A preparation method of rifamycin S sodium salt

Abstract

The present application discloses a preparation method of rifamycin S sodium salt. The fermentation broth containing rifamycin SV is successively oxidized, acidified, and subjected to air flotation, and then allowed to stand for layering. After separating the acid water, an extraction agent is added for extraction, and then solid-liquid separation is carried out. The obtained liquid is allowed to stand for layering, and the acid water layer is removed. An alkali solution is added to the extraction agent layer, and then the pH is adjusted with alkali and stirred for crystallization. After separation and drying, rifamycin S sodium salt is obtained. The method of the present invention greatly reduces the consumption of the extraction agent, produces less wastewater, has simple operation steps, low impurity content, and is suitable for automated production.

Description

Technical Field

[0001] The present invention belongs to the field of compound purification and preparation, and particularly relates to a preparation method of rifamycin S sodium salt. Background Art

[0002] Rifamycin antibiotics are a class of broad-spectrum antibiotics, and the main varieties include rifamycin sodium, rifampicin, rifandin, rifabutin, rifaximin, rifapentine, etc. They have high activity against Mycobacterium tuberculosis and can be used to treat tuberculosis and other diseases caused by drug-resistant mycobacteria. Rifamycin compounds exert their antibacterial effect by inhibiting the activity of bacterial RNA polymerase, thereby preventing the synthesis of bacterial RNA, further blocking protein synthesis, and ultimately leading to the death of bacteria. Rifamycin is usually used in combination with other anti-tuberculosis drugs (such as isoniazid, pyrazinamide, etc.) to reduce the generation of drug-resistant strains and improve the treatment effect. Due to its broad-spectrum antibacterial effect, rifamycin is sometimes also used to treat other infections, such as brucellosis and lymphoma virus infection.

[0003] Different strains of bacteria can produce different components of rifamycin after fermentation, including A, B, C, D, E, O, S, and SV, etc.

[0004] Rifamycin is a semi-synthetic antibiotic, and usually the parent rifamycin S sodium salt is obtained by fermentation and extraction. Starting from it, rifamycin sodium, rifampicin, rifapentine, rifabutin, etc. are synthesized.

[0005] Rifamycin is fermented by Nocardia mediterranei. The fermentation broth is filtered through a plate and frame to remove impurities such as bacterial residues and proteins. The filtrate can obtain rifamycin S sodium salt through two process flows: (1) adding an oxidant to the filtrate for oxidation, and then extracting with butyl acetate. The extract is demulsified, washed, concentrated, crystallized, centrifuged, and dried to obtain rifamycin S-Na; (2) first extracting rifamycin SV in the filtrate with butyl acetate, and then oxidizing to obtain rifamycin S. The oxidation solution is demulsified, washed, concentrated, and crystallized to obtain rifamycin S-Na. The difference between the two processes lies in the different order of extraction and oxidation, and the other steps are generally the same.

[0006] However, in the process of removing bacterial residues from the fermentation broth by plate and frame filtration, usually the fermentation broth is pressed into the receiving tank for refining, and a flocculant is added and stirred. Then it is pressed into the plate and frame for filtration. This process takes several hours. Bacteria suffocate and die, decompose and are damaged, and metabolize abnormally during this process. When the fermentation is severely contaminated, since only the filtrate after plate and frame filtration can be used for the next step of refining. At this time, it often has to be scrapped in whole or in part because it cannot be filtered.

[0007] Therefore, it is necessary to provide a preparation process of rifamycin S sodium salt with good process stability, simple operation, and low cost. Summary of the Invention

[0008] Objective of the Invention: The technical problem to be solved by the present invention is to provide a preparation method of rifamycin S sodium salt in view of the deficiencies of the prior art.

[0009] To solve the above technical problem, the present invention discloses a preparation method of rifamycin S sodium salt. The fermentation broth containing rifamycin SV is successively oxidized, acidified, air floated and then allowed to stand for stratification. After separating the acid water, an extraction agent is added for extraction, and then solid-liquid separation is carried out. The obtained liquid is allowed to stand for stratification, the acid water layer is removed, an alkali solution is added to the extraction agent layer, and then the pH is adjusted with alkali and stirred for crystallization, and separated and dried to obtain rifamycin S sodium salt.

[0010] Among them, the fermentation broth containing rifamycin SV can be from the fermentation workshop, containing mycelium, culture medium and rifamycin SV. In some embodiments, the content of rifamycin SV is generally 0.3% - 0.6%, dissolved in the fermentation broth. The solid components of the mycelium and the culture medium are about 2%, and other soluble substances are about 3%, and the rest is water.

[0011] Among them, the oxidant used for the oxidation is any one or a mixture of several of hydrogen peroxide, bleaching powder, sodium hypochlorite, sodium nitrite, trichloroisocyanuric acid, potassium ferricyanide, chlorine dioxide or peracetic acid; the amount of the oxidant used is calculated by molar number, and the ratio of rifamycin s to the oxidant is 1 - 5, and the oxidation time is 2 - 3 h.

[0012] The acidifying agent used for the acidification is any one of acetic acid, hydrochloric acid, sulfuric acid or oxalic acid, and the addition of the acidifying agent makes the pH of the system 1 - 4.

[0013] The air flotation is to introduce compressed air and stir. Among them, during the stirring process, the vortex inhales air and the gas generated by the chemical reaction can also play a role in air flotation. The amount of air introduced for air flotation is calculated by allowing the gas to be wrapped by the feed liquid. Usually, the ventilation volume is 10 - 12 cubic meters of air per cubic meter of feed liquid per hour. During oxidation and acidification, the introduction of air is maintained, and during this process, the volume of the feed liquid gradually increases.

[0014] The extraction agent is any one of butyl acetate, chloroform or dichloromethane, and the amount of the extraction agent used is 10 - 20 times the mass of rifamycin sv.

[0015] The solid-liquid separation is carried out by any one of a horizontal screw sedimentation centrifuge, an automatic discharge centrifuge or a disc centrifuge.

[0016] Among them, the alkali solution added to the extraction agent layer is an aqueous solution of sodium bicarbonate; the alkali used for adjusting the pH is sodium hydroxide or sodium carbonate.

[0017] Preferably, the pH is adjusted with alkali to 8 - 12 for stirring crystallization.

[0018] Preferably, after adding alkali to adjust the pH and stirring for crystallization, the temperature is lowered to below 10°C and separated by a centrifuge, and then vacuum dried below 100°C.

[0019] Advantages: Compared with the prior art, the present application has the following advantages:

[0020] (1) By directly adding an oxidant to the fermentation broth, rapid sterilization can be achieved, avoiding the decomposition of unnecessary related impurities in catabolism (i.e., homologues of the product, such as deacetyl rifamycin S and demethyl rifamycin S);

[0021] (2) When severe contamination occurs during fermentation, in the conventional process, since the filtrate after plate and frame filtration is required for the next step of refining, it often has to be scrapped in whole or in part because the filtration cannot be carried out. In the present application, by using the air flotation method, since there is no filtration step, the refining can still be carried out normally without being affected;

[0022] (3) Compared with the traditional production method, the present application removes the fermentation broth filtration link, directly oxidizes the fermentation broth, and uses the air flotation method to separate the mycelium and rifamycin S. After separating the wastewater, the solid is extracted with an extractant such as butyl acetate or dichloromethane, and only a small amount of wastewater containing organic solvents is produced. The traditional method uses plate and frame filtration with high labor intensity and low efficiency, and 1.5 - 2 cubic meters of filtrate is produced per cubic meter of fermentation broth. If all is extracted with butyl acetate, a large amount of wastewater containing butyl acetate will be produced. The present application greatly reduces the production wastewater of rifamycin, and only 20% of the wastewater in the traditional method contains organic solvents. At the same time, the process of the present invention is suitable for automated production, and the number of personnel used is only 10 - 30% of the traditional method;

[0023] (4) The consumption of the extractant in the present application is also greatly reduced: since the extractant does not contact a large amount of water and does not dissolve in water, the consumption of the extractant is also significantly reduced, to less than 50% of the traditional method. Description of the Drawings

[0024] Figure 1 The left figure is a schematic diagram of the fermentation broth in the state of air flotation stirring, and the right figure is a state diagram of the fermentation broth after air flotation and standing to separate the acid water;

[0025] Figure 2 It is the liquid phase characterization diagram of rifamycin S prepared in Example 1. In order to clearly show the content of other impurities, the peak of the main peak (i.e., rifamycin S) is not fully shown. Detailed Embodiments

[0026] The following further specifically describes the present invention in conjunction with the drawings and specific embodiments, and the above and / or other advantages of the present invention will become clearer.

[0027] In the following examples, the fermentation broth is from the fermentation workshop, contains mycelium, culture medium and rifamycin SV, and the content of rifamycin SV is 0.3% - 0.6%, dissolved in the fermentation broth. The solid components of the mycelium and the culture medium are about 2%, other soluble substances are about 3%, and the rest is water. Those skilled in the art should know that the fermentation broth containing rifamycin SV from other fermentation workshops is also applicable to the method of this application, and no specific limitations are imposed on the specific properties of the fermentation broth.

[0028] Example 1 Preparation of sodium rifamycin S.

[0029] To 1000 L of the fermentation broth containing rifamycin SV, add 0.5 kg of bleaching powder and 0.4 kg of trichloroisocyanuric acid for oxidation per kg of rifamycin sv, and introduce air for stirring and air flotation. The ventilation rate is 10 cubic meters of air per cubic meter of liquid per hour, as shown in Figure 1 the left figure. Add 30% hydrochloric acid to acidify to pH 2.5, and continuously introduce air during the oxidation and acidification processes, then let it stand for stratification to separate the acid water. The state diagram of the fermentation broth after separating the acid water is as shown in Figure 1 the right figure. It can be seen from the figure that after the fermentation broth is treated by air flotation stirring and stratified, the water content drops significantly. Add 20 kg of butyl acetate per kg of rifamycin sv and stir. Use a horizontal spiral sedimentation centrifuge to separate the solid and liquid, and let the obtained liquid stand for stratification. The lower layer is the acid water layer, and the upper layer is the butyl acetate layer. Discard the acid water layer, prepare an aqueous phase with 3% sodium bicarbonate solution with the same volume as butyl acetate, adjust the pH to 9.5 with 5% sodium hydroxide by mass concentration and stir for crystallization, cool down to below 10 degrees and separate with a centrifuge, and vacuum dry below 100 degrees to obtain sodium rifamycin S.

[0030] Characterize and detect the content of the obtained rifamycin S, and the results are shown in Table 1. Among them, the content detection is determined by the external standard method, and is determined with reference to the high performance liquid chromatography method (General Principles 0512). Among them, a C19 stainless steel column with a particle size of 5 μm is used, the flow rate is 2.0 mL / min, the injection volume is 20 μL, the detection wavelength is 254 nm, and the column temperature is 30 °C. The mobile phase is a mixture of 0.025 mol / L disodium hydrogen phosphate (dihydrate) - acetonitrile (51:49), and the pH is adjusted to about 6.9 with phosphoric acid.

[0031] Among them, the liquid phase result diagram is as shown in Figure 2 shown.

[0032] Table 1

[0033] Inspection items Inspection results Rifamycin SV 1.82％ 25-Desacetyl-27-demethyl rifamycin S 0.34％ 16-Hydroxymethyl rifamycin S 0.51％ 27-Demethyl rifamycin S 0.67％ 25desacetyl rifamycin S (25-Desacetyl rifamycin S) 0.77％ Sum of peaks (Total impurities excluding 25-Desacetyl rifamycin S) 3.9％ Loss on drying (moisture) 4.9％ Content 84.7％ Solvent impurities 5.33％

[0034] Among them, the total impurities include rifamycin SV, 25 - deacetyl - 27 - demethyl rifamycin S, 16 - hydroxymethyl rifamycin S, 27 - demethyl rifamycin S and other unspecified impurities.

[0035] Example 2

[0036] The basic steps are the same as those in Example 1. The difference is that 0.1 kg of hydrogen peroxide is used for oxidation per kg of rifamycin sv.

[0037] Example 3

[0038] The basic steps are the same as those in Example 1. The difference is that 0.6 kg of trichloroisocyanuric acid is used for oxidation per kg of rifamycin sv.

[0039] Example 4

[0040] The basic steps are the same as those in Example 1. The difference is that solid oxalic acid is used for acidification to pH 3.0. After separating the acid water by air flotation stratification, the solid part is dehydrated to a water content of less than 50% by an automatic discharge centrifuge, stirred with butyl acetate, and the butyl acetate layer is separated by standing. A 3% sodium bicarbonate solution with the same volume as the butyl acetate is used as the aqueous phase, and the pH is adjusted to 9.5 with 5% sodium hydroxide for stirring crystallization. After cooling to below 10 degrees, it is separated by a centrifuge and vacuum dried below 100 degrees to obtain sodium rifamycin S.

[0041] Example 5

[0042] The basic steps are the same as those in Example 1. The difference is that after separating the acid water by air flotation stratification, the solid part is dehydrated to a water content of less than 50% by an automatic discharge centrifuge. 20 kg of chloroform is added and stirred per 1 kg of rifamycin sv, and then allowed to stand for stratification. The chloroform is in the lower layer. After separating the chloroform, a 3% sodium bicarbonate solution with the same volume as the chloroform is used as the aqueous phase, and the pH is adjusted to 9.5 with 5% sodium hydroxide for stirring crystallization. After cooling to below 10 degrees, it is separated by a centrifuge and vacuum dried below 100 degrees to obtain sodium rifamycin S.

[0043] Example 6

[0044] The basic steps are the same as those in Example 2. The difference is that after separating the acid water by air flotation stratification, chloroform is added and stirred. 20 kg of chloroform is added and stirred per 1 kg of rifamycin sv, and then allowed to stand for stratification. The chloroform is in the lower layer. After separating the chloroform, a 3% sodium bicarbonate solution with the same volume as the chloroform is used as the aqueous phase, and the pH is adjusted to 9.5 with 5% sodium hydroxide for stirring crystallization. After cooling to below 10 degrees, it is separated by a centrifuge and vacuum dried below 100 degrees to obtain sodium rifamycin S.

[0045] Example 7

[0046] The basic steps are the same as those in Example 1. The difference is that after separating the acidic water by air flotation stratification, dichloromethane is added and stirred. Then it is allowed to stand for stratification. Dichloromethane is in the lower layer. After separating dichloromethane, a sodium bicarbonate solution with a concentration of 3% and the same volume as dichloromethane is prepared as the aqueous phase, and the pH is adjusted to 9.5 with 5% sodium hydroxide for stirring crystallization. After cooling to below 10 degrees, it is separated by a centrifuge and vacuum dried below 100 degrees to obtain rifamycin S sodium salt.

[0047] The test data of each example are shown in Table 1.

[0048] Table 1

[0049]

[0050]

[0051] Comparative example

[0052] 1.5 kg of zinc sulfate is added to 1000 L of fermentation broth and stirred for 10 minutes, then it enters the plate and frame filtration. After all the fermentation broth enters the plate and frame, it is rinsed with tap water until the volume reaches 1500 L. The concentration of the filtrate is checked. For every kg of rifamycin sv, 1 kg of bleaching powder and 20 kg of butyl acetate are added, and air is introduced for stirring oxidation. After checking the completion of oxidation, the pH is adjusted to 1 - 4 with 30% hydrochloric acid, and water is separated after standing for several hours. When emulsification occurs, dodecyltrimethylammonium chloride is added and stirred to break the emulsification and separate water. The butyl acetate solution is washed with tap water, 1% sodium bicarbonate solution, and 1% hydrochloric acid solution respectively. A solution with 50% of the volume of butyl acetate and 3% sodium bicarbonate is prepared as the crystallization water, heated to above 40 degrees, the pH is adjusted to 9.5 with 5% sodium hydroxide and stirred for 2 hours, then cooled to below 10 degrees. After standing for several hours, it is filtered by a centrifuge to separate the solvent. The solid is vacuum dried in a vacuum drying oven below 100 degrees.

[0053] The obtained rifamycin S was characterized and detected, and the results are shown in Table 2.

[0054] Table 2

[0055]

[0056]

[0057] In summary, the present application first proposes a preparation idea for rifamycin S sodium salt that can achieve rapid sterilization by directly adding an oxidant to the fermentation broth and avoid catabolic impurities that are not required. There are many methods and ways to specifically implement this technical solution. The above description is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention. Each component not clearly defined in this embodiment can be implemented by existing technologies.

Claims

1. A method for preparing rifamycin S sodium salt, characterized in that, The fermentation broth containing rifamycin SV is successively oxidized and acidified, while continuous air flotation is carried out during the oxidation and acidification processes, then left to stand for stratification. After separating the acid water, an extractant is added for extraction, and then solid-liquid separation is performed. The obtained liquid is left to stand for stratification, the acid water layer is removed, an alkali solution is added to the extractant layer, and then the pH is adjusted with alkali and stirred for crystallization. After separation and drying, rifamycin S sodium salt is obtained.

2. The preparation method according to claim 1, characterized in that, The oxidant used for the oxidation is any one or a mixture of several of hydrogen peroxide, bleaching powder, sodium hypochlorite, sodium nitrite, trichloroisocyanuric acid, potassium ferricyanide, chlorine dioxide or peracetic acid; the amount of the oxidant used is calculated by molar number, and the ratio of rifamycin s to the oxidant is 1 - 5, and the oxidation time is 2 - 3 h.

3. The preparation method according to claim 1, characterized in that, The acidifying agent used for the acidification is any one of acetic acid, hydrochloric acid, sulfuric acid or oxalic acid, and the addition of the acidifying agent makes the pH of the system 1 - 4.

4. The preparation method according to claim 1, characterized in that, The air flotation is to introduce compressed air and stir.

5. The preparation method according to claim 1, wherein The extractant is any one of butyl acetate, chloroform or dichloromethane, and the amount of the extractant used is 10 - 20 times the mass of rifamycin sv.

6. The preparation method according to claim 1, characterized in that, The solid-liquid separation is carried out using any one of a horizontal spiral sedimentation centrifuge, an automatic discharge centrifuge or a disc centrifuge.

7. The preparation method according to claim 1, wherein The alkali solution added to the extractant layer is an aqueous solution of sodium bicarbonate; the alkali used for adjusting the pH is sodium hydroxide or sodium carbonate.

8. The preparation method according to claim 1, characterized in that, The pH is adjusted with alkali to 8 - 12.

9. The preparation method according to claim 1, wherein After adjusting the pH with alkali and stirring for crystallization, the temperature is lowered to below 10 °C and separated by a centrifuge.

10. The preparation method according to claim 1, characterized in that, The drying condition is vacuum drying at below 100 °C.

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