Biotransformation of cubenene to 7-hydroxycalamenene in cultured cells of the liverwort, Heteroscyphus planus

Article abstract of DOI:10.1016/S0031-9422(98)00737-7

The biosynthesis of 7-hydroxycalamenene using suspension cultured cells of the liverwort, Heteroscyphus planus, is described. The biotransformation of cubenene, but not calamenene, to 7-hydroxycalamenene suggests that it is formed via hydroxylation of the methylene carbon between its unconjugated double bonds. Thus followed by oxidation, thereby explaining the formation of 7-hydroxycalamenene without invoking an NIH shift.

Full text of DOI:10.1016/S0031-9422(98)00737-7

Phytochemistry 51 (1999) 389±394
Biotransformation of cubenene to 7-hydroxycalamenene in
cultured cells of the liverwort, Heteroscyphus planus
Makoto Hashimoto, Reiko Hozumi, Masateru Yamamoto, Kensuke Nabeta*
Department of Bioresource Science, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro 080-0855, Japan
Received 27 May 1998; received in revised form 22 September 1998
Abstract
The biosynthesis of 7-hydroxycalamenene using suspension cultured cells of the liverwort, Heteroscyphus planus, is described.
The biotransformation of cubenene, but not calamenene, to 7-hydroxycalamenene suggests that it is formed via hydroxylation of
the methylene carbon between its unconjugated double bonds. Thus followed by oxidation, thereby explaining the formation of
7-hydroxycalamenene without invoking an NIH shift. # 1999 Elsevier Science Ltd. All rights reserved.
Keywords: Heteroscyphus planus; Jungemanniales; Cultured cells; Liverwort; 7-Hydroxycalamenene; Calamenene; Cubenene; Deuterium label;
Biotransformation; NIH shift
1. Introduction
Calamenenes and the less saturated sesquiterpenes,
cadalenes, which are hydroxylated at the aromatic
rings, occur in terrestrial plants and aquatic invert-
ebrates (Bowden, Coll, Engelhardt, Topiolas, & White,
1986). In vascular plants, calamenene and cadalene de-
rivatives, which are substituted at the aromatic ring
are widely distributed in liverworts of Jungemanniales
(Nabeta, Katayama, Nakagawara, & Katoh, 1993;
Asakawa, 1995; Nabeta, 1995). In paticular, hydroxyl-
ated calamenenes and cadalenes are frequently found
in liverworts (Asakawa, 1995) and in the leaves and
cotyledons of cotton during the hypersensitive re-
sponse to bacterial pathogens (Essenberg, & Pierce,
1995). Phytoalexins of the hydroxylated cadalene-type
were isolated from the leaves and the cotyledons of
cotton (Gossypium spp.). Evidence supporting the
intermediacy of 6E-farnesyl diphosphate (FPP, 1) in
the biosynthesis of gossypol was obtained in two stu-
dies (Maciadri, Angst, & Arigoni, 1985; Stipanovic et
al., 1986). Davis, and Essenberg, 1995 recently pro-
vided evidence that (+)-d-cadinene is an enzymatic in-
termediate in the biosynthesis of cotton phytoalexins.
However, the `missing links' in the biosynthesis of
oxygenated calamenenes and cadalenes from hydro-
carbons of the cadinane type or corresponding carbo-
cations in infected cotton plants remain to be
identi®ed.
Cultured cells of the liverwort Heteroscyphus planus
produce 7-hydroxycalamenene (2) together with 7-
methoxycalamenene (3), 7-hydroxy-1,2-dihydrocada-
lene (4), 7-methoxy-1,2-dihydrocadalene (5) and 7-
methoxycadalene (6). 1, 2- and 1, 3-hydride shifts in
2
their formation were veri®ed by distribution of H and
13C atoms in the biosynthetically labeled compounds
incorporating various H- and 13C-labeled mevalonates
2
(Nabeta, Mototani, Tazaki,
&
Okuyama, 1994;
Nabeta, Ishikawa, Kawae, & Okuyama, 1994) (Fig. 1).
The formation of 7-hydroxycalamenene (2) may be the
®rst step in biosynthesis of hydroxylated derivatives.
However, the mode of aromatization and introduction
of a hydroxy group in the aromatic ring still remain to
be established. No NIH shift was observed at the 7
and 8 positions in the biosynthesis of 2 with feeding of
deuterated mevalonic acid (Nabeta et al., 1994). Thus,
it is unlikely that compound 2 is directly formed from
* Corresponding author.
0031-9422/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(98)00737-7

390
M. Hashimoto et al. / Phytochemistry 51 (1999) 389±394
Fig. 2. Chemical conversion of ( )-a-cubebene 2 with TFA.
Treatment a€orded compound 7 with a 10-fold molar
excess of TFA produced a mixture of cis- and trans-
calamenene (11) in a ratio of 1:1, which was deter-
mined by 1H NMR (Heymes, Plattier, & Teisseire,
1974; Ladwa, Joshi, & Kulkarni, 1978) (Fig. 2). No
deuterium incorporation into the products was
observed when the reactions were carried out with
deuterated TFA.
[2H6]-Calamenene (13) was prepared from [2H7]-4'-
methyl acetophenone (12) by the method reported by
Condon, and West, 1980. Birch reduction of com-
pound 13 did not produce deuterated cubenene (10),
and gave predominately the unconjugated diene 14
(Fig. 3). [2H2]-Cubenene (17) was obtained instead by
the dehydration (Connolly, Phillips, & Haneck, 1982)
of [2H2]-epicubenol (16), which was prepared by a
slight modi®cation of the method reported by Cane,
and Tandon, 1994a, 1994b. Pyridinium chlorochro-
mate was used for oxidation of compound 15, instead
of the Dess±Martin reaction (Fig. 4).
Fig. 1. Proposed biosynthesis of hydroxylated calamenenes and cada-
lenes from FPP in cultured cells of Heteroschypnus planus.
calamenene via hydroxylation with an NIH shift. An
unconjugated diene sesquiterpene, cubenene 10, which
is a cometabolite in cultured cells of H. planus, and is
speci®cally produced from 2E, 6E-FPP by a cell-free
extract of cultured cells (Nabeta et al., 1995, 1997), is
the most likely precursor for 2, since 2 may be formed
from cubenene via hydroxylation and then oxidation
without the NIH shift. In the present study, tests of
the conversion of cubenene 10 and calamenene 11 to
7-hydroxycalamenene 2 in suspension cultured cells of
H. planus provided direct evidence for biotransform-
ation of cubenene 10, but not calamenene 11, to form
7-hydroxycalamenene 2.
2.2. Biotransformation of cubenene and calamenene
Biotransformation of cubenene (10, 50 mg) and cala-
menene (11, 50 and 500 mg) was carried out in 1 ml of
28-day-old cultures (0.04 g fr. wt.) of H. planus. The
cultures were further incubated at 258C with stirring at
110 rpm under continuous light for 12 h. The resulting
suspension was then centrifuged at 3000g, and the pre-
cipitated cells were washed with distilled water and
ether to remove possible precursors adhering to the
2. Results and discussion
2.1. Preparation of precursors
Chemical conversion of ( )-a-cubebene (7) was car-
ried out using a slight modi®cation of the methods of
Ohta, Ohara, and Hirose, 1968 and Anderson, Syrdal,
and Graham (1972) for the preparation of cubenene
(10) and calamenene (11) (Fig. 2). A mixture of three
dienes, ( )-cadina-4,6(1)-diene (8), (+)-d-cadinene (9)
and ( )-cubenene (10) were prepared from equimolar
amounts of ( )-a-cubebene 2 and TFA in a ratio of
8:9:10 of 4:1:2, avoiding the formation of hydroxylated
sesquiterpenes such as epicubenol (Ohta et al., 1968).
Fig. 3. Synthesis of [2H6]-calamenene (13) and its Birch reduction
product (14): (i) 1-bromo-4-methyl pentane, Mg, 08C (36%); (ii)
P2O5, 1208C (54%); (iii) Li, ethylenediamine, EtOH, THF, 08C
(22%).

M. Hashimoto et al. / Phytochemistry 51 (1999) 389±394
391
(17) and [2H6]-calamenene (13). The MS spectra of 7-
hydroxycalamenene incorporating
[2H2]-cubenene,
[2H6]-calamenene and no exogenous substrate are
shown in Fig. 5. Thus the 7-hydroxycalamenene incor-
porating [2H2]-cubenene (17) gave a base ion peak at
m/z 177 (relative intensity to unlabelled product (M+)
ion at m/z 175: 13.7%), which was formed by cleavage
is the C-4/C-9 bond, indicative of two 2H atoms
attached to a dihydronaphthalene ring. Moreover, the
MS ion peak monitored at m/z 177 was only observed
in the MS chromatogram of 7-hydroxycalamenene
incorporating the [2H2]-cubenene (Fig. 6). On the other
hand the MS spectrum of 7-hydroxycalamenene from
the cells fed [2H6]-calamenene (13) did not give a base
ion peak at m/z 180, again supporting the previous
®nding that [2H6]-calamenene was not converted into
7-hydroxycalamenene.
Thus, in the biosynthesis of hydroxylated calame-
nenes and cadalenes in suspension cultured cells of H.
planus, was shown to be an intermediate for 7-hydro-
xycalamenene. Thus, in turn, is presumed to be an in-
termediate in the biosynthesis of cadalene 6 via
compounds 3 or via the sequential formation of 4 and
5 (Fig. 1). The hypothetical biosynthetic pathway is
clearly in contrast to an intermediacy of d-cadinene in
the biosynthesis of cadalenes in infected cotton plants
(Essenberg, & Pierce, 1995). The position of the
hydroxy group may be directed by the positions of the
double bonds (Fig. 7). In preliminary experiments,
compound 14, a geometrical isomer of cubenene, was
also converted to monohydroxylated calamenene.
Fig. 4. Synthesis of [2H2]-cubenene (17): (i) Pyridinium chlorocho-
mate, CH2Cl2, room temp. (41%); (iii) m-chloroperbenzoic acid,
CH2Cl2, room temp. (41%); (iv) HF-pyridine, THF, 08C (79%); (v)
methyltriphenylphosphonium bromide, NaH, DMSO, 08C (66%);
(vi) NaBD4, CoCl2Á6H2O, CD3OD, 08C (38%); (vii) SOCl2, pyridine,
08C, then 10% NaHCO3 (56%).
cells. The harvested cells were extracted with MeOH at
48C, and the MeOH soln was partitioned with n-pen-
tane. To determine if cubenene-, calamenene- and 7-
hydroxycalamenene were either taken into cells, or bio-
transformed within them, the cells were extracted im-
mediately after inoculation (indicated as 0 h in Tables
1 and 2). The n-pentane soln was concentrated and
then analyzed by GC and GC±MS analyses. No sig-
ni®cant increase (>0.1 mg/0.04 g fr. wt.) in amounts
of cubenene, calamenene and 7-hydroxycalamenene
was observed during even a 12 h incubation without
precoursors being added (Table 1). When cultured cells
were incubated with 50 mg/ml cubenene, however, the
amounts of 7-hydroxycalamenene and calamenene
increased approximately two- and threefold, respect-
ively (Table 1). In contrast, the amount of 7-hydroxy-
calamenene did not increase when cultured cells were
incubated with calamenene at 50 or 500 mg/ml (Table
2). Moreover, a higher amount of calamenene detected
in cultured cells harvested immediately after incubation
with feeding calamenene at 500 mg/ml may not simply
be ascribed to uptake of calamenene, but may indicate
that calamenene was easily absorbed onto the cell sur-
face of H. planus with the adhering calamenene not
being easily removed with Ether. Thus, 7-hydroxycala-
menene (2) apparently was formed from cubenene, but
not from calamenene.
3. Experimental
3.1. General
( )-a-Cubebene (98%) was purchased from Fluka
Chemie (Buchs, Switzerland). GC and GC±MS were
performed on a Shimadzu GC-17A and a Hitachi M-
80B spectrometer equipped with an FS/WCOT column
(Ulbon H-1, Shinwa Chemical Industries; 50 mÂ0.25
mm, temp. was elevated from 150 to 2208C at 28C
Direct evidence for conversion of exogenously sup-
plied cubenene, but not calamenene, to 7-hydroxycala-
menene was provided administering [2H2]-cubenene
Table 1
Biotransformation of cubenene (50 mg) in the suspension cultured cells of H. planus. Values are expressed as mg per ml culture medium (0.04 g fr.
wt.) and represent the mean2S.D. (n=2)
Incubation time (h)
Cubenene (mg/ml)
7-Hydroxycalamenene (mg/ml)
Calamenene (mg/ml)
0
12
0.4620.09
2.2020.20
1.74
0.1220.02
0.3520.08
0.23
0.6820.05
1.2020.10
0.52
(12±0)
12a (without substrate)
0.4020.02
0.1420.01
0.6020.03
a Signi®cant increase (>0.1 mg/0.04 g fr.wt.) in amounts of cubenene, calamenene and 7-hydroxycalamenene was not observed during 12-h in-
cubation without precursors.

392
M. Hashimoto et al. / Phytochemistry 51 (1999) 389±394
Table 2
Blotransformation of calamenene (50 and 500 mg) in the suspension cultured cells of H. planus. Values are expressed as mg per ml culture medium
(0.04 g fr. wt) and represent the mean2S.D. (n=2)
Amount (mg/ml)
Incubation time (h)
Calamenene (mg/ml)
7-Hydroxycalamenene (mg/ml)
50
0
12
0.8020.05
5.2020.15
4.40
0.01120.003
0.01020.002
±
(12±0)
0
12
500
9.1520.64
25.0825.05
15.93
0.01020.003
0.00620.002
±
(12Ð0)
min 1). To estimate concentrations of sesquiterpenes,
the calibration curves for 10, 11 and 2 were deter-
mined. 1H NMR spectra were recorded on a JEOL
GX-270 spectrometer with CDCl3 as internal standard.
Reversed-phase HPLC was performed by a JASCO
system equipped with a Finepak SIL C18T-5 column
volume of H2O and then extracted with n-pentane, fol-
lowed by salting out with NaCl.
3.2. Precursor preparation
3.2.1. ( )-Cadina-4,6(1)-diene (8), (+)-d-cadinene
(9) and ( )-cubenene (10)
(JASCO, 250Â4.6 mm, 5 mm particles) at a ¯ow rate
1
of 1 ml min
were detected with
{CH3CN±H2O (3:1)}. Sesquiterpenes
a
( )-a-Cubebene (7, 21.6 mg, 110 mmol) in n-pentane
(500 ml) was treated with equimolar of TFA (8.1 ml,
110 mmol) at 08C. The reaction mixture was stirred at
08C for 0.5 h and quenched with satd NaHCO3 (500
ml). The organic layer was sepd, dried over dry
Na2SO4, ®ltrated, and conc. with N2 ¯ow to yield a
UV spectrophotometer
(UVIDEC-100-V, JASCO) at 215 nm. The fractions
containing sesquiterpenes were diluted with an equal
Fig. 5. Mass spectra of 7-hydroxycalamenene 2 synthesized in sus-
pension cultured cells of H. planus fed with [2H2]-cubenene 17 (a),
[2H6]-calamenene 13 (b) and without precursor (c).
Fig. 6. Segments of GC±MS ion chromatograms of 7-hydroxycala-
menene (m/z 175±177) incorporating [2H2]-cubenene 17 (a) and with-
out cubenene 17 (b).

M. Hashimoto et al. / Phytochemistry 51 (1999) 389±394
393
none (12), as described in Heymes et al. (1974) and
Ladwa et al. (1978). EIMS 70 eV, m/z (rel. int): 208
[M]+ (4), 206 (9), 165 (100), 164 (95), H NMR (270
1
MHz, CDCl3); d 0.69 (trans ), 0.74 (cis ) {total 3H, d,
J=6.9 Hz, -CH(CH3)2}, 0.98 (trans ), 1.01 (cis ) {total
3H, d, J=6.9 Hz, -CH(CH3)2}, 1.21 (3H, d, J=6.9
Hz, >CHCH3), 1.60 (4H, m, CH2Â2), 1.85 (1H, m),
2.30 (1H, m), 2.80 (1H, m).
Fig. 7. Postulated biosynthesis of 7-hydroxycalamenene from cube-
nene 10 in the suspension cultured cells of H. planus.
3.2.4. Birch reduction of 13
[2H6]-Calamenene (13) (40.0 mg, 0.20 mmol) and
lithium (7.0 mg, 1.01 mmol) were suspended in THF
(0.15 ml). Ethylenediamine (200 mg, 3.33 mmol) in
EtOH (0.15 ml) was added to the suspension at 08C.
The reaction mixture was stirred at 08C for 4 h,
quenched with 1 N HCl, and extracted with n-hexane.
The organic layer was washed with satd NaHCO3 and
brine, dried over dry Na2SO4, ®ltrated, concd under a
N2 atmosphere and puri®ed using reversed phase
HPLC to yield 14 (8.7 mg, 22 %). EIMS 70 eV, m/z
(rel. int): 210 [M]+ (5), 208 (10), 206 (12), 167 (100),
166 (98), 165 (90), 1H NMR (270 MHz, CDCl3); d
0.68, 0.72 {total 3H, d, J=6.3 Hz, -CH(CH3)2}, 0.94
(3H, d, J=6.3 Hz, >CHCH3), 0.97, 1.0 {total 3H, d,
J=6.3 Hz, -CH(CH3)2}, 1.25 (1H, m), 1.50 (2H, m),
1.70 (1H, m), 1.90 (2H, m), 2.10 (1H, m), 2.5 (1H, m),
2.10 (1H, m).
colorless oil (15.2 mg, 70%). The ratio of compounds
8, 9 and 10 in the reaction mixture was estimated by
GC±MS analysis to be 4:1:2. Further puri®cation was
performed with reversed-phase HPLC to obtain pure 8
(4.2 mg, 19%), 9 (1.0 mg, 5%) and 10 (2.5 mg, 12%).
8: [a]D 70.18 (c 0.3, CHCl3, lit. Ohta et al., 1968:
67.88), EIMS 70 eV, m/z (rel. int): 204 [M]+ (100),
189 (39), 161 (91), 119 (24), 105 (27), 1H NMR (270
MHz, CDCl3); d 0.85 (3H, d, J=6.3 Hz, -CH(CH3)2),
0.90 (3H, d, J=6.3 Hz, -CH(CH3)2), 1.03 (3H, d,
J=6.3 Hz, >CHCH3), 1.10 (1H, m, CH), 1.25 (2H,
m, CH2), 1.60 (3H, s, -C(CH3).CH-), 1.70 (2H, m,
CH2), 1.92 (1H, m, CH), 2.02 (1H, m, CH), 2.15 (2H,
m, CH2), 2.20 (2H, m, CH2), 5.42 (1H, s, .C±
CH.C(CH3)-), 9: [a]D +81.88 (c 0.4, CHCl3, lit. Ohta
et al., 1968: +84.78), EIMS: 204 [M]+ (75), 189 (18),
161 (100), 134 (45), 119 (30), 105 (27), 1H NMR; d
1.78 (3H, s, -CH±CH.C(CH3)), 5.30 (1H, s, -CH±
CH.C(CH3)-), 10: [a]D 20.28 (c 0.2, CHCl3, lit. Ohta
et al., 1968: 23.28), EIMS: 204 [M]+ (51), 161 (64),
119 (100), 105 (55), 1H NMR; d 1.68 {3H, s, -CH±
CH.(C(CH3)}, 5.25 (1H, br s, CH2CH.C<), 5.48
{1H, br s, -CH±CH.C(CH3)}.
3.2.5. [2H2]-Cubenene (17)
[2H2]-Epicubenol was prepared by a slight modi®-
cation of the method reported by Cane, and Tandon,
1994a, 1994b. Pyridinium chlorochomate was used for
oxidation of 15, instead of the Dess±Martin reaction.
A mixture of cis- (50%) and trans- (50%) [2H2]-cube-
nene was prepared from 16 by the published procedure
(Connolly et al., 1982). EIMS 70 eV, m/z (rel. int): 206
[M]+ (8), 205 (18), 163 (60), 162 (40), 121 (100), 120
3.2.2. Calamenene (11)
Compound 7 (10.4 mg, 51.0 mmol) in n-pentane (150
ml) was treated with 10 molar equivalents of TFA (39
ml, 506 mmol) at 08C. The reaction mixture was stirred
at 08C for 0.5 h and quenched with satd NaHCO3 soln
(500 ml). The organic layer was extracted with n-pen-
tane (350 ml), dried over dry Na2SO4, concentrated
under a N2 atmosphere, and puri®ed by silica gel col-
umn chomatography {n-hexane±Et2O (9:1)} to yield 11
(6.2 mg, 60%). EIMS 70 eV, m/z (rel. int): 202 [M]+
1
(85), H NMR (270 MHz, CDCl3); d 0.80, 0.85 (total
3H, d, J=6.3 Hz, -CH(CH3)2), 0.90, 0.94 (total 3H, d,
J=6.3 Hz, -CH(CH3)2), 1.0 (2H, m, >CDCH2D),
1.25 (1H, m), 1.50 (2H, m), 1.65 (3H, m), 1.70 (1H,
m), 1.90 (2H, m), 2.10 (1H, m,), 2.10 (1H, m,), 5.15
(1H, br s), 5.38 (1H, br s).
3.2.6. Feeding experiment
1
(9), 159 (100), 145 (5), H NMR (270 MHz, CDCl3); d
The origins of H. planus, as well as the medium and
conditions for suspension culture have been described
earlier (Takeda, & Katoh, 1981; Nabeta et al., 1993).
One ml of 28 day-old culture was incubated without
or with precursors (50 mg for 10, and 50 or 500 mg for
11) in n-pentane (10 ml). The liquid suspensions were
agitated continuously at 110 rpm at 258C under con-
tinuous light of 2000 lux for 0 or 12 h. Duplicate ex-
periments were carried out to determine amounts of
products by GC.
0.69 (trans ), 0.74 (cis ) (total 3H, d, J=7 Hz, -
CH(CH3)2), 0.98 (trans ), 1.01 (cis ) (total 3H, d, J=7
Hz, -CH(CH3)2), 1.21 (3H, d, J=7 Hz, >CHCH3),
2.29 (3H, s, CH3Ar), 6.94 (1H, d, J=7 Hz, ArH), 7.06
(1H, s, ArH), 7.12 (1H, d, J=7 Hz, ArH).
3.2.3. [2H6]-Calamenene (13)
A mixture of cis- (40%) and trans- (60%) [2H6]-cala-
menene was prepared from [2H7]-4'-methyl acetophe-

394
M. Hashimoto et al. / Phytochemistry 51 (1999) 389±394
Bowden, B. F., Coll, J. C., Engelhardt, L. M., Topiolas, D. M., &
White, A. H. (1986). Aust. J. Chem., 39, 103.
3.2.7. Extraction
The cell culture suspension was centrifuged at 3000g
for 10 min and the supernatant was decanted. The ppt.
cells were washed with H2O and then Et2O, and
extracted with MeOH (1 ml) at 48C for 12 h. The
MeOH extract was partitioned with n-pentane (1 ml).
The n-pentane soln was conc. to 10 ml under N2 ¯ow
and then subjected to GC and GC±MS analyses.
Cane, D. E., & Tandon, M. (1994a). Tetrahedron Lett., 35, 5351.
Cane, D. E., & Tandon, M. (1994b). Tetrahedron Lett., 35, 5355.
Condon, F. E., & West, D. L. (1980). J. Org. Chem., 45, 2006.
Connolly, J. D., Phillips, W. R.,
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&
Haneck, S. (1982).
Davis, G. D., & Essenberg, G. (1995). Phytochemistry, 39, 553.
Essenberg, M., & Pierce, M. L. (1995). In M. Daniel, & P. R.
Purkayastha, Handbook of phytoalexin metabolism and action (p.
183). New York: Marcel Decker, Inc.
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Acknowledgements
Maciadri, R., Angst, W., & Arigoni, D. (1985). J. Chem. Soc. Chem.
Commun., 1573.
We thank Dr. Noriyuki Nakajima (Toyama
Prefectural University) for discussion of substrate syn-
thesis. This work was supported by Hokkaido
Foundation for the Promotion of Scienti®c and
Nabeta, K., Katayama, K., Nakagawara, S., & Katoh, K. (1993).
Phytochemistry, 32, 117.
Nabeta, K. (1995). Dev. Food. Sci., 37A, 951.
Industrial Technology and
a
Grant-in-Aid for
Nabeta, K., Mototani, Y., Tazaki, H., & Okuyama, H. (1994).
Phytochemistry, 35, 915.
Scienti®c Research (A, No. 0836021 and C, No.
08660125) from the Ministry of Education, Science
and Culture, Japan.
Nabeta, K., Ishikawa, T., Kawae, T., & Okuyama, H. (1994). J.
Chem. Soc. Perkin Trans., 1, 3277.
Nabeta, K., Kigure, K., Fujita, M., Nagoya, T., Ishikawa, T.,
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