Sesquiterpenoid constituents of the liverwort Marsupella aquatica

Article abstract of DOI:10.1016/j.tetasy.2007.05.020

Nine new amorphane sesquiterpenoids, (+)-7β-hydroxyamorpha-4,11-diene, (-)-9α-hydroxyamorpha-4,7(11)-diene, (-)-3α-hydroxyamorpha-4,7(11)-diene, (-)-3α-acetoxyamorpha-4,7(11)-diene, (-)-amorpha-4,7(11)-dien-3-one, (+)-2,8-epoxyamorpha-4,7(11)-diene, (+)-5,9-epoxyamorpha-3,7(11)-diene, (-)-2α-hydroxyamorpha-4,7(11)-diene and (-)-2β-acetoxyamorpha-4,7(11)-diene, were isolated from the essential oil of the liverwort Marsupella aquatica, collected near Gaschurn/Montafon, Austria. The isolated compounds and their chemical transformations were investigated using enantioselective GC and extensive spectroscopic studies (HRMS, ¹H, ¹³C and 2D NMR). The absolute configuration of most of the isolated compounds were established by conversions to known compounds. In addition, ¹H, and ¹³C NMR data of (-)-myltayl-4-ene are reported for the first time.

Full text of DOI:10.1016/j.tetasy.2007.05.020

Tetrahedron: Asymmetry 18 (2007) 1245–1253
Sesquiterpenoid constituents of the liverwort Marsupella aquatica
Adewale M. Adio,a,* Stephan H. von Reuß,a Claudia Paul,a Hermann Muhleb
and Wilfried A. Ko¨niga,z
aInstitut fu¨r Organische Chemie, Universita¨t Hamburg, D-20146 Hamburg, Germany
¨
Abteilung Systematische Botanik und Okologie, Universita¨t Ulm, D-89091 Ulm, Germany
b
Received 23 April 2007; accepted 17 May 2007
Dedicated to Professor Dr. Dr. h-c. W. Francke on the occasion of his 65th birthday
Abstract—Nine new amorphane sesquiterpenoids, (+)-7b-hydroxyamorpha-4,11-diene, (À)-9a-hydroxyamorpha-4,7(11)-diene, (À)-3a-
hydroxyamorpha-4,7(11)-diene, (À)-3a-acetoxyamorpha-4,7(11)-diene, (À)-amorpha-4,7(11)-dien-3-one, (+)-2,8-epoxyamorpha-
4,7(11)-diene, (+)-5,9-epoxyamorpha-3,7(11)-diene, (À)-2a-hydroxyamorpha-4,7(11)-diene and (À)-2b-acetoxyamorpha-4,7(11)-diene,
were isolated from the essential oil of the liverwort Marsupella aquatica, collected near Gaschurn/Montafon, Austria. The isolated
compounds and their chemical transformations were investigated using enantioselective GC and extensive spectroscopic studies (HRMS,
1H, 13C and 2D NMR). The absolute configuration of most of the isolated compounds were established by conversions to known
1
compounds. In addition, H, and 13C NMR data of (À)-myltayl-4-ene are reported for the first time.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
2. Results and discussion
The liverwort Marsupella emarginata var. aquatica is
closely related to M. emarginata, as indicated by the pres-
ence of longipinane type sesquiterpenoids. However, the
two species clearly differ in their morphological properties1
and chemical composition.2 Oxygenated longipinane and
gymnomitrane derivatives have been isolated from the
ether extract of the French M. aquatica,3,4 while four
oxygenated amorphane derivatives have been isolated from
the Scottish liverwort M. emarginata var. aquatica.5 In our
previous report, we have isolated (+)-amorpha-4,11-diene
1, (À)-amorpha-4,7(11)-diene 2, (À)-2a-acetoxyamorpha-
4,7(11)-diene 3, and (À)-myltayl-4(12)-ene 4 from the
essential oil of the Austrian M. aquatica.6 In continua-
tion of our work on the liverworts, we herein report our
results on the investigation of the constituents of the essen-
tial oil of the Austrian M. aquatica.
2.1. Composition of the essential oil of M. aquatica
The essential oil of the liverwort M. aquatica was prepared
by hydrodistillation and analysed by GC and GC–MS.
The following known sesquiterpene hydrocarbons could
be identified by comparison of their mass spectra and
retention indices with a spectra library established under
identical experimental conditions:7,8 b-elemene (0.4%), a-
barbatene (0.4%), isobazzanene (0.5%), b-barbatene
(1.5%), (À)-myltayl-4(12)-ene
4 (3.2%), b-acoradiene
(1.3%), (+)-amorpha-4,11-diene 1 (9.6%), (À)-amorpha-
4,7(11)-diene 2 (25.2%) and traces of a-longipinene, cyclo-
myltaylane 5, anastreptene, a-copaene, b-copaene, cala-
rene, a-chamigrene, cadina-1,4-diene and d-cuprenene.
2.2. Structure elucidation of (À)-myltayl-4-ene 6
(À)-Myltayl-4-ene 6 (0.5%), an irregular sesquiterpene
hydrocarbon, elutes before b-elemene from a non-polar
dimethylpolysiloxane column. Myltayl-4(12)-ene 4, the
double bond isomer of 6, has previously been isolated from
the same M. aquatica oil.6 The mass spectrum of 6 exhibits
a molecular ion signal at m/z 204 corresponding to the
*
Corresponding author. Tel.: +1 306 4771231; e-mail: adio20002000@
yahoo.com
z Passed away on November 19, 2004. His scientific achievements keep
him among us.
0957-4166/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2007.05.020

1246
A. M. Adio et al. / Tetrahedron: Asymmetry 18 (2007) 1245–1253
molecular formula of C15H24 with four units of unsatura-
pounds 4–6 in the essential oils of the liverworts Mylia tayl-
orii and Kurzia trichoclados.12
tion, as determined by HR-EIMS ([M+] m/z 204.1891).
1
The H NMR (C6D6) showed three methyl singlets at d
0.88 (3H, s), 0.94 (3H, s), 0.99 (3H, s) and one downfield
13
H3C
methyl at d 1.62 (3H, s) indicating the attachment to an ole-
15
H3C
1
finic carbon. The 2D H–1H COSY, HMQC and HMBC
H
spectra confirmed the structure of 6. The relative configura-
tion of 6 was deduced from the NOESY spectrum which
showed spatial interactions for the methyl group protons
(H-15) with methine H-5, and H-10b/H-9a. Spatial interac-
tions of H-13 protons with H-3, H-1b, H-2b, H-8a and
H-14 protons were observed. In addition, proton H-5 also
interacts with H-15 and H-9a/10b (Fig. 1). A biogenetic
pathway from the chamigrenyl cation via a Wagner–
Meerwein rearrangement following Markownikoff’s rule
is proposed (Scheme 1)9 as an alternative to the anti-Mar-
kownikoff route.10 The co-occurrence of 4, 6 and cyclo-
myltaylane 511 support the myltaylanyl cation as an
intermediate in both routes. We have also identified com-
5
H3C
14
3
4
H
12
2
Figure 1. NOE correlations of 6.
2.3. Structure elucidation of (+)-(1R,6S,10S)-7b-hydroxy-
amorpha-4,11-diene 7
Compound 7 (0.7%), showed a molecular ion signal at m/z
220, while the molecular formula was determined as
CH
A
junicedranyl carbonium ion
B
C
C
chamigrenyl carbenium ion
A
B
C
H
12
5
C
4
6
3
1
2
myltaylanyl carbonium ion
- H+
- H+
- H+
cyclomyltaylane 5
Scheme 1. Proposed biogenetic pathway for 4–6.
myltayl-4-ene 6
myltayl-4(12)-ene 4

A. M. Adio et al. / Tetrahedron: Asymmetry 18 (2007) 1245–1253
1247
Table 1. 13C NMR (125.7 MHz, C6D6) d (ppm) data of amorphane derivatives
Compd no.
7
9
10
12
13
46.0
43.0
197.8
136.0
147.9
41.7
14
37.3
71.7
39.0
130.1
122.7
32.9
134.5
66.8
36.8
29.6
120.8
20.1
19.1
16
48.6
73.3
36.0
132.7
125.9
41.4
134.5
26.6
37.1
28.1
121.0
19.9
20.3
17
45.3
70.7
31.8
130.5
126.0
34.9
134.1
27.1
36.4
30.2
122.2
20.0
20.6
1
2
3
4
5
6
7
8
9
36.4
25.5
26.5
39.5
26.0
25.5
41.1
34.8
67.4
135.6
129.3
41.2
135.4
27.5
37.1
30.4
121.5
20.6a
20.7a
20.2
40.6
31.8
69.2
134.9
132.2
40.7
134.9
27.6
36.8
30.0
121.8
20.6a
20.8a
20.3
134.4
121.8
43.4
75.6
32.1
30.5
27.8
150.9
18.3
111.5
19.8
23.9
134.4
125.7
40.4
132.7
37.0
133.8
27.6
36.1
76.0
37.0
10
11
12
13
14
15
16
17
29.8
123.2
20.3a
20.7a
15.5
122.3
20.2a
20.4a
19.8
20.9
23.9
22.3
23.0
20.0
23.5
23.9
21.1
21.9
16.2
170.1
21.0
170.0
21.3
All assignments were confirmed by HMBC and HMQC.
a Interchangeable.
C15H24O by HR-EIMS ([M+] m/z 220.1832). The 1H NMR
spectrum (C6D6) of compound 7 exhibited signals of three
methyl groups at d 0.92 (3H, d, H-14, J = 6.3 Hz), 1.57
(3H, br s, H-15) and 1.81 (3H, s, H-12). The signals at d
4.85 (1H, s, H-13a), 4.90 (1H, s, H-13b) and 1.81 (3H, s,
H-12) suggested the existence of an isopropenyl group.
The olefinic carbon signals at d 150.9 (s) and 111.5 (t) con-
firmed the exomethylene double bond. The signal at d 75.6
was assigned to the tertiary carbon attached to the hydr-
to the hydroxy group at C-7. In addition, the assigned
(7S)-configuration is consistent with (+)-amorpha-4,11-
diene 1 and (+)-1-epi-cubenol 813 isolated and character-
ised in the same M. aquatica oil.
2.4. Structure elucidation of (+)-(1R,6S,7S,10S)-1-epi-
cubenol 8
(+)-1-epi-Cubenol (8, 1.5%) was isolated and characterised.
The spectral data of 8 were consistent with those reported
in the literature.13 Its absolute configuration was deter-
mined by the treatment of 8 with an acidic ion exchange
resin (Amberlyst) for 2 h at room temperature to afford
(+)-cadina-1,4-diene (85%), as well as the oxidation prod-
uct (+)-trans-calamenene (5%), as determined by GC–MS
and enantioselective GC with authentic standards.
1
oxyl group. The 2D H–1H-COSY, HMQC and HMBC
spectra, in addition to the 13C NMR (Table 1) confirmed
the structure. Its relative configuration resulted from the
NOESY spectrum (Fig. 2). The absolute configuration
was determined by a comparison of the fully hydrogenated
products of (À)-amorpha-4,7(11)-diene 2 with those of
(+)-7 using enantioselective GC on a modified cyclodextrin
stationary phase. The four fully hydrogenated derivatives
of 7 are identical in GC–MS characteristics and retention
times on achiral polysiloxane and chiral cyclodextrin
derived GC phases with those of compound 2. Therefore,
the absolute configuration of 7 at C-1, C-6 and C-10 was
concluded to be (1R,6S,10S). The (7S)-configuration was
inferred from the four hydrogenation products which
suggested that proton H-6 must have a cis-configuration
2.5. Structure elucidation of (À)-(1S,6S,9R,10R)-9a-
hydroxyamorpha-4,7(11)-diene 9
Compound 9 has the same bicyclic sesquiterpene skeleton
as 7 and exhibits a molecular ion signal at m/z 220. Its
molecular formula was determined to be C15H24O by
1
HR-EIMS ([M+] m/z 220.1816). The H NMR spectrum
(C6D6) of 9 showed four methyl groups at d 1.03 (3H, d,
H-14, J = 6.6 Hz), 1.58 (3H, s, H-15) and 1.66 (6H, br s,
H-12 and H-13). The signal at d 2.98 (1H, dt, H-9,
J = 4.1, 11.4 Hz) was assigned to the methine proton
H-9. Additional useful structural information was obtained
from the 13C NMR (Table 1). The relative configuration of
9 was deduced from the NOESY spectrum (Fig. 3), and a
(1S,6S,9R,10R)-configuration was assigned by using the
same method as for 7, that is, hydrogenation to yield four
diastereomers that matched the hydrogenation products of
(À)-2 in GC–MS characteristics and coelute on achiral and
chiral cyclodextrin derived GC-phases. The a-orientation
of the secondary hydroxyl group was deduced from the
spatial interactions of H-9 and H-14. Additionally, H-9
14
CH3
H
10
1
6
15
7
H3C
OH
H
12
H3C
Figure 2. NOE correlations of 7.

1248
A. M. Adio et al. / Tetrahedron: Asymmetry 18 (2007) 1245–1253
262 corresponding to the molecular formula of C17H26O2.
1
The H NMR spectrum of 12 showed three signals corres-
CH3
H
OH
ponding to five methyl groups at d 0.86 (3H, d, H-14,
J = 6.3 Hz), 1.65 (9H, br s, H-12, H-13 and H-15) and
1.74 (3H, s, H-17). The signal at d 5.33 (1H, br d, H-3,
J = 5.0 Hz) was assigned to the methine proton connected
to the acetoxy group. Additional structural information
was obtained through 13C NMR (Table 1). The relative
configuration of 12 resulted from its NOESY spectrum,
which is similar to that of 10 (Fig. 4). The hydrolysis of
12 using K2CO3/MeOH afforded 10. Therefore, 12 should
have the same (1R,3R,6S,10S) configuration as 10.
H
1
9
6
H
H3C
Figure 3. NOE correlations of 9.
has two large couplings, which suggest that it must be axial
with two antiperiplanar vicinal protons, hence it must be b
in structure 9.
2.8. Structure elucidation of (À)-(1R,6S,10S)-amorpha-
4,7(11)-dien-3-one 13
Compound 13 (1.5%), an a,b-unsaturated ketone with
amorphane skeleton, showed a molecular ion signal at
m/z 218 corresponding to the molecular formula of
C15H22O, as determined by HR-EIMS ([M+] m/z
218.1668). The 1H NMR spectrum (C6D6) indicated signals
for four methyl signals at d 0.72 (3H, d, H-14, J = 6.3 Hz),
1.59 (6H, br s, H-12 and H-13) and 1.82 (3H, s, H-15). The
proton signal at d 5.91 was assigned to the methine proton
H-5 with a downfield carbon signal at d 147.9 (d). The 2D
1H–1H COSY, HMQC and HMBC in addition to the 13C
NMR (Table 1) spectra confirmed the structure of 13. Its
relative configuration was determined from the NOESY
spectrum, again similar to that of 10 (Fig. 4). Treatment
of 10 with pyridinium dichromate (PDC) in dry dichloro-
methane gave a product identical (same GC–MS charac-
teristics and same retention times on achiral polysiloxane
and cyclodextrin derived GC phases) to (À)-13. Conse-
2.6. Structure elucidation of (À)-(1R,6S,9R,10R)-3a-
hydroxyamorpha-4,7(11)-diene 10
Compound 10 (0.5%), C15H24O (m/z 220.1826 [M+]) exhi-
bits a similar 1H NMR as 9 except that the a-oriented
secondary hydroxyl group is now at C-3 at d 67.4 (d). The
1
2D H–1H-COSY, HMQC and HMBC data of 10 were
consistent with the assigned structure. For 13C NMR
(C6D6) data see Table 1. The relative configuration of 10
was determined from the NOESY spectrum (Fig. 4). Direct
hydrogenation of 10 afforded identical products as in 7 and
9. In addition, alcohol 10 dehydrates at the injector port
(200 ꢁC) of the preparative GC during purification to afford
(+)-amorpha-2,4,7(11)-triene 11. Compound 11 showed a
molecular ion signal at m/z 202 corresponding to the mole-
cular formula of C15H22. The 1H NMR spectrum (C6D6) of
11 indicated the loss of the secondary hydroxyl at C-3 when
compared to 10, which was replaced by the formation of an
endocyclic double bond at d 5.85 (1H, d, J = 9.5 Hz) and
6.08 (1H, dd, J = 6.0, 9.4 Hz). The relative configuration
of 11 was deduced from its NOESY spectrum, which is
similar to that of 10 (1R,6S,9R,10R) (Fig. 4). The fully
hydrogenated products of 11 were identical to those
obtained from 2, 7, 9 and 10. Hence, 11 was assigned a
(1S,6S,10S)-configuration.
quently, identical to 10, 13 shows
a (1R,6S,10S)-
configuration.
2.9. Structure elucidation of (+)-(1R,2S,6R,8S,10S)-2,8-
epoxyamorpha-4,7(11)-diene 14
Compound 14 (0.7%), showed a molecular ion signal at
m/z at 218 corresponding to C15H22O as determined by
1
HR-EIMS ([M+] m/z 218.1680). The H NMR spectrum
(C6D6) showed signals of four methyl groups at d 0.87
(3H, d, H-14, J = 7.3 Hz), 1.39 (3H, s, H-13), 1.58 (3H, s,
H-12) and 1.61 (3H, br s, H-15). The signals at d 4.04
(1H, s) and 4.49 (1H, s) are assigned to the two methine
protons at the oxygenated C-2 and C-8, respectively. The
2D 1H–1H-COSY, HMQC and HMBC spectra in addition
to the 13C NMR extracted from the HMBC and HSQC
data (Table 1) confirmed the structure. Its relative configu-
ration resulted from the NOESY spectrum (Fig. 5). Direct
rigorous hydrogenation of 14 yielded products identical to
those obtained from (À)-2 as well as from 7, 9, 10 and 11.
Thus, 14 was assigned to show a (1R,2S,6R,8S,10S)-con-
figuration. The a-orientation of the ring was deduced by
the spatial interactions of H-2 and H-1. Treatment of 14
with an acidic ion exchange resin (Amberlyst) for 2 h at
room temperature gave cadalene and two sesquiterpenes,
the major one being identical to a-calacorene and the
minor one could not be identified.
CH3
H
H
HO
3
1
6
H3C
H
Figure 4. NOE correlations of 10.
2.7. Structure elucidation of (À)-(1R,3R,6S,10S)-3a-acet-
oxyamorpha-4,7(11)-diene 12
Compound 12 (0.7%), the acetate of 10, was isolated from
the same essential oil. It showed a molecular ion signal at

A. M. Adio et al. / Tetrahedron: Asymmetry 18 (2007) 1245–1253
1249
HMQC and HMBC spectra in addition to the 13C NMR
(Table 1) confirmed the structure. The relative configura-
tion of 16 was determined by NOE spectroscopy (Fig. 7).
Its absolute configuration was deduced from the direct
hydrogenation of 16 and comparison with the hydrogena-
tion products of 3 using enantioselective GC. Thus, a
(1R,2S,6R,10S)-configuration was assigned to 16.
H
CH3
H
2
1
O
8
6
H
H
CH3
H
CH3
HO
H
Figure 5. NOE correlations of 14.
2
1
6
2.10. Structure elucidation of (+)-(1S,5S,6R,9R,10R)-5,9-
epoxyamorpha-3,7(11)-diene 15
H
Compound 15 was obtained as a trace component (0.05%)
and showed a molecular ion signal at m/z at 218 corres-
ponding to C15H22O as determined by HR-EIMS (m/z
H3C
1
218.1679 [M+]). It also exhibited a similar H NMR spec-
Figure 7. NOE correlations of 16.
trum (C6D6) as 14, except that the position of the two
methine protons at the oxygenated carbons shifted to d
3.70 (1H, s, H-9) and 3.93 (1H, s, H-5). In addition, the
endocyclic double bond was situated at C3 compared to
C4 in 14. Additional structural information was obtained
from HMBC and HMQC which confirmed the presence
of two carbon–oxygen-linkages with signals at d 72.9 (d,
C-9) and 73.9 (d, C-5). In addition, the methine carbon
at d 120.0 (d, C-3) and three quaternary carbons at d
121.9 (s, C-11), 129.8 (s, C-7) and 137.8 (s, C-4) were con-
firmed from the HMBC spectrum. The relative configu-
ration of 15 was deduced from the NOESY spectrum,
which indicated the interactions of protons H-1 with
H-14 and H-6. In addition, proton H-5 also interacts with
H-6 (Fig. 6). The hydrogenation of 15 yielded the same
products as 14, establishing the compound to show
(1S,5S,6R,9R,10R)-configuration. Subjected to acidic ion
exchange resin (Amberlyst) treatment, 10% of compound
15 formed similar compounds as 14. Regarding the yield
of degradation, product 15 was relatively stable when com-
pared to 14.
2.12. Structure elucidation of (À)-(1R,2R,6R,10S)-2b-acet-
oxyamorpha-4,7(11)-diene 17
(À)-2b-Acetoxyamorpha-4,7(11)-diene 17 (1.2%) has the
molecular formula C17H26O2. Although this could not be
confirmed by mass spectrometry (the largest ion detected
under EI conditions corresponds to C15H22 due to immedi-
ate loss of acetic acid). The 13C NMR spectrum (Table 1)
1
revealed the presence of 17 carbon resonances. H NMR
and HMBC demonstrated that compound 17 had a total
of 26 protons 23 protons of which are directly attached
1
to the carbon skeleton. The H NMR and 13C NMR were
identical to those of (À)-2a-acetoxyamorpha-4,7(11)-diene
3 except for slight chemical shift changes. The 2D
1H–1H-COSY, HMQC and HMBC spectra confirmed the
structure of 17. The spatial interactions of protons were de-
rived from the NOESY spectrum, which is similar to that
of 3 (Fig. 7), except for the lack of spatial interactions be-
tween protons H-2 and H-6. The absolute configuration of
17 was confirmed by deacetylation, followed by pyr-
idiniumdichromate (PDC) oxidation. The oxidised product
(+)-amorpha-4,7(11)-dien-2-one 18, gave identical 1H
NMR data, the same GC–MS characteristics and the same
retention times on achiral polysiloxane and chiral cyclodex-
trin derived GC phases as the deacetylated and PDC oxi-
dised product of (À)-3. Compound 18 could also be
identified as a trace constituent in the essential oil. As a re-
sult, the stereochemistry at the stereogenic centres C-1, C-
2, C-6 and C-10 was confirmed as (1R,2R,6R,10S) for 17.
CH3
H
1
H
9
O
6
4
H
H
3. Conclusion
Figure 6. NOE correlations of 15.
In conclusion, the co-existence of compounds 1, 2, 3, 7,
9, 10, 12, 13, 14, 15, 16 and 17 with (À)-(1R,2S,6R,10S)-
2a-acetoxy-11-methoxyamorpha-4,7-diene 19 and (À)-
(1R,2S,6R,10S)-2a-acetoxyamorpha-4,7(11)-dien-8-one 205
which were identified on the basis of their mass spectral
fragmentation patterns and retention indices, suggested
that the essential oil of the Austrian M. aquatica consists
2.11. Structure elucidation of (À)-(1R,2S,6R,10S)-2a-
hydroxyamorpha-4,7(11)-diene 16
Compound 16, C15H24O (m/z 220.1809 [M+]), which is the
deacetylated form of (À)-2a-acetoxyamorpha-4,7(11)-diene
1
36 also occurs in the essential oil. The 2D H–1H-COSY,

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A. M. Adio et al. / Tetrahedron: Asymmetry 18 (2007) 1245–1253
14
R2
H
H
H
OH
R3
R1
10
7
3
1
6
15
H
H
H
OH
H
2 R1 = R2 = R3 = H
3 R1 = R3 = H; R2 = OAc
1
7
8
1
2
3
9
10
R = OH; R = R = H
R = R = H; R = OH
H
H
H
1
2
3
12 R1 = R2 = H; R3 = OAc
13 R1 = R2 = H; R3 = =O
16 R1 = R3 = H; R2 = OH
O
O
H
H
H
1
3
2
17
18
β
R = R = H; R = -OAc
1
3
2
R = R = H; R = = O
11
14
15
AcO
H
AcO
H
O
H
H
OCH3
19
20
of mainly amorphanes, traces of cadinane/muurolane, for
example, (+)-epi-cubenol, few gymnomitrane based com-
pounds (a- and b-barbatene) with traces of longipinane
(a-longipinene).
Analytical 70-250S magnetic field mass spectrometer. All
compounds were identified by comparison of their mass
spectra and gas chromatographic retention indices with a
spectral library established under identical experimental
conditions.7,8
4. Experimental
4.1.4. NMR spectroscopy. NMR measurements were
carried out with a Bruker WM 500 (500 MHz) instrument
in C6D6 using TMS as internal standard. NMR-experiment
4.1. General experimental procedures
1
acquisition for H–1H COSY was with 4096 (F2) and 256
4.1.1. Gas chromatography. Carlo Erba HRGC 5300
Mega series instrument fitted with 25 m fused silica capil-
laries coated with polysiloxane CPSil-5 and polysiloxane
CPSil-19 (Chrompack); Carlo Erba Fractovap 2150 or
4160 gas chromatographs with 25 m fused silica capillaries
coated with octakis(2,6-di-O-methyl-3-O-pentyl)-c-cyclo-
dextrin,14 heptakis(2,6-di-O-methyl-3-O-pentyl)-b-cyclo-
dextrin14 or heptakis(6-O-tert-butyldimethylsilyl-2,3-di-O-
methyl)-b-cyclodextrin15 in OV 1701 (50%, w/w), split
injection; split ratio approx. 1:30; FID; carrier gas 0.5 bar
H2; injector and detector temperatures were 200 and
250 ꢁC, respectively.
(F1) data points. Delay was 1.1 s. F2 and F1 were each
processed as Qsine with a sinus shift of 0. HSQC was
recorded as a phase sensitive experiment with TD = 4096
(F2) and TD = 128 (F1) data points and with a delay of
1/4 145 Hz. Processing was done as an exponential multi-
*
plication in F2 with line broadening of 4.00 Hz and with
QSINE in F1 with a sinus shift of 2. HMBC was recorded
as a J filtered experiment with TD = 4096 (F2) and
TD = 256 (F1). Delay was 0.065 s. F2 and F1 were each
processed as Qsine with a sinus shift of 4. NOESY was
recorded as phase sensitive with TD = 4096 (F2) and
TD = 400 (F1) data points, respectively. Mixing time was
600 ms. F2 and F1 were each processed as Qsine with a
sinus shift of 2.
4.1.2. Preparative GC. Modified Varian 1400 and 2800
instruments, equipped with stainless steel columns
(1.85 m · 4.3 mm) with 10% polydimethylsiloxane SE-30
on Chromosorb W-HP or with 2.5% octakis(2,6-di-O-
methyl-3-O-pentyl)-c-cyclodextrin in OV-1701 (50%, w/w)
on Chromosorb G-HP or with 6% heptakis(6-O-tert-butyl-
dimethylsilyl-2,3-di-O-methyl)-b-cyclodextrin in SE-52
(50%, w/w) on Chromosorb W-HP; FID; helium as carrier
gas at a flow rate of 120 ml/min; injector and detector
temperatures were 200 and 250 ꢁC, respectively.16
4.1.5. Polarimetry. Measurements were performed with a
polarimeter 341 (Perkin–Elmer) at 589 nm at 20 ꢁC. Due to
the very small amounts of isolated compounds, only the
direction of the optical rotation is given in order to avoid
inaccuracies.
4.1.6. Thin-layer chromatography. Thin-layer chromato-
graphy was carried out using glass and aluminium plates
coated with silica 60 F254 (Merck). An ethanolic solution
of sulfuric acid (10%) and anisaldehyde was used as the
spray reagent. The solvent system used was n-hexane/ethyl
4.1.3. GC–MS. Electron impact (70 eV) and chemical
ionisation (NH3) GC–MS was carried out on a Hewlett
Packard HP 5890 gas chromatograph coupled with a VG

A. M. Adio et al. / Tetrahedron: Asymmetry 18 (2007) 1245–1253
1251
acetate (3:1, v/v). The Rf values were also determined in
this solvent system.
H-9b), 1.62 (3H, s, H-12), 1.71–1.76 (1H, m, H-1b), 1.77–
1.82 (1H, m, H-2b), 1.88–1.94 (2H, m, H-3, H-8b), 5.47
(1H, s, H-5); 13C NMR (125.7 MHz, C6D6): d 16.1 (q,
C-12), 20.0 (t, C-9), 21.5 (q, C-13), 23.0 (q, C-14), 25.2 (t,
C-1), 25.4 (t, C-2), 29.4 (q, C-15), 30.3 (t, C-8), 33.2 (s,
C-11), 39.3 (t, C-10), 56.1 (s, C-7), 58.5 (d, C-3), 60.8 (s,
C-6), 131.0 (d, C-5), 142.4 (s, C-4); MS (EI, 70 eV), m/z
(rel. int.): 204 [M+] (16), 176 (44), 161 (100), 147 (8), 133
(12), 119 (21), 105 (18), 91 (17), 77 (8), 55 (8), 41 (15).
4.1.7. Reactions. Hydrogenation reactions were performed
by bubbling hydrogen gas through a stirred solution of ca.
1 mg of sample in 1 ml n-hexane and 0.5 mg Pd/C at room
temp. for 1 h. The reaction mixture was filtered, and the
reaction products analysed by GC–MS and by GC on sev-
eral capillary columns with modified cyclodextrin
derivatives.
4.5. (+)-(1R,6S,7S,10S)-7b-Hydroxyamorpha-4,11-diene 7
Colourless oil, RICPSIL 5 = 1614; Rf = 0.75; sense of optical
Dehydration was carried out with ca. 1 mg of sample in
0.5 ml of pyridine and 1 drop of phosphoryl chloride under
ice cooling. After 1 h of stirring at room temperature the
reaction was quenched by adding a few drops of water,
and the mixture was extracted three times with n-hexane.
The organic phase was washed several times with water
and dried over Na2SO4.
1
rotation (benzene): (+); H NMR (500 MHz, C6D6): 0.92
(3H, d, H-14, J = 6.3 Hz), 1.34–1.46 (4H, m, H-2a, H-8a,
H-9a, H-10), 1.47–1.52 (1H, m, H-9b), 1.54–1.62 (1H, m,
H-8b), 1.57 (3H, br s, H-15), 1.62–1.68 (1H, m, H-3a),
1.75–1.82 (1H, m, H-3b), 1.81 (3H, s, H-12), 1.84–1.93
(2H, m, H-1, H-2b), 2.49 (1H, s, H-6), 4.85 (1H, s,
H-13a), 4.90 (1H, s, H-13b), 5.15 (1H, s, H-5); 13C NMR
(125.7 MHz, C6D6): see Table 1. MS (EI, 70 eV), m/z
(rel. int.): 220 [M+] (15), 202 (85), 187 (100), 173 (17),
159 (80), 146 (33), 145 (94), 134 (60), 132 (75), 133 (62),
121 (39), 119 (65), 105 (52), 91 (53), 81 (38), 79 (47), 77
(46), 69 (30), 55 (40), 41 (75); EIHRMS calcd for
C15H24O1 [M+] m/z 220.1827, found [M+] m/z 220.1832.
Oxidation reactions were carried out using ca. 1 mg of
sample in dry CH2Cl2 and ca. 2 mg of PDC. After 3 h of
stirring at room temperature Et2O (5 ml) was added, and
the resulting mixture was filtered through a short column
packed with florisil.
Deacetylation reactions were carried out by treating
ca. 1 mg of samples in methanol (1 ml) with ca. 2 mg of
K2CO3. The reaction mixture was stirred at room temper-
ature for 12 h. After work-up, the product was analysed by
GC and GC–MS.
4.6. (À)-(1S,6S,9R,10R)-9a-Hydroxyamorpha-4,7(11)-diene
9
White, viscous oil; RICPSIL 5 = 1680; Rf = 0.56; sense of
optical rotation (benzene): (À); 1H NMR (500 MHz,
C6D6): d 1.03 (3H, d, H-14, J = 6.6 Hz), 1.24–1.29 (1H,
m, H-1), 1.44–1.55 (3H, m, H-2, H-10), 1.58 (3H, s,
H-15), 1.61–1.68 (1H, m, H-3a), 1.66 (6H, br s, H-12,
H-13), 1.78–1.86 (2H, m, H-3b, H-8a), 2.71 (1H, dd,
H-8b, J = 4.1, 12.6 Hz), 2.98 (1H, dt, H-9, J = 4.1,
11.4 Hz), 3.36 (1H, br s, H-6), 5.09 (1H, s, H-5); 13C
NMR (125.7 MHz, C6D6): see Table 1. MS (EI, 70 eV),
m/z (rel. int.) 220 [M+] (63), 202 (60), 187 (100), 173 (30),
159 (52), 147 (60), 145 (65), 131 (42), 119 (48), 105 (59),
91 (61), 77 (43), 55 (42), 41 (80); EIHRMS calcd for
C15H24O1 [M+] m/z 220.1827, found [M+] m/z 220.1816.
Acidic transformations were carried out by treatment of ca.
0.3 mg of samples in hexane with a few granules of Amber-
lystꢂ 15 at room temperature for about 2 h. The reaction
mixture was monitored by GC.
4.2. Origin of M. aquatica
M. aquatica was collected near Gaschurn/Montafon,
Austria, in July 2001. A plant voucher is deposited at the
¨
Botanik und Okologie department Universita¨t Ulm,
D-89091 Ulm, Germany.
4.3. Isolation of compounds
The hydrodistillate of M. aquatica was submitted to flash
column chromatography (column packed dry with silica
gel; elution with n-hexane to yield the hydrocarbon fraction
and gradient elution with ethyl acetate in hexane to yield
the oxygenated fractions). Oxygenated fractions were
further subjected to prep-TLC and final isolations were
carried out using GC Varian 1400 and 2800 on a packed
column with SE-30- and/or SE-52-columns combined with
at least one cyclodextrin phase column.
4.7. (À)-(1R,3R,6S,10S)-3a-Hydroxyamorpha-4,7(11)-diene
10
Colourless oil; RICPSIL 5 = 1666; Rf = 0.68; sense of optical
rotation (benzene): (À); 1H NMR (500 MHz, C6D6): d
0.88–0.97 (1H, m, H-9a), 1.01 (3H, d, H-14, J = 6.6 Hz),
1.24–1.28 (1H, m, H-1), 1.48–1.53 (1H, m, H-2a), 1.60–
1.65 (1H, m, H-9b), 1.67 (3H, d, H-12, J = 0.9 Hz), 1.68
(3H, d, H-13, J = 2.2 Hz), 1.73 (3H, br s, H-15), 1.80–
1.88 (1H, m, H-8a), 1.99–2.05 (1H, m, H-10), 2.09–2.15
(1H, m, H-2b), 2.52–2.57 (1H, m, H-8b), 3.30 (1H, br s,
H-6), 3.68 (1H, s, H-3), 5.16 (1H, s, H-5); 13C NMR
(125.7 MHz, C6D6): see Table 1. MS (EI, 70 eV), m/z
(rel. int.): 220 [M+] (8), 218 (12), 202 (43), 187 (27), 177
(48), 159 (77), 145 (60), 131 (39), 119 (47), 105 (61), 91
(75), 77 (49), 67 (31), 53 (41), 41 (100). HRMS calcd for
C15H24O1 [M+] m/z 220.1827, found [M+] 220.1826.
4.4. (À)-Myltayl-4-ene 6
Colourless oil, RICPSIL 5 = 1380; sense of optical rotation
1
(benzene): (À); H NMR (500 MHz, C6D6): d 0.88 (3H,
br s, H-13), 0.94 (3H, s, H-14), 0.99 (3H, s, H-15), 0.98–
1.04 (2H, m, H-1a, H-2a), 1.21–1.26 (1H, m, H-10a),
1.39–1.48 (2H, m, H-9a, H-10b), 1.52–1.61 (2H, m, H-8a,

1252
A. M. Adio et al. / Tetrahedron: Asymmetry 18 (2007) 1245–1253
4.8. (+)-(1S,6S,10S)-Amorpha-2,4,7(11)-triene 11
41 (48); EIHRMS calcd for C15H24O1 [M+] m/z 218.1671,
found [M+] m/z 218.1680.
Colourless oil, RICPSIL 5 = 1449; sense of optical rotation
1
(benzene): (+); H NMR (500 MHz, C6D6): d 0.76–0.85
4.12. (+)-(1R,5S,6R,9R,10R)-5,9-Epoxyamorpha-3,7(11)-
diene 15
(1H, m), 0.87 (3H, d, J = 6.3 Hz), 1.49–1.57 (2H, m),
1.62 (3H, d, J = 1.9 Hz), 1.66 (3H, s), 1.69 (3H, br s),
1.70–1.76 (1H, m), 1.85–1.92 (1H, m), 2.59—2.64 (1H,
m), 3.77 (1H, s), 5.20 (1H, s), 5.85 (1H, d, J = 9.5 Hz),
6.08 (1H, dd, J = 6.0, 9.4 Hz); MS (EI, 70 eV), m/z (rel.
int.): 202 [M+] (82), 187 (25), 173 (8), 160 (32), 159 (100),
145 (82), 131 (42), 119 (39), 105 (51), 91 (38), 77 (22), 67
(20), 53 (18), 41 (39).
Colourless oil; RICPSIL 5 = 1595; sense of optical rotation
1
(benzene): (+); H NMR (500 MHz, C6D6): d 0.71 (3H,
d, H-14, J = 6.9 Hz), 1.21–1.32 (1H, m, H-1), 1.57 (3H, s,
H-12), 1.59 (3H, s, H-13), 1.75 (1H, d, H-2a, J = 17.0),
1.88 (3H, s, H-15), 1.87–1.95 (1H, m, H-10), 2.05–2.13
(1H, d, H-2b, J = 18.0 Hz), 2.15–2.23 (1H, d, H-8a,
J = 16.4 Hz), 2.42 (1H, s, H-6), 2.45 (1H, d, H-8b,
J = 13.6 Hz), 3.70 (1H, s, H-9), 3.93 (1H, s, H-5), 5.32
(1H, s, H-3); MS (EI, 70 eV), m/z (rel. int.): 218 [M+]
(98), 203 (5), 185 (12), 175 (11), 157 (13), 145 (16), 135
(100), 119 (69), 105 (30), 93 (32), 91 (35), 77 (26), 55 (21),
41 (49); EIHRMS calcd for C15H22O1 [M+] m/z 218.1671,
found [M+] m/z 218.1679.
4.9. (À)-(1R,3R,6S,10S)-3a-Acetoxyamorpha-4,7(11)-diene
12
Colourless oil; RICPSIL 5 = 1780; Rf = 0.94; sense of optical
rotation (benzene): (À); 1H NMR (500 MHz, C6D6): d 0.86
(3H, d, H-14, J = 6.3 Hz), 0.91 (1H, dt, H-9a, J = 3.5,
13.6 Hz), 1.14–1.20 (1H, m, H-1), 1.52–1.61 (2H, m,
H-2a, H-9b), 1.65 (9H, br s, H-12, H-13, H-15), 1.74
(3H, s, H-17), 1.75–1.84 (1H, m, H-8a), 2.02–2.11 (1H,
m, H-10), 2.29 (1H, dd, H-2b, J = 1.9, 15.5 Hz), 2.54
(1H, br dd, H-8b, J = 1.6, 13.9 Hz), 3.25 (1H, s, H-6),
5.24 (1H, br s, H-5), 5.33 (1H, br d, H-3, J = 5.0 Hz);
13C NMR (125.7 MHz, C6D6): see Table 1; MS (EI,
70 eV), m/z (rel. int.): 262 [M+] (20), 220 (4), 202 (98),
187 (50), 177 (21), 160 (55), 159 (100), 145 (70), 131 (33),
119 (42), 105 (44), 91 (38), 77 (23), 67 (18), 55 (23), 43 (63).
4.13. (À)-(1R,2S,6R,10S)-2a-Hydroxyamorpha-4,7(11)-
diene 16
Colourless oil; RICPSIL 5 = 1684; Rf = 0.56; sense of optical
rotation (benzene): (À); 1H NMR (500 MHz, C6D6): d
0.98–1.05 (1H, m, H-9a), 1.21 (3H, d, H-14, J = 6.6 Hz),
1.31–1.35 (1H, m, H-1), 1.56 (3H, br s, H-15), 1.56–1.60
(1H, m, H-9b), 1.67 (3H, s, H-12), 1.70 (3H, d, H-13,
J = 1.9 Hz), 1.70–1.82 (2H, m, H-8a, H-10), 1.94–2.09
(2H, m, H-3), 2.49 (1H, br d, H-8b, J = 13.9 Hz), 3.40
(1H, s, H-6), 3.78-3.82 (1H, m, H-2), 4.97 (1H, s, H-5);
13C NMR (125.7 MHz, C6D6): see Table 1; MS (EI,
70 eV), m/z (rel. int.): 220 [M+] (100), 205 (22), 202 (24),
187 (78), 177 (33), 159 (85), 145 (53), 131 (29), 121 (30),
119 (31), 105 (38), 91 (42), 55 (39), 41 (76); EIHRMS calcd
for C15H24O1 [M+] m/z 220.1827, found [M+] m/z
220.1809.
4.10. (À)-(1R,6S,10S)-Amorpha-4,7(11)-dien-3-one 13
Colourless oil; RICPSIL 5 = 1676; Rf = 0.86; sense of optical
rotation (benzene): (À); 1H NMR (500 MHz, C6D6): d 0.72
(3H, d, H-14, J = 6.3 Hz), 0.73–0.81 (1H, m, H-9a), 1.33–
1.40 (1H, m, H-1), 1.41–1.58 (3H, m, H-8a, H-9b, H-10),
1.59 (6H, s, H-12, H-13), 1.82 (3H, s, H-15), 2.12 (1H,
dd, H-2a, J = 4.7, 16.1 Hz), 2.41 (1H, br d, H-8b,
J = 13.2 Hz), 2.72 (1H, dd, H-2b, J = 2.5, 16.1 Hz), 3.52
(1H, br s, H-6), 5.91 (1H, s, H-5); 13C NMR (125.7 MHz,
C6D6): see Table 1; MS (EI, 70 eV), m/z (rel. int.): 218
[M+] (100), 203 (12), 189 (4), 176 (25), 175 (89), 161 (49),
147 (28), 133 (30), 119 (41), 107 (35), 105 (47), 91 (64), 77
(48), 67 (26), 55 (40), 41 (68); EIHRMS calcd for
C15H22O1 [M+] m/z 218.1671, found [M+] m/z 218.1668.
4.14. (À)-(1R,2R,6R,10S)-2b-Acetoxyamorpha-4,7(11)-
diene 17
Colourless oil; RICPSIL 5 = 1721; Rf = 0.94; sense of optical
rotation (benzene): (À); 1H NMR (500 MHz, C6D6): d 0.86
(3H, d, H-14, J = 6.6 Hz), 0.87–0.94 (1H, m, H-9a), 1.21–
1.32 (1H, m, H-10), 1.46–1.51 (1H, m, H-9b), 1.54 (3H,
br s, H-15), 1.56–1.62 (1H, m, H-1), 1.67 (3H, s, H-12),
1.71 (3H, s, H-17), 1.75 (3H, s, H-13), 1.75–1.83 (1H, m,
H-8a), 1.95–2.08 (2H, m, H-3), 2.52 (1H, br d, H-8b,
J = 13.6 Hz), 3.83 (1H, s, H-6), 5.18 (1H, s, H-5), 5.49
(1H, s, H-2); 13C NMR (125.7 MHz, C6D6): see Table 1;
MS (EI 70 eV), m/z (rel. int.): 262 [M+], 202 (98), 187
(89), 174 (11), 159 (98), 145 (97), 131 (41), 119 (32), 105
(55), 91 (50), 77 (30), 67 (21), 55 (29), 43 (100).
4.11. (+)-(1R,2S,6R,8S,10S)-2,8-Epoxyamorpha-4,7(11)-
diene 14
Colourless oil; RICPSIL 5 = 1597; Rf = 0.75; sense of optical
rotation (benzene): (+); 1H NMR (500 MHz, C6D6): d 0.87
(3H, d, H-14, J = 7.3 Hz), 1.02–1.06 (1H, m, H-9a), 1.37–
1.40 (1H, m, H-1), 1.39 (3H, s, H-13), 1.58 (3H, s, H-12),
1.61 (3H, br s, H-15), 1.75–1.81 (1H, m, H-10), 1.95 (1H,
br d, H-3a, J = 18.0 Hz), 2.18 (1H, d, H-3b,
J = 18.6 Hz), 2.21–2.26 (1H, m, H-9b), 3.07 (1H, s, H-6),
4.04 (1H, s, H-2), 4.49 (1H, s, H-8), 5.72 (1H, d, H-5,
J = 6.0 Hz); 13C NMR (125.7 MHz, C6D6): see Table 1;
MS (EI, 70 eV), m/z (rel. int.): 218 [M+] (49), 200 (9), 185
(12), 175 (7), 157 (12), 143 (10), 138 (11), 133 (22), 121
(100), 119 (66), 105 (28), 93 (36), 91 (32), 77 (26), 55 (21),
4.15. (+)-(1R,6R,10S)-Amorpha-4,7(11)-dien-2-one 18
Colourless oil; RICPSIL 5 = 1645; sense of optical rotation
1
(benzene): (+); H NMR (500 MHz, C6D6): d 0.79 (3H,
d, J = 6.6 Hz), 1.16 (3H, dd, J = 10.4, 17.7 Hz), 1.41 (3H,
d, J = 0.9 Hz), 1.49 (3H, d, J = 1.9 Hz), 1.58 (3H, d,
J = 0.9 Hz), 2.12 (1H, dd, J = 5.0, 11.4 Hz), 2.41–2.62
(4H, m), 3.69 (1H, br s), 5.02 (1H, s); MS (EI 70 eV),

A. M. Adio et al. / Tetrahedron: Asymmetry 18 (2007) 1245–1253
1253
m/z (rel. int.): 218 [M+] (100), 203 (21), 190 (8), 185 (8), 175
(82), 161 (51), 147 (48), 133 (38), 119 (42), 105 (43), 91 (41),
77 (28), 55 (27), 41 (67).
4. Nagashima, F.; Ishimaru, A.; Asakawa, Y. Phytochemistry
1994, 37, 777–779.
5. Leong, Y.-W.; Harrison, L. J.; Connolly, J. D.; Rycroft, D.
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Acknowledgements
7. Joulain, D.; Ko¨nig, W. A. The Atlas of Spectral Data of
Sesquiterpene Hydrocarbons; E.B.: Hamburg, 1998.
We gratefully acknowledge the financial support of DAAD
(scholarship for A. M. Adio) and the Fonds der Chemis-
chen Industrie. We also thank Dr. V. Sinnwell for his
support in recording the NMR spectra and Mrs. A. Mein-
ers and Mr. M. Preusse for GC–MS measurements.
8. Hochmuth, D. H.; Joulain, D.; Konig, W. A. MassFinder 3.1,
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Software and Data Bank; University of Hamburg, 2004
www.massfinder.com.
9. Adio, A. M. Ph.D. Thesis. University of Hamburg, 2005.
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