Beyerane derivatives and a sesquiterpene dimer from Japanese Cypress (Chamaecyparis obtusa)

Article abstract of DOI:10.1248/cpb.56.1030

In the course of studies on biological active constituents from woody plants, we previously reported the isolation of many lignan derivatives as neurite outgrowth-promoting compounds from an ethyl acetate soluble fraction of Japanese Cypress (Chamaecyparis obtusa). Further chemical investigation on the residual parts of the ethyl acetate soluble fraction of a methanol extract of Japanese Cypress resulted in the isolation of four new beyerene type derivatives and a novel sesquiterpene dimer formed between cryptomeridiol and hinokiic acid. Their structures were elucidated as 18-O-(Z)-p-coumaroylbeyer-15-ene-18-ol (1), 18-O-(E)-p-coumaroylbeyer-15-ene-18-ol (2), 18-O-(E)-p-coumaroylbeyer-15- ene-11β,18-diol (3), 18-O-(Z)-p-coumaroylbeyer-15-ene-11β,18-diol (4) and ent-cryptomeridiol-4-yl-hinokiiate (5) by ¹H-NMR, ¹³C-NMR, 2D-NMR, and HR-MS spectral analysis.

Full text of DOI:10.1248/cpb.56.1030

1030
Notes
Chem. Pharm. Bull. 56(7) 1030—1034 (2008)
Vol. 56, No. 7
Beyerane Derivatives and a Sesquiterpene Dimer from Japanese Cypress
(Chamaecyparis obtusa)
Hui Yuan GAO,a Li Jun WU,a Norio MUTO,b Hiroyuki FUCHINO,c Takahisa NAKANE,d Osamu SHIROTA,e
Toshikazu SANO,f and Masanori KUROYANAGI*,b
a School of Traditional Chinese Medicines, Shenyang Pharmaceutical University; 110016, No. 103, Wenhua Road, Shenhe
Shenyang, People’s Republic of China: b Faculty of Life and Environmental Sciences, Prefectural University of Hiroshima;
562 Nanatsuka, Shobara, Hiroshima 727–0023, Japan: c Research Center for Medicinal Plant Resources, National
Institute of Biomedical Innovation; 1 Hachimandai, Tsukuba, Ibaraki 305–0843, Japan: d Showa Pharmaceutical
University; 3–3165 Higashi-Tamagawagakuen, Machida, Tokyo 194–8543, Japan: e Faculty of Pharmaceutical Sciences at
Kagawa Campus, Tokushima Bunri University; 1314–1 Shido, Sanuki, Kagawa 769–2193 Japan: and f Hiroshima
Prefectural Forestry Research Center; 168–1 Tokaichi, Miyoshi, Hiroshima 728–0015, Japan.
Received March 4, 2008; accepted April 23, 2008
In the course of studies on biological active constituents from woody plants, we previously reported the iso-
lation of many lignan derivatives as neurite outgrowth-promoting compounds from an ethyl acetate soluble frac-
tion of Japanese Cypress (Chamaecyparis obtusa). Further chemical investigation on the residual parts of the
ethyl acetate soluble fraction of a methanol extract of Japanese Cypress resulted in the isolation of four new
beyerene type derivatives and a novel sesquiterpene dimer formed between cryptomeridiol and hinokiic acid.
Their structures were elucidated as 18-O-(Z)-p-coumaroylbeyer-15-ene-18-ol (1), 18-O-(E)-p-coumaroylbeyer-15-
ene-18-ol (2), 18-O-(E)-p-coumaroylbeyer-15-ene-11b,18-diol (3), 18-O-(Z)-p-coumaroylbeyer-15-ene-11b,18-diol
(4) and ent-cryptomeridiol-4-yl-hinokiiate (5) by 1H-NMR, 13C-NMR, 2D-NMR, and HR-MS spectral analysis.
Key words Chamaecyparis obtusa; Cupressaceae; beyerene derivative; sesquiterpene dimer
Social problems resulting from aging and degeneration of characteristic olefinic protons coupled with each other [dH
brain function have drawn attention around the world and es- 5.45 (1H, d, Jꢂ5.4 Hz), 5.67 (1H, d, Jꢂ5.4 Hz)] for H-15
pecially in Japan. Biologically active constituents from plant and H-16 of beyer-15-ene skeleton, and one oxygenated
resources are expected to suppress the development of these methylene protons [dH 3.72 (1H, d, Jꢂ11.4 Hz), 3.91 (1H, d,
problems. We have studied various biologically active con- Jꢂ11.4 Hz)] along with other alkyl protons. The 13C-NMR
stituents from woody plants collected in the Chugoku area of spectrum of 1 gave 27 carbon signals and showed the pres-
Japan.1—3) In our previous work, the ethyl acetate (EtOAc) ence of a p-coumaroyl moiety (dC 115.0ꢃ2, 132.5ꢃ2, 117.3,
soluble fraction of Japanese Cypress (Chamaecyparis 127.5, 143.4, 156.9, 166.9), two characteristic olefinic car-
obtusa) showed neurite outgrowth-promoting activity on neu- bons (dC 135.1, 136.4) for C-15 and C-16 of beyer-15-ene
ronal PC12 cells, and we isolated two novel compounds, 9- skeleton, three methyl carbons (dC 24.9, 17.7, 15.5), and an
1
O-acetoxydihydrosesamin and a lignan-sesquiterpene conju- oxymethyl group (dC 73.0). These characteristic olefinic H-
gate, together with eleven known compounds, and some of NMR signal5—8) and 13C-NMR signal5,7,8) showed the pres-
the compounds showed potent neurite outgrowth activity.4) In ence of beyer-15-ene skeleton in the structure. So compound
order to find more new constituents from this fraction, fur- 1 should be composed of a (Z)-p-coumaroyl and a beyer-15-
ther chemical investigations of the residual fraction were car- ene units through an ester linkage. This expectation and the
ried out and four new beyerene type derivatives 1—4 (named position of (Z)-p-coumaroyl linkage were confirmed by
obtsurins A—D) and a new sesquiterpene dimer ent-cryp- HMBC analysis of 1 (Fig. 2). In the HMBC spectrum of 1,
tomeridiol-4-yl-hinokiate (5), were obtained. Their structures H3-19 (dH 0.84) showed correlations to C-18, C-3 and C-5
were determined by means of spectral analysis including 2D- (dC 73.0, 35.9, 49.8), and H-18 (dH 3.72, d, Jꢂ11.4 Hz, 3.91,
NMR and high-resolution (HR)-MS. This paper deals with d, Jꢂ11.4 Hz) showed correlations to C-5, Me-19, and C-9ꢄ
the isolation and structural elucidation of the new com- (dC 49.8, 17.7, 166.9). Other HMBC correlations are shown
pounds from C. obtusa.
in Fig. 2. This data indicates that the (Z)-p-coumaroyl group
connects to a hydroxymethyl at C-4 through an ester bond.
Alkaline hydrolysis of 1 yielded 1a and p-coumaric acid. The
Results and Discussion
Compound 1 was obtained as colorless needles; mp 115— 1H-NMR, 13C-NMR, 2D-NMR, and MS data showed that 1a
117 °C. The molecular formula of 1 was determined to be was beyer-15-ene-18-ol.9) The relative stereochemistry of 1
C29H38O3 based on the molecular ion peak at m/z 434.2823 was determined by ROESY analysis (Fig. 2). NOE correla-
[M]ꢀ in the HR-EI-MS. The IR spectrum of 1 showed ab- tions of H3-20 to H-15 and H3-19; H3-19 to H-18 and H3-20;
sorption bands at 3340, 2931, 1632, 1687, 1606, and H-18 to H-5 and H3-19; and H3-17 to H-14 and H-16 were
1514 cmꢁ1 ascribable to the hydroxyl, olefin, conjugated car- observed. Thus, the relative configuration of 1 was deter-
1
bonyl, and phenyl functions. The H-NMR spectrum of 1 mined as shown in Fig. 1. The absolute configuration of 1a
showed the presence of one (Z)-p-coumaroyl moiety [dH 5.87 was determined from an opposite optical rotation of 1a ([a]D
(1H, d, Jꢂ12.6 Hz), 6.86 (1H, d, Jꢂ12.6 Hz), 7.62 (2H, d, ꢁ23.2°) to the reported optical rotation data ([a]D ꢀ33.9°)9)
Jꢂ8.4 Hz), 6.79 (2H, d, Jꢂ8.4 Hz)], three singlet methyl of the ent-isomer. Thus, the structure of 1 was determined to
groups [dH 0.77 (3H, s), 0.84 (3H, s), 0.99 (3H, s)], and two be 18-O-(Z)-p-coumaroylbeyer-15-ene-18-ol and was named
∗ To whom correspondence should be addressed. e-mail: kuroyang@pu-hiroshima.ac.jp
© 2008 Pharmaceutical Society of Japan

July 2008
1031
obtsurin A.
Thus, the structure of 2 was determined to be 18-O-(E)-p-
Compound 2 was obtained as an amorphous solid. The coumaroylbeyer-15-ene-18-ol and was named obtsurin B.
molecular formula of 2 was determined to be C29H38O3 based
Compound 3 was obtained as a colorless amorphous solid.
on the molecular ion peak at m/z 434.2832 [M]ꢀ in the HR- The molecular formula of 3 was determined as C29H38O4
1
EI-MS. The H- and 13C-NMR spectral data of 2 showed based on the molecular ion peak at m/z 450.2784 in the HR-
similar signal patterns to those of 1 (Tables 1, 2) except for a EI-MS. The 1H-NMR spectrum of 3 showed the existence of
1
small difference in the p-coumaroyl moiety. The H-NMR a (E)-p-coumaroyl moiety [dH 6.32 (1H, d, Jꢂ15.5 Hz), 7.62
spectrum of 2 showed the presence of an (E)-p-coumaroyl (1H, d, Jꢂ15.5 Hz), 6.85 (2H, d, Jꢂ8.5 Hz), 7.45 (2H, d,
unit [dH 6.32 (1H, d, Jꢂ16.2 Hz), 7.62 (1H, d, Jꢂ16.2 Hz), Jꢂ8.5 Hz)], three singlet methyl groups [dH 1.23 (3H, s),
7.45 (2H, d, Jꢂ9.0 Hz), 6.86 (2H, d, Jꢂ9.0 Hz)], which indi- 1.06 (3H, s), 0.96 (3H, s)], two olefinic protons coupled to
cated 2 was a E-isomer of 1. The 2D-NMR spectra, including each other [dH 6.23 (1H, d, Jꢂ6.0 Hz), 5.79 (1H, d,
1
HMQC, HMBC, and the H–1H COSY showed the same re- Jꢂ6.0 Hz)] characteristic for an beyer-15-ene skeleton, an
sult as those of 1. NOESY analysis of 2 also showed the oxygenated methylene group [dH 3.98 (1H, d, Jꢂ10.5 Hz),
same result as that of 1. Alkaline hydrolysis of 2 gave 1a 3.77 (1H, d, Jꢂ10.5 Hz)], and an oxygenated methine group
and (E)-p-coumaric acid. The optical rotation of 1a ([a]D (dH 4.23, 1H, dt, Jꢂ11.5, 5.0 Hz), which was coupled with a
ꢁ19.7°) from 2 was identified with 1a ([a]D ꢁ23.2°) from 1. hydroxyl proton (dH 2.39, 1H, d, Jꢂ12.0 Hz). The 13C-NMR
spectrum of 3 (Table 2) showed a similar signal pattern to
that of 2 except for the presence of an additional hydroxyl
group. These data indicated that 3 was a hydroxy derivative
of 2. In the 1H–1H COSY spectrum of 3 H-9 (dH 1.29, 1H, d,
Jꢂ5.0 Hz) and H-12a (dH 1.80, 1H, dd, Jꢂ14.0, 5.0 Hz)
showed correlations to H-11. In the HMBC spectrum of 3,
H-9 (dH 1.29, 1H, d, Jꢂ5.0 Hz) exhibited long-range correla-
tions with C-11 (dC 73.2). Other HMBC correlations in 3 are
showed in Fig. 2. Alkaline hydrolysis of 3 gave 3a and (E)-p-
coumaric acid. The molecular formula of 3a was determined
to be C20H32O2 based on the molecular ion at m/z 304.2395
1
in the HR-EI-MS. The H-NMR, 13C-NMR, and 2D-NMR
spectra of 3a showed that the structure of 3a had a beyer-15-
ene-diol structure. The position of the hydroxyl groups and
relative configuration of 3a were examined by 2D-NMR in-
cluding HMBC and ROESY analyses. The HMBC spectrum
of 3a showed correlations of H-11 (dH 4.21, br t, Jꢂ5.4 Hz)
Fig. 1. Structures of 1—5, 1a and 3a
to C-8, C-9, C-12, and C-13 (dC 48.3, 59.7, 45.3, 42.5); H-18
Fig. 2. Important HMBC Correlations of 1, 3 and 5, and ROESY Correlations of 1, 3a and 5

1032
Vol. 56, No. 7
Table 1. 1H-NMR Spectrum of 1—4 and 3a (CDCl3, 500 MHz)a)
Position
1
2
3
4
3a
H-1
2
0.81 (1H, dd, 12.6, 3.6)
1.59 (1H, overlap)
1.51 (1H, overlap)
1.40 (1H, overlap)
1.33 (1H, overlap)
1.29 (1H, overlap)
1.10 (1H, dd, 10.8, 3.0)
1.39 (2H, overlap)
0.86 (1H, dd, 12.6, 3.0)
1.61 (1H, overlap)
1.55 (1H, overlap)
1.46 (1H, overlap)
1.38 (1H, overlap)
1.42 (1H, overlap)
1.18 (1H, dd, 11.4, 2.4)
1.41 (1H, overlap)
1.47 (1H, overlap)
1.34 (1H, dd, 13.8, 4.8)
1.63 (1H, overlap)
1.04 (1H, overlap)
1.25 (1H, overlap)
1.52 (1H, overlap)
1.27 (1H, overlap)
1.29 (1H, overlap)
1.06 (1H, d, 10.2)
1.44 (1H, overlap)
5.69 (1H, d, 5.4)
5.46 (1H, d, 5.4)
0.99 (3H, s)
1.06 (1H, overlap)
1.99 (1H, br d, 12.5)
1.47 (1H, overlap)
1.66 (1H, overlap)
1.38 (1H, overlap)
1.48 (1H, overlap)
1.13 (1H, dd, 11.5, 2.5)
1.50 (1H, overlap)
1.55 (1H, overlap)
1.38 (1H, overlap)
1.70 (1H, overlap)
1.29 (1H, d, 5.0)
1.02 (1H, dt, 3.6, 13.0)
1.97 (1H, dt, 13.0, 4.0)
1.51 (1H, overlap)
1.62 (1H, m)
1.30 (1H, overlap)
1.38 (1H, dt, 4.2, 13.8)
1.95 (1H, br d, 12.5)
3
5
6
1.00 (1H, dd, 11.5, 2.5) 1.16 (1H, dd, 11.4, 2.4)
1.45 (1H, ovrlap)
1.49 (1H, overlap)
1.40 (1H, overlap)
1.69 (1H, overlap)
1.31 (1H, d, 4.8)
7
1.27 (1H, overlap)
1.58 (1H, overlap)
0.99 (1H, overlap)
1.25 (1H, overlap)
1.50 (1H, overlap)
1.24 (1H, overlap)
1.29 (1H, overlap)
1.01 (1H, d, 9.6)
1.43 (1H, overlap)
5.67 (1H, d, 5.4)
5.45 (1H, d, 5.4)
0.99 (3H, s)
9
11
1.23 (1H, d, 4.5)
4.21 (1H, dt, 11.5, 5.0)
4.23 (1H, dt, 11.5, 5.0)
4.21 (1H, br t, 5.4)
12
14
1.70 (1H, overlap)
1.80 (1H, dd, 14.0, 5.0)
1.22 (1H, d, 10.0)
1.68 (1H, d, 10.0)
6.23 (1H, d, 6.0)
5.79 (1H, d, 6.0)
1.06 (3H, s)
3.77 (1H, d, 10.5)
3.98 (1H, d, 10.5)
0.96 (3H, s)
1.70 (1H, dd, 14.5, 2.0) 1.71 (1H, dd, 13.8, 5.4)
1.79 (1H, dd, 14.5, 5.0) 1.79 (1H, dd, 13.8, 2.4)
1.22 (1H, d, 9.6)
1.64 (1H, dd, 9.6, 3.6)
15
16
17
18
6.21 (1H, d, 5.5)
5.77 (1H, d, 5.5)
1.06 (3H, s)
3.71 (1H, d, 11.0)
3.89 (1H, d, 11.0)
0.90 (3H, s)
6.22 (1H, d, 6.0)
5.78 (1H, d, 6.0)
1.06 (3H, s)
3.10 (1H, d, 10.8)
3.39 (1H, d, 10.8)
0.84 (3H, s)
3.72 (1H, d, 11.4)
3.91 (1H, d, 11.4)
0.84 (3H, s)
3.79 (1H, d, 10.8)
3.98 (1H, d, 10.8)
0.89 (3H, s)
19
20
0.77 (3H, s)
0.80 (3H, s)
1.23 (3H, s)
1.20 (3H, s)
1.22 (3H, s)
2ꢄ,6ꢄ
3ꢄ,5ꢄ
7ꢄ
7.62 (2H, d, 8.4)
6.79 (2H, d, 8.4)
6.86 (1H, d, 12.6)
5.87 (1H, d, 12.6)
7.45 (2H, d, 9.0)
6.86 (2H, d, 9.0)
7.62 (1H, d, 16.2)
6.32 (1H, d, 16.2)
7.45 (2H, d, 8.5)
6.85 (2H, d, 8.5)
7.62 (1H, d, 15.5)
6.32 (1H, d, 15.5)
2.39 (1H, d, 12.0)
7.61 (2H, d, 8.5)
6.79 (2H, d, 8.5)
6.85 (1H, d, 13.0)
5.85 (1H, d, 13.0)
2.35 (1H, d, 12.0)
8ꢄ
OH at C-11
a) 1H-NMR assignments of 1—3 and 3a were obtained from studies of 2D-NMR, but the assignment of 4 was not perfect because of no 2D-NMR data was obtained due to the
low sample amount.
Table 2. 13C-NMR Data of 1—4 and 3a (125 MHz in CDCl3)
(dH 3.39, d, Jꢂ10.8 Hz, 3.10, d, Jꢂ10.8 Hz) to C-3, C-4, C-
Position
1
2
3
4
3a
5, and C-19 (dC 35.1, 37.4, 49.0, 18.0). NOE analysis
showed correlations of H3-19 (dH 0.84, 3H, s) to H-18 and
H3-20 (dH 1.22, 3H, s); H3-20 to H3-19, H-15 (dH 6.22, d,
Jꢂ6.0 Hz); H-9 (dH 1.31, d, Jꢂ4.8 Hz) to H-5 (dH 1.16, 1H,
dd, Jꢂ11.4, 2.4 Hz) and H-11; H-11 to H-9 and H-12 (dH
1.79, dd, 13.8, 5.4 Hz, 1.71, 1H, dd, Jꢂ13.8, 5.4 Hz); and H-
16 (dH 5.78, d, Jꢂ6.0 Hz) to H3-17 (dH 1.06, 3H, s) and H-15
as shown in Fig. 2. The configuration of the hydroxyl group
at C-11 was suggested to be b-axial from the coupling con-
stant (Jꢂ4.8 Hz) of H-9 of 3a. This was confirmed from the
NOE correlations of H-11 to H-9, H-12a, H-12b and H-1b
as shown in Fig. 2. These data indicated that the structure of
3a was determined to be beyer-15-ene-11b,18-diol. This
compound has not been reported previously, and the stereo-
chemistry of 3 was confirmed as shown in Fig. 1. The ab-
solute configuration of 3 has not been confirmed, but should
be supposed to be the same with as of 1, because both com-
pounds were isolated from the same source. Based on the
above analysis, compound 3 was established as 18-O-(E)-p-
coumaroylbeyer-15-ene-11b,18-diol and was named obtsurin
C.
C-1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
1ꢄ
2ꢄ, 6ꢄ
3ꢄ, 5ꢄ
4ꢄ
7ꢄ
8ꢄ
38.5
17.8
35.9
36.5
49.8
20.0
36.9
48.9
52.7
37.1
20.1
33.1
43.6
38.6
17.8
36.0
36.9
50.0
20.1
36.7
48.9
52.8
37.2
20.2
33.1
43.6
39.2
17.6
35.9
36.7
50.3
20.5
36.7
48.3
59.9
38.3
69.3
45.3
42.6
39.1
17.6
35.8
36.6
50.0
20.4
36.6
48.3
59.8
38.3
69.3
45.3
42.6
39.1
17.6
35.1
37.4
49.0
20.1
36.6
48.3
59.7
38.1
69.1
45.3
42.5
63.3
141.3
140.8
24.7
72.2
18.0
18.7
60.1
61.1
63.3
63.3
135.1
136.4
24.9
73.0
17.7
135.1
136.4
24.9
73.1
17.7
140.9
141.4
24.7
73.2
18.1
140.9
141.4
24.8
73.2
18.1
15.5
15.5
18.7
18.7
127.5
132.5
115.0
156.9
143.4
117.3
166.9
127.2
130.0
115.8
157.7
144.2
115.7
167.6
127.3
130.0
115.9
157.7
144.3
115.8
167.5
127.6
132.2
115.1
156.7
143.3
117.4
166.8
Compound 4 was obtained as an amorphous solid. The
molecular formula of 4 was determined to be C29H38O4 based
on the molecular ion peak at m/z 450.2789 in the HR-EI-MS.
This molecular formula was identical with that of 3. The 1H-
NMR data of 4 showed the presence of a (Z)-p-coumaroyl
moiety [dH 5.85 (1H, d, Jꢂ13.0 Hz), 6.85 (1H, d, Jꢂ
9ꢄ

July 2008
1033
13.0 Hz), 7.61 (2H, d, Jꢂ8.5 Hz), 6.79 (2H, d, Jꢂ8.5 Hz)] Table 3) except for a small difference in the chemical shifts
and a beyer-15-ene-11b,18-diol structure. The 13C-NMR of the carboxyl, an oxygenated quaternary carbon, and
spectrum data of 4 were almost identical with those of 3 ex- olefinic carbons. This data indicated that 5 should be a
cept for the (Z)-p-coumaroyl moiety (see Table 2). Thus, the sesquiterpene dimmer between hinokiic acid10) and crip-
structure of 4 was determined to be 18-O-(Z)-p-coumaroyl- tomeridiol.11) These two compounds were previously isolated
beyer-15-ene-11b,18-diol and was named obtsurin D.
from the same source and their structures and NMR data as-
Compound 5 was isolated as colorless needles, mp 56— signments were fully resolved.4) This expectation was con-
58 °C. The molecular formula of 5 was determined to be firmed by the upfield shift of C-12 (from dC 171.4 to 166.1)
C30H46O3 based on the molecular ion peak at m/z 456.3586 and the different olefinic carbon chemical shift at C-7 (from
[M]ꢀ in the HR-EI-MS. The IR spectrum of 5 showed ab- dC 131.2 to 133.5) and C-8 (from dC 135.6 to 131.4) related
sorption at 3454, 2930, 1703, 1650, 1470, and 1385 cmꢁ1 as- to hinokiic acid, and the downfield shift of C-4ꢄ (from dC
cribable to the hydroxyl, olefin, carbonyl, and alkyl groups. 73.1 to 85.6) and upfield shifts of C-3ꢄ (from dC 41.3 to 38.1)
The 1H-NMR spectrum of 5 showed the presence of a cyclo- and C-15ꢄ (from dC 30.3 to 18.8). Therefore, 5 was a
propane ring [dH 0.67 (1H, t, Jꢂ4.8 Hz), 0.77 (1H, dd, sesquiterpene dimmer at C-12 of hinokiic acid and C-4ꢄ of
Jꢂ9.0, 4.8 Hz)], seven singlet methyl groups [dH 0.67 (3H, criptomeridiol. Confirmation of the structures of the two
s), 0.93 (3H, s), 1.11 (3H, s), 1.17 (3H, s), 1.20 (3H, s), 1.21 parts were carried out by HMBC analysis as shown in Fig. 2.
(3H, s) 1.48 (3H, s)], and an olefinic proton [dH 6.34 (1H, The relative stereochemistry of each sesquiterpene part was
br d, Jꢂ6.0 Hz)] together with many alkyl proton signals determined by ROESY analysis (see Fig. 2). The absolute
(Table 3). The 13C-NMR spectrum of 5 gave 30 carbon peaks configuration should be same as those of hinokiic acid and
and showed the presence of a carboxyl group (dC 166.1), two criptomeridiol previously isolated from the same source.
olefinic carbons (dC 133.5, 131.4), two oxygenated quater- Thus, the structure of 5 was determined to be ent-cryp-
nary carbons (dC 85.6, 72.8), four nonoxygenated quaternary tomeridiol-4-yl-(ꢁ)-hinokiate as shown in Fig. 1. Compound
carbons (dC 31.4, 33.9, 34.6, 34.7), seven methyls (dC 18.8, 5 was stable under alkaline hydrolysis conditions. Compound
19.1, 26.8, 27.1, 27.2, 28.3, 29.0), and three methines (dC 5 was reacted with 5% KOH in MeOH under reflux for 3 h to
16.6, 49.9, 52.2) among other signals. The 13C-NMR chemi- recover 5 and no other product on TLC analysis. This should
cal shifts of 5 were almost same as the summation of the 13C- be based on the structure of 5, which is ester of the tertiary
NMR spectral data of hinokiic acid and criptomeridiol (see alcohol.
Partial cis–trans isomerizations between 1 and 2 and be-
tween 3 and 4 were observed after long term preservation at
room temperature. Thus the cis or tran isomers should be ar-
Table 3. The 1H- and 13C-NMR Data of Compound 5 (in CDCl3, 1H-
600 MHz, 13C-150 MHz)
tifacts through extraction and purification procedure.
Hinokiic Cripto-
acid meridiol
5
Position
Experimental
Melting points were measured with a Yanaco MP-3 micro-melting point
apparatus and the temperatures were not corrected. Optical rotations were
recorded on a JASCO P-1010 polarimeter at 25 °C. UV and IR spectra were
recorded on a U-2001 Spectrophotometer (Hitachi) and FT-IR Spectroscope
(Perkin Elmer), respectively. NMR spectra were recorded on a Bruker-DRX
(1H-NMR; 600 MHz, 13C-NMR; 150 MHz) and a JEOL-a-500 (1H-NMR;
500 MHz, 13C-NMR; 125 MHz) spectrometers using CDCl3 as the solvent
and tetramethylsilane (TMS) as an internal standard. HR-EI-MS data were
recorded on a JEOL-HX110 mass spectrometer. Preparative and analytical
HPLC were performed on reverse phase columns (Mighty sil RP-18 and 8,
Kantho Chemical Co., Ltd) with CH3CN–H2O and MeOH–H2O solvent sys-
tems. Silica gel 60 (Merck) was used for column chromatography. Analyti-
cal and preparative thin layer chromatography (PLC) were carried out on
precoated Kieselgel 60 F254 (0.25 mm and 0.5 mm thick, Merck).
dC
dH
dC
dC
1
2
3
4
5
6
7
8
9
36.9 1.14 (1H, overlap), 1.28 (1H, overlap)
19.4 1.76 (1H, qt, 13.8, 3.6), 1.45 (1H, m)
40.0 1.42 (1H, overlap), 1.25 (1H, m)
33.9
37.1
19.4
40.1
33.9
34.7
16.6
131.2
135.6
41.4
34.6
16.8 2.04 (1H, dd, 9.0, 4.8)
133.5
131.4 6.34 (1H, br d, 6.0)
41.4 1.68 (1H, dd, 18.0, 7.2)
1.89 (1H, dd, 18.0, 2.4)
31.4
11.4 0.67 (1H, t, 4.8), 0.77 (1H, dd, 9.0, 4.8) 11.5
166.1
29.0 0.67 (3H, s)
26.8 1.11 (3H, s)
10
11
12
13
14
15
1ꢄ
2ꢄ
3ꢄ
4ꢄ
5ꢄ
6ꢄ
7ꢄ
8ꢄ
9ꢄ
10ꢄ
11ꢄ
31.5
Plant Material The leaves of C. obtusa were collected in the mountain-
ous northern part of Hiroshima prefecture, Japan by Mr. T. Sano of Hi-
roshima Prefectural Forestry Research Center in October 2003, and a
voucher specimen was deposited in the Faculty of Life and Environmental
Sciences, Prefectural University of Hiroshima.
171.4
26.8
29.0
28.3
28.3 1.17 (3H, s)
40.5 1.13 (1H, overlap), 1.40 (1H, overlap)
19.7 1.58 1H, (overlap), 1.60 (1H, overlap)
38.1 2.75 (1H, br d, 10.5), 1.66 (1H, overlap)
85.6
52.2 1.66 (1H, overlap)
22.2 1.92 (1H, br s), 1.11 (1H, overlap)
49.8 1.40 (1H, overlap)
22.4 1.62 (1H, overlap), 1.32 (1H, overlap)
44.8 1.47 (1H, m), 1.23 (1H, overlap)
34.7
41.4
18.0
41.3
72.1
51.6
21.3
49.9
22.4
43.7
33.6
73.0
26.7
27.4
18.6
30.3
Extraction and Isolation The process for the extraction and isolation
was carried out as shown in our previous work.1) Air-dried leaves of C. ob-
tusa (3.9 kg) were refluxed with MeOH and the filtrate was evaporated to
give a residue (538 g). The extract was suspended in water and partitioned
successively with EtOAc and n-BuOH to afford an ethyl acetate soluble frac-
tion (270 g), a n-BuOH soluble fraction (251 g) and an aqueous residue. The
EtOAc fraction (110 g) was chromatographied on a silica gel column with a
gradient CHCl3–MeOH solvent system to afford ten fractions (Fr. 1—10).
Purification of some parts of the fractions was reported in our previous re-
port.1) Residual fraction Fr. 3 (26.5 g) was further separated by silica gel col-
umn chromatography using a hexane–EtOAc solvent system to give nine
fractions (Fr. 3-1—3-9). Fr. 3-4 (4.11 g) was further purified by preparative
HPLC with reverse phase columns and preparative layer chromatography
(PLC) to give compounds 1 (37.0 mg), 2 (18.7 mg), 5 (73.0 mg). Preparative
HPLC purification of Fr. 3-5 (0.50 g) gave compounds 3 (46.0 mg), and 4
72.8
12ꢄ, 13ꢄ 27.1 1.20 (3H, s)
27.2 1.21 (3H, s)
19.1 0.93 (3H, s)
18.8 1.48 (3H, s)
14ꢄ
15ꢄ

1034
Vol. 56, No. 7
(14.0 mg).
Alkaline Hydrolysis of 2 Compound 2 (9.0 mg) was hydrolyzed under
Compound 1: Colorless needles, mp 115—117 °C (MeOH). [a]D21 ꢀ20.8° alkaline condition to give 1a {[a]D ꢁ19.7° (cꢂ0.025)} (2.7 mg) and (E)-p-
(cꢂ0.037; CHCl3). HR-EI-MS: m/z 434.2823 [M]ꢀ (Calcd for C29H38O3,
coumaric acid (3.0 mg). (E)-p-coumaric acid was identified with the authen-
434.2821). IR nmax cmꢁ1 (KBr): 3340, 2931, 1687, 1632, 1606, 1514. UV tic compound by TLC and HPLC.
lmax nm (e) (MeOH): 226 (7184), 298 (8658), 310 (8623). 1H- and 13C-
NMR spectral data, see Tables 1 and 2.
Alkaline Hydrolysis of 3 Compound 3 (15.0 mg) was dissolved in 3 ml
of 3% KOH–MeOH and refluxed for 30 min. The reaction solution was
poured into ice water and extracted with EtOAc to give a EtOAc solution.
Compound 2: Colorless amorphous solid, mp 176—178 °C (MeOH).
[a]D21 ꢁ28.2° (cꢂ0.044; CHCl3). HR-EI-MS m/z: 434.2832 [M]ꢀ (Calcd for The EtOAc solution was evaporated and purified by PLC to give 3a (10 mg).
C29H38O3, 434.2821). IR nmax cmꢁ1 (KBr): 3372, 2931, 1679, 1632, 1605, The aqueous fraction was acidified and extracted with CHCl3 to give (E)-p-
1
1516. UV lmax nm (e) (MeOH): 227 (7068), 298 (9982), 311 (10798). H- coumaric acid (3 mg). (E)-p-Coumaric acid was identified with the authentic
and 13C-NMR spectral data, see Tables 1 and 2.
sample by TLC and HPLC.
Compound 3: Colorless amorphous solid. [a]D ꢁ22.3° (cꢂ0.014,
3a: An amorphous solid. HR-EI-MS m/z: 304.2395 [M]ꢀ (Calcd for
MeOH). HR-EI-MS m/z: 450.2784 [M]ꢀ (Calcd for C29H38O4, 450.2770). IR C20H32O2, 304.2402). [a]D ꢁ13.1° (cꢂ0.034, MeOH). IR nmax cmꢁ1 (KBr):
nmax cmꢁ1 (KBr): 3384, 2928, 1694, 1606, 1515, 1167. UV lmax nm (e) 3429, 2925, 1455, 1166, 1047. UV lmax nm (e) (MeOH): 244 (8591). H-
1
(MeOH): 241 (10480), 291 (8780), 323 (8080). H- and 13C-NMR spectral NMR spectral data, see Table 1 and 13C-NMR spectral data, see Table 2.
1
data, see Tables 1 and 2.
Compound 4: Colorless amorphous solid. HR-EI-MS m/z: 450.2798 [M]ꢀ References
(Calcd for C29H38O4, 450.2770). IR nmax cmꢁ1 (KBr): 3355, 2928, 1709,
1606, 1515, 1167. UV lmax nm (e) (MeOH): 242 (10260), 293 (8350), 316
(8090). 1H- and 13C-NMR spectral data, see Tables 1 and 2.
1) Xiao D., Kuroyanagi M., Itani T., Matsuura H., Udayama M., Mu-
rakami M., Umehara K., Kawahara N., Chem. Pham. Bull., 49, 1479—
1481 (2000).
Compound 5: Colorless needles, mp 56—58 °C (MeOH), [a]D21 ꢁ94.1°
(cꢂ0.037, CHCl3). HR-EI-MS m/z: 456.3586 [M]ꢀ (Calcd for C30H46O3,
456.3603 ). IR nmax cmꢁ1 (KBr): 3454, 2930, 1703, 1650, 1470, 1385, 1255,
1090. UV lmax nm (e) (MeOH): 242 (4115). 1H- and 13C-NMR spectral
data, see Table 2.
2) Gao H., Wu L., Kuroyanagi M., Harada K., Kawahara N., Nakane T.,
Umehara K., Hirasawa A., Nakamura Y., Chem. Pham. Bull., 51,
1318—1321 (2003).
3) Kuroyanagi M., Shimomae M., Nagashima Y., Muto N., Okuda T.,
Kawahara N., Nakane T., Sano T., Chem. Pharm. Bull., 53, 1519—
1523 (2005).
4) Kuroyanagi M., Ikeda R., Gao H., Muto N., Otaki T., Sano T., Kawa-
hara N., Nakane T., Chem. Pharm. Bull., 56, 60—63 (2008).
5) Anjaneyulu A. S. R., Rao V. L., Sreedhar K., J. Nat. Prod., 65, 382—
385 (2002).
6) Delgado G., De Vivar A. R., Cardenas J., Pereda-Miranda R., Hueta
E., Phytochemistry, 23, 2285—2288 (1984).
7) Konishi T., Konoshima T., Fujiwara Y., Kiyosawa S., J. Nat. Prod., 63,
344—346 (2000).
8) Ansell S. M., Pegel K. H., Taylor D. A. H., Phytochemistry, 32, 953—
959 (1993).
9) McCrindle R., Martin A., Murray R. D. H., J. Chem. Soc. (C), 1968,
2349—2354 (1968).
10) Takeshita H., Sato T., Muroi T., Ito S., Tetrahedron Lett., 10, 3095—
3096 (1969).
Alkaline Hydrolysis of 1 Compound 1 (7.4 mg) was dissolved in 3%
KOH/MeOH (4 ml) and heated at 85 °C for 3 h. The reaction solution was
poured into ice water and extracted with EtOAc. The EtOAc solution was
evaporated and purified by PLC to give 1a (2.6 mg). The aqueous fraction
was acidified and extracted with CHCl3. The CHCl3 solution was evaporated
to give p-coumaric acid (1.5 mg). The structure of 1a was analyzed by
NMR, HR-EI-MS spectra data, and optical rotation.
Compound 1a: Colorless amorphous solid. FAB-MS; m/z 311 [MꢀNa]ꢀ
1
C20H32O, [a]D21 ꢁ23.2° (cꢂ0.026, CHCl3), H-NMR (CDCl3) dH: 0.78 (3H,
s, Me-20), 0.79 (3H, s, C-19), 0.99 (3H, s, CMe-17), 1.03 (1H, d, Jꢂ9.8 Hz,
H-14), 1.16 (1H, dd, Jꢂ11.2, 2.9 Hz, H-5), 1.44 (1H, dd, Jꢂ9.8, 2.7 Hz, H-
14), 3.11 (1H, d, Jꢂ11.0 Hz, H-18), 3.41 (1H, d, Jꢂ11.0 Hz, H-18), 5.45
(1H, d, Jꢂ5.5 Hz, H-15), 5.69 (1H, d, Jꢂ5.5 Hz). 13C-NMR (CDCl3) dC:
15.6 (C-20), 17.8 (C-19), 17.9 (C-2), 19.9 (C-6), 20.2 (C-11), 24.9 (C-17),
33.2 (C-12), 35.4 (C-3), 37.0 (C7), 37.1 (C-10), 37.5 (C-4), 38.7 (C-1), 43.6
(C-13), 48.9 (C-8), 49.0 (C-5), 52.8 (C-9), 61.2 (C-14), 72.3 (C-18), 135.2
(C-15), 136.4 (C-16).
11) Morita M., Nakanishi H., Morita H., Mihashi S., Itokawa H., Chem.
Pharm. Bull., 44, 1603—1606 (1996).