Synthesis of caffeic acid phenethyl ester analogues and their cytotoxicities against human cancer cells

Article abstract of DOI:10.14233/ajchem.2014.15974

Although caffeic acid phenethyl ester exhibits strong antitumor property, but its pharmacophore has not been elaborated clearly as yet. Seventeen caffeic acid phenethyl ester analogues were synthesized via simple and easy procedures and their antiprolifer

Full text of DOI:10.14233/ajchem.2014.15974

Asian Journal of Chemistry; Vol. 26, No. 9 (2014), 2686-2690
ASIAN JOURNAL OF CHEMISTRY
http://dx.doi.org/10.14233/ajchem.2014.15974
Synthesis of Caffeic Acid Phenethyl Ester Analogues and
Their Cytotoxicities Against Human Cancer Cells
1
1
1
1
1
YAN-HUI WANG , QU-SHENG LI , PENG-LONG WANG , KUO XU , YA-TAO CHENG ,
1
1
2
1,*
XIN XU , QIANG LI , YU-ZHONG ZHANG and HAI-MIN LEI
1School of Chinese Pharmacy, Beijing University of Chinese Medicine, Beijing 100102, P.R. China
2School of Basic Medical Sciences, Beijing University of Chinese Medicine, Beijing 100102, P.R. China
*Corresponding author: Tel: +86 10 8473 8640, E-mail: hm_lei@126.com
Received: 29 June 2013;
Accepted: 26 August 2013;
Published online: 28 April 2014;
AJC-15097
Although caffeic acid phenethyl ester exhibits strong antitumor property, but its pharmacophore has not been elaborated clearly as yet.
Seventeen caffeic acid phenethyl ester analogues were synthesized via simple and easy procedures and their antiproliferative effects were
evaluated toward Bel-7402 and MCF-7 cancer cell lines. Compared to compounds 1-11, 12, 13, 14, 15 showed cytotoxic effects. The IC50
values (µM) of compound 14 (41.87, 51.98) were about equal to caffeic acid phenethyl ester’s (34.60, 42.26). Structure-activity relationship
analysis demonstrated that the α, β-unsaturated double bond and the phenolic hydroxyl group in caffeic acid phenethyl ester were
essential for the antitumor activities. Besides, the cytotoxic properties of these compounds were determined by the methoxy groups on the
phenyl ring and the location of aromatic hydroxyl groups. These results may be of great benefit to people in screening new anticancer drug
candidates.
Keywords: Caffeic acid phenethyl ester analogues, Pharmacophore, Cytotoxic properties.
To find the pharmacophore, we designed and synthesized
seventeen caffeic acid phenethyl ester analogues (Scheme-I),
which are easily obtained. We tested their in vitro antitumor
activities by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetra-
zolium bromide (MTT) assay on hepatocellular cancer cell
lines (Bel-7402) and human breast cancer cell lines (MCF-7).
INTRODUCTION
Caffeic acid phenethyl ester (CAPE, Fig. 1) is a natural
ingredient extracted from propolis1. It is known to exhibit
antiinflammatory, antioxidant, antineoplastic and immunomo-
dulatory properties2-5. In particular, its anticancer effects have
been highly investigated, which have been demonstrated in a
number of cancer models, such as breast cancer, hepatocellular
cancer, oral cancer, cholangio carcinoma, human melanoma,
EXPERIMENTAL
All commercially available solvents and reagents were
used without further purification. Melting points were deter-
mined with a BÜCHI Melting Point B-450 apparatus (Büchi
Labortechnik, Flawil, Switzerland). The mass spectra were
obtained by using an ion trap-mass spectrometer (3200 QTRAP;
glioblastoma multiforme, prostate cancer and lung cancer6-12
.
Caffeic acid phenethyl ester shows strong anticancer effect
via its apoptotic effects and modulation of NF-κB6,11
.
1
O
Applied Biosystems, Foster City, California, USA), the H
NMR signals were obtained using a 500 MHz Bruker (Bruker
Corporation, Billerica, Massachusetts, USA), 13C NMR were
obtained in a Mercury-200 at 125 MHz (Varian Inc., PaloAlto,
California, USA). All the reactions were monitored by thin-
layer chromatography (TLC) on precoated silica gel G plates,
which were observed at 254 nm under a UV lamp. TLC was
performed using precoated silica gel GF254 (0.2 mm; Chemical
Industry Institute,Yantai, China) and column chromatography
was performed using silica gel (100-200 mesh; Haiyang Chemical
Co., Ltd, Qingdao, China). Caffeic acid phenethyl ester was
HO
O
HO
Fig. 1. Structure of caffeic acid phenethyl ester
Because of CAPE's simplicity of structure and interesting
cytotoxic property, several researchers have started to explore
strategies for the synthesis of CAPE or CAPE analogues and
have studied their activities13-15. However, there is scant lite-
rature to illustrate CAPE's structure-activity relationship.

Vol. 26, No. 9 (2014)
Synthesis of Caffeic Acid Phenethyl Ester Analogues 2687
7'
4'
Compounds 2-11 were prepared in the same manner as
described for compound 1.
O
6'
5'
O
3
9
1'
R
4
3'
2'
R
8
7
4
4
OH
a/b
b
1
O
2
3-(3,4-Dimethoxy-phenyl)acrylic acid phenethyl ester
(2): White solid, yield 90 %, m.p.: 101.2-102.1 °C; 1H NMR
(500 MHz, CDCl3) δ 7.65 (d, J = 15.9Hz, 1H), 7.35 (t, J = 7.4
Hz, 2H), 7.28 (dd, J = 15.2, 7.6 Hz, 3H), 7.10 (dd, J = 25.5,
4.9 Hz, 2H), 6.88 (t, J = 9.8 Hz, 1H), 6.33 (d, J = 15.9 Hz,
1H), 4.45 (t, J = 7.1 Hz, 2H), 3.94 (s, 6H), 3.05 (t, J = 7.0 Hz,
2H); 13C NMR (126 MHz, CDCl3) δ 167.13 (C-1), 151.16 (C-
6), 149.24 (C-7), 144.79 (C-3), 137.96 (C-3'), 128.93 (C-5',
C-7'), 128.53 (C-4), 127.42 (C-4', C-8'), 126.56 (C-6'), 122.67
(C-9), 115.75 (C-8), 111.05 (C-2), 109.64 (C-5), 64.88 (C-
1'), 55.99 (C-3,OCH3), 55.91 (C-4,OCH3), 35.26 (C-2'); MS
(ESI) m/z: 313 [M + H]+, calcd. for C19H20O4 312.14.
3-(3,4,5-Trimethoxy-phenyl)acrylic acid phenethyl
ester (3): White solid, yield 92 %, m.p.: 98.7-99.8 °C;1H NMR
(500 MHz, DMSO-d6) δ 7.62 (d, J = 15.9 Hz, 1H), 7.39 - 7.25
(m, 5H), 6.77 (s, 2H), 6.36 (d, J = 15.9 Hz, 1H), 4.46 (t, J =
7.0 Hz, 2H), 3.90 (t, J = 6.6 Hz, 9H), 3.05 (t, J = 7.0 Hz, 2H).
13C NMR (126 MHz, DMSO-d6) δ 166.84 (C-1), 153.47 (C-6,
C-8), 144.84 (C-3), 137.88 (C-3'), 129.90 (C-7), 128.91
(C-4), 128.54 (C-5', C-7'), 126.58 (C-4', C-8'), 117.29 (C-2),
105.35 (C-5, C-9), 64.97 (C-1'), 60.96 (C-7, OCH3), 56.19
(C-6, OCH3; C-8, OCH3), 35.23 (C-2'); MS (ESI) m/z: 343
[M + H]+, calcd. for C20H22O5 342.15.
5
R
R
1
3
R
R
3
1
6
R
R
2
2
7'
4'
6'
5'
8'
3'
O
7
1'
O
2
6
O
1
2'
OH
3
R
6
5
4
R
6
R
5
R
5
Reagents and conditions: (a): 2-phenyl-ethanol/CH2Cl2/EDCI/DMAP,
r.t., overnight. (b): (2-bromo-ethyl)-benzene/DMI/Na2CO3/KI,
50-60 °C, overnight
NO.
1
2
3
4
5
6
7
8
R1
H
R2
H
OCH3
OCH3
H
OCH3
H
H
OCH3
H
OCH3
H
OH
H
OH
H
R3
H
OCH3
OCH3
OCH3
H
CH3
H
H
R4
H
H
H
H
H
OCH3
H
OCH3
H
OCH3
H
OCH3
OCH3
H
H
H
H
H
9
H
OCH3
H
10
11
12
13
14
15
OCH3
N(CH3)2
H
OH
OCH3
H
H
H
H
H
H
H
H
3-(4-Methoxy-phenyl)acrylic acid phenethyl ester (4):
1
OH
H
White solid, yield 95 %, m.p.: 56.7-57.6 °C; H NMR (500
NO.
16
R5
OCH3
OH
R6
OH
H
MHz, DMSO-d6) δ 7.66 (d, J = 16.0Hz, 1H), 7.50 (d, J = 8.6
Hz, 2H), 7.38- 7.24 (m, 5H), 6.93 (d, J = 8.6 Hz, 2H), 6.33 (d,
J = 16.0 Hz, 1H), 4.45 (t, J = 7.1Hz, 2H), 3.87 (s, 3H), 3.05 (t,
J = 7.0 Hz, 2H). 13C NMR (126 MHz, CDCl3) δ 167.24 (C-1),
161.41 (C-7), 144.53 (C-3), 137.99 (C-3'), 129.76 (C-5', C-
7'), 128.96 (C-4', C-8'), 128.52 (C-5,C-9), 127.17 (C-4), 126.56
(C-6'), 115.55 (C-2), 114.34 (C-6, C-8), 64.88 (C-1'), 55.39
(C-7, -OCH3), 35.27 (C-2'); MS (ESI) m/z: 283 [M + H]+, calcd.
for C18H18O3 282.13.
17
Scheme-I:The structure of compounds 1-17 and synthetic route of caffeic
acid phenethyl ester analogues
purchased from Sigma Chemical Co. (St Louis, Missouri,
USA).
Fetal bovine serum (FBS) was obtained from Gibco. Peni-
cillin and streptomycin were obtained from Thermo Scientific.
Hepatocellular cancer cell lines and human breast cancer cell
ines were obtained from the Institute of Medicine of Peking
Union of Beijing.
3-(2, 3-Dimethoxy-phenyl)-acrylic acid phenethyl ester
1
(5): White solid, yield 95 %, m.p.: 38.3-39.2 °C; H NMR
(500 MHz, CDCl3) d 8.02 (d, J = 16.2 Hz, 1H), 7.37-7.26 (m,
5H), 7.18 (d, J = 7.9 Hz, 1H), 7.09 (t, J = 8.0 Hz, 1H), 6.97 (d,
J = 8.0 Hz, 1H), 6.49 (d, J = 16.2 Hz, 1H), 4.45 (t, J = 7.0 Hz,
Synthesis
2H), 3.90 (d, J = 12.3 Hz, 6H), 3.06 (t, J = 7.0 Hz, 2H). 13
C
3-Phenyl-acrylic acid phenethyl ester (1): A solution
of 2-phenyl-ethanol (0.989 g, 8.10 mmol) in 25 mL of CH2Cl2
was stirred at room temperature. EDCI (0.989g) and DMAP
(0.329 g) were added into the solution while stirring. After
0.5 h, 3-phenyl-acrylic acid (1.000 g, 6.76 mmol) was added
to the reaction mixture and allowed to stay overnight. The
solution was evaporated to a small volume, then purified by
column chromatography (silica gel). Elution with petroleum-
acetone (30:1, v/v) produced pure compound 1. White solid,
yield 92 %. m.p.: 54.3-55.3 °C; 1H NMR (500 MHz, CDCl3) δ
7.71 (d, J = 16.0 Hz, 1H), 7.58-7.54 (m, 2H), 7.44-7.24 (m,
8H), 6.46 (d, J = 16.0 Hz, 1H), 4.46 (t, J = 7.1Hz, 2H), 3.06 (t,
J = 7.0 Hz, 2H); 13C NMR (126 MHz, CDCl3) δ 166.90 (C-1),
144.87 (C-3), 137.91 (C-3'), 134.44 (C-4), 130.30 (C-5', C-7')
128.96 (C-6, C-8), 128.90 (C-4', C-8'), 128.54 (C-7), 128.10
(C-5, C-9), 126.60 (C-6'), 118.09 (C-2), 65.04 (C-1'), 35.24
(C-2'); MS (ESI) m/z: 253 [M + H]+, calcd. for C17H16O2 252.12.
NMR (126 MHz, CDCl3) δ 167.05 (C-1), 153.15 (C-6), 139.53
(C-5), 137.99 (C-3), 128.99 (C-3'), 128.64 (C-5', C-7'), 128.52
(C-4', C-8'), 126.55 (C-6'), 124.19 (C-4, C-8), 119.37 (C-9),
119.21 (C-2), 113.96 (C-7), 65.01 (C-1'), 61.35 (C-5,-OCH3),
55.90 (C-6,-OCH3), 35.26 (C-2'). MS (ESI) m/z: 313 [M +
H]+, calcd. for C19H20O4 312.14.
3-p-tolyl-acrylic acid phenethyl ester (6): White solid,
yield 90 %, m.p.: 67.4-68.4 °C; 1H NMR (500 MHz, CDCl3) δ
7.68 (d, J = 16.0 Hz, 1H), 7.45 (d, J = 8.0 Hz, 2H), 7.36 (t, J =
7.4 Hz, 2H), 7.32-7.26 (m, 3H), 7.22 (d, J = 7.8 Hz, 2H), 6.42
(d, J = 16.0 Hz, 1H), 4.45 (t, J = 7.1 Hz, 2sH), 3.05 (t, J = 7.0
Hz, 2H), 2.41 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 167.10
(C-1), 144.87 (C-3), 140.72 (C-3'), 137.96 (C-7), 131.71 (C-
4), 129.63 (C-6,C-8), 128.96 (C-5',C-7'), 128.53 (C-4',C-8'),
128.10 (C-5,C-9), 126.57 (C-6'), 116.98 (C-2), 64.95 (C-1'),
35.26 (C-2'), 21.48 (C-7,OCH3); MS (ESI) m/z: 267 [M + H]+,
calcd. for C18H18O2 266.13.

2688 Wang et al.
Asian J. Chem.
3-(2-Methoxy-phenyl)acrylic acid phenethyl ester (7):
in 6 mL of 1,3-dimethyl-2-imidazolidinone (DMI). 350 mg
exsiccated sodium carbonate (Na2CO3) was added while stirring.
Stirring continued for 0.5 h.At the end of this period a solution
of 0.43 mL of phenethyl bromide dissolved in 1 mL DMI was
added slowly to the reaction mixture over a period of 0.5 h.
Following the addition, a small amount of potassium iodide
(KI) was added to the reaction mixture. This mixture was
stirred at room temperature for 9 h after which the temperature
was reduced to 15 °C and the reaction mixture was stirred for
another 12 h. The reaction mixture was slowly added to 30 mL
of ice water while stirring and extracted with ethyl acetate
(3 × 15 mL). The aqueous layer was acidified by dropwise
addition of 1 M aqueous HCl and extracted with ethyl acetate.
The combined organic extract was washed with saturated
sodium chloride solution (3 × 15 mL). The organic layer was
dried over anhydrous Na2SO4, filtered and concentrated under
reduced pressure followed by purification of the crude products
by column chromatography, thereby producing 3-(3-hydroxy-
phenyl)acrylic acid phenethyl ester. White solid, yield 80 %,
m.p.: 100.8-101.8 °C; 1H NMR (500 MHz, CDCl3) δ 7.66 (d,
J = 16.0 Hz, 1H), 7.32 (dt, J = 16.9, 7.9Hz, 6H), 7.14-7.02 (m,
2H), 6.92 (d, J = 7.9Hz, 1H), 6.43 (d, J = 16.0 Hz, 1H), 6.01
(d, J = 5.8Hz, 1H), 4.47 (t, J = 7.0 Hz, 2H), 3.05 (t, J = 7.0 Hz,
2H). 13C NMR (126 MHz, CDCl3) δ 167.35 (C-1), 156.24 (C-
6), 145.00 (C-3), 137.78 (C-3'), 135.84 (C-4), 130.13 (C-8),
128.95 (C-5', C-7'), 128.57 (C-4', C-8'), 126.64 (C-6'), 120.76
(C-9), 118.21 (C-2), 117.67 (C-7), 114.65 (C-5), 65.30
(C-1'), 35.18 (C-2'); MS (ESI) m/z: 269 [M + H]+, calcd. for
C17H16O3 268.11.
Colourless oil, yield 80 %, 1H NMR (500 MHz, CDCl3) δ 8.03
(d, J = 16.2 Hz, 1H), 7.52 (t, J = 12.0 Hz, 1H), 7.40-7.23 (m,
6H), 7.02-6.90 (m, 2H), 6.55 (d, J = 16.2 Hz, 1H), 4.45 (t, J =
7.1Hz, 2H), 3.92 (s, 3H), 3.06 (t, J = 7.1 Hz, 2H). 13C NMR
(126 MHz, CDCl3) δ 167.41 (C-1), 158.36 (C-4), 140.28 (C-
3), 138.03 (C-3'), 131.49 (C-7), 129.00 (C-5', C-7'), 128.90
(C-4', C-8'), 128.51 (C-9), 126.53 (C-6'), 123.44 (C-8), 120.71
(C-4), 118.55 (C-2), 111.17 (C-6), 64.91 (C-1'), 55.50 (C-
5,OCH3), 35.29 (C-2'); MS (ESI) m/z: 283 [M + H]+, calcd.
for C18H18O3 282.13.
3-(3-Methoxy-phenyl)acrylic acid phenethyl ester (8):
1
White solid, yield 85 %, m.p.: 43.4-44.4 °C; H NMR (500
MHz, CDCl3) δ 7.67 (d, J = 16.0 Hz, 1H), 7.38-7.25 (m, 6H),
7.14 (d, J = 7.6 Hz, 1H), 7.07 (s, 1H), 6.97 (dd, J = 8.2, 2.0
Hz, 1H), 6.45 (d, J = 16.0 Hz, 1H), 4.46 (t, J = 7.1 Hz, 2H),
3.86 (s, 3H), 3.06 (t, J = 7.1 Hz, 2H). 13C NMR (126 MHz,
CDCl3) δ 166.84 (C-1), 159.92 (C-6), 144.79 (C-3), 137.90
(C-3'), 135.80 (C-4), 129.90 (C-8), 128.95 (C-5', C-7'), 128.55
(C-4', C-8'), 126.60 (C-6'), 120.82 (C-9), 118.38 (C-2), 116.17
(C-7), 112.98 (C-5), 65.04 (C-1'), 55.32 (C-6,OCH3), 35.23
(C-2'); MS (ESI) m/z: 283 [M + H]+, calcd. for C18H18O3 282.13.
3-(2,5-Dimethoxy-phenyl)acrylic acid phenethyl ester
(9): Yellow oil, yield 80 %, 1H NMR (500 MHz, DMSO-d6) δ
8.00 (d, J = 16.1 Hz, 1H), 7.37-7.24 (m, 4H), 7.07 (d, J = 2.6
Hz, 1H), 6.96-6.85 (m, 2H), 6.52 (dd, J = 16.1, 12.3 Hz, 1H),
4.45 (t, J = 7.1Hz, 1H), 3.82 (dd, J = 25.1, 17.1 Hz, 8H), 3.05
(t, J = 7.1 Hz, 1H). 13C NMR (126 MHz, DMSO-d6) δ 167.25
(C-1), 153.53 (C-8), 152.85 (C-5), 140.01 (C-3), 137.99 (C-
3'), 128.98 (C-5', C-7'), 128.51 (C-4', C-8'), 126.53 (C-6'),
124.00 (C-4), 118.55 (C-2), 117.14 (C-6), 117.11 (C-7), 113.33
(C-9), 64.93 (C-1'), 56.11 (C-5, OCH3), 51.61 (C-8, OCH3),
35.27 (C-2'); MS (ESI) m/z: 313 [M + H]+, calcd. for C19H20O4
312.14.
Compounds 13-17 were prepared in the same manner as
described for compound 12.
3-(4-Hydroxy-phenyl)acrylic acid phenethyl ester (13):
1
White solid, yield 75 %, m.p.: 90.5-91.5 °C; H NMR (500
MHz, CDCl3) δ 7.65 (d, J = 16.0 Hz, 1H), 7.44 (d, J = 8.4 Hz,
2H), 7.36-7.24 (m, 5H), 6.89 (d, J = 8.5 Hz, 2H), 6.57-6.38
(m, 1H), 6.31 (d, J = 15.9 Hz, 1H), 4.46 (t, J = 7.0 Hz, 2H),
3.05 (t, J = 7.0 Hz, 2H); 13C NMR (126 MHz, CDCl3) δ 167.77
(C-1), 158.19 (C-7), 144.97 (C-3), 137.89 (C-3'), 130.05
(C-5', C-7'), 128.95 (C-4', C-8'), 128.55 (C-5, C-9), 126.94
(C-4), 126.60 (C-6'), 115.96 (C-2), 115.15 (C-6, C-8), 65.09
(C-1'), 35.23 (C-2'); MS (ESI) m/z: 268.9 [M + H]+, calcd. for
C17H16O3 268.11.
3-(2,3,4-Trimethoxy-phenyl)acrylic acid phenethyl
1
ester (10): White solid, yield 90 %, m.p.: 54.3-55.3 °C; H
NMR (500 MHz, CDCl3) δ 7.90 (d, J = 16.1 Hz, 1H), 7.39-
7.23 (m, 6H), 6.71 (d, J = 8.8 Hz, 1H), 6.43 (d, J = 16.1 Hz,
1H), 4.44 (t, J = 7.1 Hz, 2H), 3.96-3.86 (m, 9H), 3.05 (t, J =
7.1 Hz, 2H). 13C NMR (126 MHz, CDCl3) δ 167.38 (C-1),
155.55 (C-5), 153.32 (C-7), 142.40 (C-3), 139.76 (C-3'),
138.06 (C-6), 128.98 (C-5', C-7'), 128.50 (C-4', C-8'), 126.52
(C-6'), 123.13 (C-9), 121.52 (C-2), 116.95 (C-4), 107.63 (C-
8), 64.85 (C-1'), 61.43 (C-5, OCH3), 60.90 (C-6, OCH3), 56.08
(C-7, OCH3), 35.29 (C-2'). MS (ESI) m/z: 343 [M + H]+, calcd.
for C20H22O5 342.15.
3-(4-Dimethylamino-phenyl)acrylic acid phenethyl
ester (11): Yellow solid, yield 85 %, m.p.: 117.4-118.4 °C; 1H
NMR (500 MHz, CDCl3) δ 7.65 (d, J = 15.8Hz, 1H), 7.45 (d,
J = 8.7 Hz, 2H), 7.37-7.23 (m, 5H), 6.71 (d, J = 8.4 Hz, 2H),
6.25 (d, J = 15.8 Hz, 1H), 4.47-4.39 (m, 2H), 3.09-2.98 (m,
8H). 13C NMR (126 MHz, CDCl3) δ167.77 (C-1), 151.73 (C-
7), 145.34 (C-3), 138.14 (C-3'), 129.77 (C-5', C-7'), 128.98
(C-4', C-8'), 128.49 (C-5, C-9), 126.49 (C-6'), 112.46 (C-2),
111.91 (C-6, C-8), 64.65 (C-1'), 40.20 (C-7, N (CH3)2), 35.35
(C-2'); MS (ESI) m/z: 296 [M + H]+, calcd. for C19H21NO2
295.16.
3-(4-Hydroxy-3-methoxy-phenyl)acrylic acid phenethyl
1
ester (14): Colourless oil, yield 70 %; H NMR (500 MHz,
CDCl3) δ 7.63 (d, J = 15.9 Hz, 1H), 7.35 (t, J = 7.4 Hz, 2H),
7.28 (dd, J = 14.6, 7.7 Hz, 3H), 7.10 - 7.03 (m, 2H), 6.95 (d,
J = 8.1 Hz, 1H), 6.31 (d, J = 15.9 Hz, 1H), 5.95 (s, 1H), 4.45
(t, J = 7.1 Hz, 2H), 3.95 (s, 3H), 3.05 (t, J = 7.0 Hz, 2H). 13C
NMR (126 MHz, CDCl3) δ 167.20 (C-1), 148.02 (C-6), 146.80
(C-3), 144.96 (C-7), 137.97 (C-3'), 128.94 (C-4), 128.53 (C-
5', C-7'), 127.01 (C-4', C-8'), 126.56 (C-6'), 123.12 (C-9),
115.44 (C-2), 114.74 (C-8), 109.37 (C-5), 64.88 (C-1'), 55.97
(C-6, OCH3), 35.26 (C-2'); MS (ESI) m/z: 298.9 [M + H]+,
calcd. for C18H18O4 298.12.
3-(2-Hydroxy-phenyl)acrylic acid phenethyl ester (15):
White solid, yield 75 %, m.p.: 102.1-103.1 °C; 1H NMR (500
MHz, CDCl3) δ 8.10 (d, J = 16.2 Hz, 1H), 7.49 (d, J = 7.7 Hz,
1H), 7.39-7.24 (m, 6H), 7.02-6.84 (m, 3H), 6.68 (d, J = 16.2
Hz, 1H), 4.49 (t, J = 7.1 Hz, 2H), 3.07 (t, J = 7.1 Hz, 2H). 13C
3-(3-Hydroxy-phenyl)acrylic acid phenethyl ester (12):
500 mg of 3-(3-hydroxy-phenyl)-acrylic acid was dissolved

Vol. 26, No. 9 (2014)
Synthesis of Caffeic Acid Phenethyl Ester Analogues 2689
TABLE-1
IC50 VALUES OF SYNTHETIC CAFFEIC ACID PHSENETHYL ESTER ANALOGUES AGAINST BEL-7402 AND MCF-7 (µM)
NO.
1-11
> 200
> 200
12
13
14
15
16
17
CAPE
34.6
42.26
Bel-7402
MCF-7
51.36
67.11
55.67
92.87
41.87
51.98
44.59
58.55
80.13
101.60
81.26
> 200
NMR (126 MHz, CDCl3) δ 168.50 (C-1), 155.51 (C-5), 141.02
(C-3), 137.86 (C-3'), 131.54 (C-7), 129.24 (C-5', C-7'), 128.98
(C-9), 128.57 (C-4', C-8'), 126.62 (C-6'), 121.70 (C-4), 120.71
(C-8), 118.12 (C-2), 116.48 (C-6), 65.27 (C-1'), 35.23 (C-2');
MS (ESI) m/z: 269 [M + H]+, calcd. for C17H16O3 268.11.
4-Hydroxy-3-methoxy-benzoic acid phenethyl ester
(16): Colourless oil, yield 70 %, 1H NMR (500 MHz, CDCl3)
δ 7.65 (dd, J = 8.3, 1.5 Hz, 1H), 7.54 (d, J = 1.3 Hz, 1H), 7.41-
7.23 (m, 5H), 6.96 (d, J = 8.3 Hz, 1H), 6.13 (d, J = 4.6 Hz,
1H), 4.54 (t, J = 7.0 Hz, 2H), 3.94 (s, 3H), 3.10 (t, J = 7.0 Hz,
2H); 13C NMR (126 MHz, CDCl3) δ 166.31 (C-1), 150.05 (C-
4), 146.17 (C-5), 138.05 (C-3'), 129.01 (C-5',C-7'), 128.53
(C-4', C-8'), 126.58 (C-6'), 124.18 (C-2), 122.41 (C-7), 114.10
(C-3), 111.77 (C-6), 65.32 (C-1'), 56.07 (C-4, OCH3), 35.33
(C-2'); MS (ESI) m/z: 273 [M + H]+, calcd. for C16H16O4 272.10.
3-Hydroxy-benzoic acid phenethyl ester (17): White
solid, yield 80 %, m.p.: 78.4-79.1 °C; 1H NMR (500 MHz,
CDCl3) δ 7.65-7.55 (m, 2H), 7.42-7.20 (m, 6H), 7.10 (dd, J =
8.1, 1.3 Hz, 1H), 6.12 (s, 1H), 4.56 (t, J = 7.0 Hz, 2H), 3.10 (t,
J = 6.9 Hz, 2H); 13C NMR (126 MHz, CDCl3) δ 166.79 (C-1),
155.93 (C-4), 137.76 (C-3'), 131.49 (C-2), 129.74 (C-6),
128.97 (C-5', C-7'), 128.59 (C-4', C-8'), 126.66 (C-6'), 121.89
(C-7), 120.37 (C-5), 116.39 (C-3), 65.82 (C-1'), 35.18 (C-2');
MS (ESI) m/z: 243 [M + H]+, calcd. for C15H14O3 242.09.
Biological evaluation: Bel-7402 and MCF-7 cells were
cultured in DMEM containing 10 % (v/v) fetal bovine serum
(HyClone, Logan, UT, USA), 1 % penicillin and 1 % strepto-
mycin at a humidified atmosphere with 5 % CO2 at 37 °C. The
cells were plated in 96 well microplates at a density of 2 × 104
cells/mL (100 µl per well).After 24 h of incubation to allow for
cell attachment, different concentrations of compounds 1-17
were added and stock solutions of the synthesized compounds
(10 mmol/mL) were prepared by dissolving them in DMSO.
Appropriate dilutions of the synthesized compounds were
prepared in growth medium and added to the cells. The viable
cell growth after incubation for 72 h with synthesized com-
pounds was determined using the MTT assay16. The absorbance
was determined at 490 nm using a microplate reader.
literature was replaced by DMI because of its carcino-
genicity14,19. Besides the low toxicity of DMI, it was a cheap
and a good aprotic polar solvent. Moreover, the temperature
(50-60 °C) was a critical factor to the experiment and at room
temperature, 12-17 were not obtained.
Biological activities: Caffeic acid phenethyl ester together
with its seventeen analogues (1-17) were tested for their cytoto-
xicities activities against two different human cancer cell lines,
that is, Bel-7402 and MCF-7. The antiproliferative activities
of 1-17 are summarized in terms of their IC50 values in Table-1.
Interestingly, the CAPE analogues possessed similar
strength of activity toward the two tested cell lines. The order
of their IC50 values was CAPE < 14 < 15 < 12 < 13 < 16 < 17
≤ 1-11.
Compound 14 showed stronger antiproliferative activity
than other analogues. The inhibitory activities of compound
12 (with m-hydroxy-phenyl) against two cell lines are stronger
than that of compound 13 (with p-hydroxy-phenyl) while are
weaker than that of compound 15 (with o-hydroxy-phenyl).
This indicated that the methoxy groups on the phenyl ring may
enhance antiproliferative activity on condition that the ring
had the adjacent hydroxyl.
Comparison of compounds 1-11 and compounds 12-15
revealed that 12-15 possessed stronger activity against the two
tested cell lines. On the other hand, the effects of 16 and 17 on
Bel-7402 were obvious but on MCF-7 were significantly less.
The results indicated that the presence of both phenolic hydroxyl
groups and α,β-unsaturated double bond could be playing an
important role in their antiproliferative activities.
Conclusion
This study investigated the structure-activity relationship
of CAPE and its analogues. Seventeen caffeic acid phenethyl ester
analogues were prepared in order to test their antiproliferation
activities on the two cells. The simultaneous cytotoxicity evalu-
ation on the two cells of compounds 1-17 provides valuable
information regarding the prospective lead compounds.
Among those, CAPE and compounds 12, 13, 14, 15, 16,
17 presented obvious antitumor activities against Bel-7402
with IC50 (µM) values (51.36, 55.67, 41.87, 44.59, 80.13,
81.26) and that CAPE and compounds 12, 13, 14, 15 possessed
strong antitumor activities against MCF-7 with the values
(42.26, 67.11, 92.87, 51.98, 58.55). The results indicated that
compounds with phenolic hydroxyl groups and α,β-unsatu-
rated double bond exhibited greater inhibitory activities on
the two cancer cell lines than those compounds without them.
The methoxy groups on the phenyl ring and the location of
aromatic hydroxyl groups were critical factors for the comp-
ounds' activities.
RESULTS AND DISCUSSION
The synthetic routes for the desired compounds 1-11 and
12-17 are outlined in Scheme-I. Caffeic acid phenethyl ester
(CAPE) has been synthesized previously by several steps1,14,17,18
.
Among those synthetic methods, we selected one-pot esterifi-
cation of caffeic acid with (2-bromo-ethyl)benzene in dichloro-
methane (CH2Cl2) to prepare compounds 12-1714. Compounds
1-11 were prepared by conventional procedures as following.
Compounds 12-17 with a hydroxy group on the benzene
ring made the structure more unstable compared with com-
pounds 1-11. Therefore, the yields of compounds 12-17 (about
75 %) were lower than that of compounds 1-11 (about 90 %).
In the course of obtaining 12-17, HMPA which was used in
ACKNOWLEDGEMENTS
This part of work is partially supported by the National
Natural Science Foundation of China (No.81073017; No.

2690 Wang et al.
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Beijing University of Chinese Medicine named 'Lead Com-
pounds Discovering and Developing Innovation Team Project
Foundation' (No. 2011-CXTD-15).
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