Impurity compounds in raw materials for preparing varenicline tartrate and preparation, application and detection methods thereof

Abstract

The present invention provides an impurity compound in the raw material for preparing varenicline tartrate and its preparation, application and detection method. The impurity compound is shown in formula P06. The impurity compound will be transformed into a nitrosamine impurity P08 with genotoxicity as the reaction progresses, and it is used as a standard, reference substance or detection item for varenicline tartrate or its raw material, impurity research, quality control, etc. of intermediates, to achieve qualitative and / or quantitative detection of the impurity, so as to further adopt active and effective technical means, and the nitrosamine genotoxic impurity content in the varenicline tartrate bulk drug or preparation is controlled at a lower level, thereby meeting safety requirements and drug product regulatory regulations, and better ensuring patient medication safety.

Description

Technical Field

[0001] The invention belongs to the field of medicines, and in particular relates to impurity compounds in raw materials for preparing varenicline tartrate and preparation, application and detection methods thereof. Background Art

[0002] Varenicline is an aromatic fused nitrogen polycyclic compound that modulates cholinergic function and is sold as a tartrate salt under the trade name or It can bind to neuronal nicotinic acetylcholine specific receptor sites and can be used for the following diseases:

[0003] Treatment of inflammatory bowel disease (including but not limited to: ulcerative colitis, pyoderma gangrenosum and Crohn's disease), irritable bowel syndrome, spastic dystonia, chronic pain, acute pain, celiac sprue, pouchitis, vasoconstriction, anxiety, panic disorder, depression, bipolar disorder, autism, sleep disorders, jet lag, amyotrophic lateral sclerosis (ALS), cognitive dysfunction, drug / toxin-induced cognitive impairment (e.g., caused by alcohol, barbiturates, vitamin deficiency, recreational drugs, lead, arsenic, mercury), disease-induced cognitive impairment (e.g., caused by Alzheimer's disease, vascular dementia, dementia), Parkinson's disease, multiple sclerosis, AIDS, encephalitis, trauma, renal and hepatic encephalopathy, hypothyroidism, Pick's disease, Korsakoff's syndrome, and frontal and subcortical dementia), hypertension, bulimia, anorexia, obesity, cardiac arrhythmias, gastric acid hypersecretion, ulcers, pheochromocytoma, progressive supramuscular palsy, chemical dependence and addiction (e.g., to nicotine (and / or tobacco products), alcohol, benzodiazepines, Dependence and addiction to narcotics, barbiturates, opioids or cocaine), headache, migraine, stroke, traumatic brain injury (TBI), obsessive-compulsive disorder (OCD), psychosis, Huntington's chorea, tardive dyskinesia, hyperkinesia, dyslexia, schizophrenia, multiple sclerosis dementia, age-related cognitive decline, epilepsy, including petit mal absence epilepsy, attention deficit hyperactivity disorder (ADHD), Tourette's Syndrome; in particular, dependence on, addiction to and withdrawal from nicotine, including use in smoking cessation treatment (see: CN 1509174A).

[0004] The preparation method of varenicline and the resolution method of its racemate are disclosed in US Patent Application No. 6,410,550 and International Patent Application No. WO 01 / 62736, the contents of which are also incorporated herein by reference.

[0005] Nitrosamine compounds, including nitroso (N(R1)(R2)-N=O) structures bound to amines, are genotoxic substances in some animals, and some have been listed as possible or potential human carcinogens by the International Agency for Research on Cancer (IARC).

[0006] These nitrosamine compounds are referred to as “group of concern” compounds in the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidance document M7(R1), Evaluation and Control of DNA-Reactive (Mutagenic) Impurities in Pharmaceutical Products to Limit Potential Carcinogenic Risk (March 2018). The ICH guidance recommends that any known mutagen / carcinogen, including nitrosamine compounds, be controlled to a level of intake that would be of negligible risk to humans of cancer.

[0007] Currently, the U.S. FDA has identified seven nitrosamine impurities that could theoretically be present in drugs because of the use of manufacturing processes and materials that could lead to the formation of nitrosamines: N,N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitroso-N-methyl-4-aminobutyric acid (NMBA), N-nitrosoisopropylethylamine (NIPEA), N-nitrosodiisopropylamine (NDIPA), N-nitrosodibutylamine (NDBA), and N-nitrosomethylaniline (NMPA). Five of these impurities (NDMA, NDEA, NMBA, NIPEA, and NMPA) have actually been detected in APIs or drugs.

[0008] For example, some preliminary results from FDA trials indicate that NDMA levels in some ranitidine products exceed acceptable levels. In recent years, the FDA has also found NDMA impurities in some metformin products, with levels of NDMA detected in some batches exceeding the FDA's recommended acceptable intake limit.

[0009] In view of this, the present invention is proposed to control the nitrosamine impurity content in the varenicline tartrate raw material or preparation to a lower level that can meet the safety requirements. Summary of the invention

[0010] In view of the problems and / or shortcomings of the prior art, one of the purposes of the present invention is to provide a new compound related to varenicline tartrate: a compound shown in formula P06. The new compound will be transformed into a genotoxic nitrosamine impurity P08 as the reaction progresses, and needs to be detected and controlled throughout the synthesis of varenicline tartrate, so as to further adopt active and effective technical means to control the nitrosamine genotoxic impurity content in the varenicline tartrate raw material or preparation at a low level to meet safety requirements.

[0011] The present invention provides a compound represented by formula P06:

[0012]

[0013] The present invention also provides a method for preparing a compound of formula P06, comprising the following steps: compound M01 and nitrite undergo nitrosamination reaction in the presence of an acid and a solvent to generate a compound of formula P06;

[0014]

[0015] Preferably,

[0016] The nitrite is potassium nitrite or sodium nitrite;

[0017] The solvent is water and tetrahydrofuran, and the volume ratio of water to tetrahydrofuran is 1:4-6;

[0018] The acid is acetic acid;

[0019] More preferably,

[0020] The molar ratio of compound M01 to nitrite is 1:0.5-5, and more preferably 1:2-4;

[0021] The volume mass ratio of the solvent to the compound M01 is 5 to 20 mL / g, and preferably 10 to 15 mL / g;

[0022] The mass ratio of the acid to the compound M01 is 0.1 to 1, and more preferably 0.4 to 0.8;

[0023] The reaction temperature of the nitrosamination reaction is 30-60°C.

[0024] The present invention also provides the use of the compound represented by formula P06 as a standard substance, reference substance or detection item in the impurity research, quality control or detection method of substance X; preferably, the detection method is high performance liquid chromatography;

[0025]

[0026] The substance X is R0 is hydrogen or an amino protecting group; preferably, R0 is trifluoroacetyl.

[0027] Another object of the present invention is to provide a method for detecting the compound represented by formula P06 in substance X.

[0028] The method for detecting the compound represented by formula P06 in substance X provided by the present invention uses the compound represented by formula P06 as a standard or reference substance and adopts high performance liquid chromatography for detection;

[0029]

[0030] The substance X is R0 is hydrogen or an amino protecting group; preferably, R0 is trifluoroacetyl.

[0031] Furthermore,

[0032] In any of the above technical solutions (method for detecting the compound represented by formula P06 in substance X), the chromatographic conditions of the high performance liquid chromatography method include:

[0033] Chromatographic column: C18 column, preferably Kromasil 100-5C18;

[0034] Mobile phase: phosphoric acid aqueous solution-methanol, the volume ratio of phosphoric acid aqueous solution to methanol is 30%:70% to 50%:50%, preferably the volume ratio of phosphoric acid aqueous solution to methanol is 40%:60%;

[0035] Detection wavelength: 200~320nm.

[0036] Furthermore,

[0037] In any of the above technical solutions (the detection method of the compound represented by the formula P06 in substance X), the specifications of the chromatographic column are: 4.6 mm×250 mm, 5 μm.

[0038] Furthermore,

[0039] In any of the above technical solutions (the detection method of the compound represented by the formula P06 in substance X), the concentration of the phosphoric acid aqueous solution is 0.01% to 0.1%, preferably 0.05%.

[0040] Furthermore,

[0041] In any of the above technical solutions (method for detecting the compound represented by formula P06 in substance X), the chromatographic conditions of the high performance liquid chromatography method include:

[0042] Column temperature: 15-35°C, preferably 20°C, 25°C, 30°C or 32°C;

[0043] Flow rate: 0.5~2ml / min, preferably 0.5ml / min, 0.6ml / min, 0.7ml / min, 0.8ml / min, 0.9ml / min, 1ml / min, 1.2ml / min or 1.5ml / min;

[0044] Injection volume: 1-35μ1, preferably 2μ1, 3μ1, 4μ1, 5μ1, 6μ1, 7μ1, 8μ1, 9μ1, 10μ1, 15μ1, 20μ1, 25μ1 or 30μ1.

[0045] Furthermore,

[0046] In any of the above technical solutions (method for detecting the compound represented by formula P06 in substance X), the detection method is qualitative detection or quantitative detection, the solvent for the standard, reference and substance X is acetonitrile-water, and the volume ratio of acetonitrile to water is 50%:50% to 70%:30%, preferably 60%:40%.

[0047] Furthermore,

[0048] In any of the above technical solutions (method for detecting the compound represented by formula P06 in substance X), quantitative detection is performed using an internal standard method or an external standard method;

[0049] Preferably,

[0050] The external standard method is as follows: using the compound shown in formula P06 as a standard or reference substance, preparing standard solutions or reference solutions of different concentrations, injecting samples for detection, obtaining corresponding peak area data, performing linear regression between the concentration and the peak area, and obtaining a linear equation; then substituting the peak area detection data of the compound shown in formula P06 in the detection sample solution of substance X into the linear equation, and obtaining the concentration data of the compound shown in formula P06 in the detection sample solution; and then dividing the data by the concentration data of the detection sample solution, and obtaining a quantitative detection result.

[0051] The beneficial effects of the present invention mainly lie in that the compound represented by formula P06 provided by the present invention will be transformed into nitrosamine impurity P08 with genotoxicity as the reaction progresses, and the compound can be used as a standard substance, a reference substance or a detection item for the impurity research and quality control of varenicline tartrate or its raw materials and intermediates, so as to achieve qualitative and / or quantitative detection of the impurity, so as to further adopt positive and effective technical means to control the nitrosamine genotoxic impurity content in varenicline tartrate raw materials or preparations at a lower level, thereby meeting the safety requirements and drug product regulatory provisions, and better ensuring the safety of patients' medication. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 This is a chromatogram of the nitrosamine impurity compound P06 in compound A detected by high performance liquid chromatography. DETAILED DESCRIPTION

[0053] The present invention will be clearly and completely described below in conjunction with specific embodiments. Those skilled in the art will understand that the embodiments described below are only some embodiments of the present invention, rather than all embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of protection of the present invention.

[0054] In the present invention, if no specific conditions are specified, the experiments are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0055] Regarding the definitions of terms used in the present invention, unless otherwise stated, the initial definitions provided for the terms in this document apply to the terms throughout the text; for terms that are not specifically defined herein, the meaning that a person skilled in the art would give them should be given based on the disclosure and / or context.

[0056] Example 1

[0057] Preparation of Nitrosamine Compound P06

[0058]

[0059] In a 100mL three-necked flask, add 5g of compound M01 and 52mL of tetrahydrofuran (THF), and stir to dissolve; add 10mL of NaNO2 aqueous solution (concentration of 0.4g / mL) and 3g of acetic acid, heat to 50°C for reaction, monitor the completion of the reaction by TLC or HPLC, concentrate, add 50mL of dichloromethane (DCM) and 50mL of saturated brine, stir to dissolve, let stand, separate the liquids, take the organic phase, wash once with saturated brine, dry over anhydrous sodium sulfate, filter, and concentrate to dryness to obtain nitrosamine compound P06 with a purity of 93.69% (area normalization method).

[0060]

[0061] Nitrosamine compound P06 1 H-NMR, 13 C-NMR and IR spectrum data are shown in Tables 1 to 3, respectively; M+H + =279.10; UV: The maximum absorption wavelength in acetonitrile solution is 223.80nm.

[0062] Table 1. Nitrosamine compound P06 1 H-NMR spectrum

[0063]

[0064] Table 2. Nitrosamine compound P06 13 C-NMR spectrum

[0065]

[0066] Table 3. IR spectrum of nitrosamine compound P06

[0067] <![CDATA[Absorption wave number (cm -1 )]]> Attribution 3034.5 Ar-H 2920.2,2879.0 Saturated CH stretching vibration 1667.2 Benzene ring C=C stretching vibration 1546.9,1428.2,1344.9 N=O, NO stretching vibration 845.2 Benzene ring CH bending vibration

[0068] Example 2

[0069] Preparation of nitrosamine compound P07

[0070]

[0071] To a 100 mL three-necked flask, add 4.0 g of compound P06 and 50 mL of dichloromethane (DCM), stir to dissolve; add 1.0 g of palladium carbon catalyst (Pd / C), replace with nitrogen first, then continue to pass hydrogen, raise the temperature to 25-30 ° C, react overnight, monitor the completion of the reaction by TLC, filter, and concentrate to dryness to obtain nitrosamine compound P07 with a purity of 96.59% (area normalization method).

[0072]

[0073] Nitrosamine compound P07 1 H-NMR, 13 C-NMR and IR spectrum data are shown in Tables 4 to 6, respectively; M+H + =219.20; UV: The maximum absorption wavelength in acetonitrile: water = 1:1 solution is 212.00nm.

[0074] Table 4. Nitrosamine compound P07 1 H-NMR spectrum

[0075]

[0076] Table 5. Nitrosamine compound P07 13 C-NMR spectrum

[0077]

[0078]

[0079] Table 6. IR spectrum of nitrosamine compound P07

[0080] <![CDATA[Absorption wave number (cm -1 )]]> Attribution 3432.6 NH stretching vibration 3008.2 Ar-H 2942.2,2870.9 Saturated CH stretching vibration 1655.5 Benzene ring C=C stretching vibration 1495.2 N=O stretching vibration 866.5 Benzene ring CH bending vibration

[0081] Example 3

[0082] Preparation of Nitrosamine Compound P08

[0083]

[0084] In a 100-mL three-necked flask, add 4.0 g of compound M02 (varenicline) and 45 mL of tetrahydrofuran (THF), and stir to dissolve; add 8.2 mL of NaNO2 aqueous solution (concentration of 0.4 g / mL) and 2.9 g of acetic acid, and heat to 52°C. The reaction is completed after TLC monitoring, and solids are precipitated. Filter and vacuum dry to obtain nitrosamine compound P08 with a purity of 98.87% (area normalization method).

[0085]

[0086] Nitrosamine compound P08 1 H-NMR, 13 C-NMR and IR spectrum data are shown in Tables 7 to 9, respectively; M+H + =241.20; UV: The maximum absorption wavelength in acetonitrile solution is 204.40nm.

[0087] Table 7. Nitrosamine compound P08 1 H-NMR spectrum

[0088]

[0089]

[0090] Table 8. Nitrosamine compound P08 13 C-NMR spectrum

[0091]

[0092] Table 9. IR spectrum of nitrosamine compound P08

[0093] <![CDATA[Absorption wave number (cm -1 )]]> Attribution 3048.8 Ar-H 2961.4,2931.8 Saturated CH stretching vibration 1925.2 Benzene ring C=C stretching vibration 1575.2 N=O stretching vibration 885.2 Benzene ring CH bending vibration

[0094] Example 4

[0095] The entire contents of Chinese patent application No. 202010698570.8 (Invention Title: A method for preparing a varenicline intermediate, varenicline and its salts) filed on July 20, 2020 are incorporated herein by reference in their entirety.

[0096] 1. Preparation of Varenicline Intermediates

[0097]

[0098] ①, 3 kg (about 8.69 mol) of compound A and 0.35 kg of palladium carbon (Pd / C, Pd 5%) catalyst were added to a reactor containing 36 kg of isopropanol and 15 kg of purified water, the reaction temperature was controlled at 25-35° C., hydrogen was passed through and stirred for 6 h, and then the reaction was stopped (HPLC detected that the residual amount of compound A in the reaction solution was ≤0.5%), and the palladium carbon catalyst was removed by filtration with diatomaceous earth to obtain a reaction solution containing compound B;

[0099]

[0100] ②, under the protection of inert gas (nitrogen in this embodiment), to the reaction solution obtained in step ①, add 75g of sodium bicarbonate (about 0.89mol, alkaline), and then slowly add an aqueous solution of glyoxal (0.556kg (about 9.58mol) of glyoxal and 6.5kg of water, acidic), control the reaction temperature at 20-30°C, stir the reaction for 8h (including the addition time of the glyoxal aqueous solution), and then stop the reaction (HPLC detection of the remaining amount of compound B in the reaction solution ≤0.5 %), distill under reduced pressure (vacuum degree ≤-0.09MPa, 40-50°C) until almost no fraction is produced, add 90kg of purified water, control the temperature in the kettle at 20-30°C, keep warm for 2h, then centrifuge and dry (45-50°C) to obtain 2.34kg of varenicline intermediate (compound C) as an off-white solid with HPLC purity of 99.1%. Related substance detection items: compound A ≤0.1%, compound B ≤0.1%, and other maximum single unknown impurity content ≤0.1%.

[0101] 2. Preparation of Varenicline Tartrate

[0102]

[0103] a. Under the protection of inert gas (nitrogen in this embodiment), 20 kg of purified water, 0.81 kg (about 20.25 mol) of sodium hydroxide, 8 kg of dichloromethane and 2 kg (about 6.51 mol) of compound C (prepared in the previous step) were added to the reactor, the reaction temperature was controlled at 20-35° C., the reaction was stirred for 8 h, and then the reaction was stopped (the residual amount of compound C in the reaction solution was ≤0.5% by HPLC), and then 18.5 kg of ethanol (about 20.25 mol) was added to the reactor. g dichloromethane, stirred, allowed to stand, separated, the organic phase was taken, the aqueous phase was extracted with dichloromethane (15kg×2), the organic phases were combined, 5kg of anhydrous sodium sulfate was added to the organic phase for drying, filtered, the filtrate was subjected to reduced pressure distillation (vacuum degree ≤-0.09MPa, 40-50°C) until substantially no fraction was produced, 10kg of anhydrous ethanol was added, and reduced pressure distillation (vacuum degree ≤-0.09MPa, 40-50°C) was continued until substantially no fraction was produced to obtain a mother liquor containing varenicline;

[0104]

[0105] b. Slowly add 30 kg of anhydrous ethanol and 1.16 kg (about 7.73 mol) of L-(+)-tartaric acid to the mother liquor containing varenicline obtained in step a, control the reaction temperature at 20 to 30 ° C, stir the reaction for 16 h (including the addition time of anhydrous ethanol and L-(+)-tartaric acid), then stop the reaction, centrifuge, and dry (70 to 75 ° C) to obtain 2.08 kg of varenicline tartrate as an off-white solid.

[0106] Example 5

[0107] According to the requirements of the FDA (Food and Drug Administration), nitrosamine impurities in drugs need to be tested, and are generally required to be controlled at ≤7.5ppm.

[0108] To this end, we creatively developed an ultra-high performance liquid chromatography triple quadrupole mass spectrometry (UPLC-MS / MS) method for qualitative and / or quantitative detection of nitrosamine impurities (compound P08) in the varenicline tartrate bulk drug of the present invention. The chromatographic conditions and mass spectrometry conditions of UPLC-MS / MS are specifically shown in Table 10.

[0109] Table 10. Chromatographic conditions and mass spectrometry conditions of UPLC-MS / MS

[0110]

[0111] After testing, the content of nitrosamine impurity compound P08 in varenicline tartrate prepared in Example 4 was 114 ppm (the test results showed that the retention time of the impurity was about 3.47 min); this indicates that if varenicline tartrate is prepared according to the conventional method without control and treatment, the content of nitrosamine impurity compound P08 in the obtained product is usually at the level of hundreds of ppm, which cannot meet the FDA's limit requirements for nitrosamine impurities.

[0112] Example 6

[0113] 1. Preparation of Varenicline Intermediates

[0114] ①, 3 kg of compound A (after testing, the content of nitrosamine impurity compound P06 in compound A is 722 ppm) and 0.35 kg of palladium carbon (Pd / C, Pd 5%) catalyst are added to a reactor containing 36 kg of isopropanol and 15 kg of purified water, the reaction temperature is controlled at 25-35 ° C, hydrogen is passed and stirred to react, HPLC detection of the remaining amount of compound A in the reaction solution is ≤0.5% to stop the reaction, diatomaceous earth is used for filtration to remove the palladium carbon catalyst, and a reaction solution containing compound B is obtained;

[0115] ②, under the protection of inert gas (nitrogen), to the reaction solution obtained in step ①, add 75g of sodium bicarbonate, then slowly add an aqueous solution of glyoxal (0.56kg of glyoxal and 6.5kg of water), control the reaction temperature at 20-30°C, stir the reaction, HPLC detects the remaining amount of compound B in the reaction solution ≤0.5% to stop the reaction, distill under reduced pressure (vacuum degree ≤-0.09MPa, 40-50°C) until substantially no fraction is produced, add 90kg of purified water, control the temperature in the kettle at 20-30°C, keep warm for 2h, then centrifuge and dry (45-50°C) to obtain compound C.

[0116] 2. Preparation of Varenicline Tartrate

[0117] a. Under the protection of inert gas (nitrogen), add 20kg purified water, 0.81kg sodium hydroxide, 8kg dichloromethane and 2kg compound C (prepared in the previous step) to the reaction flask, control the reaction temperature at 20-35°C, and stop the reaction when the residual amount of compound C in the reaction solution is ≤0.5% by HPLC. Then add 18.5kg dichloromethane to the reactor, stir, stand, separate the liquids, take the organic phase, extract the aqueous phase with dichloromethane (15kg×2), combine the organic phases, add 5kg anhydrous sodium sulfate to the organic phases for drying, filter, and distill the filtrate under reduced pressure (vacuum degree ≤-0.09MPa, 40-50°C) until there is basically no fraction produced, add 10kg anhydrous ethanol, and continue to distill under reduced pressure (vacuum degree ≤-0.09MPa, 40-50°C) until there is basically no fraction produced to obtain a mother liquor containing varenicline;

[0118] b. Slowly add 30 kg of anhydrous ethanol and 1.16 kg of L-(+)-tartaric acid to the mother liquor containing varenicline obtained in step a, control the reaction temperature at 20 to 30 ° C, stir the reaction for 16 hours, then stop the reaction, centrifuge, and dry (70 to 75 ° C) to obtain varenicline tartrate;

[0119] Then, the prepared varenicline tartrate is added to the pulping solvent according to the solvent dosage of 8 ml per gram of varenicline tartrate, and pulped at 30-40°C for 1.5 hours, then centrifuged and dried. After testing, the content of nitrosamine impurity compound P08 in varenicline tartrate after dichloromethane pulping is 5.94 ppm, and the contents of nitrosamine impurity compounds P06 and P07 are not detected (less than the detection limit), which meets the FDA's limit requirements for nitrosamine impurities.

[0120] Example 7

[0121] 1. Preparation of Varenicline Intermediates

[0122] ①, 3 kg of compound A (in order to better ensure product quality, it is generally required that: the content of nitrosamine impurity compound P06 in compound A is ≤500 ppm, and the detection value is 485 ppm) and 0.35 kg of palladium carbon (Pd / C, Pd 5%) catalyst are added to a reactor containing 36 kg of isopropanol and 15 kg of purified water, the reaction temperature is controlled at 25-35° C., hydrogen is passed through and stirred to react, and the reaction is stopped when the residual amount of compound A in the reaction solution is ≤0.5% by HPLC, and the palladium carbon catalyst is removed by filtration with diatomaceous earth pad to obtain a reaction solution containing compound B;

[0123] ②, under the protection of inert gas (nitrogen), to the reaction solution obtained in step ①, add 75g of sodium bicarbonate, then slowly add an aqueous solution of glyoxal (0.56kg of glyoxal and 6.5kg of water), control the reaction temperature at 20-30°C, stir the reaction, HPLC detects the remaining amount of compound B in the reaction solution ≤0.5% to stop the reaction, distill under reduced pressure (vacuum degree ≤-0.09MPa, 40-50°C) until substantially no fraction is produced, add 90kg of purified water, control the temperature in the kettle at 20-30°C, keep warm for 2h, then centrifuge and dry (45-50°C) to obtain compound C.

[0124] 2. Preparation of Varenicline Tartrate

[0125] a. Under the protection of inert gas (nitrogen), add 20kg purified water, 0.81kg sodium hydroxide, 8kg dichloromethane and 2kg compound C (prepared in the previous step) to the reaction flask, control the reaction temperature at 20-35°C, and stop the reaction when the residual amount of compound C in the reaction solution is ≤0.5% by HPLC. Then add 18.5kg dichloromethane to the reactor, stir, stand, separate the liquids, take the organic phase, extract the aqueous phase with dichloromethane (15kg×2), combine the organic phases, add 5kg anhydrous sodium sulfate to the organic phases for drying, filter, and distill the filtrate under reduced pressure (vacuum degree ≤-0.09MPa, 40-50°C) until there is basically no fraction produced, add 10kg anhydrous ethanol, and continue to distill under reduced pressure (vacuum degree ≤-0.09MPa, 40-50°C) until there is basically no fraction produced to obtain a mother liquor containing varenicline;

[0126] b. Slowly add 30 kg of anhydrous ethanol and 1.16 kg of L-(+)-tartaric acid to the mother liquor containing varenicline obtained in step a, control the reaction temperature at 20 to 30 ° C, stir the reaction for 16 hours, then stop the reaction, centrifuge, and dry (70 to 75 ° C) to obtain varenicline tartrate;

[0127] Then, the prepared varenicline tartrate is added to the pulping solvent according to the solvent dosage of 8 ml per gram of varenicline tartrate, and pulped at 30-40°C for 1.5 hours, followed by centrifugation and drying. After testing, the content of nitrosamine impurity compound P08 in the varenicline tartrate after ethyl acetate pulping was 3.04 ppm, and the contents of nitrosamine impurity compounds P06 and P07 were not detected (less than the detection limit).

[0128] Example 8

[0129] Preparation of Varenicline Tartrate

[0130] a. Under the protection of inert gas (nitrogen), 20 kg of purified water, 0.81 kg of sodium hydroxide, 8 kg of dichloromethane and 2 kg of compound C (prepared in Example 7) were added to the reaction flask, the reaction temperature was controlled at 20-35 ° C, and the residual amount of compound C in the reaction solution was detected by HPLC to be ≤0.5% to stop the reaction, then 18.5 kg of dichloromethane was added to the reactor, stirred, allowed to stand, separated, the organic phase was taken, the aqueous phase was extracted with dichloromethane (15 kg × 2), the organic phases were combined, 5 kg of anhydrous sodium sulfate was added to the organic phase for drying, filtered, and the filtrate was distilled under reduced pressure (vacuum degree ≤-0.09 MPa, 40-50 ° C) until substantially no fraction was produced, 10 kg of anhydrous ethanol was added, and the distillation under reduced pressure (vacuum degree ≤-0.09 MPa, 40-50 ° C) was continued until substantially no fraction was produced to obtain a mother liquor containing varenicline;

[0131] b. Slowly add 30 kg of anhydrous ethanol and 1.16 kg of L-(+)-tartaric acid to the mother liquor containing varenicline obtained in step a, control the reaction temperature at 20 to 30 ° C, stir the reaction for 16 hours, then stop the reaction, centrifuge, and dry (70 to 75 ° C) to obtain varenicline tartrate;

[0132] Then, the prepared varenicline tartrate is added to the pulping solvent according to the solvent dosage of 8 ml per gram of varenicline tartrate, and pulped at 30-40°C for 1.5 hours, followed by centrifugation and drying. After testing, the content of nitrosamine impurity compound P08 in varenicline tartrate after dichloromethane pulping was 4.47 ppm, and the contents of nitrosamine impurity compounds P06 and P07 were not detected (less than the detection limit).

[0133] Example 9

[0134] Preparation of Varenicline Tartrate

[0135] a. Under the protection of inert gas (nitrogen), 20 kg of purified water, 0.81 kg of sodium hydroxide, 8 kg of dichloromethane and 2 kg of compound C (prepared in Example 7) were added to the reaction flask, the reaction temperature was controlled at 20-35 ° C, and the residual amount of compound C in the reaction solution was detected by HPLC to be ≤0.5% to stop the reaction, then 18.5 kg of dichloromethane was added to the reactor, stirred, allowed to stand, separated, the organic phase was taken, the aqueous phase was extracted with dichloromethane (15 kg × 2), the organic phases were combined, 5 kg of anhydrous sodium sulfate was added to the organic phase for drying, filtered, and the filtrate was distilled under reduced pressure (vacuum degree ≤-0.09 MPa, 40-50 ° C) until substantially no fraction was produced, 10 kg of anhydrous ethanol was added, and the distillation under reduced pressure (vacuum degree ≤-0.09 MPa, 40-50 ° C) was continued until substantially no fraction was produced to obtain a mother liquor containing varenicline;

[0136] b. Slowly add 30 kg of anhydrous ethanol and 1.16 kg of L-(+)-tartaric acid to the mother liquor containing varenicline obtained in step a, control the reaction temperature at 20 to 30 ° C, stir the reaction for 16 hours, then stop the reaction, centrifuge, and dry (70 to 75 ° C) to obtain varenicline tartrate;

[0137] Then, the prepared varenicline tartrate is added to the pulping solvent according to the solvent dosage of 8 ml per gram of varenicline tartrate, and pulped at 30-40°C for 1.5 hours, followed by centrifugation and drying. After testing, the content of nitrosamine impurity compound P08 in varenicline tartrate after isopropanol pulping was 2.89 ppm, and the contents of nitrosamine impurity compounds P06 and P07 were not detected (less than the detection limit).

[0138] Example 10

[0139] Preparation of Varenicline Tartrate

[0140] a. Under the protection of inert gas (nitrogen), 20 kg of purified water, 0.81 kg of sodium hydroxide, 8 kg of dichloromethane and 2 kg of compound C (prepared in Example 7) were added to the reaction flask, the reaction temperature was controlled at 20-35 ° C, and the residual amount of compound C in the reaction solution was detected by HPLC to be ≤0.5% to stop the reaction, then 18.5 kg of dichloromethane was added to the reactor, stirred, allowed to stand, separated, the organic phase was taken, the aqueous phase was extracted with dichloromethane (15 kg × 2), the organic phases were combined, 5 kg of anhydrous sodium sulfate was added to the organic phase for drying, filtered, and the filtrate was distilled under reduced pressure (vacuum degree ≤-0.09 MPa, 40-50 ° C) until substantially no fraction was produced, 10 kg of anhydrous ethanol was added, and the distillation under reduced pressure (vacuum degree ≤-0.09 MPa, 40-50 ° C) was continued until substantially no fraction was produced to obtain a mother liquor containing varenicline;

[0141] b. Slowly add 30 kg of anhydrous ethanol and 1.16 kg of L-(+)-tartaric acid to the mother liquor containing varenicline obtained in step a, control the reaction temperature at 20 to 30 ° C, stir the reaction for 16 hours, then stop the reaction, centrifuge, and dry (70 to 75 ° C) to obtain varenicline tartrate;

[0142] Then, the prepared varenicline tartrate is added to the pulping solvent according to the solvent dosage of 8 ml per gram of varenicline tartrate, and pulped at 30-40° C. for 1.5 hours, then centrifuged and dried. After testing, the content of nitrosamine impurity compound P08 in varenicline tartrate after n-heptane pulping is 24.29 ppm.

[0143] Embodiment 11

[0144] Preparation of Varenicline Tartrate

[0145] a. Under the protection of inert gas (nitrogen), 20 kg of purified water, 0.81 kg of sodium hydroxide, 8 kg of dichloromethane and 2 kg of compound C (prepared in Example 7) were added to the reaction flask, the reaction temperature was controlled at 20-35 ° C, and the residual amount of compound C in the reaction solution was detected by HPLC to be ≤0.5% to stop the reaction, then 18.5 kg of dichloromethane was added to the reactor, stirred, allowed to stand, separated, the organic phase was taken, the aqueous phase was extracted with dichloromethane (15 kg × 2), the organic phases were combined, 5 kg of anhydrous sodium sulfate was added to the organic phase for drying, filtered, and the filtrate was distilled under reduced pressure (vacuum degree ≤-0.09 MPa, 40-50 ° C) until substantially no fraction was produced, 10 kg of anhydrous ethanol was added, and the distillation under reduced pressure (vacuum degree ≤-0.09 MPa, 40-50 ° C) was continued until substantially no fraction was produced to obtain a mother liquor containing varenicline;

[0146] b. Slowly add 30 kg of anhydrous ethanol and 1.16 kg of L-(+)-tartaric acid to the mother liquor containing varenicline obtained in step a, control the reaction temperature at 20 to 30 ° C, stir the reaction for 16 hours, then stop the reaction, centrifuge, and dry (70 to 75 ° C) to obtain varenicline tartrate;

[0147] Then, the prepared varenicline tartrate is added to the pulping solvent according to the solvent dosage of 8 ml per gram of varenicline tartrate, and pulped at 30-40°C for 1.5 hours, followed by centrifugation and drying. After testing, the content of nitrosamine impurity compound P08 in the varenicline tartrate after ethanol pulping was 2.60 ppm, and the contents of nitrosamine impurity compounds P06 and P07 were not detected (less than the detection limit).

[0148] Examples 12-14

[0149] By further controlling the content of nitrosamine impurity compounds in the starting materials of the present invention (for example, controlling the content of nitrosamine impurity compound P06 in compound A within the range of ≤250ppm, ≤200ppm, ≤150ppm, ≤100ppm or lower), and increasing the number of pulping treatments, we also prepared varenicline tartrate with nitrosamine impurity compound P08 contents of 0.511ppm, 0.205ppm and 0.118ppm, respectively.

[0150] If the content of nitrosamine impurity compound P06 in compound A is too high, the content of P06 can be reduced by the following pulping treatment method, for example: add 3.3kg of compound A (the content of nitrosamine impurity compound P06 is 722ppm) to a pulping solvent (8.00kg (11.7L) of n-heptane and 2.70kg (3L) of ethyl acetate), pulp and stir at 15-35°C for 4-5h, then centrifuge, wash the filter cake with water, centrifuge again, and dry. It has been tested that the content of nitrosamine impurity compound P06 in compound A after pulping treatment is 0.03% (315ppm). This shows that the content of nitrosamine impurity compound P06 in compound A can be reduced by at least half through the above pulping treatment.

[0151] The working principle of pulping treatment is: the pulping solvent can dissolve the trace impurities in the target compound so that they remain in the liquid, while the insoluble target compound remains in solid form, and then solid-liquid separation is performed to reduce the impurity content in the target compound.

[0152] Embodiment 15

[0153] UPLC-MS / MS was used to detect the nitrosamine impurity compound P08 in the raw material of varenicline tartrate and to verify the methodology.

[0154] 1. Solution preparation

[0155] Diluent: Purified water

[0156] Reference substance stock solution: weigh 19.970 mg of nitrosamine compound P08 (purity ≥ 98%), place in a 10 ml volumetric flask, add 8 ml of acetonitrile, then add diluent to dissolve and dilute to the scale, and mix well.

[0157] Reference substance stock solution: Measure 37.5 μl of reference substance stock solution into a 10 ml volumetric flask, dilute to the mark with diluent, and mix well.

[0158] 2. System Applicability

[0159] System suitability solution (100% limit level reference substance solution): Measure about 100.0 μl of the reference substance stock solution into a 10 ml volumetric flask, dilute to the mark with diluent, and mix well.

[0160] Six injections of the system suitability solution were injected continuously, and the RSD of the peak area of ​​the target compound in the six injections of the system suitability solution was calculated. The result was 4%, indicating that the system suitability was good.

[0161] 3. Linear

[0162] Standard curve solution: Measure 50.0μl, 100.0μl, 150.0μl, and 200.0μl of the mother solution of the reference substance into four 10ml volumetric bottles, dilute to the mark with the diluent, mix well, and use them as linear solutions L-3, L-4, L-5, and L-6, respectively. Measure 800μl of the L-3 solution into a 10ml volumetric bottle, dilute to the mark with the diluent, mix well, and use them as L-2. Measure 1000μl of the L-2 solution into a 10ml volumetric bottle, dilute to the mark with the diluent, mix well, and use them as L-1.

[0163] The standard curve solutions were sampled and analyzed separately, and the concentration of the target (X) was used as the horizontal coordinate (unit: ng / ml) and the peak area (Y) was used as the vertical coordinate. Linear regression was performed to calculate the linear equation and the linear correlation coefficient r. The results showed that the linear equation was: Y=7370X-12900, and the linear correlation coefficient was r=0.999. The linearity was good, and the linearity was good in the concentration range of 0.2951ng / ml to 147.6ng / ml (i.e.: 0.02951ppm to 14.76ppm).

[0164] 4. Limit of Detection (LOD)

[0165] LOD solution: Measure 5000μ1 of L-1 solution into a 10ml volumetric flask, dilute to the mark with diluent, mix well, and use as LOD solution.

[0166] The LOD solution was injected twice continuously, and the S / N (signal-to-noise ratio) of the target peak was 11 and 8 respectively, meeting the requirement of not less than 3, and the method was sensitive; LOD = 0.01476 ppm.

[0167] 5. Limit of Quantitation (LOQ)

[0168] The quantitative limit solution (L-1 solution) was injected 6 times continuously, and the S / N of the target peak were 23, 24, 17, 12, 24, and 14, respectively, meeting the requirement of not less than 10, and the method was sensitive; LOQ = 0.02951 ppm.

[0169] 6. Accuracy

[0170] Sample solution: Weigh about 100 mg of sample (e.g. purchased or synthesized varenicline tartrate API) into a 10 ml volumetric flask, add diluent to dilute to scale, and mix. Prepare 2 copies in parallel.

[0171] Accuracy solution: weigh about 100 mg of sample and place it in a 10 ml volumetric bottle, add 50.0 μl (50% limit level), 100.0 μl (100% limit level), and 150.0 μl (150% limit level) of the mother solution of the reference substance, dilute to the scale with diluent, mix well, and the solution is ready. Prepare 3 copies for each accuracy level in parallel.

[0172] The results showed that the recovery rate of the 50% limit level accuracy solution was between 95% and 102%, with an RSD of 4%; the recovery rate of the 100% limit level accuracy solution was between 90% and 103%, with an RSD of 7%; the recovery rate of the 150% limit level accuracy solution was between 98% and 100%, with an RSD of 1%, which met the acceptance criteria and had good accuracy.

[0173] 7. Repeatability

[0174] Weigh about 100 mg of the sample into a 10 ml volumetric flask, add 100.0 μl of the reference substance stock solution, dilute to the mark with the diluent, and mix well. Prepare 6 copies in parallel.

[0175] The results showed that the RSD of the nitrosamine impurity compound P08 content in 6 replicate solutions was 6%, which met the acceptance criteria and had good repeatability.

[0176] 8. Exclusivity

[0177] Take blank solution (diluent), 100% limit level reference solution, sample solution (same as "repeatability" sample solution), and 100% limit level accuracy solution for analysis.

[0178] The results showed that the retention time of the target substance in the sample solution, 100% limit level reference solution, and 100% limit level accuracy solution was 3.47 min. There was no obvious interference at the peak position of the target substance, which met the separation requirements and had good specificity.

[0179] 9. Intermediate precision

[0180] Intermediate precision solution: weigh about 100 mg of sample and place it in a 10 ml volumetric flask, add 100.0 μl of the mother solution of the reference substance, dilute to the mark with the diluent, and mix well. Prepare 6 portions in parallel. Add the 6 portions of solution in "7. Repeatability".

[0181] The RSD of the concentration of 12 solutions, including 6 intermediate precision solutions and 6 repeatability solutions, was calculated to be 9%, which met the requirement of no more than 15%, met the acceptance criteria, and the intermediate precision was good.

[0182] 10. Solution stability

[0183] Take 100% limit level accuracy solution and place it at 15℃, inject and analyze it at regular intervals, calculate the concentration ratio of each time point to the zero point, and the ratio should be between 96% and 113% within 21 hours.

[0184] 11. Durability

[0185] The concentration values ​​of the target substance in the solution with 100% limit level accuracy were investigated when the chromatographic column temperature changed by ±2°C, the initial proportion of the organic phase by ±1%, and the flow rate changed by ±0.01ml / min. Two injections were made under each condition, and the RSD of the target substance concentration values ​​in the six solutions under the same parameter fluctuation was calculated to be between 4% and 6%, indicating that the method has good durability.

[0186] The content of nitrosamine impurity compound P08 in the sample (varenicline tartrate API) was calculated according to the following formula. The test was performed twice and the average value was taken:

[0187] P08 content: C (ppm) = P08 concentration in sample solution (ng / ml) / [m (mg) / V (ml)]

[0188] m: sample mass, mg;

[0189] V: sample dilution volume, ml.

[0190] Example 16

[0191] Liquid chromatography-tandem mass spectrometry under the conditions described in Table 11 was used for qualitative and / or quantitative detection of the nitrosamine impurity compound P07 in the varenicline tartrate bulk drug of the present invention, and the methodological validation was performed and passed.

[0192] Table 11. Chromatographic conditions and mass spectrometry conditions for detecting nitrosamine impurity compound P07

[0193]

[0194]

[0195] 1. Solution preparation

[0196] Blank (diluent): water-acetonitrile (95:5)

[0197] Reference substance stock solution: Weigh 25 mg of nitrosamine compound P07 (purity ≥ 95%), place in a 50 ml volumetric flask, add acetonitrile to dissolve, dilute to scale, and mix well (fixed solution I). Take 1.0 ml of fixed solution I, place in a 100 ml volumetric flask, add diluent to dilute to scale, mix well, and obtain the reference substance stock solution.

[0198] Reference substance solution: Take 0.45 ml of reference substance stock solution, place it in a 100 ml volumetric flask, add diluent to the mark, mix well, and the solution is ready.

[0199] Sample solution: Take about 100 mg of varenicline tartrate API and place it in a 10 ml volumetric flask. Add diluent to dissolve and dilute to the mark, and mix well.

[0200] 2. System Applicability

[0201] For 5 consecutive tests on the reference solution, the RSD of the peak area of ​​the nitrosamine impurity compound P07 was ≤15%, and the RSD of the retention time was ≤2.0%. It was well separated from the adjacent peaks, and the system had good applicability.

[0202] 3. Linear

[0203] Prepare reference substance solutions with concentrations of 0.002688 μg / ml, 0.020157 μg / ml, 0.033594 μg / ml, 0.053751 μg / ml, 0.067189 μg / ml, and 0.100783 μg / ml (based on nitrosamine compound P07);

[0204] The above-mentioned reference solution of different concentrations was sampled and analyzed respectively, and the concentration of the target (X) was used as the horizontal coordinate (unit: μg / ml) and the peak area (Y) was used as the vertical coordinate. Linear regression was performed to calculate the linear equation and the linear correlation coefficient r. The results showed that the linear equation was: Y=3000000X+2671.7, and the linear correlation coefficient was r=0.998. The linearity was good, and the linearity was good in the concentration range of 0.002688μg / ml to 0.100783μg / ml (i.e.: 0.2688ppm to 10.0783ppm).

[0205] 4. Limit of Detection (LOD)

[0206] LOD = 0.1344 ppm, the S / N (signal-to-noise ratio) of the target peak is 14.8, 13.1, etc., which meets the requirement of being greater than 3, and the method is sensitive.

[0207] 5. Limit of Quantitation (LOQ)

[0208] LOQ = 0.2688 ppm, the S / N of the target peaks are 20.5, 24.9, etc., meeting the requirement of being greater than 10, and the method is sensitive.

[0209] 6. Exclusivity

[0210] The results showed that the retention time of the nitrosamine impurity compound P07 was about 14.487 min, there was no interference between adjacent peaks, the separation requirements were met, and the specificity was good.

[0211] also,

[0212] It has been verified that the accuracy, precision, repeatability, and durability of the detection method also meet the regulations and requirements of methodological validation, and can well achieve the qualitative and / or quantitative detection of nitrosamine impurity compound P07 in varenicline tartrate raw materials.

[0213] Embodiment 17

[0214] Liquid chromatography-tandem mass spectrometry under the conditions described in Table 12 was used for qualitative and / or quantitative detection of the nitrosamine impurity compound P06 in the varenicline tartrate bulk drug of the present invention, and the methodological validation was performed and passed.

[0215] Table 12. Chromatographic conditions and mass spectrometry conditions for detecting nitrosamine impurity compound P06

[0216]

[0217] 1. Solution preparation

[0218] Blank (diluent): purified water

[0219] Reference substance stock solution: Weigh 25 mg of nitrosamine compound P06 (purity ≥ 92%), place in a 50 ml volumetric flask, add acetonitrile to dissolve, dilute to scale, and mix well (fixed solution II). Take 1.0 ml of fixed solution II, place in a 100 ml volumetric flask, add diluent to dilute to scale, mix well, and obtain the reference substance stock solution.

[0220] Reference substance solution: Take 4.5 ml of reference substance stock solution and place it in a 50 ml volumetric flask, add diluent to make up to the mark, mix well, and the solution is ready.

[0221] Sample solution: Take about 1000 mg of varenicline tartrate API and place it in a 20 ml headspace bottle. Accurately measure 5.0 ml of diluent to dissolve and mix.

[0222] 2. System Applicability

[0223] For 5 consecutive tests on the reference solution, the RSD of the peak area of ​​the nitrosamine impurity compound P06 was ≤15%, and the RSD of the retention time was ≤2.0%. It was well separated from the adjacent peaks, and the system had good applicability.

[0224] 3. Linear

[0225] The reference solution of different concentrations was sampled and analyzed separately. The concentration of the target substance (X) was used as the horizontal coordinate (unit: μg / ml) and the peak area (Y) was used as the vertical coordinate. Linear regression was performed to calculate the linear equation and linear correlation coefficient r. The results showed that the linear correlation coefficient r>0.99, and the linear relationship was good.

[0226] 4. Limit of Detection (LOD)

[0227] LOD = 0.0145 ppm, the S / N (signal-to-noise ratio) of the target peak is 5.8, which meets the requirement of being greater than 3, and the method is sensitive.

[0228] 5. Limit of Quantitation (LOQ)

[0229] LOQ = 0.0289 ppm, the S / N of the target peak is 12.0, which meets the requirement of being greater than 10, and the method is sensitive.

[0230] 6. Exclusivity

[0231] The results showed that the retention time of the nitrosamine impurity compound P06 was about 14.648 min, there was no interference between adjacent peaks, the separation requirements were met, and the specificity was good.

[0232] also,

[0233] It has been verified that the accuracy, precision, repeatability, and durability of the detection method also meet the regulations and requirements of methodological validation, and can well achieve the qualitative and / or quantitative detection of nitrosamine impurity compound P06 in varenicline tartrate raw materials.

[0234] Embodiment 18

[0235] In order to better ensure the limit requirements of nitrosamine impurity compounds in varenicline tartrate API or preparation (including: P06, P07, P08; wherein, P08 is converted from P06 via the intermediate product P07; therefore, P08 is the main form of nitrosamine impurities in varenicline tartrate API or preparation, P06 is the main form of nitrosamine impurities in raw materials such as compound A, and P07 is an intermediate product in the preparation process), it is necessary to conduct qualitative and / or quantitative detection of the nitrosamine impurity compound P06 in the starting raw material compound A, for example: by high performance liquid chromatography, and its chromatographic conditions are shown in Table 13.

[0236] Table 13. Chromatographic conditions for detecting nitrosamine impurity compound P06 in compound A

[0237]

[0238]

[0239] 1. Make a standard curve

[0240] Blank (diluent): acetonitrile-water (60:40)

[0241] Reference solution: Weigh 25 mg of nitrosamine compound P06 (purity ≥ 92%), place in a 25 ml volumetric flask, add diluent to dissolve and dilute to the mark, mix well, and obtain a reference solution.

[0242] Prepare reference substance solutions with concentrations of 0.0001394 mg / ml, 0.0004645 mg / ml, 0.0007432 mg / ml, 0.000929 mg / ml, 0.001115 mg / ml, 0.001394 mg / ml, and 0.001858 mg / ml (based on nitrosamine compound P06) respectively;

[0243] The above-mentioned reference solution of different concentrations was sampled and analyzed respectively, and the concentration of the target (X) was used as the horizontal coordinate (unit: mg / ml) and the peak area (Y) was used as the vertical coordinate. Linear regression was performed to calculate the linear equation and the linear correlation coefficient r; the results showed that the linear equation was: Y=228.94X+0.0025, and the linear correlation coefficient was r=0.999, which showed good linearity within the concentration range.

[0244] Detection limit: LOD = 0.004% (ie 40 ppm), the S / N (signal-to-noise ratio) of the target peak is 6.4, which meets the requirement of being greater than 3, and the method is sensitive.

[0245] Limit of quantification: LOQ = 0.014% (ie 140 ppm), the S / N of the target peak is 18.9, which meets the requirement of being greater than 10, and the method is sensitive.

[0246] Specificity: The test results of the mixed solution of samples and reference substances showed that the retention time of nitrosamine impurity compound P06 was about 4.592 min (separation R=13.18), the retention time of compound A was about 7.587 min (separation R=6.82), the retention time of impurity M01 was about 9.373 min (separation R=7.24), and the retention time of impurity M02 was about 12.45 min (separation R=5.25). There was no interference between the peaks, which met the separation requirements and had good specificity.

[0247] also,

[0248] It has been verified that the accuracy, precision, repeatability, durability, etc. of the detection method also meet the regulations and requirements of methodological validation, and can be used for the qualitative and / or quantitative detection of nitrosamine impurity compound P06 in compound A.

[0249] 2. Test the samples

[0250] Sample solution: Take about 25 mg of compound A, place it in a 25 ml volumetric flask, add diluent to dissolve and dilute to the mark, and mix well.

[0251] The sample solution is injected and tested to obtain its peak area data and chromatogram (see: Figure 1 ), and the peak area data is substituted into the linear equation obtained from the reference solution to calculate the concentration data of the nitrosamine impurity compound P06 in the test sample solution.

[0252] Then, the content of nitrosamine impurity compound P06 in the sample (compound A) is calculated according to the following formula:

[0253] P06 content: C (expressed in % or ppm) = P06 concentration in sample solution (mg / ml) / [m (mg) / V (ml)]

[0254] m: sample mass, mg;

[0255] V: sample dilution volume, ml.

[0256] Embodiment 19

[0257] Test whether the nitrosamine impurity compound P08 is genotoxic (capable of causing mutations or carcinogenesis).

[0258] 1 Materials and Methods

[0259] 1.1 Experimental strains

[0260] Salmonella typhimurium TA97a, TA98, TA100, TA102, and TA1535 were purchased from Shanghai Baolu Biotechnology Co., Ltd. and were from MOLT OX, USA. The test was conducted using the identified and qualified Salmonella typhimurium strains.

[0261] 1.2 Metabolic Activation System

[0262] Rat liver S9, source: Shanghai Baolu Biotechnology Co., Ltd.

[0263] 1.3 Test substances

[0264] Name: Nitrosamine impurity compound P08, Properties: white powder.

[0265] 1.4 Main instruments and reagents

[0266] XSR-204 electronic balance (Mettler, Switzerland); high pressure steam sterilizer (SANYO, Japan); water-proof constant temperature incubator (Shanghai Yiheng Technology Co., Ltd.); Stuart temperature-controlled shaking incubator (Stuart, UK); BHC-1300II A2 biological safety cabinet (Suzhou Antai Air Technology Co., Ltd., Sujing Group).

[0267] 6-phosphoglucose, source: Beijing Bailingwei Technology Co., Ltd.; coenzyme II, source: Sinopharm Chemical Reagent Co., Ltd.; nutrient broth, source: Beijing Luqiao Technology Co., Ltd.; technical agar powder, source: Guangdong Huankai Microbiological Technology Co., Ltd.; sterile water for injection, source: Chenxin Pharmaceutical Co., Ltd.; dichlorobenzene, source: CHEMSERVICE; methyl methanesulfonate, source: Beijing Bailingwei Technology Co., Ltd.; 2-aminofluorene, source: Shanghai Jingchun Reagent Co., Ltd.; 1,8-dihydroxyanthraquinone, source: SIGMA-ALDRICH, USA; cyclophosphamide, source: SIGMA-ALDRICH, USA; sodium azide, source: RIEDEL-SEELIE.

[0268] 1.5 Test method (Ames plate incorporation method)

[0269] The experiment adopted the plate incorporation method.

[0270] 1.5.1 Dose selection and test substance preparation

[0271] Dosage design: The test was conducted at 5 mg, 2.5 mg, 1.25 mg, 0.625 mg, 0.3125 mg, and 0.15625 mg per dish.

[0272] Preparation of test substance: Prepare a 50 mg / ml solution of this product with dimethyl sulfoxide, and dilute it with dimethyl sulfoxide to 25 mg / ml, 12.5 mg / ml, 6.25 mg / ml, 3.125 mg / ml, and 1.5625 mg / ml in sequence.

[0273] 1.5.2 Test method.

[0274] 0.1ml of frozen bacterial liquid TA97a, TA98, TA100, TA102, and TA1535 were inoculated into 10ml nutrient broth medium, and cultured at 37℃ with shaking (100 times / min) for 10h. 2.0ml of the top culture medium containing 0.5mmol / L histidine-0.5mmol / L biotin was dispensed into test tubes, sterilized at 0.103MPa for 20min, and kept warm in a 50℃ water bath. Then 0.1ml of the test solution, 0.5ml of S9 mixed solution (when metabolic activation is required) and 0.1ml of bacterial liquid were added to each tube in turn, mixed thoroughly, and quickly poured onto the bottom agar plate, and the plate was rotated to make it evenly distributed. After being placed horizontally for condensation and solidification, it was inverted in a 37℃ incubator for incubation for 48h, and the number of reverted colonies in each dish was counted. Three parallel plates were made for each dose, and a spontaneous reverted group, a solvent control group, and a positive control group were set up at the same time.

[0275] 1.6 Test data statistics

[0276] Data are presented as mean ± standard deviation SPSS 19.0 was used for data analysis and calculation.

[0277] 1.7 Result determination

[0278] If the number of reverted colonies of the test substance is two times or more than that of the solvent control, and there is a dose-response relationship, the test substance is judged as positive for mutagenesis; if the test substance shows a positive reaction under any dose condition and is repeatable, the test substance is judged as positive for mutagenesis. If the test substance is negative after being tested with the above test strains, whether under the conditions of adding S9 or not adding S9, the test substance is negative for mutagenesis.

[0279] 2 Test results

[0280] The results are shown in Table 14.

[0281] Table 14. Ames test results

[0282]

[0283] Table 14 Ames test results

[0284]

[0285] The results of the Ames test show that when the dose of the nitrosamine impurity compound P08 was 625 μg / dish without S9, the number of TA1535 colonies was more than twice that of the solvent control group. When S9 was added, the number of TA98 colonies was more than twice that of the solvent control group at a dose of 2500 μg / dish, and the number of TA1535 colonies was more than twice that of the solvent control group at doses of 5000, 2500, 1250, and 625 μg / dish, and the relationship was dose-response. This shows that the nitrosamine impurity compound P08 is mutagenic to Salmonella typhimurium with or without the addition of a metabolic activation system.

[0286] Of course, the present invention may have many other embodiments. Without violating the spirit and essence of the present invention, those skilled in the art may make various corresponding changes and / or deformations based on the present invention. These corresponding changes and / or deformations should all fall within the scope of protection of the claims attached to the present invention.

Claims

1. A method for preparing a compound represented by formula P06, characterized in that: The method comprises the following steps: compound M01 reacts with nitrite in the presence of an acid and a solvent to undergo a nitrosamination reaction to generate a compound represented by formula P06; The solvent is water and tetrahydrofuran, and the volume ratio of water to tetrahydrofuran is 1:4-6; The acid is acetic acid.

2. A method for preparing a compound represented by formula P06, characterized in that: The method comprises the following steps: compound M01 reacts with nitrite in the presence of an acid and a solvent to undergo a nitrosamination reaction to generate a compound represented by formula P06; The molar ratio of compound M01 to nitrite is 1:0.5-5; The volume mass ratio of the solvent to the compound M01 is 5 to 20 mL / g; The mass ratio of the acid to the compound M01 is 0.1 to 1; The reaction temperature of the nitrosamination reaction is 30-60°C.

3. The method for preparing the compound of formula P06 according to claim 2, characterized in that: The molar ratio of compound M01 to nitrite is 1:2-4.

4. The method for preparing the compound of formula P06 according to claim 2, characterized in that: The volume mass ratio of the solvent to the compound M01 is 10 to 15 mL / g.

5. The method for preparing the compound of formula P06 according to claim 2, characterized in that: The mass ratio of the acid to the compound M01 is 0.4 to 0.

8.

6. Use of the compound represented by formula P06 as a standard substance, reference substance or test item in the impurity research, quality control or detection method of substance X; The substance X is R0 is trifluoroacetyl.

7. Use of the compound represented by formula P06 according to claim 6 as a standard substance, reference substance or test item in the impurity research, quality control or detection method of substance X; characterized in that, The detection method is high performance liquid chromatography.

8. A method for detecting a compound represented by formula P06 in substance X, characterized in that: The compound represented by formula P06 is used as a standard or reference substance and detected by high performance liquid chromatography; The substance X is R0 is trifluoroacetyl.

9. The method for detecting the compound represented by formula P06 in substance X according to claim 8, characterized in that: The chromatographic conditions of the high performance liquid chromatography include: Chromatographic column: C18 column; Mobile phase: phosphoric acid aqueous solution-methanol, the volume ratio of phosphoric acid aqueous solution to methanol is 30%:70% to 50%:50%; Detection wavelength: 200~320nm.

10. The method for detecting the compound represented by formula P06 in substance X according to claim 9, characterized in that: The chromatographic column was Kromasil 100-5C18.

11. The method for detecting the compound represented by formula P06 in substance X according to claim 9, characterized in that: The volume ratio of the phosphoric acid aqueous solution to methanol is 40%:60%.

12. The method for detecting the compound represented by formula P06 in substance X according to claim 9, characterized in that: The specifications of the chromatographic column are: 4.6 mm×250 mm, 5 μm.

13. The method for detecting the compound represented by formula P06 in substance X according to claim 9, characterized in that: The concentration of the phosphoric acid aqueous solution is 0.01% to 0.1%.

14. The method for detecting the compound represented by formula P06 in substance X according to claim 13, characterized in that: The concentration of the phosphoric acid aqueous solution is 0.05%.

15. The method for detecting the compound represented by formula P06 in substance X according to any one of claims 9 to 14, characterized in that: The chromatographic conditions of the high performance liquid chromatography include: Column temperature: 15-35°C; Flow rate: 0.5~2ml / min; Injection volume: 1~35μ1.

16. The method for detecting the compound represented by formula P06 in substance X according to claim 15, characterized in that: Column temperature: 20℃, 25℃, 30℃ or 32℃.

17. The method for detecting the compound represented by formula P06 in substance X according to claim 15, characterized in that: Flow rate: 0.5ml / min, 0.6ml / min, 0.7ml / min, 0.8ml / min, 0.9ml / min, 1ml / min, 1.2ml / min or 1.5ml / min.

18. The method for detecting the compound represented by formula P06 in substance X according to claim 15, characterized in that: Injection volume: 2μ1, 3μ1, 4μ1, 5μ1, 6μ1, 7μ1, 8μ1, 9μ1, 10μ1, 15μ1, 20μ1, 25μ1 or 30μ1.

19. The method for detecting the compound represented by formula P06 in substance X according to any one of claims 9 to 14, characterized in that: The detection method is qualitative detection or quantitative detection, the solvent of the standard substance, the reference substance and the substance X is acetonitrile-water, and the volume ratio of acetonitrile to water is 50%:50% to 70%:30%.

20. The method for detecting the compound represented by formula P06 in substance X according to claim 19, characterized in that: The volume ratio of acetonitrile to water is 60%:40%.

21. The method for detecting the compound represented by formula P06 in substance X according to claim 19, characterized in that: The internal standard method or external standard method was used for quantitative detection.

22. The method for detecting the compound represented by formula P06 in substance X according to claim 21, characterized in that: The external standard method is: using the compound represented by formula P06 as a standard or reference substance, preparing standard solutions or reference solutions of different concentrations, injecting and testing, obtaining corresponding peak area data, performing linear regression between concentration and peak area, and obtaining a linear equation; Then, the peak area detection data of the compound represented by formula P06 in the detection sample solution of substance X is substituted into the linear equation to obtain the concentration data of the compound represented by formula P06 in the detection sample solution; Then divide the data by the concentration data of the test sample solution to obtain the quantitative test result.

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