DIPHENYLPYRAZOLE COMPOUND, AND PREPARATION METHOD THEREFOR AND APPLICATION THEREOF
20240083871 ยท 2024-03-14
Inventors
Cpc classification
C07D417/12
CHEMISTRY; METALLURGY
C07D405/12
CHEMISTRY; METALLURGY
C07D231/10
CHEMISTRY; METALLURGY
C07D403/12
CHEMISTRY; METALLURGY
C07D231/14
CHEMISTRY; METALLURGY
International classification
C07D401/12
CHEMISTRY; METALLURGY
C07D417/12
CHEMISTRY; METALLURGY
C07D231/10
CHEMISTRY; METALLURGY
C07D405/12
CHEMISTRY; METALLURGY
C07D403/12
CHEMISTRY; METALLURGY
Abstract
Provided is a diphenylpyrazole-based compound of following formula (I), or its pesticidally acceptable salts, which is a novel inhibitor targeting pest GSTs, has broad inhibitory activities against GSTs in various pests, and can effectively delay the metabolism of insecticides by GSTs in pests, thereby reducing the metabolic resistance of pests to insecticides, and is suitable for use as a synergist for insecticides.
##STR00001##
Claims
1-10. (canceled)
11. Use of a diphenylpyrazole-based compound of following formula (I), or its pesticidally acceptable salts as a GSTs inhibitor, ##STR00135## wherein R.sup.1 is: (1) an amido group NHCH(O) which is unsubstituted or substituted by one or more of the following substituents; (a) C.sub.16 alkyl-R.sup.3, C.sub.38 cycloalkyl-R.sup.3, C.sub.26 alkenyl-R.sup.3, C.sub.26 alkynyl-R.sup.3, NHR.sup.3, N(R.sup.3).sub.2, C(O)R.sup.3, NHC.sub.16 alkyl-R.sup.3, C.sub.16 alkyl-NHR.sup.3, C.sub.16 alkyl-N(R.sup.3).sub.2, C.sub.16 alkyl- OR.sup.3, C.sub.38 cycloalkyl-OR.sup.3, OC.sub.16 alkyl-R.sup.3, C.sub.16 alkyl-OC.sub.16 alkyl-R.sup.3, C(O)NHR.sup.3, C(O)N(R.sup.3).sub.2, NHC(O)R.sup.3, C.sub.16 alkyl-NHC(O)R.sup.3, C.sub.16 alkyl-NHC(O)OR.sup.3, NHC(O)C.sub.16 alkyl-R.sup.3, NHC(O)C.sub.16 alkyl-OR.sup.3, C(O)NHC.sub.16 alkyl-R.sup.3 or C.sub.16 alkyl-SR.sup.3; R.sup.3 is each independently selected from: H, O, S, NH, amino, halogen, cyano, C.sub.16 alkyl, C.sub.38 cycloalkyl, SC.sub.16 alkyl, SOH, SO.sub.2C.sub.16 alkyl, -614 membered aryl, -514 membered heterocyclic group, -514 membered heteroaryl and -adamantyl; R.sup.3 is unsubstituted or substituted by one or more of the following substituents: O, OH, halogen, cyano, nitro, CH(O), amino, C.sub.16 alkyl, C.sub.26 alkenyl, C.sub.26 alkynyl, OC.sub.16 alkyl, nitro, sulfonic acid group, -C.sub.16 alkyl-halogen, C.sub.16 alkyl-HS, C.sub.16 alkyl-NH.sub.3.sup.+, C.sub.16 alkyl-OH, C.sub.16 alkyl-NHC.sub.16 alkyl, or C.sub.16 alkyl-N(C.sub.16 alkyl).sub.2; (b) -614 membered aryl, -514 membered heterocyclic group or -514 membered heteroaryl; the 614 membered aryl, the 514 membered heterocyclic group or the 514 membered heteroaryl is unsubstituted or substituted by one or more of the following substituents: O, OH, halogen, cyano, nitro, CH(O), amino, C.sub.16 alkyl, OC.sub.16 alkyl, C.sub.16 alkyl-NHC.sub.16 alkyl, C.sub.16 alkyl-N(C.sub.16 alkyl).sub.2, C(O)OC.sub.16 alkyl, NHC(O)C.sub.16 alkyl, SO.sub.2C.sub.16 alkyl, SO.sub.2NH.sub.2, SO.sub.2NHC.sub.16 alkyl, SO.sub.2N(C.sub.16 alkyl).sub.2, -614 membered aryl, -514 membered heterocyclic group, or -514 membered heteroaryl; (2) N atom and C atom of the amido group NHCH(O) are connected to form a ring structure via C.sub.36 alkylene-, NHC.sub.26 alkylene-, NHC(O)C.sub.16 alkylene-, C.sub.16 alkylene-NHC(O), NHC.sub.16 alkylene-C(O) or C.sub.16 alkylene-C(O); the ring structure is optionally substituted by the following substituents: C.sub.16 alkyl, C.sub.38 cycloalkyl, C.sub.38 cycloalkyl-C.sub.16 alkyl, OC.sub.16 alkyl, -614 membered aryl, -514 membered heterocyclic group, -514 membered heteroaryl, C.sub.16 alkyl-614 membered aryl, C.sub.16 alkyl-514 membered heterocyclic group, C.sub.16 alkyl-514 membered heteroaryl or trifluoroethyl; the 614 membered aryl, the 514 membered heterocyclic group and the 514 membered heteroaryl are optionally substituted by OC.sub.16 alkyl; or, the ring structure is further combined with 58 membered aryl, 58 membered heterocyclic group or 58 membered heteroaryl to fuse into a fused ring; the heterocyclic group contains 14 heteroatoms selected from N, S and O; the heteroaryl contains 14 heteroatoms selected from N, S and O; R.sup.2 is selected from: H, -halogen, -amino, NO.sub.2, CF.sub.3, C.sub.16 alkyl, C.sub.16 alkyl-OH, OR.sup.4, C(O)R.sup.4, C(O)NH.sub.2, NHC(O)R.sup.4, C(O)OR.sup.4 or C(O)ON(R.sup.4).sub.2; R.sup.4 is selected from: H or C.sub.16 alkyl.
12. Use according to claim 11, wherein R.sup.1 is: (1) an amido group NHCH(O) which is substituted by one or more of the following substituents, (a) C.sub.13 alkyl-R.sup.3, C.sub.36 cycloalkyl-R.sup.3, C.sub.25 alkenyl-R.sup.3, C.sub.13 alkyl-NHR.sup.3, C.sub.13 alkyl-OR.sup.3 or OC.sub.13 alkyl-R.sup.3; R.sup.3 are each independently selected from: H, O, S, NH, amino, halogen, cyano, C.sub.13 alkyl, C.sub.36 cycloalkyl, -614 membered aryl, -514 membered heterocyclic group or -514 membered heteroaryl; R.sup.3 is unsubstituted or substituted by one or more of the following substituents: O, OH, halogen, cyano, nitro, CH(O), SOH, amino, C.sub.13 alkyl, C.sub.25 alkenyl, C.sub.25 alkynyl, OC.sub.13 alkyl, nitro, sulfonic acid group, C.sub.13 alkyl-halogen or C.sub.13 alkyl-OH; (b) -614 membered aryl, -514 membered heterocyclic group or -514 membered heteroaryl; the 614 membered aryl, the 514 membered heterocyclic group or the 514 membered heteroaryl is unsubstituted or substituted by one or more of the following substituents: O, OH, halogen, cyano, nitro, CH(O), amino, C.sub.13 alkyl, OC.sub.13 alkyl, C.sub.13 alkyl-NHC.sub.13 alkyl, C.sub.13 alkyl-N(C.sub.13 alkyl).sub.2, C(O)OC.sub.13 alkyl, NHC(O)C.sub.13 alkyl, SO.sub.2C.sub.13 alkyl, SO.sub.2NH.sub.2, SO.sub.2NHC.sub.13 alkyl or SO.sub.2N(C.sub.13 alkyl).sub.2; (2) N atom and C atom of the amido group NHCH(O) are connected to form a ring structure via C.sub.36 alkylene-, NHC.sub.24 alkylene-, NHC(O)C.sub.13 alkylene-, C.sub.13 alkylene-NHC(O), NHC.sub.13 alkylene-C(O) or C.sub.13 alkylene-C(O); the ring structure is optionally substituted by the following substituents: C.sub.13 alkyl, C.sub.36 cycloalkyl, C.sub.36 cycloalkyl-C.sub.13 alkyl, -614 membered aryl, -514 membered heterocyclic group, -514 membered heteroaryl, C.sub.13 alkyl-614 membered aryl, C.sub.13 alkyl-514 membered heterocyclic group, C.sub.13 alkyl-514 membered heteroaryl or trifluoroethyl; the 614 membered aryl, the 514 membered heterocyclic group and the 514 membered heteroaryl are optionally substituted by OC.sub.13 alkyl; the heterocyclic group contains 13 heteroatoms selected from N, S and O; the heteroaryl contains 13 heteroatoms selected from N, S and O; R.sup.2 is: H or halogen.
13. Use according to claim 11, wherein R.sup.1 is: (1) NHC(O)C.sub.13 alkyl-R.sup.3; R.sup.3 is selected from: -610 membered aryl, -510 membered heterocyclic group, -510 membered heteroaryl; R.sup.3 is unsubstituted or substituted by one or more of the following substituents: O, OH, halogen, cyano, nitro, CH(O), SOH, amino, C.sub.13 alkyl, OC.sub.13 alkyl, nitro, sulfonic acid group, C.sub.13 alkyl-halogen, C.sub.13 alkyl-HS, C.sub.13 alkyl-NH.sub.3.sup.+ or C.sub.13 alkyl-OH; (2) NHC(O)-610 membered aryl, NHC(O)-510 membered heterocyclic group or NHC(O)-510 membered heteroaryl; the 610-membered aryl, the 510-membered heterocyclic group or the 510-membered heteroaryl is unsubstituted or substituted by one or more of the following substituents: O, OH, halogen, cyano, nitro, CH(O), amino, C.sub.13 alkyl or OC.sub.13 alkyl; the heterocyclic group contains 13 heteroatoms selected from N, S and O; the heteroaryl contains 13 heteroatoms selected from N, S and O; R.sup.2 is H.
14. Use according to claim 11, wherein R.sup.1 is selected from: ##STR00136##
15. Use according to claim 11, wherein the diphenylpyrazole-based compound of formula (I) or its pesticidally acceptable salts is following compound or its pesticidally acceptable salts: ##STR00137## ##STR00138## ##STR00139## ##STR00140## ##STR00141## ##STR00142## ##STR00143## ##STR00144##
16. Use according to claim 11, wherein the diphenylpyrazole-based compound of formula (I) or its pesticidally acceptable salts is following compound or its pesticidally acceptable salts: ##STR00145##
17. Use according to claim 11, wherein the GSTs inhibitor is a composition comprising the diphenylpyrazole-based compound of formula (I), or its pesticidally acceptable salts, and pesticidally acceptable excipients.
18. Use according to claim 17, wherein the composition comprises one or more insecticides.
19. Use according to claim 11, wherein the GSTs include PxGST1, PxGST3, PxGST1, PxGST2, PxGST4, PxGST1, PxGST1 and PxGST1.
20. Use according to claim 11, wherein the GSTs include PxGST1, PxGST1, PxGST2 and PxGST3.
21. Use according to claim 11, wherein the GSTs include PxGST1 and PxGST3.
22. Use of the diphenylpyrazole-based compound of formula (I), or its pesticidally acceptable salts of claim 11 in preparation of pesticides.
23. Use of the diphenylpyrazole-based compound of formula (I), or its pesticidally acceptable salts of claim 11 in preparation of insecticide synergists.
24. Use according to claim 23, wherein the insecticide synergist delays or reduces the resistance of pests to insecticides.
25. Use according to claim 23, wherein the insecticides include ryanodine receptor modulator insecticides and voltage-dependent sodium ion channel blocker insecticides.
26. Use according to claim 23, wherein the insecticides include diamide insecticides and oxadiazine insecticides.
27. Use according to claim 23, wherein the insecticides include chlorantraniliprole and indoxacarb.
28. Use according to claim 23, wherein the pests include field crop pests and economic crop pests.
29. Use according to claim 23, wherein the pests include Plutella xylostella, Mythimna separata, Pyrausta nubilalis, Chilo suppressalis, Nilaparvata lugens, Spodoptera frugiperda, Helicoverpa armigera, Carposina sasakii and Spodoptera litura.
30. Use according to claim 22, wherein the diphenylpyrazole-based compound of formula (I), or its pesticidally acceptable salts inhibits GSTs.
Description
DESCRIPTION OF THE DRAWINGS
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DETAILED EMBODIMENTS
[0129] The present invention will be described below through examples, but the present invention is not limited thereto. The experimental methods shown in the following examples are conventional methods unless otherwise specified. The reagents and materials shown are all commercially available.
Preparation Example 1 Preparation of Compound PXG1
[0130] ##STR00106##
[0131] Method 1:
[0132] (1) Preparation of hydrazide compound C1: 10 g of compound A1 (A1 was prepared by reference to J. Org. Chem. 2010, 75, 3, 984-987; HNMR (400 MHz, CDCl.sub.3): 3.83 (s, 3H), 7.33-7.36 (m, 2H), 7.41-7.50 (m, 7H), 7.87 (m, 2H). the same below), 100 ml of absolute ethanol, 20 ml of hydrazine hydrate was first added into 100 ml three-necked flask, heated to reflux overnight, cooled and filtered by suction to obtain 9.5 g of compound C1, yield 95%.
[0133] (2) Compound F1 (carboxylic acid) (2.5 g, 1 eq), DMF (10 ml), EDCI (4.14 g, 1.2 eq), potassium carbonate (6.21 g, 2.5 eq) were added into 100 ml three-necked flask, stirred for 10 minutes and then added with compound C1 (hydrazide) (5.5 g, 1.1 eq), stirred overnight at room temperature, the reaction was monitored by TLC until completion. The resultant was diluted with water, the formed precipitate was filtered off and dried, the crude compound was purified by reverse phase HPLC without additional treatment with the eluent methanol-water, the selected fractions were combined and concentrated to obtain the target product PXG1.
[0134] Or, method 2:
[0135] (1) Preparation of compound B1: 10 g of compound A1, 100 ml of absolute ethanol, 28.7 ml (2 eq) of 10% NaOH was first put into 100 ml three-necked flask, heated to reflux for 5 hours, the reaction was completely cooled, and was adjusted to pH=2-3 with 10% hydrochloric acid, filtered by suction to obtain 8 g of compound B1, yield 85%.
[0136] (2) Compound B1 (carboxylic acid) (6.5 g, 1 eq), DMF (10 ml), EDCI (5.75 g, 1.2 eq), potassium carbonate (8.63 g, 2.5 eq) were added into 100 ml three-necked flask, stirred for 10 minutes and then added with compound G1 (hydrazide) (4.21 g, 1.1 eq), stirred overnight at room temperature, the reaction was monitored by TLC until completion. The resultant was diluted with water, the formed precipitate was filtered off and dried, the crude compound was purified by reverse phase HPLC without additional treatment with the eluent methanol-water, the selected fractions were combined and concentrated to obtain the target product PXG1.
[0137] MS(ES-API) cacld. for C.sub.22H.sub.17N.sub.5O.sub.3 found 400.2 [M+1].sup.+. (
Preparation Example 2 Preparation of Compound PXG2
[0138] ##STR00107##
[0139] Method 1: Compound PXG2 was prepared using the same method and steps as in Preparation Example 1, except that compound F2 (carboxylic acid) (2.83 g, 1 eq) was added into the 100 ml three-neck flask in step (2). Or,
[0140] Method 2: Compound PXG2 was prepared using the same method and steps as in Preparation Example 1, except that compound G2 (hydrazide) (4.70 g, 1.1 eq) was added after stirring for 10 minutes in step (2).
[0141] MS(ES-API) cacld. for C.sub.22H.sub.19N.sub.5O.sub.2S found 418.2 [M+1].sup.+. (
Preparation Example 3 Preparation of Compound PXG3
[0142] ##STR00108##
[0143] Method 1: Compound PXG3 was prepared using the same method and steps as in Preparation Example 1, except that compound F3 (carboxylic acid) (2.72 g, 1 eq) was added into the 100 ml three-neck flask in step (2). Or,
[0144] Method 2: Compound PXG3 was prepared using the same method and steps as in Preparation Example 1, except that compound G3 (hydrazide) (4.54 g, 1.1 eq) was added after stirring for 10 minutes in step (2).
[0145] MS(ES-API) cacld. for C.sub.24H.sub.21N.sub.5O.sub.2 found 412.2 [M+1].sup.+. (
Preparation Example 4 Preparation of Compound PXG4
[0146] ##STR00109##
[0147] Method 1: Compound PXG4 was prepared using the same method and steps as in Preparation Example 1, except that compound F4 (carboxylic acid) (3.81 g, 1 eq) was added into the 100 ml three-neck flask in step (2). Or,
[0148] Method 2: Compound PXG4 was prepared using the same method and steps as in Preparation Example 1, except that compound G4 (hydrazide) (6.22 g, 1.1 eq) was added after stirring for 10 minutes in step (2).
[0149] MS(ES-API) cacld. for C.sub.26H.sub.24N.sub.4O.sub.5 found 473.2 [M+1].sup.+. (
Preparation Example 5 Preparation of Compound PXG5
[0150] ##STR00110##
[0151] Method 1: Compound PXG5 was prepared using the same method and steps as in Preparation Example 1, except that compound F5 (carboxylic acid) (2.74 g, 1 eq) was added into the 100 ml three-neck flask in step (2). Or,
[0152] Method 2: Compound PXG5 was prepared using the same method and steps as in Preparation Example 1, except that compound G5 (hydrazide) (4.57 g, 1.1 eq) was added after stirring for 10 minutes in step (2).
[0153] MS(ES-API) cacld. for C.sub.24H.sub.20N.sub.4O.sub.3 found 413.2 [M+1].sup.+. (
Preparation Example 6 Preparation of Compound PXG6
[0154] ##STR00111##
[0155] Method 1: Compound PXG6 was prepared using the same method and steps as in Preparation Example 1, except that compound G6 (hydrazide) (5.04 g, 1 eq) was added into the 100 ml three-neck flask in step (2).
[0156] MS(ES-API) cacld. for C.sub.24H.sub.23N.sub.5O.sub.3 found 430.2 [M+1]+. (
Preparation Example 7 Preparation of Compound PXG7
[0157] ##STR00112##
[0158] Method 1:
[0159] (1) Preparation of hydrazide compound C2: 10 g of compound A2 (A2 was prepared by reference to J Org. Chem. 2010, 75, 3, 984-987), 100 ml of absolute ethanol, 20 ml of hydrazine hydrate was first added into 100 ml three-necked flask, heated to reflux overnight, cooled and filtered by suction to obtain 7.5 g of compound C2, yield 75%.
[0160] (2) Compound F7 (carboxylic acid) (2.10 g, 1 eq), DMF (10 ml), EDCI (4.14 g, 1.2 eq), potassium carbonate (6.21 g, 2.5 eq) were added into 100 ml three-necked flask, stirred for 10 minutes and then added with compound C2 (hydrazide) (6.0 g, 1.1 eq), stirred overnight at room temperature, the reaction was monitored by TLC until completion. The resultant was diluted with water, the formed precipitate was filtered off and dried, the crude compound was purified by reverse phase HPLC without additional treatment with the eluent methanol-water, the selected fractions were combined and concentrated to obtain the target product PXG7.
[0161] Method 2:
[0162] (1) Preparation of compound B2: 10 g of compound A2, 100 ml of absolute ethanol, 28.7 ml (2 eq) of 10% NaOH was first added into 100 ml three-necked flask, heated to reflux for 5 hours, the reaction was completely cooled, and was adjusted to pH=2-3 with 10% hydrochloric acid, filtered by suction to obtain 6.8 g of compound B2, yield 70%.
[0163] (2) Compound B2 (carboxylic acid) (7.0 g, 1 eq), DMF 10 ml, EDCI (5.75 g, 1.2 eq), potassium carbonate (8.63 g, 2.5 eq) were added into 100 ml three-necked flask, stirred for 10 minutes and then added with compound G7 (hydrazide) (3.48 g, 1.1 eq), stirred overnight at room temperature, the reaction was monitored by TLC until completion. The resultant was diluted with water, the formed precipitate was filtered off and dried, the crude compound was purified by reverse phase HPLC without additional treatment with the eluent methanol-water, the selected fractions were combined and concentrated to obtain the target product PXG7.
[0164] MS(ES-API) cacld. for C.sub.22H.sub.16ClN.sub.5O.sub.2 found 418.0[M+1].sup.+. (
Preparation Example 8 Preparation of Compound PXG8
[0165] ##STR00113##
[0166] Method 1: Compound PXG8 was prepared using the same method and steps as in Preparation Example 1, except that compound F8 (carboxylic acid) (3.24 g, 1 eq) was added into the 100 ml three-neck flask in step (2).
[0167] Method 2: Compound PXG8 was prepared using the same method and steps as in Preparation Example 1, except that compound G8 (hydrazide) (5.34 g, 1.1 eq) was added after stirring for 10 minutes in step (2).
[0168] MS(ES-API) cacld. for C.sub.25H.sub.20N.sub.4O.sub.4 found 441.0[M+1].sup.+. (
Preparation Example 9 Preparation of Compound PXG9
[0169] ##STR00114##
[0170] Method 1: Compound PXG9 was prepared using the same method and steps as in Preparation Example 1, except that compound F9 (carboxylic acid) (4.12 g, 1 eq) was added into the 100 ml three-neck flask in step (2). Or
[0171] Method 2: Compound PXG9 was prepared using the same method and steps as in Preparation Example 1, except that compound G9 (hydrazide) (6.68 g, 1.1 eq) was added after stirring for 10 minutes in step (2).
[0172] MS(ES-API) cacld. for C.sub.25H.sub.23N.sub.5O.sub.4S found 490.2[M+1].sup.+. (
Preparation Example 10 Preparation of Compound PXG10
[0173] ##STR00115##
[0174] Method 1: Compound PXG10 was prepared using the same method and steps as in Preparation Example 1, except that compound F10 (carboxylic acid) (3.00 g, 1 eq) was added into the 100 ml three-neck flask in step (2).
[0175] Method 2: Compound PXG10 was prepared using the same method and steps as in Preparation Example 1, except that compound G10 (hydrazide) (4.95 g, 1.1 eq) was added after stirring for 10 minutes in step (2).
[0176] MS(ES-API) cacld. for C.sub.25H.sub.22N.sub.4O.sub.3 found 427.2[M+1].sup.+. (
Preparation Example 11 Preparation of Compound PXG11
[0177] ##STR00116##
[0178] Method 1: Compound PXG11 was prepared using the same method and steps as in Preparation Example 1, except that compound F11 (carboxylic acid) (2.20 g, 1 eq) was added into the 100 ml three-neck flask in step (2). Or
[0179] Method 2: Compound PXG11 was prepared using the same method and steps as in Preparation Example 1, except that compound G11 (hydrazide) (3.74 g, 1.1 eq) was added after stirring for 10 minutes in step (2).
[0180] MS(ES-API) cacld. for C.sub.23H.sub.18N.sub.4O.sub.2 found 383.2[M+1].sup.+. (
Preparation Example 12 Preparation of Compound PXG12
[0181] ##STR00117##
[0182] Method 1: Compound PXG12 was prepared using the same method and steps as in Preparation Example 1, except that compound G12 (hydrazide) (5.64 g, 1 eq) was added into the 100 ml three-neck flask in step (2).
[0183] MS(ES-API) cacld. for C.sub.26H.sub.21N.sub.5O.sub.3 found 452.2[M+1].sup.+. (
Preparation Example 13 Preparation of Compound PXG13
[0184] ##STR00118##
[0185] Method 1: Compound PXG13 was prepared using the same method and steps as in Preparation Example 1, except that compound G13 (hydrazide) (4.32 g, 1 eq) was added into the 100 ml three-neck flask in step (2).
[0186] MS(ES-API) cacld. for C.sub.22H.sub.21N.sub.5O.sub.3 found 404.2[M+1].sup.+. (
Preparation Example 14 Preparation of Compound PXG14
[0187] ##STR00119##
[0188] Method 1: Compound PXG14 was prepared using the same method and steps as in Preparation Example 1, except that compound G14 (hydrazide) (6.47 g, 1 eq) was added into the 100 ml three-neck flask in step (2).
[0189] MS(ES-API) cacld. for C.sub.27H.sub.23N.sub.5O.sub.4 found 482.2[M+1].sup.+. (
Preparation Example 15 Preparation of Compound PXG15
[0190] ##STR00120##
[0191] Method 1: Compound PXG15 was prepared using the same method and steps as in Preparation Example 1, except that compound G15 (hydrazide) (6.41 g, 1 eq) was added into the 100 ml three-neck flask in step (2).
[0192] MS(ES-API) cacld. for C.sub.28H.sub.25N.sub.5O.sub.3 found 480.2[M+1]+. (
Preparation Example 16 Preparation of Compound PXG16
[0193] ##STR00121##
[0194] Method 1: Compound PXG16 was prepared using the same method and steps as in Preparation Example 1, except that compound G16 (hydrazide) (5.42 g, 1 eq) was added into the 100 ml three-neck flask in step (2).
[0195] MS(ES-API) cacld. for C.sub.25H.sub.25N.sub.5O.sub.3 found 444.2[M+1].sup.+. (
Preparation Example 17 Preparation of Compound PXG17
[0196] ##STR00122##
[0197] Method 1: Compound PXG17 was prepared using the same method and steps as in Preparation Example 1, except that compound G17 (hydrazide) (6.47 g, 1 eq) was added into the 100 ml three-neck flask in step (2).
[0198] MS(ES-API) cacld. for C.sub.27H.sub.23N.sub.5O.sub.4 found 482.2[M+1].sup.+. (
Preparation Example 18 Preparation of Compound PXG18
[0199] ##STR00123##
[0200] Method 1: Compound PXG18 was prepared using the same method and steps as in Preparation Example 1, except that compound F18 (carboxylic acid) (3.28 g, 1 eq) was added into the 100 ml three-neck flask in step (2). Or
[0201] Method 2: Compound PXG18 was prepared using the same method and steps as in Preparation Example 1, except that compound G18 (hydrazide) (5.39 g, 1.1 eq) added put after stirring for 10 minutes in step (21.
[0202] MS(ES-API) cacld. for C.sub.25H.sub.22N.sub.4O.sub.4 found 443.2[M+1].sup.+. (
Preparation Example 19 Preparation of Compound PXG19
[0203] ##STR00124##
[0204] Method 1: Compound PXG19 was prepared using the same method and steps as in Preparation Example 1, except that compound F19 (carboxylic acid) (3.19 g, 1 eq) was added into the 100 ml three-neck flask in step (2). Or
[0205] Method 2: Compound PXG19 was prepared using the same method and steps as in Preparation Example 1, except that compound G19 (hydrazide) (5.25 g, 1.1 eq) was added after stirring for 10 minutes in step (2).
[0206] MS(ES-API) cacld. for C.sub.25H.sub.19N.sub.5O.sub.3 found 438.2[M+1]+. (
Preparation Example 20 Preparation of Compound PXG20
[0207] ##STR00125##
[0208] Method 1: Compound PXG20 was prepared using the same method and steps as in Preparation Example 7, except that compound F20 (carboxylic acid) (2.09 g, 1 eq) was added into the 100 ml three-neck flask in step (2). Or
[0209] Method 2: Compound PXG20 was prepared using the same method and steps as in Preparation Example 7, except that compound G20 (hydrazide) (3.47 g, 1.1 eq) was added after stirring for 10 minutes in step (2).
[0210] MS(ES-API) cacld. for C.sub.22H.sub.16ClN.sub.5O.sub.2 found 418.2[M+1].sup.+. (
Preparation Example 21 Preparation of Compound PXG21
[0211] ##STR00126##
[0212] Method 1: Compound PXG21 was prepared using the same method and steps as in Preparation Example 1, except that compound F21 (carboxylic acid) (3.67 g, 1 eq) was added into the 100 ml three-neck flask in step (2). Or
[0213] Method 2: Compound PXG21 was prepared using the same method and steps as in Preparation Example 1, except that compound G21 (hydrazide) (6.00 g, 1.1 eq) was added after stirring for 10 minutes in step (2).
[0214] MS(ES-API) cacld. for C.sub.22H.sub.16ClN.sub.5O.sub.2 found 465.2[M+1].sup.+. (
Preparation Example 22 Preparation of Compound PXG22
[0215] ##STR00127##
[0216] Method 1: Compound PXG22 was prepared using the same method and steps as in Preparation Example 1, except that compound F22 (carboxylic acid) (3.40 g, 1 eq) was added into the 100 ml three-neck flask in step (2). Or
[0217] Method 2: Compound PXG22 was prepared using the same method and steps as in Preparation Example 1, except that compound G22 (hydrazide) (5.58 g, 1.1 eq) was added after stirring for 10 minutes in step (2).
[0218] MS(ES-API) cacld. for C.sub.26H.sub.19N.sub.5O.sub.3 found 450.2[M+1].sup.+. (
Preparation Example 23 Preparation of Compound PXG23
[0219] ##STR00128##
[0220] Method 1: Compound PXG23 was prepared using the same method and steps as in Preparation Example 1, except that compound F23 (carboxylic acid) (3.19 g, 1 eq) was added into the 100 ml three-neck flask in step (2). Or
[0221] Method 2: Compound PXG23 was prepared using the same method and steps as in Preparation Example 1, except that compound G23 (hydrazide) (5.25 g, 1.1 eq) was added after stirring for 10 minutes in step (2).
[0222] MS(ES-API) cacld. for C.sub.25H.sub.19N.sub.5O.sub.3 found 438.2[M+1].sup.+. (
Preparation Example 24 Preparation of Compound PXG24
[0223] ##STR00129##
[0224] Method 1: Compound PXG24 was prepared using the same method and steps as in Preparation Example 1, except that compound F24 (carboxylic acid) (1.62 g, 1 eq) was added into the 100 ml three-neck flask in step (2). Or
[0225] Method 2: Compound PXG24 was prepared using the same method and steps as in Preparation Example 1, except that compound G24 (hydrazide) (2.86 g, 1.1 eq) was added after stirring for 10 minutes in step (2).
[0226] MS(ES-API) cacld. for C.sub.19H.sub.18N.sub.4O.sub.3 found 351.2[M+1].sup.+. (
Preparation Example 25 Preparation of Compound PXG25
[0227] ##STR00130##
[0228] Method 1: Compound PXG25 was prepared using the same method and steps as in Preparation Example 1, except that compound F25 (carboxylic acid) (2.50 g, 1 eq) was added into the 100 ml three-neck flask in step (2). Or
[0229] Method 2: Compound PXG25 was prepared using the same method and steps as in Preparation Example 1, except that compound G25 (hydrazide) (4.21 g, 1.1 eq) was added after stirring for 10 minutes in step (2).
[0230] MS(ES-API) cacld. for C.sub.22H.sub.17N.sub.5O.sub.3 found 400.2[M+1].sup.+. (
Preparation Example 26 Preparation of Compound PXG26
[0231] ##STR00131##
[0232] Method 1: Compound PXG26 was prepared using the same method and steps as in Preparation Example 1, except that compound F26 (carboxylic acid) (3.49 g, 1 eq) was added into the 100 ml three-neck flask in step (2). Or
[0233] Method 2: Compound PXG26 was prepared using the same method and steps as in Preparation Example 1, except that compound G26 (hydrazide) (5.72 g, 1.1 eq) was added after stirring for 10 minutes in step (2).
[0234] MS(ES-API) cacld. for C.sub.28H.sub.30N.sub.4O.sub.2 found 455.2[M+1].sup.+. (
Preparation Example 27 Preparation of Compound PXG27
[0235] ##STR00132##
[0236] Method 1: Compound PXG27 was prepared using the same method and steps as in Preparation Example 1, except that compound F27 (carboxylic acid) (2.52 g, 1 eq) was added into the 100 ml three-neck flask in step (2). Or
[0237] Method 2: Compound PXG27 was prepared using the same method and steps as in Preparation Example 1, except that compound G27 (hydrazide) (4.24 g, 1.1 eq) was added after stirring for 10 minutes in step (2).
[0238] MS(ES-API) cacld. for C.sub.21H.sub.16N.sub.6O.sub.3 found 455.2[M+1]+. (
Preparation Example 28 Preparation of Compound PXG28
[0239] ##STR00133##
[0240] Method 1: Compound PXG28 was prepared using the same method and steps as in Preparation Example 1, except that compound F28 (carboxylic acid) (2.05 g, 1 eq) was added into the 100 ml three-neck flask in step (2). Or
[0241] Method 2: Compound PXG28 was prepared using the same method and steps as in Preparation Example 1, except that compound G28 (hydrazide) (3.52 g, 1.1 eq) was added after stirring for 10 minutes in step (2).
[0242] MS(ES-API) cacld. for C.sub.22H.sub.22N.sub.4O.sub.2 found 375.2 [M+1].sup.+. (
Preparation Example 29 Preparation of Compound PXG29
[0243] ##STR00134##
[0244] Method 1: Compound PXG29 was prepared using the same method and steps as in Preparation Example 1, except that compound F29 (carboxylic acid) (4.12 g, 1 eq) was added into the 100 ml three-neck flask in step (2). Or
[0245] Method 2: Compound PXG29 was prepared using the same method and steps as in Preparation Example 1, except that compound G29 (hydrazide) (6.68 g, 1.1 eq) was added after stirring for 10 minutes in step (2).
[0246] MS(ES-API) cacld. for C.sub.25H.sub.23N.sub.5O.sub.4S found 490.2[M+1].sup.+. (
Experimental Example 1: Inhibitory Activity Assay of the Compounds PXG1-PXG29 (29 Compounds) of the Present Invention Against the Total GSTs Enzyme of 6 Kinds of Field Crop Pests and 3 Kinds of Economic Crop Pests
[0247] Experiment: About 50 mg of larvae/nymphs of nine major pests such as Plutella xylostella, Mythimna separata, Pyrausta nubilalis, Chilo suppressalis, Nilaparvata lugens, Spodoptera frugiperda, Helicoverpa armigera, Carposina sasakii, Spodoptera litura were taken, grounded in a mortar pre-cooled at 80 C., and then 1 mL of 100 mM sodium phosphate buffer (1 mM EDTA, pH 7.2) was added and fully homogenized. The homogenate was transferred to a 1.5 mL centrifuge tube at 4 C., and centrifuged at 14,000 g for 30 minutes, and the supernatant was transferred to a new centrifuge tube as the GSTs total enzyme. The protein concentrations of the above total enzymes were determined using the BCA method.
[0248] 100 L of PBS buffer (100 mM, pH 7.2) containing 1 mM GSH was taken and placed in a 96-well plate, 5 g of insect total GSTs enzymes and an appropriate concentration of compounds PXG1-PXG29 (29 compounds) of the present invention were added to each well, after uniformly mixing, and allowed to stand at 30 C. for 10 min. Then 100 L of PBS buffer (100 mM, pH 7.2) containing 1 mM CDNB was added to each well. Immediately after mixing, the mixture was placed in a microplate reader to measure the absorbance at a wavelength of 340 nm (A 340) once every 1 min, the measurement was carried out continuously for 5 min, the light absorption values of each measuring point were recorded and the inhibition rate in vitro of the compounds with different concentrations against insects total GSTs enzymes were calculated. Each group of assays was repeated three times, and total GSTs enzymes inactivated at 100 C. for 5 min were used as negative controls, and GTX was used as positive controls. GraphPad Prism 5 software was used for analysis and calculation of IC.sub.50. Relative inhibition was calculated using the following formula:
[0250] Results: The experimental results are shown in Table 1, Table 2, and
TABLE-US-00003 TABLE 1 The inhibitory effect of compounds PXG1-PXG29 of the present invention on the total GSTs enzymes of 3 kinds of economic crop pests Plutella xylostella GSTs Spodoptera litura GSTs 100 M Carposina sasakii GSTs 100 M Inhibition IC.sub.50 100 M IC.sub.50 Inhibition IC.sub.50 Index rate % (M) Inhibition rate % (M) rate % (M) PXG1 70.90 27.57 71.91 52.63 80.06 17.67 PXG2 69.27 18.09 35.12 >100 57.13 >100 PXG3 25.47 >100 23.07 >100 17.09 >100 PXG4 10.64 >100 2.34 >100 17.52 >100 PXG5 11.58 >100 23.22 >100 37.80 >100 PXG 6 21.15 >100 18.21 >100 6.90 >100 PXG 7 15.46 >100 48.15 >100 57.87 >100 PXG 8 35.73 >100 19.83 >100 44.25 >100 PXG 9 3.94 >100 12.55 >100 12.58 >100 PXG 10 17.71 >100 32.54 >100 3.87 >100 PXG 11 22.90 >100 76.09 >100 24.52 >100 PXG 12 15.08 >100 17.94 >100 4.60 >100 PXG 13 26.60 >100 25.90 >100 9.91 >100 PXG 14 10.33 >100 20.64 >100 2.49 >100 PXG 15 16.83 >100 17.01 >100 5.22 >100 PXG 16 8.64 >100 6.98 >100 0.15 >100 PXG 17 1.63 >100 12.40 >100 4.14 >100 PXG 18 32.94 >100 13.69 >100 7.95 >100 PXG 19 23.03 >100 19.42 >100 31.70 >100 PXG 20 1.50 >100 10.99 >100 7.09 >100 PXG 21 16.96 >100 35.52 >100 5.00 >100 PXG 22 60.50 52.98 42.77 >100 56.09 43.1 PXG 23 26.35 >100 19.76 >100 12.89 >100 PXG 24 29.72 >100 22.33 >100 28.38 >100 PXG 25 31.66 >100 48.54 >100 22.37 >100 PXG 26 25.09 >100 18.11 >100 15.89 >100 PXG 27 42.68 >100 43.24 >100 45.08 >100 PXG 28 22.40 >100 49.51 >100 46.09 >100 PXG 29 9.32 >100 33.57 >100 4.60 >100 GTX 73.72 12.9 95.39 6.87 85.61 15.19
TABLE-US-00004 TABLE 2 The inhibitory effect of compounds PXG1-PXG29 of the present invention on the total GSTs enzymes of 9 kinds of field crop pests Mythimna Pyrausta Chilo Nilaparvata Spodoptera Helicoverpa separata nubilalis suppressalis lugens frugiperda armigera GSTs GSTs GSTs GSTs GSTs GSTs 100 M 100 M 100 M 100 M 100 M 100 M Inhibition IC.sub.50 Inhibition IC.sub.50 Inhibition IC.sub.50 Inhibition IC.sub.50 Inhibition IC.sub.50 Inhibition IC.sub.50 Index rate % (M) rate % (M) rate % (M) rate % (M) rate % (M) rate % (M) PXG1 95.19 7.20 87.80 12.11 92.32 8.24 100.60 3.62 58.40 62.38 77.22 52.78 PXG2 90.46 15.69 64.92 42.01 52.60 47.88 91.51 2.18 64.72 40.16 57.53 52.13 PXG3 42.96 >100 17.35 >100 26.63 >100 35.05 >100 35.46 >100 20.74 >100 PXG4 0.00 >100 7.23 >100 10.90 >100 5.99 >100 7.57 >100 4.07 >100 PXG5 47.24 >100 14.08 >100 28.17 >100 23.90 >100 31.42 >100 9.57 >100 PXG6 38.85 >100 13.70 >100 15.98 >100 1.46 >100 11.37 >100 23.21 >100 PXG7 61.50 36.12 63.59 47.19 70.09 18.3 25.89 >100 45.78 >100 46.85 >100 PXG8 38.06 >100 14.10 >100 29.11 >100 53.31 >100 35.01 >100 24.81 >100 PXG9 2.72 >100 5.71 >100 34.79 >100 10.48 >100 5.56 >100 5.25 >100 PXG10 2.50 >100 3.85 >100 5.85 >100 19.24 >100 20.93 >100 9.75 >100 PXG11 31.76 >100 23.84 >100 54.79 >100 8.16 >100 20.95 >100 8.09 >100 PXG12 19.42 >100 19.48 >100 29.54 >100 3.30 >100 12.52 >100 5.00 >100 PXG13 40.68 >100 34.93 >100 46.18 >100 22.14 >100 19.41 >100 0.86 >100 PXG14 13.21 >100 10.91 >100 28.65 >100 17.12 >100 9.38 >100 12.41 >100 PXG15 4.29 >100 4.69 >100 18.72 >100 2.97 >100 0.41 >100 1.48 >100 PXG16 10.50 >100 0.41 >100 10.53 >100 1.00 >100 1.72 >100 2.28 >100 PXG17 4.94 >100 12.14 >100 5.11 >100 16.13 >100 2.26 >100 9.63 >100 PXG18 2.16 >100 23.84 >100 23.00 >100 45.31 >100 28.67 >100 3.02 >100 PXG19 47.16 >100 15.64 >100 22.03 >100 10.00 >100 19.64 >100 6.30 >100 PXG20 30.36 >100 41.65 >100 25.43 >100 19.64 >100 32.18 >100 2.90 >100 PXG21 18.46 >100 17.75 >100 11.87 >100 32.48 >100 0.49 >100 5.68 >100 PXG22 60.97 19.01 42.38 >100 58.55 59.29 95.14 1.72 62.87 77.05 84.32 32.8 PXG23 22.92 >100 15.55 >100 22.83 >100 24.74 >100 22.33 >100 0.43 >100 PXG24 35.70 >100 44.66 >100 41.15 >100 42.77 >100 19.70 >100 35.93 >100 PXG25 31.23 >100 36.57 >100 51.03 >100 41.85 >100 15.80 >100 33.02 >100 PXG26 42.48 >100 0.99 >100 17.07 >100 28.56 >100 16.01 >100 24.75 >100 PXG27 45.15 >100 46.93 >100 68.13 34.48 66.99 39.08 46.58 >100 44.63 >100 PXG28 55.24 >100 55.49 >100 58.02 60.53 45.44 >100 45.15 >100 23.64 >100 PXG29 4.90 >100 10.83 >100 13.81 >100 10.71 >100 4.82 >100 10.99 >100 GTX 87.20 16.33 86.35 23.46 99.97 6.65 95.14 2.31 95.20 14.41 96.30 14.05
[0251] Conclusion: the compounds PXG1, PXG2 and PXG22 of the present invention have obvious inhibitory activity on the total GSTs enzymes of various field crop pests and economic crop pests, and can reduce metabolic activity towards insecticides of the total GSTs enzymes of field crop pests such as Mythimna separata, Pyrausta nubilalis, Chilo suppressalis, Nilaparvata lugens, Spodoptera frugiperda and Helicoverpa armigera, and economic crop pests such as Plutella xylostella, Carposina sasakii and Spodoptera litura.
Experimental Example 2: Enzyme Kinetic Assay of 8 Kinds of Known GSTs Recombinant Proteins in Plutella xylostella
[0252] Experiment: There are currently 8 known Plutella xylostella GSTs (PxGSTs), namely PxGST1, PxGST3, PxGST1, PxGST2, PxGST4, PxGST1, PxGST1 and PxGST1. The recombinant proteins of the 8 PxGSTs were obtained by prokaryotic expression, and the enzyme kinetics of the recombinant PxGSTs proteins were determined by the CDNB method. 1 g of PxGST recombinant protein was added to 200 L of PBS buffer (100 mM, pH 7.2) containing gradient concentrations of CDNB (0.05-1.60 mM) and 1 mM GSH. The absorbance at a wavelength of 340 nm (A.sub.340) was measured in every 1 min at 30 C. using a microplate reader, and the measurement was continued for 5 min. The obtained light absorption value was converted into molar CDNB conjugated/min/mg, and the extinction coefficient 340=9600 M.sup.1 cm.sup.1. Michaelis-Menten plots were generated according to different concentrations of CDNB to obtain enzyme kinetic parameters.
[0253] Results: By deterring the enzyme kinetics of the recombinant protein with CDNB as substrate, it was found that only PxGST1, PxGST1, PxGST2 and PxGST3 have catalytic activity on CDNB, and the remaining four PxGSTs (PxGST4, PxGST1, PxGST1 and PxGST1) have no catalytic activity on CDNB. The experimental results are shown in Table 3 and
TABLE-US-00005 TABLE 3 Kinetic parameters of Pxlutella xylostella PxGSTs recombinant protease PxGSTs recombinant K.sub.m V.sub.max protein (mM) (mol min.sup.1mg.sup.1) PxGST1 0.032 7.64 PxGST3 0.37 0.49 PxGST1 0.12 1.7 PxGST2 0.19 0.26 PxGSTo4 N/A N/A PxGST1 N/A N/A PxGSTu1 N/A N/A
[0254] Conclusion: Among the 8 kinds of PxGSTs, only 4 kinds of PxGSTs of PxGST1, PxGST1, PxGST2 and PxGST3 have GSH binding sites, which belong to the GST type with insecticide metabolic activity in Plutella xylostella.
Experimental Example 3: Inhibitory Activity Assay of Compound PXG1-PXG29 of the Present Invention to 4 Kinds of GSTs Having GSH Binding Site of Plutella xylostella
[0255] Experiment: 100 L of PBS buffer (100 mM, pH 7.2) was taken and added to a 96-well plate, 1 g of PxGSTs recombinant protein and an appropriate concentration of the compounds PXG1-PXG29 of the present invention were added to each well, after uniformly mixing, and allowed to stand at 30 C. for 10 min. Then 100 L of PBS buffer (100 mM, pH 7.2) containing 1 mM CDNB and 1 mM GSH was successively added to each well. Immediately after mixing, the mixture was placed in a microplate reader to measure the absorbance at a wavelength of 340 nm (A.sub.340) once every 1 min, the measurement was continued for 5 min, the light absorption values of each measuring point were recorded and the inhibition rate in vitro of the compounds PXG1-PXG29 of the present invention with different concentrations against PxGSTs recombinant protein were calculated. Each group of assays was repeated three times, and PxGSTs recombinant protein inactivated at 100 C. for 5 min was used as negative control, and GTX was used as positive control. GraphPad Prism 5 software was used for analysis and calculation of IC.sub.50. Relative inhibition was calculated using the following formula:
[0257] Results: The experimental results are shown in Table 4 and
TABLE-US-00006 TABLE 4 The inhibitory effect of compounds PXG1-PXG29 of the present invention on 4 kinds of PxGSTs recombinant protein PxGST1 PxGSTl PxGST2 PxGST3 100 M 100 M 100 M 100 M Inhibition IC.sub.50 Inhibition IC.sub.50 Inhibition IC.sub.50 Inhibition IC.sub.50 Index rate % (M) rate % (M) rate % (M) rate % (M) PXG1 68.91 40.25 25.54 >100 58.07 57.77 17.64 >100 PXG2 87.75 31.41 25.12 >100 14.56 >100 4.00 >100 PXG3 72.39 31.58 36.06 >100 27.59 >100 1.11 >100 PXG4 18.41 50.56 43.63 >100 21.57 >100 8.88 >100 PXG5 81.66 17.78 35.57 >100 24.51 >100 2.97 >100 PXG6 12.21 >100 49.08 >100 33.65 >100 4.85 >100 PXG7 55.74 65.37 35.15 >100 20.06 >100 5.93 >100 PXG8 69.47 30.64 37.78 >100 42.39 >100 3.30 >100 PXG9 30.42 >100 28.59 >100 31.45 >100 3.88 >100 PXG10 91.02 24.25 29.36 >100 19.17 >100 2.03 >100 PXG11 35.34 >100 60.66 38.25 22.64 >100 7.28 >100 PXG12 40.17 >100 48.30 >100 33.65 >100 10.90 >100 PXG13 14.11 >100 34.22 >100 22.76 >100 18.43 >100 PXG14 14.93 >100 52.24 53.23 36.41 >100 15.32 >100 PXG15 7.86 >100 42.48 62.13 25.58 >100 4.62 >100 PXG16 5.35 >100 70.49 49.3 39.29 >100 4.12 >100 PXG17 2.22 >100 48.92 >100 43.85 >100 4.43 >100 PXG18 82.05 29.9 31.74 >100 13.48 >100 2.47 >100 PXG19 58.74 40.79 45.88 82.12 32.46 >100 5.57 >100 PXG20 23.01 >100 71.80 >100 29.56 >100 15.67 >100 PXG21 7.82 >100 44.93 >100 11.65 >100 2.67 >100 PXG22 78.78 25.03 45.71 80.61 76.68 27.15 65.84 36.89 PXG23 77.66 32.92 35.86 >100 26.95 >100 6.04 >100 PXG24 23.09 >100 14.94 >100 19.66 >100 28.32 >100 PXG25 23.75 >100 1.47 >100 15.27 >100 2.07 >100 PXG26 21.58 >100 67.92 35.95 24.71 >100 7.12 >100 PXG27 41.66 >100 25.10 >100 42.39 >100 17.56 >100 PXG28 70.89 63.53 19.53 >100 23.38 >100 0.96 >100 PXG29 16.73 >100 53.27 54.96 44.66 >100 0.03 >100 GTX 66.50 36.02 67.29 66.85 84.82 7.75 50.71 95.41
[0258] Conclusion: The compound PXG22 of the present invention has obvious inhibitory activity against 4 kinds of PxGSTs recombinant proteins with GSH binding sites, wherein the inhibitory activity of the compound PXG22 of the present invention against PxGST1 and PxGST3 recombinant proteins exceeds the inhibitory activity of the positive control GSTs inhibitor GTX. The remaining compounds of the present invention have different degrees of inhibitory activity against PxGST1 and PxGST1 recombinant proteins, and only the compound PXG1 of the present invention has obvious inhibitory activity against PxGST2 recombinant protein.
Experimental Example 4: Insecticidal Synergistic Activity Assay of the Compound PXG22 of the Present Invention with Chlorantraniliprole
[0259] Experiment: 96% chlorantraniliprole (Chl) original drug was prepared into mother liquor with a concentration of 50000 mg/L. During the test, 6 treatment solutions with concentrations of 500, 250, 125, 62.5, 31.25 and 15.63 mg/L were prepared. 200 mg/L of the compound PXG22 of the present invention was added to each treatment solution, 200 mg/L of diethyl maleate (DEM) was used as the control synergist, and water was used as another control. The Plutella xylostella (Linnaeus) larvae was collected from a vegetable field in Yunnan Province. Healthy 3rd instar larvae of the same age were selected for indoor biological activity assay.
[0260] Clean wild cabbage leaves were cut into discs with a diameter of 6.5 cm (avoiding main veins). The leaves were soaked in the pesticide solution for 10 seconds, then taken out, dried at 25 C., and placed in a petri dish with a diameter of 6.5 cm. 10 larvae of the resistant strains of Plutella xylostella at the early stage of 3rd instar were placed in each petri dish, which was covered with double-layer absorbent roll paper, and covered by the upper cover of the petri dish. It was placed face up in an incubator at a temperature of 251 C., a relative humidity of 65%-70%, and a light ratio (L: D) of 16: 8 h. Each treatment was replicated 4 times with 10 larvae per replicate. After 48 hours, mortality was checked, and the LC.sub.50 and resistance ratio were calculated. During observation, the worm body was gently touched with a small brush or sharp tweezers, if the worm body did not respond or could not coordinate movements, it was considered dead. The test process is strictly in accordance with the standard: NY/T 1154.7-2006.
[0261] Experimental data analysis was carried out using practical statistical analysis and its computer processing platform (DPS) (TANG Qiyi et al., 1997). The toxicity regression equation of each agent was established, LC.sub.50 value and 95% confidence limit were calculated.
[0262] Results: The experimental results are shown in Table 5. After the addition of the compound PXG22 of the present invention, the LC.sub.50 of the chlorantraniliprole solution to the resistant strains of Plutella xylostella decreases from 110.17 mg/L to 14.04 mg/L, and the resistance ratio of resistant strains of Plutella xylostella to chlorantraniliprole falls from 479 to 61.04. For the chlorantraniliprole solution added with the control synergist DEM, its LC.sub.50 and resistance ratio decreases to 50.02 mg/L and 217.48, respectively.
TABLE-US-00007 TABLE 5 Synergistic effect of compounds of the present invention and DEM with chlorantraniliprole Correlation treatment regression LC.sub.50 confidence coefficient Resistance group equation (mg/L) interval R.sup.2 ratio Chl Y = 0.95X 1.94 110.17 67.72-172.48 0.927 479 Chl + DEM Y = 1.15X 1.95 50.02 34.53-75.11 0.984 217.48 Chl + PXG22 Y = 1.75X 2.01 14.04 0.45-22.73 0.881 61.04 Note: Chl: Chlorantraniliprole
[0263] Conclusion: The compound PXG22 of the present invention has obvious insecticidal synergistic activity with chlorantraniliprole, and its synergistic activity exceeds that of the control synergist DEM.
Experimental Example 5: Insecticidal Synergistic Activity Assay of the Compounds PXG1, PXG2 and PXG22 of the Present Invention with Indoxacarb
[0264] Experiment: 95% indoxacarb (Ind) original drug was prepared into mother liquor with a concentration of 50000 mg/L. During the test, 6 treatment solutions with concentrations of 500, 250, 125, 62.5, 31.25 and 15.63 mg/L were prepared. 200 mg/L of the compounds PXG1, PXG2 and PXG22 of the present invention were added to each treatment solution, 200 mg/L of diethyl maleate (DEM) was used as the control synergist, and water was used as another control. The Plutella xylostella (Linnaeus) larvae was collected from a vegetable field in Huizhou city, Guangdong Province. Healthy 3rd instar larvae of the same age were selected for indoor biological activity assay.
[0265] Clean wild cabbage leaves were cut into discs with a diameter of 6.5 cm (avoiding main veins). The leaves were soaked in the pesticide solution for 10 seconds, then taken out, dried at 25 C., and placed in a petri dish with a diameter of 6.5 cm. 10 larvae of the resistant strains of Plutella xylostella at the early stage of 3rd instar were placed in each petri dish, which was covered with double-layer absorbent roll paper, and covered by the upper cover of the petri dish. It was placed face up in an incubator at a temperature of 251 C., a relative humidity of 65%-70%, and a light ratio (L: D) of 16: 8 h. Each treatment was replicated 4 times with 10 larvae per replicate. After 48 hours, mortality was checked, and the LC.sub.50 and resistance ratio were calculated. During observation, the worm body was gently touched with a small brush or sharp tweezers, if the worm body did not respond or could not coordinate movements, it was considered dead. The test process is strictly in accordance with the standard: NY/T 1154.7-2006.
[0266] Experimental data analysis was carried out using practical statistical analysis and its computer processing platform (DPS) (TANG Qiyi et al., 1997). The toxicity regression equation of each agent was established, LC.sub.50 value and 95% confidence limit were calculated.
[0267] Results: The experimental results are shown in Table 6. After the addition of the compound PXG1 of the present invention, the LC.sub.50 of the indoxacarb to the resistant strains of Plutella xylostella decreases from 74.72 mg/L to 59.13 mg/L, and the resistance ratio of resistant strains of Plutella xylostella to indoxacarb falls from 143.70 to 113.71; After the addition of the compound PXG2 of the present invention, the LC.sub.50 of the indoxacarb to the resistant strains of Plutella xylostella decreases from 74.72 mg/L to 39.93 mg/L, and the resistance ratio of resistant strains of Plutella xylostella to indoxacarb falls from 143.70 to 76.78; After the addition of the compound PXG22 of the present invention, the LC.sub.50 of the indoxacarb to the resistant strains of Plutella xylostella decreases from 74.72 mg/L to 51.21 mg/L, and the resistance ratio of resistant strains of Plutella xylostella to indoxacarb falls from 143.70 to 98.48; For the indoxacarb solution added with the control synergist DEM, its LC.sub.50 and resistance ratio decrease to 54.11 mg/L and 104.06, respectively.
TABLE-US-00008 TABLE 6 Synergistic effect of compounds of the present invention and DEM with indoxacarb Correlation treatment regression LC.sub.50 confidence coefficient Resistance group equation (mg/L) interval R.sup.2 ratio Ind Y = 1.32X 2.47 74.72 48.82-106.92 0.860 143.70 Ind + DEM Y = 1.16X 2.01 54.11 29.03-79.18 0.903 104.06 Ind + PXG1 Y = 1.111X 1.97 59.13 32.67-86.20 0.955 113.71 Ind + PXG2 Y = 1.17X 1.88 39.93 18.05-61.39 0.997 76.78 Ind + PXG22 Y = 1.14X 1.94 51.21 26.43-76.19 0.948 98.48 Note: Ind: Indoxacarb
[0268] Conclusion: tresistance ratiohe compounds PX1, PXG2 and PXG22 of the present invention have obvious synergistic activity with indoxacarb, and PXG2 and PXG22 have significant effects, which can make the resistance of resistant strains of Plutella xylostella to indoxacarb from high resistance to moderate resistance.
Experimental Example 6: Insecticidal Synergistic Activity Assay of the Compound PXG22 of the Present Invention with Chlorantraniliprole Using the Ultra-High Resistant Strains of Plutella xylostella (Huizhou Insect Source) as the Tested Insect
[0269] Experiment: The method was the same as in Experimental Example 4. The tested Plutella xylostella was ultra-high resistance strains to chlorantraniliprole (Huizhou insect source). The compound PXG22 of the present invention was added at a concentration of 200 mg/L. The control synergist diethyl maleate (DEM) was added at a concentration of 200 mg/L.
[0270] Results: The experimental results are shown in Table 7. After the addition of compound PXG22 of the present invention, the LC.sub.50 of the chlorantraniliprole solution to the ultra-high resistant strains of Plutella xylostella (Huizhou insect source) decreases from 503.38 mg/L to 148.45 mg/L, and the resistance ratio of ultra-high resistant strains of Plutella xylostella (Huizhou insect source) to chlorantraniliprole falls from 2188.61 to 645.43. For the chlorantraniliprole solution added with the control synergist DEM, its LC.sub.50 and resistance ratio decrease to 442.81 mg/L and 1925.26, respectively.
TABLE-US-00009 TABLE 7 Synergistic effect of compound PXG22 of the present invention and DEM with chlorantraniliprole Correlation treatment regression LC.sub.50 confidence coefficient Resistance group equation (mg/L) interval R.sup.2 ratio Chl Y = 2.39X 6.46 503.38 375.19-719.83 0.860 2188.61 Chl + DEM Y = 2.87X 7.86 442.81 369.82-540.47 0.903 1925.26 Chl + PXG22 Y = 1.53X 3.33 148.45 72.60-235.24 0.918 645.43 Note: Chl: Chlorantraniliprole
[0271] Conclusion: The compound PXG22 of the present invention significantly enhances the insecticidal activity of chlorantraniliprole to ultra-high resistant strains of Plutella xylostella (Huizhou insect source), and its synergistic activity with chlorantraniliprole is significantly higher than that of the control synergist DEM.
Experimental Example 7: Insecticidal Synergistic Activity Assay of the Compound PXG22 of the Present Invention Having Different Concentrations with Chlorantraniliprole Using the High Resistant Strains I of Plutella xylostella (Lianzhou Insect Source) as the Tested Insect
[0272] Experiment: The method was the same as in Experimental example 4, and the tested Plutella xylostella was the high resistant strains I against chlorantraniliprole (Lianzhou source). The added concentrations of the compound PXG22 of the present invention were 200 mg/L, 100 mg/L and 50 mg/L, and the added concentration of diethyl maleate (DEM) of the control synergist was maintained at 200 mg/L.
[0273] Results: The experimental results are shown in Table 8. After the addition of 200 mg/L, 100 mg/L and 50 mg/L of the compound PXG22 of the present invention, the LC.sub.50 of the chlorantraniliprole solution to the high resistant strains I of Plutella xylostella (Lianzhou insect source) decreases from 134.26 mg/L to 40.44 mg/L, 21.16 mg/L and 49.89 mg/L, respectively, and the resistance ratio of high resistant strains I of Plutella xylostella (Lianzhou insect source) to chlorantraniliprole falls from 583.74 to 175.82, 92 and 216.91 respectively. For the chlorantraniliprole solution added with the control synergist DEM, its LC.sub.50 and resistance ratio decrease to 53.36 mg/L and 232, respectively.
TABLE-US-00010 TABLE 8 Synergistic effect of compound PXG22 of the present invention and DEM with chlorantraniliprole Correlation regression LC.sub.50 confidence coefficient Resistance treatment group equation (mg/L) interval R.sup.2 ratio Chl Y = 0.99X 2.10 134.26 69.30-208.45 0.983 583.74 Chl + 200 mg/L PXG22 Y = 1.26X 2.03 40.44 6.45-75.75 0.989 175.82 Chl + 100 mg/L PXG22 Y = 1.04X 1.38 21.16 8.37-33.80 0.979 92 Chl + 50 mg/L PXG22 Y = 1.30X 2.20 49.89 28.11-71.00 0.901 216.91 Chl + DEM Y = 0.79X 1.36 53.36 16.57-95.03 0.967 232 Note: Chl: Chlorantraniliprole
[0274] Conclusion: The compound PXG22 of the present invention significantly enhances the insecticidal activity of chlorantraniliprole against high resistant strains I of Plutella xylostella (Lianzhou insect source), and when the compound PXG22 of the present invention is added with a concentration of 100 mg/L, its synergistic activity with chlorantraniliprole is the strongest, which is significantly higher than that of the control synergist DEM.
Experimental Example 8: Insecticidal Synergistic Activity Assay of the Compound PXG22 of the Present Invention Having Different Concentrations with Chlorantraniliprole Using the High Resistant Strains II of Plutella xylostella (Lianzhou Insect Source) as the Tested Insect
[0275] Experiment: The method was the same as in Experimental example 4, and the tested Plutella xylostella was the high resistant strains II against chlorantraniliprole (Lianzhou source). The added concentrations of the compound PXG22 of the present invention were 200 mg/L, 100 mg/L, 50 mg/L and 25 mg/L, and the added concentration of diethyl maleate (DEM) of the control synergist was maintained at 200 mg/L.
[0276] Results: The experimental results are shown in Table 9. After the addition of 200 mg/L, 100 mg/L, 50 mg/L and 25 mg/L of the compound PXG22 of the present invention, the LC.sub.50 of the chlorantraniliprole solution to the high resistant strains II of Plutella xylostella (Lianzhou insect source) decrease from 145.19 mg/L to 55.92 mg/L, 44.86 mg/L, 71.88 mg/L and 78.54 mg/L, respectively, and the resistance ratio of high resistant strains II of Plutella xylostella (Lianzhou insect source) to chlorantraniliprole fall from 631.26 to 243.13, 195.04, 312.52 and 341.48 respectively. For the chlorantraniliprole solution added with the control synergist DEM, its LC.sub.50 and resistance ratio decrease to 107.99 mg/L and 469.52, respectively.
TABLE-US-00011 TABLE 9 Synergistic effect of compound PXG22 of the present invention and DEM with chlorantraniliprole Correlation regression LC.sub.50 confidence coefficient Resistance treatment group equation (mg/L) interval R.sup.2 ratio Chl Y = 1.54X 3.33 145.19 111.97-192.13 0.940 631.26 Chl + 200 mg/L PXG22 Y = 1.67X 2.91 55.92 38.60-73.32 0.964 243.13 Chl + 100 mg/L PXG22 Y = 1.56X 2.58 44.86 27.78-61.46 0.984 195.04 Chl + 50 mg/L PXG22 Y = 1.76X 3.26 71.88 48.99-95.51 0.993 312.52 Chl + 25 mg/L PXG22 Y = 1.31X 2.47 78.54 48.23-111.47 0.958 341.48 Chl + DEM Y = 1.49x 3.032 107.99 82.46-140.86 0.932 469.52 Note: Chl: Chlorantraniliprole
[0277] Conclusion: The compound PXG22 of the present invention significantly enhances the insecticidal activity of chlorantraniliprole against high resistant strains II of Plutella xylostella (Lianzhou insect source), and when the compound PXG22 of the present invention is added with a concentration of 100 mg/L, its synergistic activity with chlorantraniliprole is the strongest, which is significantly higher than that of the control synergist DEM.
[0278] Based on the above description of the summary of the invention, those skilled in the art will be able fully apply the present invention, and all the same principles or similar modifications should be considered to be within the scope of the present invention.