Synthesis of a natural chalcone and its prenyl analogs - Evaluation of tumor cell growth-inhibitory activities, and effects on cell cycle and apoptosis

Article abstract of DOI:10.1002/cbdv.201100190

Six prenyl (= 3-methylbut-2-en-1-yl) chalcones (=1,3-diphenylprop-2-en-1- ones), 2-7, and one natural non-prenylated chalcone, 1, have been synthesized and evaluated for their in vitro growthinhibitory activity against three human tumor cell lines. A pron

Full text of DOI:10.1002/cbdv.201100190

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
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Synthesis of a Natural Chalcone and Its Prenyl Analogs – Evaluation of Tumor
Cell Growth-Inhibitory Activities, and Effects on Cell Cycle and Apoptosis
by Marta P. Nevesa)b), Raquel T. Limaa)c), Kanthima Choosangd), Panee Pakkonge),
Maria de Sa˜o Jose´ Nascimentoa)f), M. Helena Vasconcelosc)f), Madalena Pintoa)b),
Artur M. S. Silvag), and Honorina Cidade*a)b)
a) Centro de Quꢀmica Medicinal da Universidade do Porto (CEQUIMED-UP), Rua Anꢀbal Cunha, 164,
PT-4050-047 Porto (phone: þ351-222-078916; fax: þ351-222-003977; e-mail: hcidade@ff.up.pt)
b
´
ˆ
ˆ
ˆ
) Laboratorio de Quꢀmica Organica e Farmaceutica, Departamento de Ciencias Quꢀmicas, Faculdade de
´
Farmacia, Universidade do Porto, Rua Anꢀbal Cunha, 164, PT-4050-047 Porto
c) IPATIMUP – Instituto de Patologia e Imunologia Molecular da Universidade do Porto,
Rua Dr. Roberto Frias s/n, PT-4200-465 Porto
d) Inter Departmental Multidisciplinary Graduate Program in Bioscience, Faculty of Science,
Kasetsart University, Bangkok 10900, Thailand
e) Applied Radiation and Isotopes Department, Faculty of Science, Kasetsart University,
Bangkok 10900, Thailand
f
´
ˆ
´
´
) Laboratorio de Microbiologia, Departamento de Ciencias Biologicas, Faculdade de Farmacia,
Universidade do Porto, Rua Anꢀbal Cunha 164, PT-4050-047 Porto
) Departamento de Quꢀmica & QOPNA, Universidade de Aveiro, Campus Universitario de Santiago,
g
´
PT-3810-193 Aveiro
Six prenyl (¼ 3-methylbut-2-en-1-yl) chalcones (¼1,3-diphenylprop-2-en-1-ones), 2–7, and one
natural non-prenylated chalcone, 1, have been synthesized and evaluated for their in vitro growth-
inhibitory activity against three human tumor cell lines. A pronounced dose-dependent growth-
inhibitory effect was observed for all prenylated derivatives, except for 7. The chalcone possessing one
prenyloxy group at C(2’), i.e., 2, was the most active derivative against the three human tumor cell lines
(5.9<GI50 <7.7 mm). The majority of compounds caused an increase in percentage of apoptotic cells and/
or they interfered with cell cycle distribution in the MCF-7 cell line.
Introduction. – Flavonoids represent an outstanding class of naturally occurring
compounds with interesting biological activities, with the antitumor effect being one of
the most reported in the literature [1–4]. Chalcones (¼1,3-diphenylprop-2-en-1-ones),
which are intermediate precursors to all flavonoid compounds, have been widely
reported for their antitumor activity against a variety of tumor cell lines [5][6]. Among
flavonoids, prenylated derivatives have been attracting the attention of the scientific
community because of their myriad biological activities [7–9]. In fact, it has been
demonstrated that isoprenylation of flavonoids significantly increased their growth
inhibitory effect on human tumor cell lines [7–13]. Regarding O-substitutions, an
aliphatic chain in the ortho-position of the phenyl residue adjacent to the C¼O group
seems to contribute significantly to increase this activity [12]. Natural prenylated
chalcones, such as xanthohumol, although being structurally simple, exhibit numerous
pharmacological properties [14]. Indeed, xanthohumol has been considered as a cancer
ꢁ 2012 Verlag Helvetica Chimica Acta AG, Zꢂrich

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
chemopreventive agent with an exceptionally broad spectrum of inhibitory mecha-
nisms which influence carcinogenesis [15]. In the extensive range of biological activities
that prenylchalcones may exert, inhibition of cell cycle and induction of apoptotic cell
death are among the most intensively studied strategies for this family of compounds
regarding cancer prevention and therapy [16–19].
Although the prenylation pattern may play an important role with respect to the
biological activities [7–12], the pharmacological properties of chalcones were found to
be also related to the presence, number, and position of OH and MeO groups in both
the A and B rings [20]. In fact, MeO groups in ring A commonly occur in several natural
and synthetic flavonoids, probably constituting a key element for their growth-
inhibitory activities against tumor cell lines, since compounds with a higher degree of
methoxylation are significantly more potent than the less methoxylated ones [21]. In
addition, the presence of at least three adjacent MeO groups in the molecule appeared
to be favorable for this activity [12]. Moreover, several studies have revealed an
interesting correlation between cell-cycle regulation and methoxylation pattern. In
fact, polymethoxyphenyl moieties are commonly found in a number of naturally
occurring anticancer agents, such as colchicine and combretastatin A [20].
Considering this, we decided to synthesize prenylated chalcones with polymethoxy-
phenyl moieties. Here, we report the synthesis of a naturally occurring chalcone, 1, and
six prenylated analogs, 2–7 (Fig. 1). All synthesized compounds were evaluated for
their ability to inhibit the in vitro growth of three human tumor cell lines, MCF-7
(breast adenocarcinoma), NCI-H460 (non-small cell lung cancer), and A375-C5
(melanoma). Further studies were also carried out to clarify if the growth inhibitory
effect was associated with cell-cycle arrest and/or induction of apoptosis. Chalcone 1
was previously isolated from several plants [22–24] and synthesized by other groups
[25][26], but, to the best of our knowledge, no studies related to tumor cell-growth
inhibition have been conducted. As far as we know, four of the six prenylchalcones, 2, 3,
5, and 7, are described here for the first time.
Results and Discussion. – Synthesis. One naturally occurring chalcone, 1, and six
prenyl analogs, 2–7, were synthesized. The synthetic strategies applied for the synthesis
of new chalcones were outlined in Schemes 1–3.
Chalcone 1 was synthesized in analogy to the procedures described in [25][26] by
base-catalyzed aldol condensation of suitable acetophenone with 3,4,5-trimethoxybenz-
aldehyde to afford the desired chalcone in 90% yield. Compound 1 was then used as
precursor for the synthesis of the O-prenylated derivatives 2 and 3. This synthetic
approach was based on the O-alkylation of 1 with prenyl or geranyl bromide in
presence of tetrabutylammonium hydroxide (TBAOH; Bu4NOH) at room temper-
ature [12] (Scheme 1), leading to (prenyloxy)chalcone 2 (35% yield) and (geranyl-
oxy)chalcone 3 (62% yield), respectively.
The C-prenylated derivatives 4–7 were prepared by base-catalyzed aldol con-
densation of different prenylacetophenones with 3,4,5-trimethoxybenzaldehyde. The
treatment of 2’,4’,6’-trihydroxyacetophenone with prenyl or geranyl bromide was
performed as reported in [27][28] to yield, as major products, the expected C-
prenylated acetophenones [27–30]. These acetophenones were then regioselectively
protected with ClCH2OMe according to the method described in [27][28] to give the

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
1135
Fig. 1. Structures of non-prenylated and prenylated chalcones (1 and 2–7, resp.; the numbering used for
NMR assignments)
predictable protected compounds [27][28][30][31] in ca. 30% yield. The condensation
of the partially protected acetophenones with 3,4,5-trimethoxybenzaldehyde was
attempted by treatment with several bases, for instance, NaOH in EtOH, and NaH in
THF; however, such strategies failed (data not shown). Hence, the synthesis of 4 and 5
was achieved by aldol condensation of the corresponding protected acetophenones
with 3,4,5-trimethoxybenzaldehyde, using an excess of (iPr)2NLi (LDA) [32], to afford

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Scheme 1. Synthesis of O-Prenylated Derivatives 2 and 3
a) Prenyl bromide (¼1-bromo-3-methylbut-2-ene), Bu4NOH (TBAOH), CH2Cl2/toluene, r.t., 20 h.
b) Geranyl bromide (¼(E)-1-bromo-3,7-dimethylocta-2,7-diene), TBAOH, CH2Cl2/toluene, r.t., 20 h.
the prenylated chalcones 4 [33] and 5 (Scheme 2) in reasonable yields (46 and 47%,
resp.).
Compounds 6 and 7 were also prepared by base-catalyzed aldol condensation of the
appropriately substituted acetophenones with 3,4,5-trimethoxybenzaldehyde. First, 2’-
hydroxy-4’,6’-dimethoxyacetophenone was prenylated by the reaction with prenyl
bromide in alkaline medium in analogy to [29] to afford 2’-hydroxy-4’,6’-dimethoxy-3’-
prenylacetophenone [34], or with geranyl bromide using tBuONa as a base to afford 3’-
Scheme 2. Synthesis of the C-Prenylated Chalcone 5
a) (iPr)2NLi (LDA), THF, ꢀ788, r.t., overnight.

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
1137
geranyl-2’-hydroxy-4’,6’-dimethoxyacetophenone. Then, these isoprenylated acetophe-
nones were condensed with 3,4,5-trimethoxybenzaldehyde by treatment with LDA [32]
to give the prenylated chalcones 6 [34] and 7 (in 17 and 14% yield, resp., Scheme 3).
Scheme 3. Synthesis of C-Prenylated Chalcone 7
a) Geranyl bromide, tBuONa, DMF, 808, overnight. b) LDA, THF, ꢀ788, r.t., overnight.
The structures of compounds 1–7 were established on the basis of IR, NMR, and
HR-MS techniques. 13C-NMR Assignments were achieved by 2D heteronuclear single
quantum correlation (HSQC) and heteronuclear multiple bond correlation (HMBC)
experiments. For chalcone 7, the signal of the OH group did not appear, probably due to
some exchange with DCl in the solvent used (CDCl3). The coupling constants of the
vinylic system (J¼15.5–16.0 Hz) confirmed the (E)-configuration of the chalcone
moiety.
Biological Activity. The effects of the synthesized chalcones on the in vitro growth of
three human tumor cell lines, MCF-7 (breast adenocarcinoma), NCI-H460 (non-small
cell lung cancer), and A375-C5 (melanoma), were evaluated according to the
procedure adopted by the National Cancer Institute (NCI, USA) that uses the protein-
binding dye sulforhodamine B (SRB) to assess cell growth [35][36]. By this procedure,
a doseꢀresponse curve was obtained for each cell line and each test compound. The
concentration that caused cell growth inhibition of 50% (GI50) was determined as
described in [36]. The results are compiled in Table 1 and discussed below.
From Table 1, it can be seen that, among all prenylated derivatives, the 2’-
(prenyloxy)chalcone 2 exhibited the lowest GI50 values (< 8 mm) against the three
human tumor cell lines studied. Comparing the prenylated chalcones 2, 3, and 6 with
the non-prenylated chalcone 1, it can be concluded that the presence of a prenyl or
geranyloxy group at C(2’) (compounds 2 and 3) or a prenyloxy group at C(3’)
(compound 6) of the chalcone scaffold was associated with an increase in the growth-
inhibitory effect on the MCF-7 cells, and an appearance of activity against NCI-H460
and A375-C5 cells. On the contrary, the presence of a geranyl group at C(3’) of
compound 7 was associated with a complete loss of activity (GI50 >150 mm) against the
MCF-7 cell line.

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Table 1. Effect of Chalcone 1 and Prenylated Chalcones 2–7 on the Growth of Human Tumor Cell
Linesa)
Compound
GI50b) [mm]
MCF-7
NCI-H460
A375-C5
1
2
3
4
5
6
7
33.0ꢂ4.1
6.2ꢂ0.8
>150
>150
5.9ꢂ0.5
7.7ꢂ0.6
24.2ꢂ1.6
8.5ꢂ0.6
22.0ꢂ1.6
21.0ꢂ3.1
28.0ꢂ0.8
20.3ꢂ4.2
15.0ꢂ1.8
19.3ꢂ1.1
>150
>150
>150
13.5ꢂ2.6
>150
86.0ꢂ9.6
53.0ꢂ6.1
>150
86.0ꢂ9.6
Doxorubicinc)
68.8ꢂ15.2
a) Data represent meanꢂSEM from at least three independent experiments performed in duplicate.
b) Concentration that was able to cause 50% of cell growth inhibition after a continuous exposure of
48 h. c) Doxorubicin was used as positive control, and the concentrations are presented in nm.
For chalcones 4 and 5, the results revealed a pronounced growth-inhibitory effect
against MCF-7 cells (GI50 ꢁ15 mm), but a lower activity (compound 4) or an absence of
activity (compound 5) against NCI-H460 and A375-C5 cell lines.
The comparison of the GI50 values of prenylated compounds 2, 4, and 6 with those of
the corresponding geranylated derivatives 3, 5, and 7 indicates that the prenylation
instead of geranylation is a better strategy to improve cell growth-inhibitory activity of
this family of compounds.
To clarify the mechanism of action of the most active compounds (GI50 <25 mm),
flow cytometric analysis of the cell-cycle profiles and of levels of apoptosis in the MCF-
7 cells treated with those compounds was carried out. Regarding the cell-cycle analysis,
results showed that all tested compounds interfered with the normal cell-cycle
distribution of the MCF-7 cell line. All the compounds increased the sub-G1 peak,
indicating DNA degradation, characteristic for apoptosis (Fig. 2). Among all the
compounds studied, 5 was the most potent, increasing the sub-G1 peak more than 20%
with respect to the DMSO control. To confirm this observation, the most active
compounds, 2–6, were evaluated concerning their capability to induce apoptosis in the
MCF-7 cell line. Results showed that all the compounds studied (except 4) increased
the levels of apoptosis. The geranylated derivative 5 was the most active chalcone, with
the levels of apoptosis rising from 15.9% (DMSO control) to 30.6% after treatment
with the GI50 concentration of this compound (Table 2).
Conclusions. – Prenylchalcone-type compounds 2–7 were synthesized by molecular
modification of the naturally occurring chalcone 1 or via base-catalyzed aldol
condensation of prenylated acetophenones with 3,4,5-trimethoxybenzaldehyde. These
synthetic routes provided six biologically interesting prenylchalcones in reasonable
yields. Greater insights into the antitumor mechanism of the action of prenylchalcones
were achieved. Regarding the cell-cycle profile studies, all the tested compounds
caused an increase in the levels of the sub-G1 population of cells when compared with

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
1139
Fig. 2. Cell-cycle analysis of MCF-7 cells treated with the chalcones with GI50 values <25 mm. Cells were
cultured with the indicated GI50 concentrations of the tested compounds 2–6 for 48 h. Appropriate
controls were included: untreated cells (blank) and cells treated with the highest concentration of
DMSO used to dissolve the compounds. Results represent the meanꢂSEM of three independent
experiments. *: pꢁ0.05. and **: pꢁ0.01, when comparing the effect of each compound with the blank
(control).
Table 2. Apoptosis Levels of MCF-7 Cells Treated with the Chalcones with GI50 values <25 mm
Compounda)
Apoptotic cellsb) (% of total cells)
Blankc)
12.9ꢂ1.29
15.9ꢂ3.03
20.8ꢂ2.61*
19.3ꢂ3.14
13.3ꢂ3.54
30.6ꢂ0.49**
20.8ꢂ1.12**
DMSOc)
2 (6.2 mm)
3 (24.2 mm)
4 (8.5 mm)
5 (15.0 mm)
6 (19.3 mm)
a) Cells were cultured with the indicated GI50 concentrations of the tested compounds 2–6 for 48 h.
b) Results represent the meanꢂSEM of three independent experiments. *: pꢁ0.05 and **: pꢁ0.01, when
c
comparing the effect of each compound with the blank (control). ) Appropriate controls were included:
untreated cells (blank) and cells treated with the highest concentration of DMSO used to dissolve the
compounds.
DMSO control, which was in accordance with the results obtained by the annexin-V-
FITC/PI double staining assay. Using the MCF-7 cells, it was possible to conclude that
the effect of these compounds on cellular growth could be at least in part caused by an
apoptotic response. These results have confirmed the pro-apoptotic properties of
prenylated chalcones, supporting their further study as possible chemopreventive and
therapeutic agents against cancer.
ˆ
This work is supported through funds from FCT – Fundaca˜o para a Ciencia e a Tecnologia, the
project CEQUIMED – PEst-OE/SAU/UI4040/2011, by FEDER funds through the COMPETE program
under the projects FCOMP-01-0124-FEDER-011057 and FCOMP-01-0124-FEDER-015752, and by U.
Porto and Santander-Totta. The authors also thank the Portuguese National NMR Network (RNRMN)
for financial support. M. P. N. and R. T. L. thank also FCT for Ph.D. and postdoctoral grants (SFRH/BD/

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
21770/2005 and SFRH/BPD/68787/2010, resp.). K. C. thanks for the scholarships of Rangsit University
and the postgraduate program of Kasetsart University. The authors are also indebted to the National
Cancer Institute, Bethesda, MD, USA, for the generous gift of the NCI-H460 cell line.
Experimental Part
Chemistry. General. All reagents were from commercial sources and used without purification.
Column chromatography (CC): Macherey-Nagel silica gel 60 (SiO2; 0.04–0.063 mm), GraceResolvꢃ SiO2
cartridges (5 g/25 ml). TLC: Macherey-Nagel SiO2 60 (GF254) plates. M.p.: Kofler microscope;
uncorrected. IR Spectra: ATI Mattson Genesis series FT-IR (software: WinFirst, v. 2.10) spectropho-
tometer; KBr microplates; ˜n in cmꢀ1. 1H- and 13C-NMR spectra: Bruker Avance 300 and 500 instruments;
at r.t. in CDCl3; d in ppm rel. to Me4Si as internal standard, J in Hz. HR-EI-MS: GC-TOF mass
spectrometer (Waters); in m/z; HR-ESI-MS: APEX-Qe FT-ICR MS mass spectrometer (Bruker
Daltonics) or microTOF (focus) mass spectrometer (Bruker Daltonics); in m/z.
(2E)-1-{2,4-Dimethoxy-6-[(3-methylbut-2-en-1-yl)oxy]phenyl}-3-(3,4,5-trimethoxyphenyl)prop-2-
en-1-one (2). To a soln. of chalcone 1 (0.20 g, 0.53 mmol) [25][26] and TBAOH·30 H2O (0.87 g,
1.07 mmol) in CH2Cl2/toluene 10 :7 (17 ml) was added prenyl bromide (93.4 ml, 0.80 mmol), and the
reaction was allowed to proceed for 20 h at r.t. under gentle stirring. The reaction was quenched with H2O
and the mixture was extracted with AcOEt (3ꢃ50 ml). The combined org. layers were washed with H2O,
dried (Na2SO4), and evaporated under reduced pressure [12]. The resulting residue was purified by flash
column chromatography (FC; SiO2, hexane/AcOEt) and then by prep. TLC (SiO2; toluene/AcOEt 8 :2)
to afford 2 (0.08 g, 35%). M.p. 95–998. IR (KBr): 2996, 2927, 2840, 1643, 1602, 1580, 1500, 1456, 1415,
1380, 1336, 1314, 1271, 1244, 1221, 1200, 1154, 1121. 1H-NMR (300 MHz, CDCl3): 7.24 (d, J¼16.0,
HꢀC(b)); 6.86 (d, J¼16.0, HꢀC(a)); 6.74 (s, HꢀC(2,6)); 6.16 (br. s, HꢀC(3’,5’)); 5.36–5.32 (m,
HꢀC(2’’)); 4.50 (d, J¼6.5, CH2(1’’)); 3.87 (2s, MeOꢀC(3,5)); 3.85 (s, MeOꢀC(4)); 3.79 (s, MeOꢀC(4’));
3.76 (s, MeOꢀC(6’)); 1.68 (s, HꢀC(5’’)); 1.65 (s, HꢀC(4’’)). 13C-NMR (75 MHz, CDCl3): 194.3 (C¼O);
162.2 (C(4’)); 158.8 (C(6’)); 158.1 (C(2’)); 153.3 (C(3,5)); 144.2 (C(b)); 140.0 (C(4)); 137.5 (C(3’’)); 130.6
(C(1)); 128.6 (C(a)); 119.6 (C(2’’)); 111.6 (C(1’)); 105.4 (C(2,6)); 92.0 (C(3’)); 90.8 (C(5’)); 65.8 (C(1’’));
60.9 (MeOꢀC(4)); 56.1 (MeOꢀC(3,5)); 55.9 (MeOꢀC(6’)); 55.4 (MeOꢀC(4’)); 25.7 (C(5’’)); 18.2
(C(4’’)). HR-ESI-MS: 443.2064 ([MþH]þ , C25H31O7þ ; calc. 443.2070).
(2E)-1-(2-{[(2E)-3,7-Dimethylocta-2,6-dien-1-yl]oxy}-4,6-dimethoxyphenyl)-3-(3,4,5-trimethoxy-
phenyl)prop-2-en-1-one (3). To a soln. of 1 (0.14 g, 0.40 mmol) and TBAOH · 30 H2O (0.66 g,
0.80 mmol) in CH2Cl2/toluene 1:1 (20 ml) was added geranyl bromide (115.0 ml, 0.60 mmol), and the
reaction was allowed to proceed for 20 h at r.t. under gentle stirring. The reaction was quenched with
H2O, and the mixture was extracted with AcOEt (3ꢃ50 ml). The combined org. layers were washed with
H2O, dried (Na2SO4), and evaporated under reduced pressure [12]. The resulting residue was purified by
FC (SiO2, petroleum ether (PE)/AcOEt 8 :2) to afford 3 (0.13 g, 62%). IR (KBr): 2926, 2843, 1645, 1599,
1500, 1456, 1418, 1381, 1333, 1268, 1244, 1152, 1120. 1H-NMR (300 MHz, CDCl3): 7.24 (d, J¼15.6,
HꢀC(b)); 6.87 (d, J¼15.6, HꢀC(a)); 6.74 (s, HꢀC(2,6)); 6.16 (br. s, HꢀC(3’,5’)); 5.36–5.32 (m,
HꢀC(2’’)); 5.04–4.99 (m, HꢀC(7’’)); 4.53 (d, J¼6.2, CH2(1’’)); 3.87 (s, MeOꢀC(3,5)); 3.85 (s,
MeOꢀC(4)); 3.79 (s, MeOꢀC(4’)); 3.77 (s, MeOꢀC(6’)); 2.05–1.95 (m, CH2(5’’,6’’)); 1.65 (s, Me(4’’));
1.63 (s, Me(9’’)); 1.55 (s, Me(10’’)). 13C-NMR (75 MHz, CDCl3): 194.3 (C¼O); 162.1 (C(4’)); 158.7
(C(6’)); 158.0 (C(2’)); 153.3 (C(3,5)); 144.2 (C(b)); 140.6 (C(3’’)); 139.9 (C(4)); 131.7 (C(8’’)); 130.5
(C(1)); 128.6 (C(a)); 123.6 (C(7’’)); 119.5 (C(2’’)); 112.0 (C(1’)); 105.4 (C(2,6)); 92.0 (C(3’)); 90.8
(C(5’)); 65.8 (C(1’’)); 60.9 (MeOꢀC(4)); 56.1 (MeOꢀC(3,5)); 55.9 (MeOꢀC(6’)); 55.4 (MeOꢀC(4’));
39.4 (C(5’’)); 26.2 (C(6’’)); 25.6 (C(9’’)); 17.6 (C(10’’)); 16.6 (C(4’’)). HR-ESI-MS: 511.2706 ([MþH]þ
,
C30H39Oþ7 ; calc. 511.2696).
(2E)-1-{3-[(2E)-3,7-Dimethylocta-2,6-dien-1-yl]-2-hydroxy-4,6-bis(methoxymethoxy)phenyl}-3-
(3,4,5-trimethoxyphenyl)prop-2-en-1-one (5). To a soln. of LDA (3 mmol) in dry THF (14 ml) was added
a soln. of acetophenone 8 (1 mmol) in THF (3 ml) at ꢀ788. The mixture was stirred at ꢀ788 for 1 h, and
3,4,5-trimethoxybenzaldehyde (9; 3 mmol) in THF (3 ml) was added at ꢀ788. The resulting mixture was
stirred at ꢀ788 for 1 h, and then allowed to reach r.t. overnight. Sat. NH4Cl soln. (30 ml) was added

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
1141
dropwise, and the aq. layer was extracted with AcOEt (3ꢃ50 ml). The combined org. layers were
washed with brine, dried (Na2SO4), and evaporated under reduced pressure [32]. The resulting residue
was purified by CC (SiO2 cartridges; hexane/AcOEt) to afford 5 (0.14 g, 47%). IR (KBr): 3600–3300,
1
2959, 2923, 2841, 1618, 1578, 1499, 1451, 1412, 1313, 1273, 1124. H-NMR (300 MHz, CDCl3): 13.81 (s,
HOꢀC(2’)); 7.87 (d, J¼15.5, HꢀC(a)); 7.72 (d, J¼15.5, HꢀC(b)); 6.87 (s, HꢀC(2,6)); 6.38 (s, HꢀC(5’));
5.29 (s, CH2OꢀC(6’)); 5.26 (s, CH2OꢀC(4’)); 5.26–5.22 (m, HꢀC(2’’)); 5.14–5.07 (m, HꢀC(7’’)); 3.96 (s,
MeOꢀC(4)); 3.92 (s, MeOꢀC(3,5)); 3.54 (s, MeOCH2OꢀC(6’)); 3.50 (s, MeOCH2OꢀC(4’)); 3.36 (d, J¼
7.0, CH2(1’’)); 2.12–2.03 (m, CH2(6’’)); 2.00–1.95 (m, CH2(5’’)); 1.81 (s, Me(4’’)); 1.67 (s, Me(9’’)); 1.60 (s,
Me(10’’)). 13C-NMR (75 MHz, CDCl3): 193.0 (C¼O); 163.8 (C(2’)); 160.8 (C(4’)); 158.1 (C(6’)); 153.4
(C(3,5)); 142.2 (C(b)); 140.0 (C(4)); 135.0 (C(3’’)); 131.2 (C(8’’)); 131.1 (C(1)); 127.1 (C(a)); 124.4
(C(7’’)); 122.3 (C(2’’)); 112.2 (C(3’)); 107.7 (C(1’)); 105.5 (C(2,6)); 95.7 (CH2OꢀC(6’)); 93.9
(CH2OꢀC(4’)); 92.3 (C(5’)); 61.0 (MeOꢀC(4)); 57.0 (MeOCH2OꢀC(6’)); 56.3 (MeOCH2OꢀC(4’));
56.0 (MeOꢀC(3,5)); 39.8 (C(5’’)); 26.7 (C(6’’)); 25.7 (C(9’’)); 17.6 (C(10’’)); 16.1 (C(4’’)). HR-ESI-MS:
571.2889 ([MþH]þ , C32H43Oþ9 ; calc. 571.2907).
1-{3-[(2E)-3,7-Dimethylocta-2,6-dien-1-yl]-2-hydroxy-4,6-dimethoxyphenyl}ethanone (11). A mix-
ture of acetophenone 10 (0.34 g, 1.53 mmol), geranyl bromide (0.6 ml, 3.06 mmol), and tBuONa (0.31 g,
3.06 mmol) in dry DMF (10 ml) was gently stirred at 808 overnight. The mixture was acidified to pH of ca.
2 (10% HCl), and diluted with H2O (30 ml), and the aq. layer was extracted with AcOEt (3ꢃ50 ml).
The combined org. layers were washed with brine, dried (Na2SO4), and evaporated under reduced
pressure. The resulting residue was purified by FC (SiO2; PE/Et2O 6 :4) to afford 11 (0.24 g, 47%). IR
(KBr): 3600–3200, 2953, 2910, 2838, 1605, 1580, 1444, 1407, 1352, 1258, 1204, 1141, 1100. 1H-NMR
(300 MHz, CDCl3): 14.05 (s, HOꢀC(2’)); 5.95 (s, CH2(5’)); 5.29–5.19 (m, HꢀC(2’’)); 5.17–5.08 (m,
HꢀC(7’’)); 3.89 (s, MeOꢀC(6’)); 3.85 (s, MeOꢀC(4’)); 3.26 (d, J¼7.1, CH2(1’’)); 2.61 (s, Me(1)); 2.11–1.91
(m, CH2(5’’,6’’)); 1.76 (s, Me(4’’)); 1.68 (s, Me(9’’)); 1.60 (s, Me(10’’)). 13C-NMR (75 MHz, CDCl3): 203.1
(C¼O); 163.6 (C(2’)); 163.2 (C(4’)); 162.8 (C(6’)); 134.9 (C(3’’)); 131.2 (C(8’’)); 124.5 (C(7’’)); 122.4
(C(2’’)); 109.8 (C(3’)); 105.9 (C(1’)); 85.7 (C(5’)); 55.4 (MeOꢀC(4’,6’)); 39.7 (C(5’’)); 33.1 (C(1)); 26.7
(C(6’’)); 25.6 (C(9’’)); 21.2 (C(1’’)); 17.6 (C(10’’)); 16.0 (C(4’’)). HR-ESI-MS: 333.2059 ([MþH]þ
,
C20H29Oþ4 ; calc. 333.2066).
(2E)-1-{3-[(2E)-3,7-Dimethylocta-2,6-dien-1-yl]-2-hydroxy-4,6-dimethoxyphenyl}-3-(3,4,5-trimeth-
oxyphenyl)prop-2-en-1-one (7). To a soln. of LDA (1.0 ml, 1.81 mmol) in dry THF (10 ml) was added a
soln. of 11 (0.21 g, 0.60 mmol) in THF (3 ml) at ꢀ788. The mixture was stirred at ꢀ788 for 1 h, and 9
(0.36 g, 1.81 mmol) in THF (3 ml) was added at ꢀ788. The resulting mixture was stirred at ꢀ788 for 1 h
and then allowed to reach r.t. overnight. Sat. NH4Cl soln. (30 ml) was added dropwise, and the aq. layer
was extracted with AcOEt (3ꢃ50 ml). The combined org. layers were washed with brine, dried
(Na2SO4), and evaporated under reduced pressure [32]. The resulting residue was purified by CC (SiO2
cartridges; PE/AcOEt 7:3) to afford 7 (0.04 g, 14%). M.p. 183–1868. IR (KBr): 3500–3200, 2953, 2923,
2841, 1624, 1573, 1498, 1455, 1406, 1376, 1316, 1269, 1237, 1216, 1113. 1H-NMR (500 MHz, CDCl3): 7.80
(d, J¼15.5, HꢀC(a)); 7.70 (d, J¼15.5, HꢀC(b)); 6.86 (s, HꢀC(2,6)); 6.03 (s, HꢀC(5’)); 5.22 (t, J¼7.0,
HꢀC(2’’)); 5.12–5.07 (m, HꢀC(7’’)); 3.96 (s, MeOꢀC(6’)); 3.93 (s, MeOꢀC(3,4’,5)); 3.92 (s, MeOꢀC(4));
3.33 (d, J¼7.0, CH2(1’’)); 2.09–2.04 (m, CH2(6’’)); 2.00–1.98 (m, CH2(5’’)); 1.80 (s, Me(4’’)); 1.67 (s,
Me(9’’)); 1.60 (s, Me(10’’)). 13C-NMR (125 MHz, CDCl3): 192.7 (C¼O); 164.1 (C(2’)); 163.4 (C(4’)); 161.2
(C(6’)); 153.4 (C(3,5)); 141.9 (C(b)); 139.9 (C(4)); 134.9 (C(3’’)); 131.2 (C(1)); 131.1 (C(8’’)); 127.3
(C(a)); 124.5 (C(7’’)); 122.4 (C(2’’)); 110.3 (C(3’)); 106.5 (C(1’)); 105.4 (C(2,6)); 86.5 (C(5’)); 61.0
(MeOꢀC(4)); 56.1 (MeOꢀC(3,5)); 55.8 (MeOꢀC(6’)); 55.5 (MeOꢀC(4’)); 39.8 (C(5’’)); 26.7 (C(6’’));
25.7 (C(9’’)); 21.3 (C(1’’)); 17.7 (C(10’’)); 16.1 (C(4’’)). HR-ESI-MS: 511.2704 ([MþH]þ , C30H39O7þ ; calc.
511.2696).
Biological Activity. Stock solns. of compounds 2–7 were prepared in DMSO and stored at ꢀ208.
Appropriately diluted solns. were freshly prepared just prior to the assays. Three human tumor cell lines
were used: MCF-7 (breast adenocarcinoma, ECACC, UK), NCI-H460 (non-small cell lung cancer, a
kind gift from NCI, Bethesda, USA), and A375-C5 (melanoma, ECACC, UK). The human tumor cell
lines were grown as monolayer and routinely maintained in RPMI-1640 medium supplemented with 5%
heat-inactivated fetal bovine serum (FBS) at 378 in a humidified atmosphere containing 5% CO2.

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Growth-Inhibition Assay. The effect of chalcones on the in vitro growth of three human tumor cell
lines, MCF-7, NCI-H460, and A375-C5 (melanoma), was evaluated according to the procedure adopted
by the National Cancer Institute (NCI, USA) that uses the protein-binding dye sulforhodamine B (SRB)
to assess cell growth [35][36]. Cells were plated in 96-well plates at appropriate densities to ensure
exponential growth throughout the experimental period (5.0ꢃ103 cells/well for MCF-7 and NCI-H460,
and 7.5ꢃ103 cells/well for A375-C5) and then allowed to adhere overnight. Attached cells were exposed
for 48 h to five serial dilutions of each compound (150, 75, 37.5, 18.75, and 9.37 mm for compounds 1, 6, and
7, or 150, 50, 16.67, 5.56, and 1.85 mm for compounds 2–5). After this incubation period, adherent cells
were fixed in situ with Cl3CCOOH (TCA), washed, and stained with SRB [36][37]. The bound stain was
solubilized, and the absorbance was measured at 492 nm with a plate reader (Biotek Instruments Inc.
PowerWave XS, Winooski, USA). A doseꢀresponse curve was obtained for each cell line with each test
compound, and the concentration that caused cell-growth inhibition of 50% (GI50, i.e. concentration of
compound that inhibited 50% of the net cell growth), was determined as described in [36]. The effect of
the vehicle solvent (DMSO) on the growth of these cell lines was evaluated in preliminary experiments
by exposing untreated control cells to the maximum concentration of DMSO used in each assay. Final
concentrations of DMSO did not interfere with the biological activity tested (data not shown).
Cell-Cycle Analysis. MCF-7 Cells were plated in 6-well plates (5.0ꢃ104 cells/ml) and incubated at
378 for 24 h. Exponentially growing cells were then incubated with the compounds 2–6 at their respective
GI50 concentrations (previously determined with the SRB assay; cf. data in Table 1). Untreated cells
(control) or cells treated with the compoundꢄs solvent (DMSO) were included. DMSO was used at the
highest concentration used in the experiments. Following 48-h treatment, cells were centrifuged and fixed
in 70% EtOH at 48 for at least 12 h and subsequently resuspended in PBS containing 0.1 mg/ml RNase A
and 5 mg/ml propidium iodide. Cellular DNA content, for cell cycle distribution analysis, was measured
by flow cytometry with an Epics XL-MCL Coulter flow cytometer (Brea, CA, USA) plotting at least
20,000 events per sample, as described in [38]. The percentage of cells in the sub-G1, G1, S, and G2/M
phases of the cell cycle was determined by using the FlowJo 7.2 software (Tree Star, Inc., Ashland, OR,
USA) after cell-debris exclusion.
Analysis of Cellular Apoptosis. MCF-7 Cells were incubated with the compounds as described
previously for cell cycle-analysis assay. Cells were then harvested and the Human Annexin-V-FITC/PI
apoptosis kit (Bender MedSystems, A-Vienna) was used according to the manufacturerꢄs instructions to
detect apoptotic cells. Flow cytometry was carried out with an Epics XL-MCL Coulter flow cytometer
(Brea, CA, USA) plotting at least 20,000 events per sample. Data obtained from the flow cytometer was
analyzed by using the FlowJo 7.2 software (Tree Star, Inc., Ashland, OR, USA) as described in [39][40].
Statistical Analysis. All exper. data are presented as meansꢂSEM from at least three independent
experiments (most of them performed in duplicate). Statistical analyses was carried out using an
unpaired Studentꢄs t-test. All analyses were performed comparing cells treated with compounds with
blank cells (cells treated with medium only).
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Received May 31, 2011