A method for preparing a metabolite of avermectin B1a / B2a

Abstract

The application relates to the technical field of pesticide production, and particularly discloses a preparation method of an avermectin B1a / B2a metabolite. The method uses hydroxyl-protected avermectin B1a / B2a as a starting material, adopts a catalytic system of a copper salt or a manganese salt transition metal salt and pyridine, completes a selective oxidation reaction of a mother nucleus 8a position under the condition of oxygen pressurization at room temperature, and then removes a hydroxyl protective group to obtain a target 8a position carbonyl metabolite. The application uses clean and easily obtained oxygen as an oxygen source, replaces a high amount of chromium heavy metal oxidant in a traditional process, significantly reduces the amount of a catalyst, has mild reaction conditions, has excellent reaction region selectivity, is stable in product yield and purity, is simple in process operation and easy to industrialize, can provide a reliable material preparation path for pesticide residue standard preparation, environmental risk assessment and new pesticide lead compound screening, and has good industrial application prospect.

Description

Technical Field

[0001] This invention relates to the field of pesticide production technology, and in particular to a method for preparing avermectin B1a / B2a metabolites. Background Technology

[0002] Avermectins are a class of sixteen-membered macrocyclic lactone compounds produced by the fermentation of *Streptomyces avermitilis*. Since their commercialization, they have become a core pesticide in the global fields of agricultural pest control and livestock deworming. Their environmental fate, biological metabolic pathways, and the safety of their metabolites have always been key research areas in pesticide risk assessment. The Food and Agriculture Organization of the United Nations (FAO), in its pesticide assessment report ABAMECTIN (177), systematically elucidated the metabolic pathways of avermectins in soil, plants, and animals, clarifying the chemical structures and formation pathways of more than ten characteristic metabolites. Among them, the avermectin metabolite with carbonyl substitution at the 8a position (structural formula below) is a key component in the environmental degradation and biological metabolism of avermectins, playing an irreplaceable role in the establishment of residue detection methods, environmental toxicology evaluation, and dietary risk assessment.

[0003] The avermectin molecule contains multiple reactive sites, including carbon-carbon double bonds, hydroxyl groups, and lactone bonds. Under conventional oxidation conditions, these sites are at risk of side reactions. The carbon-hydrogen bond at the 8a position is relatively less reactive. The core technical challenge in the synthesis of this type of metabolite is how to achieve carbonylation at the 8a position without oxidizing and destroying other functional groups.

[0004] A prior art method discloses the preparation of the 8a-carbonylated product of avermectin, which uses pyridinium chlorochromate (PCC) as an oxidant. The amount of oxidant added is 10 to 20 times the molar amount of the substrate, the reaction time is 12 to 48 hours, and the yield of the target product is 70% to 80%. However, this process requires the use of a large amount of chromium-containing oxidant. On the one hand, chromium is a heavy metal pollutant, and the treatment cost of chromium-containing waste residue and waste liquid generated after the reaction is high, posing a high environmental safety risk. On the other hand, the use of excessive oxidant can easily cause side reactions such as over-oxidation, increasing the difficulty and production cost of subsequent separation and purification processes, making it difficult to meet the dual requirements of economic efficiency and environmental protection for large-scale preparation.

[0005] Therefore, developing a green, efficient, and highly selective method for synthesizing the 8a metabolite of avermectin is not only a necessary prerequisite for conducting physicochemical characterization, toxicological studies, environmental risk assessment, and preparation of residue standards for this metabolite, but also provides a material basis for screening and structural modification of novel pesticide molecules using natural metabolites as lead compounds. It has significant scientific research value and industrial application significance. Summary of the Invention

[0006] To address the problems of existing methods for preparing avermectin 8a-position carbonyl metabolites, which require large doses of chromium-containing oxidants, leading to high costs and significant environmental risks in the treatment of chromium-containing waste residues and liquids, and the potential for excessive oxidation and other side reactions due to excessive oxidants, this invention provides a method for preparing avermectin B1a / B2a metabolites. This invention uses a specific transition metal salt and pyridine complex as a catalyst to achieve a highly selective carbonylation reaction at the 8a position of avermectin. While ensuring product yield and purity, it eliminates dependence on heavy metal reagents, significantly improving the greenness of the process and its potential for industrial application.

[0007] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows: A method for preparing an abamectin B1a / B2a metabolite, comprising the following steps: Using the compound shown in formula (I) as a raw material, and with transition metal salt and pyridine as catalysts, the 8a position is oxidized under oxygen pressure to obtain the compound shown in formula (II); wherein the transition metal salt is one or both of copper salt and manganese salt. R is a hydroxyl protecting group; XY is CH=CH or CH2-CH(OH).

[0008] Compared to existing technologies, the method for preparing avermectin B1a / B2a metabolites provided by this invention uses a catalytic oxidation system composed of copper or manganese transition metal salts and pyridine, with oxygen as the oxidant. This replaces the chromium-based heavy metal oxidants such as pyridinium chlorochromate used extensively in traditional processes, eliminating the environmental safety risks and waste treatment costs associated with heavy metal pollutant emissions at the source. This catalytic system exhibits excellent regioselectivity for the oxidation reaction at the 8a position of the avermectin core, effectively avoiding side reactions such as over-oxidation and oxidation at non-target sites, and directionally completing the carbonylation conversion at the 8a position with high yield and purity of the target product. Simultaneously, the overall reaction conditions are mild, the operation process is simple, the catalytic reagents are inexpensive, and the post-processing steps are straightforward, combining good environmental benefits and economic applicability. It can provide a stable source of materials for laboratory-level studies of metabolic physicochemical properties, toxicological evaluations, and the preparation of residual standards, and also possesses good potential for large-scale industrial application.

[0009] It should be noted that the metabolite described in this invention is the 8α-carbonyl avermectin metabolite.

[0010] It should be noted that the preparation of the avermectin B1a / B2a derivative with protected hydroxyl groups at the 5- and 4″ positions, as shown in formula (Ⅰ) of the starting material used in this invention, is a mature prior art in the field, and this invention does not impose any special limitations. Specifically, it can be conveniently prepared by selective silylation reaction using commercially available or fermented avermectin B1a / B2a as the starting material, referring to the method disclosed in Chinese Patent ZL201310063563.0.

[0011] Specifically, this method uses dimethyl tert-butylchlorosilane (TBDMSCl) or trimethylchlorosilane (TMSCl) as hydroxyl protecting agents, and reacts with avermectin B1a / B2a in the presence of an organic base and in an aprotic solvent. This allows for the highly selective introduction of corresponding silane protecting groups at the 5-position and 4″-position hydroxyl groups, thereby obtaining compound (I). The above reaction is typically carried out in a temperature range of -10℃ to 30℃. After the addition is complete, stirring continues until the reaction is complete. After washing with water, drying, and concentration, compound (I) is obtained.

[0012] As a typical implementation method, when avermectin B2a is used as a substrate and dimethyl tert-butylchlorosilane is used as a protecting agent, 4″,5-dimethyl tert-butylsilyl avermectin B2a (i.e., R in formula (I) is dimethyl tert-butylsilyl, and the parent nucleus is B2a) can be prepared by reacting in dichloromethane in the presence of tetramethylethylenediamine at -5°C to 5°C. 4″,5-trimethylsilyl avermectin B2a can be prepared by replacing dimethyl tert-butylchlorosilane with trimethylchlorosilane using the same method. Alternatively, the hydroxyl-protected product corresponding to avermectin B1a can be prepared using the same method.

[0013] Furthermore, the method for preparing the avermectin B1a / B2a metabolite further includes: removing the hydroxyl protecting groups at the 5 and 4'' positions of the compound shown in formula (II) to obtain the compound shown in formula (III); Formula (III).

[0014] It should be noted that after completing the above selective oxidation at the 8a position, the 5- and 4'' hydroxyl protecting groups in the obtained intermediate need to be removed to obtain the 8a-carbonyl avermectin metabolite shown in formula (Ⅲ). In the synthesis method described in this invention, the step of removing the 5- and 4'' hydroxyl protecting groups is as follows: a deprotection reaction is carried out in methanol solvent in the presence of an acidic catalyst.

[0015] Specifically, the acidic catalyst is p-toluenesulfonic acid or methanesulfonic acid, with p-toluenesulfonic acid being preferred.

[0016] In some specific embodiments of the present invention, the specific steps of the deprotection method are as follows: dissolve the obtained oxidation product in methanol, add p-toluenesulfonic acid or methanesulfonic acid, stir the reaction in a temperature range of 10℃~40℃, monitor the reaction until the end of thin-layer chromatography, remove methanol by vacuum distillation, wash, dry, and obtain the target product shown in formula (Ⅲ).

[0017] In some specific embodiments of the present invention, the preparation method of the avermectin B1a / B2a metabolite specifically includes the following steps: The compound shown in formula (I), a transition metal salt, and pyridine were added to an organic solvent and oxidized under oxygen pressure to obtain the compound shown in formula (II). The hydroxyl protecting groups at the 5 and 4'' positions of the compound represented by formula (II) are removed to obtain the compound represented by formula (III).

[0018] The present invention provides a method for preparing avermectin B1a / B2a metabolites. Using avermectin derivatives with protected 5- and 4'' hydroxyl groups as shown in formula (I) as starting materials, a catalytic system composed of transition metal salts and pyridine is employed. Selective oxidation at the 8a-position is carried out in an organic solvent under oxygen pressure to obtain the 8a-carbonyl protected intermediate shown in formula (II). Further acid-catalyzed deprotection treatment yields the target metabolite shown in formula (III). Compared with existing processes using large doses of pyridinium chlorochromate (PCC), this method uses oxygen as the terminal oxidant, avoiding the generation of chromium-containing heavy metal pollutants at the source. This significantly reduces wastewater treatment costs and significantly improves environmental safety, providing reliable technical support for the large-scale preparation of 8a-carbonyl avermectin metabolites, the development of residue detection standards, and systematic research in environmental toxicology.

[0019] Furthermore, the amount of pyridine added is 1% to 10% of the mass of the compound shown in formula (I), and the amount of transition metal salt added is 0.1% to 1% of the mass of the compound shown in formula (I).

[0020] Compared to the existing PCC process where the oxidant needs to be added in excess at a molar ratio of 10 to 20 times, the amount of catalyst added in this invention is significantly reduced, thereby reducing the amount of waste generated at the source and significantly alleviating the environmental burden and economic cost of subsequent waste liquid treatment. On the other hand, the synergistic effect of pyridine and transition metal salt can efficiently activate the inert C-H bond at the 8a position under oxygen pressurization conditions, while avoiding the interference of side reactions of other groups in the core structure, ensuring the chemical selectivity of the oxidation process and the stable and controllable purity of the product.

[0021] Furthermore, the oxidation reaction is carried out at a temperature of 10°C to 30°C for a reaction time of 1 hour to 4 hours.

[0022] Preferably, the oxidation reaction is carried out at a temperature of 20°C to 30°C.

[0023] The oxidation reaction can be carried out smoothly at room temperature, is simple to operate and has low energy consumption, making it particularly suitable for industrial-scale production. Furthermore, the short-time reaction at room temperature can effectively avoid the damage to non-reactive sites in the avermectin core structure caused by long-term high-temperature operation, thus reducing the occurrence of side reactions.

[0024] Furthermore, the oxygen pressurization pressure is 0.4 MPa to 1.0 MPa, preferably 0.5 MPa to 0.7 MPa.

[0025] The optimal reaction pressure can effectively avoid excessive oxidation side reactions that are prone to occur under high pressure while ensuring the reaction rate and conversion degree, ensuring the regioselectivity of the 8a carbonylation reaction, and taking into account both preparation efficiency and product purity.

[0026] Furthermore, the organic solvent is N,N-dimethylformamide.

[0027] Furthermore, the amount of organic solvent added is 3 to 6 times the mass of the compound shown in formula (I).

[0028] Furthermore, the transition metal salt is at least one of manganese sulfate, copper sulfate, or copper acetate.

[0029] Furthermore, the hydroxyl protecting group is dimethyl tert-butylsilyl or trimethylsilyl.

[0030] In summary, this invention discloses a method for preparing avermectin B1a / B2a metabolites. This method uses hydroxyl-protected avermectin B1a / B2a as the starting material and employs a catalytic system of copper or manganese transition metal salts combined with pyridine. The selective oxidation of the 8a-position of the parent nucleus is completed under oxygen pressure at room temperature, followed by removal of the hydroxyl protecting group to obtain the target 8a-position carbonyl metabolite. This invention uses clean and readily available oxygen as the oxygen source, replacing the high amounts of chromium-based heavy metal oxidants used in traditional processes. The catalyst dosage is significantly reduced, the reaction conditions are mild, and the reaction cycle is short. It also exhibits excellent regioselectivity, effectively avoiding side reactions involving intramolecular double bonds, lactone rings, and non-target hydroxyl groups. This eliminates the risk of heavy metal pollution at the source, significantly reducing waste treatment costs and production energy consumption. The product yield and purity are stable, the process is simple to operate, and it is easy to scale up industrially. This method can provide a reliable material preparation route for pesticide residue standard preparation, environmental risk assessment, and screening of novel pesticide lead compounds, demonstrating good environmental benefits and promising industrial application prospects. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0032] Existing technologies for preparing avermectin 8a carbonyl metabolites still have significant limitations: while heavy metal oxidation systems, such as pyridinium chlorochromate, can achieve the target conversion, they require large amounts of reagents, have a high environmental impact, and incur high costs for waste treatment. Furthermore, multiple active sites on the avermectin core, such as double bonds, hydroxyl groups, and lactones, are prone to side reactions under strong oxidative conditions, making product purification processes cumbersome and difficult to balance economic efficiency, environmental friendliness, and the need for large-scale preparation. To address these problems, this invention provides a method for preparing avermectin B1a / B2a metabolites. This method aims to achieve highly selective carbonylation at the 8a position through the construction of a novel oxidative catalytic system and optimization of process conditions. While ensuring product yield and purity, it eliminates dependence on heavy metal reagents, significantly improving the greenness of the process and its potential for industrial application.

[0033] The method for preparing avermectin B1a / B2a metabolites provided in this application includes the following steps: S1, the compound shown in formula (I), the transition metal salt and pyridine are added to an organic solvent and oxidized at 10℃~30℃ under oxygen pressure to obtain the compound shown in formula (II); S2, under acidic conditions, the hydroxyl protecting groups at the 5 and 4'' positions of the compound represented by formula (II) are removed to obtain the compound represented by formula (III).

[0034] This preparation method employs a process route of transition metal salt and pyridine synergistic catalysis and oxygen pressurization oxidation. Using clean and readily available oxygen as the oxygen source replaces the high-volume chromium-based heavy metal oxidants used in traditional processes, eliminating the risk of heavy metal pollution at the source and significantly reducing the cost of waste treatment. The catalytic system exhibits excellent regioselectivity for the 8a position of the avermectin core. Combined with the pre-introduced hydroxyl protecting groups at the 5 and 4'' positions on the compound of formula (I), side reactions involving intramolecular double bonds, lactone rings, and other active hydroxyl groups are effectively avoided, ensuring the directional conversion efficiency and product purity of the oxidation reaction. The overall process consists of two steps: oxidation and deprotection. The reaction pathway is clear and controllable. The deprotection step can be achieved using mature conventional processes in this field. The post-processing is simple, the product yield is stable, and it possesses both excellent green and environmentally friendly attributes and potential for industrial-scale application.

[0035] To better illustrate the present invention, further examples are provided below.

[0036] In the following examples, the content of avermectin B1a+B1b is ≥98% (since B1a:B1b>30:1, the derivative of B1b component was not characterized separately by NMR), and the content of B2a is ≥98%.

[0037] Magnetic resonance spectra were measured using JEOL (400MHz), and mass spectrometry was performed using Agilent 1100 LC / MSD; all reagents used were analytical grade or chemically pure.

[0038] Example 1 Preparation of 4″,5-dimethyl-tert-butylsilyl avermectin B2a (Ⅰa): 100 g (0.11 mol) of avermectin B2a (B2a content 98%) was dissolved in 1 L of dichloromethane and cooled to -5 °C. Simultaneously, 37.7 g (0.25 mol) of dimethyl tert-butylchlorosilane and 13.9 g (0.12 mol) of tetramethylethylenediamine were slowly added dropwise over 1 h. After the addition was complete, the reaction was carried out for 2 h. After the reaction was completed, the system temperature was raised to 0-5 °C, and 500 mL of saturated sodium bicarbonate aqueous solution was added. The mixture was stirred for 30 min, allowed to stand and separate into layers, and the organic phase was dried over anhydrous magnesium sulfate. The solvent was removed under reduced pressure to obtain compound Ia.

[0039] The structural formula of compound Ia is: Ⅰa Following the same silyl etherification protection process described above, by replacing the corresponding types of avermectin substrates and silicon-based protecting reagents, 4″,5-dimethyltert-butylsilyl avermectin B1a (compound Ib), 4″,5-trimethylsilyl avermectin B1a (compound Ic) and 4″,5-trimethylsilyl avermectin B2a (compound Id) can be prepared respectively.

[0040] Example 2 This embodiment provides a method for preparing 8a-carbonyl avermectin B2a, comprising the following steps: S1. Take 45g (40 mmol) of compound Ia prepared in Example 1 and place it in a 500mL autoclave. Add 135g of N,N-dimethylformamide, 0.025g of anhydrous manganese sulfate, 0.02g of anhydrous copper sulfate and 4.5g of pyridine. After sealing the autoclave and confirming that it is connected to the oxygen cylinder, open it to purge oxygen three times and then introduce oxygen to 0.4MPa. Stir and react at 10℃ for 4h. After the reaction is completed and the pressure is released, take out the reaction solution and add 500mL of water and 100mL of ethyl acetate. Stir for 30min, let stand and separate the layers. Wash the organic layer again with 500mL of water, separate the layers, and dry the organic layer to obtain the solid. S2, the solid obtained in step S1 was dissolved in 100 g of anhydrous methanol, and 15.5 g (90 mmol) of p-toluenesulfonic acid was added. The mixture was stirred at room temperature for 1 h until the reaction was complete. The solution was concentrated to dryness under reduced pressure at 30 °C. Then, 80 g of acetonitrile was added and stirred for 30 min. The mixture was filtered and dried to obtain 28.0 g of a white solid. HPLC: 98.5%, yield 76.2%.

[0041] Melting point: 184.1~185.8℃; MS [M+H + [ (m / z): 906.10, the calculated M+H value of 8a-carbonyl avermectin B2a is 906.10.]

[0042] 1 H NMR (400 MHz, CDCl3) δ: 5.78-5.82(m, 1H), 5.73-5.76 (m, 2H), 5.63(d, J = 8.3 Hz, 1H), 5.52(s, 1H), 5.34-5.44 (m, 1H), 4.92(t, J = 8.3 Hz, 2H), 4.77-4.82 (d, J = 13.2 Hz, 2H), 4.72(t, J = 10.8 Hz, 2H), 4.42(d, J = 9.5 Hz, 1H), 4.33(d, J = 9.8 Hz, 1H), 4.23(dd, J 1 = 8.2 Hz, J 2= ​​12.4 Hz, 1H), 3.70-3.97(m, 2H), 3.50-3.62(m, 3H), 3.43(s, 3H), 3.30(t, J = 9.7 Hz, 1H), 3.23(dd, J = 11.4 Hz, 7.0Hz, 2H), 2.60(t, J = 6.4 Hz, 1H), 2.50-2.57(m, 4H), 2.46-2.48(m, 3H), 2.10-2.18(m, 2H), 1.88(dd, J= 13.3 Hz, 11.2 Hz, 2H), 1.56-1.76(m, 6H), 1.45-1.54 (m,8H), 1.30-1.39(m, 2H), 1.13-1.34(m, 4H), 0.90(dd, J = 15.8 Hz, 8.8 Hz, 8H),0.80-0.88(m, 6H). 13 C NMR (100.5 MHz, CDCl3) δ: 176.72, 172.83, 143.80, 136.70, 125.18,124.41, 121.44, 120.91, 119.10, 118.53, 100.74, 99.51, 98.92, 91.15, 86.61,83.53, 78.33, 76.11, 73.82, 70.77, 69.60, 68.54, 67.77, 66.68, 64.54, 62.74,61.19, 60.37, 59.98, 59.84, 58.81, 56.62, 55.52, 54.47, 49.87, 41.19, 38.68, 37.81, 35.35, 29.97, 30.51, 28.33, 24.79, 21.17, 19.95, 17.23, 11.53, 10.18. 8a-Carbonylavermectin B2a structural formula: Example 3 This embodiment provides a method for preparing 8a-carbonyl avermectin B2a, comprising the following steps: S1, Take 45g (40 mmol) of compound Ia prepared in Example 1 and place it in a 500mL autoclave. Add 180g N,N-dimethylformamide, 0.45g anhydrous manganese sulfate and 0.45g pyridine. After sealing the autoclave and confirming that it is connected to the oxygen cylinder, open it to purge oxygen three times and then introduce oxygen to 0.6MPa. Stir and react at 20℃ for 2h. After the reaction is completed and the pressure is released, take out the reaction solution and add 660mL water and 150mL ethyl acetate. Stir for 30min, let it stand and separate into layers. Wash the organic layer again with 200mL water, separate into layers, and dry the organic layer to obtain the solid. S2, the solid obtained in step S1 was dissolved in 100g of anhydrous methanol, and 15.5g (90 mmol) of p-toluenesulfonic acid was added and stirred at room temperature for 1h until the reaction was complete. The solution was then concentrated to dryness under reduced pressure at 30℃, and 80g of acetonitrile was added and stirred for 30min. The solution was filtered and dried to obtain 30.2g of white solid. HPLC: 90.2%, yield 75.2%.

[0043] Example 4 This embodiment provides a method for preparing 8a-carbonyl avermectin B2a, comprising the following steps: S1, Take 45g (40 mmol) of compound Ia prepared in Example 1 and place it in a 500mL autoclave. Add 270g of N,N-dimethylformamide, 0.225g of anhydrous copper sulfate and 2.25g of pyridine. After sealing the autoclave and confirming that it is connected to the oxygen cylinder, open it to purge oxygen three times and then introduce oxygen to 1.0MPa. Stir and react at 30℃ for 1h. After the reaction is completed and the pressure is released, take out the reaction solution and add 1 L of water and 300mL of ethyl acetate. Stir for 30min, let it stand and separate into layers. Wash the organic layer again with 300mL of water, separate into layers, and dry the organic layer to obtain the solid. S2, the solid obtained in step S1 was dissolved in 100 g of anhydrous methanol, and 15.5 g (90 mmol) of p-toluenesulfonic acid was added and stirred at room temperature for 1 h until the reaction was complete. The solution was then concentrated to dryness under reduced pressure at 30 °C, and 80 g of acetonitrile was added and stirred for 30 min. The solution was filtered and dried to obtain 30.9 g of white solid. HPLC: 89.5%, yield 76.3%.

[0044] Example 5 This embodiment provides a method for preparing 8a-carbonyl avermectin B1a, comprising the following steps: S1, 42g (40 mmol) of compound Ic prepared in Example 1, 252g of N,N-dimethylformamide, 0.42g of anhydrous copper acetate and 0.42g of pyridine were added to a 500mL autoclave, heated to 30°C, the autoclave was sealed, and after confirming that the connection with the oxygen cylinder was intact, the valve was opened to purge three times. Then oxygen was introduced to raise the pressure to 1.0MPa, and the reaction was stirred for 1.0h. After depressurization, the reaction solution was taken out and 1L of water and 100mL of ethyl acetate were added. The mixture was stirred for 30min, allowed to stand and separate into layers, the organic layer was washed again with 500mL of water, and the layers were separated. The organic layer was dried to obtain a solid. S2, the solid obtained in step S1 was dissolved in 100 g of anhydrous methanol, and 8.6 g (90 mmol) of methanesulfonic acid was added and stirred at room temperature for 1 h until the reaction was complete. The solution was then concentrated to dryness under reduced pressure at 30 °C, and 80 g of acetonitrile was added and stirred for 1 h. The mixture was filtered and dried to obtain 30.0 g of white solid. HPLC: 97.4%, yield: 82.3%.

[0045] The melting point is 178.6~180.1°C.

[0046] 1 H NMR (400 MHz, CDCl3) δ: 6.67(s, 1H), 5.71-5.80 (m, 2H), 5.66(m,2H), 5.54(t, J = 10.6 Hz, 1H), 4.91-5.18 (m, 2H), 4.83(t, J =9.7 Hz, 2H), 4.55-4.80(m, 2H), 4.44-4.50(m, 2H), 4.33-4.39(m, 2H), 4.15-4.20(t, J = 10.8 Hz, 1H),3.76-3.89(m, 2H), 3.53-3.77(m, 2H), 3.40(s, 3H), 3.33(s, 3H), 2.51-2.65 (m,2H), 2.44-2.49(m, 2H), 2.0-2.10(m, 2H), 1.92(dd, J = 16.2 Hz, 13.4 Hz, 2H),1.70-1.83(m, 6H), 1.44-1.54 (m, 6H), 1.39-1.43(m, 4H), 1.23-1.30(m, 4H), 1.15(dd, J = 12.3 Hz, 9.6 Hz, 9H), 0.86-1.05(m, 6H). 13C NMR (100.5 MHz, CDCl3) δ:177.13, 175.91, 131.12, 126.47, 123.31,121.72, 109.63, 100.48, 92.23, 88.89, 84.51, 82.97, 81.87, 79.45, 76.52,74.88, 73.01, 72.16, 71.83, 70.71, 69.82, 68.56, 68.06, 67.93, 67.02, 66.66,65.17, 63.93, 62.10, 60.32, 59.52, 55.53, 48.87, 42.56, 41.78, 40.15, 38.63, 36.64, 35.42, 31.86, 25.45, 21.64, 19.68, 18.64, 17.75, 16.49, 15.05, 12.55. 8a-carbonyl avermectin B1a structural formula: Example 6 This embodiment provides a method for preparing 8a-carbonyl avermectin B1a, comprising the following steps: S1, 42g (40 mmol) of compound Ic prepared in Example 1, 126g of N,N-dimethylformamide, 0.021g of anhydrous copper sulfate, 0.021g of anhydrous manganese sulfate and 4.2g of pyridine were added to a 500 mL autoclave. The autoclave was sealed, and after confirming that the connection with the oxygen cylinder was intact, the valve was opened to purge three times. Then oxygen was introduced to raise the pressure to 0.4MPa, and the reaction was stirred at 10°C for 4.0h. After depressurization, the reaction solution was taken out and 500mL of water and 100mL of ethyl acetate were added. The mixture was stirred for 30min, allowed to stand and separate into layers. The organic layer was washed again with 100mL of water, separated into layers, and the organic layer was dried to obtain a solid. S2, the solid obtained in step 1 was dissolved in 100g of anhydrous methanol, and 8.6g (90 mmol) of methanesulfonic acid was added and stirred at room temperature for 1h until the reaction was complete. The solution was then concentrated to dryness under reduced pressure at 30℃, and 80g of acetonitrile was added and stirred for 1h. The solution was filtered and dried to obtain 26.7g of white solid. HPLC: 89.4%, yield 67.2%.

[0047] Example 7 This embodiment provides a method for preparing 8a-carbonyl avermectin B1a, comprising the following steps: S1, 42g (40 mmol) of compound Ic prepared in Example 1, 210g of N,N-dimethylformamide, 0.21g of anhydrous manganese sulfate and 2.1g of pyridine were added to a 500 mL autoclave. The autoclave was sealed, and after confirming that the connection with the oxygen cylinder was intact, the valve was opened to purge three times. Then oxygen was introduced to raise the pressure to 0.6MPa, and the reaction was stirred at 20°C for 2.0h. After depressurization, the reaction solution was taken out and 800mL of water and 300mL of ethyl acetate were added. The mixture was stirred for 30min, allowed to stand and separate into layers. The organic layer was washed again with 300mL of water, separated into layers, and the organic layer was dried to obtain a solid. S2, the solid obtained in step 1 was dissolved in 100g of anhydrous methanol, and 8.6g (90 mmol) of methanesulfonic acid was added. The mixture was stirred at room temperature for 1 h until the reaction was complete. The solution was concentrated to dryness under reduced pressure at 30°C, and then 80g of acetonitrile was added and stirred for 1 h. The mixture was filtered and dried to obtain 30.5g of white solid. HPLC: 91.2%, yield 78.4%.

[0048] Comparative Example 1 This comparative example differs from Example 2 in that it does not include pyridine, and specifically includes the following steps: S1. Take 45g (40 mmol) of compound Ia prepared in Example 1 and place it in a 500mL autoclave. Add 135g of N,N-dimethylformamide, 0.025g of anhydrous manganese sulfate, and 0.02g of anhydrous copper sulfate. After sealing the autoclave and confirming that it is connected to the oxygen cylinder, open it to purge oxygen three times and then introduce oxygen to 0.4MPa. Stir and react at 10℃ for 4h. After the reaction is completed and the pressure is released, take out the reaction solution and add 500mL of water and 100mL of ethyl acetate. Stir for 30min, let it stand to separate into layers, wash the organic layer with 500mL of water again, separate into layers, and dry the organic layer to obtain a solid. S2, the solid obtained in step S1 was dissolved in 100 g of anhydrous methanol, and 15.5 g (90 mmol) of p-toluenesulfonic acid was added. The mixture was stirred at room temperature for 1 h until the reaction was complete. The solution was concentrated to dryness under reduced pressure at 30 °C. Then, 80 g of acetonitrile was added and stirred for 30 min. The mixture was filtered and dried to obtain 34.4 g of a white solid. HPLC: 78.5%, yield 75.9%.

[0049] The product structure was determined to be 23-carbonylavermectin B2a.

[0050] Melting point: 180.4~192.1℃; MS [M+H + [ (m / z): 890.08, the calculated M+H value of 23-carbonylavermectin B2a is 890.08.]

[0051] 1H NMR (400 MHz, CDCl3) δ: 6.17-6.22(m, 1H), 6.03-5.96 (m, 2H), 5.83(d, J = 9.2 Hz, 1H), 5.63(s, 1H), 5.54-5.60 (m, 1H), 5.02(t, J = 7.8 Hz, 2H),4.88-4.92(d, J = 15.3 Hz, 2H), 4.78(t, J = 8.2 Hz, 2H), 4.62(d, J = 9.3 Hz, 1H),4.51(d, J = 10.9 Hz, 1H), 4.31(dd, J 1 = 6.3 Hz, J 2= 11.2 Hz, 2H ), 3.77 -4.17(m,2H), 3.65-3.70 (m, 3H), 3.53(s, 3H), 3.42(dd, J = 13.3 Hz, 9.0 Hz, 2H),3.06(t, J = 9.6 Hz, 1H), 2.65-2.85(m, 4H), 2.56-2.58(m, 3H), 2.31-2.38(m, 1H), 2.18(dd, J = 12.1 Hz, 10.2 Hz, 2H), 1. 65-1.79(m, 6H), 1.55-1.64 (m, 8H), 1.43-1.49(m,2H), 1.23-1.39(m, 4H), 1.09(dd, J = 13.2 Hz, 9.4 Hz, 8H), 0.86-0.98(m, 6H). 13C NMR (100.5 MHz, CDCl3) δ: 189.2, 176.67, 144.18, 133.17, 126.68,123.21, 122.08, 121.42, 120.61, 119.73, 105.54, 101.51, 99.02, 97.55, 89.71,86.74, 81.34, 79.18, 76.02, 74.44, 71.16, 69.53, 68.57, 67.18, 66.0, 65.74,64.78, 63.97, 60.92, 59. 18,58.82, 56.77, 55.84, 50.51, 46.77, 40.92, 36.64, 36.01, 35.95, 30.17, 29.51, 28.88, 24.19, 23.57, 21.95, 20.03, 18.63, 16.34. 23-Carbonylavermectin B2a structural formula: Comparative Example 2 The only difference between this comparative example and Example 2 is that the catalyst is replaced with pyridine-N-oxide, and the following steps are included: S1, Take 45g (40 mmol) of compound Ia prepared in Example 1 and place it in a 500mL autoclave. Add 135g of N,N-dimethylformamide and 0.5g of pyridine-N-oxide. After sealing the autoclave and confirming that it is connected to the oxygen cylinder, open it to purge oxygen three times and then introduce oxygen to 0.4MPa. Stir and react at 10℃ for 4h. After the reaction is completed and the pressure is released, take out the reaction solution and add 500mL of water and 100mL of ethyl acetate. Stir for 30min, let it stand and separate into layers. Wash the organic layer again with 500mL of water, separate into layers, and dry the organic layer to obtain a solid. S2, the solid obtained in step S1 was dissolved in 100g of anhydrous methanol, and 15.5g (90 mmol) of p-toluenesulfonic acid was added. The mixture was stirred at room temperature for 1 h until the reaction was complete. The solution was concentrated to dryness under reduced pressure at 30°C, and then 80g of acetonitrile was added and stirred for 30 min. The mixture was filtered and dried to obtain 32.5g of a white solid. HPLC: 62.3%, yield 56.8%.

[0052] The product structure was determined to be 23-carbonylavermectin B2a.

[0053] Melting point: 180.8~192.0℃; MS [M+H + [ (m / z): 890.08, the calculated M+H value of 23-carbonylavermectin B2a is 890.08.]

[0054] 1 H NMR (400 MHz, CDCl3) δ: 6.21-6.37(m, 1H), 6.13-6.16 (m, 2H), 6.03(d, J = 10.3 Hz, 1H), 5.86(s, 1H), 5.65-5.69 (m, 1H), 5.35(m, 2H), 5.08-5.12(d, J = 12.5 Hz, 2H), 4.98(t, J = 10.2 Hz, 2H), 4.86(d, J = 11.9 Hz, 1H), 4.75(d, J =11.8 Hz, 1H), 4.63(dd, J 1 = 16.3 Hz, J 2= 10.2 Hz, 2H ), 4.17-4.44(m, 2H), 3.76-4.07 (m, 3H), 3.65(s, 3H), 3.52(d, J = 13.3 Hz, 2H), 3.43(t, J = 11.6 Hz, 1H),2.75-3.15(m, 4H), 2.66-2.68(m, 3H), 2.43-2.49(m, 1H), 2.26(d, J = 14.3 Hz, 2H),1. 75-2.09(m, 6H), 1.61-1.70 (m, 8H), 1.41-1.59(m, 2H), 1.31-1.35(m, 4H),1.29(dd, J = 13.2 Hz, 9.5 Hz, 8H), 0.96-1.18(m, 6H). 13C NMR (100.5 MHz, CDCl3) δ: 181.1, 177.77, 151.11, 132.27, 129.61, 125.63, 123.81, 122.22, 121.32, 114.53, 108.74, 104.57, 100.32, 94.52, 90.34,88.62, 85.35, 81.25, 78.67, 76.24, 73.36, 72.03, 66.27, 67.78, 66.6, 65.5664.42, 63.67, 61.96, 58. 98, 58.12, 57.77, 56.24, 54.41, 43.27, 41.24, 38.14, 37.11, 33.35, 31.57, 30.53, 29.38, 25.39, 22.13, 20.67, 19.41, 17.56, 14.23. 23-Carbonylavermectin B2a structural formula: Comparative Example 3 The only difference between this comparative example and Example 2 is that the catalyst is replaced with a mixture of pyridine, cobalt tetroxide, and cobalt chloride, and the steps include: S1, Take 45g (40 mmol) of compound Ia prepared in Example 1 and place it in a 500mL autoclave. Add 135g of N,N-dimethylformamide, 0.025g of cobalt tetroxide, 0.02g of cobalt chloride and 4.5g of pyridine. After sealing the autoclave and confirming that it is connected to the oxygen cylinder, open it to purge oxygen three times and then introduce oxygen to 0.4MPa. Stir and react at 10℃ for 4h. After the reaction is completed and the pressure is released, take out the reaction solution and add 500mL of water and 100mL of ethyl acetate. Stir for 30min, let stand and separate the layers. Wash the organic layer again with 500mL of water, separate the layers, and dry the organic layer to obtain the solid. S2, the solid obtained in step S1 was dissolved in 100 g of anhydrous methanol, and 15.5 g (90 mmol) of p-toluenesulfonic acid was added and stirred at room temperature for 1 h. After the reaction was completed, the solution was concentrated to dryness under reduced pressure at 30 °C, and then 80 g of acetonitrile was added and stirred for 30 min. The solution was filtered and dried to obtain 29.7 g of white solid. HPLC: 65.7%, yield 54.9%.

[0055] The product structure was determined to be 23-carbonylavermectin B2a.

[0056] Melting point: 181.5~192.7℃; MS [M+H +[ (m / z): 890.08, the calculated M+H value of 23-carbonylavermectin B2a is 890.08.]

[0057] 1 H NMR (400 MHz, CDCl3) δ: 6.20-6.40(m, 1H), 6.15-6.17 (m, 2H), 6.00(d, J = 11.6 Hz, 1H), 5.88(s, 1H), 5. 56-5.70 (m, 1H), 5.45(m, 2H), 5.18-5.22(d, J = 10.2Hz, 2H), 5.08(t, J = 11.1 Hz, 2H), 4.88(d, J = 13.2 Hz, 1H), 4.73(d, J =13.4 Hz, 1H), 4.66(dd, J 1 = 15.4 Hz, J 2= ​​13.3 Hz, 2H), 4.21-4.53(m, 2H), 3.86-4.10 (m, 3H), 3.76(s, 3H), 3.65(d, J = 14.4 Hz, 2H), 3.54(t, J = 12.2 Hz, 1H),2.95-3.35(m, 4H), 2.56-2.76(m, 3H), 2.33-2.49(m, 1H), 2.28(d, J = 13.4 Hz, 2H),1. 70-2.19(m, 6H), 1.56-1.65 (m, 8H), 1.41-1.50(m, 2H), 1.30-1.36(m, 4H),1.24(dd, J = 15.2 Hz, 11.5 Hz, 8H), 0.83-1.13(m, 6H). 13C NMR (100.5 MHz, CDCl3) δ: 186.47, 182.73, 165.19, 143.67, 132.44, 126.54, 124.18, 123.22, 120.78, 118.72, 110.31, 106.75, 103.28, 96.22,92.45,90.32, 87. 53, 83.52, 80.76, 78.43, 75.63, 74.53, 68.77, 67.86, 66.64, 65.61, 64.92, 63.71, 62.06, 59.89, 58.65, 57.77, 56.84, 55.61, 47.72, 45.48, 41.14, 38.11, 36.53, 30.76, 30.23, 28.43, 27.73, 26.53, 22.26, 20.17, 19.06, 16.56. 23-Carbonylavermectin B2a structural formula: The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing avermectin B1a / B2a metabolites, characterized in that, Includes the following steps: Using the compound shown in formula (I) as a raw material, and with transition metal salt and pyridine as catalysts, the 8a position is oxidized under oxygen pressure to obtain the compound shown in formula (II); wherein the transition metal salt is one or both of copper salt and manganese salt. R is a hydroxyl protecting group; XY is CH=CH or CH2-CH(OH).

2. The method for preparing avermectin B1a / B2a metabolites as described in claim 1, characterized in that, Also includes: The hydroxyl protecting groups at the 5 and 4'' positions of the compound shown in formula (II) are removed to obtain the compound shown in formula (III); Formula (III).

3. The method for preparing avermectin B1a / B2a metabolites as described in claim 2, characterized in that, Specifically, the steps include the following: The compound shown in formula (I), a transition metal salt, and pyridine were added to an organic solvent and oxidized under oxygen pressure to obtain the compound shown in formula (II). The hydroxyl protecting groups at the 5 and 4'' positions of the compound represented by formula (II) are removed to obtain the compound represented by formula (III).

4. The method for preparing avermectin B1a / B2a metabolites as described in claim 1 or 3, characterized in that, The amount of pyridine added is 1% to 10% of the mass of the compound shown in formula (I), and the amount of transition metal salt added is 0.1% to 1% of the mass of the compound shown in formula (I).

5. The method for preparing avermectin B1a / B2a metabolites as described in claim 3, characterized in that, The oxidation reaction is carried out at a temperature of 10℃ to 30℃ for 1 hour to 4 hours.

6. The method for preparing avermectin B1a / B2a metabolites as described in claim 1 or 3, characterized in that, The oxygen is pressurized at a pressure of 0.4 MPa to 1.0 MPa.

7. The method for preparing avermectin B1a / B2a metabolites as described in claim 3, characterized in that, The organic solvent is N,N-dimethylformamide.

8. The method for preparing avermectin B1a / B2a metabolites as described in claim 7, characterized in that, The amount of organic solvent added is 3 to 6 times the mass of the compound shown in formula (I).

9. The method for preparing avermectin B1a / B2a metabolites as described in claim 1 or 3, characterized in that, The transition metal salt is at least one of manganese sulfate, copper sulfate, or copper acetate.

10. The method for preparing avermectin B1a / B2a metabolites as described in claim 1, characterized in that, The hydroxyl protecting group is dimethyl tert-butylsilyl or trimethylsilyl.

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