Related impurity compounds of varenicline tartrate, their preparation, applications and detection methods

Abstract

The present invention provides a related impurity compound of varenicline tartrate, and its preparation method, application method and detection method. The impurity compound is shown in Formula II, wherein R 11 , R 12 are independently selected from nitro (-NO2) or amino (-NH2). This impurity compound will be converted into a genotoxic nitrosamine impurity P08 during the reaction process. Using this impurity compound as a reference standard, control or detection item for impurity research and quality control of varenicline tartrate or its raw materials and intermediates can achieve qualitative and / or quantitative detection of this impurity, so as to further take active and effective technical measures to control the content of nitrosamine genotoxic impurities in varenicline tartrate raw materials or preparations at a relatively low level, thus meeting the safety requirements and drug product regulatory regulations and better ensuring the medication safety of patients. #imgabs0#

Description

Technical Field

[0001] The present invention belongs to the field of drugs, and particularly relates to related impurity compounds of varenicline tartrate, and their preparation, application and detection methods. Background Art

[0002] Varenicline, an aryl-fused azapolycyclic compound, can modulate cholinergic function and is sold as the tartrate salt under the trade name or It can bind specifically to neuronal nicotinic acetylcholine receptor sites and can be used for the following diseases:

[0003] Treating inflammatory bowel diseases (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, Parkinson's disease, multiple sclerosis, AIDS, (encephalitis), trauma, renal encephalopathy and hepatic encephalopathy, hypothyroidism, Pick's disease, Korsakoff's syndrome, and frontal and subcortical dementia), hypertension, hyperphagia, anorexia, obesity, arrhythmia, hyperacidity, ulcers, pheochromocytoma, progressive supranuclear palsy, chemical dependence and addiction (e.g., to nicotine (and / or tobacco products), alcohol, benzodiazepines dependence and addiction to benzodiazepines, barbiturates, opioids or cocaine), headache, migraine, stroke, traumatic brain injury (TBI), obsessive-compulsive disorder (OCD), psychosis, Huntington's disease, tardive dyskinesia, hyperkinesia, dyslexia, schizophrenia, multi - sclerosis dementia, age - related cognitive decline, epilepsy, including petit mal absence epilepsy, attention deficit hyperactivity disorder (ADHD), Tourette's Syndrome; in particular, dependence, addiction and withdrawal from nicotine, including its use in smoking cessation treatment (see: CN 1509174A).

[0004] The preparation method of varenicline and the resolution method of its racemate have been disclosed in US Patent Application US 6,410,550 and International Patent Application WO 01 / 62736, the contents of which are also incorporated herein by reference.

[0005] Nitrosamine compounds, including the nitroso group (N(R1)(R2)-N = O) structure combined with an amine, are genotoxic substances in certain 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 Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidance document M7(R1), which assesses and controls DNA - reactive (mutagenic) impurities in pharmaceuticals to limit potential carcinogenic risks (March 2018). The ICH guideline recommends controlling any known mutagen / carcinogen, including nitrosamine compounds, at an intake level where the human cancer risk is negligible.

[0007] Currently, the US FDA has identified seven nitrosamine impurities that may theoretically be present in pharmaceuticals due to the use of manufacturing processes and materials that can lead to nitrosamine formation: N,N - dimethylnitrosamine (NDMA), N - nitrosodiethylamine (NDEA), N - nitroso - N - methyl - 4 - aminobutyric acid (NMBA), N - nitrosopropylisopropylamine (NIPEA), N - nitrosodiisopropylamine (NDIPA), N - nitrosodibutylamine (NDBA) and N - nitrosomethylaniline (NMPA). Among them, five impurities (NDMA, NDEA, NMBA, NIPEA and NMPA) have actually been detected in drug substances or pharmaceuticals.

[0008] For example, some preliminary results of FDA tests indicate that the NDMA content in certain ranitidine products has exceeded the acceptable level. In recent years, the FDA has also found that some metformin products contain NDMA impurities, and the detected NDMA content in certain batches is higher than the acceptable intake limit recommended by the FDA.

[0009] In view of this, the present invention is specifically proposed to control the content of nitrosamine impurities in varenicline tartrate API or preparations at a lower level that can meet safety requirements. Summary of the Invention

[0010] Aiming at the problems and / or deficiencies existing in the prior art, one of the objectives of the present invention is to provide a new compound related to varenicline tartrate: the compound shown in Formula II. This new compound will transform into a genotoxic nitrosamine impurity P08 during the reaction process, and it is necessary to detect and control it throughout the entire synthesis process of varenicline tartrate, so as to further adopt active and effective technical means to control the content of genotoxic nitrosamine impurities in varenicline tartrate API or preparations at a lower level, meeting safety requirements.

[0011] The present invention provides a compound shown in Formula II:

[0012]

[0013] Wherein, R 11 、R 12 are independently selected from nitro (-NO2) or amino (-NH2).

[0014] The present invention also provides a preparation method of the compound shown in Formula II, including the following steps: Compound M01 undergoes a nitrosation reaction with nitrite in the presence of an acid and a mixed solvent to form Compound P06; Optionally, it includes: Compound P06 undergoes a reduction reaction with a reducing agent in the presence of a catalyst and a halogenated hydrocarbon solvent to form Compound P07;

[0015]

[0016] The compound shown in Formula II is Compound P06 or Compound P07;

[0017] Preferably,

[0018] The nitrite is potassium nitrite or sodium nitrite;

[0019] The acid is acetic acid;

[0020] The mixed solvent is water and tetrahydrofuran, and the volume ratio of water to tetrahydrofuran is 1:4 - 6;

[0021] The reducing agent described above is hydrogen;

[0022] The catalyst described above is palladium on carbon catalyst;

[0023] The halogenated hydrocarbon solvent is a liquid halogen-substituted C1-C4 alkane, more preferably dichloromethane;

[0024] More preferably,

[0025] The molar ratio of compound M01 to nitrite is 1:0.5 - 5, more preferably 1:2 - 4;

[0026] The volume-mass ratio of the mixed solvent to compound M01 is 5 - 20 mL / g, more preferably 10 - 15 mL / g;

[0027] The mass ratio of the acid to compound M01 is 0.1 - 1, more preferably 0.4 - 0.8;

[0028] The reaction temperature of the nitrosation reaction is 30 - 60 °C;

[0029] The mass ratio of compound P06 to the catalyst is 1:0.1 - 0.5, more preferably 1:0.2 - 0.3;

[0030] The volume-mass ratio of the halogenated hydrocarbon solvent to compound P06 is 5 - 20 mL / g, more preferably 10 - 15 mL / g;

[0031] The reaction temperature of the reduction reaction is 10 - 40 °C.

[0032] The present invention also provides an application of the compound shown in Formula II as a reference standard, control or detection item in the impurity research, quality control or detection method of varenicline tartrate; preferably, the detection method is liquid chromatography-tandem mass spectrometry (LC-MS / MS);

[0033]

[0034] Among them, R 11 、R 12 are independently selected from nitro or amino.

[0035] The present invention also provides a detection method for the compound shown in Formula II in varenicline tartrate. Using the compound shown in Formula II as a reference standard or control, it is detected by liquid chromatography-tandem mass spectrometry;

[0036]

[0037] Among them, R 11 、R 12 are independently selected from nitro or amino.

[0038] In a certain embodiment of the present invention, the chromatographic conditions of the liquid chromatography-tandem mass spectrometry include:

[0039] Chromatographic column: C18 chromatographic column, preferably Waters Xbridge C18;

[0040] Mobile phase: Mobile phase A - Mobile phase B, the volume ratio of Mobile phase A to Mobile phase B is 55%:45% to 98%:2% (for example, 60%:40%, 65%:35%, 70%:30%, 80%:20%, 90%:10%, 95%:5%, etc.); Mobile phase A is formic acid-purified water or ammonium acetate aqueous solution, and Mobile phase B is acetonitrile;

[0041] Preferably,

[0042] The chromatographic conditions of the liquid chromatography-tandem mass spectrometry further include:

[0043] The specification of the chromatographic column is: 4.6mm×150mm, 3.5μm;

[0044] Column temperature: 15 - 35°C, more preferably 20°C, 25°C, 30°C or 32°C;

[0045] Flow rate: 0.1 - 2 ml / min, more preferably 0.2 ml / min, 0.3 ml / min, 0.4 ml / min, 0.5 ml / min, 0.6 ml / min, 0.7 ml / min, 0.8 ml / min, 0.9 ml / min, 1 ml / min, 1.2 ml / min or 1.5 ml / min;

[0046] Injection volume: 1 - 35 μl, more preferably 2 μl, 3 μl, 4 μl, 5 μl, 6 μl, 7 μl, 8 μl, 9 μl, 10 μl, 15 μl, 20 μl, 25 μl or 30 μl.

[0047] In a certain embodiment of the present invention, the mass spectrometry conditions of the liquid chromatography-tandem mass spectrometry include:

[0048] Ion source: ESI (electrospray ionization source) or APCI (atmospheric pressure chemical ionization source), preferably ESI;

[0049] Positive ion mode;

[0050] Scanning mode: SIM (single ion monitoring), SRM (selective reaction monitoring) or MRM (multi reaction monitoring), preferably SIM or MRM;

[0051] Preferably,

[0052] The mass spectrometry conditions of the liquid chromatography - tandem mass spectrometry also include: ion source temperature: 350 °C; capillary voltage: 6000 V; spray gas flow rate: 13 L / min.

[0053] In a certain embodiment of the present invention, the compound shown in formula II is (P06), the volume ratio of mobile phase A to mobile phase B is 55%:45% to 75%:25%, the mobile phase A is formic acid - purified water, and the volume ratio of formic acid to purified water is 0.05 - 0.2:100 (for example, 0.06:100, 0.07:100, 0.08:100, 0.09:100, 0.1:100, 0.11:100, 0.12:100, 0.15:100, etc.); preferably, the volume ratio of mobile phase A to mobile phase B is 63%:37%, and the volume ratio of formic acid to purified water is 0.1:100.

[0054] In a certain embodiment of the present invention, the compound shown in formula II is (P07), the volume ratio of mobile phase A to mobile phase B is 90%:10% to 98%:2%, the mobile phase A is an ammonium acetate aqueous solution, the concentration of the ammonium acetate aqueous solution is 0.005 - 0.02 mol / L (for example, 0.006 mol / L, 0.007 mol / L, 0.008 mol / L, 0.009 mol / L, 0.01 mol / L, 0.011 mol / L, 0.012 mol / L, 0.015 mol / L, etc.), and the pH is adjusted to 2 - 3 with formic acid; preferably, the volume ratio of mobile phase A to mobile phase B is 95%:5%, the concentration of the ammonium acetate aqueous solution is 0.01 mol / L, and the pH is adjusted to 2.5 with formic acid.

[0055] In a certain embodiment of the present invention, the detection method is qualitative detection or quantitative detection, and the solvents for the standard, reference substance, and varenicline tartrate are water or water - acetonitrile, and the volume ratio of water to acetonitrile is 90%:10% to 98%:2%, preferably 95%:5%.

[0056] In a certain embodiment of the present invention, quantitative detection is carried out by the internal standard method or the external standard method;

[0057] Preferably,

[0058] The external standard method described above is as follows: Using the compound shown in Formula II as a reference standard or control, preparing reference standard solutions or control solutions with different concentrations, injecting samples for detection, obtaining corresponding peak area data, performing linear regression on the concentration and peak area to obtain a linear equation; then substituting the peak area detection data of the compound shown in Formula II in the varenicline tartrate test sample solution into the linear equation to obtain the concentration data of the compound shown in Formula II in the test sample solution; and then dividing this data by the concentration data of the test sample solution to obtain the quantitative detection result.

[0059] The beneficial effects of the present invention mainly lie in: The compound shown in Formula II provided by the present invention will transform into a genotoxic nitrosamine impurity P08 during the reaction process. Using it as a reference standard, control, or detection item for impurity research and quality control of varenicline tartrate or its raw materials and intermediates, qualitative and / or quantitative detection of this impurity can be achieved, so as to further adopt active and effective technical means to control the content of nitrosamine genotoxic impurities in varenicline tartrate raw materials or preparations at a relatively low level, thus meeting safety requirements and drug product regulatory regulations and better ensuring the medication safety of patients. Description of the Drawings

[0060] Figure 1 It is a chromatogram for detecting nitrosamine impurity compound P06 in compound A by high performance liquid chromatography. Detailed Embodiments

[0061] The present invention will be clearly and completely described below in conjunction with specific embodiments. Those skilled in the art will understand that the following embodiments are 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 protection scope of the present invention.

[0062] In the present invention, for those not specified with specific conditions, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments not specified with the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0063] Regarding the definitions of the terms used in the present invention, unless otherwise specified, the initial definitions provided for the terms in this article apply to the terms throughout the text; for terms not specifically defined in this article, meanings that can be given by those skilled in the art should be given according to the disclosure content and / or context.

[0064] Example 1

[0065] Preparation of Nitrosamine Compound P06

[0066]

[0067] Into a 100 mL three-necked flask, add 5 g of compound M01 and 52 mL of tetrahydrofuran (THF), and stir until dissolved; add 10 mL of an aqueous NaNO2 solution (concentration 0.4 g / mL), and 3 g of acetic acid, heat to 50 °C for reaction, monitor the reaction by TLC or HPLC until completion, concentrate, add 50 mL of dichloromethane (DCM) and 50 mL of saturated brine, stir until dissolved, let stand, separate the layers, take the organic phase, wash once with saturated brine, dry over anhydrous sodium sulfate, filter, concentrate to dryness to obtain the nitrosamine compound P06 with a purity of 93.69% (area normalization method).

[0068]

[0069] For the nitrosamine compound P06 1 1H-NMR, 13 13C-NMR and IR spectral data are shown in Tables 1 - 3 respectively; M+H + = 279.10; UV: The maximum absorption wavelength in acetonitrile solution is 223.80 nm.

[0070] Table 1. 1H-NMR spectrum of the nitrosamine compound P06 1 1H-NMR spectrum

[0071]

[0072]

[0073] Table 2. 13C-NMR spectrum of the nitrosamine compound P06 13 13C-NMR spectrum

[0074]

[0075] Table 3. IR spectrum of the nitrosamine compound P06

[0076] <![CDATA[Absorption wave number (cm -1 )]]> Attribution 3034.5 Ar-H 2920.2,2879.0 Saturated C-H stretching vibration 1667.2 Benzene ring C=C stretching vibration 1546.9,1428.2,1344.9 N=O, N-O stretching vibration 845.2 Benzene ring C-H bending vibration

[0077] Example 2

[0078] Preparation of the nitrosamine compound P07

[0079]

[0080] Into a 100 mL three-necked flask, add 4.0 g of compound P06 and 50 mL of dichloromethane (DCM), and stir until dissolved; add 1.0 g of palladium on carbon catalyst (Pd / C), first displace with nitrogen and then continuously pass hydrogen, heat to 25 - 30 °C, react overnight, monitor the reaction by TLC until completion, filter, concentrate to dryness to obtain the nitrosamine compound P07 with a purity of 96.59% (area normalization method).

[0081]

[0082] Of the nitrosamine compound P07 1 H-NMR, 13 C-NMR and IR spectral data are shown in Tables 4 to 6 respectively; M+H + = 219.20; UV: The maximum absorption wavelength in the acetonitrile:water = 1:1 solution is 212.00 nm.

[0083] Table 4. H-NMR spectrum of the nitrosamine compound P07 1 H-NMR spectrum

[0084]

[0085] Table 5. C-NMR spectrum of the nitrosamine compound P07 13 C-NMR spectrum

[0086]

[0087] Table 6. IR spectrum of the nitrosamine compound P07

[0088]

[0089]

[0090] Example 3

[0091] Preparation of the nitrosamine compound P08

[0092]

[0093] Into a 100 mL three-necked flask, 4.0 g of compound M02 (varenicline) and 45 mL of tetrahydrofuran (THF) were added and stirred until dissolved; 8.2 mL of an aqueous NaNO2 solution (concentration 0.4 g / mL) and 2.9 g of acetic acid were added, and the temperature was raised to 52 °C. After monitoring the reaction by TLC and completion, a solid precipitated. It was filtered and dried under vacuum to obtain the nitrosamine compound P08 with a purity of 98.87% (area normalization method).

[0094]

[0095] Of the nitrosamine compound P08 1 H-NMR, 13 C-NMR and IR spectral data are shown in Tables 7 to 9 respectively; M+H + = 241.20; UV: The maximum absorption wavelength in the acetonitrile solution is 204.40 nm.

[0096] Table 7. H-NMR spectrum of the nitrosamine compound P08 1 H-NMR spectrum

[0097]

[0098] Table 8, 13 C-NMR spectrum of nitrosamine compound P08

[0099]

[0100]

[0101] Table 9, IR spectrum of nitrosamine compound P08

[0102] <![CDATA[Absorption wave number (cm -1 )]]> Attribution 3048.8 Ar-H 2961.4,2931.8 Saturated C-H stretching vibration 1925.2 Benzene ring C=C stretching vibration 1575.2 N=O stretching vibration 885.2 Benzene ring C-H bending vibration

[0103] Example 4

[0104] Chinese Patent Application No. 202010698570.8 (Title of Invention: A Preparation Method of a Varenicline Intermediate, Varenicline and Its Salt) with an application date of July 20, 2020, the entire content of which is incorporated herein by reference in its entirety.

[0105] 1. Preparation of the varenicline intermediate

[0106]

[0107] ① Add 3 kg (about 8.69 mol) of compound A and 0.35 kg of palladium on carbon (Pd / C, Pd 5%) catalyst to a reaction kettle containing 36 kg of isopropanol and 15 kg of purified water. Control the reaction temperature at 25 - 35°C, introduce hydrogen and stir for 6 h, then stop the reaction (the remaining amount of compound A in the reaction solution detected by HPLC ≤ 0.5%). Filter through diatomaceous earth to remove the palladium on carbon catalyst to obtain a reaction solution containing compound B.

[0108]

[0109] ② Under the protection of inert gas (nitrogen in this example), 75 g of sodium bicarbonate (about 0.89 mol, alkaline) was added to the reaction solution obtained in step ①, and then slowly added to an aqueous solution of glyoxal (0.556 kg (about 9.58 mol) of glyoxal and 6.5 kg of water, acidic). The reaction temperature was controlled at 20 - 30 °C, and the reaction was stirred for 8 h (including the addition time of the aqueous glyoxal solution), and then the reaction was stopped (the remaining amount of compound B in the reaction solution detected by HPLC ≤ 0.5%). It was distilled under reduced pressure (vacuum degree ≤ -0.09 MPa, 40 - 50 °C) until basically no distillate was produced. 90 kg of purified water was added, the temperature in the kettle was controlled at 20 - 30 °C, and it was kept warm for 2 h. Then it was centrifuged and dried (45 - 50 °C) to obtain 2.34 kg of varenicline intermediate (compound C), which was an off-white solid with an HPLC purity of 99.1%. For the related substances detection items: compound A ≤ 0.1%, compound B ≤ 0.1%, and the content of the largest single unknown impurity ≤ 0.1%.

[0110] 2. Preparation of Varenicline Tartrate

[0111]

[0112] a. Under the protection of inert gas (nitrogen in this example), 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 reaction kettle. The reaction temperature was controlled at 20 - 35 °C, and the reaction was stirred for 8 h, and then the reaction was stopped (the remaining amount of compound C in the reaction solution detected by HPLC ≤ 0.5%). Then 18.5 kg of dichloromethane was added to the reaction kettle, stirred, allowed to stand, and separated. The organic phase was taken, and 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 basically no distillate was produced. 10 kg of absolute ethanol was added, and it was continuously distilled under reduced pressure (vacuum degree ≤ -0.09 MPa, 40 - 50 °C) until basically no distillate was produced to obtain the mother liquor containing varenicline;

[0113]

[0114] b. To the mother liquor containing varenicline obtained in step a, 30 kg of absolute ethanol and 1.16 kg (about 7.73 mol) of L-(+)-tartaric acid were slowly added. The reaction temperature was controlled at 20 - 30 °C, and the reaction was stirred for 16 h (including the addition time of absolute ethanol and L-(+)-tartaric acid), and then the reaction was stopped. Then it was centrifuged and dried (70 - 75 °C) to obtain 2.08 kg of varenicline tartrate, which was an off-white solid.

[0115] Example 5

[0116] According to the requirements of the FDA (Food and Drug Administration, referring to the U.S. Food and Drug Administration), it is necessary to detect nitrosamine impurities in drugs, and generally the requirement is to control them at ≤7.5 ppm.

[0117] For this reason, we creatively developed an ultra-high performance liquid chromatography triple quadrupole mass spectrometry method (UPLC-MS / MS) for the qualitative and / or quantitative detection of nitrosamine impurities (Compound P08) in the varenicline tartrate API of the present invention. The chromatographic conditions and mass spectrometry conditions of UPLC-MS / MS are specifically shown in Table 10.

[0118] Table 10 Chromatographic conditions and mass spectrometry conditions of UPLC-MS / MS

[0119]

[0120]

[0121] After detection, the content of nitrosamine impurity Compound P08 in the varenicline tartrate prepared in Example 4 was 114 ppm (the test results showed that the retention time of this impurity was about 3.47 min); this indicates that if not controlled and treated, and varenicline tartrate is prepared by the conventional method, the content of nitrosamine impurity Compound P08 in the obtained product is usually in the hundreds of ppm level, which cannot meet the FDA's limit requirements for nitrosamine impurities.

[0122] Example 6

[0123] 1. Preparation of varenicline intermediate

[0124] ① Add 3 kg of Compound A (after detection, the content of nitrosamine impurity Compound P06 in Compound A was 722 ppm) and 0.35 kg of palladium on carbon (Pd / C, Pd 5%) catalyst to a reaction kettle containing 36 kg of isopropanol and 15 kg of purified water. Control the reaction temperature at 25 - 35°C, introduce hydrogen and stir to carry out the reaction. Stop the reaction when the remaining amount of Compound A in the reaction solution detected by HPLC is ≤0.5%. Filter through diatomaceous earth to remove the palladium on carbon catalyst to obtain a reaction solution containing Compound B;

[0125] ② Under the protection of inert gas (nitrogen), 75 g of sodium bicarbonate was added to the reaction solution obtained in step ①, and then slowly added to an aqueous solution of glyoxal (0.56 kg of glyoxal and 6.5 kg of water). The reaction temperature was controlled at 20 - 30 °C, and the reaction was stirred. When the residual amount of compound B in the reaction solution detected by HPLC was ≤ 0.5%, the reaction was stopped. The mixture was distilled under reduced pressure (vacuum degree ≤ -0.09 MPa, 40 - 50 °C) until no distillate was produced basically. 90 kg of purified water was added, the temperature in the kettle was controlled at 20 - 30 °C, and the mixture was kept warm for 2 h. Then, it was centrifuged and dried (45 - 50 °C) to obtain compound C.

[0126] 2. Preparation of Varenicline Tartrate

[0127] 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 the previous step) were added to a reaction flask. The reaction temperature was controlled at 20 - 35 °C. When the residual amount of compound C in the reaction solution detected by HPLC was ≤ 0.5%, the reaction was stopped. Then, 18.5 kg of dichloromethane was added to the reaction kettle, and the mixture was stirred, left to stand, and separated. The organic phase was taken, and 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, and then filtered. The filtrate was distilled under reduced pressure (vacuum degree ≤ -0.09 MPa, 40 - 50 °C) until no distillate was produced basically. 10 kg of absolute ethanol was added, and the mixture was continuously distilled under reduced pressure (vacuum degree ≤ -0.09 MPa, 40 - 50 °C) until no distillate was produced basically to obtain the mother liquor containing varenicline.

[0128] b. To the mother liquor containing varenicline obtained in step a, 30 kg of absolute ethanol and 1.16 kg of L-(+)-tartaric acid were slowly added. The reaction temperature was controlled at 20 - 30 °C, and the reaction was stirred for 16 h. Then, the reaction was stopped, and then it was centrifuged and dried (70 - 75 °C) to obtain varenicline tartrate.

[0129] Then, according to the solvent consumption of 8 ml per gram of varenicline tartrate, the prepared varenicline tartrate was added to the pulping solvent, and pulping was carried out at 30 - 40 °C for 1.5 h. Then, it was centrifuged and dried. After detection, in the varenicline tartrate treated by dichloromethane pulping, the content of nitrosamine impurity compound P08 was 5.94 ppm, and the contents of nitrosamine impurity compounds P06 and P07 were not detected (less than the detection limit), meeting the FDA limit requirements for nitrosamine impurities.

[0130] Example 7

[0131] 1. Preparation of Varenicline Intermediate

[0132] ①. Add 3 kg of Compound A (in order to better ensure product quality, generally required: the content of nitrosamine impurity Compound P06 in Compound A ≤ 500 ppm, the detected value is 485 ppm) and 0.35 kg of palladium-carbon (Pd / C, Pd 5%) catalyst into a reaction kettle containing 36 kg of isopropanol and 15 kg of purified water. Control the reaction temperature at 25 - 35 °C, introduce hydrogen gas and stir to carry out the reaction. Stop the reaction when the remaining amount of Compound A in the reaction solution detected by HPLC ≤ 0.5%. Filter through diatomaceous earth to remove the palladium-carbon catalyst, and obtain a reaction solution containing Compound B;

[0133] ②. Under the protection of an inert gas (nitrogen), add 75 g of sodium bicarbonate to the reaction solution obtained in step ①, and then slowly add it to an aqueous solution of glyoxal (0.56 kg of glyoxal and 6.5 kg of water). Control the reaction temperature at 20 - 30 °C, stir and react. Stop the reaction when the remaining amount of Compound B in the reaction solution detected by HPLC ≤ 0.5%. Carry out vacuum distillation (vacuum degree ≤ -0.09 MPa, 40 - 50 °C) until basically no distillate is produced. Add 90 kg of purified water, control the temperature in the kettle at 20 - 30 °C, keep warm for 2 h, then centrifuge and dry (45 - 50 °C) to obtain Compound C.

[0134] 2. Preparation of Varenicline Tartrate

[0135] a. Under the protection of an inert gas (nitrogen), add 20 kg of purified water, 0.81 kg of sodium hydroxide, 8 kg of dichloromethane and 2 kg of Compound C (prepared in the previous step) into a reaction flask. Control the reaction temperature at 20 - 35 °C. Stop the reaction when the remaining amount of Compound C in the reaction solution detected by HPLC ≤ 0.5%. Then add 18.5 kg of dichloromethane into the reaction kettle, stir, let it stand, separate the layers, take the organic phase, extract the aqueous phase with dichloromethane (15 kg × 2), combine the organic phases, add 5 kg of anhydrous sodium sulfate to dry the organic phase, filter, and carry out vacuum distillation on the filtrate (vacuum degree ≤ -0.09 MPa, 40 - 50 °C) until basically no distillate is produced. Add 10 kg of absolute ethanol, and continue vacuum distillation (vacuum degree ≤ -0.09 MPa, 40 - 50 °C) until basically no distillate is produced to obtain a mother liquor containing varenicline;

[0136] b. Slowly add 30 kg of absolute 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 - 30 °C, stir and react for 16 h, then stop the reaction, and then centrifuge and dry (70 - 75 °C) to obtain varenicline tartrate;

[0137] Then, according to the solvent dosage of 8 ml per gram of varenicline tartrate, add the prepared varenicline tartrate to the pulping solvent, pulp at 30-40°C for 1.5 h, then centrifuge and dry to obtain the product. After detection, in the varenicline tartrate treated by ethyl acetate pulping, the content of nitrosamine impurity compound P08 is 3.04 ppm, and the contents of nitrosamine impurity compounds P06 and P07 are not detected (less than the detection limit).

[0138] Example 8

[0139] Preparation of Varenicline Tartrate

[0140] a. Under the protection of an inert gas (nitrogen), add 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) to the reaction flask. Control the reaction temperature at 20-35°C. Stop the reaction when the residual amount of compound C in the reaction solution detected by HPLC is ≤0.5%. Then add 18.5 kg of dichloromethane to the reaction kettle, stir, stand still, separate the liquid, take the organic phase, extract the aqueous phase with dichloromethane (15 kg×2), combine the organic phases, add 5 kg of anhydrous sodium sulfate to the organic phase for drying, filter, and distill the filtrate under reduced pressure (vacuum degree ≤ -0.09 MPa, 40-50°C) until basically no distillate is produced. Add 10 kg of absolute ethanol and continue to distill under reduced pressure (vacuum degree ≤ -0.09 MPa, 40-50°C) until basically no distillate is produced to obtain the mother liquor containing varenicline;

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

[0142] Then, according to the solvent dosage of 8 ml per gram of varenicline tartrate, add the prepared varenicline tartrate to the pulping solvent, pulp at 30-40°C for 1.5 h, then centrifuge and dry to obtain the product. After detection, in the varenicline tartrate treated by dichloromethane pulping, the content of nitrosamine impurity compound P08 is 4.47 ppm, and the contents of nitrosamine impurity compounds P06 and P07 are not detected (less than the detection limit).

[0143] Example 9

[0144] Preparation of Varenicline Tartrate

[0145] a. Under the protection of inert gas (nitrogen), add 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) into the reaction flask. Control the reaction temperature at 20 - 35 °C. Stop the reaction when the remaining amount of Compound C in the reaction solution detected by HPLC is ≤ 0.5%. Then add 18.5 kg of dichloromethane into the reaction kettle, stir, let it stand, separate the liquid, take the organic phase. The aqueous phase is extracted with dichloromethane (15 kg × 2), and the organic phases are combined. Add 5 kg of anhydrous sodium sulfate to the organic phase for drying, filter, and distill the filtrate under reduced pressure (vacuum degree ≤ -0.09 MPa, 40 - 50 °C) until basically no distillate is produced. Add 10 kg of absolute ethanol and continue distilling under reduced pressure (vacuum degree ≤ -0.09 MPa, 40 - 50 °C) until basically no distillate is produced to obtain the mother liquor containing varenicline;

[0146] b. Slowly add 30 kg of absolute 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 - 30 °C, stir and react for 16 h, then stop the reaction. Then centrifuge and dry (70 - 75 °C) to obtain varenicline tartrate;

[0147] Then, according to the solvent dosage of 8 ml per gram of varenicline tartrate, add the prepared varenicline tartrate into the pulping solvent, pulp at 30 - 40 °C for 1.5 h, then centrifuge and dry. After detection, the content of nitrosamine impurity Compound P08 in varenicline tartrate after isopropanol pulping treatment is 2.89 ppm, and the contents of nitrosamine impurities Compound P06 and P07 are not detected (less than the detection limit).

[0148] Example 10

[0149] Preparation of Varenicline Tartrate

[0150] a. Under the protection of inert gas (nitrogen), add 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) into the reaction flask. Control the reaction temperature at 20 - 35 °C. Stop the reaction when the remaining amount of Compound C in the reaction solution detected by HPLC is ≤ 0.5%. Then add 18.5 kg of dichloromethane into the reaction kettle, stir, let it stand, separate the liquid, take the organic phase. The aqueous phase is extracted with dichloromethane (15 kg × 2), and the organic phases are combined. Add 5 kg of anhydrous sodium sulfate to the organic phase for drying, filter, and distill the filtrate under reduced pressure (vacuum degree ≤ -0.09 MPa, 40 - 50 °C) until basically no distillate is produced. Add 10 kg of absolute ethanol and continue distilling under reduced pressure (vacuum degree ≤ -0.09 MPa, 40 - 50 °C) until basically no distillate is produced to obtain the mother liquor containing varenicline;

[0151] b. Slowly add 30 kg of absolute 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 - 30 °C, stir and react for 16 h, then stop the reaction, and then centrifuge and dry (70 - 75 °C) to obtain varenicline tartrate;

[0152] Then, according to the solvent dosage of 8 ml per gram of varenicline tartrate, add the prepared varenicline tartrate to the slurrying solvent, slurry at 30 - 40 °C for 1.5 h, then centrifuge and dry to obtain the product; after detection, the content of nitrosamine impurity compound P08 in varenicline tartrate after n-heptane slurrying treatment is 24.29 ppm.

[0153] Example 11

[0154] Preparation of Varenicline Tartrate

[0155] a. Under the protection of inert gas (nitrogen), add 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) to the reaction flask, control the reaction temperature at 20 - 35 °C, stop the reaction when the residual amount of compound C in the reaction solution detected by HPLC ≤ 0.5%, then add 18.5 kg of dichloromethane to the reaction kettle, stir, stand still, separate the liquid, take the organic phase, extract the aqueous phase with dichloromethane (15 kg × 2), combine the organic phases, add 5 kg of anhydrous sodium sulfate to the organic phase for drying, filter, distill the filtrate under reduced pressure (vacuum degree ≤ -0.09 MPa, 40 - 50 °C) until basically no distillate is produced, add 10 kg of absolute ethanol, and continue to distill under reduced pressure (vacuum degree ≤ -0.09 MPa, 40 - 50 °C) until basically no distillate is produced to obtain the mother liquor containing varenicline;

[0156] b. Slowly add 30 kg of absolute 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 - 30 °C, stir and react for 16 h, then stop the reaction, and then centrifuge and dry (70 - 75 °C) to obtain varenicline tartrate;

[0157] Then, according to the solvent dosage of 8 ml per gram of varenicline tartrate, add the prepared varenicline tartrate to the slurrying solvent, slurry at 30 - 40 °C for 1.5 h, then centrifuge and dry to obtain the product; after detection, the content of nitrosamine impurity compound P08 in varenicline tartrate after ethanol slurrying treatment is 2.60 ppm, and the contents of nitrosamine impurity compounds P06 and P07 are not detected (less than the detection limit).

[0158] Examples 12 - 14

[0159] 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 ≤250 ppm, ≤200 ppm, ≤150 ppm, ≤100 ppm or lower), and increasing the number of times of pulping treatment, etc., we also prepared varenicline tartrate with the content of nitrosamine impurity compound P08 being 0.511 ppm, 0.205 ppm, and 0.118 ppm respectively.

[0160] 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 3.3 kg of compound A (the content of nitrosamine impurity compound P06 is 722 ppm) to the pulping solvent (8.00 kg (11.7 L) of n-heptane and 2.70 kg (3 L) of ethyl acetate), stir for pulping at 15 - 35 °C for 4 - 5 h, then centrifuge, wash the filter cake with water, centrifuge again, and dry. After detection, the content of nitrosamine impurity compound P06 in compound A after pulping treatment is 0.03% (315 ppm). This shows that through one-time above-mentioned pulping treatment, the content of nitrosamine impurity compound P06 in compound A can be reduced by at least more than half.

[0161] The working principle of the pulping treatment is: Utilize that the pulping solvent can dissolve the trace impurities in the target compound and make them remain in the liquid, while the insoluble target compound remains in solid form, and then through solid-liquid separation, thereby reducing the impurity content in the target compound.

[0162] Example 15

[0163] Use UPLC-MS / MS to detect nitrosamine impurity compound P08 in varenicline tartrate bulk drug and conduct method validation.

[0164] 1. Solution preparation

[0165] Diluent: Purified water

[0166] Reference stock solution: Weigh 19.970 mg of nitrosamine compound P08 (purity ≥98%), place it in a 10 ml volumetric flask, add 8 ml of acetonitrile, and then add diluent to dissolve and dilute to the scale, and mix well.

[0167] Reference mother solution: Pipette 37.5 μl of the reference stock solution into a 10 ml volumetric flask, dilute to the scale with diluent, and mix well.

[0168] 2. System suitability

[0169] System suitability solution (100% limit level reference solution): Pipette approximately 100.0 μl of the reference mother solution into a 10 ml volumetric flask, dilute to the scale with diluent, and mix well.

[0170] Six consecutive injections of the system suitability solution were made, and the RSD of the peak areas of the target in the six injections of the system suitability solution was calculated, with the result being 4%, indicating good system suitability.

[0171] 3. Linearity

[0172] Standard curve solutions: 50.0 μl, 100.0 μl, 150.0 μl, and 200.0 μl of the stock reference solution were separately taken and placed in four 10-ml volumetric flasks, diluted to the mark with the diluent, and mixed well, serving as linear solutions L-3, L-4, L-5, and L-6 in sequence. 800 μl of the L-3 solution was taken into a 10-ml volumetric flask, diluted to the mark with the diluent, and mixed well, serving as L-2. 1000 μl of the L-2 solution was taken into a 10-ml volumetric flask, diluted to the mark with the diluent, and mixed well, serving as L-1.

[0173] The standard curve solutions were separately injected for analysis. With the concentration (X) of the target as the abscissa (unit: ng / ml) and the peak area (Y) as the ordinate, 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 r = 0.999, indicating good linearity, and it showed good linearity in the concentration range of 0.2951 ng / ml to 147.6 ng / ml (i.e., 0.02951 ppm to 14.76 ppm).

[0174] 4. Limit of Detection (LOD)

[0175] LOD solution: 5000 μl of the L-1 solution was taken into a 10-ml volumetric flask, diluted to the mark with the diluent, and mixed well, serving as the LOD solution.

[0176] Two consecutive injections of the LOD solution were made, 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, indicating that the method was sensitive; LOD = 0.01476 ppm.

[0177] 5. Limit of Quantitation (LOQ)

[0178] Six consecutive injections of the quantitation limit solution (L-1 solution) were made, and the S / N of the target peak was 23, 24, 17, 12, 24, and 14 respectively, meeting the requirement of not less than 10, indicating that the method was sensitive; LOQ = 0.02951 ppm.

[0179] 6. Accuracy

[0180] Sample solution: Approximately 100 mg of the sample (e.g., varenicline tartrate API purchased or synthesized) was weighed and placed in a 10-ml volumetric flask, diluted to the mark with the diluent, and mixed well. Two parallel samples were prepared.

[0181] Accuracy solution: Weigh approximately 100 mg of the sample and place it in a 10-ml volumetric flask. Add 50.0 μl of the reference stock solution (50% limit level), 100.0 μl (100% limit level), and 150.0 μl (150% limit level) respectively, then dilute to the mark with the diluent and mix well. Three replicates are prepared for each accuracy level.

[0182] The results showed that the recovery rate of the accuracy solution at the 50% limit level was between 95% and 102%, and the RSD was 4%; the recovery rate of the accuracy solution at the 100% limit level was between 90% and 103%, and the RSD was 7%; the recovery rate of the accuracy solution at the 150% limit level was between 98% and 100%, and the RSD was 1%, meeting the acceptance criteria and indicating good accuracy.

[0183] 7. Repeatability

[0184] Weigh approximately 100 mg of the sample and place it in a 10-ml volumetric flask. Add 100.0 μl of the reference stock solution, dilute to the mark with the diluent, and mix well. Six replicates are prepared.

[0185] The results showed that the RSD of the content of nitrosamine impurity compound P08 in the six repeatability solutions was 6%, meeting the acceptance criteria and indicating good repeatability.

[0186] 8. Specificity

[0187] Inject the blank solution (diluent), the reference solution at the 100% limit level, the sample solution (the same as the sample solution in "7. Repeatability"), and the accuracy solution at the 100% limit level for analysis respectively.

[0188] The results showed that the retention time of the target substance in the sample solution, the reference solution at the 100% limit level, and the accuracy solution at the 100% limit level was all 3.47 min, and there was no obvious interference at the peak position of the target substance, meeting the requirements of resolution and indicating good specificity.

[0189] 9. Intermediate precision

[0190] Intermediate precision solution: Weigh approximately 100 mg of the sample and place it in a 10-ml volumetric flask. Add 100.0 μl of the reference stock solution, dilute to the mark with the diluent, and mix well. Six replicates are prepared. Add the six solutions in "7. Repeatability".

[0191] Calculate the RSD of the concentrations of the twelve solutions in total, including the six intermediate precision solutions and the six repeatability solutions, which is 9%, meeting the requirement of not exceeding 15% and the acceptance criteria, indicating good intermediate precision.

[0192] 10. Solution stability

[0193] The 100% limit level accuracy solution was placed at 15°C and sampled for analysis at regular intervals. The concentration ratio of each time point to the zero point was calculated, and this ratio was between 96% and 113% within 21 hours.

[0194] 11. Durability

[0195] The concentration values of the target substance in the 100% limit level accuracy solution were investigated respectively when the chromatographic column temperature changed by ±2°C, the initial proportion of the organic phase changed by ±1%, and the flow rate changed by ±0.01 ml / min. Two injections were made under each condition, and the RSD of the concentration values of the target substance in 6 solutions was between 4% and 6% during the fluctuation of the same parameter, indicating good method durability.

[0196] According to the following formula, the content of nitrosamine impurity compound P08 in the sample (varenicline tartrate API) was calculated, and the detection was carried out twice and the average value was taken:

[0197] Content of P08: C (ppm) = Concentration of P08 in the sample solution (ng / ml) / [m (mg) / V (ml)]

[0198] m: Mass of the sample, mg;

[0199] V: Dilution volume of the sample, ml.

[0200] Example 16

[0201] The liquid chromatography-tandem mass spectrometry method under the conditions described in Table 11 was used for the qualitative and / or quantitative detection of nitrosamine impurity compound P07 in the varenicline tartrate API of the present invention, and the method validation was carried out and passed.

[0202] Table 11. Chromatographic conditions and mass spectrometry conditions for detecting nitrosamine impurity compound P07

[0203]

[0204] 1. Solution preparation

[0205] Blank (diluent): Water - acetonitrile (95:5)

[0206] Reference stock solution: Weigh 25 mg of nitrosamine compound P07 (purity ≥ 95%), place it in a 50 ml volumetric flask, dissolve it with acetonitrile, and make up to the mark and mix well (solution Ⅰ). Take 1.0 ml of solution Ⅰ and place it in a 100 ml volumetric flask, add the diluent to make up to the mark and mix well to obtain the reference stock solution.

[0207] Reference solution: Take 0.45 ml of the reference stock solution, place it in a 100 ml volumetric flask, add the diluent to make up to the mark and mix well to obtain it.

[0208] Sample solution: Take about 100 mg of varenicline tartrate raw material, place it in a 10-ml volumetric flask, add diluent to dissolve and make up to the mark, and mix well.

[0209] 2. System suitability

[0210] For 5 consecutive injections of the reference solution, the RSD of the peak area of nitrosamine impurity compound P07 ≤ 15%, the RSD of the retention time ≤ 2.0%, and it can be well separated from the adjacent peaks, indicating good system suitability.

[0211] 3. Linearity

[0212] Prepare reference 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 (calculated as nitrosamine compound P07);

[0213] Inject the above reference solutions with different concentrations for analysis respectively. Take the concentration (X) of the target substance as the abscissa (unit: μg / ml) and the peak area (Y) as the ordinate for linear regression, and calculate the linear equation and linear correlation coefficient r; the results show that the linear equation is: Y = 3000000X + 2671.7, and the linear correlation coefficient r = 0.998, indicating good linearity, and it shows good linearity in the concentration range of 0.002688 μg / ml to 0.100783 μg / ml (i.e., 0.2688 ppm to 10.0783 ppm).

[0214] 4. Limit of detection (LOD)

[0215] LOD = 0.1344 ppm, and the S / N (signal-to-noise ratio) of the target peak is 14.8, 13.1, etc., meeting the requirement of being greater than 3, indicating that the method is sensitive.

[0216] 5. Limit of quantitation (LOQ)

[0217] LOQ = 0.2688 ppm, and the S / N of the target peak is 20.5, 24.9, etc., meeting the requirement of being greater than 10, indicating that the method is sensitive.

[0218] 6. Specificity

[0219] The results show that the retention time of nitrosamine impurity compound P07 is about 14.487 min, and there is no interference between adjacent peaks, meeting the requirement of resolution, indicating good specificity.

[0220] In addition,

[0221] It has been verified that the accuracy, precision, repeatability, durability, etc. of this detection method also meet the regulations and requirements of methodological verification, and can well achieve the qualitative and / or quantitative detection of nitrosamine impurity compound P07 in varenicline tartrate raw material drug.

[0222] Example 17

[0223] The liquid chromatography-tandem mass spectrometry method under the conditions described in Table 12 was used for the qualitative and / or quantitative detection of nitrosamine impurity compound P06 in the varenicline tartrate raw material drug of the present invention, and methodological verification was carried out and passed.

[0224] Table 12. Chromatographic conditions and mass spectrometry conditions for detecting nitrosamine impurity compound P06

[0225]

[0226] 1. Solution preparation

[0227] Blank (diluent): Purified water

[0228] Reference stock solution: Weigh 25 mg of nitrosamine compound P06 (purity ≥ 92%), place it in a 50 ml volumetric flask, dissolve it with acetonitrile, make up to the mark, and mix well (solution II). Take 1.0 ml of solution II, place it in a 100 ml volumetric flask, add diluent to make up to the mark, and mix well to obtain the reference stock solution.

[0229] Reference solution: Take 4.5 ml of the reference stock solution and place it in a 50 ml volumetric flask, add diluent to make up to the mark, and mix well to obtain it.

[0230] Sample solution: Take about 1000 mg of varenicline tartrate raw material drug, place it in a 20 ml headspace vial, accurately measure 5.0 ml of diluent to dissolve it, and mix well.

[0231] 2. System suitability

[0232] For 5 consecutive injections of the reference solution, the RSD of the peak area of nitrosamine impurity compound P06 ≤ 15%, the RSD of the retention time ≤ 2.0%, and it can be well separated from the adjacent peaks, and the system suitability is good.

[0233] 3. Linearity

[0234] Inject reference solutions with different concentrations for analysis respectively. Take the concentration (X) of the target substance as the abscissa (unit: μg / ml) and the peak area (Y) as the ordinate for linear regression, and calculate the linear equation and linear correlation coefficient r; the results show that the linear correlation coefficient r > 0.99, and the linear relationship is good.

[0235] 4. Limit of detection (LOD)

[0236] The LOD is 0.0145 ppm, and the S / N (signal-to-noise ratio) of the target peak is 5.8, meeting the requirement of being greater than 3, indicating that the method is sensitive.

[0237] 5. Limit of Quantitation (LOQ)

[0238] The LOQ is 0.0289 ppm, and the S / N of the target peak is 12.0, meeting the requirement of being greater than 10, indicating that the method is sensitive.

[0239] 6. Specificity

[0240] The results show that the retention time of the nitrosamine impurity compound P06 is about 14.648 min, and there is no interference between adjacent peaks, meeting the requirement of resolution, indicating good specificity.

[0241] In addition,

[0242] After verification, the accuracy, precision, repeatability, durability, etc. of this detection method also meet the regulations and requirements of the methodology verification, and can well achieve the qualitative and / or quantitative detection of the nitrosamine impurity compound P06 in the varenicline tartrate API.

[0243] Example 18

[0244] In order to better ensure the limit requirements of nitrosamine impurity compounds (including: P06, P07, P08; among them, P08 is converted from P06 via the intermediate P07; therefore, P08 is the main existing form of nitrosamine impurities in the varenicline tartrate API or preparation, P06 is the main existing form of nitrosamine impurities in raw materials such as compound A, and P07 is an intermediate in the preparation process) in the varenicline tartrate API or preparation, it is necessary to conduct qualitative and / or quantitative detection of the nitrosamine impurity compound P06 in the starting material compound A. For example, high performance liquid chromatography is used, and the chromatographic conditions are shown in Table 13.

[0245] Table 13. Chromatographic conditions for detecting the nitrosamine impurity compound P06 in compound A

[0246]

[0247] 1. Preparation of the standard curve

[0248] Blank (diluent): Acetonitrile - water (60:40)

[0249] Reference solution: Weigh 25 mg of the nitrosamine compound P06 (purity ≥ 92%), place it in a 25 ml volumetric flask, add the diluent to dissolve and make up to the mark, and mix well to obtain the reference solution.

[0250] Prepare reference 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 (calculated based on nitrosamine compound P06) respectively;

[0251] Inject the above reference solutions with different concentrations for analysis respectively. Using the concentration (X) of the target substance as the abscissa (unit: mg / ml) and the peak area (Y) as the ordinate, perform linear regression to calculate the linear equation and the linear correlation coefficient r. The results show that the linear equation is: Y = 228.94X + 0.0025, and the linear correlation coefficient r = 0.999, indicating good linearity and a good linear relationship within this concentration range.

[0252] Limit of detection: LOD = 0.004% (i.e., 40 ppm), and the S / N (signal-to-noise ratio) of the target peak is 6.4, meeting the requirement of being greater than 3, so the method is sensitive.

[0253] Limit of quantitation: LOQ = 0.014% (i.e., 140 ppm), and the S / N of the target peak is 18.9, meeting the requirement of being greater than 10, so the method is sensitive.

[0254] Specificity: The test results of the mixed solution of the sample and the reference solution show that the retention time of nitrosamine impurity compound P06 is about 4.592 min (resolution R = 13.18), the retention time of compound A is about 7.587 min (resolution R = 6.82), the retention time of impurity M01 is about 9.373 min (resolution R = 7.24), and the retention time of impurity M02 is about 12.45 min (resolution R = 5.25). There is no interference between each peak, meeting the requirement of resolution, and the specificity is good.

[0255] In addition,

[0256] After verification, the accuracy, precision, repeatability, durability, etc. of this detection method also meet the regulations and requirements of the methodology verification, and it can be used for the qualitative and / or quantitative detection of nitrosamine impurity compound P06 in compound A.

[0257] 2. Detect the sample

[0258] Sample solution: Take about 25 mg of compound A and place it in a 25-ml volumetric flask. Add diluent to dissolve and make up to the mark, and mix well.

[0259] Inject the sample solution for detection to obtain its peak area data and chromatogram (see: Figure 1) Substitute the peak area data into the linear equation obtained from the reference solution above to calculate the concentration data of nitrosamine impurity compound P06 in the test sample solution.

[0260] Then, calculate the content of nitrosamine impurity compound P06 in the sample (compound A) according to the following formula:

[0261] P06 content: C (expressed in % or ppm) = concentration of P06 in the sample solution (mg / ml) / [m (mg) / V (ml)]

[0262] m: mass of the sample, mg;

[0263] V: dilution volume of the sample, ml.

[0264] Example 19

[0265] Detect whether nitrosamine impurity compound P08 has genotoxicity (able to cause mutations or cancer).

[0266] 1 Materials and Methods

[0267] 1.1 Experimental Strains

[0268] Salmonella typhimurium TA97a, TA98, TA100, TA102, TA1535, source: MOLT OX Company, USA, purchased from Shanghai Baolu Biotechnology Co., Ltd. Use the Salmonella typhimurium strains that meet the requirements after identification for the test.

[0269] 1.2 Metabolic Activation System

[0270] Rat liver S9, source: Shanghai Baolu Biotechnology Co., Ltd.

[0271] 1.3 Test Substances

[0272] Name: Nitrosamine impurity compound P08, property: white powder.

[0273] 1.4 Main Instruments and Reagents

[0274] X SR-204 electronic balance (Mettler Company, Switzerland); high-pressure steam sterilizer (SANYO Company, Japan); water-bath constant temperature incubator (Shanghai Yiheng Technology Co., Ltd.); Stuart temperature-controlled shaking incubator (Stuart Company, UK); BHC-1300 II A2 biological safety cabinet (Suzhou Antai Air Technology Co., Ltd., Sujing Group).

[0275] Glucose-6-phosphate, source: Beijing J&K Scientific Ltd.; Coenzyme II, source: Sinopharm Chemical Reagent Co., Ltd.; Nutrient Broth, source: Beijing Land Bridge Technology Co., Ltd.; Technical Agar Powder, source: Guangdong Huankai Microbial Sci. & Tech. Co., Ltd.; Sterile Water for Injection, source: Chenxin Pharmaceutical Co., Ltd.; Dexon, source: CHEMSERVICE; Methyl Methanesulfonate, source: Beijing J&K Scientific Ltd.; 2-Aminofluorene, source: Shanghai Jingchun Reagent Co., Ltd.; 1,8-Dihydroxyanthraquinone, source: SIGMA-ALDRICH; Cyclophosphamide, source: SIGMA-ALDRICH; Sodium Azide, source: RIEDEL-SEELIE.

[0276] 1.5 Test method (Ames test plate incorporation method)

[0277] The plate incorporation method was used in the experiment.

[0278] 1.5.1 Dose selection and preparation of test substances

[0279] Dose design: The tests were conducted at 5 mg, 2.5 mg, 1.25 mg, 0.625 mg, 0.3125 mg, and 0.15625 mg per dish.

[0280] Preparation of test substances: This product was dissolved in dimethyl sulfoxide to form a 50 mg / ml solution, and then serially diluted with dimethyl sulfoxide to 25 mg / ml, 12.5 mg / ml, 6.25 mg / ml, 3.125 mg / ml, and 1.5625 mg / ml.

[0281] 1.5.2 Test method.

[0282] 0.1 ml of the frozen stock solutions of TA97a, TA98, TA100, TA102, and TA1535 were respectively inoculated into 10 ml of nutrient broth medium and cultured at 37 °C with shaking (100 times / min) for 10 h. 2.0 ml of the top agar medium containing 0.5 mmol / L histidine - 0.5 mmol / L biotin was dispensed into test tubes, sterilized at 0.103 MPa for 20 min, and kept warm in a 50 °C water bath. Then, 0.1 ml of the test substance solution, 0.5 ml of the S9 mixture (when metabolic activation was required), and 0.1 ml of the bacterial suspension were successively added to each tube, mixed well, and quickly poured onto the bottom agar plate. The plate was rotated to make the mixture evenly distributed. After being placed horizontally until solidified by condensation, it was inverted and incubated in a 37 °C incubator for 48 h, and the number of revertant colonies per dish was counted. Three parallel plates were made for each dose, and a spontaneous revertant group, a solvent control group, and a positive control group were also set up.

[0283] 1.6 Statistical analysis of test data

[0284] Data were expressed as mean ± standard deviation It is indicated that SPSS 19.0 is used for data analysis and calculation.

[0285] 1.7 Result determination

[0286] If the number of revertant colonies of the test substance is twice or more than twice that of the solvent control and shows a dose-response relationship, the test substance is determined to be mutagenic positive; if a positive reaction appears under any dose condition of the test substance and is reproducible, the test substance is determined to be mutagenic positive. If the test substance is negative under both the conditions with and without S9 after being measured by the above test strains, the test substance is mutagenic negative.

[0287] 2 Test results

[0288] The results are shown in Table 14.

[0289] Table 14. Results of the Ames test

[0290]

[0291] Continued Table 14. Results of the Ames test

[0292]

[0293] As can be seen from the results of the Ames test, under the condition without S9, when the dose of the nitrosamine impurity compound P08 is 625 μg / plate, the number of colonies of TA1535 exceeds twice that of the solvent control group. Under the condition with S9, when the dose is 2500 μg / plate, the number of colonies of TA98 exceeds twice that of the solvent control group. When the doses are 5000, 2500, 1250, and 625 μg / plate, the number of colonies of TA1535 exceeds twice that of the solvent control group and shows a dose-response relationship. This indicates that the nitrosamine impurity compound P08 is mutagenic positive to Salmonella typhimurium with or without the metabolic activation system.

[0294] Certainly, the present invention can also have many other implementation manners. Without departing from the spirit and essence of the present invention, those skilled in the art can make various corresponding changes and / or deformations according to the present invention, and these corresponding changes and / or deformations should all fall within the protection scope of the appended claims of the present invention.

Claims

1. A preparation method of a compound represented by Formula II, characterized in that, It includes the following steps: Compound M01 undergoes a nitrosation reaction with nitrite in the presence of an acid and a mixed solvent to form compound P06; optionally, it includes: compound P06 undergoes a reduction reaction with a reducing agent in the presence of a catalyst and a halogenated hydrocarbon solvent to form compound P07; The compound shown in Formula II is compound P06 or compound P07.

2. The method for preparing the compound shown in Formula II according to claim 1, wherein The nitrite is potassium nitrite or sodium nitrite; The acid is acetic acid; The mixed solvent is water and tetrahydrofuran, and the volume ratio of water to tetrahydrofuran is 1:4 - 6; The reducing agent is hydrogen; The catalyst is palladium on carbon catalyst; The halogenated hydrocarbon solvent is a liquid halogen-substituted C1 - C4 alkane.

3. The preparation method of the compound represented by Formula II according to claim 2, characterized in that, The halogenated hydrocarbon solvent is dichloromethane.

4. The method for preparing the compound shown in Formula II according to claim 2, wherein The molar ratio of compound M01 to nitrite is 1:0.5 - 5; The volume-mass ratio of the mixed solvent to compound M01 is 5 - 20 mL / g; The mass ratio of the acid to compound M01 is 0.1 - 1; The reaction temperature of the nitrosation reaction is 30 - 60 °C; The mass ratio of compound P06 to the catalyst is 1:0.1 - 0.5; The volume-mass ratio of the halogenated hydrocarbon solvent to compound P06 is 5 - 20 mL / g; The reaction temperature of the reduction reaction is 10 - 40 °C.

5. The preparation method of the compound represented by Formula II according to claim 4, wherein The molar ratio of compound M01 to nitrite is 1:2 - 4.

6. The preparation method of the compound represented by Formula II according to claim 4, wherein, The volume-mass ratio of the mixed solvent to compound M01 is 10 - 15 mL / g.

7. The preparation method of the compound shown in Formula II according to claim 4, characterized in that, The mass ratio of the acid to compound M01 is 0.4 - 0.

8.

8. The preparation method of the compound shown in Formula II according to claim 4, characterized in that, The mass ratio of compound P06 to the catalyst is 1:0.2 - 0.

3.

9. The preparation method of the compound represented by Formula II according to claim 4, characterized in that, The volume-mass ratio of the halogenated hydrocarbon solvent to compound P06 is 10 - 15 mL / g. The application of the compound shown in Formula II as a reference standard, control or detection item in the impurity research, quality control or detection method of varenicline tartrate; Among them, R 11 and R 12 are independently selected from nitro or R 11 and R 12 are independently selected from amino; The detection method is liquid chromatography-tandem mass spectrometry.

11. Detection method of the compound shown in Chinese style II of varenicline tartrate, characterized in that, Using the compound shown in Formula II as a reference standard or control, and detecting by liquid chromatography-tandem mass spectrometry; Among them, R 11 , R 12 are independently selected from nitro or R 11 , R 12 are independently selected from amino.

12. The detection method of the compound shown in Form II of varenicline tartrate according to claim 11, characterized in that, The chromatographic conditions of the liquid chromatography-tandem mass spectrometry include: Chromatographic column: C18 chromatographic column; Mobile phase: mobile phase A - mobile phase B, the volume ratio of mobile phase A to mobile phase B is 55%:45% - 98%:2%; mobile phase A is formic acid-purified water or ammonium acetate aqueous solution, and mobile phase B is acetonitrile.

13. The detection method of the compound shown in the Chinese style II of varenicline tartrate according to claim 12, wherein, The chromatographic column is Waters Xbridge C18.

14. The detection method of the compound shown in the Chinese formula II of varenicline tartrate according to claim 12, characterized in that, The chromatographic conditions of the liquid chromatography-tandem mass spectrometry also include: The specification of the chromatographic column is: 4.6 mm × 150 mm, 3.5 μm; Column temperature: 15 - 35 °C; Flow rate: 0.1 - 2 ml / min; Injection volume: 1 - 35 μl.

15. The detection method of the compound shown in Chinese style II of varenicline tartrate according to claim 14, characterized in that, The column temperature is 20 °C, 25 °C, 30 °C or 32 °C.

16. The detection method of the compound shown in the Chinese formula II of varenicline tartrate according to claim 14, characterized in that, The flow rate is 0.2ml / min, 0.3ml / min, 0.4ml / min, 0.5ml / min, 0.6ml / min, 0.7ml / min, 0.8ml / min, 0.9ml / min, 1ml / min, 1.2ml / min or 1.5ml / min.

17. The detection method of the compound shown in Chinese style II of varenicline tartrate according to claim 14, characterized in that, The injection volume is 2μ1, 3μ1, 4μ1, 5μ1, 6μ1, 7μ1, 8μ1, 9μ1, 10μ1, 15μ1, 20μ1, 25μ1 or 30μ1.

18. The detection method of the compound shown in Chinese formula II of varenicline tartrate according to any one of claims 11 to 17, characterized in that, The mass spectrometry conditions of the liquid chromatography-tandem mass spectrometry method include: Ion source: ESI or APCI; Positive ion mode; Scan mode: SIM, SRM or MRM.

19. The detection method of the compound shown in Form II of varenicline tartrate according to claim 18, characterized in that, The ion source was ESI.

20. The detection method of the compound shown in Chinese style II of varenicline tartrate according to claim 18, characterized in that, The scanning mode was SIM or MRM.

21. The detection method of the compound shown in Chinese formula II of varenicline tartrate according to claim 18, characterized in that, The mass spectrometry conditions of the liquid chromatography-tandem mass spectrometry method also include: ion source temperature: 350° C.; capillary voltage: 6000 V; and spray gas flow rate: 13 L / min.

22. The detection method of the compound shown in Chinese style II of varenicline tartrate according to any one of claims 11 to 17, characterized in that, The compound shown in Formula II is When, the volume ratio of mobile phase A to mobile phase B is 55%:45% to 75%:25%, and the mobile phase A is formic acid-purified water, and the volume ratio of formic acid to purified water is 0.05 to 0.2:

100.

23. The detection method of the compound shown in the Chinese style II of varenicline tartrate according to claim 22, characterized in that, The volume ratio of mobile phase A to mobile phase B was 63%:37%, and the volume ratio of formic acid to purified water was 0.1:

100.

24. The detection method of the compound shown in Chinese formula II of varenicline tartrate according to any one of claims 11 to 17, characterized in that, The compound shown in Formula II is When, the volume ratio of mobile phase A to mobile phase B is 90%:10% to 98%:2%, the mobile phase A is an aqueous ammonium acetate solution, the concentration of the aqueous ammonium acetate solution is 0.005 to 0.02 mol / L, and the pH is adjusted to 2 to 3 with formic acid.

25. The detection method of the compound shown in Form II of varenicline tartrate according to claim 24, characterized in that, The volume ratio of mobile phase A to mobile phase B is 95%:5%, the concentration of the aqueous ammonium acetate solution is 0.01 mol / L, and formic acid is used to adjust the pH to 2.

5.

26. The detection method of the compound shown in Formula II of varenicline tartrate according to any one of claims 11 to 17, characterized in that, The detection method is qualitative detection or quantitative detection, the solvents of the standard substance, the reference substance and varenicline tartrate are water or water-acetonitrile, and the volume ratio of water to acetonitrile is 90%:10%-98%:2%.

27. The detection method of the compound shown in Chinese formula II of varenicline tartrate according to claim 26, characterized in that, The volume ratio of water to acetonitrile was 95%:5%.

28. The detection method of the compound shown in Chinese formula II of varenicline tartrate according to claim 26, characterized in that, The internal standard method or external standard method was used for quantitative detection.

29. The method for detecting the compound of formula II of varenicline tartrate according to claim 28, characterized in that: The external standard method is: using the compound represented by formula II 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 II in the varenicline tartrate test sample solution is substituted into the linear equation to obtain the concentration data of the compound represented by Formula II in the test 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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