CN113801237A - Preparation method of caspofungin acetate impurity E - Google Patents
Preparation method of caspofungin acetate impurity E Download PDFInfo
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Abstract
A preparation method of caspofungin acetate impurity E comprises the following steps: 1) preparing a solution of crude caspofungin acetate impurity E and filtering; 2) separating and purifying the filtered crude product solution through a chromatographic column in a preparation system, and collecting eluent; 3) and (3) freeze-drying the qualified eluent to obtain solid powder of caspofungin acetate impurity E with the purity of more than 95%. According to the invention, by adopting a method for preparing a chromatogram, a mixed solution of 0.1% acetic acid and acetonitrile is used as a mobile phase for isocratic and gradient elution, and finally, impurity E solid powder with the purity of more than 95% is obtained, so that the preparation method of the high-purity caspofungin acetate impurity E is provided, and the requirements of enterprises and markets can be met; the preparation method has stable conditions, is suitable for large-scale production and has high yield.
Description
Technical Field
The invention belongs to the field of chemistry, and particularly relates to a preparation method of caspofungin acetate impurity E.
Background
Caspofungin acetate is the first echinocandin antifungal drug, is approved by the Food and Drug Administration (FDA) to be marketed at 26.1.2001, is mainly used for treating invasive candidiasis, invasive aspergillosis which is ineffective or intolerant to other treatments, and treating suspected fungal infection of patients with neutropenia and fever empirically, and has obvious advantages of high selectivity, good antibacterial activity, high safety, low drug resistance and the like as a representative of a new class of echinocandin antifungal drugs.
The caspofungin acetate is prepared by fermenting Glarea Lozoyensis to obtain a fermentation product, then performing solid-liquid separation, leaching, adsorption and desorption, concentration and drying on the fermentation product to obtain a nemadectin B0 crude product, and then performing 3-step synthesis and 1-step refining on the nemadectin B0 as a starting raw material to obtain a caspofungin acetate crude drug finished product.
The No. 10 carbon atom of caspofungin acetate is connected with two nitrogen atoms, so that the caspofungin acetate is electropositive, hydroxide ions in water molecules can easily attack the No. 10 carbon atom, a C-N bond is broken to form an amide structure intermediate, the No. 10 carbon atom still shows electropositive at the moment, ethylenediamine in the other caspofungin acetate has a lone pair electron, and can easily bond with the No. 10 carbon atom of the molecule and remove the water molecules to obtain a dimer structure, an isomer precursor of an impurity E can be generated under the condition, and the impurity E is generated through the next reaction. The structure of impurity E is shown as formula (I):
（I）
in the field of drug detection, caspofungin acetate impurity E is required as a reference substance, and the literature shows that
Leonard W R, Belyk K M, Conlon D A, et al, Synthesis of The anti β -1, 3-glucan synthase inhibitor CANCIDAS (carbohydrate acetate) from pneumococcandin B0[ J ]. The Journal of organic chemistry, 2007, 72(7): 2335-2343. it is mentioned that The hydroxyl isomerism contributes to The stabilization of The intermediate, so this configuration will be produced and finally The impurity E will be produced, Fushiji, Silong, preparation and quality control of caspofungin for injection [ J ]. chemical man-hours, 2018 (2018): 23-27, 34 "" reports a method for detecting The impurity A, B, D, E, F, G, H, I in caspofungin acetate samples.
In the prior art, no report is available about the preparation of high-purity caspofungin acetate impurity E, and therefore, a method for preparing high-purity caspofungin acetate impurity E needs to be developed to meet the needs of enterprises and markets for a reference substance of caspofungin acetate impurity E.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for preparing caspofungin acetate impurity E shown as a formula (I), which specifically comprises the following steps:
（I）
1) preparing a solution of crude caspofungin acetate impurity E and filtering;
2) separating and purifying the filtered crude product solution through a chromatographic column in a preparation system, and collecting eluent;
3) and (3) freeze-drying the qualified eluent to obtain solid powder of caspofungin acetate impurity E with the purity of more than 95%.
Further, in the step 1), the crude caspofungin acetate impurity E is dissolved in 0.1% acetic acid water solution, and is filtered by using a 0.45 μm filter membrane.
Further, the crude caspofungin acetate impurity E in the step 1) is obtained by concentrating a part of solution with higher impurity E content in the preparation solution of the intermediate product (crude caspofungin acetate impurity).
Further, in the step 2), the filtered crude caspofungin acetate impurity E solution is firstly put on a chromatographic column, the chromatographic column is a medium-low pressure liquid phase chromatographic column, the diameter of the chromatographic column is 30mm or 50mm, and the filler of the chromatographic column is SP-C18-100-8-ODS-P; then, a mobile phase containing 10% of acetonitrile is used for balancing the chromatographic column, and the dosage of the mobile phase is 2 times of the column volume; then, carrying out gradient elution by using a mobile phase containing 10-30% of acetonitrile, wherein the using amount of the mobile phase is 2 times of the column volume; then gradient elution is carried out by 30% -36% acetonitrile, the dosage of the mobile phase is 4-5 times of the column volume, gradient eluent in the stage is collected, and HPLC detection is carried out.
Further, the flow rate of the eluent in the step 2) is 3-4 times of the column volume/hour.
Further, the eluent which is not collected in the step 2) is treated as waste liquid.
Further, after gradient elution in the step 2) is finished, regeneration elution is carried out by using edible ethanol, the using amount of the ethanol is 4-8 times of the column volume, the flow rate is 3-5 times of the column volume/hour, eluent is not collected, and waste liquid is treated.
Further, the mobile phase in the step 2) consists of a mobile phase A and a mobile phase B, the sum of the volumes of the mobile phase A and the mobile phase B is 100%, wherein the mobile phase A is 0.1% acetic acid water solution, and the mobile phase B is acetonitrile.
Further, in the step 3), the qualified eluent is firstly frozen, and then the solid eluent is directly sublimated in a vacuum state, so that the solid powder of the caspofungin acetate impurity E is obtained.
Compared with the prior art, the invention has the following beneficial effects: 1) according to the invention, by adopting a method for preparing a chromatogram, a mixed solution of 0.1% acetic acid and acetonitrile is used as a mobile phase for isocratic and gradient elution, and finally, impurity E solid powder with the purity of more than 95% is obtained, so that the preparation method of the high-purity caspofungin acetate impurity E is provided, and the requirements of enterprises and markets can be met; 2) the preparation method has stable conditions, is suitable for large-scale production and has high yield.
Drawings
FIG. 1 is a finished detection chart of impurity E of example 1;
FIG. 2 is a finished detection chart of impurity E of example 2;
FIG. 3 is a finished inspection chart of impurity E of example 3;
FIG. 4 is a finished inspection chart of impurity E of example 4;
FIG. 5 is a graph showing the detection results of impurity E in example 5.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The reagents used in the following examples are all commercially available.
Example 1
First, the mobile phase, the packing and the elution gradient were determined.
Firstly, acid water is used as a mobile phase A, acetonitrile is used as a mobile phase B, and the preparation stability of the super antibiotic product needs to be further examined due to the poor stability of the super antibiotic product, so that the super antibiotic product is prepared by adopting aqueous solutions with different acidity, and the results are shown in Table 1.
TABLE 1 Effect of different acidity of mobile phase A on the purity of impurity E in the collected fractions
According to the data in table 1, the impurity E shows different stabilities under different acidic and basic conditions, wherein the impurity E shows stronger stability under acidic conditions, and the product is extremely unstable and has a significantly reduced purity under basic conditions, so that 0.1% acetic acid aqueous solution is selected as the aqueous phase mobile phase a, and acetonitrile is selected as the mobile phase B.
2. Determination of the filling
According to the product characteristics, reversed phase preparative chromatography is adopted for separating the impurity E, and a conventional C18 packing is adopted for screening, such as: SP-C18-100-8-ODS-P and SP-C18-120-10-ODS-BP, the results are shown in Table 2.
TABLE 2 preparative chromatographic separation of different packings
As shown in Table 2, the separation of SP-C18-100-8-ODS-P was the best and 95.2% of the fraction was obtained. Therefore, SP-C18-100-8-ODS-P was selected as the filler for this process.
3. Determination of elution gradient
After selecting the composition of a mobile phase and a filler phase, firstly carrying out a gradient crude test, and finding that an impurity E generates a peak when the concentration of acetonitrile is about 36%; in an analysis liquid phase spectrogram, the relative retention time of the impurity E, namely RRT, is 1.68, and is relatively far away from the peak emergence time of a main peak, so that other substances with short retention time need to be washed out firstly, and then the organic phase ratio is increased to obtain a target substance; setting the gradient interval of elution as 10% (acetonitrile content), gradient eluting for 2 times of column volume, isocratic eluting for 2 times of column volume with 10% -30% (acetonitrile content), and finally eluting for impurity E with 30% -36% gradient.
In summary, the mobile phase, the filler and the elution gradient were determined as shown in table 3.
Table 3: chromatographic conditions for example 1
Dissolving 6g of crude product E with the purity of 48% in 0.1% acetic acid aqueous solution, filtering with a 0.45-micron filter membrane to remove insoluble substances, pumping the filtered E solution into a chromatographic column for preparing a chromatogram, wherein the filler of the chromatographic column is SP-C18-100-8-ODS-P, the size of the column is 50mm x 500mm, the particle size of the filler is 8 microns, the loading amount is 5g/L, and the mobile phase is 0.1% acetic acid aqueous solution and acetonitrile, and firstly adopting the mobile phase containing 10% acetonitrile and 2 times of the volume of the column to balance the chromatographic column; then, carrying out gradient elution by using a mobile phase containing 10-30% of acetonitrile and 2 times of column volume; then gradient elution is carried out by 30% -36% acetonitrile, the eluent at the stage is collected, after HPLC detection, the content of the impurity E in the collected gradient eluent is over 98%, 1.32g of solid powder of the impurity E is obtained after freeze-drying, the total yield of the product is 46%, and the purity of the product is 95.41%.
Example 2
According to the same method and chromatographic conditions of example 1, 4g of crude product E with the purity of 48% is dissolved in 0.1% acetic acid aqueous solution, the content of impurity E in the finally collected gradient eluent is more than 95% in purity, and 1.1g of solid powder of impurity E is obtained after freeze-drying, wherein the total yield of the product is 55% and the purity of the product is 96.46%.
Example 3
According to the same method and chromatographic conditions of example 1, 5g of crude E with the purity of 48% is dissolved in 0.1% acetic acid aqueous solution, the content of impurity E in the finally collected gradient eluent is more than 95% of purity, and 1.4g of solid powder of impurity E is obtained after freeze-drying, the total yield of the product is 57%, and the purity of the product is 95.83%.
Example 4
According to the same method and chromatographic conditions of example 1, 7g of crude product E with the purity of 48% is dissolved in 0.1% acetic acid aqueous solution, the content of the impurity E in the finally collected gradient eluent is more than 95% in purity, and 1.8g of solid powder of the impurity E is obtained after freeze-drying, the total yield of the product is 55%, and the purity of the product is 95.45%.
Example 5
According to the same method and chromatographic conditions of example 1, 3g of crude product E with the purity of 48% is dissolved in 0.1% acetic acid aqueous solution, the content of the impurity E in the finally collected gradient eluent is more than 95% in purity, and 0.62g of solid powder of the impurity E is obtained after freeze-drying, the total yield of the product is 43%, and the purity of the product is 95.77%.
Claims (6)
1. A preparation method of caspofungin acetate impurity E shown as a formula (I) is characterized by comprising the following steps:
（I）
1) preparing a solution of crude caspofungin acetate impurity E and filtering;
2) separating and purifying the filtered crude product solution through a chromatographic column in a preparation system, and collecting eluent;
3) and (3) freeze-drying the qualified eluent to obtain solid powder of caspofungin acetate impurity E with the purity of more than 95%.
2. The method for preparing caspofungin acetate impurity E according to claim 1, wherein in step 1), crude caspofungin acetate impurity E is dissolved in 0.1% acetic acid aqueous solution and filtered through 0.45 μm filter membrane.
3. The preparation method of caspofungin acetate impurity E according to claim 1 or 2, wherein in the step 2), the filtered crude caspofungin acetate impurity E solution is firstly put on a chromatographic column, the chromatographic column is a medium-low pressure liquid phase chromatographic column, the diameter of the chromatographic column is 30mm or 50mm, and the packing of the chromatographic column is SP-C18-100-8-ODS-P; then, a mobile phase containing 10% of acetonitrile is used for balancing the chromatographic column, and the dosage of the mobile phase is 2 times of the column volume; then, carrying out gradient elution by using a mobile phase containing 10-30% of acetonitrile, wherein the using amount of the mobile phase is 2 times of the column volume; then gradient elution is carried out by 30% -36% acetonitrile, the dosage of the mobile phase is 4-5 times of the column volume, gradient eluent in the stage is collected, and HPLC detection is carried out.
4. The method for preparing caspofungin acetate impurity E according to claim 3, wherein after the gradient elution in step 2) is completed, the elution is regenerated with edible ethanol, the amount of ethanol is 4-8 times the column volume, and the flow rate is 3-5 times the column volume/hour.
5. The method for preparing caspofungin acetate impurity E according to claim 3, wherein the mobile phase in step 2) is composed of mobile phase A and mobile phase B, the sum of the volumes of the mobile phase A and the mobile phase B is 100%, wherein the mobile phase A is 0.1% acetic acid solution in water, and the mobile phase B is acetonitrile.
6. The method for preparing caspofungin acetate as impurity E according to claim 1, wherein the qualified eluate is frozen and then the solid eluate is directly sublimated under vacuum to obtain solid powder of caspofungin acetate as impurity E.
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| CN114276416A (en) * | 2021-12-24 | 2022-04-05 | 苏州第四制药厂有限公司 | Preparation process of caspofungin acetate impurity |
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| CN114276416A (en) * | 2021-12-24 | 2022-04-05 | 苏州第四制药厂有限公司 | Preparation process of caspofungin acetate impurity |
| CN114276416B (en) * | 2021-12-24 | 2022-09-30 | 苏州第四制药厂有限公司 | Preparation process of caspofungin acetate impurity |
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