Gymnomitrane-type sesquiterpenes of the liverworts Gymnomitrion obtusum and Reboulia hemisphaerica

Article abstract of DOI:10.1016/S0031-9422(99)00205-8

The sesquiterpene constituents of the liverworts Gymnomitrion obtusum Lindb. and Reboulia hemisphaerica (L.) Raddi were investigated. In addition to many known compounds, a new sesquiterpene hydrocarbon, (-)-gymnomitra- 3(15),4-diene was isolated and identified. Gymnomitrone, a sesquiterpene ketone, was found in nature for the first time.

Full text of DOI:10.1016/S0031-9422(99)00205-8

Phytochemistry 52 (1999) 1501±1505
Gymnomitrane-type sesquiterpenes of the liverworts Gymnomitrion
obtusum and Reboulia hemisphaerica
Ute Warmers, Wilfried A. Konig*
Institut fur Organische Chemie, Universitat Hamburg, Martin-Luther-King-Platz 6, D-20146 Hamburg, Germany
È
È
Received 27 January 1999; received in revised form 10 March 1999; accepted 22 March 1999
Abstract
The sesquiterpene constituents of the liverworts Gymnomitrion obtusum Lindb. and Reboulia hemisphaerica (L.) Raddi were
investigated. In addition to many known compounds, a new sesquiterpene hydrocarbon, ( )-gymnomitra-3(15),4-diene was
isolated and identi®ed. Gymnomitrone, a sesquiterpene ketone, was found in nature for the ®rst time. # 1999 Elsevier Science
Ltd. All rights reserved.
Keywords: Gymnomitrion obtusum; Jungermanniales; Reboulia hemisphaerica; Marchantiales; Liverwort; Sesquiterpene; ( )-Gymnomitra-3(15),4-
diene; Gymnomitrone; Enantioselective gas chromatography
1. Introduction
types with di€erent constituents (Asakawa & Matsuda,
1982; Wei & Wu, 1995).
Gymnomitrion obtusum is
a
leafy liverwort
(Hepaticae ) of the order Jungermanniales and
Reboulia hemisphaerica a thalloid liverwort of the
order Marchantiales (Frahm & Frey, 1992). G. obtu-
sum and R. hemisphaerica are rich sources of sesquiter-
penes with the gymnomitrane skeleton, which are
typical constituents of liverworts. ( )-b-Barbatene,
(+)-gymnomitrol and (+)-gymnomitrol acetate are
known as sesquiterpene constituents of G. obtusum
(Connolly, Harding, & Thornton, 1972, 1974), (+)-
gymnomitr-3(15)-en-4a-ol, (+)-3b-hydroxygymnomi-
tran-4-one, R-( )-1,4-dihydro-a-cuparenone, R-( )-a-
cuparenone as sesquiterpene constituents of R. hemi-
sphaerica (Morais, Harrison & Becker, 1988, 1991;
Morais & Becker, 1991). (Here the farnesane number-
ing system was used). Three chemotypes of R. hemi-
sphaerica are known: an European chemotype
containing several sesquiterpenes with gymnomitrane
skeleton (as described above) and two Asian chemo-
2. Results and discussion
We have reinvestigated the sesquiterpene constitu-
ents of G. obtusum and R. hemisphaerica collected in
Europe. Samples of plant volatiles were obtained by
hydrodistillation or solvent extraction (in the case of
R. hemisphaerica ) and analyzed by gas chromatog-
raphy (GC) and GC±mass spectrometry (GC±MS).
There were only small di€erences in the relative per-
centages of single constituents in the hydrodistillation
and in the solvent extraction products. Individual com-
ponents were isolated by preparative GC and investi-
gated by NMR (1H, 13C, 1H1H-COSY, HMQC,
HMBC, NOESY). The absolute con®guration was de-
rived by polarimetric measurements and enantioselec-
tive GC using cyclodextrin phases. The following
compounds were identi®ed as constituents of the essen-
tial oil of R. hemisphaerica: ( )-gymnomitra-3(15),4-
diene (1), a-barbatene, ( )-b-barbatene, gymnomitr-
3(15)-en-4-one, gymnomitran-4-one, (+)-gymnomitr-
3(15)-en-4a-ol, gymnomitrol, isobazzanene, b-bazza-
* Corresponding author.
E-mail address: wkoenig@chemie.uni-hamburg.de (W.A. Konig).
0031-9422/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(99)00205-8

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U. Warmers, W.A. Konig / Phytochemistry 52 (1999) 1501±1505
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Scheme 1.

U. Warmers, W.A. Konig / Phytochemistry 52 (1999) 1501±1505
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Fig. 1. Substructures A and B of gymnomitra-3(15),4-diene (1).
nene, cuparene, a-cuprenene, d-cuprenene and b-acora-
diene (Scheme 1).
lation. The major compound of R. hemisphaerica is
(+)-gymnomitr-3(15)-en-4a-ol (2). Dehydration of 2
yields the rearrangement product gymnomitran-4-one
(3) (Morais et al., 1988) and the hydrocarbon ( )-gym-
nomitra-3(15),4-diene (1) (Fig. 2). So the relative and
absolute con®guration of 1 has to be identical with 2.
The dehydration product and the natural product 1
were compared by GC±MS and enantioselective GC
using cyclodextrin phases.
Futhermore, 1 was correlated with ( )-b-barbatene
(4), another constituent of R. hemisphaerica.
Hydrogenation of 1 and 4 yields the same products
(Fig. 3).
( )-Gymnomitra-3(15),4-diene (1) is a new sesquiter-
pene hydrocarbon. 1 is also a constituent of the liver-
worts Plagiochasma rupestre and Gymnomitrion
corallioides. The mass spectrum exhibits a molecular
ion signal at m/z 202 and an elemental composition of
C15H22. The 1H NMR spectrum shows singlets for
three methyl groups at quaternary carbon atoms at d
0.84, 0.91 and 1.03, broad singlets for two geminal ole-
®nic protons at d 4.72 and 4.85, doublets for two vic-
inal ole®nic protons at d 5.59 and 6.02 and a broad
doublet for an allylic proton at d 2.32.
The connection of the groups could be derived from
1
The following sesquiterpenes were identi®ed as con-
stituents of the essential oil of G. obtusum: a-barba-
the H1H-COSY NMR spectrum. The two vicinal ole-
®nic protons at d 5.59 (CH-5) and 6.02 (CH-4) couple
with each other and with the two geminal ole®nic pro-
tons at d 4.72 and 4.85 (CH2-15). The allylic proton at
d 2.32 (CH-2) has to be bonded to the quaternary car-
bon atom of the exocyclic double bond (C-3). This
proton couples to the protons of a methylene group at
d 1.62 and 1.83±1.93 (CH2-1). This suggests substruc-
ture A (Fig. 1).
The protons of three methylene groups at d 1.04±
1.14, 1.20±1.28, 1.54±1.70, 1.65±1.81, 1.83±1.93 and
1.86±1.99 (CH2-8, CH2-9, CH2-10) couple with each
other. This suggests the existence of a chain of three
methylene groups as shown in substructure B (Fig. 1).
The three quaternary methyl groups at d 0.84, 0.91
and 1.03 (CH3-12, CH3-13, CH3-14) have to be located
at the ends of these two substructures A and B, so
that they could be combined to a tricyclic molecule
with the structure 1.
tene,
( )-b-barbatene,
gymnomitrone
(5),
isogymnomitrol, (+)-gymnomitrol, (+)-gymnomitrol
acetate, isobazzanene and cuparene (Scheme 1).
Gymnomitrone (5) has not been found before in
nature and was only known as a synthetic product
(Han & Paquette, 1979; Coates, Shah, & Mason, 1979,
1982; Paquette & Han, 1981; O'Neil, Quickley, &
Snider, 1997). The spectral data are identical to those
reported for the synthetic product. The absolute con-
®guration was obtained by a correlation reaction.
Reduction of 5 yields (+)-gymnomitrol (6) (O'Neil et
al., 1997), the major constituent of G. obtusum (Fig.
4). So the absolute con®guration of 5 at C-2, C-6, C-7
and C-11 must be identical to 6. The reduction pro-
duct and the natural product 6 were compared by
GC±MS and enantioselective GC using cyclodextrin
phases.
The identi®cation of 1 and 5 as new constituents of
liverworts complements the already know variety of
The structure of 1 was veri®ed by a chemical corre-
Fig. 2. Dehydration and rearrangement of (+)-gymnomitr-3(15)-en-4a-ol (2) to ( )-gymnomitra-3(15),4-diene (1) and gymnomitran-4-one (3).

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U. Warmers, W.A. Konig / Phytochemistry 52 (1999) 1501±1505
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Fig. 3. Hydrogenation of ( )-gymnomitra-3(15),4-diene (1) and ( )-b-barbatene (4) to saturated gymnomitranes.
structures with gymnomitrane skeleton as typical con-
stituents of the Hepaticae.
3.3. Extraction
Samples of plant volatiles were prepared by extrac-
tion (7 days, 208C) of fresh and green plants with
diethyl ether.
3. Experimental
3.4. Gas chromatography
3.1. Plant material
Orion Micromat 412 double column instrument with
25 m fused silica capillaries with polysiloxane CpSil 5
and polysiloxane CpSil 19 (Chrompack); Carlo Erba
Fractovap 2150, 4160 instrument with 25 m fused
silica capillaries with heptakis(2,6-di-O-methyl-3-O-
pentyl)-b-cyclodextrin or heptakis(6-O-tert-butyldi-
methylsilyl-2,3-di-O-methyl)-b-cyclodextrin in OV 1701
(50 wt%); split injection; ¯ame ionization detection;
carrier gas 0.5 bar H2.
Fresh plant material of R. hemisphaerica was col-
lected at the river Argout near Burlats and Les
Salvages (France) by U. Heseler, St. Ingbert. A tissue
culture of R. hemisphaerica was obtained from
Professor Dr. H. Becker, Universitat des Saarlandes,
Saarbrucken, Germany. R. hemisphaerica was ident-
i®ed by Professor Dr. R. Mues, Universitat des
Saarlandes, Saarbrucken, Germany. In the natural and
cultured plant material of R. hemisphaerica identical
constituents are found. G. obtusum was collected at the
mount Meiûner (Germany) and identi®ed by Dr. H.
Muhle, Universitat Ulm, Germany.
3.5. Isolation
The isolation was carried out by preparative GC.
3.2. Hydrodistillation
3.6. Preparative GC
The essential oil was prepared by hydrodistillation
(2 h) of aqueous homogenates of fresh and green
plants using n-hexane as collection solvent. Because of
the di€erent drying state of the plants the material was
not weighed.
Modi®ed Varian 1400 instrument, equipped with a
stainless steel column (1.85 m  4.3 mm) with 10%
polydimethylsiloxane SE 30 on Chromosorb W-HP;
¯ame ionization detection; helium as carrier gas at a
¯ow rate of 240 ml/min (Hardt & Konig, 1994).
3.7. NMR spectroscopy
NMR measurements were carried out with a Bruker
WM 500 instrument.
3.8. Mass spectrometry
Fig. 4. Reduction of gymnomitrone (5) to (+)-gymnomitrol (6).
GC±MS measurements were run on a HP 5890 gas

U. Warmers, W.A. Konig / Phytochemistry 52 (1999) 1501±1505
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chromatograph coupled to a VG Analytical 70-250S
mass spectrometer.
to the hydrogenation of 1. The reaction products were
analyzed by GC±MS and by GC on several capillary
columns. The hydrogenated compounds were identical
with the reaction products of the hydrogenation of 1.
3.9. ( )-Gymnomitra-3(15),4-diene (1)
1H NMR (500 MHz, C6D6): d 0.84 (3H, s, H-12/13/
14), 0.91 (3H, s, H-12/13/14), 1.03 (3H, s, H-12/13/14),
1.04±1.14 (1H, m, H-8/10), 1.20±1.28 (1H, m, H-8/10),
1.54±1.70 (1H, m, H-9), 1.62 (1H, d, J = 11.1 Hz, H-
1), 1.65±1.81 (1H, m, H-9), 1.83±1.93 (2H, m, H-1, H-
8/10), 1.86±1.99 (1H, m, H-8/10), 2.32 (1H, d, J = 4.4
Hz, H-2), 4.72 (1H, bs, H-15), 4.85 (1H, bs, H-15),
5.59 (1H, d, J = 9.5 Hz, H-5), 6.02 (1H, d, J = 9.5
Hz, H-4); MS (EI, 70 eV): m/z (rel. int.) 202 (4) [M+],
107 (19), 106 (94), 105 (17), 96 (21), 95 (22), 91 (100),
81 (17), 79 (13), 77 (14), 65 (9), 55 (15), 41 (19).
Acknowledgements
We thank Professor Dr. H. Becker, Professor Dr. R.
Mues, U. Heseler and Dr. H. Muhle for the plant ma-
terial and Dr. V. Sinnwell, Universitat Hamburg and
his team for the NMR measurements. The ®nancial
support of the Fonds der Chemischen Industrie is grate-
fully acknowledged.
3.10. Dehydration of (+)-gymnomitr-3(15)-en-4a-ol
(2)
References
Asakawa, Y., & Matsuda, R. (1982). Phytochemistry, 21, 2143.
Frahm, J.-P.,
Verlag.
To a sol. of 1 mg of 2 in 1 ml pyridine 0.1 ml SOCl2
was added and the reaction mixture was stirred at 08C
for 10 min. n-Hexane and H2O were added. The or-
ganic phase was washed three times with H2O. The
reaction product was analyzed by GC±MS and by GC
on various capillary columns with cyclodextrin phases.
It was identical with 1.
& Frey, W. (1992). Moos¯ora. Stuttgart: Ulmer-
Coates, R. M., Shah, S. K., & Mason, R. W. (1979). Journal of the
American Chemical Society, 101, 6765.
Coates, R. M., Shah, S. K., & Mason, R. W. (1982). Journal of the
American Chemical Society, 104, 2198.
Connolly, J. D., Harding, A. E., Thornton, I. M. S. (1972). Journal
of the Chemical Society Chemical Communications, 1321.
Connolly, J. D., Harding, A. E., Thornton, I. M. S. (1974). Journal
of the Chemical Society Perkin Transactions 1, 2487.
Han, Y. K., & Paquette, L. A. (1979). Journal of Organic Chemistry,
44, 3731.
3.11. Hydrogenation of ( )-gymnomitra-3(15),4-diene
(1)
Hardt, I., & Konig, W. A. (1994). Journal of Chromatography A,
666, 611.
To a sol. of 1 mg of 1 in 1 ml n-hexane, 0.5 mg Pd/
C was added. The suspension was treated with H2 and
stirred under H2 at room temp. for 1 h. The reaction
mixture was ®ltered and the reaction products were
analyzed by GC±MS and by GC on various capillary
columns.
Morais, R. M. S. C.,
Naturforschung, 46c, 28.
& Becker, H. (1991). Zeitschrift fur
È
Morais, R. M. S. C., Harrison, L. J., & Becker, H. (1988). Journal of
Chemical Research Synopses, 380.
Morais, R. M. S. C., Harrison, L. J.,
Phytochemistry, 30, 1013.
& Becker, H. (1991).
O'Neil, S. V., Quickley, C. A., & Snider, B. B. (1997). Journal of
Organic Chemistry, 62, 1970.
3.12. Hydrogenation of ( )-b-barbatene (4)
Paquette, L. A., & Han, Y. K. (1981). Journal of the American
Chemical Society, 103, 1831.
Wei, H. C., & Wu, C. L. (1995). Phytochemistry, 39, 91.
The hydrogenation of 4 was performed analogously