Synthesis and biological evaluation of 3′,4′,5′-trimethoxychalcone analogues as inhibitors of nitric oxide production and tumor cell proliferation

Article abstract of DOI:10.1016/j.bmc.2009.10.022

A series of 23 3′,4′,5′-trimethoxychalcone analogues was synthesized and their inhibitory effects on nitric oxide (NO) production in LPS/IFN-γ-treated macrophages, and tumor cell proliferation has been investigated. 4-Hydroxy-3,3′,4′,5′-tetramethoxychalco

Full text of DOI:10.1016/j.bmc.2009.10.022

Bioorganic & Medicinal Chemistry 17 (2009) 7909–7914
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of 30,40,50-trimethoxychalcone analogues
as inhibitors of nitric oxide production and tumor cell proliferation
Yerra Koteswara Rao a, Shih-Hua Fang b, Yew-Min Tzeng a,
*
a Institute of Biochemical Sciences and Technology, Chaoyang University of Technology, Wufeng, Taiwan, ROC
b Institute of Athletes, National Taiwan Sport University, Taichung, Taiwan, ROC
a r t i c l e i n f o
a b s t r a c t
A series of 23 30,40,50-trimethoxychalcone analogues was synthesized and their inhibitory effects on nitric
Article history:
Received 15 September 2009
Revised 9 October 2009
Accepted 13 October 2009
Available online 28 October 2009
oxide (NO) production in LPS/IFN-c-treated macrophages, and tumor cell proliferation has been investi-
gated. 4-Hydroxy-3,30,40,50-tetramethoxychalcone (7), 3,4-dihydroxy-30,40,50-trimethoxychalcone (11),
3-hydroxy-30,4,40,50-tetramethoxychalcone (14), and 3,30,40,50-tetramethoxychalcone (15) were the most
potent growth inhibitory agents on NO production, with an IC50 value of 0.3, 1.5, 1.3 and 0.3 lM, respec-
tively. The tumor cells proliferation assay results revealed that several compounds exhibited potent
inhibition activity against different cancer cell lines. The chalcone 15 was the most potent anti-prolifer-
Keywords:
30,40,50-Trimethoxychalcone analogues
Synthesis
ative compound in the series with IC50 values of 1.8 and 2.2 lM toward liver cancer Hep G2 and colon
cancer Colon 205 cell lines, respectively. 2,3,30,40,50-Pentamethoxychalcone (1), 3,30,4,40,5,50-hexameth-
oxychalcone (3), 2,30,4,40,5,50-hexamethoxychalcone (5), 2-hydroxy-3,30,40,50-tetramethoxychalcone
(10), 11 and 14 showed significant anti-proliferation actions in Hep G2 and Colon 205 cells with an
Nitric oxide
Tumor cell proliferation
IC50 values ranging between 10 and 20
NO production inhibition (IC50 = 0.3 M), while has no effect on tumor cell proliferation (IC50
>100
M). 3,30,4,40,50-Pentamethoxychalcone (2) showed selective anti-proliferation effect in Hep G2
lM. Among the tested agents, compound 7 showed selective
l
l
cells, in addition to its potent NO inhibition, however has no such response in Colon 205 cells. In contrast,
3-formyl-30,40,50-trimethoxychalcone (22) showed moderate growth inhibition in Colon 205 cells, while
has no such effect on NO production and Hep G2 cells proliferation. These results provide insight into the
correlation between some structural properties of 30,40,50-trimethoxychalcones and their in vitro anti-
inflammatory and anti-cancer differentiation activity.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
In previous communications the authors of this study reported
the preparation of 20-hydroxychalcone derivatives and correspond-
Chalcones, constitute an important group of natural products
and serve as precursors for the synthesis of different classes of
flavonoids, which are common substances in plants.1 Chalcones
are open-chain flavonoids in which two aromatic rings are joined
ing flavones, and evaluation of their biological activities.10,11
Research into the anti-tumor properties of chalcones has received
significant attention over the last few years, particularly with the
discovery that these compounds possess a similar mode of action
to the structurally related combretastatins. Combretastatin A-4
(CA-4, Fig. 1), a natural cis-stilbene product has received major
attention owing to its strong inhibition of tubulin polymerization
and selective targeting of tumor vascular systems (cancer vascular
disrupting), which cuts off the tumor blood flow, leading to hemor-
rhagic necrosis as well as cancer antiangiogenesis.12 A reported
critical structural requirement for the activity of these compounds
is the cis-configuration of the double bond and the 3,4,5-trime-
thoxyphenyl ring (ring A).13 Taking into account of these reports,
and an attempt to discover a potent compound that suppresses
both the inflammation and cancer was an attractive idea.14 It is
reasonable that the dual inhibitors of both inflammatory mediators
such as NO and tumor cell proliferation were more effective than
single inhibitors as blocking multiple signaling pathways in cancer
by a three carbon
a,b-unsaturated carbonyl system (1,3-diphe-
nyl-2-propen-1-ones).1 Chalcone derivatives have received a great
deal of attention due to their relatively simple structures, and wide
variety of pharmacological activities reported for these compounds
include anti-inflammatory,2 anti-bacterial,1 anti-fungal,3–5 and
anti-tumor activities.6–9 These activities are largely attributed
due to the a,b-unsaturated ketone moiety. Introduction of various
substituents into the two aryl rings is also a subject of interest be-
cause it leads to useful structure–activity relationship (SAR) con-
clusions and thus helps to synthesize pharmacologically active
chalcones.6
* Corresponding author. Tel.: +886 4 23323000x4471; fax: +886 4 23395870.
E-mail address: ymtzeng@cyut.edu.tw (Y.-M. Tzeng).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.10.022

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Y. K. Rao et al. / Bioorg. Med. Chem. 17 (2009) 7909–7914
Table 1
OMe
30,40,50-Trimethoxychalcone analogues 1–23 prepared via Aldol condensation reaction
MeO
O
OMe
MeO
O
R1
2
R2
R3
H3CO
H3CO
MeO
1
B
A
O
OMe
OH
5
MeO
MeO
R1
R4
OCH3
OMe
Combretastatin A-4 (CA-4)
R
Compound R1
R2
R3
R4
Formula
Yielda (%) Ref.
OMe
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
OMe OMe
H
H
H
C20H22O6
C20H22O6
88
90
86
78
87
75
83
88
58
72
57
70
89
85
92
77
N
H
H
OMe OMe
OMe OMe
15
15
24
N
24. R = OH, R1 = H
25. R = OH, R1 = OCH3
26. R = H, R1 = OH
27. R = H, R1 = OCH3
OMe C21H24O7
C20H23NO4
OMe C21H24O7
H
OMe
H
H
H
H
N(CH3)2
OMe
OH
H
H
H
H
OH
H
H
H
H
H
H
H
H
H
H
NO2
H
C
18H18O5
N
H
OMe OH
C19H20O6
C20H22O6
C18H18O6
C19H20O6
C18H18O6
C18H18O5
C19H20O5
C19H20O6
C19H20O5
C18H17FO4
C18H17BrO4 75
15
25
26
N
Figure 1. The Michael adducts (24–27) obtained from the Claisene Schmidt
condensation between 3,4,5-trimethoxyacetophenone and corresponding benzal-
dehyde under reflux.
OMe
OH
OH
H
H
H
H
H
OMe
OH
OH
H
OMe
H
H
OH
H
OMe
OMe
H
F
Br
NO2
Me
H
H
H
N
15
24
15
25
24
24
15
25
25
24
24
24
therapy and could be beneficial to overcome drug resistance. With
the goal of identifying such a new anti-inflammatory and anti-can-
cer strategy, therefore, we intended to synthesize 30,40,50-trimeth-
oxychalcone analogues with different substituent on B-ring. We
now present the synthesis of 30,40,50-trimethoxychalcones ana-
logues using a classical base catalyzed condensation reaction and
their inhibitory effects on nitric oxide production and tumor cell
proliferation.
H
H
H
OH
OMe
H
H
H
H
H
C18H17NO6
C19H20O4
C18H17NO7
C19H18O5
68
82
66
70
72
75
H
H
OH
CHO
H
H
H
CHO
H
H
C19H18O5
H
H
CHO C19H18O5
2. Results and discussion
2.1. Chemistry
a
Isolated yield including recovered starting material. N, new compound.
The preparation of the 30,40,50-trimethoxychalcone analogues
(Table 1) was carried out via Claisen–Schmidt condensation
(Scheme 1).10 Thus, an appropriate commercially available aryl
aldehydes were reacted with inexpensive 3,4,5-trimethoxyace-
tophenone in ethanol/KOH at 0–5 °C. Upon completion, the cooled
reaction mixture was poured into ice water and treated with HCl
yielded the desired 30,40,50-trimethoxychalcone analogues (1–23)
with an average yield of 57–92% (Table 1).10 When needed, the
crude product was purified by silica gel column chromatography.
The structure of compounds 1–23 were established with NMR
and mass spectrometry measurements. Coupling constants (J) from
the proton nuclear magnetic resonance (1H NMR) spectra clearly
indicated that compounds 1–23 were geometrically pure and were
exclusively trans (E) isomers (JtransC@C = 15–16 Hz). This aldol con-
densation via a one-step reaction produced five new substituted
30,40,50-trimethoxychalcones (1, 5, 6, 10 and 11) and eighteen
known derivatives (2–4, 7–9 and 12–23) as listed in Table 1. Our
attempt to shorten the reaction time by refluxing a reaction mix-
ture of 3,4,5-trimethoxyacetophenone and p-hydroxybenzalde-
hyde for 20 min resulted in only one product different from the
expected chalcone. Its 1H NMR spectrum showed no characteristic
2.2. Biological evaluation
2.2.1. Inhibition of nitric oxide (NO) production
Macrophages are the main pro-inflammatory cells responsible
for invading pathogens by releasing many pro-inflammatory mol-
ecules, including the free radical nitric oxide (NO) which is pro-
duced endogenously by the nitric oxide synthase (NOS).16
Activated macrophages transcriptionally express inducible nitric
oxide synthase (iNOS), which is responsible for the prolonged
and profound production of NO. The aberrant release of NO can
lead to amplification of inflammation as well as tissue injury.16
Therefore, inhibition of neutrophils and/or macrophages activation
and the following release of inflammatory mediator NO provide a
promising strategy for the development of potential anti-inflam-
matory agents.
A number of synthetic and naturally occurring chalcone deriva-
tives exhibit potent anti-inflammatory activities,2,17 although the
mechanism of action is not yet fully understood. Anti-inflammatory
activity of chalcones are associated with suppression of inflamma-
tory mediators including NO.17 In this study, all synthesized
30,40,50-trimethoxychalcone analogues were screened for their activ-
ities on NO production in the mouse macrophages, stimulated by
signals of the
a,b-olefinic protons and eight aromatic protons in-
stead of the six expected ones. In addition, two more signals were
observed—two doublets of doublets for four protons at d 3.47 and
3.18, and a pentet for one proton at d 3.90. This information
showed that at heating the reaction runs fast to receive the
chalcone, which was further attacked by acetophenone to give
the Michael adduct 24 (Fig. 1).15 The compounds 25–27 were also
prepared in this same manner from the corresponding substituted
benzaldehydes, and their structures (Fig. 1) were additionally con-
firmed by the 13C NMR and mass spectral analyses. To our knowl-
edge these compounds has not been described in the literature up
till now. Their spectral data are presented herein.
lipopolysaccharide (LPS, 2 lg/mL) plus interferon (IFN)-c (10 U/
mL). As shown in Table 2, among 23 synthesized chalcones, four
compounds including 4-hydroxy-3,30,40,50-tetramethoxychalcone
(7), 3,4-dihydroxy-30,40,50-trimethoxychalcone (11), 3-hydroxy-
30,4,40,50-tetramethoxychalcone (14), and 3,30,40,50-tetramethoxy-
chalcone (15) showed most potent inhibitory effect on NO produc-
tion with an IC50 values of 0.3, 1.5, 1.3 and 0.3
(Table 2). A similar trend was observed for chalcones 1, 3, and 10
with an IC50 value of 2.4, 2.8 and 3.0 M, respectively (Table 2). Fur-
thermore, the chalcones 2, 5, 6, 19 and 20 also exhibited potent NO
growth inhibition with an IC50 values ranging between 4 and 5
lM, respectively
l
l
M

Y. K. Rao et al. / Bioorg. Med. Chem. 17 (2009) 7909–7914
7911
O
R1
2
O
R1
R2
R3
H3CO
H3CO
H3CO
OHC
R2
R3
1
3'
a
b
1'
+
H3CO
5'
OCH3
5
R4
OCH3
R4
a. 50% KOH, EtOH, r.t., 24 h; b. 2N HCl
Scheme 1. General synthetic route for the synthesis of 30,40,50-trimethoxychalcone analogues.
Table 2
icity, since tested treatment did not affect cell viability under these
experimental conditions, as assessed by MTT assay (data not
shown). Although the small number of examples in this study pre-
cludes us from discerning a clear SAR, these encouraging results
have motivated the synthesis and biological evaluation of an ex-
panded series of analogs for further structural optimization of
these chalcones as anti-inflammatory agents.
Growth inhibition of nitric oxide production and tumor cell proliferation by 30,40,50-
trimethoxychalcone analogues 1–23 as calculated from dose–response curvesa
M)c
b
Compound
IC50
(
l
NO
Hep G2
Colon 205
1
2
3
4
5
6
7
8
9
2.4 0.3
4.5 0.5
2.8 0.2
27.0 4.0
4.6 1.1
5.0 0.7
0.3 0.1
>50
11.5 1.4
20.3 1.9
19.6 2.1
30.0 3.8
16.1 2.9
16.0 1.8
>100
13.2 2.0
>100
19.2 1.7
75.0 5.5
18.6 3.5
29.7 4.3
>100
2.2.2. Inhibition of tumor cell proliferation
We next evaluated the chalcones 1–23 for their in vitro anti-
proliferation screening using a 72-h continuous exposure MTT as-
say. Concentrations of compounds that inhibited tumor cell
growth by 50% relative to an untreated control (DMSO), or IC50
>100
>100
82.5 5.0
>100
>50
10
11
12
13
14
15
16
17
18
19
20
21
22
23
3.0 0.2
1.5 0.4
13.5 1.2
7.6 1.9
1.3 0.3
0.3 0.1
17.6 3.6
20.0 4.1
30.0 4.5
4.5 1.2
4.4 0.7
27.0 2.5
>50
13.5 0.8
14.9 2.1
94.0 4.6
49.5 3.6
10.6 1.3
1.8 0.3
22.0 2.5
80.0 4.9
>100
52.0 4.1
>100
>100
>100
>100
13.0 1.2
19.8 2.7
49.0 3.2
46.4 5.2
11.2 2.4
2.2 0.7
25.7 2.8
22.5 3.2
43.0 5.0
41.5 3.2
76.0 4.7
88.0 5.1
22.5 2.3
68.8 5.4
(lM) values for both human cancer cell lines HepG2 (human hep-
atoma cancer cell line), and Colon 205 (human colonic cancer cell
line) are shown in Table 2. The IC50 values were determined by
interpolation from dose–response curves. A total of 100 lM was
chosen as maximal concentration because much higher doses
do not normally reach the blood plasma,19 and we are looking
for compounds with high activity (low IC50) which could eventu-
ally be used in the future as a drug for chemotherapy with very
low to no side effects.
Anti-proliferation results for Hep G2 cells treated with each
compound indicate that chalcone 15 (3,30,40,50-tetramethoxychal-
>50
cone) showed the highest activity with IC50 value of 1.8 lM. The
a
Sigmoidal dose–response curves (variable slopes) were generated using GRAPH-
PAD PRISM V. 4.02 (GRAPHPAD Software Inc.).
Tested compound concentration required to inhibit cell proliferation by 50%
after 48 h of treatment compared with vehicle control DMSO.
compounds displaying the potent anti-proliferation effect in Hep
G2 cells were 1 (2,3-di-OMe), 2 (3,4-di-OMe), 3 (3,4,5-tri-OMe), 5
(2,4,5-tri-OMe), 6 (4,-OH), 10 (2-OH, 3-OMe), 11 (3,4-di-OH) and
14 (3-OH, 4-OMe), with IC50 value of 11.5, 20.3, 19.6, 16.1, 16.0,
b
c
Values are the mean of triplicate of at least three independent experiments.
13.5, 14.9 and 10.6
with an additional methoxyl or hydroxyl group at C-4 showed less
potency (IC50 = 20.3 M) or inactive (IC50 >100 M), respectively
lM, respectively (Table 2). Compound 2 or 7
(Table 2). The opposite situation was found in compounds 8, 9, 22
l
l
and 23 with the IC50 values greater than 50 lM (Table 2). The new
compounds 1, 5, 6, 10 and 11 with a methoxyl and/or hydroxyl
groups at C-2 and/or C-3 and/or C-4 showed potent inhibition activ-
ity against NO production with an IC50 values ranging 1.5–5.0 lM.
than that of 15 with C-3 methoxyl group on ring-B (Table 2).
Replacement the 3-methoxy group of ring-B with hydroxyl group
(6), also displayed a decrease in the anti-proliferation activity by
ten orders of magnitude against Hep G2 cells. Chalcones 7–9, 18
and 20–23 were found to exhibit negligible Hep G2 cell growth
With respect to different substituted groups on B-ring, oxygen-
ated groups on B ring in which one group substituted at 3-position
and the other substituted at 2- or 4-position played an important
role in the different IC50 trends against the NO production. As
shown in Table 2, a strong electron withdrawal group of F, Br,
NO2 and CHO in 16, 17, 18 and 23, respectively at C-4 position sig-
nificantly decreased NO production inhibition. However, the pres-
ence of electron donating group of OH, OCH3 and CH3 in 6, 13 and
19, respectively at the same position exhibited a stronger activity
than that of electron withdrawal group containing compounds
(16–18 and 23). This result indicated that the hydroxyl group at
the para-position of the benzene ring is enhanced to delocalize
the electron density based on a resonance effect and, generally
showed increased suppression of NO production.18 Interestingly,
compound 15 with a methoxyl group at C-3 showed the best activ-
inhibitory activity with an IC50 value greater than 100 lM. This re-
sult implies that the methoxyl group at the C-3 position of ring-B is
important for the anti-proliferation activity profile in the tested
compounds. Shifting the methoxyl group at R2 from the meta-posi-
tion (15) to para-position (13) on the ring-B decreased the anti-
proliferation activity. Introduction of a weak electron-withdrawing
group, a para-fluorine atom (16), and increasing the size of the hal-
ogen from fluorine to bromine (17) decreased the anti-proliferative
activity. The replacement of the para-fluoro group with a nitro
moiety, to give 18, eliminated anti-proliferative activity (IC50
>100 l
M).13 Turning to the effects of an electron releasing group
on the para-position, we found that replacement of p-methoxyl
(13) with a p-methyl (19) group caused only a minor decrease in
anti-proliferative activity, while the trimethoxy derivatives 3 and
5 were more active than 13. Future investigation of chalcone 15
and the other structural modifications noted herein as Hep G2 cell
growth inhibitors may provide further SAR insights.
ity with an IC50 value of 0.3
than that of 12 with a hydroxyl group at C-3 (IC50 = 13.5
The inhibitory effect of tested compounds was not due to cytotox-
lM and was 45-times more potent
lM).

7912
Y. K. Rao et al. / Bioorg. Med. Chem. 17 (2009) 7909–7914
In the Colon 205 cells, the results followed a similar trend; com-
pound 15 was a good substrate for these cells inhibition, being pro-
vided with the lowest IC50 value (2.2 M) among the prepared
compounds. However, replacement of methoxyl group of 15 to
hydroxyl group of 12 and aldehyde group of 22 resulted in the de-
crease of the anti-proliferative effect (Table 2). This might be due to
disfavored interactions between the hydrogen-bond acceptor of 12
and 22, and their molecular targets that counteract the overall
activity in Colon 205 cells.20 Compounds 1, 10 and 14 showed
moderate anti-proliferation activity in Colon 205 cells with an
of anti-inflammation and anti-cancer. Compound 2 selectively
inhibited NO production and Hep G2 proliferation; while 22
showed selective anti-proliferation affect in colon cancer Colon
205 cells. Taken together, these results indicates that 30,40,50-tri-
methoxychalcone analogues may consider as potential anti-
inflammatory and anti-cancer agents. Further studies will be re-
quired to better understand signaling mechanisms of the subject
compounds, and needs to be generalized to a larger series bearing
other substitution patterns on the ring-B. On the other hand, the
most active compounds should be analyzed for effects on normal
human cells, in order to determine potential therapeutic windows
supporting further studies on experimental tumor-bearing animals
finalized to verify possible in vivo anti-cancer activity.
l
IC50 value of 13.2, 13.0 and 11.2 lM, respectively (Table 2). Partic-
ularly, the chalcone 14 which has substituent similar to that of CA-
4 showed moderate anti-proliferation activity in both Hep G2 and
Colon 205 cells, but the presence of a methoxyl group (in 2) instead
of hydroxyl (in 14) at C-4 of ring-B is detrimental to activity in Co-
lon 205 cells (Table 2). When a methoxyl group is present in the
meta-position of the B-ring, increased activity is seen, in both
Hep G2 and Colon 205 cell lines, compared with the same group
in the para-position for the 30,40,50-trimethoxychalcones (e.g., 13
vs 15). However, this effect was in opposite when a hydroxyl group
is present in these positions (6 vs 12, Table 2). Interestingly, C-4-
substituted analog with either electron-withdrawing groups such
as 4-fluoro (16), 4-bromo (17), and 4-nitro (18) or an electron
donating group like 4-methoxyl (13) and 4-methyl (19) exhibited
moderate activities with IC50 values of 25.7, 22.5, 43.0, 46.4 and
4. Experimental section
4.1. General
Melting points (mp) were determined on a Buchi B-540 appara-
tus and were uncorrected. NMR spectra were measured on Bruker
Avance 500 MHz spectrometer, using TMS as an internal standard.
Abbreviations for signal coupling are as follows: s, singlet; d, dou-
blet; t, triplet; q, quartet; m, multiplet. Coupling constants J are re-
ported (Hz). Silica gel for column chromatography (CC), (0.063–
0.200 mm), was a product of Merck Company. TLC was performed
on Merck TLC plates (0.23 mm thickness), with compounds visual-
ized by spraying with 8% (v/v) H2SO4 in EtOH and then heating on a
hot plate.
41.5 lM, respectively. Furthermore, compound 7 bearing 3-meth-
oxy-4-hydroxy group and 9 bearing 2,5-dihydroxy group exhibited
negligible anti-proliferation response in both the cell lines Hep G2
and Colon 205. These results are in consistent with previous re-
ports on SAR studies on chalcone derivatives, which showed that
even a subtle modification of chalcone chemical structure may pro-
duce significant changes in their biological potency.18 Together,
these results indicated plausible correlations between structural
characteristics of the tested compounds with their anti-prolifera-
tive effects on the Colon 205 cells. Similar to data indicated in
Lipopolysaccharide (LPS, Escherichia coli 055: B5), bovine serum
albumin (BSA), phosphate-buffered solution (PBS), 3-(4,5-dimeth-
ylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) and Gri-
ess reagent were purchased from Sigma Chemical (St. Louis, MO,
USA). Recombinant interferon-
roTech (Margravine, London, England). RPMI-1640 medium,
Hank’s balanced salt solution (HBSS), penicillin, streptomycin,
c (IFN-c) was purchased from Pep-
L-
the Hep G2 cells, compound 15 (IC50 = 1.8 lM) demonstrated most
glutamine and fetal calf serum were purchased from Gibco BRL
(Grand Island, NY, USA). Colon 205 (human colon cancer cell line),
and HepG2 (human hepatoma cancer cell line) were obtained from
American Type Culture Collection (Rockville, MD), and cultured
with complete RPMI-1640 medium (Gibco BRL) supplemented
potent activity against Colon 205 cells among modified ring-B-
30,40,50-trimethoxychalcone analogs. The most prevalent form of
cancer among Taiwanese people is colorectal cancer, topping the
list of the five most common cancers in Taiwan according to a
2006 cancer report released by the Department of Health. About
15.03 per 100,000 individuals die per year of colorectal cancer,
and the current clinical therapies are limited to surgery, radiother-
apy, general chemotherapy, and gene therapy.21 So that there is a
need to identify new therapeutic agents for the treatment of colon
cancer. These preliminary results for our novel compounds may
useful to discover the lead derivatives. Taking into account both
anti-inflammatory and anti-proliferative data from this study, the
tested chalcones might decrease tumor cells proliferation through
inhibition of the inflammatory mediators such as NO. This is con-
sistent with results from other studies that tested the anti-tumor
activity of anti-inflammatory chalcones.14,22,23
with 10% fetal calf serum, antibiotics, L-glutamine.
4.2. General procedures for the preparation of the aldol
condensation products of 3,4,5-trimethoxyacetophenone with
aldehydes (1–23)
A stirred solution of 3,4,5-trimethoxyacetophenone (6.0 mmol)
in ethanol (30 mL) was added to KOH (1.2 mmol), and the mixture
was stirred at 0–5 °C temperature for 1 h. The aldehydes (6.0 mmol)
were added to the mixture, and the resulting mixture was stirred at
room temperature for 24 h. The mixture was concentrated under re-
duced pressure, and the residue was treated with water (35 mL). The
aqueousmixturewasneutralizedbytheadditionofaqueous10%HCl
solution and extracted with ethylacetate (2 ꢀ 30 mL). The organic
phase was washed with aqueous saturated NH4Cl solution (30 mL)
and brine (30 mL). The organic layer was separated and dried over
anhydrous MgSO4, filtered, and concentrated under reduced pres-
sure to give the crude product, which was purified by flash silica
chromatography to produce the pure substituted 30,40,50-trimeth-
oxychalcone analogues 1–23. For the known compounds spectral
data, see the Supplementary data and Table 1.15,24–26
3. Conclusions
The synthesis and biological evaluation of 30,40,50-trimethoxy-
chalcone analogues 1–23 has been described. The synthetic
approach involves Aldol condensation of 3,4,5-trimethoxyacetoph-
enone and aldehydes in the presence of KOH to generate 30,40,50-tri-
methoxychalcone analogues in high yields, in addition to the novel
Michael adducts. The prepared compounds 1–23 were evaluated
for suppression of LPS/IFN-c-induced NO generation, and human
4.2.1. 2,3,30,40,50-Pentamethoxychalcone (1)
tumor cell proliferation in vitro. Compound 15 exhibited potent
inhibition NO production as well as tumor cell proliferation, and
may provide a template to design new novels with a dual activity
Pale yellow solid, mp 98–99 °C; 1H NMR (500 MHz, CDCl3): d
8.02 (d, J = 15.5, 1H), 7.51 (d, J = 15.5, 1H), 7.24 (s, 2H), 7.22 (d,

Y. K. Rao et al. / Bioorg. Med. Chem. 17 (2009) 7909–7914
7913
J = 8.0, 1H), 7.06 (t, J = 8.0, 1H), 6.94 (d, J = 8.0 Hz, 1H), 3.90 (s, 6H,
2 ꢀ –OCH3), 3.88 (s, 3H, –OCH3), 3.86 (s, 3H, –OCH3), 3.84 (s, 3H, –
OCH3). 13C NMR (CDCl3, 125 MHz): d 190.0 (C@O), 153.4, 153.3,
149.1, 142.6, 139.9, 133.8, 129.3, 124.5, 123.8, 120.1, 114.4,
106.4, 61.5, 61.2, 56.6, 56.1. MS (ESI): 359.1 (M++1).
(dd, J = 8.5, 1.5 Hz, 1H), 6.60 (d, J = 8.5 Hz, 1H), 3.84 (s, 12H, 4 ꢀ –
OCH3), 3.80 (p, 1H), 3.73 (s, 6H, 2 ꢀ –OCH3), 3.70 (s, 3H, –OCH3),
3.40 (m, 4H). 13C NMR (DMSO-d6, 125 MHz): d 197.8 (C@O),
152.7, 147.2, 144.8, 141.8, 135.0, 132.2, 119.7, 115.1, 112.1,
105.6, 60.1, 56.0, 55.6, 44.4, 37.0. MS (ESI): 543.1 (M++1).
4.2.2. 2,30,4,40,5,50-Hexamethoxychalcone (5)
4.3.3. 3-(3-Hydroxyphenyl)-1,5-di-(3,4,5-trimethoxyphenyl)-
pentane-1,5-dione (26)
Orange yellow solid, mp 135–136 °C; 1H NMR (500 MHz,
CDCl3): d 8.04 (d, J = 16.0, 1H), 7.36 (d, J = 16.0, 1H), 7.24 (s, 2H),
7.10 (s, 1H), 6.51 (s, 1H), 3.93 (s, 3H, –OCH3), 3.92 (s, 6H, 2 ꢀ –
OCH3), 3.91 (s, 3H, –OCH3), 3.89 (s, 3H, –OCH3), 3.87 (s, 3H, –
OCH3). 13C NMR (CDCl3, 125 MHz): d 190.5 (C@O), 154.9, 153.3,
152.8, 143.5, 142.3, 140.5, 134.4, 120.7, 115.7, 112.1, 106.4, 61.2,
56.6, 56.5, 56.3, 56.2. MS (ESI): 389.1 (M++1).
Amorphous powder, yield 71%, mp 132–133 °C; 1H NMR (CDCl3,
500 MHz): d 7.27 (s, 4H), 7.15 (t, J = 7.5 Hz, 1H), 6.83 (d, J = 7.5 Hz,
1H), 6.79 (s, 1H), 5.39 (s, 1H), 4.05 (p, 1H), 3.92 (s, 18H, 6 ꢀ –OCH3),
3.47 (dd, J = 16.0, 7.0 Hz, 2H), 3.25 (dd, J = 16.0, 7.0 Hz, 2H). 13C
NMR (CDCl3, 125 MHz): d 197.8 (C@O), 156.2, 153.3, 145.7,
142.9, 132.2, 130.1, 119.9, 114.7, 114.1, 106.0, 61.2, 56.6, 44.9,
38.1. MS (ESI): 513.1 (M++1).
4.2.3. 4-Hydroxy-30,40,50-trimethoxychalcone (6)
Orange solid, mp 198–199 °C; 1H NMR (CD3OD, 500 MHz): d
7.77 (d, J = 15.5 Hz, 1H), 7.65 (d, J = 8.0 Hz, 2H), 7.57 (d,
J = 15.5 Hz, 1H), 7.38 (s, 2H), 6.84 (d, J = 8.0 Hz, 2H), 3.93 (s, 6H,
2 ꢀ –OCH3), 3.85 (s, 3H, –OCH3). 13C NMR (CD3OD, 125 MHz): d
191.5 (C@O), 162.2, 154.7, 147.0, 144.0, 135.3, 132.1, 127.9,
119.5, 117.1, 107.5, 61.4, 57.0. MS (ESI): 315.1 (M++1).
4.3.4. 3-(3-Methoxyphenyl)-1,5-di-(3,4,5-trimethoxyphenyl)-
pentane-1,5-dione (27)
Amorphous powder, yield 69%, mp 210–211 °C; 1H NMR (CDCl3,
500 MHz): d 7.24 (s, 4H), 7.03 (t, J = 8.0 Hz, 1H), 6.82 (d, J = 7.5 Hz,
1H), 6.77 (s, 1H), 5.19 (s, 1H), 4.06 (p, 1H), 3.87 (s, 12H, 4 ꢀ –OCH3),
3.77 (s, 6H, 2 ꢀ –OCH3), 3.73 (S, 3H, –OCH3), 3.28 (m, 4H). 13C NMR
(CDCl3, 125 MHz): d 206.1 (C@O), 159.7, 153.2, 152.6, 143.7, 141.2,
136.5, 134.4, 129.6, 121.5, 114.9, 112.8, 106.3, 61.0, 56.6, 55.3,
42.6, 38.5. MS (ESI): 527.1 (M++1).
4.2.4. 2-Hydroxy-3,30,40,50-tetramethoxychalcone (10)
Yellow solid, mp 178–179 °C; 1H NMR (500 MHz, DMSO-d6): d
8.13 (d, J = 15.5, 1H), 7.81 (d, J = 15.5, 1H), 7.54 (d, J = 8.0, 1H)
7.39 (s, 2H), 7.04 (d, J = 8.0, 1H), 6.85 (t, J = 8.0 Hz, 1H), 3.89 (s,
6H, 2 ꢀ –OCH3), 3.84 (s, 3H, –OCH3), 3.76 (s, 3H, –OCH3). 13C
NMR (CDCl3, 125 MHz): d 188.1 (C@O), 152.9, 148.0, 146.5,
141.8, 138.9, 133.3, 121.8, 120.8, 119.6, 119.0, 113.6, 106.0, 60.1,
56.2, 56.0, MS (ESI): 345.1 (M++1).
4.4. Macrophage cultures and nitric oxide determination
The inhibitory assay for inflammatory mediator NO induced by
LPS/IFN-c stimulants in murine peritoneal macrophage cells was
performed by the methods as we described previously.27,28
4.2.5. 3,4-Dihydroxy-30,40,50-trimethoxychalcone (11)
Pale yellow solid, mp 158–159 °C; 1H NMR ((CD3)2CO,
500 MHz): d 7.68 (d, J = 15.5, 1H), 7.62 (d, J = 15.5, 1H), 7.43 (s,
2H), 7.31 (d, J = 2.0, 1H), 7.17 (dd, J = 8.0, 2.0, 1H), 6.90 (d,
J = 8.0 Hz, 1H), 3.94 (s, 6H, 2 ꢀ –OCH3), 3.82 (s, 3H, –OCH3). 13C
NMR (CDCl3, 125 MHz): d 188.7 (C@O), 154.4, 149.0, 146.4,
145.3, 143.4, 134.9, 128.5, 123.6, 119.8, 116.4, 115.8, 107.0, 60.7,
56.7 MS (ESI): 331.1 (M++1).
4.5. Tumor cell growth inhibition assay
The anti-proliferation effect of compounds 1–23 against human
hepatoma cancer Hep G2 and human colonic cancer Colon 205
cells obtained from American Type Culture Collection (ATCC, Rock-
ville, MD), were performed by the method as we described previ-
ously.29–31
4.6. Statistical analysis
4.3. General procedure for the preparation of Michael adducts
(24–27)
Each experiment was performed in triplicate and repeated three
times (n = 9). The results were expressed as means SD. Statistical
comparisons were made by means of one-way analysis of variance
(ANOVA), followed by a Duncan multiple-comparison test.
30,40,50-Trimethoxyacetophenone (1 mmol) and aryl benzalde-
hyde (1 mmol) were dissolved in 10 mL ethanol and to this solu-
tion 10 mL of 50% KOH was added. The reaction mixture was
then refluxed for 20 min, and then cooled in an ice-water bath.
The solution was diluted with distilled water (100 mL) and acidi-
fied with 10 mL concd HCl. The product obtained was purified with
column chromatography over silica using n-hexane/ethylacetate
increasing polarity to yield the corresponding Michael adducts.
Acknowledgement
This study was supported by National Science Council of Taiwan
(NSC 97—2811-M-324-001 and NSC 98—2811-M-324-001).
Supplementary data
4.3.1. 3-(4-Hydroxyphenyl)-1,5-di-(3,4,5-trimethoxyphenyl)pen-
tane-1,5-dione (24)
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.10.022.
Amorphous powder, yield 76%, mp 163–164 °C; 1H NMR (CDCl3,
500 MHz): d 7.24 (s, 4H), 7.08 (d, J = 8.0 Hz, 2H), 6.67 (d, J = 8.0 Hz,
2H), 3.90 (p, 1H), 3.89 (s, 18H, 6 ꢀ –OCH3), 3.47 (dd, J = 16.0, 7.0 Hz,
2H), 3.18 (dd, J = 16.0, 7.0 Hz, 2H). 13C NMR (CDCl3, 125 MHz): d
198.2 (C@O), 154.9, 153.3, 142.9, 135.3, 132.2, 128.8, 115.8,
106.0, 61.2, 56.6, 45.4, 37.9. MS (ESI): 513.1 (M++1).
References and notes
1. Avila, H. P.; Smania, E. F.; Monache, F. D.; Smania, A., Jr. Bioorg. Med. Chem.
2008, 16, 9790.
2. Cheng, J. H.; Hung, C. F.; Yang, S. C.; Wang, J. P.; Won, S. J.; Lin, C. N. Bioorg. Med.
Chem. 2008, 16, 7270.
3. Sortino, M.; Delgado, P.; Juarez, S.; Quiroga, J.; Abonia, R.; Insuasty, B.;
Nogueras, M.; Rodero, L.; Garibotto, F. M.; Enriz, R. D.; Zacchino, S. A. Bioorg.
Med. Chem. 2007, 15, 484.
4. Vargas, M. L. Y.; Castelli, M. V.; Kouznetsov, V. V.; Urbina, G. J. M.; Lopez, S. N.;
Sortino, M.; Enriz, R. D.; Ribas, J. C.; Zacchino, S. Bioorg. Med. Chem. 2003, 11, 1531.
4.3.2. 3-(4-Hydroxy-3-methoxyphenyl)-1,5-di-(3,4,5-trime-
thoxyphenyl)pentane-1,5-dione (25)
Amorphous powder, yield 78%, mp 170–171 °C; 1H NMR
(DMSO-d6, 500 MHz): d 7.23 (s, 4H), 6.87 (d, J = 1.5 Hz, 1H), 6.68

7914
Y. K. Rao et al. / Bioorg. Med. Chem. 17 (2009) 7909–7914
5. Lopez, S. N.; Castelli, M. V.; Zacchino, S. A.; Dominguez, J. N.; Lobo, G.; Charris-
Charris, J.; Cortes, J. C.; Ribas, J. C.; Devia, C.; Rodriguez, A. M.; Enriz, R. D. Bioorg.
Med. Chem. 2001, 8, 1999.
6. Katsori, A. M.; Hadjipavlou-Litina, D. Curr. Med. Chem. 2009, 16, 1062.
7. Achanta, G.; Modzelewska, A.; Feng, L.; Khan, S. R.; Huang, P. Mol. Pharmacol.
2006, 70, 426.
18. Nowakowska, Z. Eur. J. Med. Chem. 2007, 42, 125.
19. Dupuis, C.; Mercier, C.; Yang, C.; Monjanel-Mouterde, S.; Ciccolini, J.;
Fanciullino, R.; Pourroy, B.; Deville, J. L.; Duffaud, F.; Bagarry-Liegey, D.;
Durand, A.; Iliadis, A.; Favre, R. Anticancer Drugs 2008, 19, 267.
20. Shaw, A. Y.; Chang, C. Y.; Liau, H. H.; Lu, P. J.; Chen, H. L.; Yang, C. N.; Li, H. Y.
Eur. J. Med. Chem. 2009, 44, 2552.
8. Modzelewska, A.; Pettit, C.; Achanta, G.; Davidson, N. E.; Huang, P.; Khan, S. R.
Bioorg. Med. Chem. 2006, 14, 3491.
21. Durai, R.; Yang, S. Y.; Seifalian, A. M.; Winslet, M. C. J. Gastrointest. Liver Dis.
2008, 17, 59.
9. Kumar, S. K.; Hager, E.; Pettit, C.; Gurulingappa, H.; Davidson, N. E.; Khan, S. R. J.
Med. Chem. 2003, 46, 2813.
22. Epifano, F.; Genovese, S.; Menghini, L.; Curini, M. Phytochemistry 2007, 68, 939.
23. Cabrera, M.; Simoens, M.; Falchi, G.; Lavaggi, M. L.; Piro, O. E.; Castellano, E. E.;
Vidal, A.; Azqueta, A.; Monge, A.; Lopez de Cerain, A.; Sagrera, G.; Seoane, G.;
Cerecetto, H.; Gonzalez, M. Bioorg. Med. Chem. 2007, 15, 3356.
24. Edwards, M. L.; Stemerick, D. M.; Sunkara, P. S. J. Med. Chem. 1990, 33, 1948.
25. Batovska, D.; Parushev, S.; Slavova, A.; Bankova, V.; Tsvetkova, I.; Ninova, M.;
Najdenski, H. Eur. J. Med. Chem. 2007, 42, 87.
10. Rao, Y. K.; Fang, S. H.; Tzeng, Y. M. Bioorg. Med. Chem. 2004, 12, 2679.
11. Rao, Y. K.; Fang, S. H.; Tzeng, Y. M. Bioorg. Med. Chem. 2005, 13, 6850.
12. Alloatti, D.; Giannini, G.; Cabri, W.; Lustrati, I.; Marzi, M.; Ciacci, A.; Gallo, G.;
Tinti, M. O.; Marcellini, M.; Riccioni, T.; Guglielmi, M. B.; Carminati, P.; Pisano,
C. J. Med. Chem. 2008, 51, 2708.
13. Tron, G. C.; Pagliai, F.; Del Grosso, E.; Genazzani, A. A.; Sorba, G. J. Med. Chem.
2005, 48, 3260.
26. Nam, N. H.; Hong, D. H.; You, Y. J.; Kim, Y.; Bang, S. C.; Kim, H. M.; Ahn, B. Z.
Arch. Pharm. Res. 2004, 27, 581.
14. Won, S. J.; Liu, C. T.; Tsao, L. T.; Weng, J. R.; Ko, H. H.; Wang, J. P.; Lin, C. N. Eur. J.
Med. Chem. 2005, 40, 103.
15. Batovska, D.; Parushev, S.; Stamboliyska, B.; Tsvetkova, I.; Ninova, M.;
Najdenski, H. Eur. J. Med. Chem. 2009, 44, 2211.
16. Tripathi, P.; Tripathi, P.; Kashyap, L.; Singh, V. FEMS Immunol. Med. Microbiol.
2007, 51, 443.
27. Fang, S. H.; Rao, Y. K.; Tzeng, Y. M. Bioorg. Med. Chem. 2005, 13, 2381.
28. Rao, Y. K.; Fang, S. H.; Hsieh, S. C.; Yeh, T. H.; Tzeng, Y. M. J. Ethnopharmacol.
2009, 121, 292.
29. Rao, Y. K.; Fang, S. H.; Tzeng, Y. M. J. Ethnopharmacol. 2007, 114, 78.
30. Geethangili, M.; Rao, Y. K.; Fang, S. H.; Tzeng, Y. M. Phytother. Res. 2008, 22,
1336.
17. Ko, H. H.; Tsao, L. T.; Yu, K. L.; Liu, C. T.; Wang, J. P.; Lin, C. N. Bioorg. Med. Chem.
2003, 11, 105.
31. Yeh, C. T.; Rao, Y. K.; Yao, C. J.; Yeh, C. F.; Li, C. H.; Chuang, S. E.; Luong, J. H.; Lai,
G. M.; Tzeng, Y. M. Cancer Lett. 2009, 285, 73.