WO2008018645A1

WO2008018645A1 - Pesticides

Info

Publication number: WO2008018645A1
Application number: PCT/KR2006/003139
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: 2006-08-10
Filing date: 2006-08-10
Publication date: 2008-02-14

Abstract

Disclosed herein are novel compounds and salts thereof having inhibitory activity against the biosynthesis of triglycerides, pesticidal compositions comprising the same as active ingredients, and a method for killing insects using the same. Functioning to inhibit the activity of diacyl CoA: glycerol acyltransferase, the novel compounds deny insects triglycerides, which are essential for their growth, thereby having a potent pesticidal effect. The compounds are safe for humans and may be used as environment- friendly pesticides.

Description

PESTICIDES

Technical Field

[1] The present invention relates to novel derivatives having inhibitory activity against the biosynthesis of triglycerides, pesticidal compositions comprising the same as an active ingredient, and pesticidal methods using the same.

[2]

Background Art

[3] Agricultural chemicals have been used around the world to protect crops from diseases and harmful insects and thus to improve the production yield of crops. However, the continuous use and abuse of agricultural chemicals for several decades has resulted in many adverse effects, including residual toxicity, environmental contamination, etc., on wildlife and ecosystems. In this regard, international conferences have been held with the aim of restricting the use of highly-toxic synthetic organic chemicals to ensure the health of humans. International agreements stipulate that the use of synthetic organic chemicals, particularly those affecting humans and livestock, must be gradually reduced. It was also agreed in 2004 that the production and the domestic use of synthesized organophosphorus and organochloride pesticides should be reduced to 50% of the total amount of such pesticides consumed a decade ago, and that the amount consumed should be reduced by 50% again by the year 2010. Despite the many worldwide efforts of researchers, however, safe pesticides having activity based on novel mechanisms have not yet been developed. 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.

[4]

[5] Insecticides are introduced into insects via various routes, including the mouth, skin and spiracles. When insecticides arrive at their targets in insects, some of them are degraded into 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, not all of the insecticide that is used exerts its insecticidal activity on its target.

[6]

[7] Various barriers which the applied insecticide encounters while being introduced into the body of the insects allow only a portion of the insecticide to arrive at its action site to then interrupt physiological and biochemical functions of the insects, eventually killing the insects. When using or developing insecticides, therefore, the sites and mechanisms of action and the metabolism thereof, affecting their effective concentrations in the bodies of insects, should be carefully considered.

[8]

[9] Insecticides currently used around the world are classified according to their 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. And there are sterol metabolism inhibitors, which are developed by the present inventors. Most of the currently used insecticides target the nervous system, which plays a critical role in sustaining life, or enzymes involved in energy production.

[10]

[11] Since insects can be killed suddenly when their nervous systems are aberrantly stimulated, excited, or suppressed, neural transmission inhibitors display toxicity targeting the nervous system.

[12]

[13] A neuron, the fundamental unit of the nervous system, usually has one long thin fiber, called an axon, projecting from the cell body. 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. Upon the arrival of the nerve impulse at the axon terminal, a neurotransmitter, acetylcholine, is immediately released from the synaptic vesicles into the synapse between pre-synaptic and postsynaptic membranes. The released acetylcholine 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.

[14]

[15] Immediately after transmitting the nerve impulse from the pre-synaptic membrane to the postsynaptic membrane, the acetylcholine released from the synaptic vesicles is hydrolyzed by acetylcholin esterase, which is released from the postsynaptic membrane. Acetylcholin esterase has two functions: one is to degrade negatively charged ions and esters, and the other is to hydrolyze acetylcholine.

[16]

[17] The accumulation of acetylcholine at the postsynaptic membrane in a state of binding to its receptor after transmission of the nerve impulse to the postsynaptic neuron may cause hyper excitability and convulsions. Thus, acetylcholine is converted to choline and acetic acid by the action of acetylcholin esterase. The choline is taken into the pre-synaptic membrane for re-use, and is converted to acetylcholine in the synaptic vesicles.

[18] [19] For the above reasons, when insecticides inhibiting the activity of acetylcholin esterase, responsible for the degradation of the neurotransmitter acetylcholine, which are mainly organophosphates and carbamates, are applied to insects, acetylcholine accumulates in the synapse to induce abnormal nerve impulse transmission, resulting in convulsions, paralysis and eventually death. Organophosphate and carbamate insecticides have been known to inhibit acetylcholine degradation mainly by acting on the active site of acetylcholin esterase. These chemicals penetrate relatively rapidly into the skin of insects and attach to the surface of the nervous system to cause abnormal nerve functioning, which is expressed as hypersensitivity, severe convulsions, paralysis and finally death after a latency period.

[20]

[21] Among insecticides are insect growth regulators, which inhibit the construction of the exoskeleton and the biosynthesis of chitin in insects. Insects have to molt to accommodate their gradual growth, during which the biosynthesis of the exoskeleton is very important to the physiological functions of the insects. The insect exoskeleton is a multi-layered structure with four functional regions. The exoskeleton is roughly divided into the basal membrane and the epidermis, which is further divisible into the epicuticle and procuticle. The procuticle of insects is based on a polymer (chitin), consisting of units of N-acetyl glucosamine. Because this polymer does not exist in vertebrates, but is a main component of insect's exoskeleton, insects can be killed when the biosynthesis of chitin is inhibited.

[22]

[23] With a mechanism different from that of neural transmission inhibitors, chitin synthesis inhibitors, when introduced into the insect body via the mouth or stigmas, cause incomplete exoskeleton formation, incapacitating the insects from molting normally. In this regard, the chitin synthesis inhibitors have no influence on the form ation of the epicuticle, but inhibit the synthesis of chitin in the procuticle layer. The chitin synthesis inhibitors, although their precise mechanisms have to be proven, are known to have inhibitory activity against enzymes responsible for the polymerization of UDP-N-acetyl glucosamine.

[24]

[25] Besides, insecticides having inhibitory activity against functions unique to insects, such as juvenile hormone inhibitors or mating disruptors are also being developed. Sex-attraction pheromones secreted from female insects are studied to capture male insects, but are not yet commercially applicable as they have failed in field tests.

[26]

[27] Many researchers have studied the physiology of insects, especially metabolism- associated enzymes or receptors, using molecular biological techniques, in an effort to develop pesticides. However, little study has been made of hormone transportation or lipid storage in insects. Because insects are unable to synthesize lipids, they require sterols as essential nutrients. Most insects convert plant lipids into suitable lipids necessary therefor.

[28]

[29] In this regard, based on the fact that most insects require lipids as essential nutrients for their growth because of lack of de novo synthesis, decompose ingested lipids into metabolites through various metabolisms, and utilize ingested lipids as energy sources, the present inventors intended to synthesize materials having inhibitory activity toward enzymes involved in lipid metabolism in order to develop safe insecticides.

[30]

Disclosure of Invention Technical Problem

[31] In the present invention, novel active materials were synthesized through chemical synthesis utilizing a glycerol acylation enzyme, which has been known to play an important role in the production of lipids for storage in insect lipid 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.

[32]

[33] Accordingly, it is an object of the present invention to provide a pesticidal composition comprising a compound having inhibitory activity against the biosynthesis of triglycerides in insects.

[34]

[35] It is another object of the present invention to provide a method for killing insects using the pesticidal composition to inhibit the biosynthesis of triglycerides within insects.

[36]

[37] It is a further object of the present invention to provide a compound having inhibitory activity against the biosynthesis of TGA in insects, particularly against diacylCoA: glycerol acyltransferase.

[38]

[39] It is still a further object of the present invention to provide a method for inhibiting the biosynthesis of TGA in insects using this compound. Brief Description of the Drawings

[41] FIG. 1 is a graph showing the diacyl CoA:glycerol acyltransferase inhibitory activity of compounds according to the present invention. [42]

Best Mode for Carrying Out the Invention

[43] In one aspect, the present invention pertains to a pesticidal composition comprising a compound that inhibits the biosynthesis of triglycerides (TGAs) in insects.

[44] In insects, lipids show various functions. First, lipids are one of the most important energy sources. Of lipids, neutral lipids are mainly responsible for the role of energy sources and are accumulated in adipose cells. Also, lipids are used, in combination with phospholipids and sterols, as components of cell membranes. Besides, lipids are implicated in the production of molting hormones, juvenile hormones, and pheromones. However, insect lipids are almost water-insoluble, but are dissolved in organic solvents, and are classified into a chemical group the chemical properties of which are unknown. Lipids, consisting mainly of fatty acid esters or derivatives thereof, are known to be synthesized from nutrients absorbed into the mid-intestine, including fatty acids and glycerols, amino acids, or simple carbohydrates. Among the lipids of insects, triglycerides are the most prevalent, with hemolymph most abundant in diglycerides. For example, the adipose cells of male Saturniidae contain triglycerides to 95% by weight thereof with diglycerides, amounting to 65% by weight of hemolymph. In insects, fatty acids are, in general, provided from their diet, but some insects, especially insects which live on low-fat plants, can synthesize fatty acids in adipose cells. Since most insects require lipids as essential nutrients, lipid metabolism is very important therefor. Accordingly, based on the concept that the inhibition of triglyceride biosynthesis in insects by interrupting the lipid metabolism thereof brings about a pesticidal effect, the present invention provides a pesticidal composition comprising a compound that inhibits the biosynthesis of triglycerides.

[45] In insects, triglycerides are synthesized from sn-l,2-diacylglycerol and fatty acyl

CoA with the aid of diacyl CoA: glycerol acyltransferase.

[46] Diacyl CoA:glycerol acyltransferase catalyzes the final step of the glycerol

3-phosphate pathway, functioning to synthesize triglycerides by using sn- 1,2-diacylglycerol and fatty acyl CoA as substrates. In greater detail, ingested fats are decomposed into fatty acids and monoglycerides by lipase secreted from the pancreas, and are absorbed into intestinal epithelial cells, in which the conversion into triglyceride by DGAT occurs. Generally, the biosynthesis of triglycerides uses a glycerol 3-phosphate pathway (in the liver and adipose tissues) and a monoa- cylglycerol pathway (in intestinal epithelial cells). [47] In an embodiment thereof, thus, the present invention pertains to the inhibition of triglyceride biosynthesis in insects with an inhibitor against glycerol acylation enzymes, especially diacyl CoA: glycerol acyltransferase (DGAT), thereby killing harmful insects.

[48] The term inhibition of triglyceride biosynthesis, as used herein, is intended to refer to the interruption of triglyceride synthesis in insects or to a decrease in the efficiency of triglyceride biosynthesis. Especially, in the preferred example, by inhibiting related enzymes activity.

[49] The term "inhibition of diacyl CoA: glycerol acyltransferase (DGAT)", as used herein, is intended to refer to interrupting the enzymatic reaction of triglyceride synthesis or decreasing the efficiency of triglyceride biosynthesis. The inhibition of the enzymatic activity induces various cellular reactions in insect cells. For example, when DGAT activity is inhibited, molting hormone or juvenile hormone is not produced, thus leading to the inhibition of insect growth and finally to the death of the insects.

[50] Accordingly, in this invention, a mechanism involved in the storage or transportation of lipids within insects, especially a change in biological activity upon the inhibition of triglyceride (TGA) biosynthesis in insects, is examined and used for the killing of insects.

[51] In accordance with another embodiment thereof, the present invention pertains to a compound having inhibitory activity against triglyceride biosynthesis, and more particularly, to a compound, represented by the following Chemical Formula 1, which inhibits the activity of diacyl CoA: glycerol acyltransferase.

[52] [Chemical Formula 1]

[53]

[54] Wherein R is C ~C alkyl or alkenyl.

[55]

[56] In the practice of the present invention, even a low concentration of the compound of Chemical Formula 1 was found to inhibit DGAT activity. Also, the compound of Chemical Formula 1 displayed sustained insecticidal effects on greenhouse whitefly larvae and Plutella xylostella L. larvae in a dose-dependent manner over time. Thus, the compound of Chemical Formula 1 inhibits the activity of DGAT to block TGA biosynthesis, showing pesticidal effects.

[57] [58] In accordance with another embodiment thereof, the present invention pertains to a method for the inhibition of triglyceride biosynthesis within insects using the compound of Chemical Formula 1. As a way of inhibiting triglyceride biosynthesis, the inhibition of diacyl CoA: glycerol acyltransferase is effective. In this way, triglyceride, an essential ingredient for insects, can be lessened and depleted so as to kill insects.

[59] The compound of Chemical Formula 1 may be prepared as illustrated in the following Reaction Scheme 1.

[60] [61] [Reaction Scheme 1] [62]

chemical formula 1

Reagents and conditions, (a) NaOH, (CHs)₂SO₄, rt 2h; (b) NBS, rt, 3h (c) CH₃O Na, CuI, reflux 3Oh, (d) CAN, rt, "I h, (e) RSH, Na₂Cr₂O₇, H₂SO₄, rt 4h, (f) MCPBA, 0°C , 1 h.

[63] [64] The processes of Reaction Route 1 are elucidated as follows. [65] First, 1,5-dihydroxynaphthalene (1) is reacted with dimethyl sulfate to yield 1,5-dimethoxynaphthalene (2), which is in turn brominated in the presence of NBS (N-bromosuccinimide) to give 4,8-dibromo-l,5-dimethoxynaphtalene (3). This brominated compound (4,8-Dibromo-l,5-dimethoxynaphtalene) (3) is subjected to substitution reaction with sodium methoxide in the presence of copper(I) iodide to give 1,4,5,8-tetramethoxynaphthalene (4). Reaction between

1,4,5,8-tetramethoxynaphthalene (4) and cerium diammonium nitrate is followed by extraction with an organic solvent to yield 5,8-dimethoxy-l,4-naphthoquinone. After the reaction of 5,8-dimethoxy-l,4-naphthoquinone with alkyl mercaptan, sodium bicarbonate and sulfuric acid are droplet added, and then extraction with an organic solvent is carried out to produce the compound of Chemical Formula 1.

[66] [67] However, the method according to Reaction Scheme 1 is only one of many possible ways of preparing the compound of Chemical Formula 1. The reaction conditions, such as solvent, base, reactant amounts, etc., are not limited to those described above. In addition to the method of Reaction Scheme 1, various methods known to those who are skilled in the art can be used to synthesize the compound of Chemical Formula 1.

[68]

[69] For use as an effective ingredient of an insecticide, the compound of Chemical

Formula 1 in accordance with the present invention may be in its original form or as a salt thereof, with no addition of other ingredients. However, the compound of Chemical Formula 1 is typically mixed with solid carriers, liquid carriers, gaseous carriers or bait, or is absorbed into base materials, for example, porous ceramic plates or nonwoven 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, liquids, well-flowing granules, dusts, aerosols, fuming preparations such as fogs, evaporable preparations, combustible preparations, poisonous bait, and sheet or resin preparations for controlling mites.

[70]

[71] In each of the above formulations, the compound of the present invention may be present as an effective ingredient in an amount of 0.01% to 95% by weight.

[72]

[73] Examples of solid carriers usable in the formulations include fine powders or granules of clays (e.g., kaolin clay, diatomaceous earth, synthetic hydrated silicon oxide, bentonite, fubasami clay and acid clay), talc, 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).

[74]

[75] As for the liquid carriers, they may be exemplified by 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).

[76]

[77] Suitable as the gas carriers or propellants are Freon gas, butane gas, LPG (liquefied petroleum gas), dimethyl ether and carbon dioxide gas.

[78]

[79] For use as base materials for the poisonous bait, there are examples including bait components (e.g., grain flour, vegetable oils, sugar, and crystalline cellulose), antioxidants (e.g., dibutylhydroxytoluene and nordihydroguaiaretic acid), preservatives (e.g., dehydroacetic acid), agents for preventing children from eating poisonous bait by mistake (e.g., red pepper powders), and attractive flavors (e.g. cheese flavor or onion flavor).

[80] As the surfactants suitable for the formulations, alkyl sulfates, alkylsulfonates, alky- larylsulfonates, alkylaryl ethers and polyoxyethylenated derivatives thereof, poly ethylenegly col ethers, polyvalent alcohol esters and sugar alcohol derivatives may be used.

[81]

[82] Like adhesive agents and dispersants, auxiliaries may be used for the formulations of the compound, and are further exemplified by 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 poly aery lie acids.

[83]

[84] 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 esters thereof.

[85]

[86] When the compound of the present invention is used as an agricultural pesticide, acarid killer or nematocide, the application amount thereof typically ranges from 0.1 g to 100 g over an area of 10 acres. In the case in which preparations such as emulsified concentrates, wettable powders or well-flowing granules are used after being diluted with water, the application concentration thereof is usually in the range of 1 ppm to 1,000 ppm. Granules, dusts and the like are applied without dilution. When the compound of the present invention is used as a pesticide, acarid killer or nematocide for the prevention of epidemics, the emulsified concentrates, wettable powders, well- flowing granules and other formulations thereof are applied after being diluted to 0.1 to 500 ppm with water, but the oil sprays, aerosols, fuming preparations, poisonous bait, 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. [87]

[88] Upon application as a pesticide or acaricide for controlling parasites of animals, including livestock, such as cattle and pigs, and pets, such as cats and dogs, the compound of the present invention 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, by spraying oily or aqueous solutions, pouring-on or spotting-on; or for non-systemic pest control with the aid of formed articles such as collars, ear tags, and the like. In these cases, the compound of the present invention may be applied in an amount of 0.01 to 100 mg per kg body weight of host animals.

[89]

[90] The compound of the present invention may be used in admixture with, or sequentially along with, other insecticides, nematocides, acaricides, bacteriocides, fungicides, herbicides, plant growth regulators, synergists, fertilizers, soil conditioners and/or animal feeds.

[91]

[92] 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.

[93]

Mode for the Invention

[94] PREPARATION EXAMPLES

[95] Chemical Synthesis of DGAT Inhibitors

[96]

[97] PREPARATION EXAMPLE 1: Preparation of

2-Methylsulfinyl-5,8-dimethoxy-l,4-naphthoquinone (Chemical Formula 2)

[98] [Chemical Formula 2]

[99]

[100]

[101] Preparation Example 1-1 : Synthesis of 1,5-Dimethoxynaphthalene [103] lOOg (0.62 mol) of 1,5-dihydroxynaphthalene) (1) was dissolved in 500ml

(1.25mol) of 10% NaOH in the presence of nitrogen gas in a 2L two-neck round- bottom flask, followed by the slow addition of 156g (1.24 mol) of dimethyl sulfate over 1 hour. After reaction for 2 hours, the precipitates thus formed were filtered in a vacuum, washed twice with 200 ml of 5% KOH and then three times with 200 ml of distilled water and dried. The resulting residue was completely dissolved at 8O⁰C in 1.5 L of benzene, containing 300 g of active carbon, and the hot solution was filtered and left, thus yielding 73 g of the title compound as a white crystal. The properties of the product are as described below.

[104]

[105] Yield : 63%, m.p.: 181.9-182.3 ⁰C

[106] Rf: 0.49 [hexane:ethylacetate(5:l)]

[107] IH-NMR (CDCl , 400MHz): δ 7.70(d, J=8.8Hz, 2H), 7.38(t, J=8.0Hz, 2H), 6.98(d,

J=8.0Hz, 2H), 3.94(s, 6H)

[108]

[109] Preparation Example 1-2: Synthesis of

4,8-Dibromo-l,5-dimethoxynaphthalene

[HO]

[111] 1Og (0.05mol) of the 1,5-dimethoxynaphthalene, synthesized in Preparation

Example 1-1, was dissolved in 160 ml of acetonitrile in a 1,000 ml round-bottom flask. A solution of 21g (0.12mol) of N-bromosuccinimide (NBS) in 180ml of acetonitrile was droplet added to the flask with stirring. After stirring at room temperature for 3 hours, the vacuum filtration of the reaction mixture formed precipitates which were washed with acetonitrile and then twice with hexane and dried to give 12.7 g of the title compound as white power. The properties of the product are as described below.

[112]

[113] Yield: 69.02%, m.p.: 187-188 ⁰C

[114] Rf: 0.20 [hexane:ethylacetate(50: 1)]

[115] IH-NMR (CDCl , 400MHz): δ 7.68(d, J=8.4Hz, 2H), 6.72(d, J=8.4Hz, 2H), 3.91(s,

6H)

[116]

[117] Preparation Example 1-3: Synthesis of 1,4,5,8-Tetramethoxynaphthalene

[118]

[119] 14.5g (0.04mol) of the 4,8-dibromo- 1,5-dimethoxynaphthalene was dissolved, along with 7.5g (0.14mol) of sodium methoxide and 26.3g (0.14mol) copper(I) iodide, in 700 ml of a solvent mixture of dimethylformamide 50% and methanol 50% and fluxed for 30 hours. The reaction mixture was cooled and added to 1 L of ice water to form precipitates which were then filtered and washed. These precipitates were dissolved in 1 L of methylene chloride, followed by filtration to remove insoluble material. The filtrate was concentrated in a vacuum and the residue was re-crystallized in benzene to produce 6.5 g of the title compound as a white needle-like crystal. The properties of the product are as described below.

[120]

[121] Yield: 62.5%, m.p.: 168-169 ⁰C

[122] Rf: 0.14 [hexane : methylene chloride (1:4)]

[123] IH-NMR (CDCl , 400MHz): δ 6.85(s, 4H), 3.90(s, 12H)

[124]

[125] Preparation Example 1-4: Synthesis of 5,8-Dimethoxy-l,4-naphthoquinone

[126]

[127] 10 g (40.28 mmol) of the 1,4,5,8-tetramethoxynaphthalene, prepared in Preparation

Example 1-3, was dissolved in a mixture of 450 ml of acetonitrile and 150 ml of chloroform in a 250 ml one-neck round-bottom flask, and a solution of 54g (98.5mmol) of cerium diammonium nitrate in 250 ml of water was added droplet to the flask over 30 min. After reaction for an additional 30 min, the reaction mixture was mixed with 500 ml of distilled water, extracted three times with 500 ml of chloroform, and dehydrated with sodium sulphate, followed by filtration. The filtrate was concentrated in a vacuum and the residue was re-crystallized in methanol to give 4.80 g of the title compound as a reddish brown needle-like crystal. The properties of the product are as described below.

[128]

[129] Yield : 54.6%, m.p.: 122-123 ⁰C

[ 130] Rf: 0.22 [hexane : ethylacetate (1:2)]

[131] IH-NMR (CDCl , 400MHz): δ 7.33(s, 2H), 6.79(s, 2H), 3.97(s, 6H)

[132]

[133] Preparation Example 1-5: Synthesis of

2-Methylthio-5,8-dimethoxy-l,4-naphthoquinones

[134]

[135] 1.38 mmol of the 5,8-dimethoxy-l,4-naphthoquinone prepared in Preparation

Example 1-4 was dissolved in 30 ml of anhydrous methanol in a 100 ml one-neck round-bottom flask, and reacted with 1.65 mmol of methylmercaptan for 4 hours with stirring. To this reaction mixture was added droplet a solution of 0.23 mmol of sodium dichromate and 0.76 mmol of sulfuric acid in solution, followed by stirring at room temperature for 3 min at room temperature. 50 ml of a saturated sodium chloride solution was added to the reaction mixture before three rounds of extraction with 50 ml of chloroform. The organic layer was dried over sodium sulfate and filtered. The filtrate was concentrated in a vacuum and the residue was re-crystallized in methanol to give the title compound as a reddish brown crystal.

[136]

[137] Preparation Example 1-6: Synthesis of

2-Methylsulfinyl-5,8-dimethoxy-l,4-naphthoquinones (Chemical Formula 2)

[138]

[139] 0.21 mmol of the 2-methylthio-5,8-dimethoxy-l,4-naphthoquinone prepared in

Preparation Example 1-5 was dissolved at O⁰C in 20 ml of chloroform. To this solution were added five aliquots of 0.32 mmol of 77% MCPBA, followed by reaction for 4 hours. The reaction mixture was mixed with an aqueous saturated sodium bicarbonate solution and 50 ml of an aqueous sodium chloride solution and extracted three times with 50 ml of chloroform. The organic layer was dehydrated with sodium sulfate and filtered. The filtrate was concentrated in a vacuum and the residue was purified using column chromatography to give the title compound (Chemical Formula 2) as a reddish brown material. The properties of the product are as described below.

[140]

[141] Yield: 66.2%, m.p.: 222-223 ⁰C

[142] Rf: 0.06 [hexane : ethyl acetate ( 1 :4)]

[143] IH-NMR (CDCl , 400MHz): δ 7.42(d, J=9.2Hz, IH), 7.34(d, J=9.2Hz, IH), 7.35(s,

IH), 3.99(s, 6H), 2.95(s, 3H)

[144]

[145] Preparation Example 2: Preparation of

2-Ethylsulfinyl-5,8-dimethoxy-l,4-naphthoquinone (Chemical Formula 3)

[146]

[147] The same procedure as in Preparation Example 1, with the exception that ethylmercaptan was used instead of methylmercaptan, which was used in Preparation Example 1-5, was carried out to produce the compound of Chemical Formula 3. The properties of the product are as described below.

[148] [Chemical Formula 3]

[149]

[150] Yield: 71.1%, m.p.: 145-146 ⁰C

[151] Rf : 0.09 [hexane : ethylacetate( 1 :4)]

[152] IH-NMR (CDCl , 400MHz): δ 7.42(d, J=9.6Hz, IH), 7.37(d, J=9.2Hz, IH), 7.28(s,

IH), 3.99(s, 6H), 3.32~3.23(m, IH), 3.03~2.95(m, IH), 1.31(t, J=7.2Hz, 3H) [153] [154] Preparation Example 3: Preparation of 2-Propylsulfinyl- 5,8-dimethoxy-l,4-naphthoquinone) (Chemical Formula 4)

[155] [156] The same procedure as in Preparation Example 1, with the exception that propy- lmercaptan was used instead of methylmercaptan, which was used in Preparation Example 1-5, was carried out to produce the compound of Chemical Formula 4. The properties of the product are as described below.

[157] [Chemical Formula 4] [158]

[159] Yield: 41.9%, m.p.: 120-121 ⁰C [160] Rf: 0.18 [hexane : ethylacetate(l:4)] [161] IH-NMR (CDCl , 400MHz): δ 7.35(d, J=9.2Hz, IH), 7.30(d, J=9.6Hz, IH), 7.22(s, IH), 3.92(s, 3H), 3.91(s, 3H), 3.18~3.10(m, IH), 2.86~2.79(m, IH), 1.94-1.84(m, IH), 1.71~1.63(m, IH), 1.01(t, J=7.2Hz, 3H)

[162] [163] PREPARATION EXAMPLE 4: Preparation of 2-Butylsulfinyl-5,8-dimethoxy-l,4-naphthoquinone) (Chemical Formula 5)

[164] [165] The same procedure as in Preparation Example 1, with the exception that butylmercaptan was used instead of methylmercaptan, which was used in Preparation Example 1-5, was carried out to produce the compound of Chemical Formula 5. The properties of the product are as described below.

[166] [chemical Formula 5] [167]

[168] [169] Yield: 81.6%, m.p.: 127-128 ⁰C [170] Rf: 0.18 [hexane : ethylacetate(l:4)] [171] IH-NMR (CDCl , 400MHz): δ 7.41(d, J=9.6Hz, IH), 7.37(d, J=9.6Hz, IH), 7.31(s,

IH), 3.99(s, 6H), 3.28~3.20(m, IH), 2.95~2.88(m, IH), 1.95~1.85(m, IH), 1.70~1.60(m, IH), 1.54~1.42(m, 2H), 0.95(t, J=7.2Hz, 3H)

[172]

[173] PREPARATION EXAMPLE 5: Preparation of

2-Pentylsulfinyl-5,8-dimethoxy-l,4-naphthoquinone) (Chemical Formula 6)

[174]

[175] The same procedure as in Preparation Example 1, with the exception that pentylmercaptan was used instead of methylmercaptan, which was used in Preparation Example 1-5, was carried out to produce the compound of Chemical Formula 6. The properties of the product are as described below.

[176] [Chemical Formula

[177]

[178]

[179] Yield: 40.2%, m.p.: 125-126 ⁰C

[180] Rf: 0.24 [hexane: ethylacetate(l:4)]

[181] IH-NMR (CDCl , 400MHz): δ 7.41(d, J=9.6Hz, IH), 7.36(d, J=9.6Hz, IH), 7.31(s,

IH), 3.99(s, 6H), 3.27~3.20(m, IH), 2.94~2.87(m, IH), 1.94~1.89(m, IH),

1.71~1.66(m, IH), 1.51~1.29(m, 6H), 0.90(t, J=7.6Hz, 3H) [182] [183] PREPARATION EXAMPLE 6: Preparation of

2-Hexylsulfinyl-5,8-dimethoxy-l,4-naphthoquinone) (Chemical Formula 7) [184] [185] The same procedure as in Preparation Example 1, with the exception that hexylmercaptan was used instead of methylmercaptan, which was used in Preparation

Example 1-5, was carried out to produce the compound of Chemical Formula 7. The properties of the product are as described below. [186]

[187] [Chemical Formula 7]

[188]

[189] [190] Yield: 76.7%, m.p.: 120-121 ⁰C [191] Rf: 0.18 [hexane: ethylacetate(l:4)] [192] IH-NMR (CDCl , 400MHz): δ 7.42(d, J=9.6Hz, IH), 7.37(d, J=9.6Hz, IH), 7.30(s, IH), 4.00(s, 3H), 3.99(s, 3H), 3.27~3.20(m, IH), 2.95~2.88(m, IH), 1.96-1.88(m, IH), 1.71~1.65(m, IH), 1.49-1.37 (m, 2H), 1.33-1.27 (m, 4H), 0.87(t, J=7.2Hz, 3H)

[193] [194] PREPARATION EXAMPLE 7: Preparation of 2-Heptylsulfinyl-5,8-dimethoxy-l,4-naphthoquinone (Chemical Formula 8)

[195] [196] The same procedure as in Preparation Example 1, with the exception that hep- tylmercaptan was used instead of methylmercaptan, which was used in Preparation Example 1-5, was carried out to produce the compound of Chemical Formula 8. The properties of the product are as described below.

[197] [198] [Chemical Formula 8] [199]

[200] [201] Yield: 46.9%, m.p.: 106-107 ⁰C [202] Rf: 0.27 [hexane: ethylacetate(l:4)] [203] IH-NMR (CDCl , 400MHz): δ 7.42(d, J=9.6Hz, IH), 7.37(d, J=9.2Hz, IH), 7.30(s, IH), 3.99(s, 6H), 3.27~3.20(m, IH), 2.94~2.87(m, IH), 1.96~1.86(m, IH), 1.49~1.37(m, 2H), 1.35~1.27(m, 6H), 0.87(t, J=6.8Hz, 3H)

[204] [205] PREPARATION EXAMPLE 8: Preparation of 2-Octylsulfinyl-5,8-dimethoxy-l,4-naphthoquinone (Chemical Formula 9)

[206] [207] The same procedure as in Preparation Example 1, with the exception that octylmercaptan was used instead of methylmercaptan, which was used in Preparation Example 1-5, was carried out to produce the compound of Chemical Formula 9. The properties of the product are as described below.

[208] [209] [Chemical Formula 9] [210]

[211] Yield: 63.2%, m.p.: 110-111 ⁰C [212] Rf: 0.26 [hexane: ethylacetate(l:4)] [213] IH-NMR (CDCl , 400MHz): δ 7.41(d, J=9.2Hz, IH), 7.36(d, J=9.6Hz, IH), 7.23(s, IH), 3.99(s, 6H), 3.27~3.20(m, IH), 2.94~2.87(m, IH), 1.93~1.88(m, IH), 1.73~1.60(m, 2H), 1.50-1.88(m, 9H), 0.87(t, J=7.2Hz, 3H)

[214] [215] PREPARATION EXAMPLE 9: Preparation of 2-Nonylsulfinyl-5,8-dimethoxy-l,4-naphthoquinone (Chemical Formula 10)

[216] [217] The same procedure as in Preparation Example 1, with the exception that nonylmercaptan was used instead of methylmercaptan, which was used in Preparation Example 1-5, was carried out to produce the compound of Chemical Formula 10. The properties of the product are as described below.

[218] [219] [Chemical Formula 10] [220]

[221] Yield: 74%, m.p.: 100-101 ⁰C [222] Rf: 0.26 [hexane: ethylacetate(l:4)] [223] IH-NMR (CDCl₃, 400MHz): δ 7.42(d, J=9.6Hz, IH), 7.37(d, J=9.6Hz, IH), 7.31(s, IH), 4.00(s, 3H), 3.99(s, 3H), 3.27~3.20(m, IH), 2.94~2.87(m, IH), 1.97-1.86(m, IH), 1.71~1.61(m, 2H), 1.49~1.25(m, HH), 0.87(t, J=7.2Hz, 3H) [224] [225] PREPARATION 10: Preparation of 2-Decylsulfinyl-5,8-dimethoxy-l,4-naphthoquinone (Chemical Formula 11)

[226] [227] The same procedure as in Preparation Example 1, with the exception that de- cylmercaptan was used instead of methylmercaptan, which was used in Preparation Example 1-5, was carried out to produce the compound of Chemical Formula 11. The properties of the product are as described below.

[228] [229] [Chemical Formula 11] [230]

[231] Yield: 92.4%, m.p.: 127-128 ⁰C [232] Rf: 0.27 [hexane: ethylacetate(l:4)] [233] IH-NMR (CDCl , 400MHz): δ 7.41(d, J=9.6Hz, IH), 7.37(d, J=9.2Hz, IH), 7.30(s, IH), 3.99(s, 6H), 3.28~3.18(m, IH), 2.94~2.84(m, IH), 1.95~1.85(m, IH), 1.70~1.58(m, 2H), 1.45~1.20(m, 13H), 0.87(t, J=7.6Hz, 3H)

[234] [235] EXPERIMENTAL EXAMPLE: Preparation of DGAT Source [236] [237] The liver was removed from male Sprague-Dawley rats (250-300 g) washed with buffer A (0.25 M sucrose, 1.0 mM EDTA, 10 mM Tris-HCl. pH 7.4) and homogenized using a glass homogenizer equipped with a Teflon rod. The homogenate was centrifuged at 14,000 x g at 4 ⁰C for 15 min. The supernatant was further centrifuged at 100,000 x g at 4 ⁰C for 1 hour. To the pellet was added buffer B (0.25 M sucrose, 10 mM Tris-HCl. pH 7.4), followed by centrifugation at 100,000 x g at 4 ⁰C for 1 hour to isolate microsomes containing DGAT. The pellet was dissolved again in buffer B (4 ml) and analyzed for protein concentration with bovine serum albumin, serving as a standard material. The enzyme source solution thus obtained was diluted to a protein concentration of 10 mg/ml and divided in vials which were stored at -70 ⁰C until use in assay for DGAT activity.

[238] [239] EXPERIMENTAL EXAMPLE 2: Assay of Compounds for Inhibitory Activity against DGAT [240]

[241] The compounds of the present invention were assayed for inhibitory activity against

DGAT as follows. The [ C] triacylglycerol, which was synthesized from 1,2-diacylglycerol and [ C] palmitoyl-CoA, with the rat microsomal protein serving as an enzyme source, was measured for radioactivity. In detail, a reaction solution containing 175 mM Tris-HCl (pH 8.0), 20 μl of bovine serum albumin (10 mg/ml), 8 mM MgCl , 30 μM [¹⁴C]palmitoyl CoA (0.02 mCi, Amersham), and 200 μM 1,2-dioleoyl glycerol was added with 10.0 μl of a solution of a sample in methanol or DMSO and with 100-200 mg of the microsomal protein, followed by reaction for 10 min at 25⁰C. The reaction was terminated with the addition of 1.5 ml of a solution (2-propanol / heptane / water = 80 / 20 / 2, v/v/v). The reaction mixture was mixed with 1 ml of heptane and 0.5 ml of H O and vortexed. To 1 ml of the supernatant was added 2 ml of an alkaline ethanol solution (ethanol / 0.5 N sodium hydroxide / water = 50 / 10 / 40, v/v/v) before vortexing. 0.65ml of the supernatant, containing [ C] triacyl glycerol, was measured for radioactivity using LSC (liquid scintillation counter). The inhibition activity of the sample against DGAT was calculated according to Equation 1, below.

[242]

[243] [Equation 1]

[244]

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

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

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

[248]

[249] Also, the other compounds (Chemical Formulas 2 to 11) chemically synthesized according to the present invention were measured for inhibitory activity against DGAT. As a result, DGAT activity was inhibited by 13% at 50 μg/ml of the compound of Chemical Formula 2, by 15% at 50 μg/ml of the compound of Chemical Formula 3, by 24% at 50 μg/ml of the compound of chemical Formula 4, by 63% at 50 μg/ml of the compound of Chemical Formula 5, and by 81% and 51%, respectively, at 50 μg/ml and 25 μg/ml of the compound of Chemical Formula 6. The compounds of Chemical Formulas 7 to 11 were measured to inhibit 50% of DGAT activity at 18.9 μg/ml, 14.2 μg/ml, 11.8 μg/ml, 9.2 μg/ml, and 5.5 μg/ml, which were calculated as IC50 values of 53.9 μM, 39.0 μM, 31.2 μM, 23.4 μM, and 13.5 μM, because their molecular weights are 350.43, 364.43, 378.48, 392.51, and 406.54, respectively. In FIG. 1, inhibitory activities of the compounds of Chemical Formulas 7 to 11 toward DGAT were plotted against concentrations.

[250] [251] EXPERIMENTAL EXAMPLE 3: Assay of Compounds for Toxicity against Greenhouse whitefly Larval Development and Egg Hatching

[252] [253] The compounds according to the present invention were tested for their insecticidal effects on greenhouse whitefly larvae (Trialeurodes vaporariorum) on May 2006 in the Agrobiology Department, Agrobiological Environment College, Chungbuk National University, Cheongju city, Chung-cheong-buk-do. After the compounds of the present invention, identified as having inhibitory activity against DGAT, were weighed precisely, a suitable 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 active compound solutions.

[254] 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 in two replicates. The results are given in Table 1, below.

[255] 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, which were not treated with the present active compound but were treated merely with a mixture of 10% acetone and nine volumes of a 100 ppm Triton X-100 solution. This leaf-disk bioassay was performed in triplicate, and LC50 (50% lethal concentration) was calculated. The results are summarized in Table 2, below.

[256] [257] Table 1 Inhibitory Activity against Egg Hatching of Greenhouse whitefly

[258] Table 2

Pesticidal Activity on Greenhouse Whitefly Nymphs

[259] [260] It is apparent from the data of Tables 1 and 2 that the DGAT inhibitor of Chemical Formula 9 sustained insecticidal effects over time in a dose-dependent manner compared to the control as measured for mortality at regular intervals of one day after it was applied to greenhouse whitefly at concentrations of 1, 10 and 100 ppm.

[261] [262] EXPERIMENTAL EXAMPLE 3: Assay of Compounds for toxicity against Plutella xylostella L. larvae

[263] [264] The compounds of the present invention were evaluated for insecticidal effects on larvae of Plutella xylostella L. A leaf-disk bioassay was conducted on May 2006 in the Agrobiology Department, Agrobiological Environment College, Chungbuk National University, Cheongju city, Chung-cheong-buk-do. After the compounds, identified as DGAT inhibitors, were weighed accurately, a suitable 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 active compound solutions. 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 under 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 instars were placed on each leaf disc using a soft brush, taking care not to damage the larvae. Each sample was prepared in triplicate. The larvae were grown in an incubator (25+1 ⁰C, 40-45% relative humidity, 16L:8D), and mortality was recorded after 24 and 48 hrs. A control group was grown on leaf disks which were not treated with the present compound but treated merely with a mixture of 10% acetone and nine volumes of a 100 ppm Triton X-100 solution. This leaf-disk bioassay was repeated three times, and LC50 (50% lethal concentration) was calculated.

[265] The results are given in Table 3, below.

[266]

[267]

[268] [Table 3]

[269] Pesticidal Activity on P. xylostella larvae

[270]

[271] [272] It is apparent from the data of Table 3 that the DGAT inhibitors of Chemical Formulas 8 to 10 sustained insecticidal effects over time in a dose-dependent manner, compared to the control, as measured for mortality at regular intervals of one day after they were applied in amounts of 1, 10 and 100 ppm to P. xylostella larvae. Also, it was observed that the larvae were more reluctant to eat the feed treated with the compounds of the present invention as compared to the control.

[273] Industrial Applicability The compounds of the present invention, as described hitherto, inhibit the biosynthesis of triglycerides in insects, particularly, the activity of diacyl CoA: glycerol acyltransferase, inducing the lack of triglycerides, essential for insects, thus leading to potent insecticidal effects. Also, the compounds of the present invention are safe for humans and may be used as environment-friendly pesticides.

Claims

[1] A pesticidal composition, comprising a compound an inhibitor against biosynthesis of triglycerides in insects as an effective ingredient.

[2] The pesticidal composition according to claim 1, wherein the inhibitor has inhibitory activity against diacyl CoA: glycerol acyltransferase.

[3] The pesticidal composition according to claim 1, wherein the inhibitor is a compound represented by the following Chemical Formula 1 : [Chemical Formula 1]

Wherein R is a C ~ C alkyl or alkenyl.

[4] The pesticidal composition according to claim 3, wherein R is C ~ C alkyl.

[5] The pesticidal composition according to claim 1, further comprising a carrier.

[6] A method for killing insects, using the pesticidal composition of one of claims 1 to 5.

[7] The method according to claim 6, wherein the composition is sprayed or administered to insects.

[8] A compound, represented by the following Chemical Formula 1 : Chemical Formula 1

Wherein R is a C ~ C alkyl or alkenyl.

[9] The compound according to claim 8, wherein R is C ~ C alkyl. [10] A method of inhibiting triglyceride biosynthesis in insects, using the compound of claim 8.

[H] The method according to claim 10, wherein the inhibiting triglyceride biosynthesis results from inhibiting diacyl CoA: glycerol acyltransferase.