Anti-tumor promoting diterpenes from the stem bark of Thuja standishii (Cupressaceae)

Article abstract of DOI:10.1016/S0968-0896(01)00099-2

Three new labdane-type diterpenoids, labda-8(17), 13-dien-15,2R-olid-19-oic acid (1), 12S-hydroxylabda-8(17),13(16),14-trien-19-oic acid (2) and 13-ethoxylabda-8(17), 11,14-trien-19-oic acid (3), along with known diterpenoids, trans-communic acid (4), totarol (5), 12-methoxyabieta-8,11,13-trien-11-ol (6), and 7α,8α-epoxy-6α-hydroxyabieta-9(11),13-dien-12-one (7) were isolated from the stem bark of Thuja standishii. The structures of 1-3 were established by spectroscopic methods and chemical conversion. These compounds together with standishii. (8), 12-hydroxy-6,7-seco-abieta-8,1,13-trien-6,7-dial (9) and 6α-hydroxysugiol (10) were tested for their inhibitory efects on Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), as a test for potential cancer chemopreventive agents. Compound 10 showed strong inhibitory effect on EBV-EA induction. In addition, 15-oxolabda-8(17),11Z,13E-trien-19-oic acid (11) was found to exhibit the anti-tumor promoting activity in two-stage mouse skin carcinogenesis test using 7,12-dimethylbenz[a]anthracene and TPA. Copyright

Full text of DOI:10.1016/S0968-0896(01)00099-2

Bioorganic & Medicinal Chemistry 9 (2001) 1911–1921
Anti-tumor Promoting Diterpenes from the Stem Bark
of Thuja standishii (Cupressaceae)
Manabu Iwamoto,a Hironori Ohtsu,a Harukuni Tokuda,b Hoyoku Nishino,b
Shunyo Matsunagaa and Reiko Tanakaa,*
aDepartment of Medicinal Chemistry, Osaka University of Pharmaceutical Sciences,
4-20-1Nasahara, Takatsuki, Osaka 569-1094, Japan
bDepartment of Biochemistry, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto 602-8414, Japan
Received 1 March 2001; accepted 31 March 2001
Abstract—Three new labdane-type diterpenoids, labda-8(17),13-dien-15,12R-olid-19-oic acid (1), 12S-hydroxylabda-
8(17),13(16),14-trien-19-oic acid (2) and 13-ethoxylabda-8(17),11,14-trien-19-oic acid (3), along with known diterpenoids, trans-
communic acid (4), totarol (5), 12-methoxyabieta-8,11,13-trien-11-ol (6), and 7a,8a-epoxy-6a-hydroxyabieta-9(11),13-dien-12-one
(7) were isolated from the stem bark of Thuja standishii. The structures of 1–3 were established by spectroscopic methods and che-
mical conversion. These compounds together with standishinal (8), 12-hydroxy-6,7-seco-abieta-8,11,13-trien-6,7-dial (9) and 6a-
hydroxysugiol (10) were tested for their inhibitory effects on Epstein–Barr virus early antigen (EBV-EA) activation induced by 12-
O-tetradecanoylphorbol-13-acetate (TPA), as a test for potential cancer chemopreventive agents. Compound 10 showed strong
inhibitory effect on EBV-EA induction (100% inhibition at 1000 mol ratio/TPA), and compounds 2 and 6 showed moderate inhi-
bitory effects on EBV-EA induction. In addition, 15-oxolabda-8(17),11Z,13E-trien-19-oic acid (11) was found to exhibit the anti-
tumor promoting activity in two-stage mouse skin carcinogenesis test using 7,12-dimethylbenz[a]anthracene and TPA. # 2001
Elsevier Science Ltd. All rights reserved.
Introduction
this tree showed significant anti-tumor promoting
activity of in vivo two-stage mouse-skin carcinogenesis
assay using 7,12-dimethylbenz[a]anthracene (DMBA) as
an initiator and TPA as a tumor promotor.6
Cancer chemoprevention is regarded as one of the effi-
cient strategies for cancer prevention.1 Inhibition of the
tumor promotion stage in the multistage of chemical
carcinogenesis has been regarded as the most promising
method for cancer chemoprevention.2 In the search for
cancer chemopreventive agents, the inhibitory effects on
Epstein–Barr virus early antigen (EBV-EA) induction by
the tumor promotor, 12-O-tetradecanoylphorbol-13-ace-
tate (TPA), have been studied as a primary screening test.3
Recently, we reported that the novel carbon skeletal diter-
penoid, standishinal [6a,12-dihydroxy-6(7!11)abeo-
abieta-8,11,13-trien-7-al].4 and three new compounds, 15-
oxolabda-8(17),11Z,13E-trien-19-oic acid, 15-oxolabda-
8(17),11Z,13Z-trien-19-oic acid, and 12-oxo-11-nor-drim-
8-en-14-oic acid.5 from the stem bark of Thuja standishii
(Gord.) Carr (Cupressaceae), and 15,16-bisnor-13-oxo-
labda-8(17),11E-dien-19-oic acid from the stem bark of
Further careful examination of this extract has led to
the isolation of three new labdane-type diterpenoids, 1–
3, besides four known diterpenoids. The structures of
the new compounds (1–3) were determined on the basis
of spectroscopic data and chemical conversion. The
known compounds were identified as trans-communic
acid (4),7,8 totarol (5),9 12-methoxyabieta-8,11,13-trien-
11-ol (6),10 and 7a,8a-epoxy-6a-hydroxyabieta-9(11),13-
dien-12-one (7)11 by comparison of their spectral data
with those reported in the literature.
This paper deals with the structure determination of 1–3
and results of in vitro and in vivo anti-tumor promoting
activities of compounds 1–7, along with standishinal (8),
12-hydroxy-6,7-seco-abieta-8,11,13-trien-6,7-dial (9), 6a-
hydroxysugiol (10)4 and 15-oxolabda-8(17),11Z,13E-
trien-19-oic acid (11)5,6 using 7,12-dimethylbenz[a]an-
thracene and TPA.1
*Corresponding author. Tel.: +81-726-90-1084; fax: +81-726-90-
1084; e-mail: tanakar@oysun01.oups.ac.jp
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00099-2

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M. Iwamoto et al. / Bioorg. Med. Chem. 9 (2001) 1911–1921
1913

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M. Iwamoto et al. / Bioorg. Med. Chem. 9 (2001) 1911–1921
1
Results and Discussion
involving HMQC, HMBC, H/1H COSY and NOESY
spectra. In the HMBC spectrum (Fig. 1), H-12 was
correlated with C-9, C-11, C-13, C-14 and C-16. In the
1H–1H COSY spectrum (Fig. 1), H-12 oxymethine sig-
nal (dH 4.87) was correlated with H-11a and H-11b. On
the other hand, methylation of 1 with trimethylsilyldia-
zomethane gave a methyl ester (1a) and subsequent
alkaline hydrolysis furnished 12-oxo-13-methyl-15-oic
acid (1b) (Table 1) whose C-13 methyl group was epi-
meric mixture. The relative stereostructure was deter-
mined by NOESY spectrum (Fig. 2). Hence, compound
1 was proved to be labda-8(17),13-dien-15,12-olid-19-
oic acid, and the configuration of C-12 was determined
as R because the negative cotton effect curve was
observed at 214 nm (ÁeÀ32.4).12 To the best of our
knowledge, evillosin is a labdane-type diterpenoid pos-
sessing a 15,12-lactone ring was isolated from Eupa-
trium villosum (Compositae),13 thus, compound 1 was
the second instance of labdane-type diterpene having a
15,12-lactone.
The CHCl3 extract of the stem bark of T. standishii was
carefully chromatographed over Si gel, Sephadex LH-
20, and then fractionated by medium-pressure liquid
chromatography to give three new (1–3) and four
known diterpenoids (4–7). The known compounds were
confirmed as trans-communic acid (4),7,8 totarol (5),9
12-methoxyabieta-8,11,13-trien-11-ol (6),10 and 7a,8a-
epoxy-6a-hydroxyabieta-9(11),13-dien-12-one
(7).11
Compound 4 was reported in ref 6, but no chemical
description was written, in this study, we synthesized
compound 2 from compound 4.
The molecular formula of compound 1 was assigned as
C20H28O4 based on HREI-MS. The IR spectrum of 1
exhibited bands indicative of terminal methylene group,
ab-unsaturated g-lactone ring and carboxyl group. The
1H and 13C NMR spectra (Table 1) exhibited two ter-
tiary methyl groups, six methylenes, two methine
groups, an oxymethine [dH 4.87 (d); dC 83.4 (d)], a b-
methyl-ab-unsaturared g-lactone [dH 2.10(3H, brs),
5.78 (quintet); dC 13.9 (q), 116.5 (d), 169.3 (s), 173.1 (s)],
a terminal methylene group [dH 4.44 (s), 4.94 (d); dC
106.3 (t), 147.9 (s)] and a carboxyl group [dC 183.3 (s)].
Its EI-MS (Fig. 1) showed a prominent ion peak at m/z
98 (b-methyl-ab-unsaturated-g-lactone) and a base ion
peak at m/z 235 corresponding to a M+Àb-methyl-ab-
unsaturared-g-lactone moiety. The gross structure of 1
was established by extensive 2D NMR experiments
Compound
2
was isolated as the N-(chloro-
methyl)phthalimide (NMP) ester14 of 2 (2a), and its
molecular formula was assigned as C20H29O3 [m/z
317.2111, M+ÀN-(methyl) phthalimide)] by HREI-MS.
The IR spectrum showed absorption bands for exo-
cyclic methylene group and hydroxyl group. The 1H and
13C NMR spectra (Table 2) showed two tertiary methyl
groups, six methylene groups, two methine groups, two
sp3 quaternary carbons, a hydroxymethine [dH 4.41
Table 1. 1H and 13C NMR data for compounds 1 and 1b (CDCl3)a
1
1b
Position
dC
dH
dC
dH
1a
1b
2a
2b
3a
3b
4
38.6t
1.20 ddd (4.0, 13.0, 13.3)
39.15 t, 39.19 t
1.15 ddd (4.0, 13.0, 13.3)
1.64 m
1.51 m
1.81 m
1.63 m
1.52 m
1.84 m
19.7 t
37.7 t
19.9 t
38.1 t
1.07 ddd (4.0, 13.5, 13.5)
2.15 m
1.06 ddd (4.0, 13.5, 13.5)
2.18 m
44.3 s
55.9 d
25.9 t
44.3 t
55.96 d, 55.98 d
25.76 t, 25.84 t
5
1.42 m
2.10m
1.92 m
2.44 m
1.98 m
1.41 t (3.0), 1.43 t (3.0)
6a
6b
7a
7b
8
2.01 m
1.78 m
2.41 m
2.06 m
38.4 t
38.01 t, 38.04 t
147.9s
51.7 d
148.4s, 148.8s
49.7 d, 50.1 d
9
2.22 brd (11.5)
39.43 s, 39.46 s
1.44 m
2.45 brd (11.0)
1040
11A
11B
12
13
14
28.0t
36.8 t, 37.0t
2.27 m
2.76 m
1.93 m
4.87 d (11.0)
83.4 d
169.3 s
116.5 d
177.7 s
41.9 d, 42.4 d
37.5 t, 37.8 t
3.06 m
2.66 m
2.74 m
5.78 quintet (1.0)
15
16
17A
17B
18
19
20
OMe
OMe
173.1 s
13.9 q
106.3 q
211.8 s, 211.9 s
17.1 q, 17.4 q
105.9 t, 106.5 t
2.10 brs
4.94 d (1.0)
4.44 s (11.0)
1.26 s
1.14 d (7.0), 1.15 d (7.0)
4.75 d (1.0), 4.77 brs
4.32 brs, 4.39 brs
1.20s
29.0q
183.3 s
12.8 q
28.8
172.71 s, 172.75 s
12.95 q, 12.96 q
51.2 q
0.61 s
3.62 s
3.639 s, 3.642 s
0.54 s
51.7 q
aOperated at 500 and 125 MHz for 1H and 13C NMR experiments, respectively; d in ppm, J (in parentheses) Hz; assignments made from 1H–1H
COSY, HMQC, HMBC, and NOESY data.

M. Iwamoto et al. / Bioorg. Med. Chem. 9 (2001) 1911–1921
1915
(dd), dC 72.4 (d)], three exocyclic methylene groups [dH
4.70(brs), 4.88 (d), dC 106.9 (t), 148.7 (s); dH 5.11 (d),
5.15 (brs), dC 115.0(t), 149.2 (s); dH 5.10(d), 5.41 (dd),
dC 115.1 (t), 135.7 (d)], and a NMP ester group [dH 4.69
(d), 5.64 (d), 7.79 (m), 7.93 (m); dC 60.6 (t), 123.9 (d),
131.7 (s), 134.6 (d), 166.7 (s)]. The above data showed 2
should be a myrcerocommunic acid15 derivative having
a secondary hydroxyl group. The secondary hydroxyl
group was attached at C-12 based on the cross peaks
between C-12 with H-9a, H-11a, H-11b, H-16A and H-
16B in the HMBC spectrum, and cross peaks between
The result determined the S configuration for the
asymmetric center at C-12, and consequently, led to the
absolute stereo structure of 2.
Compound 3 was also isolated as the NMP ester of 3
(3a), and its molecular formula was assigned as
C22H34O3 (m/z 345.2431, M+ÀN-(methyl)-phthalimide)
by HREI-MS. The IR spectrum showed absorption
bands for cis-ditsubstituted olefin, exocyclic methylene
1
groups, ester group and ether oxygen. The H and 13C
NMR spectra (Table 3) showed three tertiary methyl
groups, five methylene groups, two sp3 methine groups,
three sp3 quaternary carbons, Á11,12-disubstituted olefin
[dH 5.607 (dd), 5.45 (d) dC 128.4 (d), 136.63 (d); dH 5.614
(dd), 5.45 (d), dC 128.5 (d), 136.64 (d)], two exocyclic
methylene groups [dH 4.72 (d), 4.41 (d), dC 149.4 (s),
108.0 (t); dH 4.72 (d), 4.45 (d), dC 108.1 (t), 149.5 (s)], a
tertiary ethoxy group [dH 1.15 (2H, t), 3.367 (3H, quar-
tet), dC 16.0(q), 58.2 (t); dH 1.16 (2H, t), 3.370(3H,
quartet), dC 16.0(q), 58.3 (t)] and a NMP ester group
[dH 5.65 (d), 5.71 (d), 7.80(m), 7.94 (m), dC 60.9 (t),
123.9 (d), 131.7 (d), 134.6 (d), 166.7 (s)]. An ethoxy
group was attached at C-13, since C-13 correlated with
H-10, H-11, H-12, H-14, H-15 and Me-16 in the HMBC
spectrum (Table 3). Compound 3 gave a mixture of two
stereoisomers on ethoxy groups at C-13S and C-13R as
is shown in Table 3.
1
H-12 with H-11a and H-11b in the H–1H COSY spec-
trum of 2a (Fig. 3). In the NOESY spectrum of 2a
(Table 2) NOEs were observed for Me-18 with H-3a, H-
5a, and for Me-20with H-1 b, H-2b, H-11b, since the
carboxylic acid should be placed at C-19. Accordingly,
the structure of 2 was assumed to be 12-hydroxy-labda-
8(17),13(16),14-trien-19-oic acid.
The absolute stereo structure was determined by the
synthesis of 2a from trans-communic acid methyl ester
(4a) and the use of the modified Mosher method. M-
CPBA oxidation of 4a gave a 12,13-epoxy derivative
(4b), and subsequent treatment with aluminium iso-
proxide16 afforded compounds 4c and 12-epi hydroxyl
derivative (4d). Compound 4c was hydrolysed with
potassium t-butoxide to give compound 4e, and then
esterified with N-(chloromethyl)phthalimide to give
NMP ester (4f). The above synthetic 4f was identical
with 2a in all respects. On the other hand, the absolute
configuration of C-12 hydroxyl group was determined by
the modified Mosher method. The (S)- and (R)-2-meth-
oxy-2-phenyl-2-(trifluoromethyl)-acetic acid (MTPA)
esters (4g and 4h) were prepared from 4a by a standard
method.17 The 1H chemical-shift differences between (S)-
and (R)-MTPA esters 4g and 4h) are shown in Figure 4.
Antitumor-promoting activity
Compounds 1–10 were tested for their inhibitory effects
on Epstein–Barr virus early antigen (EBV-EA), induced
by the tumor promotor, TPA, in Raji cells. Their inhi-
bitory effects on the activation of the early antigen and
the viability of Raji cells are shown in Table 4.
The potency of compound 10 was stronger than that of
a representative control b-carotene18 at every con-
centration. Compounds 2 and 11 showed strong inhibi-
tory effect on EBV-EA induction, even at 10mol ratio/
TPA (100% inhibition activity at 1000 mol ratio/TPA,
and more than 25% even at 100 mol ratio/TPA, respec-
tively), and compounds 5 and 6 showed moderate inhi-
bitory effects (more than 28% even at 100 mol ratio/
TPA, respectively) on EBV-EA induction. The viability
percentages of Raji cells treated with the test
compounds (1–11) were mostly 60% at the highest con-
centration of 1000 mol ratio/TPA; indicating that the
cytotoxicities of these compounds seem to be con-
siderably moderate against in vitro cell lines (Table 4).
Figure 1. EI-MS, 1H–1H COSY and HMBC correlations for 1.
Figure 2. NOESY correlations for 1.

1916
M. Iwamoto et al. / Bioorg. Med. Chem. 9 (2001) 1911–1921
Table 2. 1H, 13C and NOESY data for compound 2 (CDCl3)a
Position
dC
38.8 t
dH
NOESY
1a
1b
2a
2b
3a
3b
0.93 m
1.72 m
1.46 m
1.78 m
2.13 m
1.00 m
9a
20
1a, 2a
19.9 t
37.9 t
18
4
5
44.5 s
56.4 d
1.26 m
1.93 m
1.75 m
1.80m
6a, 9a, 18
5a, 18
6a
6b
7a
7b
26 t
7b
38.6 t
5a
6a, 6b, 17A
2.36 m
8
9
1040
148.7 s
52.5 d
1.54 m
7a, 11b, 12R
11A
11B
12
13
14
15A
15B
16A
16B
17A
17B
18
30.7 t
1.73 m
1.81 m
4.41 dd (6.0, 8.5)
1a, 17A
18
9a, 15A, 15B
72.4 d
149.2 s
135.7 d
115.1 t
6.32 ddd (1.0, 11.0, 17.5)
5.10d (11.0)
5.41 dd (1.0, 18.0)
5.11 d (1.5)
5.15 brs
4.70 brs
4.88 d (1.5)
1.18 s
15A, 15B, 16B
14
12, 14, 16B
115.0t
9 a, 15
14, 15
11A, 12R
7b
106.9 t
28.8 q
176.0s
3a, 5a, 6a, 7b
19
.5120s12.7 q
N-(Chloromethyl)phthalimide
1 0
b, 2b, 11b, 17A, 17B
1b, 2b, 11b, 17A, 17B
60.6 t, 123.9 d, 131.7 s,
134.6 d, 166.7 s
5.64 d (10.5), 4.69 d (10.5),
7.79 m, 7.93 m
aOperated at 500 and 125 MHZ for 1H and 13C NMR experiments, respectively; d in ppm, J (in parentheses) Hz; assignments made from 1H–1H
COST, HMQC, HMBC, and NOESY data.
was obtained as a minor product. No significant toxic
effects, such as inflammation and lesional damages, on
the areas of mouse skin topically treated with the test
compound was observed, except for the formation of
papillomas, at the end of the treatment, and also the
body weight gains was not influenced during the treat-
ment. As demonstrated in Figure 5, the percentage of
papilloma bearers in the control group (DMBA and
TPA only) increased rapidly from week 6 and reached
100% after week 8, whereas the treatment with com-
pound 11 along with the initiator and the promotor
reduced the percentage of papilloma-bearing mice to
approximately 25% during weeks 7–11 and thereafter
73.3% over the period of week 20. As shown in Figure
6, in the control group, the number of papillomas per
mouse formed increased rapidly after 6 and reached
10.0 papillomas/mouse at week 20. On the other hand,
the mice treated with compound 11 bore 2.4 papillomas
over the period of week 20.
Konishi et al. reported the inhibitory effect of a beyer-
ane-type diterpene, ent-3b-hydroxy-15-beyeren-2-one,
isolated from Excoecaria agallocha in the in vivo test
under similar experimental conditions described above;
when treated with ent-3b-hydroxy-15-beyeren-2-one, the
percentage of papilloma-bearing mice was approxi-
Figure 3. 1H–1H COSY and HMBC correlations of 2a.
The results of the in vitro experiments prompted us to
examine the effect of compound 11 on the in vivo two-
stage carcinogenesis bioassay on mouse skin using
DMBA as an initiator and TPA as a promotor. Com-
pound 2 was not tested in this in vivo assay because it
mately 70% and the number of papilloma/mouse was
18
about 4.0at week 18.
Thus, as demonstrated above,
the in vivo anti-tumor promoting activity of compound
11 seemed to be stronger than that of the beyerene
diterpenoid.

M. Iwamoto et al. / Bioorg. Med. Chem. 9 (2001) 1911–1921
1917
The labdane or abietane-type diterpenoids including
Experimental
compound 11 and 15,16-bisnor-13-oxolabda-8(17),11E-
dien-19-oic acid6 can be considered to become appro-
priate lead compounds for developing more potent
agents with anti-tumor promoting activity for clinical
employment.
General
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. UV spectra were recorded
using a Hitachi 150-20 spectrophotometer. IR spectra
were recorded using a Perkin-Elmer 1720X FTIR spec-
trophotometer. 1H and 13C NMR spectra were obtained
on a Varian INOVA 500 spectrometer with standard
pulse sequences, operating at 500 and 125 MHz, respec-
tively. CDCl3 was used as the solvent and TMS as the
internal standard. EIMS were recorded on a Hitachi
4000H double-focusing mass spectrometer (70 eV).
Column chromatography was carried out over Si gel
(70–230 mesh, Merck) and medium-pressure liquid
chromatography (MPLC) was conducted with Si gel
(230–400 mesh, Merck) and Cosmocil 40C18-PREP
(ODS, Nacalai Tesque). Fractions obtained from col-
umn chromatography were monitored by TLC (silica
gel 60F 254, Merck). Preparative TLC was carried out
on Merck silica gel F254 plates (20Â20cm, 0.5 mm
thick).
Plant material
The stem bark of T. standishii (Gord.) Carr. was col-
lected at Hashimoto City, Wakayama Prefecture,
Japan, in September 1995. A voucher specimen (TS-95-
01) is deposited at the Herbarium of the Laboratory of
Figure 4. 1H Chemical-shift differences (Ád=dSÀdR) between the (S)-
and (R)-MTPA esters 4g and 4h of 4a. Ád values are expressed in Hz
(500 MHz).
Table 3. 1H, 13C and HMBC NMR data for compounds 3a (CDCl3)a
Position
dC
dH
HMBC (C!H)
2a, 3b
1a
1b
2a
2b
3a
3b
19.7 t
1.43 m
1.75 m
1.03 m
1.51 m
2.16 m
1.04 m
40.6 t
38.1 t
20
18
4
5
6a
6b
44.4 s
55.9 d
24.9 t
2a, 3b, 5, 18
1 b, 6b, 18, 20
18
1.30dd (2.5, 7.5)
1.93 m
1.77 m
7a
7b
37.2 t
1.98 m
2.40m
17A, 17B
8
149.4 s, 149.5 s
60.2 d
7a, 7b, 9
12, 17A, 17B, 20
b, 5, 9, 20
9
1039.2 s, 39.3 s
2.30 brd (10.0)
1
11
12
13
14
15A
15B
16
17A
128.4 d, 128.5 d
136.63 d, 136.64 d
77.6 s, 77.7 s
142.5 d, 142.6 d
113.7 t, 113.8 t
5.607 dd (10.0, 16.0), 5.1614 dd (10.0, 16.0)
5.45 d (16.0)
9, 12
11, 16
10, 11, 12, 14, 15A, 15B, 16
10, 12, 15B
16
5.871 dd (11.0, 17.5), 5.875 dd (11.0, 17.5)
5.107 dd (1.0, 10.5), 5.108 (1.0, 10.5)
5.16 dd (1.0, 17.5), 5.17 dd (1.0, 17.5)
1.36 s
24.6 q, 24.7 q
108 t, 108.1 t
12
6a, 7b, 9
4.72 d (1.5)
4.41 d (1.5), 4.45 d (1.5)
1.21 s
17B
18
28.7 q
176.1 s
2a, 3b
2a, 3b, 5, 18
19
2013.41 q, .1634.4s3 q
0
2
a, 5, 9
10
58.2 t, 58.3 t
16.0 (2H) q
60.9 t, 123.9 d, 131.7 d,
134.6 d, 166.7 s
3.367 quartet (7.0), 3.370 quartet (7.0)
1.15 t (7.0), 1.16 t (7.0)
5.65 d (10), 5.71 d (10), 7.80 m, 7.94 m
20
10
20
N-(Chloromethyl)phthalimide
aOperated at 500 and 125 MHz for 1H and 13C NMR experiments, respectively; d in ppm, J (in parentheses) Hz; assignments made from 1H–1H
COSY, HMQC, HMBC, and NOESY data.

1918
M. Iwamoto et al. / Bioorg. Med. Chem. 9 (2001) 1911–1921
Medicinal Chemistry, Osaka University of Pharmaceu-
tical Sciences.
derived from Burkitts lymphoma) were cultured in 10%
fetal bovine serum (FBS) in RPMI-1640medium (Nis-
sui). 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, 1Â106/mL) were incubated at 37 ꢀC for
48 h in 1 mL of a medium containing n-butyric acid
(4 mmol), TPA (32 pmol=20ng in dimethylsulfoxide
(DMSO), as inducer and various amounts of test com-
pound in 5 mL DMSO. Smears were made from the cell
suspension, and the activated cells that were stained by
EBV-EA positive serum from NPC patients were detec-
ted by an indirect immunofluorescence technique.2 In
each assay, at least 500 cells were counted, the number
of stained 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 expres-
sed as a relative ratio to the control experiment (100%)
which was carried out only with n-butylic acid (4 mmol)
plus TPA (32 pmol). EBV-EA induction was ordinarily
around 35%. The viability of treated Raji cells was
assayed by the Tripan Blue staining methods.
Bioassays
Inhibition of EBV-EA activation test. EBV-EA positive
serum from a patient with nasopharyngeal carcinoma
(NPC) was a gift from Dr. Y. Zaizen, the Department
of Biochemistry, Oita Medicinal University. The EBV
genome-carrying lymphoblastoid cells (Raji cells
Table 4. Relative ratioa of EBV-EA activation with respect to posi-
tive control (100%) in the presence of compounds 1–11 and related
compounds (1a, 3a, 8a)
Concentration (mol ratio/TPA)b
Compounds
1000
500
100
10
% to control (% viability)
1
1a
2
3a
4
5
6
7
8
8a
9
10
19.4(60)c
26.6(60)
0(60)
64.8
67.9
50.6
55.9
46.2
40.4
30.7
50.9
68.0
69.5
54.0
22.7
45.1
34.0
84.1
88.5
72.7
87.4
75.3
71.5
68.4
88.9
83.3
88.9
82.7
63.9
72.0
82.0
100.0
100.0
92.6
100.0
90.5
89.9
97.0
100
100
100
100
92.6
88.3
100.0
13.4(60)
4.4(60)
3.5(70)
2.3(70)
3.1(70)
14.1(60)
15.5(60)
11.7(70)
0(70)
Two-stage mouse-skin carcinogenesis test
Specific pathogen-free female ICR mice (6 weeks old
body weight approx. 30g) were obtained from Japan
SLC Inc., Shizuoka, Japan, and the animals were
housed, 5 per polycarbonate cage, in a temperature-
controlled room at 24Æ2 ꢀC and given food and water
ad libitum throughout the experiment. Animals were
divided into three experimental groups containing 15
mice each. The back (2Â8 cm2) of each mouse was
11
b-Carotene
0(60)
9(90)
aValues represent percentages relative to the positive control value
(100%).
bTPA concentration was 20ng/ml (32 pmol/mL).
cValues in parentheses are the viability percentages of Raji cells.
Figure 6. Inhibition of TPA-induced tumor promotion by multiple of
15-oxolabda-8(17),11Z,13E-trien-19-oic acid (11). All mice initiated
with DMBA (390nmol) and promoted with 1.7 nmol of TPA, given
twice weekly starting 1 week after initiation. Average number of
papillomas per mouse. *, Control (TPA alone); ~, TPA+85 nmol of
11. Papillomas per mouse of 11 treatment were significantly different
from the positive control at 20weeks after promotion, p<0.005.
Figure 5. Inhibition of TPA-induced tumor promotion by multiple
application of 15-oxolabda-8(17),11Z,13E-trien-19-oic acid (11). All
mice were initiated with DMBA (390nmol) and promoted with
1.7 nmol of TPA, given twice weekly starting 1 week after initiation.
Percentage of mice bearing papillomas. *, Control (TPA alone); ~,
TPA+85 nmol of 11.

M. Iwamoto et al. / Bioorg. Med. Chem. 9 (2001) 1911–1921
1919
shaved with surgical clippers, and the mice were topi-
cally treated with DMBA (100 mg, 390nmol) in acetone
(0.1 mL) as an initiating treatment. One week after the
initiation, papilloma formation was promoted twice
weekly by the application of TPA (1 mg, 1.7 nmol) in
acetone (0.1 mL) to the skin. One hour before each
treatment, the mice were treated with the samples
(85 nmol) in acetone (0.1 mL). The incidence of papillo-
mas was examined weekly over a period of 20weeks.
12.6 (q, C-20), 13.9 (q, C-16), 19.8 (t, C-2), 26.1 (t, C-6),
28.0(t and s C-11 and C-18), 28.8 (q, C-18), 38.0(t, C-
3), 38.5 (t, C-7), 38.7 (t, C-1), 40.0 (s, C-10), 44.3 (s, C-
4), 51.2 (q, OMe), 55.9 (d, C-5), 83.4 (d, C-12), 106.2 (t,
C-17), 116.5 (d, C-14), 148.1 (s, C-8), 169.3 (s, C-13),
173.1 (s, C-15), 177.6 (s, C-19); EI-MS m/z (rel. int.) 346
(18) [M]+, 286 (25), 249 (75), 217 (26), 189 (100), 161
(16), 147 (14), 133 (22), 121 (93), 107 (28), 41 (17).
Alkaline hydrolysis of 1a. Compound 1 (5.1 mg) was
refluxed with a solution of 1 N KOH/MeOH on a steam
bath for 24 h. Evaporation of the solvent under reduced
pressure afforded a residue which was purified by PTLC
(n-hexane–CHCl3, 1:2) to afford compound 1b (5.0mg).
Colorless oil; [a]D25 À34 (c 0.18, CHCl3); IR (CHCl3)
nmax cmÀ1 2932, 2874, 2850, 2360, 2243, 1723
(COOMe), 1645 and 989 (>C¼CH2), 1462, 1379, 847;
1H and 13C NMR, see Table 1; EI-MS m/z (rel. int.) 378
(34) [M]+, 346 (15), 287 (15), 234 (49), 175 (24), 129
(100), 121 (48), 59 (31).
Extraction and isolation. Preliminary Si gel column
chromatography of the CHCl3 extract (558.8 g) of the
chopped stem bark (5.3 kg) of T. standishii has been
reported previously, with separation into 13 (residues
A–M) fractions.5 Residue C (fraction nos 9–11, 28.7 g)
was rechromatographed over Si gel (1 kg) eluting with
CHCl3 to give compound 4 (12.78 g). Twice rechroma-
tography over Si gel (500 and 100 g) of residue E (frac-
tion nos 21–31, 15.4 g) eluting with CHCl3 gave a crude
oil (17.6 mg) followed by esterification with N-(chlor-
omethyl)phthalimide (NCMP) followed by preparative
TLC with CHCl3/MeOH (50:1) to give compound 2
NMP ester (2a) (4.1 mg). Repeated CC of residue G
(fraction nos 47–53, 53.2 g) over Si gel, Sephadex LH-
20, and ODS column chromatography has already
reported,5 and afforded 15-oxolabda-8(17),11(Z),13(E)-
trien-19-oic acid, 15-oxolabda-8(17),11(Z),13(Z)-trien-
19-oic acid, and 15-nor-14-oxolabda-8(17),12(E)-dien-
19-oic acid. At this stage, we had collected diterpene
mixture (residue c, fraction nos 31–33, 155.0mg), which
was subjected to preparative TLC with CHCl3/MeOH
(50:1) to afford compound 1 (50.4 mg). Residue H
(fraction nos 54–59, 15.5 g) was rechromatographed
over silica gel eluting with n-hexane/EtOAc (50:1) to
yield a crude oil (10.6 mg), which was esterification with
N-(chloromethyl)phthalimide, and this was purified by
HPLC with ODS, eluting with 85% MeOH to give
compound 3-NMP ester (3a) (4.3 mg).
Esterification of 12S-hydroxylabda-8(17),13(16),14-trien-
19-oic acid (2) with N-(chloromethyl)phthalimide. A
mixture of crude compound 2 (17.6 mg) in MeCN
(0.5 mL) solution and Et3N (24 mg) and N-(chlor-
omethyl)phthalimide (35.6 mg) was stirred at 60 ꢀC for
1 h. Evaporation of the solvent under reduced pressure
afforded a residue which was purified by PTLC (n-hex-
ane–CHCl3–EtOH, 10:1:1) followed by HPLC (ODS)
using 70% CH3CN to give compound 2a. Colorless oil;
[a]2D5 +13 (c 0.15, CHCl3); HR-EI-MS m/z 317.2111
(C20H29O3, M+ÀN-(methyl) phthalimide, calcd for m/z
317.2115); UV lmax nm 218, 292 (log e 4.4, 3.1); IR
(CHCl3) nmax cmÀ1 3650(OH), 1733 (–COO), 1637 and
902 (>C¼CH2); 1H and 13C NMR, see Table 2; EI-MS
m/z (rel. int.): 446 (1) [M]+, 400 (1), 317 (2) [MÀN-
(methyl)phthalimide]+, 299 (5) [317–H2O], 271 (3), 253
(3), 160 (100).
Labda-8(17),13-dien-15,12R-olid-19-oic acid (1). Col-
ourless oil; [a]2D5 À150( c 0.72, CHCl3), HR-EI–MS m/z
332.1981 [M]+ (C20H28O4 requires 332.1986), IR nmax
cmÀ1 2942 br and 1693 (COOH), 1760and 1693 ( ab-
Esterification of 13-ethoxylabda-8(17),11,14-trien-19-oic
acid (3) with N-(chloromethyl)phthalimide. A mixture of
crude compound 3 (12.6 mg) in MeCN (0.4 mL) solu-
tion and Et3N (18 mg) and N-(chloromethyl)phthalimide
(22.4 mg) was stirred at 60 ꢀC for 1 h. Evaporation of
the solvent under reduced pressure afforded a residue
which was purified by HPLC (ODS) using 85% MeOH
to give compound 3a. Colorless oil; [a]2D5 À5 (c 0.15,
CHCl3); HR-EI-MS m/z 505.2836 (C31H39NO5, calcd
for 505.2826), m/z 345.2431 (C22H33O3, M+ÀN-
(methyl)-phthalimide, calcd for m/z 345.2428); UV lmax
nm 217, 283 (log E 4.5, 3.3); IR (CHCl3) nmax cmÀ1 1733
1
unsaturated g-lactone), 1643 and 960( >C¼CH2); H
and 13C NMR, see Table 1; EI-MS m/z (rel. int) 332
[M]+ (4), 286 (6), 235 (100), 217 (17), 189 (72), 121 (47),
98 (53), 81 (53), 41 (42). CD nm (c 4.156Â10À5 M,
EtOH) (Áe) 238 (0), 214 (À32.4).
Methyl labda-8(17),13-dien-15,12R-olid-19-oate (1a). A
MeOH (2 mL) and C6H6 (1 mL) solution of compound
1 (14.7 mg) was added a trimethylsilyldiazomethane
2.0M solution in n-hexane (TMSCHN2) (0.2 mL) for
20h at room temperature. Evaporation of the solvent
under reduced pressure afforded a residue which was
purified by PTLC (n-hexane–CHCl3, 1:2) to afford
compound 1a (5.1 mg). Colorless powder; [a]2D5 À69 (c
0.10, CHCl3); IR (CHCl3) nmax cmÀ1 2342, 1762 (ab-
unsaturated g-lactone), 1723 (COOMe), 1645 and 959
(>C¼CH2), 1309, 1134, 1094, 1076, 1043, 959, 915,
1
(–COO), 1632 and 891 (>C¼CH2), 1222 (–C–O); H
and 13C NMR, see Table 3; EI–MS m/z (rel. int.) 505 (3)
[M]+, 490(1) [M ÀMe]+, 459 (1), 345 (4)[MÀN-
(methyl)phthalimide]+, 300 (7), 160 (100), 99 (40).
Methylation of trans-communic acid (4). A solution of
diazomethane in ether was added to a solution of crude
compound 4 (2.55 g) in ether (100 mL), and the mixture
was allowed to stand over night. The removal of the
solvent gave a residue (2.66 g) which was purified by Si
gel CC (n-hexane–EtOAc, 5:1), and recrystallized from
MeOH–ether to give 4a (1.67 g). [a]2D5 +48 (c 1.26,
1
875; H NMR d 0.51 (3H, s, H-20), 1.20 (3H, s, H-18),
2.10(3H, m, H-16), 3.62 (3H, s, COOMe), 4.43 (1H, s,
H-17A), 4.94 (1H, d, J=1.5 Hz, H-17B); 13C NMR d

1920
M. Iwamoto et al. / Bioorg. Med. Chem. 9 (2001) 1911–1921
CHCl3); IR (CHCl3) nmax cmÀ1 1644 and 889
1 h. Evaporation of the solvent under reduced pressure
afforded a residue which was purified by PTLC (n-hex-
ane–CHCl3–EtOH, 10:1:1) to give compound 4f. Col-
orless oil; [a]2D5 +13 (c 0.08, CHCl3). The synthetic 4f
was identified by direct comparison with an authentic
sample of 2a.
1
(>C¼CH2), 1446, 1385, 1318, 1229, 1153; H NMR: d
1.19 (3H, s, H-18), 3.62 (3H, s, OMe).
M-CPBA oxidation of compound 4a. A CH2Cl2 solution
of compound 4a (402.5 mg) was gradually added to a
80% m-CPBA (411.3 mg) and NaHCO3 (160.2 mg) with
stirring at À10 ꢀC for 1.5 h. The reaction mixture was
washed with saturated NaHCO3 and H2O. Evaporation
of the solvent under reduced pressure afforded a residue
(457.1 mg) that was purified by silica gel CC (n-hexane–
EtOAc, 5:1) to yield 12,13-epoxylabda-8(17),14-dien-19-
oic acid (4b) (281.5 mg), C20H30O3, [a]2D5 +35 (c 0.19,
CHCl3); IR (CHCl3) nmax cmÀ1 3583, 2945, 2874, 2849,
1727 (COOMe), 1644 and 891 (>C¼CH2), 1230, 1154,
Formation of the (S)- and (R)-MTPA ester 4e and 4f
from 4a. (S)-MTPA (4.0mg), dicyclohexylcarbodiimide
(DCC)
(3.5 mg)
and
4-(dimethylamino)pyridine
(DMAP) (1.5 mg) were added to a dry CH2Cl2 solution
(200 mL) of compound 4a (1.4 mg), and the reaction
mixture was left at room temperature for over night.
The solvent was evaporated off under reduced pressure,
and the residue was purified by HPLC (ODS) using
95% MeOH to afford (S)-MTPA ester of 4a (4g)
(1.0mg). The same reaction with 4a (1.8 mg) using (R)-
MTPA ester (3.8 mg) gave (R)-MTPA ester of 4a (4h)
(0.5 mg).
1
1135; H NMR: d 3.74 (m), 4.28 (m); EI-MS m/z (rel.
int.) 332 (2) [M]+, 317 (3), 249 (9), 203 (19), 189 (35),
161 (21), 121 (100), 107 (31), 93 (34).
Synthesis of methyl-12(R), (S)-hydroxylabda-8(17),
13(16),14-trien-19 oate (4c), (4d) from 4b. A mixture of
dry toluene solution (10mL) of compound 4b (65.5 mg)
and Al(O-i-Pr)3 (56.2 mg) was refluxed for 4.5 h. Eva-
poration of the solvent under reduced pressure afforded
a residue which was resolved in EtOAc (25 mL) and the
organic layer was washed with 1 M HCl (15 mL), H2O
and NaHCO3. The crude residue (65.4 mg) was purified
by PTLC (n-hexane–ether, 4:1), followed by HPLC (ODS)
eluting with 85% MeOH to give methyl-12S-hydro-
xylabda-8(17),11Z,13(16)-trien-19-oate (4c) (13.1 mg) and
methyl-12R-hydroxylabda-8(17),11Z,13(16)-trien-19-oate
(4d) (6.6 mg). (4c): C21H32O3; [a]2D5 +41 (c 0.70, CHCl3);
IR (CHCl3) nmax cmÀ1 1725 (COOMe), 1645 and 901
1
(S)-MTPA ester (4g). Amorphous powder; H NMR: d
1.16 (3H, s, H-18), 5.13 and 5.43 (each 1H, d, J=11.5,
17.5 Hz, H-15), 5.75 (1H, dd, J=4.0, 11.0 Hz, H-12),
6.29 (1H, ddd, J=1.0, 11.5, 18.0 Hz, H-14).
1
(R)-MTPA ester (4h). Amorphous powder; H NMR: d
1.16 (3H, s, H-18), 5.06 and 5.36 (each 1H, d, J=11.5,
17.5 Hz, H-15), 5.69 (1H, dd, J=4.5, 11.0Hz, H-12),
6.22 (1H, dd, J=11.5, 18.0Hz, H-14).
Acknowledgements
1
(>C¼CH2), 1466, 1449, 1229, 1154, 1134. H NMR :d
The authors are grateful to Mr. M. Fujiwara, National
Osaka Forestry Bureau, 1-8-75 Temmabashi, Kita-ku,
Osaka 530–0042, Japan, for the supply of the plant
material. Thanks are also due to Mrs. M. Fujitake and
Mr. K. Minoura of this University, for MS and NMR
measurements.
0.51 (3H, s, H-20), 1.17 (3H, s, H-18), 4.44 (1H, dd,
J=5.4, 8.1 Hz, H-12R), 4.74 and 4.91 (each 1H, s, H-
17), 5.12 (1H, d, J=11.4 Hz, H-15), 5.14 and 5.17 (each
1H, s, H-16), 5.43 (1H, d, J=18 Hz, H-15), 6.34 (1H,
dd, J=11.1, 18.0Hz, H-14); EI-MS m/z (rel. int.) 332
(7) [M]+, 328 (10), 314 (7), 299 (7), 255 (14), 189 (21),
121 (100), 81 (37), 52 (39). (4d): C21H32O3; [a]2D5 +41 (c
0.27, CHCl3); IR (CHCl3) nmax cmÀ1: 1724 (COOMe),
References and Notes
1
1645 and 891 (>C¼CH2), 1448, 1229, 1154, 1036. H
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(1H, dd, J=5.7, 11.4 Hz, H-12S), 4.53 (1H, s, H-17),
4.89 (1H, d, J=1.5 Hz, H-17), 5.12 (1H, d, J=11.0Hz,
H-15), 5.14 and 5.21 (each 1H, s, H-16), 5.42 (1H, d,
J=18 Hz, H-15), 6.34 (1H, dd, J=11.0, 18.0 Hz, H-14);
EI-MS m/z (rel. int.) 332 (7) [M]+, 314 (21), 262 (21),
189 (21), 132 (32), 121 (100), 81 (37), 52 (39).
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(0.5 mL) and H2O (0.2 mL) at 0 ꢀC, and then stirred at
room temperature for 26 h. Work up as described above
gave a residue which was purified by HPLC (ODS, 80%
CH3CN) to afford compound 4e (1.1 mg). Colorless oil;
EI-MS m/z 318 [M+, C20H30O3].
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