3′-Hydroxy-3,4′-dimethoxyflavone blocks tubulin polymerization and is a potent apoptotic inducer in human SK-MEL-1 melanoma cells

Article abstract of DOI:10.1016/j.bmc.2017.09.043

Flavonoids are naturally occurring polyphenolic compounds and are among the most promising anticancer agents. A series of flavonols and their 3-methyl ether derivatives were synthesized and assessed for cytotoxicity. It was found that 3′-hydroxy-3,4′-dime

Full text of DOI:10.1016/j.bmc.2017.09.043

Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
30-Hydroxy-3,40-dimethoxyflavone blocks tubulin polymerization
and is a potent apoptotic inducer in human SK-MEL-1 melanoma cells
Francisco Estévez-Sarmiento a, Mercedes Said a, Ignacio Brouard b, Francisco León b, Celina García c,
José Quintana a, Francisco Estévez a,
⇑
a Departamento de Bioquímica y Biología Molecular, Unidad Asociada al Consejo Superior de Investigaciones Científicas (CSIC), Instituto Universitario de Investigaciones
Biomédicas y Sanitarias, Universidad de las Palmas de Gran Canaria, Paseo Blas Cabrera Felipe s/n, 35016 Las Palmas de Gran Canaria, Spain
b Instituto de Productos Naturales y Agrobiología, CSIC, Avenida Astrofísico Francisco Sánchez 3, 38206 La Laguna, Tenerife, Spain
c Instituto Universitario de Bio-Orgánica ‘‘Antonio González” (IUBO-AG), Departamento de Química Orgánica, Universidad de La Laguna, Avda. Astrofísico Francisco Sánchez 2,
38206 La Laguna, Tenerife, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Flavonoids are naturally occurring polyphenolic compounds and are among the most promising anti-
cancer agents. A series of flavonols and their 3-methyl ether derivatives were synthesized and assessed
for cytotoxicity. It was found that 30-hydroxy-3,40-dimethoxyflavone (flavonoid 7a) displayed strong
cytotoxicity against human SK-MEL-1 melanoma cells and blocked tubulin polymerization, but had no
significant cytotoxic effects against quiescent or proliferating human peripheral blood mononuclear cells.
Our analyses showed that flavonoid 7a induces G2-M cell cycle arrest and apoptosis in melanoma cells
which is associated with cytochrome c release and activation of both extrinsic and intrinsic apoptotic
pathways of cell death.
Received 27 July 2017
Revised 26 September 2017
Accepted 30 September 2017
Available online xxxx
Keywords:
Apoptosis
Caspases
Cytotoxicity
Flavonoids
Melanoma
Ó 2017 Published by Elsevier Ltd.
1. Introduction
pathway, apoptosis is mediated by death receptors and involves
caspase-8 activation. Both caspase-8 and caspase-9 activate cas-
Natural products are important sources of potential new anti-
cancer agents. Approximately 63% of anticancer drugs introduced
over the last 25 years are natural products or can be traced back
to a natural products source.1 Flavonoids are secondary metabo-
lites with low molecular weight and phenolic structure, occurring
ubiquitously in food plants, and being a common component in the
human diet. Many flavonoids display a vast array of biological
activities including antitumor properties.2,3 These properties of fla-
vonoids are mediated by different types of cell cycle arrest and
induction of apoptosis. This kind of cell death is characterized by
DNA fragmentation, chromatin condensation, and formation of
apoptotic bodies. It is mediated by the activation of a class of cys-
teine proteases, known as caspases, by two main pathways.4,5 In
the intrinsic pathway, diverse pro-apoptotic signals induce mito-
chondrial cytochrome c release to the cytosol, and promote apop-
tosome assembly and caspase-9 activation. In the extrinsic
pase-3, which is responsible for cleaving specific cellular proteins
during apoptosis.
Previous research has shown that flavonoids with a hydroxy
group at position 3 are potential anticancer agents and that methy-
lation of these compounds increases their antiproliferative activity
against human tumor cells.6–8 Knowledge of the effects of flavo-
noid substitution profiles on their antitumoral and tubulin poly-
merization inhibitory activity has witnessed much progress in
recent years. Casticin and centaureidin (Fig. 1), are two important
members of the family of flavones which show two methoxy
groups at the C40 and C3 positions and one hydroxy at the C30 posi-
tion and are promising anticancer agents due to their high cytotox-
icity against a variety of cancer cells.
Centaureidin was the first cytotoxic flavone described as a tubu-
lin interactive agent and this was followed by the identification of
a flavone which incorporated the same substitution profile as cen-
taureidin and that inhibited the incorporation of tubulin into
microtubules.9,10 Recently, methoxylated flavones have been used
as the lead compounds in the design of new analogues as tubulin
inhibitors.11 Analyses of structure and activity showed that i) com-
pounds that contain the previously indicated substitution profile
Abbreviations: MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide; PI, propidium iodide.
⇑
Corresponding author.
E-mail address: francisco.estevez@ulpgc.es (F. Estévez).
https://doi.org/10.1016/j.bmc.2017.09.043
0968-0896/Ó 2017 Published by Elsevier Ltd.
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2
F. Estévez-Sarmiento et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
structural requirements for a flavone to induce cell death in the
human melanoma cell line SK-MEL-1. We focused mainly on the
B ring substituents and the C-3 position (C ring). Four compounds
were designed to contain the same pair of 30-hydroxy-40-methoxy
substituents (B ring) and all four possible combinations of meth-
oxy/hydrogen groups at position C-7 (5a, 5b) and hydroxy/meth-
oxy groups at C-3 (7a, 7b). Additionally, the same compounds
were prepared with a 40-hydroxy-30-methoxy pair of substituents
in the B ring (5c, 5d and 7c, 7d). Finally, the collection was com-
pleted with similar examples containing just one hydroxy group
in the B ring (positions 40 and 20). Two acetophenones and four
aldehydes were used as starting material for this collection of fla-
vones (Scheme 1).
The synthetic route did not differ from the traditional one. An
aldol condensation reaction in basic media was used to generate
eight compounds with a chalcone skeleton (3a-h) which were sub-
sequently subjected to the Algar-Flynn-Oyamada cyclization under
oxidative conditions to produce eight benzylated flavonols.14 These
compounds were submitted to hydrogenation reaction conditions
to obtain the free alcohols (5a-h).15 The methylation reaction
employing MeI provided methyl ethers (6a-g) which were then
deprotected in order to obtain the free alcohols (7a-g).16
Fig. 1. Chemical structure of anticancer 3-methoxyflavones.
were more active as inhibitors of tubulin polymerization than
those that did not, ii) the substitution of a methoxy in C3 for a
hydroxy group dramatically decreased the activity of the com-
pound, and iii) substitutions in the A ring do not seem to be rele-
vant at all.12 Studies in our research group confirmed that the
substitution profile (methoxy on C3, C40 and hydroxy on C30) leads
to compounds with antileukemic activity and significant capacity
to inhibit tubulin polymerization.13
The potential significance of flavonoids containing this substitu-
tion pattern in antimelanoma therapy is largely unexplored to
date. The main aim of this work was to investigate the cytotoxic
effects of 15 synthetic flavonoids against SK-MEL-1 melanoma cells
and elucidate the mechanism of cell death for the most effective
compounds. The results show 30-hydroxy-3,40-dimethoxyflavone,
7a to be the most cytotoxic compound against this cell line. We
provide new evidence that shows for the first time that this flavo-
noid induces apoptotic cell death which is mediated by caspase
activation and tubulin polymerization inhibition.
2.2. Biological evaluation
2.2.1. A synthetic phenylbenzopyrone derivative, 30-hydroxy-3,40-
dimethoxyflavone, inhibits the growth and cell viability of human SK-
MEL-1 melanoma cells
Previous studies have demonstrated that the methoxyflavones
have higher antiproliferative activity than the corresponding
hydroxylated derivatives and that the presence of 30-hydroxy-40-
methoxy groups on the B ring of the flavonoid skeleton enhances
the cytotoxicity.7,12,13,17
2. Results and discussion
2.1. Chemistry
In this manuscript, the design and synthesis of 15 flavonoids
was developed with the purpose of determining the minimum
In the present study, the potential cytotoxic properties of a
series of 15 flavonoids, including flavonols and 3-methyl ether
Scheme 1. Synthesis of the intermediate chalcones and methyl ethers.
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F. Estévez-Sarmiento et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
3
derivatives with one or two substituents on the B ring (2-phenyl
Compound 7a (µM)
(A)
group) and an additional substituent on the A ring were evaluated
using the human melanoma cell line SK-MEL-1 (Scheme 1, Table 1).
We tested antiproliferative activity with an MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay.
The compounds containing two substituents on the B ring were
found to show significant differences in their cytotoxicity between
the flavonols and their corresponding 3-methoxy derivatives (5c vs
7c and 5a vs 7a). The 3-methylether derivative 7a is more cytotoxic
than the corresponding flavonol 5a. The IC50 values (the concentra-
tions that induce a 50% inhibition of cell growth) for 7a against SK-
MEL-1 cells was 6.9 1.1 mM, while the IC50 value for 5a was
32.6 7.1 mM. The effects of the flavonoid 7a on SK-MEL-1 cells
viability were visualized by phase contrast microscopy. While con-
trol cells appeared perfectly round and healthy, there was a clear
reduction in the number of cells and they appeared unhealthy after
treatment with increasing concentrations of 7a (Fig. 2A).
0
3
10
30
(B)
125
(C)
125
PBMC
PBMC+PHA
SK-MEL-1
100
75
100
75
50
25
0
50
25
0
Although there were no differences between the flavonols 5c
and 5a, the position of the hydroxy and methoxy groups on the B
ring seems to play a key role in determining the potency of 3-
methoxyflavonols on cell viability (7a vs 7c).
0
3 10 30 0 3 10 30
0
0.1 1 10 100
Compound 7a (µM)
Compound 7a (µM)
For the flavonols containing one substituent at the C7 position
on the A ring and one hydroxy group on the B ring (5f and 5h),
the position of the hydroxy group on the B ring does not seem to
be important, since the 40-hydroxy derivative was inactive as well
as the 20-hydroxy derivative (5f vs 5h) and methylation of the
hydroxy group located at the C3 position did not affect antiprolif-
erative activity either (5f vs 7f). The presence of a methoxy at the
C7 position on the A ring apparently is not a major determinant of
cytotoxicity (5g vs 5h and 7e vs 7f).
Fig. 2. Effects of 7a on SK-MEL-1 melanoma cells viability. (A) Cells were incubated
with vehicle or the described concentrations of 7a for 24 h and images were
obtained with an inverted phase-contrast microscope. (B) Cells were cultured in the
presence of the described concentrations of 7a for 72 h, and thereafter cell viability
was determined by the MTT reduction assay. The results are representative of those
obtained in at least three independent experiments. (C) Effect of 7a on cell viability
of peripheral blood mononuclear cells (PBMCs). Quiescent and phytohemagglu-
tinine-activated PBMC cells from healthy human origin were cultured in the
presence of the specified concentrations of 7a for 24 h. Values represent means SE
of three independent experiments each performed in triplicate. *P < 0.05, signifi-
cantly different from the untreated control.
In the case of flavonols (5b vs 5d) and their corresponding 3-
methylethers (7b and 7d) that contained one substituent in C7
on the A ring and two substituents on the B ring, there were signif-
icant differences in cytotoxicity against melanoma cells between
the position of the hydroxy and methoxy groups on the B ring
(5d vs 5b). In the case of 3-methylether derivatives (7b vs 7d),
the substitution pattern of the latter groups on the B ring seems
to play a key role in conferring cytotoxicity since the 30-hydroxy-
2.2.2. 7a induces G2-M arrest and apoptosis on human SK-MEL-1
melanoma cells
To determine whether 7a-induced cytotoxicity involves
changes in cell cycle progression, we evaluated the effect of 7a
on cell-cycle phase distribution by flow cytometry. As shown in
Table 2 and Fig. 3A, 7a caused a significant G2–M arrest at the
expense of G1 phase cell population in SK-MEL-1 cell line at all con-
centrations analysed (3–30 mM) and an increase in the percentage
of sub-G1 cells (apoptotic cells) as determined by flow cytometry
after propidium iodide labeling. Flavonoid 7a increased apoptosis
and this effect is concentration-dependent. The percentage of
sub-G1 increased up to 22% after 24 h of treatment with 30 mM
7a (Fig. 3A). The morphological changes characteristic of apoptotic
cells (condensed and fragmented chromatin) after treatment with
7a were also visualized by fluorescent microscopy (Fig. 3B). Taken
together, these results indicate that 7a induces cell-cycle arrest in
the G2–M phase and apoptosis on human melanoma cells.
40-methoxy derivative (7b) exhibited
40-hydroxy-30-methoxy derivative 7d.
a higher potency than
In the case of flavonoids containing 30-hydroxy-40-methoxy
substituents on the B ring, the 3-hydroxy-7-methoxy flavonoid
(5b) was more potent than the 3,7-dimethoxy derivative (7b). In
all cytotoxicity assays the chemotherapeutic etoposide was used
as a positive control (Table 1).
The cytotoxicity experiments revealed that 30-hydroxy-3,40-
dimethoxyflavone (7a) displays strong cytotoxic activity against
the human melanoma cell line SK-MEL-1 with an IC50 value as
low as 6.9 mM (Table 1, Fig. 2B). Moreover, we also assayed the
effects of 7a on the growth of the human lymphocytes and found
that it did not significantly affect cell viability of quiescent and pro-
liferating cells (Fig. 2C). Since 7a was the most cytotoxic compound
we decided to investigate whether this flavonoid displays its cyto-
toxic action on SK-MEL-1 cells through activation of the apoptotic
pathway.
2.2.3. 7a induces cell death by a caspase-dependent pathway in SK-
MEL-1 cells
To analyze whether the effect of 7a is mediated by caspases, we
examined whether this flavonoid induces the enzymatic activities of
caspase-3-like proteases (caspase-3/7) and of caspase-8 and -9. To
Table 1
Effects of flavonoids on the growth of human SK-MEL-1 melanoma cells.
Compound
IC50 M)
Compound
IC50 M)
5a
32.6 7.1
7a
5b
5c
5d
5e
>100
5f
>100
7f
5g
>100
7g
5h
(
l
15.5 2.8
7b
22.0 5.8
7c
37.0 5.0
7d
>100
7e
Etoposide
8.0 4.1
(l
6.9 1.1
26.2 1.9
79.0 13.1
52.0 8.2
91.0 11.1
>100
>100
Cells were cultured for 72 h in presence of the indicated compounds and the IC50 values were calculated using a colorimetric MTT assay. The data shown represent the
mean SEM of 2–3 independent experiments with three determinations in each.
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F. Estévez-Sarmiento et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Table 2
Effect of different durations of treatment with 7a on cell cycle phase distribution of human SK-MEL-1 melanoma cells.
% Sub-G1
% G1
% S
% G2-M
Control
4.5 1.6
15.9 1.1*
9.2 1.7*
61.5 1.7
24.4 0.5*
14.4 1.6*
30.7 0.9*
17.7 0.4
21.4 1.2*
23.7 0.4
26.8 1.8*
16.1 0.2
37.9 2.6*
52.2 3.9*
20.2 10.1*
3 mM 7a
10 mM 7a
30 mM 7a
21.9 7.5*
Cells were cultured with increasing concentrations of 7a for 24 h and the cell cycle phase distribution was determined by flow cytometry. The values are means SE of three
independent experiments with three determinations in each. Asterisks indicate a significant difference (P < 0.05) compared with the corresponding controls.
this end, cells were treated with increasing concentrations of 7a and
cell lysates were assayed for the cleavage of the tetrapeptides DEVD-
0.7
pNA, IETD-pNA and LEHD-pNA as specific substrates for caspase-
Taxol
0.6
0.5
0.4
0.3
0.2
0.1
0.0
3/7, caspase-8 and caspase-9, respectively. All these caspase activi-
ties were significantly detectable after 24 h of treatment with 7a
(Fig. 3C). Dose-response experiments show that a low concentration
(3 mM) of 7a was sufficient to induce caspase-3/7 activation, while
caspase-8 and -9 caused lower levels of activation.
To determine whether 7a-triggered apoptosis involves the pro-
cessing of caspase-3, cells were treated with increasing concentra-
tions of the flavonoid for 24 h and this caspase was analysed by
Western blot. As shown in Fig. 3D, 7a stimulated the cleavage of
the main executioner caspase. It was also studied whether this
apoptotic effect of 7a involves the release of mitochondrial cyto-
chrome c. The results showed a significant increase in the amount
of this proapoptotic protein in the cytosol after treatment with 7a
in a concentration-dependent manner (Fig. 3D). Taken together
these data show that both the intrinsic and the extrinsic pathways
play an important role in cell death induced by 7a in SK-MEL-1
melanoma cells.
Control
10 µM 7a
Colchicine
30 µM 7a
0
10
20
30
40
50
Time (min)
Fig. 4. Flavonoid 7a blocks the tubulin polymerization in vitro. Purified tubulin
protein in a reaction buffer was incubated at 37 °C in the absence (control) or in the
2.2.4. Effect of 7a on tubulin polymerization
As shown before, concentrations of flavonoid 7a that inhibited
SK-MEL-1 proliferation caused an increase in the number of cells
in the G2–M phase at 24 h, taking into account that these cells have
presence of taxol (10
lM), colchicine (5 lM) or the indicated concentrations of 7a
and the absorbance at 340 nm was measured using a microplate reader.
Fig. 3. Effects of 7a on apoptosis induction in SK-MEL-1 melanoma cells. (A) Cells were incubated with increasing concentrations of 7a for 24 h, subjected to flow cytometry
using propidium iodide labeling and the percentage of cells in the sub-G1 region (apoptotic cells) determined. (B) Photomicrographs of representative fields of SK-MEL-1 cells
stained with bisbenzimide trihydrochloride to evaluate nuclear chromatin condensation (i.e. apoptosis) after treatment for 24 h with 30 lM of 7a. (C) Activation of caspases
in response to 7a. The cells were treated with the described concentrations of 7a and cell lysates were assayed for caspase 3/7, -8 and -9 activities. Results are expressed as
factorial increases in caspase activity relative to the control. Values represent the means SEs of three independent experiments each performed in triplicate. *P < 0.05,
significantly different from untreated control. (D) Effects of 7a on processing of pro-caspase-3 and mitochondrial cytochrome c release. Cells were incubated in the presence
of the described concentrations of 7a, and whole cell lysates or cytosolic fractions (in the case of cytochrome c) were probed with antibodies raised against the indicated
proteins by immunoblotting. b-Actin was used as a loading control.
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F. Estévez-Sarmiento et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
5
long doubling times of approximately 72 h. Several flavonoids have
been reported to exert their antiproliferative activity by targeting
microtubules through tubulin binding.12 One of the structural
requirements usually found in flavones for tubulin interaction is
the presence of a methoxy group at C-3. Since 7a contains a meth-
oxy group at C-3, G2–M arrest induced by this flavonoid might be
explained by the inhibition of microtubule formation or by changes
in the expression and/or activity of G2–M cell-cycle regulators. The
flavonoid quercetin has been shown to inhibit cancer cell prolifer-
ation by depleting cellular microtubules and inhibiting micro-
tubule polymerization through tubulin binding.18 To confirm this
hypothesis we used purified tubulin (containing no microtubule-
associated proteins) in our assay and found that 7a led to a concen-
tration-dependent inhibition of tubulin assembly. Colchicine and
taxol were used as positive controls of inhibition and promotion
of tubulin polymerization, respectively (Fig. 4). Further studies
are needed to determine the effect of this compound on these reg-
ulators such as the cyclin-dependent kinase-1, cyclin-dependent
kinase inhibitor p21Cip1, B-type cyclin isoforms, and cdc25C.
500 MHz for 1H and 100 and 125 MHz for 13C, respectively. Chem-
ical shifts (d) are reported in parts per million relative to the resid-
ual solvent signals, and coupling constants (J) are reported in hertz.
High resolution ESI mass spectra were obtained from a Fourier
transform ion cyclotron resonance (FT-ICR) mass spectrometer,
an RF-only hexapole ion guide, and an external electrospray ion
source. Flash column chromatography was carried out using silica
gel 60 (230–400 mesh), and analytical thin layer chromatography
(TLC) was performed using silica gel aluminum sheets. All com-
mercially available chemicals were used without further
purification.
4.1.1. General procedure for the synthesis of chalcones (3a-3h)
A mixture of the acetophenone (5–10 mmol, 1 equiv) and the
corresponding aldehyde (1 equiv) in EtOH (20–40 mL) was stirred
at room temperature and a 50% aqueous solution of NaOH (5–
8 mL) was added. The reaction mixture was stirred at room tem-
perature until aldehyde consumption. After that, HCl (10%) was
added until neutrality. Precipitated chalcones were generally fil-
tered and crystallized from MeOH, although in some cases the pro-
duct was purified using column chromatography. Chalcones 3a, 3c,
3e and 3f have been previously described.21–25
3. Conclusions
We have synthesized 15 flavonoids, including eight flavonols
and seven 3-methyl ethers, with one or two substituents on the
B ring and an additional substituent on the A ring, and evaluated
their effects on cell viability of the malignant cell line SK-MEL-1.
The B ring substituents included the pairs of 30-hydroxy-40-meth-
oxy or 40-hydroxy-30-methoxy or just one hydroxy group. The 3-
methoxyflavone 7a, containing a 30-hydroxy-40-methoxy pair of
substituents in the B ring, was the most cytotoxic compound
against melanoma cells but had low cytotoxicity against human
lymphocytes. The fact that human melanoma SK-MEL-1 cells were
sensitive to 7a is extremely interesting given that melanoma is the
most aggressive and lethal skin cancer and frequently resists
chemotherapy.
Relatively few studies have analysed the potential of flavonoids
in antimelanoma therapy. Tamarixetin (3,5,7,30-tetrahydroxy-40-
methoxyflavone) and dihydromyricetin are two known flavonoids
that have been reported to display antiproliferative capacity
against melanoma cells.19,20 Tamarixetin exhibits cytotoxic proper-
ties on SK-MEL-1 cells but the pathways involved in cell death
remain to be explored,19 and comparative studies indicate that
its cytotoxic capacity is three times lower than 7a
(IC50 = 23.1 2.1 mM vs. 6.9 1.1 mM). Dihydromyricetin suppress
cell proliferation of SK-MEL-28 melanoma cells by inducing cell
cycle arrest at the G1 phase and apoptosis, although this occurs
at higher concentrations than were used for flavonoid 7a.
In summary, the present study on the human melanoma cells
SK-MEL-1 demonstrates that the flavonoid 7a arrested the cell
cycle at the G2-M phase, induced apoptotic cell death and blocked
tubulin polymerization. Apoptosis was confirmed by i) fluorescent
microscopy, ii) the appearance of a subdiploid DNA content and iii)
caspase-3 activation. The antiproliferative effect of 7a is associated
with cytochrome c release and with the activation of the initiator
caspases involved in the extrinsic and the intrinsic pathways of cell
death.
4.1.1.1. 3-Benzyloxy-20-hydroxy-4,40-dimethoxychalcone (3b).
Orange solid, mp 145–146 °C (65%). IR (KBr, cmꢀ1
) mmax: 2956,
1716, 1683, 1635, 1607, 1558, 1540, 1508, 1456, 1436, 1374,
1260, 1236, 1156, 1130, 1020, 846. 1H NMR (500 MHz, CDCl3)
d = 13.44 (s, 1H, OH); 7.69 (d, 1H, J = 15.6 Hz, H-b); 7.67 (d, 1H,
J = 8.3 Hz, H-60); 7.40 (d, 2H, J = 7.5 Hz, 2xH-Ph); 7.31 (tt, 2H,
J = 7.2, 1.5 Hz, 2xH-Ph); 7.26–7.22 (m, 2H, H-Ph and H-a); 7.15
(dd, 1H, J = 8.3, 1.9 Hz, H-6); 7.10 (d, 1H, J = 2.0 Hz, H-2); 6.83 (d,
1H, J = 8.3 Hz, H-5); 6.41–6.37 (m, 2H, H-50 and H-30); 5.12 (s, 2H,
CH2-Ph); 3.85 (s, 3H, CH3O-C-4); 3.77 (s, 3H, CH3O-C-40). 13C
NMR (125 MHz, CDCl3) d = 191.7 (C@O), 166.6 (C-20), 166.0 (C-40),
152.3 (C-4), 148.4 (C-3), 144.4 (C-b), 136.8 (C-Ph), 131.1 (C-60),
128.7 (C-Ph), 128.1 (C-Ph), 127.7 (C-1), 127.4 (C-Ph), 123.6 (C-6),
118.1 (C-a
), 114.1 (C-10), 113.4 (C-2), 111.6 (C-5), 107.6 (C-50),
101.0 (C-30), 71.3 (CH2-Ph), 56.0 (CH3O-C-4), 55.6 (CH3O-C-40).
HRMS (ESI-FT-ICR) m/z: 413.1371 [M+Na]+; calcd. for C24H22O5Na:
413.1365.
4.1.1.2. 4-Benzyloxy-20-hydroxy-3,40-dimethoxychalcone (3d).
Orange solid, mp 153–155 °C (70%). IR (KBr, cmꢀ1
) mmax: 2936,
4. Experimental
1633, 1571, 1508, 1454, 1443, 1420, 1370, 1338, 1297, 1259,
1213, 1128, 1019, 959, 846. 1H NMR (500 MHz, CDCl3) d = 13.58
(s, 1H, OH); 7.87 (d, 1H, J = 15.3 Hz, H-b); 7.85 (d, 1H, J = 8.8 Hz,
4.1. General information
H-60); 7.49–7.47 (m, 2H, 2xH-Ph); 7.44–7.42 (m, 3H, H-
a and
Compounds used as starting materials and reagents were
obtained from Aldrich Chemical Co., or other chemical companies
and utilized without further purification. Melting points are uncor-
rected. 1H NMR and 13C NMR spectra were recorded at 400 and
2xH-Ph); 7.36 (t, 1H, J = 7.5 Hz, Ph); 7.22–7.21 (m, 2H, H-2 and
H-6); 6.95 (d, 1H, J = 8.8 Hz, H-5); 6.53–6.51 (m, 2H, H-50 and H-
30); 5.24 (s, 2H, CH2-Ph); 4.00 (s, 3H, CH3O-C-3); 3.88 (s, 3H,
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F. Estévez-Sarmiento et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
CH3O-C-40). 13C NMR (125 MHz, CDCl3) d = 191.7 (C@O), 166.6
(C-20), 166.1 (C-3 or C-40), 150.7 (C-4), 149.8 (C-3 or C-40), 144.5
(C-b), 136.5 (Ph), 131.1 (C-60), 128.6 (Ph), 128.1 (C-1), 128.1 (Ph),
J = 8.4 Hz, 2xH-Ph); 7.45–7.42 (m, 2xH-Ph); 7.39–7.34 (m, 1H,
Ph); 7.05 (d, 1H, J = 8.6 Hz, H-50); 7.01 (dd, 1H, J = 8.9, 2.2 Hz, H-
6); 6.92 (d, 1H, J = 2.3 Hz, H-8); 5.29 (s, 2H, CH2-Ph); 4.00 (s, 3H,
127.2 (Ph), 123.0 (C-2 or C-6), 118.1 (C-a
), 114.1 (C-10), 113.5
CH3O-C-40); 3.97 (s, 3H, CH3O-C-7). 13C NMR (125 MHz, CDCl3)
d = 172.4 (C-4), 164.1 (C-7), 157.1 (C-9), 151.2 (C-40), 147.9 (C-30),
144.4 (C-2), 137.3 (C-3), 136.9 (Ph), 128.6 (Ph), 128.0 (Ph), 127.6
(Ph), 126.7 (C-5), 123.7 (C-10), 121.6 (C-60), 114.6 (C-6), 114.6 (C-
(C-5), 110.9 (C-2 or C-6), 107.6 (C-50), 101.0 (C-30), 70.9 (CH2-Ph),
56.1 (CH3O-C-3), 55.6 (CH3O-C-40). HRMS (ESI-FT-ICR) m/z:
413.1370 [M+Na]+; calcd. for C24H22O5Na: 413.1365.
10), 113.7 (C-20), 111.4 (C-50), 99.8 (C-8), 71.3 (CH2-Ph), 56.0
(CH3O-C-40), 55.8 (CH3O-C-7). HRMS (ESI-FT-ICR) m/z: 427.1162
4.1.2. General procedure for the synthesis of flavonols
[M+Na]+; calcd. for C24H20O6Na: 427.1158.
A solution of the corresponding 2-hydroxychalcone (2–4 mmol)
in 3.0 M KOH in MeOH (20–30 mL) was cooled at 0 °C. An aqueous
solution of H2O2 (30%) (5–8 mL) was added to the chalcone solu-
tion. The resulting mixture was stirred at room temperature, until
the starting material was totally consumed, as evidenced by TLC.
The reaction mixture was cooled in an ice bath and distilled water
(2–4 mL) was added. HCl (2M) was added until pH 2 and the pre-
cipitate was filtered and washed with distilled water and extracted
with EtOAc. The organic layer was washed with brine until neutral-
ity and dried with anhydrous MgSO4. The solvent was evaporated
in vacuo and the residue was purified using a chromatographic col-
umn [SiO2, petroleum ether:EtOAc (1:0–7:3)]. Flavonols 4c and 4e
have been previously described.26,27
4.1.2.3. 2-[4-(Benzyloxy)-3-methoxyphenyl]-3-hydroxy-7-methoxy-
4H-chromen-4-one (4d).
Yellow pale solid, mp 160–162 °C (55%). IR (KBr, cmꢀ1
) mmax:
4.1.2.1. 2-[3-(Benzyloxy)-4-methoxyphenyl]-3-hydroxy-4H-chromen-
4-one (4a).
3422, 2923, 2851, 2009, 1651, 1616, 1506, 1455, 1401, 1262,
1213, 1012. 1H NMR (500 MHz, CDCl3) d = 8.11 (d, 1H, J = 8.8 Hz,
H-5); 7.84 (d, 1H, J = 2.0 Hz, H-20); 7.77 (dd, 1H, J = 8.6, 2.1 Hz, H-
60); 7.46 (d, 2H, J = 7.2 Hz, 2xH-Ph); 7.38 (t, 2H, J = 7.4 Hz, 2xH-
Ph); 7.35–7.29 (m, 1H, Ph); 7.00 (d, 1H, J = 8.6 Hz, H-50); 6.98 (dd,
1H, J = 8.9, 2.3 Hz, H-6); 6.92 (d, 1H, J = 2.4 Hz, H-8); 5.24 (s, 2H,
CH2-Ph); 3.99 (s, 3H, CH3O-C-30); 3.92 (s, 3H, CH3O-C-7). 13C
NMR (125 MHz, CDCl3) d = 172.6 (C-4), 164.1 (C-7), 157.1 (C-9),
149.6 (C-40), 149.4 (C-30), 144.5 (C-2), 137.4 (C-3), 136.6 (Ph),
128.6 (Ph), 128.0 (Ph), 127.2 (Ph), 126.7 (C-5), 124.2 (C-10), 120.9
(C-60), 114.7 (C-6), 114.6 (C-10), 113.3 (C-50), 111.1 (C-10), 99.8
(C-8), 70.8 (CH2-Ph), 56.2 (CH3O-C-30), 55.8 (CH3O-C-7). HRMS
(ESI-FT-ICR) m/z: 427.1153 [M+Na]+; calcd. for C24H20O6Na:
427.1158.
Yellow pale solid, mp 202–203 °C (79%). IR (KBr, cmꢀ1
)
m
max:3249, 2929, 1612, 1560, 1515, 1480, 1438, 1413, 1336,
1267, 1204, 1176, 1144, 1124, 1017. 1H NMR (500 MHz, CDCl3)
d = 8.27 (dd, 1H, J = 8.0, 1.4 Hz, H-5); 7.95–7.93 (m, 2H, H-20 and
H-60); 7.73 (ddd, 1H, J = 8.5, 7.1, 1.5 Hz, H-7); 7.58–7.55 (m, 3H,
H-8 and 2xH-Ph); 7.46–7.42 (m, 3H, H-6 and 2xH-Ph); 7.36 (tt,
1H, J = 7.4, 1.9 Hz, Ph); 7.08 (d, 1H, J = 9.1 Hz, H-50); 7.02 (s, 1H
4.1.2.4. 2-[4-(Benzyloxy)phenyl]-3-hydroxy-7-methoxy-4H-chromen-
4-one (4f).
OH); 5.30 (s, 2H, CH2-Ph); 4.01 (s, 3H CH3O-C-40). 13C NMR
(125 MHz, CDCl3) d = 173.1 (C-4), 155.2 (C-9), 151.5 (C-40), 148.0
(C-30), 145.1 (C-2), 137.7 (C-3), 136.9 (C-7), 133.4 (Ph), 128.6
(Ph), 128.0 (Ph), 127.5 (Ph), 125.4 (C-5), 124.4 (C-6), 123.6 (C-10),
122.0 (C-60), 120.6 (C-10), 118.1 (C-8), 113.6 (C-20), 111.4 (C-50),
71.3 (CH2-Ph), 56.0 (CH3O-C-40000000). HRMS (ESI-FT-ICR) m/z:
397.1048 [M+Na]+; calcd. for C23H18O5Na: 397.1052.
4.1.2.2. 2-[3-(Benzyloxy)-4-methoxyphenyl]-3-hydroxy-4H-chromen-
4-one (4b).
Yellow pale solid, mp 163–164 °C (52%). IR (KBr, cmꢀ1
) mmax:
3274, 1603, 1560, 1508, 1452, 1423, 1401, 1378, 1315, 1257,
1209, 1179, 1170, 1119, 1027, 1015, 953, 882, 832. 1H NMR
(500 MHz, CDCl3) d = 8.24 (d, 2H, J = 8.9 Hz, H-20 and H-60); 8.16
(d, 1H, J = 8.9 Hz, H-5); 7.50 (d, 2H, J = 7.2 Hz, 2xH-Ph); 7.46–7.43
(m, 2H, 2xH-Ph); 7.39 (tt, 1H, J = 6.2, 1.2 Hz, Ph); 7.15 (d, 2H,
J = 9.0 Hz, H-30 and H-50); 7.02 (dd, 1H, J = 8.9, 2.3 Hz, H-6); 6.97
(d, 1H, J = 2.3 Hz, H-8); 5.19 (s, 2H, CH2-Ph); 3.96 (s, 3H, CH3O-C-
7). 13C NMR (125 MHz, CDCl3) d = 172.5 (C-4), 164.1 (C-7), 160.0
(C-40), 157.2 (C-9), 144.6 (C-2), 137.3 (C-3), 136.5 (Ph), 129.2 (C-
20 and C-60), 128.7 (Ph), 128.1 (Ph), 127.5 (Ph), 126.7 (C-5), 123.9
(C-10), 114.9 (C-30 and C-50), 114.7 (C-6 and C-10), 99.8 (C-8),
Yellow pale solid, mp 192–193 °C (71%). IR (KBr, cmꢀ1
) mmax:
3243, 1608, 1560, 1505, 1453, 1403, 1261, 1210, 1176, 1165,
1144, 1122, 1019, 841. 1H NMR (500 MHz, CDCl3) d = 8.15 (d, 1H,
J = 8.9 Hz, H-5); 7.90–7.78 (m, 2H, H-20 and H-60); 7.56 (d, 2H,
70.1 (CH2-Ph), 55.8 (CH3O-C-40). HRMS (ESI-FT-ICR) m/z:
397.1059 [M+Na]+; calcd. for C23H18O5Na: 397.1052.
Please cite this article in press as: Estévez-Sarmiento F., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.09.043

F. Estévez-Sarmiento et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
7
4.1.2.5. 2-[2-(Benzyloxy)phenyl]-3-hydroxy-4H-chromen-4-one (4g).
Yellow pale solid, mp 104–106 °C (65%). IR (KBr, cmꢀ1
) mmax:
2931, 2839, 1637, 1615, 1601, 1560, 1511, 1468, 1438, 1378,
1329, 1267, 1243, 1212, 1171, 1134, 1020, 974, 891, 812. 1H
NMR (500 MHz, CDCl3) d = 8.24 (dd, 1H, J = 7.9, 1.7 Hz, H-5);
7.79–7.76 (m, 2H, H-20 and H-60); 7.65 (ddd, 1H, J = 8.6, 7.1,
1.7 Hz, H-7); 7.50–7.46 (m, 3H, H-8 and 2xH-Ph); 7.41–7.35 (m,
3H, H-6 and 2xH-Ph); 7.32 (t, 1H. J = 7.4 Hz, Ph); 7.02 (d, 1H,
Yellow pale solid, mp 138–139 °C (64%). IR (KBr, cmꢀ1
) mmax:
3289, 3066, 3029, 2929, 2872, 1949, 1910, 1815, 1633, 1614,
1606, 1567, 1486, 1469, 1417, 1382, 1347, 1287, 1211, 1135,
1102, 1050, 1024, 900, 859. 1H NMR (500 MHz, CDCl3) d = 8.18
(dd, 1H, J = 8.1, 1.1 Hz, H-5); 7.57–7.54 (m, 2H, H-40 or H-60 and
H-7); 7.37 (d, 1H, J = 8.4 Hz, H-8); 7.36 (dt, 1H, J = 9.0, 1.5 Hz, H-
60 or H-40); 7.31 (t, 1H, J = 7.4 Hz, H-6); 7.27 (d, 2H, J = 7.1 Hz,
Ph); 7.20–7.13 (m, 3H, 3xH-Ph); 7.03–7.00 (m, 2H, H-3 and H-50);
J = 8.4 Hz, H-50); 5.26 (s, 2H, CH2-Ph); 3.98 (s, 3H, CH3O-C-40);
3.73 (s, 3H, CH3O-C-3). 13C NMR (125 MHz, CDCl3) d = 174.9 (C-
4), 155.3 (C-2), 155.1 (C-9), 151.8 (C-40), 147.7 (C-30), 140.9 (C-3),
136.8 (Ph), 133.2 (C-7), 128.6 (Ph), 128.0 (Ph), 127.2 (Ph), 125.8
(C-5), 124.5 (C-6), 124.1 (C-10), 123.3 (C-10), 122.5 (C-60), 117.8
6.52 (s, 1H, OH); 5.08 (s, 2H, CH2-Ph). 13C NMR (125 MHz, CDCl3)
d = 173.3 (C-4), 156.6 (C-20), 156.0 (C-9), 145.9 (C-2), 138.9 (C-3),
136.7 (Ph), 133.3 (C-7), 132.0 (C-40 or C-60), 131.1 (C-60 or C-40),
128.5 (Ph), 127.8 (Ph), 126.9 (Ph), 125.5 (C-5), 124.3 (C-6), 121.4
(C-10), 120.9 (C-50), 120.2 (C-10), 118.4 (C-8), 113.4 (C-30), 70.5
(C-8), 114.3 (C-20), 111.3 (C-50), 71.1 (CH2-Ph), 59.8 (CH3O-C-3),
56.0 (CH3O-C-40). HRMS (ESI-FT-ICR) m/z: 411.1219 [M+Na]+;
calcd. for C24H20O5Na: 411.1208.
(CH2-Ph). HRMS (ESI-FT-ICR) m/z: 367.0944 [M+Na]+; calcd. for
C22H16O4Na: 367.0946.
4.1.3.2. 30-Benzyloxy-3,7,40-trimethoxyflavone (6b).
4.1.2.6. 2-[2-(Benzyloxy)phenyl]-3-hydroxy-7-methoxy-4H-chromen-
4-one (4h).
Yellow pale solid, mp 154–155 °C (68%). IR (KBr, cmꢀ1
) mmax:
3014, 2931, 2847, 1611, 1513, 1445, 1382, 1364, 1327, 1266,
1256, 1215, 1170, 1133, 1103, 1022, 953, 836. 1H NMR
(500 MHz, CDCl3) d = 8.06 (d, 1H, J = 8.9 Hz, H-5); 7.68–7.65 (m,
2H, H-20 and H-60); 7.41 (d, 2H, J = 7.2 Hz, 2xH-Ph); 7.31 (t, 2H,
J = 7.5 Hz, 2xH-Ph); 7.24 (t, 1H, J = 7.3 Hz, Ph); 6.94 (d, 1H,
J = 8.5 Hz, H-50); 6.88 (dd, 1H, J = 8.9, 2.2 Hz, H-6); 6.77 (d, 1H,
White solid, mp 161–162 °C (60%). IR (KBr, cmꢀ1
) mmax: 3275,
3032, 2936, 2841, 1608, 1567, 1505, 1454, 1408, 1318, 1276,
1257, 1230, 1204, 1168, 1130, 1112, 1048, 1026, 995, 954, 885,
835. 1H NMR (500 MHz, CDCl3) d = 8.20 (d, 1H, J = 8.9 Hz, H-5);
7.68 (dd, 1H, J = 7.5, 1.6 Hz, H-60); 7.49 (dt, 1H, J = 7.7, 1.7 Hz, H-
40); 7.42 (d, 2H, J = 6.9 Hz, 2xH-Ph); 7.35–7.28 (m, 3H, 3xH-Ph);
7.18–7.14 (m, 2H, H-30 and H-50); 7.03 (dd, 1H, J = 8.9, 2.3 Hz, H-
6); 6.87 (d, 1H, J = 2.3 Hz, H-8); 6.62 (s, 1H, OH); 5.22 (s, 2H,
CH2-Ph); 3.90 (s, 3H, CH3O-C-7). 13C NMR (125 MHz, CDCl3)
d = 172.7 (C-4), 164.0 (C-7), 157.9 (C-9), 156.5 (C-20), 145.1 (C-2),
138.5 (C-3), 136.8 (Ph), 131.8 (C-40), 131.1 (C-60), 128.4 (Ph),
127.8 (Ph), 126.9 (Ph), 126.8 (C-5), 120.9 (C-30 or C-50), 120.3 (C-
10), 115.3 (C-10), 114.7 (C-6), 113.5 (C-50 or C-30), 99.8 (C-8), 70.5
J = 2.3 Hz, H-8); 5.18 (s, 2H, CH2-Ph); 3.90 (s, 3H, CH3O-C-40);
3.84 (s, 3H, CH3O-C-7); 3.64 (s, 3H, CH3O-C-3). 13C NMR
(125 MHz, CDCl3) d = 174.4 (C-4), 163.9 (C-7), 156.8 (C-9), 154.8
(C-2), 151.7 (C-40), 147.7 (C-30), 140.7 (C-3), 136.9 (Ph), 128.6
(Ph), 127.9 (Ph), 127.2 (Ph), 127.1 (C-5), 123.4 (C-10), 122.3 (C-60),
118.0 (C-10), 114.2 (C-6 or C-20), 114.2 (C-20 or C-6), 111.2 (C-50),
99.9 (C-8), 71.1 (CH2-Ph), 59.9 (CH3O-C-40), 56.0 (CH3O-C-7), 55.8
(CH3O-C-3). HRMS (ESI-FT-ICR) m/z: 419.1483 [M+H]+; calcd. for
C25H23O6: 419.1495.
(CH2-Ph), 55.8 (MeO-C-7). HRMS (ESI-FT-ICR) m/z: 397.1059 [M
+Na]+; calcd. for C23H18O5Na: 397.1052.
4.1.3.3. 40-Benzyloxy-3,30-dimethoxyflavone (6c).
4.1.3. General procedure for the 3-methylation of flavonols
Methyl iodide (1.2 equiv.) and potassium carbonate (2 equiv.)
were added to a solution of the flavonols (1 equiv.) in acetone
(0.1 M) under argon atmosphere and stirred. After standing over-
night, the resulting mixture was filtered and the solvent was
removed under vacuum to leave a yellow pale solid, which was
purified by flash column chromatography or recrystallized from
EtOH. 3-Methoxyflavones 6d-f have been previously described.28
White solid, mp 128–129 °C (80%). IR (KBr, cmꢀ1
) mmax: 3064,
3005, 2931, 2849, 1733, 1638, 1615, 1601, 1561, 1511, 1469,
1438, 1419, 1379, 1330, 1270, 1241, 1212, 1170, 1135, 1012,
4.1.3.1. 30-Benzyloxy-3,40-dimethoxyflavone (6a).
Please cite this article in press as: Estévez-Sarmiento F., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.09.043

8
F. Estévez-Sarmiento et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
972, 915, 889, 879. 1H NMR (500 MHz, CDCl3) d = 8.30 (dd, 1H,
J = 7.9,1.5 Hz, H-5); 7.81 (d, 1H, J = 1.9 Hz, H-20); 7.74 (dd, 1H,
J = 8.5, 1.9 Hz, H-60); 7.70 (ddd, 1H, J = 8.5, 7.2, 1.5 Hz, H-7); 7.56
(d, 1H, J = 8.3 Hz, H-8); 7.51 (d, 2H, J = 7.2 Hz, 2xH-Ph); 7.45–7.42
(m, 3H, H-6 and 2xH-Ph); 7.36 (t, 1H, J = 7.4 Hz, Ph); 7.05 (d, 1H,
OH-C-3); 9.34 (s, 1H, OH-C-30); 8.10 (dd, 1H, J = 8.0, 1.5 Hz, H-5);
7.79–7.75 (m, 2H, H-7 and H-20); 7.72–7.70 (m, 2H, H-8 and H-
60); 7.44 (dt, 1H, J = 7.9, 0.9 Hz, H-6); 7.10 (d, 1H, J = 8.7 Hz, H-50);
3.85 (s, 3H, CH3O-C-40). 13C NMR (125 MHz, DMSO d6) d =172.5
(C-4), 154.3 (C-9), 149.2 (C-40), 146.1 (C-30), 145.6 (C-2), 138.1
(C-3), 133.4 (C-7), 124.6 (C-5), 124.3 (C-6), 123.7 (C-10), 121.2 (C-
10), 119.7 (C-60), 118.1 (C-8), 114.7 (C-20), 111.7 (C-50), 55.5
J = 8.6 Hz, H-50); 5.30 (s, 2H, CH2-Ph); 4.02 (s, 3H, CH3O-C-30);
3.93 (s, 3H, CH3O-C-3). 13C NMR (125 MHz, CDCl3) d = 174.9
(C-4), 155.4 (C-2), 155.1 (C-9), 150.4 (C-40), 149.2 (C-30), 141.0
(C-3), 136.5 (C-Ph), 133.3 (C-7), 128.7 (C-Ph), 128.1 (C-Ph), 127.2
(C-Ph), 125.8 (C-5), 124.6 (C-6), 124.2 (C-10), 123.7 (C-10), 122.0
(CH3O-C40). HRMS (ESI-FT-ICR) m/z: 285.0768 [M+H]+; calcd. for
C16H13O5: 285.0763.
(C-60), 117.8 (C-8), 113.1 (C-50), 112.1 (C-20), 70.8 (CH2-Ph), 60.0
4.1.4.2. 3-Hydroxy-2-(3-hydroxy-4-methoxyphenyl)-7-methoxy-4H-
chromen-4-one (5b).
(CH3O-C-3), 56.2 (CH3O-C-30). HRMS (ESI-FT-ICR) m/z: 389.1385
[M+H]+; calcd. for C24H21O5: 389.1389.
4.1.3.4. 20-Benzyloxy-3-methoxyflavone (6g).
White solid, mp 179–181 °C (82%). IR (KBr, cmꢀ1
) mmax: 3389,
3013, 2936, 2842, 1614, 1563, 1557, 1506, 1455, 1407, 1335,
1261, 1212, 1167, 1138, 1121, 1027, 1010, 960, 836. 1H NMR
(500 MHz, DMSO d6) d = 9.27 (s, 1H, OH-C-30); 9.23 (s, 1H, OH-C-
3); 7.98 (d, 1H, J = 8.9 Hz, H-5); 7.74 (d, 1H, J = 2.2 Hz, H-20); 7.70
(dd, 1H, J = 8.6, 2.2 Hz, H-60); 7.23 (d, 1H, J = 2.3 Hz, H-8); 7.09 (d,
1H, J = 8.7 Hz, H-50); 7.03 (dd, 1H, J = 8.85, 2.3 Hz, H-6); 3.91 (s,
3H, CH3O-C-7), 3.85 (s, 3H, CH3O-C40). 13C NMR (125 MHz,
DMSO d6) d = 172.0 (C-4), 163.4 (C-7), 156.2 (C-9), 149.0 (C-40),
146.1 (C-30), 144.9 (C-2), 137.8 (C-3), 126.0 (C-5), 123.8 (C-10),
119.4 (C-60), 115.0 (C-10), 114.6 (C-20), 114.4 (C-6), 111.7 (C-50),
White solid, mp 115 °C (59%). IR (KBr, cmꢀ1
) mmax: 3064, 3033,
3005, 2936, 1643, 1622, 1610, 1581, 1570, 1489, 1466, 1450,
1385, 1278, 1254, 1230, 1212, 1171, 1148, 1111, 1048, 1008,
955, 900. 1H NMR (500 MHz, CDCl3) d = 8.34 (dd, 1H, J = 8.0,
1.6 Hz, H-5); 7.69 (ddd, 1H, J = 8.6, 7.1, 1.6 Hz, H-7); 7.55 (dd, 1H,
J = 7.4, 1.5 Hz, H-60); 7.51–7.48 (m, 2H, H-8 and H-40); 7.44 (ddd,
1H, J = 8.7, 7.9, 0.9, Hz, H-6); 7.38 (d, 2H, J = 7.1 Hz, 2xH-Ph);
7.33–7.27 (m, 3H, 3xH-Ph); 7.15–7.12 (m, 2H, H-30 and H-50);
5.21 (s, 2H, CH2-Ph); 3.83 (s, 3H, CH3O-C-20). 13C NMR (125 MHz,
CDCl3) d = 174.9 (C-4), 156.5 (C-20), 156.2 (C-2), 155.7 (C-9),
142.0 (C-3), 136.6 (Ph), 133.2 (C-7), 131.9 (C-40), 131.0 (C-60),
128.5 (Ph), 127.8 (Ph), 126.9 (Ph), 125.9 (C-5), 124.6 (C-10),
124.5 (C-6), 120.7 (C-30 or C-50), 120.7 (C-10), 118.1 (C-8), 113.1
100.1 (C-8), 56.0 (CH3O-C-7), 55.6 (CH3O-C-40). HRMS (ESI-FT-
ICR) m/z: 337.0680 [M+Na]+; calcd. for C17H14O6Na: 337.0688.
4.1.4.3. 3-Hydroxy-2-(2-hydroxyphenyl)-7-methoxy-4H-chromen-4-
one (5h).
(C-50 or C-30), 70.4 (CH2-Ph), 60.5 (CH3O-C20). HRMS (ESI-FT-ICR)
m/z: 381.1107 [M+Na]+; calcd. for C23H18O4Na: 381.1103.
4.1.4. General procedure for debenzylation
20 mg of 10% Pd/C was added to a solution of the required com-
pound in THF (0.1 M). The reaction mixture was stirred at room
temperature for 1 h under an atmosphere of 1.5 atm H2(g) after
replacement of the air by nitrogen. The Pd/C was filtered through
Celite and the solvent was evaporated under reduced pressure.
The crude was purified by flash column chromatography or recrys-
tallized from EtOH. Flavonoids 5b, 5c, 5d, 5e, 5f, 5g, 7c, 7d and 7f
have been previously described.26,29–32
White solid, mp 165–166 °C (75%). IR (KBr, cmꢀ1
) mmax: 3233,
2929, 1618, 1610, 1507, 1450, 1406, 1260, 1206, 1169, 1131,
1111, 1026, 996, 954, 885, 834. 1H NMR (500 MHz, DMSO d6)
d = 9.32 (brs, 1H, OH); 8.02 (d, 1H, J = 8.9 Hz, H-5); 7.43 (dd, 1H,
J = 7.6, 1.5 Hz, H-60); 7.34 (dt, 1H, J = 8.3, 1.6 Hz, H-40); 7.10 (d,
1H, J = 2.2 Hz, H-8); 7.04 (dd, 1H, J = 8.9, 2.2 Hz, H-6); 6.98 (d, 1H,
4.1.4.1. 3-Hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-chromen-4-
one (5a).
J = 7.9 Hz, H-30); 6.94 (t, 1H, J = 7.4 Hz, H-50); 3.88 (s, 3H, CH3O-C-
7); 3.35 (brs, 1H, OH). 13C NMR (125 MHz, DMSO d6) d = 172.1
(C-4), 163.3 (C-7), 156.9 (C-9), 155.3 (C-20), 146.7 (C-2), 138.5 (C-
3), 131.2 (C-40), 130.8 (C-60), 126.1 (C-5), 118.6 (C-50), 118.3 (C-
10), 116.3 (C-30), 115.8 (C-10), 114.3 (C-6), 100.1 (C-8), 55.9
(CH3O-C-7). HRMS (ESI-FT-ICR) m/z: 285.0766 [M+H]+; calcd. for
C16H13O5: 285.0763.Anal. Calcd for C16H12O5: C 67.60; H 4.25.
Found: C 67.58; H 4.50.
White solid, mp 201–203 °C (80%). IR (KBr, cmꢀ1
) mmax: 3296,
3028, 3205, 3028, 1584, 1537, 1523, 1487, 1464, 1417, 1369,
1350, 1329, 1291, 1257, 1207, 1182, 1142, 1121, 1111, 1041,
1026, 883, 811. 1H NMR (500 MHz, DMSO d6) d = 9.38 (s, 1H,
4.1.4.4. 2-(3-Hydroxy-4-methoxyphenyl)-3-methoxy-4H-chromen-4-
one (7a).
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F. Estévez-Sarmiento et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
9
7.0, 1.6 Hz, H-7); 7.71 (dd, 1H, J = 8.4, 0.6 Hz, H-8); 7.46 (ddd, 1H,
J = 8.0, 7.0, 1.0 Hz, H-6); 6.96 (d, 2H. J = 8.9 Hz, H30 and H-50);
3.80 (s, 3H, CH3O-C-3). 13C NMR (125 MHz, DMSO d6) d =173.5
(C-4), 159.9(C-2), 155.2 (C-40), 154.5 (C-9), 139.7 (C-3), 133.7 (C-
7), 130.1 (C-20 and C-60), 124.8 (C-5 or C-6), 124.8 (C-6 or C-5),
123.4 (C-10), 120.9 (C-10), 118.2 (C-8), 115.5 (C-30 and C-50), 59.3
(CH3O-C-3). HRMS (ESI-FT-ICR) m/z: 269.0818 [M+H]+; calcd. for
C
16H13O4: 269.0814. Anal. Calcd for C16H12O4: C 71.64; H 4.51.
White solid, mp 138–139 °C (78%). IR (KBr, cmꢀ1
) mmax: 3358,
Found: C 71.91; H 4.76.
2933, 2840, 1613, 1557, 1509, 1469, 1438, 1385, 1337, 1275,
1253, 1213, 1172, 1126, 1035, 1023, 1011, 979, 892. 1H NMR
(400 MHz, CDCl3) d = 8.24 (dd, 1H, J = 8.0, 1.8 Hz, H-5); 7.76–7.74
(m, 2H, 20 and H-60); 7.65 (ddd, 1H, J = 8.5, 7.2, 1.4 Hz, H-7); 7.51
(d, 1H, J = 8.2 Hz, H-8); 7.37 (t, 1H, J = 7.8 Hz, H-6); 6.96 (d, 1H,
4.1.4.7. 2-(2-Hydroxyphenyl)-3-methoxy-4H-chromen-4-one (7g).
J = 9.2 Hz, H-500); 5.88 (s, 1H, OH); 3.97 (s, 3H, CH3O-C-3); 3.89 (s,
3H, CH3O-C-40). 13C NMR (100 MHz, CDCl3) d = 175.3 (C-4), 155.2
(C-2 or C-9), 155.1 (C-9 or C-2), 148.9 (C-3), 145.6 (C-30), 141.3
(C-40), 133.2 (C-7), 125.7 (C-5), 124.5 (C-6), 124.2 (C-10 or C-10),
124.1 (C-10 or C-10), 121.6 (C-60), 117.8 (C-8), 114.6 (C-20), 110.4
(C-50), 59.8 (CH3O-C-40), 56.0 (CH3O-C-3). HRMS (ESI-FT-ICR) m/z:
321.0725 [M+Na]+; calcd. for C17H14O5Na: 321.0739. Anal. Calcd
for C17H14O5: C 68.45; H 4.73. Found: C 68.41; H 5.09.
White solid, mp 191–192 °C (77%). IR (KBr, cmꢀ1
) mmax: 3235,
3007, 2940, 1612, 1560, 1466, 1454, 1388, 1293, 1252, 1212,
1147, 1111, 1037, 1007, 955, 901. 1H NMR (500 MHz, DMSO d6)
d = 9.96 (s, 1H, OH); 8.13 (dd, 1H, J = 8.1, 1.6 Hz, H-5); 7.80 (ddd,
1H, J = 8.6, 7.1, 1.6 Hz, H-7); 7.63 (d, 1H, J = 8.4 Hz, H-8); 7.50
(ddd, 1H, J = 8.0, 7.3, 0.9 Hz, H-6); 7.41 (dd, 1H, J = 7.6, 1.6 Hz, H-
60); 7.37 (ddd, 1H, J = 8.2, 7.5, 1.6 Hz, H-40); 7.00 (d, 1H, J = 8.2 Hz,
4.1.4.5.
men-4-one (7b).
2-(3-Hydroxy-4-methoxyphenyl)-3,7-dimethoxy-4H-chro-
H-30); 6.94 (dt, 1H, J = 7.5, 0.8 Hz, H-50); 3.73 (s, 3H, CH3O-C-3).
13C NMR (125 MHz, DMSO d6) d = 173.6 (C-4), 156.4 (C-2), 155.3
(C-20), 155.1 (C-9), 141.1 (C-3), 133.8 (C-7), 131.7 (C-40), 130.4
(C-60), 124.9 (C-5), 124.8 (C-6), 123.9 (C-10), 118.7 (C-50), 118.3
(C-8), 118.0 (C-10), 116.1 (C-30), 59.5 (CH3O-C-3). HRMS (ESI-FT-
ICR) m/z: 269. [M+H]+; calcd. for C16H13O4: 269.0814. Anal. Calcd
for C16H12O4: C 71.67; H 4.51. Found: C 71.30; H 4.44.
White solid, mp 174–175 °C (75%).IR (KBr, cmꢀ1
) mmax: 3234,
2932, 2840, 1605, 1560, 1508, 1438, 1386, 1367, 1274, 1255,
1213, 1169, 1128, 1103, 1017, 982, 952, 836. 1H NMR (400 MHz,
CDCl3) d = 8.06 (d, 1H, J = 8.8 Hz, H-5); 7.65 (dd, 1H, J = 6.8,
3.5 Hz, H-60); 7.63 (brs, 1H, H-20); 6.97 (d, 1H, J = Hz, H-50); 6.96
(d, 1H, J = Hz, H-6); 6.81 (d, 1H, J = 2.3 Hz, H-8); 5.86 (s, 1H, OH);
4.2. Biological assays
4.2.1. Cell culture and cytotoxicity assays
SK-MEL-1 melanoma cells were obtained from the German Col-
lection of Microorganisms and Cell Cultures (Braunschweig, Ger-
3.89 (s, 3H, CH3O-C-40); 3.83 (s, 3H, CH3O-C-7); 3.80 (s, 3H,
CH3O-C-3). 13C NMR (100 MHz, CDCl3) d = 174.5 (C-4), 163.9 (C-
7), 156.8 (C-9), 154.8 (C-2), 148.5 (C-40), 145.5 (C-30), 140.9 (C-3),
127.0 (C-5), 124.1 (C-10), 121.3 (C-60), 118.0 (C-10), 114.4 (C-20),
many) and grown in RPMI 1640 containing 2 mM L-glutamine
supplemented with 10% (v/v) heat-inactivated fetal bovine serum
and 100 units/mL penicillin and 100 mg/mL streptomycin at 37 °C
in a humidified atmosphere containing 5% CO2. Human peripheral
blood mononuclear cells (PBMCs) were isolated from heparin-anti-
coagulated blood of healthy volunteers by centrifugation with
Ficoll-Paque Plus (GE Healthcare Bio-Sciences AB; Uppsala, Swe-
den). PBMCs were also stimulated with phytohemagglutinine
114.2 (C-6), 110.3 (C-50), 99.9 (C-8), 59.9 (CH3O-C-3), 56.0 (CH3O-
C-40), 55.7 (CH3O-C-7). HRMS (ESI-FT-ICR) m/z: 351.0844 [M]+;
calcd. for C18H16O6: 351.0845. Anal. Calcd for C18H16O6: C 65.85;
H 4.91. Found: C 65.85; H 5.09.
(PHA, 2 lg/mL) for 48 h before experimental treatment.
4.1.4.6. 2-(4-Hydroxyphenyl)-3-methoxy-4H-chromen-4-one (7e).
The cytotoxicity of compounds was evaluated by using a colori-
metric 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide (MTT) assay. Briefly, 1 ꢁ 104 exponentially growing cells
were seeded in 96-well microculture plates with various com-
pounds concentrations. After the addition of MTT (0.5 mg/mL),
cells were incubated at 37 °C for 4 h. Sodium dodecyl sulfate
(10% w/v) in 0.05 M HCl was added to the wells and then incubated
at room temperature overnight under dark conditions. The absor-
bance was measured at 570 nm. Concentrations inducing a 50%
inhibition of cell growth (IC50) were determined graphically for
each experiment by a nonlinear regression using the curve-fitting
routine implemented within the software Prism 4.0 (GraphPad).
Values are means SE from at least three independent experi-
ments, with each performed in triplicate.
White solid, mp 220 °C (80%). IR (KBr, cmꢀ1
) mmax: 3217, 2917,
2849, 1603, 1555, 1509, 1469, 1385, 1283, 1242, 1213, 1177,
1149, 1110, 1034, 1007, 953, 898, 811. 1H NMR (500 MHz,
DMSO d6) d = 10.2 (brs, 1H, OH); 8.07 (dd, 1H, J = 7.9, 1.6 Hz, H-
5); 7.98 (d, 2H, J = 8.9 Hz, H-20 and H-60); 7.79 (ddd, 1H, J = 8.5,
Please cite this article in press as: Estévez-Sarmiento F., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.09.043

10
F. Estévez-Sarmiento et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
4.2.2. Evaluation and quantification of apoptosis and analysis of the
cell cycle
4.2.6. Statistical analysis
Student’s t test was used to determine the statistical signifi-
cance of differences between means of control and treated sam-
ples, with p values of <0.05 considered significant.
Fluorescent microscopy was used to identify nuclear changes
associated with apoptosis as previously described.33 To study
changes in the cell DNA content, flow cytometric analysis of pro-
pidium iodide-stained nuclei was carried out using a BD FACS-
VerseTM cytometer (BD Biosciences, San Jose, CA, USA) as
described.8 Briefly, cells were collected and centrifuged at 500ꢁg,
Acknowledgments
Funding: This research was supported in part by the Spanish
Ministerio de Economía y Competitividad (MINECO) and the Euro-
pean Regional Development Fund (CTQ2015-63894-P).
washed with PBS and resuspended in 50 lL of PBS. Following drop-
wise addition of 1 mL of ice-cold 75% ethanol, fixed cells were
stored at ꢀ20 °C for 1 h. Samples were then centrifuged at 500ꢁg
and washed with PBS and finally resuspended in 1 mL of PBS con-
Conflicts of interest
taining 50 lg/mL propidium iodide and 100 lg/mL RNase A and
incubated for 1 h at 37 °C in the dark. The percentage of cells with
decreased DNA staining, composed of apoptotic cells resulting
from either fragmentation or decreased chromatin, was counted
(minimum of 10,000 cells per experimental condition). Cell debris
was excluded from analysis by selective gating based on anterior
and right angle scattering.
None.
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.bmc.2017.09.043.
4.2.3. Western blot analysis
Immunoblot analysis of caspase-3 and cytochrome c was per-
formed as previously described.34 Briefly, after treatments, cells
were washed twice with PBS and then resuspended in ice-cold
buffer [20 mM HEPES (pH 7.5), 1.5 mM MgCl2, 10 mM KCl,
1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.1 mM phenyl-
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