Novel inhibitors of the mitochondrial respiratory chain: Oximes and pyrrolines isolated from Penicillium brevicompactum and synthetic analogues

Article abstract of DOI:10.1021/jf058075f

The capacity of inhibition of the mammalian mitochondrial respiratory chain of brevioxime 5a, a natural insecticide compound isolated from Penicillium brevicompactum culture broth, and another 15 analogue compounds, other oximes 5b and 5c; two diastereome

Full text of DOI:10.1021/jf058075f

8296 J. Agric. Food Chem. 2005, 53, 8296−8301
Novel Inhibitors of the Mitochondrial Respiratory Chain:
Oximes and Pyrrolines Isolated from Penicillium
brevicompactum and Synthetic Analogues
AÄ NGEL CANTIÄN,† M. PILAR LOÄ PEZ-GRESA,† M. CARMEN GONZAÄ LEZ,§
PILAR MOYA,*,† MIGUEL AÄ NGEL MIRANDA,† JAIME PRIMO,† VANESSA ROMERO,‡
‡
EVA PERIS,‡ AND ERNESTO ESTORNELL
Centro de Ecolog´ıa Qu´ımica Agr´ıcola, Universidad Polite´cnica de Valencia, Campus de Vera, Edificio
9B, Laboratorio 111, 46022 Valencia, Spain, and Departament de Bioqu´ımica i Biologia Molecular,
Facultat de Farma`cia, Universitat de Valencia, Avgda Vicent Andre´s Estelle´s s/n, 46100 Burjassot,
Valencia, Spain
The capacity of inhibition of the mammalian mitochondrial respiratory chain of brevioxime 5a, a natural
insecticide compound isolated from Penicillium brevicompactum culture broth, and another 15
analogue compounds, other oximes 5b and 5c; two diastereomeric pyrrolidines 1c′ and 1c′′; five
pyrrolines 3c′, 3c′′ (diastereomers between them), 3a, 3b, and 6; two oxazines 4c′ and 4c′′ (also
diastereomers between them); and four pyrrol derivatives 7-10, are analyzed in this paper.
Compounds 3b, 3c′, 3c′′, 4c′, 4c′′, 5b, 5c, 6, and 10 were found to be inhibitors of the integrated
electron transfer chain (NADH oxidase activity) in beef heart submitochondrial particles (SMP),
establishing that all of them except compound 3b and 6 only affected to complex I of the mitochondrial
respiratory chain. The most potent product was 5b, with an IC50 of 0.27 µM, similar to the IC50 values
of other known complex I inhibitors. The diastereomeric pairs 1c′/1c′′, 3c′/3c′′, 4c′/4c′′, and 5c have
not been previously described. Chemical characterization, on the basis of spectral data, is also shown.
KEYWORDS: Penicillium brevicompactum; brevioxime; oximes; oxazines; pyrrols; pyrrolines; pyrro-
lidines; inhibitors of mitochondrial respiratory chain; complex I; insecticide; fungicide
INTRODUCTION
as an inhibitor of juvenile hormone biosynthesis (Figure 1) (8).
Thereafter, derivative compounds of 5a, showing important
insecticidal and fungicidal activities, were found (9-14). The
interesting nature of these compounds has encouraged other
research groups to synthesize 5a and other analogues (15-20).
In this report, we analyze whether brevioxime and analogues
can inhibit the mitochondrial electron transport system.
Over the past years, new potent natural and synthetic
insecticides have been described as inhibitors of complex I of
the mitochondrial respiratory chain (1-7). The present knowl-
edge of complex I is primarily based on studies with bovine
heart and Neurospora crassa enzymes, whereas little is known
about the structure of insect complex I (1). Enzymatic and
immunochemical evidence indicate a high degree of similarity
to their mammalian and fungal counterparts. The study of
mammalian complex I inhibition by new potential insecticides
is relevant by two opposite reasons. On one hand, an inhibitory
effect would explain their mechanism of action similarly to other
previously described insecticides. On the other hand, lack of
inhibition could represent low toxicity against mammals, at this
level.
MATERIALS AND METHODS
General Experimental Procedures. Chemicals were obtained from
commercial suppliers and used without further purification. IR spectra
were obtained with a 710FT spectrophotometer (Nicolet, Madison, WI).
1H, 13C, and COSY H-H NMR spectra were recorded on a Gemini
300 MHz instrument (Varian, Walnut Creek, CA). The assignment of
13C signals is supported by DEPT experiments. Mass spectra were
obtained under electron impact with a VG AutoSpec spectrometer
(Fisons, Manchester, United Kingdom). Thin-layer chromatography was
run on silica gel F254 precoated plates (Merck), and spots were detected
under UV light.
In 1997, Moya et al. isolated from Penicillium breVicompac-
tum a natural product, brevioxime 5a, which was demonstrated
Biological Material. Submitochondrial particles (SMP) for biological
assays were obtained from beef heart following Fato et al. (21).
Extensive ultrasonic disruption of frozen-thawed mitochondria was
carried out to produce open membrane fragments.
Biological Methods. The inhibitory potency of the compounds was
assayed by using SMPs from beef heart. SMPs were diluted to 0.5
* To whom correspondence should be addressed. Tel: 34 96 3879058.
Fax: 34 96 3879059. E-mail: mmoyasa@ceqa.upv.es.
† Universidad Polite´cnica de Valencia.
‡ Universitat de Valencia.
§ Present address: Instituto Valenciano de Investigaciones Agrarias,
Departamento de Citricultura, Carretera Na´quera-Moncada, km. 4.5, 46113,
Moncada, Valencia, Spain.
10.1021/jf058075f CCC: $30.25
© 2005 American Chemical Society
Published on Web 09/23/2005

New Inhibitors of the Mitochondrial Respiratory Chain
J. Agric. Food Chem., Vol. 53, No. 21, 2005 8297
activity) was measured by changing NADH for 12.5 mM succinate
and following the reduction of DB at 378 nm (ꢀ 14.0 mM-1 cm-1).
SMPs were preincubated for 10 min in the presence of 1.25 mM
succinate to fully activate complex II.
Synthesis of Natural Products and Their Analogues. The synthesis
of compounds 3a, 3b, and 6-10 was carried out as described in our
previous work (9-11, 13). In addition, Scheme 1 shows the new
brevioxime synthetic route, which is based on that previously described
by Clark (18) and from which byproducts 1c′/1c′′, 3c′/3c′′, 4c′/4c′′,
5a, 5b, and 5c were obtained. This route presents the following five
steps.
First Step (i, ii): Synthesis of Hydroxyketoamides (1). To a 2.0
M solution of lithium diisopropylamide (LDA) in anhydrous tetrahy-
drofuran (THF) (30 mmol) at -78 °C was slowly added 3-triethylsi-
liloxy-N-propionylpyrrolidine (20 mmol) dissolved in anhydrous THF
(15 mL) (13). After the mixture was stirred for 2 h, the corresponding
ester (10 mmol) was added dissolved in anhydrous THF (10 mL)
shaking for 6 h. Then, the mixture was quenched by addition of a
saturated solution of ammonium chloride, warmed at ambient temper-
ature, and extracted with diethyl ether. At that point, â-ketoamide signals
were observed in the NMR spectra; therefore, no further purification
was carried out. Thus, the crude was dissolved in anhydrous THF (60
mL), and acetic acid (10 mmol) and a 1.0 M solution of Bu4NF (10
mmol) were added. The mixture was stirred at ambient temperature
before being diluted with ethyl acetate and washed with water and a
saturated solution of NaHCO3. The combined organic extracts were
concentrated and purified by column chromatography over silica gel
(gradient elution with mixtures of Hex and EtOAc, with 50-100%
EtOAc). According to this step, two compounds, 1c′/1c′′, were
synthesized.
3-Hydroxy-N-[2-methyl-4-(3-phenoxyphenyl)-3-oxopentanoyl]-
pyrrolidine (1c). In these conditions, only two diastereomeric alcohols
could be clearly resolved. The first eluted diastereomer (1c′, Rf ) 0.78
in EtOAc) was obtained with a 18% yield and exhibited the following
spectral data. HRMS m/z 367.1717 (C22H25NO4 requires 367.1783).
IR: γmax 3500-3100, 2970, 2929, 2878, 1721, 1613, 1495, 1429, and
1
1244. H NMR: δH 7.3-6.8 (m, 9H, Ar-H), 4.3 (m, 1H, H-3), 3.8
(m, 1H, H-2a), 3.5-2.8 (m+m+m, 5H, H-2b, H-5, H-2′, H-4′), 1.8
(m, 2H, H-4), 1.4, and 1.3 (d+d, J ) 7 Hz, 6H, 2 × CH3). 13C NMR:
δC 206.3 (C3′), 169.6 (C1′), 157.7, 156.6, 142.8 (C1′′, C3′′, C1′′′), 130.3,
129.8, 123.7, 122.8, 118.9, 118.9, 117.2 (C2′′, C4′′-C6′′, C2′′′-C6′′′), 70.9
(C3), 54.4 (C2), 51.1, 49.7 (C2′, C4′), 44.5 (C5), 34.0 (C3), 18.4, and
13.2 (2 × CH3). MS m/z 367 (M+, 100), 224 (6), 197 (27), 170 (29),
143 (72), and 114 (84).
Figure 1. Structures of assayed oximes (5a
(1c and 1c′′), pyrrolines [3c , 3c′′ (diastereomers), 3a, 3b, and 6],
diastereomer oxazines (4c and 4c′′), and pyrrols (7 10).
−c), diastereomer pyrrolidines
′
′
The second eluted diastereomer (1c′′, Rf ) 0.73 in EtOAc) obtained
in 40% yield showed the following spectral data. HRMS m/z 367.1784
(C22H25NO4 requires 367.1783). IR: γmax 3500-3100, 2970, 2929,
′
−
1
2878, 1721, 1628, 1495, 1429, and 1239. H NMR: δH 7.4-6.8 (m,
mg/mL in 250 mM sucrose and 10 mM Tris-HCl buffer, pH 7.4, and
treated with 300 µM NADH in order to activate complex I before
starting the experiments. The enzymatic activity was assayed at 22 °C
in 50 mM potassium phosphate buffer, pH 7.4, and 1 mM EDTA with
SMP diluted to 6 µg/mL. Stock solutions (15 mM in absolute EtOH)
of 1c′, 1c′′, 3a, 3b, 3c′, 3c′′, 4c′, 4c′′, 5a, 5b, 5c, and 6-10 were
prepared and kept in the dark at -20 °C. Each compound was added
to the diluted SMP preparation and incubated, during 5 min, in ice.
NADH oxidase activity was measured as the aerobic oxidation of 75
µM NADH. Reaction rates were calculated from the linear decrease of
NADH concentration (λ 340 nm, ꢀ 6.22 mM-1 cm-1) in an end window
photomultiplier spectrophotometer ATI-Unicam UV4-500. For each
compound, three experiments were carried out.
9H, Ar-H), 4.4 (m, 1H, H-3), 4.0 (m, 1H, H-2a), 3.7-2.9 (m+m+m,
5H, H-2b, H-5, H-2′, H-4′), 1.9 (m, 2H, H-4), 1.4, and 1.3 (d+d, J )
7 Hz, 6H, 2 × CH3). 13C NMR: δC 206.0 (C3′), 171.0 (C1′), 158.1,
157.0, 143.5 (C1′′, C3′′, C1′′′), 130.6, 130.2, 124.0, 123.2, 119.3, 118.9,
117.9 (C2′′, C4′′-C6′′, C2′′′-C6′′′), 71.3 (C3), 55.0 (C2), 50.8, 50.5 (C2′, C4′),
45.0 (C5), 34.7 (C3), 18.8, and 13.5 (2 × CH3). MS m/z 367 (M+, 100),
224 (8), 197 (32), 170 (30), 143 (69), and 114 (75).
Second Step (iii): Synthesis of Mesylates (2). To a solution of the
corresponding alcohol (1.0 mmol) in anhydrous CH2Cl2 (7.0 mL) was
added Et3N (1.3 mmol) and methane sulfonyl chloride (1.1 mmol). The
mixture was stirred at ambient temperature overnight and then diluted
with 1.0 M HCl. After it was extracted with CH2Cl2, the crude was
purified by column chromatography over silica gel (gradient elution
with mixtures of Hex and EtOAc, with 50-100% EtOAc). According
to this step, two mesylates, 2c′/2c′′, were synthesized.
Cytochrome c reductase activity sustained by succinate (integrated
activity of complexes II and III) was measured while monitoring the
reduction of 40 µM ferricytochrome c at 550 nm (ꢀ 19.1 mM-1 cm-1
)
in the presence of 2 mM KCN to block its reoxidation. The reaction
was started with 13.5 mM succinate.
3-Methanesulfonyloxy-N-[2-methyl-4-(3-phenoxyphenyl)-3-oxo-
pentanoyl]pyrrolidine (2c). The mesylates corresponding to the
previously described alcohols were obtained with 80-85% yields. The
mesylate obtained from the first described alcohol (2c′, Rf ) 0.63 in
EtOAc) showed as spectral data. HRMS m/z 445.1591 (C23H27NO6S
requires 445.1559). IR: γmax 2970, 2934, 2883, 1726, 1644, 1480, 1429,
NADH:ubiquinone oxidoreductase (specific complex I activity) was
measured with 75 µM NADH and 30 µM decylubiquinone (DB) as a
soluble short chain analogue of ubiquinone in the presence of 2 µM
antimycin and 2 mM potassium cyanide to block any reaction
downstream of complex I, following the NADH oxidation rate.
Similarly, succinate:ubiquinone oxidoreductase (specific complex II
1
1352, 1249, 1162, and 886. H NMR: δH 7.4-6.8 (m, 9H, Ar-H),
5.2 (m, 1H, H-3), 3.9 (m, 1H, H-2a), 3.8-3.2 (m, 5H, H-2b, H-5, H-2′,

8298 J. Agric. Food Chem., Vol. 53, No. 21, 2005
Cant´ın et al.
Scheme 1. General Scheme of the New Synthesis of Brevioxime 5a and the Other Analogues, Based on the Clark Method
Reagents and conditions: (i) 3-Triethylsililoxy-N-propionylpyrrolidine, LDA, THF,
−78 °C. (ii) Bu4NF, AcOH, THF. (iii) MeSO2Cl, Et3N, CH2Cl2. (iv) t-BuOK,
Martin periodinane, CH2Cl2. (viii) NH2OH HCl, Et3N, MeOH.
DMSO. (v) Oxone, NaHCO3, CF3COCF3, MeOH. (vi) PPTS, toluene. (vii) Dess
−
H-4′), 3.0 (s, 3H, SO2CH3), 2.2 (m, 2H, H-4), 1.4, and 1.3 (d+d, J )
7 Hz, 6H, 2 × CH3). 13C NMR: δC 205.0 (C3′), 166.8 (C1′), 156.3,
155.3, 140.4 (C1′′, C3′′, C1′′′), 128.7, 128.4, 122.2, 121.4, 117.5, 117.4,
116.1 (C2′′, C4′′-C6′′, C2′′′-C6′′′), 77.8 (C3), 50.6 (C2), 49.6, 48.5 (C2′, C4′),
42.2 (C5), 37.3 (SO2CH3), 31.4 (C3), 16.8, and 11.9 (2 × CH3). MS
m/z 445 (M+, 100), 248 (44), 221 (86), and 192 (96).
50.6, 45.5 (2 × CHCH3), 45.2 (C5), 28.1(C4), 18.3, and 12.9 (2 × CH3).
MS m/z 349 (M+, 35), 279 (6), 242 (10), 224 (18), 211 (10), 197 (100),
and 83 (43).
Fourth Step (v, vi): Synthesis of Alcohols (4). 1,1,1-Trifluoroac-
etone (1 mL), sodium bicarbonate (1.48 mmol), and oxone (1.00 mmol)
were added to a 0° C solution of acyl enamine (1.00 mmol) in methanol
(10 mL). The resulting mixture was warmed at room temperature and
stirred overnight. Then, it was diluted with water, extracted with CH2-
Cl2, and dried over Na2SO4. Without further purification, the mixture
was dissolved in toluene (40 mL) and PPTS (0.12 mmol) was added
to the resulting solution, which was heated at reflux for 3 h. After it
was cooled, the mixture was concentrated and purified by column
chromatography over silica gel using gradient elution with mixtures
of Hex and EtOAc (30-100% EtOAc). According this step, we
synthesized the compounds 4a′/4a′′, 4b′/4b′′, and 4c′/4c′′ [4a′/4a′′ and
4b′/4b′′ were synthesized from compounds 3a and 3b, respectively,
which we had previously obtained by other known route (9-11)].
2-Hept-5-enyl-6,7-dihydro-8(8aH)-hydroxy-3-methyl-4H-pyr-
role[2,1-b]-[1.3]oxazine-4-one (4a). In the above-described conditions,
two different alcohols were obtained. Their spectral data were fully
coincident with those described by Clark (18).
The spectral data of the mesylate obtained from the second alcohol
(2c′′, Rf ) 0.58 in EtOAc) were as follows. HRMS m/z 445.1590
(C23H27NO6S requires 445.1559). IR: γmax 2990, 2929, 2868, 1730,
1
1644, 1480, 1429, 1352, 1249, 1168, and 896. H NMR: δH 7.4-6.8
(m, 9H, Ar-H), 5.2 (m, 1H, H-3), 4.0 (m, 1H, H-2a), 3.9-3.3 (m, 5H,
H-2b, H-5, H-2′, H-4′), 3.0 (s, 3H, SO2CH3), 2.2 (m, 2H, H-4), and
1.3 (m, 6H, 2 × CH3). 13C NMR: δC 206.9 (C3′), 168.7 (C1′), 158.2,
157.1, 142.2 (C1′′, C3′′, C1′′′), 130.4, 130.3, 124.0, 123.2, 119.4, 118.8,
117.9 (C2′′, C4′′-C6′′, C2′′′-C6′′′), 79.7 (C3), 52.5 (C2), 52.3, 50.5 (C2′, C4′),
44.1 (C5), 39.3 (SO2CH3), 33.3 (C3), 18.7, and 14.0 (2 × CH3). MS
m/z 445 (M+, 10), 221 (28), 197 (76), and 149 (100).
Third Step (iv): Synthesis of Pyrrolines (3). To a solution of the
corresponding mesylate (0.6 mmol) in dimethyl sulfoxide (5 mL) was
added t-BuOK (12.6 mmol), and the mixture was stirred at ambient
temperature for 4 h. Ice was then added followed by a saturated solution
of ammonium chloride. The mixture was extracted twice with ethyl
ether, and the combined organic extracts were washed with water. After
that, the organic extract was purified by column chromatography over
silica gel (gradient elution with mixtures of Hex and EtOAc, with 50-
100% EtOAc). According to this step, two compounds, 3c′/3c′′, were
synthesized.
N-[2-Methyl-4-(3-phenoxyphenyl)-3-oxopentanoyl]-2-pyrroline (3c).
The pyrrolines corresponding to the previously described mesylates
were obtained in 30-40% yields as oils. The spectral data of the first
one (3c′, Rf ) 0.41 in Hex/EtOAc:70/30) were as follows. HRMS m/z
349.1646 (C22H23NO3 requires 349.1677). IR: γmax 3067, 2990, 2924,
2863, 1721, 1644, 1590, 1490, 1255, and 1152. 1H NMR: δH 7.3-6.8
(m, 9H, Ar-H), 6.3 (m, 1H, H-2), 5.3 (m, 1H, H-3), 4.0 (m, 1H, H-4′),
3.7-3.4 (m, 2H, H-5), 3.1 (m, 1H, H-2′), 2.6 (m, 2H, H-4), and 1.3
(m, 6H, 2 × CH3). 13C NMR: δC 205.6 (C3′), 166.0 (C1′), 158.6, 145.2,
142.0 (C1′′, C3′′, C1′′′), 130.2, 130.1, 123.7, 122.7, 119.4, 118.1, 117.4,
109.4 (C2-C3, C2′′, C4′′-C6′′, C2′′′-C6′′′), 52.5, 51.2 (2 × CHCH3), 41.8
(C5), 30.5(C4), 17.8, and 14.6 (2 × CH3). MS m/z 349 (M+, 36), 242
(5), 224 (14), 211 (9), 197 (87), and 83 (100).
The second 2-pyrroline obtained (3c′′, Rf ) 0.25 in Hex/EtOAc:70/
30) showed as spectral data. HRMS m/z 349.1601 (C22H23NO3 requires
349.1677). IR: γmax 2990, 2925, 1735, 1660, 1655, 1587, 1501, 1298,
and 1190. 1H NMR: δH 7.3 (m, 2H, H-3′′′+H-5′′′), 7.2 (m, 1H, H-5′′),
7.1 (t, J ) 8 Hz, 1H, H-4′′′), 7.0-6.8 (m, 5H, H-2′′, H-4′′, H-6′′, H-2′′′,
H-6′′′), 6.2 (m, 1H, H-2), 5.2 (m, 1H, H-3), 3.9 (q, J ) 7 Hz, 1H,
H-4′), 3.8-3.6 (m, 2H, H-5), 3.5 (q, J ) 7 Hz, 1H, H-2′), 2.5 (m, 2H,
H-4), and 1.4 (d+d, J ) 7 Hz, 6H, 2 × CH3). 13C NMR: δC 205.6
(C3′), 166.0 (C1′), 157.6, 142.4, 142.1 (C1′′,C3′′, C1′′′), 130.0, 129.8, 127.9,
123.5, 122.6, 118.9, 118.4, 117.1, 112.9 (C2-C3, C2′′, C4′′-C6′′, C2′′′-C6′′′),
2-Heptyl-6,7-dihydro-8(8aH)-hydroxy-3-methyl-4H-pyrrole[2,1-
b]-[1.3]oxazine-4-one (4b). In these conditions, two different alcohols
were obtained; the less polar one (4b′, Rf ) 0.32 in EtOAc) was
obtained with a 56% yield for the two steps. It showed as spectral
1
data. H NMR: δH 5.0 (d, J ) 4 Hz, 1H, H-8a), 4.5 (m, 1H, H-8),
3.6-3.4 (m 2H, H-6), 2.3-2.1 (m, 2H, H-7), 2.0 (m, 2H, H-1′), 1.8 (s,
3H, CH3), 1.5 (m, 2H, H-2′), 1.3 [m, 8H, (CH2)4CH3], and 0.9 (t, J )
7 Hz, 3H, CH2CH3). 13C NMR: δC 171.1 (C4), 163.6 (C2), 106.0 (C3),
92.2 (C8a), 74.9 (C8), 60.3 (C6), 41.8 (C1′), 31.6, 30.5, 28.9, 26.7, 22.5,
20.9 (C7, C2′-C6′), 14.0, and 9.9 (2 × CH3).
The more polar isomer (4b′′, Rf ) 0.23 in EtOAc) was obtained
with a 24% yield for the two steps. It showed as spectral data. 1H
NMR: δH 5.2 (d, J ) 4 Hz, 1H, H-8a), 4.4 (m, 1H, H-8), 3.7-3.5 (m
2H, H-6), 2.4-2.0 (m, 4H, H-7, H-1′), 1.8, (s, 3H, CH3), 1.5 (m, 2H,
H-2′), 1.3 [m, 8H, (CH2)4CH3], and 0.9 (t, J ) 7 Hz, 3H, CH2CH3).
13C NMR: δC 171.0 (C4), 163.0 (C2), 106.4 (C3), 87.6 (C8a), 70.4 (C8),
60.3 (C6), 41.5 (C1′), 31.6, 30.5, 28.8, 22.5, 20.9 (C7, C2′-C6′), 14.0,
and 9.8 (2 × CH3).
2-[1-(Phenoxyphenyl)ethyl]-8(8aH)-hydroxy-3-methyl-4H-pyr-
role[2,1-b]-[1.3]oxazine-4-one (4c). In these conditions, two different
alcohols were obtained; the less polar one (4c′, Rf ) 0.19 in Hex/EtOAc:
5/5) was obtained with a 24% yield for two steps. It showed the
following spectral data. HRMS m/z 365.161259 (C22H23NO4 requires
365.162708). IR: γmax 3400-3100, 2888, 1634, 1485, 1444, 1234, 922,
and 692. 1H NMR: δH 7.4-6.8 (m, 9H, Ar-H), 4.9 (d, 1H, J ) 3 Hz,
H-8a), 4.5 (m, 1H, H-8), 3.9 (q, 1H, J ) 7 Hz, H-1′), 3.6 (m, 2H,
H-6), 2.5 (m, 1H, H-7a), 2.2 (m, 1H, H-7b), 1.8 (s, 3H, CH3), and 1.4
(d, 3H, J ) 7 Hz, CH3). 13C NMR: δC 165 (C4), 158 (C2),145, 144,
130, 125, 124, 123, 122, 119, 118, 117 (C1′′-C6′′, C1′′′-C6′′′), 107 (C3),

New Inhibitors of the Mitochondrial Respiratory Chain
J. Agric. Food Chem., Vol. 53, No. 21, 2005 8299
93 (C8a), 75 (C8), 42 (C6), 40 (C1′), 30 (C7), 17 (CH3), and 10 (CH3).
MS m/z 365 (M+, 21), 280 (10), 252 (15), 224 (34), and 197 (100).
Table 1. Inhibitory Potency of Pirrolidines, Pyrrolines, Oxazines,
Oximes, and Pyrrol Compoundsa
The more polar isomer (4c′′, Rf ) 0.13 in Hex/EtOAc:5/5) was
obtained with a 15% yield for the two steps and showed as spectral
data. HRMS m/z 365.160703 (C22H23NO4 requires 365.162708). IR:
γmax 3400-3100, 2883, 1644, 1486, 1440, 1245, 922, and 687. 1H
NMR: δH 7.4-6.8 (m, 9H, Ar-H), 5.0 (d, 1H, J ) 3 Hz, H-8a), 4.4
(m, 1H, H-8), 4.0 (q, 1H, J ) 7 Hz, H-1′), 3.6 (m, 2H, H-6), 2.4-2.2
(m, 2H, H-7), 1.8 (s, 3H, CH3), and 1.4 (d, 3H, J ) 7 Hz, CH3). 13C
NMR: δC 164 (C4), 158 (C2),143, 130,123, 122, 119, 118, 117 (C1′′-
C6′′, C1′′′-C6′′′), 107 (C3), 93 (C8a), 75 (C8), 42 (C6), 40 (C1′), 30 (C7),
18 (CH3), and 9 (CH3). MS m/z 365 (M+, 39), 280 (15), 252 (19), 224
(35), and 197 (100).
NADH:ubiquinone
oxidoreductase
activity IC50 (µM)
NADH oxidase
activity IC50 M)
inhibitors
(µ
pyrrolidine skeleton
1c
1c′′
3c
3c′′
3a
3b
6
′
41.50
36.25
3.92
±
±
±
±
14.85
1.06
0.57
0.10
ND
ND
2-pyrroline skeleton
′
6.57
16.45
±
±
2.39
0.10
4.40
inactive
4.55
6.82
5.48
3.13
±
±
±
±
0.85
0.37
0.49
0.32
ND
ND
oxazine skeleton
oxime skeleton
4c
′
14.31
11.93
±
±
1.40
1.27
Fifth Step (vii, vii): Synthesis of Oximes (5). To a solution of the
less polar alcohol (0.55 mmol) in CH2Cl2 (10.0 mL), a 15 wt % solution
of Dess-Martin periodinane in CH2Cl2 (0.60 mmol) was added. After
it was stirred at room temperature for 41 h, the mixture was concentrated
and extracted with diethyl ether. The resulting crude was dissolved in
methanol (10 mL) and stirred overnight after adding hydroxylamine
hydrochloride (0.66 mmol) and triethylamine (0.66 mmol). The mixture
was diluted with ethyl acetate, washed with water, and dried over Na2-
SO4. A white dust was obtained in this manner, which was recrystallized
with Hex/EtOAc mixtures. According to this last step, the compounds
5a, 5b, and 5c were synthesized.
4c′′
5a
5b
5c
7
8
9
10
inactive
0.27
0.92
±
±
0.06
0.09
1.52
3.44
±
±
0.84
0.14
pyrrol skeleton
inactive
inactive
214.50
8.60
±
±
6.30
0.20
ND
14.30
±
0.14
a Values are means
± standard deviation of three assays; ND, nondetermined.
could be new inhibitors of the respiratory chain and more
specifically which complexes in the chain were affected (Figure
1).
Nine of the 16 assayed products, compounds 3b, 3c′, 3c′′,
4c′, 4c′′, 5b, 5c, 6, and 10, were found to be inhibitors of the
integrated electron transfer chain (NADH oxidase activity) in
beef heart SMPs with IC50 values lesser than 10 µM (Table 1).
Oximes 5b and 5c were the most potent inhibitors.
2-Hept-5-enyl-6,7-dihydro-3-methyl-4H-pyrrole[2,1-b]-[1.3]oxazine-
4,8(8aH)-dione 8-Oxime (5a). It was obtained with a 27% yield (two
steps). All data of the resulting product were coincident with the
literature (18).
2-Heptyl-6,7-dihydro-3-methyl-4H-pyrrole[2,1-b]-[1.3]oxazine-
4,8(8aH)-dione 8-Oxime (5b). It was obtained with a 23% yield (two
steps). Again, data of the resulting product were coincident with the
literature (16).
In the integrated electron transfer chain, electrons are first
carried from complex I (NADH:ubiquinone oxidoreductase) or
from complex II (succinate:ubiquinone oxidoreductase) to
complex III (ubiquinol:cytochrome c oxidoreductase) by ubiquino-
ne and then from complex III to complex IV (cytochrome c
oxidase) by the peripheral membrane protein cytochrome c. The
effect of 3c′, 3c′′, 4c′, 4c′′, 5b, 5c, and 10 on the activity of
complex I or complex II was assayed by using DB, following
the method of Estornell et al. (22). None of them affected the
specific activity of complex II, but they strongly reduced the
specific activity of complex I (Table 1). The effect of 3b and
6 on both complex I and complex II using DB could not be
determined, because both compounds showed a poor inhibitory
potency.
2-[1-(Phenoxyphenyl)ethyl]-3-methyl-4H-pyrrole[2,1-b]-[1.3]ox-
azine-4,8(8aH)-dione 8-Oxime (5c). It was obtained with a 13% yield
(two steps). Compound 5c was purified by semipreparative high-
performance liquid chromatography using the following conditions:
25.0 cm × 1.0 cm i.d., 5 µm, Kromasil column; mobile phase, Hex/
EtOAc (3/7, v/v); flow, 2 mL/min; detection by photodiode array and
refraction index, simultaneously; retention time (Rt) ) 17.1 min. It had
the following spectral data. HRMS m/z 378.156342 (C22H22N2O4
requires 378.157957). IR: γmax 3400-3100, 2970, 2924, 1644, 1588,
1
1495, 1434, 1250, 1209, 1163, 978, 758, and 702. H NMR: δH 7.9
(s, 1H, N-OH), 7.4-6.8 (m, 9H, Ar-H), 5.4 (s, 1H, H-8a), 4.2-4.0
(m, 2H, H-6), 3.4 (q, 1H, J ) 7 Hz, H-1′), 2.9 (m, 2H, H-7), 1.9 (s,
3H, CH3), and 1.4 (d, 3H, CH3). 13C NMR: δC 164 (C2), 163 (C4), 158
(C2),157, 144,130, 123, 121, 119, 117 (C1′′-C6′′, C1′′′-C6′′′), 107 (C3), 84
(C8a), 42 (C6), 40 (C1′), 24 (C7), 17 (CH3), and 9 (CH3). MS m/z 378
(M+, 12), 280 (9), 252 (12), 224 (19), and 197 (100).
In addition, we could establish for other assays that 3c′, 3c′′,
4c′, 4c′′, 5b, 5c, 10, 3b, and 6 did not affect complex III. To
demonstrate this, these nine compounds were added in the
presence of succinate as a substrate to complex II; in these
conditions, succinate cytochrome c was not affected.
RESULTS AND DISCUSSION
Recently, the isolation of brevioxime (5a) and other bioactive
metabolites from the P. breVicompactum culture broth has been
reported (8). During the chemical synthesis of these compounds
in order to confirm their structures, a number of intermediates
showing interesting insecticidal and fungicidal activities were
obtained (Figure 1) (8-14). The important nature of these
compounds has encouraged other research groups to synthesize
5a and other analogues (15-20).
In the past years, a new series of natural and synthetic
products with important biological activities have been described
as inhibitors of mitochondrial respiratory chain complex I
(NADH oxidase activity) (3-7). In this context, it has been
studied here whether the oxime 5a (a mixture of E And Z-oxime)
and two natural analogues from P. breVicompactum, 3a (11)
and 3b (9), as well as 13 synthetic analogues [three new pairs
of diastereomers 1c′/1c′′, 3c′/3c′′, and 4c′/4c′′, oxime 5c, and
six already known compounds 5b (16), 6 (13), and 7-10 (10)],
In summary, compounds 3c′, 3c′′, 4c′, 4c′′, 5b, 5c, 10, 3b,
and 6 indeed inhibit the mitochondrial electron transfer chain
and the first seven compounds affect specifically to complex I.
These seven compounds were slightly more active on the couple
NADH oxidase as compared to the NADH:DB oxidoreductase.
Furthermore, as the substrate concentration was higher in DB-
dependent NADH oxidation than in the coupled NADH oxidase
reaction, it seems to indicate that these inhibitors compete with
ubiquinone binding to complex I. In fact, these compounds have
a structural similarity with ubiquinone, with a cyclic “head”
resembling the ubiquinone ring and a hydrophobic “tail”. It is
the case of some potent natural inhibitors of complex I, as for
example, piericidin A (4). In this way, oxime, pyrroline, oxazine,
and the pyrrol nucleus in 5b, 5c, 3c′, 3c′′, 3b, 6, 4c′, 4c′′, and
10, respectively, should likely be needed for establishing the
interaction with the enzyme, since they could mimic the quinoid

8300 J. Agric. Food Chem., Vol. 53, No. 21, 2005
Cant´ın et al.
head of ubiquinone, whereas the aliphatic part favors the passage
through the hydrophobic enviromment of the ubiquinone
catalytic site.
IC50 value similar to those shown by diastereomeric pyrrolines
3c′/3c′′ and 6. These results confirm that the oxazine skeleton,
as pyrroline nucleus, favors the interaction with the respiratory
mitochondrial chain. However, the fact that 4c′/4c′′, 3c′/3c′′,
and 6 were 3-6 times less active than 5c demonstrates that the
oxime nucleus is much more active to complex I than pyrroline
or oxazine nucleus.
Finally, the fourth group was formed by four synthetic pyrrol
compounds (7-10). Compounds 7 and 8 did not show inhibition
on the mitochondrial chain of mammalian cell because they have
a very short alkyl chain and, thus, they cannot interfere with
the respiratory chain. Compound 9 showed a very high IC50
value inhibiting the respiratory chain, whereas 10 exhibited a
level of activity similar to that of 6. As in the case of compound
6, 9 and 10 also show fungicidal activities against 13 phyto-
pathogen fungi, and in view of the present results, a possible
mode of action could be the inhibition of the fungal respiratory
chain (10). The structural difference between 9 and 10 is the
presence, in the latter, of a long alkyl chain, substituted in 9
for a phenyl group. This confirms again that the alkyl chain is
very important to promote the union with the enzyme, because
10, 5b, and 3b, all of them with this alkyl chain, are able to
inhibit the electron transfer.
To establish some preliminary structure-activity relationship
(SAR) of 1c′, 1c′′, 3c′, 3c′′, 4c′, 4c′′, 3a, 3b, 5a, 5b, 5c, and
6-10 as inhibitors of the mitochondrial complex I, compounds
were classified in four groups, according to structural similarity.
The first group was formed by brevioxime 5a, a compound
with a heterocyclic oxime structure, 5b, its synthetic 5′,6′-
dihydro derivative, and 5c, another oxime with a phenoxyphenyl
group as hydrophobic anchor. Whereas 5a was not active on
the respiratory chain, 5b and 5c exhibited an interesting
bioactivity inhibiting respiratory mitochondrial chain with an
IC50 of 0.27 and 0.92 µM, respectively. These results seem to
suggest that the aliphatic chain in the oxime products plays an
important role in the interaction with complex I, taking into
account that only the presence of a double bond at the C-5
position in the aliphatic chain (5a) removed the inhibitory
activity exhibited by 5b. It is unusual for other inhibitors as
acetogenins, very active natural compounds with long aliphatic
chains, sometimes with double bonds (23). It indicates that the
important structural factors that affect the inhibitory potency
are not necessarily obvious for each inhibitor (5).
It is worth noticing that neither 5a nor 3a inhibited the
respiratory chain of mammalian cells while, according to
previous assays (8, 11, 12), 5a is able to prevent spontaneous
juvenile hormone synthesis in vitro and 3a shows antijuvenile
hormone in vivo activity. This would make these compounds
very promising for future application in pest control fields,
because they would not induce damage in mammalian mito-
chondria.
Also, we previously reported the insecticidal activity of 7
and 8 against the hemipteran Oncopeltus fasciatus (10). As 7
and 8 did not inhibit the mammalian respiratory chain, both of
them could be used as potential insecticides without affecting
mammals or as lead molecules for the design of new, more
potent insecticides.
In addition, the activity of 5c seems to demonstrate that the
phenoxyphenyl group promotes the interaction with the respira-
tory chain, even though 5b is almost three times more active
than 5c. The IC50 values of 5b and 5c are similar to those shown
by other natural complex I inhibitors as benzopyrans and
almuheptolides (24, 25). Compounds 5b and 5c are placed in a
middle range with respect to the most potent complex I
respiratory inhibitors, as rotenone, with an IC50 of 4.4 nM (6).
The second group was formed by two diastereomeric pyrro-
lidines, 1c′ and 1c′′, and five pyrrolines, 3c′ and 3c′′ (diaster-
eomers), 3a, 3b, and 6. All of them, except 3a, were able to
inhibit the respiratory chain; 3c′, 3c′′, 3b, and 6 were slightly
less active than 5b and 5c, whereas 1c′ and 1c′′ (diastereomers)
exhibited weak activities.
Finally, compound 3b, which showed antijuvenile hormone
in vivo activity (9), could be used as insecticide. Although it
inhibits the complex I mitochondrial chain, its IC50 is in the
micromolar concentration range. In general, the toxicity of
mitochondrial chain inhibitors is described for compounds with
IC50 values in the nanomolar concentration range, such as
rotenone (26). Between the brevioxime analogues, only the
oxymes 5b and 5c would come close to the area of toxicological
concern.
Again, the only structural difference between 3a and 3b is
the presence, in the former, of a double bond at the C-8′ position.
This confirms that the existence of an insatured chain impedes
the appropriate interaction of 3a with the enzyme, as in the case
of 5a.
Diastereomeric pyrrolines 3c′/ 3c′′ and compound 3b turned
out to be slightly more potent than 6 showing inhibition on
respiratory chain (Table 1). The only structural difference
between 6 and 3c′/3c′′ is the presence in the latter compounds
of another 1-carbonyl-2-methylethyl unit in the hydrophobic tail.
This seems to demonstrate that a longer hydrophobic tail in the
inhibitor favors their interaction with mitochondrial chain. Also,
6 showed a potent fungicidal activity in previous assays, and
according to the current results, its mechanism of action could
be the inhibition of the fungal respiratory chain (10).
In addition, diastereomers 1c′ and 1c′′ have the same
hydrophobic chain as 3c′ and 3c′′, but the first ones showed
lower affinity for the respiratory chain. This should be explained
by the substitution of pyrroline nucleus in 3c′ and 3c′′ for a
pyrrolidine nucleus with an OH group in 1c′ and 1c′′. This
hydroxy-pyrrolidine nucleus difficults the interaction with the
enzyme.
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Received for review March 23, 2005. Revised manuscript received July
17, 2005. Accepted July 18, 2005. We acknowledge the Fundacio´n Jose´
y Ana Royo for a postdoctoral grant to M.C.G., and the Conserjer´ıa
de Educacio´n y Ciencia de la C. Valenciana for the doctoral grant to
M.P.L. This work has been supported by “Oficina de Ciencia y
Tecnolog´ıa. I+D projects of the Generalitat Valenciana” (Project GV01-
293), the “Conselleria d’Agricultura, Pesca i Alimentacio´” (Project GV-
CAPA00-0529), and Fondos Europeos de Desarrollo Regional (FEDER-
FSE) of the European Union through the Fondo de Investigacio´n
Sanitaria (FIS 01/0406).
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