Encephaline isomer impurity, and preparation method, detection method and application thereof

Abstract

The invention belongs to the technical field of pharmaceuticals, and relates to an ensafenetine isomer impurity as well as a preparation method, a detection method and application thereof, the ensafenetine isomer impurity provided by the invention can provide a reference substance for quality detection of ensafenetine, and has very important guiding significance for medication safety of ensafenetine.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of pharmacy, and particularly relates to an enoximone isomer impurity and a preparation method, detection method and application thereof. BACKGROUND

[0002] Enoximone, also known as ensitifene, is a new type of first inhaled PDE3 and PDE4 dual inhibitor with anti-inflammatory activity and bronchodilator effect. Inhibition of PDE can cause smooth muscle relaxation by regulating cyclic guanosine phosphate levels. In a parallel controlled phase 3 clinical trial, enoximone administered by a standard nebulizer significantly improved lung function and dyspnea.

[0003] In the synthesis process of enoximone, when bromine substitutes the primary amine in intermediate 1 during the substitution reaction of intermediate 1 and N-(2-bromoethyl) phthalimide, bromine may also attack the oxygen of the amide to generate an ether impurity, which will participate in the subsequent reaction to generate derivative impurity compound B of enoximone.

[0004]

[0005] Currently, there is a report of enoximone oxygen isomer (CN 109641891A). The secondary amine of intermediate 1 reacts with an alkyl halide under alkaline conditions to generate a tertiary amine intermediate 2, and the carbonyl group often undergoes enol isomerization to form an enol structure. The hydroxyl group of the enol structure will undergo substitution reaction with the halogenated alkane to generate an ether compound, forming an oxygen isomer impurity. However, there are unverified impurities in the process. The existence of impurities poses a great risk to the quality of the drug and the safety of patients. Therefore, determining the structure and detection method of unknown impurities is of great significance to improve the quality of the drug substance. SUMMARY

[0006] To solve the above technical problems, the application provides an enoximone isomer impurity, a preparation method, a detection method and an application thereof. The enoximone isomer impurity provided by the application can provide a control sample for enoximone quality detection, and has very important guiding significance for enoximone drug safety.

[0007] To achieve this purpose, the application adopts the following technical solutions:

[0008] In a first aspect, the application provides an enoximone isomer impurity, which has the structure of compound A:

[0009] .

[0010] There is a report of enantiomers of entacapone (CN 109641891A). The inventors accidentally discovered another isomer impurity during the process development. The impurity was confirmed as a nitrogen isomer impurity by nuclear magnetic resonance, mass spectrometry and microcrystalline diffraction. The generation of the nitrogen isomer impurity is similar to the generation of the oxygen isomer impurity in CN 109641891A. The generation mechanism of the nitrogen isomer impurity obtained by the present application may be that the imine also undergoes an enol isomerization phenomenon similar to the carbonyl group, the imine is converted into an enamine isomer, and the secondary amine of the enamine undergoes a substitution reaction with an alkyl halide to generate the nitrogen isomer impurity. The polarity of the isomer impurity is very similar to that of the drug substance. In the analysis and detection, the impurity cannot be separated from the drug substance peak, which leads to the inability to effectively control the residual amount of the impurity, and poses a great risk to the quality of the drug and the safety of the patients.

[0011] The present application prepares a reference substance of the impurity and optimizes the analysis method of the drug substance using the reference substance, so that the residual amount of the impurity can be effectively detected in the drug substance, thereby realizing the quality control of the isomer impurity and ensuring the safety, effectiveness and controllable quality of the drug substance. Therefore, the present application has important significance for improving the quality of the drug substance by determining the structure of the isomer impurity, analyzing its source and generation mechanism, and further optimizing the process parameters to control its content.

[0012] In a second aspect, the present application provides a preparation method of the entacapone isomer impurity according to the first aspect, which comprises the following steps:

[0013] (1) reacting compound 1 and compound 2 to obtain compound 3 and compound 3-1;

[0014] (2) reacting compound 3 in step (1) with hydrazine hydrate to obtain compound 4;

[0015] (3) reacting compound 4 in step (2) with cyanate to obtain the entacapone isomer impurity compound A;

[0016] The reaction formula of the preparation method is as follows:

[0017] .

[0018] Preferably, in step (1), the molar ratio of compound 1 to compound 2 is 1: (3-6) (for example, it can be 1:3, 1:4, 1:5, 1:6, etc.).

[0019] Preferably, in step (1), the reaction is carried out in the presence of a catalyst.

[0020] Preferably, the catalyst comprises potassium carbonate and sodium iodide.

[0021] Preferably, the molar ratio of compound 1, potassium carbonate and sodium iodide is 1:(1-9):(2.5-9).

[0022] Preferably, the catalyst comprises sodium acetate and sodium iodide.

[0023] Preferably, the molar ratio of compound 1, sodium acetate and sodium iodide is 1:(1-9):(2.5-9).

[0024] The values ​​1-9 can be 1, 2, 4, 6, 8, 9, etc., independently; 2.5-9 can be 2.5, 4, 6, 8, 9, etc., independently.

[0025] Preferably, in step (1), the reaction is carried out in the presence of a solvent.

[0026] Preferably, the solvent includes any one or a combination of at least two of 2-butanone, toluene, or chlorobenzene.

[0027] Preferably, in step (1), the reaction temperature is 80-135℃ (e.g., 80℃, 100℃, 120℃, 135℃, etc.), and the time is 24-69h (e.g., 24h, 36h, 48h, 69h, etc.).

[0028] Preferably, in step (1), the reaction further includes a post-processing step: filtering the reaction solution and purifying the filtrate by column chromatography to obtain compound 3.

[0029] Preferably, the eluent used in the column purification includes dichloromethane and methanol.

[0030] Preferably, the volume ratio of dichloromethane to methanol is (20-50):1 (for example, it can be 20:1, 25:1, 30:1, 35:1, 50:1, etc.).

[0031] Preferably, in step (2), the molar ratio of compound 3 to hydrazine hydrate is 1:(5.7-11.5) (for example, it can be 1:5.7, 1:7, 1:8, 1:10, 1:11.5, etc.).

[0032] Preferably, in step (2), the reaction is carried out in the presence of a solvent.

[0033] Preferably, the solvent includes any one or a combination of two of dichloromethane or ethanol.

[0034] Preferably, in step (2), the reaction temperature is 25-35℃ (e.g., 25℃, 28℃, 30℃, 35℃, etc.), and the time is 2-6h (e.g., 2h, 3h, 4h, 5h, 6h, etc.).

[0035] Preferably, in step (2), the reaction further includes a post-processing step: filtering the reaction solution, concentrating the filtrate to dryness, re-dissolving, filtering again, and concentrating the filtrate to dryness again to obtain compound 4.

[0036] Preferably, in step (3), the cyanate is sodium cyanate.

[0037] Preferably, in step (3), the molar ratio of compound 4 to sodium cyanate is 1:(2.1-4.5) (for example, it can be 1:2.1, 1:2.5, 1:3, 1:3.5, 1:4.5, etc.).

[0038] Preferably, in step (3), the reaction is carried out in the presence of an acid solution, wherein the acid is hydrochloric acid.

[0039] Preferably, in step (3), the reaction temperature is 70-80℃ (e.g., 70℃, 72℃, 75℃, 78℃, 80℃, etc.), and the time is 2-4h (e.g., 2h, 2.5h, 3h, 3.5h, 4h, etc.).

[0040] Preferably, in step (3), the reaction further includes a post-processing step: mixing and extracting the reaction solution and an organic solvent, and then performing column purification on the organic phase to obtain the encefentin isomer impurity compound A.

[0041] Preferably, the eluent used in the column purification includes dichloromethane and methanol.

[0042] Preferably, the volume ratio of dichloromethane to methanol is (20-50):1 (for example, it can be 20:1, 25:1, 30:1, 35:1, 50:1, etc.).

[0043] Thirdly, the present invention provides a method for detecting encefentin isomer impurities according to the first aspect, the method comprising: detecting encefentin isomer impurities by liquid chromatography to obtain the content of the encefentin isomer impurities.

[0044] Preferably, the mobile phase of the liquid chromatography includes mobile phase A and mobile phase B, wherein mobile phase A is an aqueous solution of ammonium acetate at 8-12 mmol (e.g., 8 mmol, 9 mmol, 10 mmol, 11 mmol, 12 mmol, etc.), and mobile phase B is acetonitrile.

[0045] Preferably, the elution procedure of the liquid chromatography is isocratic elution.

[0046] Preferably, the volume ratio of mobile phase A to mobile phase B is 40:60-55:45 (for example, it can be 40:60, 45:55, 50:50, 55:45, etc.).

[0047] Preferably, the chromatographic column used in the liquid chromatography is a phenylbutyl column based on an organic hybrid silica matrix.

[0048] Fourthly, the present invention provides an application of the encefentin isomer impurities according to the first aspect in the quality detection of encefentin.

[0049] Compared with the prior art, the present invention has at least the following beneficial effects:

[0050] The encefentin isomer impurities provided by this invention can serve as a reference standard for the quality testing of encefentin, which is of great guiding significance for the safety of encefentin use. At the same time, obtaining the structure of unknown impurities is of great significance for determining their source and generation mechanism, thereby optimizing the process to control their content. Attached Figure Description

[0051] Figure 1 This is the mass spectrometry result of compound A.

[0052] Figure 2 This is the NMR spectrum of compound A.

[0053] Figure 3 This is an MRI scan of Enfertin.

[0054] Figure 4 This is the absolute configuration diagram of compound A.

[0055] Figure 5 This is the chromatogram of the test sample in Application Example 1.

[0056] Figure 6 This is the chromatogram of the test sample in Application Example 2.

[0057] Figure 7 This is a chromatogram of encefentin in Comparative Application Example 1.

[0058] Figure 8 This is a chromatogram of compound A in Comparative Application Example 1. Detailed Implementation

[0059] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0060] In this invention, the reaction formula for preparing the encefenstein isomer impurity compound A is as follows:

[0061]

[0062] Compound 1 is chemically named 9,10-dimethoxy-2-(2,4,6-trimethylaniline)-6,7-pyrimidin[6,1-A]isoquinoline-4-one, and is sourced from commercially available sources.

[0063] Compound 2 is chemically named N-(2-bromoethyl)phthalimide and is sourced from commercially available sources.

[0064] Example 1

[0065] This embodiment provides a method for preparing an encefenstein isomer impurity compound A, comprising the following steps:

[0066] (1) 30g of compound 1 (0.077mol), 95.5g of potassium carbonate (0.691mol), 68.5g of sodium iodide (0.457mol), 117g of compound 2 (0.460mol), and 750mL of 2-butanone were added to the reaction flask. The mixture was heated to 80℃ and refluxed for 48 hours. After cooling to 25℃, the mixture was filtered. The filtrate was concentrated and passed through a column. After elution with dichloromethane / methanol = 50 / 1 (v / v), the mixture was dried to obtain 0.8g of compound 3.

[0067] (2) Add 0.8g (1.4mmol) of compound 3, 12mL of dichloromethane, 20mL of ethanol and 0.5g (8mmol) of hydrazine hydrate to the reaction flask, react at 25℃ for 2h, add 0.5g of hydrazine hydrate and continue the reaction for 4 hours, filter, concentrate the filtrate to dryness, add dichloromethane, filter again, concentrate the filtrate to dryness to obtain 0.6g of compound 4;

[0068] (3) Add 0.6 g of compound 4 (1.38 mmol), 20 mL of water, 3 mL of 1 M hydrochloric acid, and sodium cyanate aqueous solution (0.2 g, 3.1 mol sodium cyanate dissolved in 3.5 mL of water) to the reaction flask, heat to 80 °C and react for 2 hours, cool to 25 °C, extract twice with dichloromethane, 20 mL each time, combine and concentrate the organic phases and pass through a column, elute with dichloromethane / methanol = 33 / 1 (v / v) to obtain 0.25 g of encefentin isomer impurity compound A, yield 37.9%, purity 99.17%.

[0069] Mass spectrometry was performed on compound A and encefentin, respectively. The mass spectra of compound A are shown below. Figure 1 Like encefentin, it also yielded an ion peak at M+1=478, consistent with the molecular weight of encefentin (477). NMR analysis was performed on compound A and encefentin respectively. Figure 2 and Figure 3 As shown, the two compounds are basically identical, except that a methylene group at position 4.0 of encefentin moves to position 3.9 in compound A and superimposes with the hydrogen of the methoxy group.

[0070] The above mass spectrometry and NMR analysis confirmed that compound A is an isomer of encefentin, but its absolute configuration could not be determined. Therefore, cryo-electron microscopy was performed on compound A to obtain its absolute configuration ( Figure 4 ).

[0071] Example 2

[0072] This embodiment provides a method for preparing an encefenstein isomer impurity compound A, comprising the following steps:

[0073] (1) 30g of compound 1 (0.077mol), 6.3g of sodium acetate (0.077mol), 28.9g of sodium iodide (0.193mol), 58.7g of compound 2 (0.231mol) and 750mL of toluene were added to the reaction flask. The mixture was heated to 110℃ and refluxed for 24 hours. After cooling to 25℃, the mixture was filtered. The filtrate was concentrated and passed through a column. After elution with dichloromethane / methanol = 50 / 1 (v / v), the mixture was dried to obtain 0.5g of compound 3.

[0074] (2) Add 0.5g (0.9mmol) of compound 3, 12mL of dichloromethane, 20mL of ethanol and 0.5g (8mmol) of hydrazine hydrate to the reaction flask, react at 35°C for 2 hours and filter. Concentrate the filtrate to dryness, add dichloromethane, filter again, and concentrate the filtrate to dryness to obtain 0.3g of compound 4.

[0075] (3) Add 0.3g of compound 4 (0.7mmol), 20mL of water, 3mL of 1M hydrochloric acid, and sodium cyanate aqueous solution (0.2g, 3.1mol sodium cyanate dissolved in 3.5mL of water) to the reaction flask, heat to 70℃ and react for 3 hours, cool to 25℃, extract twice with dichloromethane, 20mL each time, combine and concentrate the organic phases and pass through a column, elute with dichloromethane / methanol = 33 / 1 (v / v) to get 0.06g of encefentin isomer impurity compound A, yield 18.2%, purity 96.12%.

[0076] Example 3

[0077] This embodiment provides a method for preparing an encefenstein isomer impurity compound A, comprising the following steps:

[0078] (1) 30g of compound 1 (0.077mol), 56.8g of sodium acetate (0.693mol), 103.9g of sodium iodide (0.693mol), 117g of compound 2 (0.460mol), and 750mL of chlorobenzene were added to the reaction flask. The mixture was heated to 135℃ and refluxed for 69 hours. After cooling to 25℃, the mixture was filtered. The filtrate was concentrated and passed through a column. After elution with dichloromethane / methanol = 50 / 1 (v / v), the mixture was dried to obtain 1.2g of compound 3.

[0079] (2) 1.2 g (2.1 mmol) of compound 3, 12 mL of dichloromethane, 20 mL of ethanol and 1.5 g (24 mmol) of hydrazine hydrate were added to the reaction flask. After reacting at 25 °C for 4 hours, the mixture was filtered, the filtrate was concentrated to dryness, dichloromethane was added, the mixture was filtered again, and the filtrate was concentrated to dryness to obtain 0.9 g of compound 4.

[0080] (3) Add 0.9 g of compound 4 (2.07 mmol), 20 mL of water, 4.5 mL of 1 M hydrochloric acid, and sodium cyanate aqueous solution (0.3 g, 4.5 mol sodium cyanate dissolved in 3.5 mL of water) to the reaction flask. Heat to 80 °C and react for 4 hours. Cool to 25 °C and extract twice with dichloromethane, 20 mL each time. Combine and concentrate the organic phases and pass through a column. Elute with dichloromethane / methanol = 33 / 1 (v / v) to obtain 0.32 g of encefentin isomer impurity compound A. The yield is 32.4% and the purity is 95.32%.

[0081] Application Example 1

[0082] This application example provides a method for detecting compound A in enstatin active pharmaceutical ingredient, including the following steps:

[0083] (1) Preparation of test sample

[0084] Diluent: Acetonitrile

[0085] Solution of compound A: Weigh 5 mg of compound A into a 25 mL volumetric flask, add acetonitrile and sonicate to dissolve, then dilute to the mark and shake well;

[0086] Encerfentin raw material solution: Weigh 5 mg of encerfentin raw material into a 25 mL volumetric flask, add acetonitrile and sonicate to dissolve, then dilute to the mark and shake well;

[0087] (2) Liquid chromatography detection: The test sample is tested under the following conditions:

[0088] Mobile phase A: 10 mmol ammonium acetate; Mobile phase B: acetonitrile;

[0089] Chromatographic column: YMC-Triart Phenyl 250×4.6mm, 3μm, 12nm;

[0090] Column temperature: 35℃

[0091] Elution gradients are shown in Table 1:

[0092] Table 1

[0093]

[0094] The results are as follows Figure 5As shown, compound A had a peak elution time of 4 min, and encerfentin had a peak elution time of 6 min. The two components were completely separated, and the running time could be controlled within 10 min, saving running time and mobile phase.

[0095] Application Example 2

[0096] This application example provides a method for detecting compound A in enstatin active pharmaceutical ingredient, which differs from application example 1 only in step (2):

[0097] Elution gradients are shown in Table 2:

[0098] Table 2

[0099]

[0100] Other applications can be referenced in Example 1.

[0101] The results are as follows Figure 6 As shown, the elution time of compound A was 4 min, and the elution time of encerfentin was 6 min, which was not significantly different from the chromatographic conditions in application example 1.

[0102] Comparative Application Example 1

[0103] This comparative application example provides a method for detecting compound A in enstatin active pharmaceutical ingredient, which differs from application example 1 only in step (2):

[0104] The conditions are as follows:

[0105] Mobile phase A: water, mobile phase B: acetonitrile;

[0106] Chromatographic column: YMC-Triart Phenyl 250×4.6mm, 3μm, 12nm;

[0107] Column temperature: 35℃;

[0108] Elution gradients are shown in Table 3:

[0109] Table 3

[0110]

[0111] Encerfentin API chromatogram as follows Figure 7 As shown, the spectrum of compound A is as follows: Figure 8 As shown, ensifine did not produce a peak in this system.

[0112] The above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. An encefenstein isomer impurity, characterized in that, The encefentin isomer impurity has the structure shown in compound A: 。 2. A method for preparing the encefenstein isomer impurity according to claim 1, characterized in that, The preparation method includes the following steps: (1) Compound 1 and compound 2 react to give compound 3 and compound 3-1; (2) Take compound 3 from step (1) and react it with hydrazine hydrate to obtain compound 4; (3) React compound 4 from step (2) with cyanate to obtain the encefentin isomer impurity compound A; The reaction formula for the preparation method is as follows: 。 3. The preparation method according to claim 2, characterized in that, In step (1), the molar ratio of compound 1 to compound 2 is 1:(3-6). In step (1), the reaction is carried out in the presence of a catalyst, the catalyst comprising potassium carbonate and sodium iodide, wherein the molar ratio of compound 1, potassium carbonate and sodium iodide is 1:(1-9):(2.5-9); or the catalyst comprises sodium acetate and sodium iodide, wherein the molar ratio of compound 1, sodium acetate and sodium iodide is 1:(1-9):(2.5-9). In step (1), the reaction is carried out in the presence of a solvent, which includes any one or a combination of at least two of 2-butanone, toluene, or chlorobenzene.

4. The preparation method according to claim 2, characterized in that, In step (1), the reaction temperature is 80-135℃ and the time is 24-69h.

5. The preparation method according to claim 2, characterized in that, In step (2), the molar ratio of compound 3 to hydrazine hydrate is 1:(5.7-11.5). In step (2), the reaction is carried out in the presence of a solvent, which includes any one or a combination of two of dichloromethane or ethanol; In step (2), the reaction temperature is 25-35℃ and the time is 2-6h; In step (2), the reaction also includes a post-processing step: filtering the reaction solution, concentrating the filtrate to dryness, re-dissolving, filtering again, and concentrating the filtrate to dryness again to obtain compound 4.

6. The preparation method according to claim 2, characterized in that, In step (3), the cyanate is sodium cyanate; the molar ratio of compound 4 to sodium cyanate is 1:(2.1-4.5). In step (3), the reaction is carried out in the presence of an acid solution, wherein the acid is hydrochloric acid.

7. The preparation method according to claim 2, characterized in that, In step (3), the reaction temperature is 70-80℃ and the time is 2-4h.

8. A method for detecting encerfentin isomer impurities according to claim 1, characterized in that, The detection method includes: using liquid chromatography to detect encefentin isomer impurities, and obtaining the content of the encefentin isomer impurities.

9. The detection method according to claim 8, characterized in that, The mobile phase of the liquid chromatography method includes mobile phase A and mobile phase B, wherein mobile phase A is an 8-12 mmol aqueous solution of ammonium acetate and mobile phase B is acetonitrile; The elution program of the liquid chromatography is isocratic elution, and the volume ratio of mobile phase A to mobile phase B is 40:60-55:

45. The chromatographic column used in the liquid chromatography method is a phenylbutyl column based on an organic hybrid silica matrix.

10. The application of the encefentin isomer impurity according to claim 1 in the quality detection of encefentin.

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