The synthetic flavanone 6-methoxy-2-(naphthalen-1-yl)chroman-4-one induces apoptosis and activation of the MAPK pathway in human U-937 leukaemia cells

Article abstract of DOI:10.1016/j.bioorg.2019.103450

Synthetic flavonoids containing a naphthalene ring have attracted attention as potential cytotoxic compounds. Here, we synthesized ten chalcones and their corresponding flavanones and evaluated their antiproliferative activity against the human tumour cel

Full text of DOI:10.1016/j.bioorg.2019.103450

BioorganicChemistryxxx(xxxx)xxxx
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
The synthetic flavanone 6-methoxy-2-(naphthalen-1-yl)chroman-4-one
induces apoptosis and activation of the MAPK pathway in human U-937
leukaemia cells
Ester Saavedraa, Henoc Del Rosarioa, Ignacio Brouardb, Judith Hernández-Garcésc,
⁎
Celina Garcíac, José Quintanaa, Francisco Estéveza,
a Departamento de Bioquímica y Biología Molecular, Unidad Asociada al Consejo Superior de Investigaciones Científicas (CSIC), Instituto Universitario de Investigaciones
Biomédicas y Sanitarias (IUIBS), Universidad de las Palmas de Gran Canaria, Spain
b Instituto de Productos Naturales y Agrobiología, CSIC, La Laguna, Tenerife, Spain
c Instituto Universitario de Bio-orgánica AG, Departamento de Química Orgánica, Universidad de La Laguna, Tenerife, Spain
A R T I C L E I N F O
A B S T R A C T
Keywords:
Synthetic flavonoids containing a naphthalene ring have attracted attention as potential cytotoxic compounds.
Here, we synthesized ten chalcones and their corresponding flavanones and evaluated their antiproliferative
activity against the human tumour cell line U-937. This series of chalcone derivatives was characterized by the
presence of a naphthalene ring which was kept unaltered- and attached to the β carbon of the 1-phenyl-2-
propen-1-one framework. The structure-activity relationship of these chalcone derivatives and their corre-
sponding cyclic compounds was investigated by the introduction of different substituents (methyl, methoxy,
benzyloxy, chlorine) or by varying the position of the methoxy or benzyloxy groups on the A ring. The results
revealed that both the chalcone containing the methoxy group at 5′ position of the A ring as well as its corre-
sponding flavanone [6-methoxy-2-(naphthalen-1-yl)chroman-4-one] were the most cytotoxic compounds, with
Apoptosis
Structure-activity relationship
Caspase
Cell cycle
Cytotoxicity
Flavanone
IC50 values of 2.8
0.2 and 1.3
0.2 μM, respectively, against U-937 cells. This synthetic flavanone was as
cytotoxic as the antitumor etoposide in U-937 cells and displayed strong cytotoxicity against additional human
leukaemia cell lines, including HL-60, MOLT-3 and NALM-6. Human peripheral blood mononuclear cells were
more resistant than leukaemia cells to the cytotoxic effects of the flavanone. Treatment of U-937 cells with this
compound induced G2-M cell cycle arrest, an increase in sub-G1 ratio and annexin-V positive cells, mitochondrial
cytochrome c release, caspase activation and poly(ADP-ribose)polymerase processing. Apoptosis induction
triggered by this flavonoid was blocked by overexpression of the anti-apoptotic protein Bcl-2. This flavanone
induces phosphorylation of p38 mitogen-activated protein kinases, extracellular-signal regulated kinases and c-
jun N-terminal kinases/stress-activated protein kinases (JNK/SAPK) following different kinetics. Moreover, cell
death was attenuated by the inhibition of mitogen-activated extracellular kinases and JNK/SAPK and was in-
dependent of reactive oxygen species generation.
1. Introduction
treatment protocols, including the discovery of targeted therapies. The
5-year relative survival rate for all cancers combined improved from
Cancer is the second most common cause of death among children
aged 1 to 14 years in the United States and leukaemia accounts for 29%
for all childhood cancers. Cancer survival has improved especially for
hematopoietic and lymphoid malignancies due to improvements in
58% during the mid-1970s to 83% during 2007 through 2013 for
children. The lowest survival rate corresponds to acute myeloid leu-
kaemia with a value of 65.1% between 2007 and 2013 [1]. In ac-
cordance with the GLOBOCAN estimates of incidence and mortality
Abbreviations: ERK, extracellular signal-regulated kinase; FL, 6-methoxy-2-(naphthalen-1-yl)chroman-4-one; 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; ROS, reactive oxygen species
⁎ Corresponding author at: Departamento de Bioquímica y Biología Molecular, Universidad de Las Palmas de Gran Canaria, Paseo Blas Cabrera Felipe s/n, Las
Palmas de Gran Canaria 35016, Spain.
E-mail address: francisco.estevez@ulpgc.es (F. Estévez).
https://doi.org/10.1016/j.bioorg.2019.103450
Received 14 June 2019; Received in revised form 19 September 2019; Accepted 14 November 2019
0045-2068/©2019ElsevierInc.Allrightsreserved.
Pleasecitethisarticleas:EsterSaavedra,etal.,BioorganicChemistry,https://doi.org/10.1016/j.bioorg.2019.103450

E. Saavedra, et al.
BioorganicChemistryxxx(xxxx)xxxx
Scheme 1. Synthesis of chalcones and flavanones.
worldwide for 2018, the estimated numbers of new cases and deaths
from leukaemia were 437,033 and 309,006, respectively, indicating
that mortality rates for this disease are still very high [2], and providing
an urgent need for improved treatments.
properties that illustrate the capacity of these systems to form func-
tional complexes [7–9]. In particular, recent studies using small mole-
cules carrying naphthalene groups describe the interaction of such a
ring system with a hydrophobic pocket located in the Ras oncogene as
causing the inhibition of that oncogene. Naphthalene also has the
ability to generate strong π-π stacking interactions, providing high af-
finity ligands for more than one type of receptor, and making it a pri-
vileged scaffold. These findings suggest that the naphthalene ring could
become a propitious building block for the development of new ther-
apeutic agents [10].
Natural products represent realistic options as potential cytotoxic
compounds against cancer cells [3]. Flavonoids are secondary poly-
phenolic metabolites present in almost all plants and the human diet.
These compounds exhibit a wide spectrum of pharmacological prop-
erties that have been recently reviewed [4,5]. Flavonoids are able to
interfere in all phases of cancer progression by modulating key proteins
involved in proliferation, differentiation, apoptosis, angiogenesis, me-
tastasis and reverse multidrug resistance process. Apoptosis is a form of
regulated cell death which results in destruction of target cell with
minimal inflammatory response. Essential executioners of apoptosis are
the caspases, a family of cysteine proteases which are expressed in cells
as inactive zymogens known as procaspases. These enzymes pre-
dominantly cleave their substrates on the C-terminal side of aspartate.
Proteolytic cleavage leads to important changes in cell morphology
such as membrane blebbing, DNA fragmentation, phosphatidylserine
exposure at the cell surface, and formation of apoptotic vesicles. Two
main apoptotic pathways have been described, the mitochondrial and
the extrinsic pathways [6]. The mitochondrial or intrinsic pathway is
initiated by perturbations of the intracellular or extracellular micro-
environment, demarcated by mitochondrial outer membrane permea-
bilization and precipitated mainly by caspase-3. The extrinsic pathway
is initiated by perturbations of the extracellular microenvironment that
are detected by plasma membrane receptors (propagated by caspase-8
and precipitated by executioner caspases, mainly caspase-3).
Naphthalene-based chalcones, in which one of the aryl rings is re-
placed by naphthalene, have been explored as potential anticancer
agents. The aim of this study was to synthesize a series of chalcone
derivatives and their corresponding cyclic compounds containing a
naphthalene ring to explore the effect of different substituents (methyl,
methoxy, benzyloxy, chlorine) or changes in the position of the
methoxy or benzyloxy groups on the phenyl ring on cytotoxicity against
the human tumour U-937 cells. This cell line was selected as it is the
most frequently used cell line in biomedical research for the study of
neoplasia and therapeutics and has made important contributions to the
disciplines of cancer, hematology, and immunology [11,12]. In addi-
tion, we investigated the signal transduction pathways of cell death
triggered by one of the most cytotoxic compounds, the flavanone 6-
methoxy-2-(naphthalen-1-yl)chroman-4-one (FL).
2. Results
2.1. Chemistry
It is well established that mutual molecular recognition of host and
guest depends on non-covalent interactions (H-bonding, stacking in-
teractions, cation-π interactions, ionic interactions and hydrophobic
interactions) which are the basis of the functional properties of most
molecules. Inter and intramolecular interactions involving aromatic
ring systems are key processes in chemical and biological recognition
and knowledge of these helps explain and predict the functionality of
different structures.
In the present study, we explored the cytotoxic effects of a collection
of 19 chalcones and flavanones containing a naphthalene core against
human U-937 leukaemia cells. The chalcones (1–10) were prepared by
a standard aldolic condensation procedure involving pH > 12 condi-
tions. The aldolic condensation is one of the most versatile of organic
chemical reactions because of its ability to use small molecules in the
synthesis of larger ones under Claisen-Schmidt conditions. If this re-
action is performed at lower pH (9–10) a reversible interconversion
between the chalcone and its constitutional isomer flavanone is ob-
served depending on the range of pH and on the substitution on the A
ring [13]. The flavanones (11–19) were subsequently synthesized by
Interactions between aromatic platforms and sugar and aminoacid
residues play a crucial role in determining the specificity of drug-re-
ceptor interaction processes. The planarity of the aromatic systems to-
gether with their polarizability and their multipolar moment are
2

E. Saavedra, et al.
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Scheme 2. Acetophenone derivatives and 1-naphthaldehyde used in the synthesis of the chalcones (1–10) and flavanones (11–19).
cyclization reactions of the above-mentioned chalcones in the presence
of an excess of sodium acetate under reflux (Scheme 1). In the parti-
cular case of chalcone 10, it was not possible to isolate the desired
flavanone. Chalcones (1–10) and their corresponding flavanones were
designed to contain one or two substituents in the A ring (Scheme 2).
the methoxy or the benzyloxy group at the 6′ position. To this end, U-
937 cells were treated with increasing concentrations of each com-
pound and the IC50 values (the concentration that induces a 50% in-
hibition of cell growth) were determined by the MTT assay.
The results indicated that the introduction of a methoxy group at
position 5′ on the A ring (chalcone 3) or at position 6 in the A ring of the
corresponding flavanone (flavanone 13) led to a significant improve-
2.2. Biology
ment in cytotoxic activity, both in chalcones [(IC50 = 9.7
2.0 μM for
chalcone 1 vs IC50 = 2.8
0.2 μM for chalcone 3); 3.5-fold increase in
3.1 μM for flavanone 11
2.2.1. Screening of synthetic flavonoids. The synthetic 6-methoxy-2-
(naphthalen-1-yl)chroman-4-one (FL) inhibits the viability of human
leukaemia cells
cytotoxicity] and flavanones [(IC50 = 10.4
vs. IC50 = 1.3
0.2 μM for flavanone 13); 8-fold increase in cyto-
toxicity]. In contrast, the introduction of the methyl group or an atom
of chlorine in this position appears to be irrelevant in determining cy-
totoxicity against U-937 cells. The presence of an additional chlorine
atom at position 3′ did not change the cytotoxicity of the chalcone (8,
The SARs (structure-activity relationship) of naphthalene-based
chalcones and their corresponding flavanones were investigated by
varying the substituent on the A ring or the location of the methoxy
and/or benzyloxy groups at the 4′ or 5′ position on the A ring. In this
series of naphthalene-chalcones we maintained the naphthyl ring at-
tached to the β carbon of the 2-propen-1-one system and added various
substituents (hydrogen, methyl, chlorine, methoxy or benzylxoxy) at
the 5′ position on the phenyl ring. In addition, we included another
chlorine atom or another benzyloxy group and changed the position of
IC50 = 13.4
7.2 μM) or the flavanone (18, IC50 = 8.2
3.0 μM)
skeleton. Moreover, the introduction of a methoxy group at position 4′
of the chalcone skeleton (chalcone 4) did not enhance the potency
against cell growth inhibition compared with chalcone 1.
The introduction of one or two benzyloxy groups at the 4′ and 5′
3

E. Saavedra, et al.
BioorganicChemistryxxx(xxxx)xxxx
Scheme 2. (continued)
positions on the A ring of chalcones or at positions C6 or C7 and C8 of
flavanones significantly decreased cytotoxic activity (chalcones:
chalcone 3 and the flavanone 13 with IC50 values of 2.8
1.3 0.2 μM, respectively for U-937 cells (Table 1).
Since flavanone 13 [6-methoxy-2-(naphthalen-1-yl)chroman-4-one,
0.2 and
IC50 > 30 μM for 7 and 9 and IC50 = 20.3
9.0 μM for 6, flava-
nones: IC50 > 30 μM for 17, 16 and 19). The bulky groups on the A
ring appear to interfere with the cytotoxic activity and might be ex-
plained for their low solubility due to the presence of the aromatic rings
and exacerbated by the naphthyl group.
FL] was found to be one of the most cytotoxic compounds, it was se-
lected for further experiments. Comparison with additional human
leukaemia cells revealed that FL displays strong cytotoxic properties
against three human leukaemia cell lines, including the human acute
myeloid leukaemia HL-60, the human B cell precursor leukaemia
NALM-6 and the acute lymphoblastic leukaemia MOLT-3, with IC50
values between 1 and 3 μM (Table 2). For comparison, the standard
antitumor agent etoposide was included as a positive control for U-937
Since there are no large differences in cytotoxic activity between
chalcones and flavanones, these results suggest that the major de-
terminant of cytotoxicity is the presence and the position of a methoxy
group on the A ring, and that the most cytotoxic compounds were the
Table 1
Effects of flavonoids on the growth of human tumour cell line U-937.
Chalcone
IC50 (μM)
Flavanone
IC50 (μM)
1
2
3
4
5
6
7
8
9
10
9.7
2.0
8.3
2.2
2.8
0.2
8.5
0.9
5.0
7.5
5.7
1.6
2.1
20.3
9.0
> 30
17
13.4
8.2
7.2
3.0
> 30
19
12.6
3.9
11
12
13
14
15
16
18
10.4
3.1
22.6
3.0
1.3
0.2
15.3
> 30
> 30
> 30
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 3 independent experiments with three determinations in each.
4

E. Saavedra, et al.
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Table 2
with 10 μM FL during increasing periods of time. In addition, quiescent
and proliferating peripheral blood mononuclear cells isolated from
healthy volunteers were more resistant to the cytotoxic effects of FL
than human leukaemia U-937 cells (Fig. 1F). These results indicate that
FL exhibits strong cytotoxic properties against leukaemia cells but has
only weak cytotoxic effects against peripheral blood mononuclear cells
(PBMCs).
Effects of FL on cell viability of human leukaemia cell lines.
IC50 (µM)
HL-60
2.0
NALM-6
3.1 1.0
MOLT-3
1.1 0.2
1.5
Cells were cultured for 72 h and IC50 values were calculated as described in the
Experimental Section. Representable values are means SE from 3 to 5 in-
dependent experiments with determinations performed in triplicate.
2.2.2. FL induces G2-M arrest and apoptosis in human myeloid leukaemia
cells
(IC50 = 1.2
(IC50 = 0.2
0.2 μM), HL-60 (IC50 = 0.4
0.1 μM).
0.1 μM) and MOLT-3
To determine whether the effects of FL on cell viability were due to
alterations in cell cycle progression, cells were incubated with in-
creasing concentrations for different time periods (6–24 h), stained with
propidium iodide and analyzed by flow cytometry. As shown (Table 3,
Fig. 2A) an exposure at a concentration as low as 3 μM FL caused a
significant G2-M arrest at the expense of G1 phase cell population which
was evident after 6 h of treatment. The percentage of cells in G2-M
phase increased from ~21% in control cells to ~36% after treatment
with FL for 12 h. Moreover, the percentage of hypodiploid cells (i.e.
Treatment of the human histiocytic lymphoma U-937 cells with this
flavanone resulted in a concentration-dependent inhibition of cell via-
bility (Fig. 1A), induced significant morphological changes and caused
an important reduction in the number of cells (Fig. 1B). Studies using
the trypan blue exclusion method showed that FL displays cytotoxicity
but it is not cytostatic against U-937 cells. Fig. 1C–E show the number
of total cells, live cells and the percentage of live cells after treatment
A
B
100
75
50
25
0
3
10
0
1
0.1
0
1 10 100
FL (µM)
FL (µM)
C
D
E
20
15
10
5
20
15
10
5
Control
FL
Control
FL
Control
FL
125
100
*
75
50
25
0
*
*
24h
*
24h
*
72h
*
48h
*
72h
*
*
0
0
48h
72h
24h
48h
U-937
PBMC
PBMC + PHA
F
125
125
100
75
125
100
75
50
25
0
100
75
50
25
0
*
*
50
25
0
0
3
10 30
0
3
10 30
0
3
10 30
FL (µM)
FL (µM)
FL (µM)
Fig. 1. FL is cytotoxic against human leukaemia U-937 cells but not against peripheral blood mononuclear cells. (A) Dose-response study of FL on U-937 cells
viability. Cells were incubated with increasing concentrations of FL for 72 h, and thereafter cell viability was determined by the MTT assay. (B) Photomicrographs of
morphological changes visualized with inverted phase contrast microscopy after treatment with the indicated concentrations of FL for 24 h. (C–E) Time-course of FL-
mediated cell death. Cells were incubated with FL (10 μM) for the indicated times and the number of total cells (C), live cells (D) and the percentage of live cells were
determined by the trypan blue exclusion method using a TC10 counter (Bio-Rad, Hercules, CA). (F) Differential effects of FL on cell viability of U-937 and quiescent
and phytohemagglutinine (PHA)-activated healthy human peripheral blood mononuclear cells (PBMC). Cells were incubated in the presence of the specified con-
centrations of FL for 24 h and thereafter cell viability was determined by the MTT assay. Values represent means
SE of two independent experiments each
performed in triplicate. *P < 0.05, significantly different from the untreated control.
5

E. Saavedra, et al.
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Table 3
with respect to control) were observed at 24 h with 10 μM FL in U-937
(Table 3, Fig. 2B). FL treatment also led to the exposure of phosphati-
dylserine on the outside of the plasma membrane as detected by An-
nexin V-FITC staining in U-937 cells (Fig. 2C). In addition, fluorescence
microscopy experiments clearly demonstrate increases in condensed
and fragmented chromatin, which is typical of apoptotic cells in U-937
(Fig. 2D). Taken together, these results indicate that FL induces cell
cycle arrest in the G2-M phase and apoptosis on human myeloid leu-
kaemia U-937 cells.
Effect of FL on cell cycle phase distribution of U-937 leukaemia cells.
(μM)
% Sub-G1
%G1
%S
%G2-M
6 h
0
1.4
2.9
4.2
3.3
0.2
52.5
45.6
29.0
19.8
2.2
24.4
26.7
33.4
34.5
1.0
21.7
24.7
33.3
42.3
3.3
1
0.2
0.4
0.3
1.0
0.4
1.1
3
1.1*
0.7*
0.4*
1.2*
1.4*
2.0*
10
12 h
24 h
0
0.9
1.5
8.0
7.4
0.1
51.5
50.6
29.0
7.3
0.8
26.5
24.7
27.4
31.7
0.6
0.1
3.4
1.2*
21.0
23.1
35.6
53.6
0.9
1
0.2
0.4
0.4
3
1.8*
0.5*
1.5*
3.7*
2.1*
10
0.5*
2.2.3. Effects of FL on caspases- and PARP cleavage
To determine whether caspases were associated with the cell death
triggered by FL, we examined the proteolytic processing of these pro-
teases and PARP [poly(ADP-ribose) polymerase] cleavage. To this end,
U-937 cells were treated with increasing concentrations of FL for 24 h,
and caspases were determined by western blot (Fig. 3A). The results
indicate that FL stimulates the cleavage of the main effector pro-caspase
(pro-caspase-3) into 18–20 kDa fragments as well as the other execu-
tioner pro-caspases (pro-caspases-6 and -7). These effector caspases
perform downstream execution of apoptosis. Interestingly, FL stimu-
lated pro-caspase-4 processing as determined by a significant decrease
in the zymogen levels. Processing of the main initiator caspases (pro-
caspases-8 and -9) - involved in the extrinsic and the intrinsic apoptotic
pathways - was detected by the visualization of the active 35–37 kDa
fragment (pro-caspase-9) and a decrease of pro-caspase-8 levels. This
flavanone also induced poly(ADP-ribose) polymerase (PARP) cleavage
in accordance with caspase activation, generating the 85 kDa fragment.
Protein loading was checked by reprobing the membranes with β-actin
antibody.
0
1.4
0.1
55.6
51.0
17.0
17.3
0.2
27.6
29.4
45.8
24.5
0.9
0.1
2.6*
14
15.8
17.0
11.1
18.6
0.9
1
2.6
0.3
0.6
0.4*
1.9*
4.9
3
25.9
39.2
3.4*
0.6*
0.6*
10
9.2*
Cells were cultured with the four concentrations of FL (0, 1, 3, 10 μM) over
three time periods (6, 12, 24 h) and the percentages of cells in the four phases of
the cell cycle (%Sub-G1, %G1, %S, %G2-M) were determined by flow cytometry.
The values are means
S.E. of two independent experiments with three de-
terminations in each. Asterisks indicate a significant difference (P
< 0.05)
compared with the corresponding controls.
A
Control
6h
12h
24h
1.4%
4.2%
8.0%
25.9%
To confirm that caspases processing correlates with activity, enzy-
matic activities of caspase-3-like protease (caspase-3/7), caspase-8 and
caspase-9 were also investigated in extracts of U-937 cells treated with
increasing concentrations of FL during 24 h. To this end, cell lysates
were assayed for cleavage of the tetrapeptide substrates DEVD-pNA,
IETD-pNA and LEHD-pNA as specific substrates for caspase-3/7, -8 and
-9, respectively. As shown (Fig. 3B), dose-response experiments showed
that a low concentration (1 μM) of FL was sufficient to induce caspases
activation. A 2-fold increase in caspases-8 and -9 activities was ob-
served in cells treated with 3 μM FL.
Propidium iodide fluorescence (→)
B
C
50
0%
0.9% 0.2%
1.0%
*
40
30
20
10
*
95.1%
3.9% 66.7%
32.1%
0
Confirmation that FL-triggered apoptosis requires the activation of
caspases was carried out with cells pre-treated with the broad-spectrum
caspase inhibitor z-VAD-fmk (100 μM). The results (Fig. 3C) showed
that apoptosis was significantly suppressed in the presence of the in-
hibitor, which suggests that FL induces cytotoxicity by a caspase de-
pendent mechanism. To determine which caspases are important in the
mechanism of cell death triggered by this flavanone, we determined the
effect of two sets of caspase inhibitors, including eCHO and –fmk de-
rivatives. The eCHO derivatives included DEVD-CHO, LEVD-CHO,
IETD-CHO and LEHD-CHO (all at 10 μM, except z-LEHD-CHO which
was assayed at 50 μM, results not shown) and the –fmk derivatives
included DEVD-fmk, IETD-fmk and LEHD-fmk (all at 50 μM, Fig. 3C).
None of these inhibitors was able to decrease the percentage of hypo-
diploid cells induced by the flavanone. Fig. 3D shows the representative
histograms of annexin V-FITC and propidium iodide-stained U-937 cells
to confirm the effect of the pan-caspase inhibitor z-VAD-fmk. The lack
of effect of the specific caspase inhibitors (z-DEVD-fmk, caspase-3/7
inhibitor; z-LEVD-CHO, caspase-4 inhibitor; z-IETD-fmk, caspase-8 in-
hibitor and z-LEHD-fmk, caspase-9 inhibitor) was also confirmed by
flow cytometry after double staining with annexin V-FITC and propi-
dium iodide (results not shown).
0
1
3 10
Annexin V-FITC →
FL (µM)
D
0
1
3
10
FL (µM)
Fig. 2. Effect of FL on apoptosis on human myeloid leukaemia U-937 cells. (A)
Cells were incubated in the presence/absence of 3 μM FL for the indicated times
and subjected to flow cytometric analysis using propidium iodide labeling. The
percentages of hypodiploid cells (apoptotic cells) are shown. (B) U-937 cells
were incubated in the presence of the indicated concentrations of FL for 24 h
and percentages of hypodiploid cells were quantified by flow cytometry. (C)
Cells were treated with 10 μM FL for 24 h and subjected to flow cytometry
analysis after annexin V-FITC and propidium iodide staining. (D)
Photomicrographs of representative fields of U-937 cells stained with Hoechst
33,258 to evaluate nuclear chromatin condensation after treatment with the
specified concentrations of FL for 24 h.
2.2.4. Effects of FL on mitochondrial proteins release and on Bcl-2 family
members
sub-G1 fraction) increased about 18-fold in FL-treated U-937 compared
with control cells after 24 h (Fig. 2A).
Release of cytochrome c from mitochondria to cytosol is a central
event in apoptotic signalling. To determine whether FL-induced apop-
tosis involves release of mitochondrial proteins, cytosolic preparations
Maximal levels of apoptotic cells (approximately 30-fold increase
6

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kDa
A
B
Caspase-3/7 Caspase-8
Caspase-9
D
8
7
6
5
4
3
2
1
0
Pro-caspase-3
36
2.2%
1.6%
*
*
18-20
35
Pro-caspase-7
Pro-caspase-6
Pro-caspase-4
Control
19
34
22
*
*
*
*
*
*
*
94.5%
0.4%
1.5%
5.6%
0 1 3 10 0 1 3 10 0 1 3 10
FL (µM)
43
FL
47
35-37
C
Pro-caspase-9
60
50
40
30
63.7%
0.4%
30.1%
5.0%
*
55-57
Pro-caspase-8
PARP
116
85
#
FL
+
20
10
0
zVAD
β-Actin
42
88.3%
6.1%
0
1
3
10
Annexin V-FITC →
FL (µM)
+ FL
Fig. 3. Involvement of caspases in the mechanism of cell death triggered by FL in U-937 cells. (A) Immunoblotting for the cleavage of caspases and PARP after 24 h of
treatment with the specified concentrations of FL. The protein β-actin was used as a loading control. (B) Activation of caspases in response to FL. Cells were incubated
as above and cell lysates were assayed for caspase-3/7, caspase-8 and caspase-9 activities using the colorimetric substrates DEVD-pNA, IETD-pNA and LEHD-pNA,
respectively. Results are expressed as the fold-increase in caspase activity compared with control. (C) Effect of cell-permeable caspase inhibitors on FL-induced
apoptosis. Cells were incubated with 10 μM FL for 24 h, in presence or absence of the broad-spectrum caspase inhibitor z-VAD-fmk (z-VAD, 100 μM), the caspase-3/7
inhibitor z-DEVD-fmk (iC3, 50 μM), the caspase-4 inhibitor z-LEVD-CHO (iC4, 50 μM), the caspase-8 inhibitor z-IETD-fmk (iC8, 50 μM) and the caspase-9 inhibitor z-
LEHD-fmk (iC9, 50 μM) and percentages of hypodiploid cells were quantified by flow cytometry. Bars represent the mean
SE of two independent experiments
#
each performed in triplicate. *P < 0.05, significantly different from untreated control. P < 0.05, significantly different from FL treatment alone. (D) Flow
cytometry analysis of Annexin V-FITC and propidium iodide-stained cells after 24 h of treatment with 10 μM FL in absence or in presence of the pan-caspase inhibitor
z-VAD-fmk (100 μM). Representative data from two independent experiments are presented.
A
B
kDa
22
kDa
57
C
U-937
U-937/Bcl-2
FL
(µM)
0.0%
1.1% 0.1%
2.2%
Bax
DR4
0
Bcl-2
57
57
42
26
DR5
93.1%
0.0%
5.8% 93.0%
5.3% 0.2%
4.7%
2.5%
TRAIL
22
15
22
Bid
Cyt c
Smac
3
β-Actin
74.0%
0.4%
21.1% 93.2%
4.6% 0.1%
4.2%
2.1%
0
1
3
10
FL (µM)
10
β-Actin
42
53.3%
42% 91.5%
6.3%
0
1
3
10
Annexin V-FITC →
FL (µM)
Fig. 4. Effect on Bcl-2 family proteins, release of mitochondrial proteins and expression of death receptors. (A) Cells were incubated with increasing concentrations of
FL and the expression of Bcl-2 family members and release of cytochrome c and Smac/DIABLO were analyzed by western blot in U-937 cells. (B) Cells were treated as
in (A) and expression of death receptors was detected by immunoblotting. β-Actin was used as a loading control. (C) U-937 and the cell line overexpressing Bcl-2, U-
937/Bcl-2, were incubated with the specified concentrations of FL and apoptosis was determined by flow cytometry after double staining with annexin V-FITC and
propidium iodide. The experiment was replicated twice.
were analyzed by immunoblotting of U-937 cells. The results showed a
significant increase in the amount of cytochrome c in the cytosol in a
concentration-dependent manner (Fig. 4A). There was also a con-
centration-dependent release of the mitochondrial apoptogenic factor
Smac/DIABLO (second mitochondrial activator of caspases/Diablo IAP-
binding mitochondrial protein).
We also investigated the expression of the Bcl-2 family proteins in
FL-treated cells. The results revealed that flavanone induces
7

E. Saavedra, et al.
BioorganicChemistryxxx(xxxx)xxxx
downregulation of Bcl-2 and a clear increase in Bax levels (Fig. 4A). In
addition, there was a clear decrease in Bid protein, which is a substrate
of caspase-8, indicating processing of this protein.
These results suggest that activation of ERK1/2 and JNK/SAPK is in-
volved in FL–induced apoptosis.
Activation of the MAPK pathway can be mediated by the generation
of reactive oxygen species (ROS). Hence the ability of FL to increase
ROS levels was investigated using the fluorescent dye 2′, 7′-di-
chlorodihydrofluorescein diacetate after 1, 2 and 3 h of incubation, as
well as the effect of different antioxidants [20 μM vitamin E, 2 mM
trolox, 10 mM N-acetyl-L-cysteine, 0.5–1 μM diphenyleneiodonium
chloride (a NADPH oxidase inhibitor); 500 units/ml catalase; 400
units/ml superoxide dismutase]. However, ROS were not generated at
assayed times and the antioxidants did not reduce the cell death, either
(results not shown). These results suggest that cell death is not de-
pendent on ROS generation.
Since FL induces activation of caspase-8, it is possible that death
receptors and/or their ligand might be involved in cell death.
Therefore, we explored the effects of FL on the expression of DR4 (death
receptor 4), DR5 (death receptor 5) and TRAIL (tumor necrosis factor-
related apoptosis-inducing ligand) in U-937 cells. Cells were treated
during 24 h and cell lysates were subjected to immunoblot analysis. As
shown in Fig. 4B, FL up-regulates the expression of DR4, DR5 and
TRAIL.
To clarify whether the anti-apoptotic protein Bcl-2 is involved in the
activation of intrinsic pathway by FL treatment, we compared the effect
on apoptosis in the U-937 cell line overexpressing human Bcl-2 protein
(U-937/Bcl-2) and the parental U-937 cell line. As shown in Fig. 4C,
Bcl-2 over-expression blocked apoptosis triggered by FL, suggesting
that Bcl-2 is involved in FL-induced apoptosis.
3. Discussion
Despite the potential of flavonoids in human medicine its clinical
use is limited due to challenges associated with their effective use, in-
cluding, among others, their pharmacokinetic and pharmacodynamic
properties. Low solubility, poor oral absorption and extensive hepatic
metabolism are responsible for the unfavorable pharmacokinetics of
flavonoids. The modification of the flavonoid structure to generate new
derivatives constitutes an important strategy to optimize their phar-
macokinetic and pharmacodynamic properties. Previous studies have
shown that methylation and/or the introduction of some lipophilic
moieties in specific flavonoids significantly increase the potency and
bioavailability and also enhance the stability and effectiveness by
preventing chemical and metabolic hydrolysis [14–16].
2.2.5. FL activates MAPKs (mitogen-activated protein kinases) and cell
death is independent on ROS (reactive oxygen species) generation
Whether FL induces the activation of the MAPK pathway or not was
investigated because this signal transduction pathway plays a crucial
role in cell fate. Incubation of U-937 cells with FL leads to a fast
phosphorylation (0.5 h) of JNK/SAPK (c-jun N-terminal kinases/stress-
activated protein kinases) and p38 MAPK while the activation of ERK1/2
(extracellular signal-regulated kinase) was not detected until 6 h
(Fig. 5A). The activation of p38 MAPK decreased after 1 h, while the level
of phosphorylated JNK/SAPK occurs in a biphasic manner. These re-
sults indicate that FL treatment of U-937 cells leads to activation of
JNK/SAPK, p38MAPK and ERK1/2 following different kinetics. To de-
termine whether the phosphorylation of MAPKs plays a key role in FL-
induced apoptosis, we examined the effects of p38MAPK, JNK/SAPK, and
mitogen-activated extracellular kinases 1/2 (MEK 1/2) inhibitors
(Fig. 5B). Pretreatment of U-937 cells with the specific p38MAPK in-
hibitor SB 203,580 (2 μM) did not affect the rate of FL-mediated
apoptosis. These results suggest that activation of p38MAPK is not in-
volved in FL-induced cell death. Treatment of cells with U0126 or
PD98059, inhibitors of MEK1/2, and with the JNK/SAPK inhibitor
SP600125 significantly decreased FL-induced cell death. The percen-
tage of apoptotic cells (hypodiploid cells) decreased from ~37% in FL-
treated cells to 17% and 22% with U0126 and PD98059, respectively.
The JNK/SAPK inhibitor attenuated FL-induced cell death from ~37%
of apoptotic cells to 22% in the combination group (FL + SP600125).
In the present report, we attempted to find new potent anti-
proliferative molecules inspired by natural products. The results re-
vealed that both the chalcone containing the methoxy group at 5′ po-
sition of the A ring as well as its corresponding flavanone were the most
cytotoxic compounds, with IC50 values of 2.8
0.2 and
1.3
0.2 μM, respectively, against U-937 cells. This is the first time to
date that the effects on cell viability and the mechanism involved in cell
death induction have been investigated for this specific flavanone.
In a previous study, we showed that the synthetic chalcone 6′-
benzyloxy-4-bromo-2′-hydroxychalcone is cytotoxic against seven
human leukaemia cells and induces caspase-8- and reactive oxygen
species-dependent apoptosis [17]. These results prompted us to design
additional analogues of chalcone containing additional substituents
other than halogen chalcones. When the 4-bromobenzyl group was
replaced by a naphthyl group, the chalcone was unexpectedly cyclized
Fig. 5. Role of the MAPK pathway on FL-in-
duced apoptosis in U-937 cells. (A)
Representative Western blots show the time-de-
pendent phosphorylation of p38 MAPK, ERK1/2
A
B
kDa
38
P-p38
p38
50
40
30
20
10
0
*
and JNK/SAPK by FL. Cells were incubated with
38
10 μM FL for the time periods shown. Protein
extracts were prepared and analysed on Western
#
#
42/44
42/44
blots probed with specific antibodies to ascertain
the phosphorylation of MAPKs. Membranes were
stripped and reprobed with total MAPK anti-
bodies as loading controls. (B) Effect of MAPKs
inhibitors on FL-induced cell death. Cells were
preincubated with the MEK1/2 inhibitors U0126
(10 μM) and PD98059 (10 μM), the JNK/SAPK
P-ERK 1/2
ERK 1/2
#
46/54
46/54
P-JNK/SAPK
JNK/SAPK
MAPK
inhibitor SP600125 (10 μM) and the p38
inhibitor SB203580 (2 μM) for 1 h and then
treated with FL. Apoptosis was quantified by
flow cytometry as described in Materials and
0
0.5
1
2
4
6
+ FL
Time (h)
Methods. Bars represent the mean
S.E. of
three independent experiments each performed
in triplicate. *P < 0.05, significantly different
from untreated control. #P < 0.05, significantly
different from FL treatment alone.
8

E. Saavedra, et al.
BioorganicChemistryxxx(xxxx)xxxx
to generate a flavanone (FL). This cyclic compound was found to have
strong cytotoxic properties with IC50 values of approximately 2 μM
against the four human leukaemia cell lines tested, including U-937,
HL-60, NALM-6 and MOLT-3. Dose-response experiments revealed that
U-937 cells were more sensitive to FL than quiescent and proliferating
PBMC, as determined by the MTT assay. This flavanone is able to in-
duce a fast cell cycle arrest at the G2-M phase, starting at 6 h, and a
delayed (later) increase in the percentage of hypodiploid cells. The
arrest of cells in the G2-M phase of the cell cycle by FL could be ex-
plained by microtubule formation or by changes in the expression and/
or activity of G2-M cell cycle regulators. Previous studies have shown
that some flavonoids exert their anti-proliferative activity by targeting
microtubules through tubulin binding [18] and we have reported that
some naturally occurring and synthetic cytotoxic flavonoids block tu-
bulin polymerization [19–21]. Further studies are needed to determine
the effect of FL on tubulin polymerization and on G2-M cell cycle reg-
ulators such as the cyclin-dependent kinases (Cdk) inhibitor p21Cip1/
WAF1, Cdk1, Cdc25C phosphatase and B-type cyclin isoforms.
Although further experiments are needed to determine whether FL is
able to amplify the sensitivity to TRAIL these results are of potential
importance because they demonstrate that FL is a death receptor in-
ducer.
The mitogen activated protein kinase (MAPK) signalling pathway
regulates the expression of a large number of proteins involved in the
control of cell proliferation, differentiation and apoptosis. Activation of
the MAPK signalling pathway is known to cause the transformation of
normal cells to tumor phenotype [29,30]. Therefore, targeting any
component in the MAPK signalling pathway has the potential to arrest
tumor growth. Here we found that FL effectively activates the MAPK
pathway and interestingly the activation of ERK1/2 and JNK/SAPK is
involved in FL–induced apoptosis, since the MEK1/2 and JNK/SAPK
inhibitors were able to block partially cell death. These results indicate
a key role for the MAPK signalling cascade in the mechanism of cyto-
toxicity triggered by this flavanone. We have previously shown that
betuletol 3-methyl ether, a naturally occurring flavonoid, induces the
activation of mitogen-activated protein kinases. However, FL is a
greater apoptotic inducer than betuletol 3-methyl ether and induces a
fast phosphorylation of JNK/SAPK [31]. In addition, we have described
that a flavonoid derivative, trifolin acetate, induces cell death in human
leukaemia cells and that this was attenuated by MEK1/2 inhibition.
However, this was different to the flavanone described here: the JNK/
SAPK inhibitor SP600125 amplified cell death [32]. These results reveal
that these flavonoid derivatives differentially modulate the MAPK
pathways and could be used to enhance the cell death triggered by
these specific compounds. An amplification of the cleavage of caspase-3
and PARP by JNK/SAPK inhibition has been also described in the me-
chanism of cell death triggered by the prenylated flavonoid daphne-
giravone D in human hepatocellular carcinoma cells [33].
Our results indicate that FL induces cell death by apoptosis, a pro-
cess of programmed cell death that can occur with or without activation
of caspases, but in this case cell death was dependent on caspases since
the pan-caspase inhibitor z-VAD-fmk was able to significantly reduce
both the percentage of hypodiploid cells and the percentage of annexin
V-FITC positive cells. Our results showed that the flavanone induces the
activation and processing of multiple caspases in U-937 cells. Caspase-6
which is believed to be an effector caspase that is activated downstream
of caspase-3 and -7 in apoptosome-mediated apoptosis [22] and the
effector caspase-7 which is substrate for initiator caspases in extrinsic or
intrinsic apoptotic pathways were also processed. However, the specific
caspase inhibitors against caspase-3/7, -4, -8 and -9 were unable to
block the cell death. This raises the possibility that other specific cas-
pases such as caspase-6 and/or additional pathways may play a key role
in the mechanism of cell death. The potential involvement of caspase-6
is supported by recent data which indicate that caspase-6 may be in-
volved in regulated cell death initiation [23–25].
Future studies addressing the bioavailability – including the devel-
opment of micro- and nanodelivery systems – as well as efficacy of in
vivo concentrations of this specific compound are necessary to de-
termine its potential beneficial effects for human health.
The synthetic flavanone induced the release of mitochondrial
apoptogenic proteins including cytochrome
c
and Smac/DIABLO.
4. Conclusions
Cytochrome c in the cytosol binds to apoptotic peptidase activating
factor 1 (Apaf-1) and pro-caspase-9 to form the apoptosome which is
responsible for caspase-9 activation [26]. Smac/DIABLO in the cytosol
precipitates apoptosis by binding with members of the inhibitor of
apoptosis (IAP) protein family [27]. Although the specific caspase-9
inhibitor was unable to significantly block the cell death triggered by
FL, the results seem to indicate that the intrinsic pathway, which is
activated in response to cellular stress and is controlled by the Bcl-2
protein family, plays a key role. Treatment with the flavanone induced
a strong downregulation of Bcl-2 protein in U-937. In addition, over-
expression of Bcl-2 was able to block the cell death triggered by FL
suggesting that Bcl-2 itself is a potential target in the mechanism of cell
death. Furthermore, the Bax protein that promotes apoptosis was in-
creased in U-937 cells, demonstrating that the ratio between proa-
poptotic and anti-apoptotic Bcl-2 members plays a major role in de-
termining susceptibility of cells to apoptotic stimuli, as suggested by
previous studies [28].
The SAR of naphthalene-based chalcones and their corresponding
flavanones are displayed graphically in Fig. 6. The most relevant results
are (i) some chalcones are more potent than their corresponding fla-
vanones, while other chalcones show similar potency to flavanones,
indicating that flexible structures are important in determining cyto-
toxicity; (ii) the introduction of a methoxy group at a specific position
(position 5′ on the A ring in chalcone or at position 6 on the A ring of
the corresponding flavanone) improved the cytotoxicity against U-937
cells and (iii) the introduction of a methyl group or an atom of chlorine
appears to be irrelevant, while the presence of one or two benzyloxy
groups on the A ring completely abrogates cytotoxicity, except for the
6′-benzyloxy derivative in the chalcone skeleton that showed a similar
potency to the parent chalcone.
We show that the synthetic flavanone FL is cytotoxic against four
human leukaemia cells but not against human peripheral blood
mononuclear cells. FL-induced cytotoxicity (i) involves the activation
and processing of the initiator (caspase-8 and -9) and the executioner
caspases (caspases-3, -6 and -7); (ii) is attenuated by the pan-caspase
inhibitor z-VAD-fmk; (iii) is associated with the phosphorylation of the
We found that FL also induces activation of caspase-8 which is in-
volved in death receptor-mediated apoptosis. Caspase-8 cleaves Bid to a
truncated form (tBid), which engages the mitochondrial pathway to
amplify the apoptotic response. We did not observe a truncated form of
Bid but the Western blot experiments revealed a clear decrease in Bid
levels mainly at 10 μM FL in accordance with caspase-8 processing and
activation. Since caspase-8 activation is mediated by ligand-binding
and activation of the death-domain-containing tumor necrosis receptor
superfamily we explored the involvement of death receptors. FL was
found to effectively stimulate the expression of TRAIL, DR4 and DR5.
mitogen-activated protein kinases ERK1/2, JNK/SAPK and p38MAPK
;
and (iv) is reduced by the inhibition of MEK1/2 and JNK/SAPK. The
IC50 values in human leukaemia cells were comparable with those ob-
tained with the antitumor compound etoposide. The studies shown here
open new avenues of research into the potential of this compound or
derivatives in the development of new therapeutic strategies against
leukaemia cells.
9

E. Saavedra, et al.
BioorganicChemistryxxx(xxxx)xxxx
Fig. 6. Structure-activity relationship of naphthyl-chalcone/flavanone analogues.
5. Experimental
CA, USA). All other chemicals were obtained from Sigma (Saint Louis,
MO, USA).
5.1. General method and reagents
5.2. General procedure for the synthesis of chalcones (1–10)
1H and 13C NMR spectra were obtained on a Bruker model AMX-500
spectrometer with standard pulse sequences operating at 500 MHz in
1H and 126 MHz in 13C NMR. Chemical shifts (δ) are given in ppm
upfield from tetramethylsilane as internal standard, and coupling con-
stants (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
recorded on a Micromass model Autospec (70 eV) spectrometer.
Column chromatography was carried out on silica gel 60 (Merck
230–400 mesh) and analytical thin layer chromatography (TLC) was
performed using silica gel aluminum sheets. HPLC analyses were per-
formed on a Waters 600 Controller using a Chiralcel OD-H column
(iPrOH:hexane) and a Silica gel 100 Å LC column (250 × 4.6 mm –
Hexane:ethyl acetate). Elemental analyses were performed on a Leco
TruSpec Micro equipment. The purity of compounds used in biological
testing was > 98%, as determined by elemental analysis or high-per-
formance liquid chromatography. The inhibitors benzyloxycarbonyl-
Val-Ala-Asp(OMe) fluoromethyl ketone (z-VAD-fmk), benzylox-
ycarbonyl-Asp(OMe)-Glu(O-Me)-Val-Asp(O-Me) fluoromethyl ketone
(z-DEVD-fmk), benzyloxycarbonyl-Ile-Glu-Thr-Asp(OMe) fluoromethyl
ketone (z-IETD-fmk), benzyloxycarbonyl-Leu-Glu-His-Asp(OMe) fluor-
omethyl ketone (z-LEHD-fmk), Ac-LEVD-CHO (N-acetyl-Leu-Glu-Val-
Asp-CHO), PD98059, U0126, SP600125 and SB203580 were purchased
from Sigma (Saint Louis, MO, USA). Acrylamide, bisacrylamide, am-
monium persulfate and N,N,N′,N′-tetramethylethylenediamine were
from Bio-Rad (Hercules, CA, USA). Antibodies for caspase-3, caspase-7,
caspase-8 and caspase-9 were purchased from Stressgen-ENZO
(Victoria, British Columbia, Canada). Anti-caspase-6 and anti-caspase-4
monoclonal antibodies were from Medical & Biological Laboratories
(Nagoya, Japan). Monoclonal anti-β-Actin (clone AC-74) was purchased
from Sigma (Saint Louis, MO, USA). Monoclonal anti-human Bcl-2 was
from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Polyclonal anti-
human Bax and Bid antibodies and monoclonal anti-cytochrome c and
poly(ADP-ribose) polymerase (PARP) antibodies were from BD
Pharmingen (San Diego, CA, USA). Monoclonal antibody for Smac/
DIABLO was from BD Transduction Laboratories. Anti-JNK/SAPK, anti-
phospho-JNK/SAPK (T183/Y185), anti-p44/42 MAP kinase (ERK1/2),
anti-phospho-p44/42 MAP kinase (T202/Y204), anti-p38MAPK and a
phosphorylated form (T180/Y182) of p38MAPK were purchased from
New England BioLabs (Cell Signaling Technology, Inc, Beverly, MA,
USA). Secondary antibodies were from GE Healthcare Bio-Sciences AB
(Little Chalfont, UK). PVDF membranes were from Millipore (Temecula,
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 temperature until the
starting materials had been consumed. HCl (10%) was then added until
neutrality was reached. Precipitated chalcones were generally filtered
and crystallized from MeOH although in some cases, the product was
purified using column chromatography.
5.2.1. (E)-1-(2-hydroxyphenyl)-3-(naphthalen-1-yl)prop-2-en-1-one (1)
Yellow amorphous solid (65%). IR (cm−1): 1694, 1640, 1607, 1581,
1566, 1491, 1472, 1462, 1439, 1365, 1342, 1304, 1278, 1209, 1159,
1072, 1024, 977, 901. 1H NMR (500 MHz, CDCl3): δ = 12.86 (s, 1H);
8.79 (d, J = 15.2 Hz, 1H); 8.29 (dd, J = 8.5, 1.0 Hz, 1H); 8.01–7.89
(m, 4H); 7.76 (d, J = 15.3 Hz, 1H); 7.62 (ddd, J = 8.4, 6.8, 1.5 Hz,
1H); 7.60–7.47 (m, 3H); 7.06 (dd, J = 8.4, 1.1 Hz, 1H); 6.97 (ddd,
J = 8.2, 7.2, 1.2 Hz, 1H). 13C NMR (126 MHz, CDCl3): δ = 193.6,
163.7, 142.4, 136.5, 133.8, 132.1, 131.8, 131.2, 129.7, 128.8, 127.2,
126.4, 125.4, 125.3, 123.4, 122.8, 120.0, 118.9, 118.7. HRMS (ESI-FT-
ICR) m/z: 297.0895 [M+Na]; calcd. for C19H14NaO2: 297.0891.
5.2.2. (E)-1-(2-hydroxy-5-methoxyphenyl)-3-(naphthalen-1-yl)prop-2-en-
1-one (2)
Orange crystalline solid (82%), mp 112–113 °C. IR (cm−1): 1635,
1566, 1479, 1396, 1337, 1323, 1289, 1273, 1249, 1207, 1176, 1162,
1020, 1001, 979, 849. 1H NMR (500 MHz, CDCl3): δ = 12.68 (s, 1H);
8.77 (d, J = 15.2 Hz, 1H); 8.32–8.24 (m, 1H); 7.95 (t, J = 7.7 Hz, 2H);
7.93–7.86 (m, 1H); 7.75 (d, J = 15.3 Hz, 1H); 7.73 (s, 1H); 7.62 (ddd,
J = 8.4, 6.8, 1.5 Hz, 1H); 7.59–7.50 (m, 2H); 7.34 (dd, J = 8.6, 2.2 Hz,
1H); 6.97 (d, J = 8.5 Hz, 1H); 2.36 (s, 3H). 13C NMR (126 MHz, CDCl3):
δ = 193.5, 161.6, 142.1, 137.6, 133.8, 132.2, 131.8, 131.1, 129.4,
128.8, 128.0, 127.1, 126.4, 125.4, 125.3, 123.4, 122.9, 119.7, 118.4,
20.6. HRMS (ESI-FT-ICR) m/z: 311.1043 [M+Na]; calcd. for
C20H16NaO2: 311.1048.
5.2.3. (E)-1-(2-hydroxy-5-methoxyphenyl)-3-(naphthalen-1-yl)prop-2-en-
1-one (3)
Orange crystalline solid (78%), mp 111–112 °C. IR (cm−1):1644,
1581, 1566, 1495, 1417, 1346, 1271, 1188, 1166, 1048, 1017, 975,
960, 844. 1H NMR (500 MHz, CDCl3): δ = 12.42 (s, 1H); 8.78 (d,
J = 15.2 Hz, 1H); 8.32–8.24 (m, 1H); 7.96 (d, J = 8.2 Hz, 1H);
10

E. Saavedra, et al.
BioorganicChemistryxxx(xxxx)xxxx
7.94–7.88 (m, 2H); 7.69 (d, J = 15.2 Hz, 1H); 7.62 (ddd, J = 8.5, 6.8,
1.4 Hz, 1H); 7.60–7.51 (m, 2H); 7.40 (d, J = 3.0 Hz, 1H); 7.17 (dd,
J = 9.1, 3.0 Hz, 1H); 7.01 (d, J = 9.1 Hz, 1H); 3.84 (s, 3H). 13C NMR
(126 MHz, CDCl3): δ = 193.2, 158.1, 151.8, 142.5, 133.8, 132.1,
131.8, 131.2, 128.8, 127.2, 126.4, 125.4, 125.3, 124.1, 123.4, 122.9,
119.6, 119.4, 112.8, 56.1. HRMS (ESI-FT-ICR) m/z: 327.0999 [M+Na];
calcd. for C20H16NaO3: 327.0997.
975, 968, 866, 844, 821. 1H NMR (500 MHz, CDCl3): δ = 13.41 (s, 1H);
8.87 (d, J = 15.1 Hz, 1H); 8.27 (dd, J = 8.5, 1.1 Hz, 1H); 8.03–7.94
(m, 2H); 7.92 (dd, J = 8.5, 1.2 Hz, 1H); 7.85 (d, J = 2.5 Hz, 1H);
7.69–7.60 (m, 3H); 7.60–7.52 (m, 2H). 13C NMR (126 MHz, CDCl3):
δ = 192.4, 158.1, 144.4, 135.8, 133.8, 132.0, 131.8, 131.4, 128.9,
127.5, 127.4, 126.5, 125.7, 125.4, 124.2, 123.3, 123.2, 121.3, 121.0.
HRMS (ESI-FT-ICR) m/z: 341.0136 [M−H]; calcd. for C1935Cl2H11O2:
341.0136; m/z: 343.0121 [M−H]; calcd. for C1935Cl37Cl H11O2:
343.0107.
5.2.4. (E)-1-(2-hydroxy-4-methoxyphenyl)-3-(naphthalen-1-yl)prop-2-en-
1-one (4)
Pale yellow crystalline solid (58%), mp 114–115 °C. IR (cm−1):
1583, 1573, 1500, 1356, 1249, 1204, 1171, 1121, 1088, 1020, 1005,
958, 849, 828. 1H NMR (500 MHz, CDCl3): δ = 13.48 (s, 1H); 8.74 (d,
J = 15.2 Hz, 1H); 8.28 (d, J = 8.5 Hz, 1H); 8.00–7.82 (m, 4H);
7.72–7.64 (m, 1H); 7.61 (ddt, J = 8.3, 6.9, 1.4 Hz, 1H); 7.58–7.46 (m,
5.2.9. (E)-1-(3,4-bis(benzyloxy)-2-hydroxyphenyl)-3-(naphthalen-1-yl)
prop-2-en-1-one (9)
Yellow amorphous solid (54%). IR (cm−1): 1635, 1564, 1500, 1453,
1431, 1342, 1306, 1294, 1259, 1228, 1128, 1091, 1060, 1017, 986,
972. 1H NMR (500 MHz, CDCl3): δ = 13.28 (s, 1H); 8.75 (d,
J = 15.2 Hz, 1H); 8.28 (d, J = 8.4 Hz, 1H); 7.94 (d, J = 8.2 Hz, 1H);
7.92–7.87 (m, 2H); 7.71–7.64 (m, 2H); 7.61 (ddd, J = 8.4, 6.8, 1.5 Hz,
1H); 7.58–7.49 (m, 4H); 7.42–7.28 (m, 8H); 6.55 (d, J = 9.1 Hz, 1H);
5.20 (s, 2H); 5.17 (s, 2H). 13C NMR (126 MHz, CDCl3): δ = 206.9,
192.3, 158.9, 158.0, 141.6, 137.6, 136.2, 133.7, 132.3, 131.8, 131.0,
128.8, 128.6, 128.6, 128.2, 128.1, 127.9, 127.2, 127.1, 126.4, 126.1,
125.4, 125.2, 123.5, 123.0, 115.7, 104.6, 74.8, 70.8. HRMS (ESI-FT-
ICR) m/z: 509.1727 [M+Na]; calcd. for C33H26NaO4: 509.1729.
2H); 6.50 (ddd, J = 4.6, 2.5, 1.3 Hz, 2H); 3.87 (d, J = 1.2 Hz, 3H). 13
C
NMR (126 MHz, CDCl3): δ = 191.7, 166.8, 166.3, 141.4, 133.7, 132.3,
131.8, 131.3, 130.9, 128.8, 127.0, 126.3, 125.4, 125.2, 123.5, 123.0,
114.1, 107.8, 101.1, 55.6. HRMS (ESI-FT-ICR) m/z: 327.1000 [M+Na];
calcd. for C20H16NaO3: 327.0997.
5.2.5. (E)-1-(5-chloro-2-hydroxyphenyl)-3-(naphthalen-1-yl)prop-2-en-1-
one (5)
Yellow crystalline solid (88%), mp 165–165 °C. IR (cm−1): 1642,
1556, 1462, 1403, 1365, 1358, 1332, 1327, 1275, 1242, 1207, 1190,
1024, 1013, 984, 826. 1H NMR (500 MHz, CDCl3): δ = 12.77 (s, 1H);
8.82 (d, J = 15.1 Hz, 1H); 8.28 (d, J = 8.9 Hz, 1H); 8.02–7.94 (m, 2H);
7.92 (dt, J = 3.4, 1.9 Hz, 2H); 7.72–7.60 (m, 2H); 7.60–7.50 (m, 2H);
7.46 (dd, J = 8.9, 2.5 Hz, 1H); 7.02 (d, J = 8.9 Hz, 1H). 13C NMR
(126 MHz, CDCl3): δ = 192.7, 162.2, 143.4, 136.3, 133.8, 131.8,
131.7, 131.6, 128.9, 128.9, 127.3, 126.5, 125.6, 125.4, 123.6, 123.3,
122.0, 120.6, 120.3. HRMS (ESI-FT-ICR) m/z: 307.0522 [M−H]; calcd.
for C1935ClH12O2: 307.0526; m/z: 309.0499 [M−H]; calcd. for
C1937ClH12O2: 309.0496.
5.2.10. (E)-1-(2-(benzyloxy)-6-hydroxyphenyl)-3-(naphthalen-1-yl)prop-
2-en-1-one (10)
Bright yellow crystalline solid (80%), mp 145–146 °C. IR (cm−1):
1630, 1555, 1460, 1446, 1353, 1233, 1209, 1169, 1065, 1024, 968,
918, 849. 1H NMR (500 MHz, CDCl3): δ = 13.41 (s, 1H); 8.62 (d,
J = 15.4 Hz, 1H); 8.25 (dd, J = 8.5, 1.2 Hz, 1H); 7.95 (d, J = 15.4 Hz,
1H); 7.88–7.80 (m, 2H); 7.58–7.49 (m, 2H); 7.49–7.44 (m, 2H); 7.40 (t,
J = 8.4 Hz, 1H); 7.38–7.34 (m, 1H); 7.32–7.27 (m, 2H); 7.21 (dd,
J = 8.2, 7.2 Hz, 1H); 7.08 (dt, J = 7.2, 0.9 Hz, 1H); 6.68 (dd, J = 8.4,
1.0 Hz, 1H); 6.56 (dd, J = 8.3, 1.0 Hz, 1H); 5.14 (s, 2H). 13C NMR
(126 MHz, CDCl3): δ = 194.5, 165.3, 160.3, 139.6, 136.0, 135.6,
133.6, 132.2, 131.8, 130.3, 130.0, 128.8, 128.6, 128.5, 128.5, 126.8,
126.1, 125.5, 125.0, 123.4, 112.0, 111.3, 102.4, 71.4. HRMS (ESI-FT-
ICR) m/z: 403.1302 [M+Na]; calcd. for C26H20NaO3: 403.1310.
5.2.6. (E)-1-(5-benzyloxy)-2-hydroxyphenyl)-3-(naphthalen-1-yl)prop-2-
en-1-one (6)
Orange crystalline solid (76%), mp 140–141 °C. IR (cm−1): 1637,
1566, 1486, 1415, 1358, 1339, 1327, 1282, 1235, 1214, 1192, 1176,
1013, 965, 849, 837. 1H NMR (500 MHz, CDCl3): δ = 12.41 (s, 1H);
8.76 (d, J = 15.2 Hz, 1H); 8.27 (d, J = 8.4 Hz, 1H); 7.97 (d,
J = 8.2 Hz, 1H); 7.90 (dd, J = 13.9, 7.6 Hz, 2H); 7.67–7.51 (m, 4H);
7.51–7.30 (m, 6H); 7.23 (dd, J = 9.1, 2.9 Hz, 1H); 7.00 (d, J = 9.0 Hz,
1H); 5.08 (s, 2H). 13C NMR (126 MHz, CDCl3): δ = 193.2, 158.2, 150.9,
142.4, 136.8, 133.8, 132.1, 131.8, 131.3, 128.8, 128.7, 128.2, 127.6,
127.2, 126.4, 125.4, 125.3, 125.0, 123.4, 122.8, 119.6, 119.4, 114.6,
71.3. HRMS (ESI-FT-ICR) m/z: 403.1314 [M+Na]; calcd. for
5.3. General procedure for the synthesis of flavanones (11–19)
To a solution of a chalcone (0.12–0.2 mmol) in ethanol (1–2 mL),
sodium acetate (10 equiv.) was added. The reaction was heated in re-
fluxing for 24–48 h and then was allowed to cool to room temperature,
poured into ice water (10 mL) and extracted with CH2Cl2 (3 × 5 mL).
The combined organic phase was washed with brine, dried over
Na2SO4, and then concentrated in vacuo. The flavanones were purified
by crystallization (MeOH) or column chromatography.
C
26H20NaO3: 403.1310.
5.2.7. (E)-1-(4-benzyloxy)-2-hydroxyphenyl)-3-(naphthalen-1-yl)prop-2-
en-1-one (7)
5.3.1. 2-(naphthalen-1-yl)chroman-4-one (11)
Bright yellow crystalline solid (80%), mp 74–75 °C. IR (cm−1):
1678, 1607, 1581, 1474, 1465, 1410, 1339, 1304, 1226, 1147, 1117,
1067, 1027, 984, 918, 901, 856, 840. 1H NMR (500 MHz, CDCl3):
δ = 8.11–8.03 (m, 1H); 8.01 (dd, J = 8.0, 1.8 Hz, 1H); 7.97–7.86 (m,
2H); 7.79 (d, J = 7.1 Hz, 1H); 7.56 (m, 4H); 7.16–7.05 (m, 2H); 6.24
(dd, J = 13.4, 2.8 Hz, 1H); 3.28 (dd, J = 17.0, 13.4 Hz, 1H); 3.11 (dd,
J = 17.0, 2.8 Hz, 1H). 13C NMR (126 MHz, CDCl3): δ = 192.3, 161.9,
136.3, 134.3, 134.0, 130.3, 129.5, 129.2, 127.3, 126.8, 126.1, 125.5,
124.0, 122.9, 121.9, 121.2, 118.3, 77.0, 44.1. HRMS (ESI-FT-ICR) m/z:
297.0901 [M+Na]; calcd. for C19H14NaO2: 297.0891.
Yellow amorphous solid (60%). IR (cm−1): 1633, 1566, 1507, 1363,
1289, 1254, 1218, 1190, 1128, 1020, 970, 830. 1H NMR (500 MHz,
CDCl3): δ = 13.45 (s, 1H); 8.75 (d, J = 15.2 Hz, 1H); 8.28 (d,
J = 8.5 Hz, 1H); 7.98–7.84 (m, 4H); 7.67 (d, J = 15.2 Hz, 1H);
7.64–7.49 (m, 3H); 7.49–7.32 (m, 5H); 6.64–6.53 (m, 2H); 5.13 (s, 2H).
13C NMR (126 MHz, CDCl3): δ = 191.7, 166.7, 165.4, 141.4, 135.8,
133.7, 132.3, 131.8, 131.4, 130.9, 128.8, 128.7, 128.3, 127.6, 127.0,
126.4, 125.4, 125.2, 123.5, 123.0, 114.3, 108.3, 102.1, 70.3. HRMS
(ESI-FT-ICR) m/z: 403.1007 [M+Na]; calcd. for
C26H20NaO3:
403.1310.
5.3.2. 6-methyl-2-(naphthalen-1-yl)chroman-4-one (12)
5.2.8. (E)-1-(3,5-dichloro-2-hydroxyphenyl)-3-(naphthalen-1-yl)prop-2-
en-1-one (8)
White crystalline solid (42%), mp 137–138 °C. IR (cm−1): 1687,
1618, 1488, 1417, 1292, 1228, 1181, 1131, 1048, 982, 908. 1H NMR
(500 MHz, CDCl3): δ = 8.09–8.02 (m, 1H); 7.96–7.85 (m, 2H);
7.83–7.75 (m, 2H); 7.62–7.47 (m, 3H); 7.35 (dd, J = 8.4, 2.4 Hz, 1H);
Orange crystalline solid (83%), mp 188–189 °C. IR (cm−1): 1633,
1557, 1436, 1339, 1320, 1271, 1240, 1211, 1166, 1128, 1048, 1029,
11

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7.00 (d, J = 8.4 Hz, 1H); 6.20 (dd, J = 13.3, 2.8 Hz, 1H); 3.25 (dd,
J = 17.0, 13.4 Hz, 1H); 3.08 (dd, J = 16.9, 2.9 Hz, 1H); 2.36 (s, 3H).
13C NMR (126 MHz, CDCl3): δ = 192.5, 159.9, 137.3, 134.3, 133.9,
131.2, 130.2, 129.3, 129.1, 126.7, 126.7, 125.9, 125.4, 123.8, 122.8,
120.7, 118.0, 76.8, 44.0, 20.5. HRMS (ESI-FT-ICR) m/z: 311.1049 [M
+Na]; calcd. for C20H16NaO2: 311.1048.
3H); 7.37–7.25 (m, 5H); 6.67 (dd, J = 8.8, 2.4 Hz, 1H); 6.55 (d,
J = 2.3 Hz, 1H); 6.15 (dd, J = 13.2, 2.8 Hz, 1H); 5.04 (s, 2H); 3.16 (dd,
J = 17.0, 13.3 Hz, 1H); 2.97 (dd, J = 17.0, 2.9 Hz, 1H). 13C NMR
(126 MHz, CDCl3): δ = 190.8, 165.3, 163.7, 135.8, 134.2, 133.9,
130.2, 129.4, 129.1, 128.9, 128.7, 128.3, 127.5, 126.7, 126.0, 125.4,
123.9, 122.8, 115.2, 110.9, 102.0, 77.3, 70.3, 43.6. HRMS (ESI-FT-ICR)
m/z: 403.1313 [M+Na]; calcd. for C26H20NaO3: 403.1310.
5.3.3. 6-methoxy-2-(naphthalen-1-yl)chroman-4-one (13)
White crystalline solid (70%); mp 164–165 °C. IR (cm−1): 1682,
1611, 1484, 1429, 1334, 1278, 1209, 1178, 1057, 1027, 982, 892, 866,
818. 1H NMR (500 MHz, CDCl3): δ = 8.06 (d, J = 8.0 Hz, 1H); 7.92 (d,
J = 8.4 Hz, 1H); 7.89 (d, J = 8.1 Hz, 1H); 7.78 (d, J = 7.1 Hz, 1H);
7.55 (m, 3H); 7.43 (d, J = 3.2 Hz, 1H); 7.15 (dd, J = 9.0, 3.2 Hz, 1H);
7.04 (d, J = 9.0 Hz, 1H); 6.19 (dd, J = 13.4, 2.8 Hz, 1H); 3.85 (s, 3H);
3.25 (dd, J = 17.1, 13.4 Hz, 1H); 3.09 (dd, J = 17.0, 2.8 Hz, 1H); 13C
NMR (126 MHz, CDCl3): δ = 192.3, 156.5, 154.4, 134.3, 133.9, 130.2,
129.3, 129.1, 126.7, 125.9, 125.4, 125.4, 123.8, 122.8, 120.9, 119.5,
107.5, 77.2, 55.9, 43.9. HRMS (ESI-FT-ICR) m/z: 327.0999 [M+Na];
calcd. for C20H16NaO3: 327.0997.
5.3.8. 6,8-dichloro-2-(naphthalen-1-yl)chroman-4-one (18)
Pale yellow crystalline solid (56%), mp 172–173 °C. IR (cm−1):
1694, 1592, 1455, 1337, 1266, 1254, 1233, 1214, 1181, 1166, 1003,
982, 968, 878, 852, 809. 1H NMR (500 MHz, CDCl3): δ = 7.96 (d,
J = 8.2 Hz, 1H); 7.89–7.82 (m, 2H); 7.80 (d, J = 2.6 Hz, 1H); 7.71 (d,
J = 7.2 Hz, 1H); 7.56–7.44 (m, 4H); 6.23 (dd, J = 12.2, 3.5 Hz, 1H);
3.20 (dd, J = 17.2, 12.2 Hz, 1H); 3.13 (dd, J = 17.2, 3.5 Hz, 1H). 13C
NMR (126 MHz, CDCl3): δ = 190.3, 155.9, 135.7, 133.9, 133.0, 130.1,
129.7, 129.1, 126.9, 126.8, 126.1, 125.3, 125.2, 124.4, 123.9, 122.7,
122.6, 77.6, 43.2. HRMS (ESI-FT-ICR) m/z: 365.0109 [M+Na]; calcd.
for C1935Cl2H12O2: 365.0112; m/z: 367.0089 [M+Na]; calcd. for
C1935Cl37ClNaH12O2: 367.0083
5.3.4. 7-methoxy-2-(naphthalen-1-yl)chroman-4-one (14)
White amorphous solid (48%). IR (cm−1): 1697, 1671, 1611, 1600,
1578, 1443, 1379, 1360, 1292, 1259, 1202, 1159, 1121, 1053, 1041,
1013, 991, 949, 892, 873, 828. 1H NMR (500 MHz, CDCl3):
δ = 8.11–8.03 (m, 1H); 7.98–7.85 (m, 3H); 7.77 (d, J = 7.2 Hz, 1H);
7.55 (dd, J = 8.5, 6.6 Hz, 3H); 6.67 (dd, J = 8.8, 2.4 Hz, 1H); 6.54 (d,
J = 2.4 Hz, 1H); 6.23 (dd, J = 13.3, 2.9 Hz, 1H); 3.85 (s, 3H); 3.23 (dd,
J = 16.9, 13.3 Hz, 1H); 3.04 (dd, J = 17.0, 2.9 Hz, 1H). 13C NMR
(126 MHz, CDCl3): δ = 190.9, 166.2, 163.7, 134.2, 133.9, 130.2,
129.4, 129.1, 128.9, 126.7, 126.0, 125.4, 123.9, 122.8, 115.0, 110.4,
101.0, 77.2, 55.7, 43.6. HRMS (ESI-FT-ICR) m/z: 327.1007 [M+Na];
calcd. for C20H16NaO3: 327.0997.
5.3.9. 7,8-bis(benzyloxy-2-(naphthalen-1-yl)chroman-4-one (19)
White amorphous solid (40%). IR (cm−1): 1686, 1641, 1592, 1505,
1446, 1384, 1312, 1293, 1261, 1194, 1120, 1090, 1073, 1013. 1H NMR
(500 MHz, CDCl3): δ = 8.05 (dd, J = 6.1, 3.3 Hz, 1H); 7.93 (dd,
J = 6.3, 3.4 Hz, 1H); 7.90 (d, J = 8.3 Hz, 1H); 7.77 (d, J = 7.2 Hz, 1H);
7.73 (d, J = 8.9 Hz, 1H); 7.59–7.49 (m, 3H); 7.45–7.31 (m, 5H); 7.27
(m, 2H); 7.18 (dd, J = 8.4, 6.3 Hz, 1H); 7.15–7.06 (m, 2H); 6.74 (d,
J = 8.9 Hz, 1H); 6.14 (dd, J = 12.3, 3.4 Hz, 1H); 5.21 (s, 2H); 5.01 (s,
2H); 3.16 (dd, J = 17.1, 12.3 Hz, 1H); 3.08 (dd, J = 17.0, 3.4 Hz, 1H).
13C NMR (126 MHz, CDCl3): δ = 205.9, 190.2, 157.3, 155.0, 136.2,
135.3, 135.1, 133.3, 132.7, 129.1, 128.1, 128.0, 127.6, 127.5, 127.1,
127.0, 126.8, 126.2, 125.6, 124.9, 124.3, 122.7, 122.0, 121.8, 115.4,
106.5, 74.2, 69.9, 42.8. HRMS (ESI-FT-ICR) m/z: 509.1729 [M+Na];
calcd. for C33H26NaO4: 509.1732.
5.3.5. 6-chloro-2-(naphthalen-1-yl)chroman-4-one (15)
Bright yellow crystalline solid (80%), mp 137–138 °C; IR (cm−1):
1687, 1604, 1467, 1424, 1408, 1337, 1275, 1221, 1209, 1171, 1131,
982, 911, 901, 892, 852, 833. 1H NMR (500 MHz, CDCl3) δ 8.06–8.00
(m, 1H); 7.96 (d, J = 2.6 Hz, 1H); 7.95–7.87 (m, 2H); 7.76 (dt, J = 7.3,
1.0 Hz, 1H); 7.61–7.51 (m, 3H); 7.48 (dd, J = 8.8, 2.7 Hz, 1H); 7.06 (d,
J = 8.8 Hz, 1H); 6.23 (dd, J = 13.2, 2.9 Hz, 1H); 3.26 (dd, J = 17.1,
13.2 Hz, 1H); 3.12 (dd, J = 17.1, 2.9 Hz, 1H). 13C NMR (126 MHz,
CDCl3) δ 191.2, 160.3, 136.2, 134.0, 133.8, 130.3, 129.7, 129.3, 127.5,
126.9, 126.6, 126.2, 125.5, 124.0, 122.8, 122.0, 120.1, 77.2, 43.7.
HRMS (ESI-FT-ICR) m/z: 331.0505 [M+Na]; calcd. for
C1935ClH13NaO2: 331.0502; m/z: 333.0463 [M+Na]; calcd. for
C1937ClH13NaO2: 333.0472
5.4. Cell culture and cytotoxicity assays
U-937, HL-60, NALM-6 and MOLT-3 cells were from DSMZ (German
Collection of Microorganisms and Cell Cultures, Braunschweig,
Germany). U-937/Bcl-2 cells were kindly provided by Dr. Jacqueline
Bréard (INSERM U749, Faculté de Pharmacie Paris-Sud, Châtenay-
Malabry, France). The U-937 is a pro-monocytic, human myeloid leu-
kaemia cell line which was isolated from a histiocytic lymphoma. HL-60
is an acute myeloid leukaemia cell line. NALM-6 is a human B cell
precursor leukaemia. MOLT-3 is an acute lymphoblastic leukaemia cell
line.
5.3.6. 6-benzyloxy-2-(naphthalen-1-yl)chroman-4-one (16)
Cells were cultured in RPMI 1640 medium containing 10% (v/v)
heat-inactivated fetal bovine serum, 100 μg/ml streptomycin and 100
U/ml penicillin, incubated at 37 °C in a humidified atmosphere con-
taining 5% CO2 as described [17]. The doubling times of the cell lines
were 30 h for U-937 and U-937/Bcl-2, 24 h for HL-60 and 40 h for
NALM-6 and MOLT-3. Human peripheral blood mononuclear cells
(PBMC) were isolated from heparin-anticoagulated blood of healthy
volunteers by centrifugation with Ficoll-Paque Plus (GE Healthcare Bio-
Sciences AB, Uppsala, Sweden). PBMC were also stimulated with phy-
tohemagglutinine (2 μg/mL) for 48 h before the experimental treat-
ment. The trypan blue exclusion method was used for counting the cells
by a hematocytometer and the viability was always greater than 95% in
all experiments. The cytotoxicities of chalcones and flavanones were
evaluated by colorimetric 3-(4,5-dimethyl-2-thiazolyl-)-2,5-diphenyl-
2H-tetrazolium bromide (MTT) assays as previously described [34].
Chalcones and flavanones were dissolved in DMSO and kept under dark
conditions at 25 °C. Before each experiment, compounds were dissolved
in culture media at 37 °C. The final concentration of DMSO did not
exceed 0.3% (v/v).
White crystalline solid (46%), mp 159–160 °C. IR (cm−1): 1682,
1484, 1453, 1431, 1382, 1337, 1271, 1181, 1155, 1131, 1062, 1039,
1027, 982, 963, 894, 861, 837. 1H NMR (500 MHz, CDCl3): δ = 8.05
(d, J = 7.8 Hz, 1H); 7.97–7.83 (m, 2H); 7.78 (d, J = 7.1 Hz, 1H);
7.61–7.50 (m, 4H); 7.46 (d, J = 7.4 Hz, 2H); 7.41 (dd, J = 8.4, 6.6 Hz,
2H); 7.37–7.29 (m, 1H); 7.23 (dd, J = 9.0, 3.2 Hz, 1H); 7.05 (d,
J = 8.9 Hz, 1H); 6.19 (dd, J = 13.5, 2.8 Hz, 1H); 5.10 (s, 2H); 3.25 (dd,
J = 17.1, 13.5 Hz, 1H); 3.09 (dd, J = 17.1, 2.8 Hz, 1H). 13C NMR
(126 MHz, CDCl3): δ = 192.2, 156.6, 153.5, 136.6, 134.2, 133.9,
130.2, 129.3, 129.1, 128.6, 128.1, 127.6, 126.7, 126.0, 126.0, 125.4,
123.8, 122.8, 121.0, 119.6, 109.0, 77.0, 70.6, 43.9. HRMS (ESI-FT-ICR)
m/z: 403.1310 [M+Na]; calcd. for C26H20NaO3: 403.1310.
5.3.7. 7-benzyloxy-2-(naphthalen-1-yl)chroman-4-one (17)
Pale yellow amorphous solid (50%). IR (cm−1): 1668, 1607, 1571,
1500, 1443, 1377, 1353, 1334, 1294, 1252, 1185, 1166, 1119, 1060,
1034, 998, 984, 930, 828. 1H NMR (500 MHz, CDCl3): δ = 7.98 (d,
J = 7.7 Hz, 1H); 7.90–7.79 (m, 3H); 7.72–7.66 (m, 1H); 7.52–7.43 (m,
12

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5.5. Fluorescent microscopy analysis
and lysed 10 times with a 22-gauge needle. The lysates were cen-
trifuged (1000g, 5 min, 4 °C). The supernatants were centrifuged at
105,000g for 45 min at 4 °C, and the resulting supernatant was used as
the soluble cytosolic fraction and analysed by immunoblotting with
specific antibodies against Smac/DIABLO and cytochrome c.
Cells were washed with PBS and fixed in 3% paraformaldehyde for
10 min at room temperature. The paraformaldehyde was removed by
centrifugation (12,000g, 1 min, 25 °C) and the samples were stained
with 20 μg/mL of bisbenzimide trihydrochloride (Hoechst 33258) in
PBS at 25 °C during 15 min. An aliquot of 10 μL of the mixture was used
to observe the stained nuclei with fluorescent microscopy (Zeiss-
Axiovert).
5.10. Intracellular reactive oxygen species (ROS) determination
Intracellular ROS were detected by flow cytometry using the probe
2′,7′-dichlorodihydrofluorescein diacetate (H2-DCF-DA). Flow cyto-
metric analysis was carried out using a BD FACSVerseTM cytometer (BD
Biosciences, San Jose, CA, USA) and has been described in detail else-
where [36].
5.6. Quantification of hypodiploid cells and flow cytometry analysis of
annexin V-FITC and propidium iodide-stained cells
Flow cytometric analysis of propidium iodide-stained cells was
performed as previously described [35]. Briefly, cells were centrifuged
for 10 min at 500g, washed with cold PBS, fixed with ice-cold 75%
ethanol and stored at −20 °C for at least 1 h. Samples were then cen-
trifuged at 500g for 10 min at 4 °C, washed with PBS, resuspended in
200 μL of PBS containing 100 μg/mL RNase A and 50 μg/mL propidium
iodide and incubated for 1 h in the dark. The DNA content was analyzed
by flow cytometry with a BD FACSVerse™ cytometer (BD Biosciences,
San Jose, CA, USA). Flow cytometric analysis of annexin V-FITC and
propidium iodide-stained cells was performed as described [35].
5.11. Statistical methods
Statistical differences between means were tested using (i) Student’s
t-test (two samples) or (ii) one-way analysis of variance (ANOVA) (3 or
more samples) with Tukey’s test used for a posteriori pairwise compar-
isons of means. A significance level of P < 0.05 was used.
Acknowledgements
We thank Dr. Jacqueline Bréard (INSERM U749, Faculté de
Pharmacie Paris-Sud., Châtenay-Malabry, France) for supplying U-937/
Bcl-2 cells.
5.7. Assay of caspase activity
Caspase activity was determined by measuring proteolytic cleavage
of the chromogenic substrates Ac-DEVD-pNA (for caspase-3 like pro-
tease activity), Ac-IETD-pNA (for caspase-8 activity) and Ac-LEHD-pNA
(for caspase-9 activity) as previously described [35].
Funding
This work was supported in part by the Spanish Ministry of Science,
Innovation and Universities and the European Regional Development
Fund (PGC2018-094503-B-C21).
5.8. Western blot analysis
Cells were harvested by centrifugation (500g, 10 min, 4 °C) and
pellets were resuspended in lysis buffer [1% Triton X-100, 10 mM so-
dium fluoride, 2 mM EDTA, 20 mM Tris-HCl (pH 7.4), 2 mM tetra-
sodium pyrophosphate, 10% glycerol, 137 mM NaCl, 20 mM sodium β-
glycerophosphate], with the protease inhibitors phenylmethylsulfonyl
fluoride (PMSF, 1 mM), aprotinin, leupeptin, and pepstatin A (5 μg/mL
each) and kept on ice during 15 min. Cells were sonicated on ice five
times (5 s each, with intervals between each sonication of 5 s) with a
Braun Labsonic 2000 microtip sonifier and centrifuged (11,000g,
10 min, 4 °C). Bradford’s method was used to determine protein con-
centration. The samples that were loaded in sodium dodecyl sulphate-
polyacrylamide gel (from 7.5 to 15% depending on the molecular
weight of interest) were prepared with the same amount of protein and
boiled for 5 min. The proteins were transferred to a poly(vinylidene
difluoride) membrane for 20 h at 20 V. The membrane was blocked
with 10% nonfat milk in Tris-buffered saline [50 mM Tris-HCl (pH 7.4),
150 mM NaCl] containing 0.1% Tween-20 (TBST) for 1 h, followed by
incubation with specific antibodies against caspase-3, caspase-4, cas-
pase-6, caspase-7, caspase-8, caspase-9, Bax, Bcl-2, Bid, DR4, DR5,
TRAIL, MAPKs, β-actin and poly(ADP-ribose)polymerase overnight at
4 °C. Membranes were washed three times with TBST and incubated for
1 h with the specific secondary antibody and the antigen-antibodies
complexes were visualized by enhanced chemiluminescence using the
manufacturer’s protocol.
Conflict of interest
The authors declare that there is no conflict of interest.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2019.103450.
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