Synthesis, biological evaluation, and structure-activity relationship study of novel cytotoxic aza-caffeic acid derivatives

Article abstract of DOI:10.1016/j.bmc.2010.07.016

Three series of aza-caffeic acid derivatives with different linkers were designed and synthesized. Each of the synthesized derivatives was then used in cytotoxicity screening on either 8 or 12 human cancer cell lines. The structure-activity relationships on three structural regions A, B, and C are analyzed in detail, indicating that a nine bond linker B, containing a piperazine unit, is the most favorable linker leading to the generation of molecules with potent cytotoxicities. Compound (E)-1-(4-(3,4-dichlorobenzyl) piperazin-1-yl)-3-(4-(4-ethoxybenzyloxy)-3,5-dimethoxyphenyl)prop-2-en-1-one (80) exhibited the most significant and selective cytotoxicity to KB, BEL7404, K562, and Eca109 cell lines, with IC₅₀ values of 0.2, 2.0, 1.7, and 1.1 μM, respectively, stronger than that seen for caffeic acid phenethyl ester (CAPE) and cisplatin (CDDP). Flow cytometric and western blot analysis indicate that compound 80 plays a role in mitochondria-dependent apoptosis activity by suppressing K562 cell proliferation in a concentration- and time-dependent manner.

Full text of DOI:10.1016/j.bmc.2010.07.016

Bioorganic & Medicinal Chemistry 18 (2010) 6351–6359
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, biological evaluation, and structure–activity relationship
study of novel cytotoxic aza-caffeic acid derivatives
a,
Hongbin Zou a, Hao Wu a, Xiangnan Zhang b, Yu Zhao a, Joachim Stöckigt a,c, Yijia Lou b, , Yongping Yu
*
*
a Institute of Material Medica, College of Pharmaceutical Sciences, Zhejiang University, 383 Yu Hang Tang Road, Hangzhou 310058, China
b Institute of Pharmacology, Toxicology, and Biochemical Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, 383 Yu Hang Tang Road, Hangzhou 310058, China
c Lehrstuhl für Pharmazeutische Biologie, Institut für Pharmazie, Johannes-Gutenberg Universität Mainz, Staudinger Weg 5, D-55099 Mainz, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Three series of aza-caffeic acid derivatives with different linkers were designed and synthesized. Each of
the synthesized derivatives was then used in cytotoxicity screening on either 8 or 12 human cancer cell
lines. The structure–activity relationships on three structural regions A, B, and C are analyzed in detail,
indicating that a nine bond linker B, containing a piperazine unit, is the most favorable linker leading
to the generation of molecules with potent cytotoxicities. Compound (E)-1-(4-(3,4-dichlorobenzyl)pip-
erazin-1-yl)-3-(4-(4-ethoxybenzyloxy)-3,5-dimethoxyphenyl)prop-2-en-1-one (80) exhibited the most
significant and selective cytotoxicity to KB, BEL7404, K562, and Eca109 cell lines, with IC50 values of
Received 4 May 2010
Revised 7 July 2010
Accepted 8 July 2010
Available online 13 July 2010
Keywords:
Aza-caffeic acid derivatives
Cytotoxicity
Mitochondria-dependent apoptosis
SAR analysis
Linker B
0.2, 2.0, 1.7, and 1.1 lM, respectively, stronger than that seen for caffeic acid phenethyl ester (CAPE)
and cisplatin (CDDP). Flow cytometric and western blot analysis indicate that compound 80 plays a role
in mitochondria-dependent apoptosis activity by suppressing K562 cell proliferation in a concentration-
and time-dependent manner.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
regions (A, B, and C, shown in Fig. 1). In order to investigate the
structure–cytotoxicity relationship of aza-caffeic acid derivatives,
Caffeic acid phenethyl ester (CAPE, 1, Fig. 1), which was first iso-
lated from Hungarian propolis,1 is a well known caffeic acid deriv-
ative with strong cytotoxic activity.2,3 Petasiphenol (2, Fig. 1),
another caffeic acid derivative isolated from Petasites japonicum,
also possesses antimutagen activity.4 Due to their structural sim-
plicity and interesting cytotoxic properties, the synthesis and bio-
logical evaluation of CAPE, petasiphenol, and their derivatives have
been the subject of studies by several groups.4–7 In addition to caf-
feic acid derivatives, sinapyl acid derivatives possessing the same
different substituents in region A, C, and variant linker B with a dif-
ferent position and number of nitrogen atoms were synthesized to
constitute three series of 32 compounds in total based on the
structures of compounds 1, 2, and 3 (Fig. 2). In addition, com-
pounds 91 and 88 were synthesized as analogous of series 2 and
3 (Fig. 2). The synthetic procedures are reported herein. The syn-
thesized aza-caffeic acid derivatives were then used to screen 8
(37–51, 59–63, Table 1) and 12 (73–84, 88, 91, Table 2) cancer cell
lines, respectively. The results showed that the compounds have
cytotoxic potential. Moreover, the SAR discussion on cytotoxicity
effect by structural variations in regions A, B, and C is reported
herein.
a
-, b-unsaturated carbonyl as an important Michael acceptor anti-
cancer pharmacophore have also been previously reported.8,9 Sina-
pyl acid 3 (Fig. 1) displays potent cytotoxicity against the KB cell
line with an IC50 value of 14 l
M.9
The aza-derivatives of caffeic acid, in particular, the aza-CAPE
(N-caffeoylphenalkylamide) derivatives have also been found to
possess intensive activities.10–12 However, a cytotoxicity study of
the aza-caffeic acid derivatives has rarely been reported.13 To
systematically assess the effect of structural variations on the
compound’s activity, the structure of CAPE was divided into three
2. Results and discussion
2.1. Chemistry
The synthesis of target compounds was accomplished according
to the reaction sequence reported in Schemes 1–3. The aza-caffeic
acid derivatives (31–44, 59–63) with an eight bond linker (B) were
coupled by potassium salts of substituted cinnamic acids (12–14,
56–58) and N-substituted 2-chloro acetamides (23–30) under the
activation of potassium iodine with yields of 56–87% (Scheme 1).14
Compounds 9–11, which were further hydrolyzed by potassium
* Corresponding authors. Tel./fax: +86 571 88208452 (Y.Y.); tel./fax: +86 571
88208403 (Y.L.).
E-mail addresses: yijialou@zju.edu.cn (Y. Lou), yyu@zju.edu.cn, yyongping@
tpims.org (Y. Yu).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.07.016

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H. Zou et al. / Bioorg. Med. Chem. 18 (2010) 6351–6359
Figure 1. Caffeic acid derivatives and regions A, B, and C for structure modulation.
hydroxide to afford the intermediate potassium salts (12–14, 56–
58), were prepared by a Wittig reaction from 5, 6, and 8 as previously
described.9 The aldehydes 5–7 were synthesized by alkylation of the
hydroxy group of protocatechualdehyde (4) with methyl, methoxy-
methyl, and benzyl substituents, respectively, in the presence of
potassium carbonate in which 7 was further alkylated by methoxy-
methyl to afford 8.9 N-substituted 2-chloro acetamides (23–30)
were synthesized by coupling the corresponding amines (15–22)
to chloroacetyl chloride.15 The preparation of 53–55 was achieved
as previously described by the appropriate substitution of benzyl-
bromides and syringaldehyde (52).9 Hydrolyzation of compounds
31–36 and 44 were conducted in the presence of hydrochloric acid
(1 M) to afford compounds 46–51 with two free hydroxy groups
and 45 with one free hydroxy.16
Figure 2. Designed and synthesized aza-caffeic acid derivatives.
Table 1
Inhibitory results of 37–51 and 59–63 to 8 human cancer cell linesa
Compound
IC50
(l
M)
PC-3
BEL7404
HL-60
HeLa
CNE
K562
Eca109
LS174T
b
37
38
39
40
—
46.4 4.1
31.9 2.7
92.5 7.3
—
193 14.3
45.4 3.7
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
120 9.3
60.2 4.8
0.8 0.04
—
81.9 6.3
—
—
—
—
41
42
43
—
—
50.4 4.9
82.2 4.8
—
—
—
—
—
—
—
—
—
—
32.4 1.6
0.3 0.01
—
143 9.13
—
—
—
—
—
130 8.7
44
133 9.6
—
155 11.7
—
—
—
—
—
45
46
47
48
49
50
51
59
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
56.1 3.3
56.8 4.1
—
—
—
—
—
—
—
30.3 2.3
92.6 6.5
8.1 0.5
72.1 5.2
97.4 7.1
—
73.9 6.6
—
9.8 0.9
6.4 0.4
117 7.1
145 6.7
108 5.8
124 8.3
69.4 4.0
—
72.4 5.3
—
9.9 0.7
15.6 0.8
—
195 10.3
—
187 10.6
—
—
—
89.6 6.3
—
170 9.7
—
—
—
160 8.8
172 9.1
155 6.3
—
—
—
99.4 5.8
79.1 5.1
121 8.3
—
—
—
175 8.1
190 9.0
131 8.4
—
—
—
97.8 7.6
—
—
14.5 1.2
5.6 0.2
60
61–63
CAPE
DDP
8.8 0.7
5.0 0.3
54 2.6
6.7 0.2
91 4.3
4.0 0.2
46 1.6
11.6 0.8
42.6 0.9
7.6 0.5
a
Key to cell lines: BEL7404, human hepatocellular carcinoma line; HL-60, human promyelocytic leukemia cell line; HeLa, human cervical carcinoma cell line; CNE,
nasopharyngeal carcinoma cell line; PC-3, human prostate cancer cell line; K562, human erythroleukemia cells; Eca109, human esophageal carcinoma cell line; LS174T,
human colon carcinoma cells.
b
IC50 values greater than 200 lM were considered as inactive and omitted here.

H. Zou et al. / Bioorg. Med. Chem. 18 (2010) 6351–6359
6353
Table 2
Inhibitory results of compounds 73–84, 88, and 91 to 12 human cancer cell linesa
Compound
IC50 (lM)
KB
A549
BEL7404
HL-60
HeLa
CNE
PC-3
K562
BGC-823
Eca109
HO8910
LS174T
—
165 8.3
8.4 0.3
b
73–77
78
79
80
81
82
83
84
88
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
157 8.4
—
—
195 10.3
—
1.7 0.1
143 11.0
—
—
31.2 2.8
—
64.4 5.9
—
154 9.8
—
1.1 0.06
17.0 1.1
—
7.4 0.4
—
103 7.0
41.4 2.6
42.6 0.9
7.6 0.5
85.7 5.1
0.2 0.01 63.4 2.5
124 7.9
—
2.7 0.2
—
2.0 0.1
—
20.7 1.6
69.2 2.8
3.7 0.02
—
103 6.4
20.1 1.2
45.2 2.3
4.9 0.3
173 8.8 44.8 2.6 82.6 3.2 73.9 3.5
—
107 7.3
—
139 6.4 67.1 3.6 99.4 4.7 84.8 4.5
72.6 3.3 21.2 1.0 39.3 2.1 73.1 2.9 43.7 3.6 40.9 2.8
26.8 1.6 14.5 1.2
7.5 0.4 5.6 0.2
12.8 1.1 14.1 0.4
70.8 3.9
7.4 0.4 19.3 0.8
45.4 2.5
121 6.6
69.7 3.0
—
34.1 1.7
—
139 10.1
50.4 2.3
67.2 3.6
85.7 3.2
—
—
140 5.8 76.6 5.8
31.0 1.3 29.1 1.7
165 7.8
—
—
—
—
—
—
78.8 4.4
—
91
CAPE
DDP
0.9 0.04 46.0 3.0
190 11.1 20.1 1.0
25.5 1.0
6.0 0.2
9.8 0.9
6.4 0.4
8.8 0.7 54.2 2.6 91.4 4.3 46.1 1.6
5.0 0.3 6.7 0.2 4.0 0.2 11.6 0.8
19.3 1.3
5.0 0.4
9.9 0.7
15.6 0.8
a
Key to cell lines: KB, human oral epithelial cell line; A549, human lung adenocarcinoma cell line; BGC-823, human gastric cancer cell line; HO8910, human ovarian cancer
line; the rest human tumor cell lines seen from Table 1.
b
IC50 values greater than 200 lM were considered as inactive and omitted here.
The aza-caffeic acid derivatives (73–76, 78) with a seven bond
linker B length were achieved by coupling dicyclohexylcarbodiim-
ide (DCC) activated cinnamic acids (69–72) with commercially
available corresponding substituted ethylamines in the yields of
46–86% (Scheme 2).17 Target compounds 77 and 79 with two free
hydroxy groups were obtained by hydrolyzation of the corresponding
Scheme 1. Synthesis of aza-caffeic acid derivatives 37–51 and 59–63. Reagents and conditions: (a) for 5: MOMCl, K2CO3, acetone, reflux, 4 h, 86%; for 6–7: RBr, K2CO3,
acetone, reflux, 3 h; (b) MOMCl, K2CO3, acetone, reflux, 4 h, 89%; (c) Ph3PCH@CO2Et, benzene, reflux, 2 h; (d) KOH, EtOH, H2O, reflux, 3 h; (e) ClCH2COCl, K2CO3, CH2Cl2, reflux,
4 h; (f) KI, DMF, reflux, 4 h; (g) HCl, MeOH, 50 °C, 1 h.

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H. Zou et al. / Bioorg. Med. Chem. 18 (2010) 6351–6359
Scheme 2. Synthesis of aza-caffeic acid derivatives 73–84 and 88. Reagents and conditions: (a) MOMCl, K2CO3, acetone, reflux, 4 h, 86%; (b) malonic acid, pyridine, reflux, 3 h;
(c) K2CO3, piperazine, acetone, reflux, 2 h, 92%; (d) DCC, CHCl3, 45 °C, 1 h; RCH2CH2NH2, reflux, 5 h; (e) HCl, MeOH, 50 °C, 1 h.
Scheme 3. Synthesis of aza-caffeic acid derivative 91. Reagents and conditions: (a) NH2NH2ÁH2O, EtOH, reflux, 82%; (b) trans-cinnamic acid, SOCl2, CHCl3, 0–76 °C, 86%.
76 and 78.16 Compounds 69–72 were prepared by Knoevenagel
condensation of malonic acid and the aldehyde group of corre-
sponding intermediates (64–66, 68) which were synthesized from
protocatechualdehyde and syringaldehyde as described previ-
ously.9,17 In order to introduce two nitrogens with a nine bond
linker B length, piperazine, which was coupled with 3, and 4-
dichlorobenzyl bromide (85), was utilized to construct 80–83,
followed by hydrolyzation of 83 to afford 84.9,16 3,4-Dichloroben-
zyl piperazine (86) was also used to form 88, which has one more
carbonyl as a prolonged conjugated system, by coupling with
benzoylacrylic acid (87).17
In order to obtain significant insight into the impact of activity
on molecular diversity, compound 91, possessing two connected
nitrogens in a seven bond linker length, was synthesized via a
two step reaction starting from salicylic acid methyl ester (89,
Scheme 3).18 The structures of these compounds were confirmed
both by mass spectrometry and 1H NMR spectral data.
Analysis of the MTT assay results, suggest that analogues of series
3 with a piperazine linker were generally more potent than those
from series 1 and 2. The bioassay results also suggest that HL-60
and K562 cell lines and, in particular K562, are much more sensitive
to the synthesized aza-caffeic acid derivatives. Four compounds (39,
42, 80, 91) showed significant cytotoxicity, with IC50 values in the
micromolar range against the K562 cell line, 10 and 50 times higher
than the control CDDP and CAPE values, respectively.
In series 2, compounds 73–77 were found to have no cytotoxic
activity towards all 12 cell lines tested, while compounds 78 and
79 were selectively cytotoxic against a number of cancer cell lines.
These results suggest that series 2 compound cytotoxicity may be
contained within the electron rich aromatic system of region C, as
opposed to the electron poor heterocycle like morpholinyl. Com-
paring compounds 79, 74, and 78, it indicates that various series
2 substituents of region A are crucial for activity: compound 79,
possessing two free hydroxy groups, showed selective and signifi-
cant cytotoxicity, particularly with respect to HeLa and LS174T cell
2.2. Biological activity tests
lines, with IC50 values of 2.7 and 8.4
The bioassay results of 81–84 demonstrated the crucial effect of
double substituents of region on activity as follows: 83
(OMOM) P 81 (H) > 82 (OMe) > 84 (OH), this trend is dissimilar
to that observed for analogues of series 2 (74, 78, and 79). Com-
pared to 81, compound 88, which possesses 10 linker B bonds
including an additional conjugated carbonyl, lost cytotoxicity
against PC-3, K562, and HO8910 cell lines and showed weaker
activity to the remaining cell lines; this confirms that the most
lM, respectively.
In order to perform the biological activity tests, caffeic phenyl
ethyl ester (CAPE) and cisplatin (CDDP) were chosen as positive
controls. Compounds 37–51, 59–63, and 73–84, 88, 91 were eval-
uated against 8 and 12 cancer cell lines, respectively, by the color-
imeter assay MTT.19–21 Results showing the concentrations
required to inhibit cell growth by 50% (IC50 values) are presented
in Tables 1 and 2.
A

H. Zou et al. / Bioorg. Med. Chem. 18 (2010) 6351–6359
6355
favorable linker B length is nine bonds. Also of note is the finding
that hydrazide 91, possessing a seven bond linker B length, exhib-
ited cytotoxicity against all 12 of the cancer cell lines, the best with
in Figure 3D, 48 h treatment with compound 80 dramatically in-
creased the Bax/Bcl-2 ratio in a concentration dependent manner,
indicating that mitochondria dysfunction was involved in com-
pound 80 induced cell apoptosis.
an IC50 value of 0.9 lM being observed for the K562 cell. Among
these aza-caffeic acid derivatives, compound 80 of series 3 was
found to be the most potent and selective cytotoxic agent, showing
IC50 values of 0.2, 2.0, 1.7, and 1.1 lM against KB, BEL7404, K562,
3. Conclusions
and Eca109 cell lines, respectively, higher than those observed
for the two control compounds CAPE and CDDP.
Three series of 34 aza-caffeic acid derivatives, including com-
pounds 88 and 91 were synthesized. Compounds 37–51, 59–63
and 73–84, 88, 91 were used to screen 8 or 12 cultured human can-
cer cell lines, respectively. It was found that HL-60 and K562 cell
lines, in particular K562, were especially sensitive to the synthe-
sized compounds. Four compounds (39, 42, 80, and 91) showed
significant cytotoxicity, with IC50 values on K562 cell line in the
micromolar range, 10 and 50 times better than the control com-
pounds CDDP and CAPE, respectively. The aza-caffeic acid deriva-
tives with nine bond linkers show better cytotoxicity to the
cancer cell lines than the other series of compounds, which indi-
cates that the linker with nine bonds might be the best linker
length in aza-caffeic acid derivatives against the investigated can-
cer cell lines. Yet this observation still needs to be confirmed by
testing N-containing caffeic acid derivatives with different linker
lengths. The structure–cytotoxicity relationship of these analogues
based on three structural variations of region A, B, and C was dis-
cussed. This extensive SAR analysis will guide us in the design of
optimized aza-caffeic derivatives. Among these aza-caffeic acid
derivatives compound 80 is the most potent and selective cyto-
toxic agent against KB, BEL7404, K562, and Eca109 cell lines with
In order to determine the possible mechanism involved in the
cytotoxicity, the K562 cell line treated with compound 80 was cho-
sen for further biological analysis. The results show a concentration-
and time-dependency for both the cell viability and apoptosis rate
by MTT assay and flow cytometric analysis, respectively (Fig. 3A
and B). With treatment of compound 80 at 8.0 lM for 72 h, the
K562 cell viability was reduced to 37.9 1.4% of the control
(Fig. 3A) and 20.4% of the cells were detected to be apoptotic cells
(Fig. 3B). The apoptotic cells in both early and late stage were de-
tected by 48 h treatment of compound 80 while only apoptosis in
late stage was observed at 72 h with double positive dyeing of An-
nexin V and PI. Simultaneously, the apoptotic protein caspase-3
expression level was also up-regulated detected by western blot
after 48 h treatment with compound 80 (Fig. 3C). These results indi-
cate that compound 80 suppresses K562 cell viability by, at least in
part inducing cell apoptosis.
Bcl-2 and Bax are suggested to be key Bcl-2 family members in
determining K562 cell survival.22 The Bax/Bcl-2 ratio is an index
suggesting cell apoptosis tendency. Bcl-2 family proteins are up-
stream regulators responsible for controlling mitochondria-depen-
dent cell apoptosis. Both Bax and Bcl-2 protein expression was de-
tected after 48 h incubation with the compounds. As results shown
IC50 values of 0.2, 2.0, 1.7, and 1.1 lM, respectively, which is stron-
ger than CAPE and CDDP. Based on a series of biological studies,
compound 80 demonstrates a role in mitochondria-dependent
Figure 3. Apoptosis involved in compound 80 cytotoxicity on K562 cells. K562 cells were exposed to indicate concentrations of compound 80 for 48 or 72 h. (A) Cell viability
was determined by MTT assay with 48 and 72 h compound 80 incubation, respectively. (B) Cell apoptosis rate following Annexin V/propidium iodide staining was quantified
by flow cytometric analysis after 72 h compound 80 exposure. Cell apoptosis rate in each group was marked. (C) Western blot analysis was carried out to determine caspase-
3, Bax, and Bcl-2 expression. The treatment duration was indicated. (D) Bands were semi-quantified by densitometric measurement. Data were expressed as mean S.D. from
at least three independent experiments. **P <0.01, ***P <0.001 versus control.

6356
H. Zou et al. / Bioorg. Med. Chem. 18 (2010) 6351–6359
apoptosis activity by suppressing K562 cell proliferation in a con-
centration- and time-dependent manner. These results shed light
on the possibility of developing compound 80 as a lead compound
for a promising antitumor agent especially for leukemia.
and purified by column chromatography (pet. ether/EtOAc 7:1 to
2:1) to afford 31–44 and 59–63 (56–87%).
4.2.2.1. (2E)-3-(3,4-Dimethoxyphenyl)-2-oxo-2-(phenylamino)-
2-propenoic acid ethyl ester (37). Yield: 76%; yellow powder;
mp 165–166 °C; Rf (CH2Cl2/MeOH 20:1) 0.42; 1H NMR (400 MHz,
CDCl3): d 7.87 (1H, br s), 7.78 (1H, d, J = 16.0 Hz), 7.57 (2H, d,
J = 8.0 Hz), 7.36 (3H, m), 7.17 (1H, d, J = 7.2 Hz), 7.10 (1H, s), 6.91
(1H, d, J = 8.0 Hz), 6.43 (1H, d, J = 15.6 Hz), 4.84 (2H, s), 3.95 (6H,
s); ESI-MS m/z [M+H]+ 342.
4. Experimental section
4.1. Materials
Melting points were measured on a Perkin-Taike X-4 apparatus
and then corrected. 1H NMR and 13C NMR spectra were recorded
on a Varian INOVA 400 spectrometer with TMS as an internal stan-
dard and CDCl3 as solvent. ESI-MS data were recorded on a Bruker
Esquire 3000+ spectrometer and EI-MS was performed on a Varian
MAT-95 MS instrument. TLC was performed on Silica Gel (GF254).
Column chromatography was carried out on silica gel H (10–
4.2.2.2.
(2E)-3-(3,4-Dimethoxyphenyl)-2-oxo-2-(4-chlorophe-
nylamino)-2-propenoic acid ethyl ester (38). Yield: 80%; yellow
powder; mp 167–169 °C; Rf (CH2Cl2/MeOH 20:1) 0.44; 1H NMR
(400 MHz, CDCl3): d 7.88 (1H, br s), 7.78 (1H, d, J = 16.0 Hz), d
7.53 (2H, d, J = 8.8 Hz), 7.32 (2H, d, J = 8.4 Hz), 7.18 (1H, d,
J = 8.4 Hz), 7.09 (1H, s), 6.91 (1H, d, J = 8.4 Hz), 6.43 (1H, d,
J = 16.0 Hz), 5.94 (2H, s), 3.95 (6H, s); ESI-MS m/z [M+H]+ 376.
40 lm). All of the Silica Gel GF254 and Silica Gel H were purchased
from Qingdao Marine Chemical Factory, China.
4.2.2.3.
(2E)-3-(3,4-Dimethoxyphenyl)-2-oxo-2-[(2-furanylm-
4.2. Synthesis
ethyl)amino]-2-propenoic acid ethyl ester (39). Yield: 79%; yel-
low powder; mp 145–146 °C; Rf (CH2Cl2/MeOH 20:1) 0.40; 1H NMR
(400 MHz, CDCl3): d 7.76 (1H, d, J = 16.0 Hz), 7.50 (1H, d, J = 0.8 Hz),
7.14 (1H, d, J = 8.4 Hz), 7.08 (1H, s), 6.91 (1H, d, J = 8.4 Hz), 6.52
(1H, br s), 6.40 (1H, d, J = 16.0 Hz), 6.33 (1H, dd, J = 2.8, 0.8 Hz),
6.28 (1H, dd, J = 2.8, 1.8 Hz), 5.94 (2H, s), 4.53 (2H, d, J = 6.0 Hz),
3.95 (6H, s); ESI-MS m/z [M+H]+ 346.
4.2.1. General procedure for the preparation of intermediates
4.2.1.1. General procedure for the preparation of substituted
cinnamic acid potassium salts 12–14 and 56–58. A solution of
KOH (1 M, 4.3 mL) was added to ethanol (20 mL) dissolved substi-
tuted cinnamic acid ethyl ester derivatives 9–11 and 53–55
(4.0 mmol), which were prepared as described previously using
protocatechualdehyde and syringaldehyde as starting material.9,17
The mixture was refluxed for 3 h and then cooled to room temper-
ature. The solvent was removed and the concentrate was dried
overnight by vacuum drying oven to obtain 12–14 and 56–58 as
intermediates for the synthesis of 31–44 and 59–63, respectively.
4.2.2.4. (2E)-3-(3-Methoxymethoxy-4-phenylmethoxy)-2-oxo-
2-[(4-chlorophenyl)amino]-2-propenoic acid ethyl ester (40).
Yield: 87%; white powder; mp 132–133 °C; Rf (pet. ether/EtOAc 3:1)
0.22; 1H NMR (400 MHz, CDCl3): d 7.82 (1H, br s), 7.74 (1H, d,
J = 16.0 Hz), 7.53 (2H, d), 7.44–7.31 (8H, m), 7.15 (1H, d, J = 8.0 Hz),
6.93 (1H, d, J = 8.8 Hz), 6.41 (1H, d, J = 15.6 Hz), 5.28 (2H, s), 5.22
(2H, s), 4.82 (2H, s), 3.55 (3H, s); ESI-MS m/z [M+H]+ 482.
4.2.1.2. General procedure for the preparation of N-substituted-
2-chloro-acetamides 23–30. K2CO3 (25 mmol) was added to a
solution of substituted amines (10 mmol) in CH2Cl2 (60 mL) and
the suspension was stirred for 30 min at room temperature. Chlo-
roacetyl chloride (0.88 mL, 11 mmol) was slowly added to the mix-
ture and was refluxed for 4 h after 10 min stirring. After cooling to
room temperature the filtrate was concentrated to obtain the res-
idue which was recrystallized in hexane to afford 23–30 (69–91%).
4.2.2.5. (2E)-3-(3-Methoxymethoxy-4-phenylmethoxy)-2-oxo-
2-[(4-morpholinyl)amino]-2-propenoic acid ethyl ester (41).
Yield: 61%; white powder; mp 117–119 °C; Rf (pet. ether/EtOAc
3:1) 0.26; 1H NMR (400 MHz, CDCl3): d 7.73 (1H, br s), 7.64 (1H, d,
J = 16.0 Hz), 7.49 (2H, d, J = 6.8 Hz), 7.43–7.35 (3H, m), 7.39 (1H, s),
7.25 (1H, d, J = 8.4 Hz), 7.10 (1H, d, J = 8.8 Hz), 6.40 (1H, d,
J = 16.0 Hz), 5.26 (2H, s), 5.20 (2H, s), 4.04 (2H, s), 3.81 (4H, m),
3.46 (3H, s), 2.85 (4H, m); ESI-MS m/z [M+H]+ 457.
4.2.1.3. General procedure for the preparation of substituted
cinnamic acids 69–72. Compounds 64–66 and 68 were synthe-
sized as described previously.9,17 Malonic acid (2.1 g, 20 mmol)
and two drops of piperidine were added to corresponding alde-
hydes 64–66 and 68 (16 mmol) in pyridine (20 mL). The mixture
was refluxed for 3–4 h and evaporated to remove pyridine. The res-
idue was suspended in H2O (30 mL) and extracted with EtOAc
(2 Â 50 mL) which was further purified by column chromatogra-
phy (pet. ether/EtOAc/HCOOH 4:1:0.05 to 3:1:0.05) to afford 69–
72 (71–92%).
4.2.2.6. (2E)-3-(3-Methoxymethoxy-4-phenylmethoxy)-2-oxo-
2-[(2-furanylmethyl)amino]-2-propenoic acid ethyl ester (42).
Yield: 60%; white powder; mp 120–122 °C; Rf (pet. ether/EtOAc
3:1) 0.23; 1H NMR (400 MHz, CDCl3): d 7.67 (1H, d, J = 15.6 Hz),
7.44–7.31 (7H, m), 7.13 (1H, d, J = 8.4 Hz), 6.92 (1H, d, J = 8.0 Hz),
6.52 (1H, br s), 6.35 (1H, d, J = 16.0 Hz), 6.33 (1H, d, J = 2.0 Hz),
6.28 (1H, d, J = 2.8 Hz), 5.27 (2H, s), 5.21 (2H, s), 4.72 (2H, s), 4.53
(2H, d, J = 6.0 Hz), 3.54 (3H, s); ESI-MS m/z [M+H]+ 452.
4.2.2. General procedure for the preparation ofN-substituted-3-
phenyl-2-oxo-2-amino-2-propenoic acid ethyl ester derivatives
31–44, 59–63
4.2.2.7. (2E)-3-(3-Methoxymethoxy-4-phenylmethoxy)-2-oxo-2-
(2-thiazolylamino)-2-propen-oic acid ethyl ester (43). Yield:
63%; white powder; mp 133–134 °C; Rf (pet. ether/EtOAc 3:1)
0.26; 1H NMR (400 MHz, CDCl3): d 7.74 (1H, d, J = 16.0 Hz), 7.48–
7.31 (7H, m), 7.15 (1H, d, J = 8.4 Hz), 7.04 (1H, s), 6.90 (1H, d,
J = 8.4 Hz), 6.45 (1H, d, J = 15.6 Hz), 5.25 (2H, s), 5.18 (2H, s), 4.95
(2H, s), 3.52 (3H, s); ESI-MS m/z [M+H]+ 455.
KI (91 mg, 0.55 mmol) was added to N-substituted-2-chloro-
acetamides 23–30 (0.50 mmol) in dry DMF (10 mL), and the sus-
pension stirred at 40 °C for 20 min. Dry potassium salts 12–14
and 56–58 (0.58 mmol) prepared as described in Section 4.2.1.1
was added to the suspension and refluxed for 4 h followed by evap-
oration under reduced pressure to remove DMF. The residue was
extracted with EtOAc (2 Â 20 mL) and the organic layer washed
to neutral by water (2 Â 15 mL) and brine (2 Â 10 mL) followed
by overnight drying over Na2SO4. The filtrate was concentrated
4.2.2.8. (2E)-3-(3-Methoxymethoxy-4-phenylmethoxy)-2-oxo-
2-[(4-methoxyphenyl)amino]-2-propenoic acid ethyl ester (44).
Yield: 80%; white powder; mp 129–130 °C; Rf (pet. ether/EtOAc
3:1) 0.24; 1H NMR (400 MHz, (CD3)2CO): d 9.18 (1H, br s), 7.67

H. Zou et al. / Bioorg. Med. Chem. 18 (2010) 6351–6359
6357
(1H, d, J = 16.4 Hz), 7.56–7.30 (9H, m), 7.14 (1H, d, J = 8.0 Hz), 6.86
(2H, d, J = 8.8 Hz), 6.49 (1H, d, J = 16.0 Hz), 5.25 (2H, s), 5.21 (2H, s),
4.74 (2H, s), 3.74 (3H, s), 3.46 (3H, s); ESI-MS m/z [M+H]+ 478.
4.2.2.15.
(2E)-3-(3,4-Dihydroxy)-2-oxo-2-(4-pentylamino)-2-
propenoic acid ethyl ester (51). This compound was prepared
in the same way as 46 from 36. Yield: 71%; white powder; mp
168–169 °C; Rf (CH2CH2/MeOH 20:1) 0.46; 1H NMR (400 MHz,
(CD3)2CO): d 8.61 (1H, br s), 8.37 (1H, br s), 7.58 (1H, d,
J = 16.0 Hz), 7.43 (1H, br s), 7.14 (1H, s), 7.03 (1H, d, J = 6.8 Hz),
6.86 (1H, d, J = 8.4 Hz), 4.58 (2H, s), 3.21 (2H, m), 1.47 (2H, m),
1.28 (4H, m), 0.87 (3H, t, J = 6.8 Hz); ESI-MS m/z [M+H]+ 308.
4.2.2.9. (2E)-3-(3-Hydroxy-4-phenylmethoxy)-2-oxo-2-[(4-meth-
oxyphenyl)amino]-2-propen-oic acid ethyl ester (45). Hydro-
chloric acid (1 M, 0.3 mL) was added to 44 (124 mg, 0.26 mmol)
in methanol (5 mL) and stirred at 55 °C for 30 min. After cooling
to room temperature it was concentrated and the residue purified
by column chromatography with CH2Cl2/MeOH (70:1) as eluting
system to get 45 (82 mg). Yield: 73%; yellow powder; mp 173–
174 °C; Rf (pet. ether/EtOAc 4:3) 0.36; 1H NMR (400 MHz,
4.2.2.16. (2E)-3-[3,5-Dimethoxy-4-(3,4-dichlorophenylmethoxy)]-
2-oxo-2-[(4-methoxy-phenyl)amino]-2-propenoic acid ethyl ester
(59). Yield: 72%; white powder; mp 151–153 °C; Rf (hexane/EtoAc
3:2) 0.36; 1H NMR (400 MHz, (CD3)2CO): d 9.20 (1H, br s), 7.77
(1H, d, J = 1.6 Hz), 7.70 (1H, d, J = 16.0 Hz), 7.58–7.56 (3H, m,
J = 8.8 Hz), 7.48 (1H, dd, J = 8.0, 1.6 Hz), 7.10 (2H, s), 6.88 (2H, d,
J = 8.4 Hz), 6.40 (1H, d, J = 16.0 Hz), 5.06 (2H, s), 4.78 (2H, s), 3.92
(6H, s), 3.76 (3H, s); ESI-MS m/z [M+H]+ 546.
(CD3)2CO):
d 9.12 (1H, br s), 8.08 (1H, br s), 7.64 (1H, d,
J = 15.6 Hz), 7.55 (2H, d, J = 8.8 Hz), 7.47 (2H, d, J = 8.8 Hz), 7.40–
7.31 (3H, m), 7.22 (1H, s), 7.14–7.07 (2H, m), 6.85 (2H, d,
J = 9.2 Hz), 6.45 (1H, d, J = 15.6 Hz), 5.21 (2H, s), 4.73 (2H, s), 3.74
(3H, s); ESI-MS m/z [M+H]+ 434.
4.2.2.17. (2E)-3-[3,5-Dimethoxy-4-(3,4-dichlorophenylmethoxy)]-
2-oxo-2-[(4-cholorphenyl)-amino]-2-propenoic acid ethyl ester
(60). Yield: 70%; yellow powder; mp 164–166 °C; Rf (hexane/EtoAc
5:2) 0.30; 1H NMR (400 MHz, (CD3)2CO): d 9.44 (1H, br s), 7.77 (1H,
d, J = 2.0 Hz), 7.70 (2H, d, J = 8.8 Hz), 7.68 (1H, d, J = 16.0 Hz), 7.56
(1H, d, J = 8.0 Hz), 7.47 (1H, d, J = 8.0 Hz), 7.34 (2H, m), 7.10 (2H, s),
6.63 (1H, d, J = 16.0 Hz), 5.06 (2H, s), 4.81 (2H, s), 3.92 (6H, s); ESI-
MS m/z [M+H]+ 550.
4.2.2.10. (2E)-3-(3,4-Dihydroxy)-2-oxo-2-[(4-morpholinyl)amino]-
2-propenoic acid ethyl ester (46). HCl (1 M, 0.6 mL) was added to
31 (102 mg, 0.25 mmol) in methanol (5 mL) and stirred at 55 °C for
30 min followed by evaporation to remove methanol. The residue
was purified by column chromatography (CH2Cl2/MeOH 55:1) to
get 46 (45 mg). Yield: 56%; white powder; mp 145–146 °C; Rf
(CH2Cl2/MeOH 20:1) 0.33; 1H NMR (400 MHz, CDCl3): d 7.67 (1H, d,
J = 16.0 Hz), 7.11 (1H, s), 7.06 (1H, d, J = 8.4 Hz), 6.83 (1H, d,
J = 8.4 Hz), 6.39 (1H, d, J = 16.4 Hz), 5.05 (1H, br s), 4.64 (2H, s), 3.80
(4H, m), 2.86 (4H, m); ESI-MS m/z [M+H]+ 323.
4.2.2.18. (2E)-3-[3,5-Dimethoxy-4-(4-ethoxyphenylmethoxy)]-
2-oxo-2-[(4-cholorphenyl)-amino]-2-propenoic acid ethyl ester
(61). Yield: 67%; white powder; mp 180–182 °C; Rf (hexane/EtoAc
5:2) 0.21; 1H NMR (400 MHz, (CD3)2CO): d 9.49 (1H, br s), 7.70 (2H,
d, J = 8.8 Hz), 7.69 (1H, d, J = 16.0 Hz), 7.40 (2H, d, J = 8.0 Hz), 7.34
(2H, m), 7.07 (2H, s), 6.88 (2H, d, J = 7.6 Hz), 6.61 (1H, d,
J = 16.0 Hz), 4.96 (2H, s), 4.80 (2H, s), 4.04 (2H, q, J = 7.2 Hz), 3.90
(6H, s), 1.35 (3H, t, J = 7.2 Hz); ESI-MS m/z [M+H]+ 526.
4.2.2.11.
(2E)-3-(3,4-Dihydroxy)-2-oxo-2-(4-phenylamino)-2-
propenoic acid ethyl ester (47). This compound was prepared
in the same way as 46 from 32. Yield: 74%; yellow powder; mp
165–166 °C; Rf (CH2Cl2/MeOH 20:1) 0.42; 1H NMR (400 MHz,
(CD3)2CO): d 9.29 (1H, br s), 8.60 (1H, br s), 8.31 (1H, br s), 7.65
(2H, d, J = 7.2 Hz), 7.31–7.27 (2H, m), 7.15 (1H, s), 7.08–7.04 (3H,
m), 6.87 (1H, d, J = 7.2 Hz), 6.39 (1H, d, J = 16.0 Hz), 4.76 (2H, s);
ESI-MS m/z [M+H]+ 314.
4.2.2.19. (2E)-3-[3,5-Dimethoxy-4-(4-ethoxyphenylmethoxy)]-2-
oxo-2-(phenylamino)-2-propenoic acid ethyl ester (62). Yield:
70%; white powder; mp 133–134 °C; Rf (hexane/EtoAc 3:2) 0.40;
1H NMR (400 MHz, (CD3)2CO): d 9.33 (1H, br s), 7.70 (1H, d,
J = 16.0 Hz), 7.67 (2H, m), 7.40 (2H, d, J = 8.0 Hz), 7.32 (2H, dd,
J = 7.6, 7.6 Hz), 7.09 (1H, m), 7.08 (2H, s), 6.88 (2H, d, J = 7.6 Hz),
6.63 (1H, d, J = 16.0 Hz), 4.95 (2H, s), 4.80 (2H, s), 4.04 (2H, q,
J = 6.8 Hz), 3.90 (6H, s), 1.35 (3H, t, J = 6.8 Hz); 13C NMR (CDCl3,
100 MHz): d 166.6, 166.3, 159.7, 154.8, 154.0 146.6, 140.1, 139.4,
130.8, 130.6, 129.5, 124.6, 120.4, 117.2, 114.7, 106.6, 74.8, 63.8,
63.5, 56.5, 15.0; ESI-MS m/z [M+H]+ 492.
4.2.2.12.
(2E)-3-(3,4-Dihydroxy)-2-oxo-2-[(4-methylphenyl)-
amino]-2-propenoic acid ethyl ester (48). This compound was
prepared in the same way as 46 from 33. Yield: 75%; yellow pow-
der; mp 202–203 °C; Rf (CH2Cl2/MeOH 20:1) 0.44; 1H NMR
(400 MHz, (CD3)2CO): d 9.11 (1H, br s), 8.51 (1H, br s, OH-7), 8.23
(1H, br s), 7.52 (1H, d, J = 16.0 Hz), 7.46 (2H, d, J = 7.6 Hz), 7.11
(1H, s), 7.04–6.99 (3H, m), 6.80 (1H, d, J = 7.6 Hz), 6.27 (1H, d,
J = 15.6 Hz), 4.67 (2H, s), 2.18 (3H, s); ESI-MS m/z [M+H]+ 329.
4.2.2.20. (2E)-3-[3,5-Dimethoxy-4-(phenylmethoxy)]-2-oxo-2-[(4-
methoxyphenyl)amino]-2-propenoic acid ethyl ester (63). Yield:
68%; white powder; mp 155–156 °C; Rf (hexane/EtoAc 3:2) 0.33; 1H
NMR (400 MHz, (CD3)2CO): d 9.20 (1H, br s), 7.70 (1H, d, J = 16.0 Hz),
7.57 (2H, d, J = 8.8 Hz), 7.52 (2H, d, J = 7.6 Hz), 7.36 (2H, dd, J = 7.2,
7.2 Hz), 7.30 (1H, dd, J = 7.2, 7.2 Hz), 7.09 (2H, s), 6.88 (2H, d,
J = 8.8 Hz), 6.63 (1H, d, J = 16.0 Hz), 5.03 (2H, s), 4.78 (2H, s), 3.91
(6H, s), 3.76 (3H, s); ESI-MS m/z [M+H]+ 478.
4.2.2.13. (2E)-3-(3,4-Dihydroxy)-2-oxo-2-[(4-chlorophenyl)amino]-
2-propenoic acid ethyl ester (49). This compound was prepared in
the same way as 46 from 34. Yield: 78%; yellow powder; mp 167–
169 °C; Rf (CH2Cl2/MeOH 20:1) 0.44; 1H NMR (400 MHz, (CD3)2CO):
d 7.67 (2H, d, J = 8.8 Hz), 7.52 (1H, d, J = 16.0 Hz), 7.25 (2H, d,
J = 8.4 Hz), 7.08 (1H, s), 7.00 (1H, d, J = 8.0 Hz), 6.80 (1H, d,
J = 8.4 Hz), 6.31 (1H, d, J = 15.6 Hz), 4.69 (2H, s); ESI-MS m/z [M+H]+
348.
4.2.3. General procedure for the preparation of N-substituted-3-
phenyl-2-propenamide derivatives 73–76, 78 and 4-(3,4-dichlor-
ophenylmethyl)-1-(phenyl-1-oxo-2-propenyl)-piperazine
derivatives 80–83
DCC (512 mg, 2.0 mmol) was added to substituted cinnamic
acid derivatives 69–72 (2.0 mmol) in CHCl3 (15 mL) and was stir-
red at 45 °C for 1 h. Substituted amines (2.2 mmol) was added
and the reaction mixture refluxed for 8 h. After cooling to room
temperature, it was filtrated and concentrated to obtain a residue
4.2.2.14. (2E)-3-(3,4-Dihydroxy)-2-oxo-2-[(4-methoxyphenyl)-
amino]-2-propenoic acid ethyl ester (50). This compound was
prepared in the same way as 46 from 35. Yield: 70%; yellow pow-
der; mp 202–203 °C; Rf (CH2Cl2/MeOH 20:1) 0.36; 1H NMR
(400 MHz, (CD3)2CO): d 9.13 (1H, br s), 8.59 (1H, br s), 8.34 (1H,
br s), 7.61 (1H, d, J = 16.0 Hz), 7.55 (2H, d, J = 8.8 Hz), 7.17 (1H, s),
7.06 (1H, d, J = 8.4 Hz), 6.78 (3H, m), 6.34 (1H, d, J = 16.0 Hz),
4.72 (2H, s), 3.74 (3H, s); ESI-MS m/z [M+H]+ 344.

6358
H. Zou et al. / Bioorg. Med. Chem. 18 (2010) 6351–6359
which was further purified by column chromatography using
CH2Cl2/MeOH (100:1 to 30:1) as the eluting system, to afford
73–76, 78 and 80–83 (46–86%).
4.2.3.9. (E)-4-(3,4-Dichlorophenylmethyl)-1-[1-oxo-3-phenyl-2-
propenyl]-piperazine (81). Yield: 67%; white powder; mp 114–
116 °C; Rf (CH2Cl2/MeOH 12:1) 0.58; 1H NMR (400 MHz, CDCl3):
d 7.68 (1H, d, J = 15.6 Hz), 7.53 (2H, dd, J = 8.0, 2.0 Hz), 7.46 (1H,
s), 7.40 (1H, d, J = 8.0 Hz), 7.38–7.36 (3H, m), 7.18 (1H, d,
J = 8.4 Hz), 6.87 (1H, d, J = 15.6 Hz), 3.76–3.66 (4H, m), 3.48 (2H,
s), 2.47 (4H, t, J = 4.4 Hz); ESI-MS m/z [M+H]+ 375.
4.2.3.1.
(2E)-3-(3,4-Dimethoxyphenyl)-N-[2-(4-morpholinyl)-
ethyl]-2-propenamide (73). Yield: 67%; white powder; mp
173–174 °C; Rf (CH2Cl2/MeOH 12:1) 0.49; 1H NMR (400 MHz,
CDCl3): d 7.58 (1H, d, J = 15.6 Hz), 7.11 (1H, dd, J = 8.4, 1.6 Hz),
7.05 (1H, d, J = 1.6 Hz), 6.87 (1H, d, J = 8.4 Hz), 6.31 (1H, d,
J = 15.6 Hz), 6.15 (1H, br s), 3.93–3.92 (6H, s), 3.75 (4H, t,
J = 4.4 Hz), 3.51 (2H, q, J = 5.6 Hz), 2.56 (2H, t, J = 6.0 Hz), 2.49
(4H, m); ESI-MS m/z [M+H]+ 321.
4.2.3.10.(E)-4-(3,4-Dichlorophenylmethyl)-1-[3-(3,4-dimethoxy-
phenyl)-1-oxo-2-propenyl]-piperazine (82). Yield: 56%; pale yel-
low oil; Rf (CH2Cl2/MeOH 12:1) 0.58; 1H NMR (400 MHz, CDCl3): d
7.63 (1H, d, J = 15.2 Hz), 7.46 (1H, s), 7.40 (1H, d, J = 8.0 Hz), 7.18 (1H,
d, J = 8.0 Hz), 7.11 (1H, dd, J = 8.4, 1.6 Hz), 7.03 (1H, s), 6.86 (1H, d,
J = 8.4 Hz), 6.73 (1H, d, J = 15.2 Hz), 3.92–3.91 (6H, s), 3.76–3.67
(4H, m), 3.48 (2H, s), 2.47 (4H, m); ESI-MS m/z [M+H]+ 435.
4.2.3.2. (2E)-3-(3,4-Dimethoxyphenyl)-N-[2-(1H-indol-3-yl)ethyl]-
2-propenamide (74). Yield: 54%; yellow powder; mp 150–151 °C;
Rf (CH2Cl2/MeOH 12:1) 0.66; This compound was identical to that re-
ported by Michalet et al.12
4.2.3.11. (E)-4-(3,4-Dichlorophenyl-methyl)-1-[3-(3,4-dimeth-
oxymethoxyphenyl)-1-oxo-2-propenyl]-piperazine (83). Yield:
51%; pale yellow oil; Rf (CH2Cl2/MeOH 12:1) 0.57; 1H NMR
4.2.3.3. (2E)-3-[3,5-Dimethoxy-4-(4-ethoxyphenylmethoxy)]-N-
[2-(4-morpholinyl)ethyl]-2-propenamide (75). Yield: 59%; white
powder; mp 162–164 °C; Rf (CH2Cl2/MeOH 12:1) 0.45; 1H NMR
(400 MHz, CDCl3): d 7.55 (1H, d, J = 15.6 Hz), 7.37 (2H, d, J = 8.8 Hz),
6.85 (2H, d, J = 8.4 Hz), 6.72 (2H, s), 6.33 (1H, d, J = 15.6 Hz), 6.17
(1H, br s), 4.97 (2H, s), 4.02 (2H, q, J = 6.8 Hz), 3.85 (6H, s), 3.74 (4H,
t, J = 4.4 Hz), 3.50 (2H, q, J = 6.0 Hz), 2.56 (2H, t, J = 6.0 Hz), 2.49 (4H,
m), 1.41 (3H, t, J = 6.8 Hz); ESI-MS m/z [M+H]+ 471.
(400 MHz, CDCl3):
d 7.60 (1H, d, J = 15.6 Hz), 7.46 (1H, d,
J = 1.2 Hz), 7.40 (1H, d, J = 8.4 Hz), 7.34 (1H, d, J = 1.2 Hz), 7.18
(1H, dd, J = 8.0, 2.0 Hz), 7.15 (1H, dd, J = 8.0, 2.4 Hz), 7.12 (1H, d,
J = 8.4 Hz), 6.73 (1H, d, J = 15.2 Hz), 5.27 (4H, s), 3.75–3.66 (4H,
m), 3.52 (6H, s), 3.49 (2H, s), 2.46 (4H, m); ESI-MS m/z [M+H]+ 495.
4.2.3.12. (E)-4-(3,4-Dichlorophenylmethyl)-1-[3-(3,4-dihydroxy-
phenyl)-1-oxo-2-propenyl]-piperazine (84). This compound
was prepared in the same way as 42 from 80; yield: 54%; white
powder; mp 197–199 °C; Rf (CH2Cl2/MeOH 12:1) 0.39; 1H NMR
(400 MHz, CD3OD): d 7.81 (1H, br s), 7.73 (1H, d, J = 8.4 Hz), 7.56
(1H, d, J = 15.2 Hz), 7.52 (1H, d, J = 8.4 Hz), 7.11 (1H, s), 7.04 (1H,
d, J = 8.0 Hz), 6.94 (1H, d, J = 15.2 Hz), 6.82 (1H, d, J = 8.0 Hz), 4.38
(4H, m), 3.48 (2H, s), 3.47 (4H, m); ESI-MS m/z [M+H]+ 407.
4.2.3.4. (2E)-3-(3,4-Dimethoxymethoxy)-N-[2-(4-morpholinyl)-
ethyl]-2-propenamide (76). Yield: 58%; white powder; mp 155–
157 °C; Rf (CH2Cl2/MeOH 12:1) 0.45; 1H NMR (400 MHz, CDCl3): d
7.56 (1H, d, J = 15.6 Hz), 7.38 (1H, d, J = 1.6 Hz), 7.16 (1H, d,
J = 8.4 Hz),7.12 (1H, dd, J = 8.4, 1.6 Hz), 6.31 (1H, d, J = 15.6 Hz),
6.16 (1H, br s), 5.29–5.27 (2H, s), 3.76 (4H, t, J = 4.4 Hz), 3.55–
3.52 (6H, s), 3.49 (2H, q, J = 6.0 Hz), 2.55 (2H, t, J = 6.0 Hz), 2.49
(4H, d, J = 4.4 Hz); ESI-MS m/z [M+H]+ 381.
4.2.3.13. (E)-1-[3-Butenyl-1,4-dioxo-4-phenyl]-4-(3,4-dichlor-
ophenylmethyl)-piperazine (88). Yield: 46%; pale yellow oil; Rf
(hexane/EtOAc 3:1) 0.38; 1H NMR (400 MHz, CDCl3): d 8.04 (2H,
d, J = 8.0 Hz), 7.90 (1H, dd, J = 15.6 Hz), 7.63 (1H, dd, J = 7.6,
7.6 Hz), 7.51 (1H, s), 7.50 (1H, d, J = 15.6 Hz), 7.48 (2H, dd, J = 8.0,
7.6 Hz), 7.40 (1H, d, J = 8.4 Hz), 7.17 (1H, d, J = 8.4 Hz), 3.76–3.65
(4H, m), 3.48 (2H, s), 2.48 (4H, br s); ESI-MS m/z [M+H]+ 403.
4.2.3.5.
(2E)-3-(3,4-Dihydroxy)-N-[2-(4-morpholinyl)ethyl]-2-
propenamide (77). This compound was prepared in the same
way as 42 from 73; yield: 42%; white powder; mp 101–103 °C; Rf
(CH2Cl2/MeOH 12:1) 0.31; 1H NMR (400 MHz, CD3OD): d 8.42
(2H, br s), 7.96 (1H, s), 7.45 (1H, d, J = 15.2 Hz), 7.05 (1H, s), 6.95
(1H, d, J = 6.0 Hz), 6.81 (1H, d, J = 8.4 Hz), 6.42 (1H, d, J = 15.6 Hz),
3.83 (4H, m), 3.58 (2H, m), 2.86 (6H, m); ESI-MS m/z [M+H]+ 293.
4.2.3.14. (E)-2-[1-Oxo-3-phenyl-2-propenyl]-2-hydroxy-benzoic
acid hydrazide (91). Sulfoxide chloride (0.24 mL, 3.4 mmol) was
added to trans-cinnamic acid (503 mg, 3.4 mmol) in chloroform
(15 mL) containing triethylamine (0.49 mL, 3.4 mmol) at 0 °C over
a 5 min period with stirring. The solution was refluxed for 2 h and
slowly cooled to ambient temperature. Upon addition of salicyl-
hydrazide (380 mg, 2.5 mmol) (prepared from salicylic acid methyl
ester and NH2NH2ÁH2O) a white suspension immediately ap-
peared.24 After additional stirring for 30 min, the white precipitate
obtained was filtered and washed with hexane (2 Â 20 mL) and
water (2 Â 25 mL), followed by overnight drying in vacuum oven
to get 91 (819 mg). Yield: 86%; this compound was identical to that
reported by Moon et al.18
4.2.3.6. (2E)-3-(3,4-Dimethoxymethoxy)-N -[2-(1H-indol-3-yl)-
ethyl]-2-propenamide (78). Yield: 48%; yellow oil; Rf (CH2Cl2/
MeOH 12:1) 0.66; 1H NMR (400 MHz, CDCl3): d 8.13 (1H, br s),
7.65 (1H, d, J = 8.0 Hz), 7.53 (1H, d, J = 15.6 Hz), 7.41 (1H, d,
J = 8.0 Hz), 7.32 (1H, s), 7.22 (1H, d, J = 7.2 Hz), 7.15–7.10 (3H, m),
7.09 (1H, br s), 6.18 (1H, d, J = 15.6 Hz), 5.37 (1H, br s), 5.26 (4H,
s), 3.76 (2H, m), 3.52 (6H, s), 3.06 (2H, m); ESI-MS m/z [M+H]+ 411.
4.2.3.7. (2E)-3-(3,4-Dihydroxy)-N -[2-(1H-indol-3-yl)ethyl]-2-
propenamide (79). This compound was prepared in the same
way as 42 from 75. Yield: 54%; this compound was identical to that
reported by Park and Chen.23
4.3. Cell culture
4.2.3.8. (E)-4-(3,4-Dichlorophenylmethyl)-1-[3-(3,5-dimethoxy-
4-(4-ethoxyphenyl methoxy)-phenyl-1-oxo-2-propenyl]-piper-
azine (80). Yield: 57%; pale yellow oil; Rf (CH2Cl2/MeOH 12:1)
0.57; 1H NMR (400 MHz, CDCl3): d 7.58 (1H, d, J = 15.2 Hz3), 7.46
(1H, s), 7.40 (1H, d, J = 8.0 Hz), 7.37 (2H, d, J = 8.4 Hz), 7.18 (1H,
d, J = 8.4 Hz), 6.85 (2H, d, J = 8.4 Hz), 6.74 (1H, d, J = 15.2 Hz), 6.71
(2H, s), 4.97 (2H, s), 4.03 (2H, q, J = 7.2 Hz), 3.85 (6H, s), 3.76–
3.67 (4H, m), 3.49 (2H, s), 2.48 (4H, t, J = 4.4 Hz), 1.41 (3H, t,
J = 7.2 Hz); ESI-MS m/z [M+H]+ 585.
Various human cancer cell lines were cultured in minimum
essential medium (MEM), supplemented with 10% fetal calf serum
(Gibco Laboratories, Grand island, NY), 100 units/mL penicillin and
100 mg/mL streptomycin in a humidified atmosphere in 5% CO2 at
37 °C. Cell culture media were renewed every 3 days, up to the
confluence of the monolayer. Cell culture was passaged upon for-
mation of confluent cultures, using trypsin–EDTA to detach the

H. Zou et al. / Bioorg. Med. Chem. 18 (2010) 6351–6359
6359
cells from their culture flasks or dishes. Test compounds were
4.7. Statistical analysis
stored at À70 °C and solubilized in 100% DMSO.
All data were expressed as mean S.D. from at least three inde-
pendent experiments. Differences between groups were examined
for statistical significance using one-way ANOVA analysis with
SPSS 10.0 for WINDOWS. In all cases, P <0.05 was considered
significant.
4.4. MTT assay for cell viability
Exponentially growing cells were seeded in quadruplicate into
96-well flat-bottomed plates at a concentration of 5 Â 103 cells
per well. After 24 h incubation, the compounds studied were added
to the wells. After 72 h, 10
lL of MTT solution (5 mg/mL in phos-
Acknowledgments
phate buffered solution) was added to the culture medium and
incubated at 37 °C for a further 4 h. After removing unconverted
MTT, 200 lL of DMSO was added to each well and the plates sha-
ken to dissolve the reduced MTT crystals (formazan); the optical
density was measured on a microplate reader at a wavelength of
570 nm. The average 50% inhibitory concentration (IC50) was
determined graphically from the dose–response curves.
This study was supported by the National Natural Sciences
Foundation of China (No. 20802066), the China Postdoctoral
Science Foundation (No. 20080441268), National Science & Tech-
nology Major Project ‘‘Key New Drug Creation and Manufacturing
Program” of China (No. 2009ZX09501-010) and the Department
of Education of Zhejiang Province Foundation (No. Y200805312).
We thank Dr. Jiali Luo and Mr. Xiaoren Wang at the R&D center
of Zhejiang Hisun group for measuring of the mass data.
4.5. Detection of apoptosis by flow cytometric analysis
To determine the cell apoptosis, fluorescent probes Annexin V/
propidium iodide (PI, BioVision, CA, USA) were used. After 48 and
72 h treatment with indicated concentrations of compound 80,
K562 cells were collected by centrifuging. Cell pellets were resus-
pended in pre-warmed D-Hank’s buffer. Cell suspension was
stained by Annexin V/PI staining kit following manufacturer’s
instructions. Flow cytometry was performed on FACScan (BD Bio-
sciences, San Jose, CA, USA).
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