Oseltamivir azide derivatives, preparation method and uses thereof

Abstract

The present invention discloses an oseltamivir azide derivative, the structure of which is shown as impurity 338 or impurity 340. The two impurities were first discovered during the preparation process of oseltamivir phosphate, and have relatively obvious toxicity. Therefore, it is necessary to control the content of these two impurities in oseltamivir phosphate to be no higher than 30 ppm to reduce the safety risks existing during the use of oseltamivir phosphate.

Description

Technical Field

[0001] The present invention relates to the technical field of drug synthesis and analysis, and involves impurities, preparation, and analysis and detection thereof during the production process of bulk drugs; more specifically, it relates to an oseltamivir azide derivative, a preparation method thereof, and its use as a reference substance in quality control. Background Art

[0002] Oseltamivir phosphate (OST) is a potent antiviral drug used to treat influenza A and B. It inhibits the viral enzyme neuraminidase, preventing the virus from budding, infectivity, and replication from host cells. Oseltamivir phosphate is a prodrug ester that is metabolized by hepatic esterases in the liver to oseltamivir carboxylate; its half-life is 1.8 hours. After oral administration, 80% of the metabolite shows a therapeutic effect of approximately 30 minutes, and 20% of the drug will be present in the plasma for 3 - 4 hours.

[0003] Oseltamivir phosphate was first approved by the FDA in October 1999. The dosing method for its use in the treatment of adult influenza is twice a day, 75 mg each time, for five consecutive days, while the dosing method for its use in the prevention of adult influenza is once a day, 75 mg each time, for 10 consecutive days. The structure of oseltamivir phosphate is shown as follows:

[0004]

[0005] The USP2021 monograph of Oseltamivir Phosphate discloses an oseltamivir azide related substance with a structure as shown in Formula I. This related substance may be introduced during the synthesis and preparation of the bulk drug using sodium azide. Pikkili Viswanath et al. defined the compound shown in Formula I as a genotoxic impurity in the article "Method Development and Validation for the Trace Level Quantification of Genotoxic Impurity Oseltamivir Phosphate Related Compound - A in Oseltamivir Phosphate using LC - MS" and proposed an effective control method.

[0006]

[0007] John C. Rohloff et al. disclosed the route and method for preparing oseltamivir phosphate from AS7 as shown below in the article "Practical Total Synthesis of the Anti - Influenza Drug GS - 4104" (source: J. Org. Chem. 1998, 63, 13, 4545–4550, hereinafter referred to as "Literature 1").

[0008]

[0009] AS9 is one of the commonly used intermediates in the synthesis of oseltamivir phosphate API. It is prepared from AS7 through azide ring - opening and acetylation reactions successively. There are multiple sites on the cyclohexene ring in the structure of AS7 where nucleophilic addition reactions of azide groups can occur, thus possibly generating impurities of AS9 analogs and remaining in the oseltamivir phosphate API, which may have an unpredictable impact on the quality of oseltamivir phosphate API and its preparations. Summary of the Invention

[0010] The object of the present invention is to provide an oseltamivir azide derivative. The oseltamivir azide derivative was first discovered and identified during the preparation process of oseltamivir phosphate, and it is an impurity of oseltamivir phosphate.

[0011] Another object of the present invention is to provide a preparation method of the oseltamivir azide derivative.

[0012] Still another object of the present invention is to provide the application of the oseltamivir azide derivative as a reference substance in the quality control of oseltamivir phosphate.

[0013] Yet another object of the present invention is to provide a method for the quality control of oseltamivir phosphate.

[0014] The above objects of the present invention are achieved through the following solutions:

[0015] An oseltamivir azide derivative, whose structure is one of impurity 338 or impurity 340:

[0016]

[0017] In the process of preparing oseltamivir phosphate, two compounds, impurity 338 and impurity 340, were discovered for the first time. Even after oseltamivir is recrystallized and then made into oseltamivir phosphate, the contents of these two impurities are significantly reduced, but they may still exist in oseltamivir phosphate. In view of the obvious toxicity of these two impurities, it is necessary to detect the contents of these two impurities in oseltamivir phosphate to control their contents not to exceed the specified limits. According to the TTC method of ICH M7, it is necessary to control the contents of these two impurities in oseltamivir phosphate to be no higher than 30 ppm to meet the standard.

[0018] The present invention also protects the preparation methods of impurity 338 and impurity 340. The preparation processes of impurity 338 and impurity 340 are as follows: AS7 is mixed with ammonium chloride and sodium azide in N,N-dimethylformamide and heated under an inert gas atmosphere for reaction; after the reaction is completed, the solvent is removed, the obtained mixture is dissolved in dichloromethane, and triethylamine and acetic anhydride are added, and the reaction is carried out under low temperature conditions; after the reaction is completed, the solvent is removed and the target compound is obtained through purification.

[0019] Preferably, in the preparation processes of impurity 33 and impurity 340, the molar dosage ratio of AS7 to ammonium chloride and sodium azide is 1:1.7 - 2.2:2.0 - 3.0. More preferably, in the preparation processes of impurity 33 and impurity 340, the molar dosage ratio of AS7 to ammonium chloride and sodium azide is 1:1.9 - 2.1:2.2 - 2.7.

[0020] More preferably, in the preparation processes of impurity 33 and impurity 340, the molar dosage ratio of AS7 to ammonium chloride and sodium azide is 1:2:2.5.

[0021] Preferably, in the preparation processes of impurity 33 and impurity 340, the molar dosage ratio of AS7 to triethylamine and acetic anhydride is 1:1.0 - 1.5:0.7 - 1.2.

[0022] More preferably, in the preparation processes of impurity 33 and impurity 340, the molar dosage ratio of AS7 to triethylamine and acetic anhydride is 1:1.0 - 1.3:0.9 - 1.0.

[0023] More preferably, in the preparation processes of impurity 33 and impurity 340, the molar dosage ratio of AS7 to triethylamine and acetic anhydride is 1:1.2:0.98.

[0024] Preferably, in the preparation processes of impurity 33 and impurity 340, the temperature for heating reaction under an inert gas atmosphere is 50 - 70 °C; more preferably, the temperature is 60 °C.

[0025] Preferably, the inert gas is nitrogen.

[0026] Preferably, the temperature for reaction under low temperature conditions is 0 - 5 °C.

[0027] The present invention also protects the use of oseltamivir azide derivatives as reference substances in the quality control of oseltamivir phosphate.

[0028] Preferably, the mass of impurity 338 and impurity 340 in oseltamivir phosphate is controlled to be not higher than 30 ppm each.

[0029] The present invention also protects a method for quality control of oseltamivir phosphate, which uses impurity 338 and / or impurity 340 as reference substances for detection and analysis to control the content of impurity 338 and impurity 340 in oseltamivir phosphate.

[0030] Preferably, the mass of impurity 338 and impurity 340 in oseltamivir phosphate is controlled to be not higher than 30 ppm each.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] In the preparation process of oseltamivir phosphate, the present invention discovers two impurities, namely impurity 338 and impurity 340 for the first time, and these two impurities have obvious toxicity. Therefore, it is necessary to control the content of these two impurities in oseltamivir phosphate to be not higher than 30 ppm to reduce the safety risks existing in the use of oseltamivir phosphate. Description of the Drawings

[0033] Figure 1 It is the HPLC detection and analysis result of sample AS9 in Example 1.

[0034] Figure 2 It is the HPLC detection and analysis result of the crude oseltamivir phosphate in Example 1.

[0035] Figure 3 It is the mass spectrum of impurity 338.

[0036] Figure 4 It is the mass spectrum of impurity 340.

[0037] Figure 5 It is for impurity 338 1 1H-NMR spectrum.

[0038] Figure 6 It is for impurity 338 13 13C-NMR spectrum.

[0039] Figure 7 It is for impurity 340 1 1H-NMR spectrum.

[0040] Figure 8 It is for impurity 340 13 13C-NMR spectrum. Detailed Embodiments

[0041] The present invention will be further elaborated in detail below in conjunction with specific embodiments. The embodiments are only used to explain the present invention and are not used to limit the scope of the present invention. Unless otherwise specified, the test methods used in the following embodiments are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels.

[0042] Example 1 Discovery of Impurity 338 and Impurity 340

[0043] Oseltamivir phosphate is prepared by reacting with AS7 (AS7 is a commercial product). The specific reaction process is carried out according to the steps described in Document 1: John C. Rohloff, Kenneth M. Kent, Michael J. Postich, et al. Practical Total Synthesis of the Anti-Influenza Drug GS-4104. J. Org. Chem. 1998, 63, 4545 - 4550. The reaction route is as follows:

[0044]

[0045]

[0046] AS7, AS8, and AS9 in the above route correspond to Compound 19, Compound 6, and Compound 20 in Document 1 respectively; that is, for the preparation of AS8, AS7 is used as the reaction material and the reaction is carried out with reference to the preparation process of Compound 6 in Document 1 to obtain AS8; then, using AS8 as the reaction material, the reaction is carried out with reference to the preparation process of Compound 20 in Document 1 to obtain AS9. Finally, AS9 undergoes an Aza-Wittig reaction to obtain the free base AS10, and then is salted to prepare oseltamivir phosphate.

[0047] AS9 Analysis Method

[0048] Take the AS9 sample prepared by the above method, dissolve it with a 50% acetonitrile solution and dilute to make a solution containing about 1 mg per 1 mL as the test solution; separately take an appropriate amount of the oseltamivir phosphate intermediate AS9 reference substance (purchased externally), dissolve it with a 50% acetonitrile solution and dilute to make a solution containing about 1 mg of oseltamivir phosphate intermediate AS9 per 1 mL as the reference solution. Carry out the test according to the high performance liquid chromatography method (General Principles 0512, Volume IV, Chinese Pharmacopoeia 2015 Edition), using octadecylsilane chemically bonded silica gel as the filler (Kromasil C18 4.6 mm × 250 mm, 5 μm or a chromatographic column with equivalent efficiency); use a 0.01 mol / L potassium dihydrogen phosphate solution (adjust the pH value to 6.0 with a 1 mol / L potassium hydroxide solution)-acetonitrile (45:55) as the mobile phase; the detection wavelength is 207 nm. Precisely measure 20 μL of each of the reference solution and the test solution, inject them into the liquid chromatograph respectively, and record the chromatogram until 4 times the retention time of the main peak.

[0049] AS9 Sample Detection and Analysis Results

[0050] The HPLC detection results of the AS9 sample are shown in Figure 1 , and the retention times of 3.974 min and 9.002 min are the impurities 340 and 338 respectively.

[0051] Obtaining and Structure Confirmation of Target Impurities: Take about 2 g of the AS9 sample, separate and purify it by semi-preparative liquid phase (chromatographic column: HPLCONE-5C18C (30 mm I.D. × 250 mm), detection wavelength: 207 nm, flow rate: 20 mL / min, column temperature: 25 °C, mobile phase: acetonitrile-water (45:55)), and collect the elution components corresponding to the retention times of 3.974 min and 9.002 min in Figure 1 .

[0052] The mass spectrum of the component corresponding to RT 9.002 min is shown in Figure 3 .

[0053] The mass spectrum of the component corresponding to RT 3.974 min is shown in Figure 4 .

[0054] The 1 1H-NMR of the component corresponding to RT 9.002 min and 13 13C-NMR spectra are shown in Figure 5 and Figure 6 .

[0055] The 1 1H-NMR of the component corresponding to RT 3.974 min and 13 13C-NMR spectra are shown in Figure 7 and Figure 8 .

[0056] Upon identification, Figure 1 the structures of the corresponding components with RT 9.002 min and RT 3.974 min in Figure 1 are shown as Impurity 338 and Impurity 340 respectively:

[0057]

[0058] Analysis of Crude Oseltamivir Phosphate

[0059] Test solution: Take about 30 mg of the sample, weigh accurately, place it in a 10 mL volumetric flask, dilute to the mark with the mobile phase, shake well, and obtain the solution immediately.

[0060] Analysis method

[0061] Determine according to the high performance liquid chromatography method (General Principles 0512, Volume IV, Chinese Pharmacopoeia 2020 Edition) and the tandem mass spectrometry method (General Principles 0431, Volume IV, Chinese Pharmacopoeia 2020 Edition).

[0062] Chromatographic conditions:

[0063] Chromatographic column: Octadecylsilane chemically bonded silica gel as the filler (Kromasi1 C18 4.6 mm × 250 mm, 5 μm or a chromatographic column with equivalent efficiency);

[0064] Mobile phase: 0.01 mo1 / L potassium dihydrogen phosphate solution (adjust the pH value to 6.0 with 1 mo1 / L potassium hydroxide solution) - acetonitrile (45:55);

[0065] Detection wavelength: 207 nm;

[0066] Flow rate: 1.0 ml / min;

[0067] Column temperature: 35 °C;

[0068] Injection volume: 20 μL.

[0069] Detection and analysis results of oseltamivir phosphate sample

[0070] Figure 2 In Figure 2 , the retention time of the peak of Impurity 338 is RT 19.135 min, and the retention time of Impurity 340 is RT 12.483 min.

[0071] Preparation of Impurity 338 and Impurity 340 in Example 2

[0072] Take a certain amount of AS7, dissolve it in N,N-dimethylformamide at 20 °C (the dosage is 2 g / mL relative to AS7), then add ammonium chloride (the dosage is 2 times the molar mass of AS7) and sodium azide (the dosage is 2.5 times the molar mass of AS7), displace nitrogen, and stir at 60 °C for 42 h. After the reaction is completed, remove the solvent by distillation under reduced pressure. The obtained solid mixture is dissolved in dichloromethane (the dosage is 3.2 times v / w relative to the solid mixture), add triethylamine (the dosage is 1.2 times the molar mass of AS7), cool down to 0 - 5 °C, and dropwise add acetic anhydride (the dosage is 0.98 times the molar mass of AS7). The dropping takes about 1 h, and the reaction is completed in 2 - 3 h. Remove the solvent by distillation under reduced pressure. The obtained product is separated and purified by semi-preparative liquid phase (chromatographic column: HPLCONE-5C18C (30 mm I.D.×250 mm), detection wavelength: 207 nm, flow rate: 20 mL / min, column temperature: 25 °C, mobile phase: acetonitrile - water (45:55)), and impurity 338 and impurity 340 are obtained successively. The yields (relative to AS7) are 30.2% and 28.7% respectively.

[0073] In vitro cytotoxicity experiment of impurity 338 and impurity 340 in Example 3

[0074] The cytotoxicity of impurity 338 and impurity 340 was tested by the method disclosed in the prior art, and the results are shown in Table 1.

[0075] Table 1 Cytotoxicity results of impurity 338 and impurity 340

[0076]

[0077] As can be seen from the above table, impurity 338 and impurity 340 have significant toxicity to at least one of the liver L02 cells, myocardial H9c2 cells, embryonic kidney HEK-293 cells, and nerve SH-SY5Y cells. If their concentrations in the drug are not controlled, it is very likely to produce certain toxic side effects.

[0078] It should also be noted that although Figure 1 the contents of various impurities are all lower than 0.1%, in the actual process, many factors will cause fluctuations in the impurity content. These factors include but are not limited to: ① equipment modification or upgrade; ② proficiency of process personnel; ③ change of batch, etc. And in view of the obvious toxicity of impurity 338 and impurity 340, according to the TTC method of ICH M7, it is necessary to control the contents of these two impurities in oseltamivir phosphate not to exceed 30 ppm to meet the standard. Therefore, for safety reasons, it is necessary to detect the contents of these two impurities in oseltamivir phosphate to control their contents to meet the standard. This requires using impurity 338 and impurity 340 as impurity reference standards for detection and analysis.

[0079] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description and ideas. It is not necessary and impossible to enumerate all implementation manners here. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. An oseltamivir azido derivative, characterized in that: Its structure is one of impurity 338 or impurity 340:

2. The method for preparing the oseltamivir azido derivative according to claim 1, characterized in that: The preparation process of impurity 338 and impurity 340 is as follows: AS7 is mixed with ammonium chloride and sodium azide in N,N-dimethylformamide, and heated to react under an inert gas atmosphere; after the reaction is completed, the solvent is removed, the resulting mixture is dissolved in dichloromethane, and triethylamine and acetic anhydride are added, and the reaction is carried out under low temperature conditions; after the reaction is completed, the solvent is removed, and the target compound is obtained after purification; The reaction temperature under the low temperature condition is 0-5°C.

3. The method for preparing the oseltamivir azido derivative according to claim 2, characterized in that: During the preparation of impurities 338 and 340, the molar ratio of AS7 to ammonium chloride and sodium azide is 1:1.7-2.2:2.0-3.

0.

4. The method for preparing the oseltamivir azido derivative according to claim 2, characterized in that: During the preparation of impurity 338 and impurity 340, the molar ratio of AS7 to triethylamine and acetic anhydride is 1:1.0-1.5:0.7-1.

2.

5. The method for preparing the oseltamivir azido derivative according to claim 2, characterized in that: During the preparation of impurities 338 and 340, the heating reaction temperature is 50-70° C. under an inert gas atmosphere.

6. Use of the oseltamivir azido derivative according to claim 1 as a reference substance in the quality control of oseltamivir phosphate, characterized in that: The masses of impurity 338 and impurity 340 in oseltamivir phosphate are controlled to be no higher than 30 ppm.

7. A method for quality control of oseltamivir phosphate, characterized in that: The impurity 338 and / or impurity 340 described in claim 1 are used as reference substances for detection and analysis, and the content of impurity 338 and impurity 340 in oseltamivir phosphate is controlled to be no higher than 30 ppm.

Images