Synthesis, antitumor activity, and mechanism of action of 6-acrylic phenethyl ester-2-pyranone derivatives

Article abstract of DOI:10.1039/c5ob00007f

Based on the scaffolds of caffeic acid phenethyl ester (CAPE) as well as bioactive lactone-containing compounds, 6-acrylic phenethyl ester-2-pyranone derivatives were synthesized and evaluated against five tumor cell lines (HeLa, C6, MCF-7, A549, and HSC-2). Most of the new derivatives exhibited moderate to potent cytotoxic activity. Moreover, HeLa cell lines showed higher sensitivity to these compounds. In particular, compound 5o showed potent cytotoxic activity (IC₅₀ = 0.50-3.45 μM) against the five cell lines. Further investigation on the mechanism of action showed that 5o induced apoptosis, arrested the cell cycle at G2/M phases in HeLa cells, and inhibited migration through disruption of the actin cytoskeleton. In addition, ADMET properties were also calculated in silico, and compound 5o showed good ADMET properties with good absorption, low hepatotoxicity, and good solubility, and thus, could easily be bound to carrier proteins, without inhibition of CYP2D6. A structure-activity relationship (SAR) analysis indicated that compounds with ortho-substitution on the benzene ring exhibited obviously increased cytotoxic potency. This study indicated that compound 5o is a promising compound as an antitumor agent.

Full text of DOI:10.1039/c5ob00007f

Organic &
Biomolecular Chemistry
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Synthesis, antitumor activity, and mechanism of
action of 6-acrylic phenethyl ester-2-pyranone
derivatives†
Cite this: Org. Biomol. Chem., 2015,
13, 4714
Sai Fang,‡a Lei Chen,‡a Miao Yu,a Bao Cheng,a Yongsheng Lin,a
Susan L. Morris-Natschke,b Kuo-Hsiung Lee,b,c Qiong Gu*a,b and Jun Xu*a
Based on the scaffolds of caffeic acid phenethyl ester (CAPE) as well as bioactive lactone-containing
compounds, 6-acrylic phenethyl ester-2-pyranone derivatives were synthesized and evaluated against
five tumor cell lines (HeLa, C6, MCF-7, A549, and HSC-2). Most of the new derivatives exhibited moderate
to potent cytotoxic activity. Moreover, HeLa cell lines showed higher sensitivity to these compounds. In
particular, compound 5o showed potent cytotoxic activity (IC50 = 0.50–3.45 μM) against the five cell
lines. Further investigation on the mechanism of action showed that 5o induced apoptosis, arrested the
cell cycle at G2/M phases in HeLa cells, and inhibited migration through disruption of the actin cytoskele-
ton. In addition, ADMET properties were also calculated in silico, and compound 5o showed good ADMET
properties with good absorption, low hepatotoxicity, and good solubility, and thus, could easily be bound
to carrier proteins, without inhibition of CYP2D6. A structure–activity relationship (SAR) analysis indicated
that compounds with ortho-substitution on the benzene ring exhibited obviously increased cytotoxic
potency. This study indicated that compound 5o is a promising compound as an antitumor agent.
Received 4th January 2015,
Accepted 10th March 2015
DOI: 10.1039/c5ob00007f
www.rsc.org/obc
Introduction
Cancer is a major public health problem in many parts of the
world, accounting for 23% of all deaths and was second only
to heart diseases in 2014.1 Among various cancers, lung cancer
is the most common cause of deaths, accounting for more
than one-quarter of all cancer deaths in men and women.
Other cancers, such as prostate in men and breast in women,
are also very common causes of deaths from cancer.2
Caffeic acid phenethyl ester (CAPE) (Fig. 1) is the main con-
stituent of propolis, a resinous substance used in folk medi-
cine for treating various ailments. CAPE was widely reported to
possess anti-inflammatory, antibacterial, antiviral, and anti-
tumor activities.3 Moreover, CAPE selectively inhibited prolifer-
ation of several types of carcinoma cell lines, but showed
almost no toxic effects on normal peripheral blood cells or
Fig. 1 Chemical structure of CAPE.
normal hepatocytes. Some CAPE derivatives showed potent
activity against lung cancer, prostate cancer, melanoma,4
uterine corpus cancer, breast cancer,5 glioma,6 leukemia,7 and
oral cancer.8 Omene and co-authors9 also demonstrated that
CAPE induced cell cycle arrest and apoptosis and inhibited
angiogenesis.
In our continuing studies to modify antitumor natural pro-
ducts for increased potency, we aimed to determine whether
the catechol moiety is essential for the cytotoxic activity of
CAPE and its related derivatives. Our plan was to change the
catechol ring to a lactone ring, specifically a 4-methoxy-2H-
pyran-2-one. Lactone ring systems are found in many bioactive
natural products obtained from plants, animals, marine organ-
isms, bacteria, and insects,10 including those that exhibit
various pharmacological activities, such as HIV protease
inhibitory,11 anticonvulsant,12 antimicrobial,13 antitumor,14,15
and other effects. Examples include styryl-2-pyrone,16 isorum-
brin,17 bufadienolide,18 neo-tanshinlactone,19 wortmannin,20
aResearch Center for Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-sen
University, Guangzhou 510006, People’s Republic of China.
E-mail: junxu@biochemomes.com, guqiong@mail.sysu.edu.cn
bNatural Products Research Laboratories, UNC Eshelman School of Pharmacy,
University of North Carolina, Chapel Hill, North Carolina 27599, USA
cChinese Medicine Research and Development Center, China Medical University and
Hospital, Taichung, Taiwan
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c5ob00007f
‡These authors contributed equally to this work.
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Scheme 1 Synthesis of 5a–u. Reagents and conditions: (a) Me2SO4, K2CO3,
acetone, r.t., 16 h, 84%; (b) SeO2, dioxane, 160 °C, 4 h, 91%; (c) CH2-
(COOH)2, pyridine, piperidine, reflux, 3 h, 47%; (d) phenethyl alcohols or
heterocyclic EtOHs, EDC·HCl, DMAP, CH2Cl2, r.t., overnight, 49%–71%.
Fig. 2 Chemical structures of several natural products with lactone
rings.
was obtained in 47% yield through a Knoevenagel conden-
sation reaction with the reaction temperature set at 110 °C. As
we know, the general condition of the Knoevenagel conden-
sation reaction to afford cinnamic acid derivatives is with
piperidine as the base, pyridine as the solvent, and the reac-
tion temperature is at room temperature. Since the lactone
ring in the target compound was not stable in strong basic
solution, we explored several bases, such as K2CO3, Et3N,
EtONa, and piperidine. As a result, piperidine was found to be
the best. To discover the best solvent, we explored methanol,
ethanol, and pyridine. Pyridine is a commonly used solvent for
Knoevenagel condensation, and we found that it was also the
best solvent to afford our desired compound 4. The reaction
temperature was also optimized. Room temperature, 60 °C,
and reflux temperature were investigated, and we found that
the reflux reaction temperature was the best. So we chose
piperidine as the base, pyridine as the solvent, and the reac-
tion temperature was at reflux. Finally, our desired series-5
compounds were prepared from 4 through an esterification
reaction using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
Fig. 3 Design for the 6-acrylic phenethyl ester-2-pyranone derivatives.
and camptothecin,21 as shown in Fig. 2. Based on numerous
modification studies, such as those performed on campto-
thecin and its derivatives, a lactone ring can be an important
pharmacophore in anticancer drugs.22
Therefore, based on the antitumor activity of CAPE and
various lactone-containing compounds, we combined both
CAPE and styryl-2-pyrone scaffolds as chemical starting points
to design chemically modified CAPE analogues, which contain
variously substituted acrylic phenethyl esters and a 4-methoxy-
2-pyranone moiety (Fig. 3). Subsequently, 21 new CAPE deriva-
tives were synthesized and evaluated against five tumor cell
lines. Preliminary structure–activity relationship (SAR) corre-
lations were also obtained, and the antitumor mechanism of
action was also investigated.
hydrochloride (EDC·HCl) as
a condensing reagent. The
obtained yields were varied from 49% to 71%.
Antitumor activity against five human cancer cell lines
To evaluate the effects of 5-derivatives on the growth of human
cancer cells, the growth inhibitory potential was evaluated
using a MTT assay in five human cancer cells. Initially, 12
compounds (5a–l) were screened at 20 μM. Only compounds
5b and 5e exhibited significant in vitro antitumor activity
(greater than 50% growth inhibition) against the tested
human cancer cell lines at this concentration. Table 1 lists the
Results and discussion
Chemistry
The series-5 target compounds were synthesized according to IC50 values for the active compounds. Interestingly, both 5b
the approaches illustrated in Scheme 1. Compounds 2 and 3 and 5e were substituted at C-2 of the benzene ring, while the
were synthesized according to literature methods.23 Initially, inactive compounds (5a, 5c, 5e, 5f, and 5g) were substituted at
compound 2 was obtained by methylation of commercially the C-3 or C-4 position or contained a heterocyclic rather than
obtained 4-hydroxy-6-methyl-2H-pyran-2-one (1). Then, the a benzene ring (5h–l). While substitution at C-2 sharply
methyl group at C-6 of 2 was oxidized with selenium dioxide in affected the antitumor activity, both chlorine and methyl
1,4-dioxane in a sealed tube at 160 °C to yield 3. The inter- groups had similar impacts (compare 5b vs. 5e). Moreover,
mediate (E)-3-(4-methoxy-2-oxo-2H-pyran-6-yl)acrylic acid (4) both new lactone-containing CAPE-derivatives were more
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Table 1 In vitro cytotoxicity data for 5a–l against five human tumor cell lines
IC50 (μM)
Compound
Structure
CAPE
MCF-7
NAb
C6
HSC-2
NAb
HeLa
NAb
A549
NAb
CAPE
5a
NAb
a
a
a
a
a
—
—
—
—
—
5b
5c
6.56
NAb
1.24
1.14
NAb
a
a
a
a
a
—
—
—
—
—
a
a
a
a
a
5d
5e
—
—
—
—
—
3.13
6.77
0.92
2.31
10.3
a
a
a
a
a
5f
—
—
—
—
—
a
a
a
5g
5h
5i
NAb
—
—
8.27
—
a
a
a
a
a
—
—
—
—
—
a
a
a
a
a
—
—
—
—
—
a
a
a
a
a
5j
—
—
—
—
—
a
a
a
a
a
5k
—
—
—
—
—
a
a
a
a
a
5l
—
—
—
—
—
a Growth inhibition did not reach 50% at 20 μM. b NA = IC50 > 10 μM.
active than CAPE, especially against MCF-7, HSC-2, and HeLa with multiple substituents on the phenyl ring, the 2,4,6-tri-
cancer cell lines. methylated compound (5t) showed significant activity against
On the basis of the preliminary results, nine additional the MCF-7, HSC-2, and HeLa cell lines. A comparison of 5o,
5-derivatives were synthesized to more fully evaluate the effects 5b, and 5s indicated that the former compound with a 2,6-
of substituents at C-2 on the cytotoxicity. Compounds 5m–u disubstituted benzene ring displayed better activity against all
contained –F, –Br, –OCH3, –CF3, and –NO2 substituents, as five tested cell lines than the latter compounds with 2- or 2,4-
well as multiply substituted phenyl rings or a naphthyl ring. substitution. Finally, when the benzene ring was changed to a
The assay results are shown in Table 2. Compounds with a 2-F naphthalene ring (5u), no cytotoxicity was observed. This
or 2-NO2 group (5m and 5r, respectively) were inactive against finding indicated that greater steric bulk could lead to
all cancer cell lines, whereas the derivative with a 2-Br group decreased antitumor activity.
(5n) was active against four cell lines. These results demon-
strated that stronger electron-withdrawing inductive effects against the five tested human tumor cell lines (IC50: 2.61, 3.45,
decreased the antitumor activity. Compounds with 1.19, 0.50, and 1.15 μM). Moreover, the lowest IC50 value for 5o
Among all 21 compounds, 5o showed most potent activity
a
2-methoxy (5p) or 2-trifluoromethyl (5q) group were active only (0.50 μM) was obtained in HeLa cells compared with the other
against the HeLa cell lines, which appeared to have the great- cell lines. The effect of 5o on cell proliferation in HeLa cell
est sensitivity to this compound class. Among the compounds lines was also assayed as shown in Fig. 4. Cells treated with 5o
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Table 2 In vitro cytotoxicity data for 5m–u against five human tumor cell lines
IC50 (μM)
Compound
Structure
MCF-7
C6
HSC-2
HeLa
A549
a
a
a
a
a
5m
—
—
—
—
—
5n
5o
5.01
2.61
NAb
3.45
2.62
1.19
1.47
0.50
7.03
1.15
5p
NAb
NAb
NAb
NAb
5.63
9.13
NAb
a
a
a
5q
5r
5s
5t
—
—
—
a
a
a
a
a
—
—
—
—
—
NAb
7.99
NAb
NAb
9.45
3.52
6.26
1.49
NAb
NAb
a
a
a
a
a
5u
—
—
—
—
—
a Growth inhibition did not reach 50% at 20 μM. b NA = IC50 > 10 μM.
(0.5 and 1 μM) displayed morphological changes and showed results suggested that 5o inhibited cell growth by arresting the
distinctly rounded shapes compared with the control cells cell-cycle at the G2/M phase in a concentration-dependent
(Fig. 4B). This result prompted us to study the mechanism of manner. Furthermore, sub-G0/G1 phase cells were observed
action of 5o in HeLa cell lines.
after 5o exposure (Fig. 5A), which indicated that 5o induced
apoptosis of HeLa cells.
Cell cycle progression is driven by cyclins and cyclin-depen-
dent kinases (Cdks).24 Generally, Cdc25c, Cdc2 kinases and
Cell cycle effects of 5o
Based on previous investigation, cell proliferation is associated CyclinB1 are primarily activated at the G2/M phase.25 The cell
with regulation of three phases (G0/G1, S, and G2/M) of the cycle transition from the G2 phase to the M phase is controlled
cell cycle. To investigate if growth inhibition induced by 5o by Cdc2/CyclinB1 kinase complex activity, which is activated
was associated with regulation of the cell cycle, cycle distri- by Cdc25C. Cdc25C activity is a rate-limiting process for G2/M
bution of HeLa cells with or without 5o treatment was ana- phase transition.24b Subsequent experiments were performed
lyzed by flow cytometry. As shown in Fig. 5B, the untreated to analyze changes in the expression of key G2/M checkpoint
group of HeLa cells had a low proportion of cells in G2/M regulatory proteins using western blot. As shown in Fig. 5D,
(39.17%), while the experimental groups treated with 5o (0.5, cells treated with 5o for 48 h had decreased levels of CyclinB1,
1, and 2 μM) for 24 h showed increased proportions of G2/M mitotic cyclin-dependent kinase Cdc2, and mitotic Cdc25C in
phase cells (51.62%, 68.45%, and 73.82%, respectively). These a dose-dependent manner. These results are consistent with
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cantly increased the level of cleaved caspase-3 (active form of
caspase-3) in a dose-dependent manner compared with the
control group. At the same time, total full-length PARP
(116 kDa) was cleaved into a large fragment (89 kDa), and
cleaved PARP also increased remarkably in a dose-dependent
manner. These findings corresponded with the activation of
caspase-3 and the data demonstrated that 5o induced HeLa
cell apoptosis through activation of the caspase-mediated
pathway.
5o Induced morphological changes by actin cytoskeleton
disruption
As shown in Fig. 4B, significant changes in cell morphology
were observed. HeLa cells treated with 5o (0.5 and 1 μM) for
24 h exhibited a distinctly round shape and detachment com-
pared to the untreated cells. Cell morphology and adhesion
are usually associated with the actin cytoskeleton.31 Therefore,
immunofluorescence microscopy was used to explore the
effect of 5o on the actin cytoskeleton. In the control group,
actin was diffusely distributed, whereas in the 5o-treated group
(0.5 μM), actin was locally clustered. When HeLa cells were
treated with 1 μM of 5o, actin aggregation was enhanced and
actin disruption was also generated (Fig. 7). In addition, apop-
totic bodies (denoted by red mark), which are consistent with
apoptosis-induced conclusion, were observed in HeLa cells
treated with 1 μM of 5o. These results suggested that 5o
induced actin aggregation and further disrupted the actin
Fig. 4 The inhibitory effects of 5o on HeLa cell growth. (A) Cells were
treated or untreated with increasing concentrations (0.5–50 μM) of 5o
for 48 h. (B) Effects on cell morphology after treatment with 5o
(0.5, 1 μM) under a phase contrast microscope at 100× magnification.
***Significant difference compared to the control group (P < 0.001).
G2/M arrest and demonstrated that 5o inhibited the prolifer-
ation of HeLa cells by G2/M-phase arrest.
Induction of apoptosis by 5o in HeLa cell lines
As a natural process of programmed cell death (PCD), apopto- cytoskeleton. Because a disequilibrium of the actin dynamics
sis plays an important role in cell clearance. Based on current between G-actin and F-actin can result in actin aggregation
studies, extensive apoptosis is observed in regressing tumor and influence cell shape and cell adhesion,32 we speculated
cells and also in those cells treated with chemotherapeutic that 5o induced actin aggregation and actin cytoskeleton dis-
agents.26 There is a lot of evidence to show that apoptosis is ruption through disturbing the actin dynamic equilibrium.
the most crucial and renowned mechanism for clearance of Actin dynamics is essential for cell division. G2/M-phase block-
tumor cells.27 Apoptosis tolerance may result in treatment age of 5o may also contribute to the abnormal actin dynamics,
resistance.28 In the previous analysis of the 5o’s effect on the but the detailed mechanism should be further confirmed.
cell cycle, a sub-G0/G1 peak was observed (Fig. 5A). In order to
Inhibition of cell migration and invasion
confirm that the sub-G0/G1 peak was caused by apoptosis
rather than by cell debris, quantitative apoptotic analysis was Malignant tumor invasion and metastasis have been hot and
performed by using an Annexin V-FITC/PI Apoptosis Detection difficult research topics in oncology. Tumor cells use their
Kit (Becton Dickinson, USA). As shown in Fig. 6A, in HeLa intrinsic migratory ability to invade adjacent tissues and the
cells treated with 5o at indicated concentrations for 24 h, the vasculature and, ultimately, to metastasize.33 Migration and
percentages of early/late apoptotic cells were 7.52%/1.66%, invasion of cancer cells are dependent on the actin cytoskele-
7.18%/2.25%, and 10.2%/2.50% compared to control (1.05%/ ton,34 which are driven by the polymerization of actin within
0%). These results indicated that 5o induced HeLa cell apopto- two distinct structures, lamellipodia and filopodia, and attach-
sis, especially early apoptosis.
ment to the extracellular matrix through actin-rich adhesive
To further confirm that 5o induced apoptosis, the structures.35 In previous confocal assays, we observed that 5o
expressions of key apoptosis-related proteins, such as caspase- disturbed the actin dynamics and disrupted the actin cytos-
3 and PARP, were detected by western blot assay. Caspase pro- keleton. Subsequently, we conducted invasion and migration
teins play a central role in the execution-phase of cell apopto- assays to explore whether 5o influenced the migration and
sis. Among caspase proteins, caspase-3 is a critical executioner invasion abilities of HeLa cells. In Fig. 8, HeLa cells with or
of apoptosis. Caspase-3 can be activated both by extrinsic without treatment of different doses of 5o were stained by
(death ligand) and intrinsic (mitochondrial) apoptotic path- crystal violet. As shown in Fig. 8, in a Matrigel-coated (for
ways.29 Caspase-3 activation is dominant and reflected in the migration ability) or -noncoated (for invasion ability) Transwell
cleavage of PARP, which is a substrate of caspase-3.30 Cleavage assay, the numbers of HeLa cells passing through the filter
of PARP results in DNA repair inhibition and DNA degra- were significantly decreased in response to increasing concen-
dation. As shown in Fig. 6B, treatment with 5o for 48 h signifi- trations of 5o. These results demonstrated that 5o could mark-
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Fig. 5 5o Induced G2/M arrest by modulating the expression of cell cycle-related proteins in the G2/M phase. (A) Cells were treated with 5o (0, 0.5,
1 and 2 μM) for 24 h, and stained with PI and RNase A, then analyzed by flow cytometry to determine sub-G1 and cell-cycle phases. (B) Distribution
of cells (%) treated with 5o in cell-cycle phases (sub-G0/G1, G0/G1, S, and G2/M). (C) The histogram analysis of (B). (D) After 5o (0, 0.5, 1 and 2 μM)
treatment for 48 h, G2/M phase related protein (cyclin B1, Cdc2, and Cdc25C) levels were detected by western blot.
edly inhibit both migration and invasion abilities of HeLa barrier (BBB) penetration and decreased liver toxicity were cal-
cells.
culated for 5o in comparison with CAPE. In addition, calcu-
lations indicated that 5o had good absorption and solubility
and could easily be bound to carrier proteins in the blood,
while showing no inhibition of CYP2D6. However, 5o should
still be evaluated experimentally and further optimized regard-
ing its pharmacokinetics properties.
Preliminary ADMET study
To investigate the drug-like profiles of the synthesized com-
pounds, we subjected several 5-series compounds for prelimi-
nary ADMET prediction (Table 3). Improved blood–brain
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Fig. 8 Compound 5o inhibited HeLa cell line migration and invasion in
a Transwell assay. Invasion potential of HeLa cells passing through
Matrigel-coated Transwell after treatment with the indicated concen-
tration of 5o for 24 h. Migration potential is without Matrigel. These
images show the number of HeLa cells passing through the filter with/
without Matrigel. As the 5o dose increased, the number of cells on the
undersurface of the filter decreased.
Conclusion
In summary, the acrylic phenethyl ester portion of CAPE was
combined with the 4-methoxy-2H-pyran-2-one of styryl-2-
pyrone, and a series of 6-acrylic phenethyl ester-2-pyranone
derivatives were designed and synthesized. Among the new
compounds, 5o exhibited significantly more anti-proliferation
Fig. 6 5o Induces apoptosis via a caspase-dependent pathway. (A)
HeLa cell lines were treated with 5o (0, 0.5, 1 and 2 μM) for 24 h, and
stained with Annexin-V FITC/PI, then analyzed by flow cytometry to activity than CAPE against five human cancer cell lines. Par-
evaluate apoptosis. (B) Western blot analysis of apoptosis-related
protein (caspase-3 and PARP) levels in HeLa cell lines exposed to 5o (0,
0.5, 1 and 2 μM) for 48 h.
ticular efficacy was observed against the HeLa cell lines (IC50
=
0.50 μM). Flow cytometric analysis of the cell cycle demon-
strated an increased percentage of G2/M phase cells in 5o-
treated cells. More importantly, 5o induced HeLa cell apopto-
sis via a caspase-3-dependent manner. Moreover, further study
indicated that 5o influenced invasion and migration in HeLa
cells by disrupting the actin cytoskeleton. In addition, based
on in silico ADMET predictions, compound 5o is likely to
demonstrate good ADMET properties with low hepatotoxicity.
These findings suggest that 5o is a promising compound for
further study to generate a clinical trial candidate for the treat-
ment of cancer.
Experimental
Synthesis
Materials and equipment. Reagents were used without
further purification unless otherwise specified. Solvents were
dried and redistilled prior to use by usual methods. The 1H
NMR and 13C NMR spectra were recorded using TMS as the
internal standard on a Bruker BioSpin GmbH spectrometer
(Avance III, Switzerland) at 400 MHz and 100 MHz. Coupling
constants are given in Hz. High-resolution mass spectra were
obtained using a Shimadzu LCMS-IT-TOF mass spectrometer.
Flash column chromatography was performed using silica gel
(200–300 mesh) purchased from Qingdao Haiyang Chemical
Co., Ltd or alumina from Sinopharm Chemical Reagent Co.,
Fig. 7 Immunofluorescence microscopy analysis of the actin cytoskele-
ton in HeLa cell lines with 5o treatment. Cells were either untreated or
treated with 5o (0.5, 1 μM) for 24 h, and then immunofluorescence assay
was conducted as described in the Experimental section. The β-actin
(green) was revealed by single-immunofluorescence staining. Nuclei
(blue) were stained by using Hoechst 33342. Scale bar, 20 μM.
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Table 3 The parameters of ADMET descriptors of 5 series
Compound
BBBa
Absorptionb
Solubilityc
Hepatotoxicityd
CYP2D6e
PPBf
5b
5e
5g
5n
5o
5p
5q
5s
2
2
2
2
1
2
2
1
1
2
0
0
0
0
0
0
0
0
0
0
3
3
3
3
2
3
2
2
2
3
0
1
1
1
0
1
1
1
0
1
0
0
0
0
0
0
0
0
0
0
2
2
2
2
2
2
1
2
1
2
5t
CAPE
a BBB is blood–brain barrier penetration and predicts blood–brain penetration after oral administration. 1 (high), 2 (medium). b Absorption
predicts human intestinal absorption (HIA) after oral administration. 0 (good). c Solubility predicts the solubility of each compound in water at
25 °C. 2 (low), and 3 (good). d Hepatotoxicity predicts potential liver toxicity of compounds. 0 (Nontoxic), 1 (toxic). e CYP2D6 predicts CYP2D6
enzyme inhibition using the 2D chemical structure. 0 (Non-inhibitor). f PPB predicts whether a compound is likely to be highly bound to carrier
proteins in the blood. 1 (Binding ability >90%), 2 (binding ability >95%).
Ltd. All reactions were monitored by thin layer chromato- The crude product was purified by flash chromatography
graphy using silica gel.
(MeOH–dichloromethane = 3/50) to afford the acrylic acid (4)
(1.84 g, 47%) as a yellow solid. H NMR (400 MHz, DMSO): δH
1
Synthesis of 4-methoxy-6-methyl-2H-pyran-2-one (2)23
7.23 (1H, dd, J = 15.7, 2.6 Hz), 6.71 (1H, s), 6.41 (1H, dd, J =
15.7, 2.8 Hz), 5.79 (1H, s), 3.84 (3H, s); 13C NMR (100 MHz,
DMSO): δC 169.9, 166.3, 162.0, 155.3, 133.9, 124.1, 106.3, 91.2,
56.6; MS (ESI): m/z 195.0 [M − H]−; HRMS (ESI): m/z calcd for
C9H8O5 [M − H]−, 195.0299; found, 195.0293.
To a suspension of K2CO3 (62.4 g, 452.4 mmol) in anhydrous
acetone (200 mL) under nitrogen was added Me2SO4 (9.8 mL,
103.2 mmol). Then, the substrate 4-hydroxy-6-methyl-2H-
pyran-2-one (1) (10.0 g, 79.36 mmol) was added in one portion.
The reaction mixture was stirred at room temperature for 16 h.
Then, the system was filtered, the obtained solid was washed
with acetone, and the solvent was removed under reduced
pressure. After purification by flash chromatography (EtOAc–
General procedure for the preparation of (E)-phenethyl-3-(4-
methoxy-2-oxo-2H-pyran-6-yl)acrylate and its derivatives
(5a–u)37
petroleum ether
= 1/4) and recrystallization of EtOAc– To a stirred solution of acrylic acid (4) (0.3 mmol) in CH2Cl2
petroleum ether, the methylated product 4-methoxy-6-methyl- (3 mL) were added EDC·HCl (1.2 eq.), DMAP (0.1 eq.), and the
1
2H-pyran-2-one (9.3 g, 84%) was obtained as a white solid. H corresponding phenethyl alcohol (1.2 eq.), and the resulting
NMR (400 MHz, CDCl3): δH 5.74 (1H, d, J = 2.0 Hz), 5.37 (1H, mixture was stirred at room temperature overnight. The
d, J = 2.0 Hz), 3.75 (3H, s), 2.17 (3H, s).
solvent was removed under reduced pressure, and the residue
was purified by flash chromatography (EtOAc–petroleum ether
= 1/3) to give the corresponding ester (5a–u).
Synthesis of 4-methoxy-2-oxo-2H-pyran-6-carbaldehyde (3)23
Under nitrogen, 4-methoxy-6-methyl-2H-pyran-2-one (2) (5.6 g,
40 mmol) and SeO2 (22 g, 200.0 mmol) were dissolved in dry
dioxane (150 mL), inside a sealed tube. The mixture was
Synthesis of (E)-phenethyl-3-(4-methoxy-2-oxo-2H-pyran-6-yl)-
acrylate (5a)
heated to 160 °C for 4 h. Then, the reaction was cooled to Following the general procedure, 5a was obtained as a white
1
room temperature and filtered, and the filter cake was washed solid (54.1 mg, 60%). H NMR (400 MHz, CDCl3): δH 7.22–7.33
with EtOAc (30 mL). The crude product was purified by flash (5H, m), 7.06 (1H, d, J = 15.6 Hz), 6.70 (1H, d, J = 15.5 Hz), 6.10
chromatography (EtOAc–petroleum ether = 1/1) to afford the (1H, d, J = 2.0 Hz), 5.58 (1H, d, J = 2.1 Hz), 4.41 (2H, t, J = 6.9
aldehyde (3) (5.6 g, 91%) as a yellowish solid. 1H NMR Hz), 3.83 (3H, s), 2.99 (2H, t, J = 6.9 Hz); 13C NMR (100 MHz,
(400 MHz, CDCl3): δH 9.51 (1H, s), 6.67 (1H, s), 5.74 (1H, s), CDCl3): δC 169.9, 165.6, 162.8, 155.6, 137.6, 133.8, 128.9, 128.5,
3.86 (3H, s).
126.6, 124.5, 106.1, 91.3, 65.6, 56.2, 35.0; MS (ESI): m/z 301.1
[M + H]+; HRMS (ESI): m/z calcd for C17H16O5Na [M + Na]+,
323.0890; found, 323.0921.
Synthesis of (E)-3-(4-methoxy-2-oxo-2H-pyran-6-yl)acrylic acid
(4)36
Synthesis of (E)-2-chlorophenethyl-3-(4-methoxy-2-oxo-2H-
pyran-6-yl)acrylate (5b)
A solution of aldehyde (3) (3.08 g, 20.0 mmol), malonic acid
(2.08 g, 20.0 mmol) in pyridine (10 ml, 120 mmol) and piper-
idine (0.1 ml) was heated to reflux for 3 h. The resulting solu- Following the general procedure, 5b was obtained as a white
1
tion was poured into 2 M aq. HCl and cooled to room solid (68.1 mg, 68%). H NMR (400 MHz, CDCl3): δH 7.37 (1H,
temperature. The solid was filtered and washed with water. dd, J = 7.2, 1.9 Hz), 7.28–7.18 (3H, m), 7.07 (1H, d, J = 15.6 Hz),
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6.69 (1H, d, J = 15.5 Hz), 6.10 (1H, d, J = 2.1 Hz), 5.58 (1H, d, Synthesis of (E)-4-methoxyphenethyl-3-(4-methoxy-2-oxo-2H-
J = 2.2 Hz), 4.44 (2H, t, J = 6.8 Hz), 3.84 (3H, s), 3.14 (2H, t, J = pyran-6-yl)acrylate (5g)
6.8 Hz); 13C NMR (100 MHz, CDCl3): δC 169.9, 165.6, 162.8,
Following the general procedure, 5g was obtained as a white
solid (63.9 mg, 71%). H NMR (400 MHz, CDCl3): δH 7.15 (2H,
155.6, 135.3, 134.2, 133.9, 131.2, 129.6, 128.2, 126.9, 124.4,
106.2, 91.4, 63.9, 56.2, 32.8; MS (ESI): m/z 335.1 [M + H]+;
HRMS (ESI): m/z calcd for C17H15ClO5Na [M + Na]+, 357.0500;
found, 357.0522.
1
d, J = 8.6 Hz), 7.06 (1H, d, J = 15.6 Hz), 6.86 (2H, d, J = 8.6 Hz),
6.71 (1H, d, J = 15.5 Hz), 6.10 (1H, d, J = 2.1 Hz), 5.58 (1H, d,
J = 2.1 Hz), 4.37 (2H, t, J = 6.9 Hz), 3.84 (3H, s), 3.80 (3H, s),
2.93 (2H, t, J = 6.9 Hz); 13C NMR (100 MHz, CDCl3): δC 170.0,
165.6, 162.8, 158.4, 155.7, 133.8, 129.9, 129.6, 124.6, 114.0,
106.1, 91.3, 65.8, 56.2, 55.2, 34.2; MS (ESI): m/z 353.2 [M + Na]+;
HRMS (ESI): m/z calcd for C18H18O6Na [M + Na]+, 353.0996;
found, 353.1026.
Synthesis of (E)-3-chlorophenethyl-3-(4-methoxy-2-oxo-2H-
pyran-6-yl)acrylate (5c)
Following the general procedure, 5c was obtained as a white
1
solid (52.1 mg, 52%). H NMR (400 MHz, CDCl3): δH 7.28–7.20
(3H, m), 7.12 (1H, dt, J = 6.7, 1.7 Hz), 7.06 (1H, d, J = 15.6 Hz),
6.69 (1H, d, J = 15.5 Hz), 6.12 (1H, d, J = 2.1 Hz), 5.59 (1H, d,
J = 2.2 Hz), 4.40 (2H, t, J = 6.8 Hz), 3.84 (3H, s), 2.97 (2H, t, J =
6.8 Hz); 13C NMR (100 MHz, CDCl3): δC 169.9, 165.5, 162.8,
155.6, 139.7, 134.0, 134.0, 129.8, 129.0, 127.1, 126.9, 124.3,
106.3, 91.4, 65.1, 56.2, 34.7; MS (ESI): m/z 335.1 [M + H]+;
HRMS (ESI): m/z calcd for C17H15ClO5Na [M + Na]+, 357.0500;
found, 357.0529.
Synthesis of (E)-2-(piperidin-1-yl)ethyl-3-(4-methoxy-2-oxo-2H-
pyran-6-yl)acrylate (5h)
Following the general procedure, 5h was obtained as a white
1
solid (50.0 mg, 51%). H NMR (400 MHz, CDCl3): δH 7.10 (1H,
d, J = 15.6 Hz), 6.74 (1H, d, J = 15.6 Hz), 6.12 (1H, d, J = 2.1
Hz), 5.58 (1H, d, J = 2.2 Hz), 4.33 (2H, t, J = 6.0 Hz), 3.84 (3H,
s), 2.67 (2H, t, J = 6.0 Hz), 2.47(4H, t, J = 4.9 Hz), 1.59 (4H, dt,
J = 11.1, 5.6 Hz), 1.44 (2H, dt, J = 10.3, 4.7 Hz); 13C NMR
(100 MHz, CDCl3): δC 169.9, 165.6, 162.8, 155.7, 133.8, 124.6,
106.1, 91.3, 62.9, 57.2, 56.1, 54.8, 25.9, 24.1; MS (ESI): m/z
308.1 [M + H]+.
Synthesis of (E)-4-chlorophenethyl-3-(4-methoxy-2-oxo-2H-
pyran-6-yl)acrylate (5d)
Following the general procedure, 5d was obtained as a white
1
solid (65.1 mg, 65%). H NMR (400 MHz, CDCl3): δH 7.28 (2H,
d, J = 8.4 Hz), 7.17 (2H, d, J = 8.3 Hz), 7.06 (1H, d, J = 15.5 Hz),
6.69 (1H, d, J = 15.5 Hz), 6.11 (1H, d, J = 2.0 Hz), 5.59 (1H, d,
J = 2.1 Hz), 4.38 (2H, t, J = 6.8 Hz), 3.84 (3H, s), 2.96 (2H, t, J =
6.8 Hz); 13C NMR (100 MHz, CDCl3): δC 169.9, 165.5, 162.8,
155.6, 136.1, 134.0, 132.5, 130.2, 128.7, 124.3, 106.2, 91.4, 65.2,
56.2, 34.4; MS (ESI): m/z 335.0 [M + H]+; HRMS (ESI): m/z calcd
for C17H15ClO5Na [M + Na]+, 357.0500; found, 357.0519.
Synthesis of (E)-2-(pyridin-2-yl)ethyl-3-(4-methoxy-2-oxo-2H-
pyran-6-yl)acrylate (5i)
Following the general procedure, 5i was obtained as a white
1
solid (45.0 mg, 50%). H NMR (400 MHz, CDCl3): δH 8.56 (1H,
d, J = 4.8 Hz), 7.64 (1H, td, J = 7.7, 1.8 Hz), 7.24–7.12 (2H, m),
7.06 (1H, d, J = 15.6 Hz), 6.67 (1H, d, J = 15.5 Hz), 6.10 (1H, d,
J = 2.1 Hz), 5.58 (1H, d, J = 2.1 Hz), 4.60 (2H, t, J = 6.6 Hz), 3.83
(3H, s), 3.17 (2H, t, J = 6.6 Hz); 13C NMR (100 MHz, CDCl3):
δC 170.0, 165.6, 162.8, 157.8, 155.6, 149.5, 136.5, 133.8, 124.4,
123.5, 121.7, 106.1, 91.3, 64.2, 56.2, 37.3. MS (ESI): m/z 302.1
[M + H]+; HRMS (ESI): m/z calcd for C16H21NO5Na [M + Na]+,
324.0842; found, 324.0860.
Synthesis of (E)-2-methylphenethyl-3-(4-methoxy-2-oxo-2H-
pyran-6-yl)acrylate (5e)
Following the general procedure, 5e was obtained as a white
1
solid (51.8 mg, 55%). H NMR (400 MHz, CDCl3): δH 7.20–7.12
(4H, m), 7.07 (1H, d, J = 15.6 Hz), 6.70 (1H, d, J = 15.5 Hz), 6.10
(1H, d, J = 2.1 Hz), 5.58 (1H, d, J = 2.2 Hz), 4.38 (2H, t, J = 7.2
Hz), 3.83 (3H, s), 3.00 (2H, t, J = 7.2 Hz), 2.36 (3H, s); 13C NMR
(100 MHz, CDCl3): δC 169.9, 165.6, 162.8, 155.7, 136.4, 135.6,
133.8, 130.4, 129.6, 126.8, 126.1, 124.5, 106.1, 91.4, 64.7, 56.1,
32.3, 19.3; MS (ESI): m/z 315.1 [M + H]+; HRMS (ESI): m/z calcd
for C18H18O5Na [M + Na]+, 337.1046; found, 337.1062.
Synthesis of (E)-2-morpholinoethyl-3-(4-methoxy-2-oxo-2H-
pyran-6-yl)acrylate (5j)
Following the general procedure, 5j was obtained as a white
1
solid (52.9 mg, 57%). H NMR (400 MHz, CDCl3): δH 7.10 (1H,
d, J = 15.5 Hz), 6.74 (1H, d, J = 15.5 Hz), 6.13 (1H, d, J = 2.1
Hz), 5.59 (1H, d, J = 2.2 Hz), 4.34 (2H, t, J = 5.7 Hz), 3.84 (3H,
s), 3.72 (4H, t, J = 4.6 Hz), 2.70 (2H, t, J = 5.7 Hz), 2.54 (4H, t,
J = 4.6 Hz); 13C NMR (100 MHz, CDCl3): δC 169.9, 165.6, 162.7,
155.6, 134.0, 124.4, 106.2, 91.4, 66.8, 62.3, 57.0, 56.2, 53.8; MS
(ESI): m/z 310.2 [M + H]+; HRMS (ESI): m/z calcd for C15H19NO6
[M + H]+, 310.1285; found, 310.1281.
Synthesis of (E)-3-methylphenethyl-3-(4-methoxy-2-oxo-2H-
pyran-6-yl)acrylate (5f)
Following the general procedure, 5f was obtained as a white
1
solid (52.8 mg, 56%). H NMR (400 MHz, CDCl3): δH 7.20 (1H,
t, J = 7.8 Hz), 7.02–7.08 (4H, m), 6.71 (1H, d, J = 15.5 Hz), 6.10
(1H, d, J = 2.1 Hz), 5.58 (1H, d, J = 2.2 Hz), 4.40 (2H, t, J = 7.0
Hz), 3.83 (3H, s), 2.95 (2H, t, J = 7.0 Hz), 2.34 (3H, s); 13C NMR
(100 MHz, CDCl3): δC 169.9, 165.6, 162.8, 155.7, 138.1, 137.5,
Synthesis of (E)-2-(thiophen-2-yl)ethyl-3-(4-methoxy-2-oxo-2H-
pyran-6-yl)acrylate (5k)
133.8, 129.7, 128.4, 127.4, 125.9, 124.6, 106.1, 91.3, 65.7, 56.1, Following the general procedure, 5k was obtained as a white
1
35.0, 21.3; MS (ESI): m/z 315.2 [M + H]+; HRMS (ESI): m/z calcd solid (63.4 mg, 69%). H NMR (400 MHz, CDCl3): δH 7.17 (1H,
for C18H18O5Na [M + Na]+, 337.1046; found, 337.1069.
dd, J = 5.1, 1.1 Hz), 7.10 (1H, d, J = 15.6 Hz), 6.95 (1H, dd, J =
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5.1, 3.5 Hz), 6.88 (1H, dd, J = 3.4, 0.9 Hz), 6.73 (1H, d, J = 15.5 56.2, 30.4; MS (ESI): m/z 391.0 [M + Na]+; HRMS (ESI): m/z
Hz), 6.12 (1H, d, J = 2.1 Hz), 5.59 (1H, d, J = 2.1 Hz), 4.42 (2H, calcd for C17H14Cl2O5Na [M + Na]+, 391.0111; found, 391.0131.
t, J = 6.7 Hz), 3.84 (3H, s), 3.21 (2H, t, J = 6.6 Hz); 13C NMR
(100 MHz, CDCl3): δC 169.9, 165.5, 162.7, 155.6, 139.6, 134.0,
Synthesis of (E)-2-methoxyphenethyl-3-(4-methoxy-2-oxo-2H-
pyran-6-yl)acrylate (5p)
126.9, 125.6, 124.4, 124.1, 106.2, 91.4, 65.2, 56.1, 29.2; MS
(ESI): m/z 307.0 [M + H]+; HRMS (ESI): m/z calcd for C15H14O5
SNa [M + Na]+, 329.0454; found, 329.0465.
Following the general procedure, 5p was obtained as a white
1
solid (68.3 mg, 69%). H NMR (400 MHz, CDCl3): δH 7.25–7.14
Synthesis of (E)-2-(4-methylthiazol-5-yl)ethyl-3-(4-methoxy-2-
oxo-2H-pyran-6-yl)acrylate (5l)
(2H, m), 7.05 (1H, d, J = 15.6 Hz), 6.92–6.83 (2H, m), 6.69 (1H,
d, J = 15.6 Hz), 6.09 (1H, d, J = 2.1 Hz), 5.58 (1H, d, J = 2.1 Hz),
4.39 (2H, t, J = 6.9 Hz), 3.82 (6H, s), 3.01 (2H, t, J = 6.9 Hz); 13
C
Following the general procedure, 5l was obtained as a white
1
NMR (100 MHz, CDCl3): δC 170.0, 165.7, 162.9, 157.6, 155.7,
133.6, 130.8, 128.0, 125.8, 124.7, 120.4, 110.3, 106.0, 91.3, 64.5,
56.1, 55.2, 29.9; MS (ESI): m/z 331.2 [M + H]+; HRMS (ESI): m/z
calcd for C18H18O6Na [M + Na]+, 353.0996; found, 353.1017.
solid (60.7 mg, 63%). H NMR (400 MHz, CDCl3): δH 8.60 (1H,
s), 7.10 (1H, d, J = 15.5 Hz), 6.71 (1H, d, J = 15.5 Hz), 6.13 (1H,
d, J = 2.1 Hz), 5.60 (1H, d, J = 2.1 Hz), 4.37 (2H, t, J = 6.6 Hz),
3.84 (3H, s), 3.16 (2H, t, J = 6.6 Hz), 2.43 (3H, s); 13C NMR
(100 MHz, CDCl3): δC 169.9, 165.4, 162.7, 155.5, 150.0, 149.9,
134.3, 126.4, 124.0, 106.4, 91.5, 64.6, 56.2, 25.8, 14.9; MS (ESI):
m/z 344.1 [M + Na]+; HRMS (ESI): m/z calcd for C15H15NO5SNa
[M + Na]+, 344.0563; found, 344.0581.
Synthesis of (E)-2-(trifluoromethyl)phenethyl-3-(4-methoxy-2-
oxo-2H-pyran-6-yl)acrylate (5q)
Following the general procedure, 5q was obtained as a white
1
solid (56.3 mg, 51%). H NMR (400 MHz, CDCl3): δH 7.66 (1H,
Synthesis of (E)-2-fluorophenethyl-3-(4-methoxy-2-oxo-2H-
d, J = 7.8 Hz), 7.51 (1H, t, J = 7.4 Hz), 7.43–7.32 (2H, m), 7.08
(1H, d, J = 15.6 Hz), 6.70 (1H, d, J = 15.6 Hz), 6.12 (1H, d, J =
2.0 Hz), 5.59 (1H, d, J = 2.1 Hz), 4.43 (2H, t, J = 6.8 Hz), 3.84
(3H, s), 3.19 (2H, t, J = 6.7 Hz); 13C NMR (100 MHz, CDCl3): δC
169.9, 165.5, 162.8, 155.6, 136.1, 134.0, 131.8, 131.8, 128.8,
126.9, 126.2 (q, J = 5.6 Hz, 11.1 Hz), 124.3, 106.2, 91.4, 64.9,
56.2, 31.8; MS (ESI): m/z 369.2 [M + H]+; HRMS (ESI): m/z calcd
for C18H15F3O5Na [M + Na]+, 391.0764; found, 391.0798.
pyran-6-yl)acrylate (5m)
Following the general procedure, 5m was obtained as a white
1
solid (60.1 mg, 63%). H NMR (400 MHz, CDCl3): δH 7.26–7.18
(2H, m), 7.12–6.99 (3H, m), 6.68 (1H, d, J = 15.5 Hz), 6.11 (1H,
d, J = 2.1 Hz), 5.59 (1H, d, J = 2.1 Hz), 4.41 (2H, t, J = 6.8 Hz),
3.83 (3H, s), 3.04 (2H, t, J = 6.7 Hz); 13C NMR (100 MHz, CDCl3
): δC 169.9, 165.5, 162.7 (d, J = 33.6 Hz), 160.0, 155.6, 133.9,
131.2 (d, J = 4.5 Hz), 128.5 (d, J = 8.2 Hz), 124.5 (d, J = 15.8 Hz),
124.4, 124.1 (d, J = 3.5 Hz), 115.3 (d, J = 21.8 Hz), 106.2, 91.3,
64.3, 56.2, 28.5; MS (ESI): m/z 319.1 [M + H]+; HRMS (ESI): m/z
calcd for C17H15FO5Na [M + Na]+, 341.0796; found, 341.0825.
Synthesis of (E)-2-nitrophenethyl-3-(4-methoxy-2-oxo-2H-pyran-
6-yl)acrylate (5r)
Following the general procedure, 5r was obtained as a white
solid (72.5 mg, 70%). H NMR (400 MHz, CDCl3): δH 7.97 (1H,
Synthesis of (E)-2-bromophenethyl-3-(4-methoxy-2-oxo-2H-
pyran-6-yl)acrylate (5n)
1
d, J = 8.16 Hz), 7.58 (1H, td, J = 7.5, 1.1 Hz), 7.46–7.39 (2H, m),
7.06 (1H, d, J = 15.6 Hz), 6.66 (1H, d, J = 15.6 Hz), 6.14 (1H, d,
J = 2.0 Hz), 5.59 (1H, d, J = 2.1 Hz), 4.51 (2H, t, J = 6.4 Hz), 3.84
(3H, s), 3.32 (2H, t, J = 6.3 Hz); 13C NMR (100 MHz, CDCl3): δC
170.0, 165.4, 162.8, 155.5, 149.6, 134.1, 133.1, 132.8, 132.8,
128.0, 125.0, 124.0, 106.3, 91.4, 64.5, 56.2, 32.3; MS (ESI): m/z
346.2 [M + H]+; HRMS (ESI): m/z calcd for C17H15NO7Na
[M + Na]+, 368.0741; found, 368.0772.
Following the general procedure, 5n was obtained as a white
solid (77.1 mg, 68%). H NMR (400 MHz, CDCl3): δH 7.55 (1H,
1
d, J = 7.9 Hz), 7.27 (2H, d, J = 4.3 Hz), 7.14–7.05 (2H, m), 6.69
(1H, d, J = 15.6 Hz), 6.11 (1H, d, J = 2.1 Hz), 5.59 (1H, d, J = 2.1
Hz), 4.44 (2H, t, J = 6.8 Hz), 3.83 (3H, s), 3.15 (2H, t, J = 6.8
ĆHz); 13C NMR (100 MHz, CDCl3): δC 169.9, 165.5, 162.8,
155.6, 137.0, 133.9, 132.9, 131.2, 128.5, 127.6, 124.6, 124.3,
106.2, 91.4, 64.0, 56.2, 35.2; MS (ESI): m/z 379.1 [M + H]+;
HRMS (ESI): m/z calcd for C17H15BrO5Na [M + Na]+, 400.9995;
found, 401.0025.
Synthesis of (E)-2,4-dichlorophenethyl-3-(4-methoxy-2-oxo-2H-
pyran-6-yl)acrylate (5s)
Synthesis of (E)-2,6-dichlorophenethyl-3-(4-methoxy-2-oxo-2H-
pyran-6-yl)acrylate (5o)
Following the general procedure, 5s was obtained as a white
solid (71.8 mg, 65%). H NMR (400 MHz, CDCl3): δH 7.39 (1H,
1
Following the general procedure, 5o was obtained as a white s), 7.20 (2H, d, J = 1.4 Hz), 7.07 (1H, d, J = 15.5 Hz), 6.67 (1H,
1
solid (69.6 mg, 63%). H NMR (400 MHz, CDCl3): δH 7.31 (2H, d, J = 15.5 Hz), 6.12 (1H, d, J = 2.1 Hz), 5.59 (1H, d, J = 2.2 Hz),
d, J = 8.0 Hz), 7.14 (1H, d, J = 7.9 Hz), 7.09 (1H, d, J = 15.7 Hz), 4.41 (2H, t, J = 6.7 Hz), 3.84 (3H, s), 3.10 (2H, t, J = 6.7 Hz); 13
C
6.67 (1H, d, J = 15.5 Hz), 6.11 (1H, d, J = 2.0 Hz), 5.59 (1H, d, J NMR (100 MHz, CDCl3): δC 169.9, 165.5, 162.8, 155.5, 134.8,
= 2.1 Hz), 4.45 (2H, t, J = 6.8 Hz), 3.84 (3H, s), 3.37 (2H, t, J = 134.0, 133.9, 133.3, 131.9, 129.4, 127.2, 124.1, 106.3, 91.4, 63.6,
6.8 Hz); 13C NMR (100 MHz, CDCl3): δC 169.9, 165.5, 162.8, 56.2, 32.2; MS (ESI): m/z 369.0 [M + H]+; HRMS (ESI): m/z calcd
155.7, 135.9, 133.9, 133.6, 128.5, 128.3, 124.4, 106.2, 91.3, 62.6, for C17H14Cl2O5Na [M + Na]+, 391.0111; found, 391.0143.
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Synthesis of (E)-2,4,6-trimethylphenethyl-3-(4-methoxy-2-oxo-
2H-pyran-6-yl)acrylate (5t)
various concentrations (0.5, 1, 2 μM). Cells were harvested with
trypsin and fixed with ice-cold 70% EtOH at 4 °C overnight.
EtOH was washed with ice-cold PBS buffer. Finally, samples
were stained with a Cell Cycle and Apoptosis Analysis Kit
(Beyotime, China) for 30 min at 37 °C. The DNA contents of
11 000 events were measured by flow cytometry (Beckman
Coulter, Epics XL, USA).
Following the general procedure, 5t was obtained as a white
1
solid (50.3 mg, 49%). H NMR (400 MHz, CDCl3): δH 7.09 (1H,
d, J = 15.5 Hz), 6.85 (2H, s), 6.70 (1H, d, J = 15.5 Hz), 6.11 (1H,
d, J = 2.0 Hz), 5.59 (1H, d, J = 2.1 Hz), 4.27 (2H, t, J = 7.7 Hz),
3.84 (3H, s), 3.02 (2H t, J = 7.7 Hz), 2.34 (6H, s), 2.25 (3H, s); 13
C NMR (100 MHz, CDCl3): δC 169.9, 165.7, 162.8, 155.7, 136.8,
136.0, 133.9, 130.6, 129.1, 124.5, 106.2, 91.4, 63.8, 56.2, 28.5,
20.8, 19.9; MS (ESI): m/z 365.2 [M + Na]+; HRMS (ESI): m/z
calcd for C20H22O5Na [M + Na]+, 365.1359; found, 365.1390.
Cell apoptosis assay
Cell apoptosis assay was assessed by using an Annexin V-FITC/
PI staining assay kit (Becton Dickinson, USA). The cells were
treated with 5o at concentrations of 0.5, 1 and 2 μM for 24 h.
Both detached and attached cells were harvested with trypsin,
washed with cold phosphate-buffered saline (PBS), and then
resuspended in 100 μL binding buffer. Five μL each of Annexin
V-FITC solution and PI were added. The cells were incubated
at room temperature for 15 min and analyzed by flow cyto-
metry within 1 h.
Synthesis of (E)-2-(naphthalen-1-yl)ethyl-3-(4-methoxy-2-oxo-
2H-pyran-6-yl)acrylate (5u)
Following the general procedure, 5u was obtained as a white
1
solid (60.9 mg, 58%). H NMR (400 MHz, CDCl3): δH 8.10 (1H,
d, J = 8.3 Hz), 7.86 (1H, d, J = 7.8 Hz), 7.76 (1H, d, J = 7.8 Hz),
7.58–7.36 (4H, m), 7.02 (1H, d, J = 15.6 Hz), 6.69 (1H, d, J =
15.6 Hz), 6.06 (1H, d, J = 2.0 Hz), 5.57 (1H, d, J = 2.1 Hz), 4.53
(2H, t, J = 7.2 Hz), 3.82 (3H, s), 3.46 (2H, t, J = 7.2 Hz); 13C
NMR (100 MHz, CDCl3): δC 169.9, 165.6, 162.6, 155.6, 133.9,
133.5, 132.0, 130.9, 128.9, 127.5, 127.1, 126.2, 125.7, 125.5,
124.4, 123.5, 106.2, 91.4, 65.1, 56.1, 32.1; MS (ESI): m/z 351.2
[M + H]+; HRMS (ESI): m/z calcd for C21H18NO5Na [M + Na]+,
373.1046; found, 373.1072.
Immunofluorescence assay
HeLa cells (4 × 104 cells per well) were seeded on glass-bottom
cell culture dishes for 24 h. After incubation in the presence or
absence of 5o (0.5, 1 μM), cells were fixed in 4% paraformalde-
hyde, permeabilized with 0.5% Triton X-100 and 0.05% Tween-
20, blocked for 1 h and immunostained with rabbit anti-
β-actin (1 : 400; Cell Signaling Technology) overnight at 4 °C.
Subsequently, the antibodies were removed and the cells were
washed with PBS. The cells were incubated with CF488-conju-
gated goat-anti-rabbit IgG (1 : 700), highly cross-absorbed
(Biotium, USA). Nuclei were stained with Hoechst 33342 (5 mg
ml−1). Fluorescence images were captured under a Zeiss fluo-
rescence microscope (Zeiss, LSM710, Germany).
Cell culture
HeLa, HSC-2, and A549 cell lines were kindly provided by Pro-
fessor Li Haiou (Japan). MCF-7 and C6 were purchased from
the Center of Experiment Animal of Sun Yat-sen University
(Guangzhou, China). Cells were cultured in Dulbecco’s modi-
fied Eagle’s medium (Gibco) supplemented with 10% (v/v)
fetal bovine serum (Hyclone), 100 U mL−1 of penicillin, and Migration and invasion assay
100 μg mL−1 of streptomycin (Hyclone). These cells were grown
In vitro migration and invasion assays were carried out using
in a humidified incubator containing 5% CO2 at 37 °C and
subcultured every three days.
24-well plates with 8 μm pore size inserts (Transwell, Corning,
USA). Only for the invasion assay, the upper chamber was
coated with 100 μL diluted Matrigel (0.5 mg mL−1). A serum-
free DMEM cell suspension (100 μL, 3 × 105 cells mL−1) with/
without 5o (0.5, 1 μM) was added in the upper chamber.
A medium (600 μL) with 10% FBS was added to the lower
chamber. After 36 h of incubation, cells were fixed with 4% for-
maldehyde and stained with 0.1% crystal violet. Then, cells on
the upper membrane surface were removed with cotton swabs.
Stained cells were photographed under a microscope.
Antiproliferative assays
These derivatives were evaluated for their antiproliferative
activities in five different cancer cell lines by MTT assay. Cells
in log-phase were plated into 96-well plates with a density of
3.5 × 104 cells mL−1 overnight at 37 °C. Cells were then treated
with various concentrations of compounds and incubated at
37 °C for an additional 48 h. The antiproliferative activities of
compounds were evaluated by MTT assay. In brief, 20 μL of
MTT reagent was added to each well and the plates continued
to incubate for another 4 h at 37 °C. Formazan crystals were
dissolved in 120 μL of DMSO. The optical density was assayed
at 490 nm with an automated micro-plate reader (Thermo,
Multiskan Ascent, USA) and the 50% inhibition concentration
was determined from a dose–response curve.
Western blot assay
Treated cells were lysed with RIPA lysis buffer (P0013, Beyo-
time, China). The total protein quality was determined by
using a BCA Protein Assay Kit (Pierce; Rockford IL, USA). Pro-
teins were denatured and separated by 10% SDS-PAGE and
then electrotransferred to polyvinylidene difluoride mem-
branes (Immobilon PVDF, Millipore, Bedford). The mem-
branes were washed with Tris-Buffered Saline and Tween 20
Cell cycle analysis
HeLa cells (2 × 105 cells per well) were seeded into 6-well (TBST) and then blocked for 2 h at room temperature. The
plates for 24 h. Compound 5o was added into each well in membranes were then incubated with special antibodies
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Acknowledgements
This work was funded in part by the National Natural Science
Foundation of China (no. 81173470, 81473138, 81172941), the
National High-tech R&D Program of China (863 Program)
(2012AA020307), and the introduction of Innovative R&D Team
Program of Guangdong Province (no. 2009010058). Partial
support is also due to NIH grant CA177584 from the National
Cancer Institute awarded to K.H. Lee.
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