Antifungal activity of 4-methyl-6-alkyl-2H-pyran-2-ones

Article abstract of DOI:10.1021/jf052792s

A number of 4-methyl-6-alkyl-α-pyrones were synthesized and characterized on the basis of ¹H NMR and mass spectroscopy. These compounds were tested in vitro against pathogenic fungi, namely, Sclerotium rolfsii Saccardo, Rhizoctonia bataticola (Taub.) Butler, Pythium aphanidermatum (Edson) Fitz., Macrophomina phaseolina (Tassi), Pythium debaryanum (Hesse), and Rhizoctonia solani Nees. Lower homologues were less effective, whereas compounds such as 4-methyl-6-butyl-α-pyrone, 4-methyl-6-pentyl-α-pyrone, 4-methyl-6-hexyl-α-pyrone, and 4-methyl-6-heptyl-α-pyrone were found effective against all of the test fungi. They inhibited mycelial growth by approximately 50% (ED₅₀) at 15-50 μg/mL. 4-Methyl-6-hexyl-α- pyrone, which was found most effective, was tested against S. rolfsii in a greenhouse at 1, 5, and 10% concentrations. The 10% aqueous emulsion of 4-methyl-6-hexyl-α-pyrone suppressed disease development in tomato by 90-93% as compared with the untreated infested soil in the greenhouse after 35 days of treatment.

Full text of DOI:10.1021/jf052792s

J. Agric. Food Chem. 2006, 54, 2129−2133 2129
Antifungal Activity of 4-Methyl-6-alkyl-2H-pyran-2-ones
TARUN KUMAR CHATTAPADHYAY AND PREM DUREJA*
Division of Agricultural Chemicals, Indian Agricultural Research Institute, New Delhi 110012, India
1
A number of 4-methyl-6-alkyl-R-pyrones were synthesized and characterized on the basis of H NMR
and mass spectroscopy. These compounds were tested in vitro against pathogenic fungi, namely,
Sclerotium rolfsii Saccardo, Rhizoctonia bataticola (Taub.) Butler, Pythium aphanidermatum (Edson)
Fitz., Macrophomina phaseolina (Tassi), Pythium debaryanum (Hesse), and Rhizoctonia solani Nees.
Lower homologues were less effective, whereas compounds such as 4-methyl-6-butyl-R-pyrone,
4
-methyl-6-pentyl-R-pyrone, 4-methyl-6-hexyl-R-pyrone, and 4-methyl-6-heptyl-R-pyrone were found
effective against all of the test fungi. They inhibited mycelial growth by approximately 50% (ED50) at
5-50 µg/mL. 4-Methyl-6-hexyl-R-pyrone, which was found most effective, was tested against S.
1
rolfsii in a greenhouse at 1, 5, and 10% concentrations. The 10% aqueous emulsion of 4-methyl-6-
hexyl-R-pyrone suppressed disease development in tomato by 90-93% as compared with the
untreated infested soil in the greenhouse after 35 days of treatment.
KEYWORDS: 4-Methyl-6-alkyl-r-pyrones; antifungal activity; Sclerotium rolfsii; Rhizoctonia bataticola;
Pythium aphanidermatum; Macrophomina phaseolina; Pythium debaryanum; Rhizoctonia solani; green
house testing
INTRODUCTION
has been found costly. Therefore, its cost is an apparent obstacle
to further development. Earlier structure-activity relationships
determining the antifungal activity of a range of synthetic
analogues of 6-PAP indicated that structural requirements for
antifungal activity appeared to be stringent. Shortening of the
The yield of major food and cash crops is reduced nearly
20% by pathogenic fungi. Among these are Sclerotium rolfsii
and Rhizoctonia bataticola, devastating soil borne fungi with a
wide host range. They infect seeds, seedlings, and mature plants
in the field causing collar rot, wilt, damping off, dry root rot,
etc. A large number of chemical crop protectants used to control
these organisms are detrimental to the environment and human
health and need to be replaced by safe, biodegradable products.
Lactones are of considerable significance to the fragrance and
food industries due to their potent fragrant and organoleptic
characteristics, which may convey either desirable or objection-
able qualities (1). It has been reported that 6-alkyl-R-pyrones
show a smooth progressive change in flavor with increasing
molecular weight. The lower homologues are coumarin-coconut,
while the higher homologues are waxy, green, and floral. Of
the 6-alkyl-R-pyrones evaluated, 6-pentyl-R-pyrone (6-PAP) was
considered to have the most pleasant flavor (2). Besides its
organoleptic properties, the antifungal activity of the natural
product 6-PAP, a fungal metabolite isolated from various
Trichoderma spp., is well-known and its in vitro activity against
a range of phytopathogens has been reported (3-7). As a natural
product, 6-PAP is innately biodegradable. Combined with its
established use as a food additive and assuming low toxicity,
6
-alkyl substituents resulted in a marked loss of activity, as did
2
saturation of the ∆ -bond of the pyrone ring (8). The 6-pent-
-enyl-substituted analogues showed similar antifungal activity.
1
It was found that 4-methyl-6-pentyl-2H-pyran-2-one, a substi-
tuted analogue of 6-PAP, showed comparable activity in vitro
and could be prepared with relative ease and economy relative
to 6-PAP (9).
To further study the structure-activity relationship, several
-methyl-6-alkyl-R-pyrones were prepared. In this paper, we
4
report the in vitro and in vivo fungicidal activities of 4-methyl-
-alkyl-2H-pyran-2-ones against a number of soil borne patho-
6
gens.
MATERIALS AND METHODS
Chemicals and Reagents. Aliphatic acids, thionyl chloride, alumi-
num chloride, and 3,3-dimethyl acrylic acid were procured locally.
Thionyl chloride was distilled before use. The organic solvents used
for syntheses were analytical grade and distilled and dried before use.
Plant Pathogenic Fungi. Plant pathogenic fungi such as S. rolfsii
Saccardo, R. bataticola (Taub.) Butler, Pythium aphanidermatum
6-PAP has been found as an attractive candidate for development
as an agricultural fungicide. Prior to its identification as a natural
product, the chemical synthesis of 6-PAP and its structural
analogues received attention; however, the synthesis of 6-PAP
(
(
Edson) Fitz., Macrophomina phaseolina (Tassi), Pythium debaryanum
Hesse), and Rhizoctonia solani Nees were collected from the Indian
Type Culture, Division of Plant Pathology, Indian Agricultural Research
Institute (New Delhi, India). Pathogenic fungi were maintained on
potato dextrose agar (PDA) at 25 °C and were subcultured on PDA
Petri dishes for 5-6 days at 28 °C prior to use as inoculums.
*
To whom correspondence should be addressed. E-mail:
Pd_dureja@yahoo.com.
1
0.1021/jf052792s CCC: $33.50
© 2006 American Chemical Society
Published on Web 02/23/2006

2130 J. Agric. Food Chem., Vol. 54, No. 6, 2006
Chattapadhyay and Dureja
Table 1. Antifungal Activity of 4-Methyl-6-alkyl-R-pyrones against Soil Borne Pathogenic Fungi
ED50 (µ
g mL-1)a
b
pathogenic fungi
compd
no.
R
Sr
Rs
Rb
Mp
Pa
Pd
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
CH
CH
n(CH
i(CH
n(CH
i(CH
n(CH
n(CH
n(CH
n(CH
n(CH
n(CH
3
2
712.63
665.30
250.87
313.37
77.99
39.45
22.45
15.10
30.7
121.84
396.98
562.21
349.00
180.01
403.82
46.93
492.98
690.40
149.46
437.17
50.08
48.69
34.49
18.66
45.04
115.90
130.32
267.34
183.66
205.99
293.51
22.75
1185.66
625.53
562.39
403.49
212.34
68.44
34.16
24.53
47.04
132.36
212.75
649.00
303.13
373.88
360.99
111.23
328.36
4.36
823.52
267.49
328.62
1207.04
235.02
47.26
36.77
30.96
82.15
163.89
352.43
548.37
261.94
337.54
276.02
72.08
450.75
407.62
572.71
475.25
227.19
52.40
39.12
16.61
74.64
123.78
203.93
287.12
276.96
337.06
102.18
121.94
205.86
8.47
1140.96
580.33
288.13
437.09
142.79
37.03
20.56
14.91
71.71
174.64
283.65
594.38
150.97
188.84
95.49
101.99
264.23
12.30
CH
3
2
)
2
2
CH
CH
CH
CH
CH
3
2
)
3
)
2 3
3
2
)
3
3
2
)
2
)
2
)
2
)
2
)
2
)
4
3
5
6
7
8
9
CH
CH
CH
CH
CH
3
3
3
3
1
1
1
1
1
1
1
1
1
3
CH(C
C(CH
2
H
5
2 3 3
)(CH ) CH
)dCH
3 2
CH
ClCH
CH
d
CHCH
3
2
d
C(CH
3
)
2
322.57
18.34
286.01
12.96
377.48
10.53
hexaconazole
(standard)
a
Average of five replicates. b Sr, S. rolfsii; Rs, R. solani; Rb, R. bataticola; Mp, M. phaseolina; Pa, P. aphanidermatum; and Pd, P. deberianum.
Chromatography and Spectroscopy. Thin-layer chromatography
TLC) was performed on 250 µm silica gel G plates preactivated at
identity of the product was confirmed by comparison of their NMR,
IR, and gas chromatography-mass spectrometry reported in the
literature (11).
(
120 °C for 2 h. The plates were developed in chloroform and visualized
either by iodine vapor or spraying with an alcoholic solution of 2,4-
dinitrophenyl hydrazine. Gas liquid chromatographic (GLC) analyses
were performed on a Hewlett-Packard model 5890 A, gas liquid
chromatograph fitted with a HP-17 capillary column (10 m × 0.53
mm i.d., 2.54 µm film thickness) and equipped with a flame ionization
detector and a Hewlett-Packard 3390A integrator. The operating
Antifungal Assay. The antifungal activity was tested in vitro against
S. rolfsii, R. solani, R. bataticola, M. phaseolina, P. aphanidermatum,
and P. debaryanum and in vivo on tomato seedlings infested with S.
rolfsii.
In Vitro Antifungal Activity. The above-synthesized compounds
were tested for their ability to inhibit above soil borne pathogenic fungi
against the standard fungicide hexaconazole. The concentrations of the
latter were those recommended by the manufacturer. The fungicidal
activity of synthesized compounds was evaluated at various concentra-
tions by the poisoned food technique using PDA media. The ready-
made PDA medium (39 g) was suspended in distilled water (1000 mL)
and heated to boiling until completely dissolved. The medium and Petri
dishes were autoclaved at 120 °C for 30 min. These compounds were
tested at concentrations of 250, 125, 100, 50, 25, and 10 µg/mL. A
stock solution of 1000 µg/mL was prepared, which was further diluted
with acetone to give the required concentrations. Acetone (1 mL) was
used as the control. These solutions were added to the media (65 mL)
contained in conical flasks to obtain the desired concentrations of the
test compounds in the media. The medium was poured into a set of
two Petri dishes (90 cm in diameter) under aseptic conditions in a
laminar flow hood. The plates were kept under UV light in the laminar
flow chamber for solidification of the media. After solidification, a 5
mm × 5 mm mycelial plug cut from the actively growing front of a 2
week old colony of the desired pathogenic fungus was then placed with
the inoculum side down in the center of each treatment plate, aseptically.
Treated Petri dishes were then incubated at 28 °C till the fungal growth
was almost complete in the control plates. All experiments were in
quadruplet for each treatment against each fungus.
conditions were as follows: The column temperature was programmed
from 70 °C for 1 min to 250 °C at 10 °C min-1 with injector
temperatures of 300 °C. Nitrogen was used as a carrier gas with a flow
-
1
rate of 20 mL min . Infrared (IR) spectra were recorded on a Nicolet
1
(
Impact 400) FT-IR spectrophotometer as neat. The H NMR spectra
were recorded on a Varian EM 360 L (60 MHz) and on a Bruker 300
AC (300 MHz) spectrometer using tetramethylsilane as the internal
reference. Mass spectra were recorded on a HRGC-MEGA 2 series
gas chromatograph coupled to a FISONS-TRIO 1000 ion trap mass
spectrometer and connected with a Panasonic KX-P1150 multimode
printer. The ionization potential was 70 eV. The gas chromatograph
was fitted with a HP-17 capillary column (30 m × 0.25 mm i.d.; film
thickness, 0.1-0.15 µm). Helium was used as a carrier gas at a flow
-
1
rate of 2 mL min
.
General Procedure of Synthesis of 4-Methyl-6-alkyl-2H-pyran-
-ones. 4-Methyl-6-alkyl-2H-pyran-2-ones were prepared from methyl-
-methyl-2-butenoate, which in turn was prepared from 3,3-dimethyl
2
3
acrylic acid by esterification (10) using methanol and sulfuric acid and
1
was purified by distillation (bp 130-131 °C; yield, 93-95%). H NMR
(
3
2
3
CDCl ): δ 2.2 (d, 6H, J ) 10 Hz), 3.99 (s, 3H), 5.70 (s, 1H). Methyl-
-methyl-2-butenoate was converted to methyl-3-methyl-5-oxo-5-alkyl-
-pentenoates (keto esters) by Friedel-Crafts acylation using aluminum
chloride and dichloromethane as the solvent in the presence of different
acyl chlorides. The crude keto ester thus obtained was further purified
by fractional distillation under reduced pressure. These keto esters were
converted to 4-methyl-6-alkyl-R-pyrones by lactonization using a
mixture of glacial acetic acid and sulfuric acid (9:1). The 4-methyl-6-
alkyl-R-pyrones thus obtained were further purified from the crude
products by fractional vacuum distillation at reduced pressure. The
Recording of Observations. The mycelial growth of fungus (cm)
in both treated (T) and control (C) Petri dishes was measured
diametrically. The mean and standard errors were calculated from the
four replicates of each treatment, and the percentage inhibition of growth
(I) was calculated using the following formula

Antifungal Activity of 4-Methyl-6-alkyl-2H-pyran-2-ones
J. Agric. Food Chem., Vol. 54, No. 6, 2006 2131
C - T
C
corrected inhibition (% I) )
× 100
Calculation of ED50 Values. For calculation of ED50 values
(effective dose required for 50% inhibition of growth), the percent
inhibition was converted to corrected percent inhibition by using
Abbott’s formula:
%
I - CF
corrected inhibition (%) )
× 100
1
00 - CF
where CF is the correction factor obtained by the equation
9
- C
C
correction factor (CF) )
× 100
where 9 is the diameter of the Petri dish in cm and C is the diameter
of growth of the fungus in control plates. From the concentration (ppm)
and corresponding corrected percentage inhibition data of each
compound, the ED50 (ppm) value was calculated statistically by Probit
analysis with the help of Probit package of MSTATC software using
a personal computer. ED50 values were calculated (effective dose for
-1
5
0% inhibition µg mL ) for inhibition of growth using the Basic LD50
program version 1.1.
In Vivo Antifungal Activity. The most active compound of the
series was tested in vivo for antifungal activity against S. rolfsii in
pots. S. rolfsii was chosen because it has an extensive host range; at
least 500 species in 100 families are susceptible. It primarily attacks
host stems, although it may infect any part of a plant under favorable
environmental conditions including roots, fruits, petioles, leaves, and
flowers. The first signs of infection, although usually undetectable, are
dark-brown lesions on the stem at or just beneath the soil level; the
first visible symptoms are progressive yellowing and wilting of the
leaves. Following this, the fungus produces abundant white, fluffy
mycelia on infected tissues and soil. Seedlings are very susceptible
and die quickly once they become infected. Older plants that have
formed woody tissue are gradually girdled by lesions and eventually
die. Invaded tissues are pale brown and soft but not watery.
Inoculum Preparation. Mycelial mats of S. rolfsii were grown in
potato dextrose broth in 250 mL conical flasks and incubated
horizontally at 27 °C. After 7 days, the medium was decanted and 200
mL of sterile deionized water was added to each bottle and incubated
vertically at 20 °C for 3-4 weeks. Chlamydospores were harvested
by rinsing the cultures two or three times with deionized water and
homogenizing in a small blender for 1 min. The mycelial suspension
then was ground in a glass tissue grinder to further break up the mycelia.
The suspension was filtered through two layers of cheesecloth and
sonicated in a water bath for two 30 s periods to disrupt any remaining
viable mycelia. Microscopic examination verified that no cytoplasm
remained in the mycelial fragments. Inoculum from 10 flasks was
thoroughly incorporated into 2.5 kg of composted soil previously sifted
through a 2 mm sieve and sterilized by autoclave at 120 °C at 15 psi
for 15 min. The infested soil was incubated in the laboratory at room
temperature (approximately 25 °C) for 5-7 days. Sterilized, autoclaved,
uninfested soil was used as a control in all experiments.
Figure 1. Effect of different concentrations of 6-hexyl-4-methyl-2H-yran-
2-ones on the mycelial growth of test fungi (250, 125, 100, 50, 25, and
10
µ
g/mL). Key (
µg/mL): 1, 250; 2, 125; 3, 100; 4, 50; 5, 25; 6, 10; 7,
6.25; and C, control.
on the greenhouse bench. Disease characteristics (wilting) and mortality
were assessed 7, 14, 21, 28, and 35 days after transplanting and weekly
thereafter. The number of symptomless plants was recorded for each
treatment at each assay date and expressed in terms of the proportion
of symptomless plants. Six trials of the experiment with all concentra-
tions were repeated. Disease severity was rated daily after inoculation
based on a scale from 0 to 5 as follows: 0 for no visible disease
symptoms; 1 for slightly wilted leaves, with brownish lesions beginning
to appear on the stems; 2 for 30-50% of the entire plant diseased; 3
for 50-70% of the entire plant diseased; 4 for 70-90% of the entire
plant diseased; and 5 for a dead plant. Data are the means of 20 plants
per treatment.
Analysis. Data from the above experiments in the greenhouse were
transformed as the arcsine of the square root of the proportion of the
symptomless plant stand. It was analyzed as repeated measure design
and analysis of variance determined using MSTATC software (Statisti-
cal Package). The significance level was determined before analysis
based on the observed variation in plant growth among trials due to
external greenhouse variables.
Treatments. The experimental treatments for the disease control
experiment in the greenhouse were (i) uninfected soil, moistened with
water as a check; (ii) infested soil, only water added; (iii) infested and
moistened soil treated with the formulated compound (1, 5, and 10%
concentration); and (iv) infested and moistened soil treated with the
RESULTS AND DISCUSSION
A number of 4-methyl-6-alkyl-2H-pyran-2-ones were pre-
pared by the lactonization of methyl-3-methyl-5-oxo-5-alkyl-
2
-pentenoates or ethyl-3-methyl-5-oxo-5-alkyl-2- pentenoates
commercial fungicide metalaxyl (0.364, 0.728, and 1.46 mL a.i. per
using a suitable concentrated acid (conveniently a mixture of
glacial acetic acid and concentrated sulfuric acid). They were
purified from the crude products by fractional vacuum distil-
lation (yield, 68-78%). Boiling points are similar to those
reported in the literature (11). The purity of these compounds
was further confirmed by TLC and GLC. The FT-IR spectra of
3
1
50 cm of the soil).
Disease Control in Greenhouse. After a 5-7 day incubation period,
the infested soil was treated by incorporating 84 mL of 1, 5, and 10%
aqueous emulsion of formulated compound into 2.5 kg of soil at the
3
rate of 5.0 mL of aqueous emulsion in 150 cm of the soil. The treated
soil was placed in double polyethylene bags that were then closed tightly
4
-methyl-6-alkyl-2H-pyran-2-ones showed absorption at 1720-
and incubated for 7 days. Metalaxyl was incorporated similarly at 0.364,
-
1
3
1740 (CdO) and 1630-1640 and 1550-1560 cm (CdC),
characteristic of R-pyrone.
0
.728, and 1.46 mL a.i. per 150 cm of the soil as above. After the
incubation period, soil from each treatment was placed in 20 6.6 cm
diameter standard plastic pots, and one 4-6 week old tomato seedling
was transplanted into the soil in each pot. Pots were placed randomly
1
The H NMR spectra of 4-methyl-6-substituted-2H-pyran-2-
ones showed ring protons as singlets at δ 5.80 and δ 5.90 (one

2132 J. Agric. Food Chem., Vol. 54, No. 6, 2006
Chattapadhyay and Dureja
Figure 2. Comparison of antifungal activity of different analogues of 4-methyl-6-alkyl-R-pyrones against Sr, S. rolfsii; Rs, R. solani; Rb, R. bataticola; Mp,
M. phaseolina; Pa, P. aphanidermatum; and Pd, P. deberianum.
proton each). They also indicated a normal aliphatic chain and
three vinyl protons typical of an ABX spin system. The mass
spectra of 4-methyl-6-alkyl-2H-pyran-2-ones showed, besides
the molecular ion peak, various fragment ion peaks. The mass
spectra showed major ions at m/z 95 and 109 arising from R
cleavage of the C-6 alkyl group with or without the loss of the
+
C4 methyl group, respectively, and m/z 53 (C4H5 ) and m/z 27
+
(
C2H3 ).
Antifungal Activity. Substituted 4-methyl-6-alkyl-2H-pyran-
2-ones were tested for fungicidal activity against above-reported
five soil borne pathogenic fungi by the poisoned food technique.
A smooth progressive increase in fungicidal activity was
observed with increasing molecular weight (Table 1). The lower
homologues such as 6-methyl-, 6-ethyl-, and 6-propyl-substituted
compounds resulted in a marked loss of activity (Table 1), while
compounds, namely, 6-butyl-4-methyl-2H-pyran-2-one, 6-isobu-
tyl-4-methyl-2H-pyran-2-one, 6-pentyl-4-methyl-2H-pyran-2-
one, 6-hexyl-4-methyl-2H-pyran-2-one, and 6-heptyl-4-methyl-
Figure 3. Percent healthy tomato plants after treatment of S. rolfsii infested
soil with 6-hexyl-4-methyl- -pyron and metalaxyl. Each point represents
R
the mean of repeated trials of the experiment with 20 replications (one
plant per pot) per trial. Soil was treated with 1, 5, and 10% aqueous
emulsion of the formulated compound and with metalaxyl at 0.364, 0.728,
3
and 1.46 mL a.i. per 150 cm of the soil, respectively. *Control, uninfested
soil.
2
H-pyran-2-one, were found to be very effective against all of
the tested fungi (Table 1). Compounds with 6-octyl, 6-nonyl,
and 6-decanoyl side chains showed a marked decrease in activity
6
-methyl and 6-ethyl analogues with the corresponding chain
length. The antifungal activity of 6-alkyl-4-methyl-2H-pyran-
2-one followed the order methyl < ethyl < propyl < butyl <
pentyl < hexyl < heptyl > octyl > nonyl in straight chain
compounds (Table 1 and Figure 2).
(
Table 1). The 6-hexyl-4-methyl-2H-pyran-2-one showed maxi-
-
1
mum activity against S. roflsii (ED50 ) 15.10 µg mL ), P.
-1
debaryanum (ED50 ) 14.91 µg mL ), and P. aphanidermatum
-
1
(
ED50 ) 16.61 µg mL ) (Table 1). Figure 1 shows the
inhibition of mycelial growth at different concentrations (250,
25, 100, 50, 25, 10, and 6.25 µg/mL) as compared to control
Disease Control in the Greenhouse. Among the compounds
tested for antifungal activity in vitro, 6-hexyl-4-methyl-R-pyrone
1
(compound 8 in Table 1 and Figure 1) was found to be most
by 6-hexyl-4-methyl-2H-pyran-2-one, when tested in vitro. A
complete inhibition of mycelial growths of the different
pathogenic fungi was observed at 250 and 150 µg/mL concen-
trations (Figure 1) as compared to control (full growth).
Within this and similar length conditions, compounds with a
branch(es), unsaturation, or heteroatom(s) were prepared to
examine additional effects. Branching at the â-position to the
pyrone ring was found to enhance activity; that is, the activity
of 6-isopropyl-4-methyl-2H-pyran-2-one (compound 4, ED50 )
effective against all of the test pathogenic fungi. Therefore, it
was thought worthwhile to study the antifungal activity of
6
for control in tomatoes (Lycopersicon esculentum Mill.) at
different concentrations (5.0 mL of 1, 5, and 10% aqueous
-hexyl-4-methyl-R-pyrone against S. rolfsii in the greenhouse
3
emulsion in 150 cm of the soil). Metalaxyl, a commercial
fungicide, was used for comparison.
The occurrence of symptoms in different treatments was
recorded at each assay date and expressed in terms of the
proportion of symptomless (healthy) plant stands. The results
are shown in Figure 3.
The untreated control resulted in 100% disease incidence (0%
healthy plant stands) 35 days after transplanting. The treatment
of soil with 1% aqueous emulsion of the compound resulted in
only 20% healthy plant stands after 7 days of transplanting,
whereas no healthy plant stands were observed after 35 days of
transplanting. Soil treated with metalaxyl at 0.364 mL a.i. per
3
6
13.37 µg/mL, Table 1) was considerably higher than that of
-ethyl-4-methyl-2H-pyran-2-one (compound 2, ED50 ) 665.30
µg/mL) with the corresponding length, and similarly, 6-iso-
butyl-4-methyl-2H-pyran-2-one (compound 6, ED50 ) 39.45 µg/
mL) had a significantly higher activity than 6-propyl-4-methyl-
2
H-pyran-2-one (compound 3, ED50 ) 250.87 µg/mL) against
S. rolfsii.
The unsaturated 6-alkyl substituents (compounds 14, 15, and
7) resulted in loss of fungicidal activity. The 6-chloromethyl-
-methyl-R-pyrone (compound 16) was found more active than
1
4
3
150 cm of the soil resulted in 34.3% healthy plant stands 35

Antifungal Activity of 4-Methyl-6-alkyl-2H-pyran-2-ones
J. Agric. Food Chem., Vol. 54, No. 6, 2006 2133
LITERATURE CITED
days after transplanting; however, because of experimental
variations, the healthy plant stands were not significant (P )
(
1) Nobuhara, A. Synthesis of unsaturated lactones; Part IV:
0
.103).
Flavorous nature of some aliphatic γ-lactones. Agric. Biol. Chem.
The treatment of soil with 5% aqueous emulsion of the
1
970, 34, 1745-1747.
compound showed survival of more than 60% healthy plant
stands after 35 days of transplanting. However, the healthy plant
stands were not significantly (P > 0.1) different from the soil
treated with a medium dose of metalaxyl (at 0.728 a.i. per 150
(2) Collins, R. P.; Halim, A. F. Characterization of major aroma
constituent of the fungus Trichoderma viride (Pers.). J. Agric.
Food Chem. 1972, 20, 437-439.
(
3) Claydon, N.; Allan, M.; Hanson, J. R.; Avent, A. G. Antifungal
3
alkyl pyrones of Trichoderma harzianum. Trans. Br. Mycol. Soc.
cm ; 60% healthy plant stands, Figure 3).
1
987, 88 (4), 503-513.
The treatment of soil with 10% aqueous emulsion of the
compound resulted in significantly (P < 0.1) greater healthy
plant stands (92.4%) than that obtained with metalaxyl (90.3%)
(
4) Cutler, H. G. Biologically active natural products from fungi.
In Templates for Tomorrow’s Pesticides, in Bioregulators,
Chemistry and Uses; Ory, R. L., Rittig, F. R., Eds.; American
Chemical Society: Washington DC, 1984.
3
5 days after transplanting (Figure 3). Thus, significant
(
5) Cutler, H. G.; Cox, R. L.; Crumley, F. G.; Cole, P. D. 6-Pentyl-
R-pyrone from Trichoderma harzianum; its plant growth inhibi-
tory and antimicrobial properties. Agric. Biol. Chem. 1986, 50
differences existed among concentration and time for disease
control experiments in the greenhouse. The 10% aqueous
emulsion of the compound prevented tomato damping off
significantly. It allowed a plant stand comparable to the
(11), 2943-2945.
(
6) Parker, S. R.; Cutler, H. G,; Jacyno, J. M.; Hill, R. A. Biological
activity of 6-pentyl-2-H-pyran-2-one and its analogues. J. Agric.
Food Chem. 1997, 45, 2774-2776.
uninfested control and metalaxyl (at 0.728 and 1.46 mL a.i. per
3
1
50 cm of the soil).
In conclusion, among 4-methyl-6-alkyl-R-pyrones, 6-butyl-,
(7) Scarselletti, R.; Faull, J. L. In vitro activity of 6-pentyl-R-pyrone,
a metabolite of Trichoderma harzianum in the inhibition of
Rhizoctonia solani and Fusarium oxysporum f.sp. lycopersici.
Mycol. Res. 1984, 98 (10), 1207-1209.
6
-pentyl-, 6-hexyl-, and 6-heptl-substituted 4-methyl pyrones
showed antifungal activity against S. rolfsii Saccardo, R.
bataticola (Taub.) Butler, P. aphanidermatum (Edson) Fitz., M.
phaseolina, P. debaryanum, and R. solani Nees in vitro. The
(
8) Moss, M. O.; Jackson, R. M.; Roger, D. The characterization of
6-(pent-1-enyl)-R-pyrone from Trichoderma Viride. Phytochem-
4
-methyl-6-hexyl-R-pyrone, which was found to be the most
istry 1975, 14, 2706.
effective of the series, reduced disease development on tomato
caused by S. rolfsii. Experimental conditions were optimized
for survival of S. rolfsii in soil and disease development in the
greenhouse. Despite favorable conditions for the pathogens, the
aqueous formulation of 4-methyl-6-hexyl-R-pyrone reduced the
pathogen population and resulted in higher healthy plant stands
as compared to the control (pathogen only).
(
9) Cutler, H. G,; Parker, S. R.; Hill, R. A. Fungicide comprising
4-methyl-6-pentyl-2H-pyran-2-one. U.S. Patent 6,251,832, 2001.
(10) Vogel’s Text Book of Practical Organic Chemistry, 5th ed.;
Furniss, B. S., Hanford, A. J., Smith, P. W. G., Tatchell, A. R.,
Eds.; ELBS: 1996.
11) Pittet, A. O.; Klaiber, E. M. Synthesis and flavour properties of
some alkyl substituted R-pyrones derivatives. J. Agric. Food
Chem. 1975, 23, 1189-1195.
(
Received for review November 10, 2005. Revised manuscript received
January 30, 2006. Accepted January 31, 2006. We thank DBT for
financial support.
ACKNOWLEDGMENT
We thank Dr. B. S. Parmar, Head, Division of Agricultural
Chemicals, IARI, New Delhi, for providing all facilities.
JF052792S