Synthesis, growth inhibition, and cell cycle evaluations of novel flavonoid derivatives

Article abstract of DOI:10.1016/j.bmc.2005.07.062

As a continuation of our search for potential new anticancer agents, a series of ten flavonoid derivatives has been synthesized by cyclization of substituted chalcones. Target compounds were evaluated for their biological activity. Among them, compounds 1

Full text of DOI:10.1016/j.bmc.2005.07.062

Bioorganic & Medicinal Chemistry 13 (2005) 6850–6855
Synthesis, growth inhibition, and cell cycle evaluations
of novel flavonoid derivatives
Yerra Koteswara Rao,a Shih-Hua Fangb and Yew-Min Tzenga,*
aInstitute of Biotechnology, Chaoyang University of Technology, Wufeng 413, Taiwan, ROC
bDepartment of Microbiology, School of Medicine, China Medical University, Taichung 400, Taiwan, ROC
Received 14 June 2005; revised 25 July 2005; accepted 26 July 2005
Available online 2 September 2005
Abstract—As a continuation of our search for potential new anticancer agents, a series of ten flavonoid derivatives has been
synthesized by cyclization of substituted chalcones. Target compounds were evaluated for their biological activity. Among them,
compounds 1–4 and 9 displayed a significant growth inhibitory action against a panel of tumor cell lines including Jurkat, PC-3,
and Colon 205. On treatment with an equitoxic (IC50) concentration, compounds 1–5 and 7–9 blocked cells in the G2/M phase
of the Jurkat cell cycle, whereas compound 6 blocked the same in the G0/G1 phase.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
cascade in hepatocellular carcinoma SK-HEP-1 cells
and human leukemia HL-60 cells.7,8 Further, two pro-
spective cohort studies in Finland, where average flavo-
noid intakes are relatively low, found that men with the
highest dietary intakes of flavonols and flavones had a
significantly lower risk of developing lung cancer than
those with the lowest intakes.9 More recently, flavone-
8-acetic acid derivatives have been reported as reversible
inhibitors of aminopeptidase N/CD13.10 Importantly, a
flavonoid derivative flavopiridol was widely used in tra-
ditional medicine and was a novel semisynthetic flavone
analog of rohitukine, a leading anticancer compound
derived from an Indian tree. Flavopiridol has been
found to inhibit cyclin-dependent kinases (Cdks), induce
apoptosis, suppress inflammation, and modulate the im-
mune response,11 and was the first compound with this
activity to have entered the clinic as an anticancer drug.
Many clinically successful anticancer drugs were them-
selves either naturally occurring molecules or have been
developed from their synthetic analogs. Great interest is
currently being paid to natural products for their inter-
esting anticancer activities. Flavonoids are prominent
plant secondary metabolites that are consumed by
humans as dietary constituents in amounts exceeding
0.1 g/day. A large number of biological activities have
been attributed to these compounds including antican-
cer.1 The interaction of dietary flavonoids with the gut
has numerous implications for human health, and flavo-
noid compounds in the diet may act as chemopreventive
agents against the development of cancer in the alimen-
tary tract.2 In fact, a number of studies have demon-
strated anticancer activity associated with flavonoids;3
for example, flavone has been shown to be a potent
apoptosis inducer in human colon carcinoma cells, and
this effect appears to be restricted to cancer cells.4 Sim-
ilarly, selective induction of apoptosis in human prostate
cancer cells by apigenin5 and in human myeloid leuke-
mia HL-60 cells by luteolin has been reported recently.6
Induction of apoptosis by wogonin and fisetin has been
demonstrated to involve activation of the caspase 3
As part of our continuing search for potential anticancer
drug candidates, we have recently described the growth
inhibitory properties and cell cycle analysis of novel
flavonoid (chalcone) derivatives using a panel of cancer
cell lines.12 Based on the diverse biological activities of
flavonoid derivatives, in the present study we have syn-
thesized a series of natural flavonoids and examined
their in vitro cytotoxic activity on human tumor cell
lines, namely Jurkat (human lymphocytic cancer cell
line), PC-3 (human prostate cancer cell line), HepG2
(human hepatoma cancer cell line), Colon 205 (human
colonic cancer cell line), and the normal peripheral
blood mononuclear cells (PBMCs). In addition, to
Keywords: Flavonoids; Natural product synthesis; Cytotoxicity; Cell
cycle analysis.
*
Corresponding author. Tel.: +886 4 23323000x3003; fax: +886 4
23304896; e-mail: ymtzeng@cyut.edu.tw
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.07.062

Y. K. Rao et al. / Bioorg. Med. Chem. 13 (2005) 6850–6855
6851
obtain some preliminary insights into the mechanism (s)
of action, these compoundsÕ effects on Jurkat cell cycle
analysis were also examined.
5-Hydroxy 7,20,30-trimethoxyflavone (9) and 5-hydroxy
7,20,30-trimethoxyflavanone (10) were prepared from
the corresponding 5-methoxy compounds 4 and 8 by
demethylation with AlCl3 in CH3CN because this meth-
od can selectively cleave the methyl ether b to the car-
bonyl group.16 However, we have not studied further
various products or relative yields from demethylation
reactions.
2. Results and discussion
2.1. Chemistry
Compounds described in this study were prepared fol-
lowing a straightforward chemistry, and are shown in
Figure 1. The starting compounds (chalcones) were syn-
thesized by Claisen–Schmidt condensation between
substituted 20-hydroxyacetophenones and benzaldehy-
des in alkaline medium, according to our previously de-
scribed method.12 Studies with the model compound 4
on cyclization of these chalcones revealed that the reac-
tion was affected by temperature. When these chalcones
were oxidized with 2 equiv of DDQ (2,3-dichloro-5,6-
dicyano-1,4-benzoquinone) in dioxane at 110 ꢁC, a sin-
gle compound, flavone, was generated in 90% yield, as
reported previously.13–15 In fact, our aim was to high-
light the possibility of obtaining several products by
gradual variation of reaction conditions. Therefore,
when the temperature-controlled at 60 ꢁC, the chalcones
were cyclized with DDQ in dioxane to the correspond-
ing flavones and flavanones. Despite modest yield, this
procedure involves mild conditions, a one-step reaction,
does not need any protection of hydroxyl groups, and
affords a range of natural flavones and flavanones.
The majority of flavonoids as shown in Figure 1, occur
in nature confirming the versatility of our method for
the synthesis of natural flavonoids. Compounds 1, 3–5,
and 8–10 were natural products found in Andrographis
spp.17–19 Compound 2 was reported from the fruits of
Neoraputia magnifica.20 Flavonoids 6 and 7 were other
natural compounds and can be isolated from Caesalpi-
nia pulcherrima.21 All the synthesized flavonoid deriva-
tives were chemically characterized by melting point
(mp), infrared (IR) and nuclear magnetic resonance
(1H NMR) spectra, as well as mass spectra (MS).
2.2. Cytotoxicity
The newly synthesized flavonoid derivatives were
examined for their cytotoxic properties in a panel of
four human tumor cell lines containing examples of lym-
phocytic (Jurkat), prostate (PC-3), hepatoma (HepG2),
and colonic (Colon 205). To determine the degree of
toxicity of these compounds toward healthy cells,
experiments were also carried out under the same
R5
R2
R5
R2
R6
R4
R3
R5
R6
R4
R3
R6
R4
R3
H3CO
OH
O
H3CO
O
H3CO
O
(i)
+
R2
R1
R1
O
R1
O
2
3
4
5
6
1. R1 R = OH, R , R , R , R = H
2. R1 = OMe, R2, R5, R6 = H,
R3, R4 = -OCH2O-
,
= OMe, R2, R3, R4, R5, R6 = H
1
6.
R
7. R1 = OMe, R2, R5, R6 = H,
R3, R4 = -OCH2O-
3. R1, R2, R5 = OMe, R3, R4, R6 = H
4. R1, R2, R3 = OMe, R4, R5, R6 = H
5. R1, R2, R4, R6 = OMe, R3, R5 = H
8. R1, R2, R3 = OMe, R4, R5, R6 = H
H3CO
O
H3CO
O
OCH3
OCH3
(ii)
OCH3
OCH3
OH
O
OCH3 O
9
4
H3CO
O
H3CO
O
OCH3
OCH3
(ii)
OCH3
OCH3
OH
O
OCH3 O
10
8
Figure 1. Reagents and conditions: (i) DDQ, 1,4-dioxane, 60 ꢁC, 6 h, (ii) AlCl3, CH3CN, reflux, 1 h.

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Y. K. Rao et al. / Bioorg. Med. Chem. 13 (2005) 6850–6855
experimental conditions in normal PBMCs. All the re-
sults presented were obtained by two independent meth-
ods: cell density measurement—Trypan blue exclusion
method and cellular viability assessment—MTT colori-
metric assay. A treatment period of 3 days was selected
since the control cells were still in the exponential
growth phase at this time. The IC50 (drug concentration
required to reduce the initial cell number by 50%) val-
ues, calculated from the dose-survival curves obtained
after 72 h of compound treatment from MTT test, are
shown in Table 1. These results showed that flavones
1–4 and 9 exhibited significant cytotoxic activity, with
IC50 values below 100 lM, against all of the cell lines,
except the hepatoma cancer cell line HepG2, confirming
the importance of the C2–C3 double bond for flavonoid
cytotoxicity.22 A comparison of the potencies of 4 and 9
suggests that substitution of the hydroxyl with a meth-
oxy group reduces the activity; in addition, 4 possesses
the hydrogen bond of the 5-hydroxyl group with the
4-ketonic oxygen and was therefore more hydrophobic
than 9.22 On the other hand, the flavanones 6–8 and
10 had negligible cytotoxic effects against all cell lines
tested, as their IC50 values were generally higher than
100 lM, which suggested that these compounds dis-
played growth inhibitory activities to human tumor cells
and caused neither normal nor cancer cell death, even
when the concentrations of these compounds were as
high as 100 lM. This finding may be useful to under-
stand the major drawback in the application of antican-
cer drugs to clinical therapies, bringing a host of side
effects due to a large number of normal functional cells
being destroyed by the increasing dosages of antitumor
agents. This phenomenon was unusual and meaningful
to put forward medical therapies on human cancer dis-
eases, whereas drugs with higher cytotoxic activities al-
ways inflicted a the higher damage to normal cells.
Further, the data in this study indicate that the tumor
cell lines were more sensitive to the tested compounds,
while normal cells (PBMCs) are relatively resistant to
these drugs. In the presence of these compounds, cell
growth of the human cancer cell lines Jurkat, PC-3,
and Colon 205 was inhibited, which was indicated by
a range of low IC50 values, whereas the cell growth in
the normal PBMCs was less inhibited as shown by high-
er IC50 values (Table 1).
2.3. Cell cycle analysis
The eukaryotic cell cycle was a series of tightly regulated
events that result in the transmission of genetic material
from one generation to the next. Fidelity of these pro-
cesses was monitored by cell cycle checkpoints that in-
duce cell cycle arrest whenever any damage was
detected. If the damage cannot be rectified, these check-
points may also provoke cells to undergo apoptosis.
Many chemotherapeutic agents take advantage of these
checkpoint controls to preferentially kill rapidly divid-
ing cancer cells. However, there were several problems
common to the existing cancer chemotherapies which
mainly result from a narrow therapeutic index leading
to any undesired side effects. Increased drug resistance
in tumors was another problem.23 Thus, there is still
need for effective drugs with an improved safety profile.
Parallel to the cytotoxic studies described above, a cell-
cycle analysis was performed for compounds 1–10 to
determine whether these flavonoid derivatives influence
the progression of cell cycle of the Jurkat cell line. As
shown in Table 2, Jurkat cells were exposed to the vehi-
cle solvent (DMSO) as control, and IC50 concentrations
of compounds 1–10 were added to the cell line. After
exposure of the compounds for 24 h, attached cells were
Table 2. Cell cycle perturbations induced by flavonoid derivatives
1–10a
Compound
G0/G1
S
G2/M
Control
33.17
82.62
1.21
1
2
34.15*
35.67*
32.32*
28.89*
29.19*
66.07***
32.32*
32.37*
41.02**
29.28*
0.25***
2.01***
9.76***
26.87**
2.47***
31.6**
9.76***
0.5***
65.6***
62.32***
57.92***
44.24***
68.34***
2.33
3
4
5
6
7
57.92***
67.13***
50.89***
2.16
8
9
10
8.09***
68.55*
*,**,
and ***Represents p < 0.05, 0.01, and 0.001, respectively, com-
pared to control.
a Cells were treated with an IC50 value of each compound for 24 h and
then the cell cycle was analyzed by flow cytometry analysis.
Table 1. Cell growth inhibition in the presence of newly synthesized flavonoid derivatives
Compound
IC50 (lM) SD
Jurkat
PC-3
HepG2
Colon 205
PBMCs
1
2
20
15
50
20
200
>200
45
25
150
>200
>200
200 (85%)a
150
3
35
25
45
>200
90 (75%)a
50
4
50
200 (85%)a
>200
>200
5
60
100 (85%)a
>200
>200
200 (85%)a
30
100 (80%)a
>200
6
100
100
100
15
>200
>200
>200
>200
>200
7
>200
>200
200 (85%)a
>200
>200
200 (85%)a
30
200 (80%)a
8
9
10
100
>200
a Percentage of inhibition when compared with control values.

Y. K. Rao et al. / Bioorg. Med. Chem. 13 (2005) 6850–6855
6853
analyzed by flow cytometry. The majority of control
cells exposed to DMSO were either in the G0/G1 phase
(33.17%) or the S phase (82.62%) of the cell cycle and
only a few cells in the G2/M phase were detected
(1.21%) (Table 2). After treatment with compounds 1–
10 for 24 h, an accumulation of G2/M stage cells in
the 1–5 and 7–9 samples was observed. The percentage
of cells in the G2/M phase increased in a range from
1.21% to 68.34% and 67.13% after treatment of Jurkat
cells with 5 and 8. A similar G2/M blockage was also in-
duced by compounds 1–4, 7, and 9. It was assumed that
G2/M phase arrest was possible due to the inhibition of
enzymes essential for G2/M progression or mitosis.24 In
contrast, compound 6 increased the proportion of cells
in the G0/G1 phase, while it decreased the percentage
of those in the S phase, the cells found in the G0/G1
phase was 66.07%, compared to 33.17% for untreated
cells after 24 h of treatment. This finding indicates either
(a) an inhibition of progression through the G1 phase or
an inhibition of transition from G1 into the S phase or
(b) that the cells exited from the cell cycle and entered
the G0 phase.25 Unfortunately, none of the flavonoid
derivatives showed any significant S-phase arrest in Jur-
kat cells at their IC50 concentration, and only compound
10 had significant effects on cell cycle progression. To
the best of our knowledge, this is the first report of the
compounds 1–10 for their cell cycle analysis on the
Jurkat cell line.
chromatography (CC) (0.063–0.200 mm), was a product
of Merck. TLC was performed on Merck TLC plates
(0.23 mm thickness), with compounds visualized by
spraying with 8% (v/v) H2SO4 in EtOH and then heating
on a hot plate.
3.2. General procedure for the synthesis of flavonoid
derivatives (1–8)15
Substituted chalcone (0.01 mol) was dissolved in 1,4-
dioxane (10 mL). To the solution was added DDQ
(0.01 mol). The ensuing mixture was stirred for 6 h at
60 ꢁC. The solvent was removed in vacuo. Water
(50 mL) was added and the mixture was extracted with
EtOAc (3 · 50 mL), dried over MgSO4, and evaporated.
The residue was purified by column chromatography
over silica gel, eluting with gradient of n-hexane/EtOAc
to yield the corresponding substituted flavonoids (1–8)
(Fig. 1).
3.2.1. 5,20-Dihydroxy 7-methoxyflavone (1). This com-
pound was prepared by the general method of 2 with
2,20,60-trihydroxy 40-methoxychalcone as a starting
material, yield 1.68 g, 58.7%, white solid; mp 258–
260 ꢁC; UV (MeOH) kmax (loge): 262 (4.32), 330 (4.10)
1
nm; IR (KBr) mmax: 3350, 2990, 1660, 1615, 1520; H
NMR (600 MHz, CDCl3): d 12.80 (OH-5), 10.82 (OH-
20), 7.88 (1H, dd, J = 7.8, 3.0 Hz, H-60), 7.35 (1H, dt,
J = 7.8 Hz, H-40), 7.15 (1H, s, H-3), 6.95-7.02 (2H, m,
H-30, 50), 6.70 (1H, d, J = 3.0 Hz, H-8), 6.31 (1H, d,
J = 3.0 Hz, H-6), 3.87 (3H, s, OMe-7); ESI-MS (positive
mode) m/z 308.1 [M+Na]+.
In summary, the present work describes the first report
on the synthesis of natural flavonoid derivatives (1–10)
and evaluation of their cytotoxic activities. Representa-
tive compounds inhibited the in vitro growth of various
human cancer cells including lymphocytic, colon, and
prostate. These results revealed that the alkenone moiety
plays an important role in cytotoxicity. Further, this
study also revealed several specific G2/M phase blocker
lead compounds. A more detailed biological evaluation
of these and related compounds will be reported in due
course.
3.2.2. 5,7-Dimethoxy 30,40-methylenedioxyflavone (2).
This compound was prepared by the general method
of 2 with 20-hydroxy 3,4-methylenedioxy 40,60-dimeth-
oxychalcone as a starting material, yield 1.71 g, 52.1%,
white solid, mp 160–162 ꢁC; UV (MeOH) kmax (loge):
260 (4.12), 325 (3.96) nm; IR (KBr) mmax: 1655, 1610,
1495; 1H NMR (500 MHz, CDCl3): d 7.45 (1H, d,
J = 3.0 Hz, H-20), 7.41 (1H, dd, J = 8.0, 3.0 Hz, H-60),
7.15 (1H, s, H-3), 6.96 (1H, d, J = 8.0 Hz, H-50), 6.82
(1H, d, J = 2.5, H-8), 6.40 (1H, d, J = 2.5 Hz, H-6),
6.14 (2H, s, -OCH2O-), 3.89 (3H, s, OMe-7), 3.81 (3H,
s, OMe-5); ESI-MS (positive mode) m/z 350.1 [M+Na]+.
3. Materials and methods
3.1. Chemistry
Unless otherwise noted, chemicals were commercially
available and used as received without further purifica-
tion. Melting points were determined with a Buchi-510
melting point apparatus and are not corrected. Infrared
spectra were recorded on KBr disks on a Perkin-Elmer
FT-IR paragon 500 spectrometer. UV spectra were ob-
tained in MeOH on a Varian Cary Win UV-50 spectro-
photometer. ESI-MS spectra were recorded on a
Thermo-Finnigan LCQ Advantage system. 1H NMR
spectra were measured on a Varian Unity Inova-600
VXR-300/51 spectrometer, using the indicated solvents;
chemical shifts were reported in d (ppm) downfield from
TMS as an internal reference. Coupling constants are
given in Hertz. In case of multiplets, the chemical shift
quoted was measured from an approximate center. Inte-
grals corresponded satisfactorily to those expected on
the basis of compound structure. Silica gel for column
3.2.3. 7,20,50-Trimethoxyflavone (3). This compound was
prepared by the general method of 2 with 20-hydroxy
2,40,5-trimethoxychalcone as a starting material, yield
1.90 g, 60.5%, yellow amorphous powder; mp 136–
138 ꢁC; UV (MeOH) kmax (loge): 255 (4.24), 330 (4.13)
1
nm; IR (KBr) mmax: 2940, 1645, 1618, 1584; H NMR
(600 MHz, CDCl3): d 8.13 (1H, d, J = 9.0 Hz, H-5),
7.44 (1H, d, J = 3.6 Hz, H-60), 7.11 (1H, s, H-3), 7.03
(1H, dd, J = 9.0, 3.6 Hz, H-40), 6.98 (2H, m, H-30, 8),
6.92 (1H, d, J = 2.4 Hz, H-6), 3.92 (3H, s, OMe-7),
3.89 (3H, s, OMe-20), 3.86 (3H, s, OMe-50); ESI-MS
(positive mode) m/z 313.3 [M+H]+.
3.2.4. 5,7,20,30-Tetramethoxyflavone (4). This compound
was prepared by the general method of 2 with 20-hy-
droxy 2,3,40,60-tetramethoxychalcone as a starting mate-
rial, yield 1.86 g, 54.0%, yellow crystals, mp 150–152 ꢁC;

6854
Y. K. Rao et al. / Bioorg. Med. Chem. 13 (2005) 6850–6855
UV (MeOH) kmax (loge): 260 (3.98), 310 (3.76) nm;
IR (KBr) mmax: 2930, 1656, 1612, 1575; 1H NMR
(600 MHz, CDCl3): d 7.32 (1H, dd, J = 7.8, 1.2 Hz, H-
60), 7.17 (1H, t, J = 7.8 Hz, H-50), 7.05 (1H, dd,
J = 7.8, 1.2 Hz, H-40), 6.83 (1H, s, H-3), 6.51 (1H, d,
J = 3.0 Hz, H-8), 6.38 (1H, d, J = 3.0 Hz, H-6), 3.96
(3H, s, OMe-5), 3.92 (3H, s, OMe-30), 3.90 (3H, s,
OMe-7), 3.88 (3H, s, OMe-20); ESI-MS (positive mode)
m/z 366.1 [M+Na]+.
3.3. General procedure for the preparation of compounds
9 and 1016
A mixture of substituted 5-methoxyflavonoid derivative
(0.1 mmol) and anhydrous aluminium chloride
(0.4 mmol) in acetonitrile (5 mL) was refluxed for 1 h.
After standing at room temperature for 2 h, the mixture
was poured into ice water and then neutralized with di-
lute HCl. The aqueous phase was extracted with EtOAc
(3 · 50 mL). The combined organic layer was washed
with a saturated NaCl, dried, filtered, and concentrated.
The residue was purified by column chromatography
over silica gel, eluting with a gradient of n-hexane/
EtOAc to yield the corresponding substituted flavonoids
9 and 10 (Fig. 1).
3.2.5. 5,7,20,40,60-Pentamethoxyflavone (5). This com-
pound was prepared by the general method of 2 with
20-hydroxy 2,4,40,6,60-pentamethoxychalcone as a start-
ing material, yield 2.01 g, 53.7%, pale yellow solid, mp
192–194 ꢁC; UV (MeOH) kmax (loge): 256 (4.38), 304
1
(4.18) nm; IR (KBr) mmax: 2940, 1660, 1612, 1585; H
3.3.1. 5-Hydroxy 7,20,30-trimethoxyflavone (9). This
compound was prepared by the general method of 3
with 5,7,20,30-tetramethoxyflavone (4) as a starting
material, yield 0.30 g, 88.7%, yellow amorphous solid,
mp 190–192 ꢁC; UV (MeOH) kmax (loge): 245 (3.68),
330 (3.00) nm; IR (KBr) mmax: 3160, 2920, 1656, 1610;
1H NMR (600 MHz, CDCl3): d 12.77 (1H, s, OH-5),
7.32 (1H, dd, J = 7.8, 1.2 Hz, H-60), 7.17 (1H, t,
J = 7.8 Hz, H-50), 7.06 (1H, dd, J = 7.8, 1.2 Hz, H-40),
6.86 (1H, s, H-3), 6.43 (1H, d, J = 2.4 Hz, H-8), 6.36
(1H, d, J = 2.4 Hz, H-6), 3.91 (3H, s, OMe-20), 3.87
(3H, s, OMe-7), 3.85 (3H, s, OMe-30); ESI-MS (positive
mode) m/z 352.1 [M+Na]+.
NMR (600 MHz, CDCl3): d 6.44 (1H, d, J = 2.4 Hz,
H-8), 6.33 (1H, d, J = 2.4 Hz, H-6), 6.22 (1H, s, H-3),
6.14 (2H, s, H-30, 50), 3.93 (3H, s, OMe-5), 3.84 (3H,
s, OMe-40), 3.83 (3H, s, OMe-7), 3.75 (6H, s, OMe-20,
60); ESI-MS (positive mode) m/z 395.1 [M+Na]+.
3.2.6. 5,7-Dimethoxyflavanone (6). This compound was
prepared by the general method of 2 with 20-hydroxy
40,60-dimethoxychalcone as a starting material, yield
1.2 g, 42.2%, white needles, mp 98–100 ꢁC; UV
(MeOH) kmax (loge): 282 (4.20), 325 sh (3.85) nm; IR
(KBr) mmax: 3140, 1645, 1600, 1505 cmꢀ1 1H NMR
;
(600 MHz, CDCl3): d 7.47 (2H, dd, J = 9.0, 1.8 Hz,
H-20, 60), 7.42 (2H, dt, J = 9.0, 1.8 Hz, H-30, 50), 7.37
(1H, m, H-40), 6.17 (1H, d, J = 2.4 Hz, H-8), 6.10
(1H, d, J = 2.4 Hz, H-6), 5.41 (1H, dd, J = 13.2,
3.0 Hz, H-2), 3.90 (3H, s, OMe-7), 3.82 (3H, s, OMe-
8), 3.02 (1H, dd, J = 16.2, 13.2 Hz, H-3ax), 2.80 (1H,
dd, J = 16.2, 3.0 Hz, H-3eq); ESI-MS (positive mode)
m/z 285.1 [M+H]+.
3.3.2. 5-Hydroxy 7,20,30-trimethoxyflavanone (10). This
compound was prepared by the general method of 3
with 5,7,20,30-tetramethoxyflavanone (8) as a starting
material, yield 0.31 g, 90.1%, colorless solid, mp 148–
150 ꢁC; IR (KBr) mmax: 3390, 2910, 1655, 1628,
1
1585 cmꢀ1; H NMR (600 MHz, CDCl3): d 12.07 (1H,
s, OH-5), 7.13 (2H, d, J = 9.0 Hz, H-40, 60), 6.92 (1H,
t, J = 9.0 Hz, H-50), 6.06 (1H, d, J = 2.4 Hz, H-8), 6.03
(1H, d, J = 2.4 Hz, H-6), 5.74 (1H, dd, J = 13.2,
3.0 Hz, H-2), 3.87 (3H, s, OMe-20), 3.86 (3H, s, OMe-
7), 3.79 (3H, s, OMe-30), 3.03 (1H, dd, J = 16.2,
13.2 Hz, H-3ax), 2.79 (1H, dd, J = 16.2, 3.0 Hz, H-3eq);
ESI-MS (positive mode) m/z 329.1 [M+H]+.
3.2.7. 5,7-Dimethoxy 30,40-methylenedioxyflavanone (7).
This compound was obtained together with compound
2 in the same reaction as colorless needles, yield
0.90 g, 27.4%; IR (KBr) mmax: 3090, 1665, 1620,
1575 cmꢀ1
;
1H NMR (600 MHz, CDCl3): d 7.05
(1H, d, J = 1.8 Hz, H-20), 6.96 (1H, dd, J = 8.4,
1.8 Hz, H-60), 6.86 (1 H, d, J = 8.4 Hz, H-50), 6.20
(1H, d, J = 2.4 Hz, H-8), 6.13 (1H, d, J = 2.4 Hz, H-
6), 6.10 (2H, s, -OCH2O-), 5.39 (1H, dd, J = 13.2,
3.0 Hz, H-2), 3.91 (3H, s, OMe-7), 3.84 (3H, s,
OMe-5), 3.03 (1H, dd, J = 15.6, 13.2 Hz, H-3ax), 2.78
(1H, dd, J = 15.6, 3.0 Hz, H-3eq); ESI-MS (positive
mode) m/z 329.1 [M+H]+.
3.4. Biological data
3.4.1. Chemicals. Compounds were dissolved in dimeth-
ylsulfoxide just before the experiments; calculated
amounts of drug solution were added to the growth
medium containing cells to a final solvent concentration
of 0.5%, which had no discernible effect on cell killing.
MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoli-
um bromide and L-glutamine were obtained from Sigma
Chemical Co., St. Louis, USA.
3.2.8. 5,7,20,30-Tetramethoxyflavanone (8). This com-
pound was obtained together with compound 4 in the
same reaction as colorless needles, yield 1.21 g,
35.1%; mp 164–166 ꢁC; UV (MeOH) kmax (loge): 264
(4.20), 335 sh (3.85) nm; IR (KBr) mmax: 1665, 1610,
1
1595 cmꢀ1; H NMR (600 MHz, CDCl3): d 7.13 (2H,
3.4.2. Cell culture. Jurkat, PC-3, Colon 205, and HepG2
cell lines were obtained from ATCC (Rockville, MD).
Blood was collected from healthy volunteers and periph-
eral blood mononuclear cells (PBMCs) were isolated by
Ficoll–Hypaque (Pharmacia) density gradient centrifu-
gation.26 The cell numbers were determined with a
hemocytometer, and viabilities were assessed by Trypan
blue dye exclusion. Cell lines were maintained in
m, H-50, 60), 6.92 (1H, dd, J = 7.8, 1.8 Hz, H-40), 6.12
(1H, d, J = 2.4 Hz, H-8), 6.08 (1H, d, J = 2.4 Hz, H-
6), 5.75 (1H, dd, J = 13.2, 3.0 Hz, H-2), 3.89 (3H, s,
OMe-50), 3.87 (3H, s, OMe-5), 3.84 (3H, s, OMe-30),
3.80 (3H, s, OMe-7), 2.96 (1H, dd, J = 16.2, 13.2 Hz,
H-3ax), 2.76 (1H, dd, J = 16.2, 3.0 Hz, H-3eq); ESI-
MS (positive mode) m/z 368.1 [M+Na]+.

Y. K. Rao et al. / Bioorg. Med. Chem. 13 (2005) 6850–6855
6855
logarithmic phase at 37 ꢁC in a 5% carbon dioxide atmo-
References and notes
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3.5. Statistics
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Acknowledgments
This research was supported by National Science Coun-
cil of Taiwan (NSC 91-2622-E-324-003-CC3; NSC
92-2811-M-324-002) and China Medical University
(CMU-93-M-06).