Selective antiproliferative activity of caffeic acid phenethyl ester analogues on highly liver-Metastatic murine colon 26-L5 carcinoma cell line

Article abstract of DOI:10.1016/S0968-0896(02)00138-4

Caffeic acid phenethyl ester (CAPE, 2) and its 20 analogues (1, 3-21) were prepared. These esters were tested by MTT assay on growth of murine colon 26-L5 carcinoma, murine B16-BL6 malonoma, murine Lewis lung carcinoma, human HT-1080 fibrosarcoma, human lung A549 adenocarcinoma, and human cervix HeLa adenocarcinoma cell lines. It was found that CAPE analogues possessed selective antiproliferative activity toward highly liver-metastatic murine colon 26-L5 carcinoma cell line. Among them, 4-phenylbutyl caffeate (4), (Z)-8-phenyl-7-octenyl (10a) and (E)-8-phenyl-7-octenyl (10b) caffeate showed the most potent antiproliferative activity (EC₅₀ value, 0.02μM). In addition, CAPE (2) induced DNA fragmentation at concentrations of 1 to 10μg/mL towards murine colon 26-L5 carcinoma cells. Copyright

Full text of DOI:10.1016/S0968-0896(02)00138-4

Bioorganic & Medicinal Chemistry 10 (2002) 3351–3359
Selective Antiproliferative Activity of Caffeic Acid Phenethyl
Ester Analogues on Highly Liver-Metastatic Murine Colon
26-L5 Carcinoma Cell Line
Takema Nagaoka, Arjun H. Banskota, Yasuhiro Tezuka,
Ikuo Saiki and Shigetoshi Kadota*
Institute of Natural Medicine, Toyama Medical and Pharmaceutical University, 2630-Sugitani, Toyama 930-0194, Japan
Received 1February 2002; accepted 10 April 2002
Abstract—Caffeic acid phenethyl ester (CAPE, 2) and its twenty analogues (1, 3–21) were prepared. These esters were tested by
MTT assay on growth of murine colon 26-L5 carcinoma, murine B16-BL6 malonoma, murine Lewis lung carcinoma, human HT-
1080 fibrosarcoma, human lung A549 adenocarcinoma, and human cervix HeLa adenocarcinoma cell lines. It was found that
CAPE analogues possessed selective antiproliferative activity toward highly liver-metastatic murine colon 26-L5 carcinoma cell line.
Among them, 4-phenylbutyl caffeate (4), (Z)-8-phenyl-7-octenyl (10a) and (E)-8-phenyl-7-octenyl (10b) caffeate showed the most
potent antiproliferative activity (EC50 value, 0.02 mM). In addition, CAPE (2) induced DNA fragmentation at concentrations of 1
to 10 mg/mL towards murine colon 26-L5 carcinoma cells. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
Thus, we synthesized CAPE (2) and twenty analogues,
including benzyl and cinnamyl caffeates (1, 3–21), and
examined their antiproliferative activities toward six
tumor cell lines.
Caffeic acid phenethyl ester (CAPE), which was firstly
isolated from Hungarian propolis,1 is a well known con-
stituent with interesting biological properties.2 Grunberger
et al. reported the significant cytotoxic effect of CAPE
against various tumor cell lines.3 Because of its simpli-
city in structure and of interesting cytotoxic property, its
antitumor activity was further studied.4ꢀ9 Besides cyto-
toxic and antitumor properties, CAPE also possessed
inhibitory activity against HIV-1integrase, cyclooxygen-
ase and lipoxygenase.10ꢀ12 It has also reported to block
the activation of nuclear factor NF-kB by tumor necrosis
factor and completely block the production of reactive
oxygen species (ROS) in human neutrophils.13,14
Results and Discussion
Chemistry
Caffeic acid phenethyl ester (CAPE) has been synthe-
sized previously by several groups.3ꢀ5,14,16 Among the
synthetic methods, we selected one-pot esterification of
caffeic acid and alcohol with thionyl chloride (SOCl2) in
dioxane, reported by Lee et al.5 to prepare CAPE and
its analogues except for methyl (13) and ethyl caffeates
(14). The percentage yield of individual compounds are
given in Table 1. Methyl (13) and ethyl caffeates (14)
were prepared by classical acid-catalyzed esterification
of caffeic acid with methanol and ethanol, respectively.
In our recent work, we have isolated CAPE (2) together
with benzyl caffeate (1) and cinnamyl caffeate (9) from
The Netherlands propolis as potent antiproliferative
agents.15 They showed selective antiproliferative activity
toward highly liver-metastatic murine colon 26-L5 car-
cinoma cells, which encouraged us to further investigate
the structure–activity relationship on CAPE analogues.
Alcohols 23–26, corresponding to esters 7, 8, 10 and 11,
are not commercially available, and thus we synthesized
them from the corresponding bromoalcohol and benz-
aldehyde through Wittig reaction (Scheme 1). First, the
hydroxyl group of commercially available 7-bromohept-
anol (22a) and 11-bromoundecanol (23a) was protected as
*Corresponding author. Fax: +81-76-434-5059; e-mail: kadota@ms.
toyama-mpu.ac.jp
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00138-4

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T. Nagaoka et al. / Bioorg. Med. Chem. 10 (2002) 3351–3359
Scheme 1. (a) Synthetic procedure of alcohols 23–26; (b) equation of esterificatin of caffeic acid and alcohols.
Antiproliferative activity
Table 1. Yields of CAPE analogues
Caffeic acid phenethyl ester (CAPE) together with its
twenty analogues (1, 3–21) were tested for their anti-
proliferative activities toward six different tumor cell
lines, that is, murine colon 26-L5 carcinoma (colon 26-
L5),17 murine B16-BL6 malonoma (B16-BL6),18 murine
Lewis lung carcinoma (LLC),19 human HT-1080 fibro-
sarcoma (HT-1080),20 human lung A549 adenocarcinoma
(A549),21 and human cervix HeLa adenocarcinoma
(HeLa) cell lines.22 The antiproliferative activities of 1–21
are summarized in terms of their EC50 values in Table 2.
All these esters showed stronger antiproliferative activ-
ity than caffeic acid, and all the compounds except for
20 and 21 showed the strongest activity toward colon
26-L5 cell line. Interestingly, the CAPE analogues
revealed no cytotoxic effect toward primary cultured
mouse hepatocytes up to 100 mM concentration. This
indicates that CAPE and its analogues possessed selec-
tive antiproliferative activity toward colon 26-L5 cell
line. Especially, the activities of compounds 4, 10a, 10b
and 12 (EC50: 0.02, 0.02, 0.02 and 0.03 mM, respectively)
were stronger than those of 5-fluorouracil (EC50:
0.06 mM) and doxorubicin (EC50: 0.04 mM), which were
used as positive controls. Antiproliferative activity of
the cis (10a and 11a) and trans (10b and 11b) isomers of
10 and 11 possessed similar strength of activity toward
all tested cell lines. Moreover, almost all esters pos-
sessed stronger antiproliferative activities than those of
5-fluorouracil in B16-BL6 and LLC cell lines, but less
active than doxorubicin.
Product
R
Yield (%)
1
2
3
4
5
6
7
8
–CH2Ph
–(CH2)2Ph
–(CH2)3Ph
–(CH2)4Ph
–(CH2)5Ph
–(CH2)6Ph
–(CH2)8Ph
–(CH2)12Ph
75
55
55
57
53
56
41
54
36
40
50
74
40
17
58
76
64
48
57
45
35
9
–CH2CH¼CHPh
–(CH2)6CH¼CHPh
–(CH2)10CH¼CHPh
–(CH2)2-c-Hex
–CH3
10
11
12
13
14
15
16
17
18
19
20
21
–CH2CH3
–(CH2)2CH3
–(CH2)3CH3
–(CH2)7CH3
–(CH2)9CH3
–(CH2)11CH3
–(CH2)13CH3
–(CH2)15CH3
a tetrahydropyranyl (THP) ether. The protected bro-
mides 22b and 23b were reacted with triphenylphos-
phine to yield phosphonium salts, followed by Wittig
reaction with benzaldehyde to give olefins 22c and 23c,
respectively. These alkenyl products were reduced by
using Pd/C catalyst, and the products 22d and 23d were
deprotected by p-toluenesulfonic acid (p-TsOH) to
afford the saturated alcohols 22 and 23, respectively. On
the other hand, unsaturated alcohols 24 and 25 were
prepared through deprotection of 22c and 23c as a 77/
23 mixture of cis/trans isomers, which were used to
prepare the esters 10 and 11 as a mixture. The cis/trans
mixtures of 10 and 11 were separated by using HPLC
and the antiproliferative activity of the individual
compounds were examined.
Considering the range of the potent cytotoxic agent
(EC50<4 mg/mL) made by Geran et al.,23 almost all
CAPE analogues fall within the potent cytotoxic range
against colon 26-L5, B16-BL6 and LLC cell lines. In
addition, nearly 50% of the esters also possessed inter-
esting antiproliferative activity toward human HT-1080
fibrosarcoma and HeLa cell lines (EC50<4 mg/mL) but

T. Nagaoka et al. / Bioorg. Med. Chem. 10 (2002) 3351–3359
Table 2. Antiproliferative activities of caffeic acid esters
3353
alcoholic part: esters having an alkyl group with a phenyl
group at the end of the alkyl chain (1–8, group 1), esters
having an alkyl group with a stylyl group at the end of the
alkyl chain (9–11, group 2), an ester having an alkyl group
with a cyclohexyl group at the end of the alkyl chain (12,
group 3) and esters having a straight alkyl chain (13–21,
group 4). Comparison of group 1and group 2 com-
pounds (3, 7, 8 vs 9, 10, 11) revealed that both group
possessed almost equal extent of activity against all the
tested cell lines. On the other hand, in a comparison of
group 1and group 3 compounds ( 2 vs 12), ester 12 showed
stronger antiproliferative activity than ester 2 against all
the tested cell lines, which indicates that conversion of a
phenyl group into a cyclohexyl group may enhance anti-
proliferative activity. Moreover, group 1compounds
seem to be more potent than group 4 compounds.
Compd
EC50 (mM)
Colon 26-L5 LLC B16 BL-6 HT-1080 A549 HeLa
1
2
3
4
5
6
7
8
9
10a
10b
11a
11b
12
13
14
15
16
17
18
19
20
21
1.34
0.15
0.10
0.02
0.08
0.08
0.09
1.75
0.22
0.02
0.02
1.17
2.49
0.03
3.27
1.14
1.52
0.27
0.22
0.25
0.29
5.73
2.57
2.32
2.29
1.27
1.40
0.84
8.11
2.16
0.88
1.01
1.93
2.42
0.74
4.66.171
4.39
3.30
2.48
1.19
1.15
0.77
2.56
7.90
2.18
2.16
1.99
2.12
1.85
1.81
12.1
14.4
18.1
13.3
19.6 25.3
32.4 10.7
23.5 22.0
31.6 20.0
21.9 10.6
7.38
10.4
20.2
62.8
17.1
10.51 22.3
10.21 33.5
37.3
38.4
11.8
21.4
22.4
9.11
2.61
17.2
21.2 18.4
50.0 11.5
2.83
1.77
1.49
7.20
7.23
2.00
1.93
1.91
7.30
8.59
9.54
38.1
37.5
28.1
35.2
43.7 26.8
33.4 61.9 24.4
42.9 21.9
4.64
3.87
2.78
2.23
2.06
1.80
On careful examination of the EC50 values of group 1
compounds (1–8) against colon 26-L5, B16-BL6 and LLC
cell lines, it was concluded that the strength of anti-
proliferative activity was increased from benzyl caffeate
(1) to 8-phenyloctyl caffeate (7). The EC50 values of
8-phenyloctyl caffeate (7) were 0.09, 0.84, 1.81 mM
toward colon 26-L5, B16-BL6 and LLC cell lines,
respectively. 12-Phenyldodecanyl caffeate (8) showed less
antiproliferative activity than 8-phenyloctyl caffeate (7)
toward all the tested cell lines, indicating further elonga-
tion of alkyl chain may decrease the antiproliferative
activity.
17.9
20.2
20.0
14.2
19.1
19.9
36.9
42.2
34.6
21.4
21.4
4.02
2.40
1.85
2.00
16.1
10.2
43.6
0.06
0.04
14.7
15.6
314
8.76
0.22
18.5 10.0
23.7 14.6
288 300
3.610.76
0.20 0.12
13.3
318
4.69
0.09
Caffeic acid
5-FU
Doxorubicin
257
5.00
0.06
showed less activity toward A549 cell line (EC50>4 mg/
mL). Colon 26-L5, B16-BL6 and LLC cell lines were
derived from murine and the others (HT-1080, HeLa
and A549) were derived from human. It indicates that
CAPE analogues selectively inhibit murine tumor cell
lines rather than human tumor cell lines. Colon 26-L5,
B16-BL6, LLC and HT-1080 cell lines are highly meta-
static cell lines and are frequently used for metastatic
experiments.24ꢀ26 In this study, CAPE analogues
showed the significant activity toward these cell lines,
except for HT-1080, suggesting that CAPE analogues
can be used for anti-metastatic drugs.
In the EC50 values of group 4 compounds, having a
straight chain alkyl group in the alcoholic part, gave
more sharp structure–activity relationship in the tested
cell lines. The EC50 values decreased from methyl caffe-
ate (13) having only one carbon on the alcoholic part to
n-dodecanyl caffeate (18) having a C10 alkyl chain, while
further increase of the chain length of the alkyl part
increased the EC50 values (Fig. 1). It should be noted
here that there is only small differences in the EC50
values of 16, 17 and 18, having a C4, C8 and C10 alkyl
chain, toward colon 26-L5, B16-BL6 and LLC cell lines.
Similar trends were also observed for the other cell lines,
although their EC50 values were high (Fig. 1). Etzen-
houser et al. reported that n-octyl ester of caffeic acid is
ten times more toxic than CAPE towards L1210 leuke-
mia and MCF-7 breast cancer cell lines.28 In the present
study, however, we found the identical antiproliferative
activities between CAPE (2) and n-octyl caffeate (17).
These different results may be due to the difference in
the cell lines taken in two different experiments. Inter-
estingly, 8-phenyloctyl caffeate (7) possessed 5–10 times
stronger antiproliferative activities than CAPE (2) in all
the tested cell lines, except for HT-1080 cell line.
In previous reports, CAPE and its analogues showed
significant cytotoxic effect against B16, HCT116, A431,
HL-60 and oral cancer cell lines.4,5,27 The EC50 values
of CAPE analogues toward these cell lines were at least
10 to 100 times higher than those against colon 26-L5
carcinoma cell line, in the present study. Thus, CAPE
analogues are expected to be good candidates for anti-
cancer agent for colon cancer. In fact, CAPE at a diet-
ary level decreased tumor formation by 63% in C57BL/
6J-Min/+ mice, which bear a germline mutation in the
Apc gene and spontaneously develop numerous intest-
inal adenomas by 15 weeks of age.7
Structure–activity relationship
DNA fragmentation
Considering the structure–activity relationship, neither
caffeic acid alone nor the alcohol of an individual esters
possessed any significant antiproliferative activity (data
not shown), suggesting that the ester structure should be
essential for the antiproliferative effect of CAPE analo-
gues. In this study, the esters are mainly divided into
four different groups according to the nature of their
It was well documented that DNA fragmentation is one
of the marker for induction of apoptosis.29 Thus, we
further examined DNA fragmentation to clarify whe-
ther CAPE analogues induced apoptosis or not. For this
experiment, we selected CAPE (2) as a model com-
pound and performed on murine colon 26-L5 cell line.
Consequently, CAPE (2) at concentrations of 1–10 mg/

3354
T. Nagaoka et al. / Bioorg. Med. Chem. 10 (2002) 3351–3359
Figure 1. Structure–activity relationship of straight chain alkyl esters 13–21 on increasing alkyl chain. &=murine colon 26-L5 carcinoma,
&=murine B16-BL6 malonoma, ~=murine Lewis lung carcinoma, ~=human HT-1080 fibrosarcoma, *=human lung A549 adenocarcinoma,
*=human cervix HeLa adenocarcinoma.
reactive species are thought to act as second messengers
for signal transduction pathways that regulate cell pro-
liferation.30 Thus, by the reducing intracellular peroxides,
antioxidants are expected to inhibit cancer. Therefore, one
of the possible mechanisms for inhibition of cell pro-
liferation by CAPE analogues may be involvement of
their antioxidative properties and induction of apoptosis.
Conclusion
In conclusion, we synthesized 21CAPE analogues,
which were tested by MTT assay on growth of six dif-
ferent tumor cell lines. Among the tested cell lines,
CAPE analogues possessed selective antiproliferative
activity toward colon 26-L5 cell line, especially, esters 4,
10a, 10b and 12 showed stronger activity than positive
controls, 5-fluorouracil and doxorubicin. Considering
the structure–activity relationship, ester itself seems to
Figure 2. CAPE induced DNA fragmentation in murine colon 26-L5
be important for antiproliferative activity, and the
carcinoma cells. After the cells were cultured for 24 h with various
structure of the alcoholic part, that is, phenyl group,
conjugate double bond and cyclohexyl group, bring
only small changes in their activity. However, the
strength of antiproliferative activity was increased from
benzyl caffeate (1) to 8-phenyloctyl caffeate (7) against
colon 26-L5, B16-Bl6 and LLC cell lines, and further
elongation of the alkyl chain decreased the anti-
proliferative activities. In esters with a straight alkyl
chain, the C8–C12 alcoholic chain appeared to be more
effective structure for antiproliferative activity.
concentration of CAPE, the fragmented DNA was isolated, electro-
phoresed on 1.5% agarose gel, and then visualized by ethidium bro-
mide staining. Lanes 1–5: treated with 10, 5, 2, 1, 0.1 mg/mL of CAPE,
respectively; lane 6: normal; lane 7: 100 base pair ladder marker.
mL caused the DNA fragmentation, indicating the
induction of apoptosis in tumor cells (Fig. 2).
In our previous study, we observed CAPE (2), benzyl
caffeate (1) and cinnamyl caffeate (9) isolated from the
Netherlands propolis possessed scavenging activities
toward DPPH free radical equal to those of a-toco-
pherol and ascorbic acid, well known antioxidants.15
Moreover, CAPE was reported to completely block the
production of ROS in human neutrophils.14 These
Experimental
All reagents and solvents were reagent grade or were
purified by standard methods before use. IR spectra were

T. Nagaoka et al. / Bioorg. Med. Chem. 10 (2002) 3351–3359
3355
obtained on a Shimadzu IR-408 spectrophotometer. EI-
MS and high-resolution FAB-MS (HRFABMS) data
were obtained by a Jeol JMS-700T spectrometer. H-
and 13C NMR spectra were taken on a Jeol JNM-
LA400 spectrometer with tetramethylsilane (TMS) as an
internal standard; chemical shifts are recorded in d
values. Column chromatography was performed by
using Wakogel C-200 silica gel. TLC was carried out on
precoated Merck silica gel F254 plates (0.25 or 0.5 mm).
8-Phenyloctyl caffeate (7). IR (KBr, cmꢀ1): 3500, 3340,
1675, 1595, 1535, 1445, 1360, 1270, 1170, 975, 850, 810;
1H NMR (CD3OD) d 7.53 (1H, d, J=15.9 Hz), 7.26–
7.08 (5H, m), 7.04 (1H, d, J=1.9 Hz), 6.92 (1H, dd,
J=8.2, 1.9 Hz), 6.77 (1H, d, J=8.2 Hz), 6.24 (1H, d,
J=15.9 Hz), 4.14 (2H, t, J=6.6 Hz), 2.57 (2H, t, J=
7.7 Hz), 1.67 (2H, quintet, J=6.6 Hz), 1.60 (2H, quintet,
J=7.7 Hz), 1.44–1.24 (8H, m); 13C NMR (CD3OD) d
169.3 (s), 149.5 (s), 146.7 (d), 143.9 (s), 129.3 (d), 129.2
(d), 127.7 (s), 126.6 (s), 122.9 (d), 116.5 (d), 115.2 (d),
115.1 (d), 65.5 (t) 36.9 (t), 32.6 (t), 30.4 (t), 30.3 (t), 30.2
(t), 29.8 (t), 27.0 (t); HRFABMS calcd for C23H29O4
[M+H]+: 369.2066; found: 369.2084.
1
General procedure for preparation of caffeic acid esters
A solution of caffeic acid (36 mg, 0.2 mmol) in dioxane
(2 mL) under argon was treated with thionyl chloride
(44 mL, 0.6 mmol). The mixture was stirred at 100 ꢁC
for 3 h. Then alcohol (0.3 mmol) was added dropwise to
the mixture, and the mixture was stirred at 100 ꢁC for
6 h. After removal of the solvent under reduced pres-
sure, the residue was subjected to preparative TLC with
CHCl3–MeOH (90:10) to give the desired caffeic acid
esters. The known esters 1–3, 16 and 17 were identified
by comparisons of their spectral data with those in
literature.28
12-Phenyldodecanyl caffeate (8). IR (KBr, cmꢀ1): 3460,
3300, 1685, 1600, 1525, 1445, 1385, 1270, 1175, 970,
855, 810; 1H NMR (CD3OD) d 7.53 (1H, d, J=
15.9 Hz), 7.25–7.10 (5H, m), 7.03 (1H, d, J=1.9 Hz), 6.92
(1H, dd, J=8.2, 1.9 Hz), 6.77 (1H, d, J=8.2 Hz), 6.24
(1H, d, J=15.9 Hz), 4.15 (2H, t, J=6.6Hz), 2.57 (2H, t,
J=7.7Hz), 1.68 (2H, quintet, J=6.6 Hz), 1.58 (2H, quin-
tet, J=7.7 Hz), 1.44–1.22 (16H, m); 13C NMR (CD3OD)
d 169.3 (s), 149.5 (s), 146.8 (d), 143.9 (s), 129.4 (d), 129.3
(d), 127.7 (s), 126.6 (s), 122.9 (d), 116.5 (d), 115.2 (d),
115.1 (d), 65.5 (t) 36.1 (t), 32.7 (t), 30.7 (t), 30.6 (t),
30.30 (t), 30.28 (t), 29.8 (t), 27.0 (t); HRFABMS calcd
for C27H37O4 [M+H]+: 425.2692; found: 425.2703.
4-Phenylbutyl caffeate (4). IR (KBr, cmꢀ1): 3490, 3340,
1685, 1595, 1530, 1440, 1355, 1270, 1170, 975, 845, 810;
1H NMR (CD3OD) d 7.52 (1H, d, J=15.9 Hz), 7.30–
7.10 (5H, m), 7.03 (1H, d, J=1.9 Hz), 6.93 (1H, dd,
J=8.2, 1.9 Hz), 6.77 (1H, d, J=8.2 Hz), 6.24 (1H, d, J=
15.9 Hz), 4.17 (2H, t, J=6.8 Hz), 2.66 (2H, t, J=
6.8 Hz), 1.78–1.64 (4H, m); 13C NMR (CD3OD) d 169.3
(s), 149.5 (s), 146.8 (d), 143.4 (s), 129.4 (d), 129.3 (d),
127.7 (s), 126.8 (s), 122.9 (d), 116.7 (d), 115.14 (d), 115.10
(d), 65.3 (t), 36.4 (t), 29.4 (t), 29.0 (t); HRFABMS calcd
for C19H21O4 [M+H]+: 313.1440; found: 313.1440.
Cinnamyl caffeate (9). IR (KBr, cmꢀ1): 3450, 3300,
1675, 1630, 1595, 1530, 1445, 1355, 1270, 1165, 965,
845, 810; 1H NMR (CD3OD) d 7.59 (1H, d, J=
15.9 Hz), 7.42 (2H, br d, J=7.2 Hz), 7.31(2H, br t, J=
7.2 Hz), 7.23 (1H, br t, J=7.2 Hz), 7.05 (1H, d,
J=1.9 Hz), 6.95 (1H, dd, J=8.2, 1.9 Hz), 6.78 (1H, d,
J=8.2 Hz), 6.70 (1H, d, J=15.9 Hz), 6.38 (1H, dt,
J=15.9, 6.2 Hz), 6.30 (1H, d, J=15.9 Hz), 4.82 (2H, d,
J=6.2 Hz); 13C NMR (CD3OD) d 169.0 (s), 149.6 (s),
147.1 (d), 146.8 (s), 137.8 (s), 135.0 (d), 129.6 (d), 129.0
(s), 127.7 (d), 124.6 (s), 123.0 (d), 116.5 (d), 115.1 (d),
114.9 (d), 66.0 (t); HRFABMS calcd for C18H17O4
[M+H]+: 297.1127; found: 297.1129.
5-Phenylpentyl caffeate (5). IR (KBr, cmꢀ1): 3450, 3290,
1675, 1595, 1530, 1440, 1355, 1265, 1170, 975, 845, 810;
1H NMR (CD3OD) d 7.52 (1H, d, J=15.9 Hz), 7.26–
7.08 (5H, m), 7.04 (1H, d, J=1.9 Hz), 6.92 (1H, dd,
J=8.2, 1.9 Hz), 6.78 (1H, d, J=8.2 Hz), 6.23 (1H, d, J=
15.9 Hz), 4.14 (2H, t, J=6.6 Hz), 2.61(2H, t, J=
7.6 Hz), 1.70 (2H, quintet, J=7.3 Hz), 1.65 (2H, quintet,
J=7.6 Hz), 1.41 (2H, quintet, J=7.6 Hz); 13C NMR
(CD3OD) d 169.3 (s), 149.5 (s), 146.7 (d), 143.6 (s),
129.4 (d), 129.2 (d), 127.7 (s), 126.7 (s), 122.9 (d), 116.5
(d), 115.2 (d), 115.1 (d), 65.4 (t), 36.7 (t), 32.2 (t), 29.62
(t), 26.58 (t); HRFABMS calcd for C20H23O4 [M+H]+:
327.1569; found: 327.1582.
2-cycloHexylethyl caffeate (12). IR (KBr, cmꢀ1): 3460,
3290, 1675, 1595, 1530, 1445, 1360, 1260, 1160, 975,
850, 810; 1H NMR (CD3OD)
d 7.52 (1H, d,
J=15.9 Hz), 7.03 (1H, d, J=1.9 Hz), 6.94 (1H, dd,
J=8.2, 1.9 Hz), 6.77 (1H, d, J=8.2 Hz), 6.24 (1H, d,
J=15.9 Hz), 4.20 (1H, t, J=6.7 Hz), 1.83–1.62 (6H, m),
1.58 (2H, q, J=6.7 Hz), 1.48–1.35 (1H, m), 1.35–1.13
(2H, m), 1.04–0.91 (2H, m); 13C NMR (CD3OD) d
169.4 (s), 149.5 (s), 146.8 (d), 127.7 (s), 122.9 (d), 116.5
(d), 115.2 (d), 115.1 (d), 63.6 (t), 37.3 (t), 35.9 (d), 34.3
(t), 27.6 (t), 27.3 (t); HRFABMS calcd for C17H23O4
[M+H]+: 291.1596; found: 291.1598.
6-Phenylhexyl caffeate (6). IR (KBr, cmꢀ1): 3480, 3250,
1675, 1595, 1530, 1445, 1360, 1270, 1175, 975, 850, 810;
1H NMR (CD3OD) d 7.53 (1H, d, J=15.9 Hz), 7.26–
7.08 (5H, m), 7.04 (1H, d, J=2.2 Hz), 6.93 (1H, dd,
J=8.2, 2.2 Hz), 6.77 (1H, d, J=8.2 Hz), 6.24 (1H, d,
J=15.9 Hz), 4.14 (2H, t, J=6.6 Hz), 2.60 (2H, t, J=
7.7 Hz), 1.68 (2H, quintet, J=6.6 Hz), 1.63 (2H, quintet,
J=7.7 Hz), 1.48–1.32 (4H, m); 13C NMR (CD3OD) d
169.4 (s), 149.5 (s), 146.8 (d), 143.8 (s), 129.4 (d), 129.2
(d), 127.7 (s), 126.6 (s), 123.0 (d), 122.9 (d), 116.5 (d),
115.2 (d), 115.1 (d), 65.5 (t), 36.8 (t), 32.6 (t), 29.9 (t),
29.8 (t), 26.6 (t); HRFABMS calcd for C21H25O4
[M+H]+: 341.1753; found: 341.1780.
n-Propyl caffeate (15). IR (KBr, cmꢀ1): 3450, 1660,
1600, 1530, 1445, 1315, 1270, 1180, 1110, 1035, 980,
870, 810; 1H NMR (CD3OD) d 7.53 (1H, d, J=
15.9 Hz), 7.04 (1H, d, J=1.9 Hz), 6.94 (1H, dd, J=8.2,
1.9 Hz), 6.78 (1H, d, J=8.2 Hz), 6.25 (1H, d, J=
15.9 Hz), 4.12 (2H, t, J=7.2 Hz), 1.71 (2H, sextet, J=
7.2 Hz), 0.99 (3H, t, J=7.2 Hz); 13C NMR (CD3OD) d
169.4 (s), 149.5 (s), 146.7 (d), 127.7 (s), 122.9 (d), 116.5

3356
T. Nagaoka et al. / Bioorg. Med. Chem. 10 (2002) 3351–3359
(d), 115.2 (d), 115.1 (d), 67.0 (t), 23.1 (t), 10.7 (q);
HRFABMS calcd for C12H15O4 [M+H]+: 223.0970;
found: 223.0980.
(Z)-8-Phenyl-7-octenyl caffeate (10a). Retention time:
34.5 min; IR (KBr, cmꢀ1): 3480, 3300, 1680, 1635, 1595,
1530, 1445, 1365, 1270, 1170, 975, 855, 810; H NMR
1
(CD3OD) d 7.52 (1H, d, J=15.9 Hz), 7.36–7.10 (5H, m),
7.03 (1H, d, J=1.7 Hz), 6.92 (1H, dd, J=8.2, 1.7 Hz),
6.77 (1H, d, J=8.2 Hz), 6.40 (1H, d, J=11.7 Hz), 6.23
(1H, d, J=15.9 Hz), 5.64 (1H, dt, J=11.7, 7.2 Hz), 4.13
(2H, t, J=6.7 Hz), 2.32 (2H, q, J=7.2 Hz), 1.68 (2H,
quintet, J=7.0 Hz), 1.58–1.25 (6H, m); 13C NMR
(CD3OD) d 169.3 (s), 149.5 (s), 146.8 (d), 139.0 (s),
133.6 (d), 130.2 (d), 129.7 (d), 129.1 (d), 127.7 (s), 127.5
(d), 122.9 (d), 116.5 (d), 115.2 (d), 115.1 (d), 65.5 (t) 30.8
(t), 29.9 (t), 29.8 (t), 29.3 (t), 26.9 (t); HRFABMS calcd
for C23H27O4 [M+H]+: 367.1909; found: 367.1918.
n-Decanyl caffeate (18). IR (KBr, cmꢀ1): 3460, 3290,
1675, 1600, 1530, 1445, 1360, 1270, 1170, 1105, 1010,
975, 860, 810; 1H NMR (CD3OD) d 7.53 (1H, d,
J=15.9 Hz), 7.04 (1H, d, J=1.9 Hz), 6.93 (1H, dd,
J=8.2, 1.9 Hz), 6.78 (1H, d, J=8.2 Hz), 6.24 (1H, d,
J=15.9 Hz), 4.16 (2H, t, J=6.6 Hz), 1.68 (2H, quintet,
J=7.0 Hz), 1.46–1.21 (14H, m), 0.89 (3H, t, J=7.0 Hz);
13C NMR (CD3OD) d 169.4 (s), 149.5 (s), 146.8 (d),
127.7 (s), 122.9 (d), 116.5 (d), 115.2 (d), 115.1 (d), 65.6
(t), 33.0 (t), 30.6 (t), 30.4 (t), 30.3 (t), 29.8 (t), 27.1(t),
23.7 (t), 14.4 (q); HRFABMS calcd for C19H29O4
[M+H]+: 321.2066; found: 321.2057.
(E)-8-Phenyl-7-octenyl caffeate (10b). Retention time:
38.0 min; IR (KBr, cmꢀ1): 3480, 3300, 1680, 1635, 1595,
1
n-Dodecanyl caffeate (19). IR (KBr, cmꢀ1): 3490, 3310,
1680, 1605, 1535, 1445, 1360, 1275, 1175, 1110, 1025,
975, 860, 815; 1H NMR (CD3OD) d 7.43 (1H, d,
J=15.9 Hz), 6.93 (1H, d, J=1.9 Hz), 6.83 (1H, dd,
J=8.2, 1.9 Hz), 6.67 (1H, d, J=8.2 Hz), 6.14 (1H, d,
J=15.9 Hz), 4.06 (2H, t, J=6.7 Hz), 1.59 (2H, quintet,
J=6.7 Hz), 1.37–1.02 (18H, m), 0.79 (3H, t, J=6.9 Hz);
13C NMR (CD3OD) d 169.3 (s), 149.5 (s), 146.8 (d),
127.7 (s), 122.9 (d), 116.5 (d), 115.2 (d), 115.1 (d), 65.5
(t), 33.0 (t), 30.73 (t), 30.65 (t), 30.6 (t), 30.4 (t), 30.3 (t),
29.8 (t), 27.1(t), 23.7 (t), 14.5 (q); HRFABMS calcd for
C21H33O4 [M+H]+: 349.2379; found: 349.2381.
1530, 1445, 1365, 1270, 1170, 975, 855, 810; H NMR
(CD3OD) d 7.52 (1H, d, J=15.9 Hz), 7.36–7.10 (5H, m),
7.03 (1H, d, J=1.7 Hz), 6.92 (1H, dd, J=8.2, 1.7 Hz),
6.77 (1H, d, J=8.2 Hz), 6.36 (1H, d, J=15.9 Hz), 6.23
(1H, dt, J=15.9, 7.0 Hz), 6.23 (1H, d, J=15.9 Hz), 4.13
(2H, t, J=6.7 Hz), 2.21(2H, q, J=7.0 Hz), 1.68 (2H,
quintet, J=7.0 Hz), 1.58–1.25 (6H, m); 13C NMR
(CD3OD) d 169.4 (s), 149.5 (s), 146.8 (d), 139.2 (s),
131.6 (d), 131.3 (d), 129.4 (d), 127.8 (d), 127.7 (s), 126.9
(d), 122.9 (d), 116.5 (d), 115.2 (d), 115.1 (d), 65.5 (t) 33.9
(t), 30.4 (t), 29.9 (t), 29.8 (t), 27.0 (t); HRFABMS calcd
for C23H27O4 [M+H]+: 367.1909; found: 367.1956.
n-Tetradecanyl caffeate (20). IR (KBr, cmꢀ1): 3480,
3300, 1675, 1600, 1530, 1445, 1355, 1275, 1175, 1105,
975, 855, 815; 1H NMR (CD3OD) d 7.43 (1H, d,
J=15.9 Hz), 6.93 (1H, d, J=2.0 Hz), 6.83 (1H, dd,
J=8.2, 2.0 Hz), 6.67 (1H, d, J=8.2 Hz), 6.14 (1H, d,
J=15.9 Hz), 4.06 (2H, t, J=6.7 Hz), 1.60 (2H, quintet,
J=6.7 Hz), 1.40–1.10 (22H, m), 0.80 (3H, t, J=6.8 Hz);
13C NMR (CD3OD) d 169.3 (s), 149.5 (s), 146.8 (d), 127.7
(s), 122.9 (d), 116.4 (d), 115.2 (d), 115.1 (d), 65.5 (t), 33.0
(t), 30.74 (t), 30.71(t), 30.63 (t), 30.59 (t), 30.4 (t), 30.3 (t),
29.8 (t), 27.1(t), 23.7 (t), 14.5 (q); HRFABMS calcd for
C23H37O4 [M+H]+: 377.2692; found: 377.2678.
(Z)-12-Phenyl-11-dodecenyl caffeate (11a). Retention
time: 34.1min; IR (KBr, cmꢀ1): 3440, 3290, 1685, 1630,
1595, 1525, 1445, 1365, 1270, 1170, 975, 855, 810; H
1
NMR (CD3OD) d 7.43 (1H, d, J=15.9Hz), 7.24–7.00
(5H, m), 6.94 (1H, d, J=1.9 Hz), 6.83 (1H, dd, J=8.2,
1.9Hz), 6.67 (1H, d, J=8.2 Hz), 6.29 (1H, d, J=11.8 Hz),
6.14 (1H, d, J=15.9 Hz), 5.53 (1H, dt, J=11.8, 7.2 Hz),
4.05 (2H, t, J=6.7 Hz), 2.19 (2H, q, J=7.2Hz), 1.57 (2H,
quintet, J=7.0 Hz), 1.45–1.00 (14H, m); 13C NMR
(CD3OD) d 169.4 (s), 149.5 (s), 146.8 (d), 139.1 (s), 133.8
(d), 130.0 (d), 129.7 (d), 129.1 (d), 127.7 (s), 127.5 (d), 122.9
(d), 116.5 (d), 115.2 (d), 115.1 (d), 65.5 (t) 30.9 (t), 30.5 (t),
30.4 (t), 30.3 (t), 29.8 (t), 29.4 (t), 27.0 (t); HRFABMS
calcd for C27H35O4 [M+H]+: 423.2535; found: 423.2519.
n-Hexadecanyl caffeate (21). IR (KBr, cmꢀ1): 3490,
3300, 1680, 1605, 1530, 1445, 1360, 1280, 1175, 1110,
1
1020, 975, 860, 810; H NMR (CD3OD) d 7.43 (1H, d,
(E)-12-Phenyl-11-dodecenyl caffeate (11b). Retention
time: 36.9 min; IR (KBr, cmꢀ1): 3440, 3290, 1685, 1630,
J=15.9 Hz), 6.93 (1H, d, J=2.0 Hz), 6.83 (1H, dd,
J=8.2, 2.0 Hz), 6.67 (1H, d, J=8.2 Hz), 6.14 (1H, d,
J=15.9 Hz), 4.06 (2H, t, J=6.7 Hz), 1.59 (2H, quintet,
J=6.7 Hz), 1.38–1.10 (26H, m), 0.79 (3H, t, J=6.8 Hz);
13C NMR (CD3OD) d 169.3 (s), 149.5 (s), 146.8 (d),
127.7 (s), 122.9 (d), 116.4 (d), 115.2 (d), 115.1 (d), 65.5
(t), 33.0 (t), 30.8 (t), 30.7 (t), 30.6 (t), 30.4 (t), 30.3 (t),
29.8 (t), 27.1(t), 23.7 (t), 14.5 (q); HRFABMS calcd for
C25H41O4 [M+H]+: 405.3005; found: 405.3003.
1
1595, 1525, 1445, 1365, 1270, 1170, 975, 855, 810; H
NMR (CD3OD) d 7.43 (1H, d, J=15.9 Hz), 7.24–7.00
(5H, m), 6.94 (1H, d, J=1.9 Hz), 6.83 (1H, dd, J=8.2,
1.9 Hz), 6.67 (1H, d, J=8.2 Hz), 6.25 (1H, d,
J=15.9 Hz), 6.14 (1H, dt, J=15.9, 7.0 Hz), 6.14 (1H, d,
J=15.9 Hz), 4.05 (2H, t, J=6.7 Hz), 2.09 (2H, q,
J=7.0 Hz), 1.57 (2H, quintet, J=7.0 Hz), 1.45–1.00
(14H, m); 13C NMR (CD3OD) d 169.4 (s), 149.7 (s),
146.8 (d), 139.3 (s), 131.8 (d), 131.2 (d), 129.4 (d), 127.8
(d), 127.7 (s), 127.5 (d), 122.9 (d), 116.5 (d), 115.2 (d),
115.1 (d), 65.6 (t) 34.1 (t), 30.6 (t), 30.5 (t), 30.3 (t), 29.8
(t), 29.4 (t), 27.1(t); HRFABMS calcd for C 27H35O4
[M+H]+: 423.2535; found: 423.2504.
Separation of compounds 10 and 11. Separations of cis/
trans isomers of 10 and 11 were conducted by HPLC
with a Shimadzu LC-5A system using a Discovery C18
column (21.2 mm i.d.Â25 cm; Supelco, USA). The
mobile phase was MeOH–H2O (78:22) for the separa-
tion of 10 and MeOH–H2O (87:13) for the separation of
11, and UV (254 nm) was used for detection.
Preparation of 13 and 14. A solution of caffeic acid
(180.16 mg, 1.0 mmol) in methanol or ethanol (30 mL)

T. Nagaoka et al. / Bioorg. Med. Chem. 10 (2002) 3351–3359
3357
containing p-TsOH (19.0 mg, 0.1 mmol) was stirred
under reflux. After removal of the solvent under
reduced pressure, the residue was subjected to silica gel
column chromatography using CHCl3–MeOH (90:10)
to give methyl caffeate (13)28 and ethyl caffeate (14).28
d, J=15.9 Hz, trans), 6.22 (1H, dt, J=15.9, 6.8 Hz, trans),
5.66 (1H, dt, J=11.6, 7.2 Hz, cis), 4.57 (1H, dd, J=4.4,
2.6 Hz), 3.87 (1H, ddd, J=11.1, 7.2, 3.6 Hz), 3.73 (1H, dt,
J=9.6, 6.8 Hz), 3.53–3.46 (1H, m), 3.38 (1H, dt, J=9.6,
6.8 Hz), 2.32 (2H, q, J=7.2Hz, cis), 2.20 (2H, q,
J=6.8 Hz, trans), 1.90–1.20 (22H, m); HREIMS calcd
for C23H36O2 [M+]: 344.2715; found: 344.2696.
Preparation of tetrahydropyranyl ethers 22b and 23b. A
solution of bromoalcohol (4.0 mmol) and dihydropyran
(6.0 mmol) in dry 1,2-dichloroethane (20 mL) containing
p-TsOH (0.4 mmol) was stirred for 4 h at room tem-
perature. After removal of the solvent under reduced
pressure, the residue was subjected to silica gel column
chromatography using hexane–AcOEt (97:3) to give the
tetrahydropyranyl ethers.
Reduction of 22c and 23c
The ether (0.8 mmol) was dissolved in MeOH (10 mL)
and hydrogenated over 10% palladium on charcoal
(100 mg) at room temperature for 2 h. After the cata-
tyst was filtrated off and the solvent was removed
under reduced pressure, the residue was subjected to
silica gel column chromatography using hexane–
AcOEt (97:3) to give phenylalkyl tetrahydropyranyl
ethers.
7-Bromoheptyl tetrahydropyranyl ether (22b). Yield:
94%; 1H NMR (CDCl3) d 4.57 (1H, dd, J=4.5, 2.8 Hz),
3.91–3.83 (1H, m), 3.73 (1H, dt, J=9.6, 6.6 Hz), 3.54–
3.47 (1H, m), 3.40 (2H, t, J=6.7 Hz), 3.38 (1H, dt,
J=9.6, 6.6 Hz), 1.86 (2H, quintet, J=7.0 Hz), 1.84–1.30
(14H, m); HREIMS calcd for C12H22O2Br [M+ꢀH,
79Br]: 277.0803; found: 277.0786, calcd for C12H22O2Br
[M+ꢀH, 81Br]: 279.0784; found: 277.0777.
8-Phenyloctyl tetrahydropyranyl ether (22d). Yield:
1
95%; H NMR (CDCl3) d 7.22–7.06 (5H, m), 4.49 (1H,
dd, J=4.1, 2.6 Hz), 3.79 (1H, ddd, J=11.3, 7.2, 3.6 Hz),
3.65 (1H, dt, J=9.6, 6.8 Hz), 3.45–3.38 (1H, m), 3.30
(1H, dt, J=9.6, 6.8 Hz), 2.52 (2H, t, J=7.7 Hz), 1.80–
1.40 (10H, m), 1.32–1.20 (8H, m); HREIMS calcd for
C19H30O2 [M+]: 290.2246; found: 290.2244.
11-Bromoundecanyl tetrahydropyranyl ether (23b). Yield:
1
96%; H NMR (CDCl3) d 4.57 (1H, dd, J=4.6, 2.4 Hz),
3.89 (1H, ddd, J=11.2, 7.5, 3.5 Hz), 3.73 (1H, dt, J=9.6,
6.8 Hz), 3.54–3.46 (1H, m), 3.40 (2H, t, J=6.8Hz), 3.38
(1H, dt, J=9.6, 6.8 Hz), 1.85 (2H, quintet, J=7.0Hz),
1.84–1.20 (22H, m); HREIMS calcd for C16H30O2Br
[M+ꢀH, 79Br]: 333.1429; found: 333.1435, calcd for
C16H30O2Br [M+ꢀH, 81Br]: 335.1410; found: 335.1412.
12-Phenyldodecyl tetrahydropyranyl ether (23d). Yield:
1
95%; H NMR (CDCl3) d 7.31–7.12 (5H, m), 4.57 (1H,
dd, J=4.4, 2.6 Hz), 3.87 (1H, ddd, J=11.3, 7.2, 3.6 Hz),
3.73 (1H, dt, J=9.6, 6.8 Hz), 3.53–3.46 (1H, m), 3.38
(1H, dt, J=9.6, 6.8 Hz), 2.60 (2H, t, J=7.7 Hz), 1.88–
1.45 (10H, m), 1.40–1.20 (16H, m); HREIMS calcd for
C23H38O2 [M+]: 346.2872; found: 346.2889.
Preparation of phenylalkenyl tetrahydropyranyl ethers
22c and 23c. A mixture of THP ethers (3.0 mmol) and
triphenylphosphine (3.0 mmol) was stirred for 1h at
120 ꢁC. After cooling, the reaction mixture was dec-
anted into hexane and ether. Then, to a stirred suspen-
sion of the decanted residue (ca. 1.7 mmol) in THF
(10 mL) under argon, n-BuLi (2 mmol) was added at
ꢀ10 ꢁC, and the mixture was stirred at ꢀ10 ꢁC for 5 min.
Benzaldehyde (2 mmol) in THF (10 mL) was added to
the mixture and stirred at ꢀ10 ꢁC for 2 h. After poured
Deprotection of 22c, 23c, 22d and 23d. A solution of
ether (0.5 mmol) in MeOH (10 mL) containing p-TsOH
(0.5 mmol) is stirred for 1h at room temperature. After
removal of the solvent under reduced pressure, the resi-
due was subjected to preparative TLC with CHCl3–
MeOH (95:5) to give each alcohol.
1
8-Phenyloctanol (22). Yield: 92%; H NMR (CDCl3) d
ꢁ
into 1N HCl at 0 C, the product was extracted with
7.30–7.15 (5H, m), 3.61 (1H, t, J=6.8 Hz), 2.60 (2H, t,
J=7.2 Hz), 1.61 (2H, quintet, J=7.2 Hz), 1.55 (2H,
quintet, J=6.8 Hz), 1.40–1.26 (8H, m); HREIMS calcd
for C14H22O [M+]: 206.1671; found: 206.1675.
CH2Cl2, and the CH2Cl2 layer was dried over Na2SO4,
and evaporated. The residue was subjected to silica gel
column chromatography using hexane–AcOEt (95:5) to
give phenylalkenyl tetrahydropyranyl ethers.
12-Phenyldodecanol (23). Yield: 91%; 1H NMR
(CDCl3) d 7.35–7.10 (5H, m), 3.62 (1H, t, J=6.8 Hz),
2.59 (2H, t, J=7.2 Hz), 1.61 (2H, qu, J=7.2 Hz), 1.55
(2H, quintet, J=6.8 Hz), 1.40–1.20 (16H, m);
HREIMS calcd for C18H30O [M+]: 262.2297; found:
262.2274.
8-Phenyl-7-octenyl tetrahydropyranyl ether (22c). Yield:
1
25% (cis/trans=77/23); H NMR (CDCl3) d 7.40–7.15
(5H, m), 6.40 (1H, d, J=11.6 Hz, cis), 6.37 (1H, d,
J=15.9 Hz, trans), 6.21(1H, dt, J=15.9, 6.8 Hz, trans),
5.65 (1H, dt, J=11.6, 7.2 Hz, cis), 4.57 (1H, dd, J=4.4,
2.8 Hz), 3.86 (1H, ddd, J=11.1, 7.2, 3.6 Hz), 3.72 (1H,
dt, J=9.4, 6.8 Hz), 3.54–3.46 (1H, m), 3.37 (1H, dt,
J=9.6, 6.8 Hz), 2.32 (2H, q, J=7.2 Hz, cis), 2.21(2H, q,
J=6.8 Hz, trans), 1.90–1.30 (14H, m); HREIMS calcd
for C19H28O2 [M+]: 288.2089; found: 288.2072.
1
8-Phenyl-7-octenol (24). Yield: 97%; H NMR (CDCl3)
d 7.36–7.17 (5H, m), 6.41 (1H, d, J=11.6 Hz, cis), 6.37
(1H, d, J=15.9 Hz, trans), 6.21(1H, dt, J=15.9, 6.8 Hz,
trans), 5.65 (1H, dt, J=11.6, 7.2 Hz, cis), 3.60 (1H, t,
J=6.8 Hz), 2.33 (2H, q, J=7.2 Hz, cis), 2.21(2H, q,
J=6.8 Hz, trans), 1.62–1.26 (8H, m); HREIMS calcd
for C14H20O [M+]: 204.1514; found: 204.1521.
12-Phenyl-11-dodecenyl tetrahydropyranyl ether (23c).
Yield: 23% (cis/trans=77/23); 1H NMR (CDCl3) d
7.38–7.15 (5H, m), 6.40 (1H, d, J=11.6 Hz, cis), 6.37 (1H,

3358
T. Nagaoka et al. / Bioorg. Med. Chem. 10 (2002) 3351–3359
12-Phenyl-11-dodecenol (25). Yield: 94%; 1H NMR
(CDCl3) d 7.28–7.07 (5H, m), 6.32 (1H, d, J=11.6 Hz,
cis), 6.29 (1H, d, J=15.9 Hz, trans), 6.14 (1H, dt,
J=15.9, 6.8 Hz, trans), 5.58 (1H, dt, J=11.6, 7.2 Hz,
cis), 3.54 (1H, t, J=6.8 Hz), 2.24 (2H, q, J=7.2 Hz, cis),
2.12 (2H, q, J=6.8Hz, trans), 1.48 (2H, quintet, J=
7.2 Hz), 1.37 (2H, quintet, J=6.8 Hz), 1.32–1.12 (12H,
m); HREIMS calcd for C18H28O [M+]: 260.2140;
found: 260.2142.
concentration of test specimen was added to each well
and incubated for 72 h under the same conditions. After
fixation with 25% glutaraldehyde solution (20 mL), the
cells were stained with 0.5% crystal violet in 20%
methanol/water for 30 min. After gentle rinsing with
water, the retained crystal violet was extracted with
30% acetic acid and measured spectrophotometrically
at 590 nm.
Each esters was dissolved in a bit of DMSO, and then
diluted by the medium; final concentration of DMSO
was less than 0.25%. 5-FU and doxorubicin were used
as positive controls, and EC50 values were calculated
from the mean values of data from four wells.
Cells
Highly liver metastatic murine colon 26-L5 carcinoma cell
line was established by one of the authors (I. Saiki).17
Highly liver metastatic murine B16-BL6 melanoma cell
line, obtained by an in vivo selection procedure for inva-
sion,18 was kindly provided by Dr. I. J. Fidler (M.D.
Anderson Cancer Center, Houston, TX, USA). Highly
lung metastatic murine Lewis lung carcinoma (LLC) cell
line, originated spontaneously from murine lung,19 was
kindly provided by Dr. K. Takeda (Juntendo University,
Tokyo, Japan). Highly metastatic human HT-1080 fibro-
sarcoma cell line (ATCC # CCL-121)20 was obtained from
American Type Culture Collection (Rockville, MD,
USA). Human lung A549 adenocarcinoma (RCB0098)21
and human cervix HeLa adenocarcinoma (RCB0007)22
cell lines were purchased from Riken Cell Bank (Tsukuba,
Japan).
Cytotoxicity of CAPE analogues towards primary
cultured mouse hepatocytes
Mouse liver parenchymal cells were isolated according
to the procedure described previously.33,34 In brief, the
liver was perfused with Ca2+-free Hank’s balanced salt
solution (HBSS) containing 0.5% bovine serum albu-
min (BSA) and 5 mM ethyleneglycol-O,O-bis(2-ami-
noethyl)-N,N,N0,N0-tetraacetic acid (EGTA), then
recirculated with collagenase solution composed of
Ca2+-free free HBSS, 0.075% collagenase, 4 mM CaCl2,
and 0.005% trypsin inhibitor. Isolated hepatocytes were
cultured in William’s E medium supplemented with
10% FCS, 100 IU/mL penicillin G, 100 mg/mL strepto-
mycin, 100 mM dexamethasone and 50ng/mL insulin and
incubated in 96-well plastic plate (2Â104 cells/well). After
2 h pre-incubation, the medium was replaced with fresh
medium containing test specimens at various concentra-
tions. After 72h incubation, the hepatocytes viability was
assessed by MTT method.
B16-BL6, LLC, HT-1080, A549 and HeLa cell lines were
maintained in 75-cm2 cell culture flasks in Eagle’s mini-
mum essential medium (EMEM) supplemented with 10%
heat inactivated fetal calf serum (FCS), 2 mM l-(+)-glu-
tamine and 0.1% sodium hydrogen carbonate at 37ꢁC
under a humidified 5% carbon dioxide. Colon 26-L5 cell
line was maintained in RPMI 1640 medium containing
the same supplement under the same conditions.
DNA fragmentation
DNA was isolated and detected by the procedure
described previously.35,36 Briefly, murine colon 26-L5
cell (>2Â106 cells) was preincubated in RPMI medium
for 24 h, and then cultured with various concentrations
of test specimen in serum free Dulbecco’s modified
MEM (DMEM/F-12) medium containing 0.1% BSA,
100 IU/mL penicillin G and 80IU/mL streptomycin for
24h. At end of the incubation, cells were pelleted and
lysed in 600 mL of lysis buffer (10mM Tris–HCl buffer, pH
8.0, 10mM EDTA and 0.2% Triton X-100) for 10min on
ice. After the lysate was centrifuged at 14,000rpm for
10min, the supernatant was extracted with TE buffer
(10 mM Tris–HCl buffer, pH 8.0, 1 mM EDTA)-saturated
phenol, and then centrifuged at 14,000rpm for 10min.
After the upper layer was extracted with CIAA solution
(chloroform–isoamylalcohol=24:1), DNA in the upper
layer (500 mL) was precipitated with 3 M NaCl (50 mL)
and cold ethanol (1000 mL) at ꢀ20 ꢁC overnight. After
drying, DNA was dissolved in TE buffer. Contamina-
tion of RNA was removed by incubation with 1mg/mL
RNase at 37 ꢁC for 30 min. Following the addition of
loading buffer, fragmented DNA was electrophoresed
on 1.5% agarose gel in TAE (40 mM Tris, 20 mM acetic
acid, 1mM EDTA) at 100 V for 30 min and visualized
by ethidium bromide staining.
Antiproliferative activity
Viability of cells other than LLC, in the presence or
absence of compounds, was determined using the stan-
dard MTT assay31 as described previously.32 In brief,
exponentially growing cells were harvested and 100 mL
medium per well with 2Â103 cells suspended was plated
in 96-well plate. After 24 h incubation at 37 ꢁC under a
humidified 5% carbon dioxide to allow cell attachment,
the cells were treated with varying concentrations of test
specimens in their respective medium (100mL) and incu-
bated for 72h under the same conditions. After 2 h of the
MTT (0.4–0.5 mg/mL, 100 mL) addition, the formazan
formed was extracted with dimethyl sulfoxide (DMSO)
and its amount was measured spectrophotometrically at
550 nm with Perkin-Elmer HTS-7000 Bio Assay Reader
(Norwalk, CT, USA).
In the case of LLC cells, standard crystal violet stain-
ing assay was used in following the literature proce-
dure.25 In brief, exponentially growing cells were
harvested and 100 mL medium per well with 1Â103 cells
suspended was plated in 96-well plate. After 24 h of
incubation at 37 ꢁC under a humidified 5% carbon
dioxide atmosphere, 100-mL medium containing various

T. Nagaoka et al. / Bioorg. Med. Chem. 10 (2002) 3351–3359
3359
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