Ancistrobertsonines B, C, and D as well as 1,2- didehydroancistrobertsonine D from Ancistrocladus robertsoniorum

Article abstract of DOI:10.1016/S0031-9422(99)00130-2

From the East African Liana Ancistrocladus robertsoniorum, four new naphthylisoquinoline alkaloids have been isolated using High Speed Countercurrent Chromatography (HSCCC) and High Performance Liquid Chromatography (HPLC). Their stereostructures were established by spectroscopic, chemical, and chiroptical methods. Two of the new compounds, ancistrobertsonines B and C, constitute O- and N-permethylated 5,1'-coupled and 5,8'-coupled regioisomeric naphthylisoquinolines, while ancistrobertsonine D and its 1,2-didehydro analogue are based on a 7,1'- coupling mode between the isoquinoline and naphthalene moieties. Biological tests revealed that some of the isolated alkaloids possess moderate antimalarial activities.

Full text of DOI:10.1016/S0031-9422(99)00130-2

Phytochemistry 52 (1999) 321±332
Ancistrobertsonines B, C, and D as well as
1,2-didehydroancistrobertsonine D from
1
Ancistrocladus robertsoniorum
Gerhard Bringmanna,*, Friedrich Teltschika, Manuela Michela, Stefan Busemanna,
Markus Ruckerta, Rene Hallerb, Sabine Barb, S. Anne Robertsonc,
Ronald Kaminskyd
aInstitut fur Organische Chemie, Am Hubland, D-97074, Wurzburg, Germany
È
È
bBaobab Farm Ltd, PO Box 819995, Mombasa, Kenya
cPO Box 162, Malindi, Kenya
dSchweizerisches Tropeninstitut, Socinstrasse 57, CH-4002, Basel, Switzerland
Received 21 December 1998; received in revised form 22 February 1999; accepted 24 February 1999
Abstract
From the East African Liana Ancistrocladus robertsoniorum, four new naphthylisoquinoline alkaloids have been isolated using
High Speed Countercurrent Chromatography (HSCCC) and High Performance Liquid Chromatography (HPLC). Their
stereostructures were established by spectroscopic, chemical, and chiroptical methods. Two of the new compounds,
ancistrobertsonines
B
and C, constitute O- and N-permethylated 5,1'-coupled and 5,8'-coupled regioisomeric
naphthylisoquinolines, while ancistrobertsonine D and its 1,2-didehydro analogue are based on a 7,1'-coupling mode between
the isoquinoline and naphthalene moieties. Biological tests revealed that some of the isolated alkaloids possess moderate
antimalarial activities. # 1999 Elsevier Science Ltd. All rights reserved.
Keywords: Ancistrocladus robertsoniorum; Ancistrocladaceae; Ancistrobertsonine B; Ancistrobertsonine C; Ancistrobertsonine D; 1,2-didehy-
droancistrobertsonine D; Naphthylisoquinoline alkaloids; Naturally occuring biaryls; High Speed Countercurrent Chromatography (HSCCC);
Stereochemistry; Structural elucidation
1. Introduction
ary metabolites, the Ancistrocladaceae and the closely
related Dioncophyllaceae produce a unique class of
natural biaryls, the naphthylisoquinoline alkaloids,
being remarkable with respect to their intriguing struc-
tures, their unprecedented biosynthesis, promising bio-
logical activities, and chemotaxonomic implications
(Bringmann & Pokorny, 1995; Bringmann, Francois,
Ake Assi & Schlauer, 1998). Early investigations on A.
robertsoniorum showed this plant to form the naphtho-
quinone droserone in a crystalline form when wounded
(Bringmann, Kehr, Dauer, Gulden, Haller, Bar et al.,
1993; Peters et al., 1995). First systematic phytochem-
ical investigations carried out recently by our group
(Bringmann, Teltschik, Scha€er, Haller, Haller, Bar et
al., 1998) revealed the presence of the 5,1'-coupled al-
Ancistrocladus
robertsoniorum
Leonard
(Ancistrocladaceae) (Leonard, 1984; Bringmann,
Haller, Bar, Isahakia & Robertson, 1994), a tropical
liana indigenous to Kenya, belongs to the small mono-
generic family of Ancistrocladaceae, which consists of
20 species (Gereau, 1997). As a rich source of second-
* Corresponding author. Tel.: +49-931-888-5323; fax: +49-931-
888-4755.
E-mail
Bringmann)
address:
bringman@chemie.uni-wuerzburg.de
(G.
1 Part 122 in the series `Acetogenic Isoquinoline Alkaloids'. For
Part 121, see Bringmann, Ruckert, Messer, Schupp & Louis, 1999.
0031-9422/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(99)00130-2

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G. Bringmann et al. / Phytochemistry 52 (1999) 321±332
kaloid ancistrocladine (1a) and its atropisomer, hama-
tine (1b), as well as the 5,8'-linked naphthylisoquino-
lines ancistrobrevine B (2) and ancistrobertsonine A
(3) as the major alkaloids of A. robertsoniorum. In this
paper, the isolation of four minor naphthylisoquino-
lines, named ancistrobertsonines B (4), C (5), and D
(7) as well as 1,2-didehydroancistrobertsonine D (6,
alias 6-O-demethylancistrocladisine), i.e. 5,1'-, 5,8'-
and 7,1'-coupled alkaloids, is described. With the iso-
lation of these four new metabolites, a total of eight
naphthylisoquinoline alkaloids (of which ®ve are new)
have been identi®ed from this species, allowing a more
exact chemotaxonomic classi®cation of this interesting
plant (Fig. 1).
method as previously elaborated (Bringmann,
Teltschik et al., 1998) was used, viz the extraction of
the dried and ground plant material with 1 N
H2SO4ÐMeOH (5:1), followed by liquid±liquid par-
tition and further resolution by High Speed
Countercurrent Chromatography (HSCCC) (Conway,
1990). In total, six fractions were obtained. The iso-
lation and characterization of the major alkaloids 1a,
1b, 2, and 3 of fractions 3±5 have already been
described earlier (Bringmann, Teltschik et al., 1998).
Further analysis of fractions 1, 3, and 6 yielded four
Dragendor€-positive compounds.
1H NMR investigations of fraction 1 showed the
presence of two very similar, N-methylated
Ancistrocladaceae-type naphthylisoquinoline alkaloids,
which were separated by HPLC on a chiral stationary
phase (Chiralcel OD) after puri®cation by column
chromatography and preparative TLC on silica gel.
The molecular formula of both compounds was shown
to be C27H33NO4 by High Resolution Mass
Spectrometry (HRMS).
2. Results and discussion
A. robertsoniorum was collected in Kenya in
November 1992 and in August 1993. For the extrac-
tion and separation of the alkaloids the same isolation
Fig. 1. Naphthylisoquinoline alkaloids of A. robertsoniorum.

G. Bringmann et al. / Phytochemistry 52 (1999) 321±332
323
1
The H NMR spectrum of the slightly less polar al-
kaloid, named ancistrobertsonine B, resembled the
spectrum of ancistrocline (8), a 5,1'-coupled alkaloid
from A. tectorius (Bringmann & Kinzinger, 1992).
A ®rst hint at the position of the biaryl axis in the
naphthalene part of the molecule was obtained from
the chemical shift of the Me-2' group (2.05 ppm) (see
Fig. 2(a)). Like in 8, this signal is high-®eld shifted,
whereas the resonances of MeO-4' (4.00 ppm) and
MeO-5' (3.97 ppm) remain in their `normal' positions,
excluding a 3'- and a 6'-position of the biaryl axis. A
series of NOE and ROESY (Rotating Frame
Overhauser E€ect Spectroscopy) measurements (see
Fig. 2(b)), as well as the spin system pattern also
exclude the biaryl axis to be situated in the 6'-, 7'-, or
8'-position and con®rm the coupling site to be C-1' in
the naphthalene moiety.
In contrast to ancistrocline (8), ancistrobertsonine B
bears a methoxy (instead of a hydroxy) group at C-6.
The signal of MeO-6 appears up-®eld shifted (3.62
ppm), while the resonance of MeO-8 remains unaf-
fected at 3.92 ppm, which indicates the biaryl axis to
be situated at C-5. This conclusion is con®rmed by the
high-®eld shift of the protons at C-4 (1.93 and 2.27
ppm) and Me-3 (1.00 ppm) (see Fig. 2(a)). In addition,
a C-7 coupling site can be excluded because of
ROESY interactions of H-7 with both MeO-6 and
MeO-8 groups and Heteronuclear Multiple Bond
Correlations (HMBC) of H-7 with C-6 and C-8 as
shown in Fig. 2(b). The relative con®guration of C-1
vs. C-3 was concluded to be cis, from the chemical
shifts of H-1 (3.81 ppm) and H-3 (2.38 ppm) as well as
from NOE interactions between these two spin sys-
tems. Further NOE experiments also showed an inter-
action between H-8' in the naphthalene part and H-4ax
(which was identi®ed as such by the large coupling
constant of Jax=9.4 Hz over to H-3) in the isoquino-
line part, proving both spin systems to be on the same
side of the molecule as depicted in Fig. 2(c).
Fig. 2. Constitution of ancistrobertsonine B (4) from (a) selected 1H
NMR chemical shifts (d values in ppm), as well as (b) NOE,
ROESY (double arrows) and relevant HMBC (unidirectional arrows)
interactions; its relative stereostructure (c) as determined by further
characteristic NOE interactions.
logues) were stereochemically analyzed by Gas
Chromatography with Mass Sensitive Detection (GC-
MSD) after derivatization with R-Mosher chloride.
The formation of 3 S-(N-methylamino)butyric acid
showed the alkaloid clearly to be S-con®gured at C-3.
For cis-con®gured tetrahydroisoquinolines of this
type less reliable information can be obtained for C-1,
due to the formation of N-methylalanine from both of
the two (here heterochiral) stereocenters, C-1 and C-3
(Bringmann, God & Scha€er, 1996). Therefore the ab-
The absolute con®guration at C-3 was determined
ruthenium-mediated
by
oxidative
degradation
(Bringmann, God & Scha€er, 1996). The degradation
products, 3-(N-methylamino)butyric acid and N-
methylalanine (as well as their N-demethylated ana-

324
G. Bringmann et al. / Phytochemistry 52 (1999) 321±332
Fig. 3. CD spectra of ancistrobertsonine B (4) (Ð), hamatine (1b)
(Á Á Á), and ancistrocline (8) (± ±).
solute con®guration of C-1 was more conveniently
deduced from the relative con®guration of C-1 vs C-3
as established through NMR (see above) and the now
known absolute con®guration at C-3, unambiguously
indicating C-1 to be R-con®gurated.
Likewise based on the relative con®guration as
shown in Fig. 2(c) and the given absolute con®gur-
ation at C-3, the axial con®guration of ancistrobertso-
nine
B
was attributed as M. This result is
corroborated by comparison of the CD spectrum with
that of hamatine (1b, see Fig. 3), which also shows a
®rst negative and a second positive Cotton e€ect,
while the CD spectrum of the P-con®gured ancistro-
cline (8) is, as expected, virtually opposite. The new,
thus M-con®gured alkaloid, ancistrobertsonine B,
must consequently have stereostructure 4.
The more polar compound from the same fraction,
named ancistrobertsonine C, is a naphthylisoquinoline
alkaloid with a similar 1H NMR spectrum as com-
pared to that of 4. In contrast to 4, however, a `nor-
mal', i.e. not high-®eld shifted signal is observed for
Me-2' (2.32 ppm), and a di€erent multiplicity for the
signals of the four aromatic protons (2 singlets and 2
doublets) in the naphthalene part of ancistrobertsonine
C (see Fig. 4(a)). Like for ancistrobertsonine B, MeO-
4' (3.97 ppm) and MeO-5' (4.00 ppm) were found to
have `normal' shifts, which exclude the biaryl axis to
be located at C-3' or C-6'. This was con®rmed by
ROESY experiments (see Fig. 4(b)), showing inter-
actions between H-1', Me-2', H-3' and MeO-4' as well
as between MeO-5', H-6' and H-7', leaving only the
8'-position open for the biaryl axis. Like in ancistro-
bertsonine B (4), the up-®eld shift of the signals of
MeO-6 (3.62 ppm), Me-3 (0.99 ppm), Heq-4 and Hax-4
(1.99 and 2.27 ppm) indicate these protons to be in
close proximity of the biaryl axis, which must thus be
Fig. 4. Selected 1H NMR shifts (d values in ppm) (a), HMBC (single
arrows) and ROESY interactions (double arrows) (b) relevant for
the constitution; relative con®guration at the stereogenic centers and
the biaryl axis (c) of ancistrobertsonine C (5) through long-range
NOE interactions.
located at C-5. The 5,8'-linkage of the two parts of the
molecule is further con®rmed by HMBC experiments,
showing C-5 to have crosspeaks with H-7 in the iso-
quinoline part and H-7' in the naphthalene part (see
Fig. 4(b)).
Like for 4 the relative con®guration at C-1 vs C-3
was again established as cis by NOE-measurements
(see Fig. 4(c)): Irradiation of H-1 (3.81 ppm) resulted
in a signi®cant enhancement of the signal of H-3 (2.39

G. Bringmann et al. / Phytochemistry 52 (1999) 321±332
325
Fig. 5. CD comparison of ancistrobertsonine C (5) (Ð) and koru-
pensamine D (9) (± ±).
Fig. 6. Constitution 6 of 1,2-didehydroancistrobertsonine D from (a)
selected 1H NMR chemical shifts (d values in ppm), as well as (b)
ROESY and relevant HMBC interactions.
ppm). This relative cis-con®guration is in agreement
with the chemical shifts of these two protons (see
above), which are typical of cis-substituted 1,3-
with a constitution related to that of ancistrocladisine
(10, cf Scheme 1) from A. heyneanus (Govindachari,
Parthasarathy & Desai, 1972) and A. hamatus (Desai
et al., 1976).
The most obvious di€erence between these two com-
pounds is the lack of an O-methyl group in the new al-
kaloid 6 compared with 10, from which the molecular
formula was deduced to be C25H27NO4. This was con-
dimethyltetrahydroisoquinolines
(Bringmann
&
Pokorny, 1995). Again, the absolute con®guration at
C-3 was established as S, by the above-mentioned
degradation method with GC-MSD analysis of the
Mosher derivative of 3-aminobutyric acid. With this
fact and the NOE interaction between H-1 and H-3,
the absolute con®guration at C-1 unequivocally has to
be R. The con®guration of the biaryl axis as the third
stereogenic element was determined as P by long-range
NOE interactions between the protons Hax-4 and H-7'
as well as between Heq-4 and H-1' (see Fig. 4(c)).
This axial P-con®guration was further corroborated
by comparison of the CD spectra of this new alkaloid,
ancistrobertsonine C (5), with that of korupensamine
D (9) (Hallock et al., 1994) (see Fig. 5), which is like-
wise 1R,3S,5P-con®gured. Due to the same con®gur-
ation of the stereogenic elements the new alkaloid 5
also can be classi®ed as 6,8,5'-O,O,O-trimethylkoru-
pensamine D.
From the third HSCCC fraction, a polar yellow
solid was obtained by preparative TLC and HPLC,
which revealed to be a naphthylisoquinoline alkaloid,
Scheme 1. Transformation of 1,2-didehydroancistrobertsonine (6)
into ancistrocladisine (10). (i) CH2N2, MeOH±H2O (9:1)
(Organikum, 1988).

326
G. Bringmann et al. / Phytochemistry 52 (1999) 321±332
the con®guration of its stereogenic center at C-3 was
established as S.
The last structural information required was the
con®guration at the sterically hindered biaryl linkage.
Attempts to elucidate the absolute con®guration at the
biaryl axis by long-range NOE interactions
(Bringmann, Koppler, Scheutzow & Porzel, 1997) were
not successful, whereas the comparison of the CD
spectra of the new compound and the closely related
alkaloid ancistrocladisine (10) (Govindachari et al.,
1972; Bringmann, Pokorny, Stablein & Ake Assi,
1993) gave a ®rst hint at a P-con®guration at the axis
(see Fig. 7).
For an unambiguous elucidation of the axial chiral-
ity and of the full stereostructure of the new com-
pound in general, the alkaloid was reacted with
diazomethane (de Boer & Baker 1963; Organikum,
1988), to give its 6-O-methyl derivative (see Scheme 1),
which proved to be fully identical with natural (from
A. heyneanus ) and synthetic (Bringmann & Reuscher,
1989) ancistrocladisine (10), in all its physical, spectro-
scopic, and chromatographic properties, thus con®rm-
ing the full stereostructure of the novel alkaloid as 6,
i.e., 6-O-demethylancistrocladisine or (see below) 1,2-
didehydroancistrobertsonine D (6).
Workup of the sixth fraction of the HSCCC separ-
ation by preparative TLC on silica gel and reversed
phase HPLC yielded a pale yellow amorphous solid,
homogeneous according to analytical TLC. By HRMS
of the [M Me]+ peak, the compound, subsequently
named ancistrobertsonine D, was found to correspond
to the molecular formula C25H29NO4. Again the con-
stitution of this new naphthylisoquinoline was estab-
lished by 1H NMR and 13C NMR spectroscopy. A
7,1'-position of the axis was deduced i.a. from the
high-®eld shifted signals of Me-2' (2.22 ppm) and
MeO-8 (3.07 ppm) (see Fig. 8(a)), indicating these two
groups to be adjacent to the biaryl axis, and ROESY
interactions observed between Me-1 and MeO-8, Me-
2', H-3', and MeO-4' as well as between MeO-5', H-
6', H-7' and H-8' (see Fig. 8(b)). A 5,1'-coupling
between the two halves can be excluded by the `nor-
mal' chemical shift of Hax-4 (2.54 ppm) and Heq-4
(2.75 ppm) and by a ROESY interaction between Heq-
4 and the isolated aromatic proton (6.58 ppm), H-5
(see Fig. 8(a/b)).
Fig. 7. CD comparison of 1,2-didehydroancistrobertsonine D (6)
(Ð) and ancistrocladisine (10) (± ±).
®rmed by HRMS analysis of the [M]+ peak at m/z
405.
The ancistrocladisine-like constitution of the new al-
kaloid 6 was clearly deduced from NMR data (see
Fig. 6(a)). The spin coupling pattern of H-3, Heq-4
and Hax-4, the signal of Me-3, which appears as a
doublet, and the broad singlet of Me-1 (2.73 ppm)
strongly indicated the presence of a naphthyl-3,4-dihy-
droisoquinoline. For the Me-1 signal a slow H/D-
exchange was observed in D2O or CD3OD. The reson-
ances of Me-3 (1.42 ppm), H-3 (3.91 ppm), Heq-4 and
Hax-4 (2.75 and 2.97 ppm) did not appear high-®eld
shifted. This fact and the ROESY interactions
observed between H-4eq and H-5 (see Fig. 6(b))
excluded the biaryl axis to be situated in the 5-position
in the isoquinoline part of the molecule. Further analy-
sis of the ROESY experiment (marked interaction
between Me-1 and MeO-8, but no interactions between
H-5 and any of the methoxy groups) revealed a HO-6/
MeO-8 substitution pattern in the isoquinoline part.
The high-®eld shift of MeO-8 (3.13 ppm) and the
HMBC interaction between H-5 and C-7 (see Fig.
6(b)) clearly showed C-7 to be the coupling position in
the isoquinoline part of the molecule. In the naphtha-
lene part of the molecule the biaryl axis is attached to
C-1', which is deduced from the high-®eld shift of the
protons of Me-2' (2.22 ppm), the una€ected, i.e. not
high-®eld shifted resonances of MeO-4' and MeO-5'
(4.00 and 3.97 ppm) (see Fig. 6(a)), the spin system
pattern observed for the aromatic protons, and
ROESY interactions between Me-2', H-3' and MeO-4'
as well as between MeO-5', H-6', H-7' and H-8' (Fig.
6(b)).
The relative con®guration at the centers and the axis
was established by ROESY experiments. A weak, but
signi®cant ROESY interaction between H-1 (4.31
ppm) and H-3 (3.01 ppm) revealed the relative con-
®guration of C-1 vs C-3 to be cis. The same exper-
iment also showed a distinct interaction between Me-1
in the isoquinoline and Me-2' in the naphthalene part,
indicating both groups to be on the same side of the
molecule, which should thus have the (relative) stereo-
structure 7 shown in Fig. 8(c).
From the S-con®guration of the Mosher derivative
of the 3-aminobutyric acid formed in the oxidative
degradation (Bringmann et al., 1996) of the alkaloid,

G. Bringmann et al. / Phytochemistry 52 (1999) 321±332
327
Table 1
IC50 values of the compounds 2, 3, 4, 5, and 7 (isolated from A.
robertsoniorum) as well as 10, 11a, and 11b (partial synthetic origin)
on chloroquine resistant (K1-strain) and susceptible (NF54-strain)
strains of Plasmodium falciparum
Compound
K1-strain IC50 [mM]
NF54-strain IC50 [mM]
2
2.0
15.9
9.0
4.5
±
4.7
>23.7
>23.0
10.1
4.8
3
4
5
7
10
11a
11b
1.4
1.7
1.0
5.9
5.0
4.9
as evident from NMR, the absolute con®guration of
C-1 must be R as depicted in Fig. 8(c).
With the absolute con®guration at the stereocenters
in the isoquinoline part established and the relative
con®guration of centers vs axis again known from
NMR (see also Fig. 8(c)), the absolute con®guration at
the biaryl axis was unambiguously assigned as P, lead-
ing to 7 as the absolute stereostructure of ancistrobert-
sonine D. This structure, including its absolute
con®guration (1R,3 S,7P)2 was con®rmed by synthetic
transformations. Reduction of ancistrocladisine (10)
with LiAlH4/AlMe3 gave two stereoisomeric tetrahy-
droisoqinolines, 11a and 11b, of which the cis-isomer
11b proved to be identical with the O-methylation pro-
duct of ancistrobertsonine D (7) by co-chromatog-
raphy on reversed phase HPLC, physical (mp and
[a]D), and spectroscopic (1H NMR and CD) data
(Scheme 2).
An alkaloid with the same constitution as 7 (cf in
Fig. 8(a/b)), named `ancistrine', had previously been
isolated by Foucher, Pousset & Cave, (1975) from the
roots of the Central African liana, Ancistrocladus
ealaensis. However, neither the relative nor the absol-
ute con®guration was determined for that alkaloid.
Moreover, the physical (mp and [a ]D) data and some
characteristic 1H NMR shifts of `ancistrine' do not
correspond to those of 7, and no more authentic ma-
terial of `ancistrine' is available (A. Cave, personal
communication) so that, given the uncertain identity
of `ancistrine', ancistrobertsonine D (7) has to be
regarded as a new compound.
In contrast to some other naphthylisoquinolines iso-
lated from Anstrocladaceae and Dioncophyllaceae in
West and Central Africa (Bringmann, Francois et al.,
1998), the alkaloids 2, 3, 4, 5, and 7 isolated from A.
robertsoniorum as well as the partial-synthetic com-
pounds 10, 11a, and 11b showed only moderate anti-
Fig. 8. Constitution 7 of ancistrobertsonine D from (a) selected 1H
NMR chemical shifts (d values in ppm), as well as (b) ROESY inter-
actions; its relative stereostructure (c) by further characteristic
ROESY interactions.
From a ruthenium-mediated degradation experiment
(Bringmann et al., 1996) the con®guration at C-3 was
established to be S. Given the relative cis-con®guration
malarial
activities
in
vitro.
Interestingly,
2 The absolute stereostructure was further corroborated by quan-
tumchemical circular dichroism calculations, by methods described
earlier (Bringmann & Busemann, 1998)
antiplasmodial activity was more pronounced against
the chloroquinine resistant K1 strain and to a 2±5 fold

328
G. Bringmann et al. / Phytochemistry 52 (1999) 321±332
Scheme 2. Con®rmation of the absolute con®guration of the new alkaloid 7 by conversion into the cis-dihydro derivative 11b of ancistrocladisine
(10). (i) LiAlH4±AlMe3, THF (abs.) (Bringmann, Jansen & Rink, 1986) (ii) CH2N2, MeOH±H2O (9:1) (Organikum, 1998).``
lesser extent against the susceptible NF54 strain of
Plasmodium falciparum (see also Table 1). It is note-
worthy that the studied compounds expressed cyto-
toxic e€ects on mammalian cells only at or above
90 mg/ml (highest concentration tested). Thus, the anti-
malarial activity, although only moderate, is speci®c
against intraerythrocytic forms of P. falciparum.
Compound 6 was not tested due to the low quantities
isolated and because of its instability.
between African and Asian representatives of the
Ancistrocladaceae family.
3. Experimental
3.1. General
Mps uncorr. Optical rotations: 258, 10 cm cell,
CHCl3. CD: 258, EtOH. IR: KBr. 1H NMR:
(200 MHz, 400 MHz or 600 MHz) and 13C NMR
(63 MHz, 100 MHz or 150 MHz) were recorded in
CDCl3, with the solvent as int. standard (CDCl3, d
7.26 and d 77.01, resp.). Proton detected, heteronuclear
correlations were measured using Heteronuclear
Multiple Quantum Correlation (HMQC, optimized for
1JHC=150 Hz) and HMBC (optimized for
nJHC=7 Hz). EIMS: 70 eV. TLC: precoated silica gel
60 F254 plates (Merck), deactivated with NH3. Spots
were visualized under UV light and by Dragendor€'s
reagent. Prep. TLC: plates with a layer thickness of
2 mm and a concentration zone (Merck) were used.
80±100 mg samples were applied and recovered with
25% MeOH in CHCl3 after resoln. HPLC (analytical):
Novapak C18 (4 mm, 3.9 Â 150 mm, Waters), ¯ow
1.0 ml min 1; Chiralcel OD (10 mm, 4.6 Â250 mm,
Daicel), ¯ow 1.0 ml min 1; HPLC (preparative):
Novapak C18 (6 mm, 7.8 Â 200 mm, Waters), ¯ow
4.0 ml min 1; Novapak C18 (6 mm, 25 Â200 mm,
Waters), ¯ow 12.0 ml min 1; Chiralcel OD (10 mm,
10 Â 250 mm, Daicel) ¯ow 4.0 ml min 1; UV detection
280 nm (tunable UV detector) and 200±400 nm
(photodiode array detector). HSCCC: CHCl3±MeOH±
In summary, four new naphthylisoquinoline alka-
loids were isolated from the East African Liana
Ancistrocladus robertsoniorum. The new compounds 4,
5, 6 and 7 and the alkaloids 1a, 1b, 2 and 3 isolated
previously from this plant are all S-con®gured at C-3
and have an oxygen function at C-6, thus representing
pure `Ancistrocladaceae-type' (Bringmann & Pokorny,
1995) alkaloids. In this respect A. robertsoniorum
therefore resembles the Asian Ancistrocladus species,
which have been found to produce `Ancistrocladaceae-
type' alkaloids only, whereas all the West and Central
African species investigated so far produce
`Dioncophyllaceae-type' alkaloids (i.e. with R at C-3
and without oxygen at C-6), too, as well as `hybrid
types' (Bringmann & Pokorny, 1995). On the other
hand, the identi®cation of 5,8'-coupled alkaloids like
2, 3, and 5, which have never been found in Asian
Ancistrocladus species (Bringmann & Pokorny, 1995),
reveals a clear chemotaxomic relationship to the West
and Central African representatives of this genus.
The work described here thus con®rms the assump-
tion (Bringmann, Teltschik et al., 1998) that A. robert-
soniorum is the geographic and chemotaxonomic link

G. Bringmann et al. / Phytochemistry 52 (1999) 321±332
329
0.1
M
HCl (5:5:3), mobile phase: lower phase,
OMe-6), 3.81 (1H, q, J = 6.4 Hz, H-1), 3.92 (1H, s,
OMe-8), 3.97 (3H, s, OMe-5'), 4.00 (3H, s, OMe-4'),
6.50 (1H, s, H-7), 6.77 (1H, dd, Jortho=7.9 Hz,
Jmeta=1.1 Hz, H-6'), 6.80 (1H, s, H-3'), 6.90 (1H, dd,
Jortho=8.3, Jmeta=1.1 Hz, H-8'), 7.16 (1H, dd,
Jortho=8.3 Hz, Jortho=7.9, H-7'). 13C NMR (150 MHz,
CDCl3): d 20.63 (Me-2'), 20.85 (Me-3), 22.40 (Me-1),
35.21 (C-4), 41.10 (N-Me), 46.89 (C-3), 55.32 (OMe-8),
56.05 (OMe-6), 56.41 (OMe-4'), 56.46 (OMe-5'), 57.12
(C-1), 93.80 (C-7), 105.37 (C-6'), 108.96 (C-3'), 116.32
(C-10'), 118.46 (C-8'), 118.85 (C-5), 125.38 (C-1'),
126.15 (C-9), 126.35 (C-7'), 134.85 (C-2'), 136.54 (C-
10), 136.86 (C-9'), 156.03 (C-4'), 156.12 (C-8), 156.24
(C-6), 157.26 (C-5'). EIMS m/z (rel. int.): 435 [M]+
(2), 420 [M Me]+ (100), 404 [M OMe]+ (7). HRMS
m/z 435.241 [M]+, (C27H33NO4 requires: 435.241).
(H) 4 T, Triple Coil, 1.7 Â 950 mm (large coil), TLC
detection (see above), ¯ow 2.0 ml min 1, 850 min
.
1
3.2. Plant material
Stems and leaves of A. robertsoniorum Leonard were
collected in November 1992 and in August 1993 in the
Buda Ma®sini Forest in Kenya, identi®ed by one of us
(R.H.) and con®rmed by Dr J. Schlauer (Wurzburg).
A voucher specimen has been deposited at the
Herbarium Bringmann (No. 8), Wurzburg.
3.3. Extraction and isolation
The air dried stems (1.1 kg) were ground and
extracted exhaustively with 1 N H2SO4±MeOH (5:1) as
described earlier (Bringmann, Teltschik et al., 1998).
After the removal of MeOH, the aq. soln was prefrac-
tionated by liquid±liquid partition using n-hexane,
CHCl3, and (after basi®cation with conc. NH3 soln of
pH 8) n-BuOH. 1H NMR and the Dragendor€ test
proved the CHCl3 fr. to be the only alkaloid contain-
ing fr. After evapn of the solvent the residue (ca 11.5 g)
was subjected to HSCCC (1 g/run). Portions of 4 ml
were collected, monitored by TLC, and combined. In
the following, the isolation of the alkaloids in the
order of increasing polarity is described.
3.6. Ancistrobertsonine C (5)
Mp 148±1498. [a]2D5 38 (CHCl3; c 0.09). CD: DE200
13.3, DE216 4.0, DE228 16.7, DE240+19.0, DE310 1.4
(EtOH, c 0.02). IR nmax cm 1: 2950, 2920, 2820 (C±
H), 1605, 1572 (C1C), 1270, 1250, 1090, 1070 (C±O).
1H NMR (200 MHz, CDCl3):
d 0.99 (3H, d,
J = 5.1 Hz, Me-3), 1.47 (3H, d, J = 6.4 Hz, Me-1),
1.99 (1H, dd, Jgem=14.9 Hz, Jeq=1.9 Hz, Heq-4), 2.27
(1H, dd, Jgem=14.8 Hz, Jax=10.9 Hz, Hax-4), 2.32
(3H, s, Me-2'), 2.39 (1H, mc, H-3), 2.45 (3H, s, N-Me),
3.62 (3H, s, OMe-6), 3.81 (1H, q, J = 6.4 Hz, H-1),
3.93 (1H, s, OMe-8), 3.97 (3H, s, OMe-5'), 4.00 (3H, s,
OMe-4'), 6.49 (1H, s, H-7), 6.68 (1H, s, H-3'), 6.69
(1H, s, H-1'), 6.84 (1H, d, Jortho=8.0, H-6'), 7.15 (1H,
d, Jortho=8.0, H-7'). 13C NMR (150 MHz, CDCl3): d
20.85 (Me-3), 21.99 (Me-2'), 22.48 (Me-1), 36.35 (C-4),
41.20 (N-Me), 55.18 (OMe-6), 55.54 (C-3), 56.06
(OMe-8), 56.24 (OMe-4'), 56.58 (OMe-5'), 57.36 (C-1),
93.57 (C-7), 105.22 (C-6'), 108.52 (C-3'), 115.89 (C-
10'), 117.86 (C-1'), 120.31 (C-5), 120.52 (C-9), 126.48
(C-8'), 128.93 (C-7'), 135.77 (C-2'), 136.27 (C-9'),
137.17 (C-10), 156.03 (C-8), 156.41 (C-5'), 156.51 (C-
6), 157.12 (C-4'). EIMS m/z (rel. int.): 435 [M]+ (3),
420 [M Me]+ (100), 404 [M OMe]+ (5). HRMS m/z
435.241 [M]+, (C27H33NO4 requires: 435.241).
3.4. Isolation of ancistrobertsonines B (4), and C (5)
For removal of the main part of brown by-products,
fr. 1 from the HSCCC sepn was subjected to CC on
silica gel, using methyl-tert-butylether as the eluent.
Further puri®cation on prep. TLC yielded 70 mg of a
yellow oil as a mixt. of two alkaloids. Resolution of
these compounds was ®nally achieved analytically and
preparatively by HPLC on a chiral stationary phase
(Chiralcel OD) using n-hexane±i-propanol±triethyla-
mine (96:4:0.1) as eluent. After removal of the eluent
under reduced pressure, the alkaloids were recrystal-
lized separately from n-hexane±i-propanol to give
13 mg of 4 and 51 mg of 5.
3.5. Ancistrobertsonine B (4)
3.7. Isolation of 1,2-didehydroancistrobertsonine D (6)
Mp 192±1938. [a ]2D5+48 (CHCl3;
DE200 15.5, DE202 15.6, DE230 40.4, DE244 10.8,
c
0.09). CD:
Fr. 3 of the HSCCC sepn was further resolved
by prep. TLC using CHCl3±MeOH (19:1) as
eluent. Puri®cation of the most polar compound
on prep. HPLC with MeOH±H2O (40:60) 10 min,
1
DE282+0.8, DE306 1.7 (EtOH, c 0.02). IR nmax cm
:
3050 (1C0H), 2962, 2930, 2820 (C±H), 1585, 1570,
1475 (C1C), 1258, 1200, 1070 (C±O). 1H NMR
(200 MHz, CDCl3): d 1.00 (3H, d, J = 6.4 Hz, Me-3),
1.32 (3H, d, J = 6.4 Hz, Me-1), 1.93 (1H, dd,
Jgem=16.0 Hz, Jeq=3.4 Hz, Heq-4), 2.05 (3H, s, Me-
2'), 2.06 (1H, dd, Jgem=16.0 Hz, Jax=9.4 Hz, Hax-4),
2.39 (1H, mc, H-3), 2.45 (3H, s, N-Me), 3.62 (3H, s,
(40:60 4 46:54) in
(46:54 4 52:48) in
2
5
min, (46:54)
min, (52:48) 10 min,
8
min,
(52:48 4 40:60) in 3 min, gave 7 mg of 6 as microcrys-
talline powder after recrystallization from MeOH±
H2O. Mp 185±1878. [a]2D5 308 (CHCl3; c 0.04). CD:
DE200 119.0, DE207 79.0, DE210 83.0, DE231+34.5,

330
G. Bringmann et al. / Phytochemistry 52 (1999) 321±332
DE244+9.0, DE273 18.6, DE302 3.7 (EtOH, c 0.03). IR
nmax cm 1: 3380 (O±H), 2940, 2920, 2820 (C±H), 1570,
1490 (C1C), 1270, 1260, 1070 (C±O). 1H NMR
(200 MHz, CDCl3): d 1.42 (3H, d, J = 6.1 Hz, Me-3),
2.22 (3H, s, Me-2'), 2.73 (3H, s, Me-1), 2.75 (1H, dd,
Jgem=16.5 Hz, Jax not measurable due to partial over-
lap by signal of Me-2', Hax-4), 2.97 (1H, dd,
Jgem=16.5 Hz, Jeq=5.2 Hz, Heq-4), 3.13 (1H, s, OMe-
8), 3.91 (1H, mc, H-3), 3.97 (3H, s, OMe-5'), 4.00 (3H,
s, OMe-4'), 6.80 (1H, s, H-3'), 6.81 (1H, s, H-5), 6.82
(1H, dd, Jortho=8.4 Hz, Jmeta=1.3 Hz, H-6'), 6.91 (1H,
dd, Jortho=8.4 Hz, Jmeta=1.3 Hz, H-8'), 7.15 (1H,
pseudo-t, Jortho=8.2 Hz, H-7'). 13C NMR (150 MHz,
CDCl3): d 17.49 (Me-3), 20.74 (Me-2'), 23.53 (Me-1),
34.35 (C-4), 48.25 (C-3), 56.36 (OMe-4'), 56.48 (OMe-
5'), 60.36 (OMe-8), 106.35 (C-6'), 108.83 (C-3'), 111.56
(C-5), 111.96 (C-9), 116.64 (C-10'), 117.19 (C-8'),
117.87 (C-1'), 118.81 (C-7), 128.06 (C-7'), 136.16 (C-
9'), 138.03 (C-2'), 140.61 (C-10), 157.94 (C-5'), 158.35
(C-4'), 162.99 (C-8), 163.40 (C-6), 174.18 (C-1). EIMS
m/z (rel. int.): 405 [M]+ (100), 390 [M Me]+ (91), 375
[M OMe+H]+ (11). HRMS m/z 405.194 [M]+,
(C25H27NO4 requires: 405.194).
3), 22.21 (Me-1), 37.67 (C-4), 49.16 (C-3), 50.49 (C-1),
56.33 (OMe-4'), 56.44 (OMe-5'), 59.71 (OMe-8),
105.82 (C-6'), 109.15 (C-3'), 110.38 (C-5), 116.39 (C-
10'), 117.56 (C-7), 117.74 (C-8'), 119.39 (C-1), 122.80
(C-9), 127.52 (C-10), 127.71 (C-7'), 136.30 (C-9'),
138.43 (C-2'), 152.61 (C-6), 156.55 (C-8), 157.54 (C-
4'), 157.64 (C-5'). EIMS m/z (rel. int.): 407 [M]+ (8),
392 [M Me]+ (100), 377 [M OMe+H]+ (12).
HRMS m/z 392.186 [M±Me]+, (C24H26NO4 requires:
392.186).
3.9. Oxidative degradation of 4, 5, 6 and 7
The degradation, the derivatization of the amino
acids, and the subsequent GC-MSD analysis were car-
ried out as described previously (Bringmann et al.,
1996).
3.10. Preparation of ancistrocladisine (10) from 1,2-
didehydro-ancistrobertsonine (6)
To a soln of 2.4 mg (6.0 mmol) of 1,2-didehydroan-
cistrobertsonine (6) in MeOH±H2O (9:1) freshly prepd
CH2N2 in Et2O (de Boer & Baker, 1963) was added
until the generation of N2 ceased and the resulting
mixt. remained yellow (Organikum, 1988). After addn
of CH2N2 the mixt. was left to stand 10 min at room
temp. to complete the reaction. After that time the sol-
vent was removed under reduced pressure to yield
2.5 mg (6.0 mmol, quantitative yield) ancistrocladisine
(10) after recrystallization from CH2Cl2. Mp 1768;
(Bringmann & Reuscher, 1989): 1788 (synthetic ancis-
trocladisine); (Govindachari et al., 1972): 178±1808
(natural product from A. heyneanus ). [a]2D5+68
(CHCl3; c 0.04);. (Bringmann & Reuscher, 1989)
:+7.88. CD: DE200 13.2, DE215 45.0, DE230+18.0,
DE248+10.9, DE262+9.0, DE312 2.7 (EtOH, c 0.05).
3.8. Isolation of ancistrobertsonine D (7)
From HSCCC fr. 6 ancistrobertsonine D was iso-
lated by prep. TLC using i-PrOH±MeOH (19:1) as elu-
ent. The crude alkaloid obtained was further puri®ed
by prep. HPLC with MeOH±H2O (4:6)
5 min,
(4:6 4 6:4) in 15 min, (6:4) 5 min, (6:4 4 4:6) in 1 min,
(4:6) 10 min. Attempts to recrystallize the compound
from di€erent solvents being not succesful, the alkaloid
was solved in MeOH±H2O. After the removal of
MeOH under reduced pressure, the aq. soln was lyo-
philized, yielding 8 mg of an amorphous, yellow pow-
der. Mp 133±13483 [a ]D25+828 (CHCl3; c 0.51); (Data
1
Chromatographic behaviour as well as IR, H NMR
and MS data were identical to those of natural and
for `ancistrine' from A. ealaensis (Foucher et al., 1975):
20
578
Mp
230±231.
[a]
358).
CD:
DE200+32.5,
synthetic
ancistrocladisine
obtained
earlier
DE213 86.8, DE239+68.8, DE282 32.0, DE305+8.7
(EtOH, c 0.01). IR nmax cm 1: 3400 (O±H), 2950,
2920, 2840 (C±H), 1600, 1585, 1570 (C1C), 1260,
(Govindachari et al., 1972; Bringmann & Reuscher,
1989). 13C NMR (100 MHz, CDCl3): d 21.00 (Me-3),
23.70 (Me-2'), 29.70 (Me-1), 34.91 (C-4), 56.29 (OMe-
6), 56.29 (OMe-4'), 56.38 (OMe-5'), 60.44 (OMe-8),
62.99 (C-3), 96.32 (C-5), 105.47 (C-6'), 106.44 (C-3'),
108.57 (C-7), 113.81 (C-9), 116.15 (C-10'), 117.78 (C-
1'), 118.39 (C-8'), 129.70 (C-7'), 133.00 (C-2'), 135.95
(C-2'), 144.50 (C-10), 157.14 (C-5'), 157.50 (C-4'),
157.86 (C-8), 167.54 (C-6), 178.10 (C-1).
1
1070 (C±O). H NMR (200 MHz, CDCl3): d 1.28 (3H,
d, J = 6.3 Hz, Me-3), 1.51 (3H, d, J = 6.4 Hz, Me-1),
2.22 (3H, s, Me-2'), 2.54 (1H, dd, Jgem=16.0 Hz,
Jax=10.8 Hz, Hax-4), 2.75 (1H, dd, Jgem=16.0 Hz,
Jeq=3.7 Hz, Heq-4), 3.01 (1H, mc, H-3), 3.07 (1H, s,
OMe-8), 3.98 (3H, s, OMe-5'), 4.02 (3H, s, OMe-4'),
4.31 (1H, q, J = 6.4 Hz, H-1), 6.58 (1H, s, H-5), 6.82
(1H, dd, Jortho=7.8 Hz, Jmeta=1.0 Hz, H-6'), 6.86 (1H,
s, H-3'), 7.06 (1H, dd, Jortho=8.4, Jmeta=1.0 Hz, H-
8'), 7.29 (1H, pseudo-t, Jortho=7.9 Hz, H-7'). 13C
NMR (150 MHz, CDCl3): d 20.53 (Me-2'), 20.86 (Me-
3.11. Reduction of ancistrocladisine (10) (Bringmann et
al., 1986)
Ancistrocladisine (60 mg, 0.14 mmol), AlMe3 (76 mg,
2.1 mmol solved in 1.1 ml n-hexane) and LiAlH4
(40 mg, 2.1 mmol) were added to 8.0 ml of cold
3 `Melting point' of the amorphous powder after lyophilization.

G. Bringmann et al. / Phytochemistry 52 (1999) 321±332
331
( 788) abs. THF under Ar. The mixt. was stirred vig-
orously while the temp. was held at 788 for 30 min.
During a period of 4 h, the temp. was slowly raised to
08. After that time, 12 ml of abs. THF and NaF (1.3 g,
30 mmol) were added and the mixt. was left to stir for
another 5 min and hydrolyzed with 0.3 ml of H2O.
After additional stirring for 30 min, the precipitate
was ®ltered o€ and washed with CH2Cl2 repeatedly.
After removal of the solvent under reduced pressure,
30.5 mg of a mixt. of trans- and cis-1,2-dihydroancis-
trocladisine (11a and 11b, respectively) were obtained.
The resoln of this mixt. of epimers by preparative
TLC [silica gel, CHCl3±MeOH (19:1)] yielded 18.4 mg
(43.0 mmol, 31%) of 11a as the major product, along
with 6.6 mg of 11b (15.0 mmol, 11%). The separated
compounds were recrystallized from CHCl3±MeOH
(3H, s, OMe-8), 3.12 (1H, dd,
J
gem=13.7 Hz,
Jax=7.8 Hz, Hax-4), 3.31 (1H, mc, H-3), 3.60 (3H, s,
OMe-6), 3.97 (3H, s, OMe-5'), 4.00 (3H, s, OMe-4'),
4.62 (1H, q, J = 6.4 Hz, H-1), 6.53 (1H, s, H-5), 6.77
(1H, d, Jortho=8.3 Hz, H-6'), 6.81 (1H, s, H-3'), 6.91
(1H, d, Jortho=8.5 Hz, H-8'), 7.29 (1H, dd,
Jortho=8.5 Hz, Jortho=8.3 Hz, H-7'). 13C NMR
(100 MHz, CDCl3): d 21.07 (Me-2'), 21.75 (Me-3),
22.56 (Me-1), 37.83 (C-4), 49.98 (C-3), 51.13 (C-1),
56.28 (OMe-6), 56.78 (OMe-4'), 56.93 (OMe-5'), 60.02
(OMe-8), 95.53 (C-5), 105.08 (C-6'), 106.21 (C-3'),
108.60 (C-7), 108.84 (C-10), 118.20 (C-10'), 121.29 (C-
8'), 125.28 (C-9), 126.39 (C-1), 134.39 (C-7'), 134.84
(C-9'), 135.76 (C-2'), 155.24 (C-6), 155.61 (C-8),
156.07 (C-4'), 156.64 (C-5'). EIMS m/z (rel. int.): 421
[M]+ (14), 407 [M Me+H]+ (27), 406 [M Me]+
(100), 390 [M OCH3] (8)
3.11.1. trans-1,2-Dihydroancistrocladisine (11a)
Mp 260±2628; (Bringmann & Reuscher, 1989) : 263±
2668. tR=18.9 min (same elution pro®le as for the iso-
lation of 7 was used). [a]2D5=+43 (CHCl3, c 0.13); ref.
3.12. Methylation of ancistrobertsonine D (7)
O-methylation of 0.9 mg (2.0 mmol) of ancistrobert-
sonine D (7) was performed in analogy to the prep-
aration of ancistrocladisine (10) from 6 (see above),
yielding 0.9 mg of pure 6-O-methylancistrobertsonine
D after recrystallization from CHCl3±MeOH (2.0 mmol,
quantitative). Mp 2158. tR=16.9 min (same elution
pro®le as for the isolation of 7 was used). [a]2D0+72
(Bringmann
&
Reuscher, 1989) :+49.7. CD:
DE200 28.6, DE205 15.9, DE212 33.6, DE231+53.5,
DE245 3.6, DE284 12.2, DE305+0.9 (EtOH, c 0.02). IR
nmax cm 1: 3450 (O±H), 2960, 2920, 2850 (C±H), 1605,
1580, 1575 (C1C), 1260, 1075 (C±O). 1H NMR
(200 MHz, CDCl3): d 1.54 (3H, d, J = 6.4 Hz, Me-3),
1.62 (3H, d, J = 6.1 Hz, Me-1), 2.19 (3H, s, Me-2'),
2.93-3.11 (2H, m, Hax-4, Heq-4; Jgem, Jax, Jeq not mea-
surable due to peak overlap), 3.10 (3H, s, OMe-8),
3.61 (1H, mc, H-3), 3.62 (3H, s, OMe-6), 3.96 (3H, s,
OMe-5'), 4.00 (3H, s, OMe-4'), 4.63 (1H, q,
J = 6.4 Hz, H-1), 6.49 (1H, s, H-5), 6.77 (1H, d,
Jortho=7.3 Hz, H-6'), 6.79 (1H, s, H-3'), 6.89 (1H, d,
Jortho=7.6 Hz, H-8'), 7.29 (1H, dd, Jortho=7.6 Hz,
Jortho=7.3 Hz, H-7'). 13C NMR (63 MHz, CDCl3): d
19.75 (Me-2'), 20.09 (Me-3), 20.94 (Me-1), 35.25 (C-4),
43.77 (C-3), 48.04 (C-1), 55.87 (OMe-6), 56.38 (OMe-
4'), 56.53 (OMe-5'), 59.83 (OMe-8), 94.13 (C-5),
105.53 (C-6'), 106.20 (C-3'), 108.87 (C-7), 116.30 (C-
10'), 118.57 (C-1'), 120.87 (C-8'), 122.64 (C-9), 126.30
(C-10), 132.67 (C-7'), 136.00 (C-9'), 136.49 (C-2'),
155.95 (C-6), 156.59 (C-8), 157.29 (C-4'), 157.69 (C-
5'). EIMS m/z (rel. int.): 421 [M]+ (2), 407
[M Me+H]+ (2), 406 [M Me]+ (8)
(CHCl3;
c
0.12). CD: DE200 18.6, DE208 50.0,
DE229+19.1, DE246 10.9, DE267+5.5, DE281 10.9,
1
DE300 3.6 (EtOH, c 0.04). IR, H NMR and MS data
were fully identical to those of cis-1,2-dihydroancistro-
cladisine 11b (see above). Due to the small amount of
substance available, no 13C NMR spectrum could be
obtained.
3.13. Biological experiments
Activity against P. falciparum was tested by the
semiautomated microdilution assay against intraery-
throcytic forms derived from asynchronous stock cul-
tures as previously described (Desjardins, Can®eld,
Haynes & Chulay, 1979) with minor modi®cations
(Ridley et al., 1996). The strains used were K1
(Thailand; resistant to chloroquine and pyrimetha-
mine) and NF54 (an airport strain of unknown origin;
susceptible to standard antimalarials). Cytotoxicity
was tested against rat skeletal muscle myoblast (L-6)
cells as described previously (Kaminsky & Brun, 1998).
The activities are given as IC50 values (mM).
3.11.2. cis-1,2-Dihydroancistrocladisine (11b)
Mp 212±2138. tR=16.9 min (same elution pro®le as
for the isolation of 7 was used). [a ]2D5=+77 (CHCl3, c
0.14). CD: DE200+5.0, DE208 49.9, DE227+28.3,
DE243 3.2, DE261+4.9, DE285 10.0 (EtOH, c 0.04). IR
nmax cm 1: 3400 (O±H), 2960, 2920, 2840 (C±H), 1600,
1580, 1575 (C1C), 1280, 1070 (C±O). 1H NMR
(400 MHz, CDCl3): d 1.63 (3H, d, J = 5.8 Hz, Me-3),
1.78 (3H, d, J = 6.4 Hz, Me-1), 2.16 (3H, s, Me-2'),
2.83 (1H, dd, Jgem=13.7 Hz, Jeq=3.7 Hz, Heq-4), 3.01
Chloroquine was used as
a
standard [IC50
(K1)=0.30 mM, IC50 (NF54)=0.01 mM].
Acknowledgements
We wish to thank Dr D. Koppler for performing the

332
G. Bringmann et al. / Phytochemistry 52 (1999) 321±332
Robertson, M. A., & Isahakia, M. A. (1998). Phytochemistry, 47,
31.
HMQC, HMBC and ROESY experiments. This work
was supported by the Deutsche
Forschungsgemeinschaft (Graduiertenkolleg
`Magnetische Kernresonanz in vitro und in vivo fur
die biologische medizinische Grundlagenforschung'
and SFB 251 `Okologie, Physiologie und Biochemie
p¯anzlicher und tierischer Leistung unter Streû') and
by the Fonds der Chemischen Industrie.
Bringmann, G., Kehr, C., Dauer, U., Gulden, K-P., Haller, R., Bar,
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