Synthesis and biological evaluation of new analogues of the active fungal metabolites N-(2-methyl-3-oxodecanoyl)-2-pyrroline and N-(2-methyl-3- oxodec-8-enoyl)-2-pyrroline

Article abstract of DOI:10.1021/jf990115q

To evaluate the effect of simplifying the β-ketoamide system present in active isolated metabolites from Penicillium brevicompactum (2 and 3) on the activity, new analogues with a monocarbonylic amide functionality have been obtained. In this way, the insecticidal and fungicidal activities have been improved in relation to the natural products taken as lead molecules. Thus, two of the synthetic analogues (5a and 5b) showed very important insecticidal activities against third-instar nymphs of Oncopeltus fasciatus Dallas, with acute LD₅₀ values of 3.0 and 1.5 μg/cm², respectively. Moreover, some analogues showed good levels of fungicidal activity against a wide range of commercially important and taxonomically diverse fungi; remarkably, compound 7c has proved to be highly active against Colletotrichum gloesporoides and Colletotrichum coccodes, with ED₅₀ values of 2.04 and 11.7 μg/mL, respectively.

Full text of DOI:10.1021/jf990115q

3866
J. Agric. Food Chem. 1999, 47, 3866−3871
Syn th esis a n d Biologica l Eva lu a tion of New An a logu es of th e Active
F u n ga l Meta bolites N-(2-Meth yl-3-oxod eca n oyl)-2-p yr r olin e a n d
N-(2-Meth yl-3-oxod ec-8-en oyl)-2-p yr r olin e
Pilar Moya, AÄ ngel Cant´ın, Miguel A. Miranda, J aime Primo, and Eduardo Primo-Yu´fera*
Instituto de Tecnolog´ıa Qu´ımica UPV-CSIC and Departamento de Qu´ımica,
Universidad Polite´cnica de Valencia, 46022 Valencia, Spain
To evaluate the effect of simplifying the â-ketoamide system present in active isolated metabolites
from Penicillium brevicompactum (2 and 3) on the activity, new analogues with a monocarbonylic
amide functionality have been obtained. In this way, the insecticidal and fungicidal activities have
been improved in relation to the natural products taken as lead molecules. Thus, two of the synthetic
analogues (5a and 5b) showed very important insecticidal activities against third-instar nymphs of
Oncopeltus fasciatus Dallas, with acute LD50 values of 3.0 and 1.5 µg/cm2, respectively. Moreover,
some analogues showed good levels of fungicidal activity against a wide range of commercially
important and taxonomically diverse fungi; remarkably, compound 7c has proved to be highly active
against Colletotrichum gloesporoides and Colletotrichum coccodes, with ED50 values of 2.04 and
11.7 µg/mL, respectively.
Keyw or d s: Amide; fungicidal; insecticidal
INTRODUCTION
The independent synthesis of bioactive natural prod-
ucts is often necessary to confirm their structures and
activities as these compounds are secondary metabolites
usually isolated in minimal quantities. Such work also
leads to a series of synthetic intermediates, chemically
related to the natural compounds, which are potentially
active; sometimes these analogues are even more active
than the isolated compounds. Thus, active natural
products can be used as lead molecules to design dif-
ferent derivatives with similar functionalities but with
improved biological activities.
Recently, we have achieved the isolation and identi-
fication of a new family of bioactive metabolites from
fungal extracts. Thus, brevioxime (1), isolated from
Penicillium brevicompactum, exhibits a very high activ-
ity as a juvenile hormone (J H) biosynthesis inhibitor
(Moya et al., 1997; Castillo et al., 1998). The related
compounds, N-(2-methyl-3-oxodecanoyl)-2-pyrroline (2),
N-(2-methyl-3-oxodec-6-enoyl)-2-pyrroline (3), and 2-hept-
5-enyl-3-methyl-4-oxo-6,7,8,8a,-tetrahydro-4H-pyrrolo-
[2,1-b]-1,3-oxazine (4), isolated from the same source,
show in vivo J H antagonistic and insecticidal activity
(Moya et al., 1998; Cant´ın et al., 1999). Two inactive
pyrrolic metabolites, presumably belonging to the same
biosynthetic pathway, were used as starting points to
obtain active analogues, upon introduction of simple
structural changes (Cant´ın et al., 1998).
improving the activities exhibited by these natural
products.
Now we wish to report the synthesis and biological
activities of several derivatives of the enamides 2 and
3, which were prepared as part of a program aimed at
MATERIALS AND METHODS
* Address correspondence to this author at the Instituto de
Tecnolog´ıa Qu´ımica UPV-CSIC, Universidad Polite´cnica de
Valencia, Avenida de los Naranjos s/n, Apartado 22012, 46022
Valencia, Spain (fax 34-6-3877809; telephone 34-6-3877807;
e-mail eprimo@itq.upv.es).
All chemicals were obtained from commercial suppliers and
used without further purification. IR spectra were obtained
as liquid films; νmax is given for the main absorption bands.
1H and 13C NMR spectra were recorded at 300 and 75 MHz,
respectively, in CDCl3 solvent; chemical shifts are reported in
10.1021/jf990115q CCC: $18.00 © 1999 American Chemical Society
Published on Web 08/17/1999

Bioactive Analogues of Fungal Metabolites
J. Agric. Food Chem., Vol. 47, No. 9, 1999 3867
δ (parts per million) values, using tetramethylsilane (TMS)
as internal standard. The assignment of 13C signals is sup-
ported by DEPT experiments. Mass spectra were obtained
under electron impact or chemical ionization; the ratios m/z
and the relative intensities are reported. Isolation and puri-
fication were done by flash column chromatography on silica
gel 60 (230-400 mesh). Analytical thin layer chromatography
(TLC) was carried out on precoated plates (silica gel 60 F254),
and spots were visualized with ultraviolet (UV) light and in
an I2 chamber.
1240, 1160, 1110, 1040, 990, 910, and 830; 1H NMR δH 5.4
and 5.0 (d + d, J ) 4 Hz, 1H, H-2), 3.6 (m, 2H, H-5), 3.4 and
3.3 (s + s, 3H, OMe), 2.3 (m, 2H, H-2′), 2.2-1.8 (m, 4H, H-3 +
H-4), 1.6 (m, 2H, H-3′), 1.3 [br s, 8H, (CH2)4CH3], and 0.9 (t, J
) 7 Hz, 3H, CH3); 13C NMR δC 173.5 (C1′), 88.7 and 86.9 (C2),
56.4 and 53.9 (OMe), 46.1 and 45.4 (C5), 34.6, 34.0, 31.6, 31.3,
30.9, 29.4, 29.3, 29.0, 25.1, 24.5, 22.9, 22.5, 21.0 (C3, C4, C2′-
C7′), and 14.0 (CH3); MS (CI) m/z 228 (M + H+, 61), 214 (74),
195 (73), 184 (8), 180 (7), 173 (9), 142 (12), 129 (100), 113 (29),
and 111 (85).
Gen er a l Syn th etic P r oced u r es. Synthesis of N-Acylpyr-
rolidines. To a mixture of pyrrolidine (14.1 mmol) with 1.7 M
KOH (9.0 mL) was added a solution of acyl chloride (14.0
mmol) in CH2Cl2 (9.0 mL) dropwise (10 min). After being
stirred at room temperature for 5.5 h, the mixture was
extracted with CH2Cl2; the resulting organic extracts were
washed with brine, dried over Na2SO4, and concentrated to
dryness to give the N-acylpyrrolidine in a straightforward
manner as oils.
N-Octanoylpyrrolidine (5a ): 91% yield; obtained as an oil;
HRMS (EI) m/z 197.1774 (C12H23NO requires 197.1779); IR
νmax 2900, 2860, 2840, 1605, 1410, 1330, 1230, 1160, 1090,
1030, 905, and 830; 1H NMR δH 3.4 (m, 4H, H-2 + H-5), 2.2 (t,
J ) 7 Hz, 2H, H-2′), 2.0-1.8 (m, 4H, H-3 + H-4), 1.6 (m, 2H,
H-3′), 1.3 [br s, 8H, (CH2)4CH3], and 0.9 (t, J ) 7 Hz, 3H, CH3);
13C NMR δC 171.8 (C1′), 46.5 (C2), 45.5 (C5), 34.8 (C2′), 31.6,
29.4, 29.0, 26.0, 24.9, 24.3, 22.5 (C3, C4, C3′-C7′), and 14.0
(CH3); MS m/z 197 (M+, 11), 168 (12), 154 (9), 140 (13), 126
(73), 113 (100), 98 (46), 85 (56), 71 (72), 70 (80), 57 (30), and
55 (65).
2-Methoxy-N-oct-6-enoylpyrrolidine (6b): 25% yield; obtained
as an oil; HRMS (CI) m/z 226.1813 (M + H+, C13H24NO2
requires 226.1807); IR νmax 2920, 2860, 1645, 1400, 1350, 1310,
1230, 1170, 1090, 1070, 1060, 960, 910, 810, and 720; 1H NMR
δH 5.4 (m, 2H, H-6′ + H-7′), 5.0 (d, J ) 4 Hz, 1H, H-2), 3.6 (m,
2H, H-5), 3.4 and 3.3 (s + s, 3H, OMe), 2.3 (m, 2H, H-2′), 2.1-
1.8 (m, 6H, H-3 + H-4 + H-5′), 1.6 (m, 5H, H-3′ + H-8′), and
1.4 (m, 2H, H-4′); 13C NMR δC 173.1 and 173.0 (C1′), 131.0 and
130.9 (C6′), 124.9 and 124.8 (C7′), 88.6 and 86.8 (C2), 56.3 and
53.8 (OMe), 46.0 and 45.3 (C5), 34.4, 33.8, 32.2, 31.3, 30.8, 29.2,
29.1, 24.5, 23.9, 22.8, 20.9 (C3, C4, C2′-C5′), and 17.8 (CH3);
MS (CI) m/z 226 (M + H+, 9), 212 (22), 194 (100), 165 (5), 142
(10), 129 (16), 124 (9), and 111 (10).
2-Methoxy-N-[2-(3-phenoxyphenyl)propionyl]pyrrolidine (6c):
two diastereomers; combined yield 27%; obtained as oils.
Spectral data of the first eluted diasteromer 6c1: HRMS (EI)
m/z 325.1689 (C20H23NO3 requires 325.1678); IR νmax 3060,
2975, 2931, 2888, 1658, 1581, 1488, 1403, 1238, 1163, 1083,
1
917, and 694; H NMR δH 7.4-6.8 (m, 9H, Ar-H), 5.5 and 4.8
(d + d, J ) 5 Hz, 1H, H-2), 3.9 (q, J ) 7 Hz, 1H, H-2′), 3.6-3.3
(m, 2H, H-5), 3.3 and 3.2 (s + s, 3H, OMe), 2.1-1.7 (m, 4H,
H-3 + H-4), and 1.4 (m, 3H, CH3); 13C NMR δC 173.8 and
173.3 (C1′), 157.5, 156.9, 143.9 (C1′′, C3′′, C1′′′), 130.1, 130.0,
123.5, 122.2, 118.8, 117.1, 117.0 (C2′′, C4′′-C6′′, C2′′′-C6′′′),
88.0, 87.4 (C2), 56.5, 55.9 (OMe), 45.9, 45.7 (C5), 44.7, 44.0
(C2′), 31.3, 30.6 (C3), 22.9 (C4), 20.4, and 19.8 (CH3); MS m/z
325 (M+, 23), 310 (41), 294 (72), 224 (15), 197 (46), 181 (10),
128 (100), 103 (18), 91 (20), 85 (84), 77 (30), 70 (32), and 55
(29).
N-Oct-6-enoylpyrrolidine (5b): 85% yield; obtained as an oil;
HRMS (EI) m/z 195.1627 (C12H21NO requires 195.1623); IR
ν
max 2910, 2845, 1640, 1430, 1330, 1250, 1220, 1190, 1160, and
1
960; H NMR δH 5.4 (m, 2H, H-6′ + H-7′), 3.4 (m, 4H, H-2 +
H-5), 2.2 (t, J ) 7 Hz, 2H, H-2′), 2.0-1.7 (m, 6H, H-3 + H-4 +
H-5′), 1.6 (m, 5H, H-3′ + H-8′), and 1.4 (m, 2H, H-4′); 13C NMR
δC 171.5 (C1′), 130.9 (C6′), 124.7 (C7′), 46.4 (C2), 45.4 (C5), 34.5,
32.2, 29.2, 25.9, 24.2 (C3, C4, C2′-C5′), and 17.7 (CH3); MS
m/z 195 (M+, 87), 180 (6), 166 (12), 152 (7), 140 (30), 127 (95),
126 (57), 113 (58), 99 (36), 98 (65), 85 (42), 70 (100), and 55
(85).
Spectral data of the second eluted diasteromer 6c2: HRMS
(EI) m/z 325.1672 (C20H23NO3 requires 325.1677); IR νmax 3060,
2973, 2931, 2884, 1658, 1579, 1489, 1442, 1400, 1242, 1084,
N-[2-(3-Phenoxyphenyl)propionyl]pyrrolidine (5c): 83% yield;
1
mp 80-83 °C (from hexane); HRMS (EI) m/z 295.1577 (C19H21
-
926, and 694; H NMR δH 7.4-6.8 (m, 9H, Ar-H), 5.4 (d + d,
NO2 requires 295.1572); IR νmax 3040, 2960, 2860, 1700, 1630,
1575, 1480, 1420, 1360, 1330, 1240, 1160, 1060, 1020, 950, 920,
750, and 695; 1H NMR δH 7.3 (m, 2H, H-3′′′ + H-5′′′), 7.2 (t, J
) 8 Hz, 1H, H-5′′), 7.1 and 6.9 (m, 5H, H-2′′ + H-6′′ + H-2′′′ +
H-4′′′ + H-6′′′), 6.8 (ddd, J ) 8, 3, and 1 Hz, 1H, H-4′′), 3.7 (q,
J ) 7 Hz, 1H, H-2′), 3.5 and 3.4 (m + m, 4H, H-2 + H-5), 1.8
(m, 4H, H-3 + H-4), and 1.4 (d, J ) 7 Hz, 3H, CH3); 13C NMR
δC 171.5 (C1′), 157.1, 156.8, 143.4 (C1′′, C3′′, C1′′′), 129.7, 129.4,
122.9, 122.0, 118.4, 117.9, 116.6 (C2′′, C4′′-C6′′, C2′′′-C6′′′), 46.0
(C2), 45.7 (C5), 44.4 (C2′), 25.7 (C3), 23.8 (C4), and 19.8 (CH3);
MS m/z 295 (M+, 81), 242 (5), 224 (3), 197
(18), 181 (4), 104 (8), 103 (7), 98 (100), 91 (7), 77 (10), and 55
(33).
An od ic Oxid a tion of N-Acylp yr r olid in es. A solution of
amide (1.6 mmol) in methanol (60.0 mL) containing tetrabu-
tylammonium p-toluenesulfonate (4.4 mmol) as a supporting
electrolyte was placed into an electrolysis cell equipped with
carbon electrodes (8.5 cm2). A constant current (20 mA) was
passed through the solution. After 4.0 F/mol of electricity had
been passed, the solvent was evaporated under reduced
pressure. Water was added to the residue, and the product
was extracted with CH2Cl2. The combined organic layer was
dried over anhydrous sodium sulfate. Thereafter, the drying
agent was removed by filtration, the solvent was evaporated
to dryness, and the residue was filtered through silica gel using
ethyl acetate as eluent, to eliminate the supporting electrolyte.
The solvent was evaporated under reduced pressure, and the
residue was purified by column chromatography on silica gel,
to afford the methoxylated amide.
J ) 5 Hz, 1H, H-2), 3.9 (q, J ) 7 Hz, 1H, H-2′), 3.8-3.5 (m,
2H, H-5), 3.4 and 3.1 (s, 3H, OMe), 2.2-1.7 (m, 4H, H-3 +
H-4), and 1.4 (m, 3H, CH3); 13C NMR δC 173.5 (C1′), 157.4,
144.1, 143.6 (C1′′, C3′′, C1′′′), 130.0, 129.6, 123.2, 122.3, 118.6,
118.2, 117.1 (C2′′, C4′′-C6′′, C2′′′-C6′′′), 88.5, 87.6 (C2), 56.9 (OMe),
45.7 (C5), 44.9 (C2′), 31.1 (C3), 22.7 (C4), 20.7, and 19.8 (CH3);
MS m/z 325 (M+, 34), 310 (15), 294 (10), 224 (19), 197 (30),
181 (7), 128 (100), 103 (10), 91 (10), 85 (50), 77 (12), 70 (13),
and 55 (8).
Syn t h esis of E n a m id es. The corresponding methoxy
derivative (0.05 mmol) and silica gel (0.05 mmol) were heated
at 150-160 °C in a flask, under reduced pressure and nitrogen
atmosphere. After 2.75 h, water was added to the residue and
the slurry was extracted with CH2Cl2. The combined organic
layer was dried over anhydrous sodium sulfate. The drying
agent was then removed by filtration, the solvent was evapo-
rated to dryness, and the residue was purified by column
chromatography on silica gel. Under those conditions enamides
were obtained; when the reaction was carried out with
â-oxoamides, bicyclic oxazines were also formed.
N-Octanoyl-2-pyrroline (7a ): 30% yield; obtained as an oil;
HRMS (EI) m/z 195.1619 (C12H21NO requires 195.1623); IR
νmax 2960, 2920, 2860, 1630, 1550, 1410, 1350, 1160, 1110,
1
1050, and 840; H NMR δH 7.0 and 6.5 (m + m, 1H, H-2), 5.2
(m, 1H, H-3), 3.8 (dd, J ) 9 Hz, 2H, H-5), 2.7 and 2.6 (m + m,
2H, H-4), 2.3 and 2.2 (t + t, J ) 7 Hz, 2H, H-2′), 1.6 (m, 2H,
H-3′), 1.3 [m, 8H, (CH2)4CH3], and 0.9 (t, J ) 7 Hz, 3H, CH3);
13C NMR δC 169.1 (C1′), 129.3 and 128.9 (C2), 111.3 and 111.0
(C3), 45.5 and 44.7 (C5), 34.5, 34.2 (C4), 31.6, 29.3, 29.0, 25.0,
22.5 (C4, C2′-C7′), and 14.0 (CH3); MS m/z 195 (M+, 12), 156
(5), 145 (33), 141 (98), 129 (48), 127 (52), 111 (39), 98 (26), 86
(45), 70 (73), 69 (64), 57 (100), and 55 (37).
2-Methoxy-N-octanoylpyrrolidine (6a ): 45% yield; obtained
as an oil; HRMS (CI) m/z 228.1965 (M + H+, C13H26NO2
requires 228.1963); IR νmax 2910, 2840, 1650, 1410, 1350, 1330,

3868 J. Agric. Food Chem., Vol. 47, No. 9, 1999
Moya et al.
N-Oct-6-enoyl-2-pyrroline (7b): 28% yield; obtained as an oil;
HRMS (EI) m/z 194.1463 (C12H19NO requires 193.1466); IR
νmax 2920, 2850, 1647, 1224, 1333, 1106, 964, 734, and 612;
1H NMR δH 7.0 and 6.5 (m + m, 1H, H-2), 5.4 (m, 2H, H-6′ -
H-7′), 5.2 (m, 1H, H-3), 3.8 (dd, J ) 9 Hz, 2H, H-5), 2.7 and
2.6 (m + m, 2H, H-4), 2.3 and 2.2 (t + t, J ) 7 Hz, 2H, H-2′),
2.0 (m, 4H, H-4 + H-5′), 1.6 (m, 5H, H-3 ′+ H-8′), and 1.4 (m,
2H, H-4′); 13C NMR δC 169.0 (C1′), 130.9 (C6′), 130.9 and 129.2
(C2), 125.0 (C7′), 111.4 and 110.1 (C3), 45.5 and 44.8 (C5), 34.1-
(C4), 32.2, 30.1, 29.2, 24.4 (C2′-C5′), and 17.9 (CH3); MS m/z
193 (M+, 37), 138 (5), 124 (21), 111 (17), 97 (11), 96 (11), 84
(18), 81 (35), 69 (72), 68 (100), and 55 (98).
N-[2-(3-Phenoxyphenyl)propionyl]-2-pyrroline (7c): 29% yield;
obtained as an oil; HRMS (EI) m/z 293.1416 (C19H19NO2
requires 293.1416); IR νmax 3060, 2931, 2888, 1647, 1573, 1489,
1
1420, 1258, 1110, and 610; H NMR δH 7.4-6.8 (m, 9H, Ar-
H), 6.5 (m, 1H, H-2), 5.2 and 5.1 (m + m, 2H, H-3), 4.0-3.5
(m, 3H, H-5 + H-2′), 2.8-2.5 (m, 2H, H-4), and 1.5 (d, J ) 7
Hz, 3H, CH3); 13C NMR δC 169.1 (C1′), 157.4, 157.3, 143.9 (C1′′,
C3′′, C1′′′), 130.1, 129.7, 128.4, 123.2, 122.0, 118.7, 117.2, 111.8,
110.4 (C2, C3, C2′′, C4′′-C6′′, C2′′′-C6′′′), 45.2 (C5), 44.9 (C2′), 27.9
(C4), 19.9, and 19.7 (CH3); MS m/z 293 (M+, 52), 250 (2), 197
(72), 104 (14), 91 (17), 77 (20), and 69 (100).
Syn th esis of Im id es. A mixture of 2-pyrrolidinone (2.7
mmol), acyl chloride (1.8 mmol), and Et3N (2.0 mmol) in
benzene (20 mL) was refluxed for 8 h. The reaction mixture
was extracted with CH2Cl2; the resulting organic extracts were
washed with brine, dried over Na2SO4, and concentrated to
dryness, providing a residue that was purified by column
chromatography on silica gel to afford the corresponding imide.
N-Octanoyl-2-pyrrolidinone (8): 68% yield; obtained as an
oil; HRMS (EI) m/z 211.1570 (C12H21NO2 requires 211.15.72);
IR νmax 2952, 2922, 2853, 1738, 1694, 1460, 1360, 1324, 1250,
1
and 593; H NMR δH 3.8 (t, J ) 7 Hz, 2H, H-5), 2.9 (t, J ) 7
Hz, 2H, H-3), 2.6 (t, J ) 8 Hz, 2H, H-2′), 2.0 (m, 2H, H-4), 1.6
(m, 2H, H-3′), 1.3 [br s, 8H, (CH2)4CH3], and 0.9 (t, J ) 7 Hz,
3H, CH3); 13C NMR δC 175.4 (C2), 174.4 (C1′), 45.4 (C5), 36.7,
33.7, 31.6, 29.1, 29.0, 24.1, 22.5, 17.1 (C3, C4, C2′-C7′), and 14.0
(CH3); MS m/z 211 (M+, 35), 182 (7), 154 (23), 140 (98), 127
(100), 112 (14), 99 (88), 86 (53), 69 (26), 57 (91), and 55 (58).
Biologica l Activity. Insects. Oncopeltus fasciatus Dallas
were maintained at 28 ( 1 °C, 50-60% relative humidity, and
16 h/8 h (light/dark) photoperiod on a diet based on sunflower
seeds.
Target Microorganisms. Fungicidal activity was measured
against 13 agronomically important phytopathogens: As-
pergillus parasiticus (CECT 2681), Geotrichum candidum
(CCM 245), Alternaria tenuis (CECT 2662), Colletotrichum
gloesporoides (CECT 2859), Colletotrichum coccodes (CCM
327), Fusarium oxysporum ssp. gladioli (CCM 233), Fusarium
oxysporum ssp. niveum (CCM 259), Fusarium culmorum (CCM
172), Penicillium italicum (CECT 2294), Trichoderma viride
(CECT 2423), Trichothecium roseum (CECT 2410), Rosellinia
necatrix (CCM 297), and Verticillium dahliae (CCM 269).
The strains were provided by the “Coleccio´n Espan˜ola de
Cultivos Tipo” (CECT) or by the “Coleccio´n de la Ca´tedra de
Microbiolog´ıa” (CCM) of the Department of Biotechnology
(Universidad Polite´cnica de Valencia).
Entomotoxicity and Anti-J H Activity. The test was carried
out basically according to the contact method of Bowers et al.
(1976). Briefly, 15 third-instar O. fasciatus nymphs were
confined to a 9 cm Petri dish coated, across the bottom, with
10 µg/cm2 of the product. Chemicals showing high activity at
10 µg/cm2 were retested at 7.5, 6, 5, 4, 2.5, and 1 µg/cm2.
Toxicity effects were considered according to the number of
insects dead after 72 h of exposure to the chemicals, and probit
analysis (Finney, 1971) was applied to determine the LD50
.
The surviving nymphs were transferred to a 500 cm3 glass
flask and held at standard conditions. After metamorphosis
occurred and reproduction was successful with the production
of viable offsprings, the tests were finished. The tests were
considered positive for J H antagonistic activity when preco-
cious metamorphosis occurred. Controls were run in parallel
and received the same amount of acetone as treated insects.

Bioactive Analogues of Fungal Metabolites
J. Agric. Food Chem., Vol. 47, No. 9, 1999 3869
Sch em e 1. Syn th etic Sequ en ce w ith Mon oca r bon ylic Sid e Ch a in s
Antifungal Activity. The products, dissolved in acetone, were
added to PDA, in a concentration 100 µg/mL. PDA plates
containing only acetone were used as control plates, and a
positive control with benomyl [methyl-1-(butylcarbamoyl)-2-
benzimidazolecarbamate; Sigma, Germany] at 2.5 µg/mL was
performed to appraise the level of activity of the synthesized
compounds. Spores from 7-day-old cultures of each fungus on
PDA plates were used as an inoculum onto the control and
test plates. The radial mycelial growth was measured and the
percentage of inhibition was calculated on the basis of growth
in control plates, after 4 days of incubation (6 days for R.
necatrix and V. dahliae), at 28 °C. The antifungal activity of
each product was determined three times. When minimun
inhibitory concentration (MIC) values were <100 µg/mL, the
effective dose to inhibit 50% (ED50) of the mycelial growth was
estimated by linear regression analysis of the percentage of
inhibition versus log of compound concentration. Analysis of
variance (ANOVA) was performed for fungicidal data (Table
1), and the least significant difference (LSD) test was used to
compare means (Statgraphics Plus 2.1).
Biologica l Activities. Insecticidal and Anti-J H Ac-
tivity. Two of the products (5a and 5b) showed potent
insecticidal activity against O. fasciatus. In the case of
5a , acute LD50 and LD90 values for third-instar nymphs,
exposed to the chemical by the contact method, were
3.0 and 5.0 µg/cm2, respectively. The corresponding
values for compound 5b were 1.5 and 2.0 µg/cm2. Thus,
the latest compound was 2-fold more active than 5a ;
these toxicity data unambiguously demonstrate that
introduction of an unsaturation in the side chain is
associated with an improved entomotoxicity. Insects
were unaffected, at test levels, by the other synthetic
analogues. Thus, deeper modifications of the side chain
or the five-membered ring produce an adverse effect on
the toxicity.
Insecticidal activity has been shown to be closely
associated with the pyrrolidine moiety. In our previous
work on the isolation, synthesis, and biological activity
of compound 2 (Moya et al., 1998), the study of the
insecticidal activity of its synthetic precursors showed
that the presence of the pyrrolidine ring was essential
for the activity. Particularly, N-(3-oxodecanoyl)pyrroline
and N-(2-methyl-3-oxodecanoyl)pyrroline, both with the
â-ketoamide functionality, showed LD50 values of 7.0
and 3.75 µg/cm2, respectively. As mentioned above, our
current results show the same tendency, but in this case
there has been a significant improvement of entomo-
toxicity.
RESULTS AND DISCUSSION
The sequence of reactions previously used to synthe-
size 2-4 was modified to prepare the related compound
7, to ascertain whether the â-ketoamide functionality
is essential for the activity. Thus, the side chain present
in the natural products was maintained in the mono-
carbonylic analogues. A phenoxyphenyl group was
introduced because it is very common in pesticides.
As shown in Scheme 1, the employed synthetic
sequence implied formation of amides by means of a
Schotten-Baumann reaction, taking pyrrolidine and
the corresponding acid chloride as starting materials in
the first step. Subsequent anodic oxidation of the
pyrrolidine ring using the method described by Shono
(Shono, 1984; Shono et al., 1981a,b, 1982a,b) followed
by elimination of MeOH upon acid catalysis and heating
at 150-160 °C (Slomczynska et al., 1996; Cornille et
al., 1994, 1995; Moeller et al., 1992, 1994) afforded the
corresponding enamides. This worked in a satisfactory
way in the case of 7a and 7b; however, obtention of 7c
implied in the anodic oxidation a diastereomeric mix-
ture, which appeared joined with a series of oxidation
products of aromatic rings. This mixture of diastereo-
mers was resolved by chromatographic column. Both
diastereomers were used in the elimination reaction
affording the desired enamide 7c.
Although natural enamides 2 and 3 show an impor-
tant in vivo anti-J H activity, neither their previously
reported synthetic precursors nor the new analogues
assayed in the present work proved to retain such
activity; no precocious metamorphosis or even slighter
symptoms of J H deficiency, as delayed growth or altered
fertility, were detected. It seems that this activity has
a very specific structural requirement.
Finally, to obtain further analogues, 2-pyrrolidinone
was introduced as a five-membered ring instead of
pyrrolidine. This was achieved by simple heating of
octanoyl chloride with 2-pyrrolidinone and triethyl-
amine in refluxing benzene (Ostrovskaya et al., 1993;
Sasaki et al., 1991).
Fungicidal Activity. At first sight, it is interesting to
note that the introduction of an amide group, instead

3870 J. Agric. Food Chem., Vol. 47, No. 9, 1999
Moya et al.
of the â-ketoamide functionality present in the natural
products and their synthetic precursors, resulted in an
important increase of the fungicidal activity [for com-
parative purposes see Moya et al. (1998) and Cant´ın et
al. (1998)]. However, comparatively, the levels of activity
are clearly lower that those of a conventional fungicide
such as benomyl (Table 1).
Fungicidal activity of the new analogues, expressed
as the percentage of growth inhibition against 13
agronomically important plant pathogens, is shown in
Table 1.
In general, the products possessing the phenoxyphen-
yl substituent showed the best fungicidal activity with
regard to the percentage of growth inhibition and the
number of affected species; compound 7c has proved to
be highly active against C. gloesporoides [ED50 ) 2.04
µg/mL; 95% confidence interval of (1.26, 4.12)] and C.
coccodes [ED50 ) 11.7 µg/mL with a 95% confidence
interval of (7.3, 20.7)], although these results still
compare unfavorably with those found for benomyl
[ED50 ) 0.05 µg/mL; 95% confidence interval of (0.04,
0.08) against C. gloesporoides and ED50 ) 0.13 µg/mL
with a 95% confidence interval of (0.12, 0.17) against
C. coccodes]. Besides, compound 7c strongly affected the
growth of T. roseum and P. citrophthora and, very
interestingly, A. tenuis, one of the benomyl-resistant
species; all of the other fungi were also inhibited to some
extent. The improvement obtained with this product
appears to be important and warrants further work. It
will be used as a starting point in the search for more
active analogues and also for studies on its mechanism
of action.
pound 5c for Colletotrichum, A. tenuis, and T. viride,
but the other fungi were less affected. Previously, a
similar 2-pyrrolidone derivative with antibiotic activity
was described (Takeuchi and Yonehara, 1964). The
product, named variotin, was isolated from the culture
broth of the fungus Paecilomyces varioti and exhibits
activity against different kinds of fungi with MIC
values in the range 10-160 µg/mL. It is interesting
to note that, as in our case, variotin is especially effec-
tive against various species of the Colletotrichum gen-
era, with MIC values from 0.2 to 2.0 µg/mL (Yonehara
et al., 1959; Takeuchi et al., 1959; Abe et al., 1959).
In summary, good insecticidal and fungicidal activi-
ties have been achieved and preliminary structure-
activity relationships have been established. In this
context, the improvements obtained so far on the
biological activities and the simplicity of structures are
encouraging to pursue the search of new, more effective,
analogues as promising pesticidal candidates.
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Received for review February 5, 1999. Revised manuscript
received J une 4, 1999. Accepted J une 5, 1999. We acknowledge
the financial support of Institucio´ Valenciana d’Estudis i
Investigacio´ (fellowship to A.C.), Comisio´n Interministerial de
Ciencia y Tecnolog´ıa (CICYT), and Consejer´ıa de Agricultura
P. y A. de la C. Valenciana.
J F990115Q