Pomalidomide related substances and uses thereof

Abstract

The present application provides an isolated compound which is a related substance of paritaprevir mesylate. The present application also provides the use of said compound for the preparation of paritaprevir mesylate or a composition comprising paritaprevir mesylate.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology. Specifically, this invention relates to related substances of paradefovir mesylate and their applications. Background Technology

[0002] Paradefovir mesylate is a bioequivalent liver-targeted adefovir (PMEA) prodrug created by constructing an aryl phosphate cyclic diester structure within the target active drug using HepDirect technology. Its structural formula is as follows:

[0003]

[0004] Paradefovir mesylate is effectively converted to PMEA by the hepatic cytochrome P450 isoenzyme CYP3A4. PMEA can effectively bind to the sites of hepatitis B virus infection and replication in the liver to achieve a therapeutic effect. This drug is a potential effective treatment for hepatitis B.

[0005] Chinese Patent Nos. 200510098771.X and 201210344333.7 disclose the preparation of paradefovir mesylate (active pharmaceutical ingredient). However, the inventors have discovered that due to the structural characteristics of paradefovir mesylate, related substances are easily generated during its storage, transportation, and preparation into a pharmaceutical formulation. However, prior art reports on related substances of paradefovir mesylate are scarce.

[0006] Without any prior technological inspiration or guidance, the inventors tackled the challenge head-on, conducting extensive and arduous research. They discovered numerous related substances in paradefovir mesylate, which not only characterize the quality of paradefovir but, more surprisingly, can also indicate anomalies encountered during actual production, thereby improving production processes and enhancing the quality of paradefovir mesylate. Furthermore, the inventors investigated the excipient ratios and dosage design of paradefovir mesylate, developing a drug formulation and dosage that combines safety and efficacy. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a method for detecting related substances in paradefovir mesylate.

[0008] Specifically, in a first aspect, the present invention provides isolated compounds represented by one or more of the following formulas 1 to 10.

[0009]

[0010]

[0011] The compound of the first aspect of the present invention is a paradefovir mesylate related substance. The compound of the first aspect of the present invention may be a compound represented by one of formulas 1 to 10, or a compound represented by 2, 3, 4, 5, 6, 7, 8, 9 or 10 of formulas 1 to 10, the latter being actually a composition of compounds represented by 2, 3, 4, 5, 6, 7, 8, 9 or 10 of formulas 1 to 10.

[0012] If present in the active pharmaceutical ingredient or formulation of paradefovir mesylate, the compound of the first aspect of the present invention exhibits stable chromatographic behavior (see the two tables below). For example, the compound represented by Formula 1 can be represented as a compound of paradefovir mesylate separated by the chromatographic method described in Example 6, having a localized retention time of 14.474 min; and as well, the compounds represented by Formulas 2 and 3 can be represented as compounds of paradefovir mesylate separated by the chromatographic method described in Example 6, having localized retention times of 9.679 min and 9.892 min, respectively.

[0013] Separation of components in active pharmaceutical ingredient

[0014]

[0015]

[0016] In a second aspect, the present invention provides the use of the compound of the first aspect of the present invention in the preparation of paradefovir mesylate; in addition, the present invention provides the use of the compound of the first aspect of the present invention in the preparation of a composition comprising paradefovir mesylate, preferably in the preparation of a pharmaceutical composition comprising paradefovir mesylate.

[0017] In this document, unless otherwise specified, "preparation" refers to large-scale preparation under actual production conditions. Those skilled in the art will understand that this differs from preparation under ideal or near-ideal conditions (such as strict control of environment, equipment, conditions, reagents, and operators regardless of cost) (e.g., small-scale preparation by research experts in a high-level laboratory). Large-scale preparation under these conditions is more prone to anomalies due to equipment malfunctions, less stringent environmental or conditional controls, operator errors, and / or even factors with unidentifiable causes, thus affecting the quality of paradefovir mesylate. Therefore, in the application of the second aspect of this invention, the preparation process includes the step of detecting paradefovir mesylate-related substances. Typically, the preparation process of paradefovir mesylate includes the following steps in sequence:

[0018] (1) Synthesize and purify paradefovir mesylate; and

[0019] (2) Detection of paradefovir mesylate-related substances synthesized and purified in step (1).

[0020] Typically, the process for preparing a composition containing paradefovir mesylate includes the following steps:

[0021] (1) Optional detection of paradefovir mesylate-related substances;

[0022] (2) Mix any paladefovir mesylate tested in step (1) with an acceptable excipient (e.g., a pharmaceutically acceptable excipient); and

[0023] (3) Detect paradefovir mesylate-related substances in the composition obtained in step (2).

[0024] In this document, unless otherwise indicated by contradiction, the terms "pharmaceutical composition" and "pharmaceutical formulation" are used interchangeably, referring to a combination of a pharmaceutically active substance and a pharmaceutically acceptable excipient. In this document, "pharmaceuticalally acceptable excipient" means non-toxic fillers, stabilizers, diluents, carriers, solvents, or other pharmaceutical excipients.

[0025] Preferably, in the application of the second aspect of the invention, the compound of the first aspect of the invention is used as a reference. For example, the compound of the first aspect of the invention can be used as a reference for the HPLC detection method described in the specific embodiments of the invention to determine the peak position (retention time) and amount of the compound. Of course, the compound of the first aspect of the invention has a wider range of uses as a reference for other detection methods (especially HPLC detection methods with different conditions).

[0026] More preferably, in the application of the second aspect of the invention, the compound of Formula 1 is added to demonstrate that paradefovir mesylate is destroyed by high temperatures. The inventors have found that, although the possibility of excessively high temperatures during the preparation and storage of paradefovir mesylate or its formulations cannot be ruled out, this is usually caused by excessively high temperatures occurring during the preparation and storage of paradefovir mesylate or its formulations. Therefore, it is necessary to detect, regulate, and control the temperature of the equipment, repair damaged or malfunctioning components, and simultaneously monitor and control the temperature during production, transportation, and storage to ensure it remains within a suitable temperature range.

[0027] More preferably, in the application of the second aspect of the invention, the compound of Formula 2 is added to demonstrate that paradefovir mesylate or its formulations are destroyed by light, oxidation, and / or oxidative heating. The inventors have found that this is usually caused by exposure to light, oxidation, or high oxidative temperatures during the preparation or storage of paradefovir mesylate or its formulations, thus requiring improvements in light protection, heat insulation, sealing, or oxidant isolation during the aforementioned processes. In this case, the compound of Formula 3 is not added.

[0028] More preferably, in the application of the second aspect of the invention, the compounds of Formula 2 and Formula 3 are added to demonstrate that paradefovir mesylate or its preparations are destroyed by acids, alkalis, or high temperatures. The inventors have found that this is usually caused by contact with acids, alkalis, or high temperatures during the production, transportation, and storage of paradefovir mesylate and its preparations. Although the possibility of excessive addition of acidic reagents or excipients during the preparation of paradefovir mesylate or its preparations cannot be ruled out, this is usually caused by uneven distribution of acidic reagents or excipients and excessively high local concentrations during the preparation of paradefovir mesylate or its preparations. Therefore, it is necessary to reduce the rate of addition of acidic reagents or excipients, reduce the concentration of the acidic reagents to be added, and / or increase the mixing power of the mixing equipment. Furthermore, the inventors have found that contact with alkaline reagents or excipients should be completely avoided for paradefovir mesylate and its preparations, as even a very small amount of alkaline reagents or excipients can cause a significant increase in the concentration of Formulas 2 and 3. Simultaneously, storage conditions for paradefovir mesylate and its preparations, such as ambient temperature, are also required.

[0029] Preferably, in the application of the second aspect of the invention, the detection is HPLC detection. For example, the detection can be performed using a high-performance liquid chromatograph with gradient elution on a reversed-phase column, and the elution products can be detected by HPLC.

[0030] The packing material for the reversed-phase chromatography column can be octadecylsilane-bonded silica gel, such as a Shimadzu Intersil-ODS-SP column.

[0031] The length of the chromatographic column can be 150-250 mm, the diameter can be 4-5 mm, and the packing particle size can be 1.8-5 μm. In a specific embodiment of the present invention, for example, the length of the chromatographic column is 250 mm, the diameter is 4.6 mm, and the packing particle size is 3-5 μm.

[0032] The mobile phase consists of mobile phase A and mobile phase B; wherein, mobile phase A is a mixed solution of buffer solution and organic solvent, and mobile phase B is an organic solvent;

[0033] The organic solvent can be methanol or acetonitrile, or a mixture of both;

[0034] The buffer solution is a phosphate buffer solution. Preferably, the phosphate is selected from one or more of sodium phosphate, ammonium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, ammonium dihydrogen phosphate, diammonium hydrogen phosphate, dipotassium hydrogen phosphate, and potassium dihydrogen phosphate, such as a mixture of ammonium dihydrogen phosphate and diammonium hydrogen phosphate. Preferably, the phosphate buffer solution is a 22 mmol / L to 28 mmol / L potassium dihydrogen phosphate solution.

[0035] Gradient elution procedures can be:

[0036] Time: 0 min; Mobile phase A: 90%; Mobile phase B: 10%;

[0037] Time: 25 min; Mobile phase A: 40%; Mobile phase B: 60%;

[0038] Time: 30 min; Mobile phase A: 40%; Mobile phase B: 60%;

[0039] Time: 30.1 min; Mobile phase A: 90%; Mobile phase B: 10%;

[0040] The detection wavelength can be 206nm to 222nm, such as 220nm;

[0041] The gradient elution flow rate is 0.8–1.2 ml / min, preferably 1.0 ml / min.

[0042] The inventors obtained the relevant substances in the actually prepared paradefovir mesylate and its formulations, resolved their structures, and studied their absorption wavelengths. As shown in the table below, their maximum absorption wavelengths are basically in the range of 206–222 nm. However, since 206 nm is a near wavelength, gradient elution can easily lead to baseline instability. Considering the response values ​​of each component, the inventors preferred a detection wavelength of 220 nm.

[0043] Maximum UV absorption wavelength of each component

[0044] Ingredient Absorption maximum nm Formula 1 260 nm with a shoulder at 210 nm Formula 2 262 nm with a shoulder at 210 nm Formula 3 214 nm Formula 4 206 nm, 261 nm Formula 5 206 nm, 260 nm Formula 6 206 nm, 261 nm Formula 7 218 nm, 254 nm Formula 8 261 nm with a shoulder at 210 nm Formula 9 211 nm Formula 10 222 nm, 244 nm Pardafloxate mesylate 260 nm with a shoulder at 210 nm

[0045] Using the HPLC detection method of the present invention, the separation between each related substance peak and between the main peak of paradefovir and its adjacent related substance peaks all achieved baseline separation, with good specificity, as shown in the table below. The HPLC detection method of the present invention has high sensitivity, low detection limit and good linearity.

[0046] Table of sensitivity and linear correction factor results for each component

[0047]

[0048]

[0049] The preferred HPLC detection method of the second aspect of this invention, namely, the method for detecting related substances of paradefovir mesylate, includes using a high-performance liquid chromatograph with a reversed-phase column for gradient elution, and performing HPLC detection on the elution products. Paradefovir mesylate can be either the active pharmaceutical ingredient (API) or a formulation of paradefovir mesylate. Long-term stability tests were conducted on paradefovir mesylate (API) and its formulations under conditions of protection from strong light, maintaining room temperature, isolation from oxidants, and absence of other reagents such as acids and alkalis. The results of this HPLC detection method are shown in the following two tables.

[0050] Related substance stability results of paradefovir mesylate (active pharmaceutical ingredient)

[0051]

[0052] Related substance stability results of paradefovir mesylate formulation

[0053]

[0054] The results show that the compounds of the first aspect of the present invention are of great advantage for the storage of paradefovir mesylate and its preparations. Their application in improving the storage conditions (including storage during the preparation process) of paradefovir mesylate and its preparations can effectively avoid a significant increase in related substances. Even after long-term storage, the related substances of paradefovir mesylate (active drug) hardly increase, and the increase in related substances of its preparations is also very small.

[0055] Preferably, in the application of the second aspect of the invention, the (pharmaceutical) composition containing paradefovir mesylate can be prepared from the following parts by weight:

[0056] Paradefovir mesylate, 10–400 parts;

[0057] 200-550 parts of siliconized microcrystalline cellulose;

[0058] 30-300 parts of pregelatinized starch;

[0059] 1-10 parts of silicon dioxide;

[0060] Stearic acid 1-10 parts;

[0061] 1-20 parts of coating solution.

[0062] More preferably, the (pharmaceutical) composition containing paradefovir mesylate is prepared from the following parts by weight:

[0063] 55 parts of paradefovir mesylate;

[0064] 140 parts of siliconized microcrystalline cellulose;

[0065] 70 parts of pregelatinized starch;

[0066] Two parts silicon dioxide;

[0067] Stearic acid 2.5 parts;

[0068] 13.5 parts coating solution

[0069] The preparation of the (pharmaceutical) composition containing paradefovir mesylate includes the following steps:

[0070] Siliconized microcrystalline cellulose, pregelatinized starch, and silica are dried.

[0071] Take paradefovir mesylate, pulverize and sieve it, and mix it with silica.

[0072] The mixture of paradefovir mesylate, silica, silicified microcrystalline cellulose, and pregelatinized starch is then added, followed by further mixing, tableting, and coating.

[0073] Also preferably in the application of the second aspect of the invention, the (pharmaceutical) composition containing paradefovir mesylate contains 5 to 120 mg of paradefovir, preferably 5, 10, 30, 45, 60, or 75 mg, and most preferably 45 mg.

[0074] In a third aspect, the present invention provides a method for preparing related substances of paradefovir mesylate. The method is capable of directionally synthesizing related substances of paradefovir mesylate, with fewer steps, mild conditions, and high purity. It can be used as a reference standard for the analysis of related substances of paradefovir mesylate raw materials and its preparations, thereby improving the level of quality control.

[0075] Specifically, the present invention provides a method for preparing the compound of Formula 1.

[0076] The preparation reaction of compound 1 is shown below:

[0077]

[0078] The preparation method includes the following steps:

[0079] a. Under nitrogen protection, adefovir, N,N-diethylformamide, and dichloromethane were added to a three-necked flask. Then, oxaloyl chloride was added dropwise. The addition was slightly exothermic and hydrochloric acid gas was generated. After the addition was completed, the temperature was raised to reflux. The system was allowed to become clear, and then concentrated to dryness under reduced pressure at ≤40℃. Dichloromethane was then added to dissolve the substance. The temperature was lowered to ≤10℃, and pyridine was added. The color changed from brown to dark green. The mixture was stirred and left to stand to obtain 2-(6-((diethylamino)methyleneamino)-9H-purine-9-yl)ethoxy)methylphosphoryl dichloride.

[0080] b. In another 30L three-necked flask, add (S)-1-(3-chlorophenyl)propyl-1,3-diol, then add dichloromethane, then add triethylamine, then cool to ≤-60℃, and begin to add a dichloromethane solution of 2-(6-((diethylamino)methyleneamino)-9H-purine-9-yl)ethoxy)methylphosphoryldichloro, until the addition is complete.

[0081] c. Then, warm the mixture in a water bath to ≥-5℃, stir for at least 30 minutes, then add water at ≤0℃, stir and separate the liquids. Add ethyl acetate to the organic phase, then wash with saturated brine. Dry the organic phase with anhydrous sodium sulfate. Filter, and concentrate the filtrate to dryness under reduced pressure at ≤40℃.

[0082] d. The optical isomer was obtained after column chromatography purification.

[0083] Preferably, the organic base mentioned in step a includes triethylamine, diethylamine, ammonia, N,N-diisopropylethylamine, N,N-diethylformamide, N,N-dimethylformamide, N,N-dimethylacetamide, and more preferably N,N-diethylformamide.

[0084] Preferably, the halogenating agent in step a can be acetyl chloride or oxaloyl chloride, with oxaloyl chloride being the most preferred.

[0085] Preferably, the ratio of (S)-1-(3-chlorophenyl)propyl-1,3-diol to 2-(6-((diethylamino)methyleneamino)-9H-purine-9-yl)ethoxy)methylphosphoryldichloro in step b is between 1:1 and 20, preferably 1:1.

[0086] Preferably, the organic solvent in step c can be one or more of methyl tert-butyl ether, petroleum ether, ethyl acetate, methyl acetate, methyl isobutyl ketone, n-butanol, and tert-amyl alcohol, with ethyl acetate being preferred.

[0087] Preferably, step d further includes column chromatography; more preferably, the elution solvent used for chromatography is one or more of dichloromethane, chloroform, n-hexane, ethyl acetate, methyl tert-butyl ether, methanol, and ethanol, preferably a mixture of dichloromethane and methanol in a mixing ratio of 100 to 20:1, more preferably 20:1.

[0088] This invention also provides methods for preparing compounds of formulas 2 and 3, comprising:

[0089] Paradefovir mesylate was reacted with an inorganic base in an organic solvent-water system to produce a mixture of Formula 2 and Formula 3. The individual compounds of Formula 2 or Formula 3 were then separated.

[0090] Preferably, the organic solvent-water system can be a mixed solution of acetonitrile, chloroform, dichloromethane, acetone and water, and more preferably acetonitrile-water.

[0091] Preferably, the organic solvent-water volume ratio is 1 to 3:1, more preferably 1:1 or 2:1, such as 1:1.

[0092] Preferably, the inorganic base can be sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, or lithium hydroxide, with lithium hydroxide being the most preferred.

[0093] Preferably, the ratio of paradefovir mesylate to inorganic base can be 1:1 to 1:5, preferably 1:1.

[0094] Preferably, the separation is performed by HPLC.

[0095] The beneficial effects of this invention are that it provides related substances in paradefovir mesylate and methods for preparing some of these related substances. This not only characterizes the quality of paradefovir mesylate but also reveals abnormalities that occur during actual production, thereby improving the production of the raw material or formulation of paradefovir mesylate and enhancing its drug quality. Furthermore, the HPLC detection method provided by this invention can perform baseline separation of paradefovir mesylate and its related substances, exhibiting good specificity, high sensitivity, low detection limit, and good linearity.

[0096] For ease of understanding, the present invention will be described in detail below with reference to specific embodiments and accompanying drawings. It should be particularly noted that these descriptions are merely illustrative and do not constitute a limitation on the scope of the invention. Many variations and modifications of the invention will be apparent to those skilled in the art based on the discussion in this specification.

[0097] In addition, this invention cites published documents that are used to describe the invention more clearly, and their entire contents are incorporated herein by reference as if they had been repeated herein. Attached Figure Description

[0098] Figure 1 This is a chromatogram of the system suitability solution in Example 3.

[0099] Figure 2 This is a chromatogram of related substances in paradefovir mesylate from Example 4.

[0100] Figure 3 The chromatogram of related substances in the paradefovir mesylate preparation in Example 5 is shown.

[0101] Figure 4The chromatogram overlay of related substances in paradefovir mesylate tablets shows that the spectral lines from bottom to top represent the undegraded solution, the strongly acid-degraded solution, the strongly alkali-degraded solution, the oxidatively degraded solution, the oxidatively heated degraded solution, the heated degraded solution, and the light-degraded solution, respectively. Detailed Implementation

[0102] The present invention is further illustrated by the following examples. Unless otherwise specified, the technical terms and descriptions used in the examples are well known and understood by those skilled in the art, and the compounds not specifically described for synthesis are all commercially available chemically pure or analytically pure products.

[0103] Example 1

[0104] Paradefovir Mesylate Tablets Formula Composition

[0105]

[0106] 1) Weighing and preparing materials: Dry the siliconized microcrystalline cellulose, pregelatinized starch, and silica in an oven at 105°C for 3 hours. Weigh and pulverize paradefovir mesylate, and pass it through an 80-mesh sieve; mix it with silica.

[0107] 2) Mixing: The mixed raw materials and silica mixture are then mixed with silicified microcrystalline cellulose and pregelatinized starch for 10 minutes. Stearic acid is then added and mixed for 8 minutes before discharging.

[0108] 3) Tableting: Control the tablet weight according to the intermediate content and compress into tablets.

[0109] 4) Coating: Coating the uncoated sheets into film-coated sheets.

[0110] Example 2

[0111] Phase II clinical trials and preferred dosage

[0112] A randomized, double-blind, positive-drug parallel-controlled, multicenter, phase II dose-exploration clinical trial design was adopted: using tenofovir disoproxil fumarate tablets as a control, the efficacy of different doses of paradefovir mesylate tablets in the treatment of chronic hepatitis B was evaluated, the safety of different doses of paradefovir mesylate tablets was evaluated, and the optimal safe and effective dose of paradefovir mesylate tablets was explored.

[0113] The 240 subjects were divided into 5 groups: a positive control group receiving 30 mg / dose, 45 mg / dose, 60 mg / dose, and 75 mg / dose of paradefovir mesylate, and 300 mg / dose of tenofovir disoproxil fumarate. The medication was administered orally once daily. The screening period was 14 days, the double-blind trial period was 24 weeks, and the subjects were observed for 4 weeks after the end of the treatment course.

[0114] The primary efficacy endpoint is the logarithmic decrease in serum HBV DNA (PCR method) from baseline after 24 weeks of medication.

[0115] Safety indicators mainly include: vital signs (body temperature, respiration, heart rate, blood pressure); complete blood count and urinalysis;

[0116] Liver function; kidney function; serum electrolytes; coagulation panel; 12-lead electrocardiogram; creatine kinase, creatine kinase isoenzymes, serum amylase, fasting blood glucose, and other possible adverse events.

[0117] Data processing and statistical analysis were performed using SAS 9.4 software. Statistical reports primarily present the analysis results in tables, graphs, and tables (TFLs). Statistical charts are self-explanatory, meaning they have titles, headings, and case numbers. Major / important analyses are supplemented with necessary statistical interpretations, judgments, conclusions, and relevant notes.

[0118] The results show:

[0119] The efficacy of tenofovir disoproxil fumarate tablets at doses of 75 mg, 60 mg, and 45 mg was superior to that of the positive control. The 45 mg group showed a greater advantage in HBeAg seroconversion / serological conversion. The 30 mg group had comparable efficacy to the positive control.

[0120] No major adverse events were observed in any of the studies, and the incidence of adverse events indicated that 30 mg and 45 mg showed the best safety profile.

[0121] Based on the results of the above clinical trials, the optimal formulation strength for this paradefovir mesylate drug composition is 45 mg.

[0122] Example 3

[0123] Preparation of (+)-trans-9-{2-[4-[(S)-(3-chlorophenyl)-2-oxo-1,3,2-dioxophosphahexane-2-methylene]-1-ethyl}adenine methanesulfonate (Formula 1):

[0124]

[0125] Adefovir (29 g), N,N-diethylformamide (13.2 ml), and dichloromethane (370 ml) were added to a 1 L three-necked flask. Oxaloyl chloride (40 ml) was added dropwise with stirring. The system became white and turbid. After reflux for 2.5 h, the system became clear. The solution was concentrated at 40 °C to obtain a yellow semi-solid / semi-liquid. Dichloromethane (230 ml) was added to dissolve the solution. The temperature was lowered to below 10 °C, and pyridine (10.4 ml) was added in portions. The color changed from brown to dark green. The solution was stirred until ready for use, yielding 2-(6-((diethylamino)methyleneamino)-9H-purine-9-yl)ethoxy)methylphosphoryl dichloride.

[0126] Add (S)-1-(3-chlorophenyl)propyl-1,3-diol (20 g) and dichloromethane (280 ml) to another 1 L three-necked flask. Cool the mixture to -30 °C, control the temperature below -20 °C, and slowly add a dichloromethane solution of 2-(6-((diethylamino)methylamino)-9H-purin-9-yl)ethoxymethylphosphorodichloridate to the system. After the addition, warm the mixture to -5 to 5 °C and stir for 30 min. Then, control the temperature below 0 °C and add water (320 ml) to quench the reaction. Separate the organic layer, add ethyl acetate (280 ml) to the organic layer, wash it with saturated brine (40 ml × 1), dry it over anhydrous sodium sulfate, and concentrate it to dryness at 40 °C to obtain a brownish-red oily substance. Add isopropanol:ethanol (95:5, v / v, 280 ml) and acetic acid (32 ml) to the obtained substance, and reflux the mixture for 5 h. Cool the reaction mixture to room temperature, add methanesulfonic acid (13.2 ml), and stir the mixture at room temperature overnight to crystallize. Filter the crystals by suction, concentrate the filtrate to dryness at 85 °C, perform column chromatography (dichloromethane:methanol = 100:1 to 20:1), monitor the eluent by HPLC, collect the eluent, and concentrate it to dryness to obtain a light brown oily substance (about 10 g). Cool the obtained substance to room temperature, add water (50 ml) and acetone (30 ml), and extract the mixture with methyl tert-butyl ether (30 ml × 3). Slowly adjust the pH of the aqueous layer to 7 with saturated sodium bicarbonate solution, and a large amount of off-white solid will precipitate. Stir the mixture at room temperature for 30 min, filter the crystals by suction, wash them with a small amount of water, and air-dry them to obtain an off-white solid (1.1 g).

[0127] EM (calculated value): 423.79; MS (ESI) m / e (M + H) + : 424; m / e (M + H) - : 422

[0128] HPLC purity: 95.85%

[0129] 1 H NMR (600 MHz, DMSO-d 6 ): δ 8.22 (1H, s), δ 8.15 (1H, s), δ 7.24 - 7.43 (6H, m), δ 5.35 - 5.37 (1H, d, J = 12H Z [[ID=...]] ), δ 3.94 - 4.42 (8H, m), δ 2.09 (1H, s), δ 1.82 - 1.84 (1H, d, J1 = 12H Z ).

[0130] 13 C NMR (600 MHz, DMSO-d 6): δ156.47, 152.99, 150.17, 142.5, 141.52, 133.74, 131.02, 128.76, 125.80, 1 24.78, 119.09, 80.15~80.19, 71.33~71.43, 68.47, 66.64, 65.60, 33.74~33.79.

[0131] Example 4

[0132] Preparation of dimethyl (S)-((S)-3-(3-chlorophenyl)-3-hydroxypropyl)hydro(2-(6-amino-9H-purine-9-yl)ethoxy)methylphosphonate (Formula 2):

[0133] Take 2.2 g of paradefovir mesylate, add 110 ml of acetonitrile and 110 ml of water, and add 0.22 g of LiOH dropwise. Stir at 25 °C for 2 h. After neutralization with 1 N HCl and concentration, prepare formula 2 (0.65 g, yield: 28%) and formula 3 (0.72 g, yield: 31%) by Prep-HPLC.

[0134] EM (calculated value): 441.81; MS(ESI)m / e(M+H) + :442.1;

[0135] HPLC purity: 92.40%;

[0136] 1 H NMR (400MHz, DMSO-d) 6 ): δ8.41 (1H, s), δ8.34 (1H, s), δ7.35-7.24 (4H, m), δ4.76 (1H, t, J=6.8H Z ), δ4.53(2H, t, J1=4.8H Z ), δ4.11-4.09 (1H, m), δ4.04-3.97 (3H, m), δ3.82-3.80 (2H, d, J=8.8H Z ), δ1.95-1.91(2H, dd, J1=6.0H Z J1 = 12.4H Z ).

[0137] Example 5

[0138] Preparation of dimethyl methylphosphonate (S)-((S)-1-(3-chlorophenyl)-3-hydroxypropyl)hydro(2-(6-amino-9H-purine-9-yl)ethoxy)methylphosphonate (Formula 3)

[0139] Take 2.2 g of paradefovir mesylate, add 110 ml of acetonitrile and 110 ml of water, and add 0.22 g of LiOH dropwise. Stir at 25 °C for 2 h. After neutralization with 1 N HCl and concentration, prepare formula 2 (0.65 g, yield: 28%) and formula 3 (0.72 g, yield: 31%) by Prep-HPLC.

[0140] EM (calculated value): 441.81; MS(ESI)m / e(M+H) + :442.1.

[0141] HPLC purity: 94.64%.

[0142] 1 H NMR (400MHz, DMSO-d) 6 ): δ8.335 (1H, s), δ8.305 (1H, s), δ7.219-7.368 (4H, m), δ5.38 (1H, d, J=4.8H Z ), δ4.403-4.428(2H, t, J=6.0H Z ), δ3.775-3.800(8H, t, J=3.6H Z ), δ3.513-3.627(4H, m), δ2.01-1.91(2H, m).

[0143] Example 6

[0144] High Performance Liquid Chromatography (HPLC) and Preparation:

[0145] An octadecylsilane-bonded silica gel column (250 × 4.6 mm, 5 μm) was used; 25 mmol / L potassium dihydrogen phosphate solution (3.4 g of potassium dihydrogen phosphate was dissolved in 1000 ml of water by sonication, and the pH was adjusted to 2.5 with phosphoric acid) was used as mobile phase A, and acetonitrile was used as mobile phase B for gradient elution at a flow rate of 1.0 ml / min, a detection wavelength of 220 nm, and a column temperature of 35 °C.

[0146] The gradient elution process is as follows: from 0 to 25 min, the volume ratio of mobile phase A to mobile phase B gradually changes from 90:10 to 40:60 at a constant rate; from 25 to 30 min, the volume ratio of mobile phase A to mobile phase B is 40:60; and at 30.1 min, the volume ratio of mobile phase A to mobile phase B immediately changes to 90:10.

[0147] Sample preparation:

[0148] System suitability solutions: Accurately weigh approximately 10 mg of each of the reference standards of formulas 4, 5, 6, 7, and 8, place them in a 50 ml volumetric flask, dissolve them in methanol and dilute to the mark, and shake well. These are the stock solutions for each reference standard. Take approximately 5 mg of each of the reference standards of formulas 1, 2, 3, and 9, place them in a 10 ml volumetric flask, dissolve them in 3 ml of methanol, dilute to the mark with water, and shake well. These are the stock solutions for each reference standard. Take approximately 5 mg of the reference standard of formula 10, place it in a 10 ml volumetric flask, dissolve it in 2 ml of acetonitrile, dilute to the mark with water, and shake well. This is the stock solution for reference standard 10. Separately, take approximately 5 mg of paradefovir mesylate, place it in a 10 ml volumetric flask, add 50 μl of each of the above impurity stock solutions, dilute to the mark with water, and shake well.

[0149] Experimental procedure: Inject 20 μl of the system suitability solution and record the chromatogram.

[0150] Typical chromatograms are shown below. Figure 1 ,Depend on Figure 1 It can be seen that the resolution between each related substance peak, and between the main peak of paradefovir mesylate and its adjacent related substance peaks, is greater than 1.5, indicating good baseline separation.

[0151] Example 7

[0152] The high-performance liquid chromatography conditions are the same as in Example 6.

[0153] Sample preparation:

[0154] Test solution: Weigh approximately 10 mg of paradefovir mesylate (raw material), place it in a 50 ml volumetric flask, add an appropriate amount of water to dissolve and dilute to the mark, shake well, and use it as the test solution.

[0155] Test procedure: Inject 20 μl of the test solution and record the chromatogram. See [link to typical chromatogram]. Figure 2 .

[0156] Example 8

[0157] The high-performance liquid chromatography conditions are the same as in Example 6.

[0158] Sample preparation:

[0159] Test solution: Take 10 tablets of 45mg paradefovir mesylate from Example 1, grind them into a fine powder and mix them evenly. Take an appropriate amount of the fine powder (containing about 10mg of paradefovir), weigh it accurately, put it in a 50ml volumetric flask, add an appropriate amount of water, sonicate to dissolve and dilute to the mark, shake well, and use it as the test solution.

[0160] Test procedure: Inject 20 μl of the test solution and record the chromatogram. See [link to typical chromatogram]. Figure 3 .

[0161] Example 9

[0162] Preparation of forced degradation test solution:

[0163] Undegraded sample: Weigh an appropriate amount of finely powdered paradefovir mesylate tablets (containing approximately 10 mg of paradefovir), place it in a 25 ml volumetric flask, add water to dissolve and dilute to the mark, shake well, take 1 ml, place it in a 2 ml volumetric flask, add water to dilute to the mark, shake well, centrifuge, and the sample is obtained.

[0164] Acid degradation sample: Weigh an appropriate amount of finely powdered paradefovir mesylate tablets (containing approximately 10 mg of paradefovir), place it in a 5 ml volumetric flask, add water to dissolve and dilute to the mark, shake well, take 1 ml, place it in a 10 ml volumetric flask, add 1 ml of 1 mol / L hydrochloric acid, let it stand at room temperature for 1 hour, add 1 ml of 1 mol / L sodium hydroxide to neutralize, add water to dilute to the mark, shake well, centrifuge, and the sample is obtained.

[0165] Alkali-degraded sample: Weigh an appropriate amount of finely powdered paradefovir mesylate tablets (containing approximately 10 mg of paradefovir), place it in a 5 ml volumetric flask, add water to dissolve and dilute to the mark, shake well, take 1 ml, place it in a 10 ml volumetric flask, add 0.5 ml of 0.1 mol / L sodium hydroxide, let it stand at room temperature for 2 minutes, add 0.5 ml of 0.1 mol / L hydrochloric acid to neutralize, add water to dilute to the mark, shake well, centrifuge, and the sample is obtained.

[0166] Oxidative degradation sample: Weigh an appropriate amount of finely powdered paradefovir mesylate tablets (containing approximately 10 mg of paradefovir), place it in a 5 ml volumetric flask, add water to dissolve and dilute to the mark, shake well, take 1 ml, place it in a 10 ml volumetric flask, add 1 ml of 30% hydrogen peroxide, let it stand at room temperature for 1 hour, add water to dilute to the mark, shake well, centrifuge, and the sample is obtained.

[0167] Oxidative heating degradation of samples: Weigh an appropriate amount of finely powdered paradefovir mesylate tablets (containing approximately 10 mg of paradefovir), place it in a 5 ml volumetric flask, add water to dissolve and dilute to the mark, shake well, take 1 ml, place it in a 10 ml volumetric flask, add 1 ml of 30% hydrogen peroxide, place it at room temperature for 1 hour, place it in a 60℃ oven for 30 minutes, add water to dilute to the mark, shake well, centrifuge, and the sample is obtained.

[0168] Thermally degraded sample: Weigh an appropriate amount of finely powdered paradefovir mesylate tablets (containing approximately 10 mg of paradefovir), place it in a 25 ml volumetric flask, add water to dissolve and dilute to the mark, shake well, take 1 ml, place it in a 2 ml volumetric flask, place it at 105℃ for 30 min, add water to dilute to the mark, shake well, centrifuge, and the sample is obtained.

[0169] Photodegradation sample: Weigh an appropriate amount of finely powdered paradefovir mesylate tablets (containing approximately 10 mg of paradefovir), place it in a 25 ml volumetric flask, add water to dissolve and dilute to the mark, shake well, take 1 ml, place it in a 2 ml volumetric flask, irradiate under strong light (4000 lx ± 500 lx) for 24 hours, add water to dilute to the mark, shake well, centrifuge, and the sample is obtained.

[0170] Experimental procedure: Take 20 μl of each of the above solutions and inject them separately. The chromatographic conditions are as described in Example 6, and the chromatograms are recorded.

[0171] A superimposed diagram of the chromatograms of the various degraded solutions is shown in the appendix. Figure 4 From bottom to top, these represent undamaged solutions, strongly acid-damaged solutions (with a significant increase in compounds of formulas 2 and 3), strongly base-damaged solutions (with a significant increase in compounds of formulas 2 and 3), oxidatively damaged solutions, oxidatively heated solutions (with a significant increase in formula 2), solutions heated (with a significant increase in formulas 1, 2, and 3), and light-damaged solutions (with a significant increase in formula 2).

[0172] Therefore, in paradefovir mesylate, the addition of compound 1 can indicate its degradation by heating; compound 2 alone can be used to indicate its degradation by light, oxidation, and oxidative heating; the combined addition of compounds 2 and 3 can be used to indicate its degradation by acid, alkali, and high temperature, indicating that during the preparation of paradefovir mesylate, there may be excessive addition of alkaline or acidic reagents, uneven distribution of alkaline or acidic reagents resulting in excessively high local concentrations, or excessively high temperatures. Based on these results, after step (2) in the process of preparing paradefovir mesylate or step (1) or (3) in the process of preparing a composition containing paradefovir mesylate, it is determined to replace paradefovir mesylate with paradefovir mesylate to continue the subsequent steps, or to improve the previous preparation steps or storage conditions and repeat the preparation.

[0173] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. The use of one or more of the compounds represented by formulas 1 to 10 isolated below in the preparation of paradefovir mesylate or compositions containing paradefovir mesylate, wherein, The preparation process includes a step of detecting paradefovir mesylate related substances, wherein one or more compounds represented by formulas 1 to 10 are paradefovir mesylate related substances. 。 2. The application according to claim 1, wherein, The detection method is HPLC detection.

3. The application according to claim 1 or 2, wherein, The isolated compounds, one or more of the following formulas 1 to 10, were used as reference standards.

4. The application according to claim 3, wherein, The addition of the compound in Formula 1 was used to demonstrate that paradefovir mesylate is destroyed by high temperatures.

5. The application according to claim 3, wherein, The addition of compounds in Formula 2 is used to demonstrate that paradefovir mesylate is destroyed by light, oxidation, and / or oxidative heating.

6. The application according to claim 3, wherein, Compounds of Formulas 2 and 3 are added to demonstrate that paradefovir mesylate is destroyed by acids, alkalis, high humidity and / or high temperatures.

7. The application according to claim 1, wherein, The composition containing paradefovir mesylate is prepared from the following parts by weight: Paradefovir mesylate, 10–400 parts; 200-550 parts of siliconized microcrystalline cellulose; 30-300 parts of pregelatinized starch; 1-10 parts of silicon dioxide; Stearic acid 1-10 parts; and 1-20 parts of coating solution.

8. The application according to claim 7, wherein, The composition containing paradefovir mesylate is prepared from the following parts by weight: 55 parts of paradefovir mesylate; 140 parts of siliconized microcrystalline cellulose; 70 parts of pregelatinized starch; Two parts silicon dioxide; Stearic acid 2.5 parts; and 13.5 parts of coating solution.

9. The application according to claim 7 or 8, wherein the composition comprising paradefovir mesylate contains 5, 10, 30, 45, 60 or 75 mg of paradefovir.

10. The application of claim 9, wherein the composition comprising paradefovir mesylate contains 45 mg of paradefovir.

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