Synthetic cinnamylphenol derivatives as cancer chemopreventive agents

Article abstract of DOI:10.1016/j.ejmech.2006.12.024

Several substituted cinnamylphenol (1,3-diphenylpropene) derivatives were synthesized and tested for their inhibitory activities against in vitro Epstein-Barr virus early antigen activation induced by 12-O-tetradecanoylphorbol-13-acetate in Raji cells. The prenylated cinnamylphenols were found to show remarkably potent activity. Furthermore, prenylated cinnamylphenols (19 and 25) exhibited a marked inhibitory effect on mouse skin tumor promotion in an in vivo two-stage carcinogenesis test. These results indicate that some prenylated cinnamylphenols might be valuable as potential cancer chemopreventive agents (anti-tumor promoters).

Full text of DOI:10.1016/j.ejmech.2006.12.024

European Journal of Medicinal Chemistry 42 (2007) 902e909
http://www.elsevier.com/locate/ejmech
Original article
Synthetic cinnamylphenol derivatives as cancer chemopreventive agents
a
a
Chihiro Ito a, Masataka Itoigawa b, , Tetsufumi Kanematsu , Yuuki Imamura ,
*
Harukuni Tokuda c, Hoyoku Nishino c, Hiroshi Furukawa a,
*
a Faculty of Pharmacy, Meijo University, Tempaku-ku, Nagoya 468-8503, Japan
b Faculty of Human Wellness, Tokai Gakuen University, 2-901 Nakahira, Tempaku-ku, Nagoya 468-8514, Japan
c Department of Molecular Biochemistry, Kyoto Prefectural University of Medicine, Kamigyou-ku, Kyoto 602-0841, Japan
Received 26 August 2006; received in revised form 16 December 2006; accepted 21 December 2006
Available online 13 January 2007
Abstract
Several substituted cinnamylphenol (1,3-diphenylpropene) derivatives were synthesized and tested for their inhibitory activities against in
vitro Epstein-Barr virus early antigen activation induced by 12-O-tetradecanoylphorbol-13-acetate in Raji cells. The prenylated cinnamylphenols
were found to show remarkably potent activity. Furthermore, prenylated cinnamylphenols (19 and 25) exhibited a marked inhibitory effect on
mouse skin tumor promotion in an in vivo two-stage carcinogenesis test. These results indicate that some prenylated cinnamylphenols might be
valuable as potential cancer chemopreventive agents (anti-tumor promoters).
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Cinnamylphenols; Cancer chemopreventive agents; Anti-tumor promoters; Epstein-Barr virus activation; Two-stage mouse skin carcinogenesis
1. Introduction
herein the active structures, synthetic methods, and anti-tumor
promotion results from this study of cinnamylphenol
derivatives.
We prepared a series of cinnamylphenols, one of the chem-
ical constituents of the Dalbergia species (Leguminosae) for
evaluation as cancer chemopreventive agents (anti-tumor pro-
moters). Cinnamylphenols possess the same chemical subunits
(C6 þ C3 þ C6) biogenetically analogous to flavonoids [1].
We have previously reported that naturally occurring flavo-
noids [2,3], isoflavonoids [4e6] and cinnamylphenols [7]
might be valuable as potential anti-tumor promoters. However,
because of the continuing need for new cancer chemopreven-
tive agents with novel structures and mechanisms of action,
we also screened the synthetic cinnamylphenols for anti-
tumor promoting activity. Several prenylated cinnamylphenols
showed marked inhibitory activities against Epstein-Barr virus
early antigen (EBV-EA) activation induced by 12-O-tetrade-
canoylphorbol-13-acetate (TPA) in Raji cells. We report
2. Chemistry
Schemes 1e3 show the synthetic methods and structures of
target compounds 1e28. Methoxyphenol, methoxycatechol,
dimethoxyphenol and trimethoxybenzene were reacted with
3-phenyl-2-propen-1-ol in 90% formic acid at 60 ꢀC for
30 min to give cinnamylphenol derivatives 1e12 (Scheme 1)
[8]. In Scheme 2, it has been shown that vinyl quinone methide
prepared from eugenol by treatment with Ag2O in CHCl3, eas-
ily reacts with 3-methoxycatechol in the presence of catalytic
amounts of p-toluenesulfonic acid ( p-TsOH) to give cinna-
mylphenol derivatives 21 and 22 [9]. Cinnamylphenol deriva-
tives 27 and 28 were obtained by reacting coniferyl diacetate
and 3-methoxycatechol in 90% formic acid at 60 ꢀC for 7.5 h
(Scheme 3). Finally, prenylated cinnamylphenol derivatives
13e20 and 23e26 were prepared from each corresponding
cinnamylphenol by treatment with prenyl bromide in the
* Corresponding authors. Tel.: þ81 52 801 1201; fax: þ81 52 804 1044.
E-mail address: itoigawa@tokaigakuen-u.ac.jp (M. Itoigawa).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2006.12.024

C. Ito et al. / European Journal of Medicinal Chemistry 42 (2007) 902e909
903
Scheme 1. Structures of synthetic cinnamylphenol derivatives 1e20. (a) 90% formic acid, 60 ꢀC, 30 min, (b) prenyl bromide, K2CO3/acetone, 80 ꢀC, 1 h.
presence of anhydrous potassium carbonate (K2CO3) in ace-
tone at 80 ꢀC for 1 h, respectively.
the indicator, n-butyric acid as the trigger, TPA as the EBV-ac-
tivator and the test substance. Their inhibitory effects on acti-
vation of the virus-genome, the viability of the Raji cells and
the 50% inhibitory concentration (IC50) values are shown in
Table 1. Of the cinnamylphenol derivatives shown in Scheme
1, both phenols (1e10) and non-phenolic methyl ethers (11
and 12) showed weaker activity than that of curcumin
(IC50 ¼ 341) [10,11], a compound used as a reference in can-
cer prevention studies. Also, the inhibitory effects of phenols
21 and 22 (Scheme 2) and 27 and 28 (Scheme 3) were weaker
than that of curcumin. Prenylation of one or two of the hy-
droxy groups in these phenols increased the inhibitory activity.
3. Results and discussion
The synthesized cinnamylphenol derivatives were tested for
their ability to inhibit tumor-promoting activity using a short-
term in vitro assay for TPA-induced EBV-EA activation in
Raji cells. The activation of EBV-EA expression in an EBV
genome-carrying human lymphoblastoid cell line has been
used to detect tumor promoter or anti-tumor promoter activity.
This assay system is composed of EBV-non-producer cells as

904
C. Ito et al. / European Journal of Medicinal Chemistry 42 (2007) 902e909
Scheme 2. Structures of synthetic cinnamylphenol derivatives 21e26. (a) Ag2O/CHCl3, (b) PTSA/CHCl3, (c) prenyl bromide, K2CO3/acetone, 80 ꢀC, 1 h.
All prenylated cinnamylphenols (13e20 and 23e26) inhibited
EBV-EA activation even at 1 ꢁ 10 mol ratio/TPA and fully
carcinogenesis test focusing on mouse skin papillomas in-
duced by DMBA as an initiator and TPA as a promoter. The
results from the in vivo bioassay using these compounds are
shown in Fig. 1. The control animals showed a 100% inci-
dence of papillomas in 10 weeks after DMBA-TPA tumor pro-
motion, while treatment with non-phenolic derivatives (19)
with two prenyl groups in the A-ring along with the initiator
and promoter remarkably reduced the percentage of tumor-
bearing mice to 77% and 30% after 10 and 20 weeks, respec-
tively (Fig. 1A). The inhibitory activity of phenolic derivative
(25), with two prenyl groups in the A- and B-rings, resulted in
a lower incidence of papilloma-bearing mice to 87% and 53%
after 10 and 20 weeks, respectively (Fig. 1A). As shown in
Fig. 1B, the number of papillomas per mouse was reduced
by 44e48% after 20 weeks in mice treated with derivatives
19 and 25 as compared to untreated mice.
In previous studies, we reported that the presence of
a prenyl moiety in the flavanones [3] and isoflavonoids [4,6]
containing a C6 þ C3 þ C6 structural unit plays an important
role in the inhibition of EBV-EA induction. In view of the
present findings, a hydrophobic prenyl moiety on the cinna-
mylphenol nucleus might be an important factor for producing
the observed chemopreventive effects against chemical-in-
duced carcinogenesis.
blocked EBV-EA activation at
a
high concentration
(1 ꢁ 103 mol ratio/TPA) without causing a decrease in viabil-
ity (>70% survival) of the Raji cells. In the cinnamylphenols
with only one prenyl side chain on the A-ring, these values
corresponded to an IC50 value of 277e300 mol ratio/TPA.
The inhibitory effect of 3,300-dimethoxy-2,400-diprenyloxy-
6-cinnamylphenol (25) (IC50 ¼ 277) was present even at
1 ꢁ 10 mol ratio/TPA (10.9%) and the activation was fully
blocked at 1 ꢁ 103 mol ratio/TPA. Furthermore, the corre-
sponding IC50 values of the non-phenolic derivatives (17, 19,
and 26), with two prenyl side chains on the A-ring, were
225e278 mol ratio/TPA. Prenylated non-phenolic derivatives
(19 and 26) showed the most potent inhibitory activity
(IC50 ¼ 225 and 226, respectively). From the viewpoint of
structureeactivity relationships, an essential feature for the ac-
tivity of the cinnamylphenols examined in the present study is
the presence of two prenyl groups on the A-ring of the cinna-
mylphenol skeleton.
Based on the results obtained in vitro, we studied the inhib-
itory effect of two diprenylated cinnamylphenols (19 and 25),
as representative compounds of more active non-phenolic and
phenolic derivatives in vitro, in an in vivo two-stage
Scheme 3. Structures of synthetic cinnamylphenol derivatives 27 and 28. (a) 90% formic acid, 60 ꢀC, 7.5 h.

C. Ito et al. / European Journal of Medicinal Chemistry 42 (2007) 902e909
905
Table 1
Inhibitory effects of synthetic cinnamylphenol derivatives on TPA-induced EBV-EA activationa
b
Compound
EBV-EA-positive cells (% viability)
IC50
Compound concentration (mol ratio/32 pmol TPA)
1000
500
100
10
1
20.7 ꢂ 1.2 (60)
18.3 ꢂ 0.6 (60)
12.8 ꢂ 0.5 (60)
18.1 ꢂ 0.6 (60)
10.8 ꢂ 0.5 (60)
10.2 ꢂ 0.3 (60)
13.3 ꢂ 0.4 (60)
12.5 ꢂ 0.3 (60)
15.9 ꢂ 0.3 (60)
14.1 ꢂ 0.4 (60)
14.2 ꢂ 0.5 (60)
12.6 ꢂ 0.4 (60)
45.1 ꢂ 1.3 (>80)
43.7 ꢂ 1.8 (>80)
41.5 ꢂ 1.1 (>80)
44.7 ꢂ 1.4 (>80)
42.5 ꢂ 1.3 (>80)
41.3 ꢂ 1.7 (>80)
45.5 ꢂ 1.8 (>80)
44.3 ꢂ 1.3 (>80)
46.4 ꢂ 1.1 (>80)
41.2 ꢂ 1.1 (>80)
41.5 ꢂ 1.8 (>80)
39.8 ꢂ 1.4 (>80)
73.7 ꢂ 2.0 (>80)
70.5 ꢂ 2.1 (>80)
68.2 ꢂ 1.9 (>80)
73.2 ꢂ 1.8 (>80)
71.5 ꢂ 2.0 (>80)
71.0 ꢂ 1.5 (>80)
76.2 ꢂ 2.4 (>80)
74.0 ꢂ 2.0 (>80)
75.6 ꢂ 1.9 (>80)
76.9 ꢂ 1.9 (>80)
76.6 ꢂ 2.7 (>80)
75.2 ꢂ 2.0 (>80)
100.0 ꢂ 0.2 (>80)
100.0 ꢂ 0.3 (>80)
100.0 ꢂ 1.1 (>80)
100.0 ꢂ 0.9 (>80)
100.0 ꢂ 0.7 (>80)
100.0 ꢂ 0.4 (>80)
100.0 ꢂ 0.2 (>80)
100.0 ꢂ 0.5 (>80)
100.0 ꢂ 0.4 (>80)
100.0 ꢂ 0.5 (>80)
100.0 ꢂ 0.5 (>80)
100.0 ꢂ 0.5 (>80)
486
430
420
433
439
432
430
440
438
401
425
419
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
0.0 ꢂ 0.7 (70)
0.0 ꢂ 0.3 (70)
0.0 ꢂ 0.6 (70)
0.0 ꢂ 0.5 (70)
0.0 ꢂ 0.2 (70)
0.0 ꢂ 0.4 (70)
0.0 ꢂ 0.6 (70)
0.0 ꢂ 0.3 (70)
29.9 ꢂ 1.8 (>80)
30.8 ꢂ 1.2 (>80)
29.3 ꢂ 1.1 (>80)
30.6 ꢂ 1.3 (>80)
24.6 ꢂ 1.2 (>80)
27.6 ꢂ 1.3 (>80)
22.6 ꢂ 1.0 (>80)
29.8 ꢂ 1.4 (>80)
67.8 ꢂ 2.1 (>80)
68.9 ꢂ 2.0 (>80)
65.2 ꢂ 1.3 (>80)
65.6 ꢂ 1.3 (>80)
62.2 ꢂ 1.2 (>80)
64.5 ꢂ 1.5 (>80)
64.5 ꢂ 2.2 (>80)
68.2 ꢂ 2.0 (>80)
91.7 ꢂ 0.6(>80)
92.7 ꢂ 0.4 (>80)
87.6 ꢂ 0.6 (>80)
90.8 ꢂ 0.7 (>80)
88.6 ꢂ 0.3 (>80)
90.6 ꢂ 0.5 (>80)
88.9 ꢂ 1.3 (>80)
91.5 ꢂ 1.5 (>80)
280
282
284
291
278
280
225
282
21
22
23
24
25
26
8.7 ꢂ 0.5 (60)
7.5 ꢂ 0.5 (60)
0.0 ꢂ 0.6 (70)
0.0 ꢂ 0.4 (70)
0.0 ꢂ 0.0 (70)
0.0 ꢂ 0.3 (70)
45.5 ꢂ 1.9 (>80)
43.7 ꢂ 1.8 (>80)
28.3 ꢂ 1.0 (>80)
27.8 ꢂ 1.4 (>80)
27.5 ꢂ 1.5 (>80)
23.6 ꢂ 1.2 (>80)
73.2 ꢂ 2.0 (>80)
70.2 ꢂ 2.0 (>80)
64.2 ꢂ 1.5 (>80)
63.2 ꢂ 1.2 (>80)
68.6 ꢂ 2.1 (>80)
61.5 ꢂ 2.0 (>80)
95.7 ꢂ 0.3 (>80)
95.7 ꢂ 0.3 (>80)
87.1 ꢂ 0.7 (>80)
88.1 ꢂ 0.8 (>80)
89.1 ꢂ 0.5 (>80)
83.3 ꢂ 0.3 (>80)
435
420
300
294
277
226
27
28
10.0 ꢂ 0.7 (60)
37.6 ꢂ 1.3 (>80)
74.6 ꢂ 2.3 (>80)
100.0 ꢂ 0.3 (>80)
407
404
9.4 ꢂ 0.5 (60)
36.1 ꢂ 1.6 (>80)
74.0 ꢂ 2.4 (>80)
100.0 ꢂ 0.4 (>80)
Curcuminc
0.0 ꢂ 0.5 (60)
22.8 ꢂ 1.8 (>80)
81.7 ꢂ 1.9 (>80)
100.0 ꢂ 0.2 (>80)
341
a
Mol ratio/TPA (32 pmol ¼ 20 ng/ml), 1000 mol ratio ¼ 32 nmol, 500 mol ratio ¼ 16 nmol, 100 mol ratio ¼ 3.2 nmol, and 10 mol ratio ¼ 0.32 nmol. Values are
EBV-EA activation (%) ꢂ SD in the presence of test compound relative to the positive control (100%). Values in parentheses represent the surviving Raji cells
measured by Trypan Blue staining. A minimum 60% survival rate of Raji cells two days after treatment with the compounds is required for an accurate result.
b
IC50 represents the mol ratio to TPA that inhibits 50% of the positive control (100%) activated with 32 pmol of TPA.
Positive control substance.
c
Fig. 1. Inhibitory effects of prenylated cinnamylphenols (19 and 25) on DMBA-TPA mouse skin carcinogenesis. Tumors were initiated in all mice with DMBA
(390 nmol) and promoted with TPA (1.7 nmol) twice weekly starting one week after initiation. A: Percentage of mice with papillomas. B: Average number of
papillomas per mouse. C, control TPA alone; B, TPA þ 85 nmol of 19; 6, TPA þ 85 nmol of 25. After 20 weeks of tumor promotion, a significant difference
in the number of papillomas per mouse between the groups treated with each test compound and the control group was evident ( p < 0.05).

906
C. Ito et al. / European Journal of Medicinal Chemistry 42 (2007) 902e909
4. Experimental protocols
4.1. General experimental procedures
4.3.2. 3-Hydroxy-2,6-dicinnamylphenol methyl ether (3)
Pale yellow oil; IR (CHCl3) nmax 3596, 3345 (br),
1
1599 cmꢃ1; H NMR (CDCl3): 7.35 (2H, d, J ¼ 7.7 Hz), 7.33
(2H, d, J ¼ 7.7 Hz), 7.28 (2H, t, J ¼ 7.7 Hz), 7.27 (2H, t,
J ¼ 7.7 Hz), 7.19 (2H, t, J ¼ 7.7 Hz), 7.03 (1H, d, J ¼ 8.4 Hz),
6.62 (1H, d, J ¼ 8.4 Hz), 6.50 (1H, d, J ¼ 15.8 Hz), 6.44 (1H,
d, J ¼ 15.8 Hz), 6.40 (1H, dt, J ¼ 15.8, 6.6 Hz), 6.35 (1H, dt,
J ¼ 15.8, 6.6 Hz), 5.00 (1H, br s, OH), 3.76 (3H, s, OCH3),
3.65 (2H, d, J ¼ 6.6 Hz), 3.53 (2H, d, J ¼ 6.6 Hz); HRMS m/z
356.1730 (calcd for C25H24O2: 356.1776).
1H and 13C NMR, COSY, HMQC, HMBC (J ¼ 8 Hz), and
NOE were measured using a JNM A-400, A-600 and/or ECP-
500 (JEOL) spectrometer. Chemical shifts are shown in
d (ppm) with tetramethylsilane (TMS) as an internal reference.
All mass spectra were taken under EI conditions, unless other-
wise stated, using an HX-110 (JEOL), and/or JMS-700 (JEOL)
spectrometer with a direct inlet system. UV spectra were re-
corded on an UVIDEC-610C double-beam spectrophotometer
(JASCO) in MeOH, IR spectra were recorded on an IR-230
(JASCO) in CHCl3. Preparative TLC was done on Kieselgel
60 F254 (Merck).
4.4. Synthesis of cinnamylphenols 5 and 6
Following the method I, 3-methoxycatechol (700 mg) and
3-phenyl-2-propen-1-ol (670 mg) afford compound 5 (196 mg)
and hydroxyobtustyrene 6 (120 mg).
4.2. General procedure for the synthesis of
cinnamylphenol derivatives
4.5. Synthesis of cinnamylphenols 7e9
Following the method I, 2,3-dimethoxyphenol (700 mg) and
3-phenyl-2-propen-1-ol (609 mg) afford compound 7 (107 mg),
mucronustyrene 8 (79 mg), and compound 9 (89 mg).
Method I: Methoxyphenol, methoxycatechol, dimethoxy-
phenol and trimethoxybenzene were reacted with 3-phenyl-
2-propen-1-ol in 90% formic acid at 60 ꢀC for 30 min to
give cinnamylphenols 1e12 [8].
Method II: Vinyl quinone methide, prepared from eugenol
by treatment with Ag2O in CHCl3, easily reacts with 3-me-
thoxycatechol in the presence of catalytic amounts of p-tolue-
nesulfonic acid to give cinnamylphenols 21 and 22 [9].
Method III: Cinnamylphenols 27 and 28 were obtained by
reacting coniferyl diacetate and 3-methoxycatechol in 90%
formic acid at 60 ꢀC for 7.5 h.
Method IV: Prenylated cinnamylphenols 13e20 and 23e26
were prepared from each corresponding cinnamylphenol by
treatment with prenyl bromide in the presence of potassium
carbonate (K2CO3) in acetone at 80 ꢀC for 1 h, respectively.
All cinnamylphenols were named on the basis of cinnamyl-
phenol. Seven of the known compounds (1, 4e8, and 10) were
synthesized according to usual methods and fully character-
ized by comparison of their physical and spectroscopic data
with those previously reported in the literatures [8,12e15].
4.5.1. 2,3-Dimethoxy-4,6-dicinnamylphenol (9)
Yellow oil; IR (CHCl3) nmax 3527, 3293 (br), 1598 cmꢃ1
;
1H NMR (CDCl3): 7.34 (4H, m), 7.26 (4H, t, J ¼ 7.7 Hz),
7.18 (2H, t, J ¼ 7.7 Hz), 6.74 (1H, s), 6.45 (1H, d, J ¼
15.8 Hz), 6.41 (1H, d, J ¼ 15.8 Hz), 6.34 (2H, m), 5.76 (1H,
s, OH), 3.93 (3H, s, OCH3), 3.83 (3H, s), 3.49 (2H, d,
J ¼ 6.6 Hz), 3.45 (2H, d, J ¼ 6.6 Hz); HRMS m/z 386.1839
(calcd for C26H26O3: 386.1882).
4.6. Synthesis of cinnamylphenol 10
Following the method I, 2,6-dimethoxyphenol (70 mg) and
3-phenyl-2-propen-1-ol (65 mg) afford isomucronustyrene 10
(11.3 mg).
4.7. Synthesis of cinnamylphenols 11 and 12
Following the method I, trimethoxybenzene (70 mg) and 3-
phenyl-2-propen-1-ol (65 mg) afford cinnamylphenols 11
(48 mg) and 12 (7 mg).
4.3. Synthesis of cinnamylphenols 1e4
Following the method I, m-methoxyphenol (620 mg) and
3-phenyl-2-propen-1-ol (670 mg) afford cinnamylphenols 1
4.7.1. 2,3-Dimethoxy-6-cinnamylphenol methyl ether (11)
Pale yellow oil; IR (CHCl3) nmax 1599 cmꢃ1 1H NMR
;
(241 mg) 2 (20 mg) 3 (14.7 mg), and obtustyrene 4
(174.7 mg).
(CDCl3): 7.35 (2H, d, J ¼ 7.7 Hz), 7.27 (2H, t, J ¼ 7.7 Hz),
7.19 (1H, t, J ¼ 7.7 Hz), 6.88 (1H, d, J ¼ 8.4 Hz), 6.63 (1H,
d, J ¼ 8.4 Hz), 6.41 (1H, d, J ¼ 15.8 Hz), 6.36 (1H, dt,
J ¼ 15.8, 6.6 Hz), 3.88 (6H, s, OCH3), 3.85 (3H, s, OCH3),
3.49 (2H, d, J ¼ 6.6 Hz); HRMS m/z 284.1407 (calcd for
C18H20O3: 284.1412).
4.3.1. 3-Methoxy-2-cinnamylphenol (2)
Colorless oil; IR (CHCl3) nmax 3599, 3312 (br), 1596 cmꢃ1
;
1H NMR (CDCl3): 7.32 (2H, d, J ¼ 7.7 Hz), 7.26 (2H, t,
J ¼ 7.7 Hz), 7.17 (1H, t, J ¼ 7.7 Hz), 7.08 (1H, t, J ¼
8.0 Hz), 6.51 (1H, d, J ¼ 8.4 Hz), 6.48 (1H, d, J ¼ 8.4 Hz),
6.46 (1H, d, J ¼ 16.1 Hz), 6.34 (1H, dt, J ¼ 16.1, 6.6 Hz),
4.99 (1H, br s, OH), 3.82 (3H, s, OCH3), 3.60 (2H, d,
J ¼ 6.6 Hz); HRMS m/z 240.1151 (calcd for C16H16O2:
240.1150).
4.7.2. 2,3-Dimethoxy-4,6-dicinnamylphenol methyl
ether (12)
Pale yellow oil; IR (CHCl3) nmax 1599 cmꢃ1 1H NMR
;
(CDCl3): 7.34 (2H, d, J ¼ 7.7 Hz), 7.26 (2H, t, J ¼ 7.7 Hz),

C. Ito et al. / European Journal of Medicinal Chemistry 42 (2007) 902e909
907
1
7.18 (1H, t, J ¼ 7.7 Hz), 6.78 (1H, s), 6.43 (1H, d,
J ¼ 15.8 Hz), 6.32 (1H, dt, J ¼ 15.8, 6.6 Hz), 3.92 (3H, s,
OCH3), 3.87 (6H, s, OCH3), 3.48 (2H, d, J ¼ 6.6 Hz);
HRMS m/z 400.2006 (calcd for C27H28O3: 400.2039).
4.11.1. 3-Methoxy-2-prenyloxy-6-cinnamylphenol (16)
Colorless oil; IR (CHCl3) nmax 3516, 1619, 1596 cmꢃ1; H
NMR (CDCl3): 7.51 (1H, s, OH), 7.36 (2H, d, J ¼ 7.7 Hz),
7.27 (2H, t, J ¼ 7.7 Hz), 7.17 (1H, t, J ¼ 7.7 Hz), 6.79 (1H,
d, J ¼ 8.5 Hz), 6.46 (1H, d, J ¼ 8.5 Hz), 6.42 (2H, m), 5.50
(1H, m), 4.52 (2H, d, J ¼ 7.3 Hz), 3.81 (3H, s, OCH3), 3.45
(2H, d, J ¼ 6.6 Hz), 1.69 (3H, s), 1.61 (3H, s); HRMS m/z
324.1707 (calcd for C21H24O3: 324.1725).
4.8. Synthesis of cinnamylphenol 13
Following the method IV, cinnamylphenol 1 (11.3 mg) and
prenyl bromide (8.4 mg) afford cinnamylphenol 13 (8.2 mg).
4.11.2. 3-Methoxy-2-prenyloxy-6-cinnamylphenol
prenyl ether (17)
4.8.1. 5-Methoxy-2-cinnamylphenol prenyl ether (13)
Colorless oil; IR (CHCl3) nmax 1598 cmꢃ1 1H NMR
;
Colorless oil; IR (CHCl3) nmax 1610 cmꢃ1 1H NMR
;
(CDCl3): 7.37 (2H, d, J ¼ 7.7 Hz), 7.28 (2H, t, J ¼ 7.7 Hz),
7.17 (1H, t, J ¼ 7.7 Hz), 6.88 (1H, d, J ¼ 8.4 Hz), 6.70 (1H,
d, J ¼ 8.4 Hz), 6.40 (2H, m), 5.54 (1H, m), 5.53 (1H, m),
4.56 (2H, d, J ¼ 7.0 Hz), 4.48 (2H, d, J ¼ 7.0 Hz), 3.80 (3H,
s, OCH3), 3.47 (2H, d, J ¼ 6.6 Hz), 1.73 (6H, s), 1.66 (6H,
s); HRMS m/z 392.2336 (calcd for C26H32O3: 392.2351).
(CDCl3): 7.33 (2H, d, J ¼ 7.7 Hz), 7.27 (2H, t, J ¼ 7.7 Hz),
7.17 (1H, t, J ¼ 7.7 Hz), 7.07 (1H, d, J ¼ 8.1 Hz), 6.47 (1H,
d, J ¼ 2.2 Hz), 6.43 (1H, dd, J ¼ 8.1, 2.2 Hz), 6.39 (1H, d,
J ¼ 15.8 Hz), 6.37 (1H, dt, J ¼ 15.8, 6.6 Hz), 5.49 (1H, m),
4.51 (2H, d, J ¼ 6.6 Hz), 3.79 (3H, s, OCH3), 3.47 (2H, d,
J ¼ 6.6 Hz), 1.78 (3H, s), 1.72 (3H, s); HRMS m/z 308.1747
(calcd for C21H24O2: 308.1777).
4.11.3. 2-Hydroxy-3-methoxy-6-cinnamylphenol
prenyl ether (18)
Colorless oil; IR (CHCl3) nmax 3539, 1619 cmꢃ1; 1H NMR
(CDCl3): 7.37 (2H, d, J ¼ 7.7 Hz), 7.27 (2H, t, J ¼ 7.7 Hz),
7.17 (1H, t, J ¼ 7.7 Hz), 6.65 (2H, m), 6.41 (2H, m), 5.54
(1H, m), 4.58 (2H, d, J ¼ 7.0 Hz), 3.80 (3H, s, OCH3), 3.47
(2H, d, J ¼ 6.6 Hz), 1.72 (3H, s), 1.64 (3H, s); HRMS m/z
324.1727 (calcd for C21H24O3: 324.1726).
4.9. Synthesis of cinnamylphenol 14
Following the method IV, cinnamylphenol 2 (10.6 mg) and
prenyl bromide (7.8 mg) afford cinnamylphenol 14 (7.6 mg).
4.9.1. 3-Methoxy-2-cinnamylphenol prenyl ether (14)
Colorless oil; IR (CHCl3) nmax 1594 cmꢃ1 1H NMR
;
4.12. Synthesis of cinnamylphenol 19
(CDCl3): 7.32 (2H, d, J ¼ 8.0 Hz), 7.25 (2H, t, J ¼ 8.0 Hz),
7.14 (2H, t, J ¼ 8.0 Hz), 6.58 (1H, d, J ¼ 8.0 Hz), 6.56 (1H,
d, J ¼ 8.0 Hz), 6.41 (1H, d, J ¼ 15.8 Hz), 6.33 (1H, dt,
J ¼ 15.8, 6.6 Hz), 5.50 (1H, m), 4.54 (2H, d, J ¼ 6.2 Hz),
3.84 (3H, s, OCH3), 3.58 (2H, d, J ¼ 6.6 Hz), 1.79 (3H, s),
1.72 (3H, s); HRMS m/z 308.1740 (calcd for C21H24O2:
308.1776).
Following the method IV, cinnamylphenol 6 (5.2 mg) and
prenyl bromide (6.7 mg) afford cinnamylphenol 19 (10.7 mg).
4.12.1. 2,3-Diprenyloxy-6-cinnamylphenol methyl ether (19)
Pale yellow oil; IR (CHCl3) nmax 1599 cmꢃ1 1H NMR
;
(CDCl3): 7.34 (2H, d, J ¼ 7.7 Hz), 7.28 (2H, t, J ¼ 7.7 Hz),
7.18 (1H, t, J ¼ 7.7 Hz), 6.84 (1H, d, J ¼ 8.4 Hz), 6.62 (1H,
d, J ¼ 8.4 Hz), 6.41 (1H, d, J ¼ 15.8 Hz), 6.34 (1H, dt,
J ¼ 15.8, 6.6 Hz), 5.59 (1H, m), 5.51 (1H, m), 4.53 (2H, d,
J ¼ 7.0 Hz), 4.51 (2H, d, J ¼ 7.0 Hz), 3.89 (3H, s, OCH3),
3.48 (2H, d, J ¼ 6.6 Hz), 1.77 (3H, s), 1.76 (3H, s), 1.72
(3H, s), 1.70 (3H, s); HRMS m/z 392.2313 (calcd for
C26H32O3: 392.2351).
4.10. Synthesis of cinnamylphenol 15
Following the method IV, cinnamylphenol 4 (13.6 mg) and
prenyl bromide (8.4 mg) afford cinnamylphenol 15 (10.3 mg).
4.10.1. 5-Prenyloxy-2-cinnamylphenol methyl ether (15)
Colorless oil; IR (CHCl3) nmax 1620 cmꢃ1 1H NMR
;
(CDCl3): 7.34 (2H, d, J ¼ 7.7 Hz), 7.27 (2H, t, J ¼ 7.7 Hz),
7.18 (1H, t, J ¼ 7.7 Hz), 7.06 (1H, d, J ¼ 8.4 Hz), 6.49 (1H,
d, J ¼ 2.2 Hz), 6.45 (1H, dd, J ¼ 8.4, 2.2 Hz), 6.40 (1H, d,
J ¼ 15.8 Hz), 6.35 (1H, dt, J ¼ 15.8, 6.6 Hz), 5.50 (1H, m),
4.49 (1H, d, J ¼ 7.0 Hz), 3.81 (3H, s, OCH3), 3.46 (2H, d,
J ¼ 6.6 Hz), 1.80 (3H, s), 1.74 (3H, s); HRMS m/z 308.1770
(calcd for C21H24O2: 308.1777).
4.13. Synthesis of cinnamylphenol 20
Following the method IV, cinnamylphenol 8 (9.0 mg) and
prenyl bromide (6.0 mg) afford cinnamylphenol 20 (4.5 mg).
4.13.1. 2-Methoxy-3-prenyloxy-6-cinnamylphenol
methyl ether (20)
Pale yellow oil; IR (CHCl3) nmax 1598 cmꢃ1 1H NMR
;
4.11. Synthesis of cinnamylphenols 16e18
(CDCl3): 7.35 (2H, d, J ¼ 7.7 Hz), 7.28 (2H, t, J ¼ 7.7 Hz),
7.19 (1H, t, J ¼ 7.7 Hz), 6.86 (1H, d, J ¼ 8.4 Hz), 6.64 (1H,
d, J ¼ 8.4 Hz), 6.43 (1H, d, J ¼ 15.8 Hz), 6.34 (1H, dt,
J ¼ 15.8, 6.6 Hz), 5.51 (1H, m), 4.55 (2H, d, J ¼ 7.0 Hz),
3.89 (3H, s, OCH3), 3.49 (2H, d, J ¼ 6.6 Hz), 1.79 (3H, s),
Following the method IV, cinnamylphenol 5 (10 mg) and
prenyl bromide (11.6 mg) afford cinnamylphenols 16 (8.8 mg),
17 (1.4 mg), and 18 (2.2 mg).

908
C. Ito et al. / European Journal of Medicinal Chemistry 42 (2007) 902e909
1.74 (3H, s); HRMS m/z 338.1843 (calcd for C22H26O3:
338.1882).
4.15.3. 3,300-Dimethoxy-2,400-diprenyloxy-6-cinnamylphenol
(25)
Pale yellow oil; IR (CHCl3) nmax 3517, 1602 cmꢃ1 1H
;
NMR (CDCl3): 6.90 (1H, d, J ¼ 1.5 Hz), 6.85 (1H, dd,
J ¼ 8.4, 1.8 Hz), 6.80 (1H, d, J ¼ 8.4 Hz), 6.78 (1H, d,
J ¼ 8.4 Hz), 6.42 (1H, d, J ¼ 8.4 Hz), 6.35 (1H, d,
J ¼ 15.8 Hz), 6.22 (1H, dt, J ¼ 15.8, 6.6 Hz), 5.94 (1H, s,
OH), 5.52 (1H, m), 5.51 (1H, m), 4.57 (2H, d, J ¼ 7.0 Hz),
4.56 (2H, d, J ¼ 7.0 Hz), 3.86 (3H, s, OCH3), 3.84 (3H, s,
OCH3), 3.47 (2H, d, J ¼ 6.6 Hz), 1.76 (6H, s), 1.72 (3H, s),
1.68 (3H, s); HRMS m/z 438.2401 (calcd for C27H34O5:
438.2407).
4.14. Synthesis of cinnamylphenols 21 and 22
Following the method II, 3-methoxycatechol (1 g) and eu-
genol (1.17 g) afford cinnamylphenols 21 (126 mg) and 22
(34 mg).
4.14.1. 2,400-Dihydroxy-3,300-dimethoxy-6-cinnamylphenol
(21)
Colorless oil; IR (CHCl3) nmax 3549, 1632, 1601 cmꢃ1; H
1
NMR (CDCl3): 6.88 (1H, d, J ¼ 1.5 Hz), 6.85 (1H, dd, J ¼ 8.4,
1.5 Hz), 6.82 (1H, d, J ¼ 8.4 Hz), 6.67 (1H, d, J ¼ 8.4 Hz),
6.43 (1H, d, J ¼ 8.4 Hz), 6.37 (1H, d, J ¼ 15.8 Hz), 6.21
(1H, dt, J ¼ 15.8, 6.6 Hz), 5.55 (1H, s), 5.40 (2H, s), 3.87
(3H, s, OCH3), 3.86 (3H, s, OCH3), 3.49 (2H, d, J ¼
6.6 Hz); HRMS m/z 302.1139 (calcd for C17H18O5: 302.1154).
4.16. Synthesis of cinnamylphenol 26
Following the method IV, cinnamylphenol 23 (5.5 mg) and
prenyl bromide (4.9 mg) afford cinnamylphenol 26 (3.6 mg).
4.16.1. 3,300-Dimethoxy-2,400-diprenyloxy-6-cinnamylphenol
prenyl ether (26)
4.14.2. 2,3,400-Trihydroxy-300-methoxy-6-cinnamylphenol
methyl ether (22)
Colorless oil; IR (CHCl3) nmax 3545, 3265 (br), 1613,
Pale yellow oil; IR (CHCl3) nmax 1601 cmꢃ1 1H NMR
;
(CDCl3): 6.90 (1H, d, J ¼ 1.5 Hz), 6.88 (1H, d, J ¼ 8.4 Hz),
6.84 (1H, dd, J ¼ 8.4, 1.5 Hz), 6.79 (1H, d, J ¼ 8.4 Hz), 6.62
(1H, d, J ¼ 8.4 Hz), 6.36 (1H, d, J ¼ 15.8 Hz), 6.19 (1H, dt,
J ¼ 15.8, 6.6 Hz), 5.59 (1H, m), 5.57 (1H, m), 5.51 (1H, m),
4.56 (4H, d, J ¼ 7.0 Hz), 4.52 (2H, d, J ¼ 7.0 Hz), 3.86 (3H,
s, OCH3), 3.83 (3H, s, OCH3), 3.49 (2H, d, J ¼ 6.6 Hz),
1.77 (3H, s), 1.76 (6H, s), 1.72 (3H, s), 1.69 (3H, s), 1.68
(3H, s); HRMS m/z 506.3020 (calcd for C32H42O5: 506.3032).
1
1509 cmꢃ1; H NMR (CDCl3): 6.87 (1H, br s), 6.84 (2H, s),
6.68 (2H, s), 6.34 (1H, d, J ¼ 15.8 Hz), 6.15 (1H, dt,
J ¼ 15.8, 6.6 Hz), 5.57 (1H, s), 5.50 (1H, s), 5.22 (1H, br s),
3.88 (3H, s, OCH3), 3.83 (3H, s, OCH3), 3.48 (2H, d,
J ¼ 6.6 Hz); HRMS m/z 302.1135 (calcd for C17H18O5:
302.1154).
4.15. Synthesis of cinnamylphenols 23e25
4.17. Synthesis of cinnamylphenols 27 and 28
Following the method IV, cinnamylphenol 22 (130 mg) and
prenyl bromide (70 mg) afford cinnamylphenols 23 (15.3 mg),
24 (8.5 mg) and 25 (6.0 mg).
Following the method III, 3-methoxycatechol (700 mg) and
coniferyl diacetate (670 mg) afford cinnamylphenols 27
(121 mg) and 28 (91 mg).
4.15.1. 400-Hydroxy-3,300-dimethoxy-2-prenyloxy-6-
cinnamylphenol (23)
4.17.1. 400-Acetoxy-2-hydroxy-3,300-dimethoxy-6-
cinnamylphenol (27)
Pale yellow oil; IR (CHCl3) nmax 3552, 3321 (br), 1761,
Pale yellow oil; IR (CHCl3) nmax 3536, 1616 cmꢃ1 1H
;
1632, 1602 cmꢃ1
;
1H NMR (CDCl3): 6.94 (1H, d,
NMR (CDCl3): 6.87 (1H, d, J ¼ 1.5 Hz), 6.83 (2H, s), 6.82
(1H, d, J ¼ 8.4 Hz), 6.42 (1H, d, J ¼ 8.4 Hz), 6.34 (1H, d,
J ¼ 15.8 Hz), 6.20 (1H, dt, J ¼ 15.8, 6.6 Hz), 5.94 (1H, s,
OH), 5.55 (1H, s, OH), 5.52 (1H, m), 4.57 (2H, d,
J ¼ 7.0 Hz), 3.87 (3H, s, OCH3), 3.84 (3H, s, OCH3), 3.47
(2H, d, J ¼ 6.6 Hz), 1.76 (3H, s), 1.67 (3H, s); HRMS m/z
370.1773 (calcd for C22H26O5: 370.1780).
J ¼ 1.5 Hz), 6.93 (1H, d, J ¼ 8.4 Hz), 6.91 (1H, dd, J ¼ 8.4,
1.5 Hz), 6.66 (1H, d, J ¼ 8.4 Hz), 6.43 (1H, d, J ¼ 8.4 Hz),
6.40 (1H, d, J ¼ 15.8 Hz), 6.32 (1H, dt, J ¼ 15.8, 6.6 Hz),
5.42 (2H, s, OH), 3.86 (3H, s, OCH3), 3.82 (3H, s, OCH3),
3.50 (2H, d, J ¼ 6.6 Hz), 2.30 (3H, s, COCH3); HRMS m/z
344.1224 (calcd for C19H20O6: 344.1260).
4.15.2. 2,400-Dihydroxy-3,300-dimethoxy-6-cinnamylphenol
prenyl ether (24)
4.17.2. 400-Acetoxy-2,3-dihydroxy-300-methoxy-6-
cinnamylphenol methyl ether (28)
Pale yellow oil; IR (CHCl3) nmax 3547, 3335 (br), 1761,
1
Yellow oil; IR (CHCl3) nmax 3540, 1612 cmꢃ1; H NMR
(CDCl3): 6.87 (1H, d, J ¼ 1.5 Hz), 6.83 (2H, s), 6.69 (1H, d,
J ¼ 8.4 Hz), 6.61 (1H, d, J ¼ 8.4 Hz), 6.34 (1H, d, J ¼
15.8 Hz), 6.16 (1H, dt, J ¼ 15.8, 6.6 Hz), 5.58 (1H, m), 5.57
(1H, s, OH), 5.55 (1H, s, OH), 4.54 (2H, d, J ¼ 7.0 Hz),
3.87 (6H, s, OCH3), 3.50 (2H, d, J ¼ 6.6 Hz), 1.78 (3H, s),
1.69 (3H, s); HRMS m/z 370.1780 (calcd for C22H26O5:
370.1780).
1603 cmꢃ1
;
1H NMR (CDCl3): 6.94 (1H, d, J ¼ 8.4 Hz),
6.93 (1H, d, J ¼ 1.5 Hz), 6.91 (1H, dd, J ¼ 8.4, 1.5 Hz), 6.67
(1H, d, J ¼ 8.4 Hz), 6.65 (1H, d, J ¼ 8.4 Hz), 6.37 (1H, d,
J ¼ 15.8 Hz), 6.27 (1H, dt, J ¼ 15.8, 6.6 Hz), 5.62 (1H, s,
OH), 5.36 (1H, s, OH), 3.82 (6H, s, OCH3), 3.81 (3H, s,
OCH3), 3.49 (2H, d, J ¼ 6.6 Hz), 2.30 (3H, s, COCH3);
HRMS m/z 344.1242 (calcd for C19H20O6: 344.1260).

C. Ito et al. / European Journal of Medicinal Chemistry 42 (2007) 902e909
909
4.18. In vitro and in vivo biological assays
acetone (0.1 ml) 1 h before each TPA treatment. The incidence
of papillomas was observed weekly for 20 weeks.
The inhibition of EBV-EA activation was assayed using
Raji cells (virus non-producer type) as previously described
[2e7]. The inhibition of mouse skin tumor promotion in an
in vivo two-stage carcinogenesis test was also assayed using
the same method as described previously [3,4,6].
Acknowledgments
We thank Mr. Yoshitaka Ito for measurements of some high
resolution mass spectra. This work was supported in part by
Grants-in-Aid for Scientific Research (C) from the Japan So-
ciety for the Promotion of Science, High-Tech Research Cen-
ter Project from the Ministry of Education, Culture, Sports,
Science and Technology (MEXT) of Japan, and also by grant
from the National Cancer Institute (CA17625) USA.
4.18.1. Short-term in vitro bioassay for anti-tumor
promoters
The inhibition of EBV-EA activation was assayed using the
same method as described previously [2e7]. In brief, Raji
cells were grown to a density of 106 cells/mL, harvested by
centrifugation and resuspended in RPMI 1640 medium (Sigma
Chemical Co., St. Louis, USA) with 10% FCS containing
4 mM n-butyric acid as inducer, 32 pmol of TPA (20 ng/mL
in DMSO), and 32, 16, 3.2 or 0.32 nmol of the test compound
(DMSO solutions). The cells were incubated at 37 ꢀC for 48 h,
and cell number and viability were determined after 48 h by
means of a hemocytometer (Trypan Blue staining method).
EBV-EA inhibitory activity of the test compounds was esti-
mated on the basis of the percentage of positive cells com-
pared to that observed in a control without the test product.
In each assay, at least 500 cells were counted and the results
were read blind. As regards the effects of acridone alkaloids
on this assay, the IC50 values were estimated by the probit
transformation technique.
References
[1] W. Heller, G. Forkmann, in: J.B. Harborne (Ed.), The Flavonoids, Chap-
man and Hall, London, 1988, pp. 399e425 Chapter 11.
[2] C. Ito, M. Itoigawa, Y. Miyamoto, K.S. Rao, J. Takayasu, Y. Okuda,
T. Mukainaka, H. Tokuda, H. Nishino, H. Furukawa, J. Nat. Prod. 62
(1999) 1668e1671.
[3] M. Itoigawa, C. Ito, M. Ju-ichi, T. Nobukuni, E. Ichiishi, H. Tokuda,
H. Nishino, H. Furukawa, Cancer Lett. 176 (2002) 25e29.
[4] C. Ito, M. Itoigawa, H.T.W. Tan, H. Tokuda, X.Y. Mou,
T. Mukainaka, T. Ishikawa, H. Nishino, H. Furukawa, Cancer Lett.
152 (2000) 187e192.
[5] C. Ito, M. Itoigawa, T. Kanematsu, N. Ruangrungsi, T. Mukainaka,
H. Tokuda, H. Nishino, H. Furukawa, Phytochemistry 64 (2003)
1265e1268.
[6] C. Ito, M. Itoigawa, N. Kojima, H. Tokuda, T. Hirata, H. Nishino,
H. Furukawa, J. Nat. Prod. 67 (2004) 1125e1130.
[7] C. Ito, M. Itoigawa, T. Kanematsu, N. Ruangrungsi, H. Higashihara,
H. Tokuda, H. Nishino, H. Furukawa, J. Nat. Prod. 66 (2003)
1574e1577.
4.18.2. In vivo two-stage mouse skin carcinogenesis test
Female SENCAR mice were obtained at 5e6 weeks of age
from SLC Co., Ltd. (Shizuoka, Japan), and maintained in
Kyoto Prefectural University of Medicine, Animal Center.
Groups of animals (15 animals per group) were housed in sub-
groups of five in polycarbonate cages. Mice were permitted
free access to an MF solid diet (Oriental yeast Co., Ltd. Chiba,
Japan) and drinking water at all times during the study. The
back of each mouse was shaved with surgical clippers before
the first day of initiation. Tumors on the back of the mice were
initiated with dimethylbenz[a]anthracene (DMBA; 390 nmol)
in acetone (0.1 ml). One week after initiation, they were pro-
moted twice a week by the application of TPA (1.7 nmol) in
acetone (0.1 ml). The mice in the test compound treated
groups were treated with the test compounds (85 nmol) in
[8] Y. Goda, F. Kiuchi, M. Shibuya, U. Sankawa, Chem. Pharm. Bull. 40
(1992) 2452e2457.
[9] A. Zanarotti, Tetrahedron Lett. 23 (1982) 3963e3964.
[10] M.-T. Huang, W. Ma, Y.-P. Lu, R.L. Chang, C. Fisher, P.S. Manchand,
H.L. Newmark, A.H. Conney, Carcinogenesis 16 (1995) 2493e2497.
[11] J. Ishida, M. Kozuka, H.-K. Wang, T. Konoshima, H. Tokuda, M. Okuda,
X.Y. Mou, H. Nishino, N. Sakurai, K.-H. Lee, M. Nagai, Cancer Lett.
159 (2000) 135e140.
[12] S. Mageswaran, W.D. Ollis, R.J. Roberts, I.O. Sutherland, Tetrahedron
Lett. 34 (1969) 2897e2900.
[13] Y. Goda, M. Katayama, K. Ichikawa, M. Shibuya, F. Kiuchi, U. Sankawa,
Chem. Pharm. Bull. 33 (1985) 5606e5609.
[14] K. Kurosawa, W.D. Ollis, I.O. Sutherland, O.R. Gottlieb, A.B. Oliveira,
Phytochemistry 17 (1978) 1389e1394.
[15] H. Achenbach, M. Stocker, M.A. Constenla, Phytochemistry 27 (1988)
1835e1841.