A new seco-abietane-type diterpene from the stem bark of Picea glehni

Article abstract of DOI:10.1055/s-2004-827241

A new seco-abietane-type diterpenoid, 13S-hydroxy-9-oxo-9,10-seco-abiet- 8(14)-en-18,10α-olide (1) along with a known lignan compound, pinoresinol (2) was isolated from the stem bark of Picea glehni (Fr. Schm.) Masters. Spectroscopic methods and chemical conversions were used to establish the structure of 1. In order to assess their cancer chemopreventive potential, the inhibition of Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol 13-acetate (TPA) was examined for compound 1, its synthetic analogue, 9,10-seco-8S,13S-epoxy-abiet-8(14)-en-18,10α-olide (1a) and 2. The inhibitory effect of 1a on EBV-EA induction was strong (0, 20.7, 67.1 and 89.2% inhibition at 1000, 500, 100 and 10 mol ratio/TPA). The IC ₅₀ of 1a was 226 mol ratio/32 pmol/TPA.

Full text of DOI:10.1055/s-2004-827241

A New seco-Abietane-Type Diterpene
from the Stem Bark of Picea glehni
Reiko Tanaka1, Shun-ichi Wada1, Yoshitaka Kinouchi1,
Harukuni Tokuda2, Shunyo Matsunaga1
Abstract
A new seco-abietane-type diterpenoid, 13S-hydroxy-9-oxo-9,10-
seco-abiet-8ꢀ14)-en-18,10a-olide ꢀ1) along with a known lignan
compound, pinoresinol ꢀ2) was isolated from the stem bark of
Picea glehni ꢀFr. Schm.) Masters. Spectroscopic methods and che-
mical conversions were used to establish the structure of 1. In
order to assess their cancer chemopreventive potential, the inhi-
bition of Epstein-Barr virus early antigen ꢀEBV-EA) activation in-
duced by 12-O-tetradecanoylphorbol 13-acetate ꢀTPA) was ex-
amined for compound 1, its synthetic analogue, 9,10-seco-
8S,13S-epoxy-abiet-8ꢀ14)-en-18,10a-olide ꢀ1a) and 2. The inhibi-
tory effect of 1a on EBV-EA induction was strong ꢀ0, 20.7, 67.1 and
89.2% inhibition at 1000, 500,100 and 10 mol ratio/TPA). The IC50
of 1a was 226 mol ratio/32 pmol/TPA.
From the stem bark of Picea glehni ꢀFr. Schm.) Masters ꢀPinaceae),
we isolated three new diterpenes, 19ꢀ4®3)abeo-8a,13S-epoxy-
labda-4ꢀ18),14-diene, 19-nor-abieta-4ꢀ18),8,11,13-tetraen-7-one
and 12-hydroxydehydroabietic acid along with nine known di-
terpenes [1], and two new triterpenes, 3a-methoxyserrat-14-
en-21b-yl formate, and 24-methylcycloartanone together with
three known triterpenes [2]. In order to assess usefulness of this
plant as a source of natural agents for cancer chemoprevention,
the extract was further examined and a new 13S-hydroxy-9-
oxo-9,10-seco-abiet-8ꢀ14)-en-18,10a-olide ꢀ1) was isolated to-
gether with the known pinoresinol ꢀ2).
877
HR-EI-MS assigned the molecular formula of 1 as C20H30O4. The
IR spectrum of 1 showed a hydroxy group ꢀnmax = 3459 cm±1), a
g-lactone ꢀnmax = 1770 cm±1), and an a,b-unsaturated six-mem-
bered ring ketone ꢀnmax = 1675 cm±1). The 1H- and 13C-NMR
spectra ꢀTable 1) exhibited two tertiary methyl groups, an isopro-
pyl group, seven sp3 methylenes, an sp3 methine, an sp3 quaten-
ary carbon, a tertiary carbon bearing a hydroxy group [dC = 72.3
ꢀs)], a g-lactone ring [dC = 85.4 ꢀs), 180.3 ꢀs)], a trisubstituted
double bond [dH = 6.50 ꢀ1H, d); dC = 139.0 ꢀs), 148.4 ꢀd)], and a
conjugated ketone [dC = 198.9 ꢀs)]. The unsaturation of com-
pound 1 suggested that it is a B-ring seco-abietane-type diterpe-
noid and possesses a lactone ring at positions C-10a and C-18re-
Affiliation: 1 Department of Medicinal Chemistry, Osaka University of Pharma-
2
ceutical Sciences, Osaka, Japan ´ Department of Biochemistry, Kyoto Prefec-
tural University of Medicine, Kyoto, Japan
Correspondence: Dr. Reiko Tanaka ´ Department of Medicinal Chemistry ´ Osa-
ka University of Pharmaceutical Sciences ´ 4±20±1 Nasahara ´ Takatsuki ´ Osaka
569±1094 ´ Japan ´ Phone: +81-72-690-1084 ´ Fax: +81-72-690-1084 ´ E-mail:
tanakar@gly.oups.ac.jp
Received: January 14, 2004 ´ Accepted: May 15, 2004
Bibliography: Planta Med 2004; 70: 877±880 ´ ꢁ Georg Thieme Verlag KG
Stuttgart ´ New York ´ DOI 10.1055/s-2004-827241 ´ ISSN 0032-0943
Letter¼ Planta Med 2004;70: 877±880

Table 1 1H and 13C-NMR data for compounds 1, 1a, 1b and 1c $CDCl3).a
1
1a
1b
1c
Position
dC
dH
dC
dH
dC
dH
dC
dH
1a
1b
2a
2b
3a
3b
4
27.8 t
1.59 m
1.59 m
1.59 m
1.68 m
1.52 m
1.39 m
27.8 t
1.61 m
1.61 m
1.61 m
1.69 m
1.50 m
1.40 m
27.9 t
1.61 m
1.61m
1.61 m
1.68 m
1.55 m
1.42 m
27.9 t
1.62 m
1.62 m
1.62 m
1.74 m
1.56 m
1.42 m
18.5 t
26.2 t
18.5 t
26.3 t
18.5 t
26.3 t
18.6 t
26.3 t
46.7 s
54.0 d
24.2 t
46.7 s
53.8 d
23.5 t
46.7 s
53.6 d
26.9 t
46.6 s
54.5 d
22.2 t
5a
6A
6B
7A
1.80 t $6.1)
1.46 m
1.82 t $6.5)
1.54 m
1.88 t $6.1)
1.68 m
1.95 t $7.0)
2.30 $2H) m
1.53 m
1.60 m
1.78 m
29.5 t
2.20 dddd $13.5, 10.8,
5.9, 0.9)
32.2 t
2.10 ddd $14.3, 10.5,
5.5)
35.1 t
2.67 m
23.3 t
2.14 m
2.30 m
7B
2.28 dddd $13.5, 10.8,
5.9, 0.9)
2.40 dddd $14.3, 10.5,
5.5, 1.1)
2.67 m
8
9
139.0 s
198.9 s
85.4 s
143.6 s
68.6 d
85.6 s
29.7 t
141.2 s
125.7 d
85.4 s
137.7 s
70.7 d
85.4 s
30.9 t
4.08 m
7.00 d $7.5)
7.24 t $7.5)
4.21 dd $6.5, 3.5)
10
11a
34.1 t
2.69 ddd $17.2, 10.3,
5.3)
2.03 ddt $12.3, 3.5,
5.5)
128.5 d
1.84 m
11b
12a
12b
2.44 ddd $17.2, 6.4,
4.8)
1.78 m
1.66 m
1.66 m
1.78 m
30.8 t
1.96 ddd $13.5, 10.3,
4.8)
28.7 t
124.3 d
7.09 d $7.5)
121.9 d
5.37 t $7.5)
2.14 dddd $13.5, 6.4,
5.3, 1.4)
2.14 dddd $13.5, 6.4,
5.3, 1.4)
13
14
15
16
17
18
19
20
72.3 s
148.4 d
37.0 d
16.4 q
17.4 q
180.3 s
20.2 q
25.6 q
72.4 s
129.4 d
37.6 d
16.4 q
17.7 q
180.3 s
20.1 q
24.8 q
149.3 s
126.4 d
34.1 d
24.0 q
24.0 q
180.3 s
20.2 q
24.5 q
149.1 s
114.3 d
35.5 d
21.4 q
22.2 q
180.2 s
20.0 q
24.5 q
6.50 d $0.9)
5.41 s
7.02 s
6.08 s
1.92 septet $6.9)
1.03 d $6.9)
1.75 septet $7.0)
0.96 d $7.0)
0.89 d $7.0)
2.89 septet $7.0)
1.25 d $7.0)
1.25 d $7.0)
2.35 septet $6.5)
1.08 d $6.5)
1.08 d $6.5)
0.99 d $6.9)
878
1.16 s
1.41 s
1.15 s
1.43 s
1.16 s
1.39 s
1.15 s
1.38 s
a Assignments were made by 1H-1H COSY, HMQC, HMBC and NOESY data.
sulting from dehydration between a C-10 hydroxy group and a C- Table 2 lists inhibitory effects of compounds 1, 1a and 2 on Ep-
18carboxylic acid. The HMBC spectrum showed the following stein-Barr virus early antigen ꢀEBV-EA) induced by the tumour
correlations: between Me-16 and C-13, C-15, C-17; Me-17 and promotor, TPA and the associated viability of Raji cells. The via-
C-13, C-15, C-16; Me-19 and C-3, C-4, C-5, C-18; Me-20 and C-1, bility of Raji cells treated with the test compounds ꢀ1, 1a, and 2)
C-5, C-10; H-14 and C-8, C-9, C-12, C-13, C-15. In the 1H/1H COSY was over 70% at the highest concentration of 1000 mol ratio/
spectrum correlations were seen between H-7a and H-6a, H-6b, TPA; suggesting that these compounds had moderate cytotoxi-
H-7b; H-7b and H-6a, H-6b, H-7a, although H-14 correlated with cities against in vitro cell lines ꢀTable 2). On comparison of the
no peak. Therefore, the tertiary hydroxy group is attached at C- anti-tumour promoting activities, 1a showed a stronger effect
13. These data suggested that the structure of 1 was a 13-hydro- than 1. It is interesting to note that the presence of an ether
xy-9-oxo-9,10-seco-abiet-8ꢀ14)-en-18,10a-olide. Reduction of 1 group in 1a seems to enhance its activity against tumour pro-
with LiAlH4 gave 1a, [M]+m/z = 318, in quantitative yield and its motion as is the case with 13a,14a-epoxy-3b-methoxyserra-
1H- and 13C-NMR spectra showed new signals at dH = 4.08ꢀ1H,
m) and dC = 68.6 ꢀd). NaBH4 reduction of 1 also gave the same
compound ꢀ1a), and the following HOAc treatment gave a known
tan-21b-ol [3].
compounds 1b and 1c. The NOESY spectrum of 1 ꢀFig. 2) showed Materials and Methods
that isopropyl methyl groups ꢀMe-16 and -17) were correlated
not with Me-20 but with Me-19, and the relative configuration Plant material: The stem bark of Picea glehni ꢀFr. Schm.) Masters
of 1 was determined as shown in Fig.1. Therefore, compound 1 ꢀPinaceae) was collected in the mountainous terrain under the
is a new seco-abietane-type diterpenoid, 13S-hydroxy-9-oxo- control of National Hokkaido Bureau, Iwamizawa City, Japan, in
9,10-seco-abiet-8ꢀ14)-en-18,10a-olide.
October 1997. A voucher specimen ꢀPG-9710±1) is deposited at
Letter¼ Planta Med 2004;70: 877±880

the Herbarium of the Department of Medicinal Chemistry, Osaka
University of Pharmaceutical Sciences.
Isolation procedure: Preliminary silica gel column chromatogra-
phy separated the CHCl3 extract ꢀ412.4 g) of the chopped stem
bark ꢀ9.0 kg) of P. glehni into 13 ꢀresidues I ± XIII) fractions as re-
ported previously [1]. Residue XI ꢀfraction nos 81 ± 85, 1.958 g)
was rechromatographed over silica gel ꢀ300 g) eluting with
CHCl3:EtOAc ꢀ10:1) to give an amorphous gum ꢀfraction nos 50
± 68, 379.6 mg, 3.8 L), which has a UV absorption band on the TLC
plate ꢀ254 nm). This material was rechromotographed over silica
gel ꢀ30 g) using CHCl3:EtOAc ꢀ20:1) to give pinoresinol ꢀ2) [4]
ꢀfraction no 64, 18.8 mg, 50 mL) and crude compound 1 ꢀfraction
nos 72 ± 77, 75.9 mg, 300 mL), which was subjected to PTLC ꢀn-
hexane:EtOAc:MeOH, 50:50:2) to give pure compound 1 ꢀ57.0
mg). Compounds 1 and 2 had purities of over 98%.
13S-Hydroxy-9-oxo-9,10-seco-abiet-8%14)-en-18,10a-olide ꢀ1):
Colourless oil; [a]D23: ±4.88 ꢀc 0.46, CHCl3); HR-EI-MS: m/z =
334.2143 [M]+ ꢀC20H30O4 requires 334.2142); IR ꢀfilm):
nmax = 3459 ꢀOH), 2960, 2933, 1770 ꢀg-lactone), 1675 ꢀC = C-
C = O), 923 cm±1; EI-MS: m/z = 334 ꢀ7), 316.2058ꢀ16) [M ±
H2O]+, 291.1596 ꢀ33) [M ± C3H7]+, 273.1501 ꢀ16), 245.1545 ꢀ86),
227.1437 ꢀ18), 167 ꢀ14), 150 ꢀ15), 137 ꢀ13), 123 ꢀ24), 122 ꢀ21), 109
ꢀ100); 1H- and 13C-NMR: see Table 1.
Reduction of 1 with LiAlH4: To a solution of compound 1 ꢀ16.9 mg)
in absolute ether ꢀ10 mL), LiAlH4 ꢀ20 mg) was added and the mix-
ture stirred at room temperature for 6 h. The reaction mixture
was worked up as usual to give 1a ꢀ15.8mg).
Fig. 1 Chemical structures of compounds 1, 1a ± c, and 2.
9S,13S-Epoxy-9,10-seco-abiet-8%14)-en-18,10a-olide ꢀ1a): M.p.
156 ± 158 8C; [a]D23: ±21.78 ꢀc 0.35, CHCl3); EI-MS: m/z = 318ꢀ2)
[M]+; 1H- and 13C-NMR: see Table 1.
879
Reduction of 1 with NaBH4: To a solution of 1 ꢀ14.1 mg) in MeOH
ꢀ2 mL), NaBH4 ꢀ2.0 mg) was added and the mixture was allowed
to stand at room temperature for 1 h. The reaction product was
confirmed as 1a by TLC ꢀCHCl3:MeOH, 19:1). One drop of HOAc
was added into the reaction mixture, followed by usual work-up.
Evaporation of the solvent under reduced pressure afforded a re-
sidue ꢀ13.9 mg), which was subjected to PTLC ꢀCHCl3:MeOH,
19:1) to give compounds 1b ꢀ5.2 mg) and 1c ꢀ4.3 mg).
Fig. 2 NOESY correlations of 1.
Table 2 Relative ratioa of EBV-EA activation with respect to positive control $100%) in the presence of compounds 1, 1a and 2.
Compounds
EBV-EA positive cells ꢀ% viability)
Compound concentration ꢀmol ratio/32 pmol TPA)
IC50
1000
500
100
10
ꢀmol ratio/32 pmol TPA)
1
3.7 $70)b
0 $70)
22.5
20.7
40.6
22.8
70.7
67.1
72.0
81.7
90.3
273
226
398
341
1a
89.2
100.0
100.0
2
11.5 $70)
0 $60)
Curcuminc
a Values represent percentages relative to the positive control value $100%).
b Values in parentheses are the viability percentages of Raji cells.
c Positive control substance [8].
Letter¼ Planta Med 2004;70: 877±880

9,10-seco-Abieta-8,11,13-trien-18,10a-olide ꢀ1b): Colourless oil; References
[a]D20
+ 8.58 ꢀc 0.41, CHCl3); IR ꢀfilm): nmax = 3459 ꢀOH),
:
1
1675 cm±1; EI-MS: m/z = 300 [M]+; 1H- and 13C-NMR: see Table 1.
Compound 1b was identified from published data including EI-
MS, 1H- and 13C-NMR chemical shift values and its [a]D value
{1b: [a]D20: + 8.58 ꢀc 0.41, CHCl3), synthetic sample: [a]D20: + 9.58
ꢀc 0.11, CHCl3) [5]; [a]D20: + 9.28 ꢀc 1.85, CHCl3) [6]}.
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9S-Hydroxy-9,10-seco-abieta-11,13-dien-18,10a-olide ꢀ1c): Col-
ourless oil; [a]D23: + 8.78 ꢀc 0.10, CHCl3); EI-MS: m/z = 318[M] ;
+
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1H- and 13C-NMR: see Table 1.
Pinoresinol ꢀ2): Colourless oil; [a]D23: + 53.18 ꢀc 0.53, CHCl3).
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was assayed according to Ito et al. [7]. The inhibiting activities
of the test compounds were estimated on the basis of the percen-
tage of the number of positive cells compared with that of a con-
trol without the test compound. The viability of the cells was as-
sayed by the trypan-blue staining method.
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Acknowledgements
The authors are grateful to Mrs. Mihoyo Fujitake and Mr. Katsu-
hiko Minoura of this University, for MS and NMR measurements.
This study was supported by a Grant-in-Aid for High Technology
from the Ministry of Education, Science, Sports and Culture, Ja-
pan.
880
Letter¼ Planta Med 2004;70: 877±880