Methylenedioxy flavonoids: Assessment of cytotoxic and anti-cancer potential in human leukemia cells

Article abstract of DOI:10.1016/j.ejmech.2014.07.003

A new series of chalcones, flavanones and flavones with methylenedioxy group at the 3,4 position in chalcone, 7,8 position in flavanones and flavones with mono-, di- and trimethoxy groups in the benzaldehyde ring have been assessed for their effect on pro

Full text of DOI:10.1016/j.ejmech.2014.07.003

European Journal of Medicinal Chemistry 84 (2014) 173e180
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Methylenedioxy flavonoids: Assessment of cytotoxic and anti-cancer
potential in human leukemia cells
,
b
c,
Barbora Orlikova a , Jose C.J.M.D.S. Menezes d, Seungwon Ji a, Shrivallabh P. Kamat d,
ꢀ
c
a, *
ꢀ
Jose A.S. Cavaleiro , Marc Diederich
a Department of Pharmacy, College of Pharmacy, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul 151-742, South Korea
b
ꢀ
^
Laboratoire de Biologie Moleculaire et Cellulaire du Cancer, Fondation de Recherche Cancer et Sang, Hopital Kirchberg, 9 Rue Edward Steichen, 2540
Luxembourg, Luxembourg
c Department of Chemistry & QOPNA, University of Aveiro, 3810-193 Aveiro, Portugal
d Department of Chemistry, Goa University, Taleigao Plateau, Goa 403 206, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of chalcones, flavanones and flavones with methylenedioxy group at the 3ʹ,4ʹ position in
chalcone, 7,8 position in flavanones and flavones with mono-, di- and trimethoxy groups in the benz-
aldehyde ring have been assessed for their effect on proliferation, cytotoxic potential and apoptosis in
human leukemia cells. Among the tested compounds, the chalcone series showed the best activity and
chalcone 3 (mono methoxy group at the ortho position in A-ring) showed a significant effect on down-
regulation of cancer cell proliferation and viability in three different leukemia cell lines (K562, Jurkat,
U937). The executioner caspase cleavage analyses indicated that the cytotoxic effect mediated by chal-
cone 3 is due to induction of apoptotic cell death. Interestingly, the cytotoxic effect was cell type-specific
and targeted preferentially cancer cells as peripheral blood mononuclear cells (PBMCs) from healthy
donors were less affected by the treatment compared to K562, Jurkat and U937 leukemia cells. Altogether
our results indicate a potential drug candidate with interesting differential toxicity obeying Lipinski's
rule of five.
Received 25 March 2014
Received in revised form
19 June 2014
Accepted 2 July 2014
Available online 3 July 2014
Keywords:
Chalcones
Flavanones
Flavones
Cytotoxic
Leukemia
Anti-inflammatory
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
activated by the tumor selective catalytic activity of the cytochrome
P450 enzyme CYP1B1 [12]. This chalcone, which behaves as a pro-
Flavonoids are polyphenolic compounds, which are naturally
present in fruits, vegetables as well as in beverages, such as red
wine and tea. The influence of this large class of dietary compounds
on various biological activities is well known [1e3]. Special atten-
tion has been focused on potential chemotherapeutic effects of
flavonoids in cancer [4,5].
The methylenedioxy group has a special emphasis on cancer drug
designwhereinitspresence[6,7]orinclusionhasamarkedincreasein
cytotoxicity [8e10]. Although the number of chalcones, flavanones
and flavones having the methylenedioxygroup is scarce, the naturally
occurring chalcones, flavanones and flavones, which present avariety
of biological activities, bear the methylenedioxy group at 40,50 or 3,4
positions in chalcone, 40,50 in flavanone and at 6,7 or 30,40 positions in
flavones [11]. A synthetic chalcone, 3,4-methylenedioxy-30,40,50-tri-
methoxy chalcone was found to be a potent anticancer prodrug
drug, had low intrinsic toxicity and was only activated by the CYP1B1
enzyme to give the free 3,4-dihydroxy group which turned it into a
potent broad spectrum tyrosine kinase inhibitor [13].
Substitution with the methylenedioxy group at positions 30,40 in
chalcones and positions 7,8 in flavanones and flavones is scarce in
nature. With our interest in the synthesis of natural and synthetic
methylenedioxy flavonoids [14], it was pertinent to explore and
synthesize newer compounds of the same series [15] to assess their
biological potential. This study aims at exploring the anti-
proliferative and cell death-inducing potential of these flavonoids
in human leukemia (U937, Jurkat and K562) cells.
2. Results and discussion
2.1. Chemistry
* Corresponding author.
The flavonoid compounds (1e12) used in this study were pre-
E-mail
addresses:
marcdiederich@snu.ac.kr,
marc.diederich@lbmcc.lu
(M. Diederich).
pared according to the previously described method [14] and are
http://dx.doi.org/10.1016/j.ejmech.2014.07.003
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

174
B. Orlikova et al. / European Journal of Medicinal Chemistry 84 (2014) 173e180
Scheme 1. Methylenedioxy flavonoids 1e12 used in this study.
shown in Scheme 1. Briefly, the 20-hydroxy-30,40-methylenediox-
yacetophenone was condensed with the appropriate benzaldehyde
derivatives in basic ethanolic solution to afford the corresponding
chalcones (1, 3, 5 and 7). The chalcones were refluxed with tri-
fluoroacetic acid (TFA) to give the flavanone derivatives (2, 4, 6 and
9). The flavones (8, 10e12) were obtained by refluxing the corre-
sponding chalcones (1, 5, 3 and 7) in DMSO containing catalytic
amount of iodine. All the synthesized flavonoid derivatives were
characterized by melting point (mp), infrared (IR) and nuclear
magnetic resonance (1H and 13C NMR) spectra, and studied by
tandem mass spectra (ESI-MS/MS and HRMS) [15].
about 20% at 72 h, but did not significantly affect K562 cells (Fig 2B).
PBMCs from healthy donors were affected to a much lesser extent
compared to leukemia cells underlining excellent differential
toxicity of chalcones 3 and 7 (Fig 2C).
2.5. Chalcone 3 induces apoptotic cell death
As reduced viability could correspond to induction of apoptotic
cell death, effect of chalcone 3 was investigated on activation of
executioner caspases. Results showed that chalcone 3 induced
cleavage of caspase 3 and -7 after 48 h starting from 20 mM in Jurkat
cells confirming the apoptotic nature of cell death mediated by this
2.2. Biological assays
compound (Fig. 3).
We screened the 12 flavonoid derivatives to measure inhibition
of leukemia cell viability. Results showed that chalcones 3 and 7
2.6. Structure activity relationships
were most efficient after 72 h at 10 mM (Table 1) and hence were
investigated in more detail.
Screening of 12 flavonoids of the chalcone, flavanone and fla-
vones series indicated only chalcones (3 and 7) to have strongest
activity against human leukemia (U937, Jurkat and K562) cell lines
tested in this study without affecting cells from health donors. The
methylenedioxy group has a clear and marked effect in cancer drug
design being pointed out in earlier studies [12,13], wherein the
methylenedioxy group in the A-ring of chalcone and trimethoxy
groups in the B-ring contributed to superior toxicity. Another study
with several chalcones identified the methoxy group, its position
2.3. Chalcones modulate leukemia cell proliferation
Chalcone 3 showed the most powerful potential to inhibit pro-
liferation of the 3 cancer cell lines (Fig. 1A) with a significant
cytotoxic effect in Jurkat and U937 cells starting at 20
estingly, chalcone 3 inhibited cell proliferation of K562 cells at
20 M and mediated a cytostatic effect at 30 M and higher.
Chalcone 7 reduced cell proliferation in a dose-dependent
manner in Jurkat cells reaching up to 70% of inhibition potential
at 50 M and 72 h (Fig. 1B). Similarly, chalcone 7 reduced prolif-
eration in U937 cells reaching a plateau effect starting at 30 M by
mM. Inter-
m
m
and the a,b-double bond as a prerequisite for heme-oxygenase-I
activity [16]. Considering the observed effects in the present
study, it is clear that the methylenedioxy group present in all tested
compounds in the B-ring of chalcones and A-ring of flavanones and
flavones is not the only decisive feature in these set of compounds,
but the methoxy groups also have an equal weightage. It was
observed that the toxicity in the chalcone series is increased by the
positioning of the methoxy group on the A-ring of the chalcone
system, where it is evident that the 2-methoxy group may place the
molecule in the right conformation for interaction with receptor
binding pockets in cellular macromolecules by hydrogen bonding.
m
m
inhibition of 41% of cell proliferation (Fig. 1B). Chalcone 7 did not
significantly inhibit proliferation of K562 cells (Fig 1B).
2.4. Chalcones reduce cancer cell viability
Based on previous results, we investigated the effect of chal-
cones on viability. Chalcone 3 inhibited dose- and time-dependent
viability of Jurkat, U937 and K562 leukemia cells with IC50s of
2.7. Drug-likeness properties
13.7 0.8 mM, 11.4 0.6 mM and 42.3 6.5 mM after 72 h, respec-
tively (Fig 2A). Chalcone 7 dose-dependently inhibited viability of
Jurkat cells and induced a cytotoxic effect in U937 cells by
decreasing about 35% of cellular metabolic activity at 48 h and
Interestingly all flavonoids follow the Lipinski's ‘rule of five’ [17]
(Table 2), which indicates high drug-likeness property. All flavo-
noids possess an adequate number of proton acceptors but only the
Table 1
Inhibition of leukemia cell viability by methylenedioxy flavonoids. Cells were treated with or without compounds for 72 h and viability was determined by trypan blue staining.
Control (Co) corresponds to DMSO-treated cells. Values represent viable cells normalized to control (%).
Co
Flavonoids (10 mM)
1
2
3
4
5
6
7
8
9
10
11
12
Jurkat (72 h)
U937 (72 h)
100.0
100.0
80.6
80.9
88.2
81.7
59.0
55.4
83.2
74.0
77.5
69.3
74.9
80.4
56.1
64.4
81.2
76.5
69.7
68.3
87.9
82.5
91.6
82.7
76.0
90.5

B. Orlikova et al. / European Journal of Medicinal Chemistry 84 (2014) 173e180
175
Fig. 1. Effect of flavonoids on cancer cell proliferation. Effect of chalcone 3 (1A) and chalcone 7 (1B) on proliferation Jurkat, U937 and K562 cells assessed by trypan blue exclusion
test. Each value is the mean SD of three determinations. The asterisk indicates a significant difference compared to control (Co) analyzed by t-test (*p < 0.05).
chalcone series have one proton donor group, which will ensure
efficient interaction with the hydrogen bonding groups of the re-
ceptors. This fact corroborates well with the experimentally
observed activity of the chalcone series in cellular assays.
The number of rotatable bonds in a molecule defines the mo-
lecular flexibility for membrane permeation. In the present series
all flavonoids have 2e6 rotatable bonds, whereas the chalcone
series have higher number (4e6) and hence all flavonoids will have
good probability of oral bioavailability. The topological polar sur-
face area (TPSA) has been shown to correlate well with the drug
transport properties [18] and chalcone compounds have moderate
(ꢀ60 Å2) to high (ꢀ140 Å2) TPSA values that also indicate high
probability of good oral bioavailability and absorption.
potent chalcone 3 are underway. A structural analysis into the
substitution pattern in the A-ring of chalcone and its effect on the
activity was derived. All analyzed compounds obey Lipinski's rule
of five indicating high probability of oral bioavailability and
permeability of compounds.
4. Materials and methods
4.1. Chemistry
4.1.1. General experimental details
Melting points were measured with a Büchi B-540 melting point
apparatus (heating rate 5 ꢁC/min) and are uncorrected. IR spectra
were recorded on a Shimadzu (IR Prestige-21) FTIR and Bruker
Tensor 27 spectrometer. 1H NMR spectra were recorded at 300 or
500 MHz and 13C NMR spectra were recorded at 75.5 or 125.8 MHz
with Bruker Avance 300 and Bruker DRX 500 spectrometers. CDCl3
3. Conclusions
Various chalcone derivatives have been previously described to
possess potent anticancer properties [19,20]. In the present study,
we demonstrated that chalcone 3 showed a significant effect on
down-regulation of cancer cell proliferation and viability in
different cancer cell lines (K562, Jurkat and U937). Moreover, the
cytotoxic effect was cell type specific targeting preferentially cancer
cells whereas non-transformed PBMCs were significantly less
affected by the treatment. Results from caspase cleavage analyses
indicate, that the cytotoxic effect mediated by chalcone 3-treat-
ment is due to induction of apoptotic cell death in leukemia cells.
Detail cell death mechanisms to ascertain the mode of action of the
was used as solvent. Chemical shifts (d) are expressed in parts per
million (ppm) relative to tetramethylsilane. The coupling constants
(J) are given in Hertz (Hz). Unequivocal 1H assignments were made
using 2D COSY experiment (mixing time of 800 ms) while 13C as-
signments were made on the basis of DEPT-135 and 2D HSQC and
HMBC experiments (delay for long-range J C/H couplings were
optimized for 7 Hz). HREIMS was recorded on Micromass-GCT
(voltage-70 eV) at CACTI-Vigo, Spain. All yields refer to isolated
products unless stated. Preparative thin layer chromatography was
carried out on 20 ꢂ 20 cm glass plates coated with silica gel (1 mm

176
B. Orlikova et al. / European Journal of Medicinal Chemistry 84 (2014) 173e180
Fig. 2. Effect of flavonoids on viability. Effect of chalcone 3 (2A) and chalcone 7 (2B) on metabolic activity of Jurkat, U937, K562 and PBMCs (2C) analyzed after 24, 48 and 72 h.
Control (Co) corresponds to untreated cells. Etoposide (Eto) served as a positive inhibitory control. Each value is the mean SD of three determinations. The asterisk indicates a
significant difference compared to control analyzed by t-test (*p < 0.05).

B. Orlikova et al. / European Journal of Medicinal Chemistry 84 (2014) 173e180
177
(compounds 1, 3 and 5) or extracted with CHCl3 (4 ꢂ 5 mL). The
combined extracts were washed with water, brine and dried over
Na2SO4, they were then filtered and concentrated to give a residue.
The residue was crystallized to give chalcones 1, 3 and 5. For
chalcone 7 the residue was purified by silica gel column chroma-
tography [CHCl3:EtOAc (95:5)].
4.1.2.1. 20-Hydroxy-30,40-methylenedioxy-2-methoxychalcone
3.
Yellow solid; m.p. 169.9e170.6 ꢁC (CHCl3:PE); yield-89%; IR (KBr,
cmꢃ1): 3455, 2919, 1660 (CO), 1561, 1454, 1320, 1246, 1164, 1069,
743. 1H NMR (500 MHz, CDCl3):
d 3.94 (s, 3H, OCH3), 6.10 (s, 2H,
OCH2O), 6.52 (d, 1H, J ¼ 8.4 Hz, H-50), 6.96 (dd, 1H, J ¼ 8.4, 0.8 Hz, H-
3), 6.98e7.04 (m, 1H, H-5), 7.40 (ddd, 1H, J ¼ 8.4, 7.4, 1.7 Hz, H-4),
7.59 (d, 1H, J ¼ 8.4 Hz, H-60), 7.63 (dd, 1H, J ¼ 7.4, 1.7 Hz, H-6), 7.69
(d, 1H, J ¼ 15.6 Hz, H-80), 8.19 (d, 1H, J ¼ 15.6 Hz, H-90), 13.05 (s, 1H,
OH). 13C NMR (125 MHz, CDCl3):
d
55.6 (OCH3), 100.6 (C-50), 102.6
(C-100), 111.3 (C-3), 117.4 (C-10), 120.8 (C-5), 120.9 (C-80), 123.6 (C-1),
125.7 (C-60), 129.6 (C-6), 132.1 (C-4), 134.6 (C-30), 140.6 (C-90), 148.1
(C-20), 153.8 (C-40), 158.9 (C-2), 193.3 (C-70). HREIMS: m/z 298.0845
ꢄ
ꢄ
found for [M]þ ; Calcd for C17H14Oþ5 : 298.0841.
4.1.2.2. 3,4-Dimethoxy-20-hydroxy-30,40-methylenedioxychalcone 5.
Yellow solid; m.p. 194.6e194.9 ꢁC (CHCl3:PE); yield-84%; IR (KBr,
cmꢃ1): 3003, 2937, 1643 (CO), 1556, 1441, 1317, 1267, 1138, 1068,
Fig. 3. Caspase cleavage by chalcone 3. Jurkat cells were incubated with different
concentrations of chalcone 3 at 48 h. Western blot analysis shows dose-dependent
effect of chalcone 3 on cleavage of pro-caspases-3 and -7. (Co) corresponds to un-
789. 1H NMR (300 MHz, CDCl3):
d 3.95 (s, 3H, OCH3), 3.97 (s, 3H,
OCH3), 6.11 (s, 2H, OCH2O), 6.53 (d, 1H, J ¼ 8.4 Hz, H-50), 6.92 (d, 1H,
J ¼ 8.4 Hz, H-5), 7.16 (d, 1H, J ¼ 2.0 Hz, H-2), 7.26 (dd, 1H, J ¼ 8.4,
2.0 Hz, H-6), 7.42 (d, 1H, J ¼ 15.3 Hz, H-80), 7.59 (d, 1H, J ¼ 8.4 Hz, H-
60), 7.85 (d, 1H, J ¼ 15.3 Hz, H-90), 13.03 (s, 1H, OH). 13C NMR
treated cells, (Coþ) corresponds to Etoposide-treated cells at 100
mM for 4 h.
thick, Merck). Analytical TLC was carried out on precoated sheets
with silica gel (0.2 mm thick, Merck). Petroleum ether (PE) refers to
hydrocarbon fraction boiling in the range 60e80 ꢁC. All other sol-
vents and reagents were used without further purification. The
derivatives 1, 5 (only 13C NMR reported [21]), 2 and 8 are known
compounds and spectral data matched well with literature [14].
The purity of all synthesized compounds was assessed by 1D and
2D NMR, and high-resolution mass spectrometry (HRMS).
(75 MHz, CDCl3): d
56.0 (OCH3), 56.01 (OCH3), 100.6 (C-50), 102.6 (C-
100), 110.2 (C-2), 111.1 (C-5), 117.3 (C-10), 117.9 (C-80), 123.5 (C-6),
125.5 (C-60), 127.6 (C-1), 134.6 (C-30), 145.09 (C-90), 148.04 (C-20),
149.31 (C-3), 151.74 (C-4), 153.86 (C-40), 192.58 (C-70). HREIMS: m/z
ꢄ
ꢄ
328.0948 found for [M]þ ; Calcd for C18H16Oþ6 : 328.0947.
4.1.2.3. 20-Hydroxy-30,40-methylenedioxy-3,4,5-trimethoxychalcone
7. Orange-yellow solid; m.p. 209.5e210.5 ꢁC (CHCl3:PE); yield-81%
(based on recovered acetophenone derivative); IR (KBr, cmꢃ1):
3096, 2939, 1655 (CO), 1560, 1452, 1292, 1130, 1016, 800. 1H NMR
4.1.2. General procedure for the synthesis of chalcones (1, 3, 5 and
7)
To a mixture of 20-hydroxy-30,40-(methylenedioxy)acetophe-
none (1.1 mmol), appropriate benzaldehyde derivative (1.3 mmol)
in EtOH (5 mL), was added aq. KOH (2.5 g in 5 mL H2O) dropwise
and the reaction mixture was stirred for 1 h at 45 ꢁC. Stirring was
further continued overnight. The yellow/orange mixtures obtained
were diluted with water (10 mL) and acidified with conc. HCl. The
yellow solids obtained were filtered, washed with water and dried
(300 MHz, CDCl3):
d
3.91 (s, 3H, OCH3), 3.94 (s, 6H, OCH3 ꢂ 2), 6.11
(s, 2H, OCH2O), 6.54 (d, 1H, J ¼ 8.5 Hz, H-50), 6.87 (s, 2H, H-2, 6), 7.43
(d, 1H, J ¼ 15.3 Hz, H-80), 7.59 (d, 1H, J ¼ 8.5 Hz, H-60), 7.81 (d, 1H,
J ¼ 15.3 Hz, H-90), 12.94 (s,1H, OH). 13C NMR (75 MHz, CDCl3):
d 56.2
(OCH3 ꢂ 2), 61.0 (OCH3), 100.7 (C-50), 102.7 (C-100), 105.8 (C-2, 6),
117.2 (C-10), 119.4 (C-80), 125.7 (C-60), 130.1 (C-1), 134.7 (C-30), 140.8
(C-4), 145.1 (C-90), 148.0 (C-20), 153.5 (C-3, 5), 154.0 (C-40), 192.5 (C-
Table 2
Drug-likeness property/Lipinski's ‘rule of five’ parameters calculated for flavonoids 1e12.a
Compound
Molecular weight
miLogP
n-ROTB
n-ON acceptors
n-ONNH
Volume
TPSA
donors
1
3
5
7
2
4
6
9
298.294
298.294
328.32
358.346
298.294
298.294
328.32
358.346
296.278
326.304
296.278
356.33
3.467
3.239
3.057
3.042
3.106
3.058
2.696
2.68
3.66
3.25
3.612
3.234
4
4
5
6
2
2
3
4
2
3
2
4
5
5
6
7
5
5
6
7
5
6
5
7
1
1
1
1
0
0
0
0
0
0
0
0
259.345
259.345
284.891
310.437
255.683
255.683
281.229
306.775
249.47
65.001
65.001
74.235
83.469
54.007
54.007
63.241
72.475
57.913
67.147
57.913
76.381
8
10
11
12
275.016
249.47
300.562
a
n-ROTB, number of rotatable bonds; n-ON, number of hydrogen acceptors; n-OHNH, number of hydrogen bond donors; TPSA, topological polar surface area.

178
B. Orlikova et al. / European Journal of Medicinal Chemistry 84 (2014) 173e180
ꢄ
ꢄ
70). HREIMS: m/z 358.1051 found for [M]þ ; Calcd for C19H18O7þ
358.1052.
:
Na2SO4. Evaporation of the solvent afforded flavones (8, 10, 11 and
12) which were recrystallized from petroleum etherechloroform
mixture or ethanol.
4.1.3. General procedure for the synthesis of flavanones (2, 4, 6 and
9)
4.1.4.1. 7,8-Methylenedioxy-20-methoxyflavone 11. Colorless solid;
m.p. 214.2e215.4 ꢁC (CHCl3:PE); yield-65%; IR (KBr, cmꢃ1): 3455,
2916, 1652 (CO), 1598, 1454, 1390, 1257, 1090, 1023, 762. 1H NMR
The appropriate chalcone derivative (0.3 mmol) and trifluoro-
acetic acid (5 mL) were refluxed for 2 h (monitored by TLC). The
reaction mixture was diluted with H2O (5 mL) and extracted with
CHCl3 (4 ꢂ 5 mL). The combined extracts were washed with satu-
rated NaHCO3 solution, brine and dried over Na2SO4. Filtered and
concentrated to give a residue. Purification of this residue with
silica gel column chromatography using hexane:EtOAc (95:5) gave
unreacted chalcone. Further elution with hexane:EtOAc (9:1)
afforded flavanones which were recrystallized from petroleum
ether-chloroform mixtures.
(300 MHz, CDCl3): d 3.95 (s, 3H, OCH3), 6.21 (s, 2H, OCH2O), 6.96 (d,
1H, J ¼ 8.5 Hz, H-6), 7.04 (d, J ¼ 8.4 Hz, 1H, H-30), 7.10 (s, 1H, H-3),
7.11 (td, 1H, J ¼ 6.6, 1.0 Hz, H-50), 7.48 (ddd, J ¼ 8.4, 7.4, 1.8 Hz, 1H, H-
40), 7.82 (d, 1H, J ¼ 8.5 Hz, H-5), 7.92 (dd, J ¼ 7.8, 1.7 Hz, 1H, H-60). 13C
NMR (125 MHz, CDCl3): d 55.7 (OCH3), 103.1 (C-9), 106.9 (C-6), 111.7
(C-30), 112.1 (C-3, C-10), 119.9 (C-4a), 120.1 (C-5), 120.8 (C-50), 129.2
(C-60), 132.4 (C-40), 134.9 (C-8), 141.5 (C-8a), 152.2 (C-7), 159.9 (C-2,
ꢄ
C-20), 177.9 (C-4). HREIMS: m/z 296.0686 found for [M]þ ; Calcd for
ꢄ
C
17H14Oþ5 : 296.0685.
4.1.3.1. 7,8-Methylenedioxy-20-methoxyflavanone 4. Colorless solid;
160.8e162.1 ꢁC (CHCl3:PE); yield-68%; IR (KBr, cmꢃ1): 3491, 2897,
1677 (CO), 1633, 1487, 1383, 1303, 1256, 1083, 1021, 763. 1H NMR
4.1.4.2. 30,40-Dimethoxy-7,8-methylenedioxyflavone
10.
Colorless solid; m.p. 235.3e236.0 ꢁC (CHCl3:PE); yield-83%; IR (KBr,
(300 MHz, CDCl3):
d
2.86e2.99 (m, 2H, H-3eq and H-3ax), 3.83 (s,
cmꢃ1): 3088, 2961,1659 (CO),1591,1414,1391,1275,1147,1084, 812.
3H, OCH3), 5.89 (dd, 1H, J ¼ 11.0, 5.0 Hz, H-2), 6.08 (d, 2H, J ¼ 5.8,
1.2 Hz, OCH2O), 6.62 (d, 1H, J ¼ 8.4 Hz, H-6), 6.91 (dd, 1H, J ¼ 8.0,
0.5 Hz, H-30), 7.04 (td, 1H, J ¼ 8.0, 0.8 Hz, H-50), 7.30e7.37 (ddd, 1H,
J ¼ 8.0,1.5 Hz, H-40), 7.61 (d,1H, J ¼ 8.4 Hz, H-5), 7.63 (dd,1H, J ¼ 8.0,
1H NMR (300 MHz, CDCl3):
d 3.97 (s, 3H, OCH3), 3.98 (s, 3H, OCH3),
6.23 (s, 2H, OCH2O), 6.68 (s, 1H, H-3), 6.96 (d, 1H, J ¼ 8.4 Hz, H-6),
6.98 (d, 1H, J ¼ 8.5 Hz, H-50), 7.39 (d, 1H, J ¼ 2.1 Hz, H-20), 7.57 (dd,
1H, J ¼ 8.5, 2.1 Hz, H-60), 7.81 (d, 1H, J ¼ 8.4 Hz, H-5). 13C NMR
1.5 Hz, H-60). 13C NMR (125 MHz, CDCl3):
d
43.9 (C-3), 55.3 (OCH3),
(75 MHz, CDCl3):
d
56.0, 56.1 (OCH3 ꢂ 2), 103.2 (C-9), 105.8 (C-3),
75.5 (C-2), 102.6 (C-9), 103.3 (C-6), 110.4 (C-30), 117.8 (C-4a), 120.9
(C-50), 122.6 (C-5), 126.4 (C-60), 127.1 (C-10), 129.4 (C-40), 134.6 (C-8),
146.0 (C-8a), 154.1 (C-7), 155.7 (C-20), 190.9 (C-4). HREIMS: m/z
107.0 (C-6), 108.7 (C-20), 111.1 (C-50), 119.91 (C-4a), 119.95 (C-60),
120.1 (C-5), 123.9 (C-10), 134.7 (C-8), 141.1 (C-8a), 149.2 (C-30), 152.1
(C-40), 152.3 (C-7), 162.6 (C-2), 177.4 (C-4). HREIMS: m/z 326.0793
ꢄ
ꢄ
ꢄ
ꢄ
298.0845 found for [M]þ ; Calcd for C17H14Oþ5 : 298.0841.
found for [M]þ ; Calcd for C18H14Oþ6 : 326.0790.
4.1.3.2. 30,40-Dimethoxy-7,8-methylenedioxyflavanone
6.
4.1.4.3. 7,8-Methylenedioxy-30,40,50-trimethoxyflavone
12.
Colorless solid; 175.3e176.0 ꢁC (CHCl3:PE); yield-80%; IR (KBr,
Colorless solid; m.p. 254.0e255.0 ꢁC (EtOH); yield- 90%; IR (KBr,
cmꢃ1): 3442, 2914, 1691 (CO), 1632, 1525, 1460, 1358, 1293, 1075,
cmꢃ1): 3057, 2947, 1662 (CO), 1583, 1344, 1132, 1091, 852. 1H NMR
1021, 806. 1H NMR (300 MHz, CDCl3):
d
2.88 (dd,1H, J ¼ 16.8, 2.9 Hz,
(300 MHz, CDCl3):
d
3.93 (s, 3H, OCH3), 3.96 (s, 6H, OCH3 ꢂ 2), 6.23
H-3eq), 3.12 (dd, 1H, J ¼ 16.8, 12.6 Hz, H-3ax), 3.90 (s, 3H, OCH3),
3.92 (s, 3H, OCH3), 5.47 (dd, 1H, J ¼ 12.6, 2.9 Hz, H-2), 6.08 (dd, 2H,
J ¼ 5.8, 1.2 Hz, OCH2O), 6.62 (d, 1H, J ¼ 8.4 Hz, H-6), 6.89 (d, 1H,
J ¼ 8.8 Hz, H-50), 6.99e7.02 (m, 2H, H-60, 20), 7.60 (d, 1H, J ¼ 8.4 Hz,
(s, 2H, OCH2O), 6.69 (s, 1H, H-3), 6.97 (d, 1H, J ¼ 8.4 Hz, H-6), 7.14 (s,
2H, H-20, 60), 7.81 (d, 1H, J ¼ 8.4 Hz, H-5). 13C NMR (75 MHz, CDCl3):
d
56.3 (OCH3 ꢂ 2), 61.0 (OCH3), 103.2 (C-9), 103.6 (C-20, 60), 106.7 (C-
3), 107.2 (C-6), 119.9 (C-4a), 120.1 (C-5), 126.7 (C-10), 137.3 (C-8),
H-5). 13C NMR (75 MHz, CDCl3):
d
44.6 (C-3), 55.9 (OCH3 ꢂ 2), 80.3
141.1 (C-40, C-8a),152.5 (C-7),153.5 (C-30, 50), 162.4 (C-2), 177.4 (C-4).
(C-2), 102.6 (C-9), 103.4 (C-6), 109.5 (C-20), 111.0 (C-50), 117.7 (C-4a),
118.9 (C-60), 122.6 (C-5), 130.7 (C-10), 134.6 (C-8), 145.5 (C-8a), 149.2
(C-30), 149.5 (C-40), 154.2 (C-7), 190.3 (C-4). HREIMS: m/z 328.0955
HREIMS: m/z 356.0898 found for [M]þꢄ; Calcd for C19H16Oþ7 ꢄ
:
356.0896.
ꢄ
ꢄ
found for [M]þ ; Calcd for C18H16Oþ6 : 328.0947.
4.2. Compounds and purification
4.1.3.3. 7,8-Methylenedioxy-30,40,50-trimethoxyflavanone
9.
TNFa was purchased from SigmaeAldrich (Bornem, Belgium)
Colorless solid; 204.2e204.9 ꢁC (CHCl3:PE); yield-72%; IR (KBr,
and dissolved to a concentration of 10 mg/mL in 1 ꢂ PBS supple-
mented with 0.5% (w/v) BSA, according to the manufacturer's
instructions.
The methylenedioxy flavonoids were solubilized in 100% DMSO
(SigmaeAldrich, Bornem, Belgium) to obtain a stock concentration
of 100 mM. This stock solution was then further diluted in DMSO to
obtain working aliquots. Both stock and working solutions were
frozen at ꢃ20 ꢁC. Control cells were treated with equivalent
amounts of DMSO.
cmꢃ1): 3443, 2938, 1688 (CO), 1628, 1595, 1465, 1360, 1293, 1128,
1077, 810. 1H NMR (300 MHz, CDCl3):
d
2.88 (dd, 1H, J ¼ 17.0, 2.9 Hz,
H-3eq), 3.10 (dd, 1H, J ¼ 17.0, 12.7 Hz, H-3ax), 3.86 (s, 3H, OCH3),
3.89 (s, 3H, OCH3 ꢂ 2), 5.44 (dd, 1H, J ¼ 12.7, 2.9 Hz, H-2), 6.09 (dd,
2H, J ¼ 4.5, 1.3 Hz, OCH2O), 6.63 (d, 1H, J ¼ 8.4 Hz, H-6), 6.69 (s, 2H,
H-20,60), 7.60 (d, 1H, J ¼ 8.4 Hz, H-5). 13C NMR (75 MHz, CDCl3):
d
44.8 (C-3), 56.2 (OCH3 ꢂ 2), 60.8 (OCH3), 80.6 (C-2), 102.7 (C-9),
103.4 (C-20, 60), 103.4 (C-6), 117.7 (C-4a), 122.7 (C-5), 133.8 (C-10),
134.7 (C-8), 138.3 (C-40), 145.3 (C-8a), 153.5 (C-30, 50), 154.3 (C-7),
ꢄ
190.0 (C-4). HREIMS: m/z 358.1061 found for [M]þ ; Calcd for
4.3. Cell culture
ꢄ
C
19H18Oþ7 : 358.1052.
K562 (human chronic myelogenous leukemia), U937 (histiocytic
lymphoma) and Jurkat (T-cell leukemia) cells (DSMZ) were cultured
in RPMI medium (Lonza) supplemented with 10% (v/v) fetal calf
serum (Lonza) and 1% (v/v) antibioticeantimycotic (Bio-Whittaker)
at 37 ꢁC and 5% CO2. The cells were harvested every 3 days. After
thawing, the cells were kept in normal culture conditions for 10
days before experiments. Healthy blood samples were kindly
donated as buffy coats by the Red Cross (Luxembourg,
4.1.4. General procedure for the synthesis of flavones 8, 10, 11 and
12
The appropriate chalcone derivative (0.21 mmol) was dissolved
in DMSO (5 mL) and a crystal of I2 added to it. The mixture was
refluxed for 45 min, cooled to room temperature, diluted with
water and extracted with ethyl acetate. The organic extracts were
washed with aqueous sodium thiosulphate, water and dried over

B. Orlikova et al. / European Journal of Medicinal Chemistry 84 (2014) 173e180
179
Luxembourg). Diluted (1/3) blood in RPMI 1640 was layered onto
4.8. Statistical analysis
Ficoll and centrifuged (400 ꢂ g, 20 min) to isolate mononuclear
cells. The isolated peripheral blood mononuclear cells (PBMCs)
were kept in culture at 37 ꢁC and 5% CO2 for 24 h before they were
subjected to treatments.
2. Data are expressed as a mean standard deviation. Significance
was determined by the Student's t-tests. p-values below 0.05
were considered as statistically significant. Asterisks indicate p-
values corresponding to *p < 0.05.
4.4. Assessment of metabolically active cells
Cell survival percentages were evaluated using Promega's
CellTiter-Glo® Luminescent Cell Viability Assay kit according to
the manufacturer's instructions. Briefly, the assay determines the
number of viable cells based on the quantification of ATP as an
indicator of metabolic activity. ATP is measured using the lucif-
erase catalysis of luciferin to oxyluciferin and light in the presence
of Mg2þ, ATP and oxygen. The lyophilized enzyme/substrate
mixture was reconstituted with the provided buffer. Then, an
equal volume of the reaction buffer was added to medium con-
taining K562, U937, Jurkat or PBMC cells (untreated or treated
with flavonoids at the indicated concentrations and times). The
mixture was incubated for 10 min in the dark at RT. The lumi-
nescence signal, which is proportional to the amount of ATP
present, was quantified using an Orion microplate luminometer
and converted into the number of viable cells according to the
manufacturer's instructions. Data were normalized to the control
and reported as percentage of viable cells.
4.9. Calculation of drug-likeness properties
The parameters for drug-likeness were evaluated according to
the Lipinski's ‘rule-of-five’, using the Molinspiration WebME Editor
3.81. [http://www.molinspiration.com].
Acknowledgments
We gratefully acknowledge the research facilities at University
of Aveiro and Goa University. Thanks are due to Fundaçao para a
~
^
Ciencia e a Tecnologia (FCT, Portugal), European Union, QREN,
FEDER and COMPETE for funding the QOPNA research unit (project
PEst-C/QUI/UI0062/2013; FCOMP-01-0124-FEDER-037296); the
Portuguese National NMR Network. JCJMDS Menezes thanks
QOPNA for a research grant. Research at the Laboratoire de Biologie
ꢀ
Moleculaire et Cellulaire du Cancer (LBMCC) is financially sup-
ported by “Recherche Cancer et Sang” foundation, by «Recherches
€
Scientifiques Luxembourg» asbl, by «Een Haerz fir Kriibskrank
4.5. Cell viability assessment
Kanner» association and the Action Lions “Vaincre le Cancer”
Luxembourg. MD and BO are supported by the National Research
Foundation (NRF) by the MEST of Korea for Tumor Microenviron-
ment Global Core Research Center (GCRC) grant, [grant number
2012-0001184]; by the Seoul National University Research grant, by
the Research Settlement Fund for the new faculty of SNU and by the
Research Institute of Pharmaceutical Sciences.
Jurkat and U937 cells were incubated with 10 mM of different
flavonoids during 72 h. Cancer cell viability was assessed by trypan
blue exclusion test.
4.6. Extraction of cellular proteins
1. After the indicated incubation times with chalcone 3, Jurkat cells
were lysed, and the total protein extracts were prepared as
previously reported [22]. Briefly, 107 cells per sample were lysed
in a hypertonic detergent medium containing protease inhibitor
cocktail (Complete®, Roche, Luxembourg). The extraction was
performed on ice to avoid protein degradation. The suspension
was put on a vertical agitation shaker for 20 min at þ4 ꢁC and
was then centrifuged at 15,000 ꢂ g for 15 min at þ4 ꢁC for
suspension clearing. Afterward, supernatants were removed
and aliquots were stored at ꢃ80 ꢁC until use.
Appendix A. Supplementary data
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.ejmech.2014.07.
003. These data include MOL file and InChiKeys of the most
important compounds described in this article.
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