New synthetic flavone derivatives induce apoptosis of hepatocarcinoma cells

Article abstract of DOI:10.1016/j.bmc.2010.07.019

Natural flavonoids have broad biological activity, including anticancer. In this study, a series of novel flavone derivatives were synthesized with the substitutions of chlorine, isopropyl, methoxy, and nitro groups on the benzene ring of flavone skeleton to develop effective anticancer agents. Antiproliferative assays showed that the synthesized chemicals possess notable activity against hepatocarcinoma cells (HepG-2); in particular, the compound 6f with chlorine and dimethoxy modifications at the two benzene rings showed an IC₅₀ at 1.1 μM to HepG-2. The 6f also displayed marked anticancer activity towards a panel of cancer cells, including nasopharyngeal carcinoma cells (CNE-2 and CNE-1), breast adenocarcinoma cell (MCF-7), and epithelial carcinoma cells (Hela). Exposing HepG-2 cells to compound 6f at 10 μM induced chromatin condensation, nuclear disassembly, and DNA fragmentation. In 6f-treated HepG-2 cells, the sub-G₀ population was remarkably increased; and in these cells, both caspase-8 and caspase-9 activity was significantly increased, which in turn activated caspase-3. In addition, proapoptotic Bax was upregulated by compound 6f while the antiapoptotic Bcl-2 was downregulated. Taken together, our data suggest that the new flavonoid derivative 6f triggers apoptosis through both death-receptor and mitochondria-dependent intrinsic pathways, being a potent therapeutic agent against hepatocarcinoma.

Full text of DOI:10.1016/j.bmc.2010.07.019

Bioorganic & Medicinal Chemistry 18 (2010) 6322–6328
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New synthetic flavone derivatives induce apoptosis of hepatocarcinoma cells
Huachen Liu a, , Aijun Dong a, , Chunmei Gao a, Chunyan Tan a, Zhenhua Xie a, Xuyu Zu a,
Long Qu a, Yuyang Jiang a,b,
*
a The Key Laboratory of Chemical Biology, Guangdong Province, Graduate School at Shenzhen, Tsinghua University, Lishui Road, Shenzhen 518055, PR China
b School of Medicine, Tsinghua University, Beijing 100084, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Natural flavonoids have broad biological activity, including anticancer. In this study, a series of novel fla-
vone derivatives were synthesized with the substitutions of chlorine, isopropyl, methoxy, and nitro
groups on the benzene ring of flavone skeleton to develop effective anticancer agents. Antiproliferative
assays showed that the synthesized chemicals possess notable activity against hepatocarcinoma cells
(HepG-2); in particular, the compound 6f with chlorine and dimethoxy modifications at the two benzene
Received 28 May 2010
Revised 7 July 2010
Accepted 8 July 2010
Available online 13 July 2010
rings showed an IC50 at 1.1 lM to HepG-2. The 6f also displayed marked anticancer activity towards a
panel of cancer cells, including nasopharyngeal carcinoma cells (CNE-2 and CNE-1), breast adenocarci-
noma cell (MCF-7), and epithelial carcinoma cells (Hela). Exposing HepG-2 cells to compound 6f at
10 lM induced chromatin condensation, nuclear disassembly, and DNA fragmentation. In 6f-treated
HepG-2 cells, the sub-G0 population was remarkably increased; and in these cells, both caspase-8 and
caspase-9 activity was significantly increased, which in turn activated caspase-3. In addition, proapoptot-
ic Bax was upregulated by compound 6f while the antiapoptotic Bcl-2 was downregulated. Taken
together, our data suggest that the new flavonoid derivative 6f triggers apoptosis through both death-
receptor and mitochondria-dependent intrinsic pathways, being a potent therapeutic agent against
hepatocarcinoma.
Keywords:
Flavone derivatives
Structure–activity relationship
Antiproliferative activity
Apoptosis
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
dence have shown that flavonoid compounds are able to induce
apoptosis in various human cancer cell lines.27,29 However, the
Apoptosis is essential for normal development and the mainte-
nance of homeostasis.1 It plays a necessary role to protect against
carcinogenesis by eliminating damaged cells.2 Many studies have
demonstrated that the dysregulation of apoptosis results in cancer
and this provides an approach to develop therapeutic agents via
inducing apoptosis. Design and development of small molecules
that can induce cancer cells to apoptosis is an attractive approach
for the development of new anticancer agents.3–6
Flavonoids are an extensive group of polyphenolic compounds
which have been found in plants and dietary components, such as
fruits, soy beans, vegetables, and red wine.7–9 A large number of bio-
logical effects have been attributed to these compounds, including
anticancer,10–14 antifungal,15,16 antiviral,17,18 anti-inflammatory,19
antioxidant,19,20 anti-osteoporotic,21,22 and anticarcinogenic activi-
ties.23 In particular, flavonoid compounds in the diet may act as che-
mopreventive agents against the development of cancers in the
alimentary tract.24,25 In this regard, a number of flavone derivatives
have been synthesized and their antiproliferative effects and antitu-
mor mechanisms have been investigated.26–28 Many lines of evi-
mechanism of flavonoids’ antitumor effects is not fully understood
because flavonoids with different structures display diverse biolog-
ical potency and mechanisms of action.
In this study, we synthesized a series of new flavonoid deriva-
tives with different substitutional groups and studied the struc-
ture–activity relationship. The results indicated that most of the
new synthetic derivatives displayed noticeable antiproliferative
activity against hepatocarcinoma cells (HepG-2) and the most ac-
tive compound 6f was selected to investigate the mechanisms of
action. Results showed that the synthetic new flavonoid deriva-
tives are able to induce cancer cell apoptosis via a caspase-depen-
dent pathway.
2. Results and discussion
2.1. Synthesis of new flavone derivatives
The synthesis route and structures of new flavone derivatives
investigated in this study were illustrated in Scheme 1. Synthetic
reactions involved an acetylation,
modified Schotten–Baumann reaction, and a Baker–Venkataraman
rearrangement, followed by cyclization catalyzed by acid.30
a Fries rearrangement, a
* Corresponding author. Tel./fax: +86 755 2603 2094.
E-mail address: jiangyy@sz.tsinghua.edu.cn (Y. Jiang).
Authors with equal contributions.
A
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.07.019

H. Liu et al. / Bioorg. Med. Chem. 18 (2010) 6322–6328
6323
R
1
O
O
OH
O
O
O
Z
Z
d
e
R
1
O
O
R
R
1
Z
4i-4l
6i-6l
5i-5l
1
R
R
O
5
O
O
R
R
R
6
4
OH
O
O
R
R
O
R
O
OH
OH
2
O
R
R
3
a
c
O
d
b
e
3
2
R
R
R
R
R
1
R
6
2
6
4
O
R
1
R
5
R
1
1
1
5
3
R
4
1
4a-4h
6a-6h
2
3
5a-5h
O
O
O
O
OH
O
OH
O
O
O
Z
Z
d
b
c
e
O
O
Z
4m-4n
5m-5n
6m-6n
Scheme 1. R1-R6, A and Z are defined in Tables 1 and 2. Reagents: (a) Ac2O, H2O, NaOH. (b) AlCl3, NaCl. (c) Substituted or non-substituted benzoyl chloride, pyridine. (d) KOH,
pyridine. (e) H2SO4, AcOH.
Table 2
series of
a-naphthalin compounds were synthesized in parallel
Cytotoxic activity of compounds 6i–6n against HepG-2 cells
(Scheme 1). Products were purified by column chromatography
a
and characterized by NMR and mass spectral analysis.
Compd
R1
Z
IC50 (lM)
6i
6j
6m
6k
6l
CH(CH3)2
Cl
—
CH(CH3)2
Cl
—
Cyclopentyl
Cyclopentyl
Cyclopentyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
8.6 1.1
13.4 4.7
6.1 3.8
18.0 5.2
20.0 7.6
2.9 0.9
2.2. Antiproliferative activity of new flavone derivatives
The antiproliferative activity of all newly synthesized flavone
derivatives were evaluated with HepG-2 cells in vitro. Viable cells
were determined using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide] assay after exposure for 72 h.33 As
summarized inTables 1 and 2, most chemicals displayed promising
antiproliferative activity against HepG-2 cells, except for 6g and 6h
6n
a
Viable cells were determined by MTT proliferation assays after incubation with
tested compound for 72 h.
an –OMe group at 30-position showed the most antiproliferative
activity. It is probably due to the more prominent resonance effect
than 40-position and 20-position. It is noteworthy that the introduc-
tion of two more methoxyl groups to the compound 6d, forming
compound 6e, enhanced up to three folds of the antiproliferative
activity, compared to the parental compound 6d; (3) use of a nitro
group as a substituent on the benzene ring (compounds 6g and 6h)
inactivated the flavone antiproliferative activity against HepG-2,
which suggests that electron-negativity in the benzene ring greatly
affected the compound activity; (4) finally, not only the substituent
groups, but also the backbone ring (benzene vs naphthalene)
played significant influence on the antiproliferation activity of
compounds, as indicated by the comparative data of compounds
(6m vs 6i/6j and 6n vs 6k/6l). Therefore, the naphthalene group
may improve the antiproliferation activity, which provides a new
idea for the development of novel flavone derivatives.
(IC50 >50 lM). A structure–activity analysis revealed several
findings as follows: (1) flavone antiproliferative activity depended
on the backbone of 2-substituent group, as suggested by the results
of compounds 6a, 6i, and 6k; (2) number and position of substitu-
ent methoxy groups greatly affected the antiproliferative potency
of flavones. Among compounds 6b, 6c, and 6d, the 6c with
Table 1
Cytotoxic activity of compounds 6a–6h against HepG-2 cells
a
Compd
R1
R2
R3
R4
R5
R6
IC50
(
lM)
6a
6b
6c
6d
6e
6f
CH(CH3)2
CH(CH3)2
CH(CH3)2
CH(CH3)2
CH(CH3)2
Cl
H
OCH3
H
H
H
H
H
H
H
H
H
H
H
OCH3
OCH3
H
H
NO2
H
H
H
H
OCH3
OCH3
H
H
H
H
H
H
H
H
H
6.4 1.8
5.4 1.7
2.1 0.7
9.9 1.9
3.6 0.6
1.1 0.3
>50
OCH3
H
OCH3
OCH3
NO2
H
Among the 14 compounds, compound 6f showed most promis-
ing antiproliferative activity against HepG-2 with an IC50 at 1.1 lM,
6g
6h
CH(CH3)2
CH(CH3)2
H
>50
and thus was tested for its activity towards a panel of other four
tumor cell lines. As shown in Figure 1A, compound 6f showed
significant cytotoxicity to all tested cell lines in a dose-dependent
a
Cell viability was determined by the MTT assay, IC50 denotes the concentration
of drug required for 50% inhibition of cell growth (HepG-2). Results are the means of
three independent determinations.

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H. Liu et al. / Bioorg. Med. Chem. 18 (2010) 6322–6328
the endonucleolytic DNA cleavage was checked by agarose gel
electrophoresis to further confirm 6f-induced cell apoptosis. Cleav-
age of DNA at the internucleosomal linker sites yielding DNA frag-
ments in multiples fragments (180–200 bp) was regarded as a
biochemical characteristic of apoptosis.32 As shown in Figure 2B,
DNA fragmentation occurred in a dose–response manner in 6f-
treated cells, whereas no obvious DNA ladders were observed in
the control cells.
Compound 6f-induced apoptosis of HepG-2 cells was further
proven by a FACScan analysis. In the flow cytometry analysis, the
sub-G0 cell population is considered as apoptotic cells.33 With pro-
pidium iodide (PI) staining for DNA-content histogram, we as-
sessed the sub-G0 populations of HepG-2 cells in the presence of
the compound 6f at different concentrations. The results demon-
strated that the sub-G0 phase of the cell cycle in HepG-2 cells con-
sisted of 3.41%, 21.54%, 34.54%, 55.83%, and 79.32% at 0, 5, 10, 25,
and 50 lM of compound 6f, respectively; accordingly, cells in the
G1, S, and G2 phases were concomitantly decreased. Together these
data suggested that the compound 6f efficiently induced apoptosis
of HepG-2 cells.
2.4. Compound 6f-induced apoptosis is mediated via caspase-
dependent pathways
Apoptosis is triggered by either intrinsic mitochondrial or death-
receptor pathways; the mitochondrial pathway includes caspase-
dependent or independent apoptosis.34,35 Caspases, a family of
cystein proteases, are capable of cleaving essential cellular sub-
strates after aspartic residues and are critical for the initiation and
execution phases of apoptosis.36–38 To understand the underlying
pathway(s) of compound 6f-induced apoptosis, we examined cas-
pase-3 activity via measuring Ac-DEVD-pNA cleavage of HepG-2 cell
lysates collected at 0, 4, 8, 12, 16, and 24 h after compound 6f treat-
ment. As shown in Figure 3A-a, the activity of caspase-3 was in-
creased with time and peaked at 12 h after treatment. Thereafter,
caspase-3 activity gradually decreased, which may be due to
massive loss of cells. These results indicated that the activation of
caspase-3 was involved in the compound 6f-induced cell apoptosis.
It is understood that caspase-8 is involved in the death-receptor
pathway while caspase-9 mediates the mitochondrial pathway.39
Once activated, both caspase-8 and caspase-9 activates down-
stream caspase-3, triggering cell apoptosis. Therefore, we mea-
sured the activity of caspase-8 and caspase-9, respectively, and
the results showed that both caspase-8 and caspase-9 were acti-
vated dramatically with an activity peak at 8 h after treatment
with the compound 6f, suggesting that both mitochondrial and
death-receptor pathways were involved in the 6f-induced apopto-
sis (Fig. 3A-b). Nevertheless, compared to caspase-8, the activity of
caspase-9 was higher and lasted longer in response to the 6f, sug-
gesting that the apoptosis proceeded mainly via a caspase-depen-
dent intrinsic mitochondria pathway.
Figure 1. Antiproliferative activity of compound 6f. (A) Effect of 6f on the viability
of HepG-2 (j), Hela (d), MCF-7 (N), CNE-1 (.), and CNE-2 (ꢀ) cells. (B) Effect of 6f
on colony formation of HepG-2 cells.
manner. Although the antiproliferative activity varied with cell
lines, the effective IC50 is at approximate 10
for CNE-2 cells (Table 3).
lM or less, except
Antiproliferative activity of compound 6f was further confirmed
by the clonogenic growth of HepG-2 cells, a critical feature of tu-
mor cells (Fig. 1B). Results showed that compound 6f at a concen-
tration of low up to 2.5 lM greatly decreased the colony formation
capability of HepG-2 cells. Together these data suggest that 6f is a
potent anticancer agent.
2.3. Compound 6f induces HepG-2 cell apoptosis
HepG-2 cells were most sensitive to the compound 6f, and thus
were used to investigate the molecular mechanism of action.
Apoptosis is a programmed cell death, characterized with chro-
maqtin condensation and internucleosomal DNA fragmentation.31
To understand whether compound 6f induces apoptosis of HepG-
2 cells, we examined cell morphology and DNA fragmentation
when exposed to compound 6f at 10 lM. As shown in Figure 2A,
compound 6f led to significant chromatin condensation, nuclear
deformation, and disassembly, compared to control cells. Next,
Bcl-2 family genes have a central role in controlling the mito-
chondrial pathway.4 Therefore, we examined the expression of
Bcl-2, an apoptotic inhibitor, and Bax, an apoptotic inducer using
semi-quantitative RT-PCR. The results indicated that the com-
pound 6f led to a dose-dependent suppression of Bcl-2 expression,
accompanied by concomitant increase of Bax expression (Fig. 3B).
These results suggest that compound 6f induces apoptosis of
HepG-2 cells via both death-receptor and the mitochondria-depen-
dent apoptotic cascade.
Table 3
Growth inhibition compound 6f on different tumor cells
Cell line
Hela
3.0
MCF-7
7.4
CNE-1
10.4
CNE-2
12.9
3. Conclusion
a
IC50
(lM)
a
We have designed and synthesized a series of new flavone
derivatives with potent antiproliferative activity against HepG-2
Viable cells were determined by MTT proliferation assays after incubation with
tested compound for 72 h.

H. Liu et al. / Bioorg. Med. Chem. 18 (2010) 6322–6328
6325
Figure 2. Apoptosis of HepG-2 cells induced by compound 6f. (A) Fluorescent staining of nuclei by Hoechst 33258. (B) DNA fragmentation. HepG-2 cells were treated with
compound 6f at 0, 5, 10, 25, and 50 M for 36 h and DNA fragments were prepared. (C) Dose-dependent effects of 6f on cell cycle phase distributions. Cells were treated with
0, 5, 10, 25, and 50 M 6f for 36 h and analyzed by flow cytometry.
l
l
cells in vitro. The cytotoxic activity of the new synthetic flavone
derivatives was dependent on the nature and position of the sub-
stituents on the two benzene rings. Among the synthetic com-
pounds, the 6f is the most active, inducing death-receptor and
mitochondrial-dependent apoptosis via triggering the caspase cas-
cade. Therefore, the compound 6f may be a potent lead compound
for further optimization as a chemotherapeutic agent for hepato-
carcinoma therapy.
13C NMR with tetramethylsilane as the internal standard. Melting
points were determined by the open capillary method on a SGW
X-4 electrothermal melting apparatus and are uncorrected. Reac-
tion progress was monitored using analytical thin layer chroma-
tography (TLC) on pre-coated silica gel plates and the spots were
detected under UV light (254 nm). High-resolution mass spectra
were obtained with Waters micromass Q-Tof Premier mass spec-
trometer. All other reagents and analytical grade solvents were
bought from commercial sources and used without further purifi-
cation unless indicated. Pyridine was distilled from anhydrous
potassium hydroxide (KOH) immediately prior to use.
4. Experimental section
4.1. Chemistry
4.1.1. Synthesis of acetic acid 4-isopropyl-phenyl ester (2)
10 mmol of 1 (4-isopropyl-phenol) was added into the NaOH
(15 mmol) of ice water solution keeping rapidly stirring, then the
1H NMR and 13C NMR spectra were obtained in CDCl3 at
300 MHz or 500 MHz for 1H NMR and 75 MHz or 125 MHz for

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H. Liu et al. / Bioorg. Med. Chem. 18 (2010) 6322–6328
Figure 3. Mechanism of compound 6f-induced apoptosis. (A) Effect of 6f on caspase activity. (a) Caspase-3 activity was detected by Ac-DEVD-pNA at 0, 4, 8, 12, 16, and 24 h
after treated by 6f at 50 M. (b) Caspase-8 and (c) capase-9 activities detected by Ac-IETD-pNA and Ac-LEHD-pNA at 0, 4, 8, 12, and 16 h of compound 6f treatment at 50 M.
(B) Effects of 6f on Bcl-2 and Bax expression. Cells were treated with 0, 5, 10, 25, and 50 M for 26 h and then harvested. The total RNA was extracted, and then cDNA was
synthesized from total RNA (1 g) which was amplified using the sets of primers for examining the expression of Bcl-2 and Bax.
l
l
l
l
acetic anhydride was dropped into foregoing solution at ice bath,
keeping the dropped in an appropriative velocity. The mixture
was stirred for 3 h and extracted with EtOAc, the organic layer
was collected, dried over anhydrous NaSO4, and evaporated under
vacuum to obtain the desired crude product 2, which was used
without further purification. Yield: 90%.
precipitate formed was collected by filtration and purified by
chromatography.
4.1.4. Synthesis of compounds 6a–6n
All of the compounds in the 6 series were synthesized according
to the literature as outlined in Scheme 1.30,40 Concentrated sulfuric
acid (0.2 ml), compound 5 (0.5 mmol) and 5 ml of glacial acetic
acid were stirred at 80 °C for 2 h. After the reaction mixture was
cooled to room temperature, 25 ml ice water was slowly added
with rapidly stirring. The crude products were collected by filtra-
tion, washed with water, and purified by chromatography.
4.1.2. Synthesis of 1-(2-hydroxy-5-isopropyl-phenyl)-ethanone
(3)
In
a 100-ml round-bottomed flask, 5 mmol compound 2,
10 mmol NaCl, and 10 mmol anhydrous AlCl3 were added. The
temperature of the reaction mixture was heated and maintained
at 110 °C for 1.5 h while being stirred continuously. Then ice was
added into flask slightly. The mixture was extracted with 20 ml
EtOAc, the organic layer was dried over anhydrous NaSO4 and
the dried solution was concentrated to get the crude product,
which on purification by column chromatography afforded pure
compound 3. Yield: 67%.
4.1.4.1. 6-Isopropylflavone (6a). Yield: 67%; slight yellow solid,
mp: 83–86 °C; 1H NMR (CDCl3, 300 MHz) d 1.30 (d, J = 6.90 Hz,
6H), 3.03 (m, 1H), 6.82 (s, 1H), 7.50 (m, 5H), 7.89 (m, 2H), 8.07
(m, 1H); 13C NMR (CDCl3, 75 MHz) d 23.93, 33.78, 107.33, 117.96,
122.38, 123.63, 126.22, 128.99, 131.50, 131.81, 132.73, 146.14,
154.68, 163.23, 178.69; HRMS m/z calcd for [C18H17O2]+:
265.1229; found: 265.1219.
4.1.3. General synthesis of compounds 530
Compound 4 (1.0 mmol) was dissolved in 5 ml of anhydrous
pyridine containing finely powdered potassium hydroxide
(84 mg, 1.5 mmol) and stirred at 50 °C until TLC indicated com-
pletion of reaction. After the mixture was cooled to room tem-
perature and 25 ml of 10% acetic acid (aq) was added, the
4.1.4.2. 6-Isopropyl-20-methoxylflavone (6b). Yield: 76%; slight
yellow oil; 1H NMR (CDCl3, 500 MHz) d 1.30 (d, J = 6.90 Hz, 6H),
3.03 (m, 1H), 3.91 (s, 3H), 7.01 (m, 1H), 7.07 (m, 1H), 7.13 (s, 1H),
7.43 (m, 2H), 7.53 (m, 1H), 7.87 (m, 1H), 8.08 (m, 1H); 13C NMR
(CDCl3, 125 MHz) d 23.79, 33.64, 55.49, 111.64, 112.29, 117.75,

H. Liu et al. / Bioorg. Med. Chem. 18 (2010) 6322–6328
6327
120.56, 120.83, 122.15, 123.39, 129.09, 132.14, 132.29, 145.63,
154.80, 157.83, 160.59, 178.90; HRMS m/z calcd for [C19H19O3]+:
295.1334; found: 295.1329.
3H), 1.78 (m, 1H), 1.88 (m, 2H), 2.04 (m, 2H), 2.53 (m, 1H), 6.17
(s, 1H), 7.39 (m, 1H), 7.57 (m, 1H), 8.13 (m, 1H); 13C NMR (CDCl3,
125 MHz) d 25.87, 30.44, 42.83, 107.91, 119.66, 124.79, 125.10,
130.77, 133.62, 154.85, 173.82, 177.46; HRMS m/z calcd for
[C14H14ClO2]+: 249.0682; found: 249.0688.
4.1.4.3. 6-Isopropyl-30-methoxylflavone (6c). Yield: 81%; white
solid, mp: 114–116 °C; 1H NMR (CDCl3, 500 MHz) d 1.31 (d,
J = 6.90 Hz, 6H), 3.04 (m, 1H), 3.90 (s, 3H), 6.82 (s, 1H), 7.07 (m,
1H), 7.43 (m, 2H), 7.50 (m, 2H), 7.58 (m, 1H), 8.80 (m, 1H); 13C
NMR (CDCl3, 75 MHz) d 23.95, 33.82, 55.47, 107.64, 111.74,
117.10, 117.99, 118.72, 122.42, 123.70, 130.11, 132.75, 133.28,
146.22, 154.71, 160.02, 163.08, 178.72; HRMS m/z calcd for
[C19H19O3]+: 295.1334; found: 295.1336.
4.1.4.11. 2-Cyclohexyl-6-isopropyl-chromen-4-one (6k). Yield:
59%; white oil; 1H NMR (CDCl3, 300 MHz) d 1.29 (d, J = 6.90 Hz,
6H), 1.40 (m, 5H), 1.77 (m, 1H), 1.87 (m, 2H), 2.03 (m, 2H), 2.53
(m, 1H), 3.02 (m, 1H), 6.20 (s, 1H), 7.37 (m, 1H), 7.53 (m, 1H),
8.03 (m, 1H); 13C NMR (CDCl3, 125 MHz) d 23.79, 25.62, 25.73,
30.46, 33.63, 42.76, 107.54, 117.55, 122.25, 123.35, 132.18,
145.63, 154.85, 173.25, 178.90; HRMS m/z calcd for [C18H23O2]+:
271.1698; found: 271.1705.
4.1.4.4. 6-Isopropyl-40-methoxylflavone (6d). Yield: 78%; white
solid, mp: 134–136 °C; 1H NMR (CDCl3, 300 MHz) d 1.32 (d,
J = 6.90 Hz, 6H), 3.03 (m, 1H), 3.90 (s, 3H), 6.78 (s, 1H), 7.03 (m,
2H), 7.49 (m, 1H), 7.58 (m, 1H), 7.90 (m, 2H), 8.07 (m, 1H); 13C
NMR (CDCl3, 75 MHz) d 23.96, 33.81, 55.50, 106.01, 114.43,
117.84, 122.39, 123.63, 124.14, 127.96, 132.51, 146.01, 154.63,
162.33, 163.27, 178.66; HRMS m/z calcd for [C19H19O3]+:
295.1334; found: 295.1336.
4.1.4.12. 2-Cyclohexyl-6-chloro-chromen-4-one (6l). Yield:
64%; white solid, mp: 101–105 °C; 1H NMR (CDCl3, 300 MHz) d
1.76 (m, 8H), 2.08 (m, 2H), 3.01 (m, 1H), 6.21 (s, 1H), 7.38 (m,
1H), 7.56 (m, 1H), 8.10 (m, 1H); 13C NMR (CDCl3, 75 MHz): d
25.53, 31.01, 44.15, 108.18, 119.62, 124.67, 124.97, 130.66,
133.51, 154.81, 173.17, 177.20; HRMS m/z calcd for [C15H16ClO2]+:
263.0839; found: 263.0842.
4.1.4.5. 6-Isopropyl-30,40,50-trimethoxylflavone (6e). Yield: 55%;
brown oil; 1H NMR (CDCl3, 500 MHz) d 1.32 (d, 6H, J = 6.95 Hz), 3.05
(m, 1H), 3.94 (s, 3H), 3.96 (s, 6H), 6.81 (s, 1H), 7.14 (s, 2H), 7.52 (d, 1H,
J = 8.60 Hz), 7.58 (m, 1H), 8.07 (d, 1H, J = 2.152 Hz); 13C NMR (CDCl3,
125 MHz) d 23.76, 33.63, 56.19, 60.84, 103.66, 106.99, 117.77,
122.23, 123.44, 126.92, 132.55, 141.08, 146.08, 153.42, 154.50,
162.98, 178.49; HRMS m/z calcd for [C21H23O5]+: 355.1546; found:
355.1538.
4.1.4.13. 2-Cyclopentyl-benzo[h]chromen-4-one (6m). Yield:
70%; white solid, mp: 85–87 °C; 1H NMR (CDCl3, 300 MHz) d 1.85
(m, 6H), 2.18 (m, 2H), 3.16 (m, 1H), 6.40 (s, 1H), 7.68 (m, 3H),
7.91 (m, 1H), 8.13 (m, 1H), 8.44 (m, 1H); 13C NMR (CDCl3,
125 MHz) d 25.69, 31.40, 44.15, 109.63, 119.96, 120.85, 122.14,
124.04, 124.84, 126.97, 128.12, 129.04, 135.82, 153.80, 171.74,
178.32; HRMS m/z calcd for [C18H17O2]+: 265.1229; found:
265.1226.
4.1.4.6. 6-Chloro-30,50-dimethoxylflavone (6f). Yield: 62%; slight
yellow solid, mp: 175–179 °C; 1H NMR (CDCl3, 300 MHz) d 3.87 (s,
6H), 6.62 (m, 1H), 6.78 (s, 1H), 7.01 (m, 2H), 7.52 (m, 1H), 7.64 (m,
1H), 8.18 (m, 1H); 13C NMR (CDCl3, 75 MHz) d 55.62, 103.68,
104.51, 107.84, 119.87, 124.92, 125.16, 131.25, 133.28, 134.01,
154.53, 161.23, 163.50, 171.22; HRMS m/z calcd for [C17H14ClO4]+:
317.0581; found: 317.0571.
4.1.4.14. 2-Cyclohexyl-benzo[h]chromen-4-one (6n). Yield:
75%; white solid, mp: 117–121 °C; 1H NMR (CDCl3, 500 MHz) d
1.29 (m, 1H), 1.45 (m, 2H), 1.57 (m, 2H), 1.80 (m, 1H), 1.92
(m,2H), 2.13 (m, 2H), 2.67 (m, 1H), 6.32 (s, 1H), 7.66 (m, 3H),
7.88 (m, 1H), 8.12 (m, 1H), 8.46 (m, 1H); 13C NMR (CDCl3,
125 MHz) d 25.81, 25.91, 30.74, 42.73, 109.20, 119.98, 120.82,
122.16, 124.06, 124.80, 126.92, 128.08, 129.01, 135.81, 153.70,
172.27, 178.41; HRMS m/z calcd for [C19H19O2]+: 279.1385; found:
279.1377.
4.1.4.7. 6-Isopropyl-30-nitroflavone (6g). Yield: 76%; white solid,
mp: 183–185 °C; 1H NMR (CDCl3, 300 MHz) d 1.33 (d, J = 6.90 Hz,
6H), 3.07 (m, 1H), 6.91 (s, 1H), 7.61 (m, 1H), 7.74 (m, 1H), 8.06 (m,
1H), 8.23 (m, 1H), 8.39 (m, 1H), 8.82 (s, 1H); 13C NMR (CDCl3,
75 MHz) d 23.91, 33.83, 108.66, 118.05, 121.25, 122.57, 123.65,
125.82, 130.25, 131.72, 133.29, 133.80, 146.80, 148.81, 154.61,
160.36, 178.29; HRMS m/z calcd for [C18H16NO4]+: 310.1079; found:
310.1086.
4.2. Bioassay
4.2.1. Cell culture and reagents
Caspase-3, caspase-8, and caspase-9 activity assay kits were
purchased from Beyotime. Monoclonal antibody for b-actin was
purchased from Beyotime.
4.1.4.8. 6-Isopropyl-40-nitroflavone (6h). Yield: 70%; slight yel-
low solid, mp: 212–216 °C; 1H NMR (CDCl3, 500 MHz) d 1.33 (d,
J = 6.90 Hz, 6H), 3.07 (m, 1H), 6.91 (s, 1H), 7.54 (d, 1H, J = 8.60 Hz),
7.63 (d, J = 8.20 Hz, 1H), 8.10 (m, 3H), 8.38 (m, 2H); 13C NMR (CDCl3,
125 MHz) d 23.75, 33.69, 109.32, 117.87, 122.46, 124.08, 127.06,
133.16, 137.67, 146.71, 149.28, 154.52, 160.29, 172.20, 178.19;
HRMS m/z calcd for [C18H16NO4]+: 310.1079; found: 310.1080.
The human cell lines used in the present study included HepG-2
(hepatocellular carcinoma), Hela, CNE-1, CNE-2, and MCF-7 cells.
All cells were maintained in Dulbecco’s modified Eagle’s medium
(DMEM) with 10% fetal bovine serum (FBS), 100
and 100 g/ml streptomycin at 37 °C in a humidified atmosphere
of 5% CO2.
lg/ml penicillin,
l
4.2.2. Cell proliferation assay and colony formation assay
4.1.4.9. 2-Cyclopentyl-6-isopropyl-chromen-4-one (6i). Yield:72%;
white oil; 1H NMR (CDCl3, 500 MHz) d 1.29 (d, J = 6.90 Hz, 6H), 1.77
(m, 6H), 2.07 (m, 2H), 3.00 (m, 2H), 6.20 (s, 1H), 7.36 (m, 1H), 7.51 (m,
1H), 8.03 (m, 1H); 13C NMR (CDCl3, 75 MHz) d 23.97, 25.55, 31.13,
33.77, 44.19, 108.06, 117.77, 122.27, 123.46, 132.33, 145.69,
154.99, 172.65, 178.76; HRMS m/z calcd for [C17H21O2]+:
257.1542; found: 257.1548.
Cells were seeded at a density of 5 ꢀ 103/100
ll medium in 96-
well microtiter plate and treated with the synthesized compounds
at different concentrations, ranging from 0.5 to 50
were incubated with MTT (5 mg/ml) for 5 h and formazan precip-
itate was dissolved in 100 l DMSO and the absorbance at 490 nm
lM. Viable cells
l
was measured by Multimode Detector DTX880 (Beckman Coulter).
For colony formation assays, HepG-2 cells at the exponential
phase were plated into 24-well culture plate (200–300 cells/well)
and allowed to adhere for 10 h before treatment. Culture medium
4.1.4.10. 2-Cyclopentyl-6-chloro-chromen-4-one (6j). Yield: 64%;
white solid, mp: 65–68 °C; 1H NMR (CDCl3, 300 MHz) d 1.43 (m,
containing compound 6f ranging from 0 to 50 lM was added to

6328
H. Liu et al. / Bioorg. Med. Chem. 18 (2010) 6322–6328
cells and incubated for 14 days. Cells were then rinsed with PBS,
stained with 1.0% crystal violet and photographed with a digital
camera (Bio-Rad).
ware. Results were presented as normalized fold changes in
relation to control.
Acknowledgments
4.2.3. Fluorescent morphological assay
HepG-2 cells were seeded into 24-well culture plates and trea-
The work was supported in part by the Ministry of Science and
Technology of China (2007AA02Z160, 2009ZX09501-004), the Chi-
nese National Natural Science Foundation (20872077 and
90813013).
ted with 10
fixed in MeOH–HAc (3:1, v/v) for 10 min at 4 °C, and stained with
Hoechst 33258 (5 g/ml) for 5 min at room temperature and then
lM compound 6f for 24 h. Cells were washed with PBS,
l
examined under a fluorescent microscope.
References and notes
4.2.4. DNA fragmentation assay
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to a standard procedure. HepG-2 cells (1 ꢀ 106) were seeded in 6-
well plate and treated with 0, 5, 10, 25, and 50
l
M 6f for 36 h. The
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Total RNA was extracted from cells using Trizol reagent (Invit-
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by optical density measurements at 260 and 280 nm using ultravi-
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for reverse transcription. Primers were obtained from invitrogen
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ll) of RT product was used for
PCR amplification in a total volume of 25
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by electrophoresis on 1.2% (w/v) agarose gel and were visualized
by ethidium bromide staining under ultraviolet light. Densitomet-
ric analysis of the images was conducted with Quantity One soft-