Potential antitumor promoting diterpenoids from the stem bark of Thuja standishii

Article abstract of DOI:10.1055/s-2003-37037

Six diterpenes, including one new natural product, were isolated from a CHCl₃ extract of the stem bark of Thuja standishii. The new compound has been characterized as 15-oxolabda-8(17),13Z-dien-19-oic acid. The known compounds were identified as ferruginol (2), sugiol (3), isocupressic acid (4), sandaracopimaric acid (5) and 15-oxolabda-8(17),13E-dien-19-oic acid (6). Compounds 2-5 and the derivatives 4a and 4b were tested for their inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol 13-acetate (TPA). Compounds 2, 3, 4 and 5 showed strong inhibitory effect on EBV-EA induction (100% inhibition at 1000 mol ratio/TPA).

Full text of DOI:10.1055/s-2003-37037

Potential Antitumor Promoting
Diterpenoids from the Stem Bark of
Thuja standishii
Manabu Iwamoto1, Toshifumi Minami1, Harukuni Tokuda2,
Hironori Ohtsu1, Reiko Tanaka1
Abstract
Six diterpenes, including one new natural product, were isolated
from a CHCl3 extract of the stem bark of Thuja standishii. The new
compound has been characterized as 15-oxolabda-8(17),13Z-
dien-19-oic acid. The known compounds were identified as fer-
ruginol (2), sugiol (3), isocupressic acid (4), sandaracopimaric
acid (5) and 15-oxolabda-8(17),13E-dien-19-oic acid (6). Com-
pounds 2±5 and the derivatives 4a and 4b were tested for their
inhibitory effects on Epstein-Barr virus early antigen (EBV-EA)
activation induced by 12-O-tetradecanoylphorbol 13-acetate
(TPA). Compounds 2, 3, 4 and 5 showed strong inhibitory effect
on EBV-EA induction (100% inhibition at 1000 mol ratio/TPA).
In our previous study of the stem bark of Thuja standishii (Gord.)
Carr, we reported the isolation of the novel carbon skeletal diter-
penoid, standishinal [1], and five labdane-type diterpenoids [2],
[3]and a nor-drimane-type sesquiterpene [2]. In addition, two
labdane-type diterpenoids, 15,16-bisnor-13-oxolabda-8(17),11E-
dien-19-oic acid [4]and 15-oxolabda-8(17),11 Z,13E-trien-19-oic
acid [3]showed significant antitumor promoting activities in an
in vivo two-stage mouse-skin carcinogenesis assay using 7,12-di-
methylbenz[a]anthracene (DMBA) as an initiator and 12-O-tet-
radecanoylphorbol 13-acetate (TPA) as a tumor promotor. Fur-
ther careful examination of this extract has led to the isolation
of a new labdane-type diterpene (1), besides five known diterpe-
noids, ferruginol (2) [5] , sugiol (3) [6], isocupressic acid (4) [7], [8],
sandaracopimaric acid (5) [9], and 15-oxolabda-8(17),13E-dien-
19-oic acid (6) [10]. Known compounds were identified by com-
parison of their physical and spectral data with those already
published.
69
Compound 1 was obtained as a colorless oil, and the molecular
formula was assigned as C20H30O3 by HREIMS. Its UV and IR spec-
tra showed an a,b-unsaturated aldehyde group [lmax = 238 nm
(log e 4.2); nmax = 1672 cm±1 (>C = C-CHO)]and a carboxyl group
[nmax = 3200±2800, 1961 cm±1 (COOH)]. The 1H- and 13C-NMR
spectra exhibited two tertiary methyl groups, a trisubstituted
double bond [dH = 5.89 (d), dC = 129.2 (d), 164.6 (s)], an exo-
Affiliation: 1 Department of Medicinal Chemistry, Osaka University of Pharma-
2
ceutical Sciences, Takatsuki, Osaka, Japan ´ Department of Biochemistry,
Kyoto Prefectural University of Medicine, Kyoto, Japan
Correspondence: Reiko Tanaka ´ Department of Medicinal Chemistry ´ Osaka
University of Pharmaceutical Sciences ´ 4-20-1 Nasahara ´ Takatsuki ´ Osaka
569-1094 ´ Japan ´ E-mail: tanakar@oysun01.oups.ac.jp ´ Fax: +81-726-90-1084
Received: April 8, 2002 ´ Accepted: September 7, 2002
Bibliography: Planta Med 2003; 69: 69±72 ´ ꢀ Georg Thieme Verlag Stuttgart ´
New York ´ ISSN 0032-0943
Letter¼ Planta Med 2003; 69:69±72

Table 1 Relative ratioa of EBV-EAactivation with respect to positive
control (100%)in the presence of compounds 2, 3, 4, 4a, 4b
and 5
Concentration (mol ratio/TPA)
Compound
1000
500
100
10
2
0 (70)
35.2
37.4
45.1
50.1
49.2
31.4
22.8
73.6
75.5
76.3
81.3
79.1
68.5
81.7
91
3
0 (70)
93.4
97.3
4
3.2 (60)
8.2 (60)
7.3 (60)
0 (70)
4a
100
100
91.4
100
4b
5
Curcuminb
0 (60)
a
Values represent relative percentages compared with the positive control value. TPA(32
pmol, 20 ng) = 100%. Values in parentheses are viability percentages of Raji cells.
b Positive control substance [12].
cyclic methylene group [dH = 4.56 (s), 4.93 (s), dC = 106.8 (t),
147.5 (s)], an a,b-unsaturated aldehyde group [dH = 9.83 (d), highest concentration of 1000 mol ratio/TPA; indicating that the
dC = 191.1 (d)], and a carboxyl group [dC = 182.2 (s)]. The struc- cytotoxicities of these compounds seem to be quite moderate
ture of 1 was established by detailed 2D NMR experiments invol- against cell lines in vitro (Table 1). Compounds 4a and 4b were
ving HMQC, HMBC, 1H/1H COSY and NOESY spectra. In the HMBC weaker than 4, thus, the COOH group at C-19 is more effective
spectrum (Fig.1), an aldehyde proton correlated with C-13 and C- in the enhancing antitumor promoting activity. Labdane, abie-
14, Me-16 with C-12, C-13 and C-14, Me-18 with C-3, C-4, C-5 and tane, and pimarane-type diterpenes such as 2, 3, 4 and 5 may be
C-19, Me-20 with C-1, C-5, C-9 and C-10, and an exomethylene lead compounds to more potent agents with antitumor promot-
proton with C-7, C-8 and C-9, respectively. In the NOESY spec- ing activity for clinical use.
trum, NOE's were observed for H-15 with H-12b, Me-16 with H-
14, Me-18 with H-5a, and Me-20 with H-11b. Thus, the relative
stereostructure of 1 was established as 15-oxolabda-8(17),13Z- Materials and Methods
dien-19-oic acid. Compound 1 was transformed into the mixture
of compounds 1 and 6 (1:1) by exposure to air for about 2 hours. The plant material (stem bark) used in this study was collected in
On the other hand, in CHCl3 solution, the pure compound 1 was September, 1995 at Hashimoto City, Wakayama Prefecture, Ja-
immediately changed to a mixture of compounds 1 and 6. Com- pan. A voucher specimen (TS-95-01) is deposited at the Herbar-
pounds 1 and 6 exist as a equimolar mixture and the ratio does ium of the Laboratory of Medicinal Chemistry, Osaka University
70
not change.
of Pharmaceutical Sciences.
The inhibitory effects of 2, 3, 4, 4a, 4b and 5 on EBV-EA activation 1H- and 13C-NMR were recorded in CDCl3 on a Varian INOVA 500
induced by TPA were examined to screen for antitumor promot- spectrometer with standard pulse sequences, operating at 500
ing activities, and the results are shown in Table 1. Compound 1 and 125 MHz, respectively. EIMS were recorded on a Hitachi
was not tested in this assay because it was present in a mixture 4000H double-focusing mass spectrometer (70 eV). Column
with 6. The potencies of compounds 2, 3, 4 and 5 were stronger chromatography was carried out over silica gel (70±230 mesh,
than that of the control curcumin [11]at concentrations of 100
Merck), and MPLC were performed on silica gel (230±400
and 10 mol ratio/TPA (Table 1). The viability percentages of Raji mesh, Merck) and Cosmocil 40 C18-PREP (ODS, Nacarai Tesque).
cells treated with the test compounds were mostly 60% at the TLC and PTLC were carried out on Merck silica gel F254 plates. Pre-
liminary silica gel column chromatography of the CHCl3 extract
(558.8 g) of the chopped stem bark (5.3 kg) of T. standishii has
Fig. 1 1H-1H COSY
nn ) and HMBC cor-
relations (®) of 1.
been reported previously, with separation into 13 (residues A -
M) fractions [3]. Residue B (frs. No. 5±8, 28.7 g) was rechromato-
graphed over silica gel (1 kg) eluting with n-hexane:CHCl3 (1:1)
to give residues B-a (frs. No. 22±54, 4.9 g) and B-b (frs. No. 55±
70, 11.3 g). Residue B-a was rechromatographed over silica gel
(200 g) eluting with n-hexane:EtOAc (10:1) to afford com-
pounds 2 (35.3 mg) and 3 (22.1 mg) from the fractions 66±81
and 85±90, respectively. Rechromatography (2”) over silica gel
(1 kg and 100 g) of residue F (frs. No. 32±46, 22.2 g) eluting with
CHCl3 gave a crystalline solid, which was recrystallized from
MeOH-CHCl3 to give compound 5 (1.13 g). Further elution of this
column with CHCl3 afforded an oil (40.4 mg) followed by HPLC
(ODS) eluting with 80% MeOH to give compounds 1 (11.2 mg)
(
Letter¼ Planta Med 2003; 69:69±72

and 6 (8.2 mg), respectively. Residue I (frs. No. 60±71,12.5 g) was identical in all respects with authentic 15-oxolabda-8(17),13E-
rechromatographed twice over silica gel (500 g and 100 g) elut- dien-19-oic acid (Lit. [10], [a]D: + 47.5).
ing with CHCl3 :EtOAc (10:1) to give compound 4 (1.77 g). Com-
pounds 2±5 had the purities of over 99%.
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
15-Oxolabda-8(17),13Z-dien-19-oicacid (1): Colorless oil; [a]D25
:
+ 27.3 (c 0.28, CHCl3); HREIMS: m/z = 318.2206 (C20H30O3, re- University. The EBV genome-carrying lymphoblastoid cells
quires 318.2193); UV (MeOH): lmax = 238 nm (log e 4.2); IR (Raji cells derived from Burkitt's lymphoma) were cultured in
(film): nmax = 3200±2800 and 1961 (COOH), 1672 (> C = C- 10% fetal bovine serum (FBS) in RPMI-1640 medium (Nissui).
CHO), 1647 and 889 (> C = CH2), 2937, 1448, 1395, 1263, 1165 Spontaneous activation of EBV-EA in our sub-line Raji cells
cm±1 1H-NMR (500 MHz, CDCl3): d = 0.62 (3H, s, Me-20), 1.24 was less than 0.1%. The inhibition of EBV-EA activation was as-
;
(3H, s, Me-18), 1.98 (3H, d, J = 1.1 Hz, Me-16), 4.56 and 4.93 sayed using Raji cells (virus non-producer type) as described
(each 1H, s, H-17), 5.89 (1H, d, J = 8.2 Hz, H-14), 9.83 (1H, d, previously [3]. The indicator cells (Raji cells, 1”106/mL) were
J = 8.2 Hz, H-15); 13C-NMR (125 MHz, CDCl3): d = 12.9 (C-20), incubated at 37 8C for 48 h in 1 mL of a medium containing n-
19.9 (C-2), 22.4 (C-11), 24.8 (C-16), 26.0 (C-6), 28.9 (C-18), 31.1 butyric acid (4 mmol), TPA (32 pmol = 20 ng) in DMSO, as in-
(C-12), 37.9 (C-8), 38.5 (C-9), 39.1 (C-1), 40.4 (C-10), 44.1 (C-4), ducer and various amounts of test compound in 5 mL DMSO.
55.0 (C-9), 56.1 (C-5), 106.8 (C-17), 129.2 (C-14), 147.5 (C-17), Smears were made from the cell suspension, and the activated
164.6 (C-13), 182.2 (C-19), 191.1 (C-15); EIMS: m/z (rel. cells that were stained by EBV-EA positive serum from NPC pa-
int.): = 318 [M]+ (2), 303 [M±Me]+ (9), 300 (2), 285 (3), 274 tients were detected by an indirect immunofluorescence tech-
(12), 235 (51), 189 (100), 121 (78).
nique [11]. In each assay, at least 500 cells were included, and
the number of stained cells (positive cells) present was record-
ed. Triplicate assays were performed for each compound. The
Ferruginol (2): C20H30O, [a]D25: + 39.3 (c 0.70, CHCl3); EIMS: m/z
= 286 [M]+. Compound 2 was identical in all respects with au- average EBV-EA induction of the test compounds was expressed
thentic ferruginol (Lit. [5], [a]D25: + 40.6).
as a relative ratio to the control experiment (100%) which was
carried out only with n-butyric acid (4 mmol) plus TPA (32
pmol). EBV-EA induction was ordinarily around 35%. The viabi-
Sugiol (3): C20H28O2, m.p. 289±291 8C (n-hexane-EtOAc); [a]D25
:
+ 24.8 (c 0.44, EtOH); EIMS: m/z = 300 [M]+. Compound 3 was lity of treated Raji cells was assayed by the trypan blue staining
identical in all respects with authentic sugiol (Lit. [6], m.p. method.
292±294 8C, [a]D25: + 26).
Isocupressic acid (4): C20H32O3, [a]D25: + 51.0 (c 0.90, CHCl3); Acknowledgements
HREIMS: m/z = 320.2334 [M]+ (C20H32O3, calcd. for 320.2349).
Compound 4 was identical in all respects with authentic isocu- The authors are grateful to Mr. K. Minoura and Mrs. M. Fujitake,
71
pressic acid (Lit. [7], [a]D25: + 52.9).
Osaka University of Pharmaceutical Sciences, for NMR and MS
measurements.
Methyl isocupressate (4a): To a MeOH (2 mL) and C6H6 (1 mL) so-
lution of compound 4 (15.0 mg) was added a 2.0 M trimethyl-
silyldiazomethane solution in n-hexane (TMSCHN2) (0.2 mL) for References
20 h at room temperature. Evaporation of the solvent under re-
1
Ohtsu H, Iwamoto M, Ohishi H, Matsunaga S, Tanaka R. Standishinal, a
novel carbon skeletal diterpene from the bark of Thuja standishii
(Gord.) Carr. Tetrahedron Letters 1999; 40: 6419±22
duced pressure afforded a residue which was purified by PTLC
(CHCl3) to give 4a (13.8 mg), C21H34O3, [a]D25: + 48.9 (c 0.90,
CHCl3); EIMS: m/z = 334 [M]+. Compound 4a was identical in all
2 Iwamoto M, Ohtsu H, Matsunaga S, Tanaka R. Labdane-type diter-
penes and a nordrimane-type sesquiterpene from the stem bark of
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respects with authentic methyl isocupressate (Lit. [7], [a]D25
:
+ 51.2).
O-Acetylisocupressic acid (4b): Compound 4 (15.0 mg) was acety-
lated with Ac2O-pyridine (1:1, 2 mL) at room temperature for
24 h. Usual work-up afforded a residue, which was purified by
PTLC (CHCl3) to give 4b (15.1 mg), C22H34O4, [a]D25: + 47.8 (c 0.40,
CHCl3); EIMS: m/z = 362 [M]+. Compound 4b was identical in all
respects with authentic acetyl isocupressic acid (Lit. [8], [a]D:
+ 49).
4 Tanaka R, Ohtsu H, Iwamoto M, Minami T, Tokuda H, Nishino H, Mat-
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m.p. 170±172 8C (MeOH-CHCl3); EIMS: m/z = 302 [M]+. Com-
pound 5 was identical in all respects with authentic sandaracopi-
maric acid (Lit. [9], [a]D25: -19.8, m.p. 165±168 8C).
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15-Oxolabda-8(17),13E-dien-19-oicaidc
+ 45.2 (c 0.12, CHCl3); EIMS: m/z = 318 [M]+. Compound 6 was
(6): C :
20H30O3, [a]D25
Letter¼ Planta Med 2003; 69:69±72

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Letter¼ Planta Med 2003; 69:69±72