Besudil mesylate impurity, preparation method and detection method

Abstract

The invention belongs to the technical field of medicine synthesis, and particularly relates to a series of impurities of besudil mesylate and a synthesis process thereof. The besudil mesylate series impurity provided by the invention can be used as a standard substance for determining the content of impurities in API (American Petroleum Institute) by a specific analysis method (such as HPLC (High Performance Liquid Chromatography)), and meets the requirements in the field. The besudil sulfonate impurity disclosed by the invention is mild in preparation process condition, high in yield, high in product purity and beneficial to large-scale production; besides, by developing an HPLC (High Performance Liquid Chromatography) detection method, the series of impurities can be effectively quantitatively or qualitatively analyzed, and the method has important significance on quality control of the bulk drug besudil mesylate.

Description

Technical Field

[0001] The invention belongs to the technical field of drug synthesis, and particularly relates to a series of impurities of besudil mesylate, and a preparation method and a detection method thereof. Background Art

[0002] Belumosudil (trade name: REZUROCK), chemical name 2-[3-[4-[(1H-indazol-5-yl)amino]quinazolin-2-yl]phenoxy]-N-isopropylacetamide, is a selective ROCK2 (Pho-associated proteinKinase2, Rho-associated protein kinase 2) inhibitor, which has been recognized as a breakthrough therapy by NMPA and is intended for the treatment of patients with chronic graft-versus-host disease (cGVHD) who have undergone at least one line of systemic treatment. In the United States, it is approved for the treatment of pediatric patients (age ≥12 years) and adult patients with chronic graft-versus-host disease (cGVHD) who have previously failed at least two systemic therapies.

[0003] The purity of pharmaceutical active substances has always been considered an important factor in ensuring drug safety and quality. It is well known in the art that, similar to any synthetic compound, besudil mesylate may contain foreign compounds or impurities from a variety of origins, and also impurities with structures closely related to the active pharmaceutical ingredient (API). In addition to affecting the reduction of content, these impurities may also cause undesirable side effects, thereby adversely affecting the safety of medication.

[0004] At certain stages of API processing, analytical methods (HPLC, LC-MS, etc.) must be used to ensure suitability for further processing and final use in drugs. APIs do not need to be absolutely pure, because absolute purification is usually an ideal goal that is difficult to achieve. It is more appropriate to set purity standards to ensure that the API is as free of impurities as possible, so that it can be safely used clinically. According to the ICH guidelines, it is recommended to limit the amount of some impurities to less than 0.1%.

[0005] To date, there are almost no reports on impurities in besudiol mesylate. Given the importance of obtaining active substances with high purity levels, it is necessary to prepare and identify each impurity present in the active substance and / or drug combination. Because the response factor of the impurity to a specific analytical method (such as HPLC) is different from that of the active substance for the active substance in question, standard substances (known chemical structure) are required for the correct determination of their content. By developing detection methods, impurities can be effectively analyzed quantitatively or qualitatively, which is of great significance to the quality control of the raw material besudiol mesylate. Summary of the invention

[0006] In order to overcome the problem of lack of research on besudil mesylate impurities in the prior art, the present invention provides a series of high-purity besudil mesylate impurities, preparation and detection methods, and provides standard impurity reference substances for quality research of besudil mesylate; by developing a detection method, the besudil mesylate series impurities disclosed in the present invention are effectively quantitatively or qualitatively analyzed, and the adverse effects of unknown impurities in drugs are further reduced.

[0007] The technical solution adopted by the present invention is: In one aspect, the present invention provides a besudil mesylate process impurity having a structure shown in Formula I:

[0008] Wherein, R is independently selected from H, methyl, ethyl, tert-butyl.

[0009] Specifically, compounds of formula V, formula VI, formula VII, and formula VIII:

[0010] The original patent reported the following reaction formula for the synthesis of besudil mesylate:

[0011] In the synthesis process of besudiol mesylate, solvents such as methanol, ethanol, tert-butyl alcohol, and water are used, and alcoholysis or hydrolysis side reactions occur in the synthesis of intermediates IN3 / IN4, thereby deriving compounds shown in the impurity formulas V, VI, VII, and VIII of the besudiol mesylate series disclosed in the present invention. At the same time, the structures of the impurities in this series are similar. If there is no reference substance, it is difficult to pay attention to whether the impurities are separated from the raw material drug besudiol mesylate under the HPLC method, thereby affecting the product quality.

[0012] The present invention also provides a preparation process of the above-mentioned besudil mesylate impurity, comprising: 1) The compound of formula III and the compound of formula IV undergo a substitution reaction under the action of a basic catalyst to generate a compound of formula II; 2) the compound of formula II is reacted with a deprotecting agent to generate a compound of formula I; The structural formulas of Formula II, Formula III and Formula IV are:

[0013] X is independently selected from Cl, Br, I, preferably, X is selected from Br.

[0014] According to an embodiment of the present invention, in step 1), the catalyst is selected from sodium hydroxide, potassium hydroxide, potassium carbonate, preferably potassium carbonate.

[0015] According to an embodiment of the present invention, the solvent for the reaction in step 1) is selected from 1,4-dioxane, dimethyl sulfoxide, N,N-dimethylformamide, preferably N,N-dimethylformamide.

[0016] According to an embodiment of the present invention, the reaction temperature in step 1) is selected from 20-60°C, preferably 20-30°C.

[0017] According to an embodiment of the present invention, in step 1), the molar ratio of the compound of formula III to the compound of formula IV is 1:1.1-1.5, preferably 1:1.2-1.3.

[0018] According to an embodiment of the present invention, in step 2), the deprotection reagent is selected from methanesulfonic acid, trifluoroacetic acid, hydrogen chloride / methanol solution, hydrogen chloride / 1,4-dioxane solution, preferably trifluoroacetic acid.

[0019] According to an embodiment of the present invention, the reaction solvent in step 2) is selected from dichloromethane, 1,4-dioxane, acetone, tetrahydrofuran, preferably dichloromethane.

[0020] According to an embodiment of the present invention, in step 2), the molar ratio of the compound of formula II to the deprotection reagent is 1:20-40, preferably 1:30-35.

[0021] Finally, the present invention also provides a method for detecting impurities of besudil mesylate, wherein the impurities are compounds represented by the impurity formula V, formula VI, formula VII, and formula VIII disclosed in the present invention, and high performance liquid chromatography is used to perform quantitative or qualitative analysis on the impurities in besudil mesylate, and the detection conditions include: Chromatographic column: reversed-phase octadecylsilane bonded silica gel column; preferably HORIZON Aurashell C18 ULTRA chromatographic column, specification 4.6*100mm, 2.7μm; Detection wavelength: selected from 210 to 400 nm, preferably 254 nm; Column temperature: 30°C; Flow rate: selected from 0.8 to 1.2 ml / min, preferably 1.0 ml / min; Mobile phase A: 0.1% phosphoric acid aqueous solution and acetonitrile, volume ratio 95:5; Mobile phase B: acetonitrile; The mobile phase was eluted by gradient elution, and the elution procedure was as follows:

[0022] Compared with the prior art, the present invention has the following beneficial effects: 1) The besudil mesylate series impurities provided by the present invention can be used as standard substances for determining the content of impurities in API using specific analytical methods (such as HPLC), thus meeting the requirements of the art; 2) The preparation process of the besudiol mesylate series impurities provided by the present invention has mild reaction conditions, high yield and high product purity. By developing a detection method (HPLC), the series of impurities can be effectively quantitatively or qualitatively analyzed, which is of great significance to the quality control of the raw material drug besudiol mesylate. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The compound of besudil mesylate impurity formula V 1 H-NMR spectrum; Figure 2 It is the compound of the impurity formula VI of besudil mesylate 1 H-NMR spectrum; Figure 3 It is the compound of formula VII of besudil mesylate impurity 1 H-NMR spectrum; Figure 4 is the compound of besudil mesylate impurity formula VIII 1 H-NMR spectrum; Figure 5 is the MS graph of the besudil mesylate impurity compound of formula V; Figure 6 is the MS graph of the besudil mesylate impurity compound of formula VI; Figure 7 is the MS graph of the besudil mesylate impurity compound of formula VII; Figure 8 is the MS graph of the besudil mesylate impurity compound of formula VIII; Fig. 9 This is the HPLC chart of the detection of impurities added to besudil mesylate. DETAILED DESCRIPTION

[0024] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] The related substance detection method described in the present invention is shown in Table 1.

[0026] Table 1 HPLC related substances detection method

[0027] The nuclear magnetic resonance hydrogen spectrum (1H-NMR) described in the embodiment of the present invention adopts a BRUKER 500MHz nuclear magnetic resonance spectrometer, and the test conditions and methods are as follows: the solvent is deuterated DMSO, the internal standard is TMS, and its nuclear magnetic resonance hydrogen spectrum is measured.

[0028] Example 1: Preparation of Compound V In a reaction bottle, add 10 g of compound III, 90 ml of DMF, 3.7 g of methyl bromoacetate, and 4.6 g of potassium carbonate, control the temperature at 20-30°C, and react for 24 hours; After the heat preservation is completed, the temperature is lowered to 10-20°C, ethyl acetate: 200ml, water: 100ml are added to the reaction bottle, stirred for 0.5h, allowed to stand, separated, the organic phase is collected, and concentrated under reduced pressure to obtain a residue of 11.2g; Add 50 ml of dichloromethane and 85.5 g of trifluoroacetic acid to the above residue, stir, and react at 20-30°C for 1-2 hours; After the insulation is completed, the temperature is lowered to 0-10°C, and 100 ml of MTBE is added dropwise to the system over a period of about 0.5 h. The mixture is stirred for 0.5-1 h, and filtered to collect the filter cake, which is the crude product of the compound of formula V. Crystallization: Take the crude product of the compound of formula V, add 70 ml of methanol, heat to 65-75°C, keep warm for 2-3 hours, cool to 10-20°C, keep warm for about 1 hour, filter, rinse the reaction bottle with 10 ml of methanol, wash the filter cake, and vacuum dry at 40-50°C to obtain 8.02 g of the compound of formula V, purity: 97.82%, yield 85.3%, 1 The results of H-NMR test are shown in Figure 1 , MS test results are shown in Figure 5 .

[0029] Example 2: Preparation of Compounds of Formula V In a reaction bottle, add 10 g of compound III, 90 ml of 1,4-dioxane, 3.7 g of methyl bromoacetate, and 1.3 g of sodium hydroxide, control the temperature at 20-30°C, and react for 10 hours; After the insulation is completed, the temperature is lowered to 10-20°C, ethyl acetate: 200 ml, water: 100 ml are added to the reaction bottle, stirred for 0.5 h, allowed to stand, separated, the organic phase is collected, and concentrated under reduced pressure to obtain a residue of 10.4 g; Add 50 ml of 1,4-dioxane and 150 ml of 4N hydrogen chloride / 1,4-dioxane solution to the above residue, stir, and react at 20-30°C for 1-2 hours; After the insulation is completed, the temperature is lowered to 0-10°C, and 100 ml of MTBE is added dropwise to the system over a period of about 0.5 h. The mixture is stirred for 0.5-1 h, and filtered to collect the filter cake, which is the crude product of the compound of formula V. Crystallization: Take the crude product of the compound of formula V, add 70 ml of methanol, raise the temperature to 65-75°C, keep warm for 2-3 h, cool to 10-20°C, keep warm for about 1 h, filter, rinse the reaction bottle with 10 ml of methanol, wash the filter cake, and dry in vacuo at 40-50°C to obtain 7.08 g of compound of formula V, purity: 96.33%, yield 75.3%.

[0030] Example 3: Preparation of Compounds of Formula V In a reaction bottle, add 10 g of compound III, 90 ml of dimethyl sulfoxide, 3.7 g of methyl bromoacetate, and 1.9 g of potassium hydroxide, control the temperature at 20-30°C, and react for 8 hours; After the insulation is completed, the temperature is lowered to 10-20°C, ethyl acetate: 200 ml, water: 100 ml are added to the reaction bottle, stirred for 0.5 h, allowed to stand, separated, the organic phase is collected, and concentrated under reduced pressure to obtain a residue of 10.4 g; Add 50 ml of acetone and 150 ml of 4N hydrogen chloride / methanol solution to the above residue, stir, and react at 20-30°C for 1-2 hours; After the insulation is completed, the temperature is lowered to 0-10°C, and 100 ml of MTBE is added dropwise to the system over a period of about 0.5 h. The mixture is stirred for 0.5-1 h, and filtered to collect the filter cake, which is the crude product of the compound of formula V. Crystallization: Take the crude product of the compound of formula V, add 70 ml of methanol, raise the temperature to 65-75°C, keep warm for 2-3 h, cool to 10-20°C, keep warm for about 1 h, filter, rinse the reaction bottle with 10 ml of methanol, wash the filter cake, and dry in vacuo at 40-50°C to obtain 7.32 g of compound of formula V, purity: 97.25%, yield 77.9%.

[0031] Example 4: Preparation of Compounds of Formula V In a reaction bottle, add 10 g of compound III, 90 ml of DMF, 3.7 g of methyl bromoacetate, and 4.6 g of potassium carbonate, control the temperature at 20-30°C, and react for 24 hours; After the heat preservation is completed, the temperature is lowered to 10-20°C, ethyl acetate: 200ml, water: 100ml are added to the reaction bottle, stirred for 0.5h, allowed to stand, separated, the organic phase is collected, and concentrated under reduced pressure to obtain a residue of 11.2g; Add 50 ml of tetrahydrofuran and 85.5 g of trifluoroacetic acid to the above residue, stir, and react at 20-30°C for 1-2 hours; After the insulation is completed, the temperature is lowered to 0-10°C, and 100 ml of MTBE is added dropwise to the system over a period of about 0.5 h. The mixture is stirred for 0.5-1 h, and filtered to collect the filter cake, which is the crude product of the compound of formula V. Crystallization: Take the crude product of the compound of formula V, add 70 ml of methanol, raise the temperature to 65-75°C, keep warm for 2-3 h, cool to 10-20°C, keep warm for about 1 h, filter, rinse the reaction bottle with 10 ml of methanol, wash the filter cake, and dry in vacuo at 40-50°C to obtain 7.20 g of compound of formula V, purity: 95.27%, yield 76.6%.

[0032] Example 5: Preparation of the compound of formula VI In a reaction bottle, add 10 g of compound III, 90 ml of DMF, 4.1 g of ethyl bromoacetate, and 4.6 g of potassium carbonate, control the temperature at 20-30°C, and react for 24 hours; After the insulation is completed, the temperature is lowered to 10-20°C, ethyl acetate: 200 ml, water: 100 ml are added to the reaction bottle, stirred for 0.5 h, allowed to stand, separated, the organic phase is collected, and concentrated under reduced pressure to obtain a residue of 11.7 g; Add 50 ml of dichloromethane and 85.5 g of trifluoroacetic acid to the above residue, stir, and react at 20-30°C for 1-2 hours; After the insulation is completed, the temperature is lowered to 0-10°C, and 100 ml of MTBE is added dropwise to the system over a period of about 0.5 h. The mixture is stirred for 0.5-1 h, and filtered to collect the filter cake, which is the crude product of the compound of formula VI. Crystallization: Take the crude product of the compound of formula VI, add 70 ml of methanol, heat to 65-75°C, keep warm for 2-3 hours, cool to 10-20°C, keep warm for about 1 hour, filter, rinse the reaction bottle with 10 ml of methanol, wash the filter cake, and dry in vacuo at 40-50°C to obtain 8.06 g of compound of formula VI, purity: 99.22%, yield 83.2%, 1 The results of H-NMR test are shown in Figure 2 , MS test results are shown in Figure 6 .

[0033] Example 6: Preparation of compound of formula VII In a reaction bottle, add 10 g of compound III, 90 ml of DMF, 4.7 g of tert-butyl bromoacetate, and 4.6 g of potassium carbonate, control the temperature at 20-30°C, and react for 24 hours; After the heat preservation is completed, the temperature is lowered to 10-20°C, ethyl acetate: 200ml, water: 100ml are added to the reaction bottle, stirred for 0.5h, allowed to stand, separated, the organic phase is collected, and concentrated under reduced pressure to obtain a residue of 12.1g; Add 50 ml of dichloromethane and 150 ml of 4N HCl / 1,4-dioxane solution to the above residue, stir, and react at 20-30°C for 1-2 hours; After the insulation is completed, the temperature is lowered to 0-10°C, and 100 ml of MTBE is added dropwise to the system over a period of about 0.5 h. The mixture is stirred for 0.5-1 h, and filtered to collect the filter cake, which is the crude compound VII. Crystallization: Take the above compound of formula VII, add methanol: 70 ml, heat to 65-75 ° C, keep warm for 2-3 hours, cool to 10-20 ° C, keep warm for about 1 hour, filter, rinse the reaction bottle with methanol: 10 ml, wash the filter cake, and vacuum dry at 40-50 ° C to obtain 8.16 g of compound of formula VII, purity: 98.38%, yield 79.2%, 1 The results of H-NMR test are shown in Figure 3 , MS test results are shown in Figure 7 .

[0034] Example 7: Preparation of Compound VIII In a reaction bottle, add 10 g of compound III, 90 ml of DMF, 4.7 g of tert-butyl bromoacetate, and 4.6 g of potassium carbonate, control the temperature at 20-30°C, and react for 24 hours; After the heat preservation is completed, the temperature is lowered to 10-20°C, ethyl acetate: 200ml, water: 100ml are added to the reaction bottle, stirred for 0.5h, allowed to stand, separated, the organic phase is collected, and concentrated under reduced pressure to obtain a residue of 12.1g; Add 85.5 g of trifluoroacetic acid to the above residue, stir, and react at 50-60°C for 1-2 hours; After the insulation is completed, the temperature is lowered to 0-10°C, and 100 ml of MTBE is added dropwise to the system over a period of about 0.5 h. The mixture is stirred for 0.5-1 h, and filtered to collect the filter cake, which is the crude product of the compound of formula VIII. Crystallization: Take the crude product of the compound of formula VIII, add 70 ml of methanol, heat to 65-75°C, keep warm for 2-3 hours, cool to 10-20°C, keep warm for about 1 hour, filter, rinse the reaction bottle with 10 ml of methanol, wash the filter cake, and vacuum dry at 40-50°C to obtain 8.16 g of the compound of formula VIII, purity: 97.89%, yield 79.2%, 1 The results of H-NMR test are shown in Figure 4 , MS test results are shown in Figure 8 .

[0035] Example 8: Detection of compounds of formula V, formula VI, formula VII, and formula VIII in besudil mesylate API Sample solution preparation: accurately weigh besudil mesylate API: 100.112 mg, formula V: 2.991 mg, formula VI: 2.024 mg, formula VII: 2.019 mg, formula VIII: 5.031 mg and place in a 50 ml volumetric flask, add acetonitrile to the mark; measure 5 ml of the above solution, place in a 10 ml volumetric flask, and dilute to the mark with acetonitrile / water = 1:1.

[0036] Chromatographic conditions: using octadecylsilane bonded silica gel as filler (HORIZON Aurashell C18 ULTRA-4.6*100mm, 2.7μm or chromatographic column of equivalent performance); using 0.1% aqueous phosphoric acid: acetonitrile (95:5 V / V) as mobile phase A, and acetonitrile as mobile phase B, with linear gradient elution as shown in the table below; flow rate is 1.0ml / min; column temperature is 30℃; detection wavelength is 254nm; injection volume is 10μl.

[0037]

[0038] Conclusion: The separation between each chromatographic peak is greater than 1.5, and the results meet the requirements. See the spectrum. Fig. 9 .

[0039] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. Besudil mesylate process impurities, characterized in that, The besudil mesylate process impurity has a structure shown in Formula I: , wherein R is independently selected from H, methyl, ethyl, and tert-butyl.

2. A process for preparing the besudil mesylate process impurity as claimed in claim 1, characterized in that: The preparation process comprises: 1) The compound of formula III and the compound of formula IV undergo a substitution reaction under the action of a basic catalyst to generate a compound of formula II; 2) the compound of formula II is reacted with a deprotecting agent to generate a compound of formula I; The structural formulas of Formula II, Formula III and Formula IV are: , wherein X is independently selected from Cl, Br, I, preferably, X is selected from Br.

3. The process for preparing besudil mesylate process impurities according to claim 2, characterized in that: The alkaline catalyst in step 1) is selected from sodium hydroxide, potassium hydroxide, potassium carbonate, preferably potassium carbonate.

4. The process for preparing besudil mesylate process impurities according to claim 2, characterized in that: The solvent for the reaction in step 1) is selected from 1,4-dioxane, dimethyl sulfoxide, and N,N-dimethylformamide, preferably N,N-dimethylformamide.

5. The process for preparing besudil mesylate process impurities according to claim 2, characterized in that: The reaction temperature in step 1) is selected from 20-60°C, preferably 20-30°C.

6. The process for preparing besudil mesylate process impurities according to claim 2, characterized in that: In the step 1), the molar ratio of the compound of formula III to the compound of formula IV is 1:1.1-1.5, preferably 1:1.2-1.

3.

7. The process for preparing besudil mesylate process impurities according to claim 2, characterized in that: The deprotection reagent in step 2) is selected from methanesulfonic acid, trifluoroacetic acid, hydrogen chloride / methanol solution, hydrogen chloride / 1,4-dioxane solution, preferably trifluoroacetic acid.

8. The process for preparing besudil mesylate process impurities according to claim 2, characterized in that: The reaction solvent in step 2) is selected from dichloromethane, 1,4-dioxane, acetone, tetrahydrofuran, preferably dichloromethane.

9. The process for preparing besudil mesylate process impurities according to claim 2, characterized in that: In the step 2), the molar ratio of the compound of formula II to the deprotection reagent is 1:20-40, preferably 1:30-35.

10. A method for detecting impurities in the process of besudil mesylate according to any one of claims 1 to 9, characterized in that: The detection method is a high performance liquid chromatography detection method, and the detection conditions include: Chromatographic column: reversed-phase octadecylsilane bonded silica gel column; preferably HORIZON Aurashell C18 ULTRA chromatographic column, specification 4.6*100mm, 2.7μm; Detection wavelength: selected from 210 to 400 nm, preferably 254 nm; Column temperature: 30°C; Flow rate: selected from 0.8 to 1.2 ml / min, preferably 1.0 ml / min; Mobile phase A: 0.1% phosphoric acid aqueous solution and acetonitrile, volume ratio 95:5; Mobile phase B: acetonitrile; The mobile phase was eluted by gradient elution, and the elution procedure was as follows: 。

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