Agricultural pesticides, properly used, are essential in supplying the food requirements of the world’s ever growing population. The use of synthetic pesticides affects the health of human being. The indiscriminate use of pesticides has adversely affected the health of the soil. The residual pesticides in the soil not only affect the soil quality but also the water quality, as they get leached into the ground water. Due to these reasons, role of biopesticides are very important for sustainable agriculture. The use of biopesticides for sustainable agriculture is a complex issue that at times is difficult to comprehend and plan. This is the first book of its kind which provides different parameters for manufacturing of biopesticides. The book will not only be useful for new entrepreneurs but will also help the technocrats, research scholars and those who willing to know more about biopesticides.
Synthesis
of Triazino Benzimidazol as Biopesticides
[1,2,4]Triazino [4,3-a]
benzimidazol-4(10 H)-ones(4) have been obtained by the reaction of
2-hydrazinobenzimidazole with ethyl pyruvate in neutral medium followed
by hydrolysis and cyclization. Further, it has been found that these
compounds exist in two tautomeric forms due to the labile hydrogen. The
compounds display promising activity when screened for their
antibacterial and antifungal activities.
Various 2-substituted
benzimidazoles possessing antimicrobial activity have been reported by
several group of workers. 2-Hydrazinobenzimidazoles are highly reactive
compounds; some of the derivatives have been used as azodyes while some
other derivatives show tuberculostatic and influenza virus inhibition
activities. In pursuit of our search for new and better antimicrobial
agents, we have now synthesized [1,2,4] triazinobenzimidazol-4(10
H)-ones(4) by the reaction of 2-hydrazinobenzimidazoles with ethyl
pyruvate. This procedure which involved three steps (Scheme 1) gave 4
in better yields than that reported by other workers from the reaction
of ethyl pyruvate with other hydrazines.
2-Hydrazinobenzimidazoles on
refluxing with ethyl pyruvate in ethanol for 6-7 hr gave ethyl
a-oxopropionate benzimidazol-2-ylhydrazones(2). Alkaline hydrolysis of
2 in 50% ethanol gave a-oxopropionic acid
benzimidazol-2-ylhydrazones(3) which underwent cyclization with gl.
acetic acid affording 4 (Scheme 1).
In the PMR spectrum of 4d,
the aromatic protons appeared as a multiplet in the region d. 7,1 -7.3,
as in the case of hydrazone 3d. This indicates the cyclization to be
linear giving 4 and not the angular isomer (4'). If 4' had been formed,
C9-H would have appeared slightly downfield due to deshielding effect
of the carbonyl group.
The
characterization data of all the new compounds are given in Table 1.
The IR, PMR, 13C NMR and mass spectral data of only representative
compounds have been given (see Experimental).
Methylation of 3-methyl-[1,
2,4] triazino [4,3-a] benzimidazol-4(10H)-one (4a) in alkaline medium
with methyl iodide, gave two compounds (5a, 5b). One of them was found
to be 3,10-dimethyl-[1,2,4] triazino [4,3-a] benzimidazol-4 (10H)-one
(5a) and the other as 1,3-dimethyl-[1,2,4] triazino [4,3-a]
benzimidazol-4 (10 H)-one(5b) on the basis of PMR data. The N-methyl
protons in 5b appeared at d 3.5 as compared to 5a where these appeared
at 4.0, the data being in agreement with the respective environment.
The formation of these products prompted us to make a new and important
conclusion that [1,2,4] triazino [4,3-a] benzimidazol-4(10H)-ones exist
in two tautomeric forms A and B. The former being the predominent form
as indicated by the high yield of 5a.
Antimicrobial activity
Compounds 4a and 4c-4e of the
series were evaluated for their antimicrobial activity following the
method of Gould using streptomycin in antibacterial and mycostatin in
antifungal activity as reference compounds.
All compounds were active
against gram positive bacteria Staphylococcu saureus while none was
active against gram negative bacteria. Compound 4e showed maximum zone
of inhibition (15.0 mm) against Staphylococcus aureus. Its enhanced
activity may be attributed to the presence of fluorine. Compounds 4d
and 4e were active against all the fungi tested. All the compounds
showed maximum activity against Aspergillus flavus. Compound 4a showed
maximum zone of inhibition (10.0 mm) against the fungus Drechslera
tetramera. The results are recorded in Table 2.
Experimental
IR spectra in KBr (nmax in
cm–1) were recorded on a Perkin-Elmer 577 spectrophotometer, PMR
spectra in TFA on a Jeol FX 90Q spectrometer (89.55 MHz) using TMS as
internal reference and 13CNMR spectra in DMSO-d6, at 22.49 MHz
(chemical shifts in both cases in d, ppm). The mass spectra were
recorded on MS-30 and MS-50 Krantos mass spectrometers at an ionisation
potential of 70 eV. Melting points were determined in open glass
capillaries on a Gallenkamp melting point apparatus and are
uncorrected. Elemental analyses were performed at CDRI, Lucknow.
Studies on Coumarin Derivatives
On the synthesis and
evaluation of biological activities of coumarins, we report here the
synthesis of 1-aroyl-1, 2-dihydro-3H-naphtho [2, 1-b] pyran-3-ones (2)
and the reactivity of their C-2 methylene groups toward some organic
reagents aiming to synthesis new naphthopyrane derivatives which might
have enhanced biological activities. Coumarin derivatives are known to
exhibit bactericidal, bacterostatic, anticoagulant, anticancerogenic,
rodenticidic and antihelminthic activities.
Condensation of b-(4-methoxy-
or 3,4-dimethyl-benzoyl) acrylic acid with 2-naphthol in the presence
of 75% sulphuric acid gave 1-(4-methoxy- or 3,4-dimethylbenzoyl)-1,
2-dihydro-3H-naphtho [2,1-b]-pyran-3-one (2a or 2b) (Scheme 1) via ring
closure of the intermediate
b-aroyl-b-(2-hydroxynaphthalen-1-yl)propionic acid (1), which gave
positive acidity test and was formed exclusively and predominantly in
the presence of 50% sulphuric acid.
Claisen-Schmidt condensation
of the naphthopyrones (2) with aromatic aldehydes such as benzaldehyde,
2- and 4-chloro-benzaldehydes, and 3- and 4-nitrobenzaldehydes in the
presence of piperidine afforded 1-aroyl-2-arylidene-1,
2-dihydro-3H-naphtho [2,1-b] pyran-3-ones (3a-f; Scheme 2) (Table 1).
Bromination of 2 in carbon
tetrachloride at room temperature resulted in electrophilic
substitution at both C-1 and C-2 to give 1,2-dibromo derivatives (4a
and 4b; Table 1), while bromination of 2 in boiling acetic acid gave
1-aroyl-5-bromo-3H-naphtho [2,1-b] pyran-3-ones (5a and 5b) (Scheme 2)
which probably resulted from bromination at C-1 followed by
dehydrobromination and subsequent bromination of the more reactive
aromatic C-5.
Base-catalyzed addition of 2
to 4-methoxybenzalacetophenone at 25° in the presence of sodium
ethoxide yielded the expected Michael adducts 1-aroyl-2 -
(1-p-methoxyphenyl-3-oxo-3-phenyl-prop-1 -yl)-1, 2-dihydro-3H-naphtho
[2,1-b] pyran-3-ones(6a and 6b) while in the presence of isobutylamine,
2 underwent cycloaddition to give 12-aroyl-8, 12-dihydro-8-isobutyl-11
-(p-methoxyphenyl)-9-phenyl-11H-naphtho [1, 2':5, 6]-pyrano[2,3-b]
pyridines (7a and 7b).
On the other hand at 120° the
naphthopyrones (2) underwent base-catalyzed cycloaddition with
4-methoxybenzalacetophenone and/or ethyl benzoylacrylate to give the
corresponding cyclic Michael adducts depending upon the type of base
used. In the presence of sodium ethoxide, the reaction yielded
1-aryl-2-benzoyl-3a, 11 c-dihydro-3-(p-methoxy-phenyl/or carbethoxy)
cyclopenta [d] naphtho [2,1-b]- pyran-4(3H)-ones (8a-c) while in the
presence of ammonium acetate it gave 12-aroyl-8,
12-dihydro-11-(p-methoxyphenyl)-9-phenyl-11 H-naphtho [1', 2':
5,6]-pyrano [2,3-b] pyridines (7c and 7d) and in the presence of
isobutylamine-4,7-dihydro-3-(2-hydroxynaphthyl)-1,7-diisobutyl-4-(p-methoxyphenyl)-6-phenyl-2-(3,4-xylyl)-1H-pyrrolo[2,3-b]-pyridine
(9) was obtained.
Experimental Procedure
Melting points recorded are
uncorrected. IR spectra were recorded on a Unicam SP 1200
spectrophotometer using KBR wafer technique (nmax in cm–1), and PMR
spectra on a Varian T-60 instrument using TMS as internal standard
(chemical shifts in d-scale).
b-(p-Anisoyl)-b-(2-hydroxynaphthalen-1
-yl)propionic acid(1)
A mixture of 2-naphthol (7.2
g, 0.05 mol), b-(p-anisoyl)acrylic acid (10.3 g, 0.05 mol) and
sulphuric-acid (80 ml, 50%) was warmed on a water-bath for 2 hr,
cooled, and poured into 200 ml cold water. The solid separated was
filtered, washed with water, dried and crystallised from benzene to
give 1 as brown crystals, yield 80%.
1 -Aroyl-1,2-dihydro-3H-naphtho
[2,1-b]pyran-3-ones(2)
A mixture of 2-naphthol (7.2
g, 0.05 mol), b-(p-anisoyl)acrylic acid/or b-(3,4-dimethylbenzoyl)
acrylic acid (0.05 mol) and sulphuric acid (80 ml, 75%) was warmed on a
water-bath for 2 hr, cooled and poured into 200 ml cold water. The
solid separated was filtered, washed with water, dried and crystallised
from benzene-light petrol (80-100) to give 2a or 2b as orange crystals,
yield 71-75%.
1 -A royl-2-arylidene-
1,2-dihydro-3H-naphtho[2,1-b]pyran-3-ones(3)
A solution of 2 (0.01 mol),
an aromatic aldehyde (benzaldehyde, 2- or 4-chlorobenzaldehyde, 3- or
4-nitrobenzaldehyde) (0.01 mol), piperidine (a few drops) and ethanol
(50 ml) was refluxed for 10 hr. The reaction mixture was cooled and
poured into dil. HCl (100 ml, 10%). The solid product was filtered,
washed with water, dried and crystallised from an appropriate solvent
(cf. Table 1 ).
1-Aroyl-1,2-dibromo-1,2-dihydro-3H-naphtho-[2,1-b]pyran-3-ones
(4a, b)
To a stirred solution of
naphthopyrone (2a, b) (0.006 mol) in carbon tetrachloride (40 ml),
bromine (0.006 mol) in CCl4 (10 ml) was added dropwise during 1 hr at
25°. The reaction mixture was left to stand for 48 hr at room
temperature. The solid product which separated after evaporation of
CCl4 was crystallised from benzene to give 4a or 4b as yellow crystals
(cf. Table 1 ).
1-Aroyl-5-bromo-3H-
naphtho[2, 1-b]pyran-3-ones (5a,b)
A solution of naphthopyrone
(2a, b) (0.006 mol), bromine (0.006 mol) and acetic acid (30 ml) was
heated under reflux for 5 hr. The solid product which separated after
concentration and cooling was filtered and crystallised from acetic
acid to give 5a or 5b as reddish crystals (cf. Table 1).
Base-catalyzed addition and
cycloaddition of naphthopyrones (2a, b) to
p-methoxybenzalacetophenone/or ethyl benzoylacrylate. General procedures (A) At 25°: Formation
of(6a, b) and (7a, b).
A mixture of naphthopyrone (2a, b)
(0.015 mol), p-methoxy-benzalacetophenone (0.01 mol) and sodium
ethoxide (0.02 mol) or isobutylamine (2 ml) in abs. ethanol (30 ml) was
left to stand at 25° for 3 days and then poured into HCl (50 ml, 10%).
The solid product was filtered, washed with water, dried and
crystallised from a suitable solvent to give 6 or 7 (cf. Table 1).
Synthesis
and Insect Growth Regulating Activity of Benzoylphenylureas
Disruption of metamorphosis
with the use of insect growth regulators has been recognized as an
attractive target for insecticidal action because the process is vital
to the development of invertebrates. The renewed interest in cuticle
biochemistry, particular ly in chitin synthesis stems from the
discovery of bioactive benzoylphenylureas more than a decade ago. A
thorough quantitative structure-activity study was recently conducted
for a large number of benzoylphenylureas (BPUs), with larvicidal
activity and inhibition of cuticle formation in cultured integuments
used as probing parameters. A number of insect growth regulators (IGRs)
have been synthesized and evaluated in the laboratory and field
conditions against Dipterous insects of medical and economic
importance, particularly against mosquitoes.
The specific larvicidal
spectra of certain compounds in this series were suggested to be due to
innate differences in the metabolic mechanism besides differences in
the physicochemical substituent effects on the ultimate activity. In
this paper, we report the synthesis of a structurally modified class of
BPU compounds by incorporating a methylene spacer between the benzamido
and anilido segments of benzoylphenylurea.
These compounds were screened
for their insect growth regulating activity against three different
species of mosquito vectors.
Synthesis of the compounds
with the desired structures was achieved through the new method. The
yield of the product in most of the cases was around 80%. All the
compounds were crystalline solids with high melting points (>
200°) and limited solubility in organic solvents. The IR spectra of the
compounds showed two distinct carbonyl absorptions, one around 1650 and
the other between 1675 and 1720 cm–1. The absorption at 1650 cm–1 may
be due to the carbonyl function flanked between methylene and amino
groups. The PMR spectra showed a doublet around d4.33 corresponding to
the methylene group incorporated between benzamido and anilide
moieties. The details of the spectral data are given in Table 1.
The biological activity of
the compounds was determined against three vector species of
mosquitoes. The preliminary screening results show that
N-[(4-methylphenylamino) carbonylmethyl] benzamide (BGA-3),
N-[(phenylamino) carbonyl-methyl]- 3,4-methylenedioxybenzamide (BGA-4)
and N-[(3-trifluoromethylphenylamino) carbonylmethyl] benzamide (BGA-7)
are effective against Cx. quinquefasciatus. On testing at lower
concentrations the respective El50 values of the above compounds were
found to be 0.4714, 0.3821 and 0.1850 mg/litre, respectively.
The treatment showed various
abnormalities in the larvae, pupae and emerging adults as frequently
observed in the case of diflubenzuron (a benzoylphenylurea compound)
treatment. The compound BGA-11, which differs from diflubenzuron
(dimilinR) by a methylene spacer between the benzamido and anilide
moieties, was found to be ineffective at the preliminary screening
against all the three species tested. Hence, it could be stated that
the biologi cal activity of the effective parent compound was lost
during the incorporation of the methylene spacer. But some structural
analogues of the same class of compounds are able to show the
biological activity. So, further structural modifications may result in
a derivative with optimal biological activity. The insect growth
regulating activity of the compounds against Cx. quinquefasciatus in
comparison with dimilin is given in Table 2.
The cost analysis of the compounds in
comparison to the parent compound dimilin was carried out at the
laboratory scale synthesis. The analysis shows that the most effective
compound (BGA-7) of the class is ten times cheaper than dimilin.
Moreover, this compound could be synthesized at a single stage reaction
between hippuric acid and 3-aminobenzotrifluoride, without the
involvement of an acid chloride intermediate. A suitable formulation of
this compound may play a role in the control of the mosquito vector Cx.
quinquefasciatus and thereby the reduction in the diseases transmitted
by the same.
Melting points are
uncorrected. Purity of the compounds was checked by TLC using pet.
ether ethyl acetate (1:1) as irrigant. PMR spectra were recorded in a
mixture of CDCl3 and DMSO-d6 on a Hitachi R-600 spectrometer using TMS
as internal standard and IR spectra in KBr on a Perkin-Elmer 783
spectrophotometer.
Experimental Procedure
Synthesis
The synthesis of BGA type of
compounds with substitution in the methylene spacer was described as an
intermediate in the preparation of polypeptides and proteins involving
a complex mechanism by condensing four different chemical components
such as carboxylic acid, isonitrile, aldehyde and amine. We have
modified the pathway for the synthesis of the compounds containing
unsubstituted methylene spacer with starting materials as carboxylic
acid, glycine and amine, by avoiding the involvement of highly toxic
isonitriles. Synthesis of the compounds was carried out by reacting the
substituted benzoyl chloride with glycine in alkali. Twelve compounds
were synthesized by appropriate substituting the benzoyl and
aminophenyl rings. The resultant benzoylglycines were purified by
extracting the reaction product with hot carbon tetrachloride to remove
the free benzoic acids and recrystallising from boiling water.
The second
stage of reaction involved condensation of the benzoyl glycines with an
equimolar quantity of an appropriate aniline at 135 ± 5° in an oil-bath
for 2 hr using a Dean-Stark phase separator. The product obtained was
extracted with hot chloroform followed by washing with 1N hydrochloric
acid (3 x 25 ml), water, saturated sodium bicarbonate solution and
finally with water. The solvent was removed by distillation and the
product recrystallised from a hot mixture of diethyl ether and
chloroform (1:1).
Insect growth
regulating (IGR) activity against mosquito vectors
Third instar larvae of Culex
quinquefasciatus, Aedes aegypti and Anopheles stephensi were obtained
from the insectaries maintained at the Centre and used for the
evaluation of IGR activity. The compound was dissolved in acetone to
get 1% stock solution from which further dilutions were made. To
achieve the required concentration, 1 ml of the stock solution of
appropriate concentration was added to 249 ml of tap water in 500 ml
beaker and stirred vigorously to ensure thorough mixing. Twenty five
larvae were added to the test solution by means of a strainer or
dropper. The larvae in each beaker were provided with food (yeast
powder + dog biscuit). The larvae were exposed continuously till
pupation and mortality of larvae and pupae was recorded and removed
regularly. Pupae from the treated water were collected in small vials,
kept in netted cages and observed for normal emergence, morphological
aberrations, incomplete emergence and death.
Synthesis
and Pesticidal Activities of Thiadiazolo-s-Triazine and Imidazol
The 1,3,4-thiadiazole ring is
associated with diverse biological activities probably by virtue of
incorporating a toxophorictsâ€â€N = C–S– linkage, the importance of which
has been well stressed in many pesticides. Likewise, symtriazine and
imidazole derivatives are well known for their herbicidal,
insecticidal, bactericidal and fungicidal activities.
These observations coupled
with our interest in the synthesis of heterocyclic compounds and the
fact that compactness and planarity of a molecule might augment its
other biocidal activities as it does with herbicidal activity, the
biolabile-s-triazine and imidazole nuclei were fused with
1,3,4-thiadiazole nuclei respectively to yield the titled compounds
(Illa-g) and (Va-g) with the hope of achieving compounds of better
biocidal action.
The required
2-amino-5-aryloxymethyl-1,3,4-thiadiazoles (la-g) were prepared by the
method of Maffii et al. The condensation of la-g with furfural in
methanol furnished the compounds Ila-g which were converted into the
titled compounds (Illa-g) by 1,4-cycloaddition of phenyl isothiocyanate
in xylene. On the other hand treatment of la-g with chloroacetyl
chloride in cold furnished the compounds IVa-g which were converted
into the titled compounds (Va-g) by stirring with dry pyridine at room
temperature (Scheme 1).
Fungicidal activity
All the seven compounds were
screened for their fungicidal activity against Aspergillus niger and Helminthosporium
oryzae at 1000, 100 and 10 ppm concentrations. Carbendazim, a
commercial fungicide was also tested for comparison. Compounds lIb, c,
f and g were found to be promising fungicides while Va-g were nearly
inactive. The activity of the compound IIlf (86% at 1000 ppm) was quite
comparable with carbendazim (92% at 1000 ppm), and hence further
screening of this compound on a wider range of fungi as well as at more
dilution is desirable. It is to be noted that all the four compounds
(IIb, c, f and g) contain a polar chloro group in the phenyl ring on
1,3,4-thiadiazole nucleus indicating that the presence of halogen atom
enhances the fungi-toxicity of this series of compounds.
Bactericidal activity
The antibacterial activity of
all the compounds (III and V) evaluated against the bacteria
Staphylococcus aureus and Bacillus subtilis at 1000, 750, and 500
mg/litre concentrations. None of these compounds showed any notable
activity except the compound (Illf) which was active against both the
species (82% and 80% against S. aureus and B. subtilis, respectively at
500 mg/litre concentration). This shows that association of hydrocarbon
residue (CH3) with a chloro group in the phenoxy moiety could enhance
the activity.
Herbicidal activity
All the compounds were
subjected to primary post-emergent and pre-emergent herbicide
evaluation at the rates of 8.0, 4.0, 2.0, 1.0 and 0.5 kg/ ha. The test
species were wheat, cocklebur, sunflower, velvet-leaf and sicklepod.
None of the compounds except IIlf was found to be promising herbicide.
The activity of Illf was found to be 100% at 8.0 kg/ha and 80% at 0.5
kg/ha. Its activity is probably due to the presence of a
2,4-dichlorophenoxy moiety on 1,3,4-thiadiazole ring.
Although, various toxophoric
groups and structures have been combined in the titled molecules with a
hope of achieving compounds of better biocidal potency, the results are
not very encouraging. This indicates that the activity of any compound
may not necessarily be related to the numerical sum of all toxophores
present in the molecule.
Synthesis
and Fungicidal Activity of Thiazolidine and 2-Aryl Indoles
ÂÂ
The synthesis and biological
activities of novel heterocycles such as [1,3,4]
oxadiazoloquinazolones, [1, 2, 4] triazolothiazoles, [1,3,4]
thiadiazoloquinazolones 1,3,4-thiadiazolo-s-triazines, [1,2,4]
triazoloquinazolones and [l,3,4] oxadiazolyl-2-azetidinones was
reported. ln continuation of our this work we wish to report herein a
convenient synthesis of another fused heterocyclic system [1,3,4]
oxadiazino [5,6-b] indoles and a novel spiro heterocyclic
spiro-[3H-indole-3,2'-thiazolidine]-2,2'(1H)-diones. The fungicidal
activity of these compounds is also reported.
The key compounds
isatine-b-(aryloxyacetylhydrazones) (I) and isatin-b-(aroylhydrazones)
(II) were prepared by condensation of isatine with
aryloxyacetylhydrazines and aroylhydrazines, res pectively. The
cycloaddition of 2-mercaptoacetic acid to I in dioxane furnished the
corresponding spiro [3H-indole-3,2'-thiazolidines] (III). The absence
of > C = N peak in the IR spectra of III suggested the
conversion of I into III. On the other hand, the synthon II on
refluxing with aq. KOH furnished the desired compounds, 2-aryl-[1,3,4]
oxadiazino-[5,6-b]indoles (III) (Scheme 1).
The absence of -NH and
> C = O peaks in the IR spectra of IV suggested the cyclization
of II to IV.
Fungicidal activity
Compounds Ila-IIg and IVa-lVg
were screened for their antifungal activity against Aspergillus flavus
and Helminthosporium oryzae at 1000, 100 ppm and 10 ppm concentrations.
The results have been compared with carbendazim, a commercial
fungicide, tested under similar conditions.
Compound IIa showed better
antifungal activity than IV. However, the activity of II decreased
considerably upon dilution. Compounds lIb and IIe were found to show
much better activity than any of the compounds of this series. However,
compound lIe was more potent than lIb. The activity of II was almost
comparable (89% of 1000 ppm) with that of the commercial fungicide
carbendazim (97% at 1000 ppm). Further investigation of this compound
on wider range of fungi as well as at more dilution is in progress.
Synthesis
and Biological Activity of
Benzothiazoles/Benzoxazoles/Benzimidazoles/imidazolidines
In view of the fact that a
large number of derivatives of thia-zoles, benzoxazoles,
benzothiazoles, benzimidazoles and imidazoles have been found to
exhibit a wide variety of pharmacological activity and our interest in
thiazole nucleus, it was considered worthwhile to synthesize compounds
bearing a thiazole nucleus linked to the benzothiazole, benzoxazole,
benzimidazole and imidazole nuclei through an NH linkage and evaluate
their antifungal and antibacterial activities. Only a limited number of
molecules where an NH group is linked to two heterocyclic moieties has
been described in literature.
The synthetic route is
outlined in Scheme 1. Reaction of 2-amino-4-arylthiazoles (1a-d) with
carbon disulphide and methyl iodide in dimethyl-formamide in the
presence of strong sodium hydroxide solution gave the corresponding
dimethyl N-(4-aryl-2-thiazolyl) dithiocarbonimidodithioates (Ila-d).
These compounds (Ila-d) on reaction with 2-aminothiophenol in refluxing
dimethylformamide in the presence of one equivalent sodium hydroxide
afforded Illa-d in very good yield. The high nucleophilicity of the
thiolate anion, generated in the reaction, led the formation of III in
good yields and in a short period. In a similar way 2-heteroaryl-amino
derivatives of benzoxazoles (IVa-d) were prepared from o-aminophenol
and II under nitrogen atmosphere to avoid darkening. In the preparation
of V and VI the reaction conditions depended upon the diamine used.
With o-phenylenediamine a 1:1 mixture of reactants was heated under
reflux until no more methylmercaptan was evolved. The nature of
substituents on II affected the reaction period (10-16 hr generally).
In case of ethylenediamine a threeÂÂfold excess of it was used and the
reaction started at room temperature which was completed at 100° after
10-12 hr. Structures of all the compounds were established by elemental
analysis and spectral data.
Biological activity
All the compounds were tested
for their antifungal and antibacterial activities. The antifungal
activity was determined against Aspergillus niger and Aspergillus
flavus at 10 and 25,mg/ml concentrations using the modified Czapeck Dox
nutrient medium. The antifungal data revealed that all the compounds
were mode-rately toxic to the fungal species. Only compounds IIa,b and
IIId were comparable with commercial fungicides (Carbendazim and
Capta-fol).
The antibacterial activity
was evaluated by the cup plate agar diffusion technique at 10 and 25
mg/ml concentrations against E. coli and S. aureus. DMF was used as
control in both the cases. All the compounds tested showed only
marginal inhibition but better inhibition was observed at 25 mg/ml
concentration in comparison to that at 10 mg/ml.
Antimicrobial Agents
The discovery of meconazole,
clotrimazole, bifonazole, etc. in the treatment of topical and systemic
fungal infections has created a great impetus in imidazole derivatives
as antifungal agents. Furthermore, several benzofuran and benzoxazine derivatives also exhibit
marked antibacterial, antimycotic and other pharmacological activities.
In view of this, it was considered of interest to synthesize some new
l-[(benzofuran-2-yl)(3-oxo-1,4-benzoxazin-6-yl)methyl]-1 H-imidazoles
(5a-f) with a view to evaluating their antifungal activity in vitro.
The key starting materials
chloroacetylbenzoxazinones (2) were obtained by chloroacetylation of
various benzoxazinones (1) under Friedel-Crafts reaction conditions.
Condensation of 2 with salicylaldehyde in acetone-K2CO3 afforded the
benzofuranoyl-benzoxazinones (3) in good yields. The ketones 3 were
reduced to the corresponding carbinols (4) with sodium borohydride in
methanol. The alcohols 4 thus obtained were subjected to the imidazole
transfer reaction using N,N’-thionylimidazole to obtain the desired
imidazole derivatives 5 in fair yields. Two carbinols (4a and 4d) did
not undergo the imidazole transfer reaction owing to their poor
solubility in dichloromethane. However, these were converted to the
respective imidazole derivatives via their chlorides. Thus, the
carbinols (4a and 4d) were treated with thionyl chloride to give the
corresponding chlorides (6a and 6d) which in turn were treated in situ
with imidazole to give the respective imidazole derivatives (5a and 5d)
(Scheme 1).
The structures of 3, 4 and 5
were confirmed by their elemental analyses (Table 1) and IR and PMR
spectra (see experimental).
Antifungal activity
All the title imidazole
derivatives (5a-e) were tested for their antifungal activity in vitro
against dermatophytes (Microsporum canis, Microsporum gypseum,
Epidermophyton floccosum, Trichophyton rubrum and Trichophyton
mentagrophytes), yeasts (Candida Albicans, Cryptococcus neoformans) and
systemic fungi (Aspergillus fumigatus and Sporotrichun schenkii) using
two-fold agar dilution method according to the method described earlier.
Three compounds in this
series (5b, 5c and 5e) were found to possess moderate antifungal
activity in vitro and the activity was mainly restricted towards
dermatophytes only. Thus, 5c was found to be the most potent compound
of the series. It inhibited the growth of all the dermatophytes
employed in the screening at 62 mg/ml. However, 5e was active against
M. gypseum, T. mentagrophytes and E. floccosum only, with MIC values of
62 mg/ml. Compound 5b was less active as compared to 5c and 5e and the
fungi, M. gypseum and E. floccosum only were found to be sensitive
towards this compound at 62 mg/ml. All the other dermatophytes were
inhibited by 5b and 5e at 125-250 mg/ml.
In addition to the above,
compound 5c was also found to possess a low order of activity against
systemic fungi by inhibiting their growth at 250 mg/ml. None of the
compounds reported herein displayed any significant activity against
yeasts at test concentration (250 mg/ml).
Synthesis
and Antimicrobial Activities of Pyrazoles
Keeping in view the
biological properties associated with pyrazoles, sulphonamides and
pyrazoles with sulphamides moiety attached at different positions, it
was considered of interest to synthesize some new
1-substituted-5-aryl-3-methyl-4-(N’-substituted
p-sulphamylbenzene-azo)pyrazoles (11-26) and 1-substituted
3-(2'-hydroxyaryl)-5-phenylpyrazoles (30-41) as potential antimicrobial
agents.
3-(2',4'-Dimethoxy-
3’ -methylphenyl)-1 -methyl-propane-l,3-dione (1) and
3-(2',4'-dimethoxy-6'-methylphenyl)-1-methylpropane-l,3-dione (2)
(prepared by the Claisen condensation of
2,4-dimethoxy-3-methylacetophenone and
2,4-dimethoxy-6-methylacetophenone respectively with ethyl acetate in
the presence of pulverised sodium) on coupling with diazotised solution
of different sulphonamides gave 3-(2',4'-dimethoxy-3'-methylphenyl)- 1
-methyl-2-(N’ -substituted p-sulphamyl-benzeneazo)propane-1,3-diones
(3-6) and 3 -(2' ,4' -dimethoxy-6 ‘-methylphenyl)-1 -methyl-
2-(N’-substituted        ÂÂ
p-sulphamylbenzeneazo)- propane-1,3-diones (7-10),
respectively. These on cyclisation with 4-methylphenylhydrazine
hydrochloride and 4-pyridylhydrazide gave 1-substituted-5-(2 ‘,
4‘-dimethoxy-3-methylphenyl)-3-methyl-4-(N'-substituted
p-sulphamylbenzeneazo)- pyrazoles (11-18) and
1-substituted-5-(2'-4'-dimethoxy-6'-methylphenyl)-3-methyl-4-(N'-substitutedp-
sulphamylbenzeneazo)pyrazoles (19-26) (Table 1).
Similarly,
2-benzoyloxy-4-methoxyacetophenone,
2-benzoyloxy-4-methoxy-3-methylacetophenone and
2-benzoyloxy-4,6-dimethoxyacetophenone (prepared by Schotten Baumann
reaction of the corresponding acetophenones with benzoyl chloride in
the presence of pyridine) on Baker-Venkataraman transformation in
alkaline medium gave w-benzoyl-2-hydroxy-4-methoxyacetophenone (27),
w-benzoyl-2-hydroxy-4-methoxy-3-methylacetophenone (28) and
w-benzoyl-2-hydroxy-4,6-dimethoxyacetophenone (29), respectively. These
b-diketones on cyclocondensation with hydrazine hydrate,
phenylhydrazine, 4-methylphenylhydrazine hydrochloride and
2,4-dinitrophenylhydrazine, gave the corresponding pyrazoles (30-41)
(Table 1).
The structures of all the
compounds were established on the basis of elemental analysis and PMR
spectral data.
Biological testing
All the title compounds
prepared were screened in vitro against the bacteria Escherichia coli
and Pseudomonas pyocyanea using cup-plate agar diffusion method at 10,
25 and 50 mg/ml concentrations. Compounds 13, 14, 17, 18, 22, 26, 30,
32, 33, 34 and 39 showed moderate activity against both the bacterial
species.
These compounds were also
screened for their antifungal activity against, Aspergillus niger and
Aspergillus flavous using Czapeck Dox nutrient medium at 10, 25 and 50
mg/ml concentrations. Compounds 11, 17, 18, 22, 39 and 41 inhibited the
growth of A. niger to the extent of 72.2, 75.3, 80.1, 71.6, 59.1 and
82.3% respectively at 50 ug/ ml concentration. Compounds 11, 17, 18,
39, 40 and 41 inhibited the growth of A. flavous to the extent of 68.4,
68.9, 75.8, 49.5, 59.3 and 88.5% respectively at 50 mg/rnl
concentration. Bavistin was used as the standard which showed 88%
inhibition under similar conditions.
Experimental Procedure
Melting points were taken in
sulphuric acid-bath and are uncorrected. PMR spectra were recorded on a
Perkin-Elmer R-32 (90 MHz) spectrometer using TMS as internal standard
(chemical shifts in d, ppm).
3-(2’,4’-Dimethoxy-3’-methylphenyl)-1-methylpropane-1,
3-dione (1)
ÂÂ
Synthesis
and Insecticidal Activity of New Substituted Hydroxyacetophenones
Acetophenones have been
extensively used recently in the synthesis of different classes of
compounds which behave as hypolipidemic, antibacterial and
antiinflammatory agents and also as passive cutaneous anaphylaxis (PCA)
inhibitors and anti-juvenile hormones. Some of them show photochromism.
The insecticidal and other biological activities of thiophosphorylated
acetophenones prompted us to synthesise more compounds in this series
with the hope of getting insecticides of low toxicity and less harmful
effects. We wish to report in this paper the synthesis of
3-(N,N-dialkylaminomethyl)-4-hydroxyacetophenones (4-9) and their
corresponding thiophosphorylated compounds. The intra-molecular
H-bonding arising due to lone pair of nitrogen and hydroxy group of
these compounds has also been discussed.
Chloromethylation of
4-hydroxyacetophenone (1) by the method of Trave gave the corresponding
chloromethylated derivative (2) which was converted into different
N,N-substituted aminomethyl derivatives (4-9; Table 1) by refluxing
with different amines in dry benzene. Compound 2 was also hydrolysed
with alkali to get the 3-hydroxymethyl derivative (3). The reaction of
O,O-diethylthio-phosphoryl chloride with 3-9 in the presence of acetone
and potassium carbonate at room temperature gave the corresponding
thiophosphorylated derivatives (11-16; Table 1). The structural
assignments of all these compounds were based on elemental analyses,
mass IR and PMR spectral data.
In 4-hydroxyacetophenone (1)
the carbonyl group is known to appear at a lower frequency! (1635). It
was observed that when a chloromethyl or hydroxymethyl side chain was
introduced at 3-position, the carbonyl band shifted to 1655 and 1663
respectively. Similarly in 3-(N,N-dialkylaminomethyl) derivatives
(4-10) the carbonyl bands appeared at a higher frequency compared to
that in 1 (1640). In hydroxy frequency region the bands were observed
for both free (3600-3660) and intramolecular bonded (3375-3450)
hydroxyl groups in the case of 4, 7 and 9; however compounds 5,6 and 8
exhibited a band for free OH at 3450-3398.
From a study of the carbonyl
band frequency of o-hydroxyacetophenone and o-methoxyacetophenone
Hergert and Kurth arrived at a conclusion that the increased stability
of the dipolar form of o-hydroxy acetophenone and the consequent
lowering in vCO band depend upon the greater ability of the hydroxyl
group to donate electrons to the ring. Since the carbonyl bands of 2, 3
and 4-9 appeared at different frequencies, it can be expected that the
carbonyl group must have different degrees of s-orbital character of
the C-O s bond. This in turn gives different force constants of the
carbonyl group and thus changes the frequency of nCO.
It is also known that the
appearance of nCO at a lower frequency in 4-hydroxyacetophenone
compared to acetophenone is attributed to the conjugation of C = O
group with unsaturated chromophore11 and stabilization of the
consequent dipolar form (1a)10.
Therefore, it is the capacity
of the O - Z(Z = H, CH3 etc) group to donate electrons to the ring that
finally determines the participation of the carbonyl group in
conjugation and consequently the degree of s-orbital character in the
above compounds. Thus, it is clear from the above discussion that the
capacity of the hydroxyl group in 2-9 to donate electron to the ring is
affected.
The formation of
intramolecular hydrogen bond in 2-hydroxybenzyl alcohol is well
documented12. The formation of intramolecular hydrogen bond in
o-halophenols was investigated by Pauling and other workers and it was
observed that a strong repulsion between the proton donor and acceptor
exists. And from the examination of atomic population and bond order of
this class of compounds it was observed that a major part of the
electron density shifts from the phenolic proton to the proton
accepting halogen. Dietrich has also suggested that in
o-halogenophenols the intramolecular interactions are mainly determined
by Hâ€â€halogen (X) interaction in cis-conformer and Oâ€â€X repulsion in cis-
and trans-conformers and the O-Hâ€â€X angle. Baker and Shulgin have shown that steric
interactions become important in 2-dimethylaminophenol where the methyl
groups force the nitrogen lone pair orbital towards the hydroxyl group.
Rao have observed that in pyridine-methanol system a slight increase in
the electron density of the nitrogen atom occurs due to hydrogen bond
formation.
The IR spectra of 2-9
indicated that the 4-OH group forms intramolecular hydrogen bonding
with different hydrogen acceptors, i.e. chlorine in 2, oxygen in 3 and
nitrogen in 4-9. It is expected that depending upon the steric factors.
O–Hâ€â€X angle and basicity of the hydrogen acceptor, the degree of
intramolecular hydrogen bond changes which in turn affects the acidity
of the phenolic O  H group and consequently its capacity to donate
electron to the ring.
4-Hydroxy-3-(N,N-diphenylaminomethyl)aceto-phenone
(9) exhibited two intense bands at 3600 and 3375 due to free and
intramolecular hydrogen bonded OH respectively. From the intensity of
the free hydroxyl bond it can be assumed that due to the two bulky
phenyl rings, the less bulky free electron pair orbital points in such
a way that it is not available as an acceptor in hydrogen bonding.
Therefore, it is assumed that 9 exists as an equilibrium mixture of 9a,
9b. 9c and 9d. Similarly compounds 4-8 also exist as equilibrium
mixtures of forms a-d.
In both 6 and 9 the carbonyl
band appears near the VC–O frequency of 4-hydroxyacetophenone(l), i.e.
at a lower frequency (1652 and 1650 respectively) compared to that in
4, 5, 7 and 8, because the strong electron-donating (towards the ring)
capacity of O–H group is in these two cases are not affected much by
small intramolecular hydrogen bonding.
The formation of six-membered
ring-B through hydrogen bond may cause certain rigidity in the
molecules (4-9). From the PMR spectra (Table 2) it can be observed that
in compounds 4-8 the H-2 doublet appears at a higher field than the
doublet of doublets (dd) for H-6, but the trend reverses in compounds
10-14 where the intramolecular hydrogen bonding is removed by
methylation or thiophosphorylation of the 4-OH group. Therefore, the
upfield shift of H-2 proton must arise from the steric effect of either
the benzylic methylene protons or from the groups attached to nitrogen
atoms. When Dreiding models for 4,5,6 and 9 were constructed, it was
found that both the rings A and B remained almost on the same plane.
The two alkyl groups and the two benzylic protons remained one each
above and below the plane of the rings A and B. There was a probability
that the b-alkyI group might come over the H-2 proton and cause steric
hindrance resulting in the upfield shift of the latter. Therefore we
examined the model for a rigid system in 7 [N(R)=piperidino] and found
that both rings A and B were almost planar and the b-equiterial proton
of a-carbon atom to nitrogen could not exert any influence on the H-2
proton. Thus, the steric hindrance on H-2 and the resulting upfield
shift of its PMR signal must arise from the two benzylic methylene
protons with which it is making an angle of about 60°.
To confirm our above views we
examined the PMR spectra of
6-acetyl-2-methoxy-4H-1,3,2-benzodioxaphosphorine-2-sulphide (17) and
2-methoxy-6-nitro-4H-1,3,2-benzodioxapho-sphorin-2-sulphide (18) and
found that there also the H-2 proton doublet appeared at a higher field
compared to doublet of doublets for 6-H. On examining the model for 17
or 18 it was found that in these models the ring-A and the two oxygen
atoms of ring-B remained on the same plane and the H-2 proton was
making an angle of about 60° with the two benzylic methylene protons as
in 4-9.
From the structural point of
view, the compounds 2-9 have an sp3 carbon. atom between the aromatic
ring and the proton acceptor X to minimise the delocalization between
OH and X and does not make much impact on the dipole moment.
The 3-chloromethyl and
3-hydroxymethyl compounds (2 and 3) were found to be insoluble in CDCl3
and their PMR were recorded in acetone-d6. It was observed that unlike
in 4-8, the H-2 proton doublet in 2 and 3 appeared at a lower field
compared to H-6 proton in the PMR spectrum. Since 2 and 3 also form
hydrogen bonding with chlorine or oxygen atom of 3-substituted side
chain, it was expected that H-2 proton would appear at a higher field
compared to H-6 as in 4-8. To examine the solvent effect of acetone-d6,
we recorded the spectrum of 4 in this solvent and found that H-2 proton
appeared at a relatively higher field compared to H-6 (see Table 2).
Further, there was no difference in the pattern of the PMR spectrum
from that recorded in CDCl3. Although it is premature at this stage to
predict, without knowing in detail about the intramolecular hydrogen
bonding in compounds 2-4 and the solvent effect of acetone-d6 on them,
we would like to suggest that the downfield shift of H-2 proton in 2
and 3 might arise from the electronegative chlorine or oxygen atom of
the side chain.
Insecticidal activity
All the compounds (4-16) were
screened for their insecticidal activity against Drosophila
melanogaster (vinegar fly) by residue film evaporation method.at 1%,
0.5% and 0.2% concentrations. The mortality was observed after 24 hr.
The screening results, recorded in Table 3, show that almost all the
thiophosphorylated compounds (11-16) have significant insecticidal
activity. Rest of the compounds have either poor or no activity.
Further, the screening
results clearly indicate that the activity of compounds 11-16 lies only
in the organophosphorus part of the molecules. Since, the 4-methoxy
derivative (10) did not exhibit any appreciable activity, it was clear
that the 4-OH group as such or the hydrogen bonding caused by it did
not contribute to the insecticidal properties of the compounds.
Similarly, the nitrogen part of the molecule was also proved
ineffective because compound 15 showed the same activity as 11-14 and
16.
Syntheses
of Sulfanilyl Derivatives
The present study forms part
of our program on the chemistry of aromatic sulfonyl derivatives and
their evaluation as candidate pesticides. A survey of the literature
reveals that not much work has been reported on N4-(alkyl or aryl
sulfonyl) sulfanilyl derivatives.
Reaction of o-dichlorobenzene
with excess chlorosulfonic acid in boiling chloroform, gave the
3,4-dichlorobenzenesuIfonyl chloride in 57% yield as compared with 81%
reported earlier. Similarly m- and p-dichlorobenzene on reaction with
excess of chlorosulfonic acid with or without the solvent afforded 2,4-
and 2,5-dichloro-benzene-sulfonyl chlorides respectively in very good
yields (80-90%). These dichlorobenzenesulfonyl chlorides, on
condensation with excess aniline, gave the corresponding
dichloro-benzenesulfonanilides. Further reaction of these anilides with
warm excess chlorosulfonic acid under the conditions employed for the
successful chlorosulfonation of the analogous, dichlorobenzoic acid
anilides caused N  S bond cleavage to give the corresponding
dichlorobenzenesulfonyl chlorides. However, when the reaction was
carried out at lower temperature (-10° to 10°), good yields (65-85%) of
the dichlorobenzenesulfonyl sulfanilyl chlorides (1, 8. 19) (Scheme 1)
were obtained (cf. ref. 5). With sulfonylanilides, N â€â€S bond cleavage
was observed when chlorosulfonation was carried out at temperatures
above 10°, although similar chlorosulfonation of carboxylic acid
anilides occurred at 50-60° without appreciable
N-S bond cleavage.
The difference in behaviour
is presumably largely a reflection of the greater strength of the Câ€â€N
bond (184kcal/mol) as compared with the N-S bond (111 kcal/mol).
N4-(2,4 Dichlorobenzene-sulfonyl)sulfanilyl chloride (1) was condensed
with nucleophiles such as dimethylamine and hydrazine under standard
conditions to give the derivatives (2,3). The hydrazide(3) was treated
with acetone and benzaldehyde to afford the hydrazones (4, 5). The
hydrazides (3, 11, 21) on refluxing with acetylacetone in ethanol for 3
hr furnished the 3,5-dimethylpyrazole (6). 1 on reaction with sodium
azide gave the azide (7), which on treatment with triethyl phosphite
(Imol) in toluene at 0° furnished the phosphinimine (8). The
2,5-dichlorosulfanilyl chloride (9) was similarly reacted with
nucleophilic reagents to give the derivatives (10-16,18); the aziridine
(17) was obtained by reaction of the azide (15) with norbornene(1
mol)in toluene for 6 hr (cf. ref. 8b). Analogous reactions of
3,4-dichlorosulfanilyl chloride (19) gave the derivatives (20-25).
Attempts to prepare
N4-acetamidobenzene-sulfonylsulfanilyl derivatives (Table 1) via the
chlorosulfonation of N4-acetylsulfanilyl anilide by treatment with
excess chlorosulfonic acid failed. However, reaction of
N4-acetylsulfanilyl chloride with the dimethylamine, morpholine and
sodium azide gave the N4-acetylsulfanilyl derivatives which were
deacetylated with hydrochloric .acid to give the corresponding
sulfanilyl compounds. Thus N4-acetylsulfanilyl-dimethylamide was
deacetylated to N,N-dimethylsulfanilylamide (50%), PMR(DMSO-d6): d
7.20-6.56 (m, 4ArH), 5.90 (s, 2H, NH2, exchangeable with D2O), 2.05 [s,
6H, N(CH3)2]; N4-acetylsulfanilyl-morpholidate to the morpholidate12
(58%), PMR (DMSO-d6): d 7.40-6.60(m, 4ArH), 6.0 (s, 2H, NH2 exchangable
with D2O), 3.6-2.8 (m, 8H, morpholino H) and N4-acetylsulfanilyl azide
to the azide (61%). These were condensed with N4-acetylsulfanilyl
chloride to afford the required products (26,27,30) (Table 1). The
condensation could be carried out either in the presence of
triethylamine in acetonitrile or sodium hydrogen carbonate in acetone;
the latter system afforded a higher yield of a purer product. The azide
(30) was obtained by the condensation of N4-acetylsulfanilyl chloride
with sulfanilyl azide (1 mol) in the presence of sodium bicarbonate (1
mol) in aq. acetone for 3 hr. This, on reaction with triethylphosphite
(1 mol) in toluene at 80° for 1 hr gave (31) and on treatment with
norbornene (1 mol) in boiling THF for 6 hr furnished the aziridine(32).
Acetone and benzaldehyde sulfanilyl hydrazones reacted with
N4-acetylsulfanilyl chloride in pyridine to give low yields of the
required products (28, 29).
Methanesulfonanilide on
treatment with excess chloro-sulfonic acid at â€â€10" to 10" gave
N4-(methanesulfonyl)sulfanilyl chloride (Table 1, 33) which was
characterized as the derivatives (34-39). Reaction with aniline
afforded N4-(methanesulfonyl)- sulfanilylanilide, which like the
corresponding N4-acetyl derivative, failed to form the sulfonyl
derivatives with chlorosulfonic acid, under conditions successfully
used for the dichlorobenzene and methyl sulfonanilides (Table 1).
However, benzenesulfonanilide with excess chlorosulfonic acid at -10°
to 0° gave the corresponding sulfonyl chloride (54%).
In the PMR spectra of the
dichloro- and acetamido benzene sulfonylsulfanilyl compounds (Table 1),
the lowest field proton resonance is assigned to the SO2NH group due to
the combined anisotropic effect of the two attached benzene rings, e.g.
the acetone hydrazones (4,28). In the methane sulfonyl series (Table
1), the methylsulfamoyl proton appears at the lowest field and the
SO2NH  N proton resonance is merged with the aromatic proton
resonances, e.g. compound (37), which is confirmed by the disappearance
of part of the aromatic multiplet after treatment with deuterium oxide.
The mass spectra of the compounds generally showed the molecular ions
(M+), and fragment ions corresponding to M-X, M-SO2X, and M
-NHC6H4SO2X. In the acetamido series (Table 1) cleagage of the
acetamido group was also observed.
Biological activity
Antibacterial screening was
carried out by innoculation of agar plates as described by Steers et
al. and the results were compared with penicillin as standard (100%
control). The in vitro antifungal screening was performed using the
standard glass slide spore germination test as described by Kirby and
Frick.
In the preliminary in vitro
antibacterial screening tests against Streptococcus faecalis,
Clostridium perfringens, and Staphylococcus aureus at 50ppm, compounds
5, 10, 12, 13, 14, 15, 20 showed complete inhibition of the bacteria.
The compounds were also screened against Botrytis cinerea in vitro at
50ppm, high activity was observed for compounds 4,12, 26, 36. Compounds
were examined against Rhizoctonia solani and Pythium aphanodermatum in
vivo by soil incorporation at 16 kg/ha, the best antifungal activity
was shown by the azides (7, 24, 30).
Qsar of Fluridone
At least eight chemically
different classes of herbicidal inhibitors have been demonstrated to
affect phytoene desaturase as their essential mode of action. At the
moment, qualitative and/or quantitative structure-activity correlations
(QSAR) are available only for five groups, namely phenoxybenzamides,
phenylpyrida-zinones phenylfuranones and phenoxynicotinamides. For a
better understanding of the essential structural elements of a
phytoene-desaturase inhibitor and favorable substitution patterns more
structure-activity investigations with chemically different inhibitors
are needed.
The phenylpyridinone,
fluridone
(1-methyl-3-phenyl-5-(3-(trifluoromethyl)phenyl-(1H)-pyridinone),
belongs to the same type of bleaching herbicides as indicated above. In
a preceding publication it has been demonstrated that this compound
directly interacts with phytoene desaturase as a reversible
noncompetitive inhibitor. In the present study our attempts are
continued to analyze the structural requirements of typical
phytoene-desaturase inhibitors. Accordingly, a range of fluridone
derivatives have been investigated for their inhibitory activity either
in vivo or in vitro. Furthermore, quantitative structure-activity
relations have been calculated for fluridone analogs substituted at
position 3 of the pyridinone ring.
MATERIALS AND METHODS
Aphanocapsa (= Synechococcus
PCC 6714) was cultivated for 48 hr. Freshly harvested algal cells grown
in the presence of bleaching compounds were directly used for
carotenoid extraction or membrane preparation. Fusarium SG 4 mycelium
grown for 5 days as described was freeze-dried and stored at - 15°C.
Carotenoids were extracted with hot methanol (20 min, 65°C) and
partitioned into 10% (v/v) diethyl ether/petrol. Total carotenoids were
quantitated from this extract by their absorbance at 445 nm and related
to dry weight of cells. I50 values for intact cells were determined by
a Dixon-type plot with 5 to 7 concentrations around the l50 value.
For in vitro carotenogenesis,
Aphano-capsa cells were resuspended in 0.1 M
tris(hydroxymethyl)-aminomethane (Tris)-HCl buffer, pH 8.0, containing
5 mM dithiothreitol (DTT), and broken in a French press at 500 bar.
After centrifugation
(12,000g, 15 min), the membrane pellet was resuspended in the same
buffer. Fusarium SG 4 extracts, which form [14C]geranylgeranyl
pyrophosphate from [14C]mevalonic acid, were obtained by suspending 0.1
g of powdered material in 0.8 ml of 0.4 M Tris-HCl buffer, pH 8.0,
containing 5 mM DTT, and centrifugation (10.000g, 10 min). The
incubation mixture contained 0.2 ml of this supernatant, 0.1 ml of
Aphanocapsa (thylakoid) mem branes equivalent to 0.15 mg of
chlorophyll, ATP (5 mol), NAD+ (1 mmol), Mn2+ (3 mmol), and Mg2+ (2
mmol) made up to a total volume of 0.5 ml with water. The reaction was
terminated after 2 hr by addition of 2.5 ml of methanol containing 6%
KOH. After saponification for 20 min at 65°C, the carotenoids were
partitioned into 10% (v/v) diethyl ether/petrol and separated by HPLC.
The system used was a 25-cm Spherisorb ODS-1 5 mm column with
iso-cratic elution with acetonitrile/methanol/ propanol (85:10:5; v/v)
at a flow rate of 1 ml/min. Radioactivity of the elution peaks was
determined by a radioactive flow detector Ramona LS (Raytest,
Strauben-hardt, Germany). Inhibition ratios (IR) for incubations with a
certain inhibitor concentration were calculated from the ratio of
radioactivity accumulated in phytoene versus radioactivity in
b-carotene, related to the corresponding ratio of an untreated control
(4-3). In a previous publication this value has been verified as a
suitable in vitro parameter for QSAR. i50 and IR values are the means
of three to five experiments. The standard deviation was in the range
of 10%.
Details on the
phenylpyridones employed including the procedure of their synthesis are
given elsewhere. Quantitative structure-activity analysis was carried
out with R.A. Coburn’s multiple linear regression program, QSAR-PC,
from Biosoft. Physicochemical parameters (Hansch-Fujita hydrophobicity
constants p and Hammett para constants sp) were provided by the data
base of this program, except for the -C6H4F-p substituent which were
estimated by comparison with the data for similar substituents. The
Hammett para constants are derived from pka values of substituted
benzoic acid.
RESULTS AND DISCUSSION
As it has been established,
that most bleaching herbicides target the same enzyme reaction of
questions about common structural elements in the bleaching molecules
and about their interaction with the same or a similar inhibition site
may be brought closer to an answer by modification of their structures
and comparing their inhibitory properties. In case of phenylpyridinones
two sets of derivatives could be investigated. For all compounds of
Table 1, I50 values of bleaching activity have been determined from
decreased contents of colored carotenoids in the cells. The most active
compound was fluridone (no. 1). Replacement of the -CF3 group by less
lipophilic -Cl (no. 2) moderately decreased its activity and
replacement by -COOH (no. 8) decreased it strongly. Inhibitory activity
disappeared completely when one of the phenyl rings was missing and the
aromatic nature of the pyrimidinone ring was lost simultaneously (nos.
10, 11). Replacement of one of the phenyl rings by phenoxy (no. 6)
resulted in lower but substantial inhibition. Modification of the
fluridone molecule from a N-methyl to its N-ethyl derivative (no. 5)
was also accompanied by a negative effect on its bleaching activity.
When the pyridinone ring of fluridone was completely saturated, the
resulting piperidinone (no. 4) still showed reasonable activity with an
I50 value of about 25 mm. The structure of this compound allows for a
keto-enol equilibrium. In this case, a charge separation between the
heterocyclic nitrogen (positive) and the oxygen (negative) can be
imagined, which might exert an additional effect on the inhibitory
activity.
From the results of Table 1
it can be concluded that the aromatic nature of the pyridinone
structure influences the level of activity but is not essential for the
fluridone derivatives to interact as inhibitors of phytoene desaturase.
Apparently, both phenyl rings at positions 3 and 5 are favorable. It
has recently been reported for phenyltet rahydropyrimidines, which
contain a non-aromatic ring structure similar to a fluridone-related
piperidinone, that at least one of the two phenyl rings is essential
for activity. As an N-ethyl group instead of N-methyl strongly affects
the inhibitory activity in a negative way, the bulkiness of certain
substituents and the overall shape of the substituted pyridinone moiety
seems to be an important feature to influence herbicidal activity.
With respect to the
substitution of the keto group at position 4 by other heteroatoms, a
replacement by  OCH3 increased the I50 value of the resulting
pyridylium cation (no. 7) by a factor of about 100. A similar decrease
of activity was observed for - N(CH3)2 (no. 3). A thione group (no. 9)
or even -Cl (no. 12) led to very weak inhibition or to a compound which
had lost all its inhibitory activity. These results are difficult to
interpret. However, there seems to be a tendency of decreasing
inhibitory activity depending on how strongly electrons are withdrawn
from the heterocyclic nitrogen. Furthermore, electron density on the
various substitutents at position 4 could exhibit an additional
influence on activity.
All compounds in Tables 2 and
3 differ by a variation of the phenyl substituent at position 3 of the
pyridinone ring. For this series of derivatives, I50 values with intact
Aphanocapsa cells and the in vitro IR have been determined. These
values have been compared to physicochemical parameters for the
corres-ponding substituents and lipophilicity p and their electronic
properties sp were found to be important.
Only one compound, the
C6H4F-p analog, showed slightly better inhibitory activity than
fluridone. All other substituents were less effective (Table 2). The
dominant factor which determines their inhibitory properties is
lipophilicity. A plot of pI50 versus it gave a good linear relationship
with a correlation of 0.99 (Fig. 1A). However, the -OC6H5 analog did
not fit into this relationship. This might be due to the bulkiness of
this group which has e.g., a B2 STERIMOL constant of 3.11 as compared
to 1.71 for a phenyl group.
In a multiple regression
analysis, lipophilicity p and the electronic parameter sp as
independent variables gave a significant contribution to explain pl50.
The resulting QSAR equation is given in Table 4A. The validity of the
regression was confirmed by an F test and the significant contribution
of both independent variables by a t test. Other parameters of the
substituents did not improve this calculation.
In order to elucidate the
relevance of the results from this QSAR analysis for the interaction of
the pyridinone inhibitors with their target site, the phytoene
desaturase, in vitro inhibition of the compounds of Table 2 was
determined. For this purpose, the 10 compounds had to be divided into
high-and low-activity groups. Then, the inhibition ratios have been
measured for fixed concentrations of 0.3 and 10 mM, respectively (Table
3). Again, a dependency of inhibitory properties on lipophilicity could
be demonstrated. The log IR from the first group gave a very good (r =
0.98) linear correlation with lipophilicity -p (Fig. 1B). As shown for
in vivo inhibition (pI50 values of Fig. 1A), the -OC6H5 analog is an
outlier and does not fit into this correlation.
Interference of Fluridone
Fluridone was one of the
first developed bleaching herbicides and has been successfully used
during the last 15 years for weed control in cotton. Its primary mode
of action is inhibition of carotene interconversion at the level of
phytoene. This colorless carotene is accumulated together with
phytofluene in leaves after fluridone treatment, and formation of
b-carotene is impaired. As a consequence of the decreased levels of
colored carotenoids, chlorophyll is destroyed and disruption of the
chloroplast structure is observed in the light.
Accumulation of phytoene in
the presence of fluridone indicates inhibition of the desaturation
reaction. Direct interaction with phytoene desaturase at the enzyme
level has been reported for several other bleaching herbicides and also
for fluridone. It is a goal to find common features as well as
differences of the known bleaching herbicides that interfere with
phytoene desaturase. Helpful information may be obtained by comparing
their binding sites, looking at their types of inhibition, and
performing structure-activity investigations with chemically modified
compounds. Increasing our knowledge of inhibitor-enzyme interaction may
facilitate rational design of new potent phytoene desaturase
inhibitors. In this context, the present publication concentrates on an
enzyme-kinetic study of fluridone inhibition of phytoene desaturase
from Aphanocapsa. The results obtained allow the type of inhibition
exhibited by this herbicide to be determined. Furthermore, it was
demonstrated that Aphanocapsa membranes represent a good assay system
for in vitro ki determinations of different substituted fluridone
derivatives with a broad range of inhibitory activity.
MATERIALS AND METHODS
Aphanocapsa 6714 (=
Synechocystis PCC 6714) and Fusarium SG 4 were cultivated for 48 hr and
5 days, respectively, as described. Carotenoids were extracted from
harvested cells by suspending them in methanol containing 6% KOH and
then heating them for 20 min at 60°C. This step and subsequent ones
were all carried out in very dim light and under nitrogen, if possible.
Carotenoids were partitioned into 10% (v/v) diethyl ether in petrol (bp
35-80°C). Total carotenoids were determined from this extract by
absorbance at 445 nm and calculated with an average extinction
coefficient of
2500. Subsequently, the carotenoid extracts were evaporated to dryness,
resuspended in acetone, and sub jected to HPLC separation. The
reversed-phase HPLC system employed a Spherisorb ODS-1 5 mm column 25
mm in length and an isocratic solvent system of
acetonitrile/methanol/2-propanol 85/10/5 (v/v/v) (7) with a flow rate
of 1 ml/min. Carotenoids were identified with appropriate standards.
Chlorophyll was extracted with hot methanol from cells in a manner
similar to that used for carotenoids. Then, chlorophyll was quantitated
spectrophotometri-cally according to Mackinney.
In vitro carotenogenesis was
carried out with membranes from Aphanocapsa prepared by French-press
treatment (500 bar) of cells suspended in 0.1 M
Tris(hydroxy-methyl)aminomethane (Tris)-HCI buffer, pH 8.0, containing
5 mM dithiothreitol (DTT). Membranes were collected by centrifugation
(12,000g, 15 min) and resuspended in the same buffer to a final
chlorophyll concentration of 1.5 mg/ml. [14C]Geranylgeranyl
pyrophosphate, the substrate for the Aphanocapsa membranes, was
generated from [14C]mevalonic acid by a preparation from the fungal
mutant Fusarium SG 4. Two hundred milligrams of powdered mycelium was
mixed with 1.6 ml of 0.4 M Tris-HCl buffer, pH 8.0, containing 5 mM
DTT, and centrifuged for 10 min at 10.000g. Together with the
Aphanocapsa membrane suspension (0.15 ml), 0.1 ml of the supernatant (3
mg protein/ml) was used in the incubation mixture which additionally
contained K-[2-l4C]mevalonic acid (0.5 mCi), ATP (5 mol), NAD+ (1
mmol), Mg2 + (2 mmol), and Mn2+ (3 mmol) in a total volume of 0.5 ml.
Herbicides were applied in 5 ml methanol. The kinetics of Fig. 2 show
that, in a first step, [14C]geranylgeranyl pyrophosphate was formed by
preincubation of Fusarium SG 4 extracts with [14C]mevalonic acid and
all cofactors present for 2 h. Varied amounts of the resulting crude
geranylgeranyl pyrophosphate solution were used as substrate for the
subsequent phytoene desaturase reaction with AphanoÂÂcapsa membranes.
The reaction was terminated after 2 h by addition of 2.5 ml of methanol
and 0.3 ml of 60% KOH solution. Heating, partitioning, and HPLC
separation were done as described above for unlabeled carotenoids. The
radioactivity in the phytoene and b-carotene peaks was de termined
on-line with the radioactivity monitor Ramona LS (Raytest,
Straubenhardt, Germany). As the Aphanocapsa membranes catalyze a
reaction sequence from geranylgeranyl pyrophosphate to b-carotene with
phytoene as the only detectable intermediate, the sum of radioactivity
in phytoene + b-carotene accumulated over the total reaction period was
used as the substrate concentration value and the radioactivity in
b-carotene as the product value. Binding of [14C]fluridone (6.68
mCi/mmol) was carried out by incubation of Aphanocapsa membranes in 0.1
M Tris-HCl buffer, pH 8.0, with 0.1 nmol of radioactive herbicide in a
total volume of 1 ml for 1 h. After centrifugation (10,000g, 10 min),
residual radio-activity was determined from the supernatant. Then the
pellet was resuspended in 0.5 ml of the same buffer, and centrifugation
was repeated.
The data in Tables 1 and 2
are means from three experiments, the standard deviation is ± 10%. In
Figs. 1 to 3 data of typical experiments are shown. The carotenogenic
activity of the membranes used for them may vary from one preparation
to another. However, the overall picture and the bioÂÂchemical
information is always the same. The km values calculated from Fig. 2
and the ki values from Fig. 3 are very reproducible with standard
deviations of less than 10% from three to five determinations. The
herbicidal compounds used in this study are fluridone ( = EL-171;
1-methyl-3-phenyl-5-(3-(trifluoromethyl)phenyl)-4(1H)-pyridinone),
compound A
(1-methyl-3-(3-(ethylacetyl)phenyl)-5-(3-(trifluoromethyl)-phenyl)-4(1H)-pyridinone),
and compound B (1-methyl-3-phenyl-2,3-dehydropiperidi-none.
RESULTS
Application of the herbicide
fluridone to cultures of the cyanobacterium Aphanocapsa resulted in a
decrease of all the major colored carotenoids present in this organism.
Depending on the fluridone concentration of up to 1 mM, the acyclic
glycoside myxoxanthophyll, b-carotene and its hydroxylated and keto
derivatives zeaxanthin and echinenone, all decreased more or less to
the same extent (Table 1). With the highest herbicide concentration
used, only 20% of the colored carotenoids of the control were retained.
Simultaneous to the decrease of total colored carotenoids, phytoene,
found at a very low level in the control, accumulated. At least at low
concentrations of fluridone (0.1 mm), which only moderately affect
colored carotenoids, the sum of colored carotenoids plus phytoene is
similar to the control value. From the values of total colored
carotenoids, a ki, value in the range of 0.2 mM was estimated. In
addition to the decrease in carotenoids, lower levels of chlorophyll
were observed in the presence of various concentrations of fluridone.
Direct interference of
fluridone with phytoene desaturase was demonstrated in the in vitro
experiment of Fig. 1. [l4C]Gera-nylgeranyl pyrophosphate is converted
by Aphanocapsa membranes and the labeled products of the carotenogenic
pathway detected were phytoene and b-carotene exclusively. The results
are in parallel with the in vivo data. With increasing concentrations
of fluridone less radioactivity was found in b-carotene and more
radioactivity was accumulated in phytoene. The nature of this
inhibition of phytoene desaturase by fluridone was analyzed by enzyme
kinetic studies and is presented in a Lineweaver-Burk plot (Fig. 2).
The substrate was varied and the radioactivity in the reaction product
was determined in three sets of experiments: one as a herbicide-free
control, the two others with different concentra-tions of fluridone.
For all of them, straight lines were obtained in this double-reciprocal
plot with a common intersection at the abscissa which corresponds to
noncompetitive nature for fluridone interaction. It is difficult to
determine a km value for a heterogenous enzyme reaction. In our case, a
suspension of membranes, in which the desaturation takes place and in
which phytoene is generated, is employed. As the site of substrate
conversion and product formation is exclusively in the Aphanocapsa
membranes, the packed volume of the membranes per incubation was
determined as 12 ml. Based on this value, a km value of 0.3mM was
calculated for phyotene.
To find out whether fluridone
binding is reversible, binding and replacement experiments were carried
out with 14C-labeled herbicide (Table 2). Phytoene desaturase is
located in the thylakoid membranes. Therefore, an amount of fluridone
with a radioactivity of about 1500 dpm was added to various amounts of
thylakoid membranes that were quantitated by their chlorophyll content.
Depending on the quantity of thylakoids, up to 30% of fluridone was
bound. Reversibility of the binding was shown by washing the
radioactive fluridone from the membranes. One wash released about 85%
of the radioactive fluridone and a second wash released the rest of the
bound fluridone.
For the noncompetitive and
reversibly bound fluridone and two other pyridinone derivatives, ki
values have been determined by a Dixon plot of inhibitor concentration
versus inverse product formation at a constant substrate concentration
(Fig. 3). The intersections of the straight lines with the abscissa
gave a ki value of 0.08 mM for fluridone and 0.4 mM for compound A
which carries an ethylacetyl group at position 3 instead of the
unsubstituted phenyl ring. In the case of compound B in which both the
3-phenyl ring and the 3,4-double bond are lost, inhibitory activity is
ex tremely low with a ki value of about 100 mM.
Absorption
and Metabolism of Clomazone
Clomazone is a selective
biopesticide used in soybean to control certain grass and broadleaved
weeds. Clomazone is thought to inhibit an early step in the synthesis
of terpenoids resulting in the absence of chlorophyll, carotenoids, and
other terpenoids. Specifically, clomazone has been observed to inhibit
either isopentenyl pyrophosphate isomerase or prenyl transferase,
resulting in failure to produce plastidic terpenoids. However,
clomazone inhibition of isopentenyl isomerase and prenyl transferase
has not been observed in a different study. Past research has shown
that both tolerant and susceptible plant species have the capacity to
metabolize clomazone. Therefore, clomazone detoxi-cation does not
appear to account for soybean tolerance to the herbicide. Selectivity
to clomazone has been speculated to involve either differential
bioactivation of clomazone by sensitive species or differences of
clomazone sensitivity at the site of action.
Although metabolism of
clomazone has been studied in plants and plant cells, characterization
of the metabolites produced is lacking. In seedlings of both soybean
(tolerant) and velvetleaf (susceptible), we have demonstrated that
clomazone metabolism occurs by oxidative cleavage followed by the
benzyl moiety conjugating with glucose.
Cell cultures have been used
to evaluate absorption and metabolism of herbicides in plant cells, and
results have usually correlated well with observations made in intact
plants. Advantages of cell cultures for this research were the uniform
exposure of the cells to herbicide, absence of translocation, the
relative high capacity of the cells to produce clomazone metabolites
for iden tification purposes, and the possible absence of clomazone
phytotoxicity in heterotrophic cells. The objectives of this study were
to characterize the absorption and metabolism of clomazone by
suspension-cultured cells of soybean and velvetleaf and to characterize
the major metabolites produced.
MATERIALS AND METHODS
Chemicals. Analytical
clomazone (99.0% purity), [14C]CAR-C1 (1.04 GBq/mmol; 98% purity),
[14C]MET-C (1.15 GBq/mmol; 98% purity), 5-OH-clomazone, and
5-keto-clomazone were provided by FMC Corp. 2-CBA and b-glucosidase
were purchased from Sigma Chemical Co.
Cell
culture. Cell suspension cultures of soybean (cv. Corsoy 79) and
velvetleaf were established and cultured in a modified Gamborg B5
medium as described previously (8). Medium consisted of Gamborg B5
major and minor salts supplemented with 0.3, 2.0, 0.6, and 100 mg
liter–1 of kinetin, IAA (indole-3-acetic acid), picloram (4-amino-3 ,5
,6-trichIoro-2-pyridinecarboxcylic acid), and myoinositol,
respectively, and 25 g liter–1 sucrose. Suspension cells, established
from callus produced from hypocotyl sections of each species, were
subcultured every 14 days and grown on a rotary shaker at 25°C and 125
rpm.
Clomazone absorption and
metabolism. Past research had indicated that clomazone metabolism may
occur, in part, by oxidative cleavage. Therefore, we utilized two 14C
labels of clomazone to follow each half of the clomazone molecule.
MET-C and
CAR-C labeled the benzyl and
hetero-cyclic moieties of the clomazone molecule, respectively.
Soyabean and velvetleaf cells (10-12 days after subculturing) were
treated with either 1 mM MET-C or 1 mM CAR-C. Preliminary experiments
indicated that ex posure to 1 (M clomazone for 14 days had no effect
on the growth of either soybean or velvetleaf heterotrophic cells.
Aliquots of 20 ml (containing 0.8-1.2 g fresh wt of cells) were taken
at 1, 3, 6, 12, 24, and 48 hr after treatment. Cells were collected by
vacuum filtration on glass-fiber filter discs (WhatÂÂman GF/A, VWR
Scientific, Chicago, IL) and immediately rinsed with 10 ml of ice-cold
incubation medium containing 1 mM unlabeled clomazone to remove
unab-sorbed [MC]clomazone. Cells were collected again by vacuum
filtration. Cell fresh weights were determined and 4 ml 100% methanol
was added. Cells were stored at -20°C prior to homogenization. Ten
milliliters of 80% methanol was added and cells were homogenized using
a Ten-Broeck homogenizer.
Homogenized cells were
prepared for chromatographic analysis as described previously. The
homogenate was filtered through a glass-fiber filter. The filtrate was
concentrated by evaporation under a stream of N2 gas at room
temperature and filtered through a 0.2-mm-pore fluoropolymer membrane
(Arco LC13, Gelman Sci ences, Inc., Ann Arbor, MI). Radiolabel
remaining in the aqueous, suspension-cell upÂÂtake medium (30 ml) was
partitioned three times against 20 ml CH2Cl2. The CH2Cl2 phases were
combined, a subsample was taken from both the aqueous and the CH2Cl2
phases, and the radioactivity present was determined by LSS. The
remaining CH2Cl2 phase was evaporated to dryness in vacuo at 30°C and
precipitates were dissolved in 1.5 ml methanol prior to chromatographic
analysis.
Radiolabel remaining in the
cellular debris was determined by oxidation and collection of I4CO2
Radioactivity collected was quantified by LSS. In all cases greater
than 85% of the applied radioactivity was recovered.
Metabolite characterization.
Cells were homogenized and [14C]extracted as described above. Methanol
was removed in vacuo at 35°C and the aqueous concentrate (adjusted to
20 ml H2O) was partitioned three times with 15 ml CH2Cl2 (nonconjugate
fraction). The three CH2CI2 phases were combined for I4C analysis. The
aqueous phase (Polar I) was concentrated to 2 ml in vacuo at 50°C. Five
milliliters of 0.1 M Na-acetate buffer (pH 5.0) containing 50 U
b-glucosidase (E.G. 3.2.1.21) was added to cleave (b-1,6-sugar
conjugates. After incubation for 12 hr at 37°C, another 50 U of
b-glucosidase was added. Following incubation at 37°C for another 12
hr, the aglycones were partitioned into CH2Cl2 (3 x 25 ml). The
remaining aqueous phase (Polar II) was adjusted to pH 1.5 with 1 N HCl
and partitioned immediately against CH2Cl2 (3 x 25 ml). All CH2Cl2
phases were individually evaporated in vacuo at 30°C to dryness.
Samples were dissolved and stored in 0.5 ml of methanol prior to
chromatographic analysis.
Chromatographic analysis.
Separation of clomazone and clomazone metabolites was done using HPLC
with a H2O/acetonitrile gradient and a C,8 reverse-phase HPLC column.
Gradient steps were: (a) 0 to 20% acetonitrile from 0 to 15 min, (b) 20
to 50% acetonitrile over the next 5 min, (c) 50% acetonitrile for the
next 13 min, (d) 50 to 95% acetonitrile over the next 5 min, and (e) 95
to 0% acetonitrile over the next 5 min. Fractions were collected at
1-min intervals and radioactivity was determined by LSS. With this
solvent system, clomazone and 2-CBA had retention times of 31.2 and
25.5 min, respectively.
GC/MS data on HPLC-purified
metabolites were obtained using a Varion 5890 gas chromatograph coupled
to a Finnigan MAT ITD 800 ion trap detector mass spectrometer. The GC
was equipped with a fused silica capillary column 15 m long by 0.25 mm
i.d. containing a 0.25-mm bonded phase of Durabond-5 (J and W
Scientific, Folsom, CA). The GC column was coupled directly to the ion
trap manifold through the transfer line. The transfer line was
maintained at 280ºC. The linear velocity of helium through the column
was 26 cm sec–1. Splitless injections of 1 ml were made at an injection
port temperature of 280°C. The GC oven was maintained at 50°C for 4 min
and was increased at 6°C min–1 to a maximum of 300°C. The multiplier
voltage was set at 1450 V. The ion trap detector was repetitively
scanned from 50 to 450 amu in 1.0 sec.
Statistical analysis. All
experiments were conducted twice with at least two replications of each
treatment per experiment. Analysis of variance were performed on data
expressed as nmol g–1 fresh wt. Means were compared with Fisher’s least
significant difference test.
Results
Clomazone uptake and
metabolite retention. Cells of both soybean and velvetleaf retained
more total radioactivity when treated with MET-C than with CAR-C (Figs.
1A-1D). Retention of I4C increased in both soybean and velvetleaf over
time. Soybean treated with MET-C initially had higher concentrations of
radiolabeled compounds than did velvetleaf cells, but by 48 hr
velvetleaf had higher concentrations than did soybean. Compared to
velvetleaf, soybean cells treated with CAR-C had slightly higher
concentrations of I4C compounds at all times except 48 hr.
Clomazone metabolism. Soybean
cells treated with MET-C had clomazone concentrations of 0.7 to 1.0
nmol g–1 fresh wt throughout the experiment (Fig. 1A). AlÂÂthough the
metabolite concentration increased over time, clomazone was metabolized
more rapidly from 0 through 6 hr than from 6 through 48 hr.
Velvetleaf cells treated with
MET-C had clomazone concentrations from 0.1 to 0.3 nmol g–1 fresh wt
(Fig. IB). This was lower than for soybean cells treated with MET-C.
Metabolite concentration in velvetleaf cells increased at a constant
rate through 24 hr. A decrease in the rate of metabolite accumulation
was observed between 24 and 48 hr. The metabolite concentration made up
a higher percentage of total labeled compounds in velvetleaf cells than
in soybean cells (93% versus 82% at 48 hr, respectively).
Soybean cells treated with
CAR-C had clomazone concen-trations similar to those observed with
soybean cells treated with MET-C (0.7 to 1.0 nmol g–1 fresh wt) (Fig.
1C). However, the metabolite concentration was lower than that observed
for MET-C soybean cells. Thus, the percentÂÂage of labeled metabolites
in CAR-C soybean cells was lower than that observed for MET-C-treated
soybean cells (55% versus 82% at 48 hr, respectively).
A similar difference between
MET-C-and CAR-C-treated cells on uptake and metabolism was observed
with velvetleaf (Figs. IB and ID). Clomazone concentrations were equal
between CAR-C and MET-C velvetleaf cells; however, CAR-C-treated cells
had lower metabolite concentrations than MET-C-treated cells. Thus, as
was observed in soybean cells, velvetleaf cells treated with CAR-C had
a lower percentage of radiolabel as metabolites when compared to cells
treated with MET-C (85% versus 97% at 48 hr, respectively).
Extracellular metabolites.
Both MET-C-and CAR-C-treated cells of soybean and velvetleaf had
detectable levels of metaboÂÂlites in the media (Fig. 2). However,
greater amounts of water-soluble clomazone metabolites were present in
the media of CAR-C-treated cells than in MET-C-treated cells of both
velvetleaf and soybean. Concentrations of metabolites in the media
increased over time. No differences in the amounts of extracellular
clomazone metabolites were observed between soybean and velvetleaf
treated with MET-C.
Metabolite characterization.
Soybean and velvetleaf cells produced the same clomazone metabolites
based on HPLC elu-tion profiles (Fig. 3). Because velvetleaf
metabolized clomazone more rapidly than soybean did (Fig. 1), higher
concentrations of total metabolites were observed in velvetleaf than in
soybean (Table 1). However, the differences in percentage distributions
of the clomazone metabolites, if observed, were small between soybean
and velvetleaf cells whether they were treated with CAR-C or MET-C
(Table 1).
All of the metabolites were
more polar than clomazone (Fig. 3). Velvetleaf produced higher
concentrations of H2O-soluble (Polar I) metabolites than soybean did
(Table 2). Since more H2O-soluble metabolites leaked out of the
CAR-C-treated cells than the MET-C treated cells (Fig. 2), lower
amounts of H2O-soluble metabolites were detected in the cells of each
species with CAR-C than MET-C treatments.
A majority (>85%) of
the radiolabel present in the nonconjugate fraction (initial CH2Cl2
phase) was clomazone for both soybean and velvetleaf cells.
Confirmation of the identity of clomazone in this fraction was
demonstrated by HPLC and mass spectral analysis. Mass spectra (El) of
the isolated products from the different cells and labels were nearly
identical to clomazone reference standard (Fig. 4). Characteristic
molecular (M+) and base peak ions were observed at m/z 240 and m/z 125,
respectively. Clomazone may also lose Cl to give (M-Cl) m/z 204. The
other minor metabolite had a retention time of 22 min and was present
in both MET-C and CAR-C cells of soybean and velvetleaf.
Antidote Mode of Action
Studies have suggested that
antidotes (safeners) for chloroacetamide herbicides protect crops by
inducing glutathione conjugation of these herbicides. Antidote activity
has been correlated with enhanced herbicide metabolism and with
enÂÂhanced glutathione S-transferase (GST)2 activity. Until recently the
levels of parent herbicide and metabolites in tissues of plants grown
in soil had not been assayed, raising the question of whether enhanced
herbicide metabolism played a role in antidote action in vivo, and if
so, whether enhanced metabolism was the primary mechanism of antidote
action.
The first study in this
series indicated that BAS 145-1383 (BAS) protected corn (Zea mays L.)
from metazachlor injury by reducing the concentration of parent
metazachlor in growing tissues, especially in the developing leaves.
This decrease was attributed to (i) enhanced metabolism of metazachlor,
(ii) decreased mobility of [14C]metazachlor and/or its metabolites, and
(iii) slightly decreased absorption of metazachlor. The enhanced
metabolism was not measured directly but was suggested as an
explanation for the reduced amount of parent [14C]metazachlor as a
percentage
ofÂÂ
total radioactivity
in BAS-treated corn tissues .
The decreased mobility and
absorption of radioactivity observed in BAS-treated corn plants does
not rule out the possibility that enhanced metabolism may be the
primary or sole mechanism of antidote action. The reduced mobility of
radioactivity in BAS-treated plants could be due to the reduced levels
of parent metazachlor, since the parent is more mobile than the
metabolites, and especially the conjugates, of most herbicides. The
reduced absorption of metazachlor could also be the consequence of
reduced levels of parent metazachlor (i.e., the consequence of enhanced
metazachlor metabolism) in BAS-treated corn plants, since
chloroacetamide herbicides inhibit cuticular wax development and since
herbicide absorption can be increased by herbicide treatments that
inhibit cuticle development (or
conversely, herbicide absorption may be decreased by treatments such as
BAS that decrease levels of chloroacetamide herbicides in plant
tissues). Preservation of cuticle development by a herbicide antidote
has been previously reported. Thus, we evaluated the possibility that
the reduced mobility and absorption of radioactivity in corn plants
treated with BAS and [14C]metazachlor were the consequence of enhanced
metazachlor metabolism.
Corn cultivars DeKalb XL-25A
and XL-55A were not effectively protected from EPTC by the antidote,
dichlormid. Since dichlormid and BAS protect corn from metazachlor to
the same extent and
since both antidotes are dichloroacetamides, it seemed likely that
these corn varieties would not be effectively protected from
metazachlor injury by BAS. Dean el al. have further noted that the two
major GST isozymes induced by the dichloroacetamide antidote,
CGA-154281
[4-(di-chloroacetyl)-3,4-dihydro-3-methyl-2H-1,4-benzoxazine], appeared
to have activity on both chloroacetamide (metolachlor) and
thiocarbamate (EPTC) herbicides. FurtherÂÂmore, wheat seemed likely to
be poorly protected from metazachlor by BAS because wheat has not been
reported to be protected from chloroacetamide herbicides by dichlormid.
If our hypotheses were correct, the reduced antidote responses could be
verified using wheat and the two corn varieties, thereby allowing us to
determine whether the decreased antidotal activity was also correlated
with an altered effect on herbicide metabolism.
Experiments were conducted to
further investigate the significance of metabolism, mobility, and
absorption of metazachlor in the mode of action of BAS. The objectives
of this study were to (i) directly assay the rate of metazachlor
metabolism in the growing tissues of corn seedlings; (ii) evaluate
metazachlor phytotoxicity and metabolism in plants with reduced
responses to dichloroacetamide antidotes, including certain corn
cultivars and wheat; (iii) evaluate the mobility of metazachlor
metabolites; (iv) evaluate the effect of metazachlor and BAS treatments
on subsequent metazachlor absorption in corn; and (v) evaluate the
effect of BAS on glutathione (GSH) levels and GST activity.
MATERIALS AND METHODS
General
Experiments were conducted
with corn cultivar Northrup King PX9144 unless otherwise indicated.
Corn was grown in soil as previously describedÂÂ
at 21°C with incorporated treatments of 0 or 5 parts per
million by weight (ppmw) metazachlor and 0 or 1 ppmw BAS. Metazachlor
at 5 ppmw inhibited corn growth and BAS at 1 ppmw prevented most of
this inhibition. [14C]Metazachlor [pyrazol-U-14C, sp act 11.6 mCi/mmol
(1.55 MBq/mmol)] was used in all studies. All experiments were repeated.
[14C] Metazachlor metabolism studies
Four-day-old unemerged
seedlings having shoots 3.0 to 4.5 cm long were removed from the soil
and washed with water, and 2-cm root or apical shoot sections were
excised. Fifteen root sections (0.30-0.35 g) or six shoot sections
(0.40-0.45 g) were placed in 18 x 70-mm test tubes and pulse-labeled
for 30 sec by vacuum infiltration with 2.5 ml of aqueous 5 mM
[14C]metazachlor. The shoots and solution were stirred gently with a
vortex mixer during vacuum infiltration. After vacuum infiltration, the
[14C]metazachlor solution was immediately removed by aspiration and the
tissue was rinsed three times with 3-ml portions of water. The water
rinses were also removed by aspiration and the moist tissue was
incubated in the original test tubes at 25°C for the indicated periods.
After the incubation, the sample was frozen on aÂÂ
dry ice-acetone bath and stored at -20°C. Samples were
ground with a polytron in 3.5 ml of 70% acetone and filtered through
Whatman No. 3 filter paper. The polytron and filter were sequentially
rinsed with 3.5 ml of’35% acetone and 3.5 ml of water. Extracts were
partitioned three times with 10 ml of methylene chloride. The 14C in
the aqueous phase of partitioned extracts was shown by thin-layer
chromatography to be metabolites of [14C]metazachlor and the 14C in the
methylene chloride phase was confirmed to be parent [14C]metazachIor.
Radioactivity in the two phases was quantified by liquid scintillation
spectrometry. There were two replications per treatment.
Effect of BAS on Metazachlor Metabolism in Excised Tissues
Four
studies were conducted:
Effect of metazachlor
concentration. Plants were grown in soil treated with 5 ppmw
metazachlor and 0 or 1 ppmw BAS. These treatments were chosen both here
and in the time course (below) because they were the same as those used
in the previous study. The effect of [ 14C]metazachlor concentration
(0.5-50 mM) during the pulse-label period on the rate of metazachlor
metabolism was evaluated using a 30-min incubation.
Effect of soil treatments.
Plants were grown in soil treated with 0 or 5 ppmw metazachlor with or
without 1 ppmw BAS or 1 ppmw dichlormid. The 0 ppmw metazachlor
treatment was included so that the effect of the antidote alone could
be evaluated. Metazachlor metabolism in shoots and roots was evaluated
using a 30-min incubation.
Time course. Plants were
grown in soil treated with 5 ppmw metazachlor and 0 or 1 ppmw BAS.
Atime course for metazachlor metabolism in shoots and roots was
conducted for incubation periods ranging from 1 to 60 min.
Comparison of shoot tissues.
Plants were grown in soil treated with 0 or 1 ppmw BAS. Metazachlor was
omitted from the soil treatment in this experiment because metazachlor
caused the leaves to adhere tightly to the coleoptile, thus making the
dissection difficult. The shoot was cut at the coleoptile node, a 2-cm
section of mesocotyl was excised, and the leaves were dissected from
the coleoptile. Six shoots were dissected per replication. Metazachlor
metabolism was evaluated separately for each tissue.
Effect of BAS on Metazachlor Phytotoxicity atÂÂ
Metabolism in Three Corn Cultivars and Wheat.
Growth studies were conducted
with three corn cultivars, Northrup King PX9144, DeKalb XL-25A, and
DeKalb XL-55A, and one wheat cultivar, Olaf. Fifteen corn seeds or 25
wheat seeds were planted in vermiculite and grown under conditions
previously described. Treatments were applied in the initial watering
solution. Metazachlor was applied at concentrations ranging from 0 to
300 mM for corn and from 0 to 10 mm. for wheat. BAS was applied at 0,
1, or 10 mm. Treatments which contained both metazachlor and BAS were
applied as a single solution. Shoot height was evaluated 12 days after
planting for corn cultivars Northrup King PX9144 and DeKalb XL-25A and
13 days after planting for corn cultivar DeKalb XL-55A and wheat.
Untreated plants were at the two-leaf growth stage at the time of
harvest. There were two replications of each treatment.
Metabolism studies were
conducted using separate plants grown as described above and treated
with 0,1, or 10 mM BAS. Northrup King PX9144 seedlings were 4 days old
and other seedlings were 5 days old at the time of evaluation; all
seedlings were evaluated prior to emergence. Metabolism of
[14C]metazachlor in apical shoot sections was evaluated as described
above except that the incubation period was 15 min and 15 apical shoot
sections of wheat (approximately 0.40 g) were used.
Mobility of Metazachlor Metabolites.
Corn seedlings were grown for
3.5 days at 21°C in vermiculite treated with 0 or 2 mM aqueous BAS.
Metazachlor was omitted because of the previously described
difficulties it causes in dissection. Seedlings with 2.5- to 4-cm-long
shoots were removed and washed. Five seedlings were placed in a 50-ml
test tube for each replication. Seedlings were pulse-labeled by
submerging and aerating them in 25 ml of aqueous 5 mM [14C]metazachlor
for 12 min. Seedlings were rinsed three times with 25 ml water, placed
between layers of moist germination paper with the shoots protruding,
and incubated at 21°C under an inverted 800-ml beaker lined with moist
paper towels to maintain a high humidity. The germination paper was
changed at 0.5, 1, 2, and 4 hr to minimize reabsorption of parent
[l4C]metazachlor that
may have diffused into the germination paper. The shoots were dissected
into coleoptile, leaves, and mesocotyl. The tissues were weighed and
frozen after 4 and 36 hr. Tissues were analyzed for parent metazachlor
and metabolites as previously described. There were two replications of
each treatment.
In a second experiment, the
ability of metazachlor metabolites to diffuse out of corn shoot apical
sections into water was evaluated. Corn shoot apical sections were
excised, pulse-labeled with [14C]metazachlor, and incubated for 30 min,
as described for studies of metabolism in excised shoot tissues. This
was followed by a 30-min incubation in 5 ml water to allow diffusion of
metazachlor and metabolites out of the shoot. The levels of parent
metazachlor and metabolite in corn tissue and water were analyzedÂÂ
as described forÂÂ
metabolism studies.
Effect of BAS and Metazachlor on [14C] Metazachlor Absorption.
Corn was grown in soil
treated with 0 to 15 ppmw metazachlor and 0 or 1 ppmw BAS. Four-day-old
unemerged seedlings with shoots 3 to 4.5 cm long were removed from the
soil, washed, and placed between moist paper towels. Each seedling was
placed in a 5 ml scintillation vial and treated with 10-1.5ml droplets
containing 10 mM aqueous [14C]metazachlor (no organic solvent present).
Five droplets were applied to the coleoptile and five were applied to
the mesocotyl. Treated seedlings were incubated for 10 min under an
inverted 800-ml beaker lined with moist paper towels to maintain high
humidity. The shoot was then rinsed for 5 sec under a stream of acetone
and 5 sec under a stream of chloroform to remove cuticular waxes and
any associated 14C.
The shoot was excised and absorbed radioactivity was determined by
oxidation and liquid scintillation spectrometry. There were three
seedlings per replication and three replications per treatment.
Effect of BAS on GSH Levels and GST Activity.
Corn was grown in soil
treated with 0 or 1 ppmw BAS. GSH was assayed using equine GST and the
substrate 1-chloro-2,4-dinitrobenzene as previously described (6).
There were three replications for each treatment. GST was assayed as
previously described with minor modifications. GST assays were
conducted at 30°C and the reaction was started by adding
[14C]metazachlor (5 mM final concentration). Enzymatic activity values
were corrected for nonenzymatic and Time 0 controls. There were two
replications for each time point.
RESULTS AND DISCUSSION
Effect of BAS on Metazachlor Metabolism in Excised Tissues
Effect of metazachlor
concentration. The concentration of [14C]metazachlor used for
pulse-labeling did not significantly affect metazachlor metabolism in
excised shoots when expressed as percentage of 14C absorbed, nor did
metazachlor concentration affect the differences between antidoted and
control treatments (Fig. 1). Results with roots were virtually
identical (data not shown). The 5 mM metazachlor concentration was used
in all subsequent studies.
Effect of soil treatments.
BAS and chlormid increased the rate of metazachlor metabolism in corn
shoots (Table 1). This correlates with the protection from metazachlor
injury conferred by these two antidotes. Metazachlor soil treatment
slightly increased the rate of [14C]metazachlor metabolism in corn
shoots whether or not BAS was present in the soil. The effects of BAS
and metazachlor treatments on metazachlor metabolism in the roots were
similar to the effects in shoots (data not shown). The effect of
dichlormid on roots was not evaluated. A similar stimulation of the
metabolism of the chloroacetamide herbicide metolachlor by itself was
previously reported.
Time course. Metazachlor was
metabolized rapidly in unantidoted shoot and root tissues of corn but
metabolism was even more rapid in tissues grown in BAS-treated soil
(Fig. 2). The half-life of metazachlor was 58 and 14 min in roots (Fig.
2A) and 33 and 13 min in shoots (Fig. 2B) from unantidoted and
antidoted plants, respectively. This is consistent with the previous
dissection and growth studies which showed that BAS protected both
roots and shoots and indirectly indicated that BAS enhanced the rate of
metabolism of metazachlor in both tissues.
Comparison of shoot tissues.
BAS soil treatment enhanced [14C]metazachlor metabolism in the
coleoptile, developing leaves, and mesocotyl (Fig. 3). It appears that
the effects of BAS on metabolism are not tissue specific. This suggests
that metabolism of metazachlor in tissue adjacent to the developing
leaves, i.e., the coleoptile and mesocotyl, could reduce the amount of
metazachlor reaching the leaves and that the leaves themselves could
further decrease the level of metazachlor present in BAS-treated
plants. This corresponds with measurements of metazachlor levels in
corn plants grown in soil, in which BAS reduced the level of
metazachlor in the mesocotyl, coleoptile, and leaves.
Effect of BAS on Metazachlor Phytotoxicity and Metabolism in
Three Corn Cultivars and Wheat
All three corn cultivars
showed substantial increases in the concentration of metazachlor
required for 50% inhibition of growth (I50) as the BAS concentration
increased from 0 to 10 mM (Table 2). These increases in tolerance were
associated with increases in metazachlor metabolism rate. Wheat was
much less tolerant of metazachlor than corn, as indicated by the lower
I50 values for wheat. Wheat also was not protected from metazachlor by
BAS, as indicated by the constantÂÂ
I50 values as BAS concentration increased. Metazachlor
metabolism rates were correspondingly slower in wheat and these rates
did not increase as BAS concentration increased (Table 2).
The corn cultivars, DeKalb
XL-25A and XL-55A, were protected from metazachlor by BAS, contrary to
our previously discussed expectation (see Introduction). It is possible
that the previously reported apparent lack of dichloroacetamide
antidote activity in these two corn cultivars is not repeatable, since
no indication was given of the number of plants evaluated, the number
of replications, the variability, or whether the experiment was
repeated. The data reported here indicate that the level of antidotal
activity is correlated with the rate of herbicide metabolism. When
there is no antidotal activity, as in the case of wheat, there is
likewise no effect of the antidote on herbicide metabolism rate (Table
2).
Mobility of Metazachlor Metabolites
[14C]Metazachlor absorbed by
corn shoots should be almost entirely metabolized within 4 hr because
it has a half-life of only 33 min or less (Fig. 2). Therefore, if
metabolites are immobile, as we have proposed, radiolabel should not
move after 4 hr, even if a concentration gradient exists.
Most [14C]metazachlor was
converted to metabolites in corn seedlings after incubation for 4 hr
(Table 3). A strong concentration gradient (dpm/mg) of metabolites was
present at 4 hr among the three tissues evaluated; the coleoptile had
the highest concentration and the developing leaves had the lowest
concentration. The concentration of radioactivity (dpm/mg) declined
between 4 and 36 hr in all tissues as a result of growth but the
concentration gradients remained. The total amount of radioactivity
(dpm) remained constant in these tissues between 4 and 36 hr despite
the concentraÂÂtion gradient. Similar results were obtained in both
untreated and BAS-treated plants (Table 3). Thus, it appears likely
that the glutathione conjugate of metazachlor and its short-term
catabolic products are relatively immobile.
In a second experiment, the
ability of metazachlor and its metabolites to diffuse out of corn shoot
apical sections into water was also evaluated. Only 2.2% of the
metabolites present in corn shoots diffused into water, versus 14.7% of
the parent metazachlor. This observation also suggests that metazachlor
metabolites are less mobile than the parent. The plasmalemma and/or
tonoplast presumably act as barriers to movement of polar metabolites.
Effect of BAS and Metazachlor on [14C]Metazachlor Absorption
As the rate of unlabeled
metazachlor applied to the soil increased, the amount of
[14C]metazachlor absorbed increased (Fig. 4). BAS reduced the effect of
soil-applied metazachlor on [14C]metazachlor absorption, but when no
metazachlor was applied to the soil (i.e., at 0 ppmw metazachlor in
Fig. 4). BAS did not decrease absorption. This suggests that BAS
reduced absorption indirectly; perhaps metazachlor alone inhibited
synthesis of cuticular waxes, and BAS prevented this effect by
enhancing the detoxification of metazachlor. These obser-vations are
consistent with previous reports that chloroacetamides inhibit
cuticular wax development, that herbicide-treated plants absorb more
herbicide and that antidote treatment maintains cuticular wax integrity
in the presence of the herbicide. These observations are consistent
with the hypothesis that the apparent slight BAS-induced decrease in
metazachlor absorption previously observed in corn seedlings grown in
the soil actually represented protection from a metazachlor-induced
increase in [14C] metazachlor absorption.
Effect of BAS on GSH Levels and GST Activity
Metazachlor is metabolized to
the glutathione conjugate in corn. An antidote that induces metazachlor
metabolism might do so either by increasing GSH levels or by increasing
GST activity.
Untreated and BAS-treated
corn shoots contained 3.0 and 3.1 (mmol GSH/g fresh wt, respectively.
Thus, the antidote had no significant effect on GSH levels.
GST activity was low in
extracts of plants grown in untreated soil (Fig. 5), suggesting that a
significant portion of metazachlor metabolism observed in untreated
plants may be nonenzymatic. However, the relative importance of
enzymatic and non-enzymatic metabolism in unantidoted corn remains
inconclusive because the concentration of the enzyme in the assay was
diluted 70-fold compared to its concentration in vivo, and because some
loss of activity could have occurred prior to the assay. Antidote
treatment enhanced GST activity severalfold (Fig. 5). BAS-induced
metaza-chlor metabolism is probably due to induction of GST activity.
Diclofop Resistance in Avena Fatua
The herbicide
diclofop-methyl, a member of the aryloxy-phenoxypropionate class of
herbicides, is commonly used in western Canada to control grass weeds,
including wild oat (Avena fatua L.), in cereal grain crops. Selectivity
between wild oat and wheat is based on differences in metabolism of
diclofop-methyl in the two species. In wheat, the herbicide is rapidly
converted to an O-glycoside following ring hydroxylation; this product
is considered to be an irreversible detoxification product. In
susceptible species, such as wild oat, the predominant metabolite is a
glucose ester of diclofop (the parent acid), which can be hydrolyzed in
vivo to regenerate the active form, diclofop.
The target site of diclofop
and related herbicides is acetyl-coenzyme A carboxylase (ACCase;3 EC
6.4.1.2), a key enzyme in acyl lipid biosynthesis. Many dicotyÂÂledonous
plants are resistant to diclofop and other aryloxyphenoxypropionate
herbicides, basedÂÂ
on the low sensitivity of dicot ACCase to these
herbicides. These species are also resistant to cyclo-hexanedione
herbicides, such as tralkoxydim and sethoxydim, which act on the same
target enzyme. One grass species, Festuca rubra (red fescue), is
resistant to both aryloxyphenoxypropionate and cyclo-hexanedione
herbicides; resistance is based on reduced sensitivity of F. rubra
ACCase to these herbicides compared to that from susceptible species.
A diclofop-resistant biotype
of annual ryegrass (Lolium rigidum) has been identified in Australia,
but the mechanism of resistance has not been determined. Several
biotypes of wild oat have been identified in western Canada and
elsewhere that are resistant to diclofop-methyl. Recent
characterization of some of the Canadian biotypes indicates that they
are initially injured by the herbicide after application, but recover
within 9 days after treatment.
The objective of this study
was to examine the physiological and/or biochemical basis for
resistance in two of these wild oat biotypes. This included examination
of foliar absorption of diclofop-methyl, translocation within the
plants, metabolism of diclofop-methyl, and comparison of the
sensitivities of ACCase from the two resistant biotypes and a
susceptible biotype to diclofop and tralkoxydim, a cyclohexanedione
ACCase inhibitor.
MATERIALS AND METHODS
Plant material. Wild oat and
wheat seeds were germinated in 9-cm petri dishes lined with Whatman No.
1 filter paper moistened with distilled water. After 2 to 3 days, seeds
with emerged radicles were transferred to 195-ml styrofoam cups
containing coarse silica sand or vermiculite. The sand cultures were
subirrigated with half-strength Hoagland’s solution. The plants were
grown in a growth
cabinet maintained at 22/18°C day/night temperatures, in a 16-hr
photoperiod at 325 mE m–2 s–1. Plants were treated at the three-leaf
stage of development in all experiments, except where noted.
Diclofop-methyl uptake and
translocation. Solutions of [l4C]diclofop-methyl
([U-14C]dichlorophenyl; sp act, 690.8 MBq g–1) were prepared in 10%
aqueous ethanol so that 10 ml contained approximately 840 Bq. The
herbicide solution (10 ml) was applied as 5 to 10 droplets to the
second leaf using a Wiretrol capillary micropipette (Drumond Scientific
Co.), At various time intervals after application, unabsorbed
[14C]diclofop-methyl was removed from the leaf surface by washing the
treated area three times with 5 ml 10% aqueous ethanol. The I4C content
of the leaf washes was determined by liquid scintillation spectrometry
(LSS). The plants were then divided into the treated leaf, remainder of
shoot, and root; the treated leaf was further subdivided into the
treated area, the upper portion (i.e., toward the leaf tip), and the
basal portion. The plant tissue was air-dried at room temperature and
combusted in a biological sample oxidizer and the 14C content of the
different plant parts quantified by LSS.
Uptake of diclofop-methyl was
calculated as the total amount of radioactivity recovered in the plant,
expressed as a percentage of the total applied. Translocation was
calculated as 14C recovered in various plant parts, expressed a
percentage of the total radioactivity taken up by the plants.
Metabolism of
[14C]diclofop-methyl. The metabolism of [l4C]diclofop-methyl in the
different wild oat lines was examined both by thin-layer chromatography
(TLC) and by high-pressure liquid chromatography (HPLC). In the TLC
experiments, plants were treated as described above and sampled 24 and
72 hr after treatment. The treated leaf was washed (as above) and the
treated area homogenized and extracted twice with 5 ml 80% methanol.
The ho-mogenate was filtered through Whatman No. 1 filter paper and
dried under nitrogen in a heating block at 37°C. The dried residue was
redissolved in 400 ml 80% methanol, and a 50-mi.l aliquot removed and
counted by LSS to determine total radioactivity content. The remainder
of the sample was applied in a band to a plastic-backed silica gel TLC
plate (15 cm by 5 cm) and allowed to dry at air temperature. The plates
were run in baths containing approximately 150 ml solvent (toluene:
acetic acid:ethanol, 150:7:7) until
the solvent front had advanced to the top of the plates. The plates
were then cut into 15 I-cm strips, each strip was placed in a
scintillation vial, and 14C content determined by LSS. Counting
efficiency was not significantly affected by the presence of the
plastic strip in the scintillation vial.
The Rf values were calculated
based on the location of 14C activity on the plates and were compared
to standards of diclofopmethyl, diclofop, and ring-hydroxylated
diclofop. The Rf values of these compounds were determined by running
standards using the same solvent system and locating the compounds on
the plates by ultraviolet fluorescence. The Rf values of
diclofopmethyl, diclofop, and aryl-hydroxylated diclofop were 0.85,
0.35, and 0.15, respectively.
An attempt was made to
identify polar metabolites (i.e., those with an Rf 60,000-fold resistant to
topically applied abamectin. This resistance could not be suppressed by
the synergists piperonyl butoxide or S,S,S-tributyl phosphorotrithioate
and did not confer cross-resistance to lindane, dieldrin, crotoxyphos,
dichlorvos, dimethoate, permethrin, or tetrachlorvinphos. In this paper
we investigated the biochemical mechanisms and the genetic control of
the >60,000-fold abamectin resistance found in the AVER strain
of house fly.
MATERIALS AND METHODS
Insects. Three strains of
house fly were used in this study: S + is a laboratory susceptible
strain originally obtained from Dr. F. W. Plapp, Jr., of Texas
A&M University, College Station; aabys is an insecticide
susceptible strain from Dr. T. Hiroyoshi, University of Osaka, Japan,
which has the recessive morphological markers ali-curve, aristapedia,
brown body, yellow eye, and snip wing on autosomes 1, 2, 3, 4, and 5,
respectively; and the AVER strain that has high levels
(>60,000-fold) of resistance to topically applied abamectin and
moderate levels (35-fold) of resistance to formulated abamectin by
residual exposure.
To examine the inheritance of
abamectin resistance in the AVER strain we crossed AVER to the
susceptible aabys strain en mass. Females of the appropriate strain
were isolated every 8 hr to be certain they had not mated.
Chemicals. Abatmectin,
abamectin 8,9-oxide, MK-243 (4"-deoxy-4"-epimethyI-amino avermectin
B1), and [5-3H]abamectin (sp act 10.8 Ci/mmol) were gifts from Merck
Sharp and Dohme Research Laboratories (Rahway, NJ). Purity of
[3H]abamectin was 99-4% by thin-layer chromatography (TLC). All other
compounds were purchased from commercial sources.
Insecticide bioassays.
Insecticide bioassays were carried out by topical application and
injection. Topical application was conducted using 3- to 5-day-old
female flies. Injection was carried out as follows: 0.25 ml of
abamectin solution in acetone was injected into the metathoracic notum
of 3- to 5-day-old female flies using a 10-ml syringe (No. 701,
Hamilton Co., Reno, NV). Each replicate consisted of 20 flies/dose and
at least four doses. All tests were run at 25°C and were replicated six
times. Mortality was assessed after 48 hr. Probit analysis was by the
method of Finney as modified by Raymond.
Thin-layer chromatography and
radiography. TLC analysis was conducted on silica gel plates (60-F254,
Merck). The solvent system used was ethyl acetate:toluene:2-propanol
(10:3:1). The Rf value of abamectin in this solvent system was 0.61.
Radioactive metabolites were detected by autoradiography, and unlabeled
abamectin was detected by us visualization. Radioactive zones scraped
off TLC plates were quantified by liquid scintillation counting (LSC).
Liquiscint (National diagnostics. Palmetto, FL) was used as
scintillation fluid and gave 47% counting efficiency.
Penetration studies. The time
course of penetration was determined by applying 0.85 ng [3H] abamectin
in 0.5 ml acetone to the thoracic notum of female flies (4 days old) of
the AVER and S + strains and placing them, in groups of five, into
scintillation vials. At this dose none of the flies showed symptoms of
poisoning. At selected times the flies were transferred to another
scintillation vial and rinsed twice in 5 ml of acetone for about 10 sec
with gentle shaking. The body rinses were combined, evaporated, and
analyzed by LSC. The holding vials were also analyzed by LSC.
Percentages of unchanged [3H]abamectin contained in the body rinses and
holding vials were identified using TLC. Each time point was replicated
three times per experiment. Data are the average of four experiments.
Metabolism studies. In vivo
metabolism of [3H]abamectin was conducted using an injection technique.
A dose of 0.425 ng [3H]abamectin in 0.25 ml acetone was injected into
the metathoracic notum of female flies (4 days old) of the AVER and S +
strains using a 10-mI syringe (No. 701, Hamilton Co.). Groups of five
treated flies were placed into scintillation vials. At this dose, none
of the flies showed symptoms of poisoning. At selected times the flies
were transferred to a new scintillation vial and homogenized in 10 ml
of an ethyl acetate: water mixture (1:1) using a Biohomogenizer
(Biospec Products, Inc., Bartlesville, OK). The homogenized solutions
were then centrifuged at 1000 g for 5 min, and the 3H content in the
ethyl acetate-soluble and the water-soluble fractions was measured by
LSC. The ethyl acetate-soluble fraction was reduced in vacuo and
analyzed using TLC. The holding vials were also analyzed by LSC and TLC
as described above. Unextractable materials were digested with 1 ml of
Protosol for 12 hr and analyzed by LSC.
Receptor binding. Receptor
binding was carried out by the methods of Schaeffer and Haines with
slight modification. Adult house flies (4 days old) of the AVER and S +
strains were frozen at - 80°C for 1 hr. Immediately upon removal from
the freezer, the container was shaken vigorously, and the frozen body
parts were sifted through a 1.7-mm-mesh sieve, which removed most of
the heads, legs, and wings. Retained body parts were placed in a
1-liter beaker and gently swirled in 400 ml ice-cold distilled water.
Thoraces floated to the surface and were collected. The thoraces were
first homogenized in 10 vol of ice-cold 50 mM Hepes buffer (pH 7.4)
using a Biohomogenizer, and then rehomogenized using a Wheaton Potter
glass-teflon homogenizer. The homogenate was filtered over two layers
of muslin and the filtrate was centrifuged for 5 min at 1000g. The
pellet was discarded, and the supernatant fraction was centrifuged for
20 min at 40,000g. The resulting pellet was resuspended in Hepes buffer
to approximately 5 mg protein/ml. The membrane preparations (1.0 ml)
were incubated with various concentrations (0.125-3.0 nM) of
[3H]abamectin at 22°C for 45 min in the presence (nonspecific binding)
or absence (total binding) of 1.25 p.M unlabeled abamectin in glass
tubes (13 x 100 mm). For the equilibrium binding experiments, 1 mg
protein/ml was used. The incubation was terminated by rapid filtration
over Whatman GF/B filters (presoaked with 0.15% poly-ethyleneimine and
0.5% Triton X-100). The filters were immediately rinsed with 15 ml (3 x
5 ml) of ice-cold Hepes buffer containing 0.25% Triton X-100. The
filters were placed into scintillation vials containing 10 ml of
Liquiscint for 16 hr, and the radioactivity was determined by LSC.
Specific binding was calculated by subtracting nonÂÂspecific from total
binding. Protein concentration was determined according to the method
of Bradford.
Results
Inheritance ofabamectin
resistance. Reciprocal crosses between the aabys and AVER strains
produced an F1 generation that gave nearly identical dose-response
lines (Table 1), indicating that abamectin resistance was not sex
linked or due to cytoplasmic factors. Abamectin resistance was also
highly recessive, having only a 3.5- to 5.4-fold resistance in the F1
compared to >60,000-fold for the AVER strain. While other
recessive resistance mechanisms (ex. kdr) are known, the tremendous
change in abamectin resistance between the F1 and parental AVER strain
is quite remarkable. Males generally had lower LD50 values than
females, probably due to the slightly smaller size of the males. The S+
strain was slightly less sensitive to abamectin compared to the aabys
strain, although this difference is commonly noted between these
strains and is most likely due to the larger size of the S + flies.
Given the steep slopes of the dose-response lines for aabys and the S+
strains, it is likely that these strains have little variability in
their response to abamectin. Since the log dose-response lines for the
F1 progeny (aabys x AVER) also had steep slopes, this suggests that the
AVER strain may be homozygous for the major mechanisms of resistance.
Toxicity tests. Toxicity
tests by topical application and by injection were conducted against
the S + and AVER strains. Results are shown in Table 2. The AVER strain
showed only a 35-fold resistance to abamectin by injection. Since the
AVER strain showed high levels (>60,000-fold) of resistance to
topically applied abamectin, the dramatic change suggests that
decreased cuticular penetration is an important mechanism of resistance
in the AVER strain.
The AVER strain was found to
be >4000-fold cross-resistant to abamectin oxide with LD50
values of 0.02 and >100 mg/fly for the S+ (susceptible) and
resistant AVER strains, respectively. MK-243 was reasonably toxic to S
+ house flies with an LD50 of 0.03 mg/fly. However, the AVER strain was
very heterogeneous in its response to this compound, having a
dose-response line that was very flat: ranging from 5.7% kill at 0.03
mg/fly to 90% kill at 60 mg/fly. The approximate LDso was 0.4 mg/fly.
Although the resistance ratio for this compound (»13) is much less than
found toward abamectin oxide, the heterogeneity of response in the AVER
strain suggests that high levels of resistance could be selected for by
the use of abamectin oxide.
Penetration
studies. The rate of penetration of radiolabeled abamectin is shown in
Fig. 1. The penetration of radiolabeled abamectin was relatively slow,
reaching only 53% after 8 hr in the S + strain. The rate constant for
penetration of radiolabel was 2.4-fold slower in AVER than in S + with
values of 1.37 and 3.28 x 10–2 hr–1, respectively. This suggests that
decreased cuticular penetration is one of the mechanisms of resistance
in the AVER strain. TLC and LSC analysis of the body rinses and holding
vials at 8 hr after topical application showed that >98% of
radiolabel recovered was [3H]abamectin in AVER and S+ strains. Nearly
all of the applied radio-label was recovered from the body rinses at
all time points, while there was very little radiolabel remaining in
the holding vials (i.e., less than 3% of total radiolabel even after 8
hr).
Metabolism
studies. Because we lacked unlabeled standards of abamectin
metabolites, no attempt was made to identify me tabolites. The
metabolites of [3H]abamectin were, therefore, simplified to four
groups: water-soluble metabolites, abamectin in the ethyl
acetate-soluble fraction, unknown metabolites in the ethyl
acetate-soluble fraction, and unextractable materials. The results are
summarized in Table 3. Almost 90% of the radiolabel was recovered from
the body homogenates while less than 11% was recovered from the holding
vials even 8 hr after treatment in both strains. In the body
homogenates, the radiolabel from the water-soluble fraction and the
unknown metabolites in the ethyl acetate-soluble fraction were
constantly about two times higher in the S + strain than in the AVER
strain. The radiolabel from the unextractable materials was also higher
in the S + strain than in the AVER strain. These data suggest that
increased metabolism is not a mechanism of abamectin resistance in the
AVER strain. In the holding vials, unchanged abamectin was detected at
slightly higher levels in the AVER strain than in the S+ strain.
However, this is probably due to more abamectin leaking from the wound
caused by the needle rather than as a result of true excretion because
abamectin is lipophilic and unlikely to be excreted unchanged.
Receptor binding. Specific
binding of [3H]abamectin at 2.5 nM to the membrane preparations of the
AVER and S + strains increased linearly as a function of tissue protein
concentration up to 1 mg protein/ml, and the specific binding was
constantly about 1.8 times higher in the S + strain than in the AVER
strain (Fig. 2). Nonspecific binding increased linearly, but was only
7% of total binding at 3 mg protein/ml. Specific [3H]abamectin binding
to the membrane preparations was saturable with increasing
concentrations of [3H]abamectin in both strains, and the saturated
specific binding in the S + strain was about 1.5 times higher than that
in the AVER strain (Fig. 3). The Scatchard analysis (Fig. 4) of these
data yielded a straight line in both strains, indicating that both
strains have a single class of [3HJabamectin binding sites. The
equilibrium dissociation constant (x ± SD) was not significantly
different between the AVER (KD = 0.74 ± 0.03 nM) and the S + (KD = 0.72
± 0.02 nM). strains. However, the maximum number of binding sites
(Bmaxi) was significantly different between the two strains, with the
Bmax for the S+ strain (0.113 ± 0.008 pmol/mg protein) being 1.5 times
higher than that of the AVER strain (0.077 ± 0.006 pmol/mg protein).
Chlorimuron
Ethyl Metabolism in Corn
Chlorimuron ethyl,
N-(4-chloro-6-methoxypyrimidine-2-yl)-N’-(2-ethoxycarbonyl-benzenesulfonyl)urea,
is a sulfonylurea herbicide used for weed control in soybean. Like
other sulfonylurea herbicides, the primary mechanism of action of
chlorimuron ethyl has been related to its inhibition of acetolactate
synthase. Chlorimuron ethyl is metabolized at a much more rapid rate in
tolerant soybean than in susceptible cock-lebur or pigweed. Two
products of chlorimuron ethyl metabolism in soybean have been
identified: the free acid formed by hydrolysis of the ester and the
homoglutathione conjugate formed by displacement of chlorine. Both
products are inactive as inhibitors of acetolactate synthase and it has
been concluded that the selectivity of chlorimuron ethyl between
tolerant soybean and susceptible cocklebur and pigÂÂweed is related to
metabolism.
Corn is susceptible to
chlorimuron ethyl, and except for an indication that it may undergo
hydroxylation, the metabolism of chlorimuron ethyl in corn has not been
reported. Several reports have suggested that corn can be protected
from sulfonylurea injury by herbicide safeners. However, these
sulfonylurea herbicides are of types known to be metabolized in plants
by oxidation followed by conjugation with glucose. More recently,
chlorimuron ethyl injury to corn was partially alleviated by the use of
the herbicide safener BAS 145 138 (l-dichloroacetylhexahydro-3,3,8
a-trimethylpyrrolo [1,2-a] pyrimidine-6-(2H-one). Chlorimuron ethyl
metabolism in soybean does not involve oxidation and BAS 145 138 is
known to stimulate metabolism by conjugation with reduced glutathione
(GSH).
The purpose of this study was
to elucidate the routes of chlorimuronÂÂ
ethyl metabolism
in corn. Since the primary route of metabolism of chlorimuron ethyl in
soyÂÂbean involves conjugation with homoglutathione, it seemed likely
that metabolism in corn might involve conjugation with GSH. Soybean and
several other legumi-nous species contain homoglutathione rather than
GSH and form homoglutathione conjugates by a process that is assumed to
be analogous to GSH conjugation in corn. If chlorimuron ethyl was found
to be metabolized primarily by conjugation with GSH in corn, it would
then be possible to determine if BAS 145 138 protects corn from
chlorimuron ethyl by stimulating the formation of a nontoxic GSH
conjugate. The mechanisms of action of herbicide safeners that
stimulate GSH conjugation have been well studied with herbicides that
do not have a precisely known mechanism of action, such as the
chloroacetamides and thiocarbamates. It is difficult to prove the
mechanism of action of safeners using herbicides that do not have a
known mechanism of action and whose metabolism has been studied
primarily in tolerant species.
In this paper, the metabolism
of chlorimuron ethyl was studied primarily in the roots of corn. The
inhibition of acetolactate synthase in the roots of young seedlings is
of particular importance because this causes a stunting of root growth
and under mild stress can result in the death of the seedling.
Metabolism studies were of short term, not exceeding 11 hr, because the
primary objective of this study was to determine the rate and routes of
initial metabolism. The objective of the second part of this study will
be to determine the mode of action of BAS 145 138 as a safener for
chlorimuron ethyl in corn.
MATERIALS AND METHODS
Chemicals
Chemicals were obtained from
the following sources: chlorimuron ethyl (99.7%) and
[phenyl-14C]chlorimuron ethyl (3.48 mCi/mmol, radiochemical purity 99%)
(E.I. Du Pont de Nemours and Co., Wilmington, DE), analytical grade BAS
145 138 herbicide safener
(1-dichloroacetylhexahydro-3,3,8a-trimethylpyrrolo[l,2-a]-pyrimidin-
6-(2H-one) (98.4%) (BASF Corp.,
Research Triangle Park, NC), b-glucosidase (EC 3.2.1.21) from almonds
(Type II) and reduced glutathione (Sigma Chemical Co., St. Louis, MO),
high-performance liquid chromatography (HPLC) grade acetonitrile. HPLC
grade methylene chloride from this source did not cause the oxidation
of sulfide conjugates to the corresponding S-oxides. The
[phenyl–14C]chlorimuron ethyl used in these studies was usually diluted
to a specific activity of 0.45 mCi/mmol with analytical grade
chlorimuron ethyl.
2-Ethoxycarbonylbenzene
sulfonamide was prepared by hydrolysis of chlorimuron ethyl at pH 3 for
24 hr at 40°C. It was purified by extraction into methylene chloride
and by chromatography with HPLC System A [retention time (Rt) 10.5
min]. The GSH conjugate of chlorimuron ethyl
(N-(4-[S-glutathionyl]-6-methoxypyrimidine-2-yl)-N'-(2-ethoxycarbonylbenzenesulfonyl)
urea was synthesized in 25% yield by the method of Brown and Neighbors
(2). It was purified by HPLC System B (Rt 48 min).
Plant Treatment
Corn roots. Corn seed (Zea
mays L., Northrup
King hybrid PX 9144) was placed between layers of paper toweling and
moistened with 0 or 20 ppm BAS 145 138 in aqueous 0.2% acetone. The
toweling was rolled into cylinders, placed vertically into beakers
containing 0 or 20 ppm BAS 145 138 in 0.2% acetone, and placed in a
growth chamber. After incubation for 4 days (21°C, 50% relative
humidity, 14-hr photoperiod), the seedlings were removed and selected
on the basis of uniformity of size and appearance (root length 5.5 ±
0.7 cm). Selected seedlings were placed horizontally on grated racks 2
cm above a water
bath (22°C) and 12.3 ± 0.61 nmol of [14C]chlorimuron ethyl (0.45
mCi/mmoI) in 2.5 ml of 70% acetone was streaked onto the roots of each
seedling. After 7 hr of incubation in the covered water bath, the
seedlings were removed and dip rinsed in acetone (20 ml for 5 sec). The
roots (60 ± 4 mg fresh wt/seedling) were excised into 1-cm sections,
placed into tared centrifuge tubes containing ice water, and frozen at
-20°C. Eighty seedlings were used in this experiment and the experiment
was repeated three times. In each experiment, 40 seedlings were
germinated in the presence of BAS 145 138 and 40 were germinated in the
absence of BAS 145 138. Several additional experiments were conducted
with seedlings treated with a lower dose (1.3 nmol/root) of a higher
specific activity [14C]chlorimuron ethyl (3.48 mCi/mmol).
Plant Treatment
Corn coleoptiles. Corn seed
was germinated for 5 days between layers of paper as previously
described. After 5 days, nine coleoptiles were excised under water (1 g
fresh wt), placed cut ends down into 2 ml of aqueous 5.3 mm [14C]chlorimuron ethyl,
and incubated at 22°C. After 20 hr, the coleoptiles were removed and
frozen in 1.8 ml of water. The frozen coleoptiles were extracted three
times with 6 vol of 70% acetonitrile. The extract contained 47% of the
14C originally used in the treatment. The residual treating solution
contained 50.6%.
Corn shoots. Corn seed was
germinated between layers of paper as previously described. After 10 days, the resulting
shoots were excised under water. The cut ends of the shoots were
partially immersed (cut ends down) in 2 ml of 25.9 mM [14C]chlorimuron
ethyl in aqueous 0.5% acetone (4 shoots/2.0 g fresh wt tissue/test
tube). Three tubes of seedlings were incubated under fluorescent lights
at 22°C. After 2.5 hr, the treating solution was removed by aspiration,
the shoots were rinsed with 1.0 ml water, and 2.0 ml of water was
added. The shoots were incubated for an additional 0.5, 3.0, and 5.5
hr. After incubation, the shoots were diced into 40-ml Teflon
centrifuge tubes containing 6 ml of ice water and frozen.
Soybean roots. Soybean seeds
[Glycine max (L.) Merr.] variety Evans were germinated between layers
of moist paper toweling partially immersed in water as described for
corn. After 5 days, 40 seedlings were treated by application of [14C]
chlorimuron ethyl to the roots of each seedling (13.6 nmol/2.5 ml 70%
acetone/seedling). The seedlings were incubated as previously described
for corn. After 7 hr, the seedlings were rinsed with acetone and the
roots (4.28 g) were excised and extracted. The extract from the roots
contained 118 nmol of herbicide and metabolites/g fresh wt of roots.
Extraction of Tissue
Frozen tissue was thawed by
adding acetonitrile to bring the concentration of acetonitrile in the
extraction medium to 70%. Tissue was extracted three times with a 10:1
(v/w) ratio of 70% acetonitrile (4°C) using a Polytron homogenizer
equipped with a PTA-10TS generator (Brinkman Instruments, Westbury,
NY). Extracts were separated from cell debris by centrifugation at
3200g for 5 min. The cell debris was washed with acetone and the 14C
present in the cell debris and in the 70% acetonitrile extracts was
quantified by liquid scintillation spectrometry. The 70% acetonitrile
extracts from each sample were combined, concenÂÂtrated to ca. 5 ml
under vacuum at 37°C, diluted with water to 40 ml, and partitioned
three times against 20-ml volumes of methylene chloride. The methylene
chloride and water-soluble fractions were quantified by liquid
scintillation spectrometry and concentrated under vacuum. The methylene
chloride fractions were dissolved in 15% acetonitrile for analysis by
HPLC as described later. The water-soluble fractions were diluted to 15
ml with aqueous 5% acetonitrile, the 14C was quantified, and the
solutions were applied to columns containing 3 g of a preparative 55-
to 105-mm C18 liquid chromatography packing (Waters Division, Millipore
Corp., Milford, MA). The columns were washed with 7 ml of water, eluted
with 15 ml of aqueous 67% acetonitrile, and regenerated with 100%
acetonitrile followed by water. Recovery of 14C was 99.2 ± 0.8% (six
replicates), and 97.4 ± 1.2% of the recovered 14C was in the 67%
acetonitrile eluate. The acetonitrile eluates were concentrated to
dryness and dissolved in 15% acetonitrile for HPLC. In several cases,
partitioning with methylene chloride was excluded and the 70%
acetonitrile extracts were concentrated and fractionated directly on
columns containing 3 g of C18 liquid chromatography packing.
Chromatography
Substrates and metabolites
were analyzed or purified by HPLC on 3.9 mm x 30 cm columns of 10-mm
C18 mBondapak (Waters Division, Millipore Corp.) eluted at 1.5 ml/min.
Solvent System A consisted of a linear gradient from aqueous 25%
acetonitrile/1% glacial acetic acid to aqueous 55% acetonitrile/1%
glacial acetic acid in 30 min. Solvent System B consisted of a 30-min
isocratic elution with aqueous 20% acetonitrile/1% glacial acetic acid,
a 30-min linear gradient to aqueous 40% acetonitrile/1% glacial acetic
acid, and a 10-min linear gradient to 99% acetonitrile/1% glacial
acetic acid. Analyses were terminated by washing the HPLC columns with
a 99% organic solvent. In addition, various isocratic HPLC systems were
employed as described under Results. Column effluents were monitored
for radioactivity with a Model LB 503 Berthold radioactivity monitor
equipped with a -400-ml solid scintillator flow cell and also for uv at
254 nm with a Pharmacia dual path monitor UV-2. Thin-layer
chromatography (TLC) was performed on silica gel plates (250 mm x 5 cm
x 20 cm Anasil-HF silica gel plates, Analabs, Norwalk, CT) developed in
butanol:glacial acetic acid: water (12:3:5, v/v/v). Radioactivity on
these chromatograms was detected with a Bioscan System 200 imaging
scanner and nonradioactive compounds were detected by uv at 254 nm.
Purification of Metabolites for Mass Spectrometry
Chlorimuron
ethyl and metabolites of chlorimuron ethyl eluted from the open column
of C18 were further purified by HPLC using solvent Systems A and B.
HPLC System A was used in the initial separation of chlorimuron ethyl
and two nonpolar metabolites from the polar metabolites. The individual
metabolites resolved by HPLC Systems A and B were further purified as
discussed in conjunction with the identification of these metabolites.
Mass Spectrometry
Metabolites were analyzed by
mass spectrometry (MS) with a Varian MAT CH-5DF mass spectrometer. For
fast atom bom bardment mass Spectrometry (FAB MS), samples (1.5 to 5
mg) dissolved in a matrix of 0.2 ml glycerol, 0.1 ml methanol, and 10
mg oxalic acid were applied to a copper probe tip. An lonTek
Saddlefield gun was used to produce a xenon atom beam. Electron impact
mass spectra were obtained using a solid-sample probe with an El
source. Precise mass measurements were made by high resolution peak
matching.
Nuclear Magnetic Resonance (NMR) Spectrometry
Proton NMR Spectrometry was
performed on a Brucker AM 400 M Hz NMR spectrometer. The samples (25
mg) were dissolved in 30 ml of methanol-d4 and tet-ramethylsilane was
used as the internal standard.
b-Glucosidase Hydrolysis
Metabolites suspected of
being glucosides were treated with b-glucosidase Type II from almonds
using the procedures rec ommended by the Sigma Chemical Co.
Approximately 5.6 nmol of metabolite in 400 ml water was added to 100
ml of b-glucosidase (5.6 enzyme units, 1 mg protein) in 0.5 N, pH 5.0,
sodium acetate buffer and incubated at 30°C. After 6.5 hr, the
solutions were acidified with 1.5 ml of 1% glacial acetic acid and
partitioned twice with 4-ml portions of methylene chloride. The 14C in
the aqueous and methylene chloride-soluble fractions was measured and
the methylene chloride-soluble fractions were concentrated and analyzed
by HPLC and/or mass spectrometry. Little or no hydrolysis was observed
in no-enzyme controls.
Results
Uptake and Metabolism of Chlorimuron Ethyl
[l4C]
Chlorimuron ethyl was readily absorbed by the roots of intact corn
seedlings treated by surface application of the herbicide to the roots.
Uptake was nearly complete in 1 hr. When chlorimuron ethyl was applied
to the leaves in a similar manner, very little uptake was observed
after 24 hr (data not shown). [14C]Chlorimuron ethyl was metabolized
rapidly in the roots of corn seedlings treated with 21 nmol chlorimuron
ethyl/g fresh wt of roots. Metabolism occurred at a linear rate of 2.3
nmol/g fresh wt/hr and the half-life of the absorbed chlorimuron ethyl
was ca. 4 hr (Fig. 1). [14C]Chlorimuron ethyl was also metabolized
rapidly in excised leaves that were partially immersed in a dilute
aqueous solution of [14C]chlorimuron ethyl. Following pulse treatment
for 2.5 hr, the concentration of 14C in the leaves, expressed as
[14C]chlorimuron ethyl, was 4.58 nmol/g fresh wt leaves. Metabolism of
chlorimuron ethyl in the leaves was not linear, but the rate of
metabolism, based on the half-life of the herbicide (ca. 0.9 hr), was
estimated to be ca. 2.5 nmol/g fresh wt/hr (Fig. 1). Because of ‘lack of uptake’
[14C]chlorimuron ethyl metabolism was not monitored in intact corn
leaves. The data in Fig. 1 are not a true comparison of the relative
abilities of the roots and the leaves to metabolize chlorimuron ethyl
since the methods of treatment and the levels of [14C]chlorimuron ethyl
in the roots and shoots were different. However, it is clear that
chlorimuron ethyl is metabolized at an appreciable rate in both organs.
[14C]Chlorimuron ethyl
appeared to be metabolized by similar routes in both the roots and the
leaves of corn. Chlorimuron ethyl and seven or eight metabolites were
detected in both the roots and the shoots 7 hr following exposure to
the herbicide (Fig. 2). Only the metabolites from the roots were
actually isolated and identified. In the roots, the average recovery of
I4C 7 hr following treatment with [l4C]chlorimuron ethyl was 96.2%, and
the bound residue accounted for only 1.4% of the total residue. In the
leaves, the average recovery of 14C 3 to 8 hr following treatment was
93.2%, and the bound residue accounted for an average of 1.0% of the
total residue.
Detection of Metabolites
Three
nonpolar, radioactive compounds were detected by HPLC of the methylene
chloride-soluble extracts of roots from corn treated with
[14C]chlorimuron ethyl (Fig. 3). Six polar, radioactive metabolites
were detected by HPLC of the corresponding water-soluble extracts (Fig.
4). A radioactive peak observed during the HPLC of the water-soluble
fraction in HPLC System B (Rt 25 min, Fig. 4) was resolved into two
major metabolites by HPLC with water:
Chlorimuron Ethyl
The methylene
chloride-soluble product with a retention time of 30 min in HPLC System
A (Fig. 3) accounted for 50.5% of the radioactivity in the roots of
corn seedlings 7 hr following treatment with [14C]chlorimuron ethyl. It
was identified as chlorimuron ethyl by comparison to the standard by
HPLC in System A and by FAB MS and El MS.
Metabolite I
Metabolite I accounted for
18.5% of the total 14C or 40.5% of the l4C-labeled metabolites detected
in the roots 7 hr following treatment with chlorimuron ethyl. It was
purified from the methylene chloride fraction by successive HPLC with
System A (Rt 20 min), with methanol:water:acetic acid (49.5:49.5:1) (Rt
29 min) and with water:acetonitrile (70:30) (Rt 23.2 min).
The positive ion FAB MS of
metabolite I is shown in Fig. 5A. The intense ion cluster at m/z
431/433 [MH]+ is consistent with the presence of one chlorine and a
molecular weight of 430. The ion at m/z 397 correspends to the
reductive dechlorination of the MH+ ion. Reductive dechlorination
during positive ion FAB MS has been previously reported and was also
observed in this study during FAB MS of chlorimuron ethyl. The ion
fragments of metabolite I at m/z 219, 202, and 175 appeared to be due
to fragmentation of the hydroxylated pyrimidine moiety (Fig. 5A) while
ions at m/z 213 and 197 appeared to be due to the substituted phenyl
ring. The electron impact mass spectrum of metabolite I contained ion
fragments derived from the hydroxylated pyrimidine ring (m/z 175, 70%)
and the phenyl ring (m/z 210, 100%; m/z 184/185, 60%; m/z 120, 50%; and
mtz 104, 100%), but a molecular ion was not observed. It was concluded
that metabolite I was
N-(4-chloro-5-hydroxy-6-methoxypyrimidine-2-yl)-N-(2-ethoxy-carbonylbenzenesulfonyl)
urea.
Metabolite II
Metabolite
II accounted for 4.1% of the total 14C or 9.0% of the 14C-labeled
metabolites in the roots 7 hr following treatment with chlorimuron
ethyl. It was purified from the methylene chloride fraction by
chromatography with HPLC System A (Rt 10 min) and by isocratic HPLC
with water: acetonitrile:acetic acid (79:20:1) (Rt 14.3 min).
Metabolite II cochromatographed with standard 2-ethoxycarbonylbenzene
sulfonamide and the FAB MS of metabolite II and 2-ethoxycarbonylbenzene
sulfonamide were nearly identical. The FAB MS of metabolite II is shown
(Fig. 6). The spectra of metabolite II and the standard were
characterized by a series of quasi-molecular ions at m/z 230, 252, 268,
322, 344, and 360 that established the molecular weight of metabolite
II to be 229. The intense ions at m/z 213 and 184 confirmed the
identity of metabolite II to be 2-ethoxycarbonylbenzene sulfonamide.
Metabolite III
Metabolite III accounted for
1.5% of the total I4C or 3.3% of 14C-labeled metabolites detected in
the roots 7 hr following treatment with chlorimuron ethyl. It was
isolated from the roots of corn plants that had been treated with
[14C]chlorimuron ethyl and with [14C]chlorimuron ethyl plus BAS 145 138
safener. Based upon chromatographic evidence obtained with HPLC System
B, it was concluded that metabolite III was produced by both
treatments. Metabolite III was isolated by successive HPLC with System
B (Rt 25 min) by isocratic HPLC with water:acetonitrile:acetic acid
(83:16:1) and by a final step with HPLC System B. Metabolite III was
conÂÂtaminated with metabolite VI after the iniÂÂtial purification with
HPLC System B, but these metabolites were resolved by HPLC with water:
acetonitrile:acetic acid (83:16:1) (metabolite III, Rt 54 min and
metabolite VI, Rt 64 min).
Metabolite III was hydrolyzed
in a 91% yield by b-glucosidase. The methylene chloride-soluble,
radioactive product from this hydrolysis was tentatively characterized
as metabolite I by HPLC System A. Subsequently, metabolite I was
converted to metabolite III in vivo in corn and an enzyme preparation
from corn hydrolyzed metabolite III to metabolite I .
The FAB MS of metabolite III
was characterized by a series of quasi-molecular ions at m/z 593/595,
615/617, and 631/633 (Fig. 5B). These ions were consistent with the
presence of one chlorine and a molecular weight of 592. The intense ion
at m/z 431 (corresponds to the loss of the glucose moiety) and the
similarity of this FAB MS to the FAB MS of metabolite I in the region
from m/z 100 to 431 are evidence that metabolite III is the
corresponding glucoside of metabolite I. The precise mass of the ion at
m/z = 202.00200 agreed to within 0.0001 mass units to the calculated
mass for the ion shown in Fig. 5B and confirmed its elemental
composition. The ions at m/z 559 and 397 appear to be reductively
clechlorinated forms of the ions at m/zÂÂ
593 and 431, respectively. Metabolite III was concluded to
be N-(4-chloro-5-[O-b-D-glucosyl]-6-methoxy-pyrimidine-2-yl)-N'-(2
ethoxycarbonylbenzenesulfonyl) urea.
Metabolite IV
Metabolite IV accounted for
3.4% of the total 14C or 7.4% of the l4C-labeled metabolites detected
in corn roots 7 hr following treatment with chlorimuron ethyl. It was
purified from corn that had been treated with [14C]chlorimuron ethyl or
with [14C)chlorimuron ethyl plus BAS 145 138. Based upon analysis with
HPLC System B, it was concluded that metabolite IV was present in
extracts from both treatments. Metabolite IV was purified by successive
chromatography with HPLC System B (Rt 48 min), isocratic HPLC with
methanol: water.glacial acetic acid (49.5:49.5:1) (Rt 19-21 min), and
by HPLC System B (Rt 48-49 min).
When metabolite IV, purified
as described above, was chromatographed in HPLC System B in the absence
of 1% acetic acid, four radioactive peaks were eluted between Rt 24 and
41 min. When these peaks were combined, concentrated, and
chromato-graphed in HPLC System B in the presence of 1% acetic acid, a
single peak at 48 min was observed. Rechromatography of this peak in
HPLC System B in the absence of 1% acetic acid resulted in the elution
of multiple peaks. A similar phenomena occurred when corn tissue was
spiked with the synthetic 14C-labeled GSH conjugate of chlorimuron
ethyl, extracted, and analyzed by HPLC System B in the presence or
absence of 1% acetic acid. Metabolite IV and the synthetic GSH
conjugate chromatographed with retention times of 48 min in HPLC System
B. The homoglutathione conjugate of chlorimuron ethyl, isolated from
soybean root, also chromatographed in HPLC System B with a retention
time of 48 min.
The FAB MS ofÂÂ
the synthetic GSH conjugate and metabolite IV are compared
(Figs. 7A and 7B). Metabolite IV produced a series of quasi-molecular
ions at m/z 686, 708. and 724; but the synthetic GSH conjugate, which
apparently contained less salt, produced a single quasi-molecular ion
at mlz 686. The key fragmentation ions in the spectrum of the standard
GSH conjugate (m/z 557, 431, 413, 225, 184, and 158) were also present
in the spectrum of metabolite IV. The FAB MS of the homoglutathione
conjugate, isolated from soybean, contained a series of quasi-molecular
ions at m/z = 738, 722, and 700 (14 mass units higher than metabolite
IV) and fragmentation ions comparable to metabolite IV (Fig. 7C). ion
fragments from the homoglutathione conjugate that contained the
b-alanine moiety occurred at 14 mass units higher than ion fragments
from metabolite IV that contained the glycine moiety; therefore, it was
possible to determine which ion fragments contained the C-terminal
b-alanine or glycine moieties. It was concluded that metabolite IV was
N-(4-[S-glutathionyl]-6-methoxypyrimidine-2-yl)-N’-(2-ethoxycarbonyl-benzenesulfonyl)urea.
Metabolite V
The FAB MS of metabolite IV
contained two moderately intense ions (m/z 500,16%; and m/z 522, 6%)
which were not in the spectrum of the standard GSH conjugate and which
had no equivalent at the same mass or at 14 mass units higher in the
spectrum of the homoglutathione conjugate isolated from soybean (Figs.
7A, 7B, and 7C). Subsequently, TLC of this preparation of metabolite IV
showed that two radioactive metabolites were actually present,
metabolite IV (the GSH conjugate, Rf 0.42, 80%) and a minor component,
metabolite V (Rf 0.55,20%).
The Rf of metabolite V was consistent with that of a cysteine
conjugate. When metabolites IV and VÂÂ
were isolated from corn roots that had been incubated with
chlorimuron ethyl for 11 hr instead of 7 hr, the ratio of metabolite IV
to V changed from 4:1 to 2:1. It was concluded that the ions at m/z 500
and 522 (Fig. 7B) were the MH+ and MNa+ quasi-molecular ions of
metabolite V,
N-(4-[S-cysteinyl]-6-methoxypyrimidine-2-yl)-N’-(2-ethoxy-carbonylbenzene-sulfonyl)urea.
Metabolite VI
Metabolite
VI accounted for 3.4% of the total 14C or 7.4% of the 14C-labeled
metabolites in the roots 7 hr following treatment with chlorimuron
ethyl. Metabolite VI co-chromatographed with metabolite III in HPLC
System B, but after HPLC System B, it was resolved from metabolite III
by isocratic HPLC with water.acetonitrile:acetic acid (83:16:1)
(metabolite III, Rt 54 min and metabolite VI, Rt 63 min). Metabolite VI
was rechromatographed on HPLC System B prior to analysis by FAB MS and
NMR.
The FAB MS of this metabolite
was characterized by the following series of quasi-molecular ions:
[MNa] + , 724, 6%; [MK]+, 740, 8%; [MNaK-H]+ , 762, 6%; [MK2-H], 778,
5%; [MNa2K-H2]+, 784, 6%; and [MNaK2-H2], 800, 7%. These ions are
consistent with a molecular weight of 701 and might be expected from
either a hydroxylated GSH conjugate or the S-oxide of the GSH conjugate
of chlorimuron ethyl. Attempts to prepare the S-oxide of the GSH
conjugate of chlorimuron ethyl by oxidation of the synthetic GSH
conjugate with hydrogen peroxide or with m-chloroperbenzoic acid were
not successful; however, these methods frequently fail with aryl GSH
conjugates. A product with the same HPLC System B retention time as
metabolite VI was prepared in low yield (4%) by the reaction of
N-(4-chloro-5-hy-droxy6-methoxypyrimidine-2-yI)-N’-(2-ethoxycarbonylbenzene-sulfonyl)
urea with GSH under the same conditions as used in the synthesis of the
GSH conjugate of chlorimuron ethyl. This synthetic product, metabolite
VI, metabolite IV, the homoglutathione conjugate of chlorimuron ethyl,
S-(2.4-dinitrophenyl)-glutathione, and the GSH conjugate of propachlor
have TLC Rf values of 0.38 to 0.42 in butanol:acetic acid-:water
(12:3:5). The cysteine conjugates of propachlor and chlorimuron ethyl
have Rf values of 0.55 in this TLC system.
Proton NMR spectra (400 MH)
were obtained from metabolite VI, synthetic metabolite IV (GSH
conjugate), chlorimuron ethyl, primisulfuron, and three other
substituted pyrimidinyl derivatives. Preliminary evidence indicated
that metabolite VI was hydroxylated in the 5-position of the pyrimidine
ring. The chemical shift of the H-5 pyrimidine proton of chlorimuron
ethyl and metabolite IV was 6.29 ppm and in four other substituted
pyrimidinyl standards it ranged from 6.06 to 6.33 ppm. In contrast, no
protons with chemical shifts of 5.4 to 6.4 ppm were observed in the
spectrum of metabolite VI. further suggesting that the 5-position of
metabolite VI was hydroxylated. The chemical shifts of the protons on
the phenyl rings of these metabolites and standards were from 7.5 to
8.0 ppm due to the sulfonyl and carboxylic ester substituents on the
phenyl ring. Therefore, there were no interfering bands in this region
of the spectra. That portion of the spectrum of metabolite VI due to
the peptide side chain (1.9 to 4.6 ppm) was consistent with a GSH
conjugate and was similar to the spectrum of metabolite IV, but this
portion of the spectrum was not sufficiently resolved to be
unambiguous. However, the methylene protons attributed to the cysteinyl
residue of a GSH conjugate, usually observed between 2.8 and 3.1 ppm
were present at 2.8 ppm in the spectra of both metabolites IV and VI.
If metabolite VI was the S-oxide of the GSH conjugate, a significant
chemical shift of these methylene protons would be expected. Therefore,
based on synthetic evidence, MW data from FAB MS, chromatographic
comparisons with standards, and NMR spectrometry, it was concluded that
metabolite VI was
N-(5-hydroxy-4-[S-glutathionyl]-6-methoxypyrimidine-2-yl)-N’-(2-ethoxycarbonylbenzenesulfony)
urea.
Metabolite VII
Metabolite VII accounted for
1.4% of the total 14C or 3.4% of the MC-IabeIed metabolites present in
the roots 7 hr following treatment with chlorimuron ethyl. Based on
HPLC evidence, metabolite VII appeared to be present in corn treated
with either chlorimuron ethyl or chlorimuron ethyl plus BAS 145 138. It
was purified from extracts pooled from both types of treatment.
Purification was accomplished by two successive chromatographic steps
with HPLC System B followed by HPLC with System B that had been
modified by elimination of the glacial acetic acid. The retention time
of metabolite VII in HPLC System B with 1% glacial acetic acid was 54
min and in the absence of 1% glacial acetic acid it was 52 min.
Metabolite VII appeared to be
resistant to mild base hydrolysis. The retention time of metabolite VII
in HPLC System B was not altered by treatment of metabolite VII with
0.5 N ammonium hydroxide at 60°C for 60 min. However, metabolite VII
was hydrolyzed by b-glucosidase from almonds. After hydrolysis for 6.5
hr, 90% of the radioactivity was soluble in methylene chloride. The
retention time of the radioactive hydrolysis product in HPLC System A
was 25.6 min and the retention times of metabolite I and chlorimuron
ethyl in HPLC System A were 20.8 and 29 min, respectively. The negative
ion FAB MS of metabolite VII was characterized by quasi-molecular ions
at 591/593 and 683/685 that confirmed the presence of one chlorine and
a molecular weight of 592 while ions at m/z 244 and 270 were consistent
with a metabolite hydroxylated in the phenyl ring (Fig. 5C). These data
are consistent with a simple O-b-D-glucoside formed by hydroxylation
and conjugation of the phenyl ring of chlorimuron ethyl.
The radioactive hydrolysis
product of metabolite VII, obtained by treatment with b-glucosidase and
extraction with methylene chloride, was purified by HPLC (1.9 u,g) and
analyzed by El MS. A molecular ion was not observed in the El MS, but
the presence of the following ion fragments is consistent with a
metabolite hydroxylated in the phenyl ring:[HO-C6H3(COOC2H5)(SO2NH2)+
at m/z 245 (10%), [HO-C6H3(CO)(SO2NHCONH2)+ at m/z 243 (11%),
[HO-C6H3(CO)-(SO2NCO)] + at m/z 226 (28%), [HO-C6H3(COOH)(SO2NH2)] + at
m/z 217 (10%), [HO-C6H3(CO)(SO2NH2)+ at m/z 200 (43%), and
[HO-C6H3(CO)(SO2)]+ at m/z 184/185 (20%). The absence of hy-droxylation
in the pyrimidine ring was indicated by the following ions:
[NH2-C4N2H(Cl)(OCH3)]+ at m/z 159 (22%) and [NH2-C4N2H2(Cl)]+ at m/z
129 (20%). The interpretation of this El spectrum was based on a
comparison with the El MS of chlorimuron ethyl, metabolite 1,
2-amino-4-chloro-6-methoxy pyrimidine, and 2-ethoxycarbonylbenzene
sulfonamide (metabolite II). There was not sufficient metabolite to
determine the position of hydroxylation on the phenyl ring by proton
NMR. Metabolite VII was concluded to be
N-(4-chloro-6-methoxypyrimidine-2-yl)-N'-(2-ethoxycarbonyI-?-[O-b-D-glucosyl]
ben-zenesulfonyl) urea.
Superoxide
Dismutase Inhibition by a-Terthienyl
Over the past few years the
secondary plant metabolite, a-terthienyl (2,2':5,2"-terthiophene), has
been a subject of consid-erable interest because of its involvement in
a wide variety of phototoxic actions. The phototoxic effect toward
mosquito larvae and
its potential application as a larvicide make its mechanism of action a
topic of considerable importance. It has been reported that
a-terthienyl acts by Type II photodynamic action by sensitizing singlet
oxygen. Recently, it has been shown that a-terthienyl generates a
superoxide anion radical in aqueous media. Mosquito larvae treated with
a-terthienyl in the presence of long wave ultraviolet light or sunlight
show an accumulation of this compound in the anal gills and occurrence
of gill membrane damage as a consequence of a-terthienyl treatment as
can be seen by the halide leakage technique. Cell/ membrane proteins
are also shown to be the targets of toxicity of a-terthienyl. The
inhibition of glucose-6-phosphate dehydrogenase and malate
dehydro-genase and acetylcholine esterase are a few examples.
Another vital enzyme reported
to be present in the organs rich in oxygen, viz. rete mirable and gas
gland epithelium of marine fishes i.e., the superoxide dismutase, has
never been located in the anal gills of the mosquito larvae. Once it
was established that a-terthienyl toxicity was oxyradical mediated, the
significance of superoxide dismutase in the anal gills became apparent.
Recently, a histo-chemical method has been introduced by Laloraya et
al. modifying the original negative staining method of Beauchamp and
Fridovich. Using this technique, nitroblue tetrazolium, a free soluble
yellow compound, upon reduction forms an intensely blue product
(formazan) that is virtually insoluble in aqueous solutions and quickly
precipitates, making it ideal for staining. In this negative staining
technique, the enzyme superoxide dismutase dismutates the superoxide
radical generated by riboflavin, thus making it unavailable for the
electron acceptor nitroblue tetrazolium and thereby inhibiting the
diformazan formation; whereas, in the absence of superoxide dismutase,
the radical generated by riboflavin promotes diformazan formation.
Thus, the achromatic zones represent areas showing superoxide dismutase
activity, because of which this is called a negative staining method.
In this study, we report our pioneer attempt to localize the enzyme
superoxide dismutase in the anal gills of mosquito larvae and its fate
after exposure to a-terthienyl, successfully applying this technique.
MATERIALS AND METHODS
Reagents. Nitroblue
tetrazolium, riboflavin, and Diethyldithio-carbamic acid were obtained
from Sigma Chemical Co. N,N,N,N,-Tetramethylethylene diamine was from
Loba Chemical Co., India, and a-terthienyl was a gift from Dr. Arnason,
University of Ottawa, Canada.
All of the four mosquito
larval stages used for this study were reared in the laboratory and
each stage was studied separately. Two different sets of experiments
were performed. The first one involved superoxide dismutase activity
and the second one determined superoxide dismutase activity after
a-terthienyl treatment.
Histochemical test for
superoxide dismutase activity. Mosquito larvae were taken and cleaned
with double-distilled water, taking care not to injure the larvae. They
were treated initially with 2.5 x 10–3 M nitroblue tetrazolium for 30
min. These were then transferred to an incubation medium containing
0.036 M potassium phosphate (pH 7.8), 0.028 M tetramethyl-ethylene
diamine, and 2.8 x 10–5 M riboflavin
for 20 min. These larvae were then illuminated for 1 hr with a 15-W
Phillips fluorescent lamp. For negative controls the incubation medium
also contained diethyl dithiocarbamic acid, a known inhibitor of
superoxide dismutase at a final concentration of 2 x 10–2 M, with all
the abovementioned compounds.
Histochemical test for
superoxide dismutase activity after a-terthienyl treatment. Mosquito
larvae were cleaned with double-distilled water. They were then treated
with a-terthienyl at a concentration of 20 ppb and illuminated with an
ultraviolet light lamp (wavelength, 366 nm) (Model UVL-21 Black-Ray
lamp, Ultraviolet Proc., Inc., San Gabriel, CA) for 1 hr. These larvae
were then processed for the histochemical test outlined above.
All of the photochemical
reactions were carried out in the absolute dark in aluminium
foil-covered petri dishes. After illumination, larvae were rinsed with
0.036 M potassium phosphate (pH 7.8) and subsequently with
double-distilled water. The anal gills were separated from the larvae
and were mounted in glycerol jelly and viewed under bright field
optics. Photographs were taken using ORWO NP 22 (ASA 125) black and
white film. Exposure time was computed by a Nikon UFX II camera monitor
unit and the film was processed using a fine grain developer and Amfix
fixer (May & Baker, India).
Discussion
Discovery of the
light-dependent toxicity of a-terthienyl against many numbers of
organisms led to a number of studies whose main aim was to (i)
determine whether the phototoxic reactions were oxygen dependent, (ii)
establish whether the photodynamic reaction is Type I or Type II, and
(iii) locate a site(s) affected in damaged cells. However, the main
emphasis has been given to Type II photodynamicÂÂ
reactions, a consequence of the long-lived triplet excited
molecule and the high quantum yield of singlet oxygen formation.
Recently, however, the recognition that Type I photodynamic reactions
also operate in vitro has extended the range of possible mechanisms of
toxicity in vivo. Unfortunately, no definitive experiments have yet
compared the participation of Type I and Type II processes for a
specific photo-toxic effect in vivo. Tuveson et al. attempted such a
comparison using several strains of Escherichia coli varying in repair
capacities and catalase proficiency. Their study demonstrated that (i)
the specific repair mechanisms tested did not play a role in protecting
the organism from inactivation and (ii) the presence of H2O2 (which
would have been generated from superoxide radical through a Type I
photodynamic reaction) was of no detectable importance in cell
lethality. In a separate study, anÂÂ
E. coli strain engineered to produce carotenoid pigments
in the membrane exhibited virtually complete resistance to the photo
sensitized damage induced by a-terthienyl. This result strongly
suggested that singlet oxygen generated by a-terthienyl was lethal to
the cells in vivo and that its target was the cell membrane.
Viruses are inactivated by
a-terthienyl in a light-dependent manner, but only if they contain a
membrane. Similarly, a-terthienyl photosensitizes the conversion of
supercoiled into open circular pBR322 DNA in vitro in a manner which
appears independent of oxygen and which does not involve singlet
oxygen, superoxide, or hydrogen peroxide when oxygen is present.
Parathion
Metabolism by Soybean Lipoxygenase
Parathion (O,O-diethyl
p-nitrophenyl phosphorothionate) is a widely used or-ganophosphorus
insecticide. It is generally accepted that the toxicity of parathion is
due to the irreversible inhibition of acetyl-cholinesterase in the
nervous system. Parathion itself is a poor inhibitor of the
acetyl-cholinesterase but it is bioactivated by oxidative desulfuration
to its oxygen analogue paraoxon, which is a potent inhibitor of
acetytcholinesterase. Metabolism of parathion by the liver microsomal
cytochrome P-450-dependent monooxygenase and serum A-esterases has been
well documented in various animal species.
 Lipoxygenase
(EC.1.13.11.12) is a group of ubiquitous enzymes found in several
plants and animals. Lipoxygenase catalyzes the incorporation of
molecular oxygen into polyunsaturated fatty acids to yield
corresponding hydroperoxy fatty acids which subsequently break down
either enzymatically or non-enzymatically into hydroxy fatty acids,
leukotrienes, lipoxins, and other products. Lipid peroxides are known
to be produced in blood cells such as reticulocytes, leukocytes, and
platelets and in tissues like lung and brain by lipoxygenases. Various
lipid radicals are reactive compounds that support oxidation of
different xenobiotics either directly or through mediation of enzymatic
systems. Our laboratory has demonstrated the cooxidation of several
hydrogen donors, epoxidation of benzo(a)pyrene-7,8-dihydrodiol and
aldrin and sulfoxidation of thiobenzamide by lipoxygenase in the
presence of
linoleic acid. In addition to 13-hydroperoxylinoleic acid (13-HPOD) the
hydroperoxides of other polyÂÂunsaturated fatty acids as well as
inorganic peroxide such as hydrogen peroxide were shown to support
xenobiotic oxidation. In this study, we examined the catalytic
potential of lipoxygenase to mediate desulfuration and dearylation of
parathion using a highly purified enzyme from soybean as a model.
MATERIALS AND METHODS
Chemicals. Soybean
lipoxygenase type V (567,400 Sigma units/mg protein; MW 108 kDa),
acetylcholinesterase (electric eel, sp act 355 units/mg protein),
acetylthiocholine, 5,5,-dithiobis-2-nitrobenzoic acid (DTNB),
[14C]parathion (uniformly ring labeled, sp act 20.0 mCl/mmol), linoleic
acid, nor-dihydro-guaiaretic acid (NDGA), phenidone, paraoxon, and
p-nitrophenol were the products of Sigma chemical company. Parathion
(98.76%) was purchased from City Chemical Company. Petroleum ether,
diethyl ether, and acetic acid were purchased from Fisher Scientific
Company. Scintillation counting fluid was the product of ICN
Biomedicals, Inc. 13-HPOD was biosynthe-sised by incubating linoleic
acid with soybean lipoxygenase and the fatty acid hydroperoxide was
separated by thin layer chromatography.
Cholinesterase inhibition
studies. Paraoxon production was estimated indirectly from the
inhibition of acetylcholinesterse. Electric eel acetylcholinesterase
was used as exogenous source of the enzyme to trap the oxon produced
during lipoxygenase-catalyzed metabolism of parathion. Parathion (25.0
mM), linoleic acid (1.0 mM), lipoxygenase (20.0 nM), and
acetylcholinesterase (0.05 units) were incubated for 5.0 min at room
temperature in 0.1 M phosphate buffer, pH 8.0. At the end of the
incubation period, the reaction mixture was assayed directly for
residual acetylcholinesterase activity by adding acetylthiocholine (1.5
mM) and DTNB (1.0 mM). The control incubation medium contained all the
components except lipoxygenase. The results were compared with the
standard curves of acetylcholinesterase inhibition by paraoxon to
quantify the amount of paraoxon produced.
Radiometric method.
Metabolism of [14C]parathion was studied in the presence of linoleic
acid or 13-HPOD and lipoxygenase. The standard assay mixture, except
otherwise stated, contained 50.0 mM Tris buffer (pH 8.0), 25.0 mM
parathion (18.75 mM unlabeled parathion plus 6.25 mM [14C]parathion),
1.0 mM linoleic acid, and 20.0 nM enzyme in a final volume of 1.0 ml.
The reaction mixture was incubated for 5 min at 37°C. The reaction was
arrested with the addition of 250 ml of 10% trichloroacetic acid. The
tubes were then frozen at -70°C until analyzed. In some experiments,
linoleic acid was replaced with the desired concentration of 13-HPOD in
the incubaÂÂtion mixture.
Quantitative analysis. The
metabolites accumulated in the reaction mixture were extracted with 1.0
ml of methylene chloride. The organic phase was separated by
centrifugation and concentrated under nitrogen. An aliquot of the
concentrated extract was spotted on silica gel coated thin layer plate
(Silica G pre-coated plastic sheets from Brinkman Instruments Co). The
plate was developed to a height of 15.0 cm using the solvent system
containing petroleum ether, ethyl ether, glacial acetic acid (55:45:5
v/v). The Rf values observed in this solvent system were 0.8 for
parathion, 0.6 for p-nitrophenol, and 0.4 for paraoxon. The plate was
removed from the developing chamber and dried and the locations of
paraoxon and p-nitrophenol were visualized under ultraviolet light. The
spots were marked; the portions of the plate containing the metabolites
were cut and placed in scintillation vial containing 10.0 ml of
scintillation fluid and counted in a Beckman liquid scintillation
counter. The data reported are corrected for the quenching and counting
efficiency.
Dioxygenase activity.
Dioxygenase activity of lipoxygenase was measured
spectrophotometrically as an increase in the absorbance at 234 nm as
described previously using assay medium containing 1.0 mM linoleic
acid, 20.0 nM lipoxygenase enzyme, and the selected concentration of
parathion in 50.0 mM Tris buffer (pH 8.0). The specific activity was
calculated using an extinction coefficient of 25 mM–1 cm–1 for 13-HPOD.
The lipoxygenase inhibitors NDGA and phenidone (at desired
concentration) were preincubated with the enzyme for 2.0 min prior to
the addition of parathion and linoleic acid.
Oxygen uptake studies. Oxygen
uptake during metabolism of parathion was monitored with Clark type
oxygen electrode and a Biological oxygen monitor (Yellow Spring
Instruments Co., Inc) at 30°C. The reaction medium (final volume of 2.0
ml) contained 50.0 mM Tris buffer, pH 8.0, 1.0 mM linoleic acid,
desired concentration of parathion, and 20.0 nM enzyme. NDGA and
phenidone (at indicated concentration) were preincubated with the
enzyme for 2.0 min prior to the addition of parathion and linoleic acid.
After several pilot runs,
each experiment was repeated two or more times and the results of a
typical experiment are presented in the tables and figures. In most
cases, the experimental variation was about 10%.
RESULTS
The data in Table 1 show that
lipoxy-genase is capable of activating parathion to the more potent
acetylcholinesterase inhibitor, paraoxon. The data also show that the
amount of oxon produced was dependent on the concentration of
parathion, linoleic acid, and the enzyme (Table 1). The maximal rate of
metabolism was observed when incubation medium contained 25.0 mM
parathion, 1.0 mM linoleic acid, and 20.0 nM soybean lipoxygenase. A
decrease in the rate of paraoxon formation was evident when relatively
higher concentrations of parathion, linoleic acid, and the enzyme were
employed (Table 1).
[14C]Parathion was used in
all other experiments. It was meta-bolized to paraoxon and
p-nitrophenol in the presence of lipoxyÂÂgenase and linoleic acid (Table
2). A measurable nonenzymatic conversion of parathion to paraoxon and
p-nitrophenol was noted in the presence of linoleic acid alone.
However, a several-fold increase in the formation of these metabolites
was observed in the presence of the enzyme. The production of
metabolites was in the ratio of approximately 1:2 for paraoxon and
p-nitro-phenol, respectively.
The data on the effects of
experimental conditions on parathion metabolism are presented in Figs.
1 and 2. As shown in Fig. 1, the rate of both paraoxon and
p-nitrophenol production increased with the incubation time up to 5 min
and leveled off thereafter. The amount of products of desulfuration and
dearylation reactions increased when the concentration of enzyme
present in the incubation medium was increased up to 30 nM (Fig. 2A).
The rate of metabolism of parathion was dependent upon the
concentration of parathion (Fig. 2B). A lack of proportionality in the
generation of metabolites was noted at parathion concentration
>25 mM and this may be due to limited solubility of parathion or
enzyme saturation. Both paraoxon and p-nitrophenol production increased
with an increase in linoleic acid concentration and maximum rate of
production of these metabolites was noted when 1.0 mM linoleic acid was
used (Fig. 2C). A marked decline in specific activity for paraoxon and
p-nitrophenol was observed when >1.0 mM linoleic acid was
present in the reaction media. The data in Fig. 2D show the effect of
pH of incubation medium on the parathion metabolism. P-nitrophenol
formation increased with increase in pH. A broad pH response curve was
noted for paraoxon and maximum rate was observed at pH 8.0.
13-HPOD
also supported the lipoxygenase-catalyzed parathion metabolism in the
absence of linoleic acid (Table 2). However, as compared to the
linoleic acid-supported reaction, significantly lower rates of
enzymatic desulfuration and dearylation were observed when the reaction
medium was supplemented with 13-HPOD (Table 2). The rate of parathion
metabolism declined when higher (>22.5 mM) concentrations of
13-HPOD were used.
The data on the dioxygenase
activity of lipoxygenase and oxygen uptake (Table 3) revealed a small
change (4-5%) when up to 25.0 mM of parathion was present in the
reaction medium. Higher parathion concentrations caused a marked
inhibition of oxygen uptake and dioxygenase activity. A slightly
greater than additive response in terms of inhibition of dioxygenase
activity and oxygen consumption was observed when either NDGA or
phenidone, the inhibitors of lipoxygenase, was present in the reaction
medium in addition to parathion. As shown in Figs. 3A and 3B, both NDGA
and phenidone, at indicated micromolar concentration, inhibited the
desulfuration and dearylation reactions by 60 to 90%.
Depolarization
Studies of Pyrethroids
Primary target sites of
insecticidal pyrethroids are the nervous system of insects. Pyrethroids
have various effects on excised nerve preparations. Nerve cord
preparations discharge repetitively upon electrical stimulus after
exposure to a certain class of pyrethroids. Other pyrethroids suppress
the action potential. We have quantitatively analyzed the relationships
between wholebody symptomatic activities against American cockroaches
and house flies and nerve activities measured extracellularly from
nerve cords excised from the cockroaches. Both repetitive and blocking
neurophysiological effects are important in the toxicological effects
on these insects. Depolarization has been observed intracellularly in
single axons. We have measured this activity with crayfish axonal
membranes used as model for the study of the mode of action of
pyrethroids. Quantitative analysis has shown that the higher the
neurophysiological activity, the higher the whole-body symptomatic
activities.
To examine more directly the
origins of the symptoms of intoxication of American cockroaches and the
nerve effects measured extracellularly, we measured the depolarizing
potency of pyrethroids with cockroach giant axons using intracellular
microelectrodes. We suggest that membrane depolarization is a major
factor for block of nerve conduction leading to intoxication of insects.
MATERIALS AND METHODS
Compounds. Test compounds are
listed in Table 1. Compounds 1-7 are the benzyl esters of the acid
moiety of kadethrin (compound 17) and are called benzyl kadethrates in
this paper. Compounds 1-6 and 8-17 are the same as those used
previously (3, 4, 6, 11). (RS)-a-Cyano-meta-phenoxybenzyl kadethrate
(compound 7) was prepared from the corresponding acid chloride and
(RS)-a-cyano-meta-phenoxybenzyl alcohol in the presence of pyridine in
dry benzene at room temperature. The structure was confirmed by PMR1
and elementary analyses. The acid moiety of compounds 1-7 and 17 had
the (1R)-cis configuration and that of compounds 8-16 had the
(1R)-trans configuration. The alcohol moiety of compounds 7, 10, and 14
was not stereochemically defined. The structures of the acid and
alcohol moieties of the esters are shown in Tables 2 and 3. Pyrethroids
and veratridine were stored as methanol solutions in a freezer at -20°C
until use. Tetrodotoxin (TTX) was purchased from Sigma and stored as an
aqueous solution in a refrigerator at 5°C.
Measurement of the membrane
potential in cockroach giant axons. Central nerve cords excised from
the adult male American cockroaches (Periplaneta americana) were used
as the material. Unless otherwise noted, the nerve cord with the intact
sheath was used. In some experiments, the sheath was partially broken
between the 4th and 5th ganglia to give a “desheathed†nerve
preparation. The preparations were immersed in a saline solution
containing 210 mM NaCl, 2.9 mM KCI, 1.8 mM CaCl2, and 2.0 mM phosphate
buffer (1.8 mM Na2HPO4 and 0.2 mM KH2PO4, pH 7.2). The membrane
potential of the giant axon between the 4th and 5th ganglia was
measured intracellularly. Glass capillary microelectrodes filled with 2
M potassium acetate with a resistance of 4-8 MW and 10-20 Mft for the
desheathed and intact nerve preparations, respectively, were used
through-out the experiments. Nerve preparations with a resting
potential more negative than -75 mV were selected for the experiments.
Test solutions were prepared by addition of the stock solution of a
test chemical into the saline solution. The final concentration of
methanol in the solution was 1% or lower (v/v). Methanol at this
concentration had no effect on the resting potential. Compounds were
applied to the nerve preparation by external perfusion. Measurements
were done at room temperature (23 ± 1°C).
RESULTS
Depolarization of the Resting Membrane
Kadethrin depolarized both
the desheathed and intact nerve membranes (Figs. 1A and IB). With
additional treatment with TTX (1.0 x 10–5 M), the membrane potential in
the desheathed preparation recovered to a level more negative than that
observed before treatment with kadethrin (Fig. 1A). TTX alone slightly
hyperpolarized the axonal membrane in desheathed nerve cords that were
not treated with kadethrin (Fig. 1C). In the intact nerve preparation
(Fig. IB), however, the rate of recovery was much slower than that in
the desheathed preparation, indicating that TTX did not readily
penetrate the sheath of the cockroach nerve cord, probably because the
toxin is highly hydrophilic. By increasing the duration and intensity
of exposure to kadethrin, the membrane potential did not completely
recover from the depola-rization when the TTX was added (Fig. 2A). This
toxin-insensitive component of the depolarization was not observed when
the compound was applied at a low concentration (Fig. 2B).
Depolarizing Activity in Axonal Membranes
To evaluate the depolarizing
potency, we measured the maximum depolarization from the original
resting potential within 60 min after the start of treatment with a
compound. Usually, the membrane potential attained a steady state by
this time even in the intact nerve preparations as shown in Fig. 2 for
kadethrin with a desheathed nerve preparation. The dose-response
relationship for compound 3 with intact nerve preparations is shown in
Fig. 3. From such a curve for each compound, the DC10 value (M), which
is the concentration needed for membrane depolarization by 10 mV, was
estimated graphically. Log (l/DC10) was used as an index of the
depolarizing potency. The log (l/DC10) values are listed in Table 1.
Relationship between Neuroblocking and Excitatory Potencies
and Depolarizing Potency
Previously, we measured
extracellularly the neuroblocking and excitatory potencies of
pyrethroids in terms of log(l/MBC) and log(1/MEC), respectively. The
MBC and MEC are the minimum concentrations to block nerve conduction
and to induce repetitive discharges in response to a single stimulus in
the central nerve cord of American cockroaches. These values are
plotted against the depolarizing potency in Fig. 4. The neuroblocking
activity is almost linearly related to the depolarizing activity (Fig.
4B). There is, however, no clear-cut relationship between the
repetitive neu-roexcitatory activity and the depolarizing activity
(Fig. 4A).
Equation [1] was derived for
the neuro blocking and depolarizing activities with the least squares
method.
In this and the following
equations, n is the number of data points, s is the standard deviation,
r is the correlation coefficient, and F is the ratio of the variances
of the observed and calculated values. The figures in parentheses are
the 95% confidence intervals. Equation [1] was improved by the addition
of the log P term, where P is the 1-octanol/water partition
coefficient, to give Eq. [2].
Equation [2] indicates that
the higher the depolarizing activity and the greater the hydrophobicity
of compounds, the higher is the neuroblocking activity. The log (1/MBC)
values calculated by Eq. [2] are listed in Table 1.
Correlation was not found
between the neuroexcitatory and depolarizing activities even when the
log P term was added.
Relationship between the Insecticidal Potency and the
Depolarizing Potency
The insecticidal activity of
several sets of pyrethroids against the American cockroaches have been
represented in terms of log(1/MLD), where MLD is the minimum lethal
dose (in moles) to kill the cockroaches within 24 hr after injection of
compound together with piperonyl butoxide (50 mg/insect) and NIA 163882
(50 mg/ insect) that inhibit oxidative and hydrolytic metabolic
mechanisms, respectively. The activity values are listed in Table 1.
The activity was measured under conditions that minimized metabolic
mechanisms; under these conditions, the insecticidal activity should
simulate the activity at the target sites. Variations in the lethal
activity were quantitatively analyzed with variations in the
depolarizing activity, giving Eq. [3] as the best correlation.
^ Top
Adhesives, Chemical, Drugs, Gums, Insecticides, Jute, Pe ...