar extinction coefficients
ε follow the
sequence 2≈11>12>13>14, which are different to observations
made in methanol solution where ε values are practically the
same for all the products (see inset Figure S1a, Supporting
Information, as an example). This behaviour is probably due to
the formation of aggregates in PBS, which occurs to various
extents. Irradiation of PBS solutions of all compounds with a 400
nm violet led at an irradiance of 7 mW/cm2 (the same irradiation
conditions were used in all the experiments described in this work)
led to the typical bleaching of the visible absorption band,
according to the release of NO.[17] (see Figure S1b, Supporting
Information, as example).
Lipophilicity
The partition coefficient (log P), between n-octanol and buffered
saline (PBS, pH 7.4), was chosen to describe the lipophilicity of
the products. Measurements were carried out using the shake-
flask technique, and results are reported in Table 1. The log P
values range from 1.99 to 3.24. An attempt to calculate the log P
of the products using the Hansch and Leo method on the CLOGP
program (Bio-Loom for Windows, Version1.5, BioByte), gave very
high values which are unrealistic in nature. This molecular
descriptor has both additive and constitutive components. An
analysis of the table shows that log P values increase, by 0.40 (11
to 12), 0.39 (12 to 13), and 0.46 (13 to 14), as we move from one
compound to the next. This pattern is due to the presence of
additional 3, 6 and 8 methylene units in 12, 13, 14, with respect
to 11. If fCH2=0.66 is taken as the CH2 fragmental constant,[29] one
must conclude that the products are much less lipophilic than the
simply additive nature of log P would provide for. It is most likely
that a significant role in this log P decrement is played by the inter-
and intramolecular hydrophobic interactions that form between
the apolar parts of the molecules. Indeed, these interactions
induce water molecules to strip off from the hydrophobic hydration
Scheme 1. Synthesis of compounds 2, 11, 12, 13 and 14: i) (CH3CO)2O, Et3N,
DCM, 0°C; ii) PPh3, CH3CN, Δ for cpds 7 and 9; iii) PPh3, toluene, Δ for cpds 8
and 10; iv) DCC, NHS, DMF; v) 1, DCM, DMF.
2
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shell (flickering cluster), and move to the bulk aqueous medium,
leading to a subsequent decrease in molecular lipophilicity.[29]
triphenylphosphonium moiety were seen to be in close contact to
form pi-pi interactions (Figure 1).
In order to estimate the extent of ligand collapse in these
molecules, the distances between the nitroaniline and
triphenylphosphonium rings were calculated (see Figure S3 in
Supporting Information). While compound 11 hardly folds back on
itself at all, compounds 12, 13 and 14 spend a large part of the
simulation time (20 ns), in collapsed globular forms. In particular,
compound 13 shows the highest tendency to assume a globular
structure, which is characterised by the presence of pi-pi contact.
Different, but still collapsed, conformations are also assumed by
the ligands when the nitroaniline folds back on the methylene unit
(see Figure S2 in Supporting Information). Water molecules were
displaced towards the bulk in all these forms, thus reducing the
solvent accessible surface area of the ligands. As mentioned, this
effect could, at least in part, explain the low log P increase
observed upon the elongation of the methylene unit. It is
interesting to note that the nitro group assumes an orthogonal
orientation, which is indispensable requisite for the NO release
mechanism.
Table 1. Partition Coefficients (log P), between n-octanol and PBS (pH 7.4), of
2, 11, 12, 13 and 14.
Compound
2
log P (±SD)
2.03 (±0.08)
11
12
13
14
1.99 (±0.05)
2.39 (±0.09)
2.78 (±0.08)
3.24 (±0.16)
Compounds conformation
Figure 2. NO release profile upon led irradiation (= 400 nm, 7mW/cm2), of 100
M solutions of 2 (green), 11 (red), 12 (fuchsia), 13 (light blue), and 14 (grey).
NO release of cpds 11-14 vs. cpds 2: * p<0.05; *** p<0.001.
NO-photorelease in PBS solution
The capacity of 2 and 11-14 to release NO over 30 min of
irradiation was evaluated at a concentration of 100 M in 50 mM
PBS, pH = 7.4, using the experimental conditions described
previously. Released NO was detected in the form of nitrite, its
main degradation product, using the Griess reaction in aerobic
aqueous solution. The results are summarized in Figure 2 and it
is clear that the NO-release of compounds is linearly correlated
with time. All of the hybrid structures are better NOPDs than
reference compound 2, and their performance can be graded as
follows: 14≈13>12>11>2.
The variety in the NO photoreleasing efficiency values observed
for the 5 compounds can be, at first sight, quite surprising as the
same NO photodonor moiety is present in all. However, we
believe that the different conformations adopted by the
investigated compounds play a key role in this respect. As
summarized in the introduction, the NO release mechanism
involves a nitro-to-nitrite rearrangement, followed by O-NO bond
cleavage and the consequent generation of NO and a phenoxy
radical, which is the key transient intermediate.[16,17] In turn, the
Figure 1. Compound 13 in the collapsed conformation, highlighting the
close contact between the nitroaniline ring and one of the phenyl groups
forming the triphenylphosphonium moiety. The ligand is shown in capped
sticks (a) and in spheres (b).
In order to better investigate the conformations assumed by the
compounds in solution, Molecular Dynamics (MD), simulations of
11, 12, 13 and 14 were performed. MD simulations were run using
Gromacs 4.6.1. as implemented in the BiKi Life Science software
suite (http://www.bikitech.com). All simulations were performed in
water solution using the TIP3P water model (see the Experimental
section and SI for further details). An increase in methylene
number generally leads the molecules to assume a more
collapsed and globular conformation. These more ordered
conformations are likely stabilized by hydrophobic interactions
that form between the hydrophobic regions of the molecules; the
nitroaniline, the triphenylphosphonium and the methylene units.
In particular, the nitroaniline and phenyl rings that form the
3
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nitro-to-nitrite photorearrangement is triggered by the overlap
between the p orbital of the nitro-group oxygen and the  orbital
of the phenyl ring.[16,17] On these grounds, the collapsed
conformation that is dominant in compounds 13 and 14 can, to
some extent, not only encourage the above orbital overlap but
also stabilize the intermediate phenoxy radical, which accounts
for the fact that their NO photorelease efficiencies are higher than
those of 2, 11 and 12.
UPLC MS. The concentration found in the cytosol was 0.068 M
for compound 2, 0.103 M for 11, 0.264 M for 12, 2.701 M for
13 and 1.502 M for 14. Mitochondrial accumulation was below
the detection threshold for model compound 2, 0.448 M for
compound 11, 1.105 M for 12, 8.530 M for 13 and 4.042 M for
14. The results are reported in Figure 3 and are normalized to mg
of proteins in the cytosolic and mitochondrial fractions.
Interestingly, while an accumulation increase occurs from 11 (log
P = 1.99), to 13 (log P = 2.78), a reduction is seen from 13 to 14
(log P = 3.24). As expected, the accumulation of reference
compound 2 was not detectable. Consequently, compound 13
and reference compound 2, which does not contain the
alkyltriphenylphosphonium moiety, were chosen for antitumor
property investigations.
NO photorelease of 2 and 13 in lung adenocarcinoma cells
The NO photorelease of 2 and 13 in lung adenocarcinoma A549
cells, which had either been kept in the dark for 30 min or
irradiated for the same time, was studied using the Griess reaction
to detect nitrite. Concentrations of 5 M, considered non-toxic in
the dark (see below), of the compounds were used in the
experiments and the results are reported in Figure 4. While none
of the compounds has the effect of increasing nitrite release in the
dark, as compared to control cells, irradiation did induce this effect.
In particular, compound 13 was a more effective NO donor than
2, which is in line with results obtained in the cell-free experiments.
Figure 3. Mitochondrial and cytosolic accumulation of compounds 2, 11-14.
Measurements were performed in triplicate and data are presented as means
± SEM. nd=not detectable. Mitochondrial vs. cytosolic accumulation ** p<0.01;
*** p<0.001.
Accumulation in the mitochondria of human lung
adenocarcinoma cells
The accumulation of 11-14 and reference 2 in mitochondria was
Cytotoxicity of 2 and 13 against human lung adenocarcinoma
cells
In order to evaluate the effect of mitochondrial accumulation on
the toxicity of 13, A549 cells were either left in the dark for 30 min
or irradiated either in the presence of the compounds in a range
of concentrations, or in their absence. Parallel experiments were
carried out with reference
2 under the same conditions.
Cytotoxicity was evaluated, 24 h after irradiation, by measuring
the release of lactate dehydrogenase (LDH), which is sensitive to
the loss of membrane integrity.[31] and correlated well with the cell
death. We have made use of the LDH release assay in our
previous works on NO-photodonors.[15,27] The LDH assaywas also
performed here since it is a sensitive index of irreversible cell
damage leading to cell death, and to allow comparisons between
the data obtained in the present work and in previous findings to
be carried out.[32] Figure 5 shows that the extent of cell death in
both irradiated, untreated cells and in irradiated cells that had
been treated with compound 2 is two times higher than that of
non-irradiated cells. Moreover, the extent of cell death in
irradiated cells that had been treated with compound 13 is 4 times
that of not-irradiated cells, whether they were untreated or treated
with either compound 2 or 13. Reference 2 did not show any
toxicity in the dark, unlike 13 which was not significantly toxic up
to a concentration of 5 µM and then showed enhanced toxicity
with increasing concentration (data not shown). This is in keeping
with the finding that phosphonium salts can display toxic activity
towards cancer cell lines.[33] When irradiated at 5 µM, 2 did not
show any toxicity, unlike 13 which was markedly more toxic, in
keeping with its accumulation into mitochondria (Figure 5).
Figure 4. NO-photorelease in A549 cells incubated with either model
compound 2 (a) or 13 (b) and either maintained in the dark or irradiated ( =
400 nm, 7mW/cm2), for 30 min. Measurements were performed in triplicate and
data are presented as means ± SD. Vs untreated cells (ctrl): * p<0.05; ***
p<0.001.
assessed by incubating 5 M solutions of each compound with
human lung adenocarcinoma A549 cells according to a previously
described procedure.[30] Briefly, the cytosolic and mitochondrial
cellular fractions were separated after 4 h of incubation and the
amounts of each compound in the two fractions was measured by
4
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Chemical shifts (), are given in parts per million (ppm), and the coupling
constants (J), in Hertz (Hz). The following abbreviations were used to
designate multiplicities: s = singlet, d = doublet, dd = doublet of doublet, t
= triplet, q = quartet, quint = quintet, m = multiplet, br = broad, bs = broad
singlet. ESI spectra were recorded on a Micromass Quattro API micro
(Waters Corporation, Milford, MA, USA), mass spectrometer. Data were
processed using a MassLynx System (Waters). The purity of the final
compounds (≥ 95%), was determined by analytical HPLC analysis on a
Merck LiChrospher C18 end-capped column (250 × 4.6 mm ID, 5 µm),
using either CH3CN/H2O or CH3CN 0.1% TFA/H2O 0.1% TFA as the
solvent. HPLC retention times (tR), were obtained at flow rates of 1.0 mL
min-1 and the column effluent was monitored using UV as the detector
(DAD  = 226, 254, 400 nm). UV-vis spectra absorption was recorded on
a Varian Cary 50BIO UV/Vis spectrophotometer (Varian Australia Pty Ltd.,
Mulgrade, Australia), in air-equilibrated solutions, using quartz cells with a
path length of 1 cm. Absorption spectral changes were monitored by
irradiating the sample in a quartz cell (1 cm path length, 3mL capacity),
using a blue led 10W lamp as the light source and an irradiance of 7
mW/cm2.
Conclusions
We have studied the influence of lipophilicity on the mitochondrial
accumulation of a series of novel compounds obtained by linking
N-(3-((4-Nitro-3-(trifluoromethyl)phenyl)amino)propyl)acetamide (2).
Acetic anhydride (1.21 mmol, 0.11 mL), and triethylamine (1.01 mmol, 0.14
mL), were added to a solution of 1 (1.01 mmol, 266 mg), in DCM (10 mL),
and stirred for 4 h at 0°C. H2O (20 mL), was then added to the reaction
mixture and the yellow solid 2 obtained was filtered and then dried under
vacuum. (160 mg, 53%). Mp = 76.6-77.8 °C. 1H NMR (600 MHz, Acetone-
d6),  7.87 (d, J=9.1 Hz, 1H), 6.90 (d, J=2.4 Hz, 1H), 6.71 (dd, J=9.1, 2.6
Hz, 1H), 3.22–3.07 (m, 4H), 1.70 (s, 3H), 1-69–1.63 (m, 2H). 13C NMR
Figure 5. Cytotoxicity in A549 cells incubated with 5 M solutions of compounds
2 and 13 and either maintained in the dark or irradiated ( = 400 nm, 7mW/cm2),
for 30 min (light). Measurements were performed in triplicate and data are
presented as means ± SEM (n=3). Vs untreated cells (CTRL): * p<0.05.
alkyltriphenylphosphonium moieties to a nitroaniline-derivative
NOPD. Interestingly, the dependence between the accumulation
and log P of the products was roughly bilinear, showing a
maximum for compound 13. MD simulations demonstrated that
methylene unit elongation generally leads the molecules to
assume a more collapsed conformation, stabilized by the
formation of hydrophobic interactions, which is probably
responsible for the low estimated log P. Compound 13 (log P=
2.78), which shows the highest tendency to assume a globular
structure, when irradiated triggered high toxicity at a 5 M
concentration, against A549 adenocarcinoma cell lines, unlike
reference model 2, which lacks the triphenylphosphonium moiety.
It is worth noting that the cytotoxicity induced by 13, via NO
release into mitochondria upon irradiation, falls in the low-
micromolar range, which is similar to that of most antitumor
chemotherapy drugs (e.g. anthracyclines, platinum-derivatives,
gemcitabine). Our results may well facilitate the design of new
efficient NOPDs which could potentially treat hypoxic tumors that
are not responsive to classical PDT. Indeed, these compounds,
2
(150 MHz, Acetone-d6),  170.6, 153.7, 135.6, 129.9, 126.3 (q, JF=32.8
Hz), 123.4 (q, 1JF= 272.6 Hz), 112.8, 111.7, 40.9, 37.0, 29.2, 22.6. ESI-MS
[M+Na]+: m/z 328.4. HPLC purity ≥ 95% (CH3CN/H2O 60/40 (v/v), flow =
1.0 mL/min, tR = 3.40 min) at 226, 254 and 400 nm.
General synthetic procedure for the preparation of aliphatic
carboxylic acids bearing the triphenylphosphonium moiety in the ω-
position (7-10). A solution of the appropriate carboxylic acid 3-6 (1.0
equiv), and PPh3 (2.0 equiv), was heated in either CH3CN or toluene (20
mL), at reflux for 24h. The reaction mixture was then purified as described
below.
(2-Carboxyethyl)triphenylphosphonium bromide (7). The reaction was
performed in CH3CN. After 24h, the solvent was removed under reduced
pressure and the obtained residue was purified by flash chromatography
using DCM and then MeOH as the eluents, to give 7 as an orange oil
(3.500g, 84%). 1H NMR (300 MHz, CDCl3),  7.89–7.60 (m, 15H), 6.14 (bs,
1H), 3.82–3.62 (m, 2H), 3.02–2.82 (m, 2H). 13C NMR (75 MHz, CDCl3) 
171.8 (d, 3JP=13.2 Hz), 135.5 (d, 4JP=3.0 Hz), 133.7 (d, 3JP=10.1 Hz), 130.8
(d, 2JP=12.6 Hz), 117.6 (d, 1JP=86.7 Hz), 28.2 (d, 2JP=2.5 Hz), 19.0 (d,
1JP=54.7 Hz). ESI-MS [M]+: m/z 335.3.
1
unlike the classical photosensitizers for PDT, do not require O2
production and therefore molecular oxygen availability in order to
be active.
(5-Carboxypentyl)triphenylphosphonium bromide (8). The reaction
was performed in toluene. After 24h, the obtained white solid 8 was filtered
and then dried under vacuum. (2.00 g, 87%). 1H NMR (300 MHz, CDCl3),
 9.68 (bs, 1H), 7.95–7.58 (m, 15H), 3.5–3.26 (m, 2H), 2.40–2.19 (m, 2H),
1.71–1.55 (m, 6H). 13C NMR (75 MHz, CDCl3),  177.6, 135.4, 133.6 (d,
3JP=10 Hz), 130.7 (d, 2JP=12.5 Hz), 117.9 (d, 1JP=86.2 Hz), 33.8, 29.7 (d,
2JP=16.2 Hz), 24.0, 22.6 (d, 1JP=51.4 Hz), 22.2 (d, 3JP=3.6 Hz). ESI-MS
[M]+: m/z 377.5.
Experimental Section
Synthesis. All reactions involving air-sensitive reagents were performed
under nitrogen in oven-dried glassware using the syringe-septum cap
technique. All solvents were purified and degassed before use.
Chromatographic separation was carried out under pressure on a Merck
silica gel 60 using flash-column techniques. Reactions were monitored
using thin-layer chromatography (TLC), carried out on 0.25 mm silica gel
coated aluminium plates (60 Merck F254), using UV light (254 nm), a
bromocresol green solution and a solution of diphenylamine in ethanol as
the visualizing agents. Unless specified, all reagents were used as
received without further purification. Dichloromethane was dried over P2O5
and freshly distilled under nitrogen prior to use. 1H NMR and 13C NMR
spectra were either recorded at room temperature on a BrukerAvance 300,
at 300 and 75 MHz, or a JEOL ECZ-R 600, at 600 and 150 MHz,
respectively, and calibrated using SiMe4 as an internal reference.
(8-Carboxyoctyl)triphenylphosphonium bromide (9). The reaction was
performed in CH3CN. After 24h, the solvent was removed under reduced
pressure to give an oily residue. Purification by flash chromatography
using DCM/ MeOH (96/4 to 85/15 v/v), as the eluent, gave 9 as a whitish
semi-solid. (582 mg, 69%). 1H NMR (600 MHz, DMSO-d6),  7.96–7.73 (m,
15H), 3.67–3.49 (m, 2H), 3.39 (bs, 1H), 2.15 (t, J=7.4 Hz, 2H), 1.58–1.39
(m, 6H), 1.31–1.14 (m, 6H). 13C NMR (150 MHz, DMSO-d6),  174.6, 134.9
(d, 4JP=3 Hz), 133.6 (d, 3JP=10 Hz), 130.3 (d, 2JP=12.5 Hz), 118.6 (d,
5
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1JP=86 Hz), 33.8, 30.7, 29.8 (d, 2JP=16.5 Hz), 28.5, 28.0, 24.5, 21.7 (d,
3JP=4 Hz), 20.1 (d, 1JP=50 Hz). ESI-MS [M]+: m/z 419.5.
29.4, 26.9, 23.5 (d, 3JP=4.4 Hz), 22.9, 22.3. ESI-MS [M]+: m/z 664.5. HPLC
purity = 100% (CH3CN 0.1% TFA/H2O 0.1% TFA 65/35 (v/v), flow = 1.0
mL/min, tR = 9.7 min), at 226, 254 and 400 nm.
(10-Carboxydecyl)triphenylphosphonium bromide (10). The reaction
was performed in CH3CN. After 24h, the solvent was removed under
reduced pressure to give an oily residue. Purification by flash
chromatography using DCM/MeOH (95/5 to 80/20, v/v), gave 10 as a
(11-((3-((4-Nitro-3-(trifluoromethyl)phenyl)amino)propyl)amino)-11-
oxoundecyl) triphenylphosphonium bromide (14). The carboxyl
function activation reaction time was 12 hours. Eluent: DCM/MeOH, 95/5
to 90/10, v/v. The target compound  was as a foamy yellow solid (415 mg,
40%). 1H NMR (300 Mz, CDCl3),  7.90 (d, J=9.1 Hz, 1H), 7.85–7.64 (m,
15H), 7.01–6.95 (m, 1H), 6.78–6.68 (m, 1H), 3.39–3.28 (m, 6H), 2.27 (t,
J=7.5 Hz, 2H), 1.87–1.76 (m, 2H), 1.72–1.47 (m, 6H), 1.35–1.10 (m, 10H).
1
whitish semi-solid. (2.200 g, 83%). H NMR (300 MHz, CD3OD),  7.90–
7.66 (m ,15H), 3.43–3.31 (m, 2H), 2.21 (t, J=7,4 Hz, 2H), 1.70–1.44 (m,
6H), 1.36–1.16 (m, 10H). 13C NMR (75 MHz, CD3OD),  176.8, 135.3 (d,
4JP=3.0 Hz), 133.8 (d, 3JP=10.0 Hz), 130.5 (d, 2JP=12.6 Hz), 119.0 (d,
1JP=86.3 Hz), 34.0, 30.5 (d, 2JP=16.2 Hz), 29.4, 29.3, 29.2, 28.8, 25.1, 22.5
(d, 3JP=4.4 Hz), 21.6 (d, 1JP=51.0 Hz). ESI-MS [M]+: m/z 447.4.
4
13C NMR (75MHz, CDCl3),  174.6, 169.4, 153.0, 135.5, (d, JP=3.0 Hz),
134.3, 133.6 (d, 3JP=9.9 Hz), 130.7 (d, 2JP=12.5 Hz), 129.5, 126.6 (q,
2JF=32.9 Hz), 122.7 (q, 1JF=272.2 Hz), 118.1 (d, 1JP=86.1 Hz), 39.9, 36.5,
2
36.3, 30.3 (d, JP=15.9 Hz), 28.9, 28.8, 28.6 (d, 3JP=3.2 Hz), 28.3, 27.1,
General synthetic procedure for the preparation of target compounds
25.7, 22.9 (d, 1JP=44.6 Hz), 22.5. ESI-MS [M]+: m/z 692.4. HPLC purity =
95% (CH3CN 0.1% TFA/H2O 0.1% TFA 65/35 (v/v), flow = 1.0 mL/min, tR
= 13.8 min), at 226, 254 and 400 nm.
(11-14).
N,N'-dicyclohexylcarbodiimide
(2.5
equiv),
and
N-
hydroxysuccinimide (1.5 equiv), were added to
a
solution of the
appropriate ω-triphenylphosphonium-substituted acid (7-10, 1 equiv), in
dry DMF (40 mL), and the reaction mixture was stirred at room temperature
for 4-12 hours. When the reaction was completed (TLC), the solvent was
removed under reduced pressure and the crude solid was triturated with
cold CH3CN and filtered under reduced pressure. The filtrate was
concentrated to dryness and dissolved in dry DCM/DMF (97:3, 31 mL). 1
(1 equiv), was added to this solution and the mixture was stirred for 72h at
room temperature. The reaction mixture was then washed with H2O (3 ×
20 mL), and brine (20 mL), dried over Na2SO4 and concentrated to dryness.
Purification by flash chromatography, eluted with a different mixture of
DCM/MeOH, gave target compounds as yellow foamy solids.
Determination of lipophilicity descriptor (log P).
The partition coefficients (log P), of the target compounds between n-
octanol and PBS (pH 7.4), were obtained using the shake-flask technique
at room temperature. In the shake-flask experiments, 50 mM phosphate
buffer saline (pH 7.4), was used as the aqueous phase and ionic strength
was adjusted to 0.15 M using KCl. The organic (n-octanol), and aqueous
phases were mutually saturated by shaking for 4 h. The compounds were
solubilised in the buffered aqueous phase at concentrations of either 0.1
mM or 0.05 mM, depending on their solubility, and appropriate amounts of
n-octanol were added. The two phases were shaken for about 20 min, by
which time the partitioning equilibrium of the solutes had been reached,
and then centrifuged (10000 rpm, 10 min). The concentration of the solutes
was measured in the aqueous phase using UV spectrophotometer (UV-
2501PC, Shimadzu). Each log D value is an average of at least six
measurements. All the experiments were performed while avoiding
exposure to light.
(3-((3-((4-Nitro-3-(trifluoromethyl)phenyl)amino)propyl)amino)-3-
oxopropyl)triphenylphosphonium bromide (11). The carboxyl function
activation reaction time was 4 hours. Eluent: DCM/MeOH, 98/2 to 95/5, v/v.
The target compound 11 was as a foamy yellow solid (yellow solid, lit.[13])
(923 mg, 58%). 1H NMR (300 MHz, DMSO-d6),  8.03 (d, J=9.1 Hz, 1H),
7.92–7.63 (m, 15H), 7.03–6.96 (m, 1H), 6.75 (dd, J=9.2, 2.2 Hz, 1H), 3.82–
3.65 (m, 2H), 3.15–2.97 (m, 4H), 2.06–2.00 (m, 2H), 1.65–1.51 (m, 2H).
13C NMR (75 MHz, DMSO-d6):  168.7 (d, 3JP=15.0 Hz), 153.2, 135.0,
133.7 (d, 3JP=10.2 Hz), 133.5, 130.3 (d, 2JP=12.5 Hz), 129.9, 126.4 (q,
1JF=248.9 Hz), 118.4 (d, 1JP=86.3 Hz), 36.6, 30.8, 27.9, 27.5, 16.9 (d,
1JP=53.4 Hz). ESI-MS [M]+: m/z 580.3. HPLC purity = 96% (CH3CN 0.1%
TFA/H2O 0.1% TFA 65/35 (v/v), flow = 1.0 mL/min, tR = 6.4 min), at 226,
254 and 400 nm.
Molecular Dynamics.
The ligands were parameterized using the ab initio RESP-charge-fitting
methodology, as implemented in the BiKi Life Science software suite
(http://www.bikitech.com). MD simulation setup was performed using BiKi.
Gromacs 4.6.1 was used to run MD simulations.[34] The water model used
was TIP3P. The solvated system was preliminarily minimized by 5000
steps of steepest descent. As in the subsequent equilibration, the
integration step was equal to 1 fs. The Verlet cut-off scheme, the
Bussi−Parrinello thermostat[35] LINCS for the constraints (all bonds), and
the particle mesh Ewald for electrostatics, with a short-range cut-off of 11
Å, were used. The system was equilibrated in four subsequent steps: 100
ps in the NVT ensemble at 100 K, 100 ps in the NVT ensemble at 200 K,
100 ps in the NVT ensemble at 300 K and a 1 ns long NPT simulation in
order to reach the pressure equilibrium condition. No restraint was applied.
The production run was carried out in the NVT ensemble at 300 K without
any restraint for 20 ns. Two replicas were simulated for each ligand upon
velocity reassignment.
(6-((3-((4-Nitro-3-(trifluoromethyl)phenyl)amino)propyl)amino)-6-
oxohexyl) triphenylphosphonium bromide (12). The carboxyl function
activation reaction time was 4 hours. Eluent: DCM/MeOH, 98/2 to 95/5, v/v.
The target compound 12 was as a foamy yellow solid (489 mg, 70%). 1H
NMR (600 MHz, DMSO-d6),  8.01 (d, J=9.1 Hz, 1H), 7.78–7.67 (m, 15H),
7.02–6.96 (m, 1H), 6.77–6.74 (m, 1H), 3.55–3.47 (m, 2H), 3.12 (dd, J=12.7,
6.8 Hz, 2H), 3.05 (q, J=6.7, 2H), 1.98 (t, J=7.2 Hz, 2H), 1.61 (quin, J=6.9
Hz, 2H), 1.49–1.34 (m, 6H). 13C NMR (150 MHz, DMSO-d6):  172.0, 153.2,
135.0 (d, 4JP=3.0 Hz), 133.6 (d, 3JP=10.1 Hz), 133.5, 130.3 (d, 2JP=12.6
Hz), 122.6 (q, 1JF=272.7 Hz), 118.6 (d, 1JP=85.5), 36.2, 35.1, 30.8, 29.6 (d,
2JP=16.3 Hz), 28.2, 24.5, 21.7 (d, 3JP=3.2 Hz), 20.2 (d, 1JP=49.8 Hz). ESI-
MS [M]+: m/z 622.3. HPLC purity = 97% (CH3CN 0.1% TFA/H2O 0.1% TFA
65/35 (v/v), flow = 1.0 mL/min, tR = 7.4 min), at 226, 254 and 400 nm.
NO-photorelease.
(9-((3-((4-Nitro-3-(trifluoromethyl)phenyl)amino)propyl)amino)-9-
oxononyl) triphenylphosphonium bromide (13). The carboxyl function
activation reaction time was 6 hours. Eluent: DCM/MeOH, 98/2 to 85/15,
v/v. The target compound 13 was as a foamy yellow solid (340 mg, 79%).
1H NMR (300 MHz, CD3OD),  7.95 (d, J=9.2 Hz, 1H), 7.89–7.66 (m, 15H),
6.93 (d, J=2.2 Hz, 1H), 6.72 (dd, J=9.2, 2.5 Hz, 1H), 3.44–3.29 (m, 2H),
3.29–3.16 (m, 4H), 2.12 (t, J=7.4 Hz, 2H), 1.87–1.71 (m, 2H), 1.69–1.42
(m, 6H), 1.35–1.15 (m, 6H). 13C NMR (75 MHz, CD3OD),  174.9, 154.4,
136.8 (d, 4JP=3.0 Hz), 134.8 (d, 3JP=10.0 Hz), 131.5 (d, 2JP=12.6 Hz), 130.4,
127.3 (q, 2JF 32.8 Hz), 124.0 (q, 1JF=273.0 Hz), 119.9 (d, 1JP=86.3 Hz),
112.9, 112.1, 41.4, 37.4 (d, 1JP=57.6 Hz), 31.5 (d, 2JP=16.3 Hz), 30.1, 29.7,
NO release was evaluated, as nitrite formed, using the Griess reaction. 5
mL of 100 µM solutions of compounds 2, 11, 12, 13 and 14 in 50 mM
pH=7.4 phosphate buffer saline solution (PBS), and 1% DMSO were
irradiated using a violet led 10W lamp. An irradiance of 7 mW/cm2 was
measured with an HD2302.0 Delta Ohm light meter equipped with a Delta
Ohm LP471RAD light probe. The presence of nitrite was determined at
regular time intervals using the Griess assay; 1.0 mL of the reaction
mixture was treated with 250 µL of Griess reagent (4% w/v sulphanilamide,
0.2 % w/v N-naphthylethylenediamine dihydrochloride, 1.47 M phosphoric
acid). After 10 min at room temperature, the reaction mixture was analysed
6
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10.1002/cmdc.201800088
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using a UV spectrophotometer Shimadzu UV-2501PC and calibration
curves obtained with standard solutions of sodium nitrite at 0.5 µM to 50
(MWD, model G1365D), integrated into the system. Data analyses were
performed using a HP ChemStation system (Agilent Technologies).
Samples were eluted on a HyPURITY Elite C18 column (250 × 4.6 mm, 5
mm, Hypersil, ThermoQuest Corporation, UK). The injection volume was
20 L (Rheodyne, Cotati, CA, USA). The mobile phase consisted of
CH3CN 0.1% TFA (solvent A), and H2O 0.1% TFA (solvent B), at a flow
rate of 1.0 mL/min with gradient conditions: 50% A up to 4 min, from 50 to
90% A between 4 and 8 min, 90% A between 8 and 12 min, and from 90
to 50% A between 12 and 15 min. The column effluent was monitored at
540 nm, referenced against an 800 nm wavelength. Data analysis was
performed using Agilent ChemStation. The values obtained from the
integration of the azo dye peak were interpolated into a calibration curve
obtained using standard solutions of sodium nitrite from 0.5 μM to 50 μM
-
µM (r2>0.99). The nitrite yield was expressed as percentage of NO2
(mol/mol, relative to the initial compound concentration) ± SEM.
Biological experiments.
The culture media was obtained from Invitrogen Life Technologies
(Carlsbad, CA), while plasticware for cell cultures was obtained from
Falcon (Becton Dickinson, Franklin Lakes, NJ). The protein content of cell
monolayers and lysates was assessed using the BCA kit from Sigma
Chemical Co (St. Louis, MO). Unless otherwise specified, all the reagents
were obtained from Sigma Chemical Co. The test compounds were
dissolved in dimethyl sulfoxide (DMSO), and then diluted in culture
medium to achieve the final concentration. The concentration of DMSO in
the culture medium in all the experiments was lower than 0.1%; a
concentration that did not affect cell viability (data not shown).
-
(r2=0.996). The nitrite yield was expressed as percent NO2 (mol/mol,
relative to the initial compound concentration), ±SEM.
Cytotoxicity. The cytotoxic effect of the compounds was measured as the
leakage of lactate dehydrogenase (LDH), activity into the extracellular
medium using a Synergy HT microplate reader (Bio-Tek Instruments,
Winooski, VT), as previously described.[31] Both intracellular and
extracellular LDH were measured and then extracellular LDH activity (LDH
out), was calculated as a percentage of the total (intracellular +
extracellular), LDH activity (LDH tot), in the dish.
Cells. Human lung adenocarcinoma A549 cells, provided by Istituto
Zooprofilattico Sperimentale “Bruno Ubertini” (Brescia, Italy), were
cultured in Ham’s F12 medium supplemented with 10% foetal bovine
serum and 1% penicillin-streptomycin. Cell cultures were kept in a
humidified atmosphere at 37°C and 5% CO2. When indicated, cells were
placed in Ham’s F12 medium without phenol red and exposed for 30
minutes to the light emitted by a 400 nm, 10W violet led, using an
irradiance of 7 mW/cm2. Non-irradiated cells were kept in a dark room for
30 minutes in Ham’s F12 medium without phenol red at room temperature.
After this period, cells were left for 19.5 h in the incubator before the
experimental procedures described below were carried out.
Statistical analysis. All data are provided as means ± SEM. Results were
analysed using one-way Analysis of Variance (ANOVA), and Turkey’s test
(software: SPSS 21.0 for Windows, SPSS Inc., Chicago, IL). p<0.05 was
considered significant.
Mitochondria accumulation. For the mitochondrial accumulation
experiments, A549 cells were incubated for 4 h with the compounds and
the mitochondria were then extracted, as described earlier.[30] A 50 μL
aliquot was sonicated and used for protein content measurements and
Western blotting. The remainder was stored at −80°C until use. 10 μg of
each sonicated sample was subjected to SDS-PAGE and probed with an
anti-VDAC/porin antibody in order to confirm the presence of mitochondrial
proteins in the extracts (Abcam; data not shown). The quantity of
compounds (2, 11-14), in the mitochondrial and cytosolic fractions was
measured using an Acquity Ultra Performance LC (Waters Corporation
Milford MA, USA), equipped with BSM, SM, CM and PDA detectors. The
analytical column was a Zorbax Eclipse XDB-C18, 150 × 4.6 mm × 5 µm.
The mobile phase consisted of CH3CN 0.1% HCOOH/H2O 0.1% HCOOH
70/30 v/v for compound 2, and CH3CN 0.1% HCOOH/H2O 0.1% HCOOH
60/40 v/v for compounds 11-14. UPLC retention time (tR), was obtained at
flow rates of 0.5 mL/min and the column effluent was monitored using a
Micromass Quattro micro API Esci with multi-mode ionization enabled, as
the detector. The molecular ions [M+H]+ for 2 and [M]+ for 11-14 were used
for the quantitative measurements of the analyte. MS conditions were as
follows: drying gas (nitrogen), heated at 350°C at a flow rate of 800 L/h;
nebulizer gas (nitrogen), at 80 L/h; capillary voltage in positive mode at
3000 V; fragmentor voltage at 30 V. The values obtained from the
integration of compound peaks were interpolated into calibration curves
obtained using standard solutions at 0.1 μM to 5 µM (r2=0.996). The
quantity of compounds in the mitochondrial and cytosolic fractions was
expressed as nmol/(mg protein).
Acknowledgements
We thank AIRC, Project IG-19859 and the University of Turin
(Ricerca Locale ex 60%) for financial support.
We thank the Centro di Competenza sul Calcolo Scientifico (C3S)
at the University of Turin (c3s.unito.it) for providing computational
time and resources.
Keywords: mitochondria • phosphonium salts • nitric oxide •
targeted therapy • photoactivation
References:
[1]
a) J. F. Kerwin Jr., J. R. Lancaster, P. L. Feldman, J. Med. Chem. 1995,
38, 4343-4362; b) J. F. Kerwin Jr., M. Heller. Med. Res. Rev. 1994, 14,
23-74.
[2]
a) Z. Huang, J. Fu, Y. Zhang, J. Med. Chem. 2017, 60, 7617–7635; b) Q.
Ma, Z. Wang, M. Zhang, H. Hu, J. Li, D. Zhang, K. Guo, H. Sha, Current
Pharmaceutical Design 2010, 16, 392-410.
[3]
[4]
[5]
A. D. Ostrowski, P. C. Ford, Dalton Trans. 2009, 48, 10660-10669.
S. Huerta, B. Chilka, B. Bonavida, J. Oncol. 2008, 33, 909-927.
See, for example: a) S. Sortino, Chem. Soc. Rev., 2010, 39, 2903; b) P.
C. Ford, Acc. Chem. Res., 2008, 41, 190; c) N. L. Fry , P. K. Mascharak,
Acc. Chem. Res., 2011, 44, 289; d) P. C. Ford, Nitric Oxide, 2013, 34,
56; e) A. Fraix, S. Sortino, Chem. Asian J., 2015, 10, 1116; E) A. Fraix,
N. Marino, S. Sortino, Top. Curr. Chem., 2016, 370, 225.
P. Agostinis, K. Berg, K. A. Cengel, T. H. Foster, A. W. Girotti, S. O.
Gollnick, S. M. Hahn, M. R. Hamblin, A. Juzeniene, D. Kessel, M.
Korbelik, J. Moan, P. Mroz, D. Nowis, J. PietteB. C. Wilson, J. Golab, CA
Cancer J. Clin. 2011, 61, 250-281.
NO-photorelease in cells. Cells were incubated and irradiated as
described in the Cells section. 1 mL of cell supernatant was then collected
and centrifuged for 10 minutes at 13000g. The presence of nitrite in the
supernatants was determined using the Griess assay; 0.5 mL of the
reaction mixture was treated with 125 mL of the Griess reagent (4% w/v
sulfanilamide, 0.2% w/v N-naphthylethylenediamine dihydrochloride, 1.47
mM phosphoric acid), after 10 min at room temperature, the sample was
analyzed by RP-HPLC in order to detect the azo dye. HPLC analyses were
performed using a HP 1200 chromatograph system (Agilent Technologies,
Palo Alto, CA, USA), equipped with a quaternary pump (model G1311A),
a membrane degasser (G1322A), and a multiple wavelength UV detector
[6]
[7]
[8]
C. Fowley, A. P. McHale, B. McCaughan, A. Fraix, S. Sortino and J. F.
Callan, Chem. Commun. 2015, 51, 81--84
a) S. Fulda, L. Galluzzi, G. Kroemer, Nature Reviews 2010, 9, 447-463;
b) M. C. Frantz, P. Wipf, Environ Mol. Mutagen 2010, 51, 462-475; c) S.
Wen, D. Zhu, P. Huang, Future Med. Chem. 2013, 5, 53-67.
7
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201800088
ChemMedChem
FULL PAPER
[9]
S. Moncada, J. D. Erusalimsky, Nat. Rev. Mol. Cell. Biol. 2002, 3, 214-
220.
[10] G. C. Brown, Front. Biosci. 2007, 12, 1024-1033.
[11] T. Horinouchi, H. Nakagawa, T. Suzuki, K. Fukuhara, N. Miyata, Bioorg.
Med. Chem. Lett. 2011, 21, 2000-2002.
[12] T. Horinouchi, H. Nakagawa, T. Suzuki, K. Fukuhara, N. Miyata, Chem.
Eur. J. 2011, 17, 4809-4813.
[13] J. Xu, F. Zeng, H. Wu, C. Hu, C. Yu, S. Wu, Small 2014, 10, 3750-
3760.
[14] J. Xu, F. Zeng, H. Wu, S. Wu, J. Mater. Chem. 2015, 3, 4904-4912.
[15] F. Sodano, E. Gazzano, A. Fraix, B Rolando, L. Lazzarato, M. Russo, M.
Blangetti, C. Riganti, R. Fruttero, A. Gasco, S. Sortino, ChemMedChem.
2018, 13, 87-96.
[16] a) T. Suzuki, O. Nagae, Y. Kato, H. Nakagawa, K. Fukuhara, N. Miyata,
J. Am. Chem. Soc. 2005, 127, 11720 –11726; b) K. Kitamura, N. Ieda, K.
Hishikaw, T. Suzuki, N. Miyata, K. Fukuhara, H Nakagawa, Med. Chem.
Lett. 2014, 24, 5660 –5662.
[17] a) E. B. Caruso, S. Petralia, S. Conoci, S. Giuffrida, S. Sortino, J. Am.
Chem. Soc. 2007, 129, 480−481; b) S. Conoci, S. Petralia, S. Sortino,
EP 2051935A1/US 20090191284, 2006.
[18] J. Zielonka, J. Joseph, A. Sikora, M. Hardy, O. Ouari, J. Vasquez-Vivar,
G. Cheng, M. Lopez, B. Kalyanaraman, Chem. Rev. 2017, 117, 10043–
10120.
[19] A.P. Trotta, J.E. Chipuk, Cell. Mol. Life Sci. 2017, 74, 1999–2017.
[20] M.C. Caino, J.C. Ghosh, Y.C. Chae, V. Vaira, D.B. Rivadeneira, A.
Faversani, P. Rampini, A.V. Kossenkov, K.M. Aird, R. Zhang, M.R.
Webster, A.T. Weeraratna, S. Bosari, L.R. Languino, D.C. Altieri, Proc.
Natl. Acad. Sci. USA. 2015, 112, 8638–8643.
[21] L. García-Ledo, C. Nuevo-Tapioles, C. Cuevas-Martín, I. Martínez-
Reyes, B. Soldevilla, L. González-Llorente, J.M. Cuezva, Front. Oncol.
2017, 7, 69.
[22] C. Riganti, E. Gazzano, G.R. Gulino, M. Volante, D. Ghigo, J. Kopecka,
Cancer Lett. 2015, 360, 219–226.
[23] I. Buondonno, E. Gazzano, S.R. Jean, V. Audrito, J. Kopecka, M. Fanelli,
I.C. Salaroglio, C. Costamagna, I. Roato, E. Mungo, C.M. Hattinger, S.
Deaglio, S.O. Kelley, M. Serra, C Riganti, Mol Cancer Ther. 2016, 15,
2640–2652.
[24] J. Modica-Napolitano, J. Aprille, J. Adv. Drug. Deliv. Rev. 2001, 49, 63–
70.
[25] A. Fraix, S. Guglielmo, V. Cardile, A. C. E. Graziano, R. Gref, B. Rolando,
R. Fruttero, A. Gasco, S. Sortino, RSC Adv. 2014, 4, 44827 –44836.
[26] A. Fraix, M. Blangetti, S. Guglielmo, L. Lazzarato, N. Marino, V. Cardile,
A. C. E. Graziano, I. Manet, R. Fruttero, A. Gasco, S. Sortino,
ChemMedChem 2016, 11, 1371-1379.
[27] K. Chegaev, A. Fraix, E. Gazzano, G. E. F. Abd-Ellatef, M. Blangetti, B.
Rolando, S. Conoci, C. Riganti, R. Fruttero, A. Gasco, S. Sortino ACS
Med. Chem. Lett. 2017, 8, 361–365.
[28] F. L. Callari, S. Sortino, Chem. Commun. 2008, 17, 1971-1973.
[29] C. Hansch, A. J. Leo in ACS Professional Reference Book. American
Chemical Society, American Chemical Society, Washington DC, 1995.
[30] I. Campia, C. Lussiana, G. Pescarmona, D. Ghigo, A. Bosia, C. Riganti,
Br. J. Pharmacol. 2009, 158, 1777-1786.
[31] E. Aldieri, I. Fenoglio, F. Cesano, E. Gazzano, G. Gulino, A. Scarano, D.
Attanasio, G. Mazzucco, D. Ghigo, B. Fubini, J. Toxicol. Environ. Health
A. 2013, 76, 1056-1071.
[32] E. Gazzano, B. Rolando, K. Chegaev, I.C. Salaroglio, J. Kopecka, I.
Pedrini, S. Saponara, M. Sorge, I. Buondonno, B. Stella, A. Marengo, M.
Valoti, M. Brancaccio, R. Fruttero, A. Gasco, S. Arpicco, C. Riganti, J.
Control. Release 2018, 270, 37-52.
[33] B. Bachowska, J. Kazmierczak-Baranska, M. Cieslak, B. Nawrot, D.
Szczesna, J. Skalik, P. Balczewski, Chemistry Open 2012, 1, 33-38.
[34] B. Hess, C. Kutzner, D. Van der Spoel, E. Lindahl, J. Chem. Theory
Comput. 2008, 4, 435−447.
[35] G. Bussi, D. Donadio, M. Parrinello, J. Chem. Phys. 2007, 126, 014101.
8
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Entry for the Table of Contents
Compound 13
NO-photodonor unit
Targeting mitochondria. A few NO-photodonor alkyltriphenylphosphonium compounds were synthesized in order to modulate their
lipophilicity and ability to release NO under the control of light irradiation. Compound 13 showed the highest tendency to assume a
globular structure. It exhibited extended accumulation in mitochondria of human lung adenocarcinoma A549 cells and displayed high
toxicity upon irradiation-mediated NO release in mitochondria.
9
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