Pinguisane and dimeric pinguisane-type sesquiterpenoids from the Japanese liverwort Porella acutifolia subsp. tosana

Article abstract of DOI:10.1016/S0031-9422(99)00451-3

Two new pinguisane-type, three new Diels-Alder reaction-type dimeric pinguisane sesquiterpenoids and known sesqui and diterpenoids were isolated from the ether extract of the Japanese liverwort Porella acutifolia subsp. tosana. Their absolute stereostructures were established by a combination of extensive 2D-NMR, CD spectra, X-ray crystallographic analysis, modified Mosher's method and chemical correlation. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0031-9422(99)00451-3

Phytochemistry 53 (2000) 593±604
www.elsevier.com/locate/phytochem
Pinguisane and dimeric pinguisane-type sesquiterpenoids from the
Japanese liverwort Porella acutifolia subsp. tosana
Toshihiro Hashimoto, Hiroshi Irita, Masami Tanaka, Shigeru Takaoka,
Yoshinori Asakawa*
Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima 770-8514, Japan
Received 1 June 1999; accepted 16 July 1999
Abstract
Two new pinguisane-type, three new Diels±Alder reaction-type dimeric pinguisane sesquiterpenoids and known sesqui and
diterpenoids were isolated from the ether extract of the Japanese liverwort Porella acutifolia subsp. tosana. Their absolute
stereostructures were established by a combination of extensive 2D-NMR, CD spectra, X-ray crystallographic analysis, modi®ed
Mosher's method and chemical correlation. # 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Porella acutifolia subsp. tosana; Jungermanniales; Hepaticae; Acutifolones A and B; Bisacutifolones A±C; Pinguisane- and dimeric
pinguisane-type sesquiterpenes; Diels±Alder reaction type; Biogenesis; Chemosystematics
1
. Introduction
tionation of the ether extract from a large amount of
P. acutifolia subsp. tosana resulted in the isolation of
two new pinguisane-type sesquiterpenoids, acutifolones
A (6) and B (7), and three dimeric pinguisane-type
sesquiterpenoids, bisacutifolones A (13), B (18) and C
(22), along with three known terpenoids (5-8) and their
structures were reported in preliminary publications
(Hashimoto, Irita, Tanaka, Takaoka & Asakawa,
1998a; Hashimoto, Irita, Tanaka, Takaoka & Asa-
kawa, 1998b). In this paper we report the absolute
stereostructures of the newly isolated compounds, their
biogenesis and describe some chemosystematic re-
lationships among the Porella species. In addition, the
structure of bisacutifolone B (18), previously reported
has been revised.
Liverworts contain oil bodies, which consist of lipo-
philic terpenoids and aromatic compounds (Asakawa,
982a, 1995). Porella species belonging to the Junger-
1
manniaceae are distributed in both northern and
southern hemispheres. From Japan, 18 Porella species
are described (Furuki & Mizutani, 1994). Porella
species are known to produce terpenoids with interest-
ing biological activities, for example, ®sh killing, anti-
microbial, ornithine decarboxylase and cathepsin B
inhibitory activity (Asakawa, 1990, 1995, 1998). The
genus Porella comprises two groups: (a) species with
pungent taste [(� )-polygodial] and (b) species without
a pungent taste. Porella acutifolia subsp. tosana
belongs to the latter group. Previously, we reported
the isolation and the structure elucidation of germa-
crane- and guaiane-type sesquiterpene lactones and
sacculatane-type diterpene dialdehyde from this liver-
wort (Toyota, Ueda & Asakawa, 1991). Further frac-
2. Result and discussion
Air-dried and ground P. acutifolia subsp. tosana col-
lected in Kochi, Japan was extracted with ether. The
crude extract was checked by TLC and GC±MS to
con®rm the presence of b-elemene (1), valencene (2),
bicyclgermacrene (3) and perrottetianal (8). The
remaining extract was subjected to column chromatog-
raphy on Silica gel and Sephadex LH-20 to a€ord two
*
Corresponding author. Tel.: +81-88-622-9611; fax: +81-88-655-
051.
E-mail address: asakawa@ph.bunri-u.ac.jp (Y. Asakawa).
3
0031-9422/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(99)00451-3

5
94
T. Hashimoto et al. / Phytochemistry 53 (2000) 593±604
Table 1
1
new pinguisane-type sesquiterpenes named acutifolone
A (6) and B (7) and three novel pinguisane-type ses-
quiterpene dimers named bisacutifolone A (13), B (18)
and (22), together with six known terpenoids (4, 5, 8).
13
H (600 MHz) and C NMR spectral data (150 MHz) for 6 and 7
a,b
(
3
in CDCl )
6
7
H
C
H
C
2
.1. 7-Oxopinguisenol-12-methyl ester (5)
1
2
2.21 m
1.68 m
45.7 3.24 m
31.4 1.55 m
2.20 m
27.3
33.2
Compound (5) is the key pinguisane-type sesquiter-
1.73 m
1.53 m
pene keto ester for the new compounds, which was
previously isolated from the title liverwort (Toyota et
al., 1991). However, their absolute stereochemistry
remained to be clari®ed. In order to establish its absol-
ute con®guration, CD spectrum was measured. It
showed a positive Cotton e€ect at 287 nm, indicating
that C-5 of 5 might have S con®guration. This
assumption was further con®rmed by application of
the modi®ed Mosher's method for a-methoxy-a-tri-
3
40.9 1.08 m
37.2
1.65 m
2.62 q (7.1)
1.77 dd (12.6, 8.0)
38.5 1.84 dq (7.1, 1.9)
160.3
4
5
6
7
8
9
1
1
45.5
84.6
50.0
203.5
72.3
52.3
5.99 s
124.3 2.49 dd (19.1, 1.9)
197.1 2.58 d (18.1)
67.3
49.9
0
1
6.46 dd (17.4, 11.0) 136.8 5.85 dd (17.4, 11.3) 134.4
5.45 d (11.0)
.69 d (17.4)
120.1 5.34 dd (11.3, 1.1)
5.48 dd (17.4,1.1)
171.2
115.9
5
¯
uoromethylphenylacetic acid (MTPA) ester (Ohtani,
1
1
2
3
168.7
20.1
19.4
12.9
Kusumi, Kashman & Kakisawa, 1991) to the diol (9)
obtained from 5. Reduction of 5 with NaBH4 gave
two alcohols (9) and (10). The NOESY spectrum of 9
indicated the NOEs between (1) H-7 and H-14 and (2)
1.25 d (7.1)
1.11 s
1.18 d (7.1)
15.6 1.41 d (7.7)
23.3 1.10 s
18.0 1.03 d (7.1)
14
15
COOMe
3.67s
51.1
H-7 and C -OH, whilst compound 10 showed the
5
a
NOE between H-7 and H-1. On the basis of the
NOEs, compound 9 possessed a 7a-hydroxyl group
and 10 a 7b-hydroxyl group. Since the H-NMR of 9
Coupling constants J (in Hz) in parentheses.
Assignments are based on DEPT, COSY, HMQC and HMBC.
b
1
showed that the hydroxyl group at C-7 was equatorial,
we applied the modi®ed Mosher's method for MPTA
ester of 9. Compound 9 was esteri®ed with (+)- and
determined by high-resolution mass spectrometry (HR-
MS) (m/z 262. 1552). The presence of an a,b,g,d-unsa-
�
1
turated ketone [269 nm ꢀlog e 4.18), 1657 cm ] and
�
1
esters (1732 cm ) was con®rmed by IR and UV spec-
(
(
� )-a-methoxy-a-tri¯uoromethylphenylacetyl chloride
1
13
MTPACl) and dimethylaminopyridine (DMAP) in
tra. The H- and C-NMR spectra (Table 1) of 6 were
similar to 7-oxopinguisenol-12-methyl ester (5) (Toyota
et al., 1991), except for the absence of one hydroxyl
group in place of a tetrasubstituted double bond, indi-
cating that 6 was the dehydrated compound of 5. The
location of the tertiary ole®n group at C-6 was estab-
lished by HMBC and NOESY experiments (Table 2).
In HMBC of 6, the H-6 proton was correlated to C-4,
C-5, C-7, C-8 and C-10 carbons. The NOESY spec-
trum showed the presence of NOEs between (1) H-6
and H-10, H-11 and (2) H-1 and H-4, indicating that 6
CH Cl to a€ord (+)-MTPA ester (11) and (-)-MTPA
2
2
ester (12), respectively. The values of di€erence of each
chemical shift ‰Dd values; dꢀ� ꢁ � dꢀ‡ꢁŠ for (+)-MTPA
ester (11) and (� )-MTPA ester (12) are shown in
Fig. 1. On the basis of the above results, the con®gur-
ation at C-7 is R, consequently that of C-5 in 5 must
have the S-con®guration.
2.2. Acutifolone A (6)
Compound 6 was obtained as colorless prisms, mp
02±1048C. The molecular formula, C H O , was
1
16
22
3
Table 2
3
HMBC and NOE correlations for 6 (600 MHz in CDCl )
H
HMBC correlation
NOE
1
2
3
4
6
C-2, C-7, C-8, C-9, C-13
C-1, C-3, C-8, C-9, C-13
C-1, C-2, C-4, C-8, C-9, C-14
C-3, C-6, C-9, C-14, C-15
C-4, C-5, C-7, C-8, C-10
C-4, C-5, C-6
H-3, H-4, H-13
H-1, H-14
H-1, H-11, H-14, H-15
H-10, H-11
1
1
1
1
1
0
H-6, H-10, H-15
H-4, H-6, H-10, H-15
H-1
1
3
4
5
C-5, C-10
C-1, C-2, C-8
C-3, C-4, C-8, C-9
C-4, C-5, C-9
H-3, H-4, H-15
H-4, H-10, H-11, H-14
Fig. 1. Dd values [d(� )� d(+)] for (+)-MTPA ester (11) and (� )-
MTPA ester (12) of 7a-hydroxypinguisenol-12-methyl ester (9).

T. Hashimoto et al. / Phytochemistry 53 (2000) 593±604
595
might be the 5,6-dehydroxylated compound of 5. Con-
clusive evidence for the absolute structure for 6 was
obtained from the chemical correlation. Dehydration
of 5 with POCl in pyridine gave a dehydrated product
3
the physical and spectral data of which were identical
to those of the natural product (6).
2.3. Acutifolone B (7)
Compound 7 was obtained as colorless prisms (mp
38±1408C). The molecular formula, C H O was
established by HR-MS (m/z 248.1406). The presence of
1
1
5
20
3
�
1
a d-lactone (1769 cm ; dC 168.7) and a ketone (1725
�
1
13
cm ; dC 203.5) was con®rmed by IR and C-NMR
1
13
spectra. The H- and C-NMR spectra (Table 1) of
compound 7 were very similar to those of 7-oxopingui-
senol-12-methyl ester (5), except for the presence of a
lactone group in place of one hydroxyl group and one
ester group, showing that 7 might be the C-12 and C-6
lactonized compound of 5. The relative stereostructure
of 7 was deduced from analysis of HMBC and
NOESY spectra (Table 3) and ®nally established by its
X-ray crystallographic analysis as shown in Fig. 2. The
absolute con®guration of 7 was suggested to be the
same as 5 and 6.
Fig. 2. ORTEP drawing of acutifolone B (7).
and one trisubstituted ole®nic group which were con-
rmed by the presence of only one ole®nic proton sig-
nal at dH 5.55 (d, J = 2.2 Hz). The location of the C-
±C-6 trisubstituted double bond was established by
the HMBC (Table 6) in which the H-6 correlated to
the C-4, C-5, C-8 and C-10 carbons and the H-10 cor-
related to the C-4, C-5, C-6. C-5', C-6', C-7' and C-
®
5
Table 4
1
2.4. Bisacutifolone A (13)
a,b
3
H NMR data for 13, 18 and 22 (600 MHz in CDCl )
Compound 13 was obtained as colorless prisms
H
13
18
22
(
was established by HR-MS (m/z 540.3058). The UV,
mp 204±2068C). The molecular formula, C H O
32
44
7
1
2
2.18 m
1.75 m
2.17 m
1.75 m
1.95 m
1.59 m
1.86 m
2.19 m
1.77 m
1.94 m
1.60 m
1.89 m
1
13
IR, H- and C-NMR spectra (Tables 4 and and 5) of
3 showed the presence of two a,b-unsaturated ketones
1
.96 m
1.59 m
.87 m
1
�
1
3
[
251 nm ꢀlog e 4.11); 1657 cm ; dC 195.1, 195.3] and
1
�
1
two methyl esters (1732 cm ; dH 3.66 s, 6H, 2 Â OMe;
4
6
2.91 dq (7.1, 2.2) 2.91 dd (7.1, 1.9) 2.91 dq (7.1, 1.9)
5.55 d (2.2) 5.82 d (2.2) 5.59 d (1.9)
3.58 dd (6.0, 5.9) 3.48 dd (5.9, 5.9) 3.55 dd (5.2, 4.7)
�
1
dC 171.2) and one allylic hydroxyl group (3513 cm
;
1
0
dH 4.35, bs; dC 68.9) which was con®rmed by the for-
1
1
1.61 m
.13 m
1.78 m
1.91 m
1.68 m
1.83 m
mation of an acetate (14) ꢀd 2.11, s, 3H; dH 5.50 bs,
H
2
1
13
1
H). The H- and C-NMR spectra of 13 also con-
1
3
4
1.06 d (6.9)
0.90 s
1.07 d (6.9)
0.91 s
1.06 d (6.9)
0.91 s
tained the signals of two tertiary methyls, four second-
ary methyls and one tetrasubstituted ole®nic group
1
15
1.29 d (7.1)
1.82 m
1.67 m
1.28 d (7.1)
1.76 m
1.64 m
1.28 d (7.1)
1.96 m
1.68 m
1
2
'
'
1
.78 m
1.56 m
.68 m
1.73 m
1.53 m
1.81 m
1.55 m
Table 3
3
'
3
HMBC and NOE correlations for 7 (600 MHz in CDCl )
1
1.81 m
2.63 d (7.0)
4.29 bs
1.73 m
2.31 q (7.1)
2.16 m
4
1
'
2.58 q (6.9)
4.35 bs
H
HMBC correlation
NOE
H-3
0'
2.36 m
1.65 m
1.81 m
1
2
3
4
6
C-2, C-7, C-8, C-9, C-13
C-1, C-3, C-8, C-9, C-13
C-1, C-2, C-4, C-8, C-9, C-14
C-3, C-6, C-9, C-14, C-15
C-4, C-5, C-7, C-8, C-10
C-5, C-6
11'
1.72 m
2.03 m
1.84 m
1.89 m
H-1, H-14
1
1
1
2
3'
4'
5'
0.97 d (6.6)
1.01 s
1.27 d (6.9)
1.00 d (6.6)
1.02 s
1.16 d (7.1)
0.98 d (6.9)
0.94 s
1.16 d (7.1)
H-6, H-14, H-15
H-4, H-10, H-11
H-6, H-11
1
1
1
1
1
0
 CO
2
Me
1
3
4
5
C-5, C-10
C-1, C-2, C-8
H-6, H-10
H-1
3.66 s
3.66 s
3.66 s
C-3, C-4, C-8, C-9
C-4, C-5, C-9
H-3, H-4, H-15
H-4, H-15
a
Coupling constants J (in Hz) in parentheses.
Assignments are based on DEPT, COSY, HMQC and HMBC.
b

5
96
T. Hashimoto et al. / Phytochemistry 53 (2000) 593±604
Table 5
13
a
3
C NMR spectral data for 13, 18 and 22 (150 MHz, CDCl )
C
13
40.5
18
40.8
22
41.1
C
13
43.8
18
44.7
22
43.1
1
2
3
4
5
6
7
8
9
1'
2'
3'
4'
5'
6'
7'
8'
30.7
35.9
40.4
168.5
124.0
195.1
69.5
52.5
37.0
24.8
171.2
14.5
19.2
13.0
51.0
30.8
36.2
40.6
169.3
124.2
195.7
69.4
52.4
37.2
25.0
171.2
14.7
19.4
13.2
51.0
30.9
36.3
40.9
169.5
124.1
195.5
69.6
52.4
36.6
27.7
171.4
14.5
19.6
13.3
51.0
31.4
41.0
40.8
159.6
133.1
195.3
67.3
49.3
68.9
29.6
171.2
13.7
23.3
18.3
50.9
31.6
42.1
40.0
159.3
133.2
195.4
66.9
48.1
65.6
28.7
171.3
13.8
24.1
18.1
51.0
31.3
40.1
42.8
159.6
132.1
194.1
67.9
49.6
30.0
18.3
171.4
13.9
22.1
16.1
50.9
b
e
b
e
9'
1
1
1
1
1
1
0
10'
11'
12'
13'
14'
15'
16'
1
2
3
4
5
c
f
h
c
f
h
Fig. 3. ORTEP drawing of bisacutifolone A (13).
CD spectrum of the p-bromobenzoate (15) of 9
showed a positive ®rst Cotton e€ect at 255 nm ꢀDe
+30.10) and a negative second Cotton e€ect at 230
nm ꢀDe � 3.21). The absolute con®guration of C-10'
was presumed to be R by the application of an exciton
chirality method (Koreeda, Harada & Nakanishi,
1974) to the conjugated enone system in CD spec-
trometry (Fig. 4). In order to obtain the more precise
establishment of the absolute con®guration of 13, the
modi®ed Mosher's method was carried out as men-
tioned earlier. Compound 13 was esteri®ed with (� )-
and (+)-MTPA chloride in dicyclohexylcarbodiimide
(DCC) and DMAP, to give (+)-MTPA ester (16) and
(� )-MTPA ester (17), respectively. The Dd values
‰dꢀ� ꢁ � dꢀ‡ꢁŠ (Fig. 5) revealed that the absolute con-
®guration at C-10' was represented as R. Thus, the
structure of 13 must be that of a Diels±Alder reaction-
type dimeric pinguisane sesquiterpenoid.
d
g
i
d
g
i
OMe
a
Assignments are based on DEPT, COSY, HMQC and HMBC.
Assignments interchangeable.
b±i
1
1' carbons. The NOESY spectrum (Table 6) of 13
indicated the presence of NOEs between (1) H-6 and
H-1', and H-10', (2) H-10 and H-4, and H-15 and (3)
H-10' and H-6, H-1', H-4', and H-15'. The additional
HMBC and NOESY spectral data are shown in Table
6
. On the basis of the above spectral evidence, a pin-
guisane-type sesquiterpene dimer (13), which could be
formed by Diels±Alder reaction of two pinguisane
ester (6), was deduced. This presumption was con-
®
shown in Fig. 3.
rmed by X-ray crystallographic analysis of 13 as
The absolute con®guration of 13 was elucidated fol-
lowing two experimental results described below. The
Table 6
3
HMBC and NOE correlation of 13 (600 MHz in CDCl )
HMBC
NOE
H-1
H-2
C-2, C-8, C-13
C-1, C-3, C-8
C-1, C-4, C-9, C-14
H-4, H-13
H-3
H-4
C-3, C-5, C-6, C-7, C-8, C-9, C-14, C-15
C-4, C-5, C-8, C-10
H-1, H-10, H-15
H-1', H-10'
H-4, H-15
H-6
H-10
H-11
H-13
H-14
H-15
H-1'
H-2'
H-3'
H-4'
H-10'
H-11'
H-13'
H-14'
H-15'
C-4, C-5, C-6, C-11, C-5', C-6', C-7', C-11'
C-5, C-6', C-10', C-11'
C-1, C-2, C-8
H-1
H-15
C-3, C-4, C-8, C-9
C-4, C-5, C-9
H-4, H-10, H-14
H-6, H-4', H-10', H-13'
C-2', C-7', C-8', C-12'
C-3', C-8', C-9', C-13'
C-1', C-2', C-8', C-9', C-14'
C-5', C-6', C-8', C-9', C-10', C-14', C-15'
C-5', C-6'
C-10, C-11, C-5', C-10'
C-1', C-2', C-8'
C-3', C-4', C-8', C-9'
C-4', C-5', C-9'
H-1', H-10', H-14', H-15'
H-6, H-1', H-4', H-15'
H-1'
H-4', H-15'
H-4', H-10', H-14'

T. Hashimoto et al. / Phytochemistry 53 (2000) 593±604
597
Table 7
NOE correlation for 18 and 22 (600 MHz in CDCl
22
H-4, H-13
3
)
18
H-1
H-4, H-13
H-4
H-6
H-1, H-10, H-15
H-1', H-10'
H-4, H-15
H-1, H-10, H-14, H-15
H-1'
H-4, H-15
H-10
H-13
H-14
H-15
H-1'
H-4'
H-10'
H-13'
H-14'
H-15'
H-1
H-15
H-1
H-4, H-15
H-4, H-10, H-14
H-6, H-4', H-10', H-13'
H-1', H-10', H-14', H-15'
H-6, H-1', H-4', H-15'
H-1'
H-4, H-10, H-14
H-6, H-4', H-10', H-13'
H-1', H-10', H-14', H-15'
H-1', H-4', H-15'
H-1'
Fig. 4. A positive Cotton e€ect for allyl benzoate (15).
2.5. Bisacutifolone B (18)
H-4', H-15'
H-4', H-10', H-14'
H-4', H-15'
H-4', H-10', H-14'
Compound 18 has the same molecular formula,
C H O (HR-MS: m/z 540.3093) as that of 13. The
3
2
44
7
ester (21), respectively. On the basis of the results of
the modi®ed Mosher's method, C-10' possessed the S
con®guration (Fig. 6), and thus, the absolute con®gur-
ation of 18 was depicted as C-10' epimer of 13. Pre-
viously, the structure of 18 was represented as C-10
isomer of 13, taking a strong hold of the result of the
positive Cotton e€ect of the benzoate (19) (Hashimoto
et al., 1998a, 1998b). But, this conclusion is ruled out
by the further experiments described above.
1
13
UV and IR absorption bands and H- and C-NMR
spectra (Tables 4 and 5) quite resembled those of 13.
13
The C-NMR spectrum of 18 was almost identical to
3, except for the C-10' and the presence of the posi-
tive ®rst Cotton e€ect at 259 nm in CD spectrum of
8 resembled that of 13. The above spectral data and
1
1
1
1
H± H COSY, HSQC, HMBC and NOESY exper-
iments (Table 7) suggested that 18 was the C-10' epi-
mer of 13. To con®rm this assumption, the CD
spectrum of p-bromobenzoate (19) of 18 was
measured. Contrary to expectation, a positive ®rst
Cotton e€ect at 259 nm was observed. This phenom-
enon could be explained by the fact that the absorp-
tion positions of Cotton e€ects of both p-
bromobenzoate and dienone are very close. Thus, the
allyl benzoate rule in CD spectrometry could not
apply to 19. To establish the absolute stereochemistry
of 18, the modi®ed Mosher's method for MTPA ester
of 18 was carried out. Compound 18 was esteri®ed
with (+)- and (� )-MTPA chloride in DCC and
DMAP to give (+)-MTPA ester (20) and (� )-MTPA
2
.6. Bisacutifolone C (22)
The molecular formula, C H O , has been estab-
44
32
6
lished by HR-MS (m/z 524.3141). The presence of an
a,b-unsaturated ketone [249 nm ꢀlog e 4.09), 1657
�
1
cm ] and an ester (1732 cm ) was con®rmed by IR
� 1
1
13
and UV spectra. The H-, C- (Tables 4 and 5), and
D-NMR spectra of compound 22 were almost identi-
cal to 13 and 18, except for the absence of one hy-
2
droxyl group in place of a methylene group, indicating
that 22 was the dehydroxylated compound of 13 and
1
1
8. This assumption was also con®rmed by 1H± H
COSY, HSQC, HMBC and NOESY spectral data
Fig. 5. Dd values [d(� )� d(+)] for (+)-MTPA ester (16) and (� )
MTPA ester (17) of bisacutifolone A (13).
Fig. 6. Dd values [d(� )� d(+)] for (+)-MTPA ester (20) and (� )
MTPA ester (21) of bisacutifolone B (18).

5
98
T. Hashimoto et al. / Phytochemistry 53 (2000) 593±604
(
Table 7) and the chemical correlation between 22 and
bisacutifolone A (13). Treatment of 13 with tosyl
chloride in pyridine did not give 10'b-tosylate (23) but
yielded 10'a-chlorinated compound (24) (C H O Cl;
32
43
6
HR-MS m/z 558.2769). It is considered that compound
24 might be obtained from tosylate (23) by attacking
of chloride anion as S 2 reaction as shown in Fig. 7.
N
Reduction with NaI and Zn in glyme (Fujimoto &
Tatsuno, 1976) a€orded a dechlorinated product the
spectral data of which including the CD spectrum were
identical to those of the natural 22. Thus, the absolute
structure of 22 was established to be dehydroxylated
bisacutifolone A (13) or B (22).
The possible biogenetic pathways of the three new
Diels±Alder reaction-type dimeric pinguisane sesquiter-
penoids (13, 18, and 22) are shown in Fig. 8. Firstly,
biological Diels±Alder cycloaddition reaction occurs
between 6±11 diene of acutifolone A (6) and 10±11
dienophile of another molecule which attacks from low
side. Then, 5'±10' double bond is isomerized to 5'-6'
Fig. 7. Formation of bisacutifolone C (22) from bisacutifolone A
13).
(
Fig. 8. Possible biogenetic pathway of bisacutifolone A±C (13, 18 and 22).

T. Hashimoto et al. / Phytochemistry 53 (2000) 593±604
599
to give bisacutifolone C (22). Furthermore, C-10' of 22
is hydroxylated to give two dimeric pinguisanes, bisa-
cutifolone A (13) and bisacutifolone B (18). In order
to con®rm the above biogenetic Diels±Alder-type
dimerization, compound 6 was treated with p-toluene-
sulphonic acid ( p-TsOH) in toluene (110±1208C, over
night) but only the starting material was recovered.
Many Diels±Alder-type dimeric cycloaddition com-
pounds have been isolated from higher plants and
fungi. Ichihara and Oikawa (1998) reported the iso-
lation of phytotoxins, solanapyrone A and B from
Alternaria solani which might be biosynthesized
through Diels±Alder-type reaction and proposed the
presence of solanapyrone synthase. Sporle, Becker,
Gupta, Veith and Huch (1989, 1991) found that the
liverwort Plagiochila moritziana produced plagiospiro-
lides (A±D) which might be biosynthesized through
Diels±Alder-type reaction from the sesquiterpene lac-
tone, diplophyllolide and the diterpene, fusicoccadiene.
The dimeric pinguisanes (13, 18, 22) might be syn-
thesized through enzymatic Diels±Alder reactions.
Pinguisane-type sesquiterpenoids are signi®cant
chemical constituents of the Porella species (Asakawa,
1982b, 1995). However, dimeric pinguisanes have not
been found in the other Porella species so far exam-
ined. It is considered that P. acutifolia subsp. tosana is
chemically di€erent from the other Porella species
because it contains pinguisane dimers as the major

6
00
T. Hashimoto et al. / Phytochemistry 53 (2000) 593±604
components. Previously, three guaianolides have been
isolated from P. acutifolia subsp. tosana collected in
di€erent localities (Toyota et al., 1991); these lactones
have not been detected in the present sample. This
result implies that there are two chemo types of P.
acutifolia subsp. tosana in Japan.
fractions. Fr. 1 (1.220 g) contained a mixture of b-ele-
mene (1), valencene (2) and bicyclogermacrene (3). Fr.
11±13 (3.469 g) was rechromatographed on silica gel
using CH Cl ±EtOAc gradient to give perrottetianal
(8) (1.350 g) and acutifolone B (7) (65 mg). Fr. 20±21
(2.236 g) was rechromatographed on silica gel using
the same solvent system described in Fr. 11±13 to
divide into two fractions, Fr. 20±21A (145 mg) and Fr.
2
2
3. Experimental
2
0±21B (248 mg). The former was chromatographed
on silica gel (n-hexane±EtOAc gradient) to a€ord acu-
tifolone A (6) (60 mg). The latter was puri®ed on silica
gel CC (n-hexane±EtOAc gradient) to yield 7-oxopin-
guisenol-12-methyl ester (5) (177 mg), acutifolone A
3
.1. General
Melting points were uncorrected. TLC was carried
out on silica gel precoated glass plates (Kieselgel 60
F254, Merck) with n-hexane±EtOAc (1:1, 2:1 and 4:1)
and CH C ±EtOAc (2:1 and 3:1). Detection was with
Godin reagent (Godin, 1954). For normal phase CC,
silica gel 60 (70±230 mm, Merck) and silica gel C-300
(230±400 mm, Wako) were used. The mixture of
CHCl ±MeOH (1:1) was used as solvent for CC on
Sephadex LH-20. UV and CD spectra were measured
in EtOH. ‰aŠ was measured in CHCl .
(
(
6) (32 mg) and bisacutifolone C (22) (10 mg). Fr. 29
1.026 g) was recrystallized from ether to give 4a,5b-
2
l2
epoxy-8-epiinnunolide (4) (664 mg). Fr. 35 (741 mg)
was chromatographed on Sephadex LH-20 using
CHCl ±MeOH (1:1), followed by silica gel CC
3
(
CH Cl ±EtOAc gradient) to give bisacutifolone A
2 2
3
(
13) (282 mg) and bisacutifolone B (18) (27 mg). Fr.
6, 37 (963 mg) was treated in the same manner
described above to give bisacutifolone A (13) (198
3
D
3
mg).
3.2. Spectral data
3
.5. Acutifolone A (6)
1
13
The H- and C-NMR spectra were obtained with a
Varian Unity 200 (200 MHz) or a Varian Unity 600
600 MHz) spectrometer. EI mass were measured at 70
19
D
Colorless prisms; mp 102±1048C; ‰aŠ +2.10 (c
+
(
1.73, CHCl ); HR-MS: m/z 262.1552, C H O
requires 262.1568; EI-MS: m/z 262 (M , 12%), 230
3
16 22
3
eV. The temperature programming of GC±MS analysis
was performed from 808C, then 80±2508C at 158C
(
(
3), 203 (4), 180 (10), 153 (3), 108 (100), 79 (9), 32
� 1
�
1
min
and ®nally isothermal at 2508C for 13 min.
15); FT-IR (KBr) cm : 1732 (COO), 1657 (C.O);
Injection temperature was 2608C. A fused silica col-
umn coated with DB-17 (30 m  0.25 mm i.d., ®lm
thickness 0.25 mm) using He as carrier gas (1 ml
1
13
UV: lmax nm ꢀlog e): 269 (4.18); H (600 MHz) and
C
NMR (150 MHz) data: (Table 1).
�
1
min ).
3
.6. Acutifolone B (7)
1
D
9
3
.3. Plant material
Colorless prisms; mp 138±1408C; ‰aŠ � 94.9 (c 0.66,
+
CHCl ); HR-MS: m/z 248.1406, C H O requires
3
3
15 20
Porella acutifolia (Lehm. et Lindenb.) subsp. tosana
Steph.) Hatt. was collected in May 1996 at Umazi,
2
48.1412; EI-MS: m/z 248 (M , 48%), 219 (28), 206
63), 178 (97), 165 (71), 150 (57), 123 (100), 95 (29), 55
1
43), 32 (21); FT-IR (KBr) cm : 1769 (COO); 1725
(
(
(
(
�
Kochi, Japan and identi®ed by YA and con®rmed by
Dr. M. Mizutani. A voucher specimen has been depos-
ited at the Faculty of Pharmaceutical Sciences,
Tokushima Bunri University.
1
13
C.O); H- (600 MHz) and C-NMR data: (Table 1);
�
3
CD: lmax nm ꢀDe): 297 (� 2.93), (c 8.1 Â 10 ); Crystal
data: Crystal dimensions = 0.5 Â 0.3 Â 0.1 mm, mono-
Ê
clinic, space group P2 with a ˆ 11:343 (0)A, b ˆ 8:296
1
Ê
Ê
3.4. Separation and isolation
(0)A, c ˆ 7:288 (0)A, b ˆ 106:507 (0)8, V ˆ 657:6
3
� 3
Ê
(
0)A , Z ˆ 2, Dx = 1.25 mg m , Dm = 1.30 mg
�
3
� 1
Porella acutifolia subsp. tosana was dried for 1 day
m
MXC 18 instrument. Final R value was 0.058 for 936
, and m(Cu Kaꢁ = 6.553 mm by Mac Science
and ground mechanically. The ground material (1.24
kg) was extracted with ether for 1 week. Filtration and
evaporation of the solvent gave a green oil (23.92 g).
A small amount of the crude extract was analyzed by
TLC and GC±MS to detect the presence of b-elemene
re¯ections.
3.7. Bisacutifolone A (13)
2
1
(
1), valencene (2), bicyclogermacrene (3) and perrotte-
Colorless prisms; mp 204±2068C; ‰aŠ +59.0 (c
+
requires 540.3087; EI-MS: m/z 540 (M , 100%), 480
D
tianal (8). The remaining extract was chromatographed
on silica gel using n-hexane±EtOAc gradient to give 37
0.51, CHCl ); HR-MS: m/z 540.3058, C H O
3
32 44
7

T. Hashimoto et al. / Phytochemistry 53 (2000) 593±604
601
1
(
(
(
40), 465 (15), 405 (12), 357 (8), 203 (8), 176 (11), 123
�
(COO); H-NMR (600 MHz): d 0.85 (3H, d, J = 6.9
Hz, H-15), 0.96 (3H, s, H-14), 1.09 (3H, d, J = 6.9
Hz, H-13), 3.77 (3H, s, COOMe ), 4.71 (1H, ddd, J =
2.5, 4.4, 12.5 Hz, H-7), 5.08 (1H, dd, J = 1.1, 10.7 Hz,
H-11), 5.21 (1H, dd, J = 1.1, 17.3 Hz, H-11), 5.83
(1H, dd, J = 10.7, 17.3 Hz, H-10).
1
15), 32 (25); FT-IR (KBr) cm : 3513 (OH), 1732
COO), 1657 (C.O); UV: lmax nm ꢀlog e): 251 (4.11);
1
13
H- (600 MHz) and
C-NMR (150 MHz) data:
(
0
Tables 4 and 5); Crystal data: Crystal dimensions =
.5 Â 0.2 Â 0.2 mm, orthorhombic; space group
Ê
Ê
P2 2 2 with a ˆ 13:720 (5)A, b ˆ 21:053 (7)A, c ˆ
1
1 1
3
Ê
Ê
9
:954 (5)A, V ˆ 2875:3 (2)A , Z ˆ 4, Dx = 1.25 mg
�
3
� 3
3.12. 7b-Hydroxypinguisenol-12-methyl ester (10)
m
mm by Mac Science MXC 18 instrument. Final R
, Dm = 1.30 mg m , and m(Cu Kaꢁ = 6.64
�
1
2
D
2
Colorless amorphous powder, ‰aŠ � 44.2 (c 0.52,
+
value was 0.046 for 2156 re¯ections. CD: lmax nm ꢀDe):
2
59 (+16.37), 226 (� 3.75) (c 9.0 Â 10� ).
5
3
16 26
CHCl ); HR-MS: m/z 282.1816, C H O requires
4
2
(
(
82.1831; EI-MS: m/z 282 (M , 3%), 246 (8), 194
15), 184 (66), 155 (100), 135 (16), 123 (29), 95 (20), 55
3
.8. Biscutifolone B (18)
�
1
39); FT-IR (KBr) cm : 3495 (OH), 1696 (COO); H-
1
2
D
0
NMR (600 MHz): d 0.88 (3H, d, J = 6.9 Hz, H-13),
Colorless oil; ‰aŠ +18.1 (c 0.74, CHCl ); HR-MS:
3
0
.97 (3H, d, J = 6.9 Hz, H-15), 1.14 (3H, s, H-14),
.79 (3H, s, COOMe ), 4.38 (1H, dd, J = 3.0, 5.2 Hz,
m/z 540.3093, C H O requires 540.3087; EI-MS: m/z
7
3
2
44
3
5
40 (100%), 522 (12), 480 (25), 462 (14), 402 (7), 368
H-7), 5.06 (1H, dd, J = 1.9, 10.7 Hz, H-11), 5.34 (1H,
dd, J = 1.9, 17.0 Hz, H-11), 5.66 (1H, ddd, J = 1.1,
10.7, 17.0 Hz, H-10).
(
(
7), 203 (8), 176 (11), 123 (16), 95 (9), 32 (52); FT-IR
�
1
KBr) cm : 3480 (OH), 1730 (COO), 1655 (C.O);
1
UV: l
nm ꢀlog e): 249 (4.15); H- (600 MHz) and
max
1
3
C-NMR (150 MHz) data: (Tables 4 and 5); CD: l
max
nm ꢀDe): 259 (+9.68), 236 (� 7.39) (c 8.6 Â 10� 5).
3
.13. Chlorination of (+)-MTPA
+)-MTPA (501 mg) in SOCl (1.5 ml) was re¯uxed
3
.9. Bisacutifolone C (22)
(
2
with NaCl (300 mg) at 908C for 50 h. The reaction
mixture was dissolved in a small amount of anhydrous
benzene. The reaction mixture was ®ltered through a
small column packed with cotton and concentrated in
vacuo to give (+)-MTPACl (438 mg) (Y. 80.8%).
2
D
1
Colorless amorphous powder; ‰aŠ +69.5 (c 0.36,
+
24.3138; EI-MS: m/z 524 (M , 100%), 464 (44), 449
CHCl ); HR-MS: m/z 524.3141, C H O requires
6
3
32 44
5
(
18), 436 (12), 389 (12), 341 (16), 229 (12), 203 (10),
1
�
1
75 (9), 123 (13), 95 (7), 40 (9); FT-IR (KBr) cm
732 (COO), 1657 (C.O), UV: l
:
1
nm ꢀlog e): 249
max
1
13
(
(
(
4.09); H- (600 MHz) and C-NMR data (150 MHz):
Tables 4 and 5); CD: lmax nm ꢀDe): 260 (+15.44), 238
3
.14. Chlorination of (� )-MTPA
�
5
� 7.82), (c 5.7 Â 10 ).
(
� )-MTPA (501 mg) in SOCl (1.5 ml) was re¯uxed
2
with NaCl (301 mg) at 908C for 25 h. The reaction
mixture was treated in the same manner described
above to give (� )-MTPA chloride (439.3 mg) (Y.
3.10. NaBH reduction of 5
4
To compound 5 (126 mg) in MeOH (10 ml) was
added NaBH (105.7 mg), and stirred at 08C for 30
min. The reaction mixture was concentrated in vacuo.
To the residue were added CH Cl (100 ml) and HCl
8
1.3%). (+)- and (� )-MTPA chlorides thus obtained
4
were used for the esteri®cation without further puri®-
cation.
2
2
(100 ml) The CH Cl layer was washed with sat. NaCl
2 2
solution and 1 N HCl and the organic layer was dried
3.15. Preparation of (+)-MTPA ester of 9
over MgSO . After removal of the solvent gave the
4
crude oil (122 mg) which was puri®ed by silica gel CC
(
To compound 9 (8.1 mg) in pyridine (0.25 ml) was
added (+)-MTPA chloride (0.05 ml), DMAP (24.4
mg) and stirred at room temperature for 2 h. The reac-
tion mixture was concentrated in vacuo and then the
residue was partitioned between CHCl and H O. The
CH Cl /EtOAc, gradient) to give 9 (44.3 mg, Y.
2 2
35.0%) and 10 (55.8 mg, Y. 44.2%).
3.11. 7a-Hydroxypinguisenol-12-methyl ester (9)
3
2
organic layer was washed with sat. NaCl solution, 1 N
HCl and 5% NaHCO3 successively and dried over
MgSO . Filtration and evaporation of solvent gave the
crude oil (119.6 mg) which was puri®ed by silica gel
CC (n-hexane/EtOAc, gradient) to a€ord (+)-MTPA
ester (11) (13.2 mg, Y. 92.3%).
2
D
2
Colorless amorphous powder; ‰aŠ � 72.1 (c 0.51,
+
CHCl ); HR-MS: m/z-H O 264.1697, C H O
16 24
3
2
3
4
requires 264.1726; EI-MS: m/z 282 (M , 1%), 264
(
18), 232 (56), 204 (34), 183 (89), 155 (49), 123 (100),
�
1
5 (28), 55 (26): FT-IR (KBr) cm : 3534 (OH), 1705
9

6
02
T. Hashimoto et al. / Phytochemistry 53 (2000) 593±604
3
.16. (+)-MTPA ester (11)
latetd with acetic anhydride (3 ml) at room tempera-
ture for 48 h. The reaction mixture was poured into
ice water and extracted with CHCl . The organic layer
3
2
1
Colorless oil; ‰aŠ +14.8 (c 0.70, CHCl ); HR-MS:
D
3
m/z 498.2194; C H O F requires 498.2230; EI-MS:
6 3
was washed with sat. NaCl solution, 1 N HCl, 5%
NaHCO and dried over MgSO and concentrated in
2
6
33
+
m/z 498 (M , 1%), 480 (6), 264 (17), 247 (87), 233
3
4
(
1
25), 205 (25), 189 (81), 187 (100), 145 (32), 121 (36),
�
vacuo to give the residue (48 mg) which was puri®ed
by prep. TLC (silica gel plate, n-hexane: EtOAc 2:1) to
furnish a monoacetate (14) (21.1 mg, Y. 37.5%).
1
05 (28), 55 (31), 32 (75); FT-IR (KBr) cm : 3536
1
(
OH), 1728 (COO); H-NMR (600 MHz): d 0.85 (3H,
d, J = 6.9 Hz, H-15), 0.94 (3H, d, J = 6.6 Hz, H-13),
.99 (3H, s, H-14), 3.68 (3H, s, COOMe), 5.10 (1H,
dd, J = 0.8, 10.7 Hz, H-11), 5.22 (1H, dd, J = 0.8,
0
3.21. Bisacutifolone A mono acetate (14)
1
D
9
1
1
7.3 Hz, H-11), 5.76 (1H, dd, J = 10.7, 17.3 Hz, H-
0), 6.20 (1H, dd, J = 4.9, 12.2 Hz, H-7).
Colorless oil; ‰aŠ +71.0 (c 0.65, CHCl ): HR-MS:
34 46 8
3
m/z 582.3212, C H O requires 582.3193; EI-MS: m/z
82 (M , 100%), 522 (35), 462 (17), 447 (10), 368
(10), 340 (11), 176 (9), 123 (15), 95 (7), 32 (10); FT-IR
+
5
3
.17. Preparation of (� )-MTPA ester of 9
�
1
(
KBr) cm : 1734 (COO), 1663 (C.O); UV l
nm
max
1
To compound 9 (7.3 mg) in pyridine (0.25 ml) was
ꢀlog e): 248 (4.30); H-NMR (200 MHz): d 0.90 (3H, s,
H-14), 0.96 (3H, d, J = 6.7 Hz, H-13'), 0.99 (3H, s,
H-14'), 1.06 (3H, d, J = 6.7 Hz, H-13), 1.17 (3H, d, J
= 6.9 Hz, H-15'), 1.29 (3H, d, J = 7.2 Hz, H-15),
2.11 (3H, s, OAc), 3.66 (3H, s, COOMe ), 3.67 (3H, s,
COOMe), 5.50 (1H, br.s, H-10'), 5.54 (1H, d, J = 2.2
added (� )-MTPA chloride (0.05 ml), DMAP (16.5 mg)
and stirred at room temperature for 2 h and the reac-
tion mixture was treated in the same manner as
described above to give the residue (110.2 mg), which
was further puri®ed by silica gel column CC (n-hex-
ane/EtOAc, gradient) to give (� )-MTPA ester (12) (8.8
mg, Y. 68.3%).
13
Hz, H-6). C-NMR (50): d 124.4 (d, C-6), 135.5 (s, C-
6'), 155.1 (s, C-5'), 167.0 (s, C-5), 170.1, 171.0, 171.2
(
s, C-12, 12' or OAc), 194.7, 195.0 (s, C-7 or 7').
3.18. (� )-MTPA ester (12)
3
.22. Preparation of p-bromo benzoate of 13
2
D
1
Colorless oil; ‰aŠ � 53.4 (c 0.81, CHCl ); HR-MS:
3
m/z 498.2229; C H O F requires 498.2230; EI-MS:
2
To compound 13 (11 mg) in pyridine (2 ml) was
6
33
6 3
+
m/z 498 (M , 1%), 480 (6), 264 (15), 247 (78), 189
90), 187 (100), 145 (38), 121 (32), 105 (33), 55 (29);
added p-bromobenzoyl chloride (104 mg), DMAP (58
mg) and the mixture stirred at room temperature for 2
h. The reaction mixture was concentrated in vacuo
and the residue partitioned between CHCl and H O.
The organic layer was washed with sat. NaCl solution,
1 N HCl, 5% NaHCO and dried by MgSO . The sol-
vent was evaporated under reduced pressure to obtain
the residue (71.6 mg), which was puri®ed by silica gel
column chromatography (n-hexane/EtOAc, gradient)
to give the benzoate (15) (15.5 mg, Y. 100%).
(
�
1
FT-IR (KBr) cm : 3530 (OH), 1728 (COO); UV lmax
1
nm ꢀlog e): 201 (4.19); H-NMR (600 MHz): d 0.85
3
2
(
3
6H, d, J = 7.1 Hz, H-13, H-15), 0.96 (3H, s, H-14),
.49 (3H, s, COOMe), 5.12 (1H, dd, J = 0.8, 10.7 Hz,
H-11), 5.24 (1H, dd, J = 0.8, 17.3 Hz, H-11), 5.80
1H, dd, J = 10.7, 17.3 Hz, H-10), 6.15 (1H, dd, J =
3
4
(
4
.9, 12.2 Hz, H-7).
3.19. Dehydration of 5
3
.23. Bisacutifolone A p-bromobenzoate (15)
To compound 5 (30.2 mg) in pyridine (2 ml) was
added POCl (0.22 ml), the mixture stirred for 1.5 h
2
D
3
Colorless oil; ‰aŠ +69.1 (c 0.55, CHCl ); HR-MS:
39 47 8
3
3
and re¯uxed for 2 days. The residue was partitioned
between CHCl and H O and the organic layer was
m/z 722.2432, C H O Br requires 722.2454; EI-MS:
+
m/z 724 100%), 722 (M , 93), 664 (18), 522 (33), 462
3
2
washed with sat. NaCl solution, 1 N HCl, 5%
NaHCO and dried over MgSO . After ®ltration and
(40), 402 (16), 368 (19), 340 (23), 185 (35), 183 (36),
�
1
123 (25), 32(21); FT-IR (KBr) cm : 1728 (COO),
1663 (C.O); UV l
3
4
evaporation of the solvent gave the residue (26 mg)
which was puri®ed by silica gel CC (n-hexane/EtOAc,
gradient) to give 6 (6.1 mg, Y. 21.6%) the physical
and spectroscopic data of which were identical to
those of natural acutifolone A (6).
nm ꢀlog e): 203 (4.27), 251 (4.23);
max
1
H-NMR (200 MHz): d 0.91 (3H, s, H-14), 0.97 (3H,
s, H-14'), 0.99 (3H, d, J = 4.4 Hz, H-13'), 1.07 (3H,
d, J = 6.7 Hz, H-13), 1.19 (3H, d, J = 7.0 Hz, H-
15'), 1.31 (3H, d, J = 7.1 Hz, H-15), 3.67 (3H, s,
COOMe), 3.68 (3H, s, COOMe ), 3.68 (1H, m, H-10),
5.58 (1H, d, J = 1.9 Hz, H-6), 5.75 (1H, br.s, H-10').
CD: lmax nm ꢀDe): 255 (+30.10), 230 (� 3.21), (c 1.5 Â
3.20. Acetylation of 13
�
3
Compound 13 (52 mg) in pyridine (3 ml) was acety-
10 ).

T. Hashimoto et al. / Phytochemistry 53 (2000) 593±604
603
3
.24. Preparation of (+)-MTPA ester of 13
2 h. The reaction mixture was treated in the same
manner as described above to furnish the benzoate
(19) (3.4 mg, Y. 65.9%).
Compound 13 (19 mg) in anhydrous CH Cl (4 ml)
2
2
was treated with (+)-MTPA (72 mg), DCC (90 mg),
DMAP (59 mg). The reaction mixture was treated in
the same manner as described in the preparation of
the MTPA ester of 11 to a€ord (+)-MTPA ester (16)
3.29. Bisacutifolone B p-bromobenzoate (19)
2
3
Colorless amorphous powder; ‰aŠ � 3.0 (c 0.10,
+
D
(
18.9 mg, Y. 69%).
CHCl ); HR-MS: m/z 722.2479, C H O Br requires
8
3
39 47
7
(84), 462 (100), 434 (27), 402 (45), 387 (33), 338 (31),
22.2454; EI-MS: m/z 724 (77%), 722 (M , 73), 522
3.25. (+)-MTPA ester (16)
2
02 (39), 200 (39), 185 (46), 183 (46), 123 (43), 91 (27);
� 1
2
D
0
Colorless oil; ‰aŠ +45.8 (c 0.71, CHCl ); HR-MS:
FT-IR (KBr) cm : 1728 (COO), 1663 (C.O); UV lmax
3
1
m/z 756.3492, C H O F requires 756.3486; EI-MS:
4
nm ꢀlog e): 247 (4.64); H-NMR (200 Mz): d 0.91 (3H,
2
51
9 3
+
m/z 756 (M , 100%), 696 (16), 522 (9), 462 (10), 368
s, H-14), 0.99 (3H, s, H-14'), 1.03 (3H, d, J = 6.0 Hz,
H-13'), 1.09 (3H, d, J = 6.8 Hz, H-13), 1.18 (3H, d, J
= 6.9 Hz, H-15'), 1.30 (3H, d, J = 7.1 Hz, H-15),
3.55 (1H, t, J = 6.9Hz, H-10), 3.66 (3H, s, COOMe ),
3.67 (3H, s, COOMe ), 5.74 (1H, br.s, H-10'), 5.83
(1H, d, J = 2.2 Hz, H-6). CD: lmax nm ꢀDe): 259
�
1
8), 189 (22), 123 (11), 32 (26); FT-IR (KBr) cm :
(
1
(
1
738 (COO), 1665 (C.O); UV lmax nm ꢀlog e): 204
1
4.05) 250 (4.10); H-NMR (600 Mz): d 0.89 (6H, s, H-
4, 14'), 0.92 (3H, d, J = 6.9 Hz, H-15'), 0.94 (3H, d,
J = 6.6 Hz, H-13'), 1.05 (3H, d, J = 6.9 Hz, H-13),
.26 (3H, d, J = 7.4 Hz, H-15), 3.64 (3H, s, COOMe),
.66 (3H, s, COOMe ), 5.51 (1H, d, J = 2.2 Hz, H-6),
.62 (1H, t, J = 4.3, 4.4 Hz, H-10').
�
3
1
3
5
(+11.56), 241 (� 10.77), (c 1.5 Â 10 ).
3.30. Preparation of (+)-MTPA reaction of 18
3
.26. Preparation of (� )-MTPA ester of 13
Compound 18 (3.2 mg) in anhydrous CH Cl was
2
2
stirred with (+)-MTPA (21 mg), DCC (23 mg),
DMAP (17 mg) (2 ml) at room temperature for 4
days. The reaction mixture was treated in the same
manner as described above to give (+)-MTPA ester
(20) (4.4 mg, Y. 99%).
To compound 13 (21 mg) in anhydrous CH Cl (4
2
2
ml) was added (� )-MTPA (37.1 mg), DCC (48.8 mg),
DMAP (24.3 mg) and stirred at room temperature for
1
day. To the reaction mixture was added (� )-MTPA
(
37.6 mg), DCC (48.6 mg), DMAP (25.3 mg) and
further stirred for 1 day. The reaction mixture was
treated in the same manner as described above to give
the crude oil (128 mg) which was puri®ed by silica gel
column chromatography (n-hexane/EtOAc, gradient)
to give (� )-MTPA ester (17) (22.7 mg, Y. 77%).
3.31. (+)-MTPA ester (20)
20
Colorless oil; ‰aŠ +9.0 (c 0.13, CHCl ); HR-MS:
D
3
m/z 756.3505, C H O F requires 756.3485; EI-MS:
9 3
42
51
+
m/z 756 (M , 53%), 755 (100), 696 (7), 522 (43), 462
39), 368 (20), 189 (55), 123 (22), 105 (22), 95 (12), 44
(
�
1
3.27. (� )-MTPA ester (17)
(14); FT-IR (KBr) cm : 1738 (COO), 1665 (C.O);
1
UV lmax nm ꢀlog e): 201 (4.38) 246 (4.31); H-NMR
2
D
0
Colorless oil; ‰aŠ +14.5 (c 0.77, CHCl ); HR-MS:
(600 MHz): d 0.83 (3H, s, H-14'), 0.89 (3H, s, H-14),
0.96 (3H, d, J = 6.3 Hz, H-13'), 1.06 (6H, d, J = 6.9
Hz, H-13, 15'), 1.26 (3H, d, J = 7.1 Hz, H-15), 3.62
(3H, s, COOMe ), 3.68 (3H, s, COOMe ), 5.61 (1H, d,
J = 2.2 Hz, H-6), 5.62 (1H, t, J = 4.8 Hz, H-10').
3
m/z 756.3482, C H O F requires 756.3485; EI-MS:
4
2
51
9 3
+
m/z 756 M , 100%), 696 (14), 522 (9), 462 (10), 368
11), 189 (29), 123 (15), 105 (10), 32 (13); FT-IR (KBr)
(
�
1
cm : 1738 (COO), 1665 (C.O); UV lmax nm ꢀlog e):
2
04 (4.16), 249 (4.20); 1H-NMR (600 MHz): d 0.71
(
(
(
3H, d, J = 6.9 Hz, H-15'), 0.79 (3H, s, H-14'), 0.89
3H, s, H-14), 0.93 (3H, d, J = 6.3 Hz, H-13'), 1.05
3H, d, J = 6.9 Hz, H-13), 1.27 (3H, d, J = 7.1 Hz,
3.32. Preparation of (� )-MTPA ester of 18
Compound 18 (2.0 mg) was stirred with (� )-MTPA
(20 mg), DCC (30 mg), DMAP (17 mg) at room tem-
perature for 5 days. Treatment of the residue (35 mg)
in the same manner as described above a€orded (� )-
MTPA ester (21) (2.6 mg, Y. 93%).
H-15), 3.62 (3H, s, COOMe ), 3.66 (3H, s, COOMe),
5
4
.53 (1H, d, J = 2.2 Hz, H-6), 5.64 (1H, t, J = 4.4,
.4 Hz, H-10').
3.28. Preparation of p-bromo benzoate of 18
3
.33. (� )-MTPA ester (21)
Compound 18 (3.8 mg) in pyridine (2 ml) was esteri-
ed with p-bromobenzoyl chloride (14.2 mg) in the
2
D
0
®
presence of DMAP (8.6 mg) at room temperature for
Colorless oil; ‰aŠ � 9.9 (c 0.13, CHCl ); HR-MS:
42 51 9 3
3
m/z 756.3466, C H O F requires 756.3485; EI-MS:

6
04
T. Hashimoto et al. / Phytochemistry 53 (2000) 593±604
+
m/z 756 (M , 52%), 755 (100), 695 (7), 522 (40), 462
� 5
)
nm ꢀDe): 260 (+10.42), 238 (� 5.71), (c 7.6 Â 10
whose HR-MS, EI-MS IR, UV, 1H- and C-NMR
were identical to those of the natural bisacutifolone C
(22).
13
(
(
36), 368 (18), 189 (49), 123 (19), 105 (20), 95 (10), 44
�
1
9); FT-IR (KBr) cm : 1736 (COO), 1665 (C.O); UV
1
lmax nm ꢀlog e): 202 (4.56), 245 (4.50); H-NMR (600
Mz): d 0.87(3H, s, H-14), 0.94 (3H, s, H-14'), 0.97
(
3H, d, J = 6.3 Hz, H-13'), 1.06 (3H, d, J = 6.9 Hz,
H-13), 1.13 (3H, d, J = 7.1 Hz, H-15'), 1.25 (3H, d, J
7.1 Hz, H-15), 3.64 (3H, s, COOMe ), 3.68 (3H, s,
3
.37. Reaction of acutifolone A (6) with p-TsOH
=
To compound 6 (21.1 mg) in toluene (2 ml) was
COOMe ), 5.55 (1H, d, J = 2.2 Hz, H-6), 5.58 (1H, t,
J = 4.1 Hz, H-10').
added p-TsOH (50 mg) and re¯uxed at 110±1208C for
4 h. The reaction mixture was extracted with CHCl3
and washed with H O, Na CO and the organic layer
2
2
2
3
3
.34. Reaction of 13 with p-toluenesulfonyl chloride
was dried over MgSO . Work-up as usual recovered
4
the starting material (6) (10.7 mg, Y. 50.7%).
To compound 13 (20.3 mg) in pyridine (1 ml) was
added p-toluenesulfonyl chloride (148 mg) and the
mixture was stirred at room temperature for 26 h. The
reaction mixture was partitioned between CHCl and
Acknowledgements
3
H O and the organic layer extracted sat. NaCl sol-
2
We thank Dr. M. Mizutani (The Hattori Botanical
Laboratory, Nichinan, Japan) for the con®rmation of
the liverwort species. This work was supported in part
by a Grant-in-Aid for Scienti®c Research (B) (No.
08459026) from the Ministry of Education, Science,
Sports and Culture of Japan.
ution, 1 N HCl and 5% NaHCO3 and dried over
MgSO . After ®ltration and evaporation of solvent
4
gave a residue (23.0 mg) which was puri®ed by silica
gel CC (CH Cl /AcOEt, gradient) to give 24 (18.6 mg,
Y. 88.6%).
2
2
3.35. 10'a-Chlorobisacutifolone C (24)
2
D
5
Colorless oil; ‰aŠ +12.4 (c 0.83, CHCl ); HR-MS:
3
References
m/z 558.2769, C H O Cl requires 558.2749; EI-MS:
3
2
43
6
+
+
m/z 560 (M +2, 5%), 558 (M , 12), 522 (98), 490
22), 462 (100), 432 (23), 402 (40), 374 (18), 338 (65),
27 (20), 176 (21), 153 (26), 123 (44), 95 (20), 3 (44);
Asakawa, Y. (1982a). In W. Herz, H. Grisebach, & G. W. Kirby,
Progress in the chemistry of organic natural products, vol. 42 (pp.
(
2
1±285). Vienna: Springer.
�
1
Asakawa, Y. (1982b). Journal Hattori Botanical Laboratory, 53, 283.
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their chemistry and chemotaxonomy (pp. 369±410). Oxford:
Clarendon Press.
FT-IR (KBr) cm : 1734 (COO), 1661 (C.O); UV lmax
1
nm ꢀlog e): 250 (4.21); H-NMR (600 MHz): d 0.90
(
(
(
3H, s, H-14), 0.99 (3H, d, J = 6.6 Hz, H-13'), 1.02
3H, s, H-14'), 1.07 (3H, d, J = 6.9 Hz, H-13), 1.15
3H, d, J = 7.1 Hz, H-15'), 1.28 (3H, d, J = 7.1 Hz,
Asakawa, Y. (1995). In W. Herz, G. W. Kirby, R. E. Moore, W.
Steglich, & Ch. Tamm, Progress in the chemistry of organic natu-
ral product, 65 (pp. 1±562). Vienna: Springer.
H-15), 3.66, 3.66 (6H, s, 2 Â COOMe ), 3.66 (3H, s,
Asakawa, Y. (1998). Journal Hattori Botanical Laboratory, 84, 91.
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Society of Japan, 6, 75.
COOMe ), 4.60 (1H, br.s, H-10'), 5.81 (1H, d, J = 2.2,
13
H-6). C-NMR (150 MHz): d 56.0 (d, C-10'), 124.4
d, C-6), 134.3 (s, C-6'), 155.6 (s, C-5'), 169.5 (s, C-5),
(
Godin, P. (1954). Nature, 174, 134.
Hashimoto, T., Irita, H., Tanaka, M., Takaoka, S., & Asakawa, Y.
1
7
71.1, 171.3 (s, C-12 or 12'), 195.2, 195.2 (s, C-7 or
').
(
1998a). Tetrahedron Letters, 39, 2977.
Hashimoto, T., Irita, H., Yoshida, M., Kikkawa, A., Toyota, M.,
Koyama, H., Motoike, Y., & Asakawa, Y. (1998b). Journal
Hattori Botanical Laboratory, 84, 309.
3.36. Reduction of 24
Ichihara, A., & Oikawa, H. (1998). Current Organic Chemistry, 2,
NaI (20.4 mg), Zn (15.8 mg) in glyme (2 ml) was
365.
added to compound 24 and stirred at 60±808C for 1 h
and then at room temperature for 16 h. The reaction
mixture was ®ltered through a short column packed
with celite and eluted with ether. The ether solution
Koreeda, M., Harada, N., & Nakanishi, K. (1974). Journal American
Chemical Society, 96, 266.
Ohtani, I., Kusumi, T., Kashman, Y., & Kakisawa, H. (1991).
Journal American Chemical Society, 113, 4092.
Sporle, J., Becker, H., Gupta, M. P., Veith, M., & Huch, V. (1989).
Tetrahedron, 45, 5003.
was washed with H O, sat. NaCl solution and dried
2
over MgSO . Work-up as usual gave the residue (7.8
4
Sporle, J., Becker, H., Allen, N. S., & Gupta, M. P. (1991).
Phytochemistry, 30, 3043.
mg), which was puri®ed by silica gel column chroma-
tography (n-hexane/EtOAc, gradient) to give (22) (1.8
Toyota, M., Ueda, A., & Asakawa, Y. (1991). Phytochemistry, 30,
567.
2
D
0
mg, Y. 18%): ‰aŠ +48.8 (c 0.05, CHCl ); CD: lmax
3