Stable AST-3424 raw material medicine ethanol composition solution

Abstract

< TableDetails number = '0001' > < Title / > < tbl: tgroup cool = '2' > < tbl: colspec cool = 'c001' cool = '46%' / > < tbl: colspec cool = 'c002' cool = '54%' / > < tbl: cool > < tbl: cool = '1' > impurity < / tbl: cool > < tbl: cool = '1' > HPLC percentage content < / tbl The mobile phase B is a 8mmol / L ammonium acetate solution, and the solvent is a mixed solvent of acetonitrile and water in a volume ratio of 95: 5; and carrying out gradient elution at a flow rate of 1.0 ml / min.

Description

TECHNICAL FIELD

[0001] The present application relates to the raw material of the compound AST-3424 disclosed in patent application PCT / US2016 / 021581, publication number WO2016145092A1, corresponding Chinese application number 2016800150788, publication number CN107530556A, belongs to the field of medicine research and development, and particularly relates to the quality control of the raw material. BACKGROUND

[0002] The DNA alkylating cancer treatment drug AST-3424 developed by our company, which targets overexpression of aldehyde ketone reductase 1C3 (AKR1C3) (see patent application: DNA alkylating agent, corresponding PCT application number PCT / US2016 / 021581, publication number WO2016 / 145092, corresponding Chinese application number 2016800150788, publication number CN107530556A, discloses the compound TH2870; (R)- and (S)-1-(3-(3-N,N-dimethylaminocarbonyl)phenoxy-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)aminophosphonate, compositions and methods of use and preparation thereof, corresponding PCT application number PCT / US2016 / 062114, publication number WO2017087428A1, corresponding Chinese application number 2016800446081, publication number CN108290911A, S-configuration compound), Chinese name (S)-1-(3-(3-N,N-dimethylaminocarbonyl)phenoxy-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)aminophosphonate, also known as OBI-3424, TH-2870 S-configuration compound), CAS number 2097713-69-2, has the following structure:

[0003]

[0004] It has been confirmed by authoritative industry literature (Kathryn Evans, Jian Xin Duan, Tara Pritchard, et al. OBI-3424, a novel AKR1C3-activated prodrug, exhibits potent efficacy against preclinical models of T-ALL [J], Clinical Cancer Research, 2019, DOI: 10.1158 / 1078-0432.CCR-19-0551; Richard B. Lock, Kathryn Evans, Raymond Yung, Tara Pritchard, Beverly A. Teicher, Jian Xin Duan, Yue long Guo, Stephen W. Erickson and Malcolm A. Smith, Abstract LB-B16: The AKR1C3-Activated Prodrug OBI-3424 Exerts Profound In Vivo Efficacy Against Preclinical Models of T-Cell Acute Lymphoblastic Leukemia (T-ALL); a Pediatric Preclinical Testing Consortium Study [C], AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; October 26-30, 2017; Philadelphia, PA, DOI: 10.1158 / 1535-7163.) that the compound is a broad-spectrum small molecule anticancer prodrug, which has efficacy against various solid tumors and hematological tumors.

[0005] In order to carry out subsequent clinical trials, a large amount of AST-3424 bulk drug needs to be prepared to prepare formulations for long-term clinical trials (2-5 years).

[0006] It is found that the substance is a light yellow oily liquid during the synthesis process, and the bulk drug is a viscous, oily semi-liquid under conventional conditions (25°C room temperature, atmospheric pressure), which is quite different from the general solid form of bulk drug, and there are great inconveniences in storage, transportation, packaging, dispensing, metering and other links. SUMMARY

[0007] The inventors' team found that AST-3424 ethanol solution with a mass percentage of 25-45% has long-term stability under certain storage conditions and has suitable fluidity, which facilitates storage, packaging, sub-packaging, metering, and solves the problem of AST-3424 compound being an oily viscous liquid.

[0008] Further, the applicant found that temperature has a direct impact on the storage stability of the above-mentioned drug substance solution: increasing temperature not only increases impurities, but also may detect new impurities, while other factors such as humidity and whether inverted have no obvious effect on the stability of the solution.

[0009] In particular, the inventors' team of the applicant found that the different impurity content ranges under specific storage conditions can ensure that the quality of the above-mentioned AST-3424 ethanol solution meets the preset requirements to a certain extent, i.e., by controlling the impurity profile (a series of impurities and their contents) of the above-mentioned ethanol solution, the quality of AST-3424 drug substance ethanol solution can be indirectly predicted or controlled, i.e., the above-mentioned impurity profile can be a marker for determining whether the quality of AST-3424 drug substance ethanol solution is qualified.

[0010] Specifically, the present application provides the following technical solutions.

[0011] The stable AST-3424 drug substance ethanol composition solution contains AST-3424 drug substance and impurities, and the mass percentage content of AST-3424 drug substance in the solution is 25-45%,

[0012] The composition solution is stored under sealed light-proof conditions at -20±5℃ or lower temperature, and after dilution with acetonitrile, the sample is detected by HPLC, and after calculation by area normalization method, the HPLC chemical purity of AST-3424 drug substance is greater than or equal to 98.0%, the main signal peak of AST-3424 with a peak time of 10.0-14.0 min is taken as a control, and the impurities are represented by RRT, then the composition solution contains the following impurities:

[0013] Impurities HPLC percentage content RRT = 1.05-1.10 ≤0.11％ RRT = 1.11-1.19 ≤0.15％ RRT = 1.21-1.25 ≤0.14％ RRT = 1.25-1.29 ≤0.06％ RRT = 1.44-1.49 ≤0.26％ RRT = 1.50-1.55 ≤0.19％ ;

[0014] or

[0015] The composition solution is stored at 5±3℃ or at 5±3℃ to -20±5℃ in sealed light-avoiding condition, the sample is diluted with acetonitrile and then detected by HPLC, and the HPLC chemical purity of AST-3424 is greater than or equal to 98.0% after area normalization calculation, the main signal peak of AST-3424 with a peak time of 10.0-14.0 min is taken as a control, and the impurities are represented by RRT, and the impurities include the following:

[0016] Impurities HPLC percentage content RRT = 1.05-1.10 ≤0.30％ RRT = 1.11-1.19 ≤0.18％ RRT = 1.21-1.25 ≤0.36％ RRT = 1.25-1.29 ≤0.30％ RRT = 1.44-1.49 ≤0.28％ RRT = 1.50-1.55 ≤0.19％

[0017] wherein,

[0018] The HPLC detection conditions are as follows:

[0019] an ultraviolet detector, a detection wavelength of 230 nm,

[0020] a chromatographic column of Waters Xbridge BEH C8, 150 mm x 4.6 mm, 3.5 μm,

[0021] a column temperature of 15℃,

[0022] mobile phase A is 10 mmol / L ammonium acetate solution, and the solvent is a mixed solvent with a volume ratio of water to acetonitrile of 95:5;

[0023] mobile phase B is 8 mmol / L ammonium acetate solution, and the solvent is a mixed solvent with a volume ratio of acetonitrile to water of 95:5;

[0024] a flow rate of 1.0 ml / min for gradient elution, and the elution gradient is as follows:

[0025] Time (min) 0 5 15 25 30 30.1 35 Volume percentage of mobile phase B 10 30 30 95 95 10 10 .

[0026] In the preparation of AST-3424, the applicant tried to remove the solvents, water and the like used in the preparation and separation process by distillation as much as possible, mixed the oil-like AST-3424 product after separation and purification with various common solvents (ethyl acetate, ethanol, methanol and the like), and then distilled under reduced pressure after heating at a relatively low temperature. It was found that ethanol could almost infinitely dissolve AST-3424, and after the ethanol was distilled off, a thick and sticky oil-like AST-3424 remained. Although methanol and ethyl acetate also showed good solubility in several experiments, methanol was more toxic and ethyl acetate was less toxic, so ethanol was finally determined to be used.

[0027] It is proved in the subsequent accelerated stability study and the stability study under long-term storage conditions that the 25-45% ethanol solution of AST-3424 has good storage stability and suitable high and low temperature impact stability, and can be stored for a long time.

[0028] AST-3424 solution with suitable concentration also has long stability under certain storage conditions. According to the existing experimental data and the inventor's experience, AST-3424 ethanol solution can still maintain good stability and suitable viscosity when the mass percentage is 20-45%.

[0029] Further, since there is ethanol in the AST-3424 ethanol solution, there is a relatively active alcohol hydroxyl group in the molecular structure of ethanol; and there are amide groups and aziridine rings in AST-3424 that can decompose or react, so in the subsequent long-term storage, high temperature (such as conventional 25°C, 5°C, etc.), high humidity, and light, contact with high molecular materials and their monomers in the packaging container, and environmental metal ions existing in the solution, these conditions can all cause new impurities in the storage process of AST-3424 bulk drug.

[0030] Obviously, at any time after production is completed, the detected impurities can be divided into two categories:

[0031] The first type of impurity is the impurity brought about by the production and purification process. These impurities exist after production is completed, and they can be related to the process during production, such as the residues or by-products of raw materials, solvents, and reaction reagents. These impurities have constant or slow changes in their content, and they can generally be reduced by changing or optimizing the production process. Generally speaking, their content is basically unchanged or changes slowly during the subsequent storage process as the storage time increases.

[0032] The second type of impurity usually increases with the extension of the storage time, especially it can be characterized by slow or not obvious growth in the early stage, and then obvious growth after a certain time. This type of impurity is mostly a reactive impurity, i.e., it can be the product of the reaction between the active groups such as amide groups and aziridine rings in AST-3424 and ethanol (including trace amounts of other alcohols, ethers, aldehydes, and acids contained therein) during storage.

[0033] AST-3424 compound is purified by chromatography. After purification, the AST-3424 chemical purity detection result calculated by area normalization method under the condition of 230 nm ultraviolet detector is greater than or equal to 98.0% (see Appendix Figure 2 ), and the remaining 2.00% is various impurities.

[0034] When there is no reference substance for the component to be tested (retention time tR1), another component in the sample or another known component added to the sample can be used as a reference substance (retention time tR2), and the relative retention time can be used as a qualitative (or positioning) method. The relative retention time generally refers to the ratio of the retention time of the component to be tested to the retention time of the main component (RRT = tR1 / tR2).

[0035] Due to different storage temperatures, the types and contents of impurities in the final product are different.

[0036] The composition solution is stored at a temperature of -20±5°C or lower in a sealed and light-protected environment. After dilution with acetonitrile, the sample is detected by HPLC. After calculation by area normalization method, the HPLC chemical purity of AST-3424 bulk drug is greater than or equal to 98.0%. The main signal peak of AST-3424 with an elution time of 10.0-14.0 min is used as a reference, and the impurities are represented by the relative elution time (also known as relative retention time) RRT. The impurities include the following:

[0037]

[0038]

[0039] In fact, since the common HPLC uses a non-chiral chromatographic column that cannot separate AST-3424 from its isomer AST-3423, a portion of the isomer / portion of the structurally similar compound cannot be completely separated in the detection by the common non-chiral HPLC. Therefore, the substances corresponding to the above-mentioned five HPLC chromatographic peaks may be a single impurity or a mixture of multiple impurities. However, after detecting multiple batches and multiple conditions of AST-3424 bulk drug ethanol solution, storing at a temperature of -20±5°C or lower in a sealed and light-protected environment, and detecting by the above-mentioned HPLC method, in addition to the main peak corresponding to AST-3424 bulk drug, the five impurity peaks are completely present (in some cases, other new impurity peaks may appear), so it can be concluded that the five HPLC chromatographic peaks correspond to five impurities or five groups of impurities.

[0040] In the subsequent continuous 24-month detection, it was found that the content of the 5 impurities or 5 groups of impurities changed slowly throughout the time, and some were almost unchanged for a period of time and then the impurity content increased sharply at a certain time, so it can be concluded that the content of the 5 or 5 groups of impurities can be detected to control the quality of AST-3424 raw material ethanol solution: that is, AST-3424 raw material ethanol solution products with impurity content within the above range can be determined as qualified products, otherwise unqualified. Because for the impurities that change slowly throughout the time, exceeding the above threshold range means that the first type of impurities in the batch of products is too high, which is likely to mean that the production process control has a problem; for the impurities that are almost unchanged for a period of time and then the impurity content increases sharply at a certain time, exceeding the above threshold is likely to mean that the temperature is over-temperature during storage, the environment changes (light failure or closed environment is damaged), so even if the subsequent product detection may still meet the qualified standard, this batch of products must still be determined as failed products and cannot be used for subsequent preparation production.

[0041] The composition solution is stored in a sealed light-proof and 5±3℃ condition or in a 5±3℃ to -20±5℃ condition, and after the sample is diluted with acetonitrile and detected by HPLC, the HPLC chemical purity of AST-3424 raw material is greater than or equal to 98.0% after area normalization calculation, and the main signal peak of AST-3424 with a peak time of 10.0-14.0 min is taken as a control, and the impurities are represented by RRT, then the following impurities are contained:

[0042]

[0043] The substances corresponding to the above 6 HPLC chromatographic peaks may be a single impurity or a mixture of multiple impurities. However, after detecting multiple batches and multiple conditions of AST-3424 raw material ethanol solution, and storing in a sealed light-proof and 5±3℃ condition or in a 5±3℃ to -20±5℃ condition, and after detecting by the above HPLC method, in addition to the main peak corresponding to AST-3424 raw material, the 6 impurity peaks / 6 groups of impurity peaks are all completely present (in some cases, other new impurity peaks may appear), so it can be concluded that the 6 HPLC chromatographic peaks correspond to 6 impurities or 6 groups of impurities.

[0044] In the subsequent continuous detection for 12 months, it was found that the content of the 6 impurities or 6 groups of impurities changed slowly and almost unchanged throughout the time, and some changed almost unchanged for a period of time and then the impurity content increased sharply at a certain time, so it can be concluded that the content of the 6 or 6 groups of impurities can be detected to determine whether the quality of AST-3424 raw material ethanol solution is qualified: that is, the AST-3424 raw material ethanol solution product with impurity content within the above range is determined to be a qualified product, otherwise it is unqualified. Because for the impurities that change slowly and almost unchanged throughout the time, exceeding the above threshold range means that the first type of impurities in the batch product is too high, which is likely to mean that the production process control has a problem; for the impurities that change almost unchanged for a period of time and then the impurity content increases sharply at a certain time, exceeding the above threshold is likely to mean that the temperature is overheated during storage, the environment changes (light failure or closed environment is damaged), so even if the subsequent product detection may still meet the qualified standard, this batch of product must still be determined as a failed product and cannot be used for subsequent preparation production.

[0045] The above certain / group of impurities ≤ a certain value actually refers to the HPLC percentage content of a certain / group of impurities between 0 and a certain value, that is, the impurity exists but the HPLC instrument cannot accurately measure (below the detection limit or the quantitative limit) at this time it is determined as 0.

[0046] The mass percentage content of AST-3424 raw material in the solution is 25-45%, which is measured and calculated using the external standard method.

[0047] (1) Single-point correction method

[0048] When the concentration of the component to be tested (cx) changes within a small range, and the approximate concentration of the component is known, a standard solution (cR) close to the content of the component to be tested can be prepared. Under the same HPLC chromatographic conditions as the component to be tested, accurately inject the same volume, measure the peak area or peak height of the control sample solution (AR) and the peak area or peak height of the component to be tested in the test sample solution (Ax), and the concentration of the component to be tested cx = cR x Ax / AR.

[0049] (2) Standard curve method

[0050] When the concentration of the component to be tested changes within a large range and the concentration is unknown, the control sample can be prepared into solutions with different concentrations, accurately injected with the same volume under the same chromatographic conditions as the component to be tested, and the peak area or peak height of each concentration solution was measured. Draw a standard curve with the peak area or peak height as the ordinate and the concentration as the abscissa, measure the peak area or peak height of the sample, and find its corresponding concentration on the standard curve. The slope of the standard curve is the absolute correction factor.

[0051] In this application, the approximate mass percentage of AST-3424 is known, so the single-point correction method is selected for measurement and calculation.

[0052] The chromatographic column is Waters Xbridge BEH C8, 150 mm x 4.6 mm, 3.5 μm, which is a chromatographic column of the Xbridge series of the British Waters Company, the filler is C8, the filler particle size is 3.5 μm, the column body length is 150 mm, and the inner diameter is 4.6 mm.

[0053] Preferably, the composition solution is stored at a sealed light-proof temperature of -20±5℃ or lower, and the impurities are represented by RRT, and the impurities include the following:

[0054]

[0055] or

[0056] The composition solution is stored at a sealed light-proof temperature of 5±3℃ or at a temperature of 5±3℃ to -20±5℃, and the impurities are represented by RRT, and the impurities include the following:

[0057]

[0058] *, which means that the impurity exists, but the HPLC instrument cannot accurately measure (below the detection limit or the quantitative limit) at this time, and it is determined as 0.

[0059] Since the ordinary HPLC uses a non-chiral chromatographic column that cannot separate AST-3424 from its isomer AST-3423, there will be a part of the isomer that cannot be reflected in the detection in the ordinary non-chiral HPLC detection. Therefore, a chiral chromatographic column needs to be used for HPLC detection. Under the condition of a 230 nm ultraviolet detector, the area normalization method calculates the chiral HPLC percentage content of the isomer AST-3423 to be less than or equal to 2.0% (i.e., the enantiomeric excess e.e.%≥96.0%, see Appendix 1). Figure 1

[0060] Preferably, the above-mentioned AST-3424 bulk drug solution is stored at a sealed light-proof temperature of -20±5℃, and the shelf life (validity period) is greater than or equal to 24 months. The 24-month storage experimental data can undoubtedly prove that after 24 months of storage, each quality index of the bulk drug solution, especially the content of the above-mentioned 5 impurities or 5 groups of impurities meets the requirements, and no over-standard impurity or group of impurities is detected, which may involve the risk of process control failure or substandard storage conditions.

[0061] ​Applicant speculates that the shelf life (validity) of the above AST-3424 drug substance ethanol solution is greater than or equal to 36 months based on the existing 24-month storage experiment results: different temperature storage experiments show that the lower the temperature, the slower the impurity growth rate, so by further reducing the temperature, the storage time of the above AST-3424 drug substance ethanol solution can be extended while the impurities and other quality indicators still meet the requirements.

[0062] Applicant speculates that the shelf life (validity) of the above AST-3424 drug substance ethanol solution is greater than or equal to 48 months based on the existing 24-month storage experiment results: different temperature storage experiments show that the lower the temperature, the slower the impurity growth rate, so by further reducing the temperature, the storage time of the above AST-3424 drug substance ethanol solution can be extended while the impurities and other quality indicators still meet the requirements.

[0063] Applicant speculates that the shelf life (validity) of the above AST-3424 drug substance ethanol solution is greater than or equal to 60 months based on the existing 24-month storage experiment results: different temperature storage experiments show that the lower the temperature, the slower the impurity growth rate, so by further reducing the temperature, the storage time of the above AST-3424 drug substance ethanol solution can be extended while the impurities and other quality indicators still meet the requirements.

[0064] Preferably, the mass percentage content of AST-3424 drug substance in the solution is 29.50-39.80%.

[0065] More preferably, the mass percentage content of AST-3424 drug substance in the solution is 29.40-30.90% or 35.80-37.70% or 36.20-39.80% or 35.50-39.30%.

[0066] Preferably, the above AST-3424 drug substance solution is stored at 5±3°C in a sealed light-proof condition, and the shelf life (validity) is greater than or equal to 12 months. The 12-month storage experiment data can undoubtedly prove that after 12 months of storage, each quality indicator in the drug substance solution, especially the content of the above 6 or 6 groups of impurities, meets the requirements, and no over-standard impurity or group of impurities is detected, which may involve the risk of process control failure or non-compliance of storage conditions.

[0067] The stable AST-3424 drug substance ethanol composition solution should meet the regulatory requirements for the limit of residual solvent mass.

[0068] The stable AST-3424 drug substance ethanol composition solution does not add water, and the water content should be controlled during the corresponding production process to be within 1.0% by mass.

[0069] Obviously, no water added means no additional water is added in the preparation process, and other reagents, solvents and the like used are anhydrous grade reagents, such as anhydrous ethanol, and water environment is avoided as much as possible or contact with water is avoided during production, that is, the water content needs to be controlled during the preparation process of the drug substance product of the application, and theoretically the lower the water content, the better the stability, but the research and development team considers the technical difficulty and the realization of industrial mass production, and the water content (determined by Karl Fischer method) is preferably within 1.0% by mass.

[0070] From the above, for the drug substance or compound product determined as AST-3424, it can be determined that the drug substance or compound product contains AST-3424 through the product specification or other analysis and detection methods, and after it is determined that the solvent of the solution is ethanol, the mass percentage content of AST-3424 drug substance in the solution is determined to be 25-45% by HPLC detection and external standard method or other methods, and then the following fixed HPLC method is used:

[0071] an ultraviolet detector with a detection wavelength of 230 nm,

[0072] a chromatographic column of Waters Xbridge BEH C8, 150 mm x 4.6 mm, 3.5 μm,

[0073] a column temperature of 15°C,

[0074] mobile phase A is 10 mmol / L ammonium acetate solution, and the solvent is a mixed solvent with a volume ratio of water to acetonitrile of 95:5;

[0075] mobile phase B is 8 mmol / L ammonium acetate solution, and the solvent is a mixed solvent with a volume ratio of acetonitrile to water of 95:5;

[0076] a flow rate of 1.0 ml / min for gradient elution, and the elution gradient is as follows:

[0077] Time (min) 0 5 15 25 30 30.1 35 Volume percentage of mobile phase B 10 30 30 95 95 10 10

[0078] After detection, area normalization method is used to detect and confirm that the HPLC chemical purity of the following impurities and AST-3424 drug substance is greater than or equal to 98.0% (as the main peak, it is used as a reference, and the peak time is 10.0-14.0 min):

[0079] Impurities HPLC percentage content RRT = 1.05-1.10 ≤0.11％ RRT = 1.11-1.19 ≤0.15％ RRT = 1.21-1.25 ≤0.14％ RRT = 1.25-1.29 ≤0.06％ RRT = 1.44-1.49 ≤0.26％ RRT = 1.50-1.55 ≤0.19％ ;

[0080] or

[0081] Impurities HPLC percentage content RRT = 1.05-1.10 ≤0.30％ RRT = 1.11-1.19 ≤0.18％ RRT = 1.21-1.25 ≤0.36％ RRT = 1.25-1.29 ≤0.30％ RRT = 1.44-1.49 ≤0.28％ RRT = 1.50-1.55 ≤0.19％

[0082] It can be confirmed that the AST-3424 mixture is the stable AST-3424 raw material ethanol composition solution provided by the present application.

[0083] Obviously, the above-mentioned stable AST-3424 raw material ethanol composition solution provided by the present application needs to be designed for corresponding packaging during long-term storage, transportation or sales. Unlike the paper barrel packaging used by the conventional solid form of raw materials, since the AST-3424 raw material product is dissolved in an ethanol solution, it is a suitable concentration of ethanol solution: too high concentration of ethanol solution, its viscosity is large, not easy to transfer; too low concentration means high ethanol content, strong volatility, large product content fluctuation, reduced safety, increased transportation risk. Correspondingly, in order to make it not deteriorate during storage, it needs to be packaged in a specially designed packaging unit.

[0084] The packaging unit includes a container assembly and the above-mentioned stable AST-3424 raw material ethanol composition solution contained in the container, and the container assembly includes a container for containing, and the material of the container is a pharmaceutical grade high-density polyethylene or a medium borosilicate glass.

[0085] Experiments have proved that using high-density polyethylene material as the material directly contacting the raw material product solution is very stable under storage conditions. In the case of using a pharmaceutical grade high-density polyethylene material, it is feasible to use a suitable packaging container to contain the raw material product solution, for example, using a bottle, and then using a bottle cap to tighten and seal.

[0086] Further, the container assembly further includes an outer structure for wrapping the container.

[0087] The outer structure is preferably a low-density polyethylene sealing bag, which is sealed by using a buckle.

[0088] The container is preferably a high-density polyethylene bottle with a sealing film on the mouth and a cap or a cap sealed with a sealing film after the cap is screwed.

[0089] Further, the packaging unit further includes a light shielding structure, which wraps the container.

[0090] Correspondingly, the light shielding structure can be an outer package, that is, an outer package of light shielding material completely wraps the inner package formed by the above-mentioned container and the outer structure. Correspondingly, an aluminum can, a paper barrel, an aluminum foil bag, etc. can be used for outer packaging.

[0091] Preferably, the light shielding structure is an aluminum foil bag, which is heat sealed.

[0092] Further, a preparation method of the above-mentioned raw material product solution is further provided.

[0093] Regardless of the process route used to prepare the AST-3424 compound, the final compound product obtained needs to be purified. Since the AST-3424 compound is a viscous oil under normal conditions, column chromatography separation is used in the prior art, so there are some residual solvents in the compound, such as reaction solvents, column chromatography mobile phase solvents, water, etc. Through experimental exploration, these residual solvents can be removed by adding ethanol to dissolve and then evaporating.

[0094] The method for preparing a stable AST-3424 bulk drug product is to mix the purified AST-3424 product with anhydrous ethanol under the condition that the temperature does not exceed 30°C, and then distill under reduced pressure until the mass percentage of AST-3424 reaches a predetermined value or additional ethanol is added until the mass percentage of AST-3424 reaches a predetermined value.

[0095] Further, the operation of mixing with anhydrous ethanol and distilling under reduced pressure is performed three times, wherein the first two times are reduced pressure concentration until there is no distillate, and the third time is distillation under reduced pressure until the mass percentage of AST-3424 reaches a predetermined value or additional ethanol is added until the mass percentage of AST-3424 reaches a predetermined value.

[0096] Preferably, the pressure of the distillation under reduced pressure is P≤-0.08 MPa.

[0097] In particular, unless otherwise specified, all numerical values in the specification have a range of ±10%, i.e. if a numerical range of 2-3 is specified in the specification, due to the existence of measurement errors, accidental errors, and systematic errors, the actual measurement result of 1.8-3.3 should also be considered to fall within the above-mentioned range of 2-3, which is also acceptable. BRIEF DESCRIPTION OF DRAWINGS

[0098] Figure 1 Chromatogram of chiral HPLC detection in enantiomeric excess detection of a sample of batch 034101 after 3 months of storage in the long-term stability experiment at 5±3℃;

[0099] Figure 2 HPLC detection chromatogram in chemical purity detection of a sample of batch 034101 after 1 month of storage in the long-term stability experiment at 5±3℃;

[0100] Figure 3 Photo of comparison of the viscosity of a 30% AST-3424 ethanol solution and the AST-3424 bulk drug product;

[0101] Figure 4 Impurity content change curve of batch 03-03P-01-37-01 AST-3424 bulk drug stored at 5℃±3℃ / -20℃±5℃, light-protected, upright, for different periods of time;

[0102] Figure 5 Impurity profile of 01-03-02-41-01 batch AST-3424 bulk drug stored at -20°C ± 5°C / 5°C ± 3°C / 25°C ± 2°C, 60 ± 5 RH, light protected, upright for different time;

[0103] Figure 6 Impurity profile of 01-03-03-41-01 batch AST-3424 bulk drug stored at -20°C ± 5°C / 5°C ± 3°C / 25°C ± 2°C, 60 ± 5 RH, light protected, upright for different time;

[0104] Figure 7 Impurity profile of 01-03-04-41-01 batch AST-3424 bulk drug stored at -20°C ± 5°C / 5°C ± 3°C / 25°C ± 2°C, 60 ± 5 RH, light protected, upright for different time;

[0105] Figure 8 Typical HPLC detection chromatogram of blank;

[0106] Figure 9 Typical HPLC detection chromatogram of sensitivity solution;

[0107] Figure 10 Typical HPLC detection chromatogram of system suitability solution;

[0108] Figure 11 Typical HPLC detection chromatogram of reference solution;

[0109] Figure 12 Typical HPLC detection chromatogram of test solution. DETAILED DESCRIPTION

[0110] The stable AST-3424 bulk drug product of the present application is essentially an ethanol solution with the mass percentage of AST-3424 being 25-45%. The use of ethanol solution is based on the solubility experimental data of AST-3424 and the long-term stability data of the solution. The following are the specific experimental data.

[0111] General detection method description

[0112] 1. HPLC detection method of AST-3424

[0113] The HPLC detection conditions are as follows:

[0114] UV detector, detection wavelength 230 nm,

[0115] The chromatographic column is Waters Xbridge BEH C8, 150 mm x 4.6 mm, 3.5 μm,

[0116] Column temperature 15℃, injection volume 5μl,

[0117] Mobile phase A is 10mmol / L ammonium acetate solution, solvent is a mixture of water and acetonitrile in the volume ratio of 95:5;

[0118] Mobile phase B is 8mmol / L ammonium acetate solution, solvent is a mixture of acetonitrile and water in the volume ratio of 95:5;

[0119] The flow rate is 1.0ml / min for gradient elution, and the elution gradient is as follows:

[0120] Time (min) 0 5 15 25 30 30.1 35 Volume percentage of mobile phase A 90 70 70 5 5 90 90 Volume percentage of mobile phase B 10 30 30 95 95 10 10

[0121] The total running time is 35 minutes.

[0122] The HPLC instrument is Agilent 1200 high performance liquid chromatograph equipped with ultraviolet detector and automatic sampler, or other equivalent instrument.

[0123] The sample is diluted with acetonitrile and then detected by HPLC. The diluted solution is stable at 2-8℃ for 3 days, and the detection should be completed within 3 days.

[0124] The mass percentage content of AST-3424 bulk drug in the solution is detected and calculated using the standard curve method-single point correction method.

[0125] The HPLC percentage content of each impurity in the AST-3424 bulk drug ethanol composition solution is detected and calculated using the area normalization method: under the above HPLC chromatographic method, the sample is diluted with acetonitrile and then detected by HPLC, and the peak area corresponding to each RRT is calculated in turn. The sum of the contents of all impurities and AST-3424 component is calculated as 100%, and the percentage of the area of a single peak to the total peak area is calculated.

[0126] Wherein, the total impurities are the percentage of the sum of the peak areas of all impurities other than AST-3424 to the total peak area; the HPLC chemical purity of AST-3424 bulk drug is the ratio of the peak area of AST-3424 to the total peak area.

[0127] Unless otherwise specified, the relevant detection in this application is detected using the above method.

[0128] 2. System suitability evaluation of HPLC detection method of AST-3424

[0129] 2.1 HPLC system suitability evaluation requirements

[0130] Before the HPLC detection of the mass percentage content of AST-3424 bulk drug in the solution, the HPLC percentage content of each impurity and the HPLC percentage content of total impurities, the HPLC system suitability evaluation should be carried out and meet the requirements.

[0131] The HPLC detection system suitability evaluation requirements are as follows:

[0132] 1) There should be no significant interfering peaks in the blank solution chromatogram at the AST-3424 peak position. If interfering peaks appear, the peak area should not be more than 1 / 2 of the AST-3424 peak peak area in the sensitivity solution.

[0133] 2) The relative standard deviation of the AST-3424 peak peak area should be not more than 2.0% for 6 injections of the working control solution.

[0134] 3) The recovery rate of the check control solution should be 98.0%-102.0%.

[0135] 4) The relative standard deviation of the target peak area in all working control solutions should be not more than 2.0%.

[0136] 5) The resolution of the impurities with RRT = 1.44-1.49, RRT = 1.50-1.55 in the system suitability solution should be not less than 1.5

[0137] 2.2 Preparation of HPLC system suitability evaluation related solutions

[0138] Blank solution: acetonitrile

[0139] Preparation of working control solution and check control solution

[0140] Take AST-3424 control solution, accurately weigh, add acetonitrile, dissolve and dilute to prepare a solution containing about 1.0 mg of AST-3424 per 1 ml, as the working control solution. Prepare a second control solution by the same method, as the check control solution. The control solution can be stable at 2-8°C and room temperature for 72 hours.

[0141] Preparation of sensitivity solution

[0142] Accurately take working control solution, dilute with acetonitrile to prepare a solution containing about 0.5 μg of AST-3424 per 1 ml, as the sensitivity solution.

[0143] System suitability solution preparation

[0144] Take RRT = 1.44 ~ 1.49, RRT = 1.50 ~ 1.55 impurity reference substance and AST-3424 reference substance, add acetonitrile to dissolve and dilute to prepare a solution containing about 0.002 mg of RRT = 1.44 ~ 1.49 impurities, 0.002 mg of RRT = 1.50 ~ 1.55 impurities and 1.0 mg of AST-3424 per 1 ml, and mark it as system suitability solution.

[0145] The RRT = 1.44 ~ 1.49, RRT = 1.50 ~ 1.55 impurity reference substances are prepared by preparing HPLC after increasing their content by high temperature and high humidity test.

[0146] Preparation of test sample solution

[0147] Take AST-3424 ethanol solution sample, accurately weigh and add acetonitrile to dissolve and dilute to prepare a solution containing about 1.0 mg of AST-3424 per 1 ml, as test sample solution, and prepare two samples in parallel.

[0148] 2.3 Determination method

[0149] Accurately take blank solution, system suitability solution, working reference solution, check reference solution and test sample solution, inject into the chromatograph respectively, and record the chromatogram. The working reference solution is injected continuously for 6 times, and the working reference solution is injected after the test sample solution is injected, as the back needle.

[0150] 3. HPLC related calculation of the mass percentage content of AST-3424 raw material, the HPLC percentage content of each impurity and the total impurities of AST-3424

[0151] 3.1 Recovery rate of check reference

[0152]

[0153] Among them,

[0154] A standard = the average peak area of AST-3424 measured by working reference solution injected continuously for 6 times,

[0155] A check = the peak area of AST-3424 in check reference solution,

[0156] C standard = the concentration of AST-3424 in working reference solution (mg / mL),

[0157] C check = the concentration of AST-3424 in check reference solution (mg / mL).

[0158] 3.2 Percentage content of AST-3424 active pharmaceutical ingredient

[0159] The content (%) of the test sample is calculated using the following formula:

[0160]

[0161] in,

[0162] A Standard =The average peak area of ​​AST-3424 obtained by continuous injection of the working reference solution over 6 injections.

[0163] A Sample = Peak area of ​​AST-3424 in the test solution

[0164] C Standard =Concentration of AST-3424 in the working control solution (mg / mL)

[0165] C Sample =Concentration of the test solution (mg / mL)

[0166] P W = Percentage content of AST-3424 in the AST-3424 reference standard.

[0167] 3.3 HPLC percentage content of specific impurities

[0168] The percentage of specific impurities in the test sample is automatically read by the calculation software built into the HPLC system, or it can be calculated using the following formula:

[0169]

[0170] Impurity (%) results are rounded to two decimal places.

[0171] 3.4 Chemical purity

[0172]

[0173] Chemical purity is automatically calculated and read out by the calculation software built into the HPLC system according to the above formula, and the purity result is retained to two decimal places.

[0174] 3.5 General Miscellaneous

[0175] %Totals impurities = 1 - A

[0176] Total impurities (%)=1-A

[0177] in,

[0178] The purity of A = AST-3424

[0179] The total miscellaneous result is rounded to two decimal places.

[0180] The following are the limits of quantitation and limits of detection for the HPLC method in this application.

[0181] Substance LOQ / limit of quantification LOD / limit of detection AST-3424 0.05％ 0.02％ RRT = 1.44-1.49 0.04％ 0.01％ RRT = 1.50-1.55 0.05％ 0.01％

[0182] 3.6 Identification

[0183] The retention time of AST-3424 in the test solution should be consistent with that in the working control solution. The ratio of the average retention time of the AST-3424 peak measured by six consecutive injections of the working control solution to the retention time of the main peak of the first injection sample should be between 0.98 and 1.02.

[0184] 3.7 Typical Atlas

[0185] Typical chromatograms for the blank, typical chromatograms for the sensitivity solution, typical chromatograms for the system suitability solution, typical chromatograms for the standards solution, and typical chromatograms for the sample solution. Figures 8-12 As shown.

[0186] 4. Method for determining the moisture percentage of AST-3424

[0187] The measurement was performed using the Karl Fischer titration method (KF), and the specific testing was conducted using the "coulometric titration method" in the Chinese Pharmacopoeia (2020 edition).

[0188] Unless otherwise specified, all tests in this application are conducted using the methods described above.

[0189] 5. HPLC method for determining the percentage content of the chiral AST-3424 isomer (AST-3423).

[0190]

[0191] The chiral HPLC detection conditions are as follows:

[0192] UV detector, detection wavelength 230 nm,

[0193] Chromatographic column: Chiralcel OJ-H, 5 μm, 250 mm x 4.6 mm,

[0194] Column temperature: 25 °C,

[0195] The mobile phase is a mixture of n-hexane and ethanol with a volume ratio of 70:30.

[0196] The flow rate is 1.0 ml / min, and the total running time is 20 minutes.

[0197] The HPLC instrument is an Agilent 1200 high-performance liquid chromatograph equipped with a UV detector and an automatic sampler, or other equivalent instruments.

[0198] The sample is diluted with ethanol and then detected by HPLC. The diluted solution is stable at 2-8 °C for 3 days, and the detection should be completed within 3 days.

[0199] Isomer chiral HPLC percentage content detection calculation: under the above chiral HPLC chromatographic method, the sample is diluted with ethanol and then detected by chiral HPLC. The peak areas corresponding to AST-3424 and isomer AST-3423 are calculated in turn, and then the isomer chiral HPLC percentage content calculation formula is

[0200]

[0201] Unless otherwise specified, the relevant detection in this application is detected by the above method.

[0202] I. Solubility study of AST-3424

[0203] 1.1 Solubility test

[0204] Take AST-3424 (hereinafter referred to as drug substance or API) in an appropriate container, gradually add solvent, shake well, and observe the process of turbidity (presence of excess AST-3424 oil droplets) to clarity (AST-3424 completely dissolved) of the solution. Record the volume of the solvent that makes the AST-3424 mixed solution clear, and calculate the solubility.

[0205] 1.2 Test results

[0206] Table 1: Solubility study results of AST-3424 (mg / ml)

[0207] Solvent Ethanol Propylene glycol Ethyl acetate Water Solubility (mg / ml) ＞1000 ＞100 ＞1000 About 20 Phenomenon Very easily soluble (miscible) Easily soluble Very easily soluble (miscible) Slightly soluble

[0208] Table 1 summarizes the solubility of AST-3424 in different alcohol solvents at 25°C. Ethanol, propylene glycol and other alcohol solvents have good solubility for the drug substance. In addition, water was also used for the experiment, and the results showed that AST-3424 was slightly soluble in water.

[0209] 1.3 Stability Study

[0210] AST-3424 solutions (about 90 mg / ml) dissolved in different solvents were subjected to high temperature destruction (40°C, 3 days), and the purity was detected and compared with samples that were not subjected to destruction (40°C, 3 days) to investigate the stability of AST-3424 in different solvents.

[0211] The purity was determined by HPLC method:

[0212] UVDAD detector wavelength 230 nm, C18 column, column temperature 25°C.

[0213] Mobile phase:

[0214] A: 10 mmol / L ammonium acetate solution of ammonium acetate dissolved in 95% water and 5% acetonitrile by volume ratio of mixed solvent;

[0215] B: 8 mmol / L ammonium acetate solution of ammonium acetate dissolved in 95% acetonitrile and 5% water by volume ratio of mixed solvent;

[0216] Gradient elution was performed.

[0217] Table 2: Results of chemical purity study of AST-3424 solutions in different solvents at 40°C for 3 days (mg / ml)

[0218]

[0219] The e.e% value was detected by chiral HPLC method.

[0220] AST-3424 has poor stability in water, and is more stable in anhydrous ethanol, propylene glycol, ethyl acetate and acetonitrile. AST-3424 dissolved in propylene glycol solution has a viscosity significantly higher than other solvents.

[0221] AST-3424 is a viscous oil, which is not convenient to transfer and sub-pack, and needs to be dissolved with a solvent to reduce the viscosity. See Appendix Figure 3 .

[0222] AST-3424 is the raw material of injection, and the solvents that can be used for injection include water, anhydrous ethanol and propylene glycol, etc. Through experiments, it is proved that the solubility and stability of AST-3424 in water are poor, and the solubility and stability of AST-3424 in ethanol and propylene glycol are good, but the viscosity of propylene glycol is high and not suitable, so the final choice is anhydrous ethanol, and the concentration of AST-3424 is diluted to 30-40% by mass percentage.

[0223] II. Preparation of AST-3424 raw material ethanol solution and preliminary stability study

[0224] 2.1 Preparation

[0225] Using the disclosed process ((R)- and (S)-1-(3-(3-N,N-dimethylaminocarbonyl) phenoxy-4-nitrophenyl)-1-ethyl-N,N'-bis(ethoxycarbonyl) phosphoramidate, compositions and uses and methods of making thereof, corresponding to PCT application number PCT / US2016 / 062114, publication number WO2017087428A1, corresponding to Chinese application number 2016800446081, publication number CN108290911A, the S configuration compound TH3424) is synthesized and prepared, after obtaining a yellow oil using a silica gel column chromatography, the obtained oil mixture is mixed with anhydrous ethanol (pharmaceutical grade) and then distilled under reduced pressure until the mass percentage of AST-3424 reaches the predetermined value to obtain the AST-3424 raw material product solution. The temperature of the reduced pressure distillation should not be higher than 30°C, and the pressure of the reduced pressure distillation should be P≤-0.08 MPa.

[0226] 2.2 Preliminary stability study under different storage temperatures, in the dark and under light

[0227] Using the above preparation method, distillation is carried out until the final volume is 0.5 to 1 L, and three batches (under GMP conditions) are prepared, and the data of each batch is as follows:

[0228] 024101 batch, equivalent to 0.32 kg of AST-3424 (0.8938 kg of ethanol solution with a content of 35.8%), packaged in HDPE bottles and screw caps and packaged in LDPE bags with aluminum foil;

[0229] 034101 batch, equivalent to 0.35 kg of AST-3424 (0.9668 kg of ethanol solution with a content of 36.2%), packaged in HDPE bottles and screw caps and packaged in LDPE bags with aluminum foil;

[0230] 044101 batch, equivalent to 0.31 kg of AST-3424 (0.8732 kg of ethanol solution with a content of 35.5%), packaged in HDPE bottles and screw caps and packaged in LDPE bags with aluminum foil;

[0231] A preliminary stability experiment was performed using the solution product of batch 034101.

[0232] The photodegradation study was designed to evaluate any potential effects of light exposure on the ethanol solution of AST-3424 drug substance. The ethanol solution of AST-3424 drug substance was transferred to clear glass vials and exposed to 1.08 x 104 lux of light (4500 lux of visible light and 600 lux of UV light) for 10 hours per day (10 days at 4500 lux). Control samples were prepared in separate clear glass vials placed inside an aluminum foil bag (25°C + 4500 ± 500 Lx + covered). 6 lxhours (i.e. 10 days at 4500 lx). Control samples were prepared in separate clear glass vials placed inside an aluminum foil bag (25°C + 4500 ± 500 Lx + covered).

[0233] In addition to the test and control samples, two additional positive control samples were prepared in separate clear vials and stored in a 25°C and 40°C dark room (25°C + natural light, 40°C + natural light). The purpose of storing in a 25°C and 40°C dark room was to observe the temperature effects behind the photodegradation group.

[0234] The samples were inspected for appearance, assay, related substances, chemical purity, and enantiomeric excess on day 0, day 5, and day 10.

[0235] The samples were placed under the corresponding storage conditions to study the data changes of the corresponding content, chemical purity, enantiomeric excess, etc. at different time nodes, and the results are shown in Table 3 below.

[0236] The three anhydrous ethanol mixing and vacuum distillation operations were performed, the first two vacuum concentration operations were stopped until no distillate was obtained, and the third vacuum distillation operation was performed until the mass percentage of AST-3424 reached the predetermined value, and the mass percentage of AST-3424 in the solution was predetermined.

[0237] The mass percentage content was quantitatively detected by HPLC external standard method, and the HPLC method was the method in section 1.3.

[0238] The HPLC chemical purity of the solution AST-3424 was detected by HPLC, the HPLC method was the method in section 1.3, and the area normalization method was used for calculation.

[0239] The isomer chiral HPLC percentage content (enantiomeric excess) detection was detected by chiral HPLC method.

[0240] Table 3: Data of content (mass percentage of AST-3424 in solution), chemical purity, enantiomeric excess, etc. of the solution product of batch 034101 at different storage temperatures, whether light-protected conditions, at different time nodes

[0241]

[0242]

[0243] 40°C + natural light, placed in the laboratory's natural light environment (dark room) at a temperature of 40°C without direct sunlight.

[0244] 25°C + natural light, placed in the laboratory's natural light environment (dark room) at a temperature of 25°C without direct sunlight.

[0245] 25°C + 4500 ± 500 Lx, placed in a pharmaceutical stability test chamber at a temperature of 25°C with light set at 4500 ± 500 Lx.

[0246] 25°C + 4500 ± 500 Lx + covered, placed in a pharmaceutical stability test chamber at a temperature of 25°C with light set at 4500 ± 500 Lx, and the samples were completely wrapped in aluminum foil.

[0247] Analysis of experimental data can find:

[0248] From the 5th day, only the test sample exposed to 4500 lx was observed to change color from light yellow to light brown. There was no difference between the 4500 Lx ± 500 Lx sample and the 25°C dark room control sample in other test items including content determination, chemical purity, and enantiomeric excess.

[0249] Impurity increases (chemical purity decreases) were observed in both 4500 ± 500 Lx and 40 & 25°C dark room samples. These impurities were caused by temperature increases during the photodegradation study.

[0250] Overall, the impact of exposure to light on content determination, chemical purity, individual impurities and total impurities, and enantiomeric excess was small, and it is recommended to store in the dark to maintain the stability of the physicochemical properties (such as color) of AST-3424 bulk drug ethanol solution.

[0251] Preliminary stability experiment conclusion:

[0252] AST-3424 bulk drug ethanol solution showed no significant changes when stored at 25°C, light and dark conditions for 5 days and 10 days, but the sample stored at 40°C for 10 days had significant changes.

[0253] The experiment preliminarily revealed that temperature is an important factor affecting stability, and low temperature will be beneficial to stability during storage; light has an impact, but the impact is not as significant as temperature. Therefore, in long-term stability studies, the stability of solution products should be tested under different low-temperature conditions in a light-free environment.

[0254] III. Preparation of AST-3424 bulk drug ethanol solution and long-term stability study

[0255] 3.1 Preparation

[0256] The synthesis preparation was carried out using the disclosed process ((R)- and (S)-1-(3-(3-N,N-dimethylaminocarbonyl)phenoxy-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylenyl)aminophosphate, compositions thereof, and methods of use and preparation thereof, corresponding to PCT application number PCT / US2016 / 062114, publication number WO2017087428A1, corresponding to Chinese application number 2016800446081, publication number CN108290911A, S configuration compound TH3424) and after using silica gel column chromatography to obtain yellow oil, the obtained oil mixture was mixed with anhydrous ethanol (pharmaceutical grade) and then distilled under reduced pressure until the mass percentage of AST-3424 reached a predetermined value to obtain the AST-3424 bulk drug product solution, the temperature of the reduced pressure distillation should not be higher than 30°C, and the pressure of the reduced pressure distillation should be P≤-0.08 MPa.

[0257] When the anhydrous ethanol mixing and reduced pressure distillation operation was carried out three times, the first two times were reduced pressure concentration until there was no distillate, and the third time was reduced pressure distillation until the mass percentage of AST-3424 reached a predetermined value.

[0258] The solution was transferred to an HDPE bottle, sealed with a sealing film, and then tightly capped with a screw cap, and then wrapped with a low-density polyethylene (LDPE) sealing bag, and the whole was packaged in an LDPE bag with an aluminum foil, and stored in the dark.

[0259] 3.2 Long-term stability study of different batches of AST-3424 bulk drug ethanol combination under different storage temperatures, light and light

[0260] Using the above preparation method, distillation was carried out until the final volume was 0.5 to 1 L, and four batches were prepared (under GMP conditions): 03-03P-01-37-01 batch, 01-03-02-41-01 batch, 01-03-03-41-01 batch, and 01-03-024-41-01 batch (03-03P-01-37-01 batch was produced by A workshop of A factory, and the remaining three batches were produced by B workshop of A factory), and storage stability experiments at different temperatures were carried out.

[0261] Transfer about 2 g of sample into a 10 mL HDPE bottle, and seal with a closure film. Put the HDPE bottle into a LDPE bag, and seal with 2 clips at each end. Then put the LDPE bag into a food-grade aluminum foil bag, and heat seal. Repeat the above steps to prepare multiple samples for stability study: 6 bags for each sampling time point for the long-term stability study at -20±5℃; 3 bags for each sampling time point for the accelerated stability study at 5±3℃; 2 bags for each sampling time point for the long-term stability study at 25±2℃.

[0262] Sample at the specified time: 1M, 3M, 6M, 9M, 12M, 18M and 24M and subsequent predetermined time periods, the time sampling time shall not exceed the specified time ± 3 days. The samples stored at 5±3℃ are temporarily placed in the environment of -20±5℃ after taking out, the samples stored at -20±5℃ are temporarily placed in the environment of -20±5℃ after taking out until all tests are completed, and the samples stored at 25±2℃ / 60±5RH are temporarily placed in the environment of -20±5℃ after taking out until all tests are completed. The detection of the stability test samples at 6 months and above is completed within 2 days after the samples are taken out; the detection of the stability test samples below 6 months is completed within 1 day after the samples are taken out. The specific detection results are as follows in Tables 4 to 14.

[0263] The mass percentage content of AST-342 in the solution, the HPLC percentage content of impurities, and the like are determined by “General Detection Method Description-1, HPLC Detection Method of AST-3424”; the mass percentage of water is determined by “General Detection Method Description-2, Mass Percentage Detection Method of Water of AST-3424”; and the chiral HPLC percentage content is determined by “General Detection Method Description-3, Chiral HPLC Percentage Content Detection Method of Isomer (AST-3423) of AST-3424”.

[0264] 3.2.1, 03-03P-01-37-01 batch

[0265] The specific detection results are as follows in the table. If / is filled in the table, it means that it is not applicable or below the detection limit, and the same applies below.

[0266] Table 4: Stability test result statistical table of AST-3424 bulk drug of 03-03P-01-37-01 batch stored at -20℃±5℃, light-avoiding and upright

[0267]

[0268] Table 5: Stability test result statistical table of AST-3424 bulk drug of 03-03P-01-37-01 batch stored at 5℃±3℃, light-avoiding and upright

[0269]

[0270]

[0271] The corresponding figures were made according to the data in Tables 4 and 5 Figure 4 (R represents below the detection limit or the limit of quantification, according to the value 0, the same below), RRT = 1.05-1.10 corresponding to impurities A, RRT = 1.11-1.19 corresponding to impurities B, RRT = 1.21-1.25 corresponding to impurities C, RRT = 1.28-1.29 corresponding to impurities D, RRT = 1.44-1.49 corresponding to impurities E, RRT = 1.50-1.55 corresponding to impurities F.

[0272] The stability data of the 03-03P-01-37-01 batch stored at different temperatures and can be known as follows:

[0273] The storage stability at 5°C±3°C, light-avoiding and upright is worse than that at -20°C±5°C, light-avoiding and upright.

[0274] Specifically, under the condition of -20°C±5°C, light-avoiding and upright, only 5 impurities of ABCEF are detected, the D impurity is below the detection limit and has a small content; and with the extension of storage time, the content of impurities ABCEF has almost no change or the content rising trend is not obvious.

[0275] Specifically, under the condition of 5°C±3°C, light-avoiding and upright, 6 impurities of ABCDEF are detected, and with the extension of storage time, the rising trend of the content of impurities ABCDEF is very obvious; in comparison, the growth rate of impurities CD is obviously faster than that of impurities ABEF. In comparison with the impurity growth and total impurity, the total impurity will exceed the standard after 9 months, and the subsequent storage impurities will be more and more, so the product of this batch is stored at this temperature condition, and the maximum storage time is 10 months.

[0276] 3.2.2, 01-03-02-41-01 batch:

[0277] The specific test results are as follows.

[0278] Table 6: 01-03-02-41-01 batch AST-3424 API 5°C±3°C, light-avoiding and upright storage stability test result statistical table

[0279]

[0280] Table 7: 01-03-02-41-01 batch AST-3424 API -20°C±5°C, light-avoiding and upright storage stability test result statistical table

[0281]

[0282]

[0283] Table 8: Statistical Table of 01-03-02-41-01 Batch AST-3424 Bulk Drug Storage Stability Test Results at 25±2℃ / 60±5RH, Light-Proof, Upright

[0284]

[0285] According to the corresponding data table of Table 6, Table 7 and Table 8, the corresponding figures are made Figure 5 , record RRT = 1.05-1.10 corresponding to impurities A, RRT = 1.11-1.19 corresponding to impurities B, RRT = 1.21-1.25 corresponding to impurities C, RRT = 1.28-1.29 corresponding to impurities D, RRT = 1.44-1.49 corresponding to impurities E, RRT = 1.50-1.55 corresponding to impurities F.

[0286] Analysis of the stability data of 01-03-02-41-01 batch stored at different temperatures and can be known:

[0287] Comparing 25±2℃ / 60±5RH, 5℃±3℃, -20℃±5℃, three storage temperatures, the lower the temperature, the more stable the storage.

[0288] Specifically, under the condition of -20℃±5℃, light-proof, upright, only 5 impurities ABCEF were detected, D impurity was below the detection limit and had a small amount; moreover, with the extension of storage time, under the storage of 42 months, the content of impurities ABCEF had almost no change or the content increasing trend was not obvious, only impurity E slowly increased; accordingly, it can be inferred that the continued storage is still stable.

[0289] Specifically, under the condition of 5℃±3℃, light-proof, upright, 6 impurities ABCDEF were detected, moreover, with the extension of storage time, the content increasing trend of impurities ABCDE was very obvious, F had almost no change; by comparison, the growth rate of impurities CDE was faster. By comparing the impurity growth and total impurity, it can be known that the total impurity will exceed the standard after 12 months, and the impurities will be more and more in the subsequent storage, therefore, the storage time of this batch product at this temperature condition is at most 15 months.

[0290] Specifically, under the conditions of 25±2℃ / 60±5RH, light avoidance, and upright storage, impurities ABCDEF were detected, and the content of impurities ABCDEF showed a very obvious upward trend with the extension of storage time; in comparison, the growth rate of impurity ACD was faster and uniform, and in particular, the growth of impurity CD was particularly fast. By comparing the growth of impurities and the total impurities, it can be seen that the total impurities will exceed the standard after 1 month of storage, and the subsequent storage impurities will be more and more, so the storage time of this batch of product under this temperature condition is at most 1 month.

[0291] 3.2.3, 01-03-03-41-01 batch:

[0292] The specific test results are as follows.

[0293] Table 9: 01-03-03-41-01 batch AST-3424 API 5℃±3℃, light avoidance, upright storage stability test result statistical table

[0294]

[0295] Table 10: 01-03-03-41-01 batch AST-3424 API -20℃±5℃, light avoidance, upright storage stability test result statistical table

[0296]

[0297] Table 11: 01-03-03-41-01 batch AST-3424 API 25±2℃ / 60±5RH, light avoidance, upright storage stability test result statistical table

[0298]

[0299]

[0300] According to the data table of Table 9, Table 10 and Table 11, the corresponding figures are made Figure 6 , RRT=1.05-1.10 corresponds to impurity A, RRT=1.11-1.19 corresponds to impurity B, RRT=1.21-1.25 corresponds to impurity C, RRT=1.28-1.29 corresponds to impurity D, RRT=1.44-1.49 corresponds to impurity E, and RRT=1.50-1.55 corresponds to impurity F.

[0301] Analysis of the stability data of 01-03-03-41-01 batch stored at different temperatures shows that:

[0302] By comparing the three storage temperatures of 25±2℃ / 60±5RH, 5℃±3℃, and -20℃±5℃, the lower the temperature, the more stable the storage.

[0303] Specifically, at -20℃±5℃, in the light-avoiding and upright condition, only the 5 impurities of ABCEF were detected, the D impurity was below the detection limit and had a low content; moreover, with the extension of storage time, under the storage for 42 months, the content of the impurities ABCEF had almost no change or the content rising trend was not obvious, only the impurity E had a slow growth; accordingly, it can be inferred that the continued storage is still stable.

[0304] Specifically, at 5℃±3℃, in the light-avoiding and upright condition, the 6 impurities of ABCDEF were detected, moreover, with the extension of storage time, the rising trend of the content of the impurities ACDE was very obvious, BF had almost no change; by comparison, the growth rate of the impurities CDE was faster. By comparing the impurity growth and total impurity, it can be known that the total impurity will exceed the standard after 12 months, and the impurities will be more and more in the subsequent storage, therefore, the storage of the product at the temperature condition, the maximum storage time is probably 15 months.

[0305] Specifically, at 25±2℃ / 60±5RH, in the light-avoiding and upright condition, the 6 impurities of ABCDEF were detected, moreover, with the extension of storage time, the rising trend of the content of the impurities ABCDEF was very obvious; by comparison, the growth rate of the impurities ACD was faster and uniform, especially, the growth of the impurities CD was particularly fast. By comparing the impurity growth and total impurity, it can be known that the total impurity will exceed the standard after 1 month, and the impurities will be more and more in the subsequent storage, therefore, the storage of the product at the temperature condition, the maximum storage time is 1 month.

[0306] 3.2.4, 01-03-04-41-01 batch:

[0307] The specific test results are as follows.

[0308] Table 12: 01-03-04-41-01 batch AST-3424 API 5℃±3℃, light-avoiding and upright storage stability test result statistical table

[0309]

[0310]

[0311] Table 13: 01-03-04-41-01 batch AST-3424 API -20℃±5℃, light-avoiding and upright storage stability test result statistical table

[0312]

[0313] Table 14: Statistics table of 01-03-04-41-01 batch AST-3424 bulk drug 25±2℃ / 60±5RH, light-avoiding, upright storage stability test results

[0314]

[0315]

[0316] According to the corresponding data table of Table 12, Table 13, and Table 14, the corresponding figures are prepared Figure 7 , and RRT = 1.05-1.10 corresponds to impurity A, RRT = 1.11-1.19 corresponds to impurity B, RRT = 1.21-1.25 corresponds to impurity C, RRT = 1.28-1.29 corresponds to impurity D, RRT = 1.44-1.49 corresponds to impurity E, and RRT = 1.50-1.55 corresponds to impurity F.

[0317] Analyzing the stability data of 01-03-04-41-01 batch stored at different temperatures, it can be seen that:

[0318] Comparing 25±2℃ / 60±5RH, 5℃±3℃, and -20℃±5℃, the lower the temperature, the more stable the storage.

[0319] Specifically, under the condition of -20℃±5℃, light-avoiding, upright, only 5 impurities ABCEF were detected, and the content of D impurity was below the detection limit and was less; moreover, with the extension of storage time, under the storage of 42 months, the content of impurities ABCEF hardly changed or the content rising trend was not obvious, only impurity E had a slow growth; accordingly, it can be inferred that the continued storage is still stable.

[0320] Specifically, under the condition of 5℃±3℃, light-avoiding, upright, 6 impurities ABCDEF were detected, and moreover, with the extension of storage time, the rising trend of impurities ACDE content was very obvious, and BF hardly changed; in comparison, the growth rate of impurities CDE was faster. Comparing the impurity growth and total impurity, it can be seen that the total impurity will exceed the standard after 12 months, and the impurities will be more and more in subsequent storage, therefore, the storage time of this batch product at this temperature condition is probably 17 months at most.

[0321] Specifically, under the conditions of 25±2℃ / 60±5RH, light avoidance, and upright storage, impurities ABCDEF were detected, and the content of impurities ABCDEF showed a very obvious upward trend with the extension of storage time; in comparison, the growth rate of impurities ACD was faster and uniform, and in particular, the growth of impurities CD was particularly fast. By comparing the impurity growth and total impurity, it can be seen that the total impurity will exceed the standard after 1 month of storage, and the subsequent storage impurities will be more and more, so the maximum storage time of the product under this temperature condition is 1 month.

[0322] In particular, the 18-month test data of the above-mentioned 01-03-02-41-01, 01-03-03-41-01, and 01-03-04-41-01 batches showed an operation error after investigation.

[0323] Further comparison, the impurity changes of 01-03-02-41-01, 01-03-03-41-01, and 01-03-04-41-01 batches produced by B workshop have a greater difference from 03-03P-01-37-01 batch produced by A workshop, and in comparison, the storage stability of 01-03-02-41-01, 01-03-03-41-01, and 01-03-04-41-01 batches under the conditions of 5℃±3℃, light avoidance, and upright storage may be better than that of 03-03P-01-37-01 batch.

[0324] IV. Inverted stability study of AST-3424 drug substance ethanol solution

[0325] About 2g sample was transferred to a 10mL HDPE bottle, and sealed with a sealing film. The HDPE bottle was placed in a LDPE bag, and the two ends were each sealed with 2 drawfasts. Then the LDPE bag was placed in a food-grade aluminum foil bag, heat-sealed, and then placed upside down. Repeat the above steps to prepare multiple samples for stability study: 6 bags for each sampling time point for -20±5℃ long-term stability study; 3 bags for each sampling time point for 5±3℃ accelerated stability study; 2 bags for each sampling time point for 25±2℃ long-term stability study.

[0326] The above-mentioned three batches of drug substance ethanol composition solutions (01-03-02-41-01, 01-03-03-41-01, and 01-03-04-41-01) were subjected to 5±3℃ / -20±5℃, light avoidance, and inverted storage stability experiments, and the results are shown in the following table.

[0327] Table 15: Statistics of 5±3℃, light avoidance, and inverted storage stability test results of AST-3424 drug substance of 01-03-02-41-01, 01-03-03-41-01, and 01-03-04-41-01 batches

[0328]

[0329]

[0330] Table 16: Statistics table of AST-3424 bulk drug storage stability test results at -20±5℃, light-avoiding, inverted storage

[0331]

[0332] The analysis data show that the inverted storage of the ethanol composition solution of the bulk drug does not affect the storage stability, and the detection indexes are still stable after one month of inversion, so the jolt and inversion during storage and transportation do not adversely affect storage and transportation.

[0333] The average retention time of the main peak of AST-3424 actually detected in the above Tables 4 to 16 is in the range of 11.4-12.7 min, and the above values are obtained by detection in an Agilent 1200 high-performance liquid chromatograph equipped with an ultraviolet detector and an automatic sampler. Considering other different instruments, it is also reasonable that the actual retention time detection is 10.0-14.0 min.

[0334] V. Failed Batch of AST-3424 Bulk Drug Ethanol Solution

[0335] Another batch was produced by B factory using the same process, with batch number 03-ZZQ-0919-04 and production date 2021.06.04. After production, it was stored at -20±5℃ under light-avoiding conditions, and samples were taken. The results are shown in Table 17 below.

[0336] Table 17: Statistics table of AST-3424 bulk drug storage stability test results at -20±5℃, light-avoiding, upright storage

[0337]

[0338]

[0339] Obviously, although the total impurities, chemical purity, mass percentage content, isomers, and mass percentage of moisture of the product of this batch all meet the standards, the impurities ABCEF all exceed the standards, and the total impurities are close to exceeding the standards, so the product of this batch is determined to be unqualified. After investigation, it was confirmed that the reason for the unqualification of this batch was that the personnel did not strictly follow the operation guidelines during the column chromatography purification process, resulting in the unqualification of the product of this production batch.

Claims

1. A stable AST-3424 drug substance ethanol composition solution, wherein the solute in the solution comprises AST-3424 drug substance and impurities, and the mass percentage content of the AST-3424 drug substance in the solution is 25-45%, The composition solution is stored in a sealed light-proof environment at-20±5℃ or lower, and after the test sample is diluted with acetonitrile and detected by HPLC, the HPLC chemical purity of AST-3424 drug substance is greater than or equal to 98.0% after calculation by area normalization method, and the main signal peak of AST-3424 with a peak time of 10.0-14.0 min is taken as a control, and the impurities are represented by RRT, then the impurities comprise: ； Or The composition solution is stored in a sealed light-proof environment at 5±3℃ or at 5±3℃ to-20±5℃, and after the test sample is diluted with acetonitrile and detected by HPLC, the HPLC chemical purity of AST-3424 is greater than or equal to 98.0% after calculation by area normalization method, and the main signal peak of AST-3424 with a peak time of 10.0-14.0 min is taken as a control, and the impurities are represented by RRT, then the impurities comprise: Wherein, The HPLC detection conditions are as follows: UV detector, detection wavelength 230 nm, The chromatographic column is Waters Xbridge BEH C8, 150 mm×4.6 mm, 3.5 μm, Column temperature 15℃, The mobile phase A is 10 mmol / L ammonium acetate solution, and the solvent is a mixed solvent with a volume ratio of water to acetonitrile of 95:5; The mobile phase B is 8 mmol / L ammonium acetate solution, and the solvent is a mixed solvent with a volume ratio of acetonitrile to water of 95:5; The gradient elution is carried out at a flow rate of 1.0 ml / min, and the elution gradient is as follows: 。 2.The stable AST-3424 drug substance ethanol composition solution of claim 1, The composition solution is stored in a sealed light-proof environment at-20±5℃ or lower, and the impurities are represented by RRT, then the impurities comprise: Or The composition solution is stored in a sealed light-proof environment at 5±3℃ or at 5±3℃ to-20±5℃, and the impurities are represented by RRT, then the impurities comprise: 。 3. The stable AST-3424 drug substance ethanol composition solution of claim 1, wherein, The percentage content of isomer chiral HPLC in AST-3424 drug substance is less than or equal to 2.0%. 4.The stable AST-3424 drug substance ethanol composition solution of claim 1, which is stored in a sealed light-proof environment at-20±5℃, and has a shelf life of greater than or equal to 24 months. 5.The stable AST-3424 drug substance ethanol composition solution of claim 1, which is stored in a sealed light-proof environment at-20±5℃, and has a shelf life of greater than or equal to 36 months; or a shelf life of greater than or equal to 48 months; or a shelf life of greater than or equal to 60 months. 6.The stable AST-3424 drug substance ethanol composition solution of claim 1, wherein the mass percentage content of AST-3424 drug substance in the solution is 29.50-39.80%.

7. The stable AST-3424 drug substance ethanol composition solution of claim 1, wherein the mass percentage of AST-3424 drug substance in the solution is 29.40-30.90% or 35.80-37.70% or 36.20-39.80% or 35.50-39.30%.

8. The stable AST-3424 drug substance ethanol composition solution of claim 1, wherein the shelf life of the solution is greater than or equal to 12 months when stored at 5±3℃ in the dark.

9. The stable AST-3424 drug substance ethanol composition solution of claim 1, wherein, The mass percentage of water in the solution is not greater than 1.0%.

10. A packaging unit comprising a container assembly and the stable AST-3424 drug substance ethanol composition solution of any one of claims 1-9 contained in the container, wherein the container assembly comprises a container for containing, and the container is made of pharmaceutical grade high density polyethylene or borosilicate glass.

11. The packaging unit of claim 10, wherein the container assembly further comprises an outer structure for wrapping the container, the outer structure is preferably a low density polyethylene seal bag, the container is preferably a high density polyethylene bottle sealed by a cap and a sealing film or sealed by a cap and a sealing film after the cap is twisted.

12. The packaging unit of claim 10, further comprising a light shielding structure for wrapping the container assembly, and preferably the light shielding structure is an aluminum foil bag.

13. A method for preparing the stable AST-3424 drug substance ethanol composition solution of any one of claims 1-9, wherein the purified AST-3424 product is mixed with anhydrous ethanol at a temperature not higher than 30℃, and then distilled under reduced pressure until the mass percentage of AST-3424 reaches a predetermined value or additional ethanol is added until the mass percentage of AST-3424 reaches a predetermined value. Preferably, the operation of mixing with anhydrous ethanol and distilling under reduced pressure is performed three times, wherein the first two times are distilled under reduced pressure until no distillate is obtained, and the third time is distilled under reduced pressure until the mass percentage of AST-3424 reaches a predetermined value or additional ethanol is added until the mass percentage of AST-3424 reaches a predetermined value. More preferably, the pressure of distillation under reduced pressure is P≤-0.08 MPa.

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