Epoxy-acetogenins and other polyketide epoxy derivatives as inhibitors of the mitochondrial respiratory chain complex I

Article abstract of DOI:10.1055/s-2000-8545

Annonaceous acetogenins (ACG), an extensive group of cytotoxic natural products, are antitumor agents whose main mode of action is inhibition of the mammalian mitochondrial complex I. Herein we describe the importance of the different chemical groups along the alkyl chain for optimal inhibitory potency, discussing the structurally relevant factors present in these compounds. For this purpose, a series of epoxide derivatives from α- linolenic acid were prepared and their activity compared with that of epoxy- acetogenins and tetrahydrofuranic (THF) acetogenins isolated from Rollinia membranacea.

Full text of DOI:10.1055/s-2000-8545

318
Original Paper
Epoxy-Acetogenins and other Polyketide Epoxy Derivatives as
Inhibitors of the Mitochondrial Respiratory Chain Complex I
1
1
1
2
1,*
JosØ R. Tormo , M. C. Zafra-Polo , Angel Serrano , Ernesto Estornell , Diego Cortes
1
Departament de Farmacologia, Farmacognòsi i Farmacodinàmia, Facultat de Farmàcia, Universitat de Val›ncia, Burjassot, Val›ncia, Spain
2
Departament de Bioquímica i Biologia Molecular, Facultat de Farmàcia, Universitat de Val›ncia, Burjassot, Val›ncia, Spain
Rev Ri sei oc en i va ec dc e: pJ ut el yd :2 D8 ,e 1c 9e 9m 9 b; e Ar c 5c ,e 1p 9t 9e 9d ;: RD ee c ceei vme bd e: rJ u 5l y, 12 98 9, 91 999
Abstract: Annonaceous acetogenins (ACG), an extensive group
of cytotoxic natural products, are antitumor agents whose
main mode of action is inhibition of the mammalian mitochon-
drial complex I. Herein we describe the importance of the dif-
ferent chemical groups along the alkyl chain for optimal inhibi-
tory potency, discussing the structurally relevant factors pres-
ent in these compounds. For this purpose, a series of epoxide
derivatives from a-linolenic acid were prepared and their activi-
ty compared with that of epoxy-acetogenins and tetrahydrofur-
anic (THF) acetogenins isolated from Rollinia membranacea.
Materials and Methods
Chemical experimental procedures
Opticalrotations were measured with a Perkin-E lm er 241 po-
larimeter. IR spectra (film) were recorded on a Perkin-Elmer
843 infrared spectrophotometer. CIMS and LSIMS were deter-
mined on a VG Auto Spec Fisons spectrometer. NMR spectra
were taken on a Varian Unity-400 spectrometer at 400 MHz
for 1H, and 100 MHz for C, using CDCl3 (d 7.26 ppm and d
13
13
77.0 ppm) as reference standard. Multiplicities of C-NMR
Key words: Annonaceae, Rollinia membranacea, epoxy-aceto-
genins, tripoxyrollin, diepomuricanin A, membrarollin, a-lino-
lenic acid derivatives, inhibitors of mitochondrial complex I.
resonances were assigned by DEPT experiments. COSY 45 and
HMQC correlations were performed using a Varian Unity-
400 MHz instrument. Column chromatography was carried
out over silica gel 60H (Merck 7736). Analytical TLC was per-
formed on Merck precoated silica gel 60 F254 plates and spots
were detected by spraying with phosphomolybdic acid.
Introduction
Extraction and isolation from plant material
Annonaceous acetogenins (ACG) have attracted much interest
because of their cytotoxic, antitumor, parasitic and insecticide
activities (1±3). These biological effects have been related
with the ability to inhibit the NADH:ubiquinone oxidoreduc-
tase (complex I) of the mitochondrial electron transport chain
Tripoxyrollin (1), diepomuricanin-A (2), and membrarollin (4)
were isolated by our group from Rollinia membranacea (Anno-
naceae) seeds, as previously described (8±10).
(
4±6). To study the structurally relevant factors for the inhibi-
Other products
tory potency of these compounds we decided to select a series
of epoxy ACG, potentialprecursors of the THF-ACG (7), and a
series of alkyl chain epoxide derivatives without the methyl-
g-lactone characteristic of the ACG. This is the first study that
describes the inhibitory action of epoxy-ACG: tripoxyrollin (1)
and diepomuricanin A (2) (8, 9), and compares their potency
with the ones observed for THF-acetogenins (4), and alkyl
chain epoxide derivatives (3b±3d) semisynthesized from the
unsaturated fatty acid, a-linolenic acid, (3) (Fig.1). The rele-
vance of the number of epoxy groups and the presence or the
absence of the lactone ring is discussed in an attempt to
establish a structure-activity relationship (SAR).
Pure a-linolenic acid (3) and rotenone (5) were purchased
from Sigma Chemical Co. (St. Louis, MO, USA). Salts and sol-
vents were from Merck (Darmstadt, Germany).
Chemical transformations of a-linolenic acid (3)
Methylation of 3: Concentrated H SO (5 drops) was added to
2
4
a solution of 3 (117 mg, 0.42 mmol) in MeOH (20 ml) and the
mixture stirred and refluxed for 2 h. After concentration of
the solvent, 5% NaHCO3 (aq.) up to pH 7 was added to the
mixture which was extracted with CH Cl2 (3 ” 25 ml). Then
2
the organic layer was washed with H O, dried over anhydrous
2
Na SO and evaporated to yield 3a (115 mg, 95%).
2
4
Epoxidation of 3a: m-CPBA (306 mg) in dry CH Cl (20 ml) was
2
2
added dropwise during 1 h to a stirred solution of 3a (107 mg,
.36 mmol) in dry CH Cl (20 ml) at 0 8C. The reaction mixture
0
2
2
Planta Medica 66 (2000) 318±323
was then maintained at room temperature for 2 h, washed
ꢀ
Georg Thieme Verlag Stuttgart
·
New York
with 10% Na CO3 (2 ” 30 ml), saturated NaCl (20 ml), dried
2
ISSN: 0032-0943
over anhydrous NaO4 and evaporated. The residue (125 mg)

Epoxy-Acetogenins and other Polyketide Epoxy Derivatives
Planta Med. 66 (2000) 319
Fig.1 Structures of compounds assayed as complex I
inhibitors.
was purified by CC on silica gel with n-hexane-EtOAc (8:2) to
yield 3b (7 mg, 5.8%) and 3c (77.7 mg, 63.9%).
Methyl 9,10,12,13,15,16-triepoxyoctadecanoate (triepoxy-a-li-
nolenic methyl ester, 3c): C H O ; Optically inactive color-
19
32
5
±1
less oil; IR (CH Cl ): n
= 1735 cm ; LSI-MS: m/z = 341
2
2
max
+
+
Opening of the 12,13-oxirane ring in 3c: The triepoxide 3c
[MH] ; CIMS: m/z = 341 [MH] , 323, 305, 291, 275, 273, 268,
(
(
40 mg, 0.117 mmol) in MeOH/H O (8:2; 10 ml) and 1 N NaOH
2 ml) was stirred at 50 8C for 3 h. The mixture was cooled to
255, 243, 237, 225, 221, 213, 199, 185, 167, 155, 141, 137, 127,
109, 97, 85, 71; H-NMR (400 MHz) and C-NMR (100 MHz),
2
1
13
room temperature, and neutralized with 5% HCl. Then it was
saturated with NaCland extracted with CH 2Cl2 (3 ” 25 ml).
The extracts were dried over anhydrous Na SO and concen-
Tables 1 and 2.
12,13-Diacetoxy-9,10,15,16-diepoxyoctadecanoic acid (diepoxy-
diacetylated a-linolenic acid, 3d): C H O ; Optically inactive
2
4
trated to dryness. Pyridine (1.5 ml) and Ac O (3 ml) were add-
2
22 36
8
±1
ed and the mixture was allowed to stand for 12 h. A usual
work-up gave a residue (50 mg) which was purified by flash
chromatography with CH Cl :MeOH (98:2) to provide 3d
colorless oil; IR (CH Cl ): n
= 1734 cm ; LSI-MS: m/z = 429
2
2
max
+
+
+
[MH] ; CIMS: m/z = 429 [MH] , 411 [MH ± H O] , 401 [MH ±
2
+
+
+
CO] , 369 [MH ± AcOH] , 341 [401 ± AcOH] , 309 [MH ±
2
2
+
1
(
42 mg, 81.2%).
2AcOH] , 291, 281, 249, 211, 185, 155, 109; H-NMR (400 MHz),
Table 1; 13C-NMR (100 MHz): d 179.4 (COOH), 170.7 and 170.6
(OCOMe), 21.1 and 21.0 (OCOMe).
Physical properties
Methyl Z,Z,Z-octadeca-9,12,15-trienoate (a-linolenic methyl es-
Bioassay experimental procedures
ter, 3a): Colorless viscous oil; IR (CH Cl ): n
= 1733 (CO es-
2
2
max
±1
+
1
13
ter) cm ; LSI-MS: m/z = 293 [MH] ; H-NMR and C-NMR,
Tables 1 and 2.
The bioactivity of the compounds was assayed using submito-
chondrialpartic el s (SMP) from beef heart. These SMP were
obtained by extensive ultrasonic disruption of frozen-thawed
mitochondria in such a way to produce open membrane frag-
ments where permeability barriers to substrates were lost. Fi-
nally, they were ultracentrifugated, resuspended in 250 mM
sucrose, 10 mM Tris-HClpH 7.4 buffer, and stored frozen at
±80 8C (11). For the inhibitor titrations, compounds were ini-
tially diluted in absolute ethanol at 15 mM. These stock solu-
tions were kept in the dark at ±20 8C. Appropriate dilutions
Methyl 9,10,15,16-diepoxy-octadec-12(Z)-enoate (diepoxy-a-li-
nolenic methyl ester, 3b): C H O ; Optically inactive color-
1
9
32
4
±
1
less oil; IR (CH Cl ): n
= 1735 cm ; LSI-MS: m/z = 325
MH] ; CIMS: m/z = 325 [MH] , 309, 307, 293, 275, 253, 239,
21, 199, 185, 167, 155, 137, 125, 109, 97; H-NMR (400 MHz)
and C-NMR (100 MHz), Tables 1 and 2.
2
2
max
+
+
[
1
2
13

3
20 Planta Med. 66 (2000)
JosØ R. Tormo et al.
Table 1 1H-NMR spectral data of compounds 3a±3d (CDCl
d in ppm).
, J in Hz,
3
between 2 and 10 mM were made before the titrations. Beef
±
1
heart SMP were diluted to 0.5 mg´ml in sucrose-Tris buffer
and treated with 300mM NADH to activate complex I. Increas-
ing concentrations of ethanolic solution of inhibitor were
added to this preparation with 5 min incubation on ice be-
tween each addition. Maximalethanolconcentration never
exceeded 2% of volume and control activity was not affected
by this concentration (5).
Proton Compounds
o
N
3
a
3
b
3
2
3
4
8
9
2.29 t (7.6)
1.61 m
2.30 t (7.6) 2.25 t (7.5) 2.31 t (7.6)
1.61 m
1.32 m
1.56 m
2.93 m
2.93 m
2.23 m
5.60 m
5.60 m
2.23 m
2.93 m
2.93 m
1.56 m
1.60 m
1.28 m
1.60 m
2.92 m
3.07 m
1.75 m
3.12 m
3.12 m
1.75 m
3.07 m
2.92 m
1.49 m
1.60 m
1.31 m
1.60 m
2.96 m
3.09 m
1.72 m
4.21 m
4.21 m
1.72 m
3.09 m
2.96 m
1.49 m
±7
1.30 m
2.06 m
After each addition of inhibitor the enzymatic activity was
±
1
5.35 m
measured at 22 8C in SMP diluted to 6±7 mg´ml in the cuv-
ette with 50 mM potassium phosphate buffer, pH 7.4, 1 mM
EDTA, as both, NADH oxidase and NADH:ubiquinone
10
11
12
5.35 m
2.80 brt (5.6)
5.35 m
(
NADH:DB) oxidoreductase activities. NADH oxidase activity
was measured as the aerobic oxidation of 75 mM NADH
following the decrease in absorbance at 340 nm (e = 6.22
mM±1 ´cm ) in an end-window photo-multiplier double-
beam spectrophotometer ATI-Unicam UV4-500. NADH:ubi-
quinone oxidoreductase was measured in the same manner
but with 30mM decylubiquinone (DB) as ubiquinone ana-
logue, in presence of 2 mM KCN and 2mM antimycin A (12).
Rotenone sensitivity was routinely assessed in all samples for
both assays (11, 12). Data from four titrations were pooled
and fitted for graphics. IndividualIC 50 for each titration were
used for the statistic means and the standard deviations.
1
1
1
1
1
1
3
4
5
6
7
8
5.35 m
2.80 brt (5.6)
5.35 m
±1
5.35 m
2.06 m
0.97 t (7.6)
1.05 t (7.6) 1.03/1.00 t 1.03/0.87 t
(
7.6)
(7.6)
OMe
3.66 s
3.66 s
3.61 s
OCOMe
OCOMe
2.03 s
2.06 s
Results and Discussion
13
Table 2
ppm)*.
C-NMR chemical shifts of compounds 3a±3c (CDCl ; d in
3
Chemistry
Carbon
N
Compounds
3
a-Linolenic acid (3) was taken as an appropriate substrate for
epoxidation. So, after methylation with MeOH and concentrat-
ed sulfuric acid the methyl ester derivative (3a) was epoxi-
dized with m-CPBA. Two compounds, 3b and 3c, were found
in the reaction mixture and further purified by column chro-
matography.
o
a
3
b
3
1
2
3
174.2
34.1
174.2
34.0
174.2
34.0
24.9
24.8
24.8
4
8
9
±7
29.1±29.5
27.1
29.0±29.3
27.4b
56.3a
57.1a
26.4
28.9±29.2
27.5
Compound 3b was obtained as a colorless oil. The LSI-MS
+
+
56.9/56.6a
53.7/54.1a
27.3b
showed ion peaks at m/z 347 [M + Na] and m/z 325 [MH] , in-
127.1
131.9
25.5
dicating a molecular formula of C H O . Extensive analysis
10
11
12
1
9
32
4
1
13
of the H- and C-NMR spectra (see Tables 1 and 2) together
with DEPT, H- H COSY and HMQC data indicated the pres-
1
1
a
128.2
128.2
25.5
126.8
126.8
53.7/54.1
ence of a diepoxide derivative. Two olefinic protons at d 5.60
53.7/54.1a
27.0b
13
14
15
16
17
18
1
in the H-NMR spectra and a multiplet resonance at d 2.93, in-
26.4
58.3a
56.3a
27.7b
10.6
51.4
tegrating for four protons, also indicated the presence of two
epoxy rings. A unique carbon signalresonance at d 126.8 es-
tablished the double bond between C-12 and C-13 and the
placement of the two epoxy groups at C-9, C-10 and C-15, C-
53.7/54.1a
58.1/57.8a
127.7
130.2
20.5
27.8
14.2
10.4/10.3
51.4
16. This was also supported by the presence of two pairs of
OMe
51.4
fragment ions at m/z 167/155 and 137/125 towards the termi-
nalmethylgroup, and a peak at m/z 253 (cleavage at C-14/C-
*
Assignments from DEPT andHMQC da ta.
15 towards the ester group).
a, b
Assignments may be interchangedwithin the column.
Compound 3c, observed as only one spot in TLC, was obtained
+
as an oil. The LSI-MS exhibited peaks at m/z = 363 [M + Na]
dation because the two faces of the olefin are interconverted
by rotation. Therefore 3b presumably was the meso-diaster-
eomer. We found no way to separate these meso-derivatives
and so they were assayed as the mixture for their biological
activity.
+
and m/z 341 [MH] which agreed with a molecular formula
C H O and with the formation of a tri-epoxy derivative. Ex-
19
32
5
1
13
amination of both H- and C-NMR evidenced a series of dou-
ble signal resonances (see Tables 1 and 2), suggesting that 3c
could be a mixture of meso-triepoxides obtained from the
diepoxide 3b during the epoxidation reaction of 3a. As de-
scribed by Hoye and Suhadolnik (13), if 3b is the all-S- or the
all-R-diastereomer only one compound will arise from epoxi-
The triepoxide 3c treated first with NaOH and then acetylated
by pyridine/Ac O, yielded 3d. The CIMS of 3d showed a peak
2
+
at m/z 429 [MH] for a molecular formula of C H O . Se-
22
36
8

Epoxy-Acetogenins and other Polyketide Epoxy Derivatives
Planta Med. 66 (2000) 321
+
quentia lol sses of two 60 mass units from the MH
in the
Fig. 2 Titrations of a-lino-
lenic acid de rivatives as res-
piratory chain inhibitors.
1
13
CIMS, as well as the H- and C-chemicalshifts at d 2.03/2.06
and d 170.7/170.6, respectively, suggested the presence of a di-
acetoxy derivative. Carefulexamination of fragment ions in
the CIMS and the 1H- H COSY spectraldata confirmed the
opening of the oxirane ring placed at C-12/C-13.
(
(
n) a-linolenic methyl ester
3a); (l) di-epoxy-a-linolen-
1
ic methyl ester (3b); (~)
tri-epoxy-a-linolenic methyl
ester (3c); (^) di-epoxy-di-
acetylated-a-linolenic acid
Bioassays of a-linolenic acid derivatives
(
(
3d). Control acitivities
±
1
±1
mmol´min ´mg )
were
It has been well established that the ACG are potent specific
inhibitors of the mitochondrialcomp le x I (NADH:ubiquinone
oxidoreductase (4±6). Complex I is a limiting step in the pro-
duction of cell energy by the mitochondria. Therefore, inhibi-
tion of this enzyme compromises the tumor cell metabolism
because of the high demand of energy production needed for
tumor growth (1±3).
0
.96  0.09 for NADH oxi-
dase and 0.57  0.11 for
NADH:DB oxidoreductase.
The study of the different epoxy moieties along the alkyl
chain of ACG was the main purpose of this research. In that
way, the a-linolenic acid derivatives obtained (3b±3d), two
natural epoxy-acetogenins, tripoxyrollin (1) and diepomurica-
nin A (2), and a bis-THF ACG, membrarollin (4), were biologi-
cally tested as inhibitors of mitochondrial respiratory chain. A
classical inhibitor of the mitochondrial complex I, rotenone
(5), was also used for reference.
The assays included not only the whole respiratory chain, but
also the complex I specific activity. In the first case, an NADH
oxidase assay was performed. It represents an integrated ac-
tivity in which NADH is oxidized, and the electrons are trans-
ferred along the respiratory chain to be finally accepted by
molecular oxygen. As acetogenins only inhibit mitochondrial
complex I (4±6), the inhibition of this activity is directly at-
tributed to the inhibition of complex I. Measurement of com-
plex I specific activity was performed as the NADH:ubiqui-
none oxidoreductase assay with decylubiquinone (DB) as an
ubiquinone analogue. This activity, referred to as the
NADH:DB assay, is a less physiological assay because a high
quantity of the quinone is added. This measurement presents
lesser-observed enzymatic activities compared with those of
the NADH oxidase assay, probably due to a limited solubility
of the DB in the assay media (14). Moreover, inhibitors are
partially displaced by DB (15, 16) showing greater IC50 values
compared with those obtained in the NADH oxidase assay.
Nevertheless, the NADH:DB assay is a good measurement of
the complex I specific activity (12, 14) and it is needed in the
studies of complex I inhibitors for adequate comparisons (16)
despite its limitations.
It has been suggested that hydrophobicity is a key factor to
understand the SAR of a complex I inhibitor (15). Differential
solubility in the lipophilic membrane bilayer could affect the
access of the inhibitor to the enzyme. A too hydrophilic com-
pound will not be able to get dissolved in the membrane in
sufficient amounts to be active. A too hydrophobic compound
will be retained by the membrane and will not reach the en-
zyme core. We found by TLC that all 3a±3d compounds have
a similar hydrophobicity. This indicates that the differences
observed in the inhibitory potencies were produced by the
presence or the absence of the epoxy groups along the alkyl
chain. Therefore it was not merely an effect of a hydrophobici-
ty factor.
The IC50 values indicated that the presence of two distanced
epoxy moieties along the alkyl chain (3b) increases signifi-
cantly the potency of the inhibitor in the NADH oxidase assay
with regard to 3a. This pattern was also observed in the
NADH:DB assay but with less significant differences. More-
over, if an additionalepoxy ring is present ( 3c), the inhibitory
potency is clearly increased in both assays (Table 3). It is im-
portant to note that this structuralchange increased the
NADH:DB oxidoreductase inhibition by five fold for 3c with
respect to 3b, avoiding the decylubiquinone displacement by
one half (NADH:DB oxidoreductase/NADH oxidase ratio de-
creased from 4.7 to 2.4) which is indicative of a more tight
binding to the enzyme (16). The cleavage of the central epoxy
ring (3d) effected not only loss of potency but also a null and
void inhibition of the enzyme at the experimentalrange,
probably due to an esteric effect along the other epoxy groups
produced by the acetoxy moieties (see Table 3 for tentative
IC50 values for compound 3d).
Figure 2A shows the titration curves of a-linolenic acid deriv-
atives (3a±3d) against the NADH oxidase activity. All the four
compounds showed typicalhyperbo il c curves with a micro-
molar (mM) level potency. The triepoxide (3c) was the most
potent of the series. However the opening of the centralepoxy
ring in addition to the loss of the methyl ester group de-
creased the potency of the compound with the result that 3d
was the weakest inhibitor of the study (Table 3). Compounds
3
a and 3b exhibited an intermediate potency. The titration
curves for compounds 3a±3d against the specific complex I
activity are represented in Fig. 2B. All the a-linolenic acid de-
rivatives had similar tendencies except 3b and 3a that
showed crossing curves. This little difference could be due to
interference with the externally added quinone (16).
Bioassays of epoxy-acetogenins
Isolated linear acetogenins are claimed as the biogenetic pre-
cursors of the epoxy-acetogenins (7, 8), and differ in the de-

3
22 Planta Med. 66 (2000)
JosØ R. Tormo et al.
Table 3 IC50 values for compounds assayed as complex I inhibitors.
Inhibitor
NADH
NADH:DB
Ratio
oxidase
oxidoreductase
A. Annonaceous acetogenins
tripoxyrollin (1)
IC50 (nM)
0.83  0.16
17.0  1.4
0.29  0.02
19.3  5.4
>250
23
>15
2.9
diepomuricanin-A (2)
membrarollin (4)
0.83  0.18
B. a-Linolenic acid derivatives
a-linolenic methyl ester (3a)
di-epoxy-a-linolenic methyl ester (3b)
tri-epoxy-a-linolenic methyl ester (3c)
di-epoxy-diacetylated-a-linolenic (3d)
C. Other complex I inhibitors
rotenone (5)
IC50 (mM)
3.69  0.11
2.85  0.37
1.17  0.16
>30
15.0  3.6
13.5  0.6
2.82  0.91
>60
4.1
4.7
2.4
±
IC50 (nM)
5.1  0.9
28.8  1.5
5.6
Fig. 3 Titrations of aceto-
genins androtenone as res-
piratory chain inhibitors.
gree of unsaturation and hydroxylation of their alkyl chains
1±3). They are similar to the a-linolenic derivatives of our
(
study. Naturalepoxy-acetogenins, originated by oxidation of
linear olefinic acetogenins, are also key metabolites in the bio-
synthesis pathway of mono-, bis- or tri-THF acetogenins. The
existence of three epoxy groups at the proper carbon distance
affords in biogenetic conditions an a,a¢-dihydroxylated-bis-
THF acetogenin (7). For that reason we also compared the in-
hibitory potency values found for tripoxyrollin (1) with those
of membrarollin (4), an a,a¢-dihydroxylated-bis-THF acetoge-
nin with a threo/cis/threo/cis/erythro relative configuration
(
n) tripoxyrollin (1); (l) die-
pomuricanin-A (2);
(~)
membrarollin (4); (^) rote-
none (5). Control activities
±
1
±1
(
0
mmol´min ´mg )
were
.94  0.09 for NADH oxi-
dase and 0.56  0.09 for
NADH:DB oxidoreductase.
(
10). From IC50 values (Table 3) we can deduce that the tetra-
hydrofuran formation increases the potency against both res-
piratory-chain activities.
Figure 3 shows titrations of both NADH oxidase (3A), and
NADH:DB oxidoreductase (3B) activities. All natural ACG
showed inhibitory potency at the nano-molar (nM) level. The
presence of the terminalmethy -l g-lactone moiety (as in 1, 2,
and 4) increased the potency of the compounds by three or-
ders of magnitude with regard to 3a±3d. This demonstrated
that the lactone moiety is a key factor in the interaction of the
acetogenin with the enzyme, in agreement with recent stud-
ies on the membrane conformations of severalACG and their
cytotoxic effects (17). The same SAR found for the a-linolenic
acid derivatives was present in the epoxy-acetogenins because
the presence of a third epoxy ring increased inhibition of both
NADH oxidase and NADH:DB oxidoreductase activities. More-
over, it is important to note that the presence of the lactone
moiety gives the compound the ability to inhibit the mammali-
an complex I at a range similar to that found for other classical
potent complex I inhibitors like rotenone (5) (Tabl e3).
This study contributes to our knowledge of the natural Anno-
naceous acetogenins. SAR conclusions open a new perspective
in the study of these compounds and focus the research ef-
forts on the study of the rules that make the methyl-g-lactone,
tetrahydrofuran, and epoxy rings along the alkyl chain so im-
portant, and in that order, for improving the drug design of
this family of future antitumor drugs.
All these data can be summarized in the following structure-
activity relationship conclusions: (i) An increasing number of
epoxy rings improves the inhibitory potency of both, acetoge-
nins and alkyl chain derivatives without a terminal methyl-g-
lactone, (ii) tetrahydrofuranic acetogenins are more potent
than epoxy-acetogenins, being the last ones followed in po-
tency by alkyl chain epoxides without the lactone moiety, and
Acknowledgements
This research was supported by the Spanish CICYT under
grant SAF97-0013. We wish to thank the Spanish Ministerio
de Educación y Cultura for the scholarship grant to J. R. T.
(
iii) the terminalmethy l- g-lactone of the ACG is a relevant fac-
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Departamento de Farmacología
Facultad de Farmacia
Avda. Vicent AndrØs EstellØs s/n
3
46100-Burjassot
4
Valencia
Spain
E-mail: dcortes@uv.es
Fax: +34-96-3864943
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