Synthesis of 2-styrylchromones as a novel class of antiproliferative agents targeting carcinoma cells

Article abstract of DOI:10.1016/j.ejmech.2009.01.034

A series of 2-styrylchromone analogs were synthesized and examined for their antiproliferative effects on a panel of carcinoma cells. Among the tested agents, only 4m exhibited a moderate activity with an IC₅₀ value of 28.9 μM against PC-3 cells which indicates the selectivity of PC-3 cells in response to 2-styrylchromones. In addition, 4q demonstrated the most antiproliferative effect with an IC₅₀ value of 4.9 μM against HeLa cells. Flow cytometric analysis and DAPI staining revealed that HeLa cells exposed to 4q as low as 5 μM induced cell death through sub-G1 arrest and DNA fragmentation. Furthermore, CoMFA analysis of tested 2-styrylchromones resulted in a q² of 0.459 to generate a 3D-QSAR model on BT483 cell line. Together, these results suggest a potential structural optimization and pharmacological study of 2-styrylchromones.

Full text of DOI:10.1016/j.ejmech.2009.01.034

European Journal of Medicinal Chemistry 44 (2009) 2552–2562
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of 2-styrylchromones as a novel class of antiproliferative agents
targeting carcinoma cells
,
a
a
b
b
Arthur Y. Shaw a , Chun-Yi Chang , Hao-Han Liau , Pei-Jung Lu , Hui-Ling Chen ,
*
Chia-Ning Yang c, Hao-Yi Li c
a Department of Chemistry, Tamkang University, Tamsui 251, Taiwan
b Institute of Clinical Medicine, National Cheng Kung University, Tainan 701, Taiwan
c Institute of Biotechnology, National University of Kaohsiung, Tainan 700, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 2-styrylchromone analogs were synthesized and examined for their antiproliferative
Received 2 July 2008
Received in revised form
20 January 2009
Accepted 27 January 2009
Available online 6 February 2009
effects on a panel of carcinoma cells. Among the tested agents, only 4m exhibited a moderate activity
with an IC50 value of 28.9
response to 2-styrylchromones. In addition, 4q demonstrated the most antiproliferative effect with an
IC50 value of 4.9 M against HeLa cells. Flow cytometric analysis and DAPI staining revealed that HeLa
cells exposed to 4q as low as 5
mM against PC-3 cells which indicates the selectivity of PC-3 cells in
m
m
M induced cell death through sub-G1 arrest and DNA fragmentation.
Furthermore, CoMFA analysis of tested 2-styrylchromones resulted in a q2 of 0.459 to generate a 3D-
QSAR model on BT483 cell line. Together, these results suggest a potential structural optimization and
pharmacological study of 2-styrylchromones.
Keywords:
2-Styrylchromone
Antiproliferation
Carcinoma cells
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
against P388 lymphocytic leukemia and HL 60 human promyelotic
leukemia cell lines, while 6-desmethoxyhormothamnione exhibi-
Naturally occurring chromones (1-benzopyran-4-ones) are
widely distributed throughout the plant kingdom [1–5]. These
compounds have attracted a great deal of attention due to their
substantial activities, including antioxidative and anticancer effects
[6–11]. Accordingly, many synthesized chromone derivatives have
been extensively studied for the development of novel anticancer
agents. Molecular mechanisms of anticancer effects mediated by
chromones such as flavonoids could be attributed to anti-
proliferation, induction of apoptosis, cell cycle arrest, promotion of
differentiation, inhibition of angiogenesis, and modulation of
multidrug resistance [12]. Among these chromone derivatives,
flavopiridol (I) has been identified as a cyclin-dependent kinase
(CDK) inhibitor and it has entered in Phase II clinical trial [13,14]. 2-
Styrylchromones are a small group of chromones with only two
natural 2-styrylchromones hormothamnione (II) and 6-desme-
thoxyhormothamnione (III) are known (Fig. 1).
ted antitumor activity against 9 kb and colon 38 tumor cells,
respectively. A number of synthesized 2-styrylchromone deriva-
tives have shown to exhibit antiallergic [17], antiviral [18], anti-
tumor [19], anticancer [20,21], and antioxidant activities [22,23],
and to serve as antagonism of A3 adenosine receptor [24], and
xanthine oxidase inhibitors [25]. Among all biological activities, the
anticancer effect is of particular interest that we decided to
synthesize and evaluate the antiproliferation of a series of 2-styr-
ylchromones against a panel of carcinoma cell lines. Herein, we
report the synthesis of 2-styrylchromones based on the modifica-
tions of ring-B and ring-A as shown in Fig. 1. Mechanistic study of
antiproliferative effect mediated by the representative compound
4q is examined in this paper. Moreover, 3D-QSAR model estab-
lished by CoMFA analysis on BT483 cell line is studied as well.
Hormothamnione was isolated in 1986 from the marine cryp-
tophyte Crysophaeum taylori [15]. Its 6-desmethoxy analog was
characterized in 1989 from the extracts of the same blue-green
algae [16]. Hormothamnione demonstrated potent cytotoxicity
2. Results and discussion
2.1. Chemistry
Various strategies have been developed for the synthesis of
2-styrylchromones, all including the formation of a carbon–
carbon bond as the key step [26,27]. As shown in Scheme 1, our
initial goal was to synthesize the 2-styrylchromone scaffold with
* Corresponding author. Tel.: þ886 2 2621 5656x2842; fax: þ886 2 2620 9924.
E-mail address: shaw299@mail.tku.edu.tw (A.Y. Shaw).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.01.034

A.Y. Shaw et al. / European Journal of Medicinal Chemistry 44 (2009) 2552–2562
2553
Fig. 1. Chemical structures of chromone derivatives.
5,7-dimethoxy 2-styrylchromones with different substituents in
the ring-B moiety designated in Fig. 1.
were required to accomplish the reaction. In contrast, benzaldehydes
with electron-withdrawing groups such as 4-fluoro (4c), 4-chloro
(4d), 4-bromo(4d)4-trifluoromethyl(4i),4-trifluoromethoxy(4j)and
4-nitro (4k) are more reactive that condensation could be carried out
at ambient temperature and reduced reaction time.
Modification of ring-A indicated in Fig. 1 was focused on the
introduction of methoxy groups at different positions in the chro-
mone scaffold. The preparation of all the analogs was carried out
according to the synthetic route shown in Scheme 2.
Compounds 4m–4q were synthesized according to their
corresponding acetophenones 2m–2q. In addition, we employed
the 4-trifluoromethoxy (4-OCF3) group in ring-B as a serial modi-
fication because preliminary antiproliferation screens indicated
that 4j exhibited the most potent activity against A431 skin carci-
To carry out the synthesis of 2-styrylchromone analogs 4a–4l,
commercially available 20,40,60-trihydroxyacetophenone (1a) was
subjected to dimethylation reaction reported by Gray et al. [28] with
slight modification (Scheme 1). Treatment of 1a with two equivalents
of dimethylsulfate in the presence of potassium carbonate in acetone
solution at room temperature for 18 h afforded 20,40-dimethoxy-6-
hydroxyacetophenone (2a) in good yields. We attempted to synthe-
size 2-methyl-5,7-dimethoxychromone (3a) via a Claisen condensa-
tion of 2a with ethyl acetate but obtained low conversion rates.
Therefore, an alternative method was adopted in which 2a was dis-
solvedintheneatethylacetatesolutionwithsixequivalents ofsodium
at room temperature to give 1,3-diketone intermediate (monitored by
TLC plate) [29]. Without further purification, 1,3-diketone interme-
diate was treated with couple drops of concentrated HCl in methanol
to obtain 2-methyl-5,7-dimethoxychromone (3a) with good yield
(67% in two steps). With the crucial chromone intermediate 3a in
hand, a series of 2-styrylchromone derivatives (Table 1) were
successfully synthesized under similar conditions [30]. For example,
4a was prepared by the condensation of 3a with benzaldehyde in the
presence of sodium methoxide and methanol at reflux for 18 h. It is
noteworthy that benzaldehydes bearing electron-donating groups
such as 4-methoxy (4f), 1,2-methylenedioxy (4g) retarded the
condensation that an appreciable heat and extended reaction time
noma cancer cells with 95% growth inhibition at 25 mM treatment
(data not shown). Therefore, as chromone intermediates 3m–3q
were prepared, the final condensation to obtain 2-styrylchromone
analogs 4m–4q were successfully achieved and ready for the
antiproliferation study.
2.2. Antiproliferation study
In this study 2-styrylchromone derivatives 4a–4q were
screened in vitro against a panel of five human carcinoma cell lines,
including PC-3 (prostate carcinoma cell), A549 (non-small cell lung
Scheme 1. Reagents and conditions: (i) Me2SO4/K2CO3, acetone; (ii) Na/EtOAc; (iii) conc. HCl/MeOH; (iv) MeONa/ArCHO/MeOH.

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A.Y. Shaw et al. / European Journal of Medicinal Chemistry 44 (2009) 2552–2562
Table 1
4a and its 4-fluoro counterpart 4c exhibited comparably
moderate antiproliferative activity with IC50 values of 21.5 and
Chemical structures of 2-styrylchromone derivatives 4a to 4l
18.6 mM, respectively. However, replacement of phenyl group of
4a to 4-pyridiyl group of 4b resulted in the loss of the anti-
proliferative effect. This might be due to disfavored interactions
between the hydrogen-bond acceptor of 4b and its molecular
targets that counteract the overall activity. Compared to 4c
bearing
a 4-fluoro group in ring-B, analogs with bulkier
substituents such as 4-chloro (4d), 4-bromo (4e), 4-methoxy
(4f) and 4-trifluoromethyl (4i) did not show any anti-
proliferative effects on A549 cells. Interestingly, 4-substituted
analogs with either electron-withdrawing groups such as 4-tri-
fluoromethoxy (4j) and 4-nitro (4k) or an extended side chain
like 4-cyclopropylmethoxy (4l) exhibited moderate activities
Compound
Ar
Compound
Ar
4a
4g
with IC50 values of 24.2, 19.7 and 20.9
Furthermore, 4h bearing 3,5-dimethoxy group (IC50, 15.6
rather than 4g (IC50, >40 M) with 3,4-dioxomethylene group
m
M, respectively.
m
M)
4b
4h
m
exhibited relatively better activity against A549 cells. Together,
these results indicated plausible correlations between structural
characteristics of the tested compounds with their anti-
proliferative effects on the A549 cells. Similar to data indicated
4c
4d
4e
4f
4i
4j
4k
4l
in the PC-3 cells, only 4m (IC50
, 15.6 mM) demonstrated
a moderate activity against A549 cells among modified ring-A 2-
styrylchromone analogs.
Results of BT483 cells exposed to the tested compounds showed
that most compounds exhibited moderate potency with IC50 values
in a range of 15.5–30.9 mM. In contrast to the results shown in A549,
HeLa and SKHep cells, 4a exhibited no activity against BT483 cells.
Additionally, 4e bearing 4-bromo group of ring-B was not active
against the tested carcinoma cells. On the other hand, 3,5-dime-
thoxy group of 4h and an extended side chain of 4l exhibited
relatively higher activities against BT483 cells with IC50 values of
16.6 and 16.5
mM respectively, which were 1.7-fold more potent
than their counterpart 4f (IC50, 29.6
mM), suggesting that certain
space of ring-B is amenable to structural optimization. Among
modified ring-A analogs, most of the tested compounds exhibited
moderate activities except for 4o with 6-methoxy group, this might
be due to the repulsive interactions such as the steric hindrance
between 4o and its molecular targets. Instead, 4m bearing no
substituent showed the most potent antiproliferative activity with
an IC50 value of 15.5 mM.
adenocarcinoma cell), BT483 (mammary gland adenocarcinoma
cell), HeLa (cervical epithelioid carcinoma cell) and SKHep (hepa-
tocellular carcinoma cell). The MTT (3-[4,5-dimethylthiazol-2-yl]-
2,5-diphenyltetrazolium bromide) assay [31] was employed for
these antiproliferation studies and the IC50 values are summarized
in Table 2. The compound concentration causing a 50% cell growth
inhibition (IC50) was determined by interpolation from dose–
response curves.
Interestingly, HeLa cells seemed to be the most sensitive to the
tested compounds among five carcinoma cell lines. Most
compounds displayed better antiproliferative effects on HeLa
cells. Among them, compound 4q exhibited the most potent
antiproliferative activity with an IC50 value of 4.9 mM while 4j
with 4-trifluoromethoxy was not active among the ring-A modi-
fied analogs. Basically, no distinct antiproliferative structure–
activity relationship was observed in this series of 2-styr-
ylchromones against HeLa cells. SKHep cells were not as sensitive
as HeLa cells toward the tested compounds in which 4j and 4q
showed better antiproliferative activity with IC50 values of 12.5
Surprisingly, among all tested agents, only 4m (IC50, 28.9 mM)
exhibited moderate activity against PC-3 cells. For example,
modifications of 2,5-dimethoxy 2-styrylchromone analogs in ring-
B with different stereochemical properties (4a–4l) did not suppress
the cell proliferation, indicating neither steric nor electronic effects
of various substituents in the ring-B show significant effect on PC-3
cells. It is noteworthy that 4m without any methoxy groups in the
ring-A exhibited the most antiproliferative effect, suggesting either
steric or electronic property of methoxy group could decrease the
antiproliferative activity of 2-styrylchromones. Therefore, we
concluded that selective antiproliferation of PC-3 cells to the tested
compounds could be mainly attributed to both steric and electronic
effects of ring-A rather than ring-B moiety in the 2-styrylchromone
structure.
and 12.4 mM respectively.
In an effort to further investigate the antiproliferative effects
mediated by 4q on the HeLa cells, total 30 h period of growth time
was monitored. The growth curves showed that 4q can suppress
the cell proliferation in a dose-dependent manner. After 30 h of
treatment shown in Fig. 2, 4q exhibited about 60% and 15% of cell
proliferation at 10 and 3 mM, respectively. Fig. 3 demonstrates the
incubation of the HeLa cells with different concentrations of 4q
after 24 h treatment with significant morphological changes
including cell rounding and detachment from the substratum.
Recently, synthetic 4-methoxy-2-styrylchromone has been
reported to be a microtubule-stabilizing antimitotic agent that
blocks G2/M phase of the cell cycle [20]. To examine the association
Exposure of A549 cells to the tested compounds also
revealed certain degree of selectivity. For instance, compound

A.Y. Shaw et al. / European Journal of Medicinal Chemistry 44 (2009) 2552–2562
2555
Scheme 2. Reagents and conditions: (i) Me2SO4/K2CO3, acetone; (ii) Na/EtOAc; (iii) HCl/MeOH; (iv) MeONa/4-OCF3C6H4CHO/MeOH.
between 2-styrylchromone-induced antiproliferation and cell cycle
arrest, we selected 4a, 4i and 4q to analyze the cell cycle distri-
assays were examined. As shown in Fig. 5B, treatment of 4q at
10 M did not interfere with microtubule assembly compared to
m
bution by flow cytometry. Exposure of HeLa cells to 20
m
M of 4a and
untreated control (Mock). Moreover, tubulin polymerization assay
(Fig. 5C) revealed that 4q did not exhibit microtubule-stabilizing
effect that is correspondent with the observations in flow cyto-
metric analysis. In summary, these results suggest that some of our
compounds could induce the antiproliferative effect on HeLa cells
through distinct mechanisms such as G1 phase cell cycle arrest and
DNA fragmentation.
4i for 24 h was found to exhibit a certain amount of G1 phase cell
cycle arrest as shown in Fig. 4E and F, respectively. Nevertheless,
4q-induced sub-G1 phase arrest was observed in a dose-dependent
manner as shown in Fig. 4C and D, indicating that antiproliferative
effect on HeLa cells mediated by 4q may be attributed to the DNA
fragmentation and resulting in cell death.
To further demonstrate 4q-mediated antiproliferation via the
induction of DNA fragmentation, DAPI staining of HeLa cells at 5
and 10 M of 4q treatment was carried out. As shown in Fig. 5A,
DNA fragmentation effect was clearly observed upon 10 M of 4q
treatment. Moreover, to investigate whether 4q exhibits microtu-
bule-stabilizing effect as similar to that of reported in the literature
[20], immunocytochemistry and in vitro tubulin polymerization
mM
2.3. 3D-QSAR on BT483 cell line
m
m
To gain insight into the structure–activity relationship between
2-styrylchromones and carcinoma cells tested in this study,
Table 2
Cytotoxicity of 2-styrylchromones against carcinoma cell lines. IC50 are presented as
the mean ꢀ sem (standard error of the mean) from four to six separated
experiments.
Compound
IC50(mM)
PC-3
A549
BT483
HeLa
SKHep
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
21.5 ꢀ 4.1
>40
>40
10.1 ꢀ 1.9
18.8 ꢀ 2.2
14.2 ꢀ 1.2
14.2 ꢀ 1.4
>40
17.8 ꢀ 2.5
16.4 ꢀ 2.8
16.7 ꢀ 2.9
19.0 ꢀ 1.5
>40
9.6 ꢀ 1.6
10.9 ꢀ 2.2
15.9 ꢀ 2.5
9.1 ꢀ 2.5
9.0 ꢀ 2.0
9.9 ꢀ 1.6
4.9 ꢀ 1.0
26.0 ꢀ 0.9
>40
29.2 ꢀ 0.8
26.9 ꢀ 7.1
>40
25.2 ꢀ 1.7
19.9 ꢀ 2.2
26.1 ꢀ 2.2
>40
29.6 ꢀ 1.4
29.3 ꢀ 2.9
16.6 ꢀ 2.4
>40
26.6 ꢀ 3.0
25.5 ꢀ 1.9
16.6 ꢀ 1.3
15.5 ꢀ 1.9
18.2 ꢀ 2.3
>40
18.6 ꢀ 0.6
>40
>40
>40
>40
>40
23.7 ꢀ 7.6
23.7 ꢀ 3.0
20.6 ꢀ 4.0
12.5 ꢀ 2.9
22.0 ꢀ 1.3
22.1 ꢀ 0.4
>40
15.6 ꢀ 2.6
>40
24.2 ꢀ 1.5
19.7 ꢀ 4.6
20.9 ꢀ 5.9
15.6 ꢀ 1.9
>40
>40
>40
>40
28.9 ꢀ 0.7
>40
21.0 ꢀ 0.4
Fig. 2. Antiproliferative effect of 4q on HeLa cells. Adherent cells proliferated in 96-
well plates (104 cells/well) were incubated with different concentrations of 4q (1.0, 3.0
>40
>40
>40
>40
20.1 ꢀ 2.2
>40
12.4 ꢀ 1.5
and 10
mM) compared to untreated control (Mock) and 0.1% DMSO treatment, and
30.9 ꢀ 1.1
determined by MTT assay at various time intervals.

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A.Y. Shaw et al. / European Journal of Medicinal Chemistry 44 (2009) 2552–2562
Fig. 3. Morphological analysis for the effects of 4q on HeLa cells after 24 h incubation. (A) Untreated control. (B) DMSO treatment. (C) 4q at 20
treatment. (E) 4q at 5 M treatment.
mM treatment. (D) 4q at 10 mM
m
comparative molecular field analysis (CoMFA) was performed by
Sybyl 8.0 (Tripos International, 1699 South Hanley Road, St. Louis,
Missouri 63144, USA) to investigate how substituents influence the
activities of the BT483 cell line. We chose to investigate BT483
results rather than other cell lines due to its higher sensitivity and
selectivity toward the studied compounds according to our
empirical data. The studied 17 structures with charge assignment
done with Gasteiger–Marsili method were generated for CoMFA
analysis and by applying 100 steps in steepest descent algorithm
followed by 500 steps in conjugate gradient algorithm for geometry
minimization without constraints on the internal geometry of the
molecules. The alignment of compounds plays a critical role in
determining structure–activity relationship. For the binding site is
unknown, we assumed that each compound would adopt
a conformation in its lowest energy. Database alignment protocol in
Sybyl 8.0 was utilized to pairwise align the structures according to
the core conformational template specified in ball and stick as
shown in Fig. 6A. To build CoMFA model, the CoMFA descriptors,
steric (Lennard–Jones 6–12 potential) and electrostatic (Coulombic
potential) field energies were calculated with an sp3 carbon atom
carrying þ1.0 charge to serve as a probe atom using Sybyl default
parameters.
200
200
untreated
Cont'l DMSO
A
B
0
0
Restricted to the small size of our data set, the 17 compounds
were all included in training set to build a CoMFA model. Model and
the statistical results are listed in Fig. 6B. The optimal number of
components (ONC ¼ 5) was recommended after a leave-one-out
cross-validated run with a q2 of 0.459 which is acceptable for
a small group of compounds. Generally a q2 value close to or larger
than 0.5 indicates the model is internally predictive and the future
model built without cross-validation shall be accountable. The final
training set model and coefficients such as R2 and F-value were
obtained by using the optimal number of components and all the
data points in the training set. The obtained R2 value of 0.860 is
higher than the criteria value 0.6 for a fairly good model. To further
ensure the predictive ability of the CoMFA model built by the
training set, we carried out a series of test modeling (Pred-1, -2, -3,
-4, and -5 columns in Fig. 6B) by deleting at random one data point
from the training set to form new models and then predict the
deleted compounds’ pIC50 values. The test set predictions are given
in bold in each Pred-column. Comparing the training and test set
results, we found that the fractions of steric and electrostatic
contributions to each model are similar and both contributions are
nearly equally important.
2n
4n
2n
4n
200
200
D
C
4q 20µM
4q 5µM
subG1
0
0
2n
4n
2n
4n
200
200
E
F
4i 20µM
4a 20µM
0
0
2n
4n
2n
4n
DNAcontents
Since 4a–4l differ in substituents on ring-B, the lower-right
fields derived from CoMFA model are applied for comparing their
activities. Likewise, the left side fields surrounding ring-A are
suitable to explain the inhibition performance of 4m–4q. In Fig. 6A,
the lower-right green region indicates a preference of bulky group
Fig. 4. Flow cytometric analysis of HeLa cells. Untreated HeLa cells were taken as blank
group (A). Cells were treated with 0.5% DMSO as the control (B); compound 4q 5 M;
(C) compound 4q 20 M; (D) compound 4a 20 M (E); or compound 4i 20 M (F). Cells
were harvested after 24 h treatment and followed by fixation, propidium iodide
staining as described in Section 4 prior to the flow cytometric analysis. Sub-G1 phase
indicated the DNA fragmentation and cell death.
m
m
m
m
that explains 4l has a higher activity with an IC50 of 16.6 mM in

A.Y. Shaw et al. / European Journal of Medicinal Chemistry 44 (2009) 2552–2562
2557
Fig. 5. DNA fragmentation and microtubule effect of compound 4q on HeLa cells. (A) DAPI staining to examine the DNA fragmentation effect by compound 4q. Untreated HeLa
(Mock) or cells were treated with 0.5% DMSO as the control or treated with compound 4q 5 M or 10 M. (B and C) Microtubule effect of compound 4q. HeLa cells were fixed after
24 h 4q treatment and followed by immunocytochemistry examination using anti- -tubulin antibody, FITC-conjugated secondary antibody and DAPI staining as described in
Section 4. Compound 4q did not affect the tubulin polymerization in vitro by light scattering assay (C). CIL compound was used as positive control.
m
m
b
response to the longest side chain compared to other compounds.
The lower-right red regions which prefer negatively charged
slightly longer –OCF3 group in 4j allows its negatively charged
fluorine atoms to reach the red regions. As to the left side fields, the
upper red region that prefers a negatively charged group is related
to the R5-substitution whereas the lower blue region that prefers
groups or atoms explain why 4j (IC50, 26.6
mM) is better than 4i
(IC50 > 40 M). That is, compared to the –CF3 group in 4i, the
m

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A.Y. Shaw et al. / European Journal of Medicinal Chemistry 44 (2009) 2552–2562
Fig. 6. A) Steric and electrostatic fields generated with the CoMFA model with training set data: yellow indicates regions where bulky groups decrease activity (contribution level of
80%) whereas green indicates regions where bulky groups increase activity (contribution level of 20%); red indicates regions where negatively charged groups increase activity
(contribution level of 20%), whereas blue indicates regions where positively charged groups increase activity (contribution level of 80%). The core used in alignment protocol of
Sybyl 8.0 program is specified in ball and stick. (B) Statistics of CoMFA analysis. (For interpretation of the references to color in this figure legend, the reader is referred to the web
version of this article.)

A.Y. Shaw et al. / European Journal of Medicinal Chemistry 44 (2009) 2552–2562
2559
a positively charged group is related to the R3-substitution. These
two regions can be used to interpret the different levels of activities
of compounds 4n, 4p, and 4q that have methoxy group on R3 or R5
site. Obviously, the negatively charged character of methoxy group
in 4n’s R5 site fits in the red region and shows a better inhibition
activity whereas the methoxy group in 4p and 4q’s R3 sites violate
the positive charge preference and show lower inhibition toward
the BT483 cell line. Moreover, since the oxygen atom on methoxy
group, by virtue of its high electronegativity, can inductively
The methanol was removed in vacuo to get the residue, followed by
the addition of ethyl acetate (50 ml) and washed with brine (50 ml).
The organic layer was dried, evaporated in vacuo and purified with
silica gel chromatography to obtain 3a (2.95 g, 67% in two steps).
M.p. 120–121 ꢁC. 1H NMR (300 MHz, CDCl3)d 2.27 (s, 3H), 3.87 (s,
3H), 3.92 (s, 3H), 6.00 (s, 1H), 6.33 (d, J ¼ 2.3 Hz, 1H), 6.41 (d,
J ¼ 2.3 Hz,1H) ppm. 13C NMR (75 MHz, CDCl3)
d 19.7, 55.7, 56.3, 87.0,
92.6, 95.8, 108.7, 111.8, 135.5, 160.1, 160.8, 163.0, 163.7, 177.4,
207.9 ppm.
withdraw electron through
s-bonds. Consequently, among 4m–4q,
compound 4m without any methoxy group exhibits the highest
4.1.1.3. (E)-5,7-Dimethoxy-2-styryl-4H-chromen-4-one (4a). Sodium
(0.69 g, 30.0 mmol) was gradually added to dry methanol (30 ml)
and the mixture was stirred until the solution reached room
temperature. 3a (1.1 g, 5.0 mmol) and benzaldehyde (0.64 g,
6.0 mmol) were added and the resulting mixture was allowed to stir
at reflux for 18 h. After this period, the solution was poured into iced
water and the pH was adjusted to 4 with HCl. The yellow solid was
removed by filtration, taken up in DCM and purified with silica gel
chromatography (eluent DCM:ethyl acetate ¼ 4:1) to give 4a (1.1 g,
activity in accordance with the red regions around the two fused
rings that indicate a negative charge preference.
p
3. Conclusion
In conclusion, the present work described the synthesis of
a series of 2-styrylchromone analogs as well as the evaluation of
antiproliferative effects on five carcinoma cell lines. Only 4m
1
exhibited a moderate activity with an IC50 value of 28.9
mM against
71%) as a white solid. M.p. 186–188 ꢁC. H NMR (300 MHz, CDCl3)
PC-3 cells which indicates the selective sensitivity of PC-3 cells in
response to 2-styrylchromones. In addition, 4q exhibited the most
d
3.88 (s, 3H), 3.90 (s, 3H), 6.13 (s,1H), 6.31 (d, J ¼ 2.3 Hz,1H), 6.49 (d,
J ¼ 2.3 Hz, 1H), 6.55 (d, J ¼ 16.2 Hz, 1H), 7.32 (m, 3H), 7.52 (d,
antiproliferative effect with an IC50 value of 4.9
mM against HeLa
J ¼ 16.2 Hz,1H), 7.69 (m, 2H) ppm. 13C NMR (75 MHz, CDCl3)
d 55.87,
cells. Further flow cytometric analysis and DAPI staining revealed
that 4q showed its cytotoxic effects on HeLa cells through sub-G1
arrest and DNA fragmentation. Moreover, CoMFA analysis to study
3D-QSAR on BT483 cell line gave a q2 value of 0.459. Taken together,
these results suggest a potential structural optimization and more
detailed pharmacological study of 2-styrylchromones.
56.48, 92.87, 96.10, 112.35, 120.03, 127.62, 129.68, 135.29, 135.62,
135.79, 159.18, 159.75, 160.99, 164.15, 177.71 ppm. HRMS (M)þ calcd
for C19H16O4 308.3279; found 308.1039. Anal. Calcd for C19H16O4: C,
74.01; H, 5.23. Found: C, 73.89; H, 5.32.
4.1.1.4. (E)-5,7-Dimethoxy-2-(2-pyridin-4-yl-vinyl)-chromen-4-one
(4b). Compound 4b was synthesized from the procedure described
for compound 4a. M.p. 212–213 ꢁC. 1H NMR (300 MHz, CDCl3)d 3.91
(s, 3H), 3.92 (s, 3H), 6.24 (s, 1H), 6.35 (d, J ¼ 16.0 Hz, 1H), 6.37 (d,
J ¼ 2.3 Hz, 1H), 6.54 (d, J ¼ 2.3 Hz, 1H), 6.85 (d, J ¼ 16.0 Hz, 1H), 7.42
(m, 2H), 8.65 (m, 2H) ppm. 13C NMR (75 MHz, CDCl3)d 55.9, 56.5,
92.9, 96.3, 113.9, 119.6, 124.3, 129.1, 132.4, 142.5, 150.7, 157.9, 159.7,
161.1, 164.4, 177.5 ppm. HRMS (M)þ calcd for C18H15NO4 309.1001;
found 309.0994. Anal. Calcd for C18H15NO4: C, 69.89; H, 4.89.
Found: C, 69.76; H, 4.81.
4. Experimental
4.1. Chemistry
Chemical reagents and organic solvents were purchased from
TCI and Alfa Aesar unless otherwise mentioned. Melting points
were determined by Fargo MP-2D. Nuclear magnetic resonance
spectra (1H NMR) were measured on a Bruker AC-300 instrument.
Chemical shifts (d) are reported in parts per million relative to the
TMS peak. Mass spectra were obtained by FAB on a Jeol JMS-700
instrument. Flash column chromatography was performed with
silica gel (230–400 mesh). Elemental Analysis was carried out on
a Heraeus VarioEL-III C, H, N analyzer.
4.1.1.5. 2-[2-(4-Fluoro-phenyl)-vinyl]-5,7-dimethoxy-chromen-4-one
(4c). Compound 4c was synthesized from the procedure described
for compound 4a. M.p.169–171 ꢁC.1H NMR (300 MHz, CDCl3)d 3.85
(s, 3H), 3.94 (s, 3H), 6.17 (s, 1H), 6.35 (d, J ¼ 2.3 Hz, 1H), 6.53 (d,
J ¼ 2.3 Hz, 1H), 6.62 (d, J ¼ 16.0 Hz, 1H), 7.10 (m, 2H), 7.43 (d,
J ¼ 16.0 Hz, 1H), 7.54 (m, 2H) ppm. 13C NMR (75 MHz, CDCl3)d 55.9,
56.7, 92.9, 96.2, 109.6, 112.4, 116.1, 116.4, 119.9, 129.5, 131.6, 134.6,
159.1, 159.8, 161.9, 164.3, 177.8 ppm. HRMS (Mþ1)þ calcd for
C19H15FO4 326.0954; found 326.0952. Anal. Calcd for C19H15FO4:
C, 69.93; H, 4.63. Found: C, 69.04; H, 4.58.
4.1.1. General procedure
4.1.1.1. 1-(2-Hydroxy-4,6-dimethoxy phenyl)ethanone (2a). To a solu-
tion of 20,40,60-trihydroxyacetophenone (1a, 5.0 g, 30.0 mmol),
Me2SO4 (5.3 ml, 56 mmol), and anhydrous K2CO3 (8.2 g, 56 mmol) in
acetone (90 ml) was stirred at room temperature for 18 h. The
reaction mixture was filtered and evaporated in vacuo, followed by
recrystallization from ether–hexane to afford 2a (4.9 g, 83%) as
a yellowish solid. M.p. 78–80 ꢁC (lit [32] 78.5–79.5 ꢁC). 1H NMR
(300 MHz, CDCl3)d 2.58 (s, 3H), 3.81 (s, 3H), 3.84 (s, 3H), 5.91 (d,
J ¼ 2.4 Hz, 1H), 6.05 (d, J ¼ 2.3 Hz, 1H), 14.0 (s, 1H) ppm. 13C NMR
(75 MHz, CDCl3)d 32.9, 55.6, 90.7, 93.6, 106.1, 163.0, 163.2, 167.6,
203.3 ppm.
4.1.1.6. 2-[2-(4-Chloro-phenyl)-vinyl]-5,7-dimethoxy-chromen-4-one
(4d). Compound 4d was synthesized from the procedure described
for compound 4a. M.p. 171–172 ꢁC. 1H NMR (300 MHz, CDCl3)d 3.91
(s, 3H), 3.95 (s, 3H), 6.19 (s, 1H), 6.35 (d, J ¼ 2.3 Hz, 1H), 6.54 (d,
J ¼ 2.3 Hz, 1H), 6.67 (d, J ¼ 16.0 Hz, 1H), 7.37 (d, J ¼ 8.5 Hz, 2H), 7.45
(d, J ¼ 16.0 Hz, 1H), 7.49 (d, J ¼ 8.5 Hz, 2H) ppm. 13C NMR (75 MHz,
CDCl3)d 55.9, 56.6, 92.9, 95.2, 112.7, 120.7, 127.1, 128.7, 129.4, 133.9,
134.4, 135.5, 158.9, 159.8, 161.1, 164.3, 177.6 ppm. HRMS (M)þ calcd
for C19H15ClO4 342.0659; found 342.0660. Anal. Calcd for
C19H15ClO4: C, 66.58; H, 4.41. Found: C, 65.95; H, 4.52.
4.1.1.2. 5,7-Dimethoxy-2-methyl-4H-chromen-4-one (3a). A solu-
tion of 2a (3.92 g, 20.0 mmol) in dry ethyl acetate (30 ml) was
added sodium (2.76 g, 120 mmol), the reaction mixture was stirred
at room temperature for 18 h. Cold 0.5 N HCl (30 ml) was added and
the aqueous layer was separated, the remained organic layer was
dried and evaporated in vacuo to obtain the crude diketone. A
solution of the crude diketone with couple drops of concentrated
HCl in methanol (50 ml) was stirred at room temperature for 4 h.
4.1.1.7. 2-[2-(4-Bromo-phenyl)-vinyl]-5,7-dimethoxy-chromen-4-one
(4e). Compound 4e was synthesized from the procedure described
for compound 4a. M.p. 172–173 ꢁC. 1H NMR (300 MHz, CDCl3)d 3.91
(s, 3H), 3.94 (s, 3H), 6.19 (s, 1H), 6.35 (d, J ¼ 2.3 Hz, 1H), 6.53 (d,

2560
A.Y. Shaw et al. / European Journal of Medicinal Chemistry 44 (2009) 2552–2562
J ¼ 2.3 Hz, 1H), 6.68 (d, J ¼ 16.0 Hz, 1H), 7.42 (d, J ¼ 8.5 Hz, 2H), 7.43
(d, J ¼ 16.0 Hz, 1H), 7.54 (d, J ¼ 8.5 Hz, 2H) ppm. 13C NMR (75 MHz,
CDCl3)d 55.7, 56.4, 92.6, 95.9, 112.5, 120.5, 123.6, 128.8, 131.6, 134.1,
134.3, 158.7, 159.6, 160.9, 164.1, 174.3, 177.6 ppm. HRMS (Mþ1)þ
calcd for C19H16BrO4 388.2314; found 388.0143. Anal. Calcd for
C19H15BrO4: C, 58.93; H, 3.90. Found: C, 58.42; H, 3.92.
4.1.1.13. 5,7-Dimethoxy-2-[2-(4-nitro-phenyl)-vinyl]-chromen-4-one
(4k). Compound 4k was synthesized from the procedure described
for compound 4a. M.p. 255–257 ꢁC. 1H NMR (300 MHz, CDCl3)d 3.93
(s, 3H), 3.95 (s, 3H), 6.25 (s, 1H), 6.37 (d, J ¼ 2.3 Hz, 1H), 6.55 (d,
J ¼ 2.3 Hz, 1H), 6.83 (d, J ¼ 16.0 Hz, 1H), 7.52 (d, J ¼ 16.0 Hz, 1H), 7.69
(d, J ¼ 8.6 Hz, 2H), 8.26 (d, J ¼ 8.6 Hz, 2H) ppm. 13C NMR (75 MHz,
CDCl3)d 56.0, 56.7, 93.0, 96.4, 109.4, 114.1, 118.7, 124.5, 128.2, 133.0,
141.6, 148.2, 158.0, 159.8, 161.3, 164.6, 177.6 ppm. HRMS (M)þ calcd
for C19H15NO6 353.0899; found 353.0895.
4.1.1.8. 5,7-Dimethoxy-2-[2-(4-methoxy-phenyl)-vinyl]-chromen-4-
one (4f). Compound 4f was synthesized from the procedure
described for compound 4a. M.p. 175–176 ꢁC. 1H NMR (300 MHz,
CDCl3)d 3.85 (s, 3H), 3.92 (s, 3H), 3.94 (s, 3H), 6.06 (s, 1H), 6.35 (d,
J ¼ 2.3 Hz, 1H), 6.54 (d, J ¼ 2.3 Hz, 1H), 6.56 (d, J ¼ 16.0 Hz, 1H), 6.91
(d, J ¼ 8.5 Hz, 2H), 7.49 (d, J ¼ 16.0 Hz, 1H), 7.51 (d, J ¼ 8.5 Hz, 2H)
ppm. 13C NMR (75 MHz, CDCl3)d 55.5, 55.8, 56.5, 92.8, 96.0, 109.5,
111.6, 114.5, 117.6, 119.6, 123.1, 128.1, 129.2, 135.5, 159.7, 160.9, 164.1,
177.8 ppm. HRMS (M)þ calcd for C20H18O5 338.1154; found
338.1154. Anal. Calcd for C20H18O5: C, 70.99; H, 5.36. Found: C,
70.23; H, 5.29.
4.1.1.14. 2-[2-(4-Cyclopropylmethoxy-phenyl)-vinyl]-5,7-dimethoxy-
chromen-4-one (4l). Compound 4l was synthesized from the
procedure described for compound 4a. M.p. 214–215 ꢁC. 1H NMR
(300 MHz, CDCl3)d 0.35 (m, 2H), 0.65 (m, 2H), 1.32 (m, 1H), 3.84 (d,
J ¼ 6.9 Hz, 2H), 3.91 (s, 3H), 3.94 (s, 3H), 6.14 (s, 1H), 6.34 (d,
J ¼ 2.3 Hz, 1H), 6.53 (d, J ¼ 2.3 Hz, 1H), 6.54 (d, J ¼ 16.0 Hz, 1H), 6.92
(d, J ¼ 8.8 Hz, 2H), 7.43 (d, J ¼ 16.0 Hz, 1H), 7.48 (d, J ¼ 8.8 Hz, 2H)
ppm. 13C NMR (75 MHz, CDCl3)d 3.16, 10.16, 55.7, 56.3, 72.8, 92.7,
95.8, 109.3, 111.4, 114.9, 117.4, 127.7, 128.9, 135.3, 159.5, 160.2, 160.8,
163.9, 177.6 ppm. HRMS (Mþ16)þ calcd for C23H22O6 394.1416;
found 394.1414. Anal. Calcd for C23H22O6: C, 73.00; H, 5.86. Found:
C, 71.45; H, 5.45.
4.1.1.9. 2-(2-Benzo[1,3]dioxol-5-yl-vinyl)-5,7-dimethoxy-chromen-4-
one (4g). Compound 4g was synthesized from the procedure
described for compound 4a. M.p. 217–218 ꢁC. 1H NMR (300 MHz,
CDCl3)d 3.87 (s, 3H), 3.89 (s, 3H), 6.00 (s, 2H), 6.12 (s, 1H), 6.33 (d,
J ¼ 3.8 Hz, 1H), 6.50 (d, J ¼ 15.9 Hz, 1H), 6.51 (d, J ¼ 2.2 Hz, 1H), 6.81
(d, J ¼ 7.9 Hz, 1H), 6.99 (d, J ¼ 7.9 Hz, 1H), 7.02 (d, J ¼ 3.8 Hz, 1H),
7.38 (d, J ¼ 15.9 Hz, 1H) ppm. 13C NMR (75 MHz, CDCl3)d 55.9, 56.6,
87.2, 92.9, 96.1, 101.7, 106.2, 108.8, 111.9, 119.5, 123.7, 129.9, 135.6,
148.6, 149.2, 159.5, 159.8, 161.1, 164.2, 177.8 ppm. HRMS (M)þ calcd
for C20H16O6 352.0947; found 352.0946. Anal. Calcd for
C20H16O6: C, 68.18; H, 4.58. Found: C, 68.04; H, 4.44.
4.1.1.15. 2-Methyl-chromen-4-one (3m). Compound 3m was
synthesized from the procedure described for compound 3a. M.p.
55–56 ꢁC. 1H NMR (300 MHz, CDCl3)d 2.37 (s, 3H), 6.16 (s, 1H), 7.33
(dd, J ¼ 7.7, 1.6, 1H), 7.36 (dd, J ¼ 7.8, 1.5 Hz, 1H), 7.62 (d, J ¼ 7.7, 1H),
8.17 (dd, J ¼ 7.8, 1.5 Hz, 1H) ppm. 13C NMR (75 MHz, CDCl3)d 20.5,
110.4, 117.7, 123.4, 124.8, 125.5, 133.4, 156.4, 166.2, 178.1 ppm.
4.1.1.16. 2-[2-(4-Trifluoromethoxy-phenyl)-vinyl]-chromen-4-one
(4m). Compound 4m was synthesized from the procedure
described for compound 4a. M.p. 169–171 ꢁC. 1H NMR (300 MHz,
CDCl3) 6.34 (s, 1H), 6.75 (d, J ¼ 16.1 Hz, 1H), 7.25 (d, J ¼ 7.4 Hz, 2H),
7.37 (m, 1H), 7.52 (d, J ¼ 7.4 Hz, 2H), 7.55 (d, J ¼ 16.1 Hz, 1H), 7.61 (m,
2H), 8.18 (d, J ¼ 7.9 Hz, 1H) ppm. 13C NMR (75 MHz, CDCl3) 110.9,
115.2, 117.7, 118.6, 119.3, 122.0, 124.1, 125.0, 128.9, 133.6, 133.7, 135.0,
149.9, 155.9, 161.1, 178.3 ppm. HRMS (M)þ calcd for C18H11F3O3
332.0660; found 332.0667. Anal. Calcd for C18H11F3O3: C, 65.06; H,
3.34C. Found: C, 64.48; H, 3.41.
4.1.1.10. 2-[2-(3,5-Dimethoxy-phenyl)-vinyl]-5,7-dimethoxy-chromen-
4-one (4h). Compound 4h was synthesized from the procedure
described for compound 4a. M.p. 188–189 ꢁC. 1H NMR (300 MHz,
CDCl3)d 3.83, 3.84 (s, 3H), (s, 3H), 3.92 (s, 3H), 3.94 (s, 3H), 6.17 (s,
1H), 6.35 (d, J ¼ 2.3 Hz, 1H), 6.48 (d, J ¼ 2.3 Hz, 1H), 6.54 (s, 1H), 6.65
(d, J ¼ 16.0 Hz, 1H), 6.69 (s, 2H), 7.42 (d, J ¼ 16.0 Hz, 1H) ppm. 13C
NMR (75 MHz, CDCl3)d 55.6, 55.9, 55.6, 92.9, 96.2, 102.2, 105.7,
109.5, 112.5, 119.7, 120.6, 135.7, 137.2, 159.2, 159.8, 161.3, 164.3,
177.8 ppm. HRMS (M)þ calcd for C21H20O6 368.1260; found
368.1263. Anal. Calcd for C21H20O6: C, 68.18; H, 4.58. Found: C,
68.04; H, 4.44.
4.1.1.17. 1-(2-Hydroxy-6-methoxy-phenyl)-ethanone (2n). Compound
2n was synthesized from the procedure described for compound
2a. M.p. 55–58 ꢁC. 1H NMR (300 MHz, CDCl3)d 2.67 (s, 3H), 3.89 (s,
3H), 6.39 (d, J ¼ 4.5 Hz,1H), 6.56 (d, J ¼ 5.6 Hz,1H), 7.35 (m,1H),13.3
(s, 1H) ppm. 13C NMR (75 MHz, CDCl3)d 33.8, 56.8, 101.3, 110.8, 111.4,
135.7, 136.2, 161.7, 164.8, 205.3 ppm.
4.1.1.11. 5,7-Dimethoxy-2-[2-(4-trifluoromethyl-phenyl)-vinyl]-chro-
men-4-one (4i). Compound 4i was synthesized from the procedure
described for compound 4a. M.p. 201–202 ꢁC. 1H NMR (300 MHz,
CDCl3)d 3.92 (s, 3H),3.95 (s, 3H), 6.22 (s, 1H), 6.37 (d, J ¼ 2.3 Hz, 1H),
6.55 (d, J ¼ 2.3 Hz, 1H), 6.78 (d, J ¼ 16.0 Hz, 1H), 7.51 (d, J ¼ 16.0 Hz,
1H), 7.65 (m, 4H) ppm. 13C NMR (75 MHz, CDCl3)d 55.9, 56.6, 75.4,
77.4, 92.7, 96.0, 109.4, 113.1, 119.3, 122.0, 122.4, 134.0, 138.5, 158.2,
159.5, 160.9, 164.1, 177.4 ppm. HRMS (M)þ calcd for C20H15F3O4
376.0922; found 376.0921. Anal. Calcd for C20H15F3O4: C, 63.83; H,
4.02. Found: C, 63.75; H, 4.13.
4.1.1.18. 5-Methoxy-2-methyl-chromen-4-one (3n). Compound 3n
was synthesized from the procedure described for compound 3a.
M.p. 97–98 ꢁC. 1H NMR (300 MHz, CDCl3)d 2.27 (s, 3H), 3.93 (s, 3H),
6.04 (s, 1H), 6.75 (d, J ¼ 8.2 Hz, 1H), 6.94 (d, J ¼ 8.5 Hz, 1H), 7.27 (dd,
J ¼ 8.5, 8.2 Hz, 1H) ppm. 13C NMR (75 MHz, CDCl3)d 19.9, 56.4, 106.3,
109.9, 112.1, 114.2, 133.5, 158.6, 159.7, 163.8, 178.3, 196.1 ppm.
4.1.1.12. 5,7-Dimethoxy-2-[2-(4-trifluoromethoxy-phenyl)-vinyl]-chro-
men-4-one (4j). Compound 4j was synthesized from the procedure
described for compound 4a. M.p. 155–156 ꢁC. 1H NMR (300 MHz,
CDCl3)d 3.92 (s, 3H), 3.94 (s, 3H), 6.19 (s, 1H), 6.35 (d, J ¼ 2.3 Hz, 1H),
6.54 (d, J ¼ 2.3 Hz, 1H), 6.66 (d, J ¼ 16.1 Hz, 1H), 7.25 (d, J ¼ 6.9 Hz,
2H), 7.53 (d, J ¼ 16.1 Hz, 1H), 7.57 (d, J ¼ 6.9 Hz, 2H) ppm. 13C NMR
(75 MHz, CDCl3)d 55.7, 56.2, 75.4, 92.6, 95.9, 109.3, 112.5, 118.6,
121.1, 122.0, 125.4, 133.7, 149.7, 158.5, 159.5, 160.8, 164.0, 177.4 ppm.
HRMS (M)þ calcd for C20H15F3O5 392.0872; found 392.0876. Anal.
Calcd for C20H15F3O5: C, 61.23; H, 3.85. Found: C, 61.07; H, 3.89.
4.1.1.19. 5-Methoxy-2-[2-(4-trifluoromethoxy-phenyl)-vinyl]-chromen-
4-one (4n). Compound 4n was synthesized from the procedure
described for compound 4a. M.p. 169–171 ꢁC. 1H NMR (300 MHz,
CDCl3)d 3.95 (s, 3H), 6.25 (s, 1H), 6.67 (d, J ¼ 16.1 Hz, 1H), 6.81 (d,
J ¼ 7.8 Hz, 1H), 7.09 (d, J ¼ 8.0 Hz, 1H), 7.24 (d, J ¼ 8.0 Hz, 2H), 7.55
(d, J ¼ 16.1 Hz, 1H), 7.61 (m, 3H) ppm. 13C NMR (75 MHz, CDCl3)d
56.5, 106.5, 109.9, 112.6, 114.7, 118.8, 119.5, 120.9, 121.3, 128.9, 133.8,
134.5, 149.9, 158.0, 159.1, 159.8, 178.3 ppm. HRMS (M)þ calcd for
C19H13F3O4 362.0766; found 362.0757. Anal. Calcd for C19H13F3O4:
C, 62.99; H, 3.62. Found: C, 62.45; H, 3.74.

A.Y. Shaw et al. / European Journal of Medicinal Chemistry 44 (2009) 2552–2562
2561
4.1.1.20. 1-(2-Hydroxy-5-methoxy-phenyl)-ethanone (2o). Compound
2o was synthesized from the procedure described for compound
2a. M.p. 48–50 ꢁC. 1H NMR (300 MHz, CDCl3)d 2.56 (s, 3H), 3.79 (s,
3H), 6.89 (d, J ¼ 8.9 Hz, 1H), 7.09 (d, J ¼ 8.9 Hz, 1H), 7.16 (s, 1H), 11.85
(s, 1H) ppm. 13C NMR (75 MHz, CDCl3)d 11.7, 26.9, 56.1, 107.5, 113.6,
119.3, 124.3, 135.7, 151.8, 156.9, 194.8, 204.2 ppm.
procedure described for compound 4a. M.p. 181–182 ꢁC. 1H NMR
(300 MHz, CDCl3)d 4.00 (s, 3H), 4.05 (s, 3H), 6.28 (s, 1H), 6.77 (d,
J ¼ 16.0 Hz, 1H), 7.03 (d, J ¼ 9.0 Hz,1H), 7.25 (d, J ¼ 8.6 Hz, 2H), 7.59 (d,
J ¼ 16.0 Hz,1H),7.62(d,J ¼ 8.6 Hz,2H), 7.93(d, J ¼ 9.0 Hz,1H)ppm.13C
NMR (75 MHz, CDCl3)d 56.6, 61.8,109.9,110.7,118.8,119.0,121.3,121.5,
128.2, 133.9, 135.2, 136.9, 150.2, 150.5, 157.0, 161.2, 178.2 ppm. HRMS
(M)þ calcd forC20H15F3O5 392.0872;found392.0867. Anal. Calcd for
C20H15F3O5: C, 61.23; H, 3.85. Found: C, 60.89; H, 3.78.
4.1.1.21. 6-Methoxy-2-methyl-chromen-4-one (3o). Compound 3o
was synthesized from the procedure described for compound 3a.
M.p. 109–110 ꢁC. 1H NMR (300 MHz, CDCl3)d 2.37 (s, 3H), 3.74 (s,
3H), 6.15 (s, 1H), 7.22 (m, 1H), 7.33 (dd, J ¼ 9.0, 2.8 Hz, 1H), 7.53 (d,
J ¼ 2.4 Hz, 1H) ppm. 13C NMR (75 MHz, CDCl3)d 20.5, 55.8, 104.8,
109.7, 119.1, 123.2, 124.0, 151.2, 156.9, 165.9, 177.9 ppm.
4.2. Antiproliferative activity
4.2.1. Cell culture
Cancer cells were purchased from Bioresource Collection and
Research Center in Taiwan. Each cell line was maintained in the
standard medium and grown as a monolayer in Dulbecco’s Modi-
fied Eagle Medium (DMEM) containing 10% fetal bovine serum,
2 mM glutamine, 100 units/ml penicillin, and 100 g/ml strepto-
mycin. Cultures were maintained at 37 ꢁC with 5% CO2 in
a humidified atmosphere.
4.1.1.22. 6-Methoxy-2-[2-(4-trifluoromethoxy-phenyl)-vinyl]-chromen-
4-one (4o). Compound 4o was synthesized from the procedure
described for compound 4a. M.p. 152–153 ꢁC. 1H NMR (300 MHz,
CDCl3)d 3.89 (s, 3H), 6.26 (s, 1H), 6.75 (d, J ¼ 16.0 Hz, 1H), 7.28 (m,
3H), 7.48 (d, J ¼ 16.0 Hz, 1H), 7.59 (m, 4H) ppm. 13C NMR (75 MHz,
CDCl3)d 55.9, 105.0, 110.3, 118.8, 119.3, 121.3, 122.2, 123.7, 124.7,
129.0, 133.7, 134.8, 150.1, 150.8, 156.9, 161.0, 178.2 ppm. HRMS (M)þ
calcd for C19H13F3O4 362.0766; found 362.0768. Anal. Calcd for
C19H13F3O4: C, 62.99; H, 3.62. Found: C, 61.38; H, 3.60.
4.2.2. Antiproliferative assay
Cell proliferation and viability were measured by MTT assay.
Compound stock solution (10 mM in DMSO) waspreparedandstored
at ꢂ20 ꢁC, and was diluted with DMSO to 0.1–1 mM range at room
temperature before experiment. The final percentage of DMSO in the
reaction mixture was less than 1% (v/v). Cancer cells (1 ꢃ104 cells/
well) were plated in the 96-well plates and incubated in medium for
12 h. Serial dilutions of individual compounds were added. 3-[4,5-
Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, (2 mg/ml)
(MTT, 20 ml) wasadded tothe culturesandincubatedduringthefinal
1.5 h. The resultant tetrazolium salt was then dissolved by the addi-
tion of dimethylsulfoxide. Color was measured spectrophotometri-
cally in a microtiter plate reader at 570 nm and used as a relative
measure of viable cell number. The percentage of growth inhibition
was calculated by using the equation: percentage growth inhib-
ition ¼ (1ꢂAt/Ac) ꢃ 100, where At and Ac represent the absorbance in
treated and control cultures, respectively. The compound concen-
tration causing a 50% cell growth inhibition (IC50) was determined by
interpolation from dose–response curves.
4.1.1.23. 1-(2-Hydroxy-4-methoxy-phenyl)-ethanone (2p). Compound
2p was synthesized from the procedure described for compound 2a
as a liquid. 1H NMR (300 MHz, CDCl3)d 2.56 (s, 3H), 3.84 (s, 3H), 6.52
(s, 1H), 6.54 (d, J ¼ 7.8 Hz, 1H), 7.63 (d, J ¼ 7.8 Hz, 1H), 12.7 (s, 1H) ppm.
13C NMR (75 MHz, CDCl3)d 26.3, 29.4, 55.7, 58.7, 100.9, 107.7, 114.0,
132.5, 165.4, 166.3, 202.8 ppm.
4.1.1.24. 7-Methoxy-2-methyl-chromen-4-one (3p). Compound 3p
was synthesized from the procedure described for compound 3a.
M.p. 106–108 ꢁC. 1H NMR (300 MHz, CDCl3)d 2.34 (s, 3H), 3.88
(s, 3H), 6.08 (s, 1H), 6.79 (d, J ¼ 2.4 Hz, 1H), 6.92 (dd, J ¼ 8.9, 2.4 Hz,
1H), 8.06 (d, J ¼ 8.9 Hz, 1H) ppm. 13C NMR (75 MHz, CDCl3)d 20.5,
55.8, 100.2, 110.4, 114.1, 117.4, 126.9, 158.2, 163.9, 165.7, 177.7 ppm.
4.1.1.25. 7-Methoxy-2-[2-(4-trifluoromethoxy-phenyl)-vinyl]-chromen-
4-one (4p). Compound 4p was synthesized from the procedure
described for compound 4a. M.p. 170–172 ꢁC. 1H NMR (300 MHz,
CDCl3)d 3.94 (s, 3H), 6.23 (s, 1H), 6.74 (d, J ¼ 16.0 Hz, 1H), 6.94 (m,
2H), 7.26 (m, 2H), 7.52 (d, J ¼ 16.0 Hz, 1H), 7.62 (m, 2H), 8.08 (d,
J ¼ 8.9 Hz, 1H) ppm. 13C NMR (75 MHz, CDCl3)d 55.9, 100.5, 111.2,
114.3, 118.1, 118.8, 121.4, 122.3, 127.2, 129.1, 133.9, 134.6, 150.1, 157.8,
160.9, 164.4, 177.9 ppm. HRMS (M)þ calcd for C19H13F3O4 362.0766;
found 362.0771. Anal. Calcd for C19H13F3O4 : C, 62.99; H, 3.62.
Found: C, 62.54; H, 3.57.
4.2.3. Flow cytometric analysis
Apoptosis and cell cycle profile were assessed by DNA fluores-
cence flow cytometry. HeLa cells treated with DMSO or 4q at
a concentration of 10 mM for 24 h or 48 h were harvested, rinsed in
PBS, resuspended and fixed in 80% ethanol, and stored at ꢂ20 ꢁC in
fixation buffer until ready for analysis. Then the pellets were
suspended in 1 ml of fluorochromic solution (0.08 mg/ml PI (pro-
pidium iodide), 0.1% TritonX-100 and 0.2 mg/ml RNase A in
1 ꢃ PBS) at room temperature in the dark for 30 min. The DNA
content was analyzed by FACScan flow cytometer (Becton
Dickinson, Mountain View, CA) and CellQuest software (Becton
Dickinson). The population of apoptotic nuclei (subdiploid DNA
peak in the DNA fluorescence histogram) was expressed as the
percentage in the entire population.
4.1.1.26. 1-(2-Hydroxy-3,4-dimethoxy-phenyl)-ethanone (2q). Com-
pound 2q was synthesized from the procedure described for
compound 2a. M.p. 75–76 ꢁC. 1H NMR (300 MHz, CDCl3)d 2.56 (s,
3H), 3.87 (s, 1H), 3.92 (s, 3H), 6.47 (d, J ¼ 9.0 Hz, 1H), 7.48 (d,
J ¼ 9.0 Hz, 1H), 12.55 (s, 1H) ppm. 13C NMR (75 MHz, CDCl3)d 26.3,
56.1, 60.5, 102.9, 115.2, 127.1, 136.3, 156.9, 158.5, 203.3 ppm.
4.2.4. Immunofluorescence analysis
4.1.1.27. 7,8-Dimethoxy-2-methyl-chromen-4-one (3q). Compound
3q was synthesized from the procedure described for compound
3a. M.p. 86–88 ꢁC. 1H NMR (300 MHz, CDCl3)d 2.41 (s, 3H), 3.91 (s,
3H), 3.96 (s, 3H), 6.10 (s, 1H), 6.99 (d, J ¼ 9.0 Hz, 1H), 7.90 (d,
J ¼ 9.0 Hz, 1H) ppm. 13C NMR (75 MHz, CDCl3)d 20.7, 56.5, 61.6,
109.8, 110.1, 118.4, 121.0, 135.6, 136.6, 150.9, 156.4, 166.0, 178.0 ppm.
HeLa Cells were seeded on cover glasses in 12-well plates with
the drug treatment for 24 h were used for DAPI staining. After
incubation, cells were washed with 1 ꢃ PBS twice and fixed in 4%
paraformaldehyde for 1 h. Then, cells were washed with PBS
containing 0.1 M Glycine for 5 min and permibilized with solution
containing 2% FBS and 0.4% TritonX-100 in PBS at room tempera-
ture for 15 min. After permibilization, cells were stained with a-
4.1.1.28. 7,8-Dimethoxy-2-[2-(4-trifluoromethoxy-phenyl)-vinyl]-
chromen-4-one (4q). Compound 4q was synthesized from the
tubulin monoclonal antibody (Santa Cruza 1:1000) at 4 ꢁC over-
night. After primary antibody incubation, cells were washed with

2562
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Financial support from the National Science Council of the
Republic of China (NSC 96-2113-M-032-007-MY2) and Tamkang
University to A.Y.S. is gratefully acknowledged. We are indebted to
Professor Kuo-Wei Huang (NUS) for useful discussion.
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