γ-lactone-functionalized antitumoral acetogenins are the most potent inhibitors of mitochondrial complex I

Article abstract of DOI:10.1016/S0960-894X(01)00036-1

To study the relevance of the terminal α,β-unsaturated γ-methyl-γ-lactone moiety of the antitumoral acetogenins of Annonaceae for potent mitochondrial complex I inhibition, we have prepared a series of semisynthetic acetogenins with modifications only in this part of the molecule, from the natural rolliniastatin-1 (1) and cherimolin-1 (2). Some of the hydroxylated derivatives (1b, 1d and 1e) in addition to two infrequent natural β-hydroxy γ-methyl γ-lactone acetogenins, laherradurin (3) and itrabin (4), are more potent complex I inhibitors than any other known compounds.

Full text of DOI:10.1016/S0960-894X(01)00036-1

Bioorganic & Medicinal Chemistry Letters 11 (2001) 681±684
ꢀ-Lactone-Functionalized Antitumoral Acetogenins are the Most
Potent Inhibitors of Mitochondrial Complex I
Jose R. Tormo,a Ernesto Estornell,b Teresa Gallardo,a M. Carmen Gonzalez,a
Adrien Cave,c Susana Granell,a Diego Cortesa,* and M. Carmen Zafra-Poloa
a
Â
Departamento de Farmacologõa, Farmacognosia y Farmacodinamia, Universidad de Valencia, 46100 Burjassot, Valencia, Spain
b
Â
Departamento de Bioquõmica y Biologia Molecular, Facultad de Farmacia, Universidad de Valencia, 46100 Burjassot, Valencia, Spain
c
Â
Â
Centre de Biochimie Structurale, Faculte de Pharmacie, 34060 Montpellier Cedex 1, France
Received 6 September 2000; revised 6 December 2000; accepted 8 January 2001
AbstractÐTo study the relevance of the terminal a,b-unsaturated g-methyl-g-lactone moiety of the antitumoral acetogenins of
Annonaceae for potent mitochondrial complex I inhibition, we have prepared a series of semisynthetic acetogenins with modi®ca-
tions only in this part of the molecule, from the natural rolliniastatin-1 (1) and cherimolin-1 (2). Some of the hydroxylated deriva-
tives (1b,1d and 1e) in addition to two infrequent natural b-hydroxy g-methyl g-lactone acetogenins, laherradurin (3) and itrabin
(4), are more potent complex I inhibitors than any other known compounds. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
It is thought that this THF system mainly mimics the
quinone head of the natural substrate of the complex I,
the ubiquinone, whereas the g-lactone moiety allows a
second binding point if it is properly positioned by the
alkyl spacer and its oxygenated groups.6,7d,e However,
the relevance of the g-lactone moiety itself for potent
complex I inhibition has not been studied yet. With this
aim, we have prepared two series of semisynthetic ACG
with modi®cations only in the terminal a,b-unsaturated
g-lactone moiety and the close 4-hydroxy group of the
alkyl chain spacer. The ®rst series was obtained from
the adjacent bis-THF ACG, rolliniastatin-1 (1), isolated
from Rollinia membranacea seeds.7c The second series
was obtained from the non-adjacent bis-THF ACG,
cherimolin-1 (2), isolated from Annona cherimolia
seeds.8a The inhibitory potency of the new derivatives
was additionally compared with laherradurin (3) and
itrabin (4), also isolated from A. cherimolia seeds,8b
which present a scarce b-hydroxylated saturated g-lac-
tone moiety. We have found that some of these lactone-
modi®ed ACG are more potent complex I inhibitors
than any other known compound.
The acetogenins of Annonaceae (ACG) are very inter-
esting compounds because they have well-known
biological e€ects such as potent cytotoxic, antitumor,
parasitic and insecticide activities.1 All these diverse
biological activities are explained by the inhibition of
the enzyme NADH:ubiquinone oxidoreductase or com-
plex I of the mitochondrial respiratory chain.2 Although
complex I is probably the ®rst enzyme regarding num-
ber of inhibitors,3 some natural ACG are the most
potent inhibitors known to date.1,4
ACG are members of a class of polyketide derivatives
characterized by the presence of one or two tetra-
hydrofuranic (THF) rings in the center of a long alkyl
chain bearing a terminal g-lactone.1,4 Therefore, they
have longer and more ¯exible structures than most
complex I inhibitors, an unusual characteristic that has
hindered the unambiguous establishment of a structure±
activity relationship.5,6 Recent studies have shown that
the two functional units, the central THF system
¯anked by hydroxyl groups and the terminal a,b-unsa-
turated g-lactone, and also the alkyl chain that acts as a
spacer between them, play signi®cant roles in inhibiting
the enzyme.6,7
Chemistry
When the catalytic hydrogenation of the 4-hydroxylated
a,b-unsaturated g-lactone ACG was performed in a
MeOH solution with 10% Pd/C a 1:1 mixture of two
diastereoisomers (2,36-cis and -trans) was obtained, due
*Corresponding author. Tel.: +34-96-386-49-75; fax: +34-96-386-49-
43; e-mail: dcortes@uv.es
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00036-1

682
J. R. Tormo et al. / Bioorg. Med. Chem. Lett. 11 (2001) 681±684
to a stabilization of the molecules by H-bonding that
allows both faces to be similarly accessible.9 In these
conditions the 2,35-dihydrogenated derivatives, 1a and
2a,10 were prepared in quantitative yields from 1 and 2
(Scheme 1).
The implementation of two hydroxyl groups in the
a,b-unsaturated g-methyl-g-lactone was carried out
by treatment of 1 and 2 with osmium tetroxide and
N-methylmorpholine N-oxide monohydrate to regener-
ate the oxidizing agent,11 a€ording 1b (75%) and 2b
(60%),12 respectively, as mixtures of the corresponding
cis-dihydroxylated derivatives.
It has been well established that the alkaline treatment
of a,b-unsaturated g-methyl-g-lactone ACG hydroxy-
lated at 4 position, a€ords in high yields the translacto-
nized 2-acetonyl saturated g-lactone derivatives, as a
mixture of 2,4-cis and 2,4-trans diastereoisomers usually
called `isoacetogenins'.13 Thus, the alkaline treatment
(DEA) of a MeOH solution of 1 and 2 and further
puri®cation of the crude by preparative HPLC
(CH3CN/H2O 80:20) at a speci®c wavelength (263 nm),
a€orded 1c and 2c in a good yield (ꢀ85%).14
Recently, we have reported that the hydroxylimino
derivatives prepared from ketonic bis-THF ACG in
which a nitrogen atom is inserted in the molecule,
increase the complex I inhibitory potency.7a,e Therefore
the ketonics 1c and 2c were employed as starting mate-
rial for preparing the corresponding 36-deoxo-36-
hydroxylimino derivatives, 1d and 2d,15 respectively,
obtained as a mixture of 2,4-cis and 2,4-trans diaster-
eoisomers in an ꢀ80% yield.
Scheme 1. Reagents and conditions: (i) H2, 10% Pd/C, 1 atm, rt, 2 h;
(ii) NMO/OsO4, rt, 24 h, followed by 2% NaHSO3, rt, 1 h; (iii) DEA,
.
re¯ux, 24 h; (iv) H2NOH HCl/Pyr, re¯ux, 1.5 h; (v) NaBH4/MeOH, rt,
1 h.

J. R. Tormo et al. / Bioorg. Med. Chem. Lett. 11 (2001) 681±684
Table 1. Inhibitory potency of natural and semisynthetic ACGs
683
IC50 (nM)
NADH oxidase NADH:DB oxidoreductase IC50 DB/Ox ratio Relative potencya
Compound
1
Rolliniastatin-1
0.60Æ0.04
0.43Æ0.07
0.21Æ0.05
0.33Æ0.02
0.23Æ0.06
0.18Æ0.01
1.84Æ0.27
1.22Æ0.28
8.47Æ1.31
0.83Æ0.38
0.54Æ0.11
2.48Æ0.32
0.18Æ0.02
0.21Æ0.03
0.74Æ0.04
0.57Æ0.06
0.56Æ0.02
1.21Æ0.23
0.22Æ0.04
0.21Æ0.04
5.18Æ0.63
3.32Æ1.22
38.3Æ8.1
1.2
1.3
2.7
3.7
1.0
1.2
2.8
2.7
4.6
3.5
2.6
1.9
1.6
3.4
3.3
2.4
1.2
1.8
1.3
1.0
10.2
6.8
47.1
4.6
3.0
13.8
1.0
1.2
1a
1b
1c
1d
1e
2
2a
2b
2c
2d
2e
3
2,35-Dihydrorolliniastatin-1
2,35-Dihydro-2,35-dihydroxy-rolliniastatin-1
Isorolliniastatin-1
36-Deoxo-36-hydroxylimino-isorolliniastatin-1
36-Dihydroisorolliniastatin-1
Cherimolin-1
2,35-Dihydrocherimolin-1
2,35-Dihydro-2,35-dihydroxy-cherimolin-1
Isocherimolin-1
36-Deoxo-36-hydroxylimino-isocherimolin-1
36-Dihydroisocherimolin-1
Laherradurin
2.88Æ0.04
1.42Æ0.13
4.64Æ0.44
0.29Æ0.01
0.72Æ0.10
4
Itrabin
aRelative potency compared with the NADH oxidase IC50 of the most potent compounds 1e and 3 (the lowest value, the highest potency).
The reduction of the ketonic group of the iso-
acetogenins was prepared to elucidate the in¯uence in
the inhibitory potency of the 2-acetonyl group. NaBH4
reduction of 1c and 2c produced 36-dihydrogenated
derivatives (1e and 2e)16 in good yields (Scheme 1).
logue. Therefore, it reveals that the binding site of the
terminal lactone moiety of the ACG is also related with
an ubiquinone binding site in the complex I.6,18
Three out of ®ve cherimolin-1 derivatives (2a, 2c and
2d) were also more potent than their head of series (2).
But all of them were less potent than rolliniastatin-1
derivatives. The di€erent THF system and the con-
sequent di€erent length of the alkyl spacer between this
and the terminal lactone moiety were then probably less
appropriate for potent inhibition.
Bioactivity
The inhibitory potency of the natural and semi-syn-
thetic ACG was evaluated against the integrated
NADH oxidase activity of beef heart open sub-
mitochondrial particles as well as against the speci®c
complex I NADH:ubiquinone oxidoreductase activity
using decylubiquinone (DB) as arti®cial ubiquinone-like
substrate (NADH:DB oxidoreductase activity),6,17
which is a good indicator of tight binding of the inhi-
bitor to the enzyme.3,6,18 Results are shown in Table 1.
All rolliniastatin-1 derivatives (1a±1e) were more potent
than the natural compound (1). Isoacetogenin deriva-
tives (1c±1e) were more potent probably because of a
shorter distance between the THF system and the term-
inal g-lactone in accordance with previous results
obtained with rollimembrin,19 an ACG with 2 C shorter
as 1. Moreover, the presence of a hydroxylimino group
(1d) increased the potency, according to previous results
with other ACG series.7a,e It is worth noting that 36-
dihydro-isorolliniastatin-1 (1e) gave the strongest inhi-
bitory potency ever found for a complex I inhibitor. It
seems that the presence of hydroxyl groups in the core
of the lactone itself gives an extreme potency in these
compounds.
In conclusion, we have found that some g-lactone-func-
tionalized ACG derivatives as well as two natural
compounds with an uncommon b-hydroxylated saturated
g-lactone moiety are the most potent complex I inhibi-
tors known up to date. Moreover, we have shown that
the terminal lactone of the long and ¯exible ACG
structure contributes signi®cantly for tight binding to the
enzyme and potent inhibitory activity. Therefore, our
results are highly interesting for an optimal design of
antitumoral drugs with this new mechanism of action.
Acknowledgements
This research has been supported by the Spanish DGI-
CYT under grant SAF 97-0013. We also wish thank the
Spanish MEC for the scholarship grant to J.R.T.
References and Notes
For that reason we decided to assay the b-hydroxylated
g-lactone ACG, laherradurin (3) and itrabin (4). These
compounds also showed an extremely high potency,
con®rming the relevance of the hydroxyl group placed
in the terminal saturated g-lactone moiety, especially at
a proper length from the THF system.
1. (a) Zafra-Polo, M. C.; Figadere, B.; Gallardo, T.; Tormo,
J. R.; Cortes, D. Phytochemistry 1998, 48, 1087. (b) Alali,
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504.
2. Londershausen, M.; Leicht, W.; Lieb, F.; Moeschler, H.;
Weiss, H. Pestic. Sci. 1991, 33, 427.
Nevertheless, compounds 1b and 1c gave IC50 DB/Ox
ratios higher than the remaining derivatives of this ser-
ies. This is an indication of their weaker binding to the
enzyme and easier displacement by the substrate ana-
3. Degli Esposti, M. Biochim. Biophys. Acta 1998, 1364, 222.
4. Tormo, J. R.; Gallardo, T.; Gonzalez, M. C.; Bermejo, A.;
Cabedo, N.; Andreu, I.; Estornell, E. Curr. Top. Phytochem.
1999, 2, 69.

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1365, 443.
HRLSIMS m/z 673.492257 MH+ (calcd 673.489074 for
C37H69O10).*The assignments were made by COSY 45, DEPT,
TOCSY, HMQC and HMBC.
6. Tormo, J. R.; Gallardo, T.; Aragon, R.; Cortes, D.; Estor-
nell, E. Chem.±Biol. Interact. 1999, 122, 171.
13. (a) Hoye, T. R.; Hanson, P. R. J. Org. Chem. 1991, 56,
5092. (b) Duret, P.; Laurens, A.; Hocquemiller, R.; Cortes, D.;
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14. (a) Abreo, M. J.; Sneden, A. T. J. Nat. Prod. 1990, 53,
983. (b) Duret, P.; Gromek, D.; Hocquemiller, R.; Cave, A.;
Cortes, D. J. Nat. Prod. 1994, 57, 911.
7. (a) Gallardo, T.; Saez, J.; Granados, H.; Tormo, J. R.;
Velez, I. D.; Brun, N.; Torres, B.; Cortes, D. J. Nat. Prod. 1998,
61, 1001. (b) Shimada, H.; Grutzner, J. B.; Kozlowski, J. F.;
McLaughlin, J. L. Biochemistry 1998, 37, 854. (c) Gonzalez,
M. C.; Lavaud, C.; Gallardo, T.; Zafra-Polo, M. C.; Cortes, D.
Tetrahedron 1998, 54, 6079. (d) Kuwabara, K.; Takada, M.;
Iwata, J.; Tatsumoto, K.; Sakamoto, K.; Iwamura, H.;
Miyoshi, H. Eur. J. Biochem. 2000, 267, 2538. (e) Gallardo, T.;
Zafra-Polo, M. C.; Tormo, J. R.; Gonzalez, M. C.; Franck, X.;
Estornell, E.; Cortes, D. J. Med. Chem. 2000, 43, 4793.
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Phytochemistry 1993, 32, 1475. (b) Cortes, D.; Myint, S. H.;
Leboeuf, M.; Cave, A. Tetrahedron Lett. 1991, 32, 6133.
9. Cortes, D.; Myint, S. H.; Harmange, J. C.; Sahpaz, S.;
Figadere, B. Tetrahedron Lett. 1992, 33, 5225.
10. 2,35-Dihydrogenated ACG derivatives. Compound 1a:
C37H68O7; [a]D +20ꢁ (c 3.1, EtOH); LSIMS m/z 647
M+Na+, 625 MH+. Compound 2a: see ref 8a. The elucida-
tions were made by EIMS, COSY 45, DEPT and HMQC.
11. Elomri, A.; Mitaku, S.; Michel, S.; Skaltsounis, A. L.;
Tillequin, F.; Koch, M.; Pierre, A.; Guilbaud, N.; Leonce, S.;
Kraus-Berthier, L.; Rolland, Y.; Atassi, G. J. Med. Chem.
1996, 39, 4762.
15. 36-Hydroxylimino isoacetogenin derivatives: relevant
values. Compound 1d: [a]D +31ꢁ (c 0.3, EtOH); HRLSIMS
1
m/z 638.498581 MH+ (calcd 638.499579 for C37H68O7N); H
NMR* (CDCl3, 400 MHz) d 4.50 (m, H-4 trans), 4.35 (m, H-4
cis), 3.01±2.94 (m, H-2 trans), 2.92±2.87 (m, H-2 cis), 2.83 (dd,
J=3.4, 16 Hz, H-35b cis), 2.72 (m, H-35b trans), 2.53±2.47 (m,
H-3b cis), 2.34 (dd, J=10.4, 16.0 Hz, H-35a trans), 2.26 (dd,
J=10.4, 16.0 Hz, H-35a cis), 2.09±2.04 (m, H-3b trans), 1.88
(s, H-37), 1.55±1.40 (m, H-3a); 13C NMR* (CDCl3, 100 MHz)
d 178.7 (C-1 trans), 178.2 (C-1 cis), 155.5 (C-36 cis), 155.3 (C-
36 trans), 79.2 (C-4 cis), 79.0 (C-4 trans), 38.2 (C-2 cis), 36.6
(C-2 trans), 36.5, 35.4, 35.3 (C-3, C-5 and C-35 cis), 35.5, 35.2,
32.9 (C-3, C-5 and C-35 trans), 13.8 (C-37 cis), 13.7 (C-37
trans). Compound 2d: HRLSIMS m/z 654.494133 MH+
(calcd 654.494494 for C37H68O8N). *The assignments were
made by COSY 45, DEPT and HMQC.
16. 36-Dihydrogenated isoacetogenin derivatives. Compound
1e: [a]D +27ꢁ (c 3.1, EtOH); HRLSIMS m/z 647.484177 (calcd
647.486275 for C37H68O7+Na). Compound 2e: [a]D +8.4ꢁ (c
3.2, EtOH); HRLSIMS m/z 641.496251 (calcd 641.499245 for
C37H69O8). The elucidations were made by EIMS, COSY 45,
DEPT, TOCSY, HMQC and HMBC.
12. 2,35-Dihydroxylated ACG derivatives: relevant values.
Compound 1b: [a]D +30ꢁ (c 0.1, EtOH); HRLSIMS m/z
1
657.495061 MH+ (calcd 657.4941 for C37H69O9); H NMR*
(CDCl3, 400 MHz) d 4.70 and 4.10 (2m, H-4), 4.36 and 3.95
(2m, H-36), 3.77 and 3.75 (2d, J=8, 6.4 Hz, H-35), 2.26, 2.18,
2.10 and 1.77 (4dd, J=6, 14 Hz, H-3), 1.47 and 1.35 (2d,
J=6.8, 6.0 Hz, H-37); 13C NMR* (CDCl3, 100 MHz) d 176.9
and 175.2 (C-1), 80.2 and 75.0 (C-2), 79.7 and 69.6 (C-36), 79.2
and 68.9 (C-4), 77.9 and 73.4 (C-35), 40.5 and 36.4 (C-3), 18.0
and 20.5 (C-37). Compound 2b: [a]D +6ꢁ (c 3.3, EtOH);
17. Estornell, E.; Fato, R.; Palotti, F.; Lenaz, G. FEBS Lett.
1993, 332, 127.
18. Tormo, J. R.; Gonzalez, M. C.; Cortes, D.; Estornell, E.
Arch. Biochem. Biophys. 1999, 369, 119.
19. Gonzalez, M. C.; Tormo, J. R.; Bermejo, A.; Zafra-Polo,
M. C.; Estornell, E.; Cortes, D. Bioorg. Med. Chem. Lett.
1997, 7, 1113.