Coronavirus protease inhibitor related substance as well as preparation method and detection method thereof

Abstract

The invention relates to a related substance of a coronavirus 3CLpro protease inhibitor compound as shown in a formula (IV), a preparation method of the related substance and application of the related substance as an impurity reference substance in detection of the related substance of the compound as shown in the formula (IV), and the scientificity and accuracy of impurity detection of the compound as shown in the formula (IV) can be effectively improved by taking the related substance as the reference substance. The impurity content of the compound of the formula (IV) and the preparation thereof can be effectively and conveniently monitored, and the quality of the compound of the formula (IV) and the preparation thereof is favorably controlled, so that the safety and effectiveness of the compound and the preparation thereof are ensured.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical chemical industry, and in particular to a related substance of a coronavirus 3CLpro protease inhibitor, a preparation method and a detection method thereof, and a use thereof as a control. BACKGROUND

[0002] Coronavirus, abbreviated as CoV, is a kind of RNA virus that can cause diseases in mammals and birds, and can cause mild to severe respiratory infections, including SARS, MERS, and SARS-CoV-2 (COVID-19) and other fatal diseases. 3CLpro (3C-like protease, also known as 3C-like protease) is a major protease produced by coronavirus, which can hydrolyze viral polyproteins pp1a and pp1ab to produce functional proteins required for viral replication. Therefore, 3C-like protease is essential for viral replication, and inhibition of 3CLpro activity can effectively inhibit viral replication and infection.

[0003] WO2022150962A1 discloses a coronavirus 3CLpro protease non-covalent binding inhibitor, wherein the compound N-((1S,2R)-2-((4-bromo-2-(methylcarbamoyl)-6-nitrophenyl)amino)cyclohexyl)isoquinoline-4-carboxamide hydrochloride (i.e. the compound of formula (IV)) has good inhibitory activity on SARS-CoV-2 3CLpro protease, and exhibits significant antiviral activity in various cell models and in ACE2 transgenic mice. Research papers (ACS Cent. Sci. 2023, 9, 217-227) systematically introduce that the compound has high exposure and bioavailability, and good safety. Phase I, II, and III clinical studies (NCT06205329; NCT06205329; NCT06205329) further prove the safety and effectiveness of the compound of formula (IV) in treating coronavirus infection, and are in the process of marketing application, and are expected to be developed into a clinical drug, and have obtained the WHO International Nonproprietary Name (INN) of Iscartrelvir (CAS: 2921711-74-0), the structure of which is shown in the following formula (IV):

[0004]

[0005] WO2022150962A1 describes the preparation route of the compound of formula (IV):

[0006]

[0007] For the above-mentioned compound IV, the impurities thereof are not known in the prior art, and there is no reliable quality control method at present. In order to further improve the quality standard, improve the quality and safety of the drug, reduce the possible toxic side effects, provide a control or standard, it is necessary to study the related substances thereof and provide a reliable quality control method, so as to analyze the impurity content and determine the reasonable impurity limit. This plays a crucial role in the safety detection of clinical medication, the improvement of the accuracy of product quality research, and the improvement of product safety and reliability. SUMMARY

[0008] The purpose of the present application is to provide the related substances of the 3CLpro protease inhibitor compound of formula (IV), its preparation method and its use as a control, which directly and effectively monitors the change of the impurities of the compound of formula (IV), helps to improve the quality standard of the compound of formula (IV), and better controls the product quality.

[0009] The present application can be described from different aspects, and the inventions described in these aspects and any embodiments are independent of each other and are related to each other, which together constitute the content of the present application.

[0010] In a first aspect of the present application, the related substances of the 3CLpro protease inhibitor compound of formula (IV) are provided, the structure of which is shown in formula (I), formula (II) and formula (III):

[0011]

[0012] The inventors found in the research that the related substances of the prepared compound of formula (IV) are mainly the starting materials, intermediates, polymers and by-products brought in during the production process, and the compound of formula (IV) is stable during storage. The three impurities with relatively large content contained therein are separated and identified, and the structures thereof are shown in formula (I), formula (II) and formula (III).

[0013] The reason for the generation of the compound impurity of formula I is that in the last step of the reaction, in order to make the intermediate as completely as possible into the final product, an excess of the starting material 4-isoquinoline carboxylic acid (CAS: 7159-36-63, i.e. formula (I)) is added, and formula (I) is difficult to completely remove during the purification of the final product.

[0014] The reason for the generation of the compound of formula II impurity is as follows: the key starting material 3-bromo-2-fluoro-5-nitrobenzoic acid (CAS: 1526564-53-3) in the production process of formula (IV) is prepared by bromination of 2-fluoro-3-nitrobenzoic acid (CAS: 317-46-4). The bromination reaction cannot be completely converted, and the remaining raw material 2-fluoro-3-nitrobenzoic acid is difficult to completely remove finally, so that a small amount of 2-fluoro-3-nitrobenzoic acid exists in the key starting material 3-bromo-2-fluoro-5-nitrobenzoic acid. In the production process of formula (IV), 2-fluoro-3-nitrobenzoic acid can finally generate formula (II) impurity through four steps of aromatic nucleophilic substitution reaction, acid amine condensation reaction, Boc deprotection and acid amine condensation reaction according to the following process route, or formula (IV) generates formula (II) impurity through debromination reaction

[0015]

[0016] The reason for the generation of the compound of formula III impurity is as follows: there is a trace amount of enantiomeric impurity ((1S,2R)-2-aminocyclohexyl) tert-butyl carbamate (CAS: 365996-30-1) which is difficult to completely remove in the key starting material ((1R,2S)-2-aminocyclohexyl) tert-butyl carbamate (CAS: 364385-54-6) in the production process of formula IV compound. In the production process of formula (IV), ((1S,2R)-2-aminocyclohexyl) tert-butyl carbamate finally generates the compound of formula III impurity according to the following process route.

[0017]

[0018] In order to study and control the quality of the compound of formula IV, the compounds of formula I, II and III are prepared as impurity reference substances for research.

[0019] In a second aspect of the present application, a preparation method of the compounds of formula I, II and III is provided.

[0020] In some embodiments of the present application, the preparation method of the compound of formula (I) is as follows:

[0021]

[0022] The compound of formula (I-1) undergoes a cyanylation reaction to obtain an aryl cyanide of formula (I-2), and the compound of formula (I) is obtained by hydrolysis of the compound of formula (I-2). The cyanylation reaction mainly uses inorganic cyanide selected from KCN, NaCN, CuCN and K4[Fe(CN)6], and CuCN is preferred.

[0023] In some embodiments of the present application, the preparation method of the compound of formula (II) is as follows:

[0024]

[0025] The compound of formula (II-1) and the compound of formula (IV-2) are subjected to an aromatic nucleophilic substitution reaction to obtain the compound of formula (II-2), the compound of formula (II-2) and methylamine hydrochloride are subjected to an acid-amine condensation to obtain the compound of formula (II-3), the compound of formula (II-3) is subjected to a Boc deprotection to obtain the compound of formula (II-4), and the compound of formula (II-4) and the compound of formula (I) are subjected to an acid-amine condensation to obtain the compound of formula (II).

[0026] In some embodiments of the present application, the compound of formula (III) is prepared by the following method:

[0027]

[0028] The compound of formula (IV-1) and the compound of formula (III-1) are subjected to an aromatic nucleophilic substitution reaction to obtain the compound of formula (III-2), the compound of formula (III-2) and methylamine hydrochloride are subjected to an acid-amine condensation to obtain the compound of formula (III-3), the compound of formula (III-3) is subjected to a Boc deprotection to obtain the compound of formula (III-4), and the compound of formula (III-4) and the compound of formula (I) are subjected to an acid-amine condensation to obtain the compound of formula (III).

[0029] In some embodiments of the present application, the aromatic nucleophilic substitution reaction is promoted by a base selected from an organic base, an inorganic base or a combination thereof, and the base includes triethylamine (TEA), N,N-diisopropylethylamine (DIPEA), pyridine, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), Na2CO3, K2CO3, Cs2CO3, NaH, LiOH, NaOH, KOH, t-BuOK, t-BuONa. Preferably, the base is N,N-diisopropylethylamine or K2CO3.

[0030] In some embodiments of the present application, the organic base is selected from triethylamine (TEA), N,N-diisopropylethylamine (DIPEA), pyridine, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), t-BuOK or t-BuONa.

[0031] In some embodiments of the present application, the organic base is selected from one or more of triethylamine, N,N-diisopropylethylamine or N-methylmorpholine in any combination, and preferably the organic base is N,N-diisopropylethylamine.

[0032] In some embodiments of the present application, the inorganic base is selected from Na2CO3, K2CO3, Cs2CO3, NaH, LiOH, NaOH, KOH.

[0033] In some embodiments of the present application, a condensing agent is used in the acid-amine condensation reaction, and the condensing agent is selected from 2-(7-azobenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), O-benzotriazol-tetramethyluronium hexafluorophosphate (HBTU), 6-chlorobenzotriazol-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), or 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), oxalyl chloride, dichlorosulfoxide, isopropyl chloroformate.

[0034] In some embodiments of the present application, a condensing agent is used in the acid-amine condensation reaction, and the condensing agent is preferably 2-(7-azobenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) or isopropyl chloroformate.

[0035] In some embodiments of the present application, the solvent used in the aromatic nucleophilic substitution reaction and the acid-amine condensation reaction is selected from N,N-dimethylformamide, N,N-dimethylacetamide, or N-methylpyrrolidone, or a combination thereof.

[0036] In some embodiments of the present application, the temperature of the amide condensation reaction is -5°C to 10°C.

[0037] In some embodiments of the present application, the temperature of the amide condensation reaction is 0°C to 25°C.

[0038] In a third aspect of the present application, there is provided a use of a compound of formula (I), a compound of formula (II), and a compound of formula (III) as a control sample for detecting related substances of a protease inhibitor compound of formula (IV).

[0039] In some embodiments of the present application, the HPLC chromatographic conditions for detecting related substances of the compound of formula I and formula II are as follows: HSS T3 or an equivalent column thereof, ghost peak trapping column, 0.03% trifluoroacetic acid-water solution as mobile phase A, 0.03% trifluoroacetic acid-acetonitrile solution as mobile phase B, linear gradient elution.

[0040] In some embodiments of the present application, the HPLC chromatographic conditions for detecting related substances are as follows: octadecylsilane-bonded silica gel as the filler (Waters HSS T3 4.6mm×150mm, 3.5μm or equivalent chromatographic column, pre-column: Chiralpak, 4.6mm×50mm, 40Mpa); 0.03% trifluoroacetic acid-water solution as mobile phase A, 0.03% trifluoroacetic acid-acetonitrile solution as mobile phase B, linear gradient elution according to the following table; the flow rate is 1.2ml per minute; the column temperature is 45℃; the detection wavelength is 220±5nm; the injection volume is 10μl.

[0041] Time (min) Mobile phase A (%) Mobile phase B (%) 0 100 0 2 100 0 20 5 95 23 5 95 23.1 100 0 35 100 0

[0042] In some embodiments of the present application, the HPLC chromatographic conditions for detecting the related substances of the compound of formula III are as follows: CHIRAL ART Cellulose-SC or equivalent chromatographic column, hexane: 0.1% diethylamine in ethanol solution (85:15) as mobile phase, isocratic elution.

[0043] In some embodiments of the present application, the HPLC chromatographic conditions for detecting the related substances are as follows: cellulose-tris (3, 5-dichlorophenyl carbamate) bonded silica gel as the filler (YMC CHIRAL ART Cellulose-SC 4.6mm×150mm, 3μm); hexane: 0.1% diethylamine in ethanol solution (85:15) as the mobile phase; the flow rate is 1.0ml per minute; the column temperature is 35℃; the detection wavelength is 248±5nm; the injection volume is 15μl.

[0044] The present application has the beneficial technical effects that the three impurities generated in the preparation process of the compound of formula (IV) directly affect the drug quality of the compound of formula (IV), and have a crucial influence on the effectiveness, drug property and safety of the drug. A large number of experimental researches are carried out for the process optimization and quality control of the compound of formula (IV), and the compound of formula (I), the compound of formula (II) and the compound of formula (III) are obtained, and the synthesis method of the compound of formula (I), the compound of formula (II) and the compound of formula (III) is unexpectedly obtained. The impurity can be used as the impurity control for detecting the related substances of the compound of formula (IV), which can effectively and conveniently monitor the related substances in the compound of formula (IV). The quality of the compound of formula (IV) is effectively controlled, so that the safety and effectiveness of the compound of formula (IV) in the drug use process are ensured. BRIEF DESCRIPTION OF DRAWINGS

[0045] Figure 1 The hydrogen spectrum of the compound of formula (I);

[0046] Figure 2 The hydrogen spectrum of the compound of formula (II);

[0047] Figure 3hydrogen spectrum of a compound of formula (III);

[0048] Figure 4 hydrogen spectrum of a compound of formula (III);

[0049] Figure 5 detection of compounds of formula (I) and (II) in the first batch of the representative spectrum of a compound of formula (IV);

[0050] Figure 6 detection of compounds of formula (I) and (III) in the first batch of the representative spectrum of a compound of formula (IV);

[0051] Figure 7 detection of compounds of formula (I) and (II) in the second batch of the representative spectrum of a compound of formula (IV);

[0052] Figure 8 detection of compounds of formula (III) in the second batch of the representative spectrum of a compound of formula (IV). DETAILED DESCRIPTION

[0053] Example 1 Preparation of a compound of formula (IV)

[0054]

[0055] First step: ((1R,2S)-2-Aminocyclohexyl)carbamic acid tert-butyl ester (81.18 g) and 3-bromo-2-fluoro-5-nitrobenzoic acid (100.0 g) were dissolved in N-methylpyrrolidine (510 ml) and N,N-diisopropylethylamine (102.81 g) under nitrogen at room temperature. The reaction temperature was then increased to 60-80 °C and stirring was continued for 5 hours. The sample HPLC monitored the compound of formula (IV-1) / compound of formula (IV-3) (%) and the target was <1.0%. When the target was reached, the reaction temperature was adjusted to 10-30 °C and water (1500 ml) was added dropwise under stirring and stirring was continued for 1 hour. The reaction temperature was then adjusted to 10-30 °C and aqueous hydrochloric acid (487.2 g, concentrated hydrochloric acid 47.2 g: water 440 g) was added dropwise and stirring was continued for 2 hours. The reaction mixture was then filtered and the filter cake was washed with water and isopropanol and the wet product was dried in a vacuum at 40-50 °C for 12 hours to give 115.1 g of the compound of formula (IV-3).

[0056] Second Step: Dissolve compound of formula (IV-3) (100.0 g) in N-methyl pyrrolidone (306.0 g, 3.0 V) under nitrogen protection at room temperature, then add DIEA (98.7 g). Adjust the temperature of the reaction system to -5-10 °C, and drop isopropyl chloroformate (40.1 g) into the reaction system. After stirring the reaction system at -5-10 °C for 5 hours, sample and monitor: TLC spotting monitoring, 254 nm; developing agent: EtOAc (2 mL + 0.1 mL of HCl / EtOAc); raw material R f = 0.75, product R f = 0.95, TLC monitoring raw material reaction complete. Then add hydrochloric acid salt of methylamine (22.1 g) into the reaction system in batches, and continuously control the reaction temperature at -5-10 °C to stir for 5 hours. Sample HPLC monitoring compound of formula (IV-3) / compound of formula (IV-4) (%) ≤ 10.0%, reaction termination. After termination, add water (2375.0 g) into the reaction system and adjust the temperature to 10-30 °C, and stir for 1 hour. Filter and rinse the filter cake with water and n-hexane. After drying the wet product at 40-50 °C under vacuum for 12 hours, obtain compound of formula (IV-4) 105.5 g.

[0057] Third Step: Dissolve compound of formula (IV-4) (100.0 g) in dichloromethane (690 g) under nitrogen protection at room temperature. Adjust the temperature of the reaction system to 0-10 °C, and drop 4M HCl / EtOAc (249.3 g) into the reaction system. Stir the reaction system at 15-25 °C for 5 hours, sample HPLC monitoring: compound of formula (IV-4) / compound of formula (IV-5) (%) ≤ 2.0%, reaction termination. Add dichloromethane (330.0 g) and n-hexane (683.0 g) into the reaction system to crystallize and filter. Rinse and dry to obtain compound of formula (IV-5) 65.7 g.

[0058] Fourth Step: Dissolve compound of formula (IV-5) (100.0 g) and compound of formula (I) (51.0 g) in N,N-dimethylformamide (350 g) under nitrogen protection at room temperature. Adjust the temperature of the reaction system to 0-10 °C, and drop N,N-diisopropyl ethylamine (95.1 g) into the reaction system, and add HATU (111.9 g) in batches, control the internal temperature at 0-20 °C. Stir the reaction system at 15-25 °C for 5 hours, sample HPLC monitoring: compound of formula (IV-5) / compound of formula (IV) (%) ≤ 1.0%, reaction termination. Add N,N-dimethylformamide (50.0 g) and water (2500 g) into the reaction system, and stir at 15-25 °C for 2 hours, then filter. Rinse and dry to obtain compound of formula (IV) 115.8 g.

[0059] The compound of formula (IV) prepared in the example was identified, and the results are as follows:

[0060] ESI-MS (m / z): 526.0;

[0061] 1 H NMR (400 MHz, DMSO-d6) δ 9.84 (s, 1H), 8.89 (d, J = 8.6 Hz, 1H), 8.79 (q, J = 4.2 Hz, 1H), 8.67 (s, 1H), 8.49 (d, J = 8.3 Hz, 1H), 8.32 (d, J = 9.9 Hz, 1H), 8.24 (d, J = 8.5 Hz, 1H), 8.15 (d, J = 2.4 Hz, 1H), 8.09-8.13 (m, 1H), 7.98 (t, J = 7.3 Hz, 1H), 7.75 (d, J = 2.4 Hz, 1H), 4.35 (br 1H), 3.75 (br 1H), 2.76 (d, J = 4.6 Hz, 3H), 1.33-1.80 (m, 8H).

[0062] Separation and identification of impurities of Example 2

[0063] The compound of formula (IV) prepared in Example 1 was subjected to related substance test, and three impurities with relatively large content were found. The HPLC-MS method was used for detection, and the method was as follows:

[0064] Chromatographic conditions: octadecylsilane-bonded silica gel as the filler (Waters XSelect HSS T3, 4.6 mm x 150 mm, 3.5 μm or a chromatographic column with equivalent performance); mobile phase A: 0.03% trifluoroacetic acid-water solution, V / V; mobile phase B: 0.03% trifluoroacetic acid-acetonitrile solution, V / V; column temperature: 45°C; flow rate: 1.5 mL / min; detection wavelength: 215 nm, and the elution program is shown in Table 1.

[0065] Table 1 Gradient elution program

[0066] Time (min) MPB (%) 0.0 0 2.0 0 20.0 95 23.0 95 23.1 0 30.0 0

[0067] Mass spectrometry parameters: ESI source positive and negative full scan mode;

[0068] Resolution: 70000;

[0069] Scan range: 100-1500.

[0070] After the detection, the data analysis is carried out by extracting the molecular weight of the impurity corresponding to the retention time, and it is judged to be the impurity of formula (I), formula (II) and formula (III). The compound of formula (I) is a key starting material, which can be directly purchased or synthesized. The reference substances of the impurities of formula (I), formula (II) and formula (III) are prepared by the method of targeted synthesis, see examples 3, 4 and 5, and the prepared impurities are detected and confirmed by HPLC method to determine that the synthesized is the target compound of formula (I), formula (II) and formula (III).

[0071] Preparation, structure confirmation and calibration as reference substance of the impurity (I) of example 3

[0072] The preparation route of the compound of formula (I) is as follows:

[0073]

[0074] The compound of formula (I-2) can be obtained by cyanation of aryl bromide using CuCN as a cyanide source, and then the compound of formula (I) can be obtained by hydrolysis in an alkaline environment using 4-bromoisoquinoline of formula (I-1) as a starting material.

[0075] The structure confirmation and calibration as reference substance of the compound of formula (I) prepared in example 3 are as follows:

[0076] Structure confirmation

[0077] UV absorption spectrum

[0078] 1) Test conditions

[0079] Instrument: Shimadzu UV-2550

[0080] Solvent: methanol, 20.2 μg / ml

[0081] 2) Test data and analysis

[0082] The UV analysis results are shown in the following table 2:

[0083] Table 2 UV detection results of the compound of formula (I)

[0084] Wavelength (nm) Absorbance 324 0.181 286 0.177 223 1.920

[0085] 3) Conclusion

[0086] From the above determination results, the structure of the sample is consistent with the structure of the compound of formula (I).

[0087] Infrared absorption spectrum

[0088] 1) Test conditions

[0089] Instrument: Shimadzu IR Affinity-1

[0090] Method: KBr pellet method

[0091] 2) Test data and assignment analysis

[0092] The infrared analysis results are shown in the following Table 3:

[0093] Table 3 Infrared detection results of the compound of formula (I)

[0094] Observed values Absorption peak wave number (cm -1 )]]> Vibration type and group assignment 3042,2791 C-H stretching vibration 1709 C=0 stretching vibration 1566,1501 C=C stretching vibration, C=N stretching vibration 1377,1290,1215,1105 C-N stretching vibration, C-O stretching vibration 799,746 C-H out-of-plane bending vibration

[0095] 3) Conclusion

[0096] From the characteristic absorption peaks of the infrared spectrum, the structure of the sample is consistent with that of the compound of formula (I).

[0097] Nuclear magnetic resonance (NMR)

[0098] 1) Test conditions

[0099] Instrument: Bruker Biospin AG AV NEO 400 spectrometer

[0100] Solvent: DMSO-d6 (D, 99.9%)

[0101] 2) Test data and assignment analysis

[0102] Hydrogen spectrum:

[0103]

[0104] Table 4 Hydrogen spectrum detection results of the compound of formula (I)

[0105] Proton Chemical shift (ppm) Multiplicity (coupling constant) Number of protons [H1] 7.77-7.81 m 1 [H2] 7.93-7.97 m 1 [H6] 8.26 d (J = 8.1 Hz) 1 ​ 8.91 d (J = 8.6 Hz) 1 [H8] 9.10 s 1 [H9] 9.53 s 1 -COOH 13.54 s 1

[0106] Carbon spectrum:

[0107] Table 5 Carbon spectrum detection results of the compound of formula (I)

[0108] Assignment Chemical shift (ppm) [C7] 120.9 [C3] 124.6 [C2] 127.8 [C5] 128.2 [C6] 128.6 [C2] 132.3 [C4] 133.0 [C8] 146.1 [C9] 156.9 [C 10 ]]> 167.6

[0109] 3) Conclusion

[0110] From the above detection results, the structure of the sample is consistent with that of the compound of formula (I).

[0111] Mass spectrometry (MS)

[0112] 1) Test conditions

[0113] Instrument: Agilent 1290 UPLC + 6125 CMS

[0114] Solvent: acetonitrile

[0115] 2) Test data and analysis

[0116] The mass spectrometry results are shown in the following table.

[0117] Table 6 Mass spectrometry results of the compound of formula (I)

[0118] Observed mass-to-charge ratio (m / z) Theoretical mass-to-charge ratio (m / z) Remarks 172.2 172.0 [[M-H] - ]]

[0119] 3) Analysis and conclusion

[0120] From the above test results, the structure of the sample is consistent with that of the compound of formula (I).

[0121] In summary, the structure of the reference compound of formula (I) is:

[0122]

[0123] Calibration procedure

[0124] High performance liquid chromatography (HPLC)

[0125] Purity

[0126] 1) Solution preparation

[0127] Diluent: ACN:H2O = 1:1, V / V

[0128] Blank solution: diluent

[0129] Test solution: about 25 mg of the reference compound to be calibrated was accurately weighed into a 100.0 mL volumetric flask, dissolved and diluted to the mark with the diluent, shaken well, and the test solution concentration was about 0.25 mg / mL. Three portions were prepared in parallel.

[0130] Sensitivity solution: 1.0 mL of the first test solution was accurately transferred into a 100.0 mL volumetric flask, diluted to the mark with the diluent, shaken well; and 0.5 mL of the above solution was accurately transferred into a 10.0 mL volumetric flask, diluted to the mark with the diluent, shaken well. The sensitivity solution concentration was 0.000125 mg / mL, which was equivalent to 0.05% of the test solution concentration.

[0131] Note: The preparation of the test solution and sensitivity solution can be scaled up or down in volume proportionally, while keeping the final concentration unchanged.

[0132] 2) Chromatographic conditions

[0133] Instrument: Agilent 1260 Infinity II or similar liquid chromatograph, which can be connected to a ghost peak trapping column, Yuwu 4.6 x 50 mm or equivalent column

[0134] Column: Waters XSelect HSS T3 4.6mm x 150mm, 3.5μm or other equivalent column

[0135] Column temperature: 35℃

[0136] Sample chamber temperature: room temperature

[0137] Mobile phase A: 0.03% trifluoroacetic acid in water, V / V

[0138] For example: accurately weigh about 0.3 mL of trifluoroacetic acid into 1000 mL of water, mix well and degas by ultrasonic.

[0139] Mobile phase B: 0.03% trifluoroacetic acid in acetonitrile, V / V

[0140] For example: accurately weigh about 0.3 mL of trifluoroacetic acid into 1000 mL of acetonitrile, mix well and degas by ultrasonic.

[0141] Gradient:

[0142] Time (min) Mobile phase A (% V / V) Mobile phase B (% V / V) 0 98 2 2 98 2 12 20 80 15 20 80 15.1 98 2 25 98 2

[0143] Flow rate: 1.5 mL / min

[0144] Injection volume: 5μL

[0145] Detector wavelength: UV 215nm

[0146] Run time: 25min

[0147] Equivalent columns have the same or similar separation characteristics and column efficiency as the aforementioned column, but can be achieved with different packing materials and stationary phases.

[0148] 3) System suitability test

[0149] Inject blank solution 1 needle or more until the system is stable without interference or significant interference. Significant interference means that the peak area of the interference peak should not exceed 10% of the peak area of the compound of formula (I) in the sensitivity solution.

[0150] Inject sensitivity solution 1 needle, and the signal-to-noise ratio of the peak of the compound of formula (I) should not be less than 10.

[0151] Retention time:

[0152] Substance name Retention time RT (min) about Compound of formula (I) 5.9

[0153] 4) Determination

[0154] After the system suitability test is passed, start the analysis determination:

[0155] Inject 3 portions of 1 needle of the test sample solution each, and record the chromatogram.

[0156] 5) Calculation

[0157] Total impurities (%) = sum of individual impurities

[0158] Purity (%) = 100 - total impurities

[0159] The average value of purity is calculated and reported.

[0160] Note: Reporting limit (RL): 0.05%, peaks below 0.025% are not integrated.

[0161] Purity factor

[0162] Primary standard: Purity factor f = (100 - loss weight) x Purity / 100 / 100

[0163] Preparation method, structure confirmation and calibration as a standard of impurity (II)

[0164] The preparation route of impurity (II) is as follows:

[0165]

[0166] The compound 2-fluoro-3-nitrobenzoic acid of formula (II-1) is used to replace the compound 3-bromo-2-fluoro-5-nitrobenzoic acid of formula (IV-1) in Example 1 as a starting material. The specific experimental operation process is the same as that of Example 1, and the impurity of formula (II) can be prepared directionally.

[0167] The compound of formula (II) prepared in Example 4 is subjected to structure confirmation and calibration as a standard, and the results are as follows:

[0168] Structure confirmation

[0169] UV absorption spectrum

[0170] 1) Test conditions

[0171] Instrument: Shimadzu UV-2550

[0172] Solvent: methanol, 10.0 μg / ml

[0173] 2) Determination data and analysis

[0174] The UV analysis results are shown in the following table.

[0175] Table 7 UV detection results of the compound of formula (II)

[0176] Wavelength (nm) Absorbance 220.3 1.392

[0177] 3) Conclusion

[0178] From the above determination results, the sample structure is consistent with the structure of the compound of formula (II).

[0179] Infrared absorption spectrum

[0180] 1) Test conditions

[0181] Instrument: Shimadzu IRAffinity-1

[0182] Method: KBr tabletting method

[0183] 2) Test data and attribution analysis

[0184] The infrared analysis results are shown in the following table.

[0185] Table 8 Infrared detection results of the compound of formula (II)

[0186] 3) Conclusion

[0187] From the characteristic absorption peaks of the infrared spectrum, the sample structure is consistent with the structure of the compound of formula (II).

[0188] Nuclear magnetic resonance (NMR)

[0189] 1) Test conditions

[0190] Instrument: Bruker Biospin AG AVANCE III 400 spectrometer

[0191] Solvent: CDCl3(D, 99.8%)

[0192] 2) Test data and attribution analysis

[0193] Hydrogen spectrum:

[0194]

[0195] Table 9 Hydrogen spectrum detection results of the compound of formula (II)

[0196] Proton Chemical shift (ppm) Multiplicity (coupling constant) Number of protons H 23 ]]> 8.970 s 1 H 25 ]]> 8.254 s 1 H 28 ,H 29 ,H 30 ,H 31 ]]> 8.102-7.026 m 4 H 13 ]]> 8.102-8.046 m 1 [H8] 7.822-7.782 m 1 H 11 ]]> 7.619-7.545 m 1 H7, H 18 ]]> 7.103-7.026 m 2 H 12 ]] 6.801-6.762 m 1 H6 4.430 s 1 [H5] 3.880 s 1 H 19 ]]> 2.990,2.979 d (J = 4.40 Hz) 3 [H1, H2, H3, H4] 1.835-1.510 m 8

[0197] Carbon spectrum:

[0198] Table 10 Carbon spectrum detection results of the compound of formula (II)

[0199] Assignment Chemical shift (ppm) C 16 ]]> 168.61 C 20 ]]> 167.31 C 23 ]]> 154.21 [C9, C 10 ,C 14 ]]> 143.44,138.24,127.79 C 25 ]]> 140.64

[0200] Assignment Chemical shift (ppm) [C 11 ]]> 135.97 [C 21 ,C 26 ,C 27 ]]> 132.69,128.04,127.60 [C 28 ,C 29 ,C 30 ,C 31 ]]> 131.70,127.89,124.48 C 13 ]]> 128.50 [C 12 ]]> 117.84 [C5] 56.76 [C6] 50.34 [C1,C2,C3,C4] 29.30,28.29,22.53,21.80 C 19 ]]> 26.85

[0201] 3) Conclusion

[0202] In summary, the NMR data of the sample is consistent with the structure of the compound of formula (II) shown.

[0203] Mass spectrum

[0204] 1) Test conditions

[0205] Instrument: Agilent 1200 HPLC + 6530 Q-TOF

[0206] Solvent: acetonitrile

[0207] 2) Test data and analysis

[0208] The mass spectrum analysis results are shown in the following table.

[0209] Table 11 Mass spectrum detection results of the compound of formula (II)

[0210] Observed mass-to-charge ratio (m / z) Theoretical mass-to-charge ratio (m / z) Remarks 448.2022 447.1907 [[M+H] + ]]>

[0211] 3) Conclusion

[0212] From the above detection results, the structure of the sample is consistent with the structure of the compound of formula (II).

[0213] In summary, the structure of the reference substance compound of formula (II) is correct, which is:

[0214]

[0215] Calibration procedure

[0216] High performance liquid chromatography (HPLC)

[0217] Purity

[0218] 1) Solution preparation

[0219] Diluent: acetonitrile-water (1:1, V / V)

[0220] For example: take 500 mL of acetonitrile and 500 mL of purified water into the mobile phase bottle and mix well.

[0221] Blank solution: diluent

[0222] Test sample solution: accurately weigh about 25 mg of the sample to be calibrated and placed in a 50.0 mL volumetric flask, ultrasonically dissolved with diluent and diluted to the mark, shake well, the concentration of the test sample solution is about 0.5 mg / mL, prepare 3 parallel samples.

[0223] Sensitivity solution: precisely pipette 1.0 mL of the first test solution into a 100.0 mL volumetric flask, dilute to the mark with diluent, and shake well; then precisely pipette 0.5 mL of the above solution into a 10.0 mL volumetric flask, dilute to the mark with diluent, and shake well. The concentration of the sensitivity solution is 0.00025 mg / mL, which is equivalent to 0.05% of the concentration of the test solution.

[0224] 2) Chromatographic conditions

[0225] Chromatograph: Agilent 1260 Infinity II or similar liquid chromatograph, which can be connected to a ghost peak trapping column, YMC 4.6 x 50 mm or other equivalent column

[0226] Chromatographic column: Infinity poroshell 120Aq-C18 4.6 mm x 150 mm x 2.7 μm or other equivalent chromatographic column

[0227] Column temperature: 45°C

[0228] Sample chamber: room temperature

[0229] Mobile phase A: 0.03% trifluoroacetic acid-water solution, V / V

[0230] Preparation method of mobile phase A, for example: accurately weigh about 0.3 mL of trifluoroacetic acid, and place it in 1000 mL of water, mix well and ultrasonic degassing.

[0231] Mobile phase B: 0.03% trifluoroacetic acid-acetonitrile solution, V / V

[0232] Preparation method of mobile phase B, for example: accurately weigh about 0.3 mL of trifluoroacetic acid, and place it in 1000 mL of acetonitrile, mix well and ultrasonic degassing.

[0233] Gradient:

[0234] Time (min) Mobile phase A (% V / V) Mobile phase B (% V / V) 0 100 0 2 100 0 20 5 95 23 5 95 23.1 100 0 35 100 0

[0235] Flow rate: 1.2 mL / min

[0236] Run time: 35 min

[0237] Detection wavelength: UV 215 nm

[0238] Injection volume: 10 μL

[0239] Equivalent chromatographic columns have the same or similar separation characteristics and column efficiency as the aforementioned chromatographic columns, but different fillers and stationary phases can be used to achieve them.

[0240] 3) System suitability test

[0241] Injection of blank solution 1 needle or more until the system is stable and free of interference or no significant interference, no significant interference means the peak area of interference should not exceed 10% of the peak area of the compound of formula (II) in the sensitivity solution;

[0242] Injection of sensitivity solution 1 needle, the signal-to-noise ratio of the peak of the compound of formula (II) should not be less than 10.

[0243] Retention time:

[0244] Substance name Retention time RT (min) about Compound of formula (II) 11.5

[0245] 4) Determination

[0246] After the system suitability test is passed, start the analysis determination:

[0247] Injection of test sample solution 3 times, 1 needle each time, record the chromatogram.

[0248] 5) Calculation

[0249] Total impurities (%) = sum of individual impurities

[0250] Purity (%) = 100 - total impurities

[0251] Calculate and report the average value of the purity.

[0252] Note: Report threshold (RL): 0.05%, do not integrate peaks below 0.025%.

[0253] Purity factor

[0254] Primary control: purity factor f = purity / 100

[0255] Example 5: Preparation, structure confirmation and calibration of impurity (III) as a control

[0256] The preparation route of impurity (III) is shown in the following figure:

[0257]

[0258] Use tert-butyl ((1S,2R)-2-aminocyclohexyl)carbamate instead of tert-butyl ((1R,2S)-2-aminocyclohexyl)carbamate as the starting material in Example 1. The specific experimental operation process is the same as that of Example 1, and the compound of formula (III) can be prepared directly. The compound of formula (III) is an enantiomer of the compound of formula (IV), and the name of the compound of formula (III) is: N-((1R,2S)-2-((4-bromo-2-(methylcarbamoyl)-6-nitrophenyl)amino)cyclohexyl)isoquinoline-4-carboxamide.

[0259] The compound of formula (III) prepared in Example 5 was structurally confirmed and labeled as a reference sample, and the results are as follows:

[0260] Structural confirmation

[0261] UV absorption spectrum

[0262] 1) Test conditions

[0263] Instrument: Shimadzu UV-2550

[0264] Solvent: methanol, 10.0 μg / ml

[0265] 2) Test data and analysis

[0266] The UV analysis results are shown in the following table:

[0267] Table 12 UV detection results of the compound of formula (III)

[0268] Wavelength (nm) Absorbance 322 0.084 250 0.351 220 1.101

[0269] 3) Conclusion

[0270] From the above determination results, the structure of the sample is consistent with that of the compound of formula (III).

[0271] Infrared absorption spectrum

[0272] 1) Test conditions

[0273] Instrument: Thermo IRAffinity-1

[0274] Method: KBr tabletting method

[0275] 2) Test data and analysis

[0276] The infrared analysis results are shown in the following table:

[0277] Table 13 Infrared detection results of the compound of formula (III)

[0278]

[0279] 3) Conclusion

[0280] From the characteristic absorption peaks of the infrared spectrum, the structure of the sample is consistent with that of the compound of formula (III).

[0281] Nuclear magnetic resonance (NMR)

[0282] 1) Test conditions

[0283] Instrument: Bruker Biospin AG AV400 NEO spectrometer

[0284] Solvent: DMSO-d6(D, 99.9%)

[0285] 2) Test data and attribution analysis

[0286] Hydrogen spectrum:

[0287]

[0288] Table 14 Hydrogen spectrum detection results of the compound of formula (III)

[0289] Proton Chemical shift (ppm) Multiplicity (coupling constant) Number of protons H 10 ,H 11 ,H 12 ,H 13 ]]> 1.33-1.79 m 8 [H1] 2.77 d (J = 4.5 Hz) 3 [H9] 3.76 br 1 H 14 ]]> 4.34 br 1 H4, H 23 ]]> 7.71-7.74 m 2 H 22 ]]> 7.77-7.81 m 1 H 21 ]]> 8.06 d (J = 8.4 Hz) 1 H6, H 24 ]]> 8.16-8.18 m 2 -NH- (b) ]]> 8.32 d (J = 10.1 Hz) 1 H 17 ]]> 8.47 s 1 -NH- (c) ]]> 8.65 d (J = 8.6 Hz) 1 -NH- (a) ]]> 8.70 q (J = 4.6 Hz) 1 H 18 ]]> 9.37 s 1

[0290] Carbon spectrum:

[0291] Table 15 Carbon spectrum detection results of the compound of formula (III)

[0292] Assignment Chemical shift (ppm) C 12 ]] 21.0 C 11 ]]> 22.6 [C1] 26.3 C 10 ,C 13 ]]> 27.6,28.4 C 14 ]]> 48.8 [C9] 54.3 [C5] 105.7 C 21 ]]> 124.4 [C3, C 16 ,C 19 ]]> 127.5,127.8 [C6, C 23 ]] 127.6,127.9 C 24 ]]> 129.5 [C 22 ]]> 131.2 [C 20 ]]> 132.3 [C7] 136.6 [C4] 137.3 C 17 ]]> 141.7 [C8] 142.0 [C 18 ]]> 153.9 [C 15 ]]> 166.7 [C2] 166.9

[0293] 3) Conclusion

[0294] From the above detection results, it can be seen that the structure of the sample is consistent with the structure of the compound of formula (III).

[0295] Mass spectrum (MS)

[0296] 1) Test conditions

[0297] Instrument: Agilent 1290 UPLC + 6125 CMS

[0298] Solvent: acetonitrile

[0299] 2) Test data and analysis

[0300] The mass spectrum analysis results are shown in the following table.

[0301] Table 16 Mass spectrum detection results of the compound of formula (III)

[0302] Observed mass-to-charge ratio (m / z) Theoretical mass-to-charge ratio (m / z) Remarks 526.0,528.0 526.1,528.1 [[M+H] + ]]>

[0303] 3) Analysis and conclusion

[0304] From the above detection results, it can be seen that the structure of the sample is consistent with the structure of the compound of formula (III).

[0305] In summary, the structure of the reference compound of formula (III) is:

[0306]

[0307] Calibration procedure

[0308] High performance liquid chromatography (HPLC)

[0309] Chiral purity

[0310] 1) Solution Preparation

[0311] Diluent: Ethanol

[0312] Blank Solution: Diluent

[0313] Test Solution: Accurately weigh about 25 mg of the reference substance of compound of formula (III) into a 25.0 mL volumetric flask, dissolve and dilute to the mark with diluent, shake well. The concentration of the test solution is about 1.0 mg / mL. Prepare three parallel solutions.

[0314] Sensitivity Solution: Accurately pipette 1.0 mL of the first test solution into a 100.0 mL volumetric flask, dilute to the mark with diluent, shake well. Accurately pipette 0.5 mL of the above solution into a 10.0 mL volumetric flask, dilute to the mark with diluent, shake well. The concentration of the sensitivity solution is 0.0005 mg / mL, which is equivalent to 0.05% of the concentration of the test solution.

[0315] Positioning Solution: Accurately weigh about 10 mg of the reference substance of compound of formula (IV) or reference substance, the reference substance of compound of formula (III) into a 100.0 mL volumetric flask, dissolve and dilute to the mark with diluent, shake well. Accurately pipette 1.0 mL of the above solution into a 10.0 mL volumetric flask, dilute to the mark with diluent, shake well. The concentration of the positioning solution is about 0.01 mg / mL.

[0316] Note: The solution preparation can be proportionally enlarged or reduced in volume, keeping the final concentration unchanged.

[0317] 2) Chromatographic Conditions

[0318] Chromatograph: Agilent 1260 High Performance Liquid Chromatograph or similar instrument

[0319] Chromatographic Column: YMC CHIRAL ART Cellulose-SC 4.6x150mm, 3μm or other equivalent chromatographic column

[0320] Column Temperature: 35℃

[0321] Sample Chamber Temperature: Room Temperature

[0322] Mobile Phase A: Hexane

[0323] Mobile Phase B: 0.1% Diethylamine in ethanol (V / V)

[0324] Mobile Phase: Mix mobile phase A and mobile phase B in the ratio of 85:15 by volume

[0325] Gradient: Isocratic elution

[0326] Flow rate: 1.0 mL / min

[0327] Run time: 25 min

[0328] Detection wavelength: UV 248 nm

[0329] Injection volume: 15 μL

[0330] An equivalent column has the same or similar separation characteristics and column efficiency as the aforementioned column, but can employ different packing and stationary phase to achieve.

[0331] 3) System suitability test

[0332] Inject blank solution 1 needle or more until the system is stable and free of interference or significant interference, significant interference means that the peak area of the interference peak should not exceed 10% of the peak area of the compound of formula (III) in the sensitivity solution;

[0333] Inject sensitivity solution 1 needle, the signal-to-noise ratio of the peak of the compound of formula (III) should not be less than 10;

[0334] Inject positioning solution 1 needle, record the retention time of the peak of the compound of formula (IV) and the peak of the compound of formula (III).

[0335] 4) Determination

[0336] After the system suitability test is passed, start the analysis determination:

[0337] Inject 3 portions of 1 needle of the test sample solution, and record the chromatogram.

[0338] 5) Calculation

[0339] Calculate the chiral purity of the compound of formula (III) in the test sample solution by area normalization method.

[0340] Calculate and report the average value of the chiral purity.

[0341] Purity

[0342] 1) Solution preparation

[0343] Diluent: ACN:H2O = 1.1, V / V

[0344] Blank solution: diluent

[0345] Test sample solution: accurately weigh about 25 mg of the compound of formula (III) to be marked reference substance, place it in a 50.0 mL volumetric flask, ultrasonically dissolve and dilute to the mark with diluent, shake well, the concentration of the test sample solution is about 0.5 mg / mL, prepare 3 portions in parallel.

[0346] Sensitivity solution: precisely pipette 1.0 mL of the first test solution into a 100.0 mL volumetric flask, dilute to the mark with diluent, and shake well; then precisely pipette 0.5 mL of the above solution into a 10.0 mL volumetric flask, dilute to the mark with diluent, and shake well. The concentration of the sensitivity solution is 0.00025 mg / mL, which is equivalent to 0.05% of the concentration of the test solution.

[0347] Note: The solution preparation can be proportionally enlarged or reduced in volume, while the final concentration remains unchanged.

[0348] 2) Chromatographic conditions

[0349] Instrument: Agilent 1260 Infinity II or similar liquid chromatograph, which can be connected to a ghost peak trapping column, YMC 4.6 x 50 mm or other equivalent column

[0350] Column: Waters XSelect HSS T3 4.6 x 150 mm, 3.5 μm or other equivalent column

[0351] Sample chamber temperature: room temperature

[0352] Mobile phase A: 0.03% trifluoroacetic acid-water solution, V / V

[0353] Preparation method of mobile phase A, for example: accurately weigh about 0.3 mL of trifluoroacetic acid, and place it in 1000 mL of water, mix well and ultrasonic degassing.

[0354] Mobile phase B: 0.03% trifluoroacetic acid-acetonitrile solution, V / V

[0355] Preparation method of mobile phase B, for example: accurately weigh about 0.3 mL of trifluoroacetic acid, and place it in 1000 mL of acetonitrile, mix well and ultrasonic degassing.

[0356] Gradient:

[0357]

[0358]

[0359] Flow rate: 1.5 mL / min

[0360] Injection volume: 5 μL

[0361] Detector wavelength: UV 220 nm

[0362] Run time: 35 min

[0363] Equivalent columns have the same or similar separation characteristics and column efficiency as the aforementioned columns, but different fillers and stationary phases can be used to achieve them.

[0364] 3) System suitability test

[0365] Injection of blank solution 1 needle or more until the system is stable without interference or without significant interference, without significant interference means that the peak area of interference peak should not exceed the peak area of compound of formula (III) in sensitivity solution;

[0366] Injection of sensitivity solution 1 needle, the signal-to-noise ratio of the peak of compound of formula (III) should not be less than 10.

[0367] Retention time:

[0368] Substance name Retention time RT about (min) Compound of formula (III) 8.2

[0369] 4) Determination

[0370] After the system suitability test, start the analysis determination:

[0371] Injection of test sample solution 3 times, 1 needle each time, record the chromatogram.

[0372] 5) Calculation

[0373] Total impurities (%) = sum of individual impurities

[0374] Purity (%) = 100 - total impurities

[0375] Calculate and report the average value of purity.

[0376] Note: report limit (RL): 0.05%, do not integrate the peak below 0.025%.

[0377] Purity factor

[0378] Primary control: purity factor f = (purity - loss weight) / 100

[0379] Primary control: chiral purity factor f = (purity - loss weight) x chiral purity / 100 / 100

[0380] Example 6: Detection method for determining related substances of compound of formula (IV) using impurities (I), (II) and (III) as control

[0381] Chromatographic conditions

[0382] Instrument: Agilent 1260 Infinity II or similar liquid chromatograph

[0383] Chromatographic column: Waters HSS T3 4.6mm x 150mm x 3.5μm or other equivalent chromatographic column

[0384] Ghost peak collection column: Yusheng, 4.6mm x 50mm, 40Mpa

[0385] Column temperature: 45 °C

[0386] Sample compartment temperature: room temperature

[0387] Mobile phase A: 0.03% trifluoroacetic acid in water, V / V

[0388] Preparation method of mobile phase A, for example: take trifluoroacetic acid 0.3 ml accurately, put into 1000 ml water, stir to mix, ultrasonic degassing, stand.

[0389] Mobile phase B: 0.03% trifluoroacetic acid in acetonitrile, V / V

[0390] Preparation method of mobile phase B, for example: take trifluoroacetic acid 0.3 ml accurately, put into 1000 ml acetonitrile, stir to mix, ultrasonic degassing, stand.

[0391] Gradient:

[0392] Time (min) Mobile phase A (% V / V) Mobile phase B (% V / V) 0 100 0 2 100 0 20 5 95 23 5 95 23.1 100 0 35 100 0

[0393] Flow rate: 1.2 ml / min

[0394] Injection: automatic injector

[0395] Injection volume: 10 μl

[0396] Detector wavelength: UV 220 nm

[0397] Run time: 35 min

[0398] Needle washing reagent: acetonitrile: water = 1:1, V / V

[0399] An equivalent column has the same or similar separation characteristics and column efficiency as the aforementioned column, but can be achieved using different packing materials and stationary phases.

[0400] Solution preparation

[0401] Diluent 1: acetonitrile: water = 3:7, V / V

[0402] Diluent 2: acetonitrile: water = 2:1, V / V

[0403] Blank solution: diluent 1

[0404] 1% control solution: take about 50.0 mg of compound of formula (IV) control, accurately, put into a 50 ml volumetric flask, dissolve and dilute to the mark with diluent 1, shake well. Take 1.0 ml accurately, put into a 100 ml volumetric flask, dilute to the mark with diluent 1, shake well, mark as STD-1% (about 0.01 mg / ml).

[0405] LOQ solution: Pipette 0.6 ml of 1% reference solution into a 20 ml volumetric flask, dilute to the mark with diluent 1, and shake to homogeneity. The LOQ solution has a concentration of 0.3 μg / ml, which is equivalent to 0.03% of the concentration of the test solution.

[0406] Compound (I) orientation solution: Weigh accurately about 12.5 mg of the compound (I) orientation reference substance into a 50 ml volumetric flask, dissolve and dilute to the mark with diluent 2, and shake to homogeneity.

[0407] Compound (II) orientation solution: Weigh accurately about 12.5 mg of the compound (II) orientation reference substance into a 50 ml volumetric flask, dissolve and dilute to the mark with diluent 2, and shake to homogeneity.

[0408] Mixed impurity orientation solution: Weigh accurately about 25 mg of the compound (IV) reference substance into a 50 ml volumetric flask, transfer 1 ml of the compound (I) orientation solution and 1 ml of the compound (II) orientation solution into the volumetric flask, respectively, dilute to the mark with diluent 1, and mix well.

[0409] Test solution: Weigh accurately about 50.0 mg of the compound (IV) test substance into a 50 ml volumetric flask, dissolve and dilute to the mark with diluent 1, and shake to homogeneity (about 1.0 mg / ml).

[0410] The preparation volumes of the above-mentioned compound (I) orientation solution, compound (II) orientation solution, mixed impurity orientation solution, and test solution can be enlarged in proportion according to the analysis needs.

[0411] The 1% reference solution is stable at room temperature for 48.5 hours, and stable at refrigeration (2-8°C) for 47.5 hours; the test solution is stable at room temperature for 34 hours, and stable at refrigeration (2-8°C) for 33.5 hours.

[0412] System suitability test

[0413] 1) Inject the blank solution, and the blank should have no interference. If there is an interference peak at the compound (IV) and known impurities, the interference peak area should be no more than the peak area of the compound (IV) in the LOQ solution;

[0414] 2) Inject the LOQ solution 1 time, and the signal-to-noise ratio of the MPS peak should be ≥10;

[0415] 3) Inject the mixed impurity orientation solution 1 time, and the separation degree R of each component peak from the adjacent impurity peak should be ≥1.5;

[0416] 4) Inject the 1% reference solution 5 times, and the RSD of the peak area of the compound (IV) should be ≤5.0%, and the RSD of the retention time of the compound (IV) should be ≤1.0%.

[0417] Determination

[0418] After the system suitability test is passed, the determination is started:

[0419] 1) Inject 1 needle of the test sample solution, record the chromatogram;

[0420] 2) Continue to inject other batches of test sample;

[0421] 3) After injecting 6 needles of test sample solution each time and 1 needle of 1% control sample solution at the end of the sequence, ensure that the RSD of the peak area of the compound of formula (IV) in all 1% control sample solutions should be ≤5.0%.

[0422] Retention time:

[0423] Substance name Retention time RT (min) about Compound of formula (I) 7.8 Compound of formula (II) 11.7 Compound of formula (IV) 13.2

[0424] Calculation

[0425] The content of each impurity in the test sample solution is calculated according to the following formula:

[0426]

[0427] In the formula:

[0428] A R The average value of the peak area of the compound of formula (IV) in 5 needles of 1% control sample solution;

[0429] A S The peak area of each impurity in the test sample solution;

[0430] W R The weight of the compound of formula (IV) control sample when preparing the 1% control sample solution, mg;

[0431] f The purity factor of the compound of formula (IV) control sample when preparing the 1% control sample solution;

[0432] W S The weight of the test sample when preparing the test sample solution, mg;

[0433] CF The correction factor of each impurity (0.54 for the compound of formula (I), 0.80 for the compound of formula (II), and 1.00 for unknown impurities).

[0434] Total impurities = the sum of the contents of all impurities

[0435] Note: Report limit: 0.03%, do not integrate peaks below 0.015%.

[0436] Enantiomer

[0437] Chromatographic conditions

[0438] Chromatograph: Agilent 1260 high performance liquid chromatograph or similar instrument

[0439] Column: YMC CHIRAL ART Cellulose-SC 4.6x150mm, 3μm or other equivalent column

[0440] Column temperature: 35℃

[0441] Mobile phase:

[0442] A: Hexane

[0443] B: 0.1% diethylamine in ethanol (V / V)

[0444] Mix A and B at a ratio of 85:15 by volume.

[0445] Prepare mobile phase B by, for example, pipetting 1.0ml diethylamine into 1000ml ethanol, mix well and degas by sonication.

[0446] Elution: Isocratic elution

[0447] Flow rate: 1.0ml / min

[0448] Run time: 25min

[0449] Detection wavelength: UV 248nm

[0450] Injection volume: 15μl

[0451] Needle wash solvent: Ethanol

[0452] Injector: Autosampler

[0453] Equivalent columns have the same or similar separation characteristics and column efficiency as the aforementioned column but can be achieved using different packing materials and stationary phases.

[0454] Solution preparation

[0455] Diluent: Ethanol

[0456] Blank solution: Diluent

[0457] Stock solution of reference: Accurately weigh about 7.0mg of reference compound of formula (III) into a 100ml volumetric flask, dissolve and dilute to the mark with diluent, mix well.

[0458] Solution of reference: Pipette 12.5ml of the stock solution of reference into a 250ml volumetric flask, dilute to the mark with diluent, mix well.

[0459] Sensitivity solution: Pipette 8.6 ml of the reference solution into a 100 ml volumetric flask, dilute to the mark with the diluent, and mix.

[0460] Test solution: Accurately weigh about 25.0 mg of MPS test sample into a 25 ml volumetric flask, dissolve and dilute to the mark with the diluent, and mix.

[0461] System suitability test

[0462] 1) Inject the blank solution 1 or more times until the system is stable and there is no interference;

[0463] 2) Inject the sensitivity solution 1 time, and the signal-to-noise ratio of the peak of compound of formula (III) should not be less than 10;

[0464] 3) Inject the reference solution 6 times in succession, and record the retention time and peak area of the peak of compound of formula (III). The RSD of the peak area of the peak of compound of formula (III) in 6 injections of the reference solution should not be greater than 10.0%, and the RSD of the retention time should not be greater than 1.0%.

[0465] Retention time:

[0466] Solvent name Retention time RT (min) about Compound of formula (III) 14.4

[0467] Determination

[0468] After the system suitability test is passed, start the analysis determination:

[0469] 1) Inject the test solution 1 time, and record the chromatogram. Identify the peak of compound of formula (III) on the chromatogram of the test solution according to the retention time;

[0470] 2) Inject the test solution every 6 times, and inject the reference solution 1 time at the end of the sequence. The RSD of the peak area of the peak of compound of formula (III) in all the reference solutions in the sequence should not be greater than 10.0%.

[0471] Calculation

[0472] The content of compound of formula (III) in the test solution is calculated according to the following formula:

[0473]

[0474] In the formula:

[0475] A R The average value of the peak area of compound of formula (III) in 6 injections of the reference solution in the system suitability test;

[0476] A S The peak area of compound of formula (III) in the test solution;

[0477] WR —When preparing the reference standard stock solution, the sample weight of the compound reference standard of formula (III) is in mg;

[0478] f——Purity factor of compound (III) reference standard when preparing reference standard stock solution;

[0479] W S —The sample weight, in mg, when preparing the test solution.

[0480] Note: Reporting limit (RL): 0.03%, no points are awarded for values ​​below 0.015%.

[0481] Table 17 shows the results of the determination of related substances in different batches of compound (IV) using impurity compounds (I), (II) and (III) as reference standards.

[0482] Table 17 Results of Related Substance Detection for Compound (IV)

[0483]

[0484] Note: The atlas numbered 1 is attached. Figure 5 and Figure 6 As shown, the atlas numbered 2 is attached. Figure 7 and Figure 8 As shown, in Figures 5-8 In the original spectrum, compound (I) is C220104010-SM3; compound (II) is C220104010-FP_impurity4; compound (III) is C220204010-FP_impurity1 and compound (IV) is C220104010-G.

Claims

1. A related substance of a compound of formula IV, characterized in that, Including compounds of formula I, formula II and formula III:

2. The related substance as described in claim 1, characterized in that: The preparation method of compound I is as follows: Compound I-1 undergoes a cyanidation reaction with an inorganic cyanide to give aryl cyanide I-2, and aryl cyanide I-2 undergoes a hydrolysis reaction to give compound I.

3. The relevant substance as described in claim 1, characterized in that: The preparation method of compound II is as follows: Compound II-1 and compound IV-2 are reacted via an aromatic nucleophilic substitution reaction to give compound II-2. Compound II-2 is reacted with methylamine hydrochloride via acid-amine condensation to give compound II-3. Compound II-3 is deprotected by the Boc protecting group to give compound II-4. Compound II-4 is reacted with compound I via acid-amine condensation to give compound II.

4. The related substance as described in claim 1, characterized in that: The preparation method of compound III is as follows: Compound IV-1 and Compound III-1 are reacted via an aromatic nucleophilic substitution reaction to give Compound III-2. Compound III-2 is reacted with methylamine hydrochloride via an acid-amine condensation reaction to give Compound III-3. Compound III-3 is deprotected by the Boc protecting group to give Compound III-4. Compound III-4 is reacted with Compound I via an acid-amine condensation to give Compound III.

5. The preparation method according to claim 2, characterized in that: The inorganic cyanide is selected from KCN, NaCN, CuCN or K4[Fe(CN)6], with CuCN being preferred.

6. The preparation method according to any one of claims 3-4, characterized in that: Aromatic nucleophilic substitution reactions are promoted by using a base, wherein the base is selected from inorganic bases, organic bases, or combinations thereof; Preferably, the organic base is selected from triethylamine (TEA), N,N-diisopropylethylamine (DIPEA), pyridine, 1,8-diazobispirocyclo[5.4.0]undec-7-ene (DBU), t-BuOK, or t-BuONa; the inorganic base is selected from Na2CO. 3， K2CO3, Cs2CO3, NaH, LiOH, NaOH, KOH; More preferably, the alkali is selected from N,N-diisopropylethylamine or K2CO3.

7. The preparation method according to any one of claims 3-4, characterized in that: In the acid-amine condensation reaction, a condensing agent is used, which is selected from 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU), 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU), benzotriazole-1-yl-oxytripyrrolylphosphonate hexafluorophosphate (PyBOP), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), oxaloyl chloride, sulfoxide, or isopropyl chloroformate; preferably 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) or isopropyl chloroformate.

8. The preparation method according to any one of claims 3-4, characterized in that: The temperature for acid-amine condensation reaction is -5℃ to 10℃ or 0℃ to 25℃.

9. The preparation method according to any one of claims 3-4, characterized in that: The solvents used in the aromatic nucleophilic substitution reaction and amide condensation reaction are selected from N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone or combinations thereof.

10. The use of the related substances of the compound of formula IV as a standard reference for the quality control of the compound of formula IV, characterized in that: The relevant substances of compound IV were determined by high-performance liquid chromatography (HPLC): Compounds of Formula I, Formula II or Formula III.

11. The use as described in claim 10, characterized in that: Compound I and Compound II are used HSS T3 or equivalent columns were connected to ghost peak trapping columns and gradient elution was performed.

12. The use as described in claim 11, characterized in that: Gradient elution is employed, with mobile phase A being a trifluoroacetic acid-water solution and mobile phase B being a trifluoroacetic acid-acetonitrile solution; preferably, mobile phase A is a 0.03% trifluoroacetic acid-water solution and mobile phase B is a 0.03% trifluoroacetic acid-acetonitrile solution.

13. The use as described in claim 12, characterized in that, The elution gradient is: 。 14. The use as described in claim 10, characterized in that: Compound III was eluted isocratically using a CHIRAL ART Cellulose-SC or equivalent column.

15. The use as described in claim 14, characterized in that: The isocratic elution conditions are that mobile phase A is hexane and mobile phase B is an ethanol solution of diethylamine; preferably, the ratio of mobile phase A to mobile phase B is 85:15 (V / V), and mobile phase B is a 0.1% ethanol solution of diethylamine.

16. The use as described in claim 11, characterized in that, The detection wavelength is 220± 5nm.

17. The use as described in claim 14, characterized in that, The detection wavelength is 248± 3nm.

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