Synthesis and structure-activity relationship studies of cytotoxic cinnamic alcohol derivatives

Article abstract of DOI:10.1080/14786410902830175

Three series of di- and trisubstituted derivatives of cinnamic alcohol and its conjugated dienol analogues were designed and synthesised. The derivatives were screened for cytotoxicity against nine tumour cell lines: KB, A549, Hela, CNE, PC-3, BEL-7404, HL-60, BGC823 and P388D1. Most of the cinnamic alcohol derivatives showed cytotoxic activity. The compound 7-(4',5'-dichlorobenzyloxy)- 6,8-dihydroxycinnamic alcohol (55) exhibited significant cytotoxicity to seven human tumour cell lines on a micromolar range, especially with regard to the KB and P388D1 cell lines, showing IC50 values of 0.4 and 0.5μ M, respectively. The structure-activity relationships of the derivatives are discussed.

Full text of DOI:10.1080/14786410902830175

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Synthesis and structure–activity
relationship studies of cytotoxic
cinnamic alcohol derivatives
Hong-Bin Zou a b , Liang Zhang a , Lei-Xiang Yang a , Liu-Qing Yang
a b , Yu Zhao a , Yong-Ping Yu a & Joachim Stöckigt a b
a Institute of Materia Medica, College of Pharmaceutical Sciences,
Zhejiang University, 383 Yu Hang Tang Road, Hangzhou 310058,
China
b Lehrstuhl für Pharmazeutische Biologie, Institut für Pharmazie,
Johannes-Gutenberg Universität Mainz, Staudinger Weg 5,
D-55099 Mainz, Germany
Version of record first published: 05 Jul 2010.
To cite this article: Hong-Bin Zou, Liang Zhang, Lei-Xiang Yang, Liu-Qing Yang, Yu Zhao, Yong-Ping
Yu & Joachim Stöckigt (2011): Synthesis and structure–activity relationship studies of cytotoxic
cinnamic alcohol derivatives, Natural Product Research: Formerly Natural Product Letters, 25:3,
203-221
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Natural Product Research
Vol. 25, No. 3, February 2011, 203–221
Synthesis and structure–activity relationship studies of
cytotoxic cinnamic alcohol derivatives
Hong-Bin Zouab, Liang Zhanga, Lei-Xiang Yanga, Liu-Qing Yangab, Yu Zhaoa,
Yong-Ping Yua and Joachim Stockigtab
*
¨
aInstitute of Materia Medica, College of Pharmaceutical Sciences, Zhejiang University,
b
383 Yu Hang Tang Road, Hangzhou 310058, China; Lehrstuhl fur Pharmazeutische
¨
Biologie, Institut fur Pharmazie, Johannes-Gutenberg Universitat Mainz, Staudinger
¨
Weg 5, D-55099 Mainz, Germany
¨
(Received 18 January 2009; final version received 19 February 2009)
Three series of di- and trisubstituted derivatives of cinnamic alcohol and its
conjugated dienol analogues were designed and synthesised. The deriva-
tives were screened for cytotoxicity against nine tumour cell lines: KB,
A549, Hela, CNE, PC-3, BEL-7404, HL-60, BGC823 and P388D1. Most of
the cinnamic alcohol derivatives showed cytotoxic activity. The compound
7-(40,50-dichlorobenzyloxy)-6,8-dihydroxycinnamic alcohol (55) exhibited
significant cytotoxicity to seven human tumour cell lines on a micromolar
range, especially with regard to the KB and P388D1 cell lines, showing IC50
values of 0.4 and 0.5 mM, respectively. The structure–activity relationships
of the derivatives are discussed.
Keywords: cinnamic alcohol derivatives; cytotoxicity; structure–activity
relationship
1. Introduction
Cinnamic alcohols are a widespread group of natural phenylpropanoids and
a number of those originating from higher plants have been shown to be highly
bioactive, possessing significant cytotoxic activity (Choi et al., 2004; Cis, Nowak,
Horoszkiewicz-Hassan, & Kisiel, 2003; Ito et al., 2000; Zhao et al., 2002). Despite
their structural simplicity and interesting cytotoxic properties, their structure–
cytotoxicity relationship has rarely been described (Zou et al., 2006). Our previously
reported studies have shown that the 6,8-dimethoxy-7-(50-ethoxybenzyloxy)-
cinnamic alcohol (1, Figure 1), a derivative of the natural geranyloxysinapyl alcohol
isolated from Ligularia nelumbifolia, shows potent cytotoxicity to several human
tumour cell lines, and is metabolically stable due to substitution of the arylalkyloxy
substituent instead of the geranyloxy unit (Zou et al., 2006). Comparison of the
previously reported cinnamic alcohol derivative (2) with its methylated derivatives
indicates that the phenolic groups, instead of the methoxy group, on C6 and C8 may
enhance the cytotoxic effect on a number of selected human tumour cell lines
*Corresponding author. Email: stoeckig@uni-mainz.de
ISSN 1478–6419 print/ISSN 1029–2349 online
ß 2011 Taylor & Francis
DOI: 10.1080/14786410902830175
http://www.informaworld.com

204
H.-B. Zou et al.
O
5
6
MeO
4
HO
O
O
O
1
3
OH
OEt
OH
2
3
6
′
′
1′
7
2′
9
4
′
O
8
OMe
OH
7
′
5
′
EtO
3
2
1
Figure 1. Cinnamic alcohol derivatives.
(Zou et al., 2006). Previous 3D-QSAR studies of the sinapyl alcohol derivatives lead
to the conclusion that the bulky negative charged substituents at the C1 of cinnamic
alcohol confer increased cytotoxicity (Zou et al., 2006). In 2000, Ito et al. reported
that 6,7-(methylenedioxy)-cinnamic alcohol (3), isolated from the root of Boronia
pinnata, inhibits Epstein-Barr virus early antigen (EBV-EA) activation (Ito et al.,
2000), which is considered to be one of the causative agents of nasopharyngeal
carcinoma. This suggests that the trisubstituted unit of cinnamic alcohol is not
necessary for its cytotoxic activity.
In order to further investigate the structure–cytotoxicity relationship of cinnamic
alcohol derivatives, disubstituted derivatives with different substitution positions
(Scheme 1), trisubstituted derivatives with variant substituents (Scheme 2) and their
conjugated dienols (Scheme 3) were synthesised resulting in three series of 20
compounds based on the structures of compounds 1–3 (Figure 1). The synthetic
procedures are reported herein. The synthesised cinnamic alcohol derivatives and
some of the intermediates were then used to screen nine tumour cell lines. The results
showed that the compounds possess cytotoxic potential. Moreover, the structure–
activity relationship discussion on the cytotoxicity effect of structural variations in
substituents, substitution position and charge properties are also discussed as the
basis for further systematic research in this field.
2. Results and discussion
2.1. Synthesis of cinnamic alcohol derivatives
Three series of sinapyl alcohol derivatives were synthesised (Schemes 1–3) and some
of them were included in a Chinese Patent (Zhao, Wang, Zeng, Wu, & Bai, 2008a;
Zhao et al., 2008b). The synthetic route to double substituted cinnamic alcohol
derivatives (series 1) is illustrated in Scheme 1. Compounds 4–7 were subjected to an
alkylation of the corresponding hydroxy group by 4-ethoxy-benzyl bromide in the
presence of potassium carbonate, to provide the 3,4-disubstituted aldehydes 8–11
(K. Park, Han, & J. Park, 2001). The 3,5-disubstituted aldehyde 15 was prepared via
three steps from starting material 12, including alkylation with 4-ethoxy-benzyl
bromide to obtain 13 (Park et al., 2001). Treatment of 13 with hydrazine afforded
14 (Macaev et al., 2005) and an oxidation with potassium ferrocyanide gave the
aldehyde 15 (Chida, Vani, Chandrasekharam, Srinivasan, & Singh, 2001).
2,4-Dihydroxybenzalaldehyde (18) was obtained by Vilsmeier–Haack reaction
(Mendelson & Hayden, 1996) from resorcinol (16) and a further alkylation of 18
with 4-ethoxy-benzyl bromide afforded 19 (Park et al., 2001). Further condensation
of 8–11, 15 and 19 directly with monoethyl malonate afforded compounds 20–25

Natural Product Research
205
2
5
4: R1= H, R2= OH
3
1
CHO
R1
R2
R1
R2
CHO
8: R1= H, R2= 4-EtOC6H4CH2O
5: R1= OH, R2= OMe
6: R1= OMe, R2= OH
7: R1= OH, R2= OH
a
c
9: R1= 4-EtOC6H4CH2O, R2= OMe
10: R1= OMe, R2= 4-EtOC6H4CH2O
11: R1= R2= 4-EtOC6H4CH2O
4
6
R
COOMe
R
CONHNH2
R
CHO
CHO
HO
COOMe
b
d
g
R
R
R
OH
12
14
15
13
R
CHO
OH
e
f
N
Cl
HO
OH
HO
OH
R
HO
16
18
19
17
O
5
5
26: R1= H, R2= 4-EtOC6H4CH2O
27: R1= 4-EtOC6H4CH2O, R2= OMe
28: R1= OMe, R2= 4-EtOC6H4CH2O
29: R1= R2= 4-EtOC6H4CH2O
R1
R2
6
4
6
4
R1
R2
3
1
3
OEt
1
OH
OH
2
2
7
7
9
9
8
-
8
20 23
O
R
R
i
h
OEt
OEt
30
31
24
R
R
R
R
O
OH
R= 4-EtOC6H4CH2O
R
R
25
Scheme 1. Reagents and conditions: (a) RBr, K2CO3, acetone, reflux, 4 h; (b) same as (a),
96%; (c) 85% NH2NH2 ꢀ H2O, EtOH, reflux, 3 h, 63%; (d) K3Fe(CN)6, Bu4NBr, NH3 ꢀ H2O,
benzene, H2O, room temperature, 30 min, 66%; (e) DMF, (COCl)2, MeCN, ꢁ20–25ꢂC, 3.5 h;
(f) NaS2O3, H2O, 52ꢂC, 53% for (e) and (f); (g) same as (a), 68%; (h) monoethyl malonate,
pyridine, piperidine, reflux, 4 h; (i) LiAlH4, Et2O, 10ꢂC, 2 h.
(List, Doehring, Hechavarria Fonseca, Job, & Rios Torres, 2005), which were
reduced with lithium aluminumhydride in ether to give the corresponding
disubstituted cinnamic alcohol derivatives 26–31 (Ghera & Ben-David, 1988).
The trisubstituted cinnamic alcohol derivatives 1 and 44–47 (series 2) were
synthesised from the starting material syringaldehyde (32), which was alkylated with
corresponding bromides (Park et al., 2001) to afford aldehydes 33–37 (Scheme 2).
The aldehydes were subjected to Knoevengel reaction (List et al., 2005) to
afford cinnamic acid ethyl esters 38–42, of which 42 was further hydrolysed
(Oyama & Kondo, 2004) to get 43 with a free ꢀ-D-glucopyranosyl substituent.
Reduction of 38–41 and 43 with lithium aluminumhydride (Ghera & Ben-David,
1988) afforded the cinnamic alcohols 1 and 44–47. The 7-alkyloxy-6,8-dihydroxy
substituted cinnamic alcohol derivatives 54–55 were obtained by reduction (Ghera &
Ben-David, 1988) of corresponding esters (50, 53) which were synthesised from
syringaldehyde via five steps, as previously reported (Zou et al., 2006).

206
H.-B. Zou et al.
O
MeO
RO
CHO
MeO
HO
CHO
a
d
b
OH
OEt
OMe
OMe
1, 44 47
−
33: R= 4-EtOC6H4CH2
38 41
−
32
34: R=3,4-OCH2OC6H4CH2
35: R=3,4-(MeO)2C6H4CH2
36: R=2,3-(MeO)2C6H4CH2
37: R=Ac- -D-glucose
42: R=Ac- -D-glucose
43: R= -D-glucose
c
e
O
R1O
RO
CHO
R1O
RO
HO
RO
HO
HO
CHO
h
OEt
j
f
g
OH
OR2
OR2
OH
OH
49: R1=H, R2=Ac, R=4-EtOC6H4CH2
50: R1= R2=H, R=4-EtOC6H4CH2
51: R1=R2=Ac, R=3,4-Cl2C6H4CH2
52: R1=H, R2=Ac, R=3,4-Cl2C6H4CH2
53: R1=R2=H, R=3,4-Cl2C6H4CH2
48
54: R=4-EtOC6H4CH2
55: R=3,4-Cl2C6H4CH2
i
i
Scheme 2. Reagents and conditions: (a) RBr, K2CO3, acetone, reflux, 4 h; (b) monoethyl
malonate, pyridine, piperidine, reflux, 4 h; (c) NaOMe, MeOH, room temperature, 1 min;
(d) LiAlH4, Et2O, 0–10ꢂC, 2 h; (e) AlCl3, CH2Cl2, room temperature, 48 h, 31%; (f) Ac2O,
Et3N, 0ꢂC then room temperature, 4 h, 48%; (g) RBr, K2CO3, DMF, 45ꢂC, 12 h;
(h) Ph3P¼CHCO2Et, benzene, reflux, 2 h; (i) K2CO3, MeOH, room temperature, 1.5 h;
(j) LiAlH4–AlCl3, Et2O, 10ꢂC, 2 h.
O
MeO
O
MeO
O
1
5
3
OH
1
5
3
OEt
4
2
a
b
4
2
33
OMe
OMe
EtO
EtO
58
56 (2E, 4E)
MeO
O
OH
OH
OMe
EtO
EtO
MeO
O
O
59
b
a
OEt
MeO
O
OMe
EtO
57 (2Z, 4Z)
OMe
60
Scheme 3. Reagents and conditions: (a) (Ph3Pþ¼CHCH¼CHCO2Et)Brꢁ, Na, EtOH, THF,
reflux, 4 h; (b) LiAlH4–AlCl3, Et2O, 0ꢂC, 1.5 h.
Wittig reaction of 3,5-dimethoxy-4-(50-ethoxybenzyloxy)benzaldehyde (33) and
the phosphorus ylide, prepared from 4-bromocrotonic acid ethyl ester and
triphenylphosphine (Mazur, Hope-Ross, Kadla, Sederoff, & Chang, 2007), afforded
two dienoic acid ethyl esters 56 (2E, 4E) and 57 (2Z, 4Z) (Scheme 3). Further
reduction of 56 and 57 with lithium aluminumhydride and aluminum chloride

Natural Product Research
207
complex afforded two dienols 58 (2E, 4E), 59 (2Z, 4Z) and one enol 60 (2Z) (series 3).
For all these compounds, TLC on silica gel (GF254) was used for analysis and
column chromatography was carried out on silica gel H (10–40 mm).
2.2. Structure–activity relationship of synthesised cytotoxic cinnamic
alcohol derivatives
The targeted cinnamic alcohol derivatives and their intermediates (8–60) were
evaluated against nine tumour cell lines by the colourimeter assay MTT (Horowitz
& King, 2000; Putnam, Bombick, & Doolittle, 2002). Although the binding sites
of both the anticancer drug cisplatin (DDP) and the series of described cinnamic
alcohol derivatives might not be identical, DDP was used as a reference
compound because of its well-established clinical application in cancer treatment.
7-(50-Ethoxybenzyloxy)-6,8-dimethoxycinnamic alcohol (1) was also used as
a control. Results showing the concentrations required to inhibit cell growth by
50% (IC50 values) are presented in Table 1.
Analysis of the results of the MTT assay indicated that most of the analogues
of series 1 showed moderate cytotoxicity to the nine tumour cell lines. They also
suggest that analogues of series 2 with two free hydroxyl groups (53–55) were
generally more potent than those from series 1 and 3. Compound 55 displayed
significant cytotoxicity to eight of the tumour cell lines, especially to the KB and
P388D1 cell lines, with IC50 values of 0.4 and 0.5 mM, which was 50 and 30 times
higher than that observed for the control compound 1 and DDP values, respectively.
The bioassay results also suggest that the cytotoxic activity of the dienols from series
3 is tightly linked to the configuration of their double bond.
By thorough analysis of analogues 8–31 (series 1), the 6,8-disubstituted cinnamic
alcohol 30 and the 5,7-disubstituted one (31) showed intensive cytotoxicity to most
of the nine tumour cell lines tested, especially 31 to P388D1, with an IC50 value
of 1.2 mM. In contrast, the 6,7-disubstituted cinnamic alcohol derivatives (27–29),
cinnamic acid esters (21–23) and their intermediate aldehydes (9–11) showed weak
cytotoxicity to the nine tumour cell lines, which suggests that for the cytotoxicity of
disubsitituted cinnamic alcohols, positions C-6 and C-7 are not favoured for
substitution by an alkyloxy group. The mono-subsitituted cinnamic alcohol 26 alone
showed potent cytotoxic activity to seven of the selected tumour cell lines, in
particular to KB and HL-60 cell lines. Interestingly, the intermediate hydrazide 14
and aldehyde 15 exhibited efficient cytotoxicity in the micromolar range on some
tumour cell lines, especially for Hela and CNE cell lines.
In series 2, the 6,8-dimethoxy-7-substituted cinnamic alcohols 44–47 were
found to have no cytotoxic activity towards all the tested tumour cell lines,
while compound 6,8-dimethoxy-7-(50-ethoxybenzyloxy)cinnamic alcohol (1) was
selectively cytotoxic against a number of tumour cell lines. These results might
indicate that the small substituent around the aromatic cycle of C-7 confers
the series 2 compound cytotoxicity. Comparing compounds 54, 55, 1 and
6,8-dimethoxy-7-(40,50-dichlorobenzyloxy)cinnamic alcohol, which we have pre-
viously reported (Zou et al., 2006), it can also be deduced that the two free
hydroxy groups at C-6 and C-8 are crucial for activity of series 2: compounds 54
and 55 showed significant cytotoxicity to most of the tumour cell lines,

208
H.-B. Zou et al.

Natural Product Research
209

210
H.-B. Zou et al.
particularly 55 with respect to KB and P388D1 cell lines, with IC50 values of 0.4
and 0.5 mM, respectively.
The bioassay results of the cinnamic dienols 58 and 59 of series 3 demonstrated
that compound 59, possessing two conjugated double bonds (Z-configuration),
showed relatively high cytotoxicity to all nine tumour cell lines. By comparison to the
dienoic acid ester derivatives 56 and 57 we found that ester 56 with E-configuration
revealed no cytotoxic activity to any tested tumour cell lines, while the ester 57 with
Z-configuration exhibited potent cytoxicity to seven of the selected tumour cell lines,
in particular to P388D1, with an IC50 value of 1.6 mM. These results further indicate
that the two conjugated double bonds with Z-configuration are necessary for the
cytotoxicity of dienol derivatives (series 3). Compared to 59, compound 60, which
has one of the conjugated double bonds reduced, lost cytotoxicity against A549, Hela
and CNE cell lines and showed weaker activity to the remaining cell lines. This result
suggests that a negatively charged unit in this region will lead to an increase of
bioactivity.
3. Experimental
3.1. Materials
3.1.1. Materials for synthesis
All of the synthetic reagents used in this research work are commercially available.
The silica gel GF254 and silica gel H were purchased from Qingdao Marine Chemical
Factory, Qingdao, China.
3.1.2. Tumour cell lines
The nine tumour cell lines were obtained from the Shanghai Institute of
Biochemistry and Cell Biology, Chinese Academy of Sciences. The 96-well cell
culture plates (Falcon) for the biological screening were bought from BD Company,
Bedford, USA.
3.2. Methods
3.2.1. Synthetic approaches
Three series of sinapyl alcohol derivatives have been synthesised as outlined in
Schemes 1–3, which were described in Section 2.1.
4-(50-Ethoxybenzyloxy)benzaldehyde
(8):
4-Hydroxybenzaldehyde
(131 mg,
1.08 mmol) and 4-ethoxybenzyl bromide (13.0 mmol), which was formed by
bromination of 4-ethoxybenzyl alcohol under HBr and conc. H2SO4 catalysis,
were dissolved in 3 mL of acetone. This was added to a suspension of K2CO3
(290 mg, 21.6 mmol) and acetone (10 mL) in a dry flask. The mixture was refluxed for
3 h and cooled to room temperature. The reaction was monitored by TLC using
petro ether : EtOAc (3 : 1) as the mobile phase. The solvent was removed and the
residue was diluted with water, adjusted with 1 M HCl to pH 9, extracted with Et2O
(3 ꢄ 30 mL) and dried over anhydrous Na2SO4. After removal of the solvent under
reduced pressure the residue was purified by column chromatography (petro
ether : EtOAc 4 : 1) to afford 7. Yield: 86.3%; white crystalline solid; m.p. ¼ 80–82ꢂC;

Natural Product Research
211
Rf (hexane : EtOAc 3 : 1) 0.26; 1H NMR (400 MHz, CDCl3): ꢁ 9.89 (1H, s, CHO), 7.84
(2H, d, J ¼ 8.8 Hz, H-2,6), 7.35 (2H, d, J ¼ 8.8 Hz, H-30, H-70), 7.08 (2H, d,
J ¼ 8.8 Hz, H-40, H-60),
6.93
(2H, d, J ¼ 8.8 Hz, H-3, 5),
5.07
(2H, s, H-10),
4.05 (2H, q, J ¼ 7.2 Hz, OCH2CH3), 1.43 (3H, t, J ¼ 7.2 Hz, OCH2CH3); ESI-MS m/
z [M þ H]þ 257.
3-(50-Ethoxybenzyloxy)-4-methoxybenzaldehyde (9): This aldehyde was prepared
from 3-hydroxy-4-methoxybenzaldehyde in a similar way as described for the
synthesis of compound 8. Yield: 80.8%; white crystalline solid; m.p. ¼ 71–72ꢂC;
1
Rf (hexane : EtOAc 1 : 1) 0.70; H NMR (400 MHz, CDCl3): ꢁ 9.82 (1H, s, CHO),
7.47 (1H, d, J ¼ 1.6 Hz, H-2), 7.46 (1H, dd, J ¼ 8.4,1.6 Hz, H-6), 7.38 (2H, d,
J ¼ 8.4 Hz, H-30, H-70), 6.99 (1H, d, J ¼ 8.4 Hz, H-5), 6.90 (2H, d, J ¼ 8.4 Hz, H-40,
H-60), 5.12 (2H, s, H-10), 4.04 (2H, q, J ¼ 7.2 Hz, OCH2CH3), 3.95 (3H, s, OCH3),
1.42 (3H, t, J ¼ 7.2 Hz, OCH2CH3); ESI-MS m/z [M þ Na]þ 309.
4-(50-Ethoxybenzyloxy)-3-methoxybenzaldehyde (10): This compound was prepared
from 4-hydroxy-3-methoxybenzaldehyde in a similar way as described for the
synthesis of compound 8. Yield: 83.6%; white crystalline solid; m.p. ¼ 100–101ꢂC;
1
Rf (hexane : EtOAc 1 : 1) 0.70; H NMR (400 MHz, CDCl3): ꢁ 9.84 (1H, s, CHO),
7.42 (1H, d, J ¼ 2.0 Hz, H-2), 7.40 (1H, dd, J ¼ 8.0,2.0 Hz, H-6), 7.36 (2H, d,
J ¼ 8.4 Hz, H-30, H-70), 7.01 (1H, d, J ¼ 8.0 Hz, H-5), 6.90 (2H, d, J ¼ 8.4 Hz, H-40,
H-60), 5.17 (2H, s, H-10), 4.04 (2H, q, J ¼ 7.2 Hz, OCH2CH3), 3.94 (3H, s, OCH3),
1.42 (3H, t, J ¼ 7.2 Hz, OCH2CH3); ESI-MS m/z [MꢁH]ꢁ 285.
3,4-Di-(50-ethoxybenzyloxy)benzaldehyde (11): Compound 11 was prepared in a
similar way as described for the synthesis of compound 8 from 3,4-dihydrox-
ybenzaldehyde and a double amount of 4-ethoxybenzyl bromide. Yield: 73.2%;
1
white crystalline solid; m.p. ¼ 100–102ꢂC; Rf (hexane : EtOAc 1 : 1) 0.73; H NMR
(400 MHz, CDCl3): ꢁ 9.84 (1H, s, CHO), 7.48 (1H, d, J ¼ 1.6 Hz, H-2), 7.41 (1H, dd,
J ¼ 8.4,1.6 Hz, H-6), 7.36 (2H, d, J ¼ 8.4 Hz, H-300, H-700), 7.34 (2H, d, J ¼ 8.4 Hz,
H-30, H-70), 7.02 (1H, d, J ¼ 8.4 Hz, H-5), 6.89 (2H, d, J ¼ 8.4 Hz, H-400, H-600), 6.88
(2H, d, J ¼ 8.4 Hz, H-40, H-60), 5.17 (2H, s, H-100), 5.12 (2H, s, H-10), 4.04 (2H, q,
J ¼ 6.8 Hz, 500-OCH2CH3), 4.02 (2H, q, J ¼ 6.8 Hz, 50-OCH2CH3), 1.43 (6H, m,
OCH2CH3-50, 500); ESI-MS m/z [MꢁH]ꢁ 405.
3,5-Di-(50-ethoxybenzyloxy)benzoic acid hydrazide (14):
A
suspension of
3,5-dihydroxy-benzoic acid methyl ester (1.0 g, 6.0 mmol), 4-ethoxybenzyl bromide
(4.5 g, 18.0 mmol) and K2CO3 (2.48 g, 18.0 mmol) were refluxed in acetone (100 mL)
for 10 h followed by concentration to remove acetone. The concentrate was purified
by column chromatography (hexane : EtOAc 8 : 1) to afford 3,5-di-(50-ethoxybenzy-
loxy)benzoic acid methyl ester (13) (2.4 g, 5.5 mmol, 93.3%) and was dissolved in
20 mL ethanol and refluxed with 85% hydrazine hydrate (1.76 g, 30.3 mmol) for 3 h.
The mixture was evaporated and the residue was extracted by ethyl acetate
(50 mL ꢄ 3). The extract was dried overnight by Na2SO4 and the filtrate was
concentrated to get crude solid followed by crystallisation with hexane and ethyl
acetate to give 14 (1.5 g). Yield: 63.2%; m.p. 151–153ꢂC; Rf (CHCl3 : MeOH 10 : 1)
0.59; 1H NMR (400 MHz, CDCl3): ꢁ 9.67 (1H, s, NHNH2), 7.34 (4H, d,
J ¼ 8.4 Hz, H-30, 70, 300, 700), 7.05 (2H, d, J ¼ 2.0 Hz, H-2,6), 6.92 (4H, d, J ¼ 8.4 Hz,
H-40, 60, 400, 600), 6.75 (1H, dd, J ¼ 2.4,2.0 Hz, H-4), 5.02 (4H, s, H-10, 100), 4.45 (2H, d,

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H.-B. Zou et al.
J ¼ 9.6 Hz, NHNH2-7), 4.03 (4H, q, J ¼ 7.2 Hz, OCH2CH3-50, 500), 1.32 (6H, t,
J ¼ 7.2 Hz, OCH2CH3-50, 500); ESI-MS m/z [MꢁH]ꢁ 435.
3,5-Di-(50-ethoxybenzyloxy)benzaldehyde (15): 3,5-Di-(50-ethoxybenzyloxy)-benzoic
acid hydrazide (1.05 g, 2.4 mmol) and tetrabutyl ammonium bromide
(776 mg, 2.4 mmol) were suspended in 25% ammonia (2.5 mL), water (1.25 mL)
and benzene (20 mL). K3Fe (CN)6 (1.66 g, 5.06 mmol) in 4 mL water was added to
this suspension under stirring for 30 min and the mixture was filtered. The filtrate
was separated and the aqueous layer was extracted by benzene (10 mL ꢄ 2). The
organic layer was combined, washed with water (5 mL ꢄ 2), brine (10 mL ꢄ 2) and
dried over Na2SO4 overnight. Removal of benzene afforded a crude product which
was further purified by column chromatography (hexane : EtOAc 7 : 1) to get 15
(750 mg). Yield: 66.2%; white crystalline solid; m.p. ¼ 97–99ꢂC; Rf (hexane : EtOAc
5 : 1) 0.40; 1H NMR (400 MHz, CDCl3): ꢁ 9.89 (1H, s, CHO), 7.34 (4H, d, J ¼ 8.4 Hz,
H-30, 70, 300, 700), 7.09 (2H, d, J ¼ 2.0 Hz, H-2,6), 6.92 (4H, d, J ¼ 8.4 Hz, H-40, 60, 400, 600),
6.84 (1H, dd, J ¼ 1.6,2.0 Hz, H-4), 5.01 (4H, s, H-10, 100), 4.05 (4H, q, J ¼ 7.2 Hz, OCH2
CH3-50, 500), 1.42 (6H, t, J ¼ 7.2 Hz, OCH2CH3-50, 500); ESI-MS m/z [M ꢁ H]ꢁ405.
2,4-Di-(50-ethoxybenzyloxy)benzaldehyde (19): The product was prepared in the same
way as described for the synthesis of compound 11 from 2,4-dihydroxybenzaldehyde,
which was prepared from resorcinol by VilsmeierꢁHaack reaction (Mendelson &
Hayden, 1996). Yield: 68.0%; m.p. ¼ 116–118ꢂC; Rf (hexane : EtOAc 5 : 1) 0.28;
1H NMR (400 MHz, CDCl3): ꢁ 10.34 (1H, s, CHO), 7.82 (1H, d, J ¼ 8.8 Hz, H-6), 7.32
(4H, d, J ¼ 8.8 Hz, H-30, 70, 300, 700), 6.92 (4H, d, J ¼ 8.4 Hz, H-40, 60, 400, 600), 6.63 (1H,
dd, J ¼ 8.8,1.6 Hz, H-5), 6.59 (1H, d, J ¼ 1.6 Hz, H-3), 5.05ꢁ5.03 (4H, s, H-10, 100), 4.05
(4H, q, J ¼ 7.2 Hz, OCH2CH3-50, 500), 1.42 (6H, t, J ¼ 7.2 Hz, OCH2CH3-50, 500);
ESIꢁMS m/z [MꢁH]ꢁ 405.
7-(50-Ethoxybenzyloxy)cinnamic acid ethyl ester (20): Monoethyl malonate
(121 mg, 0.92 mmol) and piperidine (0.1 mL) were added to 4-(50-ethoxybenzyloxy)-
benzaldehyde (8) (117 mg, 0.46 mmol) in 5 mL pyridine. The mixture was refluxed for
6 h. After cooling to room temperature, diluted HCl (1 M) was added to adjust pH 5
followed by extraction with EtOAc. The extract was concentrated to give a residue,
which was purified by column chromatography to afford 20 (133 mg). Yield: 89.2%;
m.p. ¼ 103–104ꢂC; Rf (hexane : EtOAc 3 : 1) 0.30; 1H NMR (400 MHz, CDCl3): ꢁ 7.65
(1H, d, J ¼ 16.0 Hz, H-3), 7.48 (2H, d, J ¼ 8.8 Hz, H-5,9), 7.35 (2H, d, J ¼ 8.8 Hz,
H-30, 70), 6.97 (2H, d, J ¼ 8.8 Hz, H-6,8), 6.92 (2H, d, J ¼ 8.8 Hz, H-40, 60), 6.32
(1H, d, J ¼ 16.0 Hz, H-2), 5.02 (2H, s, H-10), 4.26 (2H, q, J ¼ 7.2 Hz, OCH2CH3-1),
4.05 (2H, q, J ¼ 7.2 Hz, OCH2CH3-50), 1.43 (3H, t, J ¼ 7.2 Hz, OCH2CH3-50), 1.34
(3H, t, J ¼ 7.2 Hz, OCH2CH3-1); ESI-MS m/z [M þ H]þ 327.
Compounds 21–25 and 38–42 were synthesised by treatment of corresponding
aldehydes 9–11, 15, 19 and 33–37 with monoethyl malonate according to the
procedure performed for 20.
6-(50-Ethoxybenzyloxy)-7-methoxycinnamic acid ethyl ester (21): Yield: 81.7%;
1
white crystalline solid; m.p. ¼ 82–83ꢂC; Rf (hexane : EtOAc 1 : 1) 0.77; H NMR
(400 MHz, CDCl3): ꢁ 7.58 (1H, d, J ¼ 16.0 Hz, H-3), 7.36 (2H, d, J ¼ 8.8 Hz, H-30, 70),
7.12 (1H, d, J ¼ 2.0 Hz, H-5), 7.10 (1H, dd, J ¼ 8.8,2.0 Hz, H-9), 6.90 (2H, d,
J ¼ 8.8 Hz, H-40, 60), 6.88 (1H, d, J ¼ 8.8 Hz, H-8), 6.24 (1H, d, J ¼ 16.0 Hz, H-2),

Natural Product Research
213
5.09 (2H, s, H-10), 4.25 (2H, q, J ¼ 7.2 Hz, OCH2CH3-1), 4.04 (2H, q, J ¼ 6.8 Hz,
OCH2CH3-50), 3.90 (3H, s, OCH3), 1.42 (3H, t, J ¼ 6.8 Hz, OCH2CH3-50), 1.34
(3H, t, J ¼ 6.8 Hz, OCH2CH3-1); ESI-MS m/z [M þ H]þ 357.
7-(50-Ethoxybenzyloxy)-6-methoxycinnamic acid ethyl ester (22): Yield: 83.1%; white
crystals; m.p. ¼ 100–102ꢂC; Rf (hexane : EtOAc 1 : 1) 0.81; 1H NMR (400 MHz,
CDCl3): ꢁ 7.61 (1H, d, J ¼ 16.0 Hz, H-3), 7.35 (2H, d, J ¼ 8.4 Hz, H-30, 70), 7.06 (1H, d,
J ¼ 2.0 Hz, H-5), 7.04 (1H, dd, J ¼ 8.4,2.0 Hz, H-9), 6.89 (2H, d, J ¼ 8.4 Hz, H-40, 60),
6.88 (1H, d, J ¼ 8.4 Hz, H-8), 6.30 (1H, d, J ¼ 16.0 Hz, H-2), 5.31 (2H, s, H-10), 4.26
(2H, q, J ¼ 7.2 Hz, OCH2CH3-1),
[M þ H]þ 357.
4.03 (2H, q, J ¼ 6.8 Hz, OCH2CH3-50),
1.41
(3H, t, J ¼ 6.8 Hz, OCH2CH3-50), 1.34 (3H, t, J ¼ 7.2 Hz, OCH2CH3-1); ESI-MS m/z
6,7-Di-(50-ethoxybenzyloxy)cinnamic acid ethyl ester (23): Yield: 88.6%; white
crystals; m.p. ¼ 96–98ꢂC; Rf (hexane : EtOAc 5 : 1) 0.36; 1H NMR (400 MHz,
CDCl3): ꢁ 7.57 (1H, d, J ¼ 15.6 Hz, H-3), 7.33 (4H, dd, J ¼ 8.4,2.0 Hz, H-30, 70, 300, 700),
7.11 (1H, d, J ¼ 1.6 Hz, H-5), 7.06 (1H, dd, J ¼ 8.4,1.6 Hz, H-9), 7.05 (1H, d,
J ¼ 8.4 Hz, H-8), 6.91 (2H, d, J ¼ 8.4,2.0 Hz, H-400, 600), 6.88 (2H, d, J ¼ 8.4,2.0 Hz,
H-40, 60), 6.24 (1H, d, J ¼ 15.6 Hz, H-2), 5.11 (2H, s, H-100), 5.08 (2H, s, H-10), 4.25
(2H, q, J ¼ 7.2 Hz, OCH2CH3-1), 4.04 (2H, q, J ¼ 7.2 Hz, OCH2CH3-500), 4.03 (2H, q,
J ¼ 7.2 Hz, OCH2CH3-50), 1.43 (6H, m, J ¼ 7.2 Hz, OCH2CH3-50, 500), 1.33 (3H, t,
J ¼ 7.2 Hz, OCH2CH3-1); ESI-MS m/z [M þ H]þ 477.
6,8-Di-(50-ethoxybenzyloxy)cinnamic acid ethyl ester (24): Yield: 93.1%; white
crystals; m.p. ¼ 99–100ꢂC; Rf (hexane : EtOAc 5 : 1) 0.34; 1H NMR (400 MHz,
CDCl3): ꢁ 7.58 (1H, d, J ¼ 16.0 Hz, H-3), 7.33 (4H, d, J ¼ 8.4 Hz, H-30, 70, 300, 700), 6.91
(4H, d, J ¼ 8.4 Hz, H-40, 60, 400, 600), 6.75 (2H, d, J ¼ 2.4 Hz, H-5,9), 6.63 (1H, dd,
J ¼ 2.4,2.0 Hz, H-7), 6.38 (1H, d, J ¼ 16.0 Hz, H-2), 4.97 (4H, s, H-10, 100), 4.26 (2H,
q, J ¼ 7.2 Hz, OCH2CH3-1), 4.04 (4H, q, J ¼ 7.2 Hz, OCH2CH3-50, 500), 1.43 (6H, t,
J ¼ 7.2 Hz, OCH2CH3-50, 500), 1.34 (3H, t, J ¼ 7.2 Hz, OCH2CH3-1); ESI-MS m/z
[M þ H]þ 477.
5,7-Di-(50-ethoxybenzyloxy)cinnamic acid ethyl ester (25): Yield: 90.6%; white
crystalline solid; m.p. ¼ 88–91ꢂC; Rf (hexane : EtOAc 5 : 1) 0.32; 1H NMR
(400 MHz, CDCl3): ꢁ 7.97 (1H, d, J ¼ 16.0 Hz, H-3), 7.46 (1H, dd, J ¼ 8.4,2.4 Hz,
H-9), 7.33 (4H, dd, J ¼ 8.4,2.0 Hz, H-30, 70, 300, 700), 6.90 (4H, d, J ¼ 8.4,2.0 Hz,
H-40, 60, 400, 600), 6.57 (2H, m, H-6,8), 6.42 (1H, d, J ¼ 16.0 Hz, H-2), 5.04 (2H, s,
H-10), 4.97 (2H, s, H-100), 4.24 (2H, q, J ¼ 7.2 Hz, OCH2CH3-1), 4.04 (4H, q,
J ¼ 7.2 Hz, OCH2CH3-50, 500), 1.42 (6H, t, J ¼ 7.2 Hz, OCH2CH3-50, 500), 1.31 (3H, t,
J ¼ 7.2 Hz, OCH2CH3-1); ESI-MS m/z [M þ H]þ 477.
7-(50-Ethoxybenzyloxy)cinnamic alcohol (26): LiAlH4 (28 mg, 0.72 mmol) was added
to anhydrous ether (8 mL) with vigorous stirring in an ice-salt bath. Compound 20
(94 mg, 0.29 mmol) in ether (5 mL) was added dropwise to the mixture at ꢁ10ꢂC in
2 min and the solution was stirred below 0ꢂC for 30–60 min. About 2 mL of water
was added to destroy the excessive LiAlH4, and the solution was acidified to pH 5.
The aqueous layer was extracted with ether (3 ꢄ 15 mL) and the combined organic
layer was washed with brine and dried over anhydrous Na2SO4 for 10 h. After
removal of the solvent, the residue was purified through column chromatography
with hexane : EtOAc (10 : 3) to give 26 (53 mg). Yield: 64.8%; white crystals;

214
H.-B. Zou et al.
m.p. ¼ 132–134ꢂC; Rf (hexane : EtOAc 5 : 2) 0.34; 1H NMR (400 MHz, CDCl3): ꢁ 7.48
(2H, d, J ¼ 8.8 Hz, H-5,9), 7.35 (2H, d, J ¼ 8.0 Hz, H-30, H-70), 6.96 (2H, d,
J ¼ 8.8 Hz, H-6,8), 6.91 (2H, d, J ¼ 8.0 Hz, H-40, 60), 6.56 (1H, d, J ¼ 16.0 Hz, H-3),
6.25 (1H, dt, J ¼ 16.0,6.0 Hz, H-2), 4.99 (2H, s, H-10), 4.31 (2H, t, J ¼ 6.0 Hz, H-1),
4.04 (2H, q, J ¼ 7.2 Hz, OCH2CH3-50), 1.44 (3H, t, J ¼ 7.2 Hz, OCH2CH3-50); ESI-MS
m/z [M þ H]þ 285. Compounds 27–31 and 44–47 were synthesised by treatment of
corresponding cinnamic acid esters 21–25, 39–41 and 43 with LiAlH4 according to
the procedure used for 26.
6-(50-Ethoxybenzyloxy)-7-methoxycinnamic alcohol (27): Yield: 60.1%; white
crystalline solid; m.p. ¼ 89–91ꢂC; Rf (hexane : EtOAc 1 : 1) 0.48; 1H NMR
(400 MHz, CDCl3): ꢁ 7.37 (2H, d, J ¼ 8.4 Hz, H-30, 70), 6.99 (1H, d, J ¼ 2.0 Hz, H-5),
6.93 (1H, d, J ¼ 8.4 Hz, H-9), 6.91 (2H, d, J ¼ 8.4 Hz, H-40, 60), 6.84 (1H, d,
J ¼ 8.0 Hz, H-8), 6.50 (1H, d, J ¼ 15.6 Hz, H-3), 6.18 (1H, dt, J ¼ 15.6,6.0 Hz, H-2),
5.08 (2H, s, H-10), 4.29 (2H, d, J ¼ 6.0 Hz, H-1), 4.04 (2H, q, J ¼ 7.2 Hz, OCH2CH3-
50), 3.89 (3H, s, OCH3), 1.42 (3H, t, J ¼ 7.2 Hz, OCH2CH3-50); ESI-MS m/z
[M þ H]þ 315.
7-(50-Ethoxybenzyloxy)-6-methoxycinnamic alcohol (28): Yield: 57.7%; white crystal-
line solid; m.p. ¼ 76–78ꢂC; Rf (hexane : EtOAc 1 : 1) 0.42; 1H NMR (400 MHz,
CDCl3): ꢁ 7.35 (2H, d, J ¼ 8.4 Hz, H-30, 70), 6.96 (1H, d, J ¼ 1.6 Hz, H-5), 6.88 (2H, d,
J ¼ 8.4 Hz, H-40, 60), 6.86 (1H, dd, J ¼ 8.4,1.6 Hz, H-9), 6.84 (1H, d, J ¼ 8.4 Hz, H-8),
6.54 (1H, d, J ¼ 16.0 Hz, H-3), 6.24 (1H, dt, J ¼ 16.0,6.0 Hz, H-2), 5.08 (2H, s, H-10),
4.31 (2H, d, J ¼ 6.0 Hz, H-1), 4.03 (2H, q, J ¼ 7.2 Hz, OCH2CH3-50), 3.90 (3H, s,
OCH3), 1.41 (3H, t, J ¼ 7.2 Hz, OCH2CH3-50); ESI-MS m/z [M þ H]þ 315.
6,7-Di-(50-ethoxybenzyloxy)cinnamic alcohol (29): Yield: 67.4%; white crystalline
1
solid; m.p. ¼ 60–62ꢂC; Rf (hexane : EtOAc 5 : 2) 0.22; H NMR (400 MHz, CDCl3):
ꢁ 7.34 (4H, d, J ¼ 8.4 Hz, H-30, 70, 300, 700), 7.00 (1H, s, H-5), 6.88 (1H, d, J ¼ 8.4 Hz,
H-9), 6.87 (1H, d, J ¼ 8.4 Hz, H-8), 6.87 (4H, d, J ¼ 8.4 Hz, H-40, 60, 400, 600), 6.50
(1H, d, J ¼ 16.0 Hz, H-3), 6.17 (1H, dt, J ¼ 16.0,6.0 Hz, H-2), 5.07 (4H, s, H-10, 100),
4.29 (2H, dd, J ¼ 6.0,1.2 Hz, H-1), 4.10 (4H, m, OCH2CH3-50, 500), 1.42 (6H, m,
OCH2CH3-50, 500); ESI-MS m/z [M þ H]þ 435.
6,8-Di-(50-ethoxybenzyloxy)cinnamic alcohol (30): Yield: 64.8%; white crystalline
1
solid; m.p. ¼ 65–67ꢂC; Rf (hexane : EtOAc 3 : 1) 0.13; H NMR (400 MHz, CDCl3):
ꢁ 7.33 (4H, d, J ¼ 8.4 Hz, H-30, 70, 300, 700), 6.91 (4H, d, J ¼ 8.4 Hz, H-40, 60, 400, 600), 6.63
(2H, s, H-5,9), 6.53 (1H, d, J ¼ 16.0 Hz, H-3), 6.52 (1H, s, H-7), 6.33 (1H, dd,
J ¼ 16.0,5.6 Hz, H-2), 4.95 (4H, s, H-10, 100), 4.32 (2H, d, J ¼ 5.6 Hz, H-1), 4.05
(4H, q, J ¼ 6.8 Hz, OCH2CH3-50, 500), 1.42 (6H, t, J ¼ 6.8 Hz, OCH2CH3-50, 500);
ESI-MS m/z [M þ H]þ 435.
5,7-Di-(50-ethoxybenzyloxy)cinnamic alcohol (31): Yield: 65.8%; white crystalline
1
solid; m.p. ¼ 67–86ꢂC; Rf (hexane : EtOAc 5 : 2) 0.25; H NMR (400 MHz, CDCl3):
ꢁ 7.46 (1H, dd, J ¼ 8.4,2.4 Hz, H-9), 7.31 (4H, d, J ¼ 8.4 Hz, H-30, 70, 300, 700), 6.90
(4H, d, J ¼ 8.4 Hz, H-40, 60, 400, 600), 6.59 (2H, m, H-6, 8), 6.53 (1H, d, J ¼ 16.0 Hz, H-
3), 6.33 (1H, dd, J ¼ 16.0,5.6 Hz, H-2), 4.95 (4H, s, H-10, 100), 4.32 (2H, d,
J ¼ 5.6 Hz, H-1), 4.05 (4H, q, J ¼ 6.8 Hz, OCH2CH3-50, 500), 1.42 (6H, t, J ¼ 6.8 Hz,
OCH2CH3-50, 500); ESI-MS m/z [M þ H]þ 435.

Natural Product Research
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3,5-Dimethoxy-4-(50-ethoxybenzyloxy)benzaldehyde (33): This compound was synthe-
sised by treatment of 3,4-methylenedioxybenzyl bromide and syringaldehyde (32)
according to the method previously reported in Zou et al. (2006).
3,5-Dimethoxy-4-(40,50-methylenedioxybenzyloxy)benzaldehyde (34): Compound 34
was obtained by treatment of 3,4-methylenedioxybenzyl bromide and 32 similar
to the method described for 8. Yield: 73.7%; white crystalline solid; m.p. ¼ 86–88ꢂC;
Rf (hexane : EtOAc 5 : 2) 0.42; ꢁ 9.87 (1H, s, CHO), 7.12 (2H, s, H-2,6), 7.03 (1H, d,
J ¼ 1.6 Hz, H-30), 6.90 (1H, dd, J ¼ 8.0,1.6 Hz, H-70), 6.76 (1H, d, J ¼ 8.0 Hz, H-60),
5.96 (2H, s, OþCH2O), 5.03 (2H, s, H-10), 3.92 (6H, s, OCH3-3,5); ESI-MS m/z
[M þ Na þ H] 339.
3,5-Dimethoxy-4-(40,50-dimethoxybenzyloxy)benzaldehyde (35): This compound was
prepared by treatment of 3,4-dimethoxybenzyl bromide and 32 similar to the method
described for 8. Yield: 65.8%; white crystalline solid; m.p. ¼ 81–83ꢂC;
Rf (hexane : EtOAc 5 : 2) 0.49; ꢁ 9.87 (1H, s, CHO), 7.17 (1H, dd, J ¼ 8.0,1.6 Hz, H-
70), 7.13 (2H, s, H-2,6), 7.03 (1H, d, J ¼ 1.6 Hz, H-30), 6.97 (1H, d, J ¼ 8.0 Hz, H-60),
5.18 (2H, s, H-10), 3.89 (3H, s, OCH3-40), 3.88 (6H, s, OCH3-3,5), 3.87 (3H, s, OCH3-
50); ESI-MS m/z [M þ Na]þ 355.
3,5-Dimethoxy-4-(30,40-dimethoxybenzyloxy)benzaldehyde (36): Compound 36 was
synthesised by treatment of 3,4-dimethoxybenzyl bromide and 32 similar to the
method described for 8. Yield: 67.7%; white crystalline solid; m.p. ¼ 86–88ꢂC;
1
Rf (hexane : EtOAc 5 : 2) 0.43; H NMR (400 MHz, CDCl3): ꢁ 9.87 (1H, s, CHO),
7.17 (1H, dd, J ¼ 8.0,1.6 Hz, H-70), 7.13 (2H, s, H-2,6), 7.06 (1H, dd, J ¼ 8.0,8.0 Hz,
H-60), 6.89 (1H, dd, J ¼ 8.0,1.6 Hz, H-50), 5.18 (2H, s, H-10), 3.89 (3H, s, OCH3-30),
3.88 (6H, s, OCH3-3, 5), 3.87 (3H,s,OCH3-40); ESI-MS m/z [M þ Na]þ 355.
3,5-Dimethoxy-4-(20,30,40,60-tetra-O-acetyl-b-D-glucopyranosyloxyl)benzaldehyde (37):
This compound (Delay & Delmotte, 1990) was synthesised by treatment of 20,30,40,
60-tetra-O-acetyl-ꢀ-D-glucopyranosyloxyl bromide and 32 similar to the method
used for 8.
6,8-Dimethoxy-7-(50-ethoxybenzyloxy)cinnamic acid ethyl ester (38): Compound 38
was identical to that we have previously reported (Zou et al., 2006).
6,8-Dimethoxy-7-(40,50-methylenedioxybenzyloxy)cinnamic acid ethyl ester (39): Yield:
77.2%; white crystalline solid; m.p. ¼ 104–106ꢂC; Rf (hexane : EtOAc 5 : 1) 0.26;
1H NMR (400 MHz, CDCl3): ꢁ 7.60 (1H, d, J ¼ 15.6 Hz, H-3), 7.05 (1H, d,
J ¼ 1.6 Hz, H-30), 6.90 (1H, dd, J ¼ 8.0,1.6 Hz, H-70), 6.76 (1H, d, J ¼ 8.0 Hz, H-60),
6.74 (2H, s, H-5,9), 6.35 (1H, d, J ¼ 15.6 Hz, H-2), 5.95 (2H, s, OCH2O), 4.94
(2H, s, H-10), 4.27 (2H, q, J ¼ 7.2 Hz, OCH2CH3-1), 3.87 (6H, s, OCH3-6,8), 1.34
(3H, t, J ¼ 7.2 Hz, OCH2CH3-1); ESI-MS m/z [M þ Na]þ 409.
6,8-Dimethoxy-7-(40,50-dimethoxybenzyloxy)cinnamic acid ethyl ester (40): Yield:
76.7%; white crystalline solid; m.p. ¼ 81–83ꢂC; Rf (hexane : EtOAc 5 : 1) 0.10;
1H NMR (400 MHz, CDCl3): ꢁ 7.60 (1H, d, J ¼ 16.0 Hz, H-3), 7.11 (1H, d,
J ¼ 1.6 Hz, H-30), 6.94 (1H, dd, J ¼ 8.0,1.6 Hz, H-70), 6.81 (1H, d, J ¼ 8.0 Hz, H-60),
6.74 (2H, s, H-5,9), 6.35 (1H, d, J ¼ 16.0 Hz, H-2), 5.01 (2H, s, H-10), 4.24 (2H, q,
J ¼ 7.2 Hz, OCH2CH3-1), 3.90 (3H, s, OCH3-50), 3.87 (6H, s, OCH3-6,8), 3.86 (3H, s,
OCH3-40), 1.34 (2H, t, J ¼ 7.2 Hz, OCH2CH3-1); ESI-MS [M þ Na]þ 425.

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6,8-Dimethoxy-7-(30,40-dimethoxybenzyloxy)cinnamic acid ethyl ester (41): Yield:
84.2%; white crystalline solid; m.p. ¼ 6–78ꢂC; Rf (hexane : EtOAc 5 : 1)
0.21; 1H NMR (400 MHz, CDCl3): ꢁ 7.61 (1H, d, J ¼ 16.0 Hz, H-3), 7.19 (1H, dd,
J ¼ 8.0,1.2 Hz, H-70), 7.07 (1H, dd, J ¼ 8.0,8.0 Hz, H-60), 6.89 (1H, dd, J ¼ 8.0,1.2 Hz,
H-50), 6.87 (2H, s, H-5,9), 6.36 (1H, d, J ¼ 16.0 Hz, H-2), 5.10 (2H, s, H-10), 4.28
(3H, t, J ¼ 7.2 Hz, OCH2CH3-1), 3.87 (6H, s, OCH3-6,8), 3.85 (3H, s, OCH3-30), 3.84
(3H, s, OCH3-40), 1.35 (3H, t, J ¼ 7.2 Hz, OCH2CH3-1); ESI-MS m/z [M þ Na]þ 425.
6,8-Dimethoxy-7-(20,30,40,60-tetra-O-acetyl-b-D-glucopyranosyloxyl)cinnamic
acid
ethyl ester (42): Yield: 68.7%; white crystalline solid; m.p. ¼ 153–156ꢂC;
Rf (hexane : EtOAc 4 : 5) 0.62; 1H NMR (400 MHz, CDCl3): ꢁ 7.58 (1H, d,
J ¼ 15.6 Hz, H-3), 6.74 (2H, s, H-5,9), 6.35 (1H, d, J ¼ 15.6 Hz, H-2), 5.24ꢁ5.33
(3H, m, H-10, 20, 40), 5.12 (1H, d, J ¼ 6.8 Hz, H-30), 4.25 (3H, m, OCH2CH3-1, H-60ꢂ),
4.12 (1H, dd, J ¼ 12.0,2.4 Hz, H-60ꢀ), 3.85 (6H, s, OCH3-6, 8), 3.71 (1H, m, H-50),
2.03 (12H, s, CH3CO), 1.34 (3H, t, J ¼ 6.8 Hz, OCH2CH3-1). ESI-MS m/z
[M þ Na]þ 605.
6,8-Dimethoxy-7-b-D-glucopyranosyloxylcinnamic acid ethyl ester (43): This com-
pound was synthesised by treatment of 42 with sodium methoxide as previously
reported by Mazur, Hope-Ross, Kadla, Sederoff and Chang (2007). Yield:
63.4%; white crystalline solid; m.p. 180–182ꢂC; Rf (CHCl3 : MeOH 4 : 1) 0.67;
1H NMR (400 MHz, CDCl3): ꢁ 7.88 (1H, d, J ¼ 16.0 Hz, H-3), 7.03 (2H, s, H-5,9),
6.71 (1H, d, J ¼ 16.0 Hz, H-2), 5.98 (1H, d, J ¼ 6.8 Hz, H-10), 4.44 (1H, d,
J ¼ 10.8 Hz, H-60ꢂ), 4.38ꢁ4.25 (6H, m, H-20, 30, 40, 60ꢀ, OCH2CH3-1), 4.01 (1H, m,
H-50), 3.80 (6H, s, OCH3-6, 8), 1.23 (3H, t, J ¼ 7.2 Hz, OCH2CH3-1); ESI-MS m/z
[M þ Na]þ 437.
6,8-Dimethoxy-7-(40,50-methylenedioxybenzyloxy)cinnamic alcohol (44): Yield:
50.4%; white crystalline solid; m.p. ¼ 89–90ꢂC; Rf (hexane : EtOAc 5 : 2) 0.13;
1H NMR (400 MHz, CDCl3): ꢁ 7.05 (1H, d, J ¼ 1.6 Hz, H-3%), 6.91 (1H, dd, J ¼ 8.0,
1.6 Hz, H-7%), 6.76 (1H, d, J ¼ 8.0 Hz, H-6%), 6.59 (5.94 (2H, s, H-5, 9), 6.52
(1H, d, J ¼ 15.6 Hz, H-3), 6.27 (1H, dt, J ¼ 15.6,6.0 Hz, H-2), 5.93 (2H, s, OCH2O),
4.88 (2H, s, H-1%), 4.30 (2H, dd, J ¼ 5.6,0.8 Hz, H-1), 3.84 (6H, s, OCH3-6,8);
ESI-MS m/z [M þ H]þ 345.
6,8-Dimethoxy-7-(40,50-dimethoxybenzyloxy)cinnamic alcohol (45): Yield: 49.3%;
1
white crystalline solid; m.p. ¼ 99–100ꢂC; Rf (hexane : EtOAc 5 : 2) 0.14; H NMR
(400 MHz, CDCl3): ꢁ 7.12 (1H, d, J ¼ 1.6 Hz, H-30), 6.95 (1H, dd, J ¼ 8.0,2.0 Hz, H-70),
6.81 (1H, d, J ¼ 8.0 Hz, H-60), 6.60 (2H, s, H-5,9), 6.54 (1H, d, J ¼ 15.6 Hz, H-3), 6.29
(1H, dt, J ¼ 15.6,6.0 Hz, H-2), 4.97 (2H, s, H-10), 4.32 (2H, dd, J ¼ 5.6,0.9 Hz, H-1),
3.90 (3H, s, OCH3-50), 3.87 (3H, s, OCH3-40), 3.84 (6H, s, OCH3-6,8); ESI-MS m/z
[M þ H]þ 361.
6,8-Dimethoxy-7-(30,40-dimethoxybenzyloxy)cinnamic alcohol (46): Yield: 51.7%;
white crystalline solid; m.p. ¼ 66–71ꢂC; Rf (hexane : EtOAc 5 : 2) 0.12; 1H NMR
(400 MHz, CDCl3): ꢁ 7.22 (1H, d, J ¼ 7.6 Hz, H-70), 7.07 (1H, dd, J ¼ 8.0,7.6 Hz, H-60),
6.89 (1H, d, J ¼ 8.0 Hz, H-50), 6.62 (2H, s, H-5, 9), 6.54 (1H, d, J ¼ 16.0 Hz, H-3), 6.30
(1H, dt, J ¼ 16.0,6.0 Hz, H-2), 5.08 (2H, s, H-10), 4.32 (2H, dd, J ¼ 5.6,0.6 Hz, H-1),
3.88 (3H, s, OCH3-30), 3.87 (3H, s, OCH3-40), 3.82 (6H, s, OCH3-6, 8); ESI-MS m/z
[M þ H]þ 361.

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217
6,8-Dimethoxy-7-b-D-glucopyranosyloxylcinnamic alcohol (47): Yield: 30.4%;
ESI-MS m/z [M þ Na]þ 395; Rf (CHCl3 : MeOH 4 : 1) 0.44; this compound was
identical to that reported by Delay and Delmotte (1990).
6-Acetoxy-8-hydroxy-7-(50-ethoxybenzyloxy)cinnamic acid ethyl ester (49): This
compound was prepared according to the procedure previously reported in Zou
et al. (2006) by three steps from the starting material 4-ethoxybenzyl bromide and
3,4,5-trihydroxybenzaldehyde (48). Yield 70%; white crystalline solid; m.p. ¼ 73–
1
75ꢂC; Rf (hexane : EtOAc 3 : 1) 0.29; H NMR (400 MHz, (CD3)2CO): ꢁ 8.74 (1H,
brs, OH-8), 7.52 (1H, d, J ¼ 16.0 Hz, H-3), 7.37 (2H, d, J ¼ 8.4 Hz, H-30, 70), 7.11 (1H,
d, J ¼ 2.4 Hz, H-9), 6.98 (1H, d, J ¼ 2.4 Hz, H-5), 6.91 (2H, dd, J ¼ 8.4,2.0 Hz,
H-40, 60), 6.39 (1H, d, J ¼ 16.0 Hz, H-2), 5.01 (2H, s, H-10), 4.17 (2H, q, J ¼ 7.2 Hz,
OCH2CH3-1), 4.05 (2H, q, J ¼ 5.6 Hz, OCH2CH3-50), 2.22 (3H, s, CH3CO-6), 1.36
(3H, t, J ¼ 7.2 Hz, OCH2CH3-50), 1.27 (3H, t, J ¼ 7.2 Hz, OCH2CH3-1); ESI-MS
m/z [M]þ 400.
6,8-Dihydroxy-7-(50-ethoxybenzyloxy)cinnamic acid ethyl ester (50): This compound
was synthesised by treatment of 49 with potassium carbonate (Zou et al., 2006). Yield:
70.4%; white crystalline solid; m.p. ¼ 90–92ꢂC; Rf (hexane : EtOAc 3 : 1) 0.21;
1H NMR (400 MHz, CDCl3): ꢁ 7.52 (1H, d, J ¼ 16.0 Hz, H-3), 7.30 (2H, d, J ¼ 8.4 Hz,
H-30, 70), 6.89 (2H, d, J ¼ 8.4 Hz, H-40, 60), 6.84 (2H, s, H-5, 9), 6.29 (1H, d, J ¼ 16.0 Hz,
H-2), 5.59 (2H, brs, OH-6,8), 5.00 (2H, s, H-10), 4.26 (2H, q, J ¼ 7.2 Hz, OCH2CH3-1),
4.03 (2H, q, J ¼ 7.2 Hz, OCH2CH3-50), 1.42 (3H, t, J ¼ 7.2 Hz, OCH2CH3-50), 1.34
(3H, t, J ¼ 7.2 Hz, OCH2CH3-1); ESI-MS m/z [M]þ 358.
6,8-Diacetoxy-7-(40,50-dichlorobenzyloxy)cinnamic acid ethyl ester (51): This com-
pound (69%), accompanied by 52 (31%), were synthesised as previously reported in
Zou et al. (2006) by three steps from the starting material 3,4-dichlorobenzyl
bromide and 3,4,5-trihydroxybenzaldehyde (48). Yield: 68.2%; white crystalline
solid; Rf (hexane : EtOAc 3 : 1) 0.44; 1H NMR (400 MHz, CDCl3): ꢁ 7.55 (1H, d,
J ¼ 16.0 Hz, H-3), 7.51 (1H, d, J ¼ 2.0 Hz, H-30), 7.44 (1H, dd, J ¼ 8.0,2.0 Hz, H-70),
7.41 (1H, d, J ¼ 8.0 Hz, H-60), 7.16 (2H, s, H-5, 9), 6.77 (1 H, d, J ¼ 12.4 Hz, H-3), 6.33
(1H, d, J ¼ 16.0 Hz, H-2), 5.95 (0.25 H, d, J ¼ 12.4 Hz, H-2), 4.96 (2H, s, H-10), 4.25
(2H, q, J ¼ 7.2 Hz, OCH2CH3-1), 2.24 (6H, s, CH3CO-6, 8), 1.33 (3H, t, J ¼ 7.2 Hz,
OCH2CH3-1); ESI-MS m/z [M]þ 466.
6-Acetoxy-8-hydroxy-7-(40,50-dichlorobenzyloxy)cinnamic acid ethyl ester (52): Yield:
74.2%; white crystalline solid; m.p. 150–152ꢂC; Rf (hexane : EtOAc 3 : 1) 0.33;
1H NMR (400 MHz, CDCl3): ꢁ 7.52 (1H, d, J ¼ 16.0 Hz, H-3), 7.50 (1H, d, J ¼ 2.0 Hz,
H-30), 7.46 (1H, d, J ¼ 8.0 Hz, H-60), 7.20 (1H, dd, J ¼ 8.0,2.0 Hz, H-70), 7.00 (1H, d,
J ¼ 2.4 Hz, H-5), 6.83 (1H, d, J ¼ 2.4 Hz, H-9), 6.31 (1H, d, J ¼ 16.0 Hz, H-2), 4.96
(2H, s, H-10), 4.25 (2H, q, J ¼ 7.2 Hz, OCH2CH3-1), 2.26 (3H, s, CH3CO-6), 1.33
(3H, t, J ¼ 7.2 Hz, OCH2CH3-1); ESI-MS m/z [M]þ 424.
6,8-Dihydroxy-7-(40, 50-dichlorobenzyloxy)cinnamic acid ethyl ester (53): Compound
53 was obtained by treatment of 51 and 52 with potassium carbonate (Zou et al.,
2006). Yield: 74.2%; white crystalline solid; m.p. ¼ 150–152ꢂC; Rf (hexane : EtOAc
1
3 : 1) 0.33; H NMR (400 MHz, CDCl3): ꢁ 7.52 (1H, d, J ¼ 16.0 Hz, H-3), 7.50 (1H, d,
J ¼ 2.0 Hz, H-30), 7.46 (1H, d, J ¼ 8.0 Hz, H-60), 7.20 (1H, dd, J ¼ 8.0,2.0 Hz, H-70),
6.85 (2H, s, J ¼ 2.4 Hz, H-5,9), 6.31 (1H, d, J ¼ 16.0 Hz, H-2), 4.95 (2H, s, H-10), 4.26

218
H.-B. Zou et al.
(2H, q, J ¼ 7.2 Hz, OCH2CH3-1), 1.34 (3H, t, J ¼ 7.2 Hz, OCH2CH3-1); ESI-MS m/z
[M]þ 424.
7-(50-Ethoxybenzyloxy)-6,8-dihydroxycinnamic alcohol (54): Yield: 44.6%; white cry-
stalline solid; m.p. ¼ 102–104ꢂC; Rf (hexane : EtOAc 2 : 3) 0.46;1H NMR (400 MHz,
CDCl3): ꢁ 7.39–7.36 (5H, m, H-30–70), 6.50 (2H, s, H-2,6), 6.38 (1H, d, J ¼ 16.0 Hz,
H-3), 6.19 (1H, dt, J ¼ 16.0,6.0 Hz, H-2), 5.83 (2H, brs, OH-6, 8), 5.01 (2H, s, H-10),
4.26 (2H, d, J ¼ 6.0 Hz, H-1); ESI-MS m/z [M]þ 272.
7-(40, 50-Dichlorobenzyloxy)-6,8-dihydroxycinnamic alcohol (55): Yield: 44.6%; white
crystalline solid; m.p. ¼ 102–104ꢂC; Rf (hexane : EtOAc 2 : 3) 0.46;1H NMR
(400 MHz, CDCl3): ꢁ 7.39ꢁ7.36 (5H, m, H-30ꢁ70), 6.50 (2H, s, H–2,6), 6.38 (1H, d,
J ¼ 16.0 Hz, H–3), 6.19 (1H, dt, J ¼ 16.0,6.0 Hz, H-2), 5.83 (2H, brs, OH-6, 8), 5.01
(2H, s, H-10), 4.26 (2H, d, J ¼ 6.0 Hz, H-1); ESI-MS m/z [M]þ 272.
(2E, 4E) 5-[30,50-Dimethoxy-40-(500-ethoxybenzyloxy)phenyl]-2,4-pentadienoic acid
ethyl ester (56): Sodium (85 mg, 3.67 mmol) was added to absolute ethanol (2 mL)
and stirred to an even mixture. After cooling to room temperature an additional
10 mL of tetrahydrofuran and [(2E)-4-ethoxy-4-oxo-2-buten-1-yl]triphenyl-phospho-
nium bromide (1.84 g, 4.04 mmol) were added to give a yellow suspension.
Compound 33 (1.16 g, 3.67 mmol) was added and the resulting mixture was stirred
for 2 h. Addition of small portions of water quenched the reaction and 1 M HCl was
used to adjust to pH 7. The solvent was evaporated and the residue was purified by
chromatography to give two isomers 56 (main product) and 57. Data for 56: Yield:
68.8%; white crystalline solid; m.p. ¼ 112–114ꢂC; Rf (hexane : EtOAc 5 : 2) 0.25;
1H NMR (400 MHz, CDCl3): ꢁ 7.49 (1H, dd, J ¼ 15.6,9.2 Hz, H-3), 7.39 (2H, d,
J ¼ 8.4 Hz, H-30, 70), 6.85 (2H, d, J ¼ 8.4 Hz, H-40, 60), 6.76 (2H, m, H-4,5), 6.66
(2H, s, H-7,11), 5.98 (1H, d, J ¼ 15.6 Hz, H-2), 4.97 (2H, s, H-10), 4.22 (2H, q,
J ¼ 7.2 Hz, OCH2CH3-1), 4.03 (2H, q, J ¼ 7.2 Hz, OCH2CH3-50), 3.85 (6H, s, OCH3-
8,10), 1.43 (3H, t, J ¼ 7.2 Hz, OCH2CH3-50), 1.32 (3H, t, J ¼ 7.2 Hz, OCH2CH3-1);
ESI-MS m/z [M þ H]þ 413.
(2Z, 4Z) 5-[30,50-Dimethoxy-40-(500-ethoxybenzyloxy)phenyl]-2,4-pentadienoic acid
ethyl ester (57): Yield: 12.1%; white crystalline solid; m.p. ¼ 75–77ꢂC; Rf
(hexane : EtOAc 5 : 2) 0.31; 1H NMR (400 MHz, CDCl3): ꢁ 7.46 (1H, t,
J ¼ 11.6 Hz, H-3), 7.39 (2H, d, J ¼ 8.4 Hz, H-30, 70), 7.07 (1H, t, J ¼ 11.6 Hz, H-5),
6.86 (2H, d, J ¼ 8.4 Hz, H-40, 60), 6.83 (1H, t, J ¼ 11.6 Hz, H-4), 6.50 (2H, s, H-7,11),
5.78 (1H, d, J ¼ 11.6 Hz, H-2), 4.98 (2H, s, H-10), 4.22 (2H, q, J ¼ 7.2 Hz, OCH2
CH3-1), 4.04 (2H, q, J ¼ 7.2 Hz, OCH2CH3-50), 3.82 (6H, s, OCH3-8,10), 1.43
(3H, t, J ¼ 7.2 Hz, OCH2CH3-50), 1.33 (3H, t, J ¼ 7.2 Hz, OCH2CH3-1); ESI-MS m/z
[M þ H]þ 413.
(2E, 4E) 5-[30, 50-Dimethoxy-40-(500-ethoxybenzyloxy)phenyl]-2,4-pentadien-1-ol (58):
Compound 58 was synthesised by treatment of 56 with LiAlH4–AlCl3 complex
similar to the preparation of 26. Yield: 52.4%; white crystalline solid; m.p. ¼ 108–
1
109ꢂC; Rf (hexane : EtOAc 5 : 2) 0.15; H NMR (400 MHz, CDCl3): ꢁ 7.37 (2H, d,
J ¼ 8.4 Hz, H-30, 70), 6.84 (2H, d, J ¼ 8.4 Hz, H-40, 60), 6.68 (1H, t, J ¼ 15.6 Hz, H-3),
6.59 (2H, s, H-7,11), 6.46 (1H d, J ¼ 15.6 Hz, H-5), 6.46 (1H d, J ¼ 15.6 Hz, H-4), 5.95
(1H, dt, J ¼ 15.6,5.2 Hz, H-2), 4.93 (2H, s, H-10), 4.25 (2H, d, J ¼ 5.2 Hz, H-1), 4.02

Natural Product Research
219
(2H, q, J ¼ 7.2 Hz, OCH2CH3-50), 3.85 (6H, s, OCH3-8,10), 1.23 (3H, t, J ¼ 7.2 Hz,
OCH2CH3-50); ESI-MS m/z [M þ H]þ 371.
(2Z, 4Z) 5-[30, 50-Dimethoxy-40-(500-ethoxybenzyloxy)phenyl]-2,4-pentadien-1-ol (59):
Compound 59 was synthesised by treatment of 57 with LiAlH4–AlCl3 complex
similar to the method used for 26. Over-reduction of 57 also afforded compound 60
with only one double bond. Yield: 43.4%; white crystalline solid; m.p. ¼ 66–68ꢂC; Rf
(hexane : EtOAc 5 : 2) 0.20; 1H NMR [400 MHz, (CD3)2CO]: ꢁ 7.38 (2H, d,
J ¼ 8.4 Hz, H-30, 70), 6.83 (2H, d, J ¼ 8.4 Hz, H-40, 60), 6.67 (2H, s, H-7,11), 6.53
(1H, d, J ¼ 10.8 Hz, H-5), 6.43 (1H, dd, J ¼ 12.0,11.2 Hz, H-3), 5.63 (1H, dt, J ¼ 12.0,
5.2 Hz, H-2), 4.84 (1H, dd, J ¼ 11.2,10.8 Hz, H-4), 4.83 (2H, s, H-10), 4.16 (2H, d,
J ¼ 5.2 Hz, H-1), 4.03 (2H, q, J ¼ 7.2 Hz, OCH2CH3-50), 3.83 (6H, s, OCH3-8,10), 1.36
(3H, t, J ¼ 7.2 Hz, OCH2CH3-50); ESI-MS m/z [M þ H]þ 371.
(2Z) 5-[30, 50-Dimethoxy-40-(500-ethoxybenzyloxy)phenyl]-2-penten-1-ol (60): Yield:
13.7%; white crystalline solid; m.p. ¼ 60–62ꢂC; Rf (hexane : EtOAc 5 : 2) 0.22;
1H NMR [400 MHz, (CD3)2CO]: ꢁ 7.40 (2H, d, J ¼ 8.4 Hz, H-30, 70), 6.88 (2H, d,
J ¼ 8.4 Hz, H-40, 60), 6.54 (2H, s, H-7,11), 6.53 (1H, dt, J ¼ 11.2,6.8 Hz, H-3), 5.59
(1H, m, H-2), 4.83 (2H, s, H-10), 4.24 (2H, d, J ¼ 5.2 Hz, H-1), 4.03 (2H, q,
J ¼ 6.8 Hz, OCH2CH3-50), 3.80 (6H, s, OCH3-8, 10), 2.41 (1H, t, J ¼ 7.6 Hz, H-5),
2.22 (1H, m, H-4), 1.36 (3H, t, J ¼ 6.8 Hz, OCH2CH3-50); ESI-MS m/z [M þ H]þ 373.
3.2.2. Structure elucidation
The structures of these compounds were confirmed by both mass spectrometry and
1H NMR spectral data. Melting points were measured on a Perkin-Taike X-4
apparatus and then corrected. 1H NMR spectra were recorded on a Varian INOVA
400 spectrometer with TMS as the internal standard and CDCl3 as the solvent.
ESI-MS data were recorded on a Bruker Esquire 3000þ spectrometer and EI-MS
was performed on a Varian MAT-95 MS instrument.
3.2.3. Cytotoxicity evaluation
Exponentially growing cells were seeded in quadruplicate into 96-well flat-bottomed
plates at a concentration of 5 ꢄ 103 cells per well. After 24 h incubation,
the compounds studied were added to the wells. After 72 h, 10 mL of MTT solution
(5 mg mLꢁ1 in phosphate buffered solution) as added to the culture medium and
incubated at 37ꢂC for further 4 h. Following removal of unconverted MTT, 200 mL
of DMSO was added to each well and the plates were shaken 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.
4. Conclusions
Three series of di- and tri-substituted derivatives of cinnamic alcohol and its
conjugated dienol analogues were synthesised, and their cytotoxicity was evaluated
on nine tumour cell lines. Most of the cinnamic alcohol derivatives showed cytotoxic
activity. Most of the synthesised disubstituted cinnamic alcohols exhibited moderate

220
H.-B. Zou et al.
cytotoxicity to the tested tumour cell lines, while the trisubstituted derivatives with
two free hydroxy groups showed potential cytotoxic activity. In particular,
compound 55, with a 40,50-dichlorobenzyloxy group at C-7, exhibited significant
cytotoxicity to seven human tumour cell lines in the micromolar range, especially to
KB and P388D1 cell lines, with IC50 values of 0.4 and 0.5 mM, respectively, which
was 20 times higher than the control DDP. The finding that the configuration of the
cinnamic dienol analogues is also crucial for its cytotoxicity, indicating that the both
Z-configurated conjugated double bonds will enhance the bioactivity, is also to be
noted. The structure relationship of these three series was discussed. This extensive
structure–activity relationship analysis will guide us in designing optimised cytotoxic
cinnamic derivatives, and compound 55 will serve as the lead compound for the
synthesis of compounds with better and more selective cytotoxicity to human tumour
cell lines.
Acknowledgements
This work was in part financially supported by the Special Foundation 985 from Zhejiang
University, the German Science Foundation (Bad Godesberg) and Zhejiang University K.P.
Chao’s Foundation. The authors are grateful to Prof. Handong Sun and Prof. Jun Zhou (KIB,
´
CAS), as well as to Franc¸ oise Gueritte (ICSN, CNRS) for their interest in this research topic.
We thank Dr Jiali Luo and Mr Xiaoren Wang at the R&D Centre of Zhejiang Hisun Group
for measuring the mass data.
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