Continuous flow synthesis of lipophilic cations derived from benzoic acid as new cytotoxic chemical entities in human head and neck carcinoma cell lines

Article abstract of DOI:10.1039/d0md00153h

Continuous flow chemistry was used for the synthesis of a series of delocalized lipophilic triphenylphosphonium cations (DLCs) linked by means of an ester functional group to several hydroxylated benzoic acid derivatives and evaluated in terms of both reaction time and selectivity. The synthesized compounds showed cytotoxic activity and selectivity in head and neck tumor cell lines. The mechanism of action of the molecules involved a mitochondrial uncoupling effect and a decrease in both intracellular ATP production and apoptosis induction. This journal is

Full text of DOI:10.1039/d0md00153h

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RESEARCH ARTICLE
Continuous flow synthesis of lipophilic cations
derived from benzoic acid as new cytotoxic
chemical entities in human head and neck
carcinoma cell lines†
Cite this: DOI: 10.1039/d0md00153h
Mabel Catalán,‡a Vicente Castro-Castillo,‡§b Javier Gajardo-de la Fuente,b
Jocelyn Aguilera,c Jorge Ferreira,a Ricardo Ramires-Fernandez,d Ivonne Olmedo,e
Alfredo Molina-Berríos,c Charlotte Palominos,a Marcelo Valencia,a
f
c
Marta Domínguez, José A. Souto f and José A. Jara
*
*
Continuous flow chemistry was used for the synthesis of
a
series of delocalized lipophilic
Received 13th May 2020,
Accepted 30th July 2020
triphenylphosphonium cations (DLCs) linked by means of an ester functional group to several hydroxylated
benzoic acid derivatives and evaluated in terms of both reaction time and selectivity. The synthesized
compounds showed cytotoxic activity and selectivity in head and neck tumor cell lines. The mechanism of
action of the molecules involved a mitochondrial uncoupling effect and a decrease in both intracellular
ATP production and apoptosis induction.
DOI: 10.1039/d0md00153h
rsc.li/medchem
mitochondrial transmembrane potential (ΔΨm) and ATP
concentrations, consequently inducing apoptosis and
1. Introduction
Delocalized lipophilic triphenylphosphonium cations (DLCs)
linked to gallic acid (2,3,4-trihydroxybenzoic acid) and its
derivatives have been synthesized and evaluated previously by
our group, resulting in their characterization as antineoplastic
compounds in a breast adenocarcinoma mouse model. All this
work has allowed the identification of several lead compounds,
with promising results both in vivo and in vitro.1 It has been
demonstrated that these gallic acid (GA) alkyl esters block the
electron transport chain by inhibiting OXPHOS (oxidative
phosphorylation).2 Moreover, DLC–GA derivatives induce a
decoupling effect, decreasing the NADH/NAD+ ratio, the
antitumor effects.1 On the other hand, DLC-benzoic acid
derivatives with two hydroxyl groups, such as protocatechuic
acid (3,4-dihydroxybenzoic acid) and gentisic acid (2,5-
dihydroxybenzoic acid), have shown chemopreventive, cytotoxic
and antiangiogenic activities in human tumor cells.3–6
Furthermore, DLC-2,3-dihydroxybenzoic acid has shown
antioxidant and antifungal activities.7,8 Despite the promising
biological results obtained, the low overall yields associated
with the synthesis of these derivatives together with the long
reaction times needed for their preparation are far from an
optimal drug delivery process and have prevented intensive
biological characterization of lead compounds (Fig. 1).
Therefore, the development of robust, efficient and
reproducible synthetic protocols for the preparation of active
molecules at a very early stage in the compound development
pipeline warrants large quantities of material needed for the
different stages of biological evaluation while increasing the
speed of the eventual process of scaling up lead compounds.9
Continuous flow processing stands out among the enabling
technologies available (e.g. microwave assisted synthesis,
high throughput catalyst screening platforms) for the
synthesis of active pharmaceutical ingredients (APIs).9–12
Advantages of this technique, compared to traditional batch
procedures, include the observed enhancement of mass and
heat transfer or the use of high-temperature and high-
pressure reaction conditions, among others, which results in
more efficient mixing of reagents and precise control of
a Clinical and Molecular Pharmacology Program, Institute of Biomedical Sciences
(ICBM), Faculty of Medicine, Universidad de Chile, Santiago, 8380453, Chile
b Department of Organic and Physical Chemistry, Faculty of Chemical and
Pharmaceutical Sciences, Universidad de Chile, Santos Dumont 964, Santiago
8380494, Chile
c Institute for Research in Dental Sciences (ICOD), Faculty of Dentistry, Universidad
de Chile, Santiago, 8380492, Chile. E-mail: jsandovalj@u.uchile.cl;
Tel: +56 2 29781730
d Dentistry School, Universidad Mayor, Santiago 8340585, Chile
e Physiopathology Program, Institute of Biomedical Sciences (ICBM), Faculty of
Medicine, Universidad de Chile, Santiago 8380453, Chile
f Departamento de Química Orgánica, Facultad de Química, CINBIO and IIS
Galicia Sur, Universidade de Vigo, E-36310, Vigo, Spain. E-mail: souto@uvigo.es
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0md00153h
‡ These authors contributed equally in this manuscript.
§ In memoriam of Dr. Vicente Castro-Castillo (1981–2019).
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Fig. 1 A selection of DLCs previously synthesized and biologically characterized.
reaction parameters.13–18 This results in high performance of
Consequently, these treatments markedly lead to a
the developed reaction in terms of reaction selectivity,
product yield, low waste production and reduced
cost.11,13,19,20
Head and neck squamous cell carcinoma (HNSCC) is one
of the most common types of cancer worldwide with over
500 000 new cases reported annually.21
HNSCCs are a high incidence decease, and despite the
progress in cancer research and therapies, the survival rate
has not improved significantly in the last years, representing
a continuing challenge for biomedical science, thus is a
highly critical important problem for global public health,
especially for dental health.
significant deterioration in the patient quality of life.
Furthermore, the treatment with chemotherapy has high
rates of resistance. In this clinical context, the search of new
molecules with antineoplastic potential it is crucial for
efficacy improvement and reduce drug resistance.
Given the previously mentioned need to obtain products
in high yield with short reaction times, easy purification, and
chemical scalability for their biological evaluation, in the
present work, we developed and optimized a telescoped,
continuous-flow synthetic methodology to prepare different
derivatives of poly-hydroxy-DLC esters bearing a variable
number of phenolic groups.
Although most of time HNSCC is considered a preventable
condition, due to the possibility of early detection and
treatment, the mainstream of these cancers are diagnosed in
a late phase (in state III or IV). Unfortunately, in state III or
IV, a combination of surgery and/or chemo/radiation therapy
provides the best treatment available, these therapies
produce serious adverse effects, like difficulties in talking,
swallowing and performing oral hygiene, as well as increased
dental caries and ulceration.22
2. Results and discussion
2.1 Chemistry
In our seminal work on the synthesis and biological
characterization of DLC derivatives of polyhydroxy benzoates,
we have been able to synthesize
a broad number of
compounds resulting in the identification of several lead
molecules. That synthetic pathway contemplated as a first
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step the formation of
a
phosphonium salt using
the reactor was increased (Table 1, entries 4 and 5).
triphenylphosphine and different bromoalcohols with various
chain lengths (n = 8, 10, 11 and 12). The target compounds
were isolated in moderate yields (31–70%) within 48 h.
Subsequently, Steglich esterification resulted in the isolation
of the desired esters, with yields ranging from 37 to 44%
Surprisingly, increasing the concentration of the starting
materials to near saturation resulted in higher yields for our
reaction (Table 1, entries 6 and 7). Finally, an increase in the
reactor temperature afforded the desired product in 92%
isolated yield with a residence time of 200 min and 2.2 g h−1
of reaction throughput. Furthermore, the system was allowed
to run for more than 5 h without any observed clogging of
the reactor.
Next, we focused on the development of the esterification
reaction under continuous-flow regime (Fig. 3).
For that purpose, and based on a previously described
batch procedure,1 an equimolar mixture of phosphonium salt
3a and gallic acid 4a in DMF merged with a solution of
after 24
h of reaction. Furthermore, in both steps,
chromatographic separation was necessary in order to purify
the products from the secondary compounds formed during
the reaction.1
In order to access to enough material of previously
described lead compounds for further biological
characterization, we initiated a study on the optimization of
the previously described batch reactions under continuous
flow conditions. For optimization purposes only, the
commercially available and inexpensive 11-bromoundecanol
2a was selected as the starting material (Fig. 2).
As an initial point, previously reported batch reaction
conditions1 were extrapolated to create a continuous flow
regime. Hence, 2a was dissolved in acetonitrile, and
equimolar amounts of triphenylphosphine 1 were added. The
resulting mixture was introduced into a 5 mL coiled reactor
(0.25 mL min−1, 20 min residence time), obtaining the
desired product, albeit in low yield (Table 1, entry 1).
Increasing the residence time to 40 min by means of
reducing the flow rate and increasing the concentration of
reagents slightly improved the yield of the reaction (Table 1,
entries 2 and 3). Further improvement was observed when
the flow rate was reduced to 0.1 mL min−1 and the volume of
dicyclohexylcarbodiimide
(DCC)
and
4-N,N-
dimethylaminopyridine (DMAP) in DMF, and the outstream
liquid was introduced in a 15 mL coiled reactor at room
temperature. Under these conditions, 5aa was isolated in low
yield together with secondary products that might arise by
the self-reaction of activated gallic acid. Furthermore, the
formation of a white precipitate caused reactor blockage after
a long operation time (Table 2, entry 1). The use of CH3CN as
the solvent and a decrease in residence time did not afford
satisfactory yields (Table 2, entries 2 and 3). A dramatic
increase in reaction performance was obtained when a
solution of 4a in CH3CN was reacted with another solution
containing 3a, DCC and DMAP in CH3CN. However, despite
the formation of the desired product in 70% yield, the
precipitation of di-cyclohexyl urea clogged the reactor,
Fig. 2 Continuous flow set-up for the formation of the phosphonium salt 3a.
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Table 1 Optimization of the formation of phosphonium salt 3a
Entry
φ (mL min−1
)
[1] (M)
[2a] (M)
V (mL)
T (°C)
Yielda (%)
1
2
3
4
5
6
7
8
0.25
0.125
0.125
0.1
0.1
0.1
0.12
0.12
0.19
0.19
0.19
0.38
0.74
0.74
0.12
0.12
0.19
0.19
0.19
0.38
0.74
0.74
5
5
5
15
20
20
20
20
110
110
110
110
110
110
110
120
8
15
21
25
54
74
92
0.1
0.1
96 (92)
a
NMR yields. Isolated yield between brackets.
Fig. 3 Continuous flow formation of ester 5aa.
Table 2 Optimization of the esterification reaction
Entry
Solvent
LA (x, M)
LB (x, M)
V (mL)
T (°C)
Yielda (%)
1
2
3
4
5
6
7
8
DMF
3a, 4a; 0.1, 0.1
3a, 4a; 0.1, 0.1
3a, 4a; 0.1, 0.1
4a; 0.1
4a; 0.1
4a; 0.1
DCC, DMAP; 0.1, 0.001
DCC, DMAP; 0.1, 0.001
DCC, DMAP; 0.1, 0.001
3a, DCC, DMAP; 0.1, 0.1, 0.001
3a, DCC, DMAP; 0.1, 0.1, 0.001
3a, T3P, TEA; 0.1, 0.1, 0.2
3a, T3P, TEA; 0.1, 0.1, 0.2
3a, T3P, TEA; 0.1, 0.1, 0.2
15
15
5
5
5
5
10
10
25
25
25
25
50
25
25
50
11b
CH3CN
CH3CN
CH3CN
CH3CN
CH3CN
CH3CN
CH3CN
13b
17b
70b,c
74b,c
82
4a; 0.1
4a; 0.1
89 (85)
76
a
b
NMR yields. Isolated yield between brackets. Reactor blockage. c Ultrasonic irradiation.
preventing the reaction from being carried out in this way
(Table 2, entry 4). Any attempts to overcome these problems,
applying different solutions previously reported in the
literature to avoid clogging issues derived from particle
aggregation,23–26 were unsuccessful (Table 2, entries 4 and 5).
Therefore, the coupling agent was changed to
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propanephosphonic acid
Research Article
anhydride
(T3P®),
and
exhibit compared to derivatives of 2a,1
a short but
triethylamine (TEA) was used as the base.27 Under these
conditions, we were able to isolate 5aa in 82% yield, without
any blockage of the reactor noticed (Table 2, entry 6).
Increasing the residence time by doubling the reactor volume
had a beneficial effect on the yield of the reaction, while
increasing the temperature did not afford better results
(Table 2, entries 7 and 8).
representative set of already described lead DLC
benzoylesters was prepared. Thus, the reaction of 2b with
different benzoic acid derivatives (4a–e) under the above
described conditions afforded the targeted molecules,
demonstrating the robustness of our methodology while
affording enough material of most of the active compounds
previously reported1,3,28 for further biological characterization
to be carried out (Fig. 5).
To our delight, the reaction conditions independently
optimized for each step were straightforwardly telescoped for
the continuous production of 5aa without isolation of the
2.2 Biology
intermediate phosphonium salt 3a. Hence,
a solution
containing equimolar amounts of 1 and 2a in acetonitrile
was injected at a flow rate of 0.1 mL min−1 into a 20 mL
reactor at 120 °C. The reactor outstream liquid merged into a
0.5 mL coiled reactor with a solution of T3P (1 equiv.) and
TEA (2 equiv.) in acetonitrile (0.1 mL min−1) prior to reacting
with a third solution of gallic acid (1 equiv.) in acetonitrile
(0.2 mL min−1). The resulting mixture was introduced into a
10 mL reactor at room temperature and finally collected to
afford desired product 5aa in 74% isolated yield. Under the
described reactor setup, the reactor ran for up to 5 h without
obstruction (Fig. 4).
2.2.1. Cytotoxic effect of benzoic acid-derived lipophilic
cations in HNSCC cell lines. The cytotoxicity results for all
cell lines were plotted on a semilogarithmic graph (Fig. S1†),
which allowed us to determine the concentration needed to
obtain 50% cell viability (IC50) for each compound assayed at
24, 48 and 72 h in human HNSCC lines and in a normal
human oral keratinocyte cell line (Table 3).
The results shown in Table 3 suggest that all assayed
compounds had similar IC50 values, approximately 2–5 μM in
Cal 27 cells, 2–4.5 μM in SCC-15 cells, and 2–12 μM in HEp-2
cells. Additionally, the hydroxylated benzoic acid derivatives
of this series were more potent than cisplatin in all the tumor
cell lines tested. On the other hand, the IC50 values for oral
keratinocyte cells (OKF6/TERT, nontumor cells) were
With the optimized conditions in hand for the continuous
flow formation of DLC esters and considering the higher
activity that DLC esters derived from 10-bromodecanol 2b
Fig. 4 Two-step flow sequence for the telescoped preparation of DLCs esters.
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Fig. 5 Set of DLCs esters synthesized under flow regime.
significantly higher than those observed in the Cal-27 tumor
cell line, thus showing that hydroxylated benzoic acid esters
linked to TPP+ are selective for the Cal-27 and SCC-15 cell
lines (Table 4). In the case of PIA-TPP+C10 (5ba) in HEp-2
cells, there is clearly no selectivity, as the IC50 values for
laryngeal tumor cells are close to the IC50 values in the OKF-
6/TERT non-tumor cells (Table 4).
Table 3 The cytotoxic effect of TPP+-benzoates on HNSCC cell lines
(Cal27, HEp-2, SCC-15) and oral keratinocyte (OKF-6/TERT), and
selectivity index IC50 = the concentration required to induce cytotoxicity
effects in 50% of HNSCC cells and oral keratinocyte cells after 48 h of
treatment. Compounds BA-C10 (benzoic acid decyl ester), and PIA-C10
(2,3-dihydroxybenzoic acid decyl ester) were used as controls to evaluate
the effects of inclusion of TPP+ moiety in compounds (structures in
Fig. 1). Each assay was performed in triplicate. Key: *p ≤ 0.05 with respect
to the effect of TPP+C10 (3,4,5-trihydroxybenzoic acid decyl ester) at the
same cell line time; ***p ≤ 0.001 with respect to the effect of TPP+C10 at
the same cell line and time; a = p ≤ 0.05 respect to the effect at the
same time in Cal-27 cell line
The results obtained in the SCC line suggest that every
compound assayed exerted a cytotoxic effect with similar IC50
values (<2 μM after 72 h of incubation, Table S1 in the ESI†)
and had similar selectivity. However, the IC50 values did not
correlate with the number and position of the hydroxyl
groups present on the benzoic acid moiety (pharmacophore
group), although the values were significantly different
compare to non-tumor cells ( p < 0.05). According to results
obtained previously for these compounds in breast cancer
cells, it has been suggested that cytotoxic activity is achieved
with the presence of at least one hydroxyl group as a
substituent on benzoic acid.3 Apparently, modification of the
number of hydroxyl groups in the benzoic acid ring and their
position does not significantly affect the cytotoxic activity in
the tumor cells assessed. Furthermore, triphenylphosphine
does not have cytotoxic activity in the cell lines assessed in
this work (data not shown). Furthermore, the IC50 values of
the hydroxylated benzoic acid derivatives were similar or
IC50 at 48 h (μM)
Cell lines
Compounds
Cal-27
HEp-2
SCC-15
OKF-6/TERT
SA-TPP+C10 (5be) 2.3 0.06* 4.28 0.12 3.2 0.2 12.7 0.2a
GA-TPP+C10
(5bd)
2.2 0.02* 5.66 1.09 2.7 0.1* 13.2 0.4a
1.6 0.01* 8.28 0.27 2.9 0.07 7.5 0.1a
PIA-TPP+C10
(5bb)
TPP+C10 (5ba)
Cisplatin (CDDP) 4.7 0.9
3.4 0.1
12.52 0.94 3.0 0.09 13.1 0.4
5.2 0.7 12.5 1.2 3.2 0.2
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Table 4 Selectivity index for benzoates-TPP+ derivatives at 48 h values were calculated from IC50 ratio between non-tumor cell and tumor cell lines
from the respective sigmoidal dose–response curves
Selectivity index (48 h)
Compounds
OKF-6 TERT/Cal-27
OKF-6 TERT/HEp-2
OKF-6 TERT/SCC-15
SA-TPP+C10 (5be)
GA-TPP+C10 (5bd)
PIA-TPP+C10 (5bb)
TPP+C10 (5ba)
5.5
6.0
4.7
3.0
2.3
0.9
4.0
4.9
2.6
3.8
1.0
4.4
Cisplatin (CDDP)
<1.0
<1.0
<1.0
lower than the IC50 values for cisplatin, a gold standard
therapy in HNSCC tumors (Table 3). Moreover, our
compounds were more selective than cisplatin for tumor
versus nontumor cells (Table 4).
from the OCR experiments (Fig. 7) suggest that all
compounds were able to induce a mitochondrial uncoupling
effect in a concentration-dependent manner in the tongue
carcinoma cell line Cal-27 but with different efficacies
(Fig. 7B–D). Indeed, SA-TPP+C10 (5be) and TPP+C10 (5ba) did
not accomplish the maximal uncoupling effect induced by
the classical uncoupling agent CCCP (Fig. 7B and E).
However, GA-TPP+C10 (5bb) and PIA-TPP+C10 (5bd) did reach
the maximal effect induced by CCCP (Fig. 7C and D). These
uncoupling effects may be implicated in the selectivity of
each compound, but more experiments are needed to
establish the relationship.
The results show that the compounds exhibited OXPHOS
uncoupling effects. However, the uncoupling effect of SA-
TPP+C10 (5be) and TPP+C10 (5ba) was less potent than that of
CCCP, the classical uncoupling agent. This could be
favourable because it implies low toxicity of SA-TPP+C10 (5be)
2.2.2. Anticlonogenic effect of benzoic acid-derived
lipophilic cations in Cal 27 cell line. In order to evaluate the
consequences of inducing an antiproliferative effect in the
Cal 27 cell line, we determined the effects of the benzoate
derivatives on the clonogenic capacity of these cells. Fig. 6
shows that all the compounds tested were able to inhibit this
capacity in a concentration-dependent manner (Fig. 6A–D).
2.2.3. Mechanism of action of benzoic acid-derived
lipophilic cations in HNSCC cell lines. To determine the
mechanism of action of the benzoic acid derivatives, we
evaluated the mitochondrial oxygen consumption rate (OCR),
ΔΨm and intracellular levels of ATP after treatment of the
cells with the different compounds (Fig. 7 and 8). The results
Fig. 6 Effects of benzoate-TPP+ derivatives on the colony formation of HNSCC cells (Cal 27 cell line). Figures A–D shows quantification of 3
independent experiment of SA-TPP+C10 (5be), GA-TPP+C10 (5bb), PIA-TPP+C10 (5bd), and TPP+C10 (5ba), respectively. Data were compared to the
control group through ANOVA followed by the Bonferroni post-test. Key: *p < 0.05, **p < 0.01 ***p < 0.001.
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Fig. 7 Oxygen consumption rate. Tumour cell respiration was measured using a Clark electrode coupled to a personal computer. 5 × 106 Cal-27
cells were used per experimental measurement. 0.6 mL cell suspension in PBS (pH 7.4) at 25 °C, and then 8.3 mM of L-glutamine were added in
the electrode chamber. Respiration rates were inhibited by 2.5 μg ml−1 oligomycin, and OXPHOS was fully uncoupled by 0.133 μM FCCP, used as a
control of all experiments. Graph A shows a representative sequence of events for each experiment. Figures B–E show the uncoupling effects of
SA-TPP+C10 (5be), GA-TPP+C10 (5bb), PIA-TPP+C10 (5bd) and TPP+C10 (5ba), respectively. The results shown are the averages of three independent
assays SD. Data were compared to the control group (ANOVA) followed by the Bonferroni post-test. Key: *p < 0.05, **p < 0.01 ***p < 0.001.
and TPP+C10 (5ba) in nontumor cells, as it is known that
powerful uncoupling compounds such as FCCP, CCCP or
2,4-dinitrophenol produce nonselective necrotic or apoptotic
death of normal cells, since all these compounds have been
demonstrated low selectivity, with reduced effective
concentration range.29,30 However, recently it has been
described new uncoupler agents with high potency and low
selectivity, but this compound are chemically different to our
benzoic acid lipophilic cations.31 The mechanism of action
proposed is different from that described for gallic acid
derivatives because diverse authors have shown an apoptotic
effect of GA derivatives through an increase in ROS
production.29 On the other hand, these results are
comparable with the effects observed for the same molecules
evaluated in parallel against colorectal cancer in vitro.28
Fig. 8 shows the decrease in both ΔΨm and intracellular
ATP levels caused by the action of the benzoic acid
derivatives in the Cal-27 and HEp-2 cell lines. Fig. 8A and C
show
a decrease in ΔΨm for all compounds assessed
independent of the concentration used. All molecules
induced similar potential decreases to 0.5 μM CCCP
(maximal uncoupling effect). Nevertheless, the effects of the
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Fig. 8 Effects of hydroxybenzoate-TPP+ derivatives on mitochondrial transmembrane potential and intracellular ATP levels. Panel A shows the
representative histograms of the effects of hydroxybenzoate-TPP+ derivatives on the mitochondrial transmembrane potential in CAL-27 cells.
Panel B shows the corresponding effects in HEp-2 cells. Panels C and D represent the percent ratio of the concentration-dependent effects of all
lipophilic cations with respect to controls in the Cal-27 and Hep-2 cell lines, respectively. Panels E and F similarly show the percent ratios of the
effects for 4 h of incubation of each cation on ATP levels in Cal-27 and HEp-2 cells, respectively. The values shown are the mean values SD of at
least three independent experiments. Data were compared to the control group through ANOVA followed by the Bonferroni post-test. Key: *p <
0.05, **p < 0.01 ***p < 0.001. N.S.: not statistical differences between groups.
lipophilic cations on the mitochondrial membrane potential
of HEp-2 cells was less potent than that observed in Cal-27
cells (Fig. 8B and D). SA-TPP+C10 (5be) and PIA-TPP+C10 (5bd)
showed a marked effect on mitochondrial potential, similar
to that observed with CCCP. Moreover, the intracellular ATP
contents decreased significantly in the Cal-27 cell line at each
concentration tested for GA-TPP+C10 (5bb) and PIA-TPP+C10
(5bd) but not in a concentration-dependent manner (Fig. 8E).
Similarly, the ATP levels observed in HEp-2 cells were
significantly reduced for all molecules, but the effects were
not proportional to the molecule concentration under any of
the conditions tested (Fig. 8F). Moreover, the data showed
that uncoupling causes a decrease in ATP levels, but this
decrease is lower than expected to induce cell death. This
behaviour could be explained by the activation of
compensatory mechanisms triggered in conditions of energy
deprivation, such as the activation of adenylate kinase or
AMPK (AMP-activated protein kinase).32,33 Indeed, the
uncoupling effects and the decrease in ATP levels and
mitochondrial transmembrane potential, which are elicited
by the TPP+ derivatives, induce energy stress and selectively
trigger tumor cell apoptosis. This cytotoxic mechanism may
represent a new alternative for inducing cell death in tumor
cells that are frequently multiresistant to chemotherapy and
new biological drugs.34–36
2.2.4. Benzoic acid-derived lipophilic cations induce
caspase 3 activation and apoptosis in HNSCC cell lines.
Finally, we determined whether the hydroxybenzoate
derivatives were able to induce apoptosis in the Cal-27 and
HEp-2 cell lines. We used the IC50 and
2 × IC50
concentrations for this experiment, to guaranty the cell death
effect, as we saw in the cytotoxicity assay. The results showed
that all compounds were able to induce apoptosis at a similar
level to the positive control staurosporine in the Cal-27 cell
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Fig. 9 Apoptosis and caspase 3 activation induced by hydroxybenzoate-TPP+ compounds. Representative dot-plots of PI and annexin V double
staining in Cal-27 cells (panel A) in the presence of 2.5 μM of each cation or 10 μM staurosporine (STS) as a positive control during 48 h of
treatment. Panel C shows the quantification of apoptotic Cal-27 cells. Panel B shows representative dot-plots of PI and annexin V double staining
in HEp-2 cells in the presence of 2.5 μM of each cation or 10 μM STS during 48 h of treatment. Panel D shows the quantification of apoptotic
HEp-2 cells. The activation of caspase 3 was evaluated at 48 h of incubation at two concentrations of the compounds E) SA-TPP+C10 (5be) F) PIA-
TPP+C10 (5bd) G) GA-TPP+C10 (5bb) and H) TPP+C10 (5ba), respectively. The values shown are the means
SD of at least three independent
experiments. The error bars show the 95% confidence interval. Data were compared to the control group through ANOVA followed by the
Bonferroni post-test. Key: *p < 0.05, **p < 0.01 ***p < 0.001; ns = no statistically significant difference.
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line (Fig. 9A and C). The apoptosis death in Cal 27 seems not
to be dependent of concentration, since at double
concentrations did not increase the cell death twice
(Fig. 9A and C). The percent of necrotic cells was not
significant (approximately 5% after 48 h), except in the case
of treatment with PIA-TPP+C10 (5bd) and GA-TPP+C10 (5bb)
(approximately 10% for both compounds after 48 h, p <
0.05). Furthermore, the effects of hydroxybenzoic acid
derivatives in HEp-2 cells were weaker than those induced by
the classical apoptosis inducer staurosporine but significant
with respect to the control (Fig. 9B and D). Similar to what
was seen in Cal-27 cells, the apoptosis-inducing effects in
HEp-2 cells was not concentration-dependent under our
experimental conditions.
To evaluate whether the compounds triggered the classical
apoptotic response, we also evaluated caspase 3 activation
through flow cytometry. Fig. 9E–H show that each compound
was able to induce caspase 3 activation in the Cal-27 cell line,
which was not dependent on the concentration. These results
suggest that hydroxybenzoate-TPP+ derivatives induce
apoptosis by activation of caspase 3 through the intrinsic
pathway.
it is known that tumor cells are frequently multiresistant to
both classical chemotherapeutic drugs and monoclonal
antibodies. Furthermore, metabolic reprogramming appears
to be a generalized phenomenon observed in many tumor
cells and an important feature in multidrug resistant cells,
which are present in solid tumors. In conclusion, our results
offer a series of molecules that are able to exert potent
cytotoxic effects in human tumor cell lines through the
induction of energy stress. These compounds might be
assessed in vivo either alone or in combination with old and
new chemotherapeutic drugs in order to evaluate the
possibility of improving current treatments.
Materials and methods
Chemicals and reagents
General procedures. Commercially available, laboratory
grade reagents were used without further purification. High
resolution mass spectrometry (HRMS, ESI+) was measured
with an Apex III FT ICR mass spectrometer (Bruker, Billerica,
USA). 1H-Nuclear magnetic resonance (NMR) spectra were
recorded in CDCl3, acetone-d6, and DMSO-d6 at room
temperature with a Bruker AMX-400 spectrometer (Bruker,
Billerica, USA) operating at 400.16 MHz with residual protic
solvent as the internal reference (DMSO-d6, δ = 2.50 ppm);
chemical shifts (δ) are given in parts per million (ppm) and
coupling constants ( J) are given in Hertz (Hz). The proton
spectra are reported as follows: δ (multiplicity, coupling
constant J, number of protons). 13C-NMR spectra were
recorded in CDCl3, and DMSO-d6 at room temperature with
the same spectrometer operating at 101.62 MHz with the
central peak of DMSO-d6 (δ = 39.50 ppm) as the internal
reference. DEPT-135 pulse sequences were used to aid in the
assignment of signals in the 13C-NMR spectra. 31P-NMR
spectra were recorded in DMSO-d6 at room temperature with
the same spectrometer operating at 162.13 MHz with
triphenylphosphine (δ = −7.00 ppm) as the internal reference.
Analytical TLC was performed on Merck silica gel 60 F254
chromatofoils. The purification of all products was carried
out on silica gel and the solvents used are specified in each
case.
3. Conclusion
Regarding the relevance of reproducing the in vitro effects
obtained and observing a significant antineoplastic effect in
animal models for which more product with high purity is
necessary, it is currently relevant to optimize synthetic routes
for new organic compounds with biological activity,
principally those that may reach effects at lower
concentrations, which may be easily achieved in systemic
circulation or in tissues. These kinds of molecules would be
useful for the treatment of pathologies that actually present
higher mortality and drug resistance rates, such as head and
neck cancer. In this work, we optimized the continuous flow
synthesis of up to five lipophilic cations, obtaining higher
yields and better selectivity than the previous route while
embracing the well-known advantages of performing
reactions in a continuous fashion.
With respect to the biological results, it is known that
head and neck cancers are highly lethal diseases, principally
because they are diagnosed in their later stages, thus
reducing the probability of successful treatments such as
surgery, radiotherapy and chemotherapy. Currently, a few
classical antitumor drugs are available as chemotherapy,
which can be used in the advanced stages of the tumors.
Indeed, in these patients, multimodal intervention is
DMEM
culture
medium,
carbonylcyanide
m-chlorophenylhydrazone (CCCP), glutamine, glucose, 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT),
dimethyl sulfoxide (DMSO), fetal bovine serum (FBS),
staurosporine, and trypan blue 0.4% solution, were
purchased from Sigma Chemical Co. (St. Louis, MO).
Cisplatin was purchased from Santa Cruz Biotechnology.
Penicillin/streptomycin and trypsin, were purchased from
Hyclone Laboratories (South Logan, UT). All other reagents
were purchased from Sigma (Sigma-Aldrich, USA).
frequently
required,
involving
the
use
of
new
chemotherapeutic drugs (such as monoclonal antibodies)
that have a pivotal role. Unfortunately, the new strategies
used in combination treatments for head and neck
squamous cell carcinoma have experienced unexpected
resistance. However, according to our results, energy stress
induction, as a mechanism of action, may represent an
alternative approach to induce death of tumor cells. Indeed,
Triphenyl(11-ol-undecyl)phosphonium bromide (3a)
General procedure for the continuous-flow synthesis of
phosphonium
salts.
A
solution
containing
triphenylphosphine (0.74 M in CH3CN) and 11-bromo-1-
undecanol (0.74 M in CH3CN) was reacted at 120 °C in a 20
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mL PFA reactor coil (0.1 mL min−1). The outcome stream was
collected during 5 min (steady state regime) and solvent was
evaporated under reduced pressure to isolate 187 mg (98%)
of colourless oil identified as triphenyl(11-ol-undecyl)
phosphonium bromide (n). 1H NMR (400 MHz, CDCl3): δ
7.94–7.69 (m, 15H), 3.67 (t, J = 6.6 Hz, 2H), 1.65–1.57 (m, 6H),
1.30–1.24 (m, 14H) ppm. 31P NMR (162 MHz, DMSO-d6): δ
24.04 ppm. MS (ESI) m/z (%) 435.27, 434.27, 433.27 (100, M+–
Br).
The combined stream was then combined in a T-piece with a
solution gallic acid (0.387 M in CH3CN) (0.2 mL min−1) and
reacted at rt in a 10 mL PFA reactor coil. The outstream
solution was collected during 5 min (steady state regime) and
the residue purified by column chromatography (silica gel,
DCM/MeOH 5%) to isolate 182 mg of a light yellow oil (74%
isolated
yield)
identified
as
triphenyl(11-((3,4,5-
bromide
trihydroxybenzoyl)oxy)-undecyl)phosphonium
(TPP+C11, 5aa).
Triphenyl(10-ol-decyl)phosphonium bromide (3b). Following
the general procedure previously described for the
continuous-flow synthesis of phosphonium salts the reaction
of triphenylphosphine (0.74 M in CH3CN) and 10-bromo-1-
decanol (0.74 M in CH3CN) afforded, after collection during 5
min (steady state regime) 181 mg (99%) of a colourless oil
identified as triphenyl(10-ol-decyl)phosphonium bromide (n).
1H NMR (400 MHz, CDCl3): δ 7.91–7.59 (m, 15H), 3.62 (t, J =
6.7 Hz, 2H), 1.70–1.11 (m, 18H) ppm. 31P NMR (162 MHz,
DMSO-d6): δ 24.03 ppm. MS (ESI) m/z (%) 421.26, 420.26,
419.25 (100, M+–Br).
Triphenyl(10-((3,4,5-trihydroxybenzoyl)oxy)-decyl)
phosphonium bromide (TPP+C10, 5ba). Following the general
procedure for the preparation of lipophilic phosphonium
bromide derivatives, the reaction of triphenylphosphine, 10-
bromo-1-decanol, T3P, Et3N, and gallic acid, afforded after
collecting during 5 min (steady state regime) 190 mg (79%)
of
a
colorless oil identified as triphenyl(10-((3,4,5-
trihydroxybenzoyl)ozy)-decyl)phosphonium
bromide
(TPP+C10, 5ba).
1H NMR (400 MHz, DMSO-d6): δ 7.90–7.73 (m, 15H, ArH),
6.98 (s, 2H, ArH), 4.12 (t, J = 6.2 Hz, 2H), 1.62–1.17 (m, 18H)
ppm. 13C NMR (100 MHz, DMSO-d6): δ 170.87 (s), 166.36 (s),
146.00 (s), 138.80 (s), 135.30 (d, JC–P = 3.0 Hz), 134.05 (d, JC–P
= 10.0 Hz), 130.68 (d, JC–P = 12.0 Hz), 120.04 (s), 119.05 (s,
JC–P = 85.0 Hz), 109.04 (d), 64.30 (t), 30.13 (t, JC–P = 17.0 Hz),
29.19 (t), 29.04 (t), 28.99 (t), 28.71 (t), 28.55 (t), 25.92 (t), 22.22
(t, JC–P = 4.0 Hz), 20.72 (t, JC–P = 5.0 Hz) ppm. 31P NMR (162
MHz, DMSO-d6): δ 24.03 ppm. HRMS calcd for C35H40O5P:
571.2608, found: 571.2610.
Triphenyl(11-((3,4,5-trihydroxybenzoyl)oxy)-undecyl)
phosphonium bromide (TPP+C11, 5aa)
General procedure for the stepwise continuous-flow synthesis
of lipophilic phosphonium bromide-benzoic acid derivatives.
A
solution containing triphenylphosphine (0.74 M in CH3CN)
and 11-bromo-1-undecanol (0.74 M in CH3CN) was reacted at
120 °C in a 20 mL PFA reactor coil (0.1 mL min−1). The
outcome stream was collected during 5 min (steady state
regime) and solvent was evaporated under reduced pressure
to isolate 176 mg (92%) of colourless oil identified as
triphenyl(11-ol-undecyl)phosphonium bromide (n). The crude
was then redissolved in CH3CN (3.2 mL, 0.1 M in CH3CN)
and T3P® (0.20 mL, 50% w/w in DMF) and triethylamine
(0.09 mL) were added. The resulting mixture was flowed (0.2
mL min−1) to react with a solution of gallic acid (4a) (57 mg)
in CH3CN (3.4 mL, 0.1 M in CH3CN) in a 10 mL coiled
reactor at room temperature. The outstream solution was
collected, solvent was removed under reduced pressure and
the obtained oily residue purified by chromatographic
column (silica gel, DCM/MeOH 5%) to isolate 192 mg of a
light yellow oil (85% isolated yield) identified as triphenyl(11-
((3,4,5-trihydroxybenzoyl)oxy)-undecyl)phosphonium bromide
(TPP+C11, 5aa).
Triphenyl(10-((2,5-dihydroxybenzoyl)oxy)-decyl)phosphonium
bromide (GA- TPP+C10, 5bb). Following the general procedure
for the preparation of lipophilic phosphonium bromide
derivatives, the reaction of triphenylphosphine, 10-bromo-1-
decanol, T3P, Et3N, and gentisic acid, afforded after
collecting during 5 min (steady state regime) 150 mg (73%)
of
a
colourless oil identified as triphenyl(10-((2,5-
dihydroxybenzoyl)oxy)-decyl)phosphonium bromide (GA-
TPP+C10, 5bb).
1H NMR (400 MHz, DMSO-d6): δ 9.99 (s, 1H), 9.25 (s, 1H),
7.83–7.76 (m, 15H, ArH), 7.18 (d, J = 1.8 Hz, 1H, ArH), 7.00
(dd, J = 8.7 and 1.8 Hz, 1H, ArH), 6.80 (d, J = 8.6 Hz, 1H),
4.27 (t, J = 6.2 Hz, 2H), 1.70–1.21 (m, 18H). 13C NMR (100
MHz, DMSO-d6): δ 169.45 (s), 153.75 (s), 150.08 (s), 135.31 (d,
JC–P = 3.0 Hz), 134.06 (d, JC–P = 10.0 Hz), 130.68 (d, JC–P = 13.0
Hz), 124.37 (d), 119.06 (s, JC–P = 85.1 Hz), 118.53 (d), 114.55
(d), 112.92 (s), 65.52 (t), 30.19 (t, JC–P = 16.0 Hz), 29.19 (t),
1H NMR (400 MHz, DMSO-d6): δ 7.95–7.76 (m, 15H, ArH),
7.10 (s, 2H, ArH), 4.22 (t, J = 6.3 Hz, 2H), 1.74–1.29 (m, 20H)
ppm. 31P NMR (162 MHz, DMSO-d6): δ 24.04 ppm. MS (ESI)
m/z (%) 587.27, 586.27, 585.27 (100, M+–Br).
General procedure for the telescoped synthesis of lipophilic
phosphonium bromide-benzoic acid derivatives. A solution
containing triphenylphosphine (0.74 M in CH3CN) and 11-
bromo-1-undecanol (0.74 M in CH3CN) was reacted at 120 °C
in a 20 mL PFA reactor coil (0.1 mL min−1). The outcoming
stream was combined in a T-piece (each stream running at
0.1 mL min−1) with a solution containing a previously mixed
combination of T3P (0.74 M in CH3CN) and Et3N (1.5 M in
CH3CN) and allowed to mix at rt in a 0.5 mL PFA reactor coil.
29.06 (t), 28.99 (t), 28.53 (t), 28.42 (t), 25.83 (t), 22.20 (t, JC–P
4.0 Hz), 20.69 (t, JC–P = 5.0 Hz) ppm. 31P NMR (162 MHz,
DMSO-d6): 24.07 ppm. HRMS calcd for C35H40O4P:
=
δ
555.2659, found: 555.2668.
Triphenyl(10-((2,3-dihydroxybenzoyl)oxy)-decyl)phosphonium
bromide (PIA-TPP+C10, 5bd). Following the general procedure
for the preparation of lipophilic phosphonium bromide
derivatives, the reaction of triphenylphosphine, 10-bromo-1-
decanol, T3P, Et3N, and 2,3-dihydroxybenzoic acid, afforded
after collecting during 5 min (steady state regime) 146 mg
(71%) of a colourless oil identified as triphenyl(10-((2,3-
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dihydroxybenzoyl)oxy)-decyl)phosphonium bromide (PIA-
TPP+C10, 5bd).
by using trypsin-EDTA and seeded in culture plates for
different experiments.
1H NMR (400 MHz, DMSO-d6): δ 7.12–6.90 (m, 15H), 6.51
(dd, J = 8.1 and 1.6 Hz, 1H), 6.20 (dd, J = 7.9 and 1.6 Hz, 1H),
5.93 (t, J = 8.0 Hz, 1H), 4.26 (t, J = 6.5 Hz, 2H), 1.06–0.30 (m,
18H) ppm. 13C NMR (100 MHz, DMSO-d6): δ 162.30 (s),
Cell viability assay
Cells were seeded into a 96-well plate (1 × 104 cell per well).
After 24 h, benzoate-TPP+ compounds were added at
increasing concentrations into the wells and incubated for
24, 48 and 72 h. After this time, the cells were washed twice
with PBS (phosphate buffered saline) 1×, and then 100 μL of
0.5 mg mL−1 MTT solution was added to each well. After 2 h
of incubation, MTT was removed and the formazan crystals
were dissolved in 40 μL of DMSO. Absorbance was measured
at 570 nm with a microplate ELISA reader (Infinite F50®
Tecan Group Ltd., Swiss).
141.77 (s), 137.59 (s), 126.74 (d, JC–P = 3.0 Hz), 125.3 (d, JC–P
=
9.9 Hz), 122.03 (d, JC–P = 12.5 Hz), 111.93 (s, JC–P = 50.6 Hz),
110.93 (d), 110.58 (d), 110.08 (d), 104.56 (s), 57.10 (t), 22.02 (t,
JC–P = 16.0 Hz), 20.81 (t), 20.66 (t, JC–P = 4.3 Hz), 20.30 (t),
20.05 (t), 17.47 (t), 14.02 (t, JC–P = 4.6 Hz), 13.48 (t), 12.97 (t)
ppm. 31P NMR (162 MHz, DMSO-d6): δ 24.04 ppm. HRMS
calcd for C35H40O4P: 555.2659, found: 555.2668.
Triphenyl(10-((2-hydroxybenzoyl)oxy)-decyl)phosphonium
bromide (SA- TPP+C10, 5be). Following the general procedure
for the preparation of lipophilic phosphonium bromide
derivatives, the reaction of triphenylphosphine, 10-bromo-1-
decanol, T3P, Et3N, and salicylic acid, afforded after
collecting during 5 min (steady state regime) 165 mg (72%)
Oxygen consumption assay
The cell respiration rates were measured polarographically.2
Tumour cells were cultured at 80% of confluence and then
of
a
colourless oil identified as triphenyl(10-((2-
trypsinized and counted. 5 ×
106 cells were used for
hydroxybenzoyl)oxy)-decyl)phosphonium
bromide
(SA-
experimental measurement. 0.6 mL of cell suspension in PBS
(pH 7.4) at 25 °C was added to the electrode chamber with
L-glutamine (8.3 mM). The respiration rate which accounts
for the OXPHOS system was firstly inhibited by 2.5 μg mL−1
oligomycin. Then, the full uncoupled respiration rates were
assessed adding 0.133 μM of CCCP (carbonyl cyanide
m-chlorophenylhydrazine), which was used as control. The
increase of oxygen consumption rates (OCR) caused by each
compound under study were measured by adding increasing
concentrations of each. The results were compared with the
OCR for CCCP.
TPP+C10, 5be).
1H NMR (400 MHz, DMSO-d6): δ 10.56 (s, 1H), 7.91–7.73
(m, 16H, ArH), 7.51–7.47 (m, 1H), 6.98–6.93 (m, 1H), 6.91–
6.89 (m, 1H), 4.27 (t, J = 6.3 Hz, 2H), 1,68–1,14 (m, 18H) ppm.
13C NMR (100 MHz, DMSO-d6): δ 169.39 (s), 160.63 (s), 135.30
(d, JC–P = 2.0 Hz), 134.07 (d, JC–P = 10.0 Hz), 130.68 (d, JC–P
=
13.0 Hz), 130.28 (d), 119.84 (d), 119.07 (s, JC–P = 86.1 Hz),
117.82 (d), 113.46 (s), 65.61 (t), 30.17 (t, JC–P = 17.0 Hz), 29.19
(t), 29.07 (t), 28.99 (t), 28.58 (t), 28.40 (t), 25.82 (t), 22.26 (t,
JC–P = 4.0 Hz), 20.76 (t, JC–P = 5.0 Hz) ppm. 31P NMR (162
MHz, DMSO-d6): δ 24.03 ppm. HRMS calcd for C35H40O3P:
539.2710, found: 539.2706.
Mitochondrial transmembrane potential (ΔΨm)
Changes in transmembrane potential (ΔΨm) were determined
using tetramethylrhodamine methyl ester (TMRM) as
a
Cell lines and cell culture
probe. Cells were seeded into 24-well plates (1 × 105 cells per
well) for 24 h and then incubated with 200 nM TMRM. Cells
were immediately exposed to increasing concentrations of
each compound (2.5 to 30 μM) for 30 min. After washing with
PBS, cells were detached and suspended in cold PBS for
analysis by flow cytometry FACS (FACSAria® III, BD
Biosciences) at a wavelength of 540Ex/595Em nm.
The human oral squamous cell lines, Cal-27 (ATCC®
CRL2095™) and SCC15 (ATCC® CRL1623™) were acquired
from the American type culture collection (ATCC). The
laryngeal cell line HEp-2 was acquired from the Instituto de
Salud Pública de Chile. Normal human oral keratinocyte cells
OKF-6/Tert-2, were kindly donated by Dr. Denise Bravo
(Faculty of Dentistry, Universidad de Chile). Cal-27 and HEp-
2 cell lines were grown in DMEM supplemented with 10%
Intracellular ATP levels
FBS, 100
U
mL−1 of penicillin and 100 μg mL−1 of
streptomycin in a humidified 5% CO2 atmosphere at 37 °C.
SCC-15 cells were grown in a 1 : 1 mixture of Dulbecco's
modified Eagle's medium and Ham's F12 medium containing
1.2 g L−1 sodium bicarbonate, 2.5 mM Lglutamine, 15 mM
HEPES and 0.5 mM sodium pyruvate supplemented with 400
ng mL−1 hydrocortisone and fetal bovine serum 10%. Oral
keratinocytes were grown in keratinocyte serum-free medium
(KSFM) (Gibco®) supplemented with bovine pituitary extract,
hEGF (0.2 ng mL−1), CaCl2 (0.3 mM) and 100 U mL−1
penicillin, and 100 μg mL−1 streptomycin in a humidified 5%
CO2 atmosphere at 37 °C. For passages, cells were detached
The tumour cell ATP levels were measured using the
CellTitle-Glo Luminescent Assay (Promega, Madison, WI)
according to the manufacturer's instructions. Briefly, 1 × 104
cells per well were seeded into a 96-well plate, and were
cultured during 24 h. Each well was stimulated with the
compounds at different concentrations for 4 h. Then, 100 μL
of the cell suspension were transferred to an opaque 96-well
plate and incubated at room temperature for 10 min in the
dark. Finally, the luminescence was captured using
a
Thermo-Scientific Varioskan Flash spectral scanning reader.
As a control assay for cell viability and cell membrane
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integrity for the measurement of intracellular ATP content, PI Funding
incorporation was assessed by flow cytometry (FACS Canto,
This work was supported by U-INICIA grant from the
Vicerrectoría de Investigación y Desarrollo, Universidad de
Chile. [Grant U-INICIA-2014-82379] (JAJ), Fondecyt Iniciación
Grant No. 11180533 (JAJ), Fondecyt Iniciación grant No.
11160281 (MC), Fondecyt Iniciación Grant No. 11170962 and
Fondecyt Regular grant No. 1180296 (JF and JAJ).
BD Biosciences, San Jose, CA)37 according to previously
described.1
Colony assay
For this assay 1 × 103 cells were cultured in a 6-well plate.
Then, the cells were incubated during 24 h at 37 °C under
5% CO2. Later the cells were treated for 24 h with different
concentrations of each compound. Then, the culture medium
was replaced with culture medium without compound, and
the cells were incubated for further 120 h. Finally, crystal
violet (0.5% in methanol) was applied to the cells for 30 min.
The number of colonies was photographed and determined
by using ImageJ software program (NIH, Bethesda, MD,
USA).38
Role of the funding source
The funders did not interfere in any of the stages of the
preparation of this manuscript.
Statement of contributions
VCC, JG and MD planned and performed, and JAS planned
and coordinated the organic chemical synthesis of the
compounds. MC, JA, IO, CP, MV, AM and JAJ performed
biological evaluation. JF, AM and RRF support the experiment
realized. MC and JAJ planned and coordinated the biological
assays. All authors contributed to manuscript revision, read
and approved the submitted version.
Apoptosis
The induction of apoptosis by the benzoic acid derivatives
was determined through flow cytometry using annexin V and
PI probes (annexin V-FITC apoptosis detection kit, Abcam),
according to the manufacturer's instructions (FACS Canto,
BD Biosciences). Briefly, 1 × 105 cells per mL were incubated Conflicts of interest
with the lipophilic cations for 48 h at 37 °C under 5% CO2.
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that
could be construed as a potential conflict of interest.
The cells were suspended in 500 μL of 1× annexin V-binding
buffer, 5 μL of both annexin V and PI were added to the
samples which then were incubated for 5 min at room
temperature. The samples were measured at Ex/Em = 488/530
nm for annexin V-FITC and Ex/Em = 488/575 nm for PI (Miao
et al., 2014)39 and 10 000 events were recorded. The results
were analyzed using the Cyflogic program (non-commercial
version, CyFlo Ltd.).
Acknowledgements
MD and JAS acknowledge Prof. Robert Stockman for the generous
loan of a R2–R4 Vapourtec Flow Reactor, and Prof. Rosana
Álvarez and Prof. Ángel R. de Lera for their continuous support.
Caspase 3 activation
Notes and references
Activation of caspase 3 was determined using a PE Active
Caspase-3 apoptosis kit (BD, Franklin Lakes, NJ) according to
the manufacturer's protocol. Briefly, 5 × 105 cells per well
were seeded in 24-well plates for 24 h, at 37 °C. Cells were
then incubated with various concentrations of the
compounds for 24 h. Cells were detached and suspended in
cell permeability/fixation buffer and incubated for 30 min in
the dark at 4 °C. Cells were centrifuged and washed once
with cell-staining buffer, then centrifuged and suspended in
anti-caspase PE antibody and incubated for 30 min in the
dark. Finally, cells were washed once with cell-staining buffer
and analyzed by flow cytometry at 488Ex/617Em nm and the
data were analyzed using Cyflogic software (non-commercial
version, CyFlo Ltd.).
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