Semisynthesis of new tetrahydrofuranic alkyl ester and furano-pyrone derivatives as inhibitors of the mitochondrial complex I

Article abstract of DOI:10.1016/S0040-4020(01)01234-0

Methoxymethylation of altholactone (1) led to the corresponding O-methoxymethyl derivative (3) in addition to the unexpected 6,7-dihydro-7-methoxy analogue (4), and two original tetrahydrofuranic (THF) alkyl esters (5,6). Moreover, when we accomplished a new method for the preparation of the furano-pyrone goniofupyrone (7) through 7-hydroxylation of 1 in acid medium, a minor compound (8) with an identical skeleton to that of compounds 5 and 6 was identified. Careful examination of the published spectral data of the reported styryl-lactones with an heptolide skeleton reveals that those structures possess also a THF alkyl ester skeleton. The revision of those structures was confirmed by chemical correlation. All altholactone derivatives assayed proved to be specific inhibitors of the mitochondrial complex I.

Full text of DOI:10.1016/S0040-4020(01)01234-0

TETRAHEDRON
Pergamon
Tetrahedron 58 (2002) 1335±1342
Semisynthesis of new tetrahydrofuranic alkyl ester and furano-
pyrone derivatives as inhibitors of the mitochondrial complex I
a
b
c
a
d
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Eva Peris, Adrien Cave, Ernesto Estornell, M. Carmen Zafra-Polo, Bruno Figadere,
Diego Cortesa and Almudena Bermejoa,p
a
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Laboratorio de Farmacognosia, Departamento de Farmacologõa, Farmacognosia y Farmacodinamia, Facultad de Farmacia,
Universidad de Valencia, 46100 Burjassot, Valencia, Spain
Faculte de Pharmacie, Centre de Biochimie Structurale, 34060 Montpellier cedex 1, France
b
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Departamento de Bioquõmica y Biologõa Molecular, Facultad de Farmacia, Universidad de Valencia, 46100 Burjassot, Valencia, Spain
d
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Laboratoire de Pharmacognosie, Faculte de Pharmacie, Universite Paris-Sud, 92296 Chatenay-Malabry, France
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Received 17 September 2001; revised 22 November 2001; accepted 12 December 2001
AbstractÐMethoxymethylation of altholactone (1) led to the corresponding O-methoxymethyl derivative (3) in addition to the unexpected
6,7-dihydro-7-methoxy analogue (4), and two original tetrahydrofuranic (THF) alkyl esters ( 5,6). Moreover, when we accomplished a new
method for the preparation of the furano-pyrone goniofupyrone (7) through 7-hydroxylation of 1 in acid medium, a minor compound (8) with
an identical skeleton to that of compounds 5 and 6 was identi®ed. Careful examination of the published spectral data of the reported styryl-
lactones with an heptolide skeleton reveals that those structures possess also a THF alkyl ester skeleton. The revision of those structures was
con®rmed by chemical correlation. All altholactone derivatives assayed proved to be speci®c inhibitors of the mitochondrial complex I.
q 2002 Elsevier Science Ltd. All rights reserved.
1. Introduction
identi®ed a minor compound (8), which presents an
identical skeleton to those of compounds 5 and 6, and the
natural goniothalesdiol (9).6,7 The careful examination of
the published spectral data of styryl-lactones with an hepto-
lide skeleton (gonioheptolide-A8 and almuheptolide-A3)
revealed that those structures possess also a THF alkyl
ester skeleton. The revision of those structures was con-
®rmed by chemical correlation.
Styryl-lactones represent a class of natural and synthetic
compounds, with signi®cant cytotoxicity against several
human tumor cell lines.1 We have recently showed that
the mechanism of cytotoxicity of furano-pyrones, altho-
lactone (1), O-acetylaltholactone (2)2 and other related
styryl-lactones3 isolated from Goniothalamus arvensis, is
based on the inhibition on mammalian mitochondrial
respiratory chain. These results stimulated us to prepare
several semisynthetic altholactone analogues in order to
establish their structure±activity relationship as inhibitors
of the cellular respiration.2±4
In this work we also report the activity of several prepared
altholactone derivatives and accomplished the structure±
activity relationship as speci®c inhibitors of mitochondrial
complex I.
There is evidence that several protecting groups affect the
potency of cytotoxic activity.5 Thus, the introduction in 1 of
a methoxymethyl group (MOM) led to the corresponding
O-methoxymethyl derivative (3), in addition to the
unexpected 6,7-dihydro-7-methoxy analogue (4) and two
original tetrahydrofuranic (THF) alkyl esters (5,6). More-
over, when we accomplished a new method for the prepa-
ration of the furano-pyrone goniofupyrone (7) through
7-hydroxylation of altholactone (1) in acid medium, we
Keywords: tetrahydrofuran alkyl esters; furano-pyrones; styryl-lactones;
mitochondrial complex I inhibitors.
p
Corresponding author. Tel.: 134-96-386-4975; fax: 134-96-386-4943;
e-mail: bermejoa@uv.es
0040±4020/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(01)01234-0

1336
E. Peris et al. / Tetrahedron 58 (2002) 1335±1342
The two other minor compounds (5 and 6) were consistent
with an original structure without lactone moiety and they
are characterised by the presence of a methyl-ester alkyl
chain linked to a tetrasubstituted THF ring. Compound 5
was obtained as yellow oil. The molecular formula
C18H24O7 was established by the ion observed at m/z
353.161374 [MH]
1
in the HRLSIMS, and by liquid
chromatography with mass spectrometry detection (LC±
MS). Inspection of the 1D (1H and 13C NMR spectra), 2D
homonuclear (1H±1H COSY 45), 2D heteronuclear corre-
lation spectroscopy (HMQC and HMBC) and NOE
experiments, revealed the existence of a THF ring substi-
tuted by two MOM groups, an ole®nic chain, and a phenyl
moiety. Moreover, an IR absorption band at 1721 cm21
suggested a methyl ester function which was con®rmed by
the NMR signals at dH 3.75 (COOCH3) and dC 166.0
(COOCH3).
The NMR spectral data of 5 revealed the presence of a
THF ring with four oxygen-linked methines. The more
deshielded proton at dH 5.55 (HMQC, dC 79.2) is correlated
in COSY 45 with both a THF methine proton at dH 4.51
(H-5), and an ole®nic proton at dH 6.56 (H-3). Moreover the
Scheme 1. Semisynthesis of compounds 3±6 from altholactone (1).
3
carbon at dC 79.2 possesses a J correlation (HMBC) with
the other methine ole®nic proton at dH 5.99 (H-2). All those
results allowed to place the ole®nic chain over one of the
oxygen-bearing carbons (position 4) on the THF ring.
Indeed, the HMBC correlations observed between the car-
bonyl of the terminal ester function (C-1) with both the
methyl ester protons and one of the ole®nic protons (H-3),
were consistent with a THF alkyl ester skeleton for the
compound 5, identical to that described for the natural
goniothalesdiol (9) (see Fig. 1 and Table 1).6
The combined analysis of HMQC, HMBC and NOE's
experiments allowed the placement in the THF system, of
two O-MOM groups, and a phenyl ring at 5±7 positions,
respectively (Fig. 1).
2. Results and discussion
We envisaged that introduction of MOM group to the C-3
position of the furano-pyrone (1)-altholactone (1) would
increase the cytotoxicity of this class of bioactive
compounds.5 The methoxymethylation of the secondary
alcohol in 1 was carried out with dimethoxymethylether
and tri¯uoromethane sulfonic acid at room temperature
during 6 h, under nitrogen atmosphere.9 The expected
O-MOM derivative (3) was obtained in good yield.
However when the reaction conditions were modi®ed
(re¯uxing for 24 h), three minor compounds (4±6) were
obtained, in addition to compound 3 (Scheme 1).
The relative stereochemistry of the chiral centres in 5 was
deduced from both 1H-coupling constant values and
NOEDIFF experiments. The NOEs observed between H-4/
H-7 and H-4/H-5 were in agreement with a cis-relationships
for these three protons. The relative con®guration of 5 is
therefore postulated on the basis of the known con®guration
of the starting furano-pyrone altholactone (1) as 4,5-cis, 5,6-
trans, and 6,7-trans. In conclusion, the novel compound 5
was identi®ed as 3-(5,6-dimethoxymethyl-7-phenyl)-tetra-
hydrofuranyl acrylic methyl ester.
Inspection of their mono- and bi-dimensional NMR spectra
revealed that 3 and 4 present a furano-pyrone skeleton
(altholactone type). The most signi®cant structural dif-
ference between these two compounds appears in the satu-
ration degree of the d-lactone ring. Similar to 1, compound 3
presents an a,b-unsaturated d-lactone moiety, whereas 4
has in a 7-methoxy-saturated d-lactone. The mechanism of
7-alkoxylation of unsaturated furano-pyrones was described
as a Michael-type addition of an alcohol molecule (as
nucleophilic agent) in acid medium, across the a,b-unsatu-
rated d-lactone double bond from the less electron dense
position (b of the carbonyl group).3,10 So, the 7-methoxy-
saturated d-lactone (4) is probably generated from 3 in the
procedure of methoxymethylation.
Compound 6 was also obtained as a yellow oil. Its molecular
formula C19H28O8 was deduced from the HRLSIMS by a
peak at m/z 385.18643 [MH]1 and by LC±MS (API-ES) in
positive mode which showed an intensive positive ion at m/z
407 corresponding to [M1Na]1. Inspection of the NMR and
mass spectral data indicated the existence in 6 of similar
THF substituents to those of 5, two O-MOM groups, a
phenyl ring, and a methyl ester terminal chain. An
additional methoxyl group on the saturated alkyl chain,
was evidenced in HRLSIMS by a fragment ion peak at
m/z 353 [M2OCH3]1 and con®rmed by 1D and 2D NMR
experiments, where a proton resonance at dH 3.57 correlated
with the carbon signal at dC 59.7, was observed. Compound

E. Peris et al. / Tetrahedron 58 (2002) 1335±1342
1337
6 was identi®ed as 3-(5,6-dimethoxymethyl-7-phenyl)-
tetrahydrofuranyl-3-methoxy propionic methyl ester, and
an identical stereochemistry to that of 5 was established.
In order to explain the mechanism operating in the forma-
tion of the minor THF alkyl ester compounds (5 and 6)
during this reaction, we propose that the generation of
those compounds could be produced from the corresponding
furano-pyrone precursors 3 and 4.
In the hypothesis depicted in Scheme 2, we suggest that the
mechanism operating in the formation of THF alkyl ester
compounds would be considered as follows; i.e. the acidic
medium (CF3SO3H, CH2(OCH3)2) liberaties CH3OH that
opens up the lactone ring and subsequently gives rise to
methoxymethyl etheri®cation. Because the presence of an
ole®nic chain in 5 is consistent with the a,b-unsaturated-d-
lactone in 3, compound 5 could be generated via this furano-
pyrone. In this way, compound 4, resulting of the meth-
oxylation by a Michael-type addition of 1, seems to be the
Figure 1. 1H±13C long-range correlations (3J) established from HMBC, and
NOE of 5.
Table 1. 1D and 2D NMR experiments of 5 (CDCl3, 600 MHz)
Position
1H NMR and COSY 45
1H±1H COSY 45
13C NMR/DEPT, HMQC and HMBC
d
H (J Hz)
d
C (multiplicity)
HMQC (1J)
HMBC (3J)
1
2
3
4
5
6
7
8
9,13
10,12
11
1-OCH3
5-OMOM
±
±
H-3
H-2, H-4
H-3, H-5
H-4, H-6
H-5, H-7
H-6
±
H-10, H-12
166.0 (C)
H-3, OCH3
H-4
±
H-2
OCH2 of 5-OMOM
OCH2 of 6-OMOM
H-9, H-13
H-10, H-12
Ar-H
Ar-H
H-9, H-13
±
H-5, CH3 of 5-OMOM
CH2 of 5-OMOM
H-6, CH3 of 6-OMOM
CH3 of 6-OMOM
5.99 dd (11.7; 1.6)
6.56 dd (11.7; 6.6)
5.55 ddd (6.6; 3.9; 1.6)
4.51 dd (3.9; 0.7)
4.16 dd (3.9; 0.7)
4.80 d (3.9)
±
7.47 d (7.6)
7.34 t (7.6)
7.27 d (7.6)
3.75 s
CH2: 4.61 d/4.53 d (6.8)
CH3: 3.34, s
CH2: 4.77 d/4.68 d (6.7)
CH3: 3.19, s
120.3 (CH)
146.2 (CH)
79.2 (CH)
83.1 (CH)
87.9 (CH)
86.2 (CH)
140.3 (C)
126.3 (2 CH)
128.4 (2 CH)
127.6 (CH)
51.4 (CH3)
95.7 (CH2)
55.5 (CH3)
95.7 (CH2)
55.5 (CH3)
H-2
H-3
H-4
H-5
H-6
H-7
±
H-9, H-13
H-10, H-12
H-11
H-9, H-11, H-13
H-9, H-10, H-12, H-13
±
±
±
±
±
OCH3
CH2 of 5-OMOM
CH3 of 5-OMOM
CH2 of 6-OMOM
CH3 of 6-OMOM
6-OMOM
Scheme 2. Proposed mechanism for THF alkyl esters formation.

1338
E. Peris et al. / Tetrahedron 58 (2002) 1335±1342
Scheme 3. (a) H2O±dioxane, H2SO4, re¯ux; (b) (CH3O)2CH2, CF3SO3H, CH2Cl2, rt; (c) Ac2O, pyr, rt; (CH3O)2CH2, CF3SO3H, CH2Cl2, re¯ux; (e) MeOH,
H2SO4, re¯ux; (f) NaBH4, MeOH, re¯ux.
Table 2. 1D and 2D NMR experiments of 8 (CDCl3, 600 MHz)
Position
1H NMR and COSY 45
1H±1H COSY 45
13C NMR/DEPT, HMQC and HMBC
dH (J Hz)
dC (multiplicity)
HMQC (1J)
HMBC (2J and 3J)
1
2a
2b
3
4
5
6
7
8
9,13
10,12
11
1-OCH3
Oac
±
±
H-2b, H-3
H-2a, H-3
H-2a, H-2b, H-4
H-3, H-5
H-4, H-6
H-5, H-7
H-6
±
H-10, H-12
H-9, H-11, H-13
H-9, H-10, H-12, H-13
±
±
±
±
±
±
170.3 (C)
35.0 (CH2)
OCH3, H-2a, H-2b
±
±
H-2a, H-2b
±
H-4, H-6
H-5, H-7
H-9, H-13
H-10, H-12
H-11, H-9
Ar-H
2.77 dd (16.1; 5.3)
2.67 dd (16.1; 7.2)
5.57 dt (7.2; 5.3; 5.2)
4.52 t (5.4; 5.2)
5.39 dd (5.4; 3.8)
5.20 dd (5.2; 3.8)
4.90 d (5.2)
±
7.47 d (7.6)
7.35 t (7.6)
7.29 d (7.6)
H-2a, H-2b
68.3 (CH)
79.0 (CH)
76.1 (CH)
81.7 (CH)
83.3 (CH)
138.4 (C)
126.1 (2 CH)
128.4 (2 CH)
128.1 (CH)
52.0 (CH3)
21.1 (CH3)
170.0 (C)
H-3
H-4
H-5
H-6
H-7
±
H-9, H-13
H-10, H-12
H-11
OCH3
CH3
H-9, H-13
3.69 s
2.12a s
Oac
Oac
2.10a s
1.90a s
20.8 (CH3)
169.8 (C)
20.6 (CH3)
169.6 (C)
CH3
CH3
a
Assignments for three acetyl exchangeable groups.

E. Peris et al. / Tetrahedron 58 (2002) 1335±1342
1339
Figure 2. Revised structure of heptolide-type compounds.
precursor of 6. This procedure constitutes a new method for
preparing this class of compounds.
3-(5,6-di-O-acetyl-7-phenyl)-tetrahydrofuranyl-3-O-acetyl
propionic methyl ester.
The reaction, in both cases, proceeded in low yields but with
high stereoselectivity because no other lactone-ring-opened
products were observed. Thus, 5 and 6 were prepared with
complete retention of the stereochemical con®guration of
the starting phenyl-THF centres of altholactone (1).
To con®rm this new structural hypothesis, we carried out the
synthesis of 6,7-dihydro-7-methoxyaltholactone (10) from
1, under the same previously described conditions (conc.
H2SO4/MeOH).3,13 The minor compound obtained in this
reaction (11), previously considered as a heptolide,3,13
showed the same spectral features that were observed for
compounds 5, 6 and 8 described above. The chemical corre-
lation between compounds 11 and 6 (see Scheme 3),
allowed us to con®rm unambiguously a THF alkyl ester
skeleton for the related natural and synthetic heptolides
(Fig. 2). Finally, a supplementary proof that the THF alkyl
ester compounds have not lactone function, but a methyl
ester, was reported by reduction of both compound 10 (a
saturated d-lactone) and 11 (a THF alkyl ester) with NaBH4
in re¯uxing MeOH. The identi®cation of the acetylated
reductive derivatives (12) corroborates this structural
hypothesis.
In view of the good results obtained in the preparation of
7-alkoxylated furano-pyrone derivatives,3,10 we decided to
apply this procedure to the synthesis of goniofupyrone
(7) an 7-hydroxylated analogue isolated in 1991 from
G. giganteus.11 So, we semisynthesised 7 from 1 by a
single-step method through selective hydroxylation at C-7
position in concentrated acid medium, together with a not
isolated intermediate, which was acetylated to led to
compound 8. Spectral and physical data of 7 were in all
identical to those previously reported for the natural
goniofupyrone (Scheme 3).11
Inspection of the spectral data revealed that structure of 8
was closely related to that of 5 and 6. A THF alkyl ester
skeleton was established for 8 by careful examination of the
2D homonuclear (COSY 45) and 2D heteronuclear corre-
lation (HMQC and HMBC) NMR spectra (see Table 2).
Comparison of the spectral data of 8 with those of the
previously reported triacetyl-gonioheptolide-A8 suggested,
as it has been recently described,12 that the heptolide-type
skeleton (a-oxocanone) proposed for gonioheptolide-A and
related natural and semisynthetic heptolides,3,8 should now
be revised to their corresponding THF alkyl ester ones
(Scheme 3 and Table 2). Thus compound 8 was identi®ed as
The inhibitory activity of these compounds against mam-
malian respiratory chain was systematically assayed to
identify their mode of action as described in Section 3.
Cytochrome oxidase activity (complex IV) and succinate/
cytochrome c reductase activity (integrated complexes II
and III activity) were not affected by these compounds.
Instead, NADH oxidase activity (integrated complexes I,
III and IV activity) and NADH/cytochrome c reductase
activity (integrated complexes I and III activity) were
inhibited. Therefore, compounds were speci®c inhibitors
of the mitochondrial complex I. It was assessed by
inhibition of the NADH/ubiquinone oxidoreductase
activity, the isolated complex I activity, measured as
described in Section 3. However, as potency criterion IC50
was taken against the integrated NADH oxidase activity of
beef heart open submitochondrial particles (SMP),14 which
evaluates the complex I activity in a more physiological
environment. Results are shown in Table 3.
Table 3. Inhibitory potency of styryl-lactones against NADH oxidase
Inhibitors
CI50 (mM)
Furano-pyrone skeleton
1
2
24.80^7.5
4.70^1.6
All derivatives were complex I inhibitors more potent that
the starting product, altholactone (1). It is noteworthy that
protection of the hydroxyl groups increased the inhibitory
potency lowering the IC50 of the compounds. The acetylated
derivative 2 was found to be the most potent inhibitor of this
series. Taking into account the hydrophobic nature of the
inhibitor binding site (s) of complex I, likely placed inside
3
7
7a
11.22^3.32
18.17^3.39
9.82^2.42
THF alkyl ester skeleton
5
6
12.77^3.91
15.07^4.44

1340
E. Peris et al. / Tetrahedron 58 (2002) 1335±1342
the inner mitochondrial membrane or near the interface, and
in accordance with previous studies with other complex I
inhibitors,14 it is thought that protection of the hydroxyl
groups improves the access of the inhibitor to the site of
action in the mitochondrial complex I.
100 MHz): d 160.9 (C-5), 139.5 (C-7), 138.1 (C-8), 128.6
(C-10,12), 128.4 (C-11), 126.3 (C-9,13), 124.3 (C-6), 96.3
(OCH2OCH3), 88.1 (C-3a), 85.6 (C-2), 84.5 (C-3), 68.7
(C-7a), 55.8 (OCH2OCH3).
3.4. Semisynthesis of compounds 4±6
3. Experimental
Using a similiar procedure as described earlier for prepa-
ration of 3, but re¯uxing in this case for 24 h, afforded 4
(12 mg, 7%), 5 (20 mg, 10%) and 6 (8 mg, 4%).
3.1. General instrumentation
Optical rotations were determined with a Perkin±Elmer 241
polarimeter. IR spectra were run in ®lm using NaCl plates
on a Perkin±Elmer 1750 FTIR spectrometer. EIMS,
LSIMS, HREIMS and HRLSIMS were recorded on a VG
Auto Spec Fisons spectrometer instruments. Liquid chroma-
tography with mass spectrometry detection (LC±MSD)
with API (atmospheric pressure ionisation) source con®gu-
rated as APCI (atmospheric pressure chemical ionisation) or
API-ES (electrospray ionisation) in positive mode, were
3.4.1. 6,7-Dihydro-7-methoxy-3-O-methoxymethylaltho-
lactone (4). C16H20O6; [a]Dˆ1208 (c 1.0, EtOH); IR nmax
(®lm) cm21: 2936, 2895, 2828, 2359, 2341, 1744, 1496,
1455, 1371, 1236, 1200, 1152, 1103, 1034, 973, 918, 761,
700; LC±MSD API-ES mode positive m/z: 331 [M1Na]1;
1H NMR (CDCl3, 400 MHz): d 7.35 (m, 5H, H-9-13), 4.92
(dd, Jˆ4.2, 1.6 Hz, 1H, H-3a), 4.78 (d, Jˆ5.5 Hz, 1H, H-2),
4.74 and 4.63 (2d, Jˆ6.6 Hz, 2H, OCH2OCH3), 4.34 (t,
Jˆ4.2, 3.9 Hz, 1H, H-7a), 4.25 (dd, Jˆ5.5, 1.6 Hz, 1H,
H-3), 3.95 (m, 1H, H-7), 3.47 (s, 3H, OCH3), 3.28 (s, 3H,
OCH2OCH3), 2.87 (dd, Jˆ16.4, 3.9 Hz, 1H, H-6a), 2.72 (dd,
Jˆ16.4, 5.8 Hz, 1H, H-6b); 13C NMR (CDCl3, 100 MHz): d
168.2 (C-5), 138.1 (C-8), 128.7 (C-10,12), 128.3 (C-11),
126.3 (C-9,13), 96.4 (OCH2OCH3), 87.6 (C-3a), 85.5
(C-2), 84.5 (C-3), 76.1 (C-7a), 74.6 (C-7), 57.2 (OCH3),
55.7 (OCH2OCH3), 32.6 (C-6).
1
determined on a Hewlett±Packard (HP-1100). H and 13C
NMR spectra were recorded with CDCl3 as solvent on a
Bruker AC-400, Bruker AC-600, Varian-Unity-300 or
Varian-Unity-400. Multiplicities of 13C NMR resonances
were assigned by DEPT experiments. NOEDIFF irradia-
tions, COSY 45, HMQC, HSQC and HMBC correlations
were recorded at 400 or 600 MHz. All reactions were
monitored by analytical TLC with silica gel 60 F254
(Merck 5554). The residues were puri®ed through 60H
silica gel column (5±40 mm, Merck 7736) or by ¯ash
chromatography (230±400 mm, Merck 9385).
3.4.2.
3-(5,6-Dimethoxymethyl-7-phenyl)-tetrahydro-
furanyl acrylic methyl ester (5). C18H24O7; [a]Dˆ11798
(c 1.0, EtOH); IR nmax (®lm) cm21: 2950, 2891, 2824, 2360,
2341, 1721, 1439, 1203, 1151, 1032, 919, 827, 761, 700;
HRLSIMS m/z: 353.161374 [MH]1 (calcd 353.160028); 1H
NMR (CDCl3, 600 MHz) and 13C NMR (CDCl3, 150 MHz)
see Table 1.
3.2. Natural starting styryl-lactones
Altholactone (1) and 3-O-acetylaltholactone (2), were iso-
lated from G. arvensis Scheff (Annonaceae) stem bark.2,15
3.4.3.
3-(5,6-Dimethoxymethyl-7-phenyl)-tetrahydro-
furanyl-3-methoxy propionic methyl ester (6). C19H28O8;
[a]Dˆ1348 (c 1.0, EtOH); IR nmax (®lm) cm21: 2947, 2893,
2826, 2359, 2340, 1739, 1439, 1151, 1104, 1030, 917,
761, 700; HRLSIMS m/z: 385.186432 [MH]1 (calcd
385.186243), 353 [M2OCH3]1, 229 [M2OCH322£HO-
CH2OCH3]1; LC±MSD API-ES mode positive m/z: 407
3.3. General procedure for O-methoxymethylation
3.3.1. Semisynthesis of 3-O-methoxymethylaltholactone
(3). To a solution of altholactone (1, 130 mg, 0.56 mmol)
in dry dichloromethane (30 mL) and (CH3O)2CH2 (30 mL,
3.39£1024 mmol), were added 4 drops of tri¯uoromethane
sulfonic acid (CF3SO3H). The reaction mixture was stirred
at rt for 6 h under nitrogen atmosphere. The excess
CF3SO3H was destroyed by dropwise addition of NH4Cl
15% aq (4 mL), and the resulting solution was concentrated
and extracted with CH2Cl2, washed with brine and H2O, and
dried. The residue was subjected to column chromatography
on silica gel 60H, eluting with hexane±EtOAc (70:30), to
give 80 mg of 3-O-methoxymethylaltholactone (3, 52%).
C15H16O5; [a]Dˆ1488 (c 1.0, EtOH); IR nmax (®lm)
cm21: 3064, 3033, 2950, 2894, 2826, 1736, 1640, 1495,
1454, 1368, 1246, 1152, 1101, 1038, 970, 918, 763, 700;
EIMS m/z (%): 232 [M2OCH2OCH3]1 (4), 231 (20), 214
(88), 170 (23), 141 (100), 107 (75), 97 (67), 77 (27); LC±
1
[M1Na]1; H NMR (CDCl3, 300 MHz): d 7.45 (d, Jˆ
7.5 Hz, 2H, H-9,13), 7.33 (t, Jˆ7.5 Hz, 2H, H-10,12),
7.26 (d, Jˆ7.5 Hz, 1H, H-11), 4.84 (d, Jˆ3.6 Hz, 1H,
H-7), 4.72 and 4.67 (2d, Jˆ6.8 Hz, 2H, OCH2OCH3-6),
4.64 and 4.57 (2d, Jˆ7.2 Hz, 2H, OCH2OCH3-5), 4.10
(m, 4H, H-3-6), 3.72 (s, 3H, COOCH3), 3.57 (s, 3H,
OCH3), 3.34 (s, 3H, OCH2OCH3-6), 3.21 (s, 3H,
OCH2OCH3-5), 2.69 (dd, Jˆ15.4, 3.2 Hz, 1H, H-2a), 2.57
(dd, Jˆ15.4, 8.1 Hz, 1H, H-2b); 13C NMR (CDCl3,
75 MHz): d 171.8 (C-1), 140.6 (C-8), 128.2 (C-10,12),
127.5 (C-11), 126.2 (C-9,13), 96.0 (OCH2OCH3-6), 95.8
(OCH2OCH3-5), 87.0 (C-6), 86.3 (C-7), 83.7 (C-5), 82.2
(C-4), 77.4 (C-3), 59.7 (OCH3), 56.1 (OCH2OCH3-6), 55.5
(OCH2OCH3-5), 51.7 (COOCH3), 36.6 (C-2).
1
MSD API-ES negative mode, m/z 275 [M2H]1; H NMR
(CDCl3, 400 MHz): d 7.35 (m, 5H, H-9-13), 7.00 (dd,
Jˆ9.8, 5.1 Hz, 1H, H-7), 6.25 (d, Jˆ9.8 Hz, 1H, H-6),
4.98 (dd, Jˆ4.9, 1.7 Hz, 1H, H-3a), 4.83 (d, Jˆ5.1 Hz,
1H, H-2), 4.76 and 4.66 (2d, Jˆ6.8 Hz, 2H, OCH2OCH3),
4.57 (t, Jˆ4.9, 5.1 Hz, 1H, H-7a), 4.39 (dd, Jˆ5.1, 1.7 Hz,
1H, H-3), 3.31 (s, 3H, OCH2OCH3); 13C NMR (CDCl3,
3.5. General procedure for hydroxylation
3.5.1. Semisynthesis of goniofupyrone (7) and 3-(5,6-di-
O-acetyl-7-phenyl)-tetrahydrofuranyl-3-O-acetyl-pro-
pionic methyl ester (8). To a solution of altholactone (1,

E. Peris et al. / Tetrahedron 58 (2002) 1335±1342
1341
100 mg, 0.43 mmol) in dioxane (2 mL) and H2O (13.5 mL),
was added dropwise concentrated H2SO4 (2 mL). After the
mixture was stirred and re¯uxed for 9 h, water was added
and the reaction mixture was extracted with EtOAc. The
organic solution after usual workup was puri®ed by silica
gel 60H column chromatography (eluting with CH2Cl2±
EtOAc 50:50), affording goniofupyrone (7, 54 mg, 50%)
and a minor polyhydroxylated compound. This last com-
pound was O-acetylated (Ac2O/pyridine), and puri®ed by
silica gel 60H column chromatography (hexane±EtOAc
60:40), affording the corresponding triacetate 8 (15 mg,
11%).
3.6.2. 6,7-Dihydro-7-methoxyaltholactone (10). C14H16O5;
[a]Dˆ269.28 (c 1.3, EtOH); EIMS m/z: 264 [M]1 (see
Refs. 3,13).
3.6.3. 3-(5,6-Dihydroxy-7-phenyl)-tetrahydrofuranyl-3-
methoxy propionic methyl ester (11). C15H20O6; [a]Dˆ
115.08 (c 0.8, EtOH); LSIMS m/z: 297 [MH]1 (see Refs.
3,13).
3.7. Chemical correlations
3.7.1. Di-O-methoxymethylation of compound 11 (semi-
synthesis of 6 from 11). Compound 11 (4 mg, 0.0135
mmol) was reacted using the same procedure described
above for 1 (CH2(OCH3)2/CF3SO3H), to afford the corre-
sponding 5,6-dimethoxymethyl derivative, which was
found to be in all identical respects to the previously
prepared compound 6.
3.5.2. Goniofupyrone (7). Spectral and physical data of
compound 7 were identical to those reported for the natural
goniofupyrone.11
3.5.3. 3-(5,6-Di-O-acetyl-7-phenyl)-tetrahydrofuranyl-3-
O-acetyl propionic methyl ester (8). C20H24O9; [a]Dˆ
1208 (c 0.5, EtOH); HRLSIMS m/z (%): 431 [M1Na]1
(18), 409.150309 [MH]1 (calcd 409.149858) (100), 349
[MH2CH3COOH]1 (20), 289 [MH22£CH3COOH]1 (5),
229 [MH23£CH3COOH]1 (15), 154 [MH23£OCOCH3±
Ph]1 (58); EIMS m/z (%): 348 [M2CH3COOH]1 (2), 288
[M22£CH3COOH]1 (20), 228 [M23£CH3COOH]1 (100),
197 [M2HOCH323£CH3COOH]1 (3), 173 (39), 161 (29),
120 [M2Ph±OCH323£CH3COOH]1 (7), 107 (10), 105
3.7.2. Reduction of compounds 10 and 11 (semisynthesis
of compound 12). To a solution of 10 (40 mg, 0.15 mmol)
in dry MeOH (2 mL) was added an excess of NaBH4 (3 mg).
After the reaction mixture was stirred for 3 h at 908C, water
was added and the resulting solution was concentrated to
dryness. The residue obtained was O-acetylated using dry
pyridine (1 mL) and Ac2O (2 mL) to yield after usual
workup the triacetylated derivative 12 (42.4 mg, 73.6%).
The same procedure was used starting from 11 (5 mg,
0.016 mmol) to afford compound 12 (5 mg, 76.5%).
C20H26O8; LC±MSD API-ES mode positive m/z: 417
1
(12), 98 (3), 91 (13), 77 (5); H NMR (CDCl3, 600 MHz)
and 13C NMR (CDCl3, 150 MHz) see Table 2.
3.5.4. Di-O-methoxymethylation of 7. Goniofupyrone (7,
95 mg, 0.38 mmol) were treated using the same procedure
described above for 1. After applied silica gel 60 H
column chromatography (hexane±CH2Cl2±EtOAc 60:5:
35), afforded 3,7-di-O-methoxymethyl-goniofupyrone (7a,
10 mg, 8%). C17H22O7; EMIE m/z (%): 293 [M2
CH2OCH3]1 (100), 276 (25), 247 (3), 214 (6), 187 (38),
1
[M1Na]1, 395 [MH]1; H NMR (CDCl3, 300 MHz): d
7.42 (m, 5H, H-9-13), 5.24 (dd, Jˆ3.4, 1.3 Hz, 1H, H-5),
5.03 (dd, Jˆ3.0, 1.3 Hz, 1H, H-6), 4.92 (d, Jˆ3.0 Hz, 1H,
H-7), 4.23 (m, 3H, CH2-1, H-4), 3.70 (m, 1H, H-3), 3.58 (s,
3H, OCH3), 2.13, 2.07 and 1.94 (3s, 9H, 3£OCOCH3), 1.78
(m, 2H, CH2-2); 13C NMR (CDCl3, 100 MHz): d 170.7 and
169.7 (3£OCOCH3), 138.9 (C-8), 128.1 (C-10,12), 127.7
(C-11), 126.0 (C-9,13), 85.3 (C-7), 83.8 (C-4), 82.8 (C-6),
76.9 (C-3), 76.6 (C-5), 60.6 (C-1), 59.5 (OCH3), 30.1 (C-2),
20.9, 20.8 and 20.5 (3£OCOCH3).
1
151 (40), 144 (75), 115 (14), 105 (20), 77 (10), 51 (2); H
NMR (CDCl3, 400 MHz): d 7.32 (m, 5H, H-9-13), 5.00 (dd,
Jˆ4.4, 1.6 Hz, 1H, H-3a), 4.80 (d, Jˆ5.4 Hz, 1H, H-2), 4.75
(m, 2H, OCH2OCH3), 4.72 (m, 1H, OCH2OCH3), 4.62 (m,
1H, OCH2OCH3), 4.35 (m, 1H, H-7a), 4.31 (m, 1H, H-7),
4.25 (dd, Jˆ5.4, 1.6 Hz, 1H, H-3), 3.40 and 3.30 (2s, 6H,
OCH2OCH3), 2.90 (dd, Jˆ16.8, 3.6 Hz, 1H, H-6a), 2.00 (dd,
Jˆ16.8, 4.5 Hz, 1H, H-6b); 13C NMR (CDCl3, 100 MHz): d
167.9 (C-5), 138.1 (C-8), 128.7 (C-10, 12), 128.4 (C-11),
126.2 (C-9,13), 96.2 and 95.9 (2£OCH2OCH3), 87.8 (C-3),
84.8 (C-3a), 85.5 (C-2), 75.6 (C-7), 70.9 (C-7a), 55.8 and
55.7 (2£OCH2OCH3), 33.3 (C-6).
3.8. Biochemical methods
3.8.1. Styryl-lactone derivatives and other reagents. Styryl-
lactone derivatives were either isolated and puri®ed from
the plant material, or prepared by semisynthesis as des-
cribed earlier. Rotenone, antimycin-A, decylubiquinone
and other biochemical reagents were purchased from
Sigma Chemical Co. Stock solutions (15 mm in absolute
ethanol) of compounds were prepared and kept in the dark
at 2208C. Appropiate dilutions (3±6 mm) were made
before the experiments.
3.6. General procedure for alkoxylation
3.6.1. Semisynthesis of 6,7-dihydro-7-methoxy-altho-
lactone (10) and 3-(5,6-dihydroxy-7-phenyl)-tetrahydro-
furanyl-3-methoxy propionic methyl ester (11). To a
solution of altholactone (1, 60 mg, 0.258 mmol) in MeOH
(10 mL), was added dropwise at 08C concentrated H2SO4
(1.5 mL). After the mixture was stirred and re¯uxed for
3 h, water was added and the reaction mixture was extracted
with CH2Cl2. The organic solution after usual workup was
puri®ed by silica gel 60 H column chromatography (eluting
with CH2Cl2±EtOAc 70:30), affording 10 (20 mg, 28%) and
11 (34 mg, 40%).
3.8.2. Preparation of beef-heart submitochondrial par-
ticles. Inverted SMP from beef heart were obtained by
extensive ultrasonic disruption of frozen-thawed mito-
chondria in such a way to produce open membrane frag-
ments where permeability barriers to substrates were lost.16
After ultracentrifugation they were ®nally resuspended 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.17

1342
E. Peris et al. / Tetrahedron 58 (2002) 1335±1342
5. Tsubuki, M.; Kanai, K.; Nagase, H.; Honda, T. Tetrahedron
1999, 55, 2493±2514.
NADH oxidase was measured as the aerobic oxidation of
75 mM NADH in the absence of the quinone substrate and
other inhibitors of the respiratory chain. NADH/ubiquinone
oxidoreductase was measured with 75 mM NADH and
30 mM decylubiquinone (DB) as soluble short-chain ubi-
quinone analogue in the presence of 2 mM antimycin and
2 mm potassium cyanide to block any reaction downstream
of complex I.18 Cytochrome c reduction by both NADH and
succinate, and the cytochrome c oxidase activity were
measured as previously described.19,20
6. Cao, S-G.; Wu, X-H.; Sim, K-Y.; Tan, B. K. H.; Pereira, J. T.;
Goh, S.-H. Tetrahedron 1998, 54, 2143±2148.
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Â
10. Bermejo, A.; Blazquez, M. A.; Serrano, A.; Zafra-Polo, M. C.;
Inhibitor titrations were made as previously described in
detail.4,17,21 The inhibitory concentration 50 (IC50) was
taken as the ®nal compound concentration in the assay
cuvette that yielded 50% of the initial activity. Data from
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deviations.
Cortes, D. J. Nat. Prod. 1997, 60, 1338±1340.
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Tetrahedron 1991, 47, 9751±9758.
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Â
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Acknowledgements
Â
This research was supported by the Spanish Direccion
General de Investigacion Cientõ®ca y Tecnica (DGICYT)
Â
15. Bermejo, A.; Lora, M. J.; Blazquez, M. A.; Rao, K. S.; Cortes,
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D.; Zafra-Polo, M. C. Nat. Prod. Lett. 1995, 7, 117±122.
16. Fato, R.; Estornel, E.; Di Bernardo, S.; Palloti, F.; Parenti-
Castelli, G.; Lenaz, G. Biochemistry 1996, 35, 2705±2716.
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under grant SAF 97-0013.
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