Echinocandin drug impurity, preparation and purification method and application thereof

Abstract

The application provides a echinocandin drug impurity, a preparation and purification method thereof and application. The echinocandin drug impurity has a structure shown in formula I: the echinocandin drug impurity is obtained by reacting micafungin sodium and a protonic acid aqueous solution, and high-purity echinocandin drug impurity is obtained after chromatographic purification. The echinocandin drug impurity obtained by the application can be used for quality control of echinocandin drugs as a control sample for establishing an analysis method.

Description

Field of Invention:

[0001] This invention relates to the field of pharmaceutical technology, and provides an echinocandin drug impurity, its preparation, purification method, and application. Background technology:

[0002] Micafungin sodium is a new generation of echinocandin antifungal drug, mainly used to treat fungemia caused by Aspergillus and Candida, respiratory fungal infections, and gastrointestinal fungal infections. It is also used to prevent Aspergillus and Candida infections in hematopoietic stem cell patients. It is a type of echinocandin antifungal drug that inhibits the synthesis of β(1,3)-glucan in filamentous fungi and yeasts.

[0003] In recent years, the incidence and mortality rates of fungal infections have been on the rise, posing a particularly deadly threat to critically ill patients. Micafungin sodium, a type of echinocandin, is the first-line drug for treating infections caused by Candida or Aspergillus due to its excellent antifungal activity. It is effective in treatment, has minimal impact on human cells, and exhibits low toxicity and high efficacy in clinical practice.

[0004] Echinocandin drug impurity (shown in Formula 1) is a byproduct of micafungin degradation. Through in-depth research on this impurity, it was found that the structure of this impurity is similar to that of micafungin sodium, with multiple chiral centers and multiple functional groups such as peptide bonds. It is extremely unstable to acid, alkali, heat and light. There are currently no literature reports on the preparation method of this impurity.

[0005] Impurity analysis is crucial in drug development. Impurities are closely related to the quality, safety, and stability of drugs. Preparing and structurally confirming impurities can reveal their formation pathways, providing a basis for improving drug synthesis routes and manufacturing processes. Impurities can be used in drug quality control, serving as reference standards for establishing analytical methods. For pharmaceutical manufacturers, strict control over impurities is essential. Research on impurities in echinocandins can be used for the qualitative and quantitative analysis of impurities in micafungin sodium production, thereby providing technical support for subsequent quality studies of micafungin sodium and improving its quality standards.

[0006] Therefore, there is an urgent need to provide an efficient and convenient method for preparing echinocandin drug impurities (as shown in Formula 1). Summary of the Invention

[0007] The inventors conducted in-depth research on the preparation process of impurities in echinocandin drugs. Referring to the literature [Journal of Synthetic Organic Chemistry 2006, 64, 12] and patents [US 6107458, US 7199248], during the preparation of micafungin sodium, an unknown impurity was found at position RRT 1.1 in the HPLC spectrum. The peak area ratio of this impurity to micafungin sodium was 0.2%. Furthermore, this impurity could not be effectively removed during the subsequent recrystallization stage, and a certain proportion of this unknown compound remained. During the experiment, it was unexpectedly discovered that placing the micafungin sodium sample in an acidic aqueous solution showed a tendency for the content of this unknown compound impurity to increase. Therefore, this invention was conceived, and a synthetic experiment was designed to react micafungin sodium with acid to prepare this impurity.

[0008] The inventors used micafungin sodium as a raw material and reacted it with hydrochloric acid aqueous solution at room temperature, obtaining an unknown impurity, RRT 1.1, with a low purity of only 3%. Through extensive experiments, the inventors optimized the reaction conditions and finally determined the optimal process for synthesizing the unknown impurity, RRT 1.1. When using column chromatography to purify the impurity, it was found that the impurity was extremely prone to degradation during purification, making purification very difficult. Through optimization of purification conditions, the optimal purification process was determined, yielding the unknown impurity, RRT 1.1, with an HPLC purity of over 90%. Structural identification of this unknown impurity confirmed it to be the compound shown in Formula 1. The structural difference between it and micafungin sodium was found to be that the hydroxyl group at C-21 has an S configuration, while the hydroxyl group at C-21 of micafungin sodium has an R configuration. This impurity can be generated under acidic conditions during the preparation process, and analysis showed that the generation pathway of this impurity is the racemic product of the C-21 hydroxyl group under acidic conditions.

[0009]

[0010] The purpose of this invention is to provide an echinocandin drug impurity, its preparation, purification method, and application.

[0011] The preparation method of this invention uses readily available commercially available raw and auxiliary materials. Using commercially available micafungin as the starting material, it was unexpectedly discovered that adjusting the pH to an acidic environment significantly improved the HPLC purity of the echinocandin drug impurity in the reaction solution. This invention also provides a further purification method. During the purification process, macroporous adsorption resin and silica gel column purification are used sequentially, further improving the HPLC purity of the echinocandin drug impurity in the reaction solution, reaching a maximum HPLC purity of over 90%, providing a new approach for industrial production. The requirement that the purity of the drug impurity reference standard reach over 90% is a well-known requirement in the art. This invention is the first to prepare an echinocandin drug impurity with a purity of over 90%, which can be used as a drug quality control reference standard.

[0012] The echinocandin drug impurity provided by this invention has the chemical name: 5-[(1S,2S)-2-[(3S,6S,9S,11R,15S,18S,20R,21S,24S,25S,26S)-3-[(R)-2-carbamoyl-1-hydroxyethyl]-11,20,21,25-tetrahydroxy-15-[(R)-1-hydroxyethyl]-26-methyl-2,5,8,14,17,23-hexaoxo-18-[4-[5-(4-pentoxyphenyl)isoxazol-3-yl]benzoylamino]-1,4,7,13,16,22-hexaazatricyclo[22.3.0.09,13]heptadecyl-6-yl]-1,2-dihydroxyethyl]-2-hydroxyphenyl sulfate sodium, and its structure is shown in Formula I.

[0013]

[0014] The preparation method of this invention includes the following steps:

[0015] (a) Prepare an aqueous solution of protic acid and adjust the pH value of the aqueous solution.

[0016] (b) Add micafungin sodium to a protic acid aqueous solution and heat.

[0017] As a further improvement to the above preparation method, the protic acid described in step (a) can donate a proton (H). + ) molecules or ions.

[0018] As a further improvement to the above preparation method, the protic acid aqueous solution in step (a) is selected from any one or a combination of formic acid, sulfuric acid, hydrochloric acid, phosphoric acid, acetic acid, and sodium dihydrogen phosphate; the pH of the solution is adjusted to 1.0-6.0.

[0019] As a further improvement to the above preparation method, the protic acid aqueous solution in step (a) is selected from acetic acid; the pH of the solution is adjusted to 2.0-3.0.

[0020] As a further improvement to the above preparation method, the reaction temperature in step (b) is 10-80℃.

[0021] As a further improvement to the above preparation method, the reaction temperature in step (b) is 20-50℃.

[0022] As a further improvement to the above preparation method, the reaction time in step (b) is 1-48 h.

[0023] As a further improvement to the above preparation method, the reaction time in step (b) is 10-24 h.

[0024] The preferred preparation method for echinocandin drug impurities provided by this invention results in a reaction product with an HPLC purity greater than 20%.

[0025] The present invention also provides a further purification method to obtain high-purity echinocandin drug impurities by column chromatography.

[0026] As a further improvement to the above preparation method, the column chromatography purification employed includes macroporous adsorption resin purification and silica gel purification.

[0027] Macroporous adsorption resin purification specifically includes the following steps:

[0028] (a) The reaction products were adsorbed using a macroporous adsorption resin;

[0029] (b) Elution with an organic solvent to obtain a collected solution;

[0030] (c) The liquid is then eluted with an organic solvent to obtain a collection solution, which is then concentrated at low temperature.

[0031] As a further improvement to the above purification method, the macroporous adsorption resin used in step (a) is based on a styrene-divinylbenzene framework.

[0032] As a further improvement to the above purification method, the macroporous adsorption resin used in step (a) includes HP20, HP20SS, HP21, SP70, SP700, SP825L, SP850, CHP20, CHP55 or a mixture thereof, with HP20SS macroporous adsorption resin being preferred in this invention.

[0033] As a further improvement to the above purification method, the organic solvent in step (b) is selected from methanol, ethanol, propanol, butanol, acetone, acetonitrile, or mixtures thereof.

[0034] As a further improvement to the above purification method, the volume percentage of the organic solvent in step (b) is 0%-50% relative to the total volume of the eluent.

[0035] As a further improvement to the above purification method, the volume percentage of the organic solvent in step (b) is 0%-40% relative to the total volume of the eluent.

[0036] As a further improvement to the above purification method, the volume percentage of the organic solvent in step (c) is preferably 70%-100%.

[0037] As a further improvement to the above purification method, the volume percentage of the organic solvent in step (c) is preferably 70%-95%.

[0038] As a further improvement to the above purification method, the purification temperature is controlled at 10-30℃.

[0039] As a further improvement to the above purification method, the purification temperature is controlled at 15-25℃.

[0040] As a further improvement to the above purification method, the flow rate is 0.1-10 column volumes per hour.

[0041] As a further improvement to the above purification method, the flow rate is 0.5-2 column volumes per hour.

[0042] As a further improvement to the above purification method, the low-temperature concentration temperature in step (c) is 5-25℃, to obtain a concentrated solution containing the drug impurity component of echinocandin.

[0043] Silica gel purification specifically includes the following steps:

[0044] (a) After diluting the above concentrate, prepare the sample loading solution and adsorb it through a silica gel column.

[0045] (b) Elution was performed using an acidic aqueous solution of an organic solvent;

[0046] As a further improvement to the above purification method, the silica gel is silica gel with hydrophilic interaction chromatography mode (HILIC).

[0047] As a further improvement to the above purification method, the proportion of organic solvent in the sample loading solution in step (a) shall not exceed 5%.

[0048] As a further improvement to the above purification method, the silica gel is selected from... ChromatorexARG Silica, Click XIon, Inertsil or a mixture thereof.

[0049] 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.

[0050] As a further improvement to the above purification method, the volume percentage of organic solvent to water is 20%-100%.

[0051] As a further improvement to the above purification method, the volume percentage of organic solvent to water is 50%-100%.

[0052] As a further improvement to the above purification method, the acidic solution is selected from aqueous solutions of formic acid, acetic acid, hydrochloric acid, phosphoric acid, trifluoroacetic acid, or mixtures thereof.

[0053] As a further improvement to the above purification method, the pH value of the acidic solution is 2.0-7.0.

[0054] As a further improvement to the above purification method, the pH value of the acidic solution is 3.0-5.0.

[0055] As a further improvement to the above purification method, the HPLC purity of the echinocandin drug impurity obtained after purification by chromatography column is above 90%.

[0056] The collected liquid components were concentrated at a temperature below 20°C and then freeze-dried to obtain the finished product of the echinocandin drug impurity.

[0057] A high-purity echinocandin drug impurity with a structure of Formula I can be used for the quality control of echinocandin drugs and as a reference standard for establishing analytical methods. High-purity echinocandin drug impurities refer to those with an HPLC purity of 90% or higher. Attached Figure Description

[0058] Figure 1 This is the 1H NMR spectrum of the echinocandin drug impurity in Example 5. 1 H NMR).

[0059] Figure 2 This is the carbon NMR spectrum of the echinocandin drug impurity in Example 5. 13 (C NMR).

[0060] Figure 3 This is a high-resolution mass spectrum (HRMS) of the echinocandin drug impurities in Example 5.

[0061] Figure 4 This is the HPLC chromatogram of the echinocandin drug impurity in Example 5. Detailed Implementation

[0062] The present invention will be further described below with reference to specific embodiments.

[0063] Comparative Example 1

[0064] Referring to the literature [Journal of Synthetic Organic Chemistry 2006, 64, 12] and patents [US6107458, US 7199248], 100 g of micafungin sodium side chain was dissolved in 2.0 L of DMF, cooled to 0 °C, and 22.3 ml of DIPEA was added. After stirring for 5 min, 100 g of FR-179642 was added, and the reaction was maintained at this temperature for 1.5 h. 1.0 L of methanol / acetone (V:V = 1:2) was added, followed by 12 L of ethyl acetate. After stirring for 12 h, the mixture was filtered to obtain the micafungin compound. Purification with UBK resin yielded a drug impurity containing 0.2% echinocandin. The collected solution was then crystallized by HP20SS column chromatography to obtain micafungin sodium. The purity of the echinocandin drug impurity in the HPLC chromatogram was 0.1%.

[0065] Example 1

[0066] Add 100 mL of water to the reaction flask and slowly add concentrated hydrochloric acid until the pH reaches 1.0. Add 1.0 g of micafungin sodium and stir until the solid dissolves. Control the temperature of the reaction solution at 45 °C and stir for 24 hours. Take a sample for analysis. The HPLC purity of the echinocandin drug impurity in the reaction solution is 3%.

[0067] Example 2

[0068] Add 100 mL of water to the reaction flask and slowly add concentrated sulfuric acid until the pH reaches 2.0. Add 1.0 g of micafungin sodium and stir until the solid dissolves. Control the temperature of the reaction solution at 50 °C and stir for 24 hours. Take a sample for analysis. The HPLC purity of the echinocandin drug impurity in the reaction solution is 8%.

[0069] Example 3

[0070] Add 100 mL of water to the reaction flask, slowly add phosphoric acid until the pH reaches 4.0, add 1.0 g of micafungin sodium and stir until the solid dissolves. Control the temperature of the reaction solution at 55 °C and stir for 24 hours. Take a sample for analysis. The HPLC purity of the echinocandin drug impurity in the reaction solution is 12%.

[0071] Example 4

[0072] Add 100 mL of water to the reaction flask, slowly add acetic acid until the pH reaches 3.0; add 1.0 g of micafungin sodium and stir until the solid dissolves. Control the temperature of the reaction solution at 45 °C and stir for 36 hours. The HPLC purity of the echinocandin drug impurity in the reaction solution is 23%.

[0073] Example 5

[0074] 100 mL of water was added to the reaction flask, and acetic acid was slowly added dropwise until the pH reached 2.8. 1.0 g of micafungin sodium was added and stirred until the solid dissolved. The reaction solution temperature was controlled at 50 °C, and stirring was performed for 24 hours. The HPLC purity of the echinocandin drug impurity in the reaction solution was 21%. After the reaction, the solution was first adsorbed using 20 mL of HP20ss resin, washed with 40 mL of 50% methanol, and eluted with 60 mL of 90% methanol. The solution was collected, concentrated by vacuum distillation, and a collected solution containing the echinocandin drug impurity was obtained. Then, Click Xion silica gel chromatography was performed, eluted with a 60% acetonitrile aqueous solution at approximately pH 4.0, yielding the echinocandin drug impurity with an HPLC purity of 92%. After lyophilization, 30 mg of a white solid was obtained.

[0075] 1 H NMR (DMSO- d6,400MHz)δ:0.91(t,J=8.0Hz,3H),0.99(d,J=8.0Hz,3H),1.27(d,J=4.0Hz,3H),1.29-1.45(m,4H),1.73-1.77(m,3H),1.87-1.93(m,2H),2.20-2.30(m,4H),3.22(t,J=4.0Hz,1H),3.58-3.60(m,1H),3.78(s,2H),3.94(m,1H),4.06-4.12(m,5H),4.22(m,2H),4.36-4.41(m,3H),4.59(m,2H),4.86-4.88(m,2H),4.98(s,1H),5.21-5.30(m,3H),5.33(d,J=4.0Hz,1H),5.64(d,J=4.0Hz,1H),6.76(d,J=8.0Hz,1H),6.85(d,J=8.0Hz,1H),6.99(s,1H),7.05(s,1H),7.08-7.20(m,4H),7.45(b,1H),7.55(b,2H),7.84(d,J=8.0Hz,2H),8.00-8.04(m,4H),8.46(d,J=8.0Hz,1H),8.86(s,1H).

[0076] 13 C NMR(DMSO- d6 ,400MHz)δ:11.30,14.39,19.74,22.35,28.13,28.74,33.37,37.62,37.62,38.61,49.07,51.60,54.78,56.80,57.11,57.66,60.54,66.30,68.23,69.20,69.20,69.79,72.92,73.86,74.01,74.77,75.71,97.73,115.67,115.67,117.25,119.76,122.20,123.87,126.85,126.85,127.79,127.79,128.60,128.60,131.59,134.11,136.05,140.91,148.96,160.91,162.43,165.47,169.23,169.34,170.56,170.56,171.70,171.90,171.90,172.95.

[0077] HRMS(ESI):calcd.For C 56 H 70 O23 N9Na2S(M+Na + ):1314.41089,Found:1314.4095.

[0078] Table 1. Composition of Impurities in Echinocandin Drug HPLC Spectra in Example 5

[0079]

[0080] Example 6

[0081] 100 mL of water was added sequentially to the reaction flask, and formic acid was slowly added dropwise until the pH reached 3.0. 1.0 g of micafungin sodium was added and stirred until the solid dissolved. The reaction solution temperature was controlled at 45°C, and stirring was performed for 48 hours. The HPLC purity of the echinocandin drug impurity in the reaction solution was 20%. After the reaction, the solution was first adsorbed using 20 mL of HP20ss resin, washed with 40 mL of 30% methanol, and eluted with 60 mL of 85% methanol. The solution was collected, concentrated by vacuum distillation, and a collected solution containing the echinocandin drug impurity was obtained. Then, the solution was separated by Chromatorex ARG Silica silica gel chromatography, eluted with 70% acetonitrile aqueous solution at approximately pH 6.0, yielding the echinocandin drug impurity with an HPLC purity of 90%. After lyophilization, 60 mg of a white solid was obtained.

[0082] Example 7

[0083] 100 mL of water was added sequentially to the reaction flask, followed by slow dropwise addition of acetic acid until the pH reached 3.0. 1.0 g of micafungin sodium was added and stirred until the solid dissolved. The reaction mixture was kept at 45°C and stirred for 24 hours. The HPLC purity of the echinocandin drug impurity in the reaction solution was 8%. After the reaction, the mixture was first adsorbed using 20 mL of SP207 resin, washed with 40 mL of 1% ethanol, and eluted with 60 mL of 95% propanol. The solution was collected, concentrated by vacuum distillation, and a collected solution containing the echinocandin drug impurity was obtained. This solution was then separated by Inertsil silica gel chromatography, eluted with a 60% acetonitrile aqueous solution at approximately pH 3.0, yielding the echinocandin drug impurity with an HPLC purity of 91%. After lyophilization, 30 mg of a white solid was obtained.

[0084] Example 8

[0085] Add 100 mL of water to the reaction flask sequentially, then slowly add phosphoric acid dropwise until the pH reaches 4.0. Add 1.0 g of micafungin sodium and stir until the solid dissolves. Maintain the reaction temperature at 80°C and stir for 48 hours. The HPLC purity of the echinocandin drug impurity in the reaction solution is 12%. After the reaction, adsorption is performed using 20 mL of HP20SS resin, followed by washing with 40 mL of 50% methanol and elution with 70 mL of 95% ethanol. Collect the solution, distill under reduced pressure, and concentrate to obtain a collected solution containing the echinocandin drug impurity. Then use... The drug impurity, echinocandin, was separated by silica gel chromatography and eluted with a 60% acetonitrile aqueous solution at a pH of approximately 3.0. The HPLC purity was 91%, and after lyophilization, 32 mg of a white solid was obtained.

Claims

1. A method for preparing a high-purity echinocandin drug impurity, characterized in that: The structure of the echinocandin drug impurity is shown in Formula I: the HPLC purity of the high-purity echinocandin drug impurity reaches over 90%. Formula I The preparation method includes: 100 mL of water was added to the reaction flask, and acetic acid was slowly added dropwise until the pH reached 2.

8. 1.0 g of micafungin sodium was added and stirred until the solid dissolved. The reaction solution temperature was controlled at 50 °C, and the mixture was stirred for 24 hours. The HPLC purity of the echinocandin drug impurity in the reaction solution was 21%. After the reaction, the mixture was first adsorbed using 20 mL of HP20ss resin, washed with 40 mL of 50% methanol, and eluted with 60 mL of 90% methanol. The mixture was collected, distilled under reduced pressure, and concentrated to obtain a collection solution containing the echinocandin drug impurity. Then, ClickXion silica gel was used for chromatography separation, and eluted with 60% acetonitrile aqueous solution at a pH of approximately 4.0 to obtain the echinocandin drug impurity with an HPLC purity of 92%.

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