Enantiospecific semisynthesis of (+)-almuheptolide-A, a novel natural heptolide inhibitor of the mammalian mitochondrial respiratory chain

Article abstract of DOI:10.1021/jm9706574

The development of novel styryl lactone derivatives as bioactive compounds and the semisynthesis of both 4,5-dialkoxylated eight-membered- ring lactones with a heptolide skeleton (almuheptolide-A (1) type) and 7- alkoxylated 5-lactones with a saturated fu

Full text of DOI:10.1021/jm9706574

5
158
J . Med. Chem. 1998, 41, 5158-5166
Articles
En a n tiosp ecific Sem isyn th esis of (+)-Alm u h ep tolid e-A, a Novel Na tu r a l
Hep tolid e In h ibitor of th e Ma m m a lia n Mitoch on d r ia l Resp ir a tor y Ch a in
†
‡
Almudena Bermejo, J os e´ R. Tormo, Nuria Cabedo, Ernesto Estornell, Bruno Figad e` re, and Diego Cortes*
Departamento de Farmacolog ı´ a, Farmacognosia y Farmacodinamia and Departamento de Bioqu ´ı mica y Biolog ı´ a Molecular,
Facultad de Farmacia, Departamento de Bioqu ı´ mica y Biolog ı´ a Molecular, Universidad de Valencia, 46100 Burjassot,
Valencia, Spain, and Laboratoire de Pharmacognosie, Facult e´ de Pharmacie, Universit e´ Paris-Sud, Ch aˆ tenay-Malabry, France
Received September 29, 1997
The development of novel styryl lactone derivatives as bioactive compounds and the semisyn-
thesis of both 4,5-dialkoxylated eight-membered-ring lactones with a heptolide skeleton
(
almuheptolide-A (1) type) and 7-alkoxylated δ-lactones with a saturated furanopyrone skeleton
(etharvensin (8) type) have been successfully achieved from the chiral unsaturated R-pyrone
altholactone (7). This new method is a direct and one-step enantiospecific alkoxylation of
altholactone (7) in concentrated acid medium, followed by formation of the eight-membered-
ring ú-lactone. The reaction mechanism operating in the synthesis of the heptolide skeleton is
postulated to be a direct Michael-type addition. Concerted opening of both the R-pyrone and
tetrahydrofuran rings and subsequent intramolecular rearrangement with the ring closure
lead to almuheptolide-A (1). This compound (1) and its diacetated derivative (1a ) showed potent
and selective inhibitory activity toward mammalian mitochondrial respiratory chain complex
I. This mechanism of action, reported here for the first time, provides a possible explanation
for the cytotoxic and antitumor activities previously described for related natural compounds.
In tr od u ction
Several plants of the Annonaceae family are known
as a source of natural, potent, biologically active com-
pounds. As a part of our search for new cytotoxic
compounds in Annonaceous plants,1-4 series of styryl
lactones were isolated from the methanolic extract of
Goniothalamus arvensis stem barks.5
-7
In this paper
we report the isolation and structure elucidation of (+)-
almuheptolides-A (1) and -B (2), new natural heptolides
from G. arvensis, and the first method for the prepara-
tion of pentasubstituted heptolides from furano-R-
pyrone structures (Figure 1). (+)-Almuheptolide-A (1)
possesses an unusual skeleton similar to that of the
8
known (+)-gonioheptolides-A (3) and -B (4) and related
to that described for the natural marine products
octalactins-A (5) and -B (6) (Figure 1).9
The cytotoxic activities previously reported for eight-
membered-ring ú-lactones8,9 and the structural resem-
blance of these compounds to antimycin A (a nine-
membered dilactone with a salicylamide moiety and a
1
0
well-known inhibitor of the respiratory chain) prompted
us to assay the (+)-almuheptolide-A (1) and its diacetate
F igu r e 1. Natural eight-membered-ring lactones.
(
1a ) against the mammalian mitochondrial respiratory
chain activities. The current research on respiratory
chain inhibitors has provided very interesting com-
pounds with high potential for biomedical basic re-
search,11 antitumor therapy,
1-3,12,13
and agrochemical
1
4
pest control. We describe herein the selective inhibi-
tory action against the mitochondrial complex I (NADH:
ubiquinone oxidoreductase) of the (+)-almuheptolide-A
(1) and its diacetate derivative (1a ). Therefore, in
addition to the new rapid and convenient method for
semisynthesis, the mechanism of action of this type of
*
To whom correspondence should be addressed. Phone: (34)
9
63.86.49.75. Fax: (34) 963.86.49.43. E-mail: dcortes@uv.es.
†
Departamento de Bioqu ´ı mica y Biolog ´ı a Molecular, Universidad
de Valencia.
‡
Universit e´ Paris-Sud.
1
0.1021/jm9706574 CCC: $15.00 © 1998 American Chemical Society
Published on Web 12/03/1998

Enantiospecific Semisynthesis of Almuheptolide-A
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 26 5159
Ta ble 1. 1D ( H and C NMR) and 2D ( H- H COSY 45, 1H-13C HMQC, and HMBC) Experiments of 1 (CDCl3, 400 MHz)
1
13
1
1
1H NMR and COSY 45
13C NMR, HMQC, and HMBC
1
1
1
2
3
position
2
δH (J , Hz)
H- H COSY 45
δC (multiplicity) HMQC ( J )
HMBC ( J and J )
H-3a, H-3b, H-4
H-3a, H-3b H-4, H-5
171.80 (C)
36.38 (CH2)
3
3
4
5
6
7
8
9
1
1
1
1
1
1
1
a
b
2.84 dd (16.0, 6.0)
2.75 dd (16.0, 6.4)
H-3b, H-4
H-3a, H-4
4.24 ddd (6.4, 6.0, 4.4) H-3a, H-3b, H-5
75.39 (CH)
80.62 (CH)
79.58 (CH)
84.72 (CH)
85.41 (CH)
140.00 (C)
H-4
H-5
H-6
H-7
H-8
H-3a, H-3b
H-3a, H-3b, H-7
4.17 dd (4.9, 4.4)
4.22 dd (4.9, 3.9)
4.08 dd (5.1, 3.9)
4.62 d (5.1)
H-4, H-6
H-5, H-7
H-6, H-8
H-7
H-8
H-10, H-14
H-7, H-8, H-11, H-13
0,14
1,13
7.45 dd (7.6, 1.9)
7.35 dt (7.6, 1.9)
7.23 dd (7.6, 1.9)
4.14 q (7.0)
1.26 t (7.0)
3.68 dq (6.8)
1.20 t (6.8)
H-11, H-13, H-12
H-10, H-14, H-12
H-10, H-14, H-11, H-13
16-CH3
15-CH2
18-CH3
126.26 (2 CH)
128.36 (2 CH)
127.76 (CH)
60.78 (CH2)
14.14 (CH3)
65.66 (CH2)
15.55 (CH3)
H-10, H-14 H-8, H-12 (C-10fH-14 and C-14fH-10)
H-11, H-13 C-11fH-13 and C-13fH-11
2
5
6
7
8
H-12
H-10, H-14
16-CH3
15-CH2
18-CH3
17-CH2
15-CH2
16-CH3
17-CH2
18-CH3
17-CH2
1
1
1
Ta ble 2. 1D ( H NMR) and 2D ( H- H COSY 45) Experiments in CDCl3 of 1a (400 MHz), 2, and 2a (250 MHz)
diacetylalmuheptolide-A (1a )
almuheptolide-B (2)
diacetylalmuheptolide-B (2a )
1
1
1
1
1
1
proton
δH (J , Hz)
H- H COSY 45
δH (J , Hz)
H- H COSY 45
δH (J , Hz)
H- H COSY 45
2
3
3
4
4
5
6
7
8
9
1
1
1
1
1
1
1
6
7
a
b
a
b
2.53 m
2.51 m
4.19 m
H-3b, H-4
H-3a, H-4
2.64 dd (16.7, 6.6)
2.47 dd (16.7, 7.2)
H-3b, H-4a, H-4b 2.52 m
H-3a, H-4a, H-4b
H-4a, H-4b
H-3a, H-3b, H-5 2.10 ddd, 2H (15.1, 7.2, 6.6) H-3a, H-3b, H-5 2.15 m
1.93 m
H-4, H-6
H-3a, H-3b, H-4b
H-3a, H-3b, H-4a, H-5
H-4a, H-4b, H-6
4.31 brd (3.9)
5.27 dd (3.9, 2.0) H-5, H-7
5.09 dd (3.5, 2.0) H-6, H-8
4.23 m
4.20 m
4.07 m
4.63 m
H-4a, H-4b, H-6 4.23 brd (4.0)
H-5, H-7
H-6, H-8
H-7
5.24 dd (4.0, 1.1) H-5, H-7
5.03 dd (3.5, 1.1) H-6, H-8
4.81 d (3.5) H-7
4.92 d (3.5)
H-7
0,14
1,13
7.48 dd (7.3, 1.7) H-11,13, H-12
7.35 dt (7.3, 1.7) H-10,14, H-12
7.30 dd (7.3, 1.7) H-10,14, H-11,13 7.32 dd (7.4, 1.2)
4.16 q (7.0)
1.27 t (7.0)
3.79 dq (6.8)
1.19 t (6.8)
7.43 dd (7.4, 1.2)
7.36 dt (7.4, 1.2)
H-11,13, H-12
H-10,14, H-12
7.44 dd (7.1, 1.8) H-11,13, H-12
7.31 dt (7.1, 1.8) H-10,14, H-12
2
5
6
7
8
H-10,14 H-11,13 7.34 dd (7.1, 1.8) H-10,14, H-11,13
16-CH3
15-CH2
18-CH3
17-CH2
4.15 q (7.0)
1.25 t (7.0)
16-CH3
15-CH2
4.14 q (7.0)
1.26 t (7.0)
16-CH3
15-CH2
-OCOCH3 2.12 s
-OCOCH3 1.94 s
2.13 s
2.00s
was indicated by an IR band at 3422 cm- and cor-
1
cytotoxic compounds is established for the first time,
which opens a guideline for potential applications.
roborated by the preparation of a diacetate derivative
(
1a ). Two ethoxyl groups in 1 were evidenced by the
Resu lts a n d Discu ssion
+
EIMS peaks m/z 278 [M - HOCH2CH3] and 232 [M -
2 HOCH2CH3] . The H NMR spectrum resonances at
δ 4.14 (q, 2H), 1.26 (t, 3H) and δ 3.68 (dq, 2H), 1.20 (t,
3H) and the C NMR spectrum resonances at δ 60.78
(CH2), 14.14 (CH3) and δ 65.66 (CH2), 15.55 (CH3)
confirmed the presence of these ethoxyl groups (Table
1). A saturated ú-lactone moiety in 1 was suggested by
+
1
Isola tion a n d Ch em istr y. (+)-Almuheptolides-A (1)
and -B (2) are members of a small class of natural
products which contain a saturated eight-membered-
ring lactone (ú-lactone). Both 1 and 2 have the same
1
3
heptolide skeleton as the bioactive natural compounds
8
3
and 4 previously isolated from G. giganteus and a
-
1
lactone system similar to 5 and 6, cytotoxic metabolites
both an IR carbonyl absorption band at 1716 cm and
isolated from marine actinomycetes belonging to the
Streptomyces genus (Figure 1).
a weak downfield quaternary carbon signal at δ 171.80
9
13
(C-2) in the C NMR spectrum. The presence of a
1
(
+)-Almuheptolide-A (1) was first isolated from G.
methylene in the ring of 1 was indicated in the H NMR
1
13
arvensis stem bark. Inspection of the 1D ( H and
NMR spectra), 2D homonuclear ( H- H COSY 45), and
D heteronuclear correlation spectroscopy (HMQC,
C
spectrum by two resonances at δ 2.84 (dd, H-3a) and
2.75 (dd, H-3b) with J 3a,3b ) 16.0 Hz and by a signal at
1
1
1
3
2
δ 36.38 (C-3) in the C NMR spectrum.
1
1
proton-detected heteronuclear multiple-quantum coher-
ence, and HMBC, proton-detected multiple-bond het-
eronuclear multiple-quantum coherence) (Table 1) re-
vealed that 1 has a heptolide skeleton (8-phenyl-2-
oxocanone) similar to that of (+)-gonioheptolides-A (3)
H- H COSY 45 spectra of 1 and 1a (Tables 1 and 2)
revealed the correlation between the methylene at C-3
and a methine at C-4 and the successive correlations of
five oxygenated methine protons (CH-4 to CH-8), as well
as with their corresponding carbons in the HMQC
spectrum (Table 1). Indeed, NOEs observed in 1a
between H-8 and the aromatic ring protons H-10 and
H-14 (Figure 2) are consistent with a phenyl group
placed at position 8, excluding the location of the
8
and -B (4). Compound 1 is an optically active, yellow-
ish oil, [R]D +11.7° (EtOH, c 1.8), with a molecular
formula C17H24O6, as was indicated by an EIMS peak
+
at m/z 324 [M] . The presence of two hydroxyl groups

5
160 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 26
Bermejo et al.
F igu r e 3. Perspective of the fully energy-minimized structure
of (+)-almuheptolide-A (1) by MM2 calculations.
Ta ble 3. 13C NMR Data in CDCl3 of 1a (100 MHz), 2, and 2a
(62.5 MHz)
δC (DEPT)
carbon
1a
2
2a
1
13
3
F igu r e 2. H- C long-range correlations ( J ) established
from HMBC of 1 and NOE of 1a .
2
3
4
5
6
7
8
9
172.00 (C)
174.50 (C)
173.08 (C)
37.34 (CH2)
74.95 (CH)
82.60 (CH)a
77.10 (CH)
82.61 (CH)a
84.85 (CH)
139.00 (C)
30.80 (CH2)
23.50 (CH2)
81.00 (CH)
79.00 (CH)
85.00 (CH)
86.00 (CH)
140.00 (C)
126.00 (2 CH)
128.50 (2 CH)
127.80 (CH)
61.40 (CH2)
14.00 (CH3)
30.88 (CH2)
23.93 (CH2)
79.78 (CH)
77.52 (CH)
83.73 (CH)
84.83 (CH)
138.87 (C)
126.24 (2 CH)
128.28 (2 CH)
127.91 (CH)
60.51 (CH2)
14.20 (CH3)
aromatic ring at other positions. Moreover, 2D long-
range correlations HMBC observed between the aro-
matic ring protons, H-10 and H-14, and the carbon
nucleus at C-8 and between H-7 and C-9 supported the
placement of the aromatic moiety at C-8 of the saturated
ú-lactone system. The combined analysis of HMBC and
HMQC data (Table 1 and Figure 2) indicated the
linkages between the constitutive parts of 1 and the
allowed resonance assignments. Figure 2 shows the
cross-peaks observed due to the three-bond ( J ) hetero-
nuclear coupling.
The relative stereochemistry of the chiral centers in
was deduced from both the H-coupling constant data
10,14
11,13
2
5
6
7
126.15 (2 CH)
128.20 (2 CH)
127.83 (CH)
60.70 (CH2)
14.18 (CH3)
67.14 (CH2)
15.58 (CH3)
1
1
1
1
3
18
6
7
-OCOCH3
169.00 (C)b
0.89 (CH3)
169.70 (C)d
20.93 (CH3)
169.69 (C)
20.67 (CH3)
c
e
2
b
d
-OCOCH3
168.50 (C)
1
1
0.90 (CH3)c
e
2
and the NOEDIFF experiments for 1a . NOEs between
H-5/H-6 and H-5/H-8 (Figure 2) were in agreement with
a cis-relationship for these three protons. The relative
configuration of 1 is therefore cis-4,5, cis-5,6, trans-6,7,
and trans-7,8, and thus, it is similar to that of the known
compounds 3 and 4.8 To observe this stereochemistry
of 1, a 3D representation of the fully energy-minimized
structure was built using a molecular modeling program
with the MM2 derived force field (Figure 3). In conclu-
sion, the novel (+)-almuheptolide-A (1) is the 4,5-
diethoxy-6,7-dihydroxy-8-phenyl-2-oxocanone (Figure 1),
where its stereochemistry (4R,5R,6R,7R,8R) is postu-
lated on the basis of the known absolute configuration
of the starting semisynthetic furanopyrones altholactone
a-e
Exchangeable values.
comparisons of the 1H NMR, 13C NMR, and COSY
spectra of 2 and its diacetate derivative (2a ) with those
of 1 and 1a suggested that these compounds had the
same plain heptolide skeleton and relative stereochem-
istry, but 2 had only one ethoxyl group at C-5 and
therefore two adjacent methylene carbons placed at 3
and 4 (Tables 2 and 3). The H- H COSY 45 spectrum
of 2 revealed the correlations between the spin reso-
nance at δ 2.10 (ddd, 2H, H-4a,4b) and the proton
signals at δ 2.64 (dd, H-3a), 2.47 (dd, H-3b), and 4.23
1
1
(
m, H-5), as well as the consecutive proton correlations
7) and etharvensin (8).6
1
(
between four methines (H-5 to H-8). Indeed, the H
NMR spectrum of 2a showed that, upon acetylation, the
chemical shifts of H-6 and H-7 moved downfield in
agreement with the location of the two hydroxyl groups
at these positions. In conclusion, (+)-almuheptolide-B
(2) is the 6,7-dihydroxy-5-ethoxy-8-phenyl-2-oxocanone,
with a similar stereochemistry to that of 1 (Figure 1).
Almuheptolide-B (2) was also isolated from G. arven-
sis as a yellowish oil, [R]D +5.6° (EtOH, c 1.6). Its
molecular weight was indicated by a peak in the EIMS
+
at m/z 280 [M] corresponding to the molecular formula
C15H20O5. The IR, UV, MS, and NMR spectra of 2 were
very similar to those of 1. The assignments and

Enantiospecific Semisynthesis of Almuheptolide-A
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 26 5161
Sch em e 1. Preparation of 1 and 8 from 7
Sch em e 2. General Procedure for Alkoxylation:
Semisynthesis of 4,5-Dialkoxylated Heptolides and
7
-Alkoxyfuranopyrones from 7
Sem isyn th esis. Because octalactin-A (5) has an
attractive structure and a potent biological activity,
several research groups have attempted its total syn-
thesis.1
5,16
Therefore, in view of the original and unusual eight-
membered-ring lactone of 1, similar to that of 5, we
decided to carry out an enantiospecific semisynthesis
of 1 starting from (+)-altholactone (7), an unsaturated
furano-R-pyrone, using ethanol and concentrated H2SO4
To corroborate this mechanistic hypothesis, we have
carried out the solvolysis using other alcohols as nu-
cleophilic agents in the acid medium, MeOH/H2SO4 and
i-PrOH/H2SO4. Thus, with compound 7, under the same
conditions, mixtures of 7a ,b or 7c,d were obtained,
respectively. Scheme 2 illustrates the general semi-
synthetic route used for the preparation of compounds
1, 8, and 7a -d .
(Scheme 1). Almuheptolide-A (1) was obtained as a
single diastereoisomer (yield 40%) when the mixture
was refluxed for 3 h, via the intermediate furanopyrone
skeleton etharvensin (8). This reaction was carried out
under a variety of conditions, such that only 8 was
This protocol constitutes a new enantiospecific method
for the preparation of this class of compounds. In
addition, we have shown that the substituted eight-
membered-ring lactone can be achieved using a simple
alkoxylation procedure involving a concerted opening
of the δ-lactone and tetrahydrofuran rings to yield a
ú-lactone by direct translactonization. On the other
hand, the natural starting material for this semisyn-
thesis, altholactone (7), is a compound isolated in large
6
obtained (yield 90%) when 7 was refluxed for 1.5 h,
while 1 was prepared in a yield of 62% when 8 was
refluxed for 5 h (Scheme 2).
In our semisynthetic plan, depicted in Scheme 1, we
envisaged that a saturated ú-lactone unit (2-oxocanone
ring) could be easily obtained from the unsaturated
6
δ-lactone 7. Formation of intermediate etharvensin (8)
suggested that the mechanism operating in this trans-
formation, and the key connection between 7 and 1
reported in this paper, involved alkoxylation of 7 by
Michael-type addition of an EtOH molecule across the
R,â-unsaturated δ-lactone double bond from the less
electron dense position (â of the CdO group). Such
addition took place under acidic conditions at the less
hindered face to afford intermediate 8. Subsequent
nucleophilic attack with a second EtOH molecule by a
SN2 mechanism would lead to an inversion of configuar-
tion of C-7a of 7 (C-5 in 1), followed by a concerted
opening of the R-pyrone and tetrahydrofuran rings. This
allowed the rotation, rearrangement, and subsequent
intramolecular eight-ring closure.
5
quantity from G. arvensis, and therefore, it is a good
material for the preparation of other related compounds.
Although several total syntheses of macrocyclic lactones
1
5-18
have been previously reported,
none of them are
easily available. This is the first report of the semisyn-
thesis of pentasubstituted eight-membered-ring lac-
tones, constituting a novel, efficient, and direct method
for the preparation of 8-phenyl-2-oxocanone compounds
from a readily available substrate.
Bioa ctivities. Almuheptolide-A (1) and its diacetate
(1a ) were initially found to be inhibitors of the inte-
grated mitochondrial electron-transport chain, mea-
sured as aerobic NADH oxidation, with similar potency,

5
162 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 26
Bermejo et al.
F igu r e 4. Titration of (+)-almuheptolide-A (1) and the
diacetate derivative (1a ) against NADH oxidase activity in
bovine heart submitochondrial particles. Control activity was
-
1
-1
0
.95 µmol‚min ‚mg .
as shown in Figure 4. Almuheptolide-A (1) showed an
IC50 of 4.4 ( 0.2 µM, whereas its diacetate (1a ) gave an
IC50 of 3.9 ( 0.2 µM. Taking into account that most of
the respiratory chain inhibitors with potential biomedi-
cal interest act within this order of magnitude on
mammalian submitochondrial particles,1
,19-21
both com-
pounds were effective inhibitors of the whole respiratory
chain. However, the NADH oxidase activity involves
all three energy-conserving enzymatic complexes of the
respiratory chain, and thus the inhibitory action of the
compounds might be affecting one or more of these
electron-transfer chain components.
To define the target enzyme within the respiratory
chain, the time course of the reduction of ferricyto-
chrome c by both NADH and succinate was followed
after addition of almuheptolide-A (1) as shown in Figure
5
. The inhibitor did not affect the succinate:cytochrome
F igu r e 5. Effect of (+)-almuheptolide-A (1) on the time-course
reduction and oxidation of cytochrome c in bovine heart
submitochondrial particles. Succinate- and NADH-induced
cytochrome c reductase activities were 0.12 and 0.15 µmol‚
c reductase activity (Figure 5A) at a concentration that
fully inhibited the NADH oxidase activity, whereas the
reduction of cytochrome c was abolished when antimycin
A (specific inhibitor of the ubiquinol:cytochrome c re-
ductase or complex III) was added. In turn, almuhep-
tolide-A (1) inhibited the reduction of ferricytochrome
c by NADH (Figure 5B), but when succinate was added,
the cytochrome c was further reduced. Finally, the
aerobic oxidation of ferrocytochrome c was not affected
by the compound (Figure 5C), but it was fully inhibited
by cyanide, a common inhibitor of the cytochrome c
oxidase or complex IV. Similar results were obtained
with the diacetate derivative (1a ).
-
1
-1
min ‚mg , respectively. Cytochrome c oxidase activity was
-
1
-1
0
.42 µmol‚min ‚mg . (+)-Almuheptolide-A was added at 15
µM, antimicyn A at 5 µM, and cyanide at 2 mM.
activity assay which is added at high concentration with
respect to the natural content of the endogenous
2
4
ubiquinone.
The most recent mechanistic hypotheses on mito-
chondrial complex I function have established three
ubiquinone binding sites within the transmembrane
2
5-27
segment of the complex.
Although not completely
These results indicate that the heptolides act prima-
rily by inhibiting the mitochondrial complex I. To
confirm the target enzyme of these compounds, indi-
vidual activity of respiratory chain enzymes was mea-
sured in the presence of almuheptolide-A (1). Neither
succinate:ubiquinone reductase (complex II) nor
ubiquinol:cytochrome c reductase (complex III) were
affected by the compound, even at relatively high
concentration (more than 97% activity with more than
proved, inhibitors of the enzyme could act by binding
independently to one of these sites. Inhibition kinetic
pattern is useful to classify the complex I inhibi-
1
2,21,28
tors.
Figure 6A shows that almuheptolide-A
behaved as a noncompetitive inhibitor with regard to
the ubiquinone analogue substrate, and thus, it mim-
icked the rotenone behavior. Although most of the
noncompetitive inhibitors of the complex I act by
overlapping the rotenone binding site, some inhibitors
could act at a different site with the same inhibitor
1
00 µM). Indeed, almuheptolide-A showed a selective
2
5
inhibition of the NADH:ubiquinone reductase (complex
I) with an IC50 of 32 ( 3 µM. This loss of potency or
weakened affinity with respect to the integrated NADH
kinetics. To determine the mutual exclusivity between
both inhibitors, we have done the titration of one of
them in the presence of the other to reveal the kinetic
1
2,28
oxidase assay is commonly observed with many complex
displacement.
Figure 6B shows Dixon plots ob-
I inhibitors,2
0,22,23
and it is probably due to a partial
tained by titration of almuheptolide-A in the presence
of rotenone. As observed in the graphic, rotenone
competition with the ubiquinone analogue used for the

Enantiospecific Semisynthesis of Almuheptolide-A
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 26 5163
of complex I are expanded with a new interesting type
of molecular structures as the heptolides described
herein.
Con clu sion
In this paper we have described for the first time a
fast, easy, and stereospecific semisynthesis method for
a high-yield obtention of pentasubstituted eight-mem-
bered-ring lactones (1,7a ,c). One of them, a new
8
-phenyl-2-oxocanone (almuheptolide-A, 1) isolated from
G. arvensis, was found to be a noncompetitive inhibitor
of the mammalian respiratory chain complex I. Inhibi-
tion of the transmembrane segment (ubiquinone region)
of the complex I has been correlated with oxidative
damage of the cell. Electrons trapped in the peripheral
part of the complex are shunted to radical oxygen
2
9-32
species formation,
and thus, cellular damage is
produced at two levels: ATP depletion by inhibition of
the oxidative phosphorylation and free-radical attack
to mitochondrial components. The high metabolic rate
of tumoral cells might explain their sensitivity to
mitochondrial electron-transfer chain inhibitors. Al-
though some complex I inhibitors such as rotenoids,
3
,12,28
piericidins, and Annonaceous acetogenins
are highly
potent (with IC50 1 order of magnitude below), the
antitumor trials have not been successful due to the
extreme toxicity of these compounds. Instead, the
current tendency for searching for new antitumoral
agents targeting complex I is moving toward less potent
3
3
inhibitors which act at the micromolar range in in
vitro assays, an intermediate potency matched by al-
muheptolide-A.
F igu r e 6. Inhibition kinetics and kinetic displacement of (+)-
almuheptolide-A (1) and rotenone. (A) Reciprocal plots of the
NADH:ubiquinone oxidoreductase activity in the presence of
This new 8-phenyl-2-oxocanone, isolated first from the
natural source G. arvensis, is easily obtained by the fast,
stereospecific, and efficient method for the semisynthe-
sis of pentasubstituted eight-membered-ring lactones
described herein. Therefore, almuheptolide-A is a very
interesting molecule that might be exploited in the
future for antitumor chemotherapy, biomedical re-
search, and insect control.
(+)-almuheptolide-A (45 µM) and rotenone (18.5 nM). (B)
Dixon plots of (+)-almuheptolide-A titrated at different fixed
concentrations of rotenone (only the lineal part of the plot is
shown).
modified the Ki of the new inhibitor (x-axis intercept),
and thus, both compounds compete by the same binding
site. Therefore, we can assess that almuheptolide-A
selectively binds the energy-coupling site of the complex
Exp er im en ta l Section
I, namely, the “rotenone site”,22 site B, or site PI.
27
30
Gen er a l In str u m en ta tion . Optical rotations were deter-
mined on a Perkin-Elmer 241 polarimeter. IR spectra were
run in film using NaCl plates on a Perkin-Elmer 843 spec-
trometer. UV spectra were obtained on a Perkin-Elmer
lambda computer 15 UV/vis spectrophotometer. Mass spectra
were recorded with a VG Auto Spec Fisons spectrometer, and
the liquid secondary ion mass spectrum (LSIMS) was obtained
The initial hypothesis of the present work was that
the newly synthesized compound almuheptolide-A might
be a complex III inhibitor due to its resemblance with
the antimycin A molecule. Surprisingly, it did not
inhibit this segment of the respiratory chain. Close
scrutiny of the chemical structure of almuheptolide-A
shows that it lacks the functional groups that bear the
antimycin A and its derivatives that have been recently
characterized as involved in the binding to the Qn
pocket of the complex III.10 Therefore, this is additional
evidence of the relevance of the colateral chemical
groups to drive selective inhibition of respiratory com-
plexes.
1
13
by fast ion bombardment. H NMR (250 and 400 MHz),
C
NMR, and DEPT (62.5 and 100 MHz) spectra were recorded
on Bruker AC-250, Varian Unity-300, and Varian Unity-400
instruments. Silica gel TLC plates were observed under UV
light (254 nm) and after spraying with anisaldehyde-sulfuric
acid. Preparative TLC was performed using silica gel (0.5 mm
plates).
P la n t Ma ter ia l. G. arvensis Scheff. (Annonaceae) was
collected in the National Park of Varirata, located in the
Central Province of Papua New Guinea. A voucher specimen
was deposited in the herbarium of the University of Papua
New Guinea.
Extr a ction a n d Isola tion . Dried and powdered stem bark
of G. arvensis (368 g) was macerated with MeOH at room
temperature. The concentrated MeOH extract was partitioned
between hexane (extract A) and 50% aqueous MeOH. The
2 2
aqueous extract was fractionated successively into CH Cl and
EtOAc (extracts B and C, respectively). Extract B (7 g) was
applied to a silica gel 60H column (Merck 7736) and eluted
Instead, we have found that almuheptolide-A is a very
specific inhibitor of the site B of the complex I. Accord-
ing to previous studies, this ubiquinone binding site of
the complex should be wide enough to accommodate a
great variety of chemical structures acting as inhibi-
1
9,22
tors.
Note that neither eight-membered-ring lac-
tones nor related structures have ever been described
as complex I inhibitors up to date, and thus, inhibitors

5
164 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 26
Bermejo et al.
with CH
phy of some fractions afforded 1 (5 mg) and 2 (8 mg).
+)-Alm u h ep tolid e-A (1): C17 ; [R] +11.7° (EtOH, c
2
Cl
2
-EtOAc (60:40). Repeated column chromatogra-
3422, 2932, 2103, 1735, 1635, 1493, 1452, 1381, 1239, 1198,
+
1156, 1096, 1053, 919, 760, 699; LSIMS m/z (%) [MH] 297
+
(
H
24
O
6
D
(100), 265 [MH - HOCH
3
]
(23); EIMS m/z (%) 264 [M -
3
+
+ 1
1
ν
1
.8); UV λmax EtOH nm (log ꢀ) 229 (2.82) and 257 (2.35); IR
HOCH
3
]
(2), 230 [MH - OCH
2 3
- 2H O] ; H NMR (CDCl ,
-
1
max (film) cm 3422, 3059, 3026, 2974, 2926, 1716, 1602, 1492,
250 MHz) δ 7.45 (d, H-10,14), 7.35 (t, H-11,13), 7.29 (d, H-12),
449, 1370, 1282, 1182, 1085, 1042, 857, 757, 699; EIMS m/z
4.63 (d, H-8), 4.23 (dd, H-4), 4.20 (dd, H-6), 4.14 (brd, H-5),
+
+
+
(
(
%) 324 [M] , 306 [M - H
2
O] (8), 288 [M - 2H
2
O] (3), 279
4.09 (dd, H-7), 3.71 (s, 3H, 15-CH
3 3
), 3.49 (s, 3H, 16-CH ), 2.77
+
22), 278 [M - HOCH
2
CH
3
] (64), 262 (30), 260 [M - HOCH
2
-
(dd, H-3b), 2.86 (dd, H-3a); J 3a,3b ) 16.0 Hz, J 3a/3b,4 ) 6.3 Hz,
+
+
CH
3
- H
2
O] (20), 232 [M - 2HOCH
2
CH
3
] (30), 173 (34), 162
J 4,5 ) 4.5 Hz, J 5,6 ) 5.0 Hz, J 6,7 ) 4.0 Hz, J 7,8 ) 5.1 Hz, J 10,11
+
(88), 145 (85), 133 (100), 117 (85), 103 (89), 97 [C
5
H
5
O
2
] (72),
) J 11,12 ) J 12,13 ) J 13,14 ) 6.9 Hz, J 10,12 ) J 11,13 ) J 12,14 ) 1.5
1
13
13
9
1 (92), 77 (66); H and C NMR (CDCl
3
, 400 and 100 MHz)
3
Hz; C NMR (CDCl , 100 MHz) δ 172.05 (C-2), 139.76 (C-9),
Table 1.
+)-6,7-Dia cetyla lm u h ep tolid e-A (1a ). 1 (3.9 mg) was
treated with pyridine (1 mL) and Ac O (1.5 mL). The reaction
mixture was stirred for 12 h at room temperature. After the
usual workup the diacetate 1a (4.0 mg, 82%) was obtained:
128.39 (C-11,13), 127.80 (C-12), 126.20 (C-10,14), 85.39 (C-8),
4.67 (C-7), 80.59 (C-5), 79.49 (C-6), 76.00 (C-4), 57.75 (15-
CH ), 51.88 (16-CH ), 35.50 (3-CH ).
Com p ou n d 7b: C14 ; [R] -69.2° (EtOH, c 1.3); UV
8
(
3
3
2
2
H
16
O
5
D
λmax EtOH nm (log ꢀ) 217 (2.7) and 257 (1.9); IR νmax (film)
-
1
C
ꢀ
1
21
H
28
O
8
; [R]
) 218 (3.2) and 260 (2.4); IR νmax (film) cm 2975, 2925, 1742,
448, 1368, 1232, 1093, 1038, 940, 758, 698; LSIMS m/z (%)
D
+22.31° (EtOH, c 1.3); UV λmax EtOH nm (log
cm 3429, 2928, 2108, 1724, 1639, 1492, 1437, 1368, 1282,
-1
+
1200, 1171, 1084, 1044, 759, 700; EIMS m/z (%) [M] 264 (7),
+
+
232 [M - HOCH
3
]
(3), 215 [M - OCH
3
- H
O] (100), 107 (29), 105 (40), 97
(20), 91 (89), 77 (42); H NMR (CDCl , 250 MHz) δ 7.38-7.32
(m, H-9 to H-13), 4.88 (dd, H-3a), 4.70 (d, H-2), 4.39 (brt, H-7a),
4.28 (brt, H-3), 3.93 (dd, H-7), 3.46 (s, 3H, CH ), 2.86 (dd, H-6b),
2.73 (dd, H-6a); J 2,3 ) 6.0 Hz, J 3,3a ) 2.1 Hz, J 3a,7a ) 4.5 Hz,
7a,7 ) 4.0 Hz, J 7,6b ) 3.8 Hz, J 7,6a ) 5.7 Hz, J 6a,6b ) 16.7 Hz;
13
2
O] (3), 177 (3),
+
+
+
+
[MH] 409 (100); CIMS m/z (%) [MH] 409 (50), 408 [M] (20),
162 (20), 144 (34), 133 [C H
9 9
+
+
1
3
2
49 [MH - HOCOCH
3
]
(36), 289 [MH - 2HOCOCH
3
]
(12),
(65), 107 (69); H and
, 400 and 100 MHz) Tables 2 and 3.
+)-Alm u h ep tolid e-B (2): C15 ; [R] +5.6° (EtOH, c
.6); UV λmax EtOH nm (log ꢀ) 227 (2.65) and 260 (2.0); IR νmax
3
+
2 3
- HOCH CH ]
1
42 [M - 2HOCOCH
3
1
3
C NMR (CDCl
(
3
3
H
20
O
5
D
1
J
-1
(film) cm 3422, 2921, 1708, 1451, 1370, 1262, 1035, 758, 699;
C NMR (CDCl
128.67 (2 CH), 128.36 (CH), 126.04 (2 CH), 86.91 (C-3a), 86.05
(C-2), 83.56 (C-3), 75.25 (C-7a), 74.53 (C-7), 57.32 (14-CH ),
32.56 (6-CH ).
3
, 62.5 MHz) δ 169.13 (C-5), 138.07 (C-8),
+
+
+
EIMS m/z (%) 280 [M] (1), 279 [M - 1] (2), 262 [M - H
2
O]
+
+
(
[
(
(
4), 244 [M - 2H
M - OCH CH
2), 167 [M - 2H
2
O] (19), 234 [M - HOCH
2
CH
3
]
(16), 217
3
+
+
2
3
- H
2
2
O] (11), 198 [M -2H
2
O - HOCH
2
CH
3
]
2
+
O - Ph] (5), 107 (73), 97 (16), 91 (100), 77
3. Sem isyn th esis of Com p ou n d 7c. A similar procedure
(i-PrOH/H SO , 9 h) and workup afforded 7c (20 mg, 0.057
1
13
55); H and C NMR (CDCl , 250 and 62.5 MHz) Tables 2
3
2
4
and 3.
+)-6,7-Dia cetyla lm u h ep tolid e-B (2a ). 2 (7.9 mg) was
treated with pyridine (2 mL) and Ac O (2.5 mL). The reaction
mixture was stirred for 12 h at room temperature. After the
usual workup the diacetate 2a (7.1 mg, 69%) was obtained:
mmol, 16.5%) and 7d as synthetic intermediate (40 mg, 0.137
mmol, 40.0%).
(
2
Com p ou n d 7c: C H O ; [R] +17.5° (EtOH, c 4.0); UV λ
1
9
28
6
D
max
EtOH nm (log ꢀ) 225 (2.7) and 257 (2.5); IR νmax (film) cm-1
3423, 3405, 2975, 2927, 2084, 1708, 1640, 1492, 1450, 1372,
C
19
H
24
O
7
; [R]
D
+15.0° (EtOH, c 2.0); UV λmax EtOH nm (log ꢀ)
1243, 1196, 1104, 1042, 824, 757, 699; LSIMS m/z (%) [MH]
+
-
1
+
1
2
1
26 (3.0) and 256 (3.0); IR νmax (film) cm 2918, 2341, 2327,
740, 1720, 1541, 1449, 1369, 1224, 1117, 1037, 698; LSIMS
353 (100), 233 [MH - 2HOCH(CH ) ] (15); H NMR (CDCl ,
3
2
3
400 MHz) δ 7.46 (d, H-10,14), 7.34 (t, H-11,13), 7.28 (d, H-12),
4.63 (d, H-8), 4.36 (dd, H-4), 4.26 (dd, H-6), 4.21 (brd, H-5),
4.09 (dd, H-7), 3.87 (sept, 15-CH), 5.02 (sept, 18-CH), 2.88 (dd,
3
+
+
m/z (%) 365 [MH] (100); EIMS m/z (%) 365 [MH] (2), 319
+
+
[
MH - HOCH
2
CH
3
] (54), 260 [M - HOCH
2
CH
3
- OCOCH ]
3
+
(50), 245 [MH - 2HOCOCH
3
]
(98), 244 (85), 243 (76), 231
H-3a), 2.71 (dd, H-3b), 1.29-1.23 (ddd, 19,20-CH ), 1.22-1.19
(30), 217 (56), 215 (72), 199 (64), 128 (60), 125 (54), 115 (51),
(ddd, 16,17-CH ); J
) 16.3 Hz, J
) 6.1 Hz, J ) 10.1
3
3a,3b
3a/3b,4
4,5
1
13
1
2
05 (83), 99 (47), 91 (70), 77 (47); H and C NMR (CDCl
50 and 62.5 MHz) Tables 2 and 3.
3
,
Hz, J 5,6 ) 4.9 Hz, J 6,7 ) 3.3 Hz, J ) 5.3 Hz, J
) J 11,12
)
7,8
10,11
) 1.7 Hz; 13
J 12,13 ) J 13,14 ) 7.1 Hz, J
10,12
) J
11,13
) J
12,14
C
Gen er a l P r oced u r e for Hep tolid e Sk eleton P r ep a r a -
tion : 1. Sem isyn th esis of (+)-Alm u h ep tolid e-A (1). To
an ethanol solution (10 mL) of altholactone (7; 60 mg, 0.258
3
NMR (CDCl , 100 MHz) δ 171.81 (C-2), 140.00 (C-9), 128.38
(C-11,13), 127.90 (C-12), 126.20 (C-10,14), 84.87 (C-8), 84.80
(C-7), 80.43 (C-5), 79.75 (C-6), 77.80 (C-4), 68.21 (18-CH), 68.20
mmol) was added dropwise at 0 °C concentrated H
.5 mL). After the mixture was stirred and refluxed for 3 h,
water was added and the reaction mixture was extracted into
CH Cl . The organic solution after usual workup was purified
by silica gel 60H column chromatography (eluting with
CHCl -EtOAc (70:30)), affording 1 (34 mg, 0.105 mmol, 40%)
and 8 as synthetic intermediate (20 mg, 0.072 mmol, 28%).
Compound 1 was obtained in a yield of 62% when 8 was
refluxed for 5 h and was found to be identical in all respects
2
SO
4
(96%,
(15-CH), 23.23 (16,17-CH
Com p ou n d 7d : C16
EtOH nm (log ꢀ) 219 (2.9) and 257 (2.3); IR νmax (film) cm
3
), 21.78 (19,20-CH
3 2
), 37.17 (3-CH ).
1
H
20
O
5
; [R] -3.1° (EtOH, c 1.9); UV λmax
D
-1
2
2
3
1
417, 3031, 2970, 2923, 2124, 1737, 1635, 1493, 1451, 1380,
236, 1174, 1142, 1079, 1054, 973, 924, 855, 759, 700; EIMS
+
+
2
3
3
m/z (%) [M] 292 (3), 232 [M - HOCH(CH ) ] (3), 215 [M -
OCH(CH
1
2
H-2), 4.30 (brt, H-7a), 4.25 (td, H-3), 4.19 (dd, H-7), 3.80 (sept,
4-CH), 2.81 (dd, H-6b), 2.63 (dd, H-6a), 1.18 (d, 15-CH ), 1.16
+
+
3
)
2
- H
15 (9.5), 107 (15), 105 (15), 91 (45), 77 (12); H NMR (CDCl
50 MHz) δ 7.33-7.30 (m, H-9 to 13), 4.87 (dd, H-3a), 4.68 (d,
2 9 9
O] (1), 162 (53), 133 [C H O] (100), 120 (16),
1
3
,
D
to the previously isolated (+)-almuheptolide-A ([R] , NMR, MS
data).
1
3
Com p ou n d 1 ((+)-Alm u h ep tolid e-A): see extraction and
isolation.
Com p ou n d 8 ((-)-Eth a r ven sin ): C15
EtOH, c 2.0); IR (film) νmax 3416 (OH), 1740 (CdO); HREIMS
(d, 16-CH ); J ) 5.7 Hz, J 3,3a ) 1.6 Hz, J 3a,7a ) 4.4 Hz, J
3
2,3
7a,7
) 4.0 Hz, J 7 ) 3.7 Hz, J
,6b
7,6a
) 5.2 Hz, J
6a,6b
) 16.6 Hz; 13C
H
18
O
5
; [R]
D
-6.5°
3
NMR (CDCl , 62.5 MHz) δ 169.28 (C-5), 138.32 (C-8), 128.55
(2 CH), 128.15 (2 CH), 125.95 (CH), 87.29 (C-3a), 86.10 (C-2),
83.33 (C-3), 75.87 (C-7a), 70.99 (C-7), 70.09 (14-CH), 33.63 (6-
(
+
m/z (%) [M] 278.1148 (calcd 278.1154 for C15
H
18
O
5
) (8), [M -
) (12), 133.0653
O) (100); H NMR (CDCl , 400 MHz),
, 100 MHz), COSY 45, HMQC, and other
physical and chemical data, see ref 6.
. Sem isyn th esis of Com p ou n d 7a . A similar procedure
MeOH/H SO , 6 h) and workup afforded 7a (24 mg, 0.080
+
HOEt] 232.0740 (calcd 232.0735 for C13
H
12
O
4
2 3 3
CH ), 22.41 (15-CH ), 22.41 (16-CH ).
1
(
calcd 133.0653 for C
C NMR (CDCl
9
H
9
3
Bioch em ica l Meth od s. Antimycin A, decylubiquinone,
and other biochemical reagents were purchased from Sigma
Chemical Co. (St. Louis, MO). Stock solutions (15 mM in
absolute ethanol) of the compounds were prepared and kept
in the dark at -20 °C. Appropriate dilutions (3-6 mM) were
made before the experiments. Inverted submitochondrial
particles (SMP) from beef heart were obtained by extensive
ultrasonic disruption of frozen-thawed mitochondria to pro-
duce open membrane fragments where permeability barriers
1
3
3
2
(
2
4
mmol, 32%) and 7b as synthetic intermediate (24 mg, 0.09
mmol, 36%).
Com p ou n d 7a : C15
EtOH nm (log ꢀ) 217 (3.2) and 257 (2.02); IR νmax (film) cm
H
20
O
6
; [R] +15° (EtOH, c 0.8); UV λmax
D
-
1

Enantiospecific Semisynthesis of Almuheptolide-A
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 26 5165
to substrates were lost.24 Beef heart SMP were diluted to 0.5
mg/mL in 250 mM sucrose, 10 mM Tris-HCl buffer, pH 7.4,
and treated with 0.3 mM NADH to activate complex I before
starting experiments. The enzymatic activities were assayed
at 22 °C in 50 mM potassium phosphate buffer, pH 7.4, 1 mM
EDTA with the SMP diluted to 6 µg/mL in the cuvette.
(8) Fang, X. P.; Anderson, J . E.; Qiu, X. X.; Kozlowski, J . F.; Chang,
C. J .; McLaughlin, J . L. Gonioheptolides-A and -B: novel eight-
membered-ring lactones from Goniothalamus giganteus (An-
nonaceae). Tetrahedron 1993, 49, 1563-1570.
1
2
(
9) Tapiolas, D. M.; Roman, M.; Fenical, W.; Stout, T. J .; Clardy, J .
Octalactins-A and -B: cytotoxic eight-membered-ring lactones
from a marine bacterium, Streptomyces sp. J . Am. Chem. Soc.
1
991, 113, 4682-4683.
NADH oxidase was measured as the aerobic oxidation of
(
(
(
10) Miyoshi, H.; Tokutake, N.; Imaeda, Y.; Akagi, T.; Iwamura, H.
A model of antimycin A binding based on structure-activity
studies of synthetic antimycin A analogues. Biochim. Biophys.
Acta 1995, 1229, 149-154.
7
5 µM NADH in the absence of the quinone substrate and
other inhibitors of the respiratory chain. NADH:ubiquinone
oxidoreductase was measured with 75 µM NADH and 30 µM
decylubiquinone (DB) as soluble short-chain ubiquinone ana-
logue in the presence of 2 µM antimycin and 2 mM potassium
cyanide to block any reaction downstream of the complex I.34
Cytochrome c reduction by both NADH and succinate and the
cytochrome c oxidase activity were measured as previously
11) Ramsay, R. R.; Salach, J . I.; Dadgar, J .; Singer, T. P. Inhibition
of mitochondrial NADH dehydrogenase by pyridine derivatives
and its possible relation to experimental and idiopathic parkin-
sonism. Biochem. Biophys. Res. Commun. 1986, 135, 269-275.
12) Degli Esposti, M.; Ghelli, A.; Ratta, M.; Cortes, D.; Estornell, E.
Natural substances (acetogenins) from the family Annonaceae
are powerful inhibitors of mitochondrial NADH dehydrogenase
4
,35
described.
Ubiquinol:cytochrome c reductase (complex III)
(Complex I). Biochem. J . 1994, 301, 161-167.
and succinate:ubiquinone reductase (complex II) activities
were assayed in the conditions previously repored.36
(13) Oberlies, N. H.; Croy, V. L.; Harrison, M. L.; McLaughlin, J . L.
The Annonaceous acetogenin bullatacin is cytotoxic against
multidrug-resistant human mammary adenocarcinoma cells.
Cancer Lett. 1997, 115, 73-79.
Inhibitor titrations were made as previously described in
3
,12
detail.
Data from four titrations in the same conditions
(
14) Hollingworth, R. M.; Ahammadsahib, K. I.; Gadelhak, G.;
McLaughlin, J . L. New inhibitors of complex I of the mitochon-
drial electron transport chain with activity as pesticides. Bio-
chem. Soc. Trans 1994, 22, 230-233.
were pulled and fitted for graphics. The inhibitory concentra-
tion 50 (IC50) was taken as the final compound concentration
in the assay cuvette that yielded 50% of the initial activity.
Data from individual titrations were used to assess the means
and standard deviations. Inhibition kinetics were evaluated
against the NADH:ubiquinone oxidoreductase activity by
varying the concentration of the ubiquinone analogue at a fixed
(15) Andrus, M. B.; Argade, A. B. Synthesis of octalactin lactone and
side chain. Tetrahedron Lett. 1996, 37, 5049-5052.
(
(
(
16) Kodama, M.; Matsushita, M.; Terada, Y.; Takeuchi, A.; Yoshio,
S.; Fukuyama, Y. Enantioselective synthesis of octalactin-A.
Chem. Lett. 1997, 117-118.
1
2,28
concentration of the compound.
Kinetic displacement
17) McNaughton-Smith, G. A.; Taylor, R. J . H. The preparation of
between inhibitors was evaluated by the Dixon plot using
5
9
-substituted ∆ -octenolides: synthesis of 8-deoxy-pseudo-as-
2
8
decylbenzoquinone as the complex I substrate.
cidiatrienolide-C. Tetrahedron 1996, 52, 2113-2124.
18) Doyle, M. P.; Protopopova, M. N. Macrocyclic lactones from
dirhodium (II)-catalyzed intramolecular cyclopropanation and
carbon-hydrogen insertion. J . Am. Chem. Soc. 1995, 117, 7281-
7282.
Ack n ow led gm en t. This research was supported by
the Spanish Direcci o´ n General de Investigaci o´ n Cien-
t ´ı fica y T e´ cnica (DGICYT) under Grant SAF 97-0013.
We are grateful to Mr. P. Wanganigi and Mr. Max
Kuduk of the Biology Department of the University of
Papua New Guinea for identification of the plant and
Prof. Kundar S. Rao of the University of Papua New
Guinea for help in obtaining the plant material. We are
also grateful to Prof. M. M. Midland for allowing us to
use his PC Model version 2.0 (PC Model, Serena
Sofware, Box 3076, Bloomington, IN 47402-3076). We
also thank the Spanish Ministerio de Educaci o´ n y
Cultura for the predoctoral fellowship grant to J . R.
Tormo.
(
19) Miyoshi, H.; Inoue, M.; Okamoto, S.; Ohshima, M.; Sakamoto,
K.; Iwamura, H. Probing the ubiquinone reduction site of
mitochondrial complex I using novel cationic inhibitors. J . Biol.
Chem. 1997, 272, 16176-16183.
20) Gluck, M. R.; Krueger, M. J .; Ramsay, R. R.; Sablin, S. O.; Singer,
T. P.; Nicklas, W. J . Characterization of the inhibitory mecha-
nism of 1-methyl-4-phenylpyridinium and 4-phenylpyridine
analogues in inner membrane preparations. J . Biol. Chem. 1994,
(
2
69, 3167-3174.
(
21) Friedrich, T.; Van Heek, P.; Leif, H.; Ohnishi, T.; Forche, E.;
Kunze, B.; J ansen, R.; Trowitzsch-Kienast, W.; H o¨ fle, G.; Re-
ichenbach, H.; Weiss, H. Two binding sites of inhibitors in
NADH: ubiquinone oxidoreductase (complex I). Relationship of
one site with the ubiquinone-binding site of bacterial glucose:
ubiquinone oxidoreductase. Eur. J . Biochem. 1994, 219, 691-
6
98.
(
22) Singer, T. P.; Ramsay, R. R. The reaction sites of rotenone and
ubiquinone with mitochondrial NADH dehydrogenase. Biochim.
Biophys. Acta 1994, 1187, 198-202.
23) Andreani, A.; Rambaldi, M.; Leoni, A.; Locatelli, A.; Ghelli, A.;
Ratta, M.; Benelli, B.; Degli Esposti, M. Thienylimidazo[2,1-b]-
thiazoles as inhibitors of mitochondrial NADH dehydrogenase.
J . Med. Chem. 1995, 38, 1090-1097.
(24) Fato, R.; Estornell, E.; Di Bernardo, S.; Pallotti, F.; Parenti-
Castelli, G.; Lenaz, G. Steady-state kinetics of the reduction of
coenzyme Q analogues by complex I (NADH:ubiquinone oxi-
doreductase) in bovine heart mitochondria and submitochondrial
particles. Biochemistry 1996, 35, 2705-2716.
Refer en ces
(
(
1) Zafra-Polo, M. C.; Figad e` re, B.; Gallardo, T.; Tormo, J . R.; Cortes,
D. Natural acetogenins from Annonaceae, synthesis and mech-
anisms of action. Phytochemistry 1998, 48, 1087-1117.
(
2) Cav e´ , A.; Figad e` re, B.; Laurens, A.; Cortes, D. Acetogenins from
Annonaceae. In Progress in the Chemistry of Organic Natural
Products; Herz, W., Kirby, G. W., Moore, R. E., Steglich, W.,
Tamm, Ch., Eds.; Springer-Verlag: New York, 1997; pp 81-
2
88.
(
(
(
(
3) Gonz a´ lez, M. C.; Tormo, J . R.; Bermejo, A.; Zafra-Polo, M. C.;
Estornell, E.; Cortes, D. Rollimembrin, a novel acetogenin
inhibitor of mammalian mitochondrial complex I. Bioorg. Med.
Chem. Lett. 1997, 7, 1113-1118.
(25) Degli Esposti, M. Inhibitors of NADH-ubiquinone reductase: an
overview. Biochim. Biophys. Acta 1998, 1364, 222-235.
(26) Brandt, U. Proton-translocation by membrane-bound NADH:
ubiquinone-oxidoreductase (complex I) through redox-gated
ligand conduction. Biochim. Biophys. Acta 1997, 1318, 79-91.
(27) Degli Esposti, M.; Ghelli, A. The mechanism of proton and
electron transport in mitochondrial complex I. Biochim. Biophys.
Acta 1994, 1187, 116-120.
(28) Estornell, E.; Tormo, J . R.; Cortes, D. Cherimolin-1, new selective
inhibitor of the first energy-coupling site of the NADH: ubiquino-
ne oxidoreductase (complex I). Biochem. Biophys. Res. Commun.
1997, 240, 234-238.
(29) Cornelissen, J .; Van Kuilenburg, A. B. P.; Vo uˆ te, P. A.; Van
Gennip, A. H. The effect of the neuroblastoma-seeking agent
meta-iodobenzylguanidine (MIGB) on NADH-driven superoxide
formation and NADH-driven lipid peroxidation in beef heart
submitochondrial particles. Eur. J . Cancer 1997, 33, 421-424.
(30) Turrens, J . F.; Boveris, A. Generation of superoxide anion by
the NADH dehydrogenase of bovine heart mitochondria. Bio-
chem. J . 1980, 191, 421-427.
4) Zafra-Polo, M. C.; Gonz a´ lez, M. C.; Tormo, J . R.; Estornell, E.;
Cortes, D. Polyalthidin: new prenylated benzopyran inhibitor
of the mammalian mitochondrial respiratory chain. J . Nat. Prod.
1
996, 59, 913-916.
5) Bermejo, A.; Lora, M. J .; Bl a´ zquez, M. A.; Rao, K. S.; Cortes, D.;
Zafra-Polo, M. C. (+)-Goniotharvensin, a novel styryl-lactone
from the stem bark of Goniothalamus arvensis. Nat. Prod. Lett.
1
995, 7, 117-122.
6) Bermejo, A.; Bl a´ zquez, M. A.; Serrano, A.; Zafra-Polo, M. C.;
Cortes, D. Preparation of 7-alkoxylated furano-pyrone skel-
etons: Semisynthesis of (-)-etharvensin, a new styryl-lactone
from Goniothalamus arvensis. J . Nat. Prod. 1997, 60, 1338-
1
340.
(7) Bermejo, A.; Bl a´ zquez, M. A.; Rao, K. S.; Cortes, D. Styryl-
pyrones from Goniothalamus arvensis. Phytochemistry 1998, 47,
1
375-1380.

5
166 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 26
Bermejo et al.
(
31) Degli Esposti, M.; Ngo, A.; Ghelli, A.; Benelli, B.; Carelli, V.;
McLennan, H.; Linnane, A. W. The interaction of Q analogues,
particularly hydroxydecyl benzoquinone (idebenone), with the
respiratory complexes of heart mitochondria. Arch. Biochem.
Biophys. 1996, 330, 395-400.
(34) Estornell, E.; Fato, R.; Pallotti, F.; Lenaz, G. Assay conditions
for the mitochondrial NADH: coenzyme Q oxidoreductase. FEBS
Lett. 1993, 332, 127-131.
35) Estornell, E.; Fato, R.; Catelluccio, C.; Cavazzoni, M.; Parenti-
Castelli, G.; Lenaz, G. Saturation kinetics of coenzyme Q in
NADH and succinate oxidation in beef heart mitochondria.
FEBS Lett. 1992, 311, 107-109.
(36) Estornell, E.; Tormo, J . R.; Barber, T. A deficiency in respiratory
complex I in heart mitochondria from vitamin A-deficient rats
is counteracted by an increase in coenzyme Q. Biochem. Biophys.
Res. Commun. 1997, 233, 451-454.
(
(
32) Turrens, J . F. Superoxide production by the mitochondrial
respiratory chain. Biosci. Rep. 1997, 17, 3-8.
(
33) Oberlies, N. H.; Chang, C.; McLaughlin, J . R. Structure-activity
relationships of diverse Annonaceous acetogenins against mul-
tidrug resistant human mammary adenocarcinoma (MCF-7/Adr)
cells. J . Med. Chem. 1997, 40, 2102-2106.
J M9706574