Synthesis and effects on cell viability of flavonols and 3-methyl ether derivatives on human leukemia cells

Article abstract of DOI:10.1016/j.ejmech.2014.07.010

Flavonoids are polyphenolic compounds which display an array of biological activities and are considered potential antitumor agents. Here we evaluated the antiproliferative activity of selected synthetic flavonoids against human leukemia cell lines. We found that 4′-bromoflavonol (flavonol 3) was the most potent. This compound inhibited proliferation in a concentration-dependent manner, induced apoptosis and blocked cell cycle progression at the S phase. Cell death was found to be associated with the cleavage and activation of multiple caspases, the activation of the mitogen-activated protein kinase pathway and the up-regulation of two death receptors (death receptor 4 and death receptor 5) for tumor necrosis factor-related apoptosis-inducing ligand. Moreover, combined treatments using 4′-bromoflavonol and TRAIL led to an increased cytotoxicity compared to single treatments. These results provide a basis for further exploring the potential applications of this combination for the treatment of cancer.

Full text of DOI:10.1016/j.ejmech.2014.07.010

European Journal of Medicinal Chemistry 84 (2014) 30e41
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and effects on cell viability of flavonols and 3-methyl ether
derivatives on human leukemia cells
a
a
a
b
ꢀ
Olga Burmistrova , María Teresa Marrero , Sara Estevez , Isabel Welsch ,
b
a
a, *
ꢀ
ꢀ
Ignacio Brouard , Jose Quintana , Francisco Estevez
a Departamento de Bioquímica y Biología Molecular, Unidad Asociada al Consejo Superior de Investigaciones Científicas (CSIC), Instituto Canario de
ꢀ
ꢀ
Investigacion del Cancer, Universidad de Las Palmas de Gran Canaria, Plaza Dr. Pasteur s/n, 35016 Las Palmas de Gran Canaria, Spain
b
ꢀ
Instituto Productos Naturales y Agrobiología, CSIC, Avenida Astrofísico Francisco Sanchez 3, 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 polyphenolic compounds which display an array of biological activities and are considered
potential antitumor agents. Here we evaluated the antiproliferative activity of selected synthetic flavo-
noids against human leukemia cell lines. We found that 40-bromoflavonol (flavonol 3) was the most
potent. This compound inhibited proliferation in a concentration-dependent manner, induced apoptosis
and blocked cell cycle progression at the S phase. Cell death was found to be associated with the cleavage
and activation of multiple caspases, the activation of the mitogen-activated protein kinase pathway and
the up-regulation of two death receptors (death receptor 4 and death receptor 5) for tumor necrosis
factor-related apoptosis-inducing ligand. Moreover, combined treatments using 40-bromoflavonol and
TRAIL led to an increased cytotoxicity compared to single treatments. These results provide a basis for
further exploring the potential applications of this combination for the treatment of cancer.
© 2014 Elsevier Masson SAS. All rights reserved.
Received 10 January 2014
Received in revised form
27 May 2014
Accepted 3 July 2014
Available online 4 July 2014
Keywords:
Apoptosis
Flavonoids
Caspases
Cell cycle
Cytotoxicity
Death receptors
1. Introduction
features such as internucleosomal DNA fragmentation, the trans-
location of phosphatidylserine to the outside of the plasma mem-
Flavonoids are naturally occurring polyphenolic compounds
which may have beneficial effects for health and might be
considered as potentially protective or therapeutic agents against
cancer [1]. Among the different bioflavonoids, quercetin
(3,30,40,5,7-pentahydroxyflavone) is one of the best studied and is
widely distributed in nature. This natural product induces
apoptosis in a variety of tumors [2e4], including leukemia [5].
Antitumor properties of flavonoids are mediated by different types
of cell cycle arrest and induction of apoptosis, an active process of
cell death which displays an essential role in the development and
survival. It is also an important response to many chemothera-
peutic agents. Cells that suffer this kind of cell death display
brane, apoptotic bodies formation and chromatin condensation.
Caspases are fundamental executioners of apoptosis. They
comprise a family of cysteine proteases which are synthesized as
zymogens and are activated by proteolysis [6]. There are two main
apoptotic pathways [7]. In the extrinsic pathway, apoptosis is
mediated by death receptors (such as receptors for tumor necrosis
factor-a, Fas and TRAIL) and involves caspase-8 activation. In the
intrinsic pathway pro-apoptotic signals induce mitochondrial cy-
tochrome c release to the cytosol, and promote the apoptosome
assembly and caspase-9 activation. Both caspase-8 and caspase-9
activate caspase-3 which is responsible for specific cellular pro-
tein destruction during apoptosis [8]. The cellular response to this
chemically-induced apoptosis is associated with the inactivation of
the protein kinases involved in cell survival and with the activation
of protein kinases that promote apoptosis. One of the most
important aspects of apoptosis regulation is that it requires
mitogen-activated protein kinases (MAPKs). MAPKs are a family of
serine/threonine protein kinases directed to proline and function as
intracellular mediators in response to diverse stimuli. They are
activated by phosphorylation in threonine and tyrosine residues
[9]. In mammals the MAPKs are mainly represented by the cascades
Abbreviations: ERK, extracelular signal-regulated kinase; IC50, 50% inhibition of
cell growth; JNK/SAPK, c-jun N-terminal kinases/stress-activated protein kinases;
MAPK, mitogen-activated protein kinases; MEK, mitogen-activated extracellular
kinases; MTT, 3-(4,5-dimethyl-2-thiazolyl-)-2,5-diphenyl-2H-tetrazolium bromide;
p38MAPK, p38 mitogen-activated protein kinases; TRAIL, tumor necrosis factor-
related apoptosis-induced ligand.
* Corresponding author.
ꢀ
E-mail address: festevez@dbbf.ulpgc.es (F. Estevez).
http://dx.doi.org/10.1016/j.ejmech.2014.07.010
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

O. Burmistrova et al. / European Journal of Medicinal Chemistry 84 (2014) 30e41
31
ERK (extracellular signal-regulated protein kinases) 1/2, the c-Jun
2. Chemistry
NH2-terminal kinases/stress-activated protein kinases (JNK/SAPK)
and the p38 mitogen-activated protein kinases (p38MAPK). The
activation of ERK can trigger both anti-apoptotic and pro-apoptotic
effects, depending on the stimuli and cell type [10]. In contrast, JNK/
SAPK and p38MAPK are mainly activated by cytotoxic stimuli and are
associated with pro-apoptotic actions in many cell types [11].
However, there are multiple exceptions to this rule.
A selected combination of nine commercially available alde-
hydes (containing halogen, methoxy, methyl or aromatic groups)
with two hydroxyacetophenones provided nine flavonols (1e9)
and ten 3-methyl ether flavonols derivatives. The synthetic com-
pounds differ in terms of their substituents on the B ring (2-phenyl
group) and minor changes on the A ring (Fig. 1).
Recent studies indicate that the combination of conventional
cytotoxic compounds and new drugs directed at molecular targets
can enhance the cell death induced by the tumor necrosis factor
(TNF)-related apoptosis-inducing ligand (TRAIL) in tumor cells [12].
The identification of compounds that activate death receptors for
TRAIL or that block anti-apoptotic effectors might constitute an
important therapeutic advance. However, very little is known about
the synergy between semi-synthetic compounds derived from
natural products and apoptosis-inducing ligands such as tumor
The synthesis takes advantage of the well-known two
steps procedure for flavonols which combines
a
Clai-
seneSchmidt condensation of 2-hydroxyacetophenones and
benzaldehydes with NaOH as base followed by a cyclization
under AlgareFlynneOyamada reactions conditions (Scheme 1)
[20]. Treatment of flavonols with 5 equiv. of iodomethane led to
new methyl ethers 11, 13, 15, 17 and 18 in high yield. The spec-
troscopic data obtained for known compounds were compared
with those described in the literature in order to confirm the
structures 1e5 [19], 7 [21], 9 [22], 10 [19], 12 [23], 14 [24], 16
[25], 19 [19], 20 [26]. The structure of new synthetic flavonols (6,
8) and the methyl ether derivatives (11, 13, 15, 17 and 18) was
determined using 1D and 2D NMR experiments, high resolution
mass spectroscopy and IR. Compound 19 was synthesized from
quercetin by partial methylation reaction as previously
described [21].
necrosis factor
a (TNF-a), Fas and TRAIL. The search for compounds
that can enhance TRAIL sensitivity in human leukemia cells is
therefore of undoubted biomedical interest.
TRAIL induces selective apoptosis in several tumor cell lines and
shows low toxicity against normal cells [13]. This distinctive feature
has inspired the development of drugs to combat malignant dis-
eases. Recombinant TRAIL and agonist antibodies that bind to death
receptors have been used in clinical trials in phase IeII [14]. TRAIL
can bind to the death receptors DR4 (death receptor 4) and DR5
(death receptor 5), which contain a cytoplasmic death domain, and
to the decoy receptors DcR1 and DcR2, which lack a functional
death domain. Decoy receptors compete with death receptors for
the ligands and therefore block the apoptotic signals.
3. Biological results
3.1. Effect of the synthetic flavonoids on the growth and cell
viability of human leukemia cell lines
The results from several studies suggest that flavonoids with a
hydroxy group at carbon 3 are potential anticancer agents [15] and
also the presence of 30-hydroxy-40-methoxy groups on the B ring
enhances cytotoxicity [16]. The justification for investigating
methoxylated derivatives is provided by their chemopreventive
properties, which are much greater than those seen in the more
common unmethylated flavonoids, and the fact that they show
increased metabolic stability [17]. In a previous study of naturally
occurring and semi-synthetic flavonoids, we showed that methyl-
ation of hydroxyl groups of quercetin yields a compound with a
higher antiproliferative activity against human cancer cell lines
[18]. Moreover, it has recently been reported that molecules with
halogen substituents in the B ring show improved anticancer ac-
tivity relative to methoxylated, methylated or hydroxylated analogs
[19].
Comparative studies that examine methylation of hydroxy
group at C3 and the introduction of different substituents on the B
ring and minor changes on the A ring, will greatly enhance
knowledge of their impact on cytotoxicity. Here we synthesized a
series of flavonols and methyl ether derivatives carrying varying
substitutions at positions 5, 7, 20, 30, 40, 50. The substitutions
included different electron-donating and electron-withdrawing
groups, such as Cl, Br, CH3O, CH3, and OH, and allowed us to
compare their anticancer activity with similar compounds tested
against other tumor cell lines. We studied the effects of these
synthetic flavonoids on viability of human leukemia cells and then
examined the effects of the most potent, 40-bromoflavonol, on
apoptosis induction. This semi-synthetic flavonoid has been
recently assessed for cytotoxicity against the human colorectal
carcinoma cell line HCT116 [19] but, so far, its potential use in
antileukemia therapy is largely unexplored. We have evaluated
whether caspase activation and the MAPK cascade are involved in
the mechanism of action. Finally, we have investigated whether 40-
bromoflavonol induces death receptors expression and the poten-
tial modulation of cell death in combination with TRAIL.
In the present study, the potential cytotoxic properties of a se-
ries of 20 flavonoids, including nine flavonols and eleven 3-methyl
ethers with different substituents on the B ring (2-phenyl group)
and minor changes on the A ring were evaluated using HL-60 cells
(Table 1). The results indicated that 40-bromoflavonol (flavonol 3)
was the most cytotoxic compound with an IC50 value (the con-
centration that induces a 50% inhibition of cell growth) of
3.3 0.7 mM. A 30-fold increase in antiproliferative activity was
detected for 40-substituted flavonols by increasing the size of the
halogen from chlorine (flavonol 1) to bromine (flavonol 3). The
properties of the bromine atom, including its higher volume and its
polarizability together with the lower electron withdrawal at the
benzene moiety compared with the chlorine atom, seem to play a
key role in determining the potency of this compound on cell
viability. The differences between flavonol 1 and flavonol 3 suggest
that the higher potency of the latter might be due to steric and
electronic properties conferred by the identity of the halogen on
the B ring and are important in binding to the molecule target.
The introduction of a chlorine atom (flavonol 5) or a methoxy
group (flavonol 4) on position 20on the B ring improved the anti-
proliferative activity as compared with flavonol 1, however,
flavonol 5 was more potent than flavonol 4. The position of chlorine
on the B ring is important in conferring cytotoxicity, since 20-
chloroflavonol (flavonol 5) exhibited a higher potency than 40-
chloroflavonol (flavonol 1). The methylation of the hydroxyl group
at position C3 of flavonol 1 to produce flavonol 12 improved the
cytotoxicity. In contrast, methylation of the 3-hydroxy group of
flavonol 3 to produce flavonol 13 did not enhance the potency
against cell growth inhibition. The hydroxy group located at the C3
position seems to play a key role in determining the potency of
flavonol 3 on cell viability since this is significantly more potent (7-
fold) than the 3-methoxy derivative 13. It thus seems unlikely that
the C3 methoxy group is a major determinant of cytotoxicity.
Although the exact target and binding site of this flavonoid has not

32
O. Burmistrova et al. / European Journal of Medicinal Chemistry 84 (2014) 30e41
Fig. 1. Chemical structures of synthetic flavonoids.
yet been determined, the hydroxy group could stabilize the binding
by forming hydrogen bonds with the target.
ring: flavonol 16 was less potent than flavonol 10, but flavonol 18
was more potent than flavonol 15. Among the 3,7-dimethoxy de-
rivatives, the least potent was flavonol 16 containing only an
additional methoxy group at position 40 on the B ring, although the
introduction of two (flavonol 19) or three (flavonol 20) additional
methoxy groups on the B ring increased the antiproliferative ac-
tivity (Table 1). These results are in accordance with the fact that
higher lipophilicity can facilitate penetration through the cell
membrane and increase the cytotoxicity in vitro.
Among the 3-methoxy flavonols, the presence of an additional
methoxy group at position 40 on the B ring (flavonol 10) improved
the antiproliferative activity compared with the compound con-
taining the methoxy group at position 20 (flavonol 14). The most
potent 3-methoxy flavonols were those containing a 40-chlor-
ophenyl (flavonol 12), a 40-bromophenyl (flavonol 13) and a
naphthyl group (flavonol 17) at position 2 of the phenylbenzo-g-
pyrone core. Differences in effects on cell growth might derive from
differences in drug uptake, retention, and/or metabolism by the
cells or from differences in compound stability in tissue culture
medium.
The effect of the introduction of a methoxy group at C7 in 3-
methoxy flavonols depends on the substitution pattern on the B
The antitumor agent etoposide and the flavonoid quercetin were
used as positive controls in the cytotoxicity assays, and the IC50
values were 0.6 0.1
m
M and 47
8 mM, respectively.
The IC50 value for flavonol 3 in HL-60 cells was similar to
the values obtained for the human leukemia cell line resistant
to the antitumor compound mitoxantrone, HL-60/MX1

O. Burmistrova et al. / European Journal of Medicinal Chemistry 84 (2014) 30e41
33
Scheme 1. Synthesis of the intermediate chalcones and methyl ethers.
(IC50 ¼ 6.5 0.1
(IC50 ¼ 5.8 0.1
at position 20 (20-chloroflavonol) and displayed similar cytotoxic
m
M) and for the Burkitt lymphoma cell line Raji
apoptotic HL-60 cells increased approximately 14-fold (51.8 3.9%
vs 3.8 0.6%), and 4-fold (14.9 3.0% vs 3.8 0.6%) after 24 h
exposure to 40-bromoflavonol (flavonol 3) and 20-chloroflavonol
(flavonol 5), respectively. Similar results were obtained for U937
cells (Fig. 2B). These results are in accordance with the qualitative
assessment of DNA damage on agarose gels (Fig. 2C) and the fluo-
rescence microscopy experiments (Fig. 2D). For this reason, 40-
bromoflavonol (flavonol 3) was selected for further experiments.
mM) (Fig. 2A). Flavonol 5 contains a chlorine atom
potency against HL-60 cells (IC50 ¼ 10.7 2.9
m
m
M) and low cyto-
toxicity against HL-60/MX1 and Raji (IC50 > 30
M). However, 40-
bromoflavonol (flavonol 3) had a greater apoptotic effect than 20-
chloroflavonol (flavonol 5), as determined by evaluation of the
number of hypodiploid cells by flow cytometry. The percentage of
Table 1
Effects of flavonoids on the growth of human tumor cell line HL60.
Compound
IC50 M)
Compound
IC50 M)
1
2
3
4
5
6
7
8
9
10
39
20
21
(
m
102
11
34
7
138 18
3.3 0.7
30
14
98
6
2
10.7 2.9
15
32
16
3
148 14
34
18
27
1
4
53
19
28
7
2
1
4
12
13
17
(m
7
26
3
21
3
96 13
122 18
16
4
Cells were cultured for 72 h in presence of the indicated compounds and the IC50 values were calculated as described in the Experimental Section. The data shown represent
the mean SEM of 2e3 independent experiments with three determinations in each.

34
O. Burmistrova et al. / European Journal of Medicinal Chemistry 84 (2014) 30e41
Fig. 2. (A) Doseeresponse of flavonol 3 on human HL-60 cell viability. The results of a representative experiment are shown. Each point represents the mean of three replicates.
Similar results were obtained with HL-60/MX1 cell line and Raji cells. (B) Cells were incubated with 30 M of the indicated flavonol and the percentage of cells in the sub-G1 region
m
was quantified using flow cytometry after propidium iodide staining. (C) HL-60 cells were incubated in the presence of the indicated concentrations of compounds for 24 h and
genomic DNA was extracted, separated on an agarose gel and visualized under UV light by ethidium bromide staining. (D) Photomicrographs of representative fields of HL-60 cells
stained with Hoechst 33258 to evaluate nuclear chromatin condensation (i.e. apoptosis) after treatment for 24 h with 30 mM of the indicated compounds.
3.2. Flavonol 3 induces apoptosis in human leukemia cells
this synthetic flavonoid induces cell cycle arrest in the S phase and
apoptosis on human myeloid leukemia HL-60 and U937 cells.
To determine whether apoptosis was involved in the cytotoxic
effects of flavonol 3, we used complementary methods such as
fluorescence microscopy after DNA staining with Hoechst 33258,
the evaluation of DNA fragmentation and the quantification of
hypodiploid cells by flow cytometry. When cells were incubated
with flavonol 3, nuclei displayed condensation of chromatin and
the appearance of apoptotic bodies. The DNA showed the typical
fragmentation patterns caused by internucleosomal hydrolysis of
chromatin (Fig. 2C and D). To determine whether the inhibition of
cell growth triggered by flavonol 3 is mediated by alterations in the
progression of the cell cycle, we studied the effect of this compound
on the distribution of the different phases of the cell cycle at
different time periods in HL-60 and U937 cell lines by flow
cytometry (Fig. 3 and Table 2). The treatment of HL-60 cells with
10 mM flavonol 3 for 24 h caused an arrest of ~33% cells in S phase of
the cell cycle which was accompanied by a concomitant decrease in
the proportion of cells in the G2-M and G1 phases of the cell cycle.
The treatment of HL-60 cells with 3 mM flavonol 3 for 48 h caused
an arrest of ~27% cells in S phase which was accompanied by a
concomitant decrease of cells in the G1 and G2-M phases of the cell
3.3. Flavonol 3 induces cell death mediated by caspases activation
Caspases are cysteine aspartate-specific proteases which are
essential executioners of apoptosis. To determine whether caspases
were involved in the cell responses to flavonol 3, this compound
was examined for the production of proteolytic processing of cas-
pases. Initial experiments were performed to determine the hier-
archical relationship between the initiator caspases, caspases-8 and
-9. To this end, kinetic assays were performed and analyzed by
Western blots using antibodies which bind both the proenzyme
(caspase precursors) and the cleaved caspases. The results show
that pro-caspase-9 was processed before procaspase-8 (Fig. 4).
In order to identify the caspases activated and early mechanism
of action of flavonol 3 on human leukemia cells, we performed
doseeresponse experiments using concentrations up to 10-fold
higher than the antiproliferative IC50 value. To this end, HL-60,
U937 and Molt-3 cells were treated with increasing concentra-
tions of flavonol 3 for 24 h, and initiator caspases (caspase-8 and -9)
were determined by Western blots (Fig. 5). The results show that
cycle. Flavonol 3 (3
mM) showed a similar trend in S arrest in U937
flavonol 3 stimulated the cleavage of procaspase-9 to the
cells and this effect was sustained for 48 h. There was no arrest of
HL-60 or U937 cells at the S phase of the cell cycle for flavonol 3 at
35e37 kDa fragment in HL-60 and Molt-3 cells, and also induced
pro-caspase-9 processing in U937 cells. As shown in Fig. 5, flavonol
3 also induced pro-caspase-8 cleavage in all three leukemia cells.
Moreover, this compound stimulated the proteolytic processing of
executioner caspases-7 and -6, while the hydrolysis of pro-caspase-
3 was only observed in HL-60 and Molt-3, but not in U937 cells. We
also evaluated the capacity of flavonol 3 to induce cleavage of pro-
caspase-4, which is involved in endoplasmic reticulum stress [27].
concentrations below 3
apoptotic cells increased to 20% (6-fold) and 56% (15-fold) in
response to 10 M flavonol 3 after 24 and 48 h of treatment,
mM. In HL-60 cells, the percentage of
m
respectively (Table 2, Fig. 3). In U937 cells, the percentage of
apoptotic cells increased to ~14% (~4-fold) and ~46% (~7-fold) after
24 and 48 h of treatment, respectively. These results indicate that

O. Burmistrova et al. / European Journal of Medicinal Chemistry 84 (2014) 30e41
35
Fig. 3. Effects of flavonol 3 on apoptosis induction and on cell cycle phase distribution of HL-60 and U937 cells. (A) Cells were incubated with the indicated concentrations of
flavonol 3 and apoptotic cells were quantified as percentage of cells in the sub-G1 region using flow cytometry. Values represent means SEs of three independent experiments
each performed in triplicate; * indicates P < 0.05 for comparison with untreated control. (B) Representative histograms of cell cycle. Cells were cultured with 10
indicated periods of time and subjected to DNA flow cytometry.
mM flavonol 3 for the
We observed a reduction (Molt-3) and processing (HL-60 and
U937) of this zymogen after 24 h of treatment (Fig. 5).
concentration (3 mM) of flavonol 3. Moreover, the flavonoid induced
a decrease (HL-60 and Molt-3) or processing (U937) of Bid in
accordance with caspase-8 processing (Fig. 5).
The enzymatic activities of caspases-3/-7, -8 and -9 were also
evaluated since they are not always associated with pro-caspase
processing. To this end, HL-60, U937 and Molt-3 cells were
The expression of the Bcl-2 family members which control the
intrinsic pathway of apoptosis was also investigated. Although no
change in the expression of Bcl-2 and Bax was observed, in Molt-3
cells an 18 kDa cleavage fragment generated from the full length
23 kDa Bax was detected even after treatment with a very low
treated with 10 mM flavonol 3 for 24 h, and cell lysates were assayed
for cleavage of the tetrapeptides DEVD-pNA, IETD-pNA, and LEHD-
pNA as specific substrates for caspase-3/7, caspase-8, and caspase-
9, respectively. In HL-60 cells, caspase-3/7 activity increased 4.5-
fold while caspases-9 and -8 activities increased 1.8-fold and 1.7-
fold over the control, respectively. A similar trend in caspase ac-
tivity was observed in U937 and Molt-3 cells (Fig. 6).
Table 2
Effect of different durations of treatment with flavonol 3 on cell cycle phase dis-
tribution of human leukemia cells.
% Sub-G1
%G1
%S
%G2-M
HL-60 12 h Control
6.1 0.5
5.6 0.2
6.3 0.3
8.9 0.8
3.4 0.3
4.1 0.5
5.9 0.4
57.2 0.9
56.2 0.7
56.7 0.6
58.2 1.0
53.0 1.1
49.5 0.5
50.1 1.9
14.7 0.3
15.6 0.3
14.8 0.4
15.9 1.0
17.9 0.6
20.7 0.9
17.6 0.7
18.5 0.5
19.4 0.2
18.2 0.3
13.4 0.6
22.5 0.9
22.5 0.9
21.8 1.8
1
3
m
m
M
M
3.4. Flavonol 3 activates mitogen-activated protein kinases
10
24 h Control
mM
Since MAPKs pathways play a crucial role in a diverse array of
cellular functions, including apoptosis, we evaluated the effect of
this flavonoid on the activation of this signal transduction pathway.
The results show a fast phosphorylation (2 h) of these MAPKs,
phospho-ERK 1/2 remained elevated for at least 6 h, while the level
1
3
m
m
M
M
10
m
M
20.4 2.8* 37.3 1.8* 24.1 0.9* 12.6 0.7*
48 h Control
3.7 0.1
3.4 0.4
60.4 0.4
57.6 1.1
15.3 0.1
15.5 0.5
17.9 0.7
20.8 0.7
1
3
m
m
M
M
14.3 1.9* 40.4 1.2* 22.1 0.4* 18.0 0.8
10
m
M
56.7 8.2* 18.4 5.4*
7.3 1.8*
20.5 0.8
21.5 0.4
22.4 0.2
24.9 0.4
18.6 0.3
18.9 0.3
24.7 0.3
5.5 1.6*
24.2 1.2
25.1 0.3
23.9 0.4
19.6 0.6
22.8 1.6
25.2 1.0
21.7 0.8
18.7 0.6
19.1 1.1
19.9 0.1
19.0 0.7
7.0 0.8*
U937
12 h Control
4.6 0.9
3.3 0.5
3.1 0.4
4.3 0.5
3.9 0.8
3.7 0.4
5.5 0.3
45.8 0.8
45.8 0.6
46.6 0.7
47.6 0.5
49.8 1.5
47.4 0.5
42.3 2.0
1
3
m
m
M
M
mM
10
24 h Control
1
3
m
m
M
M
m
10
M
13.8 2.2* 40.2 0.7* 23.7 1.6
48 h Control
6.7 0.3
7.0 0.2
51.9 0.9
49.0 0.7
18.9 0.2
19.6 0.2
1
3
m
m
M
M
16.3 1.2* 38.5 1.0* 21.2 0.1
45.7 3.9* 41.4 18.0 11.9 2.4*
10 mM
Cells were cultured with the concentrations indicated of flavonol 3 for the indicated
period of times and the cell cycle phase distribution was determined by flow
cytometry. The values are means SEs of two independent experiments with three
Fig. 4. Kinetics and doseeresponse of pro-caspases-8 and -9 processing in response to
flavonol 3. Cells were treated with the indicated concentrations, harvested at the
indicated times and the cleavage of caspases was determined by immunoblotting.
determinations in each. Asterisks indicate
compared with the corresponding controls.
a significant difference (P < 0.05)

36
O. Burmistrova et al. / European Journal of Medicinal Chemistry 84 (2014) 30e41
Fig. 5. Effect of flavonol
3 on caspases cleavage and on Bcl-2 family members
expression. The cells were incubated in the presence of the indicated concentrations of
flavonol 3, harvested at 24 h and cell lysates were assayed by immunoblotting.
Fig. 7. Flavonol 3 induces phosphorylation of MAPKs. Representative Western blots
show the time-dependent phosphorylation of JNK/SAPK, p38MAPK and ERK 1/2 by
flavonol 3. HL-60 cells were incubated with flavonol 3 for the indicated time points and
protein extracts were analyzed by Western blots probed with specific antibodies to
ascertain the phosphorylation of MAPKs. Membranes were stripped and reprobed with
total JNK/SAPK, p38MAPK and ERK 1/2 antibodies as loading controls.
of phosphorylated JNK/SAPK and phospho-p38MAPK was transient
(Fig. 7). This indicates that treatment of cells with flavonol 3 leads
to activation of ERK 1/2, JNK/SAPK and p38MAPK following different
kinetics.
To determine whether the phosphorylation of MAPK plays a key
role in flavonol 3-induced apoptosis, we examined the effects of
specific inhibitors. Neither the selective JNK/SAPK inhibitor
SP600125 nor the p38MAPK inhibitor SB203580 attenuated or
influenced cell death (results not shown) suggesting that activation
of these MAPKs is not required for apoptosis. In contrast, the
mitogen-activated extracellular kinases (MEK) 1/2 inhibitors,
PD98059 and U0126, enhanced the apoptosis induced by this
flavonoid. In HL-60 cells, combination of flavonol 3 and PD98059
HL-60
U937
Molt-3
6
4
3
2
1
0
8
*
Casp-8
Casp-9
Casp-3
Casp-8
Casp-8
*
7
6
5
4
3
2
1
0
*
5
4
3
2
1
0
Casp-9
Casp-3
Casp-9
Casp-3
*
*
*
*
*
*
Control Flavonol 3
Control Flavonol 3
Control Flavonol 3
Fig. 6. Activation of caspases in response to flavonol 3. Cells were incubated with 10 mM flavonol 3 for 24 h and cell lysates were assayed for caspase-3/7, -8 and -9 activities. Results
are expressed as n-fold increases in caspase activity compared with control. Values represent the means SEs of three independent experiments each performed in triplicate; *
indicates P < 0.05 for comparison with untreated control.

O. Burmistrova et al. / European Journal of Medicinal Chemistry 84 (2014) 30e41
37
(10
m
M) or U0126 (10
m
M) at least doubled the rate of cell death
also cytotoxic against the mitoxantrone resistant, HL-60/MX1, and
against the Burkitt lymphoma cell line Raji. Moreover, flavonol 3
was a more apoptotic inducer than flavonol 5 in HL-60 and U937
cells lines. Cell cycle analysis performed in both cell lines showed
that inhibition of cell viability by flavonol 3 was caused by a sig-
nificant cell cycle arrest at the S phase and accompanied by an
increase in sub-G1 fraction indicating apoptotic cell death.
when compared with flavonol 3 alone (results not shown).
3.5. Flavonol 3 up-regulates death receptors DR4 and DR5 and the
combination of flavonol 3 and TRAIL enhances apoptosis
Since flavonol 3 also induces activation of caspase-8, it is
possible that death receptors and/or their ligand might be involved
in this cell death pathway. Therefore we first explored the effects of
flavonol 3 on the expression of TRAIL, DR4 and DR5, in human
leukemia cells. HL-60 and U937 were treated during 24 h and cell
lysates were subjected to immunoblot analysis. As shown in Fig. 8A,
flavonol 3 up-regulates expression of TRAIL, DR4 and DR5 in both
cell lines.
Additional experiments were performed to determine whether
the up-regulation of death receptors by flavonol 3 could contribute
to the amplification of cell death in the combination treatment with
TRAIL. To this end, U937 leukemia cells were treated with TRAIL
The results also show that flavonol 3 induced the processing of
initiator caspases, the caspase-8 and the caspase-9, suggesting that
this synthetic flavonoid stimulates the two major pathways of
apoptosis, the extrinsic and intrinsic pathways of cell death.
Moreover, the endoplasmic reticulum stress signaling pathway
appears to be involved since flavonol 3 induced caspase-4 cleavage.
Induction of caspase-8 and -9 as well as caspase-3 like activities
was significantly increased in response to flavonol 3 in accordance
with the processing of the corresponding pro-caspases as deter-
mined by Western blots. The results clearly demonstrated that this
flavonoid also stimulates the proteolytic processing of other
executioner caspases, namely caspase-7 and caspase-6 to form
activated enzymes. Although flavonol 3 induced the lowest acti-
vation of caspase-3 in U937 cells, as determined by Western blot, it
induced a clear caspase-3/7 activation as determined by enzymatic
activity. It appears that the substrate used (DEVD-pNA) also de-
termines caspase-7 activity, and, as shown, flavonol 3 also induced
a processing of caspase-7 in these cells.
(0.5
m mM), and TRAIL and flavonol 3
g mLꢀ1), flavonol 3 (30
simultaneously for 48 h or were incubated with flavonol 3 for 24 h,
followed by TRAIL for 24 h. Cell viability was assessed by the MTT
assay. The results show that the combination treatment, as well as
the pretreatment with flavonol 3 followed by TRAIL, enhances cell
death in comparison with both non-combination treatments
(Fig. 8B).
The intrinsic pathway of apoptosis is tightly regulated by B-cell
lymphoma
2 (Bcl-2) family proteins that control the per-
4. Discussion
meabilization of the outer mitochondria membrane [28]. Previous
studies have shown that flavonoids such as luteolin [29] and
astragalin heptaacetate [30] induce cleavage of Bcl-2. However,
flavonol 3 did not induce any Bcl-2 cleavage or any change in Bcl-2
levels. There were no changes in Bax levels in HL-60 and U937 cells
but this protein was cleaved in Molt-3 cells. Previous studies have
shown that a fragment of 18 kDa generated by Bax cleavage is more
potent at promoting mitochondrial membrane permeabilization
than the full-length protein [31]. The levels of Bid were reduced and
Bid processing occurred which is consistent with the finding of
caspase-8 activation.
In previous studies with naturally occurring and semi-synthetic
phenylbenzo-g-pyrones, we showed that methylation of the hy-
droxy group at position C3 of the quercetin yields a compound with
a higher antiproliferative activity against several cancer cell lines
[18]. In this study, we have synthesized a series of 20 flavonoids and
evaluated their potential cytotoxic properties using the human HL-
60 tumor cell line, which is a useful model to study cell growth
inhibition of leukemia cells by chemical agents. The anti-
proliferative studies indicate that flavonols containing chlorine or
bromine atoms at positions 20 (flavonol 5) and 40 (flavonol 3) of the
B ring, respectively, were the most cytotoxic compounds against
HL-60 cells. Although both flavonols displayed similar IC50 values in
this cell line, additional experiments revealed that flavonol 3 was
We have previously described the potentiation of cell death by
the combined treatment of a quercetin derivative and specific
mitogen-activated extracellular kinases 1/2 inhibitors [32]. Here we
Fig. 8. (A) Flavonol 3 up-regulates the expression of the TRAIL and TRAIL receptors DR4 and DR5 in human leukemia cells. Cells were treated with increasing concentrations of
flavonol 3 for 24 h and cell lysates were assayed by immunoblotting. (B) Increase in TRAIL sensitivity by flavonol 3 in U937 cells. Cells were incubated for 48 h with TRAIL
(0.5
m
g mLꢀ1), flavonol 3 (30
m
M), 3 þ TRAIL (simultaneous addition TRAIL and flavonol 3), or TRAIL (0.5
m mM) for 24 h
g mLꢀ1) was added after incubation with flavonol 3 (30
(3 / TRAIL). Bars represent the mean SE of three independent experiments each performed in triplicate; * indicates P < 0.05 for comparison with untreated control, # indicates
P < 0.05 for comparison with TRAIL treatment alone.

38
O. Burmistrova et al. / European Journal of Medicinal Chemistry 84 (2014) 30e41
found that flavonol 3 induced the phosphorylation and activation of
ERK 1/2, p38MAPK and JNK/SAPK following different kinetics. Our
results also indicate that MEK 1/2 inhibitors PD98059 and U0126
enhanced the apoptotic effects of flavonol 3.
Reactive oxygen species (ROS) have been implicated as second
messengers in multiple signaling pathways, including cell death via
MAPK activation [33e35]. However the cytotoxic and/or apoptotic
effects of flavonol 3 seem to be ROS independent since neither N-
crystallized from MeOH although in some cases the product was
purified using column chromatography.
A solution of the corresponding 2-hydroxychalcone (2e4 mmol)
in 3.0 M KOH in MeOH (20e30 mL) was cooled at 0 ꢁC. An aqueous
solution of H2O2 (30%) (5e8 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
(2e4 mL) was added. HCl (2 M) was added until pH 2 and the
precipitate was filtered and washed with distilled water and
extracted with EtOAc. The organic layer was washed with brine
until neutrality and dried with anhydrous MgSO4. The solvent was
evaporated in vacuo and the residue was purified using a chro-
matographic column (SiO2, petroleum ether): EtOAc (1:0e7:3).
acetyl-L-cysteine (an antioxidant agent) nor trolox (an analog of
vitamin E acting specifically as ROS scavenger) blocked cell death
(data not shown).
Since we demonstrated that apoptosis induced by flavonol 3 is
associated with caspase-8 activation and Bid cleavage, it is possible
that in the mechanism of action of this flavonoid might involve
death receptors. The results demonstrate that flavonol 3 stimulated
the expression of TRAIL, DR4 and DR5 and, at least in U937 cells,
potentiating the sensitivity to TRAIL. Although further studies are
necessary, these results are of potential importance by demon-
strating the effect of this flavonoid against tumor cells that are
resistant to TRAIL.
6.1.2.1. 20-Hydroxy-2,4-dimethoxy-3-methylchalconeꢀ1 (chal
6).
Orange solid, mp 118e120 ꢁC (92%). IR (KBr, cm
) nmax: 3449,
3002, 2941, 2837, 1685, 1634, 1596, 1564, 1488, 1463, 1442, 1411,
1362,1342,1313,1275,1247,1212,1156,1109,1029, 990, 967, 863. 1H
NMR (400 MHz, CDCl3):
d
13.02 (s, OH); 8.15 (1H, d, J ¼ 15.4 Hz);
7.94 (1H, d, J ¼ 8.0 Hz); 7.72 (1H, d, J ¼ 15.4 Hz); 7.56 (1H, d,
J ¼ 8.7 Hz); 7.50 (1H, ddd, J ¼ 1.4, 7.4, 7.4 Hz); 7.04 (1H, d, J ¼ 8.4 Hz);
6.95 (1H, dd, J ¼ 7.6, 7.6 Hz); 6.73 (1H, d, J ¼ 8.6 Hz); 3.91 (3H, s);
5. Conclusions
3.80 (3H, s); 2.20 (3H, s). 13C NMR (100 MHz, CDCl3):
d
¼ 194.5,
In this paper a series of 20 flavonoids, including nine flavonols
and eleven 3-methyl ethers were synthesized and assessed for
cytotoxicity against human leukemia cells. A flavonol derivative,
containing an atom of bromine at position 40 of the B ring, was
selected since it displayed cytotoxic properties showing IC50 values
163.9, 161.7, 160.0, 141.8, 136.3,129.9,127.8, 125.2,121.3, 120.7, 119.2,
119.0, 118.9, 107.0, 61.8, 56.1, 9.2. HRMS (ESI-FT-ICR) m/z: 299.1286
[MþH]þ; calcd. for C18H20O4: 299.1283.
6.1.2.2. 3-Hydroxy-20,40-dimethoxy-30-methylflaꢀvo1ne
(6).
) nmax: 3416, 3260,
of approximately 5
resistant HL-60/MX1 cell line. The cytotoxic effects of this flavonol
were accompanied by concentration- and time-dependent
mM, in tumor cells including the mitoxantrone
Yellow solid, mp 208e210 ꢁC (69%). IR (KBr, cm
2364, 1614, 1595, 1569, 1482, 1454, 1417, 1404, 1345, 1269, 1213,
a
1172, 1133, 1106, 1052, 1022, 940, 881, 809. 1H NMR (400 MHz,
appearance of apoptosis, associated with the activation of multi-
ple caspases and the mitogen-activated protein kinase pathway and
potentiated the sensitivity to TRAIL. The studies shown here open
new avenues of research into the roles of signal transduction
pathways in the death of the human leukemia cells, and suggest
that this compound or derivatives might be useful in the devel-
opment of new therapeutic strategies in the fight against cancer.
CDCl3):
d
8.31 (1H, d, J ¼ 8.0 Hz); 7.70 (1H, dd, J ¼ 7.6, 7.3 Hz); 7.55
(1H, d, J ¼ 8.7 Hz); 7.52 (1H, d, J ¼ 8.8 Hz); 7.44 (1H, dd, J ¼ 7.1,
7.1 Hz); 6.81 (1H, d, J ¼ 8.5 Hz); 6.64 (1H, s, OH); 3.92 (3H, s); 3.74
(3H, s); 2.25 (3H, s). 13C NMR (100 MHz, CDCl3):
d
¼ 173.5, 161.2,
157.8,156.2,146.9,138.8,133.6,129.2,125.9,124.7,121.8,121.0,118.7,
116.8, 106.4, 61.7, 56.2. HRMS (ESI-FT-ICR) m/z: 335.0886 [MþNa]þ;
calcd. for C18H16O5Na: 335.0895.
6. Experimental protocols
6.1.2.3. 20-Hydroxy-3,4,5,40-tetramethoxychalcone
Orange solid, mp 89e91 ꢁC (85%). IR (KBr, cmꢀ1
2941, 2841, 2738, 2638, 1714, 1682, 1643, 1633, 1622, 1614, 1582,
1574, 1558, 1504, 1463, 1455, 1417, 1371, 1333, 1273, 1217, 1153, 1128,
1069, 1020, 1004, 976, 960, 837. 1H NMR (400 MHz, CDCl3):
(chal
nmax: 3534, 3003,
8).
)
6.1. Chemistry
6.1.1. Materials and methods
1H and 13C NMR spectra were obtained on a Bruker model AMX-
400 spectrometer with standard pulse sequences operating at
d
¼ 13.47 (s, OH); 7.84 (1H, d, J ¼ 8.7 Hz); 7.80 (1H, d, J ¼ 15.4 Hz);
7.45 (1H, d, J ¼ 15.3 Hz); 6.87 (2H, s); 6.50e6.47 (2H, m); 3.93 (6H,
s); 3.91 (3H, s); 3.86 (3H, s). 13C NMR (100 MHz, CDCl3):
400 MHz in 1H and 100 MHz in 13C NMR. Chemical shifts (
d) are
d
¼ 192.0,
given in ppm upfield from tetramethylsilane as internal standard,
and coupling constants (J) are reported in hertz. IR spectra were
recorded using a Bruker model IFS-55 spectrophotometer. Melting
points were determined on a Büchi B-540 apparatus and are un-
corrected. EIMS and HREIMS were taken on a Micromass model
Autospec (70 eV) spectrometer. Column chromatography was car-
ried out on silica gel 60 (Merck 230e400 mesh) and analytical thin
layer chromatography (TLC) was performed using silica gel
aluminum sheets.
167.0, 166.6, 153.9, 144.9, 132.6, 131.5, 130.6, 119.9, 114.5, 108.0,
107.9, 106.4, 101.5, 101.3, 61.3, 56.6, 55.9. HRMS (ESI-FT-ICR) m/z:
345.1337 [MþH]þ; calcd. for C19H21O4: 345.1338.
6.1.2.4. 3-Hydroxy-7,30,40,50-tetramꢀe1thoxyflavone (8). Yellow solid,
mp 177e180 ꢁC (77%). IR (KBr, cm
) nmax: 3422, 3247, 2939, 1602,
1567, 1505, 1457, 1417, 1401, 1379, 1337, 1283, 1263, 1213, 1180, 1163,
1125, 1102, 1015, 848, 831, 820. 1H NMR (400 MHz, CDCl3):
d
¼ 8.04
(1H, d, J ¼ 8.9 Hz); 7.42 (2H, s); 6.91 (1H, dd, J ¼ 2.1, 8.9 Hz); 6.85
(1H, d, J ¼ 2.0 Hz); 3.88 (6H, s); 3.86 (3H, s); 3.86 (3H, s). 13C NMR
6.1.2. General procedure for the synthesis of flavonols
(100 MHz, CDCl3):
d
¼ 173.0, 164.7, 157.5, 153.6, 144.5, 138.2, 127.1,
A mixture of the acetophenone (5e10 mmol, 1 equiv.) and the
corresponding aldehyde (1 equiv.) in EtOH (20e40 mL) was stirred
at room temperature and a 50% aqueous solution of NaOH (5e8 mL)
was added. The reaction mixture was stirred at room temperature
until all aldehyde had been consumed. HCl (10%) was then added
until neutrality. Precipitated chalcones were generally filtered and
126.7, 115.1, 114.9, 105.8, 105.1, 100.3, 61.3, 56.7, 56.2. HRMS (ESI-FT-
ICR) m/z: 359.1128 [MþH]þ; calcd. for C19H19O7: 359.1131; 381.0949
[MþNa]þ; calcd. for C19H19O7Na: 381.0950.
6.1.2.5. 20-Hydroxy-2,4-dimethoxychalcone (chal 9). Orange solid,
mp 103e105 ꢁC (89%). IR (KBr, cmꢀ1
) nmax: 3011, 2966, 2940, 2836,

O. Burmistrova et al. / European Journal of Medicinal Chemistry 84 (2014) 30e41
39
2747, 2628, 1634, 1606, 1573, 1565, 1553, 1504, 1488, 1465, 1443,
1419, 1367, 1343, 1306, 1278, 1204, 1157, 1119, 1024, 983, 940, 863,
2843, 1643, 1623, 1610, 1591, 1488, 1463, 1438, 1406, 1386, 1297,
1274, 1258, 1222, 1172, 1148, 1107, 1052, 1002, 985, 920, 883. 1H
831. 1H NMR (400 MHz, CDCl3):
d
¼ 13.1 (1H, s, OH); 8.18 (1H, d,
NMR (400 MHz, CDCl3):
d
8.32 (1H, d, J ¼ 8.0 Hz); 7.67 (1H, dd,
J ¼ 15.5 Hz); 8.11 (1H, dd, J ¼ 2.1, 6.9 Hz); 7.05 (2H, dd, J ¼ 2.1,
6.9 Hz); 6.99 (1H, dd, J ¼ 2.3, 8.8 Hz); 6.93 (1H, d, J ¼ 2.3 Hz); 3.94
(3H, s); 3.92 (3H, s); 3.90 (3H, s). 13C NMR (100 MHz, CDCl3):
J ¼ 7.1, 8.3 Hz); 7.49 (1H, d, J ¼ 8.4 Hz); 7.42 (1H, dd, J ¼ 7.2, 7.3 Hz);
7.36 (1H, d, J ¼ 8.5 Hz); 6.77 (1H, d, J ¼ 8.5 Hz); 3.92 (3H, s); 3.84
(3H, s); 3.74 (3H, s); 2.23 (3H, s). 13C NMR (100 MHz, CDCl3):
d
¼ 194.6, 163.9, 163.8, 161.1, 141.6, 136.1, 131.8, 129.9, 120.7, 118.9,
d
¼ 175.2, 161.1, 158.0, 157.4, 155.9, 142.1, 133.5, 129.0, 126.2, 125.0,
118.8, 118.5, 117.3, 106.0, 98.9, 56.0, 55.8. HRMS (ESI-FT-ICR) m/z:
285.1127 [MþH]þ; calcd. for C17H17O4: 285.1127.
124.8, 120.8, 118.4, 117.4, 106.1, 61.7, 60.7, 56.2. HRMS (ESI-FT-ICR)
m/z: 349.1052 [MþNa]þ; calcd. for C19H18O5Na: 349.1051.
6.1.2.6. 3-Hydroxy-20,40-dimethoxyflavone (9). Yellow solid, mp
6.1.3.5. 3,7,40-Trimethoxyflavone (16). White solid, mp 142e144 ꢁC
187e189 ꢁC (72%). IR (KBr, cmꢀ1
)
nmax: 3396, 3311, 1609, 1569, 1507,
1467, 1425, 1411, 1284, 1209, 1162, 1138, 1114, 1041, 1029, 921, 893,
834. 1H NMR (400 MHz, CDCl3):
(75%). IR (KBr, cmꢀ1
)
nmax: 3443, 2360, 2341, 2097, 1634, 1508, 1445,
1382, 1256, 1180, 1118, 1031, 1015, 953, 834. 1H NMR (400 MHz,
CDCl3):
¼ 8.18 (1H, d, J ¼ 8.88 Hz); 8.11 (2H, d, J ¼ 6.9 Hz); 7.05 (2H,
d, J ¼ 6.9 Hz); 6.99 (1H, dd, J ¼ 2.3, 8.8 Hz); 6.93 (1H, d, J ¼ 2.3 Hz)
13C NMR (100 MHz, CDCl3):
d
¼ 8.29 (1H, d, J ¼ 1.4, 8.0 Hz); 7.68
d
(1H, ddd, J ¼ 1.6, 7.0, 8.6 Hz); 7.54 (1H, dd, J ¼ 8.5, 11.6 Hz); 7.42 (1H,
ddd, J ¼ 0.8, 7.0, 8.0 Hz); 6.66 (1H, dd, J ¼ 2.3, 8.5 Hz); 6.44 (1H, brs),
d
¼ 174.8, 164.3, 161.7, 157.3, 133.4,
3.90 (3H, s); 3.88 (3H, s). 13C NMR (100 MHz, CDCl3):
d
¼ 194.6,
130.4, 127.5, 123.8, 118.5, 114.5, 114.3, 106.7, 100.3, 60.3, 56.1, 55.7.
HRMS (ESI-FT-ICR) m/z: 313.1068 [MþH]þ; calcd. for C18H17O5:
313.1076.
163.9, 163.8, 161.1, 141.6, 136.1, 131.8, 129.9, 120.7, 118.9, 118.8, 118.5,
117.3, 106.0, 98.9, 56.0, 55.8. HRMS (ESI-FT-ICR) m/z: 285.1127
[MþH]þ; calcd. for C17H17O4: 285.1127.
6.1.3.6. 3-Methoxy-2-(1-naphthalenyl)-4H-1-benzopyran-4-one (17)
6.1.3. General method for the preparation of 3-methoxy-flavones
K2CO3 (1.8 equiv.) and iodomethane (1.2 equiv.) were added to a
solution of flavonol (0.10e0.20 mmol) at 40 ꢁC in dry CH3CN
(1e2 mL) until the starting material was totally consumed (as
evidenced by TLC). The reaction mixture was then, partitioned
between ethyl acetate and water. The ethyl acetate layer was
washed with brine, dried over MgSO4, filtered and concentrated.
The crude material was purified by column chromatography to
yield methyl ethers.
[37]. Pale yellow solid, mp 165e166 ꢁC (68%). IR (KBr, cmꢀ1
) nmax:
3431, 3066, 3011, 1645, 1614, 1569, 1506, 1467, 1441, 1374, 1337,
1293, 1265, 1239, 1211, 1172, 1148, 1109, 1081, 1002, 942, 896, 805.
1H NMR (400 MHz, CDCl3):
J ¼ 8.2 Hz); 7.97 (1H, d, J ¼ 7.5 Hz); 7.86 (1H, d, J ¼ 8.0 Hz); 7.76 (1H,
d, J ¼ 7.0 Hz); 7.71 (1H, dt, J ¼ 1.0, 7.1, 8.4 Hz); 7.63 (1H, dd, J ¼ 7.6,
7.7 Hz); 7.60e7.46 (4H, m); 3.72 (3H, s). 13C NMR (100 MHz, CDCl3):
d
8.39 (1H, d, J ¼ 7.9 Hz); 8.06 (1H, d,
d
¼ 17, 157.8, 156.1, 133.5, 130.9, 128.5, 128.2, 127.0, 126.4, 126.0,
125.2, 124.9, 124.8, 118.2, 60.6, HRMS (ESI-FT-ICR) m/z: 303.1021
[MþNa]þ; calcd. for C20H15O3Na: 303.1021.
6.1.3.1. 3,20,40-Trimethoxyflavoꢀn1e (11) [36]. Pale yellow solid, mp
119e120 ꢁC (70%). IR (KBr, cm
)
nmax: 3441, 2097, 1643, 1624, 1507,
6.1.3.7. 20-Hydroxy-2,4,40-trimethoxy-3-methylcꢀh1alcone (chal 18).
1466, 1436, 1381, 1307, 1267, 1211, 1162, 1122, 1029. 1H NMR
Orange solid, mp 161e163 ꢁC (92%). IR (KBr, cm
) nmax: 3710, 3003,
(400 MHz, CDCl3):
d
8.20 (1H, dd, J ¼ 1.6, 8.0 Hz); 7.55 (1H, ddd,
2944, 2836, 1630, 1564, 1508, 1488, 1463, 1412, 1365, 1311, 1276,
1252, 1228, 1222, 1208, 1185, 1154, 1130, 1109, 1022, 989, 959, 857,
J ¼ 1.6, 7.0, 8.4 Hz); 7.36 (1H, d, J ¼ 8.0 Hz); 7.33e7.27 (2H, m); 6.53
(1H, dd, J ¼ 2.3, 8.3 Hz); 3.79 (3H, s); 3.75 (3H, s); 3.74 (3H, s). 13
C
827. 1H NMR (400 MHz, CDCl3):
d 13.62 (s, OH); 8.10 (1H, d,
NMR (100 MHz, CDCl3):
d
¼ 175.2, 163.2, 159.1, 156.5, 156.1, 142.3,
J ¼ 15.5 Hz); 7.85 (1H, d, J ¼ 9.6 Hz); 7.64 (1H, d, J ¼ 15.5 Hz); 7.54
(1H, d, J ¼ 8.6 Hz); 6.73 (1H, d, J ¼ 8.7 Hz); 6.51e6.49 (2H, m); 3.90
(3H, s); 3.87 (3H, s); 3.80 (3H, s); 2.20 (3H, S). 13C NMR (100 MHz,
133.4, 132.0, 126.2, 125.0, 124.7, 118.4, 113.9, 105.2, 99.3, 60.6, 56.1,
55.9. HRMS (ESI-FT-ICR) m/z: 335.0904 [MþNa]þ; calcd. for
C
18H16O5Na: 303.1021.
CDCl3):
d
¼ 192.7, 167.0, 166.3, 161.5, 159.8, 140.7, 131.5, 127.6, 121.5,
120.9, 119.4, 114.7, 107.8, 107.0, 101.5, 63.2, 61.7, 56.1, 55.9, 9.2. HRMS
6.1.3.2. 40-Bromo-3-methoxyflavone (13) [19]. Pale yellow solid, mp
115e116 ꢁC (69%). IR (KBr, cmꢀ1
nmax: 3446, 2360, 2341, 2066,1643,
1615, 1487, 1468, 1439, 1399, 1379, 1340, 1291, 1238, 1213, 1147, 1110,
1072, 1009, 950, 898, 826. 1H NMR (400 MHz, CDCl3):
(ESI-FT-ICR) m/z: 299.1286 [MþH]þ; calcd. for C18H20O4: 299.1283.
)
6.1.3.8. 3-Hydroxy-7,20,40-trimethoxy-30-methylflavonꢀe1 (flavonol-
d
¼ 8.28 (1H,
18). Yellow solid, mp 197e199 ꢁC (70%). IR (KBr, cm
) nmax: 3734,
d, J ¼ 7.9 Hz); 8.02 (2H, d, J ¼ 8.6 Hz); 7.72e7.67 (3H, m); 7.55 (1H, d,
3005, 2942, 2839, 1682, 1610, 1505, 1490, 1452, 1408, 1273, 1256,
J ¼ 8.3 Hz); 7.43 (1H, dd, J ¼ 7.8, 7.8 Hz); 3.93 (3H, s). 13C NMR
1226, 1203, 1174, 1132, 1109, 1022, 1000, 834. 1H NMR (400 MHz,
(100 MHz, CDCl3):
d
¼ 175.3, 155.5, 154.6, 142.0, 133.9, 132.2, 130.3,
CDCl3):
d
8.19 (1H, d, J ¼ 8.9 Hz); 7.49 (1H, d, J ¼ 8.6 Hz); 7.85 (1H, d,
130.2, 126.2, 125.6, 125.1, 124.6, 118.3, 106.6, 60.4. HRMS (ESI-FT-
J ¼ 9.6 Hz); 7.01 (1H, dd, J ¼ 1.4, 8.9 Hz); 6.92 (1H, s); 6.80 (1H, d,
79
ICR) m/z: 330.9966 [MþH]þ; calcd. for C BrH12O3: 330.9970; calcd.
J ¼ 8.6 Hz); 6.62 (H, s); 3.91 (6H, s); 3.71 (3H, s); 2.24 (3H, s). 13
C
16
for C1861BrH12O3: 332.9949.
NMR (100 MHz, CDCl3):
d
¼ 173.0, 164.4, 158.2, 157.8, 146.1, 138.5,
129.2, 127.2, 120.9, 116.8, 115.0, 106.4, 101.4, 100.3, 61.6, 56.1, 9.3.
HRMS (ESI-FT-ICR) m/z: 343.1178 [MþH]þ; calcd. for C19H19O6:
343.1182.
6.1.3.3. 3,20-Dimethoxyflavone (14). White solid, mp 146e147 ꢁC
(74%). IR (KBr, cmꢀ1
) nmax: 3449, 3002, 2938, 2840,1642, 1624,1581,
1569, 1492, 1466, 1437, 1386, 1339, 1300, 1278, 1257, 1233, 1212,
1171, 1147, 1111, 1048, 1023, 1008, 955, 900. 1H NMR (400 MHz,
6.1.3.9. 3,7,20,40-Tetramethoxy-30-methylflꢀa1vone (18). Pale yellow
CDCl3):
d
¼ 13.1 (1H, s, OH); 8.18 (1H, d, J ¼ 15.5 Hz); 8.11 (1H, dd,
solid, mp 153e154 ꢁC (60%). IR (KBr, cm
) nmax: 3442, 1626, 1488,
J ¼ 2.1, 6.9 Hz); 7.05 (2H, dd, J ¼ 2.1, 6.9 Hz); 6.99 (1H, dd, J ¼ 2.3,
8.8 Hz); 6.93 (1H, d, J ¼ 2.3 Hz); 3.94 (3H, s); 3.92 (3H, s); 3.90 (3H,
1443, 1406, 1385, 1363, 1273, 1248, 1225, 1203, 1168, 1109, 1048,
1024, 1004, 989, 951. 1H NMR (400 MHz, CDCl3):
d
8.12 (1H, d,
s). 13C NMR (100 MHz, CDCl3):
d
¼ HRMS (ESI-FT-ICR) m/z:
J ¼ 8.9 Hz); 7.25 (1H, d, J ¼ 8.8 Hz); 6.90 (1H, dd, J ¼ 2.3, 8.9 Hz);
6.78 (1H, d, J ¼ 2.3 Hz); 6.67 (1H, d, J ¼ 8.5 Hz); 3.83 (3H, s); 3.81
(3H, s); 3.74 (3H, s); 3.63 (3H, s); 2.14 (3H, s). 13C NMR (100 MHz,
283.0964 [M]þ; calcd. for C17H14O4: 283.0970.
6.1.3.4. 3,20,40-Trimethoxy-30-methylflavone (15). Pale yellow solid,
CDCl3):
d
¼ 174.7, 164.3, 161.0, 157.9, 157.7, 129.0, 127.5, 120.7, 118.9,
mp 176e178 ꢁC (71%). IR (KBr, cmꢀ1
) nmax: 3710, 3014, 2970, 2944,
117.4,114.6,106.1,100.4, 95.6, 61.6, 60.7, 56.1, 9.4. HRMS (ESI-FT-ICR)

40
O. Burmistrova et al. / European Journal of Medicinal Chemistry 84 (2014) 30e41
m/z: 379.1151 [MþNa]þ; calcd. for C20H20O6Na: 379.1158; 357.1333
[MþH]þ; calcd. for C20H21O6: 357.1338.
6.2.7. Statistical analysis
Statistical significance of differences between means of control
and treated samples were calculated using Student's t-test. P values
of <0.05 were considered significant.
6.1.3.10. 3,7,30,40,50-Pentamethoxyflavone (20). Pale yellow solid,
mp 125e127 ꢁC (65%). IR (KBr, cmꢀ1
) nmax: 3448, 3003, 2973, 2940,
2840, 1636, 1621, 1503, 1445, 1415, 1382, 1363, 1328, 1253, 1213,
Acknowledgments
1171, 1126, 1104, 1052, 1002, 953, 839. 1H NMR (400 MHz, CDCl3):
d
¼ 8.09 (1H, d, J ¼ 8.8 Hz); 7.31 (2H, s); 6.91 (1H, dd, J ¼ 2.3, 8.9 Hz);
This work was supported by the Spanish Ministry of Science
and Innovation and the European Regional Development Fund
6.84 (1H, d, J ¼ 2.3 Hz); 3.88 (6H, s); 3.87 (3H, s); 3.86 (3H, s). 13
C
NMR (100 MHz, CDCl3):
d
¼ 174.7, 164.4, 157.2, 156.4, 155.2, 153.5,
ꢀ
(SAF2010-21380) and CSIC (Proyecto Intramural de Incorporacion e
141.6, 127.6, 126.4, 118.5, 114.6, 106.6, 100.4, 61.3, 60.5, 56.7, 56.2.
HRMS (ESI-FT-ICR) m/z: 373.1280 [MþH]þ; calcd. for C20H21O7:
373.1287; 395.1101 [MþNa]þ; calcd. for C20H20O7Na: 395.1107.
2007022). O. B. was supported by a research studentship from the
ꢀ
Fundacion Canaria Universitaria de Las Palmas.
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