37 Pyrethroid Metabolism: Microsomal Oxidase Metabolites of (S)-Bioallethrin and the Six Natural Pyrethrins.
Metabolism of the chrysanthemates (
S)-bioallethrin, cinerin I, jasmolin I, and pyrethrin I by NADPH-dependent oxidases of mouse liver microsomes yields 13?18 metabolites in each case oxidized at the methyl, methylene, and alkenyl substituents to form alcohols, aldehydes, carboxylic acids, epoxides, and dihydrodiols. Rat microsomes are more specific than mouse microsomes in hydroxylating the (
E)-methyl substituent of the 2-methylpropenyl moiety compared with other molecular sites. Metabolites in the urine of allethrin-treated rats include compounds modified in both the 2-methylpropenyl and allyl moieties as free carboxylic acids and glucuronides. The pyrethrates cinerin II, jasmolin II, and pyrethrin II undergo microsomal hydrolysis of the methoxycarbonyl group and oxidation of the butenyl, penteny, and pentadienyl substituents to alcohols, epoxides, and dihydrodiols. Metabolites of these chrysanthemates and pyrethrates are tentatively identified by chemical ionization mass spectrometry following treatment with diazomethane or diazoethane and bis(trimethylsilyl)acetamide and separation by high-resolution gas chromatography with hydrogen as the carrier gas.
38 Identification of the Sex Pheromone of the Mulberry Pyralid, Glyphodes pyloalis Walker (Lepidoptera: Pyralidae).).
The major component of the female sex pheromone of
G. pyloalis is (
E,E,Z)-10,12,14-hexadecatrienyl acetate, as evidenced by analyses of ovipositor extracts by GLC and HPLC, and comparison of EAG responses of the male moths to synthetic compounds. The amount of pheromone produced was 1-2 ng/female. No peak for any other isomers of the compound was observed on GLC analysis. Field tests of all possible geometric isomers of synthetic 10,12,14-hexadecatrienyl acetate showed that only (
E,E,Z)-configuration had activity. Traps baited with a silicone rubber septum impregnated with 0.3 mg of the (
E,E,Z)-isomer, which was protected with an antioxidant, caught more
G. pyloalis males than traps baited with virgin female moths.
39 Tritritionikkomycin Z, [uracil-5-3H,pyridyl-2,4-3H2]: Radiolabeling of a Potent Inhibitor of Fungal and Insect Chitin Synthetase.
Nikkomycin Z (NZ) (a potent fungicide, insecticide, miticide, and inhibitor of fungal and insect chitin synthetase) was converted to a mixture of specific mono-, di-, and tribromo derivatives (BrNZ, Br
2NZ, and Br
3NZ, respectively) on reaction with
N-bromosuccinimide in
N,N-dimehylformamide. Substitution by bromine occurred first at the 2-position of the 3-hydroxypyridyl moiety, second at the 5-position of the uracil moiety, and finally at the 4-position of the 3-hydroxypyridyl moiety as observed both for NZ and for mixtures of uridine and 3-hydroxy-6-methylpyridine as model compounds representative of the moieties of NZ. Following fractionation of the various bromonikkomycin derivatives by HPLC, their structures were assigned by NMR, MS, and UV analyses. Catalytic reductive debromination of Br
3NZ with tritium gas over palladium on carbon gave [
uracil-5-
3H,pyridyl-2,4-
3H
2]NZ. This material has sufficiently high specific activity (~ 60 Ci/mmol) and suitable positions of labeling to study its uptake, distribution, metabolism, and possible target site interactions in fungal and insect systems.
42 Hormonal Regulation of Pheromone Biosynthesis in the Silkworm Moth, Bombyx mori (Lepidoptera: Bombycidae).
In the silkworm moth (
Bombyx mori L.), the sex pheromone [bombykol, (10
E,12
Z)-10,12-hexadecadien-1-ol] became undetectable 24 hr after decapitation, but its production was restored by the injection of a brain hormone (pheromone biosynthesis activating neuropeptide, PBAN). In order to understand the mode of action of this hormone,
14C-incorporation into pheromone precursors and related compounds was examined by treating the pheromone glands of normal, decapitated, and decapitated and hormone-injected virgin females with
14C-hexadecanoic acid. In the decapitated female moth, conversion of the
14C-acid into bombykol as well as other C
16-fatty alcohols was very low. The levels of
14C-incorporation observed in normal females were partially recovered in the decapitated female by hormone injection. However, the amount of unsaturated C
16-fatty acyl moieties and their levels of
14C-incorporation was not decreased as much by decapitation. This suggests that the main regulatory role of PBAN is in the reduction of an acyl moiety in the bombykol biosynthetic pathway rather than effecting unsaturation steps. Further, when the gland, which was removed from the decapitated female, was incubated in a Grace’s medium with PBAN, the pheromone was again produced indicating the direct regulation of the pheromone gland by this hormone.
43 Epoxychrysanthemates: Two-Dimensional NMR Analyses and Stereochemical Assignments.
Two-dimensional NMR analyses of two pyrethroid insecticides, (
S)-bioallethrin and (1
R)-
cis-phenothrin, and their diastereomeric 7,8-epoxy derivatives resulted in complete assignments of the
1H signals of the acid moieties by COSY, long-range COSY, and NOSY measurements. The relevant
13C signals of the six compounds were them assigned by C-H COSY experiments. Subsequent analyses of long-range C-H COSY spectra established that correlations between methyl carbons and adjacent methane protons involve those in a
cis rather than a trans relationship, making this technique useful in assigning methyl resonances on the cyclopropyl and epoxy ring of related pyrethroids. The known stereochemical preference of Sharpless asymmetric epoxidation was used to assign the stereochemistry of the epoxy ring for the 7,8-epoxy-10-hydroxy derivatives of these two pyrethroids. Comparison of the NMR data of the 7,8-epoxy and 7,8-epoxy-10-hydroxy derivatives confirms that the configurations of C
7 of the principal 7,8-epoxy metabolites are
R and
S from microsomal oxidation of
trans- and
cis-chrysanthemates, respectively.
44 Epoxy and Hydroxy Derivatives of (S)-Bioallethrin and Pyrethrins I and II: Synthesis and Metabolism.
The 2-methypropenyl group of (
S)-bioallethrin (
A) and the pentadienyl group of pyrethrin II (
PII) are selectively oxidized by
m-chloroperoxybenzoic acid in dichloromethane to yield the 7,8-epoxide (
1) from
A and a mixture of the 8’,9’- and 10’,11’-epoxides (
7 and
8) from
PII. These epoxides are hydrated in aqueous acid to the corresponding diols and other hydroxyl derivatives produced by opening of the cyclopropyl ring or migration of the adjacent double bond. The epoxy and hydroxyl derivatives are identified by two-dimensional NMR techniques. Mouse liver enzymes do not detectable hydrate epoxide
1 but quickly hydrate epoxides
7 and
8 without migration of the double bond. HPLC analyses of the microsomal metabolites of pyrethrins I and II identify the 10’,11’-diols as major metabolites and the 8’,9’-diols as minor products.
46 N-Terminal Modified Analogs of C-Terminal Fragments of PBAN with Pheromonotropic Activity.
Various analogs corresponding to the C-terminal portion of pheromone biosynthesis activating neuropeptide of silkmoth,
Bombyx mori (Bom-PBAN), were synthesized and tested for pheromonotropic activity. Analogs modified at
N-terminal amino group,
N-acyl,
N-alkyl, D-alanyl and pyroglutamyl hexapeptides, exhibited much greater activity than the corresponding peptides with a free
N-terminal amino group. Some of them exhibited higher activity than the unoxidized Bom-PBAN. These blocked peptides were stable in diluted haemolymph from
B. mori in vitro, while the non-blocked peptide decomposed rapidly. These results suggest that the stability of the blocked peptides in haemolymph might account for their enhancement of biological activity in the
in vivo assay.
49 Synthesis of Fluorinated Pyrethroids: Conversion of Pyrethroid Metabolites into Some Insecticidal Fluorinated Derivatives.
New fluorinated pyrethroids were designed to block microsomal oxidation at the C-10 methyl group of biophenothrin and at the C-7' methylene of bioallethrin. By using diethylaminosulfur trifluoride and/or hexafluoropropene diethylamine, 10,10-difluorobiophenothrin and 7'-fluorobioallethrin were synthesized from the oxidized derivatives of the parent insecticides, which have been recognized as microsomal metabolites. While 10-hydroxybiophenothrin was not successfully converted into the corresponding 10-fluoro derivative by either fluorination reagent, other monofluorinated compounds were formed and their structures were elucidated.
50 Three-Bond 13C?1H Coupling Constants for Chrysanthemic Acid and Phenothrin Metabolites: Detection by Two-Dimensional Long Range 13C?1H J-Resolution.
Two-dimensional long-range
13C?
1H
J-resolution spectroscopy (LRCJR) was used to measure three-bond
13C?
1H coupling constants [
3J(C,H)] for
trans- and
cis-chrysanthemic acid, methyl
trans-pyrethrate and some microsomal metabolites of the
trans-chrysanthemate biophenothrin. The carbon of the methyl
cis-disposed to an attached proton shows a larger
3J(C,H) value than does the
trans-carbon for the dimethyl-substituted cyclopropane and epoxide rings. The reverse situation applies for the analogous dimethyl vinyl grouping. The
3J(C,H) values are not altered on conversion of one of the olefinic geminal methyl groups to a hydroxymethyl or methoxycarbonyl functionality, but increase on transformation to an aldehyde. These
3J(C,H) values are in agreement previous results from long-range C?H COSY experiments, and provide a useful method for determining the stereochemistry of chrysanthemic acid derivatives.
51 13C NMR Assignments of Brassinosteroids by Two-Dimensional Techniques.
1H NMR spectra of eight naturally occurring brassinosteroids, plant hormones with a steroid skeleton, and five synthetic analogues were analysed by COSY and long-range COSY measurements. Based on the
1H resonances, the
13C signals were assigned by a combination of C?H COSY and long-range C?H COSY spectra detecting
13C nuclei, or by HMQC and HMBC spectra detecting
1H nuclei. Although the steroids showed complex
1H signals around 1?2 ppm, the resonances for the two angular methyl groups and the other methyl groups in the side-chain could be assigned unambiguously and used as a reference point for the other assignments. Strong correlation peaks between the methyl protons and carbons connected with the protons through two or three bonds were observed in the long-range C?H COSY or the HMBC spectra. These spectra provide valuable information for assigning the
13C NMR resonances of this class of compounds.
52 Sex Pheromone Candidates with Conjugated Triene System: Synthesis and Chemical Characterization.
Sex pheromone candidates with a conjugated triene system, 8,10,12-, 9,11,13- and 11,13,15-hexadecatrienyl acetates (double bond positional isomers of the
Glyphodes pyloalis pheromone, 10,12,14-triene) were synthesized by introducing an
E configuration stereospecifically to two of three double bonds and rather nonspecifically to another double bond, so as to obtain two geometrical isomers;
E,E,E and
E,E,Z or
E,Z,E isomers. The two geometrical isomers of each triene acetate were separated on a reverse-phase HPLC column and characterized by
1H NMR analysis. The
13C NMR signals in the olefinic region of each isomer were assigned by two-dimensional NMR techniques and also by an empirical role based on the changes of the chemical shifts by converting the configuration. Based on the assignments, substituent parameters for calculating the chemical shifts of 1,6-dialkyl conjugated trienes were generated. Electron impact mass spectrometry showed characteristic fragment ions that enabled the double bond positional isomers to be distinguished from each other.
53 Hydrocarbons with a Homoconjugated Polyene System and Their Monoepoxy Derivatives: Sex Attractants of Geometrid and Noctuid Moths Distributed in Japan.
Although several sex pheromones of the family Geometridae have been characterized, investigations on Japanese species are limited. In order to obtain more information, screening using known sex pheromones and their analogs was carried out. The (
Z,Z,Z)-3,6,9-triunsaturated and (
Z,Z)-6,9-diunsaturated hydrocarbons with straight C
19-C
21 chains were synthesized by the Grignard coupling reaction as a key step starting from linolenic and linoleic acids, respectively. Oxidation of the homoconjugated trienes with
m-chloroperoxybenzoic acid yielded a 1 : 1 : 1 mixture of three monoepoxy derivatives that could be separated by silica gel chromatography. The chemical structure of each positional isomer was confirmed using two-dimensional NMR techniques and MS measurements, which enabled characteristic fragment ions from the isomers to be identified. Field tests using lures incorporating only one of the above six hydrocarbons or nine epoxides were carried out in a forest in Tokyo. Consequently, attraction of male moths of 14 geometrid species in addition to four species in another family, the Noctuidae, was observed. It was concluded that hydrocarbons with a homoconjugated polyene system and the monoepoxy derivatives are important components of sex pheromones produced by Japanese lepidopterous insects, particularly the geometrid moths.
54 Structure and Activity of Bombyx PBAN.
Two structurally related molecular species of pheromone biosynthesis activating neuropeptides (PBANs), PBAN-I and -II, were isolated from adult heads of the silkworm,
Bombyx mori, and characterized. PBAN-I is a carboxyl-terminally amidated 33-residue peptide. Structure-activity relationship studies revealed that 1) its carboxyl-terminal pentapeptide is the smallest size showing activity, 2) the carboxyl-terminal amide is indispensable for activity, and 3) oxidation of three Met residues in PBAN-I to Met(O) (methionine sulfoxide) caused marked enhancement of activity, and the three Met(O) residues contribute equally to the enhancement of activity. Molecular design of PBAN analogs using a carboxyl-terminal hexapeptide showed that modification of the amino-terminal amino group brought about a dramatic increase in activity. This increase was presumed to be mainly due to the increased stability in hemolymph. PBANs share the common carboxyl-terminal sequence, -Phe-Xaa-Pro-Arg-Leu-NH2, with myotropic peptides isolated from locust and cockroach. Examination of cross-activity of these two groups of peptides revealed that PBAN and its analogs exhibited myotropic activity comparable to myotropic peptides, while myotropic peptides showed extremely high pheromonotropic activity. In
B. mori, PBAN activates sex pheromone (bombykol) production presumably by promoting the reduction reaction from acyl to alcohol, which is the last step in the biosynthesis of bombykol.
55 Metabolism and Chemical Reactivities of Pyrethroids: Stereochemistry of 7-Hydroxy Metabolite of (S)-Bioallethrin and Chemical Oxidation of Rethronyl Acetates.
Alkenyl side chains in the alcohol moiety of natural pyrethroids and (
S)-bioallethrin are easily oxidized by mixed function oxidases (mfo) and chemical oxidants, but the products have not been defined in detail. Thus, the absolute configuration of the 7-hydroxy derivative of (
S)-bioallethrin, a major metabolite by mouse and rat liver microsomes, was examined by a modified Mosher's method which compared the
1H NMR spectra of the esters with both enantiomers of
α-methoxy-
α-(trifluoromethyl)phenylacetic acid, and the (7
R)-configuration was assigned for it indicating that the mfo attacked a (
pro-R)-hydrogen at the 7-position of the pyrethroid. Further, oxidized products of the alcohol moieties by Se0
2 and
m-chloroperoxybenzoic acid (MCPBA) were analyzed by NMR and GC-MS after their purification by HPLC. Both the regioselectivity of these reagents and their relative reactivity on the four different alkenyl side chains were similar to those of mammalian and insect mfo. This result indicated that Se0
2 and MCPBA oxidation could be used as a model reaction for metabolic studies of pyrethroids.
56 Reduction of the Acyl Group: The Critical Step in Bombykol Biosynthesis Regulated in Vitro by Neuropeptide Hormone in the Pheromone Gland of Bombyx mori.
The sex pheromone of
Bombyx mori, bombykol [(10
E,12
Z)-10,12-hexadecadien-1-ol], can be biosynthesized in four steps: construction of a hexadecanoic moiety from acetyl CoA, Δ-11-desaturation, Δ-10,12-desaturation, and reduction of the acyl group. This biosynthesis is regulated by a hormone named the pheromone biosynthesis activating neuropeptide (PBAN). To examine the steps that are accelerated by this neurohormone, pheromone glands excised from decapitated females were incubated
in vitro with either
14C-labeled sodium acetate or one of three fatty acids [hexadecanoic acid, (
Z)-11-hexadecenoic acid, or (10
E,12
Z)-10,12-hexadecadienoic acid]. After analyzing the radioactivity that was incorporated from each precursor into bombykol and the biosynthetic precursors, it was observed that the first three steps proceeded in glands both treated and untreated with synthetic PBAN of
B. mori; however, the last step proceeded only in the treated glands. From this
in vitro experiment, it can be concluded that the main regulatory role of PBAN is in the reduction of the acyl group in
B. mori, as was shown by our previous
in vivo experiment.
57 Insecticidal Activity of New Fluorinated Pyrethroids and Their Stability Toward Chemical Oxidation and Photoreaction.
The insecticidal activity of several new fluorinated pyrethroids was measured using houseflies untreated and treated with piperonyl butoxide (PB). 10,10-Difluorophenothrin and 10,10-difluoroallethrin, which include fluorine atoms at a position most easily attacked by mixed function oxidases (mfo) in the acid moiety, showed smaller topical LD
50 values on the PB-untreated flies than the parent insecticides but rather large values on the PB-treated flies, suggesting that the introduction of fluorine prevents metabolic oxidation, although it has a negative effect on the interaction with a target site. Monofluorinated analogues of phenothrin, in which the structure of chrysanthemic acid was rearranged, still had moderate insecticidal activity, but flies were scarcely killed by 7’-fluoroallethrin into which fluorine was introduced at a position most easily oxidized by mfo in the alcohol moiety.
Competitive oxidation of the fluorinated derivatives and the parent compounds showed that fluorine at both the 10- and 7’-positions stabilizes the alkenyl side-chains of the pyrethroids against selenium dioxide and m-chloroperoxybenzoic acid. Photochemical studies with 10,10-difluorophenothrin indicated that the photo-instability of the cyclopropanecarboxylic part could not be improved by this modification.
