Sesquiterpenoids, hopanoids and bis(bibenzyls) from the argentine liverwort Plagiochasma rupestre

Article abstract of DOI:10.1016/S0031-9422(99)00452-5

A new cadinane-type sesquiterpene alcohol named rupestrenol has been isolated from the Argentine liverwort Plagiochasma rupestre (Forst.) Steph., belonging to the Aytoniaceae along with the known sesquiterpenoids marsupellone, β-longipinene and caryophyllene oxide, three known hopanoids, and four known bis(bibenzyl) derivatives as well as other common plant constituents. The structure and stereochemistry of rupestrenol was established by a combination of extensive NMR experiments and X-ray crystallographic analysis.

Full text of DOI:10.1016/S0031-9422(99)00452-5

Phytochemistry 52 (1999) 1323±1329
Sesquiterpenoids, hopanoids and bis(bibenzyls) from the
Argentine liverwort Plagiochasma rupestre
Alicia Bardona, Norma Kamiyaa, Masao Toyotab, Shigeru Takaokab,
Yoshinori Asakawab,*
a
     Â
Instituto de Quõmica Organica, Facultad de Bioquõmica, Quõmica y Farmacia, Universidad Nacional de Tucuman, Ayacucho 471, Tucuman 4000,
Argentina
bFaculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima 770-8514, Japan
Received 14 May 1999; accepted 2 June 1999
Abstract
A new cadinane-type sesquiterpene alcohol named rupestrenol has been isolated from the Argentine liverwort Plagiochasma
rupestre (Forst.) Steph., belonging to the Aytoniaceae along with the known sesquiterpenoids marsupellone, b-longipinene and
caryophyllene oxide, three known hopanoids, and four known bis(bibenzyl) derivatives as well as other common plant
constituents. The structure and stereochemistry of rupestrenol was established by a combination of extensive NMR experiments
and X-ray crystallographic analysis. # 1999 Elsevier Science Ltd. All rights reserved.
Keywords: Plagiochasma rupestre; Aytoniaceae; Liverworts; Hepaticae; Rupestrenol; Cadinane; Caryophyllane; Longipinane; Sesquiterpenoids;
Hopanoids; Bis(bibenzyls); Chemosystematics
1. Introduction
(=P. intermedium ) (Hashimoto, Irita & Asakawa,
1998) and Markham & Porter (1978) reported the pre-
sence of three ¯avonoid glycosides from P. rupestre.
Harrison, Becker, Connolly & Rycroft (1992) found in
P. rupestre the longipinane-type sesquiterpenoid, mar-
supellone (7), two elemane-type sesquiterpenoids, and
a hopane-type triterpenoid zeorin (11) together with a
¯avone methyl ether.
In order to study the comparative biochemistry of
the species belonging to the Aytoniaceae, we reinvesti-
gated the chemical constituents of P. rupestre from
Argentine and report here the isolation and structure
of a new cadinane-type sesquiterpenoid named rupes-
trenol (1), together with known sesqui- and triter-
penoids and bis(bibenzyls).
Liverworts produce not only various pungent and
bitter terpenoids but also benzyls and bis(bibenzyl) de-
rivatives which show interesting biological activity,
and which are valuable for the chemosystematics of
the Hepaticae (Asakawa, 1982, 1990, 1993, 1995, 1997,
1998). The Plagiochasma genus (Aytoniaceae) includes
P. appendiculata, P. australe, P. japonicum, P. pteros-
permum (=P. intermedium ) and P. rupestre. We have
reported the isolation and the structure elucidation of
a new bis(bibenzyl), marchantin H (Tori, Toyota,
Harrison, Takikawa & Asakawa, 1985) and the pre-
sence of elemane-, caryophyllane-, barbatane-, bazza-
nane-type sesquiterpenoids and the isolation of (+)-
gymnomitr-8(12)-en-9a-ol and two bis-bibenzyls,
pakyonol and riccardin C (13) from P. pterospermum
2. Results and discussion
P. rupestre is widespread in humid rocky mountains
of northern Argentina. The air-dried and ground P.
rupestre was extracted with ether and then methanol.
* Corresponding author. Tel.: +81-88-622-9611; fax: +81-88-655-
3051.
E-mail address: asakawa@ph.bunri-u.ac.jp (Y. Asakawa).
0031-9422/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(99)00452-5

Â
A. Bardon et al. / Phytochemistry 52 (1999) 1323±1329
1324
Table 2
13C-NMR spectral data for compounds 1 and 5 (CDCl3-TMS)
The ether extract was chromatographed on silica gel
and Sephadex LH-20, and each fraction was puri®ed
by HPLC to give rupestrenol (1), a new cadinane-type
sesquiterpene alcohol, the known sesquiterpenoids
marsupellone (7) (Matsuo, Uto, Uchio, Nakayama &
Hayashi, 1979), caryophyllene oxide (8) (Asakawa,
Toyota, Bischler, Campbell & Hattori, 1984) and b-
longipinene (6) (Andersen et al., 1977), the norhopa-
Carbon
1a
1b
5c
46.3
1
46.5
35.3
2
26.5
30.9
17.0
31.3
26.2
26.4
3
4
134.6
123.5
44.1
134.1
125.0
39.6
137.5
125.5
51.1
5
noid adiantone (9) (Hveding-Bergseth, Bruun
&
6
Kjùsen, 1983), the hopanoids diplopterol (10) (Ageta,
Iwata & Otake, 1963) and zeorin (11) (Huneck &
Lehn, 1963), the known bis(bibenzyls) marchantin K
(12) (Asakawa, Tori, Masuya & Frahm, 1990), march-
antin B (13) (Asakawa, Toyota, Matsuda, Takikawa &
7
47.5
34.1
43.5
29.6
42.6
32.5
8
9
35.9
36.3
34.3
42.0
35.7
36.5
10
11
12
13
14
15
152.2
64.9
152.9
69.5
148.4
19.1
Takemoto, 1983), riccardin
C
(15) (Asakawa
&
109.1
19.6
23.5
108.2
19.5
23.5
111.6
19.7
67.4
Matsuda, 1982) and isoriccardin C (16)(Asakawa,
Tori, Takikawa, Krishnamurti & Kanti Kar, 1987).
The methanol extract was treated in the same manner
as described above to a€ord 1, 10, 12 and 16.
a Present study.
b Garbarino et al. (1993).
c Wei et al. (1995).
The structure of rupestrenol (1) was deduced by
spectroscopic data, along with X-ray crystallographic
analysis of the 2,4-dinitrophenylhydrazone of the alde-
hyde (2) derived from 1. The EI-mass spectrum of 1
gave a molecular ion peak at m/z 220 and its HR-mass
spectrum indicated a molecular formula C15H24O that
accounted for four degrees of unsaturation. The IR
primary allylic alcohol was present. The signals of 3
vinyl protons and the corresponding 4 ole®nic carbons
which accounted for two double bonds, led to the
assumption of a bicyclic molecule. The H±1H COSY,
1
HMQC and HMBC spectra provided evidence for the
presence of 4,11(13)-cadinadienol. The antiperiplanar
relationships of the four protons at C-1, C-2, C-6 and
C-10 were indicated by the large coupling constants
J1, 2ax ˆ 12 Hz, J1, 6 ˆ J1, 10 ˆ 10 Hz. A compound
with the structure and stereochemistry we hereby pro-
pose for 1 had been previously reported from the
Chilean Nolanaceae Nolana coelestis (Garbarino,
Chami, Montagna & Gambaro, 1993) but the chemical
shifts of H-1, C-1, C-2, C-6, C-7, C-8 and C-10 in the
corresponding NMR spectra (Table 1 and Table 2) as
well as the reported value for the speci®c optical ro-
tation (Table 1) of the compound assigned formula 1
in the above reference di€er from those of our com-
pound 1. The 13C-NMR spectral data of 1 resembled
1
spectrum showed the broad band at 3300 cm of a
hydroxyl group along with the bands at 1645 and 895
1
cm related to ole®nic systems. The broad singlet at d
4.10 (2H) in the 1H-NMR spectrum suggested that a
Table 1
1H-NMR data for compound 1 (CDCl3-TMS)
1a ꢀ‰aŠ +43.9)
1b ꢀ‰aŠ
177:3†
D
D
(600 MHz)
(360 MHz)
H-1
0.76
dddd (12, 12, 10, 2)c
1.20 ddd (11, 9, 2.0)
H-2eq 1.92±2.02
H-2ax 1.18
m
dddd (12, 12, 4.7, 1.5)
H-3eq 1.92±2.02
H-3ax 1.92±2.02
m
m
quin (1.6)
H-5
H-6
H-7
5.26
1.8±1.82
1.7±1.82
5.39 br s
1.73 md (9, 11)
1.88 ddd (9.0,9.0, 2.5)
m
m
H-8eq 1.7±1.82
H-8ax 1.38
m
dddd (13, 3.6, 3.6, 3.6)
H-9eq 1.7±1.82
H-9ax 1.08
m
dddd(13, 13, 13, 4)
H-10
H-12
1.20
4.10
m
br s
4.00 br s
H-13a 4.91
H-13b 5.15
q (1.4)
q (1.6)
d (6.3)
m
4.93 d (1.0)
5.11 d (1.0)
0.93 d (5)
H-14
H-15
0.92
1.61
1.61
s
a Present study.
b Garbarino et al. (1993).
c Coupling constants (J in parentheses) were given in Hz;.
d Obscured due to overlapping signals.
Fig. 1. Partial NOEs observed for 1.

Â
A. Bardon et al. / Phytochemistry 52 (1999) 1323±1329
1325
those of (1R,6S,7S,10S)-15-hydroxy-cadina-4,11(13)-
diene (5) (Table 1), isolated from the Taiwanese liver-
wort, Reboulia hemisphaerica (Wei, Ma & Wu, 1995),
indicating that 1 possessed the same relative stereo-
chemistry as 5. This assumption was further con®rmed
by the NOE experiment of 1 and the X-ray crystallo-
graphic analysis of the 2,4-dinitrophenylhydrazone (4)
of 2, which was obtained by oxidation of 1 with pyri-
dinium dichromate (PDC). The partial NOESY spec-
trum showed NOEs between (i) H-1 and H-9ax, (ii) H-
1 and H-14, (iii) H-5 and H-12, (iv) H-5 and H-13,
and (v) H-5 and H-12, as shown in Fig. 1. The
ORTEP drawing of 4 is shown in Fig. 2. Oxidation
and benzoylation of 1 to the corresponding aldehyde
(2) and p-bromobenzoate (3) provided additional
chemical evidence. Consequently, the structure of
rupestrenol was established to be (+)-(1RÃ,
From the methanolic extract, a minor bis-bibenzylic
compound, which was assigned as marchantin K (12)
was isolated. This compound has been isolated from
Marchantia polymorpha collected in Germany and its
strcuture elucidated by the 1H-, 13C-NMR and NOE
spectral data of the permethylated product (14) of 12
1
(Asakawa et al., 1990). In Tables 3 and 4, the H- and
13C-NMR and HMBC spectral data of the original
marchantin K have been shown. These data further
con®rmed the structure of 12. Previously, the 13C-
NMR data for isoriccardin C (16) has been reported
(Asakawa et al., 1987). In Table 4, their assignments
are newly added.
The structures of the previously known terpenoids
and bis(bibenzyls) were established by comparison of
spectral and other physical data with those of auth-
entic samples.
In Table 5, the chemical constituents of the
Plagiochasma have been shown. A previous report on
the French P. rupestre indicated the presence of com-
pounds 7 and 11 in addition to elemane-type sesqui-
terpenoids (Harrison et al., 1992). Bis(bibenzyls) were
6SÃ,7SÃ,10SÃ)-15-hydroxy-cadina-4,11(13)-diene
(1).
The absolute con®guration of 1 remained to be clari-
®ed. Thus the structure assigned to compound 1
reported by Garbarino et al. (1993) might need to be
revised.
Fig. 2. ORTEP drawing for 4.

Â
A. Bardon et al. / Phytochemistry 52 (1999) 1323±1329
1326
Table 3
1H-NMR (600 MHz, CDCl3-TMS) data for 12, and 13C-NMR data
for 12 and 16 (150 MHz, CD3OD-TMS)
Grammes, Burkhardt
&
Becker, 1994), lichens
(Huneck & Yoshimura, 1996) and ferns (Murakami
& Tanaka, 1988). They have also been isolated from
sedimentary organic matter of most varied origin and
are ubiquitous molecular fossils derived from cellular
constituents of microorganisms (Ourisson, Albrecht &
Rohmer, 1979). The Argentine and French P. rupes-
tre and the Japanese P. pterospermum produce com-
monly zeorin (11). Thus, this triterpene alcohol is one
of the chemical markers of the genus Plagiochasma.
The Argentine P. rupestre is chemically similar to P.
pterospermum, since both species produce cyclic bis(-
bibenzyl), riccardin C (15) and the similar bis(biben-
zyls) (12, 13, 16) to those of marchantin H and
pakyonol (Hashimoto et al., 1998). There is chemical
anity between the Plagiochasma species and
Reboulia hemisphaerica which belongs to the
Aytoniaceae, because both genera biosynthesize bar-
batane-, and cadinane-type sesquiterpenoids and
marchantin- and riccardin-type bis(bibenzyls) (Wei et
al., 1995).
12
12
16
H
J
C
C
2,6
3,5
7a
6.50
6.91
br d
br d
m
8
8
1
155.2
122.7
130.6
140.6
130.6
122.7
37.1
153.2
121.7a
130.8
137.2
130.3
121.7a
34.5
2
3
3.00±3.05
2.90±2.97
2.80
7b,8a
8b
10
m
4
ddd
d
15, 10, 2
5
6.83
6.75
8.5
8.5
2
6
11
3'
5'
d
7
4.96
6.52
d
8
30.8
36.1
d
2
9
128.3
121.3
113.5
145.8
139.1
142.2
134.8
148.9
109.2
132.1
107.0
147.5
85.9
143.3
116.6
153.4
117.0
130.5
122.6
143.6
147.8
114.7
133.6
121.6a
114.6
38.0
7'
4.06
3.03
dd
dd
dd
d
10,4
12,4
12,10
2
10
11
12
13
14
1'
2'
3'
4'
5'
6'
7'
8'
9'
10'
11'
12'
13'
14'
8a'
8b'
10'
12'
13'
14'
OMe
2.56
6.68
6.46
6.85
ddd
t
8,3,1
8
6.02
3.26
br d
s
8
It is also noteworthy that adiantone (9) and
diplopterol (10) and their related hopanoids have
been isolated from in vitro cultured liverwort
Fossombronia alaskana and F. pusilla belonging to
the order Metzgeriales (Grammes et al., 1994),
although these species are morphologically di€erent
from the Plagiochasma classi®ed into the order
Marchantiales.
45.0
36.5
139.9
118.1
158.9
114.0
129.0
124.6
121.6
120.0
154.0
113.3
130.1
142.9
a Assignments may be reversed.
Table 5
Distribution of terpenoids and phenolic compounds in Plagiochasma
species
absent in this collection. The elemane-type sesquiter-
penoids have not been detected in the present species.
Hopanoids are triterpenes widely distributed among
bacteria and cyanobacteria and have been found in a
few genera of liverworts (Toyota & Asakawa, 1993;
Compounds
P. rupestrea
P. rupestreb
P. pterospermumc
Sesquiterpenoids
Barbatanes
Bazzananes
Cadinanes
+
+
+
+
Table 4
C±H correlation for 12 observed by HMBC spectrum
Caryophyllanes
Elemanes
+
+
+
Longipinanes
Hopanoids
Adianton
+
C
H
C
H
+
+
+
1
2,6,3,5
2,6,3,5
1'
2'
3'
4'
5'
6'
7'
8'
3', 5'
3'
5'
3', 5', 8a', 8b',
3'
5'
3', 8b', OMe
10', 14'
8a', 8b', 13'
12', 14'
Zeorin
+
+
+
2,6
3,5
4
Diplopterol
Bis(bibenzyls)
Marchantin B
Marchantin H
Marchantin K
Pakyonol
3, 5, 7a,7b
2, 6, 7a, 7b, 8a, 8b,
3, 5, 8a, 8b
7a, 7b, 10
8a, 8b, 10
8a, 8b
+
+
7
8
9
+
+
10
12
13
14
Riccardin C
Isoriccardin C
+
+
10, 11
11
8a, 8b, 10
9'
10'
11'
12'
13'
14'
10', 12'. 13'
10', 13', 14'
14'
a Present study.
b Harrison et al. (1992).
c Tori, Toyota, Harrison, Takikawa
Hashimoto et al. (1998).
& Asakawa (1985) and
8a', 8b', 10', 12'

Â
A. Bardon et al. / Phytochemistry 52 (1999) 1323±1329
1327
ential refractometer were used. Flow rate was 2 ml
min 1. Retention times were measured from the sol-
vent peak. A mixture of CH2Cl2±MeOH (1:1) was
used for separation with Sephadex LH-20.
3.2. Spectral data
NMR spectra were recorded at 150 or 50 MHz for
13C and 600 or 200 MHz for 1H. GC±MS was per-
formed at 70 eV using a fused silica column coated
with DB-17 (30 m  0.25 mm i.d., ®lm thickness 0.25
mm).
3.3. Plant material
Plagiochasma rupestre (Forst.) Steph. was collected
in February 1996 in La Heladera, Tucuman province,
Argentina. A voucher specimen is on deposit at
Faculty of Pharmaceutical Sciences, Tokushima Bunri
University.
3.4. Extraction and isolation
The mechanically ground air dried liverwort was
successively extracted with Et2O (15 days) and MeOH
(20 days) at room temp. Evaporation of the Et2O
extract in vacuo gave a residue (9.30 g) which was
chromatographed on silica gel using n-hexane with
increasing amounts of EtOAc (0±100%) to give 4 frac-
tions (I±V). Fr. I (1.13 g) was chromatographed on
Sephadex LH-20 to give a mixture further rechromato-
graphed on silica gel using n-hexane with increasing
amounts of EtOAc (0±20%) to give fractions 1 to 3.
HPLC of Fr. 1 with n-hexane±EtOAc (95:5) yielded
marsupellone (7) (332 mg, Rt 4 min) and adiantone (9)
(12.4 mg, Rt 8 min), Fr. 2 gave a mixture containing
caryophyllene oxide (8) the retention time and mass
spectrum (measured by GC±MS) of which were identi-
cal to those of an authentic sample. Fr. 3 gave diplop-
terol (10) (130 mg). Fr. II (1.40 g) was subjected to CC
on Sephadex LH-20, followed by CC on silica gel
using n-hexane and increasing amounts of EtOAc (10±
50%) to give a new portion of 10 (84 mg), stigmasterol
(71 mg) and rupestrenol (1) (239 mg). Fr. III (202 mg)
was chromatographed on Sephadex LH-20 to give a
mixture further processed by HPLC using n-hexane±
EtOAc (80:20), to give zeorin (11) (46 mg, Rt 3 min).
Fr IV (1.00 g) was subjected to CC on Sephadex LH-
20 and the mixture further chromatographed on silica
gel to give 2 fractions. Fr. 1 gave marchantin B (13)
(630 mg) and Fr. 2 was processed by HPLC (n-hex-
ane-EtOAc 70:30) to give riccardin C (15) (12 mg, Rt
14 min). Evaporation of MeOH extract in vacuo gave
a residue (2.05 g) which was chromatographed on
silica gel using n-hexane and increasing amounts of
EtOAc (20±100%) to give 3 fractions (I±III). All frac-
3. Experimental
3.1. General
For separation of a mixture of the chemical con-
stituents, a silica gel column and HPLC with a di€er-

Â
A. Bardon et al. / Phytochemistry 52 (1999) 1323±1329
1328
tions were separately subjected to CC on Sephadex
LH-20. Fr. I was then rechromatographed on silica gel
to give 10 (26 mg) and 1 (13 mg). HPLC of Fr. II (n-
hexane-EtOAc 80:20) gave 10 (2 mg, Rt 16 min), 1 (22
mg, Rt 32 min) and phytol (7 mg, Rt 38 min). Fr. III
was subjected to HPLC (n-hexane-EtOAc 1:1) to give
isoriccardin C (16) (8 mg, Rt 10 min) and a mixture
further subjected to HPLC (n-hexane-EtOAc 70:30) to
give marchantin K (12) (5 mg, Rt 19 min).
spectrum (600 MHz, CDCl3): d 7.93 (2H, d, J =8.5
Hz, H-2', H-6'), 7.58 (2H, d, J =8.5 Hz, H-3', H-5'),
5.34 (brs, H-5), 5.23 (q, J =1.5 Hz, H-13), 5.04 (d, J
=1 Hz, H-13'), 4.74 (ddd, J =13.5, 1, 0.5 Hz, H-12),
4.77 (ddd, J =13.5, 1, 0.5 Hz, H-12'), 1.92±2.24 (3H,
m1, H-2eq, H-3eq, H-3ax), 1.82±1.88 (2H, m1, H-6, H-
7), 1.81 (ddd, J =13, 3.5, 3.5 Hz, H-8eq), 1.75 (ddd, J
=13, 3.5, 3.5 Hz, H-9eq), 1.62 (3 H, s, H-15), 1.44
(dddd, J =13, 13, 13, 4, H-8ax), 1.21 (dddd, J =12, 10,
6.5, 3.5 Hz, H-10), 1.15 (dddd, J =13, 13, 8, 1.5 Hz,
H-2ax), 1.09 (dddd, J = 13, 13, 13, 3.5 Hz, H-9ax),
0.92 (3H, d, J =6.5 Hz, H-14), 0.78 (dddd, J = 12, 12,
10, 2.5 Hz, H-1). 13C-NMR spectrum (150 MHz,
CDCl3): d 165.5 (C'1O), 146.8 (C-11), 134.6 (C-4),
131.7 (C-3', C-5'), 131.2 (C-2', C-6') 129.22 (C-1'),
128.02 (C-4'), 123.6 (C-5), 113.2 (C-13), 66.8 (C-12),
47.9 (C-7), 46.5 (C-1), 44.1 (C-6), 36.3 (C-10), 35.9 (C-
9), 33.9 (C-8), 30.9 (C-3), 26.5 (C-2), 23.5 (C-15), 19.6
(C-14).
3.5. Rupestrenol [(+)-(1RÃ,6SÃ,7SÃ,10SÃ)-12-hydroxy-
4,11(13)-cadinadiene] (1)
Colourless solid; mp 50±528; ‰aŠ +43.9 (CHCl3; c
D
0.85). FT±IR (neat) nmax: 3300, 1645, 1378, 1050 and
895 cm 1. HR±MS: found 220.1820 C15H24O, requires
220.1829. EI±MS m/z (rel.int.): 220 [M]+ (52), 202
(65), 189 (40), 187 (41), 132 (55), 121(100), 119 (72),
105 (37), 93 (48), 81 (35). 1H- (600 MHz) and 13C-
NMR spectral data (150 MHz, CDCl3) (see Table 1
and 2).
3.8. 2,4-Dinitrophenylhydrazone of 2
To 2 (6 mg) in EtOH, was added dropwise 1 ml of a
solution that contains 2,4-dinitrophenylhydrazine (150
mg) dissolved in H2SO4 (1 ml), EtOH (3.3 ml) and
H2O (7.6 ml). An orange solid precipitated immedi-
ately. After ®ltration and puri®cation by CC on silica
gel using C6H6, the hydrazone (4) was recrystallized
from C6H6.
3.6. Oxidation of 1 with pyridinium dichromate (PDC)
To compound (1) (18 mg) in CH2Cl2 (5 ml) PDC
(60 mg) was added and the mixture stirred for 6 h at
room temperature. After puri®cation by HPLC (n-hex-
ane-EtOAc 95:5) 4,11(13)-cadinadien-12-al (2) (7.2 mg)
was obtained. Colourless oil, FT±IR (neat) nmax: 2699,
1694 and 1620 cm 1, UV lmax (EtOH) nm ꢀlog e): 221
1
3.9. X-ray crystal data of 4
(3.80). H-NMR spectrum (600 MHz, C6D6): d 9.33 (s,
H-12), 5.71 (s, H-13), 5.41 (s, H-13'), 5.17 (t, J =1
Hz, H-5), 1.80±1.92 (5H, m,1, H-6, H-8eq, H-2eq, H-
3ax, H-3eq), 1.69 (ddd, J =13, 13, 3.3 Hz, H-7), 1.56
(3H, dd, J =2, 1 Hz, H-15),1.56 (dddd, J =12, 3, 3,
3Hz, H-9eq), 0.96±1.11 (m1, H-8ax, H-9ax, H-10), 1.08
(dddd, J =13, 13, 8, 1.5 H-2ax), 0.84 (3H, d, J =6.5
Hz, H-14), 0.74 (dddd, J =12, 12, 10, 2 Hz, H-1). 13C-
NMR spectrum (150 MHz, CDCl3): d 193.7 (C-12)
154.1 (C-11), 134.4 (C-4), 133.4 (C-13), 123.7 (C-5),
46.5 (C-1), 44.2 (C-6), 36.5 (C-10), 35.9 (C-9), 34.1 (C-
7), 31.2 (C-3), 26.8 (C-8, C-2), 23.6 (C-15), 19.8 (C-14).
Data collection: MXC (MAC Science). Cell re®ne-
ment: MXC (MAC Science). Data reduction:
CRYSTAN. Program used to solve structure:
CRYSTAN SIR 92. Re®nement: Full matrix least
square. Di€ractometer: Mac Science MX C18.
C21H26O4N4, MW=398.00, Triclinic, P1, a = 6.082
(7) A, b = 7.495 (3) A, c = 23.03 (1) A, V =1033.93
(1) A3, Z =2, Dx = 1.278 Mg m 3, Dm = 1.300 Mg
m
3, Cu±Ka radiation, l ˆ 1:54178 A, y ˆ 20 258,
m ˆ 7:003 mm 1, Refractions: 1570, Parameters: 520,
Rint =0.0061, R = 0.101, oR ˆ 0:127, S= 2.092.
3.7. Benzoylation of 1
Acknowledgements
To compound 1 (25 mg) in C5H5N (2 ml) p-bromo-
benzoyl chloride (500 mg) was added and the mixture
was stirred for 72 h at room temperature. The result-
ing product was puri®ed by CC on silica gel and by
HPLC (n-hexane±EtOAc 95:5) to give p-bromobenzo-
Authors thank Dr. M. Schiavone from Facultad de
Ciencias Naturales, Universidad Nacional de
Tucuman, Argentina, for taxonomic determination of
plant material and Dr. T. Hashimoto (TBU) for his
useful discussion. A. Bardon thanks a grant from
Matsumae International Foundation, Japan. This
work was supported in part by a Grant-in-Aid for
Scienti®c Research (No. 11309012) from Japan
Society, for the Promotion of Science.
ate (3) (8 mg), colourless gum, FT±IR (neat) nmax
1725, 1591, 1269, 1013, 897 and 756 cm . H-NMR
:
1
1
1 Obscured due to overlapping signals.
2 Interchangeable carbons.

Â
A. Bardon et al. / Phytochemistry 52 (1999) 1323±1329
1329
Asakawa, Y. (1998). Journal Hattori Botanical Laboratory, 84, 91.
Harrison, L., Becker, H., Connolly, J. & Rycroft, D. (1992), Journal
Chemical Research (S), 74.
References
Ageta, H., Iwata, K.,
Pharmaceutical Bulletin, 11, 407.
& Otake, Y. (1963). Chemical and
Hashimoto, T., Irita, H., & Asakawa, Y. (1998). Journal Hattori
Botanical Laboratory, 85, 239.
Andersen, N. H., Bissonette, P., Liv, C. B., Shunk, B., Ohta, Y.,
Tseng, C. W., Moore, A., & Huneck, S. (1977). Phytochemistry,
16, 1731.
 Â
Huneck, S., & Lehn, J.M. (1963). Bulletin de la Societe Chimique de
France, 1702.
Huneck, S., & Yoshimura, I. (1996). In Identi®cation of Lichen
Substances (p. 364). Berlin: Springer.
Asakawa, Y. (1982). In W. Herz, H. Grisebach, & G. W. Kirby,
Progress in the Chemistry of Organic Natural Products, vol. 42 (p.
1). Vienna: Springer.
Hveding-Bergseth, N., Bruun, T.,
Phytochemistry, 22, 1826.
&
Kjùsen, H. (1983).
Asakawa, Y., & Matsuda, R. (1982). Phytochemistry, 21, 2143.
Asakawa, Y., Toyota, M., Matsuda, R., Takikawa, K.,
Garbarino, J. A., Chami, M. C., Montagna, M. P., & Gambaro, V.
(1993). Phytochemistry, 32, 987.
&
Takemoto, T. (1983). Phytochemistry, 22, 1413.
Asakawa, Y., Toyota, M., Bischler, H., Campbell, E., & Hattori, S.
(1984). Journal Hattori Botanical Laboratory, 57, 383.
Grammes, C., Burkhardt, G., & Becker, H. (1994). Phytochemistry,
35, 1293.
Markham, K. R., & Porter, L. J. (1978). In L. Reinhold, J. B.
Harborne, & T. Swain, Progress in Phytochemistry, vol. 5 (p.
181). Oxford: Pergamon Press.
Asakawa, Y., Tori, M., Takikawa, K., Krishnamurti, H. G., &
Kanti Kar, S. (1987). Phytochemistry, 26, 1811.
Asakawa, Y., Tori, M., Masuya, T.,
Phytochemistry, 29, 1577.
& Frahm, J.-P. (1990).
Matsuo, A., Uto, S., Sakuda, K., Uchio, Y., Nakayama, M., &
Hayashi, S. (1979). Chemistry Letters, 73.
Asakawa, Y. (1990). In H. D. Zinsmeister, & R. Mues, Bryophytes:
Their Chemistry and Chemotaxonomy (p. 369). Oxford:
Clarendon.
Murakami, T., & Tanaka, N. (1988). In W. Herz, H. Grisebach, G.
W. Kirby, & Ch. Tamm, Progress in the Chemistry of Organic
Natural Products, vol. 54 (p. 72). Vienna: Springer.
Ourisson, G., Albrecht, P., & Rohmer, M. (1979). Pure and Applied
Chemistry, 51, 709.
Asakawa, Y. (1993). In S. M. Colegate, & R. J. Molyneux, Bioactive
and
Natural
Determination (p. 319). Boca Raton: CRC Press.
Products:
Detection,
Isolation
Structural
Tori, M., Toyota, M., Harrison, L. J., Takikawa, K., & Asakawa,
Y. (1985). Tetrahedron Letters, 26, 4735.
Asakawa, Y. (1995). In W. Herz, G. W. Kirby, R. E. Moore, W.
Steglich, & Ch. Tamm, Progress in the Chemistry of Organic
Natural Product, vol. 65 (p. 1). Vienna: Springer.
Toyota, M., & Asakawa, Y. (1993). Phytochemistry, 32, 1235.
Wei, H.-C., Ma, S.-J., & Wu, C.-L. (1995). Phytochemistry, 39, 91.
Asakawa, Y. (1997). Heterocycles, 46, 795.