3-Acetylaltholactone and related styryl-lactones, mitochondrial respiratory chain inhibitors

Article abstract of DOI:10.1016/S0031-9422(00)00104-7

A novel furano-pyrone, 3-acetylaltholactone, and two other known styryl- lactones, altholactone and 5-acetoxyisogoniothalamin oxide, have been isolated from Goniothalamus arvensis (Annonaceae) stem bark. We report here the isolation and structural elucida

Full text of DOI:10.1016/S0031-9422(00)00104-7

Phytochemistry 54 (2000) 311±315
www.elsevier.com/locate/phytochem
3-Acetylaltholactone and related styryl-lactones, mitochondrial
respiratory chain inhibitors
Eva Perisa, Ernesto Estornellb, Nuria Cabedoa, Diego Cortesa, Almudena Bermejoa,*
a
Â
Departamento de Farmacologõa, Laboratorio de Farmacognosia, Universidad de Valencia, 46100 Burjassot, Valencia, Spain
 Â
Departamento de Bioquõmica y Biologõa Molecular, Facultad de Farmacia, Universidad de Valencia, 46100 Burjassot, Valencia, Spain
b
Received 26 October 1999; received in revised form 9 March 2000
Abstract
A
novel furano-pyrone, 3-acetylaltholactone, and two other known styryl-lactones, altholactone and 5-
acetoxyisogoniothalamin oxide, have been isolated from Goniothalamus arvensis (Annonaceae) stem bark. We report here the
isolation and structural elucidation of these compounds with furane-pyrone and styryl-pyrone skeletons, postulating also for the
®rst time their mechanism of cytotoxicity based on inhibition on mammalian mitochondrial respiratory chain. 7 2000 Elsevier
Science Ltd. All rights reserved.
Keywords: Goniothalamus arvensis; Annonaceae; Furano-pyrones; 3-Acetylaltholactone; Mitochondrial respiratory chain inhibitors
1. Introduction
tors of the cell-cycle on L-1210 leukemia line (Bermejo
et al., 1999b). In a recent biochemical work, we have
reported the ability of the bioactive heptolide-type-
styryl-lactones to inhibit complex I of the mitochon-
drial respiration chain (Bermejo et al., 1998b).
Styryl-lactones are an interesting group of bioactive
agents with signi®cant cytotoxicities against several
human tumor cell lines, many of which have been iso-
lated from Goniothalamus species (Annonaceae) (Blaz-
quez et al., 1999). In previous publications, we have
described for the ®rst time several styryl-lactones from
Goniothalamus arvensis stem bark. Four furano-pyr-
ones, (+)-goniotharvensin (Bermejo et al., 1995), ( )-
etharvensin (Bermejo et al., 1997), arvensin and (+)-2-
epialtholactone (Bermejo et al., 1999a); two styryl-pyr-
ones, (+)-garvensintriol and (+)-etharvendiol (Ber-
mejo et al., 1998a); and two heptolides, (+)-
almuheptolide-A and (+)-almuheptolide-B (Bermejo et
al., 1998b). Several furano-pyrone semisynthetic de-
rivatives have been found both cytotoxic and inhibi-
We describe herein both the isolation from G. arven-
sis stem bark methanolic extract, and the structure elu-
cidation of the novel natural 3-acetylaltholactone (1),
with a furano-pyrone skeleton, and two other known
compounds previously isolated from several Goniotha-
lamus species, altholactone (2) (Loder and Nearn,
1977) and 5-acetoxyisogoniothalamin oxide (3) (Hasan
et al., 1994), with furano-pyrone and styryl-pyrone
skeletons, respectively. In addition, to gain new
insights on their cytotoxicity mechanism of action,
these natural compounds (1±3) were tested for inhi-
bition of the mitochondrial respiratory chain. Our
results indicate that they are highly e€ective to block
this important bioenergetic process, and thus, they
might be exploited for biomedical research, antitumor
therapy and agrochemical pest control like other res-
piratory chain inhibitors.
* Corresponding author. Tel.: +34-96-386-4975; fax: +34-96-386-
4943.
E-mail address: bermejoa@uv.es (A. Bermejo).
0031-9422/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(00)00104-7

312
E. Peris et al. / Phytochemistry 54 (2000) 311±315
2. Results and discussion
2.1. Isolation and chemistry
resonance at dH 2.15 (3H, COCH3) and a deshielded
dd proton at dH 5.38. The existence of a phenyl group
in 1 was suggested by the proton resonances at dH 7.34
(m, 5H, H-9 to H-13), IR absorptions at 763 and 700
cm 1, and by the carbon signals at dC 137.4 (C-8),
126.2 (C-9 and C-13), 128.4 (C-11) and 128.6 (C-10
and C-12). Four resonances due to oxygen-bearing car-
bons ꢀdC 86.1: C-3a; dC 83.6: C-2; dC 83.5: C-3; dC
69.1: C-7a) and the corresponding four carbinol pro-
tons between dH 4.62 and 5.38, were observed in the
NMR spectra (Fig. 1).
The results consigned above are in agreement with a
furano-pyrone skeleton in 1. The relative stereochemis-
try of the four stereogenic centers in the tetrahydro-
furan ring was evidenced by the 1H±1H coupling
constant data and 2D homonuclear experiments.
COSY 45 spectrum of 1 showed correlations between
ole®nic protons (H-6 at dH 6.28 and H-7 at dH 7.04)
and the consecutive methine oxygenated ones, consist-
ent with the placement of the acetyl group at C-3 pos-
ition (see Fig. 2). Therefore, a 2,3-trans ꢀJ2,3 ˆ 3:5 Hz),
3,3a-trans ꢀJ3,3a ˆ 1:0 Hz) and 3a,7a-cis ꢀJ3a,7a ˆ 4:1
Hz) relative con®guration was established for 1, as it
occurs in altholactone (2) and other furano-pyrone
compounds (Blazquez et al., 1999). The acetylation of
2 gave 1 identical to natural one in all respects,
suggesting the structure of 1 as 3-acetylaltholactone.
The biological active compound 1, which we have
assigned the trivial name of 3-acetylaltholactone, was
isolated as transparent needles, m.p. 140±1428C and
1
‰a]D + 166.68 (c 0.3, EtOH). Analysis of the UV, H-
and 13C-NMR spectra of 1, revealed that it has a
characteristic furano-pyrone skeleton, closely related to
that of altholactone (2), a 3-hydroxy-2-phenyl-tetrahy-
drofurano-pyr-5-one derivative with an a,b-unsatu-
rated d-lactone (Loder et al., 1977; El-Zayat et al.,
1985).
Molecular weight of
1 was indicated in the
HRCIMS by a prominent peak at m/z 275.09178
[M+H]+ (calc. 275.09194), corresponding to the mol-
ecular formula C15H14O5, which is in agreement with
15 carbon signals observed in the 13C-NMR spectrum.
The mass fragmentation pattern of 1 was very close to
that of 2 (Bermejo et al., 1995). The presence of two
carbonyl groups in 1 was established by carbon reson-
ances at dC 160.4 (a,b-unsaturated d-lactone) and dC
169.4 (acetyl group), and by two strong IR absorptions
at 1742 and 1722 cm 1. The acetyl group in 1 was evi-
denced in HRCIMS by successive losses of COCH3
and OCOCH3 from [M+H]+, and by a singlet proton
Fig. 1. 1H- and 13C-NMR (in parentheses) of compounds 1±3.

E. Peris et al. / Phytochemistry 54 (2000) 311±315
313
Fig. 2. 1H±1H COSY 45 of 1.
Thus, the absolute con®guration of 1 is [2R, 3R, 3aR,
7aS ], as occurs in 2, whose con®guration was deter-
mined by X-ray crystallographic analysis (El-Zayat et
al., 1985).
The known compounds 2 and 3 (Fig. 1) were iso-
lated as yellowish oils. Comparison of their spectro-
scopic properties with those related compounds
previously isolated supported their structural assign-
ments (Loder et al., 1977; El-Zayat et al., 1985; Hasan
et al., 1994; Bermejo et al., 1995).
Fig. 3. Inhibition of NADH:oxidase activity in beef-heart submito-
chondrial particles by: 3-acetylaltholactone (1), altholactone (2), and
5-acetoxyisogoniothalamin oxide (3).
Table 1 shows both the half-inhibition (IC50) and
full-inhibition concentrations (IC100) of each com-
pound 1 and 3 gave IC50 in the mM range, and thus,
they were e€ective inhibitors of the respiratory chain
with similar potency to other inhibitors with potential
biomedical interest. Instead, 2 was less potent. Ti-
tration curves showed in Fig. 3 evidenced that 1 and 3
yielded a rapid decay of the NADH oxidase activity
with increasing concentration, but ®nal inhibition
stages, below 15% activity, were slower. Contrarily,
compound 2 gave a more hyperbolic curve and thus,
IC100 of the three compounds were closer than IC50.
Comparison of compounds 1 and 2 shows that
acetylation of C-3 hydroxy group clearly increases the
inhibitory potency. Compound 3 is as potent as 1 and
it also bears an acetyl group spatially near to epoxy
ring. It is thought that these functional groups placed
in this part of the molecule play an important role to
modulate the inhibitory action of this type of com-
pounds against the mitochondrial respiratory chain,
accounting for the cytotoxic and antitumor activities
previously described for several styryl-lactone deriva-
tives.
2.2. Bioactivity
Natural and synthetic styryl-lactone derivatives have
been found to possess signi®cant cytotoxic activities
against several human tumor cell lines (Cao et al.,
1998; Tsubuki et al., 1999). We have recently reported
the enantiospeci®c semisynthesis of almuheptolide-A, a
novel natural heptolide, a selective inhibitor of the
complex I of the mammalian mitochondrial respiratory
chain. Almuheptolide-A, a 8-phenyl-2-oxocanone de-
rivative also isolated from G. arvensis was synthesized
from altholactone (2) via the 6,7-dihydro-7-ethoxy-
altholactone (etharvensin) (Bermejo et al., 1997,
1998b). Taking into account such precedents, styryl-
lactone derivatives obtained here were expected to
inhibit the mitochondrial respiratory chain as cytotox-
icity mechanism, and thus, we assayed compounds 1±3
against the NADH oxidase activity of beef-heart sub-
mitochondrial particles, a model of mammalian respir-
atory chain.
Table 1
Inhibitory potency of compounds 1±3 against the NADH oxidase activity of mammalian respiratory chain
Compound
Half inhibition IC50 (mM)
Full inhibition IC100 (mM)
3-Acetylaltholactone (1)
Altholactone (2)
5-Acetoxyisogoniothalamin oxide (3)
4.721.6
2527
3.020.3
3224
8426
2222

314
E. Peris et al. / Phytochemistry 54 (2000) 311±315
3. Experimental
assigned by 1D and 2D NMR experiments: see Figs. 1
and 2.
3.1. General experimental procedures
3.5. Acetylation of 2
Optical rotations were determined with a Perkin-
Elmer 241 polarimeter. IR spectra were run in ®lm on
a Perkin-Elmer 843 spectrometer. UV spectra were
obtained on a Perkin-Elmer Lambda 15 UV/Vis spec-
trophotometer. Mass spectra were performed with
direct injection at 70 eV on a Nermay-Sidar instrument
or a VG Auto Spec Fisons spectrometer. 1H-NMR
and 13C-NMR 400 MHz Variant (Unity 400). Multi-
plicities of 13C-NMR resonances were obtained by
DEPT experiments. COSY 45 correlations were run
using a Bruker AC-250 (250 MHz).
Fifty mg of altholactone (2) were treated with Ac2O
(2 ml) and pyridine (1 ml) at room temperature for 3
h. Usual work up gave a compound in quantitative
yield identical to 1.
3.6. Bioactivity assays
Inhibition of mitochondrial respiratory chain was
assayed using titration of the compounds against the
aerobic oxidation of 75 mM NADH using beef-heart
submitochondrial particles obtained by ultrasonic dis-
ruption (6±7 mg/ml). Reaction rates were calculated
from the linear decrease of NADH concentration
ꢀl ˆ 340 nm, e ˆ 6:22 mM 1cm 1) in presence of
increasing amount of the compound in an end-window
photomultiplier spectrophotometer ATI-Unicam UV4-
500 (Tormo et al., 1999a). The inhibitory concen-
tration (IC50) was the ®nal compound concentration in
the assay medium that yielded 50% inhibition of
NADH oxidase activity. Full-inhibition concentration
(IC100) was taken when reaction rate was less than
3% of control activity. Given values are means 2 SD
of four assays for each compound (Tormo et al.,
1999b).
3.2. Plant material
Goniothalamus arvensis Sche€. was collected in the
National Park of Varirata, located in the Central Pro-
vince of Papua New Guinea. A voucher specimen (No.
517 Varirata plot) was deposited in the herbarium of
the University of Papua New Guinea.
3.3. Extraction and isolation
Dried and powdered stem barks of G. arvensis (368
g) were macerated with methanol at room temperature.
The crude methanolic extract was partitioned between
hexane and 50% aqueous methanol. After partial
evaporation, the aqueous solution was fractioned with
CH2Cl2 to obtain 8 g of organic extract which was
applied to a ¯ash silica gel (Merck 9385) column and
eluted with CH2Cl2/EtOAc (8:2). This a€orded us the
altholactone (2, 4.5 g) and a fraction (200 mg) that
was submited to a subsequent 60H silica gel (Merck
7736) column with CH2Cl2/EtOAc (99:1), which led us
to the isolation of the new compound 3-acetylaltholac-
tone (1, 7 mg) and the known 5-acetoxyisogoniothala-
min oxide (3, 5 mg).
Acknowledgements
This research was supported by the Spanish Direc-
cion General de Investigacion Cientõ®ca y Tecnica
(DGICYT) under grant SAF 97-0013. We are grateful
to Prof. Kundar S. Rao of the University of Papua
New Guinea, for help in obtaining the plant material.
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