Hybrids of 3α-methoxyserrat-14-en-21β-ol (PJ-1) and 3β-methoxyserrat-14-en-21β-ol (PJ-2) and various anti-oxidants as cancer chemopreventive agents

Article abstract of DOI:10.1016/j.ejmech.2010.01.057

3α-Methoxyserrat-14-en-21β-ol (1) and 3β-methoxyserrat-14-en-21β-ol (2) and their conjugates with curcumin, kojic acid, quercetin, and baicalein (3-18), as well as new analogs (19-24) derived from 1 and 2, were tested for their inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). The inhibitory effects of 16 (IC₅₀ = 330 mol ratio/32 pmol/TPA), 9 (IC₅₀ = 335), 10 (IC₅₀ = 338), and 15 (IC₅₀ = 350) were stronger than those of the other compounds and the positive control, oleanolic acid (IC₅₀ = 449). Compounds 15 and 16, which are conjugates of one molecule each of 1 or 2 and quercetin, inhibited mouse skin tumor promotion in an in vivo two-stage carcinogenesis model. The in vivo two-stage mouse skin carcinogenesis test employed 7,12-dimethylbenz[a]anthracene (DMBA) as an initiator and TPA as a promoter.

Full text of DOI:10.1016/j.ejmech.2010.01.057

European Journal of Medicinal Chemistry 45 (2010) 2191–2197
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Hybrids of 3
a
-methoxyserrat-14-en-21
b-ol (PJ-1) and
3b
-methoxyserrat-14-en-21
b-ol (PJ-2) and various anti-oxidants as cancer
chemopreventive agents
,
b
Hiroko Tsujii a, Takeshi Yamada a, Tetsuya Kajimoto a, Reiko Tanaka a , Harukuni Tokuda ,
*
Junya Hasegawa c, Yoshio Hamashima c, Manabu Node c
a Laboratory of Medicinal Chemistry, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan
b Department of Complementary and Alternative Medicine, R&D Graduate School of Medical Sciences, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-8640, Japan
c Department of Pharmaceutical Manufacturing Chemistry, Kyoto Pharmaceutical University, Misasagi, Yamashina, Kyoto 607-8414, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
3a-Methoxyserrat-14-en-21b-ol (1) and 3b-methoxyserrat-14-en-21b-ol (2) and their conjugates with
Received 22 July 2009
Received in revised form
20 January 2010
Accepted 21 January 2010
Available online 29 January 2010
curcumin, kojic acid, quercetin, and baicalein (3–18), as well as new analogs (19–24) derived from 1 and
2, were tested for their inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced
by 12-O-tetradecanoylphorbol-13-acetate (TPA). The inhibitory effects of 16 (IC50 ¼ 330 mol ratio/
32 pmol/TPA), 9 (IC50 ¼ 335), 10 (IC50 ¼ 338), and 15 (IC50 ¼ 350) were stronger than those of the other
compounds and the positive control, oleanolic acid (IC50 ¼ 449). Compounds 15 and 16, which are
conjugates of one molecule each of 1 or 2 and quercetin, inhibited mouse skin tumor promotion in an in
vivo two-stage carcinogenesis model. The in vivo two-stage mouse skin carcinogenesis test employed
7,12-dimethylbenz[a]anthracene (DMBA) as an initiator and TPA as a promoter.
Keywords:
3
3
a
-Methoxyserrat-14-en-21
b
b
-ol
-ol
b
-Methoxyserrat-14-en-21
Ó 2010 Elsevier Masson SAS. All rights reserved.
Quercetin
Epstein-Barr virus early antigen (EBV-EA)
activation
In vivo two-stage mouse skin
carcinogenesis test
1. Introduction
anti-tumor promoting activities in an in vivo two-stage mouse skin
carcinogenesis test using 7,12-dimethylbenz[a]anthracene (DMBA)
In recent years, the demand for more effective and safer agents
for the chemoprevention of human cancer has been a great surge,
and natural products from plants and their synthetic analogs are
expected to play a vital role in creating new and better chemo-
preventive agents [1,2]. In particular, there is a need for chemo-
preventive agents that target the promotion stage of carcinogenesis
in the two- or multi-stage theory [3] because it is not possible to
completely avoid carcinogens in daily life and tumor promotion is
a long and reversible process that can be efficiently suppressed [4].
The genus Picea consists of approximately 40 species world-
wide. Its number is next to those of Pinus and Abies in the order
Pinales. Picea species, however, do not have any important use
besides construction materials. In our fervent search for biologi-
cally active constituents from natural sources, we previously
reported several serratane-type triterpenoids that show significant
as an initiator and 12-O-tetradecanoylphorbol-13-acetate (TPA) as
a promoter. These include 3 -methoxyserrat-14-en-21 -ol (1) [5],
13 ,14 -epoxy-3 -methoxyserratan-21 -ol and 21 -hydroxy-3
methoxyserrat-14-en-29-al [6], serrat-14-en-3 ,21 -diol [7], and
14 ,15 -epoxy-3 -methoxyserratan-21 -ol [8], which were iso-
lated from the bark of Picea jezoensis (Sieb. et Zucc) Carr. var. jez-
oensis (Pinaceae, Japanese name: Ezomatsu), and P. jezoensis (Sieb.
et Zucc) Carr. var. hondoensis (Mayr) Rehder (Pinaceae, Japanese
a
b
a
a
b
b
a
b
b-
b
b
b
b
b
name: Touhi).
3
a-Methoxyserrat-14-en-21
b
-ol (1) and
3b-
methoxyserrat-14-en-21
b-ol (2) are the most abundant triterpe-
noids in the above two Picea plants and Picea glehni (Fr. Schm.)
Masters (Japanese name: Akaezomatsu) [9]. Recently, we reported
that 1 significantly decreased the proliferation of adenomas and
total tumors in a revised rat multi-organ carcinogenesis (DMBDD)
model [10]. In addition, triterpenoids are widely distributed in
vegetables [11] and fruits [12] and are attractive compounds
because they have various physiological activities yet little toxicity.
As active triterpenoids, Kawamori et al. reported that oleanolic acid
* Corresponding author. Tel./fax: þ81 72 690 1084.
E-mail address: tanakar@gly.oups.ac.jp (R. Tanaka).
0223-5234/$ – see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.01.057

2192
H. Tsujii et al. / European Journal of Medicinal Chemistry 45 (2010) 2191–2197
can reduce AOM-induced ACF, colonic mucosal ODC activity, and
AgNOR number in the colonic epithelium [13], and Senthil et al.
indicated that ursolic acid has a protective effect against myocardial
ischemia induced by isoproterenol in rats [14]. Chen et al. reported
that the nitric oxide releasing derivatives of oleanolic acid inhibit
HCC tumor growth in vivo [15]. On the other hand, betulonic acid-
acetylene hybrids showed hepatoprotective and anti-inflammatory
activities [16]. The conjugation of two bioactive compounds is an
effective strategy that is now well accepted [17]. Natural product
hybrids, including triterpenoids, play an important role in the
development of drugs for the treatment of infections and cancer, as
well as the development of immunosuppressive compounds [18].
Meanwhile, well-known phenolic natural products, curcumin [19],
kojic acid [20], quercetin [21], and baicalein [22], possess excellent
anti-tumor and anti-oxidative activities. Therefore, based on the
diverse bioactivities of the terpenoids as well as the anti-oxidants
described above, we initiated a study of triterpenoid-anti-oxidant
conjugation. As regards well-established triterpenoid conjuga-
tions with other substances, a number of studies of hybrid
connections of triterpenoids are available. Iveson and Parke
We report herein the results of in vitro and in vivo anti-tumor
promoting activities of 3–24. EBV-EA is activated by tumor
promoters, producing viral early antigen (EA), and the evaluation of
its inhibitors is used as a primary screen for in vivo anti-tumor
promoting activities [30]. The results of the in vitro EBV-EA acti-
vation assay are shown in Table 1. Synthetic 3–24 had purities
>99%. Their effects on the viability of Raji cells and their 50%
inhibitory concentration (IC50) values are shown in Table 1. All
compounds exhibited dose-dependent inhibitory activities and the
viability of Raji cells treated with test compounds 3–24 ranged from
60 to 70% except 20 at the highest concentration of 1000 mol ratio/
32 pmol/TPA, suggesting that these compounds showed moderate
cytotoxicities against in vitro cell lines (Table 1). The inhibitory
activities of 16 (IC50 ¼ 330 mol ratio/32 pmol/TPA), 9 (IC50 ¼ 335),
10 (IC50 ¼ 338), 15 (IC50 ¼ 350), 7 (IC50 ¼ 354), and 8 (IC50 ¼ 358)
were stronger than those of the other compounds. The relative
ratios of 16 and 15 (conjugates of one molecule of 1 or 2 and
quercetin using succinic acid as linker) with respect to TPA (100%)
were 2.2, 30.1, 71.9, and 90.3% and 5.1, 32.3, 73.0, and 91.7% at the
concentrations of 1000, 500, 100, and 10 mol ratio/32 pmol/TPA,
respectively (Table 1). This means 97.8, 69.9, 28.1, and 9.7%
(compound 16) inhibition and 94.9, 67.7, 27.0, and 8.3% (compound
15) inhibition of the TPA-induced EBV-EA activation, respectively.
On the other hand, compounds 9 and 10 showed 98.4, 68.3, 29.9,
and 8.4%, and 98.3, 68.9, 29.7, and 8.9% inhibition of TPA-induced
EBV-EA activation at concentrations of 1000, 500, 100, and 10 mol
ratio/32 pmol/TPA, respectively. Compounds 9 and 10 are conju-
gates of two molecules of 1 or 2 and one molecule of curcumin
using malonic acid as linker and compounds 7 and 8 are conjugates
of one molecule of 1 or 2 and one molecule of curcumin using
malonic acid as linker. As hybrids of 1 or 2 and curcumin (Fig. 3),
compound pairs 9, 10 and 7, 8 showed stronger inhibitory activity
reported
b-glycyrrhizic acid – glucuronyl esters [23]. Baltina
disclosed glycyrrhizic acid
–
glucopeptide conjugates having
immune response [24]. Tatsuzaki et al. reported glycyrrhizic acid –
dehydrozingerone conjugates as cytotoxic agents [25] and Ma et al.
indicated oleanane-type triterpene – azidothymidine (AZT) or
FK-3000 conjugates that inhibit the proliferation of HIV-1 and its
protease [26].
In our newly published paper, we reported the synthesis of 3–18
and evaluated their anti-HIV-1 reverse transcriptase (RT) activities
in infected C8166-CCR5 cells, a human CD4þ T-lymphocyte cell line.
Among them, 13, which is a conjugate of two molecules of
3
a
-methoxyserrat-14-en-21
exerted significant anti-HIV RT activity with an EC50 value of
0.12 g/mL [27]. Herein, we report the in vitro and in vivo
b-ol (1) and one molecule of kojic acid,
than compound pairs
3
(IC50 ¼ 393),
4
(IC50 ¼ 460), and
5
m
(IC50 ¼ 462), 6 (IC50 ¼ 395) that used succinic acid as linker. This
may mean that malonic acid is a better linker than succinic acid.
Compounds 11 (IC50 ¼ 370) and 12 (IC50 ¼ 371) are conjugates of
one molecule of 1 or 2 and one molecule of kojic acid using succinic
acid as linker, while 13 (IC50 ¼ 463) and 14 (IC50 ¼ 461) are conju-
gates of two molecules of 1 or 2 and one molecule of kojic acid
using succinic acid as linker. Compounds 11 and 12 showed
stronger inhibitory activity than 13 and 14. Compounds 15 and 16
are conjugates of one molecule of 1 or 2 and one molecule of
quercetin using succinic acid as linker, and 17 (IC50 ¼ 370) and 18
(IC50 ¼ 375) are conjugates of one molecule of 1 or 2 and one
molecule of baicalein using succinic acid as linker. Compounds 19–
24 are new analogs of compound 2. Compounds 19 (IC50 ¼ 459) and
20 (IC50 ¼ 468) are phenylcarbamate and 2-chlorophenylcarbamate
derivatives, respectively. Compounds 21 (IC50 ¼ 470) and 22
(IC50 ¼ 461) are carbamate and N-[(4-methylphenyl)sulfonyl]
carbamate derivatives, respectively. Compounds 23 (IC50 ¼ 460)
and 24 (IC50 ¼ 473) are phenylcarbamothioate and chloroacetate
derivatives, respectively. The EBV-EA activation ratios of 19–24
were high.
In our past work, we found that the inhibitory effects on EBV-EA
induction by TPA correlated well with the anti-tumor promoting
activity in vivo [5–8]. Among the 22 conjugates, we selected 15 and
16 to examine their effects on the in vivo two-stage mouse skin
carcinogenesis using mouse skin papillomas induced by DMBA as
an initiator and TPA as a promoter. The experimental protocol is
shown in Fig. 4. During the in vivo test, the body-weight gains of
the mice were not influenced by the treatment with the test
compounds and no toxic effects, such as lesional damage and
inflammation (edema, erosion, and ulcer), were observed on the
areas of mouse skin topically treated with the test compounds. As
shown in Fig. 5A, papilloma-bearing mice in the positive control
anti-tumor promoting activity of 3–18 and new synthetic analogs
(19–24) derived from 1 and 2. The screening methods employed
were a convenient primary in vitro assay to estimate the inhibitory
effect on Epstein-Barr virus early antigen (EBV-EA) activation
induced by a well-known tumor promoter, TPA [28], and an in vivo
two-stage mouse skin carcinogenesis test using DMBA as an initi-
ator and TPA as a tumor promoter [5].
2. Results and discussion
3a
-Methoxyserrat-14-en-21
b-ol (1) and 3b-methoxyserrat-14-
en-21 -ol (2) were the predominant triterpenoids in the chloro-
b
form extracts of P. jezoensis Carr. var. jezoensis and P. jezoensis Carr.
var. hondoensis. Compounds 1 and 2 constituted 25% and 10% of
P. jezoensis Carr. var. jezoensis extract and 18% and 30% of P. jez-
oensis Carr. var. hondoensis extract, respectively [29]. Although,
oleanolic acid, ursolic acid, and betulinic acid were often modified
into various forms, serratane-type triterpenoids, such as 1 and 2,
were not modified. Therefore, based on the diverse bioactivities of
the triterpenoids as well as phenolic compounds having
anti-oxidant activity, such as curcumin, kojic acid, quercetin, and
baicalein, we studied the conjugation of triterpenoids and
phenolic compounds on the basis of the hybrid drug strategy,
and synthesized 16 compounds (3–18) (Fig. 1) as described in
a recent paper [27]. In addition, six new compounds (19–24) were
synthesized from 2 and subjected to the in vitro EBV-EA activation
assay. Compound 19 is compound 2-phenylcarbamate, 20 is
compound 2-2-chlorophenylcarbamate, 21 is compound 2-carba-
mate, 22 is compound 2-N-[(4-methylphenyl)sulfonyl]carbamate,
23 is compound 2-phenylcarbamothioate, and 24 is compound 2-
chloroacetate (Section 3, Fig. 2).

H. Tsujii et al. / European Journal of Medicinal Chemistry 45 (2010) 2191–2197
2193
Fig. 1. 3a-Methoxyserrat-14-en-21b-ol (1) and 3b-methoxyserrat-14-en-21b-ol (2) and their curcumin, kojic acid, quercetin, and baicalein conjugates (3–18).
group treated with DMBA (390 nmol) and TPA (1.7 nmol, twice/
week) appeared as early as at week 6, and the percentage of
papilloma bearers increased rapidly to reach 100% after week 10.
On the other hand, treatment with 15 and 16 (85 nmol) along with
DMBA/TPA reduced the percentage of papilloma-bearing mice to
26.6–46.6% and 20–53.3% during weeks 10–15, respectively, and
the percentage reduction was 73.3% and 86.6% at week 20. As
shown in Fig. 5B, in the positive control group treated with DMBA/
TPA, the number of papillomas formed per mouse increased rapidly
after week 6 to reach 8.6 papillomas/mouse at week 20, whereas
mice treated with 16 bore 4.7 papillomas (tumor length
26 ꢀ 1.0 mm, tumor width 24 ꢀ 0.7 mm) and those treated with 15
bore 5.0 papillomas (tumor length 28 ꢀ 1.0 mm, tumor width
25 ꢀ 0.7 mm) even at week 20. As illustrated in Fig. 5A, B, in the
group treated with 1 and quercetin (each 1 mol) plus DMBA/TPA,
the percentages of papilloma-bearing mice reached 93.3% and the
number of papillomas/mouse was 7.0 (tumor length 28 ꢀ 1.0 mm,
tumor width 25 ꢀ 0.7 mm) at week 20. In the in vivo two-stage
mouse skin carcinogenesis test, 15 and 16 were found to delay
papilloma formation. The inhibitory effects of 16 and 15 were
stronger than that of the positive control, i.e., oleanolic acid and an
admixture of 1 mol each of 1 and quercetin. The results of in vitro

2194
H. Tsujii et al. / European Journal of Medicinal Chemistry 45 (2010) 2191–2197
Fig. 2. New
3a
-methoxyserrat-14-en-21
b-ol (1) and
3b-methoxyserrat-14-en-21b-ol (2) and their phenylcarbamate, 2-chlorophenylcarbamate, carbamate, N-[(4-methyl-
phenyl)sulfonyl]carbamate, phenylcarbamothioate, and chloroacetate (19–24).
EBV-EA induction and in vivo two-stage mouse skin carcinogenesis
test suggest that serratane-type triterpenoids 1 or 2 conjugated
with quercetin, such as 15 and 16, are useful as cancer chemo-
preventive agents. Further in vivo experiments of remaining
effective compounds 7–10 are under way.
spectra were recorded using a Perkin-Elmer 1720X FTIR spectro-
photometer. 1H and 13C NMR spectra were obtained on a Varian
INOVA 500 spectrometer with standard pulse sequences operating
at 500 and 125 MHz, respectively. CDCl3 was used as solvent and
TMS, as the internal standard. EIMS were recorded on a Hitachi
4000H double-focusing mass spectrometer (70 eV). Column chro-
matography was carried out over silica gel (70–230 mesh, Merck)
and medium-pressure liquid chromatography (MPLC) was carried
out with silica gel (230–400 mesh, Merck). HPLC was run on
a JASCO PU-1586 instrument equipped with a differential refrac-
tometer (RI 1531). Fractions obtained from column chromatog-
raphy were monitored by TLC (silica gel 60 F254, Merck). Preparative
TLC was carried out on Merck silica gel F254 plates (20 ꢁ 20 cm,
0.5 mm thick).
3. Experimental
3.1. Chemistry
Melting points were determined on a Yanagimoto micro-
melting point apparatus and are uncorrected. Optical rotations
were measured using a JASCO DIP-1000 digital polarimeter. IR
Table 1
Relative ratioa of EBV-EA activation with respect to positive control (100%) in the
presence of compounds 1–24, compound 1 plus quercetin, and oleanolic acid.
3.1.1. Chemicals
Phenyl isocyanate, chlorosulfonyl isocyanate, and p-toluene-
sulfonyl isocyanate were purchased from Wako Pure Chemical
Industries, Ltd. (Osaka, Japan). 2-Chlorophenyl isocyanate and
phenyl isothiocyanate (GR) were obtained from Tokyo Chemical
Industry (TCI) Ltd. (Tokyo, Japan). N,N-Dimethylaminopyridine
(DMAP) and chloroacetyl chloride (GR) were purchased from
Sigma–Aldrich Co. (USA). Cell culture reagents, n-butyric acid, and
other reagents for the bioassay were purchased from Nacalai
Tesque, Inc. (Kyoto, Japan). TPA and DMBA were obtained from
Sigma Chemical Co. (St. Louis, MO, USA).
Compound
Concentration (mol ratio/32 pmol/TPA)
IC50
1000
500
100
10
96.2
100
100
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
6.5
13.9
14.3
6.2
2.9
3.1
1.6
1.7
4.9
4.8
13.1
12.7
5.1
2.2
6.5
(60)b
(60)
(60)
(60)
(60)
(60)
(60)
(60)
(70)
(70)
(60)
(60)
(60)
(60)
(70)
(70)
(60)
(50)
(60)
(60)
(60)
(60)
(60)
(70)
35.6
47.9
48.6
35.2
33.1
34.5
31.7
31.1
32.0
32.1
46.8
47.0
32.3
30.1
36.0
37.6
46.2
47.7
48.2
46.3
46.1
48.2
33.1
30.0
75.4
79.7
81.0
73.9
71.7
72.8
70.1
70.3
73.1
73.8
81.1
80.0
73.0
71.9
75.3
76.1
81.3
83.6
80.7
82.1
81.1
84.3
71.7
80.0
393
460
462
395
354
358
335
338
370
371
463
461
350
330
370
375
459
468
470
461
460
473
352
449
96.8
92.3
93.7
91.6
91.1
92.1
92.6
100
100
91.7
3.2. Test compounds
Natural serratane-type triterpenoids, 3
a
-methoxyserrat-14-en-
-ol (2), were isolated
90.3
95.7
96.9
21
b
-ol (1) and 3 -methoxyserrat-14-en-21b
b
from the stem bark of P. jezoensis Carr. var. jezoensis (Pinaceae) that
was collected in Sapporo City, Hokkaido, Japan. The isolation and
characterization of 1 and 2 had been reported previously [29].
A voucher specimen has been deposited at the Laboratory of
Medicinal Chemistry, Osaka University of Pharmaceutical Sciences.
The synthesis, physical properties, and spectral data of compounds
3–18 were described in a recent paper [27].
6.3
19
20
21
22
23
24
11.7
13.6
14.2
12.3
12.0
14.9
2.9
100
100
100
100
100
100
1 þ Quercetin (1:1)
93.4
100
Oleanolic acidc
12.7
3.2.1. 3
To a solution of compound 2 (46 mg, 0.1 mmol) in pyridine
(1 mL) was added phenyl isocyanate (100 L, 0.9 mmol) and the
b-Methoxyserrat-14-en-21b-yl phenylcarbamate (19)
a
Values represent percentages relative to the positive control value (100%).
Values in parentheses are the percentage viability of Raji cells.
Positive control.
b
c
m

H. Tsujii et al. / European Journal of Medicinal Chemistry 45 (2010) 2191–2197
2195
Fig. 3. Structures of curcumin, kojic acid, quercetin, and baicalein.
mixture was allowed to react at room temperature for 120 min. To
the reaction mixture was added a small amount of H2O and the
whole was concentrated. To the residue was added Et2O (10 mL)
and the mixture was stirred for a while. The residue after removal of
Et2O was purified by silica gel column chromatography (CHCl3) to
3.2.3. 3
To an ice-cooled solution of compound 2 (92 mg, 0.2 mmol) in
THF (4 mL) was added chlorosulfonyl isocyanate (30 L, 0.3 mmol)
b-Methoxyserrat-14-en-21b-yl carbamate (21)
m
and the mixture was allowed to react at room temperature for
40 min. To the residue obtained after concentration was added aq.
NaHCO3 and the whole was extracted with methyl ethyl ketone
(MEK) several times. The extract was allowed to stand until the Rf
value of the extract showed a constant value by TLC. The extract
was dried and concentrated, and the residue was chromatographed
on a silica gel column (AcOEt/CHCl3 ¼ 1/1). The eluted product was
give 19 (37 mg, 64%) as a colorless powder. mp 158–160 ꢂC (MeOH–
22
CHCl3); [
a
]
D ꢃ2.0 (c 0.102, CHCl3); 1H NMR
2.63 (1H, dd, J ¼ 11.7,
d
4.2 Hz, H-3a), 4.69 (1H, m, H-21a), 5.34 (1H, m, H-15), 6.61 (1H, bs,
NH), 7.05 (1H, tt, J ¼ 7.4, 1.5 Hz, H-40), 7.30 and 7.31 (each 1H, dt,
J ¼ 7.4, 1.5 Hz, H-20 and H-60), 7.41 (2H, m, H-30 and H-50); 13C NMR
d
79.3 (C-21), 88.4 (C-3), 118.5 (C-30 and C-50), 122.0 (C-15), 123.2
rechromatographed (AcOEt/CHCl3 ¼1/3) to afford 21 (52 mg, 52%)
22
(C-40), 129.0 (C-20 and C-60), 138.1 (C-10), 138.5 (C-14), 153.4 (C]O);
MS (FAB) m/z: 598 (Mþ þ Na); HRMS calculated for C38H57NO3Na,
598.4236; found, 598.4232.
as colorless crystals. mp 279–281 ꢂC (MeOH–CHCl3); [
a
]
D ꢃ0.7 (c
), 4.56
), 4.60 (2H, bs, NH2), 5.33 (1H, m, H-15); 13
0.107, CHCl3); 1H NMR
(1H, t, J ¼ 2.8 Hz, H-21
NMR
d
2.63 (1H, dd, J ¼ 11.8, 4.2 Hz, H-3
a
a
C
d
79.1 (C-21), 88.5 (C-3), 122.0 (C-15), 138.4 (C-14), 156.8
3.2.2. 3
b
-Methoxyserrat-14-en-21
b
-yl 2-chlorophenylcarbamate
(C]O); MS (FAB) m/z: 522 (Mþ þ Na); HRMS calculated for
(20)
C32H53NO3Na, 522.3923; found, 522.3929.
To an ice-cooled solution of compound 2 (92 mg, 0.2 mmol) in
pyridine (2 mL) was added 2-chlorophenyl isocyanate (85 L,
m
3.2.4. 3b-Methoxyserrat-14-en-21b-yl N-[(4-
0.7 mmol), and the mixture was allowed to react at room temper-
ature for 6 hrs. To the residue obtained after concentration was
added AcOEt and the mixture was washed with brine, dried, and
concentrated to give a crystalline residue (200 mg). The residue
was purified by silica gel column chromatography (CHCl3) to give
methylphenyl)sulfonyl]carbamate (22)
To an ice-cooled mixture of compound 2 (92 mg, 0.2 mmol) and
triethylamine (56
nesulfonyl isocyanate (TsNCO) (40
was allowed to react at room temperature for 60 min. To the
reaction mixture was added N,N0-dimethylpropanediamine (50
L,
m
L, 0.4 mmol) in THF (4 mL) was added p-tolue-
mL, 0.26 mmol) and the mixture
20 (120 mg, 98%) as colorless crystals. mp 200–202 ꢂC (MeOH–
m
22
CHCl3); [
a
]
ꢃ22.4 (c 0.102, CHCl3); 1H NMR
d
2.63 (1H, dd,
0.4 mmol) and the whole was stirred until it became a clear solu-
tion. This solution was concentrated and extracted with AcOEt. The
extract was successively washed with 1 M HCl and brine, dried, and
concentrated. The residue was purified by silica gel column chro-
D
J ¼ 11.7, 4.1 Hz, H-3
a
), 4.71 (1H, t, J ¼ 2.3 Hz, H-21
a), 5.35 (1H, m, H-
15), 6.99 (1H, td, J ¼ 7.8, 1.5 Hz, H-40), 7.10 (1H, bs, NH), 7.26 (1H, td,
J ¼ 7.8, 1.5 Hz, H-50), 7.35 (1H, dd, J ¼ 7.8, 1.5 Hz, H-30), 8.16 (1H, bd,
J ¼ 7.8 Hz, H-60); 13C NMR
d
79.9 (C-21), 88.5 (C-3), 120.3 (C-60),
matography (AcOEt/CHCl3 ¼ 1/5) to give compound 22 (115 mg,
22
121.9 (C-15), 122.0 (C-20), 123.6 (C-40), 127.7 (C-50), 129.0 (C-30),
135.0 (C-10), 138.5 (C-14), 153.2 (C]O); MS (FAB) m/z: 632
(Mþ þ Na); HRMS calculated for C38H56NClO3Na, 632.3846; found,
632.3841.
91%) as colorless crystals. mp 218–221 ꢂC (MeOH–CHCl3); [
a]
D
ꢃ9.4 (c 0.106, CHCl3); 1H NMR
d
2.44 (3H, s, H-40-Me), 2.64 (1H, dd,
), 4.61 (1H, t, J ¼ 2.3 Hz, H-21
J ¼ 11.6, 4.1 Hz, H-3
a
a), 5.33 (1H, m, H-
15), 7.33 (2H, d, J ¼ 8.0 Hz, H-30 and H-50), 7.90 (2H, d, J ¼ 8.0 Hz, H-
Promoter
TPA (1.7 nmol)
Initiator
DMBA (390 nmol)
acetone
Positive
Control
Papillomas (%)
Papillomas/Mouse
1 week
60 min
twice a week (20 weeks)
Test Compound
(85 nmol)
TPA
(1.7 nmol)
DMBA
(390 nmol)
Test
Compound
Papillomas(%)
Papillomas/Mouse
1 week
60 min
twice a week (20 weeks)
Fig. 4. Experimental design of chemopreventive activity of compounds 15 and 16 on DMBA–TPA-induced carcinogenesis.

2196
H. Tsujii et al. / European Journal of Medicinal Chemistry 45 (2010) 2191–2197
Fig. 5. Inhibition effects of compounds 13 and 14 on DMBA–TPA mouse skin carcinogenesis. Tumor formation in all mice was initiated with DMBA (390 nmol) and promoted with
TPA (1.7 nmol) twice weekly, starting 1 week after initiation. (A) Percentage of mice with papillomas. (B) Average number of papillomas/mouse. C Control (TPA alone);
TPA þ 80 nmol of positive control (PJ-1 þ quercetin (1:1)); A TPA þ 85 nmol of 15; , TPA þ 85 nmol of 16. a,bStatistically different from the positive control (P < 0.05).
6
20 and H-60); 13C NMR
d
21.6 (C-40-Me), 82.5 (C-21), 88.5 (C-3), 121.7
3.3. Assay for inhibition of EBV-EA activation
(C-15),128.1 (C-20 and C-60), 129.6 (C-30 and C-50), 135.8 (C-10),138.5
(C-14), 144.8 (C-40), 150.2 (C]O); MS (FAB) m/z: 676 (Mþ þ Na);
HRMS calculated for C39H59NO5SNa, 676.4012; found, 676.4017.
EBV-EA-positive serum from a patient with nasopharyngeal
carcinoma (NPC) was a gift from the Department of Biochemistry,
Oita Medicinal University. Lymphoblastoid cells carrying EBV
genome (Raji cells derived from Burkitt’s lymphoma) were cultured
in 10% fetal bovine serum (FBS) in RPMI-1640 medium (Nissui). The
spontaneous activation of EBV-EA in our sub-line Raji cells was less
than 0.1%. The inhibition of EBV-EA activation was assayed using
Raji cells (virus non-producer type), as described previously [6].
The indicator cells (Raji cells, 1 ꢁ106/mL) were incubated at 37 ꢂC
for 48 h in 1 mL of a medium containing n-butyric acid (4 mmol),
3.2.5. 3
A mixture of compound 2 (92 mg, 0.2 mmol), DMAP (13 mg),
and phenyl isothiocyanate (200 L, 1.67 mmol) in pyridine (1.5 mL)
b-Methoxyserrat-14-en-21b-yl phenylcarbamothioate (23)
m
was reacted at 100 ꢂC for 48 h. To the residue obtained after
concentration was added AcOEt and the suspension was filtered.
The filtrate was washed with brine, dried, and concentrated to yield
a brown residue. CHCl3 was added and the whole was filtered. The
filtrate was concentrated and the residue was purified by silica gel
column chromatography (AcOEt/CHCl3 ¼ 1/30) to give compound
TPA (32 pmol ¼ 20 ng in dimethylsulfoxide (DMSO),
2 mL) as
inducer, and various amounts of test compounds in 5 L of DMSO.
m
23 (52 mg, 44%) as a colorless powder. mp 226–229 ꢂC (MeOH–
Smears were made from the cell suspension and the activated cells
that were stained by EBV-EA-positive serum from NPC patients
were detected by an indirect immunofluorescence technique [31].
In each assay, at least 500 cells were counted and the number of
stained cells (positive cells) present was recorded. Triplicate assays
were performed for each compound. The average EBV-EA induction
of the test compounds was expressed as percentage relative to the
control experiment (100%) that was carried out only with n-butyric
acid (4 mmol) plus TPA (32 pmol). EBV-EA induction was ordinarily
around 35%. The viability of treated Raji cells was assayed by the
trypan blue staining method.
22
CHCl3); [
a
]
þ64.3 (c 0.099, CHCl3); 1H NMR
d
2.63 (1H, dd,
D
J ¼ 11.7, 4.0 Hz, H-3
a), 5.30 (1H, m, H-15), 5.41 (1H, m, H-21a), 7.18
(1H, t, J ¼ 7.6 Hz, H-40), 7.28 and 7.31 (each 1H, d, J ¼ 7.6 Hz, H-20 and
H-60), 7.34 (2H, t, J ¼ 7.6 Hz, H-30 and H-50). 8.20 (1H, bs, NH); 13C
NMR
d
88.4 (C-3 and C-21), 121.8 (C-15), 122.9 (C-20 or C-60), 125.9
(C-40), 129.0 (C-30 and C-50), 129.0 (C-20 or C-60), 136.8 (C-10), 138.5
(C-14),188.7 (C]S); MS (FAB) m/z: 614 (Mþ þ Na); HRMS calculated
for C38H57NO2SNa, 614.4008; found: 614.4013.
3.2.6. 3
b-Methoxyserrat-14-en-21b-yl chloroacetate (24)
To an ice-cooled mixture of compound 2 (228 mg, 0.5 mmol) and
pyridine (160
chloride (120
m
L, 2 mmol) in CHCl3 (5 mL) was added chloroacetyl
3.4. Animals
mL, 1.5 mmol) and the mixture was allowed to react at
room temperature for 40 min. To the reaction mixture were added
chilled H2O and dil. NaHCO3, and the whole was extracted with CHCl3.
The extractwaswashed withbrine, dried,andconcentratedtofurnish
a brown powder. The residue was purified by silica gel column
Specific pathogen-free female ICR mice (6 weeks old, body
weight: approx. 30 g) were obtained from Japan SLC Inc., Shizuoka,
Japan. Five animals were housed in each polycarbonate cage in
a temperature-controlled room at 24 ꢀ 2 ꢂC and given food and
water ad libitum throughout the experiment.
chromatography (CHCl3) to give compound 24 (206 mg, 77%) as
22
colorless crystals. mp 231–233 ꢂC (MeOH–CHCl3); [
a]
ꢃ65.8 (c
D
0.102, CHCl3);1HNMR
J ¼ 2.8 Hz, H-21
), 4.09 (2H, s, O]CeCH2eCl), 5.33 (1H, m, H-15); 13
NMR
d
2.63(1H, dd, J ¼ 11.8, 4.3 Hz,H-3
a),4.77(1H, t,
3.5. Two-stage mouse skin carcinogenesis test
a
C
d
41.3 (O]CeCH2eCl), 80.8 (C-21), 88.5 (C-3),121.8 (C-15),138.5
Animals were divided into three experimental groups of 15 mice
(C-14), 166.9 (C]O); MS (FAB) m/z: 555 (Mþ þ Na); HRMS calculated
each. The back (2 ꢁ 8 cm2) of each mouse was shaved with surgical
for C33H53ClO3Na, 555.3581; found, 555.3586.
clippers and topically treated with DMBA (100 mg, 390 nmol) in

H. Tsujii et al. / European Journal of Medicinal Chemistry 45 (2010) 2191–2197
2197
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