JP2007031365A - Natural immune inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a stronger compound which can inhibit the natural immunization routes of insects and exterminate the insects mediating the diseases of field crops to prevent the diffusion of field crops diseases with the insects, and to provide an anti-insect agent containing the compound as an active ingredient. <P>SOLUTION: This compound represented by formula (I) (X is OH, OR, NRR' or a heterocyclic group containing N as a heterogeneous atom; R and R' are each independently H, an alkyl, or an aryl). An natural immune inhibitor, especially an anti-insect agent, containing the compound, its salt or its hydrate as an active ingredient. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

The present invention relates to an innate immunity inhibitor comprising a cyclopentanediol derivative as an active ingredient, in particular, an anti-insect agent.

One of the reasons for the spread of crop diseases is the presence of vector insects. Insects with immunity to crop diseases are transmitted as vectors, and the pathogenic viruses spread rapidly to surrounding crops, increasing the damage to crops caused by diseases.

So far, as pesticides, insecticides for controlling pests, fungicides, herbicides, rodenticides, plant growth regulators, attractants, spreading agents, natural enemies, microbial agents and the like have been used (see Non-Patent Document 1). ).

However, there has been no known drug or method that can effectively prevent the spread of crop diseases once they occur.

The immune system is a biological defense system that detects and eliminates the invasion of pathogens (such as bacteria and viruses). Among them, the innate immune system is basically a non-specific defense system against foreign substances and pathogens of living organisms, and is the only defense mechanism for insects that do not have an acquired immune system.

In order to solve the above problems, the present inventor focused on Drosophila as an insect that mediates diseases of agricultural crops, and developed a screening method for substances that inhibit the innate immune pathway using Drosophila created by genetic recombination (Patent Literature). 1) The search was performed. As a result, it was found that a specific cyclopentanediol derivative remarkably suppresses the expression of endogenous antibacterial substances by the Drosophila innate immunity pathway. Some of them are reported.

JP2004-121155A “Basic knowledge on pesticides” “http://www.maff.go.jp/nouyaku/nouyakukiso.htm”

The object of the present invention is to inhibit the insect's innate immunity pathway, to control insects that mediate crop diseases, and to prevent the spread of crop diseases by insects, and more effective compounds It is to provide an anti-insect agent or the like contained as a component.

（式（Ｉ）中、ＸはＯＨ、ＯＲ、ＮＲＲ’ 又は、Ｎをヘテロ原子として含む複素環の何れかであり、Ｒ及びＲ’ は、夫々、独立して水素、アルキル基、又はアリール基である）。
２．上記化合物、その塩又はその水和物を有効成分として含有する自然免疫阻害剤。
３．上記化合物、その塩又はその水和物を有効成分として含有する抗昆虫剤。
４．上記化合物、その塩又はその水和物を有効成分として含有するショウジョウバエの駆除剤。
５．上記化合物、その塩又はその水和物を用いた病害昆虫又は媒介昆虫の駆除方法。 That is, the present invention relates to the following aspects.
1. Compound represented by the following structural formula (I):

(In the formula (I), X is OH, OR, NRR ′ or a heterocyclic ring containing N as a hetero atom, and R and R ′ are each independently hydrogen, an alkyl group, or an aryl group. Is).
2. An innate immunity inhibitor comprising the above compound, a salt thereof or a hydrate thereof as an active ingredient.
3. An anti-insect agent containing the above compound, a salt thereof or a hydrate thereof as an active ingredient.
4). A Drosophila control agent comprising the above compound, a salt thereof or a hydrate thereof as an active ingredient.
5. A method for controlling diseased insects or vector insects using the above compounds, salts thereof or hydrates thereof.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide an effective anti-insect agent for inhibiting insect innate immunity pathways, controlling insects that mediate crop diseases, and preventing the spread of crop diseases by insects.

The compound of the present invention represented by the structural formula (I) is a cyclopentanediol derivative substance. In the formula, the alkyl group is preferably a lower alkyl group, for example, a linear or branched alkyl group having 1 to 6 carbon atoms. Examples of the aryl group include a phenyl group, a tolyl group, and a naphthyl group. Examples of the heterocyclic ring containing N as a hetero atom include a 6-membered ring and a 5-membered ring. In particular, a compound in which X is NH ₂ or NHMe in the structural formula (I) is preferable.

The compounds of the present invention can be synthesized by any method known to those skilled in the art as shown in the Examples.

Salts in the present invention include salts with mineral acids such as sulfuric acid, hydrochloric acid and phosphoric acid, salts with organic acids such as acetic acid, oxalic acid, lactic acid, tartaric acid, fumaric acid, maleic acid and methanesulfonic acid, trimethylamine and methylamine And any salts known to those skilled in the art, such as salts with amines such as sodium ions, potassium ions, and calcium ions.

When the compound of the present invention is used as an innate immunity inhibitor or anti-insect agent based on the expression of endogenous antibacterial substances in insects such as Drosophila, various conditions such as the purpose of use, the type of target insect, the type and amount of the compound are determined. Considering the form, for example, wettable powder, agar wettable powder, aqueous solvent, emulsion, liquid, flowable agent such as suspension in water / emulsifier in water, capsule, powder, granule, aerosol, etc. Can take any form known to those skilled in the art.

  The amount of the compound of the present invention contained in such various agents is also appropriately selected by those skilled in the art in consideration of various conditions such as the purpose of use, the type of insect to be controlled, the type and amount of the compound, and the form. I can do it. For example, as an example, the concentration range may be 0.1 mg to 100 g.

Each of the above agents can be used by any method known to those skilled in the art. That is, the method for controlling diseased insects or vector insects of the present invention varies depending on the type and amount of insects to be controlled, the type of crops and trees to be controlled, the cultivation form, and the growth state. A flour wettable powder, an aqueous solvent, an emulsion, a liquid, a flowable agent such as a suspension in water and an emulsifier in water, and a capsule are diluted with water and sprayed on crops, trees and the like. In addition, powders, granules, and aerosols can be sprayed in the form of a preparation.

When an anti-insect agent containing the compound of the present invention is sprayed on crops in advance, even if crop diseases occur, insects with reduced immune function will be infected with the disease without causing pathogens to propagate to healthy crops. It can be killed and damage can be minimized. In addition, diseased insects that damage crops can be easily controlled with a smaller amount of insecticide than usual because their immune functions are reduced.

  In the method of the present invention, the conditions such as the application amount / interval of various agents, the timing, etc., depend on the type of crop and the cultivation form / growth state, the type of insect to be controlled, the type / amount of compound, the form, etc. Those skilled in the art can select as appropriate.

The Drosophila endogenous antibacterial substance dipterisin expression inhibitory activity test shown in Test Example 1 below is a method described in Patent Document 1, which is an innate immune system common to higher organisms such as mammals called the Imd pathway. Specific screening method. Therefore, the disease insects or vector insects that can be controlled by the compound of the present invention or the method of the present invention include, for example, damage to crops known to those skilled in the art, such as planthoppers that feed on rice, aphids that damage vegetables and fruit trees, etc. Any disease insect that gives is included.

Hereinafter, the present invention will be specifically described with reference to Examples and Test Examples, but the technical scope of the present invention is not limited thereto.

Example 1: Production of the compound of the present invention (1)
In an ice bath, 3-Cyclopentene-1-carboxylic acid (17.2 g) was dissolved in acetonitrile (160 mL), and 1,8-Diazabicyclo [5.4.0] undec-7-ene (25.5 mL) and 1-bromobutane (18.2 g) were dissolved. mL) was added and stirred. After 4 hours, the reaction mixture was poured into 1M hydrochloric acid (400 mL) and extracted three times with ethyl acetate (500 mL). All the ethyl acetate layers were combined, washed with saturated aqueous sodium hydrogen carbonate (300 mL) and saturated brine (300 mL), dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was subjected to silica gel column chromatography, and butyl 3-cyclopentene-1-carboxylate (12.7 g) was obtained from the fraction eluted with hexane-ethyl acetate (49: 1).

In an ice bath, butyl 3-cyclopentene-1-carboxylate (7.49 g) is dissolved in a mixed solvent of water-acetonitrile-acetone (1: 1: 1), and osmium tetroxide (4% solution) (1.4 mL), 4- Methylmorpholine N-oxide (7.83 g) was added and stirred. After 1 hour, the reaction solution was poured into an aqueous sodium sulfite solution (50 mL) and extracted three times with ethyl acetate (50 mL). All the ethyl acetate layers were combined, washed with saturated aqueous sodium hydrogen carbonate (30 mL) and saturated brine (30 mL), dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was dissolved in 2,2-dimethoxypropane (73 mL), p-toluenesulfonic acid (12.5 g) was added, and the mixture was stirred at room temperature. After 1 hour, the reaction mixture was poured into saturated aqueous sodium hydrogen carbonate (50 mL) and extracted three times with ethyl acetate (50 mL). All the ethyl acetate layers were combined, washed with saturated aqueous sodium hydrogen carbonate (30 mL) and saturated brine (30 mL), dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was subjected to silica gel column chromatography, and butyl c-3, c-4- (dimethylmethylenedioxy) -r-1-cyclopentanecalboxylate (2.48 g) was obtained from the fraction eluted with hexane-ethyl acetate (4: 1). .

In an ice bath, butyl c-3, c-4- (dimethylmethylenedioxy) -r-1-cyclopentanecalboxylate (2.67 g) is dissolved in methylene chloride (33 mL) and diisobutylaluminum hydride (1.0 M in toluene) (23.2 mL) Was added and stirred. After 1 hour, acetone (1 mL) was added to the reaction solution, poured into a saturated potassium sodium tartrate solution (30 mL), and extracted three times with diethyl ether (30 mL). All the ether layers were combined, washed with water (30 mL) and saturated brine (30 mL), dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was subjected to silica gel column chromatography, and c-3, c-4- (dimethylmethylenedioxy) -r-1-cyclopentanylmethanol (1.60 g) was obtained from the fraction eluted with hexane-ethyl acetate (1: 1).

c-3, c-4- (dimethylmethylenedioxy) -r-1-cyclopentanylmethanol (395 mg) was dissolved in methylene chloride (10 mL), and pyridinium chlorochromate (1.49 g) was added and stirred. After 2 hours, the reaction mixture was poured into diethyl ether (50 mL), the solid was filtered off, and the filtrate was washed with saturated aqueous sodium hydrogen carbonate (30 mL) and saturated brine (30 mL), and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure. The residue was dissolved in toluene (10 mL), tert-butyl diethylphosphonoacetate (0.65 mL) and sodium hydride (60% mineral oil paste) (140 mg) were added and stirred. After 1 hour, the reaction mixture was poured into 1M hydrochloric acid (50 mL) and extracted three times with diethyl ether (50 mL). All the ether layers were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was subjected to silica gel column chromatography. From the fraction eluted with hexane-ethyl acetate (19: 1), tert-butyl (E) -3- [c-3 ', c-4'-(dimethylmethylenedioxy) -r -1'-cyclopentanyl] propenoate (397 mg) was obtained.

In an ice bath, tert-Butyl (E) -3- [c-3 ', c-4'-(dimethylmethylenedioxy) -r-1'-cyclopentanyl] propenoate (520 mg) was added to methylene chloride (5 mL) and trifluoro Dissolved in acetic acid (5 mL) and stirred. After 1 hour, the solvent was distilled off, and the resulting residue was dissolved in 2,2-dimethoxypropane (2.5 mL), p-toluenesulfonic acid (40 mg) was added, and the mixture was stirred at room temperature. After 1 hour, the reaction solution was directly subjected to column chromatography, and from the fraction eluted with chloroform-methanol (24: 1), (E) -3- [c-3 ', c-4'-(dimethylmethylenedioxy)- r-1'-cyclopentanyl] propenoic acid (185 mg) was obtained.

In an ice bath, dissolve (E) -3- [c-3 ', c-4'-(dimethylmethylenedioxy) -r-1'-cyclopentanyl] propenoic acid (98 mg) in tetrahydrofuran (2.5 mL). Morpholine (60 μL) and isobutyl chloroformate (70 μL) were sequentially added and stirred. After 30 minutes, 28% aqueous ammonia (90 μL) was added, and the mixture was stirred at room temperature. After 3 hours, the reaction mixture was poured into water (5 mL) and extracted three times with ethyl acetate (10 mL). All the ethyl acetate layers were combined, washed with 0.5M hydrochloric acid (5 mL), water (5 mL), saturated brine (5 mL), and dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was subjected to silica gel column chromatography, and from the fraction eluted with chloroform-methanol (97: 3), (E) -3- [c-3 ', c-4'-(dimethylmethylenedioxy) -r-1'- cyclopentanyl] propenamide (69 mg) was obtained.

In an ice bath, (E) -3- [c-3 ', c-4'-(dimethylmethylenedioxy) -r-1'-cyclopentanyl] propenamide (43 mg) was dissolved in 10% hydrogen chloride-methanol solution (1 mL). Dissolved and stirred. After 1 hour, the solvent was distilled off, and the residue was subjected to silica gel column chromatography. From the fraction eluted with chloroform-methanol (4: 1), the compound of the present invention shown in Table 1 below, TPS-17 ( 32 mg) was obtained. TPS-17 is (E) -3- (c-3 ', c-4'-dihydroxy-r-1'-cyclopentanyl) propenamide. The spectral data of this compound TPS-17 is shown below.

¹ H NMR (400MHz, CD ₃ OD) δ6.80 (1H, dd, J = 15.4, 8.5 Hz), 5.88 (1H, dd, J = 15.4, 1.1 Hz), 3.97 (2H, m), 2.63 (1H , m), 2.11 (2H, ddd, J = 14.6, 8.5, 6.0 Hz), 1.57 (2H, ddd, J = 14.6, 8.5, 6.0 Hz); ¹³ C NMR (100 MHz, CD ₃ OD) δ 172.1, 154.0, 120.5, 74.3 (2C), 38.2, 37.8 (2C); HREIMS m / z 153.0788 [MH ₂ O] ⁺ (153.0789 calculated for C ₈ H ₁₁ NO ₂ ).

Example 1: Production of the compound of the present invention (2)
In an ice bath, dissolve (E) -3- [c-3 ', c-4'-(dimethylmethylenedioxy) -r-1'-cyclopentanyl] propenoic acid (61 mg) in tetrahydrofuran (2 mL). Morpholine (35 μL) and isobutyl chloroformate (40 μL) were sequentially added and stirred. After 30 minutes, 40% aqueous methylamine solution (35 μL) was added, and the mixture was stirred at room temperature. After 3 hours, the reaction mixture was poured into water (5 mL) and extracted three times with ethyl acetate (10 mL). All the ethyl acetate layers were combined, washed with 0.5M hydrochloric acid (5 mL), water (5 mL), saturated brine (5 mL), and dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was subjected to silica gel column chromatography. From the fraction eluted with chloroform-methanol (99: 1), (E) -N-methyl-3- [c-3 ', c-4'-(dimethylmethylenedioxy) -r -1'-cyclopentanyl] propenamide (62 mg) was obtained.

In an ice bath, (E) -N-methyl-3- [c-3 ', c-4'-(dimethylmethylenedioxy) -r-1'-cyclopentanyl] propenamide (41 mg) was added to a 10% hydrogen chloride-methanol solution ( 1 mL) and stirred. After 1 hour, the solvent was distilled off, and the residue was subjected to silica gel column chromatography. From the fraction eluted with chloroform-methanol (9: 1), the compound of the present invention shown in Table 1 below, TPS-19 ( 30 mg) was obtained. TPS-19 is (E) -N-methyl-3- (c-3 ', c-4'-dihydroxy-r-1'-cyclopentanyl) propenamide. The spectral data of this compound TPS-19 is shown below.

¹ H NMR (400MHz, CD ₃ OD) δ 6.66 (1H, dd, J = 15.4, 8.3 Hz), 5.73 (1H, d, J = 15.4 Hz), 3.87 (2H, m), 2.67 (3H, s) , 2.52 (1H, m), 2.00 (2H, ddd, J = 14.2, 8.3, 5.7 Hz), 1.46 (2H, ddd, J = 14.2, 8.3, 5.7 Hz); ¹³ C NMR (100 MHz, CD ₃ OD ) δ 169.9, 151.0, 121.6, 74.4 (2C), 38.4, 37.7 (2C), 26.7; HREIMS m / z 185.1042 [M] ⁺ (185.1051 calculated for C ₉ H ₁₅ NO ₃ ).

  As mentioned above, although the synthesis example of the typical compound of this invention was shown, the other compound is compoundable by the same method.

Test Example 1 Diptericin expression inhibitory activity (“Dpt-lacZ”) in Drosophila
(Test animal)
Drosophila in which a gene having a reporter gene lacZ introduced into the transcriptional regulatory region of Drosophila's endogenous antibacterial substance (Diptericin) was bred at 18 ° C or 25 ° C by the method described in Patent Document 1, and female 3rd instar larvae were tested. Were selected and used.

(Test method)
Schneider containing 20% (v / v) fetal bovine serum, 1% (v / v) antibioticantimycotic (manufactured by SIGMA), exposing fat body which is an endogenous antibacterial substance producing organ by dissecting Drosophila larvae It was cultured at 25 ° C. for 12 hours in 'sDrosophila medium. The compound was dissolved in dimethyl sulfoxide to the desired concentration and added to the culture medium. As a positive control, lipopolysaccharide (LPS) having the ability to activate innate immunity was dissolved in purified water and added to the culture medium to a final concentration of 10 mg / mL. Six larvae were used for each sample.

As an indicator of the expression level of the dipterisin gene, β-galactosidase, which is a reporter protein, was quantified for each larva and the average value was determined. The average value when no sample was added was O, the average value of the positive control was 100, and the innate immunity suppression effect when each concentration of the compound of the present invention was added was determined. The results are shown in Table 1 as IC50 (μg) values.

Test Example 2 Toxicity test using Drosophila-derived S2 cells (S2 cell assay)
(Test method)
Drosophila-derived S2 cells were cultured at 25 ° C. for 24 hours in Schneider's Drosophila medium containing 20% (v / v) fetal calf serum and 1% (v / v) Antibiotics-Antimycotic (GIBCO). The compound was dissolved in dimethyl sulfoxide to the desired concentration and added to the culture medium. Thereafter, an MTT-like reagent (viable cell count measuring reagent SF: nacalai tesque) was added, and the absorbance immediately after (0H) and the absorbance after 3 hours of incubation at 25 ° C. (3H) were measured. 6 wells were carried out for each compound. The absorbance obtained by subtracting 0H from 3H was defined as the number of viable cells, and the average value was obtained. The cell viability when each concentration of the compound of the present invention was added was determined by setting the average value of the culture medium alone without seeding the cells to 0 and the average value of 100 when no compound was added. The results are shown in Table 1 as IC50 (μg) values.

Test Example 3 b-galactosidase expression inhibitory activity by heat stimulation (biological reaction other than infection defense: hs-lacZ assay)
(Test animal)
Using heat shock promotor (hs) that enables induction of genes by thermal stimulation, and finally using Drosophila of hs-GAL4 / UAS-lacZ system that can express b-galactosidase similar to Dpt-lacZ It was. The animals were reared at 18 or 25 ° C., and 3rd instar larvae were selected and used during the experiment.
(Test method)
Drosophila larvae were dissected under low temperature conditions (4 ° C), 37% in Schneider's Drosophila medium containing 20% (v / v) fetal calf serum, 1% (v / v) Antibiotics-Antimycotic (GIBCO), 37 After heat stimulation at 30 ° C. for 30 minutes, the cells were cultured at 25 ° C. for 18 hours. The compound was dissolved in dimethyl sulfoxide to the desired concentration and added to the culture medium. Six larvae were used for each compound. As a reaction index other than infection protection, b-galactosidase, a reporter protein induced by heat, was quantified for each larva and the average value was determined. The average value when 100 mM of T-2 toxin, a known toxic compound, was added was 0, the average value when no compound was added was 100, and b-galactosidase when each concentration of the compound of the present invention was added. Expression inhibitory activity was determined. The results are shown in Table 1 as IC50 (μg) values.

  The above results are shown in Table 1 together with the structural formula of each compound (“TPS-14” is not a compound of the present invention). From these results, the compounds of the present invention significantly inhibit the insect infection defense reaction shown by diptericin expression inhibitory activity (“Dpt-lacZ”) (low IC50), while other than Drosophila S2 cell survival and infection protection (Hs-lacZ test) was found to have virtually no effect (IC50: greater than 50 μg). In particular, the compounds of the present invention represented by TPS-17 and TPS-19 inhibit the diptericin expression inhibitory activity (“Dpt-lacZ”) at IC50: 3 μg and IC50: 1 μg, respectively, and thus have excellent natural properties. It can be seen that it has an immunosuppressive effect.

In the field of anti-insect agents, inhibition of the innate immune pathway is a new mechanism of action. The compound of the present invention is a novel type of anti-inhibitory substance that is highly specific and effective, inhibits the insect's innate immune system, and exterminates insects that mediate crop diseases to prevent the spread of damage. Development as an insecticide is expected.

Claims (10)

Compound represented by the following structural formula (I):

(In the formula (I), X is OH, OR, NRR ′ or a heterocyclic ring containing N as a hetero atom, and R and R ′ are each independently hydrogen, an alkyl group, or an aryl group. Is). The compound of Claim 1 whose alkyl group is a C1-C6 linear or branched alkyl group. The compound according to claim 1, wherein the aryl group is a phenyl group. The compound according to claim 1, wherein the heterocyclic ring containing N as a hetero atom is a 6-membered ring. The compound of claim 1, wherein X is NH ₂ . The compound of claim 1, wherein X is NHMe. The innate immunity inhibitor which contains the compound, its salt, or its hydrate as described in any one of Claims 1-6 as an active ingredient. The anti-insect agent which contains the compound, its salt, or its hydrate as described in any one of Claims 1-6 as an active ingredient. A Drosophila control agent comprising the compound according to any one of claims 1 to 6, a salt thereof or a hydrate thereof as an active ingredient. A method for controlling diseased insects or vector insects using the compound, salt or hydrate thereof according to any one of claims 1 to 6.