A preparation method and use of impurity A

Abstract

The present invention relates to a method for preparing an impurity A and its application as an impurity reference substance in the detection of compound A-51. By studying the impurities of compound A-51, the present invention obtains an impurity reference substance (impurity A) of A-51. The structure of impurity A is the deprotected hydrochloride of compound A-51. The purity of impurity A prepared by the present invention reaches more than 98%, which can be used as a reference substance for impurity research for the content determination of impurity A in compound A-51, and can effectively ensure the drug safety of the finished drug A.

Description

Technical Field

[0001] The present invention relates to but is not limited to the field of pharmaceutical chemistry, and in particular to a method for preparing a compound and its use. Background Art

[0002] The new RHO-related protein inhibitor 6-(4-((4-(1H-pyrazol-4-yl)phenyl)amino)pyrimidin-2-yl)-1-methyl-1H-indol-2-yl)(3,3-difluoroazetidine-1-yl)methanone (Formula A) is the world's first small molecule innovative drug that inhibits the infection of the new coronavirus to host cells by highly selectively inhibiting ROCK2 kinase, thereby achieving a significant effect in combating the new coronavirus. The mechanism of action of the new RHO-related protein inhibitor is to achieve antiviral effects by inhibiting viral invasion and replication by highly selectively inhibiting the ROCK2 target. This is a new antiviral mechanism that has a significant effect in inhibiting the new coronavirus in in vitro experiments and in vivo animal experiments; it also has anti-inflammatory and anti-fibrosis effects. The new RHO-related protein inhibitor is different from the traditional anti-new coronavirus drugs currently under development globally and domestically. It is a Class I small molecule oral new coronavirus treatment drug with a new mechanism, new target, and new structure.

[0003] 2-Chloro-N-(4-(1-(1-ethoxyethyl)-1H-pyridin-4-yl)phenyl)pyrimidin-4-amine (see WO2022 / 042711, intermediate A-51) is an important intermediate of the novel RHO-related protein inhibitor 6-(4-((4-(1H-pyrazol-4-yl)phenyl)amino)pyrimidin-2-yl)-1-methyl-1H-indol-2-yl)(3,3-difluoroazetidine-1-yl)methanone (raw material finished product A, referred to as "finished product A"). The quality control of this intermediate is crucial. If these impurities are not strictly controlled, the quality of the novel RHO-related protein inhibitor finished product will be seriously affected, and serious toxic side effects may be caused to the human body.

[0004] The significance of the present invention lies in guiding the process of preparing a 2-chloro-N-(4-(1-(1-ethoxyethyl)-1H-pyridin-4-yl)phenyl)pyrimidine-4-amine intermediate, strictly controlling its impurity N-[4-(1H-pyrazol-4-yl)phenyl]-2-chloropyrimidine-4-amine (impurity A), improving product quality, making product quality more controllable, and achieving more guaranteed safety of drugs.

[0005]

[0006] (6-(4-((4-(1H-pyrazol-4-yl)phenyl)amino)pyrimidin-2-yl)-1-methyl-1H-indol-2-yl)(3,3-difluoroazetidin-1-yl)methanone

[0007]

[0008] 2-Chloro-N-(4-(1-(1-ethoxyethyl)-1H-pyridin-4-yl)phenyl)pyrimidin-4-amine

[0009]

[0010] N-[4-(1H-pyrazol-4-yl)phenyl]-2-chloropyrimidin-4-amine

[0011] Patent WO 2022 / 012409A1 discloses a ROCK inhibitor, its preparation method and use, and specifically discloses the structure and preparation method of impurity A. However, this method requires the use of dichloromethane, which has a low boiling point. Long-term inhalation of dichloromethane can cause damage to the body's central nervous system and symptoms such as dizziness, headache, and nausea. At the same time, the hydrogen chloride ethanol solution used is relatively expensive, which is not in line with the concept of green economy.

[0012] Therefore, there is an urgent need for a cleaner, safer and more stable method for preparing impurity A compound to guide the process improvement of finished product A, improve product quality, make product quality more controllable, and ensure the safety of drugs. Summary of the invention

[0013] In view of the above technical status, the present invention provides a preparation method of impurity A and its use, which is beneficial to drug safety by controlling the content of the impurity in the preparation process.

[0014] The present invention provides a method for preparing impurity A, the method comprising:

[0015] (1) Add compound A-51 to the reaction flask, and then add 2-8 times the weight of C 1-6 Alcoholic solvents;

[0016] (2) adding 1-6 molar equivalents of an acid reagent to the reaction bottle of step (1) at a temperature of 0-50° C. for 1-5 hours. After the addition is complete, stirring the reaction for 1-5 hours until the solution becomes a white suspension;

[0017] (3) Filter the white suspension obtained in step (2) and add 1-3 times the weight of C-51 1-6 The filter cake is then washed with an alcohol solvent and collected;

[0018] (4) The filter cake obtained in step (3) is dried under vacuum at 30-80° C. for 1-10 h to obtain a yellow-white solid, which is impurity A;

[0019] The structure of compound A-51 is shown below:

[0020]

[0021] The structure of the impurity A is shown below:

[0022]

[0023] In the method of the present invention, as one of the embodiments, the method further comprises: C in the step (1) 1-6 The alcohol solvent is selected from methanol, ethanol, butanol, or isopropanol, preferably methanol, ethanol, and more preferably ethanol.

[0024] In the method of the present invention, as one of the embodiments, the method further comprises, in the step (1), preferably adding 3 times the weight of C 1-6 of alcohol solvents.

[0025] In the method of the present invention, as one of the embodiments, the method further comprises that the acid reagent in step (2) is selected from hydrochloric acid, sulfuric acid, nitric acid, and glacial acetic acid, preferably hydrochloric acid and sulfuric acid, and more preferably hydrochloric acid.

[0026] In the method of the present invention, as one of the embodiments, the acid reagent in step (2) is hydrochloric acid with a mass fraction of 3.65%-36%, preferably 20%-36% hydrochloric acid, and the optimal range is 32%-36%.

[0027] In the method of the present invention, as one of the embodiments, 2-4 molar equivalents, more preferably 3 molar equivalents of an acid reagent are added in step (2).

[0028] In the method of the present invention, as one of the embodiments, the method further comprises that in the step (2), the reaction temperature is 25-35°C, preferably 30°C, the dropwise addition time is 1-2h, preferably 1h, and the reaction time is 2-3h, preferably 3h.

[0029] In the method of the present invention, as one of the embodiments, the method further comprises: C in the step (3) 1-6 The alcohol solvent is the same as the alcohol solvent in step (1).

[0030] In the method of the present invention, as one embodiment, the method further comprises: in the step (3), 1 times the weight of C 1-6 Alcohol solvent elution.

[0031] In the method of the present invention, as one of the embodiments, the method further comprises that in the step (4), the drying temperature is 40-50°C, preferably 45°C, the vacuum degree of the oven is 0.07-0.095MPa, preferably 0.085-0.095MPa, more preferably 0.095MPa, and the drying time is 1-10h, preferably 6-8h, more preferably 8h.

[0032] In the method of the present invention, as one of the embodiments, the method further comprises:

[0033] (1) Add compound A-51 to a reaction flask, and then add 3 times the weight of ethanol;

[0034] (2) Add 3 mol equivalents of 32% hydrochloric acid to the reaction bottle of step (1) at 30° C. for 1 hour. After the addition is complete, stir the reaction for 3 hours to turn into a white suspension;

[0035] (3) filtering the white suspension obtained in step (2), rinsing with 1 times the weight of ethanol, and collecting the filter cake;

[0036] (4) The filter cake obtained in step (3) is dried at 45±5°C and a vacuum degree of 0.085 MPa-0.095 MPa for 6-8 h to obtain a yellow-white solid, which is impurity A.

[0037] As an exemplary explanation, the technical solution reaction equation adopted by the present invention is:

[0038]

[0039] The present invention also provides the use of the compound impurity A prepared by any of the aforementioned methods in the detection of compound A-51, wherein the impurity A is used as a reference substance for compound A-51 to detect the content of impurity A in A-51.

[0040] In the use of the present invention, as one of the embodiments, the impurity A is prepared by the following method:

[0041] (1) Add compound A-51 to a reaction flask, and then add 3 times the weight of ethanol;

[0042] (2) Add 3 mol equivalents of 32% hydrochloric acid to the reaction bottle of step (1) at 30° C. for 1 hour. After the addition is complete, stir the reaction for 3 hours to turn into a white suspension;

[0043] (3) filtering the white suspension obtained in step (2), rinsing with 1 times the weight of ethanol, and collecting the filter cake;

[0044] (4) The filter cake obtained in step (3) is dried at 45±5°C and a vacuum degree of 0.085 MPa-0.095 MPa for 6-8 h to obtain a yellow-white solid, which is impurity A.

[0045] The present invention also provides a method for detecting the content of impurity A in A-51 by using the compound impurity A prepared by any of the aforementioned methods as a reference substance, the method comprising:

[0046] (1) preparing impurity A according to any of the above methods;

[0047] (2) Determination of the content of related substance impurity A in A-51:

[0048] HPLC instrument: high performance liquid chromatography Waters e2695, detector: 2489UV, chromatographic column: octadecyl bonded silica gel chromatographic column (150×4.6mm, 3um), flow rate: 0.8ml / min, column temperature: 25℃, detection wavelength: 254nm, injection volume: 5ul;

[0049] Mobile phase: 0.05% trifluoroacetic acid water was used as mobile phase A, methanol was used as mobile phase B, and gradient elution was performed. The gradient conditions are as follows:

[0050] Time (min) Mobile phase A (%) Mobile phase B (%) 0 80 20 2 80 20 20 10 90 28 10 90 28.5 80 20 35 80 20

[0051] Test solution: Take 20 mg of A-51, weigh accurately, place in a 100 ml volumetric flask, add acetonitrile to dissolve, dilute to the mark, shake well, and obtain;

[0052] 1% self-control solution: Take 1 ml of the above A-51 test solution, place it in a 100 ml volumetric flask, add acetonitrile to dilute to the scale line, repeat the injection 6 times, the peak area RSD ≤ 2.0%, retention time ≤ 2.0%.

[0053] In the method of the present invention, as one of the embodiments, the detection limit of impurity A in the compound A-51 should be less than or equal to 0.5%, preferably less than or equal to 0.2%.

[0054] The purpose of the present invention is: Impurity A is an impurity generated during the preparation of compound A-51. Through experimental research, it is found that impurity A does not participate in the subsequent reaction, and impurity A is transferred to the finished product A by impurity transfer. It is particularly important to control impurity A during the process. At the same time, no other literature reports on the synthesis process of this impurity. By controlling the content of this impurity during the preparation process, it is beneficial to the safety of the drug.

[0055] The beneficial effects obtained by the present invention are as follows: by studying the synthesis process of the impurity reference substance (impurity A) of A-51, impurity A is prepared by using new starting materials through different synthesis routes, thereby obtaining a cleaner, safer and more stable method for preparing impurity A compounds.

[0056] The structure of impurity A is the deprotected hydrochloride of compound A-51. Its hydrogen spectrum is measured (400 MHz, DSMO): 1.03 (m, 1H, -CH) 1.05 (m, 2H, -CH) 2.06 (m, 2H, -CH) 3.91 (m, 4H, -CH) 1.00 (m, 1H, -NH) 3.86 (m, 1H, -NH). The purity of impurity A is more than 95% through HPLC. It can be used as a reference substance for impurity research and for the content determination of compound A-51, which can effectively ensure the drug safety of compound A-51. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 The HPLC spectrum of impurity A is shown in FIG.

[0058] Figure 2 This is the hydrogen spectrum of impurity A. DETAILED DESCRIPTION

[0059] The following examples are used to further illustrate the present invention, but are not intended to limit the effective scope of the present invention in any way.

[0060] In the examples of this application, the instrument models used are as follows:

[0061] NMR: 400MHz NMR (solvent: DSMO, TMS: internal standard)

[0062] HPLC instrument: High performance liquid chromatography Waters e2695, detector: 2489UV, chromatographic column: octadecyl bonded silica gel chromatographic column (150×4.6mm, 3um). Flow rate: 0.8ml / min. Column temperature: 25℃. Detection wavelength: 254nm. Injection volume: 5ul.

[0063] Mobile phase: 0.05% trifluoroacetic acid water was used as mobile phase A, methanol was used as mobile phase B, and gradient elution was performed. The gradient conditions are shown in the following table.

[0064]

[0065]

[0066] The reagents used are as follows:

[0067] name factory batch number A-51 Hebei Dingtai Pharmaceutical Co., Ltd. TDI01-51-220905P3 hydrochloric acid Modern Oriental Development Technology Co., Ltd. 202210016 Ethanol Modern Oriental Development Technology Co., Ltd. 202205015

[0068] The present invention provides the use of the impurity A as a reference substance in the quality control of the raw materials or preparations of compound A.

[0069] Example 1 Screening of hydrochloric acid concentration

[0070] Experimental methods:

[0071] A. Add compound A-51 to the reaction bottle, then add 3 times ethanol to dissolve it, and add different concentrations of 3 molar equivalents of hydrochloric acid reagent dropwise at 30°C for 1 hour. After the addition is complete, stir the reaction for 3 hours to turn it into a white suspension.

[0072] B. Filter the above white suspension, rinse with 1 times the weight of ethanol solvent, collect the filter cake, and dry it in vacuum at 45°C with a vacuum degree of 0.095 MPa for 8 hours to obtain a yellow-white solid, which is impurity A.

[0073] Experimental results: See Table 1. According to the reaction mechanism and monitoring during the reaction, there is no obvious side reaction in this process. The higher the hydrochloric acid concentration, the faster the reaction should be. Too low a hydrochloric acid concentration can easily lead to incomplete reaction.

[0074] Table 1

[0075]

[0076]

[0077] Example 2 Screening of reaction time

[0078] Experimental methods:

[0079] A. Add compound A-51 to the reaction bottle, then add 3 times ethanol to dissolve it, and add 3 molar equivalents of 36% hydrochloric acid reagent dropwise at 30°C for 1 hour. After the addition is complete, stir the reaction for 1, 2, 3, 4, and 5 hours respectively to turn it into a white suspension.

[0080] B. Filter the above white suspension, rinse with 1 times the weight of ethanol solvent, collect the filter cake, and dry it in vacuum at 45°C with a vacuum degree of 0.095 MPa for 8 hours to obtain a yellow-white solid, which is impurity A.

[0081] Experimental results: see Table 2. According to the reaction mechanism and monitoring during the reaction, the most appropriate reaction time is 3 h. A reaction time less than 3 h will result in incomplete reaction of the raw materials, while a reaction time greater than 3 h will waste resources and easily cause other side reactions.

[0082] Table 2

[0083]

[0084]

[0085] Example 3 Screening of reaction temperature

[0086] Experimental methods:

[0087] A. Add compound A-51 to the reaction bottle, then add 3 times ethanol to dissolve it, and add 3 molar equivalents of 36% hydrochloric acid reagent dropwise at 10°C, 20°C, 30°C, 40°C, and 50°C for 1 hour. After the addition is complete, stir the reaction for 3 hours to turn it into a white suspension.

[0088] B. Filter the above white suspension, rinse with 1 times the weight of ethanol solvent, collect the filter cake, and dry it in vacuum at 45°C with a vacuum degree of 0.095 MPa for 8 hours to obtain a yellow-white solid, which is impurity A.

[0089] Experimental results: see Table 3. According to the reaction mechanism and monitoring during the reaction, the most suitable reaction temperature is 30°C. When the reaction temperature is lower than 30°C, the reaction rate is very slow. When the reaction temperature is higher than 30°C, it is easy to cause an increase in side reactions and a decrease in product quality.

[0090] Table 3

[0091] Reaction temperature Yield purity Hydrochloric acid concentration Reaction time 10℃ 59％ 75.24％ 37％ 3h 20℃ 73％ 83.12％ 37％ 3h 30℃ 89％ 98.63％ 37％ 3h 40℃ 88％ 97.98％ 37％ 3h 50℃ 87％ 92.43％ 37％ 3h

[0092] Example 4 Use of impurity A as a reference substance

[0093] The compound impurity A in the present invention is a process impurity generated in the production process of A-51. Its identification and detection are of great significance for A-51 and 6-(4-((4-(1H-pyrazol-4-yl)phenyl)amino)pyrimidin-2-yl)-1-methyl-1H-indol-2-yl)(3,3-difluoroazetidine-1-yl)methanone (A), and provide important support for the quality control of the raw material finished product A.

[0094] The preparation, identification and detection of the compounds in the present invention can guide the optimization of the A-51 production process and improve the yield and product quality.

[0095] (1) Preparation of reference substance impurity A:

[0096] A. Add compound A-51 to the reaction bottle, then add 3 times ethanol to dissolve it, and add 3 molar equivalents of 32% hydrochloric acid reagent dropwise at 30°C for 1 hour. After the addition is complete, stir the reaction for 3 hours to turn it into a white suspension.

[0097] B. Filter the above white suspension, rinse with 1 times the weight of ethanol solvent, collect the filter cake, and dry it in vacuum at 45°C with a vacuum degree of 0.095MPa for 8 hours to obtain a yellow-white solid, which is impurity A. (Yield: 92%, purity 98.45%) The test results are shown in the attached Figure 1 ,1 H NMR see attached Figure 2 .

[0098] Figure 1 The details of the HPLC spectrum are shown in the following table:

[0099]

[0100]

[0101] Figure 2 The hydrogen spectrum of is analyzed as follows:

[0102] (400MHz, DSMO) 1.03 (m, 1H, -CH) 1.05 (m, 2H, -CH) 2.06 (m, 2H, -CH) 3.91 (m, 4H, -CH) 1.00 (m, 1H, -NH) 3.86 (m, 1H, -NH)

[0103] A-51 Route

[0104]

[0105] (2) Determination of the content of related substance impurity A in A-51

[0106] HPLC instrument: high performance liquid chromatography Waters e2695, detector: 2489UV, chromatographic column: octadecyl bonded silica gel chromatographic column (150×4.6mm, 3um), flow rate: 0.8ml / min, column temperature: 25℃, detection wavelength: 254nm, injection volume: 5ul;

[0107] Mobile phase: 0.05% trifluoroacetic acid water was used as mobile phase A, methanol was used as mobile phase B, and gradient elution was performed. The gradient conditions are as follows:

[0108]

[0109]

[0110] Test solution: Take 20 mg of A-51, weigh accurately, place in a 100 ml volumetric flask, add acetonitrile to dissolve, dilute to the mark, shake well, and obtain;

[0111] 1% self-control solution: Take 1 ml of the above A-51 test solution, place it in a 100 ml volumetric flask, add acetonitrile to dilute to the scale line, repeat the injection 6 times, the peak area RSD ≤ 2.0%, retention time ≤ 2.0%.

[0112] (3) By using the above content detection method to measure the content of impurity A, the quality comparison after process improvement is as follows:

[0113] name batch number Yield purity Related substance impurity A A-51 20220701 70 94.5％ 3.32％ Before improvement A-51 20220704 72 95.3％ 3.12％ Before improvement A-51 20220802 76 98.17％ 0.33％ After Improvement A-51 20220905 82 99.54％ 0.20％ After Improvement A-51 20220907 84 99.82％ 0.07％ After Improvement

[0114] It can be seen from the production data that after the structure of the impurities prepared by the present invention is determined, the mechanism of their generation in the actual production process is speculated, which guides the process optimization to improve the product yield, while avoiding the generation of the impurities and improving the product quality of A-51, which has important guiding significance for the production of A-51.

[0115] Comparative Example 1

[0116] 1. Place tert-butyl 4-(4-((2-chloropyrimidin-4-yl)amino)phenyl)-1H-pyridine-1-carboxylate in a 250ml three-necked flask and add dichloromethane to dissolve.

[0117] 2. Add ethanol solution of hydrogen chloride (33%), stir at room temperature for 2 hours, and concentrate under reduced pressure to obtain impurity A. (Yield 38.79%, purity 62.21%)

[0118] Comparative Example 2

[0119] 1. Add compound A-51 to the reaction bottle, and then add 3 times ethanol to dissolve. Add 3 molar equivalents of 27% hydrochloric acid reagent dropwise at 30°C for 1 hour. After the addition is complete, stir the reaction for 3 hours to turn into a white suspension.

[0120] 2. Filter the white suspension, rinse with 1 times the weight of ethanol solvent, collect the filter cake, and dry it under vacuum at 45°C with a vacuum degree of 0.095 MPa for 8 hours to obtain a yellow-white solid, which is impurity A. (Yield 54.87%, purity 76.74%)

[0121] The present invention can prepare impurity A compound cleanly, safely, stably and at low cost. The significance of the preparation of impurity A is to guide the improvement of A process, improve product quality, and ensure the safety of drugs.

[0122] The above description is only the best specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent substitutions or changes according to the technical solution and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for preparing impurity A, characterized in that: The method comprises: (1) Add compound A-51 to a reaction flask, and then add 2-4 times the weight of ethanol solvent; (2) adding 2-4 molar equivalents of a 32% to 36% hydrochloric acid reagent to the reaction bottle of step (1) at a temperature of 25-35° C., the addition time is controlled to be 1-2 hours, and after the addition is completed, the reaction is stirred for 2-4 hours to turn into a white suspension; (3) filtering the white suspension obtained in step (2), eluting with 1-3 times the weight of A-51 feed ethanol solvent, and collecting the filter cake; (4) The filter cake obtained in step (3) is dried under vacuum at 30-80° C. for 1-10 h to obtain a yellow-white solid, which is impurity A; The structure of compound A-51 is shown below: The structure of the impurity A is shown below:

2. The method according to claim 1, characterized in that The method further comprises adding 3 times the weight of ethanol solvent in step (1).

3. The method according to claim 1, characterized in that In the step (2), 3 molar equivalents of hydrochloric acid reagent are added.

4. The method according to claim 1, characterized in that: The reaction time in step (2) is 2-3 hours.

5. The method according to claim 4, characterized in that The dropwise addition time in step (2) is 1 h and the reaction time is 3 h.

6. The method according to claim 1, characterized in that In the step (3), 1 times the weight of ethanol solvent is used for elution.

7. The method according to claim 1, characterized in that In the step (4), the drying temperature is 40-50° C., the vacuum degree of the oven is 0.085-0.095 MPa, and the drying time is 6-8 hours.

8. The method according to claim 7, characterized in that In the step (4), the drying temperature is 45° C., the vacuum degree of the oven is 0.095 MPa, and the drying time is 8 h.

9. The method according to claim 1, characterized in that: The method specifically comprises: (1) Add compound A-51 to a reaction flask, and then add 3 times the weight of ethanol; (2) Add 3 mol equivalents of 32% hydrochloric acid to the reaction bottle of step (1) at 30° C. for 1 hour. After the addition is complete, stir the reaction for 3 hours to turn into a white suspension; (3) filtering the white suspension obtained in step (2), eluting with ethanol solvent in an amount of 1 times the weight of A-51, and collecting the filter cake; (4) The filter cake obtained in step (3) is dried at 45±5°C and a vacuum degree of 0.085 MPa-0.095 MPa for 6-8 h to obtain a yellow-white solid, which is impurity A.

10. A method for detecting the content of impurity A in compound A-51 using the compound impurity A as a reference substance according to claim 1, characterized in that: The method comprises: (1) preparing impurity A according to the method according to any one of claims 1 to 9; (2) Determination of the content of related substance impurity A in A-51: HPLC instrument: high performance liquid chromatography Waters e2695, detector: 2489UV, chromatographic column: octadecyl bonded silica gel chromatographic column 150×4.6mm, 3μm, flow rate: 0.8ml / min, column temperature: 25℃, detection wavelength: 254nm, injection volume: 5μl; Mobile phase: 0.05% trifluoroacetic acid water was used as mobile phase A, methanol was used as mobile phase B, and gradient elution was performed. The gradient conditions are as follows: Test solution: Take 20 mg of A-51, weigh accurately, place in a 100 ml volumetric flask, add acetonitrile to dissolve, dilute to the mark, shake well, and obtain; 1% self-control solution: Take 1 ml of the above A-51 test solution, place it in a 100 ml volumetric flask, add acetonitrile to dilute to the scale line, repeat the injection 6 times, the peak area RSD ≤ 2.0%, retention time ≤ 2.0%.

11. The method according to claim 10, characterized in that The detection limit of the impurity A in A-51 should be less than or equal to 0.5%.

12. The method according to claim 11, characterized in that The detection limit of the impurity A in A-51 should be less than or equal to 0.2%.

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