Diversity of Ligularia kanaitzensis in sesquiterpenoid composition and neutral DNA sequences

Article abstract of DOI:10.1016/j.tet.2008.02.036

Twenty-six eremophilane-type sesquiterpenoids, including six new compounds, were isolated from the title species. One of the new compounds, kanaitzensol, suggested the presence of an enzyme for the conversion of eremophil-7(11)-en-8-one derivative to furanoeremophilanes. The plant was collected at 15 locations in Yunnan Province of China and found to be diverse with respect to its sesquiterpenoid composition. DNA sequencing also revealed a large intraspecific diversity of the plant.

Full text of DOI:10.1016/j.tet.2008.02.036

Available online at www.sciencedirect.com
Tetrahedron 64 (2008) 4486e4495
www.elsevier.com/locate/tet
Diversity of Ligularia kanaitzensis in sesquiterpenoid composition
and neutral DNA sequences
Motoo Tori a, ,y, Aki Watanabe a, Sachie Matsuo a, Yasuko Okamoto a, Kana Tachikawa a,
*
Shigeru Takaoka a, Xun Gong b, ,z, Chiaki Kuroda c, ,x, Ryo Hanai d, ,{
*
*
*
a Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima 770-8514, Japan
b Kunming Institute of Botany, Chinese Academy of Science, Kunming 654204, China
c Department of Chemistry, Rikkyo University, Nishi-Ikebukuro, Toshima-ku, Tokyo 171-8501, Japan
d Department of Life Science, Rikkyo University, Nishi-Ikebukuro, Toshima-ku, Tokyo 171-8501, Japan
Received 8 December 2007; received in revised form 7 February 2008; accepted 12 February 2008
Available online 15 February 2008
Dedicated to Professor Kunio Iwatsuki, who has received an honorary distinction from the Japanese government for his achievements
in the field of plant systematics
Abstract
Twenty-six eremophilane-type sesquiterpenoids, including six new compounds, were isolated from the title species. One of the new
compounds, kanaitzensol, suggested the presence of an enzyme for the conversion of eremophil-7(11)-en-8-one derivative to furanoeremophi-
lanes. The plant was collected at 15 locations in Yunnan Province of China and found to be diverse with respect to its sesquiterpenoid compo-
sition. DNA sequencing also revealed a large intraspecific diversity of the plant.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Ligularia kanaitzensis; Furanoeremophilane; Internal transcribed spacers; Diversity
1. Introduction
diversity of compounds in root extract of Ligularia in order to
understand the mechanism(s) of generation of chemical diver-
sity.8e13 Ligularia in the Hengduan Mountains is suitable for
such studies because it is highly diversified and is believed to be
still evolving.14,15 Besides, because some Ligularia species pro-
duce furanoeremophilanes, survey of root chemicals in many
samples can be easily carried out by TLC and by visualization
of furans with Ehrlich’s reaction on TLC plates.16,17 We have
been also analyzing DNA sequences of non-coding regions in
order tocomplement theresultsof chemical analyseswith phylo-
genetic information.8e13 Combination of these independent
approaches has enabled us to describe diversity in more detail
andofferedsomeinsightintochemicalevolutioninsomespecies.
We have isolated furanoeremophilanes from all our samples
of Ligularia pleurocaulis (Franch.) Hand.-Mazz.,9 Ligularia
virgaurea var. virgaurea (Maxim.) Mattf.,10 Ligularia tongo-
lensis (Franch.) Hand.-Mazz.,8 and Ligularia cymbulifera
(W.W.Smith)Hand.-Mazz.,8 andfromsomesamplesofLigularia
Furanoeremophilanes and related sesquiterpenes are often
found in plants of Ligularia (Asteracea) and related genera
and have been studied since 1960s.1e7 Although hundreds of
furanoeremophilanes have been known to date, it is not known
how the diversity of these compounds has been generated
through plant evolution. We have been delineating intraspecific
* Corresponding authors. Tel.: þ81 88 602 8462; fax: þ81 88 655 3051
(M.T.); tel.: þ86 871 5223625 (X.G.); tel./fax: þ81 3 3985 2396 (C.K.);
tel./fax: þ81 3 3985 2377 (R.H.).
E-mail addresses: tori@ph.bunri-u.ac.jp (M. Tori), gongxun@mail.kib.ac.
cn (X. Gong), kuroda5000144@grp.rikkyo.ne.jp (C. Kuroda), hanai@rikkyo.
ne.jp (R. Hanai).
y
z
x
{
For structural determination.
For taxonomy.
For general information.
For genetic study.
0040-4020/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.02.036

M. Tori et al. / Tetrahedron 64 (2008) 4486e4495
4487
dictyoneura (Franch.) Hand.-Mazz.13 The composition of fura-
noeremophilanes and related sesquiterpenes, as well as DNA
sequence, was found to be diverse within most Ligularia species.
Each of L. pleurocaulis,9 Ligularia tsangchanensis (Franch.)
Hand.-Mazz.,11 and L. virgaurea10 can begrouped into two types.
The two types of L. pleurocaulis9 and L. tsangchanensis11 were
geographically separated, while those of L. virgaurea were
not.10 Intraspecific diversity was particularly high in L. dictyo-
neura.13 In contrast, L. cymbulifera was uniform.8 These results
indicated that complex mechanisms of evolution are at work to
generate the diversity in Ligularia, which led us to investigate
more species to obtain a fuller picture of the diversity.
In the present study, we focus on Ligularia kanaitzensis
(Franch.) Hand.-Mazz., which belongs to the section Ligu-
laria, series Racemiferae, and inhabits grassy slopes, swamps,
and scrubs of around 3000 m in altitude.18,19 The species are
divided into two varieties, var. kanaitzensis and var. subnudi-
caulis (Hand.-Mazz.) S.W. Liu, based on morphological differ-
ences such as the shape of foliose bract.18,19 However, in our
observation, the difference was not distinct but continuous.
Therefore, we do not distinguish the two varieties in the pres-
ent report. Wang and co-workers obtained two eremophilane
derivatives,20 as well as lignan,21 but no furanoeremophilane
from L. kanaitzensis collected near Lijiang city. Here we report
that L. kanaitzensis harbors chemical and generic diversity and
plants from other locations produce furanoeremophilanes.
Table 1
Collection locality, chemical composition, atpB-rbcL and ITS sequence of
L. kanaitzensis samples
Samplea Locality
Elevation Compositionb atpB-rbcLc ITSd
(m)
1
Jianchuan
Niujie
2800
2400
3200
2900
N
N
N
N
F
F
F
F
F
N
F
F
F
N
F
d
505A-A10
B
B
A
B
A
A
A
A
B
B
B
B
B
B
B
2
3
Sandawan
Yulongxueshan
d
605T
4
5
Baishuitai (Annan) 3000
Baishuitai (Annan) 3000
Baishuitai (Jiulong) 3600
Baishuitai (Jiulong) 3600
d
d
6
7
d
d
d
d
8
9
Luguhu
2700
3300
3200
3300
2900
3500
2400
10
11
12
13
14
15
a
Lidiping
Lidiping
Lidiping
Labadi
d
d
d
d
Jiushijiulongtan
Dali
505A-A10
Samples 2 and 7 were collected in 2002; sample 1 was collected in 2003;
samples 3 and 9 were collected in 2004; samples 4e6, 8, and 14 were
collected in 2005; samples 10e13 and 15 were collected in 2006.
b
Major components: F, furanoeremophilanes; N, non-furano eremophilanes.
‘d’: Same as deposited under accession AB281495. See text for the
c
others.
d
See Table 2 and text for details.
especially for the samples from the eastern Zhongdian area
(samples 5e8), indicating that the major furanoeremophilane
composition of these samples was the same.
2. Results
Chemical constituents of typical samples were examined.
Dried roots (samples collected in 2004e2006) were extracted
with EtOAc and fresh roots (samples collected in 2002 and
2003) were extracted with EtOH. The extracts were separated
by silica-gel column chromatography and HPLC. Twenty-six
eremophilanes 1e26, including six new compounds 1e6, were
isolated along with b-bisabolol (27),22 valeranone (28),23,24
Fifteen samples of L. kanaitzensis were collected in north-
western Yunnan Province of China in August of 2002e2006
(Table 1 and Fig. 1). The roots of each sample were extracted
with ethanol and the compounds therein were subjected to
Ehrlich’s test on TLC plates. The pattern of the TLC spots
that were positive to Ehrlich’s test was mostly the same,
Zhongdian
8
13
7
6
Changjiang River
9
5
10
12
3
11
14
4
Area of the left map
Kunming
Lijiang
1
Jianchuan
2
15
Dali
Figure 1. Locations where samples of L. kanaitzensis species (open squares) were collected. Filled triangles and open circles indicate major peaks and major cities,
respectively.

4488
M. Tori et al. / Tetrahedron 64 (2008) 4486e4495
24,44,45 25,10,46,47 and 267 were eremophilanes without a furan
ring, among which 25 has only 14 carbons (nor-eremophilane).
Although compound 10 seems to be an artifact due to extraction
with ethanol, the compound has not been reported.
H
10
H
OH OH
O
O
O
8
7
O
O
3
4
6
13
11
12
OH
O
R
2
From samples 1e3 and 14, only non-furano type of eremo-
philane compounds were isolated except for b-bisabolol
(27).22 Samples 4 and 10 contained similar compounds, together
with a small amount of furanoeremophilane(s). Samples 5e9
and 13 contained furanoeremophilanes as major components.
While enough amount of sample could not be collected for sam-
ples 11, 12, and 15, TLC analysis indicated that these samples
also include furano compounds as major components. Wang
and co-workers previously collected a L. kanaitzensis sample
in the Lijiang county, presumably not far from our collection
location of sample 3 or 4.20 Although they obtained 11-hydro-
peroxyeremophila-6,9-dien-8-one and 6-hydroxyeremophil-
7(11)-en-8,12-olide, these compounds were not isolated in the
present study. This difference may have arisen from the differ-
ence in the extraction procedure and/or in the sampling location.
The structures of the new compounds were determined as
follows.
1
R = OH
3
O
(eremofarfugin C)
(kanaitzensol)
17 R = H
OH
O
O
R
OR
O
O
6 R = Ac
8 R = H
9 R = Me
10 R = Et
4 R =
5 R =
O
O
O
11 R =
18 R = H
19 R =
O
O
O
O
12 R =
13 R =
H
O
COOH
R1 R2
Kanaitzensol (1) showed the presence of a hydroxy
(3400 cmꢀ1) and a carbonyl (1700 cmꢀ1) group in the IR spec-
trum. The CIMS spectrum exhibited the quasi-molecular ion
peak at m/z 237 and the molecular formula was deduced to be
O
O
2'
(2'S)-7 R =
(2'R)-7 R =
20 R1 = Me, R2 = H
21 R1 = H, R2 = Me
1
C15H24O2 by HRCIMS. The H NMR spectrum showed the
presence of a doublet methyl, a singlet methyl, and a singlet
methyl attached to an sp2 carbon atom as well as two sets of dou-
blet signals at d 3.78 and 3.83 (each one proton). The HMBC
spectrum clearly indicated correlations between H-15 and
C-3, 4, and 5, between H-14 and C-4, 5, 6, and 10, between
H-13 and C-7, 11, and 12, and between H-6 and C-5, 7, 8, and
11 (Fig. 2). Therefore, the planar structure was deduced to be
12-hydroxyeremophil-7(11)-en-8-one. Because the NOESY
spectrum showed correlation between H-14 and H-10, rings A
and B were judged to be cis-fused. The geometry of the 7(11)-
double bond was determined to have the E configuration by
the presence of NOE between H-12 and H-6 (Fig. 2).
O
O
O
H
O
OR
22
23
14 R = H
15 R = Me
16 R = Ac
H
O
H
H
24
25
O
O
O
Eremofarfugin C (2) exhibited absorptions at 3460 and
1800 cmꢀ1 in the IR spectrum, indicating the presence of a
hydroxy and an enol lactone groups, respectively. Because
signals in the 1H NMR spectrum were very broad at ambient tem-
perature, measurement was carried out at 60 ꢁC in C6D6. The 1H
NMR spectrum was very similar to those of eremofarfugins A48
and B.10 The HMBC spectrum at the elevated temperature clearly
showed the correlations illustrated in Figure 3, indicating the
eremophilane skeleton with an enol lactone moiety. The
A/B-cis stereochemistry with the C-11a methyl group was also
OH
26
HO
OMe
30
HO
H
H
O
27
28
HO
31
O
H
O
H
O
Br
O
H
H
O
O
O
29
O
32
HO
HMBC
Figure 2. Selected HMBC and NOE correlations detected for kanaitzensol (1).
bakkenolide (29),25,26 O-geranylconiferyl alcohol (30),27e29 and
lupeol (31).30,31 Compounds 6, 7,5 8,4 9,5 10, 11,32 12,33 13,33
14,1,34 15,35 and 1636 were furanoeremophilanes, and com-
pounds 1e5, 17,37 18,38,39 19,40 20,41 21,41 22,42 23,39,43
HO
NOE

M. Tori et al. / Tetrahedron 64 (2008) 4486e4495
4489
H
O
O
H
O
O
O
O
H
O
O
O
H
OH
OH
NOE
O
HMBC
HMBC
NOE
Figure 3. Selected HMBC and NOE correlations detected for eremofarfugin
C (2).
Figure 5. Selected HMBC and NOE correlations detected for compound 5.
1
The H NMR spectrum was similar to those of 4, 18, and 19.
The analysis of 2D NMR spectra (Fig. 5) led to the conclusion
that it was a propionyloxy derivative of 18 as depicted in the
formula including the stereochemistry.
established by the NOESY spectrum (Fig. 3). Thus, it was named
eremofarfugin C.
Compound 3 exhibited a quasi-molecular ion peak at m/z 353
and its molecular formula was deduced to be C19H28O6 by
HRMS. The IR spectrum indicated the presence of a hydroxy
group at 3350 cmꢀ1, an ester at 1730 cmꢀ1, and a lactone at
1760 cmꢀ1. The 1H NMR spectrum showed one proton singlet
at d 5.66, which is characteristic of a proton at the C-6 position.
The spectrum indicated the presence of five methyl groups, three
of which were doublet. The 13C NMR spectrum indicated sig-
nals of five methyls, four methylenes, three methines, and seven
quaternary carbons. Because the 13C NMR spectrum showed
a signal of a quaternary carbon at d 103.5, the C-8 position
was deduced to have a hydroxy group. The HMBC spectrum
showed connectivities between H-15 and C-3, 4, and 5, between
H-14 and 4, 5, 6, and 10, between H-13 and C-7, 11, and 12,
between H-30 or 40 and C-10 and 20, and between H-6 and C-10
(Fig. 4). An isobutyryl group was found to be at the C-6 position,
because the HMBC spectrum indicated correlation between H-6
and C-10. These results led to the conclusion that the compound
should be 8b,10b-dihydroxy-6b-isobutyryloxyeremophil-
7(11)-en-12,8-olide. Its stereochemistry was established by
the NOESY spectrum as depicted in the formula (Fig. 4).
Compound 4 had no furan ring but a 9-en-8-one system,
judged by NMR. Compound 19 and an angelate derivative
were reported by Bohlmann.40 A doublet methyl at the C-40
position was detected instead of a singlet methyl of 19. An ole-
finic proton was detected at d 6.95 as a quartet of quartets. The
methyl groups at d 1.42 and 1.82 had NOE cross peaks. There-
fore, the compound was determined to be a tiglate derivative.
Compound 5 exhibited a quasi-molecular ion peak at m/z 291
and the molecular formulawas deduced to be C18H26O3, indicat-
ing six degrees of unsaturation, by HRMS. The 1H NMR spec-
trum contained signals due to an ethyl group, a singlet and
a doublet methyl groups as well as a singlet methyl attached
to the double bond. Because two double bonds and two carbonyl
groups were identified by the 13C NMR spectrum, the compound
was judged to be bicyclic from the six degrees of unsaturation.
Compound 6 had a molecular formula of C17H24O4 and its 1H
NMR spectrum showed it to be an acetate. Two doublet signals
at d 2.99 and 2.71 with a coupling constant J¼17.4 Hz suggested
the presence of a hydroxy group at the C-10 position. Analysis of
the 2D NMR spectra indicated that this compound was furanoer-
emophilane-6b,10b-diol 6-O-acetate, which was known as a
derivative of 6,10-diol,4 although no report of isolation from
a natural source was found in the literature.
Compound 10 seems to be an artifact due to extraction with
EtOH. However, the data of the compound are given in
Section 5, since this compound was not found in the literature.
Although compound 7 had been reported by Tada and co-
workers5 the absolute configuration of the ester side chain was
not determined. Therefore, we synthesized both (20R)- and (20S)-
7 from diol 8. First, (20S)-7 was derived from 8 and (S)-2-methyl-
butyric anhydride. The doublet methyl group at the C-20 position
of (20S)-7 was observed at d 0.98. Then, diol 8 was treated with
rac-2-methylbutyric acid to afford a mixture of (20R)-7 (d 0.95,
H-50) and (20S)-7 (d 0.98, H-50). The H-50 of 7 from L. kanaitzensis
was at d 0.98 and the compound was established to be (20S)-7.
Although the carboxylic acids 20 and 21 were isolated in pure,
spectroscopic evidence was insufficient to assign their isomeric
configurations. Therefore, 21 was treated with p-bromophenacyl
bromide to give a crystalline compound 32. The structure was
solvedbythedirectmethodand refined byfull matrix least-square
on F2. The refinement converged to a R(all) value of 0.0438 using
all data and R(gt) value of 0.0410 for observed reflections. The
ORTEP drawing is shown in Figure 6 with the absolute structural
C(22)
C(18)
C(30)
C(19)
C(21)
O(4)
C(11)
O(12)
Br(1)
O(3)
C(6)
C(15)
C(23)
C(14)
C(17)
C(10)
O(2)
OH
HO
OH
OH
C(31)
O
C(20)
O
O
O
C(8)
C(9)
C(16)
C(27)
O
O
C(26)
C(13)
O
O
C(7)
C(5)
C(24)
HMBC
NOE
Figure 4. Selected HMBC and NOE correlations detected for compound 3.
Figure 6. ORTEP drawing of compound 32.

4490
M. Tori et al. / Tetrahedron 64 (2008) 4486e4495
parameter of ꢀ0.018. Therefore, compound 21 was established to
be 4S,5R,10R,11R-8-oxoeremophil-6-en-12-oic acid.
The other compounds were identified by comparing
spectral data with those in the literature or by analysis of 2D
NMR data.
furanoeremophilane-6b,10b-diol (8) and its 6-alkoxy (9 and
10) and 6-acyloxy derivatives (6, 7, and 11e13) as well as fur-
anoeremophilan-6b-ol (¼ligularol or petasalbin, 14) and its
derivatives (15 and 16). Samples of the other group (N in
Table 1) contained non-furano eremophilanes as major compo-
nents, such as eremophilan-8-one derivatives 17, 22, and 23.
We have been observing that production of a furan(s) appears
to be related to the ecological niche of the plant. For example,
L. cymbulifera, which produces furanoeremophilan-10b-ol
as the major component, makes extensive colonies in the
Zhongdian area.8,51 Similarly, we found that populations of
L. kanaitzensis that produce furanoeremophilane-6b,10b-diol
derivatives (samples 4e8, 11e13, and 15) were occupying a
predominant niche, whereas the other populations of the plant
(samples 1e3, and 14) were growing intermingling with other
plants (the population size at the locations of samples 9 and 10
was very small). Although the exact biochemical role(s) of
furanoeremophilanes has yet to be elucidated, this observation
reinforces our idea that production of furanoeremophilanes has
conferred an ecological advantage(s).13 It also accords with an
idea that plants that produce furanoeremophilanes must be
evolutionarily more advanced.
Analysis of the ITS1e5.8SeITS2 region revealed nucleo-
tide sites with additive bases in most samples and sequences
of different lengths in quite a few samples. Because DNA
sequencing was carried out using a single leaf of one individ-
ual, multiple ITS sequences result from differences among the
copies of the rRNA gene that exist in the thousands in higher
plants.52 The presence of multiple ITS sequences is believed to
be a result of past hybridization.53 Because we have seen
multiple ITS sequences in many samples of Ligularia species
that we have determined the sequence for10e13 hybridization
appears quite common in Ligularia, which suggests reticulate
evolution in the genus and possibly in closely related genera.54
Besides that past hybridization is clearly evident in most of the
samples of L. kanaitzensis, the present samples can be unam-
biguously divided into two groups. Moreover, when the data-
base is searched with Blast55 for sequences similar to the
ITS1e5.8SeITS2 sequence of sample 6 (type A), sequences
of L. virgaurea,10 L. tsangchanensis,11 Ligularia vellerea,56
and other plants were found to be more similar than the type
B sequence. Thus, the separation between the two types
appears as large as that expected to be observed between spe-
cies. Further studies are necessary to determine whether they
constitute separate species.
The nucleotide sequences of the atpB-rbcL intergenic
region in the plastid genome and the internal transcribed
spacers (ITSs) of the ribosomal RNA gene in the nuclear
genome were determined. Most of the samples had one
same sequence of the atpB-rbcL region and it has been depos-
ited to the database (accession AB281495). The common
sequence was mostly the same as the one we reported earlier
for L. tongolensis.8 It differed from the L. tongolensis sequence
at five sites: the 245th base is thymine (T) in place of guanine
(G); the 301st base is T in place of cytosine (C); the 469th base
is cytosine (C) in place of adenine (A); the number of Ts in
a stretch around the 390th position is 11 not 9; the number
of As in a stretch around the 510th position is 9 not 11 (the
base numbering is based on the L. tongolensis sequence).8
This type of atpB-rbcL sequence with Ts at the 245th and
the 301st, C at the 469th, and 11Ts has been seen in some
samples of Ligularia latihastata,12 Ligularia villosa,12 and L.
dictyoneura.13 Three samples had variations in the sequence
(Table 1). In samples 2 and 15, the 505th base was A in place
of C, and the number of A stretch around the 510th position
was 10 not 9. In sample 4, the 605th base was T in place of C.
The results of sequencing the ITSs with 5.8S rRNA gene
in-between (ITS1e5.8SeITS2 region) are summarized in
Table 2. A number of samples contained sequences of different
lengths. Therefore, we were unable to unambiguously deter-
mine the sequences in them. Still, the sequences clearly sepa-
rated into two groups. One consisted of samples 3 and 5e8
(type A in Table 1) and the other, the rest of the samples
(type B). L. kanaitzensis sequences in the database
(DQ272335 and AY458837) were type B. This separation
was supported by the neighbor-joining cladistic analysis using
the PAUP* program suite with a bootstrap value of 100%.49
3. Discussion
Six new compounds, kanaitzensol (1), eremofarfugin C (2),
and compounds 3e6, were obtained from L. kanaitzensis in
addition to many known compounds. Among the six new com-
pounds, kanaitzensol (1) isolated from sample 3 is especially
noteworthy. A study with rat cytochrome P450 has suggested
that a furanoeremophilane can be enzymatically synthesized
from eremophil-7(11)-en-8-one via a (Z)-12-hydroxy deriva-
tive.50 Compound 1 is a geometrical isomer of the proposed
intermediate with respect to the 7(11)-double bond. Com-
pound 1 would not form a furanoeremophilane because of geo-
metric mismatch, and in fact, no furan was isolated from
sample 3.
The spectrum of terpenoids isolated from the samples
appears more or less continuous. However, the samples can
be grouped roughly into two with respect to the major compo-
nents (Table 1). Samples of one group (F in Table 1) contained
furanoeremophilanes as major components, such as
Correlation between the chemical composition and the
DNA sequence has been seen in some Ligularia species,
most notably in L. virgaurea,10 L. dictyoneura,13 and L. vel-
lerea,56 which indicated that the chemical diversity was likely
to have been brought about by genetic alteration. In the present
case of L. kanaitzensis, no clear correlation was observed, as
was the case with L. dictyoneura,13 which was found to be
highly diverse both in chemicals and DNA sequences. No cor-
relation was observed between the DNA sequences and
morphology of the plants. We believe further accumulation
of data on other Ligularia species and on related plants will
reveal the structure of the evolution of eremophilanes.

Table 2
ITS1e5.8SeITS2 base sequence of L. kanaitzensis samplesa
ITS1
5.8S
ITS2
1
1
0
9
1
2
5
1
2
6
1
2
7
1
7
7
1
9
6
2
1
6
2
3
6
2
5
5
1
1
3
3
1
3
4
1
3
6
1
3
7
1
4
8
1
5
1
1
5
7
1
0
1
1
0
5
1
1
6
1
6
8
1
9
1
1
9
2
1
9
5
2
0
3
2
0
4
2
0
5
2
1
0
2
1
8
2
2
1
2
2
2
1
3
2
5
6
0
8
1
8
4
8
5
8
6
8
7
9
0
9
4
9
8
2
3
1
9
3
2
6
1
7
1
7
2
9
1
9
2
9
3
2
5
1
1b
2
c
a
c
a
t
g
a
t
g
t
c
g
t
c
C
C
C
C
C
C
C
C
C
S
g
g
c
g
c
c
c
c
g
c
c
c
c
c
c
t
c
g
c
c
t
a
t
a
a
c
c
t
C
S
c
t
t
c
t
c
t
c
c
C
C
C
C
C
C
C
C
c
A
A
A
A
A
A
A
A
a
C
C
C
C
Y
C
C
C
c
A
A
A
A
A
A
A
A
a
T
T
T
T
T
W
W
W
t
G
G
G
G
G
K
G
G
g
A
A
A
A
A
R
R
R
a
T
T
T
T
T
Y
T
T
t
G
G
G
G
G
K
G
G
g
T
T
T
T
T
T
T
T
t
C
C
C
C
C
C
C
C
c
G
G
G
G
G
G
G
G
k
T
T
T
T
T
T
T
T
t
C
C
C
C
C
C
C
C
c
G
G
R
G
G
G
G
G
g
G
G
G
G
G
R
R
G
g
C
C
C
C
C
C
C
C
c
G
G
G
G
G
G
G
G
g
C
C
C
C
C
C
C
C
c
Y
C
C
C
C
c
C
C
C
C
C
c
C
C
C
C
C
c
G
G
G
G
G
g
C
C
C
Y
Y
c
C
S
C
C
C
C
C
c
C
C
C
Y
C
c
C
C
C
C
C
c
C
C
C
C
C
c
T
T
T
T
T
t
C
Y
C
C
C
c
G
G
G
G
G
g
C
C
C
C
C
c
C
C
C
C
C
c
T
T
T
T
T
t
A
A
A
A
A
a
T
T
T
T
T
t
A
A
A
A
A
a
A
A
A
A
A
a
C
C
C
C
C
c
C
C
C
C
C
c
T
T
T
T
T
t
Y
Y
C
C
C
c
T
T
T
T
T
t
T
T
T
T
T
t
C
C
C
C
C
c
K
T
K
Y
T
t
Y
C
C
C
C
c
T
T
T
T
T
t
C
C
Y
C
C
c
C
C
C
C
C
c
11
9
C
C
C
M
C
C
C
C
C
C
C
C
C
C
C
C
C
c
4
13
10c
12d
15
14e
7f
c
c
c
g
c
c
c
c
c
c
t
c
g
c
c
t
a
t
a
a
c
c
t
c
t
t
y
t
c
t
c
c
C
c
C
C
C
C
C
C
Y
C
C
C
C
C
C
C
Y
C
G
G
G
G
G
G
K
G
C
C
Y
Y
Y
Y
C
C
C
C
C
C
C
C
C
C
C
C
Y
Y
Y
Y
C
C
C
C
C
C
C
C
C
C
C
C
Y
Y
Y
Y
C
C
C
C
Y
Y
Y
Y
C
C
T
T
C
C
C
C
C
T
C
C
C
C
C
C
C
C
G
G
A
A
A
A
A
G
C
C
C
C
C
C
Y
C
C
C
e
e
e
e
e
C
T
T
A
A
A
A
A
T
A
A
A
A
A
A
R
A
T
T
Y
Y
Y
Y
K
T
A
A
W
W
W
W
A
A
A
A
Y
Y
Y
Y
Y
A
C
C
T
T
T
T
T
C
C
C
Y
Y
Y
Y
Y
C
T
T
C
C
C
C
C
T
C
C
C
C
C
C
C
C
T
T
W
W
W
W
W
T
T
T
Y
Y
Y
Y
C
T
Y
Y
T
T
T
T
T
C
T
T
T
T
T
T
T
T
C
C
C
C
C
C
C
C
T
T
C
C
C
C
C
T
C
C
C
C
C
C
C
C
C
C
C
C
C
C
Y
C
y
t
c
r
t
g
a
t
g
y
y
y
y
y
T
c
g
c
t
A
A
A
A
A
C
g
g
y
e
e
e
e
e
G
y
c
5f
y
t
c
r
t
g
a
t
g
c
g
c
t
g
g
y
y
c
6f
y
t
c
r
t
g
a
t
g
c
g
c
t
g
g
y
y
c
8f
y
t
c
r
t
g
a
t
g
c
g
c
t
g
g
y
y
c
3g
Refh
a
y
t
c
r
t
g
a
t
g
y
g
c
t
g
g
y
y
c
C
A
C
A
T
G
A
T
G
C
G
T
C
G
G
C
C
C
Only the differences from the reference sequence are listed. ‘d‘ indicates deletion; K¼G+T; M¼A+C; R¼A+G; S¼C+G; W¼A+T; Y¼C+T. Bases in lower-case letters were determined from data on only one
strand because of the presence of two sequences of different lengths as noted below, making the base calling less accurate.
b
An additional sequence with six Cs in place of five Cs at the positions 155e159 in ITS1 was present.
An additional sequence with two Ts in place of three Ts at the positions 117e119 in ITS1 was present. Besides, an additional sequence with one T in place of two Ts at the positions 67 and 78 of ITS2 was
c
present.
d
An additional sequence with two As in place of one A at the 118th position of ITS2 was present.
An additional sequence with two Ts in place of three Ts at the positions 117e119 in ITS1 was present.
e
f
An additional sequence with six Cs in place of five Cs at sites 155e159 in ITS1 was present.
Sequence with six and seven Cs in place of five Cs at sites 155e159 in ITS1 was present.
Voucher 02269 (Pan and Gong; KIB); accession DQ272335.
g
h

4492
M. Tori et al. / Tetrahedron 64 (2008) 4486e4495
4. Conclusion
water-soluble starch was added for handling purposes. For the
samples collected in 2004e2006, roots were dried and extracted
with EtOAc at rt. Oily extracts were obtained by the standard
method.
Sample 1: the ethanol extract obtained from 80 g of fresh
root was separated by silica-gel column chromatography along
with HPLC to give 1737 (239.4 mg) and 2722 (3.3 mg).
Sample 2: the ethanol extract (1.3 g) was separated similarly
to give 4 (1.8 mg), 1737 (35.0 mg), 1838,39 (64.0 mg), and 1940
(2.5 mg).
Sample 3: the ethanol extract (648.8 mg) was separated sim-
ilarly to give 1 (3.0 mg), 1737 (438.8 mg), 1838,39 (5.8 mg), and
2242 (6.6 mg).
Sample 4: the EtOAc extract (1.68 g) was separated similarly
to give 1333 (4.2 mg), 1737 (58.8 mg), and 2925,26 (362.2 mg).
Sample 5: the EtOAc extract (721.7 mg) was separated sim-
ilarly to give 6 (25.2 mg), (20S)-75 (7.2 mg), 84 (9.5 mg), 1132
(29.9 mg), 1233 (4.5 mg), 141,34 (271.1 mg), 267 (1.1 mg), and
3027e29 (0.7 mg).
Six new compounds were isolated from root extracts of L.
kanaitzensis. Kanaitzensol (1) is an isomer of an intermediate
from eremophil-7(11)-en-8-one to furanoeremophilanes with
respect to the 7(11)-double bond.
The chemical composition was found to be diverse. The plant
produces furanoeremophilanes in the Baishuitai and the Lidiping
areas, where the plant was abundant. The results of sequencing of
the ITSs of the rRNA gene indicated that the populations in
BaishuitaiandthoseinLidipinghave undergonegenetic differen-
tiation. The chemical results and observation in the field seem to
support our previous premise that furanoeremophilanes, which
arelikelytobeproducedvia a 12-hydroxyderivative(s)oferemo-
phil-7(11)-en-8-ones, are evolutionarily more advanced among
eremophilanes and confer ecological advantage on plants.
5. Experimental
5.1. General
Sample 6: the EtOAc extract (612.7 mg) was separated simi-
larly to give 6 (17.6 mg), (20S)-75 (15.3 mg), 1132 (27.5 mg),
1233 (7.6 mg), 141,34 (100.9 mg), 1636 (3.2 mg), and 2040
(5.9 mg).
Sample 7: the ethanol extract obtained from 110 g of fresh
root was separated similarly to give 84 (3.1 mg), 95 (12.9 mg),
10 (10.4 mg), and 1535 (2.9 mg).
Sample 8: the EtOAc extract (2.69 g) was separated similarly
to give2 (21.3 mg), 3 (4.4 mg), 6 (10.9 mg), (20S)-75 (103.6 mg),
84 (22.9 mg), 1132 (200.8 mg), 141,34 (51.9 mg), 1636 (44.9 mg),
2041 (4.9 mg), 2141 (17.8 mg), 2510,46,47 (1 mg), 267 (2.5 mg),
2823,24 (3.5 mg), and 3027e29 (1.8 mg).
Sample 9: the ethanol extract (712.6 mg) was separated sim-
ilarly to give (20S)-75 (15.9 mg), 84 (119.2 mg), 10 (182.2 mg),
1132 (42.9 mg), 141,34 (300.2 mg), and 2444,45 (21.3 mg).
Sample 10: the ethanol extract (424.2 mg) was separated sim-
ilarly to give 4 (15.9 mg), 5 (1.0 mg), (20S)-75 (4.1 mg), 84
(2.9 mg), 1132 (5.1 mg) and 1838,39 (18.4 mg), 1940 (15.2 mg),
2339,43 (2.5 mg), and 3130,31 (1.5 mg).
Specific rotations and CDspectrawere measuredona JASCO
DIP-1000 and a JASCO J-725 auto recording polarimeters; IR
spectra, on a JASCO FT/IR-5300 spectrophotometer; H and
1
13C NMR spectra, on a Varian Unity 600 (600 MHz
and 150 MHz, respectively) and a JEOL ECP 400 (400 MHz
and 100 MHz, respectively) spectrometers. Mass spectra,
including high-resolution ones, were recorded on a JEOL
JMS-700 MStation. X-ray crystallographic analysis was carried
out on a Mac Science MXC 18 diffractometer using a DIP image
plate. Chemcopak Nucleosil 50-5 (4.6ꢂ250 mm) with a solvent
system of hexaneeethyl acetate was used for HPLC (a JASCO
pump system). Silica gel 60 (70e230 mesh, Fuji Sylisia) was
used for column chromatography. Silica gel 60 F254 plates
(Merck) were used for TLC. Ehrlich’s test on TLC was carried
out as previously described.8 Polymerase chain reaction and
purification of the products were carried out as described pre-
viously.8e10 DNA sequencing reactions were carried out with
the BigDye Terminator Ver 3.0 kit (Applied Biosystems) and
the primers described previously9,10 and analyzed on a 3130xl
sequencer (Applied Biosystems).
Sample 13: the ethanol extract (445.6 mg) was separated
similarly to give 6 (14.8 mg), (20S)-75 (24.6 mg), 84 (8.2 mg),
1132 (15.7 mg), 1333 (8.5 mg), and 3130,31 (1.0 mg).
Sample 14: the EtOAc extract (531.9 mg) was separated sim-
ilarly to give 1737 (56.5 mg) and 2339,43 (5.6 mg).
5.2. Plant materials
Samples of L. kanaitzensis were collected in August,
2002e2006 at 15 locations (Table 1 and Fig. 1). Each plant
was identified by Xun Gong, one of the authors.
5.3.2. Kanaitzensol A (1)
[a]1D6 ꢀ3.2 (c 0.3, EtOH); FTIR (KBr) 3400, 1700,
1630 cmꢀ1; MS (CI) m/z 237 [MþH]þ (base), 236, 219;
HRMS (CI) obsd m/z 237.1833 [MþH]þ (calcd for C15H25O2
237.1855); 13C NMR (600 MHz, C6D6) d 15.8 (C-15), 17.7
(C-13), 20.5 (C-2), 21.4 (C-14), 27.3 (C-1), 30.1 (C-4), 30.3
(C-3), 36.9 (C-5), 40.3 (C-6), 41.5 (C-10), 44.4 (C-9), 62.8
(C-12), 133.5 (C-7), 139.5 (C-11), 204.8 (C-8); 1H NMR
(600 MHz, C6D6) d 0.65 (3H, d, J¼6.9 Hz, H-15), 0.68 (3H, s,
H-14), 0.97 (1H, m, H-1), 1.02 (1H, m, H-3), 1.15 (1H, m,
H-2), 1.18 (1H, m, H-2), 1.38 (2H, m, H-3, 4), 1.41 (1H, m,
H-1), 1.47 (1H, dq, J¼11.0, 5.5 Hz, H-10), 1.68 (1H, d,
J¼15.1 Hz, H-6b), 2.10 (3H, d, J¼1.4 Hz, H-13), 2.17 (1H,
5.3. Extraction, purification, and structural determination
5.3.1. General procedure
For the samples collected in 2002 and 2003, roots were cut
into small pieces without drying, and immediately extracted
with EtOH at rt. The extract was filtered and concentrated to
afford an oily residue with an aqueous phase. EtOAc was added
to this oil/water mixture and the organic layer was recovered.
Evaporation of the solvent afforded an oily residue, to which

M. Tori et al. / Tetrahedron 64 (2008) 4486e4495
4493
dd, J¼15.9, 5.5 Hz, H-9b), 2.38 (1H, dd, J¼15.9, 11.0 Hz,
5.3.6. 3a-Propionyloxy-7bH-eremophila-9,11-dien-8-
H-9a), 2.50 (1H, d, J¼15.1 Hz, H-6a), 3.78 (1H, d,
J¼12.4 Hz, H-12), 3.83 (1H, d, J¼12.4 Hz, H-12).
one (5)
[a]2D0 þ15.5 (c 0.104, EtOH); FTIR (KBr) 1730, 1670,
1620 cmꢀ1; MS (CI) m/z 291 [MþH]þ (base), 217, 148, 89;
HRMS (CI) obsd m/z 291.1962 [M]þ (calcd for C18H27O3
5.3.3. Eremofarfugin C (2)
[a]1D9 þ6.8 (c 1.07, EtOH); FTIR (KBr) 3460, 1800 cmꢀ1
;
291.1960); C NMR (150 MHz, C6D6) d 9.3 (C-30), 10.3
13
MS (CI) m/z 251 [MþH]þ (base), 233; HRMS (CI) obsd m/z
251.1648 [MþH]þ (calcd for C15H23O3 251.1647); 13C
NMR (100 MHz, C6D6, 60 ꢁC) d 14.2 (C-13), 15.0 (C-15),
17.3 (C-14), 20.5 (C-2), 25.3 (C-9), 27.3 (C-1), 29.7 (C-3),
31.4 (C-4), 35.1 (C-10), 39.0 (C-11), 40.4 (C-5), 66.7 (C-6),
(C-15), 16.5 (C-14), 20.6 (C-13), 27.8 (C-20), 30.2 (C-1), 31.8
(C-2), 39.8 (C-5), 41.8 (C-6), 47.4 (C-4), 50.4 (C-7), 72.9 (C-
3), 113.7 (C-12), 125.0 (C-9), 144.2 (C-11), 164.6 (C-10),
1
173.2 (C-10), 196.5 (C-8); H NMR (600 MHz, C6D6) d 0.61
(3H, s, H-14), 0.66 (3H, d, J¼6.6 Hz, H-15), 1.00 (3H, t,
J¼7.4 Hz, H-30), 1.16 (1H, m, H-2a), 1.22 (1H, dq, J¼11.0,
6.6 Hz, H-4a), 1.32 (1H, m, H-1a), 1.62 (1H, t, J¼13.0 Hz, H-
6a), 1.67 (1H, m, H-1b), 1.71 (1H, dd, J¼13.0, 4.6 Hz, H-6b),
1.84 (3H, q, J¼0.5 Hz, H-13), 1.92 (1H, m, H-2b), 2.07 (2H,
q, J¼7.4 Hz, H-20), 2.88 (1H, dd, J¼13.0, 4.6 Hz, H-7), 4.82
(1H, m, H-12), 4.83 (1H, td, J¼11.0, 4.4 Hz, H-3b), 5.01 (1H,
t-like, J¼1.6 Hz, H-12), 5.64 (1H, d, J¼1.6 Hz, H-9); CD
(EtOH) [q]241 nm þ16,300, [q]324 nm ꢀ1600.
1
116.4 (C-7), 149.6 (C-8), 180.0 (C-12); H NMR (400 MHz,
C6D6, 60 ꢁC) d 0.66 (3H, d, J¼7.0 Hz, H-15), 0.76 (3H, s,
H-14), 1.03 (1H, m, H-1), 1.13 (3H, d, J¼7.3 Hz, H-13),
1.15e1.19 (2H, m, H-2, 3), 1.24e1.32 (2H, m, H-1, 2), 1.35
(1H, m, H-3), 1.45 (1H, m, H-4), 1.60 (1H, m, H-10), 1.80
(1H, br d, J¼15.4 Hz, H-9b), 2.02 (1H, dd, J¼15.4, 3.6 Hz,
H-9a), 3.02 (1H, m, H-11), 4.04 (1H, s, H-6).
5.3.4. 8b,10b-Dihydroxy-6b-isobutyryloxyeremophil-7(11)-
en-(12,8)-olide (3)
5.3.7. 6b-Acetoxyfuranoeremophilan-10b-ol (6)
[a]1D9 þ104.7 (c 0.44, EtOH); FTIR (KBr) 3560, 3350, 1760,
1730 cmꢀ1; MS (CI) m/z 353 [MþH]þ, 335 (base), 317, 265,
247; HRMS (CI) obsd m/z 353.1961 [MþH]þ (calcd for
C19H29O6 353.1964); 13C NMR (100 MHz, C6D6) d 8.7 (C-13),
10.6 (C-14), 16.3 (C-15), 18.7 (C-40), 18.9 (C-30), 21.7 (C-2),
29.8 (C-3), 33.2 (C-4), 34.3 (C-20), 34.8 (C-1), 43.8 (C-9), 47.4
(C-5), 71.8 (C-10), 74.2 (C-6), 103.5 (C-8), 130.0 (C-11), 152.0
[a]1D8 ꢀ39.4 (c 1.09, EtOH); FTIR (KBr) 1730, 3580 cmꢀ1
;
MS (CI) m/z 293 [MþH]þ, 275, 233 (base), 215; HRMS (CI)
obsd m/z 293.1743 [MþH]þ (calcd for C17H25O4 293.1753);
13C NMR (150 MHz, C6D6) d 8.2 (C-13), 10.0 (C-14), 16.1
(C-15), 20.2 (C-20), 22.3 (C-2), 29.2 (C-3), 33.5 (C-1), 33.8
(C-4), 34.1 (C-9), 45.5 (C-5), 70.5 (C-6), 75.0 (C-10), 115.6
(C-7), 119.6 (C-11), 139.1 (C-12), 151.9 (C-8), 169.5 (C-10);
1H NMR (600 MHz, C6D6) d 0.72 (3H, d, J¼6.0 Hz, H-15),
0.94 (1H, m, H-3), 1.00 (1H, m, H-4), 1.05 (3H, s, H-14),
1.07 (1H, m, H-3), 1.09 (1H, m, H-2), 1.36 (1H, m, H-2),
1.45 (1H, dd, J¼14.1, 3.0 Hz, H-1), 1.50 (3H, s, Ac), 1.72
(1H, m, H-1), 1.92 (3H, d, J¼1.1 Hz, H-13), 2.71 (1H, d,
J¼17.4 Hz, H-9a), 2.99 (1H, d, J¼17.4 Hz, H-9b), 6.37
(1H, s, H-6), 6.94 (1H, q, J¼1.1 Hz, H-12).
1
(C-7), 171.4 (C-12), 175.7 (C-10); H NMR (400 MHz, C6D6)
d 0.46 (3H, d, J¼6.6 Hz, H-15), 0.64 (1H, m, H-4), 0.86e0.93
(3H, m, H-2, 3, 3), 1.04 (3H, s, H-14), 1.05 (3H, d, J¼7.0 Hz,
H-40), 1.06 (3H, d, J¼7.0 Hz, H-30), 1.18 (1H, m, H-2), 1.32
(1H, br d, J¼12.5 Hz, H-1a), 1.65 (1H, td, J¼12.5, 3.6 Hz, H-
1b), 1.91 (3H, s, H-13), 2.07 (1H, d, J¼14.3 Hz, H-9a), 2.18
(1H, dd, J¼14.3, 6.2 Hz, H-9b), 2.33 (1H, m, H-20), 3.87 (1H,
br s, OH), 4.47 (1H, br, OH), 5.66 (1H, s, H-6).
5.3.8. 6b-Ethoxyfranoeremophilan-10b-ol (10)
5.3.5. 3a-Tigloyloxyeremophila-9,11-dien-8-one (4)
MS (CI) m/z 279 [MþH]þ, 233 (base), 232, 217, 152; 13C
NMR (150 MHz, C6D6) d 8.6 (C-13), 11.3 (C-14), 15.4 (C-20),
15.7 (C-15), 22.8 (C-2), 29.1 (C-3), 33.8 (C-4), 34.3 (C-1),
35.3 (C-9), 45.6 (C-5), 65.2 (C-10), 74.9 (C-10), 77.8 (C-6),
117.0 (C-7), 119.5 (C-11), 138.4 (C-12), 151.9 (C-8); 1H
NMR (600 MHz, C6D6) d 0.56 (3H, J¼6.6 Hz, H-15), 0.87
(3H, t, J¼7.1 Hz, H-20), 0.92 (1H, m, H-4), 0.94 (1H, m,
H-3), 1.12 (1H, m, H-2), 1.16 (1H, m, H-3), 1.29 (3H, s,
H-14), 1.42 (1H, m, H-2), 1.56 (1H, br d, J¼12.9 Hz, H-1a),
1.78 (3H, J¼1.2 Hz, H-13), 1.86 (1H, td, J¼12.9, 4.9 Hz,
H-1b), 2.93 (1H, d, J¼18.0 Hz, H-9b), 3.03 (1H, dq, J¼8.5,
7.0 Hz, H-10), 3.08 (1H, d, J¼18.0 Hz, H-9a), 3.36 (1H, dq,
J¼8.5, 7.0 Hz, H-10), 6.99 (1H, q, J¼1.2 Hz, H-12).
[a]1D8 þ21.7 (c 0.18, EtOH); FTIR (KBr) 1700, 1670,
1650 cmꢀ1; MS (CI) m/z 317 (base), 316 [M]þ, 305, 148;
HRMS (CI) obsd m/z 317.2105 [MþH]þ (calcd for C20H29O3
317.2116); 13C NMR (150 MHz, C6D6) d 10.4 (C-15), 12.2
(C-50), 14.2 (C-40), 16.5 (C-14), 20.7 (C-13), 30.2 (C-1), 31.8
(C-2), 39.8 (C-5), 41.9 (C-6), 47.6 (C-4), 50.4 (C-7), 73.2 (C-
3), 113.7 (C-12), 125.0 (C-9), 129.3 (C-20), 136.9 (C-30), 144.3
(C-11), 164.8 (C-10), 167.1 (C-10), 196.6 (C-8); 1H NMR
(600 MHz, C6D6) d 0.63 (3H, s, H-14), 0.70 (3H, d, J¼6.9 Hz,
H-15), 1.20 (1H, m, H-2a), 1.28 (1H, dq, J¼11.3, 6.9 Hz,
H-4), 1.42 (3H, dq, J¼7.1, 1.1 Hz, H-40), 1.64 (1H, dd,
J¼14.3, 12.9 Hz, H-6a), 1.69 (1H, m, H-1a), 1.71 (1H, dd,
J¼12.9, 4.4 Hz, H-6b), 1.82 (3H, quint, J¼1.4 Hz, H-50), 1.85
(3H, td, J¼1.4, 0.5 Hz, H-13), 1.87 (1H, dd, J¼3.4, 1.9 Hz,
H-1b), 2.02 (1H, m, H-2b), 2.89 (1H, dd, J¼14.3, 4.4 Hz,
H-7), 4.83 (1H, s, H-12), 4.93 (1H, td, J¼11.3, 4.4 Hz, H-3b),
5.00 (1H, t-like, J¼1.6 Hz, H-12), 5.64 (1H, d, J¼1.9 Hz,
H-9), 6.95 (1H, qq, J¼7.1, 1.4 Hz, H-30); CD (EtOH) [q]217 nm
þ32,800, [q]327 nm ꢀ2480.
5.3.9. 6b-((20S)-2-Methylbutyryloxy)furanoeremophilan-
10b-ol ((20S)-7)
[a]1D9 ꢀ33.0 (c 1.02, EtOH); FTIR (KBr) 3570, 3450,
1730 cmꢀ1; MS (CI) m/z 335 [MþH]þ, 334 [M]þ, 317, 249,
234, 233 (base), 232; HRMS (CI) obsd m/z 334.2144 [M]þ
(calcd for C20H30O4 334.2145); 13C NMR (100 MHz, C6D6)

4494
M. Tori et al. / Tetrahedron 64 (2008) 4486e4495
d 8.2, 10.2, 11.7, 16.0, 16.9, 22.3, 26.9, 29.2, 33.5, 33.9, 34.1,
1
41.5, 45.6, 70.3, 75.1, 115.8, 119.5, 139.2, 151.9, 175.2; H
purified by silica-gel column chromatography (hexaneeEtOAc,
in gradient) to isolate (20S)-7 (2.8 mg): [a]1D9 ꢀ28.3 (c 0.28,
EtOH).
NMR (600 MHz, C6D6) d 0.72 (3H, d, J¼7.4 Hz, H-15), 0.73
(3H, t, J¼7.4 Hz, H-40), 0.94 (1H, m, H-3), 0.98 (3H, d,
J¼6.8 Hz, H-50), 1.02 (1H, m, H-4), 1.06 (1H, m, H-3), 1.09
(1H, m, H-2), 1.10 (3H, s, H-15), 1.21 (1H, dq, J¼13.7,
6.6 Hz, H-30), 1.35 (1H, m, H-2), 1.48 (1H, br d, J¼13.5 Hz,
H-1), 1.59 (1H, dq, J¼13.7, 6.6 Hz, H-30), 1.74 (1H, m, H-1),
1.95 (3H, d, J¼1.2 Hz, H-13), 2.03 (1H, sextet, J¼6.6 Hz,
H-20), 2.74 (1H, d, J¼17.9 Hz, H-9b), 3.00 (1H, d, J¼
17.9 Hz, H-9a), 6.44 (1H, s, H-6), 6.94 (1H, q, J¼1.2 Hz, H-12).
5.5. Preparation of (20RS)-7
Diol 8 (38.4 mg) was treated with rac-2-methylbutyric acid
(37.2 mg) and DCC (64.0 mg) in CH2Cl2 (4 mL) in the presence
of DMAP (13 mg) at rt overnight. Water was added and the mix-
ture was extracted with EtOAc. The organic layer was washed
with 1 M HCl, satd NaHCO3 soln, and brine. After drying
over MgSO4, the solvents were evaporated invacuo to give a res-
idue (99.9 mg). The residue was purified by silica-gel column
chromatography (hexaneeEtOAc, in gradient) to isolate
(20R,S)-7 (26.6 mg). Attempts of separation of the 20S and 20R
derivative, by normal phase (Nucleosil 50-5, hexaneeEtOAc),
reversed phase HPLC (Cosmosil ODS, Cosmosil 5C18-Ar-II,
Cadenza CD, Cadenza Intakt: in MeOHeH2O or
CH3CNeH2O), Chiralcel OB-H, and Chiralcel OD-H (both in
hexaneeiPrOH), were not successful. 1H NMR (200 MHz,
C6D6) d 0.95 (d, J¼6.5 Hz, H-50R), 0.98 (d, J¼6.8 Hz, H-50S).
5.3.10. 8-Oxo-11S-eremophil-6-en-12-oic acid (20)
[a]1D9 þ18.7 (c 0.35, EtOH); FTIR (KBr) 3500e2500, 1710,
1680 cmꢀ1; MS (CI) m/z 251 (base) [MþH]þ, 233, 207;
HRMS (CI) obsd 251.1648 [MþH]þ (calcd for C15H23O3
251.1647); 13C NMR (100 MHz, C6D6) d 15.8 (C-15), 16.3
(C-13), 20.3 (C-14), 20.5 (C-2), 27.0 (C-1), 30.2 (C-3), 35.6
(C-4), 38.8 (C-11), 39.36 (C-5),* 39.42 (C-9, 10),* 136.8
(C-7), 156.0 (C-6), 179.1 (C-12), 197.9 (C-8) [*assignment
ambiguous]; 1H NMR (400 MHz, C6D6) d 0.60 (3H, d,
J¼7.0 Hz, H-15), 0.72 (3H, s, H-14), 0.92 (1H, m, H-3),
0.95 (1H, m, H-1), 1.07 (1H, m, H-3), 1.16 (2H, m, H-2),
1.26 (3H, d, J¼7.0 Hz, H-13), 1.28 (1H, m, H-1), 1.30 (1H,
m, H-4), 1.51 (1H, m, H-10), 2.05 (1H, J¼17.2, 4.4 Hz,
H-9b), 2.33 (1H, J¼17.2, 12.4 Hz, H-9a), 3.59 (1H, q,
J¼7.0 Hz, H-11), 6.36 (1H, s, H-6); CD (EtOH) [q]215 nm
þ8800, [q]253 nm ꢀ800, [q]340 nm þ450.
5.6. Preparation of 11R-acid phenacyl ester 32
A solution of 11R-acid (21, 16.3 mg), 2,40-dibromoacetophe-
none (77.8 mg), and Et3N (0.1 mL) in acetone (2 mL) was
stirred at rt for 1 h. Water was added and the mixture was
extracted with EtOAc. The organic layer was washed with
1 M HCl, satd NaHCO3 soln, and brine, dried (MgSO4), and
evaporated to give a residue. The residue was purified by
silica-gel column chromatography (hexaneeEtOAc) to give
a crystal (32, 24.3 mg): colorless plate; mp 134e136 ꢁC
(EtOAc); FTIR (KBr) 1740, 1700, 1670 cmꢀ1; MS (CI) m/z
449 and 447 [MþH]þ, 234, 233 (base), 232; HRMS (CI) obsd
5.3.11. 8-Oxo-11R-eremophil-6-en-12-oic acid (21)
[a]2D0 þ6.49 (c 1.34, EtOH); FTIR (KBr) 3500e2500, 1710,
1680 cmꢀ1; MS (CI) m/z 251 (base) [MþH]þ, 233, 207;
HRMS (CI) obsd 251.1646 [MþH]þ (calcd for C15H23O3
251.1647); 13C NMR (100 MHz, C6D6) d 16.1 (C-15), 16.6
(C-13), 20.7 (C-2, 14), 27.3 (C-1), 30.5 (C-3), 36.0 (C-4),
38.9 (C-11), 39.2 (C-5),* 39.6 (C-9),* 39.7 (C-11),* 136.8
(C-7), 155.8 (C-6), 178.7 (C-12), 197.6 (C-8) [*assignment
ambiguous]; 1H NMR (400 MHz, C6D6) d 0.65 (3H, d,
J¼7.0 Hz, H-15), 0.72 (3H, s, H-14), 0.96 (1H, m, H-3),
0.99 (1H, m, H-1), 1.08 (1H, m, H-3), 1.12 (1H, m, H-2),
1.16 (1H, m, H-2), 1.23 (3H, d, J¼7.0 Hz, H-13), 1.30 (1H,
m, H-1), 1.32 (1H, m, H-4), 1.54 (1H, m, H-10), 2.06 (1H,
dd, J¼17.2, 4.4 Hz, H-9), 2.36 (1H, J¼17.2, 12.1 Hz, H-9),
3.70 (1H, q, J¼7.0 Hz, H-11), 6.37 (1H, s, H-6); CD (EtOH)
[q]204 nm ꢀ3200, [q]238 nm þ8200, [q]239 nm þ900.
1
m/z 447.1175 [MþH]þ (calcd for C23H28O4Br 447.1171); H
NMR (200 MHz, C6D6) d 0.78 (3H, d, J¼6.8 Hz, H-15), 0.82
(3H, s, H-14), 1.48 (3H, d, J¼7.4 Hz, H-13), 2.17 (1H, dd,
J¼17.0, 4.8 Hz, H-9), 2.50 (1H, dd, J¼17.0, 12.0 Hz, H-9),
4.03 (1H, q, J¼7.0 Hz, H-11), 4.67 (1H, d, J¼16.2 Hz, H-10),
4.88 (1H, d, J¼16.2 Hz, H-10), 6.60 (1H, s, H-6), 7.03 (2H, d,
J¼9 Hz, H-50, 70), 7.20 (2H, d, J¼9 Hz, H-40, 80).
All diagrams and calculations were performed using maXus
(Bruker Nonius, Delft & MacScience, Japan). Mo Ka radiation,
˚
l¼0.71073 A, 3808 measured reflections, 3805 independent
reflections, 3581 observed reflections, data collection: DIP Im-
age plate, Program(s) used to refine structure: SHELXL-97
(Sheldrick, 1997). Crystal data: monoclinic, P21, a¼
5.4. Preparation of 6b-((20S)-2-methylbutyryl-
oxy)furanoeremophilan-10b-ol ((20S)-7)
10.8030(7) A, b¼8.0320(4) A, c¼12.0130(9) A, a¼90.00 ,
ꢁ
˚
˚
˚
3
ꢁ
ꢁ
˚
b¼92.954(2) , g¼90.00 , V¼1040.98 (12) A , R(all)¼0.0438,
R(gt)¼0.0410, wR(ref)¼0.1137, Flack parameter¼ꢀ0.018
(9).57 Crystallographic data (excluding structure factors) for
the structure of this compound have been deposited with
Cambridge Crystallographic Data Centre as supplementary
publication CCDC 669685. Copies of the data can be
obtained, free of charge, on application to CCDC, 12 Union
Road, Cambridge CB2 1EZ (fax: þ44-(0)1223-336033 or
e-mail:deposit@ccdc.cam.ac.uk).
Diol 8 (6.8 mg) was treated with (S)-(þ)-2-methylbutyric
anhydride (0.1 mL) in pyridine (0.1 mL) in the presence of
N,N-dimethylaminopyridine (DMAP) (5.8 mg) at rt for 2 days.
Methanol was added and the mixture was extracted with EtOAc.
The organic layer was washed with 1 M HCl, satd NaHCO3
soln, and brine. After drying over MgSO4, the solvents were evap-
orated in vacuo to give a residue (11.5 mg). The residue was

M. Tori et al. / Tetrahedron 64 (2008) 4486e4495
25. Naya, K.; Takagi, Y. Tetrahedron Lett. 1968, 629.
4495
Acknowledgements
26. Abe, N.; Onoda, R.; Shirahata, K.; Kato, T.; Woods, M. C.; Kitahara, Y.
Tetrahedron Lett. 1968, 369.
The authors wish to thank Mrs. Guowen Hu of Kunming
Institute of Botany for research coordination and Mr. Shoji
Yatsuhashi for assistance in the field. We also thank Prof.
Yuemao Shen for letting us use his laboratory facilities. This
work was conducted as part of Frontier Project ‘Adaptation
and Evolution of Extremophile’ of Rikkyo University, sup-
ported in part by the Ministry of Education, Culture, Sports,
Science and Technology of Japan. Financial support was
also obtained from JSPS (Grant-in-Aid for Scientific Research
No. 16404008).
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