Preparation method of caspofungin acetate impurity K

Abstract

The invention relates to the technical field of chemical pharmacy, in particular to a preparation method of caspofungin acetate impurity K. According to the preparation method of the caspofungin acetate impurity K provided by the invention, an intermediate (compound I) of caspofungin acetate is taken as a raw material, groups at specific positions are subjected to substitution reaction under homogeneous conditions to directly obtain a caspofungin acetate impurity K crude product, then the impurity K crude product is taken as a raw material, and the purity of the caspofungin acetate impurity K is obtained through a preparative chromatography method. According to the method, the caspofungin acetate is taken as a raw material, qualified caspofungin acetate chromatographic liquid is obtained, solid powder of caspofungin acetate impurity K with the chromatographic purity larger than 96% is obtained through a freeze-drying method, and the requirements of enterprises and the market can be met; the preparation method is simple in step, stable in condition, high in yield and suitable for large-scale production.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of chemical pharmacy, and particularly relates to a preparation method of acetic acid caspofungin impurity K. BACKGROUND

[0002] As a new type of semi-fermentation semi-synthetic echinocandin antifungal agent, acetic acid caspofungin has significant clinical application value, can be used for treating invasive aspergillosis which is ineffective or intolerable to other treatments, can be used for carrying out empirical treatment on patients with granulocytopenia accompanied by fever suspected of fungal infection, and is suitable for oropharyngeal and esophageal candidiasis and invasive candidiasis including candidaemia of neutropenic patients. Among echinocandin antifungal drugs, acetic acid caspofungin becomes an important choice for clinical treatment of related fungal infections due to its strong efficacy, unique mechanism of action, high safety and wide indications.

[0003] The negative influence of drug impurities on drug quality, safety and effectiveness cannot be ignored. On the one hand, some impurities may have potential toxicity or harmfulness, thereby causing adverse drug reactions or side effects and threatening the safety of patients; on the other hand, impurities can interfere with the physical and chemical properties of drugs, reduce the stability and quality of drugs, and affect the storage and use effect of drugs; in addition, impurities can also interfere with the drug analysis and detection process, leading to inaccurate detection results, destroying the drug compliance and quality control system, and finally having an adverse effect on the efficacy of drugs. Therefore, in-depth research on acetic acid caspofungin impurities is a key link to ensure the quality of drugs and the safety of clinical medication, and is of great significance for drug research and development, production and supervision.

[0004] In the chemical synthesis process of acetic acid caspofungin, due to the use of ethylenediamine raw material containing diethylenetriamine impurities, acetic acid caspofungin impurity K (structure as shown in formula II) will be generated. In the field of drug detection, acetic acid caspofungin impurity K is an important reference substance, but there is no specific report on acetic acid caspofungin impurity K or caspofungin impurity K in any reference at present, and there is also lack of related scheme for preparing high-purity acetic acid caspofungin impurity K in the prior art. This situation makes it impossible to meet the needs of enterprise's own drug quality control and market demand for this impurity reference substance, therefore, developing an efficient, stable and high-purity acetic acid caspofungin impurity K preparation method has become a problem to be solved in the current chemical pharmacy field. SUMMARY

[0005] The purpose of the present application is to provide a preparation method of acetic acid caspofungin impurity K, aiming at solving the problem that there is no efficient, stable and high-purity acetic acid caspofungin impurity K preparation method in the prior art.

[0006] To achieve the above application purposes, the technical scheme adopted by the present application is as follows:

[0007] In a first aspect, the present application provides a method for preparing Caspofungin Acetate Impurity K, comprising the following steps:

[0008] The raw material shown in the chemical formula I is subjected to a substitution reaction with diethylenetriamine to obtain a crude Caspofungin Acetate Impurity K shown in the chemical formula II

[0009] The crude Caspofungin Acetate Impurity K is subjected to a dissolution and filtration to obtain a crude product solution;

[0010] The crude product solution is subjected to a separation and purification, and the eluate is collected;

[0011] The eluate is subjected to a freeze-drying to obtain the Caspofungin Acetate Impurity K shown in the chemical formula III

[0012] In some embodiments, the solvent for the substitution reaction comprises a diethylenetriamine solution.

[0013] In some embodiments, the reaction temperature for the substitution reaction is 25-35°C, and the reaction time is 4-5 hours.

[0014] In some embodiments, in the step of dissolving and filtering the crude Caspofungin Acetate Impurity K, the dissolving solvent comprises a 16wt% acetic acid aqueous solution.

[0015] In some embodiments, the filtration is performed using a 0.45μm filter membrane.

[0016] In some embodiments, in the step of separation and purification, a high-pressure preparative chromatography system is used for the separation and purification, and in the chromatography system, the packing material of the chromatography column is SP-100-10-ODS-P, and the diameter of the chromatography column is 30nm or 50nm.

[0017] In some embodiments, the step of separation and purification using a high-pressure preparative chromatography system comprises:

[0018] The chromatography column is equilibrated with a first eluent, and the amount of the first eluent is 2 times the column volume;

[0019] Gradient elution is performed using a second eluent, and the amount of the second eluent is 6 times the column volume;

[0020] Elution is performed using a third eluent, and the amount of the third eluent is 12 times the column volume, and the eluate is collected.

[0021] In some embodiments, the first eluent comprises a mixture of a 1‰ acetic acid aqueous solution and a 3% acetonitrile solution.

[0022] ​​In some embodiments, the second eluent comprises a mixture of acetic acid aqueous solution with a mass percentage of 1 ‰ and acetonitrile with a mass percentage of 5-8 %.

[0023] In some embodiments, the third eluent comprises a mixture of acetic acid aqueous solution with a mass percentage of 1 ‰ and acetonitrile with a mass percentage of 10 %.

[0024] In some embodiments, the flow rate of the eluent is 3-6 times of column volume per hour.

[0025] In some embodiments, the freeze-drying comprises: pre-freezing the eluent, and then performing sublimation drying under a vacuum degree of ≤-0.090 MPa.

[0026] In some embodiments, the final chromatographic purity of the caspofungin impurity K is ≥96 %, and the recovery rate is ≥48 %.

[0027] The preparation method of the caspofungin impurity K provided in the first aspect of the present application uses the intermediate compound I of caspofungin acetate as a raw material, substitutes the group at a specific position with diethylenetriamine in a diethylenetriamine solution to directly obtain a caspofungin impurity K crude product. The method is convenient and fast in operation, high in process safety, and provides a preparation method of caspofungin impurity K. Further, the high-pressure preparation column is good in separation and purification effect of the caspofungin impurity K, and is simple and fast in operation. After separation and purification, a high-chromatographic-purity caspofungin impurity K solid powder is finally obtained, and a high-purity caspofungin impurity K preparation method is provided, which is convenient for large-scale production, can sufficiently meet the requirements of enterprises themselves and the market, and the preparation method is stable in conditions, simple in steps, strong in specificity, and high in yield, is suitable for large-scale production, and can meet the market demand. BRIEF DESCRIPTION OF DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creating any inventive labor.

[0029] Figure 1 is a high-pressure preparation separation schematic diagram of caspofungin acetate impurity K;

[0030] Figure 2 is a freeze-dried product detection diagram of caspofungin acetate impurity K of Example 1;

[0031] Figure 3 is a crude product solution detection diagram of caspofungin acetate impurity K of Example 2;

[0032] Figure 4is the test graph of the caspofungin acetate impurity K freeze-dried product of Example 2;

[0033] Figure 5 is the test graph of the caspofungin acetate impurity K freeze-dried product of Example 3;

[0034] Figure 6 is the test graph of the caspofungin acetate impurity K freeze-dried product of Example 4. DETAILED DESCRIPTION

[0035] In order to make the technical problems to be solved by the present application, technical solutions and beneficial effects clearer, the present application will be further described in detail below in combination with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and not intended to limit the present application.

[0036] In the present application, the term "and / or" describes the association relationship of the associated objects, which means that there can be three kinds of relationships, for example, A and / or B can mean that A exists alone, A and B exist together, and B exists alone. Wherein A and B can be singular or plural. The character " / " generally represents an "or" relationship between the associated objects before and after it.

[0037] In the present application, "at least one" means one or more, and "multiple" means two or more. "At least one of the following" or similar expressions means any combination of these items, including any combination of single item or multiple items. For example, "at least one of a, b, or c", or "at least one of a, b, and c", can mean a, b, c, a-b (i.e. a and b), a-c, b-c, or a-b-c, wherein a, b, and c can be single or multiple.

[0038] It should be understood that in various embodiments of the present application, the size of the sequence number of the above-mentioned processes does not mean the order of execution, and part or all of the steps can be executed in parallel or in sequence. The execution order of each process should be determined according to its function and inherent logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0039] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a" and "the" used in the embodiments of the present application and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0040] The weight of the related components mentioned in the embodiment specification of the present application can not only refer to the specific content of each component, but also represent the proportional relationship between the weights of each component. Therefore, as long as the content of the related components in the embodiment specification of the present application is scaled up or down in proportion, it is within the scope disclosed in the embodiment specification of the present application. Specifically, the mass in the embodiment specification of the present application can be μg, mg, g, kg, etc. mass units commonly known in the chemical industry.

[0041] The terms "first", "second" are only for descriptive purposes, to distinguish objects such as substances from each other, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. For example, without departing from the scope of the present application, the first XX can also be referred to as the second XX, and similarly, the second XX can also be referred to as the first XX. Therefore, the features defined with "first", "second" can explicitly or implicitly include one or more of the features.

[0042] The first aspect of the present application provides a preparation method of caspofungin acetate impurity K, comprising the following steps:

[0043] S01. The raw material shown by formula I is subjected to a substitution reaction with diethylenetriamine to obtain a crude caspofungin acetate impurity K shown by formula II .

[0044] S02. The crude caspofungin acetate impurity K is dissolved and filtered to obtain a crude solution;

[0045] S03. The crude solution is separated and purified, and the eluent is collected;

[0046] S04. The eluent is freeze-dried to obtain caspofungin acetate impurity K shown by formula III .

[0047] The preparation method of caspofungin acetate impurity K provided in the first aspect of the present application uses intermediate compound I of caspofungin acetate as raw material, and substitutes the group at a specific position with diethylenetriamine in a diethylenetriamine solution to directly obtain a crude caspofungin acetate impurity K. This method is convenient and fast to operate, has high process safety, provides a preparation method of caspofungin acetate impurity K; further, the high-pressure preparation column has good separation and purification effect on caspofungin acetate impurity K, and the operation is simple and fast. After separation and purification, a solid powder of caspofungin acetate impurity K with high chromatographic purity is obtained, providing a preparation method of high-purity caspofungin acetate impurity K, which is convenient for large-scale production, can fully meet the requirements of enterprises themselves and the market, and the preparation method has stable conditions, simple steps, strong specificity, high yield, is suitable for large-scale production, and can meet the market demand.

[0048] In step S01, a raw material of Formula I is subjected to a substitution reaction with diethylenetriamine to obtain a crude caspofungin impurity K of Formula II

[0049] In some embodiments, the solvent for the substitution reaction includes a diethylenetriamine solution. Since diethylenetriamine is both a reactant participating in the substitution reaction and a solvent providing a uniform and stable reaction environment for the reaction, this integrated selection of “reactant-solvent” not only avoids the problems of increased impurities and increased difficulty in subsequent separation caused by the introduction of additional unrelated solvents, but also promotes the full progress of the substitution reaction by increasing the concentration of the reactants, reduces the occurrence of side reactions, thereby improving the yield and purity of the crude caspofungin impurity K, reducing the pressure of the subsequent purification step, and simplifying the composition of the reaction system, facilitating the monitoring and control of the reaction process.

[0050] In some embodiments, the reaction temperature for the substitution reaction is 25-35°C, and the reaction time is 4-5 hours. Limiting the temperature range of the substitution reaction to 25-35°C and the reaction time to 4-5 hours, this temperature interval belongs to mild reaction conditions, which can not only avoid the decomposition and deterioration of raw materials or intermediates under high temperature environment, but also ensure that the reaction has enough activation energy to proceed smoothly; while ensuring the complete progress of the substitution reaction to improve the yield of the crude product, the reaction time of 4-5 hours avoids the problems of excessive reaction of the product, increase of impurities caused by too long reaction time, or incomplete reaction, reduced yield caused by too short reaction time. By precisely controlling the reaction temperature and time, the balance between reaction efficiency and product quality is achieved, effectively improving the quality stability and yield consistency of the crude product, and providing high-quality crude raw materials for the subsequent purification step.

[0051] In step S02, the crude caspofungin impurity K is dissolved and filtered to obtain a crude solution.

[0052] In some embodiments, in the step of dissolving and filtering the crude caspofungin impurity K, the dissolving solvent includes a 16wt% acetic acid aqueous solution. Using a 16wt% acetic acid aqueous solution as the solvent can not only ensure that the crude caspofungin impurity K is fully dissolved, thereby minimizing the loss of raw materials caused by insufficient dissolution, but also avoid the problems of introducing too many acidic impurities caused by too high acetic acid concentration or low dissolution efficiency caused by too low acetic acid concentration.

[0053] ​In some embodiments, the filtering is performed using a filter membrane with a pore size of 0.45 μm. The use of a filter membrane with a pore size of 0.45 μm can accurately trap insoluble impurities in the crude solution, effectively remove impurities that affect subsequent separation and purification, avoid plugging the chromatographic column or interfering with the separation of target impurities, provide a pure and clear crude solution for subsequent high-pressure preparative chromatography, and ensure the smooth progress of the separation and purification steps and the purity of the final product.

[0054] In step S03, the crude solution is separated and purified, and the eluate is collected.

[0055] In some embodiments, in the step of separation and purification, a high-pressure preparative chromatography system is used for separation and purification. In the chromatography system, the packing material of the chromatographic column is SP-100-10-ODS-P, and the diameter of the chromatographic column is 30 nm or 50 nm. The high-pressure preparative chromatography system has the advantages of high efficiency and large processing capacity, which can meet the requirements of purification efficiency and yield in the preparation process of impurity K; and the SP-100-10-ODS-P packing material has good hydrophilicity and selectivity, which can realize accurate separation according to the polarity difference between caspofungin impurity K and other impurities.

[0056] The provided chromatographic column specifications are designed by reasonable column length and column diameter to improve the sample loading capacity while ensuring separation efficiency, avoid incomplete separation caused by insufficient column efficiency, or low preparation efficiency caused by low loading capacity, effectively improve the efficiency of separation and purification, and the purity of target impurity K, which provides a key guarantee for obtaining high-quality finished products.

[0057] In some embodiments, the step of separation and purification using a high-pressure preparative chromatography system comprises:

[0058] G01. Equilibrating the chromatographic column with a first eluent, and the amount of the first eluent is 2 column volumes;

[0059] G02. Gradient elution using a second eluent, and the amount of the second eluent is 6 column volumes;

[0060] G03. Elution using a third eluent, and the amount of the third eluent is 12 column volumes, and the eluate is collected.

[0061] In step G01, the chromatographic column is equilibrated with a first eluent, and the amount of the first eluent is 2 column volumes. The use of 2 column volumes of eluent to equilibrate the chromatographic column can ensure that the chromatographic column packing and the eluent are in full contact, ensure that the column environment is stable and uniform, lay a good foundation for subsequent elution and separation, and avoid unstable separation results caused by fluctuations in the column environment.

[0062] In some embodiments, the first eluent comprises a mixture of 1 ‰ acetic acid aqueous solution and 3% acetonitrile. The 1 ‰ acetic acid aqueous solution can maintain a stable weakly acidic environment of the elution system, avoid structural changes or deterioration of the target impurity K due to pH fluctuations during elution, and help improve the stability of the retention behavior of the target impurity on the chromatographic column.

[0063] In step G02, the second eluent is used for gradient elution, and the amount of the second eluent is 6 column volumes. The use of 6 column volumes of the second eluent for gradient elution can gradually elute the impurities with large polarity differences from the crude product solution, and achieve preliminary impurity removal.

[0064] In some embodiments, the second eluent comprises a mixture of 1 ‰ acetic acid aqueous solution and 5%-8% acetonitrile. The second eluent comprises a mixture of 1 ‰ acetic acid aqueous solution and 5%-8% acetonitrile, which is mainly used to achieve accurate separation according to the polarity difference between the target impurity K and other impurities by adjusting the polarity of the eluent. The second eluent with a moderate proportion of acetonitrile can gradually elute the impurities with moderate polarity by gradient change,

[0065] In step G03, the third eluent is used for elution, and the amount of the third eluent is 12 column volumes, and the eluate is collected. The use of 12 column volumes of the third eluent for elution and collection can ensure that the target impurity K is fully and completely eluted from the chromatographic column, avoiding the problem of reduced recovery rate due to insufficient elution.

[0066] In some embodiments, the third eluent comprises a mixture of 1 ‰ acetic acid aqueous solution and 10% acetonitrile. Further, the use of a high proportion of acetonitrile in the third eluent can effectively elute the target impurity K, efficiently separate various impurities, and ensure the purity and stability of the target impurity, avoiding the problems of poor separation effect or deterioration of the target impurity due to improper composition of the eluent.

[0067] The whole elution process is progressive, which not only ensures the effective removal of impurities, but also ensures the full recovery of the target impurity, significantly improving the recovery rate and purity of the product.

[0068] In some embodiments, the flow rate of the eluent is 3-6 times the column volume per hour. The flow rate of the eluent is limited to 3-6 times the column volume per hour, and this flow rate range is precisely optimized. If the flow rate is too low, the elution time will be too long, the preparation efficiency will be low, and the target impurity may diffuse due to the excessive residence time of the target impurity in the chromatographic column, affecting the separation effect. If the flow rate is too high, the target impurity and other impurities cannot be fully separated in the chromatographic column, resulting in peak overlap and reducing the product purity. A flow rate of 3-6 times the column volume per hour can ensure that the target impurity K and other impurities have sufficient time to fully separate in the chromatographic column, ensuring the separation effect and product purity, and avoiding the problem of low efficiency due to a slow flow rate, achieving the best balance between separation effect and preparation efficiency, and effectively improving the overall quality and efficiency of the separation and purification step.

[0069] In some embodiments, the freeze-drying includes: pre-freezing the eluent, and then performing sublimation drying under a vacuum degree of ≤-0.090 MPa.

[0070] The freeze-drying specifically includes "pre-freezing and then performing sublimation drying under a vacuum degree of ≤-0.090 MPa", and the pre-freezing step can cause the water in the eluent to rapidly freeze into ice, avoiding the agglomeration or dissolution loss of the target impurity K due to the liquid flow in the subsequent drying process. The sublimation drying condition of a vacuum degree of ≤-0.090 MPa can cause the ice to directly sublimate into water vapor in a low-temperature environment, effectively avoiding the problems of thermal decomposition, structure damage, or purity reduction of the caspofungin impurity K that may be caused by traditional high-temperature drying. At the same time, the high-vacuum environment can accelerate the sublimation of water, ensuring efficient and sufficient drying, and ultimately obtaining a caspofungin impurity K product that is uniformly dried, high in purity, and complete in structure, thereby ensuring the quality stability and structural integrity of the product.

[0071] In some embodiments, the final chromatographic purity of the caspofungin impurity K is ≥96%, and the recovery rate is ≥48%.

[0072] A chromatographic purity of ≥96% means that the product purity is high, which can meet the high-purity requirements of impurity reference substances in drug quality control, and avoid the problem of inaccurate test results caused by insufficient impurity purity. A recovery rate of ≥48% means that the material utilization rate of the preparation method is high, which takes into account the economy while ensuring high purity, avoids the problems of raw material waste and high preparation cost caused by a low recovery rate, and provides reliable protection for the large-scale preparation and practical application of the caspofungin impurity K, and has important practical application value.

[0073] The following will be described in conjunction with specific embodiments.

[0074] Embodiment 1

[0075] First, the reaction solvent, reaction temperature, preparation column filler, eluent, and elution gradient are determined.

[0076] 1. Selection of reaction solvent

[0077] Take 5 g of compound I, 1 ml of diethylene triamine into the reaction bottle, then add the reaction solvent, stir the reaction at room temperature, and then quench the reaction with 60 ml of 16.67% acetic acid solution to obtain the crude solution of caspofungin impurity K. The most critical factor in the reaction process is the type of reaction solvent. Several solvents are screened, including: methanol, ethanol, acetonitrile, diethylene triamine. The conversion rate of compound I to product impurity K within the same reaction time is used as the standard for investigation. After 4 h of reaction, the conversion rate of impurity K in different reaction solvents is shown in Table 1:

[0078] Table 1 Conversion rate of caspofungin impurity K obtained after 4 h of reaction in different reaction solvents

[0079] Reaction solvent Conversion rate (%) Methanol 15 Ethanol 17 Acetonitrile 5 Diethylenetriamine 61

[0080] From the experimental results in Table 1, it can be seen that the conversion rate of caspofungin impurity K is the highest when diethylene triamine is used as the reaction solvent, which can reach 61%. Therefore, diethylene triamine is selected as the reaction solvent.

[0081] 2. Selection of reaction temperature

[0082] Take 5 g of compound I, 1 ml of diethylene triamine into the reaction bottle, then add the reaction solvent, stir the reaction at different temperatures, and then quench the reaction with 60 ml of 16.67% acetic acid solution to obtain the crude solution of caspofungin impurity K. The reaction temperature includes: 5℃, 15℃, 25℃, 35℃, 45℃. The chromatographic purity of the reaction solution of product impurity K and the remaining compound I within the same reaction time are used as the standard for investigation. After 4 h of reaction, the chromatographic purity of impurity K and the remaining compound I under different reaction temperatures are shown in Table 2:

[0083] Table 2 Conversion rate of caspofungin impurity K obtained after 4 h of reaction in different reaction solvents

[0084] Reaction temperature (°C) Chromatographic purity (%) Compound I remaining (%) 5 27 58 15 50 24 25 61 8 35 63 5 45 45 ＜1

[0085] From the experimental results in Table 2, it can be seen that within the same reaction time, the reaction rate of compound I increases with increasing reaction temperature, but the chromatographic purity of impurity K shows a trend of first increasing and then decreasing. That is, too high a reaction temperature cannot continue to improve the conversion rate of impurity K, and the product will be degraded with increasing temperature. Therefore, 25-35℃ is selected as the reaction temperature.

[0086] 3. Selection of preparation column packing

[0087] According to the characteristics of the antibiotic polypeptide, the column separation is carried out by using reversed-phase high-pressure preparation chromatography, and several C18 fillers are screened, such as SP-100-50-ODS-P, SP-100-30 / 50-ODS-P, SP-100-10-ODS-P and SP-100-30 / 50-ODS-BP,

[0088] The highest chromatographic purity of the impurity K obtained by the separation is used as the standard for the preparation, and the results are shown in Table 3:

[0089] Table 3 Chromatographic purity of caspofungin impurity K obtained after column separation with different fillers

[0090] Type of packing Chromatographic purity (%) SP-100-50-ODS-P 72 SP-100-30 / 50-ODS-P 89 SP-100-10-ODS-P ＞96 SP-100-30 / 50-ODS-BP 85

[0091] As can be seen from the experimental results in Table 3, the separation effect of SP-100-10-ODS-P is the best, and more than 96% of the chromatographic liquid can be obtained, so SP-100-10-ODS-P is selected as the high-pressure preparation filler.

[0092] 4. Selection of eluent

[0093] (I) Selection of mobile phase A

[0094] Since the stability of the antifungal antibiotic product is generally poor, it is necessary to investigate the stability of the product in the preparation system. The high-pressure preparation column with SP-100-10-ODS-P as the high-pressure preparation filler after the caspofungin impurity K crude product solution (chromatographic purity 63%) is used to flush the column with different volumes of mobile phase A, so that the impurity K is placed in the mobile phase A atmosphere at room temperature for 24 h, and then the product on the filler is regenerated with 95% ethanol, and the sample is detected by HPLC. The detection results are shown in Table 4:

[0095] Table 4 Chromatographic purity of caspofungin impurity K obtained after 24 h storage under different mobile phase A systems

[0096]

[0097]

[0098] As can be seen from the experimental results in Table 4, the chromatographic purity of caspofungin impurity K has the least difference compared with the control and almost no change after 24 h storage in 1‰ acetic acid aqueous solution, so 1‰ acetic acid aqueous solution is selected as the mobile phase A.

[0099] (II) Selection of mobile phase B

[0100] After selecting the mobile phase A, the same batch of impurity K crude solution (chromatographic purity of 63%) was used as the column loading solution to uniformly prepare the caspofungin impurity K chromatographic solution with chromatographic purity of ≥95%. Different mobile phase B was used for preparation, including methanol, ethanol, acetonitrile, ethyl acetate. The highest chromatographic purity of the prepared and separated impurity K chromatographic solution was used as the observation standard, and the results are shown in Table 5:

[0101] Table 5 Highest chromatographic purity of caspofungin impurity K obtained under different mobile phase B systems

[0102] Mobile phase B Maximum chromatographic purity (%) Methanol 88 Ethanol 92 Acetonitrile 99 Ethyl acetate 78

[0103] From the experimental results in Table 5, it can be seen that when acetonitrile is used as the mobile phase B, the chromatographic purity of the prepared and separated caspofungin impurity K is the highest, and meets the requirement of chromatographic purity of ≥95% for the prepared chromatographic solution, therefore acetonitrile is selected as the mobile phase B.

[0104] 5. Screening of elution gradient

[0105] After selecting the eluent and the combination of the fillers, gradient was first tried. It was found that when the proportion of acetonitrile was about 3%, there was a peak of acetate salt; when the proportion of acetonitrile was 5%-8%, there was a peak of caspofungin impurity K front impurity; when the proportion of acetonitrile was about 10%, there was a peak of caspofungin impurity K; when the proportion of acetonitrile was 12% or above, there were many peaks of rear impurities. Therefore, the elution program was set as follows: the column was equilibrated with the mobile phase containing 3% acetonitrile, and the amount of the mobile phase was 2 times the column volume; then the column was eluted with the mobile phase containing 5%-8% acetonitrile, and the amount of the mobile phase was 6 times the column volume; finally, the column was eluted with the mobile phase containing 10% acetonitrile, and the amount of the mobile phase was 12 times the column volume. The experiments were carried out on the chromatographic column with a diameter of 30 mm or 50 mm high-pressure preparation column, and it was found that under the elution program, caspofungin impurity K could be well separated. The column chromatography in the high-pressure preparation separation process is shown in Figure 1 .

[0106] In summary, the reaction solvent, reaction temperature, preparation column filler, eluent and elution gradient were determined, and the preparation chromatography conditions of Example 1 are shown in Table 6:

[0107] Table 6 Preparation chromatography conditions of Example 1

[0108] Item Conditions Instrument High pressure preparative chromatography system Sample Caspofungin impurity K crude solution Packing of the preparative column SP-100-10-ODS-P Eluent Mobile phase A: 1% water with acetic acid; mobile phase B: acetonitrile Elution program Isocratic: 3% acetonitrile; gradient: 5% to 8% acetonitrile; isocratic: 10% acetonitrile Elution rate 3 to 6 column volumes / hour Detector wavelength 210 nm

[0109] 10g of compound I and 10ml of diethylenetriamine were added to a reaction flask, followed by 40ml of diethylenetriamine as a reaction solvent. The reaction temperature was controlled at 25℃, and the mixture was stirred for 4 hours. After the reaction was completed, the solution was quenched with 120ml of 16.67% acetic acid aqueous solution to obtain a crude solution of caspofungin impurity K with a purity of 61%, totaling 172ml. The insoluble matter was filtered off using a 0.45μm filter membrane, and the entire filtered crude impurity K solution was pumped into a high-pressure preparative column with a column size of 450mm*50mm. After sample loading, the column was equilibrated with a mobile phase of 3% acetonitrile (2 column volumes). Gradient elution was then performed with a mobile phase containing 5%–8% acetonitrile (6 column volumes). Finally, the column was eluted with a mobile phase containing 10% acetonitrile (12 column volumes). The 10% acetonitrile fraction was collected and analyzed by HPLC. The combined chromatographic fractions with a purity of ≥95% were freeze-dried to obtain 4.88 g of a product with a purity of 98.63% (e.g., [missing information]). Figure 2 The solid powder of caspofungin acetate impurity K (shown) had a product yield of 48.8%.

[0110] Example 2

[0111] 10g of compound I and 10ml of diethylenetriamine were added to a reaction flask, followed by 40ml of diethylenetriamine as a reaction solvent. The reaction temperature was controlled at 30℃, and the mixture was stirred for 4 hours. After the reaction was completed, the mixture was quenched with 120ml of 16.67% acetic acid aqueous solution to obtain a compound with a purity of 65% (e.g., compound I). Figure 3 A crude solution of caspofungin impurity K (as shown) was prepared, totaling 175 ml. Insoluble matter was filtered off using a 0.45 μm filter membrane. The filtered crude impurity K solution was then pumped entirely into a high-pressure preparative column (400 mm * 50 mm). After loading, the column was equilibrated with a mobile phase of 3% acetonitrile (2 column volumes). Gradient elution was then performed with a mobile phase containing 5%–8% acetonitrile (6 column volumes). Finally, the column was eluted with a mobile phase containing 10% acetonitrile (12 column volumes). The 10% acetonitrile fraction was collected and analyzed by HPLC. The combined chromatographic fractions with a purity of ≥95% were freeze-dried to obtain 5.33 g of a product with a purity of 96.46% (as shown). Figure 4 The solid powder containing caspofungin K (as shown) had a product yield of 53.3%.

[0112] Example 3

[0113] Take 10 g of compound I, 10 ml of diethylene triamine into the reaction bottle, add 40 ml of diethylene triamine as the reaction solvent, control the reaction temperature at 35℃, stir for 4 h, and then quench the reaction with 120 ml of 16.67% acetic acid solution. The crude solution of caspofungin impurity K with a purity of 63% is obtained, with a total volume of 173 ml. The insoluble substances are filtered out with a 0.45 μm filter membrane, and the filtered crude solution of impurity K is pumped into a high-pressure preparation column. The size of the chromatographic column is 450 mm*50 mm. After the sample is loaded, the chromatographic column is equilibrated with a mobile phase containing 3% acetonitrile, and the amount of the mobile phase is 2 times the column volume. Then, the column is eluted with a mobile phase containing 5%-8% acetonitrile by gradient elution, and the amount of the mobile phase is 6 times the column volume. Finally, the column is eluted with a mobile phase containing 10% acetonitrile, and the amount of the mobile phase is 12 times the column volume. The chromatographic liquid containing 10% acetonitrile is collected, and the purity of the chromatographic liquid is detected by HPLC. The chromatographic liquid with a purity of more than 95% is combined, and then freeze-dried to obtain 5.09 g of solid powder of caspofungin acetate impurity K with a chromatographic purity of 96.88% (as shown in Table 1), and the mass yield of the product is 50.9%. Figure 5

[0114] Example 4

[0115] Take 5 g of compound I and 5 ml of diethylene triamine into a reaction bottle, add 20 ml of diethylene triamine as the reaction solvent, control the reaction temperature at 30℃, stir for 4 h, and then quench the reaction with 60 ml of 16.67% acetic acid solution. The crude solution of caspofungin impurity K with a purity of 65% is obtained, with a total volume of 87 ml. The insoluble substances are filtered out with a 0.45 μm filter membrane, and the filtered crude solution of impurity K is pumped into a high-pressure preparation column. The size of the chromatographic column is 500 mm*50 mm. After the sample is loaded, the chromatographic column is equilibrated with a mobile phase containing 3% acetonitrile, and the amount of the mobile phase is 2 times the column volume. Then, the column is eluted with a mobile phase containing 5%-8% acetonitrile by gradient elution, and the amount of the mobile phase is 6 times the column volume. Finally, the column is eluted with a mobile phase containing 10% acetonitrile, and the amount of the mobile phase is 12 times the column volume. The chromatographic liquid containing 10% acetonitrile is collected, and the purity of the chromatographic liquid is detected by HPLC. The chromatographic liquid with a purity of more than 95% is combined, and then freeze-dried to obtain 2.43 g of solid powder of caspofungin acetate impurity K with a chromatographic purity of 100% (as shown in Table 2), and the mass yield of the product is 48.6%. Figure 6

[0116] ​​In summary, the preparation method of caspofungin impurity K provided by the embodiments of the present application directly obtains the crude caspofungin impurity K by substituting the group at a specific position with diethylenetriamine in a diethylenetriamine solution, using the intermediate compound I of caspofungin as a raw material, which is convenient and fast in operation, high in process safety, and provides a preparation method of caspofungin impurity K. Further, the high-pressure preparation column is good in separation and purification effect, and is simple and fast in operation, and after separation and purification, the solid powder of caspofungin impurity K with high chromatographic purity is finally obtained, which provides a preparation method of high-purity caspofungin impurity K, is convenient for large-scale production, is sufficient to meet the requirements of enterprises themselves and the market, and the preparation method is stable in conditions, simple in steps, strong in specificity, and high in yield, is suitable for large-scale production, and can meet the market demand.

[0117] The above merely describes preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A process for the preparation of caspofungin impurity K, characterized in that, The method comprises the following steps: The starting material of chemical formula I is subjected to substitution reaction with diethylenetriamine to obtain chemical formula II The crude caspofungin impurity K of acetic acid is shown The crude caspofungin impurity K is dissolved and filtered to obtain a crude solution; The crude solution is separated and purified, and the eluate is collected; The eluate is subjected to freeze-drying to obtain the chemical formula III Caspofungin acetate impurity K shown in the figure.

2. The process for the preparation of Caspofungin acetate impurity K according to claim 1, characterized in that, The solvent of the substitution reaction comprises a diethylene triamine solution.

3. The process for the preparation of caspofungin impurity K according to claim 1 or 2, characterized in that, The reaction temperature of the substitution reaction is 25-35 DEG C, and the reaction time is 4-5 hours.

4. The process for the preparation of Caspofungin acetate impurity K according to claim 1, characterized in that, In the step of dissolving and filtering the crude caspofungin impurity K, the dissolving solvent comprises a 16wt% acetic acid aqueous solution; and / or, The filtering is performed by using a 0.45 μm filter membrane.

5. The process for the preparation of Caspofungin Acetate Impurity K according to claim 1, characterized in that, In the step of separating and purifying, a high-pressure preparative chromatography system is used for separating and purifying, and in the chromatography system, the packing of the chromatographic column is SP-100-10-ODS-P, and the diameter of the chromatographic column is 30 mm or 50 mm.

6. The process for the preparation of Caspofungin acetate impurity K according to claim 5, characterized in that, The step of separating and purifying by using a high-pressure preparative chromatography system comprises: The chromatographic column is equilibrated by using a first eluent, and the amount of the first eluent is 2 times the column volume; Gradient elution is performed by using a second eluent, and the amount of the second eluent is 6 times the column volume; Elution is performed by using a third eluent, and the amount of the third eluent is 12 times the column volume, and the eluate in this section is collected.

7. The process for the preparation of caspofungin acetate impurity K according to claim 6, characterized in that, The first eluent comprises a mixture of a 1 ‰ acetic acid aqueous solution and a 3% acetonitrile solution; and / or, The second eluent comprises a mixture of a 1 ‰ acetic acid aqueous solution and a 5%-8% acetonitrile solution; and / or, The third eluent comprises a mixture of a 1 ‰ acetic acid aqueous solution and a 10% acetonitrile solution.

8. The process for the preparation of caspofungin acetate impurity K according to claim 6 or 7, characterized in that, The flow rate of the eluate is 3-6 times the column volume per hour.

9. The process for the preparation of Caspofungin acetate impurity K according to claim 1, characterized in that, The freeze-drying comprises: pre-freezing the eluate, and then performing sublimation drying under the condition that the vacuum degree is ≤-0.090 MPa.

10. The process for the preparation of Caspofungin acetate impurity K according to claim 1, characterized in that, The final chromatographic purity of the caspofungin impurity K is ≥96%, and the recovery rate is ≥48%.

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