A preparation method for reducing the content of oxytetracycline impurity C

Abstract

The present invention relates to the field of medicine, and discloses a preparation method for reducing the content of oxytetracycline impurity C, comprising the following steps: S1: oxytetracycline crude product is added to a saturated alcohol aqueous solution, then substituted phenylhydrazine is added, and the reaction is stirred for 0.5-1.2h; S2, step S1 reaction solution is filtered, the filter cake is beating with a saturated alcohol aqueous solution for 1h, filtered, and the filter cake is washed with a saturated alcohol aqueous solution once; S3, step S2 solid is added to a saturated alcohol solution, hydrochloric acid solution is added to adjust the system pH to 2-3, stirring reaction, amine solution is added to adjust the pH to 5-6, and solvent is filtered out; S4, step S3 solid is placed in a vacuum drying oven and dried to constant weight to obtain a oxytetracycline finished product with low impurity C content, and the impurity C content in the finished product is as low as 0.2%. The preparation method for reducing the content of oxytetracycline impurity C provided by the present invention can effectively remove the difficult-to-remove impurity C in oxytetracycline compared to traditional purification methods, and its content can be reduced from 2-5% to less than 0.3%, greatly improving product quality and reducing adverse drug reactions.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and specifically relates to a preparation method for reducing the content of oxytetracycline impurity C. Background Art

[0002] Antibiotics, also known as antimicrobials, are a class of secondary metabolites produced by microorganisms (including bacteria, fungi, and actinomycetes) or higher animals and plants in their life activities that have anti-pathogen or other activities, and are chemical substances that can interfere with the developmental functions of other cells. Antibiotics are mainly derived from microbial fermentation broth extracts, semi-synthetic antibiotics, and chemical synthesis. Commonly used antibiotics in clinical practice include penicillins, aminoglycosides, tetracyclines, cephalosporins, macrolides, lincomycins, etc.

[0003] Oxytetracycline is a broad-spectrum antibiotic synthesized by Streptomyces chelidonus through fermentation. Its structural formula is Figure 4 As shown, it belongs to the tetracycline class. At present, the mainstream extraction process in China is to use oxalic acid as an acidifier to adjust the pH value of the fermentation liquid to 2-3, and then use sodium ferroxanthate and zinc sulfate to remove high molecular impurities, such as impurities at 430nm and 490nm absorption wavelengths, and then use a resin column for decolorization.

[0004] Oxytetracycline impurity C is a byproduct impurity produced during microbial fermentation. Its structure is as follows Figure 5 As shown in the figure, its antibacterial activity is much lower than that of oxytetracycline, and its structure is very similar to that of oxytetracycline. The only difference is that the 2-position structure is different. The 2-position structure of impurity C is acetyl, which is better than oxytetracycline in chemical activity and easier to derivatize than oxytetracycline. The content of impurity C in the fermentation broth is generally around 2-5%. The traditional purification method has a very low efficiency in removing this impurity. The content of this impurity in oxytetracycline products is high and easily exceeds the standard limit, resulting in high production costs and drug safety issues. Therefore, the development of new purification methods has great practical significance. Summary of the invention

[0005] The purpose of the present invention is to provide a preparation method for reducing the content of oxytetracycline impurity C, which solves the problem that the purification process of the preparation method disclosed in the prior art can remove impurities and pigments at absorption wavelengths of 430nm and 490nm, but has a very low removal efficiency for impurity C.

[0006] In order to solve the above technical problems, the present invention provides a preparation method for reducing the content of oxytetracycline impurity C, comprising the following steps:

[0007] S1: Add crude oxytetracycline to a saturated alcohol aqueous solution, then add substituted phenylhydrazine, and stir to react for 0.5-1.2h. The reaction formula is as follows:

[0008]

[0009] Wherein, formula I is impurity C, formula II is substituted phenylhydrazine, and formula III is the adduct of impurity C and substituted phenylhydrazine;

[0010] S2, filtering the reaction solution of step S1, beating the filter cake with fatty alcohol aqueous solution for 1 hour, filtering, and washing the filter cake with saturated alcohol aqueous solution once;

[0011] S3, adding the solid from step S2 to a saturated alcohol solution, then adding a hydrochloric acid solution to adjust the pH of the system to 2-3, stirring the reaction, then adding an amine solution to adjust the pH to 5-6, and filtering to remove the solvent;

[0012] S4. The solid from step S3 is placed in a vacuum drying oven and dried to constant weight to obtain a oxytetracycline product with a low impurity C content.

[0013] Preferably, the saturated alcohol is a C1-C4 monohydric saturated alcohol, and the volume fraction of the saturated alcohol aqueous solution is 80-95%.

[0014] Preferably, in the substituted phenylhydrazine in step S1, R1, R2, and R3 are selected from hydrogen, C1-C3 alkyl, hydroxyl, nitro, aromatic ring, and nitrogen heterocycle, wherein R1, R2, and R3 are not selected from hydroxyl or nitro at the same time.

[0015] Preferably, the reaction temperature in step S1 is 20-50°C.

[0016] Preferably, the weight ratio of the crude oxytetracycline, substituted phenylhydrazine and saturated alcohol aqueous solution in step S1 is 100:1-5:100-300.

[0017] Preferably, the beating temperature in step S2 is 30-60°C.

[0018] Preferably, in step S2, the weight ratio of the filter cake to the saturated alcohol aqueous solution is 1:1-2.

[0019] Preferably, the amine in step S3 is one or a combination of aqueous ammonia, aqueous methylamine solution, and aqueous dimethylamine solution, more preferably aqueous ammonia.

[0020] The beneficial effects of the present invention are as follows: compared with the traditional purification method, the preparation method for reducing the content of oxytetracycline impurity C provided by the present invention can effectively remove the difficult-to-remove impurity C in oxytetracycline, and its content can be reduced from 2-5% to below 0.3%, thereby greatly improving product quality, reducing adverse drug reactions, and improving drug efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the HPLC chromatogram and peak diagram of crude oxytetracycline.

[0022] Figure 2The HPLC chromatogram and peak diagram of oxytetracycline prepared in Example 1.

[0023] Figure 3 The HPLC chromatogram and peak diagram of oxytetracycline prepared in Example 4.

[0024] Figure 4 The chemical structure of oxytetracycline.

[0025] Figure 5 The chemical structure diagram of oxytetracycline impurity C. DETAILED DESCRIPTION

[0026] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] Example 1

[0028] Add 100g of oxytetracycline crude product (commercial product) into a 500ml flask. The HPLC spectrum is as follows: Figure 1 As shown, 300g 90% isopropanol aqueous solution, 4g 2,4-dimethylphenylhydrazine, control the temperature to 40°C and react for 1h. Filter, the filter cake is slurried with 100g 90% isopropanol aqueous solution for 1h, the temperature is set to 50°C, filter after the slurrying, and wash the filter cake with 50g 90% isopropanol aqueous solution. Add the filter cake to 300g isopropanol, adjust the pH to 2 with hydrochloric acid, stir for 0.5h, adjust the pH to 5 with ammonia water, and filter. The filter cake is vacuum dried at 50°C to obtain the oxytetracycline finished product 1, and its HPLC spectrum is as shown Figure 2 shown.

[0029] Example 2

[0030] Add 100g of oxytetracycline crude product into a 500ml flask. Its HPLC spectrum is as follows: Figure 1 As shown, 300g 95% ethanol aqueous solution, 4g 2-nitro-4-methylphenylhydrazine, temperature controlled to 40℃ for 1h. Filter, the filter cake is pulped with 100g 95% ethanol aqueous solution for 1h, the temperature is set to 50℃, filtered after pulping, and the filter cake is washed with 50g 95% ethanol aqueous solution. The filter cake is added to 300g ethanol, the pH is adjusted to 2 with hydrochloric acid, stirred for 0.5h, the pH is adjusted to 5 with ammonia water, and filtered. The filter cake is vacuum dried at 50℃ to obtain oxytetracycline finished product 2.

[0031] Example 3

[0032] Add 100g of oxytetracycline crude product into a 500ml flask. Its HPLC spectrum is as follows: Figure 1As shown, 300g 95% methanol aqueous solution, 4g 2,4-dinitrophenylhydrazine, temperature controlled to 30℃ for 1h. Filter, the filter cake is slurried with 100g 95% methanol aqueous solution for 1h, the temperature is set to 50℃, filtered after slurrying, and the filter cake is washed with 50g 95% methanol aqueous solution. The filter cake is added to 300g methanol, the pH is adjusted to 2 with hydrochloric acid, stirred for 0.5h, the pH is adjusted to 5 with ammonia water, and filtered. The filter cake is vacuum dried at 50℃ to obtain oxytetracycline finished product 3.

[0033] Example 4

[0034] Add 100g of oxytetracycline crude product into a 500ml flask. Its HPLC spectrum is as follows: Figure 1 As shown, 13g of oxytetracycline impurity C, 300g of 95% methanol aqueous solution, and 26g of 2,4-dinitrophenylhydrazine are added thereto, and the temperature is controlled to 50°C for reaction for 1h. Filter, and beat the filter cake with 100g of 95% methanol aqueous solution for 1h, and the temperature is set to 60°C. After beating, filter, and wash the filter cake with 50g of 95% methanol aqueous solution. Add the filter cake to 300g of methanol, adjust the pH to 2 with hydrochloric acid, stir for 0.5h, adjust the pH to 5 with ammonia water, and filter. The filter cake is vacuum dried at 50°C to obtain oxytetracycline finished product 4, and its HPLC spectrum is as shown Figure 3 shown.

[0035] Comparative Example 1:

[0036] Add 100g of oxytetracycline crude product into a 500ml flask. Its HPLC spectrum is as follows: Figure 1 As shown, 300g of 95% ethanol aqueous solution, temperature controlled to 40℃ and stirred for 1h. Filter, the filter cake is slurried with 100g of 95% ethanol aqueous solution for 1h, the temperature is set to 50℃, filtered after slurrying, and the filter cake is washed with 50g of 95% ethanol aqueous solution. The filter cake is added to 300g of ethanol, the pH is adjusted to 2 with hydrochloric acid, stirred for 0.5h, the pH is adjusted to 5 with ammonia water, and filtered. The filter cake is vacuum dried at 50℃ to obtain oxytetracycline sample 1.

[0037] The test results are shown in the following table:

[0038] Example Example 1 Example 2 Example 3 Example 4 Comparative Example 1 Finished product yield 91.3% 90.5% 91.7% 84.9% 92.6% Impurity C content 0.2% 0.3% 0.2% 1.5% 2.9% Impurity C removal efficiency 93.9% 90% 93.9% 89.7% 12.1%

[0039] Test results: The content of impurity C in the oxytetracycline finished products prepared in Examples 1-3 was significantly reduced, and the removal efficiency was as high as 93.9%. After purification, the content of impurity C in oxytetracycline was as low as 0.2%, which met the requirements of pharmacopoeias of various countries. The content of impurity C was much better than that of commercially available products (3.3%). Example 4 was a spiked test. Even if the amount of impurity C added reached 13%, after purification by the present technical solution, its residue could be reduced to about 1.5%, and the removal efficiency was still as high as 89.7%. The control example used a simple pulping method, and the removal efficiency of impurity C was extremely low, indicating that the present technical solution can effectively remove impurity C in oxytetracycline.

Claims

1. A preparation method for reducing the content of oxytetracycline impurity C, characterized in that: The following steps are involved: S1: Add crude oxytetracycline to a saturated alcohol aqueous solution, then add substituted phenylhydrazine, and stir to react for 0.5-1.2h. The reaction formula is as follows: 。 2. Among them, Formula I is impurity C, Formula II is substituted phenylhydrazine, and Formula III is the adduct of impurity C and substituted phenylhydrazine. R1 and R2 are simultaneously selected from methyl or nitro, or R1 is selected from nitro, R2 is selected from methyl, and R3 is selected from hydrogen; S2, filtering the reaction solution of step S1, beating the filter cake with fatty alcohol aqueous solution for 1 hour, filtering, and washing the filter cake with saturated alcohol aqueous solution once; S3, adding the solid from step S2 to a saturated alcohol solution, then adding a hydrochloric acid solution to adjust the pH of the system to 2-3, stirring the reaction, then adding an amine solution to adjust the pH to 5-6, and filtering to remove the solvent; S4. The solid from step S3 is placed in a vacuum drying oven and dried to constant weight to obtain a oxytetracycline product with a low impurity C content.

3. The method for reducing the content of oxytetracycline impurity C according to claim 1, characterized in that: The saturated alcohol is a monovalent saturated alcohol of C1-C4, and the volume fraction of the saturated alcohol aqueous solution is 80-95%.

4. The method for reducing the content of oxytetracycline impurity C according to claim 1, characterized in that: The reaction temperature in step S1 is 20-50°C.

5. The method for reducing the content of oxytetracycline impurity C according to claim 1, characterized in that: The weight ratio of the crude oxytetracycline, substituted phenylhydrazine and saturated alcohol aqueous solution in step S1 is 100:1-5:100-300.

6. The method for reducing the content of oxytetracycline impurity C according to claim 1, characterized in that: The beating temperature in step S2 is 30-60°C.

7. The method for reducing the content of oxytetracycline impurity C according to claim 1, characterized in that: In step S2, the weight ratio of the filter cake to the saturated alcohol aqueous solution is 1:1-2.

8. The method for reducing the content of oxytetracycline impurity C according to claim 1, characterized in that: The amine in step S3 is one or a combination of aqueous ammonia, aqueous methylamine solution, and aqueous dimethylamine solution.

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