2'-chloro-4'-aminoflavone derivatives selectively targeting hepatocarcinoma cells: Convenient synthetic process, G2/M cell cycle arrest and apoptosis triggers

Article abstract of DOI:10.1002/ardp.201100383

A series of 2'-chloro-4'-nitroflavone and 2'-chloro-4'-aminoflavone derivatives were synthesized by a convenient synthetic process. The in vitro anti-proliferation ability of these compounds was evaluated against hepatocarcinoma cells (HepG2), breast adenocarcinoma cells (MCF-7), and human chronic myelogenous leukemia cells (K562). Most of synthetic compounds possessed notable anti-proliferation activity against HepG2 cells and little activity against MCF-7 cells and K562 cells. In particular, compounds 4c and 4e exhibited high anti-proliferation activity against HepG2 cells with IC₅₀ at about 2.0 μM. Further toxicity screening toward normal human hepatocytes indicated that some compounds had low toxicity against normal liver cells, among which 4e displayed very weak effects on QSG7701 and HL7702 cells, with IC ₅₀ values >100 and 50 μM, respectively. Compound 4c, with the best anti-proliferation activity in amino-substituted flavones (IC₅₀ = 2.0 μM), was selected for further evaluation of its effects on apoptosis and the cell cycle. HepG2 cells were exposed to this compound at 10 μM, which induced nuclear disassembly and DNA fragmentation. Flow cytometry analysis suggested that the population of apoptotic cells greatly increased in the 4c-treated HepG2 cells, and the cell cycle was arrested at the G₂/M phase. A series of 2'-chloro-4'-nitroflavone derivatives and 2'-chloro-4'-aminoflavone derivatives were synthesized. Most compounds exhibited potent in vitro anti-tumor ability toward HepG2 cells, but very little effect on MCF-7 and K562 cells. The representative compound 4e displayed very weak toxicity against two human normal liver cells lines, QSG7701 and HL7702; the most active compound 4c (IC₅₀ = 2 μM) arrested the cell cycle at the G₂/M phase and induced apoptosis of HepG2 cells. Copyright

Full text of DOI:10.1002/ardp.201100383

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
1
Full Paper
20-Chloro-40-aminoflavone Derivatives Selectively Targeting
Hepatocarcinoma Cells: Convenient Synthetic Process, G2/M
Cell Cycle Arrest and Apoptosis Triggers
Feng Jin1,2, Nannan Zhang1,2, Chunyan Tan2, Dan Gao2, Cunlong Zhang2, Feng Liu2, Zhe Chen1,2
Chunmei Gao2, Hongxia Liu2, Shangfu Li1,2, and Yuyang Jiang2,3
,
1 Department of Chemistry, Tsinghua University, Beijing, China
2 The Key Laboratory of Chemical Biology, Guangdong Province, The Graduate School at Shenzhen,
Tsinghua University, Shenzhen, China
3 School of Medicine, Tsinghua University, Beijing, China
A series of 20-chloro-40-nitroflavone and 20-chloro-40-aminoflavone derivatives were synthesized by a
convenient synthetic process. The in vitro anti-proliferation ability of these compounds was evaluated
against hepatocarcinoma cells (HepG2), breast adenocarcinoma cells (MCF-7), and human chronic
myelogenous leukemia cells (K562). Most of synthetic compounds possessed notable anti-proliferation
activity against HepG2 cells and little activity against MCF-7 cells and K562 cells. In particular,
compounds 4c and 4e exhibited high anti-proliferation activity against HepG2 cells with IC50 at
about 2.0 mM. Further toxicity screening toward normal human hepatocytes indicated that some
compounds had low toxicity against normal liver cells, among which 4e displayed very weak effects on
QSG7701 and HL7702 cells, with IC50 values >100 and 50 mM, respectively. Compound 4c, with the
best anti-proliferation activity in amino-substituted flavones (IC50 ¼ 2.0 mM), was selected for further
evaluation of its effects on apoptosis and the cell cycle. HepG2 cells were exposed to this compound at
10 mM, which induced nuclear disassembly and DNA fragmentation. Flow cytometry analysis
suggested that the population of apoptotic cells greatly increased in the 4c-treated HepG2 cells,
and the cell cycle was arrested at the G2/M phase.
Keywords: Anti-tumor / Apoptosis / Cell cycle arrest / Cell selectivity / Flavonoids
Received: October 27, 2011; Revised: January 18, 2012; Accepted: February 16, 2012
DOI 10.1002/ardp.201100383
Supporting information available online
:
Introduction
tory, antioestrogenic, antimicrobial, anti-allergic, antioxi-
dant, antitumor, and cytotoxic activities [4]. The antitumor
activity of flavones was embodied by arresting cell cycle and
inducing cell apoptosis [5–8]. Apoptosis, also named pro-
grammed cell death [9], plays a vital role for eliminating
cells in a damaged or infected situation, and is an effective
and new cancer treatment strategy [10, 11]. Consequently,
the design and synthesis of novel flavone derivatives as apop-
tosis triggers is a promising research field [12–14]. In recent
years, amino-substituted flavones have attracted an increas-
ing interest [15–19], because the amino group can act as a
hydrogen donor or hydrogen bonding acceptor for binding
with target proteins. For example, 20-aminothioflavones were
reported as specific inhibitors of the ERK-MAP kinase signal-
Flavonoids are widely distributed among plant species, and
consist of more than 9000 natural compounds [1]. Owing to
their various biological activities, the study of flavonoids has
been a research focus in the past decades [2, 3]. A subset of
flavonoids, flavones, contain a C6–C3–C6 carbon framework
and possess many biological activities such as anti-inflamma-
Correspondence: Prof. Yuyang Jiang, The Key Laboratory of Chemical
Biology, Guangdong Province, The Graduate School at Shenzhen,
Tsinghua University, Shenzhen 518055, China.
E-mail: jiangyy@sz.tsinghua.edu.cn
Fax: þ86 755 26036017
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F. Jin et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
ing pathway [18], 30-aminoflavones as activators of apoptosis
[19], and 40-aminoflavones are potential inhibitors of protein
tyrosine kinases [20]. Most aminoflavone derivatives are
derived from synthetic chemistry [21–23]. The Baker–
Venkataraman routine is the most commonly used method
to synthesize various substituted flavone derivatives [24], but
highly toxic pyridine and step-by-step synthetic operations
are needed [25, 26].
2-chloro-4-nitrobenzoylchloride were employed as starting
materials, which were refluxed in dry acetone for 15 h with
anhydrous K2CO3 as catalyst. Acetone was then removed after
the reaction by evaporation. Without further purification,
glacial acetic acid and concentrated H2SO4 were added into
the resulting mixture to reflux for another 2 h. Inorganic
salts were then removed by hot filtration, and the resulting
solution was allowed to cool down to room temperature to
give the desired products as crystals. To obtain the corre-
sponding amino products, these nitro derivatives were fur-
ther treated with 4 equiv. of SnCl2 under reflux in anhydrous
ethanol for 2 h, after which the final products were purified
by column chromatography.
In our previous study, we reported the synthesis and bio-
activity evaluation of some novel flavone derivatives with 6-
chlorine or 6-isopropyl substitutions [27]. Those compounds
showed good inhibition affect on HepG2 cells probably by
induction of the apoptosis pathway, and the structure–
activity relationship of B-ring substitutions was discussed.
Herein, we continued that study and used a convenient
synthetic process to synthesize 20-chloro-40-nitroflavones,
which were then reduced to corresponding 20-chloro-40-ami-
noflavones. Some of these novel synthetic flavone derivatives
were discovered to selectively inhibit HepG2 cell growth.
Structure–activity relationships at the A-ring were disclosed.
The most active compound in amino-substituted flavones, 4c,
arrested the cell cycle at the G2/M phase, and some typical
morphological changes of apoptotic cells were observed in 4c-
treated HepG2 cells.
Selective protection of hydroxyl group on O-hydroxyaceto-
phenone derivatives is usually used to synthesize the A-ring
hydroxyl substituted flavones. Some commonly used protec-
tion groups are tetra-butyl dimethylsilyl (TBS) [28], methyl
[25, 29, 30], methoxymethyl (MOM) [31], benzoyl (Bz) [32], etc.
It is noteworthy to point out that in our synthetic route, 7-
hydroxyl-20-chloro-40-nitroflavone (3e) was conveniently
obtained by a one-pot reaction of 2,4-dihydroxylacetophe-
none 1e and 2-chloro-4-nitrobenzoylchloride 2 without pro-
tection/deprotection steps. As shown in Scheme 2, only
product 5 is obtained when 1 equiv. of 2 reacted with 1e.
When 2 equiv. of 2 reacted with 1e, the main product is 3e in
about 26% yield. A mechanism was proposed by which the
hydroxyl group on compound 5 after the first nucleophilic
substitution reacted with excess 2 to give compound 6, fol-
lowed by ring-closure and rearrangement to form the final
product 3e.
Results and discussion
Synthesis of 20-chloro-40-aminoflavone derivatives
The synthetic route of target flavone derivatives is illustrated
in Scheme 1. Briefly, O-hydroxyacetophenone derivatives and
Scheme 1. Synthesis of compounds 3a–3h and 4a–4h. Regents and conditions: (i) dry K2CO3, acetone, reflux for 15 h; then
conc. H2SO4, CH3COOH, reflux for 2 h; (ii) anhydrous SnCl2, anhydrous CH3CH2OH, reflux for 2 h.
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
Aminoflavones Selectively Targeting Hepatocarcinoma Cells
3
Scheme 2. The consecutive reaction
mechanism and intermediate products in the
synthetic processes of 3e.
Antiproliferative activity and tumor cell selectivity of
synthetic flavones
pounds. The results are shown in Table 1. An interesting
result was that all compounds displayed no effect against
MCF-7 cells (with IC50 >50 mM), and only a few 40-amino-
flavone derivatives show weak activities against K562 cells,
while 40-nitroflavone analogues had no effect against K562
The anti-proliferation ability of the synthesized compounds
20-choro-40-nitroflavone derivatives (3a–3h) and 20-choro-40-
aminoflavone derivatives (4a–4h) were evaluated against
HepG2 cells in vitro by MTT assay (MTT, 3-(4,5-dimethylthia-
zol-2-yl)-2,5-diphenyltetrazolium bromide), and the results
are summarized in Table 1. Most compounds exhibited
obvious anti-proliferation activity against HepG2 cells.
Comparison of the activity of 40-nitroflavone derivatives
and their reduction products 40-aminoflavone derivatives
indicated that the latter had more promising activities
against HepG2 cells, except for compound 3b. Introduction
of alkyl groups on the A-ring, for example, methyl and ethyl
groups as weak electron-donating groups, provided better
anti-tumor activities. However, electron-withdrawing groups
and strong electron-donating groups have less effect on anti-
tumor activities. The reason might be attributed to better
solubility and bioavailability of alkyl-substituted compounds
than fluoro- and methoxy-substituted flavone derivatives [33].
The most effective compound, 4c, was chosen to investigate
its inhibition on colony formation against HepG2 cells. As
shown in Fig. 1, HepG2 cells were strongly inhibited at
concentrations as low as 2.5 mM. Thus, both MTT and colony
formation inhibition results demonstrated the inhibition
effect of our synthetic compounds against HepG2 cells.
Breast adenocarcinoma cells (MCF-7) and human chronic
myelogenous leukemia cells (K562) were also selected to
evaluate the anti-proliferation activities of the synthetic com-
Table 1. Anti-proliferation activity of compounds against HepG2
cells, K562 cells, and MCF-7 cells
Compound
IC50 (mM)a)
HepG-2
K562
MCF-7
3a
4a
3b
4b
3c
4c
3d
4d
3e
4e
3f
4f
3g
4g
3h
7.4 ꢀ 0.7
2.5 ꢀ 0.4
1.6 ꢀ 0.2
2.5 ꢀ 1.2
6.1 ꢀ 0.3
1.8 ꢀ 0.3
nd
>50
17.0 ꢀ 0.6
>50
>50
>50
>50
>50
>50
>50
nd
>50
>50
>50
>50
>50
>50
>50
>50
>50
nd
20.4 ꢀ 2.3
>50
10.8 ꢀ 1.5
nd
10.2 ꢀ 0.8
12.3 ꢀ 1.4
>50
>50
>50
2.1 ꢀ 0.5
13.7 ꢀ 1.4
7.2 ꢀ 1.0
14.5 ꢀ 2.7
7.3 ꢀ 0.4
>50
19.4 ꢀ 2.1
1.8 ꢀ 0.4
nd
>50
36.7 ꢀ 1.8
>50
>50
>50
>50
nd
4.9 ꢀ 0.8
4h
Colchicine
Imatinib
nd
nd, not determined.
a)
All values are means of three experiments.
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F. Jin et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
6-Ethyl-20-chloro-40-aminoflavone 4c induced
apoptosis of HepG2 cells
Apoptosis is regulated by genetic mechanisms and is princi-
pally characterized by morphological and biochemical changes
in the nucleus including chromatin condensation and inter-
nucleosomal DNA fragmentation [34, 35]. In order to confirm
the existence of apoptosis in HepG2 cells treated with 6-ethyl-20-
chloro-40-aminoflavone 4c at 10 mM, typical apoptotic morpho-
logical study and flow cytometry analysis were carried out.
HepG2 cells were treated with 10 mM of 4c and 4d for 24 h
and then stained with Hoechst 33258 for fluorescence micro-
scopy observation. The observation showed some character-
istic morphological changes of apoptosis, including
membrane blebbing, nuclear condensation and fragmenta-
tion, chromatin condensation, and the formation of apop-
totic bodies as shown in Fig. 2. Another typical feature of cells
undergoing apoptosis was the loss of small DNA fragments
which could be observed by agarose gel electrophoresis. As
shown in Fig. 2, there were no obvious DNA segments in
control cells. However, different concentrations of com-
pound-treated cells displayed DNA laddering in a dose-
dependent manner.
Figure 1. Effect of 4c on colony formation of HepG2 cells.
cells. These results indicated that our synthetic compounds
might selectively target HepG2 cells.
To further identify the selectivity of synthetic compounds,
we evaluated the toxic activities of synthetic compounds
against two normal human liver cells, QSG7701 and
HL7702 cells. As shown in Table 2, compound 4e exhibited
less cytoxicity to QSG7701 cells with the inhibition rate of 21
and 15% at the concentration of 100 and 50 mM, respectively.
In addition, compounds 3c, 3e, 3f, and 4h also displayed very
weak cytoxicity against the normal human liver cells.
The Annexin-V/PI binding assay was used to further
confirm the effect of compound 4c induced apoptosis.
Annexin-V conjugated with the fluorochrome FITC serves
as a marker for apoptotic cells because it has a strong binding
affinity to phosphatidylserine (PS), which re-distributes from
the inner to the outer layer of the plasma membrane in
apoptotic cells. The dual parametric dot plots show four
quadrants as follows: (1) the lower left quadrant represents
the viable cell population (annexin-V negative and PI nega-
tive); (2) the upper right represents apoptotic cells under-
going secondary necrosis at the last stage or dead cells
(annexin-V and PI double positive); (3) the lower right
represents the early stage apoptotic cell population
(annexin-V positive and PI negative). As shown in Fig. 3, only
about 8.29% of the untreated HepG2 cells were apoptotic cells
either in early or secondary stage. However, the apoptotic cell
population increased after the treatment with 4c at different
concentrations. The dose-dependent effect can also be
observed as the concentration of 4c increased from 5 to
50 mM, in which the apoptotic cells increased from 11.9 to
56.3%. The data also suggested that an increasing number of
apoptotic cells progressed from the early stage to the late
stage resulting in either death or secondary necrosis under
the effect of 4c at higher concentrations, which confirmed
again that 4c induced the apoptosis of HepG2 cells.
Table 2. Toxicity evaluation of compounds against QSG7701 cells
and HL7702 cells
Compound
Inhibition ratea)
(100 mM)
Inhibition ratea)
(50 mM)
QSG7701
HL7702
QSG7701
HL7702
3a
4a
3b
4b
3c
4c
3d
4d
3e
4e
3f
67.6%
51.3%
54.1%
34.9%
59.9%
48.8%
60.7%
34.6%
96.4%
nd
96.1%
26.5%
21.3%
37.8%
59.0%
nd
57.2%
65.7%
63.9%
46.2%
99.2%
nd
85.2%
26.3%
54.4%
56.8%
63.2%
nd
37.3%
47.0%
54.2%
17.9%
72.3%
nd
63.9%
33.5%
15.2%
34.5%
57.9%
nd
46.2%
46.1%
47.5%
35.9%
56.6%
nd
63.3%
26.6%
37.9%
52.9%
55.9%
nd
4f
3g
4g
3h
4h
88.2%
nd
58.8%
84.3%
nd
48.6%
84.1%
nd
37.8%
72.9%
nd
39.0%
6-Ethyl-20-chloro-40-aminoflavone (4c) arrested cell
cycle in G2/M phase
The cell cycle is a vital process strictly regulated by transcrip-
tional signaling pathways [36–38], deregulation of which
nd, not determined.
a)
All values are means of three experiments.
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
Aminoflavones Selectively Targeting Hepatocarcinoma Cells
5
in a dose-dependent manner. As shown in Fig. 4, the percent
of cells at the G2/M phase increased from 16 to 31% when the
concentration was raised from 5 to 50 mM.
Conclusion
A convenient synthetic process was employed to synthesize
20-chloro-40-nitroflavone
and
20-chloro-40-aminoflavone
derivatives. These novel synthetic flavone derivatives pos-
sessed notable anti-proliferation activity with selectivity
against HepG2 cells, and structure–activity relationships at
the A-ring were disclosed. The introduction of alkyl group
into the A-ring effectively improved their anti-proliferation
ability. Compound 4c demonstrated the strongest inhibition
activity against HepG2 cell lines in amino-substituted flavone
derivatives. Further study showed that the cell cycle was
arrested at the G2/M phase and induced apoptosis in
HepG2 cells. This compound could be considered as a poten-
tial lead compound for developing new therapeutic agents
against hepatocarcinoma.
Experimental
Chemistry
Melting points were measured with a SGW X-4 electrothermal
melting apparatus, and data were uncorrected. 1H NMR and
13C NMR spectra were recorded with a Bruker spectrometer at
1
400 MHz for H NMR, at 101 MHz for 13C NMR, with TMS as an
internal standard in CDCl3, or in [D6] DMSO. High-resolution
mass spectra were recorded with a Waters Q-Tof Premier mass
spectrometer. Acetone was dried according to the standard proc-
ess. All other reagents and analytical grade solvents commer-
cially available were used without further purification.
General procedure for the synthesis of derivatives of
20-chloro-40-nitroflavones (3a–3h)
Figure 2. Fluorescent staining of nuclei by Hoechst 33258: (a) left:
control cells; right: 10 mM 4c-treated HepG2 cells; (b) left: control
cells; right: 10 mM 4d-treated HepG2 cells. DNA fragmentation
induced by 4c: (c) HepG2 cells were treated by 4c at 0, 1, 5, 10,
25, and 50 mM for 30 h, then DNA was extracted for analysis.
To a 100 mL round-bottomed flask add 2-chloro-4-nitrobenzoyl-
chloride (0.01 mol, 2.20 g) and equimolar O-hydroxyacetophe-
none derivatives, dry K2CO3 (5.52 g, 0.040 mol), 20 mL dry
acetone, and reflux the mixture for 15 h. Then, acetone was
removed by vacuum distillation, and then 20 mL ice acetate acid
and 0.50 mL concentrated sulfuric acid were added to the above
resulting solids. The solutions were refluxed for 2 h to obtain
white solids and clear solutions. The white solids were separated
from the solutions by hot filtration. The filtrates were allowed to
cool to room temperature, and crystals of derivatives of 20-chloro-
40-nitroflavones were obtained.
concerns many diseases including human cancer. The cell
cycle consists of four distinct phases: G1 phase, S phase, G2
phase, and M phase [39], the progress of which is monitored
and regulated by cell cycle checkpoints so as to ensure repair
of DNA damage and completion of each phase. Therefore,
affecting the cell cycle has been one robust cancer therapy
strategy, and some compounds exerted potent antitumor
activity through cell cycle arrest [40, 41]. Herein, we inves-
tigated the effect of 4c on the cell cycle and results showed
that this compound arrested the cell cycle at the G2/M phase
20-Chloro-40-nitroflavone (3a)
Yield 55%; white solid, mp: 143–1448C; Ms (EI) 301.00. 1H NMR
(400 MHz, CDCl3) d 8.43 (d, J ¼ 2.2 Hz, 1H), 8.28 (ddd, J ¼ 7.9, 4.6,
1.9 Hz, 2H), 7.87 (d, J ¼ 8.5 Hz, 1H), 7.75 (ddd, J ¼ 8.7, 7.2, 1.7 Hz,
1H), 7.59–7.45 (m, 2H), 6.72 (s, 1H). 13C NMR (101 MHz, CDCl3)
d 177.53, 160.13, 156.55, 149.20, 137.56, 134.41, 134.37, 131.55,
126.00, 125.97, 125.83, 123.91, 122.05, 118.20, 113.87.
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F. Jin et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
Figure 3. Flow cytometry analysis of cell
apoptosis induced by 4c. HepG2 cells were
treated by 4c at 0, 1, 5, 10, and 50 mM for 36 h.
6-Methyl-20-chloro-40-nitroflavone (3b)
7.59 (dd, J ¼ 8.6, 2.2 Hz, 1H), 7.46 (d, J ¼ 8.6 Hz, 1H), 6.70 (s,
1H), 2.80 (q, J ¼ 7.6 Hz, 2H), 1.31 (t, J ¼ 7.6 Hz, 3H). 13C NMR
(101 MHz, CDCl3) d 177.87, 160.04, 155.03, 149.16, 142.35,
137.75, 134.70, 134.44, 131.62, 126.07, 124.12, 123.69, 122.10,
118.14, 113.74, 28.51, 15.58.
Yield 62%; white solid, mp: 179–1808C; Ms (EI) 315.01. 1H NMR
(400 MHz, CDCl3) d 8.42 (d, J ¼ 1.38 Hz, 1H), 8.27 (dd, J ¼ 8.94,
1.38 Hz, 1H), 8.04 (s, 1H), 7.87 (d, J ¼ 8.94 Hz, 1H), 7.56 (dd,
J ¼ 8.94, 1.38 Hz, 1H), 7.43 (d, J ¼ 8.94 Hz, 1H), 6.70 (s, 1H),
2.50 (s, 3H). 13C NMR (101 MHz, CDCl3) d 177.79, 160.01,
154.85, 149.10, 137.69, 136.02, 135.70, 131.64, 126.04, 125.26,
123.53, 122.11, 118.04, 113.73, 21.10.
6-Methoxy-20-chloro-40-nitroflavone (3d)
Yield 35%; white solid, mp: 208–2098C; Ms (EI) 331.00. 1H NMR
(400 MHz, CDCl3) d 8.42 (d, J ¼ 2.2 Hz, 1H), 8.27 (dd, J ¼ 8.5,
2.2 Hz, 1H), 7.86 (d, J ¼ 8.5 Hz, 1H), 7.62 (d, J ¼ 3.1 Hz, 1H),
7.48 (d, J ¼ 9.2 Hz, 1H), 7.34 (dd, J ¼ 9.2, 3.1 Hz, 1H), 6.71 (s,
1H), 3.93 (s, 3H). 13C NMR (101 MHz,CDCl3) d 177.67, 159.99,
157.57, 151.48, 149.19, 137.67, 134.44, 131.65, 126.09, 124.66,
124.56, 122.12, 119.76, 113.05, 104.99, 56.14.
6-Ethyl-20-chloro-40-nitroflavone (3c)
Yield 60%; white solid, mp: 152–1538C; Ms (EI) 329.04. 1H NMR
(400 MHz, CDCl3) d 8.42 (d, J ¼ 2.2 Hz, 1H), 8.27 (dd, J ¼ 8.5,
2.2 Hz, 1H), 8.08 (d, J ¼ 2.1 Hz, 1H), 7.86 (d, J ¼ 8.5 Hz, 1H),
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
Aminoflavones Selectively Targeting Hepatocarcinoma Cells
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Figure 4. Analysis of cell cycle phase distribu-
tion induced by 4c. HepG2 cells were treated by
4c at 0, 5, 10, 25, and 50 mM for 36 h, and then
analyzed by flow cytometry.
7-Hydrox-20-chloro-40-nitroflavone (3e)
1H), 7.55 (dt, J ¼ 9.6, 4.8 Hz, 1H), 7.47 (ddd, J ¼ 9.2, 7.5,
3.1 Hz, 1H), 6.70 (s, 1H). 13C NMR (101 MHz, CDCl3) d 110.53,
110.764, 112.839, 120.11 (J ¼ 8.08 Hz), 121.816, 122.34
(J ¼ 25.65 Hz), 124.86 (J ¼ 7.37 Hz), 125.772, 131.28, 134.17,
149.03, 152.46, 158.44, 160.52 (J ¼ 78.17 Hz), 176.49.
In its special synthetic processes, twofold equivalents 2-chloro-4-
nitrobenzoylchloride (0.02 mol, 4.40 g) were needed. The other
operations were the same as the general procedure. Yield 26%;
white solid, mp >3008C; Ms (EI) 316.99. 1H NMR (400 MHz,
DMSO) d 10.96 (s, 1H), 8.48 (d, J ¼ 1.7 Hz, 1H), 8.34 (dd,
J ¼ 8.5, 1.7 Hz, 1H), 8.09 (d, J ¼ 8.5 Hz, 1H), 7.94 (d,
J ¼ 8.7 Hz, 1H), 6.98 (dd, J ¼ 8.7, 1.6 Hz, 1H), 6.90 (s, 1H), 6.63
(s, 1H). 13C NMR (101 MHz, DMSO) d 175.61, 163.12, 159.50,
157.72, 148.96, 136.90, 132.79, 132.48, 126.69, 125.18, 122.52,
115.93, 115.52, 112.77, 102.38. Intermediate product (5), white
solid, 1H NMR (400 MHz, DMSO) 12.20 (s, 1H), 8.48 (m, 1H), 8.37
(m, 2H), 8.04 (d, J ¼ 8.40 Hz, 1H), 6.98–7.03 (m, 2H), 2.67 (s, 3H).
5-Hydroxy-20-chloro-40-nitroflavone (3g)
Its synthetic procedure was more convenient than the general
procedure. Equimolar O-hydroxyacetophenone and 2-chloro-4-
nitrobenzoyl chloride, and 4 equiv. of dry K2CO3 were refluxed
for 15 h, acetone was removed by evaporation, and the resulting
brown solid was purified by rinsing with hot water. Yield 30%; light
brown solid, mp: 193–1948C; Ms (EI) 317.03. 1H NMR (400 MHz,
DMSO) d 12.39 (s, 1H), 8.52 (d, J ¼ 2.1 Hz, 1H), 8.43–8.34 (m, 1H),
8.12 (d, J ¼ 8.5 Hz, 1H), 7.74 (t, J ¼ 8.4 Hz, 1H), 7.15 (d, J ¼ 8.4 Hz,
1H), 6.99–6.84 (m, 2H). 13C NMR (101 MHz, DMSO) d 182.59, 161.61,
159.82, 156.16, 149.28, 136.50, 136.22, 132.88, 132.72, 125.24,
122.56, 111.91, 111.42, 110.16, 107.53.
6-Fluoro-20-chloro-40-nitroflavone (3f)
Yield 50%; white solid, mp: 191–1928C; Ms (EI) 319.02. 1H NMR
(400 MHz, CDCl3) d 8.43 (d, J ¼ 2.2 Hz, 1H), 8.28 (dd, J ¼ 8.5,
2.2 Hz, 1H), 7.90 (dd, J ¼ 8.0, 3.0 Hz, 1H), 7.86 (d, J ¼ 8.5 Hz,
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F. Jin et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
(d, J ¼ 8.5 Hz, 1H), 7.01–6.81 (m, 2H), 6.74 (d, J ¼ 2.0 Hz, 1H),
6.63 (dd, J ¼ 8.5, 2.1 Hz, 1H), 6.42 (d, J ¼ 17.3 Hz, 1H), 6.08 (s, 2H).
13C NMR (101 MHz, DMSO) d 175.93, 162.53, 162.31, 157.57,
152.26, 132.24, 131.75, 126.42, 116.93, 115.84, 114.83, 114.13,
112.26, 109.95, 102.25.
2-(2-Chloro-4-nitrophenyl)-4H-benzo[h]chromene-4-one
(3h)
Yield 32%; white solid, mp: 280–2828C; Ms (EI) 351.00. 1H NMR
(400 MHz, CDCl3) d 8.50 (dd, J ¼ 9.3, 5.3 Hz, 2H), 8.34 (dd, J ¼ 8.5,
2.2 Hz, 1H), 8.21 (d, J ¼ 8.7 Hz, 1H), 7.97 (dd, J ¼ 10.8, 8.2 Hz,
2H), 7.86 (d, J ¼ 8.7 Hz, 1H), 7.73 (dtd, J ¼ 16.4, 7.0, 1.2 Hz, 2H),
6.89 (s, 1H). 13C NMR (101 MHz, CDCl3) d 177.45, 159.62, 154.14,
149.30, 137.56, 136.26, 134.47, 131.77, 129.74, 128.34, 127.54,
126.21, 126.05, 124.00, 122.34, 122.24, 120.65, 120.35, 115.07.
6-Fluoro-20-chloro-40-aminoflavone (4f)
Yield 85%; yellow solid, mp: 176–1778C; HRMS (ESI) m/z calcd for
[MþH]þ 289.0306, found 289.0303. 1H NMR (400 MHz, CDCl3)
d 7.87 (dd, J ¼ 8.2, 3.1 Hz, 1H), 7.51 (dd, J ¼ 9.1, 4.2 Hz, 1H),
7.46 (d, J ¼ 8.5 Hz, 1H), 7.40 (ddd, J ¼ 9.1, 7.7, 3.1 Hz, 1H),
6.80 (d, J ¼ 2.3 Hz, 1H), 6.70–6.62 (m, 2H), 4.09 (s, 2H).
13C NMR (101 MHz, CDCl3) 177.53 (J ¼ 2.22 Hz), 163.39, 159.60
(J ¼ 247.65 Hz), 152.78, 149.77, 134.04, 131.76, 125.05
(J ¼ 7.37 Hz), 121.75 (J ¼ 25.55 Hz), 120.48 (J ¼ 53.53 Hz),
116.18, 113.14, 111.16, 110.70, 110.47.
General procedure for the synthesis of derivatives of 20-
chloro-40-aminoflavones
Derivatives of 20-chloro-40-nitroflavones and fourfold equivalents
anhydrous SnCl2 were refluxed in the anhydrous ethanol for 2 h,
ethanol was removed by evaporation, and the resulting solids
were purified by column chromatography, dichloromethane/
methanol ¼ 20:1 v/v as flush.
5-Hydroxy-20-chloro-40-aminoflavone (4g)
Yield 70%; yellow solid, mp: 167–1698C; HRMS (ESI) m/z calcd for
[MþH]þ 288.0428, found 288.0425. 1H NMR (400 MHz, DMSO)
d 12.73 (s, 1H), 7.66 (d, J ¼ 8.6 Hz, 1H), 7.54 (d, J ¼ 8.4 Hz, 1H),
7.07 (d, J ¼ 8.2 Hz, 1H), 6.80 (d, J ¼ 2.2 Hz, 1H), 6.76
(d, J ¼ 2.4 Hz, 1H), 6.63–6.66 (m, 2H), 6.23 (s, 2H). 13C NMR
(101 MHz, DMSO) 183.14, 165.09, 160.39, 156.57, 153.47,
136.32, 133.17, 132.75, 116.38, 114.92, 112.88, 111.35, 110.32,
109.04, 107.83.
20-Chloro-40-aminoflavone (4a)
Yield 90%; yellow solid, mp: 147–1488C; HRMS (ESI) m/z calcd for
[MþH]þ 272.0479, found 272.0477. 1H NMR (400 MHz, CDCl3)
d 8.24 (dd, J ¼ 8.0, 1.6 Hz, 1H), 7.68 (ddd, J ¼ 8.7, 7.1, 1.7 Hz, 1H),
7.54–7.45 (m, 2H), 7.45–7.39 (m, 1H), 6.80 (d, J ¼ 2.3 Hz, 1H),
6.72–6.62 (m, 2H), 4.07 (s, 2H). 13C NMR (101 MHz, CDCl3)
d 178.36, 163.04, 156.55, 149.52, 133.98, 133.61, 131.76,
125.69, 125.06, 123.84, 121.08, 118.11, 116.14, 113.11, 111.92.
2-(2-Chloro-4-aminophenyl)-4H-benzo[h]chromene-4-one
(4h)
6-Methyl-20-chloro-40-chloroaminoflavone (4b)
Yield 92%; yellow solid, mp: 165–1668C; HRMS (ESI) m/z calcd for
[MþH]þ 286.0636, found 286.0639. 1H NMR (300 MHz, CDCl3)
d 8.02 (s, 1H), 7.45–7.47 (m, 2H), 7.40 (d, J ¼ 4.44 Hz, 1H), 6.79
(s, 1H), 6.65 (m, 3H), 4.06 (s, 2H), 2.36 (s, 3H). 13C NMR (101 MHz,
CDCl3) d 178.50, 163.00, 154.82, 149.68, 135.01, 134.85, 133.87,
131.69, 124.93, 123.40, 120.88, 117.86, 116.09, 113.09, 111.61,
20.94.
Yield 81%; light yellow solid, mp: 238–2418C; HRMS (ESI) m/z
1
calcd for [MþH]þ 322.0636, found 322.0635. H NMR (400 MHz,
DMSO) d 8.54 (d, J ¼ 7.5 Hz, 1H), 8.17–8.08 (m, 1H), 8.01 (d,
J ¼ 8.7 Hz, 1H), 7.94 (d, J ¼ 8.7 Hz, 1H), 7.87–7.74 (m, 2H), 7.64
(d, J ¼ 8.5 Hz, 1H), 6.81 (d, J ¼ 2.0 Hz, 1H), 6.78–6.66 (m, 2H), 6.18
(br, 2H). 13C NMR (101 MHz, DMSO) d 177.00, 163.11, 153.57,
153.18, 135.91, 132.89, 132.87, 130.04, 128.77, 128.21, 125.76,
124.01, 122.59, 120.55, 119.87, 117.12, 114.95, 113.10, 111.75.
6-Ethyl-20-chloro-40-aminoflavone (4c)
Yield 88%; yellow solid, mp: 96–978C; HRMS (ESI) m/z calcd for
[MþH]þ 300.0792, found 300.0788. 1H NMR (400 MHz, CDCl3)
d 8.05 (d, J ¼ 2.0 Hz, 1H), 7.52 (dd, J ¼ 8.6, 2.2 Hz, 1H), 7.44
(dd, J ¼ 14.4, 8.5 Hz, 2H), 6.79 (d, J ¼ 2.3 Hz, 1H), 6.69–
6.62 (m, 2H), 4.07 (s, 2H), 2.77 (q, J ¼ 7.6 Hz, 2H), 1.30
(t, J ¼ 7.6 Hz, 3H). 13C NMR (101 MHz, CDCl3) d 178.34, 162.72,
154.77, 149.25, 141.16, 133.74, 133.62, 131.53, 123.61, 123.35,
121.01, 117.75, 115.92, 112.90, 111.53, 28.21, 15.37.
Biological assays
Cell culture and reagents
All human cell lines, including HepG2 cells, K562 cells, MCF-7
cells, QSG7701 cells, and HL7702 cells were obtained from Cell
Resources Center of Shanghai Institutes for Biological Science,
Chinese Academy of Science. They were maintained in
Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal
bovine serum, 100 mg mLꢁ1 penicillin, and 100 mg mLꢁ1 strep-
tomycin at 378C in a humidified atmosphere of 5% CO2.
6-Methoxy-20-chloro-40-aminoflavone (4d)
Yield 80%; yellow solid, mp: 142–1438C; HRMS (ESI) m/z calcd for
[MþH]þ 302.0585, found 302.0580. 1H NMR (400 MHz, CDCl3)
d 7.60 (d, J ¼ 3.0 Hz, 1H), 7.50–7.41 (m, 2H), 7.28 (d, J ¼ 3.1 Hz,
1H), 6.79 (d, J ¼ 2.3 Hz, 1H), 6.70–6.62 (m, 2H), 4.09 (s, 2H), 3.91
(s, 3H). 13C NMR (101 MHz, CDCl3) d 178.21, 162.82, 156.93,
151.41, 149.47, 133.93, 131.73, 124.39, 123.71, 121.15, 119.54,
116.13, 113.12, 111.14, 104.78, 55.97.
Cell growth inhibition assay
The synthesized compounds were dissolved in DMSO, and then
diluted with culture medium to the final concentrations ranging
from 0.5 to 50 mM (the final DMSO concentration was <1%) for
tumor cell assays, 50 and 100 mM for normal liver cell assays.
Hundred microliters of cell solution with the concentration of
5 ꢂ 105 cells mLꢁ1 was seeded to each well of a 96-well plate and
incubated for 24 h at 378C in a 5% CO2 incubator. The medium
was then removed from the 96-well plate. The test compound
solution was added to each well for the treatment of maintained
cells in triplicate per concentration, and incubated at 378C in a
5% CO2 incubator for 48 h. After this treatment, 10 mL MTT
7-Hydroxy-20-chloro-40-aminoflavone (4e)
Yield 72%; yellow solid, mp: 281–2838C; HRMS (ESI) m/z calcd for
[MþH]þ 288.0428, found 288.0451. 1H NMR (400 MHz, DMSO)
d 10.79 (d, J ¼ 15.6 Hz, 1H), 7.89 (t, J ¼ 8.0 Hz, 1H), 7.47
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
Aminoflavones Selectively Targeting Hepatocarcinoma Cells
9
solution (5 mg mLꢁ1) was then added to each well and incubated
for 4 h at 378C. The formazan precipitate was dissolved in 100 mL
DMSO and the absorbance at 495 nm was determined using a
Multimode Detector DTX880 (Beckman Coulter).
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For colony formation assays, approximately 300 HepG2 cells at
the exponential phase were seeded into individual wells of a
24-well plate and incubated for 12 h to allow for adhesion.
Culture medium containing compound 4c ranging from 0 to
50 mM was added to each well and incubated for 14 days. Cells
were then washed with PBS and stained with 1.0% crystal violet.
The photograph was taken by a Canon digital camera.
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The authors would like to thank for the financial support provided by the
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National Natural Science Foundation of China (Grant No. 20872077
and 90813013), and Shenzhen Sci & Tech Bureau.
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Supplementary data
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