Novel 16-membered macrolide compound as well as preparation method and application thereof in prevention and control of agricultural and forestry pests

Abstract

The invention relates to a novel 16-membered macrolide compound 13alpha-O-alpha-L-oleandrosyl-23-alpha-hydroxy-25-isopropyl milbemycin beta3, a preparation method of the novel 16-membered macrolide compound 13alpha-O-alpha-L-oleandrosyl-23-alpha-hydroxy-25-isopropyl milbemycin beta3, and an application of the novel 16-membered macrolide compound 13alpha-O-alpha-L-oleandrosyl-23-alpha-hydroxy-25-isopropyl milbemycin

Description

Technical Field

[0001] This invention belongs to the field of natural products, specifically relating to a novel sixteen-membered macrocyclic lactone compound 13α-O-α-L-oleandrosyl-23-α-hydroxy-25-isopropyl milbemycin β3, its preparation method, and its application in the preparation of drugs for controlling agricultural and forestry pests. Background Technology

[0002] Sixteen-membered macrolide insecticides mainly include two categories: abamectin and milbemycin. They are widely used in the control of pests and mites in agricultural and forestry plants, generating significant economic and social benefits. Of particular note is abamectin B1, which, due to its excellent insecticidal effect, has become the only pesticide product with annual sales exceeding 3 billion yuan. However, with the long-term and widespread use of abamectin B1, the problem of insecticide resistance is becoming increasingly prominent, necessitating the development of new insecticides.

[0003] Our research group has long been committed to the research and development of novel sixteen-membered macrocyclic lactone insecticides. Among these, nearly twenty new sixteen-membered macrocyclic lactone compounds have been obtained from *Streptomyces avermitilis* NEAU1069 and its mutant strain *Streptomyces avermitilis* NEAU1069-3. Recently, through traditional selection of *Streptomyces avermitilis* NEAU1069, we obtained the *Streptomyces avermitilis* NEAU1069-6 mutant, and further isolated a new sixteen-membered macrocyclic lactone compound from its fermentation broth. This new compound exhibits good insecticidal activity against avermectin B1-resistant strains of diamondback moth, spider mite, and pine wilt nematode, showing considerable development potential and significant importance for addressing the problem of avermectin B1 resistance. Summary of the Invention

[0004] This invention relates to compound 13α-O-α-L-oleandrosyl-23-α-hydroxy-25-isopropylmilbemycin β3, which is derived from Streptomyces. Streptomyces avermitilis The chemical structure of the new sixteen-membered macrocyclic lactone compound isolated from the NEAU1069-6 fermentation culture is shown in Formula 1 below.

[0005] This invention relates to a method for preparing compound 13α-O-α-L-oleandrosyl-23-α-hydroxy-25-isopropyl milbemycin β3, characterized by comprising the following steps: 1) The mutant strain Streptomyces avermitilis NEAU1069-6 was cultured according to the method described in the reference (Wang M, et al. J Antibiot. 2009, 62 (10): 587-591.) to obtain the fermentation broth; 2) After solid-liquid separation of the fermentation broth obtained by fermentation culture in step 1), mycelium is obtained. After extraction of mycelium with alcohol, it is concentrated to an appropriate volume and then extracted with organic solvent to obtain organic phase extract. The extract is concentrated under reduced pressure to dryness to obtain an oily substance. 3) After the oily substance obtained in step 2) is purified by silica gel column chromatography, it is further purified by preparative HPLC to obtain the target compound.

[0006] ; Chemical structure of compound 13α-O-α-L-oleandrosyl-23-α-hydroxy-25-isopropyl milbemycinβ3 (Formula 1) In step 1), the Streptomyces avermitilis NEAU1069-6 is a mutant strain obtained by mutagenesis and selection based on the doramectin-producing strain Streptomyces avermitilis NEAU1069 (China General Microbiological Culture Collection Center, registration number: CGMCC2943, Institute of Microbiology, Chinese Academy of Sciences) through mutagenesis using nitrosoguanidine, ethyl methanesulfonate, ultraviolet light, and other mutagenesis methods.

[0007] Step 2) The alcohol is methanol or ethanol or a mixture of both, and the organic solvent extraction is ethyl acetate or dichloromethane or chloroform.

[0008] Step 3) The silica gel column is eluted using a gradient of petroleum ether / ethyl acetate (95%-50%, v / v); the preparative HPLC uses C18 reversed-phase packing material, and the elution solvent is a mixture of methanol, acetonitrile, acetone, or any mixture thereof in any proportion with water. More preferably, the preparative HPLC uses C18 reversed-phase packing material, and the elution solvent is a mixture of methanol, acetonitrile, or any mixture thereof in any proportion with water. Even more preferably, the preparative HPLC column is C18, 5 µm, 250 × 20 mm id; the detection wavelength is 220 nm / 254 nm; the flow rate is 20 mL / min; and the mobile phase and elution program are a gradient elution of 50-80% acetonitrile aqueous solution for 30 min.

[0009] This invention also relates to the structural characterization of compound 13α-O-α-L-oleandrosyl-23-α-hydroxy-25-isopropylmilbemycin β3: Appearance: White powder Specific curl: [α] -197 (c 0.09, EtOH) Molecular formula: C 40 H 57 O 10 HRESIMS m / z: 697.3957 [M - H] - , calcd. 697.3952 UV absorption spectrum λmax (EtOH) nm (log ε): 246 (3.81) 1 H NMR (CDCl3, 400 MHz) and 13 The C NMR (CDCl3, 100 MHz) data are shown in Table 1.

[0010] Table 1: NMR data of compound 13α-O-α-L-oleandrosyl-23-α-hydroxy-25-isopropyl milbemycinβ3 in CDCl3 (H1N, 400 MHz; C1N, 100 MHz) ; Finally, this invention relates to the use of compound 13α-O-α-L-oleandrosyl-23-α-hydroxy-25-isopropylmilbemycin β3 and its pesticide compositions in the control of agricultural and forestry pests, including lepidopteran pests, nematodes, and mites, particularly abamectin B1 resistant strains. The compositions also contain one or more conventional carriers and / or diluents. The pesticide compositions can be formulated as water-dispersible granules, emulsifiable concentrates, aqueous suspensions, oil suspensions, microemulsions, or tablets. The controlled agricultural and forestry pests include the diamondback moth (Plutella xylostella), the pine wood nematode (Bursaphelenchus xylophilus), and the carmine spider mite (Tetranychus cinnabarinus), preferably abamectin B1 resistant strains. In addition, 13α-O-α-L-oleandrosyl-23-α-hydroxy-25-isopropyl milbemycin β3 can also be used as a precursor to synthesize other compounds with better structures for the control of agricultural and forestry pests.

[0011] The extraction, separation, and activity experiments of compound 13α-O-α-L-oleandrosyl-23-α-hydroxy-25-isopropyl milbemycin β3 are detailed in the following examples. These examples are further illustrations of the invention but do not limit the scope of the invention. Detailed Implementation

[0012] Example 1: Obtaining and culturing the mutant strain Streptomyces avermitilis NEAU1069-6 This invention relates to a mutant strain of *Streptomyces avermitilis* NEAU1069-6, obtained through mutagenesis using nitrosoguanidine, ethyl methanesulfonate, and ultraviolet light, based on the doramectin-producing strain *Streptomyces avermitilis* NEAU1069 (China General Microbiological Culture Collection Center, registration number: CGMCC2943, Institute of Microbiology, Chinese Academy of Sciences). (Methods are described in: Teng Yun et al., *Chinese Journal of Antibiotics*, 2019, 44: 197-202.) The mutant strain *Streptomyces avermitilis* NEAU1069-6 was cultured according to the method described in the reference (Wang M, et al., *JAntibiot.*, 2009, 62 (10): 587-591.).

[0013] Example 2: Extraction and separation of compound 13α-O-α-L-oleandrosyl-23-α-hydroxy-25-isopropylmilbemycin β3 30 L of the fermentation broth obtained in Example 1 was filtered to obtain mycelium, which was then extracted with 15 L of ethanol to obtain an ethanol extract. The ethanol extract was concentrated under vacuum at 50°C to approximately 1 L, and then extracted three times with equal volumes of ethyl acetate to obtain ethyl acetate extracts. The extracts were combined and concentrated to dryness under reduced pressure at 50°C to obtain 49 g of an oily substance. The obtained oily substance was subjected to silica gel column chromatography with a gradient elution of petroleum ether / ethyl acetate (95%-50%, v / v). TLC analysis showed that the combined fractions yielded five fractions (AE). Fraction C was subjected to silica gel column chromatography with a gradient elution of petroleum ether / ethyl acetate (85%-75%, v / v). TLC analysis showed that the combined fractions yielded components C1 and C2. C2 was purified by preparative HPLC (purification conditions: PRC-ODS column, 5 µm, 250 × 20 mm id; detection wavelength 254 / 220 nm; flow rate 20 mL / min; mobile phase and elution program MeCN / H2O, 0%-80% v / v, 30 min) to obtain compound 13α-O-α-L-oleandrosyl-23-α-hydroxy-25-isopropyl milbemycin β3 (t R =28.8 minutes, 23.3 mg).

[0014] Example 3: Bioactivity of compound 13α-O-α-L-oleandrosyl-23-α-hydroxy-25-isopropylmilbemycin β3 against abamectin B1 resistant strains of diamondback moth, spider mite, and pine wood nematode. In the laboratory, diamondback moth, spider mite, and pine wilt nematode were reared to breed abamectin B1 resistant populations according to the methods described in the Ministry of Agriculture standard NY / T 1859.5-2014 "Pesticide Resistance Risk Assessment Part 5: Risk Assessment of Pesticide Resistance in Diamondback Moth of Cruciferous Vegetables". The desired abamectin B1 resistant strains for diamondback moth, spider mite, and pine wilt nematode were obtained. The methods for testing the abamectin B1 activity against spider mite and pine wilt nematode are described in the reference (Huang, J., et al. Appl. Environ. Microbiol. 2015, 81, 5326–5334.), and the method for testing the abamectin B1 activity against diamondback moth is described in the reference (FAUZIAH, I., et al. Pesticide science, 1991, 33: 359–370.). The experimental results are shown in Table 2.

[0015] Table 2. Activity of compound 13α-O-α-L-oleandrosyl-23-α-hydroxy-25-isopropyl milbemycinβ3 against abamectin B1 resistant strains of Tetranychus cinnabarinus and pine wood nematode. .

Claims

1. This invention provides a sixteen-membered macrocyclic lactone compound of the following formula, the specific structure of which is as follows: 。 2. The compound according to claim 1, characterized in that... The compound was obtained by fermenting Streptomyces avermitilis NEAU1069-6 strain and then separating and purifying the fermentation broth.

3. According to the preparation method described in claim 2, the strain Streptomyces avermitilis NEAU1069-6 is a mutant strain obtained by mutagenesis and selection of the doramectin-producing strain Streptomyces avermitilis NEAU1069 (China General Microbiological Culture Collection Center, registration number: CGMCC2943, Institute of Microbiology, Chinese Academy of Sciences).

4. The preparation method according to claims 2 and 3, characterized in that... The specific method includes the following steps: 1) The fermentation broth of Streptomyces avermitilis NEAU1069-6 as described in claim 3 was obtained by culturing according to the method described in the reference (Wang M, et al. J Antibiot. 2009, 62 (10): 587-591.); 2) After solid-liquid separation of the fermentation broth obtained by fermentation culture in step 1), mycelium is obtained. After extraction of mycelium with alcohol, it is concentrated to an appropriate volume and then extracted with organic solvent to obtain organic phase extract. The extract is concentrated under reduced pressure to dryness to obtain an oily substance. 3) After the oily substance obtained in step 2) is purified by silica gel column chromatography, it is further purified by preparative HPLC to obtain the target compound.

5. The method according to claim 4, characterized in that, In step 2), the alcohol is methanol or ethanol or a mixture of both; the organic solvent extraction is ethyl acetate, dichloromethane or chloroform; in step 3), the silica gel column is eluted using a gradient of petroleum ether / ethyl acetate (95%-50%, v / v); the preparative HPLC uses C18 reversed-phase packing material, and the elution solvent is a mixture of methanol, acetonitrile, acetone or any ratio of these solvents and water.

6. The method according to claims 4 and 5, characterized in that, The preparative HPLC uses C18 reversed-phase packing material and methanol, acetonitrile, or a mixture of these solvents and water in any ratio as the elution solvent.

7. The method according to claims 4-6, characterized in that, The preparative HPLC column was a C18 column, 5 µm, 250 × 20 mm id; the detection wavelength was 220 nm / 254 nm; the flow rate was 20 mL / min; and the mobile phase and elution program were gradient elution with 50-80% acetonitrile aqueous solution for 30 min.

8. A pharmaceutical composition comprising the compound of claim 1, and one or more conventional carriers and / or diluents.

9. Use of the compounds of claims 1 and 8 and their pharmaceutical compositions in the preparation of drugs for controlling agricultural and forestry pests, wherein the pests include lepidopteran pests, nematodes and mites and their abamectin B1 resistant strains.

10. The pests controlled according to claims 1, 8 and 9 include diamondback moth (Plutella xylostella), pine wilt nematode (Bursaphelenchus xylophilus), and carmine spider mite (Tetranychus cinnabarinus) and their abamectin B1 resistant strains.

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