Dioncophylline E from Dioncophyllum thollonii, the first 7,3′-coupled dioncophyllaceous naphthylisoquinoline alkaloid

Article abstract of DOI:10.1016/S0031-9422(02)00109-7

The isolation and structural elucidation of dioncophylline E, a novel naphthylisoquinoline alkaloid from the rare West African liana Dioncophyllwn thollonii, is described. The alkaloid displays an unusual 7,3′-linkage between the naphthalene and the isoquinoline portions. Due to the resulting medium degree of steric hindrance exerted by the ortho-substituents next to the biaryl axis, the compound undergoes slow rotation about the axis at room temperature and thus is the first such alkaloid that occurs as a mixture of two configurationally semi-stable atropo-diastereomers. Dioncophylline E exhibits good antimalarial activity against both chloroquine-sensitive and -resistant strains of Plasmodium falciparum while its antitrypanosomal activity against Trypanosoma cruzi is weak and that against T. brucei rhodesiense is moderate. Furthermore, four additional naphthylisoquinoline alkaloids previously known from the related plant species Triphyophyllum peltatum, have been identified in D. thollonii.

Full text of DOI:10.1016/S0031-9422(02)00109-7

Phytochemistry 60 (2002) 389–397
www.elsevier.com/locate/phytochem
0Dioncophylline E from Dioncophyllum thollonii, the first
7,3 -coupled dioncophyllaceous naphthylisoquinoline alkaloid§
Gerhard Bringmanna,*, Kim Messera, Kristina Wolfa, Jorg Muhlbachera,
Matthias Grunea, Reto Brunb, Adriaan M. Louisc
aInstitut fur Organische Chemie der Universitat, Am Hubland, D-97074 Wurzburg, Germany
¨
¨
¨
bSchweizerisches Tropeninstitut, Socinstrasse 57, CH-4002 Basel, Switzerland
cHerbier National de Gabon, BP 4015, Libreville, Gabon
Received 13 December 2001; received in revised form 15 March 2002
Dedicated to Professor Gottfried Blaschke on the occasion of his 65th birthday.
Abstract
The isolation and structural elucidation of dioncophylline E, a novel naphthylisoquinoline alkaloid from the rare West African
liana Dioncophyllum thollonii, is described. The alkaloid displays an unusual 7,30-linkage between the naphthalene and the iso-
quinoline portions. Due to the resulting medium degree of steric hindrance exerted by the ortho-substituents next to the biaryl axis,
the compound undergoes slow rotation about the axis at room temperature and thus is the first such alkaloid that occurs as a
mixture of two configurationally semi-stable atropo-diastereomers. Dioncophylline E exhibits good antimalarial activity against
both chloroquine-sensitive and -resistant strains of Plasmodium falciparum while its antitrypanosomal activity against Trypanosoma
cruzi is weak and that against T. brucei rhodesiense is moderate. Furthermore, four additional naphthylisoquinoline alkaloids
previously known from the related plant species Triphyophyllum peltatum, have been identified in D. thollonii. # 2002 Published by
Elsevier Science Ltd.
Keywords: Dioncophyllum thollonii; Dioncophyllaceae; Dioncophylline E; Structural elucidation; Stereochemistry; Antimalarial activity
1. Introduction
(Bringmann et al., 1992b), polyketide-derived (Bring-
mann et al., 2000a) main metabolite of T. peltatum
(Bringmann et al., 1990b), is characterized by the pre-
sence of two stereocenters and a stereogenic axis
between the 7- and the 10-position of the molecular
portions. The antifungal (Bringmann and Pokorny,
1995) alkaloid dioncophylline B (2) (Bringmann et al.,
1991b), by contrast, does not form stable atropisomers,
but undergoes rapid rotation about its—now 7,60-loca-
ted—biaryl axis, which is neighbored by only two small
OH groups, while dioncophylline C (3) (Bringmann et
al., 1992a), a highly antimalarial (Francois et al., 1996)
alkaloid with a 5,10-coupling type, is again config-
urationally stable. These three natural biaryls—like the
meanwhile ca. 16 further naphthylisoquinolines isolated
from T. peltatum (Bringmann et al., 1998a, b, c,
2000b)—all have in common the characteristic combi-
nation of an R-configuration at C-3 and the absence of an
oxygen function at C-6. These structural features classify
them as ‘Dioncophyllaceae-type alkaloids’, in contrast
to the ‘Ancistrocladaceae-type’ naphthylisoquinolines,
Within the Dioncophyllaceae, a very small family of
palaeotropical lianas consisting only of three monotypic
genera, the ‘part-time’ carnivorous (Bringmann et al.,
1996b) plant Triphyophyllum peltatum is the phyto-
chemically by far best investigated species (Bringmann
et al., 1998a, b, c, 2000b). Its structurally, biosyntheti-
cally, and pharmaceutically remarkable secondary
metabolites, the naphthylisoquinoline alkaloids, have
been found throughout the Dioncophyllaceae and in the
related Ancistrocladaceae (Bringmann and Pokorny,
1995), but never outside the two plant families. Thus,
dioncophylline A (1, Fig. 1), an insect antifeedant
§
Part 150 in the series ‘‘Acetogenic isoquinoline alkaloids’’. For
part 149, see Bringmann et al. (2002a).
* Corresponding author. Tel.: + 49-931-888-5323; fax: +49-931-
888-4755.
E-mail
(G. Bringmann).
address:
bringman@chemie.uni-wuerzburg.de
0031-9422/02/$ - see front matter # 2002 Published by Elsevier Science Ltd.
PII: S0031-9422(02)00109-7

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G. Bringmann et al. / Phytochemistry 60 (2002) 389–397
which are S-configured at C-3 and oxygenated at C-6
(Bringmann and Pokorny, 1995).
indeed proved to be dioncophylline A (1) and 50-O-
demethyldioncophylline A (4), identical in all respects
with authentic material from T. peltatum (Bringmann et
al., 1998c). The example demonstrates the value of the
HSCCC methodology as an entirely different separation
principle, saving time (here three chemical protection
and deprotection steps) and material (as compared to
the unavoidable loss on normal separation columns e.g.
with SiO2 as the adsorbent).
Different from T. peltatum, very little is known phy-
tochemically about the two other Dioncophyllaceae
species. Early isolation work on D. thollonii (Lavault
and Bruneton, 1980) revealed the presence of six naph-
thylisoquinolines, but their structures were not satisfac-
torily characterized, in particular with respect to the
absolute configurations at the stereocenters (which were
arbitrarily assumed to be S) and -axes (which were not
recognized as stereogenic elements). Therefore and
because of promising hints from LC-NMR investiga-
tions on crude plant extracts (Bringmann et al., 1999a),
a reinvestigation of this plant seemed rewarding. Here
we report on the isolation and structural assignment of
the four known naphthylisoquinolines 1, 4, (P)-5, and
its atropisomer (M)-5 from D. thollonii and of the new
compound dioncophylline E (6), the first 7,30-coupled
dioncophyllaceous alkaloid. In contrast to the very few
other 7,30-coupled Ancistrocladaceae alkaloids known
(Govindachari et al., 1973; Ruangrungsi et al., 1985;
Bringmann et al., 1998d), 6 is configurationally semi-
stable at the axis. It is thus the first naphthylisoquino-
line alkaloid that exists as a pair of slowly interconvert-
ing atropo-diastereomers, (P)-6 and (M)-6, at room
temperature.
In the region less polar than 1, the second isolated
main constituent of D. thollonii coeluted on TLC with
N-methyldioncophylline A (5) and also exhibited the
corresponding NMR signals, albeit with a (1:1)-peak
doubling for some of the signals, e.g. for those of the
N-methyl (2.54 and 2.55 ppm) and the 20-methyl (2.17
and 2.20 ppm) groups, clearly indicating the presence of
a mixture of two structurally very similar, apparently
stereoisomeric naphthylisoquinolines. By our ruthe-
nium-catalyzed oxidative degradation (Bringmann et
al., 1996a), the stereocenters of the two isomers were
shown to be R-configured, both at C-1 and C-3, sug-
gesting the isomers to differ only by their axial con-
figuration. As previously for a similar mixture of (P)-5
and its likewise natural atropo-diastereomer (M)-5 from
Ancistrocladus abbreviatus (Bringmann et al., 1991a),
resolution of (P)-5 and (M)-5 succeeded again by chro-
matography on a chiral phase (Chiralcel OD), albeit
now with a slight adaptation of the separation condi-
tions to the semipreparative HPLC column. As expected
for atropisomeric naphthylisoquinoline alkaloids (even
though diastereomeric ones), the CD spectra of the
resulting pure substances were near-opposite to each
other and the physical and spectral data were fully
identical with those of the known (Bringmann et al.,
1991a) alkaloids N-methyldioncophylline A [(P)-5] and
N-methyl-7-epi-dioncophylline A [(M)-5] (see Fig. 1).
One of the more polar fractions obtained from CC
seemed to contain one pure compound according to
TLC. As described for (M)/(P)-5 above, however, the
NMR spectrum of this fraction again showed two sets
of signals for two very similar naphthyl-1,3-dimethylte-
trahydroisoquinoline structures. HPLC analysis gave
two well separated peaks with retention times differing
by ca. 10 min, even on a preparative scale. But the ana-
lysis of the two chromatographic fractions thus
obtained, showed these samples to be fully identical:
Both by HPLC and by NMR, again each of them con-
sisted of the two previous compounds, indicating their
interconversion at room temperature. This was unam-
biguously proven by an immediate investigation of the
freshly resolved single peaks as obtained by rapid ana-
lytical HPLC analysis, now clearly showing a gradual
equilibration of the two peaks, with a moderate atropi-
somerization rate at room temperature (see Fig. 2).
This interconversion could also be nicely followed by
HPLC-NMR experiments in the stopped-flow mode, by
2. Results and discussion
Air-dried and powdered root material of D. thollonii
was successively extracted with n-hexane and methanol,
of which the latter was perforated with chloroform. This
chloroform extract was then subjected to CC. By TLC
comparison, the main Dragendorff-active compound
resembled the well known (Bringmann et al., 1990a, c)
alkaloid dioncophylline A (1), but NMR investigations
revealed the presence of two compounds, apparently
resulting from the co-occurrence of 1 and its 50-O-
demethyl analog 4 (see Fig. 1). They proved to be diffi-
cult to resolve because of their nearly identical chroma-
tographic behavior on normal-phase silica gel
(Bringmann et al., 1998c). This separation problem had
previously been solved (Bringmann et al., 1998c) after
conversion of the mixture into a pair of brominated and
benzylated, now well separable, derivatives. In an
attempt to resolve the mixture directly, without chemi-
cal modification, we took advantage of their possibly
different partition coefficients in a two-phase mixture,
by using high-speed countercurrent chromatography
(HSCCC) (Ito and Conway, 1996). Indeed, after opti-
mization of the solvent mixture (CHCl3–MeOH–0.1 N
HCl 6:3:3), 70% of a starting sample was easily sepa-
rated even on a semipreparative scale, with complete
recovery of the remaining unresolved material. The
resulting two compounds thus obtained in a pure form

G. Bringmann et al. / Phytochemistry 60 (2002) 389–397
391
Fig. 1. Bioactive naphthylisoquinolines from Dioncophyllaceae: dioncophyllines A-C (1–3), 50-O-demethyldioncophylline A (4), N-methyldionco-
phylline A and its 7-epimer [(P)-5 and (M)-5], and the two configurationally semi-stable atropisomers of dioncophylline E (6) (*: configurationally
unstable axis).
watching, e.g., the well-separated signals of the 1- and
3-methyl groups (not shown) of the two—apparently
stereoisomeric—naphthylisoquinoline alkaloids present
in that mixture. Unfortunately, some of the other NMR
signals of these two species overlapped in the LC-NMR
solvent system (CH3CN–D2O), which thus did not per-
mit a secure attribution of the entire two sets of signals.
Still, from the very similar NMR signals and also from
HPLC-MS/MS experiments, giving rise to identical
mass spectra for both peaks, it became clear that the
two interconverting species were probably again atropo-
diastereomers. This was entirely unprecedented in so far
as all other naphthylisoquinoline alkaloids as yet
known, form rotational isomers that are—depending on
the substitution patterns in the proximity of the biaryl
linkage—either configurationally fully stable, such as,
e.g., dioncophyllines A (1) and C (3), or rotate very
rapidly at room temperature, as is the case for dionco-
phylline B (2). The apparent semi-stability of the axial
configuration already gave a clear hint that this slowly
atropisomerizing alkaloid had to be new and was appar-
ently based on an unusual, if not novel, coupling type.
The 1H NMR spectrum of that pair of interconverting
new alkaloids exhibited signals typical of a naphthyl-
isoquinoline with one methoxy substituent (4.00/4.04
ppm, Fig. 3a), suggesting the two other oxygen func-
tions, as evident from the molecular formula of
C23H25NO3 (from HRMS of the [MÀCH3]+ peak), to
be hydroxy groups. The aromatic region showed the
presence of a one-proton system and of groups of
two and of three adjacent protons, by H,H-COSY
Fig. 2. Analytical HPLC analysis of the interconversion of peaks A
and B of dioncophylline E (6) on reversed phase material (C18).
experiments. The group of two neighboring aromatic
protons was easily located in the 5- and 6-positions
(6.81, 7.05 and 7.06 ppm) of the isoquinoline moiety by
HMBC (H-40s ! C-5, H-5 ! C-4) and ROESY inter-
actions (H-40s $ H-5 $ H-6) (Fig. 3b). The position of
the biaryl axis between the two molecular moieties was
determined as follows: HMBC signals from H-6 to the
quaternary carbon atom C-30 in the naphthalene half of
the molecule assigned the coupling site to be C-7, which

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G. Bringmann et al. / Phytochemistry 60 (2002) 389–397
was further confirmed by interactions of H-5 with the
likewise quaternary C-7. The coupling position in the
naphthalene part was excluded to be in the methyl-free
ring, because of the existence of the above mentioned
spin system of three neighboring aromatic protons. Of
the remaining two positions of the isoquinoline sub-
stituent, C-30 or C-10, the latter was ruled out from the
fact that it bears the above-mentioned single aromatic
proton, which was assigned by an HMBC interaction
between H-80 and this—hence tertiary—C-atom C-10
and, vice versa, from H-10 to C-80, thus unequivocally
locating the biaryl axis to be at C-30. This attribution
was further confirmed by HMBC correlations from H-10
(7.24 and 7.27 ppm), HO-40 (9.71 ppm), and H-6 (7.05
and 7.06 ppm) to C-30. The 7,30-linkage thus deduced
was further supported by the high-field shifted signal of
the CH3-20 group (2.14 and 2.15 ppm) as compared to
that e.g. of the 7,60-coupled alkaloid dioncophylline B
(2), which exhibits a ‘normal’ shift of 2.46 ppm (Bring-
mann et al., 1991b). The methoxy function was proven
to be located at C-50, by HMBC interactions of the
methyl protons with C-50 and by ROESY signals with
H-60 (Fig. 3b).
The new alkaloid is unique both constitutionally,
being the first 7,30-coupled ‘Dioncophyllaceae-type’
alkaloid (i.e. with R-configuration at C-3 and without
an oxygen function at C-6) and stereochemically,
because it is configurationally semi-stable at the axis, due
to the unsubstituted C-6 position. The two only other
7,30-linked naphthylisoquinoline alkaloids as yet known
belong to the ‘Ancistrocladaceae-type’ (i.e. being 3S-
configured and 6-oxygenated), viz ancistrocladidine (7)
from Ancistrocladus heyneanus (Govindachari et al., 1973,
1975; Parthasarathy and Kartha, 1983) and A. tectorius
(Meksuriyen et al., 1990) and ancistrotectorine (8), its
N-methyl dihydro analog from A. tectorius (Ruangrungsi
et al., 1985) and from A. guineensis (Bringmann et al.,
¨
1998d), but those biaryls are configurationally fully
stable at the axis (see Fig. 4).
Because of the new coupling type within the Dionco-
phyllaceae alkaloids, the new compound 6 was hence-
forth named dioncophylline E. For the attribution of
the absolute axial configuration of the two respective
atropo-diastereomers, (M)- and (P)-dioncophylline E
[(M)- and (P)-6], an investigation of their circular
dichroism (CD) seemed to be the best method since the
two atropisomers (although diastereomers due to the
additional existence of the stereogenic centers!) should
deliver near-opposite CD spectra because the biaryl
chromophore strongly dominates the CD behavior of
such molecules (Bringmann and Busemann, 1998). The
usual stand-alone CD measurements of the previously
resolved dioncophylline E isomers turned out to be dif-
ficult because of their interconversion, so that the
method of choice was a direct CD analysis in hyphena-
tion with HPLC separation, as possible by the HPLC-
CD coupling technique, which has recently been intro-
duced into phytochemical analysis (Bringmann et al.,
1999b). Indeed, on-line CD analysis of the two HPLC
peaks obtained by resolution of dioncophylline E (6)
yielded near-opposite CD-spectra (Fig. 5, bottom, full
lines), again confirming this new alkaloid to consist of
two rotational isomers that gradually interconvert at
room temperature.
As to the stereostructure, the relative configuration of
the two methyl groups at C-1 and C-3 was deduced to
be trans by a ROESY interaction between H-3 and
CH3-1 (Fig. 3b), in agreement with the typical (Bring-
1
mann et al., 1990a; Bringmann and Pokorny, 1995) H
NMR down-field shift for H-3 (3.69–3.77 ppm). The
absolute configuration was investigated by oxidative
degradation of the new compound to give (R)-3-amino-
butyric acid (from C-3) and d-alanine (mainly from
C-1), establishing 1R,3R-configuration, which is again
in agreement with the relative 1,3-trans array proven
above by NMR.
In addition to just demonstrating the existence of two
atropisomeric forms, an interpretation of the CD spec-
tra thus measured on line, became possible by a tech-
nique improved by our group earlier, the quantum
Fig. 3. Selected NMR data of dioncophylline E (6): 1H NMR shifts
for the co-occurring two atropisomers (a), decisive HMBC (single
arrows), and ROESY interactions (broken line arrows) (b).
Fig. 4. The two other 7,30-linked—but 6-oxygenated and thus config-
urationally stable—naphthylisoquinolines known to date.

G. Bringmann et al. / Phytochemistry 60 (2002) 389–397
393
Fig. 5. LC-CD spectra (full lines) of the two rotational isomers of dioncophylline E (6) and assignment of the absolute configurations at the biaryl
axes by comparison with the calculated MD-based CD spectra (broken lines).
chemical calculation of CD spectra based on molecular
dynamics (MD) simulations (Bringmann et al., 2001).
As seen in Fig. 5, the CD spectrum calculated for the
P-atropisomer of 6 matches quite well with that of the
more rapidly eluting peak A in the decisive region
between 200 and 230 nm, while the experimental CD
spectrum for the slower peak B on reversed-phased
material is in good agreement with the one calculated
for M, clearly indicating peak A to correspond to (P)-6
and peak B to (M)-6. This assignment is also in agree-
ment with the similarity of the CD spectrum of (M)-6
with that of the—likewise 7,30-coupled and M-config-
ured—Ancistrocladaceae alkaloid ancistrotectorine (8,
see Fig. 4).
(M)-6 fits well with the slow interconversion of the two
atropisomers as room temperature. It is likewise in a
good agreement with the results from additionally per-
formed quantum chemical calculations using the semi-
empirical AM1 method as developed by Dewar et al.
¼
(1985), leading to a calculated value of ꢀH =107 kJ
molÀ1. The agreement between the predicted and the
experimental values is satisfying, the deviation being
due mainly to the fact that the calculations had to be
done ‘in vacuo’, i.e. without regarding any solvent
effects. Likewise in agreement with the experimentally
measured atropisomeric equilibrium ratio of (P)-6:(M)-
6 of 43:57 at room temperature is the calculated stability
difference of 0.8 kJ molÀ1 in favor of the M-isomer.
Besides its new structural properties, dioncophylline E
(6) is also pharmacologically interesting: While its anti-
trypanosomal activities against Trypanosoma cruzi
(Chagas disease) and T. brucei rhodesiense (African
sleeping sickness) are weak to moderate (18.4 and 0.73
mg mlÀ1) as compared to the respective standards,
benznidazole (0.68 mg mlÀ1) and melarsoprol (0.002 mg
mlÀ1), it exhibits good activity against the pathogen of
the malaria tropica, Plasmodium falciparum, with nearly
identical IC50 values on the chloroquine-sensitive
(NF54) and chloroquine-resistant (K1) strains. With
values of only 22 and 21 ng mlÀ1, the activity of the new
alkaloid ranges within those of the most active naph-
thylisoquinoline alkaloids and is only by a factor of 5–
10 weaker than that of the standards artemisine (2.8 and
With the unprecedented semi-stability of the axial
chirality of dioncophylline E (6), it seemed rewarding to
determine the atropisomerization barrier of this unusual
novel naphthylisoquinoline alkaloid. This was achieved
by measuring the decrease of the diastereomeric purity
of freshly separated samples enriched in the respective
P- or M-atropisomer (Fig. 6a). The rate constants at
four different temperatures as determined from their
logarithmic plots (Fig. 6b), delivered the corresponding
enthalpy and entropy terms by means of analysis of
slope and intersection with the coordinate axis accord-
ing to procedures established in the literature (Frost and
Pearson, 1964; Ernst, 1983).
¼
The activation enthalpy ꢀH =81.9 kJ molÀ1 (ꢀS=
60.0 J KÀ1 molÀ1) for the atropisomerization of (P)-6 to

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G. Bringmann et al. / Phytochemistry 60 (2002) 389–397
1JHC=145 Hz) and HMBC (optimized for nJHC=7 Hz).
NOE and ROE effects were measured using NOESY
and ROESY pulse sequences from the standard Bruker
pulse program library. EIMS (70 eV) and HREIMS (70
eV) were determined on Finnigan MAT 8200 and Fin-
nigan MAT 90 instruments. Analytical HPLC: Sym-
metry RP18 4.6ꢀ250 mm (Waters). Semipreparative
HPLC: Prep LC 25, Nova Pak 25ꢀ210 mm (Waters).
Chiral HPLC: Chiralcel OD 4.6ꢀ250 mm and 10ꢀ250
mm (Daicel). HPLC-CD measurements were performed
on a Jasco J-715 spectropolarimeter with a standard
flow cell. CD spectra were recorded in the stop-flow
mode. HSCCC: ‘Triple coil’, 1.68 mmꢀ37.0 m (medium
coil), 1.68 mmꢀ106.5 m (large coil), (H)!T, lower
phase as mobile phase, forward elution mode. The
absolute configuration of the stereocenters at C-1 and
C-3 of the isolated naphthylisoquinolines were deter-
mined by oxidative degradation as described previously
(Bringmann et al., 1996a).
3.2. Plant material
Plant material of D. thollonii was collected by one of
us (AML) in Rabi Kounga (Gabon) (10/1996). A vou-
cher specimen (No. 01) has been deposited at Herb.
Bringmann, University of Wurzburg.
3.3. Extraction and isolation
Fig. 6. Determination of the rotational barrier of dioncophylline E
(6): Time-dependent decrease of the diastereomeric excess of peak A
(a), logarithmic plots for the determination of the rate constants at
different temperatures by the slope (b).
D. thollonii: 1.2 kg of air-dried root material were
ground and subsequently extracted with n-hexane and
MeOH. The methanolic extract was perforated with
n-hexane and CHCl3, and the resulting chloroform
extract prefractionated by CC with CH2Cl2–MeOH
(9:1) as the eluent. The second fraction, containing
Dragendorff-active compounds, was further resolved by
CC with CHCl3–MeOH (9:1/10:3/15:1).
1.1 ng mlÀ1, respectively) and chloroquine (4.4 and 65
ng mlÀ1). These activities, including the even higher
ones of a few other naphthylisoquinolines like dionco-
phylline C (3) (Francois et al., 1996; Bringmann and
Feineis, 2000), dioncopeltine A (Francois et al., 1997),
and habropetaline A (Bringmann et al., 2002b), make it
rewarding to isolate or synthesize (Bringmann et al.,
2000b) further new representatives of this novel class of
bioactive compounds. This work is in progress.
3.4. Dioncophylline E (6)
Analytical HPLC: MeOH–H2O 60:40, 0.1% TFA, 1
ml minÀ1, 20 ꢁC, 230 nm. Semipreparative HPLC:
ꢁ
MeOH–H2O 70:30, 0.1% TFA, 6 ml minÀ1, 24 C, 230
ꢁ
3. Experimental
nm. Mp 59–60 C. [a]D25 À146ꢁ (MeOH; c 0.1). CD (fas-
ter atropisomer): [È]217 À3.3, [È]230 0.8, [È]240 À1.7,
[È]277 0.7, [È]334 À1.2. CD (slower atropisomer): [È]216
3.1. General
5.3, [È]230 À1.3, [È]240 1.7, [È]280 À0.4, [È]335 1.3. IR
max
KBr
Mps: uncorr. IR spectra were taken on a Jasco FT/
IR-410 spectrometer, CD spectra on a Jasco J-715
spectropolarimeter, and optical rotations on a Perkin-
n
cmÀ1: 3376, 2961, 2919, 2851, 1578, 1464, 1358,
1
1088, 805, 625. H NMR (600 MHz, CDCl3; ‘double’
values are due to partial spectral differences for the two
atropisomeric forms of 6): ꢀ 1.58 and 1.59 (3H, d, J=6.0
and 6.1 Hz, CH3-3), 1.71 and 1.74 (3H, d, J=6.5 and
6.7 Hz, CH3-1), 2.14 and 2.15 (3H, s, CH3-20), 2.98–3.13
(2H, m, H-4), 3.69–3.77 (1H, m, H-3), 4.00 and 4.04
(3H, s, CH3O-50), 4.89–4.96 (1H, m, H-1) 6.74 and 6.77
(1H, m, H-60), 6.81 (1H, d, J=7.9 Hz, H-5), 7.05 and
1
Elmer 241MC polarimeter. H NMR (600 MHz) and
13C NMR (150 MHz) were recorded on a Bruker DMX
600 either in CDCl3 or CD3OD with the solvent as the
int. standard (CDCl3: ꢀ 7.26 and ꢀ 77.01, CD3OD: ꢀ 3.30
and ꢀ 49.02). Proton-detected, heteronuclear corre-
lations were analyzed using HMQC (optimized for

G. Bringmann et al. / Phytochemistry 60 (2002) 389–397
395
7.06 (1H, d, J=7.8 and 7.8 Hz, H-6), 7.24 and 7.27 (1H,
s, H-10), 7.29–7.37 (2H, m, H-70 and H-80), 9.71 (1H, s,
OH-40). 13C NMR (150 MHz, CDCl3): ꢀ 17.8 and 18.1
(CH3-1), 18.4 and 18.8 (CH3-3), 20.6 and 20.8 (CH3-20),
33.8 and 33.8 (C-4), 44.1 and 44.4 (C-3), 48.1 and 48.1
(C-1), 56.2 and 56.2 (OCH3-50), 104.0 and 104.0 (C-60),
113.3 and 113.3 (C-40a), 116.9 and 117.2 (C-30), 120.0
and 120.0 (C-10), 120.6 and 121.2 (C-8a), 120.7 and
120.7 (C-5), 121.1 and 121.3 (C-80), 122.0 and 122.4 (C-
7), 126.6 and 126.6 (C-70), 130.2 and 130.5 (C-6), 131.3
and 131.4 (C-4a), 136.3 and 136.5 (C-80a), 137.4 and
138.1 (C-20), 149.5 and 149.7 (C-8), 151.8 and 151.8 (C-
40), 156.0 and 156.0 (C-50). The 13C attributions were
achieved by HMQC and HMBC experiments. EIMS
m/z (rel. int.): 363 [M]+ (5), 348 [MÀCH3]+ (100), 333
[MÀ2CH3]+ (12). HREIMS m/z: 348.1604 [MÀCH3]+
(C22H22NO3 requires 348.1600).
states were obtained by a CNDO/S-CI (Downing, 1992)
calculation, in which the CI expansion takes into
account the ground state and all n and p orbitals. These
calculations were carried out on Linux iPII and iPIII
workstations using the program package BDZDO/
MCDSPD (Downing, 1992; Del Bene and Jaffe, 1968).
For a better comparison of the theoretical CD spectrum
with the experimental one, a Gaussian band shape
function was generated over the calculated rotational
strength values.
3.8. Biological experiments
Plasmodium falciparum: Antiplasmodial activity was
determined using the strains K1 (resistant to chlor-
oquine and pyrimethamine) and NF54 (sensitive to all
known drugs). A modification of the [3H]-hypoxanthine
incorporation assay (Desjardins et al., 1979) was used
(Ridley et al., 1996). Briefly, infected human red blood
cells were exposed to ꢁserial drug dilutions in microtiter
plates for 48 h at 37 C in a gas mixture with reduced
oxygen and elevated CO2. [3H]-Hypoxanthine was
added to each well and after further incubation for 24 h
the wells were harvested on glass fiber filters and coun-
ted in a liquid scintillation counter. From the sigmoidal
inhibition curve the IC50 value was calculated. The
assays were run in duplicate and repeated at least once.
Antitrypanosomal activities were determined as fol-
lows: Trypanosoma cruzi: Rat skeletal myoblasts (L-6
cells) were seeded in 96-well microtiter plates at 2000
cells/well/100 ml in RPMI 1640 medium with 10% FBS
and 2 mM l-glutamine. After 24 h 5000 trypomasti-
gotes of T. cruzi [Tulahuen strain C2C4 containing the
galactosidase (Lac Z) gene] were added in 100 ml per
well with a serial drug dilution. The plates were incu-
bated at 37 ꢁC in 5% CO2 for 4 d. For determination of
the IC50 the substrate CPRG/Nonidet was added to the
wells. The color reaction which developed during the
following 2–4 h in the presence of live cells was read
photometrically at 540 nm. IC50 values were calculated
from the sigmoidal inhibition curve. Trypanosoma b.
rhodesiense: Minimum Essential Medium (50 ml) sup-
plemented according to Baltz et al. (1985) with 2-mer-
captoethanol and 15% heat-inactivated horse serum
was added to each well of a 96-well microtiter plate.
Serial drug dilutions were added to the wells. Then 50 ml
of trypanosome suspension (T. b. rhodesiense STIB 900)
were added to each well and the plate incubated at 37 ꢁC
under a 5% CO2 atmosphere for 72 h. Alamar Blue (10
ml) was then added to each well and incubation was
continued for a further 2–4 h. The plate was then read
using a Millipore Cytofluor 2300 at an excitation wave-
length of 530 nm and an emission wavelength of 590 nm
(Raz et al., 1997). Fluorescence development was
expressed as percentage of the control, and IC50 values
were determined.
3.5. Known naphthylisoquinoline alkaloids
Resolution of dioncophylline A (1) (Bringmann et al.,
1990a,c) and 50-O-demethyldioncophylline
A
(4)
(Bringmann et al., 1998c) by preparative HSCCC
(CHCl3–MeOH–0.1 N HCl 6:3:3) yielded 5.1 mg and
5.0 mg of the respective pure compounds. Semi-
preparative separation (n-hexane–i-PrOH 98:2, 0.1%
NEt3) of N-methyldioncophylline A and N-methyl-7-
epi-dioncophylline A (Bringmann et al., 1991a) on a
chiral phase resulted in 5.1 and 2.6 mg of the sub-
stances, reflecting a natural occurrence of 2:1. All sub-
stances were fully identical with the spectroscopic and
physical properties reported in the literature (Bring-
mann et al., 1991a).
3.6. Oxidative degradation procedure
The oxidative degradation, the derivatization of the
derived amino acids, and the subsequent GC-MSD
analyses were carried out as described previously
(Bringmann et al., 1996a).
3.7. Computational
The molecular dynamics simulation was performed
on a SGI Octane (R10000) workstation using the TRI-
POS force-field as implemented in the molecular mod-
eling package SYBYL1 with time steps of 2 fs. The
molecule was weakly coupled to a virtual thermal bath
at T=300 K (Berendsen et al., 1984), with a tempera-
ture relaxation time t=0.28 ps. The wavefunctions for
the calculation of the rotational strengths for the elec-
tronic transitions from the ground state to the excited
1
SYBYL: Tripos Associates, 1699 Hanley Road, Suite 303, St.
Louis, MO 63144, USA.

396
G. Bringmann et al. / Phytochemistry 60 (2002) 389–397
first 5,10-coupled Dioncophyllaceae alkaloid. Phytochemistry 31,
4019–4024.
Acknowledgements
Bringmann, G., Gramatzki, S., Grimm, C., Proksch, P., 1992b. Feed-
ing deterrency and growth retarding activity of the naphthyliso-
quinoline alkaloid dioncophylline A against Spodoptera littoralis.
Phytochemistry 31, 3821–3825.
This work was supported by the Fonds der Chemischen
Industrie and the Deutsche Forschungsgemeinschaft (Br
699/7–1). We wish to thank Dr. C. Gunther for stimulat-
ing discussions and M. Michel and M. Munchbach for
performing the degradation procedure. Furthermore we
thank E. Ruckdeschel for the NMR experiments, Dr. G.
Lange and F. Dadrich for the mass spectra, and Jasco
Deutschland for technical support. Particular thank is
due to E. Gobright and C. Scheurer for carrying out the
in vitro assays.
Bringmann, G., God, R., Schaffer, M., 1996a. An improved degrada-
tion procedure for determination of the absolute configuration in
chiral isoquinoline and b-carboline derivatives. Phytochemistry 43,
1393–1403.
Bringmann, G., Wenzel, M., Bringmann, H., Schlauer, J., Ake Assi,
L., 1996b. Die ‘‘Teilzeit-fleischfressende’’ Pflanze Triphyophyllum
peltatum (Dioncophyllaceae): Nutzung der Fangorgane zur Erfor-
schung der Alkaloidbildung. Der Palmengarten 60, 32–37.
Bringmann, G., Francois, G., Ake Assi, L., 1998a. The alkaloids of
Triphyophyllum peltatum (Dioncophyllaceae). Chimia, 18–28.
Bringmann, G., Wenzel, M., Rubenacker, M., Schaffer, M., Ruckert,
M., Ake Assi, L., 1998b. Dioncophylline D and 8-O-methyldionco-
phylline D, 7,80-coupled naphthylisoquinoline alkaloids from Tri-
phyophyllum peltatum. Phytochemistry 49, 1151–1155.
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