Antifungal activity of 4′-(2,6,6-Trimethyl-2-cyclohexen-1-yl)- 3′-butene-2′-ketoxime N-O-alkyl ethers

Article abstract of DOI:10.1021/jf903044j

A number of 4′-(2, 6, 6-trimethyl-2-cyclohexen-1-yl)-3′-butene- 2′-ketoxime N-O-alkyl ethers were prepared, separated into their E and Z isomers, and characterized on the basis of ¹H NMR and mass spectroscopy. These compounds were tested in vit

Full text of DOI:10.1021/jf903044j

1032 J. Agric. Food Chem. 2010, 58, 1032–1039
DOI:10.1021/jf903044j
Antifungal Activity of 40-(2,6,6-Trimethyl-2-cyclohexen-1-yl)-
30-butene-20-ketoxime N-O-Alkyl Ethers
T
IRTHANKAR
B
ANERJEE AND
P
REM
DUREJA
*
Division of Agricultural Chemicals, Indian Agricultural Research Institute, New Delhi 110 012, India
A number of 40-(2,6,6-trimethyl-2-cyclohexen-1-yl)-30-butene-20-ketoxime N-O-alkyl ethers were prepared,
separated into their E and Z isomers, and characterized on the basis of 1H NMR and mass spectroscopy.
These compounds were tested in vitro for antifungal activity against four important phytopathogenic fungi,
namely, Sclerotium rolfsii, Rhizoctonia bataticola, Macrophomina phaseolina, and Sclerotinia sclerotior-
um. E isomers of 40-(2,6,6-trimethyl-2-cyclohexen-1-yl)-30-butene-20-ketoxime N-O-propyl ether (ED50
=
32.36 μg mL-1) and 40-(2,6,6-trimethyl-2-cyclohexen-1-yl)-30-butene-20-ketoxime N-O-(100-methyl) ethyl
ether (ED50 = 35.50 μg mL-1) showed maximum antifungal activity against R. bataticola and S. rolfsii,
respectively, whereas 40-(2,6,6-trimethyl-2-cyclohexen-1-yl)-30-butene-20(Z)-ketoxime N-O-pentyl ether
was found to be active against M. phaseolina (ED50 = 31.08 μg mL-1) and S. sclerotiorum (ED50
21.39 μg mL-1), respectively. The Z isomer of 40-(2,6,6-trimethyl-2-cyclohexen-1-yl)-30-butene-20-ketox-
ime N-O-pentyl ether, which was found to be most effective, was tested against S. sclerotiorum in a
greenhouse at 1 and 5% concentrations. The 5% aqueous emulsion of 40-(2,6,6-trimethyl-2-cyclohexen-
1-yl)-30-butene-20(Z)-ketoxime N-O-pentyl ether suppressed disease development in pea by 90-95% as
compared with the untreated infested soil in the greenhouse after 21 days of treatment.
KEYWORDS: R-Ionone; antifungal; oxime ethers; Rhizoctonia bataticola; Macrophomina phaseolina;
Sclerotium rolfsii; Sclerotinia sclerotiorum
INTRODUCTION
number of oxime ethers, prepared from R-ionone and separated
into their E and Z isomers, against four widely occurring
soilborne plant pathogenic fungi, namely, Rhizoctonia bataticola
(Taub) Butler, the causal organism for damping-off of seedlings;
Macrophomina phaseolina (Tassi) Goid, responsible for charcoal
rot of soybean, peanut, and corn, stem rot of jute; Sclerotium
rolfsii (Sacc.) Curzi, which causes southern blight of cucurbitac-
eous crops; and Sclerotinia sclerotiorum, which causes water-
soaked spots of irregular shape on fruits, stems, and leaves of
beans, cabbage, carrots, cucumbers, lettuce, onions, peas, pump-
kins, squash, and tomatoes.
The terpenoids obtained from nature are a rich source of
bioactive molecules. They are considered as a bountiful domain
because of their versatility in bioactivity (1), target specificity,
environmentally benign nature, and nonpersistency. The essential
oils, which are the primary source of terpenoids, have been found
to possess antifeedant (2), repellent (3), insect growth regula-
tory (4), feeding deterrent, fungicidal fumigant (5), antifungal (6),
and antibacterial (7) activities.
A variety of terpenoid juvenile hormone (JH) analogues (8)
have been invented, and the majority of them possess sesquiter-
penoid structure (9). With the progress of research on JH
analogues, the oxime ether group of the compounds has become
the center of attraction for their insect growth regulatory activ-
ity (10,11). It has been also found that unlike the natural product
as such, their derivatives are more stable in the environment. A
few strobilurin fungicides containing an oxime ether moiety,
namely, kresoxim-methyl (12) and trifloxystrobin (13), have been
commercialized to control powdery mildew of various fruit crops,
which indicates the potential of this group for more investigation
for controlling plant pathogenic fungi.
MATERIALS AND METHODS
Chemicals and Reagents. R-Ionone (C13H20O, MW 192.30), ob-
tained from Florentine iris (I. florentina) was used as lead molecule for the
synthesis of oxime ethers and was procured from Fluka Chemika. Alkyl
halides, sodium hydride, and hydroxylamine hydrochloride were procured
locally and used directly without further purification. All of the solvents
used in synthesis as well as in chromatography were distilled and dried
before use.
Plant Pathogenic Fungi. Plant pathogenic fungi such as R. bataticola
(Taub) Butler, M. phaseolina (Tassi) Goid., S. rolfsii (Sacc.) Curzi, and S.
sclerotiorum 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.
R-Ionone is a cyclic monoterpenoid ketone naturally obtained
from the oil of orris (Iris florentina) root. As it is from natural
sources it can be considered as an effective substrate for the
synthesis of desired oxime ethers. In the present study, we report
the synthesis and in vitro and in vivo antifungal activities of a
Chromatography and Spectroscopy. Thin layer chromato-
graphy (TLC) was performed on 20 cm ꢀ 20 cm glass plates coated with
0.5 mm silica gel G, containing 10% of gypsum as binder, air-dried, and
*Author to whom correspondence should be addressed (e-mail Pd.
dureja@gmail.com).
pubs.acs.org/JAFC
Published on Web 12/28/2009
© 2009 American Chemical Society

Article
J. Agric. Food Chem., Vol. 58, No. 2, 2010 1033
Scheme 1. Scheme for the Synthesis of 40-(2,6,6-Trimethyl-2-cyclohexen-1-yl)-30-butene-20-ketoxime N-O-Alkyl Ethers
preactivated at 120 °C for 2 h before use. Hexane and acetone in the ratios
of 9:1 and 8:2 were used as developing solvent, and iodine vapor was used
as visualizing agent. E and Z isomers of 40-(2,6,6-trimethyl-2-cyclohexen-
1-yl)-30-butene-20-ketoxime N-O-alkyl ethers were separated using a glass
column (75 cm ꢀ 2 cm i.d.) packed with preactivated silica gel (50 g,
60-120 mesh) in hexane and eluted with a mixture of hexane and
chloroform in various ratios. Different fractions (25 mL) were collected
and distilled on a water bath. The fractions containing similar compounds
were combined and purified further for characterization. A gas-liquid
chromatograph (GLC) (Hewlett-Packard 5890 series II) equipped with a
flame ionization detector, HP-1 megabore column (10 m ꢀ 0.53 mm i.d.
and 2.65 μm film thickness) and HP 3390A integrator was used to
determine the ratio of E and Z isomers in the reaction mixture. The
operating conditions were oven, injector, and detector temperatures at
150, 250, and 250 °C, respectively. Nitrogen was used as carrier at a flow
rate of 20 mL min-1. The proton nuclear magnetic resonance spectra
(1H NMR) were recorded on a Varian EM 360 L (60 MHz) and on a
Bruker 400 AC (400 MHz) instrument. The solvents used were carbon
tetrachloride (CCl4) and deuteriochloroform (CDCl3) containing tetra-
methylsilane ((CH3)4Si, TMS) as the internal standard. The chemical shifts
are expressed in δ values (ppm), and coupling constants (J) are given in
hertz (Hz). Mass spectra were recorded on a HRGC-MEGA 2 series gas
chromatograph coupled to a FISONS-TRIO 1000 ion trap mass spectro-
meter 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 rate
separated, washed with water, dried over anhydrous sodium sulfate, and
concentrated in vacuum, which gave an amber-colored viscous oily liquid.
The oily residue was separated on a silica gel column with hexane and
hexane/chloroform in different ratios as eluting solvent separatingE and Z
isomers of 40-(2,6,6-trimethyl-2-cyclohexen-1-yl)-30-butene-20-ketoxime
N-O-alkyl ether. The physicochemical properties are given in Table 1.
Antifungal Activity. The antifungal activity was tested in vitro against
R. bataticola (Taub) Butler, M. phaseolina (Tassi) Goid., S. rolfsii (Sacc.)
Curzi, and S. sclerotiorum.
In Vitro Antifungal Activity. The above-synthesized compounds
were tested for their ability to inhibit the above soilborne pathogenic fungi.
The fungicidal activity of synthesized compounds was evaluated at various
concentrations by the poisoned food technique using PDA media (14).
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 then tested at concentrations of 1000, 500, 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 (9 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 mycelial disk 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 until the fungal growth was almost complete in the
control plates. All experiments were in quadruplicate for each treatment
against each fungus.
of 2 mL min-1
.
General Procedure for the Synthesis of 40-(2,6,6-Trimethyl-2-
cyclohexen-1-yl)-30-butene-20-ketoxime. Hydroxylamine hydrochlor-
ide (0.75 g, 0.011 mol) was added to a stirred solution of R-ionone (1.9 g,
0.01 mol) dissolved in dry and distilled ethyl alcohol (25 mL) (Scheme 1).
Stirring was continued for another 2 h with simultaneous monitoring by
TLC (solvent system hexane/acetone 80:20, Rf = 0.33). After completion
of the reaction, the reaction mixture was poured into water and extracted
with diethyl ether (3 ꢀ 25 mL). The solvent extracts were combined and
dried over anhydrous sodium sulfate. The ether was completely distilled
off. An amber-colored viscous liquid product was obtained and used for
further reaction.
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:
C -T
C
inhibition ð%IÞ ¼
ꢀ 100
General Procedure for the Synthesis of 40-(2,6,6-Trimethyl-2-
cyclohexen-1-yl)-30-butene-20-ketoxime N-O-Alkyl Ether. Dimethyl
sulfoxide (dried over molecular sieve, 5 mL) was slowly added with stirring
to sodium hydride (0.024 g, 0.01 mol, weighed in a drybox) in an
atmosphere of dry nitrogen. Nitrogen was dried by passing though a train
consisting of (a) a trap, (b) a wash bottle containing concentrated sulfuric
acid, and (c) a drying tube containing fresh soda lime. Stirring was further
continued for about 30 min until the evolution of hydrogen ceased. Into
the above stirred solution was added dropwise 40-(2,6,6-trimethyl-2-
cyclohexen-1-yl)-30-butene-20-ketoxime (0.01 mol), maintaining the tem-
perature at 5 °C (ice bath). The reaction mixture was stirred for another
1 h, which was followed by the addition of the appropriate alkyl halide
(0.011 mol), and the mixture was again stirred for another 2 h (Scheme 1).
The progress of the reaction was monitored by TLC (solvent system
hexane/acetone, 9:1, v/v) and iodine as visualizing agent. After completion
of the reaction, the reaction mixture was poured into ice-cold water and
extracted with ethyl acetate (3 ꢀ 25 mL). The ethyl acetate layer was
Calculation of ED50 Values. For the 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
100 -CF
corrected inhibition ð%Þ ¼
ꢀ 100
where CF is the correction factor obtained by the equation
9 -C
C
correction factor ðCFÞ ¼
ꢀ 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 (μg mL-1
)
and corresponding corrected percentage inhibition data of each com-
pound, Probit analysis was done with the help of the Probit package

1034 J. Agric. Food Chem., Vol. 58, No. 2, 2010
Banerjee and Dureja
Table 1. Physicochemical Properties of 40-(2,6,6-Trimethyl-2-cyclohexen-1-yl)-30-butene-20-ketoxime N-O-Alkyl Ether
compd
R
name
physical state molecular formula TLC (Rf)a
1a
1b
CH3
CH3
40-(2,6,6-trimethyl-2-cyclohexen-1-yl)-30-butene-20(E)-ketoxime N-O-methyl ether
40-(2,6,6-trimethyl-2-cyclohexen-1-yl)-30-butene-20(Z)-ketoxime N-O-methyl ether
40-(2,6,6-trimethyl-2-cyclohexen-1-yl)-30-butene-20(E)-ketoxime N-O-ethyl ether
40-(2,6,6-trimethyl-2-cyclohexen-1-yl)-30-butene-20(Z)-ketoxime N-O-ethyl ether
40-(2,6,6-trimethyl-2-cyclohexen-1-yl)-30-butene-20(E)-ketoxime N-O-propyl ether
40-(2,6,6-trimethyl-2-cyclohexen-1-yl)-30-butene-20(Z)-ketoxime N-O-propyl ether
40-(2,6,6-trimethyl-2-cyclohexen-1-yl)-30-butene-20(E)-ketoxime N-O-(100-methyl)ethyl ether
40-(2,6,6-trimethyl-2-cyclohexen-1-yl)-30-butene-20(Z)-ketoxime N-O-(100-methyl)ethyl ether
40-(2,6,6-trimethyl-2-cyclohexen-1-yl)-30-butene-20(E)-ketoxime-N-O-200propenyl ether
40-(2,6,6-trimethyl-2-cyclohexen-1-yl)-30-butene-20(Z)-ketoxime N-O-200-propenyl ether
40-(2,6,6-trimethyl-2-cyclohexen-1-yl)-30-butene-20(E)-ketoxime N-O-butyl ether
40-(2,6,6-trimethyl-2-cyclohexen-1-yl)-30-butene-20(Z)-ketoxime N-O-butyl ether
liquid
liquid
liquid
liquid
liquid
liquid
liquid
liquid
liquid
liquid
liquid
liquid
C14 H23 NO
C14 H23 NO
C15 H25 NO
C15 H25 NO
C16 H27 NO
C16 H27 NO
C16 H27 NO
C16 H27 NO
C16 H25 NO
C16 H25 NO
C17 H29 NO
C17 H29 NO
C17 H29 NO
C17 H29 NO
C18 H31 NO
C18 H31 NO
C18 H31 NO
C18 H31 NO
C19 H33 NO
C19 H33 NO
C20 H35 NO
C20 H35 NO
C21 H37 NO
C21 H37 NO
C23 H41 N O
C23 H41 NO
0.59
0.34
0.64
0.36
0.71
0.38
0.72
0.39
0.74
0.42
0.76
0.42
0.75
0.42
0.79
0.44
0.77
0.42
0.79
0.46
0.80
0.49
0.81
0.51
0.84
0.55
2a
2b
C2H5
C2H5
C3H7
C3H7
3a
3b
4a
4b
CH(CH3)2
CH(CH3)2
CH2CHdCH2
CH2CHdCH2
C4H9
5a
5b
6a
6b
C4H9
7a
7b
CH2CH(CH3)2
CH2CH(CH3)2
C5H11
40-(2,6,6-trimethyl-2-cyclohexen-1-yl)-30-butene-20(E)-ketoxime N-O-(200-methyl)propyl ether liquid
40-(2,6,6-trimethyl-2-cyclohexen-1-yl)-30-butene-20(Z)-ketoxime N-O-(200-methyl)propyl ether liquid
40-(2,6,6-trimethyl-2-cyclohexen-1-yl)-30-butene-20(E)-ketoxime N-O-pentyl ether
40-(2,6,6-trimethyl-2-cyclohexen-1-yl)-30-butene-20(Z)-ketoxime N-O-pentyl ether
8a
8b
liquid
liquid
liquid
liquid
liquid
liquid
liquid
liquid
liquid
liquid
liquid
liquid
C5H11
9a
9b
C2H4CH(CH3)2 40-(2,6,6-trimethyl-2-cyclohexen-1-yl)-30-butene-20(E)-ketoxime N-O-(300-methyl)butyl ether
C2H4CH(CH3)2 40-(2,6,6-trimethyl-2-cyclohexen-1-yl)-30-butene-20(Z)-ketoxime N-O-(300-methyl)butyl ether
10a
10b
11a
11b
12a
12b
13a
13b
C6H13
C6H13
C7H15
C7H15
C8H17
C8H17
C10H21
C10H21
40-(2,6,6-trimethyl-2-cyclohexen-1-yl)-30-butene-20(E)-ketoxime N-O-hexyl ether
40-(2,6,6-trimethyl-2-cyclohexen-1-yl)-30-butene-20(Z)-ketoxime N-O-hexyl ether
40-(2,6,6-trimethyl-2-cyclohexen-1-yl)-30-butene-20(E)-ketoxime N-O-heptyl ether
40-(2,6,6-trimethyl-2-cyclohexen-1-yl)-30-butene-20(Z)-ketoxime N-O-heptyl ether
40-(2,6,6-trimethyl-2-cyclohexen-1-yl)-30-butene-20(E)-ketoxime N-O-octyl ether
40-(2,6,6-trimethyl-2-cyclohexen-1-yl)-30-butene-20(Z)-ketoxime N-O-octyl ether
40-(2,6,6-trimethyl-2-cyclohexen-1-yl)-30-butene-20(E)-ketoxime N-O-decyl ether
40-(2,6,6-trimethyl-2-cyclohexen-1-yl)-30-butene-20(Z)-ketoxime N-O-decyl ether
a Solvent system: hexane/acetone (9:1).
of MSTATC software using a personal computer. ED50 values were
statistically calculated (15) for the inhibition of growth using the Basic
LD50 program version 1.1.
thoroughly incorporated into 2.5 kg of composted soil previously sifted
through a 2 mm sieve and sterilized by autoclave at 120 °Cat15psifor15min.
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.
In Vivo Antifungal Activity. The most active compound of the series,
40-(2,6,6-trimethyl-2-cyclohexen-1-yl)-30-butene-20(Z)-ketoxime N-O-pen-
tyl ether (ED50 = 31.39 μg mL-1), was tested in vivo for antifungal activity
against S. sclerotiorum in pots keeping carbendazim as standard fungicide
for comparison. The concentrations of the latter were those recommended
by the manufacturer. S. sclerotiorum was chosen because it is among the
most nonspecific, omnivorous, and successful of plant pathogens with an
extensive host range; at least 361 species in 64 families are susceptible. The
first signs of infection are water-soaked spots on the stem at or just beneath
the soil level. The symptoms are water-soaked spots on fruits, stems,
leaves, and petioles, which usually have an irregular shape. These spots
enlarge, and a cottony mycelium covers the affected area. The fungus
spreads, and the plant becomes a soft, slimy, water-soaked mass. Seedlings
are very susceptible and die quickly once they become infected. In contrast
to the water-soaked symptoms, the older plants may exhibit “dry” lesions
on the stalk, stems, or branches. The lesions enlarge, girdling the plant part
to yellowish brown in color, causing it to die.
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, with only water added; (iii) infested and
moistened soil treated with the formulated compound (1 and 5%
concentration); and (iv) infested and moistened soil treated with the
commercial fungicide carbendazim (0.364 and 0.728 mL of ai/150 cm3
of soil).
Disease Control in the Greenhouse. After a 5-7 day incubation
period, the infested soil was treated by incorporating 84 mL of 1 and 5%
aqueous emulsion of formulated compound into 2.5 kg of soil at the rate of
5.0 mL of aqueous emulsion in 150 cm3 of the soil. The treated soil was
placed in double polyethylene bags that were then closed tightly and
incubated for 7 days. Carbendazim was incorporated similarly at 0.364
and 0.728 mL of ai/150 cm3 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 pea seedling was transplanted into the
soil in each pot. Pots were placed randomly on the greenhouse bench.
Disease characteristics (soft, slimy, water-soaked spots) and mortality
were assessed 7, 14, and 21 days after transplantation and weekly there-
after. The number of symptomless plants was recorded for each treatment
at eachassay date and expressed in terms of the proportion of symptomless
plants. Six trials of the experiment with all concentrations were repeated.
Disease severity was rated daily after inoculation on the basis of a scale
from 0 to 5 as follows: 0 for no visible disease symptoms; 1 for water-
soaked spots on stems and leaves, with cottony mycelium 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
Inoculum Preparation. Mycelial mats of S. sclerotiorum were grown
in potato dextrose broth in 250 mL conical flasks and incubated horizon-
tally 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. Ascospores or sclerotia 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 twice for 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

Article
J. Agric. Food Chem., Vol. 58, No. 2, 2010 1035
diseased; and 5 for a dead plant. Data are the means of 20 plants per
treatment.
with respect to base peak has been observed in products 3E and
3Z. The mass spectrum of product 3E showed a base peak at m/z
134 (100%) along with fragment ion peaks with higher relative
abundance at m/z 249 and 193 than the other isomer. In contrast,
3Z has shown a base peak at m/z 83 (100%) formed by retro-
Diels-Alder reaction of the unsaturated ring. All other sets
of isomers have shown similar trends of mass fragmentation for
E and Z isomers. Both products showed similar fragment ion
peaks but with different relative abundance percentages at m/z
234 (Mþ - CH3), 207 (Mþ - C3H6), 206 (Mþ - C3H7), 193
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 a repeated measure design
and analysis of variance determined using MSTATC software (Statistical
Package). The significance level was determined before analysis on the
basis of the observedvariation inplant growth among trials due to external
greenhouse variables.
(Mþ - CH3 - C3H5), 178 (Mþ - 2CH3 - C3H6), 160 (Mþ
C3H7 - NOH - CH3), 150 (Mþ - C5H9NO), 136 (Mþ - C5H9-
-
RESULTS AND DISCUSSION
Synthesis. 40-(2,6,6-Trimethyl-2-cyclohexen-1-yl)-30-butene-20-
ketoxime required for the preparation of oxime ethers was
obtained by reacting 40-(2,6,6-trimethyl-2-cyclohexen-1-yl)-30-
butene-20-one (R-ionone) with hydroxylamine hydrochloride.
The oximes are usually a 1:1 mixture of cis and trans isomers
and are inseparable by conventional chromatographic technique.
40-(2,6,6-Trimethyl-2-cyclohexen-1-yl)-30-butene-20-ketoxime N-
O-alkyl ethers were synthesized by reacting 40-(2,6,6-trimethyl-
2-cyclohexen-1-yl)-30-butene-20-ketoxime with appropriate alkyl
halide (Scheme 1). These oxime ethers being a mixture of
two isomers were separated and purified on a silica gel column
eluting with hexane and hexane/chloroform (90:10) as eluting
solvent. Although it was expected that the E and Z isomers
would be produced in equal amounts, actually in the reaction
mixture the E isomer of oxime ether was found to be the
predominant product and the E/Z ratio calculated by GLC was
found to be 2:1. An unequivocal assignment of their stereochem-
NO - CH2), 134 (Mþ - 2CH3 - C3H6 - CNOH2), 119 (Mþ
-
NO - C4H9), 117 (Mþ - NO - C4H11), 107 (Mþ - NO - C5H9),
105 (Mþ - NO - C4H9 - CH2), 93 (Mþ - C5H9NO - C4H9), 91
(Mþ - 2CH3 - C3H6 - NO - C4H9 or Mþ - NO - C4H9 -
2CH2), 85 (Mþ - CH3 - C3H5 - C8H12), 83 (Mþ - C3H6 -
C9H16), 79 (Mþ - C5H9NO - C4H9 - CH2), and 77 (Mþ
-
2CH3 - C3H6 - NO - C4H9 - CH2), but their percent relative
abundances were different. Thus, on the basis of 1H NMR and
mass spectroscopy, products 3E and 3Z were characterized as
40-(2,6,6-trimethyl-2-cyclohexen-1-yl)-30-butene-20(E)-ketoxime
N-O-propyl ether and 40-(2,6,6-trimethyl-2-cyclohexen-1-yl)-30-
butene-20(Z)-ketoxime N-O-propyl ether.
Antifungal Activity. E and Z isomers of 40-(2,6,6-trimethyl-2-
cyclohexen-1-yl)-30-butene-20-ketoxime N-O-alkyl ethers were
tested for fungicidal activity against the above-reported four
soilborne pathogenic fungi by the poisoned food technique. A
smooth progressive increase in fungicidal activity was observed
with increasing molecular weight (Table 3). The lower homo-
logues such as O-methyl- and O-ethyl-substituted compounds
resulted in a marked loss of activity (Table 3), whereas the
E isomer of 40-(2,6,6-trimethyl-2-cyclohexen-1-yl)-30-butene-20-
ketoxime N-O-propyl ether was found to be more effective
against R. bataticola (ED50 = 32.36 μg mL-1) as compared to
its Z isomer (ED50 = 156.85 μg mL-1). It was also found to be
effective against other test fungi as compared to methyl and ethyl
derivatives. The E isomer of 40-(2,6,6-trimethyl-2-cyclohexen-
1-yl)-30-butene-20-ketoxime N-O-isopropyl ether was found to
be effective against S. rolfsii (ED50 = 35.50 μg mL-1) as com-
pared to its Z isomer (ED50 = 129.03 μg mL-1). Here a pronoun-
ced effect of branching of the side chain on the antifungal
activity was observed in the case the E isomer, where a 3-fold
increase in activity was observed when the n-propyl group
(ED50=101.30 μg mL-1) was replaced by and isopropyl group
(ED50 = 35.50 μg mL-1). On the other hand, 40-(2,6,6-trimethyl-
2-cyclohexen-1-yl)-30-butene-20(Z)-ketoxime N-O-pentyl ether
has been found to exhibit highest activity against M. phaseolina
(ED50 =31.08 μg mL-1) and S. sclerotiorum (ED50=21.39 μg
mL-1), respectively. There was an increase in antifungal activity
with an increase in the number of carbon atoms to a certain
extent. Compounds, namely, E and Z isomers of 40-(2,6,6-
trimethyl-2-cyclohexen-1-yl)-30-butene-20-ketoxime N-O-propyl
ether, E and Z isomers of 40-(2,6,6-trimethyl-2-cyclohexen-1-yl)-
30-butene-20-ketoxime N-O-isopropyl ether, E and Z isomers
of 40-(2,6,6-trimethyl-2-cyclohexen-1-yl)-30-butene-20-ketoxime
N-O-butyl ether, E and Z isomers of 40-(2,6,6-trimethyl-2-cyclo-
hexen-1-yl)-30-butene-20-ketoxime N-O-isobutyl ether, and E and
Z isomers of 40-(2,6,6-trimethyl-2-cyclohexen-1-yl)-30-butene-20-
ketoxime N-O-pentyl ether, were found to be very effective
against all of the tested fungi (Table 3). Both E and Z iso-
mers of 40-(2,6,6-trimethyl-2-cyclohexen-1-yl)-30-butene-20-ke-
toxime N-O with isopentyl, hexyl, heptyl, octyl, and decyl ether
showed a marked decrease in activity (Table 3). The antifungal
activity of 40-(2,6,6-trimethyl-2-cyclohexen-1-yl)-30-butene-20-ketoxime
1
istry was accomplished with H NMR and mass spectroscopy
(Table 2).
1
The H NMR spectra of all the pure compounds (Table 2)
exhibited a similar general pattern in a homologous series with
some distinct differences that were confirmatory enough to
identify each of the E and Z isomers. For example, R-ionone
oxime on reaction with propyl bromide produced 40-(2,6,6-
trimethyl-2-cyclohexen-1-yl)-30-butene-20-ketoxime N-O-propyl
ether as an amber-colored viscous liquid. The oily residue was
separated into E and Z isomers on a silica gel column eluting with
n-hexane and gave the 3E isomer, whereas elution with hexane/
chloroform (9:1) gave the 3Z isomer. Formation of oxime ether
was characterized by the appearance of a two proton triplets at
δ 4.08-4.12 due to O-CH2 (j) with a coupling constant (J) value
of 3 Hz (Table 2). The product 3E showed two three-proton
singlets at δ 0.94 and 0.92 due to CH3 (e) and CH3 (f), whereas in
product 3Z isomer one six-proton singlet was found downfield at
δ 1.26 for [CH3 (e) and CH3 (f)]. Similarly, one proton doublet
was found at δ 6.10 due to dCH (h) in the 3E isomer, but was
found downfield at δ 6.75 in the case of the 3Z isomer. The
difference in the chemical shifts in CH3 protons in the 3E and 3Z
isomers is because of the difference in anisotropic shielding in the
E and Z isomers with their fixed geometries. The presence of
protons due to CH3 (e), CH3 (f) and dCH (h) upfield in product
3E isomer as compared to the 3Z isomer indicated that in the 3E
isomer the O-propyl group is syn to the methyl group (i), whereas
in the 3Z isomer it is anti to the methyl group (i). Thus, on the
1
basis of H NMR spectra, the product 3E was assigned the E
configuration and product 3Z as the Z configuration.
The mass spectra of products 3E and 3Z showed a molecular
ion peak at m/z 249 with relative abundances of 89 and 7%,
respectively. The higher relative abundance of the molecular ion
peak indicates greater stability of 3E, which can be correlated
with the 2:1 ratio of 3E and 3Z in the reaction mixture. As both
the products showed the same molecular ion peak, these two
products are geometric isomers. A distinct difference in the
fragmentation pattern as well as in percent relative abundance

1036 J. Agric. Food Chem., Vol. 58, No. 2, 2010
Banerjee and Dureja
Table 2. Spectral Data of 40-(2,6,6-Trimethyl-2-cyclohexen-1-yl)-30-butene-20-ketoxime N-O-Alkyl Ether
compd
R
1H NMR (δ)
mass (m/z)
1a
CH3
6.04 (1H, h, d, J = 8 Hz), 5.81 (1H, g, dd, J = 3 Hz), 5.43 (1H, b, s), 3.84 221 (Mþ, 89%), 203 (17%), 207 (4%), 206 (5%), 193 (89%), 178 (48%),
(2H, j, s), 2.23 (1H, m, d, J = 3 Hz), 1.94 (3H, i, s), 1.71(1H, c2, q, J =
3 Hz), 1.58 (2H, d, t, J = 3 Hz), 1.57 (3H, a, s), 1.42 (1H, c1, q, J = 3
Hz), 0.94 (3H, e, s), 0.92 (3H, f, s)
160 (8%), 150 (49%), 136 (28%), 134 (100%), 119 (22%), 117 (13%),
107 (35%), 105 (20%), 93 (83%), 91 (60%), 85 (7%), 83 (12%), 79
(25%), 77 (29%)
1b
2a
2b
3a
CH3
6.57 (1H, h, d, J = 8 Hz), 5.79 (1H, g, dd, J = 3 Hz), 5.46 (1H, b, s), 3.87 221 (Mþ, 7%), 207 (2%), 193 (5%), 178 (4%), 150 (5%), 136 (3%), 134
(2H, j, s), 2.00 (1H, m, d, J = 3 Hz), 1.97 (3H, i, s), 1.71 (1H, c2, q, J =
3 Hz), 1.58 (2H, d, t, J = 3 Hz), 1.58 (3H, a, s), 1.45 (1H, c1, q, J = 3
Hz), 1.26 (3H, e, s), 1.26 (3H, f, s)
(10%), 119 (5%), 117 (4%), 107 (5%), 105 (3%), 93 (17%), 91 (14%),
85 (92%), 83 (100%), 79 (5%), 77 (7%)
C2H5
C2H5
C3H7
6.11 (1H, h, d, J = 8 Hz), 5.85 (1H, g, dd, J = 3 Hz), 5.42 (1H, b, s), 4.1 235 (Mþ, 89%), 217 (17%), 207 (4%), 206 (5%), 193 (89%), 178 (48%),
(2H, j, q, J = 3 Hz), 2.23 (1H, m, d, J = 3 Hz), 2.01 (3H, l, t, J = 3 Hz),
1.94 (3H, i, s), 1.71 (1H, c2, q, J = 3 Hz), 1.58 (2H, d, t, J = 3 Hz), 1.57
(3H, a, s), 1.42 (1H, c1, q, J = 3 Hz), 0.94 (3H, e, s), 0.92 (3H, f, s)
6.67 (1H, h, d, J = 8 Hz), 5.83 (1H, g, dd, J = 3 Hz), 5.4 (1H, b, s), 4.08 235 (Mþ, 7%), 207 (2%), 193 (5%), 178 (4%), 150 (5%), 136 (3%), 134
160 (8%), 150 (49%), 136 (28%), 134 (100%), 119 (22%), 117 (13%),
107 (35%), 105 (20%), 93 (83%), 91 (60%), 85 (7%), 83 (12%), 79
(25%), 77 (29%)
(2H, j, q, J = 3 Hz), 2.06 (3H, l, t, J = 3 Hz), 2.00 (1H, m, d, J = 3 Hz),
1.97 (3H, i, s), 1.71 (1H, c2, q, J = 3 Hz), 1.58 (2H, d, t, J = 3 Hz), 1.58
(3H, a, s), 1.45 (1H, c1, q, J = 3 Hz), 1.26 (3H, e, s), 1.26 (3H, f, s)
6.10 (1H, h, d, J = 8 Hz), 5.83 (1H, g, dd, J = 3 Hz), 5.43 (1H, b, s), 4.12 249 (Mþ, 89%), 234 (17%), 207 (4%), 206 (5%), 193 (89%), 178 (48%),
(10%), 119 (5%), 117 (4%), 107 (5%), 105 (3%), 93 (17%), 91 (14%),
85 (92%), 83 (100%), 79 (5%), 77 (7%)
(2H, j, t, J = 3 Hz), 2.23 (1H, m, d, J = 3 Hz), 1.94 (3H, i, s), 2.02 (2H,
k, m), 1.71(1H, c2, q, J = 3 Hz), 1.58 (2H, d, t, J = 3 Hz), 1.57 (3H, a,
s), 1.42 (1H, c1, q, J = 3 Hz), 0.94 (3H, e, s), 0.92 (3H, f, s), 0.90 (3H,
l, t, J = 3 Hz)
160 (8%), 150 (49%), 136 (28%), 134 (100%), 119 (22%), 117 (13%),
107 (35%), 105 (20%), 93 (83%), 91 (60%), 85 (7%), 83 (12%), 79
(25%), 77 (29%)
3b
C3H7
6.75 (1H, h, d, J = 8 Hz), 5.90 (1H, g, dd, J = 3 Hz), 5.45 (1H, b, s), 4.08 249 (Mþ, 7%), 207(2%), 193 (5%), 178 (4%), 150 (5%), 136 (3%), 134
(2H, j, t, J = 3 Hz), 2.01 (2H, k, m), 2.00 (1H, m, d, J = 3 Hz), 1.97
(3H, i, s), 1.71 (1H, c2, q, J = 3 Hz), 1.58 (2H, d, t, J = 3 Hz), 1.58 (3H,
a, s), 1.45 (1H, c1, q, J = 3 Hz), 1.26 (3H, e, s), 1.26 (3H, f, s), 0.91
(3H, l, t, J = 3 Hz)
(10%), 119 (5%), 117 (4%), 107 (5%), 105 (3%), 93 (17%), 91 (14%),
85 (92%), 83 (100%), 79 (5%), 77 (7%)
4a
4b
5a
CH(CH3)2
CH(CH3)2
6.19 (1H, h, d, J = 8 Hz), 5.83 (1H, g, dd, J = 3 Hz), 5.43 (1H, b, s), 4.23 249 (Mþ, 89%), 234 (17%), 207 (4%), 206 (5%), 193 (89%), 178 (48%),
(2H, j, h, J = 3 Hz), 2.23 (1H, m, d, J = 3 Hz), 2.02 (6H, l, d, J = 3 Hz),
1.94 (3H, i, s), 1.71 (1H, c2, q, J = 3 Hz), 1.58(2H, d, t, J = 3 Hz), 1.57
(3H, a, s), 1.42 (1H, c1, q, J = 3 Hz), 0.94 (3H, e, s), 0.92 (3H, f, s)
160 (8%), 150 (49%), 136 (28%), 134 (100%), 119 (22%), 117 (13%),
107 (35%), 105 (20%), 93 (83%), 91 (60%), 85 (7%), 83 (12%), 79
(25%), 77 (29%)
6.83 (1H, h, d, J = 8 Hz), 5.90 (1H, g, dd, J = 3 Hz), 5.45 (1H, b, s), 4.26 249 (Mþ, 7%), 207 (2%), 193 (5%), 178 (4%), 150 (5%), 136 (3%), 134
(2H, j, h, J = 3 Hz), 2.03 (6H, l, d, J = 3 Hz), 2.00 (1H, m, d, J = 3 Hz),
1.97 (3H, i, s), 1.71 (1H, c2, q, J = 3 Hz), 1.58 (2H, d, t, J = 3 Hz), 1.58
(3H, a, s), 1.45 (1H, c1, q, J = 3 Hz), 1.26 (3H, e, s), 1.26 (3H, f, s)
(10%), 119 (5%), 117 (4%), 107 (5%), 105 (3%), 93 (17%), 91 (14%),
85 (92%), 83 (100%), 79 (5%), 77 (7%)
CH2CHdCH2 6.12 (1H, h, d, J = 8 Hz), 6.03 (1H, k, m), 5.81 (1H, g, dd, J = 3 Hz), 5.42 247 (Mþ, 89%), 232 (17%), 207 (4%), 206 (5%), 193 (89%), 178 (48%),
(1H, b, s), 5.23 (2H, l, m), 4.12 (2H, j, t, J = 3 Hz), 2.23 (1H, m, d, J =
3 Hz), 1.94 (3H, i, s), 1.71(1H, c2, q, J = 3 Hz), 1.58(2H, d, t, J = 3
Hz), 1.57 (3H, a, s), 1.42 (1H, c1, q, J = 3 Hz), 0.94 (3H, e, s), 0.92
(3H, f, s)
160 (8%), 150 (49%), 136 (28%), 134 (100%), 119 (22%), 117 (13%),
107 (35%), 105 (20%), 93 (83%), 91 (60%), 85 (7%), 83 (12%), 79
(25%), 77 (29%)
5b
CH2CHdCH2 6.75 (1H, h, d, J = 8 Hz), 6.0 (1H, k, m), 5.90 (1H, g, dd, J = 3 Hz), 5.45 247 (Mþ, 7%), 207 (2%), 193 (5%), 178 (4%), 150 (5%), 136 (3%), 134
(1H, b, s), 5.32 (2H, l, m), 4.08 (2H, j, t, J = 3 Hz), 2.00 (1H, m, d, J =
3 Hz), 1.97 (3H, i, s), 1.71 (1H, c2, q, J = 3 Hz), 1.58 (2H, d, t, J = 3
Hz), 1.58 (3H, a, s), 1.45 (1H, c1, q, J = 3 Hz), 1.26 (3H, e, s), 1.26
(3H, f, s)
(10%), 119 (5%), 117 (4%), 107 (5%), 105 (3%), 93 (17%), 91 (14%),
85 (92%), 83 (100%), 79 (5%), 77 (7%)
6a
6b
C4H9
C4H9
6.16 (1H, h, d, J=8Hz),5.84(1H, g, dd, J=3Hz),5.4(1H, b, s),4.0(2H,j,t, 263 (Mþ, 89%), 248 (17%), 207 (4%), 206 (5%), 193 (89%), 178 (48%),
J = 3 Hz), 2.23 (1H, m, d, J = 3 Hz), 1.94 (3H, i, s), 2.02 (4H, k, m), 1.71
(1H, c2,q, J=3Hz), 1.58(2H, d, t, J=3Hz), 1.57(3H, a, s),1.42(1H, c1,
q, J = 3 Hz), 0.94 (3H, e, s), 0.92 (3H, f, s), 0.90 (3H, l, t, J = 3 Hz)
160 (8%), 150 (49%), 136 (28%), 134 (100%), 119 (22%), 117 (13%),
107 (35%), 105 (20%), 93 (83%), 91 (60%), 85 (7%), 83 (12%), 79
(25%), 77 (29%)
6.83 (1H, h, d, J = 8 Hz), 5.68 (1H, g, dd, J = 3 Hz), 5.37 (1H, b, s), 4.12 263 (Mþ, 7%), 207 (2%), 193 (5%), 178 (4%), 150 (5%), 136 (3%), 134
(2H, j, t, J = 3 Hz), 2.01 (4H, k, m), 2.00 (1H, m, d, J = 3 Hz), 1.97
(3H, i, s), 1.71 (1H, c2, q, J = 3 Hz), 1.58 (2H, d, t, J = 3 Hz), 1.58 (3H,
a, s), 1.45 (1H, c1, q, J = 3 Hz), 1.26 (3H, e, s), 1.26 (3H, f, s), 0.91
(3H, l, t, J = 3 Hz)
(10%), 119 (5%), 117 (4%), 107 (5%), 105 (3%), 93 (17%), 91 (14%),
85 (92%), 83 (100%), 79 (5%), 77 (7%)
7a
CH2CH(CH3)2 6.10 (1H, h, d, J = 8 Hz), 5.87 (1H, g, dd, J = 3 Hz), 5.4 (1H, b, s), 3.8 263 (Mþ, 89%), 248 (17%), 207 (4%), 206 (5%), 193 (89%), 178 (48%),
(2H, j, d, J = 3 Hz), 2.23 (1H, m, d, J = 3 Hz), 1.94 (3H, i, s), 2.02 (1H,
k, m), 1.71 (1H, c2, q, J = 3 Hz), 1.58 (2H, d, t, J = 3 Hz), 1.57 (3H, a,
s), 1.42 (1H, c1, q, J = 3 Hz), 0.94 (3H, e, s), 0.92 (3H, f, s), 0.90 (6H,
l, d, J = 3 Hz)
160 (8%), 150 (49%), 136 (28%), 134 (100%), 119 (22%), 117 (13%),
107 (35%), 105 (20%), 93 (83%), 91 (60%), 85 (7%), 83 (12%), 79
(25%), 77 (29%)

Article
J. Agric. Food Chem., Vol. 58, No. 2, 2010 1037
mass (m/z)
Table 2. Continued
compd
R
1H NMR (δ)
7b
CH2CH(CH3)2 6.8 (1H, h, d, J = 8 Hz), 5.90 (1H, g, dd, J = 3 Hz), 5.45 (1H, b, s), 3.84 263 (Mþ, 7%), 207 (2%), 193 (5%), 178 (4%), 150 (5%), 136 (3%), 134
(2H, j, d, J = 3 Hz), 2.01 (1H, k, m), 2.00 (1H, m, d, J = 3 Hz), 1.97
(3H, i, s), 1.71 (1H, c2, q, J = 3 Hz), 1.58 (2H, d, t, J = 3 Hz), 1.58 (3H,
a, s), 1.45 (1H, c1, q, J = 3 Hz), 1.26 (3H, e, s), 1.26 (3H, f, s), 0.91
(6H, l, d, J = 3 Hz)
(10%), 119 (5%), 117 (4%), 107 (5%), 105 (3%), 93 (17%), 91 (14%),
85 (92%), 83 (100%), 79 (5%), 77 (7%)
8a
8b
C5H11
6.23 (1H, h, d, J=8Hz), 5.84(1H, g, dd, J=3Hz), 5.42(1H, b, s), 4.0(2H, j, 277 (Mþ, 89%), 262 (17%), 207 (4%), 206 (5%), 193 (89%), 178 (48%),
t, J = 3 Hz), 2.23 (1H, m, d, J = 3 Hz), 1.94 (3H, i, s), 2.02 (6H, k, m),
1.71(1H, c2, q,J=3Hz),1.58(2H,d,t,J=3Hz),1.57(3H,a,s),1.42(1H,
c1, q, J = 3 Hz), 0.94 (3H, e, s), 0.92 (3H, f, s), 0.90 (3H, l, t, J = 3 Hz)
160 (8%), 150 (49%), 136 (28%), 134 (100%), 119 (22%), 117 (13%),
107 (35%), 105 (20%), 93 (83%), 91 (60%), 85 (7%), 83 (12%), 79
(25%), 77 (29%)
C5H11
6.96 (1H, h, d, J = 8 Hz), 5.72 (1H, g, dd, J = 3 Hz), 5.39 (1H, b, s), 4.08 277 (Mþ, 7%), 207 (2%), 193 (5%), 178 (4%), 150 (5%), 136 (3%), 134
(2H, j, t, J = 3 Hz), 2.01 (6H, k, m), 2.00 (1H, m, d, J = 3 Hz), 1.97
(3H, i, s), 1.71 (1H, c2, q, J = 3 Hz), 1.58 (2H, d, t, J = 3 Hz), 1.58 (3H,
a, s), 1.45 (1H, c1, q, J = 3 Hz), 1.26 (3H, e, s), 1.26 (3H, f, s), 0.91
(3H, l, t, J = 3 Hz)
(10%), 119 (5%), 117 (4%), 107 (5%), 105 (3%), 93 (17%), 91 (14%),
85 (92%), 83 (100%), 79 (5%), 77 (7%)
9a
9b
C2H4CH(CH3)2 6.23 (1H,h,d,J=8Hz),5.84(1H,g,dd,J=3Hz),5.42(1H, b, s),4.0(2H, j, 277 (Mþ, 89%), 262 (17%), 207 (4%), 206 (5%), 193 (89%), 178 (48%),
t, J=3Hz), 2.23(1H, m, d, J=3Hz), 1.94(3H, i, s), 2.02(3H, k, m), 1.71
(1H, c2, q,J=3Hz),1.58(2H, d, t, J=3Hz), 1.57(3H, a, s),1.42(1H, c1,
q, J = 3 Hz), 0.94 (3H, e, s), 0.92 (3H, f, s), 0.90 (6H, l, d, J = 3 Hz)
160 (8%), 150 (49%), 136 (28%), 134 (100%), 119 (22%), 117 (13%),
107 (35%), 105 (20%), 93 (83%), 91 (60%), 85 (7%), 83 (12%), 79
(25%), 77 (29%)
C2H4CH(CH3)2 6.96 (1H, h, d, J = 8 Hz), 5.72 (1H, g, dd, J = 3 Hz), 5.39 (1H, b, s), 4.08 277 (Mþ, 7%), 207 (2%), 193 (5%), 178 (4%), 150 (5%), 136 (3%), 134
(2H, j, t, J = 3 Hz), 2.01 (3H, k, m), 2.00 (1H, m, d, J = 3 Hz), 1.97
(3H, i, s), 1.71 (1H, c2, q, J = 3 Hz), 1.58 (2H, d, t, J = 3 Hz), 1.58 (3H,
a, s), 1.45 (1H, c1, q, J = 3 Hz), 1.26 (3H, e, s), 1.26 (3H, f, s), 0.91
(6H, l, d, J = 3 Hz)
(10%), 119 (5%), 117 (4%), 107 (5%), 105 (3%), 93 (17%), 91 (14%),
85 (92%), 83 (100%), 79 (5%), 77 (7%)
10a
10b
11a
11b
12a
12b
13a
13b
C6H13
C6H13
C7H15
C7H15
C8H17
C8H17
C10H21
C10H21
6.13 (1H, h, d, J = 8 Hz), 5.78 (1H, g, dd, J = 3 Hz), 5.43 (1H, b, s), 4.0 291 (Mþ, 89%), 276 (17%), 207 (4%), 206 (5%), 193 (89%), 178 (48%),
(2H, j, t, J = 3 Hz), 2.23 (1H, m, d, J = 3 Hz), 1.94 (3H, i, s), 2.02 (8H,
k, m), 1.71 (1H, c2, q, J = 3 Hz), 1.58 (2H, d, t, J = 3 Hz), 1.57 (3H, a,
s), 1.42 (1H, c1, q, J = 3 Hz), 0.94 (3H, e, s), 0.92 (3H, f, s), 0.90 (3H,
l, t, J = 3 Hz)
160 (8%), 150 (49%), 136 (28%), 134 (100%), 119 (22%), 117 (13%),
107 (35%), 105 (20%), 93 (83%), 91 (60%), 85 (7%), 83 (12%), 79
(25%), 77 (29%)
6.65 (1H, h, d, J = 8 Hz), 5.90 (1H, g, dd, J = 3 Hz), 5.45 (1H, b, s), 4.08 291 (Mþ, 7%), 207 (2%), 193 (5%), 178 (4%), 150 (5%), 136 (3%), 134
(2H, j, t, J = 3 Hz), 2.01 (8H, k, m), 2.00 (1H, m, d, J = 3 Hz), 1.97
(3H, i, s), 1.71 (1H, c2, q, J = 3 Hz), 1.58 (2H, d, t, J = 3 Hz), 1.58 (3H,
a, s), 1.45 (1H, c1, q, J = 3 Hz), 1.26 (3H, e, s), 1.26 (3H, f, s), 0.91
(3H, l, t, J = 3 Hz)
(10%), 119 (5%), 117 (4%), 107 (5%), 105 (3%), 93 (17%), 91 (14%),
85 (92%), 83 (100%), 79 (5%), 77 (7%)
6.10 (1H, h, d, J = 8 Hz), 5.87 (1H, g, dd, J = 3 Hz), 5.43 (1H, b, s), 4.0 305 (Mþ, 89%), 290 (17%), 207 (4%), 206 (5%), 193 (89%), 178 (48%),
(2H, j, t, J = 3 Hz), 2.23 (1H, m, d, J = 3 Hz), 1.94 (3H, i, s), 2.02
(10H, k, m), 1.71 (1H, c2, q, J = 3 Hz), 1.58 (2H, d, t, J = 3 Hz), 1.57
(3H, a, s), 1.42 (1H, c1, q, J = 3 Hz), 0.94 (3H, e, s), 0.92 (3H, f, s),
0.90 (3H, l, t, J = 3 Hz)
160 (8%), 150 (49%), 136 (28%), 134 (100%), 119 (22%), 117 (13%),
107 (35%), 105 (20%), 93 (83%), 91 (60%), 85 (7%), 83 (12%), 79
(25%), 77 (29%)
6.81 (1H, h, d, J = 8 Hz), 5.90 (1H, g, dd, J = 3 Hz), 5.4 (1H, b, s), 4.0 305 (Mþ, 7%), 207 (2%), 193 (5%), 178 (4%), 150 (5%), 136 (3%), 134
(2H, j, t, J = 3 Hz), 2.01 (10H, k, m), 2.00 (1H, m, d, J = 3 Hz), 1.97
(3H, i, s), 1.71 (1H, c2, q, J = 3 Hz), 1.58 (2H, d, t, J = 3 Hz), 1.58 (3H,
a, s), 1.45 (1H, c1, q, J = 3 Hz), 1.26 (3H, e, s), 1.26 (3H, f, s), 0.91
(3H, l, t, J = 3 Hz)
(10%), 119 (5%), 117 (4%), 107 (5%), 105 (3%), 93 (17%), 91 (14%),
85 (92%), 83 (100%), 79 (5%), 77 (7%)
6.16 (1H, h, d, J = 8 Hz), 5.80 (1H, g, dd, J = 3 Hz), 5.43 (1H, b, s), 4.0 319 (Mþ, 89%), 304 (17%), 207 (4%), 206 (5%), 193 (89%), 178 (48%),
(2H, j, t, J = 3 Hz), 2.23 (1H, m, d, J = 3 Hz), 1.94 (3H, i, s), 2.02
(12H, k, m), 1.71 (1H, c2, q, J = 3 Hz), 1.58 (2H, d, t, J = 3 Hz), 1.57
(3H, a, s), 1.42 (1H, c1, q, J = 3 Hz), 0.94 (3H, e, s), 0.92 (3H, f, s),
0.90 (3H, l, t, J = 3 Hz)
160 (8%), 150 (49%), 136 (28%), 134 (100%), 119 (22%), 117 (13%),
107 (35%), 105 (20%), 93 (83%), 91 (60%), 85 (7%), 83 (12%), 79
(25%), 77 (29%)
6.75 (1H, h, d, J = 8 Hz), 5.73 (1H, g, dd, J = 3 Hz), 5.4 (1H, b, s), 4.0 319 (Mþ, 7%), 207 (2%), 193 (5%), 178 (4%), 150 (5%), 136 (3%), 134
(2H, j, t, J = 3 Hz), 2.01 (12H, k, m), 2.00 (1H, m, d, J = 3 Hz), 1.97
(3H, i, s), 1.71 (1H, c2, q, J = 3 Hz), 1.58 (2H, d, t, J = 3 Hz), 1.58 (3H,
a, s), 1.45 (1H, c1, q, J = 3 Hz), 1.26 (3H, e, s), 1.26 (3H, f, s), 0.91
(3H, l, t, J = 3 Hz)
(10%), 119 (5%), 117 (4%), 107 (5%), 105 (3%), 93 (17%), 91 (14%),
85 (92%), 83 (100%), 79 (5%), 77 (7%)
6.13 (1H, h, d, J = 8 Hz), 5.87 (1H, g, dd, J = 3 Hz), 5.5 (1H, b, s), 4.0 347 (Mþ, 89%), 332 (17%), 207 (4%), 206 (5%), 193 (89%), 178 (48%),
(2H, j, t, J = 3 Hz), 2.23 (1H, m, d, J = 3 Hz), 1.94 (3H, i, s), 2.02
(16H, k, m), 1.71 (1H, c2, q, J = 3 Hz), 1.58 (2H, d, t, J = 3 Hz), 1.57
(3H, a, s), 1.42 (1H, c1, q, J = 3 Hz), 0.94 (3H, e, s), 0.92 (3H, f, s),
0.90 (3H, l, t, J = 3 Hz)
160 (8%), 150 (49%), 136 (28%), 134 (100%), 119 (22%), 117 (13%),
107 (35%), 105 (20%), 93 (83%), 91 (60%), 85 (7%), 83 (12%), 79
(25%), 77 (29%)
7.0 (1H, h, d, J=8Hz), 5.8(1H, g, dd, J=3Hz), 5.45(1H, b, s), 4.0(2H, j, t, 347 (Mþ, 7%), 207 (2%), 193 (5%), 178 (4%), 150 (5%), 136 (3%), 134
J=3Hz), 2.01(16H, k, m), 2.00(1H, m, d, J=3Hz), 1.97(3H, i, s), 1.71
(1H, c2, q, J = 3 Hz), 1.58 (2H, d, t, J = 3 Hz), 1.58 (3H, a, s), 1.45 (1H,
c1, q, J = 3 Hz), 1.26 (3H, e, s), 1.26 (3H, f, s), 0.91 (3H, l, t, J = 3 Hz)
(10%), 119 (5%), 117 (4%), 107 (5%), 105 (3%), 93 (17%), 91 (14%),
85 (92%), 83 (100%), 79 (5%), 77 (7%)
N-O alkyl ethers followed the order methyl
< propyl < butyl < pentyl > hexyl > heptyl > octyl > decyl
in straight-chain compounds (Table 3).
<
ethyl
Disease Control in the Greenhouse. Among the compounds
tested for antifungal activity in vitro, 40-(2,6,6-trimethyl-2-
cyclohexen-1-yl)-30-butene-20(Z)-ketoxime N-O-pentyl ether

1038 J. Agric. Food Chem., Vol. 58, No. 2, 2010
Banerjee and Dureja
Table 3. Fungi Toxicity of 40-(2,6,6-Trimethyl-2-cyclohexen-1-yl)-30-butene-20-ketoxime N-O-Alkyl Ethers against Rhizoctonia bataticola, Macrophomina
phaseolina, Sclerotium rolfsii, and Sclerotinia sclerotiorum
R. bataticola ED50(μg mL-1
)
M. phaseolina ED50(μg mL-1
)
S. rolfsii ED50(μg mL-1
)
S. sclerotiorumED50(μg mL-1
)
compd
R
E
Z
E
Z
E
Z
E
Z
1
CH3
387.93
392.90
32.36
412.86
245.99
156.85
116.35
113.63
58.69
306.54
731.62
181.26
126.01
100.64
102.96
94.75
310.93
393.99
235.76
197.99
173.89
100.91
31.08
237.61
410.19
101.30
35.50
258.52
414.89
132.22
129.03
88.89
441.74
294.53
257.20
181.79
107.39
94.07
516.87
261.42
219.70
190.49
163.66
79.05
2
C2H5
C3H7
3
4
CH(CH3)2
C4H9
56.57
67.89
5
113.23
45.22
43.56
6
CH2CH(CH3)2
C5H11
50.60
39.17
47.62
41.65
7
57.56
32.68
21.39
8
C2H4CH(CH3)2
C6H13
C7H15
722.57
894.89
918.55
1094.84
1943.64
125.34
326.69
566.24
1613.71
2058.44
665.58
750.68
717.34
824.24
956.86
625.23
962.75
1091.25
1109.20
1759.29
257.30
572.80
974.61
1037.26
842.46
366.53
912.11
956.26
1356.83
1107.14
273.48
427.72
392.18
837.26
681.27
375.13
243.85
419.18
584.24
477.48
9
10
11
12
C8H17
C10H21
(compound 7 in Table 3) was found to be most effective against
most of the test pathogenic fungi. Therefore, it was thought
worthwhile to study the antifungal activity of 40-(2,6,6-trimethyl-
2-cyclohexen-1-yl)-30-butene-20(Z)-ketoxime N-O-pentyl ether
against S. sclerotiorum (ED50 = 21.39 μg mL-1) in the green-
house for control in pea at different concentrations (5.0 mL of 1
and 5% aqueous emulsion in 150 cm3 of soil). Carbendazim, a
commercial fungicide, was used for comparison.
The occurrence of symptoms in different treatments was re-
corded at each assay date and expressed in terms of the proportion
of symptomless (healthy) plant stands. The results are shown in
Figure 1. The untreated control (pathogen only) resulted in 100%
disease incidence (0% healthy plant stands) 21 days after trans-
plantation. The treatment of soil with a 1% aqueous emulsion
of the compound resulted in 65% healthy plant stands at 7 days
after transplantation, whereas only 40% healthy plant stands were
observed at 21 days after transplantation. Soil treated with
carbendazim at 0.364 mL of ai/150 cm3 of soil resulted in
80% healthy plant stands after 14 days and 75% at 21 days after
transplantation. The treatment of soil with 5% aqueous emulsion
of the compound resulted in significantly (P < 0.1) greater healthy
plant stands. It showed survival of >90% healthy plant stands
at 21 days after transplantation. However, the healthy plant stands
were not significantly (P > 0.1) different from the soil treated
with carbendazim (0.728 mL of ai/150 cm3) (Figure 1). Thus,
significant differences existed among concentration and time
for disease control experiments in the greenhouse. The 5% aqueous
emulsion of the compound prevented water-soaked spots in
pea significantly. It allowed a plant stand comparable to the
uninfested control and carbendazim (at 0.728 mL of ai/150 cm3
of soil).
Figure 1. Percent healthy pea plants after treatment of S. sclerotiorum
infested soil aqueous emulsion of 40-(2,6,6-trimethyl-2-cyclohexen-1-yl)-
30-butene-20(Z)-ketoxime N-O-pentyl ether and carbendazim . Each point
represents the mean of repeated trials of the experiment with 20 replica-
tions (one plant per pot) per trial. Soil was treated with 1 and 5% aqueous
emulsion of the formulated compound and with carbendazim at 0.364 and
0.728 mL of ai/150 cm3 of soil, respectively.
found to be active against M. phaseolina (ED50=31.08 μg mL-1
)
and S. sclerotiorum (ED50 = 21.39 μg mL-1), respectively.
Experimental conditions were optimized for survival of S. scler-
otiorum in soil and disease development in the greenhouse.
Despite favorable conditions for the pathogens, the 5% aqueous
formulation of 40-(2,6,6-trimethyl-2-cyclohexen-1-yl)-30-butene-
20(Z)-ketoxime N-O-pentyl ether reduced the pathogen popula-
tion and resulted in higher healthy plant stands as compared to
nontreatment (pathogen only).
In conclusion, E isomers of 40-(2,6,6-trimethyl-2-cyclohexen-1-
yl)-30-butene-20(E)-ketoxime N-O-propyl ether (ED50 = 32.36 μg
mL-1) and 40-(2,6,6-trimethyl-2-cyclohexen-1-yl)-30-butene-20-
(E)-ketoxime N-O-(100-methyl)ethyl ether (ED50 = 35.50 μg
mL-1) showed maximum antifungal activity against R. bataticola
and S. rolfsii, respectively, whereas 40-(2,6,6-trimethyl-2-cyclo-
hexen-1-yl)-30-butene-20(Z)-ketoxime N-O-pentyl ether was
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