CN109400621B - Preparation method of high-purity milbemycins - Google Patents
Preparation method of high-purity milbemycins Download PDFInfo
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- CN109400621B CN109400621B CN201811400424.1A CN201811400424A CN109400621B CN 109400621 B CN109400621 B CN 109400621B CN 201811400424 A CN201811400424 A CN 201811400424A CN 109400621 B CN109400621 B CN 109400621B
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- C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
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Abstract
The invention provides a preparation method of high-purity milbemycins, which comprises the steps of extracting filter residues of a milbemycins fermentation liquor by alcohol with a low boiling point, concentrating the filter residues by hot water under reduced pressure, cooling, filtering and removing solid matters; adding a strong polar organic solvent into the filtrate for back extraction, and concentrating the back-extracted feed liquid to dryness after salt washing, alkali washing and water washing; adding strong polar solvent for dissolving, maintaining at a certain temperature, adding a large amount of low polar solvent under stirring, cooling under stirring, crystallizing, and filtering to obtain high-purity milbemycin. The method adopts the principles of phase transfer and solubility and polarity difference to replace the traditional macroporous resin adsorption separation technology and silica gel chromatography technology, and the technology is simple, stable and controllable, can shorten the production period, save a large amount of organic solvents, reduce a large amount of solid wastes, save energy and reduce the production cost.
Description
Technical Field
The invention relates to the technical field of biological fermentation pharmacy, in particular to a method for obtaining high-purity milbemycins by utilizing phase inversion forced crystallization.
Background
Milbemycins (mibemycins) are antibody internal and external parasite medicines of macrolides, contain two effective components A3 and A4, and are commonly used as anthelmintics for canines. Milbemycins are very effective in controlling and preventing most common parasitic diseases. Is generally used for preventing heartworm disease, controlling canine and feline diseases caused by nematodes and hookworms and controlling flagellosis of dogs. The milbemycins are intermediates for synthesizing the milbemycin oxime, and the high-purity intermediates provide efficient means for synthesizing the milbemycin oxime.
The purity of milbemycins is greatly related to the subsequent yield and the cost; the current purification method of milbemycins is macroporous resin adsorption and desorption and silica gel chromatography, which needs a large amount of organic solvents and generates a large amount of solid wastes. With the gradual enhancement of environmental awareness, it is urgent to reduce the use amount of organic solvents and reduce solid wastes in the purification process of milbemycins according to the current requirements of safety and environmental protection in response to the requirements of building an environment-friendly and resource-saving society.
Disclosure of Invention
The invention aims to overcome the technical defects of the prior art in the extraction of the milbemycins and provides a preparation method of high-purity milbemycins.
The invention skillfully adopts the idea of phase transfer to replace macroporous resin adsorption and silica gel chromatography technology, and comprises the following steps:
(1) extracting the filter residue of the milbemycin fermentation liquor by using low-boiling-point alcohol, concentrating the filter residue by using hot water under reduced pressure, cooling, filtering and removing solid matters to obtain filtrate;
(2) adding a strong polar organic solvent into the filtrate for back extraction, collecting an organic phase after the back extraction, adding an adsorbent after salt washing, alkali washing and water washing, performing filter pressing, collecting the filtrate, concentrating the filtrate under reduced pressure to dryness, and collecting solid matters;
(3) and dissolving the solid substance with a strong polar solvent, heating, keeping the temperature, adding a weak polar organic solvent while stirring, cooling, crystallizing, centrifuging and drying to obtain the high-purity milbemycin.
In the filter residue of the milbemycin fermentation liquor in the step (1), the chromatographic purity of the milbemycin is 50-55%. Specifically, the chromatographic purity of milbemycins A3+ A4 is 50% -55%.
The low-boiling-point alcohol in the step (1) comprises but is not limited to methanol and ethanol, and the amount of the added low-boiling-point alcohol is 6-8 times of the volume of the filter residue; the volume percentage of the low boiling point alcohol is 75-80%.
And (2) concentrating the hot water in the step (1) at 50-60 ℃ under reduced pressure until the alcoholic strength is 18-23% and off-white solid appears, and stopping concentrating. Preferably, the concentration in the step (1) is stopped when the off-white solid appears after the concentration is carried out under reduced pressure until the alcoholic strength is 20 percent.
And (2) the temperature reduction in the step (1) is to slowly reduce the temperature to 8-10 ℃ after the decompression concentration, and keep the temperature for 10-14 hours. Preferably, the temperature is maintained for 12 hours, the solid matter is filtered by a plate-and-frame filter, the filtrate is collected, and the chromatographic purity of the milbemycin A3+ A4 in the filtrate is 65-75%.
The strong polar organic solvent in the step (2) is dichloromethane, ethyl acetate and/or butyl acetate; the salt washing is carried out by adopting saturated saline water washing, and the alkali washing is carried out by adopting saturated sodium bicarbonate solution; the adsorbent is active carbon and anhydrous sodium sulfate.
The strong polar organic solvent in the step (3) is dichloromethane, toluene, ethyl acetate and butyl acetate; the weak polar organic solvent comprises: n-heptane, n-hexane, methylcyclohexane, petroleum ether.
And (3) adding strong polar organic solvent with the volume 1-1.5 times that of the solid, heating to 40-60 ℃, keeping the temperature, and adding weak polar organic solvent with the volume 60-80 times that of the solid while stirring.
In the step (3), after the weak-polarity organic solvent is added, the temperature is kept for 1.5 to 2 hours, the temperature is reduced to 8 to 10 ℃ according to the temperature reduction range of 5 to 7 ℃/hour, and the mixture is stirred and aged for 10 to 12 hours to separate out crystals.
The invention provides application of the preparation method in improving the purity of the milbemycins.
The high-purity milbemycins prepared by the method have the chromatographic purity of 50-55% in the fermentation mushroom dregs, the chromatographic purity of 95-98% after phase transfer crystallization, and the absolute content of 93-96%.
The method utilizes phase inversion forced crystallization to obtain high-purity milbemycins, greatly reduces the using amount of a solvent by about 70 percent compared with the using amount of the solvent in the prior art, greatly reduces the solid waste amount by about 90 percent compared with the solid waste amount in the prior art, is safe and environment-friendly, and has controllable risks (the crystallization method of the invention is utilized to maximally require 60 times of volume of organic solvent of a pure product, if the conventional silica gel chromatography and macroporous resin adsorption and desorption are adopted to minimally require 600 times of volume of organic solvent of the pure product, and in the aspect of solid waste, because the process of the invention does not produce waste silica gel and resin, only produce active carbon and anhydrous sodium sulfate with 5 percent of pure product amount). The method replaces macroporous resin chromatography and silica gel chromatography, is simple and easy to operate, has low cost, high product purity and good quality yield, reduces the production cost and the investment cost, and reduces the pollution to the environment.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.
Unless otherwise specified, the chemical reagents used in the examples are all conventional commercially available reagents, and the technical means used in the examples are conventional means well known to those skilled in the art. In the filter residue of the milbemycins fermentation liquor adopted by the embodiment of the invention, the chromatographic purity of the milbemycins is 50-55%.
Example 1
The procedure for the preparation of high purity milbemycins according to this example is as follows:
(1) extracting the filter residue of the milbemycin fermentation liquor by using 75% ethanol to extract more than 92% of milbemycin, and performing pressure filtration to obtain a filtrate, wherein the chromatographic purity of the milbemycin A3 and A4 components in the filtrate is 57%;
(2) heating with hot water at 50-60 deg.C, concentrating the filtrate obtained in step (1) under reduced pressure until about 20% of alcohol content has off-white solid, stopping concentrating, cooling to 8-10 deg.C, maintaining for 12 hr, filtering out solid with plate frame, and collecting filtrate to obtain milbemycin A3+ A4 with chromatographic purity of 67%;
(3) performing back extraction on the filtrate obtained in the step (2) by using ethyl acetate, discharging a water phase after extraction, collecting an organic phase, sequentially performing salt washing (saturated salt water), alkali washing (saturated sodium bicarbonate solution) and water washing on the organic phase, and collecting the organic phase;
(4) adding the organic phase obtained in the step (3) into needle-shaped activated carbon accounting for 5% of the volume of the pure product and anhydrous sodium sulfate accounting for 5% of the volume of the pure product, uniformly stirring, performing plate-and-frame filter pressing, and collecting clear filtrate;
(5) concentrating the clear filtrate obtained in the step (4) under reduced pressure to dryness to obtain a colloidal solid;
(6) dissolving the colloidal solid obtained in the step (5) by using ethyl acetate with the volume 1 time of that of the colloidal solid, heating to 40 ℃, keeping the temperature, slowly adding n-hexane with the volume 60 times of that of the solid at the temperature, stirring and adding the n-hexane, keeping the temperature for about 2 hours, then starting frozen water to slowly cool, cooling the n-hexane with the cooling range of 5 ℃/hour, cooling the n-hexane to about 10 ℃, stirring and aging for 12 hours, separating out crystals, and separating and drying by using a centrifugal machine. The chromatographic purity of the obtained high-purity milbemycin A3+ A4 component is 96.7%.
Example 2
The procedure for the preparation of high purity milbemycins according to this example is as follows:
the method comprises the steps of extracting filter residues of milbemycin fermentation liquor by using 80% ethanol to extract more than 93.5% of milbemycin, and performing filter pressing to obtain filtrate, wherein the chromatographic purity of components of milbemycin A3 and A4 in the filtrate is 57.6%;
heating with hot water, wherein the water temperature is not more than 55 ℃, decompressing and concentrating the filtrate obtained in the step until gray solid matters appear after the alcoholic strength is about 20%, stopping concentrating, cooling to 8-10 ℃, preserving heat for 12 hours, filtering the solid matters with a plate frame, and collecting the filtrate to obtain milbemycin A3+ A4 with the component chromatographic purity of 68%;
performing back extraction on the filtrate obtained in the step II by using a certain amount of dichloromethane, discharging a water phase after extraction, and collecting an organic phase, wherein the organic phase is sequentially subjected to salt washing (saturated salt water), alkali washing (saturated sodium bicarbonate solution) and water washing, and the organic phase is collected;
fourthly, adding the organic phase obtained in the step three into needle-shaped activated carbon accounting for 5% of the volume of the pure product and anhydrous sodium sulfate accounting for 5% of the volume of the pure product, uniformly stirring, performing plate-and-frame filter pressing, and collecting clear filtrate; step five, decompressing and concentrating the clear filtrate obtained in the step four to be dry to obtain a colloidal solid;
sixthly, dissolving the colloidal solid obtained in the step fifthly by using butyl acetate with the volume being 1.5 times that of the colloidal solid, heating to 45 ℃, keeping the temperature, slowly adding petroleum ether with the volume being 60 times that of the solid, stirring and adding the petroleum ether, keeping the temperature for about 2 hours, then starting chilled water to slowly cool, cooling the petroleum ether at the temperature of 5 ℃/hour, cooling to about 10 ℃, stirring and aging for 12 hours, separating out crystals, and separating and drying by using a centrifuge to obtain the milbemycin A3+ A4 with the high-purity component chromatography purity of 96.1%.
Example 3
The procedure for the preparation of high purity milbemycins according to this example is as follows:
extracting filter residues of milbemycin fermentation liquor by using 85% methanol to extract more than 96% of milbemycin, and performing filter pressing to obtain filtrate, wherein the chromatographic purity of components of milbemycin A3 and A4 in the filtrate is 64.8%;
heating with hot water at a water temperature of 58 ℃, concentrating the filtrate obtained in the step under reduced pressure until off-white solid matters appear in the filtrate with the alcoholic strength of about 20%, stopping concentration, cooling to 8-10 ℃, preserving heat for 12 hours, filtering the solid matters with a plate frame, and collecting the filtrate to obtain milbemycin A3+ A4 with the component chromatographic purity of 71.6%;
performing back extraction on the filtrate obtained in the step II by using dichloromethane, discharging a water phase after extraction, collecting an organic phase, and sequentially performing salt washing (saturated saline solution), alkali washing (saturated sodium bicarbonate solution) and water washing on the organic phase to collect the organic phase;
fourthly, adding the organic phase obtained in the step three into needle-shaped activated carbon accounting for 5% of the volume of the pure product and anhydrous sodium sulfate accounting for 5% of the volume of the pure product, uniformly stirring, performing plate-and-frame filter pressing, and collecting clear filtrate;
step five, decompressing and concentrating the clear filtrate obtained in the step four to be dry to obtain a colloidal solid;
sixthly, dissolving the colloidal solid obtained in the step fifthly by using ethyl acetate with the volume being 1 time that of the colloidal solid, heating to 40 ℃, keeping the temperature, slowly adding n-heptane with the volume being 60 times that of the solid at the temperature, stirring and adding the n-heptane, keeping the temperature for about 2 hours, then starting chilled water, slowly cooling, cooling the n-heptane with the cooling amplitude being 5 ℃/hour, cooling to about 10 ℃, stirring and aging for 12 hours, separating out crystals, and separating and drying by using a centrifuge to obtain the milbemycin A3+ A4 with the high-purity component chromatographic purity being 98.7%.
Example 4
The procedure for the preparation of high purity milbemycins according to this example is as follows:
extracting filter residues of milbemycin fermentation liquor by using 75% methanol to extract more than 93% of milbemycin, and performing filter pressing to obtain filtrate, wherein the chromatographic purity of components of milbemycin A3 and A4 in the filtrate is 59%;
heating with hot water at the water temperature of 56 ℃, concentrating the filtrate obtained in the step under reduced pressure until off-white solid matters appear in the filtrate with the alcoholic strength of about 20%, stopping concentration, cooling to 8-10 ℃, preserving heat for 12 hours, filtering the solid matters with a plate frame, and collecting the filtrate to obtain milbemycin A3+ A4 with the component chromatographic purity of 68%;
performing back extraction on the filtrate obtained in the step II by using butyl acetate, discharging a water phase after extraction, and collecting an organic phase, wherein the organic phase is sequentially subjected to salt washing (saturated saline solution), alkali washing (saturated sodium bicarbonate solution) and water washing, and the organic phase is collected;
fourthly, adding the organic phase obtained in the step three into needle-shaped activated carbon accounting for 5% of the volume of the pure product and anhydrous sodium sulfate accounting for 5% of the volume of the pure product, uniformly stirring, performing plate-and-frame filter pressing, and collecting clear filtrate;
step five, decompressing and concentrating the clear filtrate obtained in the step four to be dry to obtain a colloidal solid;
sixthly, dissolving the colloidal solid obtained in the step fifthly by using ethyl acetate with the volume being 1.2 times that of the colloidal solid, heating to 43 ℃, keeping the temperature, slowly adding n-heptane with the volume being 60 times that of the solid, stirring and adding the n-heptane, keeping the temperature for about 2 hours, then starting chilled water to slowly cool, cooling the n-heptane to 5 ℃/hour, cooling to 10 ℃ or so, stirring and aging for 12 hours, separating out crystals, and separating and drying by using a centrifuge to obtain the milbemycin A3+ A4 with the high-purity component chromatographic purity of 97.7%.
The above examples are only for describing the preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, and various modifications and improvements made to the technical solution of the present invention by those skilled in the art without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims (2)
1. A preparation method of milbemycin is characterized by comprising the following steps:
(1) extracting the filter residue of the milbemycin fermentation liquor by methanol or ethanol, concentrating by hot water under reduced pressure, cooling, filtering to remove solid substances, and collecting the filtrate; in the filter residue, the chromatographic purity of the milbemycins is 50% -55%; adding methanol or ethanol in an amount 6-8 times the volume of the filter residue; the volume percentage of the methanol or the ethanol is 75 to 80 percent; the temperature reduction is to slowly reduce the temperature to 8-10 ℃ after the decompression concentration, and the temperature is preserved for 10-14 hours;
(2) adding dichloromethane, toluene, ethyl acetate or butyl acetate into the filtrate for back extraction, collecting an organic phase after back extraction, adding an adsorbent after salt washing, alkali washing and water washing, performing filter pressing, collecting the filtrate, concentrating the filtrate under reduced pressure to dryness, and collecting a solid matter; the salt washing is carried out by adopting saturated saline water washing, and the alkali washing is carried out by adopting saturated sodium bicarbonate solution; the adsorbent is activated carbon and anhydrous sodium sulfate;
(3) dissolving the solid with 1-1.5 times volume of solid of dichloromethane, toluene, ethyl acetate or butyl acetate, heating to 40-60 deg.C, maintaining the temperature, adding 60-80 times volume of n-heptane, n-hexane, methylcyclohexane or petroleum ether while stirring, maintaining the temperature for 1.5-2.0 hr, cooling to 8-10 deg.C/hr according to 5-7 deg.C/hr, stirring and aging for 10-12 hr for crystallization, and centrifuging and drying to obtain milbemycin.
2. The preparation method according to claim 1, wherein after the filter residue of the milbemycin fermentation liquor is extracted by ethanol in the step (1), the hot water reduced pressure concentration means that the concentration is stopped when the alcohol content is reduced to 18-23% by hot water at 50-60 ℃.
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| 大孔树脂分离纯化米尔贝霉素工艺研究;谢捷，等;《浙江工业大学学报》;20140831;第42卷(第4期);第413-417页 * |
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Effective date of registration: 20230613 Address after: 3007, Hengqin international financial center building, No. 58, Huajin street, Hengqin new area, Zhuhai, Guangdong 519031 Patentee after: New founder holdings development Co.,Ltd. Patentee after: CHONGQING DAXIN PHARMACEUTICAL Co.,Ltd. Patentee after: Peking University Medical Management Co.,Ltd. Address before: 100871 8th floor, founder building, 298 Chengfu Road, Haidian District, Beijing Patentee before: PEKING UNIVERSITY FOUNDER GROUP Co.,Ltd. Patentee before: CHONGQING DAXIN PHARMACEUTICAL Co.,Ltd. Patentee before: PKU HEALTHCARE INDUSTRY Group |