Isolation of N-(2-Methyl-3-oxodecanoyl)pyrrole and N-(2-Methyl-3-oxodec-8-enoyl)pyrrole, Two New Natural Products from Penicillium brevicompactum, and Synthesis of Analogues with Insecticidal and Fungicidal Activity

Article abstract of DOI:10.1021/jf9800763

Two new natural products have been isolated from culture broth of Penicillium brevicompactum Dierckx. The structures have been assigned as N-(2-methyl-3-oxodecanoyl) pyrrole and N-(2-methyl-3-oxodec-8-enoyl)pyrrole on the basis of spectral data. Synthesis of analogues has been carried out by acylation of the pyrrole ring at C₂ with different acylated Meldrum's acids. Two analogues (6b and 7b) have shown interesting insecticidal activities, and three other ones (6a, 6c, and 7a) have exhibited significant broad-spectrum fungicidal activities. These synthetic products might be considered as a starting point in the search for new pesticides.

Full text of DOI:10.1021/jf9800763

4748
J. Agric. Food Chem. 1998, 46, 4748−4753
Isola tion of N-(2-Meth yl-3-oxod eca n oyl)p yr r ole a n d
N-(2-Meth yl-3-oxod ec-8-en oyl)p yr r ole, Tw o New Na tu r a l P r od u cts
fr om P en icilliu m br evicom pa ctu m , a n d Syn th esis of An a logu es w ith
In secticid a l a n d F u n gicid a l Activity
AÄ ngel Cant´ın, Pilar Moya, 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
Two new natural products have been isolated from culture broth of Penicillium brevicompactum
Dierckx. The structures have been assigned as N-(2-methyl-3-oxodecanoyl)pyrrole and N-(2-methyl-
3-oxodec-8-enoyl)pyrrole on the basis of spectral data. Synthesis of analogues has been carried out
by acylation of the pyrrole ring at C2 with different acylated Meldrum’s acids. Two analogues (6b
and 7b) have shown interesting insecticidal activities, and three other ones (6a , 6c, and 7a ) have
exhibited significant broad-spectrum fungicidal activities. These synthetic products might be
considered as a starting point in the search for new pesticides.
Keyw or d s: Penicillium brevicompactum; fungal metabolites; Meldrum’s acid; 2-(3-oxoacyl)pyrrole;
insecticide; fungicide
INTRODUCTION
The isolation and identification of fungal metabolites
have attracted considerable attention, both for the
discovery of new bioactive compounds and for taxonomic
purposes.
Particularly, the fungus Penicillium brevicompactum
Dierckx has been described as one of the most prolific
producers of secondary metabolites. These metabolites
include mycophenolic acid and related compounds (Bir-
kinshaw et al., 1952), the Raistrick phenols (Oxford and
Raistrick, 1932, 1933; Godin, 1955), the pebrolides
(McCorkindale et al., 1981), and the N-benzoyl deriva-
tives of phenylalanine, phenylalaninol, and their ester,
asperphenamate (Doerfler et al., 1981). In addition, the
fungus also produces brevigellina (McCorkindale and
Baxter, 1981), several piperazine-2,5-dione derivatives,
a drimane diterpenoid (Ayer et al., 1990), the brevian-
amides (Birch and Wright, 1970; Birch and Russell,
1972), and compactin (Brown et al., 1976).
brevioxime (1) (Moya et al., 1997), together with the
structural analogies among the three natural products
1-3, prompted us to synthesize a series of related
Recently we have reported the isolation and identi-
fication of brevioxime (1), a new metabolite from P.
brevicompactum, which exhibits a very high activity as
pyrroles and to determine their biological activities.
a juvenile hormone (J H) biosynthesis inhibitor (Moya
Moreover, other pyrrolic compounds of natural origin
et al., 1997). Its chemical structure contains an unusual
have been used as lead molecules for the development
heterobicyclic skeleton and an oxime functionality.
of commercial fungicides (Nyfeler and Ackermann,
1992). To increase the stability of the resulting ana-
logues toward hydrolysis, the acyl side chains have been
attached as C-substituents at the heterocyclic ring.
Some of the pyrroles obtained following this approach
exhibit promising insecticidal and fungicidal properties.
Now we report on the isolation of N-(2-methyl-3-
oxodecanoyl)pyrrole (2) and N-(2-methyl-3-oxodec-8-
enoyl)pyrrole (3), two new pyrrolic metabolites from P.
brevicompactum. Their chemical structures are new:
they were described neither as fungal metabolites nor
as synthetic compounds. The fact that compounds 2 and
3 were isolated from the same extract as the active
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 (or KBr plates for isolated natural products);
ν
max is given for the main absorption bands. 1H and 13C NMR
spectra were recorded at 300 and 75 MHz (or 400 and 100 MHz
10.1021/jf9800763 CCC: $15.00 © 1998 American Chemical Society
Published on Web 10/17/1998

New Products of Penicillium brevicompactum
J. Agric. Food Chem., Vol. 46, No. 11, 1998 4749
Sch em e 1. Isola tion a n d Id en tifica tion of Tw o New
Na tu r a l P r od u cts fr om P . br evicom pa ctu m
Compound 3 was assigned to be N-(2-methyl-3-oxodec-8-
enoyl)pyrrole on the basis of spectral data: [R]20D ) 23° (c 0.20,
CHCl3); obtained as an oil; HRMS, m/z 248.1661 (M + H+,
C
15H22NO2 requires 248.1650); IR νmax 3150, 2937, 2871, 1727,
1700, 1465, 1404, 1345, 1275, 1129, 1072, 965, 900, 741, and
1
587; H NMR δΗ 7.3 (m, 2H, H-2 + H-5), 6.3 (m, 2H, H-3 +
H-4), 5.4 (m, 2H, H-8′ + H-9′), 4.1 (q, J ) 7 Hz, 1H, H-2′), 2.5
and 2.4 (m + m, 2H, H-4′), 1.9 (m, 2H, H-7′), 1.6 (d, J ) 5 Hz,
3H, CHdCHCH3), 1.6-1.5 (m, 2H, H-5′), 1.5 (d, J ) 7 Hz,
CHCH3), and 1.3 (m, 2H, H-6′); 13C NMR δC 131.1 (C8′), 125.5
(C9′), 119.6 (C2 + C5), 114.2 (C3 + C4), 53.6 (C2′), 39.8 (C4′), 32.2,
28.9, 23.0 (C5′-C7′), 17.8, and 13.8 (2 × CH3); MS, m/z 229 (1),
200 (1), 180 (2), 178 (2), 165 (3), 150 (6), 125 (4), 123 (3), 97
(8), 94 (6), 83 (10), 81 (16), 67 (100), and 55 (30).
Compound 2 was assigned to be N-(2-methyl-3-oxodecanoyl)-
pyrrole on the basis of its spectral data and by comparison
with the spectral data of compound 3: [R]19 ) 14° (c 0.14,
D
+
CHCl3); obtained as an oil; HRMS, m/z 278.2121 (M + C2H5
,
C17H28NO2 requires 278.2120); IR νmax 3150, 2951, 2925, 2855,
1725, 1703, 1466, 1404, 1345, 1275, 1128, 1070, 903, and 742;
1H NMR δΗ 7.3 (m, 2H, H-2 + H-5), 6.3 (m, 2H, H-3 + H-4),
4.1 (q, J ) 7 Hz, 1H, H-2′), 2.6 and 2.5 (m + m, 2H, H-4′), 1.6
(d, J ) 7 Hz, CHCH3), 1.3 (m, 8H, (CH2)4CH3), and 0.9 (m,
3H, CH2CH3); MS, m/z 249 (2), 183 (1), 154 (1), 150 (1), 127
(11), 123 (2), 109 (2), 98 (2), 94 (5), 83 (6), 67 (100), 57 (18),
and 55 (10).
Gen er a l Syn th etic P r oced u r es. Acylation of Pyrrole.
This reaction was carried out with different acyl side chains
according to the following procedure. To a cooled solution (0
°C) of 2,2-dimethyl-1,3-dioxane-4,6-dione (1.12 mmol) in dichlo-
romethane (1.50 mL) were added pyridine (2.47 mmol) and
the corresponding acyl chloride (1.23 mmol) via syringe,
dropwise, under nitrogen. The solution was stirred at 0 °C
for 1 h, after which time it was allowed to warm to room
temperature for an additional period of 2 h. The dichlo-
romethane solution was washed with dilute HCl, water, and
brine, dried, and concentrated to dryness to give the almost
pure acylated Meldrum’s acid, which was used for the ami-
nolysis without further purification.
for isolated natural products), respectively, in CDCl3 solvent;
chemical shifts are reported in δ (parts per million) values,
using tetramethylsilane (TMS) as internal standard. Mass
spectra were obtained under electron impact (or chemical
ionization for isolated natural products); the ratios m/z and
the relative intensities are reported. Isolation and purification
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 UV light and in an I2 chamber.
Isola tion a n d Ch a r a cter iza tion of th e Com p ou n d s.
The procedure, summarized in Scheme 1, was similar to that
previously reported for brevioxime (Moya et al., 1997). Briefly,
the fungus was isolated in our laboratories and classified by
The International Mycological Institute (IMI, Surrey, U.K.)
as P. brevicompactum Dierckx. A sample of the strain is filed
in the Coleccio´n de Cultivos de la Ca´tedra de Microbiolog´ıa of
the Department of Biotechnology (Universidad Polite´cnica de
Valencia). It is codified as P79 and kept in agar slants with
potato dextrose agar (PDA) as culture medium.
The solution of the acylated Meldrum’s acid and pyrrole
(2.15 mmol) in benzene (9.00 mL) was refluxed for 14 h. The
solvent was evaporated under reduced pressure, and the
residue was purified by column chromatography on silica gel
to afford the â-diketone.
The strain was seeded in Petri dishes with PDA culture
medium and incubated for 7 days at 28 °C. Sterile distilled
water with Tween 80 (0.05%) was then used to obtain a
suspension containing ∼106 conidia/mL. This suspension (100
mL) was added to a 5 L Erlenmeyer flask with 1 L of antibiotic
test broth (composition: yeast extract, 2.0 g; bacto peptone,
3.0 g; glucose, 2.0 g; sucrose, 30.0 g; corn steep, 5.0 g; NaNO3,
2.0 g; K2HPO4‚3H2O, 1.0 g; MgSO4‚7H2O, 0.5 g; KCl, 0.2 g;
FeSO4‚7H2O, 0.01 g; distilled water, 1000 mL; pH 7) and
incubated for 15 days, in the dark, without shaking, at 28 °C.
After incubation, the culture medium was extracted three
times with CH2Cl2 (1:3, v/v). The resulting extract was dried
over CaCl2, filtered, and evaporated in vacuo. The residue (2.0
g from 20 L of culture contained in 20 flasks) was submitted
to column chromatography on silica gel (1:60, w/w) using a
stepwise gradient from CH2Cl2 to MeOH [CH2Cl2; CH2Cl2/
AcOEt (95:5); CH2Cl2/AcOEt (50:50); AcOEt; AcOEt/acetone
(90:10); AcOEt/acetone (50:50); acetone; MeOH]; 300 mL of
each mobile phase was eluted and collected in aliquots of 30
mL, which were pooled in 20 fractions according to their
similarity in TLC. These obtained fractions were systemati-
cally studied for the search of new metabolites. Fraction 3
was shown to contain the two new natural products described
here.
2-(3-Oxodecanoyl)pyrrole (6a ): 37% yield; mp 45-47 °C (from
hexane); HRMS, m/z 235.1578 (C14H21NO2 requires 235.1572);
IR νmax 3280, 3090, 2900, 2820, 1700, 1610, 1550, 1450, 1430,
1390, 1310, 1180, 1110, 1030, 940, 870, 790, and 740; 1H NMR
δH 10.0 (br s, 1H, N-H), 7.1 (m, 1H, H-5k), 7.0 (m, 1H, H-5e),
6.9 (m, 1H, H-3k), 6.8 (m, 1H, H-3e), 6.3 (m, 1H, H-4), 5.9 (s,
1H, H-2′e), 3.9 (s, 1H, H-2′k), 2.6 (t, J ) 7 Hz, 2H, H-4′k), 2.3
(m, 2H, H-4′e), 1.6 (m, 2H, H-5′), 1.3 [br s, 8H, (CH2)4CH3],
and 0.9 (t, J ) 7 Hz, 3H, CH3); 13C NMR δC 209.9 (C3′k), 191.4
(C1′k), 185.2 and 179.0 (C1′e and C3′e), 132.5 (C2k), 130.2 (C2e),
126.3 (C5k), 124.5 (C5e), 118.6 (C3k), 114.7 (C3e), 111.3 (C4k),
111.0 (C4e), 95.3 (C2′e), 53.6 (C2′k), 45.2 (C4′k), 43.2 (C4′e), 36.4,
34.0, 31.7, 31.6, 29.2, 29.0, 26.4, 24.7, 23.5, 22.6 (C5′-C9′), and
14.1 (CH3); MS, m/z 235 (M+, 63), 217 (6), 193 (4), 164 (16),
151 (100), 136 (51), 123 (8), 109 (89), 94 (78), 80 (7), 68 (13),
and 57 (14). Anal. Calcd for C14H21NO2: C, 71.49%; H, 8.94%;
N, 5.96%. Found: C, 71.34%; H, 9.35%; N, 5.54%.
2-(3-Oxopentanoyl)pyrrole (6b): 28% yield; obtained as an
oil; HRMS, m/z 165.0783 (C9H11NO2 requires 165.0789); IR νmax
3280, 3100, 2960, 2940, 2860, 1710, 1640, 1540, 1400, 1320,
1220, 1180, 1120, 1080, 1040, 990, 950, 930, 880, 810, 790,
1
and 750; H NMR δH 9.7 (br s, 1H, N-H), 7.1 (m, 1H, H-5k),
7.0 (m, 1H, H-5e), 6.9 (m, 1H, H-3k), 6.8 (m, 1H, H-3e), 6.3
(m, 1H, H-4), 5.9 (s, 1H, H-2′e), 3.9 (s, 2H, H-2′k), 2.6 (q, J )
7 Hz, 2H, H-4′k), 2.3 (q, J ) 7 Hz, 2H, H-4′e), 1.2 (t, J ) 7 Hz,
3H, CH3e), and 1.1 (t, J ) 7 Hz, 3H, CH3k); 13C NMR δC 204.8
(C3′k), 185.9 (C1′k), 183.1 and 182.1 (C1′e and C3′e), 131.5 (C2k),
130.1 (C2e), 126.4 (C5k), 124.7 (C5e), 118.6 (C3k), 114.8 (C3e),
111.1 (C4k), 110.9 (C4e), 94.4 (C2′e), 53.1 (C2′k), 36.4 (C4′k), 29.3
(C4′e), 10.4 (CH3e), and 7.5 (CH3k); MS, m/z 165 (M+, 100), 150
(2), 148 (3), 136 (58), 125 (2), 109 (30), 94 (65), 80 (7) 68 (21),
Preparative HPLC chromatographic resolution of fraction
3 (14.3 mg) was achieved using the following conditions:
column, Spherisorb W, 5 µm (25.0 × 0.7 cm); mobile phase,
hexane/AcOEt (98:2, v/v); flow, 1.5 mL/min; detection by UV
(254 nm) and refraction index, simultaneously. Two pure
products were obtained: compound 2 [retention time (tr) ) 24.3
min; 2.8 mg] and compound 3 (tr ) 29.5; 6.6 mg).

4750 J. Agric. Food Chem., Vol. 46, No. 11, 1998
Cant´ın et al.
and 57 (7). Anal. Calcd for C9H11NO2: C, 65.45%; H, 6.66%;
N, 8.48%. Found: C, 64.94%; H, 6.98%; N, 8.39%.
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). Six
different bacterial strains were used to determine bactericidal
activity: Staphylococcus aureus (CECT 86), Enterococcus
faecalis (CCM 12), Salmonella typhi (CECT 409), Erwinia
carotovora (CECT 225), Escherichia coli (CECT 405), and
Bacillus cereus (CECT 148).
2-(3-Oxo-4-phenylbutanoyl)pyrrole (6c): 42% yield; mp 115-
117 °C (from hexane); HRMS, m/z 227.0944 (C14H13NO2
requires 227.0946); IR νmax 3270, 1700, 1620, 1590, 1560, 1530,
1490, 1450, 1430, 1410, 1310, 1130, 1110, 1040, 950, 920, 870,
830, 780, 740, and 690; 1H NMR δH 9.5 (br s, 1H, N-H), 7.4-
7.3 (m, 5H, Ph), 7.1 (m, 1H, H-5k), 7.0 (m, 1H, H-5e), 6.8 (m,
1H, H-3), 6.2 (m, 1H, H-4), 5.8 (s, 1H, H-2′e), 3.9 and 3.8 (s +
s, 2H + 2H, H-2′k + H-4′k), and 3.6 (s, 2H, H-4′e); 13C NMR
δC 201.7 (C3′k), 182.9 (C1′k), 182.8 and 181.8 (C1′e and C3′e),
135.6 (C1′′), 131.5 (C2k), 130.0 (C2e), 129.6 (C3′′k), 129.2 (C3′′e),
128.7 (C2′′k), 128.6 (C2′′e), 127.2 (C4′′k), 127.0 (C4′′e), 126.2 (C5k),
124.7 (C5e), 118.4 (C3k), 115.1 (C3e), 111.1 (C4k), 111.0 (C4e),
95.9, (C2′e), 52.1 (C2′k), 50.2 (C4′k), and 42.8 (C4′e); MS, m/z
227 (M+, 53), 136 (100), 123 (5), 118 (2), 109 (40), 94 (93), 91
(26), 86 (7), and 68 (24). Anal. Calcd for C14H13NO2: C,
74.01%; H, 5.73%; N, 6.17%. Found: C, 73.70%; H, 5.78%; N,
6.24%.
2-(3-Hydroxy-5-phenylpenta-2,4-dienoyl)pyrrole (6d ): 40%
yield; obtained as an oil; HRMS, m/z 239.0950 (C15H13NO2
requires 239.0946); IR νmax 3290, 1630, 1590, 1550, 1520, 1450,
1435, 1380, 1320, 1120, 790, 730, and 690; 1H NMR δH 9.6 (br
s, 1H, N-H), 7.6 (d, J ) 16 Hz, 1h, H-5′), 7.5 (dd, J ) 7 and
2 Hz, 2H, H-2′′ + H-6′′), 7.4 (m, 3H, H-3′′-H-5′′), 7.1 (m, 1H,
H-5), 6.9 (m, 1H, H-3), 6.6 (d, J ) 16 Hz, 1H, H-4′), 6.3 (m,
1H, H-4) and 6.1 (s, 1H, H-2′); 13C NMR δC 189.5 (C1′), 172.8
(C3′), 138.0 (C1′′), 135.4 (C2), 129.6 (C4′′), 128.9 (C5′ + C3′′ + C5′′),
127.8 (C2′′ + C6′′), 124.5 (C5), 122.6 (C4′), 114.8 (C3), 111.2 (C4),
and 98.0 (C2′); MS, m/z 239 (M+, 41), 221 (7), 211 (7), 210 (6),
179 (5), 169 (13), 149 (16), 131 (27), 126 (15), 115 (9), 111 (25),
101 (20), 97 (37), 91 (62), 83 (100), 71 (35), 69 (42), 57 (62), 55
(50), and 49 (84).
Meth yla tion of â-Dik eton es 6a a n d 6b. General Proce-
dure. To a stirred slurry of prewashed NaH (60% dispersion
oil; 1.64 mmol) in dimethylformamide (DMF) (2.50 mL) at 0
°C was added a solution of the â-diketone 6a or 6b (1.26 mmol)
in DMF (4.00 mL), via double-ended needle technique, drop-
wise. After hydrogen evolution had ceased, the mixture was
warmed to room temperature, stirred for 2 h, and then recooled
to 0 °C. Iodomethane (1.77 mmol) was then added. After
being stirred at room temperature for 4 h, the mixture was
diluted with water and extracted with CH2Cl2. The combined
extracts were washed with brine, dried, and concentrated to
dryness. Chromatography of the residue on silica gel provided
the methylated 2-acylpyrrole 7a or 7b.
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
values. All assays were made three times. Because of the
small isolated quantities, the natural pyrroles were assayed
by topical application, at 10 µg/nymph, on newly molted fourth-
instar O. fasciatus nymphs. 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 precocious metamorphosis occurred.
Controls were run in parallel and received the same amount
of acetone as treated insects.
Antifungal Activity. The products, dissolved in acetone,
were added to PDA, in a concentration of 100 µg/mL. PDA
plates containing only acetone were used as control plates.
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. Analysis of variance (ANOVA)
was performed for fungicidal data (Table 2), and the least
significant difference (LSD) test was used to compare means
(Statgraphics Plus 2.1).
2-(2-Methyl-3-oxodecanoyl)pyrrole (7a ): 45% yield; obtained
as an oil; HRMS, m/z 249.1728 (C15H23NO2 requires 249.1728);
IR νmax 3280, 3100, 2905, 2840, 1710, 1620, 1540, 1445, 1400,
1
1140, 1090, 1040, 1000, 900, and 750; H NMR δH 10.0 (br s,
1H, N-H), 7.1 (m, 1H, H-5), 7.0 (m, 1H, H-3), 6.3 (m, 1H, H-4),
4.2 (q, J ) 7 Hz, 1H, H-2′), 2.5 (m, 2H, H-4′), 1.5 (m, 2H, H-5′),
1.4 (d, J ) 7 Hz, 3H, CHCH3), 1.2 (br s, 8H, (CH2)4CH3), and
0.8 (t, J ) 7 Hz, 3H, CH2CH3); 13C NMR δC 206.9 (C3′), 186.9
(C1′), 131.1 (C2), 125.9 (C5), 117.7 (C4), 111.1 (C3), 56.2 (C2′),
40.7 (C4′), 31.6, 28.9, 23.5, 22.6 (C5′-C9′), 14.0, and 13.7 (2 ×
CH3); MS, m/z 249 (M+, 24), 219 (2), 181 (2), 165 (5), 150 (2),
123 (100), 94 (34), 69 (4), 66 (5), and 57 (12).
2-(2-Methyl-3-oxopentanoyl)pyrrole (7b): 99% yield; obtained
as an oil; HRMS, m/z 179.0945 (C10H13NO2 requires 179.0946);
IR νmax 3280, 3150, 2980, 2920, 2860, 1710, 1620, 1540, 1450,
1420, 1400, 1320, 1150, 1090, 1045, 900, and 750; 1H NMR δH
10.1 (br s, 1H, N-H), 7.1 (m, 1H, H-5), 7.0 (m, 1H, H-3), 6.3
(m, 1H, H-4), 4.2 (q, J ) 7 Hz, 1H, H-2′), 2.5 (m, 2H, H-4′), 1.4
(d, J ) 7 Hz, 3H, CHCH3), and 1.0 (t, J ) 7 Hz, 3H, CH2CH3);
13C NMR δC 207.3 (C3′), 187.1 (C1′), 131.1 (C2), 126.3 (C5), 117.9
(C3), 111.1 (C4), 55.8 (C2′), 34.1 (C4′), 13.8, and 7.7 (2 × CH3);
MS, m/z 179 (M+, 67), 150 (2), 123 (73), 106 (3), 94 (100), 84
(2), 66 (11), and 57 (12).
RESULTS AND DISCUSSION
Two new natural products have been isolated in this
work, from P. brevicompactum, following the procedure
summarized in Scheme 1. The metabolite obtained in
higher amount was assigned to possess structure 3 on
the following basis. Its molecular formula, C15H21NO2,
was established by HRMS. The spectrum showed
fragment ions corresponding to cleavage of the amide
group (m/z 181.1226 [C11H17O2]) and the C2′-C3′ bond
(m/z 125.0968 [C8H13O]). The 1H NMR spectrum ex-
hibited two signals (δ 7.3 and 6.3) corresponding to the
four protons of a symmetric pyrrole ring and a broad
singlet (δ 5.4) attributable to the two protons of a
dialkyl-substituted double bond. The corresponding
carbons appeared in the 13C NMR spectrum as two sets
of signals at δ 119.6 and 114.1 (pyrrol) and δ 131.1 and
Biologica l Assa ys. Insects. Oncopeltus fasciatus Dallas
were maintained at 28 ( 1 °C, 50-60% relative humidity, and
16 h/8 h (light/dark) photoperiod and on a diet based on
sunflowers seeds.
Target Microorganisms. Fungicidal activity was measured
against 13 agronomically important phytopathogens: As-
1
125.5 (double bond). Moreover, the H NMR spectrum
presented a signal at 4.1 ppm corresponding to a
methine group connected to a methyl group. The IR
spectrum showed two bands at 1727 and 1700 cm-1
,
suggesting the presence of two carbonyl groups. This

New Products of Penicillium brevicompactum
J. Agric. Food Chem., Vol. 46, No. 11, 1998 4751
Ta ble 1. Toxicity of Active P yr r olic Com p ou n d s a ga in st
Sch em e 2. Syn th esis of 2-(3-Oxoa cyl)p yr r oles
O. fa scia tu sa
product
slope
intercept
rb
LD50 (µg/cm2)
6b
7b
5.0022
3.1583
1.3934
2.7731
0.96
0.98
5.26
5.07
a
Regression analysis, linear model: y ) ax + b; log-dose vs
probit mortality. b correlation coefficient. 6b, 2-(3-oxopentanoyl)pyr-
role; 7b, 2-(2-methyl-3-oxopentanoyl)pyrrole.
Romagnoli, 1995; Abad et al., 1997). This reaction led
to 7a ,b in a straightforward manner. In this series the
keto-enol balance is completely shifted toward the keto
form as a consequence of the introduction of the methyl
group and the lower flexibility of the side chain.
Compound 7a was a structural isomer of the natural
product 2; however, comparison of their spectral proper-
ties revealed their differences. As the symmetry of the
pyrrole ring disappears in 7a , the NMR spectrum of this
compound showed different peaks for each proton or
carbon nuclei. On the other hand, the â-ketoamide to
â-diketone transformation was accompanied by char-
acteristic changes in the IR frequencies of the carbonyl
groups and in their 13C NMR chemical sifts.
Biologica l Activities. There are a number of both
naturally occurring and synthetic pyrroles that possess
plant protection properties. In particular, most of these
compounds are effective against fungi (Nyfeler and
Ackermann, 1992; Knuppel et al., 1992; Artico et al.,
1995) and insects (Addor et al., 1992; Kuhn et al., 1992;
Kameswaran et al., 1992; Black et al., 1994). For this
reason, the natural pyrrolic products, 2 and 3, were
tested for entomotoxicity and/or fungicidal activity.
None of them showed toxicity against O. fasciatus when
assayed by topical application on newly molted fourth-
instar nymphs at the dose of 10 µg/nymph. Compound
3 did not show fungicidal activity either. A possible
reason for this lack of activity might, in principle, be
the weakness of the amide bond, which could be easily
hydrolyzed by the enzymes of the test insects and fungi.
Hence, structural modifications were introduced to
increase the stability of these molecules. The most
simple modification was to attach the side chain at one
of the carbons of the heterocyclic ring. Thus, it seemed
that this amide-to-ketone isomerization could be inter-
esting to obtain stable, potentially active isomers.
Noteworthy, two of the synthetic products (6b and 7b)
have significant insecticidal activity. Acute LD50 values
for third-instar nymphs exposed to these products by
the contact method were 5.26 and 5.07 µg/cm2, respec-
tively. Although the two products have similar LD50
values, it seems that methylation between the two
carbonyl groups, yielding compound 7b, is associated
with an important decrease in the slope of the dose-
response curve (Table 1). Thus, compound 6b produced
100% mortality at 7.5 µg/cm2, whereas 7b showed
∼73.3% of toxicity at the same dose. The most potent
insecticidal N-substituted pyrroles, typified by the
experimental AC-303,630, the original model of which
is the natural product dioxapyrrolomycin, are highly
toxic to insect (LC50 of 1-3 ppm by feeding and 1-10
µg/g fo body weight by injection) and appear as novel
broad spectrum insecticides and acaricides (Black et al.,
1994). Although, comparatively, 6a and 7a do not reach
the level of activity of the N-substituted pyrroles, results
were good enough to consider further studies to improve
the entomotoxicity and to appraise the spectrum of
insecticidal activity. On the other hand, insects were
was compatible with a â-keto amide substructure.
1
Comparison of the H NMR spectrum of 3 with that of
brevioxime (1) (Moya et al., 1997) revealed that both
compounds share the same side chain. The other
metabolite was assigned to possess structure 2. Its
molecular formula was C15H23NO2, as established by
HRMS. As in the case of 3, the mass spectrum showed
cleavage of the amide bond. The IR spectrum showed
two carbonyl groups (1725 and 1703 cm-1). The 1H
NMR spectrum exhibited signals (at 4.1 and 1.6 ppm
as a quartet and a doublet, respectively) corresponding
to a CHCH3 unit between the two carbonyl groups. The
rest of the signals and the absence of olefinic peaks
indicated that both natural products differed only in the
degree of unsaturation, due to the presence or absence
of the double bond in the side chain.
The fact that compounds 2 and 3 were isolated from
a different fraction obtained from the same extract as
the active brevioxime (1) (Moya et al., 1997), together
with the presence of a side chain of seven carbons (with
or without a double bond) in the three natural products
1-3, prompted us to synthesize other related com-
pounds to explore their biological activities.
However, the relative weakness of the C-N bond
between a carbonyl group and the N atom of pyrrole
suggested that this type of amide could be easily
hydrolyzable. For this reason, the introduction of
similar side chains as C-substituents in the pyrrole ring
would lead to more stable compounds. Thus, it seemed
that this change could be interesting to obtain new
active analogues.
To introduce this variation, we decided to start from
different acyl chlorides (5) and Meldrum’s acid (4)
(Meldrum, 1908; Davidson and Bernhardt, 1948; Oika-
wa et al., 1978; Pak et al., 1992). As acylation of pyrrole
is easier at C2 than at the nitrogen atom, reaction of
the acylated Meldrum’s acid intermediate led directly
to the desired products 6. This simple reaction was
carried out with different acyl chlorides, giving rise to
a variety of substituted derivatives (Scheme 2).
In the NMR spectra it was evident that these com-
pounds exist in solution as an equilibrium mixture of
the keto and enol forms, except for compound 6d , which
exhibits only the enol form due to its extended conjuga-
tion.
Methylation of 6 was achieved by treatment with
iodomethane after basification with NaH (Benetti and

4752 J. Agric. Food Chem., Vol. 46, No. 11, 1998
Cant´ın et al.
Ta ble 2. F u n gicid a l Activities of Active P r od u cts
percentage of radial mycelial growth inhibition,a % (mean ( SD)b
6b 6c 6d
target phytopathogen
6a c
7a
F. culmorum
F. oxysporum ssp. gladioli
F. oxysporum ssp. niveum
G. candidum
C. gloesporoides
C. coccodes
T. roseum
A. tenuis
R. necatrix
V. dahliae
0A
0A
0A
<20B
0A
0A
<20B
60.7 ( 1.2D
24.8 ( 0.3B
27.7 ( 0.9B
31.0 ( 3.4C
51.7 ( 4.3C
26.4 ( 0.4C
56.4 ( 1.0C
62.5 ( 2.3C
82.2 ( 3.6D
50.5 ( 0.5C
0A
0A
47.7 ( 5.2C
36.7 ( 3.4D
40.9 ( 2.1D
31.8 ( 1.5B
30.8 ( 2.3C
56.9 ( 2.3D
28.2 ( 2.0C
30.8 ( 2.3C
43.0 ( 1.0D
31.2 ( 0.1D
43.3 ( 0.1C
<20B
0A
0A
29.3 ( 3.5B
25.7 ( 3.8A
20.2 ( 2.3B
34.8 ( 3.0B
<20A
<20A
33.8 ( 1.3B
30.9 ( 1.3C
0 A
54.3 ( 2.7D
0A
0A
0A
0A
0A
0A
0A
<20B
0A
<20B
27.8 ( 3.6C
<20B
55.1 ( 4.8D
23.0 ( 2.0C
T. viride
P. italicum
A. parasiticus
<20B
0A
0A
0A
24.1 ( 0.1B
24.6 ( 0.1B
<20C
25.7 ( 1.4B
a
b
Assays concentration: 100 µg/mL. Each value represents the average and standard deviation of three independent experiments.
Within each line, mean values labeled with the same superscript (A-D) do not present statistically significant differences (P > 0.05).c 6a ,
2-(3-oxodecanoyl)pyrrole; 6b, 2-(3-oxopentanoyl)pyrrole; 6c, 2-(3-oxo-4-phenylbutanoyl)pyrrole; 6d , 2-(3-hydroxy-5-phenylpenta-2,4-
dienoyl)pyrrole; 7a , 2-(2-methyl-3-oxodecanoyl)pyrrole.
unaffected, at test levels, by all of the other synthetic
products, showing that the introduction of longer chains
or additional aromatic rings produces an adverse effect
on the entomotoxicity. None of the compounds showed
in vivo J H antagonistic activity.
The fungicidal activities of the synthesized products
are summarized in Table 2. Compounds 7a and 6c,
together with 6a , appear to be broad-spectrum toxi-
cants; their fungitoxic activity was determined against
fungi belonging to 10 different genera, and mycelial
growth was inhibited in all cases, albeit to different
extents. On the other hand, apart from a few excep-
tions, Colletotrichum species and T. roseum were more
sensitive to the products, whereas F. culmorum and R.
necatrix showed lesser sensitivity.
The fungicidal activity of compound 7a was ∼2-fold
that of 6a . It seems that the introduction of a methyl
group between the two carbonyls has beneficial effects
with regard to the inhibition of fungal growth. In this
context, it is interesting to note that methylation
increases the rigidity of the molecule and shifts the
keto-enol equilibrium toward the keto form. In the
nonmethylated compounds (such as 6a ), the enolic
structure predominates. This could be related to the
observed differences in the biological activities.
In addition, the chain length appears to be very
important, conferring fungicidal activity to the products.
Contrary to what was observed for insecticidal activity,
pyrroles with longer chains were the most active.
Compound 6c showed the highest fungicidal activi-
ties, with growth inhibitions >50% in almost half of the
fungi assayed. Other pyrroles containing phenyl sub-
stituents have also shown important fungicidal activi-
ties. This is the case of the natural pyrrolnitrin, a
secondary metabolite produced by different Pseudomo-
nas species. Two of its analogues have been selected
for commercial fungicide development (Nyfeler and
Ackermann, 1992).
On the other hand, compound 6d , structurally related
to 6c, was selectively effective against the Colletotri-
chum genus, particularly against C. coccodes. In this
case, the loss of activity, as indicated by the reduced
range of phytopathogens affected, could be related to
the high level of conjugation, which moves the balance
to the enol form. Again, as discussed above for com-
pound 6a , it seems that the enol forms of this series
are less active.
In summary, a significant activity increase over the
original lead molecules has been achieved by structural
modification; however, improvements are still required
to obtain new products able to achieve a more effective
control of insects and fungi. In this context, the
structures here presented are simple enough to warrant
consideration as a starting point for further synthetic
modifications.
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Received for review J anuary 26, 1998. Revised manuscript
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acknowledge the financial support of Institucio´ Valenciana
d’Estudis i Investigacio´ (fellowship to A.C.), Comisio´n Inter-
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J F9800763