Differential effects of synthesized 2′-oxygenated chalcone derivatives: Modulation of human cell cycle phase distribution

Article abstract of DOI:10.1016/j.bmc.2004.03.014

Ten structurally related 2′-oxygenated chalcone derivatives, bearing either hydroxy and/or methoxy substituents on the A and B rings, were synthesized through Claisen-Schmidt condensation. The synthesis procedure was relatively easy and had an acceptable yield. The in vitro cytotoxicities of these compounds against the human tumor cells such as Jurkat, U937 cells, and normal cells PHA stimulated PBMCs were investigated. Among those, compounds 1 (IC₅₀=2.5μM), 2 (1.7μM), and 8 (3.2μM) showed potent inhibitory activity toward Jurkat cell line. In parallel, compounds 1 (6.7μM), 2 (1.5μM), and 10 (5.3μM) showed the highest activity against U937 cell line. However, the chalcones also inhibit the PHA stimulated PBMCs cells, but the IC₅₀ values were relatively high when compared to the tumor cell line values. Studies were also on the effect of synthesized chalcones on the cell cycle phase distribution. In Jurkat cell line, compounds 7 and 9 showed the highest activity and the most striking effect in reduction of the percentage of cells in the S phase, which was associated with an increase of cells in G2/M phase. In U937 cell line, compound 3 increased the proportion of cells in the G0/G1 phase and reduced the proportion in S phase. In contrast, compounds 1, 9, and 10 showed a decrease effect on the percentage of cells in S phase and an increase effect on the percentage of cells in the G2/M phase of the cell cycle. Whereas in the case of PHA stimulated PBMCs, compounds 1, 4, 8, and 10 increased the percentage of cells in G2/M phase, which was associated with a decrease effect in the S phase of the cell cycle.

Full text of DOI:10.1016/j.bmc.2004.03.014

Bioorganic & Medicinal Chemistry 12 (2004) 2679–2686
Differential effects of synthesized 20-oxygenated chalcone
derivatives: modulation of human cell cycle phase distribution
Yerra Koteswara Rao,a Shih-Hua Fangb and Yew-Min Tzenga,*
aDepartment of Applied Chemistry, Chaoyang University of Technology, Wufeng 413, Taiwan, ROC
bDepartment of Microbiology, School of Medicine, China Medical University, Taichung 400, Taiwan, ROC
Received 22 December 2003; revised 16 February 2004; accepted 8 March 2004
Available online 2 April 2004
Abstract—Ten structurally related 20-oxygenated chalcone derivatives, bearing either hydroxy and/or methoxy substituents on the A
and B rings, were synthesized through Claisen–Schmidt condensation. The synthesis procedure was relatively easy and had an
acceptable yield. The in vitro cytotoxicities of these compounds against the human tumor cells such as Jurkat, U937 cells, and
normal cells PHA stimulated PBMCs were investigated. Among those, compounds 1 (IC50 ¼ 2.5 lM), 2 (1.7 lM), and 8 (3.2 lM)
showed potent inhibitory activity toward Jurkat cell line. In parallel, compounds 1 (6.7 lM), 2 (1.5 lM), and 10 (5.3 lM) showed the
highest activity against U937 cell line. However, the chalcones also inhibit the PHA stimulated PBMCs cells, but the IC50 values
were relatively high when compared to the tumor cell line values. Studies were also on the effect of synthesized chalcones on the cell
cycle phase distribution. In Jurkat cell line, compounds 7 and 9 showed the highest activity and the most striking effect in reduction
of the percentage of cells in the S phase, which was associated with an increase of cells in G2/M phase. In U937 cell line, compound 3
increased the proportion of cells in the G0/G1 phase and reduced the proportion in S phase. In contrast, compounds 1, 9, and 10
showed a decrease effect on the percentage of cells in S phase and an increase effect on the percentage of cells in the G2/M phase of
the cell cycle. Whereas in the case of PHA stimulated PBMCs, compounds 1, 4, 8, and 10 increased the percentage of cells in G2/M
phase, which was associated with a decrease effect in the S phase of the cell cycle.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
Chalcones are a class of anticancer agents that have
shown promising therapeutic efficacy for the manage-
ment of human cancers. Chalcones, considered as the
precursor of flavonoids and isoflavonoids, are abundant
in edible plants. Chemically they consist of open-chain
flavonoids in which the two aromatic rings are joined by
a three-carbon a,b-unsaturated carbonyl system. Recent
studies revealed that these chalcones had shown a wide
variety of anticancer,1–7 anti-inflammatory,8–10 anti-
invasive,11 antituberculosis,12 and antifungal13 activities.
Previous studies showed that the 20-oxygenated chalcone
derivatives (2POCDs) possess a wide spectrum of
activities like inhibition of MCF-7 human breast cancer
cells growth,14 prostaglandin E2 production inhibition
in rat peritoneal macrophages,15 inhibition of the iNOS
protein expression, and inhibition of cyclooxygenase-2
in RAW 264.7 cells.16 In a recent article, 20-hydroxy-
chalcones are reported as inhibitors of peroxynitrite-
mediated oxidation of low-density lipoproteins (LDL).17
Furthermore, some 20-aminochalcone derivatives were
also found to show antitumor activity.18 However, there
is not yet specific report on the cytotoxic effects of
2POCDs on human tumor cells such as Jurkat, U937,
Cancer is one of the leading causes of death in the
present society. A great deal of efforts have been
underway to treat various forms of cancer for decades;
and until recently, chemoprevention of cancer is
receiving its due share of attention. Among the small
molecule drugs developed to prevent or treat cancer,
there is a potentially important group of structurally
effective features, which contain the alkenone moiety as
the integral part of the molecule. Alkenones, acyclic, or
cyclic, are a class of organic molecules bearing a ketone
functionality and an unsaturated group (double bond).
These two functional groups are in conjugated
arrangement among the naturally occurring or synthetic
anticancer agents.
Keywords: Chalcones synthesis; Anticancer activity.
* Corresponding author. Tel.: +886-4-23323000x3003; fax: +886-4-
23304896; e-mail: ymtzeng@cyut.edu.tw
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.03.014

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Y. K. Rao et al. / Bioorg. Med. Chem. 12 (2004) 2679–2686
and normal cells PHA stimulated primary peripheral
blood mononuclear cells (PBMCs); and exploring the
relevance for a potential clinical application. The pres-
ent article is focused on synthesis of specific chalcones
and interactions of the synthesized 2POCDs with human
lymphocytic, monocytic cell lines and PHA stimulated
PBMCs to address additional important aspects of
chalcone pharmacology and toxicology.
method for the preparation of chalcones is attractive
because it specifically generates the trans-isomer (the
coupling constants of the two olefinic protons were
around 16 Hz), with a high yield. The ease of prepara-
tion and the biological activity of chalcones clearly
showed that as a class of anticancer agent, they deserved
greater attention.20 Among these, compounds 3, 8, 9,
and 10 were prepared for the first time, but the com-
pounds 1,21 2,22 4–710;23–27 had been reported earlier.
However, as the complete spectral data of some com-
pounds had not been reported so far these were recorded
here (vide Experimental).
2. Results
2.1. Preparation and characterization of compounds
2POCDs
The general synthetic strategy employed to prepare the
chalcone derivatives was based on Claisen–Schmidt
condensation, which was reported previously.19;20 As
shown in Table 1, a series of 10 chalcone derivatives (1–
10), bearing either hydroxy and/or methoxy substituents
on the A and B rings were prepared by base catalyzed
condensation of appropriately substituted acetophenone
with substituted benzaldehyde in EtOH (Scheme 1). To
a stirred mixture of substituted acetophenone and
substituted benzaldehyde in alcohol at 0 ꢁC was added a
60% solution of KOH dropwise in 60 min. The reaction
mixture was kept at room temperature for 2 days, then
diluted with water and neutralized with 2 N HCl, then
extracted with EtOAc. After purification of the residue
obtained, chalcones were obtained yields of 45–88%.
The structures of all the 10 chalcones synthesized in this
study were established on the basis of UV, IR, 1H
NMR, 13C NMR, and ESI-MS spectral data. This
2.2. Effect of 2POCDs on cell proliferation
In a preliminary feasibility study, it was aimed to
determine the concentrations of chalcones inhibiting cell
growth by 50% (IC50) in two cancer cell lines and in
PHA stimulated PBMCs. Cytotoxicity assays were car-
ried out by use of the MTT test. Exponentially growing
cultures of each cell line were exposed to a series of
increasing concentration of different chalcones. A dose-
dependent decrease in cell number was observed in all
tested samples demonstrated by IC50 values (Table 2). In
particular, compounds 1, 2, and 8 were the most active
agents against Jurkat cell line with IC50 values 2.5, 1.7,
and 3.2 lM, respectively. Furthermore, compounds 5–7
and 10 showed moderate activity (IC50 < 10 lM), and
the remaining compounds were less active toward Jurkat
cell line. Similar results were obtained with U937 cell
line, among 10 tested compounds, compounds 1, 2, and
Table 1. Structure and analytical data of 20-oxygenated chalcone derivatives
R
A
R
R
2
6
3
3'
R
3
R
R
R
R
1
5
7
2'
1'
2
4'
5'
4
B
α
1
5
8
6'
β
6
R
O
4
9
No
R1
R2
R3
R4
R5
H
R6
R7
R8
R9
Formula
Yield (%)
1
2
OMe
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
OMe
OMe
OMe
OMe
H
OMe
OMe
OMe
OMe
H
H
H
H
H
H
C
18H18O4
18H20O6
80
88
85
86
45
52
46
77
82
86
H
OMe
OMe
OMe
OH
H
OMe
OMe
H
H
H
C
3
H
OMe
OMe
H
H
H
C20H22O7
C20H22O7
C15H12O3
4
H
H
OMe
H
5
H
H
H
6
OMe
H
OMe
OH
H
H
OH
OMe
H
H
H
C17H16O5
7
H
H
OH
H
H
H
C16H14O5
C18H18O5
C18H16O6
8
H
OMe
OMe
OMe
H
OMe
H
OMe
OMe
H
H
9
10
OMe
OMe
H
–OCH2O–
OMe OMe
H
H
H
H
C
20H22O7
R2
R6
R5
R9
R6
R2
3
3'
R3
R1
R5
R7
2'
2
H
O
R3
R1
a
4'
5'
4
B
A
R7
α
+
1
1'
b
CH3
5
R8
6'
R4
β
6
R8
O
R9
R4
O
Scheme 1. General synthetic route for the synthesis of 20-oxygenated chalcone derivatives. Reagents and conditions: (a) 60% KOH, EtOH, 48 h;
(b) 2 N HCl.

Y. K. Rao et al. / Bioorg. Med. Chem. 12 (2004) 2679–2686
2681
Table 2. Cytotoxicity of chalcone derivatives against human cancer
cell lines Jurkat, U937, and PHA stimulated PBMCs [50% inhibition
concentration (IC50, lM)]
Table 4. Effect of 20-oxygenated chalcone derivatives on the distribu-
tion of different phases in U937 cell cycle
Compd
Concn (lM) G0/G1
S
G2/M
Compd
Jurkat cells
U937 cells
PBMCs
Control
1
32.61a
43.11
0ꢀꢀꢀ
24.28ꢀꢀꢀ
44.86ꢀꢀ
0ꢀꢀꢀ
1
2
3
4
5
6
7
8
9
10
2.5
1.7
6.7
1.5
6.7
5.8
6.7
1.5
39.01
36.12
98.27ꢀꢀꢀ
48.04
29.84
45.45
42.88
43.84
44.14
36.82
60.99
2
19.02ꢀꢀ
1.73ꢀꢀꢀ
33.98ꢀꢀ
16.84ꢀꢀꢀ
1.93
16.0
66.8
8.3
16.0
93.6
20.8
13.3
17.5
16.0
30.5
5.3
16.0
107.0
52.1
13.3
35.0
39.8
91.5
10.7
3
16.0
93.6
20.8
13.3
17.5
16.0
30.5
5.3
4
17.98ꢀꢀ
53.32ꢀꢀ
52.63ꢀꢀ
54.65ꢀꢀ
50.59
55.86ꢀꢀ
63.18ꢀꢀꢀ
5
6.7
7.0
6
7
2.49ꢀꢀꢀ
5.ꢀ5ꢀ8ꢀ ꢀꢀꢀ
0
3.2
8
30.5
5.3
9
10
0ꢀꢀꢀ
a Percentage of cells in different phases.
ꢀRepresents p < 0.05 compared with control.
** Represents p < 0.01 compared with control.
*** Represents p < 0.001 compared with control.
10 were found to exhibit the highest activity with IC50
values 6.7, 1.5, and 5.3 lM, respectively; followed by the
remaining compounds of less activity. Interestingly, the
inhibitory activity values (IC50) of chalcones 2, 4, 5, and
7–10 in PBMCs were quite high comparing with the
tumor cell line results (Table 2). The data in Table 2 also
showed the toxicity profile of 2POCDs and the results
implied that these chalcones were more cytotoxic to
cancer cell lines than to normal cells.
Table 5. Effect of 20-oxygenated chalcone derivatives on the distribu-
tion of different phases in PHA stimulated PBMCs cell cycle
Compd
Concn (lM)
G0/G1
S
G2/M
Control
PHA only
57.67a
92.03
30.93
11.4
4.35
0ꢀ
3.63
1
2
6.7
5.8
54.62ꢀꢀ
51.35ꢀꢀ
58.73ꢀꢀ
62.37ꢀꢀ
78.33ꢀꢀ
72.13ꢀꢀ
63.02
58.21ꢀꢀ
71.18ꢀ
34.61ꢀꢀꢀ
45.38ꢀꢀꢀ
36.04ꢀꢀ
27.41ꢀꢀ
37.34ꢀꢀꢀ
17.94ꢀꢀ
23.16
12.61ꢀ
13.86ꢀ
0.29ꢀ
3.76
4.53
11.22ꢀ
0ꢀ
2.3. Cell cycle analysis
3
16.0
107.0
52.1
13.3
35.0
39.8
91.5
10.7
4
Cell cycle analysis was performed to explore the basis
for antiproliferative properties of these synthesized
chalcones. For Jurkat cells, at the IC50 of each synthe-
sized chalcone, it was found that chalcones 7 and 9
exhibited the highest activity and caused cells arrest in
the G2/M phase with a concomitant decrease of cells in
the S phase (Table 3). In addition, compounds 5 and 8
showed less activity and cells arrest in the G2/M phase.
In contrast, compounds 1 and 2 cause accumulation in
the G2/M phase with a concomitant decrease of cells in
the S phase. For U937 cells, the results showed at the
IC50 of compound 3, it caused marked accumulation in
the G0/G1 phase with a concomitant decrease of cells in
the S phase (Table 4). However, PHA stimulated normal
PBMCs proliferation was inhibited by each synthesized
chalcone mostly through arresting cells in the S phase
(Table 5).
5
6
7
25.76ꢀꢀ
41.79ꢀꢀꢀ
28.54ꢀꢀ
58.56ꢀꢀꢀ
8
9
10
0.29ꢀ
6.84ꢀ
a Percentage of cells in different phases.
* Represents p < 0.05 compared with PHA only.
** Represents p < 0.01 compared with PHA only.
*** Represents p < 0.001 compared with PHA only.
3. Discussion
Many diverse phytochemicals exist in nature, and a
great number of them have been traditionally used for
treating specific diseases.28–31 Massive screening as well
as chemical modification of phytochemicals would lead
to the discovery of some modern drugs. However, it was
often hampered by the difficulty of obtaining a sufficient
amount of the phytochemicals. Here, the 2POCDs are
also a group of phytochemicals, which occur in very
small quantities in nature. Moreover, these compounds
have anticancer potential to interfere with targets makes
them interesting tools in cancer research.4–7;14;18;34 They
were therefore chemically synthesized, purified, and
characterized for investigating their biological activity.
The synthesis was easy, one-step reaction, no need for
protection of hydroxyl groups and had a good yield. The
synthesized compounds were structurally identical to
2POCDs in plants. From the IC50 values summarized in
Table 2, compounds 1, 2, and 8 showed significant
(IC50 < 4 lM) cytotoxic activity, followed by compounds
5–7 and 10, especially selectivity against Jurkat cell lines.
Compound 2, recently reported as natural compound
Table 3. Effect of 20-oxygenated chalcone derivatives on the distribu-
tion of different phases in Jurkat cell cycle
Compd
Concn (lM)
G0/G1
S
G2/M
Control
50.86a
44.86ꢀ
43.75ꢀ
46.87
49.35
49.23
49.75
54.1
23.72
23.08
24.44
24.57
20.61
17.78ꢀ
20.23
0ꢀꢀꢀ
25.42
32.06ꢀ
31.81ꢀ
28.56
30.03
32.98ꢀ
30.02
45.9ꢀꢀ
34.31ꢀ
55.97ꢀꢀ
31.57
1
2
3
4
5
6
7
8
9
10
2.5
1.7
16.0
66.8
8.3
6.7
7.0
3.2
49.19
44.03ꢀ
47.61
16.5ꢀ
0ꢀꢀꢀ
30.5
5.3
20.82
a Percentage of cells in different phases.
* Represents p < 0.05 compared with control.
** Represents p < 0.01 compared with control.
*** Represents p < 0.001 compared with control.

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Y. K. Rao et al. / Bioorg. Med. Chem. 12 (2004) 2679–2686
from the aerial parts of Caesalpinia pulcherrima22 con-
tained a 20-hydroxy, and methoxyl moieties at the 2-, 3-,
40-, 60-positions was the most active in this study toward
cancer cell line. Our results provide further support to
the recent findings, that 2-hydroxychalcone and 20-hy-
droxychalcone were shown to inhibit proliferation of
HeLa cells and were the most potent among many other
chalcones and related flavonoid derivatives tested.32
Furthermore, noticed that in all cases 2POCDs were
more potent against cancer cell line than corresponding
flavanones,14 and the methoxy group at the carbon-2
atom greatly enhanced the ability of chalcone to inhibit
the cell growth. In addition, it is noteworthy that the
related chalcone isoliquiritigenin (20,4,40-trihydroxy-
chalcone), which occurs naturally in bark and wood of
some leguminous trees, was found to be a very strong
antitumor agent.33 The data in this study indicate that
the cancer cell lines are more sensitive to the compounds
1–3, 5–8, and 10, while PHA stimulated PBMCs are
relatively resistant to these compounds. In the presence
of these compounds, cell growth of the human cancer
cell lines Jurkat, and U937 is inhibited, which is indi-
cated by the range of low IC50 values from 1.7 to 17.5.
Cell growth in the normal PHA stimulated PBMCs is
less inhibited, shown by higher IC50 values ranging from
5.8 to 52.1. Among the 2POCDs, compounds 4 and 9
with 2,4,6-trimethoxy and 3,4-methylenedioxy moieties
in B ring were practically showed less cytotoxicity. It
may be due to the significant steric effects induced by the
presence of methoxyl group in 2-, 6-positions of com-
pound 4 on ring B. The presence of ortho-substituents
on ring A, which affect the planarity of compound 9,
may be the cause for its lower activity, but its IC50
(30.5 lM), value toward tumor cell lines is lower than
the structurally similar compound, 20-hydroxy-3,30,4,40-
tetramethoxychalcone IC50 (40.2 lM), value against
phospholipase A2.34
S phase. Cell cycle arrest may result in cell death
(apoptosis or necrosis). In this study, we found that
2POCDs inhibited proliferation of both normal and
cancer cells of various histogenetic origin with IC50
values. This effect was mainly due to an inhibition of cell
proliferation rather than a direct cytotoxic effect, as
suggested by the limited amount of dead cells detected in
the culture by the trypan blue exclusion test, and was
associated with an inhibition of cell cycle progression.
However, this conclusion will require in-depth investi-
gation to conclusively determine whether apoptosis or
necrosis is the pathway of cell death, and for identifi-
cation of a nontoxic, potent compound activity at very
low concentrations to provide leads toward new thera-
peutic agents in cancer research.
4. Conclusion
In an attempt to generate novel anticancer structures
with significant activities against human cancer cell line
but more potent than those in use nowadays, a series of
10 chalcone derivatives were synthesized and evaluated
for their cytotoxicity against Jurkat, U937, human tu-
mor cell lines. Compounds 1, 2, and 8; 1, 2, and 10 were
identified as potent cytotoxic against Jurkat and U937
cell lines, respectively. Further, cell cycle perturbations
induced by the chalcones synthesized were also studied
on the Jurkat, U937, and PHA stimulated PBMCs cell
lines. The present study revealed that these chalcones
were potent antiproliferative agents against tumor cell
lines without being more cytotoxic to normal cells. The
results of this study may find a lead toward the devel-
opment of new therapeutic agents to fight cancer.
5. Experimental
Several studies demonstrated an association between cell
cycle regulation and cancer.35 The cell cycle machinery
regulates cell proliferation, and dysregulated cellular
proliferation is a hallmark of cancer.36;37 Many synthetic
cell cycle inhibitors such as flavopiridol, olomoucine,
roscovitine, and puvalanol B were viewed as a new
generation of anticancer drugs, and some were investi-
gated in clinical trials.38–40 Cell cycle analysis of 2POCDs
treated Jurkat cells clearly showed a G2/M arrest by the
compounds 7 and 9, no effect by the compound 3, and
only a slight effect by the compounds 5 and 8 (Table 3).
The increase in the G2/M was accompanied with a
decrease of cell numbers in the S phase. The compounds
7, 9 and 5, 8 caused G2/M arrest was irreversible.
Whereas in the case of U937, compound 3 showed a G0/
G1 arrest accompanied by a decrease of cells in the S
phase. In contrast, compounds 1, 2, and 5–10 caused
cells arrest in the G2/M phase with a concomitant de-
crease in percentage of cells in the S phase of the cell
cycle (Table 4). PHA stimulated PBMCs proliferation
also showed similar profiles (Table 5), where compounds
1 and 8 showed the percentage of cells increase in S
phase accompanied with decrease in percentage of cells
in the G2/M phase. In contrast, compound 5 showed a
G0/G1 arrest associated with the decrease of cells in the
5.1. Cell
Jurkat (human lymphocytic cell line), and U937 (human
monocytic cell line) were obtained from American Type
Culture Collection (Rockville, MD), and cultured with
complete RPMI-1640 medium (Gibco BRL) supple-
mented with 10% fetal calf serum, antibiotics, L-gluta-
mine. The cell numbers were determined with a
hemocytometer, and viabilities were assessed by trypan
blue dye exclusion. Blood was collected from healthy
volunteers and peripheral blood mononuclear cells
(PBMCs) were isolated by Ficoll-Hypaque (Pharmacia)
density gradient centrifugation as described before.41
The cells were washed twice with Hank’s balanced salt
solution (HBSS) and resuspended in RPMI 1640 com-
plete medium with 2% human AB serum prior to
immunological studies.
5.2. MTT assay
MTT [3-(40,50-dimethylthiazol-20-yl)-2,5-diphenyltetra-
zolium bromide] assay as described by Mosman42 was
widely used to determine the cytotoxicity of the anti-
cancer drugs. The growth inhibitory activity of the

Y. K. Rao et al. / Bioorg. Med. Chem. 12 (2004) 2679–2686
2683
compounds were determined in the PBMCs and human
cancer cell lines by adding MTT into each well and then
incubated at 37 ꢁC for 4 h. The MTT formazan crystals
(1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan) for-
med due to the reduction of MTT, were dissolved by
addition of acid–isopropanol and mixed at room tem-
perature. After 20 min, the optical density (OD) was
measured with a microplate reader (BIO-RAD, model
3550, USA) at 570 nm (OD570–620). The mean OD value
of the content of four wells was used for assessing the
cell viability. Percent survival was calculated by the
following formula
Chemical structures of the compounds as shown in
Table 1, were determined by the following spectral data.
UV spectra were recorded on a Varian Cary Win UV-50
spectrophotometer. IR spectra were determined in KBr
discs on a Perkin–Elmer FT-IR paragon 500 spectro-
1
meter. H NMR, 13C NMR spectra were recorded on a
Bruker Avance 500 spectrometer operating at 500.13,
125.43 MHz, respectively, in DMSO-d6 and CDCl3.
ESI-MS were recorded on a Thermo-Finnigan LCQ
Advantage system. Column chromatography (CC) sep-
arations were carried out by using Silica gel 60 (0.063–
0.200 mm) supplied by E. Merck.
General procedure for the synthesis of 2POCDs. 2-Hy-
droxy-3,4-dimethoxyacetophenone was prepared as re-
ported earlier.25 All the remaining starting materials
were commercially available. To a solution of an
appropriately substituted acetophenones (10 mmol), an
adecuated benzaldehydes (10 mmol) in EtOH (20 mL)
was added a 60% KOH (10 g) solution dropwise at 0 ꢁC
in 60 min. The reaction mixture was kept at room tem-
perature for 2 days and then water (50 mL) was added.
The mixture was neutralized with 2 N HCl (50 mL) and
extracted with EtOAc (3 · 200 mL). The combined or-
ganic layer was separated, washed with water, dried over
anhydrous MgSO4, and concentrated under reduced
pressure. The residue yielded was crude chalcone and it
was purified by CC eluted with mixtures of hexane/
EtOAc to give pure chalcone derivative.
ðOptical density of drug treated wells=
optical density of untreated wellsÞ ꢁ 100
5.3. Proliferation assay
Proliferation was measured by [3H] thymidine incorpo-
ration assay described before.43 Briefly, different chal-
cones were added to Jurkat or U937 cell culture of
2 · 106 cells/mL into 96 well microplates for 24 h.
PBMCs were stimulated with or without 5 lg/mL phy-
tohemagglutinin-L (PHA) (Sigma) in the presence of
chalcones for 72 h. During the last 18 h, cells were pulsed
with 1 lCi of [3H] thymidine (Amersham France SA, Les
Ulis, France). The result of proliferative response was
expressed as (cpm value of experiment)/(cpm value of
control) · 100%. The inhibition of 50% proliferation was
defined as IC50.
5.6.1. 20,40,60-Trimethoxychalcone (1). This was synthe-
sized using 2,4,6-trimethoxyacetophenone (2.10 g,
10 mmol) and benzaldehyde (1.06 g, 10 mmol) as starting
materials: yellow crystalline solid (2.53 g, 80%), mp 110–
112 ꢁC; UV kmax (MeOH) (log e): 305 (3.93), 238 (3.26)
5.4. Cell cycle analysis
Cell cycle analysis was performed by the method de-
scribed previously.44;45 Cells were washed in PBS and
stained with 20 lg/mL propidium iodide in 0.1% Triton
X-100, and 0.1 mM EDTA. Cell suspensions were ana-
lyzed with a FACScalibur flow cytometer (Becton-
Dickinson, San Jose, CA). The percentage of cell cycle
distribution in the G0/G1, S, and G2/M phases were
determined using the MODFIT software (Becton
Dickinson).
nm; IR (KBr) mmax: 2947, 1648, 1601, 1546, 1387 cmꢂ1
;
1H NMR (DMSO-d6): d 7.65 (2H, m, H-2, H-6), 7.41
(3H, m, H-3, H-4, H-5), 7.21 (1H, d, J ¼ 16 Hz, H-b),
6.96 (1H, d, J ¼ 16 Hz, H-a), 6.31 (2H, s, H-30, H-50);
13C NMR (DMSO-d6): d 193.2 (˜C@O), 162.0 (C-40),
158.0 (C-20, C-60), 143.5 (C-b), 134.4 (C-1), 130.4 (C-a),
129.2 (C-4), 129.0 (C-2, C-6), 128.4 (C-3, C-5), 91.0 (C-
30, C-50), 55.8 (2 · –OMe), 55.4 (OMe); ESI-MS (positive
mode) m=z 299.0 [M+H]þ.
5.5. Statistical analysis
5.6.2. 20-Hydroxy-2,3,40,60-tetramethoxychalcone (2).
This was synthesized using 2-hydroxy-4,6-dimethoxy-
acetophenone (1.96 g, 10 mmol) and 2,3-dimethoxy-
benzaldehyde (1.66 g, 10 mmol) as starting materials:
yellow needles (3.19 g, 88%), mp 120–122 ꢁC; UV kmax
All experimental data were shown as mean SD and,
statistical analysis was performed using a one-way
analysis of variance (ANOVA) followed by Dunnetts
post-hoc test, and the significant difference was set at
*p < 0.05; **p < 0.01.
(MeOH) (log e): 343 (4.69), 215 (4.23) nm; IR (KBr)
1
m
max: 2937, 1623, 1571, 1449 cmꢂ1; H NMR (DMSO-
d6): d 13.40 (1H, br s, OH-20), 7.86 (1H, d, J ¼ 16 Hz, H-
b), 7.78 (1H, d, J ¼ 16 Hz, H-a), 7.33 (1H, t, J ¼ 9 Hz,
H-5), 7.15 (2H, d, J ¼ 9 Hz, H-4, H-6), 6.16 (1H, d,
J ¼ 2 Hz, H-50), 6.13 (1H, d, J ¼ 2 Hz, H-30), 3.89 (3H,
s, OMe), 3.88 (3H, s, OMe), 3.82 (3H, s, OMe), 3.78
(3H, s, OMe); 13C NMR (DMSO-d6): d 192.4 (˜C@O),
165.6 (C-20), 165.5 (C-40), 162.0 (C-60), 152.9 (C-2), 148.1
(C-3), 136.8 (C-b), 128.5 (C-4), 128.3 (C-6), 124.5 (C-a),
119.3 (C-10), 114.9 (C-1), 96.6 (C-5), 94.0 (C-30), 91.1
5.6. Chemistry
Chemicals and solvents were reagent grade and used
without further purification. The reactions were moni-
tored by thin layer chromatography (TLC) on precoated
Merck Silica gel 60F254 aluminum plates. Melting points
were determined on a Kofler hot stage apparatus.

2684
Y. K. Rao et al. / Bioorg. Med. Chem. 12 (2004) 2679–2686
(C-50), 60.8, 56.2, 55.9, 55.7 (4 · –OMe); ESI-MS
(positive mode) m=z 345.0 [M+H]þ.
5.6.6. 20,4-Dihydroxy-30,40-dimethoxychalcone (helianone
A) (6). This was synthesized using 2-hydroxy-3,4,-di-
methoxyacetophenone (1.96 g, 10 mmol) and p-hy-
droxybenzaldehyde (1.22 g, 10 mmol) as starting
materials: yellow crystalline solid (1.65 g, 52%), mp 144–
146 ꢁC; UV kmax (MeOH) (log e): 366 (4.10), 308 (3.30)
5.6.3. 20-Hydroxy-2,3,4,40,60-pentamethoxychalcone (3).
This was synthesized using 2-hydroxy-4,6-dimethoxy-
acetophenone (1.96 g, 10 mmol) and 2,3,4-trimethoxy-
benzaldehyde (1.96 g, 10 mmol) as starting materials:
pale yellow solid (3.33 g, 85%), mp 148–150 ꢁC; UV kmax
(MeOH) (log e): 365 (4.23), 258 (3.16) nm; IR (KBr)
1
nm; IR (KBr) mmax: 3382, 1630, 1590 cmꢂ1, H NMR
(CDCl3): d 13.26 (1H, s, OH-20), 7.78 (1H, d, J ¼ 16 Hz,
H-b), 7.59 (1H, d, J ¼ 9 Hz, H-60), 7.48 (2H, d,
J ¼ 8:6 Hz, H-2, H-6), 7.36 (1H, d, J ¼ 16 Hz, H-a),
6.78 (2H, d, J ¼ 8:6 Hz, H-3, H-5), 6.50 (1H, d,
J ¼ 9 Hz, H-50), 5.54 (1H, s, OH-4), 3.91 (3H, s, OMe),
3.89 (3H, s, OMe); 13C NMR (CDCl3): d 192.4 (˜C@O),
158.3 (C-40), 158.1 (C-20), 157.9 (C-4), 144.6 (C-b), 136.2
(C-30), 130.5 (C-2, C-6), 127.0 (C-1), 125.8 (C-10), 117.4
(C-a), 116.0 (C-3, C-5), 115.2 (C-50), 102.5 (C-60), 60.2
(OMe), 56.0 (OMe); ESI-MS (positive mode) m=z 301.1
[M+H]þ.
m
max: 3260, 1632, 1580, 1555, 1406 cmꢂ1 1H NMR
;
(DMSO-d6): d 13.57 (1H, br s, OH-20), 7.80 (1H, d,
J ¼ 16 Hz, H-b), 7.74 (1H, d, J ¼ 16 Hz, H-a), 7.50 (1H,
d, J ¼ 9 Hz, H-6), 6.91 (1H, d, J ¼ 9 Hz, H-5), 6.15 (1H,
d, J ¼ 2:5 Hz, H-50), 6.12 (1H, d, J ¼ 2:5 Hz, H-30), 3.89
(3H, s, OMe), 3.86 (3H, s, OMe), 3.84 (3H, s, OMe),
3.82 (3H, s, OMe), 3.77 (3H, s, OMe); 13C NMR
(DMSO-d6): d 192.4 (˜C@O), 165.6 (C-20), 165.4 (C-40),
161.8 (C-60), 155.6 (C-4), 153.0 (C-2), 137.6 (C-3), 126.0
(C-b), 123.8 (C-6), 121.2 (C-a), 108.6 (C-10), 106.3 (C-1),
99.0 (C-5), 94.0 (C-30), 91.0 (C-50), 61.4 (2-OMe), 60.5
(3-OMe), 56.2 (4-OMe), 56.0 (60-OMe), 55.7 (40-OMe);
ESI-MS (positive mode) m=z 375.0 [M+H]þ.
5.6.7. 20,3,40-Trihydroxy-4-methoxychalcone (7). This
was synthesized using resacetophenone (1.52 g,
10 mmol) and isovanillin (1.52 g, 10 mmol) as starting
materials: yellow solid (1.40 g, 46%), mp 196–198 ꢁC;
UV kmax (MeOH) (log e): 370 (4.68), 312 (3.69), 270
(3.74) nm; IR (KBr) mmax: 3420, 3236, 2972, 1665,
5.6.4. 20-Hydroxy-2,4,40,6,60-pentamethoxychalcone (4).
This was synthesized using 2-hydroxy-4,6-dimethoxy-
acetophenone (1.96 g, 10 mmol) and 2,4,6-trimethoxy-
benzaldehyde (1.96 g, 10 mmol) as starting materials:
orange-red solid (3.37 g, 86%), mp 151–153 ꢁC; UV kmax
(MeOH) (log e): 385 (4.26), 334 (3.28), 256 (3.22) nm; IR
(KBr) mmax: 3360, 2900, 1648, 1610, 1503 cmꢂ1; 1H NMR
(DMSO-d6): d 14.25 (1H, br s, OH-20), 8.16 (1H, d,
J ¼ 15:5 Hz, H-b), 8.11 (1H, d, J ¼ 16 Hz, H-a), 6.31
(2H, s, H-3, H-5), 6.13 (1H, d, J ¼ 2 Hz, H-50), 6.09 (1H,
d, J ¼ 2 Hz, H-30), 3.91 (6H, s, 2 · –OMe), 3.89 (3H, s,
OMe), 3.85 (3H, s, OMe), 3.83 (3H, s, OMe); 13C NMR
(DMSO-d6): d 193.2 (˜C@O), 166.5 (C-20), 163.4 (C-40),
163.3 (C-60), 162.0 (C-4), 161.1 (C-2, C-6), 134.4 (C-b),
125.7 (C-a), 106.1 (C-10), 105.4 (C-1), 94.0 (C-30), 91.1
(C-3, C-5), 91.0 (C-50), 56.0 (2 · –OMe), 55.9 (OMe),
55.6 (2 · –OMe); ESI-MS (positive mode) m=z 375.0
[M+H]þ.
1
1594 cmꢂ1; H NMR (CDCl3): d 13.45 (1H, s, OH-20),
7.85 (1H, d, J ¼ 9 Hz, H-60), 7.68 (1H, d, J ¼ 16 Hz, H-
b), 7.50 (1H, d, J ¼ 16 Hz, H-a), 7.15 (1H, d, J ¼ 6 Hz,
H-2), 6.89 (1H, dd, J ¼ 2, 8 Hz, H-6), 6.48 (1H, d,
J ¼ 8 Hz, H-5), 6.31 (1H, dd, J ¼ 2, 8 Hz, H-50), 6.16
(1H, d, J ¼ 2 Hz, H-30), 4.01 (3H, s, OMe); 13C NMR
(CDCl3): d 192.9 (˜C@O), 166.4 (C-20), 162.6 (C-40),
152.0 (C-4), 150.6 (C-3), 143.8 (C-b), 132.7 (C-60), 129.2
(C-1), 125.3 (C-6), 116.9 (C-a), 116.0 (C-5), 113.9 (C-10),
109.5 (C-2), 108.1 (C-50), 101.8 (C-30), 58.1 (OMe); ESI-
MS (positive mode) m=z 287.1 [M+H]þ.
5.6.8. 20-Hydroxy-2,40,5-trimethoxychalcone (8). This
was synthesized using 2-hydroxy-4-methoxyacetophe-
none (1.66 g, 10 mmol) and 2,5-dimethoxybenzaldehyde
(1.66 g, 10 mmol) as starting materials: orange red
crystals (2.56 g, 77%), mp 108–111 ꢁC; UV kmax (MeOH)
(log e): 372 (3.68), 309 (3.74), 264 (2.97) nm; IR (KBr)
m
max: 3218, 2988, 1624, 1592, 1503 cmꢂ1 1H NMR
;
5.6.5. 2,20-Dihydroxychalcone (5). This was synthesized
using 2-hydroxyacetophenone (1.36 g, 10 mmol) and
salicylaldehyde (1.22 g, 10 mmol) as starting materials:
yellow amorphous powder (1.16 g, 45%), mp 156–
158 ꢁC; UV kmax (MeOH) (log e): 368 (3.98), 310 (2.86),
255 (2.39) nm; IR (KBr) mmax: 3406, 3240, 1640 cmꢂ1; 1H
NMR (DMSO-d6): d 12.62 (1H, br s, OH-20), 8.17 (1H,
d, J ¼ 8 Hz, H-6), 8.16 (1H, d, J ¼ 15:5 Hz, H-b), 7.97
(1H, d, J ¼ 16 Hz, H-a), 7.90 (1H, d, J ¼ 8 Hz, H-60),
7.55 (1H, dt, J ¼ 1, 8.5 Hz, H-4), 7.30 (1H, dt, J ¼ 1,
8 Hz, H-5), 6.95–7.10 (2H, m, H-30, H-50), 6.89 (1H, dt,
J ¼ 1, 8.5 Hz, H-40); 13C NMR (DMSO-d6): d 193.8
(˜C@O), 161.9 (C-20), 157.5 (C-2), 140.3 (C-b), 136.1 (C-
4), 132.5 (C-6), 130.5 (C-a), 128.9 (C-1), 121.1 (C-5),
120.7 (C-3), 120.2 (C-10), 119.4 (C-40), 119.1 (C-50), 117.7
(C-30), 115.4 (C-60); ESI-MS (positive mode) m=z 241.1
[M+H]þ.
(CDCl3): d 13.50 (1H, s, OH-20), 8.12 (1H, d,
J ¼ 15:5 Hz, H-b), 7.80 (1H, d, J ¼ 9 Hz, H-60), 7.64
(1H, d, J ¼ 15:5 Hz, H-a), 7.14 (1H, s, H-6), 6.93 (1H, d,
J ¼ 7:5 Hz, H-4), 6.86 (1H, d, J ¼ 9 Hz, H-50), 6.46 (2H,
m, H-3, H-30), 3.87 (3H, s, OMe), 3.84 (3H, s, OMe),
3.80 (3H, s, OMe); 13C NMR (CDCl3): d 192.6 (˜C@O),
166.9 (C-40), 166.3 (C-20), 153.8 (C-2), 140.1 (C-5), 131.5
(C-b), 124.6 (C-6), 121.5 (C-a), 117.5 (C-10), 114.5 (C-4),
114.2 (C-50), 112.7 (C-1), 107.9 (C-3), 101 (C-60), 97.2
(C-30), 56.4 (OMe), 56.0 (OMe), 55.7 (OMe); ESI-MS
(positive mode) m=z 315.1 [M+H]þ.
5.6.9. 20-Hydroxy-30,40-dimethoxy-3,4-methylenedioxy-
chalcone (9). This was synthesized using 2-hydroxy-3,4-
dimethoxyacetophenone (1.96 g, 10 mmol) and pipero-
nal (1.50 g, 10 mmol) as starting materials: pale yellow

Y. K. Rao et al. / Bioorg. Med. Chem. 12 (2004) 2679–2686
2685
Franco, O. R.; Salvatore, M.; Giovanni, S. Cancer
Cheomother. Pharmacol. 2000, 46, 305.
crystals (2.84 g, 82%), mp 174–176 ꢁC; UV kmax (MeOH)
(log e): 371 (4.57), 318 (sh) (3.14), 258 (3.10) nm; IR
(KBr) mmax: 3362, 2875, 1627, 1554, 1445 cmꢂ1; 1H NMR
(CDCl3): d 13.25 (1H, s, OH-20), 7.79 (1H, d, J ¼ 15 Hz,
H-b), 7.64 (1H, d, J ¼ 8:5 Hz, H-60), 7.38 (1H, d,
J ¼ 15 Hz, H-a), 7.15 (1H, s, H-2), 7.11 (1H, d,
J ¼ 8:5 Hz, H-6), 6.83 (1H, d, J ¼ 8:5 Hz, H-5), 6.50
(1H, d, J ¼ 8 Hz, H-50), 6.01 (2H, s, –OCH2O–), 3.93
(3H, s, OMe), 3.88 (3H, s, OMe); 13C NMR (CDCl3): d
192.5 (˜C@O), 158.9 (C-20), 158.5 (C-40), 150.4 (C-4),
148.7 (C-3), 144.8 (C-30), 137.0 (C-b), 129.4 (C-1), 126.1
(C-6), 125.8 (C-a), 118.4 (C-5), 115.9 (C-10), 109.0 (C-1),
106.9 (C-2), 103.0 (C-60), 101.9 (–OCH2O–), 60.9 (OMe),
56.4 (OMe); ESI-MS (positive mode) m=z 329.1
[M+H]þ.
6. Kumar, S. K.; Erin, H.; Catherine, P.; Halluru, G.;
Davidson, N. E.; Khan, S. R. J. Med. Chem. 2003, 46, 2813.
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5.6.10. 20-Hydroxy-2,3,30,4,40-pentamethoxychalcone (10).
This was synthesized using 2-hydroxy-3,4-dimethoxy-
acetophenone (1.96 g, 10 mmol) and 2,3,4-trimethoxy-
benzaldehyde (1.96 g, 10 mmol) as starting materials:
yellow solid (3.38 g, 86%), mp 98–100 ꢁC; UV kmax
(MeOH) (log e): 365 (4.12), 308 (sh) (3.35), 236 (3.32)
nm; IR (KBr) mmax: 3214, 2896, 1634, 1595, 1487 cmꢂ1
;
1H NMR (CDCl3): d 13.34 (1H, s, OH-20), 8.03 (1H, d,
J ¼ 16 Hz, H-b), 7.66 (1H, d, J ¼ 9 Hz, H-60), 7.60 (1H,
d, J ¼ 15:5 Hz, H-a), 7.35 (1H, d, J ¼ 8:5 Hz, H-6), 6.70
(1H, d, J ¼ 8:5 Hz, H-5), 6.50 (1H, d, J ¼ 9 Hz, H-50),
3.94 (3H, s, OMe), 3.93 (3H, s, OMe), 3.92 (3H, s,
OMe), 3.89 (3H, s, OMe), 3.87 (3H, s, OMe); 13C NMR
(CDCl3): d 193.1 (˜C@O), 158.6 (C-40), 158.5 (C-20),
156.3 (C-4), 154.2 (C-2), 142.7 (C-3), 140.4 (C-30), 137.0
(C-b), 126.1 (C-a), 124.6 (C-6), 122.0 (C-10), 119.6 (C-1),
116.0 (C-50), 107.8 (C-5), 103.0 (C-60), 61.6, 61.1, 60.9
(3 · –OMe), 56.3 (2 · –OMe); ESI-MS (positive mode)
m=z 375.1 [M+H]þ.
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Acknowledgements
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This research was supported by the National Science
Council of Taiwan (NSC 92-2811-M-324-001). In addi-
tion, a part of this investigation was supported by a
research grant from China Medical University (CMU-
92-M-24).
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