WO2008013336A1

WO2008013336A1 - Pyripyropene derivatives and insecticidal compositions comprising them

Info

Publication number: WO2008013336A1
Application number: PCT/KR2006/004071
Authority: WO
Inventors: Young Kook Kim; Hyun Sun Lee; Mun Chual Rho; Yong Seok Choi; Gyu Yong Song
Original assignee: Korea Research Institute Of Bioscience And Biotechnology
Priority date: 2006-07-27
Filing date: 2006-10-10
Publication date: 2008-01-31
Also published as: KR20080010563A; KR100943983B1

Abstract

Disclosed herein are novel pyripyropene derivatives and salts thereof having inhibitory activity toward acyl CoA:cholesterol acyltransferase, and insecticidal compositions comprising the same as effective ingredients. Functioning to show potent insecticidal effects by inhibiting the sterol metabolism of insect larvae, the pyripyropene derivatives or the salts thereof can be used as safe insecticides with potent acyl CoA: cholesterol acyltransferase inhibitory activity.

Description

PYRIPYROPENE DERIVATIVES AND INSECTICIDAL COMPOSITIONS COMPRISING THEM

Technical Field

[1] The present invention relates to novel pyripyropene derivatives and salts thereof having inhibitory activity toward acyl CoA: cholesterol acyltransferase, and to in- secticidal compositions comprising the same as effective ingredients.

[2]

Background Art

[3] Synthetic organic pesticides have been widely used for improving the production yields of agricultural crops and crop products, eliminating pests, and protecting forests from pests. However, continuous use and abuse of these highly toxic pesticides for several decades has resulted in many adverse effects, including abnormal occurrence of pests or the development of resistance to the pesticides, incidence of toxicity in non- target insects, and environmental contamination. In this regard, it was decided that the international production and use of such highly-toxic synthetic organic pesticides should be reduced for the health of humans. International agreements stipulate that the use of organic pesticides particularly affecting humans and livestocks must be gradually reduced. It was also agreed in 2004 that the domestic use of synthesized organophosphorus and organochloride pesticides should be reduced to 50% of the total amount of such pesticides consumed ten years ago, and that the amount consumed should be reduced by 50% again by the year 2010. However, after the agreement for reducing the production of toxic pesticides, safe pesticides, acting using a novel mechanism have not been developed to date, despite the many worldwide efforts of researchers. Thus, if safe pesticides are not developed in the near future, serious problems will be encountered due to the shortage of insecticides domestically as well as in foreign countries, and the use of highly toxic pesticides will be approved again.

[4] Insecticides penetrate insects via the mouth, skin and spiracles. When insecticides arrive at their targets in insects, some of them are degraded to nontoxic forms, while others are activated, become more toxic and accumulate in organs or are excreted outside the body. When an insecticide is applied to insects, only some of the insecticide used displays its insecticidal activity in its target. Typically, since there are several factors preventing the insecticide from arriving at its target in the body of the insects, only a portion of the insecticide that is used arrives at its action site to then destroy the physiological and biochemical functions of the insects, eventually killing the insects. Therefore, when using or developing insecticides, their action sites and action mechanisms and the metabolism affecting their effective concentrations in the body of insects should be deeply considered.

[5] Currently available insecticides are classified by mode of action into neural transmission inhibitors, energy production inhibitors, insect growth regulators and sex- attraction pheromones. Insect growth regulators are subgrouped into juvenile hormone inhibitors and chitin synthesis inhibitors.

[6] The neural transmission inhibitors kill insects by abnormally stimulating, exciting or inhibiting the nervous system.

[7] A neuron, the fundamental unit constituting the nervous system, usually has one long thin fiber projecting from the cell body, called an axon. At the axon terminal, the axon makes contact with the dendrite of another neuron, thus forming a specialized structure called a "synapse". A nerve impulse propagates along an axon. When the nerve impulse reaches the axon terminal, a neurotransmitter, acetylcholine (hereinafter, referred to as "ACh") is immediately released from the synaptic vesicles into the synapse between presynaptic and postsynaptic membranes. The released ACh binds to its receptor in the postsynaptic membrane, resulting in stimulation of the postsynaptic neuron. In this way, a nerve impulse is transmitted from one neuron to another neuron.

[8] Immediately after transmitting the nerve impulse from the presynaptic membrane to the postsynaptic membrane, the ACh released from the synaptic vesicles is hydrolyzed by acetylcholinesterase (hereinafter, referred to as "AChE"), which is released from the postsynaptic membrane. AChE has two kinds of activity: one is responsible for degradation of negatively charged ions and esters, and the other is to hydrolyze ACh.

[9] When ACh is accumulated at the postsynaptic membrane in a state of binding to its receptor after transmission of the nerve impulse to the postsynaptic neuron, hyper excitability and convulsions can be caused. Thus, ACh is converted to choline and acetic acid by the action of AChE. The choline is taken into the presynaptic membrane for reuse, and is converted to ACh in the synaptic vesicles.

[10] For the above reasons, when insecticides inhibiting the activity of AchE, acting to degrade ACh, which are mainly organophosphates and carbamates, are used for controlling insects, ACh accumulates in the synapse, and nerve impulse transmission becomes abnormal, thereby causing convulsions, paralysis and eventually death. The organophosphate and carbamate insecticides have been known to inhibit ACh degradation mainly by acting on the active site of AChE.

[11] Many researchers have studied the physiology of insects, especially metabolism- associated enzymes or receptors, using molecular biology techniques. However, hormone transport and sterol storage were rarely studied.

[12] Because insects are unable to synthesize sterols, they require sterols as essential nutrients. Most insects use plant sterols by converting them into cholesterol. Cholesterol is required for the biosynthesis of molting hormones, as well as participating in the formation of the plasma membrane together with phospholipids.

[13] Acyl CoA:cholesterol acyltransferase inhibitors are known to have the effects of preventing and treating hypertension in humans. In particular, they are under investigation as a therapeutic agent for hypertension, which has a new action mechanism related to the mechanism of the onset of arteriosclerosis. Acyl CoAicholesterol acyltransferase, which catalyzes the acylation of cholesterol, participates in the absorption of cholesterol in the small intestine, the synthesis of VLDLs (very low density lipoproteins) in the liver and the accumulation of cholesterol in an acylated form in adipose tissue and the blood vessel walls. Also, Acyl CoAxholesterol acyltransferase is known to be involved in the progress of arteriosclerosis, and is used as a target for the development of hypertension therapeutic agents, with a new action mode. Representative examples of the acyl CoA: cholesterol acyltransferase inhibitors include chemically synthesized urea, amides and phenols. Among them, some drug candidates that passed in vivo activity tests are in preclinical trials for use as therapeutic agents for arteriosclerosis. However, to date, there has been no report regarding the clinical appl ication of the acyl CoAxholesterol acyltransferase inhibitors.

[14] In this regard, based on the concept that, because insects essentially require sterols for their growth, insects can be killed by inhibiting a sterol-acylating enzyme, which participates in the storage and transport of sterols during sterol metabolization, the present inventors intended to synthesize materials having inhibitory activity toward acyl CoA:cholesterol acyltransferase in order to develop safe insecticides.

[15]

Disclosure of Invention Technical Problem

[16] In the present invention, novel active materials were synthesized through chemical synthesis utilizing a sterol-acylating enzyme, which has been known to play an important role in the production of sterols for storage or various hormones in insect sterol metabolism, as a novel concept for target- specific screening. The synthesized materials were evaluated for inhibitory activity toward the enzyme using the assay system of the present invention. The synthesized materials, identified to have inhibitory activity toward the enzyme, were found to have bioactivity in various kinds of insect larvae, thereby leading to the present invention.

[17] Accordingly, the present invention aims to provide novel pyripyropene derivatives or salts thereof having inhibitory activity toward acyl CoAxholesterol acyltransferase, and insecticidal compositions comprising the same. Brief Description of the Drawings [18] FIG. 1 is a H-NMR spectrum of a compound represented by Chemical Formula 1 according to the present invention. [19] FIG. 2 is a H-NMR spectrum of a compound represented by Chemical Formula 2 according to the present invention. [20] FIG. 3 is a ¹H-NMR spectrum of a compound represented by Chemical Formula 3 according to the present invention. [21] FIG. 4 is a graph showing the acyl CoA: cholesterol acyltransferase inhibitory activity of compounds represented by Chemical Formulas 1 to 3 according to the present invention.

[22]

Best Mode for Carrying Out the Invention

[23] In one aspect, the present invention relates to pyripyropene derivatives, represented by Chemical Formulas 1 to 3, below, or salts thereof.

[24] [25] [Chemical Formula 1] [26]

MsO^'

[27] [Chemical Formula 2] [28]

./

MsO

[29] [Chemical Formula 3] [30]

/

OMs

[31] [32] The compounds of Chemical Formulas 1 to 3 are pyripyropene derivatives having higher inhibitory activity toward acyl CoAxholesterol acyltransferase than pyripyropene A, which has been known as an inhibitor of acyl CoAxholesterol acyltransferase.

[33] The compounds represented by Chemical Formulas 1 to 3 may be in the form of salts thereof. The salts may include agro chemically acceptable salts with an inorganic acid such as hydrochloric acid or sulfuric acid, or an organic acid such as p- toluenesulfonic acid.

[34] In one detailed aspect, the compounds of Chemical Formulas 1 to 3 may be prepared according to the method described in Reaction 1 , below.

[35] However, the method is an exemplary method of preparing the pyripyropene derivatives of Chemical Formulas 1 to 3, and reaction conditions, including a reaction solvent, a base and amounts of reaction materials, are not limited to the following description. The pyripyropene derivatives of Chemical Formulas 1 to 3 may be prepared by a certain combination of several synthesis methods described in the literature in the art, as well as by the method of Reaction 1.

[36]

[37] [Reaction 1]

[38]

pyripyropene A

MsCI, TEA DMAP, CH₂CI₂

MsO'^" 5a (Ms = SO₂Ms)

MsCI TEA MsCI, TEA [WAP₁ CH₂CI₂ DMAP CH₂CI₂

[39] [40] After each reaction or all reactions are completed, the products may be isolated and purified using a common post-processing method, for example, chromatography or re- crystallization.

[41] In another aspect, the present invention relates to insecticidal compositions comprising the pyripyropene derivatives of Chemical Formulas 1 to 3 or salts thereof. [42] In detail, the insecticidal composition of the present invention has been obtained based on the fact that insects essentially require sterols for their growth, and essentially utilize a sterol-acylating enzyme participating in the storage and transport of sterols and in the activation and destruction of hormones. In Test Examples, to be described later, the present compounds were found to have insecticidal activity by inhibiting acyl CoAxholesterol acyltransferase, which participates in the storage and transport of sterols during sterol metabolization.

[43] The insecticidal composition of the present invention may have the effects of controlling pests including, but not limited to, harmful arthropods (e.g., harmful insects and harmful mites) and harmful nematodes. In particular, the composition is effective on larvae of pests. Also, the insecticidal composition may be used for effectively controlling pests having enhanced resistance to conventional insecticides. In the practice of the present invention, when the insecticidal composition comprising the pyripyropene derivatives of Chemical Formulas 1 to 3 was applied to greenhouse whitefly larvae and Plutella xylostella L. larvae, they displayed sustained insecticidal effects in a dose-dependent manner over time.

[44] When used as an effective ingredient of an insecticide, the present compounds may be used in their original forms or of as salts thereof, with no addition of other certain ingredients. However, the present compounds are typically mixed with solid carriers, liquid carriers, gaseous carriers or bait, or are absorbed into base materials, for example, porous ceramic plates or non woven fabrics, added with surfactants and, if desired, other additives, and then formulated into a variety of forms, for example, oil sprays, emulsified concentrates, wettable powders, well-flowing granules, dusts, aerosols, fuming preparations such as fogs, evaporable preparations, combustible preparations, poisonous baits, and sheet or resin preparations for controlling mites.

[45] Each of the above formulations may contain one or more of the present compounds as effective ingredients in an amount of 0.01% to 95% by weight.

[46] The solid carriers usable in the formulations may include fine powders or granules of clays (e.g., kaolin clay, diatomaceous earth, synthetic hydrated silicon oxide, bentonite, fubasami clay and acid clay), talcs, ceramics, other inorganic minerals (e.g., silicate, quartz, sulfur, active carbon, calcium carbonate and hydrated silica), and chemical fertilizers (e.g., ammonium sulfate, ammonium phosphate, ammonium nitrate, urea and ammonium chloride).

[47] The liquid carriers may include water, alcohols (e.g., methanol, ethanol, etc.), ketones (e.g., acetone and methyl ethyl ketone), aromatic hydrocarbons (e.g., benzene, toluene, xylene, ethylbenzene and methylnaphthalene), aliphatic hydrocarbons (e.g., hexane, cyclohexane, kerosene and light oil), esters (e.g., ethyl acetate and butyl acetate), nitriles (e.g., acetonitrile and isobutyronitrile), ethers (e.g., diisopropyl ether and dioxane), acid amides (e.g., N,N-dimethylformamide and N,N-dimethylacetamide), halogenated hydrocarbons (e.g., dichloromethane, trichloroethane and carbon tetrachloride), dimethyl sulfoxide, and vegetable oils (e.g., soybean oil and cottonseed oil).

[48] The gas carriers or propellants may include Freon gas, butane gas, LPG (liquefied petroleum gas), dimethyl ether and carbon dioxide gas.

[49] The base materials for the poisonous baits may include bait components (e.g., grain flour, vegetable oils, sugar, and crystalline cellulose), antioxidants (e.g., dibutylhy- droxytoluene and nordihydroguaiaretic acid), preservatives (e.g., dehydroacetic acid), agents for preventing children from eating poisonous baits by mistake (e.g., red pepper powders), and attractive flavors (e.g. cheese flavor or onion flavor).

[50] Examples of the surfactants may include alkyl sulfates, alkylsulfonates, alkylaryl- sulfonates, alkylaryl ethers and poly oxyethylenated derivatives thereof, poly ethylenegly col ethers, polyvalent alcohol esters and sugar alcohol derivatives.

[51] Examples of the other auxiliaries, such as adhesive agents and dispersants, include casein; gelatin; polysaccharides such as starch, gum Arabic, cellulose derivatives and alginic acid; lignin derivatives; bentonite; saccharides; and synthetic water-soluble polymers such as polyvinyl alcohol, polyvinylpyrrolidone and polyacrylic acids.

[52] Examples of stabilizers may include PAP (isopropyl acid phosphate), BHT

(2,6-di-tert-butyl-4-methylphenol), BHA (mixture of 2-tert-butyl-4-methoxyphenol and 3-tert-butyl-4-methoxyphenol), vegetable oils, mineral oils, surfactants, fatty acids and fatty acid esters.

[53] When the present compounds are used as an agricultural pesticide, acarid killer or nematocide, they are applied in an amount of usually 0.1 g to 100 g over an area of 10 acres. In the case that preparations such as emulsified concentrates, wettable powders or well- flowing granules are used after being diluted with water, the application concentration thereof is usually 1 ppm to 1,000 ppm. The granules, dusts and the like are applied without dilution. When the present compounds are used as a pesticide, acarid killer or nematocide for the prevention of epidemics, the emulsified concentrates, wettable powders, well-flowing granules and other formulations are applied after being diluted to 0.1 to 500 ppm with water, but the oil sprays, aerosols, fuming preparations, poisonous baits, acarid-proof sheets and the like are applied in their original form. The application amounts and concentrations may vary depending on the type of formulations, the time, site and method of application, the type of pests, damage, and other factors, and can be increased or decreased, rather than being limited to the above range.

[54] When the present compounds are used as a pesticide or acaricide for controlling ectoparasites of animals, including livestock, such as cattle and pigs, and pets such as cats and dogs, the compounds or salts thereof are used in the veterinary field in a known manner for systemic pest control, for example, by administration in the form of, for example, tablets, capsules, drenches, boli, the feed-through process or suppositories, by injections, or by administration, for example, in the form of spraying of oily or aqueous solutions, pouring-on or spotting-on; or for non-systemic pest control with the aid of molded articles such as collars, ear tags, and the like. In these cases, the present compounds are applied in an amount of 0.01 to 100 mg per kg body weight of host animals.

[55] The present compounds may be used as a mixture or individually but simultaneously with other insecticides, nematocides, acaricides, bacteriocides, fungicides, herbicides, plant growth regulators, synergists, fertilizers, soil conditioners and/or animal feeds.

[56]

Mode for the Invention

[57] A better understanding of the present invention may be obtained through the following examples, which are set forth to illustrate, but are not to be construed as the limit of the present invention.

[58]

[59] EXAMPLE 1 : Preparation of pyripyropene A

[60]

[61] The pyripyropene A, as the starting material for chemical synthesis in the present invention, was prepared using a pyripyropene A-producing strain isolated from soil collected from Ulsan, Gyeongsangbuk-do, Korea. The isolate was identified as Penicillium griseofulvum through mycological studies, and designated as "penicillium griseofulvum F1959". The isolated P. griseofulvum F1959 was deposited with KCTC (Korean Collection for Type Cultures) under KRIBB, Korea and assigned accession number of KCTC 0387BP.

[62] A frozen stock (10% glycerol, -8O⁰C) of the isolated fungus was inoculated in a 1-L baffled Erlenmeyer flask containing 100 ml of a seed culture medium: 0.5% glucose, 0.2% yeast extract, 0.5% polypeptone, 0.1% K HPO , 0.05% MgSO H O (sterilized after being adjusted to pH 5.8), and cultured with agitation at 29⁰C for 18 hrs. 20 ml of the seed culture was inoculated in a 5-L baffled Erlenmeyer flask containing 1 L of an active material production medium: 2% soluble starch, 0.4% soytone, 0.3% Pharmamedia, 0.1% K HPO , 0.05% MgSO H O, 0.3% CaCO , 0.2% NaCl (sterilized after being adjusted to pH 5.8), and was cultured at 26⁰C for 120 hrs in a rotary shaker.

[63] After the culture was completed, the fermentation fluid was extracted with an equal volume of ethyl acetate (EtOAc) with shaking and concentrated under pressure, thereby yielding an oil-phased brown extract. [64] The extract thus obtained was subjected to silica gel (Merck, 9385) column chromatography (chloroform:methanol=99:l, 98:2, 97:3, 95:5, 90: 10 V/V %, 4 volumes compared to silica gel). The collected fractions were then analyzed for distribution of materials by thin layer chromatography. The fractions containing the same materials were combined and assayed for in vitro inhibitory activity against acyl CoAxholesterol acyltransferase. The fractions with inhibitory activity were combined and eluted with chloroform/methanol (95:5 to 90: 10, v/v%). The organic solvent phase was concentrated under pressure, thereby yielding a yellowish brown oil-phased extract.

[65] The yellowish brown extract thus obtained was subjected to high-performance liquid chromatography (HPLC) in order to isolate the active starting material of the present invention, pyripyropene A. In brief, the HPLC was carried out using an YMC- ODS column (20x250 mm), and the pyripyropene A was detected at 322 nm using an UV detector.

[66] The active material pyripyropene A (Formula 1) was eluted with a solvent of ace- tonitrile and water (45:55, v/v%) at 11 min.

[67] The active fraction was concentrated under pressure and purified once more, thereby yielding a colorless crystal, pyripyropene A. The starting material for chemical synthesis was obtained at a yield of 13 mg per L of fermenting medium in 120 hr.

[68] [69] EXAMPLE 2: Synthesis of pyripyropene derivatives [70] [71]

pyripyropene A

[72] [73] NaOMe (50%, 110 ml, 8eq.) was added to a MeOH solution (5 ml) of pyripyropene A (75 mg, 0.129 mmol) and stirred at room temperature for 12 hrs. The completion of the reaction was confirmed by thin layer chromatography (TLC). After all molecules of pyripyropene A were determined to have been converted into the compound 1 , the reaction solution was supplemented with an aqueous solution of NH Cl (1 ml) and con-

4 centrated under pressure. The residue was diluted with excessive EtOAc and filtered, thereby obtaining the Compound 1 (tetraol) as a yellow solid (54 mg, 92% yield).

[74] [75]

[76] [77] Dimethoxypropane (28 ml, 10 eq) and PPTS (5.5 mg, 1 eq) were added to a DMF solution (1 ml) of the tetraol (Compound 1) (10 mg, 0.022 mmol), and incubated with stirring for 20 hrs at room temperature. The reaction mixture was concentrated under pressure to remove DMF and subjected to column chromatography (EtOAc:MeOH = 12:1), thereby obtaining acetonide (Compound 2) as a solid (11 mg, 100% yield).

[78] [79]

[80] [81] The acetonide (Compound 2; 8.5 mg, 0.017 mmol) was mixed with Et N (23 ml, 10 eq.) and a CH Cl solution (1 ml) of 4-DMAP (2 mg), cooled in an ice water bath, supplemented with valeryl chloride (4.2 mg, 2 eq.), and stirred for 3 hrs. The reaction mixture was diluted with EtOAc (10 ml), washed with water and brine, dried over MgSO , filtered, and concentrated under pressure. The residue was purified by silica gel column chromatography (EtOAc:Hex = 2: 1), and the filtrate was dried, thereby obtaining Compound 3a as a solid (9.3 mg, 94% yield).

[82] [83]

[84] [85] Also, Compound 3b (92% yield) was obtained using the same method as described above.

[86] [87]

[88] [89] The Compound 3a (12 mg, 0.0206 mmol) was mixed with 60% AcOH (1 ml) and stirred for 20 hrs at room temperature. After the reaction was completed, the reaction mixture was mixed with a NaHCO aqueous solution to neutralize AcOH, and con-

3 centrated under pressure. The concentrate was diluted with EtOAc, washed with water and brine, dried over MgSO , filtered, and concentrated under pressure. The residue

4 was purified by column chromatography (EtOAc: MeOH = 12:1), thereby obtaining a triol compound (Compound 4a) as a solid (10.5 mg, 95% yield).

[90] [91]

[92] [93] Also, Compound 4b (93% yield) was obtained using the same method as described above.

[94] [95]

[96] [97] The triol (Compound 4a; 13 mg, 0.024 mmol) was mixed with Et N (33 ml, 10 eq.) and with a solution of pyridine/CH Cl (1/2, 1 ml) and 4-DMAP (2 mg), supplemented with MsCl (4.2 mg, 1.5 eq.) at O⁰C, and stirred for 4 hrs. The reaction mixture was concentrated under pressure and purified by PTLC (EtOAc:Hex:MeOH = 20:20: 1), thereby obtaining mesylate (Compound 5a) as a solid (6.7 mg, 45% yield).

[98] [99]

[100]

[101] Also, Compound 5b (51% yield) was obtained using the same method as described above. [102] [103]

5a For mu l a 1 Formu l a 3

[104] [105] The mesylate (Compound 5a; 6.6 mg, 0.011 mmol) was mixed with Et N (16 ml, 10 eq.) and a CH Cl (1 ml) solution of 4-DMAP (2 mg), supplemented with Ac O (1.6 mg, 1.5 eq.) at O⁰C, and stirred for 4 hrs. The reaction mixture was concentrated under pressure and purified by column chromatography (EtOAc:Hex:MeOH = 20:20:1), thereby obtaining the compound of Chemical Formula 1 (6.0 mg, 85% yield) and the compound of Chemical Formula 3 (0.9 mg, 15% yield) as a solid.

[106] [107]

[108] [109] Also, the compound of Chemical Formula 2 (89% yield) was obtained using the same method as described above.

[110] [111] TEST EXAMPLE 1: Evaluation of inhibitory effects of the synthesized compounds on acyl CoA:cholesterol acyltransferase activity

[112] [113] The synthesized compounds were evaluated for inhibitory effects on acyl CoAxholesterol acyltransferase (hereinafter, referred to simply as "ACAT") through a slightly modified version of the Brecher method (Brecher. P and C. Chen, Biochimica Biophysica Acat 617:458-471, 1980). In the method, ACAT activity was determined using microsomes partially purified from the liver as a source of ACAT with substrates of cholesterol, and radiolabeled oleoyl-CoA. The radioactivity of the reaction product cholesterol ester was measured as the ACAT activity. In detail, cholesterol and Triton WR- 1339, both dissolved in acetone, were suspended in water. After acetone was removed under a stream of nitrogen gas, the suspension was supplemented with potassium-phosphate buffer (pH 7.4, final cone: 0.1 M). Bovine serum albumin was added to the mixture at a final concentration of 30 μM in order to stabilize the enzyme reaction. A sample dissolved in DMSO or methanol was added to the mixture in a predetermined amount, and was pre-incubated at 37 ⁰C for 30 min. The enzyme reaction was then initiated by adding [1- C]-oleoyl Coenzyme A to a final concentration of 0.04 μCi, followed by incubation for 30 min at 37⁰C. 1 ml of isopropanol-heptane was added to the reaction mixture in order to terminate the reaction. Then, 0.6 ml of n - heptane and 0.4 ml of KPB buffer were added to the reaction mixture. The resulting mixture was well mixed and allowed to stand under gravity for 2 min. After the resulting mixture was phase-separated, 200 μl of the supernatant was placed into a scintillation vial. 4 ml of scintillation cocktail (Lipoluma, Lumac Co.) was added to the vial, and the amount of produced cholesteryl oleate was measured using a scintillation counter (Packard Delta-200). The inhibitory activity toward ACAT was calculated according to Equation 1 , below.

[114]

[115] [Equation 1]

[116] Inhibitory activity (%) = [l-(T-B/C-B)]xlOO

[117] (T: cpm in a test reaction mixture that contains a sample (a compound of the present invention) along with an enzyme source

[118] C: cpm in a control reaction mixture that does not contain a sample but contains the enzyme source

[119] B: cpm in another control reaction mixture that contains a sample but does not contain the enzyme source)

[120]

[121] As a result, pyripyropene A was found to have an IC (the concentration of a compound that is required to inhibit 50% of ACAT activity) of 35 ng/ml, and the IC value was calculated as 60 nM because the compound has a molecular weight of 583.

[122] The synthesized pyripyropene A derivatives of Chemical Formulas 1 to 3 displayed

IC values of 4.9 ng/ml, 5.3 ng/ml and 7.8 ng/ml, respectively (FIG. 4). The IC values were calculated as 7.4 nM, 11.2 nM and 8.2 nM, respectively, because the compounds have molecular weights of 661, 697 and 647, respectively. The ACAT inhibitory effects of the pyripyropene A derivatives of Chemical Formulas 1 to 3, prepared by structurally modifying pyripyropene A through chemical synthesis, were 8.1, 5.4 and 7.3 times, respectively, higher than pyripyropene A.

[123]

[124] TEST EXAMPLE 2: Evaluation of the toxicity of the synthesized compounds against greenhouse whitefly larvae

[125]

[126] The synthesized compounds were tested for their insecticidal effects on greenhouse whitefly larvae (Trialeurodes vaporariorum). This test was conducted on May 2006 in the Agribiology Department, Agribiological Environment College, Chungbuk National University, Cheongju city, Chung-cheong-buk-do. After the pyripyropene A derivatives, identified as having ACAT inhibitory activity, were weighed accurately, a proper amount of each compound was dissolved in acetone, mixed with nine volumes of a 100 ppm Triton X-100 solution, and serially diluted in order to obtain an active compound solution.

[127] Tomato leaves containing eggs and nymphs of greenhouse whitefly were dipped in the active compound solution for 30 sec, and were dried in the shade. The rate of egg hatching and mortality of whitefly nymphs were recorded every day for a period of nine days, and this test was performed on two replicates.

[128] The active compound-treated leaf was placed onto a petri dish (55x20 mm) with a filter paper wet with distilled water. Then, the larvae treated with the active compound were grown in an incubator (25+1⁰C, 40-45% relative humidity, 16L:8D), and the mortality was recorded for a period of nine days. A control group was grown on leaf disks not treated with the present active compound but treated merely with a mixture of 10% acetone and nine volumes of a 100 ppm Trixton X-100 solution. This leaf-disk bioassay was repeated three times, and LC (50% lethal concentration) was calculated using the Probit method (Finney, 1982).

[129] The insecticidal effects of the pyripyropene A derivatives of Chemical Formulas 1 to 3 on the whitefly are given in Table 1, below.

[130] [131] TABLE 1 [132]

[133]

[134] As shown in Table 1, when the pyripyropene A derivatives of Chemical Formulas 1 to 3 were applied to greenhouse whitefly at concentrations of 1, 10 and 100 ppm and the mortality was investigated at intervals of one day, they exhibited sustained insecticidal effects over time in a dose-dependent manner compared to the control. [135] The pyripyropene A derivatives of Chemical Formula 1 showed the highest in vitro

ACAT inhibitory activity, followed by the compounds of Formula 2 and then the compound of Formula 3. The in vivo insecticidal activity was high in the order of the compounds of Formula 2 > Formula 1 > Formula 3.

[136]

[137] TEST EXAMPLE 3: Evaluation of the toxicity of the synthesized compounds against Plutella xylostella L. larvae

[138]

[139] The synthesized compounds were evaluated for insecticidal effects on larvae of

Plutella xylostella L. A leaf-disk bioassay was conducted on May 2006 in the Agribiology Department, Agribiological Environment College, Chungbuk National University, Cheongju city, Chung-cheong-buk-do. After the pyripyropene A derivatives, identified as having ACAT inhibitory activity, were weighed accurately, a proper amount of each compound was dissolved in acetone, mixed with nine volumes of a 100 ppm Triton X-100 solution and serially diluted in order to obtain an active compound solution. Leaves of uniformly grown cabbages, as feed for the larvae, were cut into leaf disks (3.0 cm in diameter), dipped in the active compound solution for 30 sec, and dried in a hood for 60 min. Each of the active compound-treated leaf disks was placed onto a petri dish (55x20 mm) with a wet filter paper. Then, ten P. xylostella larvae in the second instar were placed on each leaf disc using a soft brush, taking caution not to damage the larvae. Each sample was prepared in triplicate. The larvae were grown in an incubator (25+1⁰C, 40-45% relative humidity, 16L:8D), and mortality was recorded after 24 and 48 hrs. A control group was grown on leaf disks not treated with the present compound but treated merely with a mixture of 10% acetone and nine volumes of a 100 ppm Trixton X-100 solution. This leaf-disk bioassay was repeated three times, and LC (50% lethal concentration) was calculated using the Probit method (Finney, 1982).

[140] The compound of Chemical Formula 1 was applied in an amount of 0.1 to 10 ppm to P. xylostella larvae, and mortality was recorded with intervals of 24 hrs. The results are given in Table 2, below. The compound of Chemical Formula 1 was found to have a sustained insecticidal effect on Plutella xylostella L. in a dose-dependent manner.

[141]

[142] TABLE 2

[143]

[144]

Industrial Applicability

[145] As described hereinbefore, the present invention relates to an insecticidal composition comprising, as effective ingredients, compounds that were structurally modified through chemical synthesis using a starting material extracted from Penicillium griseofulvum F1959 and thus have higher ACAT inhibitory activity. The present compounds have enhanced ACAT inhibitory activity and have stronger insecticidal effects by inhibiting the sterol metabolism of insect larvae. Therefore, the present compounds may be used as safe insecticides with more effective insecticidal activity than conventionally known pyripyropene A.

Claims

[1] A compound selected from the group consisting of compounds of Chemical Formulas 1 to 3, below, or a salt thereof. [Chemical Formula 1]

MsO^'

[Chemical Formula 2]

MsO

[Chemical Formula 3]

OMs'

[2] An insecticidal composition comprising the compound or the salt thereof of claim 1 as an effective ingredient. [3] The composition as set forth in claim 2, wherein the compound or the salt thereof is present in an amount of 0.01% to 95% by weight based on the total weight of the composition.

[4] The composition as set forth in claim 2, which is effective on arthropods or nematodes. [5] The composition as set forth in claim 4, which is effective on larvae of arthropods or nematodes. [6] The composition as set forth in claim 2, which comprises a commonly used carrier.