Synthesis of (E)-5-methoxy-2-styryl-4-pyrones as potent growth-inhibitory agents against hepatocellular carcinoma cells

Article abstract of DOI:10.1002/jhet.1663

The present study a series of (E)-5-methoxy-2-styryl-4H-pyran-4-ones 6a, 6b, 6c, 6d, 6e, 6f, 6g, 6h, 6i, 6j was synthesized and evaluated for growth inhibitory inhibition against carcinoma cells. The growth inhibition study of eight carcinoma cell lines w

Full text of DOI:10.1002/jhet.1663

56
Synthesis of (E)-5-Methoxy-2-styryl-4-pyrones as Potent Growth-Inhibitory
Agents Against Hepatocellular Carcinoma Cells
Vol 51
Yu-Hua Chen,a Pei-Jung Lu,b Christopher Hulme,c,d and Arthur Y. Shawc,d
*
aDepartment of Chemistry, Tamkang University, New Taipei City, 251, Taiwan
bInstitute of Clinical Medicine, National Cheng Kung University, Tainan 701, Taiwan
cDepartment of Pharmacology and Toxicology, College of Pharmacy, The University of Arizona, Tucson, AZ, 85737,
USA
dBIO5 Oro Valley, The University of Arizona, Oro Valley, AZ, 85737, USA
*
E-mail: shaw.299@gmail.com
Received November 11, 2011
DOI 10.1002/jhet.1663
Published online 8 October 2013 in Wiley Online Library (wileyonlinelibrary.com).
O
O
MeO
HO
O
OH
O
R
1)
kojic acid (
(E)-5-methoxy-2-styryl-4-pyrones
The present study a series of (E)-5-methoxy-2-styryl-4H-pyran-4-ones 6a–j was synthesized and
evaluated for growth inhibitory inhibition against carcinoma cells. The growth inhibition study of eight
carcinoma cell lines was examined and demonstrated that SKHep cells exhibit significant structure-activity
relationship in response to the tested compounds. Among them, 6f showed the most potent activity against
SKHep, A549, AGS, and H460 cell lines with GI50 values of 0.17, 8.3, 3.6, 8.0 mM, respectively.
J. Heterocyclic Chem., 51, 56 (2014).
INTRODUCTION
modifications aimed at developing potential drug-like
small molecules. To the best of our knowledge, (E)-5-
methoxy-2-styryl-4-pyrones derived from kojic acid have
not yet been studied for their anticancer activity. Herein,
we take advantage of kojic acid as a molecular tem-
plate to synthesize a series of (E)-5-methoxy-2-styryl-
4H-pyran-4-ones and to examine their growth inhibitory
effect on a panel of carcinoma cell lines as shown in Figure 1.
Kojic acid (1) is a metabolic compound produced by
several species of fungi, such as Aspergillus, Acetobacter,
and Penicillium [1], Figure 1. It has been added to food
as an antioxidant [2], as a preservative to prevent formation
of warm-over flavor in beef [3]. Additionally, it serves as a
food additive for preventing enzymatic discoloration in
vegetables, crabs, and shrimps [4,5] and as a skin lightening
or bleaching agent in cosmetic preparations [6,7].
Among its pharmacological activities, kojic acid has been
demonstrated to exhibit bacteriostatic activity [8], anti-
inflammatory effects [9], insecticidal activity [10], antibi-
otic activity [11], and cytotoxic and antitumor properties
[12,13]. Particularly, kojic acid accessibly carried out-
various structural modifications, thanks to its flexibility
and amenability. For instance, both ionizable and nonio-
nizable derivatives of kojic acid have shown to exhibit
growth inhibitory effect against Escherichia coli [14].
Selenium containing kojic acid derivatives have showed
cytotoxic effects against carcinoma cells [15]. Mimosine,
a ring isomer of kojic acid, has revealed superior antipro-
liferative effects to the parent compound [16]. Moreover,
organomercury (II) complexes of kojic acids also demon-
strated improved antibacterial activity compared with
kojic acid [17]. As such, the scaffold of kojic acid shows
commitment to be a promising lead for structural
RESULTS AND DISCUSSION
Synthesis.
The synthesis of (E)-5-methoxy-2-styryl-
4H-pyran-4-ones is outlined in Scheme 1. As shown, the
C-5 hydroxyl group in kojic acid (1) was selectively
methylated by treatment of dimethyl sulfate (Me2SO4)
in the presence of 10% potassium hydroxide (KOH)
to afford 2-(hydroxymethyl)-5-methoxy-4H-pyran-4-one
2 in 70% isolated yield [18]. The hydroxyl group on
2-methylene moiety in 2 was further subjected to undergo
mesylation with mesyl chloride (MsCl) in the presence
of triethylamine (NEt3) in dichloromethane (DCM) to yield
mesylate ester 3. Without further purification, the
nucleophilic substitution of the mesylate ester 3 and sodium
bromide (NaBr) was carried out in DMF to generate 2-
(bromomethyl)-5-methoxy-4H-pyran-4-one
4
in 63%
isolated yield in two steps. Furthermore, the Arbuzov
© 2013 HeteroCorporation

January 2014
Kojic Acid-Derived Growth-Inhibitory Agents
57
reaction of 2-(bromomethyl)-5-methoxy-4H-pyran-4-one 4
and triethyl phosphite (P(OEt)3) in toluene at reflux gave
in Table 1, the tested compounds bearing electron-donating
groups on the benzene ring such as 40-methoxy (6c, GI50,
0.61 mM), 30,40-dimethoxy (6d, GI50, 0.24mM), 30,50-
dimethoxy (6e, GI50, 0.18 mM), and 30,40,50-trimethoxy
(6f, GI50, 0.17mM) displayed remarkable growth-inhibitory
effect on SKHep cells compared with 6a (GI50, 5.0 mM),
indicating that electronic effect of tested compounds play a
critical for activity. Moreover, we found that 6c (GI50,
0.61 mM) containing 4-methoxy group on the benzene ring
exhibited 4.6-fold in potency compared with its counterpart
6b (GI50, 2.8 mM) with 3-methoxy group. This finding
suggests that an appropriate position of substituent attached
to the tested compound is significantly improved to exhibit
interactions with its not yet indentified targets for growth
inhibition. Compared with its counterpart 6c (GI50, 0.61mM),
6j bearing an electron-donating 4-N, N-dimethylamino group
showed less potent activity with a GI50 value of 2.6 mM. On
the contrary, introduction of electron-withdrawing groups on
the benzene ring such as 4-trifluoromethyl (6h, GI50,
36.1 mM) and 4-trifluoromethoxy (6i, GI50, 4.7 mM) groups
resulted in a precipitate decrease in potency. According to
such interesting findings, we concluded that SKHep cells
clearly exhibited structure-activity relationship in response
to our tested compounds. On the other hand, upon exposure
of OVCA cells to the tested compounds, only 6c, 6d, 6e, 6f
and 6j showed moderate growth inhibitory effect with GI50
values of 19.4, 26.4, 17.8, 18.1 and 21.3mM, respectively.
Except for 6f with moderate activity, all tested compounds
did not revealed appreciable growth-inhibitory effect against
the rest of six cell lines as high as 40 mM treatment. As indi-
cated in Table 2, compound 6f bearing 30,40,50-trimethoxy
groups exhibited an appreciable growth inhibitory activ-
ity with GI50 values of 8.3, 3.6, 8.0 mM against A549,
AGS and H460 cell lines, respectively. Taken together,
we clearly summarized that individual cell lines indeed
revealed a distinct sensitivity in response to these
compounds.
phosphonate
5 in 68% isolated yield. Having the
phosphonate 5 in hand, a series of (E)-5-methoxy-2-styryl-4-
pyrones 6a–j was successfully synthesized by the Horner–
Emmons reaction, in which the phosphonate 5 and various
benzaldehydes underwent olefination reaction mediated by
the base sodium hydride (NaH) in the anhydrous THF
solution [19,20].
Growth inhibition. The evaluation of growth-inhibitory
effect of 6a–j was examined in a panel of eight human
carcinoma cell lines, including SKHep (hepatocellular
carcinoma cell), A549 (non-small cell lung adenocarcinoma
cell), AGS (gastric adenocarcinoma cell), PC-3 (prostate
carcinoma cell), H460 (lung large cell carcinoma cell),
SW620 (colorectal adenocarcinoma cell), HeLa (cervical
epithelioid carcinoma cell), and OVCA (ovarian carcinoma
cell. All cell lines were exposed to the tested compounds
at indicated concentration in 10% fetal bovine serum
(FBS)-supplemented Dulbecco’s Modified Eagle Medium
for a 48-h treatment. The indicated growth inhibition in
response to the tested compounds was employed by the 3-
[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
(MTT) assay.
Surprisingly, only SKHep cells exhibited significant
growth inhibition in response to 6a–j treatment with a
range of GI50 values between 0.17 and 36.1 mM. As shown
O
O
MeO
HO
O
OH
O
R
1)
kojic acid (
(E)-5-methoxy-2-styryl-4-pyrones
Figure 1. Chemical structures of kojic acid (1) and its derivatives.
Scheme 1. Preparation of (E)-5-methoxy-2-styryl-4H-pyran-4-ones 6a–j.
O
O
O
O
HO
MeO
MeO
c
a
b
OH
OH
OMs
O
O
O
2
3
kojic acid (1)
O
O
MeO
MeO
e
d
MeO
O
P
O
OEt
OEt
Br
O
O
R
4
5
6a-j
Reagents and conditions: a) Me2SO4 , 10% KOH; b) MsCl, NEt3, DCM, 0oC; c) NaBr, DMF, rt; d)
P(OEt)3, toluene, reflux; e) ArCHO, NaH, THF, 0oC to rt.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

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Y-H. Chen, P-J. Lu, C. Hulme, and A. Y. Shaw
Vol 51
Table 1
Chemical structures and growth inhibitory effect of (E)-5-methoxy-2-styryl-4-pyrones 6a–j against carcinoma cells.
a
GI50 (mM)
GI50a (mM)
Entry
Structure
Entry
Structure
SKHep
OVCA
SKHep
OVCA
18.1 ꢀ 2.2
6a
5.0 ꢀ 0.4
2.8 ꢀ 0.2
>40
6f
0.17 ꢀ 0.01
6b
>40
6g
2.9 ꢀ 0.5
>40
6c
0.61 ꢀ 0.34
0.24 ꢀ 0.05
19.4 ꢀ 1.8
26.4 ꢀ 2.2
6h
36.1 ꢀ 1.5
4.7 ꢀ 1.1
>40
>40
6d
6i
6e
0.18 ꢀ 0.02
17.8 ꢀ 1.2
6j
2.6 ꢀ 0.7
21.3 ꢀ 2.1
OVCA, Ovarian carcinoma cell.
aGI50 values are presented as the mean ꢀ standard error of the mean from four to six separated experiments.
Table 2
GI50 values of (E)-5-methoxy-2-(30, 40, 50-trimethoxy)styryl-4-pyrone 6f on a panel of human cancer cell lines.
GI50a (mM)
Cell line
Tumor type
6f
Doxorubicin
SKHep
A549
AGS
Hepatocellular carcinoma cell
Non-small cell lung adenocarcinoma cell
Gastric adenocarcinoma cell
Prostate carcinoma cell
Lung large cell carcinoma cell
Colorectal adenocarcinoma cell
Cervical epithelioid carcinoma cell
Ovarian carcinoma cell
0.17 ꢀ 0.01
8.3 ꢀ 0.3
0.09 ꢀ 0.03
2.23 ꢀ 0.3
3.6 ꢀ 0.3
0.02 ꢀ 0.01
1.76 ꢀ 0.71
0.11 ꢀ 0.02
0.03 ꢀ 0.005
0.33 ꢀ 0.04
0.95 ꢀ 0.07
PC-3
21.4 ꢀ 4.3
8.0 ꢀ 1.4
H460
SW620
HeLa
11.8 ꢀ 2.8
28.1 ꢀ 2.6
18.1 ꢀ 2.2
OVCA
aGI50 values are presented as the mean ꢀ SEM (standard error of the mean) from four to six separated experiments.
Journal of Heterocyclic Chemistry
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Kojic Acid-Derived Growth-Inhibitory Agents
59
Diethyl (5-methoxy-4-oxo-4H-pyran-2-yl)methylphosphonate
CONCLUSION
(5).
To a solution of 4 (1.1 g, 5.0 mmol) in toluene (15 mL),
triethyl phosphite (3.32 g, 20.0 mmol) was added, and the mixture
was heated at reflux. After 96 h, excess amount of triethyl
phosphite was removed in vacuo and the resulting oily residue was
purified by flash column to afford 5. Yield: 68%. 1H NMR
(300 MHz, CDCl3) d 1.28 (t, J =7.1Hz, 6H), 3.01 (s, 1H), 3.08
(s, 1H), 3.75(s, 3H), 4.11 (q, J= 7.1 Hz, 4H), 6.31(s, 1H), 7.52
(s, 1H) ppm.
The present study we take advantage of kojic acid as a
lead to synthesize a series of (E)-5-methoxy-2-styryl-4 H-
pyran-4-ones 6a-j. The growth inhibition study of eight
carcinoma cell lines was examined and demonstrated that
the SKHep cells exhibited a significant structure-activity
relationship in response to the tested compounds. Particu-
larly, compound 6f showed the most potent activity against
SKHep, A549, AGS, and H460 cell lines with GI50 values
of 0.17, 8.3, 3.6, 8.0 mM, respectively. In summary,
structural modifications of kojic acid have been carried
out and examined to show highly selective growth inhibi-
tion on SKHep cells that pave the way for further mechanis-
tic study.
General procedure for the synthesis of 5-methoxy-2-styryl-4-
pyrones.
(E)-5-Methoxy-2-styryl-4H-pyran-4-one (6a). To a
stirred solution of 5a (0.276g, 1.0 mmol) and NaH (0.08 g,
2.0 mmol, 60% suspension in mineral oil) in dry THF (20 mL), a
solution of benzaldehyde (0.106g, 1.0mmol) in THF (5 mL) was
added dropwise under Argon atmosphere at 0ꢁC. After stirring at
room temperature for 1 h, the mixture was diluted with
dichloromethane (20 mL) and washed with water (40 mL) and brine
(40 mL). The organic layer was dried over MgSO4, concentrated in
vacuo and purified by flash column chromatography (ethyl
acetate/dichloromethane = 1/9) to afford 6a. Yield: 85%. 1H NMR
(300MHz, CDCl3) d 3.77(s, 3H), 6.36(s, 1H), 6.66(d, J = 16.2Hz,
1H), 7.32 (d, J = 16.2 Hz, 1H), 7.33–7.39(m, 3H), 7.49
(d, J = 9.7Hz, 2H), 7.56(s, 1H)ppm. 13C NMR (75 MHz, CDCl3)
d 56.6, 113.3, 119.4, 127.7, 129.1, 129.9, 135.0, 136.4, 137.3,
148.8, 161.5, 174.5 ppm. HRMS (M + 1)+ Calcd for C14H12O3
228. 228.2433; found 228.2431.
EXPERIMENTAL
Chemistry.
Chemical reagents and organic solvents were
purchased from TCI and Alfa Aesar unless otherwise
mentioned. Nuclear magnetic resonance spectra (1H-NMR and
13C-NMR) were measured on a Bruker AC-300 instrument
purchased from Bruker Daltonics Inc. (Fremont, CA). Chemical
shifts (d) are reported in ppm relative to the TMS peak. High
resolution mass spectra (HRMS) were obtained by fast atom
bombardment (FAB) on a Jeol JMS-700 instrument. Flash
column chromatography was performed with Merck Kiesegel
60 Art (230–400 mesh).
(E)-5-methoxy-2-(3-methoxystyryl)-4H-pyran-4-one (6b).
1
The synthesis of 6b was similar to that of 6a. Yield: 81%. H
NMR (300 MHz, CDCl3) d 3.80(s, 3H), 3.85(s, 3H), 6.38(s, 1H),
6.66(d, J = 16.1Hz, 1H), 6.91(d, J = 7.8 Hz, 1H), 7.03(s, 1H), 7.11
(d, J = 8.0 Hz, 1H) 7.31(dd, J = 8.0, 7.8 Hz, 1H), 7.34
(d, J = 16.1 Hz, 1H), 7.57(s, 1H) ppm. 13C NMR (75MHz,
CDCl3) d 56.7, 55.5, 112.8, 113.5, 115.7, 119.8, 120.4, 130.2,
136.3, 136.5, 137.4, 148.9, 160.2, 161.5, 174.6 ppm. HRMS
(M+ 1)+ Calcd for C15H14O4 258.2693; found 258.2689.
2-(Hydroxymethyl)-5-methoxy-4H-pyran-4-one (2).
To a
well-stirred solution of kojic acid (1) (2.5 g, 17.6 mmol) and
in 10% potassium hydroxide (20 mL), redistilled dimethyl
sulfate (2.5 g, 20.09 mmol) was added. The reaction mixture
was kept below 25ꢁC by occasional cooling in an ice-bath.
Stirring was continued for an additional 30 min and the
mixture was cooled in an ice-bath and filtered, followed by
proper recrystallization from methanol to obtain 2. Yield: 70
%. 1H-NMR (300 MHz, DMSO-d6) d 3.13 (s, 3H), 4.30 (d,
J = 5.8 Hz, 2H), 5.70 (t, J = 5.8 Hz, 1H), 6.32 (s, 1H), 8.16
(s, 1H) ppm.
(E)-5-methoxy-2-(4-methoxystyryl)-4H-pyran-4-one (6c). The
1
synthesis of 6c was similar to that of 6a. Yield: 83%. H NMR
(300 MHz, CDCl3) d 3.78(s, 3H), 3.84(s, 3H), 6.33(s, 1H), 6.53
(d, J = 16.0 Hz, 1H), 6.9(d, J = 8.8 Hz, 2H), 7.31(d, J = 16.0 Hz,
1H), 7.46(d, J = 8.8 Hz, 2H), 7.54(s, 1H) ppm. 13C NMR
(75 MHz, CDCl3) d 55.6, 55.6, 112.7, 114.6, 117.1, 127.9,
129.3, 136.0, 137.2, 148.8, 161.2, 162.1, 174.6 ppm. HRMS
(M + 1)+ Calcd for C15H14O4 258.2693; found 258.2693.
(E)-2-(3,4-dimethoxystyryl)-5-methoxy-4H-pyran-4-one (6d).
(5-Methoxy-4-oxo-4H-pyran-2-yl)methyl methanesulfonate
(3).
To a stirred solution of 2 (1.56 g, 10.0 mmol) in DCM
1
(20 mL), triethylamine (1.01 g, 10.0 mmol) and methanesulfonyl
chloride (1.13 g, 10.0 mmol) was added at 0ꢁC. After stirring for
30 min at an ice-bath, the mixture was diluted with ethyl acetate
(20 mL) and washed with brine (20 mL). The organic layer was
dried over MgSO4, evaporated in vacuo to obtain crude 3.
Yield: 90%. 1H NMR (300 MHz, DMSO-d6) d 3.15 (s, 3H),
3.32 (s, 3H), 5.16 (s, 2H), 6.57 (s, 1H), 8.27 (s, 1H) ppm.
The synthesis of 6d was similar to that of 6a. Yield: 72%. H
NMR (300 MHz, CDCl3) d 3.80(s, 3H), 3.91(s, 3H), 3.93(s, 3H),
6.33(s, 1H), 6.53(d, J = 16.0Hz, 1H), 6.86(d, J = 8.3Hz, 1H), 7.03
(s, 1H), 7.07(d, J = 8.3, 1H), 7.30(d, J = 16.0 Hz, 1H), 7.54
(s, 1H) ppm. 13C NMR(75MHz, CDCl3) d 56.1, 56.1, 56.6,
109.4, 111.3, 112.7, 11.7, 122.1, 128.1, 136.2, 137.2, 148.8,
149.5, 150.9, 161.9, 174.6ppm. HRMS (M + 1)+ Calcd for
C16H16O5 288.2952; found 288.2948.
2-(Bromomethyl)-5-methoxy-4H-pyran-4-one (4).
To a
stirred solution of 3 (2.23 g, 9.0 mmol) in DMF (15 mL), NaBr
(1.02 g, 10.0 mmol) was added, and the mixture was stirred at
room temperature for 20 min. As excess amount of NaBr was
filtered, the filtrate was diluted with ethyl acetate (30 mL) and
washed with water (25 mL) and brine (20 mL). The organic
layer was dried over MgSO4, concentrated in vacuo and
purified by flash column chromatography (ethyl acetate/
dichloromethane = 3/7) to obtain 4. Yield:72%. 1H NMR
(300 MHz, CDCl3) d 3.15 (s, 3H), 5.08 (s, 2H), 6.47 (s, 1H),
7.56 (s, 1H) ppm.
(E)-2-(3,5-dimethoxystyryl)-5-methoxy-4H-pyran-4-one (6e).
The synthesis of 6e was similar to that of 6a. Yield: 79%. 1H NMR
(300 MHz, CDCl3) d 3.78(s, 3H), 3.81(s, 6H), 6.35(s, 1H), 6.46(s,
1H), 6.63(s, 2H), 6.62(d, J = 16.0 Hz, 1H), 7.27(d, J = 16.0 Hz,
1H), 7.55(s, 1H) ppm. 13C NMR (75 MHz, CDCl3) d 55.6, 55.6,
76.8, 77.2, 77.6, 102.1, 105.7, 113.5, 119.9, 136.4, 136.9, 137.3,
148.8, 161.3, 161.4 ppm. HRMS (M + 1)+ Calcd for C16H16O5
288.2952; found 288.2951.
(E)-2-(3,4,5-trimethoxystyryl)-5-methoxy-4H-pyran-4-one(6f).
The synthesis of 6f was similar to that of 6a. Yield: 77%. 1H NMR
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Y-H. Chen, P-J. Lu, C. Hulme, and A. Y. Shaw
Vol 51
(300 MHz, CDCl3) d 3.8(s, 3H), 3.87(s, 3H), 3.90(s, 6H), 6.36
(s, 1H), 6.57(d, J = 16.0Hz, 1H), 6.72(s, 1H), 7.28(d, J = 16.0Hz,
1H), 7.55(s, 1H) ppm. 13C NMR (75 MHz,CDCl3) d 56.3, 56.5,
61.1, 104.8, 113.1, 118.6, 130.5, 136.3, 137.2, 139.9, 148.7,
153.6, 161.5, 174.4 ppm. HRMS (M + 1)+ Calcd for C17H18O6
318.3212; found 318.3209.
(E)-2-(2-(benzo[d][1,3]dioxol-5-yl)vinyl)-5-methoxy-4H-pyran-
4-one (6g). The synthesis of 6g was similar to that of 6a. Yield:
72%. 1H NMR(300MHz, CDCl3) d 3.78(s, 3H), 6.00(s, 2H), 6.33
(s, 1H), 6.49(d, J = 16.0Hz, 1H), 6.81(d, J = 8.0 Hz, 1H), 6.98
(d, J = 8.0Hz, 1H), 7.02(s, 1H), 7.26(d, J = 16.0 Hz, 1H), 7.54
(s, 1H) ppm. 13C NMR (75 MHz, CDCl3) d 31.1, 56.6, 101.8,
106.2, 108.8, 112.9, 117.6, 123.8, 129.6, 136.1, 137.3, 148.7,
148.8, 161.8, 174.6 ppm. HRMS (M + 1)+ Calcd for C15H12O5
272.2528; found 272.2523.
(E)-5-methoxy-2-(4-(trifluoromethyl)styryl)-4H-pyran-4-one
(6h). The synthesis of 6h was similar to that of 6a. Yield: 86%.
1H NMR (300 MHz, CDCl3) d 3.80(s, 3H), 6.44(s, 1H), 6.76
(d, J = 16.1 Hz, 1H), 7.39(d, J = 16.1 Hz, 1H), 7.59–7.70
(m, 5H) ppm. 13C NMR (75 MHz, CDCl3) d 56.7, 114.2, 121.9,
122.2, 126.1, 126.1, 127.8, 134.6, 137.5, 138.4, 149.0, 160.8,
174.5 ppm. HRMS (M + 1)+ Calcd for C15H11F3O3 296.2412;
found 296.2411.
(MTT, 2 mg/mL) dye reduction assay in 96-well microplates
was used. The assay is dependent on the reduction of
MTT by mitochondrial dehydrogenases of viable cell to
a
blue formazan product, which can be measured
spectrophotometrically. Tumor cells were incubated in each
well with serial dilutions of the tested compounds. After
2 days of incubation (37ꢁC, 5% CO2 in a humid
atmosphere) 100 mL of MTT (2 mg/mL in phosphate
buffered saline (PBS)) was added to each well and the plate
was incubated for a further 2 h (37ꢁC). The resulting
formazan was dissolved in 100 mL DMSO and read at
570 nm. The percentage of growth inhibition was calculated
by the following equation: percentage growth inhibition = (1-
At/Ac) ꢂ 100, where At and Ac represent the absorbance in
treated and control cultures, respectively. The drug
concentration causing a 50% cell growth inhibition (GI50)
was determined by interpolation from dose–response
curves. All determinations were carried out in four to six
separated experiments.
Statistical analysis.
Data are presented as the
(E)-5-methoxy-2-(4-(trifluoromethoxy)styryl)-4H-pyran-4-
mean ꢀ standard error of the mean from four to six
separated experiments. Statistical analyses were
performed using Bonferroni t-test method after ANOVA
for multigroup comparison and Student’s t-test method
for two-group comparison, p = 0.05 was considered
significant. Analysis of linear regression (at least five
data within 20–80% inhibition) was used to calculate
GI50 value.
one (6i).
The synthesis of 6i was similar to that of 6a. Yield:
84%. 1H NMR (300MHz, CDCl3) d 3.79(s, 3H), 6.41(s, 1H),
6.65(d, J = 16.1, 1H), 7.24(d, J = 8.2 Hz, 2H), 7.34(d,
J = 16.1Hz,1H), 7.54(d, J = 8.2 Hz, 2H), 7.58(s, 1H)ppm. 13C
NMR (75MHz, CDCl3) d 56.7, 113.8, 120.5, 121.5, 129.1, 132.3,
133.7, 134.7, 137.5, 149.0. 150.2, 161.2, 174.6 ppm. HRMS
(M+ 1)+ Calcd for C15H11F3O4 312.2406; found 312.2406.
(E)-2-(4-(dimethylamino)styryl)-5-methoxy-4H-pyran-4-one (6j).
The synthesis of 6j was similar to that of 6a. Yield: 75%.
1H NMR(300 MHz, CDCl3) d 3.01(s, 6H), 3.77(s, 3H),
6.27(s, 1H), 6.43(d, J = 16.0 Hz, 1H), 6.67(d, J = 8.8 Hz, 2H),
7.28(d, J = 16.0 Hz, 1H), 7.39(d, J = 8.8 Hz, 2H), 7.51(s,
1H) ppm. 13C NMR(75 MHz, CDCl3) d 40.3, 56.5, 111.6,
112.1, 114.1, 122.9, 129.2, 136.8, 137.0, 148.6, 151.5,
162.8, 174.6 ppm. HRMS (M + 1)+ Calcd for C16H17NO3
271.3111; found 271.3108
Acknowledgment. Financial support from the National Science
Council of the Republic of China to A. Y. Shaw is gratefully
acknowledged.
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CELL CULTURE
Cancer cells were purchased from Bioresource Collec-
tion and Research Center in Taiwan. Each cell line was
maintained in the standard medium and grown as a mono-
layer in Dulbecco’s Modified Eagle Medium containing
10% fetal bovine serum, 2 mM glutamine, 100 units/mL
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alone (control) or compounds (drugs were dissolved in
DMSO previously) at the concentrations indicated. Treated
cells were further incubated for 48 h. Cell survival is
expressed as percentage of control cell growth. The 3-[4, 5-
Dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide
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