Process for the extraction and obtaining of high purity nimorazole from mycelium

Abstract

The application discloses a method for extracting and obtaining high-purity nimorazole from mycelium, and comprises the following steps: extracting nimorazole from mycelium by using an organic solvent, decolorizing the nimorazole by using activated carbon, rapidly separating the nimorazole by using a non-water-soluble organic solvent and a silica gel column chromatography, and obtaining nimorazole with a purity of 82% to 85%; then, the nimorazole is protected at a 5-position by using tert-butyl diphenyl chlorosilane, and a protection product with a purity of 93% to 96% is obtained; and then, the protection product is deprotected, and nimorazole with a purity of 93% to 96% is obtained, which can be used for preparing a nimorazole standard product. The method is simple in operation, low in cost, and can be applied to industrial production.

Description

Technical Field

[0001] This invention belongs to the field of biopesticide technology, specifically relating to the preparation of high-purity nimoxine. Background Technology

[0002] Nemadectin has the molecular formula C 36 H 52 O8, CAS number 102130-84-7, is a hexadecyl lactone produced by the fermentation of a streptomycin bacterium. It appears as a white crystalline powder or white crystals and possesses anthelmintic activity. It can be used as a broad-spectrum acaricide and is highly effective against natural infections of the gastrointestinal parasites in cattle, sheep, and dogs. It is widely used in the preparation of the novel antiparasitic drug moxixtin. The structural formula of moxixtin is as follows:

[0003]

[0004] Patent CN104628802 B discloses a method for extracting and purifying nimoxine from fermentation broth. This method involves flocculating and filtering the fermentation broth to obtain wet mycelial residue, then extracting it with a non-water-soluble organic solvent. The resulting extract is then acid-washed and alkali-washed. Next, nimoxine is derivatized using a benzene-ring-containing acyl chloride or acid anhydride, and the derivative is crystallized and separated. Finally, it is hydrolyzed and reduced to obtain nimoxine. However, the quality and purity of the nimoxine obtained by this method are not high enough.

[0005] Patent CN104292283 A discloses a purification method for nimoxetine, which involves mixing crude nimoxetine with a liquid organic salt, filtering, obtaining a filtrate, and then reacting the filtrate with C5-C... 20 Alkanes are mixed, washed, and separated to obtain a liquid organic salt solution, which is then crystallized at low temperature to obtain nimoxine. However, this method has a low crystallization success rate and is quite difficult to operate.

[0006] Patent CN103772458 A discloses a method for purifying nimoxetine. This method involves extracting nimoxetine from bacterial residue using two organic solvents of different polarities. The second solvent is less polar than the first. The extract is then concentrated and crystallized. Patent CN106046020 B also uses two organic solvents of different polarities to extract from the bacterial residue. The difference between CN106046020 B and CN103772458 A is that CN106046020 B employs a gradient cooling crystallization method. However, both methods are unsuitable for low-purity crude nimoxetine, as successful crystallization is difficult without it.

[0007] Patent CN108117562 A discloses a method for preparing nimoxetine by chromatographic separation and purification. This method uses organic solvent extraction and activated carbon decolorization to prepare high-purity nimoxetine by chromatographic separation. However, this method has problems such as poor decolorization effect of activated carbon in non-water-soluble organic solvents, high operation difficulty, high equipment requirements, and is not conducive to industrial production. Summary of the Invention

[0008] To address the aforementioned problems, this invention provides a method for extracting and obtaining high-purity nimocriptine from mycelium. This method is simple to operate, the solvent is easy to recover, and it is suitable for industrial production.

[0009] The method for extracting and obtaining high-purity nimocriptine from mycelium provided by this invention includes the following steps:

[0010] Step 1: Extract the nimoxetine mycelium with an organic solvent, filter to remove insoluble matter, and obtain nimoxetine extract;

[0011] Step 2: Remove part of the organic solvent from the nimoxetine extract, add activated carbon, decolorize at the boiling point of the organic solvent, cool and filter to remove the activated carbon, and evaporate the filtrate to obtain crude nimoxetine I.

[0012] Step 3: Dissolve crude nimoxine I in a non-water-soluble organic solvent, wash with water, dry the organic phase, and evaporate the solvent to obtain crude nimoxine II;

[0013] Step 4: Nimoxetine crude product II was rapidly separated by silica gel column chromatography to obtain nimoxetine with a purity of 82%–85%;

[0014] Step 5: The nimocriptine from step 4 is subjected to an upprotection reaction with tert-butyldiphenylchlorosilane to obtain the crude upprotected product.

[0015] Step 6: Separate the crude upper protected product by silica gel column chromatography and then crystallize it to obtain the upper protected product with a purity of 93% to 96%.

[0016] Step 7: The protected product is deprotected to obtain nimocritin with a purity of 93% to 96%.

[0017] In step 1 above, the organic solvent is preferably one or more of methanol, ethanol, ethyl acetate, dichloromethane, methyl tert-butyl ether, and n-heptane.

[0018] In step 1 above, the extraction temperature is the boiling point temperature of the organic solvent, and the extraction time is preferably 1 hour, with 1 to 2 extractions.

[0019] In step 2 above, the amount of activated carbon added is preferably 7% to 10% of the crude nimoxine mass in the extract obtained in step 1.

[0020] In step 3 above, the preferred non-water-soluble organic solvent is either ethyl acetate or dichloromethane.

[0021] In step 4 above, the preferred eluent for silica gel column chromatography is a mixture of ethyl acetate and dichloromethane in a volume ratio of 1:20 or a mixture of methanol and dichloromethane in a volume ratio of 1:80.

[0022] In step 5 above, the preferred method for the upper protection reaction is to use imidazole as the base, dichloromethane as the solvent, at a reaction temperature of 20–40°C, and for a reaction time of 3–5 hours. The reaction equation is shown below:

[0023]

[0024] In step 6 above, the preferred eluent for silica gel column chromatography is industrial-grade dichloromethane, and the solvent used for crystallization is analytical-grade acetonitrile, with crystallization occurring at room temperature.

[0025] In step 7 above, the preferred reagent for deprotection treatment is p-toluenesulfonic acid, the preferred reaction solvent is methanol, the preferred reaction temperature is -10℃ to room temperature, and the preferred reaction time is 2 to 6 hours.

[0026] The beneficial effects of this invention are as follows:

[0027] 1. In step 4 of this invention, the eluent used for separation by silica gel column chromatography is a mixture of ethyl acetate and dichloromethane in a volume ratio of 1:20, which is more effective than the traditional silica gel column chromatography separation method and increases the purity of nimoxetine by 10%.

[0028] 2. In step 5 of this invention, tert-butyldiphenylchlorosilane is used as the top protecting agent. This top protecting agent has two benzene rings and a large mass fraction, and the top protecting reaction product obtained by the top protecting agent is easier to solidify and crystallize than that obtained by the traditional top protecting agent. The top protecting reaction of nimoxetine was optimized by response surface methodology, and the optimal reaction conditions were obtained.

[0029] 3. This invention is simple to operate, has low cost, and produces nimoxetine with high purity. It can be used to prepare nimoxetine standards and can be applied to industrial production. Detailed Implementation

[0030] The present invention will be further described below with reference to the embodiments, but the scope of protection of the present invention is not limited to these embodiments.

[0031] The nimocriptine mycelium used in the examples was provided by Shandong Qifa Pharmaceutical Co., Ltd.

[0032] In the examples, the purity was determined by liquid chromatography (LC-2010A). HTShimadzu Corporation, Japan. Liquid chromatography detection conditions: Supersil ODS2 5μm packing material, 4.6mm × 15cm size; mobile phase V. 甲醇 V 水 = 95:5, detection wavelength 240nm, column temperature 30℃, mobile phase flow rate 0.7mL / min, injection volume 10μL.

[0033] The synthesis conditions of 5-oxo(tert-butyldiphenylchlorosilyl)nimoxetine were optimized using response surface methodology (RSM) with Design Expert 12. The four key factors affecting the yield of the upprotection reaction were determined using a single-factor method: time, temperature, the equivalence ratio of tert-butyldiphenylchlorosilane to nimoxetine, and the equivalence ratio of imidazole to nimoxetine. The number of RSM experiments was determined using Box-Behnken Design, and the corresponding conversion and yield were obtained according to the requirements. The results of the RSM experiments were analyzed using software, and the optimal reaction conditions for the upprotection reaction were determined to be an equivalence ratio of nimoxetine:tert-butyldiphenylchlorosilane:imidazole = 1:3.4:3.6, a reaction temperature of 29℃, and a reaction time of 3.8 h. The theoretical predicted yield was 94.6%. The experiment was repeated three times under these conditions, and the error between the actual and theoretical yields was less than 2%, indicating that using RSM to design and optimize the upprotection reaction process is feasible and the results are reliable.

[0034] Example 1

[0035] Step 1: Weigh 10g of nimoxetine mycelium, add 80mL of methanol, reflux and stir at 70℃ for 1h, filter to remove insoluble matter, and obtain nimoxetine extract. Remove the solvent from the extract by rotary evaporation to obtain 2.002g of crude nimoxetine, with a purity of 33.4% as determined by liquid chromatography.

[0036] Step 2: Dissolve the crude nimoxine from Step 1 in 50 mL of methanol, add 0.16 g of activated carbon, reflux and stir at 70 °C for 15 min, cool completely, filter to remove insoluble matter, and evaporate the filtrate to obtain 1.834 g of crude nimoxine I, with a purity of 36.4% as determined by liquid chromatography.

[0037] Step 3: Dissolve crude nimoxine I in 50 mL of ethyl acetate, add 30 mL of water for extraction, dry the organic phase with anhydrous Na2SO4, and remove the solvent by vacuum distillation to obtain 1.513 g of crude nimoxine II with a purity of 42.6%.

[0038] Step 4: Nimoxetine crude product II was separated by silica gel column chromatography with a mixture of ethyl acetate and dichloromethane in a volume ratio of 1:20 as the eluent, yielding 0.582 g of nimoxetine with a purity of 85%.

[0039] Step 5: Weigh 0.500 g (0.694 mmol, purity 85%) of nimoxetine from Step 4, dissolve it in 5 mL of dichloromethane, add 0.170 g (2.497 mmol) of imidazole, and slowly add 0.648 g (2.358 mmol) of tert-butyldiphenylchlorosilane dissolved in 3 mL of dichloromethane. React at 29 °C for 4 h. After the reaction is complete, wash with 1% hydrochloric acid aqueous solution, wash with water, dry the organic phase with anhydrous Na2SO4, and remove the solvent by vacuum distillation to obtain 1.329 g of crude 5-oxo(tert-butyldiphenylsilyl)nimoxetine, the protected reaction product. Liquid chromatography showed that the conversion rate of nimoxetine was 94.7% and the yield of the protected product was 93.9%.

[0040] Step 6: Separate the crude product of the upper protection reaction by silica gel column chromatography, using industrial-grade dichloromethane as the eluent, to obtain 0.638 g of the upper protection product with a purity of 91%. Add 3 mL of acetonitrile to the obtained upper protection product and crystallize at room temperature to obtain 0.575 g of the upper protection product crystals with a purity of 96% and a yield of 90.3%.

[0041] Step 7: Take 0.500 g (0.564 mmol, purity 96%) of the protected product crystals, add 5 mL of methanol, add 0.429 g (2.258 mmol) of p-toluenesulfonic acid, react at room temperature for 3 h. After the reaction is complete, add 30 mL of ethyl acetate, wash with saturated NaHCO3 aqueous solution, wash with water, dry the organic phase with anhydrous Na2SO4, remove the solvent by vacuum distillation to obtain 0.340 g of nimoxine, the purity of which is 96% and the yield is 95.9% as determined by liquid chromatography.

[0042] Example 2

[0043] Step 1: Weigh 10g of nimoxetine mycelium, add 80mL of ethanol, reflux and stir at 80℃ for 1h, extract twice, filter to remove insoluble matter, and combine the two extracts to obtain nimoxetine extract. Remove the solvent from the extract by rotary evaporation to obtain 2.172g of crude nimoxetine, with a purity of 29.1% as determined by liquid chromatography.

[0044] Step 2: Dissolve the crude nimoxine from Step 1 in 50 mL of ethanol, add 0.17 g of activated carbon, reflux and stir at 80 °C for 15 min, cool completely, filter to remove insoluble matter, and evaporate the filtrate to obtain 1.917 g of crude nimoxine, with a purity of 31.4% as determined by liquid chromatography.

[0045] Step 3: Dissolve crude nimoxine I in 50 mL of ethyl acetate, add 30 mL of water for extraction, dry the organic phase with anhydrous Na2SO4, and remove the solvent by vacuum distillation to obtain 1.371 g of crude nimoxine II with a purity of 35.4%.

[0046] Step 4: Nimoxetine crude product II was separated by silica gel column chromatography with methanol and dichloromethane in a volume ratio of 1:80 as the eluent to obtain 0.519 g of nimoxetine with a purity of 82%.

[0047] Step 5: Weigh 0.500 g (0.669 mmol, purity 82%) of nimoxetine from Step 4, dissolve it in 5 mL of dichloromethane, add 0.164 g (2.409 mmol) of imidazole, and slowly add 0.625 g (2.275 mmol) of tert-butyldiphenylchlorosilane dissolved in 3 mL of dichloromethane. React at 29 °C for 4 h. After the reaction is complete, wash with 1% hydrochloric acid aqueous solution, wash with water, dry the organic phase with anhydrous Na2SO4, and remove the solvent by vacuum distillation to obtain 1.195 g of crude 5-oxo(tert-butyldiphenylsilyl)nimoxetine, the protected reaction product. Liquid chromatography showed that the conversion rate of nimoxetine was 94.8% and the yield of the protected product was 92.8%.

[0048] Step 6: Separate the crude product of the upper protection reaction by silica gel column chromatography, using industrial-grade dichloromethane as the eluent, to obtain 0.598 g of the upper protection product with a purity of 91%. Add 3 mL of acetonitrile to the obtained upper protection product and crystallize at room temperature to obtain 0.549 g of the upper protection product crystals with a purity of 93% and a yield of 89.7%.

[0049] Step 7: Take 0.500 g (0.564 mmol, purity 93%) of the protected product crystals, add 5 mL of methanol, add 0.429 g (2.258 mmol) of p-toluenesulfonic acid, react at room temperature for 3 h. After the reaction is complete, add 30 mL of ethyl acetate, wash with saturated NaHCO3 aqueous solution, wash with water, dry the organic phase with anhydrous Na2SO4, remove the solvent by vacuum distillation to obtain 0.295 g of nimoxine, the purity of which is 93.2% as determined by liquid chromatography.

[0050] Comparative Example 1

[0051] Steps 1 to 3 are the same as in Example 1.

[0052] Step 4: Nimoxetine crude product II was separated by silica gel column chromatography with methanol and dichloromethane in a volume ratio of 1:80 as the eluent to obtain 0.605 g of nimoxetine with a purity of 81%.

[0053] Step 5: Weigh 0.500 g (0.661 mmol, purity 81%) of nimoxine from Step 4, dissolve it in 5 mL of dichloromethane, add 0.164 g (2.409 mmol) of imidazole, and slowly add 0.343 g (2.275 mmol) of tert-butyldimethylchlorosilane dissolved in 3 mL of dichloromethane. React at 29 °C for 4 h. After the reaction is complete, wash with 1% hydrochloric acid aqueous solution, wash with water, dry the organic phase with anhydrous Na2SO4, and remove the solvent by vacuum distillation to obtain 0.697 g of crude 5-oxo(tert-butyldimethylsilyl)nimoxine, the protected reaction product.

[0054] Step 6: Separate the crude product of the upper protection reaction by silica gel column chromatography, using industrial-grade dichloromethane as the eluent, to obtain 0.451 g of the upper protection product with a purity of 90%. Add 3 mL of acetonitrile to the obtained upper protection product, and no crystallization occurs at room temperature, with a yield of 83.5%.

[0055] Step 7: Take 0.400 g (0.496 mmol, purity 90%) of the protected product, add 5 mL of methanol, add 0.341 g (1.982 mmol) of p-toluenesulfonic acid, react at 0 °C for 4 h. After the reaction is complete, add 30 mL of ethyl acetate, wash with saturated NaHCO3 aqueous solution, wash with water, dry the organic phase with anhydrous Na2SO4, remove the solvent by vacuum distillation to obtain 0.296 g of nimoxine. The purity was determined by liquid chromatography to be 91.4%, and the yield was 89%.

Claims

1. A method for extracting and obtaining high-purity nimocriptine from mycelium, characterized in that, Includes the following steps: Step 1: Extract the nimoxetine mycelium with an organic solvent, filter to remove insoluble matter, and obtain nimoxetine extract; Step 2: Remove part of the organic solvent from the nimoxetine extract, add activated carbon, decolorize at the boiling point of the organic solvent, cool and filter to remove the activated carbon, and evaporate the filtrate to obtain crude nimoxetine I. Step 3: Dissolve crude nimoxine I in a non-water-soluble organic solvent, wash with water, dry the organic phase, and evaporate the solvent to obtain crude nimoxine II; Step 4: Nimoxetine crude product II was rapidly separated by silica gel column chromatography to obtain nimoxetine with a purity of 82%–85%; Step 5: The nimocriptine from step 4 is subjected to an upprotection reaction with tert-butyldiphenylchlorosilane to obtain the crude upprotected product. Step 6: Separate the crude upper protected product by silica gel column chromatography and then crystallize it to obtain the upper protected product with a purity of 93% to 96%. Step 7: The protected product is deprotected to obtain nimocritin with a purity of 93% to 96%.

2. The method for extracting and obtaining high-purity nimocriptine from mycelium according to claim 1, characterized in that, In step 1, the organic solvent is any one or more of methanol, ethanol, ethyl acetate, dichloromethane, methyl tert-butyl ether, and n-heptane.

3. The method for extracting and obtaining high-purity nimocriptine from mycelium according to claim 1 or 2, characterized in that, The extraction temperature is the boiling point of the organic solvent, the extraction time is 1 hour, and the extraction is performed 1 to 2 times.

4. The method for extracting and obtaining high-purity nimocriptine from mycelium according to claim 1, characterized in that, In step 2, the amount of activated carbon added is 7% to 10% of the crude nimoxetine mass in the extract obtained in step 1.

5. The method for extracting and obtaining high-purity nimocriptine from mycelium according to claim 1, characterized in that, In step 3, the non-water-soluble organic solvent is either ethyl acetate or dichloromethane.

6. The method for extracting and obtaining high-purity nimocriptine from mycelium according to claim 1, characterized in that, In step 4, the eluent used for silica gel column chromatography is a mixture of ethyl acetate and dichloromethane in a volume ratio of 1:20 or a mixture of methanol and dichloromethane in a volume ratio of 1:

80.

7. The method for extracting and obtaining high-purity nimocriptine from mycelium according to claim 1, characterized in that, In step 5, imidazole is used as the base for the upper protection reaction, dichloromethane is used as the solvent, the reaction temperature is 20-40℃, and the reaction time is 3-5 hours.

8. The method for extracting and obtaining high-purity nimocriptine from mycelium according to claim 1, characterized in that, In step 6, the eluent used for silica gel column chromatography is industrial-grade dichloromethane, and the solvent used for crystallization is analytical-grade acetonitrile, with crystallization occurring at room temperature.

9. The method for extracting and obtaining high-purity nimocriptine from mycelium according to claim 1, characterized in that, In step 7, the reagent used for deprotection treatment is p-toluenesulfonic acid, the reaction solvent is methanol, the reaction temperature is -10℃ to room temperature, and the reaction time is 2 to 6 hours.

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