Antioxidant-Inspired Drug Discovery: Antitumor Metabolite Is Formed in Situ from a Hydroxycinnamic Acid Derivative upon Free-Radical Scavenging

Article abstract of DOI:10.1021/acs.jmedchem.8b01994

Cancer cells generally possess higher levels of reactive oxygen species than normal cells, and this can serve as a possible therapeutic target. In this proof-of-concept study, an antioxidant-inspired drug discovery strategy was evaluated using a hydroxycinnamic acid derivative. The processing of oxidized mixtures of p-coumaric acid methyl ester (pcm) revealed a new antitumor lead, graviquinone. Graviquinone bypassed ABCB1-mediated resistance, induced DNA damage in lung carcinoma cells but exerted DNA protective activity in normal keratinocytes, and modulated DNA damage response in MCF-7 cells. The cytotoxic effect of pcm in MCF-7 cells was potentiated under H₂O₂-induced oxidative stress, and the formation of graviquinone was confirmed by Fenton's reaction on pcm. In silico density functional theory calculations suggested graviquinone as a kinetic product of pcm-scavenging ^?OH radicals. Our results demonstrate the pharmacological value of an in situ-formed, oxidative stress-related metabolite of an antioxidant. This might be of particular importance for designing new strategies for antioxidant-based drug discovery.

Full text of DOI:10.1021/acs.jmedchem.8b01994

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Article
Antioxidant-inspired drug discovery: antitumor metabolite is formed in
situ from a hydroxycinnamic acid derivative upon free radical scavenging
Laura Fási, Florent Di Meo, Ching-Ying Kuo, Sonja Stojkovic Buric, Ana Martins, Norbert Kúsz, Zoltán
Béni, Miklós Dékány, György T. Balogh, Milica Pesic, Hui-Chun Wang, Patrick Trouillas, and Attila Hunyadi
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01994 • Publication Date (Web): 07 Jan 2019
Downloaded from http://pubs.acs.org on January 9, 2019
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Antioxidant-inspired drug discovery: antitumor metabolite is formed in
situ from a hydroxycinnamic acid derivative upon free radical
scavenging
a,#
b,#
c,#
d
e,§
Laura Fási , Florent Di Meo , Ching-Ying Kuo , Sonja Stojkovic Buric , Ana Martins ,
a
f
f
g
d
Norbert Kúsz , Zoltán Béni , Miklós Dékány , György Tibor Balogh , Milica Pesic , Hui-
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Chun Wang , Patrick Trouillas , Attila Hunyadi *
a
Institute of Pharmacognosy, Interdisciplinary Excellence Centre, University of Szeged, Eötvös
str. 6, H-6720 Szeged, Hungary
b
INSERM UMR 1248 IPPRITT, Univ. Limoges, Faculty of Pharmacy, 2 rue du Dr Marcland,
F-87000, Limoges, France
Graduate Institute of Natural Products, Kaohsiung Medical University, Shih-Chuan 1 Rd.
c
st
1
d
00, Kaohsiung 807, Taiwan R.O.C.
Department of Neurobiology, Institute for Biological Research, University of Belgrade,
Bulevar Despota Stefana 142, 11060 Belgrade, Serbia
e
Institute of Medical Microbiology and Immunobiology, Faculty of Medicine, University of
Szeged, Dóm tér 10, H-6720 Szeged, Hungary
f
Spectroscopic Research, Gedeon Richter Plc., Gyömrői u. 19-21, H-1103 Budapest, Hungary
Compound Profiling Laboratory, Gedeon Richter Plc., Gyömrői u. 19-21, H-1103 Budapest,
g
Hungary
h
RCPTM, Faculty of Sciences, Palacký University, tr. 17. listopadu 12, 771 46 Olomouc, Czech
Republic
i
Interdisciplinary Centre for Natural Products, University of Szeged, Eötvös str. 6, H-6720
Szeged, Hungary
*
#
§
Corresponding author.
These authors contributed equally.
Permanent address: Synthetic Systems Biology Unit, Institute of Biochemistry, Biological
Research Centre, Temesvári krt. 62, 6726 Szeged, Hungary.
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Abstract
Cancer cells generally possess higher levels of reactive oxygen species than normal cells, and
this can serve as a possible therapeutic target. In this proof-of-concept study, an antioxidant-
inspired drug discovery strategy was evaluated using a hydroxycinnamic acid derivative. The
processing of oxidized mixtures of p-coumaric acid methyl ester (pcm) revealed a new
antitumor lead, graviquinone. Graviquinone bypassed ABCB1-mediated resistance, induced
DNA damage in lung carcinoma cells but exerted DNA protective activity in normal
keratinocytes, and modulated DNA damage response in MCF-7 cells. The cytotoxic effect of
pcm in MCF-7 cells was potentiated under H O -induced oxidative stress, and the formation of
graviquinone was confirmed by Fenton’s reaction on pcm. In silico DFT calculations suggested
graviquinone as a kinetic product of pcm scavenging OH radicals. Our results demonstrate the
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pharmacological value of an in situ-formed, oxidative stress-related metabolite of an
antioxidant. This might be of particular importance for designing new strategies for antioxidant-
based drug discovery.
Keywords: methyl-p-coumarate; hydroxycinnamate; reactive oxygen species (ROS); oxidative
stress; bioactive metabolite; cancer; DNA damage response; ATR / ATM; Chk1
phosphorylation; density functional theory
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Introduction
Hydroxycinnamic acids, such as p-coumaric, caffeic, ferulic and chlorogenic acids, are among
the most abundant dietary antioxidants. They occur in many vegetables (e.g., olive, potato,
lettuce, broccoli, cauliflower, chicory, cabbage), fruits (e.g., various berries, grape, date, apple),
cereals (e.g., common and hard wheat, cereal products such as bread), spices (e.g., clove,
Ceylon cinnamon, nutmeg, cumin), nuts (e.g., peanut), dark chocolate, vegetable oils, and
beverages (e.g., beers, wines, fruit juices, tea and coffee) . The antioxidant properties of these
phenolic acids have been widely described . They have been reported for a large variety of
bioactivities, including chemo-preventive and antitumor effects. For example, caffeic acid
methyl ester (cm) was shown to inhibit colon carcinogenesis through the inhibition of tyrosine
protein kinase and arachidonic acid metabolism . Another example is the chemo-preventive
activity of p-coumaric acid methyl ester (pcm; also referred to as methyl-p-coumarate), which
is enabled by the induction of Phase II metabolic enzymes such as quinone reductase and
Glutathione-S-Transferase (GST)6.
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In addition to the antitumor and antioxidant potential of hydroxycinnamic acids, some inspiring
studies about their oxidized metabolites have been published. Pieters et al. found that
Ag O-catalysed biomimetic oxidation of cm or ferulic acid methyl ester (fm) in a mixture of
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anhydrous benzene and anhydrous acetone yielded dihydrobenzofuran lignans with highly
potent in vitro antiproliferative activity on different cancer cell lines . The dimer of cm showed
great antitumor potential with growth inhibitory concentrations (GI ) lower than 10 nM against
breast cancer cell lines, MDA-MB-435, MDA-N and BT-549. Moreover, this compound was
identified as a potent antitubulin agent . These dihydrobenzofuran lignans were also reported
to exert antiangiogenic activity , and another study described similar activity of the fm-derived
lignan on angiogenesis . Recently, a dimer of cm (referred to as Q2-3) attracted great attention
for its anti-metastatic activity on mammary tumors due to interference with their
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microenvironment; namely, Q2-3 was able to induce IL-25 secretion from tumor-associated
fibroblasts10.
Antioxidants can decrease the levels of free radicals by direct scavenging, hence preventing
free radical-associated damage. In turn, this process may form oxidized metabolites with
different bioactivities. The effect of these metabolites on the overall bioactivity may be minor,
or at least difficult to discriminate with respect to that of their parental antioxidants within the
cell environment. However, these metabolites could be used as new model compounds or leads
in drug discovery. In this context, and considering the intriguing antitumor potential of
hydroxycinnamic acids, this study aims at the investigation of both the formation and the
bioactivity of oxidized metabolites of pcm.
Results and Discussion
Preparation of oxidized metabolites of pcm.
As a first step, we aimed to study the effect of various oxidative conditions on the cytotoxic
activity of pcm. The oxidation of pcm was performed by a hypervalent iodine (III) reagent,
phenyliodine bis(trifluoroacetate) (PIFA), in acetonitrile, acetonitrile – water (9:1, v/v),
methanol, or methanol – water (9:1, v/v), or with pyridinium chlorochromate (PCC) in
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dichloromethane. PIFA was selected based on two reasons. First, this reagent was previously
reported to react with p-phenols via a single electron transfer (SET) mechanism through a cation
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radical intermediate . Second, this cation radical intermediate can deprotonate in aqueous
medium and lead to the same phenoxyl radical that would form in a hydrogen atom transfer
(
HAT) reaction. Since p-coumaric acid was previously suggested to scavenge ROS either
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through SET or HAT mechanism , we expected that PIFA-mediated oxidation of pcm would
lead to a metabolite pattern like that of ROS-scavenging. Following oxidation, each product
mixture was subjected to solid-phase extraction on silica to prevent false positive results due to
residual traces of reagents. The obtained product extracts (named OX1-5) were tested for their
cytotoxic activity in comparison with that of pcm on a mouse lymphoma cell line (L5178) and
its multi-drug resistant (MDR) sub-cell line, obtained by transfection of L5178 with the human
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ABCB1 gene (L5178 ). A dramatic increase in the cytotoxic activity was observed with OX1
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and OX2 as compared to that of pcm (Figure 1A and 1B), hence these conditions were chosen
to perform scale-up in order to allow the isolation of individual active constituents. Altogether
three scale-up procedures were performed, from which 5 metabolites (1-5, see Figure 1C) were
isolated by combined chromatographic techniques. Structure elucidation of these compounds
was (i) performed by mass spectrometry and comprehensive 1- and 2D NMR methods, and then
(
ii) confirmed by comparing their chemical shifts with reported literature values13-16.
O
A
C
150
OMe
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pcm
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OMe
OMe
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pcm OX1 OX2 OX3 OX4 OX5
HO
OH
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HO
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pcm OX1 OX2 OX3 OX4 OX5
Figure 1. Cytotoxic activity of pcm and its oxidized metabolite-mixtures OX1-5 on L5178
(A) and L5178B1 (B) cancer cells, and chemical structures of pcm and its oxidized
metabolites isolated (C). Dilutions for each mixture were performed by calculating with the
molecular mass of pcm; upward arrows mark samples with IC values >150µM. Reagents and
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solvents for obtaining each mixture were as follows. OX1: PIFA, ACN; OX2: PIFA, ACN –
water (9:1); OX3: PIFA, MeOH; OX4: PIFA, MeOH – water (9:1), OX5: PCC, CH Cl
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2.
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Compound 1 has most likely been formed from pcm in a way similar to that previously
described for the dihydrobenzofuran dimers of cm or fm, followed by the elimination of a
phenol moiety and the formation of benzofuran skeleton. The formation of compounds 2 (i.e.
cm) and 3 (p-hydroxy-benzaldehyde) can intuitively be explained by the impact of the oxidative
environment. Compound 4 (methyl 2,5-dihydroxycinnamate or methyl grevillate) represents a
highly unexpected structure, which has however previously been isolated from natural
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sources . Considering the reaction mechanism of phenol oxidation by PIFA , one can only
suspect the formation and opening-up of an epoxide intermediate as an explanation for the
unexpected migration of the p-hydroxyl group to the meta-position. Compound 5 (known as
graviquinone) is also a natural product that has first been isolated from the “silky oak,”
Grevillea robusta A. CUNN, and identified as a cytotoxic compound against MCF-7, NCI-
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H460, and SF-268 cell lines in vitro . The p-quinol moiety of 5 makes it related to
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protoflavones that are known for their strong antitumor activity in vitro and in vivo , as well
as for their ability to bypass resistance mediated by the P-glycoprotein (P-gp)/ABCB1 or breast
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cancer resistance protein (BCRP)/ABCG2 efflux transporters .
Antitumor potential of oxidized pcm metabolites.
The in vitro cytotoxic activity of compounds 1-5 was tested on the above-mentioned mouse
lymphoma cell line pair (L5178 and L5178 ). Following this, compounds 4 and 5 were also
B1
tested on a diverse panel of cancer cell lines, including MCF-7, HeLa, SiHa, and several lung
carcinoma cell lines, such as A549 adenocarcinoma, a highly metastatic large cell lung
carcinoma (NCI-H661), and a non-small cell lung carcinoma (NCI-H460) and its multi-drug
resistant counterpart (NCI-H460/R) developed by continuous exposure of the initially sensitive
cell line to doxorubicin. Results of these bioassays are compiled in Table 1.
Table 1. In vitro cytotoxic activity of pcm and its oxidized metabolites 1-5. IC values are
5
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given in µM as mean ± SEM from three parallel experiments; Dox: doxorubicin.
IC50 ± SEM (µM)
L5178
L5178B1
> 150
NCI-H460
NCI-
H460/R
1043.13 ±
A549
NCI-H661
HaCaT
MCF-7
> 400
HeLa
> 150
SiHa
pcm
125.60 ±
120.01 ±
322.92 ±
289.21 ±
311.22 ±
> 150
8
.89
2.14
59.36
8.09
8.42
3.75
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> 150
> 150
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103.9 ± 3.02 112.8 ± 4.45
> 150 > 150
5.23 ± 0.29 12.38 ± 0.23 3.77 ± 0.07 7.05 ± 0.13 4.14 ± 0.09 4.19 ± 0.11 9.69 ± 0.18
8.51 ± 1.54
0.46 ± 0.09 0.62 ± 0.08 1.85 ± 0.03 2.06 ± 0.03 4.32 ± 0.09 1.44 ± 0.02 4.05 ± 0.10 6.22 ± 1.07 9.85 ± 1.03 8.89 ± 1.09
Dox 0.41 ± 0.07 11.83 ± 0.64 0.04 ± 0.01 1.89 ± 0.25 0.55 ± 0.16 0.35 ± 0.12 0.03 ± 0.01 1.08 ± 0.01 1.21 ± 0.05 0.76 ± 0.01
The parental antioxidant pcm exerted very low cytotoxic activity as measured by MTT assay,
and the NCI-H460/R cell line showed a nearly 10-fold cross resistance to this compound as
compared to the NCI-H460 cells. Some of its oxidized metabolites, however, exerted a great,
ca. 2-3 orders of magnitude stronger cytotoxic activity. Among the two identified active
metabolites (4 and 5), 2.4 times cross-resistance towards the hydroquinone analogue compound
4
was observed in the MDR cell line L5178 , suggesting that this compound is likely a
B1
substrate of the P-gp/ABCB1 efflux transporter. Nevertheless, the resistance profile of this
compound was much more favourable than that of pcm on the NCI-H460 / NCI-H460/R cell
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line, exhibiting a less than twice cross-resistance in the latter. Compound 5 bypassed resistance
of both the ABCB1-transfected cell line and that of the NCI-H460/R adapted to doxorubicin.
Its activity was as strong as that of the positive control doxorubicin in L5178 cells, but while
doxorubicin demonstrated 28.9 times higher IC50 in resistant L5178B1 compared to
corresponding sensitive L5178 cells, compound 5 was similarly active in both cell lines.
Moreover, compound 5 showed a nearly 10-times selective cytotoxicity against the lymphoma
cell lines, and a ca. 2-3 times selectivity against the NCI-460 and NCI-661 cell lines versus the
immortalized human keratinocytes (HaCaT), suggesting a good selectivity profile towards
cancer cells.
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Cell death analysis by Annexin-V/PI staining revealed that both compounds 4 and 5 induce
cellular necrosis (Table 2).
Table 2. Cell death induction by compounds 4 and 5 in human lung carcinoma cell lines
(NCI-H460, NCI-H460/R, A549, NCI-H661) and normal human keratinocytes (HaCaT).
Dox: 40 nM of doxorubicin, used as positive control. Statistical evaluation was performed by
two-way ANOVA followed by Dunnett's multiple comparisons test; *: p<0.05, **: p<0.01, ***:
p<0.001
Cell line
NCI-
H460
Sample
control
Viable cells
96.49±0.96
86.64±1.73**
89.13±2.67*
85.14±3.07*
95.76±2.87
89.65±1.79*
90.03±3.60*
96.45±2.89
88.24±2.65*
92.78±1.86
96.16±0.96
89.43±1.79*
45.43±1.36***
95.63±3.83
90.95±3.64*
91.50±1.83
78.11±2.38**
Early apoptosis
0.45±0.01
0.34±0.01
0.21±0.01
4.05±0.12**
0.22±0.01
0.16±0.01
0.12±0.01
0.48±0.01
2.36±0.07*
0.72±0.03
0.31±0.01
0.49±0.02
0.18±0.01
0.23±0.01
0.50±0.01
0.44±0.01
11.03±0.14***
Late apoptosis
0.93±0.04
5.10±0.15**
3.35±0.07**
4.11±0.23**
0.84±0.01
2.48±0.10*
2.34±0.07*
1.54±0.03
5.62±0.06**
3.93±0.16*
0.95±0.03
3.39±0.07*
9.57±0.10***
2.26±0.09
2.65±0.08
2.85±0.06
Necrosis
2.13±0.02
9.92±0.20**
7.31±0.22**
6.70±0.24**
3.17±0.13
7.71±0.08**
7.51±0.15**
1.52±0.05
3.78±0.15*
2.57±0.03*
2.58±0.05
6.69±0.20*
44.82±1.79***
1.88±0.02
5.90±0.12**
5.21±0.16**
7.82±0.12***
4
5
Dox
control
NCI-
H460/R
4
5
A549
control
4
5
NCI-
H661
control
4
5
control
4
5
Dox
HaCaT
3.04±0.07
Compound 4 exerted the strongest effect on NCI-H460 cells, which were also the most sensitive
to 4 according to the cytotoxicity assay. As the most prominent effect, compound 5 increased
the percentage of necrotic NCI-H661 cells from 2.58% (untreated control) to 44.82% (treated).
This was like our previous observations on a p-quinol flavonoid, protoapigenone, which also
induced necrosis-type cell death associated with ATR inhibition through Chk1, leading to the
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accumulation of DNA lesions . Considering the structural relationship between
protoapigenone and compound 5, our attention turned towards its potential ability to exert DNA
damage and interfere with DNA damage response at the same time.
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As a marker of DNA damage, the expression of Histone 2A.X (H2A.X) was analysed after
treatment with compound 4 or 5. The potential effect of compound 5 on DNA damage response
was evaluated with Western immunoblotting after UV irradiation of MCF7 cells (Figure 2).
A
B
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C
Figure 2. DNA damaging effect and ROS production by treatment with compounds 4 or
5, and effect of compound 5 on DNA damage response. (A) DNA damaging effects of
compounds 4 and 5 were examined by Histone 2A.X relative expression. (B) ROS production
was detected according to dihydroethidium fluorescence. Both analyses were performed by
flow cytometry, results are given as the average ± s.d. from three independent experiments.
Statistical evaluation was performed by two-way ANOVA followed by Dunnett's multiple
comparisons test; ***: p<0.001. CDDP: cisplatin. (C) Evaluation of the DNA damaging effect
of compound 5 and pcm by Western immunoblotting; MCF7 cells were pre-treated with 10 µM
2
of compound 5 or pcm for 30 minutes and exposed to UV irradiation (10 J/m ) in the following
1
hour to induce DDR. 10 µM of protoapigenone was used as positive control. Results were
analyzed by one-way ANOVA and Data represent the mean ± s.d. from three independent
experiments; *: p<0.05, **: p<0.01.
The results clearly showed the induction of DNA damage by 5 in NCI-H460 and NCI-H460/R
cells with significantly increased expression of H2A.X, which was, however, decreased in
normal HaCaT cells after treatment with 5. The results point out the selectivity of compound 5
towards DNA damaging in cancer cells. Compound 4 reduced the expression of H2A.X in most
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of the cancer cells and also in HaCaT cells, indicating its DNA protective activity (Figure 2A).
ROS production was assessed by dihydroethidium labelling that showed a significant increase
of ROS levels in NCI-H460, A549 and NCI-H661 cells after treatment with compound 5,
however it was decreased in NCI-H460/R and HaCaT cells. These results also demonstrate the
selectivity of 5 towards cancer cells. The opposite effect on NCI-H460/R cells should be
discussed in the light of our previously published results regarding the lower antioxidant
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potential of NCI-H460/R cells, as compared to that of their parental cell line NCI-H460 .
Compound 4 decreased the level of ROS in all tested cell lines including HaCaT cells (Figure
2
B). The obtained results indicate different mechanisms of action for compounds 4 and 5.
Interestingly, the positive control cisplatin (CDDP) significantly increased ROS levels in
normal HaCaT cells without affecting this measure in NCI-H460 cancer cells. This also
suggests that, at least in this regard, compound 5 has a better selectivity profile than the classical
chemotherapeutic drug cisplatin.
It is of interest that compound 5 acted as a potent modulator of the DNA damage response
through the inhibition of Chk1-S345 phosphorylation, while pcm was completely inactive in
this regard (Figure 2C). The serine-threonine checkpoint kinases 1 and 2 (Chk1 and Chk2,
respectively) have key roles in the DNA damage signalling response. Upon DNA damage, Chk1
is activated through phosphorylation by the ataxia telangiectasia and Rad3-related kinase
(ATR), and Chk2 is activated to a major extent by the ataxia telangiectasia mutated kinase
(ATM). Chk1 is especially important for the stability of stalled replication forks, and DNA
21
repair-defective tumour cells have been shown to accumulate high levels of DNA damage . A
growing body of evidence suggests that small molecule Chk1 inhibitors may act as single-
agents against cancer cells with specific potential genetic defects, as for example Fanconi
22
Anemia (FA) . In addition, the sensitivity to Chk1 inhibition is also associated with replication
stress in many cancer cell types, such as neuroblastoma cell lines or metastatic melanoma,
which were found to have higher expression of Chk1 mRNA23,24. Accordingly, our results
demonstrated that, as a Chk1 inhibitor, compound 5 has a considerable potential to specifically
target cancer cells with high levels of endogenous DNA damage. Furthermore, the increased
expression of phosphorylated H2A.X is a well-known marker of the DNA double strand breaks
25
(
DSBs) . We found that, unlike normal HaCaT cells, cancer cells treated with compound 5
showed a significantly increased expression of H2A.X (Figure 2A). Since ATM dependent
phosphorylation is also correlated with H2A.X phosphorylation, this suggests that DSBs
resulting from collapsed replication forks are responsible for the ATM activation. In Figure 2C,
our results demonstrate that compound 5 can increase Chk2-T68 phosphorylation, suggesting
that it could induce DSBs and activate the ATM-Chk2 pathway. This is an interesting difference
between compound 5 and the positive control protoapigenone, a p-quinol flavonoid, that did
not influence the ATM-dependent phosphorylation of Chk2 in accordance with our previous
1
9
findings .
Pcm was reported as a potent dietary antioxidant, counteracting lipid peroxidation in rat liver
26
microsomes with an inhibitory concentration (IC ) value of 0.4 µM . Furthermore, a recently
5
0
published spectroscopic study on the in vitro antioxidant activity of p-coumaric acid indicated
that it can efficiently scavenge DPPH, ABTS, superoxide anion and H O , as well as prevent
2
2
27
the autoxidation of linoleic acid . Since these antioxidant mechanisms are unlikely to be
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disturbed by the ester function pcm, similar activities can reasonably be expected from pcm as
well. Accordingly, our next aim was to investigate whether or not the identified bioactive
metabolites have the chance to form through ROS scavenging by pcm. In order to assess this,
•
we studied the OH radical-scavenging activity of pcm.
•
OH radical-scavenging activity of pcm
•
The OH radical scavenging capacity of pcm was performed by measuring the inhibition of the
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oxidative damage caused to 2-deoxy-D-ribose by Fenton reaction. A free radical trapping
antioxidant, NXY-059 (disufenton sodium; Cerovive) was used as the positive control; this
2
8
compound reached phase III clinical trials as a neuroprotective agent . We found that pcm is
a stronger ˙OH radical scavenger (IC =0.345 [95% C.I. 0.313–0.380] mM) in this bioassay
50
than NXY-059 (IC =1.826 [95% C.I. 1.501–2.220] mM). The results obtained for NXY-059
5
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were in good agreement with those of our previous study .
In vitro antitumor activity of pcm in the presence of H O -induced oxidative stress
2
2
MCF-7 cells were subjected to a combined treatment with pcm and H O , aiming to investigate
2
2
whether or not externally provoked oxidative stress can modulate sensitivity of cells towards
pcm (Table 3).
Table 3. Interaction between pcm and H O concerning their cytotoxicity on MCF7 cells.
2
2
Combination index (CI) values are given at 50, 75 and 90% of cytotoxicity (ED , ED and
5
0
75
ED , respectively). CI : weighted average CI value; CI = (CI + 2CI + 3CI )/6. CI < 1,
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avg
avg
50
75
90
CI = 1, and CI > 1 represent synergism, additivity, and antagonism, respectively. Dm, m, and r
represent antilog of the x-intercept, slope, and linear correlation coefficient of the median-effect
3
0
plot, respectively . Stability of pcm in the presence of H O (see Supporting information
2
2
Figure S1) suggests that the interaction is not due to a chemical reaction between them.
H O vs. pcm
CI50
0.79
0.93
1.06
CI75
0.60
0.60
0.68
CI90
0.45
0.39
0.44
Dm
m
r
CIavg
0.556
0.550
0.623
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0
.5:1
247.66
250.18
222.79
2.109
3.197
3.610
0.985
0.988
0.974
.25:1
.625:1
Synergism could clearly be observed between pcm and H O , in particularly at higher rates of
2
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inhibition that are more important in terms of antitumor activity . Moreover, there seems to be
a tendency in favour of higher H O vs. pcm ratios, further strengthening the assumption that
2
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this phenomenon might be connected to the formation of oxidized metabolites of pcm
proportionally to the level of oxidative stress. It is worth mentioning that pcm was found stable
for at least 92 hours when dissolved in MeOH (1 mg/ml) and 400 µl of hydrogen peroxide
(30 %) was added (for HPLC-PDA chromatogram, see Supporting information, Figure S1).
This indicates that the observed increase in the cytotoxic activity is not due to a simple chemical
reaction between H O and pcm.
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Fenton reaction of pcm.
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With an attempt to model the effect of oxidative stress on pcm in terms of providing possible
metabolites, pcm was subjected to Fenton reaction, which is well-known to provide excess OH
•
radicals. Following pre-purification with SPE on silica, the presence of compound 5 was
detected in the reaction mixture by RP-HPLC and HPLC-MS (for the fingerprint
chromatograms and LC-MS data, see Supporting information, Figures S2 and S3). This clearly
demonstrates that compound 5 can form when pcm scavenges hydroxyl radicals. However, it
is worth mentioning that the extreme oxidative conditions provided by Fenton’s reaction do not
allow to draw any further quantitative conclusion about the formation of metabolites that could
rise in a biological environment when hydroxyl radical (or possibly other types of ROS)
scavenging takes place. In order to obtain a deeper insight into the potential relevance of
compound 5 formation upon ROS scavenging, in silico studies were performed by means of
density functional theory (DFT) calculations.
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In silico studies about the formation of pcm metabolites in presence of OH radicals.
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In presence of highly reactive species such as OH free radical, pcm may be oxidized following
•
two mechanical sequential chemical pathways (see Figure 3). Mechanism 1 is initiated by OH
addition at various positions, leading to the formation of the corresponding radical-adducts that
are then stabilized by H-atom abstraction. Mechanism 2 is initiated by a H-atom abstraction,
•
forming a radical which then undergoes OH addition at various positions, forming the
corresponding adducts. It is important to note that compound 3 can only be obtained from
Mechanism 2, whereas compound 5 can be obtained from both Mechanisms 1 and 2. A
description of these mechanical pathways and the calculated thermodynamic analysis are shown
in Figure 3.
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Figure 3. Reactivity of pcm in presence of OH radical (A) and thermodynamics of the
reaction steps (B). HAT: hydrogen atom transfer; q-pcm and q-cm represent quinone forms of
pcm and cm, respectively. Frames mark species observed experimentally from the Fenton
reaction of pcm and/or from its oxidation with PIFA. Bond dissociation enthalpy (BDE) is
defined as the enthalpy required to the homolytic cleavage of X-H bond as follows:
•
•
BDE(R-H) = H(R , 298K) + H(H , 298K) - H(R-H, 298K), where H is the enthalpy. O-H and
-1
C-H BDE values for each HAT are given in red and blue, respectively, in kcal.mol . Gibbs
energies (ΔG, kcal.mol ) are indicated for each reaction step relative to that of pcm. Evidence
-1
for the formation of compound 5 from the Fenton reaction of pcm is shown in Supporting
Information Figures S2 and S3.
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Regardless of the mechanism, all predicted compounds are thermodynamically favourable with
respect to the parent compound (pcm) since every single step exhibits negative Gibbs energies
(
Figure 3B). The calculated global thermodynamics shows that cm and pcm-OH are the most
-1
favourable compounds (G being -105.8 and -104.7 kcal.mol , respectively), while 5 appears
-1
much less stable (G = -75.2 kcal.mol ), which can easily be explained by the loss of
aromaticity. It must be stressed that only compound 5, cm, and 3 were experimentally identified
among those that can be formed through the above mechanisms. Therefore, joint experimental
observations and calculations suggest the important role of kinetics to allow the formation of
the least thermodynamically favourable compounds such as 5.
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Mechanism 1.
Focus must be given to the OH-addition step, which may occur at several C-atom positions,
which drives regioselectivity in the adduct formation. Additions on the aromatic ring, i.e.,
positions C1ʹ and C3ʹ (see Figure 3A for numbering of pcm) can be predicted by the electronic
•
•
effect of the OH group at p-position; leading to [5-OH] and [cm-OH] . Such additions are only
slightly favourable due to the loss of aromaticity (G being -4.0 and -8.9 for C1ʹ and C3ʹ OH-
•
•
additions, respectively). The higher stability of [cm-OH] as compared to [5-OH] may be
explained by the lower geometrical disruption with the side chain while spin density
distributions are similar for the two radicals (Figure 4). Interestingly, OH-additions at C2 and
•
•
C3 (leading to [pcm-OH] and [pcm-OHʹ] ) are significantly favoured over those taking place
•
•
at C1ʹ or C3ʹ. Spin density distributions (Figure 4) in [pcm-OH] and [pcm-OHʹ] underline the
•
•
much lower stabilization of single electron with respect to [5-OH] and [cm-OH] . However,
the loss of aromaticity is energetically more expensive, which somehow explains the more
favourable OH-addition on the C-C double bond than on the aromatic ring. The addition at C2
•
and thus the formation of [pcm-OH] is the most favourable owing to the slightly more
stabilizing delocalization of free radical thanks to the aromatic ring (Figure 4A).
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Figure 4. Spin density distribution of the radicals formed by the reaction between pcm
and OH radicals. (A) Radicals formed through OH-addition, (B) radical formed through
hydrogen atom abstraction. Blue and red spheres depict positive and negative atomic spin
densities, respectively.
•
The second step of Mechanism 1 is a simple H-atom abstraction driven by the O-H bond
dissociation enthalpies (BDEs, see Figure 3A). The lower the BDE, the higher the likelihood
of the X-H breaking. It must be stressed that one or two H-atom abstractions may be considered,
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depending on the adduct. For instance, in [5-OH] and [pcm-OH´] , only OH and C3-H bonds
are expected to be broken, respectively. It must be stressed that experimental observations
•
suggest that the C-C bond is broken in [pcm-OH´] allowing rearrangement leading to 3.
•
•
Interestingly, in [cm-OH] and [pcm-OH] , H-atom may be abstracted from either the
phenolic O-H moiety or the C-H bond bearing the new OH-group (i.e., C1´ and C2 for [cm-
•
•
OH] and [pcm-OH] , respectively). The calculations exhibit very low BDEs (21.0 to 59.0
-1
kcal.mol ), suggesting that H-abstraction is not the limiting step of this process. It must be
•
•
stressed that C-H BDEs of both [cm-OH] and [pcm-OH] are systematically lower than that of
the O-H, since the former allows turning back to aromatic systems. In case of OH abstraction,
a tautomeric step is required to reach the final products cm and pcm-OH. However, such event
is known to be fast and thermodynamically favourable. Therefore, the observed difference
between BDE(O-H) and BDE(C-H) values is not expected to play a crucial role in protic
environments.
Mechanism 2.
Polyphenols are known to undergo H-atom abstraction from their phenolic OH groups. Here,
-1
OH-BDE is -82.9 kcal.mol (see Figure 3A), which lies within the typical range of polyphenol
BDEs3 . The H-atom abstraction leads to the formation of pcm-[H] free radical that can
undergo OH-addition (second step of Mechanism 2). In contrast to the OH-addition on pcm,
this step is more energetically favourable since the products turn back to closed-shell species.
The regioselectivity of OH-addition is driven by both thermodynamics and kinetics. On one
hand, thermodynamics suggests that addition preference is at C1´ < C3´ < C2, G being -38.3,
1-34
•
-1
-
42.4 and -45.8 kcal.mol for addition at C1´, C3´ and C2, respectively (see Figure 3B). On the
•
other hand, the kinetics is correlated to spin density distribution of pcm-[H] (Figure 4). The
higher spin density at C1´ suggests that OH-addition at this position is faster, rationalizing the
formation of 5. In other words, calculations suggest that 5 is the kinetic product while q-cm and
q-pcm-OH are the thermodynamic ones. Again, tautomeric steps of q-cm and q-pcm-OH are
not expected to play a key role in the global view of such mechanical pathways.
Competition between Mechanisms 1 and 2.
Interestingly, the competition between Mechanisms 1 and 2 is a matter of competition
between H-atom abstraction and OH-addition on pcm. Thermodynamically, H-atom
-1
abstraction from the p-OH (G = -36.9 kcal.mol , Figure 3B) is more favourable than OH-
-1
addition at any possible position by at least -14.7 kcal.mol . Therefore, the major pathway
likely starts with H-atom abstraction (Mechanism 2). Moreover, this explains the
experimentally observed formation of cm and 5; the former being the global thermodynamic
product while the latter is a kinetic product from the OH-addition step. It is important to note
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that experimental observation of 3 as a product again suggests the importance of kinetics. Such
a product can be formed only via the alternative Mechanism 1, i.e., OH-addition at C2 and thus
via [pcm-OH´] . The OH radical is known to be very reactive and non-specific, also
highlighting certain stochasticity in the reactivity. However, it must be stressed that the higher
Gibbs energies of OH-addition at C1´ and C3´ suggest that OH-addition on the aromatic ring is
very unlikely.
It must be noted that while theoretical calculations suggest cm as a major product, it has only
been observed from the PIFA-mediated oxidation and not from the Fenton-reaction of pcm.
This can likely be explained by the fact that the catechol moiety of cm might make it particularly
sensitive to further oxidation, leading to a rapid decomposition and consequential difficulties
of detection under the harsh experimental conditions represented by the Fenton-reaction. In
addition to this, the presence of an alternative product 4 cannot be fully explained by the
proposed mechanism. One can only suspect the formation of an epoxide intermediate and
subsequent ring opening as an explanation of the migration of the p-hydroxyl group resulting
in the hydroquinone derivative 4.
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Conclusions
By processing various oxidized mixtures of p-coumaric acid methyl ester, two active
metabolites were identified from mixtures with the highest antitumor potential. In particular,
compound 5 (graviquinone) was revealed as a prospective lead compound, with an ability to
bypass multi-drug resistance mediated by the ABCB1 transporter, in a lymphoma cell line
transfected with ABCB1 gene and an MDR non-small cell lung cancer cell line established after
continuous exposure to doxorubicin. Compound 5 showed a remarkable selectivity towards
cancer cells in respect to DNA damage induction. DNA damaging activity of compound 5 was
accompanied by its ability to interfere with components of the ATR signalling pathway.
Concerning the possible biological role of compound 5, our in vitro and in silico studies
strongly suggest that it can be formed as a kinetic product when its parental antioxidant, pcm,
scavenges ROS. The mild in vitro antitumor effect of pcm was significantly increased in the
presence of H O -induced oxidative stress, and Fenton’s reaction clearly confirmed that the
2
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•
formation of compound 5 from pcm is possible upon OH radical scavenging. Our results
provide indirect evidence for the transformation of pcm to compound 5 in a biological system
facing oxidative stress. The herein described phenomenon also suggests that the oxidative
stress-related metabolites of antioxidants represent a segment of chemical space that is rich in
valuable bioactive compounds. This would warrant systematic studies on such metabolites as a
possible novel drug discovery strategy.
Experimental Section
Chromatography
Analytical high-performance liquid chromatography (HPLC) was performed on a gradient
system of two PU-2080 pumps connected to MD-2010 Plus photodiode-array detector (Jasco
Analytical Instruments, Tokyo, Japan), on a Kinetex XB-C18 (5u, 100A, 250x4.6 mm) column
with solvent system 1. Semi-preparative HPLC was performed on a system of two Waters 600
pumps connected to Waters 600 2λ detector (Waters, Milford, Massachusetts, USA), on a
Kinetex XB-C18 (5u, 100A, AX, 250x21.2 mm) column. HPLC-MS analysis was performed
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on an Agilent 1200 liquid chromatography system, included a binary pump, a column
temperature controller and a diode array detector, connected to a 6410 triple quadrupole mass
spectrometer (QQQ-MS) equipped with an ESI source (Agilent Technologies, Palo Alto, CA,
USA). The analysis was carried out at 45 °C on a Kinetex EVO C18 (2.6u, 50x3.0 mm,
Phenomenex) column with Solvent system 2 at flow rate of 1.25 ml/min.
Solvent system 1: gradient of acetonitrile -from 30 to 70 % in water at 0-12 min, 70 % of
acetonitrile at 12-14 min, then back to 30 % of acetonitrile, total running time was 25 min.
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Solvent system 2: gradient of 2-100 % B at 0-5.9 min, 100 % of B at 5.9-7.0, from 7.01 min
back to 2 % of B. Eluent A was 0.1 % (v/v) trifluoroacetic acid (TFA) in water, eluent B was
0
.1 % TFA in the mixture of acetonitrile – water (95:5).
Flash chromatography was performed on CombiFlash Rf+ (Teledyne ISCO) with Solvent
system 3: a gradient of ethyl acetate from 0 to 100 % in n-hexane.
Oxidation of pcm for screening
As a preliminary experiment, pcm was oxidized under different conditions. 10.0 mg of pcm
was dissolved in 10 ml of ACN (OX1), ACN – water (9:1, v/v) (OX2), MeOH (OX3), MeOH
–
water (9:1, v/v) (OX4), or dichloromethane (OX5). A quantity of phenyliodine
bis(trifluoroacetate) (PIFA; 48.3 mg, 2 eq.) (OX1-4) or pyridinium chlorochromate (PCC; 18.1
mg, 1,5 eq.) (OX5) was added. The reactions were monitored by thin layer chromatography and
stopped after 1-4 hours, evaporated under vacuum, and adsorbed onto 2.0 g of silica, layered
on the top of 2.5 g of silica, eluted with 100 ml of EtOAc, and evaporated under vacuum. Each
reaction mixture was re-dissolved in MeOH and analysed by HPLC, using solvent system 1 at
flow rate of 1 ml/min.
Scale-up oxidations I and II
The starting material pcm (500 mg (A), 200 mg (B)) was dissolved in anhydrous ACN (50 ml
(A)), or in ACN:water (9:1, v/v, 10 ml (B)), and a quantity of PIFA (2 eq., 2413.3 mg (A), 965.3
mg (B)) was added to the solutions with continuous stirring, and heating on 60°C (A). After 5
hours (A), or 8 hours (B) the reaction mixtures were evaporated, re-dissolved in MeOH, and
adsorbed onto 12 g of silica. The reaction mixtures were purified by Flash chromatography
(Combi Flash Rf+) on a silica column (RediSep, 24 g), using a dry-loading technique and
solvent system 3 at flow rate of 35 ml/min. During the purifying process 233 fractions (A), and
177 fractions (B) were collected and combined based on their TLC fingerprints. Fraction 46 to
5
2 (A) gave Compound 1 in pure form (19.8 mg). From the fraction 109 to 116 (A) the
Compound 2 was purified with the help of preparative HPLC, using an isocratic elution (45 %
ACN), detected on 320 nm, was obtained 5.0 mg. From fractions 57 to 65 (B) the Compound 3
was purified with the help of preparative HPLC, using an isocratic elution (28 % ACN),
detected on 280 nm, was obtained 6.0 mg. From fraction 88 to 98 (B) the Compound 4 was
purified with the help of preparative HPLC, using an isocratic elution (30 % ACN), detected
on 280 nm, was obtained 18.9 mg. All the purified Compounds (1-4) were analysed by HPLC
to check their purity.
Scale-up oxidation III
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An initial attempt for obtaining compound 5 from pcm (200 mg in 200 ml of ACN) by using 2
eq. of PIFA led to an isolated yield of as low as 2.43% after purification with flash
chromatography and subsequent HPLC. With an aim to increase the yield, the oxidation was
repeated with phenyliodine diacetate (PIDA) as follows. After dissolving pcm (200 mg) in
ACN – water (9:1, v/v, 200 ml), PIDA (723.0 mg, 2 eq.) was added to the solution with
continuous stirring. After 1 h the reaction was stopped, evaporated under vacuum, and adsorbed
onto 3.0 g of Celite 545. The reaction mixture was processed through flash chromatography on
a silica column (Redisep Gold, 12 g), using dry-loading technique and solvent system 3 at flow
rate of 30 ml/min. Ninety-five fractions were collected and combined based on their TLC
fingerprints. Compound 5 was purified from the fraction 50 to 55 with preparative HPLC
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(isocratic elution with 42% aqueous ACN), and 43 mg (yield 19.73 %) was obtained.
Compound characterization data
Compounds 1-5 possessed a purity of >95% by means of HPLC-DAD.
HRMS and MS-MS analyses were performed on a Thermo Velos Pro Orbitrap Elite (Thermo
Fisher Scientific) system. The ionization method was ESI operated in positive ion mode. The
protonated molecular ion peaks were fragmented by CID at a normalized collision energy of
35%. For the CID experiment helium was used as the collision gas. The samples were dissolved
in methanol. The protonated molecular ion peaks were fragmented by CID at a normalized
collision energy of 35%. Data acquisition and analysis were accomplished with Xcalibur
software version 2.0 (Thermo Fisher Scientific).
NMR spectra of compounds 1, 2, 3 and 5 were recorded on a Bruker Avance NEO 500 MHz
spectrometer equipped with a Prodigy BBO 5mm CryoProbe. Compounds 1, 2 and 5 were
dissolved in acetone-d , and methanol-d was used for 3 (VWR Chemicals, Belgium). Standard
6
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one and two-dimensional spectra were processed with MestReNova v6.0.2-5475 software.
NMR spectra of compound 4 were recorded at 25°C on a Varian 800 MHz spectrometer
equipped with a 13C sensitivity enhanced salt tolerant H/ C/ N cryogenically cooled
1
13 15
probehead. Sample was dissolved in methanol-d (Eurisotop, France). Standard one and two-
4
dimensional spectra were acquired using pulse sequences available in the VNMJ 3.2 library.
Chemical shifts are reported in the delta scale using residual solvent signals (methanol-d : 3.31
4
1
13
or 49.15 ppm, acetone-d : 2.05 or 29.84 for H or C, respectively) as references. For spectral
6
analysis and data reporting the ACD/NMR Workbook 2015.2.9. software suite (ACD/Labs,
Canada) was used.
Compound 1:
1
H NMR (500 MHz, acetone-d ) δ = 8.56 (1H, s, H-2), 8.28 (1H, d, J = 1.4 Hz, H-4), 7.82 (1H,
6
d, J = 16.1 Hz, H-9), 7.79 (1H, dd, J = 8.7 Hz, 1.4 Hz, H-6), 7.68 (1H, d, J = 8.7 Hz,
H-7), 6.58 (1H, d, J = 16.1 Hz, H-10), 3.94 (3H, s, 8-OCH ), 3.77 (3H, s, 11-OCH );
3
3
1
3
C NMR (125 MHz, acetone-d ) δ = 167.5 (C-11), 163.7 (C-8), 157.4 (C-7a), 153.4 (C-2),
6
1
1
45.2 (C-9), 132.0 (C-5), 126.3 (C-6), 126.2 (C-3a), 123.0 (C-4), 118.6 (C-10), 115.4 (C-3),
13.3 (C-7), 52.0 (8-OCH ), 51.8 (11-OCH ). HRMS: M+H=261.07572 (delta=-0.1ppm;
3
3
C H O ). HR-ESI-MS-MS (CID=35%; rel. int. %): 247(2); 229(100); 215(7); 197(3).
14 13
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Compound 2:
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H NMR (500 MHz, acetone-d ) δ = 7.53 (1H, d, J = 15.9 Hz, H-3), 7.15 (1H, br s, H-5), 7.04
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(1H, dd, J = 8.2 Hz, 1.2 Hz, H-9), 6.86 (1H, d, J = 8.2 Hz, H-8), 6.27 (1H, d, J = 15.9 Hz,
H-2), 3.71 (3H, s, 1-OCH ). HRMS: M+H=195.06520 (delta=0.1ppm; C H O ). HR-ESI-
MS-MS (CID=35%; rel. int. %): 167(15); 163(100); 153(11); 138(25).
3
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Compound 3:
1
H NMR (500 MHz, methanol-d ) δ = 9.76 (1H, s, H-7), 7.76 (2H, d, J = 8.6 Hz, H-2, H-6),
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.91 (2H, d, J = 8.6 Hz, H-3, H-5); C NMR (125 MHz, methanol-d ) δ = 192.8 (C-7), 165.2
4
(C-4), 133.4 (C-2, C-6), 130.3 (C-1), 116.9 (C-3, C-5). HRMS: M+H=123.04399 (delta=-
0
.5ppm; C H O ). HR-ESI-MS-MS (CID=35%; rel. int. %): 95(100)
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Compound 4:
1
H NMR (800 MHz, methanol-d ) δ = 7.93 (1H, d, J = 16.2 Hz, H-3), 6.90 (1H, d, J = 2.6 Hz,
4
13
H-9), 6.66 - 6.74 (2H, m, H-6, H-7), 6.49 (1H, d, J = 16.1 Hz, H-2), 3.77 (3H, s, OCH ); C
NMR (201 MHz, methanol-d ) δ = 170.0 (C-1), 151.7 (C-5), 151.4 (C-8), 142.3 (C-3), 122.9
(C-4), 120.4 (C-7), 118.0 (C-2), 117.8 (C-6), 114.8 (C-9), 52.2 (OCH ). HRMS:
M+H=195.06522 (delta=0.04ppm; C H O ). HR-ESI-MS-MS (CID=35%; rel. int. %):
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67(3); 163(100); 153(9); 138(1).
Compound 5:
1
H NMR (500 MHz, acetone-d ) δ = 6.85 (2H, d, J = 10.0 Hz, H-3, H-5), 6.68 (1H, d, J = 15.7
6
Hz, H-7), 6.23 (1H, d, J = 15.7 Hz, H-8), 6.16 (2H, d, J = 10.0 Hz, H-2, H-6), 3.70 (3H, s,
9
(CID=35%; rel. int. %): 181(5); 167(30); 163(100); 153(9); 135(15); 107(5).
-OCH3). HRMS: M+H=195.06512 (delta=-0.07ppm; C H O ). HR-ESI-MS-MS
10 11 4
Chemicals
RPMI 1640 medium, Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum
(FBS), antibiotic–antimycotic solution, penicillin–streptomycin solution, L-glutamine and
trypsin/EDTA were purchased from Bioind, Beit Haemek, Israel. MTT was purchased from
Sigma, St. Louis, MO, USA. Dimethyl sulfoxide (DMSO) was obtained from Sigma-Aldrich
Chemie Gmbh, Germany. The Annexin-V-FITC (AV) apoptosis detection kit with Propidium
Iodide (PI) was purchased from Abcam, Cambridge, UK. Dihydroethidium (DHE) was
obtained from Molecular Probes, Invitrogen, USA. Phospho-histone H2A.X antibody and
secondary antibody Alexa Fluor 488 goat anti-Rabbit IgG (H + L) were purchased from Cell
Signaling Technology Inc. (Danvers, Massachusetts, USA). Doxorubicin solution was obtained
from Ebewe Arzneimittel GmbH. CDDP was obtained from Pfizer (Perth) Pty Ltd. Both drugs
were diluted in sterile water prior treatment.
Cells and cell culture
Two mouse lymphoma cell lines were used: L5178 mouse T-cell lymphoma cells (ECACC
catalog number 87111908, U.S. FDA, Silver Spring,MD, U.S.), and a multidrug resistant
3
5
(L5178 ) cell line derived from L5178 by transfection with pHa MDR1/A retrovirus . Cells
B1
were cultured in McCoy’s 5A media supplemented with nystatin, L-glutamine, penicillin,
streptomycin, and inactivated horse serum, at 37∘C and 5% CO . L5178 cell line was selected
by culturing the infected cells with 60 휇g/L colchicine (Sigma).
2
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NCI-H460, A549, NCI-H661, SiHa cell lines were purchased from the American Type Culture
Collection, Rockville, MD. HaCaT cell line was obtained from CLS-Cell Lines Service,
Eppelheim, Germany. NCI-H460/R cells were selected originally from NCI-H460 cells after 3
months of doxorubicin selective pressure . The HeLa and MCF-7 cell lines were purchased
from Bioresource Collection and Research Center, BCRC, Taiwan.
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NCI-H460, NCI-H460/R, A549 and NCI-H661 cell lines were maintained in RPMI 1640
medium supplemented with 10 % FBS, 2 mM L-glutamine and 10,000 U/ml penicillin, 10
mg/ml streptomycin, 25 μg/ml amphotericin B solution, while HaCaT cells were cultured in
DMEM supplemented with 10 % FBS, 2 mM L-glutamine, 4.5 g/l glucose, 5000 U/ml penicillin
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and 5 mg/ml streptomycin solution at 37 °C in a humidified 5 % CO atmosphere. All adherent
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cell lines were sub-cultured at 72 h intervals using 0.25 % trypsin/ EDTA and seeded into a
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fresh medium at the following densities: 8000 cells/cm for NCI-H460, NCI-H460/R and
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HaCaT, and 16 000 cells/cm for H661. MCF-7, HeLa and SiHa cell lines were maintained in
Dulbecco’s modified Eagle’s medium (Sigma-Aldrich) supplemented with 10% FBS, L-
glutamine and 4500 mg/L glucose. These cells were cultured at 37°C in a humidified 5% CO2
incubator (NuAire).
Cytotoxicity by MTT assay
Cell viability was assessed by MTT assay . In case of L5178 and L5178 cell lines, 6 × 10
37
3
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cells were added into in 96-well flat-bottom microtiter plates, treated with the samples and
2
incubated for 72 h. Adherent cells grown in 25 cm tissue flasks were trypsinized, seeded into
flat bottomed 96-well tissue culture plates (1000 cells/well for the NCI-H460, NCI-H460/R,
A549 and HaCaT cell lines, 2000 cells/well for the NCI-H661 cell line), and incubated
overnight in 100 μl of the appropriate medium. After 24 h, the cells were treated with compound
4
(1–20 μM) or 5 (1–20 μM), and incubated for 72 h.
At the end of the incubation period, MTT was added to each well in a final concentration of 0.2
2
0
mg/ ml for 4 h, as described previously . Formazan product was extracted from cells by
DMSO, and the absorbance was measured at 540 nm using an automatic microplate reader
(LKB 5060-006 Micro Plate Reader, Vienna, Austria). The values obtained for samples treated
with 4 and 5 were corrected with those of blank samples treated with the compounds’ solvent
DMSO at a maximal concentration of 1.5% v/v. IC values were defined as the concentration
5
0
of the drug that inhibited cell growth by 50% and calculated by nonlinear regression analysis
using GraphPad Prism 6. For calculated IC values, see Table 1.
5
0
Cell death analysis
The percentages of apoptotic, necrotic and viable cells were determined by AV/PI labeling. All
cell lines were incubated overnight in adherent 6-well plates (100 000 cells per well for NCI-
H460, NCI-H460/R, A549 and HaCaT cell lines, 200 000 cells per well for NCI-H661 cell
line), and then subjected to single treatments with 10 μM Compound 4 or Compound 5.
Treatment with 40 nM of doxorubicin was used as a positive control in NCI-H460 and HaCaT
cells. After 24 h, total (attached and floating) cells were collected. The cell pellet was
resuspended in 50 μl of binding buffer containing AV and PI in a ratio of 1: 1 (v/v). After the
incubation period (10 min at room temperature in the dark), 1 ml of PBS was added and AV/PI
staining was analyzed within 1 h by flow cytometry.
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The fluorescence intensity of AV (FL1-H channel) and PI (FL2-H channel) was measured on
CyFlow Space flow cytometer (Partec, Münster, Germany). A minimum of 10,000 events were
assayed for each sample. Percentages of viable (AV- PI-), early apoptotic (AV+ PI-), apoptotic
and necrotic (AV+ PI+) and dead (AV- PI+) cells were analyzed.
Flow cytometry
All cell lines were incubated overnight in adherent 6-well plates (100 000 cells per well for
NCI-H460, NCI-H460/R, A549 and HaCaT cell lines, 200 000 cells per well for NCI-H661 cell
line), and then subjected to single treatments with 10 μM compound 4 or 5 that lasted 24 h.
Treatment with 2 µM of CDDP was used as positive control.
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To detect double strand DNA breaks, cells were labeled with phospho-histone H2A.X (Ser139)
antibody coupled with secondary antibody Alexa Fluor 488 goat anti-Rabbit IgG (H + L).
Adherent cells were harvested by trypsinization, washed twice in PBS, fixed in 4%
paraformaldehyde for 10 min at room temperature. Cells were then permeabilized by adding
ice-cold 90% methanol and stored at −20 ºC overnight. After washing in PBS, cells were
blocked for 60 min with 0.5% bovine serum albumin in PBS. Cells were then re-suspended in
1
00 μl of primary antibody diluted in 0.5% bovine serum albumin (1:1000) and incubated 60
min at room temperature. After washing in PBS, cells were re-suspended in 100 μl of secondary
antibody (1:1000) and incubated for 30 min at room temperature.
To detect ROS level, cells were stained with superoxide indicator DHE. Adherent cells were
harvested by trypsinization and incubated in medium with 1 mM DHE for 30 min at 37 ºC in
the dark.
Prior to flow cytometric analysis, cells were subsequently washed and resuspended in 1 ml of
PBS. The fluorescence intensity of phospho-histone H2A.X (Ser139) antibody coupled with
secondary antibody (FL1-H channel) and DHE (FL2-H channel) were measured on CyFlow
Space flow cytometer (Partec, Münster, Germany). A minimum of 10 000 events were assayed
for each sample.
DNA Damage response studies
MCF7 cells were incubated overnight in adherent 60 mm culture dish (700 000 cells per dish),
and then pre-treated with 10 μM of compound 5 or pcm for 30 minutes. Treatment with 10 μM
of protoapigenone was used as positive control. After this, they were exposed to UV irradiation
2
(10 J/m ) in the following 1 hour to induce DDR. Protein collection and western blot assays
19
were performed in accordance with the method described by our previous study . Proteins of
the cells were extracted with lysis buffer (1 mM EDTA, 10 mM Tris, pH 7.5, 0.5% NP-40, 10%
glycerol, and 420 mM NaCl) supplemented with a cocktail of protease and phosphatase
inhibitor (Roche Applied Science), and 1 mM DL-dithiothreitol. After SDS-PAGE, the
separated proteins in the gel were electrophoretically transferred to nitrocellulose membranes.
The membranes with the transferred proteins were detected using specific primary antibodies
followed by horseradish peroxidase (HRP)-coupled secondary antibodies (Jackson
ImmunoResearch), then recognized by Enhanced chemiluminescence (ECL) reagent
(
(
Millipore). Images were captured with a LAS-4000 luminescent image analyser System
Fujifilm). The primary antibodies against Chk1-S345 and Chk2-T68 were purchased from Cell
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Signaling Technology (Danvers, USA); Chk1, Chk2 and β-actin were purchased from Santa
Cruz. The β-actin expression served as an internal control.
Hydroxyl radical scavenging activity
•
The OH scavenging capacity of the investigated compounds (pcm, and NXY-059 as positive
control) were performed by measuring the inhibition of damage to 2-deoxy-D-ribose by Fenton
3
8
reaction . Assays were carried out in 50 mM 3-(N-morpholino)propanesulfonic acid (MOPS)
buffer, pH 6.0, containing 2.8 µM 2-deoxy-D-ribose and increasing concentrations (0.002-5
mM) of samples to which 0.2 µM H O and 1.5 µM FeSO were added to start the reaction (in
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total volume of 280 µl). Then, after 3 minutes incubation at 37 °C, during which period of time
o
˙
OH formation was linear, TBA reactivity was developed by heating at 100 C for 15 minutes
after adding 400 µl of a mixture containing 15% (w/v) TCA (trichloroacetic acid), 0.25 M HCl,
.325 % (w/v) TBA and 0.06 M NaOH. The resulting chromogens were measured at
0
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wavelength of 532 nm against appropriate blanks .
Combination assay on the cytotoxicity of pcm and H O
2
2
A checkerboard microplate method was applied to study the interaction between pcm and H O
2
2
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concerning their cytotoxicity on MCF7 cells. Cells (1x10 ) were seeded to each well in a 96-
well plate. The plates were incubated at 37°C for 24 h. Standard solution of H O (Sigma-
2
2
Aldrich) and pcm were stored at -20°C until use. The stock solutions and serial two-fold
dilutions of each drug were prepared according to the recommendations of several fix pcm vs.
H O ratios to testing. Pcm was serially diluted along the ordinate, while H O was diluted
2
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2
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along the abscissa. A total volume of 100 μl (including pcm and H O ) was distributed into
2
2
each well of the microdilution plates. The plates were incubated at 37°C for 48 h, and then MTT
solution was added to each well. After incubation at 37°C for 4 h the MTT-containing medium
was removed, and 100 μl of DMSO was added to dissolve the formazan crystals. Cell viability
was determined by measuring the optical density values (absorbance) of the chromogenic
product at 550 nm with an ELISA reader (Thermo Multiskan Ascent). Three plates were
prepared on three different days, and averages of these three replicates were utilized to calculate
the combination index (CI) values at several fix pcm vs. H O ratios by using the CalcuSyn
2
2
software.
In silico calculations
Calculations were performed with the Gaussian09 package (Rev. A). Ground states geometries
were optimized using the B3P86 functional that has been shown to properly reproduce
thermodynamics of polyphenol oxidative processes. The double- basis set 6-31+G(d,p) was
used to describe C-, O-, and H-atoms. Calculations were performed including solvation
polarization effects by using conductor-like polarizable continuum model (C-PCM). Frequency
calculations were performed to ensure that the obtained geometries were in the global minimum
of the potential energy surface, as pictured by the absence of any imaginary frequency. Mulliken
spin density visualization has been carried out using the VMD program.
Acknowledgment
This work was funded by the National Research, Development and Innovation Office, Hungary
(NKFIH; K119770). Ministry of Human Capacities, Hungary grant 20391-
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3/2018/FEKUSTRAT is acknowledged. P.T. was funded by the Czech Science Foundation
(P208/12/G016) and National Program of Sustainability I from the Ministry-of-Youth,
Education and Sports of the Czech Republic (LO1305). A.H. was supported by the UNKP-18-
4 New National Excellence Program of the Ministry of Human Capacities, by a Short-Term
Scientific Mission (STSM) Grant from COST Action CM1407 “Challenging organic syntheses
inspired by nature - from natural products chemistry to drug discovery”, by the János Bolyai
fellowship of the Hungarian Academy of Sciences, and by the Kálmán Szász Prize. F.D.M. and
P.T. thank CALI “CAlcul en LImousin” for computational resources as well as Nouvelle
Aquitaine region for financial support. The authors acknowledge a bilateral mobility grant from
the Hungarian Academy of Sciences and the Ministry of Science and Technology, Taiwan
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(MOST 107-2911-I-037-502).
Abbreviations used
pcm, p-coumaric acid methyl ester; cm, caffeic acid methyl ester; fm, ferulic acid methyl
ester; GI , 50% growth inhibitory concentration; PIFA, phenyliodine bis(trifluoroacetate);
5
0
BCRP, breast cancer resistance protein; SEM, standard error of mean; MTT, 3-(4,5-
Dimethylthiazol-2-YI)-2,5-Diphenyltetrazolium Bromide; H2A.X, Histone 2A.X; ATR,
ataxia telangiectaisa and Rad3-related kinase; ATM, ataxia telangiectasia mutated kinase; FA,
Fanconi Anemia; DSBs, DNA Double strand breaks; DPPH, 2,2-diphenyl-1-picrylhydrazyl;
ABTS, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid); 95% C.I., 95% confidence
interval; CI, combination index; CI , weighted average CI value; PDA, photometric diode
avg
array; SET, single electron transfer; SPE, solid phase extraction; HAT, hydrogen atom
transfer; BDE, bond dissociation enthalpy; ACN, acetonitrile; PIDA, phenyliodine diacetate;
DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; AV, Annexin-V-
FITC; PI, propidium iodide; DHE, dihydroethidium; PBS, phosphate-buffered saline; DDR,
DNA damage response; DL, dithiothreitol; HRP, horseradish peroxidase; ECL, Enhanced
chemiluminescence; MOPS, 3-(N-morpholino)propanesulfonic acid; TBA, thiobarbituric
acid; C-PCM, conductor-like polarizable continuum model
Supporting Information Availability
The followings are available as Supporting Information: experimental description of the
stability test of pcm with ccH O (30%) and HPLC-PDA chromatogram taken after 92 hours
2
2
(Figure S1); experimental description, HPLC-PDA and LC-MS DAD1-EWC fingerprints of
the Fenton reaction of pcm, confirming the formation of compound 5 (Figures S2 and S3);
atomic numbering and NMR signal assignment for compounds 1-5 (Figure S4); Molecular
Formula strings.
Corresponding author information
E-mail: hunyadi.a@pharm.u-szeged.hu; tel.: +36 62 546456, fax: +36 62 545704.
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References
1. El-Seedi, H. R.; El-Said, A. M.; Khalifa, S. A.; Göransson, U.; Bohlin, L.; Borg-Karlson,
A. K.; Verpoorte, R. Biosynthesis, natural sources, dietary intake, pharmacokinetic
properties, and biological activities of hydroxycinnamic acids. J. Agric. Food. Chem. 2012,
60, 10877-10895.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
. Chen, J. H.; Ho C. T. Antioxidant activities of caffeic acid and its related hydroxycinnamic
acid compounds. J. Agric. Food Chem. 1997, 45, 2374–2378.
3
4
. Razzaghi-Asl, N.; Garrido, J.; Khazraei, H.; Borges, F.; Firuzi, O. Antioxidant properties
of hydroxycinnamic acids: a review of structure- activity relationships. Curr. Med. Chem.
2
013, 20, 4436-4450.
. Teixeira, J.; Gaspar, A.; Garrido, E. M.; Garrido, J.; Borges, F. Hydroxycinnamic acid
antioxidants: an electrochemical overview. Biomed. Res. Int. 2013, 2013, 251754.
5. Rao, C. V.; Desai, D.; Simi, B.; Kulkarni, N.; Amin, S.; Reddy, B. S. Inhibitory effect of
caffeic acid esters on azoxymethane-induced biochemical changes and aberrant crypt foci
formation in rat colon. Cancer Res. 1993, 53, 4182-4188.
6. Xiao, H.; Parkin, K. L. Isolation and identification of potential cancer chemopreventive
agents from methanolic extracts of green onion (Allium cepa) Phytochemistry 2007, 68,
1
059-1067.
7
8
9
1
. Pieters, L.; Van Dyck, S.; Gao, M.; Bai, R.; Hamel, E.; Vlietinck, A.; Lemière, G. Synthesis
and biological evaluation of dihydrobenzofuran lignans and related compounds as potential
antitumor agents that inhibit tubulin polymerization. J. Med. Chem. 1999, 42, 475-481.
. Apers, S.; Paper, D.; Bürgermeister, J.; Baronikova, S.; Van Dyck, S.; Lemière, G.;
Vlietinck, A.; Pieters, L. Antiangiogenic activity of synthetic dihydrobenzofuran lignans.
J. Nat. Prod. 2002, 65, 718-720.
. Basini, G.; Spatafora, C.; Tringali, C.; Bussolati, S.; Grasselli, F. Effects of a ferulate-
derived dihydrobenzofuran neolignan on angiogenesis, steroidogenesis, and redox status
in a swine cell model. J. Biomol. Screen 2014, 19, 1282-1289.
0. Yin, S. Y.; Jian, F. Y.; Chen, Y. H.; Chien, S. C.; Hsieh, M. C.; Hsiao, P. W.; Lee, W. H.;
Kuo, Y. H.; Yang, N. S. Induction of IL-25 secretion from tumour-associated fibroblasts
suppresses mammary tumour metastasis. Nat. Commun. 2016, 7, 11311.
11. Kita, Y.; Tohma, H.; Hatanaka, K.; Takada, T.; Fujita, S.; Mitoh, S.; Sakurai, H.; Oka, S.
Hypervalent iodine-induced nucleophilic substitution of para-substituted phenol ethers.
Generation of Cation Radicals as Reactive Intermediates. J. Am. Chem. Soc., 1994, 116,
3
684–3691.
2
2
ACS Paragon Plus Environment

Page 23 of 26
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
12. Urbaniak, A.; Molski, M.; Szeląg, M. Quantum-chemical calculations of the antioxidant
properties of trans-p-coumaric acid and trans-sinapinic acid. Comp. Methods Sci. Technol.
2
012, 18, 117-128.
1
1
1
3. Canevali, C.; Orlandi, M.; Pardi, L.; Rindone, B.; Scotti, R.; Sipilad, J.; Morazzoni F.
Oxidative degradation of monomeric and dimeric phenylpropanoids: reactivity and
mechanistic investigation. J. Chem. Soc., Dalton Trans., 2002, 0, 3007–3014.
4. Zhu, Y.; Zhang, L.-X.; Zhao, Y.; Huang, G.-D. Unusual sesquiterpene lactones with a new
carbon skeleton and new acetylenes from Ajania przewalskii. Food Chem. 2010, 118, 228-
238.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5. Chuang, T. H.; Chan, H. H.; Wu, T. S.; Li, C. F. Chemical constituents and biological
studies of the leaves of Grevillea robusta. Molecules 2011, 16, 9331-9339.
16. Rahman, A. U.; Shabbir, M.; Ziauddin Sultani, S.; Jabbar, A.; Choudhary, M. I.
Cinnamates and coumarins from the leaves of Murraya paniculata. Phytochemistry 1997,
4
4, 683-685.
1
1
7. Hunyadi, A.; Martins, A.; Danko, B.; Chang, F. R.; Wu, Y. C. Protoflavones: a class of
unusual flavonoids as promising novel anticancer agents. Phytochem. Rev. 2014, 13, 69-
7
7.
8. Dankó, B.; Tóth, S.; Martins, A.; Máté, V.; Kúsz, N.; Molnár, J.; Chang, F. R.; Wu, Y. C.;
Szakács, G.; Hunyadi, A. Synthesis and SAR study of novel anticancer protoflavone
derivatives - investigation of cytotoxicity and interaction with the ABCB1 and ABCG2
multidrug efflux transporters. ChemMedChem 2017, 12, 850-859.
1
2
9. Wang, H. C.; Lee, A. Y.; Chou, W. C.; Wu, C. C.; Tseng, C. N.; Liu, K. Y.; Lin, W. L.;
Chang, F. R.; Chuang, D. W.; Hunyadi, A.; Wu, Y. C. Inhibition of ATR-dependent
signaling by protoapigenone and its derivative sensitizes cancer cells to interstrand cross-
link-generating agents in vitro and in vivo. Mol. Cancer Ther. 2012, 11, 1443-1453.
0. Stanković, T.; Dankó, B.; Martins, A.; Dragoj, M.; Stojković, S.; Isaković, A.; Wang, H.
C.; Wu, Y. C.; Hunyadi, A.; Pešić, M. Lower antioxidative capacity of multidrug-resistant
cancer cells confers collateral sensitivity to protoflavone derivatives. Cancer Chemother.
Pharmacol. 2015, 76, 555-565.
21. Kastan, M. B.; Bartek, J. Cell-cycle checkpoints and cancer. Nature 2004, 432, 316-323.
2
2. Chen, C. C.; Kennedy, R. D.; Sidi, S.; Look, A. T.; D'Andrea, A. CHK1 inhibition as a
strategy for targeting Fanconi Anemia (FA) DNA repair pathway deficient tumors. Mol.
Cancer 2009, 8:24.
2
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 26
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
23. Cole, K. A.; Huggins, J.; Laquaglia, M.; Hulderman, C. E.; Russell, M. R.; Bosse, K.;
Diskin, S. J.; Attiyeh, E. F.; Sennett, R.; Norris, G.; Laudenslager, M.; Wood, A. C.; Mayes,
P. A.; Jagannathan, J.; Winter, C.; Mosse, Y. P.; Maris, J. M. RNAi screen of the protein
kinome identifies checkpoint kinase 1 (CHK1) as a therapeutic target in neuroblastoma.
Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 3336-3341.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
4. Brooks, K.; Oakes, V.; Edwards, B.; Ranall, M.; Leo, P.; Pavey, S.; Pinder, A.; Beamish,
H.; Mukhopadhyay, P.; Lambie, D.; Gabrielli, B. A potent Chk1 inhibitor is selectively
cytotoxic in melanomas with high levels of replicative stress. Oncogene 2013, 32, 788-796.
25. Kuo, L. J.; Yang, L. X. Gamma-H2AX - a novel biomarker for DNA double-strand breaks.
In Vivo 2008, 22, 305-309.
2
2
2
2
6. Kwon, Y. S.; Kim, C. M. Antioxidant constituents from the stem of Sorghum bicolor. Arch.
Pharm. Res. 2003, 26, 535-539.
7. Kiliç, I.; Yeşiloğlu, Y. Spectroscopic studies on the antioxidant activity of p-coumaric acid.
Spectrochim Acta A Mol Biomol Spectrosc. 2013, 115, 719-724.
8. Fong, J. J.; Rhoney, D. H. NXY-059: review of neuroprotective potential for acute stroke.
Ann. Pharmacother. 2006, 40, 461-471.
9. Balogh, G. T.; Vukics, K.; Könczöl, A.; Kis-Varga, A.; Gere, A.; Fischer, J. Nitrone
derivatives of trolox as neuroprotective agents. Bioorg. Med. Chem. Lett. 2005, 15, 3012-
3
015.
3
0. Chou T. C. Theoretical basis, experimental design, and computerized simulation of
synergism and antagonism in drug combination studies. Pharmacol. Rev. 2006, 58, 621-
6
81.
31. Trouillas, P.; Fagnère C.; Lazzaroni, R.; Calliste, C.; Marfak, A.; Duroux, J.-L. A
theoretical study of the conformational behavior and electronic structure of taxifolin
correlated with the free radical-scavenging activity. Food Chem. 2004, 88, 571-582.
3
2. Trouillas, P.; Marsal, P.; Siri, D.; Lazzaroni, R.; Duroux, J.-L. A DFT study of the reactivity
of OH groups in quercetin and taxifolin antioxidants: The specificity of the 3-OH site. Food
Chem. 2006, 97, 679-688.
3
3. Anouar, E.; Calliste, C. A.; Kosinová, P.; Di Meo, F.; Duroux, J. L.; Champavier, Y.;
Marakchi, K.; Trouillas P. Free radical scavenging properties of guaiacol oligomers: a
combined experimental and quantum study of the guaiacyl-moiety role. J. Phys. Chem. A.
2
009, 113, 13881-13891.
3
4. Leopoldini, M.; Russo, N.; Toscano, M. The molecular basis of working mechanism of
natural polyphenolic antioxidants. Food Chem. 2011, 125, 288-306.
2
4
ACS Paragon Plus Environment

Page 25 of 26
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
35. Pastan, I.; Gottesman, M. M.; Ueda K.; Lovelace E.; Rutherford A. V.; Willingham, M.
C. A retrovirus carrying an MDR1 cDNA confers multidrug resistance and polarized
expression of P-glycoprotein in MDCK cells. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 4486-
4
490.
36. Pesic, M.; Markovic, J. Z.; Jankovic, D.; Kanazir, S.; Markovic, I. D.; Rakic, L.; Ruzdijic,
S. Induced resistance in the human non small cell lung carcinoma (NCI-H460) cell line in
vitro by anticancer drugs. J. Chemother. 2006, 18, 66-73.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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3
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5
5
5
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5
5
5
5
5
5
6
0
1
2
3
4
5
6
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8
9
0
1
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5
6
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0
1
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3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
7.
Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to
proliferation and cytotoxicity assays. J. Immunol. Methods 1983, 65, 55−63.
8. Mathew S.; Abraham T. E. In vitro antioxidant activity and scavenging effects of
Cinnamomum verum leaf extract assayed by different methodologies. Food Chem. Toxicol.
2006, 44, 198-206.
3
3
9. Tadolini B.; Cabrini L. The influence of pH on OH scavenger inhibition of damage to
deoxyribose by Fenton reaction. Mol. Cell. Biochem. 1990, 94, 97-104.
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