High-purity echinocandin drug impurity, and preparation method therefor and use thereof

Abstract

Provided are a high-purity echinocandin drug impurity, and a preparation method therefor and a use thereof. The structure of the echinocandin drug impurity is as shown in formula I, and the purity of the echinocandin drug impurity is greater than 90%.

Description

A high-purity echinocandin drug impurity and its preparation method and application

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to the Chinese patent application filed with the China Patent Office on July 31, 2023, with application number 202310956462.X and invention name “A high-purity echinocandin drug impurity, its preparation method and application”, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present application belongs to the field of medical technology, and specifically relates to a high-purity echinocandin drug impurity and its preparation method and application. Background Art

[0004] Micafungin sodium is a new-generation echinocandin antifungal drug primarily used to treat fungemia, respiratory fungal infections, and gastrointestinal fungal infections caused by Aspergillus and Candida species. It is also used to prevent Aspergillus and Candida infections in patients with hematopoietic stem cell transplantation. It is a member of the echinocandin class of antifungals that inhibits the synthesis of β(1,3)-glucans by filamentous fungi and yeasts. In recent years, the incidence and mortality rates of fungal infections have also been increasing, posing a particularly life-threatening threat to critically ill patients. As an echinocandin, micafungin sodium, with its excellent antifungal activity, is the preferred treatment for infections caused by Candida and Aspergillus species. It is highly effective in treatment, has minimal effects on human cells, and exhibits low toxicity and high clinical efficacy.

[0005] Impurity analysis is crucial in drug development. Impurities are closely related to drug quality, safety, and stability. The preparation and structural confirmation of impurities can help understand the pathways of impurity formation, providing a basis for improving drug synthesis routes and production processes. Impurities can be used in drug quality control and as reference substances for establishing analytical methods. For pharmaceutical manufacturers, strict control of impurities is essential.

[0006] Micafungin sodium is a semisynthetic antibacterial drug. It can be obtained by chemically synthesizing FR179642 from the fermentation intermediate FR901379 through enzymatic removal of the fatty acid side chain, and then reacting it with the active side chain via a condensation reaction. During the fermentation process of FR901379, impurities with a structure similar to the FR901379 nucleus are produced. These impurities are highly similar to the structure of micafungin sodium, making their removal in subsequent purification processes ineffective. Therefore, it is necessary to control the impurity content during the fermentation and acylase fatty acid removal stages. Studies of commercially available formulations revealed the presence of multiple impurities in the HPLC spectra of the formulations. These impurities are present at relatively low levels in the bulk drug, making enrichment and purification extremely challenging. Furthermore, the multiple chiral centers and peptide bonds of echinocandin drugs make them extremely labile to acids, bases, heat, and light, making it difficult to obtain high-purity products free of these impurities.

[0007] Research on echinocandin drug impurities can be used for qualitative and quantitative analysis of impurities in the production of micafungin sodium, thereby providing technical support for subsequent quality research on micafungin sodium and improving the quality standards of micafungin sodium. Therefore, there is a need for an efficient and convenient method for separating and preparing high-purity echinocandin drug impurities.

[0008] Summary of the Invention

[0009] This application isolates and prepares an echinocandin drug impurity of Formula II from the fermentation broth of FR901379. This echinocandin drug impurity has a structure highly similar to that of micafungin sodium. Under suitable conditions, the application includes a step of removing fatty acids under the action of an acyltransferase to form a compound of Formula III. This step allows for efficient purification and isolation of the pure compound of Formula III. This preparation and purification yields a high-purity echinocandin drug impurity of Formula I. This application employs the following technical solution:

[0010] An echinocandin drug impurity, wherein the structure of the echinocandin drug impurity is as shown in Formula I, and the purity of the echinocandin drug impurity is greater than 90%;

[0011] Optionally, the purity refers to HPLC purity.

[0012] The method for preparing the above-mentioned echinocandin drug impurities comprises the following steps:

[0013] Step 1) adding an organic solvent to a microbial fermentation broth containing a compound of Formula II, mixing the mixture, and filtering the mixture. The filtrate is enriched using a first adsorption resin to obtain the compound of Formula II;

[0014] Step 2) reacting the enriched compound of formula II with an acyltransferase to obtain a compound of formula III;

[0015] Step 3) reacting the obtained compound of formula III with the side chain of micafungin sodium to obtain the echinocandin drug impurity.

[0016] Optionally, the microbial fermentation broth is FR901379 fermentation broth.

[0017] Optionally, the microbial fermentation broth is obtained by fermentation of Coleophoma sp.

[0018] Optionally, the organic solvent is selected from at least one of methanol, ethanol, propanol, acetone, and butanone;

[0019] Optionally, the volume ratio of the organic solvent to the fermentation broth is 1%-40%;

[0020] Optionally, the first adsorption resin is a non-polar aromatic adsorption resin formed by polymerization of styrene and divinylbenzene;

[0021] Optionally, the first adsorption resin is at least one of AD-1, HP20, HP20SS, HP30, SP-825, SP-850, SP207, and CHP20.

[0022] Optionally, the acyltransferase is an immobilized acyltransferase supported by a carrier.

[0023] Optionally, the carrier is selected from one or a combination of silicon dioxide, aluminum oxide, polymethacrylate-based epoxy bonded, and amino functional groups.

[0024] Optionally, in step 2), the enriched compound of formula II is reacted with acyltransferase for purification, and the purification step comprises: adsorption using a second adsorption resin and elution using an organic solvent aqueous solution at 10-30°C.

[0025] Optionally, the second adsorption resin is a macroporous adsorption resin, and adopts at least one of the resins SP207, SP207SS, UniPS10, UniPS20, and UniPS50 having styrene and divinylbenzene as matrices;

[0026] Optionally, the organic solvent in the organic solvent aqueous solution is selected from at least one of methanol, ethanol, acetone, and acetonitrile;

[0027] Optionally, the volume percentage of the organic solvent in the organic solvent aqueous solution is 1%-90%;

[0028] Optionally, the elution temperature is 15-25°C.

[0029] Optionally, the pH of the collected solution after purification needs to be adjusted to 1.0-6.0.

[0030] Optionally, the optimal pH of the collected solution after purification is adjusted to 3.0-4.0.

[0031] Optionally, the compound of formula III is reacted with the side chain of micafungin sodium at -20 to 10°C.

[0032] Optionally, the compound of formula III is reacted with the side chain of micafungin sodium in an organic solvent, wherein the organic solvent is selected from at least one of toluene, N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DME), dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), and dichloromethane.

[0033] Optionally, after the reaction of the compound of Formula III with the side chain of micafungin sodium is completed, purification is performed, wherein the purification is performed using ion exchange resin, macroporous adsorption resin, and silica gel in sequence;

[0034] Optionally, the ion exchange resin is a strongly acidic cation exchange resin, and the ion exchange resin is selected from at least one of UBK530, UBK550, and UBK555;

[0035] Optionally, the macroporous adsorption resin is selected from at least one of HP20, HP20SS, SP70, SP700, SP825L, SP850, CHP20, and CHP55;

[0036] Optionally, the silica gel is selected from at least one of pure silica gel, amino-bonded silica gel, glycol-bonded silica gel, and amide-bonded silica gel;

[0037] Optionally, the silica gel is selected from At least one of HILIC silica gel (Nanomicron), Chromatorex ARG Silica Fuji, Click XIon silica gel (Huapu Xinchuang), and Inertsil HILIC silica gel (Shimadzu, Japan);

[0038] Optionally, the organic solvent in the acidic organic solvent aqueous solution is selected from one of methanol, ethanol, acetone and acetonitrile;

[0039] Optionally, the silica gel purification step includes: adsorption using a silica gel column, followed by elution using an acidic organic solvent aqueous solution with a pH value of 2.0-7.0;

[0040] Optionally, during adsorption using a silica gel column, the proportion of organic solvent in the sample solution does not exceed 5%;

[0041] Optionally, the volume percentage of organic solvent to water in the acidic organic solvent aqueous solution is 20%-100%;

[0042] Optionally, the volume percentage of the organic solvent to water in the acidic organic solvent aqueous solution is 50%-100%.

[0043] Optionally, the acidic organic solvent aqueous solution contains at least one of formic acid, acetic acid, hydrochloric acid, phosphoric acid, and trifluoroacetic acid;

[0044] Optionally, the pH value of the acidic organic solvent aqueous solution is 3.0-5.0.

[0045] Optionally, the purity of the prepared compound of formula I is greater than 90%.

[0046] This application also proposes the use of the above-mentioned cyclic peptide compound in the quality control of echinocandin drugs.

[0047] As a specific embodiment: The echinocandin drug impurity provided in the present application has a chemical name: 5-[(1S,2S)-2-[(3S,6S,9S,11R,15S,18S,20R,21R,24S,25S)-3-[(R)-2-carbamoyl-1-hydroxyethyl]-11,20,21,25-tetrahydroxy-15-[(R)-1-hydroxyethyl]-2,5,8,14,17,23-hexahydro-18-[4-[5-(4-pentyloxyphenyl)isoxazol-3-yl]benzamido]-1,4,7,13,16,22-hexaazatricyclo[22.3.0.09,13]heptacos-6-yl]-1,2-dihydroxyethyl]-2-hydroxyphenyl sodium sulfate, and its structure is shown in Formula I.

[0048] The preparation method comprises the following steps:

[0049] (a) fermenting to obtain a fermentation broth containing FR901379 impurity (Formula II), and extracting and enriching it;

[0050] (b) using acyltransferase to convert the obtained component containing FR179642 impurity (Formula III), and purifying and isolating the obtained compound of Formula III

[0051] (c) condensing the FR179642 impurity (Formula III) with the side chain of micafungin sodium, and purifying the obtained micafungin sodium drug impurity (Formula I)

[0052] The fermentation in step (a) refers to patent CN102618604B, using Coleophoma sp as the fermentation strain, the main components of the culture medium include cottonseed powder, mannitol, sodium glutamate, and proline, and the culture is carried out at 23-28°C for 6-8 days.

[0053] As a further improvement of the above preparation method, the purification step in step (b) comprises:

[0054] 1) Use macroporous adsorption resin for adsorption

[0055] 2) At low temperature, use a certain proportion of organic solvent for elution

[0056] As a further improvement of the above preparation method, the macroporous adsorption resin in step 1) is an aromatic adsorption resin formed by polymerization of styrene / divinylbenzene, selected from one of SP207, SP207SS, UniPS10, UniPS20, or UniPS50, or a combination thereof.

[0057] As a further improvement to the above preparation method, in the purification method described in step 2), the organic solvent is selected from methanol, ethanol, acetone, acetonitrile, or a mixture thereof; the volume percentage of the organic solvent is 1%-90%, with an optimal organic solvent ratio of 1%-50%. The pH of the collected solution after purification is adjusted to 1.0-6.0, with an optimal pH of 3.0-4.0. The temperature during purification and separation is controlled at 10-30°C, with an optimal temperature of 15-25°C.

[0058] The organic solvent used in preparation step (c) is selected from one or a combination of toluene, N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DME), dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), and dichloromethane. N,N-dimethylformamide (DMF) is an optional solvent. The purification step in preparation step (c) is sequentially performed using an ion exchange resin, a macroporous adsorption resin, and silica gel.

[0059] As a further improvement of the above preparation method, the silica gel purification step used includes the following steps:

[0060] (1) After diluting the above concentrate, prepare the loading solution and adsorb it through a silica gel column.

[0061] (2) eluting with an acidic aqueous solution of an organic solvent;

[0062] The silica gel is a silica gel with a hydrophilic interaction chromatography mode (HILIC). Step (1) The proportion of the organic solvent in the sample solution does not exceed 5%; the silica gel is selected from pure silica gel, amino bonded silica gel, diol bonded silica gel, amide bonded silica gel; the silica gel is selected from HILIC silica gel (Nanomicron), Chromatorex ARG Silica Fuji, Click XIon silica gel (Huapu Xinchuang), Inertsil HILIC silica gel (Shimadzu, Japan). As a further improvement to the above purification method, the organic solvent is selected from one or a combination of methanol, ethanol, acetone, and acetonitrile. The volume percentage of the organic solvent to water is 20%-100%, and can be 50%-100%.

[0063] As a further improvement of the above purification method, after purification by chromatography, the HPLC purity of the obtained echinocandin drug impurity is above 90%. The collected liquid fraction is concentrated below 20°C and freeze-dried to obtain the echinocandin drug impurity finished product.

[0064] The high-purity echinocandin drug impurity of formula I can be used for echinocandin drug quality control and as a reference substance for establishing analytical methods. The high-purity echinocandin drug impurity refers to an HPLC purity of 90% or more.

[0065] Compared with the prior art, the present invention has the following advantages:

[0066] The preparation method of the present application is to extract and enrich the FR901379 fermentation broth containing the impurity of formula II; use acyltransferase to convert and separate to obtain the compound of formula III; and further prepare a high-purity echinocandin drug impurity (formula I).

[0067] This application also provides a further purification method, using ion exchange resin, macroporous adsorption resin, and silica gel column purification in sequence. This further improves the HPLC purity of the echinocandin drug impurities in the reaction solution, reaching a maximum HPLC purity of over 90%, providing new ideas for industrial production. The purity of drug impurity reference substances must reach over 90%, a requirement well known in the art. This application is the first to prepare echinocandin drug impurities with a purity exceeding 90%, making them suitable for use as drug quality control reference substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] FIG1 is a hydrogen nuclear magnetic resonance spectrum of the echinocandin drug impurities in Example 2 ( 1 1H NMR).

[0069] FIG2 is a carbon NMR spectrum of the echinocandin drug impurities in Example 2 ( 13 C NMR).

[0070] FIG3 is a high resolution mass spectrum (HRMS) of the echinocandin drug impurities in Example 2.

[0071] FIG4 is an HPLC spectrum of the echinocandin drug impurities in Example 2. DETAILED DESCRIPTION

[0072] To better illustrate the purpose, technical solutions and advantages of this application, the present application will be further described below in conjunction with specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are only used to explain this application and are not intended to limit this application.

[0073] Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials and reagents used are all commercially available unless otherwise specified. The fermentation strain Coleophoma sp used in each example is from Advanced Industrial Science and Technology.

[0074] Example 1:

[0075] Step 1:

[0076] Referring to patent CN102618604B (Shanghai Tianwei), Coleophoma sp was used as the fermentation strain, and the main components of the culture medium included cottonseed meal, mannitol, sodium glutamate, and proline. The culture was carried out at 23-28 ° C for 6-8 days to obtain 5L of fermentation broth containing FR901379 impurity components, about 20g of pure FR901379, 5L of methanol was added for extraction, stirred for 2h and mixed evenly, and then filtered. The filtrate was diluted with water to an organic solvent ratio of 5%, then adsorbed using HP20 resin, eluted using 80% ethanol, and enriched to obtain a collected liquid containing FR901379 impurities (Formula II structure), about 0.7g of the pure Formula II structure.

[0077] Step 2:

[0078] The collected solution from step 1 was slowly added to 2 L of pH 6.0 phosphate buffer, followed by 15 g of immobilized acyltransferase, and allowed to react at 40°C for 2 hours. Adsorption was performed using SP207SS (1 L) macroporous adsorption resin. Elution was performed with 1% and then 5% ethanol at a temperature of 10-15°C. The effluent containing the FR179642 impurity (Formula III) was collected and the pH was adjusted to 3.0. After nanofiltration and lyophilization, 0.4 g of the compound of Formula III with a purity of 90% was obtained.

[0079] Step 3:

[0080] Add 19 g of DMF to a 100 mL reaction flask, cool to -20°C, and stir for 5 minutes. Add 1.0 g of the compound of formula III and stir until clear. Then, add 0.22 g of DIPEA and 1.00 g of the side chain of micafungin sodium at -20°C, and keep the reaction warm for 1.5 hours. Maintain the temperature at 5°C, add 4 g of methanol, stir for 20 minutes, then add 23 g of acetone and stir for 1 hour. Finally, add 80 g of ethyl acetate dropwise after about 1 hour. A white solid will precipitate. Centrifuge the reaction solution to obtain 1.5 g of crude micafungin sodium drug impurity.

[0081] 1.5 g of crude micafungin sodium drug impurity product was dissolved in 50 ml of water, and ion exchange was performed using UBK550 resin (50 ml). The collected solution containing impurity components was adjusted to pH 5.5 using 0.2 M sodium bicarbonate. After enrichment using HP20 resin, elution was performed using 80% ethanol aqueous solution to obtain a collected combined solution with a purity of more than 80%. Finally, the solution was used. The product was purified by HILIC silica gel and eluted with 50% ethanol-formic acid aqueous solution (pH 4.0). Fractions with a purity of more than 90% were collected, concentrated and lyophilized to obtain 0.5 g of micafungin drug impurity (Formula I) as a white powder with a purity of 92%.

[0082] Example 2:

[0083] Step 1:

[0084] Referring to patent CN102618604B (Shanghai Tianwei), Coleophoma sp was used as the fermentation strain, and the main components of the culture medium included cottonseed meal, mannitol, sodium glutamate, and proline. The culture was carried out at 23-28 ° C for 6-8 days to obtain 5L of fermentation broth containing FR901379 impurity components, about 20g of pure FR901379, 5L of acetone was added for extraction, stirred for 30min to mix evenly, and then filtered. The filtrate was diluted with water to an organic solvent ratio of 40%, then adsorbed using HP20SS resin, eluted with 80% ethanol, and concentrated after enrichment to obtain a collected solution containing FR901379 impurity (Formula II structure), about 1.3g of pure Formula II structure.

[0085] Step 2:

[0086] The liquid collected from step 1, containing the FR901379 impurity (Formula II), was slowly added with 2 L of a 0.2 M potassium dihydrogen phosphate-sodium dihydrogen phosphate buffer, pH 6.0, and 15 g of an immobilized acyltransferase. The reaction was allowed to proceed at 40°C for 2 hours. Adsorption was then performed using SP207SS (1 L) macroporous adsorption resin. Under a controlled temperature of 10-15°C, the mixture was first washed with 1% ethanol and then eluted with 5% ethanol. The effluent, containing the FR179642 impurity (Formula III), was collected and the pH was adjusted to 3.5. After nanofiltration and lyophilization, 1.0 g of the compound of Formula III with a purity of 90% and 93% was obtained.

[0087] Step 3:

[0088] Add 19g of DMF to a 100mL reaction flask, cool to 0°C, and stir for 5 minutes. Add 1.0g of the compound of formula III and stir until clear. Then, add 0.22g of DIPEA and 1.00g of the side chain of micafungin sodium at -5°C in sequence. Maintain the temperature at 5°C and allow the reaction to proceed for 1.5 hours. Maintain the temperature at 10°C, add 4g of methanol, stir for 20 minutes, then add 23g of acetone and stir for 1 hour. Finally, add 80g of ethyl acetate dropwise after approximately 1 hour. A white solid will precipitate. Centrifuge the reaction solution to obtain 1.5g of crude micafungin sodium drug impurity.

[0089] 1.5 g of crude micafungin sodium impurity product was dissolved in 50 ml of water. Ion exchange was performed using UBK530 resin (50 ml). The collected solution containing impurity components was adjusted to a pH of approximately 5.5 using 0.2 M sodium bicarbonate. After enrichment using HP20SS resin, the solution was eluted with 60% methanol in water to obtain a pooled solution with a purity of greater than 80%. Finally, purification was performed using Click XiON silica gel and eluted with 60% methanol in hydrochloric acid (pH 4.0). Fractions with a purity of greater than 90% were collected and concentrated and lyophilized to obtain 1.0 g of micafungin impurity (Formula I) as a white powder with a purity of 96.14%.

[0090] Example 3:

[0091] Step 1:

[0092] Referring to patent CN102618604B (Shanghai Tianwei), Coleophoma sp was used as the fermentation strain, and the main components of the culture medium included cottonseed meal, mannitol, sodium glutamate, and proline. The culture was carried out at 23-28°C for 6-8 days to obtain 5L of fermentation broth containing FR901379 impurity components, about 20g of pure FR901379, 3L of propanol was added for extraction, and after stirring for 30min to mix evenly, it was filtered. The filtrate was diluted with water to an organic solvent ratio of 30%, then adsorbed using XAD-1 resin, eluted using 65% methanol, and concentrated after enrichment to obtain a collected liquid containing FR901379 impurities (Formula II structure), about 1.2g of the pure Formula II structure.

[0093] Step 2:

[0094] The aqueous solution containing the FR901379 impurity (Formula II) collected from Step 1 was diluted to 2 L. 1 L of pH 6.0 phosphate buffer and 10 g of immobilized acyltransferase were slowly added and reacted at 40°C for 2 hours. Adsorption was performed using SP207SS (1.5 L) macroporous adsorption resin at 30°C. The solution was first washed with 2% ethanol and then eluted with 30% acetonitrile. The effluent containing the FR179642 impurity (Formula III) was collected and the pH was adjusted to 4.5. After nanofiltration and lyophilization, 0.3 g of the compound of Formula III with a purity of 93% was obtained.

[0095] Step 3:

[0096] Add 10 ml of DMSO to a 100 mL reaction flask, cool to -20°C, and stir for 30 minutes. Add 0.3 g of the compound of formula III and stir until clear. Then, add 0.1 g of DIPEA and 0.2 g of the side chain of micafungin sodium at -10°C, control the temperature at 20°C, and incubate for 1.5 hours. Control the temperature at 0°C, add 4 g of ethanol, and stir for 20 minutes. Finally, add 30 g of ethyl acetate dropwise after approximately 1 hour. A white solid will precipitate. Centrifuge the reaction solution to obtain 0.3 g of crude micafungin sodium drug impurity.

[0097] 0.3 g of crude micafungin sodium drug impurity product was dissolved in 10 ml of water and ion exchanged using UBK555 resin (10 ml). The collected solution containing impurity components was adjusted to a pH of approximately 5.5 using 0.2 M sodium bicarbonate. After enrichment using HP20 resin, elution was performed using 99% methanol-water to obtain a pooled solution with a purity of greater than 80%. Finally, purification was performed using Chromatorex ARG Silica Fuji silica gel and desorption was performed using 70% acetonitrile-trifluoroacetic acid aqueous solution (pH 5.0). Fractions with a purity of greater than 90% were collected and concentrated and lyophilized to obtain 0.2 g of micafungin drug impurity (Formula I) as a white powder with a purity of 95%.

[0098] Example 4:

[0099] Step 1:

[0100] Referring to patent CN102618604B (Shanghai Tianwei), Coleophoma sp was used as the fermentation strain, and the main components of the culture medium included cottonseed meal, mannitol, sodium glutamate, and proline. The culture was carried out at 23-28 ° C for 6-8 days to obtain 20L of fermentation broth containing FR901379 impurity components, about 40g of pure FR901379, 10L of butanone / ethanol mixed solution (1:1) was added for extraction, stirred for 2h and mixed evenly, and then filtered. The filtrate was diluted with water to an organic solvent ratio of 30%, then adsorbed using HP30 resin, eluted with 90% acetonitrile, and concentrated after enrichment to obtain a collected liquid containing FR901379 impurities (Formula II structure), and the pure Formula II structure was about 2.0g.

[0101] Step 2:

[0102] The aqueous solution containing the FR901379 impurity (Formula II) collected from Step 1 was diluted to 2 L. 1 L of pH 6.0 phosphate buffer and 20 g of immobilized acyltransferase were slowly added and reacted at 40°C for 3 hours. Adsorption was performed using Unips10 resin (1.5 L). The mixture was washed with 20% acetonitrile and then eluted with 65% acetonitrile at 20°C. The effluent containing the FR179642 impurity (Formula III) was collected and adjusted to pH 3.0. After nanofiltration and lyophilization, 1.0 g of the compound of Formula III with 80% purity was obtained.

[0103] Step 3:

[0104] Add 20 ml of THF to a 100 mL reaction flask, cool to -18°C, and stir for 1 hour. Add 1.0 g of the compound of formula III and stir until clear. Then, add 0.1 g of triethylamine and 0.5 g of the side chain of micafungin sodium at -10°C, control the temperature at 5°C, and keep the reaction for 1.5 hours. Add 2 g of methanol and stir for 20 minutes. Finally, add 30 ml of ethyl acetate dropwise after about 1 hour. A white solid will precipitate. Filter and dry to obtain 0.8 g of crude micafungin sodium drug impurity.

[0105] 0.8 g of crude micafungin sodium drug impurity product was dissolved in 10 ml of water and ion exchanged using UBK530 resin (50 ml). The collected solution containing impurity components was adjusted to a pH of approximately 5.5 using 0.2 M sodium hydroxide. After enrichment using CHP20 resin, the solution was eluted with 50% methanol in water to obtain a pooled solution with a purity of greater than 90%. Finally, purification was performed using Inertsil HILIC silica gel and eluted with 50% methanol in trifluoroacetic acid (pH 6.0). Fractions with a purity of greater than 90% were collected and concentrated and lyophilized to yield 0.6 g of micafungin drug impurity (Formula I) as a white powder with a purity of 95.8%.

[0106] Example 5:

[0107] Step 1:

[0108] With reference to patent CN102618604B (Shanghai Tianwei), Coleophoma sp is used as the fermentation strain, and the main components of the culture medium include cottonseed meal, mannitol, sodium glutamate, and proline. The fermentation liquid containing FR901379 impurity components is obtained at 23-28 ° C for 6-8 days. 30L of fermentation liquid is obtained, and about 50g of pure FR901379 is obtained. 30L of methanol / ethanol mixed solution (1:1) is added for extraction. After stirring for 2h, the mixture is evenly mixed and filtered. The filtrate is diluted with water to an organic solvent ratio of 50%, then adsorbed using CHP20 resin, eluted using 90% acetonitrile, and concentrated after enrichment to obtain a collected liquid containing FR901379 impurities (Formula II structure). The pure Formula II structure is about 2.0g.

[0109] Step 2:

[0110] The aqueous solution containing the FR901379 impurity (Formula II) collected from Step 1 was diluted to 2 L. 1 L of pH 6.0 phosphate buffer and 20 g of immobilized acyltransferase were slowly added and reacted at 40°C for 3 hours. Adsorption was performed using UniPS50 (3 L) resin. The mixture was washed with 30% acetonitrile and then eluted with 70% acetonitrile at 22°C. The effluent containing the FR179642 impurity (Formula III) was collected and the pH was adjusted to 6.0. After nanofiltration and lyophilization, 1.1 g of the compound of Formula III with 86% purity was obtained.

[0111] Step 3:

[0112] Add 20 ml of DMF to a 100 mL reaction flask, cool to -15°C, and stir for 1 hour. Add 1.1 g of the compound of formula III and stir until clear. Then, add 0.1 g of triethylamine and 0.6 g of the side chain of micafungin sodium at -10°C. Incubate at 0°C for 1.5 hours. Add 50 ml of ethyl acetate dropwise to precipitate a white solid. Filter and dry to obtain 0.9 g of crude micafungin sodium drug impurity.

[0113] 0.9 g of crude micafungin sodium drug impurity product was dissolved in 10 ml of water and ion exchanged using UBK530 resin (50 ml). The collected solution containing impurity components was adjusted to pH 5.5 using 0.2 M sodium hydroxide. After enrichment using HP20SS resin, the solution was eluted with 80% methanol in water to obtain a pooled solution with a purity of 90% or greater. Finally, purification was performed using Chromatorex ARG Silica Fuji silica gel and eluted with 50% methanol in trifluoroacetic acid (pH 6.0). Fractions with a purity of 90% or greater were collected and concentrated and lyophilized to obtain 0.6 g of micafungin drug impurity (Formula I) as a white powder with a purity of 98.1%.

[0114] Take the sample prepared in Example 2 for analysis:

[0115] FIG1 is a hydrogen nuclear magnetic resonance spectrum of the echinocandin drug impurities in Example 2 ( 1 H NMR), the results are as follows:

[0116] 1 H NMR (DMSO- d6,400MHz)δ:0.91(t,J=8.0Hz,3H),1.09(d,J=8.0Hz,3H),1.35-1.45(m,4H),1.75-2.08(m,6H),2.04-2.10(m,1H),2.20-2.2 5(m,2H),2.53-2.60(m,1H),3.60-3.75(m,3H),3.88-3.97(m,3H),4.08(t,J=8Hz,2H),4.14(d,J=12Hz,1H),4.19-4.23(m,4 H),4.30-4.37(m,3H),4.49-4.51(m,1H),4.78-4.81(m,2H),4.92-4.99(m,2H),5.04-5.08(m,3H),5.18(J=8.0Hz,1H),5.20 -5.24(m,2H),5.55(d,J=8.0Hz,1H),6.75(d,J=12.0Hz,2H),6.83-6.87(m,2H),7.06(d,J=8.0Hz,1H),7.13(d,J=8.0Hz,2H), 7.26(b,1H),7.26(b,1H),7.34(d,J=8.0Hz,1H),7.39(b,1H),7.56(s,1H),7.86(d,J=8.0Hz,2H),8 .01(d, J=8.0Hz,2H),8.06-8.12(m,3H),8.27(d,J=8.0Hz,1H),8.85(s,1H),8.90(d,J=8.0Hz,1H).

[0117] FIG2 is a carbon NMR spectrum of the echinocandin drug impurities in Example 2 ( 13 C NMR), the results are as follows:

[0118] 13C NMR(DMSO-d6,400MHz)δ:14.39,19.76,22.35,28.13,28.74,33.77,34.01,37.84,3 8.75,45.73,51.52,54.03,54.51,56.30,57.34,61.35,66.79,68.23,68.50,69.66 69.66,69.86,72.27,73.01,73.31,75.51,97.75,115.67,117.12,119.75,122.52,124.24,126.82,127.80,128.90,131.79,134.30,135.64,140.84,148.95,160.93,162.40,166.09,168.34,169.91,170.47,170.60,170.94,170.94,172.09,173.35.

[0119] Figure 3 is a high resolution mass spectrometry (HRMS) of the echinocandin drug impurities in Example 2, HRMS (ESI): calcd.For C 55 H 68 N9O 23 Na2S(M+Na+):1300.3930,Found:1300.3939.

[0120] FIG4 is an HPLC spectrum of the echinocandin drug impurities in Example 2.

[0121] Based on the above test results, it can be seen that Example 2 prepared the high-purity echinocandin drug impurity represented by Formula I.

[0122] The above detection method was used to detect the products of the remaining examples. The test results showed that high-purity echinocandin drug impurities with a purity greater than 90% were prepared in each example.

[0123] Comparative Example 1:

[0124] Step 1:

[0125] Referring to patent CN102618604B (Shanghai Tianwei), Coleophoma sp was used as the fermentation strain, and the main components of the culture medium included cottonseed meal, mannitol, sodium glutamate, and proline. The culture was carried out at 23-28°C for 6-8 days to obtain 30 L of fermentation broth containing FR901379 impurity components, and about 50 g of pure FR901379 was obtained. The mixture was directly filtered and concentrated at 10°C to obtain a collected liquid containing FR901379 impurities (Formula II structure), and about 0.5 g of the pure structure of Formula II was obtained.

[0126] Step 2:

[0127] The aqueous solution containing the FR901379 impurity (Formula II) collected from Step 1 was diluted to 2 L. 1 L of pH 7.0 phosphate buffer and 20 g of immobilized acyltransferase were slowly added and reacted at 40°C for 3 hours. Adsorption was performed using UniPS50 (3 L) resin, followed by washing with 30% acetonitrile and elution with 70% acetonitrile at 31°C. The effluent containing the FR179642 impurity (Formula III) was collected and the pH was adjusted to 7.0. After nanofiltration and lyophilization, 0.3 g of the compound of Formula III with a 50% purity was obtained.

[0128] Step 3:

[0129] Add 10 ml of DMF to a 100 mL reaction flask, cool to -15°C, and stir for 1 hour. Add 0.1 g of the compound of formula III and stir until clear. Then, add 0.1 g of triethylamine and 0.6 g of the side chain of micafungin sodium at -10°C. Incubate at 0-5°C for 1.5 hours. Add 50 ml of ethyl acetate dropwise to precipitate a white solid. Filter and dry to obtain 0.4 g of crude micafungin sodium drug impurity.

[0130] 0.4 g of crude micafungin sodium impurity product was dissolved in 10 ml of water and ion exchanged using UBK530 resin (50 ml). The collected solution containing the impurity components was adjusted to pH 7.0 using 0.2 M sodium hydroxide. After enrichment using HP20SS resin, the solution was eluted with 80% methanol in water to obtain a pooled solution. Finally, the solution was purified using Click XiON silica gel and eluted with 60% methanol in hydrochloric acid (pH 4.0) to collect the impurity components primarily containing micafungin sodium. The solution was concentrated at 25°C and freeze-dried to yield 0.11 g of pure micafungin impurity (Formula I) as a white powder with a purity of 52%.

[0131] Comparative Example 2:

[0132] Step 1:

[0133] Referring to patent CN102618604B (Shanghai Tianwei), Coleophoma sp was used as the fermentation strain, and the main components of the culture medium included cottonseed meal, mannitol, sodium glutamate, and proline. The culture was carried out at 23-28 ° C for 6-8 days to obtain 30L of fermentation broth containing FR901379 impurity components, about 50g of pure FR901379, and 30L of methanol / ethanol mixed solution (1:1) was added for extraction. After stirring for 2h to mix evenly, the mixture was filtered. The filtrate was diluted with water to an organic solvent ratio of 50%, then adsorbed using HP20 resin, eluted using 90% acetonitrile, and concentrated after enrichment to obtain a collected liquid containing FR901379 impurities (Formula II structure), and the pure Formula II structure was about 2.0g.

[0134] Step 2:

[0135] The aqueous solution containing the FR901379 impurity (Formula II) collected in the above step 1 was diluted to 2 L, and sodium bicarbonate was slowly added for hydrolysis. No compound with the structure of Formula III was detected by HPLC.

[0136] Comparative Example 3:

[0137] Step 1:

[0138] With reference to patent CN102618604B (Shanghai Tianwei), Coleophoma sp is used as the fermentation strain, and the main components of the culture medium include cottonseed meal, mannitol, sodium glutamate, and proline. The fermentation liquid containing FR901379 impurity components is obtained at 23-28 ° C for 6-8 days. 30L of fermentation liquid is obtained, and about 50g of pure FR901379 is obtained. 30L of methanol / ethanol mixed solution (1:1) is added for extraction. After stirring for 2h, the mixture is evenly mixed and filtered. The filtrate is diluted with water to an organic solvent ratio of 50%, then adsorbed using CHP20 resin, eluted using 90% acetonitrile, and concentrated after enrichment to obtain a collected liquid containing FR901379 impurities (Formula II structure). The pure Formula II structure is about 2.0g.

[0139] Step 2:

[0140] The aqueous solution containing the FR901379 impurity (Formula V) collected from Step 1 was diluted to 2 L. 1 L of pH 7.0 phosphate buffer and 20 g of immobilized acyltransferase were slowly added and reacted at 40°C for 3 hours. Adsorption was performed using UniPS50 (3 L) resin, followed by washing with 30% acetonitrile and elution with 70% acetonitrile at 20°C. The effluent containing the FR179642 impurity (Formula III) was collected and the pH was adjusted to 6.0. After nanofiltration and lyophilization, 1.1 g of the compound of Formula III with 85% purity was obtained.

[0141] Step 3:

[0142] Add 20 ml of DMF to a 100 mL reaction flask, cool to -15°C, and stir for 1 hour. Add 1.1 g of the compound of formula III and stir until clear. Then, add 0.2 g of triethylamine and 0.6 g of the side chain of micafungin sodium at -10°C. Incubate at 0-5°C for 1.5 hours. Add 50 ml of ethyl acetate dropwise to precipitate a white solid. Filter and dry to obtain 0.4 g of crude micafungin sodium drug impurity.

[0143] 0.4 g of crude micafungin sodium impurity was dissolved in 10 ml of water and purified using Click XiON silica gel. Elution was performed using 60% methanol in water to collect the fraction primarily containing the micafungin sodium impurity. The fraction was concentrated at 30°C and freeze-dried to yield 0.1 g of a pale yellow powder of 47% pure micafungin impurity (Formula I).

[0144] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. An echinocandin drug impurity, characterized in that: The structure of the echinocandin drug impurity is shown in Formula I, and the purity of the echinocandin drug impurity is greater than 90%; 2. The method for preparing the echinocandin drug impurities according to claim 1, characterized in that: The steps include: Step 1) adding an organic solvent to a microbial fermentation liquid containing a compound of Formula II, mixing the mixture and filtering the mixture, and enriching the filtrate with a first adsorption resin to obtain a compound of Formula II; Step 2) reacting the enriched compound of formula II with an acyltransferase to obtain a compound of formula III; Step 3) reacting the obtained compound of formula III with the side chain of micafungin sodium to obtain the echinocandin drug impurity.

3. The method for preparing the echinocandin drug impurities according to claim 2, characterized in that: The microbial fermentation liquid in step 1) is obtained by fermentation of Coleophoma sp.; Optionally, the organic solvent is selected from at least one of methanol, ethanol, propanol, acetone and butanone; Optionally, the volume ratio of the organic solvent to the fermentation liquid is 1%-40%.

4. The method for preparing the echinocandin drug impurities according to claim 2, characterized in that: The first adsorption resin is a non-polar aromatic adsorption resin formed by polymerization of styrene and divinylbenzene; Optionally, the first adsorption resin is at least one of AD-1, HP20, HP20SS, HP30, SP-825, SP-850, SP207, and CHP20.

5. The method for preparing the echinocandin drug impurities according to claim 2, characterized in that: In the step 2), the enriched compound of formula II is reacted with acyltransferase for purification, and the purification step comprises: adsorption using a second adsorption resin and elution using an organic solvent aqueous solution at 10-30°C.

6. The method for preparing the echinocandin drug impurities according to claim 5, characterized in that: The second adsorption resin is a macroporous adsorption resin, and adopts at least one of the resins SP207, SP207SS, UniPS10, UniPS20, and UniPS50 with styrene and divinylbenzene as the matrix; Optionally, the organic solvent in the organic solvent aqueous solution is selected from at least one of methanol, ethanol, acetone and acetonitrile; Optionally, the volume percentage of the organic solvent in the organic solvent aqueous solution is 1%-90%; Optionally, the elution temperature is 15-25°C; Optionally, the acyltransferase is an immobilized acyltransferase supported by a carrier.

7. The method for preparing the echinocandin drug impurities according to claim 2, characterized in that: The compound of formula III reacts with the side chain of micafungin sodium at -20 to 10° C.; Optionally, the compound of formula III is reacted with the side chain of micafungin sodium in an organic solvent, and the organic solvent is selected from at least one of toluene, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, tetrahydrofuran, and dichloromethane.

8. The method for preparing the echinocandin drug impurities according to claim 2, characterized in that: After the reaction between the compound of formula III and the side chain of micafungin sodium is completed, purification is performed, wherein the purification is performed using ion exchange resin, macroporous adsorption resin, and silica gel in sequence; Optionally, the ion exchange resin is a strongly acidic cation exchange resin, and the ion exchange resin is selected from at least one of UBK530, UBK550, and UBK555; The macroporous adsorption resin is selected from at least one of HP20, HP20SS, SP70, SP700, SP825L, SP850, CHP20 and CHP55; The silica gel is selected from at least one of pure silica gel, amino-bonded silica gel, glycol-bonded silica gel and amide-bonded silica gel.

9. The method for preparing the echinocandin drug impurities according to claim 8, characterized in that: The silica gel purification steps used include: using a silica gel column for adsorption, and then using an acidic organic solvent aqueous solution with a pH value of 2.0-7.0 for elution; Optionally, the volume percentage of organic solvent to water in the acidic organic solvent aqueous solution is 20%-100%; Optionally, the organic solvent in the acidic organic solvent aqueous solution is selected from one of methanol, ethanol, acetone and acetonitrile; Optionally, the acidic organic solvent aqueous solution contains at least one of formic acid, acetic acid, hydrochloric acid, phosphoric acid, and trifluoroacetic acid; Optionally, the pH value of the acidic organic solvent aqueous solution is 3.0-5.

0.

10. Use of the echinocandin drug impurity according to claim 1 in the quality control of echinocandin drugs.

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